Keywords: Cancer gastrique, Cellules souches cancéreuses, LIF, EMT, Hippo, Métastases Gastric cancer, Cancer stem Cells, LIF, EMT, Hippo, Metastasis BRIC) -Inserm UMR1312 Team 4 -Helicobacter-associated digestive cancers, cancer stem cells

Ciblage de la voie de signalisation Hippo dans les cellules souches cancéreuses gastriques

Résumé:

Le cancer gastrique (GC), 4 ème cause de mortalité par cancer dans le monde, est un cancer de mauvais pronostic. Les tumeurs gastriques sont des entités hétérogènes constituées d'une petite sous population de cellules souches cancéreuses (CSCs), à l'origine de l'initiation et la croissance tumorale, ainsi que de la résistance aux traitements. Le GC est souvent diagnostiqué tardivement, à un stade métastatique, trop avancé pour les résections chirurgicales. Une partie des CSCs est aussi capable d'initier des métastases de par leur propriétés plastiques leur permettant de changer de phénotype cellulaire très rapidement. Le ciblage et la caractérisation de ces CSCs sont donc cruciaux au traitement du GC.

En conséquence, la première partie de ma thèse a consisté au ciblage des CSCs en agissant sur les voies de signalisations cellulaires importantes au maintien de leur propriétés tumorigéniques. Pour cela, les deux stratégies choisies étaient l'activation des kinases de la voie et l'inhibition des effecteurs oncogènes YAP/TAZ-TEAD. Premièrement, le Leukaemia Inhibitory Factor (LIF) a été utilisé pour activer les kinases de la voie Hippo MST1/2 et LATS1/2. L'analyse de la formation de tumeursphères, de l'invasion 2D et 3D, et de l'expression des marqueurs associés aux CSCs et à la transition épithéliomésenchymateuse (EMT) démontre que le LIF présente des propriétés anti-tumorigéniques, anti-chimiorésistantes, anti-invasives et anti-EMT sur les CSCs provenant de lignées cellulaires de GC et de cellules de tumeurs-dérivées de patients (PDX). De plus, le LIF améliore les effets anti-CSCs des chimiothérapies telles que la doxorubicine et le 5fluorouracile et réduit l'activité des facteurs de transcription associés à l'EMT, ZEB1 et SNAIL. L'utilisation d'un inhibiteur de kinases de la voie Hippo MST1/2, XMU-MP-1, a démontré la participation de la voie Hippo aux effets anti-CSC du LIF. De plus, il a été démontré que les cellules préalablement traitées au LIF ont une capacité moindre à former des métastases dans des modèles in vivo d'injection intracardiaque. Par ailleurs, la Verteporfine (VP) a été utilisée en tant que stratégie d'inhibition des effecteurs oncogéniques de la voie Hippo de par sa capacité à altérer les interactions entre YAP/TAZ et ~1~ le facteur de transcription TEAD. La VP diminue la prolifération des cellules GC ainsi que la population de CSCs et leurs marqueurs. Elle inhibe aussi la croissance tumorale dans des modèles de xénogreffes sous-cutanées et de manière intéressante les cellules résiduelles ne sont plus capables de former des tumeursphères ex vivo confirmant la diminution du pool de CSCs au sein des tumeurs. La deuxième partie de mon projet a consisté en la caractérisation des CSCs et plus particulièrement celles qui circulent, les cellules tumorales circulantes (CTCs) ; ceci dans le but de trouver de nouvelles stratégies de diagnostic, pronostic et de thérapie. Les CTCs, récupérées à partir de sang de patients atteints de GC, sont retrouvées seules ou en groupes dans le sang, par exemple regroupées avec des cellules immunitaires. En conséquence, une partie de ce projet, en collaboration avec l'équipe de N. Larmonier (Immunoconcept), a été d'explorer le rôle de l'interaction cellules myéloïdes suppressives (MDSCs)-CSCs et son importance dans les propriétés tumorigéniques et invasives des CSCs. Des expériences d'invasions et de tumeursphères ont démontré un premier effet pro-tumorigénique et proinvasif des MDSCs sur les CSC gastriques. Dans l'ensemble, mon travail de thèse a exploré le ciblage de la voie Hippo comme stratégie thérapeutique anti-CSC dans le GC. Le LIF et la VP, deux méthodes visant différentes parties de la voie, représentent des approches intéressantes dans le traitement des CSCs tumorigéniques, chimio-résistantes et invasives. Enfin, le travail sur la caractérisation des CTCs et de leurs partenaires donnent des pistes pour le développement de thérapies contre les GC métastatiques.

⁓ Introduction ⁓

Gastric Cancer

Cancer is a deadly disease accounting for approximately 10 million deaths worldwide annually 1 . This bad prognosis is related to the fact that cancer diagnosis, treatment and patient care remains a challenge. Despite the arousal of different anti-cancer strategies such as immuno-, hormone-, chemo-, radio-therapies and various biomarkers-dependent targeted approaches, cancer patients' treatment management is not very efficient, especially in some types of cancers where socio-economic impact is critical 2 .

Gastrointestinal cancers (colorectal, stomach, liver, oesophagus, pancreas) are responsible for 1/4 th cancer cases and 1/3 rd cancer deaths worldwide 3 . Among these, gastric cancer (GC) ranks 2 nd in terms of both incidence and mortality behind colorectal cancer (Figure 1), making it a major health concern.

Figure 1: Graphical representation of gastrointestinal cancers incidence and mortality.

Estimated incidence and mortality data for the World's population is represented 3 . These values were updated in 2020.

It is the 9 th cause of death worldwide among upper-middle-income countries according to WHO Global Health Estimates 2020 4 . Though GC appears as 14 th and 10 th concerning new cases and deaths respectively in France, in my country-of-origin Mauritius, it still represents the 5 th most common and deadliest cancer 1 (Figure 2). Multiple factors are accountable in this bad prognosis disease and understanding the underlying mechanisms is a priority in proper therapy research and patient management. An overview of gastric carcinogenesis is given in the following review, with a focus on actors participating in this affliction and the mechanisms behind, therapeutical strategies that could arise and potential diagnosis tools available today 2 .

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Histological and Molecular Classifications

Gastric cancer is the most common stomach malignancy among lymphomas, sarcomas, gastrointestinal stromal and neuroendocrine tumours. This heterogeneous disease has different phenotypes and most cases are gastric adenocarcinomas (GC) [1]. The two main GC, according to topographic sites, are the cardia and the non-cardia GC. The complexity of GC explains the diverse histological classification systems which exist [2,3].

The most commonly used classification is the Laurén classification which divides GC into intestinal, diffuse and intermediate subtypes, according to their histological phenotypes. Intestinal type GC, characterised by malignant cohesive epithelial cells and intestinal-type glandular differentiation infiltrating the tissue, is the most common type occurring in about 54% cases while diffuse type GC, found in about 32% cases, contains poorly differentiated and poorly cohesive tumour cells [2]. GC can also be classified since 2010, according to the WHO classification, into tubular, papillary, mucinous, poorly cohesive (including signet ring cell carcinoma) and mixed carcinomas. While the poorly cohesive including signet ring cell carcinomas corresponds to the diffuse subtype of the Laurén classification, the tubular, papillary and mucinous types correspond to the intestinal subtype [2]. The Carneiro system distinguishes 4 categories of gastric tumours: glandular, isolated cell, solid and mixed, based on their immunophenotype such that it further divides the Laurén intestinal subtype into tumours with intestinal, gastric or mixed differentiation, according to the expression of specific markers (MUC6, MUC5AC mucins and TFF1 peptide gastric markers or MUC2, CDX2, CD10 and pepsinogen-1 intestinal markers, amongst others) [3]. Finally, the Goseki classification also divides GC into 4 groups according to intracellular mucin production (poor or rich) and level of tubular differentiation (poorly or well differentiated) [2].

GC vary not only histologically, but also molecularly. The most commonly mutated gene in GC (40% cases) is the tumour suppressor gene TP53, key regulator of cell genomic stability [4]. Diffuse type GC are often characterised by somatic or hereditary mutations of CDH1 gene, coding for E-cadherin, a cell junction protein whose invalidation participates to the "independent cell" GC phenotype. Epigenetic alterations such as gene inactivationassociated hyper-methylations in CpG islands can be observed in certain cancer-related genes (APC, K-RAS, hMLH1, CDKN2A). This CpG island methylator phenotype (CIMP) is an early event in GC and can be found in contiguous normal tissue associated with H. pylori infection [2,5,6].

Based on these molecular differences, the Cancer Genome Atlas Research Network (TCGA) has proposed a molecular classification dividing GC into 4 groups: Epstein Barr Virus (EBV)-positive (EBV), microsatellite instable (MSI), genomically stable (GS) and chromosomal instability (CIN) [7]. Array-based somatic copy analysis, whole-exome sequencing, array-based DNA methylation profiling, mRNA and miRNA sequencing, reverse-phase protein assay, microsatellite instability testing and whole-genome sequencing, used on 295 tumour samples in comparison with the germline profile, showed 8.8% EBV, 21.7% MSI, 19.7% GS and 49.8% CIN cases. The last subtype is mainly associated to the intestinal-type histology [7]. The Asian Cancer Research Group (ACRG) also proposed a 4-group classification based on mRNA expression, somatic copy number and targeted gene sequencing: microsatellite instability (MSI), microsatellite stable and epithelial to mesenchymal transition phenotype (MSS/EMT), microsatellite stable and presence of TP53 (MSS/TP53+) or no TP53 signature (MSS/TP53-) [8]. These two molecular classifications use advanced molecular techniques which are not feasible in practice for individual therapy decisions. To overcome this challenge, Setia et al. proposed techniques available in routine diagnostic practice like in situ hybridisation and immunohistochemistry and identified 5 GC groups: EBV-positive (5% cases), mismatch repair-deficient (16%), aberrant E-cadherin expression (21%), aberrant p53 expression (51%) and normal p53 expression (7%) [9]. Furthermore, Li et al. used the TCGA RNA-sequencing data to identify differentially expressed set of genes in diverse tumour types for use as diagnosis biomarkers and drug development [10].

Finally, recent developments in single cell methods allows more precise deciphering of GC cells heterogeneity, though not applicable as routine diagnosis. Zhang et al. propose a GC transcriptome atlas after analysis of 22,677 cells from 3 non-tumorous samples and 9 tumours [11]. Comparison of malignant epithelium to no malignant ones put into evidence overexpression of tumour specific genes like REG4, TFF3, CLDN4 and CLDN7. Genes involved in gastric mucus and enzyme secretion like GKN1, PGC, MUC5AC and LIPF were more expressed in non-tumorous epithelium [11]. In order to compare the cellular genetic profiles obtained to known histopathological classifications, tumour samples with known histology were added to analysis. Analysed malignant cells clustered 5 main cell groups: C1 having 96.9% cells from diffuse-type sample, C2 composed of 97.1% cells from intestinal-type sample, C3 with mixed-type and intestinal-type cells, C4 containing cells mainly from one of the intestinal-type samples and C5 with cells from EBV+ patients. Interestingly, C4/C5 seem to be correspond to novel subtypes, molecularly different from C1, C2 and C3 which correspond to the Lauren classification subtypes [11].

These GC classifications (Figure 1) are the key to the comprehension of this disease and its underlying mechanisms, thus opening new pathways for novel targeted therapies since different GC types can be related to different risk factors, prognosis, and treatment management.

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since different GC types can be related to different risk factors, prognosis, and treatment management.

Epidemiology and Risk Factors

Gastric Cancer is the 5th most common and the 3rd deadliest cancer worldwide, according to the latest report of the International Agency for research on Cancer [12]. Its geographic incidence remains heterogenous with most cases occurring in Eastern Asia (619 226 in 2018), and men being twice as much affected as women. Interestingly, a decrease in GC incidence has been noted in Western Europe and North America over the past decade [13].

Different risk factors are known to affect gastric cancer incidence, among which Helicobacter pylori (H. pylori) infection is the essential one. H. pylori infection involves almost 50% of the world's population and induces chronic inflammation of the gastric mucosa, 5 to 15% of which evolve in gastric and duodenal ulcers and less than 1% in GC. The Correa cascade shows how intestinal type GC begins with H. pylori-induced chronic gastritis followed by gastric atrophy, intestinal metaplasia, and dysplasia [14]. Intestinal GC subtype is mainly caused by environmental factors while diffuse GC subtype also includes hereditary and genetic factors. About 10% intestinal GC cases are associated to infection with EBV and interestingly, EBV-positive GC are mostly associated to MSI molecular type and are related to less worst survival prognosis than diffuse/mixed GC subtypes [15,16].

The overall decrease in GC incidence can mostly be attributed to the decrease in intestinal type GC due to a lower H. pylori prevalence and modified dietary habits [13,17]. The better comprehension of intestinal type GC over the past decade has largely contributed to this, and the Correa cascade has provided a window for its prevention and early detection. Studies show that H. pylori eradication treatments, both as primary and as secondary prevention, reduces the risk of GC development. It also decreases the rate of metachronous GC in patients having early GC and improves the baseline of gastric corpus atrophy grading [18,19].

Conversely, diffuse GC subtype, not only related to H. pylori infection, is not yet well understood, and still has an increasing incidence. Due to its predominant genetic and hereditary origin, diffuse type GC seems to be more frequently described in young patients (< 45 years-old) and to correlate with worse disease-free survival while intestinal type GC with chronic gastric inflammation seems to appear mostly after 45 years-old (around 70 years-old) [20]. GC trends not only show a distinct evolution ratio between the two GC

Epidemiology and Risk Factors

Gastric Cancer is the 5th most common and the 3rd deadliest cancer worldwide, according to the latest report of the International Agency for research on Cancer [12]. Its geographic incidence remains heterogenous with most cases occurring in Eastern Asia (619,226 in 2018), and men being twice as much affected as women. Interestingly, a decrease in GC incidence has been noted in Western Europe and North America over the past decade [13].

Different risk factors are known to affect gastric cancer incidence, among which Helicobacter pylori (H. pylori) infection is the essential one. H. pylori infection involves almost 50% of the world's population and induces chronic inflammation of the gastric mucosa, 5 to 15% of which evolve in gastric and duodenal ulcers and less than 1% in GC. The Correa cascade shows how intestinal type GC begins with H. pylori-induced chronic gastritis followed by gastric atrophy, intestinal metaplasia, and dysplasia [14]. Intestinal GC subtype is mainly caused by environmental factors while diffuse GC subtype also includes hereditary and genetic factors. About 10% intestinal GC cases are associated to infection with EBV and interestingly, EBV-positive GC are mostly associated to MSI molecular type and are related to less worst survival prognosis than diffuse/mixed GC subtypes [15,16].

The overall decrease in GC incidence can mostly be attributed to the decrease in intestinal type GC due to a lower H. pylori prevalence and modified dietary habits [13,17]. The better comprehension of intestinal type GC over the past decade has largely contributed to this, and the Correa cascade has provided a window for its prevention and early detection. Studies show that H. pylori eradication treatments, both as primary and as secondary prevention, reduces the risk of GC development. It also decreases the rate of metachronous GC in patients having early GC and improves the baseline of gastric corpus atrophy grading [18,19].

Conversely, diffuse GC subtype, not only related to H. pylori infection, is not yet well understood, and still has an increasing incidence. Due to its predominant genetic and hereditary origin, diffuse type GC seems to be more frequently described in young patients (<45 years-old) and to correlate with worse disease-free survival while intestinal type GC with chronic gastric inflammation seems to appear mostly after 45 years-old (around 70 years-old) [20]. GC trends not only show a distinct evolution ratio between the two GC subtypes but also between GC localisation, with an increase in cardia GC incidence compared to distal GC which is mostly related to H. pylori infection [13].

Despite the high GC incidence, GC survival rates seem to be better in Eastern Asia with a 64.2% 5-year survival [12] compared to North America (2.7%) or Europe (8.5%).

Indeed, risk factors comprehension has helped develop strategies against GC and in Eastern Asia screening programs have been established allowing early detection of GC. More than 50% of cases are diagnosed at an early stage in Japan for example, against 27% in the USA [13]. Asian GC cases are mostly non-cardia which, again due to H. pylori eradication campaigns and programs, can be detected early. Also, secondary preventive strategies like endoscopic screening and high-risk histology surveillance has been successful and will further contribute to decrease H. pylori-induced GC [18]. In North America and Europe, non-cardia GC incidence has decreased while cardia GC rates remain stable and is possibly increasing [13]. In addition, non-cardia GC seems to decrease in older populations (>50 years-old) and increase in younger persons, especially women, which could be linked more to autoimmune gastritis and less to H. pylori-related gastritis [15]. H. pylori eradication does not affect the cardia GC trend [18] and it is important to find precursor lesions or biomarkers allowing their early detection and maybe treatment.

Still, GC incidence does not follow H. pylori prevalence, showing the role of other risk factors like the host's genetics, H. pylori strain characteristics as well as environmental factors like salty diet in this disease. Indeed, Eastern Asian populations harbour highly virulent H. pylori strains possessing variants of bacterial pathogenicity factors associated with GC such as VacA, CagA oncoprotein and other proteins including BabA and SabA outer membrane proteins [21,22]. Chronic inflammation plays an important role in gastric carcinogenesis. A recent European Prospective Investigation into Cancer and Nutrition (EPIC) study focussed on the association between inflammatory potential of diets and the risk of cardia and non-cardia GC: 476,160 subjects from 10 European countries were followed for 14 years and the results showed that the inflammatory potential of diets is associated with an increased risk of GC. Low-grade chronic inflammation induced by food intake may be associated with cardia GC risk, the pattern being less consistent for non-cardia GC [23].

Furthermore, many cancers including GC and colorectal cancers have been linked to the consumption of red and/or processed meat [24]. These contain either carcinogens like heterocyclic aromatic amines and polycyclic aromatic hydrocarbons, produced by cooking at high temperature, or N-nitroso-compounds and polycyclic aromatic hydrocarbons in cured or smoked meat. In addition, processed meat contains much salt, one of the risk factors for GC, along with tobacco smoking and low vegetable and fruit intake which are sources of antioxidant vitamins [25].

Apart from external elements, intrinsic factors such as host susceptibility also affect GC. Almost 40% hereditary diffuse GC are due to the invalidating mutation of CDH1 [26]. Moreover, whole-exome sequencing analyses show other germline mutations than CDH1, for example in tumour suppressor genes SDHB, CTNNA1, STK11 and in genes involved in DNA repair PALB2, BRCA2 and ATM. In addition, patients suffering from syndromes affecting DNA repair genes, TP53, APC as well as tumour suppressor genes (BRCA) are more likely to develop GC [2,5,6].

Gastric Carcinogenesis 3.1. A Helicobacter Disease

Helicobacter pylori, the major risk factor of GC, colonises the gastric mucosa and induces chronic gastric inflammation. Nevertheless, most infected individuals do not develop GC. Possible reasons might be the heterogeneity of the bacterial genome and its different virulence factors. The cag pathogenicity island (cag PAI) encodes the major virulent factors of the bacterium, which are CagA oncoprotein and proteins forming a type IV secretion system, normally related to the bacterial conjugation system allowing it to exchange DNA with other bacteria. Other pathogenic factors associated to GC include adhesins (BabA, SabA), outer membrane proteins (OipA, HomB), and VacA cytotoxin which is encoded by a cag-PAI independent locus [21]. Many studies have described the pro-oncogenic role of these different factors, either by being translocated into the host cells, by translocating other factors into host cells, or by facilitating gastric mucosa colonisation by H. pylori. CagA, the first described bacterial oncoprotein, is injected by the type IV secretion system into the cytoplasm of host gastric epithelial cells where it interacts with different signalling pathways. CagA destabilises cellular junctions and apico-basal cell polarisation, activates pro-inflammatory and oncogenic signalisation pathways leading to disturbance of the gastric epithelium integrity, differentiation and self-renewal [21]. Still, malignant transformation mechanisms are not fully known. Horvat et al. showed that H. pylori inhibits p14ARF tumour suppressor in a CagA dependent manner by inducing its degradation [27]. p14ARF is a critical tumour suppressor having important functions in oncogenic stress regulation. The chromosome region on which is located this gene is often deleted in primary GC and hypermethylation inactivates its expression in more than 30% of GC cases. The use of isogenic H. pylori mutants showed that CagA is responsible for the overexpression of E3 ubiquitin ligase, TRIP12, inducing ubiquitination and degradation of p14ARF protein in H. pylori infected cells and inhibition of autophagy in a p53-independent manner. TRIP12 is also overexpressed in the gastric mucosa of H. pylori-infected patients [27]. H. pylori also promotes gastric carcinogenesis in a p53-dependent manner. Costa et al. showed that this bacterium can reduce the expression of transcription factor USF1, known for its p53 stabilizing role in response to genotoxic stress [28]. The consequent p53 proteasomal degradation participates to gastric carcinogenesis promotion. In addition, H. pylori delocalises to foci nearby cell membranes and prevents USF1 /p53 nuclear translocation, altering their transcriptional function, one of which is the protection of gastric cells against H. pylori infection [28].

H. pylori infection also interferes with other tumour suppression pathways. This bacterium can downregulate the tumour suppressor Receptor for Activated C Kinase 1 (RACK1) which is physiologically involved in the modulation of NF-κB signalling pathway activity. By doing so, H. pylori promotes the upregulation of integrin β-1, leading to the upregulation of the NF-κB signalling pathway involved in H. pylori-induced carcinogenesis. In addition, this study shows that RACK1 is downregulated in GC tissue compared to normal tissue distant to tumour and is correlated to poor prognosis of patients [29].

Helicobacter pylori can use the host mechanisms to increase its virulence. It can control the activation of c-Abl kinase to maintain CagA virulence factor phosphorylation [30]. In addition, it has been recently shown that H. pylori not only induces c-Abl kinase activity but also alters the localisation of the activated protein. c-Abl becomes cytoplasmic, promotes cell migration and prevents apoptosis thus participating to H. pylori-related gastric carcinogenesis [31].

Gastric mucosal barrier disruption by H. pylori infection and inflammation also plays a role in gastric carcinogenesis [21,32]. The gastric mucosa contains epithelial cells with transmembrane and peripheral scaffolding proteins among which Claudins with a role in the regulation of tight junctions' permeability [32]. Hagen et al. showed that Claudin-18 is decreased in models of GC infected with H. pylori as it is in GC patients where this loss is associated with an aggressive phenotype and poor prognosis [33]. Targeted deletion of CLDN18 gene encoding Claudin-18 results in pre-neoplastic stomach lesions in 7-weeks mice. Interestingly, 20 weeks post-infection, H. pylori is not able to colonise Claudin-18 deficient mice but, atrophy of the gastric corpus as well as high level of dysplasia and gastrointestinal neoplasia resembling human intramucosal carcinoma are noted in sham and H. pylori-infected mice [33].

Cell adhesion molecules modifications induce Epithelial-to-Mesenchymal Transition (EMT) where epithelial cells transdifferentiate and transit from their epithelial phenotype to a mesenchymal phenotype and in so doing, migrate or invade (in case of tumours). H. pylori infection is known to initiate EMT in gastric epithelial cells [34,35]. Invalidation of IQGAP1, involved in adherent junctions stability and sequestration of β-catenin to the junctions, increases H. pylori-induced EMT in gastric epithelial cell lines in vitro and promotes H. pylori-induced pre-neoplastic lesions in vivo with 6 times more Gastric Intraepithelial Neoplasia (GIN) in mice mutated for IQGAP1 compared to wild-type mice [36]. A recent study shows that H. pylori also downregulates other adherence proteins like Afadin, having a role in tight junction stabilisation, and in so doing, induces EMT phenotype in GC cell lines. Afadin regulation is independent of CagA, type-IV secretion system and VacA virulence factors [37]. Also, H. pylori upregulates the expression and activity of various matrix metalloproteases family proteins (MMPs) in GC cells, exacerbating their invasive properties. The use of several signalling pathways inhibitors has revealed the role of Src, NFκB, ERK and JNK pathways in H. pylori-induced EMT and EGFR-modulated MMPs (MMP9, 3, 10) regulation [34,35,38].

CagA+ H. pylori infection dysregulates the Hippo signalling pathway, responsible for the control of stem cell properties and proliferation, in physiology. H. pylori induces Hippo oncogenic effector YAP1 overexpression both in vitro and in vivo in infected patients. Consequently, activation of YAP1/TEAD oncogenic pathway in gastric epithelial cells promotes EMT as well as the acquisition of intestinal metaplasia markers [39,40]. Moreover, the Hippo pathway seems to limit H. pylori-induced preneoplastic changes. The bacterial infection up-regulates oncoprotein YAP1 but also its negative regulator LATS2, a Hippo tumour suppressor kinase, in a coordinated biphasic manner, with an early temporary oncogenic YAP1 activation, followed by LATS2 activation leading to YAP1 phosphorylation and downregulation and thus, oncogenic signal restriction [40]. YAP1 paralogue TAZ is also activated after H. pylori infection, overexpressed in vivo, and is associated to EMT and to acquisition of tumorigenic and invasive properties in GC cells [41]. Furthermore, YAP1/TAZ seem to cooperate with β-catenin in Wnt signalling pathway to promote gastrointestinal neoplasia [42].

Additionally, gastric carcinogenesis can occur after H. pylori infection through the regulation of connexins (Cxs). This bacterium can upregulate transcription factors like GATA-3 and PBX-1, having a role in the expression of Cx32, which is thus inhibited [43]. In addition, it can decrease histone acetylation levels which in turn regulates the expression of Cxs. Similarly, H. pylori modulates other connexins such as Cx43, Cx26 and Cx37, expressed in gastric tissue, either by promoter hypermethylation and expression inhibition, protein delocalisation or gene polymorphism induction [43].

Helicobacter pylori also seems to protect GC cells from anti-tumoral immune response. Programmed death (PD) and programmed death ligand 1 (PD-L1) expression is one of the immune tolerance mechanisms preventing T-cells from attacking one's own tissues. PD-L1 expression was detected on tumour cells showing that tumour cells seem to exploit this immune-checkpoint pathway to escape cytotoxic T-cells-induced programmed cell death [2]. H. pylori is able to stimulate PD-L1 expression in gastric epithelial cells. It was recently found that Shh pathway is involved in this CagA dependent process. PD-L1 expression increases in H. pylori-infected organoid cultures and Shh pathway inhibitor GANT61 is able to counteract this response [44]. In addition, anti-PD-L1 treatment of gastric organoids co-cultured with autologous patient cytotoxic T lymphocytes and dendritic cells provoked organoids death. The use of this PD-L1 mechanism causes epithelial cell survival and protection against immune response, leading to GC progression and interestingly, H. pylori is not the only pathogen using this mechanism. A recent study showed that among EBV-positive tumours, those with high viral load was correlated to higher tumour cells PD-L1 expression and worse patient prognosis compared to those with lower EBV viral load [45].

H. pylori infection induces the NF-κB pathway which increases Peroxiredoxin 2 (PRDX2) expression in GC [46]. This antioxidant enzyme plays an important role in the protection of cells from oxidative stress by scavenging H2O2 and ROS from cells and seems to be used by cancer cells as a defence mechanism. PRDX2 expression is high in GC tissues and correlates with low survival of patients [46].

H. pylori not only alters the hosts' mechanisms to induce disease, but also undergoes genetic modifications in vitro and in vivo when in contact with the carcinogenic environment. Whole genome sequencing revealed a total of 180 unique single nucleotide polymorphisms (SNPs) in differently virulent H. pylori strains and in strains harvested in low-iron and high-salt carcinogenic conditions compared to reference H. pylori genome [47]. Common SNPs, including one within fur gene (FurR88H) encoding a ferric uptake regulator, were found in strains cultivated in cancer-like environment. This FurR88H variant seemed to appear after only 5 days exposure to the carcinogenic environment and fur sequencing in clinical isolates showed that 17% of strains coming from patients with premalignant lesions had the FurR88H variant compared to 6% strains from non-atrophic gastritis patients [47]. Furthermore, a genome-wide association study (GWAS) on 173 H. pylori isolates from European population revealed SNPs and genes, babA and cag pathogenicity island genes, as well as non-synonymous changes in several less well-studied genes, that differed in GC patients compared to those suffering from gastritis. The authors conclude that these bacterial factors differ enough in this pathology for a minor GWAS analysis to detect them [48].

Nonetheless, host polymorphisms can also render them more sensitive to GC development in presence of H. pylori, for example if there is less production of anti-inflammatory cytokines, activation of pro-inflammatory cyclooxygenases and oxidative damage [21].

Helicobacter pylori infection indeed affects GC status through a large variety of ways (Figure 2) and understanding the mechanisms will open paths to more targeted and efficient therapies.

tissues and correlates with low survival of patients [46].

H. pylori not only alters the hosts' mechanisms to induce disease, but also undergoes genetic modifications in vitro and in vivo when in contact with the carcinogenic environment. Whole genome sequencing revealed a total of 180 unique single nucleotide polymorphisms (SNPs) in differently virulent H. pylori strains and in strains harvested in lowiron and high-salt carcinogenic conditions compared to reference H. pylori genome [47]. Common SNPs, including one within fur gene (FurR88H) encoding a ferric uptake regulator, were found in strains cultivated in cancer-like environment. This FurR88H variant seemed to appear after only 5 days exposure to the carcinogenic environment and fur sequencing in clinical isolates showed that 17% of strains coming from patients with premalignant lesions had the FurR88H variant compared to 6% strains from non-atrophic gastritis patients [47]. Furthermore, a genome-wide association study (GWAS) on 173 H. pylori isolates from European population revealed SNPs and genes, babA and cag pathogenicity island genes, as well as non-synonymous changes in several less well-studied genes, that differed in GC patients compared to those suffering from gastritis. The authors conclude that these bacterial factors differ enough in this pathology for a minor GWAS analysis to detect them [48].

Nonetheless, host polymorphisms can also render them more sensitive to GC development in presence of H. pylori, for example if there is less production of anti-inflammatory cytokines, activation of pro-inflammatory cyclooxygenases and oxidative damage [21].

Helicobacter pylori infection indeed affects GC status through a large variety of ways (Figure 2) and understanding the mechanisms will open paths to more targeted and efficient therapies.

A Stem Cell Disease

A Stem Cell Disease

Despite the major influence of environmental factors, GC remains a heterogenous and multifactorial disease. Tumour cells are different from one another in terms of the mutations they carry, their sensitivity to drugs, their phenotype as well as their tumorigenicity. Indeed, ever since the identification of Cancer Stem Cells (CSCs), cancer cells having stemlike properties, in acute myeloid leukaemia in 1995, several studies have demonstrated the presence of such cells in solid tumours like breast, brain, colon [49][50][51][52] as well as gastric tumours [53][54][55]. GC are diverse entities not only due to the different histological phenotypes between patients but also due to the presence of different cell populations within one tumour mass, among which Gastric Cancer Stem Cells (GCSCs) form a very small proportion, ranging from 0,1 to 3,5% according to Nguyen et al. [55]. These cells have self-renewal as well as asymmetrical division capabilities allowing them to regenerate themselves and to generate new differentiated cells for proliferation and tumour mass enlargement. These highly tumorigenic cells are also known to resist to conventional chemoand radiotherapies and to migrate and cause metastasis in patients [55,56]. Studying them is crucial to decipher GC mechanisms and develop more specific and efficient therapies in GC.

Ever since their discovery, many methods have been developed taking advantage of their specific self-renewal, differentiation, and tumour initiation properties. In vitro sphere assays, in which cells are seeded at low density on non-adherent plates in the absence of serum and presence of growth factors, allow only GCSCs to survive and form spheres. In addition, only GCSCs have the capacity to generate new spheres after several passages. In vivo experiments are also used since CSCs can generate heterogenous tumours after subcutaneous xenograft in immunocompromised mice [57].

In addition, characterisation of GCSCs has allowed the discovery of markers which could be used to isolate them using Fluorescence-Activated Cell Sorting (FACS), for example. CD44, best known as being the hyaluronic acid receptor, is a marker of GCSCs as it is for many other solid tumours [50,55]. Nguyen et al. described CD44 and ALDH activity as being the most important GCSC signatures allowing the detection and the isolation of cells able to generate heterogeneous tumorspheres in vitro and tumours in vivo from noncardia gastric carcinomas. The authors described ALDH+ cells as being part of the CD44+ cell population and as possessing drug efflux properties increasing their chemoresistance properties [55]. In addition, many other markers enriched in GCSCs like CD133, CD24, CD166 were described and can be used for GCSC study. These methods have allowed to shed a new light on GC as a stem cell disease and H. pylori, being the major GC risk and having a role in GCSC emergence, many studies associate GCSCs and H. pylori.

Helicobacter pylori induces the emergence of cells with CSC phenotype and properties, through EMT, in infected patients' gastric mucosa and in cell lines in vitro. Bessède et al. showed that only CD44+ cells seem to display mesenchymal phenotype compared to CD44cells after H. pylori infection [35]. H. pylori-induced cell junction alterations also affect CSC properties. Deletion of IQGAP1 junction scaffolding protein increases neoplastic lesion in mice stomach and induces CD44 overexpression, mesenchymal phenotype, and CSC-like properties after H. pylori infection [36]. H. pylori infection also stimulates Lrig1+ gastric stem cell population, in a CagA-dependent manner, showing another way by which, this bacterium regulates gastric stemness phenotype. Lrig+ cells are enhanced in human GC tissues compared to normal mucosa [58]. CagA+ H. pylori infection induced-YAP1 and TAZ oncogenic pathway not only promotes EMT as discussed before, but also the acquisition of CSC phenotype as well as tumorigenic and invasive properties in vitro [39][40][41]. YAP1, TAZ and their target genes are significantly upregulated in GC and associated to poor prognosis [59]. Hippo pathway contribution to GCSC phenotype was demonstrated by an enrichment of YAP1 and its partners in CD44+ GC cells, and by the inhibition of GCSC tumorigenic properties in vivo after YAP1/TAZ/TEAD targeting by Verteporfin [59]. Moreover, activation of Hippo tumour suppressor kinase LATS2 by Leukaemia Inhibitory Factor (LIF), an interleukin-6 family cytokine, decreases GCSC tumorigenic properties and population of GC cell lines and patient-derived xenograft cells (PDX), again showing the implication of this pathway in GCSC phenotype [60]. High expression of LIF's receptor (LIFR) in patients is associated to better survival rates and patients having high YAP1/TAZ and target genes expressions which is correlated to bad prognosis, survive better if they also have a high LIFR expression [60], showing a protecting role of LIF/LIFR signalling in this YAP1/TAZ-pro-GCSC context.

CD44 plays a functional role in H. pylori-induced proliferation of gastric epithelial cells. It acts as a co-receptor of tyrosine kinase c-Met receptor and precipitates with phosphorylated c-Met and CagA. In this context, H. pylori fails to induce epithelial proliferation in organoids derived from CD44-deficient mice stomachs showing the importance of CD44 in H. pylori-induced gastric carcinogenesis [61]. CD44+ CSCs presence at the invasive front of GC tumours indicate poor survival of patients compared to patients with no CD44 expression at invasive front and can be used as a prognosis marker [56].

CD44 exists in multiple isoforms containing at least 20 exons due to alternative mRNA splicing. CD44 variant 8-10 (CD44v8-10) has been identified as being predominantly expressed in gastric cancer cells and less in normal tissues. Exogenous expression of CD44v8-10 increases the frequency of tumour initiation in immunocompromised mice. Finally, this CD44 variant, and not panCD44, can rescue the tumorigenic phenotype lost after panCD44 silencing [62].

Furthermore, another CD44 variant, CD44v9 is highly expressed in mouse GCSCs. Analysis of GC samples from 103 patients show a five-year survival rate lower in CD44v9positive tumours compared to CD44v9-negative ones [63]. Capping actin protein of muscle Z-line α subunit 1 (CAPZA1) is a protein having an important function in actin polymerisation. Its overexpression in gastric epithelial cells inhibits the formation of autolysosome and results in the accumulation of CagA in host cells, increasing GC risk. In human GC cell line AGS, oxidative stress increases histone H3 acetylation of CAPZA1 promoter, increasing CAPZA1 expression, nuclear accumulation of CD44 transcription factor, β-catenin, and enhancing expression of epithelial splicing regulatory protein 1 responsible for the alternative splicing of CD44 into CD44v9 [64]. Moreover, CD44 variant 6 (CD44v6) does not seem to play a role in GC progression since CD44v6 overexpression does not affect GC cells growth rate, invasion, migration, or cell-cell aggregation. However, CD44v6 cells survive better than CD44v6-cells in presence of chemotherapy drug cisplatin suggesting a role of CD44v6 in GC chemoresistance [65].

A recent study analysed the role of Human Epidermal growth factor 2 (HER2), target of trastuzumab in the treatment of HER2+ gastric and breast cancers, on GCSCs and the underlying mechanisms. The authors demonstrated that HER2 overexpressing GC cells had increased stemness and invasiveness and were regulated by the Wnt/β-catenin signalling pathway [66].

The role of microRNAs (miRNAs) in the gastric carcinogenesis and in GCSCs control is also important. miR-181a_5p and miR-22-3p have been shown to be downregulated in GC-SCs while miR-483-5p, miR-4270 and miR-16-5p are overexpressed. A recent study shows that miR-7-5p is also downregulated in GCSCs due to hypermethylation of its promoter region. miR-7-5p silencing increases GCSC invasion properties while its overexpression reduces spheroid formation and invasion by repressing Notch and Shh pathways in vitro and decreases tumour growth in vivo [67].

Due to their possible role in chemoresistance, relapse and metastasis, GCSCs comprehension (Figure 3) is a key step to understanding GC for biomarkers and therapy development.

A Microenvironment Disease

The importance of the tumour microenvironment (TME) in tumorigenesis is being more and more explored. A tumour is a dynamic entity with constant communications between the tumour cells and their rich surrounding environment, source of factors allowing and maintaining cancer cells phenotype and heterogeneity (Figure 4).

Most immune cells infiltrating tumours are Tumour Infiltrating Lymphocytes (TILs) consisting of CD3+ Tcells (CD8+ Tcytotoxic and CD4+ Thelper cells) as well as FOXP3+ Treg cells. The number of TILs present in tumours reflects the host immune response mechanism. Indeed, Yu et al. demonstrated that high CD8+ and CD3+ T cells infiltration in tumours are associated to better prognosis of patients compared to low infiltration [68]. Interestingly, CD8+ T cells seem to be less present in diffuse/mixed types GC compared to intestinal type and might be associated to worse outcome [69].

Tumour associated Neutrophils (TANs) are one of the most important stromal partners having a role in carcinogenesis. TANs are able to increase migration and invasive capacities of GC cells [70]. Increase in neutrophil blood count, in neutrophil/lymphocyte ratio as well as in TME infiltration with neutrophils reflect poor patient prognosis [71][72][73]. Li et al. demonstrate that TANs produce IL-17a which promotes EMT and GC progression by activating the JAK2/STAT3 pathway. IL-17a neutralising antibody was able to reverse TANs effect on GC progression [73].

A Microenvironment Disease

The importance of the tumour microenvironment (TME) in tumorigenesis is being more and more explored. A tumour is a dynamic entity with constant communications between the tumour cells and their rich surrounding environment, source of factors allowing and maintaining cancer cells phenotype and heterogeneity (Figure 4). Gastric cancer-associated fibroblasts (GCAFs), when activated, are able to enhance migration and invasion properties of gastric cells both in co-culture and in presence of GCAFs-conditioned medium. Activated GCAFs correlate with poor survival of patients and in vitro, paracrine action of factors from GCAFs make gastric cells resistant to 5fluorouracil (5-FU), one of the conventional chemotherapies used in GC treatment [74]. A recent study shows the role of GCAFs in stemness, transformation and chemotherapy resistance. Low expression of Secreted Protein Acidic and Rich in Cysteine (SPARC) in GCAFs seems to be associated to low cell differentiation, low 5-year survival rate and 5-FU resistance [74]. Also, GCAFs produce large amounts of IL-6 which participate to the activation of the JAK/STAT signalling pathway. The use of anti-IL-6 neutralising antibody and inhibitor of JAK2/STAT3 decreases GCAFs effect on GC and GCAFs-induced peritoneal metastasis in vivo showing the role of TME and GCAFs in GC progression through the JAK/STAT pathway [75]. IL-6 crosstalk between tumour cells and GCAFs not only supports tumour growth, but also promotes CAFs activation through IL-6Rα. Loss of IL-6 and use of inhibitors of STAT3 and MEK/ERK pathways suppress tumorigenesis in 3D organotypic and tumoroid models, showing how STAT3 and MEK/ERK pathways are also solicited in crosstalk between tumour cells and GCAFs for the maintenance of tumour integrity [76]. Loss of Trefoil Factor 1 (TFF1) is observed in human GCs, linked to STAT3 nuclear localisation and to pro-inflammatory phenotype and gastric lesions in mice. TFF1-knockout mice demonstrate a nuclear localisation of p-STAT Y705 along with an overexpression of STAT3 target genes and on the contrary, the reconstitution of the protein in GC cell lines and organoids from TFF1-knockout mice decreases p-STAT Y705 showing the role of JAK/STAT pathway in GC. TFF1 blocks the dimerization of IL6Rα and GP130 subunits and thus obstructing the binding of IL6 on its receptor and its pro-inflammatory and pro-tumorigenic consequences [77]. Furthermore, Galectin-1 (Gal-1), a β-galactosidasebinding protein, is highly expressed in GCAFs and is involved in GC progression and metastasis through the activation of signalling cascades like the Hedgehog (Shh) pathway involved in the EMT process. Gal-1 from GCAFs, binds to a carbohydrate structure in β-1 integrin and induces EMT, cell migration and invasion by regulating Glioma-associated oncogene-1 (Gli-1). High expressions of Gal-1 and Gli-1 are correlated to poor prognosis of patients [78]. GCAFs also induce the expression of Discoidin domain receptor 1 (DDR1) in GC cells leading to increased tumorigenesis both in vitro and in vivo [79]. Communications between the stroma and cancer cells are not unidirectional. Indeed, cancer cells also produce factors which modulates the stroma. GC cells produce exosomes which induce pericytes proliferation, migration, and expression of CAFs markers showing that cancer cell-derived exosomes are involved in the transition of pericytes into CAFs [80]. GC cellderived exosomes are able to induce BMP (Bone Morphogenic Protein) transfer and activate PI3K/AKT and MEK/ERK in pericytes transforming them into GCAFs. The use of BMP signalling pathway inhibitor Noggin reverses the tumour-exosome-induced pericyte-CAF transition and decreases the expression of CAFs markers by pericytes [80]. Helicobacter pylori can also participate to stroma-induced gastric carcinogenesis. In presence of the bacterium, gastric fibroblasts are activated into GCAFs with an increased secretion of TGFβ and conditioned medium from H. pylori-activated GCAFs prompts EMT-like phenotype in rat gastric epithelial cells RGM-1 [81]. Tumour associated macrophages (TAMs) can be of 2 phenotypes, M1 and M2, M2 being the cancer cell-activated form of TAMs, essential for their role in tumour growth and progression [70]. GC tissues are found to be composed mostly of M2 TAMs, which when isolated promote migration and invasion of GC cells [73]. Interestingly, recent single cell tumour microenvironment genomic characterisation demonstrate that GC TAMs genetic signatures do not match those of known M1 or M2 macrophages [82]. M1 and M2 known distinguishing genetic markers are co-expressed in the same macrophage cluster. Nevertheless, genes distinguishing these different TAM clusters seem to be related to the HSP family, THBS1, MMPs family, CCL20, CCL18 and CCL3 chemokines among others, as well as cell cycle-dependent genes. In addition, these TAMs seem to differ from PBMC monocytes but conserve some similarities with normal tissue macrophages [82]. Moreover, TAMs abundance in GC microenvironment is associated with a decrease in number and a dysfunction of Natural Killer (NK) cells, which can be reversed by TGFβ1 blockade [83]. In so doing, TAMs promote tumour immune response in GC. Furthermore, TAM programmed cell death protein 1 (PD1) expression increases as GC progresses in mice and this can also be found in primary human cancers [84]. PD1 immune checkpoint receptor is normally present on activated T cells to induce their immune tolerance. TAM PD1 expression is correlated to less phagocytosis of tumour cells which express PD1 ligand, PD-L1, as immune escape mechanism [84].

In relation with immunosuppression mechanisms, Regulatory T cells (Tregs) seem to be abundant in gastric TME [82,85]. Sathe et al. single cell analysis of gastric TME puts into light two different Treg classes marked by different expression level of proliferationassociated genes.

Dendritic cells (DCs) can also reflect GC patient prognosis. Liu et al. showed that different subsets of DCs, plasmacytoid DCs (pDCs) and myeloid CD1+ DCs (mDC1s) are increased in GC patients peripheral blood compared to healthy controls [86]. Also, high level of pDCs seems to correlate with GC stage progression while mDC1 level does not change. Furthermore, different DC subtypes are found according to GC stages. Early GC T stages present high peripheral DC2 number and tumour infiltrating DC1/DC2 ratio, while low level of tumour infiltrating DC2s and high DC1/DC2 ratio are observed at N0 GC stage [87].

Stromal R-spondin, produced by gastric myofibroblasts normally controls gastric epithelial cell and gland homeostasis by activating the proliferation of Lgr5+ gastric stem cells. H. pylori increases the expression of R-spondin 3 and causes the hyperproliferation and hyperplasia of the gastric gland [88]. Interestingly, R-spondin 3, required physiologically for the maintenance of Lgr5+ cell identity, induces differentiation into a secretory phenotype and, according to a recent study, allows these cells to develop defence mechanisms against bacterial infection. In presence of H. pylori, R-spondin 3 enables Lgr5+ cells to secrete anti-microbial protein Interlectin-1 (Itln1) which binds to the bacteria and agglutinates them. R-spondin-Lgr5 signalling in the stomach thus protects the gland base against infectious agents [89].

Finally, an acetylcholine-Nerve Growth Factor (Ach-NGF) axis can activate GC niche and promote the disease. Nerves within the gastrointestinal niche regulate both normal and neoplastic stem cell dynamics. Dclk1+ tuft cells and nerves, main Ach source in the gastric mucosa, induce a cholinergic stimulation of NGF expression, which, when overexpressed, expand enteric nerves, and promote carcinogenesis though YAP function [90].

A Microbiome Disease

Although the stomach was once thought to be a sterile environment, it is now known to house many bacterial species, leading to a complex interplay between H. pylori and other residents of the gastric microbiota. Data now show that the stomach harbours a large and diverse bacterial community with colonisation densities ranging from 10 1 to 10 3 colony forming units/g [91]. Moreover, recent advances in molecular techniques and computational analysis have provided evidence that the complex microbiota colonising the gastric epithelium in combination with H. pylori might influence gastric homeostasis and disease [92]. Currently, there are few studies that have examined differences in microbial composition and outcomes of GC. One of the key steps in the histological progression to intestinal type GC is the development of atrophic gastritis, a condition that predisposes the stomach to an increase in gastric pH due to loss of parietal cells and overgrowth of non-Helicobacter microbiota [14]. This stomach hypochlorhydria facilitates colonization by bacteria and may promote the progression towards gastric cancer, confirming that microbiota can influence gastric carcinogenesis [14].

In 2011, a study showed that in the INS-GAS mice model of spontaneous gastric cancer, gastric lesions take 13 months longer to develop when the mice are germ-free than when they are Specific Pathogens Free (SPF) [93]. Furthermore, the authors showed that H. pylori-mono association accelerates gastritis and GIN but causes less-severe gastric lesions and delays the onset of GIN compared to H. pylori infection of INS-GAS mice with complex gastric microbiota. This data proves that microbiota can influence gastric carcinogenesis and has been confirmed with further works showing that gastric colonisation with a restricted commensal microbiota can replicate the promotion of neoplastic lesions by diverse intestinal microbiota [94].

In addition, in a recent study describing GC patient microbiota and comparing it with that of chronic gastritis patients, it has been shown that GC microbiota is characterised by reduced microbial diversity, decreased abundance of Helicobacter and by the enrichment of other bacterial genera, mostly intestinal commensals. Furthermore, an analysis of the functional features of the microbiota revealed that GC patients' microbiota is compatible with the presence of a nitrosating microbial community. Thus, patients suffering from gastric carcinoma have a dysbiotic microbial community with genotoxic potential, which differs from the gastritis patients [95].

Two recent studies have described and analysed the gastric microbiome of different types of patient. A Taiwanese study profiled gastric epithelium-associated bacterial species in patients at different stages of the Correa cascade (gastritis, intestinal metaplasia and gastric cancer) [96] and found that the overall microbiota composition is similar between patients with gastritis and those with intestinal metaplasia. In GC patients, H. pylori is indeed more frequent and more abundant, corresponding to a cancer-specific signature, but, Clostridium, Fusobacterium and Lactobacillus species are also frequently abundant in those patients, though their role in gastric carcinogenesis is still not proven. Following this work, another study compared the gastric microbiota of patients presenting GC to controls having a functional dyspepsia [97]. The authors showed that several bacterial taxa are enriched in GC patients, particularly pro-inflammatory oral bacterial species, lactic acid producing bacteria and bacteria producing short fatty acids. These results are for the moment only descriptive and H. pylori still remains the only bacterium directly implicated in gastric carcinogenesis.

Although several works have shown that gastric microbiota can influence gastric carcinogenesis, there is for now no report of a potential microbiota-based therapeutic or prognosis strategy in GC context. Further work in this direction is thus warranted.

Biomarkers and Therapeutic Strategies 4.1. Biomarkers and Targeting

Gastric cancer treatment still consists of surgery and additional adjuvant or neoadjuvant radio-and chemotherapies. Early tumours can be excised endoscopically, and surgical resection remains the only curative treatment with unfortunately, a high number of relapse cases. Conventional chemotherapies consisting of leucovorin, oxaliplatin, docetaxel and 5-FU are given as perioperative chemotherapy or as palliative chemotherapy for patients having advanced metastatic disease [1,98]. Nevertheless, GC patient 5-year survival rate remains less than 5% for advanced unresectable or metastatic cases (80% of patients at diagnosis, except for Far East countries) [1].

The need for personalized targeted treatment is urgent and currently, only few targeted therapies exist. Trastuzumab (Herceptin, Roche, Germany) targets HER2+ GC and is used as first-line treatment for patients with tumours overexpressing HER2, accounting for less than 30% GC cases [98]. Ramucirumab (Cyramza, Lilly, USA) is another targeted treatment for advanced refractory GC cases. This antibody, used alone or in combination with paclitaxel, targets vascular endothelial growth factor receptor 2 (VEGFR2) thus inhibiting angiogenesis and increasing survival. However, these targeted therapies do not cater for all the different GC types having diverse aggressiveness levels and response to treatment [4,8,10].

The multiple molecular classification systems that exist indeed influence endoscopic or surgical choices but are not enough to guide precision individual treatment decisions which are urgently needed. External factors like H. pylori infection are being catered for through the multiple eradication strategies which indeed seem to be efficient [18]. Unfortunately, in patients where the disease has evolved, the establishment of genetic as well as cell phenotypic changes like the acquisition of resistant CSC and invasive properties make the tumours resistant to conventional therapies [98]. In addition, patients are diagnosed at late stages of the disease, and proper diagnosis markers development could help detect GC at earlier stages, allowing proper patient care.

Carbohydrate antigen 19-9 is a commonly used biomarker in GC allowing recurrence prediction when in combination with other tumour markers [99]. Molecules directed against Tyrosine Kinase receptors like cetuximab, rilotumumab or dovitinib could be suitable against GC cases overexpressing their respective targets. Clinical trials for antibodies against FGFR2 (for FGFR2 overexpressing GC) and EGFR (Nimotuzumab; for EGFRhigh GC) are ongoing. Similarly, mTOR pathway inhibitor Everolimus is being tested for patients with MSI type GC often having activating mutations in members of this pathway [98,[START_REF] Fireman | Helicobacter pylori: seroprevalence and colorectal cancer[END_REF].

Studies are more and more trying to understand mechanisms underlying gastric carcinogenesis, may it be induced or not by its principal risk factor H. pylori. In so doing, these studies explore molecules which are either over-or under-expressed in GC compared to non-tumorous tissues and these particular signatures could indeed be used in GC diagnosis and prognosis (Table 1). Hepatoma-derived growth factor (HDGF), overexpressed in GC patients and after H. pylori infection, seems to participate in H. pylori-induced neutrophils recruitment, gastritis and gastric carcinogenesis. HDGF level is also high in patients with intestinal metaplasia and is associated with low survival. Targeting HDGF decreases neutrophil infiltration and inflammatory responses induced by H. pylori infection, making HDGF a potential therapeutic target for H. pylori-induced GC treatment [START_REF] Georgopoulos | Hypergastrinemia is associated with increased risk of distal colon adenomas[END_REF].

Several clinical trials involving antibodies targeting the HGF receptor c-Met were also started but did not give encouraging results. However, positive results were reported from a Phase I trial with a MET specific small molecule AMS 337 showing 1 complete response and 4 partial responses out of 10 patients. MET amplification is observed in 4% GC patients only and bad trial results might be related to an incorrect selection of patients prior to trial since usual immunohistochemistry selection is not the most sensitive one [START_REF] Fireman | Helicobacter pylori: seroprevalence and colorectal cancer[END_REF]. Unfortunately, resistance to small molecule inhibitors of MET kinase is quite common. In this context, Andres et al. used the designed ankyrin repeat protein (DARPin) technology and generated MET-binding DARPins covering extracellular epitopes of MET, thus creating sets of bi-paratopic fusion proteins which efficiently inhibited MET kinase activity and downstream signalling [START_REF] Dash | Exploring the impact of Helicobacter pylori on gut microbiome composition[END_REF]. This strategy using proteins having two paratopes caused receptor downregulation and inhibition of MET induced GC cells proliferation and seems to be an interesting strategy for targeting receptors in therapy [START_REF] Dash | Exploring the impact of Helicobacter pylori on gut microbiome composition[END_REF]. Furthermore, Zhang et al. showed that exosomes could be used to deliver c-Met siRNA to GC cells thus inhibiting migration, invasion and inducing apoptosis in vitro and enhancing sensitivity to cisplatin in vitro as well as in vivo. The exosomes used were isolated from human embryonic kidney epithelial cell line, HEK293T, transfected with si-c-Met [START_REF] Kienesberger | Gastric Helicobacter pylori infection affects local and distant microbial populations and host responses[END_REF].

Bone marrow stromal cell antigen 2 (BST2) or CD317, playing a crucial role in antiretroviral defence in innate immune response, is also overexpressed in GC tissues compared to contiguous non-tumorous tissue and is correlated to tumour stage and lymphatic metastasis [START_REF] Jackson | A population-based epidemiologic study of Helicobacter pylori infection and its association with systemic inflammation[END_REF]. BST2 siRNA inhibited GC cell proliferation and motility and induced apoptosis. Furthermore, NF-κB pathway seemed to be involved in the pro-tumour function of BST2. BST2 targeting could be another possible strategy for GC treatment [START_REF] Jackson | A population-based epidemiologic study of Helicobacter pylori infection and its association with systemic inflammation[END_REF].

Moreover, the cell surface protein CEACAM6 has been identified as a potential endoscopic marker of early GC. CagA+ H. pylori induces this protein expression, which is highly present in early GC and pre-malignant lesions. Fluorescently tagged antibody against CEACAM6 was proposed to mark GC tissue for visualisation, using commercially available endoscopic methods, for use in diagnosis [START_REF] Tuomisto | Systemic inflammation in colorectal cancer: underlying factors, effects, and prognostic significance[END_REF].

GCSC Targeting

Though the major tumour mass is found to be chemo-sensitive and would respond to therapies targeting GC subtypes or immerging immunotherapies, part of the cells comprising the tumour mass, GCSCs, are believed to resist treatments and to be at the origin of cancer relapse and metastasis [98,[START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF]. GC molecular signatures are more and more being explored to propose diagnosis and prognosis markers and to try to target proteins or signalling pathways having a role in the maintenance of these GCSCs.

Several drugs have been evaluated for repositioning as anti-CSC strategy (Table 2). Verapamil, a calcium antagonist allowing the inhibition of calcium-dependent channels and used to treat angina pectoris and cardiac arrythmias, was found to block efflux mechanisms of GCSCs which normally allow these cells to evacuate chemotherapy drugs through calcium-dependent channels. The use of verapamil thus sensitized GCSCs to conventional chemotherapies [55]. Tretinoin, also known as all-trans retinoic acid (ATRA), used for the treatment of acne and acute promyelocytic leukaemia due to its pro-differentiation properties, was able to force differentiation of GCSCs thus impacting their tumorigenic self-renewal capacity [START_REF] Raderer | Association between Helicobacter pylori infection and pancreatic cancer[END_REF]. Furthermore, metformin, used for the treatment of type 2 diabetes to decrease insulin resistance and hepatic neo-glucogenesis, showed efficient anti-GCSCs effects by targeting the metabolism of these cells [START_REF] Stolzenberg-Solomon | ATBC Study, Helicobacter pylori seropositivity as a risk factor for pancreatic cancer[END_REF]. Drug repositioning was also used as strategy to target signalling pathways exacerbated in CD44+ GCSCs.

Buparlisib (BKM120, a pan-class I PI3K inhibitor), first line treatment of metastatic head and neck epidermoid carcinomas, was found to decrease GCSC tumorigenic properties in vitro and to decrease GC metastasis in vivo [START_REF] Risch | ABO blood group, Helicobacter pylori seropositivity, and risk of pancreatic cancer: a case-control study[END_REF]. Verteporfin, an FDA-approved drug for age-related macular degeneration, was repositioned in the GCSC context for its capacity to decrease YAP/TEAD transcriptional activity, cell proliferation, CD44 expression and number of tumorsphere-forming CD44+ALDHhigh GCSCs in vitro. Verteporfin also inhibits tumour growth in vivo [59]. In addition, residual tumour cells were unable to form new tumorspheres in vitro confirming the decrease of the in vivo CSC pool after verteporfin treatment [59]. Recently, LIF cytokine treatment was found target GCSCs through LATS2-induced repression of YAP1/TAZ/TEAD oncogenic activity [60]. Most GCSC-targeting based strategies are directed against CD44+ cells (panCD44) which is ubiquitously expressed in non-tumorous cells even though its expression is exacerbated in GCSCs. Thus, using anti-panCD44 as GCSC target could cause non-specificity problems. CD44 variants, which seem to be less expressed in non-tumorous tissue compared to panCD44, are an interesting alternative for GCSC targeting. Studies have shown the increased expression CD44v8-10 and CD44v9 in GCSCs but also their functional role in the maintenance of this chemo-resistant cell population [63].

Vismodegib, an antagonist of the Shh signalling pathway has been associated with leucovorin, 5-FU and oxaliplatin in the treatment of GC. It binds to SMO, in GCSCs, thus preventing the downstream activation of GLI family of transcription factors and inhibiting Shh signalling. CD44+ GC cells present an overexpression of Shh pathway proteins linked to low patient survival, which was improved after the combined treatment [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF]. Similarly, Napabucasin, a STAT3 inhibitor, repressing CSC self-renewal and inducing cell-death in GCSCs by targeting STAT3 was tested for GCSC targeting. Napabucasin was used in combination with paclitaxel in patients with advanced tumours and showed an antitumour activity. These studies show that combining chemotherapy to targeted strategies seems to be an interesting approach for anti-GCSC directed GC treatment [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF].

High production and low elimination of reactive oxygen species (ROS) by the organism is another cancer risk factor. ROS are tightly controlled under physiological conditions through antioxidant mechanisms since excessive ROS in cells can lead to DNA damage. This property of ROS makes them interesting in therapeutic strategies in order to induce cancer cell damage and death. Unfortunately, cancer cells specially CSCs seem to possess defence capacities against ROS making them resistant to therapies. CD44v9 found in GCSCs can activate xCT, glutamate-cystine exchange transporters, which help increase levels of intracellular reduced glutathione (GSH) and contribute to CSC survival in ROShigh environment [63]. Exogenous CD44v9 expression in cells increase resistance to 5-FU, a chemotherapy agent using ROS production mechanism to kill cancer cells. Inhibition of xCT involved in the anti-ROS mechanism of GCSCs enhances 5-FU anti-GC efficiency [START_REF] Martel | Helicobacter pylori infection and development of pancreatic cancer[END_REF].

Apart from their use in diagnosis, miRNA targeting, or overexpressing strategies can also be considered as GC therapeutic solutions. miR-7-5p normally exerts its tumoursuppressing effect, through the downregulation of its target genes SMO and HES1 (members of the Shh and Notch signalling pathways respectively), which is lost in GCSCs where this miRNA is under-expressed. So, targeting SMO and HES1 in GC could serve as target for GCSCs [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF]. Seed-targeting locked nucleic acid (LNA) can be used as specific miRNA inhibitors and target miR-372/373 thus decreasing GC cell growth and targeting GCSCs [START_REF] Lindkvist | A prospective study of Helicobacter pylori in relation to the risk for pancreatic cancer[END_REF].

Liquid Biopsies as Biomarkers

Certain subpopulations of GCSC, metastasis-initiating cells (MIC), overexpress MMPs, which are physiologically involved in extracellular matrix breakdown and promote invasion and metastasis. MMP10, MMP15 and MMP9 are found to be increased in GC and to correlate with poor patient prognosis [38,[START_REF] Hirabayashi | Author Correction: Helicobacter pylori infection, atrophic gastritis, and risk of pancreatic cancer: a population-based cohort study in a large Japanese population: the JPHC Study[END_REF] During the metastasis process, tumour cells originating from primary tumour or metastases can be found circulating in blood, either single or in clusters. New techniques have allowed the detection of these circulating tumour cells (CTCs) which are characteristic of disease progression and metastatic processes and can be used as surveillance markers [START_REF] Hirabayashi | Author Correction: Helicobacter pylori infection, atrophic gastritis, and risk of pancreatic cancer: a population-based cohort study in a large Japanese population: the JPHC Study[END_REF]. CTCs analysis allows the detection of early metastasis stages and are used to identify patients fit for chemotherapy after primary tumour resection. CTC properties can be evaluated to see whether they carry CSC or EMT-like properties allowing better prognosis prediction. In GC, the presence of CD44+ GC CTCs has been correlated to tumour metastasis and relapse. Some studies show that circulating cell-free DNA are more sensitive than CTCs for diagnosis and prognosis. Circulating tumour DNA (ctDNA) originates from primary tumours or metastases and allows more specific diagnosis of patients as well as the assessment of therapeutic response [START_REF] Hirabayashi | Author Correction: Helicobacter pylori infection, atrophic gastritis, and risk of pancreatic cancer: a population-based cohort study in a large Japanese population: the JPHC Study[END_REF].

In addition, miRNA differential expression in GC and high efficiency of circulating-miRNA detection assays make them interesting non-invasive biomarkers for GC [START_REF] Hirabayashi | Author Correction: Helicobacter pylori infection, atrophic gastritis, and risk of pancreatic cancer: a population-based cohort study in a large Japanese population: the JPHC Study[END_REF]. miR-192-5p and miR-9-5p are highly decreased in GC tissues compared to non-tumorous gastric tissues and can thus be candidate for GC diagnosis [START_REF] Ai | Preliminary study of pancreatic cancer associated with Helicobacter pylori infection[END_REF]. Moreover, miR-9-5p and a combined miRNA group (miR-9-5p + miR-9-3p + miR-433-3p) were found to distinguish chemo-resistant GC patients from chemo-sensitive ones [START_REF] Guarneri | Helicobacter pylori and autoimmune pancreatitis: role of carbonic anhydrase via molecular mimicry?[END_REF], confirming the interest of miRNA as novel non-invasive diagnostic tool in GC.

Furthermore, exosomes which are small vesicles produced by cells carry RNAs and miRNAs which remain protected from degradation when exposed to RNAses. Cancer cells or CAFs use exosomes to communicate and exchange material. These vesicles are a great promise for GC diagnosis and prognosis. Studies show that miRNA can be identified in serum-circulating exosomes, allowing the staging of patients. For example, exosomes containing miR-29s are found to play a suppressive role in the growth of peritoneal-disseminated tumour cells and are under-expressed in patients with peritoneal metastases [START_REF] Takayama | Effects of Helicobacter pylori infection on human pancreatic cancer cell line[END_REF]. Low expression of this miRNA in exosomes is correlated to bad prognosis of patients. The use of exosomes as biomarkers and even in therapy (as described above) seems to be an interesting strategy which will surely evolve in the next few years [START_REF] Kawaguchi | Bacteria closely resembling Helicobacter pylori detected immunohistologically and genetically in resected gallbladder mucosa[END_REF]. In addition, long non-coding RNAs (lncRNAs) can also be detected in exosomes. lncRNA MIAT, for example, has been described as overexpressed in GC patients and associated to lower survival rates. Serum exosomal MIAT can be detected, decreases post-treatment and is highly increased in patients suffering from GC relapse. This serum exosomal level of MIAT could thus be used to monitor GC progression using liquid biopsies [START_REF] Backert | Unusual manifestation of live Staphylococcus saprophyticus, Corynebacterium urinapleomorphum, and Helicobacter pylori in the gallbladder with cholecystitis[END_REF].

Immunotherapy

Immunotherapy is becoming a promising anti-cancer strategy for many cancers. PD-L1 can be used as biomarker of GC involving immune checkpoint escape. Nivolumab (Opdivo, Bristol-Myers Squibb, USA), an antibody targeting PD1 and disrupting its interaction with PD-L1 and PD-L2 and increasing T lymphocytes anti-tumoral activity has been recently approved in Japan as third-line treatment for unresectable or recurrent GC patients having already undergone 2 chemotherapeutical strategies. Furthermore, US Food and Drug Administration (FDA)-approved anti-PD-L1 antibody Pembrolizumab (Keytruda, Merck & Co., USA) is used for treating PD-L1+ recurrent or advanced GC patients with 2 or more prior lines of therapy [98,[START_REF] Boonyanugomol | Helicobacter pylori in Thai patients with cholangiocarcinoma and its association with biliary inflammation and proliferation[END_REF]. Although PD1 and PD-L1 inhibitors seem to improve the outcome of a small group of GC patients, the way to identify the patients that would respond still needs to be improved. Trials indeed show heterogenous responses and even the way PD-L1 is used as biomarker needs to be considered since qualitative or quantitative PD-L1 expression-based selected patients will not have similar responses [98,[START_REF] Boonyanugomol | Helicobacter pylori in Thai patients with cholangiocarcinoma and its association with biliary inflammation and proliferation[END_REF].

Finally, the use of CAR T-cell therapy with T-cells binding CSC-specific antigens could be an interesting path to follow. These cells could specifically target GCSCs and eliminate them. Unfortunately, GCSC markers are not specific since they are ubiquitously expressed in other non-pathologic cells and imply a non-specific targeting of tumours even if these markers are more intensively expressed in tumour cells. Despite all this, two CAR T-cell therapies have been approved for the treatment of children with acute lymphoblastic leukaemia and adults with advanced lymphomas, showing the positive evolution of this field. In GC, several in vitro and in vivo trials can be found in literature. HER2 CART-T cells showed positive response in vitro and persisted in blood circulation, specifically travelled to and accumulated in HER2 overexpressing tumours and contributed to their regression in human GC xenograft models [START_REF] Kuroki | Helicobacter pylori accelerates the biliary epithelial cell proliferation activity in hepatolithiasis[END_REF]. Another study describes the production of anti-GC cells monoclonal antibody mAb-3H11 by the hybridoma technique with spleen cells from mice immunised with five different human GC cell lines. mAb-3H11 was selected for its high specificity for GC cells and no reaction with normal cells. A single-chain variable fragment (scFV) of mAb-311, displaying the same reaction as the whole antibody, was used to design CAR-T cells which were able to kill tumour cells in vitro and GC cell lines as well as patient-derived xenograft tumours in vivo [START_REF] Kuroki | Helicobacter pylori accelerates the biliary epithelial cell proliferation activity in hepatolithiasis[END_REF]. The same in vitro and in vivo anti-tumoral effects were observed when Folate receptor 1 (FOL1), overexpressed in more than one third GC patients, coupled to CAR-T cells were used [START_REF] Cherif | Linking Helicobacter pylori with gallbladder and biliary tract cancer in Moroccan population using clinical and pathological profiles[END_REF]. Among the 38% clinical trials being performed for CAR-T cells on solid tumours, only 2.96% account for GC with 12 registered clinical trials (ClinicalTrial.gov) distributed between China and the USA. These trials target different antigens (EPCAM, MUC1, CEA, HER2, Mesothelin, BPX-601 and EGFR) and most are still in the recruiting stage [START_REF] Avenaud | Detection of Helicobacter species in the liver of patients with and without primary liver carcinoma[END_REF]. High toxicity mainly due to cytokine release syndrome, one of the main side effects which occurs due to the rapid and high activation of numerous cytokines, is what restrains this field for now [START_REF] Avenaud | Detection of Helicobacter species in the liver of patients with and without primary liver carcinoma[END_REF]. More research deserves to be carried out with the perspective of finding even more specific targets for GCSCs and limiting the toxicity [98].

New Experimental Models

Research issues are in constant evolution and so must be the strategies used to understand and resolve them. Helicobacter pylori infection has been for long known to be a major GC risk factor and the studies relating GC and this bacterium mostly depends on the use of transformed cell lines as infection models. This artificial approach can be criticised for its lack of pragmatism in terms of signalling pathways study, crucial in this context. Barker et al. found, using lineage tracing, that Lgr5+ cells were the multipotent stem cells responsible for the long-term self-renewal of the gastric epithelium and were among the first to generate organoids resembling mature pyloric epithelium using single Lgr5+ cells in vitro [START_REF] Verhoef | Detection of identical Helicobacter DNA in the stomach and in the noncirrhotic liver of patients with hepatocellular carcinoma[END_REF]. Afterwards, a new gastric primary cell culture system was developed for modelling H. pylori infection in vitro. Using gastric glands isolated from healthy human stomach tissue and growth factors-supplemented Matrigel, the authors were able to grow 3-dimensional (3D) spheroids which can differentiate into gastric organoids after the withdrawal of Wnt3A and spondin-1 from the culture medium leading to the formation of cultures of polarised epithelial cells when transferred into 2D [START_REF] Al-Soud | DNA of Helicobacter spp. and common gut bacteria in primary liver carcinoma[END_REF]. However, these structures offer only suboptimal conditions for studying consequences of bacterial infection due to their closed spherical shape. A recent study proposes a "mucosoid culture" model using antrum-derived gastric glands and air-liquid interface culture technique. The polarised epithelial monolayers formed secrete mucus at the apical surface, reproduce normal human gastric epithelium and can even differentiate into a base-like gland phenotype under the influence of Wnt signalling [START_REF] Huang | Identification of helicobacter species in human liver samples from patients with primary hepatocellular carcinoma[END_REF].

Studying gastric carcinogenesis also involve the use of proper in vivo models reflecting the underlying process. The major limitation of mouse models using H. pylori infection or carcinogens to induce gastric carcinogenesis is that they only rarely develop in situ carcinomas since most of the lesions are pre-neoplastic ones. In addition, these models do not metastasize or invade like in humans. Likewise, subcutaneous xenograft of PDX cells reflect the heterogeneity of patient tumours but still do not metastasize to distant organs [55]. In a recent study, Giraud et al. developed orthotopic PDX models in which patient-derived GC cells were xenografted directly into the stomach wall of immunodeficient mice and led after 8 weeks to distant metastases [START_REF] Risch | ABO blood group, Helicobacter pylori seropositivity, and risk of pancreatic cancer: a case-control study[END_REF]. In these pre-clinical models, luciferase-encoding GC cells were traced all through the in vivo experiment allowing the monitoring of primary tumour establishment and kinetic of GC cells spread and metastasis development. Using these models, the authors showed that Buparlisib treatment significantly inhibited GCSC properties in vitro and reduced the number of distant metastases in vivo when the treatment was done in the metastases starting time-lapse determined by the model [START_REF] Risch | ABO blood group, Helicobacter pylori seropositivity, and risk of pancreatic cancer: a case-control study[END_REF]. This preclinical mouse model of metastatic GC represents a major advance to study anti-metastatic efficiency of new GCSC-based therapies.

Conclusions

Gastric cancer, as many other cancers, is a complex and multifactorial disease. H. pylori remains the main cause for GC, despite the participation of other extrinsic and intrinsic factors. Gastric tumours are highly heterogenous both at intra-tumoral and inter-tumoral levels, with different histological and molecular subtypes and cellular hierarchy within the tumour as well as in the TME composition. The complexity of this disease makes it such that despite research advances and all the highlighted potential biomarkers and therapeutical strategies, there are still only few targeted strategies like Trastuzumab, Ramucirumab and anti-PD1/PD-L1 immunotherapies used in clinic [98]. A better understanding of this gastric disease's cellular, molecular, and infectious processes, at the basis of this tumour heterogeneity, is critical for the development of other diagnosis and therapeutic strategies against GC.

Gastric carcinogenesis & Metastasis

Gastric carcinogenesis is, as previously described, a multifactorial disease and its progression depends on its cellular composition. Indeed, GC are heterogeneous components, comprised of tumour cells with variable fates 2 . Part of the cells are well-differentiated cells and do not give rise to other tumours while others, more undifferentiated or carrying stem-like properties, will easily dedifferentiate, carry out asymmetrical division allowing them to renew themselves and, in so doing, evolve into new tumoral entities 5-7 . The latter, cancer stem cells (CSCs), described in our review Seeneevassen et al. 2 , thus gives a notion of the aggressiveness of tumours if present since they are capable, as previously described, of exceptionally hostile properties allowing them to resist conventional therapies, induce cancer relapse and give rise to metastases.

~46~ • EMT and Metastasis initiation

Metastasis is a process during which cells are able to escape primary tumours, pass through the extracellular matrix (ECM) and reach the bloodstream, before being carried to distant organs where they stay, adapt, change morphology, and colonize the tissue as secondary tumour sites. Different actors are important and necessary for each step of this complex process. Tumour cells hijack the developmental epithelial-to-mesenchymal transition (EMT) 2 process allowing plastic cells to readily transit from their epithelial state of origin to mesenchymal state and vice-versa.

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Originally, EMT plays a key role in embryogenesis, wound healing, inflammation process, fibrosis amongst others 8-12 . This cell transition program allows epithelial cells to reversibly suppress their epithelial characteristics and harbour those normally expressed in mesenchymal cells 9 . Epithelial cells are polarised cells tight to each other via lateral junctions such as adherens and tight junctions and desmosomes and to the basement membrane through hemidesmosomes and α6β4 integrins. Cytokeratins, components of epithelial intermediate filaments, also participate to desmosomes stabilisation. One of the first steps of the EMT process is the remodelling of these cellular junctions and adherences thus detaching cells from each other and from the basement membrane. Cells are no longer polarised; the basement membrane is degraded, the ECM and cytoskeleton reorganised 12 . Cells develop actin-rich membrane protrusions called lamellipodia and filopodia facilitating migration and, invadopodia having a function in ECM degradation and invasion. Proteins implicated in the epithelial architecture form part of the epithelial cell markers' toolbox allowing the follow-up of epithelial integrity and its loss. E-cadherin epithelial cell adherens junction marker for example is repressed for the benefit of its mesenchymal counterpart N-cadherin, along with vimentin (instead of cytokeratins) and fibronectin, β1 and β3 integrins 13 . Among the actors participating in EMT are the EMT transcription factors (EMT-TFs); the major ones being proteins from the ZEB, SNAIL and TWIST families, exerting a negative effect on the expression of epithelial markers while inducing mesenchymal ones. Studies have shown that SNAIL and ZEB1 repress the expression of E-cadherin 10,14-17 . ZEB family member ZEB1 also decreases the expression of polarity and adhesion molecules such as Pals1-associated tight junction protein, Crumbs3, HUGL2, Scribble, JAM1, Occludin and Claudin 7 amongst others 18-20 . Aigner et al. also show that SNAI1 knockdown (KD) is not enough to restore epithelial proteins expression in breast cancer cells compared to ZEB1 KD which re-establishes it partially giving a key role to ZEB, at least in this type of cells. EMT-TFs are also capable of inducing expression of Matrix Metalloproteases (MMPs) having an important role in the degradation of the ECM and the basement membrane and strongly facilitating cells escape from tumours. Expression of MMP-1, MMP-2, MMP-7 and MT1-MMP for example is induced by SNAIL 21,22 .

EMT-TFs are found to be induced in many cancers; colorectal, pancreatic, ovarian, breast, hepatocellular and gastric cancer for example, and to be related to poor prognosis of patients 23-33 confirming their role in carcinogenesis.

~48~ • EMT-derived CSC emergence and tumorigenicity

As described previously, EMT-TFs induce EMT program which allow epithelial cells to acquire migratory properties and escape tumours. Apart from this, studies show that they also have a crucial role in the acquisition of CSC properties by tumour cells and the related aggressiveness. Indeed, the EMT program activation in cells is capable of causing their dedifferentiation accentuating stemness properties 18,34,35 .

This was demonstrated in immortalised human epithelial mammary (HMLE) cells and colorectal cancer cells where induction of EMT gave rise to cells expressing stem cell markers and having CSC-like tumorigenic and invasive properties 34-37 . EMT repression in prostate cancer cells is related to decrease in CSC signature 38 . In GC, our team showed that the major risk factor H. pylori induces EMT which evolves to emergence of CD44+ CSCs in vivo and in vitro 39,40 . Related to the arousal of CSCs are the tumorigenic and invasive properties of CSCs. As described earlier, only CD44+ GC cells are able to initiate tumours when xenografted in immunodeficient mice 7 and EMT-TFs are found to be able to induce tumour-initiating cells (TICs) appearance in breast cancer 41 .

Yet, an important phenomenon induced by EMT is the invasion-metastasis cascade. Studies show the implication of this process and its actors in metastasis 42-46 . Overexpression of SNAIL in mammary cells provide them with metastatic capacities 44 . Furthermore, our team has demonstrated that ZEB1 is found to be expressed at the periphery and invasive fronts of gastric tumours, areas related to cell invasion 40 Though a large number of cells join the bloodstream where they intravasate, not all of them will survive as circulating tumour cells (CTCs) and reach and colonize distant organs 54- 56 . Indeed, though CSCs are aggressive entities and acquired mesenchymal phenotype have the particularity of rendering them even more resistant 11 , not all CTCs survive to the harsh conditions of bloodstream among which shear stress, immune-surveillance, oxidative stress, anoikis 57,58 . Those who do survive in blood are found alone or in clusters with each other or with immune cells 59-61 . CTC clusters, composed of around 2-50 cells, are more rare than single ones but keener to give rise to metastasis 59,62-65 . Cell clustering is found to be mediated by plakoglobin in breast cancer and presence of CTC in the bloodstream of patients has been related to poorer prognosis 66-69 . CTC clusters have a protective advantage compared to single CTCs 70 . Since cells are still attached, they are less affected by anoikis 71 which is crucial for their survival. CTC have also been found to cluster with cells from the TME 64,72 , neutrophils for example and their interaction induced proliferation of CTCs and increased metastasis formation 73 . They can also interact with CAFs and MDSCs with whom a protective relationship has been described thus promoting metastasis 74-76 . MDSCs are found to create an immunosuppressive environment for CTCs to circulate without being detected by the immune system. Moreover, CTCs have been found coated by platelets which also plays a role in metastasis efficiency 69,77-79 .

Interestingly, metastatic dissemination has been found as being a very early step, even preceding marked pancreatic tumour formation for example 42,80-82 and studies describe a latent dormancy period before which metastatic tumours appear 83-86 . Indeed, accumulation of latent disseminated tumour cells appear in some organs preferentially, for example in the bone marrow even if patients do not usually develop bone metastasis in these cancers 82,85 . Presence of these cells in the bone marrow not only predicts bone metastasis but also liver and lungs metastasis. This dormancy state can be installed by the tumour cells' environment; certain factors like TGFβ and BMP induce quiescence while type I collagen and fibronectin-rich niches inhibits it 87-89 .

~50~

• Extravasation, Homing and Colonisation Metastatic niche selection seems to be specific depending on the tumoral tissue of origin. Metastatic cells seem to be pre-programmed to colonise the liver, peritoneum, lungs, and bones in GC for example 58,90 . Liver is the preferential site with about 48% metastatic GC patients having liver metastases. Following extravasation and homing, again only part of the cells is able to colonize the distant organs 84,91 . These metastasis initiating cells (MICs) have been described as having the capacity to adapt, survive and proliferate at secondary sites 8,84 . Mesenchymal-like cells are less keen to colonisation 12 and thus need to undergo Mesenchymal to Epithelial (MET) transition allowing them to recover some epithelial characteristics necessary for them to prime to the new environment 92,93 . Tumour cells start re-expressing E-cadherin in metastases showing this phenotypic reversion 11,94,95 . MICs must thus be very plastic cells having the ability to transit quickly from one state to another. Interestingly, studies have demonstrated that some cells express both epithelial and mesenchymal markers and are thus in a hybrid state known as partial EMT 61 . This allows them to express more rapidly the suitable factors depending on the process they are undergoing 8,11,12 . Studies relate that only part of CSCs are MICs and have the capacity of seeding in distant organs (about 0.02% of disseminated cells). These are CD133+ CXCR4+ in pancreatic cancer, CD44v3-10 in breast cancer, CD44v6 and/or CD26+ in colorectal cancer and CD44v9 in GC 96-102 .

We have recently contributed to demonstrate that CD44v3 subpopulation of gastric CSCs have MICs properties in GC (Giraud, Seeneevassen et al. in preparation).

Furthermore, many studies show how the EMT process and factors are related to chemo resistant properties in several cancers 103-105 .

CSCs, with this remarkable list of aggressive characteristics, are undoubtedly a major target to take into consideration for the development of anti-cancerous therapeutical strategies. Understanding CSCs' properties is a foremost step for therapy development. The mechanistical aspects behind CSCs' impact in GC have been overviewed

in the above review. Other than being induced by Helicobacter pylori in GC, CSCs are capable of hijacking cell signalling pathways important in the maintenance of cellular integrity. The Hippo/YAP/TAZ-TEAD signalling pathway has been identified in the laboratory as being over-represented when CSCs were compared to non-CSCs in transcriptomic analyses 106,107 . Its role in the GC and GC CSC context is illustrated in the following literature review.

~51~

Hippo signalling pathway

The review has been submitted to Cancers journal.

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1.

The Hippo pathway

Overview

The Salvador-Warts-Hippo pathway (SAV1-LATS1/2-MST1/2 in mammals) is the key regulator of organ size and tissue homeostasis in physiology. Ever since its discovery in Drosophila [1,2] where its deregulation induces a dramatic tissue overgrowth, described as "hippopotamus phenotype" contributing to its name, this evolutionary conserved pathway has been described in divers physiological and pathological processes; from cell growth, proliferation, cell-cell contact, cell density and cell polarity control, stemness, shear stress, tissue homeostasis, repair and regeneration, to cancer [3][4][5][6][7][8][9][10][11][12][13]. Genetic screenings for genes involved in cell growth led to the discovery of its different members [5,6,14].

This highly conserved pathway in mammals is made up of two central groups of elements driving its role: 1) a serine-threonine kinase core comprising of Mammalian Sterile-20 like 1/2 (MST1/2) and Large tumour suppressor 1/2 (LATS1/2) kinases and, 2)

Citation

~53~

Cancers 2022, 14, x FOR PEER REVIEW 2 of 35 a transcriptional module containing Yes-associated protein (YAP) [15][16][17] and/or Transcriptional co-activator with PDZ binding motif (WWTR1 or TAZ).

YAP/TAZ are transcriptional co-activators. They do not possess DNA-binding motifs and need DNA-binding partners to accomplish their roles. The major ones are TEA domain family member 1-4 (TEAD1-4), also known as Transcriptional enhancer factor (TEF) in mammals and, Scalloped (Sd) in Drosophila [18,19]. TEAD fixation by YAP/TAZ is significant in the mediation of its biological functions. Screening of human transcription factor library, coupled with luciferase transcription reporter assays and/or yap cotransfection, allowed the identification of TEADs as the major actor of YAP binding to gene promoters and of YAP-induced transcriptional activity [6,19].

Studies show that YAP/TAZ are the effectors carrying the functional activity of the pathway. Mutations of Yorkie (Yki), YAP/TAZ ortholog in Drosophila, leads to decrease in proliferation while gigantism phenotype and liver tissue expansion are observed with its overexpression, coinciding with the phenotype observed upon Hippo kinases mutation. YAP deletion in mice decreases cell overgrowth phenotype caused by lack of MST1/2 [5,8]. Active YAP mutant transgenic expression or dysregulation, through deletion of Hippo kinase members at embryonic stage, leads to hyperproliferation of cardiomyocytes and heart enlargement [20][21][22][23].

In addition, YAP1 overactivation in the intestine causes enlargement of the pool of multipotent undifferentiated progenitor cells which undergo differentiation once YAP induction is ceased [8,11]. YAP inhibition also re-establishes cell-contact inhibition. Strict control of these effectors is thus required for proper tissue homeostasis.

Regulation Mechanisms

Hippo pathway regulators intervene at different levels for the maintenance of proper tissue homeostasis (Figure 1).

YAP/TAZ effectors regulation

Hippo effectors are indeed tightly controlled by upstream members of the pathway, the hippo kinases, and their regulatory partners Salvador Family WW Domain Containing Protein 1 (SAV1) and MOB Kinase Activator 1A/B (MOB1A/B), playing a role in the activation of MST1/2 and LATS1/2 respectively. Phosphorylation cascade of members of this kinase core induces the phosphorylation of YAP/TAZ effectors and thus represses their nuclear localisation and activity [3,6,9,18,24]. This is demonstrated in hippo kinases inhibition models where nuclear accumulation of the effectors is observed [9,25].

Among the inhibition mechanisms are cytoplasm sequestration or nuclear exclusion mechanisms. YAP/TAZ are active in the nucleus and their nuclear accumulation is thus elemental for their function as co-transcription factors. Hippo kinase LATS1/2 phosphorylate YAP and TAZ on Ser-127 (p-YAP SER127 ) and Ser-89 (p-TAZ SER89 ) residues respectively, allowing their binding to 14-3-3 proteins, thus retaining them in the cytoplasm [3,18]. 14-3-3 proteins knock-down (KD) induces nuclear accumulation, consistent with this observation.

Other 14-3-3 proteins-independent mechanisms also exist [25]. For example, LATS1/2 also phosphorylates YAP and TAZ on Ser-381 and Ser-311, respectively, modulating their protein stability by inducing their polyubiquitination and their addressing to the proteasome for degradation. Indeed, studies have shown that TAZ Ser-311 phosphorylation by LATS1/2 can prime it to be further phosphorylated on Ser-314 on their phosphodegron motif by Casein kinase 1 (CK1), allowing interaction with β-TrCP, and then with SCF E3 ubiquitin ligases, to be directed to the proteasome for degradation [10,26]. This effector inhibition mechanism is absent in the Drosophila model.

Furthermore, YAP/TAZ contains a COOH-terminal domain allowing interaction with proteins having PDZ binding domains [3,18,27]. This domain is important for YAP/TAZ regulation. Its absence causes cytoplasmic re-localisation and activity inhibition. YAP and TAZ are able to fixe Zonula occludens-2 protein (ZO-2) (and also ZO-1 for TAZ) and co-localise with it in the nucleus [28,29]. In addition, Vestigial-like 1-4 (VGLL1-4) contain Tondu domains (TDU) which have the capacity of binding TEAD. In so doing, VGLL4 for example is able to inhibit YAP-TEAD1 interaction, and target TEAD1 for degradation [30]. Likewise, Runt-related transcription factor 3 (RUNX3) physically interact with TEAD at its N-terminal region and consequently stops YAP from fixing to it and reduces its DNA-binding capacity and the downstream signalling [31]. YAP/TAZ-TEAD regulation is therefore most of the time achieved via the hippo kinase members which inhibit their function or not.

Hippo kinase core and upstream partners regulation

The Hippo kinases are also very well regulated, allowing them to sense the need to exert their inhibitory effect on YAP and TAZ. Among the major regulators of the Hippo pathway are mechanical cues like cell density, cell-cell adhesions and apico-basal polarity [3,32]. Proteins involved in the maintenance of these cellular events are thus implicated in Hippo regulation.

Merlin, coded by neurofibromatosis gene NF2, is an essential regulator of Hippo kinases. It is localised at tight and adherens junctions when cells are at high density and induce activation of core kinases, which in turn inhibit YAP/TAZ effectors to limit cell proliferation [33,34]. Merlin induces YAP phosphorylation by LATS by several means: it 1) binds with LATS, transports it to the cell membrane and stimulates a complex formation with MST and SAV and its further activation; 2) promotes the assembly of scaffold proteins to facilitate LATS-YAP interaction and phosphorylation. Actin disruption can activate this Merlin-dependent process [35]. Merlin is critical in liver homeostasis and stem cell niche regulation [36].

Another important protein in the maintenance of cell and tissue integrity and thus, having a role in the regulation of the Hippo pathway is Scribble, an adaptor protein, important in cell polarity. It is localised at cell membrane and like Merlin, is able to assemble a complex, here made of MST, LATS and TAZ, necessary for LATS activation and the consequent phosphorylation cascade [37].

Furthermore, apical proteins like Crumbs and Angiomotin family (AMOTs) as well as cadherins-actin cytoskeleton linker proteins such as α-Catenin are involved in YAP/TAZ regulation by managing their localisation through phosphorylation [38][39][40][41].

Hippo pathway regulation can also imply cell surface receptors such as G-protein coupled receptors (GPCRs) [32] and Leukaemia Inhibitory Factor receptor (LIFR) [42,43], part of the JAK/STAT signalling pathway. In addition, AMOTp130 isoform can also act as substrate for LATS1/2. AMOTp130-LATS1/2 signalling is inhibited by serum LPA through activation of Gαs-coupled GPCR signalling. In this context, LATS1/2 Ser-175 phosphorylation of AMOTp130 can disrupt the latter's interaction with F-actin and decrease stress fibres and focal adhesions. In so doing, LATS1/2 and AMOTp130 SER175 repress endothelial cell migration in vitro and angiogenesis in zebrafish models [44].

YAP/TAZ activation proteins also exist for example the Protein phosphatase 2A (PP2A) complex STRIPAK which exerts a negative control on Hippo kinases through their de-phosphorylation, thus inducing YAP/TAZ [45].

Hippo pathway crosstalk with/and regulation by other signalling pathways

o AKT/p73 related pathway p73, a p53 homologue able to induce the same anti-proliferative, pro-apoptotic and pro-differentiation p53-like properties, is found to physically interact with YAP [46]. The PPPPY motif it contains can associate with WW domain of YAP and this interaction was described in H1299 human non-small cell lung cancer (NSCLC) cells where YAP acts as co-transcriptional activator of p73 [47]. Basu et al. show that both YAP and p73 is required for the induction of p73 target gene BAX and that p73 acts downstream of YAP in human bone osteosarcoma epithelial cells (U20S) [48]. Furthermore, YAP-p73 signalling is regulated by AKT phosphorylation of YAP on Ser-127, inducing its cytoplasmic retention and the 14-3-3 protein-interaction hypothesis was emitted as explanation.

o JNK/AP1/c-Jun/p73 pathway Danovi et al. demonstrate that YAP promoter contains several AP-1 binding sites and that ectopic expression of AP-1 family member, c-Jun, in U20S cells induces YAP mRNA expression in a c-Jun dependent manner [49]. Furthermore, they confirm that YAP is important and sufficient in c-Jun dependent p73 stabilization and is a critical effector of c-Jun-induced apoptosis. c-Jun N-terminal kinase (JNK), upstream activator of c-Jun might be involved in c-Jun-related YAP upregulation. Moreover, it has been shown in U20S, MCF-7 breast cancer and BWT skin squamous cell carcinoma cells that JNK1/2 are able to directly phosphorylate YAP on 5 distinct sites; THR-119, SER-138, THR-153, SER-31 and THR362, the two latter being the most phosphorylated in vivo. In so doing YAP acts as apoptosis inductor [50].

o Src/c-Abl/p73 pathway Src family of non-tyrosine receptor kinases member, c-Abl, is found to directly phosphorylate YAP on TYR-357 in response to DNA damage and stabilize it. YAP TYR357 has better affinity for p73 and better induces its pro-apoptotic effects in HEK293 cells [51].

o Wnt/β-catenin pathway Azzolin et al. demonstrate that YAP/TAZ are components of the β-catenin destruction complex having a role in β-catenin inactivation as well as in YAP/TAZ transcriptional inhibition through cytoplasmic sequestration [52]. YAP/TAZ interacts with the complex via Axin as shown in HEK293 cells and, in the absence of Wnt signalling, βcatenin is recruited to the complex, phosphorylated by glycogen synthase kinase-3 (GSK3) and degraded by β-TrCP ubiquitin ligase [52,53]. Docking of β-TrCP to the destruction complex requires YAP/TAZ. Wnt activated signalling induces LRP6 accumulation which competes with YAP/TAZ for Axin binding. Axin thus releasing YAP/TAZ and dislodging it from the destruction complex, blocking β-TrCP recruitment. Wnt and YAP/TAZ are free to stimulate their respective target genes expression. Wnt/ β-catenin and YAP/TAZ are thus closely related in their regulations [52,54].

YAP is also able to bind SH3 domain of Src family kinase c-Yes (also called YES1) through its SH3-binding motif [15]. YAP, transcription factor TBX5 and β-catenin form a complex which can be re-localised to anti-apoptotic gene promoters following YAP phosphorylation by YES1 [55]. The authors found YAP to be crucial in the proliferation of β-catenin active cells while TAZ, also related to the Wnt pathway through its role in the inhibition of DVL1 [56], do not affect the cells proliferation when suppressed.

o TGFβ/BMP/Smad pathway YAP/TAZ are also found to act as binding proteins for Smads, acting downstream TGFβ and BMP signalling [57]. YAP interacts with PY motif of Smad7 through its WW domains and in so doing participates to Smad7-induced TGFβ inhibition [58]. Furthermore, YAP/TAZ can control the localization of Smad2/3 complexes after cell densityrelated stimuli. In conditions of high cell density, YAP/TAZ cytoplasmic localization, induced by the Hippo kinases and Crumbs elements, lead to cytoplasmic sequestration of Smad2/3-4 and abrogates TGFβ activation of Smad2/3-4 [59,60]. On the contrary, low cell density implies nuclear translocation of YAP/TAZ, TGFβ-dependent phosphorylation of Smad2/3-4 and nuclear translocation. o PI3K/AKT/mTOR pathway Communications between mTORC1 and Hippo pathways have also been demonstrated, which is not so surprising knowing that mTORC1 pathway is one of the few other pathways having a crucial role in organ size in physiology. Tumaneng et al. showed that cells overexpressing YAP had an increased phosphorylation of S6K THR389 and AKT SER473 , members of the PI3K/AKT pathway and direct substrates of mTORC1/2 while YAP KD cells presented a decrease in their phosphorylation demonstrating the crosstalk between the two pathways [61]. Furthermore, Gan et al. show that LATS1/2 phosphorylate Raptor on SER606 residue thus altering its interaction with Rheb and attenuating mTORC1 activation [62]. They thus demonstrate a balance between mTOR and Hippo pathways, one being attenuated when the other is activated to maintain proper cellular and tissular homeostasis since a deficiency in Raptor SER606 modifies cell growth, proliferation, and metabolism. o Pax8/TFF-1 pathway TAZ interacts with Paired box gene 8 (Pax8) and Thyroid transcription factor-1 (TFF-1) thus contributing to differentiation of thyroid cells both in vitro and in vivo. The interaction involves TAZ WW domain as well as the protein's N-terminal and Cterminal domains [63]. TAZ binding to Pax8 and TFF-1 transcriptional factors induce their activity on the thyroglobulin promoter, responsible for the transcription of genes involved in thyroid development for example. TAZ binding to TFF-1 also plays a role in lung formation and differentiation of respiratory epithelial cells [64].

o EGFR/RAF/MEK/ERK pathway The Hippo pathway was also found to be interconnected to the MAK/ERK pathway. Indeed, ERK1/2 inhibition is found to decrease YAP/TEAD transcriptional activity and downstream target genes expression by promoting its degradation while ERK2 overexpression promotes YAP expression, suggesting a regulation of YAP by ERK and its upstream regulator MEK [65]. Similarly, hippo effector YAP degradation and low activity is observed when ERK1/2 is suppressed by siRNA strategies confirming the role of this RAF/MEK/ERK signalling in YAP control [65]. Moreover, YAP KO can decrease EGFR expression and its downstream ERK signalling, also demonstrating ERK pathway regulation by YAP [66].

o AMPK pathway AMPK pathway is also found to interact with the Hippo pathway and participate to its regulation [67]. AMP-activated protein kinase (AMPK) phosphorylates AMOTL1 on its SER793 residue leading to its stabilisation which induces YAP inhibition.

o EMT/ZEB1/SNAIL/SLUG pathway YAP is found to interact directly with ZEB1, turning the latter into a transcriptional co-activator of a set of ZEB1-YAP target genes. Both proteins are able to bind to CTGF, CYR61, SDPR and AXL promoters and simultaneous interaction is observed at least for CTGF and CYR61 [68]. TEAD binding sites are necessary for this co-activation capacities. This interaction was shown not to exist for TAZ. Nevertheless, TAZ has been described to be able to control ZEB1 transcription by binding to its promoter in retinal pigment epithelium (RPE) cells causing their proliferation and dedifferentiation [69].

In addition, YAP/TAZ are able to interact and complex with SNAIL/SLUG and promote transcriptional activity of TEAD inducing the transcription of CTGF, ANKRD1, AXL, and DDK1 involved in cell proliferation [70]. TAZ and not YAP is able to complexify with SNAIL/SLUG to promote RUNX2 transcriptional activity and the expression of BGLAP2, OSTERIX and ALP having a role in differentiation. SNAIL/SLUG binding domains on YAP/TEAD are found to be their WW domains [70].

A proper balance between positive and negative signalling (Figure 2) seems necessary for the control of this essential cell signalling pathway and the maintenance of tissue integrity.

Hippo and Cancer

This pathway's implication in cellular and tissular vital processes makes its dysregulation highly critical. Despite the numerous levels of regulation, the hippo pathway is found to be involved in various cancers. In Drosophila, mutations altering the function of Hippo kinases induce hyperproliferation and decreased apoptosis, leading to appearance of tumours. This first demonstrated the tumour suppressor characteristics of Hippo kinases [3,12]. The liver tissue enlargement observed after mutation of Hippo kinases can be attributed to uncontrolled cell growth observed in cancers [5,8]. Furthermore, activation of YAP drosophila homologue Yki led to unrestrained growth of cells which is one prerequisite for tumour formation [5].

Studies relate the role of YAP/TAZ hyperactivation in many cancers. The critical genes regulated by these transcriptional co-activators makes it easy to go over to the dark side when disturbed, tending towards some cancer hallmarks like sustaining proliferative signalling, resisting cell death, activating invasion and metastasis, tumourpromoting inflammation among others [71,72]. Though its high functional potential, activating mutations of YAP gene are not described in cancer. Nonetheless, it is found to be localised on the 11q22 amplicon, which is amplified in various cancers, which could explain its hyperactivity [73]. Furthermore, inactive mutations or epigenetic regulations of Hippo kinases or close members are often observed in tumours [73].

Plouffe et al. show that YAP and its paralogue TAZ have distinct roles apart from their overlapping functions [74]. Hippo target genes induction can also differ depending on the effector involved. For example, CTGF and CYR61 are regulated by both YAP and TAZ since serum-induced stimulation of these genes is decreased in YAP/TAZ-KO cells. Furthermore, YAP deletion seems to have a greater effect than TAZ deletion on CTGF and CYR61 expression, though to a lower extent for the latter. This was demonstrated in 3 different cell lines including HeLa cervical cancer, MCF7 breast cancer and HEK293A human embryonic kidney cells. YAP KO, TAZ KO and YAP/TAZ KO caused the induction of LGR5, showing the role of both effectors in LGR5 repression. YAP and YAP/TAZ KO demonstrated the role of YAP in cell spreading, size, granularity, glucose uptake, proliferation, migration which decreased after its deletion compared to wild-type (WT) cells which resembled TAZ KO cells [74]. Interestingly, LATS1/2 KO cells gave the opposite of what observed in YAP KO and YAP/TAZ KO cells. Nevertheless, this study was carried out on HEK293A human embryonic kidney cells in which, despite the higher TAZ mRNA expression than YAP, the highly dynamic regulation of TAZ made its protein less than twice expressed than YAP protein [74]. This could explain why YAP deletion affects more cell size and physiology than TAZ deletion. YAP different regulation of CTGF is nonetheless a solid fact and other YAP-dependent target genes identified were AMOTL2 and Fos-like antigen 1 (FOSL1).

Colorectal (CRC), NSCLC and breast cancers, hepatocellular carcinomas (HCC) as well as melanomas for example present high expression of YAP or TAZ. In CRC, circular RNA circPPP1R22A induces YAP activation, causing tumour growth and metastasis which decreases in presence of peptide 17, an inhibitor of YAP [75].

In breast cancer-induced bone marrow metastasis, hypoxic environments influence HIF-1α interaction with TAZ. Nuclear HIF-1α is associated to epithelial-to-mesenchymal transition (EMT) process, interaction with Hippo effectors and is negatively regulated by LATS1/2 [76,77]. YAP interaction with ZEB1 discussed before contributes to the expression of target gene having a role in poor survival of patients, therapy resistance and increased metastasis in breast cancer [68].

Furthermore, breast cancer bone marrow metastasis can also be induced by crosstalk between ROR1-Her3 and Hippo-YAP through inactivation of MST1 [78]. Indeed, the phosphorylation of HER3 at Tyr1307 by ROR1 tyrosine kinase receptor induces the methylation of MST1 at Lys59 and its deactivation. MOB1a/b deletion causes breast [79] and lung tumours [80]. Furthermore, it has been demonstrated that breast cancer cells are able to promote YAP/TAZ expression and activity in cancer associated fibroblasts (CAFS), one of the components of the tumour micro-environment. This has as consequence the remodelling and stiffening of extracellular matrix, related to CAFs' protumorigenic roles, as well as angiogenesis improvement required for tumour growth [81].

YAP and TAZ overexpression in NSCLC are related to tumour development, progression, and poor patients' prognosis. Hyperactivation mutations of YAP are observed in this type of cancer as well as downregulation of LATS2 in 60% cases [82]. miR-135 is found to be highly expressed in NSCLC and is associated to poor survival outcomes. This microRNA is capable of increasing invasive and migration properties of cancer cells in vitro and metastasis in vivo through targeting of hippo kinase core members [83]. Moreover, YAP is able to activate EMT transcription factor Slug which in turn inhibits BMF pro-apoptotic factor. Cells enter a senescence-like dormant state and counter the drug-induced apoptosis [84]. RAF/MEK/ERK is found to contribute to NSCLC through YAP modulation since the anti-ERK1/2 siRNA strategy discussed earlier led to a decrease in migration and invasion of NSCLC cells along with the decrease in YAP protein expression observed [65].

In the liver, inducible YAP expression as well as deletion of NF2, SAV1 or MST1/2 leads to hepatomegaly and further to liver tumours [8,9,36,85]. HCC is characterised by high expression of miRNA-665 and miRNA-3910, which have inhibitory effects on hippo kinases like MST1, leading to decreased apoptosis and increased cell proliferation, migration, invasion and EMT [18,[86][87][88]. On the contrary, miRNA-195, having a tumour suppressor role is decreased in HCC and this is associated to low survival of patients [89,90]. YAP activation is an early event in HCC with the PDZ-binding domain, crucial for the activation of the cell proliferation gene CTGF [12]. In this type of cancer, CREB can promote transcriptional activity of YAP [91,92]. In addition, MEK1-YAP1 interaction leads to increased cell proliferation and maintenance of transformed neoplastic phenotype [93].

In melanomas, YAP is sufficient and necessary for invasion of cells and appearance of spontaneous metastasis [94]. Target genes involved in this phenomenon are AXL, THBS1 and CYR61. In skin cancers, deletion of MST1/2 surprisingly does not have any flagrant effect, though YAP activation is implicated in keratinocytes hyper-proliferation and squamous cell carcinoma that overcome following deletion of CTNNA1 encoding αcatenin [40]. Furthermore, methylation of LATS1/2 promoter contributes to oral squamous cell carcinoma (OSCC) [95,96]. And, in pancreatic cancer, YAP1 cooperates with K-RAS and induces tumour survival and EMT, involved in cancer cell metastasis [97].

Β-catenin-YAP cooperation explained above is important in the tumorigenicity of β-catenin active cells namely SW480, SNU-C1, HCT116 colon cancer cells, AGS GC cells, A549 lung cancer cells among others [55]. Interestingly, conversely to what expected, neither β-catenin partner TCF2 nor YAP partner TEAD was implicated in this protumorigenic effect [52,56]. TBX5 transcription factor is the one mediating the proproliferation signal by inducing the transcription of BCL2L1 and BIRC5 and YES1 is essential in this process.

The Hippo pathway also plays an important role in the resistance to therapy of many cancers such as gliomas, retinoblastomas, endometrial, bladder cell, pancreatic and ovarian cancers [98][99][START_REF] Fireman | Helicobacter pylori: seroprevalence and colorectal cancer[END_REF][START_REF] Georgopoulos | Hypergastrinemia is associated with increased risk of distal colon adenomas[END_REF][START_REF] Dash | Exploring the impact of Helicobacter pylori on gut microbiome composition[END_REF][START_REF] Kienesberger | Gastric Helicobacter pylori infection affects local and distant microbial populations and host responses[END_REF]. YAP-TEAD is involved in NSCLC cells escape to Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment [84]. Indeed, resistant cells are found to have high expression and activity of YAP/TEAD.

Other dysregulation observed in cancers and linked to the Hippo pathway is EMT which involves a loss of epithelial architecture. This affects Hippo membrane regulators like Scribble which gets delocalised thus activating YAP/TAZ and contributing to tumorigenesis and CSCs [37]. This has also been demonstrated in breast cancer cells lacking Ecadherin, having a role in epithelium integrity, and where Scribble delocalisation phenotype can be counteracted by Hippo kinases reactivation [42]. Indeed, perturbed Ecadherin/ α-Catenin complex leads to decreased YAP phosphorylation and induced cancer-related transcriptional activity.

The role of the Hippo pathway in tissue homeostasis is greatly responsible for tumour appearance and maintenance when dysregulated. Cancer is a complex disease and YAP/TAZ oncogenic ability seems to be involved in diverse parts of this ailment, from tumour initiation to tumour-immune cells crosstalk, extracellular matrix remodelling and dissemination [3,7,12,[START_REF] Jackson | A population-based epidemiologic study of Helicobacter pylori infection and its association with systemic inflammation[END_REF][START_REF] Tuomisto | Systemic inflammation in colorectal cancer: underlying factors, effects, and prognostic significance[END_REF]. Disbalance of Hippo-YAP/TAZ regulation is at the root of many cancers amongst which, the gastric cancer (GC) on which we will focus in the next part of this review.

2.

Hippo pathway in Gastric carcinogenesis

Hippo pathway in Helicobacter-mediated gastric carcinogenesis

GC is a major health concern, and the Hippo pathway is implicated since the very beginning of gastric carcinogenesis induced by chronic infection with Helicobacter pylori (H. pylori), classified as type 1 carcinogen and principal cause of GC [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF][START_REF] Raderer | Association between Helicobacter pylori infection and pancreatic cancer[END_REF][START_REF] Stolzenberg-Solomon | ATBC Study, Helicobacter pylori seropositivity as a risk factor for pancreatic cancer[END_REF][START_REF] Risch | ABO blood group, Helicobacter pylori seropositivity, and risk of pancreatic cancer: a case-control study[END_REF] . MST1/2 and LATS1/2 Hippo kinases are found downregulated and YAP/TAZ effectors, upregulated in GC [START_REF] Martel | Helicobacter pylori infection and development of pancreatic cancer[END_REF][START_REF] Lindkvist | A prospective study of Helicobacter pylori in relation to the risk for pancreatic cancer[END_REF][START_REF] Hirabayashi | Author Correction: Helicobacter pylori infection, atrophic gastritis, and risk of pancreatic cancer: a population-based cohort study in a large Japanese population: the JPHC Study[END_REF] (Figure 3). Nuclear expression of YAP/TAZ, related to their activity, is associated with poor prognosis in GC patients, particularly in those with intestinal type GC for YAP [32,42,[START_REF] Ai | Preliminary study of pancreatic cancer associated with Helicobacter pylori infection[END_REF][START_REF] Guarneri | Helicobacter pylori and autoimmune pancreatitis: role of carbonic anhydrase via molecular mimicry?[END_REF][START_REF] Takayama | Effects of Helicobacter pylori infection on human pancreatic cancer cell line[END_REF][START_REF] Kawaguchi | Bacteria closely resembling Helicobacter pylori detected immunohistologically and genetically in resected gallbladder mucosa[END_REF]. YAP is found overactivated in gastric carcinogenesis and H. pylori infection stimulates both Hippo downstream effectors YAP/TAZ as demonstrated by transcriptomic analyses in gastric epithelial cells after H. pylori infection [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF]. This effect is abrogated by infection with CagA-mutant strains showing the role of H. pylori in YAP hyperactivation and most particularly of the bacterium's CagA oncoprotein.

Figure 3. YAP expression in GC compared to non-tumorous gastric mucosa.

Representative images if YAP immunostaining in GC and non-tumorous tissue. A and B represent YAP-stained tissues form Diffuse and Intestinal types GC cases, respectively. KMplot™ analysis using KMplotter software (Kaplan-Meier plotter) [START_REF] Backert | Unusual manifestation of live Staphylococcus saprophyticus, Corynebacterium urinapleomorphum, and Helicobacter pylori in the gallbladder with cholecystitis[END_REF] showed bad prognosis of patients with GC, diffuse and intestinal types when YAP is highly expressed.

Furthermore, YAP inhibition affected H. pylori-induced EMT, demonstrating the pathway's contribution to H. pylori-related gastric carcinogenesis. Molina et al. have shown that H. pylori infection of gastric epithelial cells induce the expression of both YAP1 and LATS2 as from gastritis stage (early stage of the Correa's cascade of H. pyloriinduced gastric carcinogenesis) [43,[START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF], continued increasing at intestinal metaplasia and GC stages [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF]. YAP1-TEAD pro-proliferation and pro-survival-related genes were found to be increased after infection. Time-course infection with H. pylori showed that YAP1 is activated as early as 2h after the bacterial infection and causes increase in target genes including LATS2 acting as a regulatory feedback loop, thus controlling H. pyloriinduced YAP/TAZ activation and cell growth as from 5 to 24 hours post infection. An equilibrium thus exists for the maintenance of YAP1 proper balance as well as proper epithelial cell differentiation and survival in response to H. pylori infection, to limit epithelial cell identity loss [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF].

Furthermore, nuclear YAP/TAZ-TEAD activity is induced following H. pylori infection of GC cells. TAZ is co-expressed and found co-localised with EMT-related transcriptional factor ZEB1 in cells having a mesenchymal phenotype, and at invasive fronts of gastric tumours. TAZ-ZEB1 cooperation in H. pylori-induced GC is demonstrated by TAZ silencing which decreases ZEB1 expression, EMT as well as invasion and cancer stem cell (CSC) tumoursphere formation properties acquired by GC cells after H. pylori infection [START_REF] Raderer | Association between Helicobacter pylori infection and pancreatic cancer[END_REF]. This could be related to TAZ direct interaction with the promoter of ZEB1 as described in RPE cells [69].

Hippo pathway in GC & CSCs

CSCs' presence in tumours is a major problem in GC, like in many other solid tumours [START_REF] Stolzenberg-Solomon | ATBC Study, Helicobacter pylori seropositivity as a risk factor for pancreatic cancer[END_REF][START_REF] Boonyanugomol | Helicobacter pylori in Thai patients with cholangiocarcinoma and its association with biliary inflammation and proliferation[END_REF][START_REF] Kuroki | Helicobacter pylori accelerates the biliary epithelial cell proliferation activity in hepatolithiasis[END_REF][START_REF] Cherif | Linking Helicobacter pylori with gallbladder and biliary tract cancer in Moroccan population using clinical and pathological profiles[END_REF][START_REF] Avenaud | Detection of Helicobacter species in the liver of patients with and without primary liver carcinoma[END_REF][START_REF] Verhoef | Detection of identical Helicobacter DNA in the stomach and in the noncirrhotic liver of patients with hepatocellular carcinoma[END_REF][START_REF] Al-Soud | DNA of Helicobacter spp. and common gut bacteria in primary liver carcinoma[END_REF]. CSCs correspond to a subpopulation of cells within tumours at the origin of tumour initiation, growth, dissemination, and resistance to current treatments. They have asymmetrical division properties allowing their self-renewal and parallel proliferation and differentiation, at the base of tumour heterogeneity. Bessède et al. reported that, during the gastric carcinogenesis cascade, H. pylori infection induces the emergence of CD44+ cells carrying CSC properties, through EMT process [START_REF] Huang | Identification of helicobacter species in human liver samples from patients with primary hepatocellular carcinoma[END_REF]. Giraud et al. recently demonstrated through transcriptomics analysis that CD44+ gastric CSCs have a Hippo pathway-rich signature with overexpression of effectors and target genes such as AREG, CCND1, CDX2, CYR61, BIRC5, ID1, IGFBP3, JAG1, LATS2, SMAD7, MYC [START_REF] De Magalhães Queiroz | Isolation of a Helicobacter strain from the human liver[END_REF]. Interestingly, TEAD1/4 were also found upregulated in CD44+ cells compared to CD44non-CSCs while TEAD inhibitors, VGLL4 and RUNX3, were downregulated. Furthermore, the authors also demonstrated that residual chemotherapy-treated cells, which are the most resistant ones, present higher expression of YAP1, TAZ, CYR61 and CTGF, all associated to high YAP/TAZ-TEAD activity, compared to non-treated cells.

Fujimoto et al. have shown that Protease-activated receptor 1 (PAR1) stimulation sustains cells with stem-like properties, induces cell invasion and EMT through inhibition of LATS via Rho A GTPase [START_REF] Xuan | Helicobacter infection in hepatocellular carcinoma tissue[END_REF]. PAR1 induces Rho A-induced inhibition of LATS1/2 phosphorylation leading to activated YAP. Another receptor, Peroxisome proliferator-activated receptor δ (PPAR δ) is found to interact with YAP1 inducing the transcription, and upregulation of SOX9, a gastrointestinal stem cell marker, in premalignant lesions [START_REF] Rocha | Association of Helicobacter species with hepatitis C cirrhosis with or without hepatocellular carcinoma[END_REF]. SOX9 induces CSC properties in non-cancerous cells [START_REF] Wang | The association between Helicobacter pylori infection and chronic hepatitis C: a meta-analysis and trial sequential analysis[END_REF], promotes invasion and metastasis and is overexpressed in patients at advanced stages of GC [11].

Gastric carcinogenesis process was also studied independent of H. pylori infection. Overexpression of hippo effectors, through LATS1/2 knockout (KO) in mice pyloric stem cells, generated transformation of mice normal gastric epithelium to low-grade intraepithelial neoplasia followed by intramucosal carcinoma [START_REF] Esmat | Role of Helicobacter pylori in patients with HCV-related chronic hepatitis and cirrhosis with or without hepatocellular carcinoma: possible association with disease progression[END_REF]. YAP/TAZ were found to initiate GC via MYC as downstream mediator and direct transcriptional target of YAP [START_REF] Esmat | Role of Helicobacter pylori in patients with HCV-related chronic hepatitis and cirrhosis with or without hepatocellular carcinoma: possible association with disease progression[END_REF]. Inhibition of MYC interfered with YAP/TAZ-induced carcinogenesis.

Another study showed LATS1 low expression in GC patients and this was correlated with lymph node metastasis, poor prognosis, and tumour relapse [START_REF] Pellicano | Helicobacter species sequences in liver samples from patients with and without hepatocellular carcinoma[END_REF]. LATS1 is found to decrease GC cell proliferation and invasion in vitro and tumour growth and metastasis in vivo by inhibiting YAP nuclear translocation and thus, expression of proproliferative and pro-invasive target genes among which, YAP, CTGF, PCNA, MMP-2, MMP-9. TAZ overexpression, particularly in diffuse type GC is associated to EMT profile and to low survival of patients [43,[START_REF] De Magalhães Queiroz | Isolation of a Helicobacter strain from the human liver[END_REF][START_REF] Ponzetto | Helicobacter pylori seroprevalence in cirrhotic patients with hepatitis B virus infection[END_REF] RUNX3 has been shown to be inactivated in about 60% GC. Subsequently, the tumour suppressor effect of this TEAD interactor on YAP-TEAD complex is affected in GC and hyperactivation is noted [31]. RUNX3 mutation in GC impairs its activity towards a pro-YAP signalling phenotype. Likewise, RUNX2 is implicated in gastric carcinogenesis through the Hippo pathway. It is highly expressed in GC early stages and predicts poor prognosis [START_REF] Wang | Helicobacter pylori infection may increase the risk of progression of chronic hepatitis B disease among the Chinese population: a metaanalysis[END_REF][START_REF] Abdel-Razik | Helicobacter Pylori as an initiating factor of complications in patients with cirrhosis: a single-center observational study[END_REF]. Ectopic RUNX2 expression is implicated in GC invasion and metas-tasis process through binding to CXCR4 promoter thus inducing the expression of CXCR4 [START_REF] Wang | Helicobacter pylori infection may increase the risk of progression of chronic hepatitis B disease among the Chinese population: a metaanalysis[END_REF]. It also induces tumoursphere formation and tumour initiation showing an effect on gastric CSC population [START_REF] Wang | Helicobacter pylori infection may increase the risk of progression of chronic hepatitis B disease among the Chinese population: a metaanalysis[END_REF]. CXCR4's role is also described in CSC and metastasis in GC as well as in pancreatic, breast, colorectal [START_REF] Goo | Helicobacter pylori promotes hepatic fibrosis in the animal model[END_REF][START_REF] Huang | The pathological effect of Helicobacter pylori infection on liver tissues in mice[END_REF][START_REF] Ki | Helicobacter pylori accelerates hepatic fibrosis by sensitizing transforming growth factor-β1-induced inflammatory signaling[END_REF][START_REF] Roux-Goglin | Helicobacter infection induces podosome assembly in primary hepatocytes in vitro[END_REF][START_REF] Greiter-Wilke | Association of Helicobacter with C[END_REF]. Zhou et al. show the role of methyltransferase 3 (METTL3), major component of m6A methyltransferase complex, in gastric carcinogenesis [START_REF] Takemura | Helicobacter infection in the hepatobiliary system and hepatic lesions: a possible association in dogs[END_REF]. It is highly expressed in GC cases versus non tumoral tissues. METTL3 overexpression and KD show its role in GC cell proliferation, migration, and invasion in vitro and tumour growth in vivo. Interestingly, METTL3 positively controls YAP1 expression which could explain its effects [START_REF] Takemura | Helicobacter infection in the hepatobiliary system and hepatic lesions: a possible association in dogs[END_REF].

Moreover, Angiomotin Like 1 (AMOTL1) has been shown to interact with YAP1 and promote its nuclear translocation and its activity, thus contributing to gastric carcinogenesis [START_REF] Myung | Detection of Helicobacter pylori DNA in human biliary tree and its association with hepatolithiasis[END_REF]. The hippo pathway is also involved in Fibroblast growth factor receptor type 2 (FGFR2) gastric oncogenesis through indirect YAP1 activation via MAPK-c-Jun pathway [START_REF] Lee | Isolation and cultivation of spirochetes and other spiralshaped bacteria associated with the cecal mucosa of rats and mice[END_REF]. Overexpression of transcriptional core TEAD1/4 and YAP/TAZ targets CYR61 and CTGF is related to metastasis and bad prognosis. Furthermore, Tang et al. demonstrate a mechanism by which MST1/2 is turned off in some GC cases. Indeed, STRN3 was discovered as a PP2A regulatory subunit and was found to be able to recruit MST1/2 thus promoting its dephosphorylation and the consequent hippo effectors hyperactivation. dSTRIPAK forms part of STRN3 molecule family whose expression in induced in GC and associated with YAP activation and bad prognosis [START_REF] Romero | Case report of an unclassified microaerophilic bacterium associated with gastroenteritis[END_REF].

Additionally, microRNAs are also seen to intervene in hippo pathway's dysregulation in GC. MiR-125a-5p upregulates TAZ, TEAD2 and therefore, their target genes and stimulates cell survival, EMT, invasion and tumour growth [87]. MiR-125a-5p is found in high levels at late stages of GC and co-expresses with TAZ and TEAD2. Similarly, miR-375 modulation is implicated in GC. Promoter methylation and histone deacetylation represses miR-375 expression in this disease, and this is associated with bad prognosis and lymph node metastasis [START_REF] Kirkbride | Ovine abortion associated with an anaerobic bacterium[END_REF]. Kang et al. show that miR-375 directly targets YAP1, TEAD and CTGF, and that YAP1 re-expression partly stalls miR-375 tumoursuppressive effects. Furthermore, CTGF-KD demonstrates same effects as YAP1silencing and miR-375 ectopic expression on gastric tumours, demonstrating the role of Hippo effectors and their targets in GC. MiR-372 and MiR-373 are found to be implicated in GC cells AGS proliferation through inhibition of LATS2 gene expression [START_REF] Archer | Characterization of an unclassified microaerophilic bacterium associated with gastroenteritis[END_REF].

Moreover, long noncoding RNAs (lncRNAs) have been demonstrated as having a role in hippo pathway-induced gastric carcinogenesis. LncRNA RP11-323N12.5 is the most highly expressed lncRNA in GC, according to the TCGA database. RP11-323N12.5 is found to be correlated to YAP1 expression and to regulate it by acting on its promoter [START_REF] Fox | Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice[END_REF]. In so doing, it exerts pro-tumoral and pro-immunosuppressive functions. It is also regulated by YAP1/TAZ-TEAD transcription complex.

Besides, lncRNA acv3UTR is also found to be upregulated in GC. Ectopic expression of acv3UTR in GC cells demonstrated its role in cell growth promotion [START_REF] Nambiar | Progressive proliferative and dysplastic typhlocolitis in aging syrian hamsters naturally infected with Helicobacter spp.: a spontaneous model of inflammatory bowel disease[END_REF], through its negative effect on tumour suppressor miR-590-5p. Its expression correlates with that of YAP1. In addition, lncRNA LINC00649, enriched in GC is found to induce cell proliferation, migration and EMT in vitro as well as tumorigenesis in vivo [START_REF] Fox | Chronic hepatitis, hepatic dysplasia, fibrosis, and biliary hyperplasia in hamsters naturally infected with a novel Helicobacter classified in the H. bilis cluster[END_REF]. It is able to act on miR-16-5p which is connected to YAP1 mRNA at 3'UTR region. By doing so, it blocks the action of hippo kinases, which no longer phosphorylate YAP, and contributes to the expression of its pro-tumoral target genes [START_REF] Fox | Chronic hepatitis, hepatic dysplasia, fibrosis, and biliary hyperplasia in hamsters naturally infected with a novel Helicobacter classified in the H. bilis cluster[END_REF]. On the contrary, Sun et al. identified lncRNA LATS2-AS1-001 which has an anti-tumoral effect in GC through binding to Enhancer of zeste homolog 2 (EZH2). This leads to LATS2 upregulation and YAP1 phosphorylation, thus decreasing cell viability, migration, and invasion. Apparently, this lncRNA is under-expressed in GC which disables its effects [START_REF] Kaewpitoon | Helicobacter species are possible risk factors of cholangiocarcinoma, Asian Pac[END_REF].

Studies also show a relationship between cell metabolism and hippo-induced gastric carcinogenesis. Indeed, Liu et al. have shown the contribution of a nucleotide sugar transporter Solute carrier family 35 B4 (SLC35B4) having a crucial role in macromolecule glycosylation in GC [START_REF] Fox | Chronic proliferative hepatitis in A/JCr mice associated with persistent Helicobacter hepaticus infection: a model of helicobacter-induced carcinogenesis[END_REF]. YAP1-TEAD complex directly activates SLC35B4 which induce cell proliferation in vitro and in vivo. It is more expressed in cancerous tissues compared to non -cancerous ones and is related to poor prognosis through YAP1's action. In addition, Acylglycerol kinase (AGK), a lipid kinase involved in the production of physiological lysophosphatidic acid (LPA) and phosphatidic acid (PA), has been recognised for its pro-tumoral role when highly expressed [START_REF] Nagamine | Helicobacter hepaticus infection promotes colon tumorigenesis in the BALB/c-Rag2 (-/-) Apc(Min/+) mouse[END_REF]. It induces proliferation, invasion and EMT phenotype in GC cells; effects that were abrogated by its KD. YAP1 plays a positive role on its regulation through binding to TEAD and to AGK's promoter. In turn, in a positive feedback loop manner on YAP-TEAD, AGK inhibits hippo kinases, thus promoting YAP nuclear translocation and activity [START_REF] Nagamine | Helicobacter hepaticus infection promotes colon tumorigenesis in the BALB/c-Rag2 (-/-) Apc(Min/+) mouse[END_REF].

Hippo pathway in GC resistance to therapy

The Hippo pathway has also been shown to have a role in therapy resistance in GC, like in many other cancers. There is unfortunately no targeted therapy in GC except for Trastuzumab which is approved for the treatment of HER2+ metastatic GCs. However, resistance mechanisms have been discovered are also found at this level rendering the search for other targets even more vital. Trastuzumab-resistant cells are found to have an overexpression of HER4, phosphorylated-HER4 (p-HER4) and vimentin (VIM), a mesenchymal phenotype marker and decrease in epithelial markers. Shi et al. demonstrate that in these cells, HER4 acts on its target YAP1 and induce the expression of genes involved in EMT and proliferation, leading to higher cells migration and growth and decrease in HER2 and E-cadherin expression leading to HER2-therapy resistance and contributing to mesenchymal phenotype acquisition [START_REF] Rao | Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice[END_REF]. Moreover, YAP stimulates GC cell proliferation while its KD enhances cells' sensitivity to cisplatin showing the role of hippo effectors in GC chemoresistance [66]. In this context, the authors showed that this effect passes through regulation of EGFR/AKT/Extracellular signalregulated kinase ½ (ERK1/2) by YAP. TAZ overexpression, particularly in diffuse type GC is associated to EMT profile, to resistance to chemotherapy and to low survival of patients [START_REF] Ponzetto | Helicobacter pylori seroprevalence in cirrhotic patients with hepatitis B virus infection[END_REF][START_REF] Suerbaum | The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus[END_REF].

Interestingly, studies show that YAP and TAZ are not always expressed concomitantly in GC cells [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF][START_REF] Raderer | Association between Helicobacter pylori infection and pancreatic cancer[END_REF][START_REF] Bartonickova | Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential[END_REF]. TAZ is found to be more expressed in gastric signet ring cells carcinomas (SRCC), a poorly differentiated type of GC, usually attributed to Lauren diffuse type GC. This type of GC is of particularly bad prognosis, and this could be related to its high TAZ expression which is also found to be also higher in other poorly differentiated tumours compared to well differentiated ones like the Lauren intestinal type. Tiffon el al. demonstrated that in the SRCC GC cell line MKN45 and poorly differentiated GC patient-derived xenograft cells, TAZ is overexpressed, overactivated and controls the expression of TAZ-TEAD target genes including CYR61 and AXL among others and of EMT main transcription factor ZEB1 which decrease in TAZ KD cells. TAZ is also related to GC cells EMT phenotype and invasiveness in this context [START_REF] Raderer | Association between Helicobacter pylori infection and pancreatic cancer[END_REF]. Diffuse type GC can also be characterised by low KRT17 intermediate filaments expression. This decreased KRT17 expression causes E-cadherin loss, EMT phenotype arousal and metastasis in GC cells. Indeed, loss of intermediate filaments promotes cytoskeleton remodelling and reorganisation which activates YAP and induces IL6 expression linked to increased metastasis [START_REF] Moyaert | Helicobacter equorum sp. nov., a ureasenegative Helicobacter species isolated from horse faeces[END_REF].

The Hippo pathway and most particularly, YAP and TAZ oncoproteins, have a crucial role in gastric carcinogenesis and therapeutic strategies involving their targeting could be of real efficiency in this poor prognosis, high relapse disease.

Anti-GC strategies involving the Hippo pathway

Hippo pathway targeting can be achieved by either altering effectors' oncogenic function or by reinforcing kinases tumour-suppressive role. Studies have explored the potential of different pre-existing molecules in GC (Table 1). Leukaemia Inhibitory Factor Induces LATS1/2 phosphorylation by MST1/2 and through Scribble activation in some cases [43,42] 3.1. Downstream strategies : Targetting oncogenic YAP/TAZ-TEAD signaling YAP/TAZ-TEAD interaction is crucial for the function of the Hippo pathway effectors. Studies have shown the vital role of TEAD nuclear localization in this partnership. When TEAD is not localized in the nucleus, mobilizable unphosphorylated YAP is not able to translocate in the nucleus [74]. Altering this interaction could be the key against overactivation of this complex in cancer. Several strategies have been tested and some are presented thereafter.

RUNX family: Member of the RUNX family RUNX3, which has a tumour suppressor role in GC like many other cancers [START_REF] Hynes | Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters[END_REF] and is downregulated in cancers, can be used as therapeutical strategy. Its action mechanism is its ability to associate with LATS1/2phosphorylated YAP and facilitate its dissociation from TEAD transcription factor [START_REF] Chien | Identification of cdtB homologues and cytolethal distending toxin activity in enterohepatic Helicobacter spp[END_REF]. YAP-RUNX3 complex leads to decreased GC tumorigenesis making its use as therapy interesting. Ectopic RUNX3 could be used to compensate its defects in GC [31] and as competitor for TEAD binding resulting in tumour-regressing effects. Indeed, RUNX3 overexpression inhibits colony formation of AGS and MKN28 cells in vitro and tumour growth in vivo, compared to overexpression of mutated RUNX3L121H impairing RUNX3-TEAD interaction, which had no effect on GC cells.

VGLL4: Mutations of TEAD residues have shown their role and importance in tumorigenesis and their interest as targets for therapy [START_REF] Pons | Cytolethal distending toxin subunit B: a review of structure-function relationship[END_REF]. TDU domain, as described before, is important for binding TEADs and VGLL4 has been shown as a TDU domaincontaining protein having tumour suppressive roles by destabilizing YAP-TEAD interaction [30,[START_REF] Kawaguchi | Bacteria closely resembling Helicobacter pylori detected immunohistologically and genetically in resected gallbladder mucosa[END_REF]. Overexpression of VGLL4 in GC cells induces apoptosis and decreased cell viability in vitro while deletion of TDU domain impaired VGLL4's effect. Indeed, VGLL4 is in direct competition with YAP for TEAD fixation and, TDU domain interaction is necessary and sufficient to inhibit YAP-TEAD interaction and activity [START_REF] Kawaguchi | Bacteria closely resembling Helicobacter pylori detected immunohistologically and genetically in resected gallbladder mucosa[END_REF].

Super-TDU: Another strategy using endogenous-protein mechanisms is that of the super-TDU. Based on this, Jiao et al. designed an inhibitor peptide named "Super-TDU" mimicking VGLL4 anti-tumoral activity in vitro and in vivo [START_REF] Kawaguchi | Bacteria closely resembling Helicobacter pylori detected immunohistologically and genetically in resected gallbladder mucosa[END_REF]. Coimmunoprecipitation experiments show that Super-TDU is able to decrease endogenous YAP-TEAD interaction. Moreover, it is able to decrease YAP-TEAD target genes expression such as CYR61, CTGF, CDX2 as well as cell viability as well as colony formation of GC cells in vitro, compared to mutated Super-TDU. In addition, Super-TDU also impairs tumour growth in vivo [START_REF] Kawaguchi | Bacteria closely resembling Helicobacter pylori detected immunohistologically and genetically in resected gallbladder mucosa[END_REF]. This demonstrated how YAP-TEAD pro-tumoral signalling can depend on a short peptide and how this could be used as an anti-GC strategy.

Verteporfin: Consistent with the previous observations that YAP/TAZ-TEAD interaction is crucial for the hippo effectors activity and function, Verteporfin (VP) strategy works by disrupting this interaction. VP is a benzoporphyrin derivate, FDA-approved for the photodynamic therapy of ocular diseases (Vysudine) like age-related macular degeneration, pathologic myopia and presumed ocular histoplasmosis where it acts as photosensitiser through ROS generation leading to anti-angiogenic effects [START_REF] Hu | Helicobacter himalayensissp. nov. isolated from gastric mucosa ofMarmota himalayana[END_REF]. Moreover, VP competes with YAP and inhibits TEAD-YAP binding, thus limiting YAP-induced liver overgrowth in mouse model [START_REF] Hu | Genomic analysis of Helicobacter himalayensis sp. nov. isolated from Marmota himalayana[END_REF]. This potential makes VP drug repositioning of real interest in YAP-TEAD targeting in GC.

VP inhibits GC cell growth and expression of migration-associated genes and oncogenes [START_REF] Ge | Bacterial cytolethal distending toxin promotes the development of dysplasia in a model of microbially induced hepatocarcinogenesis[END_REF]. VP is found to affect FAT1 adhesion molecule, having a role in GC migration and invasion and reflect FAT1 silencing effects.

Hasegawa et al. show that cells have distinct sensitivities to VP, according to their YAP and TAZ expression levels [START_REF] Bartonickova | Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential[END_REF]. The authors demonstrate that MKN45 cells, a poorly differentiated cell line having a TAZ-dominant expression is more sensitive to VP than MKN74 cell line, moderately differentiated with a YAP-dominant expression profile. Nevertheless, both cell lines responded to VP treatment by inducing YAP/TAZ cytoplasmic re-localisation, degradation and decrease in Survivin expression, an antiapoptotic protein [START_REF] Bartonickova | Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential[END_REF].

~67~

Wang et al. demonstrate that VP-induced YAP-TEAD disruption capacity could pass through its ability to stimulate the expression of 14-3-3 family protein, 14-3-3σ, which in turn promotes YAP nuclear-cytoplasmic translocation [START_REF] Ge | Cytolethal distending toxin is essential for Helicobacter hepaticus colonization in outbred Swiss Webster mice[END_REF]. This effect requires p53 and determines the fact that VP treatment solution could better work in patients having p53 expression and allow to propose more targeted options and limit treatment non-responsiveness.

Furthermore, Giraud et al. have demonstrated the role of VP in GC CSC context. VP decreases the pool of CD44+ ALDH+ CSCs and the expression of several CSC markers in GC, thereby affecting GC cells tumoursphere-forming capacity and proliferation in vitro and tumour initiation and growth in PDX models in vivo. VP also affected the chemoresistance properties of gastric CSCs, showing its importance in complement of current chemotherapies in GC treatment [START_REF] De Magalhães Queiroz | Isolation of a Helicobacter strain from the human liver[END_REF].

VP can also be used as photodynamic therapy (PDT) in GC since it results in apoptosis of the treated gastric cell lines. This PDT implies the induction of photochemical reactions to destroy tumour cells through singlet oxygen production [START_REF] Pratt | Modulation of host immune responses by the cytolethal distending toxin of Helicobacter hepaticus[END_REF]. This implies a possible use of VP both for CSC targeting and as GC PDT strategy.

Peptide 17: Peptide 17 is able to inhibit YAP-TEAD interaction [START_REF] Ge | Helicobacter hepaticus cytolethal distending toxin promotes intestinal carcinogenesis in 129Rag2-deficient mice[END_REF]. It was shown to inhibit lung cancer cells proliferation, apoptosis as well as colony formation through its inhibitory effect on YAP-TEAD [START_REF] Dirienzo | Uptake and processing of the cytolethal distending toxin by mammalian cells[END_REF]. In addition, Peptide 17 is able to repress N6methyladenosine (m6A)'s methyltransferase 3 (METTL3) expression, leading to decrease of YAP1 mRNA expression and of the consequent tumour-promoting effects in GC [START_REF] Takemura | Helicobacter infection in the hepatobiliary system and hepatic lesions: a possible association in dogs[END_REF]. The authors show that, in AGS and MKN45 poorly differentiated cell lines overexpressing METTL3 compared to nontumoral gastric mucosal epithelial cells GES-1, METTL3 increases the expression of YAP1 gene. YAP1 inhibition by peptide 17 can abrogate METTTL3-induced effects and thus depend on YAP-TEAD interaction which can be targeted by peptide 17 [START_REF] Takemura | Helicobacter infection in the hepatobiliary system and hepatic lesions: a possible association in dogs[END_REF].

WZ35, a Curcumin analogue: Chen et al. demonstrate the anti-GC effects of WZ35, exceeding those of its analogue Curcumin. Both decrease cell survival and proliferation, though to a higher extent for WZ35 [START_REF] Frisan | Bacterial genotoxins: the long journey to the nucleus of mammalian cells[END_REF]. It plays on cell glycolysis, increases cellular ROS level, and presents anti-tumour effects in vivo. Most importantly, ROS represses YAP and activates Jun kinase (JUNK) which gets phosphorylated and promote cell apoptosis. The same effects were demonstrated in breast cancer cells and NSCLC [START_REF] Frisan | Bacterial genotoxins: the long journey to the nucleus of mammalian cells[END_REF][START_REF] Bezine | The cytolethal distending toxin effects on mammalian cells: A DNA damage perspective[END_REF][START_REF] Fahrer | Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts[END_REF]. But it is to note that in breast cancer, the authors show that ROS activates YAP and JNK and here YAP acts as tumour suppressor in a ROS-stimulating context, like in lung squamous cell carcinoma [START_REF] Lara-Tejero | A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein[END_REF]. Indeed, this dual effect of YAP could be related to its activating partners. For example, p73 related signalling confers pro-apoptotic properties while pro-growth for TEAD-associated signalling. Effect of curcumin/WZ35 was not attributed to level of differentiation but different sensitivities of GC cells to treatment according to their subtypes and to YAP and/or TAZ expression levels, as discussed above, makes this an important aspect to treat in GC drug research.

Upstream strategies: Hippo kinases and/or side-pathways stimulation

Apart from targeting YAP/TAZ-TEAD interactions and thus the functional effector core, another treatment strategy could be the modulation of the kinase core and upstream members of the hippo pathway in such a way as to stimulate YAP/TAZ phosphorylation and inhibition of the downstream effectors. Most of the strategies involving upper members of the pathway imply stimulation and crosstalk with other signalling pathways.

Dobutamine: Dobutamine is a β1-adrenoceptor agonist capable of stimulating β2 and α1-adrenoceptors too. It is used for treating patients with congestive heart failure and in dobutamine stress cardiovascular magnetic resonance, a test used in this domain [START_REF] Shenker | A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5triphosphate phosphatase activity[END_REF][START_REF] Shenker | Blockade of the PI-3K signalling pathway by the Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces macrophages to synthesize and secrete proinflammatory cytokines[END_REF][START_REF] Shenker | Cytolethal distending toxin-induced release of interleukin-1β by human macrophages is dependent upon activation of glycogen synthase kinase 3β, spleen tyrosine kinase (Syk) and the noncanonical inflammasome[END_REF]. Bao et al. demonstrated, thanks to a cell-based assay they developed, that dobutamine is capable of recruiting YAP to the cytoplasm, thus diminishing its nuclear translocation and activation of its target genes [START_REF] Guidi | Chronic exposure to the cytolethal distending toxins of Gram-negative bacteria promotes genomic instability and altered DNA damage response[END_REF]. Its action passes through Protein kinase A (PKA) signalling and LATS1/2 activation. In combination with cisplatin, it inhibits GC tumour cell growth, migration, invasion, and CSC properties [START_REF] Ceelen | Mitotic catastrophe as a prestage to necrosis in mouse liver cells treated with Helicobacter pullorum sonicates[END_REF]. It promotes GC cell death by apoptosis and causes YAP to be more phosphorylated and cytoplasmic.

Compound F10: One Hippo kinase-activation strategy is cytoskeletal remodelling, an important aspect of hippo pathway regulation. Song et al. present a new tertiary amide derivative, containing benzothiazole motifs, named Compound F10, and having an antiproliferative capacity on colorectal cancer cells HCT-116, GC cells MGC-803 and prostate cancer cells PC-3 [START_REF] Gelfanova | Cytolethal distending toxin of Haemophilus ducreyi induces apoptotic death of Jurkat T cells[END_REF]. Compound F10 represses tubulin polymerization and activates MST1/2 and the downstream signalling cascade leading to the phosphorylation and inhibition of YAP. Consequently, expression of c-Myc and Bcl-2 is affected, and cells undergo apoptosis, less proliferation and decrease in sphere-forming capacity linked to CSC properties [START_REF] Gelfanova | Cytolethal distending toxin of Haemophilus ducreyi induces apoptotic death of Jurkat T cells[END_REF].

Statins: Lovastatin and Fluvastatin were also identified as YAP/TAZ inhibitors by Oku et al. Fluvastatin, FDA-approved Lescol as anti-cholesterolemic drug, had a higher effect [START_REF] Péré-Védrenne | The cytolethal distending toxin subunit CdtB of Helicobacter Induces a Th17-related and antimicrobial signature in intestinal and hepatic cells in vitro[END_REF]. It modulates actin dynamics resulting in phosphorylation of Hippo effectors, decrease in their activity and target genes expression and in cell viability [START_REF] Péré-Védrenne | The cytolethal distending toxin subunit CdtB of Helicobacter hepaticus promotes senescence and endoreplication in xenograft mouse models of hepatic and intestinal cell lines[END_REF][START_REF] Belibasakis | Aggregatibacter actinomycetemcomitans targets NLRP3 and NLRP6 inflammasome expression in human mononuclear leukocytes[END_REF]. Fluvastatin also had an anti-CSC effect in breast cancer and promoted effects of chemotherapy drugs. Furthermore, Simvastatin was found to inhibit β-catenin expression and YAP activity and expression of target genes of both in GC cells. This resulted in decrease in cell proliferation, migration, invasion and apoptosis induction in vitro. Simvastatin effects passed through RhoA inhibition [START_REF] Shenker | Aggregatibacter actinomycetemcomitans cytolethal distending toxin activates the NLRP3 inflammasome in human macrophages, leading to the release of proinflammatory cytokines[END_REF].

Metformin: HEK293 cells treatment with Metformin induces AMPK pathway which phosphorylates Hippo adaptor protein AMOTL1 thus stabilising it. This facilitates and induces YAP phosphorylation by Hippo kinases thus inhibiting the effectors [67]. Metformin is FDA-approved as antidiabetic drug for type 2 diabetes [START_REF] Ando-Suguimoto | The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine C. Varon et al. production[END_REF]. In addition, Metformin has been shown to have anti-tumorigenic effects and specially, anti-CSC capacities in GC. The authors demonstrate how Hippo modulation by Metformin targets EMT and CSC properties both in vitro and in vivo [START_REF] Blazkova | Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling[END_REF].

Pazopanib: Pazopanib is another drug which can be interesting for YAP/TAZ-TEAD targeting in GC. This FDA-approved drug, Votrient, for the treatment of Advanced Renal cell cancer and Advanced Soft tissue sarcoma, can also be repositioned for the targeting of YAP/TAZ-TEAD signalling in GC [START_REF] Coppé | The senescence-associated secretory phenotype: the dark side of tumor suppression[END_REF]. Known for its anti-VEGFR and PDGFR activities, Oku et al. demonstrate its role in YAP/TAZ-TEAD inhibition by driving the effectors towards degradation by the ubiquitin-proteasome system [START_REF] Péré-Védrenne | The cytolethal distending toxin subunit CdtB of Helicobacter hepaticus promotes senescence and endoreplication in xenograft mouse models of hepatic and intestinal cell lines[END_REF], through an effect on Hippo kinases. Pazopanib affects breast cancer cell and CSCs growth and survival and potentiates chemotherapies.

SHAP:

In relation with the inhibitory effect of PP2A subunit STRN3 on Hippo kinases and its YAP-activation promoting effect described above [START_REF] Romero | Case report of an unclassified microaerophilic bacterium associated with gastroenteritis[END_REF], a highly selective peptide inhibitor was developed as targeted approach. The synthetised STRN3-derived Hippoactivating peptide SHAP was able to alter STRN3-PP2Aa interaction, restore MST1/2 phosphorylation capacity and kinase core tumour suppressor effect. Strong inhibition of YAP target genes was noted in presence of SHAP, and this was associated with decreased cell viability, colony formation and tumour cell growth in GC models [START_REF] Romero | Case report of an unclassified microaerophilic bacterium associated with gastroenteritis[END_REF].

Leukaemia Inhibitory Factor (LIF): LIF, an interleukin 6-family cytokine was shown to activate MST/LATS and induce the phosphorylation of YAP/TAZ in GC, resulting in the inhibition of GC cells' CSC and tumorigenic properties [43]. In addition, it blocks CSCmediated drug efflux mechanisms and sensitises CSCs to chemotherapeutical drugs. The use of XMU-MP-1 MST1/2 inhibitor confirmed the role of the hippo pathway in LIFinduced effects. LIFR was found to be under-expressed in GC compared to non-tumoral tissues and this was associated to low prognosis in patients, especially in diffuse type GC. In addition, gene co-expression analysis using KMplotter put into light the fact that patients having low expression of LIFR and high co-expression of oncogenic members of the Hippo pathway YAP/TAZ and target genes had less survival chances compared to high LIFR expressing patients [43]. This proved the protective effect of LIF/LIFR signalling against Hippo effectors induction in GC. Similarly, Chen et al. demonstrated an antimetastatic effect of LIF receptor (LIFR) in breast cancer, through the activation of hippo kinases and of adaptor protein Scribble [42].

Hippo pathway-aiming strategies, not tested in GC

Several other strategies have been tested over the years in other models apart GC and could be of real interest in GC therapy. These are resumed below and again involve strategies targeting the downstream oncogenic part of the pathway directly and those stimulating or aiming the upper kinase signalisation.

Downstream strategies : Targetting oncogenic YAP/TAZ-TEAD signaling

Digitoxin, a cardiac glycoside, was identified as having affinity with WW domains and inhibiting their interactions [START_REF] Azzi-Martin | Cytolethal distending toxin induces the formation of transient messenger-rich ribonucleoprotein nuclear invaginations in surviving cells[END_REF]. YAP/TAZ have WW domains which can be targeted and to this purpose, Sudol et al. constructed a structural model of YAP-digitoxin interaction which confirmed the binding and the interest of digitoxin in YAP-TEAD targeting [16]. Digitoxin, normally used for the treatment of cardiac arrythmias has been shown to present anti-cancer properties [16,[START_REF] He | The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/ CSDE1 and P62/SQSTM1[END_REF][START_REF] Aragon | Effect of cytolethal distending toxin on F-actin assembly and cell division in Chinese hamster ovary cells[END_REF][START_REF] Guerra | Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival[END_REF]. It is able to induce apoptosis in NSCLC [START_REF] Guerra | Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival[END_REF] and renal adenocarcinoma cells [START_REF] Varon | Helicobacter pullorum cytolethal distending toxin targets vinculin and cortactin and triggers formation of lamellipodia in intestinal epithelial cells[END_REF]. Studies show that it is able to inhibit HIF-1 which has been shown to interact with TAZ and to be associated to EMT [START_REF] Damek-Poprawa | Cytolethal distending toxin damages the oral epithelium of gingival explants[END_REF]. Its HIF-1 inhibition capacity gives it anti-tumorigenic properties in in vivo mouse models of prostate cancer cells subcutaneous xenograft [START_REF] Damek-Poprawa | Cytolethal distending toxin damages the oral epithelium of gingival explants[END_REF].

Furthermore, high-throughput screening has allowed the identification of C108, a chemical compound capable of decreasing YAP-dependent transcription activity. C108 decreases YAP protein expression, without changing its mRNA expression nor its phosphorylation [START_REF] Damek-Poprawa | Cell junction remodeling in gingival tissue exposed to a microbial toxin[END_REF]. This regulation of YAP does not involve activation of Hippo kinases nor a decrease in YAP-TEAD interaction but is dependent on ubiquitination and proteasomal degradation of YAP. Functionally, C108 decreases cell proliferation, migration, and tumour growth in vivo while apoptotic marker cleaved PARP is found to increase. Effects were shown on melanoma and lung adenocarcinoma models but could be tested in GC [START_REF] Damek-Poprawa | Cell junction remodeling in gingival tissue exposed to a microbial toxin[END_REF].

Among the different strategies are many TEAD-binding molecules having the capacity of altering YAP/TAZ-TEAD interaction. Indeed, structural understanding of YAP/TAZ binding domains (Y/TBD) of TEADs has allowed the search and/or design of molecules inhibiting YAP/TAZ-TEAD interaction. A central pocket has been discovered in TEAD's Y/TBD giving hope for its targeting by small molecule inhibitors. Pobbati et al. show that a non-steroidal anti-inflammatory drug Flufemanic acid can interact with TEAD2 at the level of this pocket and in so doing, inhibits YAP-TEAD interaction, transcriptional activity as well as the consequent effect on cell proliferation and migration in HEK293 Human embryonic kidney cells [START_REF] Levi | Bacterial genotoxins promote insideout integrin β1 activation, formation of focal adhesion complexes and cell spreading[END_REF]. Nevertheless, high concentrations of the molecule are needed for its effect take place [START_REF] Levi | Bacterial genotoxins promote insideout integrin β1 activation, formation of focal adhesion complexes and cell spreading[END_REF]. Moreover, with the aim of improving its effect, Bum-Erdene et al. used Fluefemanic acid structure as inspiration to design a small molecule, TED-347, capable of forming a covalent bond with a conserved cysteine in the YBD pocket [START_REF] Li | Loss of vinculin and membrane-bound β-catenin promotes metastasis and predicts poor prognosis in colorectal cancer[END_REF]. This covalent bond decreases YAP-TEAD4 binding affinity and irreversibly decreases YAP-TEAD transcriptional activity in a dosedependent manner and glioblastoma cells' viability [START_REF] Li | Loss of vinculin and membrane-bound β-catenin promotes metastasis and predicts poor prognosis in colorectal cancer[END_REF]. Structural studies allowing proper understanding of YAP/TAZ-TEAD interaction has permitted the discovery, development, and synthesis of a multitude of YAP/TAZ-TEAD interaction inhibitors. Compound 9 was designed following hexT21-A56 peptoid discovery and is able to inhibit YAP-TEAD4 interaction, thus decreasing YAP target gene CTGF expression in MST1/2 inhibited HEK293 Human embryonic kidney cells [START_REF] Péré-Védrenne | The nuclear remodeling induced by Helicobacter cytolethal distending toxin involves MAFB oncoprotein[END_REF]. Compound 19, an analogue of Kojic acid also inhibits YAP-TEAD4 interaction and activity by binding TEAD [START_REF] Eychène | A new MAFia in cancer[END_REF]. Moreover, Kurppa et al. developed MYF-01-37, a small molecule able to covalently bind to cysteine 380 and 359 in TEAD2 and TEAD1, respectively. YAP-TEAD interaction is inhibited by MYF-01-37 resulting in decrease in their target genes expression in PC-9 lung adenocarcinoma cells. Small molecules screening using a novel ultra-bright NanoLuc biosensor, allowing the quantification of protein-protein interactions in living cells, allowed the identification of Celastrol as YAP/TAZ-TEAD inhibitor [START_REF] Li | MicroRNA-152 inhibits cell proliferation, migration and invasion by directly targeting MAFB in nasopharyngeal carcinoma[END_REF]. When tested on breast and lung cancer cell lines, Celastrol is able to decrease TEAD target genes expression resulting in lower cell proliferation, survival, migration and in less cells with CSC characteristics. Another strategy aiming at the disruption of YAP-TEAD interactions was proposed by Zhou et al. who engineered a YAP-like peptide which can fix itself on TEAD and occupy YAPbinding region. This increases YAP-TEAD interaction disruption and seems to contribute to decreased tumour growth in HCC [START_REF] Ge | Helicobacter hepaticus cytolethal distending toxin promotes intestinal carcinogenesis in 129Rag2-deficient mice[END_REF]. This works since this engineered peptide has a much better affinity for TEAD and competes with endogenous YAP. Furthermore, studies show that cystine-dense peptides (CDP) have the capacity of altering protein-protein interactions and could be an interesting clue in the search for YAP/TEAD targeting molecules. Crook et al. identify TB1G2 as a disulfide-stabilised CDP, presenting the capacity of interrupting YAP-TEAD interactions [START_REF] Nougayrède | Escherichia coli induces DNA double-strand breaks in eukaryotic cells[END_REF]. However, tests in HeLa cells show that is not able to penetrate cells and further work is needed to try to make it cell permeable to test its functions biologically [START_REF] Nougayrède | Escherichia coli induces DNA double-strand breaks in eukaryotic cells[END_REF]. Nevertheless, the authors present a mammalian platform capable of screening CDPs of interest in the desired protein-protein interaction. Using Bristol University Docking engine (BUDE) allowing the screening of over 8 million drug-like compounds, Smith et al. identified, in silico, CPD3 as a novel TEAD interactor and inhibitor [START_REF] Terlouw | Recurrent APC splice variant c.835-8A>G in patients with unexplained colorectal polyposis fulfilling the colibactin mutational signature[END_REF]. Experiments on HeLa cells confirmed CPD3's effect as inhibitor of TEAD-dependent target gene induction, cell proliferation and migration [START_REF] Terlouw | Recurrent APC splice variant c.835-8A>G in patients with unexplained colorectal polyposis fulfilling the colibactin mutational signature[END_REF]. CPD3 protein-protein interaction inhibitor represses YAP activation of all TEAD-family members. This could be a problem since TEADs are related to diverse physiological properties and dissecting these could allow the identification of even more targeting drugs. Sturbaut et al. synthesized pyrazoles which are able to bind TEAD and among which, ethyl 1-(4-aminobutyl)-3-(3,4-dichlorophenyl)-1Hpyrazole-4-carboxylate (named Compound 6 by the authors) is able to inhibit TEADinduced target genes expression as well as cell proliferation [START_REF] Pleguezuelos-Manzano | Mutational signature in colorectal cancer caused by genotoxic pks+E. coli[END_REF].

Finally, virtual structure analysis and screening led to the discovery of DC-TEADin02, a vinylsulfonamide derivative, as TEAD auto-palmitoylation inhibitor [START_REF] Dziubańska-Kusibab | Colibactin DNA-damage signature indicates mutational impact in colorectal cancer[END_REF]. This inhibitor has low effect on YAP-TEAD interaction but acts on TEAD palmitoylation, leading to decreased TEAD transcriptional activity and low target genes expression in HCT116 colon cells and HEK293 human embryonic kidney cells. This shows another TEAD-activity related targeting of hippo signalling without affecting YAP-TEAD interaction [START_REF] Dziubańska-Kusibab | Colibactin DNA-damage signature indicates mutational impact in colorectal cancer[END_REF].

Upstream strategies: Hippo kinases and/or side-pathways stimulation

Melatonin binding to its GPCRs Melatonin receptor 1 and 2 (MT1 and MT2) suppresses TGF-β1-induced lung fibroblasts proliferation through an effect on YAP activity [START_REF] Diwan | Microbial cancer therapeutics: a promising approach[END_REF]. Melatonin decreases YAP nuclear translocation and inhibits TGF-β-promoted cell migration and proliferation, thus repressing lung fibrosis. Furthermore, Dasatinib, a small inhibitor of SRC-family protein kinases, also represents an interesting strategy. It has been FDA-approved as Sprycel for use in the treatment of Acute lymphoblastic leukaemia and Chronic myelogenous leukaemia [START_REF] Kalia | Emerging applications of bacteria as antitumor agents[END_REF] and has been shown to inhibit nuclear localization and activity of YAP/TAZ in breast cancer cells and affect the viability of the latter [START_REF] Péré-Védrenne | The cytolethal distending toxin subunit CdtB of Helicobacter hepaticus promotes senescence and endoreplication in xenograft mouse models of hepatic and intestinal cell lines[END_REF]. Like Fluvastatin, Dasatinib is able to change actin dynamics and thus induce YAP/TAZ phosphorylation by hippo kinases. It also sensitizes breast cancer cells to doxorubicin and paclitaxel chemotherapies and the effect is extended on breast CSCs [START_REF] Péré-Védrenne | The cytolethal distending toxin subunit CdtB of Helicobacter hepaticus promotes senescence and endoreplication in xenograft mouse models of hepatic and intestinal cell lines[END_REF][START_REF] Belibasakis | Aggregatibacter actinomycetemcomitans targets NLRP3 and NLRP6 inflammasome expression in human mononuclear leukocytes[END_REF]. The RAF/MEK/ERK pathway, having a role in YAP control as described above can be targeted by Trametinib, a MEK1/2 inhibitor, and FR180204 inhibiting ERK and leads to decrease in YAP protein expression, but not that of its mRNA [65]. Furthermore, a decrease in transcriptional activity of YAP-TEAD is noted in lung cancer cells. Basu et al. identify C19 compound, a small molecule, as a potential inhibitor of the Hippo pathway, but of WNT and TGF-β-associated pathways too, which are also found to be regulated by Hippo as described above [48,[START_REF] Mohan | Bacteria as a treasure house of secondary metabolites with anticancer potential[END_REF]. C19 is able to induce degradation of TAZ by acting on GSK3-β and AMPK and by activating Hippo kinases MST/LATS. Phosphorylation cascade results in inhibition of cell proliferation, migration, doxorubicin resistance as well as anti-tumour effect in vivo in melanoma and/or breast cancer models. C19 is presented as a molecule inhibiting several EMT-inducing pathways [START_REF] Mohan | Bacteria as a treasure house of secondary metabolites with anticancer potential[END_REF].

Hippo oncogenic effectors YAP/TAZ targeting can thus be achieved by different strategies involving the Hippo members or not and pathway's crosstalk in an important aspect of this signalling pathway.

Conclusion

The Hippo pathway is a crucial pathway in physiology and its dysregulation is essential in some pathologies. It has a major responsibility in the gastric carcinogenesis process and that, since the early steps of the disease. Different signalling pathways like WNT, AMPK, JNK, MAPK-c-Jun and TGF-β-associated ones are also involved in Hippoinduced carcinogenesis and/or regulation. The Hippo pathway effector core YAP/TAZ-TEAD is overactivated in most GC cases where it controls CSC tumorigenic and invasive properties. Targeting Hippo signalling is of utmost importance in GC therapy. Molecular data indicate that upregulation of YAP/TAZ-TEAD and their target genes in GC is always associated with aggressiveness of the disease and bad prognosis in patients. This is true particularly in SRCC which is particularly aggressive and for which there is cur-~72~ rently an urgent need for new therapies. Many strategies are being tested; either targeting the downstream oncogenic effectors or stimulating the upstream tumour-suppressor kinase core. Structural knowledge about protein-protein interactions has given rise to new hopes for the targeting of YAP/TAZ-TEAD interactions, with the identification and design of small molecules capable of binding TEAD and inhibiting oncogenic bindings. Nevertheless, there is still much to clarify to be able to propose targeted therapies aiming at repressing YAP/TAZ tumorigenic and metastatic effects in GC. With the goal at term of developing combinatorial therapies allowing GC and gastric CSCs' targeting, thus limiting relapse and resistance for which the Hippo pathway is greatly responsible.

Patents

Funding: The PhD fellowship of Lornella ~87~

Leukaemia Inhibitory Factor

Among the different strategies employed for the targeting of Hippo pathway oncogenic signalling is the Leukaemia Inhibitory Factor signalling, known and more and more explored for its pleiotropic aspects. My thesis work developed hereafter concerns, for a major part, its putative use as GC therapeutical strategy. Beforehand, the following review recapitulates what is known about this Interleukin-6 (IL-6) -family cytokine in general and more specifically in GC and whether it could be of interest in GC therapy.

LIF: generalities

Leukaemia inhibitory factor (LIF) is a multifaceted IL-6 family cytokine. This monomeric protein, often modified by glycosylation, binds to its specific LIF receptor (LIFRβ) GP190, allowing the recruitment of the GP130 subunit of the receptor, heterodimerization and induction of several cell signalling pathways involved in many physiological processes [1]. Indeed, LIF was simultaneously identified by several research teams and was attributed different functions as well as different names. pro-proliferation effects in the murine interleukin-3-dependent leukemic cell line DA-la [4]. Today, LIF is also known for its essential role in embryo implantation [5]. Defective embryo implantation, leading to infertility is observed in female under-expressing LIF. In addition, LIF -/-female mice infertility can be counteracted by recombinant LIF single dose injection [5]. LIF is also important for neurogenesis and tissue regeneration after brain or spinal cord injury [6] as well as for muscle stimulation and regeneration [7].

LIF/LIFR signalling

LIF signalisation (Fig. 1) is mediated by LIFRβ-GP130 subunit heterodimerization, preferentially formed when cells are in contact with LIF [8]. GP130 subunit of the receptor is shared with other members of the IL-6 family of cytokines while LIFRβ is shared only with oncostatin M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic growth factor (CNTF) and cardiotrophin-like cytokine (CLC). Like most members of IL-6 family, CT-1, CNTF and CLC require an additional LIFRα chain for mediating their signalisation while LIFRβ-GP130 is sufficient for OSM and LIF signalisation [8]. LIF/LIFR canonical pathway is the JAK-induced STAT3 phosphorylation after LIF/LIFR interaction, STAT3 homodimerization and nuclear translocation followed by the induction of target genes for example c-MYC, c-FOS, MMP2 mostly involved in pro-oncogenic processes through their role in survival, differentiation, invasion and in apoptosis inhibition [9][10][11]. The proximal parts of IL-6 receptors are constitutively bound to JAKS [8,12], but LIF fixation on its receptor can trigger many other signalling cascades. At least three of the four JAK family members (JAK1, JAK2 and TYK2) can bind to LIFR, but JAK1 seems to be the dominant one since LIF/LIFR signalling is highly affected in its absence [13]. JAK1 phosphorylates different tyrosine residues on GP130 and GP190, upon LIF fixation, which serves as scaffold for proteins allowing different signalisation processes. For instance, phosphorylation of Y767, Y814, Y905 and Y915 in GP130 and Y981, Y1001, Y1028 in GP190 allows STAT3 fixation while that of Y759 in GP130 and Y974 in GP190 allows GRB2 recruitment via its SHP2 domain for MAPK pathway [8] (Fig. 1). LIF/LIFR induces MAPK/ERK pathway through GRB2 phosphorylation leading to RAF and RAS activation followed by MEK and ERK activating phosphorylation [14]. LIF/LIFR main feedback mechanisms involve SOCS3 protein whose expression is induced by STAT3. SOCS3 can attach to JAK1 and GP130 to promote their ubiquitination or inhibit JAK activity [15,16]. Interestingly, SOCS3 fixation sites on GP130 and GP190 are the same as those of GRB2 since SOCS3 also binds via its SHP2 domain [15] (Fig. 1). SOCS3 thus also inhibits LIF-induced MAPK signalling by binding competition. LIF/LIFRβ can also trigger AKT/mTOR signalisation pathway [17] and Ali et al. demonstrate the implication of many other signalling pathways including Rap1, PI3K-AKT and Hippo signalling downstream LIF/LIFR and suggests different effects of LIF/LIFR depending on the different pathways [18]. LIF is being more and more studied in pathological context and more particularly in the cancer context where pleiotropic contradictory effects are being demonstrated. LIF/LIFR signalling is found to be anti-metastatic in breast cancer [19], hepatocellular carcinoma (HCC) [20] and clear cell renal carcinoma [21] while it stimulates melanoma cell migration [22], oesophageal adenocarcinoma treatment resistance [23] and chordoma aggressiveness [24]. In pancreatic cancer, both anti-metastatic [25] and pro-tumorigenic [26] effects of LIF were described.

LIF/LIFR in gastric cancer

Pro-tumorigenic aspects

Despite the knowledge acquired in many cancers, LIF/LIFR in gastric cancer (GC) is still not thoroughly understood. Terawaki et al. have associated cancer cachexia (CC) syndrome, where cancer subjects become frail and lose weight, with an increase in LIF plasma levels in a rat model of GC-associated CC [27] (Fig. 2). The authors demonstrate that surgical ablation of the rat tumours decrease CC symptoms as well as LIF plasma level and suggest Toll-like receptor (TLR) 5 signalling to be involved in LIF-induced CC mechanism. Another study showed that TLR2 upregulation promotes gastric carcinogenesis and is associated to GC patient low survival, thus providing another link between TLRs, GC and IL-6 family signalling [28] (Fig. 2). Here, the authors identify STAT3 as being involved in TLR2 overexpression [28].

In GC, STAT3 was shown to be pro-tumorigenic through the regulation of tumour growth and vascularization in mice models lacking STAT3 negative feedback regulators SHP2/ SOCS3 binding sites [29]. Interestingly, these mouse models mimic one of the gastric tumours-promoting capacity of carcinogen type I bacterium Helicobacter pylori, which is the disruption of intracellular SHP2 signalling by bacterium oncogenic protein CagA. SHP2 disruption leads to less feedback regulation of STAT3 and an increase in its activity [29,30] (Fig. 2). In this regard, the authors found STAT3 hyper-activation in H. pylori-infected antrum and especially in intestinal histological subtype of GC compared to non-tumorous gastric tissue. Fukui et al. found more inactivated methylated SOCS3 in non-neoplastic mucosa of patients with early GC that those without GC [31]. They also demonstrated a correlation between STAT3 activating phosphorylation, SOCS3 methylation and the proliferation marker Ki67. SOCS3 methylation seems to be linked to early GC cells proliferation. Furthermore, Zhang et al. show STAT3 over-activation in late GC as well as in GC lymph node metastasis cases [32]. STAT3-SOCS3 dysregulation can also be attributed to SOCS3 regulation by miRNAs. miR-340, seen to be overexpressed in GC, can target 3'-UTR regions of SOCS3 mRNA and lead to SOCS3 under-expression in GC compared to non-tumorous gastric tissues [33]. STAT3 feedback regulation by SOCS3 is, in this case, attenuated, leading to increase in pSTAT3-induced cell proliferation and cell survival mechanisms (Fig. 2).

Tumour development and progression is being more often linked to tumour microenvironment and associated stromal cells [34,35]. IL-6/JAK/STAT3 signalling seems to be involved in cancer associated fibroblasts-(CAFs) induced GC cells migration and epithelial to mesenchymal transition [36]. Here, JAK/STAT pathway serves as crosstalk between tumour cells and members of the associated microenvironment and stimulate GC progression.

The pro-tumorigenic effects (Fig. 2) of STAT3 in GC context described here mostly involve the GP130 subunit of the receptor, shared with other members of the IL-6 family of cytokines and demonstrate the role of IL-6 signalling in gastric carcinogenesis. However, LIF-mediated signalling Fig. 2 Pro-and anti-tumorigenic LIF signalling. Overview of LIF/LIFR pro-(red) and anti-tumorigenic (blue) properties and the related signalisation pathways. miRNA regulation of the pathways is represented by red arrows and crosses reflecting respective miRNA-induced inhibition which solicits GP190 has not been thoroughly studied in GC and the pleiotropic property of this cytokine makes it an interesting research point to dig in this context.

Induction of MAPK/ERK pathway by LIF leads to proproliferation capacities [14] and has been demonstrated as pro-tumorigenic in GC (Fig. 2). Similarly, AKT activation by LIF is found to have pro-proliferation, survival, or apoptotic roles. Pro-tumorigenic and pro-metastatic roles of LIF through AKT/mTOR pathway have been described in breast cancer [17] but have not been investigated in GC context. Despite the several signalling pathways known to be affected by LIF/LIFRβ, there is not much data assessing the role of LIF/LIFR signalling, independent of JAK/STAT3 in GC. Evidence from other types of cancer show that studying LIF/ LIFR through other pathways could undoubtedly bring to light some beneficial aspects of LIF [19][20][21]25].

Anti-tumorigenic aspects

Indeed, LIF appears to have a more positive side and its opposing effects in cancer seem to be related to the signalisation processes solicited [19,21,37]. LIFR has a tumour suppressor role in GC (Fig. 2). Zhang et al. show that LIFR is downregulated by miR-589, which is overexpressed in gastric tumour tissues and GC cells, compared to nontumorous gastric tissues and cells [37]. miR-589 decreases LIFR inhibition of PI3K-AKT-c-Jun axis and contributes to cell migration, metastasis, and invasion (Fig. 2). miR-589 oncogene expression is in turn increased by c-Jun which activates its transcription by binding to its promoter. LIFR tumour suppressor action in GC was also reported by Zhao et al. who show that long coding RNA LOWEG can increase LIFR expression and decrease GC cells invasion capacity [38]. LOWEG is found to be under-expressed in GC tissues and cell lines compared to non-tumorous gastric tissue and cell lines, and its expression is positively correlated to that of LIFR.

Several studies make the Hippo pathway one possible cell signalisation process to be exploited in the LIF/LIFR/ GC context. Cancer stem cells (CSCs) are a crucial aspect of gastric tumours, being at the origin of tumour initiation, resistance, and dissemination [39,40] and their targeting is of utmost significance. The Hippo pathway's importance in GC and cancer stem cells (CSCs) maintenance is a wellestablished fact today, making it a target of choice, and many studies involving other types of cancers relate Hippo/LIF/ LIFR signalling connection. Chen et al. describes LIF/LIFR as upstream the Hippo pathway, participating to the antimetastatic effects in breast cancer and LIFR-targeting by miR-9 as responsible for LIFR protein expression loss and associated metastasis [19]. Nandy et al. shows that miR-125a targets LIFR in breast cancer and in so doing, deregulates Hippo effector TAZ phosphorylation leading to increased tumorigenesis and CSCs maintenance [41]. Indeed, the Hippo pathway is made up of two opposing components: a tumour suppressor kinase core (MST1/2-LATS1/2) negatively phosphorylating a downstream block of oncogenic effectors (YAP/TAZ) which, when activated, can act as transcriptional co-factors for genes exacerbating CSCs' role in tumour initiation, growth, dissemination and relapse contributing to the pro-tumorigenic and pro-CSC role of this pathway [40,[42][43][44]. In clear cell renal carcinoma, the authors also demonstrate that LIF/LIFR anti-invasive effects passes through a decrease in the expression of Hippo oncogenic effector YAP [21]. In the meanwhile, Guo et al. successfully verified that LIF/LIFR pro-metastatic effects in melanoma do not implicate the Hippo signalling pathway downstream LIF/LIFR, but the canonical pathway JAK/STAT showing the opposing effects of LIF/LIFR depending on the solicited pathway and confirming Hippo pathway study interest in the LIF/LIFR context [22]. Furthermore, the recent link between Hippo, GC tumorigenesis and gastric CSC makes LIF/LIFR/ Hippo regulation study in GC even more attracting [42,43].

In addition, both Ali et al. and Nandy et al. interestingly suggest a possible crosstalk between LIF/LIFR/STAT3 and LIF/LIFR/Hippo signalling and more a regulation of STAT3 downstream LIF/LIFR/Hippo since JAK/STAT3 inhibition does not change Hippo effectors level, but inhibition of Hippo effector TAZ phosphorylation leads to increased levels of STAT3 and pSTAT3 [18,41]. Hippo pathway oncogenic effectors having transcriptional co-factor roles when activated, this suggests STAT3 as a possible target gene of Hippo oncogenic effectors and LIF/LIFR as negative regulator of this oncogenic core [19]. This appeals for more investigation on LIF/LIFR/Hippo/STAT3 and more specifically in the GC context where this has not been thoroughly inquired yet.

Nevertheless, among the few published studies exploiting LIF/LIFRβ signalling in GC, one shows LIFR as a tumour suppressor [38] and another that LIF can inhibit the proliferation of GC through a G1-phase arrest [45]. In the latter, authors show that LIF is under-expressed in GC and more specially in tissues with poorly differentiated GC cells and this is associated to poor prognosis of GC patients [45]. LIF overexpression in GC cells as well as recombinant LIF treatment upregulates p21 and decreases cyclin D1 expression and leads to delayed tumour progression in vivo [45]. This study again shows opposing effects of LIF/LIFR in cancer and though the mechanisms behind such an effect has not been investigated here, it arouses even more curiosity about LIF/LIFR potential effect and mechanism in GC and carves new paths and hypotheses for a possible anti-tumorigenic effect in this bad prognosis disease. In accordance with this, recent data from our team show that GC cell lines and patient-derived xenograft cells treatment with LIF is able to decrease gastric CSC population and properties in vitro by promoting the activity of Hippo pathway tumour suppressor kinase core and the JAK/STAT pathway does not seem to be implicated in the observed effects [44]. This hereby opens the field to more interesting research in this topic for LIF use in anti-CSC therapy, which is vital in GC where CSCs play a key role in tumour progression and relapse.

LIF: a new Gastric cancer therapeutic option?

LIF's pleiotropy makes its possibility as therapy questionable. Nevertheless, in vivo attempts with LIF can be more and more found in literature (Table 1). LIF injection in mice has shown potential in cardiomyocyte regeneration [46], type 2 immunity suppression in Duchenne muscular atrophy [47] and Th17 accumulation inhibition for intestinal epithelium repair in inflammatory bowel disease [48]. Few clinical trials have also been carried out, notably the phase II trial of Davis et al. using Emfilermin, a commercial human LIF, for the prevention of peripheral neuropathy induced by chemotherapy [49]. Although LIF did not show any positive response on peripheral neuropathy in this context, it was noted that patients did not show any side effects corroborating the fact that LIF could potentially be used as a therapeutic drug in human. Several clinical trials have also been carried out by Merck kGaA and Rambam Health Care Campus for embryo implantation improvement in in vitro fertilisation processes [50][51][52][53][54]. Despite these clinical trials, caution must be exercised, and more tests are necessary, taking into account LIF's high pleiotropy.

Furthermore, strategies have been developed for the targeted delivery of LIF, using as test LIF anti-proliferation capacity in murine leukemic M1 cells [55]. The authors conceived nanoparticles made of poly-ethylene glycolpoly(lactic acid), filled with LIF and coated with CD11b antibody allowing the targeting of activated peripheral macrophages, and capable of decreasing proliferation of M1 cells in vitro, giving hope for future use of LIF in targeted therapies, for example in the gastric CSC context. Despite its known anti-tumorigenic effects, no clinical trials using LIF in cancer therapy have been attempted yet, maybe due to its high pleiotropy.

Conclusion

LIF is indeed a remarkably interesting cytokine and LIF/ LIFR signalling a complex process with all the different pathways which can be triggered or inhibited by LIFRβinduced JAK phosphorylation. LIF/LIFR pleiotropy is highly related to the signalling pathways involved and further explorations might help elucidate LIF signalisation affinities depending on the cellular context. This review In vitro fertilisation process and LIF blood level measurement Rambam Health Care Campus et al. [50,51], Merck KGaA et al. [52,53], MD Yg et al. [54] In vitro experimentation murine leukemic M1 cells Nanoparticles made of poly-ethylene glycol-poly (lactic acid), filled with LIF Coating with CD11b antibody allows targeting of activated peripheral macrophages -anti-proliferation

Davis SM et al. [55] puts to light the complexity of LIF/LIFR biology in cancer and more particularly in GC. LIF/LIFRβ signalling has not been thoroughly investigated in the GC context, most studies involving IL-6/STAT3. This review relates the pro-and anti-tumorigenic aspects of LIF/LIFRβ signalling in GC. LIF, especially in a STAT3-independent context, seems to be promising in terms of anti-GC effects and therapy.

1 3

⁓ Strategy ⁓

In this context, my thesis work concerned the targeting of gastric CSCs, having undoubtedly a crucial role in gastric carcinogenesis, as related above. The strategy we focussed on was the targeting of signalling pathways implicated in the maintenance and properties of these CSCs, here the Hippo pathway. Indeed, our team recently demonstrated YAP/TAZ-TEAD oncogenic signalling participation to H. pylori-induced gastric carcinogenesis 107 and more particularly to CSC properties 40 . Thus, targeting this signalling pathway could be interesting in GC therapy.

As described above, Hippo oncogenic signalling inhibition can be achieved by several ways.

We hereby decided to adopt the following strategies to this aim; either activation of the upstream Hippo kinases or inhibition of YAP/TAZ-TEAD interaction. This consisted in the first part of my thesis work and was related in the chapters below. The second part of my work, also with a GC therapy developing aim, involved the characterisation of CSCs and more specially circulating ones known as circulating tumour cells (CTCs).

The work presented hereafter was carried out using different GC cell lines and in-house gastric patient-derived xenograft (PDX) cells 7,108 . In vitro models consisted of 3D tumoursphere assays allowing CSC selection and study, 2D invasion assays as well as a novel 3D collagen-invasion assay that I developed in the laboratory allowing the selection of CSCs before their collagen matrix invasion. Animal models used were divers: from subcutaneous xenografts 106 to intracardiac injection (Seeneevassen et al., in preparation) and orthotopic xenografts models 109 . In addition, tissue analysis was done using paraffin-inclusion models of the 3D collagen-invasion assay, mice tissue as well as tissue microarrays (TMA) constructed from local GC patients' tissue. Finally, CTC analysis was carried out on blood samples from GC patients.

For the first strategy, LIF's effect was deciphered on Hippo pathway in GC and further on gastric CSCs tumorigenic properties, published in Seeneevassen et al., Cancers 2020 110 and described in Chapter 1. In addition, LIF's effect was further explored on the invasive and metastatic properties of GC cells and CSCs, presented in Chapter 2 and in preparation for submission in Gut. Moreover, the second strategy consisted in blocking YAP/TAZ-TEAD interaction with Verteporfin, an FDA-approved drug. Its effect in GC was described in Giraud, Molina-Castro, Seeneevassen et al. Int. J. Cancer 2019 106 and in Chapter 3.

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In addition, CSC characterisation, an important step towards therapy development was also treated in my work. CTCs are crucial in GC metastasis, and their detection could help cater for the lack of diagnostic tools in this usually late-detected disease. Accordingly, I had the opportunity during my thesis to participate to the initiation of a project concerning GC patients' CTCs collection and characterisation. The presence of CTCs in GC blood, either single, in homophilic clusters or heterophilic ones, is of importance in patients' prognosis since clustered CTCs are found to be more metastatic than single ones 72 .

Heterophilic clusters imply interaction with other types of cells, for instance immune cells.

Thus, part of this work, in collaboration with N. Larmonier's team from Immunoconcept CNRS 5134, explores the relationship between immune cells and CSCs using MDSC-like Human Monocyte-derived Suppressor Cells (HuMoSCs) cocultured with gastric CSCs and this was related in Chapter 4.

These four Chapters are developed in the next part.

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⁓ Chapter 1 ⁓

Leukaemia Inhibitory Factor (LIF) Inhibits Cancer Stem Cells Tumorigenic Properties through Hippo

Kinases Activation in Gastric Cancer ~98~

Introduction

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related deaths worldwide with about 1,033,701 new cases and 782,685 deaths in 2018, in both sexes [1]. Most cases are gastric adenocarcinomas (GCs) which vary histologically and can be classified into three subtypes according to the Lauren classification: the intestinal, diffuse and mixed subtypes. GCs are usually detected late, most of the time at the metastatic stage. Treatment consists of surgery with additional chemo/radiotherapies, but the number of relapse cases remains high with a five-year survival rate less than 20%; less than 5% for advanced unresectable or metastatic cases which account for about 80% patients at diagnosis [2]. There exists at present no targeted therapy except for Herceptin which can be used to treat HER2+ GC cases comprising only 30% of GCs [3].

Studies are more and more incriminating cancer stem cells (CSCs) in GC bad prognosis. Experiments using different human GC cell lines and mouse models of patient-derived xenograft tumours (PDX) have demonstrated the role of this small cell subpopulation in GC initiation, growth, chemo-resistance, relapse as well as metastasis [4,5]. These cells possess unlimited self-renewal characteristics as well as asymmetric division and differentiation properties, creating the hierarchical structure of tumours. CSCs are involved in tumour recurrence due to their highly chemo-resistant properties and targeting them could help limit tumour resistance and relapse.

One characteristic of cancer cells and CSCs is the hijack of cell signalling pathways for their survival and maintenance [6,7]. Accumulating data suggest the disruption of the Hippo signalling pathway in many human cancers including GC, which often correlates with poor patient prognosis [8]. This pathway is made up of two distinct modules, a regulatory tumour suppressor kinase core composed of MST1/2 upstream LATS1/2 and a transcriptional module with the oncoproteins YAP and TAZ. The Hippo pathway and its effectors YAP/TAZ are tightly regulated physiologically in respect to their role in the maintenance of the highly ordered architecture of tissues and organs [9]. YAP/TAZ nuclear translocation and interaction with transcriptional factors from the TEAD family are responsible for the main physiological effects of the Hippo pathway in organ size during development and stemness [10]. MST1/2-phosphorylated-LATS1/2 directly phosphorylates YAP/TAZ, causing either their cytoplasmic retention or proteasomal degradation, thus regulating their activity. Dysregulation of this pathway in cancer increases the ability of YAP/TAZ oncoproteins to translocate to the nucleus and act as co-factors, inducing the expression of a core of oncogenic genes: cell-proliferative and anti-apoptotic genes, for instance [8]. The Hippo oncogenic effectors YAP/TAZ are abnormally detected in the nucleus of tumour cells in tumorous tissue where they induce growth-promoting transcriptional programs [8]. Transgenic expression of YAP in mouse liver leads to cell transformation and tumour development, and induced expression of YAP can trigger CSC properties [11]. Evidence also links the Hippo pathway to breast CSCs with YAP being overactivated in poorly differentiated breast cancers (G3) which are enriched in CSCs, compared with well-differentiated breast cancers (G1) [12].

In this respect, our team recently showed the role of the Hippo pathway in gastric carcinogenesis and gastric CSC maintenance, making this pathway a potential candidate for anti-CSC targeted therapies [13,14]. Using models previously developed in our laboratory for the study of gastric CSCs and markers like CD44, which can enrich cells with CSC properties [5,15], we demonstrated that targeting Hippo effectors through silencing RNA strategies decreased the gastric CSC population and properties [14]. Moreover, Verteporfin, an FDA-approved drug used for the treatment of age-related macular degeneration, was repositioned as a YAP/TEAD inhibitor, and demonstrated anti-tumorigenic effects via the inhibition of gastric CSC properties both in vitro and in vivo [14].

Hippo pathway regulators are diverse, from inter-cellular junctions and cell polarity, to cell surface receptors [9]. Chen et al. showed that the leukaemia inhibitory factor receptor (LIFR) acts upstream the Hippo pathway, limiting oncogenic YAP transfer to the nucleus, and is downregulated in human breast cancer, which correlates with poor clinical conditions [16,17]. Interestingly, treatment with LIFR ligand leukaemia inhibitory factor (LIF) and ectopic LIFR expression act as metastasis suppressors in breast cancer [16].

LIF, a member of the IL-6 family of cytokines, displays pleiotropic effects depending on cell types and organs. First cloned as an inducer of differentiation and inhibitor of proliferation of the myeloid leukemic cell line M1 [18], LIF also presented pro-proliferation effects on adult human T cells [19] as well as anti-differentiation properties on murine embryonic stem (ES) cells [20]. It has

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Cancers 2020, 12, 2011 3 of 24 a pivotal role in embryo implantation and uterine receptivity regulation [21], in the maintenance of hematopoietic stem cell pools, in muscle regeneration as well as in neural injury regulation, amongst others [22]. The past few years have seen an uprising interest for LIF effects in cancer. Apart from its anti-metastatic effect in breast cancer [16,17], recent studies showed that LIF/LIFR signalling could negatively regulate metastasis of pancreatic cancer (PC) and hepatocellular carcinoma (HCC) cells both in vitro and in vivo [23,24]. Conversely, and in relation to its pleiotropic effects, high LIFR expression was found to promote melanoma cell migration and unfavourable prognosis for melanoma patients [25] as well as aggressiveness of chordomas [26].

Interestingly, the role of LIF/LIFR signalling in the gastric cancer context has not yet been thoroughly investigated. Indeed, there are only few articles describing LIF/LIFR effects in gastric cancer and none of them addressed CSCs. Zhao et al. showed that the long noncoding RNA LOWEG, which is under-expressed in GC tissues and cell lines compared with non-tumorous gastric mucosa, is a tumour suppressor that can decrease GC tumorigenicity and positively affect LIF/LIFR signalling when overexpressed in GC cells [27]. In addition, Xu et al. recently showed that LIF overexpression as well as recombinant LIF treatment inhibit GC proliferation by inducing G1-phase arrest and delayed tumour formation in vivo [28].

Despite the interesting anti-tumoral effect of LIF/LIFR suggested by these studies, the cellular signalling mechanisms behind these effects have not been explored. Indeed, LIF pleiotropic effects can be attributed to its capacity to solicit different cell signalling pathways downstream its receptor. LIF binding on GP190 (LIFRβ) subunit triggers the recruitment of GP130 subunit and heterodimerisation, leading to Janus kinase (JAK) 1 phosphorylation followed by the activation of several cell signalling pathways [29]. The JAK and its transcriptional factor, Signal Transducer and Activator of Transcription (STAT), pathway is the most studied cell signalling pathway in the LIF/LIFR context. JAK1 phosphorylation induces STAT3 phosphorylation, dimerisation and nuclear translocation for the transcription of genes involved in self-renewal, cell survival, proliferation as well as differentiation. Interestingly, LIF anti-tumorigenic effects in breast cancer [16,30] and clear cell renal carcinoma [31] were found to involve the kinase core of the Hippo pathway while LIF pro-tumorigenic effects in melanoma pass through the JAK/STAT pathway activation [25], showing how the contradictory effects of LIF in cancer depend on the cellular mechanisms involved.

In this context, the aim of this study was to decipher the effect of LIF/LIFR signalling in the GC context, more particularly on the tumorigenic properties of gastric CSCs, and to explore the cellular mechanisms involved. Using GC cell lines as well as PDX cells, we hereby showed that LIF treatment can decrease the tumoursphere-forming capacity, an important CSC characteristic. In addition, LIF decreased CSC population, revealed by a decrease in CSC markers both at the mRNA and protein levels. Interestingly, besides its canonical JAK/STAT3 signalisation, LIF activated Hippo tumour suppressor kinases LATS1/2 and inhibited YAP/TAZ-TEAD-mediated oncogenic activity by decreasing nuclear translocation of Hippo effectors YAP/TAZ. Finally, the use of the Hippo kinase inhibitor XMU-MP-1 and JAK/STAT inhibitor Ruxolitinib showed that LIF anti-CSC effects involve Hippo kinase activation by LIF and not JAK/STAT, since Hippo kinase inhibition reverted LIF-induced anti-CSC effects.

Results

LIF/LIFR/JAK/STAT Pathway Is Functional in GC and Gastric CSCs

To check the relevance of the LIF treatment strategy chosen for this study, GC cells' responsiveness to LIF was verified in human GC cell lines AGS and MKN45 (Supplementary Materials Figure S1A) and PDX cells GC07. Cells were supplemented with LIF at different time intervals (0-48 h or 0-24 h) and STAT3 Tyr705 phosphorylation was used as the read-out of LIF-induced activation of the LIF/LIFR canonical pathway. As expected, GC cells treatment with LIF significantly increases p-STAT3 Tyr705 as from 30 min, especially for AGS cells (Figure 1A), it led to an increase in expression of JAK1/STAT3 target genes (Figure 1B, Supplementary Materials Figure S1B). Gastric CSCs were previously described as representing only a small proportion of GC cells [5]. JAK/STAT signature was thus checked by transcriptomic analysis on this subpopulation after CD44 based-FACS cell sorting of six different PDX-derived cells to evaluate LIF/LIFR signalisation in CD44+ gastric CSCs. Overexpression of the CSC markers CD44, ALDH1A1, CD166, CD24 and ITG6 in the CD44+ FACS-sorted cells compared with CD44-cells confirmed that the CD44 FACS-sorting was properly carried out and that the CD44+ cells were indeed CSCs (Figure 1C). CD44+ gastric PDX cells seem to present an upregulation of both JAK/STAT positive and negative regulators, showing a tightly controlled activation of this pathway in CSCs compared with non-CSCs (Figure 1C). The main transducers of the LIF/LIFR canonical JAK/STAT pathway were upregulated in CD44+ cells, including JAKs and several members of the STAT family. In addition, other JAK/STAT signalisation positive regulators like GRB2, IFNAR1 and IFNGR1 over-regulation were noted. Most JAK/STAT negative regulators, among the three major classes of inhibitors SOCS, PIAS and PTPs, were also upregulated (Figure 1C).

Those from the SOCS-family are also target genes of JAK/STAT signalling. Their expression is increased when the pathway is over-activated in order to act in turn as negative feedback regulators to retro control the pathway. In addition, the negative upregulators of the JAK/STAT pathway seemed to be more expressed than the positive regulators confirming the tight regulation of this pathway in CD44+ cells. Interestingly, LIF was significantly under-expressed in most CD44+ PDX cells analysed compared with CD44-PDX cells, strengthening the interest of LIF supplementation in GC.

Since LIF transduction implies the presence of the GP190 subunit of LIFR and since the whole GC population seems to be responsive to LIF (Figure 1A), it was important to verify the presence of LIFR-GP190 on the CSC subpopulation which would be targeted by LIF. LIFR-GP190 protein expression was examined in GC cell lines by flow cytometry. Both AGS and MKN45 cells express LIFR and interestingly, in both cell lines, CD44+ or CD44high cells, corresponding to the CSC population, expressed significantly more LIFR compared with non-CSC CD44-/low cells (Figure 1D,E). In addition, LIFR expression was not affected by LIF treatment in both CD44+/high and CD44-/low populations, suggesting that treating GC cells with LIF for 48 h does not seem to induce LIFR recycling/degrading mechanisms which might have induced non-responsiveness to LIF with time. Consequently, LIF/LIFR/JAK/STAT signal transduction observed in whole GC population after LIF treatment (Figure 1A) could be mostly attributed to that of the gastric CSC population which contains more LIFR and presents an upregulation of the JAK/STAT signature.

LIF treatment thus seems to be an appropriate strategy to target gastric CSCs since GC cells respond to LIF and CSCs show a LIF/LIFR/JAK/STAT upregulated transcriptomic signature. Besides, the LIFR-GP190 higher expression by CD44+/high cells shows that LIF/LIFR/JAK/STAT signal transduction induced after LIF treatment of a whole GC population can be attributed mostly to CSCs.

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Cancers 2020, 12, 2011 6 of 24

LIF Presents Anti-CSC Effects on GC Cell Lines and PDX Cells

LIF/LIFR signalling effects on CSC tumorigenic functional properties were then assessed after LIF treatment, through non-adherent tumoursphere-forming assays. LIF significantly decreased AGS cells' tumourspheres-forming capacity in a dose-dependent manner (Figure 2A). The dose of 50 ng/mL LIF, which was that inducing the most important effect in AGS cells, was chosen for further experiments. Results were confirmed in the MKN45 GC cell line as well as in PDX cells GC07, GC10 and GC04 (Figure 2A), showing that LIF presents anti-tumorigenic capacities in GC cell lines as well as in cells derived from GC patients.

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LIF Presents Anti-CSC Effects on GC Cell Lines and PDX Cells

LIF/LIFR signalling effects on CSC tumorigenic functional properties were then assessed after LIF treatment, through non-adherent tumoursphere-forming assays. LIF significantly decreased AGS cells' tumourspheres-forming capacity in a dose-dependent manner (Figure 2A). The dose of 50 ng/mL LIF, which was that inducing the most important effect in AGS cells, was chosen for further experiments. Results were confirmed in the MKN45 GC cell line as well as in PDX cells GC07, GC10 and GC04 (Figure 2A), showing that LIF presents anti-tumorigenic capacities in GC cell lines as well as in cells derived from GC patients. To confirm whether LIF's anti-tumorigenic effect was the consequence of CSC targeting, 48-h LIF-treated cells were analysed for the expression of the CSC-associated marker CD44 by flow cytometry. The percentage of cells expressing CD44 as well as the mean expression of CD44, determined by the mean fluorescence intensity (MFI), decreased significantly in GC07 PDX cells as well as in AGS cells (Figure 2B), but not in MKN45 cells though a decrease tendency can be noted. Nguyen et al. described CD44+ALDH+ MKN45 cells as being highly tumorigenic [5]. In this regard, ALDH activity, another marker of gastric CSCs [5], was analysed by flow cytometry in MKN45 cells upon LIF treatment. All ALDH+ cells analysed were CD44+/high in accordance with Nguyen et al.'s previous data [5] (Supplementary Figure S1C). A significant decrease in the percentage of ALDH+ cells as well as in ALDH MFI was noted after 48-h LIF treatment (Figure 2B). These results indicate that, in MKN45 cells, even if CD44 expression remained high, ALDH activity was significantly reduced by LIF treatment. These results, combined with those observed in AGS and GC07 cells, show that LIF reduces the population of cells expressing either CD44 or ALDH CSC markers in GC cell lines and patient-derived cells.

Finally, mRNA expression of gastric CSC markers, including CD44 and ALDH1A1 as well as CD24, CD166 and KLF5 as previously reported [5,14,32], also diminished after LIF treatment of AGS, MKN45 and GC06 PDX cells (Figure 2C).

Altogether, these results indicate that LIF treatment induces an anti-tumorigenic effect in GC, via a decrease in CSC properties and population in both GC cell lines and PDX cells.

LIF/LIFR Signalling Potentiates Chemotherapy Effect on Gastric CSCs

One characteristic of CSCs, making their targeting of utmost importance in cancer therapy, is their resistance to conventional chemotherapies leading to relapse cases.

Live immunofluorescence analysis was carried out for MKN45 cells to analyse ALDH activity, coupled with CD44 protein expression and Hoescht-33342 compound incorporation, in 7-day-old spheres, treated or not with LIF for 48 h. Hoechst is a nucleic acid stain, known to be cell-permeant and to have affinity for DNA. It is incorporated by live cancer cells. However, it was precedingly shown that chemo-resistant CSCs possess efflux pumps allowing them to efflux chemotherapy drugs in cancer cases and to evacuate Hoechst in vitro [5]. This live immunofluorescence technique thus allows the analysis of the different subpopulations of CSCs contributing to tumour heterogeneity. LIF treatment decreased both ALDH+ and CD44+ cells and most particularly the ALDH+Hoechst-and CD44+Hoechstsubpopulations corresponding to chemo-resistant CSCs, as previously described [5] (Figure 3A(i),(ii)).

Conversely, an increase in ALDH-Hoechst+ and CD44-Hoescht+ cells, previously described as differentiated non-CSC and non-tumorigenic cells [5], was observed (Figure 3A(i)(ii)). These results suggest that LIF reduces the population of CSCs with drug efflux properties.

To address this point, LIF treatment was tested, in combination with conventional chemotherapeutical agents, Doxorubicin (DOX) and 5-Fluorouracil (5-FU), on GC cells' tumoursphere-formation properties. In AGS and MKN45 cells as well as PDX cells, GC07, LIF, DOX and 5-FU alone significantly decreased the capacity of cells to form tumourspheres when compared with untreated controls (Figure 3B). Interestingly, when LIF was added in combination with DOX and 5-FU, the number of cells forming spheres in vitro decreased significantly compared with chemotherapy drugs alone (Figure 3B). Conversely, an increase in ALDH-Hoechst+ and CD44-Hoescht+ cells, previously described as differentiated non-CSC and non-tumorigenic cells [5], was observed (Figure 3A(i)(ii)). These results suggest that LIF reduces the population of CSCs with drug efflux properties.

To address this point, LIF treatment was tested, in combination with conventional chemotherapeutical agents, Doxorubicin (DOX) and 5-Fluorouracil (5-FU), on GC cells' tumoursphere-formation properties. In AGS and MKN45 cells as well as PDX cells, GC07, LIF, DOX and 5-FU alone significantly decreased the capacity of cells to form tumourspheres when compared with untreated controls (Figure 3B). Interestingly, when LIF was added in combination with DOX and 5-FU, the number of cells forming spheres in vitro decreased significantly compared with chemotherapy drugs alone (Figure 3B).

Combined, these results show that LIF, by reducing the population of chemo-resistant CSCs, potentiates the anti-tumorigenic efficiency of chemotherapy drugs in GC cell lines and also in cells derived from GC patients in vitro. Combined, these results show that LIF, by reducing the population of chemo-resistant CSCs, potentiates the anti-tumorigenic efficiency of chemotherapy drugs in GC cell lines and also in cells derived from GC patients in vitro.

LIF Activates Hippo Tumour Suppressor Kinases in GC

LIF/LIFR signalling was described as being upstream the Hippo pathway in breast cancer [16]. Previous studies from our team described the Hippo pathway as being involved in gastric CSC maintenance through the tumorigenic activity of its effectors YAP/TAZ/TEAD [13,14]. In order to evaluate LIF effect on the Hippo pathway in GC, which has neither been studied nor described, Hippo pathway status and especially Hippo tumour suppressor kinase LATS1/2 activation was analysed upon LIF treatment.

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Cancers 2020, 12, 2011 9 of 24 Interestingly, an increase in LATS1/2 Thr1079/1041 phosphorylation, associated with an increase in LATS1/2-mediated phosphorylation of YAP on Ser127 was found as from 30 min LIF treatment of AGS and MKN45 cells (Figure 4A). Since this LATS1/2-mediated YAP/TAZ phosphorylation leads to their cytoplasmic retention and less nuclear translocation, LIF-treated AGS and MKN45 cells were checked and quantified for nuclear YAP/TAZ. YAP and TAZ are paralogues, YAP being the main Hippo effector in AGS cells and TAZ its homologue in MKN45 cells [13,14]. Indeed, a significant decrease in nuclear YAP/TAZ was noted from 30-min LIF treatment, reflecting p-LATS1/2 Thr1079/1041 -induced phosphorylation and cytoplasmic retention of YAP/TAZ (Figure 4B,C). In accordance with these results, kinase-induced YAP/TAZ phosphorylation decreases YAP/TAZ interaction with TEAD transcription factors assessed by TEAD-luciferase reporter assay. TEAD activity significantly decreased after LIF supplementation in both AGS and MKN45 cells, as from 30 min post-LIF for the former and 2 h for the latter (Figure 4D). To confirm the above effects, mRNA expression of genes encoding targets of this pathway was analysed (Figure 4E). Both CYR61 and CTGF decreased significantly after LIF supplementation, as from 24 h for CYR61 in AGS.

LIF thus effectively activates the Hippo pathway kinase core which in turn inhibits YAP/TAZ oncogenic effectors nuclear translocation and TEAD transcriptional activity in the GC context.

LIF Anti-CSC Effects Are Linked to Hippo Pathway Tumour Suppressor Core Activation

In order to address the possible link between LIF anti-CSC effects and Hippo kinase activation, the Hippo kinase MST1/2 inhibitor XMU-MP-1 was used to inhibit LATS1/2 Thr1079/1041 phosphorylation and activation. XMU-MP-1 did not affect STAT3 and STAT3 Tyr705 phosphorylation (Supplementary Materials Figure S2(i)), while efficiently inhibiting LATS1/2 Thr1079/1041 phosphorylation, even in the presence of LIF (Figure 5A(i)). This confirmed that the LIF-induced increase in p-LATS1/2 Thr1079/1041 was due to MST1/2 activation mediating LATS1/2 phosphorylation on Thr1079/1041. TEAD-luciferase reporter assay carried out in the presence of XMU-MP-1 and LIF showed a significant increase in TEAD transcriptional activity, confirming p-LATS1/2 Thr1079/1041 functional inhibition, and therefore de-repression of YAP/TAZ/TEAD association and transcriptional activity (Figure 5B). YAP/TAZ/TEAD target genes CYR61, CTGF, AXL, TAZ and AREG's mRNA expression, which was repressed by LIF treatment, also tended to increase in the presence of XMU-MP-1. These LIF-induced effects were hindered when the inhibitor was added either in 2D (AGS cells) or 3D (MKN45 spheroids) culture conditions (Figure 5C). All this suggests that LIF-induced effects on gastric CSCs pass through Hippo core kinases activation and YAP/TAZ/TEAD effectors inhibition.

This was confirmed in functional tumoursphere-forming assays carried out in the presence of XMU-MP-1 and LIF, showing that when LATS1/2 Thr1079/1041 phosphorylation is inhibited, LIF is no longer able to decrease the number of formed spheres and it even increases it in the AGS cell line (Figure 5D). This shows that in GC cell lines AGS and MKN45 as well as in PDX cells GC07, the LIF-induced anti-CSC effect involves the activation of the Hippo core kinases.

Moreover, as the LIF/LIFR canonical pathway is the JAK1/STAT3 pathway, the implication of this signalisation for the anti-CSC effects was evaluated. JAK1-induced STAT3 Tyr705 phosphorylation was efficiently inhibited by the JAK1 inhibitor Ruxolitinib (Figure 5A(ii)) and no variation was noted in LATS2 and LATS1/2 Thr1079/1041 phosphorylation (Supplementary Materials Figure S2(ii)). A significant decrease in the number of tumourspheres was observed in the presence of LIF and Ruxolitinib in the AGS, MKN45 and GC07 cells, compared with cells treated with Ruxolitinib only. JAK1/STAT3 inhibition did not affect LIF-induced anti-CSC effects in GC (Figure 5D), showing that this LIF canonical pathway is not involved in this phenomenon. Finally, double Hippo and JAK1/STAT3 inhibition resulted in the neutralisation of LIF-induced inhibition of tumoursphere formation, confirming the role of the Hippo pathway in LIF anti-CSC properties (Figure 5D) and suggesting an antagonistic role of the two pathways in response to LIF. LIF-induced Hippo kinase activation and LIF anti-CSC effects thus seem to be linked. The LIF-induced decrease in CSC population and tumorigenic properties passes through the Hippo tumour suppressor MST1/2 and LATS1/2 kinases activation, with the consequent inhibition of Hippo YAP/TAZ/TEAD oncogenic effectors activity.

LIF-Induced Chemotherapy Potentiating Effects Pass through the Hippo Pathway

Since LIF was observed to potentiate conventional chemotherapy effects on gastric CSCs (Figure 3), the Hippo kinase inhibitor XMU-MP-1 was used to verifiy whether this effect of LIF also involved the Hippo pathway. Interestingly, LIF in combination with Doxorubicin was less efficient in decreasing tumoursphere-forming capacity when XMU-MP-1 was added to AGS, MKN45 as well as GC07 cells than in the absence of XMU-MP-1 (Figure 6A,B). The results are less obvious for the 5-FU treatment in AGS and MKN45 but remained similar in PDX cells GC07. Hippo kinase inhibition thus decreased LIF potentiating effects in chemotherapy, confirming this pathway's involvement in LIF-anti-CSC effects. In addition, inhibition of the JAK1/STAT3 pathway did not affect LIF-induced reinforcement of chemotherapeutical effects in all cells (Figure 6A,B), showing that this pathway is not involved in this phenomenon.

LIF-Induced Chemotherapy Potentiating Effects Pass through the Hippo Pathway

Since LIF was observed to potentiate conventional chemotherapy effects on gastric CSCs (Figure 3), the Hippo kinase inhibitor XMU-MP-1 was used to verifiy whether this effect of LIF also involved the Hippo pathway. Interestingly, LIF in combination with Doxorubicin was less efficient in decreasing tumoursphere-forming capacity when XMU-MP-1 was added to AGS, MKN45 as well as GC07 cells than in the absence of XMU-MP-1 (Figure 6A,B). The results are less obvious for the 5-FU treatment in AGS and MKN45 but remained similar in PDX cells GC07. Hippo kinase inhibition thus decreased LIF potentiating effects in chemotherapy, confirming this pathway's involvement in LIFanti-CSC effects. In addition, inhibition of the JAK1/STAT3 pathway did not affect LIF-induced reinforcement of chemotherapeutical effects in all cells (Figure 6A,B), showing that this pathway is not involved in this phenomenon. JAK1 inhibitor Ruxolitinib (Ruxo). Cells treated with chemotherapy drugs only are represented in light blue, while cells treated with LIF in combination with chemotherapy drugs and/or inhibitors are in light green. XMU-treated conditions are represented by dotted bars and Ruxo-treated conditions by checked bars. Treatments were carried out every 48 h and tumoursphere counting was performed after 7 days. * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001 versus untreated controls with ANOVA statistical analyses. $ p < 0.05, $$ p < 0.005, $$$ p < 0.0005 and $$$$ p < 0.0001 versus conditions treated with DOX or 5-FU alone with Student t-tests.

LIF-induced chemotherapy potentiating effects thus also involve the Hippo pathway kinases and not its canonical pathway JAK1/STAT3.

LIFR Is Underexpressed in GC Tissue and Low LIF/LIFR is Associated to Poor Survival in Diffuse Type GC

The need for new reliable biomarkers in cancer is more and more important for proper diagnosis and to predict prognosis of patients, especially in GC, where relapse is high. Since LIF supplementation and activation of LIF/LIFR signalling in the GC context seems to be beneficial in terms of CSC targetting, its capacity as a GC cancer biomarker was pondered. Consequently, the Oncomine database was queried for LIFR expression in human GC tissues compared with associated non-tumorous gastric mucosa. Both the Cui and Cho datasets show that LIFR is significantly under-expressed in GC tissues versus associated non-tumorous tissues (Figure 7A(i)). Further analysis of LIFR according to the different GC subtypes showed that it is under-expressed in all subtypes without significant difference between diffuse, intestinal and mixed GC histological subtypes (Figure 7A(ii)). The need for new reliable biomarkers in cancer is more and more important for proper diagnosis and to predict prognosis of patients, especially in GC, where relapse is high. Since LIF supplementation and activation of LIF/LIFR signalling in the GC context seems to be beneficial in terms of CSC targetting, its capacity as a GC cancer biomarker was pondered. Consequently, the Oncomine database was queried for LIFR expression in human GC tissues compared with associated non-tumorous gastric mucosa. Both the Cui and Cho datasets show that LIFR is significantly underexpressed in GC tissues versus associated non-tumorous tissues (Figure 7A(i)). Further analysis of LIFR according to the different GC subtypes showed that it is under-expressed in all subtypes without significant difference between diffuse, intestinal and mixed GC histological subtypes (Figure 7A(ii)). Interestingly, analysis of GC patients' overall survival probability using the KMplot database showed that in diffuse type GC, low LIF and LIFR expression are associated with low patient survival compared with high LIF and LIFR expression, which lead to a better prognosis of patients (Figure 7B). In intestinal type GC, this tendency remained the same for LIFR expression but is inversed for LIF since low LIF expression is correlated to high survival of patients and high expression to low survival (Figure 7B). This difference in prognosis of GC patients again shows how LIF is pleiotropic and can lead to different fates depending on the type of cancer. In addition, overall patients' survival analysis according to LIFR and LIF combined expression showed that high expression of both in diffuse type GC leads to better prognosis than low expression, and the same trend is observed for intestinal type GC, though not significant.

Furthermore, patients' median survival was analysed according to YAP1, TAZ and their target genes' expression (Table 1). High expression of these Hippo tumorigenic genes is linked to low survival of GC patients. Interestingly, when the same analysis was carried out in GC patients having low LIFR expression, YAP1, TAZ and their target genes' high expression leads to worse survival than low expression, and on the contrary, in high-LIFR-expressing patients, the less significant p-values show that this is not the case (Table 1). Finally, patients with higher expression of YAP1, TAZ and their target genes compared with LIFR expression have significantly lower survival rates than those with higher LIFR expression compared with Hippo oncogenic genes' expression (Table 2). This interestingly confirms that LIF/LIFR signalling is of better prognosis in GC. LIF/LIFR could thus represent a potential prognosis marker for diffuse type GC cases, a particularly aggressive type of GC.

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Table 2. Patients' median survival according to whether expression of YAP1, TAZ and their target genes is higher or lower than that of LIFR.

Gene

Patients' Median Survival (Months)

( Gene / LIFR ) > 1 ( Gene / LIFR )

Discussion

LIF/LIFR signalisation effect in cancer remains contradictory, with either negative or positive regulation of cancer cell properties depending on cancer types and studies [23][24][25][26]. In the GC context, LIF/LIFR signalling has been analysed only in few studies suggesting anti-tumorigenic effects [27,28], and none explored gastric CSCs.

This study focusses on the role of LIF/LIFR in CSCs, which are at the origin of GC initiation, progression and chemoresistance [4,5]. CSC targeting is of utmost importance in cancer therapy. Here, we show for the first time that LIF presents anti-CSC properties in both GC cell lines and PDX cells. LIF decreased one of the most important capacities of CSCs, which is the formation of small tumours in non-adherent conditions in vitro. It decreased the expression of the CD44 cell surface marker and ALDH activity, previously described as two of the best markers of gastric CSCs [5]. Moreover, consistent with previous studies allowing the analysis of different subpopulations of cells within GC cell lines and PDX cells [5], we hereby show that LIF acts more particularly on chemo-resistant CD44+ and ALDH+ Hoechst-CSCs by reducing their number and increasing the percentage of differentiated and non-tumorigenic CD44-and ALDH-Hoechst+ cells. Indeed, LIF has previously been described as an inducer of differentiation and inhibitor of proliferation of the myeloid leukemic cell line M1 [18] and cervical carcinoma cells [33,34]. In our AGS and MKN45 cells, LIF anti-CSC effects seems to be linked to an anti-proliferative effect (Supplementary Materials Figure S3A) coherent with Xu et al.'s recent study showing that LIF inhibits proliferation of GC cells, among which MKN45 cells, through a G1-phase, arrest induction [28]. The anti-proliferation and pro-differentiation effects of LIF could thus explain the decrease in the population of CD44 + Hoescht+ and ALDH + Hoechst+ cells corresponding to CSCs and/or proliferating progenitor cells, as previously described [5], in favour of an increase in differentiated non-proliferating and non-CSC Hoechst+ cells observed in the present study. It should however not be left aside that the LIF anti-CSC effect could also be due to a decrease in CSC viability in the presence of LIF. Indeed, the cell viability assay using uptiblue reagent showed a decrease in viability of cells forming AGS spheres after LIF treatment (Supplementary Materials Figure S3B).

Furthermore, we show that, in GC, LIF/LIFR signalling activates the Hippo kinase core MST1/2 and LATS1/2, thus inducing a phosphorylation cascade leading to the negative phosphorylation of oncogenic Hippo effectors YAP/TAZ and decreased oncogenic activity of the transcription factor TEAD, in accordance with what was observed in breast cancer [16,17].

Hippo effectors YAP/TAZ have been linked to CSC maintenance in several cancers including GC and, conversely, Hippo kinase activation is anti-tumorigenic [11,13,14,16]. Interestingly, Guo et al. have shown that LIF/LIFR pro-metastatic effects in melanoma pass through the activation of the canonical JAK/STAT pathway, which is known to be pro-oncogenic, and does not implicate the Hippo pathway. We hereby demonstrate that, in our model, LIF-induced anti-CSC effects involve Hippo pathway

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Cancers 2020, 12, 2011 16 of 24 kinases activation since the inhibition of MST1/2-mediated LATS1/2 Thr1079/1041 phosphorylation by XMU-MP-1 reverted LIF-induced anti-CSC effects. Hippo target genes' downregulation by LIF in our 3D culture condition models confirms that Hippo effectors were inhibited in LIF-treated tumourspheres, which was counteracted in the presence of XMU-MP-1. Interestingly, some Hippo pathway regulators like RASSF1A are known for their pro-differentiation capacities. RASSF1A is an MST1/2 partner and activator with important roles in the promotion of differentiation phenotypes in cells, through the YAP-p73 transcriptional programme [35][36][37], and could be involved in the LIF/Hippo effects observed in this study, making its study an interesting perspective. While the JAK1/STAT3 pathway was concomitantly activated in response to LIF, experiments performed with JAK1 inhibitor Ruxolitinib showed that the JAK1/STAT3 pathway was not involved in the LIF-mediated anti-tumorigenic effect in GC.

LIF activation of the Hippo and JAK1/STAT3 pathways in GC seems to lead to antagonistic effects since LIF had no effect on CSC properties when double Hippo-JAK1 inhibitions were carried out. When added to GC cells, LIF seems to shift the activation balance of the JAK1/STAT3 and Hippo pathways towards a higher activation of Hippo kinases and a lower activation of JAK1/STAT3 signalisation. Azmal Ali et al. and Nandy et al. suggested connections between these two pathways with STAT3 regulation being downstream LIF/LIFR/Hippo [29,30]. They demonstrated that JAK/STAT3 pharmacologicaland si-RNA-based inhibition did not change Hippo effectors expression levels, while inhibition of TAZ phosphorylation and thus higher nuclear translocation of TAZ led to induction of STAT3 expression and phosphorylation. This could be the same in our case with LIF/LIFR signalling activating Hippo kinases, thus inducing YAP/TAZ phosphorylation and reduced nuclear localisation, resulting in the possible under-regulation of both YAP/TAZ/TEAD and STAT3 oncogenic pathways. It should however be considered that LIF/LIFR solicits a large variety of signalisation pathways, which are more or less linked to the most studied JAK1/STAT3 pathway, and that signalisation cross-talks are very complex [22,38]. For instance, in HCC, LIF/LIFR anti-metastatic properties are due to the negative regulation of another LIF-regulated signalling pathway which is the PI3K/AKT pathway [24], again showing the complexity of LIF/LIFR signal transduction. LIF/LIFR/PI3K/AKT signalling has not been explored in the GC context.

LIF/LIFR signalling depends on the expression of LIFR GP190, which is an essential component of the heterodimeric LIF receptor GP130/GP190. Many studies have reported LIFR differential expression in the cancer context compared with non-tumoral conditions. Here, we show with in silico analyses that LIFR is under-expressed in GC tissues compared with adjacent non-tumorous tissues, suggesting a dysregulated LIF/LIFR signalisation in GC. Further, there was no difference in LIFR expression between the different histological subtypes according to the Lauren classification of GC, but when overall survival of patients was compared, diffuse type GC patients with low LIF and LIFR expressions had worser prognosis than those with higher LIF and LIFR expressions. Consistent with this, Zhao et al. described a low expression of long non-coding RNA-LOWEG in GC which normally upregulates LIFR [27]. This LIFR low expression could be a potential prognostic marker in GC, especially for the diffuse type cases where it reflects a low prognosis. Moreover, Zhao et al. also described LOWEG as having an anti-invasion capacity in GC and suggested a possible link between these anti-invasive effects and LOWEG-induced LIFR upregulation capacity [27]. Many other studies describing LIF/LIFR as anti-tumorigenic have also suggested an anti-metastatic role [16,24,31]. An interesting perspective could thus be to further study the role of LIF on the invasive properties of CSC in GC and most particularly on their metastatic properties.

This study presents LIF as a potential anti-CSC therapy in GC. Interestingly, we found that LIF was downregulated in CD44+ PDX cells corresponding to CSCs compared with non-CSC CD44cells. Interestingly, Xu et al. showed that the LIF protein expression level is downregulated in GC, and more importantly, that moderately and well-differentiated intestinal type GC had more LIF-positive cells compared with poorly differentiated diffuse-type GC. In addition, LIF/LIFR combined high expression in diffuse-type GC is correlated to better patients' prognosis compared with low

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Cancers 2020, 12,2011 expression. This comforts the hypothesis of LIF as a possible treatment in GC, to target chemo-resistant CSCs and palliate LIF low expression in diffuse GC cases, which are of poor prognosis. Interestingly, here we show that LIF is able to potentiate the effect of chemotherapeutical drugs on gastric CSCs of both GC cell lines and PDX cells, and that LIF-induced chemotherapy potentiating effects pass through Hippo kinase activation by LIF. CSCs are well-known for their capacity to resist conventional chemo/radiotherapies and form the small pool of residual cells responsible for post-treatment tumour relapse. LIF could be used to improve cancer therapies via its anti-CSC and chemo-potentiator properties, thus reinforcing the hypothesis of using it in GC treatment.

The use of LIF in vivo is however questionable due to its highly pleiotropic aspect. Several in vivo attempts can be found in the literature in which LIF injection or overexpression via implantation of LIF-producing cells in mouse models can promote anti-inflammatory signalling through IL-12 [39], suppress type 2 immunity in muscular dystrophic mouse model [40] and inhibit Th17 response in inflamed colon of inflammatory bowel disease models [41]. In addition, the commercial human LIF Emfilermin TM was used in a phase II clinical trial for the prevention of chemotherapy-induced peripheral neuropathy [42]. Despite the lack of positive response to LIF, patients showed no side effects, confirming the promising use of LIF for systemic treatments. Moreover, a recent study designed and synthetised LIF-loaded and vectorised nanoparticles for the targeting of activated peripheral macrophages, which were able to decrease murine leukemic cell M1 proliferation [43].

This strategy could be adapted to target gastric CSCs more specifically. Finally, the use of PDX cells and the anti-CSC effect of LIF in this model further contributes to the translational potential of this study.

Materials and Methods

Cancer Cell Lines and Patient-Derived Xenograft (PDX) Cell Culture

AGS and MKN45 diffuse GC cell lines were cultured in DMEM F12-Glutamax and RPMI 1640-Glutamax media, respectively, supplemented with 10% heat-inactivated foetal bovine serum (FBS) (all from ThermoFisher Scientific, Villebon sur Yvette, France) and 50 µg/mL vancomycin (Invitrogen, Cergy-Pontoise, France) at 37 • C in a 5% CO 2 humidified atmosphere. All cell lines, authenticated by STR profiling, were mycoplasma-free after PCR verification. AGS cells (ATCC CRL-1739 TM ) were derived from primary gastric carcinoma. Mutations found in this cell line concern CDH1 (c.1733_1734insC), CTNNB1 (c.101G > A), KRAS (c.35G > A) and PIK3CA (c.1357G > A). MKN45 is a poorly differentiated gastric adenocarcinoma cell line, derived from liver metastases. It is mutated for CDH1 (c.823_832 + 8del), BRCA1 (c.4792T > C), ERBB4 (c.82 + 8C > T) and TP53 (c.328C > T). Patient-derived xenograft (PDX) cells GC04, GC06, GC07 and GC10 were established through serial subcutaneous xenografts in NSG mice [5]. Animals were maintained at the University of Bordeaux Animal facility in accordance with institutional animal use and care committee guidelines (accreditation number B33-063-916, received on 23 May 2016). All animal experimentations were approved by the French Ethics Committee on Animal Experiments CEEA50 of Bordeaux (Authorisation n • A12005, ref 2017103118319700 v7). Molecular analysis was carried out, according to standard methods on PDX tumour tissue sections (in situ hybridisation) and on extracted DNA to determine Epstein-Barr virus (EBV) status. GC04, GC06, GC07 and GC10 were all EBV-negative. NSG colon and lung panels were used to search for mutations for KRAS, EGFR, BRAF, PI3KCA, AKT1, ERBB2, PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBX7, FGFR3, NOTCH1, ERBB4, FGFR1 and FGFR2. GC04 is mutated for PIK3CA (c.1633G > A p. (Glu545Lys)), GC06 for KRAS (c.175G > A p. (Ala59Thr) and c.38G > A p. (Gly13Asp)), GC07 for STK11 (c.1019A > G p. (Tyr340Cys)) and GC10 for TP53 (c.818G > A p. (Arg273His)).

When the tumour sizes reached 200-400 mm 3 , they were recovered and dissociated using a human tumour dissociation kit in C tubes and GentleMACS dissociator following manufacturer recommendations (All from MACS, Miltenyi, Paris, France). Cells were successively filtered using 70

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Cancers 2020, 12,2011 and 40 µm cell strainers and red blood cells were lysed. Cells could then be processed for tumoursphere formation assays and RNA experiments.

Tumoursphere Culture

AGS (500 cells per well), MKN45 (100 cells per well) and PDX (1000 cells per well) cells were seeded in 96-well culture plates, previously coated with a 10% poly-2-hydroxyethyl methacrylate (Sigma-Aldrich, Saint-Quentin Fallavier, France) solution in 95% (v/v) ethanol and left to dry overnight at 50-60 • C to make them non-adherent. Cells were incubated at 37 • C in a 5% CO 2 , humidified atmosphere in DMEM F12-Glutamax medium, supplemented with 0.3% glucose, 1:100 N2-supplement (all from ThermoFisher Scientific, Villebon sur Yvette, France), 20 ng/mL human epithelial growth factor, 20 ng/mL basic fibroblast growth factor and 5 µg/mL insulin (all from Sigma-Aldrich, Saint-Quentin Fallavier, France). MKN45 tumourspheres were grown in serum-free medium while AGS and PDX cells needed 2% FBS supplementation to grow.

Leukaemia Inhibitory Factor, Hippo Inhibitor and JAK/STAT Inhibitor Treatments

Recombinant human leukaemia inhibitory factor (LIF), produced in E. coli, was purchased from PeproTech (Neuilly-Sur-Seine, France), reconstituted in PBS and stored at -80 • C. It was used at 50 ng/mL in most experiments and 10 ng/mL in some tumoursphere assays. Adherent cells were treated after medium change for serum starvation. LIF was added either in a time-course manner (0.5, 2, 5, 24 and 48 h) or only at particular times depending on the experiments. Hippo inhibitor XMU-MP-1 (Selleckchem, Euromedex, Souffelweyersheim, France) was diluted in DMSO, stored at -80 • C and used at 0.5 µM. Ruxolitinib (Stemcell Technologies, Grenoble, France) JAK1/STAT3 inhibitor was also diluted in DMSO and stored at -20 • C before use at 1 µM. Treatments with inhibitors were carried out 30 min before LIF treatments.

Tumoursphere-Forming Assays and Chemotherapy Tests

AGS, GC06, GC10 and GC04 cells were seeded in non-adherent 3D conditions as described above and were immediately treated with 0, 10 and/or 50 ng/mL LIF or XMU-MP-1 and Ruxolitinib before LIF. Two hundred thousand MKN45 and GC07 cells were first plated in adherent conditions in 6-well plates and serum-deprived the day after before treating with LIF for 48 h. Cells were then trypsinised before plating in 3D conditions as described above. All cells' LIF and/or inhibitor treatments were repeated every 48 h before counting the number of spheroids per well at 7-8 days after the first treatment under an inverted light microscope using a ×20 objective.

For chemotherapy tests, chemotherapy drugs Doxorubicin 0.1 µM and 5-Fluorouracil 50 µM (both from Sigma-Aldrich, Saint-Quentin Fallavier, France), in combination or not with LIF, were added to AGS, MKN45 and GC07 cells upon seeding in non-adherent conditions and formed spheroids were recorded as mentioned above.

Flow Cytometry

AGS, MKN45 and GC07 cells were plated (100,000 cells) in 12-well plates, serum-deprived the day after and treated or not with 50 ng/mL LIF for 48 h. Cells were incubated with 1:25 mouse anti-human CD44-APC antibody (G44-26 clone, BD Biosciences, Le Pont de Claix, France) or isotype controls in ice-cold buffer containing PBS-0.5% bovine serum albumin (BSA, Gibco, ThermoFisher Scientific, Villebon sur Yvette, France)-2 mM EDTA (Sigma-Aldrich, Saint-Quentin Fallavier, France) for 25 min at 4 • C. ALDEFLUOR Kit (STEMCELL Technologies, Grenoble, France) was used in MKN45 cells prior to CD44 staining to detect ALDH activity, according to the manufacturer's instructions. Cells were rinsed thrice with ice-cold buffer and incubated for 10 min with buffer containing 50 µg/mL 7-Aminoactinomycin-D (7-AAD 559925, BD Biosciences, Le Pont de Claix, France). LIFR cytometry preparation was carried out as above with anti-human CD118 (LIFR)-PE antibody (12D3 clone, BD Biosciences, Le Pont de Claix, France). Cytometry analysis was performed using a BD FACSCanto II instrument and the DIVA software (BD Biosciences, Le Pont de Claix, France). Dead cells were excluded based on side scatter characteristics and 7-AAD positivity.

Luciferase Reporter Assay

AGS and MKN45 cell lines were seeded (50,000 cells) in 24-well plates and transfected with 100 ng/well TEAD (8XGTII-luciferase) or TATA box control (transcription-activator like TAL) firefly luciferase reporters (BD Biosciences, Le Pont de Claix, France) along with 10 ng/mL renilla luciferase reporter pRL-SV40 (Promega, Charbonnieres Les Bains, France). Serum starvation was then carried out before LIF and/or inhibitor treatment at different time intervals (0.5, 2, 5, 25 and/or 48 h). Firefly and renilla luciferases activities were measured using a Dual Luciferase assay kit (Promega, Charbonnieres Les Bains, France) and firefly luciferase activity values were normalised by the renilla luciferase activity values for each sample for transfection efficiency. TEAD-specific luciferase activity was then normalised by that of TAL.

Immunofluorescence Assay

AGS and MKN45 cell lines were seeded (50,000 cells) on glass coverslips in 24-well plates, serum-deprived 24 h later and treated with LIF at different time intervals (0.5, 2, 5, 24 and/or 48 h). Cells were then fixed with 3% paraformaldehyde solution in cytoskeletal buffer prior to immunofluorescence staining. After permeabilisation with 0.1% Triton X-100 solution for 1 min and blocking in 1%-BSA 2%-FBS in tris-buffered saline (TBS) solution, cells were stepwise incubated in corresponding primary antibodies for 1 h, washed 3 times in TBS and left for 30 min in secondary antibody coupled with 1:350 Alexa Fluor ® 488 goat anti-rabbit or anti-mouse IgG antibodies, 1:250 Alexa Fluor ® 647-coupled Phalloidin and 50 mg/mL 4 -6-diamino-phenyl-indol (DAPI) (all from ThermoFisher Scientific, Villebon sur Yvette, France). Primary antibodies used were 1:100 rabbit anti-YAP and 1:100 mouse anti-TAZ E5P2N (cat.4912 and cat.71192, respectively; Cell Signalling Technology, Saint-Cyr-L'École, France). Images were taken using an Eclipse 50i epi-fluorescence microscope (Nikon, Champigny sur Marne, France) with the NIS-BR acquisition software and a ×40 (numerical aperture, 1.3) oil immersion objective. Nuclear YAP/TAZ count and mean grey value measurements were carried out using the ImageJ 1.52p software (National Institutes of Health, Rockville Pike, Bethesda, MA, USA) [44].

Live Immunofluorescence Assay

MKN45 (5000) cells were plated in non-adherent 6-well plates and 5-day-old spheres were treated or not for 48 h with LIF before harvesting. ALDEFLUOR Kit (STEMCELL Technologies, Grenoble, France) was used to detect ALDH activity, according to the manufacturer's instructions, prior to spheres incubation with 1:25 anti-human CD44-PE antibody (515 clone, BD Biosciences, Le Pont de Claix, France) in ice-cold buffer containing PBS-0.5% bovine serum albumin (BSA, Gibco, ThermoFisher Scientific, Villebon sur Yvette, France)-2 mM EDTA (Sigma-Aldrich, Saint-Quentin Fallavier, France) for 25 min at 4 • C. Spheres were rinsed twice with ice-cold buffer and incubated for 30 min at RT with Hoescht-33342 dye (ThermoFisher Scientific, Villebon sur Yvette, France). Live immunofluorescence acquisition was carried out with an Eclipse 50i epi-fluorescence microscope (Nikon, Champigny sur Marne, France) using the NIS-BR acquisition software and a ×40 objective (numerical aperture, 1.3) immediately after buffer-washing and slide mounting. CSC subcellular populations were analysed using the ImageJ 1.52p software (National Institutes of Health) [44].

RNA Extraction and RTqPCR

For the 2D-RNA analysis, cells were plated (100,000 cells) in 12-well plates and serum-deprived the day after, and for 3D-RNA experiments, 2000 cells were seeded in non-adherent conditions for 4-5 days prior to treatments. All cells were treated or not with 50 ng/mL LIF either at different time intervals (0.5, 2, 5, 24 and 48 h) or at particular times. Total RNA was extracted using TRIzol TM reagent

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Cancers 2020, 12, 2011 20 of 24 (ThermoFisher Scientific, Villebon sur Yvette, France) according to manufacturer's recommendations. Reverse transcription was then carried out with the Quantitech Reverse Transcription kit (QIagen, Germantown, Netherlands) following the manufacturer's protocol. Real-time PCR was performed using SYBR-qPCR-Premix Ex-Taq (Takara, Shiga, Japan) and 0.5 µM specific primers (Supplementary Materials Table S1). Analysis of amplified RNA samples was carried out using the 2 -∆∆CT method with TBP and HPRT1 as normalisers.

Agilent Microarray

PDX cells (n = 4, in simplicates and n = 2, in triplicates) were FACS-sorted according to CD44 expression. DNA was removed and RNAs extraction was carried out using an RNeasy microkit (all from Qiagen, Germantown, the Netherlands). RNA quantification was carried out using the TapeStation system (Agilent) and RNA integrity numbers (RIN) were determined. Gene expression profiling was performed at the GeT-TRiX facility (GenoToul, Génopole Toulouse Midi-Pyrénées, Toulouse, France) with Agilent Sureprint G3 Human microarrays (8 × 60K, design 072363), following the manufacturer's instructions. One-Color Quick Amp Labelling kit (Agilent) and Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, MA, USA) were used to prepare cyanine-3 (Cy3)-labelled cRNA from 25 ng of total RNA of each sample, according to the manufacturer's conditions. Dye incorporation and cRNA yield were then verified using a Dropsense™ 96 UV/VIS droplet reader (Trinean, Ghent, Belgium). Cy3-labelled cRNA (600 ng) were hybridised on microarray slides following the manufacturer's instructions. Slides were washed and scanned immediately using an Agilent G2505C Microarray Scanner and the Agilent Scan Control A.8.5.1 software. The Agilent Feature Extraction software v10.10.1.1 was used with default parameters for fluorescence signal extraction and only the most expressed probes values were represented. T-test statistical analysis was carried out to compare differentially expressed genes.

Protein Extraction and Western Blotting

Cells were plated (200,000 cells) in 6-well plates, serum-deprived the day after and treated or not with 50 ng/mL LIF either at different time intervals (0.5, 2, 5, 24 and 48 h) or at specific times. Cells were then washed twice with ice-cold PBS and lysed with ProteoJet reagent (ThermoFisher Scientific, Villebon sur Yvette, France) supplemented with a cocktail of protease inhibitor P8340, and phosphatase inhibitors P0044 and P5726 (all from Sigma-Aldrich, Saint-Quentin Fallavier, France). Total proteins were submitted to 7.5% or 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. Membranes were blocked in 5% BSA-0.1%PBS-Tween 20 and incubated with respective primary antibodies for 1 h at room temperature (RT). Primary antibodies used at 1:2000 were: rabbit anti-YAP, anti-p-YAP Ser127 , mouse anti-TAZ E5P2N, anti-LATS2 D83D6, anti-p-LATS1/2 Thr1079/1041 D57D3, mouse anti-STAT3 124H6, p-STAT3 Tyr705 D3A7 (cat.4912, cat.4911, cat.71192, cat.5888, cat.8654, cat.9139 and cat.9145, respectively; Cell Signaling Technology, Saint-Cyr-L'École, France), mouse anti-α-tubulin (cat.T-6074; Sigma-Aldrich, Saint-Quentin Fallavier, France) and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (cat.sc-47724; Santa Cruz Biotechnology, Heidelberg, Germany). Membranes were then probed with starbright Blue700 goat anti-mouse and anti-rabbit fluorescence conjugated secondary antibodies (Bio-Rad, Marnes-la-Coquette, France) and diluted at 1:2500 for 1 h at RT. Antibody-bound proteins were detected using ChemiDoc™ Imaging Systems (Bio-Rad, Marnes-la-Coquette, France). Immunoblotting band intensity was measured using the ImageJ 1.52p software (National Institutes of Health, USA) [44].

In Silico Database Analysis and Statistics

LIFR mRNA expression patterns from GC were extracted from the Oncomine database tool (www.oncomine.org) [45]. Results were filtered for cancer versus normal tissue analysis and/or Lauren classification and gastric cancer cases in the primary filter. Cui (n = 90) and Cho (n = 160) Gastric tissue datasets were chosen and extracted for analysis.

The KMplot database tool (www.kmplot.com) [46] was used to analyse overall survival probability of non-metastatic (parameter M = 0) GC patients (n = 444) according to LIFR, LIF and combined LIFR and LIF expression. The following JetSet best probes, 225575_at (LIFR) and 205266_at (LIF), were used. Analysis was filtered for Lauren classification, to analyse intestinal and diffuse types of GC separately, and the GSE62254 dataset was excluded as suggested by the database to avoid biased results. Patients' samples were separated according to high or low expressions by the software's best cut-off value auto-setting. P-values were calculated by a log-rank test.

Results are expressed as mean ± S.E.M of at least three independent experiments. Statistical tests were carried out using the GraphPad Prism software version 8.0.2 (La Jolla, CA, USA). Mann-Whitney test or Student t-test were used for the two-groups comparisons and ANOVA with Bonferroni as a post hoc test or Kruskal-Wallis test with Dunn's test as post hoc were performed for multiple comparisons.

Conclusions

This study deciphers for the first time the role of LIF in the GC context and more specifically on the signalling pathways controlling gastric CSCs' tumorigenic properties. We show that LIF is anti-tumorigenic in GC cell lines and most importantly in patient-derived GC cells, opening further translational perspectives. Signalling pathways and molecular mechanisms involved were addressed, and the Hippo pathway was found to be at the basis of LIF-induced anti-CSC effects in GC. In this regard, this work reveals LIF as an interesting anti-CSC therapeutic potential in combination with conventional chemotherapy, which is of utmost important in this bad prognosis disease. LIF/LIFR signalling hereby represents a possible prognosis marker and therapeutic option in the management of GC.

Supplementary Materials:

The following are available online at http://www.mdpi.com/2072-6694/12/8/2011/s1, Figure S1: Effect of LIF treatment on JAKSTAT pathway and CSC markers expression in MKN45 cells, Figure S2

Supplementary Materials: Leukaemia Inhibitory Factor (LIF) inhibits Cancer Stem Cells tumorigenic properties through Hippo kinases activation in Gastric Cancer

Lornella Seeneevassen, Julie Giraud, Silvia Molina-Castro, Elodie Sifré, Camille Tiffon, Clémentine Beauvoit, Cathy Staedel, Francis Mégraud, Philippe Lehours, Océane Martin, Hélène Boeuf, Pierre Dubus and Christine Varon

Proliferation Assay

AGS and MKN45 cells were plated (50,000 cells) in 24-well plates and treated or not, the dayafter,with 50 ng/mL LIF for 48 h. Cells were then trypsinized and viable cells were counted using 0.2 mm depth Malassez counting chambers (PRECISS) in the presence of Trypan blue stain 0,4% (Gibco, ThermoFisher Scientific, Villebon sur Yvette, France).

Uptiblue Cell Viability Assay

Non-adherent 96-well cultured AGS tumourspheres were processed for cell viability analysis at day 10 after seeding. UptiBlue™ viable cell counting reagent (Interchim, Montluçon, France) (10 µL) was added to each well 2 h before analysis at λ exc//em : 570 / 590 nm which was carried out using a CLARIOstar® Plus fluorimeter (BMG Labtech, Ortenberg).

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⁓ Chapter 2 ⁓

Leukaemia Inhibitory Factor inhibits gastric Cancer Stem Cells invasive and metastatic properties

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The manuscript presented in this chapter is in preparation for submission in Gut Journal.

Introduction

Leukaemia Inhibitory Factor (LIF) is a pleiotropic member of the Interleukin-6 family of cytokines. Since its discovery, LIF was shown to be involved in a panel of effects which can be contradictory depending on the model 1 . It is pro-differentiating and anti-proliferating in murine myeloid M1 cells 2 , involved in pluripotency maintenance of murine embryonic stem cells in vitro where it contributes to maintain the epithelial-like phenotype of these cells 3 , while pro-proliferating in murine interleukin-3-dependent leukemic cell line DA-la 4 . It also has a role in embryo implantation 5 , during development for neurogenesis and in adult tissue regeneration for example brain, spinal cord 6 and muscle tissue 7 .

In cancer, LIF also presents paradoxical effects depending on the cancer type and on the signalling pathway involved 1 . LIF has been shown to promote tumorigenesis in pancreatic cancer 8 , migration in melanoma 9 , resistance in oesophageal adenocarcinoma 10 and aggressiveness in chordoma 11 . On the contrary, it has been described as anti-metastatic in hepatocellular carcinoma 12 , clear cell renal carcinoma 13 and breast cancer 14 .

LIF-LIF receptor (LIFR) canonical pathway is mediated by JAK/STAT activation, causing STAT3 phosphorylation, dimerization, nuclear translocation, and transcription of target genes known for their role in cell survival and tumorigenesis 1 . On the other hand, other cell signalisation processes have been identified downstream LIF-LIFR, which could explain its multifunctional aspect. Indeed, the Hippo signalling pathway has been identified as being activated following LIF-LIFR signalling in breast cancer leading to anti-metastatic properties in this context 14 .

The Hippo pathway is well-known for its key role in organ size during development. It is made up of two core elements, a Hippo kinase core comprising of Mammalian-Sterile20 like kinases MST1/2 which phosphorylate Large tumour suppressor kinases (LATS1/2) leading to phosphorylation and inhibition of the core effectors Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-binding motif (TAZ), both having a role in tumorigenesis 15 . YAP and TAZ are also well-described for their role in invasion and metastasis and, Hippo pathway members were found to be enriched in GC and especially in gastric cancer stem cells (CSCs) 15-17 .

GC is a deadly disease accounting for approximately 768 763 deaths worldwide in 2020, making it the 4 th leading cause of cancer-related death 18 . We have recently demonstrated that LIF decreases tumorigenic properties of gastric CSCs and this, by activating the Hippo kinase core and not the JAK/STAT pathway 19 . In addition, LIF was also found to induce G1-phase cell cycle arrest in GC cell lines 20 . Nevertheless, LIF's role in GC metastasis process is not thoroughly described.

Methods

Gastric cancer cell lines and Patient-Derived Xenograft (PDX) cell culture

GC cell lines were authenticated by STR profiling and were mycoplasma-free after PCR verification. MKN45 and AGS ATCC cells lines were cultured in RPMI 1640-Glutamax and DMEM F12-Glutamax media respectively, supplemented with 10% heat-inactivated foetal ~133~ bovine serum (FBS) (all from ThermoFisher Scientific, Villebon sur Yvette, France) and 50 µg/ml vancomycin (Invitrogen, Cergy-Pontoise, France) at 37°C in a 5% CO2 humidified atmosphere. Patient-Derived Xenograft (PDX) cells GC07 cells 17,21 were cultured in DMEM F12-Glutamax medium, supplemented with 10% FBS and 50 µg/ml vancomycin.

Production of luciferase lentiviral particles and cell transduction

The lentiviral vector used for the luciferase gene transfer in cells was pRRLsin-MND-Luc-IRES2-ZsGreen-WPRE, produced by the Bordeaux University vectorology platform, as previously described (PDOX). MKN45 GC cell line was transduced with a multiplicity of infection (MOI) of 30 lentiviral particles per cell for 24 h in RPMI 1640-Glutamax medium, supplemented with 10 mM Hepes (ThermoFisher Scientific, Villebon sur Yvette, France).

Medium was changed 12 h later and cells were amplified to be used for in vivo experiments. Transduced cells will be named pMND-Luc MKN45 cells hereafter. Transfection efficiency was verified by flow cytometry with about 100% cells ZsGreen-positive transduced cells.

Leukemia inhibitory Factor, Hippo and JAK/STAT inhibitor treatments

Recombinant Human Leukaemia Inhibitory Factor (LIF, PeproTech, Neuilly-Sur-Seine, France) reconstituted in PBS, was used at 50 ng/mL 19 . Adherent cells were submitted to serum starvation by medium change 2 h before treatments. LIF treatments were carried out for 48 h in most experiments except for cells prepared for in vivo assays which underwent 72 h treatment with a repeat 48 h after the first stimulation and cells for 3D-invasion assays in which LIF was added each 48 h. MST1/2 inhibitor XMU-MP-1 (Selleckchem, Euromedex, Souffelweyersheim, France) was diluted in DMSO and used at 0.5 µM. Ruxolitinib (Stemcell Technologies, Grenoble, France) JAK1 inhibitor was also diluted in DMSO before use at 1 µM. Inhibitors were added 30 min before LIF to ensure respective signalisation pathways blocking prior to LIF stimulation.

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Cells preparation for in vivo experiments

MKN45 pMND-Luc cells were seeded in T-25 cell culture flasks and serum starvation was carried out 24 h later. 50 ng/mL LIF treatment or not was carried out for 72 h, comprising a stimulation repeat at 48 h post treatment. Cells were then recovered with trypsin, counted and 100, 1,000 or 10,000 non-treated or LIF-treated cells were resuspended in PBS for intracardiac injection in mice.

RNA extraction and RTqPCR

100,000 MKN45 and AGS GC cell lines were plated in 12-well plates, serum-deprived the day-after and then treated or not with 50 ng/mL LIF for 48 h. Total RNA was extracted using TRIzol TM reagent (ThermoFisher Scientific, Villebon sur Yvette, France) according to manufacturer's recommendations. Reverse transcription was then carried out with the Quantitech Reverse Transcription kit (QIagen, Netherlands) following manufacturer's protocol. Real-time PCR was performed using SYBR-qPCR-Premix Ex-Taq (Takara, Shiga, Japan) and 0.5 µM specific primers (Suppl. Table S1), as previously described 17,19 . Analysis of amplified RNA samples was carried out using the 2 -∆∆CT method with TBP and HPRT1 as normalisers.

Agilent microarray

AGS, MKN45 and GC07 cells (n=1 for each cell line, n=3 GC cell lines) were treated or not with LIF for 48 h for Agilent microarray transcriptomic canalysis as previously described 15,17,19 . RNAs extraction was carried out using RNeasy microkit (all from Qiagen, Netherlands) and RNA quantification was carried out using the TapeStation system (Agilent Technologies, Santa Clara, United States) to determine RNA integrity numbers (RIN).

Agilent Sureprint G3 Human microarrays (8x60K, design 072363) were used for gene expression profiling at GeT-TRiX facility (GenoToul, Génopole Toulouse Midi-Pyrénées,

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France), following manufacturer's instructions. One-Color Quick Amp Labelling kit (Agilent) and Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, MA, USA) were used to prepare cyanine-3 (Cy3) labelled cRNA from 25 ng of total RNA of each sample, according to manufacturer's conditions. Dye incorporation and cRNA yield verification was done using Dropsense™ 96 UV/VIS droplet reader (Trinean, Ghent, Belgium). Cy3-labelled cRNA (600 ng) were hybridized on microarray slides following manufacturer's instructions.

2D and 3D Immunofluorescence assays

For 2D experiments, 50, 000 AGS and MKN45 cell lines and GC07 PDX cells were seeded on rat-tail type-I collagen-coated (Corning, New York, USA) glass coverslips in 24-well plates, serum-deprived 24 h later and treated with LIF for 48 h. Cells were then fixed with 4% paraformaldehyde solution (PFA, Electron Microscopy Sciences, Hatfield, UK) in cytoskeletal buffer prior to immunofluorescence staining, as previously described 15,16,19 . 3D immunofluorescence was carried out on tissue sections of paraffin-embedded collagen spheres. The same protocol as 2D immunofluorescence was followed apart from an antigen demasking step carried out in 96°C-hot pH6 citrate buffer for 1 h. Primary antibodies used were rabbit anti-ZEB1 (Bethyl laboratories, Montgomery, USA), rabbit anti-SNAI1 (H-130) (Santa cruz biotechnology, Bergheimer, Germany), mouse anti-TAZ (BD, Grenoble, France); all at 1:100 for 1h. This was followed by 3 times TBS washing and 30 min incubation with respective fluorophore-labelled secondary antibodies (Alexa Fluor® 488 goat anti-rabbit (1:250), Alexa Fluor® 594 donkey anti-mouse (1:200)), combined with Alexa Fluor® 647-

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Phalloidin (1:400) and 4'-6-diamino-phenyl-indol (DAPI, 50 mg/mL) (all from ThermoFisher Scientific, Villebon sur Yvette, France). Images were taken using an Eclipse 50i epifluorescence microscope (Nikon, Champigny sur Marne, France) with NIS-BR acquisition software and a x40 (numerical aperture, 1.3) oil immersion objective. ZEB1 and SNAIL mean nuclear intensity (mean Gray-value) measurement was carried out using ImageJ 1.53f51 software (National Institutes of Health, USA) 15,16,19,22 .

2-D collagen invasion assay

50, 000 AGS and MKN45 cell lines and GC07 PDX cells were seeded in 24-wells plates, serum deprived after 24 h and treated with LIF for 48 h. Cells were then trypsin-recovered and 30, 000 cells were seeded in 8-µm pored Transwell® inserts, previously coated with rattail type 1 collagen solution (all from Corning, New York, USA). Inserts were placed in 24well culture plates containing medium supplemented with 5% FBS and containing 50 ng/mL LIF or not. After 18 h incubation at 37°C, the Transwell® inserts were fixed with 4% PFA in cytoskeletal buffer. Cells from the upper part of the insert were discarded using cotton swab and the insert membrane was processed for DAPI staining (ThermoFisher Scientific, Villebon sur Yvette, France) 10 min before 3 PBS washings 15,16,23 . Invaded cells were counted on 5 different randomly chosen fields per insert, under ZOE TM Fluorescent Cell Imager (Bio-Rad, Marnes-la-Coquette, France).

3-D collagen invasion assay and paraffin inclusion

AGS (500), MKN45 (100) and GC07 (1,000) cells were seeded in 96-well culture plates, and 10 000 MKN45 cells in 6-well plates, all previously coated with a 10% poly-2-hydroxyethyl methacrylate (Sigma-Aldrich, Saint-Quentin Fallavier, France) solution in 95% (v/v) ethanol and left to dry overnight at 50-60 °C to make them non-adherent. Cells were cultured in nonadherent CSC-selective conditions as described for tumoursphere assay 19 in DMEM F12-

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Glutamax medium, supplemented with 0.3% glucose, 1:100 N2-supplement (all from ThermoFisher Scientific, Villebon sur Yvette, France), 20 ng/mL human epithelial growth factor, 20 ng/mL basic fibroblast growth factor and 5 µg/mL insulin (all from Sigma-Aldrich, Saint-Quentin Fallavier, France) at 37 °C in a 5% CO2, humidified atmosphere. MKN45 tumourspheres were grown in serum-free medium while AGS and GC07 cells needed 2% FBS supplementation to grow. After CSC selection and formation of 3-days old spheres, LIF and inhibitor treatments were carried out and 100 µL of rat-tail type 1 collagen solution, at a final concentration of 1 mg/mL, was added the day-after to each 96-plate well. Collagen was allowed to polymerize for 24 h and photos of the spheres were taken at different times (Day 1 and 5) with a camera-equipped inverted-light microscope using x20 objective (Olympus, Rungis, France). Treatments were repeated each 48 h. The invasive area was measured using ImageJ 1.53f51 software (National Institutes of Health) 22 and calculated according to the following formula: (Day 5 sphere area -Day 1 sphere area).

For sphere-paraffin inclusion, 3-days MKN45 spheres, formed in 6-well plates, were treated with LIF. The spheres were collected the day-after, low-speed centrifugated (500 rpm for 5 min) and mixed with a collagen solution containing 1 mg/mL rat-tail type 1 collagen solution, 8% NAHCO3 (Sigma-Aldrich, Saint-Quentin Fallavier, France) and sphere-specific medium before replating in 6-well plates previously coated with 1 mg/mL the same collagen solution as before. 3 wells per condition were prepared and 5 days after collagen embedding, collagen gels were recovered, each condition was pooled, rinsed with PBS before adding 2-3 drops of undiluted Indian Ink (BIC, Clichy, France) for 30 s. Collagen gels were rinsed to remove excess ink and fixed in a 30 min-4% PFA bath before transferring in 70% ethanol. Blocks were processed with classical tissue paraffin-embedding protocol 17,21,24,25 before further histological applications.

Xenograft experiments and metastasis follow up by Bioluminescence imaging (BLI)

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Animal manipulations were performed in accordance with European directives for the care and use of animals and have been approved by the local Ethics Committee CEEA50 of Bordeaux (agreement # 31293). Immunodeficient NOD/SCID/IL-2Rnull (NSG) mice (7 to 14-weeks old) were reared at the university facilities and maintained under 12-hour dark/light cycles with water and food provided ad libitum as previously reported 17,24,25 . Animal manipulations were performed on anesthetized mice using 2% isoflurane (Belmont, Nicholas Piramal Limited, London, UK). For intracardiac injection and imaging, mice were shaved with clippers. Metastasis generation was performed by intracardiac cell injection (100, 1,000 or 10,000 cells in 100 µL) in the left ventricle with ultrasound guidance (Aixplorer, Supersonic Imagine, France) 26 .

Metastasis tracking was performed by in vivo bioluminescence imaging. BLI was performed using the Lumina LT imaging system (Perkin Elmer Inc., Boston, MA, USA) at the Vivoptic platform (Univ. Bordeaux, CNRS, INSERM, TBM-Core, UMS 3427, US 5). D-luciferin (2.9 mg; 100 μL PBS, Promega) was injected intra-peritoneally. Bioluminescence acquisition (1 min, 4 x 4 binning) and photographs (100 ms) were taken 10 min after substrate injection.

Mice were monitored up to 28 days for those injected with the least number of cells. At end point, mice were sacrificed, and organs were recovered and analysed by ex vivo bioluminescence to determine the exact organs concerned by GC cells implantation. Data were analyzed with Living Image software (Perkin Elmer) and Photon quantification was performed by drawing a region of interest (ROI) 26 . Organs were sampled and processed for histological analyses following standard procedures.

Human Tissue samples collection and ethical statements

Paraffin-embedded samples, from 177 GC patients from 1999 to 2010, were obtained and processed following agreement of the Direction of Clinical Research and the Tumour and Cell

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Bank of the Bordeaux University Hospital Centre (Haut-Leveque Hospital, Pessac, France) as previously reported 16,23,24 . The Tissue microarray (TMA) blocks were produced using the MiniCore® 3 Tissue Arrayer (Excilone, Elancourt France). Areas of interest ware selected and extracted as 1mm-diameter cylindrical tissue core triplicates and re-embedded into recipient blocks at defined array coordinates. Spleen (no expression of epithelial markers) and duodenum (expression of epithelial and stem cells markers at the bottom of crypts) were used as controls (n=3 samples per bloc). The TMAs included GC of different grades and histological sub-types determined after reanalysis and classification (G. Belleannée, pathologist, Bordeaux University Hospital) according to the last WHO and Lauren's classification guidelines of gastric tumours, as well as non-tumour gastric mucosa distant from tumour sites (limits of exeresis), which may be healthy or involving pre-neoplastic lesions such intestinal metaplasia and dysplasia. Clinico-biological data including 5-years overall survival were also collected.

Immunohistochemistry

Immunostaining was carried out on 3 µm-thick tissue sections obtained from formalin-fixed paraffin-embedded human tissues or sphere inclusions. Antigen demasking step was carried out in 96°C-hot pH6 citrate buffer for 1 h. Primary antibody rabbit anti-LIFR (Abcam, Cambridge, UK) or anti-CD44 (BD Biosciences) 1:200 was used for 2 h and secondary horseradish peroxidase (HRP)-labelled HRP Horse Anti-Rabbit IgG Polymer Detection Kit ImmPRESS® (Vector Laboratories, Burlingame, United States). Immunostaining was revealed after incubation in liquid diaminobenzidine (DAB) chromogen substrate (DAKO, Agilent Technologies, Santa Clara, United States). Counterstaining was performed with Mayer′s hemalum solution (Sigma Aldrich Chimie, Saint-Quentin-Fallavier, France) before tissue dehydrating and slide mounting with Eukitt mounting medium (VWR, Fontenay-sous-Bois, France). Double-blinded scoring of percentage of positive cells was carried out ~140~ according to the following criteria and score ranges: 0: no expression, 1: 1-20%, 2: 20-50%, 3: > 50%. Subcellular localisations were noted and classified as follows: Membrane, Cytoplasmic, Nuclear (nuclear and peri-nuclear). Low expression of LIFR was defined as score ≤ 1 and high expression score > 1.

Laser microdissection and proteomics analysis by LC-MS/MS mass spectrometry

Microscopy-directed 1 mm 2 laser microdissection was carried out using a PALM type 4 automated micro-dissector (Zeiss Microscopy, France) on 5 µm-thick tissue sections previously stained haematoxylin and eosin. Areas of interest were selected beforehand from the invaded regions of MKN45 paraffin-embedded spheres treated or not with LIF (n=3).

Sample preparation as well as LC-MS/MS Fusion Lumos mass spectrometry (ThermoFisher Scientific, Villebon sur Yvette, France) was carried out as previously described 27 , followed by proteins identification using the Proteome Discoverer 2.5 software and Mascot algorithm.

In silico database analysis

KMplot database tool (www.kmplot.com) 28 was used to analyse overall survival probability of GC patients (69 < n < 249) according to ZEB1 and LIFR expression. The following JetSet best probes, 239952_at (ZEB1) and 225575_at (LIFR) were used. GSE62254 dataset was excluded during analysis as suggested by the database to avoid biased results. Patients' samples were separated according to high or low expressions by the software's best cut-off value auto-setting. P-values were calculated by a log rank test. STRING v11.5 database 29 was used to visualise possible interactions between proteins from proteomics analysis and to classify them according to their function.

Statistics

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Results are represented as mean ± S.E.M of at least three independent experiments. Statistical tests were carried out using GraphPad Prism software version 8.0.2 (La Jolla California USA). ANOVA with Bonferroni as post hoc test or Kruskal-Wallis test with Dunn's test as post hoc were performed for multiple comparisons and Mann-Whitney test or Student t-test were used for two groups comparisons. Progression-Free Survival was compared using Wilcoxon paired t-test.

Results

LIF decreased EMT and invasion-related genes expression in GC cells

Transcriptomic analysis of LIF-treated GC cells MKN45, AGS and GC07 PDX compared to non-treated ones showed that LIF decreased gastric CSC markers expression (Figure 1A), as we described previously 19 . A decrease in Hippo oncogenic signature is also noted as shown precedingly 19 , through decrease in TAZ and TEADs expression and their main target genes as well as increase in Hippo partners which are known to work with Hippo kinases to inhibit YAP/TAZ-TEAD oncogenic activity (Supplementary Figure S2A) 30 . Moreover, LIF decreased mesenchymal markers expression and increased epithelial ones (Figure 1B-C), showing a possible role on EMT process.

To confirm LIF's effect on EMT, epithelial and mesenchymal markers expression was assessed by RTqPCR. Results showed an increase in epithelial markers and a decrease in mesenchymal markers, including ZEB1 and SNAI1, EMT-TFs (Figure 1D-E), reflecting a decrease in EMT process in GC cells. In addition, several matrix metalloproteases (MMPs) linked to the capacity of cells to invade extracellular matrix (ECM) also decreased showing a possible inhibitory effect of LIF on GC cells invasion ability.

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LIF modulates EMT-TFs nuclear expression

Since EMT main transcription factors', ZEB1 and SNAIL, activity is related to their subcellular localisation, their expression was evaluated in the nucleus where they control the expression of EMT-related genes (Figure 2). LIF significantly decreased ZEB1 and SNAIL nuclear expression (Figure 2A-C), which is correlated with the reduced expression of EMTassociated genes observed in LIF-treated GC cells (Figure 1B-E). This was demonstrated in GC cell lines MKN45 and AGS, and also in PDX-derived cells GC07.

Moreover, to evaluate the implication of the Hippo and JAK/STAT signalling pathways in LIF-observed effects, cells were treated with XMU-MP-1 (XMU) or Ruxolitinib inhibiting Hippo kinase MST1/2, upstream LATS1/2 and JAK kinases, respectively. In the presence of XMU, LIF was no longer able to decrease ZEB1 and SNAIL nuclear expression in MKN45 and GC07 cells (Figure 2A-C

LIF presents anti-EMT and anti-invasive properties in GC cells

LIF effect on the EMT phenotype was verified by counting the number of GC cells carrying an elongated protrusion-rich phenotype 16,23 after LIF treatment (Figure 3A). Indeed, LIF decreased EMT phenotype in GC cell lines, effect which was blocked in presence of both inhibitors showing both Hippo and JAK/STAT pathways implication. Furthermore, since LIF was found to inhibit expression of certain invasion markers, functional 2D-invasion boyden chamber's assay was performed to validate this hypothesis (Figure 3B). The number of cells invading type I collagen-coated microporous culture inserts decreased after LIF treatment confirming the anti-invasive effect of LIF in all GC cell lines and PDX cells analysed. The Hippo pathway and not the JAK/STAT pathway was involved in LIF-dependent anti-invasive effects in AGS and MKN45 cell lines while effect in GC07 PDX cells seemed to depend on both signalisation pathways.

LIF consequently presents anti-invasive properties through a decrease in EMT and invasion process in GC cells.

LIF presents anti-invasive properties in gastric CSCs

The most aggressive component of heterogeneous gastric tumours is the rare population of CSCs which carry specific self-renewal and chemo-/radio-resistant properties 19,23-25 , allowing them to initiate tumours and cause recurrence. In the metastatic process, part of these cells is ~147~ able to acquire invasive characteristics, evade the primary tumour, disseminate, and colonise distant organs to initiate metastasis 31 .

To verify whether LIF could also affect the invasive capacity of gastric CSCs, these were selected according to previously established protocols in non-adherent conditions allowing the survival of only CSCs 19,23,24 . Type-I collagen was then added to embed the formed tumourspheres in a 3D collagen gel and collagen invasion by CSCs was measured after 1 and 5 days. Collagen-invasion area decreased in presence of LIF in MKN45 cell line and GC07 PDX cells (Figure 4) as well as in AGS cells (Supplementary Figure S1). This anti CSC-invasive effect is dependent on the Hippo and JAK/STAT pathway activation by LIF signalling in all the cells analysed (Figure 4A-C and Supplementary Figure S1A-B).

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LIF is thus able to decrease invasive properties of GC cells and more particularly that of gastric CSCs.

LIF inhibits EMT markers expression in invasive CSCs

LIF-induced decrease in CSC invasive properties was related to a decrease in ZEB1 EMT-TFs nuclear expression (Figure 5A-B). Furthermore, Hippo pathway oncogenic effector TAZ nuclear expression also decreased after LIF treatment of gastric CSCs, especially at the invasive periphery of the tumourspheres (Figure 5A). Hippo effectors YAP and TAZ were found to be associated with ZEB1 in various cancers 32 and to GC invasion properties in GC 16 .

LIF inhibitory effect on GC cells invasion could be related to both ZEB1 and TAZ downregulation observed here (Figure 5A-B). LIF also decreased CD44 expression in gastric tumourspheres (Figure 5C). Furthermore, these 3D-invasion models of gastric CSCs were analysed by laser microdissection coupled with LC-MS/MS mass spectrometry proteomics analysis 27 to explore the proteome composition of invasive edges of spheres treated or not with LIF. This showed that edges of LIF-treated spheres had a differential expression of EMT-related proteins with an increase in epithelial ones and a decrease in mesenchymal ones (Figure 5D), reflecting an inhibition of EMT. Moreover, among the pool of differentially expressed proteins were some enriched in translation of migration proteins, vesicle trafficking and actin cytoskeleton remodelling for migration and invasion which were found decreased after LIF treatment (Figure 5E) and could be related to the anti-invasive effect and anti-EMT phenotype observed. The expression of genes encoding this group of proteins was also found decreased for most in the transcriptomic analysis of LIF-treated cells (Supplementary Figure S2B).

LIF limits metastasis appearance in vivo

LIF anti-invasive effect was finally evaluated in vivo in mice models of intracardiac injection 26 , reproducing cell dissemination and implantation process in secondary organs (Figure 6A). Experiments were performed in limiting dilution with xenograft of either 10, ~151~ 000, 1000 or 100 cells. When MKN45 cells were treated with LIF, they disseminated and colonised organs at a lesser extent than non-treated cells (Figure 6B). Mice metastatic follow-up showed that metastases were detected at day 5 for both conditions when 10, 000 cells were injected, while a 4-days delay was noted for LIF-treated cells when 100-1000 cells were used. In addition, at endpoint, about 18.2% and 63.7% less mice developed metastases when 10, 000 and 100-1000 LIF-treated cells were injected, respectively (Figure 6B/E). Bioluminescence quantification of metastases revealed that overall metastasis formation was 96% and 97% lower in LIF conditions showing that LIF decreased not only the number of mice having metastases, but also the metastatic signal in these positive mice (Figure 6C/F). 1) and when those were present, their signal was lower

(Figure 6D/G).
Accordingly, LIF decreases the metastatic potency of GC cells in vivo.

LIFR represents a novel prognostic marker for GC

It has previously been demonstrated, through bioinformatical analysis 19 , that LIF is less expressed in GC and this is associated to poor patient prognosis. Here, this was confirmed using TMA from a local collection of GC patients. LIFR expression was found to be decreased in GC cases but not in preneoplastic intestinal metaplasia state, compared to nontumorous tissues (Figure 7A-C). Interestingly, LIFR expected membrane localisation was affected in GC (Figure 7A) and both in diffuse and intestinal type GC (Figure 7B), while a nuclear expression was detected, and this seemed acquired in diffuse type GC and lost in intestinal ones (Figure 7B). LIFR is thus differently expressed in different GC subtypes, classified according to the Laurèn classification, though low overall expression in observed for both and this is related to GC patients' poor 5-years progression-free survival (Figure 7D).

Interestingly, patients' overall survival analysis on KMplot database according to the expression ratio of ZEB1 EMT-TF and LIFR (ZEB1/LIFR) showed that when ZEB1 expression is higher than that of LIFR (ZEB1/LIFR > 1) GC patients have lower survival probabilities (Figure 7E). Interestingly, in all GC patients independent of LIFR expression, ZEB1 high expression in usually associated with low prognosis (Figure 7F). However, when the patients are grouped according to mean expression of LIFR 19 (Figure 7G-H), it is noted that in high LIFR patients, ZEB1 expression level is no longer related to patients' prognosis (Figure 7G) while in low LIFR expressing ones, ZEB1 high expression is of even worst prognosis (Figure 7H). This confirms with protective effect of high LIFR signalling.

Discussion

Despite LIF/LIFR signalling's known pleiotropy 31 , this pathway was found to negatively regulate GC cells proliferation 20 and gastric CSC tumorigenic properties 19 . Nevertheless, its effect on GC cells invasion and metastasis remained poorly described.

This study aims at deciphering the role of LIF in GC metastatic context, a particularly crucial aspect of gastric carcinogenesis leading to bad prognosis and in which GC patients are no ~156~ longer eligible to surgery. Metastasis can be decomplexified in a series of steps, each important in this process where tumour cells evade from the primary tumour to reach distant secondary sites. Here, we demonstrate that LIF decreases EMT-TFs expression, activity, the related expression of mesenchymal phenotype-related genes and proteins expression as well as mesenchymal phenotype. ZEB and SNAIL are two important transcription factors in the EMT process, induced in various cancers among which GC 16,33-36 and associated with poor prognosis. The EMT process is found to be responsible for the emergence of cells with stemlike properties, CSCs, largely responsible for tumour heterogeneity and aggressiveness 16,23,24,37 . We have previously reported that LIF has anti-CSC tumorigenic effects 19 in GC but a possible relationship with EMT remained to be deciphered. In this study, we show that LIF also decreased EMT and invasion markers and MMPs expression and inhibited invasion capacity of GC cells.

LIF effect on GC cells does not imply that it acts on CSCs. Indeed, CSCs are a small population of cells representing less than 3.5% of the whole GC cells population 19,23,24 . Here, we succeeded in selecting gastric CSCs, by applying a 3D culture protocol, before analysing their invasive properties in response to LIF. Interestingly, LIF did not only affect invasion of GC cells but also had an inhibitory effect on gastric CSCs invasion. LIF also affected ZEB1 transcription factor and TAZ expression in these invasive CSCs. We have previously demonstrated that ZEB1 expression is associated to that of Hippo oncogenic effector TAZ in GC. The Hippo pathway dysregulation has been related to CSC tumorigenic properties 15-17 but also invasive ones through an effect of TAZ on EMT-TFs 16 . In this context, we demonstrated ZEB1 and TAZ co-repression in invasive CSCs after LIF treatment, suggesting a possible role of activated Hippo kinases in the downregulation of TAZ and EMT-TFs axis, as described previously 16,19 .

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The Hippo pathway's role in LIF-induced effect was investigated using XMU Hippo kinase inhibitor 19 . Indeed, XMU blocked LIF inhibition of EMT and GC cells and CSC invasive properties, confirming the role of Hippo kinases in LIF-induced anti-CSC effects.

Furthermore, contrarily to what was observed for gastric tumorigenic properties 19 where JAK/STAT pathway was not involved, it seems here to be at least partly involved in anti-EMT and invasion properties of LIF in CSCs.

This study also shows that LIF acts on behaviour of GC cells in vivo. Indeed, treating cells with LIF decreased their metastasis initiation capacity, leading to less mice with metastasis and less metastasis signal. This intracardiac injection strategy 26 allows to bypass the putative effect of LIF on primary tumour growth if cells were xenografted orthotopically, in order to study only the capacity of LIF-treated GC cells survival ability as circulating tumour cells (CTC) in mice bloodstream, their specific metastatic niche homing and colonising ability.

Studies show that metastatic cells are pre-destined to reach certain organs and the liver is a preferential site in about 48% GC cases 38 . Here, we show that LIF is able to decrease liver colonisation by circulating GC cells and thus decrease liver metastasis.

Last but not least, using TMA from 177 GC patients 16,23,24 , we have demonstrated that LIFR is less expressed in GC tissues compared to non-tumorous gastric mucosa, which is correlated to bad prognosis of patients. LIFR downregulation in GC thus shows its interest as biomarker.

In addition, low LIFR-expressing patients have worst prognosis when ZEB1 expression is high. This correlates with our results suggesting that LIF/LIFR signalling is able to inhibit ZEB1 activity and have a protective effect.

In fine, this study unveils LIF possibility for the targeting of CSCs to prevent gastric metastatic disease. Nonetheless, caution should be taken considering LIF's use due to its highly pleiotropic aspects. Further studies are mandatory specially concerning the specific

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delivery of LIF to its targets which could help bypass the possible off-targets and secondary effects. Nevertheless, detection of LIFR on GC tumours remains an interesting clue for the prognosis of GC patients. S1. List of primers used for RT-qPCR analysis.

Supplementary Table

Gene

Forward Sequence (5'-3') Reverse Sequence (5'-3')

CDX2 GACGTGAGCATGTACCCTAGC GCGTAGCCATTCCAGTCCT COL1A1 GATTCCCTGGACCTAAAGGTGC AGCCTCTCCATCTTTGCCAGCA CTNNB1 CACAAGCAGAGTGCTGAAGGTG GATTCCTGAGAGTCCAAAGACAG HPRT1 TGGTCAGGCAGTATAATCCA GGTCCTTTTCACCAGCAAGCT ILK GGACATGACTGCCCGAATTAGC GCGTCTGTTTGTGTCTTCAGGC ITGB1 GGATTCTCCAGAAGGTGGTTTCG TGCCACCAAGTTTCCCATCTCC ITGB3 CATGGATTCCAGCAATGTCCTCC TTGAGGCAGGTGGCATTGAAGG ITGA5 GCCGATTCACATCGCTCTCAAC GTCTTCTCCACAGTCCAGCAAG IGGAP1 AAAACTGGGACCAACCAAAGTGT GATGTGGCCTTTGGGATTCA KRT7 GCAGCAATGCCCTGAGCT TGCGGTCCGGATGGAATA MMP3 CACTCACAGACCTGACTCGGTT AAGCAGGATCACAGTTGGCTGG MMP7 GAACGCTGGACGGATGGTA CAAGTTCATGAGTTGCAGCATACA MMP14 ACTGCCAAGCCACCCTAAGA CTGAGCAACGAAGACCCTCTCT MUC2 ACAACTACTCCTCTACCTCCA GTTGATCTCGTAGTTGAGGCA SNAI1 ACAATGTCTGAAAAGGGACTGTGA CAGACCAGAGCACCCCATT SOX9 CGGAGGAAGTCGGTGAAG CTGGGATTGCCCCGAGTGCT SPP1 CACTCCAGTTGTCCCCACAGTA TTGACCTCAGTCCATAAACCACAC TBP TGCACAGGAGCCAAGAGTGAA CACATCACAGCTCCCCACCA ZEB1 TCCCAACTTATGCCAGGCAC CAGGAACCACATTTGTCATAGTCAC ZO-1 GTCCAGAATCTCGGAAAAGTGCC CTTTCAGCGCACCATACCAACC ~165~
LIF-based strategy concerns the upper part of the Hippo signalling pathway through an action on the Hippo kinases. Targeting the downstream oncogenic core directly is also possible as described above and this was also tested using VP, known for its inhibitory role on YAP-TEAD interaction 111-114 . This work is detailed in the next chapter.

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⁓ Chapter 3 ⁓

Verteporfin targeting YAP1/TAZ-TEAD transcriptional activity inhibits the tumorigenic properties of gastric cancer stem cells

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Verteporfin targeting YAP1/TAZ-TEAD transcriptional activity inhibits the tumorigenic properties of gastric cancer stem cells

Julie Giraud 1 , Silvia Molina-Castro

Introduction

Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-related death worldwide. Therapeutic options based on surgery combined with chemotherapy are limited, leading to a poor 5-year survival rate. 1 At histological level, GC is divided in two main groups according to the Lauren classification: intestinal type and diffuse type. At molecular level, the Cancer Genome Atlas consortium classified GC into four main groups based on their mutational profile. 2 These classifications are however not currently used in clinical practice for specific targeted therapy orientation.

Tumors are heterogeneous and composed of a subpopulation of cells called cancer stem cells (CSC) able to initiate and sustain tumor growth and to resist treatments. The existence of CSC was first demonstrated in solid tumors in breast cancer 3 and more recently in GC. 4-7 CSC have self-renewal and asymmetrical division properties, allowing their division into other CSC and more differentiated cells, and thus are at the origin of tumor heterogeneity. We previously characterized cluster of differentiation-44 (CD44) and aldehyde dehydrogenase (ALDH) activity as being the most relevant markers of the subpopulation of tumorigenic and chemoresistant gastric CSC, which represent ~0.5 to 3.5% of the patient-derived primary GC cells. 7 The high mortality of GC patients has been associated with drug resistance and posttreatment tumor relapse, implicating CSC resilience. 8 Innovative approaches combine systemic chemotherapy agents (including 5-fluorouracil and cisplatin), which target the bulk of proliferative cells, with the development of new therapies specifically raised against gastric CSC. We previously proved the possibility of targeting gastric CSC self-renewal properties either by inducing their differentiation 9 or by acting on their metabolism. 10 The study and targeting of specific CSC signaling pathways could lead to new therapeutic options.

The evolutionarily conserved Hippo pathway controls stem cell homeostasis and organ size from Drosophila to mammals. 11 When the Hippo pathway is active, yes-associated protein 1 (YAP1) and the transcriptional coactivator with PDZ-binding motif (TAZ) are phosphorylated by Hippo core kinases, large tumor suppressor 1 and 2 (LATS1/2), resulting in their cytoplasmic retention and proteasomal degradation. On the contrary, when the pathway is off, YAP1 and TAZ are nuclear and preferentially bind to the TEA domain transcription factor (TEAD) to promote cell survival and proliferation through expression of target genes, including the connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (CYR61).

Very recently, Choi et al. showed in transgenic mice that YAP1/TAZ (Y/T) activation in the pyloric stem cell led to gastric tumorigenesis. 12 Several other studies pointed out the oncogenic role of the Y/T-TEAD-mediated transcription in gastric carcinogenesis and suggested that targeting Y/T-TEAD interaction could be a therapeutic strategy for GC. 13-15 For instance, Jiao et al. developed a peptide mimicking VGLL4 which directly competes with Y/T for binding TEADs, leading to an inhibition of Y/T-TEAD-mediated transcriptional activity and oncogenic transformation. 13 In a drug screening of >3,300 Food and Drug Administration (FDA)-approved drugs, Liu-Chittenden et al. identified verteporfin (VP) as an inhibitor of YAP1/TEAD interaction and were thereby able to suppress YAP1-induced liver overgrowth in murine models. 16 An antitumorigenic effect of VP was suggested in gastric cancer by suppressing adhesion molecule FAT atypical cadherin 1 (FAT1). 17 However, the properties of VP on Y/T-TEAD activity, CSC phenotype and tumorigenic properties still need to be investigated in the context of GC.

The purpose of this present study was to analyze Y/T-TEAD activity and expression of target genes in CD44+ gastric CSC, and then to evaluate the capacity of VP to target gastric CSC and inhibit tumor growth in both GC cell lines and patient-derived primary GC xenografts (PDXs).

Here, we demonstrate that Y/T-TEADs and their target genes are enriched in CD44+ GC cells and upregulated in resistant cells after chemotherapy. VP treatment inhibits Y/T-TEAD activity, expression of CSC markers and tumorsphere formation in all of the GC models studied and decreases the gastric CSC population both in vitro and in vivo, leading to tumor growth inhibition.

Materials and Methods

Gastric epithelial cell culture and patient-derived tumor xenografts

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Middlesex, Great Britain). MKN45 and MKN74 cells were cultured in RPMI 1640-Glutamax medium supplemented with 10% heat-inactivated fetal calf serum and 50 IU/ml of penicillin and 50 μg/ml of streptomycin (all from ThermoFisher Scientific, Villebon sur Yvette, France), at 37 C in a 5% CO 2 humidified atmosphere. 9 GC04, GC06, GC07, GC10, GC44 and GC35 are patient-derived GC which were successfully established by serial transplantation in NSG mice as previously described. 7 Twenty-four-hour cultures of GC cell lines were treated with VP (SML0534, Sigma-Aldrich, Saint-Quentin Fallavier, France) diluted in dimethylsulfoxyde (DMSO) (D2650, Sigma-Aldrich) or DMSO alone as control for 24-48 hr as indicated, and then cells were harvested for cell proliferation count (trypan blue exclusion) and molecular analyses.

Flow cytometry and fluorescence-activated cell sorting (FACS)-sorted cells

For cell cycle analysis, 48 hr-treated cells were dissociated by trypsinization and 100,000 cells per condition were stained with propidium iodide (P4864, Sigma-Aldrich) as described elsewhere. 9 For analysis of CD44 expression and ALDH activity, 48 hr-treated cells were recovered and dissociated and 100,000 cells per condition were stained with antihuman CD44-APC and ALDH reagent as previously reported. 9 Live immunofluorescence on tumorspheres from GC10 was conducted as previously reported. 7 Flow cytometry was performed using a BD FACSCanto II instrument and DIVA software (BD Biosciences, Franklin Lakes, NJ). Cell sorting by FACS was performed on cells dissociated from PDX tumors with human tumor dissociation enzymes and GentleMACS dissociator (all from Miltenyi, Paris, France). Cells were stained with anti-EPCAM-Vioblue antibodies (Miltenyi) and anti-CD44-APC antibodies (BD Biosciences) as described. 7 Dead cells were excluded based on light scatter properties and on positive 7AAD staining (50 μg/ml) (BD Biosciences) and EPCAM+CD44+ and EPCAM+CD44-cells were sorted using a FACS Aria II instrument (BD Biosciences).

Transfection and luciferase-reporter assay

Cells were grown in 24-well plates and transfections of plasmids were performed using Lipofectamine 2000 (ThermoFisher Scientific) as recommended. The 8xGTIIC-Luciferase reporter (100 ng/well) was kindly provided by Stefano Piccolo 18 (Addgene plasmid # 34615); the TAL-luciferase reporter (100 ng/well) was from Becton Dickinson and pRL-SV40 (10 ng/well) was from Promega (Charbonnières-les-Bains, France). Firefly and Renilla luciferase activities were measured using the Dual Luciferase Assay (Promega). Firefly luciferase activities were normalized for transfection efficiency by Renilla luciferase.

Tumorsphere assay

Two hundred cells (GC cell lines) or 1,000 cells (PDX cells) were seeded in nonadherent 96-well culture plates (previously coated with a 10% poly-2-hydroxyethyl methacrylate [Sigma-Aldrich] solution in 95% [v/v] ethanol and dried overnight at 56 C) in serum-free GlutaMAX-DMEM/F12 medium supplemented with 20 ng/ml of epidermal growth factor, 20 ng/ml of basic-fibroblast growth factor, 0.3% glucose, 5 μg/ml of insulin, 1:100 N 2 supplement, 50 IU/ml of penicillin and 50 μg/ml of streptomycin (from ThermoFisher Scientific and Sigma-Aldrich) and cultured at 37 C in a 5% CO 2 humidified atmosphere. 7,9 For preventive treatment assays, VP and DMSO were added during cell seeding and spheres were quantified 5-7 days later. For curative treatment assays, tumorspheres were grown for 3-6 days before adding treatment; tumorspheres were counted 3 days after treatment. Images of tumorspheres were taken using ZOE™ Fluorescent Cell Imager (Bio-Rad, CA) and tumor size were measured using ImageJ software.

In vivo xenograft experiments

In all, 100,000 MKN45 GC line and GC10 PDX were suspended in 100 μl of 7 mg/ml ice-cold Matrigel (Ozyme, Montigny-le-Bretonneux, France) and subcutaneously injected into the dorsal flank of 7-week-old nonobese diabetic/severe combined immunodeficiency/interleukin-2Rγ null (NSG) immunodeficient male mice. Tumors were measured three times a week using a caliper and tumor volumes were calculated as reported. 9,10 When the tumor volumes reached around 200 mm 3 , 50 μl of DMSO (control group) or VP solution was subcutaneously injected per mouse at the tumor periphery daily for 13-17 days, after which the mice were sacrificed. Tumors were collected, weighed, fixed in a 3.7% buffered-formaldehyde solution and embedded in paraffin according to standard procedures as described. 9,10 Immunodeficient mice were housed in Level 2 animal facility at the University of Bordeaux, with the agreement of the local Ethics Committee on Animal Experiments CEEA50 of Bordeaux (accreditation number 50120151-A) and the French Committee of Genetic Engineering (authorization number 4608). Conservation conditions and preparation of elements of human origin were declared at the French Ministry of Research (DC-2008-412).

Kaplan-Meier overall survival plots

Kaplan-Meier overall survival plots were performed on nonmetastatic (parameter M = 0) GC patients (n = 444) using the JetSet best probes from http://kmplot.com website. 19 Patient samples were separated into higher and lower expression groups using the following software's auto-setting of the best cutoff value. p Values were calculated by a log rank test.

Results

YAP1/TAZ-TEADs and their target genes are associated with poor overall survival in human nonmetastatic GC patients An analysis on a subset of GC patients was performed at different stages and without distant metastasis (stage M0, n = 444), using publicly available data sets including followup data (see Material and Methods section 19 ). Kaplan-Meier plots showed that high expression of YAP1, TAZ, TEAD1-4 and their main target genes CTGF and CYR61 20 was correlated to a low overall survival probability for nonmetastatic GC patients (Fig. S1). The same analysis was carried out by separating GC according to histological type and similar results were observed in both intestinal (n = 231) and diffuse (n = 192) subtypes (data not shown). These results pinpoint Y/T-TEADs and their target genes as prognostic indicators for GC patients, independently of the tumor histological type.

YAP1-TEADs and their target genes are highly expressed in CD44-positive gastric CSC High expression of the CSC marker CD44 was also found to be associated with low overall survival probability for nonmetastatic GC patients (p = 0.0018; Fig. S1). The expression level of Y/T co-transcriptional factors and target genes in the CD44-positive GC cells was compared to that of CD44-negative GC cells. Immunohistochemistry performed on serial tissue sections of primary GC showed that nuclear expression of YAP1 was heterogeneous and localized preferentially in areas of CD44+ cells (Fig. 1a). Nuclear YAP1 indicates an activation of the YAP1-TEAD transcriptional activity. To test this hypothesis, transcriptomic analyses were performed on CD44+ and CD44-FACS-sorted cells from six inhouse PDX models, including three moderately differentiated intestinal type GC (GC04, GC10 and GC44) and three poorly differentiated diffuse type GC (GC35, GC07 and GC06), according to Lauren's classification. 7 These PDX displayed a heterogeneous expression of YAP1 and low levels of TAZ (except GC07, Fig. S2a). As expected, CD44 was significantly overexpressed in CD44+ cells compared to their respective CD44-counterparts (Fig. 1b). Other CSC marker genes such as CD166, CD24, ALDH1A1, VIL1, OLFM4 and KLF5 were co-enriched in CD44+ cells (Fig. 1b). On the contrary, the expression of gastric differentiation markers such as MUC5AC and MUC6 were lower in CD44+ cells compared to CD44cells (Fig. S3a); high expressions of those genes were significantly associated with a higher overall survival probability of the nonmetastatic GC patients (Fig. S3b). Y/T target genes reported in the KEGG database and in literature, 21 including AREG, BIRC5, CCND1, CDX2, CYR61, ID1, IGFBP3, JAG1, LATS2, MYC and SMAD7, were significantly upregulated in CD44+ cells compared to CD44-cells (Fig. 1b). Conversely, the expression of BBC3, GLI2, ITGB2, SERPINE1 and SNAI2 was lower in CD44+ cells compared to CD44-cells. Interestingly, among the TEAD family, only TEAD1 and TEAD4 were upregulated in CD44+ cells compared to CD44-cells. Contrarily, VGLL4 and RUNX3 mRNAs, encoding two proteins which directly compete with Y/T for binding TEADs, 13,15 were downregulated in CD44+ cells. YAP1 was also overexpressed in the majority of CD44+ cells, contrarily to its paralogue TAZ whose expression was not significantly different compared to CD44-cells (Fig. 1b). These results were confirmed by RT-qPCR in independent experiments with GC10 FACS-sorted cells (Fig. 1c). Altogether these data indicate that Y/T-TEAD is preferentially activated in CD44+ cells in primary GC.

Chemoresistant cancer cells highly express CD44, YAP1, TAZ and their TEAD target genes

The expression of YAP1, TAZ and their TEAD target genes was analyzed in MKN74 (YAP1 high) and MKN45 (TAZ high) GC cell lines (Fig. S2b) in response to treatment with the conventional chemotherapy drugs, 5-fluorouracil (5-FU) and doxorubicin. Both GC cell lines were sensitive to the treatments which inhibited cell growth with an EC 50 of ~5 μM for 5-FU and ~0.1 μM for doxorubicin (Fig. 2a). A 48 hr treatment with cytotoxic concentrations of 50-100 μM 5-FU or 1 μM doxorubicin increased the population of chemoresistant CD44+ cells in both GC cell lines (Fig. 2b, at a dose of 50 μM 5-FU for MKN45 and 100 μM for MKN74). More importantly, the treated cells overexpressed YAP1, TAZ, CYR61 and CTGF mRNAs compared to untreated cells (Fig. 2c). Similar results were obtained for tumorspheres of GC04, GC06 and GC10 PDX cells (Fig. S4). These results demonstrate that residual chemoresistant CD44 + gastric CSC overexpressed YAP1, TAZ and their TEAD target genes CTGF and CYR61, raising the question of whether a treatment based on the inhibition of Y/T-TEADmediated transcription could be an efficient anti-CSC strategy for GC.

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VP inhibits cell proliferation and Y/T-TEAD transcriptional activity in GC cells in vitro

To address this question, the antitumor efficiency of VP, a pharmacological inhibitor of Y/T-TEAD interaction, was evaluated. 16 MKN45 and MKN74 cells were treated for 48 hr with increasing concentrations of VP or DMSO as control. In both cell lines, a decreased cell growth was observed in a dose-dependent manner (Fig. 3a). In MKN45 cells, 5 μM VP induced a cell cycle arrest with an accumulation of cells in G0/G1 phase (75.7 AE 1.4% vs. 53.6 AE 2.7% in control cells) to the detriment of S phase (12.5 AE 3.4% vs. 25.0 AE 2.9%; Fig. 3b, upper panel). Similar results were observed in MKN74 cells (Fig. 3b, lower panel). In addition, the sub-G1 population corresponding to apoptotic cells increased in VP-treated MKN74 cells (6.2 AE 0.1% vs. 1.03 AE 0.2%; Fig. 3b, lower panel). VP treatment prevented Y/T-TEAD transcriptional activity (Fig. 3c) as well as the expression of Y/T-TEAD target genes CTGF, CYR61, IGFBP3, JAGGED-1 and LATS2 which decreased in a dose-dependent manner in MKN45 and MKN74 cells (Fig. 3d). No effect was observed on the expression of AREG (except at the highest dose of VP for which an increase was noted). Interestingly, AREG and JAGGED-1 expression was not significantly associated with the overall survival probability of nonmetastatic GC patients, while a high expression of CTGF, CYR61, IGFBP3 and LATS2 was associated with a poor survival (Figs. S1 andS5). Similar results were observed after treatment of GC10 and GC04 PDXs grown as tumorspheres in vitro (Fig. 3e). VP treatment also led to TEAD1, YAP1 and/or TAZ downregulation in GC cell lines and PDX cells, suggesting that these effectors could be their own target genes as described in lung cancer. 22 TEAD4 expression was not affected by VP treatment (except at the highest dose which induced its overexpression in GC04; Figs. 3d and3e). These results showed that VP efficiently inhibited Y/T-TEAD transcriptional activity in GC cells. In order to confirm that the effect of VP passed through the targeting of Y/T-TEAD, a small interfering RNA silencing strategy targeting YAP1 and/or TAZ was used in MKN45 and MKN74 cell lines (Fig. S6). The YAP1/TAZ siRNAs significantly inhibited cell growth, tumorsphere formation as well as several Y/T-TEAD target genes expression, similarly to VP (Fig. S6a-S6d). The combined data suggest that VP, via the inhibition of Y/T-TEAD transcriptional activity, could be efficient in targeting gastric CSC.

VP treatment targets CSC properties in vitro

We previously reported that in gastric cancer, CD44+ cells contain both the CSC and the proliferative progenitor cells giving rise after division to more differentiated cells. 7 CSC are often considered to be slow growing or even dormant within cancer tissues. In order to determine if all CD44+/nuclear YAP/TAZ+ cells, which are supposed to be CSC, coexpress

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the Ki67 proliferation marker, a four-color immunofluorescent staining was performed. In MKN45 cells, which have a high CD44 expression level, Ki67 expression was associated with nuclear TAZ expression, suggesting that these CD44 +/nuclear TAZ+/Ki67+ cells are those proliferating (Fig. 4a, white arrows). Similarly, in MKN74 cells that have a lower

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CD44 expression than MKN45, the rare CD44+ cells detected were mainly positive for both nuclear YAP1 and KI67+. However, some CD44+/nuclear YAP/TAZ+ but Ki67-cells were also observed in both cell lines (Fig. 4a, yellow arrows). These results show that most of the CD44+ cells have nuclear YAP/TAZ, but they are not all Ki67+, suggesting that targeting these cells with VP would act on both Ki67+ proliferative CD44+ CSC and Ki67-non-proliferative, maybe

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dormant, CD44+ CSC. In order to confirm the hypothesis that VP could inhibit CD44+ cells proliferation properties by targeting Y/T-TEAD activity, cell proliferation experiments were performed on CD44+ FACS-sorted MKN45 and MKN74 cells. VP treatment significantly inhibited CD44+ cells proliferation as from 1 μM in both cell lines (Fig. 4b).

These results confirm that the decrease in cell growth observed in Figure 3a is mainly due to the inhibition of the subpopulation of CD44+ proliferative cells.

We then cultured GC cell lines and PDX cells under tumorsphere conditions to evaluate the impact of VP on CSC tumorigenic properties in vitro. Addition of 0.1-5 μM VP to the

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culture medium during cell seeding (preventive treatment) led to a significant dose-dependent inhibition of tumorsphere formation after 5-7 days (Fig. 4c). In a curative treatment strategy in which treatments were added on established tumorspheres, VP also decreased tumorspheres number in all cases including the two GC cell lines and five different PDX (Fig. 4c). The size of VP-treated MKN45 and GC04 tumorspheres was reduced compared to that of DMSO-treated controls (7.5-and 3.7-fold

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decrease, respectively, Figs. 4d and4e). These results suggest that VP interference with tumorsphere growth could occur by preventing CSC self-renewal. To explore this issue, VP effect was analyzed on a panel of CSC markers enriched in CD44+ cells compared to CD44-cells in the PDX cases (Fig. 1b). CD44 expression was decreased by VP treatment in a dosedependent manner in both MKN45 and MKN74 cell lines, and in GC04 and GC10 PDX cells grown as tumorspheres (Figs. 5a and5b). KLF5 was downregulated only in GC04 and GC10 PDX cells (Fig. 5b). Since 19 ALDH isoforms may contribute to ALDH activity, the global ALDH activity was measured using ALDEFLUOR reagent on live GC10-treated tumorspheres by fluorescence imaging (Fig. 5c) 7 and by flow cytometry on GC10 and MKN45 tumorsphere-dissociated cells (Fig. 5d). The number of ALDH+ cells significantly decreased with 1 μM VP in MKN45 and GC10 tumorspheres (Figs. 5c and5d). These combined data demonstrate that VP reduced the CSC pool of GC cell lines and PDX and interfered with their capacity to grow as tumorspheres in vitro.

VP treatment inhibits tumor growth in murine xenograft model and inhibits CSC properties

To evaluate the antitumor activity of VP in vivo, xenograft experiments were performed using MKN45 cells and GC10 PDX cells. Cells were subcutaneously injected into the right flank of NSG immunodeficient mice and tumor development was measured periodically as previously described. 7,10 When tumor size reached 200 mm 3 , VP in DMSO or DMSO vehicle was subcutaneously injected daily at the tumor periphery. An initial experiment was performed using 0.4 and 2 mg/kg VP on the MKN45 tumors. Mice were sacrificed at Day 13 posttreatment, when the tumors of the control group reached 600 mm 3 (Fig. 6a). MKN45 tumor growth slowed down with 0.4 mg/kg VP and was significantly inhibited with 2 mg/kg, compared to DMSO-treated controls (Fig. 6a). A significant weight decrease was observed in VP-treated MKN45 tumors compared to controls (Fig. 6b). These results were confirmed in two other independent experiments using 2 mg/kg VP on both MKN45 and GC10 PDX cells (Fig. 6b). The histological analysis of the residual MKN45 and GC10-treated tumors showed that VP treatment reduced tumor mass to smaller tumor cell foci within the stroma (Fig. 6c). In all cases, the percentage of cells expressing CD44 and ALDH1 was significantly decreased after VP treatment, particularly for GC10 tumors (Figs. 6c and6d). Proliferating cells expressing the DNA replication markers Ki67 and PCNA were significantly reduced in VP-treated GC10 tumors compared to controls (Fig. 6d). We evaluated the tumorigenic properties of the residual cells recovered from VP-and DMSOtreated tumors, by subjecting them to the in vitro tumorsphere assay (Fig. 6e). Cells from VP-treated tumors initiated less tumorspheres than DMSO-treated tumors in a dose-dependent manner, reaching a 13-fold inhibition of tumorsphere formation at the highest dose of VP (Fig. 6e). These results confirm those obtained in vitro and demonstrate that VP reduces the number of CSC and inhibits their tumorigenic properties, as assessed by the decrease in ALDH1 and CD44 expression, tumorsphere initiation and tumor growth in vivo.

Discussion

The Hippo signaling pathway controls several processes related to tumorigenesis and which define the features of cancer cells such as cell proliferation, differentiation and survival. 11 The expression of CYR61, one of the main Y/T-TEAD target genes, 20 has been associated with low survival of GC patients. 23 Here, we found by in silico analyses that the expression levels of YAP1, TAZ, TEADs and their target genes CTGF and CYR61 are inversely associated with the overall survival of nonmetastatic human GC, their overexpression being associated with a poor prognosis. In a multivariate survival analysis, Qiao et al. found that high TEAD1-4 and YAP1 expressions correlate with a poorer overall survival of GC patients. 15 Nuclear localization of YAP1 was also correlated with a poor outcome in intestinal GC, 24,25 although authors did not find a significant correlation between cytoplasmic expression of YAP1 and survival. 24 In the present study, we showed that expression of YAP1 is heterogeneous inside the primary tumors of GC patients, and we demonstrated that nuclear expression of YAP1 is preferentially found in areas of CD44+ GC cells. Using transcriptomic analyses on FACSsorted GC cells from several PDX models, we showed that CD44+ GC cells display a higher Y-T/TEAD molecular signature than CD44-GC cells.

The most significant upregulated genes in GC CD44+ cells compared to CD44-cells included BIRC5, encoding the inhibitor of apoptosis Survivin which has been associated with a poor prognosis in GC patients, 26 MYC, which drives Y/Tinduced gastric carcinogenesis in mice 12 and JAG1, CDX2, CCND1 and CYR61, all promoting cell proliferation and inhibiting apoptosis. Other cell proliferation regulators were also differentially regulated: IGFBP3, encoding an insulin growth factor (IGF) binding protein which negatively regulates IGF-induced cell proliferation, was strongly induced, and GLI2, a zinc finger transcription factor which mediates the Hedgehog oncogenic signaling pathway, was the most downregulated gene in CD44+ cells. In addition, the downregulated genes included BBC3/PUMA, a proapoptotic target gene of YAP1 associated with p73, and SMAD7, a negative regulator of Transforming Growth Factor β-induced antiproliferative effect, converging to a promotion of cell survival and proliferation in CD44+ cells.

TEAD1 and TEAD4 were upregulated in CD44+ cells, and both proteins were found to be overexpressed in GC 15,27 and associated with a low overall survival in GC (Fig. S1). Interestingly, we found that VGLL4 and RUNX3 expressions were lower in CD44+ cells compared to CD44-cells. VGLL4 is a vestigial-like protein family member which directly competes with Y/T for binding TEAD in several cancer types. 13 Similarly, RUNX3 physically interacts with TEAD preventing

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YAP1 binding and Y/T-TEAD transcriptional activity. 15 VGLL4 13,14 and RUNX3 expressions were described to be reduced in GC tissues. 28 Unlike YAP1, TAZ was not significantly overexpressed in CD44+ cells compared to CD44cells, although it was also associated with a reduced overall survival of nonmetastatic GC patients. YAP1 and TAZ are paralogue genes which display redundant effects in terms of molecular activity but may exert divergent roles in development. 29 We also showed that the number of CD44+ cells increased after conventional chemotherapies. Their chemoresistance may involve multiple mechanisms. We previously reported that CD44 +/ALDH+ gastric CSC have drug efflux properties mediating chemoresistance to conventional drugs such as 5-FU and doxorubicin used in our study. 7 In addition, the epithelial isoform of CD44 found in ~80% of CD44+ cells 6 interacts with xCT, a glutamate-cystine transporter, and may control the intracellular level of reduced glutathione which prevents reactive oxygen species damage. 30 Here, we found that the Y/T-TEAD molecular signature was enriched in the surviving chemoresistant gastric CSC in a panel of GC cell lines and PDX models. YAP1, TAZ and TEADs have also been associated with drug resilience in hepatocellular carcinoma 31 and in breast cancer. 32 Therefore, combining chemotherapeutic drugs with a pharmacological agent inhibiting Y/T-TEAD transcriptional activity could be an efficient strategy to target and counteract gastric CSC resistance to conventional chemotherapy and cure GC.

In a screening of >3,300 drugs, Liu-Chittenden et al. identified VP as a great inhibitor of YAP1/TEAD association and of YAP1-induced liver overgrowth in murine models without affecting the normal intestinal homeostasis or displaying any apparent toxicity. 16 VP is a benzoporphyrin derivative initially approved by FDA as a photosensitizer for photodynamic therapy in ophthalmology. 33 VP photosensitizer has also been tested as anticancer therapy for breast cancer, pancreatic cancer and GC. 17,34,35 Other VP light-independent effects include the regulation of reactive oxygen species production 36 and inhibition of autophagy. 37 In gastric cancer, Kang et al. found that the antiproliferative effect of VP on gastric cancer cell lines is associated to a suppression of the expression of FAT1, a marker associated with a bad prognosis in GC. 17 Moreover, authors tested neither the effect of VP on Y/T-TEAD activity and target genes expression nor on CSC phenotype and properties. In our transcriptomic analysis, FAT1 expression was not differentially expressed between CSC and non-CSC in GC (data not shown). VP inhibitory effects on CSC properties and enhancement of chemotherapy efficiency have been reported in oesophageal cancer cells. 38,39 In our study, we demonstrated that VP treatment inhibits Y/T-TEAD transcriptional activity and efficiently decreases GC cell proliferation in vitro in a dose-dependent manner. More importantly, our results demonstrate that, contrary to 5-FU and doxorubicin treatments which increase the percentage of resistant CD44+ cells, VP treatment reduces the percentage of CD44+/ALDH+ gastric CSC. At molecular level, VP inhibited the expression of several CSC markers as well as that of YAP1, TAZ, TEAD1 and the main Y/T-TEAD target genes controlling cell survival and proliferation. At functional level, VP treatment strongly inhibited tumorsphere formation and growth of all GC cases studied in vitro, in both preventive and curative treatment strategies. These antitumorigenic effects of VP were confirmed in vivo on tumor growth of GC xenografts. VP treatment reduced the percentage of tumor cells expressing CD44 and ALDH1 CSC markers in the residual tumors.

The most important finding was that the residual cells dissociated from VP-treated tumors, which could be considered as either chemoresistant and/or dormant CSC in vivo, were not able to generate new tumorspheres in vitro, confirming that the in vivo VP treatment had efficiently targeted and reduced the pool of both proliferative and resistant/dormant tumorigenic gastric CSCs.

In conclusion, we demonstrate that gastric CSC have a high Y/T-TEAD molecular signature and transcriptional activity which sustain their chemoresistance and tumorigenic properties both in vitro and in vivo. The repositioning of VP targeting YAP1/TAZ-TEAD activity could be a promising CSC-based strategy for the treatment of GC for which there is currently no specific targeted therapy. ~185~

A R E G C T G F C Y R 6 1 I G F B P 3 J A G 1 L A T S 2 T A Z T E A D 1 T E A D 4

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1 .

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M K N 4 5 m R N A r e l a t i v e e x p r e s s i o n ( c o m p a r e d t o c o n t r o l )

s i C t r l

MKN45

A R E G C T G F C Y R 6 1 I G F B P 3 T A Z T E A D 1 T E A D 4 Y

s i C t r l s i Y / T s i T 1 / T 1 s i Y 1 / T 2

Introduction

One step of the metastatic cascade is the dissemination of cells which intravasate into the bloodstream as CTCs. CTCs were first described by Dr. Ashworth in 1869 after observation of cells resembling primary tumour cells in the blood of a metastatic patient 115 . Since then, CTCs have been discovered in a panel of cancers including GC 69,116-125 . Their presence in patients is usually associated to bad prognosis since it is said to reflect advanced stage of disease 126-128 .

Interestingly, though found in rare proportions in the blood of cancer patients (about 1 -10

CTCs/mL of blood 129 , with a mean of 4 CTCs in 7.5 mL blood of GC patients 126 ), they are described as being more numerous in patients with diffuse type GC, a Lauren classification type GC known to be particularly aggressive 2,126 . Furthermore, more and more studies describe tumour cells dissemination as an early step and thus find CTCs at the very beginning of tumour formation 42,80-82 . CTCs could thus provide an interesting path in cancer diagnosis and prognosis through their detection.

Liquid biopsies allowing the detection of cancer-related markers in patients' fluids are a major advance in cancer diagnosis allowing for more non-invasive methods 2 . In GC, CTC analysis gives hope for early diagnosis of this disease, which remains asymptomatic for long. CTCs can also give information on primary tumours' molecular signature, because though molecular changes happen to allow cell dissemination, studies show certain similarities when compared to the tumour of origin 130-132 .

CTCs are undoubtedly actors of metastasis disease. Nevertheless, not all CTCs are able to form metastases following extravasation and homing. Indeed, as discussed earlier, they first need to survive to the hostile blood environment 54,55,133 . Studies in CRC demonstrate that only part of them is able to be resistant and plastic enough to carry out rapid phenotypic changes allowing them to re-acquire a more epithelial status for colonisation as MICs 99,100,134 . These MICs are found to possess CSC properties contributing to their metastasis-formation abilities. In GC, we have recently demonstrated a subpopulation of CD44v3+ CSC carrying these MIC-associated characteristics (Giraud, Seeneevassen et al. in preparation).

Concomitantly with their more resistant nature, these CTCs adopt other mechanisms contributing to their survival. They are able to interact with each other or with immune cells forming homophilic or heterophilic cell clusters 72 which are found to have higher metastatic

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potencies than single CTCs 59,62-65 . As previously discussed, CTCs can form clusters with many actors of the TME among which MDSCs 72,76,135 , found to have a protective role towards CTCs and to participate to metastasis success. MDSCs, known for their role in the suppression of innate and adaptative immunity are a heterogenous population of immature myeloid cells 135,136 .

They participate to tumour progression, neo-angiogenesis, invasion and metastasis and are correlated to poor prognosis of patients. They form part of the TME cells able to help tumour cells metastasis through the secretion of MMPs, degrading ECM. Last but not least, MDSCs also contribute to the metastatic niche preparation 135,137 . MDSCs' contribution to CTCs/MICs emergence and metastasis potency is not totally clear in GC.

Deciphering MICs and the role of MDSCs in gastric metastasis disease could help find strategies to target these specific CSCs in GC. In this context, this part of my thesis project

Methods

GC patients' blood collection and CTC filtration

Blood samples from GC patients were obtained in collaboration with Caroline Gronnier, gastric surgeon from our unit and Head of esogastric surgery unit, Department of visceral surgery of Bordeaux university hospital. Blood samples were collected in K2-EDTA tubes following

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specific precautions implying at least 10 times gentle inversion of the tube after collect. Sample was stored at 4°C and needed to be manipulated within 4 h.

CTC filtration was carried out on 3 mL of blood using Screencell® cytology filtration kits (Screencell, Sarcelles, France) according to manufacturer's recommendation. Filters were rinsed using 80µL of PBS and this step was repeated to remove all visible blood clots. They were then dried overnight at room temperature and stored at 4°C until staining. CTC enumeration was done during this step.

Gastric cancer cell lines 2D and 3D culture and Myeloid-derived Suppressor cells

MKN45 GC cells lines were cultured in RPMI 1640-Glutamax medium, supplemented with 10% heat-inactivated foetal bovine serum (FBS) (all from ThermoFisher Scientific) and 50 µg/ml vancomycin (Invitrogen, Cergy-Pontoise, France) at 37°C in a 5% CO2 humidified atmosphere.

For tumoursphere assays 7,39,40,108,110,138 , MKN45 cells (100 per well) were seeded in 96-well culture plates, previously rendered non-adherent by coating with a 10% poly-2-hydroxyethyl methacrylate (Sigma-Aldrich, Saint-Quentin Fallavier, France) solution in 95% (v/v) ethanol and left to dry overnight at 50-60°C). Specific serum-free CSC medium containing DMEM F12-Glutamax medium, supplemented with 0.3% glucose, 1:100 N2-supplement (all from ThermoFisher Scientific, Villebon sur Yvette, France), 20 ng/mL human epithelial growth factor, 20 ng/mL basic fibroblast growth factor and 5 µg/mL insulin (all from Sigma-Aldrich, Saint-Quentin Fallavier, France) was used.

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HuMoSCs, Human Monocyte-derived Suppressor cells were used to model MDSCs as described 139 .

HuMoSCs generation and phenotypic and functional characterisation

This part was carried out by Immunoconcept team for their expertise.

HuMoSC generation was carried out using blood from the French Blood Establishment (EFS).

Peripheral blood mononuclear cells (PBMC) separation was carried out using Ficoll-based gradient centrifugation method. CD14+ PBMCs were selected using a monocyte isolation kit (Militenyi Biotech SAS, Paris, France), according to the manufacturer's recommendations.

5 000 000 cells were then plated in 6-well plates in RPMI medium containing 10% FBS, 1%

Penicillin/Streptomycin, 1% glutamine, 1% AANE, 1% pyruvate and 1% Hepes. GM-CSF and IL-6 cytokines ((Militenyi Biotech SAS) were added at a dose of 10 ng/mL before incubation at 37°C in a humidified 5% CO2 atmosphere for 7 days with renewal of cytokine treatments each 72 h.

Tumoursphere promotion and initiation assays

MKN45 cells were seeded in non-adherent 96 well-plates as described above. For initiation assays allowing the evaluation of a promoting effect of HuMOSCs, the latter were added at the same time as GC cells. On the other hand, for promotion assays evaluating additive effect of MDSCs presence, HuMoSCs were added to 5-days formed spheres. Tumoursphere counting

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was carried out at 2-days and 5-days post HuMoSC stimulation using an inverted light microscope with a x20 objective 7,39,40,108,110,138 .

2D collagen invasion assays

MKN45 cell lines were seeded (200, Inserts were placed in 24-well culture plates containing medium supplemented with 5% FBS.

After 18 h incubation at 37°C, the Transwell® inserts were fixed with 4% paraformaldehyde solution (PFA, Electron Microscopy Sciences, Hatfield, UK) in cytoskeletal buffer. Cells from the upper part of the insert were discarded using cotton swab and the insert membrane was processed for DAPI staining (ThermoFisher Scientific) 10 min before 3 PBS washings 39,40 .

Invaded cells were counted on 5 different randomly chosen fields per insert, under ZOE™ Fluorescent Cell Imager (Bio-Rad, Marnes-la-Coquette, France).

3D collagen invasion assays

MKN45 (100) cells were seeded as for tumoursphere assays and HuMoSCs were added to a ratio of 1 MKN45 : 5 HuMoSCs at the same time. After CSC selection and formation 7,39,40,108,110,138 of 3-days old spheres, 100 µL of rat-tail type 1 collagen solution, at a final concentration of 1 mg/mL, was added the day-after to each 96-plate well. Collagen was allowed to polymerize for 24 h and photos of the spheres were taken at different times (Day 1 and 4) using camera-equipped inverted-light microscope using x20 objective. The invasive area was measured using ImageJ 1.53f51 software (National Institutes of Health, USA) 140 and calculated according to the following formula: (Day 4 sphere area -Day 1 sphere area). Live immunofluorescence acquisition was done, immediately after buffer-washing and slide mounting, with an Eclipse 50i epi-fluorescence microscope (Nikon, Champigny sur Marne, France) using the NIS-BR acquisition software and a x40 objective (numerical aperture, 1.3).

Preliminary results

GC patients CTC

After a long optimisation process of Screencell filters using blood from mice with artificially added GC cell lines, we have succeeded in collecting and filtrating blood from 5 GC patients (from May 2021-January 2022). The main validated staining protocol used was the one presented in the methods section. Anti-CD45 antibody was used to detect immune cells. Its broad recognition spectrum allowed us not to lose any information for these first trials. Anti-CD44 was used to both detect gastric CSCs and leucocytes and EPCAM for epithelial CTCs.

Preliminary results show that we were able to detect CTCs in the blood samples analysed (Figure 1). These CTCs were CD45-EPCAM+ and CD44+. CD44 staining was not specific of CTCs since immune cells also possess CD44 and are also stained by it, but EPCAM+CD44+ cells also demonstrate CTCs with stem-like properties. The images also show the detection of single CTCs (yellow arrows) and clustered CTCs (white arrows), here with CD45+ immune cells. Homophilic clusters were also detected (green arrows). Interestingly, some CD45-EPCAM-cells were also detected (data not shown) suggesting CTCs which could also be EPCAM-.

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MDSCs contribute to the initiation and promotion of CSCs tumorigenicity

The second part of this project was to explore the role of MDSCs on gastric CSCs tumorigenic potential. To answer this question, tumoursphere assays, in which cells were seeded in nonadherent specific conditions allowing only CSCs to survive, were carried out. Cells were subjected to MDSCs stimulation, through addition of MDSC-like HuMOSCs cells at different cell-cell ratio, either at the same time as GC cells seeding or 5-days after, to determine the effect on CSC initiation or promotion, respectively.

Live immunofluorescence analysis of MKN45 -HuMoSCs (1:5 ratio) interaction was carried out using CD44v6, CD45 and EPCAM staining. In this context, CD44v6 marks CSCs, CD45

stains MDSCs and EPCAM was used to visualise MKN45 GC cells. This demonstrated that both types of cells interacted (Figure 2A). Furthermore, preliminary results demonstrate that HuMoSCs presence in MKN45 3D cultures significantly induced CSC properties acquisition and promotion (Figure 2B). Sphere initiation is facilitated at high ratios of HuMOSC (1:5) while MDSCs presents significant spherepromoting capacities as from low ratios (1:1).

MDSCs and CSCs thus interact, and this induces CSC tumorigenic properties.

MDSCs contribute to the invasion capacity of GC cells and CSCs

Finally, to evaluate the effect of MDSCs on invasion capacity of GC cells, a 2D Boyden chamber invasion assay was carried out. Cells, previously co-cultured with a ratio of 1 MKN45

Discussion

CTCs are important actors in metastasis disease and their characterisation is an important step in the search for targeted therapies. Diverse methods exist allowing for CTC enrichment and study 123 . Here, we demonstrate with these preliminary results that CTCs can be collected from GC patients' blood samples using Screencell® filters and stained for histological Moreover, an interesting observation was the presence of CD45-EPCAM-cells. These cells could be CD44+ or not. Our hypothesis is that some CTCs might be EPCAM-as previously described for colon cancer 101 . It is important to note that CTCs are cells which, for most, have undergone EMT to arrive to the bloodstream. The most transformed ones might have lost EPCAM which is an epithelial marker in favour of mesenchymal ones explaining why they might be EPCAM-. Belthier et al. show that CD44v6, a variant of CD44 is expressed on CD45-EPCAM-colon cancer cells 101 and that CD44v6 marks a different subpopulation of CTCs than EPCAM. CD44v6+ CTCs are found to possess more CSC properties and are more aggressive in colon cancer 100 . It could be interesting to check CD44v6 expression in GC CTCs as well as CD44E or CD44v8-10 102 which is the epithelial isoform of CD44 and will not be expressed on leucocytes, in future immunostainings.

CTCs-immune cells interactions are also an important aspect of metastasis disease since studies relate those as being more prone to form metastases. Studying these interactions is thus also ~197~ mandatory in their targeting. Here we demonstrate with these preliminary results that HuMoSCs, modelling MDSCs, seem to induce CSC tumorigenic and invasive properties in GC cells. They seem to play a role both in the initiation of tumourspheres and in promoting their stemness and maintenance, which could reflect possible roles in vivo as described for ovarian and breast cancers 53,141-143 . Large number of MDSCs is needed in the tumour-initiating effect while a small number is sufficient in its promoting effects. In addition, MDSCs confer more invasive properties to GC cells and CSCs compared to absence of MDSCs showing a possible metastatic potency in vivo. This is similar to what observed previously in other cancers where MDSCs conferred EMT and invasion markers expression and metastatic properties to tumour cells 53,141 . The study of MDSC-like HuMoSCs cells' effect on the EMT characteristics of gastric CSCs is actually ongoing. All the experiments will be done also with GC patient-derived CD33+ MDSCs to see whether these have the same effect on GC CSCs.

Moreover, MDSCs also seem to interact directly with CSCs, forming clusters with them in vitro suggesting interaction sites between the two types of cells. It could be interesting to investigate these interactions and try to block them to verify whether they are necessary for MDSCs to have their effects. If it is so, this could represent an appealing path towards a potential CSC-MDSC targeting strategy.

In addition, these interactions could also suggest a CSC-MDSC communication with CSCs playing a role on the phenotype and immunosuppressive ability of HuMoSCs, as suggested by Liu et al. in breast cancer where ALDH1A1, also expressed by gastric CSCs, increases GM-CSF secretion by tumour cells leading to MDSCs cell population expansion and immunosuppression 144 . This could also be an interesting hypothesis to verify in GC context.

⁓ Discussion ⁓

Gastric cancer is a complex disease which caused approximately 768 793 deaths worldwide in 2020 1 . The 5-year survival rate is less than 20% 2 with a median survival of 3 months in case of metastatic disease 90 . Cancer stem cells are largely to be blamed in this poor prognosis 7 . Their asymmetrical division and differentiation properties are at the basis of GC heterogenicity. They confer the most aggressive characteristics to tumours namely through their tumour initiation, progression and dissemination capabilities and their resistance to conventional therapies. Since they form a rare population of cells within the tumour mass (from 0.1 -3,5% 7 ), tumour elimination by non-targeted radiotherapy methods and confirmation by imaging do not detect the residual cells that remain present, latent and awaken years after leading to tumour relapse.

GC therapy is still at its premises concerning targeted approaches. Patients are still treated with surgery and additional radio and chemotherapies such as leucovorin, oxaliplatin, docetaxel and 5-Fluorouracil 2 , conceding place to relapse cases. And a particularly critical aspect is the fact that this disease remains undetectable for long due to the lack of symptoms, that most patients are at a metastatic state at disease diagnosis and that unfortunately metastatic GC patients have unresectable tumours and are most of the time subject to palliative chemotherapies and not curative ones 2 . The search for targeted approaches is thus vital for GC treatment and this is on what most of my thesis work is focused.

My work aimed particularly at the targeting of the hostile components of gastric tumours which are, as described earlier, the gastric CSCs. Targeting these cells could give hope towards a more proper patient care by limiting resistance to treatments and relapse cases. The adopted strategy was to deviate CSCs and tumour cells capacity of hijacking cell signalling pathways to our advantage by identifying a particular pathway important for their survival and properties and targeting it.

The Hippo pathway was found to be particularly important in gastric CSCs since it has been demonstrated that GC major risk factor H. pylori is capable of inducing EMT process at the origin of CSC emergence through an effect on this signalisation, and in so doing gastric carcinogenesis 107,145 . Furthermore, a high Hippo/YAP/TAZ-TEAD effector signature was described in gastric cancer CSCs and largely contributed to invasion of GC cells 40,106 . This pathway targeting seemed to be an interesting path to follow for gastric anti-CSC therapy.

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The Hippo pathway is, as described earlier, a crucial pathway in tissue homeostasis in physiology. Balance between its tumour-suppressor and oncogenic cores is vital and this is what renders its deregulation this disastrous. During my thesis, we attempted two strategies aiming at counter-balancing Hippo pathway's dysregulation in GC.

i) Targeting Hippo upstream members and inducing Hippo tumour suppressor kinases effect ii) Targeting Hippo downstream members and inhibiting YAP/TAZ-TEAD oncogenic activity

CSC targeting i) Hippo upstream members targeting

A major part of my work focused on this aspect. We have found that Leukaemia Inhibitory

Factor LIF, an IL-6 family cytokine, is able to act on the Hippo pathway in GC through its receptor LIFR. This was already demonstrated before in breast cancer 146 where it presented anti-metastatic effects, but nothing was known for GC at that time.

In the first part of this work, we demonstrated for the first time that LIF was able to stimulate the Hippo pathway kinases MST and LATS in GC context and that LIF treatment presented anti-CSC tumorigenic effects in GC cell lines and PDX cells, through an effect on gastric main CSC markers gene and protein expression, on CD44+ and/or ALDH+ gastric CSC population and on CSC tumorigenic capacity 110 . LIF's effect on GC cells proliferation were demonstrated parallelly by Xu et al. 147 , but no description of a potential effect on CSCs had been shown. In addition, live immunofluorescence allowed us to see that LIF affected one important characteristic implicated in the chemoresistance property of CSCs which is their drug efflux ability. Indeed, this technique allowed to follow Hoechst-33342 efflux carried out by the same means as drug efflux by chemo-resistant CSCs, and showed that LIF was able to decrease this cell population. In relation with this, LIF was able to potentiate chemotherapy anti-CSC tumorigenic capacity 110 showing its potential interest in CSC targeting.

Furthermore, this first part addressed the question about the Hippo pathway implication in LIFinduced effects. Inhibitor of Hippo kinases XMU-MP-1 was used to this aim and allowed the demonstration that LIF-induced MST1/2-LATS1/2 kinases activation was related to its anti-CSC effects. LIF/LIFR is indeed a complex signalling pathway 148 LIF/LIFR/JAK/STAT exerts the exact opposite effect in melanoma, showing how complex this cell signalisation is, and care should also be taken while attributing effects to one pathway or another since all are intertwined and work in concert or in opposition in the effects observed 148 .

LIF-induced anti-CSC tumorigenic effects gave rise to the question about possible anti-invasive effects since CSCs are related to both processes in tumorigenesis. In this study, we revealed that LIF was able to decrease EMT-TFs expression as well as activity and their related EMTassociated genes expression in GC cells lines and PDX cells. EMT process modulation in presence of LIF is closely related with preceding discoveries about LIF anti-CSC effects since studies demonstrate the clear participation of EMT to arousal of cells with stem-like characteristics 39,40 . Indeed, LIF anti-CSC effects could derive from this effect on EMT. Studies show that EMT-TFs overexpression and EMT induction are able, through epithelial markers repression and induction of mesenchymal ones, to induce dedifferentiation of tumour cells, induction of stem cell markers expression and emergence of CSCs 18,34,36,37 . ZEB and SNAIL repression by LIF could thus be at the origin of LIF-induced anti-CSC effect.

Moreover, LIF effect on decrease of EMT-TFs ZEB and SNAIL activity could be related to its effect on the stimulation of Hippo effectors YAP/TAZ inhibitory phosphorylation. Indeed, YAP is found to directly interact with ZEB and induce the expression of a proper set of genes 149 . This YAP/ZEB activity is related to the capacity of these proteins to bind TEAD and in presence of LIF this is affected due to less nuclear translocation. In addition, TAZ-TEAD is found to be able to bind ZEB1 promoter and affect its transcriptional expression 150 . Less nuclear translocation of TAZ in presence of LIF is consequently closely related to less ZEB1 gene expression as shown in our work.

This study also demonstrated that LIF plays a role in modulating MMPs expression thus limiting collagen invasion process of GC cells and PDX, one key step in GC dissemination process. Even more important in the actual context is the fact that LIF not only plays a role in EMT-TFs expression and invasion of the whole population of GC cells analysed, but also in that of 3D-culture selected gastric CSCs confirming the hypothesis stipulating LIF's interest in gastric anti-CSC therapy. In concert with this, we have developed a novel tumoursphere-collagen invasion assay in the lab. It was coupled with laser microdissection and mass spectrometry-based proteomics analysis allowing to confirm the decrease in mesenchymal and increase in epithelial proteins expression at "pseudo-invasion fronts" of LIF-treated tumourspheres compared to non-treated ones. Other proteins in relation with invasion and metastasis such as transcription partners, actin cytoskeleton remodelling and vesicle trafficking forming a complex invasion-related machinery were found to be under-expressed in LIF treated conditions paving an interesting path to follow to understand more this intricate process.

LIF anti-invasion and anti-EMT effects involved the Hippo pathway as demonstrated by the use of XMU-MP-1 Hippo kinase inhibitor which blocked LIF-induced effects. What was interesting is that depending on the cells, or the EMT-TFs analysed for example, the JAK/STAT pathway was found to be implicated or not, showing the high heterogeneity among cancer cells and among each actor's regulation.

We also demonstrated LIF's effect in in vivo xenograft models of intracardial injection. GC cells, treated or not with LIF, were injected in immunodeficient mice and allowed to disseminate and metastasize. LIF treatment delayed metastasis appearance, decreased the percentage of mice developing metastases and in those where positive signals were detected, this metastatic signal was lower than in mice injected with non-treated cells. Furthermore, metastatic niche preference was altered after LIF treatment since mice developed less liver metastasis. Effect on liver metastatic niche is of particular interest since GC preferential metastatic site is the liver with 48% patients developing metastases there 90 . These patients are found to have a median survival of 2 months, even less than if metastasis had developed elsewhere. Thus, altering metastatic niche can be of real interest in patient care.

This in vivo study allowed the analysis of disseminated cells survival in bloodstream, extravasation, homing, and colonisation of secondary sites. LIF-treated cells are thus unable to well-accomplish one of these steps. LIF is found to decrease CD44-expressing cells, CSCs here in GC. CD44 is not only considered as a CSC marker but plays a role in tumorigenesis, migration and invasion. It is an adhesion molecule whose main substrate is the ECM member hyaluronic acid 151 and which is found to have a role in cell extravasation and homing 152 . CD44 plays a role in tumour cells rolling under shear stress conditions, which is exactly the type of environment to which metastatic cells are confronted. Thus, CD44 expression modulation by LIF could affect this process in our case and contribute to less metastasis.

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Nonetheless, this study does not allow the assessment of LIF's effect on whole metastatic process and more specifically whether LIF anti-invasive effect has a certain benefit in vivo since this part of the metastatic process is lacking from this model. The effect of LIF could have been studied in a more cancer-resembling model for example a GC orthotopic xenograft model.

During my thesis I got the opportunity to participate to the development of such a model where luciferase-expressing GC cells are injected in immunodeficient mice stomach sub-serosa 109 and metastasis can be followed by bioluminescence tracking. This orthotopic model allows to reproduce metastatic cascade steps in mice models but is a quite complex intervention which is also tough for the animals. However, in our case the intracardiac mice model had the advantage of bypassing the primary tumour development step, suspected to be affected by LIF treatment according to our previous in vitro data 110 . Indeed, there is a great possibility that LIF-treatedcell would not have been able to form primary tumours in the orthotopic xenograft model making it complicated to study dissemination and metastatic implantation steps which was possible with the intracardiac model.

Moreover, we also demonstrated in this part of the project that LIFR is less expressed in GC cases compared to non-tumorous tissues. This was demonstrated bioinformatically on Oncomine database 110 but also in patients tissues using TMA constructed from 177 GC patients. This is also related with poor patients' prognosis as demonstrated both in silico on KMplotter database and using patients clinico-biological data. Interestingly, LIFR seems to pass from a membranous expression, in non-tumorous tissues to a more nuclear intriguing one in tumours, especially in diffuse type GC, showing a dysregulation in LIFR localisation. Interestingly, this nuclear localisation and perinuclear localisation of LIFR has already been described in syncytial muscle cells where this nuclear translocation was induced in newly differentiated myotubes and translocated LIFR was no longer the native GP190 one, but a truncated one which might have undergone post-translational modifications 153 . Other examples of this atypical expression of LIFR were described and associated to a negative regulation of the receptor, preventing it to get stimulated by soluble LIF 154,155 . Here, in cancer tissue the nuclear/perinuclear expression of LIFR seems to be related to its cancer-associated dysregulation while its subcellular expression was not discriminant in terms of prognosis compared to overall expression. Consequently, LIFR differential expression in tumours compared to non-tumorous tissue and the related poor prognosis makes it an ideal cancer-related diagnostic and prognosis biomarker, which is necessary to detect cancer events in this disease which is usually detected late.

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In terms of Hippo targeting using LIF, though this cytokine presents interesting anti-gastric CSC tumorigenic, chemo-resistant and invasive capabilities, its high pleiotropy makes its consideration for patient treatment quite risky. As discussed earlier, it has undergone clinical trials for use in embryo implantation and though no work has been carried out concerning its use in cancer treatment, patients did not show dramatic side effects 156-161 . However, the highly diverging effects of this molecule depending on tissue types and the fact that new data are still being discovered makes it too hazardous to be tried in therapy. Secured targeted delivery as done by Davis et al. using coated nanoparticles filled with LIF could be an attractive option which could considerably minimise undesirable effects 162 . In our context, the issue of such a strategy to target gastric CSCs would be the search of specific molecules targeting them and this is still at its premises.

ii) Hippo downstream members targeting

Next part of my work concentrated on another strategy to target Hippo oncogenic signalling which is the inhibition of YAP/TAZ-TEAD signalling. As aforementioned, YAP/TAZ-TEAD signalling upregulation in GC like in many other cancers 40,107,163 , is related to bad prognosis and interaction of these Hippo co-activators with TEAD is vital for their tumorigenic activity 164,165 .

Here we used Verteporfin (VP), a benzoporphyrin derivate capable of competing with YAP and, in so doing inhibiting YAP-TEAD binding, to target this interaction 166 . VP is an FDAapproved drug for the treatment of age-related macular degeneration 166 for example and its use as therapeutical repositioning could be interesting. We show that YAP/TAZ-TEAD and their target genes is associated to poor overall survival of non-metastatic GC patients and that this signature is detected in CD44+ gastric CSCs compared to CD44-non-CSCs. Furthermore, residual resistant GC cells after doxorubicin and 5-Fluorouracil chemotherapy treatments were most CD44+ and presented high Hippo oncogenic effectors signature. This is consistent with what described precedingly about YAP/TAZ effect in GC chemoresistance. Indeed, YAP and TAZ are found to be involved in resistance of GC cells to cisplatin 167,168 and their overexpression in chemo-resistant cells is thus expected. This was shown in our context in GC cell lines as well as PDX cells. Inhibition of YAP/TAZ-TEAD interaction with VP decreased GC cell proliferation and YAP/TAZ-TEAD transcriptional activity as expected. Moreover, VP affected CSC properties and CSC markers expression in vitro confirming that YAP/TAZ-

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TEAD indeed exerted pro-CSC effects in GC and that inhibiting Hippo effectors interaction with transcription factor TEAD is sufficient to counteract their oncogenic effect. Interestingly, we showed in this study that VP is not only able to prevent CSC-tumoursphere forming capacity, but also affects already formed sphere and decreases their number, showing a role of YAP/TAZ-TEAD in tumour initiation and maintenance, main CSC properties, as described for other cancers 169 .

Finally, we demonstrate that the use of VP as peritumoral treatment in vivo in subcutaneous xenograft models of GC cell lines and PDX cells considerably inhibited tumour growth as well as the presence of residual CSCs in the tumours, demonstrated by anti-CD44 and anti-ALDH1 immunohistochemistry on collected tumours. Furthermore, this was consistent with the fact that residual tumour cells had diminished tumorigenic potential. Indeed, dissociation of tumour cells followed by tumoursphere assays after in vivo VP treatment significantly decreased tumoursphere-forming capacity of residual cells. All this showing that VP, through its YAP/TAZ-TEAD interaction disruption, is able to readily target gastric CSCs tumorigenic properties.

Deciphering effect of VP on invasive properties of CSCs would be interesting since, as described earlier, TEAD interaction with YAP and ZEB is important for promoting YAP/ZEB target genes expression 149 and with TAZ for ZEB1 gene expression 150 . Indeed, ZEB being a major EMT-TF, it is known for its epithelial genes' repression and mesenchymal genes' induction ability, among which invasion and metastasis-related genes.

Altering YAP/TAZ-TEAD interaction using VP thus presents a potential anti-gastric CSC strategy. Other molecules such as VGLL4-derived Super-TDU peptide, peptide 17 and curcumin/WZ35 exist, are being designed and tested, as described, and discussed above, with the aim of altering this crucial interaction in GC. Nevertheless, the strength of VP is that it is a drug which has already been FDA-approved since 2000 and thus has passed clinical trials and already been used in patients 170 . Repositioning VP for the targeting of CSCs would be more easily acceptable than a completely new drug and no doubt that drug repositioning takes much less time than asking for a new approval.

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CSC/CTC characterisation

Deciphering CSC characteristics and properties is a crucial step in the development of targeted therapies towards this aggressive population of cells. This is why this last part of my work focussed on the characterisation of these cells in the metastatic cascade, a key stage of the carcinogenesis process, most of the time the toughest one since patients in this phase of the disease usually do not have much time left; either because disease progression happens very fast or because no therapeutical possibilities open up for them.

Here we show the complexity of metastatic disease in GC like in all cancers and how CTCs are major actors of this stage. We managed to collect CTCs from GC patients' blood samples and 80% of the analysed cases were positive for CTC in this preliminary study. Moreover, we demonstrated that, depending on the patient, about 5-20 CTCs were enumerated per 3 mL of blood which is considerably high knowing that the mean CTCs in GC is usually 4 in 7.5 mL of blood 126 . It will be interesting to corelate this CTC count to the clinico-biological data of the patients to fully understand these results.

We also managed to carry out specific immunostaining on the cells, allowing the detection of patients' immune cells and differentiating them from CTCs. Interestingly, these were found alone or in clusters as already described in other types of cancer 59,62-65 and if clusters were present these were consistently either homophilic or heterophilic 72 .

CTCs filtration and exploration gives hope for development of new diagnosis and prognosis methods using non-invasive patient-friendly methods. What we want to do next is to narrow down the staining done so as to decipher the observed cells, their state, their interactions and interacting partners. Indeed, knowing their EPCAM status for example could give a notion of their aggressivity as described by Belthier et al. since EPCAM-cells are found as being CD44v6+ which are a particularly aggressive population of CTCs having stem-like characteristics 101 . Indeed, among the patients analysed, some had EPCAM-cells which could have been consistent with this hypothesis had we carried out a CD44v6 staining. Different stainings must thus be employed.

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Furthermore, characterisation of interacting partners is crucial since these can have a certain function in metastasis formation through their protective role amongst others 72,135 . Among the different partners could be MDSCs, a particular population of immature myeloid cells carrying pro-tumoral, pro-angiogenic, pro-invasion and pro-metastatic characteristics. One aim is to explore whether these cells are associated to the CTCs we collect from patients' blood and correlate this with a certain patients' state.

Moreover, another objective of this part of the project, independent from the patients' blood collection, was to evaluate the role of these cells on gastric CSC properties. This was initiated in tight collaboration with Nicolas Larmonier's and Charlotte Domblides' team at Immunoconcept and will be pursued.

To this purpose, MDSCs' model HuMoSCs were used and co-cultured with GC cells. CSCforming ability was assessed, and preliminary results showed that HuMoSCs were able to induce CSC properties acquisition and promotion by GC cells. This is consistent with what observed in other cancers 53,141-143 confirming the role of MDSCs in cancer aggressiveness.

Furthermore, MDSCs were able to increase invasion capacities of GC cells as well as GC CSCs, suggesting a possible role, apart from the protective one, in another step of the metastatic cascade. Indeed, MDSCs were described to induce expression of invasion-related genes in tumour cells 141 . Furthermore, in breast cancer two subsets of immunosuppressive MDSCs were described, monocytic (mMDSCs) and granulocytic (gMDSCs) having distinct and even opposing roles 53 . mMDSCs are able to induce EMT phenotype in cells and facilitate cell dissemination and invasion through the acquisition of EMT and invasion-related proteins while gMDSCs induce MET of tumour cells at metastatic sites helping their colonisation process and proliferation. This could thus be interesting to explore in GC.

Last but not least, we demonstrate in vitro clustering of HuMoSCs and GC CSCs suggesting the role of direct cell-cell interaction in CSC properties promotion capacities of MDSCs. This could serve as target with the goal of inhibiting MDSC-CSCs or MDSC-CTCs interaction in cancer context as novel CSC targeting therapeutical strategy.

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⁓ Conclusion ⁓

Overall, this work, recapitulated in Figure 5, contributed to the knowledge about gastric CSC targeting, which is of utmost importance for GC therapy and proper patient support and care. Among the different strategies, developed by our team, which can be adopted in GC CSCdirected therapy; differentiation induction using ATRA 108 , sensitisation to conventional chemotherapies with verapamil which works by blocking drug efflux mechanism 7 , metabolism alteration using Metformin 171 ; we hereby present the targeting of CSC-associated signalling pathways, here the Hippo/YAP/TAZ signalling pathway. This can be achieved either by

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activating Hippo tumour suppressor kinases or by inhibiting YAP/TAZ-TEAD oncogenic effectors.

Leukaemia Inhibitory Factor was used in the first strategy for its LATS1/2 activating properties and demonstrated anti-CSC tumorigenic, chemo-resistant, EMT, invasive and metastatic colonisation properties 110 .

Verteporfin was used in the second strategy for its capacity to alter YAP/TAZ-TEAD interaction and in so doing inhibit their transcriptional activity and downstream effect. This led to decrease in CSC tumorigenic properties in vitro and in vivo 106 . Possible anti-invasive properties were not analysed but many clues are available, rending this hypothesis plausible and interesting to explore.

This work also contributed to the knowledge about CTC and CTC-immune cells interaction, important aspects of gastric carcinogenesis and CSCs, and crucial in the development of CSCtargeting strategies. CTCs can be isolated from GC patients and visualised after specific staining of the different actors of metastatic disease. They are found single or in clusters with themselves or with immune cells. MDSCs are able to interact with CSCs and contribute to their aggressiveness through induction of tumorigenic and invasive properties.

Further characterisation of patients' CTCs and interactors, and analysis of MDSCs' role in GC will allow the elaboration of new anti-CSC strategies through targeting of CSC/CTC-immune cells interaction. Moreover, advanced CSC characterisation will enable the discovery of more specific CSC markers, allowing their appropriate targeting for the development of CSCdirected drug delivery methods, thus limiting adverse effects of drugs in patients.

Introduction

Gastric cancer has a high incidence and represents the third leading cause of cancer deaths worldwide [1]. Most of them are gastric adenocarcinoma (GC), which is the consequence of chronic infection of the gastric mucosa with Helicobacter pylori (H. pylori) [2]. GC has a poor clinical outcome, being detected most of the time too late at advanced metastatic stages. GC are highly heterogeneous both at histological and molecular levels. Except for the rare cases overexpressing the epidermal growth factor receptor HER2, which can be treated with Herceptin, there is no targeted therapy to treat GC patients. The histological criteria-based classification of GC has been recently completed by molecular classifications based on sequencing, which are expected to help in proposing targeted therapies [3][4][5][6]. Current treatment for the non-metastatic forms is based on surgery with perioperative conventional chemotherapies for non-cardia gastric cancer and neoadjuvant chemoradiotherapy for cardia gastric cancer [7][8][9]. New treatment options include the use of immune checkpoint inhibitors in resectable GC [10]. For the unresectable metastatic GC cases, the therapeutic options are usually limited to palliative chemotherapy with a five-year survival rate of less than 20% [10,11].

Targeting agents such as antibodies or small molecules against tyrosine kinases have emerged as a treatment option for GC patients. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway is commonly observed in human cancers and is critical for tumor progression and resistance to cytotoxic chemotherapy [12]. This pathway regulates essential cellular functions including cell proliferation, survival, cytoskeleton rearrangement, and metabolism [13]. PI3K signaling is initiated by the activation of tyrosine kinase receptors, including EGFR, HER2, and MET, leading to the activation of the downstream effector protein kinase B (AKT).

In GC, PI3K signaling is up-regulated through diverse mechanisms involving direct mutation through the amplification of PIK3CA or the deregulation of key components such as the monoallelic loss of phosphatase and tensin homologue (PTEN) [14,15]. Accumulating evidence indicates that the PI3K/AKT pathway plays a role in epithelial-to-mesenchymal transition (EMT) induction, which is a fundamental process implicated during embryonic development that is considered a key step toward tumor invasion and metastasis [16][17][18][19][20][21].

Using original patient-derived subcutaneous xenografts of GC (PDX) in immunodeficient mice, we previously contributed to demonstrate the existence of a rare subpopulation of gastric cancer stem cells (CSC) being at the origin of tumor initiation, growth, and chemoresistance [22]. We characterized the cell surface receptor CD44 as one of the best markers of the enrichment of gastric CSC among the cells composing the tumor mass [22]. Moreover, we have highlighted that the chronic infection of gastric epithelial cells with H. pylori induces an EMT-like transition leading to the emergence of cells with CSC properties and expressing CD44 both in vitro and in vivo in patients' derived gastric tissues and in mouse models of experimental infection [23,24]. A major limitation of the mouse models of H. pylori infection and/or carcinogens-induced gastric carcinogenesis is that they develop only gastric pre-neoplastic lesions with more rarely in situ carcinomas, but never invasive carcinomas or metastases or peritoneal carcinomatosis as in humans. Likewise, the subcutaneous tumors developed in our PDX mouse models recapitulate the heterogeneity of the primary GC of the patients, but they never metastasize to distant organs in mice [22].

The aim of this study was to develop new preclinical mouse models of GC-forming metastases in order to decipher the metastasis formation process and evaluate the anti-metastatic efficiency of a new therapeutic strategy targeting PI3K activity in GC. Here, we established three mouse models of patient-derived orthotopic xenograft of GC (PDOX) and two models of the orthotopic xenograft of GC cell lines that developed distant metastases in the liver, the lung, and the peritoneal cavity, modeling the disease as in patients. Lung and liver metastases over-expressed the CD44 gastric CSC marker, suggesting that CSC may be involved in dissemination and metastases' initiation and growth. The treatment of GC cells with the pan-class I PI3K inhibitor BKM120 displayed a cytostatic effect in a dose-dependent manner and decreased the capacity of gastric CSC to form tumorspheres in vitro. Finally, we found that, in mice orthotopically xenografted with GC, BKM120 treatment resulted in a stagnation of tumor progression and a limitation of tumor cell dissemination and metastases formation.

Results

Development of Patient-Derived Orthotopic Xenograft Mouse Models Developing Distant Metastases

We previously established several mouse models of PDX, which allowed us to characterize and study the properties of CSC in GC [22,25,26]. These PDX were amplified in mice subcutaneously, and reproduced heterogeneous tumors similar to those of patients, but never metastasized [22]. In order to reproduce the metastatic process, which is at the origin of the low prognosis of GC in humans,

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Cancers 2019, 11, 560 3 of 17 we carried out xenografts of these PDX cells directly into the stomach wall. As the engraftment success of primary GC from patients is very low in mice [22], we used four in-house PDX cases that have already been established in mice and well characterized: cases GC04, GC07, and GC10 are of the intestinal histological subtype, while case GC06 is of diffuse type, according to the Lauren histological classification criteria [22]. The GC07 case possesses the advantage of being cultivable in conventional adherent culture conditions conversely to the GC04, GC06, and GC10 cases. First, cells freshly dissociated from PDX tumors were transduced with lentiviruses encoding the luciferase gene, and were thereafter transplanted subcutaneously in NSG mice for amplification. The luciferase-encoding GC cells were recovered from these growing tumors, and 10,000 cells were subsequently xenografted into the subserosa of the stomach wall of new recipient mice (PDOX). The scheme of the experimental procedure is shown in Figure 1A. We previously established several mouse models of PDX, which allowed us to characterize and study the properties of CSC in GC [22,25,26]. These PDX were amplified in mice subcutaneously, and reproduced heterogeneous tumors similar to those of patients, but never metastasized [22]. In order to reproduce the metastatic process, which is at the origin of the low prognosis of GC in humans, we carried out xenografts of these PDX cells directly into the stomach wall. As the engraftment success of primary GC from patients is very low in mice [22], we used four in-house PDX cases that have already been established in mice and well characterized: cases GC04, GC07, and GC10 are of the intestinal histological subtype, while case GC06 is of diffuse type, according to the Lauren histological classification criteria [22]. The GC07 case possesses the advantage of being cultivable in conventional adherent culture conditions conversely to the GC04, GC06, and GC10 cases. First, cells freshly dissociated from PDX tumors were transduced with lentiviruses encoding the luciferase gene, and were thereafter transplanted subcutaneously in NSG mice for amplification. The luciferase-encoding GC cells were recovered from these growing tumors, and 10,000 cells were subsequently xenografted into the subserosa of the stomach wall of new recipient mice (PDOX). The scheme of the experimental procedure is shown in Figure 1A. In the first GC cells orthotopic xenograft experiment (experiment a, Table 1), tumor growth in the stomach and dissemination in distant organs were followed for up to 25 weeks by bioluminescence imaging on live animals (Figure 1B,C). Bioluminescence imaging was also performed on recovered organs at sacrifice (Figure 1C, bottom panel). PDX cells generated tumors in the stomach and distant metastases in the lung and the liver at variable frequencies according to the cases studied (Table 1), and were macroscopically detectable for some cases (Figure 1D). In this experiment, GC10 and GC07 were the best models of engraftment and dissemination; tumor frequency reached 100% in the stomach for both cases, 75% and 100% frequency in the lung for GC10 and GC07, respectively, and 50% in the liver for GC10 only (Table 1). GC04 generated gastric tumors and lung metastases at a lower frequency than GC10 and GC07, and no liver metastases even after 13 weeks post-xenograft. Surprisingly the diffuse case GC06, which usually forms tumors after three to four weeks post-subcutaneous engraftment, did not grow in the stomach during the 25-week follow-up period, and did not develop distant metastases. As GC10 and GC07 were the best models of engraftment in the stomach, leading to tumor growth and the development of distant metastases, a second orthotopic xenograft experiment was done with only these two cases and with more mice per GC case, which confirmed these results (experiment b, Table 1). Combined together, these experiments showed that GC10 and GC07 are good pre-clinical models to study tumor growth in the stomach and the formation of distant metastases, with tumor development for GC10 and GC07 respectively reaching 94.7% and 100% in the stomach, 73.7% and 100% in the lung, and 79% and 19% in the liver (n = 19 mice for GC10 and n = 16 mice for GC07 when combining both experiments, as shown in Table 1). At the end point, photon emission was analyzed in individually recovered organs and showed that the detection of metastatic cells was significantly more important in the lung than in the liver for both the GC07 and GC10 cases (Figure 1C, bottom panel). We also reported the detection of peritoneal tumors for the GC10 case and more rarely for the GC04 and GC07 cases, which developed locally in the case of perforative primary tumors, and were usually associated to tumor detection in the spleen and the pancreas (Table 1).

To increase the number of cases, we realized complementary experiments using three GC cell lines: one is of diffuse type (MKN45) and two are of intestinal type (MKN74 and NCI-N87). After eight to 10 weeks post-orthotopic xenograft, tumor development was determined by macroscopic observation followed by histological analyses of the expression of the human leukocyte antigen (HLA) class-I molecules, which are ubiquitously expressed in human epithelial cells (Figure 2). MKN45 and MKN74 developed tumors in the stomach and distant metastases in the lung with a frequency of 100%. MKN45 and MKN74 also developed metastases in the liver and the peritoneal cavity with a frequency ranging from 50% to 80% (Table 2). On the contrary, only three out of five mice that were xenografted with NCI-N87 cells developed a tumor in the stomach, and no distant metastases were detected up to 10 weeks post-xenograft (Table 2). In order to determine at which time post-engraftment the GC cells escaped from the stomach to disseminate and initiate distant metastases, we performed experiments of partial resection of the stomach at days 7 and 14 post-orthotopic xenograft of MKN45 luciferase-expressing cells to remove the primary tumor (Supplementary Figure S1). When the resection was performed at day 14 postxenograft, the mice still developed metastases. However, resection performed at day 7 postxenograft impaired the development of metastases compared to the non-resected control mouse after 10 days and until 52 days of follow-up (Supplementary Figure S1). Nevertheless, one resected mouse finally developed metastases and tumor in the residual stomach after 28 days, suggesting either an incomplete resection of the primary tumor or circulating tumor cells self-seeding at the primary site, as previously described in breast cancer [27]. These experiments suggest that metastases may arise from the dissemination of GC cells from the primary tumor in the stomach during the first week following orthotopic xenograft.

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Metastases Are Enriched in Cancer Stem Cells

The literature suggests that the CSC subpopulation of tumor cells is not only responsible for tumor initiation, growth, chemoresistance, and tumor heterogeneity, but also for tumor dissemination and metastases formation [28]. To evaluate this hypothesis, we analyzed the proportion of CSC in the metastases and gastric tumors of the orthotopic xenograft models. Tumor cells from stomach tumors and micrometastases formed by GC10 PDOX case and MKN45 cell line were harvested and submitted to the in vitro tumorsphere assay (Figure 3A,B). Only CSC can self- In order to determine at which time post-engraftment the GC cells escaped from the stomach to disseminate and initiate distant metastases, we performed experiments of partial resection of the stomach at days 7 and 14 post-orthotopic xenograft of MKN45 luciferase-expressing cells to remove the primary tumor (Supplementary Figure S1). When the resection was performed at day 14 post-xenograft, the mice still developed metastases. However, resection performed at day 7 post-xenograft impaired the development of metastases compared to the non-resected control mouse after 10 days and until 52 days of follow-up (Supplementary Figure S1). Nevertheless, one resected mouse finally developed metastases and tumor in the residual stomach after 28 days, suggesting either an incomplete resection of the primary tumor or circulating tumor cells self-seeding at the primary site, as previously described in breast cancer [27]. These experiments suggest that metastases may arise from the dissemination of GC cells from the primary tumor in the stomach during the first week following orthotopic xenograft.

Metastases Are Enriched in Cancer Stem Cells

The literature suggests that the CSC subpopulation of tumor cells is not only responsible for tumor initiation, growth, chemoresistance, and tumor heterogeneity, but also for tumor dissemination and metastases formation [28]. To evaluate this hypothesis, we analyzed the proportion of CSC in the metastases and gastric tumors of the orthotopic xenograft models. Tumor cells from stomach tumors and micrometastases formed by GC10 PDOX case and MKN45 cell line were harvested and submitted to the in vitro tumorsphere assay (Figure 3A,B). Only CSC can self-renew and initiate tumor

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Cancers 2019, 11, 560 6 of 17 growth in such drastic non-adherent culture conditions [23,25,26]. We observed no differences in tumorsphere formation ability between GC10 cells from the gastric primary tumors and the peritoneal metastases, suggesting the same proportion of CSC in these tumors (Figure 3A). Interestingly, distant metastases, especially those located in the liver, showed higher capacities to initiate tumorspheres than tumors of the stomach (40.5 ± 7.2% versus 19.6 ± 2.6%, respectively) (Figure 3A). The results obtained with this intestinal-type PDX case were confirmed with an opposite case, the diffuse type MKN45 GC cell line, highlighting that the histological subtype and the origin of the GC cells are not at the base of these results. In the MKN45 orthotopic xenograft model, cells from liver metastases formed more tumorspheres than cells from stomach tumors (26.2 ± 1.2% for liver metastases compared to 15.3 ± 1.6% for stomach tumors) (Figure 3B). The expression of CD44, which is one of the best markers of gastric CSC [22], was then analyzed (Figure 3C-E). Flow cytometry analyses showed a significant increase in the percentage of CD44-positive cells in lung metastases compared to the stomach primary tumors for both GC07 and GC10 PDOX cases (Figure 3C,D). Liver metastases formed by GC10 cells also contained significantly more CD44-positive cells than stomach tumors. These results were confirmed by immunohistochemistry analyses on GC10 gastric tumors and metastases (Figure 3E, upper panels). For the MKN45 cell line, in which CD44 expression is very high [22,25], immunohistochemistry analyses revealed a high expression of CD44 in most of the cells in the stomach primary tumors, peritoneal tumors, and lung and liver metastases (Figure 3E, bottom panels). Altogether, these results show that the metastases formed in PDOX models are enriched in CSC.

BKM120 Inhibits GC Proliferation and Decreases the Cancer Stem Cell Population and Properties

BKM120 Inhibits GC Proliferation and Decreases the Cancer Stem Cell Population and Properties

The PI3K pathway is involved in cell proliferation, apoptosis, cytoskeletal rearrangement, and metabolism [13]. Buparlisib (BKM120) is a pan-class I PI3K inhibitor that has shown an efficiency regarding the proliferation on GC cell lines in vitro [29][30][31]. As expected, BKM120 inhibited in a dose-dependent manner the phosphorylation of serine 473 of its downstream target, AKT (P-AKT Ser473 ), which validates the inhibitory effect of BKM120 on PI3K activity and its downstream targets (Supplementary Figure S2). Moreover, as previously reported [31], we observed a dose-dependent cell proliferation arrest of BKM120-treated MKN45 cells (Figure 4A). Cell cycle analyses revealed that the percentage of cells in G0/G1 phase decreased from 54% (control DMSO) to 28% and 13% in cells treated with 3 µmol/L and 10 µmol/L of BKM120, respectively (Figure 4B). An inversely proportional blocking in G2/M phase was observed in BKM120-treated cells (Figure 4B). This was associated to an increased expression of the cyclin-dependent kinase inhibitor p21, the DNA damage-inducible protein GADD45A, and the programmed cell death protein 4 (PDCD4) (Figure 4C), which are involved in cell cycle arrest. On the contrary, BKM120 reduces the expression of the proliferating cell nuclear antigen PCNA and the transcription factor E2F1 involved in cell cycle progression (Figure 4C).

Then, the effect of BKM120 was evaluated on the CSC subpopulation of GC. The properties of CSC to form tumorspheres and their expression of the CSC marker CD44 were evaluated in vitro in the non-adherent culture conditions of the tumorsphere assay. To overcome a potential bias related to the cytostatic effect of BKM120, we quantified both the ability of cells to form tumorspheres (Figure 4D,E), but also the proportion of CD44-positive cells in the residual tumorspheres (Figure 4F,G). The addition of BKM120 to the culture medium during cell seeding prevented the formation of MKN45 and GC10 tumorspheres in a dose-dependent manner (Figure 4D,E, respectively). MKN45 tumorsphere formation was particularly affected, with the number of tumorspheres decreasing from 81.1 ± 4.1% in the control DMSO-treated condition, to 67.2 ± 6%, 38.9 ± 3%, and 4.1 ± 0.5% at 1 µmol/L, 3 µmol/L, and 10 µmol/L of BKM120, respectively (Figure 4D). More importantly, at ~IC 50 corresponding to 3 µmol/L of BKM120, the proportion of CD44-positive cells decreased by ~twofold (Figure 4F,G) (84.1 ± 2.5% in control

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Cancers 2019, 11, 560 8 of 17 DMSO; 42.9 ± 9.8% in BKM120-treated tumorspheres). Altogether, these results demonstrate for the first time that treating GC cells with the pan-class I PI3K inhibitor BKM120 displays cytostatic effect in adherent conditions and decreased the number of CSC and their tumorigenic properties in vitro in a dose-dependent manner.

BKM120 Limits Tumor Growth and Dissemination In Vivo

To further investigate the effect of BKM120 on tumor growth and metastatic process in vivo, MKN45 orthotopically xenografted mice were treated with BKM120. Daily oral gavages started eight days after the xenograft of MKN45 cells into the stomach, when tumors became detectable in the stomach, but before metastasis detection by bioluminescence imaging (Figure 5A). One day before the first treatment, mice were randomized into two groups based on the level of photon emission by gastric tumor cells (Figure 5A, upper panels).

BKM120 Limits Tumor Growth and Dissemination In Vivo

To further investigate the effect of BKM120 on tumor growth and metastatic process in vivo, MKN45 orthotopically xenografted mice were treated with BKM120. Daily oral gavages started eight days after the xenograft of MKN45 cells into the stomach, when tumors became detectable in the stomach, but before metastasis detection by bioluminescence imaging (Figure 5A). One day before the first treatment, mice were randomized into two groups based on the level of photon emission by gastric tumor cells (Figure 5A, upper panels). In the control group, the growth of gastric tumors increased continuously and significantly from nine days of treatment with the vehicle, whereas no significant increase in gastric tumor growth was observed in the BKM120-treated group for up to 17 days of treatment (Figure 5B). At the end point, corresponding to three weeks of treatment, the group of BKM120-treated mice presented significantly ~3.8-fold less bioluminescent signals in the whole animal compared to the vehicle-treated control group (55.9 ± 24.8 versus 213.0 ± 78.4 respectively, p < 0.05) (Figure 5B). Importantly, tumor cells grew in the stomach of all the animals, but the frequency of mice bearing metastases in the lung, the liver, the spleen, and the pancreas was lower in the BKM120-treated mice compared to the vehicle-treated controls (Table 3). BKM120 treatment reduced the frequency of metastases by 12.5% in the lung (7/9 vs. 9/9), 27.8% in the liver (4/8 vs. 7/9), and 41.6% in the spleen and pancreas (2/8 vs. 6/9). Altogether, these results showed that BKM120 limits tumor growth, dissemination, and metastases formation in vivo. Table 3. Number of tumor-bearing mice per total NSG mice implanted with MKN45 and treated with BKM120 and vehicle (DMSO-treated mice). 1 Tumor development was determined by photon emission on organs at end point.

Number of Tumor-Bearing Mice

Discussion

Mouse models of H. pylori infection and/or carcinogens-induced gastric carcinogenesis do not develop invasive and metastatic GC as in humans. The best model to study the efficiency of new therapeutic strategies on primary GC and CSC remains PDX mouse models, with the limitation that tumors growing subcutaneously do not metastasize [22,32]. Furukawa et al. pioneered the PDOX nude mouse model with an orthotopic implant using the intact tissue of GC maintained by serial transplantation [33,34]. This technique using tissue samples has the disadvantage of not controlling In the control group, the growth of gastric tumors increased continuously and significantly from nine days of treatment with the vehicle, whereas no significant increase in gastric tumor growth was observed in the BKM120-treated group for up to 17 days of treatment (Figure 5B). At the end point, corresponding to three weeks of treatment, the group of BKM120-treated mice presented significantly ~3.8-fold less bioluminescent signals in the whole animal compared to the vehicle-treated control group (55.9 ± 24.8 versus 213.0 ± 78.4 respectively, p < 0.05) (Figure 5B). Importantly, tumor cells grew in the stomach of all the animals, but the frequency of mice bearing metastases in the lung, the liver, the spleen, and the pancreas was lower in the BKM120-treated mice compared to the vehicle-treated controls (Table 3). BKM120 treatment reduced the frequency of metastases by 12.5% in the lung (7/9 vs. 9/9), 27.8% in the liver (4/8 vs. 7/9), and 41.6% in the spleen and pancreas (2/8 vs. 6/9). Altogether, these results showed that BKM120 limits tumor growth, dissemination, and metastases formation in vivo. 1 Tumor development was determined by photon emission on organs at end point.

Discussion

Mouse models of H. pylori infection and/or carcinogens-induced gastric carcinogenesis do not develop invasive and metastatic GC as in humans. The best model to study the efficiency of new

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Cancers 2019, 11, 560 10 of 17 therapeutic strategies on primary GC and CSC remains PDX mouse models, with the limitation that tumors growing subcutaneously do not metastasize [22,32]. Furukawa et al. pioneered the PDOX nude mouse model with an orthotopic implant using the intact tissue of GC maintained by serial transplantation [33,34]. This technique using tissue samples has the disadvantage of not controlling the number and the viability of the tumor cells present within the grafted tissue. Then, this experiment was reproduced using GC cell lines, which makes it possible to control the number and viability of grafted tumor cells, but reflects the heterogeneity of patients' tumors less [35,36]. Here, we generated orthotopic injections of suspensions of PDX-derived cells transduced with the luciferase-encoding gene in NSG mice. Tumor growth was followed by in vivo bioluminescence imaging, as previously reported with GC cell lines [37,38]. Experiments resulted in primary tumor growth in the stomach, which was followed in three cases by the dissemination and formation of distant metastases in the same location as in humans (peritoneal cavity and liver), as well as in the lung, which is rare in humans for GC. Metastases in the liver occurred in 79% of NSG mice xenografted with GC10 and in 19% of mice xenografted with GC07. We also showed that not all PDX and GC cell lines can grow in the gastric subserosa and disseminate to form metastases. The diffuse GC06 case did not grow in the orthotopic xenograft experiments, although we previously showed that it grows and develops tumors after subcutaneous xenograft in the same way and at a similar kinetic as GC07 and GC10 cases [22]. In addition, the percentage of CD44+ cells in GC06 is high, reaching about 50% of the tumor cells, as previously reported [22,25]. Thus, its inability to form gastric tumors and metastases cannot be attributed to its diffuse, mesenchymal-like and CSC-enriched phenotype. Moreover, the diffuse GC cell line MKN45 developed stomach tumors and metastases, reinforcing the hypothesis that the histological subtype of GC is not directly responsible for their ability to grow and metastasize in the stomach or not. Thus, we hypothesized that the capacity of GC06 to grow could be linked to its dependency on certain extrinsic factors of the microenvironment in this immunodeficient mouse model, allowing its growth subcutaneously but not in the stomach. In our experiments, the NCI-N87 cell line generated tumors in the stomach in 60% of cases, but did not metastasize. However, Feng et al. reported lymph node and liver metastases in mice injected with 5 × 10 6 NCI-N87 cells [38]. We can hypothesize that a high number of cells during injection may increase tumor growth in the stomach and the frequency of metastatic development, but it could also increase the risk of leakage and unspecific dissemination related to the injection of so many cells. In our experiments, the risk of leakage was limited by the low volume of injection and the low number of injected cells (1 × 10 4 cells) compared to other studies. In addition, the time-course experiments of gastric tumor resections demonstrated that metastases are initiated during the two first weeks after GC cell xenograft in the stomach. Interestingly, in one of the gastric tumor-resected mice, the presence of tumor cells was observed in the residual stomach at sacrifice. This result should indeed be confirmed in more animals, but suggests either an incomplete tumor resection or the self-seeding of circulating tumor cells (CTC) in the primary niche. Indeed, Kim et al. demonstrated that the dissemination of cells is not unidirectional, and that aggressive CTC in the peripheral blood of patients with breast cancer, colon cancer, and melanoma can colonize their tumors of origin, being at the origin of local recurrence seeded by disseminated cells following ostensibly complete tumor excision [27].

The tumorigenic and invasive properties of CSC are closely associated to their intrinsic and extrinsic plasticity and to EMT. Brabletz et al. first described migrating CSC at the invasive front of tumors [39]. CSC properties, including tumorsphere formation capacity and CD44 high expression profiles can be acquired through the induction of EMT in breast tumors [28,40] and in GC, as we have previously shown [41]. In GC, previous studies have reported that CD44-positive cells generate gastric tumors with a higher frequency than CD44-negative cells when implanted in the stomach [42]. However, the authors did not evaluate the development of metastases in these animals. Moreover, GC patients having CD44-positive CTC are more prone to develop metastases and recurrence than patients having CD44-negative CTC [43,44]. In agreement with these studies, we show here that the proportion of CSC is increased in metastases compared to the gastric tumors formed by GC cell lines

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Cancers 2019, 11, 560 11 of 17 and patient-derived GC cases, suggesting that CSC may drive CTC dissemination and the formation of metastases. Further studies are required to fully characterize the subpopulation of CSC-derived CTC that is able to initiate metastases and may be self-seeding following surgical resection of the primary gastric tumor.

We previously showed that targeting the AMP-activated protein kinase / mammalian target of rapamycin (AMPK/mTOR) pathway, downstream of PI3K signaling, with metformin decreased the CSC population and their tumorigenic properties in GC [26]. The PI3K pathway controls cell proliferation, apoptosis, cytoskeletal rearrangement, and metabolism [13], and is being more and more linked to CSC maintenance and plasticity. Interestingly, in the molecular classification of Lei et al. distinguishing GC into proliferative, metabolic, and mesenchymal subtypes [6], tumors of the mesenchymal phenotype, which are enriched in cells with features of CSCs, are particularly sensitive to PI3K/AKT/mTOR inhibitors in vitro [6]. BKM120 is a pan-class I PI3K inhibitor, acting as a competitive ATP inhibitor targeting the catalytic activity of p110α, β, δ, and γ isoforms with the same affinity, and exhibiting its activity with or without mutation in the PIK3CA. BKM120 has shown an efficiency alone or in combination with other chemotherapies in several phase I and phase II clinical trials in patients with advanced and refractory solid tumors [45][46][47], advanced leukemia [48], ovarian and breast cancer [49], endometrial carcinoma [50], prostate cancer [51], head and neck squamous cell carcinoma [52], and in breast cancer with phase III clinical trials [53]. No clinical trials have been published concerning BKM120 in GC patients, and only three articles have shown that BKM120 inhibits the proliferation on GC cell lines in vitro [29][30][31]. In GC patients, AKT Ser-473 phosphorylation, which is a direct downstream target of PI3K, is correlated with tumor progression and a poor clinical outcome [54]. In this study, we confirmed that BKM120 treatment reduced GC cell proliferation in vitro and strongly inhibited P-AKT Ser473 . More originally, we show for the first time that BKM120 decreased the population of CD44-positive CSC and their tumorigenic properties in vitro both in GC cell lines and more importantly in patient-derived GC cases. Using the original orthotopic xenograft models that we developed, we showed that BKM120 treatment in vivo led to a significant reduction of tumor growth and a reduction in the number of metastases-bearing mice. On a more basic point of view, the resection experiments carried out before the metastasis initiation period further validated our orthotopic xenograft models as pertinent for evaluating CSC-based anti-tumorigenic and/or anti-invasive therapeutic strategies.

Materials and Methods

Cancer Cell Lines and Patient-Derived Xenograft Cell Culture

All the cell lines were mycoplasma-free (verified by PCR). MKN45 and MKN74 gastric cancer cell lines, authenticated by short tandem repeats profiling, were cultured in RPMI 1640-Glutamax supplemented with 10% heat-inactivated fetal bovine serum (10% FBS) and 50 U/mL of penicillin and 50 µg/mL of streptomycin (1:100 P/S) at 37 • C in a 5% CO 2 humidified incubator (all from Thermo Fisher Scientific, Villebon sur Yvette, France). The NCI-N87 cell line was cultured in DMEM F12-Glutamax supplemented with 10% FBS and 1:100 P/S. Cell viability was measured using trypan blue dye exclusion 48 h and 72 h after treatment. GC04, GC06, GC07, and GC10 are patient-derived GC cells successfully established by serial subcutaneous xenografts in non obese diabetous/severe combined immunodeficiency/interleukin-2Rγ null (NSG) mice, as previously described [22]. The status for Epstein-Barr Virus (EBV) was determined by molecular methods on tumor tissue sections (in situ hybridization) and on DNA extracted from PDX tumors according to standard procedures of the Haut-Lévèque hospital of University Hospital Center of Bordeaux; all GC cases were EBV-negative. Mutations have been searched using the NGS Colon and Lung Panel for KRAS, EGFR, BRAF, PI3KCA, AKT1, ERBB2, PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBX7, FGFR3, NOTCH1, ERBB4, FGFR1, and FGFR2. GC04 is PIK3CA mutated (c.1633G > A p. (Glu545Lys)), GC06 is KRAS mutated (c.175G > A p. (Ala59Thr) and c.38G > A p. (Gly13Asp)), GC07 is mutated for STK11 (c.1019A > G p. (Tyr340Cys)) and GC10 is mutated for TP53 (c.818G > A p. (Arg273His)).

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Cancers 2019, 11, 560 12 of 17 When the tumor size reached between 200-400 mm 3 , tumors were recovered and dissociated with a human tumor dissociation kit in C tubes and a GentleMACS dissociator following manufacturer recommendations (all from MACS, Miltenyi, Paris, France). Cells were filtered successively though 70 µm and 40 µm cell strainers, and red blood cells were lysed. Then, cells were processed for tumorsphere formation, flow cytometry, and lentiviral transduction, or were xenografted in mice.

Production of Lentiviral Particles and Transduction of Cells

Gene transfer of the luciferase protein was done using a pRRLsin-MND-Luc-WPRE lentiviral vector. Particles were produced by the vectorology platform of Bordeaux University. Dissociated PDX cells were transduced with a multiplicity of infection (MOI) of 30 lentiviral particles per cells overnight in DMEM F12-Glutamax medium containing 10 mM of Hepes in a 5-mL polypropylene tube. Twelve h later, cells were rinsed and prepared for subcutaneous xenograft amplification before orthotopic experiments. For the MKN45 cell line, adherent cells were transduced with pRRLsin-MND-Luc-IRES2-ZsGreen-WPRE lentiviral particles (MOI 30) in DMEM F12-Glutamax medium containing 10 mM of Hepes for 24 h.

Orthotopic Xenograft and Resection Experiments

Five-week-old NSG female mice from the Level 2 animal facility of the University of Bordeaux were maintained in accordance with the institutional guidelines of the institutional animal use and care committee (accreditation number B33-063-916, received on 23 May 2016). All of the animal experiments were approved by the French Ethics Committee on Animal Experiments CEEA50 of Bordeaux (Authorization n • A12005, ref 2017103118319700 v7).

Mice received an intraperitoneal (i.p.) injection of 300 µg/kg buprenorphine (Vetergesic, Centavet, Dinan, France) 30 min before laparotomy. Animals were anesthetized with isoflurane in sterile condition, and a 6-mm incision was made on the right side of the animal to expose the stomach. Then, 10,000 cells from the GC cell lines and PDXs cells, suspended in 30 µL of DMEM F12-Glutamax containing 1% P/S and 10% FCS, were injected into the subserosa using a 500-µL syringe supplied by a 30-gauge needle (Terumo, Guyancourt, France) under a dissection microscope. The peritoneal incision and the skin incision were sutured (5.0 Ethicon, Vicryl). The wound was disinfected with a spray of chlortetracycline (Cyclo spray, Centavet, Dinan, France). For the primary tumor resection experiment, cells were suspended in 20 µL of DMEM F12-Glutamax containing 1% P/S and 10% FCS and 7 mg/mL ice-cold matrigel (Ozyme, Montigny-le-Bretonneux, France). Seven and 15 days after orthotopic xenograft, the stomach was exposed by laparotomy and the primary tumor was excised following similar experimental surgical procedures as done during a sleeve gastrectomy.

Bioluminescence Imaging

Mice were monitored for up to 25 weeks by in vivo bioluminescence imaging (Biospace Lab, Nesles-la-Vallée, France) upon i.p. injection of 3.3 mg of D-Luciferin (Promega, Charbonnières-les-Bains, France). At the end point, mice were sacrificed, and organs were macroscopically analyzed and collected for histological analyses. In the luciferase-expressing models, photon emission on recovered organs was measured after their incubation for 5 min in a solution of 3.3 mg/mL D-luciferin.

Immunohistochemistry

Tissues were fixed 16 h in 3.7% formol and processed for paraffin embedding. Then, 3 µm tissue sections were deparaffinized in xylene and rehydrated in graded alcohol. For immunohistochemistry experiments, antigen enhancement was performed on slides in boiled citrate buffer ph6. Primary antibodies human anti-HLA class 1 (1:100e, Abcam 70328, Cambridge, UK) and human anti-CD44 (1:100e, BD Bioscience 550392, Allschwil, Switzerland) were incubated respectively for 1 h and 2 h at room temperature (RT). Then, slides were incubated with Avidin/Biotin blocking reagent (SP-2001, Vector Laboratories, Burlingame, CA, USA) followed by successive incubations of biotinylated horse

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Cancers 2019, 11, 560 13 of 17 anti-mouse Immunoglobulin G incubation and VECTASTAIN ® Elite ® ABC HRP reagent according to manufacturer' recommendations (Vector Laboratories, Burlingame, CA, USA). Immunolabeling was revealed in liquid substrate diaminobenzidine chromogen (DAKO). Slides were counterstained with hematoxylin, dehydrated, and mounted with an Eukitt-mounting medium. Slides were scanned with Pannoramic Scan (3D Histech, Budapest, Hungary).

PI3K Inhibitor Preparation and Treatments

BKM120, a pan-class I PI3K inhibitor, was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). For the in vitro experiments, BKM120 was prepared as 10 mmol/L stock solution in DMSO and conserved at -20 • C for ≤2 months. Adherent cultured cells were treated for 48 h to 72 h one day after seeding with a medium change. Three-dimensional cultured cells were treated during the seeding of the cells to analyze tumorsphere formation. Tumorspheres were treated 4 days after seeding to analyze CD44 expression. For the in vivo experiments, BKM120 was dissolved in 10% NMP (1-methyl-2-pyrrolidone) and 90% PEG300 (all from Sigma-Aldrich). Vehicle treatment consisted of a solution containing 10% NMP and 90% PEG300. Solution was administrated by oral gavage at 20 mg/kg once per day on a schedule of five days on, two days off schedule for three weeks. The GC10 PDX cell and MKN45 gastric cancer cell lines treated with BKM120 were PIK3CA wild-type.

Tumorsphere Culture and Quantification

MKN45 cells were cultured in serum-free DMEM F12-Glutamax medium containing 1:100 P/S, 0.3% glucose, N2 supplement (all from Thermo Fisher Scientific), 20 ng/mL of human epidermal growth factor, 20 ng/mL human basic growth factor and insulin 5 µg/mL (Sigma-Aldrich) onto non-adherent 96-well culture plates (previously coated with a 10% poly-2-hydroxyethyl methacrylate (Sigma-Aldrich) solution in 95% (v/v) ethanol and dried overnight at 56 • C). PDX cells (GC07 and GC10) were cultured in tumorsphere medium supplemented with 2% FBS. The formed tumorspheres, ≥50 µm, were counted 7 days post-seeding and treatment with BKM120. Images of tumorspheres were taken using a ZOE TM Fluorescent Cell Imager (Bio-Rad, Irvine, CA, USA).

Flow Cytometry and Cell Cycle Assay

MKN45 were grown for 4 days in non-adherent conditions and were then treated with 3 µmol/L of BKM120 or DMSO (control) for 48 h. Tumorspheres were dissociated with Trypsin-Ethylenediaminetetraacetic acid (EDTA) and rinsed with ice cold-buffer containing 2 mM of EDTA and 0.5% bovine serum albumin (BSA) in PBS. Cells were stained with 1:25 CD44-APC antibodies (G44-26 clone, BD Biosciences) or isotype controls for 25 min at 4 • C. Cells were rinsed three times with cold buffer and were processed for flow cytometry. Dead cells were excluded based on side scatter analysis and 7-Aminoactinomycin D (50 µg/mL, BD Pharmingen, 559925) negative staining. For cell cycle analysis, 24-h adherent cells were treated for 48 h with increased concentrations of BKM120 and DMSO as control. Cells were dissociated by trypsinization and were stained for 1 h in cold fluorochrome solution containing 50 µg/mL of propidium iodide, 0.1% (w/v) sodium citrate and 0.1% triton (v/v). Flow cytometry was performed using a BD FACSCanto II instrument and DIVA software (BD Biosciences).

RNA Extraction and RT-qPCR

Total RNA was extracted with TRIzol TM reagent (Thermo Fisher Scientific) following the manufacturer's instructions. Reverse transcription was performed using the Quantitech Reverse Transcription kit (QIagen, Venlo, The Netherlands) following the protocol provided from the manufacturer. Real-time PCR was performed using the SYBR-qPCR-Premix Ex-Taq (Takara, Shiga, Japan) and 0.3 µmol/L of specific primers (Supplementary Table S1). Amplified RNA samples were calculated using the 2 -∆∆CT method with HPRT1 and TBP as normalizers.

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Protein Isolation and Western Blotting

Treated and control whole cells (48 h) were washed twice with cold PBS and lysed in RIPA buffer containing a cocktail of protease inhibitor (P8340), phosphatase inhibitors (P0044 and P5726), and 1 mM of phenylmethylsulfonyl fluoride supplemented with 5 mmol/L of EDTA (all from Sigma-Aldrich). The lysate was centrifuged (13,000 rpm, 15 min at 4 • C), and the supernatant was stored at -80 • C until further use. Total proteins (25 µg) were electrophoresed by an 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked in 5% BSA in PBS-Tween 20 0.1% and probed overnight at 4 • C with primary antibodies to P-AKT ser473 (CST 4060), AKT (CST 4685) and GAPDH (Sc-25778). Then, IRDye 800-conjugated goat anti-rabbit secondary antibody (LI-COR, Inc., Lincoln, NE, USA) was diluted 1:5000 and probed for 60 min at room temperature. Antibody-bound proteins were detected using ChemiDoc™ Imaging Systems (Bio-Rad).

Statistical Analysis

The results are expressed as the mean ± SEM of three independent experiments. Statistical tests were performed using GraphPad Prism Version 5.0 (GraphPad, San Diego, CA, USA), using Mann-Whitney, Student's t-test or ANOVA with Bonferroni as the post-hoc test and the Kruskal-Wallis test for multiparametric comparisons. A 5% cutoff was used to validate the result significance.

Conclusions

Using these new models, we demonstrated that BKM120 treatment targets gastric CSCs and leads to a significant reduction in the number of metastases-bearing mice. The development of these new preclinical models offers the opportunity to decipher the basic mechanisms of CSC dissemination and study the efficiency of drugs targeting gastric CSC to block their growth and, most importantly, their metastatic spread, which is responsible for most of the GC-related deaths. These models could also be useful to further study and characterize the subpopulation of CSC-derived CTC initiating metastases, and their putative self-seeding mechanisms following surgical resection of the primary gastric tumor. Table S1. List of primers used for RT-qPCR analysis.

Gene

Sequence Frame (5′-3′) Sequence Reverse (5′-3′)

E2F1 GGAACTGAGGCCTGGGTGAT CCCATGGCTGTCAGTCAGTCT GADD45 GCAGGATCCTTCCATTGAGA CTCTTGGAGACCGACGCTG HPRT1 TGGTCAGGCAGTATAATCCA GGTCCTTTTCACCAGCAAGCT P21 CCTCATCCCGTGTTCTCCTTT GTACCACCCAGCGGACAAGT PDCD4 CAGTTGGTGGGCCAGTTTATT AGAAGCACGGTAGCCTTATCCA PCNA AGGGCTCCATCCTCAAGAAGG TGGTGGTTCAAATACTAGCGC TBP TGCACAGGAGCCAAGAGTGAA CACATCACAGCTCCCCACCA

Introduction

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide [1,2]. GCs are mostly non-cardia gastric adenocarcinomas, occurring in patients around 70 years old. In most situations, GC has an infectious origin, attributed to the infection by the bacterium Helicobacter pylori. Half of the world's population is infected with H. pylori, and this infection is detected in more than 92% GC cases [3,4]. Infection with H. pylori always leads to chronic inflammation of the gastric mucosa, which can potentially evolve slowly into atrophy, metaplasia, and dysplasia, and in the worst scenario leads to non-cardia gastric carcinoma after several decades [5]. The major H. pylori virulence factor is carried by the cag pathogenicity island (cagPAI), which encodes a type 4 secretion system (T4SS) and the cytotoxin associated gene A (cagA) oncoprotein (CagA). When CagA is injected into gastric epithelial cells via the T4SS, it can induce perturbations of several signaling pathways, leading to the destabilization of inter-cellular junctions, polarity complexes, and pro-inflammatory responses [6]. Previous studies, including those performed in our laboratory, have shown that H. pylori induces an epithelial-mesenchymal transition (EMT) of epithelial cells of the gastric mucosa. EMT is a very well-known pathophysiological trans-differentiation process that confers mesenchymal phenotype and properties to epithelial cells. In the gastric context, this EMT is characterized by the loss of epithelial polarity and cellular junctions and the acquisition of a mesenchymal, motile phenotype called the "hummingbird" phenotype [7][8][9][10]. The overexpression of zinc finger E-box-binding homeobox 1 (ZEB1) and Snail transcription factors and of structural components such as Vimentin, as well as migration and invasion capacities are reminiscent events of the EMT process. EMT also occurs during cancer dissemination to allow cell extravasation through blood vessels and dissemination to distant organs, thereby initiating metastases [11]. EMT can also lead to the emergence of cells with cancer stem cell (CSC) properties in different cancers including GC [12][13][14]. CSCs represent a rare cell subpopulation within the tumor that is able to initiate tumor development and dissemination to form distant metastases. CSCs are more resistant to conventional chemotherapy than the more differentiated tumor cells and can be identified by the expression of immaturity markers such as cluster of differentiation 44 (CD44) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) in GC [15][16][17]. Their recent discovery in GC [15,[17][18][19] is a very promising research axis, allowing an earlier detection of the cells at the origin of CSC in pre-neoplastic lesions, as well as the development of CSC-based targeted therapies [20,21].

Several pathways, including the Hippo signaling pathway, have been described to control CSC properties. The Hippo pathway, a highly conserved signaling pathway, from fruits flies to humans, is involved in physiology in the modulation of organ size during development and the maintenance of stemness, especially in the gastrointestinal tract. Its dysregulation, in pathological conditions, can lead to cancer emergence and progression [22][23][24][25]. The Hippo pathway is controlled by upstream regulators that activate a module of inhibitory kinases, which in turn inhibits a transducer module composed of oncogenic co-transcription factors. Upstream regulators involve components of cell/cell junctions, polarity complexes, and extracellular matrix stiffness, all acting on the regulation of the inhibitory kinases, including two serine/threonine kinases: Mammalian sterile 20-like kinase-1/2 (MST1/2) and its target the large tumor suppressor kinase 1/2 (LATS1/2). When the Hippo pathway is activated, LATS1/2 is phosphorylated, which in turn phosphorylates its downstream targets yes-associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ) on serine residues, resulting in their sequestration in the cytoplasm and subsequent degradation by the proteasome [25][26][27][28]. When the Hippo pathway is inactivated, YAP and TAZ are not phosphorylated by LATS1/2 and can therefore accumulate in the nucleus and bind to transcription factors such as the TEA domain (TEAD) transcription factor family members, their main partners. The resulting complexes activate transcriptional programs inducing cellular plasticity, proliferation, or drug resistance [29].

Recent work from our laboratory showed that the Hippo kinase LATS2 controls H. pylori-induced EMT and intestinal metaplasia [30]. YAP1/TEAD-mediated transcription is activated first following H. pylori infection and then repressed later while LATS2 accumulates. LATS2 appears to be a protective factor, limiting the loss of gastric epithelial cell identity that normally precedes neoplastic transformation and GC development. The role of YAP has been widely demonstrated in cancer initiation and progression [25][26][27], including GC [31][32][33]. Its paralogue TAZ has also been implicated in

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Cells 2020, 9, 1462 3 of 21 aggressiveness and metastasis in different cancers [34][35][36][37][38][39] and recent literature shows its involvement in GC aggressiveness, metastasis, and CSC properties [40][41][42]. In GC xenograft models, inhibition of YAP/TAZ interaction with TEADs by the pharmacological inhibitor verteporfin inhibits the tumorigenic properties of CSCs in GC [43]. TAZ is overexpressed in 66.4% GC [40], in which its overexpression is correlated with lymphatic metastasis and tumor stage [44]. In GC cell lines, studies have shown that TAZ controls cell migration, and its overexpression is associated with EMT [40,42]. Until now, the role of TAZ has never been investigated in response to H. pylori infection in the context of EMT and early steps of gastric carcinogenesis.

This study aimed to highlight TAZ implication in H. pylori-induced EMT and the gastric carcinogenesis process, both in vitro and ex vivo. For this purpose, we explored TAZ regulation in gastric epithelial cells in response to H. pylori infection and the consequences of its inhibition by interference RNA strategies on H. pylori-induced EMT and CSC-like invasive and tumorigenic properties. Furthermore, the expression of both TAZ and the major EMT marker ZEB1 were evaluated in GC patient-derived tissues and mouse models.

Materials and Methods

Cell and Bacterial Culture and Co-Culture

MKN45 (RIKEN, BRC Cell Engineering Division, Japan) and MKN74 (HSRR Bank JCRB0255) were cultivated in Roswell park memorial institute (RPMI) 1640 Glutamax medium (Thermo Fisher Scientific, Courtaboeuf, France), and NCI-N87 (ATCC CRL-5822), Kato-III (ATCC HTB-103), and GC07 were cultivated in Dulbecco's modified eagle medium (DMEM-F12) medium (Thermo Fisher Scientific), as previously described [12,15,45]. GC07 was derived from a patient-derived GC xenograft (PDX), which was successfully established by serial transplantation in non-obese diabetous severe combined immunodeficiency Interleukin 2 gamma null (NSG) mice, as previously described [15]. All cells were grown in medium supplemented with 10% heat-inactivated fetal bovine serum (Eurobio, Les Ulis, France) and 50 µg/mL vancomycin (Sandoz, Holzkirchen, Germany) at 37 • C in a 5% CO 2 humidified atmosphere. The experiments were performed with cells seeded at low density (3 × 10 4 cells per well in 24 well plates). Phase contrast microscopy images of cell cultures were taken using the ZOE fluorescent Cell Imager (BioRad, Marnes-la-Coquette, France).

The cagA-positive 7.13 wild type (WT) H. pylori strain was used for most of co-culture experiments. Several H. pylori strains were used: 7.13 WT strain and its isogenic mutant deleted for cagA (∆cagA) or cagE (∆cagE) (from Richard Peek, Vanderbilt University Medical Center, Nashville, TN, USA) and the P12 WT strain and its isogenic mutant deleted for cagA (∆cagA) or the cagPAI (∆cagPAI) (from Rainer Haas, Ludwig Maximilian University of Munich, Munich, Germany). H. pylori were cultivated on Columbia agar plates supplemented with 10% human blood under microaerophilic (5% O 2 ) conditions at 37 • C in humidified atmosphere, as previously described [8,13]. Co-culture experiments were performed at a multiplicity of infection of 1:25. The evaluation of bacterial number was estimated by spectrophotometry with OD 600nm = 1 corresponding to 2 × 10 8 bacteria/mL. DNA was extracted for gastric tissues and PCR were performed for the detection of H. pylori and cagA, as previously described [46].

Transfection of siRNA

In order to downregulate TAZ expression, we transfected cells with small interfering RNA (siRNA) designed to target human TAZ. The siRNA suspension was composed of Lipofectamine RNAiMAX (Thermo Fisher Scientific), OptiMEM medium (Thermo Fisher Scientific), and 20 nM of siRNA. Experiments were performed with three different siRNA that target TAZ: siTAZ1 (5 -ACGUUGACUUAGGAACUUU-3 , Eurofins, Dortmund, Germany) [47], siTAZ2 (5 -AGGUACCUUC CUCAAUACA-3 , Eurofins) [47], and siTAZ3 (5 -UGUGGAUGAGAUGGAUACA-3 , Eurofins). siTAZ1 and siTAZ2 are specific of TAZ sequences. siTAZ3 recognizes a common sequence of both TAZ

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Cells 2020, 9, 1462 4 of 21 and YAP mRNA. A negative control siCtrl (siCtrl 5 -GGGCAAGACGAGCGGGAAG-3 , Eurofins) with no known homology to mammalian genes was used. Two rounds of siRNA transfection were performed (separated by 24 h) in medium without antibiotics. Co-culture experiments were performed 6 h after the last transfection. Analyses were performed 30 h after the last transfection and 24 h after infection [13,30]. TAZ inhibition efficiency was evaluated by Western blot experiments.

TEAD-Luciferase Reporter Assay

TEAD reporter assay was performed by co-transfecting the Renilla luciferase reporter (pRL-SV40, Promega) and either the TEAD reporter (8xGTIIC-Luciferase reporter, gift from Stefano Piccolo [25]) or TATA box control (TAL, Becton Dickinson, Le Pont de Claix, France) firefly luciferase reporters to evaluate the relative TEAD activity. The transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific) and 12.5 ng of Renilla vector, 125 ng of pTA-Luciferase vector, or 125 ng of pTEAD-Luciferase vector. After 24 h, cells were lyzed and the luminescence quantification was measured using the Dual-Luciferase Reporter Assay system according to the manufacturer's recommendations (Promega, Charbonnières-les-bains, France). Firefly luciferase activities were normalized for transfection efficiency by Renilla luciferase for each sample, and TEAD-specific luciferase activity was then normalized to TAL activity.

((

Luci f erase Renilla ) pcDNA TEAD 8xGTIIC-Luciferase/( Luci f erase Renilla ) pcDNA pTA-Luciferase).

RNA Extraction and RTqPCR

Total RNA was extracted using TRIZOL reagent (Thermo Fisher Scientific) according to the manufacturer's recommendations and quantified by their absorbance at 260 nm. Reverse transcription was performed using the QuantiTect Reverse Transcription kit (Qiagen, Courtaboeuf, France) containing a DNA digestion step. Quantitative PCR was performed using specific primers at 0.3 µM and the SYBR-PCR-Premix Ex-Taq reagent (Thermo Fisher Scientific) (Table 1). The amplification steps contain 15 s at 94 • C, 30 s at 60 • C, and 30 s at 72 • C for 45 cycles. Relative expressions were calculated using the comparative cycle threshold (Ct) method with hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-Box binding protein (TBP) as normalizers. Primers used were: Cysteine-rich angiogenic inducer 61 (CYR61), Matrix metallopeptidase 9 (MMP9), Snail family transcriptional repressor 1 (SNAI1), Vimentin (VIM), and ZEB1. Table 1. RT-qPCR primers sequences used in this study.

Genes

Sequence Forward (5 -3 ) Sequence Reverse (5 -3 )

CYR61 ATGAATTGATTGCAGTTGGAAA TAAAGGGTTGTATAGGATGCGA HPRT1 TGGTCAGGCAGTATAATCCA GGTCCTTTTCACCAGCAAGCT MMP9 Quantitect primer assay, cat. QT00040040 SNAI1 ACAATGTCTGAAAAGGGACTGTGA CAGACCAGAGCACCCCATT TBP GGGCATTATTTGTGCACTGAGA GCCCAGATAGCAGCACGGT VIM GGATGCCCTTAAAGGAACCAA CAACGGCAAAGTTCTCTTCCAT ZEB1 TCCCAACTTATGCCAGGCAC CAGGAACCACATTTGTCATAGTCAC

Western Blot

After having been washed twice with phosphate-buffered saline (PBS), cells were lysed on ice in ProteoJET reagent (Thermo Fisher Scientific) supplemented with protease/phosphatase inhibitor cocktail (Sigma-Aldrich, St. Quentin Fallavier, France). The extracted proteins were submitted to SDS-polyacrylamide gel electrophoresis (BioRad, Marnes-la-Coquette, France) and Western blotting on nitrocellulose membranes (BioRad) for immuno-labelling. After 1 h in bovine serum albumin (BSA)-blocking solution (5%), immuno-labelling was performed using mouse anti-TAZ (1:2000, cat. 560235, BD Pharmingen, Thermo Fisher Scientific), rabbit anti-YAP1 (1:2000; cat. 4912 Santa

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Cells 2020, 9, 1462 5 of 21 Cruz Biotechnology, Heidelberg, Germany), mouse anti-Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) (1:2000; sc-47724 Santa Cruz Biotechnology), and mouse anti-α-tubulin (1:8000; cat. T-6074, Sigma-Aldrich) for 1-2 h at room temperature. Fluorescent-labelled secondary antibodies (1:2000; StarBright 700 goat anti-mouse cat. 12004158 or StarBright 700 goat anti-rabbit 12004161, BioRad) were used to detect the primary antibodies and were detected by fluorescence imaging with the BioRad Chemidoc imager. Band intensities were normalized to GAPDH or α-tubulin, and were quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) [48].

Immunofluorescence and Quantification of the Stainings

Experiments were performed as previously described [13,30] on glass coverslips at a density of 2.5 × 10 4 cells per well. Cells were fixed in a 4% paraformaldehyde solution in PBS supplemented with 1 mM CaCl 2 and 1 mM MgCl 2 for 10 min, and permeabilized in 0.1% Triton in Tris-buffered saline (TBS) supplemented with 1 mM CaCl 2 and 1 mM MgCl 2 for 1 min. After blocking with 1% bovine serum albumin and 2% fetal calf serum solution in TBS, cells were incubated with the primary antibodies mouse anti-TAZ (1:100, cat 560235, BD Pharmingen) and rabbit anti-ZEB1 (1:100, cat. A301-922, Bethyl, Souffelweyersheim, France) for 1 h at room temperature. After 3 washes in PBS solution, fluorescent-labelled secondary antibodies were incubated for 1 h at room temperature using either donkey anti-mouse Alexa-488-labelled (1:250, cat. A21202 Thermo Fisher Scientific), goat anti-rabbit Alexa-546-labelled (1:250, cat. A11010 Thermo Fisher Scientific), or goat anti-rabbit Alexa-488-labelled (1:250, cat. A32731, Thermo Fisher Scientific) secondary antibodies mixed with either Alexa-546-labelled phalloidin (1:250, cat. A22283, Thermo Fisher Scientific) or Alexa-647-labelled phalloidin (1:200, cat. A222287, Thermo Fisher Scientific) and 4 -6-diamidino-phenyl-indol (DAPI) (50 µg/mL, cat. D9542, Sigma-Aldrich). After being washed 3 times in PBS and having 1 last wash in distilled water, the coverslips were mounted on glass slides using Slowfade reagent (cat. S36936, Thermo Fisher Scientific). Images were taken using a ×40 (numerical aperture, 1.3) oil immersion objective on an Eclipse 50i epi-fluorescence microscope (Nikon, Champigny sur Marne, France) with Nis Element acquisition software. Fiji software was used for the relative quantification of the signal intensity on digital images [48]. The relative amount of TAZ and ZEB1 nuclear accumulation was quantified in the nucleus of each cells by delimiting nuclei using DAPI staining and quantifying the mean fluorescence intensity (pixels shade of grey) of TAZ and ZEB1 labelling with Fiji software [48].

In some experiments, the number of positive-vs. negative-labelled nuclei for MKN45 cells was determined. Nuclei were considered positive when the mean fluorescence intensity was higher than the mean fluorescence intensity of the uninfected cells plus two standard deviations.

Tumorsphere Assay

After 24 h of H. pylori infection and 30 h after the last siRNA transfection, we seeded 100 cells (MKN45 and NCI-N87) or 1000 cells (GC07) in non-adherent 96-well culture plates that were previously coated with a 10% poly-2 hydroxyethyl methacrylate (Sigma-Aldrich) solution in 95% (v/v) ethanol and dried overnight at 56 • C. The medium composition is serum-free GlutaMAX-DMEM/F12 medium supplemented with 20 ng/mL of epidermal growth factor, 20 ng/mL of basic-fibroblast growth factor, 0.3% glucose, 5 µg/mL of insulin, and 1:100 N 2 supplement (all from Thermo Fisher Scientific and Sigma Aldrich), and cultured at 37 • C in a 5% CO 2 humidified atmosphere [13,43]. Tumorspheres were counted 7 days after seeding under non-adherent conditions.

Invasion Assay System

After 24 h H. pylori infection and 30 h siRNA-transfection, we recovered cells by trypsinization, and 3 × 10 4 cells per condition were seeded in the upper side of 8 µm pored Transwells (Sigma-Aldrich) previously coated with rat-tail type 1 collagen (Becton Dickinson) [13]. After they were incubated for 18 h at 37 • C, inserts were fixed in 4% paraformaldehyde-PBS solution and labelled with DAPI.

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Cells 2020, 9, 1462 6 of 21 Cells from the upper part of the Transwell inserts were removed by swabbing. Cells that had invaded through the lower side of the inserts were counted using the ZOE fluorescent Cell Imager (BioRad).

Immunohistochemistry

Tissue sections that were 3 µm thick were prepared from formalin-fixed, paraffin-embedded human and mice tissues and were processed for immunohistochemistry procedures as previously described [12,30]. Primary antibodies rabbit anti-TAZ (1:80 for 1 h incubation, Sigma-Aldrich; HPA007415), rabbit anti-ZEB1 (1:800 during 2 h incubation for mice tissues and 1:100 during 2 h incubation for human tissues, Bethyl; A301-922A), mouse anti-Ki67 (1:150 during 30 min incubation for mice tissues, 652,401 clone 16A8, BioLegend, San Diego, CA, USA), or mouse anti-Ki67 (1:75 during 30 min incubation for human tissues, M7240 clone mib-1, Dako, Les Ulis, France). Secondary horseradish peroxidase (HRP)-labelled ImmPRESS anti-rabbit or anti-mouse antibodies for TAZ and ZEB1, ImmPRESS anti-rat for Ki67 from BioLegend, and VECTASTAIN Elite avidin-biotin complex (ABC) HRP kit (Peroxidase, Universal) for human Ki67 from Dako were incubated for 30 min. Immunolabeling was revealed after a 1-10 min incubation in liquid diaminobenzidine-chromogen substrate. Slides were counterstained with hematoxylin, dehydrated, and mounted with Eukitt-mounting medium (VWR, Fontenay-sous-Bois, France). Quantification scores of the relative expression of ZEB1, Ki67, and nuclear TAZ in gastric epithelial cells were determined in a blind lecture using a scale from 0 to 4 with the following criteria: 0: no staining, 1: <5% of positive epithelial cells; 2: 5 to <25% of positive cells; 3: 25 to <50% of positive cells; 4: ≥50% of positive epithelial cells.

Ethic Statements on Human and Mouse Tissue Samples

Studies on paraffin-embedded tissue samples from gastric adenocarcinoma patients were performed in agreement with the Direction for Clinical Research and the Tumour and Cell Bank of the University Hospital Centre of Bordeaux (Haut-Leveque Hospital, Pessac, France), as previously reported [13,15]. Animal experiments were performed in level 2 animal facilities of the University of Bordeaux (France), in conformity with the French Ministry of Agriculture Guidelines on Animal Care and the French Committee of Genetic Engineering and with the approval of institutional guidelines determined by the local Ethical Committee of the University of Bordeaux (approval number 4608), as described previously [30].

Statistical Analysis

Quantification values represent the mean of three or more independent experiments, each performed in duplicate or more ± standard error of the mean (SEM). Kruskal-Wallis test with Dunn's post-test or ANOVA with Dunnett's post-test were used to compare multiple groups. Statistics were performed using GraphPad Prism 8 software (San Diego, CA, USA).

Results

H. pylori Induced TAZ Overexpression and TAZ/TEAD-Dependent Transcriptional Activity

In order to select the most pertinent gastric epithelial cell lines to study TAZ implication in response to H. pylori infection, we performed a Western blot to evaluate TAZ expression compared to that of its paralogue YAP in several cell lines commonly used to study H. pylori-induced signal transduction in a gastric carcinogenesis context (Figure 1A). The MKN45 cell line showed the highest TAZ/YAP expression ratio among the different cell lines, because only TAZ was expressed and not YAP. The NCI-N87 cell line and the GC07 cell line established from patient-derived GC xenografts showed a high TAZ/YAP ratio and were also selected to perform the following experiments (Figure 1B) [45]. Then, kinetics of TAZ protein expression and TAZ/YAP-mediated TEAD transcriptional activity were performed in these three gastric cell lines in response to infection with the cagA-positive 7.13 wild type (WT) H. pylori strain (Figure 1C,D). The results showed that H. pylori stimulated both TAZ protein expression and TEAD transcriptional activity at 1-2 h post-infection in MKN45 and GC07, and at 2 h post-infection in NCI-N87. This activation was transient and was followed by a return to basal TEAD transcriptional activity at 5 h in all cell lines. After 24 h of infection, TEAD transcriptional activity remained upregulated in MKN45 but repressed in GC07 cells and to a lesser extent in NCI-N87.

infection, TEAD transcriptional activity remained upregulated in MKN45 but repressed in GC07 cells and to a lesser extent in NCI-N87.

A relevant indicator of YAP/TAZ activation is their subcellular localization. Their nuclear localization is associated with their ability to bind nuclear transcription factors such as TEADs to stimulate transcription of target genes. Immunofluorescence staining showed that the basal level of TAZ in cells is weak and localized mostly in the cytoplasm but also in the nucleus of some cells, especially in MKN45 cells (Figure 2A, arrows). Upon H. pylori infection, we clearly observed a nuclear accumulation of TAZ at 24 h post-infection in the three cell lines, which tended to start as from 2-5 h post-infection in GC07 and NCI-N87 cells (Figure 2A,B). All together, these results show that infection with the 7.13 WT H. pylori strain increased TAZ protein expression, nuclear accumulation, and activation of the TAZ-TEAD transcriptional complex activity. A relevant indicator of YAP/TAZ activation is their subcellular localization. Their nuclear localization is associated with their ability to bind nuclear transcription factors such as TEADs to stimulate transcription of target genes. Immunofluorescence staining showed that the basal level of TAZ in cells is weak and localized mostly in the cytoplasm but also in the nucleus of some cells, especially in MKN45 cells (Figure 2A, arrows). Upon H. pylori infection, we clearly observed a nuclear accumulation of TAZ at 24 h post-infection in the three cell lines, which tended to start as from 2-5 h post-infection in GC07 and NCI-N87 cells (Figure 2A,B). All together, these results show that infection with the 7.13 WT H. pylori strain increased TAZ protein expression, nuclear accumulation, and activation of the TAZ-TEAD transcriptional complex activity.

TAZ and ZEB1

Nuclear Co-Overexpression in H. pylori-Infected Gastric Epithelial Cells Was Associated with EMT and Was CagA-Dependent Kinetic co-culture experiments with H. pylori also showed the progressive acquisition of the mesenchymal "hummingbird" phenotype at 5 h and 24 h post-WT H. pylori infection in MKN45 and to a lesser extent in NCI-N87 cells, as previously reported [8,13,30] (Figure 2A,C and Figure S1A). This mesenchymal phenotype was also observed for the first time in our in-house patient-derived GC07 cell line, in which it was induced as early as 2 h and continued until 24 h post-infection (Figure 2A,C).

To confirm this EMT-like process at the molecular level, we studied the expression and subcellular localization of the EMT marker ZEB1 in parallel to that of TAZ by co-immunofluorescence microscopy over time following 7.13 WT H. pylori infection (Figure 2A). A diffuse labelling of ZEB1, mainly in the cytoplasm, was observed in the uninfected basal condition. After 24 h infection, ZEB1 expression was increased and accumulated in the nuclei of the three cell lines, with a concomitant nuclear localization of TAZ, particularly visible in GC07 and MKN45 cells harboring the mesenchymal "hummingbird" phenotype at 5 h and 24 h post-infection (Figure 2A,B). These results show the concomitant TAZ and ZEB1 expression and nuclear localization in cells having undergone EMT upon H. pylori infection (Figure 2B).

In order to determine which H. pylori-virulence factor was involved in this phenomenon, we performed co-culture experiments of MKN45 cells with the WT or the isogenic mutant of H. pylori 7.13 and P12 strains deleted for either cagA (∆cagA), or a part of the cagPAI (∆cagPAI), or cagE gene, encoding for a scaffolding protein necessary for the T4SS assembly (∆cagE) [49] (Figure S1B-D). Both ∆cagPAI and ∆cagE strains failed to assemble a functional T4SS. The ∆cagA strains have a functional T4SS but cannot express the CagA oncoprotein. Western blot analysis of coculture experiments showed that only the WT strains of H. pylori were able to increase TAZ protein expression (Figure S1B). These results were confirmed by RTqPCR assays showing that only the WT strains were able to increase the expression of CYR61, one of the main TAZ-TEAD target genes, suggesting that this effect could be CagA-dependent. The expression of the EMT markers Vimentin, ZEB1, and Snail, as well as the induction of the "hummingbird" phenotype after 24 h of infection, were also CagA-dependent, as previously reported [8,13] (Figure S1D,E). Altogether, these results show that the H. pylori-induced increase in TAZ expression and activity occurred concomitantly to EMT and could be CagA-dependent.

confirming our in vitro results. Moreover, the nuclear overexpression of TAZ and ZEB1 was detected in poorly differentiated proliferative cells in the isthmus region of H. pylori-infected gastric mucosa, which is niche for regenerative epithelial progenitor cells. These stem-like cells are stimulated upon infection to allow tissue regeneration and repair of the epithelial damages induced by chronic H. pylori infection and inflammation [13,30,46]. This proliferative region is characterized by a marked Ki67 staining that is more abundant in H. pylori-infected conditions compared to uninfected controls.

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Cells 2020, 9, 1462 10 of 21 negative-labelled nuclei for MKN45 cells (cells were considered positive when their mean fluorescence intensity was higher than that measured for the uninfected cells plus two standard deviations) and by measuring TAZ and ZEB1 nuclear intensity staining with Fiji software [48] To confirm these results in vivo, we evaluated TAZ and ZEB1 expression and subcellular localization by immunohistochemistry on serial tissue sections of gastric mucosa from uninfected and H. pylori-infected mice and patients, as well as human gastric carcinoma. Gastric tissue samples from a previously described mouse model of H. pylori-induced gastric carcinogenesis were used, corresponding to C57BL/6 mice experimentally infected with the pro-inflammatory H. pylori strain HPARE that has a functional cagPAI and cagA [12,13,30,46]. In mice, as in humans, chronic H. pylori infection leads to a sequence of pre-neoplastic lesions of the gastric mucosa evolving with time from chronic gastritis to metaplasia, dysplasia, and gastric carcinoma (gastric intraepithelial neoplasia (GIN) in mice) [5,46]. We previously reported that ZEB1 and other EMT markers were upregulated during this sequence of pre-neoplastic lesions induced by H. pylori in mice and humans; they remained upregulated in human gastric carcinoma [13,30,46]. Here, we show that TAZ and ZEB1 are strongly upregulated with a nuclear localization in gastric epithelial cells of H. pylori-infected gastric mucosa in mice and humans, when compared to healthy uninfected mucosa (Figure 3A,B,D), thus confirming our in vitro results. Moreover, the nuclear overexpression of TAZ and ZEB1 was detected in poorly differentiated proliferative cells in the isthmus region of H. pylori-infected gastric mucosa, which is niche for regenerative epithelial progenitor cells. These stem-like cells are stimulated upon infection to allow tissue regeneration and repair of the epithelial damages induced by chronic H. pylori infection and inflammation [13,30,46]. This proliferative region is characterized by a marked Ki67 staining that is more abundant in H. pylori-infected conditions compared to uninfected controls.

In human gastric carcinoma, nuclear TAZ and ZEB1 were strongly upregulated compared to healthy gastric mucosa (Figure 3C,D). Notably, the expression and localization of TAZ and ZEB1 were heterogeneous-the center of the tumor masses was less stained than the periphery, where an overexpression of both TAZ and ZEB1 was noted (Figure 3C). An important amount of Ki67-labelled proliferating cells was observed at the tumor periphery and the invasive fronts of the tumors where TAZ and ZEB1 were overexpressed. This suggests an important role for TAZ and ZEB1 in tumor invasion capacities and progression. These ex vivo results confirm those observed in vitro, i.e., that cagA-positive H. pylori strains induce a co-overexpression of TAZ and ZEB1 in gastric epithelial cells that is associated with a dedifferentiation process; their detection within areas of proliferating cells suggest their implication in both GC initiation and progression. and GC07. Apart from siTAZ1 in MKN45 cells, we observed no effect for the different siTAZ used on basal invasion properties of MKN45 and GC07 cells without H. pylori stimulation. In the context of H. pylori-induced gastric carcinogenesis, EMT has been associated with the acquisition of CSC-related properties by gastric cell lines, such as the capacity to form tumorspheres in vitro [12,13]. To address the role of TAZ in H. pylori-induced CSC-like functional properties, we performed a tumorsphere formation assay (Figure 4E,F and Figure S2E). The drastic culture conditions of this assay (non-adherent, defined culture media without fetal bovine serum) allows determination of the activity of stem cells, which represent a small subpopulation of cells within the cell lines, which will be the only ones able to survive and self-renew under these conditions. In MKN45, H. pylori infection increased the number of tumorspheres as previously reported (Figure 4E,F) [13]. This result was confirmed in NCI-N87 cells and to a lesser extent in GC07 cells (Figure 4F and Figure S2D). Remarkably, TAZ inhibition by the three siTAZ showed a full inhibition of the H. pylori-induced increase in tumorsphere number in the three cell lines (Figure 4F). There was no effect of siTAZ on the tumorsphere number under basal conditions in MKN45 and NCI-N87 cells, except for siTAZ-3 in NCI-N87. This result indicates that TAZ is not required for self-renewal capacity under basal conditions, but is mandatory in response to H. pylori stimulation in order to increase CSC-like tumorigenic properties.

Altogether, these results highlight the role of TAZ in promoting EMT and the acquisition of invasive and tumorigenic properties related to CSCs in the context of gastric carcinogenesis induced by H. pylori infection.

Discussion

H. pylori infection leads to dysregulation of gastric epithelial cell homeostasis by modulating or hijacking several cellular signaling pathways. All these modifications associated with those induced by chronic inflammation and remodeling of the gastric mucosa microenvironment contribute to the initiation and development of gastric carcinogenesis. We previously reported that cagA-positive carcinogenic strains of H. pylori upregulate the expression of the Hippo tumor suppressor LATS2 and its oncogenic target YAP in a biphasic kinetic manner [30]. Other studies have also reported an upregulation of YAP in response to H. pylori [50,51]. Although YAP and TAZ are paralogues, most studies in the field of H. pylori and GC have focused on YAP in such a way that the direct implication of TAZ remained unexplored in the context of H. pylori-induced gastric carcinogenesis. Results of this study show for the first time that TAZ is upregulated and accumulates in the nuclei of gastric epithelial cells in response to H. pylori infection where it binds to its main partners, the TEAD transcription factors. This TAZ nuclear overexpression is associated with the TAZ/TEAD oncogenic ~262~ Cells 2020, 9, 1462 15 of 21 transcriptional program illustrated by the increase in CYR61 expression (Figure S1C). CYR61 is an extracellular matrix protein involved in cell adhesion, proliferation, and migration, whose expression has been correlated with poor survival of cardia gastric adenocarcinoma patients; it can be considered a metastatic biomarker for this cancer [52]. This TAZ overexpression and TAZ/TEAD activity seems to occur in a CagA-dependent manner, concomitantly with the ZEB1 overexpression and EMT program. TAZ silencing experiments have shown that TAZ is a major regulator of ZEB1 expression and of the EMT program induced by CagA-positive H. pylori strains, and that it controls the induction of invasive and tumorigenic properties.

We also showed that TAZ is over-expressed in the gastric mucosa of human and mice infected with H. pylori. In both cases, TAZ nuclear overexpression was detected in regenerative areas with Ki67-positive epithelial cells. These areas, in the isthmus region of the gastric glands, correspond to an expansion of stem and progenitor cells that are mobilized to ensure epithelium regeneration in response to chronic injury caused by H. pylori infection [7,53,54]. Indeed, we previously showed that LATS2 is also overexpressed and nuclear in the same regenerative areas induced by chronic H. pylori infection in vivo [30,55].

According to previous study results from our laboratory and others [8,30,53], H. pylori induces an increase in EMT markers (ZEB1, Vimentin, Snail, MMP9) and leads to a progressive nuclear translocation and accumulation of ZEB1 protein in the nucleus 5-24 h post H. pylori infection in vitro. In vivo, ZEB1 is also overexpressed and nuclear in the stem/progenitor cell regenerative zone of the gastric mucosa in H. pylori-infected mice and humans [8,13].

Recent studies concerning EMT state of epithelial cells and their plasticity suggest not only an ON/OFF EMT model, but also a multilayer of EMT features induced by epigenetic events and dysregulation of signaling pathways, called the EMT hybrid state [56,57]. This body of growing evidence of EMT plasticity is well-illustrated in our cellular models of gastric cell lines, with those harboring an epithelial/mesenchymal hybrid state being more prone to a H. pylori-induced EMT-like process and acquisition of CSC-like properties. In the context of H. pylori infection, E-cadherin expression is sufficient to counteract H. pylori-mediated mesenchymal phenotype [58], and on the contrary, downregulation of E-cadherin or other cell-cell junction proteins such as IQ-domain GTPase-activating protein 1 (IQGAP1) predispose cells to overexpress ZEB1 and undergo EMT [12,59]. MKN45 and GC07 PDX-derived cells are poorly cohesive. MKN45 cells have a mutated cdh1 gene encoding a truncated and non-functional E-cadherin [60]. This may be the most likely cause of their hybrid epithelial/mesenchymal state, which makes them prone to undergoing an EMT-like process more easily than the E-cadherin functional polarized NCI-N87 cells that constrain the EMT-like process induced by H. pylori, as we previously reported [8].

Mechanistically, ZEB1 is a transcriptional repressor of epithelial differentiation genes, such as cdh1 encoding the E-cadherin adherent junction protein and other cell polarity complex genes, promoting the trans-differentiation characterized by a more or less important loss of epithelial integrity and the acquisition of a highly motile mesenchymal phenotype and invasion properties in cancer [61]. In the gastric carcinogenesis context, experiments on ZEB1 inhibition with siRNA have shown that ZEB1 is essential to the EMT induced in response to H. pylori and controls the basal mesenchymal phenotype of some GC cell lines [8,62]. In GC, ZEB1 overexpression is associated with mesenchymal type tumors that have a poor prognosis [62]. In addition to promoting EMT, ZEB1 can also act by repressing the expression of the stemness-inhibiting miR-200b microRNA conferring stemness properties, thereby linking motility and stemness towards a migrating CSC phenotype [8,13,61,63,64]. Its role in the emergence of CSC-like cells with invasive and tumorigenic properties has been demonstrated in several cancers including breast cancer [14] and GC [8,13,62], in which its inhibition by siRNA repressed CSC-like phenotype and properties. Consequently, ZEB1 has been considered as a major actor of cancer cell plasticity in tumor initiation, progression, and metastatic dissemination. A recent study shows that ZEB1, which is mostly known as a transcriptional repressor, could also act as a transcriptional activator by directly interacting with YAP to stimulate the expression of a common set of ZEB1/YAP ~263~ Cells 2020, 9, 1462 16 of 21 target genes, including CYR61, which is predictor of metastatic risk and poor survival in aggressive breast cancer [64]. However, Lehmann et al. reported in this study that ZEB1 could directly bind to YAP but not to its paralogue TAZ, due to structural differences between YAP and TAZ-TAZ possesses only one WW domain in the central part of the protein, contrary to YAP that has two WW domains. To our knowledge, the capability of TAZ to bind to ZEB1 has never been investigated in GC. In the study by Lehmann et al., the use of siYAP and siTAZ did not alter ZEB1 expression in aggressive breast cancer cell lines. On the contrary, TAZ inhibition using a lentiviral short hairpin RNA (shRNA) strategy reduced ZEB1 expression in the retinal pigment epithelial cell line (RPE) [65]. We previously showed that these RPE cells are sensitive to H. pylori infection and respond, like gastric epithelial cells, through an EMT-like transition involving YAP/TAZ/TEAD activation [30].

One of the remaining questions is how H. pylori can activate TAZ, leading to ZEB1 upregulation and induction of an EMT-like process. Different pathways and hypotheses could be associated with TAZ activation in this context. In breast cancer, the protein kinase C (PKC) is a major inducer of the EMT process [66]. H. pylori, via the T4SS, activates protein kinase C (PKC), which is responsible for c-Abl phosphorylation leading to its cytoplasmic retention via binding to 14-3-3 protein, promoting EMT-like cell elongation and migration [67]. The c-Abl kinase can phosphorylate TAZ on tyrosine residues, leading to its activation [68]. The link between PKC, c-Abl, and TAZ activation remains to be investigated in the context of H. pylori-mediated EMT and gastric carcinogenesis. Other studies have shown that H. pylori, via CagA, causes the disruption of the epithelial barrier function by altering the apical junction complex structure by recruiting the tight-junction scaffolding protein Zonula occludens-1 (ZO-1) [7]. ZO-1 and ZO-2 are negative regulators of TAZ by sequestering it at tight junctions and preventing its nuclear translocation and transcriptional activity [69]. We can hypothesize that H. pylori, via CagA-induced delocalization of ZO-1 from tight junctions, could release TAZ from the tight junctions and allow its activation and nuclear translocation to participate to EMT. Another pathway linking H. pylori-induced signaling to TAZ activation could be the Wnt/β-catenin pathway, activated in response to CagA [59,70]. CagA signaling induces the inhibition of Glycogen synthase kinase 3 beta (GSK-3β) [71] and β-catenin destruction complex, and also the disruption of E-cadherin/β-catenin complexes at cell junctions [72], both leading to β-catenin release and nuclear accumulation, and to a transcriptional program promoting gastric carcinogenesis [73]. Independently of the Hippo pathway, TAZ degradation also depends on phosphorylated β-catenin that bridges TAZ to its ubiquitin ligase Beta-transducin repeat containing E3 ubiquitin protein ligase (β-TrCP). Upon Wnt signaling, activated β-catenin impairs β-TrCP mediated TAZ ubiquitination and degradation, leading to concomitant nuclear accumulation of β-catenin and TAZ [47]. This scenario could contribute to explain the nuclear accumulation of TAZ observed in response to H. pylori infection in this study, especially in E-cadherin-deficient gastric cell lines with an epithelial/mesenchymal hybrid state.

In vivo, the nuclear co-overexpression of TAZ and ZEB1 observed in the isthmus regenerative stem and progenitor cell area of the gastric mucosa in H. pylori-infected mice and humans is consistent with the TAZ-dependent ZEB1 overexpression and EMT-like process observed in vitro in response to H. pylori infection. In vitro, loss-of-function experiments with siTAZ not only resulted in a severe inhibition of EMT marker expression, but also strongly impaired the invasive and tumorigenic properties of gastric cell lines. The nuclear co-overexpression of TAZ and ZEB1 was also detected at the invasive front of the tumors on GC tissue sections, suggesting that TAZ could constitute a marker of invasion and aggressiveness in GC. TAZ expression is a prognostic indicator in colorectal cancer and is required for metastatic activity and chemoresistance of CSC in breast cancer [34,35,38]. In GC, TAZ was reported as a biomarker of aggressiveness as it is associated with the metastatic status of GC patients, and is highly expressed in diffuse type GC associated with a mesenchymal phenotype [40,41]. Furthermore, we recently showed that inhibition of YAP/TAZ interaction with TEADs using Verteporfin inhibits CSC tumorigenic and invasive properties in vitro, as well as tumor growth in GC xenograft models [44], confirming that TAZ, like YAP, could be an interesting biomarker and target in GC.

INTRODUCTION

Gastric cancer, mainly composed of gastric adenocarcinoma (GC) represents the fourth leading cause of cancer deaths worldwide 1 . It is frequently detected too late, at advanced metastatic stages. Current treatment is based on surgery with conventional chemo and radiotherapies, but for unresectable metastatic GC cases, therapeutic options are usually limited to palliative chemotherapy with a five-year survival rate of less than 4%. GC displays molecular heterogeneity, driven by genetic and epigenetic modifications 2 , which likely gives rise to subpopulation of cells with different tumorigenic and chemoresistance properties. At the top of cellular hierarchy within tumours, the so-called cancer stem cells (CSC) give rise via asymmetrical division and differentiation properties to the more or less differentiated cells composing the heterogeneous tumour mass. CSCs biomarkers are diverse and depend on the organs of origin of the tumours. Nevertheless, CD44 transmembrane glycoproteins are one of the most relevant CSCs markers in solid cancers 3 including GC 4-6 . However, antibodies used to detect CD44 in these studies recognize epitopes located in constant regions of CD44 (panCD44) and cannot discriminate CD44 isoforms (CD44v). Human CD44 proteins are encoded by a single gene containing 19 exons, among which 12 exons can ~277~ be alternatively spliced to give rise to hundreds of CD44v proteins 7 . CD44 receptor displays multiple roles in cell-cell and cell-extracellular matrix adhesion, proliferation and migration.

CD44v have been described to act as co-receptors for growth factors and as platforms for matrix metalloproteinase activity and growth factor precursor cleavage 8 . CD44 exon v3 contains a unique and conserved 9 heparan sulfate (HS) consensus motif, located in the extracellular domain of the receptor, for attachment to HS proteoglycans. This domain can bind to HS-dependent growth factors, such as hepatocyte growth factor, heparin-binding epidermal growth factor-like, basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor 10-12 thereby increasing their local concentration for signal transduction. These properties are critical for CD44v3-containing isoforms' capacity to control cell survival, migration, tumour progression and metastatic dissemination 13-15 . The expression of panCD44 correlates with poor prognosis in patients with intestinal type GC 16,17 . Lau et al. have shown that CD44v8-10 was the predominant CD44 variant expressed in GC cells and can be a gastric CSCs marker 17 . However, panCD44 or CD44v8-10 are simply markers allowing the enrichment of gastric CSCs since only a fraction of these cells can form tumorspheres in vitro and tumours in vivo in xenograft models 4,6,17 , highlighting the need to identify more specific CSCs markers. CD44v3 is much less expressed than panCD44 and CD44v8-10 in adult tissues and is up-regulated in human colon tumours and metastases 15 . CD44v3 is expressed by CSCs in human oral squamous cell carcinoma 18 and head and neck squamous cell carcinomas (HNSCC) 19 . Nonetheless, the clinical significance of CD44v3 expression in GC was reported only in few studies with controversial results 20-22 , and no information is available regarding its functional role in gastric CSCs. variable exon) and the epithelial isoform CD44E (expressed in epithelia, containing the variable exons v8 to v10 and also named CD44v8-10), were detected, even at low levels, both in normal healthy mucosa of the stomach (fundus and antrum) and in GC cell lines and PDX-derived cells while CD44v3 expression was restricted to GC (Supplementary Fig. S1B).

CD44v3+ cells correspond to a subpopulation of CD44+ cells that is rare in healthy gastric epithelium, but present in GC

The expression of CD44v3 and panCD44 (corresponding to all CD44 isoforms) was evaluated in tissue micro arrays (TMA) composed of stage I-IV GC and paired non-tumoral gastric mucosa from 137 GC patients using immunohistochemistry (Fig. 1A-B). CD44v3+ cells were rare in the healthy gastric epithelium and detected in only 9.1% of cases compared to CD44, detected in 65% of cases (Fig. 1B). In preneoplastic lesions, CD44v3+ cases progressively increased to reach 55.1% in intestinal metaplasia and 60.6% in GC compared to CD44 which was detected in more than 96% of cases as from the stage of intestinal metaplasia. These results show that CD44v3 expression remained rare, and was detected later and in more advanced preneoplastic lesions than CD44, suggesting that it could be a more relevant biomarker of disease progression than CD44 (Fig. 1B).

The expression of CD44v3 and panCD44 was then evaluated in cells dissociated from gastric mucosa tissues, GC cell lines and PDX-derived cells by flow cytometry (Fig. 1C-G). The detection of Epithelial Cell Adhesion Molecule (EpCAM) was used to selectively analyse epithelial cells from gastric tissues. In healthy gastric mucosa, CD44v3+ epithelial cells were absent in fundus and rare in antral mucosa (<1% of cells), whereas panCD44+ epithelial cells represented 4% of cells in fundus and 14% in antrum (Fig. 1C-D). In GC, CD44v3 was corresponding to proliferative and more epithelial MET-like CSCs expressing ALDH activity 29 . These EMT-like and MET-like signatures 29 were analysed in these GC transcriptomic analyses (Fig. 3C). In GC, we previously reported that gastric CSC express CD44, CD166 (ALCAM), CD24, and ALDH activity 4,27 which was confirmed here in panCD44+ versus panCD44-cells but not in CD44v3+ versus CD44v3-cells. EMT-likeassociated transcripts such as those coding for TGF-β1, Vimentin, ZEB1 and ZEB2 were enriched in CD44v3+ cells and downregulated in panCD44+ cells. Conversely, MET-like transcripts including those encoding E-cadherin (CDH1), Occludin (OCLN), several Claudins (CLDN) and Desmoplakin (DSP) were increased in panCD44+ cells and diminished in CD44v3+ cells (Fig. 3C). Variations in the expression of a subset of those genes were confirmed by qRT-PCR in MKN45 FACS-sorted CD44v3+ cells compared to CD44v3-cells (Fig. 3D).

The proliferative state of these subpopulations was evaluated. PanCD44+ cells harboured a positive cell cycle progression molecular signature compared to panCD44-cells (upregulation of genes regulating S phase entry, DNA replication and G2/M transition, with downregulation of cyclin-dependent kinases inhibitors), but not CD44v3+ versus CD44v3cells (Fig. 3E). Flow cytometry analyses of cell proliferation confirmed that panCD44+ cells were more proliferative than panCD44-cells (Fig. 3F) whereas CD44v3+ cells were as proliferative as CD44v3-cells (Fig. 3G). Interestingly, in tissues, CD44v3+ GC cells were localized at the invasion front of GC liver metastases, whereas ALDH1+ GC cells were more centrally located within the tumour mass as described by Liu et al in breast cancer for the MET-like CSCs 29 , but both expressed Ki67 proliferation marker (Fig. 3H).

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Flow cytometry experiments confirmed that ALDH+ GC cells were mainly panCD44+ (dark orange bars compared to green bars, Fig. 4A) and CD44v3-(dark pink bars versus green bars, Fig. 4B-C). CD44v3+ cells were mainly ALDH-(light pink versus dark pink bars), and CD44v3+/ALDH+ cells (dark pink bars) accounted only for 1.3±0.2% of GC cells (Fig. 4B-D). We observed the same tendency for breast and colon cancer cell lines, in which a rare subpopulation of CD44v3+/ALDH+ was detected (1 to 5%, respectively, Fig. 4A-B). These results were confirmed on live MKN45 tumorspheres, in which CD44v3+ cells were either ALDH+ (pink arrows) or ALDH-(yellow arrows). These cells were Hoechst 33342-, illustrating their CSCs drug efflux properties as described previously 4,27 ; some Hoechst 33342+/panCD44-/ALDH-/CD44v3-cells were detected (white circle), corresponding to more differentiated non-CSC cells 4,27 (Fig. 4E).

Taken together, these results suggest that the subpopulation of GC cells expressing CD44v3 may correspond to EMT-like CSCs located at the invasive front of tumours while panCD44+ cells may correspond to more proliferative MET-like CSCs within the tumour mass.

CD44v3+ subpopulation of gastric CSC drives metastasis

Since CD44v3+ GC cells harbours an EMT-like signature and CD44v3-containing isoforms have been involved in metastatic progression in breast cancer 30,31 , we next evaluated their invasive and metastatic properties in GC. First, CD44v3 expression was evaluated by immunohistochemistry in orthotopic xenograft mouse models of GC developing distant metastases 25 . An enrichment of CD44v3+ cells in liver and lung metastases was observed compared to primary gastric tumours (Fig. 5A-B). CD44v3 was more strongly expressed in micro-metastases (Fig. 5A) than in bigger metastases, and the percentage of CD44v3+ cells ~288~ was inversely correlated with the size of lung and liver metastases (Fig. 5C). Similar results were obtained for panCD44+ cells (Fig. 5A-B, Supplemental Fig. S4). Secondly, orthotopic xenograft experiments were reproduced with MKN45 FACS-sorted CD44v3+ and CD44v3cells. CD44v3+ cells were more tumorigenic than CD44v3-cells (Fig. 5D) and generated both stomach tumours (10/10) and distant metastases (liver, 9/10; lung, 10/10) (Fig. 5E), whereas CD44v3-cells grew locally in the stomach (8/10) and disseminated at a lower frequency (liver, 3/10; lung, 4/10) (Fig. 5E). Both quantitative bioluminescence imaging and histological analysis (Fig. 5F-G) showed that liver and lung metastases derived from CD44v3+ cells were bigger than those derived from CD44v3-cells while stomach tumours were similar.

In addition, CD44v3+ cells were detected at the invasive front of macro-metastases (Fig. 3H).

Accordingly, circulating tumour cells (CTC) detected in portal venules of mice developing liver metastases expressed CD44v3 (Fig. 5H). Furthermore, this correlated with in vitro observations where CD44v3+ cells isolated by FACS were more invasive (Fig. 5I) and CD44v3 expression inhibition using RNA interference inhibited their invasive properties in collagen-coated Boyden's chamber (Fig. 5I, Supplemental Fig. S5A-C) as well as gelatine degradation properties (Supplemental Fig. S5D).

Altogether, these results suggest that GC metastatic dissemination and colonization of distant organs may originate mainly from CD44v3+ cells.

CD44v3 expression in GC cells is correlated with metastasis in GC patients and a poor overall survival

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The clinical relevance of CD44v3 expression as a putative biomarker of invasiveness and prognosis was explored by immunohistochemistry on a local collection of GC patients tissues using TMA (n=137 cases) (Fig. 6). CD44v3+ expression in GC was neither associated with tumour stage nor with lymph node invasion, but was significantly associated with the metastasis status, CD44v3 expression being detected in 79.2% of metastatic patients compared to 56.3% in non-metastatic patients (OR=2.91, P=0.0201) (Fig. 6A). CD44v3+ GC cells were detected in lymph nodes and liver metastases of GC patients, and CD44v3 expression in GC tumours was significantly associated with a poor 5-years overall survival (P=0.0078, n=91) (Fig. 6B-C).

A previous study demonstrated that CD44v6 is a marker of CSC-driving colon cancer metastasis and that its expression is correlated with poorer prognosis 32,33 . Interestingly, in our study, CD44v3+ cells represented a small sub-population of CD44v6 high cells in GC cell lines and PDX cells (Supplementary Fig. S6A,D). Similar results were observed for colon and breast cancer cell lines with the presence of a rare subpopulation of CD44v3+/v6+ cells, representing less than 10% of CD44v6+ cells (Supplemental Fig. S6B-C) and detected both in primary tumours and metastases of colon and breast cancer patients (Supplemental Fig. S7A-F).

Taken together these results suggest that CD44v3+ cells represent a subpopulation of panCD44+ and CD44v6+ cancer cells in gastric, breast and colorectal cancers, and that the detection of CD44v3+ GC cells could be a marker of aggressiveness and poor prognosis in GC.

RNA-binding proteins involved in RNA alternative splicing were significantly upregulated in CD44v3+ cells and downregulated in panCD44+ cells (supplementary results). Indeed CELF proteins are mainly considered as tumour suppressors. TCGA data showed that CELF2 expression is associated with shorter overall survival in invasive breast carcinoma, low-grade glioma and glioblastoma 45 . Their function in GC has been poorly studied 46 and their function in CD44v3+ cells remains to be investigated.

In patients with colorectal cancer, a subpopulation of CTCs bearing all the functional attributes of CSCs have been described, suggesting that CTCs may derive from CSCs having undergone EMT 33 . CTCs were almost all CD44v6+ and the proportion of CD44v6+/ALDH+ CSCs was higher in CTCs than in primary tumours 33 . These results support a previous study from Todaro et al having demonstrated that CD44v6 is a marker of CSC-driving colon cancer metastasis 32 , probably through its interaction with the c-Met tyrosine kinase receptor.

Similarly, CD44v3 splicing variant, via its HS side chain, concentrates HGF and promotes c-Met signaling 47 . Interestingly, we report here that almost all CD44v3+ cells are CD44v6+ and represent a subpopulation among the CD44v6+ cells, suggesting that the metastatic properties of CD44v6+ CSC-driving colon cancer metastasis could be attributed to the subpopulation of CD44v3+ cells among them.

CD44v3 expression has been associated with tumour progression and a poor prognosis in colon cancer 13 and in HNSCC 19 , but results from GC patients were controversial 20,22 . We demonstrate here that CD44v3 protein expression, while not correlated with advanced stage and lymph node invasion, is significantly correlated with distant metastasis, and that GC patients bearing CD44v3+ tumours have a lower 5-year overall survival.

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Targeting cell surface markers expressed by CSCs appears as an attractive therapeutic option. Proof of concept of CD44 variants targeting efficiency has emerged from previous studies. Reeder et al have shown that reducing the expression of CD44v3 and CD44v6 in colorectal cancer cell lines led to inhibition of liver metastasis in xenograft models 35 . However, Bivatuzumab Mertansine, an anti-CD44v6 antibody-drug conjugated, was stopped in phase I clinical trial for HNSCC due to fatal skin toxicity 48 . Interestingly, CD44v3 is less expressed than CD44v6 in several tissues, including the gastrointestinal tract 15 , and poorly expressed in the gastric mucosa. Targeting CD44v3 might thus be less toxic than targeting CD44v6 and could constitute an interesting strategy, in addition to conventional treatments, to target invasive CSCs and prevent metastatic dissemination in GC.

In conclusion, the subpopulation of GC cells expressing CD44v3 may correspond to EMTlike CSCs with invasive capacity, while panCD44+ GC cells may correspond to more proliferative MET-like CSCs. Further studies are required to assess molecular mechanisms involved in CD44v3 expression as putative targets to prevent metastatic dissemination. Furthermore, detecting CD44v3 expression in primary tumours and maybe blood samples of GC patients would be useful as a prognosis marker and to manage clinical evolution of the disease.

Tissue Microarray conception.

The Tissue Micro Arrays (TMA) were constructed in collaboration with Haut-Lévèque Hospital and the tumor bank of the CHU Bordeaux using formalin-fixed paraffin-embedded tissues (PET) from stomach surgical specimens from 179 patients with GC from 1999 to 2010. The TMA blocks were formed using a Tissue Arrayer MiniCore®3 (Excilone). The areas of interest were selected and cored in triplicate each 1 mm in diameter. Two control tissues were added to each slide, in order to ensure the specificity of the immunostaining: a negative spleen control (no expression of epithelial markers) and a positive duodenum control (expression of epithelial markers and of stem cells markers at the bottom of the crypts). These TMAs include gastric adenocarcinomas of different grade and histological sub-type determined after reanalysis and classification (G. Belleannée, pathologist) according to the last WHO and Lauren's classification guidelines of gastric tumors, as well as non-tumor gastric mucosa distant from the tumor site (limits of exeresis), which may be healthy or involving different pre-neoplastic lesions (chronic atrophic gastritis, intestinal signal extracted using Agilent Feature Extraction software v10.10.1.1 with default parameters.

Microarray data statistical analysis. Microarray data were analysed using R (R Development Core Team, 2008) and Bioconductor packages (www.bioconductor.org, v 3.0) 6 . Raw data (median signal intensity) were filtered, log2 transformed, corrected for batch effects (microarray washing bath serials) and normalized using quantile method 7 . A model was fitted using the limma lmFit function 8 . For panCD44 negative vs positive contrast specific to GC04 or GC10 patients, we fitted 2 models including the "source_tri" as a blocking factor. To investigate common signatures across multiple sources (patient, cell line) the expression data from 3 replicated "tri" in each of GC04 and GC10 patient were averaged. Then 3 models were fitted considering "source" as blocking factor to extract respectively panCD44 negative versus positive, CD44v3 negative vs positive signatures. A correction for multiple testing was applied using Benjamini-Hochberg procedure 9 for False Discovery Rate (FDR). Probes with FDR ≤ 0.05 were considered to be differentially expressed between conditions. Hierarchical clustering was applied to the samples and the differentially expressed probes using 1-Pearson correlation coefficient as distance and Ward's criterion for agglomeration. The clustering results are illustrated as a heatmap of expression signals for which the maximum distance method was used for the clustering of individuals.

Orthotopic tumour model and bioluminescence imaging. Detailed surgery procedures and bioluminescence imaging were previously described 10 . Briefly, mice received 300 μg/kg buprenorphine (Vetergesic, Centavet) 30 min before laparotomy. Animals were anesthetized with isoflurane in sterile condition, incision was made on the right side of the mouse and 30 µl of medium containing cells were injected into the sub-serosa. Mice were monitored up ~307~ twice a week by in vivo imaging (Biospace Lab, France) upon intraperitoneal injection of 3.3 mg D-Luciferin (Promega, Charbonnières-les-Bains, France). At end point, mice were sacrificed, and organs were macroscopically analysed and collected for histological analyses.

In MKN45 expressing luciferase, photon emission on organs was measured after 5 minutes incubation in a solution of 3 mg/mL D-luciferin.

SUPPLEMENTARY RESULTS

Enrichment in mRNA corresponding to panCD44 and CD44v3-containing isoforms was verified in FACS-sorted cells by qRT-PCR (Supplementary Fig. S3C-D). mRNA transcripts of 2,845 probes were increased in panCD44+ cells and 310 in CD44v3+ cells compared to their respective negative counterparts (Fig. 3A and Supplementary Tables S4 andS5). Among them, only CD44 and KPNA2 genes were in common (Supplementary Table 6). Similarly, S1. List of primers used for qRT-PCR analysis.

SUPPLEMENTARY TABLES

Supplementary Table

Gene

Sequence Frame (5'-3') Sequence Reverse (5'-3')

ALDH1A1 CAAGATGTCTGGAAATGGAAGAGA TGACTGTTTTGACCTCTGTATATTCATG CLDN3 GAGGGCCTGTGGATGAACTG AGCGAGTCGTACACCTTGCA CD24 TTCTCCAAGCACCCAGCA TGGAATAAATCTGCGTGGGTA CD44 CGGACACCATGGACAAGTTT GAAAGCCTTGCAGAGGTCAG CD44E AGAATCCCTGCTACCAATATGGACTC AGGTCACTGGGATGAAGGTC CD44S CATCTACCCCAGCAACCCTA CTTGGTCTCTGGTAGCAGGGA CD44v3 GCACTTCAGGAGGTTACATC CTGAGGTGTCTGTCTCTTTC CD44v3E TATCTCCAGCACCAATATGG CATCATTCCTATTGCTTGATGTC CTNNB1 CAGCTGCTGTTTTGTTCCGAA CAGCTCAACTGAAAGCCGTTT DSP CACCAGCCCCCCTCTCA AAGGAGACTCATTCCAAAATGCTT HPRT1 TGGTCAGGCAGTATAATCCA GGTCCTTTTCACCAGCAAGCT SNAI1 ACAATGTCTGAAAAGGGACTGTGA CAGACCAGAGCACCCCATT TBP GGGCATTATTTGTGCACTGAGA GCCCAGATAGCAGCACGGT TGFB1 CCTGGACACCAACTATTGCTTCA CCTTGCGGAAGTCAATGTACAG VIMENTIN GGATGCCCTTAAAGGAACCAA CAACGGCAAAGTTCTCTTCCAT ZEB1 TCCCAACTTATGCCAGGCAC CAGGAACCACATTTGTCATAGTCAC ZEB2
TGCAAGTGCCATCCTTGTACA CAAAGAACAGGGTGAGCTTAACAC Microorganisms present in our microbiota interact with our mucous membranes, influencing health and disease throughout our life. While this diverse microbial community is essential for physiological development and immunological homeostasis, alterations in this ecosystem are linked to chronic and inflammatory diseases, and cancers. In order to identify therapeutic targets and/or potential prognostic biomarkers, it is important to understand the cross-talk between our microbiota and gastrointestinal cancers [1]. More than 13% of human cancers are linked to infectious agents [2]. Though the involvement of viruses in human cancers is well recognized (herpesviruses, adenovirus, papillomaviruses, hepatitis viruses, retroviruses …), that of bacteria, on the other hand, is less studied. Nevertheless, the involvement of chronic bacterial infections in carcinogenesis has been established since 1994 with Helicobacter (H.) pylori, a bacterium classified as a type I carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) [3]. Since then, numerous Helicobacter species have been shown to drive carcinogenesis in humans and animals (Table 1).

The Helicobacter genus belongs to the Helicobacteraceae family together with Sulfuricurvum, Sulfurimonas, Sulfurovum, Thiovulum, and Wolinella genera. The Helicobacteraceae family is dominated by the Helicobacter genus, which is currently composed of 55 species, among which 46 species have been validly published [4]. However, most Helicobacter species do not only colonize the gastric mucosa but can also colonize alternative sites of the digestive tract (saliva, cecum, colon, pancreas, liver) in various hosts (mammals, birds, reptiles) and have also been identified in aqueous environments. Genotypic and phylogenetic analyses usually separate Helicobacter taxa into 2 main clades: gastric and enterohepatic species, each comprising of 18 and 30 validly published species, respectively. In the past decades, reports on bacterial chronic infection, inflammation and carcinogenesis in mammals have emerged at an increasing pace. This review focuses on Helicobacter infection and carcinogenesis in the digestive tract of animals and humans.

Gastric Helicobacters and cancer

Despite some descriptions of spiral bacteria colonizing the stomach of animals reported since the end of the 19 th century, and the human stomach as from the beginning of the 20 th century, interest in these Campylobacter-resembling organisms emerged only in 1982 when Marshall and Warren successfully cultured Helicobacter pylori from the stomach of nearly all patients with active chronic gastritis, duodenal ulcer, or gastric ulcer [5,6].

Gastric Helicobacters and Gastric cancer 2.1.1. H. pylori and gastric cancer

H. pylori is a gram-negative bacterium that colonizes exclusively the human gastric mucosa. Epidemiological surveys estimate that half of the world's population is infected by H. pylori and that chronic infection by this bacterium constitutes the predominant causative agent of gastric cancer [7]. In the early 1980's, Marshall and Warren discovered H. pylori and demonstrated the etiological role of chronic infection with this bacterium in gastritis and peptic ulcer disease [5,6]. This discovery was rewarded by the Nobel Prize in 2005. In 1994, WHO even recognized H. pylori as being the leading cause of gastric cancer, making it the first cancer-associated bacterial infection, which was confirmed in their 2012's report [3,8].

H. pylori infection is acquired during childhood mainly via a vertical intrafamilial oro-oral transmission route and persists lifelong. The prevalence of infection with H. pylori is highly heterogeneous worldwide, being the lowest in North America, Australia, and Western Europe while highest is developing countries of Africa, Latin America, and Asia. There is currently no vaccine against H. pylori infection due to its high genetic variability and its immune escape mechanisms. Symptomatic patients diagnosed for H. pylori infection can be cured by a ten days antibiotics treatment combining several antibiotics with proton pump inhibitors [9,10]. In Japan, a health policy for H. pylori eradication was launched in 2000 to prevent gastric cancer; infection prevalence decreased rapidly and gastric cancer incidence also decreased thereafter [11].

Gastric cancer is the 5 th most common diagnosed cancer and the 4 th cause of cancer-related deaths worldwide according to the latest IARC report [12]. Gastric cancers are mostly gastric adenocarcinomas (GC) that are highly heterogeneous both at the histological and the molecular level [13,14]. They are classified according to their location in the stomach, distinguishing adenocarcinomas of the corpus and antrum, mainly related to H. pylori infection, from those of the cardia that have a different etiology. The 2010 WHO classification of gastric cancer specifies five main histological subtypes, grouped into 2 main subtypes according to the Laurén classification of GC, i.e., the intestinal type and the poorly cohesive diffuse type. Although both the intestinal and diffuse types of non-cardia GC are related mainly to H. pylori infection, the intestinal type is often related to environmental factors, diet, and chronic inflammation, whereas the diffuse type is more often associated with

~332~

genetic abnormalities. Chronic infection with H. pylori is considered to be responsible for more than 93 % of non-cardia GC [15]. It is also responsible for gastric Mucosa Associated Lymphoid Tissue (MALT) B-cell lymphoma, a rare cancer, which is the first example of a cancer that can be cured by antibiotics before it evolves into an autonomously proliferating monoclonal B-cell lymphoma. In this case, antibiotics therapy leads to eradication of H. pylori that was at the origin of the sustained B-cell proliferation forming low-grade MALT lymphoma, leading to lymphoma regression [16][17][18].

Pathogenicity factors of H. pylori associated with gastric cancer

In the stomach, H. pylori infection induces a chronic inflammation of the gastric mucosa. Though most of the time asymptomatic, this inflammation can evolve in 5-10% of cases into gastric or duodenal ulcers. H. pylori infection can lead to gastric adenocarcinomas in approximately 1% and in less than 0.1 % cases to gastric MALT lymphoma (Fig. 1A). At cellular level, H. pylori infection leads to a proinflammatory response and chronic gastritis. This is followed by an increase of gastric epithelial cells apoptosis, resulting in atrophy and leading to a compensative cellular hyperproliferation with altered differentiation, at the origin of intestinal metaplasia [19]. Metaplastic lesions, can in turn evolve into dysplasia, in situ carcinoma and eventually invasive adenocarcinoma [20]. Clinical outcome is multifactorial and depends on H. pylori virulence factors and pro-inflammatory potential, strains from South-East Asia being more aggressive than strains from Africa and western countries, and on environmental factors, host genetic susceptibility and reactivity of the host immune system. Numerous studies of modulation of the Th1 versusTh2 inflammatory responses have shown that both are major players in H. pylori-induced gastric carcinogenesis and gastric MALT lymphoma [21][22][23][24].

According to epidemiological studies, numerous virulence factors of H. pylori have been associated with gastric carcinoma. Gastric Helicobacter species have the particularity of producing urease which can reduce the stomach's acidity to create a neutral pH microenvironment at the close vicinity of the bacteria [25,26]. This allows the colonization of the stomach and the development of microcolonies of H. pylori in the mucus at the surface of gastric epithelial cells [27]. The expression of certain allelic variants of adhesins and outer membrane proteins such as Blood group antigen-binding adhesin BabA, sialic-acid binding SabA, outer inflammatory protein OipA and HopQ have been associated with gastric cancer, as well as certain isoforms of the secreted vacuolating cytotoxin VacA [28][29][30][31] (Fig. 2A). OipA and VacA activate Epidermal Growth Factor Receptor (EGFR) which turns on AKT and β-catenin cascades and stimulate cell growth and oncogenic transformation through TCF/LEF-controlled transcription [32]. BabA recognizes and binds to human Lewis b epitope thus triggering cell proliferation as well as pro-inflammatory cytokines expression leading to inflammation [33]. Among all these factors, the most important one is the cytotoxin CagA, encoded by the cag pathogenicity island (cagPAI). In the Mongolian gerbil model, infection with CagA positive strains of H. pylori leads to a Fig. 1. From Helicobacter inflammation to cancer: digestive precancerous cascades in gastric and liver carcinoma. Schematic representation of the main clinical outcomes of Helicobacter pylori (A) and Helicobacter hepaticus infection (B). Cascade was created with icons from BioRender.com. A) Schematic representation of the main clinical outcomes of H. pylori infection. H. pylori infection leads to colonization of the stomach in humans. Infection is acquired during childhood. Chronic infection leads to chronic gastritis that remains asymptomic in ~85 % of cases, while in ~10 % of cases it will lead to antral gastritis and duodenal ulcer, and in ~5% of cases to corpus gastritis and gastric ulcer. Chronic atrophic gastritis (A1, diffuse inflammation of the gastric mucosa) can evolve into intestinal metaplasia (A2, enterocyte-like cells with goblet cells), dysplasia and finally gastric adenocarcinoma (image A3, moderately differencitated carcinoma) in <1% of cases. In less than 0.1% cases, H. pylori chronic infection can lead to gastric MALT. CagA is involved in most of these pathologies, and especially in gastric adenocarcinoma. Scale bars, 100 μm. B) Schematic representation of the main clinical outcomes of Helicobacter hepaticus infection. H. hepaticus infection leads to colonization of mice intestine and then of the biliary tract and liver where it induces inflammatory lesions, hepatitis (B1), the appearance of an adenoma (B2) which may undergo malignant transformation into hepatocellular carcinoma (B3). Non-transgenic mice were infected with H. hepaticus strain 3B1 for 12 months [START_REF] Roux-Goglin | Hepatic lesions observed in hepatitis C virus transgenic mice infected by Helicobacter hepaticus[END_REF] and liver specimens were fixed in 3.7 % formaldehyde solution in PBS, paraffin-embedded, sectioned, and stained with hematoxylin-eosin. B1) Image of a mouse liver following 9 months infection with H. hepaticus. Hepatitis is manifested by a periportal lymphocytic infiltrate (green arrow), accompanied by eosinophilic polynuclei (yellow arrow). B2) Image of a mouse liver following 12 months infection with H. hepaticus. Adenoma (right side of the red dotted line) is manifested by an increase in the number of cell layers in the Remak trabeculae, hepatic steatosis (lipid inclusions in the hepatocytes, yellow arrow), and the appearance of an area of tissue compression due to the pressure exerted by the adenomatous area without any sign of the healthy tissue invasion. B3) Image of a mouse liver following 12 months infection with H. hepaticus. Hepatocellular carcinoma shows disorganized, thickened trabeculae, development of arterioles (yellow arrows) with finger-like tissue projections (green arrows), and an invasion of the healthy tissue.

Scale bars, 100 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

C. Varon et al. chronic gastritis and carcinoma development after 7 months of infection, while isogenic mutant strain invalided for CagA did not [34]. CagA and gastric carcinogenesis causality link was proven in transgenic mice expressing CagA. These mice spontaneously develop gastric polyps and adenocarcinoma, demonstrating CagA's potential as the first bacterial oncoprotein [35]. The cagPAI encodes proteins assembling a type IV secretion system (T4SS), which interacts mainly via CagL at the extremity of the pilus with α5ß1 and αvß6 integrins at the plasma membrane of gastric epithelial cells. It allows the injection of different bacterial effectors, including CagA and pro-inflammatory components of the bacterial peptidoglycan directly into the cytoplasm of the host cell [36][37][38]. This triggers Integrin-Linked Kinase (ILK) activation and consequently, Raf-MEK-ERK Mitogen-Activated Protein Kinase (MAPK) cascade activation and upregulation of target genes involved in hypergastrinemia, a major GC risk factor [39]. Interaction of the bacteria with integrins located at the basolateral cell surface is facilitated by the secretion of the bacterial serine protease HtrA which degrades Occludin and Claudin-8 at tight junctions and E-cadherin at adherence cell/cell junctions [40,41]. Recent data from different teams reveals that interaction of HopQ adhesin with CEACAM receptors at the apical surface of epithelial cells does not only facilitate but is required for CagA translocation by the T4SS [42][43][44]. Once translocated into the cytoplasm by the T4SS, CagA is phosphorylated on tyrosine of EPIYA-motifs by Src and Abl kinases. It interacts in its phosphorylated or directly in its unphosphorylated form with number of different molecules, including cell-cell junction (ZO1, JAM, E-cadherin) [45,46] and polarity complex PAR1 proteins [47], the Hepatocyte Growth Factor c-Met receptor [48]. It binds to SHP2 phosphatase and to several kinases including the Focal Adhesion Kinase FAK regulating actin cytoskeleton remodeling, leading to destabilization of cell/cell junctions and loss of cellular polarity [46]. CagA also alters p53 signaling pathway and impairs DNA-damage response, leading to mitotic instability and accumulation of mutations [49][50][51][52].

CagA-induced cell signaling thereby affects several oncogenic signaling pathways such as MAPK, β-catenin, nuclear factor kappaB (NF-κB) and Hippo/YAP/TAZ pathways leading to perturbations in the cell proliferation-differentiation balance [34,[53][54][55][56][57]. This leads to alteration of the differentiation state of infected cells and to both expression of intestinal markers CDX2 and MUC2 signaling intestinal metaplasia and of mesenchymal markers such as ZEB1, SNAIL and Vimentin with the appearance of elongated cells reflecting epithelial to mesenchymal transition (EMT), and stem cells markers such as CD44 [53,58,59]. This can be observed both in vitro in coculture models and in vivo in mouse models of infection as well as in the gastric mucosa of patients infected with CagA-positive H. pylori strains. Autophagy has also been implicated in CagA-induced signaling and EMT [60,61]. VacA, throughimpairment of the autophagy pathway, leads to CagA accumulation and enhanced signaling [61].

Other proinflammatory components injected into cells by the T4SS are the peptidoglycan component muramyl di-peptide (MDP) and the H. pylori metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (βHBP) which are detected by innate immune receptors NOD1 activating NF-κB proinflammatory signaling pathway and by ALPK1 activating the TIFAsome [62,63], leading to a sustained production of pro-inflammatory cytokines mediating the innate immune response. H. pylori is also able to modify its virulent components to decrease host immune response and survive. It can produce underacetylated LPS as well as modified flagellin molecules which are less recognized by host TLR4 and TLR5 receptors, respectively, leading to moderate immune response from host cells [64,65]. The γ-glutamyl transpeptidase secreted by H. pylori and essential for its colonization of the gastric mucosa [66] is a major actor of H. pylori immune escape mechanism and can inhibit T-cell proliferation and activation. VacA can also inhibit the proliferation and function of a variety of immune cells, including T and B cells, eosinophils and neutrophils, macrophages and dendritic cells, and neutrophils [30,67]. Furthermore, H. pylori, through CagA, can increase PD-L1 expression in infected epithelial cells, allowing the cell recognition by host PD-1 receptors thus escaping immune response mechanisms [65,68].

Several epidemiological studies of gastric cancer prevention by H. pylori eradication demonstrate that in humans transformation events start at the level of metaplastic lesions which may constitute a "point of no return", after which gastric cancer prevention is no more possible [13]. In addition, data suggest that CD44+ cells at the origin of cancer stem cells (CSC) may be detectable as from this stage of pre-neoplastic metaplasia [69,53,70].

Other gastric Helicobacter species and animal models of gastric cancer

Of note, H. pylori is a human pathogen that does not naturally colonize mice. To model the disease and study the carcinogenesis process, scientists have extensively used mouse models of chronic infection with the cat and dog pathogen H. felis capable of inducing chronic inflammation of the gastric mucosa and reproducing the same sequence of pre-neoplastic lesions as that leading to gastric carcinoma in humans, i.e.chronic atrophic gastritis, metaplasia (not a real intestinal metaplasia but mucinous and pseudo-intestinal), dysplasia and gastro-intestinal neoplasia, except that they don't give rise to real invasive adenocarcinoma [69,71]. Another major limitation of this model is that H. felis has neither CagA and cagPAI nor VacA cytotoxin that are the main pathogenic factors of H. pylori associated with gastric cancer in human. So, in this model, gastric carcinogenesis induced by H. felis infection is considered to be mostly the consequence of damage caused by severe chronic inflammation. Rare mouse-adapted H. pylori strains from patients have been used to infect mice, but they induce less inflammation and less pre-neoplastic lesions compared to H. felis [69,72,73]. Mongolian gerbils are susceptible to experimental infection with some H. pylori strains and pioneer experiments have demonstrated the carcinogenic role of the inflammatory OipA, in part and mostly CagA in this model [34]. However, tools are limited to study this model and no transgenic gerbils are available for functional studies on signaling pathways involved.

Other gastric Helicobacter species have been described in animals in which they naturally colonize the stomach (Table 1). H. felis, already mentioned for its usefulness in experimental mouse models, induces chronic gastritis and gastric MALT lymphoma in cats and peptic ulcer disease in dogs [74][75][76][77]. H. heilmannii colonizes the stomach of cats, dogs, pigs and primates, and has been detected as a zoonotic pathogen in humans in whom it has been detected in patients with gastric cancer and gastric MALT lymphoma [78][79][80]. H. suis colonizes the stomach of pigs and macaques and has been associated with invasive gastric adenocarcinoma; it has also been detected in the gastric mucosa of patients with gastric MALT lymphoma [81][82][83][84]. Helicobacter mustelae, an intermediate species between gastric and enterohepatic Helicobacter species, colonizes the ferret stomach where it induces gastritis, duodenal ulcer and gastric adenocarcinoma [85][86][87][88]. It also exacerbates ulceration of the stomach and intestine [87]. Extragastric manifestations, consequence of the chronic infection of the stomach with these Helicobacter species have been studied using these animal models as described below.

Extragastric manifestations of gastric Helicobacters

Gastric Helicobacters have been shown to contribute to inflammation and cancer of the intestinal tract, as well as the hepatobiliary system of animals and humans (Table 1). Majority of literature covers the research focused on the role of H. pylori, but studies often lack the depth to the species level. Systemic manifestation of H. pylori has been reported since the beginning of the 90 s and literature implicates this gastric pathogen in the development of colorectal, pancreatic, bile duct and hepatic carcinomas. Translocation of gastric Helicobacters to extragastric locations is thought to be promoted by increased permeability of intestinal mucosal barrier, [89] and arrival to the liver may occur via biliary tract, or through the portal vein [90]. Models created with icons from BioRender.com. A) Model for multiple host responses during Helicobacter pylori infection. ① ① H. pylori adhesins and outer membrane proteins OipA, BabA, SabA interact with cell surface molecules. OipA activates EGFR which turns on the AKT-β-catenin cascade to increase cell proliferation. BabA and SabA binding to Lewis antigens triggers cell proliferation and pro-inflammatory signaling pathway. ② ② The secreted VacA cytotoxin is able to insert itself into membranes and form ion-conducting channels leading to internalization, vacuolation and induction of ROS; VacA also inhibits autophagy and activates EGFR cascade favoring proliferation and oncogenic signaling. ③ ③ H. pylori T4SS adheres to epithelial cells via α5β1 and αvβ6 integrins. HopQ interacting with CEACAM cell surface molecules facilitates these interactions and T4SS-mediated CagA translocation. ④ ④ Integrin binding triggers ILK activation and MAPK cascade and upregulation of proliferation and pro-oncogenic pathways. ⑤ ⑤ CagA interacts with cMET tyrosine kinase receptor and ⑥ ⑥ activates MAPK and β-catenin pathways stimulating cell proliferation and perturbing the differentiation state of epithelial cells. CagA also activates the Hippo/YAP pathway and ZEB1, also perturbing differentiation and leading to EMT and intestinal differentiation. ⑦ ⑦ H. pylori through CagA and various molecular mechanisms inhibits p53 signaling pathway, in favor of cell proliferation and accumulation of DNA damage promoting oncogenesis. ⑧ ⑧ H. pylori access to integrins, mainly located at the basolateral side, is facilitated by the secreted serine protease HtrA, which disrupts Occludin and Claudin-8 at tight junctions (TJ) and E-Cadherin at adherens junctions (AJ) ⑨ ⑨; CagA also destabilizes TJ and AJ by direct interactions with ZO, JAM and E-cadherin. CagA dimerizes with PAR1b polarity complex proteins and ⑩ ⑩ activates FAK, leading to loss of cell polarity and actin cytoskeleton remodeling. ⑪ ⑪ CagA and mostly MDP and βHBP, also injected by the T4SS, stimulate the NF-κB pathway and the TIFAsome leading to proinflammatory response and secretion of cytokines such as IL-8 and CCL5. ⑫ ⑫ Furthermore, CagA can increase PD-L1 expression on infected epithelial cells, allowing the cell recognition by host immune cells expressing PD-1 receptors thus escaping immune response. Wide field image of AGS gastric epithelial cells not infected (A1) and infected during 24 h with H. pylori cagA-positive strain 7.13 [53] (A2) at a multiplicity of infection of 50 bacteria/cell. Cells were stained with fluorescent labeled-phalloidin to detect F-actin (red), DAPI to counterstain the nucleus (blue) and primary monoclonal anti-CD44 antibodies detected by fluorescent labeled-secondary antibodies (green). H. pylori infection induces the appearance of CD44-positive cells with an elongated phenotype reminiscient of an EMT. Scale bars, 50 μm.

B) Model for multiple host responses during enterohepatic Helicobacter infection.

References specific to enterohepatic Helicobacters are preferentially cited. ① ① Enterohepatic Helicobacters possess potent lipopolysaccharides inducing NF-κB activation and IL-8 secretion [START_REF] Hynes | Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters[END_REF][START_REF] Varon | Study of Helicobacter pullorum proinflammatory properties on human epithelial cells in vitro[END_REF]. ② ② Some enterohepatic Helicobacters encode a type VI secretion system (T6SS) [START_REF] Suerbaum | The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus[END_REF][START_REF] Bartonickova | Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential[END_REF][START_REF] Shen | Draft genome sequences of six enterohepatic helicobacter species isolated from humans and one from rhesus macaques[END_REF] whose components modulate and exacerbate the innate pro-inflammatory effect of the chronic infection [START_REF] Bartonickova | Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential[END_REF]. Besides, ③ ③ enterohepatic Helicobacters secrete the cytolethal distending toxin (CDT). CDTs are widely distributed among many pathogenic Gram-negative bacteria and its active CdtB subunit is the most conserved subunit [START_REF] Pons | Cytolethal distending toxin subunit B: a review of structure-function relationship[END_REF]. The model presented has been established from numerous studies concerning the CdtB active subunit. The first steps of this model are discussed in [START_REF] Dirienzo | Uptake and processing of the cytolethal distending toxin by mammalian cells[END_REF][START_REF] Frisan | Bacterial genotoxins: the long journey to the nucleus of mammalian cells[END_REF]. CDT heterotrimeric complex (CdtA, CdtB and CdtC) is secreted by the bacteria. CdtA and CdtC are accessory subunits, forming the binding component that binds cholesterol-rich plasma membrane rafts. CdtA and CdtC allow the internalization of the CdtB-CdtC heterodimer in the host cell via a clathrin-dependent or independent receptor endocytosis while the CdtA subunit remains on the cell surface. Once in the cell, ④ ④ CdtB, via its phosphatidylinositol-3,4,5-triphosphatephosphatase activity, hydrolyzes PI-3,4,5-P(3) to PI-3,4-P(2) resulting in the inhibition of AKT phosphorylation and contributing to cell cycle arrest [START_REF] Shenker | A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5triphosphate phosphatase activity[END_REF]. ⑤ ⑤ CdtB-CdtC heterodimer is also delivered to the Golgi apparatus via either early or late endosomes and engulfed in COPI vesicles (originating from Golgi cisternae) to be transported by retrograde transport to the endoplasmic reticulum [START_REF] Dirienzo | Uptake and processing of the cytolethal distending toxin by mammalian cells[END_REF][START_REF] Frisan | Bacterial genotoxins: the long journey to the nucleus of mammalian cells[END_REF]. CdtC subunit is degraded in the endoplasmic reticulum and CdtB may be directly translocated from the endoplasmic reticulum lumen to the nucleoplasm through the nuclear pore complex via the recognition of its nuclear localization signal sequence [START_REF] Dirienzo | Uptake and processing of the cytolethal distending toxin by mammalian cells[END_REF][START_REF] Frisan | Bacterial genotoxins: the long journey to the nucleus of mammalian cells[END_REF][START_REF] Guerra | A novel mode of translocation for cytolethal distending toxin[END_REF]. However, CdtB may be also translocated from the endoplasmic reticulum to the cytosol before being transported to the nucleoplasm through the nuclear pore complex. ⑥ ⑥ CdtB binds DNA and owing to its DNase activity [START_REF] Lara-Tejero | A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein[END_REF][START_REF] Avenaud | Expression and activity of the cytolethal distending toxin of Helicobacter hepaticus[END_REF] induces single-strand breaks (SSBs) converted into double-strand breaks (DSBs) [START_REF] Bezine | The cytolethal distending toxin effects on mammalian cells: A DNA damage perspective[END_REF], leading to activation of DNA damage repair with subsequent ataxia telangiectasia mutated-dependent cell cycle arrest [START_REF] Bezine | The cytolethal distending toxin effects on mammalian cells: A DNA damage perspective[END_REF]. When DNA damage is beyond repair, the cell undergoes ⑦ ⑦ cell death [START_REF] Shenker | Cytolethal distending toxin-induced release of interleukin-1β by human macrophages is dependent upon activation of glycogen synthase kinase 3β, spleen tyrosine kinase (Syk) and the noncanonical inflammasome[END_REF][START_REF] Ceelen | Mitotic catastrophe as a prestage to necrosis in mouse liver cells treated with Helicobacter pullorum sonicates[END_REF][START_REF] Gelfanova | Cytolethal distending toxin of Haemophilus ducreyi induces apoptotic death of Jurkat T cells[END_REF][START_REF] Péré-Védrenne | The cytolethal distending toxin subunit CdtB of Helicobacter hepaticus promotes senescence and endoreplication in xenograft mouse models of hepatic and intestinal cell lines[END_REF] or ⑧ ⑧ either senescence [START_REF] Blazkova | Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling[END_REF][START_REF] Péré-Védrenne | The cytolethal distending toxin subunit CdtB of Helicobacter hepaticus promotes senescence and endoreplication in xenograft mouse models of hepatic and intestinal cell lines[END_REF], a process during which cells cease dividing and undergo distinctive phenotypic alterations, including profound chromatin and secretome changes, and tumour-suppressor activation. The CDT-induced senescence-associated secretory phenotype (SASP) would promote survival and tumour progression in surrounding cells (dotted arrow). Senescent cells might finally die or escape senescence [START_REF] Zheng | Polyploid cells rewire DNA damage response networks to overcome replication stress-induced barriers for tumour progression[END_REF], leading to de-polyploidisation and cell survival [START_REF] Azzi-Martin | Cytolethal distending toxin induces the formation of transient messenger-rich ribonucleoprotein nuclear invaginations in surviving cells[END_REF] (dotted arrow). ⑨ ⑨ CDT-induced cell cycle arrest is associated with a profound nuclear and actin cytoskeleton remodeling with stress fiber formation [START_REF] Aragon | Effect of cytolethal distending toxin on F-actin assembly and cell division in Chinese hamster ovary cells[END_REF][START_REF] Ceelen | The cytolethal distending toxin among Helicobacter pullorum strains from human and poultry origin[END_REF][START_REF] Young | Cytolethal distending toxin sequence and activity in the enterohepatic pathogen Helicobacter hepaticus[END_REF]. Nuclear NET1 is dephosphorylated leading to RhOA activation which subsequently phosphorylates p38 Mitogen-Activated Protein Kinase (MAPK) and its downstream effector protein MAPK-activated protein kinase 2 which promotes the formation of actin stress fibers and delays cell death [START_REF] Frisan | The Haemophilus ducreyi cytolethal distending toxin induces DNA double-strand breaks and promotes ATM-dependent activation of RhoA[END_REF][START_REF] Guerra | A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage[END_REF]. Prosurvival autophagy is also activated following CDT intoxication [START_REF] Seiwert | AKT2 suppresses pro-survival autophagy triggered by DNA doublestrand breaks in colorectal cancer cells[END_REF], leading to the selective removal of genotoxin-induced micronuclei-like structures and reducing inflammatory responses (IL-8 and NF-κB), thus protecting intoxicated cells against CDT-induced cell death [START_REF] He | The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/ CSDE1 and P62/SQSTM1[END_REF]. ⑩ ⑩ CDT can promote the formation of nucleoplasmic reticulum (NR), deeply invaginated in the nucleoplasm of giant nuclei of surviving cells [START_REF] Azzi-Martin | Cytolethal distending toxin induces the formation of transient messenger-rich ribonucleoprotein nuclear invaginations in surviving cells[END_REF]. ⑪ ⑪ CDT intoxication also leads to cell junction remodeling with disruption of adherens junctions [START_REF] Damek-Poprawa | Cytolethal distending toxin damages the oral epithelium of gingival explants[END_REF][START_REF] Damek-Poprawa | Cell junction remodeling in gingival tissue exposed to a microbial toxin[END_REF]. HtrA protease produced by some enterohepatic Helicobacter species would also cleave E-cadherin, as H. pylori HtrA does [40]. ⑫ ⑫ The release of proinflammatory cytokines following CDT intoxication involves activation of NLRP3 inflammasome and Caspase 1 [START_REF] Shenker | Cytolethal distending toxin-induced release of interleukin-1β by human macrophages is dependent upon activation of glycogen synthase kinase 3β, spleen tyrosine kinase (Syk) and the noncanonical inflammasome[END_REF][START_REF] Shenker | Aggregatibacter actinomycetemcomitans cytolethal distending toxin activates the NLRP3 inflammasome in human macrophages, leading to the release of proinflammatory cytokines[END_REF]. Dotted arrows correspond to CDT-proposed effects. Widefield image of Caco-2 intestinal epithelial cells infected during 72 h with Helicobacter pullorum strain 459-94 (CCUG 33840) (B1) and its corresponding CDT-knockout mutant strain (B2) at a multiplicity of infection of 100 bacteria/cell. Cells were stained by fluorescent labeled-phalloidin to detect F-actin (red), DAPI to counterstain the nucleus (blue), and primary monoclonal anti-vinculin antibodies (clone hVIN-1, Sigma Aldrich, Saint-Quentin Fallavier, France) detected by fluorescent labeled-secondary antibodies (green). Arrowheads indicate lamellipodia at the front edge of the cell. Scale bars, 50 μm.

Abbreviations. Ⓐ Ⓐ, CdtA; AJ, adherens junction; Ⓑ Ⓑ, CdtB; BabA, Blood group antigen-binding adhesion; Ⓒ Ⓒ, CdtC; CagA, Cytotoxin-associated gene A; CagL, cag pathogenicity island-encoded protein L; CDT, cytolethal distending toxin; DDR, DNA damage repair; DSB, double-strand breaks, EGFR, Epidermal Growth Factor Receptor; EMT, epithelial to mesenchymal transition; γGGT, γ-glutamyl transpeptidase; GSK3β, glycogen synthase kinase 3 β; ILK, Integrin-linked kinase; LPS, lipopolysaccharide; MALT, mucosa-associated lymphoid tissue; MAPK, Mitogen-Activated Protein Kinase; mRNP, messenger ribonucleoprotein; NET1, Neuroepithelial cell-transforming gene 1; NF-κB, nuclear factor kappa B; NLR, nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptors; NLRP3, NLR Family Pyrin Domain Containing 3; NR, nucleoplasmic reticulum; OipA, pylori outer inflammatory protein A; PD-L1, Programmed death-ligand 1; PI, phosphoinositide; PI-3K, phosphoinositide 3-kinase; RACK1, Receptor for activated C kinase 1; SASP, senescence-associated secretory phenotype; SSB, single-strand breaks; TJ, Tight Junctions; T4SS, type IV secretion system; T6SS, type VI secretion system; TLR4/5, Toll-like receptor 4/5; TNF-α tumor necrosis factor α; USF1, Upstream stimulatory factor 1; USF1,Upstream Transcription Factor 1; VacA, Vacuolating cytotoxin A (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Gastric Helicobacters and colorectal cancer

The prevalence of colorectal cancer (CRC) is specially high in developed countries and it is the third leading cause of cancer death in the world [12]. Several studies have indicated the association of H. pylori infection with an increased risk for CRC (reviewed in [91]). However, race/ethnicity seems to be an important aspect highlighting the fact that some populations are more vulnerable [92][93][94][95]. Seropositivity for other H. pylori-associated pathogenic factors GroEL ad CagM has also been associated with colorectal cancer risk [94]. In addition, the increased risk of CRC was correlated to serologic responses to H. pylori toxin VacA [96,95]. H. pylori DNA has been detected in human colorectal adenocarcinomas [97] and the presence of the pathogen in colon polyps has been confirmed by immunohistochemical staining [98,99] suggesting the direct role of H. pylori or its toxins in the colon carcinogenic process. Additionally, indirect roles have also been described. H. pylori infection is associated with increased release of gastrin and hypergastrinemia plays a major role in the development of colorectal cancer, known to be associated with rectal cell proliferation and enhanced growth of colorectal cancer cells [START_REF] Fireman | Helicobacter pylori: seroprevalence and colorectal cancer[END_REF][START_REF] Georgopoulos | Hypergastrinemia is associated with increased risk of distal colon adenomas[END_REF]. H. pylori has been shown to induce gut dysbiosis enabling the colonization of the gut by undesired microbes that facilitate the carcinogenic process [START_REF] Dash | Exploring the impact of Helicobacter pylori on gut microbiome composition[END_REF][START_REF] Kienesberger | Gastric Helicobacter pylori infection affects local and distant microbial populations and host responses[END_REF]. Systemic inflammation triggered by H. pylori infection might also be a risk factor for CRC as inflammation is known to promote CRC [START_REF] Jackson | A population-based epidemiologic study of Helicobacter pylori infection and its association with systemic inflammation[END_REF][START_REF] Tuomisto | Systemic inflammation in colorectal cancer: underlying factors, effects, and prognostic significance[END_REF].

Gastric Helicobacters and pancreatic malignancies

The role of H. pylori in pancreatic malignancies is controversial. 16S rDNA of H. pylori has been detected in tissue samples from patients with pancreatic cancer [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF] and serological studies have suggested H. pylori infection to be a pancreatic carcinogenic risk factor [START_REF] Raderer | Association between Helicobacter pylori infection and pancreatic cancer[END_REF][START_REF] Stolzenberg-Solomon | ATBC Study, Helicobacter pylori seropositivity as a risk factor for pancreatic cancer[END_REF][START_REF] Risch | ABO blood group, Helicobacter pylori seropositivity, and risk of pancreatic cancer: a case-control study[END_REF]. In contrast, some large cohort studies have shown no or negative correlation between H. pylori infection and pancreatic cancer [START_REF] Martel | Helicobacter pylori infection and development of pancreatic cancer[END_REF][START_REF] Lindkvist | A prospective study of Helicobacter pylori in relation to the risk for pancreatic cancer[END_REF][START_REF] Hirabayashi | Author Correction: Helicobacter pylori infection, atrophic gastritis, and risk of pancreatic cancer: a population-based cohort study in a large Japanese population: the JPHC Study[END_REF]. The presence of H. pylori virulence factor CagA has also shown controversial results, either correlating or inversely correlating with pancreatic cancer risk [START_REF] Stolzenberg-Solomon | ATBC Study, Helicobacter pylori seropositivity as a risk factor for pancreatic cancer[END_REF][START_REF] Risch | ABO blood group, Helicobacter pylori seropositivity, and risk of pancreatic cancer: a case-control study[END_REF][START_REF] Ai | Preliminary study of pancreatic cancer associated with Helicobacter pylori infection[END_REF]. Interestingly, the blood type of infected individuals has been indicated to determine the effect of H. pylori on carcinogenic processes, non-O blood type having an increased risk [START_REF] Risch | ABO blood group, Helicobacter pylori seropositivity, and risk of pancreatic cancer: a case-control study[END_REF]. Supporting this, a significant homology has been identified between human carbonic anhydrase II and H. pylori α-carbonic anhydrase, a molecular mimicry that could contribute to the development of autoimmune pancreatitis. These homologous segments contain the binding motif of HLA molecule DRB1*0405, a genetical trigger for autoimmune pancreatitis [START_REF] Guarneri | Helicobacter pylori and autoimmune pancreatitis: role of carbonic anhydrase via molecular mimicry?[END_REF]. Infection of human pancreatic cancer cells with H. pylori results in increased proinflammatory cytokine interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) secretion, as well as higher activities of NF-κB, activator protein-1, and serum response element signaling pathways promoting cell proliferation. This could suggest malignant transformation of pancreatic cells upon H. pylori infection [START_REF] Takayama | Effects of Helicobacter pylori infection on human pancreatic cancer cell line[END_REF]. More studies are needed to verify the role of gastric Helicobacters in the development of chronic pancreatitis and pancreatic cancer.

Gastric Helicobacters and hepatobiliary malignancies

The presence of H. pylori in human biliary tract has been associated with biliary tract carcinomas, including cholangiocarcinoma (CCA) and gall bladder carcinoma. Bacilli with close immunohistological and genetic resemblance to H. pylori were detected on a gallbladder mucosa of a 41-year-old woman with cholecystitis by immunohistochemical staining [START_REF] Kawaguchi | Bacteria closely resembling Helicobacter pylori detected immunohistologically and genetically in resected gallbladder mucosa[END_REF]. Moreover, H. pylori was isolated from the gallbladder of a 50-year-old male patient with acute cholecystitis, demonstrating H. pylori colonization of that site. The isolated H. pylori was genetically identical with the isolate found from the stomach of the same patient encoding Western-type CagA with ABC EPIYA-motifs [START_REF] Backert | Unusual manifestation of live Staphylococcus saprophyticus, Corynebacterium urinapleomorphum, and Helicobacter pylori in the gallbladder with cholecystitis[END_REF]. A study carried out in Thailand detected H. pylori DNA in bile samples from CCA patients and CagA was implicated as an important risk factor. Infected liver tissue from these patients showed a significantly higher inflammatory grade at the portal zone around the bile ducts, biliary epithelial cells had increased proliferation and there was a higher level of mononuclear cell infiltration in gallbladder mucosa [START_REF] Boonyanugomol | Helicobacter pylori in Thai patients with cholangiocarcinoma and its association with biliary inflammation and proliferation[END_REF]. H. pylori-induced biliary epithelial cell proliferation has also been reported in case of hepatolithiasis [START_REF] Kuroki | Helicobacter pylori accelerates the biliary epithelial cell proliferation activity in hepatolithiasis[END_REF]. A retrospective study using immunohistochemical staining for H. pylori detection suggested association of H. pylori with gallbladder cancer, possibly through the formation of bile duct stones [START_REF] Cherif | Linking Helicobacter pylori with gallbladder and biliary tract cancer in Moroccan population using clinical and pathological profiles[END_REF]. Therefore, cagA-positive H. pylori strains may be associated with the carcinogenesis of CCA, through the induction of biliary epithelial cell inflammation and proliferation.

The presence of DNA of the Helicobacter genus was first reported in humans in 2000 in the liver of patients with hepatocellular carcinoma (HCC) [START_REF] Avenaud | Detection of Helicobacter species in the liver of patients with and without primary liver carcinoma[END_REF], suggesting that these bacteria may have a role in the carcinogenic process. Interestingly, the study highlights the fact that the affected livers had unusual pathological pattern; cirrhosis was not present, and the stroma was rich in lymphocytes. 16S rDNA sequence similarity showed the closest match to H. pylori and in one case also to H. felis [START_REF] Avenaud | Detection of Helicobacter species in the liver of patients with and without primary liver carcinoma[END_REF]. Importantly, several studies have reported H. pylori-like DNA from liver tissues with HCC but with certain changes in their genomes. This might indicate the adaptation of gastric Helicobacters to other niches, making them more resistant to hepatobiliary environment [START_REF] Verhoef | Detection of identical Helicobacter DNA in the stomach and in the noncirrhotic liver of patients with hepatocellular carcinoma[END_REF][START_REF] Al-Soud | DNA of Helicobacter spp. and common gut bacteria in primary liver carcinoma[END_REF][START_REF] Huang | Identification of helicobacter species in human liver samples from patients with primary hepatocellular carcinoma[END_REF]. Helicobacter strain 99.38 % similar to that of H. pylori was isolated from a liver biopsy of a 23-year-old woman with cirrhosis caused by Wilson's disease. The bacterium was cagA-negative and possessed s2m2 vacA genotype meaning that VacA was not secreted and poorly active [START_REF] De Magalhães Queiroz | Isolation of a Helicobacter strain from the human liver[END_REF]. H.pylori-like bacteria were also cultivated from liver tissues of HCC patients and existence of mostly coccoid form of H. pylori in adjacent hepatocytes was shown with scanning electron microscopy [START_REF] Xuan | Helicobacter infection in hepatocellular carcinoma tissue[END_REF].

A retrospective study of patients at different stages of evolution of infection with hepatitis C virus (hepatitis, cirrhosis,HCC) confirmed the association between the presence of DNA from Helicobacter (H. pylori and also enterohepatic Helicobacters) and pre-malignant and malignant liver damage [START_REF] Rocha | Association of Helicobacter species with hepatitis C cirrhosis with or without hepatocellular carcinoma[END_REF]. Strong correlation between H. pylori infection and chronic hepatitis C as well as HCV-related cirrhosis and HCC was also found by a meta-analysis including 12 case-control studies with a total of 1449 patients with chronic hepatitis C and 2377 control cases reinforcing the idea that coinfection with HCV seems to enhance the severity of liver pathology ( [START_REF] Wang | The association between Helicobacter pylori infection and chronic hepatitis C: a meta-analysis and trial sequential analysis[END_REF] and references therein). H. pylori virulence factor CagA has been associated with the progress of HCV-related liver diseases [START_REF] Esmat | Role of Helicobacter pylori in patients with HCV-related chronic hepatitis and cirrhosis with or without hepatocellular carcinoma: possible association with disease progression[END_REF][START_REF] Pellicano | Helicobacter species sequences in liver samples from patients with and without hepatocellular carcinoma[END_REF]. HBV-induced liver diseases have been also related to H. pylori infection. Antibodies against H. pylori were seen in 89 % of cirrhotic patients with hepatitis B virus infection [START_REF] Ponzetto | Helicobacter pylori seroprevalence in cirrhotic patients with hepatitis B virus infection[END_REF] and meta-analysis of a Chinese population including 2977 patients with chronic hepatitis B and 1668 control cases indicated a positive correlation between H. pylori infection and HBV-induced liver disease severity [START_REF] Wang | Helicobacter pylori infection may increase the risk of progression of chronic hepatitis B disease among the Chinese population: a metaanalysis[END_REF].

A single-center prospective cohort study associated H. pylori infection with increased development of portal vein thrombosis and HCC, through elevated serum levels of C-reactive protein, tumor necrosis factor-α (TNF-α), IL-6, nitric oxide (NO), VEGF, indicating systemic inflammation and neo-agiogenesis. Moreover, the study demonstrated that effective treatment of H. pylori reduced levels of inflammatory markers and vascular mediators as well as cirrhotic complications [START_REF] Abdel-Razik | Helicobacter Pylori as an initiating factor of complications in patients with cirrhosis: a single-center observational study[END_REF]. Studies with murine models support the colonization of the hepatobiliary system by H. pylori and the induction of inflammation and liver cirrhosis and propose a mechanism through activation of resting hepatic stellate cells [START_REF] Goo | Helicobacter pylori promotes hepatic fibrosis in the animal model[END_REF][START_REF] Huang | The pathological effect of Helicobacter pylori infection on liver tissues in mice[END_REF]. In addition, in vitro models have demonstrated that H. pylori infection disturbed TGFβ1 signaling in hepatic stellate cells, resulting in uncontrolled balance between proliferation and apoptosis of hepatocytes [START_REF] Ki | Helicobacter pylori accelerates hepatic fibrosis by sensitizing transforming growth factor-β1-induced inflammatory signaling[END_REF]. TGFβ1 signaling involvement has also been shown in another study where infection of hepatocytes with H. pylori remodeled actin cytoskeleton by inducing actin-rich invasive structures called podosomes. These invasive micro-domains of infected cells mediate extracellular matrix degradation and are related to increased release of inflammatory cytokines, indicating alterations in hepatocyte functions [START_REF] Roux-Goglin | Helicobacter infection induces podosome assembly in primary hepatocytes in vitro[END_REF].

C. Varon et al. Extragastric manisfestations caused by other Helicobacters besides H. pylori has also been reported. Helicobacter DNA resembling H. pylori has been detected in liver samples of cats with cholangiohepatitis. The authors managed to reveal an Helicobacter-like bacterium presenting semicurved morphology in an intrahepatic bile duct of the cat with suppurative cholangitis [START_REF] Greiter-Wilke | Association of Helicobacter with C[END_REF]. H. heilmannii-like DNA has been identified in the hepatobiliary tract of dogs with chronic hepatic lesions. 21.2 % of liver or gallbladder samples had Helicobacter DNA detected by PCR and these samples showed chronic hepatitis associated or not with degenerative or proliferative changes. However, the authors did not manage to detect Helicobacters by Warthin-Starry staining in the liver sample positive by PCR [START_REF] Takemura | Helicobacter infection in the hepatobiliary system and hepatic lesions: a possible association in dogs[END_REF].

Difficulties in detecting gastric Helicobacters in situ in liver samples has been reported [START_REF] Avenaud | Detection of Helicobacter species in the liver of patients with and without primary liver carcinoma[END_REF][START_REF] Myung | Detection of Helicobacter pylori DNA in human biliary tree and its association with hepatolithiasis[END_REF][START_REF] Greiter-Wilke | Association of Helicobacter with C[END_REF], possibly indicating the very low amounts of bacteria in this location. Additionally, the culture of those bacteria from extragastric niches has mostly failed except in rare cases [START_REF] De Magalhães Queiroz | Isolation of a Helicobacter strain from the human liver[END_REF][START_REF] Xuan | Helicobacter infection in hepatocellular carcinoma tissue[END_REF]. Another important aspect to bring out is the fact that H. pylori strains are genetically very variable, and many studies do not consider this. Great advancement in sequencing technologies will aid in determining the genotypes involved in different pathologies. Therefore, an opened question remains whether the associations described reflect a causative role or are they a consequence of extragastric disorders that facilitate extra-gastric colonization. Further studies will be needed to verify the mechanistic approaches used by these bacteria to promote extragastric digestive malignancies.

Enterohepatic Helicobacters and cancer

In 1978, Lee et Phillips reported the isolation and culture of spiral organisms from cecal crypts of mice and rats [START_REF] Lee | Isolation and cultivation of spirochetes and other spiralshaped bacteria associated with the cecal mucosa of rats and mice[END_REF]. In 1988, stool cultures from patients with mild chronic gastroenteritis grew up an unusual microaerophilic gram-negative bacterium resembling Campylobacter species. For one patient, the same bacillus was also recovered from the stool of her asymptomatic daughter and asymptomatic young dog [START_REF] Romero | Case report of an unclassified microaerophilic bacterium associated with gastroenteritis[END_REF], suggesting transmission from animals to humans or conversely. A similar bacterium was isolated from ovine abortions [START_REF] Kirkbride | Ovine abortion associated with an anaerobic bacterium[END_REF]. All these unclassified isolates were later differentiated from Campylobacter species by transmission electron microscopy, phenotypic and protein analyses [START_REF] Archer | Characterization of an unclassified microaerophilic bacterium associated with gastroenteritis[END_REF]. These were the first descriptions of enteric Helicobacter species.

Enterohepatic Helicobacters and cancer

In 1992, Helicobacter hepaticus (H. hepaticus) was discovered as a mouse pathogen whose persistence caused hepatocarcinoma, opening new fields of investigations on non-H. pylori species and extra-gastric malignancies [START_REF] Fox | Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice[END_REF]. H. hepaticus was also involved in the development of typhlocolitis and chronic hepatitis. Since then, H. hepaticus and other Helicobacter species have been implicated in animal and human digestive diseases (Table 1). Indeed, enterohepatic Helicobacters related to the H. bilis/H. cinaedi cluster have been reported in hepatobiliary diseases of naturally infected rodents [START_REF] Nambiar | Progressive proliferative and dysplastic typhlocolitis in aging syrian hamsters naturally infected with Helicobacter spp.: a spontaneous model of inflammatory bowel disease[END_REF][START_REF] Fox | Chronic hepatitis, hepatic dysplasia, fibrosis, and biliary hyperplasia in hamsters naturally infected with a novel Helicobacter classified in the H. bilis cluster[END_REF]. Several gastric and enterohepatic Helicobacters have been identified in the bile ducts of patients with cholangiocarcinomas [START_REF] Kaewpitoon | Helicobacter species are possible risk factors of cholangiocarcinoma, Asian Pac[END_REF], in the liver of patients with HCC [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF], as well as in the pancreas, stomach, and duodenum of pancreatic cancer patients [START_REF] Nilsson | Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients[END_REF][START_REF] Rocha | Association of Helicobacter species with hepatitis C cirrhosis with or without hepatocellular carcinoma[END_REF][START_REF] Avenaud | Detection of Helicobacter species in the liver of patients with and without primary liver carcinoma[END_REF], suggesting a possible role of these bacteria in the digestive carcinogenic process [90].

H. hepaticus, colonizes the intestine of immunocompetent mice, then the bile duct and the liver, where it induces inflammatory lesions, hepatitis, and, in some older animals, hepatocarcinoma [START_REF] Fox | Chronic proliferative hepatitis in A/JCr mice associated with persistent Helicobacter hepaticus infection: a model of helicobacter-induced carcinogenesis[END_REF]. In Rag2-deficient mice, H. hepaticus promotes colon tumorigenesis [START_REF] Nagamine | Helicobacter hepaticus infection promotes colon tumorigenesis in the BALB/c-Rag2 (-/-) Apc(Min/+) mouse[END_REF], and innate immune inflammatory response against H. hepaticus induces mammary adenocarcinoma in females and enhances intestinal adenoma multiplicity by a TNF-α-dependent mechanism [START_REF] Rao | Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice[END_REF]. H. hepaticus infection provides an excellent model for the study of inflammatory bowel diseases, liver diseases and carcinogenesis, widely used in immunocompetent and immunodeficient mice and other rodent models.

Like other enterohepatic Helicobacters, H. hepaticus exhibits many divergences with H. pylori. Indeed, it does not normally colonize the stomach and lacks the major virulence factors of H. pylori, i.e. CagA and VacA, as well as the main adhesins of H. pylori, i.e. BabA, and SabA [START_REF] Suerbaum | The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus[END_REF]. None of the H. hepaticus predicted proteins contains the characteristic N-terminal motif of the Hop group of outer membrane proteins from H. pylori [START_REF] Suerbaum | The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus[END_REF]. Unlike H. pylori, which encodes a T4SS, H. hepaticus contains a large genomic pathogenicity island, named HHGI1, which encodes a type VI secretion system (T6SS) [START_REF] Suerbaum | The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus[END_REF], whose actively secreted components modulate and exacerbate innate pro-inflammatory effects of chronic infection, increasing H. hepaticus colitogenic potential [START_REF] Bartonickova | Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential[END_REF]. Some Helicobacter species genomes are not yet available. However, other enterohepatic Helicobacter species (Table 1) possess T6SS, γGGT [66,[START_REF] Moyaert | Helicobacter equorum sp. nov., a ureasenegative Helicobacter species isolated from horse faeces[END_REF], a well-known H. pylori virulence factor [66], and htrA gene encoding HtrA serine protease, known to cleave E-cadherin in H. pylori [40,41]. They also produce potent proinflammatory lipopolysaccharide (LPS, up to 10-fold higher than that of H. pylori) [START_REF] Hynes | Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters[END_REF]. More importantly, some of these enterohepatic Helicobacter species secrete a widespread toxin: the cytolethal distending toxin (CDT) [START_REF] Chien | Identification of cdtB homologues and cytolethal distending toxin activity in enterohepatic Helicobacter spp[END_REF][START_REF] Suerbaum | The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus[END_REF].

CDT and the balance between cell survival and death in cancer biology

CDTs are widely distributed among many pathogenic Gram-negative bacteria, including Aggregatibacter actinomycetemcomitans, Campylobacter species, Escherichia species, Glaesserella parasuis, Haemophilus species, Pasteurella species, Providencia alcalifaciens, Psittacicella hinzii, Salmonella species, Shigella species, Yersinia species, and some enterohepatic Helicobacter species. CDT genes are located within the bacterial genome and bacteriophage. In most enterohepatic Helicobacter species, CDT is active as a heterotrimeric complex composed of three distinct subunits, i.e., CdtA, CdtB, and CdtC, named alphabetically in the order of appearance of their coding genes in the cdt operon, i.e., cdtA cdtB and cdtC. CDT is a subclass of the AB toxin superfamily: an AB 2 toxin whose binding moiety (B2: CdtA and CdtC) allows the transport and internalization of the active subunit (A: CdtB) in the target cell. CdtB subunit is highly conserved among CDT-secreting bacteria, with 48-97 % amino acid identity among CDT-secreting bacteria, unlike CdtA and CdtC [START_REF] Pons | Cytolethal distending toxin subunit B: a review of structure-function relationship[END_REF]. Amongst the available sequenced Helicobacter genomes, CDT genes are lacking in almost all gastric Helicobacters, except for H. himalayensis, an intermediate species isolated from the gastric mucosa of Marmot in China that is nevertheless phylogenetically clustered within enterohepatic species [START_REF] Hu | Helicobacter himalayensissp. nov. isolated from gastric mucosa ofMarmota himalayana[END_REF][START_REF] Hu | Genomic analysis of Helicobacter himalayensis sp. nov. isolated from Marmota himalayana[END_REF]. Amongst the 29 available genomes of enterohepatic Helicobacter, cdtB genes are present in 20 species (Fig. 3). Interestingly, cdtB gene is present in almost all species associated with severe pathologies and, in particular with cancer, except H. macacae, and H. trogontum (Table 1). The presence of cdtB within more virulent enterohepatic Helicobacters suggests that CDT is a true virulence factor.

It has been shown that 4 and 10 months after infection with H. hepaticus and its corresponding mutant strain lacking CDT activity, immunocompetent mice develop comparable chronic hepatitis, suggesting that CDT is not necessary to induce chronic hepatitis. However, H. hepaticus-infected mice produced a significant increase in hepatic transcripts of pro-inflammatory factors (TNFα, IFNγ and, Cox2), growth factors (IL6 and TGFα), anti-apoptotic proteins (BCL2 and BCL-X L ), as well as an increase in hepatocyte proliferation. An increase in mRNAs involved in the NF-κB signaling pathway (RELA/p65, p50, GADD45β, and cIAP1) was also observed. These effects were not observed with the non-infected mice and mice infected with the corresponding strain lacking CDT activity. In addition, 10 months after infection with H. hepaticus, mice develop dysplastic nodules unlike those infected with the CDT mutant strain [START_REF] Ge | Bacterial cytolethal distending toxin promotes the development of dysplasia in a model of microbially induced hepatocarcinogenesis[END_REF], suggesting that H. hepaticus CDT displayed a major pro-inflammatory and pro-carcinogenic role in the liver [START_REF] Ge | Bacterial cytolethal distending toxin promotes the development of dysplasia in a model of microbially induced hepatocarcinogenesis[END_REF] (Fig. 1B). More importantly, the presence of CDT was meaningful for long-term colonization of H. hepaticus in mice [START_REF] Ge | Cytolethal distending toxin is essential for Helicobacter hepaticus colonization in outbred Swiss Webster mice[END_REF][START_REF] Pratt | Modulation of host immune responses by the cytolethal distending toxin of Helicobacter hepaticus[END_REF]. H. hepaticus CDT also promotes thyphlocolitis and intestinal carcinogenesis in

~338~

susceptible mice models [START_REF] Pratt | Modulation of host immune responses by the cytolethal distending toxin of Helicobacter hepaticus[END_REF][START_REF] Ge | Helicobacter hepaticus cytolethal distending toxin promotes intestinal carcinogenesis in 129Rag2-deficient mice[END_REF]. H. hepaticus infection in mice, through its CDT activity, is thus a widely used model to study inflammation and digestive carcinogenesis.

Many in vitro experiments were implemented to decipher cellular responses to CDT intoxication (Fig. 2B). It has been shown that once secreted by the bacteria, CdtB is internalized from the plasma membrane into the host cell and crosses several subcellular compartments to reach the nucleus [START_REF] Dirienzo | Uptake and processing of the cytolethal distending toxin by mammalian cells[END_REF][START_REF] Frisan | Bacterial genotoxins: the long journey to the nucleus of mammalian cells[END_REF], where it primarily causes DNA single-strand breaks (SSBs) subsequently converted into double-strand breaks (DSBs) [START_REF] Bezine | The cytolethal distending toxin effects on mammalian cells: A DNA damage perspective[END_REF], triggering a repair checkpoint comparable to that induced by ionizing radiation [START_REF] Fahrer | Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts[END_REF]. CdtB is a dual-function enzyme that can act as a DNase homolog to type I mammalian DNases [START_REF] Lara-Tejero | A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein[END_REF] and a phosphatidylinositol-3,4, 5-trisphosphate phosphatase both contributing to cell cycle arrest [START_REF] Shenker | A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5triphosphate phosphatase activity[END_REF] (G1/S and/or G2/M transition) allowing DNA repair: homologous recombination and non-homologous end-joining repair pathways [START_REF] Bezine | The cytolethal distending toxin effects on mammalian cells: A DNA damage perspective[END_REF]. This phosphatase activity leads to the inhibition of the phosphoinositide 3-kinase (PI-3 K) signaling pathway, coupled with an activation of glycogen synthase kinase 3 β (GSK3β), leading to the release of pro-inflammatory cytokines (IL-1β, IL-6, IL-18, and TNFα) [START_REF] Shenker | Blockade of the PI-3K signalling pathway by the Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces macrophages to synthesize and secrete proinflammatory cytokines[END_REF][START_REF] Shenker | Cytolethal distending toxin-induced release of interleukin-1β by human macrophages is dependent upon activation of glycogen synthase kinase 3β, spleen tyrosine kinase (Syk) and the noncanonical inflammasome[END_REF].

However, cell response to CDT intoxication may be dependent on the dose of the toxin and on the cell type. In vitro exposure of cells to sublethal doses of H. hepaticus CDT increases the frequency of mutations, including transversions and chromosomal aberrations which can lead to anchorage-independent growth, indicative of malignant transformation, with concomitant impairment of DNA damage repair (DDR). Cell viability is concomitantly maintained through the sustained activation of p38 MAP kinase [START_REF] Guidi | Chronic exposure to the cytolethal distending toxins of Gram-negative bacteria promotes genomic instability and altered DNA damage response[END_REF], suggesting that chronic infection with CDT producing bacteria may promote genomic instability and favor malignant transformation.

Still, when DNA damage is significant or beyond repair, the cell undergoes mitotic catastrophe leading to necrosis [START_REF] Ceelen | Mitotic catastrophe as a prestage to necrosis in mouse liver cells treated with Helicobacter pullorum sonicates[END_REF], apoptosis [START_REF] Gelfanova | Cytolethal distending toxin of Haemophilus ducreyi induces apoptotic death of Jurkat T cells[END_REF] and pyroptosis [START_REF] Shenker | Cytolethal distending toxin-induced release of interleukin-1β by human macrophages is dependent upon activation of glycogen synthase kinase 3β, spleen tyrosine kinase (Syk) and the noncanonical inflammasome[END_REF] associated with pro-inflammatory responses (IL-1β, IL-6, IL-18 and NF-κB) [START_REF] Shenker | Blockade of the PI-3K signalling pathway by the Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces macrophages to synthesize and secrete proinflammatory cytokines[END_REF][START_REF] Shenker | Cytolethal distending toxin-induced release of interleukin-1β by human macrophages is dependent upon activation of glycogen synthase kinase 3β, spleen tyrosine kinase (Syk) and the noncanonical inflammasome[END_REF][START_REF] Gelfanova | Cytolethal distending toxin of Haemophilus ducreyi induces apoptotic death of Jurkat T cells[END_REF]. All these responses were described for some enterohepatic Helicobacter species [START_REF] Ceelen | Mitotic catastrophe as a prestage to necrosis in mouse liver cells treated with Helicobacter pullorum sonicates[END_REF][START_REF] Péré-Védrenne | The cytolethal distending toxin subunit CdtB of Helicobacter Induces a Th17-related and antimicrobial signature in intestinal and hepatic cells in vitro[END_REF][START_REF] Péré-Védrenne | The cytolethal distending toxin subunit CdtB of Helicobacter hepaticus promotes senescence and endoreplication in xenograft mouse models of hepatic and intestinal cell lines[END_REF]. CDT-mediated proinflammatory response involves the activation of the NLRP3 inflammasome [START_REF] Belibasakis | Aggregatibacter actinomycetemcomitans targets NLRP3 and NLRP6 inflammasome expression in human mononuclear leukocytes[END_REF][START_REF] Shenker | Aggregatibacter actinomycetemcomitans cytolethal distending toxin activates the NLRP3 inflammasome in human macrophages, leading to the release of proinflammatory cytokines[END_REF] and is dependent upon caspase-1 activation [START_REF] Shenker | Cytolethal distending toxin-induced release of interleukin-1β by human macrophages is dependent upon activation of glycogen synthase kinase 3β, spleen tyrosine kinase (Syk) and the noncanonical inflammasome[END_REF][START_REF] Shenker | Aggregatibacter actinomycetemcomitans cytolethal distending toxin activates the NLRP3 inflammasome in human macrophages, leading to the release of proinflammatory cytokines[END_REF]. CDT also inhibits macrophage phagocytosis and modifies the pro-inflammatory/anti-inflammatory cytokine balance [START_REF] Ando-Suguimoto | The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine C. Varon et al. production[END_REF]. Moreover, the presence of CDT is meaningful for bacterial proliferation [START_REF] Ando-Suguimoto | The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine C. Varon et al. production[END_REF], as observed in mice for long-term colonization of H. hepaticus [START_REF] Ge | Cytolethal distending toxin is essential for Helicobacter hepaticus colonization in outbred Swiss Webster mice[END_REF][START_REF] Pratt | Modulation of host immune responses by the cytolethal distending toxin of Helicobacter hepaticus[END_REF]. Some cells with persistent DSBs survive the acute phase of CDT intoxication and enter senescence [START_REF] Blazkova | Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling[END_REF][START_REF] Péré-Védrenne | The cytolethal distending toxin subunit CdtB of Helicobacter hepaticus promotes senescence and endoreplication in xenograft mouse models of hepatic and intestinal cell lines[END_REF], a long-term cell cycle arrest. Those cells acquire a senescence-associated secretory phenotype (SASP) characterized by the release of growth factors, pro-inflammatory compounds and proteases with deleterious effects on the tissue microenvironment, favorable for tumor growth [START_REF] Coppé | The senescence-associated secretory phenotype: the dark side of tumor suppression[END_REF]. Following the genotoxic stress induced by CDT, errors made during DNA repair would therefore promote tumor initiation while CDT-senescent cells may promote, via their SASP, the survival and proliferation of surrounding transformed cells.

In some CDT-surviving cells, nuclear remodeling can be associated with the formation of nucleoplasmic reticulum (NR) corresponding to deep cytoplasmic invaginations in the nucleoplasm of giant cells whose core insulates and concentrates adaptive and dynamic ribonucleoprotein particles [START_REF] Azzi-Martin | Cytolethal distending toxin induces the formation of transient messenger-rich ribonucleoprotein nuclear invaginations in surviving cells[END_REF]. NR formation is transient and relies upon prosurvival autophagy activated following CDT intoxication [START_REF] He | The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/ CSDE1 and P62/SQSTM1[END_REF]. In this context, autophagy leads to the selective removal of genotoxin-induced micronuclei-like structures, reduces inflammatory responses (IL-8 and NF-κB) and protects intoxicated cells against the CDT-induced cell death until the genotoxic stress is relieved [START_REF] He | The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/ CSDE1 and P62/SQSTM1[END_REF].

In addition, CDT intoxication leads to profound actin cytoskeleton remodeling and cellular enlargement [START_REF] Aragon | Effect of cytolethal distending toxin on F-actin assembly and cell division in Chinese hamster ovary cells[END_REF][START_REF] Guerra | Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival[END_REF][START_REF] Varon | Helicobacter pullorum cytolethal distending toxin targets vinculin and cortactin and triggers formation of lamellipodia in intestinal epithelial cells[END_REF]. CDT intoxication also leads to the remodeling of adherens junction, E-cadherin and β-catenin [START_REF] Damek-Poprawa | Cytolethal distending toxin damages the oral epithelium of gingival explants[END_REF][START_REF] Damek-Poprawa | Cell junction remodeling in gingival tissue exposed to a microbial toxin[END_REF], and HtrA protease may also favor cell junction disruption, as reported during H. pylori infection [40,41]. CDT intoxication also provokes perinuclear relocalization of Vinculin, decreased cellular adherence and formation of cortical actin-rich large lamellipodia with an upregulation of cortactin expression [START_REF] Varon | Helicobacter pullorum cytolethal distending toxin targets vinculin and cortactin and triggers formation of lamellipodia in intestinal epithelial cells[END_REF]. CDT also promotes activation of integrin β1, leading to the enhanced assembly of focal adhesions and cell spreading through NET1, the RhoA-specific Guanine nucleotide Exchange Factor [START_REF] Levi | Bacterial genotoxins promote insideout integrin β1 activation, formation of focal adhesion complexes and cell spreading[END_REF]. Knowing that the loss of Vinculin and membrane-bound β-catenin The phylogeny presented is based on the alignment of full-length CdtB. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The optimal tree with the sum of branch length = 3.50977714 is shown. The phylogenetic analysis was generated with the Neighbor-Joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]. The tree is not rooted and drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are represented in the units of the number of amino acid substitutions per site. The analysis included 21 amino acid sequences, as H. mastomyrinus, H. mesocricetorum, and H. muridarum full CdtB sequence was not available and excluded from this analysis. All ambiguous positions were removed for each sequence pair. There was a total of 305 positions in the final dataset. Evolutionary analyses were conducted using MEGA6 [START_REF] Tamura | MEGA6: molecular evolutionary genetics analysis version 6.0[END_REF]. All sequences are labeled by species, strain name, the species from which they were isolated, and the accession number in brackets. Arrow indicates H. himalayensis, a species isolated from the gastric mucosa of Marmot in China that is phylogenetically clustered within enterohepatic species [START_REF] Hu | Helicobacter himalayensissp. nov. isolated from gastric mucosa ofMarmota himalayana[END_REF][START_REF] Hu | Genomic analysis of Helicobacter himalayensis sp. nov. isolated from Marmota himalayana[END_REF].

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promotes metastasis [START_REF] Li | Loss of vinculin and membrane-bound β-catenin promotes metastasis and predicts poor prognosis in colorectal cancer[END_REF], it is tempting to speculate that, following the loss of cell junction and adherence, CDT would promote migration and metastasis concomitantly to cell proliferation.

CDT-induced actin cytoskeleton and nuclear remodeling occur through MAFB [START_REF] Péré-Védrenne | The nuclear remodeling induced by Helicobacter cytolethal distending toxin involves MAFB oncoprotein[END_REF]. MAFB is a bona fide oncogene able to transactivate and transform primary cells [START_REF] Eychène | A new MAFia in cancer[END_REF]. It is also involved in proliferation, migration and invasion [START_REF] Li | MicroRNA-152 inhibits cell proliferation, migration and invasion by directly targeting MAFB in nasopharyngeal carcinoma[END_REF]. The involvement of MAFB oncogene in CDT effects constitutes an additional argument supporting the role of CDT in cancer promotion.

To conclude, most enterohepatic Helicobacters associated with severe pathologies secrete CDT, and the cumulative inflammatory effect of CDT with those of LPS and T6SS-secreted components would strongly modulate the host immune response leading to bacterial persistence that further leads to continuous damage and carcinoma, as demonstrated during H. hepaticus chronic infection of mice. CDT intoxication induces many hallmarks of cancer as it promotes mutations and chromosomal aberrations in intoxicated cells that do not cease to proliferate, suggesting that CDT directly promotes malignant transformation. Additionally, CDT hijacks autophagy, promotes NR formation, and activates survival pathways, that would sustain not only tumor initiation but also tumor progression. In addition to these pro-carcinogenic effects, CDT would induce metastasis via its role on vinculin and cell junctions.

Another bacterial genotoxin, colibactin, is produced primarily by some genotoxic Escherichia coli [START_REF] Nougayrède | Escherichia coli induces DNA double-strand breaks in eukaryotic cells[END_REF]. Colibactin triggers DNA damage, inducing a similar response to that observed for CDT-secreting bacteria. Numerous high-level studies carried out with animal models and in humans have revealed a colibactin-specific mutational signature, which promotes colorectal carcinogenesis and enhances the progression of colorectal cancer through cellular senescence and chromosomal abnormalities [START_REF] Terlouw | Recurrent APC splice variant c.835-8A>G in patients with unexplained colorectal polyposis fulfilling the colibactin mutational signature[END_REF][START_REF] Pleguezuelos-Manzano | Mutational signature in colorectal cancer caused by genotoxic pks+E. coli[END_REF][START_REF] Dziubańska-Kusibab | Colibactin DNA-damage signature indicates mutational impact in colorectal cancer[END_REF]. Given the numerous similarities in cellular response to CDT and colibactin, as well as the accumulating evidence regarding CDT carcinogenic potential, it is clear that chronic infection with CDT-secreting bacteria is a driver of carcinogenesis whose role will be demonstrated in the future.

Besides having an oncogenic function, bacterial genotoxins with their acute genotoxicity make them relevant candidates to be considered for the development of anti-cancer strategies. The increasing chemoresistance mechanisms due to long-term use of chemotherapeutic agents in cancer treatment makes the search of other alternatives a critical point. Indeed, a broad range of bacterial secondary metabolites, such as enzymes/proteases, toxins, and peptides, have been extensively evaluated in cancer therapy and will perhaps form part of tomorrow's chemotherapeutical strategies [START_REF] Diwan | Microbial cancer therapeutics: a promising approach[END_REF][START_REF] Kalia | Emerging applications of bacteria as antitumor agents[END_REF][START_REF] Mohan | Bacteria as a treasure house of secondary metabolites with anticancer potential[END_REF]. Bacterial toxins, i.e. Diphtheria toxin, Shiga toxin, Clostridium perfringens enterotoxin, are relevant candidates for the development of anti-cancer strategies. In line with that, it was proposed that the potent DNA damage induced by CDT in host cells can enhance the effect of ionizing radiation in radio-resistant prostate cancer [START_REF] Lai | Sensitization of radio-resistant prostate cancer cells with a unique cytolethal distending toxin[END_REF] and may constitute a promising anti-cancer therapeutic agent. These findings strongly support the use of CDT as potential anti-cancer agent and emphasize the Yin and Yang dualism of CDT toxins.

Conclusions

Helicobacters have been shown to trigger various digestive tract malignancies in animals and humans. H. pylori is the best-known member of this genus causing gastric cancer but also associated with various extragastric malignancies. However, the mechanisms behind extra-gastric carcinogenic processes are not clear due to the difficulties in detecting and in cultivating H. pylori from those sites. Further studies are needed to verify the direct role of H. pylori in extra-gastric digestive malignancies.

Nevertheless, verification of the status of H. pylori infection in people with extra-gastric diseases could be a future strategy since, eradication of H. pylori could eliminate an important risk factor. Likewise, H. pylori positive patients should be followed beyond gastric diseases. Development of accurate and rapid molecular and serological methods is needed to improve the detection and characterization of distinct Helicobacter species in various malignancies. In addition, other members of the Helicobacter genus have been associated with different hepatobiliary cancers, both in humans and vertebrates. Importantly, chronic infection with CDT-producing enterohepatic Helicobacters promotes genomic instability and carcinogenesis, bringing out CDT as an emerging carcinogenic toxin. Moreover, CDT might offer a potential approach as anticancer therapy. Le cancer de l'estomac est un cancer de mauvais pronostique dont la prise en charge reste essentiellement basée sur la résection chirurgicale associée à des traitements conventionnels de chimio et radiothérapie. Le cancer gastrique est une maladie hétérogène tant au niveau histologique que moléculaire. Les adénocarcinomes gastriques (GC) qui représentent plus de 90% des cas, sont principalement associés à l'infection par la bactérie Helicobacter pylori.

Actualités en recherche sur le cancer gastrique

Ces GC se développent principalement chez les sujets âgés, des suites de l'évolution tout au long de la vie d'une cascade de lésions pré-néoplasiques initiée par H. pylori (1), impliquant des déséquilibres du microbiote suspecté de participer au processus oncogénique, et conduisant à l'émergence de cellules souches cancéreuses (CSC) à l'origine de l'initiation et de la propagation tumorale. La compréhension de cette hétérogénéité tumorale des GC et des causes infectieuses qui en sont à l'origine est essentielle afin de pouvoir à terme adapter la stratégie thérapeutique et améliorer l'efficacité des traitements.

Le cancer gastrique : une maladie d'Helicobacter pylori

La principale cause des GC est l'infection chronique par H. pylori, classée carcinogène de classe 1 par l'IARC en 1994 et associée à plus de 93% des GC hors-cardia (2) Horvat et al. ont montré que l'infection à H. pylori inhibe l'expression du suppresseur de tumeur p14ARF en induisant sa dégradation de manière dépendante de CagA (6). La région du chromosome sur lequel est localisé ce gène est souvent délétée dans les tumeurs primaires de l'estomac. De plus, plus de 30% des GC présentent une hyper-méthylation inactivant son expression (6). La bactérie est capable d'induire la sous-expression du suppresseur de tumeur ~349~ RACK1 (Receptor for Activated C Kinase 1), impliqué dans la modulation physiologique de l'activité de la voie de signalisation NF-κB. De plus, RACK1 est sous-exprimé dans les tissus de GC par rapport à des tissus non tumoraux à distance de la tumeur et est corrélée à un pronostic vital faible (7). L'altération de la barrière muqueuse gastrique par l'infection à H. pylori et l'inflammation induite joue aussi un rôle dans la cancérogenèse gastrique (4) (8). pylori est capable, via l'oncoproétine CagA, d'initier la TEM dans les cellules épithéliales gastriques (10) (11). L'invalidation de la protéine IQGAP1, une protéine impliquée dans la stabilité des jonctions adhérentes, augmente la TEM induite par H. pylori dans des lignées de cellules épithéliales gastrique in vitro et promeut le développement de cellules CD44+ ayant des propriétés de CSC et de lésions pré-néoplasiques in vivo (11). Une étude récente montre que H. pylori inhibe également l'expression d'Afadin, une protéine de stabilisation des jonctions serrées, et ainsi induit une TEM dans des lignées cellulaires gastriques in vitro (12). (20). Plusieurs autres marqueurs tels que CD133, Lgr5, CD24, CD166 ont été décrit pour être enrichis dans les GCSCs et sont donc utilisés pour les étudier et les cibler (20) (23).

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H. pylori est connu pour sa capacité à induire un phénotype TEM ainsi que des propriétés de CSCs. De manière intéressante, seules les cellules ayant acquis le phénotype mésenchymateux et surexprimant CD44 en réponse à l'infection par H. pylori présentent des propriétés tumorigéniques et invasives de CSC (13). H. pylori, via CagA, est capable d'induire la voie oncogénique YAP1 ainsi que ses effecteurs en aval dans les cellules épithéliales gastriques et de promouvoir la TEM et l'acquisition de marqueurs de métaplasie intestinale (24) (25). Cette voie de signalisation appelée Hippo contribue au phénotype CSC avec un enrichissement de YAP1 et de ses partenaires dans des cellules tumorales gastriques CD44+ (26). La voie Hippo semble aussi limiter les changements pré-néoplasiques induits par H. pylori. L'infection par la bactérie active précocement YAP1 puis, dans un second temps, active également la kinase tumeur suppresseur LATS2 qui en phosphorylant et inactivant YAP1 va contribuer à limiter la signalisation oncogénique et préserver l'intégrité de l'épithélium gastrique (25). D'autre part, les effecteurs de la voie Hippo, YAP et TAZ collaborent avec la β-caténine dans la voie de signalisation Wnt pour promouvoir le développement de lésions néoplasiques gastrointestinales (27).

CD44 n'est pas qu'un marqueur des CSCs gastrique mais il joue aussi un rôle fonctionnel dans la prolifération des cellules épithéliales gastriques induite par H. pylori. Il interagit avec c-Met dont il joue le rôle de co-récepteur, et avec CagA. Ainsi, H. pylori n'arrive pas à induire la prolifération de cellules épithéliales d'organoïdes dérivés d'estomac de souris déficiente pour CD44, montrant l'importance de CD44 dans la cancérogenèse gastrique induite par la bactérie (28). Il existe, grâce à l'épissage alternatif, plusieurs isoformes de CD44 contenant au moins 20 exons. Le variant 8-10 du CD44 (CD44v8-10) a été identifié comme étant plus exprimé dans les cellules cancéreuses gastriques que dans le tissu normal.

L'expression exogène du CD44v8-10 augmente la fréquence d'initiation de tumeur chez des souris immunodéprimées (21). Un autre variant du CD44, CD44v9 est aussi très exprimé dans ~353~ les CSCs gastriques. L'analyse d'échantillons de GC venant de 103 patients montre un taux de survie à 5 ans plus faible pour les patients avec des tumeurs CD44v9 positives par rapport aux CD44v9 négatives (29). Une étude récente a analysé le rôle de HER2 (Human Epidermal growth factor 2), cible du trastuzumab, dans les CSCs gastrique et les mécanismes sousjacents. Les auteurs ont démontré que les cellules surexprimant HER2 avaient un phénotype plus souche et plus invasif en passant par la régulation de la voie de signalisation Wnt/βcaténine (30). D'après Tsugawa et al., le stress oxydatif augmente l'activité de la β-caténine et l'expression de la protéine « epithelial splicing regulatory protein 1 » responsable de l'épissage alternatif de CD44 en CD44v9 (31). Les altérations de jonctions cellulaires par H. pylori peut aussi affecter les propriétés des CSCs. La délétion de IQCAP1, protéine d'échafaudage au niveau des jonctions et interagissant avec la E-cadhérine, augmente les lésions néoplasiques de l'estomac chez la souris et induit le phénotype TEM ainsi que des propriétés de type CSCs après l'infection à H. pylori (11). H. pylori stimule aussi la population de cellules souches gastriques Lrig1+ de manière dépendante de CagA montrant la capacité de cette bactérie à réguler le phénotype souche des cellules gastriques. Les cellules Lrig+ sont enrichies dans les tissus humains de GC par rapport à la muqueuse gastrique normale (4).

Le rôle des CSCs gastriques dans la chimiorésistance, la récidive tumorale et les métastases font de leur compréhension une étape clé dans l'étude des GC et dans la recherche de biomarqueurs et de nouvelles thérapies.

Le cancer gastrique : le microenvironnement tumoral

Une tumeur est une entité dynamique constituée de cellules tumorales en communication constante avec l'environnement les entourant et riche en facteurs permettant et maintenant leur phénotype tumoral ainsi que leur hétérogénéité. Lorsqu'ils sont activés, les fibroblastes associés au cancer gastrique (GCAFs) sont capables de promouvoir la migration et l'invasion ~354~ des cellules cancéreuses gastriques. La présence de GCAFs est corrélée à la faible survie des patients atteints de GC et in vitro, l'action paracrine de facteurs produits par les GCAFs rend les cellules tumorales gastriques résistante au traitement conventionnel par le 5-fluorouracile (5-FU) (32) (33). L'utilisation d'anticorps neutralisant anti-IL-6, produite en grande quantité par les GCAFs, et d'inhibiteurs de la voie JAK2/STAT3 diminue l'effet des GCAFs sur les GC ainsi que les métastases péritonéales induites par les GCAFs in vivo (34) (35). La Rspondin stromale produite par les myofibroblastes gastriques contrôle l'homéostasie de l'épithélium gastriques en activant la prolifération de cellules souches gastriques Lgr5+. H. pylori augmente l'expression de R-spondin 3 causant ainsi une hyper-prolifération et une hyperplasie de la glande gastrique (36). En présence de H. pylori, R-spondin 3 permet aux cellules Lgr5+ de sécréter des protéines antimicrobiennes comme Interlectine-1 (Itln1) qui se lie à la bactérie et cause leur agglutination. La signalisation R-spondin-Lgr5 dans l'estomac protège donc la base de la glande contre les agents infectieux (36).

Les exosomes, petites vésicules extracellulaires produites par les cellules, transportent des protéines ainsi que des ARNs et des miRNAs qui restent protégés de la dégradation par les RNAses. Les cellules cancéreuses ou les CAFs utilisent les exosomes pour communiquer et échanger leur matériel. Des études montrent que les miRNAs peuvent être identifiés dans les exosomes permettant la classification des patients selon le stade de la maladie. L'utilisation des exosomes comme biomarqueurs semble être une stratégie intéressante pour le diagnostic et le pronostique des GC qui va surement évoluer dans les années à venir (37). Les spécificités du microenvironnement tumoral peuvent aussi être utilisées comme biomarqueurs et comme stratégie thérapeutique contre les GC. Les GCAFs induisent l'expression de DDR1 dans les cellules tumorales gastriques. L'inhibition pharmacologique des cellules GC DDR1+ dans des cellules surexprimant DDR1 inhibe la récurrence péritonéale induite par les GCAFs (38). Durant le processus de métastases, des cellules tumorales circulantes (CTC) provenant ~355~ de la tumeur primaire ou des métastases peuvent être retrouvées dans le sang circulant (39).

Dans le cancer gastrique, la présence de CTC CD44+ a été corrélée aux métastases et aux récidives tumorales. Certaines études montrent que l'ADN tumoral circulant a une valeur diagnostic et pronostique plus sensible que les CTCs (39).

Le cancer gastrique : le microbiote

L'estomac a longuement été considéré comme étant un environnement stérile et difficile pour la survie de microorganismes. Des données montrent que l'estomac abrite une large et diverse communauté bactérienne avec des densités de colonisations variant entre 10 1 et 10 3 unités de colonies/g (40). A ce jour, peu d'études ont examiné les différences dans la composition du microbiote et le devenir des GC. Une des étapes clé dans la progression histologique vers le GC de type intestinal est le développement de la gastrite chronique atrophique, une condition qui prédispose l'estomac à une augmentation du pH gastrique dû à la perte des cellules pariétales et à la surcroissance du microbiote non-Helicobacter (1). Cette hypochlorhydrie de l'estomac facilite la colonisation par des bactéries et peut promouvoir la progression des GC, confirmant l'influence du microbiote sur la cancérogenèse gastrique. Une étude réalisée en 2011 dans le modèle de souris INS-GAS de cancer gastrique spontané montre que les lésions gastriques mettent 13 mois de plus à se développer quand les souris sont axéniques par rapport à des souris exemptes de pathogènes spécifiques (41). De plus, les auteurs ont montré que la mono-association de H. pylori accélère la gastrite ainsi que le développement de dysplasies et de GIN tandis que l'infection à H. pylori de souris INS-GAS ayant un microbiote complexe induit des lésions gastriques moins sévères et une apparition de GIN plus tardive. Ces données prouvent que le microbiote influence la cancérogenèse gastrique et ont été confirmées par d'autres travaux montrant que la colonisation gastrique avec un microbiote commensal restreint a les mêmes effets sur les lésions néoplasiques que la présence d'un microbiote intestinal diversifié (42).

~356~

En outre, une étude récente décrivant le microbiote de patients atteints de GC et le comparant à ceux de patients atteints de gastrite chronique, démontre que le microbiote du GC est caractérisé par une diminution de la diversité microbienne, de l'abondance de Helicobacter et par un enrichissement en bactéries d'autres genres, plus particulièrement des commensales intestinales. Parallèlement, l'analyse des caractéristiques fonctionnelles du microbiote a révélé que le microbiote de patients atteints de GC était compatible avec la présence d'une communauté microbienne nitrosante. Ainsi, les patients souffrant de GC ont une communauté microbienne dysbiotique avec un potentiel génotoxique différent de ceux de patients atteints de gastrite (42). Une étude Taiwanaise a caractérisé les espèces bactériennes associées à l'épithélium gastrique de patients à différentes étapes de la cascade de Correa (gastrite, métaplasie intestinale et cancer gastrique) (43) et a montré que la composition générale du microbiote des patients atteints de gastrite était similaire à ceux atteints de métaplasie intestinale. Les patients avec un GC avaient très fréquemment et abondamment H. pylori mais aussi des Clostridiums, des Fusobacteriums et des Lactobacillus, correspondant à une signature cancer-spécifique. Une autre étude a ensuite comparé le microbiote de patients atteints de GC à celui de témoins ayant une dyspepsie fonctionnelle (44). Les auteurs ont montré que plusieurs taxons de bactéries étaient enrichis dans les patients GC, plus particulièrement des espèces bactériennes orales pro-inflammatoires, produisant de l'acide lactique et des acides gras à chaines courtes.

Bien que plusieurs études aient montré l'influence du microbiote gastrique sur la cancérogenèse gastrique, celui-ci n'est pas encore utilisé dans un but thérapeutique ou encore pronostique dans le cancer gastrique. Des études supplémentaires dans ce sens sont largement justifiées.

~357~

Le cancer gastrique : ciblage des GCSCs

Même si la majeure partie de la masse tumorale est chimio-sensible et serait sensible aux immunothérapies émergentes, une partie des cellules composants la masse tumorale, les GCSCs, sont considérées comme résistantes aux traitements et comme étant à l'origine de la récurrence tumorale et des métastases (23) (45). Les signatures moléculaires des GC sont de plus en plus explorées afin de proposer des marqueurs de diagnostic et de pronostique et d'essayer de cibler des protéines ou des voies de signalisation ayant un rôle dans le maintien de ces GCSCs. La présence de CSC CD44+ au front d'invasion des tumeurs gastriques est associée à une faible survie de ces patients et pourrait être utilisée comme marqueur de pronostique (46). La signature moléculaire YAP/TEAD (signature oncogénique de la voie de signalisation Hippo) semble être enrichie dans les GCSCs CD44+. L'effet de la Verteporfine, un médicament approuvé par la FDA, a récemment été étudié dans le contexte des GCSC. La Verteporfine diminue l'activité transcriptionnelle YAP/TEAD, la prolifération cellulaire, l'expression de CD44, le nombre de GCSCs CD44+ALDH high capable de former des tumorsphere in vitro et inhibe la croissance tumorale in vivo dans un modèle murin de xénogreffe. De plus, les cellules tumorales résiduelles sont incapables de former de nouvelles tumorspheres in vitro confirmant la diminution du pool de CSC après traitement in vivo (26).

Le Vismodegib, un antagoniste de la voie de signalisation Shh, a été associé à de la Leucovorin, au 5-FU et à l'oxaliplatine dans le traitement des GC. Les GC CD44+ présentent une surexpression des protéines de la voie Shh liée à une faible survie des patients, améliorée après le traitement combiné (45). De façon similaire, le Napabucasin, un inhibiteur de STAT3, réprimant l'autorenouvellement des CSC et induisant la mort cellulaire dans les GCSCs en ciblant STAT3, a été testé pour le ciblage des GCSCs. Le Napabucasin a été utilisé en combinaison avec le paclitaxel chez des patients avec des tumeurs avancées et a montré une activité antitumorale. Ces études montrent que la combinaison de chimiothérapies avec des stratégies ciblées contre les GCSCs semble être une approche intéressante pour le ~358~ traitement des GC (45). Une étude récente réalisée dans un modèle de xénogreffe orthotopique de GC dérivés de patients, dans lequel des cellules cancéreuses sont greffées directement dans la paroi gastrique de la souris, a évalué l'impact du Buparlisib (BKM120), un inhibiteur de la PI3K de classe 1, sur le développement de métastases à distance. Dans ce modèle, le traitement avec BKM120 inhibait significativement les propriétés des GCSCs in vitro et réduisait le nombre de métastases à distance de la tumeur in vivo (47).

Conclusion

Le cancer gastrique est une maladie multifactorielle complexe. H. pylori reste le facteur de risque majeur des GC. Les GC sont très hétérogènes au niveau histologique et moléculaire.

Une meilleure compréhension des processus fondamentaux, cellulaires et infectieux, à l'origine de cette hétérogénéité tumorale des GC est essentielle afin de pouvoir à terme adapter la stratégie thérapeutique et améliorer l'efficacité des traitements.

⁓Figure 1 :

 1 Figure 1: Graphical representation of gastrointestinal cancers incidence and mortality.

Figure 2 :

 2 Figure 2: Graphical representation of cancer incidence and mortality.

Figure 3 :

 3 Figure 3: Schematical representation of CSC tumorigenic and invasive role in gastric carcinogenesis and metastatic process.

Figure 4 :

 4 Figure 4: Schematical representation of Epithelial to Mesenchymal Transition (EMT) with some of the involved markers.

Figure 5 :

 5 Figure 5: Recapitulation of the thesis project.

Figure 2 :

 2 Figure 2: Graphical representation of cancer incidence and mortality. Estimated cancer incidence and mortality data for France and Mauritius obtained from the International Agency for Research on Cancer -Global Cancer Observatory. These values were updated in 2020.

Figure 1 .

 1 Figure 1. Gastric adenocarcinoma histological and molecular classifications.

Figure 1 .

 1 Figure 1. Gastric adenocarcinoma histological and molecular classifications.

Figure 2 .

 2 Figure 2. Gastric carcinogenesis: a Helicobacter disease.

Figure 2 .

 2 Figure 2. Gastric carcinogenesis: a Helicobacter disease.

26 Figure 3 .

 263 Figure 3. Gastric carcinogenesis: a stem cell disease.

Figure 3 .

 3 Figure 3. Gastric carcinogenesis: a stem cell disease.

  Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 26

Figure 4 .

 4 Figure 4. Gastric carcinogenesis: a microenvironment disease. Most immune cells infiltrating tumours are Tumour Infiltrating Lymphocytes (TILs) consisting of CD3+ Tcells (CD8+ Tcytotoxic and CD4+ Thelper cells) as well as FOXP3+ Treg cells. The number of TILs present in tumours reflects the host immune response mechanism. Indeed, Yu et al. demonstrated that high CD8+ and CD3+ T cells infiltration

Figure 4 .

 4 Figure 4. Gastric carcinogenesis: a microenvironment disease.

Figure 3 :

 3 Figure 3: Schematical representation of CSC tumorigenic and invasive role in gastric carcinogenesis and metastatic process 2 .

Figure 4 :

 4 Figure 4: Schematical representation of Epithelial to Mesenchymal Transition (EMT) with some of the involved markers.

:

  Lastname, F.; Lastname, F.; Lastname, F. Title. Cancers 2022, 14, x. https://doi.org/10.3390/xxxxx Academic Editor: Firstname Last-Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/).

Figure 1 .

 1 Figure 1. Hippo/YAP/TAZ-TEAD oncogenic signalling pathway. Schematical representation of Hippo signalisation network. Regulation of YAP/TAZ effectors by LATS are represented by the blue arrows, endogenous YAP/TAZ-TEAD inhibitors in green and Hippo kinases regulators and partners in pink.

Figure 2 .

 2 Figure 2. Crosstalk of YAP/TAZ with other signalling pathways than Hippo signalisation. YAP/TAZ negative regulation are represented by the blue arrows and positive regulation by the red ones.

Fig. 1

 1 Fig. 1 LIF/LIFR signalling: known aspects. Schematical representation of LIF/LIFR signalisation networks. Phosphorylation of the different receptor subunits involved in the respective signalisation processes are highlighted in red

Cancers 2020, 12 ,Cancers

 12 2011; doi:10.3390/cancers12082011 www.mdpi.com/journal/cancers ~99~

Figure 1 .

 1 Figure 1. The Leukaemia inhibitory factor (LIF)/LIF receptor (LIFR)/JAK/STAT pathway is functional in gastric cancer (GC) and upregulated in gastric cancer stem cells (CSCs). (A) p-STAT3 Tyr705 and STAT3 protein levels in AGS and MKN45 cell lines and GC07 patient-derived xenograft (PDX) cells after time course treatment with 50 ng/mL LIF. Values under each band represent quantification of relative tubulin-normalised protein expression according to band density (whole Western Blot available in Supplementary Figure S4) (B) JAK/STAT targets relative mRNA expressions after

Figure 1 .

 1 Figure 1. The Leukaemia inhibitory factor (LIF)/LIF receptor (LIFR)/JAK/STAT pathway is functional in gastric cancer (GC) and upregulated in gastric cancer stem cells (CSCs). (A) p-STAT3 Tyr705 and STAT3 protein levels in AGS and MKN45 cell lines and GC07 patient-derived xenograft (PDX) cells after time course treatment with 50 ng/mL LIF. Values under each band represent quantification of relative tubulin-normalised protein expression according to band density (whole Western Blot available in

Figure 2 .Figure 2 .

 22 Figure 2. Leukaemia inhibitory factor presents anti-CSC effects in GC. (A) 3D tumoursphere assays carried out on GC cell lines (AGS and MKN45, on the left side of the panel) and PDX cells (GC07, Figure 2. Leukaemia inhibitory factor presents anti-CSC effects in GC. (A) 3D tumoursphere assays carried out on GC cell lines (AGS and MKN45, on the left side of the panel) and PDX cells (GC07, GC10

Figure 3 .

 3 Figure 3. LIF/LIFR signalling potentiates chemotherapy effect on gastric CSCs. (A) Representative immunofluorescence images (i) and quantification (ii) of live MKN45 7-day-old spheres stained with CD44, Aldefluor reagent and Hoechst-33342 compound. White arrows point towards chemo-resistant CD44+/ALDH+Hoechst-cells and yellow arrows point towards differentiated non-CSC CD44-/ALDH-Hoechst+ cells. (B) 3D tumoursphere assays carried out on gastric cancer cell lines and patient-derived xenograft cells, treated (green) or not (blue) with 50 ng/mL LIF for 48 h. Cells were also treated or not with chemotherapy drugs Doxorubicin (DOX) and 5-Fluorouracil (5-FU). Cells treated with chemotherapy only are represented in light blue and those with combined LIF and chemotherapy treatments are in light green. LIF treatments were carried out every 48 h and tumoursphere counting was performed after 7 days. n = 3, * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001 versus respective untreated controls with ANOVA statistical analyses, colours representing the matched coloured bars comparison (A(ii)). $ p < 0.05, $$ p < 0.005, $$$ p < 0.0005 and $$$$ p < 0.0001 versus conditions treated with DOX or 5-FU alone with Student t-tests.

Figure 3 .

 3 Figure 3. LIF/LIFR signalling potentiates chemotherapy effect on gastric CSCs. (A) Representative immunofluorescence images (i) and quantification (ii) of live MKN45 7-day-old spheres stained with CD44, Aldefluor reagent and Hoechst-33342 compound. White arrows point towards chemo-resistant CD44+/ALDH+Hoechst-cells and yellow arrows point towards differentiated non-CSC CD44-/ALDH-Hoechst+ cells. (B) 3D tumoursphere assays carried out on gastric cancer cell lines and patient-derived xenograft cells, treated (green) or not (blue) with 50 ng/mL LIF for 48 h. Cells were also treated or not with chemotherapy drugs Doxorubicin (DOX) and 5-Fluorouracil (5-FU). Cells treated with chemotherapy only are represented in light blue and those with combined LIF and chemotherapy treatments are in light green. LIF treatments were carried out every 48 h and tumoursphere counting was performed after 7 days. n = 3, * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001 versus respective untreated controls with ANOVA statistical analyses, colours representing the matched coloured bars comparison (A(ii)). $ p < 0.05, $$ p < 0.005, $$$ p < 0.0005 and $$$$ p < 0.0001 versus conditions treated with DOX or 5-FU alone with Student t-tests.

25 Figure 4 .

 254 Figure 4. Leukaemia inhibitory factor activates the Hippo pathway tumour suppressor core. (A) Hippo suppressors LATS2 and p-LATS1/2 Thr1079/1041 and Hippo effectors YAP, p-YAP Ser127 and TAZ's protein expression in AGS and MKN45 cell lines. Values under each band represent quantification of relative GAPDH or tubulin-normalised protein expression according to band density (whole Western Blot available in Supplementary Figure S4)(B) Representative immunofluorescence images of AGS and MKN45 cells stained with anti-YAP or anti-TAZ antibodies (green). All cells were marked

Figure 4 .

 4 Figure 4. Leukaemia inhibitory factor activates the Hippo pathway tumour suppressor core. (A) Hippo suppressors LATS2 and p-LATS1/2 Thr1079/1041 and Hippo effectors YAP, p-YAP Ser127 and TAZ's

25 Figure 5 .Figure 5 .

 2555 Figure 5. LIF anti-CSC effects are linked to Hippo pathway core kinases activation. (A) Hippo kinase LATS2 and p-LATS1/2 Thr1079/1041 and JAK/STAT effector STAT3 and p-STAT3 Tyr705 protein expression in AGS and MKN45 cell lines. Values under each band represent quantification of relative GAPDH or tubulin-normalised protein expression according to band density (whole Western Blot available in Figure S5). (B) TEAD 8xCTIIC-luciferase reporter assay showing activity of transcription factor TEAD in AGS and MKN45 cell lines. (C) Relative Hippo target genes mRNA levels in AGS (2D) and MKN45Figure 5. LIF anti-CSC effects are linked to Hippo pathway core kinases activation. (A) Hippo kinase LATS2 and p-LATS1/2 Thr1079/1041 and JAK/STAT effector STAT3 and p-STAT3 Tyr705 protein expression

Figure 6 .

 6 Figure 6. LIF-induced chemotherapy potentiating anti-CSC effects pass through the Hippo pathway. (A-B) 3D tumoursphere assays carried out on GC cell lines (A) and patient-derived xenograft cells (B), treated (green) or not (blue) with 50 ng/mL LIF for 48 h. Cells were also treated or not with chemotherapy drugs Doxorubicin (DOX) and 5-Fluorouracil (5-FU), Hippo kinase inhibitor XMU-MP-1 (XMU) and JAK1 inhibitor Ruxolitinib (Ruxo). Cells treated with chemotherapy drugs only are represented in light blue, while cells treated with LIF in combination with chemotherapy drugs and/or inhibitors are in light green. XMU-treated conditions are represented by dotted bars and Ruxo-treated conditions by checked bars. Treatments were carried out every 48 h and tumoursphere counting was performed after 7 days. * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001 versus untreated controls with ANOVA statistical analyses. $ p < 0.05, $$ p < 0.005, $$$ p < 0.0005 and $$$$ p < 0.0001 versus conditions treated with DOX or 5-FU alone with Student t-tests.

Figure 6 .

 6 Figure 6. LIF-induced chemotherapy potentiating anti-CSC effects pass through the Hippo pathway. (A-B) 3D tumoursphere assays carried out on GC cell lines (A) and patient-derived xenograft cells (B), treated (green) or not (blue) with 50 ng/mL LIF for 48 h. Cells were also treated or not with chemotherapy drugs Doxorubicin (DOX) and 5-Fluorouracil (5-FU), Hippo kinase inhibitor XMU-MP-1 (XMU) and

Figure 7 .

 7 Figure 7. LIF and LIFR in GC and associated prognosis. (A) Oncomine data-mining analysis showing level of LIFR mRNA in (i) gastric adenocarcinoma compared with normal gastric mucosa in the Cui (n = 90) and Cho (n = 160) gastric tissue datasets and (ii) normal tissue compared with gastric cancer Lauren classification subsets (diffuse, intestinal and mixed type GC) in the Cho dataset. (B) KMplot database analysis showing overall survival probability of diffuse type and intestinal type gastric adenocarcinoma patients (23 < n < 175) according to LIFR and LIF and combined LIFR and LIF (LIFR + LIF) expression levels. * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001 versus untreated

Figure 7 .

 7 Figure 7. LIF and LIFR in GC and associated prognosis. (A) Oncomine data-mining analysis showing level of LIFR mRNA in (i) gastric adenocarcinoma compared with normal gastric mucosa in the Cui

  : Effect of JAK1 inhibition and Hippo kinase inhibition on expression of members of the Hippo and JAKSTAT pathway, Figure S3: Effect of LIF treatment on proliferation and viability of gastric cancer cells, Figure S4: raw data of Figures 1A and 4A western blots, Figure S5: raw data of Figure 5 and supplementary Figure S2 western blots, Table S1: List of primers used for RT-qPCR analysis. Author Contributions: Conceptualisation, L.S., S.M.-C., C.S., H.B., P.D. and C.V.; data curation, L.S., J.G., S.M.-C., E.S. and C.B.; formal analysis, L.S., C.B. and O.C.B.M.; funding acquisition, C.S., F.M., P.L., H.B., P.D. and C.V.; investigation, L.S., J.G., S.M.-C., E.S. and C.B.; methodology, L.S., J.G., S.M.-C., E.S., C.T., H.B., P.D. and C.V.; project administration, P.D. and C.V.; resources, J.G., E.S., C.T., F.M. and P.L.; supervision, O.C.B.M., P.D. and C.V.; validation, L.S., P.D. and C.V.; visualisation, L.S.; writing-original draft, L.S. and C.V.; writing-review and editing, J.G., C.S., P.L., O.C.B.M., H.B., P.D. and C.V. All authors have read and agreed to the published version of the manuscript.

Figure S1 .

 S1 Figure S1. Effect of LIF treatment on JAKSTAT pathway and CSC markers expression in MKN45 cells. ( A) p-STAT3 Tyr705 and STAT3 protein levels in MKN45 cell line after treatment with 50 ng/mL LIF at different time intervals (0, 0.5, 5, 24, 48 h). Values under each band represent quantification of relative tubulin-normalised protein expression according to band density. (B) JAK/STAT targets relative mRNA expressions after treatment of MKN45 cells with (green) or without (blue) LIF at different tile intervals (0, 0.5, 2, 5, 24, 48 h). (C) Dot plot representation (left panel) and quantification (right panel) of flow cytometry analysis of gastric CSCs possessing cells distribution among CD44+/high (P1) and CD44-/low (P2) cells. Mean +/-SEM is represented in each quadrant of the dot plot graphs. n = 3, *p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001 vs. untreated controls with Mann-Whitney, Student t-tests and ANOVA statistical analyses.

Figure S2 .Figure S3 .~125~Figure S4 :~126~Figure S5 .

 S2S3S4S5 Figure S2. Effect of JAK1 inhibition and Hippo kinase inhibition on expression of members of the Hippo and JAKSTAT pathway. A) p-LATS1/2 Thr1079/1041 , LATS2, p-STAT3 Tyr705 and STAT3 protein levels in AGS cell line after 24 h treatment with 0.5 µM XMU-MP-1 (XMU) (i) or 1 µM Ruxolitinib (Ruxo) (ii).

  The aim of this study was to decipher the role of LIF on invasive properties of GC cells and the signalling mechanisms involved. We have shown that LIF decreased invasive properties of GC cell lines and patient-derived xenograft (PDX) cells and more especially of gastric CSCs in vitro through activation of the Hippo kinases and repression on YAP/TAZ oncogenic signalling. LIF's anti-invasive involves the repression of the Epithelial to Mesenchymal Transition (EMT) program in CSCs, important in GC metastatic process. Finally, LIF-treated cells are less keen to form metastasis compared to non-treated cells when injected in mice blood circuit.

  Slides were washed and scanned immediately using Agilent G2505C Microarray Scanner and Agilent Scan Control A.8.5.1 software. Agilent Feature Extraction software v10.10.1.1 was used with default parameters for fluorescence signal extraction and only the most expressed probes values were represented. T-test statistical analysis was carried out to compare differentially expressed genes.

Figure 1 .

 1 Figure 1. Leukaemia inhibitory factor presents an EMT-inhibiting signature in GC cells.

Figure 2 .

 2 Figure 2. Leukaemia inhibitory factor decreases EMT-associated transcription factors

Figure 3 .

 3 Figure 3. Leukaemia inhibitory factor represses EMT phenotype and invasion capacity

Figure 4 .

 4 Figure 4. Leukaemia inhibitory factor decreases invasion of gastric Cancer Stem Cells.

Figure 5 .

 5 Figure 5. Leukaemia inhibitory factor decreases EMT markers in invasive gastric CSCs.

Figure 6 .

 6 Figure 6. LIF presents anti-metastatic properties in vivo and affects organs colonisation.

Figure 7 .

 7 Figure 7. LIFR is downregulated in GC. LIFR expression scored on TMA from GC

  Gastric carcinomas (GC) are heterogeneous tumors, composed of a subpopulation of cluster of differentiation-44 (CD44)+ tumorigenic and chemoresistant cancer stem cells (CSC). YAP1 and TAZ oncoproteins (Y/T) interact with TEA domain family member 1 (TEAD) transcription factors to promote cell survival and proliferation in multiple tissues. Their activity and role in GC remain unclear. This work aimed to analyze Y/T-TEAD activity and molecular signature in gastric CSC, and to assess the effect of verteporfin, a Food and Drug Administration-approved drug preventing Y/T-TEAD interaction, on gastric CSC tumorigenic properties. Y/T-TEAD molecular signature was investigated using bioinformatical (KmPlot database), transcriptomic and immunostaining analyses in patient-derived GC and cell lines. Verteporfin effects on Y/T-TEAD transcriptional activity, CSC proliferation and tumorigenic properties were evaluated using in vitro tumorsphere assays and mouse models of patientderived GC xenografts. High expressions of YAP1, TAZ, TEAD1, TEAD4 and their target genes were associated with low overall survival in nonmetastatic human GC patients (n = 444). This Y/T-TEAD molecular signature was enriched in CD44+ patientderived GC cells and in cells resistant to conventional chemotherapy. Verteporfin treatment inhibited Y/T-TEAD transcriptional activity, cell proliferation and CD44 expression, and decreased the pool of tumorsphere-forming CD44 + /aldehyde dehydrogenase (ALDH) high gastric CSC. Finally, verteporfin treatment inhibited GC tumor growth in vivo; the residual tumor cells exhibited reduced expressions of CD44 and ALDH1, and more importantly, they were unable to initiate new tumorspheres in vitro. All these data demonstrate that Y/T-TEAD activity controls gastric CSC tumorigenic properties. The repositioning of verteporfin targeting YAP1/TAZ-TEAD activity could be a promising CSC-based strategy for the treatment of GC.

  MKN45 and MKN74 gastric epithelial cell lines were routinely verified mycoplasma free (by PCR) and were tested and authenticated by short tandem repeat (STR) profiling (last STR profiling report, October 2015; LGC Standards, 2256 Verteporfin targets gastric cancer stem cells Int. J. Cancer: 146, 2255-2267 (2020) © 2019 UICC Molecular Cancer Biology

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Figure 1 .

 1 Figure 1. Y/T-TEAD transcriptional activity is increased in CSC CD44-positive cells. (a) Representative images of YAP1 and CD44 detection by immunohistochemistry performed on 3 μm serial tissue sections of human primary GC. GC20, GC04 and GC44 are intestinal histological subtype GC; GC06 is a diffuse type GC. Scale bars, 50 μm. (b) List of genes differentially expressed in epithelial CD44+ cells sorted by FACS compared to epithelial CD44-cells in six PDX cases. Each column corresponds to a sample (n = 1 for GC06, GC07, GC35 and GC44; n = 3 independent experiments for GC04 and GC10) and each line represents a gene. Genes were classified in three groups: CSC markers, Y/T target genes and Y/T effectors, and they were ranked according to the level of significance. p Values were determined with the paired t-test (c). Relative mRNA expression levels of CSC, Y/T target genes and effectors were determined by RT-qPCR in the GC10 case based on the expression of CD44 (CD44+ cells, black bars; CD44-cells, white bars). Data represent mean values AE SEM (n = 3). *p < 0.05 vs. CD44-cells; Mann-Whitney test. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2 .

 2 Figure 2. Chemoresistant CSC overexpressed YAP1/TAZ and their target genes. MKN45 and MKN74 cell lines were grown for 24 hr and treated without (0, control black bars) or with 0.5-100 μM 5-fluorouracil (5-FU) and 0.01-1 μM doxorubicin (DOXO) for 48 hr. (a) Percentage of viable cells was assessed by trypan blue exclusion. (b) Chemoresistant CD44+ cells were quantified by flow cytometry. The dot-plots represent the results of DOXO treatment at 1 μM and 5-FU treatment at 50 μM in MKN45 cells and 100 μM in MKN74 cells. (c) Relative mRNA expression levels of CD44, YAP1, TAZ, CTGF and CYR61 were quantified by RT-qPCR (relative to control). In (b) and (c), doses of 5-fluorouracil and doxorubicin were 50 and 1 μM, respectively. Data represent mean values AE SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; Mann-Whitney test. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3 .

 3 Figure 3. VP treatment impaired cell growth and Y/T-TEAD activity in GC cell lines and PDX. Cells were grown for 24 hr and treated with VP at different concentrations or with DMSO as a control for 24 hr (c, d) and 48 hr (a, b, e). (a) Analysis of cell growth was determined by trypan blue exclusion method. Percentage of cells vs. DMSO-treated control cells after 48 hr treatment. (b) Analysis of cell cycle progression determined after propidium iodide staining and flow cytometry analysis on MKN45 (top panel) and MKN74 (bottom panel) cell lines. (c) Effect of VP treatment on Y/T-TEAD transcriptional activity measured by TEAD-luciferase assay. (d, e) Relative mRNA expression of the main Y/T-TEAD target genes and effectors determined by RT-qPCR in MKN45 and MKN74 cell lines (d) and in GC10 and GC04 PDX cells cultured as tumorspheres (e). Data represent mean values AE SEM (n = 3-4 independent experiments). *p < 0.05 compared to DMSO-treated controls in one-way ANOVA tests.

Figure 4 .

 4 Figure 4. VP treatment impaired tumorsphere initiation and growth in GC cell lines and PDX. GC cell lines and PDX were treated with DMSO as control or with increasing VP concentrations (0.1, 1 and 5 μM). (a) Analysis of cell growth determined by trypan blue exclusion method of CD44+ and CD44-FACS-sorted cells after 48 hr of treatment. (b) Representative images of CD44, Ki67 and TAZ (on MKN45) or YAP1 (on MKN74) expression determined by multicolor immunofluorescence analysis. Colored images correspond to the merge of CD44 staining detected in red (using mouse anti-CD44-PE antibodies), with Ki67 staining detected in green (using mouse anti-Ki67 antibodies) and either TAZ staining in MKN45 or YAP1 staining in MKN74, both detected in white (using rabbit anti-TAZ or rabbit anti-YAP1 antibodies) and the nuclei detected in blue (using DAPI). Scale bars, 10 μm. (c-f ) VP or DMSO were added during cell seeding (c, e, f ) or on 3-6-day old established tumorspheres (d); the number of tumorspheres was determined 5-7 days (c) and 3 days after treatment (d). Representative images of tumorspheres formed by MKN45 and GC04 cells (e) and their relative size (f ) after treatment with DMSO or 1 μM VP. Data represent the min to max (n ≥ 9 wells). *p < 0.05 compared to DMSO-treated control in one-way ANOVA test. Scale bars, 100 μM. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5 .

 5 Figure 5. VP inhibits the expression of CSC markers in GC cell lines and PDX. (a, b) Relative mRNA expression levels of gastric CSC markers were determined by RT-qPCR on adherent cells treated for 24 hr (a) and on PDX tumorspheres treated for 48 hr (b) with increasing VP concentrations. Tumorspheres formed after 72-96 hr of culture were treated for 48 hr with 1 μM VP or DMSO as a control (c, d). (c) Representative images of CD44 expression and ALDH activity determined by immunofluorescence analysis on live GC10-treated tumorspheres. Colored images correspond to the merge of CD44 staining detected in red (using anti-CD44-APC antibodies), with the ALDH activity detected in green (using the ALDEFLUOR reagent) and the nuclei detected in blue (using DAPI). Scale bars, 10 μm. (d) Cells dissociated from MKN45 and GC10-treated tumorspheres were stained with ALDEFLUOR reagent and analyzed by flow cytometry. Representative dot-plot images of flow cytometry analysis are in the left panel and quantification in the right panel. (a, b, d) Data represents mean values AE SEM (n ≥ 3). *p < 0.05 compared to the DMSO-treated control in one-way ANOVA test (a, b) and Mann-Whitney tests (d). [Color figure can be viewed at wileyonlinelibrary.com]

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Figure 6 .

 6 Figure 6. Inhibitory effect of VP on gastric tumor growth and CSC in a murine xenograft model. In all, 100,000 cells of MKN45 GC cell line and GC10 PDX were subcutaneously xenografted into NSG mice (5 ≤ n ≤ 10). When tumors reached ~200 mm 3 , DMSO (control) or VP was injected at the tumor periphery daily for 13-17 days. (a) Tumor size was recorded in MKN45 mice treated with 0 (black curve), 0.4 (light grey curve) and 2 mg/kg of VP (dark grey curve) (n = 5 mice). At the end of the treatment, tumors were collected, weighed (b), processed for histology and stained for the expression of CSC markers, CD44 and ALDH1, and proliferation markers, Ki67 and PCNA (c). (c) Representative images and (d) quantification of the percentage of positive cells (n = 10 mice for MKN45 and n = 8 mice for GC10). MKN45-treated tumors were dissociated to evaluate the tumorsphere forming capacity of residual cells (e). (a, d) Data represent mean values AE SEM. (b, e) Data represent the min to max (n ≥ 30 wells). *p < 0.05, ***p < 0.001 compared to DMSO-treated control in Mann-Whitney tests. [Color figure can be viewed at wileyonlinelibrary.com]

  Int. J. Cancer: 146, 2255-2267 (2020) © 2019 UICC

focussed on the: 1 )

 1 Exploration and characterisation of CTCs and/or CTC clusters in GC patients' blood samples; 2) Define the role of MDSC in the acquisition or promotion of CSC tumorigenic and invasion properties in GC. My role in this project was to develop the CTC-collection method from GC patient blood sample as well as the immunofluorescence staining. The MDSC part of the project was carried out in collaboration with the Team of Nicolas Larmonier and Charlotte Domblides from Immunoconcept CNRS UMR 5164. I had the opportunity to participate to the training and work of the PhD and Master students from this team, Céline Blaye, Florent Peyraud and Thomas Boyer and share our CSC expertise. Yasmine Pobiedonoscew was another Master student from our team to whose training I participated, and who contributed to the following work in collaboration with Immunoconcept team.

2. 2

 2 CTC immunofluorescence stainingCTC staining was carried out directly on the filters. A 15 min blocking step was carried out with 1%-BSA 2%-FBS in tris-buffered saline (TBS) solution, followed by incubation with fluorophore-coupled antibodies: CD45-APC (HI30 clone, ThermoFisher Scientific, Villebon sur Yvette, France), CD44-PE (BD Biosciences, Le Pont de Claix, France) and EPCAM-FITC (VU-1D9 clone, ThermoFisher Scientific) for 1 h. The cells were then rinsed before mounting with DAPI-containing mounting medium. Filters were allowed to dry overnight at room temperature before observation with an Eclipse 50i epi-fluorescence microscope (Nikon, Champigny sur Marne, France) using the NIS-BR acquisition software and a x40 objective (numerical aperture, 1.3).

  Phenotypic characterisation was carried by flow cytometry analysis after cell staining with CD45, CD33, CD11b, CD14, CD15, HLA-DR, CD206, Lox1, a viability marker and lineage markers (CD3, CD19 and CD56). Cell and data analysis was carried out with FACSCantoII cytometer and Diva software v8.0.1 (both from BD Biosciences). Functional characterisation was done by assessing the immunosuppressive ability of HuMoSCs that is their capacity to inhibit T-cell proliferation. Total CD4+ T cells or total T cells were purified from fresh PBMCs by magnetic cell sorting with human CD4+ cell isolation kits and MidiMACS Columns (Miltenyi Biotech) and were stained with Cell Trace Violet (Life Technologies), all according to the manufacturers' procedures. Labelled cells were cultured with anti-CD3/CD28-coated T-cell expander beads (Dynabeads, Life Technologies) with or without HuMoSC at different Tcell/HuMoSC ratios (1:1, 2:1, 4:1). T-cell proliferation was detected after 5 days by flow cytometry.

~192~

2. 8 3D

 8 Live immunofluorescence AssayMKN45 (5000) cells were plated in 6-well plates in 3D non-adherent conditions and HuMoSC cells were added at the same time. 5-days spheres were recovered and incubated with 1:25 antihuman CD44v6-APC antibody (Biotechne R&D systems, Minneapolis, Canada), 1:25 antihuman CD45-PE (HI30 clone, ThermoFisher Scientific), 1:50 anti-human EPCAM-FITC (VU-1D9 clone, ThermoFisher Scientific) in ice-cold buffer containing PBS-0.5% bovine serum albumin (BSA, Gibco, ThermoFisher Scientific)-2 mM EDTA (Sigma-Aldrich, Saint-Quentin Fallavier, France) for 25 min at 4°C. Spheres were rinsed twice with ice-cold buffer and incubated for 30 min at RT with Hoescht-33342 dye (ThermoFisher Scientific).

Figure 1 :

 1 Figure 1: Representative images of CD45-EPCAM+ gastric CTCs isolated from 3 mL blood sample from 3 GC patients (PA). CD45+ immune cells are represented in yellow, CD44+ cells in red, EPCAM+ cells in green. Nuclei are stained with DAPI. Arrows point out CTCs which are either single (yellow arrows) or in clusters with other CTCs (green arrows) or with immune cells (white arrows). Scale bars: 20 µm

Figure 2 :

 2 Figure 2: MKN45 -HuMoSCs tumourspheres. (A) Tumoursphere assays evaluating initiating (i) or promoting (ii) effects on GC CSCs. MKN45: HuMoSCs ratios were compared to conditions with only MKN45 cells. (B) Representative images of live immunofluorescence

: 5

 5 HuMoSCs and FACS sorted to collect CD14-cells corresponding to GC cells, were seeded on top of collagen-coated Transwell® inserts. The number of invaded cells were counted after 18 h. Interestingly preliminary results demonstrate an increase in number of invasive cells after contact with HuMoSCs demonstrating the acquisition of an invasive phenotype by GC cells (Figure 3A (i)). Furthermore, to check whether change in invasion capacity of GC cells in presence of HuMoSCs implicated an increase in CSC invasion capacity, 3D invasion assays were accomplished after concomitant 1 MKN45 : 5 HuMoSC cell seeding and 3-days CSC selection and tumoursphere formation. Type-I collagen embedding was carried out and CSC invasion was calculated as explained earlier. HuMoSC presence induced a significant increase in invasion capacity of CSCs showing a pro-invasive role of HuMoSCs (Figure 3A (ii)).

Figure 3 :

 3 Figure 3: MKN45 -HuMoSCs invasion. (A) Invasion assays evaluating effects (i) on GC cells (ii) CSCs. MKN45: HuMOSCs ratios were compared to conditions with only MKN45 cells. n=1 for (i) n = 3 for (ii), * p < 0.05, ** p < 0.005 versus MKN45 using Mann-Whitney or T-test statistical analysis. These experiments were carried out by Yasmine Pobiedonoscew with CélineBlaye from Immunoconcept Team

  characterisation. The classical staining protocol adapted till now is broad immune cell detection by CD45, CD44 stain for gastric CSCs, but it also stains immune cells and EPCAM to detect tumoral cells. This has allowed the detection of 5-20 CTCs per 3 mL blood sample depending on the patient analysed. Further correlation with clinico-biological data is necessary to determine prognosis according to CTC enumeration. Interestingly, CTCs were detected single or in clusters with themselves or immune cells. The next step is now to test more specific immunostaining of immune cells to allow the characterisation of specific immune-CTC clusters for example those with MDSCs by using anti-CD14 and anti-CD33.

Figure 5 :

 5 Figure 5: Recapitulation of the thesis project. The main results are resumed in boxes 1-5. LIF and VP parts are presented in green and MDSC part in maroon.

Figure 1 . 15 Figure 1 .

 1151 Figure 1. Development of patient-derived orthotopic xenografts generating distant metastases. (A) Schematic representation of the injection of patient-derived subcutaneous xenografts of GC (PDX) cells into the subserosa of the stomach of NSG mice. Cells were previously transduced with lentiviruses encoding the luciferase gene (MND-luciferase) and were then amplified subcutaneously before orthotopic xenograft. (B) Representative images of bioluminescence imaging on live animals at 6.5 weeks post-orthotopic xenograft. (C) Photon quantification of GC07 and GC10 tumor growth on live animals (upper panels) or on recovered organs at end points (lower panels). GC07, n = 15 Figure 1. Development of patient-derived orthotopic xenografts generating distant metastases. (A) Schematic representation of the injection of patient-derived subcutaneous xenografts of GC (PDX) cells into the subserosa of the stomach of NSG mice. Cells were previously transduced with lentiviruses encoding the luciferase gene (MND-luciferase) and were then amplified subcutaneously before orthotopic xenograft. (B) Representative images of bioluminescence imaging on live animals at 6.5 weeks post-orthotopic xenograft. (C) Photon quantification of GC07 and GC10 tumor growth on live animals (upper panels) or on recovered organs at end points (lower panels). GC07, n = 15 mice; GC10, n = 16 mice. (D) Representative images of the tumor developed in the stomach, and of macro-metastases developed in the lung and the liver for the GC10 case (pointed out by black arrows). * p < 0.05 in one-way ANOVA test.

Figure 2 .

 2 Figure 2. Detection of gastric tumors and distant micrometastases and macrometastases by immunohistochemistry in orthotopic xenograft mice. Representative images of HLA detection by immunohistochemistry on stomach tumors and lung and liver metastases formed with MKN45 cells (A) and GC10 PDX case (B) eight and 10 weeks after orthotopic xenograft, respectively. The enlarged pictures in the lower panel show the HLA-positive gastric adenocarcinoma (GC) cells in the stomach, lung, and liver metastases. Scale bars: 2 mm in the upper panel, 200 µ m in the lower panel.

Figure 2 .

 2 Figure 2. Detection of gastric tumors and distant micrometastases and macrometastases by immunohistochemistry in orthotopic xenograft mice. Representative images of HLA detection by immunohistochemistry on stomach tumors and lung and liver metastases formed with MKN45 cells (A) and GC10 PDX case (B) eight and 10 weeks after orthotopic xenograft, respectively. The enlarged pictures in the lower panel show the HLA-positive gastric adenocarcinoma (GC) cells in the stomach, lung, and liver metastases. Scale bars: 2 mm in the upper panel, 200 µm in the lower panel.

Table 2 .1

 2 Number of tumor-bearing mice per total of NSG mice implanted with gastric cancer cell lines into gastric subserosa. Number of Tumor-Bearing Mice 1 /Number of Xenograft Tumor development was examined macroscopically after sacrifice and by histological analyses of human-specific HLA expression.

Cancers 2019, 11 , x 7 of 19 Figure 3 .

 11193 Figure 3. Cont.

Figure 3 .

 3 Figure 3. Metastases are enriched in cancer stem cells (CSC). (A,B) Quantification (left panel) and representative images (right panel) of the tumorspheres formed in vitro by GC10 CSC (A) and MKN45 CSC (B) derived from primary tumors of the stomach and distant metastases (peritoneal, lung, and liver). The arrows indicate tumorspheres. Data represent the min to max; n ≥ 3 mice with nine replicates per experiment and per mice. * p < 0.05 in one-way ANOVA and Student's t-test. Scale bars, 50 µ m. (C,D) Flow cytometry dot plots (C) and quantification (D) of the percentage of CD44-positive cells in primary stomach tumors and metastases derived from GC10 and GC07 cases. Data represent mean ± SEM. of n ≥ 3 mice in Kruskal-Wallis and Student's t-test. (E) Representative images of CD44 expression detected by immunohistochemistry on tissue sections of stomach tumors and peritoneal, lung, and liver metastases obtained in GC10 and MKN45 orthotopically xenografted mice. Scale bars: 50 µ m.

Figure 3 .

 3 Figure 3. Metastases are enriched in cancer stem cells (CSC). (A,B) Quantification (left panel) and representative images (right panel) of the tumorspheres formed in vitro by GC10 CSC (A) and MKN45 CSC (B) derived from primary tumors of the stomach and distant metastases (peritoneal, lung, and liver). The arrows indicate tumorspheres. Data represent the min to max; n ≥ 3 mice with nine replicates per experiment and per mice. * p < 0.05 in one-way ANOVA and Student's t-test. Scale bars, 50 µm. (C,D) Flow cytometry dot plots (C) and quantification (D) of the percentage of CD44-positive cells in primary stomach tumors and metastases derived from GC10 and GC07 cases. Data represent mean ± SEM. of n ≥ 3 mice in Kruskal-Wallis and Student's t-test. (E) Representative images of CD44 expression detected by immunohistochemistry on tissue sections of stomach tumors and peritoneal, lung, and liver metastases obtained in GC10 and MKN45 orthotopically xenografted mice. Scale bars: 50 µm.

Figure 4 .

 4 Figure 4. Buparlisib (BKM120) inhibits GC cell proliferation and decreases CSC population and tumorigenic properties. Adherent cells were treated with increasing concentrations of BKM120 or with DMSO as control (vehicle) for 48 h and viability assay (A), flow cytometry cell cycle assay (B), and relative mRNA expression levels of genes controlling cell-cycle progression (C) were performed. n = three independent experiments. * p < 0.05 compared to control, one-way ANOVA test. MKN45 (D) and GC10 (E) cells were cultured under non-adherent culture conditions with 1 to 10 µ mol/L BKM120 or DMSO for seven days, and the number of tumorspheres formed by 200 or 1000 seeded cells per well, respectively, were quantified (D,E). Data represent the min to max. n = three independent experiments with nine replicates per condition. * p < 0.05 in one-way ANOVA test. Cells dissociated from MKN45-treated tumorspheres were stained with anti-CD44-APC antibodies and analyzed by flow cytometry (F,G). (F) Representative dot plot images of flow cytometry analyses and (G) quantification of the percentage of CD44-positive cells. Values indicate the mean ± SEM of CD44-positive cells in three independent experiments each performed in duplicates. * p < 0.05 vs. control (DMSO alone), Mann-Whitney test.

Figure 4 .

 4 Figure 4. Buparlisib (BKM120) inhibits GC cell proliferation and decreases CSC population and tumorigenic properties. Adherent cells were treated with increasing concentrations of BKM120 or with DMSO as control (vehicle) for 48 h and viability assay (A), flow cytometry cell cycle assay (B), and relative mRNA expression levels of genes controlling cell-cycle progression (C) were performed. n = three independent experiments. * p < 0.05 compared to control, one-way ANOVA test. MKN45 (D) and GC10 (E) cells were cultured under non-adherent culture conditions with 1 to 10 µmol/L BKM120 or DMSO for seven days, and the number of tumorspheres formed by 200 or 1000 seeded cells per well, respectively, were quantified (D,E). Data represent the min to max. n = three independent experiments with nine replicates per condition. * p < 0.05 in one-way ANOVA test. Cells dissociated from MKN45-treated tumorspheres were stained with anti-CD44-APC antibodies and analyzed by flow cytometry (F,G). (F) Representative dot plot images of flow cytometry analyses and (G) quantification of the percentage of CD44-positive cells. Values indicate the mean ± SEM of CD44-positive cells in three independent experiments each performed in duplicates. * p < 0.05 vs. control (DMSO alone), Mann-Whitney test.

Cancers 2019, 11 , x 10 of 19 Figure 5 .

 11195 Figure 5. Phosphatidylinositol 3-kinase (PI3K) inhibitor BKM120 limits MKN45 tumor growth in the stomach and metastases formation in vivo. Luciferase-encoding MKN45 cells were xenografted into the stomach of NSG mice. After seven days, mice were randomized and received, one day after, 20 mg/kg BKM120 or vehicle once per day on a schedule of five days on, two days off for three weeks. Tumor development on whole animals was monitored twice a week by in vivo bioluminescence imaging. Representative images of bioluminescence imaging on live mice (A) one day before treatment (upper panels) and after 19 days of treatment (bottom panels) and quantification (B). 8 ≤ n ≤ 9 mice. * p < 0.05 vs. vehicle in Student's t-test. #, mouse identification number.

Figure 5 .

 5 Figure 5. Phosphatidylinositol 3-kinase (PI3K) inhibitor BKM120 limits MKN45 tumor growth in the stomach and metastases formation in vivo. Luciferase-encoding MKN45 cells were xenografted into the stomach of NSG mice. After seven days, mice were randomized and received, one day after, 20 mg/kg BKM120 or vehicle once per day on a schedule of five days on, two days off for three weeks. Tumor development on whole animals was monitored twice a week by in vivo bioluminescence imaging. Representative images of bioluminescence imaging on live mice (A) one day before treatment (upper panels) and after 19 days of treatment (bottom panels) and quantification (B). 8 ≤ n ≤ 9 mice. * p < 0.05 vs. vehicle in Student's t-test. #, mouse identification number.

Figure S2 .

 S2 Figure S2. Validation of BKM120 inhibition on AKT Ser473-phosphorylation. Representative western blots of active AKT (pAKT S473 ) and total AKT level with GAPDH as a loading control. MKN45 cells were treated 48 h with increasing concentrations of BKM120 (1, 3 and 10 µ M) and DMSO as a control. BKM120 induces a strong dose-dependent inhibition of AKT phosphorylation on Ser473. MW, molecular weight. Numbers indicate the densitometry intensity ratio (AKT or pAKTs473/ GAPDH compared to no BKM120-treated cells) for each band.

Cells 2020, 9 ,Cells

 9 1462; doi:10.3390/cells9061462 www.mdpi.com/journal/cells ~249~

Figure 1 .Figure 1 .

 11 Figure 1. Helicobacter pylori increased transcriptional co-activator with PDZ binding motif (TAZ) protein expression and activated TEA domain (TEAD) transcriptional activity. (A, B) Basal expression of TAZ and yes-associated protein (YAP) evaluated in five different gastric epithelial cell lines and one patient-derived xenograft cell GC07 by Western blot. Numbers under each band correspond to the relative protein expression quantification normalized to the expression of α-tubulin housekeeping protein. (B) Representation of the relative expression of TAZ compared to that of YAP in the different gastric epithelial cells. (C, D) Time-course analyses of co-cultures performed with the 7.13 wild type (WT) H. pylori strain and gastric epithelial cell lines MKN45, GC07, and NCI-N87 expressing the most important TAZ/YAP ratio. Uninfected condition corresponds to "-". (C) TAZ protein expression determined during the time-course of 7.13 WT H. pylori strain infection by Western blot. Normalization of the quantification was done in comparison to the uninfected condition and α-Figure 1. Helicobacter pylori increased transcriptional co-activator with PDZ binding motif (TAZ) protein expression and activated TEA domain (TEAD) transcriptional activity. (A,B) Basal expression of TAZ and yes-associated protein (YAP) evaluated in five different gastric epithelial cell lines and one patient-derived xenograft cell GC07 by Western blot. Numbers under each band correspond to the relative protein expression quantification normalized to the expression of α-tubulin housekeeping protein. (B) Representation of the relative expression of TAZ compared to that of YAP in the different gastric epithelial cells. (C,D) Time-course analyses of co-cultures performed with the 7.13 wild type (WT) H. pylori strain and gastric epithelial cell lines MKN45, GC07, and NCI-N87 expressing the most important TAZ/YAP ratio. Uninfected condition corresponds to "-". (C) TAZ protein expression determined during the time-course of 7.13 WT H. pylori strain infection by Western blot. Normalization of the quantification was done in comparison to the uninfected condition and α-tubulin housekeeping protein expression. (D) Relative TEAD-luciferase activity determined following 7.13 WT H. pylori infection. Bars represent means ± standard error of the mean (SEM) of fold changes relative to uninfected cells; n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. uninfected control, Kruskall-Wallis test with Dunn's post-test.

Figure 2 .

 2 Figure 2. H. pylori induced TAZ and ZEB1 co-upregulation associated with their nuclear translocation. (A) Expression and subcellular localization of TAZ and ZEB1 assessed by immunofluorescence

Figure 2 .

 2 Figure 2. H. pylori induced TAZ and ZEB1 co-upregulation associated with their nuclear translocation. (A) Expression and subcellular localization of TAZ and ZEB1 assessed by immunofluorescence during the time-course of infection with 7.13 WT H. pylori strain. Representative immunofluorescence images of TAZ (in green), ZEB1 (in red), and nuclei stained with DAPI (in blue) in MKN45, GC07, and NCI-N87 cells. Bars scale, 10 µm. White arrows show TAZ and ZEB1 co-accumulation in the nuclei. (B) The relative amount of TAZ and ZEB1 nuclear accumulation was quantified by the number of positive-vs.

  for GC07 and NCI-N87. Bars represent means ± SEM of fold changes relative to uninfected cells. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. uninfected control in Kruskall-Wallis test with Dunn's post-test. (C) Percentage of cells with mesenchymal "hummingbird" phenotype after H. pylori infection in MKN45, GC07, and NCI-N87 cells. Bars represent means ± SEM; n = 3, * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. uninfected control in ANOVA test with Dunnett's post-test.

3. 3 .

 3 TAZ and ZEB1 Were Co-Upregulated in the Gastric Mucosa of H. pylori-Infected Patients and Mice and in Human Gastric Carcinoma Specimens

Figure 3 .

 3 Figure 3. TAZ and ZEB1 were co-upregulated in gastric epithelial cells of H. pylori-infected mice and patients and in human gastric carcinoma. Representative images of the immunohistochemistry

Figure 3 .

 3 Figure 3. TAZ and ZEB1 were co-upregulated in gastric epithelial cells of H. pylori-infected mice and patients and in human gastric carcinoma. Representative images of the immunohistochemistry staining (dark brown) of TAZ, ZEB1, and Ki67 on serial tissue sections. (A) Representative images of the gastric

Figure 4 .

 4 Figure 4. TAZ conferred epithelial-mesenchymal transition (EMT) phenotype and cancer stem cell (CSC)-like properties in response to H. pylori. (A) Relative mRNA expression of EMT markers Vimentin (VIM), ZEB1, Snail family transcriptional repressor 1 (SNAI1), and Matrix metallopeptidase 9, (MMP9) measured by RT-qPCR in MKN45 and GC07 cells, presented as the ratio of mRNA expression in H. pylori-infected vs. uninfected cells for each condition of silencing RNA (siRNA) transfection. Bars represent mean fold changes ± SEM of 4-6 independent experiments relative to

Figure 4 .

 4 Figure 4. TAZ conferred epithelial-mesenchymal transition (EMT) phenotype and cancer stem cell (CSC)-like properties in response to H. pylori. (A) Relative mRNA expression of EMT markers Vimentin (VIM), ZEB1, Snail family transcriptional repressor 1 (SNAI1), and Matrix metallopeptidase 9, (MMP9) measured by RT-qPCR in MKN45 and GC07 cells, presented as the ratio of mRNA expression in H. pylori

Figure 1 .

 1 FIGURES LEGENDS

Figure 2 .

 2 Figure 2. CD44v3+ cells from GC cell lines and PDX display CSC properties. (A-B)

Figure 3 .

 3 Figure 3. panCD44 and CD44v3 identify distinct CSCs with different EMT/MET

Figure 4 .

 4 Figure 4. ALDH+ cells are mainly panCD44+ and CD44v3-. (A-B) Cumulated

Figure 5 .

 5 Figure 5. CD44v3+ cells drives GC metastasis. (A) Representative images of panCD44

Figure 6 .

 6 Figure 6. CD44v3 expression is associated with invasive stage and poor 5-year survival

Statistical analysis .

 analysis figures. Mann-Whitney test was used to compare two groups of data and Kruskal-Wallis test

Figure S5 .

 S5 Figure S5. Validation of 2 siRNA down-regulating CD44v3 expression. (A) Relative mRNA

Figure S6 .

 S6 Figure S6. CD44v3+ cells represent a subpopulation of CD44V6+ cells. (A-C) Flow

Figure S7 .

 S7 Figure S7. (A-B) Representative images of CD44v3 and CD44v6 isoforms detection by

  Figure 1

  panCD44 expression : % of positive cases / mean score 65% / 0.

  Figure 2

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Fig. 2 .

 2 Fig.2. Models for the role of gastric and enterohepatic Helicobacter species in digestive cancers. Models created with icons from BioRender.com. A) Model for multiple host responses during Helicobacter pylori infection. ① ① H. pylori adhesins and outer membrane proteins OipA, BabA, SabA interact with cell surface molecules. OipA activates EGFR which turns on the AKT-β-catenin cascade to increase cell proliferation. BabA and SabA binding to Lewis antigens triggers cell proliferation and pro-inflammatory signaling pathway. ② ② The secreted VacA cytotoxin is able to insert itself into membranes and form ion-conducting channels leading to internalization, vacuolation and induction of ROS; VacA also inhibits autophagy and activates EGFR cascade favoring proliferation and oncogenic

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Fig. 3 .

 3 Fig. 3. Evolutionary relationships of Helicobacter taxa based on the CdtB protein encoded gene.The phylogeny presented is based on the alignment of full-length CdtB. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The optimal tree with the sum of branch length = 3.50977714 is shown. The phylogenetic analysis was generated with the Neighbor-Joining method[START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]. The tree is not rooted and drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are represented in the units of the number of amino acid substitutions per site. The analysis included 21 amino acid sequences, as H. mastomyrinus, H. mesocricetorum, and H. muridarum full CdtB sequence was not available and excluded from this analysis. All ambiguous positions were removed for each sequence pair. There was a total of 305 positions in the final dataset. Evolutionary analyses were conducted using MEGA6[START_REF] Tamura | MEGA6: molecular evolutionary genetics analysis version 6.0[END_REF]. All sequences are labeled by species, strain name, the species from which they were isolated, and the accession number in brackets. Arrow indicates H. himalayensis, a species isolated from the gastric mucosa of Marmot in China that is phylogenetically clustered within enterohepatic species[START_REF] Hu | Helicobacter himalayensissp. nov. isolated from gastric mucosa ofMarmota himalayana[END_REF][START_REF] Hu | Genomic analysis of Helicobacter himalayensis sp. nov. isolated from Marmota himalayana[END_REF].

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  type strain.C.Varon et al.

  Seeneevassen, Emilie Bessède, Christine Varon INSERM U1053 BaRITON Bordeaux Research in Translational Oncology, team "Helicobacter infection, inflammation and cancer", Université de Bordeaux, Bordeaux, France. Correspondance : INSERM U1053, Université de Bordeaux, 146 rue Léo Saignat, bat. 2B RDC, 33076 Bordeaux cedex, France. Tel. direct: +33(0)5 57 57 95 75. Tel. secrétariat: +33(0)5 57 57 11 19. email: christine.varon@u-bordeaux.fr.

  Cette bactérie a été isolée pour la première fois en 1982 par Robin Warren et Barry Marshall qui ont obtenu en 2005 le prix Nobel de médecine pour cette découverte et pour avoir montré son rôle à ~348~ l'origine de la maladie ulcéreuse (3). La prévalence l'infection par H. pylori diminue progressivement du fait de l'amélioration des conditions socio-économique et d'une diminution de sa transmission ; actuellement, elle concerne environ 50% de la population mondiale et 20% de la population française. L'infection par H. pylori induit une inflammation chronique de la muqueuse gastrique, pouvant évoluer en ulcères gastriques et gastroduodénaux et dans moins d'1% des cas en GC. Néanmoins, la plupart des individus infectés ne développent pas de GC. Ceci peut être expliqué par des différences de susceptibilité de l'hôte, des co-facteurs environnementaux, et par l'hétérogénéité du génome bactérien et des différents facteurs de virulence de H. pylori. L'ilôt de pathogénicité cag (cag PAI) code pour le principal facteur de virulence de la bactérie associé au cancer, la cytotoxine CagA ; des composants proinflamatoires du peptidoglycane, des adhésines (BabA, SabA), des protéines de la membrane externe (OipA, HomB), ainsi que des toxines (VacA) et des protéases sécrétées (TrpA) contribuent également au potentiel carcinogène des souches qui les portent (4). La protéine CagA est la première oncoprotéine d'origine bactérienne décrite (5). CagA est injectée par le système de sécrétion de type IV codé par le cagPAI directement dans le cytoplasme des cellules épithéliales gastriques où elle va interagir avec différentes voies de signalisation. CagA induit une déstabilisation des jonctions cellulaires et de la polarisation apico-basale, l'activation de voies de signalisation proinflammatoires et oncogéniques conduisant à une perturbation de l'intégrité de l'épithélium gastrique, de sa différenciation, et de son autorenouvèlement (4).

D

  'après Hagen et al., la Claudin-18, une protéine des jonctions serrées, est sous-exprimée en réponse à H. pylori dans un modèle murin de GC ainsi que chez les patients atteints de GC chez qui cette perte est associée à un phénotype agressif et un faible pronostic. Des expériences de délétion ciblée du gène codant pour la Claudin-18 montrent une formation de lésions gastriques pré-néoplasiques chez des souris âgées de seulement 7 semaines ; 20 semaines après l'infection, un taux élevé de dysplasie et de néoplasie gastro-intestinale (GIN) ressemblant au carcinome intra-muqueux humain était observée(9).La modification du profil d'expression de molécules d'adhésion cellulaire induit uneTransition Epithélio-Mésenchymateuse (TEM) des cellules épithéliales qui vont perdre une partie de leurs caractéristiques épithéliales pour se transdifférencier et acquérir un phénotype mésenchymateux et motile, associé à une perte de polarité et de cohésion cellulaire, à l'envahissement du tissu et à la dissémination des cellules cancéreuses. L'infection par H.

H

  . pylori régule positivement l'expression et l'activité de nombreuses métalloprotéases ~350~ matricielles (MMPs) dans les cellules tumorales gastriques et augmentent ainsi leurs propriétés invasives. L'utilisation d'inhibiteurs de nombreuses voies de signalisation a révélé le rôle des voies Src, NF-KB, ERK et JNK dans la TEM induite par H. pylori et dans la régulation de l'expression des MMPs (10)(13)(14)(15). H. pylori semble également protéger les cellules épithéliales gastriques de la réponse immunitaire anti-tumorale. L'expression de Programmed Death Ligand 1 (PD-L1) a été détecté sur des cellules tumorales de GC, et H. pylori stimule l'expression de PD-L1 dans les cellules épithéliales gastriques et dans des cultures d'organoïdes de manière CagA dépendante (16). D'autre part, le traitement anti-PD-L1 d'organoïdes gastriques co-cultivées avec des lymphocytes T cytotoxiques de patients et des cellules dendritiques induit la mort des organoïdes. Une étude récente montre que la charge virale en EBV des tumeurs EBVpositives est corrélée à l'expression de PD-L1 et au pronostic, les tumeurs à forte charge virale étant associée à une forte expression de PD-L et à un mauvais pronostic (17). Par ailleurs, une étude de séquençage GWAS (Genome-Wide Association Study) du génome de 173 souches d'H. pylori venant de populations européennes révèle des différences de SNPs de gènes bactériens comme babA et de l'ilôt cagPAI ainsi que de changements non synonymes de plusieurs gènes peu connus, entre des patients GC et des patients souffrant de gastrites. Les auteurs montrent ainsi que les facteurs bactériens diffèrent suffisamment dans cette pathologie pour être détectés par séquençage GWAS, permettant d'associer un profil moléculaire bactérien à une pathologie (18). Le cancer gastrique : une maladie des cellules souches Malgré la forte influence des facteurs environnementaux sur cette maladie, le cancer gastrique reste une maladie hétérogène et multifactorielle tant au niveau histologique que moléculaire. Au niveau intratumoral, les tumeurs gastriques sont également hétérogènes, composées de cellules plus ou moins différenciées et d'une sous-population rare de cellules souches ~351~ cancéreuses (CSC) à l'origine de cette hétérogénéité intratumorale (19)(20)(21). Ces cellules ont des capacités d'auto-renouvellement, de division asymétrique et de différenciation, étant à l'origine de cellules plus ou moins prolifératives et différenciées qui composent la tumeur. Ces cellules, très agressives, sont capables de résister aux thérapies conventionnelles de chimio et de radiothérapies, de migrer via une TEM et d'envahir les tissus et la circulation sanguine et lymphatique pour initier des métastases à distance. Depuis leur découverte, plusieurs méthodes d'études ont été développées basées sur leur capacité à s'autorenouveler, à se différencier et à initier des tumeurs. La culture en 3 dimensions en conditions nonadhérente et dans un milieu défini et sans sérum évalue la capacité des CSC à s'autorenouveler pour former des tumorsphères hétérogènes. Les CSC sont les seules cellules à pouvoir générer de nouvelles sphères après plusieurs passages en culture dans ces conditions (22). De plus, les CSCs sont capables de générer des tumeurs hétérogènes après xénogreffe sous-cutanée chez des souris immunodéficientes in vivo (22) La caractérisation des CSCs gastriques (GCSCs) a aussi permis de recenser des marqueurs enrichis dans ces cellules et permettant de les isoler par la technique de tri cellulaire par cytométrie en flux « Fluorescence-Activated Cell Sorting » (FACS). Nguyen et al. ont décrit le CD44, connu comme étant le récepteur de l'acide hyaluronique, et l'activité aldéhyde déshydrogénase (ALDH) comme étant les principaux biomarqueurs permettant la détection et l'isolement des CSC de GC, sur la base de leur capacité à re-générer des sphères hétérogènes après ensemencement in vitro et des tumeurs après transplantation in vivo chez la souris. De plus, les cellules ALDH+/CD44+ possèdent des pompes à efflux de drogues à l'origine de leur chimiorésistance aux traitements actuels

Table 1 .

 1 Summary of molecules that can be used as potential biomarkers and therapeutic targets for GC.

	Molecule/Strategy	Targets	Known/Tested Use in GC	References
	Anti-CA19-9 antibodies	CA19-9	CA19-9-positive GC biomarker for diagnosis	Matsuoka et al. 2018 [99]
	Cetuximab	EGFR	Potential targeted therapy against tyrosine kinase receptors	Carrasco-Garcia et al. 2018 [98], Apicella et al. 2017 [100]
	Rilotumumab	HGF	Potential targeted therapy against tyrosine kinase receptors	Carrasco-Garcia et al. 2018 [98], Apicella et al. 2017 [100]
	Dovitinib	VEGFR-1/2/3; PDGFR-β; FGFR1/2/3	Potential targeted therapy against tyrosine kinase receptors	Carrasco-Garcia et al. 2018 [98], Apicella et al. 2017 [100]
	Anti-FGFR2 antibodies	FGFR2	Under clinical trial for FGFR2 overexpressing GC	Carrasco-Garcia et al. 2018 [98], Apicella et al. 2017 [100]
	Nimutuzumab	EGFR	Under clinical trial for EGFRhigh GC	Carrasco-Garcia et al. 2018 [98], Apicella et al. 2017 [100]
	Everolimus	mTOR pathway	Under clinical trial for MSI type GC with activating mutations of mTOR pathway members	Carrasco-Garcia et al. 2018 [98], Apicella et al. 2017 [100]
	Anti-HDGF antibodies	HDGF	Potential prognostic marker & target of H. pylori-induced GC	Chu et al. 2019 [101]
	AMS 337	c-Met	Positive results in Phase I clinical trial	Andres et al. 2019 [102]
	MET-binding DARPins	c-Met kinase activity	Potential receptor targeting strategy	Andres et al. 2019 [102]
	Exosomes-delivered c-Met siRNA	c-Met	Potential use as therapy in combination with chemotherapy	Zhang et al. 2020 [103]
	BST2 siRNA	BST2	Inhibits GC cell proliferation and motility -potential anti-GC therapy	Liu et al. 2018 [104]
	Anti-CEACAM6 antibodies	CEACAM6	Potential endoscopic marker for early GC diagnosis	Roy et al. 2016 [105]

Table 2 .

 2 Summary of potential Drug and/or Molecules for the targeting of GCSCs.

	Drug/Molecules	Target/Effects	Known Effects in GC	References
	Verapamil (in combination with chemotherapies)	Inhibit calcium-dependent channels	Blocks drug efflux mechanisms of CD44+ALDH+ GCSCs and prevents resistance to conventional therapies	Nguyen et al. 2017 [55]
	Tretinoin	FDA-approved drug for topical treatment of acne vulgaris; pro-differentiation properties	Forces differentiation and decreases tumorigenic properties of CD44+ALDH+ GCSCs	Nguyen et al. 2016 [107]
	Metformin	FDA-approved drug for first-line treatment of type 2 diabetes; decreases insulin resistance and hepatic neo-glucogenesis	Decreases tumorigenic properties of CD44+ GCSCs by targeting EMT and metabolism modulation	Courtois et al. 2017 [108]
	Buparlisib	Pan-class I PI3K inhibitor	Decreases CD44+ GCSC tumorigenic and metastatic capacity	Giraud et al. 2019 [109]
	Verteporfin	FDA-approved drug for age-related macular degeneration -inhibits Hippo effector YAP/TAZ-TEAD interaction	Decreases CD44+ALDH+ GCSC tumorigenic properties through Hippo pathway oncogenic effectors inhibition	Giraud et al. 2019 [59]
	LIF cytokine	Pro-differentiation properties	Decreases CD44+ALDH+ GCSCs tumorigenic properties by inducing Hippo tumour suppressor kinases activity	Seeneevassen et al. 2020 [60]
	Vismodegib (in combination with chemotherapies)	FDA-approved drug for recurrent locally advanced and/or metastatic Basal Cell carcinoma; antagonist of the Shh signalling pathway	Improves patient survival in combination with chemotherapies by targeting CD44+ GCSCs having high Shh pathway activity	Bekaii-Saab et al. 2017 [106]
	Napabucasin (in combination with chemotherapies)	FDA-approved as orphan drug for treatment of gastroesophageal junction cancer; STAT3 inhibitor	Decreases GCSCs tumorigenic properties in combination with paclitaxel in patients with advanced tumours	Bekaii-Saab et al. 2017 [106]
	Glutamate-cystine exchange transporters inhibitor (xCT)	xCT inhibition	Sensitizes GCSCs to 5-FU conventional therapy by blocking xCT anti-ROS mechanisms	Miyoshi et al. 2018 [110]

Table 1 .

 1 Recapitulation of potential anti-GC molecules targeting the Hippo pathway.

	Strategies	Molecules	Mechanism	Reference
		RUNX3	YAP-TEAD interaction competitor	[151, 152, 31]
		VGLL4	YAP-TEAD interaction competitor	[153, 30, 113]
		Super-TDU	YAP-TEAD interaction competitor	[113]
	Targeting oncogenic YAP/TAZ-TEAD signaling	Verteporfin	YAP-TEAD interaction competitor Targets FAT1 & Survivin Induces 14-3-3 proteins PDT and induces cell death through singlet oxygen production	[154, 155, 156, 149, 157, 121, 158]
		Peptide 17	YAP-TEAD interaction inhibitor, Targets N6-methyladenosine (m6A)'s methyl-transferase 3	[159, 160, 135]
		WZ35, a Curcumin analogue Cell death through increase in cellular ROS level	[161, 164]
		Dobutamine	Recruits YAP to the cytoplasm through PKA signalling	[165-169]
		Compound F10	MST1/2 activation through cytoskeletal altera-tion	[170]
		Statins (Lovastatin, Fluvas-	Modulates actin dynamics and activate Hippo
	Hippo kinases	tatin, Simvastatin)	kinases Inhibit β-catenin expression and YAP activity	[171-174]
	and/or side-
	pathways stimula-tion	Metformin	Induces AMPK which stabilizes AMOTL1 and induces Hippo kinases	[67, 175, 176]
		Pazopanib	Promotes effectors degradation by the ubiqui-tin-proteasome system	[177, 172]
		SHAP	Alters STRN3-PP2Aa interaction and restores MST1/2 activity	[138]

  Seeneevassen was funded by the French Ministry of Tertiary Education, Research, and Innovation. This work was funded by the Ligue Française Contre le Cancer/French League against Cancer.

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Table 1

 1 Known therapeutical strategies/experimentation involving LIF

	References	Kanda M et al. [46]	Welc SS et al. [47]		Zhang YS et al. [48]		Davis ID et al. [49]
	Molecules Known/tested use	LIF Cardiomyocyte regeneration	Type 2 immunity suppression. Duch-	enne muscular atrophy	Inhibition of Th17 accumulation.	Inflammatory Bowel Disease	Emfilermin, a commercial human LIF Peripheral neuropathy prevention	induced by chemotherapy		Embryo implantation improvement.
	Strategies Models	In vivo experimentation mice					Clinical trials Phase II trial: patients with solid	tumours & treated with carboplatin/	paclitaxel chemotherapy	Phase I & II trials: infertile women with	several implantation failures or women	undergoing pregnancy termination

  33. Xiao C, Hong H, Yu H, Yuan J, Guo C, Cao H, et al. MiR-340 affects gastric cancer cell proliferation, cycle, and apoptosis through regulating SOCS3/JAK-STAT signaling pathway. Immunopharmacol Immunotoxicol. 2018;40:278-83. 34. Hanahan D, Coussens LM. Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell. 2012;21:309-22. 35. Haykal MM, Nahmias C, Varon C, Martin OCB. Organotypic modeling of the tumor landscape. Front Cell Dev Biol. 2020;8:606039. 36. Wu X, Tao P, Zhou Q, Li J, Yu Z, Wang X, et al. IL-6 secreted by cancer-associated fibroblasts promotes epithelial-mesenchymal transition and metastasis of gastric cancer via JAK2/STAT3 signaling pathway. Oncotarget. 2017;8:20741-50. 37. Zhang F, Li K, Pan M, Li W, Wu J, Li M, et al. miR-589 promotes gastric cancer aggressiveness by a LIFR-PI3K/AKT-c-Jun regulatory feedback loop. J Exp Clin Cancer Res. 2018;37:152. 38. Zhao J-H, Sun J-X, Song Y-X, Chen X-W, Yang Y-C, Ma B, et al. A novel long noncoding RNA-LOWEG is low expressed in gastric cancer and acts as a tumor suppressor by inhibiting cell invasion. J Cancer Res Clin Oncol. 2016;142:601-9. 39. Nguyen PH, Giraud J, Chambonnier L, Dubus P, Wittkop L, Kanda M, Nagai T, Takahashi T, Liu ML, Kondou N, Naito AT, et al. Leukemia inhibitory factor enhances endogenous cardiomyocyte regeneration after myocardial infarction. PLoS ONE. 2016;11: e0156562. 47. Welc SS, Flores I, Wehling-Henricks M, Ramos J, Wang Y, Bertoni C, et al. Targeting a therapeutic LIF transgene to muscle via the immune system ameliorates muscular dystrophy. Nat Commun. 2019;10:2788. 48. Zhang YS, Xin DE, Wang Z, Song X, Sun Y, Zou QC, et al. STAT4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation. EMBO J. 2019;38: e99595. 49. Davis ID, Kiers L, MacGregor L, Quinn M, Arezzo J, Green M, et al. A randomized, double-blinded, placebo-controlled phase II trial of recombinant human leukemia inhibitory factor (rhuLIF, emfilermin, AM424) to prevent chemotherapy-induced peripheral neuropathy. Clin Cancer Res. 2005;11:1890-8. 50. Rambam Health Care Campus. Comparison of LIF (Leukemia Inhibitory Factor) Level Between Neonates Who Are IUGR (Intra Uterine Growth Restriction) and Those Who Are AGA (Average for Gestational Age) [Internet]. clinicaltrials.gov; 2015 Aug. Report No.: NCT02518126. Available from: https:// www. clini caltr ials. gov/ ct2/ show/ NCT02 518126 51. Rambam Health Care Campus. The Correlation Between LIF Levels in Cord Blood to Maternal Blood in Women Treated With Mg During Labor [Internet]. clinicaltrials.gov; 2015 Aug. Report No.: NCT02507817. Available from: https:// www. clini caltr ials. gov/ ct2/ show/ NCT02 507817 52. Merck KGaA, Darmstadt, Germany. A Randomised, Double-

Belleannée G, et al. Characterization of biomarkers of tumorigenic and chemoresistant cancer stem cells in human gastric carcinoma. Clin Cancer Res. 2017;23:1586-97. 40. Seeneevassen L, Bessède E, Mégraud F, Lehours P, Dubus P, Varon C. Gastric cancer: advances in carcinogenesis research and new therapeutic strategies. Int J Mol Sci. 2021;22:3418. 41. Nandy SB, Arumugam A, Subramani R, Pedroza D, Hernandez K, Saltzstein E, et al. MicroRNA-125a influences breast cancer stem cells by targeting leukemia inhibitory factor receptor which regulates the Hippo signaling pathway. Oncotarget. 2015;6:17366-78. 42. Molina Castro SE, Tiffon C, Giraud J, Boeuf H, Sifre E, Giese A, et al. The Hippo kinase LATS2 controls Helicobacter pyloriinduced epithelial-mesenchymal transition and intestinal metaplasia in gastric mucosa. Cell Mol Gastroenterol Hepatol. 2020;9:257-76. 43. Giraud J, Molina-Castro S, Seeneevassen L, Sifré E, Izotte J, Tiffon C, et al. Verteporfin targeting YAP1/TAZ-TEAD transcriptional activity inhibits the tumorigenic properties of gastric cancer stem cells. Int J Cancer. 2019;146:2255-67. 44. Seeneevassen L, Giraud J, Molina-Castro S, Sifré E, Tiffon C, Beauvoit C, et al. Leukaemia inhibitory factor (LIF) inhibits cancer stem cells tumorigenic properties through hippo kinases activation in gastric cancer. Cancers (Basel). 2020;12:2011. 45. Xu G, Wang H, Li W, Xue Z, Luo Q. Leukemia inhibitory factor inhibits the proliferation of gastric cancer by inducing G1-phase arrest. J Cell Physiol. 2019;234:3613-20. 46. blind, Placebo Controlled, Proof of Concept Study to Assess the Efficacy, Safety and Acceptability of r-hLIF for Improving Embryo Implantation Following in Vitro Fertilisation (IVF) and Embryo Transfer (ET) in Women With Recurrent Implantation Failure. [Internet]. clinicaltrials.gov; 2017 Jan. Report No.: NCT00504530. Available from: https:// www. clini caltr ials. gov/ ct2/ show/ NCT00 504530 53. Merck KGaA, Darmstadt, Germany. A Multicentre, Randomised, Double-blind, Placebo-controlled Proof of Concept Study to Compare the Efficacy and Safety of r-hLIF (Emfilermin) for Improving Embryo Implantation Following in Vitro Fertilization (IVF) and Embryo Transfer (ET) in Women With Recurrent Implantation Failure [Internet]. clinicaltrials.gov; 2017 Jan. Report No.: NCT00504608. Available from: https:// www. clini caltr ials. gov/ ct2/ show/ NCT00 504608 54. MD YG. The Correlation Between Gestational Age and Maternal and Fetal Levels of LIF and ACTH [Internet]. clinicaltrials.gov; 2017 Aug. Report No.: NCT03231904. Available from: https:// www. clini caltr ials. gov/ ct2/ show/ NCT03 231904 55. Davis SM, Reichel D, Bae Y, Pennypacker KR. Leukemia inhibitory factor-loaded nanoparticles with enhanced cytokine metabolic stability and anti-inflammatory activity. Pharm Res. 2018;35:6.

Table 1 .

 1 Patients' median survival according to expression of YAP1, TAZ and their target genes according to LIFR expression or not.

	Gene

Patients' Median Survival (Months) All Patients Independent of LIFR Expression Patients with Low LIFR Expression Patients with High LIFR Expression High Expression Low Expression p-Value High Expression Low Expression p-Value High Expression Low Expression p-Value

	YAP1	35.4	99.4	0.0011 **	21.4	41.2	0.00032 ***	36.4	113.2	0.013 **
	TAZ	35.9	46	0.085	26.2	76.2	0.009 **	32.6	87	0.24
	CTGF	35.9	85.6	0.017 *	15.2	30	0.022 *	32.6	93.2	0.0039 **
	CYR61	35.9	45.8	0.39	45.1	32.1	0.36	44.7	87	0.12
	AXL	36.4	85.6	0.04 *	26	76.2	0.027 *	36.4	99.4	0.083
	AREG	36.4	78.6	0.1	32.1	40	0.27	75.5	25.2	0.11
			*p < 0.05, **p < 0.005, ***p < 0.0005 vs. untreated controls with Log-Rank test.

  p < 0.05, ** p < 0.005, *** p < 0.0005 versus untreated controls with log-rank test.

			< 1	p-Value
	YAP1	28.7	85.6	0.0004 ***
	TAZ	32.1	85.6	0.0085 *
	CTGF	30.4	87	0.00058 **
	CYR61	34.7	85.6	0.046 *
	AXL	35.9	99.4	0.023 *
	AREG	35.9	93.2	0.033 *

*

Table S1 .

 S1 List of primers used for RT-qPCR analysis.

	Gene	Sequence Frame (5′-3′)	Sequence Reverse (5′-3′)
	ALDH1A1	TCCTGGTTATGGGCCTACAG	CAACAGCATTGTCCAAGT
	AREG	CGAACCACAAATACCTGGCTA	TCCATTTTTGCCTCCCTTTT
	AXL	CGTAACCTCCACCTGGTCTC	TCCCATCGTCTGACAGCA
	CD166	GCCTGGTTTCTGGGAGAACAC	GATGTTCATTTATCTTCCCTTTAAAGTTG
	CD24	TTCTCCAAGCACCCAGCA	TGGAATAAATCTGCGTGGGTA
	CD44	GGGCGTAACTCTGGAAGCA	TTAGCCCAGCTCACCTGAAAAT
	c-FOS	CCGGGGATAGCCTCTCTTACT	CCAGGTCCGTGCAGAAGTC
	CTGF	GCCACAAGCTGTCCAGTCTAATCG	TGCATTCTCCAGCCATCAAGAGAC
	CYR61	ATGAATTGATTGCAGTTGGAAA	TAAAGGGTTGTATAGGATGCGA
	EGR1	ACCTGACCGCAGAGTCTTTTC	GCCAGTATAGGTGATGGGGG
	HPRT1	TGGTCAGGCAGTATAATCCA	GGTCCTTTTCACCAGCAAGCT
	JUNB	TACCACGACGACTCATACACA	CGCTTTGAGACTCCGGTAGG
	KLF5	CCTGGTCCAGACAAGATGTGA	GAACTGGTCTACGACTGAGGC
	SOCS3	AGCTCGCATTCAGACTACCTA	AGTCGATCAGATGAACCACACT
	TAZ	GTTTATGGGAGAGTCCGGGAG	AGT CTA AGG GCT TCG GCT CT
	TBP	TGCACAGGAGCCAAGAGTGAA	CACATCACAGCTCCCCACCA
		~123~

Table 1 :

 1 Percentage of metastasis-bearing mice

		Metastasis-bearing mice (%)
	Conditions	Lungs	Liver	Stomach	Others	Total
	Non-treated	81.8	81.8	18.2	45.5	100
	10 000 cells
	(n = 11)
	LIF	81.8	54.5*	9.1	36.4	81.8
	Non-treated	56.3	56.3	12.5	43.8	68.8
	100-1000 cells
	(12 ≤ n ≤ 16)
	LIF	25	25	0	8.3*	25*
		~153~

  1,2 , Lornella Seeneevassen 1 , Elodie Sifré 1 , Julien Izotte 1 , Camille Tiffon 1 , Cathy Staedel 3 , Hélène Boeuf 4 , Solène Fernandez 1 , Philippe Barthelemy 3 , Francis Megraud 1,5,6 , Philippe Lehours1,5,6 , Pierre Dubus 1,5 and Christine Varon1

	1 INSERM U1053 Bordeaux Research in Translational Oncology, University of Bordeaux, Bordeaux, France
	2 INISA/School of Medicine, University of Costa Rica, San José, Costa Rica
	3 INSERM U1212, CNRS UMR5320, ARNA Laboratory, University of Bordeaux, Bordeaux, France
	4 INSERM U1026 BioTIS, University of Bordeaux, Bordeaux, France
	5 CHU de Bordeaux, Bordeaux, France

6

National Reference Center for Campylobacters and Helicobacters, Bordeaux, France

Table 1

 1

). Our preliminary results indicate that CTCs are detectable in 20% of GC patients and majority as clusters with immune cells. We are waiting for patients' clinico-biological data, including histological subtype and grade of GC to be able to carry out a proper follow up of patients' disease progression and prognosis and correlate it with CTC enumeration.

Table 1 : CTC enumeration in GC patients' blood sample.

 1

  RAF/MEK/ERK1/2 and PI3K/AKT/mTOR for example, are involved downstream LIF/LIFR signalisation, and implicating JAKs or not, and that this was not investigated in our study. Indeed, LIF/LIFR/PI3K/AKT pathway has anti-metastatic effects in HCC while

participating in numerous crosstalk implicating other pathways. Here we demonstrate that in GC, LIF/LIFR activation balance seems to shift from JAK1/STAT3 to the Hippo pathway reducing LIF/LIFR/JAK1/STAT3 pro-survival and pro-tumorigenic mechanisms towards a more LIF/LIFR/LATS1/2 anti-CSC one. Nevertheless, it should be noted that many other pathways ~200~ like RAS/

Table 1 .

 1 Number of tumor-bearing mice per total of NSG mice implanted with luciferase-encoding gastric PDX cells into gastric subserosa in two independent experiments.

	Number

of Tumor-Bearing Mice 1 /Number of Xenografted Mice

	Experiment	Case	Stomach	Lung	Liver	Peritoneal Tumors	Weeks
	a	GC04	3/4	2/4	0/4	1/4	13
		GC06	0/4	0/4	0/4	0/4	25
		GC07	4/4	4/4	0/4	0/4	8
		GC10	4/4	3/4	2/4	0/4	10
	b	GC07	12/12	12/12	3/12	1/12	8
		GC10	14/15	11/15	13/15	5/15	8

1

Tumor development was monitored by bioluminescence imaging until indicated weeks.

Table 2 .

 2 Number of tumor-bearing mice per total of NSG mice implanted with gastric cancer cell lines into gastric subserosa.

		Number of Tumor-Bearing Mice 1 /Number of Xenograft Mice
	Case	Stomach	Lung	Liver	Peritoneal Tumor	Weeks
	MKN45	5/5	5/5	4/5	3/5	8
	MKN74	4/4	4/4	2/4	2/4	8
	NCI-N87	3/5	0/4	0/5	0/5	8-10
	1 Tumor development was examined macroscopically after sacrifice and by histological analyses of
	human-specific HLA expression.

1 /Number of Xenograft Mice

	Case	Stomach	Lung	Liver	Spleen and Pancreas
	Vehicle	9/9	9/9	7/9	6/9
	BKM120	8/8	7/8	4/8	2/8

Table 3 .

 3 Number of tumor-bearing mice per total NSG mice implanted with MKN45 and treated with BKM120 and vehicle (DMSO-treated mice).

	Number of Tumor-Bearing Mice 1 /Number of Xenograft Mice
	Case	Stomach	Lung	Liver	Spleen and Pancreas
	Vehicle	9/9	9/9	7/9	6/9
	BKM120	8/8	7/8	4/8	2/8

TABLE 1 ~294~Table 1 .

 11 Gastric CSC frequencies determined on the capacity of FACS-sorted cells to develop a tumour after xenograft in limiting dilutions in NSG mice.

Table 1

 1 Non-exhaustive examples of Helicobacters associated with inflammation and/or cancer.

	C. Varon et al.
								Virulence factors
	Helicobacter species	Animal	Disease	Location	Humans/disease	Location	urease activity	cdtB	Other virulence
									factors
	Gastric Helicobacter species
	H. felis	Cat, dog, [215]	Gastritis [75,76], Gastric	Stomach, liver	Gastritis [215], Peptic ulcer	Stomach	+ [216]	cdtB-	ggt+ [66]
		rabbit, mouse	adenocarcinoma, gastric		disease [77]
			intramucosal carcinoma in
			INS-GAS mice [71], gastric
			lymphoma [74]
	H. pylori	Monkey, cat	Cholangiohepatitis [138]	Stomach, liver	Gastritis, peptic ulcer disease	Stomach, Colon, Liver	+ [217]	cdtB-	cagA+
		[138]			[5,6], gastric				iceA1+ ggt+
					adenocarcinoma [7], gastric				vacA+ T4SS
					MALT lymphoma, colorectal				[218,219,
					cancer, pancreatic cancer,				66,220]
					bile duct cancer,
					hepatocellular carcinoma
	H. heilmannii	Cat, dog, pig,	Gastritis, chronic hepatitis	Stomach,	Gastritis, gastric	stomach	+ [78]	cdtB-	iceA1+
		primate	[139]	gallbladder,	adenocarcinoma[85-88],				vacA-like
				liver [139]	gastric MALT lymphoma				[221]
					[78-80]
	H. suis	Pig, macaque	Gastritis, gastric	Stomach	Gastritis [79], peptic ulcer	Stomach	+ [83]	cdtB-	ggt+ T4SS
			adenocarcinoma, gastric		disease, gastric MALT				[222]
			MALT lymphoma [81-84]		lymphoma [82]
	Intermediate Helicobacter species
	H. mustelae	Ferret [86]	Gastritis, gastric	Stomach [86,	NA	NA	+ [85,	cdtB-	ggt+ [66]
			adenocarcinoma [85],	85,87],			223]
			duodenal ulcer [87],	intestine [87]
			exacerbates ulceration of the
			stomach and intestine [87]
	H. himalayensis Marmot [158] Gastritis [158]	Stomach [158] NA	NA	-[158,	cdtB+	/
							159]	[159]
	Enterohepatic Helicobacter species
	'H. apodemus'	Rodent	rectal prolapse and colorectal		NA	NA	+ [224]	cdtB+	/
			cancer					[225]
	H. bilis	Dog, cat,	multifocal chronic hepatitis	bile, liver, and	Colorectal cancer [229]	Colon [229]	+ [230]	cdtB+	ggt+ htrA+
		mouse, rat,	and hepatocellular tumors,	intestine				[156,231]	T6SS [66,
		hamster and	[226] cholangiocarcinoma	Sheep: livers of					204]
		sheep	[148] Colon cancer in SMAD3 -/-	aborted sheep fetuses [228]
			mice [227]
			Sheep: abortion [228]
			Chronic Hepatitis, Hepatic
			Dysplasia, Fibrosis, and
			Biliary Hyperplasia in
			Hamsters [147]
	H. canis	Dog	Feces of diarrhoeic or healthy	liver	Bacteremia	Blood [234,235]	-[232]	cdtB+	ggt+ htrA+
			dogs [232], multifocal		Immunocompromised			[156]	[66,204]
			necrotizing hepatitis [233]		humans [234,235]
	H. cholecystus	Hamster,	Hamster: cholangiofibrosis	feces,	NA	NA	-[236]	cdtB+	/
		ferret	and centrilobular pancreatitis	gallbladder and
			[236]	liver.
			Ferret: chronic
			cholangiohepatitis with
			cellular proliferation ranging
			from hyperplasia to
			neoplasia[237]
	H. cinaedi	Cat, dog,	Hamster: normal intestinal	Rodent:	Immunocompromised and	Blood,[243]	-[245]	cdtB+	htrA+ T6SS
		hamster,[238]	bacterial flora	intestine	immunocompetents	cerebrospinal fluid,		[246]	[204]
		rat, fox, and	Mice: multifocal hepatic	Rhesus monkey:	individuals/nosocomial	peritoneal fluid,
		rhesus monkey	lesion, with cholangitis and	feces, colon,	infection [240,241]	cellulitis, prosthetic
			vasculitis, evolving towards	liver, and	Bacteremia, infected	joint infection,
			bile duct hyperplasia,	mesenteric	aneurysm, pericoronary	osteomyelitis, acute or
			hepatomegaly, oval cell	lymph node	pseudotumor[242]	chronic gastroenteritis,
			hyperplasia, hepatocellular	[239]		hepatic cyst, carotid
			proliferation, and in older			atherosclerosis, thyroid
			mice hepatoma or			abscess, feces, ….[244]
			hepatocellular carcinoma
			Feces, colon, liver, and
			mesenteric lymph node of a
			Rhesus Monkey: chronic
			colitis and hepatitis [239]
			Liver lesions reminiscent of
			early liver lesions caused by
			H. hepaticus [239]
								(continued on next page)

Table 1

 1 (continued )

								Virulence factors
	Helicobacter species	Animal	Disease	Location	Humans/disease	Location	urease activity	cdtB	Other virulence
									factors
	H. fennelliae	Dog, macaque,	/	feces	Bacteremia, multifocal	Blood, non-diarrheal	-[248]	cdtB-/+	/
		[238,247]			cellulitis, cerebral cyst [241]	stool		[246]
					Bacteremia in
					immunocompromised
					humans: cervical cancer,
					esophageal cancer, pancreatic
					cancer and lymph node
					metastasis [243]
	H. hepaticus	Mice	chronic hepatitis,	feces, cecal and	Cholelithiasis, cholecystitis	Bile, gastric and	+ [250]	cdtB+	ggt+ T6SS
			hepatocarcinoma,	colonic	with gastric cancer[106,249]	duodenal samples [106,		[156,	[66,152]
			cholangiocarcinoma[148] Colon cancer in SMAD3 -/-	mucosae, liver		249]		160-163, 167,172,
			mice [227]					175,176,
								183,206,
								209]
	H. macacae	Rhesus	diarrhoea, chronic idiopathic	feces, colon and	NA	NA	-[251] cdtB-[252] htrA+ [204]
		macaque	colitis, intestinal	tumour [252]
			adenocarcinoma[251-253]
			strong pro-inflammatory gene
			expression
	H. marmotae	Woodchuck,	Woodchucks with	woodchucks:	NA	NA	-[254]	cdtB+	/
		cat [254]	hepatocellular carcinoma	liver tumour,				[156]
			[254] Cocarcinogen?	intestine
	H. mastomyrinus Rodent	Normal mice and cecum of a	liver, cecum,	NA	NA	+ [255]	cdtB+	/
			mouse with proctitis [255]	feces				[255]
			Ulcerative typhlocolitis in
			Telomerase-Deficient Mice
			[256]
	H. pullorum	Chicken	Healthy chickens and	liver,	Healthy persons and patients	Feces of healthy persons	-[248]	cdtB+	htrA+ T6SS
			chickens with gastroenteritis,	duodenum, and	with gastrointestinal disease	and patients with		[167,173,	[204,261]
			vibrionic hepatitis [248]	caecum	[257], chronic hepatitis C,	gastrointestinal disease		175,186,
					hepatocellular carcinoma	[248,257], liver [127,		208,260]
					[127,258], bacteremia [259]	258], blood [259]
	H. trogontum	Rat, sheep	Sheep: abortion [228]	Intestine	Immunocompetent patient	Blood [262]	+ [263] cdtB-[231] ggt+ [154]
				Sheep: livers of	with acute colitis [262]
				aborted sheep
				fetuses [228]
	H. japonicus	Mice	Typhlocolitis and lower	Cecum, feces	NA	NA	-[264]	cdtB+
			bowel carcinoma[264]	and the stomach				[264]
				[264]

/

cagA, cytotoxin-associated gene A; ggt, γ-glutamyl transpeptidase gene; htrA, protease that cleaves E-cadherin to disrupt intercellular adhesion

[40]

, iceA1, adherence to gastric epithelial cells induced gene 1; MALT, mucosa-associated lymphoid tissue; T4SS, type IV secretion system; T6SS, type VI secretion system; vacA, vacuolating cytotoxin.

C.

Varon et al.

Int. J. Cancer: 146, 2255-2267 (2020) © 2019 UICC Molecular Cancer Biology

Acknowledgements

The PhD fellowship of Lornella Seeneevassen was funded by the French Ministry of Tertiary Education, Research and Innovation. This work was funded by the Ligue Française Contre le Cancer/French League against Cancer.

Acknowledgments: We thank Katia Boniface (INSERM UMR1035, University of Bordeaux, France) for providing anti-Stat3 antibodies, JAK inhibitors and for helpful discussions. We also thank Jacques Robert and Elodie Darbo (INSERM U1218 ACTION, University of Bordeaux, France) for their bio-informatical counselling and discussions.

Acknowledgments

: We thank Sylvaine Di Tommaso (Oncoprot TBM-Core, Univ. Bordeaux, CNRS UMS 3427, INSERM US 005, 33076 Bordeaux, France.) for the proteomics microdissection and mass spectrometry. We thank Claire Naylies and Yannick Lippi for their contribution to microarray fingerprints acquisition and microarray data analysis carried out at GeT Genopole Toulouse Midi-Pyrénées facility (Toxalim (Research Centre in Food Toxicology, Université de Toulouse, INRAE, ENVT, INP-Purpan, UPS, Toulouse, France, https://doi.org/10.15454/1.5572370921303193E12). Acknowledgements The postdoctoral fellowship of J.G. was supported by the French "Institut National du Cancer" (grant 2014-152) and the Faculty of Medical Sciences of the University of Bordeaux. The doctoral fellowship of S.M.-C. was granted by the University of Costa Rica, the Ministry of Science, Technology, and Telecommunications of Costa Rica and the French Institute for Central America. The doctoral fellowship of C.T. was supported by the French "Ligue nationale contre le cancer." We thank Michel Moenner (IBGC CNRS UMR5095, University of Bordeaux, Bordeaux, France) and Julie Pannequin (IGF INSERM U1191 -CNRS UMR 5203, University of Montpellier, Montpellier, France) for helpful discussions and for providing antibodies, and Sarah Courtois (INSERM U1053, University of Bordeaux) and Vincent Pitard (Flow Cytometry and FACS Platform, University of Bordeaux) for technical assistance. This project received funding from the "Institut National du Cancer" (grant 2014-152) and the SIRIC BRIO (grant R16055GG, "Site de Recherche Intégrée sur le Cancer -Bordeaux Recherche Intégrée Oncologie"; grant INCa-DGOS-Inserm 6046).

Acknowledgments: We thank Julien Izotte and Lamia Azzi-Martin for technical assistance. We thank the platforms of the University of Bordeaux (Vectorology platform, Level 2 animal facility, and cytometry platform) and Violaine Moreau and Stéphanie Verbeke for supplying the antibodies. We thank Marie-Edith Lafon (service of virology, CHU of Bordeaux) for the virology analyses and determination of the EBV status of GC tumors, and Antoine Italiano and Benjamin Fourneaux from the Bergonié Institute of Bordeaux for the determination of mutations using NGS Colon and Lung Panel.

Acknowledgments: We thank R. Peek (Vanderbilt University, Nashville, TN, USA) and Rainer Haas (Ludwig Maximilian University of Munich, Germany) for the H. pylori 7.13 and P12 wild type and isogenic mutant strains, respectively, and Michel Moenner (CNRS UMR5095, University of Bordeaux, France) for providing anti-TAZ antibodies and for helpful discussions.

ACKNOWLEDGMENTS

We thank the French 'Ligue Contre le Cancer, comité Dordogne (grant number R17013GG) and comité Gironde (grant number GM/12.17/37), the 'Institut National du Cancer' (INCa, PLBio grant number 2014-152) for research funding and for the post-doctoral fellowship of Julie Giraud. We thank the UFR des sciences médicales Univ. Bordeaux for J Giraud and D Bourriez financial support, and the French ministry of national education, research and technologies for L Seeneevassen PhD salary. The PhD fellowship of Camille Tiffon was funded by the French Ligue Nationale Contre le Cancer. We thank Vincent Pitard (Flow Cytometry and FACS Platform, Univ. Bordeaux), Lamia Azzi-Martin and Quentin Jehanne for technical assistance. We thank Linda Wittkop (ISPED, USMR CHU Bordeaux), Hélène ~302~ Boeuf (INSERM U1012 Biotis, Univ. Bordeaux) and Nicolas Larmonier (CNRS UMR5134 Immunoconcept, Univ. Bordeaux) for helpful discussions and advice. We thank Olivier Augereau and Cosmin Lazaroiu (Hospital Haut-Lévêque, CHU Bordeaux) for paraffinembedded tissues selection.

Acknowledgements

We would like to acknowledge the contribution of Maya Sarieddine for the proofreading of the article. The PhD fellowship of Lornella Seeneevassen was funded by the French Ministry of Tertiary Education, Research and Innovation and that of Sadia Khalid was supported by DoRa Plus programme for doctoral Students (European Regional Development Fund). Pirjo Spuul was funded by VNF20013 from Biocodex Microbiota Foundation and PUT1130 from Estonian Research Council.

Funding:

The PhD fellowship of Lornella Seeneevassen was funded by the French Ministry of Tertiary Education, Research and Innovation. The PhD fellowship of Camille Tiffon was funded by the Ligue Nationale Française Contre le Cancer/French National League against Cancer. Silvia Elena Molina-Castro was a PhD fellowship recipient of the University of Costa Rica (San José, Costa Rica) and the Ministry of Sciences and Technologies (MICITT, Costa Rica). This research was funded by the Institut National du Cancer/French National Institute on Cancer INCa, grant PLBio 2014-152. Funding: The PhD fellowship of Lornella Seeneevassen was funded by the French Ministry of Tertiary Education, Research and Innovation. The PhD fellowship of Camille Tiffon was funded by the Ligue Nationale Française Contre le Cancer/French National League against Cancer. This research was funded by the Ligue Française Contre le Cancer/French League against Cancer and the Department of Biological and health Sciences of Bordeaux University. Funding: This research was funded by the Institut National du Cancer grant number 2014-152 and by the French Ligue Contre le Cancer comité de Gironde grant number GM/12.17/37. Julie Giraud post-doctoral research fellowship was supported by the UFR of medical sciences, University of Bordeaux. Funding: The PhD fellowship of Camille Tiffon was funded by the Ligue Nationale Française Contre le Cancer/French National League against Cancer. Silvia Elena Molina-Castro was a PhD fellowship recipient of the University of Costa Rica (San José, Costa Rica) and the Ministry of Sciences and Technologies (MICITT, Costa Rica). This research was funded by the Institut National du Cancer/French National Institute on Cancer INCa, grant PLBio 2014-152, and by a regional grant from La Ligue Contre le Cancer.

Data availability

The data that support the findings are available from the corresponding author upon request.

Abbreviations

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Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Competing Interests:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Supplementary

Statistical analysis

Quantification values represent the means of three or more independent experiments, each performed in duplicates or more AESEM. Two-tailed t-tests or Mann-Whitney tests were used to compare two groups and Kruskal-Wallis tests with Dunn's post-tests or ANOVA tests and Bonferroni post hoc tests were used for multiple comparisons. Statistics were performed using GraphPad Prism 6 software (La Jolla, CA). See Supplementary Materials and Methods for antibodies, RNA extraction and quantitative RT-PCR, Western blotting, histology, immunohistochemistry and immunofluorescence procedures, siRNA transfection, agilent microarray procedures and analysis.

Scientific communications

Oral communications in local, national and international congress meetings Biological and Medical Science Department Day. December 2021, Bordeaux (France) Local Bordeaux congress Seeneevassen L., Zaafour A., Sifré E., Genevois C., Giraud

TAZ Conferred EMT Phenotype and CSC-like Properties via ZEB1 Upregulation in Response to H. pylori Infection

In order to investigate the role of TAZ in H. pylori-induced EMT and tumor-initiating properties, we used a silencing RNA strategy (siRNA) to inhibit TAZ. The efficiency of three different siRNA used to inhibit TAZ expression under basal conditions and during H. pylori infection was confirmed by Western blot in the three gastric cell lines (Figure S2A). The inhibition of TAZ expression in cells transfected with siTAZ-1, siTAZ-2, and siTAZ-3 ranged from 46-73% compared to cells transfected with non-silencing negative control siRNAs (siCtrl). TEAD luciferase reporter assay confirmed these results, showing a strong reduction of TEAD transcriptional activity with the siTAZs even after H. pylori infection (from an 80-90% decrease in siTAZ-transfected uninfected conditions to more than 64% in siTAZ-transfected H. pylori-infected conditions, compared to siCtrl-transfected uninfected cells; Figure S2B). Contrary of MKN45 and GC07, NCI-N87 cells expressed E-cadherin, an essential component of adherent junctions, which acts as a gatekeeper of H. pylori-induced EMT to maintain epithelial polarity and integrity [8]. Furthermore, NCI-N87 cells are not invasive in vitro. The consequences of TAZ inhibition on H. pylori-induced EMT were therefore evaluated on MKN45 and GC07 cells that are more prone to EMT than NCI-N87 (Figure 4). H. pylori induced a significant increase in the expression of several EMT markers such as Vimentin, ZEB1, SNAI1 (except in GC07), and MMP9 in siCtrl-transfected cells, which was abolished or strongly reduced by TAZ silencing in siTAZ-transfected MKN45 and GC07 cells (Figure 4A). To confirm these results, ZEB1 protein expression and subcellular localization was analyzed by immunofluorescence in siRNA-transfected cells upon H. pylori infection. Results showed that H. pylori-induced ZEB1 overexpression and nuclear accumulation in siCtrl-transfected MKN45 and GC07 cells was no longer observed after TAZ silencing by siTAZ in MKN45 and strongly reduced in GC07 (Figure 4B,C and Figure S2C). These results show that TAZ activation controls ZEB1 overexpression and nuclear accumulation in response to H. pylori infection.

The invasive properties of cells were then evaluated with invasion assays using type I collagencoated microporous Transwell inserts (Figure 4D and Figure S2D). H. pylori infection stimulated invasion in MKN45 cells as previously reported [13], as well as in the GC07 cell line. The silencing of TAZ by the three different siTAZ drastically inhibited H. pylori-induced invasive properties in both MKN45 and GC07. Apart from siTAZ1 in MKN45 cells, we observed no effect for the different siTAZ used on basal invasion properties of MKN45 and GC07 cells without H. pylori stimulation.

In conclusion, the present study demonstrated for the first time a novel mechanism by which H. pylori upregulates TAZ, which is responsible for the upregulation of ZEB1, one of the most important inducers of EMT and CSC properties in the context of gastric carcinogenesis. Therefore, our findings support the fact that TAZ is a crucial inducer of EMT and invasive and tumorigenic CSC-like properties in response to H. pylori infection. The detection of TAZ co-overexpression with ZEB1 and Ki67 in H. pylori-induced pre-neoplastic lesions and in invasive GC suggests that TAZ could indeed constitute a biomarker of early transformation in gastric carcinogenesis.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. In this study, we aimed to decipher the cellular heterogeneity of GC cells in terms of CD44 variants expression, and especially to study the properties of cells expressing CD44v3.

METHODS

We declare that all authors had access to the study data and have reviewed and approved the final manuscript. GC04, GC06, GC07, GC10, and GC35 are patient-derived GC, which were successfully established by serial subcutaneous transplantation of tumour pieces in NSG immunodeficient mice as previously described 4 .

Flow cytometry and fluorescence-activated cell sorting.

For analysis of panCD44/CD44v3 expression and ALDH activity, cells were recovered and 100.000 cells per condition were stained with Aldefluor® reagent and then with anti-human CD44-PE as previously reported 4,23 and CD44v3-APC. Flow cytometry was performed using BD FACSCanto II or Fortessa instruments and DIVA analysis software (BD). FACS-sorted cells were performed after dissociation of PDX tumours with human tumour dissociation enzyme and GentleMACS dissociator (all from Miltenyi). 7-amino actinomycin (BD Biosciences) negative dead cells were excluded and EpCAM + panCD44 + /CD44v3 + and EpCAM + panCD44 - /CD44v3 -were sorted using FACS Aria II instrument. RNA extraction and RT-qPCR. RNAs were extracted using Trizol reagent (Ambion).

Reverse transcription was performed with 0.5 µg total RNA using the Quantitect Reverse Transcription kit (Qiagen). Quantitative PCR was performed using the SYBR-qPCR-Premix

Ex-Taq (TAKARA), 0.3 µmol/L of specific primers and 1:100 of RT reaction volume.

Relative expressions were calculated using the comparative Ct method with both HRPT1 and TBP as normalizers 23 . Human qPCR primer sequences are in Supplementary Table 1.

CD44 exon specific PCR. CD44 exon specific PCRs were performed as previously reported 24 . Reverse transcription was performed with 0.5 µg total RNA using the Quantitect Reverse Transcription kit (Qiagen) except that primers used for retro-transcription was specific to CD44 gene (sequence 5'-ATGCAAACTGCAAGAATC-3', primer R2). PCR was performed with primers listed in Supplementary Table S2. Amplification cycle was the following: Activation: 2' at 95°C; Amplification (35 cycles): 30" at 95°C -2'45" at 56°C -1' at 73°C; Final extension: 10' at 73°C. siRNA transfection. Cells were grown in 6-well plates and transfection of 25 nmol/L siRNA was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Courtaboeuf, France) according to manufacturer's instructions 23 . Two rounds of siRNA transfections were performed. siRNA sequences are in Supplementary Table 3.

Tumorsphere culture. 250-1,000 cells (as indicated in figure legends) were seeded in nonadherent 96-well culture plates, as previously described 4,23 , in serum-free GlutaMAX-DMEM/F12 medium supplemented with 20 ng/mL of epidermal growth factor, 20 ng/mL of basic-fibroblast growth factor, 0.3% glucose, 5 μg/mL of insulin, 1:100 N2 supplement (all from Invitrogen and Sigma) and cultured at 37 °C in a humidified 5% CO2 atmosphere.

Invasion and gelatine degradation assays. Detailed information about invasion and

gelatine degradation assay is provided in Supplemental Methods.

In vivo xenograft experiments and extreme limiting dilution assay. GC04 and GC10

PDX tumours were dissociated and EpCAM + /panCD44 + /CD44v3 -, EpCAM + / panCD44 + /CD44v3 + and EpCAM + /panCD44 -/ CD44v3 -cells freshly sorted by FACS were suspended in 100 μL of 7 mg/ mL ice-cold Matrigel (Ozyme) and subcutaneously injected into the dorsal flank of 6-week-old NSG female mice. Increasing number of cells (30; 100; 300 or 1.000 cells for GC10 and 50; 500; 1.500 and 5.000 cells for GC04) was injected in 5 mice per condition. Tumours were monitored twice a week up to 12 weeks using a calliper.

Tumours were collected, fixed in a 3.7% buffered-formaldehyde solution and embedded in paraffin following standard procedures as described 4,23,25 .

Orthotopic xenograft of FACS-sorted cells. GC10 PDX cells and MKN45 cells, previously

transduced with a lentivirus encoding luciferase gene as previously described 25 , were grown subcutaneously in NSG mice and tumours were dissociated and immuno-stained for FACS cell sorting as described in upper sections. 10.000 GC10 cells and 2.500 CD44v3+ and

~282~

CD44v3-MKN45 cells, sorted by FACS, were injected into the sub-serosa of the stomach of 6-weeks old male NSG mice 25 . Surgery procedures are detailed in Supplemental Methods.

Statistical

RESULTS

Characterization of CD44 variants in GC

The expression of CD44 isoforms was deciphered in GC cell lines and in-house patientderived xenografts (PDX) 4 using exon-specific RT-PCR 24 . Interestingly, mRNAs encoding CD44v3-containing isoforms (CD44v3) were expressed in GC cell lines and PDX-derived cells (Supplementary FigureS1A). Exon v3 was present in combination with all other exons (CD44v2/3-10) and in direct association with exons v8 to v10 (named CD44v3,8-10 or CD44v3E). Consistent with previous reports 20,21 , mRNA levels of global CD44 (CD44t) and of two common isoforms, the standard isoform CD44S (the shorter isoform, containing no expressed >3 times less than panCD44, in ≤17% of cells in the 5 GC cell lines and the 6 PDX analysed compared to panCD44 whose expression reached 98% of GC cells and 37% of PDX cells (Fig. 1E-G). Taken together, these results highlight the cellular heterogeneity inside the population of panCD44+ GC cells and show that, contrarily to panCD44, CD44v3+ cells correspond to a rare subpopulation of cells in GC that is primarily absent in healthy gastric mucosa.

The subpopulation of CD44v3+ GC cells display CSC properties

We previously reported that panCD44+ cells display all the hallmarks of CSCs since they have tumorigenic properties both in vitro (give rise to tumorspheres) and in vivo (give rise to tumours after xenograft in immunodeficient mice), and are chemo-resistant 4,26,27 . Here, we found that after 48 h treatment with 5-fluorouracil or doxorubicin, resistant GC cells highly expressed CD44v3 both at the mRNA (Supplementary Fig. S2) and protein (Fig. 2A-B) levels.

These chemoresistance properties were not restricted only to CD44v3+ cells as CD44s and CD44E expression also increased following chemotherapy (Supplementary Fig. S2). In order to evaluate tumorigenic properties associated to CSCs in vitro, panCD44+, panCD44-, CD44v3+ and CD44v3-cells were isolated by FACS and submitted to tumorsphere assay (Fig. 2C-E). Both panCD44+ and CD44v3+ cells formed significantly more tumorspheres than their negative counterparts (Fig. 2C-E). The ability of CD44v3+ cells to form tumorsphere was either higher (in GC04), the same (in GC06 and GC07) or lower (in GC10) than panCD44+ cells (Fig. 2C), suggesting that CD44v3+ cells have similar tumorigenic properties than panCD44+ cells. To confirm this result in vivo, subcutaneous xenograft experiments in immunodeficient mice were performed with GC04 and GC10 PDX FACSsorted EpCAM+ GC cells expressing or not panCD44 and CD44v3 using the extreme ~285~ limiting dilution analysis (ELDA) method as previously reported 28 (Table 1). CSCs frequency in CD44v3+ cells was significantly higher than panCD44-cells, but lower than panCD44+/CD44v3-cells (Table 1). Importantly, in both cases tumours were heterogeneous, composed of panCD44 and CD44v3 positive and negative cells (Fig. 2F-G), showing that both CD44v3+ and panCD44+/v3-cells have self-renewal and asymmetrical division and differentiation properties that are hallmarks of CSCs.

Altogether, these results show that both CD44v3+ and panCD44+/v3-GC cells have CSCs tumorigenic and differentiation properties.

CD44v3+ cells represent a subpopulation of panCD44+ cells with an epithelialmesenchymal transition molecular signature

Transcriptomic analysis was conducted on FACS-sorted subpopulations of GC cells expressing or not panCD44 or CD44v3 from 4 to 6 established PDX and MKN74 GC cell line using microarrays. Supervised hierarchical clustering of differentially expressed genes between positive and negative cells strongly separated samples in 2 groups based on their panCD44 and CD44v3 origin (Supplementary Fig. S3A-B). panCD44+ and CD44v3+ subpopulations of GC cells expressed distinct patterns of genes (detailed in supplementary materials). Among the 310 mRNA transcripts that were significantly upregulated in CD44v3+, 60 were significantly downregulated in panCD44+ (Fig. 3A-B, Supplementary properties in human oral squamous cell carcinoma 18 and HNSCC 19 , respectively.

Using transcriptomic analyses, we showed that two subpopulations of CSCs co-exist in GC, based on their EMT profile as described by Wicha's group in breast cancer 29 , but defined by different CSCs markers. In GC, panCD44+ CSCs expressed a proliferative and an epithelial MET-like signature similarly to the subpopulation of ALDH+ CSCs in breast cancer 29 , and CD44v3+ CSCs harboured an EMT-like signature like CD44+ breast CSCs 29 . In GC tissues, CD44v3+ GC cells were detected at the invasive front of metastases and in blood vessels as CTCs, whereas panCD44+ GC cells were more centrally located within the tumour mass. In GC, ALDH+ CSCs were mainly panCD44+/CD44v3-and CD44v3+ in ~1.3% of cells. Our preliminary data suggest the same results for breast and colon cancer cell lines. In addition, these panCD44+/CD44v3+ GC cells showed drug efflux-properties highlighting their CSCs chemoresistance properties.

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In vivo, both CD44v3+ cells and panCD44+/v3-cells generated heterogeneous tumours consisting of both CD44v3+ and CD44v3-cells, revealing CSCs properties of stemness and plasticity. Nevertheless, in some cases panCD44+/v3+ were less tumorigenic in vivo than panCD44+/v3-cells, while it was the contrary in vitro. This discrepancy between in vitro and in vivo results could be attributed to the influence of the tumour microenvironment on CSCs proliferation, plasticity and dormancy properties 34 . Interestingly, it has been reported that the HS site in the v3 exon is involved in metastatic tumour progression but does not affect cell proliferation 13,14,35 . Here, we found that CD44v3+ cells were as proliferative as CD44v3cells but were more invasive in vitro and highly metastatic in vivo.

In breast and ovarian cancers, physical interactions of CD44v3 with the extracellular matrix component hyaluronic acid and the cytoskeletal protein Ankyrin induce cytoskeletonmediated tumour cell migration through the stimulation of Rac1 and RhoA Rho GTPases 31,38,39 . Interestingly, CCDC125 that negatively controls RhoGTPases activity and motility 40 , was found downregulated in panCD44+ and CD44v3+ cells, suggesting that this pathway could be involved in CD44v3+ cells-associated invasive properties.

We previously demonstrated that carcinogenic strains of H. pylori alter the differentiation status of gastric epithelial cells, leading to an EMT-like process and the emergence of CD44+ cells with CSCs properties 23,42 . The EMT process is transient for cell dissemination, and after homing to distant organs, cells need to revert to a more epithelial phenotype to grow, differentiate and develop metastases 43 . EMT is an inducible and highly dynamic process modulated through diverse mechanisms including epigenetic modifications and alternative splicing 44 . Alternative splicing is a key mechanism to increase proteomic diversity and a process to rapidly adapt to microenvironment changes. CELF2 and CELF6

Supplemental Information

SUPPLEMENTARY METHODS

Gastric epithelial cell lines culture. GC cell lines were authenticated by STR profiling and were mycoplasma free (verified by PCR). Cells were cultured in media supplemented with 10% heat-inactivated foetal calf serum and 50 IU/mL of penicillin and 50 mg/L of streptomycin (all from Invitrogen), at 37 °C in a 5% CO2 humidified atmosphere. Media were RPMI 1640-Glutamax for MKN45 and MKN74 and DMEM F12-Glutamax for AGS, KATO III and NCI-N87 1(pnguy) 2 3 4(pmolin) .

Invasion and gelatine degradation assays.

Adherent cells were recovered by trypsinization and 30.000 48-h siRNA-transfected cells or 5.000 CD44v3+/-FACS-sorted cells were seeded in 8-µm pored Transwell® (Sigma-Aldrich, St. Quentin-Fallavier, France) previously coated with rat-tail type 1 collagen (Becton Dickinson, Le Pont de Claix, France). After 18 h of incubation at 37°C, inserts were fixed with 4% paraformaldehyde (PFA) and processed for DAPI staining. Cells from the upper part of the transwell inserts were removed by swabbing while invaded cells were counted using ZOE TM Fluorescent Cell Imager (Bio-Rad, California, USA). For gelatine degradation assay, 40.000 cells were seeded on glass coverslips previously coated with gelatine from pig skin (Oregon green 488 conjugate, Life Technology) 5 . Cells were fixed 24 hours later with 4% PFA, actin was stained with phalloidin-Alexa 647 and nuclei were stained with DAPI. Photos were taken using a light microscope and a 40x objective (Nikon, Champigny sur Marne, France) and gelatine degradation areas were measured using ImageJ software. metaplasia, dysplasia, scored according to the Sydney System criteria). Clinico-biological data including 5-years overall survival were collected.

Histology and immunohistochemistry staining. 3 µm-thick tissue sections were prepared from PET blocks 2 4 . Primary antibodies were incubated for 0.5 to 2 h at room temperature (RT), followed by 30 min incubation at RT with anti-mouse and anti-rabbit Labelled

Polymer-HRP DAKO Envision Systems (DAKO). Immunolabelling was revealed by 1 to 10 min incubation in liquid substrate-diaminobenzidine-chromogen (DAKO). Slides were counterstained with haematoxylin, dehydrated and mounted with Eukitt-mounting medium.

Microarray gene expression studies. FACS-sorted cells were recovered in simplicates (n=4 PDX cases and n=1 GC cell line) and in three independent replicates (n=2 PDX cases). RNAs were extracted using RNeasy microkit (All from Qiagen) containing a DNA digestion step.

RINs were determined on the TapeStation (Agilent). Gene expression profiles were performed at the GeT-TRiX facility (GénoToul, Génopole Toulouse Midi-Pyrénées) using

Agilent Sureprint G3 Human microarrays (8x60K, design 072363) following the manufacturer's instructions. For each sample, Cyanine-3 (Cy3) labelled complementary RNA (cRNA) was prepared from 25 ng of total RNA using the One-Color Quick Amp Labelling kit (Agilent) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts). Dye incorporation and cRNA yield were checked using Dropsense™ 96 UV/VIS droplet reader (Trinean, Belgium). 600 ng of Cy3-labelled cRNA were hybridized on the microarray slides following the manufacturer's instructions. Immediately after washing, the slides were scanned on Agilent G2505C Microarray Scanner using Agilent Scan Control A.8.5.1 software and fluorescence transcripts of 2,578 and 34 probes were downregulated in panCD44+ and CD44v3+ cells, respectively, compared to their respective negative cells (Supplementary Tables S4 andS5), among which, only 6 were in common (Fig. 3B and Supplementary Table S6). Enriched signalling pathways of the panCD44+ profile included ribosome biogenesis, DNA replication, RNA polymerase, genes involved in mismatch and nucleotide excision repair and metabolic pathways (pyruvate, pyrimidine and purine) and cell cycle (KEGG analysis, Supplementary Table S7). Interestingly, a significant upregulation of SLC7A11 encoding xCT, a glutamate-cysteine transporter that interacts with CD44E and controls the intracellular level of reduced glutathione and prevents reactive oxygen species-related damage 11 , was observed, which might contribute to the chemoresistance properties of these cells that we previously reported 1 . Enriched signalling pathways of CD44v3+ profile was largely different and included pathways associated with auto-immunity, innate immunity and cell adhesion molecules (Supplementary Table S8).

Among the 310 mRNA transcripts that were significantly upregulated in CD44v3+, 60 were significantly downregulated in panCD44+ (Fig. 3A-B, Supplementary Table S6).

Interestingly, many of them have been involved in EMT, invasion and tumour aggressiveness, including Zinc finger E-box-binding homeobox 2 (ZEB2) 12 , Rac Family Small GTPase 2 (RAC2) 13 , Transforming Growth Factor alpha (TGFA), Neurogenin 3 (NEUROG3), Tetraspanin 10 (TSPAN10) 14 , Myristoylated Alanine-Rich protein Kinase C Substrate (MARKS) 15-17 , ST8 Alpha-N-Acetyl-Neuraminide Alpha-2,8-Sialyltransferase 1 (ST8SIA1) 18 , the brain abundant, membrane attached signal protein 1 (BASP1) 19 , Histone Cluster 1 H2a family (HIST1H2A) 20 and variants 21 , while others are RNA binding proteins ~309~ involved in post-transcriptional modifications including pre-mRNA alternative splicing, ie, CUG-BP Elav-Like Family Members -2 (CELF2) and -6 (CELF2) 22-24 .

Altogether, these results suggest that panCD44+ and CD44v3+ subpopulations of GC cells express distinct patterns of genes, panCD44+ cells harbouring a more metabolic signature while CD44v3+ cells harboured a signature more related to EMT. The Helicobacter genus actually comprises 46 validly published species divided into two main clades: gastric and enterohepatic Helicobacters. These bacteria colonize alternative sites of the digestive system in animals and humans, and contribute to inflammation and cancers. In humans, Helicobacter infection is mainly related to H. pylori, a gastric pathogen infecting more than half of the world's population, leading to chronic inflammation of the gastric mucosa that can evolve into two types of gastric cancers: gastric adenocarcinomas and gastric MALT lymphoma. In addition, H. pylori but also non-H. pylori Helicobacter infection has been associated with many extra-gastric malignancies. This review focuses on H. pylori and its role in gastric cancers and extra-gastric diseases, as well as malignancies induced by non-H. pylori Helicobacters. Their different virulence factors and their involvement in carcinogenesis is discussed. This review highlights the importance of both gastric and enterohepatic Helicobacters in gastrointestinal and liver cancers.

SUPPLEMENTARY FIGURES

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.