Molina Castro

Correspondence. Dr Christine Varon
email: christine.varon@u-bordeaux.fr

LATS2 and YAP1 are co-upregulated in response to Helicobacter pylori infection and control cancer stem cell properties in gastric epithelial cells

Keywords: CD44, Hippo pathway, TEAD, epithelial-mesenchymal transition, gastric cancer, biomarker

Titre : Etude de la voie de signalisation HIPPO/YAP dans la carcinogenèse gastrique induite par l'infection à Helicobacter pylori

Résumé :

Le cancer gastrique (CG) est la 5 ème cause de cancer et la 3 ème cause de mortalité par cancer dans le monde. Le diagnostic est tardif et les options thérapeutiques sont limitées. Le CG est une maladie multifactorielle, fréquemment associée à l'infection chronique par des souches CagA + d'Helicobacter pylori. La transition épithélio-mésenchymateuse (EMT) est un processus réversible dans lequel une cellule épithéliale polarisée acquiert un phénotype mésenchymateux. L'EMT est à l'émergence de cellules souches cancéreuses (CSC), une minorité de cellules capables de division asymétrique et d'autorenouvèlement, à l'origine des cellules différentiées dans la masse tumorale et capables de former des nouvelles tumeurs. Dans le CG, ces CSC expriment CD44 et présentent une activité ALDH élevée. L'infection des cellules épithéliales gastriques humaines (CEGs) par CagA + H. pylori induit des cellules CD44 high avec des propriétés des CSCs via une EMT. La voie Hippo est composée par les kinases suppresseur de tumeurs MST et LATS, et leurs cibles de phosphorylation, les oncoprotéines YAP1 et TAZ. Suite à la phosphorylation inhibitrice, YAP1 et TAZ sont retenus aux jonctions cellulaires ou sont dégradées au protéasome. Au noyau, YAP1 et TAZ activés lient les facteurs TEAD pour promouvoir la croissance cellulaire et l'inhibition de l'apoptose.

Notre premier objectif était de rechercher si H. pylori change l'état d'activation de la voie Hippo et comment cette régulation modifie l'EMT et la population de cellules avec des propriétés de CSC in vitro et in vivo. Le deuxième but est la caractérisation du rôle de YAP1/TEAD dans les propriétés de CSCs gastriques in vitro et les conséquences de son inhibition pharmacologique dans la croissance des tumeurs in vivo pour le développement des thérapies co-adjuvantes pour le CG.

Pour étudier la régulation de la voie Hippo pendant l'infection par H. pylori, LATS2, YAP1 et CD44 ont été évalués dans la muqueuse gastrique de sujets non-infectés et infectés par H. pylori, qui ont été augmentés avec l'infection et leur surexpression a été associée avec la gastrite et la métaplasie intestinale. Dans les CEGs l'expression de gènes de la voie Hippo a été altérée par l'infection. La régulation de la voie Hippo par H. pylori a une cinétique diphasique et dépendante de CagA. Dans l'infection précoce, H. pylori déclenche l'activité transcriptionelle de YAP1. Cette période d'inactivité de la voie Hippo est suivi de son activation progressive, soutenue par l'accumulation de LATS2 et la phosphorylation inhibitrice de YAP1. La répression de LATS2 avec siRNAs a accéléré l'acquisition du phénotype mésenchymateux après l'infection, l'augmentation de marqueurs de l'EMT (Zeb1 et Snail1), et la diminution des miR-200 épithéliaux. Le CD44 et les capacités d'invasion et formation de sphères induites par H. pylori ont été potentialisées par l'inhibition de LATS2, ce qui suggère que LATS2 limite l'EMT et le phénotype de CSC acquis pendant l'infection. L'inhibition de LATS2 ou YAP1 diminue l'expression de ces deux protéines, révélant ainsi une boucle de régulation positive. Dans des coupes de tissu de CG, l'expression de LATS2 et YAP1 est hétérogène et positivement corrélée, fait qui a été confirmé dans 38 CEGs de la CCLE. L'expression LATS2 est fortement corrélée à celle de CTGF et CYR61, ce qui suggère que LATS2 peut aussi être un gène cible de YAP1/TEAD. La deuxième partie de l'étude correspondait à l'évaluation des propriétés anti-tumorales de la verteporfine (VP), qui est capable d'interrompre l'interaction YAP1/TEAD, et donc d'inhiber son activité transcriptionelle. In vitro, utilisant CEGs et des cellules de tumeurs de patients amplifiées chez la souris (patient-derived xenograft PDX), le traitement à la VP a diminué la croissance cellulaire, l'expression de gènes cible de YAP1/TAZ/TEAD, l'activité du rapporteur TEAD-luciférase et la capacité de formation de sphères. L'activité de la VP a été testée in vivo dans un modèle de greffe sous-cutanés des CEGs MKN45 et MKN74 et le PDX GC10 chez la souris NSG. Les tumeurs développées ont été traitées par injection péri-tumorale avec trois doses de VP ou du DMSO comme contrôle. La croissance tumorale a été diminuée à partir du jour 5-12 pour la dose intermédiaire. Le poids des tumeurs, l'analyse par IHC (CD44, ALDH, Ki67) et la capacité de formation de sphères des CSCs résiduelles ont été diminuées dans les tumeurs traités. Ces résultats montrent une activité inhibitrice de la VP sur les CSCs gastriques in vitro et in vivo.

Ce travail montre pour la première fois que l'axe LATS2/YAP1/TEAD est précocement activé pendant l'infection chronique avec H. pylori et que celui-ci contrôle l'EMT et les propriétés de CSC. Le ciblage de la voie Hippo a été montré comme étant efficace dans la prévention de la croissance tumorale, mettant en évidence le potentiel de son inhibition dans le traitement du cancer gastrique.

Mots clés : Cellules souches gastriques, transition épithélio-mésenchymateuse, CD44

Title: Study of the Hippo/YAP1/TEAD signaling pathway in gastric carcinogenesis induced by Helicobacter pylori

Abstract:

Gastric cancer (GC) is the 5 th most common cancer overall and the 3 rd cause of cancer death worldwide. Diagnosis is often late and therapeutic options are limited. It is a multifactorial disease, most frequently associated to chronic infection with CagA-positive Helicobacter pylori strains. Epithelial-to-mesenchymal transition (EMT) is reversible process in which polarized epithelial cells acquire a mesenchymal phenotype. EMT is at the origin of cancer stem cells (CSC), a few stem-like cells within the tumors with asymmetrical division and self-renewal capacities. They give rise to the differentiated tumor cells and originate new tumors. In GC, CSCs express the hyalorunate receptor CD44 and high aldehyde-dehydrogenase (ALDH) activity. Infection with H. pylori of human gastric cancer cell lines (hGECs) in vitro induces the emergence of a population of CD44 high cells with CSC-properties through an EMT process in a CagA-dependent manner. The Hippo pathway is composed by a tumor suppressor kinases MST and LATS, and their phosphorylation targets, the oncogenes YAP1 and TAZ. Upon phosphorylation by LATS, YAP1 and TAZ are retained in cell-cell junctions or undergo proteasomal degradation. Active nuclear YAP1 and TAZ bind to TEAD transcription factors to promote the expression of genes that positively regulate cell growth and inhibit apoptosis.

The first aim of this work was to investigate whether H. pylori affects the activation state of the Hippo pathway, and its effect on the EMT process and the CSC-like cell population in vitro and in vivo. Second, we intended to characterize the role of YAP1/TEAD in gastric CSC properties in vitro and the consequences of its pharmacological inhibition on tumor growth in vivo, aiming to the development of adjuvant therapies to GC.

To study the Hippo pathway regulation during infection, LATS2, YAP1 and CD44 were evaluated in gastric mucosae of either non-infected or H. pylori-infected patients. They were upregulated in infected mucosae and were associated to gastritis and intestinal metaplasia. In hGECs, expression of Hippo pathway genes was significantly altered by infection. Hippo pathway regulation by H. pylori infection has biphasic kinetics and is CagA-dependent. Early in infection, H. pylori transiently triggered YAP1 expression and co-transcriptional activity, along with LATS2. This period of Hippo pathway inactivity is followed by a progressive activation, sustained by LATS2 accumulation and inhibitory YAP1 Ser127 -phosphorylation. LATS2 siRNA-mediated repression accelerated the acquisition of the EMT-phenotype upon infection, the up-regulation of EMTmarkers ZEB1 and Snail1, and the decrease of the epithelial miR-200. H. pylori-induced CD44 upregulation, invasion and sphere-forming capacity were further enhanced upon LATS2 knockdown, suggesting that LATS2 restricts the EMT and CSC-like phenotype in hGECs upon H. pylori infection. Inhibition of either LATS2 or YAP1 reduced the expression of both proteins, revealing a positive feedback loop. In tissue sections of GC, LATS2 and YAP1 were heterogeneous and co-expressed. The positive correlation between LATS2 and YAP1 was confirmed in the 38 hGECs of the CCLE. The expression of CTGF and CYR61 was also strongly correlated to LATS2, suggesting that LATS2 could also be a YAP1/TEAD target gene.

Second, we evaluated the anti-tumor properties of verteporfin (VP), which disrupts the YAP1/TEAD interaction inhibiting its transcriptional activity. In vitro, using hGECs and cells from patient derived primary tumor xenogratfs (PDXs), we showed that treatment with VP decreased cell growth, expression of YAP1/TAZ/TEAD target genes, TEAD-luciferase reporter activity and sphere-forming capacity. The activity of VP was tested in vivo, in a model of subcutaneous graft of hGECs (MKN45 and MKN74) and PDX (GC10) in NGS mice. Resulting tumors were treated daily by peritumoral injection of three doses of VP or DMSO as control. Tumor growth was followed and a decrease starting from day 5-12 was observed at the intermediate dose. Tumor weight measurement, IHC analysis (CD44, ALDH and Ki67), and evaluation of sphere-formation capacity of residual CSCs were decreased in treated tumors. Altogether, these results show the CSCinhibitory activity of VP both in vitro and in vivo.

We showed for the first time that the LATS2/YAP1/TEAD axis is early activated during the carcinogenesis process induced by chronic H. pylori infection and controls the subsequent EMT and CSC-like features. Targeting the Hippo pathway efficiently prevented tumor growth in a PDX model, highlighting the potential of its inhibition to be implemented in gastric cancer therapy.

Keywords: Cancer stem cells, epithelial to mesenchymal transition, CD44

Unité de recherche Bordeaux Research in Translational Oncology, INSERM U1053

146 Rue Léo Saignat, Campus Carreire Zone Nord, 2B RdC 33076 Bordeaux

Substantial abstract:

Stomach cancer is the fifth most common cancer overall, and despite of a declining incidence, it remains the third leading cause of cancer death worldwide. The diagnosis of the disease is often at advances stages, the therapeutic options are limited and the survival rates are poor. Gastric cancer is a multifactorial disease, most frequently associated to the chronic infection with Helicobacter pylori. H.

pylori is a curved gram-negative bacillus present in human gastric mucosa linked to dyspeptic conditions and gastric cancer, and it is a WHO-Type I carcinogen since 1994. H. pylori infects approximately half of the world's population, especially underdeveloped countries. Upon acquisition of H. pylori, a persistent infection is established, ending only by the eradication with antibiotic treatment. Only a small percentage of cases is associated with severe pathologies (10-15 %), such as chronic atrophic gastritis, peptic ulcers and gastric malignancies (1-2 %), notably gastric adenocarcinoma and MALT lymphoma. The main virulence factor of H. pylori is the Cytotoxin associated gene A (CagA) whose expression is strongly associated with a higher virulence of the bacterium.

Epithelial-to-mesenchymal transition (EMT) is an evolutionary conserved, reversible process in which a polarized epithelial cell acquires a mesenchymal phenotype through phenotypical and molecular changes, leading to increased capacities of migration, invasion, apoptosis resistance and extracellular matrix production and remodeling. It can occur in association to cancer progression and metastasis of tumors of epithelial nature, tumor-related immunosuppression, and drug resistance. Another interesting aspect of EMT in carcinogenesis is its role in the origin of cancer stem cells (CSC). Tumors are heterogeneous entities in which CSCs represent a minority of stem-like cells with asymmetrical division and self-renewal capacities, as well as the potential to give rise to the more or less differentiated cells within the tumor. These properties combine in the ability to originate new tumors. In gastric cancer, these CSCs express, amongst other markers, the hyalorunate receptor CD44 and a high aldehyde-dehydrogenase (ALDH) activity. Infection with Helicobacter pylori of human gastric adenocarcinoma cell lines in vitro induces the emergence of a population of CD44 high cells with properties of CSC through an EMT process.

The induction of the EMT and the CD44 high cell population was demonstrated to be dependent on the bacterial oncoprotein CagA.

The Hippo pathway was first described as a size-controlling pathway in the epithelial precursors in Drosophila melanogaster. It is a highly evolutionarily conserved pathway, composed by a tumor suppressor core consisting of a module of two kinases, MST and LATS, and their regulatory proteins SAV and MOB. The second component includes the output elements, which are the targets of phosphorylation by the kinase module, the YAP1 and TAZ proteins. Upon phosphorylation by LATS, YAP1 and TAZ are retained in cell-cell junctions or undergo proteasomal degradation. This sequestration limits their activity by preventing the nuclear accumulation. Active nuclear YAP1 and TAZ function together with several partners, such as AP-1, p73, and especially TEAD family transcription factors, to promote tissue growth by increasing the expression of genes that positively regulate cell growth and inhibit apoptosis. Their capacity to regulate transcription of genes controling cell survival and proliferation make YAP1 and TAZ, oncogenes.

Using in vitro and in vivo models for the study of gastric carcinogenesis induced by H. pylori infection, the first aim of this work was to investigate whether H. pylori affects the activation state of the Hippo pathway, focusing on the roles of LAST2 and YAP1 and how this regulation influences the EMT process and the CSC-like cell population. As a second goal, the present work intended to characterize the role of YAP1/TEAD in gastric CSC properties in vitro and the consequences of its pharmacological inhibition on tumor growth in vivo in a model of human tumor xenografts, aiming to the development of adjuvant therapies to gastric cancer.

In the first part, we studied the Hippo pathway regulation during infection. LATS2, YAP1 and CD44

were evaluated in gastric mucosae of either non-infected or H. pylori-infected patients, and were found upregulated in the infected mucosa, precisely within the isthmus region of the gastric glands, where gastric epithelial stem cells reside. The over expression was associated to gastritis and intestinal metaplasia. In human gastric epithelial cell lines, expression of genes involved in the Hippo pathway was significantly altered by infection, including the Hippo kinase core genes, the transcriptional co-factors YAP1 or TAZ , the TEAD transcription-enhancer factors, and several target genes involved in cell proliferation and survival, including cyr61 and ctgf. We showed here that Hippo pathway regulation by H. pylori infection has biphasic kinetics. In the early period of infection, H. pylori transiently triggered YAP1 expression cotranscriptional activity, along with LATS2. This early period of infection corresponds to an inactive Hippo pathway and is followed by a progressive activation, likely sustained by LATS2 accumulation and the subsequent YAP1 Ser127-phosphorylation, preventing its nuclear translocation and TEAD-mediated transcriptional co-activation. Noteworthy, the activation of the Hippo pathway was CagA-dependent and thereby related to the virulence and oncogenic potential of the bacteria.

To study the role of LATS2 in the EMT and CSC-like properties induced by H. pylori in human gastric epithelial cell lines in vitro, a knock-down of LATS2 using siRNAs was performed. LATS2 repression accelerated the acquisition of the EMT-phenotype upon infection, the up-regulation of the EMT-markers ZEB1 and Snail1, as well as the decrease of the epithelial miR-200 microRNA family members. CD44

upregulation by H. pylori infection was further enhanced upon LATS2 knockdown. Invasive properties and tumorsphere-forming capacity of the cells were increased by infection and inhibition of LATS2 as well.

These results suggest that LATS2 restricts the EMT and CSC-like phenotype that human gastric epithelial cells acquire upon H. pylori infection. Moreover, LATS2 is essential for optimal growth either in monolayer or in spheroids. The siRNA experiments showed that inhibiting either LATS2 or YAP1 led to a reduced expression of both proteins, revealing a positive feedback loop between them.

In tumor tissue sections of gastric adenocarcinoma patients, LATS2 and YAP1 were found to be heterogeneous and co-expressed, regarding both subcellular localization and expression. The evaluation of the expression of LATS2, YAP1, and CD44 in the 38 gastric cancer cell lines of the Cancer Cell Line Encyclopedia confirmed the positive correlation between LATS2 and YAP1 expression. In addition, the expression of the YAP1/TEAD target genes CTGF and CYR61 is strongly correlated to LATS2 expression, suggesting that LATS2 could also be considered as a YAP1/TEAD target gene.

A second part of the study consisted in the evaluation of anti-tumor properties of verteporfin, a

protoporphyrin that is able to disrupt the YAP1/TEAD interaction, and therefore inhibits its transcriptional activity. In vitro, using the gastric cancer epithelial cell lines MKN45 and MKN74, and cells from patient derived primary tumor xenogratfs (PDXs), we showed that treatment with verteporfin decreased cell growth with an accumulation of cells in G0/G1 phase of the cell cycle. The expression of YAP1/TAZ/TEAD target genes and the activity of TEAD-luciferase reporter was also decreased upon treatment, as well as the capacity to form tumorspheres when cells were cultured in non-adherent conditions, a key feature of CSCs. The activity of verteporfin was then tested in vivo, in a model of subcutaneous PDXs in immunodeficient mice. Cells from two gastric cancer cell lines (MKN45 and MKN74) and one patient derived gastric carcinoma (GC10) were used. After a period of growth, the developed tumors were treated daily by peritumoral injection of three increasing doses of verteporfin or DMSO as control vehicle, for 15 days. The tumor growth was followed during this period at the end of which, mice were sacrificed and tumors were extracted for weight measurement, histological analysis, and seeding in non-adherent conditions to evaluate tumorsphere-formation capacity of residual CSCs. The growth rate was found to be decreased starting from day 5-12 at the intermediate dose. Tumor weight at sacrifice was also significantly lower in the treated group in comparison with the controls. Tumorsphere formation and self-renewal properties were decreased in treated cells in all cases. Finally, the immunohistochemistry evaluation of cells from treated tumors showed a heterogeneous but decreased expression of CD44 and ALDH in the tumors for the treated group, as well as a downregulation of the Ki67 proliferation marker. Altogether, these results show the CSC-inhibitory activity of verteporfin both in vitro and in vivo.

This work shows for the first time that the Hippo pathway and particularly the LATS2/YAP1/TEAD axis is early activated during the carcinogenesis process induced by chronic H. pylori infection and controls the subsequent EMT and CSC-like features. Targeting this pathway was shown to be efficient in preventing tumor growth in a PDX model, therefore highlighting the potential of its inhibition to be implemented in gastric cancer therapy.

Remerciements -Acknowledgements -Agradecimientos

To the jury members, for agreeing their precious time to analyze, criticize, and help me to improve the scientific quality of my work.

A ma directrice de thèse Mme le Dr. Christine Varon pour son guide et son soutien pendant ces quatre ans, et pour me conseiller et me permettre d'apprendre à son côté, mais aussi pour me donner la liberté de poursuivre la curiosité scientifique et être toujours ouverte au débat des idées et opinions, ce qui m'a permis de croître comme professionnel et comme personne.

A Mme le Dr. Cathy Staedel pour ses conseils, son expertise et sa gentillesse, sans lesquels ce travail n'aurait pu être possible. Merci pour être un exemple de ce qu'on aime dans la science. A Mme le Dr. Hélène Boeuf pour ses conseils, sa gentillesse et les riches discussions pour améliorer mon travail.

A M le Pr. Francis Mégraud pour son soutien, sans lequel je ne serais pas venue en France, pour son accueil chaleureux depuis mon arrivée au laboratoire, et toutes les opportunités scientifiques et personnels données dans ces années.

A M le Pr. Philippe Lehours, Mme le Dr. Emilie Bessède, Mme le Dr. Armelle Menard et Mme le Dr. Claire Roubaud pour leurs conseils, leurs critiques, et leur soutien tout au long de ma thèse.

Aux assistants ingénieurs de notre laboratoire : Elodie Sifré, Lucie Chambonnier, Julien Izotte, Alban Giese, Alice Buissonière, Astrid Ducournau, Lucie Bénéjat et Salha Ben Amor, c'était vraiment un plaisir de travailler avec vous, toujours disponibles pour partager vos compétences à la paillasse et ailleurs.

A mes chers collègues Sarah, Pauline, Christelle et Julie pour leur amitié, leur solidarité, leurs conseils et les expériences partagées, essentiels pour tenir jusqu'au bout. A Luis et Amandine, les « M2 originaux », cette expérience n'aurait été la même chose sans vous. A Camille et Solène, pour son travail et pour être les « sujets expérimentales » de mon apprentissage pédagogique, j'espère de ne pas avoir été trop décevante.

A Ann et Isabelle pour leur aide professionnel et personnel, leur gentillesse et leur amitié.

En fin, un grand merci à tous pour faire une belle ambiance de travail au laboratoire.

A Mme Lindsay Mégraud pour son aide et sa gentillesse qui m'ont fait me sentir bienvenue en France.

A mis jefes y compañeros del Programa de Epidemiología del Cáncer del INISA, Dra. Vanessa Ramirez, Dr. Clas Une, M.Sc. Wendy Malespín y Dr. Warner Alpízar, por todo el apoyo brindado, fundamental en el crecimiento profesional y personal que me permitió comenzar con la etapa que hoy termina. Gracias a la Universidad de Costa Rica y el Ministerio de Ciencia, Tecnología y Comunicaciones, así como al Instituto Francés para América Central por el apoyo financiero.

A mis papás y mis hermanitos de dos y cuatro patas, a toda mi familia y mis queridos amigos, los de siempre y los que me he ido encontrando en el camino, en Costa Rica y de este lado del charco. Les debo todo.

Table of contents

I. Introduction

20

a.

Gastric cancer i. Epidemiology

According to the latest report of the International Agency for Research on Cancer (IARC), stomach cancer is the fifth most common cancer overall, with an estimate of 952 000 new cases in 2012 1 .

Despite of a declining incidence, it remains the third leading cause of cancer death in both sexes worldwide, with approximately 723 000 stomach cancer deaths in 2012 which represent 8.8% of the death burden due to all types of cancer (except non-melanoma skin cancers). This cancer is more frequent in men than in women and more common in developing countries 1,2 . In general, gastric cancer rates are twice as high in men as in women, and the risk of gastric adenocarcinoma is lower at a young age, and gradually increases, reaching a plateau between 55 and 80 years 3 . The highest incidences are found in Eastern and Western Asian countries (Korea, Japan, and Iran) and Eastern Europe (Russia and other former URSS countries), as well as in some Latin American countries (Chile, Costa Rica, Ecuador, and Colombia) 4,5 .

In countries with very heterogeneous populations, like the United States, there are differences in the cancer incidence rates associated with the ethnical background of the population. The incidence of gastric cancer is two times higher in Hispanics, Korean Americans, and African Americans than in non-Hispanic whites and other Asian Americans 6,7 . This may be because these groups tend to have lower socioeconomic conditions, limited access to quality healthcare and screening and preventions campaigns, as well as different genetic backgrounds 6,8 .

Unfortunately, the diagnosis of the disease is often at advances stages, generally in elderly people, making the therapeutic options limited at best. Consequently, the 5-year survival is poor, usually less than 30%, and as low as 4.3-9.2% when untreated 3,9 . As expected, there is a difference between survival rates in developed and developing countries. A recent meta-analysis reported a 5-year survival in Iran, whilst values between 26 and 32% were reported for Western European countries in the SUDCAN study 10,11 .

Gastric cancer is a multifactorial disease, it can be hereditary or associated with a specific mutational profile, but most frequently, it is associated to the chronic infection with H. pylori 3 . In a study of the Eurogast-EPIC project, more than 93% of the non-cardia gastric cancer were positive for H. pylori 12 .

Moreover, of the 2.2 million new cases of cancer attributable to infectious agents in 2012, H. pylori infection occupied the first place with 770 000 cases, representing 81% of the total new cases of gastric cancer for that year and being the only bacterial pathogen included in the list 13 .

Epidemiological features of the different types of gastric adenocarcinomas vary regarding their localization and histological classification. The most frequent type of gastric adenocarcinoma is the noncardia carcinoma of intestinal type, which has the stronger association with H. pylori infection, but a tendency towards decrease has been observed 1,14 . The diagnosis for this entity is normally carried at older male patients (55-80 years old, male to female ratio 2:1), after a long history of precancerous lesions [14][15][16] .

Still in the non-cardia zone, the diffuse type incidence is, on the contrary, increasing 16,17 . The non-cardia diffuse type can be associated with the H. pylori infection but the main factor is the loss of E-cadherin, due to mutations in the CDH1 gene 17 . It is diagnosed at a younger age than the intestinal type (40-60 years old) and frequently there is an autosomal dominant pattern of inheritance, and it is equally distributed between men and women 16 . Incidence of adenocarcinomas of the cardia region is increasing as well 18 .

This type of cancer is not associated with H. pylori, but mostly with gastroesophageal reflux disease (GERD)

for which H. pylori has been described as a protective factor, and it is strongly predominant in white males (male to female ratio of 6:1) 18,19 .

ii.

Classification of gastric cancer

1.

Macro-anatomic classification

The anatomical localization of gastric adenocarcinomas often reflects the pathological mechanisms laying behind the disease. Intestinal-type adenocarcinomas usually differ in etiology regarding whether they are proximal, which are associated with reflux, or distal, associated with H. pylori infection instead, while diffuse type adenocarcinomas are usually located in the middle third and the body of the stomach 20 . The macroscopic appearance of the tumors is also a feature used for classification. Noninvasive adenocarcinomas confined to the mucosa, regardless of the presence of lymph node metastasis and size, are considered as early 21 . The Japanese system classifies these tumors into Types I to IV, and the more recent Paris classification recognizes three gross patterns for superficial neoplastic lesions in the gastrointestinal tract 21 . The prognosis of early gastric carcinoma is excellent, with a 5-year survival rate as high as 90%, contrasting with the 60% or less observed for the advanced cases, that is, when there is an invasion of the muscularis propria or beyond 21 . All three classification systems are detailed in table 1.

Advanced

Borrmann classification I Polypoid, 25% more common in the greater curvature of the corpus II Fungating, 36% of all cases, usually located in the lesser curvature of the antrum III Ulcerating, 25% more common in the greater curvature of the corpus IV Diffusely infiltrating, also referred as linitis plastica when it involves a very significant portion of the stomach and is associated with the diffuse histological type

Histological classification

There are two main systems used to classify the gastric adenocarcinomas regarding their microscopic features, the Laurén classification and the WHO system. The Laurén classification is the most widely used and was described in the 1965, based on the examination of 1344 cases of gastric cancer 22 . It distinguishes two subtypes of gastric adenocarcinomas: the intestinal type, characterized by the formation of glands with or without the production of extracellular mucin, and the diffuse, which has poorly cohesive cells that often contain intracytoplasmic mucin and are therefore known as signet ring cells 20 . Tumors with a combination of the two subtypes, known as mixed-type adenocarcinomas are included in this system as well. The WHO system, updated in 2010, recognizes four main categories for gastric adenocarcinomas: tubular, papillary, mucinous and poorly cohesive, and it includes also the possibility of mixed carcinomas when the tumors present components of the four main groups 23 .

There is a significant correlation between the two classification schemes. The Laurén's diffuse type corresponds to the poorly cohesive, and it is characterized by a proliferation of isolated cancer cells which do not form glandular structures and usually affects younger subjects (mean age of 48 years in comparison with 55 for intestinal type) 24,25 . These cancers are more associated with genetic factors of the host than with environmental triggers and are present in populations of low risk. They may be inherited in an autosomal dominant pattern due to mutations in CDH1 26 . The more prevalent intestinal subtype includes the tubular, papillary and mucinous variants. This form of adenocarcinoma is preceded by a longterm process where different types of precancerous lesions succeed each other in a model proposed by

Correa in 1975 27 . After some modifications the model recognizes the following steps from a healthy gastric mucosa to invasive adenocarcinoma: non-atrophic gastritis, multifocal atrophic gastritis without intestinal metaplasia, complete or small bowel-type intestinal metaplasia, incomplete or colonic-type intestinal metaplasia, low grade dysplasia, high grade dysplasia, and invasive adenocarcinoma 28 .

Molecular classification

The study of genetic alterations in gastric cancer is not new, and different methods have been used to this end, from detection of single nucleotide polymorphisms (SNP) to whole genome sequencing.

However, up until 2014 a systematic global assessment of the molecular features of gastric cancer was not available. In order to develop a robust molecular classification of gastric cancer The Cancer Genome Atlas (TCGA) carried on a large scale, multicenter study that included material from 295 patients 29 . Sample assessment by different techniques such as whole-exome, whole-genome, mRNA, and miRNA sequencings, SNP arrays, somatic copy number analysis, DNA-methylation profiling allowed not only the establishment of a classification system of gastric carcinomas according to molecular criteria, but also the identification of dysregulated pathways and candidate drivers for the distinct classes of gastric cancer.

The resulting classification system includes four categories: Epstein-Barr virus (EBV) infected tumors, tumors with microsatellite instability (MSI), genomically stable tumors (GS), and chromosomally unstable tumors (CIN) 29 . Further details on this classification are shown in figure 1.

Figure 1. Key features of gastric cancer subtypes according to molecular classification.

Anatomical distribution of each type in shown in pie-charts 29 .

Pathologic staging

The process of staging is the determination of how much cancer present in the body and where it is located, and it describes the severity of cancer based on criteria such as the characteristics of the primary tumor, and the spreading of cancer through the body 30 . Pathologic staging, that is, after tumor resection or exploratory surgery, follows the guidelines established by the American Joint Committee on Cancer Staging (AJCC) and the Union for International Cancer Control (UICC) known as the TNM system.

The TNM systems is based in the size of the primary tumor (T), lymph node involvement (N) and the presence or absence of distant metastasis (M) 20 . The 2010 update (7 th edition) is detailed in table 2.

Table 2. TNM-staging of gastric cancer 31

Stage

T N M Stage 0 Tis N0 M0 Stage IA T1 N0 M0 Stage IB T2 N0 M0 T1 N1 M0 Stage IIA T3 N0 M0 T2 N1 M0 T1 N2 M0 Stage IIB T4a N0 M0 T3 N1 M0 T2 N2 M0 T1 N3 M0 Stage IIIA T4a N1 M0 T3 N2 M0 T2 N3 M0 Stage IIIB T4b N0 or N1 M0 T4a N2 M0 T3 N3 M0 Stage IIIC T4b N2 or N3 M0 T4a N3 M0 Stage IV

Any T Any N M1

T category definitions. Tis: Carcinoma in situ, T1: Tumor invades lamina propria, muscularis mucosae, or submucosa, T1a: Tumor invades lamina propria or, muscularis mucosae, T1b: Tumor invades submucosa, T2: Tumor invades muscularis propria, T3: Tumor penetrates subserosal connective tissue without invasion of visceral peritoneum or adjacent structures, T4: Tumor invades serosa (visceral peritoneum) or adjacent structures, T4a: Tumor invades serosa (visceral peritoneum), T4b: Tumor invades adjacent structures (spleen, colon, liver, diaphragm, pancreas, abdominal wall, adrenal gland, kidney, small intestine, and retroperitoneum. N category definitions. N0: No regional lymph node metastasis, N1: Metastasis in 1 to 2 regional lymph nodes, N2: Metastasis in 3 to 6 regional lymph nodes, N3: Metastasis in 7 or more regional lymph nodes. M1: Positive peritoneal cytology.

iii. Pathogenesis

Multiple environmental, behavioral and genetic factors can contribute to the development of gastric cancer. Most gastric cancers are sporadic, however there are also approximately 10% of cases that are familial, associated to heritable mutations listed in table 3 andfigure

Dietary factors

Several factors associated with nutritional behavior have been associated to gastric cancer.

While an adequate intake of fruit, vegetables, and other foods rich in antioxidant compounds and dietary fiber is considered to decrease the risk of gastric cancer, the intake of high quantities of salt and nitrous compounds may increase it. There is strong epidemiological evidence for a link between increased risk of gastric cancer and the high intake of salt and salt-preserved foods, some studies have even found progressive increased risk across consumption levels 33 . Mechanistically, it has been described that intake of salted foods enhance the risk of H. pylori infection and CagA expression, therefore promoting gastric cancer, and high concentrations of sodium chloride have been shown to induce inflammatory and erosive damage in the gastric epithelium, cell death and regenerative hyperplasia in animal models 34 . Nitrous compounds are recognized mutagens and carcinogens associated with high risk of gastric cancer, and they can originated from diet or be produced endogenously, especially from red meats as nitric oxide reacts with hemoglobin and myoglobin in the acid environment of the stomach 34 . The nitrous compounds scavenging capacity of dietary fiber have been demonstrated experimentally and may be one of the reasons why an intake of dietary fiber has been described as a protective factor against gastric cancer in a dose dependent fashion 33 .

Tobacco and alcohol intake

Both tobacco smoking and high alcohol intake have been described as risk factors for gastric cancer in various prospective studies and meta-analysis. In the case of smoking, a prospective study of more than 18 000 middle-aged men in Shanghai showed a 60% more risk of gastric cancer in smokers in comparison with non-smokers after correction for the confounder variants, and a meta-analysis including 23 studies showed 50% more risk for smokers than for never smokers 33,34 . Mechanistically, tobacco smoke induced an epithelial to mesenchymal transition in the gastric epithelium in a model of BALB/c mice exposed during twelve weeks, with increased expression of Snail-1, Vimentin, and N-cadherin, and concomitantly reduced the expression of E-cadherin, ZO-1, and CK5 35 . This was mediated by an activity of ERK5 that can be inhibited with a pharmacological inhibitor 35 . In the prospective study cited before, heavy drinkers also showed a significant increase in the risk of gastric cancer, which has also been found in similar studies in European populations, especially with non-cardia intestinal-type cancer 33,34 . Not only high alcohol consumption leads to the production of acetaldehyde that may cause DNA damage incrementing the risk of gastric cancer, but it also interact with host factors such as polymorphisms in genes associated with its metabolism further elevating the risk in this individuals 36 .

Autoimmune gastritis

Autoimmune gastritis (AIG) is a chronic inflammation process in the gastric mucosa that can result in atrophy 37 . AIG is restricted to corpus and fundus, because it originates from an autoimmune reaction against parietal cells, which are absent from the antrum, and maybe totally lost during the progression of disease. In this case, the autoimmune response is mediated by CD4 + T cells against the gastric H+/K+ ATPase, causing the loss of parietal cells that results in increased pH of the stomach and loss of intrinsic factor, impairing the uptake of vitamin B12 37 . There are two proposed mechanisms for the autoimmune response: first, a cross reaction of the immune response elements generated against H. pylori infection with antigens within the proton pump, and second, a primary autoimmune response independent of H. pylori infection 38 .

Gastro-esophageal reflux disease

Gastro-esophageal reflux disease (GERD) has long been recognized as a major risk factor for esophageal adenocarcinoma as it exposes the distal esophageal mucosa to gastric juices containing acid, pepsin, and bile 39 . It has also been identified as a precursor lesion of cardia adenocarcinoma of intestinaltype resembling esophageal adenocarcinoma in H. pylori-negative patients, with OR (95% CI) ranging between 8.02 (2.25-28.58) and 10.08 (2.29-44.36) 40 . The pathophysiological mechanisms involved are suggested to be similar to those in esophageal adenocarcinoma, the reflux of gastric contents leading to a columnar intestinal metaplasia, dysplasia and adenocarcinoma.

iv. Molecular Mechanisms

Signaling pathways in cancer

a.

Wnt/β-catenin signaling pathway

The Wnt family is widely conserved family of secreted glycoproteins which includes at least 19 members in humans, and the signaling they induce upon binding to receptors of the Frizzled (Fz) family is a key mechanism that direct multiple cellular processes such as cell proliferation, differentiation, cell-fate decision, migration, and apoptosis, playing a crucial role during embryonic development and tissue homeostasis [41][42][43] . Therefore, mutations in the Wnt pathway are often linked to human birth defects, cancer, and other diseases, and abnormal activation of this pathway is a common feature of many types of cancer, including gastric adenocarcinomas 42,44,45 .

The canonical activation involves the β-catenin protein as a key signaling intermediate, therefore is known as the Wnt/β-catenin signaling pathway. In the absence of Wnt ligands, β-catenin is bound in a destruction complex with Axin, APC, CK1α, and GSK3β, where it is phosphorylated and targeted for proteosomal degradation 46,47 . On the other hand, if Wnt ligands are bound to the Fz/LRP receptor complexes, the destruction complex is dissociated by the recruitment of the Dvl protein, the phosphorylation of LRP, and the delocalization of cytoplasmic Axin 45 . These events prevent the phosphorylation of β-catenin, stabilizing it and allowing the activation of transcription mediated by the βcatenin/TCF/LEF complex, whose target genes include CCDN1, gastrin and c-myc 45 . Non-canonical or βcatenin-independent activation of Wnt signaling has also been described, including the planar cell polarity (PCP) pathway and the Wnt/Ca 2+ pathway. In the later, the intracellular calcium concentration mediates the signaling, and it is known to regulate stem cells in development 48 .

Both canonical and non-canonical Wnt signaling are not mutually exclusive and predominance of one or the other depends on the cell type, the activation signaling and the extracellular context 49 , and both have been found in association to gastric carcinogenesis 50,51 . However, the β-catenin dependent pathway seems to be the one with the stronger association, hence the more studied. β-catenin is overexpressed in most gastric cancers, especially in those of the intestinal type 52 , and a role for the Wnt/βcatenin signaling pathway in the self-renewal properties of gastric cancer stem cells has been shown 53 .

Furthermore, studies have demonstrated mutations in genes coding for pathway elements are frequent in gastric cancers, 26% of gastric cancers with nuclear accumulation of β-catenin had mutations in CTNN1 54 whilst 22% of sporadic cases presented mutations in APC 55 . Evidence suggests that H. pylori infection, the main environmental factor associated with gastric carcinogenesis, activates the Wnt/β-catenin pathway in a CagA-dependent manner 56 and induces the transcription of β-catenin and its nuclear accumulation via LRP6 phosphorylation 57 . Moreover, this pathway seems to be responsible for the upregulation of the stemness genes NANOG and OCT4 observed during H. pylori infection 58 .

b. Signaling pathways associated with growth factors

When growth factors bind to their cognate receptors, they initiate the transduction of extracellular signals through the activation of intracellular messengers or directly through receptor translocation to the nucleus [START_REF] Concha-Benavente | Oncogenic growth factor signaling mediating tumor escape from cellular immunity[END_REF] . Generally, as a result of this activation, there is a stimulation of processes that are tightly regulated in order to maintain the tissue homeostasis, such as cell proliferation and differentiation. Hence, alterations in growth factor-induced signaling lead to imbalances in the tissue equilibrium and could be implicated in carcinogenesis. Two of these pathways frequently dysregulated in gastric cancer are the fibroblast growth factor (FGF) and the epidermal growth factor (EGF) signaling pathways.

The FGF/FGFR pathway play a key role during embryogenesis and its alterations have been related to developmental disorders and carcinogenesis [START_REF] Kunath | FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from selfrenewal to lineage commitment[END_REF][START_REF] Turner | Fibroblast growth factor signalling: from development to cancer[END_REF] . The FGF proteins bind to the FGFR receptor on the cell surface, and have high affinity for heparin and the heparin sulfate glycosaminoglycans of the extracellular matrix, which protect the FGF from degradation and increase their capacity to bind the FGFR [START_REF] Knights | De-regulated FGF receptors as therapeutic targets in cancer[END_REF] . Once activated, FGFRs stimulate the intracellular PI(3)K-ERK-AKT signaling, increasing mitogenic activity and preventing apoptosis [START_REF] Dudley | Deconstructing phosphatases in limb development[END_REF] . As a consequence, FGF/FGFR are considered to be oncogenes and are overexpressed in a wide variety of cancers [START_REF] Knights | De-regulated FGF receptors as therapeutic targets in cancer[END_REF] . Amplifications in the FGFR have been reported in gastric cancer, resulting in deregulation of FGF signaling [START_REF] Hattori | K-sam, an amplified gene in stomach cancer, is a member of the heparin-binding growth factor receptor genes[END_REF][START_REF] Jang | Mutations in fibroblast growth factor receptor 2 and fibroblast growth factor receptor 3 genes associated with human gastric and colorectal cancers[END_REF] . Interestingly, H. pylori is capable of activating FGF signaling via FGFR2 upregulation and SHP2 activation [START_REF] Katoh | FGF signaling network in the gastrointestinal tract (review)[END_REF] .

EGFR is a tyrosine-kinase receptor of the ERbB family and, upon activation by its ligands EGF or transforming growth factor alpha (TGF-α), it can initiate diverse intracellular signaling cascades such as PI(3)K/Akt/mTOR and Ras/Raf/MAPK, and therefore stimulate the transcription of genes associated with cell cycle activation, cell survival, and proliferation [START_REF] Hodkinson | Targeting growth factors in lung cancer[END_REF] . EGFR and other members of the ERbB family are overexpressed in gastric cancer, especially in the advanced stages of the disease [START_REF] Galizia | Epidermal growth factor receptor (EGFR) expression is associated with a worse prognosis in gastric cancer patients undergoing curative surgery[END_REF] . H. pylori is able to induce the transcription of EGF and EGFR, causing an activation of the EGFR signaling and contributing to neoplastic transformation of gastric epithelial cells [START_REF] Kim | The Effect of Helicobacter pylori on Epidermal Growth Factor Receptor-Induced Signal Transduction and the Preventive Effect of Celecoxib in Gastric Cancer Cells[END_REF][START_REF] Wallasch | Helicobacter pyloristimulated EGF receptor transactivation requires metalloprotease cleavage of HB-EGF[END_REF] . During the infection, this activation may counterbalance the bacterium-induced apoptosis [START_REF] Yan | Epidermal growth factor receptor activation protects gastric epithelial cells from Helicobacter pylori-induced apoptosis[END_REF] .

c. Notch signaling pathway

The Notch signaling is an evolutionary conserved pathway, first described in fruits flies presenting notched wings, and controls crucial cellular processes such as self-renewal of stem cells, proliferation, differentiation, and apoptosis [START_REF] Borggrefe | The Notch signaling pathway: transcriptional regulation at Notch target genes[END_REF][START_REF] Ntziachristos | From fly wings to targeted cancer therapies: a centennial for notch signaling[END_REF] . The four Notch receptors (Notch 1,2,3, and 4) are EGF like domains containing, single pass transmembrane proteins that interact with Delta-like and Jagged ligands, which are also transmembrane proteins with EGF-like repeats [START_REF] Ntziachristos | From fly wings to targeted cancer therapies: a centennial for notch signaling[END_REF] . There are five of these ligands in mammals, Dll1, Dll3, Dll4, Jagged1, and Jagged2. Upon activation by the ligands and as a result of a tightly controlled series of proteolytic events, the intracellular portion of the Notch receptor translocates to the nucleus to form an active transcriptional complex that controls the expression of several genes including Hes family genes, NF-κB, VEGF and c-myc [START_REF] Xiao | Notch and Wnt signaling pathway in cancer: Crucial role and potential therapeutic targets (Review)[END_REF] . Hence the Notch pathway regulates expression of genes associated with carcinogenesis and, unsurprisingly, deregulations of this pathway are found in various malignant diseases including T-cell leukemia, breast, prostate, colorectal, lung, and central nervous system cancers and associated to esophageal and breast cancer stem cells [START_REF] Yuan | Notch signaling: an emerging therapeutic target for cancer treatment[END_REF] .

Regarding gastric carcinogenesis, the presence of the four types of Notch receptors and their ligands, Jagged1 and Jagged 2, has been described in gastric tumors [START_REF] Hashimoto | Involvement of epigenetically silenced microRNA-181c in gastric carcinogenesis[END_REF][START_REF] Sun | Differential Notch1 and Notch2 expression and frequent activation of Notch signaling in gastric cancers[END_REF] . Moreover, the overexpression of Jagged1 in gastric tumors is associated with a worse prognosis [START_REF] Yeh | The activated Notch1 signal pathway is associated with gastric cancer progression through cyclooxygenase-2[END_REF] . Notch1 is of particular importance as it is not present in non-cancerous gastric mucosa, but is upregulated in both intestinal and diffuse type cancers [START_REF] Sun | Differential Notch1 and Notch2 expression and frequent activation of Notch signaling in gastric cancers[END_REF] . The increased expression of Notch1 and its target gene hes1 in gastric cancer cells has been associated to the upregulation of the DLL1 and DLL4 ligands [START_REF] Li | Influence of up-regulation of Notch ligand DLL4 on biological behaviors of human gastric cancer cells[END_REF][START_REF] Piazzi | Epigenetic regulation of Delta-Like1 controls Notch1 activation in gastric cancer[END_REF] . Experimental studies have demonstrated the Notch signaling ability to promote gastric cancer progression, as cells transfected with an activated form of Notch1 presented an enhanced colony formation capacity, migration and invasion abilities and increased growth in xenografted tumors, all mediated by the induction of COX-2 expression [START_REF] Yeh | The activated Notch1 signal pathway is associated with gastric cancer progression through cyclooxygenase-2[END_REF] .

Furthermore, similar results were found for Notch2 and the effect was downregulated by an inhibitor of COX-2 [START_REF] Tseng | Notch2-induced COX-2 expression enhancing gastric cancer progression[END_REF] . Notch and other signaling pathways of the stem cell signaling signature are thought to be activated during H. pylori chronic infection [START_REF] Katoh | Dysregulation of stem cell signaling network due to germline mutation, SNP, Helicobacter pylori infection, epigenetic change and genetic alteration in gastric cancer[END_REF] , but one study suggests that, on the contrary, Notch1 and Notch2 activity is repressed during infection in a GES-1 cells in vitro model [START_REF] Liu | Helicobacter pylori Infection of Gastric Epithelial Cells Affects NOTCH Pathway In Vitro[END_REF] .

d. Epithelial to mesenchymal transition (EMT)

The transformation of epithelial cells towards mesenchymal cells was first described by Elizabeth

Hay using a chick model in the 80's and early 90's [START_REF] Greenburg | Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells[END_REF][START_REF] Hay | Role of cell-matrix contacts in cell migration and epithelial-mesenchymal transformation[END_REF][START_REF] Hay | Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced[END_REF] . Later renamed as a transition to distinguish it from a malignant transformation of the cells, it is an evolutionary conserved, reversible process in which a polarized epithelial cell acquires a mesenchymal phenotype through phenotypical and biochemical changes that leads to increased capacities of migration, invasiveness, apoptosis resistance and extracellular matrix production and remodeling [START_REF] Kalluri | The basics of epithelial-mesenchymal transition[END_REF] . Several molecular events have been described at the initiation of EMT. Transcription factors such as Snail, Slug, zinc-finger E-box binding (ZEB1/2) and FOXC2 are activated at the beginning of the process, accompanied by the expression of specific microRNAs, for instance the miR-200 family, changes in the expression of cell surface proteins, cytoskeletal reorganization, and activation of Wnt/β-catenin, growth factor-mediated, and Notch signaling [START_REF] Yeh | The activated Notch1 signal pathway is associated with gastric cancer progression through cyclooxygenase-2[END_REF][START_REF] Kalluri | The basics of epithelial-mesenchymal transition[END_REF][START_REF] Mongroo | The role of the miR-200 family in epithelial-mesenchymal transition[END_REF] . A critical feature of EMT is the downregulation of E-cadherin [START_REF] Sun | Simultaneous loss of expression of syndecan-1 and E-cadherin in the embryonic palate during epithelial-mesenchymal transformation[END_REF] , a surface glycoprotein expressed in the epithelial cells as a key component of the adherens junctions in epithelial tissues [START_REF] Rodriguez | E-cadherin's dark side: possible role in tumor progression[END_REF] . Expression of E-cadherin can be repressed directly or indirectly by multiple transcription factor including ZEB1/2, Snail, Slug, NF-B, E47, and KLF8, and also the proteins SIX1 and FOXC2 [START_REF] Chua | NF-kappaB represses Ecadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2[END_REF][START_REF] Peinado | Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype?[END_REF][START_REF] Lin | Epigenetic regulation of EMT: the Snail story[END_REF] . These and other features of cells undergoing an EMT, are used as markers of the process and are further detailed in table 4. Various signaling pathways and the cross talks between them can also influence the expression of E-cadherin such as the TGFβ signaling, hypoxiainduced response, Wnt/β-catenin, Notch, and PI3K/Akt, therefore playing a role in EMT and becoming potential targets for therapy [START_REF] Theys | E-Cadherin loss associated with EMT promotes radioresistance in human tumor cells[END_REF][START_REF] Larue | Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3' kinase/AKT pathways[END_REF] . Even if the EMT is usually depicted as a binary switch that moves cells from a fully epithelial to a fully mesenchymal state, this is a misrepresentation the normal action of the EMT program, which most frequently moves cells from a fully epithelial state to a partially mesenchymal, retaining some epithelial markers, but nonetheless has conferred a subset of mesenchymal traits with profound effects on the cell biology [START_REF] Ye | Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression[END_REF] .

The EMT process is far from exclusive of malignant transformation, it is very important in other contexts, both physiological and pathological. Hence, for the sake of clarity, a classification of EMT in three categories was proposed in 2007.

Type I EMT refers to the processes occurring during implantation of the embryo, embryogenesis, and organ development [START_REF] Kalluri | The basics of epithelial-mesenchymal transition[END_REF] . In absence of EMT, the incipient organism would not go beyond the blastula, because the epithelial cell in the blastoderm require to go through an EMT to give origin to the primary mesenchyme needed to form the mesoderm during gastrulation [START_REF] Nakaya | Epithelial to mesenchymal transition during gastrulation: an embryological view[END_REF] . Also, this type of EMT is especially important during the formation of the neural crest from the neuro-ectoderm [START_REF] Kerosuo | What is bad in cancer is good in the embryo: importance of EMT in neural crest development[END_REF] , which serves as a precursor for the nervous system, melanocytes and the suprarenal medulla, and in the cardiac morphogenesis, where there are three waves of EMT [START_REF] Nakaya | EMT in developmental morphogenesis[END_REF] . After the first stages of embryogenesis, the trophectoderm cells undergo an EMT which facilitates the invasion in the endometrium that assures the anchorage in the placenta 100 . the kidney, and the lung 106 . Myofibroblasts, a proportion of which is derived from organ epithelial cells via an EMT process, are the cells that produce ECM components to create the scar necessary to complete the wound healing, and they usually undergo apoptosis once the process is over 102 . However during pathological activation there is a dysregulation of injury trigger-EMT and TGF-β, a key regulator of wound healing, is also implicated in fibrosis.

The last category, type III EMT, is always pathologic and occurs in association to cancer progression and metastasis of tumors of epithelial nature [START_REF] Kalluri | The basics of epithelial-mesenchymal transition[END_REF] . Most of the malignancies that affect humans are of epithelial origin, and in the early stages, tumors continue to express phenotypical traits of the epithelium, such as polarization, lack of motility, and an ability to form continuous sheets. They also express markers of the untransformed cells of origin, for example cytokeratins and E-cadherin, hallmarks of the epithelial state [START_REF] Ye | Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression[END_REF] . As the tumors continue to progress to more advanced stages, the neoplastic cells start showing an "undifferentiated" phenotype where they no longer resemble the cells of origin, morphologically or otherwise, and display mesenchymal features, including motility and invasiveness. Cells with mesenchymal properties are frequently located at the invasive front of primary tumors and express mesenchymal markers such as FSP1, α-SMA, desmin, and vimentin 107 .

Tumors with these characteristics are deemed to be more aggressive, more likely to disseminate and form distant metastases and carry a worst prognostic for the patient. The acquisition of these malignant traits is proposed to be due to the reactivation within carcinoma cells of previously latent EMT programs during tumor progression 108 . The cells undergo an EMT in the primary tumor, then disseminate throughout the body, where their newly acquire features include resistance to apoptosis and anoikis, which facilitate survival after intravasation 109 . Upon arrival to distant sites, the cells reverse their mesenchymal phenotype in a process of mesenchymal-to-epithelial transition to form epithelial metastases 108 . While the signaling pathways and the transcriptional programs involved in cancer progression-associated EMT remain fundamentally the same as in other types of EMT, the heterogeneity and constant evolution of the tumor microenvironment requires that the EMT program in metastasis must be able to adapt itself to these conditions to allow the tumor to metastasize 110 . The plasticity of the epithelial cells allows a rapid response to EMT-inducing signals, but also implies that there must be molecular and cellular mechanisms operating to ensure long-term residence of the cells in one state or another [START_REF] Ye | Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression[END_REF] . Recently, mechanical signals from the tumor microenvironment have been proposed as key regulator of the induction of EMT during metastasis. A non-invasive primary tumor generates a desmoplastic stromal response that increases the stiffness of the ECM, which in turn causes the release of TGF-β, a well-known inductor of EMT, hence increasing tumor invasiveness 111 . Once in the secondary site, the unaffected local microenvironment attenuates the EMT-inducing signaling cascade allowing the reepithelialization of tumors cells and the formation of epithelial metastasis. This response to the microenvironment is accompanied by biochemical changes in order to adapt the nutrient uptake and cellular metabolism to the needs of the cells undergoing the transformation 112 . The metabolic reprogramming that favors the pathways of aerobic glycolysis, a hallmark of cancer cells, is becoming increasingly recognized as important in maintaining the undifferentiated state during EMT.

Epigenetic mechanisms also play a role in epithelial plasticity during metastasis.

Hypermethylation of E-cadherin promoter has been described as responsible to the loss of its expression during cancer progression, and the heterogeneous pattern of methylation present in some cancer cell lines is compatible with the variability of the protein expression 113 . Alternatively, hypomethylation of the promoters of transcription factors that favor EMT has also been described, as well as changes occurring in the histone marks in cell undergoing EMT during cancer progression 114 . For example, it has been shown that the EMT-associated transcription factor Snail and Slug can recruit histone deacetylases to the Ecadherin promoter region and thus repress gene transcription 115,116 .

The role of EMT in metastasis have also been disputed as it is been difficult to distinguish in vivo between the tumor cells with mesenchymal properties derived from EMT and the mesenchymal cells of the tumor stroma 117 . However, a study showed used a flox-stop-flox LacZ transgenic mouse model combined with a Cre recombinase controled by an epithelial promoter to track epithelial cancer cell fate and demonstrated that mesenchymal cells arise from an epithelial origin and can be located close to the tumors in c-Myc driven tumors. This study, together with numerous demonstrations in vitro and histologic observations of epithelial marker loss in the invasive front of the tumors, provides sufficient evidence to justify the role of EMT in cancer progression.

Besides metastasis, there are other roles of EMT in the context of cancer, for instance immunosuppression. Snail-induced EMT has been shown to impair the activation of dendritic cells and induce regulatory T cells both in vitro and in vivo through thrombospondin-1 118 , thus causing an immunosuppression that could beneficial for the tumor development and dissemination. Furthermore, ZEB1 has been found to induce the expression of PD-L1, a ligand for the inhibitory receptor PD-1 in CD8+ found to be an important and independent factor associated with aggressiveness of primary gastric cancer 128 . Furthermore, the Wnt5a-induced EMT was found to maintain CSC properties in MKN7 cells 129 , and as did hypoxia-induced EMT in BGC823 and SGC7901 cell lines 130 .

In agreement with these findings, in a model of infection with Helicobacter pylori in AGS, MKN45, and MKN74 human gastric adenocarcinoma cell lines in vitro, our group has shown the emergence of a population of CD44 high cells with properties of CSC through an EMT process 131 . The cells acquired an elongated phenotype, the expression of mesenchymal markers (Snail1, ZEB1, and vimentin) and CD44 was increased, and they showed a higher ability to migrate, invade, and form tumorspheres in vitro and tumors when grafted in immunodeficient mice, compared to the CD44 low cells. Moreover, the induction of the EMT and the CD44 high cell population was demonstrated to be dependent on the bacterial oncoprotein CagA, as isogenic mutant strains depleted for cagA or cagE were unable to produce the same result.

Recently, the production of MMP-3, a matrix metalloprotease also related to the EMT process, was showed to be not only CagA-dependent, but also to correlated with the level of phosphorylation of CagA

Genomic alterations and driver mutations

A recent study by Wang et al, outlined the genetic changes associated with a cohort of 100 gastric cancer cases and control using whole genome sequencing 134 . The authors found 3.5 million somatic mutations, of which almost 18 000 were predicted to be protein altering, and MSI tumors were the group with more SNPs. Among the MSS tumors, diffuse-type tumors had the lowest amounts of SNPs and deletions, which in turn was significantly increased with advanced age of the individual. The somatic mutation rate approached 10 SNVs per Mb, which is higher than for most solid cancers apart from lung cancer in smokers and melanoma. The study identified sixteen driver genes, whose mutations impart a survival advantage to the clone that harbors the so-called specific driver mutations in the genes, i.e.

mutations though to underlie the malignant phenotype. These include well-known alterations in gastric cancer such as ARID1A, CDH1, CTNNB1, and TP53, as well as the less studied MUC6, RNF43, CTNNA2, GLI3, ELF3, ZIC4, TGFBR2, ACVR2A, DCLK1, THBS1, SMAD4, and RHOA.

Remarkably, mutations on TP53 have been previously acknowledged as one of the most common genetic aberrations in patients with gastric cancer 135 . It is a tumor suppressor that induces cell cycle arrest, apoptosis, and senescence in response to stress cues. The mutation rate in gastric cancer cohorts varies from 35% to 73% of patients, and normally these mutations impair the protein function, contributing to its role in cancer development 135 .

Somatic copy number aberrations

Intestinal type gastric cancers display a large number of chromosomal alterations, gains or losses, in comparison to diffuse type gastric cancer and normal gastric tissue 135 . Notably, the study by Wang et al, identified disruptions in the copy number of two genes important genes in the treatment of gastric cancer: the human epidermal growth factor receptor (ERBB2 or HER2) and MET 134 .

The HER2 is a proto-oncogene that encodes for a protein of the epidermal growth factor receptor family, which, as detailed previously, is part of the signaling pathways implicated in gastric carcinogenesis.

It is tyrosine kinase receptor that transmits extracellular signals to initiate activity of MAP kinases, PI3K, PLC, PKC and STAT 136 . HER2 positivity in gastric cancer is very variable depending on the study, it has been reported to be upregulated in 4-53% of gastric tumors 135 . Contrary to the breast cancer scenario, where overexpression/amplification of HER2 is associated with increased aggressiveness, risk of distant metastases and poor prognosis in general, in gastric cancer the association is inconsistent, despite multiple studies addressing the subject 136 . However, a recent randomized clinical trial, the ToGA study, carried in 24 countries used standard chemotherapy regimens (cisplatin with either fluorouracil or capecitabine) combined with Trastuzumab, a monoclonal antibody directed to the extracellular domain of HER2 and showed that the use of the Trastuzumab resulted in longer survival times in patients with HER2 positive gastric cancer, compared to the group treated with chemotherapy alone 137 . The results were improved when the HER2 positivity threshold was set to IHC score 2+ plus positive FISH or IHC score 3+. The exact timing of the expression of HER2 in gastric cancer is also debated, but a variation between histological subtypes have been established, being much more expressed in intestinal type tumors than in the diffuse type 135 . A higher expression has been reported in proximal tumors originating from the gastroesophageal junction than in the ones originated from mid-and distal stomach 135 .

cMET is a tyrosine kinase receptor for the hepatocyte growth factor (HGF) that is activated in various human cancer, including 10-53% of gastric carcinomas 135,138 . As cited before, it can be overactivated by amplification in the copy number of the gene, but also by mutations leading to constitutive activation, upregulated transcription, or plainly by the overexpression of HGF 138 . Aberrant signaling of the cMET pathway are correlated with advanced tumor stages and poor prognosis 138 .

Interestingly, the activation of cMET is partly responsible for the acquisition of a mesenchymal-like phenotype and motility in epithelial cells infected with H. pylori 139 . The bacterial oncoprotein CagA was showed to bind c-MET, and could modulate cellular functions by deregulating c-MET receptor signaling.

Epigenetic alterations

Epigenetic regulation of gene expression has been showed to be an important phenomenon in gastric carcinogenesis. The main epigenetic mechanisms involved in gastric cancer pathogenesis are DNAmethylation and microRNAs 135 .

DNA-methylation occurs in CpG islands, sites with a high CG content that tend to cluster within the 5'-UTR regions upstream of gene promoters 135 . When these zones near the promoters are methylated, transcription factors are prevented of binding the DNA and usually gene expression as a consequence. Tumor suppressor genes, oncogenes, and genes involved in tumor progression and metastasis have been found to be regulated by DNA-methylation in gastric cancer 140 . Hypermethylation of the promoter of tumor suppressor genes such as TP53, CDKN2A, MLH1, and CDH1, as well as proapopotic proteins, is often present in gastric cancer patient samples. Albeit less frequent, hypomethylation has also been described in gastric cancer, promoting derepression of target genes 140 . For example, the oncogene MYC was showed to be hypomethylated in gastric cancer with lymph node metastasis, and gastric cancer patient samples showed hypomethylation on the ALDH2 promoter.

MicroRNAs are small (20-22 nucleotides) noncoding RNAs that can regulate the expression of target genes at the post-transcriptional level, and a single miRNA can target several messenger RNAs, affecting multiple signaling pathways 140 . MicroRNAs could be regulated by DNA-methylation as well.

Downregulation via methylation of miRNA's promoters has been described to activate growth-promoting pathways such as CDK6-VEGF and c-MYC. miR-9 downregulation via hypermethylation in gastric cancer has been found to increase proliferation, cell migration and invasion capacity. Target genes of miR-9

include NFκB, RAB34, Cyclin D1, and Ets1, so when the repression by miR-9 is lost, gastric carcinogenesis may be enhanced [140][141][142] . But independently of the mechanism, microRNAs regulation in general is associated with the pathogenesis of gastric cancer, promoting tumor growth (Let-7a, miR-375), tumor invasion and metastasis (miR21, miR-148a), and interacting with the tumor environment (miR-874) 143 .

v. Diagnosis

One of the main challenges of gastric cancer diagnosis is that the disease is usually asymptomatic until it progresses to an advanced stage. The manifestations can include gas, reflux, biliary colic, and irritable bowel, and can be confused with dyspepsia and ulcer disease 144 . However, there are some features that could lead the clinician to suspicion, such as anorexia, dysphagia, weight loss, recurrent vomiting, and early satiety. Pain tends to be mild and located in the epigastric region, but progresses in intensity and duration as the disease advances.

Since the information obtained in the physical exam is limited in early stages, the diagnosis of gastric cancer and precancerous lesions normally relays in an upper GI endoscopy, and less frequently in a barium swallow tests. The latter has been widely replaced by endoscopy, especially given the great advancement in the field, but it remains very useful in the diagnosis of linitis plastica 144 . The use of endoscopy has a 90-96% accuracy in the diagnosis and detection of gastric cancer. One of the practical advantages of endoscopy is that any suspicious lesion can be biopsied to perform further pathologic analysis and confirm the diagnosis. The Sidney system recommends two biopsies from the corpus, two from the antrum, and one from the angular notch.

Macroscopic study of the stomach via white light endoscopy is a basic step for diagnosis of gastric cancer, and it allows the subclassification of early gastric cancer based on the characteristics of the lesions of type 0 in the Japanese system 145 . In recent years, technical innovations have made possible to go deeper in the observation of subtle morphological changes, difficult to detect using the white light endoscopy 145 .

The so-called image enhanced endoscopy (IEE) includes the narrow band imaging, the blue light imaging and the i-scan. The use of IEE has improved the accuracy of early gastric cancer and increased the opportunity for endoscopic resection. Another alternative is the use of chromoendoscopy, in which a colored solution (frequently indigo carmin solution combined with acetic acid) is used to improve the visualization of superficial topography of the mucosa.

However, the method remains to be invasive and costly, and poor suited for mass screening, especially in regions with high gastric cancer risk and high prevalence of H. pylori infection. Therefore, the search for non-or minimally-invasive diagnostic methods is very important. The given the high prevalence and mortality of gastric cancer in their country, the Japanese have lead the research on this field and a mass screening program has been implemented since the decade of 1960, initially with the use of X-ray examination 146 . The screening protocol has been carried out in other populations such as a high risk region in Costa Rica, where about 7000 individuals participated in an X-ray screening, followed by videoendoscopy, gastric biopsies, and treatment when pertinent 147 . During the study, the gastric cancer mortality was decreased by half, and the X-ray intervention exhibited 88% sensitivity, 80% specificity, and 3% predictive value for individuals with two screening visits, and case survival was 85% in the intervention group after 5 years, compared to 12-35% among the controls. The main problem for the permanent implementation and scaling up of the program was its high cost, which make it unsuitable for a non-rich country like Costa Rica.

Another approach is the search for serum biomarkers for gastric cancer. Pepsinogens (PG) are the proenzymes for pepsin, and are secreted in the gastric lumen: PG-I is secreted only by chief and mucous neck cells of the fundic glands, while PG-II is also produced by the cells in the pyloric glands 148 .

About of PG can be found in serum and may function as a marker of the morphologic and functional status of the gastric mucosa. Thus, the total levels of serum pepsinogen, and more important, the PG-I:PG-II ratio can be use as predictors of gastric pathologies 148 . In recent years, a new method has been proposed, called the ABC method, which consists in the evaluation of gastric cancer risk based on the detection of H. pylori

vi. Treatment

Currently, the curative treatment of gastric cancer relays in the resection of the tumor. The procedure can be performed endoscopically when it is diagnosed in early stages (the invasion goes no deeper that the submucosa, regardless of the lymph node involvement) in patients with no or low risk of lymph node metastasis, defined by endoscopic ultrasound, computed tomography and histological analysis 151 . The endoscopic resection could be performed either as mucosal resection (EMR) or submucosal dissection (ESD), depending on several criteria such as the size and the histology of the tumor, the ulceration and the depth of invasion, and the vascular involvement. These methods represent a minimally invasive, organ-preserving approach for the treatment of premalignancies or early stage cancers, usually with successful outcomes. When the patient has a cancer in advanced stage or an early gastric cancer that does not meet the criteria for endoscopic resection, a surgical total or subtotal gastrectomy, with D2 lymphadenectomy in the advanced cancers, must be performed 152 . These procedures may be done in an open surgery or laparoscopically, and the choice of the extent of the resection, the type of reconstruction of the gastrointestinal tract, and the method used, depend on the anatomo-pathologic features of the tumor and the patient's background and condition. No matter which procedure is used, a gastric resection is an aggressive intervention that leads to severe changes in the anatomy and physiology of the gastrointestinal tract and may have severe acute complications and longterm effects.

Studies show that the outcome of treatment can be improved is chemo-and/or radiotherapy accompany the surgical procedure. In Europe and the US, the predominant strategy consists on applying three to six cycles of a combined therapy (cisplatin and 5-fluorouracil and/or epirubicin) prior to the surgery, which provides a significant improvement in 5-year disease-free survival compared to surgery alone 153 . Radiotherapy before the surgery is not a usual practice in gastric cancer treatment 154 . In the postoperative setting, capecitabicine or 5-FU monotherapy combined with radiation is the standard care 153,154 . For the unresectable locally advanced or metastatic gastric cancers the treatment is systemic therapy with a combination of a platinum and a fluoropyrimidine, with optional addition of anthracyclines or docetaxel 155 . These treatments have severe secondary effects, and even if they are the best available option, the overall treatment failure is high, with a 5-year recurrence or death rate over 60% in Western countries 156 .

There are few options of targeted treatment for gastric cancers. The major advance in the last years has been the introduction of Trastuzumab in the treatment of Her2 positive cases after the ToGA study 137 . Other monoclonal antibodies and pathway specific inhibitors have been tested in clinical trials, some of them with encouraging results, such as the ramucirumab (inhibitor of VEGFR-2), but there is still not enough evidence to include them in the routinely treatment schemes. Moreover, a recent metaanalysis of 11 targeted therapy trials reporting data about survival compared to the use of chemotherapy alone, found low quality evidence of a small effect of targeted therapies on mortality and progression free survival, even when separate in type of agent or tumor type 157 . This study highlights the need for further and deeper research for the improvement of targeted therapy in gastric cancer.

A significant challenge is the accurate identification of markers of response to a given treatment, which starts with a better understanding of the molecular events behind gastric carcinogenesis. The comprehension of the signaling pathways involved in carcinogenesis also contributes to the development of new treatments or even the use of already known molecules in an attempt to improve the overall outcome of the disease. For instance, the retinoid metabolism has been described to have a role carcinogenesis of hematologic and solid tumors and the all-trans retinoic acid (ATRA) exhibits a prodifferentiation role in acute promyelocytic leukemia. Based in the fact that gastric CSCs present a high ALDH activity and that some of the ALDH isoforms are involved in retinoid metabolism, a study by Nguyen

vii. Models of study for human gastric cancer

In vivo models

Genetically engineered mouse models are convenient tools to study multiple diseases, and gastric cancer is not an exception. Current technologies have made it possible to manipulate the genetic background of small animals with minimal difficulty and to create strains adapted to the study of different aspects of the pathology. However, it must be kept in mind that these models show some limitations, such as modest gastric pathology, slow time course, and the absence of invasive or metastatic tumors, as well as inherent factors to the animal models such as high dependency on the genetic background, gender, diet and housing conditions 159 . Animal stress associated with manipulation, overcrowding, light cycles, temperature variations, and inadequate sanitation leading to infections with rodent pathogens have great influence in the outcome of experiments in murine models 159 . The compliance of ethical standards and considerations are of great importance in all the work performed involving animal facilities.

There are significant anatomical differences between the upper gastrointestinal tract of mice and humans 160 . The murine stomach mucosa is divided in two regions, the forestomach and the glandular stomach, while humans lack the first. The mouse glandular stomach is divided into three anatomical regions: the small cardia, the fundus, and the antrum, and the humans is divided in four: cardia, fundus, body, and antrum. At the histological level, the gastric wall organization into four layers (mucosa, submucosa, muscularis propria, and serosa) and the basic cellular components are similar in both species.

Humans have two types of gastric mucosa: fundic (body and fundus) and pyloric (cardia and pylorus), and transition mucosa between the two types. In mice, the gastric pits are straight tubes similar to the human fundic mucosa. In the cardia, the glands are lined by abundant mucous cells, in the fundic region, they contain mucous neck cells, parietal cells, and chief cells, and in the pylorus the glands are composed only of mucous cells. A detailed comparison of both species is shown in table 5.

Chemical carcinogenesis

The use of carcinogenic compounds, especially nitrogen derivatives, has been used to induce carcinogenesis in mice. The induction of gastric adenocarcinoma is achieved after 5 weeks of treatment with N-methyl-N-nitrosurea (MNU) in drinking water, and the model is extensively used in the study of signaling pathways and transcription factors 159 . Its two main limitations are that the mechanism of induction of carcinogenesis by MNU is not known and that it lacks the inflammatory component present in an important proportion of gastric cancers.

b. Genetically engineered mice

The initial genetically modified mouse models of gastric cancer were based on oncogenes associated with transformation, but not specific of the stomach, such as the SV40 T antigen, which then was improved by using a stomach-specific promoter. However, this and other early models did not developed dysplasia, the malignancies did not progress following the precancerous lesions sequence, nor were they associated to chronic inflammation. Currently transgenic or knock-out mice, who overexpress or are deficient in growth factor or cytokines, or with mutations in tumor suppressor genes or oncogenes are the most used models for the study of gastric carcinogenesis. For example, the insulin-gastrin (INS-GAS) mice, where the human gastrin gene is under the control of the insulin promoter. Consequently, the serum level of gastrin is increased and the mice develop atrophy and hypochlorhydria, which progress to metaplasia and dysplasia and finally invasive gastric cancer in the corpus at 20 months of age.

Xenotransplantation in immunodeficient mice

However useful the genetically modified mice models in understanding the basic oncogenic processes, they are still significant physiological differences between human and mice that affect the outcome of the research. Albeit not perfect, the xenotransplantation of human cancer cells in immunodeficient mice allows to overcome some of these drawbacks while keeping the practical convenience of the murine models. Strains of athymic mice (nu/nu) and other immunodeficient mouse strains (severe combined immunodeficiency, SCID) allow the grafting of explants of human tumors or cell lines to grow them as xenotransplants for the study of tumor cancer biology, notably the testing of anticancer drugs 161 (Figure 3). The model allows to test a wide variety of tumor types, the possibility of genetic or therapeutic manipulation before the implantation, and it is the method of choice for the identification of CSCs, that will be discussed later in this work. All the same, the model have disadvantages that must be taking in consideration, especially because the surroundings of the tumor remain to be the host's, blood and lymphatic supply and neovascularization as well as the tumor stroma are murine 161 .

Technically, the orthotopic engraftment is difficult, so the subcutaneous transplantation is widely used, introducing an artificial compartmentalization which renders the metastatic growth very infrequent and could induce architectural and histological changes in the tumor 161 .

At present, one of the most used strains of mice in xenografts for cancer studies is the NSG strain.

Also known as NOD-scid gamma or NOD-scid IL2Rg null , in a NOD genetic background these mice carry two mutations: the scid or Rag1 null or Rag2 null , to induce immunodeficiency of the adaptive immune system, and the IL2rγ null , to prevent cytokine signaling and cause a deficiency in NK cells 162 . Thus, they are highly immunodeficient and good recipients of human cancer cells.

For gastric cancer, subcutaneous patient derived xenografts (PDXs) have been developed from surgically-obtained tissues from gastric cancer patients and from gastroscopic biopsies, with moderate success in the establishment of tumors (24.2% and 34.1%, respectively) 163,164 . In these studies, the histological type and tumor cell percentages, as well as the ex vivo and overall procedure time were shown to be determinant in the achievement of the PDXs. Remarkably, the biological characteristics of the PDXs were stable over subsequent passages and highly consistent with the primary tumors, providing a very useful tool for the preclinical testing of anticancer drugs and tailoring of targeted therapy.

Orthotopic transplantation in the stomach has been performed in the stomach with cell line suspensions and intact tumor tissue, with a greater extent of metastasis in the latter, perhaps due to the intact histology and interaction of cancer cells with the co-implanted stroma 165 . The metastasis reflected the sites in the human pathology, for instance the liver and the peritoneum.

In vitro

The wider use of in vivo models is still restricted by technical issues. The tumors can only be maintained in mice, which is costlier and requires a higher specialization than cultured cell lines 166 . There are also long latency periods from engraftment and variable success rates. Therefore, the use of cell lines cultured in vitro is relevant, especially in the initial steps of most current research and in the high scale screening of anticancer drug candidates. Commercially available gastric cancer cell lines commonly used are described in table 7.

b.

Stem cells and Cancer stem cells i. Normal stem cells

Stem cells are defined as cells that have the ability to perpetuate themselves through selfrenewal and to generate mature cells of a particular tissue trough differentiation 167 . Self-renewal allows the maintenance of the stem cell pool, as the new cells retain the properties of the cell of origin. The differentiation process, on the contrary, generates heterogeneous, more specialized cells with characteristics specific to a particular organ or tissue.

During embryogenesis, the zygote produced by the fusion of an egg and a sperm is considered to be totipotent, having the capacity of give rise to the embryonic and extra-embryonic (trophoectodermal) tissues 168 . The embryonic tissue, composed by the inner cell mass of the blastocyst is formed by the pluripotent embryonic stem cells (ESCs), which can not only give origin to other ESCs through asymmetrical division but also to all the cells that form the endoderm, mesoderm, and ectoderm 168 .

As development goes further, the pluripotent stem cells become more committed to a particular lineage and their potential to generate new cells is restricted to the specific type of tissue where they reside 168 . These cells are known as multipotent stem cells, present in small quantities in adult tissues.

These adult or somatic multipotent stem cells have been identified in practically every tissue in the body, such as the epidermis, neural tissue, gastrointestinal tract, liver, and mesenchyme. But perhaps the most studied, and the prototypical model for adult stem cells, are the hematopoietic stem cells (HSCs).

Mechanisms of cell renewal

Self-renewal is the division with maintenance of multipotency and undifferentiated state, and it is one of the most important properties of stem cells because it allows to maintain the pool of stem cells through life 167 . Yet, it is not an exclusive property of stem cells. Other types of progenitors such as lymphocytes and glial progenitors can also self-renew, but even if they share some mechanisms, the selfrenewal of stem cells can be distinguished from those of the restricted progenitors because in the latter, the potential is limited 169 .

In the case of ESC, they possess indefinite self-renewal and pluripotency, conferred by a unique transcriptional and cell cycle regulation 170 . Studies on mouse, rat, and human ESCs allowed the identification of a core subset of transcription factors of "stemness", which include Oct4, Sox2, and Nanog.

These and several other factors act in cooperation to maintain pluripotency and serve as hubs for different signaling pathways. The main signaling pathways involved in self-renewal of ESCs include LIF/STAT3, Wnt/β-catenin, FGF/ERK, TGF/SMAD and PKC signaling.

A key feature of adult stem cells is to survive and retain their proliferative potential through the lifespan of the animal, but this fact does not necessarily mean an infinite capacity to divide or that the selfrenewal is constant 171 . The frequency and timing of divisions are regulated to ensure the lifelong maintenance of the stem cell pool in a tissue, as reductions in their proliferative potential could cause tissue atrophy and premature aging, and in turn, mutations that stimulate stem cell division without a balancing differentiation could cause abnormal tissue development and malignancies 171 . The frequency of divisions depends on the nature of the tissue and the need for regeneration.

HSCs are required for the development, maintenance and regeneration of the hematopoietic system. In mice, approximately a billion of blood circulating cells are produced every day, and it has been

shown that this is a finely regulated process mainly sustained by progenitors (HPCs) committed to the myeloid and lymphoid lineages rather than HSC 171,172 . These are not used in normal hematopoiesis and are preserved for hematopoiesis following special circumstances such as blood loss or infection. Aside from this emergence hematopoiesis, most HSC are in G0 phase and divide once per month, while the homeostasis is maintained by HPCs. HPCs also have the capacity of self-renewal, and interestingly, the granulocytic lineage-committed HPCs are short-term HPCs, differing from their lymphoid counterparts that are long-term HPCs, more similar to HSCs 172 . The regulation of HSCs occurs by two mechanisms: control of G0 phase and fate determination. These processes take place in the bone marrow and are regulated by the stem cell niche, composed by different types of cells that produce extracellular signals to stimulate stem cells 172 . CXCL12 is a cytokine produced by perivascular stromal cells, among others, and it has been described as the main responsible for maintaining the G0 state in HSCs upon recognition by the CXCR4. In contrast, interferons have been identified as activators of quiescent HSCs.

The case is different for intestinal stem cells (ISCs) as the intestinal mucosa is characterized by a very rapid turnover with a high rate of loss. This arise a constant necessity for new cells, which are generated by a population of multipotent ISCs that reside at the base of the crypts 173 . ISCs give rise to transit amplifying cells that expand through multiple cycles of cell division and progressively differentiate as they migrate upwards to the tip of the villi 173 . The Wnt/β-catenin and the Notch signaling pathways have been described as crucial for the preservation of the ISC pool and a recent study in a murine model identified Bmi-1 as a downstream target of both pathways, whose deficiency resulted in reduced proliferation and self-renewal of ISCs 174 .

Gastric stem cells

The gastric mucosa, like its intestinal counterpart, is dynamic tissue with a rapid rate of selfrenewal. As in other adult tissues, this physiological process is sustained by a subpopulation of multipotent stem cells. The epithelium that constitute the gastric mucosa is glandular, and each gland is considered to be monoclonal and subdivided in to four regions: foveolus, isthmus, neck and bottom (figure 4). The characteristics and type of cells present in the glands varies depending on the zone of the stomach and the function of the gland. In the corpus region, the glands are long and they contain zymogen-producing chief cells in the bottom, acid-producing parietal cells and hormone-secreting enteroendocrine cells scattered throughout the gland, proliferative undifferentiated cells in the isthmus, and mucous-secreting cells in the neck and the pit 175 . In the antrum, the glands are shorter and composed by mucous cells and enteroendocrine hormone-secreting cells that regulate the acid and enzyme production by the glands of the corpus. Both types of glands contain a discrete quantity of adult stem cells, but their localization varies, in the corpus, they can be found in the isthmus while in the antropyloric region glands, they are located at the bottom. Putative progenitors/stem cells in the antrum and corpus of the murine stomach were first identified in the isthmus and neck regions, respectively, as a group of small undifferentiated and granulefree cells using 3H-thymidine radiolabeling by Leblond et al 176,177 . These cells give origin to the other granule cells of the gland in a bidirectional pattern of renewal, moving toward the pit and the bottom of the gastric gland as they differentiate. Further evidence was provided when using a chemical mutagenesisinduced lineage tracing strategy, Bjerkens and Cheng showed that the adult gastric epithelium harbors functional multipotent stem cells 178 . Mice expressing β-galactosidase (LacZ) under control of the Rosa26 promoter were treated with N-ethyl-N-nitrosourea (mutagen), with inactivation of the LacZ allele in some cells as a result. If a progenitor cells has the mutation, all the daughter cells will inherit the nonfunctional allele and will not be labeled, allowing the identification of clones of related cells. With this, they demonstrated the existence of a long-term, self-renewing progenitor cell population in the adult gastric epithelium. With the development of more sophisticated lineage tracing techniques, specific markers for gastric stem cells have been identified, and currently, two co-existing subpopulations of gastric stem cells can be distinguished: a population of dividing gastric stem cells recruited under "homeostatic conditions", which express CD44 or Lgr5 markers; and a rare population of quiescent cells recruited mainly upon tissue damage, expressing Villin, Troy and Mist as markers. Details on the markers of gastric stem cells are presented in table 8. ii.

Cancer stem cells

Concept of cancer stem cell and the CSC model for tumor heterogeneity

The individual cells that comprise a tumor display significant functional and morphologic heterogeneity, like those in normal tissue. The first model proposed to explain tumor heterogeneity is known as the clonal evolution or stochastic model, in which only a small number of cells within a tumor can initiate and sustain tumor growth, but all the tumor cells are biologically homogeneous and could potentially be responsible for these processes, given that they undergo the stochastic events that govern tumor initiating capacity 179 (Figure 5). These events could be intrinsic (adequate levels of critical transcription factors for example) or extrinsic to the cells (microenvironment cues). The second and more accepted model is called the hierarchical or CSC model, which proposes that, in contrast to the stochastic model, only a specific subset of tumor cells possesses the capacity to regenerate the tumor 179 .

Accordingly, is then possible to identify and isolate the cells within a tumor that have the capacity to initiate tumors and sustain their growth. These cells are the so-called CSCs, defined by similar features to those of the normal SCs, their capacity of self-renewal and asymmetric division, the potential to develop into any type of cell in the tumor, and proliferative capacity to drive the continued expansion of the tumor population. Research in the cancer field has come to support the hierarchical model in the recent years, as not only the identification and isolation of CSCs has proven attainable in hematologic and solid tumors (thanks to the identification of specific surface markers), but also because some of the key challenges in oncology have been found to be directly associated with CSCs. This is the case of treatment failure and relapse of the disease, as other property of CSCs is the resistance to both chemo-and radiotherapy.

However, some studies using high throughput, single cell, and single nucleus sequencing developed in the last years have produced results that challenge the widely accepted hierarchical model and that fit better to the clonal evolution model. The identification of different genetic subclones suggests that the clonal evolution model could also have a key role in tumor initiation, heterogeneity, progression, and metastasis. A comprehensive model that integrates both hierarchical and clonal evolution explanations of heterogeneity has been proposed by Kreso and Dick, in which favorable mutations accumulate in the rare CSC population within an early tumor as it develops, generating subclones in the CSCs, some of which achieve higher capacity for self-renewal 180 . Mutations could also occur and accumulate in cells without tumor-initiating capacities, eventually endowing them with stemness. The stemness state could also be achieved by dedifferentiation of these cells as a result of external stimuli, for example, EMT-like processes. The proposition is then of a model that considers the tumor-initiating cells not as static entities, but as ones capable of evolving, and this evolution process could be driven genetic determinants as well as epigenetics, gene expression stochasticity, the CSC niche, and the tumor microenvironment 180,181 (Figure 6). Very recently, the unified model was proposed for gastric cancer by Song et al 181 (Figure 7).

The existence of a functional hierarchy in GCSCs, the considerable variation in their function and

Evidence

The first evidence supporting the CSC model was published by the group of John Dick in 1997 182 .

Using a NOD/SCID mouse model and surface markers-based cell sorting, the authors established a hierarchy of stem cells in acute myeloid leukemia that resembled the organization observed in normal HSCs. A rare subpopulation of CD34 + /CD38 -cells with the ability to recreate the original disease in the immunodeficient mice after serial transplantation was identified. This experiment was a milestone in the CSC research, and was soon followed by studies in other types of cancer. In solid tumors, the first identification of CSCs was made by Al-Hajj in breast cancer 183 . Samples of breast tumors obtained surgically from patients were grafted in the mammary fat pads of NOD/SCID mice, and the cells of both xenograft-derived and primary tumors were analyzed for cells surface markers that allowed to distinguish a small subpopulation of CD44 + CD24 -/low cells capable of forming tumors when injected in immunodeficient mice in quantities as low as 100 cells, while the cells with other phenotypes were not. The existence of

CSCs have been since then demonstrated in tumors in other organs such as brain, colon, pancreas, lung, and liver, amongst others 179 .

In gastric cancer, the existence of CSC was demonstrated by Takaishi et al in 2009. In this study, cells of six gastric cancer cell lines were sorted regarding the expression of six surface markers 184 . CD44+ cells showed a high spheroid-forming capacity in vitro and tumor formation when grafted in skin and stomach of immunodeficient mice. These cells also exhibited a high resistance to chemotherapy in vitro.

Furthermore, the knock-down of CD44, by the means of an shRNA, diminished the tumorigenic properties of the cells. The results were supported also by the presence of CD44 in immunostaining of dysplastic lesions following H. felis infection in INS/GAS mice and human tumor tissue. A study by Fukuda et al, identified a side population (SP) in gastric cancer cell lines using the Hoescht 33342 staining, which had increased in vivo tumorigenic properties and chemoresistance when compared to the cells of the main population (MP) 185 . The study by Takaishi et al, however, failed to do so and found no significant difference between the cells in the SP and those of the MP, regarding tumor-forming capacity.

Origin of cancer stem cells

One of the main questions in the field of CSC is where do they come from. The answer to this question may have different and non-mutually exclusive answers, depending on the type of cancer of even the subpopulation of CSCs in question.

In hematologic malignancies, for example, it is considered that the transformation of normal hematopoietic stem cells gives rise to the leukemic stem cells. In some diseases, both normal and cancer stem cells share the same surface marker phenotype, like the case of acute myelocytic leukemia with the CD34 + CD38 -phenotype 182 , and in B cell acute lymphoblastic leukemia where CSCs express B220 and CD19, which are markers of B-cells 186 . But there is also evidence that more committed progenitors can also be at the origin of leukemic CSCs by the acquisition of genetic alterations. The transduction of the oncogenes MOZ-TIF2, the retroviral overexpression of the HOXB6 gene, and the translocation of the MLL gene in myeloid progenitors have been demonstrated to confer leukemic SC properties such as self-renewal and maintenance of the disease in xenograft models [187][188][189] .

In solid tumors, the role of stem cells and progenitors has been studied in intestinal cancer, among others. In a study by Barker et al, Lgr5 + intestinal stem cells were traced as intestinal cancer cell using mice a lineage tracing method, and the interbreeding of this strain with APC flox/flox mice, allowed to

show that loss of APC in these stem cells lead to their malignant transformation and was responsible for the formation of adenomas in the mouse gut, while this was not the case of short-lived transit-amplifying cells 190 . A stem cell/progenitor hierarchy was maintained in early neoplastic lesions.

a. Gastric cancer stem cells

In gastric cancer, CSC are thought to originate from multiple cell types, including gastric stem cells, epithelial cells from the glands and bone marrow-derived cells (BMDCs).

i. Bone marrow derived cells (BMDCs)

The role of BMDCs has been approached using a H. felis/C57BL/6 model of gastric cancer, and the study found that upon chronic H. felis infection, the damaged glands were repopulated by BMDCs, and their progeny later developed metaplasia, dysplasia and intraepithelial neoplasia 191 . Furthermore, in the context of H. pylori infection in C56BL/6, using GFP+ BMDCs from male mice grafted in female irradiated females and then infected with mouse adapted strains of the bacterium, Varon et al showed that 25% of the resulting dysplastic lesions included BMDCs and the bone marrow as a source of the cells was confirmed by detection of the Y chromosome 192 . Other roles for BMDCs in the gastric mucosa have been studies, such as contributing to the formation of the stem cell niche, repairing the damage caused by chronic inflammation, and promoting the proliferation and metastasis of gastric cancer cells 181 .

ii.

Gastric stem cells (GSC)

The origin of GCSCs from GSCs have been examined by Barker et al using a similar strategy to the one in the previous study in the intestine, and the authors showed that loss of APC accelerated the expansion of transformed Lgr5 + GSCs and the formation of adenomas in the pyloric region 193 . Moreover, a recent study showed that the deletion of Smad4 and PTEN in Lgr5+ GSCs resulted in intestinal type gastric cancer 194 . In other GSCs, such as Villin + cell, the depletion of the tumor suppressor klf4 induced malignant transformation and tumor formation in the antrum 195 .

iii.

Dedifferentiated epithelial cells

The

Markers of CSCs

a. General

Cell surface markers can be used to identify CSCs. Using technologies such as FACS the cells in a tumor can be separated in subpopulations based on the expression of cell surface proteins, and their stemness characteristics can be verified experimentally to determine which markers distinguish CSCs from the bulk of cells. The isolation of CSCs with the appropriate markers is of vital importance to the study of their biological properties, and it has been the subject of numerous studies aiming to identify those markers have been carried out. The cell surface markers for hematologic CSCs and solid tumors are detailed in table 9. i.

Structure and function

CD44 is a transmembrane glycoprotein that functions as a receptor for hyaluronic acid, a major component of the extracellular matrix, and also as a co-receptor for many growth factors and cytokines 199 . ii.

Role in cancer

CD44 is expressed in many cancer cell types, and besides from being used as a surface marker, studies show that it plays roles in tumor initiation and growth, cell migration, and metastatic dissemination in gastric tumors and gliomas, amongst others 199 . These are functional characteristics that associate CD44

with stemness in cancer cells, and in fact, it is one of the molecules most commonly identified as a CSC marker (alone or in combination with other cell surface proteins). The recognition of ECM components by CD44 can activate signaling pathways associated with stemness, and its role as a co-receptor have been described for many tumor-associated growth factors such as EGF, FGF, HGF, VEGF, and TGF-β 199 . In the light of the currently available evidence, CD44 seems to be a crucial mediator of the interaction of CSCs with their surroundings, and therefore, aside from the role of CSCs marker, it has become a target for cancer therapy 201 .

Methods of identification and isolation of CSCs

a.

Colony and sphere formation assays

The evaluation of the clonogenic potential of CSC evidences their capacity of self-renewal and multidifferentiation, as only CSC are able to originate colonies or spheres from a single cell. The colony formation capacity is tested by plating the cells to grow in the presence of "feeder" cells or a conditioned medium, either of which has the role of providing growth factors. The usual method consists in growing the cells in a layer of soft agar, which prevents cells to adhere to the culture plate 203 . Non-transformed cells have dependence on contact with the ECM, and if they don't have it, they are not able to survive.

This allows to infer that each colony is originated in a CSC, and therefore, permits a quantitative evaluation of cells with self-renewal capacities.

The same rationale is behind the sphere formation assay, in which the cells are seeded in low density in a defined serum-free medium, usually supplemented with growth factors (EGF, FGF, and insulin) and nutrients and in non-adherent conditions. Only cells that have lost the adhesion dependence and have self-renewal properties are able to grow in these conditions.

These techniques are widely used in the CSC research field, it is accepted as a reference in vitro method for the characterization of CSCs, but there are some limitations that must be taken in account when interpreting the results. Some cell lines have a ubiquitous capacity of growing in non-adherent serum-free conditions, and high densities of cells may promote the formation of aggregates that may be confuse with real spheres, and also an inability to detect quiescent CSCs 204 . However, a careful method and sequential dissociations and passages may be enough to overcome some of these criticisms.

b. Side-population assay

This is a flow cytometry-based method that relays on the capacity of some cells to exclude dyes such as Hoescht 33342 and rhodamine, thanks to the abundance of ABC-transporter proteins, also associated with drug resistance, such as ABCG2. The exclusion of the dyes places these cells in a side group, negative for the staining in the cytometry plot, and separated from the bulk of tumor cells that are not capable of excluding the dye and are positive. This "side population" (SP) is enriched CSCs, as was demonstrated in a study by Hirschmann-Jax et al, which isolated and evaluated the properties of side populations in primary tumor cells of neuroblastoma patients and cell lines from solid tumors 205 . The authors found that the cells from the SP were capable of self-renewal and asymmetric division.

Furthermore, the cells in the SP expressed high levels of transporter proteins such as ABCG2 and ABCA3, reflected also in the capacity to expel cytotoxic drugs, and a consequently increased drug resistance, another feature of CSCs. e.

In vivo xenotransplantation

The capacity of forming new tumors in vivo is a hallmark of CSCs. The evaluation of tumorinitiation capacity of cell when grafted in highly immunodeficient mice (NSG strain for example, detailed above) is the gold standard for identification of CSCs. This approach also allows the estimation of the proportion of CSCs present in a tumor, using the limiting-dilution method, in which the minimal number of cells required to originate a tumor when injected in mice is determined. Given that only CSCs have the ability to initiate a tumor, the number of CSC in the total cell number can be calculated from these data.

The method has been used to study CSCs of multiple solid cancers, gastric included. In a recent study by

Nguyen et al, the frequency of tumor-initiating cells was calculated between 1/29 and 1/1020 in CD44 + cells for three cases of gastric adenocarcinoma (two intestinal-type and one diffuse-type) using this method 206 .

c.

Helicobacter pylori

i. History

Due to the acidic nature of the gastric environment, it was commonly assumed that no bacterium could survive in this anatomic site. Nevertheless, the presence of spiral bacteria in the gastric mucosa was described as early as the 19 th century by the Italian pathologist Giulio Bizzozero in the stomach of dogs 207 .

This was later confirmed by Kasai and Kobayashi, who also described the colonization in other Other transmission ways such as iatrogenic, water-and food-borne have also been proposed, but they are less likely due to the characteristics of the bacterium 227,[232][233][234][235][236] . Even so, all those routes could be co-existing, and the predominant ones will depend on the particular conditions. An international study showed that in rural settings there could be a more important waterborne transmission, as opposed to urban communities where higher sanitary conditions allow a predominance in the intrafamiliar transmission 237 .

iii. Biology

According to the Bergey's Manual of Systematic Bacteriology, the genus Helicobacter belongs to the Helicobacteriaceae family, in the Campylobacterales order of the ε-Proteobacteria class 238 . These are microaerophilic gram negative helical or curved bacilli, which present characteristics such as motility by sheathed flagella, presence of a glycocalix or capsule-like structure when cultured in liquid media, and a 24-48% G+C content on chromosomal DNA. The genus comprises 36 validated species 239 , including many pathogens to humans and other animals, of which H. pylori is the type species.

H. pylori is an S-shaped bacillus of approximately 0.5 to 5 m with 5-8 polar flagella 240 . It produces circular small translucent colonies when cultured in blood agar in a microaerophilic atmosphere (3-7%) 238,241 . In old cultures, there is a predominance of coccoid forms of diminished viability 240 . H. pylori has a respiratory metabolism with glucose as the only source of carbohydrates, and grows in narrow ranges of temperature (33-40°C) and pH (6.9-8.0) 238,241 . Its main biochemical features are a strong activity of the urease enzyme and positive reactions to the oxidase and catalase tests. ions 244 , and it has been proven to be essential to the survival of H. pylori in the gastric mucosa 245,246 . Other potential roles for the urease have also been described such as inhibition of the antibacterial activity of phagocytes such as macrophages and neutrophils 247 and the induction of apoptosis of gastric epithelial cells 248 . The presence of the urease in all strains of H. pylori also makes its detection the gold standard for non-invasive diagnostic of the infection 249 and even a potential target for treatment 250,251 .

The effect of acid in the transcription regulation during chronic infection was studied by comparing the transcriptomes in vivo e in vitro, and three groups of genes were particularly increased. As expected, the transcription of the gene cluster ure (ureA, ureB and ureI) as well as rocF. The other two groups were genes related to motility and chemotaxis and those associated with virulence, notably cagA Some other genes whose expression is controlled by acid have also been described, for example wbcJ, which codes for a protein involved in LPS synthesis 255 . Mutant strains defective for wbcJ present structural alterations of the LPS and a decreased viability in acidic conditions. Wen et al. in 2003 revealed more than 300 genes which have their expression regulated by stomach acid, with functions not only in the pH homeostasis but also as diverse as transcription regulation, motility, cellular structure and pathogenicity 242 .

In H. pylori, the detection of pH is by the means of a two-component system formed by the histidine kinase type receptor ArsS and its response regulator ArsR, which stimulates the transcription of multiple genes 256 . The ArsS-ArsR system controls approximately 109 genes, including the ure cluster and the amydases AmiE and AmiF 257 . Other elements regulated by the system are related to acetone metabolism, resistance to oxidative stress and "quorum-sensing" 258 .

b. Bacterial motility and chemotaxis

Asides from the acidic environment, the motility-and chemotaxis-related genes were the second most important categories identified by a study of essential genes for gastric colonization in the Mongolian gerbil and in murine models 259,260 .

A study with motile and non-motile variants of H. pylori inoculated in gnotobiotic piglets was one of the firsts to link motility to the colonization capacity, as non-motile forms where less able to successfully colonize the animals 261 , and since then, multiple studies have confirmed the bacteria capacity to swim through the mucus layer towards the epithelial surface as a major colonization factor 262,263 . The main factor of bacterial motility are the flagella 264 , in the case of H. pylori, they are lophotrichous and individually sheathed, the number varies from two to six, they have club-shaped thickening at the ends, and they are mainly constituted by two types of flagellines, FlaA and FlaB [265][266][267] . The sheath function is most likely the protection of the flagellum from the gastric acidity and mechanical damage 268 and also adhesion, as it carries the adhesin HpaA 269 . Another determinant for the motility of H. pylori is the helical shape of the cells. A structural study showed that H. pylori's periplasm is cage-like with 18-fold symmetry that provides the necessary torque needed to swim through a high viscosity environment as the mucus layer in a corkscrew-like movement 270,271 , and the genes involved in the helical morphology of H. pylori have identified, comprising the csd family which encodes for peptidoglycan peptidases [272][273][274] , and hp0506, whose product, HpdA is a M23B family metalloprotease 275 .

In order to swim away from the acid and movement of the stomach lumen to the more favorable conditions in the mucus layer, H. pylori is guided away from low pH by a complex chemotactic machinery 276 . Chemotaxis is mainly directed by a two-component system, composed by the histidine kinase CheA (HP0392), the transductor CheY (HP1067) and other accessory proteins (CheW, CheZ and multiple proteins with CheY-like domains) [277][278][279] . In addition, four chemoreceptors have been described, TlpA, TlpB, TlpC and TlpD [280][281][282][283] . Together, these proteins account for the recognition of a large quantity of signal such as arginine, bicarbonate, urea, metals and acid [284][285][286] . The gradient of this latter is a very important factor to the spatial localization of H. pylori in the mucus layer 287 . The other signals likely diffuse from the blood through the epithelial cells thus helping the bacterium to get near them 276 . TlpB is also involved in the recognition of the quorum sensing molecule AI-2 as a repellent 288 .

Even if they have been widely studied, the motility and chemotaxis remain to be elucidated in further detail. Some of the more recent findings are a chemotaxis-related preferential localization of H.

pylori in injury sites 263 , and new putative chemotaxis arrays at the flagellar pole 271 .

c. Adhesion: Outer membrane proteins

In order to colonize epithelial cells, especially those subject to constant clearance mechanisms, bacterial pathogens have developed surface proteins which allow them to recognize and attach to molecules on these cells 289 . H. pylori is no exception, and the presence of adherent bacteria (even if the percentage is small) is very important to the establishment and persistence of the infection 290 . There are at least 63 outer membrane proteins (OMP) or putative OMPs described for H. pylori 291 . Amongst them, the "major OMP" or Hop family is the largest and includes proteins such as BabA, SabA, HopZ, OipA, and AlpA/B 292,293 . For BabA and SabA, the ligands in the host's cells are fucosylated and sialylated glycans of the Lewis and ABO antigen groups, while for the others, the ligands are still unknown 294,295 . These and other adhesines like HomB or CagL (binds to integrin α1β5) are also involved in bacterial pathogenicity and the outcome of infection 296-298 .

CagA and cagPAI

The Cytotoxin associated gene A (CagA) is the main and most studied virulence factor of H. pylori.

It is a 128 KDa protein whose expression is strongly associated with a higher virulence of the bacterium and therefore with the infection-related pathologies such as peptic ulcer, atrophic gastritis and gastric adenocarcinoma [299][300][301] . Once injected in the host cell by a type IV secretion system, CagA is phosphorylated by kinases of the Src and Abl families [302][303][304][305] . This phosphorylated form specifically binds to and deregulates the tyrosine phosphatase SHP-2, and initiates changes in the host cell signaling 306,307 . SHP-2 upregulates growth factor signals and is required for the activation of the Ras-Erk MAP kinases cascade 308,309 . SHP-2 inhibits the focal adhesion kinases (FAK), which regulates the turnover of the focal adhesion spots 310 .

Phosphorylated CagA also bind to Csk and mediated the inhibition of Src family kinases, in a negative regulation loop that seems to limit the bacterial damage to the epithelial cells and favors the persistence of infection 311 . The Src family kinases also phosphorylate other proteins such as vinculin, ezrin and cortactin, and their inhibition leads to alterations in the cytoskeleton. Notably, the dephosphorylation of cortactin induced by CagA, as well as the inhibition of the PAR1-kinase activity, have been described during the infection [312][313][314] . All these phenomena contribute to the acquisition of the so-called "hummingbird" phenotype upon infection, which is a change in the cellular morphology to an elongated, mesenchymallike shape associated with actin cytoskeleton rearrangements and increased motility.

CagA phosphorylation occurs in the carboxy-terminal end, in tyrosine phosphorylation motifs of the sequence Glu-Pro-Ile-Tyr-Ala (EPIYA) 315 . This region is polymorphic, and the variations are especially evident when comparing Western and East Asian strains, both regarding the number of phosphorylation motifs and the sequences flanking them 315,316 . Regarding the surrounding sequences, four types of EPIYA motifs are distinguished: EPIYA-A, -B, -C and -D 317,318 . EPIYA-A and -B are present in the CagA of all the positive strains, while the EPIYA-D is typical of the East Asian forms of CagA and the EPIYA-C can be present up to three times in the CagA from Western isolates. The variations alter the phosphorylation degree of CagA and its biological activity, and in consequence, the clinical outcome of the infection [319][320][321] .

CagA also exhibits activities which are independent of the tyrosine phosphorylation. CagA is able to bind to the hepatocyte growth factor receptor (HGFR or c-Met) and to the growth factor receptor bound protein 2 (Grb2), to stimulate cell growth 322,323 . Injected CagA targets the intercellular junctions of host cells leading to a disruption of epithelial junction-mediated functions and dysplastic changes in epithelial cell morphology, and both alterations have been shown to play a role in carcinogenesis 324,325 .

By studying the region upstream the cagA gene, Censini et al, described an "alien" DNA insertion on the glutamate racemase gene 326,327 . Of approximately 40 kb long, it has the features of a pathogenicity island, such as a different content of G+C (35% compared to 38-45% in the genomic DNA), flanking direct repeats, and IS elements. It contains approximately 30 genes which encode for a type IV secretion system (T4SS), homologous to the VirB system of the plant pathogen Agrobacterium tumefasciens and the Ptl system of Bordetella pertussis 328,329 . The cagPAI can be found as a continuous unit or divided in two segments, cagI and cagII, by the insertion sequence IS605, or by chromosomal DNA 326,330 . A percentage of H. pylori strains does not possess the cagPAI or it is incomplete and not functional, and the presence of a complete and functional cagPAI is directly associated with gastric pathologies 330,331 . The cagA gene is commonly used as a marker for the entire cag locus, although the presence of cagA does not necessarily signify the presence of an intact cag island 332,333 . Other genes such as cagE, cagG, and cagM have been also considered to be good markers for the presence of cagPAI 334,335 . Transcriptionally, the cagPAI is organized in at least eleven operons and five monocistronic genes and this organization seems to be conserved between different strains 336 .

This secretion system allows the translocation of the CagA protein and of the peptidoglycan from the bacterial wall to the cytosol of the epithelial cells of the gastric mucosa 337 , and consequently, the presence of a functional secretion system is necessary for CagA to exert its effects. But also, the sole presence of the cagPAI induces the activation of NF-κB, and AP-1, which are required for the proinflammatory response, in a CagA-independent manner 326,[338][339][340] . The activation of these factors is responsible for the production of the chemokine IL-8 in the gastric mucosa seen during the infection 340 .

IL-8 is the main chemotactic signal for neutrophils, therefore its levels are directly related to the strength of the inflammatory response and severity of the gastritis 341,342 . This chemokine is also related with the angiogenic process in gastric carcinogenesis 343 . Additionally, it has been reported that cagPAI -strains induced IL-10 secretion in human neutrophils 344 . Together, these alterations in cellular functions induced by CagA and the T4SS at multiple levels, provide an explanation for the association of cagPAI + strains with more severe pathologies, including gastric adenocarcinoma, and justify the denomination of CagA as the first bacterial oncoprotein 346 .

VacA

The vacuolating toxin (VacA) is a secreted protein of 87 KDa which was initially described for its capacity of inducing vacuolization in gastric epithelial cells in vitro 24,347,348 . It displays multiple effects on the host cell and is associated with gastroduodenal ulcer and gastric adenocarcinoma, hence it is considered a major virulence factor of H. pylori 349 . VacA protein is secreted by the bacteria either as monomers or as oligomers of six or seven units of the 88kDa protein. In its mature form, VacA is an AB toxin with N-terminal (p34) and C-terminal (p58) peptides separated by a protease sensitive loop 349 . At low pH, the oligomers dissociate into monomers that bind to epithelial cells, and the p58 binds to the host cell receptors and forms channels selectively permeable for anions and small neutral molecules such as urea [350][351][352][353] . Membrane-bound VacA can be endocytosed, and accumulates and forms pores in membranedelimited vacuoles which exhibit markers of late endosomes or lysosomes 354 . The influx of anions alters the permeability of the vacuoles, leading to water flux and vesicle swelling, thus forming the large vacuoles typical of VacA activity. VacA can also be translocated across the plasma membrane into the cytosol where it exerts is action 355,356 . The p34 domain is considered to be the enzymatically active part of VacA, which becomes exposed to the host cell cytosol and influences the regulation of the endosomal fusion 357 . Host cell vacuolation, which in vivo is not as evident as in vitro, may slowly lead to the eventual death of primary human gastric epithelial cells 358 . In vivo, animal models evidence the process by a loss of epithelial cells 359 .

Besides vacuolation, other effects have been attributed to VacA. One of them is the capacity to induce apoptosis both in vitro and in vivo 360,361 . Intracellular expression of VacA or the application of the purified toxin in HeLa cells has been reported to induce the release of cytochrome C from the mitochondrial intermembrane space, suggesting that VacA induces apoptosis by activating a mitochondrial-dependent pathway 362 . This process, as well as the vacuolation, are dependent in the formation of anion-selective channels 363 .

A VacA immunosuppressive activity on T lymphocytes has been described, as VacA could block the proliferation of these cells by mechanisms both dependent and independent of the toxin's poreforming capacity. In the first scenario, the formation of anion-selective channels causes membrane depolarization and prevents Ca 2+ influx from the extracellular milieu, therefore inhibiting the nuclear translocation of the NFAT transcription factor which is key for the expression of cytokines necessary to activate the cell 364,365 . On the other hand, independently of the channel formation, VacA induces the indirect activation of the GTPase Rac and to a reorganization of the actin cytoskeleton which could inhibit T cell activation and division 364 . VacA also exerts a local immunosuppressive activity by interfering with antigen presentation in B cells and phagocytes, through inhibition of the proteolytic processing of antigens and their presentation in the MHC 228,366 .

VacA seems to be implicated in the loosening of tight junctions in monolayers of polarized epithelial cells, increasing the permeability of the host cells 367 . This contributes to the capacity of H. pylori to colonize and persist in the human gastric mucosa, allowing the passage of important nutrients, as it increases the passage of small molecules and anions and the release of important substrates such as urea 351,367 . An additional increase in mucosal permeability is also present due to the induction of proinflammatory cytokines (IL-6, TNF-α) in mast cells by VacA 368 . A balance between the moderate induction of inflammation in one side, and immunosuppressive effects on the other, may be place VacA as a key factor for the maintenance of the long-term colonization of the gastric mucosa by H. pylori.

The vacA gene, which codes for VacA, is present in all the H. pylori isolates, but there are variations in expression and structure amongst them which give place to polymorphisms resulting in different levels of cytotoxicity 228,[369][370][371] . These variations are mostly in the signal region (s), where two alleles, s1 and s2, can be found, and the intermediate region (m), with the alleles m1 and m2 369,370 . All combinations of alleles are common and possible, except s2m1 which is very rare. Strains carrying the s1m1 genotype have a greater VacA activity in vitro than those carrying s1m2, while the s2m2 lacks activity 369 . The s1 type produces a fully active cytotoxin, whereas type s2 produces a toxin with a hydrophilic N-terminal extension that blocks vacuolating and pore-forming activities 372 . Variations in the m region are associated to a decreased activity because this region is part of the p58 subunit and therefore defines the cellular specificity of the toxin 373 . In cells that are recognized by the m2 form of the toxin, like the rabbit kidney line RK3, the activity is similar to that observed in other cells that bind to the m1 form, provided that the s region genotype is the same 374 . Therefore strains possessing the s1m1 genotype of vacA show an enhanced cytotoxic activity compared to s1m2 and s2m2 bearing ones, and are epidemiologically associated with peptic ulcer disease and gastric adenocarcinoma 375 . Other variable regions for VacA have been described, the intermediate region (i) and the deletion region (d) 376,377 .

LPS

The bacterial lipopolysaccharide (LPS) is an essential glycolipid of the outer membrane of Gramnegative bacteria. The classical LPS is formed by three parts: the lipid A, a hydrophobic moiety that anchors LPS to the outer leaflet of the outer membrane, the core oligosaccharide, which maintains the integrity of the outer membrane, and the O antigen polysaccharide, which consists of a polymer made of repeating oligosaccharide units in direct contact with the external milieu 378 . The LPS is also known as bacterial endotoxin, because it triggers a potent inflammatory response both in vitro and in vivo, the latter characterized by a systemic production of inflammatory cytokines, leading to tissue damage, alterations in body temperature, and lethality 379 . However, the LPS of H. pylori has been reported for a decreased toxicity, as higher quantities are needed to elicit a host response in comparison with the Escherichia coli LPS, due to modifications in the lipid A which impair its recognition by the host receptors 380 . In addition to these changes, the O-antigen contains different human Lewis antigens (Le x , Le y , Le a , Le b ) which are also expressed in gastric epithelial cells, thus providing a strategy to escape the host's immune response through molecular mimicry [381][382][383] . The Lewis antigens in the bacterium may also play a role in adherence to gastric epithelial cells, and their capacity to undergone phase variation would provide a dynamic adherent phenotype 384 .

v. Pathogenesis of gastric disease: Gastritis and precancerous lesions of the Correa cascade

In non-pathological conditions, the gastric mucosa lacks myeloid and lymphoid tissues 385 .

Gastritis is characterized by the infiltration of the lamina propria with polymorphonuclear and mononuclear leucocytes, and the presence of H. pylori is the main inductor 28 . Pro-inflammatory cytokines such as IL-1, TNF-, IL-8, and IL-6 are increased in the gastric mucosa of infected subjects 337 . The sustained inflammatory process directly and indirectly activates of different branches of the immunes system, and together with bacterial factors (mainly CagA and VacA) contribute to substantial changes in the architecture of the gastric mucosa. H. pylori stimulates the gastric epithelium, inducing the activation of dendritic cells and macrophages, as well as a Th1-predominant adaptive response 337 . The resulting immune response is incapable of eradicating the bacterium and the persisting stimulus leads to a chronic inflammation process and mucosal damage.

Multifocal atrophic gastritis is the first stage of the sequence of lesions that lead to intestinalsubtype gastric adenocarcinoma. It initiates as a chronic superficial pangastritis involving the antrum's pyloric mucosa, the body's oxyntic mucosa, and stomach fundus in a spotted pattern of multiple foci 386 .

This superficial gastritis turns to a chronic atrophy with loss of the glandular structures and their replacement with metaplastic pyloric or intestinal glands and fibrosis of the lamina propria 28,386 .

Approximately half of H. pylori-positive cases of chronic superficial gastritis will evolve to gastric chronic atrophy 387 , depending on the environmental conditions, bacterial virulence factors, and host's genetic background. Bacterial contact with epithelial cells also deregulates oncogenic signaling pathways that, along with chronic inflammation, induce genetic and epigenetic changes. For example, IL-6 production in response to infection was showed to induce STAT3 activation and stimulate ERK1/2 signaling in H. pylori-associated gastritis and during infection with H. pylori CagA + , and this aberrant activation could be one of the early events in tumor initiation and progression 388 . Another example is the H. pylori-induced hypermethylation of the promoters of genes such as p16, CDH1, DAPK, APC, MLH1, and COX2, which decrease when the bacterium is eradicated 389 .

The development of atrophy determines the course of the H. pylori infection, because the infected individuals that do not present it, have an increased probability of developing duodenal ulcer, end in the other hand, those who do develop atrophy are at greater risk of gastric adenocarcinoma 387 . As atrophic gastritis is the initial step on the Correa cascade of precancerous lesions leading to intestinal subtype gastric adenocarcinoma, its presence induces a five-fold increase in the risk of developing this type of cancer 33,390 . If atrophy constitutes or not the point of no return in the way to gastric cancer is still a subject of debate. However, additional alterations in signaling pathways have been described in metaplasia and dysplasia, which could be associated to malignant transformation 388,389 . The transition from dysplasia to adenocarcinoma is not only explained by the accumulation of mutations in transformed cells but it requires the penetration of neoplastic cells in the surrounding stroma, therefore, the acquisition of ECM degrading and migration capacities 28 , which can result from aberrant activation of signaling pathways that confer CSC-properties through an EMT 131 .

vi. Models of study

1.

In vitro models

a. Cell lines

As H. pylori naturally affects the stomach, the most used in vitro model for study of the infection are human epithelial cells from the gastric mucosa. Unfortunately, the primary culture of these type of cells is not attainable in the long term because cells loss their cohesive properties and lack the ability to renew themselves. As a consequence, in vitro studies of H. pylori infection are often carried in immortalized cell lines or, more frequently, in cell lines derived from gastric adenocarcinomas. Even if they are already transformed, these cell lines mimic the properties of a rapidly-changing, proliferative epithelium such as the gastric epithelium which is the niche of the bacterium. Therefore, the represent a convenient and accepted model for the study of the molecular aspects of the host-pathogen interaction.

The main characteristics of commonly used gastric epithelial cell lines are summarized in table 7.

b. Organoids

One of the main flaws of the conventional tumor cell culture in two dimensions (2D) is the absence of a higher three-dimensional organization (3D), which could be important in the outcome of experiments performed in this model. As an alternative strategy, culture systems that allow the gastric cell lines to grow on 3D, using extracellular matrix proteins, nanofibers, co-culture with fibroblasts, or nonadherent culture in a serum-free medium, have been developed [391][392][393] . Even if these approaches have proved very useful, they do not overcome the lack of heterogeneity that cell lines show when compared to the human stomach, much more complex than a monolayer of a single type of cells, nor they give answer to the common problems of the cell lines such as genetic and epigenetic changes in tumor suppressor genes and oncogenes, accumulated mutations due to years of culture, and even infection with viruses which could modify the cell behavior towards infection 394 . Different groups have approached this issue with strategies, such as de novo generation of human gastric organoids in vitro through the directed differentiation of pluripotent stem cells or from surgical samples of human stomach including healthy and tumor tissue and sorted stem cells 395,396 . One of many technical challenges of these methods for the study of H. pylori infection is that, as the bacterium can only interact with the apical surface of the epithelial cells, the infection has to be made by microinjection of the bacteria in the lumen of each organoid. As an alternative procedure, gastric organoids have been sheathed to establish a 2D culture of polarized gastric epithelial cells which are more suitable for infection and could be adapted to other experimental conditions 397 .

In vivo models a. Murine models

After infection with H. pylori, precancerous lesions can be observed in both C57BL/6 and BALB/c mice, but the infection does not cause the appearance of any type of gastric adenocarcinoma, only MALT lymphomas 398,399 . B6129 mice, derived from the mating of C57BL/6 with 129S6/SvEv, can develop dysplastic lesions but not adenocarcinomas upon infection 400 . In order to have a more suitable model for gastric adenocarcinoma, different strategies of transgenic mice have been developed. Mice knocked-out for TGF-β, p27 or TFF2 and infected with H. pylori are able to develop dysplastic lesions and gastric adenocarcinoma [401][402][403] . Also the overexpression of gastrin (the INS-GAS model), and to a lesser extent, of IL-1β, allow the development of gastric adenocarcinomas in mice infected with H. pylori 404,405 .

b. The Mongolian gerbil model

The Mongolian gerbil model has been so far, the only successful animal model for gastric adenocarcinoma following infection with H. pylori. The first description of H. pylori's capacity to infect this animal dates from 1991, but the follow-up was for only two months, which made it impossible to observe any lesions 406 . Then, the period of observation was increased to six months, allowing the detection of ulcers and intestinal metaplasia 407,408 . And finally, after a longer period of observation, Watanabe et al described the appearance of neoplastic gastric lesions in gerbils infected with H. pylori after 62 weeks 409 .

Since then, several studies have used the model with similar results, the developing of gastric adenocarcinoma after 62 to 90 weeks of infection [410][411][412][413] . Some studies have found earlier apparition of d.

Hippo pathway i. General description

The Salvador-Warts-Hippo pathway was first described as a size-controlling pathway in the epithelial precursors in Drosophila melanogaster, where the main components of the pathway were isolated in genetic mosaic screens for the detection of genes associated with cell growth and proliferation, by studying the mutations present in flies with organ size abnormalities 417 . It is a highly evolutionarily ii.

Upstream regulators

The Hippo pathway does not have dedicated extracellular ligand/receptor complexes like other signaling pathways. Instead, the pathway integrates multiple extracellular signals including cell polarity, contact inhibition, stress, mechanotransduction, and cell attachments, as well as response to hormones and growth factors. Some of the inputs that mediate the response to these processes are described below.

FERM-and WW-containing proteins upstream MST

Both FERM domains and WW domains are modular domains that facilitate protein-protein interactions, and their presence is a common feature amongst the various proteins that regulate the Hippo pathway. WW domains are small modules of 35 to 40 amino acid residues, which bind to proline rich binding motifs (PPxY), and include two conserved tryptophan (W) residues, therefore the name of the domain. The FERM (4.1/ezrin/radixin/moesin) domains are common in proteins that interact both with the cytoskeleton and the plasma membrane 420 . They are cysteine-rich hydrophobic protein modules of around 300 amino acid residues, able to bind a broad range of biochemically different molecules, such as phosphoinositols, glycophorins, receptors including CD44, ICAMs, actin, and other FERM-containing proteins 420 .

Merlin and angiomotins

Merlin is a FERM domain-containing, 69 kDa protein encoded by the NF2 (neurofibromin 2), which is a tumor suppressor whose mutations have been described as a cause in neurofibromatosis type 2 and as a predisposition to various tumors of the nervous system 421 . Merlin is normally located in cell junctions, in association with α-catenin or angiomotins. It is implicated in the contact-depending inhibition of cell proliferation by coupling signals from the cell juctions to growth regulatory signaling pathways, including Hippo. Although the exact mechanism remains to be confirmed, the current evidence suggests that Merlin may promote the Hippo signaling and YAP1 inactivation by assembling platforms where the LATS1/2 kinases phosphorylate and inactivate YAP1 421 .

Merlin-interacting proteins angiomotin (Amot) and angiomotin-like 1/2 (Amotl1/2) are tight junction proteins described for their roles in angiogenesis during development. Angiomotins have been identified as binding partners and negative regulators of YAP1, as they favor YAP1 cytoplasmic localization and have been describe to promote its phosphorylation and inhibit the expression of the YAP1-target genes CTGF and CYR61 421 .

Willin/FRMD6

Willin is a 71 kDa protein with a FERM domain, the human homologue of the Expanded protein of D. melanogaster, and is widely expressed in both neuronal and non-neuronal tissues 420 . Intracellularly, it is located at the plasma membrane, possibly playing a role in contact inhibition and adhesion. Willin frequently colocalizes with Merlin and interacts with YAP1. A recent study showed that Willin alone is sufficient to activate the Hippo pathway and antagonize YAP1-induced phenotype 422 .

KIBRA

KIBRA is a 125 kDa protein which contains two amino-terminal WW domains, first identified in a yeast screening as a partner for the modulator of the post-synaptic cytoskeleton Dendrin 423 , and was then shown to be a component of the Hippo signaling pathway 424 . KIBRA binds to Merlin, independently of its WW domains, and may contribute to the stabilization of the multiprotein complex and enable the transmission of signal to activate the Hippo cascade 424 . A Merlin-independent mechanism has been proposed for Hippo regulation by KIBRA. Via its WW domains, KIBRA can interact directly with LATS1/2 and increase both its proteins levels and phosphorylation, at least in part by inhibiting ubiquitination and the proteasomal degradation of LATS2, which can ultimately lead to an activation of the pathway and YAP1 phosphorylation 123 .

Cell polarity and adhesion

The modulation of the Hippo pathway, and concretely of YAP1 activity, is caused by either the increased activity and the availability of the Hippo kinases towards YAP1 or sequestration of YAP1 at cell junctions 425 . Various systems of cell polarity have been found to be involved in Hippo regulation, the apical and basolateral polarity complexes in epithelial cells, and the planar polarity regulation, whose participation is mainly restricted to the context of development of specialized structures in Drosophila and therefor will not be detailed in this work (figure 11).

a. Apical cell polarity

The Crumbs (Crb) protein is the main regulator of apical cell polarity in epithelial cells. It is a transmembrane protein that share a conserved structure consisting of a large extracellular region linked to a short intracellular tail containing FERM-and PDZ-binding domains 426 . Crumbs recruit a protein scaffold that comprises the proteins Stardust, aPKC, Moesin, Yurt, and Par6 427 . Crumbs can bind to the FERMdomain of Willin and therefore, modulate the Hippo pathway 427 . Besides, the direct binding of YAP1 and Furthermore, in a developing lung model where it was shown that changes in cell polarity dictate airway epithelial differentiation, and Crumbs is required for this process, because it promotes the interaction between YAP1 and LATS1/2, consequently inducing YAP1 phosphorylation and cytoplasmic retention 429 . b.

Basolateral cells polarity

The scribble homologue (SCRIB)-lethal giant larvae homologue (LGL)-disc-large homologue (DLG) complex defines the basolateral plasma membrane domain, and it is localized with adherens junctions 431 .

The SCRIB-LGL-DLG complex is considered a tumor suppressor, as modifications of the expression of its components promotes the acquisition of invasiveness in various cancers such as colon cancer and hepatocellular carcinoma 431 . The Lgl protein has been shown to regulate Hippo signaling in Drosophila.

However, unlike the regulation by Crumbs, the regulation is not related with the localization of the MER/KIBRA/Ex complex but directly with Hpo 428 . Furthermore, a correct localization of Scribble is required for the Hippo pathway activity and a Scribble knock-down leads to an increase of TEAD-reporter activity and a decrease in YAP1-phosphorylation 432 . Co-immunoprecipitation experiments suggest the existence of a Hippo regulatory complex formed by MST1, LATS1, Scribble, and the Scribble regulators LKB1 and MARK (PAR family) 432 .

c. Cell adhesion proteins

Cell adhesion proteins are intimately associated with cell polarity. The tight junctions (TJ) not only define the limits of polarized membrane domains, as they divide the apical from the basolateral segments of the plasma membrane, but also these and the adherens junctions (AJ) bind to polarity complex proteins and can modulate the regulation they exert on Hippo signaling 433 . Together, adhesion proteins function to sense the integrity of the epithelium, and it has been shown that disruptions and discontinuities in the epithelial cell layers induce the nuclear activity of YAP1 and TAZ 434 . Moreover, during development, even in non-polarized cells, proteins of cell adhesion such as E-cadherin, participate in the regulation of YAP1 activity.

TJ and AJ proteins sequestrate YAP1 and TAZ by interaction with several adhesion proteins. The TJ protein α-catenin interacts strongly with phosphorylated YAP1, in a binding mediated by 14-3-3 proteins, sequestering YAP1 and preventing its dephosphorylation and nuclear translocation 434 . The interaction of cell adhesion proteins with YAP1, TAZ, and with regulators of Hippo signaling such as the Mer-KIBRA-Willin has been described for PTPN14, LKB1, and ZO-1/2 434 .

E-cadherin, is a very important molecule of cell-cell adhesion in epithelial cells because its homophilic binding allows the formation of the epithelial junctional complex. Loss of E-cadherin promotes tumor progression and metastasis, and it's a hallmark of many epithelial cells cancers as well as a feature of EMT. Increased expression of E-cadherin in cancer cells blocks invasion abilities and tumor progression by increasing the cell adhesion and the β-catenin activity. A study by Kim et al, recently showed that the Hippo signaling is required for the inhibition of proliferation mediated by E-cadherin, as depletion of LATS, Merlin, and KIBRA or over expression of YAP1 blocked this inhibition 435 . Furthermore, the authors of this study found that the E-cadherin-β-catenin complex regulates the phosphorylation status and nuclear localization of YAP1 in MCF10A cells cultured in high density.

Cytoskeleton, extracellular matrix and mechanical forces

The extracellular matrix, the neighboring cells, and even its own cytoskeleton, exert mechanical forces over the cell. Cells can sense these mechanical cues and response by undergoing different processes such as proliferation, differentiation, apoptosis, and migration, and the cytoskeleton is a key element in this response 434 . The rearrangement of actin cytoskeleton is a key element in the YAP1/TAZ-mediated response to cell geometry, stiffness of the ECM, and attachment to a surface. Moreover the stabilization of F-actin using the drug jasplakinolide, was shown to induce the expression of YAP1/TEAD target genes CTGF and CYR61 and the effect of the drug was inhibited when YAP1 was knocked-down 436 . The RhoA kinase has been demonstrated to play a crucial role in the YAP1 regulation by actin, as its overexpression induces dephosphorylation of YAP1, and both RhoA activity and actin polymerization are required in PAR1mediated activation of YAP1 437 . While the YAP1/TAZ response to detachment and crowding can be inhibited by LATS activation, the response to matrix stiffness, requires not only an increase of filamentous actin but also the formation of stress fibers and seems to be independent of LATS activity 437 . In a recent study, Sun et al proposed a computational model integrating the mechano-sensing signaling network of YAP1 and TAZ, which integrated the relation of the latter and the Hippo pathway, by a synergistic effect between LATS2 and the F-action regulator protein LIMK. The study also highlighted the influence of adhesion, stiffness, and RhoA over other regulators.

iii.

Hippo kinases

MST 1/2

The gene coding for the Drosophila homologue of MST1/2, hippo (hpo) was first identified in Regarding its enzymatic activity, auto-transphosphorylation in vitro has been described in purified MST1/2 in the presence of Mg 2+ and ATP, however, the relevance of this phenomenon in vivo remains unknown 440 . On the other hand, two main regulators of the MST1/2 activity in the cell have been described: RASSF polypeptides and SAV1 (WW45, Salvador).

The RASSF family of tumor suppressor proteins are adaptor proteins that interact with GTP-Ras by their RA-domain. There are ten isoforms, and isoforms RASSF1 to RASSF6 have been described to also possess a SARAH-domain, which allows them to form complexes with MST1/2 and SAV1 441 . This

LATS1/2

The Drosophila homologue of LATS, warts (wts), was described in 1995 by Robin et al as a tumor suppressor gene whose loss resulted in overproliferation and hypertrophy of epithelail cells leading to abnormal deposit of extracellular matrix 442 . Based on the nucleotides sequence, the authors also reported a predicted serine/threonine kinase activity and homology to the human myotonic dystrophy protein kinase and the Cot-1 protein kinase from Neurospora crassa. The human homolog, identified initially as h-warts or lats1 (large tumor suppressor), was the located at chromosome 6q24-25.1 as a gene coding for a serine/threonine kinase as previously predicted, which localized to the centromeres in interphase cells and relocated dynamically during the different phases of mitosis and acted as a negative regulator of CDC2/cyclin A 443,444 . A second homologue, lats2 was the identified both in mouse and human, with high homology, especially in the kinase domain between the murine and human forms (75%) and also between human lats1 and lats2 (85%) 445 . Human lats2 was found in chromosome 13q11-12 and coded for a 125-87 116 kDa nuclear protein 445 , while a posterior study showed a cytoplasmic localization during interphase and in the mitotic apparatus during mitosis, and whose ectopic expression blocked the cell cycle in G1/S and caused a downregulation of CDK2/cyclin D 446 .

Regarding the protein structure, LATS1 and LATS2 share a high homology not only in the conserved kinase domain but also in the amino-and carboxy termini, as they both have a protein-bonding domain (PBD) upstream the catalytic domain, as well as the two LATS conserved domains LCD1 and 11), subcellular localization and conformational regulation 447,450 .

Post-transcriptional regulation by micro RNAS have been described for LATS2, notably the miR-372-373 cluster. In esophageal cancer cell lines and esophageal cancer tissue samples, miR-373 levels were shown to be inversely correlated to the expression of LATS2, and a direct inhibition of LATS2 was demonstrated in vitro in the cell lines 451

iv. Downstream transcription co-activators YAP1 and TAZ

The transcription co-activators Yes-associated protein 1 (YAP1, an orthologue of the D.

melanogaster Yorkie) and its paralogue WW domain containing transcription regulator 1 (WWTR1 or TAZ) are the two major downstream effectors of the Hippo signaling pathway, and as such, they are involved in control of organ size, development, and tumorigenesis 434 . YAP1 and TAZ have similar functions and share structural features and mechanisms of regulation, however the redundancy between them is not absolute.

Knock-out mice phenotypes illustrate some differences, as the YAP1-null mice are not viable due to defects in embryonic development, contrary to TAZ knock-out mice, which are viable but predisposed to disease.

Structure

The YAP1 gene was described in a screening of expression libraries aiming for proteins interacting with Src-homology 3 (SH3) by Sudol et al 455 . It encodes for protein of 65 kDa that has a modular structure including various domains: a WW (two conserved tryptophanes) domain that binds to PPxY (prolineproline-any amino acid-tyrosine) motifs, a PR (proline rich) domain, a TEAD-binding domain (TID), a SH3binding motif (SH3-BM), and a transcriptional activation domain (TAD) containing a PDZ-binding motif (PDZ-BM). There are two isoforms of YAP1 generated by alternative splicing, YAP1-1 and YAP1-2, the latter have an additional WW domain (Figure 12).

Using a similar strategy of screening of cDNA libraries but for 14-3-3 protein binding partners, TAZ was identified by Kanai et al as a transcriptional co-activator sharing homology with YAP 456 . TAZ is a 45 kDa protein containing a conserved WW domain, a TEAD-binding domain, and a transcriptional activating domain with a PDZ-BM, common with YAP1, but it lacks the SH3-BM and the PR-domain (Figure 13). The TAZ gene is located in chromosome 3q24.

Phosphorylation and proteasomal degradation

The most studied regulation of YAP1 and TAZ occurs by LATS1/2-mediated phosphorylation in response to Hippo pathway signaling, as these proteins have five and four consensus sites (HxRxxS) for LATS1/2-serine phosphorylation, respectively 457 . YAP1-Ser127 and TAZ-Ser89 phosphorylations are very important in YAP1/TAZ regulation as they cause YAP1/TAZ cytoplasmic retention in α-catenin/14-3-3 complexes in cell junctions, preventing their nuclear disponibility to act as transcriptional co-activators 458 .

Other kinases may also phosphorylate the YAP1-Ser127, such as Akt (for which is was originally identified), JNK, and other unknown kinases in keratinocytes and liver. Thus LATS1/2-mediated phosphorylation could be seen as a predisposing event to nuclear exclusion and not necessarily a primary inactivation event.

A second LATS-phosphorylation site, YAP1-Ser381 and TAZ-Ser311, is important in the stability of YAP1/TAZ, because it primes a following round of phosphorylation by CK1 kinases, which in turn generates a phosphodegron recognized by ubiquitin-ligase adaptators that lead to poly-ubiquitination and proteosomal degradation. YAP1 requires two CK1-mediated phosphorylations in order to be degraded and TAZ requires only one, which could explain the differences in stability observed between the two proteins. While the half-life of TAZ is short (<2h), YAP1 is more stable and its regulation is mostly by cytoplasmic retention 457 .

Physical regulation

Cytoplasmic sequestration is a crucial regulator of YAP1/TAZ co-transcriptional activities. Part of this retention is dependent of LATS1/2-mediated phosphorylation, as previously explained, but there is also cytoplasmic retention independent from the phosphorylation status of YAP1/TAZ. Angiomotins and PTPN14 have PPxY domains capable of binding directly to WW domains of YAP1/TAZ, regardless their phosphorylation status, and retaining them in the cytoplasm 458 .

Nuclear functions and TEAD-transcription factor co-transcriptional activity

As co-transcriptional regulators, YAP1 and TAZ exert their main function in the nucleus. When they are not phosphorylated in the LATS1/2-dependent phosphorylation site, YAP1/TAZ interact with and activate the TEA-domain (TEAD) transcription factors and promote the transcription of YAP1/TAZ-TEAD complex target genes. This interaction was demonstrated by the isolation of YAP1/TAZ as binding proteins of TEAD 459 and also by an increase of TEAD-luciferase reporter activity upon over expression of YAP1/TAZ 460 . In the fruit fly, the interaction has been described between the homologues of YAP1 and TEAD, Yorkie and Scalloped 461 .

TEAD-family transcription factors are evolutionary conserved transcription factors involved mainly in development, and were initially described for their capacity to bind to viral enhancers such as SV40 and HPV16 and activate the transcription of the controlled genes 462 . There are four TEAD transcription factor, TEAD1-4, and together, they are expressed in almost every tissue. The structure is conserved between the four members of the family, with a C-terminal TEA-domain, which binds DNA in the consensus sequence 5'-GGAATG-3', and an N-terminal transactivation domain that binds the coactivators 462 . Three groups of co-activators have been described for binding TEAD transcription factors:

the Hippo pathway-controlled YAP1 and TAZ, the VGLL proteins, and the p160 family, and the complexes formed have different function at the transcriptional level.

TEAD-related transcription plays an important role in development, and TEAD transcription factors are necessary for myogenesis, cardiogenesis, development of the neural crest and other embryonic tissues 462 . Gene inactivation studies in mice embryos have shown, not only that TEAD transcription factors are required for different developmental processes, but also that the roles for each TEAD are different, and that their activities may be redundant sometimes, but not necessarily identical in the same moment or in the same tissue. For example, mouse embryos null for TEAD1 do not survive due to lethal cardiac defects, proving a role of TEAD in heart development, but the silencing of TEAD4 results in a failure of endometrial implantation, but if it is silenced after implantation, the normal development is not affected, which suggest a role for TEAD4 particularly in the specification for the trophectoderm 460 .

YAP1/TAZ/TEAD target genes

The transcriptional targets of YAP1/TAZ-TEAD complexes include several genes such as CTGF, CYR61, ANKRD1, BIRC5, AXL, Ki67, surviving, c-myc, InhA, Col8a1, and others 457,462 . Remarkably, most of those are growth promoting factors, and the most important amongst them are the CCN family proteins CTGF (CCN2) and CYR61 (CCN1). Proteins of the CCN family are regulatory ECM-associated proteins, and in general, control cell migration, proliferation, cell lineage commitment and tissue specification, morphogenesis and angiogenesis, and are therefore required for embryonic development, tissue turnover, and injury repair 463 . Both CYR61 and CTGF, together with WISP2 (CCN5) are indispensable for embryonic viability, as deficiency of any of those compromised fetal development in mice, adding evidence to the importance of the YAP1/TAZ-TEAD role in this process.

Both CTGF and CYR61 share common structural features, a signal peptide and an IGFBP-domain in N-terminal, followed by a von Willebrand C (vWC)-domain, a thrombospondin-type repeat 1 (TSP-1), and a Carboxy-terminal domain with a cysteine knot motif 463 . Through these domains, they are capable of binding to integrins (αvβ3, αvβ5), growth factors such as VEGF and TGF-β, low-density lipoprotein receptor-related proteins, ECM components such as heparin sulfates, MMPs, and fibronectin, among others 463,464 . CTGF and CYR61 are co-expressed in the notochord, which suggests overlapping functions of both proteins in early stages of development. However, their relative important varies in the different tissues. For example, in the mouse CTGF is more related to development of cartilage and epithelial structures of lung and pancreas, and CYR61 is widely expresses in the cardiovascular system.

In adult tissues, over expression of CTGF and CYR61 has been associated with pathological conditions. CYR61 increased expression is associated with inflammation and tissue repair. It has been found to be increased in obese men with metabolic syndrome, colitis, rheumatoid arthritis, and atherosclerosis; and polymorphisms of the gene have been linked to higher levels of plasma cholesterol and disorder of bone metabolism 463 . Increased expression of CTGF is linked to wound healing and fibroproliferative disorders, being correlated to excessive accumulation of ECM proteins in fibrotic tissues.

While CTGF is highly expressed in some healthy tissues such as the spleen, the gastrointestinal tract, prostate, heart, amongst others, it virtually absent in others, including brain, peripheral leucocytes, and liver, except in pathological conditions 463 . A study showed that CTGF expression was elevated in hepatocellular carcinoma (HCC) samples and peritumoral non-cirrhotic and cirrhotic tissues 465 .

Furthermore, the induction of CTGF expression in HCC cell lines and primary hepatocytes increased cell growth and dedifferentiation, expression of inflammation-and carcinogenesis-related genes, and resistance to doxorubicin.

v. Hippo pathway and cancer

Several processes controlled by the Hippo pathway are important features of cancer cells (figure 14). For example, altered cell proliferation is a key aspect of tumorigenesis. It has been shown that hyperactivation of YAP1/TAZ transcriptional activity stimulates aberrant cell proliferation, not only by increasing the transcription of pro-growth factors, but also by counteracting tumor suppressor mechanisms such as RB-induced senescence 466 . LATS2 can activate p53 and its pro-apoptotic activity at least by two mechanisms: LATS2 can directly phosphorylate ASPP1 (apoptosis-stimulating protein of p53-1) to drive its translocation to the nucleus where it mediates p53 response, an effect that can be antagonized by YAP1 467 ; and it can also bind to MDM2 and inhibiting its E3 ligase activity, therefore preventing the degradation of p53 468 . Furthermore, in response to H-Ras mutation, LATS2 is activated and translocated to the nucleus where it induces p53-mediated apoptosis, and the cells that survive usually display low levels of LATS2 protein, LATS2 gene promoter hypermethylation, and genomic instability, suggesting that silencing of LATS2 expression enables tumor development and progression 469 .

This insensitivity to apoptotic cell death leads to another cancer cell property regulated by the Hippo pathway, cell survival. YAP1 over expression can promote cell survival by blocking TNF-and FAS-induced apoptosis in the mouse liver 466 . As described before, the target genes of the YAP1/TAZ/TEAD are involved mainly in growth promotion. Besides from their physiological roles, TEAD transcription factors have been involved in carcinogenesis, and their over expression has been described for various types of cancer 470 . An increasing quantity of epidemiological and experimental data supports the role of the Hippo/YAP1/TAZ signaling pathway in different types of cancer, both of epithelial and non-epithelial origin, which are thoroughly reviewed by Zanconato et al 471 (figure 15). Gastric cancer is not the exception, and even if there are less studies than in breast or colorectal cancer, evidence is available. In a study by Xu et al, the expression of MST1/2 and LATS1 was decreased in tissue sections from gastric cancer compared to those of normal gastric mucosa 473 , and several studies have revealed an upregulation of YAP1 mRNA and protein in gastric adenocarcinomas, as well as a correlation of YAP1 expression and nuclear localization with a bad outcome 471 . By studying 84 cases of gastric patients, Jiao et al 474 found an association of YAP1 mRNA levels with tumor size, differentiation and pathologic staging, and with H. pylori status, and the immunohistochemistry staining of tissue arrays revealed higher YAP1 positivity rates in gastric cancer and dysplastic lesions compared to normal tissue. Consistently, the levels of YAP1/TEAD target genes CTGF and CYR61 were also increased. Interestingly, the authors also studied the expression of the tumor suppressor VGLL4, which was significantly decreased in gastric cancer and precancerous lesions and inversely correlated with the expression of CTGF and CYR61. The YAP1/VGLL4 mRNA ratio strongly correlated with clinical features of advanced gastric cancer and to H. pylori status, leading to the propositions that the YAP1/VGLL4 ratio could be an important factor to take in account for diagnosis and classification of gastric cancer. Furthermore, gastric cancer cell lines were found to have increased YAP1

and decreased VGLL4 levels, and over expression of VGLL4 induced apoptosis, reduced cell viability and decreased YAP1 transcriptional activity, evidenced by diminished TEAD reporter activity and expression of YAP1/TEAD target genes.

A study by Li et al described a role for TEAD1 as a negative regulator of the tumor suppressor VGLL4 via the stimulation of miR-222 475 . Hypomethylation in the TEAD4 promoter and a consequent overexpression have been described in gastric cancer tissues, and its knock down impaired cell growth both in vivo and in vitro, pointing out the contribution of TEAD4 to gastric cancer development 476 .

Altogether, these studies point out the role of the Hippo signaling in gastric carcinogenesis.

Crosstalk with other oncogenic signaling pathways

Both YAP1/TAZ and TEADs are also capable of binding to other proteins, and therefore participate in other signaling mechanisms aside from their canonical role of oncogenes suppressed by the Hippo pathway activation. These pathways include Wnt/β-catenin and TGF-β/SMAD signaling, among others.

a. YAP1/TAZ -Wnt/β-catenin crosstalk

YAP1 and TAZ are components of the β-catenin destruction complex and recruit β-transducing repeat-containing protein (β-TrCP) to facilitate β-catenin degradation, which places them as critical mediators to the β-catenin degradation when the Wnt signaling is inactive 457 . When YAP1 and TAZ are released from the complex due to Wnt ligand signaling, β-catenin accumulates as it becomes undetectable to the β-TrCP, and YAP1 and TAZ are free to go to the nucleus and stimulate their target genes transcription. So, functionally cytoplasmic YAP1 and TAZ able to terminate Wnt signaling, by sequestration of β-catenin, impairing its nuclear activity, and of Dvl, further inhibiting Wnt target gene transcription. In addition, YAP1/TAZ can interact with and retain in the cytoplasm the SHP-2 phosphatase, whose nuclear localization promotes β-catenin dependent transcription 458 . In antagonizing Wnt/β-catenin, YAP1 is considered as a tumor suppressor in this context 477 . On the contrary, nuclear YAP1 and TAZ may serve as transcriptional mediators of Wnt signaling.

b.

YAP1/TAZ -TGF-β/Smad crosstalk TGF-β is characterized for having opposite roles depending on which state of tumorigenesis the signaling is activated. During tumor initiation, it has been reported to inhibit cell cycle progression, but in later stages, the TGF-β/SMAD activation promotes cancer cell proliferation, acquisition of CSC-properties through EMT, and metastasis. This dichotomy could be in part defined by the YAP1/TAZ status of the cell.

The presence of YAP1/TAZ in the nucleus enhances SMAD activity, and on the contrary, cytoplasmic accumulation of YAP1 and TAZ decreased it. According to this, in normal polarized cells where YAP1/TAZ are cytoplasmic, TGF-β/SMAD signaling is hampered whilst in cells with nuclear YAP1/TAZ, the sensitivity to TGF-β/SMAD is enhanced.

Hippo pathway targeting in cancer therapy

Both in the cancer setting and in physiological conditions, YAP1 and TAZ are key to the TEADtranscriptional activity and vice versa, as it has been shown that knock-down of TEADs or disruption of the YAP1-TEAD interaction decreased transcription of YAP1 target genes and oncogenic transformation.

Dysregulation of YAP1/TAZ has been established as a signature in multiple types of cancer, notably in the liver, the ovaries and the lungs, amongst many others, and it has been showed that the pairing with TEADs is necessary for the promotion of tumorigenesis. Therefore, the ways to disrupt YAP1/TAZ-TEAD interaction have been studied as potential strategies for cancer treatment.

a. Verteporfin

Verteporfin (VP, C41H42N4O8, molecular weight: 718.8 Da, figure 16) is a benzoporphyrin derivative initially used as photosensitizer for photodynamic therapy (PDT) in the treatment of neovascular age-related macular degeneration, choroidal neovascularization due to pathologic myopia and chronic central serous chorioretinopathy 478 . It is commercialized by Novartis under the name of Vysudine®. In PDT, the VP molecules is stimulated with a light source with a wavelength of 688-692 nm, leading to the generation of oxygen-free radical that cause cytotoxic damage 478 . Light-independent activity of VP have also been reported. VP was identified as an inhibitor of autophagosome accumulation in a screening of more than 3500 compound in MCF-7 cells 481 . VP treatment inhibited drug-and starvation-induced autophagic degradation and reduced cell viability in nutrientdepleted conditions, but it had no effect in a nutrient-rich environment.

Liu-Chittenden et al identified VP in a screening for small molecules that inhibit the YAP1/TEAD using a YAP1/TEAD luciferase reporter 482 . VP decreased luciferase reporter activity in more than 50% from concentrations as low as 2.5 µM, as a result of a selective binding of YAP1 and the possible induction of conformational changes that result in an abrogated interaction with TEAD. The result was the same when the VP treatment was tested in the Drosophila homologues Yorkie and Scalloped. The authors next examined the anti-oncogenic properties of VP in vivo using two murine models (YAP1-induced hepatomegaly and liver-specific knock-out of NF2/Merlin) treated with a 100 mg/kg systemic dose of VP and DMSO as control, and they found that in both cases VP treatment suppressed liver overgrowth resulting from YAP1 overexpression or overactivation.

The role of VP in the inhibition of the YAP1/TEAD complex formation and the effects of this inhibition in CSC properties have been studied in cells of gastrointestinal origin. In a study by Song et al in esophageal cancer cells, where the CSC population was associated with a high YAP1 activity and the expression of Sox9, VP treatment reduced the CSC properties (sphere formation and ALDH activity) in vitro and the growth of tumors derived from xenografts in immunodeficient mice 483 . Furthermore, the same group showed later that inhibition of YAP1 activity with VP sensitizes esophageal cancer cells to 5-FU et docetaxel in vitro and enhanced the inhibition of tumor growth by 5-FU in vivo 484 . Similar results have been reported in a murine model of azoxymethane/dextran sulfate sodium-induced colorectal carcinogenesis, where VP treated mice showed less numerous and minimally dysplastic tumors in treated groups versus the control group, decreased cell proliferation and expression of CTGF and CYR61, and increased apoptosis, without affecting the normal intestinal homeostasis or displaying any apparent toxicity in kidney, liver, or intestinal tissues 485 . These results were confirmed on patient-derived xenografts of colorectal cancer, in which VP inhibited tumor growth. Two other light-independent effects of VP were described in this study. First, a YAP1-independent inhibition of STAT3/IL-6 signaling leading to a decrease of tumor cell growth, which has also been reported in tubule-interstitial fibrosis of the kidney 486 . Second, the capacity of VP to induce oligomerization of high molecular weight proteins, which increases apoptotic cell death. More recently, the HMWP oligomerization mechanism has been addressed in detail and described as light-dependent 487 . Until now, the anti-oncogenic properties of VP have not been described in gastric cancer.

b. VGLL4-mimicking "Super-TDU" inhibitor peptide

Following the finding of VGLL4 as a tumor suppressor that disrupts the YAP1/TEAD activity, Jiao et al 474 deciphered the precise molecular mechanism of VGLL4-mediated disruption of the YAP1/TEAD complex, which lays on a competition between YAP1 and VGLL4 for the binding of TEADs, and while the YAP1 is an activator of transcription, VGLL4 is a repressor. Tandem Tondu (TDU) domains of VGLL4 were identified as crucial to the inhibitory activity and structural analysis allowed the identification of the precise residues, and the synthesis of an inhibitor peptide termed "Super-TDU" which targets the interaction between YAP1 and TEAD4. In vitro, the peptide inhibited cell viability and colony formation, and down-regulated the expression of CTGF and CYR61, and in vivo, the weight and size of tumors in a model of xenograft of gastric cancer cells in BALB/c mice, with acceptable levels of toxicity and specificity.

The inhibitor peptide also showed a therapeutic effect in a model of H. pylori infected mice.

a. Article 1: LATS2 and YAP1 are co-upregulated in response to Helicobacter pylori infection and control cancer stem cell properties in gastric epithelial cells

Submitted for publication

INTRODUCTION

Stomach cancer is the third cause of cancer-related deaths worldwide [1]. The most frequent are gastric adenocarcinomas (GC), which have a 5-years survival rate of <25% [2]. Current treatments include chemoand radiotherapies, with surgery for the non-metastatic forms. The chronic infection with the gramnegative bacterium Helicobacter pylori, which is a type I carcinogen [3], is a strong risk factor for GC [4][5] and nearly 93% of non-cardia GC are H. pylori-positive [6]. H. pylori specifically colonizes the stomach of half the world population and provokes a chronic inflammation of the gastric mucosa likely to evolve into ulcers and in ~1% of the cases into GC. H. pylori strains positive for the cag-pathogenicity island, encoding a type 4 secretion system (T4SS) and the virulence oncoprotein CagA, are strongly associated with malignancy development [7]. Upon H. pylori adherence on human gastric epithelial cells (hGECs), the T4SS forms a pilus, which translocates the bacterial CagA and peptidoglycans into the epithelial cytoplasm triggering cell innate immunity and other signaling pathways that alienate the mucosa homeostasis [8].

Epithelial turnover, resulting from the balance between progenitor cell proliferation and differentiated cell death, is a major host defense mechanism against pathogens and is commonly altered during bacterial infections and chronic inflammatory diseases. In an animal model, it has been demonstrated that H. pylori via CagA delivery suppresses apoptosis at the superficial gastric pit cells, delaying gastric epithelium selfrenewal [9]. In H. pylori-infected hGECs in vitro, we previously reported that H. pylori via CagA blocks cell cycle progression by up-regulating the cell cycle regulator LATS2 (Large Tumor Suppressor 2) and elicits an epithelial-to-mesenchymal transition (EMT) prominently governed by the ZEB1 transcription factor [10][11]. EMT is a developmental process characterized by loss of epithelial cell polarity and cell-cell interactions, reorganization of the cytoskeleton, and acquisition of the migratory properties of mesenchymal cells [12]. Influencing cell growth and survival, EMT in H. pylori-infected patients may contribute to a reduced renewal and an aberrant differentiation of the gastric mucosa, which leads to preneoplastic atrophic gastritis and metaplasia states [5]. EMT is also implicated in the emergence of cancer stem cells (CSC) [13]. CSCs correspond to a subpopulation of cells within the primary tumor; They express markers of immaturity, are able to self-renew and divide asymmetrically, and are at the origin of the more or less differentiated cells that constitute the tumor. CSCs are responsible for tumor initiation, progression and metastasis, and have increased resistance to conventional radio/chemo-therapies [14]. In the context of gastric carcinogenesis, we previously demonstrated that challenging hGECs with cagApositive H. pylori generates CD44 high/+ cells harboring a mesenchymal phenotype [15]. CD44, the cell surface hyaluronate receptor, has been identified as a CSC marker in many solid tumors including GC [16][17][18]. Interestingly, the H. pylori-induced CD44 high/+ hGECs share phenotypic properties with CSCs, such as the invasion, migration, tumorspheres formation in vitro and tumor formation in vivo compared to their CD44 low/-counterparts [15]. CD44 and the mesenchymal markers ZEB1 and Snail1 are overexpressed in the gastric mucosa of H. pylori-infected patients and in mouse models of experimental H. pylori infection, within the isthmus region of the gastric glands where gastric epithelial stem cells reside [15][18][19].

H. pylori-driven LATS2 upregulation and emergence of CSC-like properties prompted us to analyze the Hippo pathway, an evolutionarily conserved signaling cascade involved in stem cell homeostasis and cancers [20][21], and which has never been studied in the context of H. pylori infection. In humans and mice, the pathway activates a kinase core involving MST1/2, their cofactor Salvador, and the tumor suppressors LATS1/2. In the activated Hippo pathway, which restricts tissue overgrowth, LATS1/2 phosphorylate the transcriptional co-activator YAP1 on serine 127, inactivating YAP1 by retention within the cytoplasm and degradation by the proteasome. On the contrary, when the Hippo pathway is inactivated, YAP1 translocates into the nucleus and acts as co-activator for the transcription enhancer factors TEADs to promote cell proliferation. The Hippo pathway is connected via its components and effectors to other pathways such as Wnt/ß-catenin, Ras, junctional complexes, NF-κB, TGFß and many others [22].

In order to get a deeper insights into the molecular mechanisms governing H. pylori-mediated gastric carcinogenesis, we aimed here to determine the role of the Hippo pathway, focused on LATS2 and YAP1, on the EMT-like process and on the emergence of H. pylori-induced CSCs in vitro as well as in patientsderived gastric mucosa and GC.

RESULTS

1. LATS2 and nuclear YAP1 are co-upregulated in the gastric epithelial cells of H. pylori-infected patients LATS2, YAP1 and CD44 were evaluated by immunohistochemistry in gastric mucosae of either non-infected or H. pylori-infected patients (Fig. 1a), and their expression was scored (Table 1, Fig. 1b). Both LATS2 and YAP1 were found upregulated and strongly expressed in the nuclei of cells of the infected gastric mucosa, and are therefore associated with gastritis. Their scores were even higher in intestinal metaplasia. LATS2 and YAP1 over-expressions were found precisely within the isthmus in the fundus and in the crypts of the gastric glands in the antrum, where CD44 is also over-expressed in response to H. pylori infection, and which corresponds to the regenerative epithelial stem cells location [15].

The Hippo pathway genes are affected by H. pylori in cultured hGECs.

As primary hGECs cannot be easily isolated and expanded in tissue culture, the AGS and MKN74 established hGEC lines have been widely used to recapitulate in vitro the early events of H. pylori infection occurring within an actively replicating gastric mucosa. Both cell lines grow as monolayers of cohesive, cobblestone-shaped cells, and respond to cagA-positive H. pylori strains by an EMT and the emergence of CSC-like properties [11][15].

Global gene expression in response to H. pylori was performed using whole-genome microarrays. Genes involved in the Hippo pathway and whose expressions were significantly altered by infection are presented in Fig. S1 and summarized in Fig. 1c. In both infected AGS and MKN74, the Hippo kinase core genes were globally upregulated about twice. LATS2 was significantly up-regulated in infected MKN74 but not in AGS (Fig. S1). We previously reported that LATS2 is post-transcriptionally down-regulated by miR-372/373, which are down-regulated upon H. pylori infection leading to LATS2 upregulation and blockade of cell cycle progression [10]. In parallel, the transcriptional co-factors YAP1 or TAZ, which are both LATS2 substrates, were slightly up-regulated in AGS and MKN74, respectively. YAP1 interacts primarily with the TEAD transcription-enhancer factors, thereby promoting cell growth. VGLL4, a YAP1 negative competitor for TEAD binding [23], which prevents its oncogenic function, was also upregulated.

Other transcription factors such as members of the RunX family, the Smad family or p73 that are speculated to be interacting with YAP were variably affected according to the cell line, with the exception of RUNX1 which was upregulated in both. Several YAP1/TEAD target genes involved in cell proliferation and survival were significantly affected upon infection, including cyr61 and ctgf encoding cysteine-rich extracellular matrix proteins that function as integrin ligands and activate cell proliferation and which are among the main target genes assessing YAP/TEAD transcriptional activity [20-22] (except ctgf in MKN74).

It is noteworthy that, in infected MKN74, an outstanding increase was found in IGFBP3, an insulin growthfactor binding protein with either anti-proliferative or context-dependent EMT-and tumor-promoting activities [24].

The Hippo pathway upstream effectors as well as some components of the cell polarity complexes and intercellular junctions, which contribute to the activation of the Hippo cascade [25], were variably affected upon infection. H. pylori upregulated cdh1 encoding E-cadherin accordingly to a previous report [11], as well as crb3 encoding a component of the Crumbs complex maintaining the epithelial integrity [26], and ajuba encoding a scaffold protein of cell junctions and binding partner of LATS which negatively regulates YAP1 [27]. Thus, the analysis of the Hippo transcriptome in hGECs upon infection revealed concomitant up-regulations of the core kinase, downstream effector genes and YAP/TEAD canonical target genes.

Biphasic kinetics of Hippo activation upon H. pylori infection of hGECs in vitro

To assess how LATS2 and YAP1 are orchestrated during infection, we analyzed the time-course of their expressions in the hGECs challenged by cagA-positive H. pylori. As TEAD is considered the primary transcriptional partner of YAP1, we used a TEAD-luciferase reporter assay to sense TEAD-mediated transcription (Fig. 2a). In AGS, the TEAD transcriptional activity clearly exhibited biphasic kinetics characterized by an early and transient stimulation during the first 2 hours post infection (hpi) followed by a decreasing activity thereafter. This biphasic pattern was accompanied by parallel changes in the expression of the direct TEAD-target genes ctgf and cyr61 (Fig. 2b), indicating that H. pylori transiently inactivated the Hippo pathway triggering YAP/TEAD activity in AGS cells, and then progressively activated it rendering YAP/TEAD co-transcriptionally inactive. In MKN74, the TEAD transcriptional activity was highly variable before the first 5 hpi, but modestly decreased thereafter. Nevertheless, both ctgf and cyr61 remained upregulated during the entire infection time-course, accordingly to the transcriptomic data but not to the YAP1/TEAD activity (Fig. S1 and Fig. 2a).

H. pylori progressively triggered LATS2 and YAP expressions in hGECs at both the mRNA (Fig. 2c) and protein levels (Fig. 2d). The variations in proteins occured earlier and were more marked than in mRNAs, suggesting initial post-transcriptional activating mechanisms. LATS2 and YAP1 proteins accumulated as soon as 2 hpi in both hGECs (Fig. 2d), and YAP1 was mainly in its YAP1-P Ser127 form. YAP1 accumulation was accompanied by enhanced, although delayed, yap transcription in AGS, but not in MKN74 (Fig. 2c), in accordance with the transcriptomic data.

By immunofluorescence analyses, YAP1 was mainly observed in its nuclear active form (YAP-P ser127 negative) at 2 hpi and accumulated thereafter in the cytoplasm in its inactive form (YAP-P ser127 ) (Fig. 2e).

MKN74 exhibited a greater heterogeneity of YAP1 labeling than AGS, thereby explaining why the TEAD activity was less strikingly affected than in AGS (Fig. 2a). LATS2 progressively accumulated in both hGECs upon infection. Remarkably, LATS2 transiently accumulated in the cytoplasm of MKN74 (2 hpi) before becoming nuclear at 24 hpi, whereas LATS2 was nuclear in AGS at all time points.

Altogether these data show that LATS2 and YAP1 were regulated during H. pylori infection in a biphasic pattern. In the early period of infection, H. pylori transiently triggered YAP1 expression and cotranscriptional activity, most likely by post-transcriptional mechanisms, along with LATS2. This early period of infection corresponds to an inactive Hippo pathway and is followed by a progressive activation, most likely sustained by LATS2 accumulation and the subsequent YAP Ser127-phosphorylation, preventing its nuclear translocation and TEAD-mediated transcriptional co-activation. It should be noted that cagAdeficient H. pylori isogenic mutants affected neither TEAD transcriptional activity nor LATS2 or YAP expressions (Fig. S2). This indicates that the activation of the Hippo pathway was CagA-dependent and thereby related to the oncogenic potential of the bacteria.

LATS2 and YAP positively regulate each other's expression in response to H. pylori

To further assess to what extent LATS2 and YAP are specifically involved in hGEC responses to H. pylori, we knocked down their expression using small interfering RNAs (respectively siLATS2 and siYAP) (Fig. 3).

Under basal conditions, siLATS2 successfully knocked down LATS2 expression (Fig. 3a,c) and activity as assessed by decreased YAP-P Ser127 and enhanced TEAD activity, reflected by the stimulations of both the TEAD reporter and CYR61 and CTGF expression (except CTGF in AGS, Fig. 3c). Conversely, transfecting a constitutively active LATS2 expression vector [10] inhibited TEAD transcriptional activity by 70% compared to the control pEGFP vector in AGS (Fig. 3b).

siYAP knocked down YAP1 expression (Fig. 3a,c) and inhibited TEAD activity in both hGECs, as expected (Fig. 3b). siYAP severely down-regulated CTGF and Cyr61 in AGS, but barely affected them in MKN74 (Fig. 3b-c), suggesting that these genes could be transcribed independently of YAP/TEAD. Conversely, transfection of vectors encoding either wild type YAP1 (pYAP) or the constitutively active, unphosphorylable YAP S127A mutant (pSer127YAP) stimulated TEAD activity in AGS by 3 and 6 fold, respectively. In MKN74, only pSer127YAP but not pYAP triggered the TEAD reporter under basal conditions (Fig. 3b), suggesting a tighter regulation of YAP1 in this cell line, likely by LATS2, which is expressed at a higher level in MKN74 than in AGS under basal conditions (Fig. 2b,e).

Unexpectedly siLATS2 not only decreased YAP-P Ser127 but also total YAP protein in both hGECs (Fig. 3a) and YAP1 mRNA in MKN74 (Fig. 3c). In turn, siYAP reciprocally downregulated LATS2 under basal condition (Fig. 3a,c). Accordingly, we also found a strict co-expression of YAP1 and LATS2 in other hGECs (Fig. S3).

Under infected conditions, siLATS2 maintained the TEAD activity at a high level compared to siControl infected cells, in which it was repressed compared to non-infected hGECs. To the contrary, siYAP further decreased it (Fig. 3d). These observations are corroborated by parallel changes in CTGF and CYR61 expression (except CYR61 in AGS, Fig. 3e). Upon infection, siLATS2 and siYAP not only efficiently prevented the respective LATS2 and YAP1 H. pylori-mediated up-regulations, but siLATS2 also impaired YAP upregulation and reciprocally (Fig. 3e).

Taken together, these data show that YAP1 is the major coactivator of TEAD-mediated transcription, and that it is prominently controlled by LATS2-mediated YAP1 inhibitory phosphorylation. Remarkably, LATS2

and YAP1 reciprocally positively regulate each other's expression reciprocally both under basal conditions and in response to H. pylori infection.

LATS2 restricts the EMT and CSC-like properties induced by H. pylori in hGECs

AGS morphology was not noticeably affected by either siLATS2 or siYAP under non-infected conditions (Fig. S4a). Nevertheless, LATS2 repression accelerated the acquisition of the EMT-characteristic "hummingbird" phenotype early as 2 hpi in siLATS2 cells, in contrast to 5 hpi in siControl or siYAP AGS cells (Fig. S4a). The remarkable feature occurring specifically in siLATS2-transfected MKN74 cells was the round cell clusters loosely attached to the epithelial layer upon infection (Fig. S4b) that appeared at a much higher number than in siControl MKN74 or siYAP and also correspond to a mesenchymal phenotype [11].

The induction of the mesenchymal phenotype observed in siLATS2 compared to siControl hGECs was associated with a ZEB1 up-regulation under both basal and infected conditions, along with Snail1 EMT inducer, in siLATS2 MKN74 (Fig. 4a). The nuclear accumulation of ZEB1 in siLATS2 hGECs was confirmed by immunofluorescence (Fig. 4b). ZEB1 is reciprocally linked to the miR-200 microRNA family members in a negative feedback loop [28]. The miR-200s are produced from two microRNA clusters, miR-200b-200a-429 and miR-200c-141, the promoters of which harbor ZEB1 binding sites and are repressed by ZEB1.

Interestingly, siLATS2 decreased the expression of miR-200b and -200c in AGS and prevented their H. pylori-mediated upregulation [11] in MKN74 (Fig. 4a). Thus, the decreased miR-200 levels in infected siLATS2 hGECs may contribute to an acceleration of their EMT upon infection. The exacerbated EMT phenotype in siLATS2-treated cells was further confirmed by increased invasive properties under both basal and infected conditions, compared to the siControl (Fig. 4c).

We previously reported that H. pylori-induced EMT promotes the emergence of cells with CSC-like properties, such as high CD44 expression and the ability to grow as spheroids under non-adherent culture conditions [15]. Accordingly, CD44 was upregulated at 24 hpi in siControl hGECs (Fig. 5a). The percentage of CD44+ cells which is <1% in MKN74 cells under basal conditions (contrarily to >60% in AGS cells [15]), increased by 4 times upon LATS2 knockdown and up to 23% at 24 hpi (Fig. 5b) (along with increased mean cell fluorescence intensities, data not shown). The hGECs' ability to grow as tumorspheres was significantly increased in siLATS2 AGS cells both under basal and infected conditions, but not in siLATS2 MKN74 cells, in which this CSC property was hampered despite the CD44 enhancement (Fig. 5c). The inability of siLATS2 MKN74 to form tumorspheres may be correlated to their strong upregulation of the cyclin-dependent kinase inhibitor p21/CDKN1A along with the downregulations of S-phase markers E2F1 and PCNA compared to siControls, which did not occur in AGS cells (Fig. S5).

Collectively these results support a restricting role of LATS2 in the acquisition of EMT/CSC-like phenotype and properties upon H. pylori infection.

6. LATS2 and YAP1 are heterogeneously expressed, nuclear and co-upregulated in H. pyloriinduced gastric adenocarcinoma in cancer patients.

We previously reported that GC are heterogeneous, composed of variably differentiated cells within the primary tumor, including a subpopulation of less than 30% CD44 + cells comprising the CSCs [17]. Here we show that LATS2 and YAP1 immunolabeling is heterogeneous among the GCs and among the cells within the tumors (Fig. 6a) as far as both expression level and subcellular localization are concerned. LATS2

expression was for the majority nuclear and positively correlated to nuclear YAP1 expression on GC tissue sections (Fig. 6b), thus corroborating the LATS2 and YAP1 co-expressions in the different hGECs in vitro (Fig. S3). The transcriptomic data of the 38 GC cell lines of the Cancer Cell Line Encyclopedia (CCLE) [29] also revealed a positive correlation between LATS2 and YAP1 expression (Fig. 6c). Interestingly the CYR61 YAP1/TEAD target gene expression is also strongly correlated to LATS2 expression (R=0.758, p<0.0001, Table S1), suggesting that LATS2 could also be considered as a YAP1/TEAD target gene. Moreover, the cells overexpressing nuclear YAP1 and LATS2 were found mainly at the periphery of the tumor foci, where also the CD44 + CSCs were also detected. However, CD44 and nuclear LATS2/YAP1 expressions were not correlated neither at the protein level in GC tissue sections nor at the transcriptomic level in the CCLE GC cell lines (Fig. 6c): this further argument is in favor of LATS2 repressing CD44 as depicted in Fig. 5b.

DISCUSSION

LATS and YAP1 constitute the downstream core kinase/effector tandem within the Hippo pathway, an evolutionary conserved signaling cascade regulating tissue growth and stem cell homeostasis, and which has never been studied in the framework of H. pylori infection so far. We previously reported that on one hand carcinogenic strains of H. pylori up-regulate the tumor suppressor LATS2 [10], and on the other hand express CSC-like features [15] in hGECs. However, the role of LATS2 and its oncogenic targets YAP1/TEAD remained to be elucidated in this context.

infected and infected conditions. Moroishi et al. reported that YAP1/TEAD directly induces lats2 transcription upon binding to its promoter [39]. LATS2 in turn inactivates YAP1 via Ser 127 -phosphorylation:

this negative feedback loop is likely to be responsible for the transient YAP1 activation in H. pylorichallenged hGECs in vitro. The YAP/TEAD-dependent transcription of LATS2 observed in the hGECs of this study is also supported by the strong LATS2/YAP1 and LATS2/CTGF-CYR61 correlations that we unveiled in the CCLE data base. This leads to the conclusion that LATS2 can be considered as a YAP1 target gene aimed, as a negative feedback loop, to tightly control but not completely silence YAP1/TEAD oncogenic transcriptional activity, maintaining by the way its own expression.

LATS2 is also involved in the cell cycle control in response to microtubule stress during which LATS2 binds and inhibits Mdm2, stabilizing p53 and thereby maintaining mitotic fidelity and genomic stability [40].

During this process, LATS2 moves from the cytoplasm to the nucleus, which is also observed in MKN74 as early as 2 hpi (Fig. 2e). According to its function in the mitotic apparatus, loss of LATS2 results in mitotic defects, which could become very critical for the more differentiated hGECs like MKN74 compared to AGS for survival under extreme growth conditions such as tumospheres. This is in relation with the upregulation of p21 and the downregulation of E2F1 and PCNA observed in siLATS2 MKN74 cells, and consequently to the decrease in tumorsphere formation (Fig. 2e).

Third, LATS2 knockdown exacerbating the EMT and CSC markers ZEB1, Snail1 and CD44 indicates that LATS2 restricts the EMT and migrating-CSC phenotype in hGECs, accordingly to its tumor suppressor function. Indeed, Hippo signaling is associated with stem cell maintenance and normal epithelial architecture, characterized by specialized cell-cell junctions [35][36]. Its dysfunction leads to increased nuclear and under-phosphorylated YAP1 that promoted oncogenic transformations due to TEAD activation, revealed here by CTGF and Cyr61 upregulation. Interestingly, these YAP1/TEAD targets involved in cancer progression are also ZEB1 targets [41]: ZEB1 mechanistically and functionally binds YAP1, promoting the expression of a common ZEB1/YAP1 target oncogene set. This could be the reason why CTGF or CYR61 transcription was not totally abolished upon siYAP treatment in MKN74 like in AGS: the remnant CTGF or Cyr61 levels after YAP1 downregulation may be attributable to ZEB1. Contrarily to AGS, carcinogenesis cascade, in H. pylori-associated gastritis and metaplasia as well as in GC, therefore constituting novel additional markers of H. pylori-triggered gastric stem cell expansion leading to CSCs and GC.

MATERIALS AND METHODS

Human gastric tissues. Gastric tissue samples (tumors and non-cancerous mucosa) from consenting patients (both sexes, 68-85 years old) undergoing gastrectomy for distant non-cardia adenocarcinoma were included in the study, in agreement with the tumor bank of the Bordeaux University Hospital (France), as previously reported [17][55] [15].

Bacterial culture. H. pylori strain 7.13 and isogenic cagA-deleted mutant were cultured as previously described [11][15]. The coculture experiments were performed at a multiplicity of infection of 50 following existing protocols [11][15].

Gastric epithelial cell culture. The AGS (ATCC CRL-1739) and MKN74 (HSRR Bank JCRB0255) cell lines were cultured in DMEM/F12-Glutamax or RPMI1640-Glutamax media, respectively, supplemented with 10% heat-inactivated fetal calf serum (all from Invitrogen), at 37°C in a 5% CO2 humidified atmosphere [11][15].

The experiments were performed with cells seeded at low density (2.5x10 4 cells/well in 24 well plates).

Transfection and reporter assays. Cells were grown in 24 well plates and transfection of plasmids and siRNA were performed using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) as recommended. Two rounds of siRNA transfection were performed. Previously validated nonsilencing siRNA (siCtrl, Dharmakon, Lafayette, CO, USA) [10][19] with no known homology to mammalian genes was used as a negative control and siRNA directed against human LATS2 (Qiagen) [10] and YAP1 (Qiagen) were used at 20 nM. The 8xGTII-Luciferase, pcDNA-Flag-Yap1, and pCMV-flagS127A-YAP vectors, used at 100 ng/well were gifts from Stefano Piccolo [56], Yosef Shaul [57] and Kunliang Guan [58], respectively. TAL-luciferase (100 ng/well) reporter was from 127 Immunocytofluorescence. 25,000 cells cultured on glass coverslips were fixed with 4% paraformaldehyde solution for 10 minutes and processed for immunofluorescent staining as described [19]. Expression and subcellular localization of LATS2, total YAP and YAP-PSer127 (labelled in green) assessed by immunofluorescence using the same antibodies used in western blot, at a 1:300 dilution and anti-rabbit Invasion assay. Cells were recovered by trypsinization and 5.10 3 cells per condition were placed in the upper side of a 8-mm pore size Transwell (Corning, NY, USA) insert, previously coated with rat-tail type 1 collagen (Becton Dickinson, Le Pont de Claix, France) as described elsewhere, in 24-well culture plates with medium containing 5% fetal bovine serum. After 18h of incubation at 37C, the Transwell inserts were fixed in cold methanol and processed for hematoxylin staining, as described previously [15]. Cells having migrated through the lower side of the inserts were counted on five different randomly chosen fields per insert under light microscopy using a 20x objective.

Flow cytometry. Cells were recovered using trypsine-EDTA and 10 5 cells per condition were stained with anti-CD44-APC antibody (1:25 each, BD Pharmingen, San Jose, CA, USA) in PBS, 0.5% bovine serum albumin, 2 mM EDTA solution. Flow cytometry was performed using a FACS Canto II instrument and DIVA software (both from Becton Dickinson, Le Pont de Claix, France). Results were based on the analysis of 10000 cells.

Tumorsphere assay. Cells were recovered and 500 cells were plated on non-adherent 96-well culture plates (coated with a 10% polyHEMA (Sigma, Saint-Quentin Fallavier, France) solution in absolute ethanol and dried overnight at 56°C). After plating, cells were incubated for 5 days at 37°C in a serum-free medium consisting of DMEM-F12 Glutamax supplemented with 20ng/ml of EGF, 10ng/ml of basic-FGF, 1:100 N2supplement 100x, 0.3% glucose, 5mg/ml of gentamicin, 50IU/ml of penicillin and 2.5mg/ml of amphotericin B (all from Invitrogen and Sigma) [17]. The number of spheroids per well was counted under light microscopy using a x10 objective. i.

Introduction

The Salvador-Warts-Hippo pathway is a highly evolutionarily conserved pathway, first described as a size-controlling pathway in D. melanogaster 1 . It is composed by a tumor suppressor module of two kinases: MST1/2 and LATS1/2, their regulatory proteins SAV1 and and MOB1/2 2 , and their targets of phosphorylation, the oncoproteins YAP and TAZ. Upon phosphorylation by LATS1/2, YAP and TAZ are retained in the cytoplasm or undergo proteasomal degradation, hence limiting their nuclear activity.

Active nuclear YAP and TAZ function together with TEAD family transcription factors to promote cell survival and proliferation 2 .

Stomach cancer is the fifth most common cancer overall and the third leading cause of cancer death in both sexes worldwide 3 . The diagnosis of the disease is often at advances stages, the therapeutic options are limited, and the 5-year survival is poor 4,5 . Gastric cancer is a multifactorial disease, frequently associated to the chronic infection with H. pylori 4 . Our group has shown that H. pylori induces an activation of the Hippo signaling pathway and the Hippo pathway plays a role in the emergence of a CD44 + cell population with CSC-properties induced upon infection. An increasing body of evidence links the Hippo pathway to gastric cancer. The expression of MST1/2 and LATS1 was decreased in tissue sections from gastric cancer compared to those of normal gastric mucosa 6 , and several studies have revealed an upregulation of YAP mRNA and protein in gastric adenocarcinomas, as well as a correlation of YAP expression and nuclear localization with a bad outcome 7 . Jiao et al 8 found an association of YAP mRNA levels with tumor size, differentiation, pathologic staging, and with H. pylori status, as well as higher YAP protein positivity rates in gastric cancer and dysplastic lesions compared to normal tissue. Promoterhypomethylation and over expression of TEAD4 have been described in gastric cancer tissues, and its knock down impaired cell growth both in vivo and in vitro 9 . Altogether, these studies point out the role of the Hippo signaling in gastric carcinogenesis.

It has been shown that knock-down of TEADs or disruption of the YAP-TEAD interaction decreased transcription of YAP target genes and oncogenic transformation 8 . Therefore, the ways to disrupt YAP/TAZ-TEAD interaction have been studied as potential strategies for cancer treatment. Verteporfin (VP, C41H42N4O8, molecular weight: 718.8 Da), is a benzoporphyrin derivative initially used as photosensitizer for photodynamic therapy (PDT) in ophtalmology 10 and tested in anti-cancer therapy 11,12 . Lightindependent activity of VP have also been reported. Liu-Chittenden et al identified verteporfin as an inhibitor of YAP/TEAD, with anti-oncogenic properties in vivo in liver cancer murine models 13 . VP inhibitory effects on CSC properties and enhancement of chemotherapy effectivity have been studied in esophageal cancer cells in vitro and in vivo 14,15 , and in a model colorectal carcinogenesis without affecting the normal intestinal homeostasis or displaying any apparent toxicity 16 . Until now, the anti-oncogenic properties of VP have not been described in gastric cancer.

The aim of this work is to study the YAP/TAZ activity in gastric cancer cell lines and patient-derived primary tumor xenografts and evaluate the capacity of verteporfin to disrupt the YAP/TAZ/TEAD transcriptional activity, target CSC and inhibit tumor growth.

ii.

Results

Hippo pathway proteins are heterogeneously expressed in gastric tumors

Immunohistochemistry staining was performed in tissue samples of two poorly differentiated and four moderately differentiated gastric adenocarcinomas (Figure 1). YAP expression was mainly nuclear and the percentage of positive cells varied from 10 to 30% amongst the tumors. The YAP positive cells were often located in the invasive front of the tumors, where CSC have been described to reside.

Expression of YAP, TAZ and LATS2 mRNAs (Suppl figure 1a) and proteins (Suppl figure 1b) were evaluated in gastric cancer cell lines, healthy stomach tissue, and gastric cancer samples. YAP was detected in all samples excepting MKN45 cells, in variable levels. TAZ expression was also heterogenous but much lower than that of YAP, MKN45 cells presented the highest levels and it was absent from MKN74 and the healthy tissues. Regarding LATS2, the expression was heterogenous in the gastric cancer samples, decreased in the cell lines and higher in the healthy tissues, remarkably in the antrum.

Verteporfin inhibited cell proliferation, and YAP/TAZ/TEAD transcriptional activity in gastric cancer cell lines in vitro

MKN45 and MKN74 cell lines were treated with increasing concentrations of verteporfin and DMSO as control vehicule. In both cell lines, a significant decrease in cell growth was observed from 1 µM in a dose dependent manner (Figure 2a), without an evident alteration of cell morphology (Figure 2b). The inhibition of cell proliferation occurred along a cell cycle arrest, with a significant accumulation of cells in G0/G1 phase, and decrease of cells in phase S at the 5 µM concentration (Figure 2c-d). YAP/TAZ/TEAD transcriptional activity was studied with a TEAD-luciferase reporter and the mRNA levels of the YAP/TAZ/TEAD target genes CTGF, CYR61, IGFBP3, JAGGED1, and LATS2 (Figure 2e-f). TEAD-luciferase activity was significantly decreased from the 0.1 µM dose of verteporfin in MKN45 cells, which also showed a decreased expression of all the studied YAP/TAZ/TEAD target genes in a dose-dependent manner from 0.1 µM. In MKN74, a dose-dependent, decreasing trend of TEAD-luciferase activity from 0.1 µM, but significant only at the 5 µM concentration of verteporfin. The case was the same for the expression of LATS2 in these cells, while the other target genes showed a significantly decreased expression from the 1 µM concentration. MKN45 cells do not express YAP but they express TAZ and MKN74 cells express mainly YAP. The expressions of TAZ in MKN45 and YAP in MKN74 were decreased in the condition treated with the 5 µM dose (Figure 2f).

Verteporfin treatment targeted CSC-properties in vitro

In order to evaluate the CSC-targeting properties of verteporfin in vitro, we cultured gastric cancer cell lines and PDXs in non-adherent, nutrient defined conditions to promote tumorsphere formation and enrich the culture in CSCs and treated them with increasing doses of verteporfin. For the preventive treatment (Figure 3a), i.e. cells seeded in presence of verteporfin, a significant inhibitory effect in the formation of tumorspheres was observed in MKN45, GC04 and GC10 from the lowest concentration in comparison to the DMSO-treated control cells. The treatment was effective from 1 µM in the MKN74 and GC07, and no effect was observed for the GC06. The action of verteporfin was observed both in the total tumorsphere count (Figure 3a) and size (illustrated in Figure 3b). We also tested the curative effect of verteporfin, i.e. the treatment was added to well established tumorspheres (Figure 3c). Significant reduction on the tumorsphere counts were observed from concentration 0.1 µM in MKN74, GC04, and GC07, while a 1 µM concentration was required to observe the inhibitory effects in MKN45, GC10, GC35, and remarkably, GC06.

CD44 CSC-marker expression upon verteporfin treatment was studied by RT qPCR in MKN45, MKN74, GC04, GC06, and GC07 (Figure 4a). CD44 expression was significantly decreased in a dose dependent manner in MKN74 and GC04 from the lowest concentration, and for MKN45, GC06, and GC07, a concentration of 1 µM was required to observe the desired effect. The immunocytochemistry evaluation of CD44 in spheres from MKN45 confirmed the decrease on CD44 expression upon treatment with verteporfin 1 µM (Figure 4b).

Verteporfin treatment inhibits tumor growth in a murine xenograft model

To evaluate the antitumor activity of verteporfin, xenograft experiments were performed, where MKN45, MKN74, and GC10 cells were subcutaneously injected into the dorsal flank of immunodeficient NSG mice and tumor growth was determined by measuring tumor growth every other day. Once the tumor had reached a volume of 100 mm 3 , daily peritumoral treatment with a dose of 2 mg/kilogram of verteporfin and DMSO as a carrier vehicle for the control group was performed. For the three cell types, tumor growth slowed down in the group treated with verteporfin (Figure 5a). The strongest and fastest effect was observed in MKN45, where significant differences appeared as early as 6 days after the beginning of treatment. For MKN74 and GC10, the treatment took 25-30 days to cause and observable decrease in the tumor growth rate.

When the tumors in the control group reached a volume of 500 mm 3 , treatment was stop, mice were sacrificed and residual tumors were extracted (Figure 5b), weighted and processed for cellular dissociation and histology. A significant decreased on tumor weight was observed between controls and verteporfin-treated tumors in MKN45 and GC10, but not for MKN74 (Figure 5c). Noteworthily, an important heterogenicity was observed amongst the different tumors of the same group, in some cases pointing out a problem of distribution of the drug. Cells dissociated from the remaining treated tumors significantly initiated less tumorspheres in vitro in the verteporfin treated group compared with the control in MKN45 and MKN74, confirming that verteporfin treatment led to a decrease in the number of CSC in vivo (Figure 5d). That was not the case for GC10 where the number of tumorspheres was already low in the control condition.

The histological analysis of the residual tumors in MKN45 cells showed that after the verteporfin treatment, the tumor mass was decreased to smaller foci of cells in fibrotic tissue, compared to a more uniform mass observed in the control group. Regarding the expression of CSC markers, a slight decrease on the intensity of the CD44 staining was observed upon treatment, as well as in the percentage of cells strongly expressing ALDH staining. Proliferating cells expressing DNA replication marker Ki67 were also strongly reduced. These results support the in vitro results showing that verteporfin treatment targets CSCs, reducing proliferation and survival of tumor cells, reflected in an arrest of tumor growth.

iii.

Discussion

The Hippo signaling pathway controls several processes related to tumorigenesis, which define the features of cancer cells such as cell proliferation and survival. Deregulation of the pathway occurs at multiple levels. LATS2 can activate p53 and its pro-apoptotic activity can be antagonized by YAP 17,18 and silencing of LATS2 expression enables tumor development and progression 19 . YAP/TAZ transcriptional activity stimulates aberrant cell proliferation also by counteracting tumor suppressor mechanisms such as senescence 20 . YAP over expression can promote cell survival by blocking TNF-and FAS-induced apoptosis in the mouse liver 20 and a TEAD over expression has been involved in carcinogenesis and described in various types of cancer 21 . CTGF and CYR61, the most important YAP/TAZ/TEAD target genes, are regulatory ECM-associated proteins involved in growth promotion, cell migration, proliferation, cell lineage commitment and tissue specification, and participate in embryonic development, tissue turnover, and injury repair 22 . Their over expression has been associated with inflammatory conditions, such as metabolic syndrome, colitis, rheumatoid arthritis, wound healing and fibroproliferative disorders, being correlated to excessive accumulation of ECM proteins in fibrotic tissues, and remarkably, with cancer 22 . In hepatocellular carcinoma, CTGF was over expressed and responsible of increased cell growth and dedifferentiation, expression of inflammation-and carcinogenesis-related genes, and resistance to doxorubicin 23 .

The Hippo pathway can also control stem cell properties, including replicative potential, dedifferentiation, and drug resistance, and its deregulation has been implied in cell transformation and tumor initiation, especially in the acquisition of mesenchymal properties at the origin of CSCs 24 . Increased YAP/TEAD expression and activity has been described in various types of stem cells and stem cell compartments of different tissues, and drives the expansion of progenitor cells in the gastrointestinal tract 20 . TAZ is essential to metastatic and stem cells properties in breast cancer cell models, where it is required to sustain CSC-properties and confers self-renewal capacity 24 Given the importance of the role of YAP/TAZ/TEAD signaling in gastric carcinogenesis and CSC properties, this study evaluated the anti-tumor properties of verteporfin, showing that treatment with verteporfin decreased cell growth, YAP/TEAD transcriptional activity, and tumorsphere formation in vitro and tumor growth and CSC-properties and markers in vivo. These results show the CSC-inhibitory activity of verteporfin both in vitro and in vivo. We corroborated the results obtained by various groups in models of liver, colorectal, and esophageal carcinogenesis [13][14][15][16] , and to our knowledge, this is the first description of the anti-tumor activity of verteporfin in gastric cancer. Other mechanisms of tumor growth inhibition have been described for verteporfin 26,27 , which could also have a role in the effect found in our model.

However, even if they can contribute to the effect, our results confirm a role for the YAP/TEAD disrupting action of verteporfin, reflected in the decrease of YAP/TEAD target genes expression.

Verteporfin has are a selectivity for cancerous cells with high YAP/TEAD activity and it is an FDAapproved molecule for the routine treatment of other pathologies, making it suitable for a potential clinical application. Despite the observed significant action of verteporfin treatment in tumor reduction, the insolubility of the molecule limits its bioavailability and the possibility of reaching the adequate concentration in the tumor when administered in a systemic way, unless using very high quantities of the product which could carry systemic toxicity and increase the financial burden of the treatment. As an alternative, we used a peritumoral injection, which proved to fail in the homogeneous distribution of verteporfin around the tumor and in some cases, caused the tumor to escape the treatment, invading towards the opposite side. However, the positive results obtained despite the flaws of the model, open the possibility of exploring new alternatives for verteporfin formulation that make it more suitable for implementing the treatment in patients. Targeting the YAP/TAZ/TEAD transcriptional activity was shown to be efficient in preventing tumor growth in a PDX model, therefore highlighting the potential of its inhibition to be implemented in gastric cancer therapy.

iv.

Materials and Methods

Human gastric tissues. Gastric tissue samples (tumors and non-cancerous mucosa) from consenting patients (both sexes, 68-85 years old) undergoing gastrectomy for distant non-cardia adenocarcinoma were included in the study, in agreement with the tumor bank of the Bordeaux University Hospital (France), as previously reported 28,29 . Histology and immunohistochemistry staining. Tissue sections 3 µm thickness were prepared from formalin-fixed paraffin-embedded tissues (PET) and submitted to standard Hematoxylin/Eosin/Safran (HES) staining (all from Sigma) and immunohistochemistry protocols 28,29 . Primary mouse monoclonal antihuman CD44 antibodies 1:100 (G44-26, BD) were incubated for 30 min at room temperature, followed by 30 min incubation at room temperature with anti-mouse Labelled Polymer-HRP DAKO Envision Systems (DAKO). Immunolabeling was revealed by 10 min incubation in liquid substrate-diaminobenzidinechromogen (DAKO). Slides were counterstained with hematoxylin, dehydrated and mounted with Eukittmounting medium (Labonord, Templemars, France). Images of the staining were recorded on tissue sections using light microscopy and a x20 objective.

Ethic

Gastric epithelial cell lines culture and patient-derived tumor xenografts. MKN45 and MKN74 (HSRR Bank JCRB0255 and JCRB0255) cell lines authenticated by STR profiling and mycoplasma free (verified by PCR), were cultured in RPMI1640-Glutamax media supplemented with 10% heat-inactivated fetal calf serum (all from Invitrogen), at 37°C in a 5% CO2 humidified atmosphere 30 . GC04, GC06, GC07, GC10, and GC35 are patient-derived GC, which were successfully established by serial transplantation in NSG mice as previously described 30 . 24 hour cultures of gastric cancer cell lines were treated with verteporfin (SML0534, Sigma) diluted in DMSO (D2650, Sigma) or DMSO as control for 48 hours and then harvested for cell proliferation count, cell cycle and luciferase reports assays, and RNA extraction.

Cell cycle analysis by flow cytometry. Treated cells were dissociated by trypsination and 100 000 cells per condition were stained with propidium iodide (P4864, Sigma) as describe elsewhere. Flow cytometry was performed using a BD FACSCanto II instrument and DIVA software (BD).

Transfection and luciferase-reporter assay. Cells were grown in 24 well plates and transfection of plasmids were performed using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) as recommended. The 8xGTII-Luciferase reporter (100 ng/well) was a gift from Stefano Piccolo 31 , the TALluciferase reporter (100 ng/well) was from BectonDickinson, and pRL-SV40 (10 ng/well) was from Promega. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Assay (Promega). Firefly luciferase activities were normalized for transfection efficiency by Renilla luciferase.

RNA extraction and RT-qPCR. Cell RNAs were extracted using Trizol reagent (Invitrogen) and quantified by their absorbance at 260 nm. For RT-qPCR, retrotranscription was performed with 1 µg total RNA using the Quantitect Reverse Transcription kit (Qiagen). Quantitative-PCR was performed using the SYBR-qPCR-Premix Ex-Taq (TAKARA), 0.3 µM specific primers (Table S1) and 1:200 of the RT reaction volume. Relative expressions were calculated using the comparative Ct method with HRPT1 and TBP as normalizers.

Western blotting. Cells were lyzed on ice in ProteoJETTM reagent (Fermentas) supplemented with protease/phosphatase inhibitor cocktail (Sigma). The extracted proteins were submitted to SDS-PAGE and western blotting for immunolabeling. Immunolabelling was performed using rabbit anti-YAP or mouse anti-TAZ (each at 1:2000; Cell Signaling), rabbit anti-LATS2 (1:2000; Bethyl), mouse anti-α-tubulin (1:8000; Sigma-Aldrich), Horseradish Peroxydase-coupled anti-rabbit and anti-mouse secondary antibodies (DAKO)

and chemoluminescent detection (ECL+,Amersham).

Tumorsphere culture. 200 cells were seeded in non-adherent 96-well culture plates (previously coated with a 10% poly-2-hydroxyethyl methacrylate (polyHEMA, Sigma) 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, 50 IU/ml of penicillin and 50 μg/ml of streptomycin (from Invitrogen and Sigma) and cultured at 37 °C in a humidified 5% CO2 atmosphere. For preventive treatment, verteporfin or DMSO were added to this medium in the desired concentrations before seeding the cells and spheres were quantified 7 days after seeding. For the curative treatment, tumorspheres were grown and then treatment was added, and tumorspheres were counted 48 hours after treatment. Tumorsphere counts were performed using a Nikon TE2000 inverted light microscope using a × 10 objective (Nikon, Champigny sur Marne, France).

In vivo xenograft experiments. In all, 100 000 cells from MKN45 and MKN74 cell lines, and GC10 were suspended in 100 μl of 7 mg/ ml ice-cold Matrigel (BD Biosciences) and subcutaneously injected into the dorsal flank of randomly distributed 7-week-old NSG female mice. Tumors were measured blindly twice a week using a caliper and tumor volumes were calculated 30 . When tumor volume reached 100 mm 3 , 50 μl of DMSO (control group) or verteporfin solution per mouse was subcutaneously injected at the tumor periphery daily for 15-25 days, after which mice were sacrificed (when tumors reached 500 mm3). Tumors were collected, fixed in a 3.7% buffered-formaldehyde solution and embedded in paraffin following standard procedures as described 30 .

Statistical

a. The LATS2/YAP1/TEAD axis controls EMT and CSC-properties

A co-upregulation of LATS2 and YAP1 was found in tissues sections from H. pylori infected patients compared to non-infected tissue samples, and this increase was maintained in gastritis and metaplasia. Our findings confirm the overexpression of YAP1 in association with malignant transformation and its role as a stemness factor. However, we did not find the inverse correlation between LATS and YAP1 usually described in the literature, but a positive correlation between the two proteins instead, along with a colocalization with CD44, that is, in the stem cell compartment of the gastric mucosa.

The in vivo findings led us to the study of the molecular regulation of the Hippo pathway in gastric epithelial cell lines challenged with H. pylori. We found that H. pylori infection modulates the Hippo pathway in biphasic pattern, where YAP1 transcriptional activity is stimulated transiently during the early stage of infection and then this activity is controlled by an activation of LATS2. LATS2 phosphorylates YAP1 inducing its exclusion from the nucleus and cytoplasmic retention and degradation, therefore blocking its co-transcriptional activity (figure 17). The modulation of the Hippo pathway by H. pylori infection is CagA dependent, confirming that this pathway may play a role in the carcinogenic process induced by infection.

Repeated and chronic aggression of the gastric epithelium by H. pylori in vivo could lead to constant waves of YAP1/TEAD activation and LATS2 induction, resulting, as observed, in a co-overexpression of both and the nuclear localization of YAP1 reflecting its co-transcriptional activity.

One of the key findings of our study is the reciprocal upregulation of YAP1 and LATS2 in gastric epithelial cells in both infected and non-infected conditions, as well as the identification of LATS2 as a potential YAP1 target gene. It has been reported that YAP1/TEAD directly induces lats2 transcription upon binding to its promoter, and in turn, LATS2 inactivates YAP1 constituting a negative feedback loop.

Supporting our findings, LATS2 was correlated with CYR61 and CTGF in the CCLE database. CYR61 and CTGF are the main YAP1/TEAD target genes and their expression reflects the transcriptional activity of the complex. LATS2 knockdown with siRNA was found to exacerbate the EMT process and the CSC-properties induced by H. pylori in gastric epithelial cells, due to a failure to control YAP1/TEAD transcriptional activity evidenced by accumulation of nuclear, non-phosphorylated YAP1. The co-overexpression of LATS2/YAP1 appears at all stages of the gastric carcinogenesis cascade, in H. pylori-associated gastritis and metaplasia, and in GC. Therefore it constitutes a novel additional biomarker of H. pylori-triggered gastric stem cell expansion leading to CSC and GC. There is a first phase of early induction of YAP1/TEAD transcriptional activity, which is then repressed in a second phase by an activation of LATS2. The YAP1/TEAD transcriptional activity seems to be at last partially responsible for the induction of EMT and CSC-properties observed upon H. pylori infection of humans gastric epithelial cells. We identified LATS2 as a potential YAP1 target gene, which constitutes a negative feedback loop limiting YAP1 activity.

i.

What's next?

Is LATS2 linked with pro-inflammatory signaling pathways?

NF-B activation is a known major inducer of EMT, and also an effect of H. pylori infection. In the fruit fly, a recent study by Liu et al 488 described a role of Wts (LATS homologue) and Yki (YAP1 response and to get deeper insights in the mechanisms involved, a pilot transcriptomic study on the MKN74 cell line was performed, and various functional groups of genes were upregulated, most of them associated with inflammation or immune response (Figure 19). One of the categories that caught our attention was the NOD-like receptor (NRL) signaling pathway, which presented a four-fold increase. The NLR-signaling is activated by different pathogen-associated molecular patterns such as bacterial peptidoglycan and simple-stranded RNA virus, and its activation stimulates NF-B transcriptional activity as well as other pro-inflammatory transcription factors such as AP-1 and the Interferon-responsive elements (IRFs). Individual evaluation of the expression of genes associated with these pathway shows at least 17 genes upregulated upon inhibition of LATS2 in the context of H. pylori infection, including cytokines, chemokines, and regulators of NF-B activity. Interestingly, members of the MAPK family were also significantly increased (Figure 20). perspective of research that will be developed in our group in a near future.

Is the Hippo pathway activation status correlated with the histological subtype of gastric carcinomas?

Here we reported a positive correlation between LATS2 expression nuclear YAP1 expressions on GC tissue sections. However, when the cases were separated according to the histological classification of the tumor in intestinal and diffuse, a positive trend was observed in the intestinal category but no significant correlation was found. This is probably due to the small number of cases in each group after stratification in smaller categories. Nevertheless, it may be worth to explore if such relation exists, not only to further elucidate the molecular mechanism of regulation of the Hippo pathway in each scenario, but also to increase the value of LATS2 and YAP1 expression and localization as a biomarker for gastric cancer. In order to carry on this study, our group is currently working in the characterization of LATS2 and YAP1 expression in tissue microarrays containing samples of 178 cases of gastric cancer fully characterized for clinic, pathologic, and epidemiologic features and paired non-cancerous distant gastric mucosa (healthy tissue, gastritis, metaplasia, and dysplasia). This should allow us to determine from which lesion of the Correa cascade the markers are overexpressed and to establish them as a potential biomarker of early stages in the carcinogenic sequence.

Another possibility to approach this question lays in the use of in silico databases, such as the Cancer Cell Line Encyclopedia (CCLE) or The Cancer Genome Atlas (TCGA). In the present study, the 38 gastric cancer cell lines present in the CCLE were analyzed for the expression levels and correlation of LATS2, YAP1, CTGF, CYR61, and CD44. However, when we stratified the cell lines according to the histological type, the scenario was exactly the same we found with the tissue samples. The evaluation of the Hippo pathway genes in the TCGA database needs to be done, maybe resulting in interesting data, as a validation of YAP1 and LATS2 as biomarkers of gastric cancer, and also in the exploration of potential relations between their expression and the histological or molecular subtypes of gastric cancer.

b. Verteporfin targeting of the YAP1/TAZ/TEAD transcriptional program inhibits CSC-properties and tumor growth

Guided by the results highlighting the role of YAP1/TEAD signaling in gastric carcinogenesis and CSC properties, the second part of the study consisted in the evaluation of anti-tumor properties of verteporfin, which disrupts the YAP1/TEAD interaction 482 , inhibiting its transcriptional activity. We showed that treatment with verteporfin decreased cell growth, YAP1/TEAD transcriptional activity, and shown to possess antitumor properties in preclinical assays in hematological malignancies 491 and also the capacity of suppressing the proliferation of cells dependent on β-catenin signaling, by inhibiting YES1 and the YAP1/β-catenin/TBX5 transcriptional complex 492 . However, the in vitro results in gastric cancer cell lines were less marked than those showed by verteporfin, with a broader theoretical mechanism of action more indirectly associated with YAP1/TEAD activity, hence we decide not to pursue this line of research but it remains a possibility to explore in the future.

As mentioned before, other groups have addressed the subject, notably in the study by Jiao et al 474 where an inhibitor peptide termed "Super-TDU", targeting YAP1/TEAD4 interaction, was used successfully both in vitro and in vivo.

ii. What's next?

TAZ role in gastric cancer

For these work, we were focused on YAP1 which is the most studied of the two oncogenes in the cancer field. However, there is also TAZ, which has never been studied in the context of gastric cancer.

To study the regulation of the Hippo pathway by H. pylori infection, two cell lines were used in this work, AGS and MKN74. In these cell lines, YAP1 predominates and TAZ is expressed in a low level or not at all, therefore they do not constitute an appropriate model for the study of TAZ. However, we have identified two other gastric cancer cell lines, MKN45 and NCI-N87, with a high and intermediate expression of TAZ and a less important role of YAP1, which constitute an adequate model to study TAZ. Despite the fact that they are normally treated as identical, preliminary results indicate that the kinetics of activation in response to H. pylori maybe different between YAP1 and TAZ, but it seems that the effect of its inhibition on CSC-properties are similar. Further studies on the subject are been carried on by our group to shed more light on TAZ regulation during gastric carcinogenesis.

Hippo pathway status in response to ATRA treatment

Nguyen et al 158 recently described an antitumor and anti-CSC activity of all-trans retinoic acid (ATRA) in gastric cancer. The results are similar to those observed with verteporfin in this work, i.e. an inhibition of cell proliferation with a cell cycle arrest in G0/G1 and a decrease of tumorsphere formation in vitro, as well as an impairment of tumor growth in vivo, even if the efficacy of verteporfin seems to be slightly lower. Given the availability of biological material from the ATRA research, the evaluation of the response of the Hippo pathway to ATRA treatment would be of great interest in the cells of the residual tumors and more so, in the relapsing tumors. The relapsing tumors originate from residual CSC that where

I

  . Introduction ......................................................................................................................................... a. Gastric cancer ..................................................................................................................................

  ..................................................................................................... Table 3. Hereditary conditions associated with gastric cancer ................................................................... Table 4. Markers of EMT ............................................................................................................................ Table 5. Comparative anatomy and histology of the murine and human stomach................................... Table 6. Genetically altered murine model of gastric cancer..................................................................... Table 7. Commercially available gastric epithelial cell lines ....................................................................... Table 8. Markers of gastric stem cells identified by lineage tracing .......................................................... Table 9. CSC markers in hematological malignancies and solid tumors ....................................................

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 12345 Figure 1. Key features of gastric cancer subtypes according to molecular classification.…….………… Figure 2. Hereditary gastric tumors………………………………………………………………………………………….…… Figure 3. Xenograft murine models……………………………………………………………………………………….……… Figure 4. Architecture of the gastric glands and localization of stem/progenitor cells of the stomach.……..................................................................................................................................…… Figure 5. Models explaining the heterogeneity of tumor cells……………………………………………………….

Figure 6 .Figure 7 .

 67 Figure 6. Unified model of clonal evolution and cancer stem cells…………………………………………………Figure 7. Unified model for gastric cancer stem cells……………………………………………………………….…… Figure 8 CD44 gene and protein structure …………………………………………………………………………………….

Figure 9 .

 9 Figure 9. cagPAI T4SS structure and CagA effects on the host cells……………………..………………………… Figure 10. Hippo pathway……………………………………………………………………………………………………………… Figure 11. Cell polarity systems involved with Hippo regulation…………………………………………………….

Figure 12 .Figure 13 .

 1213 Figure 12. Structure of LATS1 and LATS2……………………………………………….……………………………………… Figure 13. Structure of YAP and TAZ……………………………………………………………………………………………… Figure 14 Features of cancer cells controlled by the Hippo pathway …………………………………………….

Figure 15 .Figure 16 .

 1516 Figure 15. YAP1/TAZ in human tumors471……………………………………………………………………….…………… Figure 16. Chemical structure of verteporfin…………………………………………………………………..…………….

Figure 17 .Figure 18 .Figure 19 .

 171819 Figure 17. Hippo pathway regulation by H. pylori infection.……………………………………………..…………… Figure 18. Regulation of anti-microbial peptide response in Drosophila……………………………………… Figure 19. Functional categories enriched upon LATS2 inhibition in MKN74 cells………………………….

Figure 20 NLR

 20 Figure 20 NLR signaling pathway associated genes differentially expressed upon LATS2 inhibition……………………………………………………………………………………………………………………………………….

2 .

 2 As mentioned before, H. pylori infection is a major risk factor for the development of cancer and the bacterial virulence factors and pathological mechanisms involved are described in detail in chapter 3.

Figure 2 .

 2 Figure 2. Hereditary gastric tumors. Germline mutations can cause hereditary gastric cancer. HDGC, hereditary diffuse gastric cancer; HNPCC, hereditary non-polyposis colorectal cancer; PPAP, polymerase proofreading-associated polyposis; FAP, familial adenomatous polyposis.

  infection and serum pepsinogens149 . The combined results place the patient in groups of low and high risk of gastric cancer, and those of the high-risk group are recommended to receive endoscopy. From 2011 to 2013 this program examined the blood of 17 000 patients, of which 3000 underwent endoscopic examination and 65 cases of gastric cancer were detected, 80% in early stages. The ABC program was established as an opportunistic screening, contrary to the organized campaigns of screening by the X-ray methods, and given the results, it could be a useful and cheaper tool to perform mass screening in high risk populations. Alternatively, a kit for serum biomarkers known as GastroPanel was developed in Finland in the late 1990's, and includes the detection of H. pylori status, serum PG-I and PG-II, and gastrin-17. A meta-analysis of 27 studies in different populations showed a sensitivities of 54,8% and 70.4% , and specificities of 76% and 92.4%, for atrophic gastritis of the antrum and the corpus, respectively 150 .

  et al showed all-trans retinoic acid (ATRA) has an inhibitory effect on gastric CSCs, making it a potential adjuvant for the treatment of gastric malignancies 158 . Part of the present work intends to characterize the role of the Hippo/YAP1 pathway in gastric CSCs to test if and how the inhibition of this pathway could contribute to the impairment of the carcinogenesis process in the stomach, aiming to the development of adjuvant therapies to gastric cancer.

Figure 3 .

 3 Figure 3. Xenograft murine models 161 . Cell lines or patient explants can be grafted subcutaneously or orthotopically in immunodeficient mice.

Figure 4 .

 4 Figure 4. Architecture of the gastric glands and localization of stem/progenitor cells of the stomach. (a) Fundic/corpus gland. (b) Antral/pyloric gastric gland. Stem/progenitor markers are indicated in bold.

Figure 5 .

 5 Figure 5. Models explaining the heterogeneity of tumor cells 179 . The stochastic model states all the cells within a tumor have the potential to regenerate a tumor, but it is a low probability stochastic event. Alternatively, the hierarchical (cancer stem cell) model postulates that a tumor possess a subset of CSCs with the unique capacity of initiating and maintaining tumor growth.

Figure 6 .

 6 Figure 6. Unified model of clonal evolution and cancer stem cells 180 . The acquisition of favorable mutations can result in clonal expansion of the founder cell. In parallel, another cell may also gain a favorable mutation and form a new subclone. Mutations can accumulate and subclones can evolve in parallel. CSCs are not static entities but can evolve as genetic changes influence CSC frequency and selfrenewal potential.

  phenotype, and the high diversity of driver mutations are features present in gastric cancer that support a model combining the stochastic and hierarchical theories, in which primitive GCSCs continually optimize their genome, improve the pattern of gene expression, and generate various subclones to adapt to the changing tumor microenvironment181 . According to this model, the most primitive GCSCs are the result of the accumulation of cancer-related mutations in stem cells derived from epithelium, mesenchyme, or dedifferentiated mature cells, and these cells gradually acquire the properties of unlimited self-renewal and multilineage differentiation, creating the hierarchical structure of the tumor. At the same time, the GCSCs evolve into different subclones, and the dominant ones generate the respective hierarchical organization, while the less fit subclones disappear. Multiple factors such as the CSC niche, epigenetic alterations, dysregulations of signaling pathways, and tumor microenvironment are proposed to have a role driving the evolution of the GCCSs.

Figure 7 .

 7 Figure 7. Unified model for gastric cancer stem cells 181 . GC stem cells originate from gastric stem cells, dedifferentiated epithelial cells or bone marrow-derived cells (A), and the accumulation of cancerassociated mutations and chromosomal aberrations in the initiating cells, induced by multiple factors, promotes the lesions leading to gastric adenocarcinoma (B). The formation, invasion and metastasis of GC is dynamically regulated by epigenetic, genetic, immune, and microenvironment-related determinants.

  third possible origin of GCSCs are dedifferentiated epithelial cells. By studying the chronology of cytokeratin-7 (CK7) expression the precancerous cascade in the Mongolian gerbil model, highly expressing CK7 dedifferentiated cells were associated to metaplastic and neoplastic changes in the precancerous sequence. These cells exhibited a low proliferation and beta-catenin accumulations, like stem cells, suggesting that dedifferentiation of cells may be associated to stemness in gastric cancer 196 . Transdifferentiation of mature chief cells was demonstrated to be at the origin of drug-and H. felisinduced metaplasia in a mice model, using lineage tracing techniques 197 . The dedifferentiation and acquisition of stemness properties was directly linked to inflammation, as it was not present in the metaplasia model induced in the absence of inflammatory mediators with the DMP-777 drug in contrast to the induction with L-635 and H. felis infection. Further evidence of the role of dedifferentiation of gastric epithelial cells in the emergence of GCSCs was provided by Bessede et al, in a model of gastric epithelial cell lines infected with H. pylori, where dedifferentiation of cells by EMT was shown to be at the origin of a population of cells with GCSCs properties 131 .

  CD44 also binds other molecules, ECM-related (collagen, fibronectin, laminin, and chondroitin sulfate) or not (serglycin, osteopontin, and mucosal addressin)200 . It mediates cell-cell and cell-ECM interaction, cell traffic and homing, uptake and intracellular degradation of HA, and signal transmission involved in hematopoiesis and apoptosis.It is encoded by the homonymous gene, located in chromosome 11 in humans 199 . The CD44 gene has 20 exons, and alternative splicing gives origin to various isoforms or variants, allowing a high heterogeneity of the protein. The cytoplasmic and the transmembrane portions of the protein are coded by the exons 18-20, while the remaining exons represent the extracellular domains 201 . The exons 1 to 5 and 16 to 20 are always present, hence they are considered as the constant exons and the resulting protein is known as the standard form (CD44s, 85 kDa). Exons from 6 to 15 are variably present in the variant forms (CD44v) 201 (Figure8). Several combinations exist, 12 of which are commonly found, and their distribution is variable amongst tissues 202 . While the CD44s is highly expressed in hematopoietic cells, the variants are more restricted. For example, the epithelial isoform or CD44E, includes exons 8 to 10, and the isoform found in keratinocytes includes exons 3 to 10. Other combinations have been found to be even more restricted to malignant cells in different types of epithelial cancers, so the study of CD44 isoforms and their use as specific markers for cancer stem cells is a very active and promising field of research. Additional variability is present due to different patterns of N-glycosylation, O-glycosylation, and glycosaminoglycanation 200 .

Figure 8 .

 8 Figure 8. CD44 gene and protein structure 123 . CD44 consists of several exons, some of which are constant region exons that are used in every CD44 mRNA and protein (green bars) and others are variant exons (red bars) that are used in the CD44 variant proteins and are selected by alternative splicing (a). Examples of alternatively spliced CD44 proteins (b). CD44 protein is composed of an extracellular link domain, a stalk-like region in the extracellular domain close to the transmembrane region, where the variant exon products (red) are inserted, the transmembrane region and the cytoplasmic tail. There are multiple sites for N-glycosylation (brown circles), O-glycosylation (orange circles), and glycosaminoglycan-binding sites (yellow circles)

  c. Aldehyde dehydrogenase (ALDH) activity assayAldehyde dehydrogenase catalyzes the oxidation of retinaldehyde to retinoic acid, which regulates gene expression in complex with the RAR/RXR receptors. Normal and cancer SC show a high ALDH activity, which is thought to have a key role in protecting the cells from oxidative stress and cytotoxic agents. In a flow cytometry-based assay, a substrate of ALDH, BODIPY-aminoacetaldehyde (BAAA), is added to the cells and then converted by the ALDH in BODIPY-aminoacetate (BAA-), which is fluorescent and allows the cells to be classified as ALDH high (SCs) or ALDH low . The presence of ALDH can also be detected by immunohistochemistry in tumor tissues. The ALDH activity can be combined with cell surface markers, such as CD44, in flow cytometry to have a more specific identification of CSCs. 206d. Isolation based on marker expressionAs detailed above, numerous cell surface proteins have been identified as markers for CSCs in different types of cancer. Based on the differential expression of these markers between the CSC and the other cells in the tumor, CSCs can be isolated using fluoresce-or magnetic-activated cell sorting platforms (FACS and MACS, respectively). The use of FACS has the possibility of performing staining with different fluorochromes, and hence, the sorting of cells based in expression of multiple markers. The determination of phenotypes or combinations of surface markers increases the specificity of the selection and enhances the isolation of CSCs. In the case of MACS, the antibodies directed against the surface markers are coupled to micro-beads, which only allows the separation based on one marker.

  animals and the bacterial reaction to Salvarsan and other compounds208 . Other investigation showed the presence of a urease activity in the stomach of carnivorous animals209 and humans, which was sensitive to antibiotics and absence in gnotobiotic animals207 . It was finally in 1984, when Warren and Marshall cultured and identified a curved gram-negative bacillus present in human gastric biopsies, and confirmed its association with antral gastritis 210 . It was initially placed as a member of the Campylobacter genus, but after a deeper study it was classified as Helicobacter pylori in the newly-created Helicobacter genus, alongside with a related bacterium of ferrets, Helicobacter mustelae 211 . After numerous studies linking the presence of H. pylori with dyspeptic conditions[212][213][214][215] and gastric cancer[216][217][218] , it was classified as a Type I carcinogen by the World Health Organization in 1994219 . Thanks to their discovery, Warren and Marshall were awarded with the Nobel Prize in Physiology and Medicine in 2005.ii. EpidemiologyH. pylori infects approximately half of the world's population 220 . The infection is more prevalent in Central and South American and Asian countries where it is present in 70-90% of the older adult population, in comparison with 20-50% in Northwestern European countries, Japan and the United States 221 . Some authors suggest that prevalence increases with age 222 , however, it is widely accepted that this is due to a cohort effect and that the seropositivity percentage in the adult population is reached around the age of 15[223][224][225] . The widespread antibiotic use and the improvements in the living conditions of the population have caused a decrease in the prevalence of infection in industrialized countries such as Germany and Australia, where the higher infection rates are found in regions with lower socioeconomic indicators and their amelioration correlates with lower prevalence226 . In more heterogeneous countries like the United States, the percentage of H. pylori-positive individuals amongst white, non-Hispanic people has decreased in a period of ten years, unlike the case of Hispanic and African American populations, in which rates remained stable 226 . The decreasing trend seen in the prevalence of the infection maybe related with factors such as the index of human development, access to safe drinking water, adequate sanitary conditions, and good healthcare 222 . The massive use of antibiotics may also play a role in this decrease220 .When a human acquires H. pylori, a persistent infection is established, ending only by the eradication with antibiotic treatment. The infection usually takes place during childhood, and the reinfection in the adult after eradication is rare222,227 . Even so, the colonization has no deleterious consequences in the host. Only a small percentage of cases is associated with severe pathologies(10-15 %), such as chronic atrophic gastritis, peptic ulcers and gastric malignancies (1-2 %), notably gastric adenocarcinoma and MALT lymphoma 228 .There are multiple transmission routes proposed for the H. pylori infection. It is accepted that it happens directly between humans, either by oral-oral, fecal-oral, or gastro-oral, none of them being mutually exclusive220,227,229 . The transmission takes place preferentially inside the family nucleus, where close contact and hygiene conditions are the most important determinants 230 . Studies carried in different countries have shown that children from infected mothers have up to a 20-fold increased risk of infection compared with those of non-infected mothers, and the same case is observed in the case of infected fathers in countries where the relation between father and child is close 227,230 . This associations where initially evidenced by epidemiological studies, and have been supported by a MLST analysis showing a similar pattern of transmission in Japan 231 .

252.

  The urease-coding genes in H. pylori are organized in the cluster ureABIEFGH, formed by two operons: ureAB and ureIEFGH. The later also presents post-transcriptional regulation by degradation of mRNA ins response to pH 253 . The first operon encodes for the UreA and UreB subunits of the enzyme whilst the second encodes for the urea channel UreI and other accessory proteins required for the maturation and the assemblage of the Ni 2+ in the apoenzyme 254 .

Figure 9 .

 9 Figure 9. cagPAI T4SS structure and CagA effects on the host cells 345 . Hypothetical model of the cagPAI T4SS machinery is shown in A. Upon injection in the host cell cytoplasm, CagA affects membrane dynamics, actin cytoskeleton, nuclear signaling and cell-to-cell junctions, among others. AJ, adherens junction; TJ, tight junction.

  conserved pathway, composed by a tumor suppressor core consisting of a module of two kinases, MST (Hippo or HPO in D. melanogaster) and LATS (Warts or WTS in D. melanogaster), and their regulatory proteins SAV (Salvador) and MOB (MATS) 418 . The second component includes the output elements, which are the targets of phosphorylation by the kinase module. In the fly, it is the transcriptional coactivator YKI (Yorkie), homolog to the YAP1 and TAZ proteins in mammals. The Hippo pathway components in mammals and their homologs in D. melanogaster are listed in table 10 and showed in figure 10.

Figure 10 .

 10 Figure 10. Hippo pathway. Component of the Hippo signaling pathway in Drosophila (left) and mammals (right). Modified from 418

  LATS, YAP1 and TAZ are bound to 14-3-3 proteins in cell-cell junctions or undergo proteasomal degradation. This sequestration limits their activity by preventing the nuclear accumulation. Active nuclear YAP1 and TAZ function together with TEAD family transcription factors to promote tissue growth by increasing the expression of genes that positively regulate cell growth and inhibit apoptosis 418 . YAP1 and TAZ can also work together with other transcription factors such as SMADfamily, TBX5, RUNT1/2, and p73 419 . Their capacity to regulate transcription of genes regulation cell survival and proliferation make YAP1 and TAZ, oncogenes.

Figure 11 .

 11 Figure 11. Cell polarity systems involved with Hippo regulation 430 . The apical and polarity complex (lefts) and cell adhesion proteins (center) in epithelial cells, and the planar polarity complex (right) are upstream signals for the Hippo pathway.

  2003 in a genetic screen of overgrowth mutants in the fruit fly, as a gene promoting both cell cycle exit and cell death 438 . It was identified at the same time by other group in a screening of homozygous mutant cells in the eyes of otherwise heterozygous fruit flies, which also detected sav and wts 439 . In the human, MST1 and MST2 are Ste20-related serine/threonine kinases, which are at the core of the Hippo kinase tumor suppressor cascade. MST1 (487 amino acids) and MST2 (491 amino acids) share more than 95% of the catalytic domains are 75% identical 440 . The most characteristic feature of these kinases is the presence of a SARAH domain (SAlvador-RASSF-Hippo), which is approximately 50 amino acids long, and mediate the homo-or heterodimerization and subsequent stabilization of MST1/2.

  mechanism is particularly important in T cells, where the MST1/RASSF5 may facilitate the phosphorylation of MST1 by allowing the continuous interaction with the activated GTPase, thus impeding dephosphorylation of the kinase and also due to a concentration of MST1 in the membrane, facilitation transphosphorylation. The other regulator, SAV1 is a 383 amino acid, SARAH-domain containing protein that has the ability of binding both MST1/2 and its substrate LATS, thanks to the WW domain that links the PPXY in LATS440 . Other stimuli can influence the activation of MST1/2. It is activated during apoptosis in the liver, by reactive oxygen species, and during the G2/M phase of the cell cycle, and on the contrary, inhibited by Akt-mediated non-canonical phosphorylation in response to EGF. MST1/2 has various substrates. Downstream in the Hippo pathway, and the most studied are the LATS1/2 kinases and its regulators Mob1A/B. MST-mediated activation of LATS occurs in a sequential process that involves the collaboration of Mob1 425 . Autophosphorylation of MST1/2 creates a site for Mob1 recruitment, and relieves Mob1 from an autoinhibitory conformation, making it accessible to LATS. This allows the formation of a MST-Mob-LATS complex in which both Mob (Threonine 35) and LATS (Threonines 1079 in LATS1 and 1041 in LATS2) are phosphorylated by MST, rending an active LATS and promoting the dissociation of the complex by phosphor-Mob1, which in addition favors the LATS autophosphorylation, also required for its activity. Besides the downstream elements of the Hippo pathway, there are other substrates of MST1/2, for example the apoptosis-related transcription factor FOXO1/3, histones H2B and H2AX, cell cycle related kinases Aurora A/B, among others.

LCD2 447 .

 447 The PBD domains binds to proteins like MOB1 and LIMK1, and while the exact function of the LCDs is unknown, they are indispensable for the tumor suppressor activity. The presence of PPxY motifs is also a common feature of both proteins, and allows the binding to YAP1 and TAZ. A schematic representation of the structure of LATS1/2 is showed in figure12.

Figure 12 .

 12 Figure 12. Structure of LATS1 and LATS2 447 . LATS1 and LATS2 share several conserved domains, including the kinase domain, but also differ in the PPxY motifs, a P-strech (present only in LATS1), and a PAPA repeat (present only in LATS2).

  YAP1 and TAZ, targets of the canonical Hippo signaling, are the main substrates of LATS1/2. Upon phosphorylation of these two proteins, LATS1/2 favor their cytoplasmic retention and degradation, acting like a tumor suppressor by blocking the pro-oncogenic transcriptional activity of the YAP1/TAZ-TEAD complex, described later in this chapter. However, there are other proteins that can be phosphorylated by LATS2. Interestingly, the EMT-associated transcription factor Snail1 has been describe as a target of LATS2 phosphorylation in the breast epithelial cell line MCF10A, where LATS2-mediated phosphorylation in Snail1 Thr203 was found to retain this protein in the nucleus, enhancing the protein stability and function, and acting, contrary to its classic role, in tumor promotion453 . Another substrate of LATS2 phosphorylation describe recently is p21, a cyclin-dependent kinase (CDK) inhibitor that regulates cell cycle and inhibits apoptosis454 . In response to UV radiation in the human osteosarcoma cell line U2OS and in HeLa cells, Lats2 phosphorylates p21 in Ser146, causing a destabilization that induces apoptosis.Noteworthy, the activity of LATS2 induced by UV radiation is independent of Mst1/2 phosphorylation, and occurs in a different amino acid residue (Ser385) by the Chk-1 kinase. Ser835-phosphorylated LATS2 is not required to phosphorylate YAP1 in the Hippo canonical Ser127 position, and that stimulation by UV may displace the classical targets of LATS2 in favor of other substrates such as p21, and represent an alternative induction of cell death via LATS2 in response to DNA damage.

Figure 13 .

 13 Figure 13. Structure of YAP and TAZ 457 . Schematic representation depicting the multiple domains of YAP1 and TAZ, the sites of interaction with other proteins and the sites of phosphorylation.

Figure 14 .

 14 Figure 14. Features of cancer cells controlled by the Hippo pathway 471 . YAP1/TAZ/TEAD transcriptional activity controls the expression of genes involved in proliferation, survival and cancer stem cell properties.

Figure 15 .

 15 Figure 15. YAP1/TAZ in human tumors 471 . YAP1/TAZ have been linked epidemiologically and functionally with several types of human cancers.

Figure 16 .

 16 Figure 16. Chemical structure of verteporfin. The benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin), chlorin-type molecule derived from porphyrin. A simplified outline of BPD-MAC, one of the two racemic regioisomers (BPD-MAC and BPD-MAD), is shown. Porphyrins have substituents in the peripheral positions of the pyrrole rings(1 -8), on the four methine carbons (mesopositions), and/or coordinated metals.

Figure 1 .

 1 Figure 1. LATS2, YAP1 and CD44 expressions in H. pylori-infected and non-infected human gastric

Figure 2 .Figure 3 .

 23 Figure 2. Biphasic kinetics of H. pylori-induced changes in LATS2 and YAP1 expressions and subcellular

Figure 4 .

 4 Figure 4. LATS2 restricts H. pylori-induced EMT in hGECs in vitro. (a) Relative mRNA levels for EMT-

Figure 5 .

 5 Figure 5. LATS2 represses CD44 and CSC-like properties in hGECs in vitro. (a) Relative mRNA levels of

Figure 6 .

 6 Figure 6. LATS2, YAP1 and CD44 expressions of in diffuse-and intestinal-type patient's GC tissue samples

  Alexa-488-labelled secondary antibody (1:250, Molecular Probes). F-Actin (in red) was marked with Alexa-546-labelled phalloidin (1:250, Molecular Probes) and nuclei (in blue) with DAPI. Images were taken using an Eclipse 50i epi-fluorescence microscope (Nikon) with Nis Element acquisition software and x40 oil immersion objective.

  Statistical analysis. Quantification values represent the means of three or more independent experiments, each performed by triplicate or more ± standard deviation, except for the tumorsphere formation experiments where five independent experiments with n=10 each were carried-on. Mann-Whitney test was used to compare between two groups and Kruskal-Wallis test with Dunn's post-test was used for multiple comparisons. Statistics were performed on GraphPadPrism 7.02 (USA). factors and other transcription factors (dark green bars), and other TEAD-target genes (orange bars) were variably affected. Only genes with statistically significant changes in expression are shown (p<0.05 in t-test with Benjamini-Hochberg correction).

Figure S2 .

 S2 Figure S2. Helicobacter pylori regulates LATS2 and YAP in a CagA-dependent manner. The infection of

Figure S3 .

 S3 Figure S3. Basal expression of LATS2 and YAP in four gastric epithelial cell lines. Basal expression of

Figure S5 .

 S5 Figure S5. Cell cycle-related gene expression upon H. pylori-infection of LATS2 knocked-down cells. p21,

  statements on human samples and animal experiments. Studies on fresh tumors from patientderived gastric adenocarcinoma were performed in agreement with the Direction for Clinical Research and the Tumor and Cell Bank of the University Hospital Center of Bordeaux (Haut-Leveque Hospital) and of the Bergonié Institute of Bordeaux. Animal experiments with non-obese diabetous/severe combined immunodeficiency /interleukin-2Rγ null (NSG) immunodeficient mice were performed in level 2 animal facilities of the University of Bordeaux, with the agreement of the local Ethic Committee on Animal Experiments CEEA50 of Bordeaux (agreement number 50120151-A) and the French Committee of Genetic Engineering (approval number 4608). Activities of conservation and preparation of elements of human origin are declared at the French Ministry of Research (DC-2008-412).

  analysis. Quantification values represent the means of three or more independent experiments, each performed by triplicate or more ± standard deviation, except for the tumorsphere formation experiments where three to five independent experiments with n=10 each were carried-on. Mann-Whitney test was used to compare between two groups and Kruskal-Wallis test with Dunn's post-test was used for multiple comparisons. Statistics were performed on GraphPadPrism 7.02 (USA).

Figure 1 .

 1 Figure 1. YAP expression in gastric adenocarcinomas. Immunohistochemistry staining for YAP was performed in tissue samples of gastric adenocarcinomas. YAP was mainly nuclear in 10-30% of positive cells (black arrows), and located in the invasive front of the tumors. Bars 20 µm.

Figure 2 .

 2 Figure 2. In vitro effect of verteporfin in MKN45 and MKN74 gastric cancer cell lines. Cell growth at 48h was decreased from 1 µM concentration in both cell lines (a), without a change in cell morphology (b).Cell cycle analysis showed a cell cycle arrest(c), with accumulation of cells in G0/G1 phase, and decrease of cells in phase S (d). TEAD-luciferase reporter (e) and mRNA expression levels of CTGF, CYR61, IGFBP3, JAGGED1, and LATS2 (f) were decreased upon treatment. * p<0.05 vs DMSO-treated control in Kruskal-Wallis test.

Figure 3 .Figure 4 .

 34 Figure 3. In vitro effect of verteporfin in tumorspheres. In vitro cultured tumorspheres from gastric cancer cell lines and PDXs were treated with verteporfin. Preventive treatment (a) was performed by adding verteporfin during the seeding of cells. Morphology and size changes of the tumorspheres upon treatment is shown for MKN45 and GC04(b). The curative effect of verteporfin, was studied by treating well established tumorspheres (c). * p<0.05, **p<0.01, ***p<0.001 vs DMSO-treated control in Kruskal-Wallis test

Figure 5 .

 5 Figure 5. Effect of verteporfin treatment on tumor growth in a murine xenograft model in vivo. MKN45, MKN74, and GC10 cells were grafted in NSG mice and tumor growth was followed for DMSO-treated control and verteporfin-treated groups, where slower growth rates were observed (a). At the end of the treatment, tumors were extracted (b) tumor weight was determined (c) and tumors were dissociated to evaluate the tumorsphere forming capacity of residual cells (d). * p<0.05, ** p<0.01 vs DMSO-treated control in Mann-Whitney test.

Figure 6 .Figure 1 .

 61 Figure 6. Effect of verteporfin treatment on CSC-and proliferation markers in a murine xenograft model in vivo. MKN45-derived subcutaneous tumors extracted from DMSO-treated control (upper panel) and verteporfin-treated (lower panel) groups were processed for histology and stained for the expression of CSC markers CD44 (left) and ALDH (center), which were decreased on the intensity and percentage of positive cells. Proliferation marker Ki67 (right) was also strongly reduced in the treated tumors.

Figure 17 .

 17 Figure 17. Hippo pathway regulation by H. pylori infection. H. pylori infection modulates the Hippo pathway in biphasic pattern in a CagA dependent manner. There is a first phase of early induction of YAP1/TEAD transcriptional activity, which is then repressed in a second phase by an activation of LATS2.The YAP1/TEAD transcriptional activity seems to be at last partially responsible for the induction of EMT and CSC-properties observed upon H. pylori infection of humans gastric epithelial cells. We identified LATS2 as a potential YAP1 target gene, which constitutes a negative feedback loop limiting YAP1 activity.

Figure 19 .

 19 Figure 19. Functional categories enriched upon LATS2 inhibition in MKN74 cells. Enrichment of genes groups according to the Kyoto Encyclopedia of Genes and Genomes (KEGG), obtained by a transcriptomic analysis (Agilent Human Microarray 8x60K).

Figure 20 .

 20 Figure 20. NLR signaling pathway associated genes differentially expressed upon LATS2 inhibition.mRNA levels expressed as Log2(siLATS2/Control), obtained by a transcriptomic analysis. Only significantly regulated genes are shown (p<0.05).

Table Index Table 1 .

 Index1 Macroanatomical classification of gastric cancer .........................................................................

Table 2 .

 2 TNM-staging of gastric cancer .

Table 10 .

 10 Hippo pathway components in Drosophila and human homologs ............................................

Table 11 .

 11 Overview of the regulation of mammalian LATS kinases by phosphorylation ............................

Table 1 . Macroanatomical classification of gastric cancer 21 Early

 1

	Japanese classification
	I	Protruding growth
	II	Superficial growth. Further classified in IIa (elevated), Iib (flat), and IIc (depressed)
	III	Excavating growth
	IV	Infiltrating growth with lateral spreading
	Paris classification
	0-I	Polypoid growth further divided in 0-Ip (pedunculated) and 0-Is (sessile)
	0-II	Non-polypoid growth further divided in 0-IIa (slightly elevated), 0-IIb (flat), and 0-IIc (slightly
		depressed)
	0-III	Excavated growth

Table 3 . Hereditary conditions associated with gastric cancer 32 .

 3

	Syndrome	Genes	Inheritance Gastric	Gastric	Features of	Onset
		(% case		cancer	cancer	gastric polyps	(years)
		association)		risk (%)	histology
	Hereditary	CDH1 (45)	Autosomal	56-70	Diffuse	-	14
	diffuse		dominant
	gastric cancer
	Hereditary	MLH1 (30),	Autosomal	2-30	Intestinal	-	-
	nonpolyposis	MSH2 (60),	dominant
	colon cancer	MSH6 (<10),
		PMS2 (<10)
	Peutz-Jeghers	STK11 (70)	Autosomal	29	Intestinal Hamartomatous	16
			dominant			polyps and
						melanocytic
						macules
						(antrum,
						pylorus)
	Juvenile	SMAD4 (4-	Autosomal	21	Intestinal	Hamartomatous	41
	polyposis	20), BMPR1A	dominant		and diffuse	and
		(20-25)				heterogenous
	Familial	BRCA1,	Autosomal	2.6-5.5	-	-	<50
	breast cancer	BCRA2	dominant
	Li-Fraumeni	TP53 (70)	Autosomal	3.1-4.9	Intestinal	-	12
			dominant		(70%) and
					diffuse
					(30%)
	Familial	APC (≤90)	Autosomal	2.1-4.2	Intestinal	FGPs	8
	adenomatous		dominant			(body/fundus)
	polyposis					and GAs
						(body/antrum
						junction)
	1.

Table 4 . Markers of EMT. Modified from 101 Proteins with increased expression Proteins increased in the nucleus

 4

	Collagen (Types 1 and 3)	β-catenin
	Fibronectin	NF-B
	FOXC2	Smad 2/3
	Goosecoid	Snail 1/2/3
	Integrins αVβ6 and α5β1	Twist
	Laminin 5	ZEB1/2
	MMP-2
	MMP-3
	MMP-9
	N-cadherin	Proteins with decreased expression
	Slug (Snail 2)	Claudins
	Snail 1	Cytokeratins
	Snail 3	Desmoplakin
	Sox10	E-cadherin
	Twist	Occludins
	Vimentin	Ostepontin 1
	ZEB1
	ZEB2

Proteins with increased activity Functional properties in vitro ILK

	promoting factors 102 . Deposition of collagen and other ECM components is part of the normal scar
	formation during tissue repair. When this deposition is excessive due to persistent injurious stimuli and
	turns pathological it is referred as fibrosis, and it occurs mainly in parenchymal organs such as the liver,
		Increased migration capacity
	GSK-3β	Increased invasion capcity
	Rho	Elongated morphology
		Apoptosis resistance
		On the other hand, type II EMT is associated with tissue regeneration and fibrosis 87 . Cutaneous
	wound-healing exhibits EMT features, especially during the process of re-epithelialization, in which the
	keratinocytes at the wound edge lose their intercellular adhesions and migrate across the wound, with
	changes that are strikingly similar to the first steps of EMT 102 . Also the myofibroblasts present in the
	wound and essential in the remodeling and maturation phases of healing, are derived from resident
	epithelial cells transformed through EMT to synthetize extracellular matrix components and contract the
	wound bed 102 . In addition to the process in skin, the involvement of EMT in wound healing has been
	described in other tissues, such as mammary epithelial cells in vitro 103 , a murine model of lacrimal gland
	injury 104 , and a myocardial injury model in zebrafish 105 . Unsurprisingly, signaling pathways activated during
	both wound healing and EMT are significantly overlapped, including activation of EGF, FGF, HGF and TGF-
	β signaling, which in turn switch on MAPK pathways to trigger simultaneously wound healing and EMT

Table 5 . Comparative anatomy and histology of the murine and human stomach 160 .

 5

		Feature	Mouse	Human
	Gross	Forestomach	Yes, keratinized squamous	No
			epithelium, thin walled right half
		Glandular	Left half of the stomach, thick	Yes: cardias, fundus, corpus, and
		stomach	mucosa: cardiac, fundic, and	pylorus
			pyloric región
		Rugal folds	Not as prominent	Prominent when stomach is
				empty
		Areae gastricae	Not described	Mucosal shallow grooves that
				don't flatten with distention
		Limiting ridge	Present projection between	No
			glandular and non-glandular
			stomach mucosa
	Tissue	Mucosa	Keratinized squamous or	Simple columnar epithelium
			glandular, simple straight glands;
			cell composition varies with
			region
		Lamina propria	Yes	Same
		Submucosa	Yes	Same
		Muscularis	Yes	Same
		mucosa
		Muscular tunic	Inner oblique, middle circular,	Same
			outer longitudinal
		Serosa	Yes	Yes
		Squamous	Amount of keratin is diet-and	No
		epithelium	fasting-dependent
	Cells	Foveolar (cardiac)	Simple columnar, mucus-	Same
		cells	secreting, lines top portion of the
			mucosa
		Parietal cells	Eosinophilic, acid-secreting,	Same
			midportion of glands
		Chief cells	Basophilic, zymogen-secreting,	Same
			near the base of glands
		Cardiac glands	Mucus glands near limiting ridge,	Same
			smallest portion
		Fundic glands	Parietal and chief cells, top	Same
			portion lined with foveolar cells
		Pyloric glands	Near duodenum, mucus cells only	Parietal cells, chief cells absent
		Enterochromaffin	Between basement membrane	Diffusely scattered in epithelium
			and chief cells

Table 6 . Genetically altered murine model of gastric cancer. Modified from 159

 6

		Cancer
	Genotype	incidence	Duration	Location	Phenotype
		(%)
	MTH1 -/-	13	18 months	Antrum	Adenocarcinoma, dysplasia, hyperplasia, lung and liver tumors
	TFF1 -/-	30	5 months	Antrum	Intramucosal carcinoma, hyperplasia, NF-κB activation
	Smad4 +/-	100	12-18 months	Corpus/ Antrum	Adenocarcinoma, dysplasia, hyperplasia, duodenal tumor
	GB-Smad4 f/f	100	12-18 months	Antrum	Dysplasia, hyperplasia
	GAS -/-	60	12 months	Antrum	Dysplasia, metaplasia, athrophy, and increased susceptibility to MNU
	Gp130 f/f	100	6 months	Antrum	Adenoma, decreased TFF1 expression
					Adenocarcinoma, dysplasia,
	IL1-β	<70	12 months Transition	metaplasia, atrophy, synergized with
					H. felis
	K19/K-ras	37.5	16 months	Corpus	Dysplasia, metaplasia, atrophy
	Wnt1/C2me	100	20 weeks	SCJ	Adenocarcinoma, dysplasia, metaplasia
	CDH1 +/-+ MNU	45.8	40 weeks	Antrum	Signet-ring cell carcinoma, adenoma
	CDH1/p53	100	12 months	Corpus	Poorly differentiated adenocarcinoma, signet-ring cell carcinoma
	RUNX3 -/-+ MNU	71	52 weeks	Corpus/ Antrum	Adenocarcinoma, metaplasia, hyperplasia
	Villin-KLF4 f/f	29	80 weeks	Antrum	Adenoma, susceptibility to MNU
	c.

The infection with H. felis or H. pylori accelerates the process. Other examples of genetically altered mice models are listed in table 6.

Table 7 . Commercially available gastric epithelial cell lines.

 7

	Cell line	Gender /Age	Origin	Gastric subtype cancer	Morphology	Genetic features
	AGS	F/54	Stomach (primary tumor)	Intestinal	Epithelial adherent, non-cohesive	Hyperploid Mutations: CDH1 (homozygous), CTNNB1 (heterozygous, function gain), KRAS (heterozygous), PIK3CA (heterozygous)
	MKN 7	H/39	Lymph node metastasi s	Well differentiate d tubular AC	Epithelial, cohesive	Mutations: TP53, APC (silent) Oncogenes: erb2 CDH1 WT, CTNNB1 WT, CD44 WT
					Epithelial,
					cohesive
	MKN 28	F/70	Lymph node metastasi s	Moderately differentiate d tubular AC	Differentiati on in cell polarity and microvilli	Mutations: TP53, APC (non-sense) Oncogenes: no gross genetic alterations CDH1 WT, CTNNB1 WT, CD44 silenced by promoter hyper metilation
					with core
					filaments
						TP53 WT, APC WT, CTNNB1 WT, CD44
	MKN 45	F/62	Liver metastasi s	Poorly differentiate d carcinoma	Multipotenti ality for differentiatio n	WT Mutations: CDH1 deletion in exon6-intron 6 junction Oncogenes: c-met (amplification), low
						bcl-2 expression
	MKN 74	M/37	Liver metastasi s	Intestinal	Epithelial, cohesive	CDH1 WT, TP53 WT, CTNNB1 WT, CD44 Oncogenes: no gross genetic alterations
	NCI-N87	M/?	Liver metastasi s	Intestinal	Epithelial, cohesive Differentiati on in cell polarity and microvilli	Near diploid CDH1 WT Oncogenes: myc, erb2 Mutations: TP53 (homozygous), SMAD4

Table 8 . Markers of gastric stem cells identified by lineage tracing

 8

	Marker Location	Features of stem cell population
	Villin	Bottom of antropyloric glands	Stem cell reservoir with a high proliferative potential
		Isthmus in the corpus
	Lgr5	Bottom of pyloric glands	Multipotent stem cells responsible of the renewal of
			gastric epithelium
	Sox2	Isthmus of glands of the corpus Long-lived stem cells
	Tff2	Isthmus of glands of the corpus Short-lived progenitors
	Troy	Base of corpus glands	Actively dividing population and small group of stem-like
			chief cells.
	Mist1	Lower third of the glands in the	Stem-like quiescent chief cells
		corpus
	eR1	Isthmus	Small population of terminally differentiated chief cells

Table 9 . CSC markers in hematological malignancies and solid tumors. Modified from 198 Hematological malignancies Solid tumors Tumor type Cell surface markers Tumor type Cell surface markers Acute myelocytic leukemia

 9

			CD34 + CD38 -	Breast	CD44 + CD24 -/low Lineage -ESA +
				Central nervous	CD133 +
				system
		B-cell acute	CD34 + CD10 -/CD34 + CD19 -	Colon	CD133 +
		lymphocytic	CD34 + CD38 -CD19 +		ESA high CD44 + Lineage -(CD166 + )
		leukemia		Ewing's sarcoma	CD133 +
		Multiple	CD34 -CD138 -	Head and neck	CD44 + Lineage -
		Myeloma		Melanoma	ABCB5 +
		T-cell acute	CD34 + CD4 -/CD34 + CD7 -	Liver	CD90 + CD45 -(CD44 + )
		lymphocytic		Ovarian	CD44 + CD117 +
		leukemia		Pancreas	CD44 + CD24 + ESA +
					CD133 +
	b.	CD44 (Cluster of differentiation 44)

  The bacterium is negative for most of the conventional tests of identification, It does not ferment or oxidizes carbohydrates, does not hydrolyze hippurate, does not reduce nitrates nor forms indol 241 .Common bacterial mechanisms of acid resistance such as proton pump activation, descarboxylases, and membrane lipid modification are not sufficient for long term survival or very low pH conditions 242 . Remarkably, the genus Helicobacter presents genes that allow further adaptation to the acid enviroment, and not just the aforementioned resistance. The urease, which catalyzes the conversion from urea to ammonium and carbon dioxide and therefore contributes to neutralization of the bacterium's surroundings, was the first protein identified with a role in adaptation to acid environment in H. pylori and it is consider a virulence factor 243 . It is composed by four trimers of heterodimers UreA-UreB with 24 nickel

	iv.	Virulence factors
	1.	Colonization
	a.	Adaptation to acid environment survival: Urease

Table 10 . Hippo pathway components in Drosophila and human homologs 418 .

 10

		Drosophila melanogaster	Human
		Fat (FT)	FAT1-FAT4
		Dachsous (DS)	DCHS1 and DCHS2
		Discs overgrown (DCO)	CK1ε and CK1δ
		Lowfat (LFT)	LIX1 and LIX1L
		Four-jointed (FJ)	FJX1
		Dachs (D)	?
		Approximated (APP)	ZDHHC9, ZDHHC14 and ZDHHC18
		Zyxin (ZYX)	Zyxin, LPP and TRIP6
		Merlin (MER)	Merlin (also known as NF2)
		Expanded (EX)	Willin/FRMD6 and FRMD1
	Upstream	Kibra	Kibra
		Crumbs (CRB)	CRB1-CRB3
		Lethal giant larvae (LGL)	LGL1 and LGl2
		Discs large (DLG)	DLG1-DLG4
		Scribble (SCRIB)	SCRIB
		aPKC	aPKCι and aPKCζ
		STRIPAK (PP2A)	PP2A (STRIPAK)
		RASSF	RASSF1-RASSF6
		Myopic (MOP)	HD-PTP
		JUB	Ajuba, LIMD1, WTIP
		TAO1	TAO1-TAO3
		Hippo (HPO)	MST1 and MST2
	Kinase core	Salvador (SAV) Mats (MTS)	SAV1

Table 11 . Overview of the regulation of mammalian LATS kinases by phosphorylation. Modified from 450

 11 . Additional evidence of this regulation has been shown in a gastric carcinogenesis model of AGS cell infected with H. pylori. Belair et al 452 reported a high expression of miR-372 in this cell line, where together with miR-373, it silences LATS2 promoting cell proliferation. Infection with H. pylori inhibits the synthesis of both microRNAs, in a CagA-dependent manner, allowing LATS2 expression and a cell cycle arrest in G1/S, and this may impair the epithelium renewal, not only representing the overcome of a major antibacterial defense mechanisms, but also potentially favoring atrophy of the mucosal epithelium (an initial step in the gastric carcinogenesis cascade).

	Kinase	Upstream kinase	Site(s)	Role of phosphorylation
	LATS1	Unknown	Ser909	Essential for kinase activity
	LATS1	MST1/2	Thr1079	Essential for kinase activity
	LATS1	Cdk1/Cyclin B	Thr490/ Ser613	Possible role in mitosis
	LATS1	NUAK1	Ser464	Controls LATS1 protein stability
	LATS2	Aurora A	Ser83/ Ser380	Plays a role in mitosis
	LATS2	CHK1/2	Ser408	Plays a role in DNA damage response
			Ser172/
	LATS2	PKA	Ser380/ Ser592/	Regulate LATS2 activity towards YAP1
			Ser598

  . Epidemiological and experimental data supports the role of the Hippo/YAP/TAZ signaling pathway in different types of cancer, both of epithelial and non-epithelial origin, which are thoroughly reviewed by Zanconato et al7 . In addition to the studies by Xu et al6 and Jiao et al8 , our group recently showed that YAP1 is upregulated and activated in the gastric epithelium upon chronic infection with H. pylori, a major risk factor for gastric cancer, and appears at all stages of the gastric carcinogenesis cascade, co-localized with the CSC and EMT marker CD44 in the gastric glands (unpublished results). Altogether, these studies point out the role of the Hippo signaling in gastric carcinogenesis.

Acknowledgments

Silvia Elena Molina Castro is a PhD fellowship recipient of the University of Costa Rica (San José, Costa Rica) and the ministry of sciences and technologies (MICIT, Costa Rica). This project and Julie Giraud and Camille Tiffon were financially supported by the French National Institute for Cancer INCa (grant 2014-152). This project was supported by the SIRIC BRIO (Bordeaux Research in Translational Oncology -Site de Recherche Intégrée sur le Cancer, grant INCa-DGOS-Inserm 6046). We thank R. Peek (Vanderbilt University, Nashville, TN, USA) for the H. pylori strains 7.13 and isogenic cagA-deleted mutant, Professor Nojima (Osaka, Japan) for the pCMVmyc-LATS2 plasmid, Yannick Lippi (GeTRix Platform, Toulouse, France) for statistical analyses of the transcriptome, and Jacques Robert (INSERM U1218, University of Bordeaux, France) for CCLE database analyses. We thank Julie Pannequin (IGF, University of Montpellier, France) and Michel Moenner (CNRS UMR5095, University of Bordeaux, France) for helpful discussions.

lesions (8 to 12 weeks), most likely linked to the use of a more virulent strain of the bacterium 56,414,415 . On the other hand, another study failed to find the neoplastic lesions in the gerbil even after a long follow up period 416 . The different results among studies may be explained partially by the genetic background of the gerbils, their microbiota, and the anatomo-pathologic assessment performed 416 . Another important fact concerning the model, is the absence of metastases and mortality of the gerbils due to the gastric adenocarcinoma, which points to important differences between the evolution of the pathology in the gerbil and in the human being.

101

II.

Results/Articles ABSTRACT Gastric carcinoma (GC), which is mostly related to infection with CagA + -Helicobacter pylori, is the third leading cause of cancer-related mortality worldwide. H. pylori infection disrupts the gastric mucosa turnover and induces, via an epithelial-mesenchymal transition (EMT), the emergence of CD44 high cells with properties of cancer stem cells (CSC) at the origin of GC. The Hippo pathway, involved in stem cell homeostasis, growth and cancer, was investigated in this context in human gastric epithelial cell lines (hGEC) and patients-derived tissues. H. pylori infection regulated Hippo signaling in a CagA-dependent manner in AGS and MKN74 hGECs, revealing concomitant up-regulations of Hippo kinases, downstream effectors and YAP/TEAD target genes. LATS2 and its target YAP1 were co-upregulated in response to H.

pylori both in vitro and in the gastric mucosa of H. pylori-infected patients with gastritis and intestinal metaplasia. In vitro, YAP1 was triggered as early as 2 hours post-infection which subsequently stimulated the transcription of YAP1/TEAD target genes among them ctgf and cyr61, and then was tightly retrocontrolled by LATS2 upregulation and phosphorylation of YAP1 on Ser127. Interestingly, we showed that LATS2 and YAP1 reciprocally maintained each other's expression both under basal and infected conditions.

Using siLATS2 in hGECs, we showed that LATS2 restricted H. pylori-induced EMT and the emergence of invasive CD44+ CSC-like cells forming tumorspheres in vitro. In GC tissues, LATS2 and YAP1 were mostly nuclear, heterogeneously expressed and colocalized in CD44 + CSC-rich areas. Their co-expression also corroborated in the GC cell lines of the Cancer Cell Lines Encyclopedia. This study points out for the first time that H. pylori, via CagA, affects the Hippo pathway and unveils an unexpected co-upregulation of LATS2 and YAP1 upon infection, which in turn tightly control gastric CSCs properties and appear as new preneoplastic biomarkers of the gastric carcinogenesis cascade leading to GC.

We show here that LATS2 and YAP1 are co-upregulated, nuclear and therefore activated in hGECs of H.

pylori chronically infected patients as early as the gastritis stage and prior to their neoplastic transformation. This observation was unexpected in regard to most data reporting an inverse correlation of LATS1/2 and YAP1 expression, with LATS1/2 downregulated while YAP1 was overexpressed and functional in the nuclei in many tumor cells [30] including those of GC [31][32]. LATS2 has been reported as a dynamic regulator of proliferation and functional maturation of stem cells via YAP1 restriction. YAP1 has been extensively portrayed as a "stemness factor" localized within the stem cell niche of adult organs [33-36] and also constitutes a Wnt/ß-catenin regulator [37][38]. YAP1 has been found to be essential to regulate stem cell growth when these cells are stirred up to ensure tissue regeneration [35], a condition fulfilled here during the H. pylori assault of the gastric mucosa. LATS2 and YAP1 co-localization with the CSC and EMT marker CD44 in the gastric glands may reveal an extension of the stem cell reservoir naturally present in the gastric mucosa. Furthermore, in the context of the H. pylori-triggered chronic inflammation, regenerative hyperplasia and metaplasia, these stem cells acquire mesenchymal and pre-neoplastic features, most likely predisposing them to transform and generate self-renewing CSCs.

Second, using hGEC lines challenged by H. pylori in vitro, we found that the bacterium affects LATS2 and YAP1 in a coordinated biphasic pattern, characterized by an early and transient YAP1/TEAD transcription stimulation and followed by LATS2 upregulation leading to YAP1 phosphorylation and nuclear exclusion, in a CagA-dependent manner. At this point, these data on infected hGECs do not support our observations in infected patients' gastric mucosa showing concomitant LATS2 and nuclear YAP1 up-regulations.

Although this wave of YAP1/TEAD activation is rapidly retro-controlled following LATS2 induction in response to H. pylori in vitro. Repeated and chronic aggression of the gastric epithelium by H. pylori in vivo could lead to constant waves of YAP1/TEAD activation and LATS2 induction, resulting, as observed, in a co-overexpression of both and the nuclear localization of YAP1 reflecting its co-transcriptional activity. The siLATS2-and siYAP1-mediated loss-of-function experiments revealed that LATS2 and YAP1 depend on each other for their expression in hGECs, and knocking down either of the two weakens the expression of the other, suggesting that LATS2 and YAP1 reciprocally maintain each other's expression under both non-MKN74 may harbor a functional YAP1/ZEB1 transcriptional relationship. ZEB1 is a NF-κB target gene during H. pylori-induced EMT in hGECs [11]. The rise in ZEB1 and Snail1 expression induced by siLATS2 (but not by siYAP, data not shown) could be explained by an indirect action of LATS2 on ZEB1 via NF-κB, which was found negatively regulated by LATS2 in non-small-cell lung carcinoma [42]. In addition, silencing LATS2 in non-transformed mammary epithelial cells increases NF-κB association with p53, thereby promoting cell migration [43]. CD44 is a Wnt/ß-catenin target gene, a pathway which is activated by H. pylori via CagA [44][45][46]. Its upregulation upon LATS2 knockdown could be explained by the ability of LATS2 to interfere with ß-catenin/BCL-9 interaction within the nucleus thereby preventing Wnt/ß-catenin signaling [47].

Finally, one open question is how are LATS2 and YAP1 alerted by H. pylori infection? In addition to the Hippo kinases MST1/2 and p53 mentioned above, LATS2 is subject to regulation by numerous proteins, including MAP4Ks, Aurora A and AJUBA, NF2/Merlin and Crb3 [30][27][54][26]. Mechanical signals in relationship with the microenvironment represent also an additional pillar for YAP1 function [48][20][35].

The destabilization of hGECs cell/cell junctions and cell shape is deeply affected by CagA, as evidenced by the "hummingbird" phenotype of infected AGS and the transcriptomic data (Fig. 1c, Fig. S1, Fig. 2e; [11][15]), thanks to its ability to binds to numbers of host proteins involved in cell junctions [45][49][50].

It is important to note that CagA targets and triggers the SHP-2 phosphatase, which can also directly interact with YAP1 and potentiate its cotranscriptional function [51][52]. This tripartite connection

CagA/SHP2/YAP may be critical in the early phases of human gastric carcinogenesis [53]. Nuclear localization of YAP1 is the sum of multiple, possibly parallel, regulatory layers including G protein-coupled

Receptor and Src kinases [30], which are also CagA-interacting proteins [8][49].

Epithelia exposed to environmental assaults, including pathogen infection, require constant stem celldriven renewal and signaling tuning to maintain tissue homeostasis. H. pylori infection constitutes the greatest risk factor for GC mainly due to its oncoprotein CagA which interacts with numerous host cell proteins, subsequently triggering several signaling pathways including, in this case, the LATS2/YAP1/TEAD axis of the Hippo pathway. The co-overexpression of LATS2/YAP1 appears at all stages of the gastric BectonDickinson, and pRL-SV40 (10 ng/well) and pEGFP-C3 (100 ng/well) were from Promega.

Firefly and Renilla luciferase activities were measured using the Dual Luciferase Assay (Promega).

Firefly luciferase activities were normalized for transfection efficiency by Renilla luciferase. -2008-412).

RNA extraction, transcriptome and RT-qPCR. Cell RNAs were extracted using Trizol reagent (Invitrogen) and quantified by their absorbance at 260 nm. For the transcriptome, RNAs were extracted using RNeasy (Qiagen), and the RINs were determined on the TapeStation (Agilent). The transcriptome was performed at the GeTRix Platform (Toulouse, France) with the G3 HGE 8x60 microarrays (Agilent). For RT-qPCR, retrotranscription was performed with 1 µg total RNA using the Quantitect Reverse Transcription kit (Qiagen). Quantitative-PCR was performed using the SYBR-qPCR-Premix Ex-Taq (TAKARA), 0.3 µM specific primers (Table S2) and 1:200 of the RT reaction volume. Relative expressions were calculated using the comparative Ct method with HRPT1 and TBP as normalizers. Mature miR-200 levels were quantified as previously described [11].

Immunohistochemistry. Tissue sections 3 µm thick were prepared from formalin-fixed paraffin-embedded human tissues and submitted to standard IHC protocols with CD44 (mouse anti-CD44 from BD, 1:100, 2 h), LATS2 (rabbit anti-LATS2 from Bethyl; 1:100, 2 h), YAP (rabbit anti-YAP from Cell Signaling; 1:100, 1 h) or and H. pylori (rabbit anti-H. pylori from DAKO; 1/100, 2 h) antibodies, and then with Horse Radish Peroxidase-labelled anti-rabbit EnVision System (DAKO; 30 min). Immunolabeling was revealed upon 10 min incubation in liquid diaminobenzidine-chromogen substrate (DAKO). Slides were counterstained with hematoxylin, dehydrated and mounted with Eukitt-mounting medium (Labonord). appeared more frequently than in siControl or siYAP (b).

Supplementary figures

Supplementary Table S1. Relative mRNA expression level of CD44 and Hippo/YAP related genes in the GC cell lines of the CCLE.

Supplementary Table S2. Primers used in qPCR experiments.

Gene

III. Discussion

homologue) in the regulation of the antimicrobial peptide production, by an indirect repression of Cactus (the orthologue of the NF-B inhibitor NFBIA/B/C), as showed in figure 18. show the CSC-inhibitory activity of verteporfin both in vitro and in vivo. We corroborated the results obtained by various groups in models of liver, colorectal, and esophageal carcinogenesis [482][483][484][485] , and to our knowledge, this is the first description of the anti-tumor activity of verteporfin in gastric cancer. Other mechanisms of tumor growth inhibition have been described for verteporfin, for example the capacity to inhibit autophagosome accumulation (reducing cell viability in nutrient-deprived conditions) 481 and STAT3/IL-6 signaling 486,487 , as well as the capacity to induce oligomerization of high molecular weight proteins, which increases apoptotic cell death 487 . All these mechanisms could also have a role in the effect found in our model, and are not exclusive. However, even if they contribute to the effect, our results confirm a role for the YAP1/TEAD disrupting action of verteporfin, reflected in the decrease of YAP1/TEAD target genes expression.

Among the advantages of verteporfin use as anti-tumor treatment, are a selectivity for cancerous cells with high YAP1/TEAD activity and the fact that it is an FDA-approved molecule for the routine treatment of ophthalmologic pathologies (age-related macular degeneration, choroidal neovascularization, and chronic central serous chorioretinopathy). But, despite the observed significant action of verteporfin treatment in tumor reduction, the insolubility of the molecule was a significant challenge. As it is not water soluble, the use of DMSO as a carrier which could introduce a bias caused by the toxic activity of DMSO. This inconvenience was, however, overcome using the appropriate control.

The other important disadvantage was also related to the product solubility, which limits its bioavailability and the possibility of reaching the adequate concentration in the tumor when administered in a systemic way, unless using very high quantities of the product which could carry systemic toxicity and increase the financial burden of the treatment. As an alternative, we used a peritumoral injection, which proved to fail in the homogeneous distribution of verteporfin around the tumor and in some cases, caused the tumor to escape the treatment, invading towards the opposite side. However, the positive results obtained despite the flaws of the model, open the possibility of exploring new alternatives for verteporfin formulation that make it more suitable for implementing the treatment in patients. More importantly, this work represents a proof of concept, in which the disruption of the YAP1/TAZ/TEAD transcriptional activity represents a strategy of great potential in the development of antitumor drugs for gastric cancer.

i. Other small molecules that modulate the Hippo pathway

Besides verteporfin, several other small molecules have been identified as modulators of the Hippo pathway with a potential use in cancer treatment and regenerative medicine, thoroughly reviewed in Johnson et al 490 . In the exploratory phase of the project we were interested in testing some of these molecules as well. One of them was the Src/Abl familiy tyrosine kinase inhibitor Dasatinib, which has been resistant to ATRA, and as the Hippo pathway deregulation has been associated with the CSC phenotype, the study of its activation status is very relevant.

In summary, this work shows for the first time that the Hippo pathway and particularly the LATS2/YAP1/TEAD axis is early activated during the carcinogenesis process induced by chronic H. pylori infection and controls the subsequent EMT and CSC-like features. Targeting this pathway was shown to be efficient in preventing tumor growth in a PDX model, therefore highlighting the potential of its inhibition to be implemented in gastric cancer therapy.

a. Deletion of IQGAP1 promotes Helicobacter pylori-induced gastric dysplasia in mice and acquisition of cancer stem cell properties in vitro

Published in Oncotarget, 2016, Vol. 7, (No. 49), pp: 80688-80699