Manon Logan

Céline Jules Claire

Arnaud, Marion Ana Benoit

Maïlys Amira Sofia

Alia Andrés

Zeinab Florian

Marylou Amie Marion

Smaranda, Thomas Bryan Amandine

Alexis Camilla

Emilie Jil

Raphael Rapetti-Mauss
email: raphael.rapetti-mauss@univ-cotedazur.fr

Jérémy Nigri

Camille Berenguier

Pascal Finetti

Sarah Simha Tubiana

Bonnie Labrum

Benoit Allegrini

Bernard Pellissier

Georgios Efthymiou

Zainab Hussain

Corinne Bousquet

Nelson Dusetti

François Bertucci

Hélène Guizouarn

Patricia Melnyk

Franck Borgese

Richard Tomasini

Olivier Soriani
email: soriani@unice.fr

Dr Pr Olivier Soriani

Baptiste Monterroso

Sameh Ben

Aicha We

Véronique Rigot

Corinne Bousqu

Richard Tomasi

SK2 channels set a signalling hub bolstering CAFtriggered tumourigenic processes in pancreatic cancer

Keywords: Wnt/β-catenin signalling pathway, mutations, organoids signaling pathways, drug targets, KCNQ1 channel, P2X7, cystic fibrosis transmembrane conductance regulator, hERG, transient receptor protein channels, ClC-2 channel

In recent years, the role of ion channels in cancer processes has emerged and the field continues to expand. It has been shown that 1) the expression of certain ion channels is deregulated in cancers, and 2) ion channels can regulate various signalling pathways. In physiology, the balance of signalling pathways must be maintained. In the epithelium of the colon, maintaining this balance is particularly crucial as cells undergo rapid renewal which relies on the combined action of multiple signalling pathways. Dysregulated activation of the Wnt/β-catenin signalling pathway, promoting cell proliferation, is a major driver of tumour development in the colon. In most cases of colorectal cancer, patients harbour mutations on the component of this pathway, contributing to its overactivation. The potassium channel KCNQ1 has diverse functions. In the colonic epithelium, it enables water secretion and maintains epithelial integrity by exerting its tumour suppressor activity through the repression of the Wnt/β-catenin pathway. Despite the impact of somatic mutations in cancers, there are no studies, to this day, exploring mutations of ion channels in this context. Therefore, we hypothesized that colorectal cancer patients carry pathogenic mutations on KCNQ1. By analysing publicly available databases, we identified 36 different mutations harboured by KCNQ1 in various cancers. We found that the most common mutations were loss-of-function. In colorectal cancer cells, the loss-of-function mutations were associated with increased Wnt/β-catenin pathway activity, independently of the canonical pathway receptors. We also observed an activation of several tyrosine kinase receptors that could directly mediate β-catenin activation. In the more physiological model of murine colonoids, lossof-function mutations in KCNQ1 also seem to increase Wnt/β-catenin pathway activity. Furthermore, these mutations had an impact on the expression of Wnt/β-catenin pathway regulators, favouring a permissive state for the pathway overactivation. KCNQ1 thus appears to restrict the signalling pathway at multiple levels: directly by sequestering β-catenin at adherens junctions, preventing its activity as a transcription factor, and indirectly by allowing the expression of regulatory proteins that inhibit the Wnt/β-catenin pathway. Our results highlight, for the first time, that ion channels can carry somatic mutations in cancers, altering channel function. Moreover, our findings demonstrate that these mutations directly influence the activity of signalling pathways involved in cancer processes. Additionally, given that KCNQ1 expression levels can be used for classifying colorectal cancers, these mutations could potentially aid in the prognosis and stratification of patients. Altogether, these findings open a new avenue of investigations to better understand the diversity of mechanisms involved in cancer processes. Numerous other ion channels or their associated subunits may carry somatic mutations in cancers, deregulating their activity and the processes they govern. Key words: ion channels, cancer

Exploration de la fonction du canal potassique KCNQ1

dans l'homéostasie de la crypte colique

Elucidating the function of the potassium channel KCNQ1 in the homeostasis of colonic crypts

JURY Présidente de Jury

Sophie Tartare-Deckert

Rapportrices

Annarosa Arcangeli

Claire Racaud-Sultan

Examinateurs

Jean-Yves le Guennec Aubin Penna

Résumé

Le rôle des canaux ioniques dans les processus cancéreux a émergé ces dernières années et constitue un domaine en plein essor. Il a notamment été montré, d'une part que l'expression de certains canaux ioniques était dérégulée dans les cancers et d'autre part que les canaux ioniques pouvaient réguler diverses voies de signalisation cellulaire. L'équilibre des voies de signalisation est essentiel en physiologie. Dans l'épithélium du colon, cet équilibre est d'autant plus crucial que les cellules sont soumises à un renouvèlement rapide, régit par l'action conjointe de plusieurs voies de signalisation. L'activation dérégulée de la voie de signalisation Wnt/β-caténine, favorisant la prolifération cellulaire, est un moteur majeur du développement tumoral dans le colon. Il a été montré que la plupart des patients atteints de cancer colorectal étaient porteurs de mutations sur des composants de cette voie, participant ainsi à sa suractivation. Le canal potassique KCNQ1 a diverses fonctions notamment dans l'épithélium du colon où il participe à la sécrétion d'eau mais également au maintien de l'intégrité épithéliale par son action de suppresseur de tumeur en réprimant la voie de signalisation Wnt/βcaténine. Malgré l'impact des mutations somatiques dans les cancers, il n'existe à ce jour aucune étude explorant les mutations portées par des canaux ioniques dans ce contexte. Nous avons donc émis l'hypothèse que des patients atteints de cancer colorectal pourraient être porteurs de mutations pathogènes sur KCNQ1. Grâce aux bases de données publiques, nous avons identifié 36 mutations portées par KCNQ1 dans divers cancers. Les mutations les plus fréquentes se sont avérées être perte de fonction. Dans les cellules de cancer colorectal, cette perte de fonction est associée à une activité accrue de la voie Wnt/β-caténine indépendamment des récepteurs canoniques de la voie. Nous avons identifié une activation de plusieurs récepteurs tyrosine kinases qui pourraient médier directement l'activation de la β-caténine. Dans le modèle des colonoïdes murins, plus physiologique, les mutations pertes de fonctions de KCNQ1 semblent également augmenter l'activité de la voie Wnt/β-caténine. De plus, ces mutations ont un impact sur l'expression de régulateurs de la voie Wnt/β-caténine, favorisant ainsi un état permissif à une suractivation pathogénique de la voie. Le canal potassique KCNQ1 restreindrait ainsi la voie de signalisation à plusieurs niveaux : un direct en séquestrant la β-caténine aux jonctions adhérentes, empêchant son activité de facteur de transcription, et un niveau indirect en permettant l'expression de protéines régulatrices inhibant la voie Wnt/β-caténine. Nos résultats mettent en évidence pour la première fois que les canaux ioniques peuvent être porteurs de mutations somatiques dans les cancers et que ces mutations peuvent altérer la fonction du canal. Nos résultats sont également les premiers à démontrer que ces mutations exercent une influence directe sur l'activité de voies de signalisation impliquées dans les processus cancéreux. De plus, les niveaux d'expression de KCNQ1 pouvant servir à la classification des cancers colorectaux, ces mutations pourraient également aider au pronostic et la stratification des patients. Ensemble, ces données ouvrent donc une nouvelle voie d'investigation permettant de mieux comprendre la diversité des mécanismes mis en place dans les processus cancéreux. De nombreux autres canaux ioniques ou leurs sous-unités associées pourraient porter des mutations somatiques dans les cancers dérégulant leur activité et les processus qui en découlent.

Mots clefs : canaux ioniques, cancer, voie Wnt/β-caténine, mutations, organoïdes

INTRODUCTION

Chapter I -Colon Physiology

1. The gastrointestinal tract

The digestive system can be decomposed into the gastrointestinal tract and the accessory digestive organs: the tongue, teeth, salivary glands, liver, gallbladder, and pancreas. The role of the digestive system is to ingest, digest, and absorb nutrients, electrolytes, and water to distribute energy to the whole organism and eliminate waste. Accessory digestive organs produce or store secretions to help the chemical breakdown of food [START_REF] Tortora | Principles of Anatomy and Physiology[END_REF]. The gastrointestinal tract (GIT) is a 5to 7-meter-long tube that runs from the mouth to the anus. It is composed of several specialised regions: the mouth, pharynx, oesophagus, stomach, small intestine, and large intestine (Figure1. 1).

Broadly, the function of the first part of the GIT is to break the ingested food into smaller pieces and further into nutrients thanks to the action of enzymes and acidic pH. The upper part of the intestine is dedicated to the absorption of nutrients, water, and electrolytes while the lower part and the rectum

Figure1. 1 -The Gastrointestinal Tract. Right lateral view of head and neck, anterior view of trunk. Organs of the gastrointestinal tract (in black) and accessory digestive organs (in red) (From Tortora et al.) are specialised in the elimination of waste with the secretion of water and electrolytes [START_REF] Silverthorn | Human Physiology: An Integrated Approach[END_REF][START_REF] Steele | The ASCRS Textbook of Colon and Rectal Surgery[END_REF].

The proper function of this system is ensured by rhythmic peristalsis, a muscular contraction of the smooth muscles that surround the GIT. This mechanism is necessary to push the bolus throughout the GIT all the way to the anus. This muscular activity is controlled by the autonomic nervous system.

Structure of the large intestine wall

The structure of the wall remains quite similar throughout the GI tract. For clarity, the large intestine will be described in this manuscript. It is composed of four layers: the mucosa, submucosa, muscularis and serosa (Figure1. 2) [START_REF] Steele | The ASCRS Textbook of Colon and Rectal Surgery[END_REF].

Mucosa

The mucosa is the inner lining of the gastrointestinal tube. The epithelium, in direct contact with the content of the lumen, rests on the lamina propria, the connective tissue above the muscularis mucosae, a thin layer of smooth muscle. The epithelium is a monolayer composed of different cell types: the columnar enterocytes/colonocytes which participate in the absorption of nutrients, water and electrolytes, the goblet cells involved in mucus secretion to lubricate and protect the mucosa [START_REF] Johansson | The gastrointestinal mucus system in health and disease[END_REF] and the neuroendocrine cells which are hormone-secreting cells. The epithelium, in addition to its roles in absorption and secretion, acts as a barrier against pathogens Figure1. 2 -Layers of the gastrointestinal tract (From Tortora, 2019) [START_REF] Peterson | Intestinal epithelial cells: regulators of barrier function and immune homeostasis[END_REF]. The lamina propria further supports the function of protection against pathogens as it contains lymphatic vessels and immune cells. It is also highly vascularised to enable the intake of nutrients and the release of hormones. The muscularis mucosae contracts to increase the surface area by stretching to ensure that all the absorptive cells are in contact with the contents of the lumen.

Submucosa

The gastrointestinal submucosa is a thick layer of connective tissue that lies just beneath the mucosa.

It is composed of loose connective tissue containing arteries, veins, lymphatic vessels, and nerve fibers.

The submucosa provides support and elasticity to the GIT. The nerve fibers belong to the Enteric Nervous System, a large network of over 100 million neurons involved in the regulation of the GIT functions. It is composed of two plexuses: the myenteric plexus or plexus of Auerbach which is in the muscularis and provides the cues for contractility and the submucosal plexus or plexus of Meissner that innervates the cells of the mucosa and muscularis mucosae to regulate the functions of the epithelium.

Muscularis

Until the middle part of the oesophagus, the muscularis is made of skeletal muscle that facilitates voluntary swallowing. In the rest of the GIT, it is composed of two sheets of smooth muscle: the inner circular layer and the outer longitudinal fibres. These muscles enable the peristaltic movement of the GIT. The circular layer thickness increases in the anal canal to form the internal anal sphincter.

Serosa

The serosa is a connective tissue with a surface mesothelial lining, surrounded by the visceral peritoneum. It secretes a lubricating fluid that prevents friction between the motile GIT organs and the surrounding viscera [START_REF] Tortora | Principles of Anatomy and Physiology[END_REF].

Anatomy of the large intestine

The large intestine is composed of the caecum, colon, rectum, and anal canal (Figure1. 3). The ileocecal sphincter separates the ileum -last part of the small intestine -and the large intestine. The caecum is a pouch-like structure about 6 cm below the ileocecal sphincter in the lower right abdomen. From the caecum originates a small finger-like structure called the appendix that contains lymphocytes. From the caecum, the large intestine extends upward as the ascending colon, across the abdomen as the transverse colon, downward as the descending colon and then curves inward as the sigmoid colon. The colon measures 1.5 meters long with a diameter of 6.5 cm, which makes it shorter but wider than the small intestine. The colon can also be separated into the proximal colon (ascending and transverse)

and distal colon (descending and sigmoid). The taeniae coli are three separate, external, longitudinal ribbons of smooth muscle that run through the length of the colon forming the haustra [START_REF] Silverthorn | Human Physiology: An Integrated Approach[END_REF]. Haustra are small pouches caused by sacculation which give the colon its segmented appearance. Haustra are important in colon motility as haustral contractions facilitate the movement of the faeces towards the rectum. The sigmoid colon connects to the rectum, a muscular chamber that stores faeces until they are expelled through the anus.

Functions of the colon

The large intestine plays a role in the digestion process. It absorbs many nutrients from the diet and from the enzymatic processes taking place in the GIT.

Figure1. 3 -The Large Intestine Anterior view of large intestine showing major regions (from Tortora, 2019) Water and Electrolytes Regulation

Each day, 1 to 1.5 litres of water enter the colon, all but 0.1 to 0.2 litres will be absorbed in physiological conditions. The rest will be eliminated in the faeces. The colon plays a major role in the water and electrolytes balance. In pathological context, disorders such as diarrhoea whether linked to an infection or a disease like Intestinal Bowel Disease (IBD) can result in severe dehydration by a defect in reabsorption of water and electrolytes.

The colonic epithelium is polarised, meaning that the plasma membrane is divided into two different entities both in their protein and lipid composition and in their function: the apical membrane facing the lumen and the basolateral membrane in contact with the blood vessels. This difference is crucial for the vectorial transport of electrolytes and water from the blood to the lumen (secretion) and from the lumen to the blood (absorption). The transport of electrolytes, osmotically active species, creates an osmotic gradient that is followed by water. Water can either move via the paracellular pathway through the tight junctions or via the transcellular pathway involving water channels called aquaporins [START_REF] Kunzelmann | Electrolyte transport in the mammalian colon: mechanisms and implications for disease[END_REF][START_REF] Laforenza | Water channel proteins in the gastrointestinal tract[END_REF]. While the paracellular transport is passive and only follows the osmotic gradient, the transcellular pathway, through the expression level of aquaporins, can be modulated by peptides and hormones (Cooke, 2000;[START_REF] Itoh | Enhancement of aquaporin-3 by vasoactive intestinal polypeptide in a human colonic epithelial cell line[END_REF].

Electroneutral absorption of sodium chloride (NaCl) occurs after eating and is mainly controlled by the Na + /H + exchangers 2 and 3 (NHE) at the apical side [START_REF] Zachos | Molecular physiology of intestinal Na+/H+ exchange[END_REF] and the Cl -/HCO3 -anionic

Basolateral

Figure1. 4 -Sodium absorption by colonocytes. Electroneutral (left) and electrogenic (right) absorption of sodium and absorption of potassium (right) (adapted from [START_REF] Kunzelmann | Electrolyte transport in the mammalian colon: mechanisms and implications for disease[END_REF] exchanger (AE) [START_REF] Rajendran | Na-H Exchanger Isoform-2 (NHE2) Mediates Butyrate-dependent Na+ Absorption in Dextran Sulfate Sodium (DSS)-induced Colitis[END_REF][START_REF] Suzuki | Basolateral Cl-/HCO3-exchange in epithelial cells from guinea pig distal colon[END_REF] in both proximal and distal colon (Figure1.

4) [START_REF] Kunzelmann | Electrolyte transport in the mammalian colon: mechanisms and implications for disease[END_REF]. The Na + absorption through NHEs is both mediated by surface and crypt cells.

Electrogenic absorption of Na + occurs exclusively in the distal colon in surface cells through the apical Epithelial Na + Channel (ENaC) [START_REF] Duc | Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry[END_REF]. The absorption of Na + requires a favourable electrochemical gradient for Na + and a negative apical membrane voltage to create a driving force for luminal Na + uptake via ENaC. To maintain this electrochemical gradient, 3 Na + are extruded from the cell in exchange for 2 K + basolaterally through the Na + /K + /ATPase. Additionally, absorption of Na + is coupled to counter ion Cl -absorption through Cl -channels on the apical membrane or via paracellular shunt. One of the most implicated Cl -channel is the Cystic Fibrosis Conductance Regulator (CFTR) (Figure1. 4) [START_REF] Kunzelmann | Electrolyte transport in the mammalian colon: mechanisms and implications for disease[END_REF][START_REF] Mall | CFTR-mediated inhibition of epithelial Na+ conductance in human colon is defective in cystic fibrosis[END_REF].

In the distal colon, K + absorption is not passive, K + is rather actively pumped out of the lumen through H + /K + /ATPases on the apical membrane and extruded basolaterally by the cotransporter KCC1 (Figure1.

4).

To maintain a hydrated mucus, secretion of electrolytes is necessary. In the colon, Cl -,K + and HCO3 - are the main secreted ions (Barrett and Keely, 2000). These secretions are crucial to create the osmotic gradient necessary for water secretion. The channels mediating the Cl -apical secretion are CFTR [START_REF] Dharmsathaphorn | Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line[END_REF][START_REF] Riordan | Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA[END_REF] and the Ca 2+ -activated Cl -channels (CACC) [START_REF] Soria | Bestrophin-1 enables Ca2+activated Cl-conductance in epithelia[END_REF][START_REF] Ousingsawat | Loss of TMEM16A causes a defect in epithelial Ca2+-dependent chloride transport[END_REF]. Cl -enters the cell basolaterally through the

K NaK AT 3 Na K KCNN4 KCNQ1 E3 Cl KCNMA1 NKC C1 K Cl Na K K CFTR Cl CACC HCO 3
Figure1. 5 -Chloride secretion in colonocytes (adapted from [START_REF] Kunzelmann | Electrolyte transport in the mammalian colon: mechanisms and implications for disease[END_REF] Na + /K + /2Cl -(NKCC1) cotransporter [START_REF] Park | Phylogenetic, structural and functional characteristics of the Na-K-Cl cotransporter family[END_REF]. Na + is recycled through the Na + /K + /ATPase and K + through basolateral K + channels such as KCNQ1:KCNE3 and KCNN4 [START_REF] Heitzmann | Physiology and pathophysiology of potassium channels in gastrointestinal epithelia[END_REF]. Apical K + secretion is mediated by the large conductance Ca 2+ -activated K + channel KCNMA1 [START_REF] Sausbier | Distal colonic K(+) secretion occurs via BK channels[END_REF]. HCO3 -is secreted into the lumen against its electrochemical gradient via CFTR, the apical Cl -/HCO3 -exchanger and the SCFA/HCO3 -exchanger (Figure1. 5) [START_REF] Kunzelmann | Electrolyte transport in the mammalian colon: mechanisms and implications for disease[END_REF].

Other roles of the colon

The colon is also the site of fermentation which, thanks to commensal bacteria from the microbiota, finishes the digestion of some proteins and starches that are resistant to digestion and absorption in the small intestine. These bacteria break down complex carbohydrates to use the nutrients themselves and produce short chain fatty acids (SCFAs) such as acetate and butyrate as a product of fermentation.

Butyrate is one of the main energy substrates for the colonic epithelium [START_REF] Bergman | Energy contributions of volatile fatty acids from the gastrointestinal tract in various species[END_REF] and studies suggest that it could reduce the risk of colorectal cancer (Dmitrieva-Posocco et al., 2022). In addition, SCFAs increase the absorption of sodium and chloride and thus help maintain a proper functioning of the colon.

Synthesis and absorption of vitamins produced by bacteria such as vitamin K and vitamin B also occurs in the colon. Vitamin K cannot be produced by the organism and thus must be provided by the diet or the production by the gut microbiota [START_REF] Ramakrishna | Role of the gut microbiota in human nutrition and metabolism[END_REF].

On a larger scale, the colon has a role in storing and moving the faeces towards the rectum. The slow, contractile waves called colonic mass movements allow the stool to progress in the colon and take part in the defecation process [START_REF] Steele | The ASCRS Textbook of Colon and Rectal Surgery[END_REF].

Colonic crypts -the functional unit of the colon

In the colon, the monolayer epithelium forms invaginations embedded in the connective tissue. These finger-like structures were first discovered by Johann Nathanael Lieberkühn in 1745 who gave his name to theses crypts. The crypts of Lieberkühn represent the functional unit of the colon. There are approximately 14 000 crypts per square centimetre in the human colon [START_REF] Ricci-Vitiani | Colon cancer stem cells[END_REF]. Apart from these crypts, the colonic epithelium is flat whereas the intestinal epithelium has villi to increase the surface area in contact with the content of the lumen for better nutrient absorption (Figure1. 6).

The harsh conditions, both chemical and mechanical, that the GIT is subjected to each day demand for a rapid renewal of epithelial cells -the lifespan of cells is about three to five days. This rapid turnover of such a large organ requires a perfect homeostasis of stem cells to maintain the integrity of the tissue.

Intestinal Stem Cells (ISCs) are located at the bottom of the crypts of Lieberkühn. In the adult colonic epithelium, approximately 200 cells per crypt are generated every day [START_REF] Sancho | Signaling pathways in intestinal development and cancer[END_REF]. Stem cells produce proliferative progenitors that themselves generate a population of Transit Amplifying (TA) cells located in the lower two thirds of the crypt. These cells migrate upward while differentiating along the way by being subjected to different signalling cues (see further description of signalling pathways involved in crypt homeostasis). Once at the surface of the crypt, differentiated cells become apoptotic and debris go into the lumen. Only the stem cells at the bottom of the crypts escape this flow of cells from the bottom to the top of the crypt (van der Flier and Clevers, 2009). At the bottom of the crypt, intermingled with ISCs, the Reg4 + cells in the colon and Paneth cells in the small intestine are deep crypt secretory cells which main role is to maintain the ISC population [START_REF] Sasaki | Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon[END_REF].

Figure1. 6 -The colonic crypt seen in scanning electron micrograph (left panel), LGR5+ stem cells at the crypt base generate rapidly proliferating TA cells in the lower half of the crypt (middle panel). TA cells subsequently differentiate into the mature lineages of the surface epithelium (goblet cells, enterocytes, enteroendocrine cells and tuft cells), as shown in the lineage tree on the right panel. (From Barker, 2014) The balance between cell proliferation and cell death is thus tightly controlled. Dysregulation would lead to abnormalities of the epithelium that could threaten its integrity. All the cell processes involved in the renewal of the colonic epithelium -stem cell self-renewal, proliferation of progenitors, cell migration, differentiation, and apoptosis -need to be finely regulated. Different signalling pathways intervene in these processes. The most widely accepted theory for the regulation of ISC is that of the niche [START_REF] Mills | The intestinal stem cell niche: there grows the neighborhood[END_REF]. This theory supports the idea that the microenvironment surrounding the ISCs plays a major role in their regulation and crypt architecture. This niche is composed of different cell types including myofibroblasts that produce growth factors and cytokines [START_REF] Powell | Myofibroblasts. II. Intestinal subepithelial myofibroblasts[END_REF]. The morphogenetic pathways Wnt/β-catenin, Notch, BMP and Hedgehog are essential in the organisation and homeostasis of the colonic crypt (Figure1. 7) (Medema and Vermeulen, 2011).

The Wnt/β-catenin pathway

The understanding of the intestinal stem cells, and the involvement of the Wnt/β-catenin pathway in their homeostasis is greatly due to work from the lab of Hans Clevers. His team has shown that ISCs can be detected by the expression of Lgr5, implicated in Wnt signalling [START_REF] Barker | Identification of stem cells in small intestine and colon by marker gene Lgr5[END_REF]. A detailed explanation of the actors and functions of this pathway is available in Chapter II. Briefly, the Wnt/βcatenin pathway has three main effects in the crypt:

Wnt is the main driving force for ISC proliferation [START_REF] Kim | Mitogenic influence of human R-spondin1 on the intestinal epithelium[END_REF][START_REF] Kuhnert | Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1[END_REF][START_REF] Pinto | Canonical Wnt signals are essential for homeostasis of the intestinal epithelium[END_REF][START_REF] Pinto | Wnt control of stem cells and differentiation in the intestinal epithelium[END_REF]. Wnt is a master switch between proliferation and differentiation (van de Wetering et al., 2002) as it represses cell differentiation at the bottom of the crypt [START_REF] Gregorieff | Expression pattern of Wnt signaling components in the adult intestine[END_REF].

Wnt participates in the differentiation of crypt cells into the secretory phenotype [START_REF] Korinek | Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4[END_REF][START_REF] Pinto | Canonical Wnt signals are essential for homeostasis of the intestinal epithelium[END_REF].

The Notch pathway

Notch ligands, Jagged and Delta, bind to the Notch receptor to induce proteolytic cleavage of Notch by γ-secretase. The released Notch IntraCellular Domain (NICD) translocates into the nucleus to activate the transcription of target genes [START_REF] Miyamoto | Role of Notch signaling in colon homeostasis and carcinogenesis[END_REF]. The Notch pathway is a gatekeeper of the progenitor compartment, it regulates cell fate decision [START_REF] Fre | Notch signals control the fate of immature progenitor cells in the intestine[END_REF][START_REF] Van Es | Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells[END_REF]. Furthermore, Notch and Wnt cooperate to keep the ISC and progenitor cell in an undifferentiated and proliferative state [START_REF] Crosnier | Organizing cell renewal in the intestine: stem cells, signals and combinatorial control[END_REF][START_REF] Sasaki | Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon[END_REF][START_REF] Van Der Flier | Stem cells, self-renewal, and differentiation in the intestinal epithelium[END_REF].

The BMP pathway

The Bone Morphogenetic Protein (BMP) pathway inhibits the proliferation of crypt cells. BMP is a member of the Transforming Growth Factor family (TGF). When these ligands bind the Serine/Threonine Kinase receptor BMPR1 and BMPR2, the cytosolic SMAD proteins are phosphorylated and translocate into the nucleus. BMP2 and BMP4 are expressed by the mesenchymal cells and repress proliferation at the crypt surface border to promote differentiation [START_REF] Hardwick | Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon[END_REF][START_REF] He | BMP signaling inhibits intestinal stem cell selfrenewal through suppression of Wnt-beta-catenin signaling[END_REF]. BMP is also involved in cell fate decision towards secretory cells [START_REF] Auclair | Bone morphogenetic protein signaling is essential for terminal differentiation of the intestinal secretory cell lineage[END_REF]. Additionally, noggin, a BMP antagonist, is expressed in the lower part of the crypt.

The Hedgehog pathway

The Hedgehog pathway (Hh) is essential for crypt organisation. Hedgehog ligands (Sonic Hh, Indian Hh, Desert Hh) bind to the patched transmembrane receptor (Ptch). This binding inhibits the repression of Ptch on the receptor Smoothened (Smo). This triggers the activation and nuclear translocation of Glioblastoma transcription factor (Gli) [START_REF] Saqui-Salces | Hedgehog signaling and gastrointestinal cancer[END_REF]. Hh is secreted by the differentiated colonocytes and signals to the mesenchyme to induce the production of BMP ligand secretion, thus activating BMP signalling and restricting Wnt signalling [START_REF] Madison | Epithelial hedgehog signals pattern the intestinal crypt-villus axis[END_REF][START_REF] Van Den Brink | Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation[END_REF][START_REF] Van Dop | Depletion of the colonic epithelial precursor cell compartment upon conditional activation of the hedgehog pathway[END_REF].

The Hippo pathway

The effectors YAP/TAZ bind to the transcription factor TEAD upon stimulation. The stimuli activating the Hippo pathway can be a range of extracellular cues as well as mechanical cues, especially through cell-cell contacts. In the GIT, YAP/TAZ activity is enhanced at the bottom of the crypt, in the stem cell niche. YAP/TAZ have been shown to maintain the pool of ISCs and promote their proliferation when required [START_REF] Camargo | YAP1 increases organ size and expands undifferentiated progenitor cells[END_REF][START_REF] Yu | Hippo pathway regulation of gastrointestinal tissues[END_REF]. The Hippo pathway engages in crosstalk with Wnt, BMP and Notch pathways to ensure the regulation of the stem cell niche [START_REF] Hong | The Hippo pathway in intestinal regeneration and disease[END_REF].

The Eph/ephrin pathway

Eph/ephrin signal constitutes the main pathway controlling cell segregation in colonic crypt. This segregation between the bottom and upper part of the crypt is necessary to maintain crypt architecture. The ligands, ephrins, are membrane-associated proteins that stay linked to the cell that produces them. The interaction between ligands and receptors lead to cell repulsion (Crosnier et al., 2006; [START_REF] Herath | The role of Eph receptors and ephrin ligands in colorectal cancer[END_REF]. Wnt signalling upregulates the expression of EphB2 and EphB3 and downregulates the expression of Ephrin 1. Therefore, the bottom of the crypt expresses the receptors while the top expresses the ligand [START_REF] Batlle | Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB[END_REF].

Apoptosis

At the top of the crypt, cells undergo apoptosis and shed into the lumen. Among the proteins that have been shown to participate in apoptosis of colonocytes are the Adenomatous Polyposis Coli (APC) a key component of the Wnt pathway (T. Chen et al., 2003), the pro-apoptotic Bcl2 family member Bak [START_REF] Duckworth | Suppression of apoptosis, crypt hyperplasia, and altered differentiation in the colonic epithelia of bak-null mice[END_REF] and BMP signalling [START_REF] Hardwick | Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon[END_REF]. Metabolites from bacterial activity such as butyrate have also been shown to play a role in apoptosis [START_REF] Hague | Sodium butyrate induces apoptosis in human colonic tumour cell lines in a p53-independent pathway: implications for the possible role of dietary fibre in the prevention of large-bowel cancer[END_REF].

All these signalling pathways must be tightly regulated to ensure crypt architecture and epithelial integrity. In addition to regulation by their ligands and specific actors of the pathway, crosstalk between pathways participates in this tight regulation and creates the balance that ensures homeostasis of the epithelium. The Wnt/β-catenin pathway, one of the major forces at play in the colonic crypt, will be our focus for this manuscript.

Chapter II -The Wnt/β-catenin pathway

Historical Background and overview

The Wnt/β-catenin pathway is highly conserved across multicellular organisms. The identification of the pathway and the first mechanical insights were made by Nusse, Peifer, Perrimon, and Wieschaus labs in the early 1980s with the genetically powerful Drosophila model and the development of DNA cloning techniques. The identified pathway genes were renamed "the Wnt gene family" in 1991 [START_REF] Nusse | Three decades of Wnts: a personal perspective on how a scientific field developed: Three decades of Wnts: a personal perspective[END_REF]. Armadillo -the Drosophila ortholog of β-catenin, the main effector of the pathway -was identified the same year [START_REF] Peifer | The segment polarity gene armadillo interacts with the wingless signaling pathway in both embryonic and adult pattern formation[END_REF]. The identification of partners of armadillo/β-catenin saw an uproar in the 1990s, providing a clearer understanding of the pathway and its regulation. In 1996, the labs of Nathans and Nusse identified the first receptor in Drosophila -namely Frizzled -able to bind the ligand wingless and increase armadillo levels [START_REF] Bhanot | A new member of the frizzled family from Drosophila functions as a Wingless receptor[END_REF]. Meanwhile, the labs of Birchmeier and Clevers made the link between β-catenin and the transcriptional activation through TCF/LEF [START_REF] Behrens | Functional interaction of beta-catenin with the transcription factor LEF-1[END_REF][START_REF] Molenaar | XTcf-3 transcription factor mediates beta-catenininduced axis formation in Xenopus embryos[END_REF]. To know more about the history of the discovery of the Wnt pathway see [START_REF] Nusse | Three decades of Wnts: a personal perspective on how a scientific field developed: Three decades of Wnts: a personal perspective[END_REF].

The Wnt/β-catenin pathway is one of the most fundamental signalling pathways implicated in several cell processes such as cell fate, proliferation, and migration as well as early developmental events such as embryonic polarity, gastrulation, and limb and organs development. In the gastrointestinal tract, and more specifically in the crypts, Wnt/β-catenin is crucial for the maintenance of the stem cell population. The pathway is most active in the lower part of the crypt and inhibited towards the centre of the lumen. A loss or an overactivation of the pathway leads to a disruption of tissue architecture and can drive tumour initiation and progression which will be discussed further.

The main effector of the Wnt/β-catenin pathway is the β-catenin which transduces the signal received by the receptor down to the nucleus for transcription. Therefore, the complexity of this pathway lies in its regulation. The main components and regulators will be discussed here to provide a broad overview of the Wnt/β-catenin pathway, but it must be noted that it can be regulated by a myriad of signals, including other signalling pathways as discussed above, and surely, we do not know the extent of it all yet.

In addition to the β-catenin-mediated pathway, β-catenin-independent Wnt pathways have been described. These non-canonical pathways have not yet been fully elucidated and there is considerable overlap between Wnt pathways making it even harder to decipher the cascade. For clarity, the two main non-canonical pathways Wnt/Planar Cell Polarity (PCP) and Wnt/Calcium will be shortly described.

Wnt/PCP

The Wnt/PCP pathway is necessary during embryonic development to determine cell polarity. Cell polarity is crucial in migration and proliferation both during development and adult physiopathology [START_REF] Barrow | Wnt/PCP signaling: a veritable polar star in establishing patterns of polarity in embryonic tissues[END_REF]. The binding of Wnt5A and Wnt11 ligands to Frizzled, ROR or RYK receptors induces Dishevelled-mediated activation of small GTPases RhoA and Rac1. These proteins in turn activate JNK (Jun N-terminal Kinase) via phosphorylation. The activation of RhoA, Rac1 and JNK induce cytoskeletal rearrangements and changes in polarisation and motility [START_REF] Akoumianakis | Non-canonical WNT signalling in cardiovascular disease: mechanisms and therapeutic implications[END_REF]. In addition, it was shown that the Wnt5A-mediated Wnt/PCP pathway is blocked by Lrp6 suggesting that activation of the canonical Wnt/β-catenin signalling downregulates the non-canonical pathways (Figure2. 1a) [START_REF] Bryja | The extracellular domain of Lrp5/6 inhibits noncanonical Wnt signaling in vivo[END_REF].

Figure2. 1 -Overview of Wnt non-canonical signalling pathways. Wnt/planar cell polarity (a) and Wnt/calcium (b). (From Akoumianakis et al., 2022) Wnt/Calcium

The Wnt/Ca ++ pathway is activated by Dishevelled which recruits Phospholipase C inducing in turn an increase of intracellular Ca ++ by calcium release of the endoplasmic reticulum and calcium entry via calcium channels. The sudden increase in Ca ++ activates a range of Ca ++ -dependent proteins including the Protein Kinase C (PKC) and the calcium/calmodulin-dependent protein kinase II (CaMKII). These two proteins in turn activate transcription factors including NFAT, CREB and NFκB. These pathways are involved in development and immune response regulation (Figure2. 1b) [START_REF] De | Wnt/Ca2+ signaling pathway: a brief overview[END_REF][START_REF] Kohn | Wnt and calcium signaling: beta-catenin-independent pathways[END_REF].

Since this research is centred on the canonical pathway, this manuscript will mainly focus on the latter.

β-catenin -a multifaceted protein

A couple of years before its identification in the Wnt pathway, β-catenin was identified by its role in cell adhesion [START_REF] Ozawa | The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species[END_REF]. It was found that three proteins of 102, 88 and 80kDa formed complexes with Ca ++ -dependent cell adhesion molecules. These three proteins were named respectively α-, β-and γ-catenin.

The dual function of β-catenin sparked some debate among the scientific community. In most living animals including mammals, drosophila, and xenopus, only one gene encodes β-catenin. At first, it was thought that two distinct pools of β-catenin were uncoupled, each with its specific function. In the early 2000s, it became clear that the junctional proteins catenins and cadherins could have functions beyond their junctional activity and that their state was highly dynamic and influenced by cell-cell junctions, cytoskeletal rearrangements, and morphogenetic pathways [START_REF] Bienz | beta-Catenin: a pivot between cell adhesion and Wnt signalling[END_REF][START_REF] Nelson | Convergence of Wnt, β-Catenin, and Cadherin Pathways[END_REF][START_REF] Perez-Moreno | Catenins: keeping cells from getting their signals crossed[END_REF]. Since then, it is broadly accepted that only one pool of β-catenin mediates both adhesion and signalling. The identification of post-translational modifications of the protein gave even more power to this second hypothesis: one β-catenin does it all. Interestingly, in Caenorhabditis elegans, four genes encode orthologs of β-catenin: BAR-1 which mediates Wnt signalling, SYS-1 and WRM-1 which are part of the Wnt/β-catenin asymmetry pathway and HMP-2 which is strictly junctional [START_REF] Sawa | Wnt signaling in C. elegans, WormBook: The Online Review of C. elegans Biology[END_REF]. Back to the other species, the localisation and role of β-catenin is thus dictated by its post-translational modifications including its phosphorylation state (Figure2. 2). A subset of these post-translational modifications will be discussed here, for review on other known modifications of β-catenin see [START_REF] Valenta | The many faces and functions of β-catenin[END_REF]. When phosphorylated on the residues Ser33, Ser37, Thr41 and Ser45, β-catenin is targeted to proteasomal degradation through ubiquitination. In the absence of these phosphorylations, β-catenin is considered "active" ie not degraded by the destruction complex and able to act as a signalling effector. Further phosphorylations can either promote its junctional activity or, on the contrary, detach β-catenin from the adherens junction and push it towards its transcriptional activity. Among these, the Tyr142 can be phosphorylated by Fyn, Fer and Met, reducing α-catenin binding, and thus impairing the adhesive function of β-catenin. Tyr654 can be phosphorylated by Receptor Tyrosine Kinases (RTKs) and Src kinases to reduce binding to E-cadherin (van Veelen et al., 2011). This phosphorylation enhances βcatenin-driven transcription and facilitates further phosphorylation by the Protein Kinase A (PKA) on residue Ser675 (Taurin et al., 2006). Those two phosphorylations have been shown to increase the recruitment of co-activators for the transcription of Wnt target genes. Upon activation of EGFR, its downstream target AKT can phosphorylate β-catenin on its Ser552 residue increasing its transcriptional activity [START_REF] Fang | Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity[END_REF]. Therefore, to investigate the function of β-catenin in cells or animal models, the phosphorylation status of β-catenin is one of the readouts. To dissect the roles of β-catenin, this manuscript will first focus on the state of β-catenin in the absence of Wnt activation -adhesion or degradation. Then, the focus will be on the ligand-dependent and -independent activation of β-catenin signalling and its downstream effect in the nucleus.

Figure2. 2 -Main phosphorylations on β-catenin, implicated kinases, and effect.

β-catenin adhesive properties -at the adherens junction

Cell-cell junctions are essential through the development and adult life for several processes and regroup different types of junctional proteins ensuring different types of junctions. In epithelia, junctions between cells are of particular importance as they are responsible for the maintenance of the barrier with the outside environment. Cell-cell junctions thus maintain the integrity of tissues [START_REF] Garcia | Cell-Cell Junctions Organize Structural and Signaling Networks[END_REF]. The adherens junctions provide tissue stability [START_REF] Meng | Adherens junction: molecular architecture and regulation[END_REF][START_REF] Oda | Evolution: structural and functional diversity of cadherin at the adherens junction[END_REF]). E-cadherin (also known as cadherin-1) is a one-pass transmembrane protein. Two cadherins of neighbouring cells form calcium-dependent extracellular bonds [START_REF] Takeichi | Functional correlation between cell adhesive properties and some cell surface proteins[END_REF][START_REF] Takeichi | Selective adhesion of embryonal carcinoma cells and differentiated cells by Ca2+-dependent sites[END_REF]. β-catenin binds to the cytoplasmic domain of E-cadherin and links this domain to αcatenin, which is anchored to the actin cytoskeleton [START_REF] Aberle | Assembly of the cadherin-catenin complex in vitro with recombinant proteins[END_REF][START_REF] Ozawa | The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species[END_REF][START_REF] Rimm | Alpha 1(E)-catenin is an actinbinding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex[END_REF]Yap et al., 1997) (Figure2. 3). γ-catenin (or plakoglobin) can also bind the cytoplasmic domain of E-cadherin similarly to β-catenin [START_REF] Knudsen | Plakoglobin, or an 83-kD homologue distinct from beta-catenin, interacts with E-cadherin and N-cadherin[END_REF]. The interaction with the cytoskeleton can also occur through other actin-binding proteins that interact with α-catenin such as vinculin, further strengthening the adherens junction (le [START_REF] Le Duc | Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II-dependent manner[END_REF]Thomas et al., 2013). The p120 catenin binds E-cadherin [START_REF] Daniel | The tyrosine kinase substrate p120cas binds directly to E-cadherin but not to the adenomatous polyposis coli protein or alpha-catenin[END_REF][START_REF] Ohkubo | p120(ctn) binds to the membrane-proximal region of the E-cadherin cytoplasmic domain and is involved in modulation of adhesion activity[END_REF] and creates the link with microtubules [START_REF] Franz | p120 catenin associates with microtubules: inverse relationship between microtubule binding and Rho GTPase regulation[END_REF][START_REF] Meng | Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell-cell contacts[END_REF][START_REF] Shahbazi | CLASP2 interacts with p120-catenin and governs microtubule dynamics at adherens junctions[END_REF]. The binding of β-catenin to E-cadherin prevents β-catenin nuclear translocation and association to TCF/LEF [START_REF] Orsulic | E-cadherin binding prevents betacatenin nuclear localization and beta-catenin/LEF-1-mediated transactivation[END_REF] and β-catenin degradation by the destruction complex. However, this junctional complex is highly dynamic, and the adhesive functions of β-catenin can be impaired by loss of E-cadherin or by phosphorylation as described above (ie on residues Tyr142 and Tyr654 for example), thus shifting βcatenin towards its signalling functions. The β-catenin destruction complex

In the absence of Wnt stimulus, the pool of β-catenin that is not tethered to the adherens junctions is bound by the destruction complex composed of the scaffold protein Axin, the tumour suppressor Adenomatous Polyposis Coli (APC), and two Serine/Threonine Kinases, the Glycogen Synthase Kinase 3 β (GSK3β) and the Casein Kinase 1 α (CK1α) (Stamos and Weis, 2013). This complex, historically called the β-catenin destruction complex is also referred to as the Axin degradasome (Figure2. 4). Free βcatenin binds to APC and Axin and is then phosphorylated by CK1α on its Ser45 residue. This "priming" phosphorylation is necessary for the subsequent phosphorylation by GSK3β on the residues Ser33, Ser37 and Thr41 [START_REF] Amit | Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway[END_REF]Liu et al., 2002). These phosphorylations result in the binding of the F-box protein β-TrCP which is part of the SCF-β-TrCP E3 ubiquitin ligase complex along with the scaffold subunit Skp1, the adaptor protein Cullin1, and the RING subunit Ring box protein 1 (Rbx1).

This complex ubiquitinylates β-catenin which is thus addressed to the proteasome for degradation (Aberle et al., 1997;[START_REF] Hart | The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell[END_REF]Latres et al., 1999). The first gene "wingless" was identified as controlling polarity in Drosophila embryos [START_REF] Sharma | Effect of the Wingless (wg1) mutation on wing and haltere development in Drosophila melanogaster[END_REF]. In mice, the gene int-1 (integretation-1) was cloned and identified as a proto-oncogene causing mammary tumours [START_REF] Van Ooyen | Structure and nucleotide sequence of the putative mammary oncogene int-1; proviral insertions leave the protein-encoding domain intact[END_REF]. It later became clear that these two genes were orthologs and the combination of the two names gave the name "Wnt".

In humans, 19 genes encoding Wnt ligands have been identified [START_REF] Mehta | The Emerging Mechanisms of Wnt Secretion and Signaling in Development[END_REF]. The members of this family have a similar length of 350 to 400 amino acids and contain a highly conserved cysteine rich domain. Phylogenetic studies show that Wnt genes are evolutionarily conserved in metazoans and cnidarians (Kusserow, 2005). Their expression is tightly controlled both spatially and temporally throughout the development and adult life. Wnts are secreted via the endoplasmic reticulum and the Golgi apparatus by proteins ensuring the necessary post-translational modifications and transport (Figure2. 5). In the endoplasmic reticulum, Wnts undergo glycosylation and lipidation [START_REF] Smolich | Wnt family proteins are secreted and associated with the cell surface[END_REF][START_REF] Yamamoto | The apical and basolateral secretion of Wnt11 and Wnt3a in polarized epithelial cells is regulated by different mechanisms[END_REF], the latter done by the acyltransferase Porcupine which attaches a palmitoleic acid [START_REF] Hausmann | Helping Wingless take flight: how WNT proteins are secreted[END_REF]Kadowaki et al., 1996). These modifications enable the secretion of Wnts. The ligands are then shuttled from the Golgi apparatus to the plasma membrane by (From MacDonald et al., 2010) Wntless/eveness (WLS/Evi) where they can be tethered to the plasma membrane or exit the cell in three different manners: direct release by solubilization (Mulligan et al., 2012), formation of exosomes (Gross et al., 2012) or in lipoprotein particles (Neumann et al., 2009). Wnt ligands can act as morphogens able to activate short-or long-range signalling. In the intestinal and colonic epithelium, they create a gradient of morphogen that is opposite that of BMP ligands (Medema and Vermeulen, 2011).

Activation of the Wnt signalosome

When Wnt ligands are present in the extracellular medium, they bind to their seven-pass transmembrane receptor Frizzled which induces the heterodimerisation with the co-receptor Lrp5/6

(Low density lipoprotein receptor-related protein) [START_REF] Janda | Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling[END_REF]. Frizzled recruits and directly binds Dishevelled [START_REF] Zeng | Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions[END_REF]. The subsequent multimerisation of Dishevelled around Frizzled and Lrp5/6 creates a hub called Wnt signalosome (Figure2. 6) [START_REF] Bilic | Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation[END_REF]Metcalfe et al., 2010;[START_REF] Schwarz-Romond | The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization[END_REF]. Dishevelled binds Axin via the interaction of their DIX domains [START_REF] Fagotto | Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization[END_REF][START_REF] Julius | Domains of axin and disheveled required for interaction and function in wnt signaling[END_REF][START_REF] Kishida | DIX domains of Dvl and axin are necessary for protein interactions and their ability to regulate beta-catenin stability[END_REF], thus sequestering the destruction complex close to the plasma membrane. GSK3β does a priming phosphorylation for CK1α on Lrp5/6 [START_REF] Davidson | Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction[END_REF][START_REF] Zeng | A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation[END_REF]. These phosphorylations provide a docking site for Axin [START_REF] Tamai | A mechanism for Wnt coreceptor activation[END_REF][START_REF] Zeng | A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation[END_REF]. In this complex, GSK3β is directly inhibited by the activated Lrp5/6 [START_REF] Cselenyi | LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of beta-catenin[END_REF][START_REF] Piao | Direct inhibition of GSK3beta by the phosphorylated cytoplasmic domain of LRP6 in Wnt/beta-catenin signaling[END_REF][START_REF] Wu | Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6[END_REF] and Axin is dephosphorylated further rendering the interaction Figure2. 6 -Overview of Wnt/β-catenin signaling A) In the absence of Wnt, cytoplasmic β-catenin forms a complex with Axin, APC, GSK3 and CK1, is phosphorylated by CK1 and subsequently by GSK3. Phosphorylated β-catenin is recognized by the E3 ubiquitin ligase β-Trcp, which targets β-catenin for proteosomal degradation. Wnt target genes are repressed by TCF-TLE/Groucho and histone deacetylases (HDAC). B) In the presence of Wnt ligand, a receptor complex forms between Fzd and LRP5/6. Dvl recruitment by Fzd leads to LRP5/6 phosphorylation, and Axin recruitment. This disrupts Axin-mediated phosphorylation/degradation of β-catenin, allowing β-catenin to accumulate in the nucleus where it serves as a co-activator for TCF to activate Wnt responsive genes. (From MacDonald et al., 2010).

with β-catenin impossible and thus abrogating the destruction of β-catenin (Kim et al., 2013). The βcatenin is then free to accumulate in the cytoplasm and translocate into the nucleus to act as a transcription factor.

Ligand-independent activation of Wnt/β-catenin signalling

Besides activation of the Wnt/β-catenin pathway by the canonical Wnt/Frizzled/Lrp complexes, βcatenin activity can be regulated upon many stimuli by the action of kinases. For our research purposes, this manuscript essentially focuses on the phosphorylation mediated by Receptor Tyrosine Kinases (RTK). To have a broader view of the mechanisms by which β-catenin can induce transcription in a Wnt-independent fashion, see the review from Shah and Kazi [START_REF] Shah | Phosphorylation-Dependent Regulation of WNT/Beta-Catenin Signaling[END_REF].

Receptor Tyrosine Kinases are a family of transmembrane receptors that function as dimers in response to external cues such as ligands, microenvironment stiffness, osmotic pressure etc. They possess a tyrosine kinase activity which is used both to do autophosphorylation of the receptor to render it active and to phosphorylate downstream targets. It has been shown that many RTKs are able, upon activation, to directly phosphorylate β-catenin (Figure2. 7) (van Veelen et al., 2011). In thyroid carcinoma, the proto-oncogene RTK RET binds to and directly phosphorylates β-catenin on its Tyr654 residue. β-catenin thus escapes degradation and accumulates in the nucleus [START_REF] Gujral | A novel RET kinase-beta-catenin signaling pathway contributes to tumorigenesis in thyroid carcinoma[END_REF]. It has been shown that in intestinal tumours in vivo, the RET-mediated β-catenin phosphorylation can be activated by mechanical stress due to dense stroma leading to an increase of tumorigenicity [START_REF] Fernández-Sánchez | Mechanical induction of the tumorigenic β-catenin pathway by tumour growth pressure[END_REF]. ErbB2 has also been shown to induce β-catenin phosphorylation by activating the soluble kinase Src. UV activation of EGFR in keratinocytes leads to a Wnt-independent localisation of β-catenin in the nucleus through a direct phosphorylation on Tyr654 [START_REF] Jean | Epidermal growth factor receptor/beta-catenin/T-cell factor 4/matrix metalloproteinase 1: a new pathway for regulating keratinocyte invasiveness after UVA irradiation[END_REF].

It has also been shown that the same phosphorylation can be mediated by MET in response to HGF treatment [START_REF] Monga | Hepatocyte growth factor induces Wnt-independent nuclear translocation of beta-catenin after Met-beta-catenin dissociation in hepatocytes[END_REF] and by Ron in response to its ligand HGFL (Wagh et al., 2011), both leading to an increase of nuclear β-catenin and β-catenin-dependent transcriptional activity. The Tyr142 residue of β-catenin can also be directly phosphorylated by a subset of RTKs, namely FGFR2, FGFR3, EGFR, and TRKA. In addition to this phosphorylation, they activate downstream MAPK pathway of which ERK phosphorylates Lrp6, enhancing the effect on Wnt/β-catenin signalling [START_REF] Krejci | Receptor tyrosine kinases activate canonical WNT/β-catenin signaling via MA kinase/LR 6 pathway and direct βcatenin phosphorylation[END_REF]. Several other RTK can lead to the phosphorylation of β-catenin through soluble kinases such as MAP kinases, src and AKT [START_REF] Fang | Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity[END_REF]Greenblatt et al., 2016;[START_REF] Lemieux | Oncogenic KRAS signalling promotes the Wnt/β-catenin pathway through LRP6 in colorectal cancer[END_REF].

In the nucleusβ-catenin as a transcription factor

In the absence of Wnt signal, TCF/LEF (T-Cell Factor/ Lymphoid Enhancing Factor) is associated with Groucho/TLE and acts as a transcriptional co-repressor of Wnt target genes [START_REF] Arce | Groucho binds two conserved regions of LEF-1 for HDACdependent repression[END_REF]Cavallo et al., 1998). When β-catenin enters the nucleus, it displaces this interaction [START_REF] Billin | Beta-catenin-histone deacetylase interactions regulate the transition of LEF1 from a transcriptional repressor to an activator[END_REF][START_REF] Daniels | Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation[END_REF] and creates a complex formed by the scaffold protein BCL9, the PHD finger protein Pygo, TCF/LEF and β-catenin itself [START_REF] Kramps | Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex[END_REF][START_REF] Thompson | A new nuclear component of the Wnt signalling pathway[END_REF]. This complex, also called Wnt enhanceosome (Figure2. 8), promotes the transcription of Wnt target genes [START_REF] Behrens | Functional interaction of beta-catenin with the transcription factor LEF-1[END_REF][START_REF] Molenaar | XTcf-3 transcription factor mediates beta-catenininduced axis formation in Xenopus embryos[END_REF]. The list of Wnt target genes can be found online ("The Wnt Homepage," n.d.). Among these target genes are c-myc [START_REF] He | Identification of c-MYC as a target of the APC pathway[END_REF], cyclinD1 [START_REF] Shtutman | The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway[END_REF][START_REF] Tetsu | Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells[END_REF] both involved in the cell cycle, Axin2 that provides a negative feedback [START_REF] Jho | Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway[END_REF][START_REF] Lustig | Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors[END_REF], and the Epithelial-to-Mesenchymal transcription factors snail, slug, ZEB1 and twist [START_REF] Howe | Twist is up-regulated in response to Wnt1 and inhibits mouse mammary cell differentiation[END_REF][START_REF] Kahlert | Activation of canonical WNT/β-catenin signaling enhances in vitro motility of glioblastoma cells by activation of ZEB1 and other activators of epithelial-to-mesenchymal transition[END_REF][START_REF] Ten Berge | Wnt signaling mediates selforganization and axis formation in embryoid bodies[END_REF]. The target genes strongly differ depending on the tissue; however, it was shown that the transcription of Axin2 is ubiquitously regulated by β-catenin/TCF and can serve as a potent marker of the pathway activity. The impact of β-catenin-induced rearrangements of chromatin to ensure the transcription of target genes have been reviewed by Mosimann [START_REF] Mosimann | Beta-catenin hits chromatin: regulation of Wnt target gene activation[END_REF].

The β-catenin/TCF complex can also repress transcription by competition with other activators or via recruitment of corepressors. Among the genes repressed by β-catenin are components of antagonists pathways of Wnt/β-catenin including Hedgehog and BMP pathways [START_REF] Baker | Wnt signaling in Xenopus embryos inhibits bmp4 expression and activates neural development[END_REF][START_REF] Kahler | Lymphoid enhancer factor-1 and beta-catenin inhibit Runx2dependent transcriptional activation of the osteocalcin promoter[END_REF][START_REF] Piepenburg | Drosophila segment borders result from unilateral repression of hedgehog activity by wingless signaling[END_REF][START_REF] Yang | decapentaplegic is a direct target of dTcf repression in the Drosophila visceral mesoderm[END_REF], and epithelial markers such as CDH1, encoding E-cadherin [START_REF] Jamora | Links between signal transduction, transcription and adhesion in epithelial bud development[END_REF]. Furthermore, the team has shown that the potassium channel KCNQ1 represses Wnt/β-catenin activity thus guaranteeing tissue homeostasis, which will be discussed further, and that the activation of Wnt/β-catenin leads to the TCF4-dependent repression of KCNQ1 transcription in colorectal cancer cells (Rapetti-Mauss et al., 2017).

Therefore, upon activation, the Wnt/β-catenin pathway regulates, at the transcriptional level, the expression of proteins involved in cell proliferation, epithelial-to-mesenchymal transition, maintenance of stemness and differentiation.

Regulation of the Wnt pathway

The Wnt/β-catenin pathway is tightly regulated to ensure proper functioning. Many proteins and complexes are involved in this regulation, the most important ones will be discussed here.

R-spondins and ZNRF3/RNF43

Zinc Ring Finger 3 (ZNRF3) and Ring Finger 43 (RNF43) are single-pass transmembrane E3 ubiquitin ligases. Upon activation of the pathway, these proteins target Frizzled for lysosomal degradation [START_REF] Koo | Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors[END_REF]. It seems that the recruitment of the ubiquitin ligases is mediated by Dishevelled when bound to Frizzled [START_REF] Jiang | Dishevelled promotes Wnt receptor degradation through recruitment of ZNRF3/RNF43 E3 ubiquitin ligases[END_REF]. In addition, it was shown that ZNRF3/RNF43 are transcriptional targets of β-catenin-mediated transcription, providing negative feedback on the pathway activity (Figure2. 9) (de [START_REF] De Lau | The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength[END_REF]Lau et al., , 2011)).

R-spondins are secreted factors that were first discovered as positive effectors of Wnt signalling [START_REF] Kazanskaya | R-Spondin2 Is a Secreted Activator of Wnt/β-Catenin Signaling and Is Required for Xenopus Myogenesis[END_REF]. R-spondins bind to their transmembrane receptor Lgr4-6 (Leucine-rich repeat containing G-protein-coupled receptor) which is in close proximity with the Frizzled/Lrp complexes [START_REF] Carmon | R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling[END_REF][START_REF] Glinka | LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling[END_REF]. When R-spondins bind to Lgr4-6, the activity of ZNRF3/RNF43 is inhibited which leads to the accumulation of Frizzled at the plasma membrane, thus enhancing Wnt activity [START_REF] Hao | ZNRF3 promotes Wnt receptor turnover in an R-spondinsensitive manner[END_REF]. In addition, it was been shown that RspoI inhibits the internalisation of Lrp6 induced by DKK1/Kremen, thus maintaining Lrp6 at the cell membrane and further enhancing Wnt/β-catenin activity [START_REF] Binnerts | R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6[END_REF].

YAP/TAZ

YAP/TAZ are transcriptional regulators of the Hippo pathway [START_REF] Pan | The hippo signaling pathway in development and cancer[END_REF] and are implicated in the Wnt pathway, providing crosstalk between these two developmental signalling activities. In the absence of Wnt ligands, YAP/TAZ is part of the destruction complex and helps recruit β-TrCP, thus further repressing Wnt signalling. However, in the presence of Wnt ligands, YAP/TAZ and β-TrCP get displaced from the destruction complex. In this isolated form, YAP/TAZ translocate into the nucleus and act as positive transcriptional effectors of Wnt signalling [START_REF] Azzolin | YAP/TAZ incorporation in the b-catenin destruction complex orchestrates the Wnt response[END_REF]Rosenbluh et al., 2012).

Soluble inhibitors

Secreted proteins can also interact with the Wnt/β-catenin pathway. Among those, WIFs (Wnt Inhibitory Protein) and sFRPs (secreted Frizzled-related proteins) can bind Wnt ligands and prevent their activation of Frizzled [START_REF] Bovolenta | Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease[END_REF][START_REF] Hoang | Primary structure and tissue distribution of FRZB, a novel protein related to Drosophila frizzled, suggest a role in skeletal morphogenesis[END_REF][START_REF] Hsieh | A new secreted protein that binds to Wnt proteins and inhibits their activities[END_REF]. Another family of Wnt inhibitors are DKKs (Dickkopf) [START_REF] Glinka | Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction[END_REF]. DKK1 for example impedes the complex formed by Frizzled/Lrp complex through its interaction with Lrp [START_REF] Bafico | Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow[END_REF][START_REF] Mao | LDL-receptor-related protein 6 is a receptor for Dickkopf proteins[END_REF][START_REF] Semënov | Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6[END_REF]. Through its binding to the receptor Kremen, DKK1 induces the internalization of Lrp5/6 for degradation therefore reducing the availability of the Wnt receptors at the plasma membrane [START_REF] Mao | Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling[END_REF].

Wnt and colorectal cancer

In 1998, a study showed that mutations in mouse TCF4 led to a loss of intestinal stem cells and disruption of tissue architecture [START_REF] Korinek | Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4[END_REF]. As Wnt/β-catenin signalling is of crucial importance in the maintenance of intestinal stem cells (for review [START_REF] Clevers | Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control[END_REF]), a dysregulation of the activity of this pathway leads to many defects including cancer. In colorectal cancer about 90% of the tumours harbour mutations on Wnt pathway components (Cancer Genome Atlas Network, 2012). Animal models showed that Wnt activation through disruption of APC or activation of β-catenin was sufficient to induce hyperproliferation and inhibit differentiation in the intestinal crypts [START_REF] Barker | Crypt stem cells as the cells-oforigin of intestinal cancer[END_REF]Dow et al., 2015;[START_REF] Sansom | Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration[END_REF]. APC min mice have become a standard model for the study of intestinal tumours.

The first Wnt gene (Wnt1) -formerly Int-1 -was identified as a gene activated by the integration of a virus inducing breast tumours in mice (Nusse and Varmus, 1982) underlying the implication of the Wnt/β-catenin pathway in cancer. In the early 1990s, the adenomatous polyposis coli (APC) gene was identified in a hereditary colon cancer syndrome called familial adenomatous polyposis (Kinzler et al., 1991;Nishisho et al., 1991). A couple years later, APC was found to interact with β-catenin [START_REF] Munemitsu | Regulation of intracellular betacatenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein[END_REF][START_REF] Rubinfeld | Association of the APC gene product with beta-catenin[END_REF][START_REF] Su | Association of the APC tumor suppressor protein with catenins[END_REF] linking APC-induced colon cancer to the Wnt/βcatenin pathway. Since then, mounting evidence have shown a link between a dysregulated Wnt/βcatenin pathway and colorectal cancer. Wnt has been thoroughly linked to progression of colorectal cancer, but a wide variety of cancers also display a dysregulation of this pathway, for review see (Clevers and Nusse, 2012;Zhan et al., 2017). The molecular basis for Wnt-induced colorectal cancer is the high frequency of mutations on genes encoding Wnt pathway components. APC is the gene that carries the highest frequency of mutations in colorectal cancer -over 70% (Wood et al., 2007).

Mutations on AXIN2 have the same effects [START_REF] Liu | Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating beta-catenin/TCF signalling[END_REF] and patients carrying hereditary Axin2 mutations display a predisposition to colorectal cancer [START_REF] Lammi | Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer[END_REF]. Mutations on β-catenin have also been found (Rubinfeld et al., 1997) and account for approximately 7% of colorectal cancers.

Studies have shown that 17% of colorectal cancers exhibit mutations on RNF43 and 10% on Rspondins and both are mutually exclusive with APC mutations [START_REF] Giannakis | RNF43 is frequently mutated in colorectal and endometrial cancers[END_REF][START_REF] Seshagiri | Recurrent R-spondin fusions in colon cancer[END_REF]. The results of different studies are continuously updated in cbioportal which grants access to the mutational landscape of tumours. With the different cohorts, the percentages of mutations on each gene harboured by colorectal cancer patients varies. The most frequent mutations in colorectal cancer and the ones on Wnt pathway component genes have been summarized in Table 1 ("cBio ortal for Cancer Genomics," n.d.).

The high frequency of mutation on TP53, KRAS and SMAD4 have for consequence an increase in tumour progression towards malignancy. Indeed, it is the global mutations harboured by a tumour cell that grants it its tumoral features. Most mutations in RNF43 and RSPO necessitate the binding of Wnt ligands and are thus sensitive to drugs blocking the production of Wnts. Lossof-function of APC and Axin reduce greatly the degradation of β-catenin thus increasing its cytosolic stability. The most common outcome of mutations on CTNNB1 encoding β-catenin is to lower the sensitivity to proteasomal degradation. Therefore mutations on Wnt component genes have a common impact: enhance Wnt/β-catenin activity mainly through the stabilization of βcatenin in the cytosol that favours its association with TCF/LEF and the subsequent transcription of target genes [START_REF] Parsons | WNT as a driver and dependency in cancer[END_REF]. There are two main paths of Wnt components mutations that lead to an overactivated pathway and ultimately to cancer. One induces the cytosolic stabilisation of β-catenin exemplified by mutations of the destruction complex proteins such as APC and Axin; and the other induces a dysregulation of the pathway activity, this is the case of mutations on ZNRF3, RNF43 [START_REF] Bugter | Mutations and mechanisms of WNT pathway tumour suppressors in cancer[END_REF].

Among the target genes, some encode proteins implicated in the cell cycle (CyclinD1, c-myc) and epithelial-to-mesenchymal transition (Snail, Twist) and stem cells markers (Lgr5). Thus, an overactivated Wnt/β-catenin pathway leads to uncontrolled proliferation, poor differentiation; in other words, it grants stemness features to cancer cells without homeostatic control. Moreover, it has been shown that colon stem cells are the origin of colorectal cancer [START_REF] Barker | Crypt stem cells as the cells-oforigin of intestinal cancer[END_REF][START_REF] Schepers | Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas[END_REF] and expression of intestinal stemness markers is associated with poor prognosis in colorectal cancer [START_REF] De Sousa E Melo | Methylation of cancer-stem-cell-associated Wnt target genes predicts poor prognosis in colorectal cancer patients[END_REF][START_REF] Merlos-Suárez | The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse[END_REF].

Interestingly, in colorectal cancer cell lines and in vivo models with APC mutation, the rescue of functional wild type APC is sufficient to downplay Wnt signalling, express differentiation markers and reduce tumour growth, even in the presence of oncogenic Kras and p53 mutations (Dow et al., 2015;[START_REF] Faux | Restoration of full-length adenomatous polyposis coli (APC) protein in a colon cancer cell line enhances cell adhesion[END_REF].

The mutational landscape of cancer cells is not the sole responsible for cancer progression in a Wntinduced manner. In the past twenty years, the role of the tumour microenvironment has proven crucial in the progression of all types of cancer, including colorectal cancer. Since the niche is of crucial importance in the maintenance of the crypt and stem cells, it is just as crucial in the cancer stem cells and has a huge impact on the development of the tumour and its progression. For example, a study showed that hepatocyte growth factor produced by the stromal myofibroblasts increases Wnt activity and induces stemness features in colorectal cancer [START_REF] Vermeulen | Wnt activity defines colon cancer stem cells and is regulated by the microenvironment[END_REF].

The activation of signalling pathways by biochemical signals such as hormones and neurotransmitters have been extensively studied; nonetheless, physical cues can also impact signalling pathways.

Mechanical pressure for example has proven to activate YAP/TAZ and integrin signalling [START_REF] Kechagia | Integrins as biomechanical sensors of the microenvironment[END_REF][START_REF] Panciera | Mechanobiology of YAP and TAZ in physiology and disease[END_REF]. In colorectal cancer, the stiffness of the tissue surrounding the tumour, mimicked by injected magnets in the crypts of APC min mice, induces the mechanical activation of the receptor tyrosine kinase RET. Activated RET directly phosphorylates β-catenin on the Tyr654 residue, promoting its nuclear translocation [START_REF] Fernández-Sánchez | Mechanical induction of the tumorigenic β-catenin pathway by tumour growth pressure[END_REF][START_REF] Ho-Bouldoires | Ret kinase-mediated mechanical induction of colon stem cells by tumor growth pressure stimulates cancer progression in vivo[END_REF].

Ion channels are mediators of the crosstalk between the environment and the cells and sense both physical and chemical cues. They have thus emerged recently as key proteins in cancer. In colorectal cancer, a subset of ion channels have been shown to impact Wnt/β-catenin signalling ((Rapetti-Mauss et al., 2020) Annex n°1). Nonetheless, these findings underline a knowledge gap in the mechanisms of signal transduction. The variety of roles held by ion channels in signal transduction is yet to be defined.

Chapter III -Ion channels

Ion channels and Membrane potential

Ion channels represent a class of pore-forming transmembrane proteins which role is to allow the passage of ions through the plasma membrane upon stimulation. Through this role, ion channels control several parameters including cell excitability, water and electrolytes transport, cell volume, hormone secretion, and signalling pathways. Ion channels can be divided into classes according to their gating, their selectivity, their molecular nature, or their pharmacology. The selectivity of ion channels for a particular ion is dependent on the selectivity filter created by the amino acids forming the pore.

Ion channels can thus be divided into chloride channels, sodium channels, potassium channels, calcium channels, proton channels and non-selective cationic channels which have poor selectivity and let through positively charges ions. Each of these families is further subdivided according to their gating properties including ligand-, voltage-, Ca ++ -dependent activation, mechanical activation and/or by their molecular structure. Each channel has a specific electrophysiological signature and can be activated or inhibited by a variety of molecules.

The plasma membrane is impermeable to ions causing a difference in their intracellular and extracellular concentrations (Table 2). The unequal distribution of ions between the inside and outside of the cell and the selective permeability of the cell membrane to these ions -electrochemical gradient

-results in an electric potential called membrane potential. Of note, these differences and the existence of an electric potential is also true for intracellular organelles such as the endoplasmic reticulum, mitochondria, and lysosomes. Each ion has an "equilibrium potential" -that can be calculated by Nernst Equation -which is the potential at which the electrical and chemical gradients are equilibrated, and the net flux is null (Equation 1). In a cell, all these equilibrium potentials are present, thus the perfect equilibrium needs to be found by considering the permeability of each ion. Two equations have been proposed to model the membrane potential equilibrium -the Goldman-Hodgkin-Katz (GHK) equation that uses the permeabilities (Equation 2) and the Hodgkin and Huxley equation using the equilibrium potential and conductance. Although these equations are simplified model that cannot integrate the wide variety of currents exhibited by ion channels, they are still widely used to calculate the theoretical resting membrane potential; the GHK equation being the most used model. To simplify and summarize, in resting state, leak potassium channels -constitutively opened channels -keep a constant efflux of potassium which is thus the main conductance at play. In these conditions, resting membrane potential is close to the equilibrium potential of potassium. In nonexcitable cells, resting membrane potential usually ranges from -70 to -50mV. Upon opening of an ion channel, the permeability and conductance change for that ion, thus impacting the membrane potential. The current that passes through the channel depends on the driving force which is the difference between the membrane potential and the equilibrium potential of the ion (Equation 3). If both are equal, the driving force is null, and no current is observed.

𝐸 𝑖𝑜𝑛 = 𝑅𝑇 𝑧𝐹 ln [𝑖𝑜𝑛]𝑒𝑥𝑡 [𝑖𝑜𝑛]int

𝐷𝑓 = 𝐸 𝑚 -𝐸 𝑖𝑜𝑛

Equation 3 -The driving force. The driving force is the equivalent of U in the Ohm law. It depends on the imposed membrane potential and the equilibrium potential of a particular ion. The driving force can be calculated for each ionic specie at play. From the Ohm law, the driving force is the multiplication of the current and the resistance.

The ion channels expressed differ according to the cell type and tissue. The ancillary subunits, able to change the properties of channels, are also expressed in different patterns. Therefore, the "electrical signature" of a cell type is unique and confers the cell its features.

Potassium Channels

Potassium channels represent the largest and most diverse family of ion channels. About 78 genes encode pore-forming subunits and about 13 genes encode ancillary subunits (Figure 3. 1) [START_REF] Heitzmann | Physiology and pathophysiology of potassium channels in gastrointestinal epithelia[END_REF]. These channels play essential roles in the maintenance of resting membrane potential in all the cells. They are necessary for cardiac and neuronal action potentials, muscle contraction, insulin secretion, water and electrolyte absorption and secretion, and cell proliferation.

The selectivity of potassium channels is conferred by three amino acids in the pore loop: G Y G [START_REF] Heginbotham | Mutations in the K+ channel signature sequence[END_REF] and four pore loops are necessary to form a functional channel [START_REF] Papazian | Potassium channels: some assembly required[END_REF]. Potassium channels can be divided into three subfamilies based on their molecular structure: 6/7 transmembrane domains with one pore loop channels (6/7TM 1P), 4 transmembrane domains and 2 pore loops channels (4TM 2P), and 2 transmembrane domains and 1 pore loop channels (2TM 1P).

2TM 1P Channels (Kir)

The KCNJ family (Kir) contains 16 members which exhibit a specific inward rectifying current. The subunits assemble in homo-or hetero-tetramer and are voltage-independent [START_REF] Heitzmann | Physiology and pathophysiology of potassium channels in gastrointestinal epithelia[END_REF]. At values below the potassium equilibrium potential, Kir channels are open and show a linear current. Upon reversal of the current, the current becomes inward rectified, very small and is almost null at depolarized values. The inward rectification is due to intracellular magnesium and polyamines that physically block the pore [START_REF] Lopatin | Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification[END_REF][START_REF] Matsuda | Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+[END_REF]. This feature enables a fine control of the resting membrane potential. Kir channels thus participate in setting membrane potential close to EK in both excitable and non-excitable cells. As an example, Kir6 forms the KATP channel when expressed with the sulfonylurea receptor (SUR) and is involved in insulin secretion in the β-cells of the islets of Langherans [START_REF] Denton | Special collection on inward rectifying K+ channels[END_REF][START_REF] Inagaki | Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor[END_REF].

4TM 2P Channels (K2P)

K2P (KCNK) channels have the particularity of possessing two pore domains. Since four pore domains are necessary to form a functional pore, these subunits assemble in homo-or hetero-dimer. KCNK are "leak" potassium channels and contribute to the background potassium conductance. KCNK family is composed of 15 members that can be regulated by a variety of stimuli such as phosphorylation, pH, and lipids [START_REF] Lesage | Molecular and functional properties of two-pore-domain potassium channels[END_REF]. These channels display a linear current and participate to setting membrane potential. In excitable cells, they hyperpolarise the current after the action potential. Their role in setting membrane potential is also crucial in non-excitable cells. For example, KCNK5 is expressed in the colonic epithelium on the basolateral side of the cells where it plays a role both in setting membrane potential and in recycling potassium for the absorption and secretion of electrolytes and water (Julio-Kalajzić et al., 018).

6/7TM 1P Channels (Kv, Kca)

Voltage-gated potassium channels are the largest and best characterized family of potassium channels.

40 genes encode these subunits composed of six transmembrane domains, one pore loop. Both Cterminal and N-terminal ends are cytosolic. A sequence of positively charged residues in the 4 th transmembrane segment (S4) senses the changes in membrane potential and is known as the voltagesensor domain (Jiang et al., 2003a(Jiang et al., , 2003b)). Upon membrane depolarisation, the conformation of the S4 shifts, modulating the channel conductance [START_REF] Catterall | Ion channel voltage sensors: structure, function, and pathophysiology[END_REF]. Because of their voltage-dependence, these potassium channels are preferably expressed in excitable cells and play an essential role in repolarisation of the plasma membrane during the action potential. Apart from their role in excitable cells, these channels are involved in a variety of cell function in non-excitable cells including substrate transport, cell volume regulation, cell migration, wound healing, proliferation, apoptosis, oxygen sensing and carcinogenesis [START_REF] Heitzmann | Physiology and pathophysiology of potassium channels in gastrointestinal epithelia[END_REF].

The second subfamily of 6/7TM 1P potassium channels is the Ca 2+ -activated K + channel group made itself of two subgroups: the small and intermediate conductance potassium channels KCNN1 to 4 and the large conductance potassium channels KCNMA1, KCNT1-2 and KCNU1. The KCNN family senses the intracellular concentration of Ca 2+ via physical interaction with the calmodulin [START_REF] Fanger | Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1[END_REF].

KCNMA1 possesses an intrinsic Ca 2+ sensor in its C-terminal domain (Bao et al., 2004). KCNN channels intervene in a wide range of processes such as red blood cell volume homeostasis [START_REF] Allegrini | New KCNN4 Variants Associated With Anemia: Stomatocytosis Without Erythrocyte Dehydration[END_REF][START_REF] Rapetti-Mauss | A mutation in the Gardos channel is associated with hereditary xerocytosis[END_REF] and cancer (Rapetti-Mauss et al., 2023) (Gueguinou et al., 2017).

Ion channels and cancer

With the wide diversity of families, each cell type has its own ion channel expression pattern. In the 1980s, the pioneer work of DeCoursey shed light on the role of potassium channels in T-lymphocytes proliferation (DeCoursey et al., 1984). In the following years, it became clear that ion channels have a role in the proliferation of cancer cells and that a pharmacological blockade of some channels results in a reduction of proliferation (Lee et al., 1988;Pancrazio et al., 1989;Taylor and Simpson, 1992;Yamashita et al., 1987). Since then, the implication of ion channels in cancer has been studied deeply and linked with cancer hallmarks. The hallmarks of cancer, described by Hanahan and Weinberg, are the key features that a cell acquires on its way towards malignancy (Figure 3. 2) (Hanahan andWeinberg, 2011, 2000). These features include evasion from cell death, sustained proliferation, promoted angiogenesis, and invasion and metastasis. Dysregulated expression or function of ion channels have been associated with all the hallmarks of cancer and could even be considered as the source of this acquired cancer feature. The term "oncochannelopathies" have been proposed to describe the hallmarks of cancer (Figure 3. 3) (Prevarskaya et al., 2018). [START_REF] Hanahan | The Hallmarks of Cancer[END_REF]. Right panel: Additional cancer hallmarks [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF].

Given the great expansion of the field of ion channels and cancer, the wide variety of ion channels and their regulation, and the different cancer types, it would be near impossible to discuss all the works in this manuscript. In a few words, ion channels can exert their effect through different mechanisms including calcium signalling, cell volume regulation, mechanosensitivity, regulation of the microenvironment, ion-permeation-independent mechanism (direct interaction with other proteins), and membrane potential regulation. For review see [START_REF] Becchetti | Ion channels and transporters in cancer. 1. Ion channels and cell proliferation in cancer[END_REF][START_REF] Besson | How do voltage-gated sodium channels enhance migration and invasiveness in cancer cells?[END_REF]Bortner and Cidlowski, 014;Déliot and Constantin, 015;Fiorio la and Munaron, 014;[START_REF] Lehen'kyi | Ion channnels and transporters in cancer. 5. Ion channels in control of cancer and cell apoptosis[END_REF]Prevarskaya et al., 2018;[START_REF] Schwab | Ion channels and transporters in tumour cell migration and invasion[END_REF]. For a review focusing specifically on the role of ion channels in gastrointestinal cancers see [START_REF] Anderson | Role of ion channels in gastrointestinal cancer[END_REF].

In addition to more or less direct roles of ion channels in signalling pathways and cancer biology, the expressed ion channels dictate a cell's resting membrane potential, which might be far more important than previously thought in non-excitable cells.

While it is broadly accepted that membrane potential changes occur in excitable cells for action potential initiation and propagation and muscle contraction, membrane potential changes in nonexcitable cells remain understudied and underappreciated. It was shown that non-excitable cells undergo changes in membrane potential -accompanied by cell volume modification -along the cell cycle [START_REF] Blackiston | Bioelectric controls of cell proliferation: ion channels, membrane voltage and the cell cycle[END_REF][START_REF] Sundelacruz | Role of membrane potential in the regulation of cell proliferation and differentiation[END_REF]. Proliferative cells thus show a more depolarised resting membrane potential than quiescent cells; suggesting that membrane potential is inversely coupled to proliferative pathways. Changes in resting membrane potential also impact directional cell polarisation, apoptosis, differentiation, and migration [START_REF] Atsuta | L-type voltage-gated Ca2+ channel CaV1.2 regulates chondrogenesis during limb development[END_REF][START_REF] Cavallo | %2Cntrk_msk_ 2019%2Cmsk_ch_2020%2Cpan_origimed_2020%2Cpancan_pcawg_2020%2Cmsk_met_2021 %2Ccoadread_dfci_2016%2Ccoadread_genentech%2Ccoadread_mskcc%2Ccoadread_mskres istance_2022%2Ccoadread_tcga%2Ccrc_apc_impact_2020%2Ccrc_dd_2022%2Ccrc_nigerian _2020%2Ccrc_msk_2017%2Crectal_msk_2019%2Ccoad_caseccc_2015%2Ccoad_cptac_2019[END_REF]. On a larger scale, bioelectrical signals have been suggested to enable tissue coordination to enable regeneration, answer to cues etc., review in [START_REF] Levin | Bioelectric signaling: Reprogrammable circuits underlying embryogenesis, regeneration, and cancer[END_REF]. The molecular mechanism linking bioelectrical changes and cell fate are yet to be understood. Nonetheless, the past few years have seen a growing body of evidence suggesting that membrane potential variations are cues for signalling pathways activation. Contrarily to previously published work on the matter, the activation of MAPK pathway was independent of Ca ++ , suggesting that depolarisation can influence signalling pathways independently of the increased calcium entry driving force [START_REF] Accardi | Lipids link ion channels and cancer[END_REF]Zhou et al., 2015).

Since then, different teams have identified links between membrane potential changes and signalling pathways activation. In xenopus embryo, a hyperpolarised membrane potential at gastrulation is necessary to downregulate mTOR signalling and thus suppress pluripotency genes to allow entry in differentiation. Although it seems that calcium might mediate part of the effect, RNA-Seq of high K + depolarised embryos vs control ones show that over 4000 genes were overexpressed including clusters involved in pluripotency maintenance and Wnt signalling [START_REF] Sempou | Membrane potential drives the exit from pluripotency and cell fate commitment via calcium and mTOR[END_REF]. Membrane potential has also been linked to Hedgehog (Hh) signalling in the developing disc of Drosophila by two independent teams [START_REF] Spannl | Glycolysis regulates Hedgehog signalling via the plasma membrane potential[END_REF]. Briefly, Hh inhibits Patched activity, thereby stabilising Smoothened at the plasma membrane. Smoothened then blocks the proteasomal degradation of the transcription factor Cubitus interruptus/Gli (in Drosophila and vertebrates respectively). Cubitus interruptus accumulates in its long form, switching from a transcriptional repressor to a transcriptional activator. The studies show that an inhibition of glycolysis reduces steady-state levels of ATP and depolarises the cells probably by lowering the ATP available for the Na + /K + /ATP-ase. The inhibition of 2015). -It must be noted that some interpretations of this commentary of Zhou, 2015[START_REF] Accardi | Lipids link ion channels and cancer[END_REF] are inaccurate: overexpression of all ion channels do not induce a depolarisation and depolarisation can be caused by loss of leak potassium channels.

There is a plethora of ways an ion channel can impact cell processes that are involved in cancer. Ectopic expression of an ion channel or loss of expression of another one can have serious impacts on cell cycle, signalling pathways, escape from apoptosis and immune system activation. These studies offer new therapeutic strategies to target ion channels and hopefully limit the progression of cancer.

Nevertheless, only down-or up-regulation of ion channels have been studied and cancer-associated mutations of ion channels have not been investigated thus far.

Chapter IV -KCNQ1: role and functions 1. KCNQ1 -a multifunctional channel

Among the voltage-gated potassium channel family, KCNQ1 -also known as KvLQT1 or Kv7.1, is a versatile channel that belongs to the KCNQ family. Contrarily to KCNQ2-5, KCNQ1 only functions as a homotetramer and is not expressed in the brain except in the choroid plexus, an epithelial tissue [START_REF] Roepke | KCNE2 forms potassium channels with KCNA3 and KCNQ1 in the choroid plexus epithelium[END_REF]. KCNQ1 is expressed in several tissues including colon and kidney epithelia, heart, and inner ear, where it assumes very different roles.

KCNQ1 was first cloned in 1996 in Long QT Syndrome (LQT) patients by Wang and co-workers using positional cloning methods [START_REF] Wang | Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias[END_REF]. This gene was described as a cardiac potassium channel which caused susceptibility to arrythmias and sudden death when mutated and thus called KvLQT1. (From Jespersen et al., 2005).

KCNQ1 is about 400kb and composed of 16 exons [START_REF] Splawski | Genomic structure of three long QT syndrome genes: KVLQT1, HERG, and KCNE1[END_REF]. Five isoforms have been identified but only isoform 1 gives a functional channel (Jespersen et al., 2005).

Isoform 1 translates in a 676 amino acid protein (68 kDa) that possesses six transmembrane domains including the S4 voltage-sensor domain and one pore loop between S5 and S6 containing the typical GYG K + selectivity filter motif. Both the N-terminal and C-terminal domains are intracellular (Figure 4. 1). These domains exhibit sites of channel regulation that are discussed further.

Expressed alone, KCNQ1 shows a voltage-dependent current, with a small conductance of 1.8 to 3.2 pS which is activated around -40mV [START_REF] Seebohm | Mutation of colocalized residues of the pore helix and transmembrane segments S5 and S6 disrupt deactivation and modify inactivation of KCNQ1 K+ channels[END_REF]. It exhibits a potassium current with a slow activation and deactivation (Figure 4. 3 A). The S4 voltage-sensor domain of KCNQ1 possess 4 positive charges compared to the usual six of other Kv [START_REF] Catterall | Ion channel voltage sensors: structure, function, and pathophysiology[END_REF]. This particularity might play a role in the KCNE-mediated modulations of voltage-sensitivity [START_REF] Panaghie | The role of S4 charges in voltage-dependent and voltageindependent KCNQ1 potassium channel complexes[END_REF]. The S4-S5 linker plays a major role in channel gating [START_REF] Franqueza | Long QT syndrome-associated mutations in the S4-S5 linker of KvLQT1 potassium channels modify gating and interaction with minK subunits[END_REF]. A small fraction of the KCNQ1 channels inactivate under a long depolarising step [START_REF] Splawski | Mutations in the hminK gene cause long QT syndrome and suppress IKs function[END_REF].

KCNQ1 is always expressed along with one of its β-subunit called KCNE1-5 [START_REF] Pongs | Ancillary subunits associated with voltage-dependent K+ channels[END_REF].

According to the associated subunit, the electrophysiological properties of KCNQ1 change drastically.

KCNQ1:KCNE1

KCNE1 was identified a few years before the identification of KCNQ1, in 1988[START_REF] Takumi | Cloning of a membrane protein that induces a slow voltage-gated potassium current[END_REF]. The expression of this protein in Xenopus oocytes displayed a voltage-dependent potassium current. It was thought that KCNE1 -then called IsK or Minimal K + channel (MinK) [START_REF] Kaczmarek | Voltage-dependent potassium channels: minK and Shaker families[END_REF] -was carrying that potassium current. However, no current was displayed in COS or CHO cells and the structure of KCNE1 was far from the usual K + channels: with only a single transmembrane pass, it was unclear how it could form a K + -selective pore. It was only after the cloning of KCNQ1 that two independent labs showed that the cardiac delayed rectified current Iks was due to the association between the α subunit KCNQ1 and the β subunit KCNE1 [START_REF] Barhanin | K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current[END_REF][START_REF] Sanguinetti | Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel[END_REF], the α subunit being expressed endogenously in Xenopus oocytes.

The co-assembly of KCNQ1 and KCNE1 gives rises to an increase in the macroscopic current and a positive shift in voltage activation threshold (around -20mV). The increase of macroscopic current is due to a four-to-seven fold increase in single channel conductance [START_REF] Pusch | Increase of the single-channel conductance of KvLQT1 potassium channels induced by the association with minK[END_REF][START_REF] Sesti | Single-channel characteristics of wild-type IKs channels and channels formed with two minK mutants that cause long QT syndrome[END_REF][START_REF] Yang | Single-channel properties of IKs potassium channels[END_REF]. In addition, KCNE1 slows the activation and deactivation kinetics of KCNQ1 and almost completely represses the inactivation (Figure 4. 3 B) [START_REF] Splawski | Mutations in the hminK gene cause long QT syndrome and suppress IKs function[END_REF].

In the heart, KCNQ1:KCNE1 contributes to ventricular repolarization in cardiac action potential by the generation of the IKs current [START_REF] Barhanin | K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current[END_REF][START_REF] Sanguinetti | Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel[END_REF]. Mutations on either KCNQ1 or KCNE1 can perturb the action potential repolarisation and lead to disorders such as the Long QT Syndrome characterized by abnormally long ventricular repolarisation resulting in arrythmias and eventually ventricular fibrillation and sudden death [START_REF] Charpentier | Delayed rectifier K(+) currents and cardiac repolarization[END_REF][START_REF] Crump | Arrhythmogenic KCNE gene variants: current knowledge and future challenges[END_REF]. The autosomal dominant form causes Romano-Ward Syndrome while the autosomal recessive form causes Jervell and Lange-Nielsen Syndrome [START_REF] George | Molecular and genetic basis of sudden cardiac death[END_REF]. KCNQ1:KCNE1 has also been found to be expressed in the inner ear where it contributes to K + secretion in endolymph of vestibular dark cells [START_REF] Bleich | The very small-conductance K+ channel KvLQT1 and epithelial function[END_REF][START_REF] Vetter | Inner ear defects induced by null mutation of the isk gene[END_REF][START_REF] Wangemann | Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro[END_REF]. Mutations in either KCNQ1

or KCNE1 genes can thus cause deafness: Jervell and Lange-Nielsen patients show a bilateral deafness in addition to the LQTS [START_REF] Schulze-Bahr | KCNE1 mutations cause jervell and Lange-Nielsen syndrome[END_REF][START_REF] Tyson | IsK and KvLQT1: mutation in either of the two subunits of the slow component of the delayed rectifier potassium channel can cause Jervell and Lange-Nielsen syndrome[END_REF].

KCNE2-5 were discovered in the late 1990s [START_REF] Abbott | MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia[END_REF][START_REF] Mccrossan | The MinK-related peptides[END_REF][START_REF] Piccini | KCNE1-like gene is deleted in AMME contiguous gene syndrome: identification and characterization of the human and mouse homologs[END_REF].

KCNQ1:KCNE2

KCNE2 is expressed in the heart and most importantly apically in gastric parietal cells where it contributes to the proton secretion to acidify the gastric content. The apical K + secretion through KCNQ1:KCNE2 is necessary for the function of the apical H + /K + /ATPase which secretes proton in the gastric lumen in exchange for K + (Grahammer et al., 2001a;[START_REF] Heitzmann | Heteromeric KCNE2/KCNQ1 potassium channels in the luminal membrane of gastric parietal cells[END_REF][START_REF] Roepke | The KCNE2 potassium channel ancillary subunit is essential for gastric acid secretion[END_REF].

When expressed with KCNE2, KCNQ1 shows a weak macroscopic current with instantaneous activation and rapid deactivation (Figure 4. 3 C). The channel becomes constitutively open over the range of membrane potentials in gastric parietal cells with a linear current voltage relationship [START_REF] Tinel | KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel[END_REF]. Furthermore, KCNQ1 is inhibited by external protons but with KCNE2, the channel becomes activated by acidic extracellular pH, thus rendering possible its activity in the gastric epithelium [START_REF] Heitzmann | KCNE beta subunits determine pH sensitivity of KCNQ1 potassium channels[END_REF]. To further support the indispensable role of KCNQ1:KCNE2 in gastric epithelium, the first KCNQ1 knockout mice exhibited gastric hyperplasia and hypochlorhydria (pH 6-7

instead of 1-2) [START_REF] Lee | Targeted disruption of the Kvlqt1 gene causes deafness and gastric hyperplasia in mice[END_REF]. Since then, it was shown that targeted disruption of KCNE2 induces gastric neoplasia and low expression of KCNE2 is associated with progression of gastric cancer [START_REF] Abbott | KCNE2 and gastric cancer: bench to bedside[END_REF][START_REF] Roepke | Targeted deletion of Kcne2 causes gastritis cystica profunda and gastric neoplasia[END_REF][START_REF] Yanglin | KCNE2, a down-regulated gene identified by in silico analysis, suppressed proliferation of gastric cancer cells[END_REF]. Thus, KCNQ1:KCNE2 is necessary for parietal cells function and seems to protect against gastric cancer. KCNQ1:KCNE2 is expressed in the thyroid where it is activated by Thyroid Stimulating Hormone (TSH)

and is required for iodide uptake. A disruption of KCNE2 in this context impairs the biosynthesis of thyroid hormones leading to hypothyroidism and all its systemic effects [START_REF] Purtell | The KCNQ1-KCNE2 K + channel is required for adequate thyroid Iuptake[END_REF][START_REF] Roepke | Kcne2 deletion uncovers its crucial role in thyroid hormone biosynthesis[END_REF].

KCNQ1:KCNE2 is also present in cardiomyocytes acting as a negative regulator of IKs current. It is even suggested that a tripartite complex composed of KCNQ1, KCNE1 and KCNE2 mediate IKs currents [START_REF] Jiang | Dynamic partnership between KCNQ1 and KCNE1 and influence on cardiac IKs current amplitude by KCNE2[END_REF][START_REF] Wu | KCNE2 is colocalized with KCNQ1 and KCNE1 in cardiac myocytes and may function as a negative modulator of I(Ks) current amplitude in the heart[END_REF]. For an in-depth view of KCNE2 roles, see for review [START_REF] Abbott | The KCNE2 K + channel regulatory subunit: Ubiquitous influence, complex pathobiology[END_REF].

KCNQ1:KCNE3

KCNQ1:KCNE3 is mostly expressed on the basolateral side of epithelial cells. In the colonic epithelium, KCNQ1:KCNE3 is coupled to the function of the Na + /K + /ATPase and participates in the recycling of K + to enable Na + absorption and cAMP-driven Cl -secretion (Julio-Kalajzić et al., 2018;Preston et al., 2010;[START_REF] Schroeder | A constitutively open potassium channel formed by KCNQ1 and KCNE3[END_REF]. In addition, KCNQ1:KCNE3, via K + recycling, participates in Cl -entry by the NKCC1 transporter [START_REF] Heitzmann | Physiology and pathophysiology of potassium channels in gastrointestinal epithelia[END_REF]. In mice airway epithelium, KCNQ1:KCNE3 may facilitate Na + reabsorption and participate in Cl -secretion (Grahammer et al., 2001b;Preston et al., 2010).

As KCNE2, the current exhibited by KCNQ1 with KCNE3 shows a rapid activation, rapid deactivation.

The channel is constitutively open with a linear current voltage relationship. The voltage-dependence of KCNQ1:KCNE3 is shifted towards hyperpolarized values (V1/2 is around -150mV). In the physiological range of membrane potentials, the open probability of the channel is already close to 1, explaining the apparent voltage-independence of this complex (Barro- [START_REF] Barro-Soria | KCNE3 acts by promoting voltage sensor activation in KCNQ1[END_REF]. The main difference between KCNE2 and KCNE3 lies in the current density, which, with KCNE3, becomes much larger than that of KCNQ1 alone (Figure 4. 3 D). With these electrophysiological characteristics, KCNQ1:KCNE3 acts as a K + leak channel (Bendahhou et al., 2005;[START_REF] Schroeder | A constitutively open potassium channel formed by KCNQ1 and KCNE3[END_REF]. KCNE3 blocks the S4 voltagesensor domain of KCNQ1 in an active state, thus locking the channel in its open conformation [START_REF] Abbott | KCNE1 and KCNE3: The yin and yang of voltage-gated K(+) channel regulation[END_REF][START_REF] Barro-Soria | KCNE1 and KCNE3 modulate KCNQ1 channels by affecting different gating transitions[END_REF], 2015;[START_REF] Sun | Structural Basis of Human KCNQ1 Modulation and Gating[END_REF].

KCNQ1:KCNE4

In the physiological voltage range, KCNE4 flattens the current exhibited by KCNQ1 to an almost complete inhibition in Xenopus oocytes and mammalian cells (Bendahhou et al., 2005;[START_REF] Grunnet | hKCNE4 inhibits the hKCNQ1 potassium current without affecting the activation kinetics[END_REF][START_REF] Grunnet | KCNE4 is an inhibitory subunit to the KCNQ1 channel[END_REF] although subcellular localisation of KCNQ1 does not seem to be impacted by the association with KCNE4 (Figure 4. 3 Figure 4. 3E) [START_REF] Grunnet | hKCNE4 inhibits the hKCNQ1 potassium current without affecting the activation kinetics[END_REF][START_REF] Grunnet | KCNE4 is an inhibitory subunit to the KCNQ1 channel[END_REF]. KCNE4 interacts with Calmodulin which is required for KCNQ1 activity [START_REF] Ciampa | KCNE4 juxtamembrane region is required for interaction with calmodulin and for functional suppression of KCNQ1[END_REF][START_REF] Ghosh | KCNQ1 assembly and function is blocked by long-QT syndrome mutations that disrupt interaction with calmodulin[END_REF]. The inhibition of KCNQ1 is exerted both directly [START_REF] Manderfield | KCNE4 domains required for inhibition of KCNQ1[END_REF] and indirectly through calmodulin binding and is thus calcium sensitive [START_REF] Ciampa | KCNE4 juxtamembrane region is required for interaction with calmodulin and for functional suppression of KCNQ1[END_REF].

KCNQ1:KCNE5

KCNE5 drastically shifts the voltage dependence of KCNQ1 to high positive values so that the channel is non-functional in the physiological range of membrane potential (Figure 4. 3 F) [START_REF] Angelo | KCNE5 induces time-and voltage-dependent modulation of the KCNQ1 current[END_REF]Bendahhou et al., 2005). The effect of KCNE5 on KCNQ1 seems to be mediated by four specific amino acids in the transmembrane domain of KCNE5 [START_REF] Angelo | KCNE5 induces time-and voltage-dependent modulation of the KCNQ1 current[END_REF]. It has been suggested that KCNE4

and KCNE5 play roles in the heart to modulate the IKs currents [START_REF] Lundquist | Expression of multiple KCNE genes in human heart may enable variable modulation of I(Ks)[END_REF].

KCNQ1 structural insights and regulation

The stoichiometry between KCNQ1 and its ancillary subunits is still debated. The studies have mostly focused on KCNE1, although it might apply to the other members of the KCNE family. It appears that the most likely stoichiometry is 4 α-subunits for 2 β-subunits (H. Chen et al., 2003;[START_REF] Plant | Individual IKs channels at the surface of mammalian cells contain two KCNE1 accessory subunits[END_REF][START_REF] Wang | Subunit composition of minK potassium channels[END_REF]. The 4α:4β stoichiometry has also been proposed [START_REF] Wang | MinK-KvLQT1 fusion proteins, evidence for multiple stoichiometries of the assembled IsK channel[END_REF][START_REF] Yu | Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels[END_REF]. It was suggested that the 4α:1β, 4α:2β, and 4α:4β stoichiometries might occur and show flexibility [START_REF] Murray | Unnatural amino acid photo-crosslinking of the IKs channel complex demonstrates a KCNE1:KCNQ1 stoichiometry of up to 4:4[END_REF][START_REF] Nakajo | Stoichiometry of the KCNQ1 -KCNE1 ion channel complex[END_REF]. For KCNQ1:KCNE3, the cryo-EM performed by Sun and MacKinnon in 2020 point to a 4α:4β stoichiometry [START_REF] Sun | Structural Basis of Human KCNQ1 Modulation and Gating[END_REF].

KCNQ1 activity is regulated by the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) [START_REF] Loussouarn | Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels[END_REF]. PIP2 is required for the activity of KCNQ1, complexes with the KCNEs [START_REF] Zaydman | PIP2 regulation of KCNQ channels: biophysical and molecular mechanisms for lipid modulation of voltage-dependent gating[END_REF] and seems to interact with the S4-S5 linker [START_REF] Sun | Cryo-EM Structure of a KCNQ1/CaM Complex Reveals Insights into Congenital Long QT Syndrome[END_REF] and the S2-S3 loop [START_REF] Sun | Structural Basis of Human KCNQ1 Modulation and Gating[END_REF]. In the absence of PIP2, and upon depolarisation, the voltage-sensor gets shifted but the pore does not open, referred to as an uncoupling of the pore and voltage sensor [START_REF] Sun | Cryo-EM Structure of a KCNQ1/CaM Complex Reveals Insights into Congenital Long QT Syndrome[END_REF][START_REF] Zaydman | Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening[END_REF]. Recently, the lab of MacKinnon has shown that PIP2 binds to KCNQ1 when in the depolarised conformation, thus promoting opening of the pore. KCNQ1 is thus both ligand-and voltage-dependent as it requires a depolarisation for the voltage-sensor to adopt its "depolarised" conformation, thus enabling binding of I in a previously hidden pocket, inducing a conformational change that opens the pore (Figure 4. 4) (Mandala and MacKinnon, 2023). The C-terminal tail of KCNQ1 shows binding sites for the N-and C-lobes of Calmodulin (CaM).

Calmodulin binding ensures a proper channel assembly and surface localisation [START_REF] Ghosh | KCNQ1 assembly and function is blocked by long-QT syndrome mutations that disrupt interaction with calmodulin[END_REF][START_REF] Shamgar | Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations[END_REF]. CaM also acts as a regulator of the channel's gating by relieving from inactivation in a Ca ++ -dependent manner [START_REF] Dvir | Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt I(KS) regulation by KA and I ₂[END_REF][START_REF] Ghosh | KCNQ1 assembly and function is blocked by long-QT syndrome mutations that disrupt interaction with calmodulin[END_REF][START_REF] Shamgar | Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations[END_REF].

Our focus being on KCNQ1:KCNE3, only the structural modifications done by KCNE3 will be discussed. Sun and MacKinnon, 2020). Upon substitution of three amino acids on KCNE3 sequence by those of KCNE1 (namely Thr71→Phe, Val72→Thr, and Gly73→Leu), KCNE3 exhibits the same properties on KCNQ1 gating as KCNE1 [START_REF] Melman | A single transmembrane site in the KCNEencoded proteins controls the specificity of KvLQT1 channel gating[END_REF][START_REF] Melman | Structural determinants of KvLQT1 control by the KCNE family of proteins[END_REF]. According to the cryo-EM structure, these amino acids interact with the S1, S4 and S5 helices [START_REF] Sun | Structural Basis of Human KCNQ1 Modulation and Gating[END_REF].

In its N-terminal region, KCNQ1 possesses basolateral targeting [START_REF] Jespersen | Basolateral localisation of KCNQ1 potassium channels in MDCK cells: molecular identification of an N-terminal targeting motif[END_REF] and Protein Kinase A (PKA) phosphorylation sites [START_REF] Marx | Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel[END_REF]. PKA phosphorylates KCNQ1 upon activation by increased levels of cAMP, guided by the A-Kinase Anchoring Protein AKAP9. AKAP9 -also called Yotiao -binds to KCNQ1 C-terminal domain to facilitate PKA phosphorylation on the Ser27 of KCNQ1 [START_REF] Marx | Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel[END_REF]. The C-terminal domain also exhibits the assembly domain which promotes selfinteraction for the homo-tetramer that forms the functional channel [START_REF] Schmitt | A recessive C-terminal Jervell and Lange-Nielsen mutation of the KCNQ1 channel impairs subunit assembly[END_REF]. In the S5-S6 extracellular loop, KCNQ1 has a glycosylation site [START_REF] Barhanin | K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current[END_REF] thought to influence the channel's gating properties [START_REF] Freeman | Glycosylation influences gating and pH sensitivity of I(sK)[END_REF].

In females, oestrogens have an anti-secretory function in the colonic epithelium (O'Mahony et al., 2009(O'Mahony et al., , 2007)). Oestrogens have a double effect in that matter: they dissociate KCNQ1 from KCNE3 and induce KCNQ1 internalisation. Through the binding to the oestrogen receptor (ER), oestrogens activate KCδ which phosphorylates KCNE3 on its Ser8 residue, dissociating β and α subunits. In turn KCδ activates the AMPK (AMP-Kinase) which recruits Nedd4.2 for KCNQ1 internalisation by Rab5. Thus, in response to oestrogens, the membrane density of KCNQ1:KCNE3 is lowered rapidly and induces a decrease of Cl -and water secretion which is sustained for a few hours. The internalised KCNQ1 rather than being degraded, is stored in early endosomes and its recycling at the plasma membrane can be divided into two phases: a rapid one through Rab4 and a slow one through Rab11 (Rapetti-Mauss et al., 2013).

KCNQ1 in cancer -timeline

In addition to the well described roles of KCNQ1, a new role has emerged recently. A body of evidence points to a tumour-suppressor action of KCNQ1 in epithelial cancers. The first evidence for this tumoursuppressor role was published in 2014 by Than and colleagues (Than et al., 2014). KCNQ1 had been identified in Sleeping Beauty DNA transposon-based forward genetic screens in mice as a GIT cancer susceptibility gene, with high frequency common insertion site [START_REF] Mann | Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma[END_REF][START_REF] March | Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis[END_REF][START_REF] Pérez-Mancera | The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma[END_REF][START_REF] Starr | A Sleeping Beauty transposon-mediated screen identifies murine susceptibility genes for adenomatous polyposis coli (Apc)-dependent intestinal tumorigenesis[END_REF][START_REF] Starr | A transposon-based genetic screen in mice identifies genes altered in colorectal cancer[END_REF]. 4. 5). This study shows that KCNQ1 is a strong prognostic marker for colorectal cancer and could be used as for stratification of colorectal cancer patients. In 2017, our team showed the molecular basis for the tumour-suppressor activity of KCNQ1 in colorectal cancer (Rapetti-Mauss et al., 2017). These findings are detailed in the following paragraph. Since then, KCNQ1 has been shown

to act as tumour suppressor in a variety of epithelial cancers. KCNQ1 is downregulated in hepatocellular carcinoma where its lowered expression is also correlated to poor overall survival (Figure 4. 5) (Fan et al., 2018). Overexpression of KCNQ1 or pharmacological activation had inhibitory effects on tumour progression both in vitro and in vivo. Mechanistic investigations showed that, as in colorectal cancer, KCNQ1 tumour-suppressor activity was mediated at least in part by its association to β-catenin and its inhibitory effect on the Wnt/β-catenin signalling pathway.

In oesophageal cancer, miR-483-5p promotes cell proliferation, migration and invasion and suppresses KCNQ1 expression. The rescue of KCNQ1 in these conditions weakens the pro-tumoral effect of miR-483-5p [START_REF] Chen | miR-483-5p promotes esophageal cancer progression by targeting KCNQ1[END_REF]. Similar effects have been observed in renal cell carcinoma with miR-140-5p negatively targeting Klf9, a transcription factor for KCNQ1 expression [START_REF] Huang | The miR-140-5p/KLF9/KCNQ1 axis promotes the progression of renal cell carcinoma[END_REF]. KCNQ1 is also downregulated in uterine endometrial cancer by succinate accumulation which promotes the progression of this cancer [START_REF] Gu | Melatonin alleviates progression of uterine endometrial cancer by suppressing estrogen/ubiquitin C/SDHB-mediated succinate accumulation[END_REF]. Several other studies point to a role of KCNQ1 in cancer. A Crispr/Cas9 knockout of KCNQ1 in Golden Syrian Hamster led to the development of several cancers linked to KCNQ1 deficiency [START_REF] Li | A novel cancer syndrome caused by KCNQ1-deficiency in the golden Syrian hamster[END_REF]; hypermethylation of KCNQ1 was also found in breast cancer patients [START_REF] Ennour-Idrissi | DNA Methylation and Breast Cancer Risk: An Epigenome-Wide Study of Normal Breast Tissue and Blood[END_REF][START_REF] Fu | DNA Methylation of Imprinted Genes KCNQ1, KCNQ1OT1, and PHLDA2 in Peripheral Blood Is Associated with the Risk of Breast Cancer[END_REF], and KCNQ1 deficient mice, as underlined earlier, also display gastric hyperplasia and metaplasia [START_REF] Elso | Heightened susceptibility to chronic gastritis, hyperplasia and metaplasia in Kcnq1 mutant mice[END_REF]. A paper published very recently explores publicly available databases from the TCGA and show that in gastric cancer, KCNQ1 expression is reduced [START_REF] Yang | Low KCNQ1 expression is associated with unfavorable outcome and metabolism of gastric cancer[END_REF]. The lower expression of KCNQ1 in gastric cancers correlates with poorer overall survival. This study only uses available data sets, an in-depth study would be of interest. Thus, KCNQ1 has been described as tumour-suppressor in a variety of cancers. In colorectal cancer, KCNQ1 exerts its tumour suppressor activity through the regulation of Wnt/β-catenin pathway. While the molecular mechanisms have not been uncovered in other tissues, the regulation of the Wnt/βcatenin pathway could well underlie the tumour suppressor activity of KCNQ1 in the other epithelia.

Bi-directional interaction KCNQ1-β-catenin

From the observation that KCNQ1 expression was correlated with epithelial features of colorectal cancer cells and inversely correlated with mesenchymal features, my thesis supervisor, Raphael

Rapetti-Mauss dug into the molecular mechanism sustaining KCNQ1 tumour-suppressor activity. This study led to important findings that are the basis for my thesis project.

In addition to being correlated with mesenchymal features, the absence of KCNQ1 was also correlated with a diffuse cytosolic localisation of β-catenin while in well-differentiated cell lines such as HT29cl19A and in rat colonic crypts KCNQ1 and β-catenin were colocalised at the basolateral plasma membrane.

In situ proximity ligation assays showed that KCNQ1 was closely associated to E-cadherin and β-catenin at the adherens junctions.

The pharmacological or molecular inhibition of KCNQ1 strongly reduced the interaction between Ecadherin and β-catenin suggesting a disruption of the adherens junctions. KCNQ1 inhibition also showed an increase of β-catenin phosphorylations on residues associated with signalling while the Ser33 (targeting for degradation) was strongly reduced. These results suggest that when KCNQ1 is inhibited or absent, β-catenin is released from the adherens junction and accumulates in the cytosol where it is no longer targeted for degradation by the destruction complex.

The inhibition of KCNQ1 in HT29cl19A cell line was associated with a switch from an epithelial to a mesenchymal phenotype with the increased expression of N-cadherin, claudin1 and β-catenin and a decreased expression of Zonula occludens 1 and E-cadherin as well as a reduced transepithelial electrical resistance. To further support the role of KCNQ1 in the maintenance of an epithelial phenotype, the molecular inhibition of KCNQ1 prevented the formation of cysts. Indeed, epithelial cells have the ability, when cultured in 3D, to spontaneously form spheres, with well-organised layer and a lumen. With a molecular extinction of KCNQ1, the cells formed bulky clusters showing little to no organisation and no lumen.

In the HCT116 mesenchymal cell line, the rescue of KCNQ1 shifted the cells to an epithelial phenotype as shown by the expression levels of markers and the rescued ability to form cysts and plasma membrane localisation of β-catenin with KCNQ1.

The results on β-catenin localisation were reproduced in HT29cl19A cell line experimentally depolarised with a high extracellular K + medium suggesting that the ion channel function of KCNQ1 is crucial for the maintenance of adherens junction and subsequent epithelial phenotype.

In addition, this study shows that while KCNQ1 controls the localisation of β-catenin, it is also a target of the Wnt/β-catenin pathway. Indeed, the transcription factor TCF4 and β-catenin bind to KCNQ1

promoter to inhibit its transcription.

Overall, these results show that KCNQ1 acts as a tumour suppressor by maintaining β-catenin at the adherens junction. Upon inhibition of KCNQ1, β-catenin is released and phosphorylated on residues promoting its signalling activity. In turn, an activated β-catenin restricts the transcription of KCNQ1, further enhancing the mesenchymal features mediated by Wnt/β-catenin signalling. In these conditions, the adherens junctions are disrupted. Β-catenin is released, accumulates into the cytosol, is able to translocate into the nucleus where it activates the transcription of Wnt target genes. Among the target genes is KCNQ1 which is repressed by β-catenin transcriptional activity, further enhancing the phenotype observed. Losing the adherens junctions and activating the Wnt/β-catenin pathway has for consequence a loss of epithelial features; mesenchymal cells aggregate but cannot form an organised sphere. In cancer, acquiring mesenchymal features is key in cancer progression and dissemination. (From Rapetti-Mauss, 2018).

Aims of the Study

The position of ion channels in cancer and the subsequent modifications of resting membrane potential have been deeply questioned the past few years. Growing evidence of the role of ion channels have brought up more questions still to be answered:

-What is the role of mutations in bioelectric regulation?

-What are the molecular mechanisms linking bioelectricity and cell behaviour?

-How is membrane potential signal integrated?

These questions have been driving my thesis research. Although not all can be answered with this work, it still offers partial answers that will help better understand the role of ion channels and membrane potential in non-excitable cells.

Mutations of KCNQ1

Since the pharmacological and molecular inhibition of KCNQ1 induce a release of β-catenin from the adherens junction, and based on the observation that Wnt/β-catenin components often carry mutations in colorectal cancer, we addressed the following questions:

-Does KCNQ1 carry mutations in colorectal cancer?

-What are the electrophysiological features of these mutations? In other words, is the channel activity lost?

-What is the effect of a loss-of-function of KCNQ1 on β-catenin localisation and more broadly on the Wnt/β-catenin pathway?

We also aimed to better characterize the link between KCNQ1 and the Wnt/β-catenin pathway and more precisely the mechanisms sustaining the activation of the pathway when KCNQ1 is mutated.

One of the questions that was raised by the previous study from my team was whether the presence or the function of KCNQ1 was mediating the effects on the signalling pathway. Indeed, it could be that KCNQ1 acts as a scaffold for the adherens junction independently of its function as a potassium channel. Although the depolarisation experiment seems to indicate that its function as a channel is crucial, the use of loss-of-function mutations enables the investigation of this hypothesis.

Publicly available datasets were searched to answer the first question. The electrophysiological recordings of KCNQ1 were performed in CHO cells, a standard model to study electrophysiological features. To answer the last question, we used two colorectal cancer cell lines: HT29cl19A, HCT116.

HT29cl19A cells are well-differentiated cells that endogenously express KCNQ1 and exhibit a low Wnt/β-catenin pathway activation despite harbouring mutations on APC. HCT116 cell line is more mesenchymal, have lost the expression of KCNQ1 and harbour a pathological heterozygous mutation on CTNNB1 encoding β-catenin.

This project will constitute my thesis first author research paper. Even though the paper is not finished, and some results are yet to be obtained, this part will be presented with explanation of the on-going experiments which might be modified along the way as new results come in.

Membrane potential

The results provided by the 2017 study and this thesis pointed towards the potassium channel function of KCNQ1 as the driver of its tumour-suppressor activity and more specifically, as the driver of the inhibition of Wnt/β-catenin pathway. In the colonic epithelium, KCNQ1:KCNE3 participate to setting the resting membrane potential. The loss of function of KCNQ1 thus depolarises cell membranes. As more studies point towards a role of the membrane potential rather than single channels, we wondered whether a non-biased depolarisation could mediate the same effects on the signalling pathway. While this big question would still benefit from further investigations, the results obtained during my thesis will be discussed in this second part.

Annex questions

From the answer to these questions, more questions arose and were developed alongside the main findings. These preliminary / side results contribute fully to the discussion, and I have chosen to present some of them independently in a third part to offer a broader perspective. Among which I will discuss the interaction between KCNQ1 and β-catenin, the possibility to pharmacologically activate KCNQ1, and the effect of Wnt3A on KCNQ1.

Of note, the team is currently investigating the precise role of KCNQ1 in the epithelial-to-mesenchymal transition in the context of gastric cancer. As this project has mainly been led by master students and Raphael, I will not detail this study nor the findings.

Part I -Cancer-associated mutations of KCNQ1 promote Wnt/β-catenin activity Colon cancer patients have a frequency of mutation on KCNQ1 ranging from 2% to 10% depending on the study, with a mean of 4,6%, similar to the percentage of mutations on other components of the Wnt pathway such as AXIN1, CTNNB1 and LRP6 for example (Table 1)(Supplementary Figure 1).

The most frequent mutations have been selected for further electrophysiological analysis. The mutations K420fs and K422Sfs*10 have for consequence a truncation of the channel in similar positions. We thus investigated the effect of the K422Sfs*10 mutation keeping in mind that surrounding mutations would have the same effect. 2. 85% of the cancer-related KCNQ1 mutations are loss-offunction.

The next step was to evaluate whether these mutations influenced the function of the channel. To do so, we cloned the thirteen most frequent mutations and expressed them in the Chinese Hamster Ovary (CHO) cells, a heterologous expression system often used in electrophysiology to characterize channel features. We expressed the constructions either alone (Supplementary Figure 2) or with their epithelial ancillary subunit KCNE3 at a ratio of 1:3 (KCNQ1:KCNE3) and evaluated protein expression by Western

Blot. The currents were recorded in the whole-cell configuration and cells were maintained at a resting membrane potential of -60mV and stimulated with a step protocol ranging from -120mV to +80mV with 20mV increments for 1000 milliseconds (to reach activation steady state) before a hyperpolarising step at -120mV for 200 msec to extract the tail current (Equation 4) and a return to the resting membrane potential (FIGURE 2 A). The voltage-current relationship shows that most mutations display little to no current (FIGURE 2 B), sustained by the analysis of the current density at 0mV (FIGURE 2 C).

The mutations E170K, R243C, A329T, R401Gfs*18, K422Sfs*10, and R518* display no current and are thus totally loss-of-function (LOF). The V185M, F339L, P343S, P448R, and R451Q mutations are still able to produce current but at a lower amplitude, therefore resulting in a loss-of-function. Out of the thirteen tested mutations only two -namely A287T and G548C -displayed no difference in current amplitude compared to the wild type channel. We then selected the mutations that still displayed a low amplitude current (threshold was set at 5 pA/pF at 0 mV) to study the open probability of the channels. Briefly, open probability was evaluated by measuring the tail current amplitude elicited at -120 mV and dividing it by the maximum tail current amplitude (Equation 4) [START_REF] Pusch | Gating of the voltage-dependent chloride channel CIC-0 by the permeant anion[END_REF].

Surprisingly, some mutations displayed a difference in the open probability curve. V1/2 of KCNQ1:KCNE3 is at -150mV, making the channel already open at the tested range of potentials (Po=0.8-1). However, at -60mV, the physiological resting membrane potential of epithelial cells, some mutations had a lower open probability. This was the case for V185M, F339L, P448R, R451Q and more surprisingly for A287T which was not considered to be loss-of-function previously (FIGURE 2 D). One of the hypotheses that could sustain these results is that the mutations impair the association with KCNE3, thus impeding the blockade of the voltage-sensor in its depolarised conformation and resulting in an in-between phenotype of voltage-sensitivity.

The G548C mutation is thus the only out of thirteen tested to have no effect on the channel.

In patients, these mutations are most likely heterozygous, we therefore questioned whether the coexpression with the wild type channel would show a dominant negative effect of the loss-of-function mutation or a rescue of the function by the wild type form. To study this question, we selected the most frequent mutations. The loss-of-function mutations prevailed on the wild type channel and a small current amplitude or no current was observed (FIGURE 2 E). This effect might be mediated by an impaired membrane targeting of the heterotetramer or a misfolding occurring when one of the α subunit carries a mutation. These results thus suggest that a heterozygous loss-of-function mutation induces a dominant negative effect on the WT channel and thus loss-of-function in the patients' cancer cells.

All in all, out of the thirteen most frequent mutations on KCNQ1, only one (G548C) has no effect on the channel properties. The other 12 tested mutations are loss-of-function when expressed with KCNE3. Some mutations have not been characterized and it could be interesting to study further all existing mutations.

We selected the most frequent loss-of-function mutations to study their effect on the Wnt/β-catenin pathway activity. From these equations, we can now write the last one: The tail current (Itail) is the current measured at the end of the stimulation protocol -a pre-conditioning pulse used to reach steady state activation. Itail is measured at a constant imposed membrane potential, just after a pre-conditioning pulse used to reach steady state activation. The current is therefore measured before deactivation, and Df is constant. This protocol elicits a tail current which is only dependent on the steady state activation (Po). When Itail reaches its maximum, Po=1. Therefore, by measuring the tail current elicited after each stimulation (from -120mV to +80mV), we obtain a value for each tail current. Divided by the highest value obtained (Itail-max) we obtain the open probability of the channel of interest. [START_REF] Pusch | Gating of the voltage-dependent chloride channel CIC-0 by the permeant anion[END_REF].

Loss-of-function mutations of KCNQ1 alter Wnt/β-catenin signalling pathway

Since the mutations had a dominant negative effect on the wild type channel, we were able to express them in HT29cl19A, a colorectal cancer cell line expressing endogenous KCNQ1 and exhibiting epithelial features as well as very low Wnt/β-catenin background activity. Three different types of mutations -missense, frameshift and stopgain -A329T, K422Sfs*10 and R518* were thus expressed in HT29cl19A by a viral vector. In order to have a broad overview of the effect of these loss-of-function mutations, we performed RNAseq on these cell lines. Since the pharmacological inhibition or molecular extinction of KCNQ1 lifted the brake on Wnt/β-catenin, we hypothesized that loss-of-function mutations of KCNQ1 would also permit the activation of the pathway. RNASeq analyses revealed 1 577, 1 022, and 808 significantly differentially expressed genes in the HT29cl19A A329T, K422Sfs*10, and R518* cell lines respectively. We then examined the list of differentially expressed genes for enrichment of functional gene-set modules. The software Amine was used to regroup gene sets into modules and corresponding Gene Ontology Term for each module was assessed with the software String. In line with our hypothesis, we found that Wnt/β-catenin was among the ten most dysregulated biological processes with all three of the LOF mutations (FIGURE 3 A). Linked biological processes were also dysregulated such as signal transduction, BMP, Hippo Ephrin receptor and GPCR signalling pathways. We plotted the differential expression of genes included in the GO Terms "Wnt signalling pathway" and "canonical Wnt signalling pathway" (accession numbers: 0016055; 0060070). The heatmap shows that many genes involved in this biological process are differentially expressed, both up-and down-regulated in the KCNQ1-LOF conditions (FIGURE 3 B).

In addition, we used a publicly available tool (muTarget) using TCGA datasets to identify differential expression of genes in samples harbouring a mutated input gene (Nagy and Győrffy, 0 1). We selected samples from colon adenocarcinoma, the cohort comprises 396 patients. We compared the differential gene expression between colon adenocarcinoma patients carrying a mutation on KCNQ1 (n=10) or not (n=386) to analyse the biological processes altered. The results show that in patients carrying a mutation, the Wnt/β-catenin signalling pathways is the fourth most modulated biological process further confirming the results obtained above (FIGURE 3 C). The volcano plot represents all the differentially expressed genes between the patients carrying or not a mutation on KCNQ1 with thresholds set at 1.44 fold change and 0.01 padj. Genes corresponding to the GO Term "Wnt signalling pathway" are represented in orange (FIGURE 3 D). While many of the genes corresponding to that GO Term are downregulated when KCNQ1 carries a mutation, a large amount encodes negative regulators of the pathway. Of note, the patients included in these data still suffer from colon adenocarcinoma and are thus likely to carry mutations on other components of the Wnt pathway such as APC (n=291/396) or mutations on BRAF or KRAS. These results thus strongly suggest that carrying a mutation on KCNQ1 is key in altering Wnt/β-catenin activity.

Using the results from the RNA-Seq, we analysed the differential expression of genes that are known Wnt/β-catenin targets and found that about half of them, including CD44, AXIN1 and MYC, were significantly upregulated. Among the downregulated Wnt targets were genes encoding proteins involved in negative regulation of the pathway such as AXIN2 and SOSTDC1 (FIGURE 3 E).

These results suggest that the Wnt/β-catenin pathway is altered upon KCNQ1 LOF. However, since promoter. In this method, luciferase is replaced by GFP, still expressed under a 7 repeat of TCF/LEF sites (FIGURE 4 C). Cells expressing an empty vector or the A329T, K422Sfs*10, or R518* mutations were fixed and imaged with a confocal microscope. We observed around three-fold increase in the relative number of GFP + expressing cells in LOF mutations-cells compared to the control cell line (FIGURE 4 D). Since the method and conditions were different, we were able to see an increase of Wnt/β-catenin transcriptional activity with the mutations without addition of Wnt3A conditioned medium further suggesting that a non-functional KCNQ1 directly induces the activation of the Wnt/βcatenin pathway.

These results show an increase of Wnt/β-catenin transcriptional activity when KCNQ1 carries loss-offunction mutations in three independent cell lines.

The levels of active β-catenin, that is the β-catenin that is not phosphorylated by CK1 and GSK3 to target for degradation, were also elevated by at least two-fold in HT29cl19A cells expressing KCNQ1 mutants (FIGURE 4 E), suggesting that more β-catenin is free in the cytosol and available for transcription.

We then quantified the protein expression of canonical Wnt target genes in HT29cl19A cell lines.

Protein expression of Axin2, CD44 and cyclinD1 were increased in the KCNQ1 mutation-expressing cells, providing further evidence of an activated Wnt/β-catenin signalling (FIGURE 4 F).

To assess the activity of the pathway in a model closer to the physiology of the colon epithelium, we have set the colon organoid model in the laboratory. Once endogenous expression of KCNQ1 was verified by immunofluorescence (Supplementary Figure 3 A), we overexpressed the mutation A329T, tagged with GFP, in the organoids. The experimental procedure is described in Supplementary Figure 3 B: in one condition, organoids are kept in the proliferation medium WENR to assess their diameter at day 6. In the second condition, at day 5 proliferation medium is replaced by differentiation medium lacking Wnt3A (ENR). Two experiments are planned, the first one a couple of days post medium substitution to assess the formation of crypts, the second at the end of differentiation to measure the length of crypts and assess their number per organoid. In addition, the mutant A329T was tagged with a GFP and a GFP plasmid was transduced into the WT organoids to ensure that puromycin selection was sufficient and validate our model (Supplementary Figure 3 C left panel) The organoids expressing the LOF mutation A329T had a larger diameter compared to the WT and a higher percentage of organoids were above the median size of the WT organoids at the same stage of development (Supplementary Figure 3 C). However, the measurement of the organoid size was only performed on two biological replicates before being interrupted by technical issues, the statistics presented here are only an indicator and should not be considered as is. Interestingly, a 2019 article showed that organoids with increased Wnt/β-catenin activity displayed larger volumes as proliferation was increased [START_REF] Cui | Wnt Signaling Mediates the Aging-Induced Differentiation Impairment of Intestinal Stem Cells[END_REF]. We are currently constructing organoids expressing A329T or K422Sfs*10 LOF mutations without GFP tag, and the 7TGC plasmid which will allow us to directly see Wnt/β-catenin transcriptional activity. We expect to obtain the same results as in the 2D cancer cell lines: increased number of GFP + cells in the presence of KCNQ1-LOF mutations. Moreover, this experiment might give us more information if the localisation of GFP + cells exhibits a specific pattern. Indeed, we could expect GFP to be preferentially expressed in the lower two thirds of the crypt in the stem, progenitor and transit amplifying cells. In addition, we will use these constructions to measure (1) the diameter, (2) assess crypt formation, and (3) crypt length and number. From our first observations, it seems that the organoids expressing the K422Sfs*10 LOF mutation display much larger sizes than WT organoids, comforting the results obtained previously. We expect that KCNQ1-LOF organoids will compensate for the lack of exogenous Wnt3A and exhibit delayed differentiation. Experiment ( 2) should show that KCNQ1-LOF organoids have formed less crypts at the same stage as WT organoids. In the third experimental procedure (3), we expect that crypts formed in the context of an LOF mutation will have an altered morphology due to an imbalance between proliferation and differentiation. 5. The LOF-KCNQ1-induced increase of Wnt activity is independent from Frizzled/Lrp but mediated by β-catenin

We then wanted to investigate whether the effect of KCNQ1 mutations on the activation of Wnt/βcatenin pathway was linked to an increased activity of the upstream Frizzled/Lrp complex. We first quantified the protein expression of the two target genes CD44 and Axin2 in cells treated with niclosamide (5 µM), a Frizzled inhibitor. The protein expression of CD44 and Axin 2 showed no change -or a slight significative increase upon treatment. Indeed, KCNQ1 mutations still exhibited higher protein expression of Wnt target genes than the WT KCNQ1 control (FIGURE 5 A). To confirm that Frizzled/Lrp were not involved, we quantified the phosphorylation of Lrp6. The overexpression of KCNQ1 mutations had no effect on the phosphorylation of LRP6 or rather seemed to induce a slight decrease (FIGURE 5 B). Therefore, the effect of the loss-of-function KCNQ1 mutations cannot be explained by an overactivation of the Wnt signalosome.

These results strongly suggest that Frizzled/Lrp are not involved in the process. However, since the levels of active-β-catenin were increased, and the Wnt target genes expression was enhanced with the mutations, we aimed to confirm that β-catenin was involved in the observed effects. To this extent, we treated HT29cl19A cells with an activator of the Axin degradasome, JW67. Should the increased pathway activity be mediated by β-catenin, increasing its degradation by the destruction complex would abolish the effect. Indeed, upon treatment with 10 µM JW67, the protein expressions of CD44 and Axin2 were decreased to an almost WT-like state when the degradasome was activated in the presence of the LOF mutations (FIGURE 5 D). Therefore, the transcription of target genes when KCNQ1 mutation is expressed is dependent -at least in majority -on β-catenin. When β-catenin is degraded, the KCNQ1 mutation-induced transcription of CD44 and Axin2 is strongly decreased or even lost.

In the control cells, activation of the Axin degradasome had little to no effect, suggesting that β-catenin shifts from being degraded in the control cells to its active-signalling mode in the KCNQ1-LOF context.

We therefore evaluated its phosphorylation status. When the loss-of-function mutations are expressed, there is a decrease of the Ser31 Ser37 and Thr41 phosphorylations that are a signal for proteasomal degradation, consistent with the results obtained with JW67. In parallel, we observed an increase of the phosphorylation on the Tyr654 residue which detaches β-catenin from E-cadherin at the adherens junctions and promotes its transcriptional activity (FIGURE 5 E). Two other phosphorylations of β-catenin important for its shift in its signalling mode -namely Ser675 and Tyr142

-are yet to be evaluated as technical issues have been encountered.

These results seem to show that in the KCNQ1-LOF context, degradation of β-catenin is decreased, and β-catenin is detached from E-cadherin. To further confirm these results, we performed proximity ligation assays between β-catenin and either KCNQ1 or E-cadherin. As shown previously (Rapetti-Mauss et al., 2017), β-catenin is in close proximity with both KCNQ1 and E-cadherin at the adherens junction. However, when the LOF mutations of KCNQ1 are expressed, β-catenin interaction with KCNQ1 seems to be reduced to its minimum (FIGURE 5 F). While this experiment must be repeated twice to perform statistical analysis, it seems that expressing a LOF mutation prevents β-catenin association with KCNQ1, even in its endogenous WT form. Strengthening this preliminary result, the number of interactions between E-cadherin and β-catenin drops down from approximately 15 interactions per cell to 2 to 3 in the presence of LOF KCNQ1 (FIGURE 5 G).

While we could argue that the LOF mutations are perhaps not present at the membrane, explaining in itself the lack of interaction with β-catenin, the decrease of interactions with E-cadherin and phosphorylation status of β-catenin strongly suggest that β-catenin is released from the adherens junctions in the LOF mutations context. To go further in this characterisation, we will assess the localisation of both KCNQ1 and β-catenin in these cells using Wheat Germ Agglutinin (WGA) as a marker of cell membrane.

All in all, these results show that when KCNQ1 carries cancer-related loss-of-function mutations, the Wnt/β-catenin pathway is activated independently of Frizzled/Lrp but requires β-catenin signalling activity. Insulin-R and IGF-IR known to activate β-catenin directly and indirectly (FIGURE 6). The activated RTKs are similar with all three mutations. These results yet need to be confirmed to ascertain that these kinases are involved in the activation of the pathway triggered by KCNQ1 LOF. Comforting our hypothesis, similar results were obtained when cells were experimentally depolarised (see Part II). We will use couples of total and phospho-antibodies to detect changes in activation of MET, RET and EGFR.

Once these results obtained, we plan on using an inhibitor of the most probable target to assess Wnt/β-catenin transcriptional activity and β-catenin phosphorylation. We hypothesize that upon inhibition of the RTK(s) involved, the pathway activity will be lowered to control levels despite the presence of LOF mutations.

From these results, we have found that most somatic mutations of KCNQ1 found in cancer were lossof-function. This loss-of-function results in an increased Wnt/β-catenin pathway activity. This mechanism is independent from the canonical Frizzled/Lrp receptors but does involve β-catenin. Βcatenin is released from the Adherens Junctions, its degradation is decreased and its Tyr654 residue To further confirm that the KCNQ1-LOF-induced decrease of negative regulators plays a major role contributes to Wnt/β-catenin overactivation, we will analyse 7TGC cells with Wnt3A and with or without addition of recombinant DKK1. Addition of DKK1 should restore the phenotype observed in the WT: no differences between Wnt unstimulated and stimulated cells.

which
These results suggest that LOF mutations of KCNQ1 have a double effect. They induce activation of Wnt/β-catenin pathway directly and in addition decrease the expression of negative regulators therefore promoting a permissive state for further activation by Wnt ligands. KCNQ1 thus protects from an overactivation of Wnt/β-catenin by restraining its activity by two mechanisms: a direct effect and an indirect effect by maintaining high expression levels of other negative regulators. The direct effect by which KCNQ1 protects from overaction of the pathway is still unclear and part II and III might offer insights into these hypotheses.

7. The regulation of Wnt/β-catenin pathway is altered with the LOF mutations of KCNQ1

We observed in the analysis of the RNASeq that among the differentially expressed genes from "Wnt signalling pathway" were some negative regulators of the pathway such as SOSTDC1, AXIN2, WIF1.

Most of them were downregulated. We therefore explored the datasets from the RNASeq and selected significantly differentially expressed genes which fell under the Gene Ontology Terms " ositive Regulators of (canonical) Wnt Signaling pathway" (accession numbers 0030177, 0090263) and "Negative Regulation of (canonical) Wnt signaling pathway" (accession numbers 0030178, 0090090).

Negative regulators were greatly downregulated in the KCNQ1-LOF conditions (FIGURE 7 A). In addition, we used the same dataset from the TCGA (muTarget) as previously described (I.3), coloured in blue, genes falling under the GO Term "Negative regulation of Wnt signaling pathway" and in red " ositive regulation of Wnt signaling pathway" in the Volcano plot representation (FIGURE 7 B).

Interestingly, negative regulators seem to be widely downregulated both in our RNASeq and in colorectal cancer patients' datasets.

We hypothesized that when KCNQ1-LOF are expressed, losing negative regulators would set a Wntpermissive state, increasing even more the activation of the pathway upon stimulation with Wnt ligand. To test this hypothesis, we used the HT29cl19A cell line expressing the 7TGC plasmid and incubated or not cells with Wnt3A conditioned medium (CM) for 30 hours prior to paraformaldehyde fixation. We then counted the relative number of GFP + cells per field which reflects the number of cells with Wnt/β-catenin transcriptional activity. Interestingly, in the WT cells, Wnt3A failed to increase the number of GFP + cells, suggesting that WT KCNQ1 is able to restrain the activation of the pathway. In the KCNQ1-LOF expressing cells however, Wnt transcriptional activity, which is already increased in basal condition, saw an additional increase with exogenous Wnt3A incubation (FIGURE 7 C).

We then evaluated the phosphorylation of Lrp6 with exogenous Wnt3A addition. While these results are still preliminary and not yet sufficient to perform statistical analysis, it seems that Lrp6 phosphorylation is not enhanced in the WT condition but increases in the LOF mutations conditions (FIGURE 7 D). In addition, the active β-catenin levels and the protein expression of Wnt target genes with or without Wnt3A addition will be assessed.

Finally, we quantified the protein expression of known negative regulators of Wnt/β-catenin signalling.

Interestingly while DKK1 mRNA expression was not significantly altered, its protein expression was greatly lowered by up to three-fold in the loss-of-function mutations conditions (FIGURE 7 E). We then assessed the protein expression of negative regulators that were significantly downregulated in our RNASeq results such as Nkd1 and WIF1. Nkd1 inhibits the pathway via its interaction with Dishevelled and β-catenin and prevents β-catenin nuclear shuttling [START_REF] Van Raay | Naked1 Antagonizes Wnt Signaling by Preventing Nuclear Accumulation of β-Catenin[END_REF] A few experiments need to be reproduced to complete this research and submit our results to a peer review journal. Nonetheless, from this first part we have shown that KCNQ1 exerts its tumour suppressor function in the colonic epithelium by two means: it restrains β-catenin from entering its "signalling mode" and it keeps high levels of Wnt negative regulators.

Most importantly, we have shown for the first time that ion channels harbour somatic mutations in cancers and that these mutations are linked to cancer physiopathology. In our case, these loss-offunction mutations might be considered pathogenic mutations or even oncogenic mutations. Many other ion channels might carry mutations in all types of cancer, leading to disruption of their function and consequently impair physiological processes.

Since KCNQ1:KCNE3 current is key in regulating resting membrane potential in colon epithelial cells, we wondered whether membrane potential had a role in these observations.

Part II -Membrane potential controls signalling pathway potential shifts towards values in between the equilibrium potential of these ions. Therefore, the membrane is depolarised. In addition to targeted disruption of a potassium leak channels, broader methods can depolarise membranes. One advantage of these techniques is that it is unbiased as it does not affect a single type of channel. Indeed, by increasing the concentration of potassium in the extracellular environment, the equilibrium potential of potassium gets shifted towards depolarised values. In addition, since extracellular K + concentration and EK+ are correlated, several concentrations can be used to achieve a range of EK+ and thus a range of resting membrane potential. Other broad option include the use of Barium, a non-permeable ion that blocks the pores of all potassium channels therefore leading to membrane depolarisation, even at very low concentrations.

Since we obtained similar results with the pharmacological inhibition, molecular extinction and LOF mutation of KCNQ1, it suggests that its activity as a potassium channel is key in sequestering β-catenin at the adherens junctions and refraining Wnt/β-catenin signalling. Furthermore, as the role of KCNQ1 in the colonic epithelium is to set membrane potential, and since β-catenin seemed to be relocated from the membrane to the cytosol upon experimental depolarisation, we hypothesised that it is not solely KCNQ1 but rather membrane potential which is responsible for the regulation of the Wnt/βcatenin pathway. To test this hypothesis, we performed similar experiments as described in part I using experimental depolarisation with high extracellular K + . In addition, according to the readout, different kinetics of depolarisation have been used: a short kinetic of time points ranging from 5 to 60 minutes and a long kinetic of 12 to 24 hours. This project is not finished, and a certain number of experiments need to be done or repeated to fully decipher the mechanisms and perhaps repeat with Barium-induced depolarisation. However, I have acquired preliminary data during my thesis that will be developed in this chapter.

Figure II-1 -Resting membrane potential changes in non-excitable cells. In physiological conditions, K+ concentrations are of approx. 4 mM outside the cell and 140 mM inside the cell, giving an equilibrium potential of -89 mV. The K+ permeability being higher, membrane potential (Vm) gets pulled towards this hyperpolarised value and is usually around -60mV. Inhibition of potassium leak channels by pharmacological inhibition, molecular extinction, or expression of LOF mutations decrease this permeability and shift Vm towards depolarised values around -30 mV (upper right panel). Experimentally, different techniques can induce an unbiased depolarisation -that is without targeting one ion channel specifically. One of these techniques is to iso-osmotically increase K+ extracellular concentrations. With an extracellular concentration of 100 mM instead of 4 mM, K+

equilibrium potential gets shifted to -10 mV. Membrane potential is therefore depolarised (lower right panel).

Depolarisation increases Wnt/β-catenin pathway activity

A short depolarisation kinetics of 10, 30 or 60 minutes induced the increase of β-catenin in its active form (Figure II-2 A). Interestingly, longer depolarisation seems to correlate with a higher increase of active β-catenin. Studying more precisely the kinetics of β-catenin would be interesting to decipher fully the link between depolarisation and activation of the pathway. HT29cl19A WT cells submitted to a 24-hour high K + medium had an increased protein expression of Wnt target genes such as c-jun, snail and slug (Figure II-2 B). The HT29cl19A 7TGC cells were then used to evaluate Wnt/β-catenin transcriptional activity in response to high K + and compared with exogenous addition of Wnt3A to the medium. High K + increased the number the GFP + cells in the same fashion as Wnt3A suggesting that depolarisation can activate Wnt/β-catenin in the same levels as its ligand. Combining exogenous Wnt3A to high K + showed an additive effect with even higher proportion of GFP + cells. Interestingly, the same effect was obtained by combining exogenous Wnt3A to HMR1556, a KCNQ1 inhibitor (Figure

II-2 C

). These results strengthen the results obtained in part I -7: loss-of-function of KCNQ1, in addition to direct activation, induce a permissive state for the activation of Wnt/β-catenin signalling by its canonical ligands. All in all, these results show that upon depolarisation, β-catenin rapidly shifts into its signalling active form under 30 minutes and transcriptional activity downstream is increased.

Furthermore, additive effects of depolarisation and Wnt3A are observed.

Depolarisation induces the phosphorylation of β-catenin

To further confirm that the increase of Wnt target genes protein expression and reporter assay were linked to an upstream activation of β-catenin signalling, we evaluated the phosphorylation of β-catenin on its Tyr654 residue. Tyr654 phosphorylation was increased when the loss-of-function mutations of KCNQ1 were expressed and when molecular extinction of KCNQ1 was performed. We thus aimed to confirm that this residue was phosphorylated upon depolarisation. To this end we subjected the cells to a short kinetic of high K + and showed that phosphorylation of β-catenin on its Tyr654 residue was significantly increased after depolarisations of 30 and 60 minutes (Figure II-3 A). To go further into the characterisation of β-catenin status, it would be interesting to study the phosphorylation status of residues Ser33, Ser37, Thr41 targeting β-catenin for degradation, and residues Tyr142 and Ser675 which improve β-catenin signalling activity. Furthermore, we isolated fresh colonic crypts from rats and performed rapid exposure to high K + . A 30-minute depolarisation induced the increase of phospho-Tyr654 as seen by Western Blot and Immunofluorescence in rat colonic crypts (Figure II-3 B,C).

Interestingly, from the immunofluorescence staining, it seems that deep crypt cells -probably stem cells or progenitor cells -are more responsive to depolarisation as the staining of phospho-Tyr654 is more important in this region. One hypothesis is that the cells at the bottom of the crypt have a higher Wnt activity and this could be the result of the additive effect shown in the previous figure. As receptor tyrosine kinases seemed to be implicated in the process, we performed PamGene PTK array after a five-minute depolarisation step. Receptor Tyrosine Kinases are predicted to be activated in response to depolarisation, among which are the family of Insulin receptors (InSR, IGF1R, IRR), ErbB family (HER2, HER3, HER4), RYK, TRKs, MET and RET. In addition, tyrosine kinases downstream of RTKs such as Src, Yes, Fyn, Lyn and JAKs are predicted to be activated (Figure .

All in all, these kinomic predictions orient towards an activation of a subset of Receptor Tyrosine Kinases and their downstream effectors, in response to experimental depolarisation, in line with the preliminary results obtained on KCNQ1 LOF mutations.

To confirm that Receptor Tyrosine Kinases are activated upon depolarisation we performed a kinomic assay. This experiment has been performed in parallel with a cell line expressing an shRNA directed against KCNQ1. In both conditions, we obtained an increased phosphorylation of c-RET, Insulin R, EGFR, This project requires additional experiments to be complete but provides insights into new mechanisms of cell biology. Our results seem to show that depolarisation in non-excitable cells trigger the activation of receptor tyrosine kinases and downstream effectors and have for effect an increase in Wnt/β-catenin signalling. It is highly probable, given the wide range of activated kinases, that other signalling pathways are turned on or off by depolarisation. A deeper understanding of the impacts of depolarisation and understanding of the triggers of depolarisation in physiological context will provide new views on physiopathological processes and perhaps lead to the development of new biomarkers and therapeutic targets.

Part III -Annex results

ML277

ML277 is a potent and highly selective pharmacological activator [START_REF] Mattmann | Identification of (R)-N-(4-(4methoxyphenyl)thiazol-2-yl)-1-tosylpiperidine-2-carboxamide, ML277, as a novel, potent and selective K(v)7.1 (KCNQ1) potassium channel activator[END_REF][START_REF] Yu | Identification of a novel, small molecule activator of KCNQ1 channels[END_REF]. Despite disputed binding pockets for ML277 on the structure of KCNQ1, it seems that it favours the open conformation of the channel [START_REF] Chen | Two smallmolecule activators share similar effector sites in the KCNQ1 channel pore but have distinct effects on voltage sensor movements[END_REF][START_REF] Eldstrom | ML277 regulates KCNQ1 singlechannel amplitudes and kinetics, modified by voltage sensor state[END_REF][START_REF] Hou | ML277 specifically enhances the fully activated open state of KCNQ1 by modulating VSD-pore coupling[END_REF][START_REF] Ma | Structural mechanisms for the activation of human cardiac KCNQ1 channel by electromechanical coupling enhancers[END_REF][START_REF] Willegems | Structural and electrophysiological basis for the modulation of KCNQ1 channel currents by ML277[END_REF][START_REF] Xu | Probing binding sites and mechanisms of action of an I(Ks) activator by computations and experiments[END_REF]. ML277 preferentially activates KCNQ1 alone and its action decreases as the stoichiometry is saturated by KCNE subunits [START_REF] Yu | Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels[END_REF]. In addition, it has been shown that ML 77 rescued specific Long QT variants and have been tested on patients' tissues and in vivo in dogs as a potential antiarrhythmic and cardioprotective drug [START_REF] Brennan | Slowly activating voltage-gated potassium current potentiation by ML277 is a novel cardioprotective intervention[END_REF][START_REF] Ma | Characterization of a novel KCNQ1 mutation for type 1 long QT syndrome and assessment of the therapeutic potential of a novel IKs activator using patient-specific induced pluripotent stem cell-derived cardiomyocytes[END_REF]van Bavel et al., 2023;[START_REF] Zou | Pharmacological rescue of specific long QT variants of KCNQ1/KCNE1 channels[END_REF]. We hypothesized that this molecule could rescue the function of some LOF mutations of KCNQ1. At a concentration of 1µM, ML277 was able to restore a small current elicited by the A329T mutant (

KCNQ1 -β-catenin interaction

Since KCNQ1 is in close proximity to the β-catenin and its current seems necessary to maintain βcatenin at the adherens junction, we wanted to evaluate whether the interaction was direct or indirect and whether this interaction had any effect on KCNQ1.

We performed GST pulldown assays and showed that KCNQ1 was able to bind to GST-fused β-catenin.

Experiments led by Loan, a Master student in the team, showed that β-catenin was binding to both the N-and C-halves of KCNQ1. Further experiments are being conducted to precisely identify the interaction sites between KCNQ1 and β-catenin. KCNQ1 being a tetramer, β-catenin could perfectly interact with different parts of each α-subunit. Additionally, β-catenin could bind to part of KCNE3.

While we know the impact of that interaction for β-catenin (stabilisation at the adherens junction), we Loan has confirmed these results in the HCT116 cell line and with β-catenin siRNAs to improve specificity. Among the next steps of this project, we will perform immunofluorescence experiments to determine if KCNQ1 is internalised in the absence of β-catenin.

While these results offer additional information, it also sheds new light on project 1. Indeed, in the context of cancer, we hypothesized that KCNQ1 was acting as a braking point on the Wnt/β-catenin pathway. When the pathway is overactivated because of the mutational landscape for example, KCNQ1 mRNA expression is repressed by TCF4. In addition, if the pathway is overactivated and βcatenin gets detached from the adherens junction, KCNQ1 could also lose its activity, further enhancing the cancerous phenotype and the activation loop of Wnt/β-catenin.

Lumen formation

At the beginning of this project, one part consisted in evaluating the epithelial-to-mesenchymal transition in cells carrying a KCNQ1 mutation. Indeed, as a partner of adherens junction and through the stabilisation of β-catenin with E-cadherin, KCNQ1 acts as a guardian of epithelial features.

Therefore, losing the activity of the channel would have a direct effect on the junction's stability and the activation of the Wnt/β-catenin pathway which promotes epithelial-to-mesenchymal transition.

While this study focuses essentially on Wnt/β-catenin signalling, some experiments on EMT have been performed. Among which, we have evaluated the ability of HCT116 to form cysts when KCNQ1 wild type is rescued or when one of the LOF mutation is expressed. Cysts are spheroid structures with an organised epithelium able to form a lumen thanks to the junctional integrity guaranteeing proper directional electrolyte and water transport. As shown in 2017, when wild type KCNQ1 is expressed, the epithelial features of these cells are rescued, and they are again able to organise and form a lumen when cultured in 3D. However, the expression of the loss-of-function mutations did not restore the ability of HCT116 to form organised spheres around a lumen . Taken together with the proximity ligation assays, these results strongly suggest that the LOF mutations of KCNQ1 impair the adherens junction and subsequently impair the epithelial features of cells.

Wnt3A inhibits KCNQ1 current

The team has shown in the previous study that activation of Wnt/β-catenin pathway led to a TCF4dependent translational repression of KCNQ1. On the other hand, KCNQ1 represses Wnt/β-catenin pathway. This antagonist bi-directional interaction comes into conflict with the tissue localisation of KCNQ1. Indeed, KCNQ1 is highly expressed at the bottom of the crypt, where Wnt pathway is at the highest levels of activation. And with KCNQ1 present, Wnt pathway should be kept low. This paradox has led us to wonder how could KCNQ1 be expressed despite Wnt activity and how could Wnt be active while KCNQ1 is expressed. One hypothesis is that despite its protein expression and apparent membrane localisation, KCNQ1 ion channel function is inhibited transiently at the bottom of the crypt to enable the activation of the pathway when needed. In addition, KCNQ1 could also be necessary in the lower crypt to maintain architecture and prevent Wnt from overactivation. Raphael has shown that, in females, oestrogen leads to a rapid internalisation-recycling of KCNQ1. Could there be a molecule that has a similar effect on KCNQ1 and that could thus enable the activation of Wnt/β-catenin pathway despite the presence of functional KCNQ1 in the lower part of the crypt? The obvious target molecule to test this hypothesis is Wnt3A. We therefore transiently transfected KCNQ1:KCNE3 in CHO cells and performed patch clamp experiments in whole cell configuration. Once cells sealed and opened, we performed a first protocol as described in part I Figure 2, added 100ng/mL Wnt3A and recorded the elicited currents. Upon Wnt3A addition, KCNQ1:KCNE3 currents were massively inhibited from a median of 23 pA/pF at 0 mV down to a median of 7 pA/pF. Moreover, current reversal (Prev) shifted from -60 mV to . Since the conductance of KCNQ1:KCNE3 decreases, the overall potassium conductance decreases. In an epithelial cell expressing all types of ion channels, it would mean that the relative permeability of potassium is lowered and thus should cause a depolarisation. As seen earlier, depolarisation creates a permissive state for the activation of Wnt/β-catenin. Therefore, we could hypothesize that in the lower part of the crypt, secretion of Wnt3A temporarily inhibits KCNQ1:KCNE3 leading to a small depolarisation allowing the activation of the pathway. To further study the physiological relevance and implications of this observation, it would first be interesting to study the effect of other growth factors on the current of KCNQ1:KCNE3 but also all ion channels expressed in this epithelium and second, to study the dynamic regulation of KCNQ1:KCNE3 in response to Wnt3A. Indeed, Wnt3A could directly inhibit KCNQ1 or lead to its internalisation, although the effect was instantaneous favouring the hypothesis of a direct inhibition, docking experiments would be interesting to know whether Wnt3A can bind KCNQ1. In addition, we have not recorded current after medium recovery and the reversibility of the effect of Wnt3A remains purely hypothetical.

My thesis project aimed at understanding the role of KCNQ1 in the homeostasis of the colonic crypt.

The team has shown that, in the colonic crypt, KCNQ1 guarantees epithelial integrity by restricting Wnt/β-catenin activity. In return, overactivation of Wnt/β-catenin signalling induces the transcriptional repression of KCNQ1 (Rapetti-Mauss et al., 2017). These results are complemented by a growing body of literature identifying KCNQ1 as a tumour suppressor in different cancer types [START_REF] Chen | miR-483-5p promotes esophageal cancer progression by targeting KCNQ1[END_REF]Fan et al., 2018;[START_REF] Gu | Melatonin alleviates progression of uterine endometrial cancer by suppressing estrogen/ubiquitin C/SDHB-mediated succinate accumulation[END_REF][START_REF] Huang | The miR-140-5p/KLF9/KCNQ1 axis promotes the progression of renal cell carcinoma[END_REF][START_REF] Yang | Low KCNQ1 expression is associated with unfavorable outcome and metabolism of gastric cancer[END_REF], and even as a prognostic factor in colorectal cancer (den [START_REF] Den Uil | Loss of KCNQ1 expression in stage II and stage III colon cancer is a strong prognostic factor for disease recurrence[END_REF]Than et al., 2014;[START_REF] Uil | KCNQ1 and lymphovascular invasion are key features in a prognostic classifier for stage II and III colon cancer[END_REF]. Therefore, understanding the mechanisms behind KCNQ1's tumour suppressor activity is necessary to consider KCNQ1 as putative therapeutical target or biomarker.

In addition, while the mutational landscape of cancers has been of great interest and ion channels are crucial in cancer processes, no investigation has been led on the possibility of somatic mutations on genes encoding ion channels. Looking into publicly available databases, we found that 2 to 11% of colorectal cancer patients carried mutations on KCNQ1.

Combining these two approaches, we selected somatic mutations to evaluate their effect on the Wnt/β-catenin pathway and dig deeper into the mechanism that links KCNQ1 and Wnt/β-catenin signalling. Therefore, we have been able to show that 1) ion channels carry somatic mutations in cancer, 2) KCNQ1 mutations are mostly loss-of-function 3) LOF mutations of KCNQ1 fail to restrict Wnt/β-catenin pathway. Concerning the mechanisms, we have shown that upon KCNQ1 LOF, Wnt/βcatenin was 1) activated independently from the canonical Frizzled/Lrp receptors, 2) dependent of βcatenin signalling activity, 3) probably activated by Receptor Tyrosine Kinases, and that 4) negative regulators of the pathway were downregulated, further lifting the brake imposed on Wnt/β-catenin pathway.

Since the role of KCNQ1 seems to be dependent on its potassium channel activity which also sets Part I -KCNQ1 Mutations

LOF mutations -expression and localisation

While we have shown that the proteins of the mutated channels were expressed in transfected CHO cells, and the studied subset in HCT116, seeing the protein expression of the truncated channels in HT29cl19A proved to be more difficult due to the high amount of endogenous WT KCNQ1 expressed by these cells. In addition, while we were able to confirm the dominant negative effect in CHO cells, we could not perform patch-clamp experiments on HT29cl19A. Indeed, these highly differentiated cells form tight islets that produce mucus making it difficult to seal properly the membrane and record current without any leak. For these reasons, we wanted to evaluate membrane potential using a voltage-sensitive probe, Disbac2( 3). This probe enters depolarised cells. Fluorescence intensity is thus correlated with depolarisation. Expression of the LOF mutation should result in a more depolarised resting membrane potential compared to the WT condition.

On a technical point of view, construction of a KCNQ1 expressing a tag in N-terminal resulted in a loss of current and no such construction exists in the literature. We could have inserted the tag between the first and second transmembrane segment, but one antibody targets an epitope located in the Nterminus of the protein allowing use to detect the truncated mutations.

In addition, the presence of the protein on a western blot does not mean that the protein is correctly addressed. We have not investigated whether the LOF channels were present at the plasma membrane by biotinlyation. Our research purposes were essentially to see if loss of function of KCNQ1 could impact the Wnt/β-catenin signalling pathway. The reason of the loss-of-function was not part of our questions for this study. However, for electrophysiological purposes, it would be interesting to know if these mutations impede the membrane targeting of KCNQ1. Some of our results and the literature can give some insight. First, the KCNQ1-A329T channel could be activated with the ML277 in an almost instantaneous fashion, suggesting a direct action on the channel that is thus already at the plasma membrane. While the basolateral targeting site is preserved in all mutants, the truncated mutant R518* has been shown to have a cytosolic localisation which was rescued at the plasma membrane upon co-expression with the WT form [START_REF] Harmer | Cellular mechanisms underlying the increased disease severity seen for patients with long QT syndrome caused by compound mutations in KCNQ1[END_REF]. It is thus highly probable that mutations inducing a truncation closer to the N-terminus (R401Gfs*18 and K422Sfs*10) display similar localisations. It could also be possible that other mutations induce a misfolding of the protein and endoplasmic reticulum retention. The mutations displaying a lower amplitude of potassium current such as the P448R for example might present a lower number of channels at the membrane resulting in a decrease of global current amplitude. Another hypothesis is that their unitary conductance is affected by the mutation. To test this hypothesis, we could perform single-channel patch clamp in the inside-out configuration providing access to channels' unitary conductance. When experimenting on the mutation A 87T, we noticed that while some cells displayed a "normal" current, some were unresponsive to stimulation as can be seen in Figure 1C with the representation of individual points.

Taken together, these currents are not statistically different from the WT currents. However, this observed phenomenon could be due to a misfolding of the channel or an alteration in the structure resulting in an impaired membrane targeting in a subset of cells.

Evaluation of Wnt/β-catenin activity

The evaluation of Wnt/β-catenin transcriptional activity by 7TGC is usually performed by flow cytometry. However, the HT29cl19A cell line poses technical challenges not compatible with this technique. This clone of HT29 parental cell line has been selected upon its epithelial features and resistance to Trypsin. Therefore, they are difficult to dissociate and tend to agglutinate despite presence of EDTA. While we have tried this method (data not shown), the analyses of the results were biased because cell clusters were forming and not passing correctly through the cell sorter. In addition, these cells produce mucus which displays auto-fluorescence in the green spectrum. Therefore, since flow cytometry analyses live cells, getting rid of this auto-fluorescence is not trivial. For these reasons, we performed confocal images of fixed cells instead of flow cytometry. To confirm the results obtained by counting the relative number of GFP+ cells, GFP expression could be assessed by ELISA Assay.

Regulators

From our different results it seems that inhibition of KCNQ1 activity through LOF mutations or experimental depolarisation led to a strong decrease of negative regulators of Wnt/β-catenin signalling. The mechanism leading to their decrease remains elusive. However, as seen in the RNASeq and in the analysis of activated kinases, other pathways seem to be impacted by a depolarisation (or KCNQ1 LOF). These pathways include BMP, MAPK, Hippo which are known to regulate Wnt/β-catenin in different manners. Therefore, the decrease of negative regulators could be the result of the activation of counter-signalling pathways. For example, expression of BAMBI -a positive regulator of Wnt/β-catenin -is increased by BMP signalling for regulation. All in all, depolarisation leads to the activation of several pathways that are intermingled resulting in an imbalance. Broad investigations will be needed to decipher the full spectrum of effects induced by depolarisation. The main limitation is the complexity of each pathway taken individually which is even more enhanced when trying to compute the different pathways at play.

Disruption of Adherens Junctions

In the proximity ligation assays, we used an antibody targeting an epitope localised in the N-terminus of the protein. Therefore, both the WT endogenous channel and overexpressed mutation are recognized by this antibody. The results obtained suggest that β-catenin was not associated with the WT channel nor the mutant when both were expressed. Different hypothesis could explain this observation. The first hypothesis is that in the presence of a LOF mutation, the heteromer is not properly addressed to the plasma membrane. Therefore, KCNQ1 is not at the adherens junction and is not in proximity with β-catenin. The first step to test this hypothesis is to perform immunocytofluorescence experiments with Wheat Germ Agglutinin as a marker of cell membrane and analyse the distribution of KCNQ1. Biotinylation experiments could also give insights on the relative amount of channel present at the plasma membrane. Should the channel be correctly addressed, we could perform proximity ligation assay between KCNQ1 and E-cadherin. A second hypothesis is that KCNQ1 and β-catenin directly interact and that mutations could disrupt the folding or lose the site of interaction. In this situation, β-catenin would be unstable at the adherens junction, unable to bind to KCNQ1 and thus released into the cytosol. We have performed preliminary GST pulldown assays

showing that KCNQ1 and β-catenin have a direct interaction and are currently investigating which residues on KCNQ1 could be important for the binding to β-catenin. Since KCNQ1 is a tetramer, it is highly probable that β-catenin binds to several parts of different α-subunits as it is the case with KCNEs for example.

In these hypotheses, we consider that β-catenin detachment from the adherens junction is the direct consequence of KCNQ1 LOF and is the cause of the activation of the pathway. However, co-existing hypotheses must be addressed. Indeed, detachment of β-catenin from the adherens junctions could be a secondary event. In this situation, depolarisation induced by KCNQ1 LOF activates Receptor Tyrosine Kinases which -directly or indirectly -phosphorylate β-catenin on its Tyr654 residue.

The best way to assess whether β-catenin release from the adherens junction is a direct consequence of KCNQ1 LOF would be to do a knock-in using Crispr-Cas9 and tag β-catenin. Experimentally depolarising the cells or using a pharmacological inhibitor of KCNQ1 and following in live imaging βcatenin would offer interesting insights into these questions. This tool, developed by a team (Massey et al., 2019), would allow us to decipher the underlying mechanisms.

From our results, we cannot say whether β-catenin release is a direct consequence of KCNQ1 LOF or rather the consequence of the upstream activation of RTKs. Nonetheless, one hypothesis would not exclude the other and both could be happening at the same time and contributing to the observed phenotype -the activation of Wnt/β-catenin signalling. The annex project described in part III concerning the understanding of this interaction from structural and functional point of views will surely give more insights into these questions.

General Perspectives

The role of ion channels in cancer processes has been studied since the 1980s (DeCoursey et al., 1984).

In the first few years of this new field, it was found that some channels were ectopically expressed in cancer tissues and blockade of those channels was decreasing proliferation. At the premises, research mostly focused on the phenotypic changes of cancer cells: proliferation, migration, invasion. More recently, it became clear that these abnormal expression of ion channels or their subunits could disturb signalling pathways and thus enhance cancer features [START_REF] Bon | SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer[END_REF][START_REF] Pillozzi | VEGFR-1 (FLT-1), beta1 integrin, and hERG K+ channel for a macromolecular signaling complex in acute myeloid leukemia: role in cell migration and clinical outcome[END_REF][START_REF] Rapetti-Mauss | SK2 channels set a signalling hub bolstering CAF-triggered tumourigenic processes in pancreatic cancer[END_REF]. However, whether ion channels carried pathogenic somatic mutations in cancer remained unexplored. In this study, we show for the first time that ion channels carry mutations in cancer, that these mutations can be loss-of-function and have an impact on the cellular processes.

A paper published in BioRxiv has also focused on mutations of KCNQ channel in GI cancers from a bioinformatic point of view [START_REF] Shorthouse | KCNQ gene family members act as both tumor suppressors and oncogenes in gastrointestinal cancers[END_REF]. From publicly available datasets, they identified two clusters of mutational burden within KCNQ1: in the S2-S3 linked and around the S6 helix.

Bioinformatic modelling predicts that A329T, P343S and L342F mutations, but not F339L, are loss-offunction. In addition, predictive analysis correlates KCNQ1 loss-of-function with higher Wnt activity.

This unpublished study thus strengthens our findings. Our results confirm the bioinformatics prediction and provide mechanistic insights.

Finally, Den Uil and colleagues had shown that expression levels of KCNQ1 could be used for stratification of patients with colorectal cancer (den [START_REF] Den Uil | Loss of KCNQ1 expression in stage II and stage III colon cancer is a strong prognostic factor for disease recurrence[END_REF][START_REF] Uil | KCNQ1 and lymphovascular invasion are key features in a prognostic classifier for stage II and III colon cancer[END_REF]. These loss-offunction mutations could thus aid in the prognosis and stratification of patients.

These findings strengthen the position of KCNQ1 as tumour suppressor and show for the first time that cancer-related mutations of ion channels can impact major signalling pathways. This first description of somatic mutations in cancer will pave the way for future research on ion channel's mutations in cancer. Many other ion channels carry somatic mutations and might therefore impact cell processes.

A better understanding of the landscape of cancer will provide new therapeutic options, particularly in an era of personalised medicine.

Mutations of KCNQ1 in cardiac physiopathology

The cancer-related mutations gathered in publicly available datasets are somatic mutations and thus do not induce defects in other tissues. Nonetheless, KCNQ1 plays a major role in cardiac physiology and a great number of studies have linked KCNQ1 mutations and Long QT Syndromes. We thus wondered whether similar mutations had been found in cardiac pathologies and if their behaviour was matching the ones we described with KCNE3. Of course, in the heart, KCNQ1 is not associated with KCNE3 but with KCNE1 which might change the features of some mutants.

Here are presented the mutations that are found in ClinVar database and associated impact and publications when applicable ("ClinVar Miner," n.d.). Of note, publications which have identified the variant in patients' cohorts but have not investigated its electrophysiological features have not been cited here but can be found through the variant accession number or ClinVar database.

The mutations V185M (rs749351255) and A329T (rs543428644) have been identified in patients with LQTS. However, while in silico analysis predicts A329T to be deleterious, the absence of functional assays has led to the classification of both mutations as of "uncertain significance". In our context, A329T alone was LOF which would suggest that it remains LOF when associated with KCNE1. However, V185M was not statistically different from the WT channel when expressed alone (Supplementary Figure 2) despite a seemingly lower amplitude of the current. Evaluating the effect of these two mutations when associated with KCNE1 might be beneficial in the context of LQT.

The R243C mutation (rs199472713) was identified in several patients with LQTS. Experimental studies showed reduced current and slower activation when expressed with KCNE1 and had decreased activation by PKA and PKC which may also contribute to disease phenotype [START_REF] Franqueza | Long QT syndrome-associated mutations in the S4-S5 linker of KvLQT1 potassium channels modify gating and interaction with minK subunits[END_REF][START_REF] Matavel | PKA and PKC partially rescue long QT type 1 phenotype by restoring channel-PIP2 interactions[END_REF]. R243C has thus been classified as pathogenic.

Mutation A287T (rs765665086) has been reported in patients with short and long QT. Patch clamp experiments with KCNE1 show an increase of macroscopic current, even in the heteromeric condition.

This study suggests that A287 is a key residue for interaction with KCNE1 which might explain the differences in the results obtained [START_REF] Rothenberg | Structural interplay of KV7.1 and KCNE1 is essential for normal repolarization and is compromised in short QT syndrome 2 (KV7.1-A287T)[END_REF]. However, taken separately, the currents we obtained with this mutant showed a wide heterogeneity with some cells exhibiting no current and other exhibiting larger amplitudes than the WT. Lacking more evidence, this mutation has been classified as "uncertain significance" in cardiac pathologies.

Mutations on F339 residue are missense into Serine and Tyrosine while in cancer the missense produced a Lysine. The mutations found in ClinVar are likely pathogenic, but it seems that F399L mutation has not been identified in LQTS patients so far.

Expression of the P343S mutation (rs199472762) and WT channel in xenopus oocytes resulted in a decrease of the current amplitude by about 90%, and reduction of IKs current of 60% (when coexpressed with KCNE1), making this variant pathogenic [START_REF] Zehelein | Identification and characterisation of a novel KCNQ1 mutation in a family with Romano-Ward syndrome[END_REF]. This mutation has been identified in several LQTS including Romano-Ward Syndrome.

Mutations resulting in a truncation of the protein R401fs (rs397508082), K422fs (rs1554895166, rs397508083) and R518Ter (rs17215500) are all predicted to be loss-of-function and thus pathogenic because of the premature protein truncation or nonsense mediated decay. R518* has been experimentally studied and displays no current when associated with KCNE1, it also has a dominant negative effect on the WT channel [START_REF] Ghosh | KCNQ1 assembly and function is blocked by long-QT syndrome mutations that disrupt interaction with calmodulin[END_REF]. Moreover, expressed with KCNE1, R518* is cytosolic and co-expression with WT KCNQ1 seems to rescue membrane targeting but not potassium current [START_REF] Harmer | Cellular mechanisms underlying the increased disease severity seen for patients with long QT syndrome caused by compound mutations in KCNQ1[END_REF].

R451Q mutation (rs199472781) has been identified in one patient with LQT but as also been found in healthy population thus orienting prediction score towards an "uncertain significance". When expressed with KCNE3, this mutant is loss-of-function.

Surprisingly, mutation P448R (rs12720449) was identified in LQT patients but considered not pathogenic by prediction scores as it is a single nucleotide polymorphism carried by 3% of the Asian population according to ALFA Allele Frequency. The amplitude of the current exhibited by this mutant when expressed with KCNE3 was lower but not totally lost. Several questions then arise: does it have any impact on KCNQ1:KCNE1 current? Are the 3% of the population carrying this SNP more prone to develop cardiac arrythmias at a later age and colorectal cancers or colon-related diseases such as inflammatory bowel disease? When transfected alone in CHO cells, P448R exhibited a smaller current that was not statistically different from the WT. erhaps this mutant impairs the channel's function only when associated with KCNE3.

All in all, several mutations have been identified in LQTS patients but only a subset have been experimentally characterized and identified as pathogenic. Confirming pathogenicity of these mutations would be beneficial to identify patients at risk by genetic screenings and perhaps offer therapeutic options such as treatment with KCNQ1 activator ML277 [START_REF] Brennan | Slowly activating voltage-gated potassium current potentiation by ML277 is a novel cardioprotective intervention[END_REF]van Bavel et al., 2023;[START_REF] Zou | Pharmacological rescue of specific long QT variants of KCNQ1/KCNE1 channels[END_REF]. In addition, since the loss-of-function mutations in cardiac pathologies are germline mutations, KCNQ1 expressed in the colon also carries the mutation. It would be interesting to study the susceptibility to colorectal -or other -cancers of these LQTS patients.

Part II -Depolarisation

Our results seem to show that acute depolarisation activates receptor tyrosine kinases, downstream effectors, and lead to the activation of Wnt/β-catenin signalling. Numerous questions arise from this project and experiments must be continued before publication. In this part I will detail some questions that remain unanswered and our hypotheses. Hedgehog signalling [START_REF] Emmons-Bell | Membrane potential regulates Hedgehog signalling in the Drosophila wing imaginal disc[END_REF][START_REF] Levin | Bioelectric signaling: Reprogrammable circuits underlying embryogenesis, regeneration, and cancer[END_REF][START_REF] Spannl | Glycolysis regulates Hedgehog signalling via the plasma membrane potential[END_REF]Zhou et al., 2015). Indeed, it will feed the growing body of literature showing that depolarisation has an effect even in non-excitable cells, which was overlooked until recently.

In a pathological context, loss of function or loss of expression of "hyperpolarisation-inducing" ion channels or on the contrary gain of function or expression of "depolarisation-inducing" ion channels can lead to a pathological depolarisation and thus dysregulation of signalling pathways creating or sustaining the pathology. However, how is this happening in a physiological context, what is the physiological relevance of this mechanism?

1. Depolarisation-induced activation of signalling pathway is conserved across species

It seems that the activation of signalling pathways in response to depolarisation is a conserved mechanism. The two papers presented in the introduction showing that Hedgehog signalling is activated in depolarised cells have been using Drosophila model. In addition, we have started to perform some experiments on a C.elegans strain resembling our 7TGC condition. Indeed, this strain called POPTOP expresses the mCherry under the control of TCF promoter. Since these results are very preliminary and much is left to do, it seems that exposing the C.elegans to high K + solution increases the mCherry fluorescence in some cells. Considering the conservation of Wnt/β-catenin signalling pathway, Receptor Tyrosine Kinases and ion channels, seeing if the depolarisation-induced signalling pathways are conserved as well would be an interesting finding.

In a pathological context

Imagining a depolarisation of non-excitable cells is easier in a pathological context. Literature shows that the expression of ion channels is highly disrupted in the context of cancer for example. Loss of function or expression of channels contributing to setting a hyperpolarised resting membrane potential (such as K2P channels) or on the contrary gain of function or expression of ion channels that tend to depolarise the membrane (such as the leak Na + channel NALCN) would lead to a depolarisation and subsequent atypical activation of signalling pathways. Activation of these signalling pathways could sustain the pathological loop.

In a physiological context

Is depolarisation in non-excitable cells relevant in a physiological context? As a first answer to this question, it has been shown that proliferating cells regulate the expression of ion channels leading to a transient depolarisation that is necessary for cell division [START_REF] Blackiston | Bioelectric controls of cell proliferation: ion channels, membrane voltage and the cell cycle[END_REF][START_REF] Sundelacruz | Role of membrane potential in the regulation of cell proliferation and differentiation[END_REF]. Is this far more complex than previously thought? Is membrane potential a key event that in addition to regulating cell volume, activates necessary signalling pathways? As we have shown, depolarisation activates Wnt/β-catenin signalling which is a proliferative pathway. Perhaps while activating some pathways, depolarisation also silences others which push more towards differentiation.

Once these questions answered, comes the question of the signal triggering depolarisation in physiological conditions. What prompts a cell to depolarise? A piece of the puzzle has been presented in part III. We have shown that in CHO cells, Wnt3A addition to the medium induces the rapid inhibition of KCNQ1. In a physiological context, Wnt3A is never alone but is rather in a mixture of different growth factors, hormones, and neurotransmitters. While their role in controlling cell processes remains extensively studied, and we know that hormones and neurotransmitters can impact ion channels expression, perhaps a direct role of these factors on ion channels activity is possible. In a condition "A" the concentrations of growth factors, hormones and neurotransmitters favour a hyperpolarisation of the membrane; and in condition "B", the concentrations differ and promote depolarisation by activation or inhibition of some ion channels. For now, this remains a hypothesis that still need to be tested. One of the experiments that could be used to test it is using Ussing chamber. Ussing chamber allows the measurement of currents through an epithelium. We could thus apply different concentrations of growth factors and see the impact on the currents generated apically and basally in an epithelium. To be even closer to what happens in vivo, one technique transforms organoids to a 2D epithelium that thus still exhibit all the different cell types.

Control of other signalling pathways

Moreover, preliminary results obtained in the team show that KCNQ1's bidirectional relationship with Wnt/β-catenin pathway could be inverted with BMP pathway. Indeed, these results seem to show that the activity of KCNQ1 favours BMP pathway activity and in return, KCNQ1 expression is upregulated by BMP transcriptional activity. If these results are confirmed, it would suggest that KCNQ1, by controlling membrane potential acts as a fine tuner of signalling pathways regulation. In addition, many kinases were predicted to be activated in the PamGene assay. For example, the MAPK pathway was among the top activated serine/threonine kinases in response to depolarisation and was also part of the most dysregulated pathway in the KCNQ1 LOF RNASeq. These results highly suggest that the MAPK pathway is sensitive to depolarisation. This observation is further strengthened by the publication from Zhou et al. on K-Ras and the phosphorylation of ERK which increases linearly with membrane depolarisation (Zhou et al., 2015).

First, the mechanism seems to be highly conserved. Indeed, depolarisation activates Hedgehog pathway during Drosophila development [START_REF] Emmons-Bell | Membrane potential regulates Hedgehog signalling in the Drosophila wing imaginal disc[END_REF][START_REF] Spannl | Glycolysis regulates Hedgehog signalling via the plasma membrane potential[END_REF] Cell Lines Culture Cells were cultured in 100mm dishes and passaged once to twice a week and prepared for experiments. Cells were grown in a humidified atmosphere containing 5% CO2 at 37°C and the medium was replaced every 2 to 3 days. The HT29cl19A cell line was a kind gift from Dr. Christian Laboisse [START_REF] Augeron | Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate[END_REF]

Plasmid transduction

cDNA coding sequence of KCNQ1 or a mutated KCNQ1 (KCNQ1mut) was subcloned into the modified mammalian expression vector pPRIG_eGFP where eGFP was exchanged for puromycin resistance sequence in order to generate pPriPu vectors. cDNA coding sequence of KCNE3 was subcloned into the modified mammalian expression vector pPRIG_eGFP where eGFP was exchanged for hygromycin resistance sequence in order to generate pPriHy vectors. Retroviruses were produced transfecting HEK 293T cells with pPriPu_KCNQ1 or pPriPu_KCNQ1mut or pPriHy_KCNE3, pCMV_VSVG, pCMV_gag-pol plasmids, using the classic calcium phosphate transfection technique. Replication-defective retroviruses were recovered in the culture medium 48h post-transfection. The retroviral supernatant was filtered through sterile 0.45 µm filters, and added directly to HCT116, HT29cl19A and CHO cells. 3 days later, puromycin (2,5 to 5 µg/mL) and/or hygromycin (75 µg/mL) was added in fresh medium to start selection of transduced cells.

Molecular Biology

Mutations of KCNQ1 were made into the pEGFP-N2_KCNQ1 plasmid by site-directed mutagenesis using Kapa HiFi HotStart according to the manufacturer's instructions and melting temperatures of oligonucleotides. The list of oligonucleotides is available in the table below.

Electrophysiology

CHO-K1 cells were plated in 60mm culture dishes and transfected using Jet Prime. For KCNQ1-KCNE3 transfections, a 1:3 ratio was used for a final quantity of 4µg of DNA (pEGFP-N2_KCNQ1:pXOOM_KCNE3) per 60mm dish. For KCNQ1 transfections, 4µg of DNA (pEGFP-N2_KCNQ1)

were transfected per 60mm dish; for the truncated mutations, 0.1µg of pIRES_GFP was added to detect transfected cells. For the dominant negative tests, CHO-K1 cells were transfected with 3 µg pXOOM KCNE3, 0,5 µg pEGFP-N2_KCNQ1 and 0,5 µg pEGFP-N2_KCNQ1_mut. After 6-8 hours, cells were rinsed, dissociated with Accutase and plated in 35mm dishes for patch clamp experiments the following day (24h post transfection). Only GFP positive cells were patched.

Electrophysiological recordings in CHO-K1 were performed at room temperature in the whole cell configuration of the patch clamp technique. The pipette (intracellular) and bath (extracellular) solutions are presented in table Table 3 pH was adjusted with KOH and NaOH respectively and consistency in osmolarity was checked with an osmometer. Soft glass patch electrodes were made on a horizontal pipette puller (Sutter Instruments) to achieve a final resistance ranging from 3 to 5 MΩ.

Electrical signals were amplified with an Axopatch 200B amplifier (Molecular Device, Foster City, CA)

and acquired with a DIGIDATA 1440 interface and pCLAMP 11.2 software (Axon Instruments). Cells

were maintained at a resting potential of -60mV, depolarised from -120mV to +80mV by increments of 20mV for 1000 msec and then hyperpolarised to -120mV for 200 sec to measure the tail current before returning to the -60mV resting potential. Elicited currents were measured at 0mV for current density analyses and at the peak current after hyperpolarisation to extract the open probability.

Experiments were conducted on at least three independent days with independent transfections.

For patch-clamp experiments with ML277, Wnt3A and DCA, after stabilization of the current, perfusion system containing the molecule was opened as close as possible to the patched cell in the extracellular bath. Currents were recorded continuously. Currents were measured upon stabilization of the effect induced by the molecule.

RNA Extraction

Cells were plated in 6-well plates and serum-starved overnight before the RNA extraction. The medium was removed and 800µL of Trizol per well was added for 10min. Cell lysates were scratched and transferred to an RNase-free tube. The same volume of 100% Ethanol was added per tube and mixed.

RNA extraction was performed using the Direct-Zol RNA Mini rep Kit according to the manufacturer's instructions. RNA was quantified using Nanodrop. 5µg of RNA were sent to Azenta-Genewiz for total RNA Sequencing. RNA Sequencing was performed on 4 biological replicates. Bioinformatic analysis to determine p-adjust and DeSeq2 was performed by Azenta-Genewiz.

Bioinformatic analyses of RNASeq results

Results from RNASeq were analysed with the software Amine [START_REF] Pasquier | Identification of active modules in interaction networks using node2vec network embedding[END_REF] to cluster differentially expressed genes by modules. Modules were then analysed with String [START_REF] Szklarczyk | The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest[END_REF] to identify corresponding Gene Ontology Terms (biological process).

TopFop Assay

TopFop Assay is a gold standard for the study of the Wnt pathway. Briefly, a gene coding for luciferase is under the control of Wnt promoter. The quantification of luciferin produced by cells is thus the direct reflection of the transcriptional activity of the Wnt/β-catenin pathway.

CHO-K1-pPriHy_KCNE3 and HCT116 cells expressing either KCNQ1 WT or a KCNQ1 mutation were plated in white 96 well plates, with 15 000 cells per well and 2 to 3 replicates per condition. The next day, HCT116 cells were transfected with Jet Prime with either M50 Super 8x TopFlash and pRL-SV40P plasmids or M51 Super 8x FopFlash and pRL-SV40P plasmids at a ratio of 10:1 for a final quantity of 0.1 µg per well. CHO-K1-pPriHy_KCNE3 cells were transfected with the same quantities of M50/pRL-SV40P

or M51/pRL-SV40P plasmids, to which was added 0.1 µg of pEGFP-N2-KCNQ1 DNA (WT or mutants) per well.

After six hours, cells were rinsed in 0% FBS medium and Wnt3A conditioned medium was added for 30 hours. Dual-Glo Luciferase Assay System was purchased from Promega and the experiment followed the manufacturer's protocol. A volume of Dual-Glo Luciferase Reagent equal to the culture medium volume was added to each well. The plate was left 10min in obscurity and the firefly luminescence was measured using the GloMax Discover Plate Reader. A volume of Dual-Glo Stop & Glo Reagent equal to the original culture medium volume was added to each well. The plate was left 10min in obscurity and the Renilla luminescence was measured using the GloMax Discover Plate Reader.

Firefly Luminesence produced in the M51 Super8x FopFlash transfected cells serves as the negative control -background noise. Renilla Luminescence produced by pRL-SV40P after addition of Dual-Glo

Stop & Glo Reagent serves as the control reporter.

The temperature of the GloMax Discover Plate Reader was set at 25°C for each experiment to avoid temperature-related shifts in luminescence over the time course of the experiments. The ratio of luminescence from the experimental reporter to luminescence from the control reporter was calculated and normalized to the ratio of a series of negative control wells. Median of duplicates/triplicates were used for statistical analysis and graph representation.

M50 Super 8x TOPFlash and M51 Super 8x FopFlash (TopFlash mutant) were gifts from Randall Moon (Addgene plasmid # 12456 ; http://n2t.net/addgene:12456 ; RRID:Addgene_12456 and Addgene plasmid # 12457 ; http://n2t.net/addgene:12457 ; RRID:Addgene_12457, respectively) [START_REF] Veeman | Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements[END_REF]. pRL-SV40P was a gift from Ron Prywes (Addgene plasmid # 27163 ; http://n2t.net/addgene:27163 ; RRID:Addgene_27163) (Chen and Prywes, 1999).

7TGC

7TGC was a gift from Roel Nusse (Addgene plasmid # 24304 ; http://n2t.net/addgene:24304 ; RRID:Addgene_24304) [START_REF] Fuerer | Lentiviral vectors to probe and manipulate the Wnt signaling pathway[END_REF]. 7TGC plasmid was stably expressed in the cells via viral transduction. HEK-293T cells were transfected with phosphate calcium and the following plasmid concentrations: 9 ug of 7TGC plasmid, 9 µg P89.1 and 2 µg pMD2G plasmids. P891 and pMD2G were kind gifts from Dr. Zeinab Rekad. Viral supernatant was collected after 48 hours, filtered through sterile 0.45 µm filters, then added directly to HT29cl19A cells. Cells were selected by fluorescent-activated cell sorting upon their expression of mCherry and then cultured in standard conditions.

To measure activity of Wnt/β-catenin transcription, cells were seeded on coverslip for 2-3 days and serum starved the night before fixation. When applicable, cells were incubated with Wnt3A / HMR1556 / high K+ solution for 12 to 30 hours after 12h serum starvation. Cells were then rinsed and fixed with 4% Paraformaldehyde and mounted on coverglass with Vectashield Vibrance DAPI mounting medium.

Imaging was performed on 710 Zeiss confocal microscope at a 63X magnification with the following lasers: 405 (DAPI), 488 (GFP), 561 (mCherry). At least 5 to 15 fields were imaged per condition and per biological replicate. The number of GFP + cells and mCherry + cells per field were counted manually.

Western Blot treatments

Part I -HT29cl19A were rinsed in 0% FBS DMEM and serum starved overnight prior to cell lysate preparation. For the experiments involving Wnt3A, HT29cl19A cells were serum starved overnight and Wnt3A CM was added for 30 hours. For experiments involving JW67 and niclosamide, cells were rinsed and incubated in 0% FBS DMEM overnight. JW67 and niclosamide were added to the 0% FBS DMEM medium.

Part II -HT29cl19A were serum starved overnight and incubated with the control solution or high K+ solution for the adequate time.

Western Blot

Protein samples were prepared from the cell lysates of CHO-K1 and HT29cl19A, using RIPA buffer (Radio-Immunoprecipitation Assay) containing anti-proteases and anti-phosphatases (Roche). Wholecell lysates were sonicated for 10 cycles of 10 seconds and centrifuged at 12 000 x g for 15 min.

Supernatant was collected and protein concentration was quantified using Bradford protein assay.

Protein samples were mixed with a 5X Laemmli buffer containing 10% β-mercaptoethanol and left at room temperature for 1 hour. Equal amounts of protein (50-75μg) were loaded onto 10% polyacrylamide gels and separated by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) at a constant voltage (80mV for 15 min and 120 mV for the remainder of the electrophoresis).

After electrophoresis, proteins were transferred from the gel onto a PVDF (polyvinylidene fluoride) membrane. The membrane was blocked with 5% non-fat dry milk or 5% BSA (Bovine Serum Albumin)

in TBST (Tris-buffered saline containing 0,1% Tween 20) or PBST (Phosphate-buffered saline containing 0,1% Tween 20) for 1 hour at room temperature.

The membrane was incubated with primary antibody (diluted in TBST or PBST containing <5% BSA or non-fat dry milk) overnight at 4°C, with gentle shaking. After washing the membrane with TBST or PBST, it was incubated with the secondary antibody conjugated with HRP (horseradish peroxidase) for 1 hour at room temperature, with gentle shaking. The membrane was then washed with TBST or PBST.

PBST was used in the conditions where phosphorylated residues were analysed.

Protein bands were detected using chemiluminescent substrates according to the manufacturer's protocol and then exposed to Fusion FX7 Edge. The intensity of the protein bands was analyzed using Image J and the results were normalized to the loading control (GAPDH or α-Tubulin).

Crypt extraction

Colonic crypts were extracted to perform depolarisation experiments and colon organoid culture.

Freshly dissected male mouse colon was everted on a glass rod and placed in 10 mL crypt isolation buffer (Table 4Table 5) at room temperature for 40 min. Crypts were detached after incubation by vigorous shaking. A sample of the solution was removed and examined under a light microscope to ensure that the crypts were isolated. Spare crypts solution was centrifuged at 1000 x g for 10 min and the pellet resuspended in Krebs solution (Table 4). The purity of isolated intact crypt was verified using microscopy. Isolations were performed at room temperature to avoid colonic crypt disintegration [START_REF] Schultheiss | Methods for the study of ionic currents and Ca2+signals in isolated colonic crypts[END_REF].

For isolated crypts experiments, crypts were seeded on coverslips precoated with Cell-TAK.

Organoid Culture

After three washes in Krebs buffer, isolated crypts were resuspended in Advanced-DMEM/F-12 supplemented with Hepes, Glutamax and 50 U/mL Penicillin/Streptomycin. Intact crypts were counted and resuspended in the proper amount of matrigel to seed 20 000 crypts per 40 µL matrigel drop. The matrigel drops containing the crypts in a 24-well plate were left to polymerise upside down for 15min at 37°C. 500uL of WENR medium containing Primocin (100 µg/mL) was then added to each well. The medium was changed the following day to remove Primocin. WENR medium was replaced every 2 to 3 days. Colon organoids were passaged every 10 to 15 days. For immunofluorescence experiments, organoids were seeded in 20 µl matrigel drops in 8-chambers Labtek.

For organoid transduction, retroviral supernatant was enriched in virus by reducing the amount of medium with HEK-293T cells during viral production. Organoids were dissociated using TrypLE, centrifuged at 200 x g for 5min and resuspended in viral supernatant with 1µM Y27632dihydrochloride to prevent anoïkis. Isolated organoid cells in viral supernatant were seeded onto 98well plate for 6 hours. Cells were then detached from the plate mechanically, centrifuged and resuspended in matrigel. Organoids were left to recover and grow for 5 days before antibiotic selection was added (1µg/mL puromycin). Viral transduction of the organoids with pPriPu-GFP and pPriPu-KCNQ1_A329T GFP plasmids was used as a proof of concept of this method.

Organoid size counting

Organoids were seeded on Labtek coverslip and imaged on the Timelapse: Axiovert 200M -Zeiss (2004) / sCMOS ANDOR Neo camera / MetaMorph software at a 5x magnification. Diameters were measured using Image J software.

Organoid medium preparation

WENR corresponds to the proliferative medium, ENR to the differentiation medium according to [START_REF] Sato | Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche[END_REF] Wnt3A conditioned medium was harvested as follows: L-Cells producing Wnt3A were a kind gift from Dr. Hans Clevers. Cells were cultured in DMEM supplemented with 10% FBS and 50 U/mL Penicillin/Streptomycin and selected using 1,25 µg/mL Zeocin. When confluency was reached, medium was replaced and left for one week. Conditioned medium was harvested, filtered using a 0,45 µm filter and stored at 4°C in sterile conditions. RspondinI conditioned medium was harvested in the same conditions, only the selection of cells was done with 3 µg/mL Zeocin.

Kit RTK

Phospho-antibody array analysis was carried out using the Proteome Profiler Human Phospho-Kinase

Array according to the manufacturer's instructions. Cells were cultured in the same conditions as for

Western Blot experiments and lysed with lysis buffer provided in the kit and agitated for 1h on ice Cell lysates were clarified by centrifugation at 12 000 g for 15 minutes and protein concentration was determined using a Bradford protein dye assay. Array membranes were blocked with blocking buffer provided in the kit and incubated overnight at 4°C with 300 µg of cell lysates. Membranes were washed to remove unbound proteins and incubated with phosphor-tyrosine antibody coupled to HRP provided in the kit. Chemiluminescent detection reagents were applied to detect spot densities. Array images were analysed using Image J software. Background noise was subtracted, and array spots integrated density were normalized to the positive control spots on each membrane to enable comparisons across different groups. The integrated density of duplicated spots representing each phosphor-RTK was determined, and data were presented as the fold change compared with the untreated or control group.

In Situ Proximity Ligation Assay

In situ proximity ligation assay is a technique used to identify the proximity between two proteins.

When this proximity is under 40 nm, the secondary antibodies coupled to probes will be close enough to perform a ligation and polymerization. Coupled to a detection reagent, this PCR reaction can be identified by dots corresponding to one protein A : protein B interaction. The number of dots per cell can thus be counted and compared between different conditions.

Bacterial and virus strains

E.Coli DH5alpha

DNA plasmids

M50 Super 8x TOPFlash Addgene (Randall Moon) Cat# 12456

INTRODUCTION

Despite decades of intensive research on its physiopathology, cancer remains a leading cause of death worldwide according to the World Health Organisation. Traditional cancer treatments including radiation therapy, chemotherapy, and surgery have substantially improved patients' conditions. However, several challenges in curing cancer remain in front of us. Side effects generated by cancer treatments significantly impact patients quality of life; high patients intrinsic heterogeneity revealed treatments resistance, and finally the efficiency of treatment used for some types of cancer remains very low such as for pancreatic cancer. These circumstances highlight the urgent need to identify new therapeutic strategies in treating cancers. Ion channels are transmembrane proteins allowing ions to cross biological membranes thereby generating electrical current and variation in membrane potential. Ion channels are classified in five main families, Potassium channels, Chloride channels, Sodium channels, Calcium channels that are selective for their specific ion, and non-selective ion channels that allow the passage of several species of ions. The functions attributed to ion channels were first confined to cell excitability and regulation of hydro-electrolytic balance. Interestingly, since the 1980's ion channels have also been described to be involved in controlling processes regulating cell behaviors such as proliferation, migration, invasion and differentiation. In 1984, the pioneer work of DeCoursey describing the involvement of potassium channels in the proliferation of T lymphocytes (DeCoursey et al., 1984) opened new perspectives in studying the biology of ion channels. The first evidence for such functions of ion channels in cancer cells emerged in the late 1980's with studies describing that the pharmacological inhibition of ion channels reduces tumor cells proliferation (Yamashita et al., 1987;Lee et al., 1988;Pancrazio et al., 1989;Taylor and Simpson, 1992). Since then, the field has greatly expanded, and it has been shown that ion channels are involved in virtually all the hallmarks of cancers described by Hanahan et al. (Prevarskaya et al., 2010;Prevarskaya et al., 2018). Although the involvement of ion channels in cancer has been clearly described, how ion channels interact with cancer-related signaling pathways from a mechanistic point of view, and how the expression of ion channels is regulated in cancers is poorly understood. However, this issue has recently emerged in the literature and a growing number of reports tackle these questions. Cancer initiation and development is often associated to altered signaling pathway involved in cell and tissue homeostasis [START_REF] Sanchez-Vega | Oncogenic Signaling Pathways in The Cancer Genome Atlas[END_REF]. Among these signaling pathways, the Wnt/b-catenin pathway emerged as the most associated pathway to ion channels in the literature. This review aims to summarize the current knowledge regarding the interplay between ion channels and the Wnt/b-catenin signaling pathway.

THE WNT SIGNALING PATHWAY

Since the first description of Wingless Int 1 (Wnt1) gene in virally induced breast tumors (Nusse and Varmus, 1982), the Wnt signaling pathway has been subjected to intense research, ranging from development to cancer. Its role in morphogenesis and adult stem cell renewal is closely linked to its involvement in cancer development [START_REF] Nusse | Wnt/b-Catenin Signaling, Disease, and Emerging Therapeutic Modalities[END_REF]. Mechanistically, Wnt signaling pathway can take mostly two directions: the canonical (Wnt/b-catenin), influencing cell-fate, proliferation, as well as self-renewal and the non-canonical (b-cateninindependent), Wnt/PCP, Wnt/Ca ++ , Wnt/STOP, influencing planar polarity [START_REF] Tree | Prickle mediates feedback amplification to generate asymmetric planar cell polarity signaling[END_REF], Ca 2+ homeostasis [START_REF] Sheldahl | Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos[END_REF], or protein stabilization [START_REF] Davidson | Cell cycle control of wnt receptor activation[END_REF], respectively. Both are involved in cancer but canonical Wnt/bcatenin remains the most studied in a large variety of tissues.

In mammals, there are more than 19 Wnt ligands which are expressed in a tissue-specific manner. After their translation into proteins Wnt ligands are modified with attachment of a palmitoleic acid (palmitoylation) by the ER-resident acyltransferase Porcupine (Kadowaki et al., 1996). This modification allows binding of Wnt ligand to the ERtransmembrane protein Evi/Wls, and therefore the transport to Golgi apparatus [START_REF] Bänziger | Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells[END_REF] assisted by the p24 protein [START_REF] Buechling | p24 proteins are required for secretion of Wnt ligands[END_REF]. Wnt ligands are either released by solubilisation (Mulligan et al., 2012), by exosomes (Gross et al., 2012), or in lipoproteins particles (Neumann et al., 2009) or linked to the plasma membrane. Importantly, after being secreted, Wnt ligands can form a gradient and act as directional growth factor, as it is the case in intestinal colonic crypts (Medema and Vermeulen, 2011) Here we will focus on Wnt canonical pathway, which depends on b-catenin stability. In the absence of Wnt ligand (Wnt OFF state), cytosolic b-catenin is sequestrated by a destruction complex (DC) composed of the tumor suppressor Axin acting as scaffold protein, the tumor suppressor Adenomatous Polyposis Coli (APC), the glycogen synthase kinase 3b (GSK3b) and casein kinase 1a (CK1a). Inside this DC, bcatenin is bound to Axin and phosphorylated by the two constitutively active kinases GSK3b and CK1a at specific Serine and Threonine residues (Liu et al., 2002). Then, bcatenin is ubiquitylated by the F-box containing protein E3 ubiquitin ligase (b-TrCP) and addressed to the proteasome for degradation (Aberle et al., 1997;Latres et al., 1999).

Initiation of the Wnt canonical (b-catenin-dependent) pathway (Wnt ON state) is mediated by engagement of Frizzled (Fz) receptor and LRP5/6 co-receptor with Wnt ligands. Activation of the pathway leads to degradation complex relocation near Fz/LRP complexes, where LRP is phosphorylated by CK1a and GSK3b. The phosphorylation of LRP triggers Dishevelled (Dvl) protein polymerisation, thus inactivating the degradation complex (Metcalfe et al., 2010). Another study suggests a direct inhibition of GSK3b by the phosphorylated-LRP co-receptor [START_REF] Stamos | Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6[END_REF]. Hence, bcatenin is not degraded, can accumulate in the cytoplasm and translocate into the nucleus. b-catenin then complexes with lymphoid enhanced factor (LEF) and T-cell factor (TCF) to act as a transcription factor activating target genes [START_REF] Li | Wnt signaling through inhibition of b-catenin degradation in an intact Axin1 complex[END_REF]. In the Wnt OFF state, TCF interacts with Groucho, preventing transcriptional activation of Wnt target genes (Cavallo et al., 1998). Interestingly, b-catenin plays a second role in maintaining epithelial junctions, by binding to the cytoplasmic tail of Ecadherin [START_REF] Peifer | The vertebrate adhesive junction proteins beta-catenin and plakoglobin and the Drosophila segment polarity gene armadillo form a multigene family with similar properties[END_REF]. Thus, presence of epithelial junctions is a limiting factor for Wnt/b-catenin activity, and reciprocally [START_REF] Huels | E-cadherin can limit the transforming properties of activating b-catenin mutations[END_REF].

The Wnt/b-catenin pathway is tightly regulated. For example, the two homologues E3 ubiquitin ligases ZNRF3/RNF43 can induce Fz receptor lysosomal degradation, thus inhibiting Wnt signaling. R-spondins are secreted proteins, interacting with their receptor LGR4-6, able to inhibit ZNRF3/RNF43 activity (de Lau et al., 2011). Furthermore, Hippo and Wnt pathway have been closely linked, since the Hippo transducer YAP/TAZ plays an integrative role inside the DC, allowing b-TrCP recruitment to the complex and subsequent b-catenin degradation [START_REF] Azzolin | YAP/TAZ incorporation in the b-catenin destruction complex orchestrates the Wnt response[END_REF]. Others regulations are nicely reviewed by Zhan et al. (2017).

As mentioned above, Wnt canonical pathway is widely implicated in cancer diseases. The first mutations have been found in the APC gene, leading to Familiar Adenomatous Polyposis (Kinzler et al., 1991;Nishisho et al., 1991), and now found in the majority of colorectal cancers (Wood et al., 2007). Mutations of Axin1 are present in hepatocellular carcinoma [START_REF] Satoh | AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1[END_REF]. Gain of function b-catenin mutations are reported in melanoma (Rubinfeld et al., 1997). Loss of function mutations in Rnf43 or Znrf3 are found in pancreatic cancer [START_REF] Wu | Whole-exome sequencing of neoplastic cysts of the pancreas reveals recurrent mutations in components of ubiquitin-dependent pathways[END_REF] and adrenocortical carcinoma [START_REF] Assie | Integrated genomic characterization of adrenocortical carcinoma[END_REF]. Wnt signaling has also been implicated in cholangiosarcoma [START_REF] Chan-On | Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infection-related bile duct cancers[END_REF], leukemia [START_REF] Wang | The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML[END_REF] and in 50% of breast cancer cases [START_REF] Lin | Beta-catenin, a novel prognostic marker for breast cancer: its roles in cyclin D1 expression and cancer progression[END_REF].

WNT SIGNALING PATHWAY INTERPLAY WITH POTASSIUM CHANNELS

With more than 80 members, the potassium channel family is the widest group of ion channels. They participate in a multitude of biological processes from cell excitability to water and electrolyte homeostasis. They are especially essential in maintaining membrane resting potential. Increasing evidence point out the involvement of this family of ion channel in the physiopathology of cancers [START_REF] Huang | Targeting potassium channels in cancer[END_REF]Pardo and Stühmer, 2014). However, the molecular mechanisms sustaining potassium channels involvement in cancer are still unclear. This section reviews recent work that report the function of potassium channels in the Wnt/b-catenin pathway.

KCNQ1

The potassium channel KCNQ1, also called Kv7.1, belongs to the family of voltage-gated potassium channel. It is constituted by the assembly of four a subunits forming the pore of the channel. This channel can associate with ancillary subunits from the KCNE family which drastically change the channel features regarding the KCNE associated (Bendahhou et al., 2005;Jespersen et al., 2005). Association with KCNE1, shows a delay in KCNQ1 activation and in this case the complex KCNQ1/ KCNE1 also called IKs, is essential to cardiac action potential repolarisation. With KCNE3 and KCNE2, KCNQ1 is no longer voltage-dependent and participates to transepithelial transport in nearly every epithelium. The effect of the last two members of the KCNE family on KCNQ1 are more discussed in the literature, however it appears that KCNE4 and KCNE5 mostly act as inhibitors of KCNQ1 activity [START_REF] Abbott | Biology of the KCNQ1 Potassium Channel[END_REF].

As aforementioned, KCNQ1 plays a key role in the physiology of epithelial transport. Transport mechanisms involve complex networks of ion channels and transporters, creating and dissipating ionic gradients required for the passage of water, electrolytes, nutrients or other substrates through the epithelium (Barrett and Keely, 2000). Thus, the Na + /K + ATPase pump activity is the main force for transepithelial transport allowing Na + output against K + entry into the cell. In this context, K + channels provide two main functions: the maintenance of membrane potential and the basolateral recycling of K + that guarantees the maintenance of electrochemical gradients. In the colon, KCNQ1, associated with its regulatory subunit KCNE3, plays a fundamental role in Leiberkühn's crypts homeostasis by maintaining the electrical driving force required for apical secretion of Cl -and water, and Na + absorption (Preston et al., 2010;[START_REF] Rapetti-Mauss | Oestrogen promotes KCNQ1 potassium channel endocytosis and postendocytic trafficking in colonic epithelium[END_REF].

Recently, studies found an unexpected link between KCNQ1 and epithelial cancers. In a first study, using mouse models, the authors have shown that the loss of KCNQ1 expression promotes the development of adenocarcinoma in the colon. They also showed that the low expression of KCNQ1 is associated with a poor survival prognosis in patients with Colorectal Cancer (CRC) showing hepatic metastases (Than et al., 2014). In line with these findings, another report has shown that in patients with stage II and III colon cancer, low expression of KCNQ1 is associated with poor survival prognosis. This channel therefore seems to be an interesting prognostic marker for the early stages of the disease as well as for the prediction of recurrences (den [START_REF] Den Uil | Loss of KCNQ1 expression in stage II and stage III colon cancer is a strong prognostic factor for disease recurrence[END_REF]. These studies also suggest that KCNQ1 acts as a tumor suppressor in CRC. Another study described the same function of KCNQ1 in Hepatocellular Carcinoma (HCC). They have shown using immunohistochemistry that HCC patients' samples show less KCNQ1 expression compared with normal liver tissues. Also, in patients with HCC, the high expression of KCNQ1 significantly correlates with a better patient's survival rate. Therefore, they reach the same conclusion as in CRC, KCNQ1 acts as a tumor suppressor in HCC (Fan et al., 2018).

It is clearly not trivial to explain the function of KCNQ1 as a tumor suppressor protein in epithelial cancers by its classical role in the physiology of epithelial transport. Therefore, could alternative functions of this channel underlie the observed correlations between KCNQ1 expression and cancers development? We and other have explored this issue to identify the molecular mechanism sustaining the tumor suppressor functions of KCNQ1 in CRC and HCC.

It appears that KCNQ1 expression is associated in CRC and HCC to epithelial cell plasticity (Rapetti-Mauss et al., 2017;Fan et al., 2018). Epithelial-to-Mesenchymal Transition (EMT) is a cellular process by which epithelial cells progressively and reversibly acquire mesenchymal features [START_REF] Ye | Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression[END_REF][START_REF] Pastushenko | EMT Transition States during Tumor Progression and Metastasis[END_REF]. This process has largely been associated to cancer development and progression by allowing loss of epithelial structure and promoting cancer cell migration, invasion and drug resistance. In CRC, the expression of KCNQ1 in a panel of cell lines showing different degrees of EMT is associated with epithelial features. Conversely, the channel's expression is lowered in most mesenchymal cell lines. These data raise the hypothesis of a functional link between KCNQ1 and the cellular process of epithelial-to-mesenchymal transition in cancer cells. Interestingly, in CRC as well as in HCC, the rescue expression of KCNQ1 in cell lines that do not express this channel, restores the expression of epithelial markers such as E-cadherin, lowers the expression of mesenchymal markers such as N-cadherin and restores the ability of cell to form spheroids in 3D culture. Moreover, the molecular silencing of KCNQ1 in epithelial CRC cell line reduces the expression of E-cadherin, increases the expression of N-cadherin and impairs the ability to grow as a spheroid in 3D culture. Therefore, the expression of KCNQ1 seems to promote epithelial features.

An important event characterising EMT is the dislocation of adherens junctions (AJ) causing the loss of cohesion between adjacent cells and therefore disrupting the epithelial integrity. The exploration of intestinal crypts by techniques coupling immunocytochemistry and in-situ immunisation (proximity ligation assay), provides data showing that KCNQ1 is directly associated with the b-catenin/E-cadherin complex in AJ in colonocytes. Interestingly, the same association between KCNQ1 and b-catenin has been established in hepatocyte cell lines. At the functional level, silencing of KCNQ1 expression in colon cell lines produces a disruption of the complex, as well as a loss of the integrity of epithelial architecture, which results in a decrease in epithelial electrical resistance. The study of the phosphorylation state of b-catenin in cell lines where KCNQ1 has been silenced by shRNA, indicates that KCNQ1 is necessary to retain b-catenin at the plasma membrane. Beside this observation it has been shown that pharmacological or molecular inhibition of KCNQ1 in CRC and in HCC, leads to a potentiation of the Wnt/b-catenin signaling pathway. Indeed, silencing of KCNQ1 in CRC induces the relocalisation of b-catenin in the cytosol and increases the expression of Wnt/b-catenin signaling pathway targets such as Cyclin D1 and C-jun. In HCC overexpression of KCNQ1 reduces Wnt target genes expression and Wnt activity using a Top/fop assay. These results indicate that KCNQ1 restricts the Wnt/b-catenin signaling pathway activity, stabilising b-catenin at the plasma membrane. These results reveal a new role for KCNQ1: in parallel with its function of transepithelial transport regulator, this channel plays a predominant role in maintaining the architectural integrity of the epithelium. These results shed new light on the correlation between the level of KCNQ1 expression and the clinical outcome of patients with CRC, since high levels of channel expression, together with the maintenance of adherens junctions guarantee the sequestration of b-catenin to the membrane, repression of the Wnt/b-catenin pathway, and finally reduces the commitment in the EMT process.

The stabilisation of the b-catenin/E-cadherin/KCNQ1 complex at the AJ constitutes an explanation to understand the role of KCNQ1 as a tumor suppressor in CRC and HCC. Would the latter only play an anchoring protein role for the b-catenin/ E-cadherin complex, or is the KCNQ1-induced hyperpolarising potassium flow important in the stabilisation of the complex? In other words, do membrane potential variations influence bcatenin localisation and activity? The answer is provided by experiments in which CRC cells expressing KCNQ1 are experimentally depolarised by increasing the extracellular K + concentration 140mM K + . This protocol causes a cytosolic accumulation of b-catenin, suggesting that the depolarisation induced by the silencing of the channel potentiates Wnt/bcatenin pathway. These results demonstrate the existence of a link between the electrical signature of the cancer cell and the signaling pathways that control cancer cells features. These data are consistent with a recent study that shows that the K-Ras/ RAF/MAPK signaling pathway is directly controlled by membrane potential (Zhou et al., 2015).

There is only few information in the literature regarding how ion channel expression is regulated in the context of cancers. It has been observed that expression of KCNQ1 is decreased in both CRC and HCC. We have tempted to address this question and we reason that the link between KCNQ1 could be bidirectional. Is KCNQ1 expression repressed by Wnt activity?

When the Wnt/b-catenin pathway is experimentally stimulated by pharmacological or molecular ways, we observed a decrease in KCNQ1 expression. We also identify that b-catenin as well as the TCF-4 transcription factor (member of the TCF/LEF family, which regulates the expression of the target genes of the Wnt pathway), bind directly to KCNQ1 promoter and cause the repression of its expression. These results thus reveal that KCNQ1 channel is a new target gene of Wnt pathway (Rapetti-Mauss et al., 2017).

By unveiling the bidirectional interaction between Wnt/bcatenin pathway and KCNQ1, these works reveal new functions of potassium channels in healthy tissue architecture, and their implication in the development of tumors. Thus, the fine understanding of these alternative functions could contribute to the characterisation of new markers, and innovative therapeutic targets. These data suggest that KCNQ1, by sequestering b-catenin at the AJ, restricts activation of Wnt signaling pathway and acts as a tumor suppressor in multiple epithelial cancers. The mechanism seems to be quite conserved suggesting a key role of KCNQ1 in epithelial homeostasis.

KCNH2

Another potassium channel has recently been associated to the Wnt/b-catenin pathway, KCNH2 also called hERG or Kv11.1 [START_REF] Breuer | Potassium channel activity controls breast cancer metastasis by affecting b-catenin signaling[END_REF]. This channel is a voltage-gated potassium channel, which role in cancer cells has largely been studied especially by the groups of Arcangeli [START_REF] Becchetti | Ion Channel Conformations Regulate Integrin-Dependent Signaling[END_REF] and Soriani (Crottès et al., 2016). This channel is associated with b1integrin when the channel is in its closed state, resulting in an increase of metastasis in breast cancer. Whereas, in the open state, the channel association with b1-integrin is decreased leading to a reduction in breast cancer metastasis [START_REF] Becchetti | The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression[END_REF].

A recent report described that an opener of hERG, the NS1643, reduces metastasis spread of breast tumor in vivo by attenuation of the Wnt/b catenin pathway signaling, suggesting that NS1643 mimics the dissociation between hERG and b1integrin [START_REF] Breuer | Potassium channel activity controls breast cancer metastasis by affecting b-catenin signaling[END_REF]. The authors observed that activating hERG channel stabilises b-catenin at the plasma membrane by inhibiting AKT (T308) and GSK3b (S9) phosphorylation. In this case the cytosolic fraction of b-catenin decreases favouring the association of b-catenin with E-cadherin. Therefore, NS1634 decreases Wnt pathway activity. This result suggests that the hyperproliferation induced by the opening of hERG reduces Wnt activity by retaining b-catenin at the plasma membrane. These data are consistent with the data from Arcangeli's lab showing that the open state of hERG lowers metastasis dissemination in breast cancer.

WNT SIGNALING PATHWAY INTERPLAY WITH CHLORIDE CHANNELS

Chloride channels regulate a variety of cell processes such as excitability and water and mucus secretion. Another important aspect of these channels, especially in cancer, is their role in cell volume regulation and pH level regulation. As for the other ion channel families, different chloride channels have been described depending on their gating properties: calcium-activated, voltagedependant, ligand-dependant. Although their role in cancer has been under study for the past years, their link with the Wnt/bcatenin pathway remains poorly investigated.

CFTR

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is widely expressed in the apical epithelial membranes. CFTR is an anion channel regulating fluid transport and pH levels. Mutations of CFTR such as DF508 result in Cystic Fibrosis (CF) causing a wide range of symptoms linked to a defect in fluid transport across the epithelial membranes. The pulmonary symptoms are welldescribed, but the earliest symptoms concern the digestive system. In addition, CF patients are at high risk of developing gastrointestinal cancers [START_REF] Yamada | Risk of gastrointestinal cancers in patients with cystic fibrosis: a systematic review and meta-analysis[END_REF]. The study from Strubberg et al. links for the first time the loss of functional CFTR and gastrointestinal cancer through the increase of the proliferation rate of crypt cells by activating Wnt/b-catenin pathway [START_REF] Strubberg | Cftr Modulates Wnt/b-Catenin Signaling and Stem Cell Proliferation in Murine Intestine[END_REF]. Indeed, the authors show that loss of functional CFTR leads to an increase of intracellular pH (pHi) levels in crypt cells as Cl -and HCO3 -secretion is impaired. In addition, it has been shown in Drosophila species that alkaline pHi (negatively charged phospholipids at the inner leaflet) facilitates Dishevelled's interaction with the Wnt receptor Frizzled by addressing Dishevelled to the negatively charged phospholipids through its DEP domain [START_REF] Simons | Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization[END_REF]. Strubberg et al. show that the same mechanism is occurring in Cftr KO cells. The inhibition of CFTR activity alkalinizes pHi in the crypt epithelium, enhancing Dishevelled-2's (Dvl-2) association at the plasma membrane and promoting binding between Fz and Dvl-2 and therefore facilitating Wnt signaling. Confirming these results, active b-catenin and LEF1 levels were higher in Cftr KO mice crypts than WT. Furthermore, CFTR has been shown to be a direct target of Wnt signaling as it exhibits an intestine-specific enhancer element positively regulated by TCF4 [START_REF] Paul | The epigenetic signature of CFTR expression is co-ordinated via chromatin acetylation through a complex intronic element[END_REF]. All in all, loss of CFTR facilitates Wnt signaling in the intestinal crypt epithelium, increasing tumorigenesis and underlying the basis of increased risk of gastrointestinal cancers in CF patients.

The main cancer risk for CF patients is gastrointestinal (GI) cancers [START_REF] Neglia | The risk of cancer among patients with cystic fibrosis. Cystic Fibrosis and Cancer Study Group[END_REF]. However, as new therapeutic strategies aim to enhance CF patients' life expectancy, and since the link between loss of functional CFTR and Wnt/bcatenin pathway has been assessed in GI cancers, other epithelial cancers could occur in CF patients following the same biological process.

CLCN2

Adherens junctions and tight junctions ensure epithelium integrity, providing a physical barrier. Adherens junctions further establish the apical-basal cell polarity. The importance of the chloride channel ClCN2, also called ClC-2, in the regulation of tight junctions' composition in the small intestine has been recently uncovered [START_REF] Nighot | Chloride channel ClC-2 modulates tight junction barrier function via intracellular trafficking of occludin[END_REF]. As ClCN2 seemed to be an important actor of epithelial integrity and maintenance of the tight junctions, Jin et al. questioned its involvement in adherens junctions (AJ) and its interaction with b-catenin [START_REF] Jin | Knockout of ClC-2 reveals critical functions of adherens junctions in colonic homeostasis and tumorigenicity[END_REF]. b-catenin binds E-cadherin in the adherens junctions. If the AJ are disrupted, b-catenin is freed from the membrane-bound E-cadherin, accumulates in the cytosol and translocates to the nucleus where it interacts with TCF/LEF1, activating the Wnt target genes.

ClCN2 is expressed physiologically in the colonic epithelium. Absence of ClCN2 alters AJ integrity, inducing an increase of nuclear b-catenin. The increase of nuclear b-catenin is linked to an upregulation of Wnt target genes. Moreover, the loss of ClCN2 alters colonic crypt homeostasis, increases proliferation and reduces differentiation of colonic cells.

Therefore, the loss of ClCN2 in colonic epithelium leads to the disruption of adherens junction and subsequent activation of Wnt pathway, promoting tumorigenicity. The involvement of other ClCs in the regulation of junctions and their link with Wnt/ b-catenin pathway has not yet been assessed. However, a study showed that absence of a regulatory protein of Calcium-activated chloride channels (CaCCs)-such as ANO1, also called TMEM16A [START_REF] Sala-Rabanal | Secreted CLCA1 modulates TMEM16A to activate Ca(2+)-dependent chloride currents in human cells[END_REF][START_REF] Sala-Rabanal | Modulation of TMEM16A channel activity by the von Willebrand factor type A (VWA) domain of the calcium-activated chloride channel regulator 1 (CLCA1)[END_REF]-CLCA1, induces an increase of b-catenin in the nucleus [START_REF] Li | CLCA1 suppresses colorectal cancer aggressiveness via inhibition of the Wnt/betacatenin signaling pathway[END_REF]. However, this study only focuses on the consequence of a knock-out of CLCA1 and not its link with CaCC channels nor its direct or indirect interaction with Wnt/bcatenin pathway. These points remain to be understood to truly consider CLCA1 as a tumor suppressor via inhibition of Wnt pathway.

WNT SIGNALING PATHWAY INTERPLAY WITH OTHER CHANNELS TRP Channels

Transient receptor protein (TRP) channels are cationic channels that generate Na + and Ca 2+ influxes in response to a great variety of stimuli [START_REF] Venkatachalam | TRP channels[END_REF]. Therefore, TRP channels enable individual cells to sense changes in their local environment, translating this information into signals that can be interpreted at a short time scale such as membrane potential and Ca 2+ homeostasis [START_REF] Shapovalov | Role of TRP ion channels in cancer and tumorigenesis[END_REF]. Several works have revealed a functional link between TRP channels and Wnt/ b-catenin pathway in cancers.

TRPV4

In gastric cancers, the Calcium receptor (CasR) is overexpressed. Its overexpression contributes to tumor growth, metastasis formation, and poor prognosis. Mechanistically, CasR stimulates TRPV4 channels. The TRPV4-dependent Ca 2+ influx increases AKT activation, which in turn phosphorylates b-catenin on its residue Ser675 and thus activating b-catenin downstream signaling [START_REF] Xie | Calcium Promotes Human Gastric Cancer via a Novel Coupling of Calcium-Sensing Receptor and TRPV4 Channel[END_REF].

TRPM4

TRPM4 channels are overexpressed in prostate and colorectal cancers where they participate to migration and invasion [START_REF] Gao | TRPM4 channel and cancer[END_REF]. In prostate cancer cells, invalidation of TRPM4 increases GSK3b activity, leading to b-catenin degradation. In LnCAP prostate cancer cells, TRPM4-associated Ca 2+ influx stimulates AKT1, which in turn increases the inhibitory Ser9 phosphorylation of GSK3b and the total amount of b-catenin [START_REF] Sagredo | TRPM4 regulates Akt/GSK3-b activity and enhances b-catenin signaling and cell proliferation in prostate cancer cells[END_REF]. In this study, Sagredo et al. suggest that the effect of TRPM4 on AKT1 is probably mediated by an alteration in the calcium/calmodulin-EGFR axis, linking TRPM4 activity with the observed effects in b-catenin-related signaling pathways.

TRPM8

A recent study driven in prostate and breast cancer cell lines has proposed that Wnt ligand-induced signaling involves TRPM8 channels. The authors propose a model in which Wnt ligands bind to both their receptors and TRPM8 channels, inducing a rapid decrease in [Ca 2+ ]i, followed by a store-operated calcium entry (SOCE). The SOCE in turn triggers calcium-dependent potassium channels (KCNN4) leading to a large membrane hyperpolarization. This study indeed suggests that control of membrane potential by Wnt ligands is an early event of the Wnt signaling pathway [START_REF] Ashmore | Wnts control membrane potential in mammalian cancer cells[END_REF].

TRPC5

TRPC5 is overexpressed, together with ATP-binding cassette, subfamily 1-member1 (ABCB1) in , in CRC cells. ABCB1 is a transport protein involved in chemoresistance by mediating efflux of cytotoxic drugs. Enforcing TrpC5 expression increases in a Ca 2+ -dependent manner nuclear accumulation of bcatenin and subsequent upregulation of Wnt target genes including ABCB1. These results suggest that TRPC5 may reinforce chemoresistance by regulating transporters controlled by the Wnt/b-catenin pathway [START_REF] Wang | Inhibition of transient receptor potential channel 5 reverses 5-Fluorouracil resistance in human colorectal cancer cells[END_REF].

Purinergic Ionotropic Receptors

P2RX7

The ionotropic ATP receptor P2RX7, (P2X7) is overexpressed in osteocarcinoma and participates to several cancer hallmarks including proliferation, migration, invasion and EMT. At the molecular level, P2X7 stimulation by receptor agonist (bzATP) increases GSK3b phosphorylation and further accumulation of b-catenin in the nucleus. Indeed, Wnt/b-catenin activation by P2X7 stimulation depends on PI3K/AKT stimulation. Interestingly, pharmacological inhibition of P2X7 decreased pro-tumoral behavior of cancer cells [START_REF] Zhang | Highly-expressed P2X7 receptor promotes growth and metastasis of human HOS/MNNG osteosarcoma cells via PI3K/Akt/GSK3b/b-catenin and mTOR/HIF1a/VEGF signaling[END_REF].

Acid Sensing Ion Channels

ASIC1a: Acid sensing ion channels (Asic) are proton-gated Na + / Ca 2+ channels involved in a wide range of physiological features including pain perception and behavior [START_REF] Hanukoglu | Epithelial sodium channel (ENaC) family: Phylogeny, structure-function, tissue distribution, and associated inherited diseases[END_REF]. Asic1a is up-regulated in liver cancer tissues and cells lines and is associated to poor prognosis. KO of Asic1a inhibits liver cancer growth in vivo and in vitro, an effect that is underlied by cell cycle arrest and increased apoptosis. Jin et al. observed occurring in tumor microenvironment, increased Asic1a activity, which in turn decreases the Ser33 phosphorylation of b-catenin, leading to its accumulation in the nucleus, its combination with LEF/TCF promoting target gene transcription associated to liver cancer proliferation [START_REF] Jin | Overexpression of ASIC1a promotes proliferation via activation of the b-catenin/ LEF-TCF axis and is associated with disease outcome in liver cancer[END_REF].

CONCLUSION AND FUTURE DIRECTIONS

In this review we have summarized the current work regarding the interplay between ion channels and the Wnt/b-catenin signaling pathway in cancers. The analysis of the literature highlighted 10 ion channels associated to the Wnt pathway (Table 1). Among these channels four are associated to the repression of Wnt pathway (KCNQ1, hERG, CFTR, and ClCN-2, Figure 1A). The loss of activity or expression of these channels leads to an increase of Wnt activity and cancer progression. Supporting this, KCNQ1, CFTR and ClCN-2 have been described as tumor suppressors in CRC. Contrarily, six ion channels are overexpressed in cancers and promote the activity of Wnt pathway (TRPC5, TRPV4, TRPM4, TRPM8, P2RX7, and ASIC1a, Figure 1B). Several conclusions can be drawn from this analysis of the literature. Firstly, the hyperpolarisation induced by the opening of potassium channel (background current from KCNQ1/ KCNE3 in colonocytes or inducing the opening of hERG in breast cancer cells) leads to the restriction of Wnt activity by a similar mechanism, i.e. the sequestration of b-catenin at the plasma membrane and probably at the adherens junctions. Hyperpolarised plasma membrane seems to be important to prevent an over activation of the Wnt pathway and stabilise bcatenin at the plasma membrane. Therefore, the remaining question is: how can membrane potential act on b-catenin stability at the membrane? This is an outstanding question which will need more investigation to be answered. Secondly, regarding the Wnt-activating ion channels, the mechanism emerging from this analysis seems to mainly involve an increase in intracellular [Ca 2+ ] due to the overexpression or up regulation of cationic channels that mediate Ca 2+ influx. The increase in intracellular [Ca 2+ ] shunts the canonical Wnt/bcatenin signaling cascade by activating AKT. AKT, in turn phosphorylates GSK3b on S9 causing its inhibition and finally the accumulation of b-catenin and the activation of Wnt pathway. Thirdly, as mentioned above, several ion channels are involved in the regulation of Wnt pathway, suggesting that interaction between them could exist. This is true especially regarding the case of KCNQ1, CFTR, and ClCN-2. It is interesting to note that KCNQ1 and ClCN-2 share the same mechanism to lower Wnt pathway activity. Both channels are associated to adherens junctions and contribute to stabilise bcatenin at the plasma membrane. This observation suggests a functional interaction between these channels which has not been investigated so far. Concerning KCNQ1 and CFTR, it is largely described that in colonic epithelium both channels are closely linked in the mechanism regulating the transepithelial transport of Cl -and water. It seems credible that this functional link is conserved in the regulation of the Wnt pathway, both channels contributing to restrict its activity by two different, but complementary mechanisms, KCNQ1 controlling the stability of b-catenin at the plasma membrane and CFTR controlling the localization of Dvl. It will be interesting to study the effect of the simultaneous inhibition of both channels on Wnt activity.

Large research efforts have been made during the last 40 years to describe and understand the functions of ion channels in controlling cellular processes. Although the involvement of ion channels in processes participating in cancer development has largely been demonstrated, the precise mechanisms sustaining these abilities remain mainly unknown. Even if several functions of ion channels in signaling pathways can be explained by classical roles, such as regulation of cell volume or modulating the intracellular calcium concentration, other implication of this class of proteins are dictated by original mechanisms. Could ion channels be necessary in the regulation of classical signaling pathway and how? Can we insert ion channels in the notebook picture of signaling pathways beside tyrosine kinases receptors and G protein coupled receptors? Answering these questions are the arising challenges in the field of ion channels in cancer biology.

Pancreas

target the communication between the stroma and PCC revealed that non-specific targeting may enhance tumour development, suggesting a complex interplay between both compartments. 1 5 6 A finer strategy consists in focusing on the specific molecular mechanisms controlling the communication between CAF and PCC, 7 8 but the landscape remains incomplete. This situation illustrates the urgent need to understand the crosstalk between CAF and PCC, its impact on PCC abilities and PDAC evolution and investigate original drug targets. SigmaR1 (Sig-1R) is a stress-activated and ligand-operated ion channel chaperone, controlling the electrical remodelling occurring in heart and brain diseases. 9-12 We showed in previous studies that Sig-1R induced electrical remodelling in cancers, promoting cell invasiveness. 13 14 In the context of PDAC, we reasoned that (1) Sig-1R may participate in intercellular communication by promoting the remodelling of PCC's ion channels activity under the influence of CAF and (2) Sig-1R regulated channels could be specifically targeted in PDAC using Sig-1R ligands.

A deep remodelling of the cells' electrical signature accompanies the development of numerous illnesses, including cardiopathies, neurodegenerative diseases, inflammation and cancers. 15 16 The role of ion channels has been appreciated in cancers, and growing evidence show their implication in cancer cell hallmarks by regulating Ca 2+ homeostasis, 15 17 cell shape 18 and, nascently, signalling transduction pathways through the control of membrane potential (ie, Wnt 19 and KRAS). 20 However, the contribution of ion channels to intercellular communication within cancer tissues remains unknown.

Therefore, we aimed to understand the significance of CAF secretome-induced ion channel regulation on PDAC progression. We show that CAF secretome triggers the activation of the calcium-dependent K + channel SK2 through its direct phosphorylation by AKT (Protein Kinase B). SK2 is thenceforth included in a β1integrin-epidermal growth factor receptor (EGFR)-AKT signalling hub and sets a positive feedback loop amplifying tumourigenic processes. The activation and spatial dynamics of SK2 on CAF secretome exposure is dependent on Sig-1R. The pharmacological inhibition of Sig-1R abrogated CAF-induced metastatic spreading and improved overall survival. Altogether, these results demonstrate the unforeseen function for SK2 channel in tuning the intercellular communication between CAF and PCC and pave the way for potent therapeutic options in PDAC.

MATERIALS AND METHODS

Cell culture and CAF conditioned media production HEK293T, PANC-1 and MiaPaCa-2 cell lines obtained from ATCC were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/mL peniciline/streptomycin under 5% CO 2 and 37°C in a humidified atmosphere. Cells were tested routinely for Mycoplasma contamination. CAFs were obtained as previously described. 3 Briefly, CAF features were verified by immunofluorescence by a positive alpha smooth muscle actin (α-SMA) staining and a negative pan cytokeratin staining. We also further used a CD45, CD326 (Epcam), pan cetokeratin (CK) and CD31 flow cytometry sorting to confirm that CAFs cultured are not immune, vascular or tumorous cells. CAFs were cultured in Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham (DMEM/F12), 10% FBS, 2 mmol/L l-glutamine, 1% antibiotic-antimycotic and 0.5 mM sodium pyruvate and used between passages 4 and 10. When the monolayer reached 70%-80% confluence, they were incubated in DMEM/F12 with 1% FBS (used as a control medium in experiments), and this conditioned media was collected every 2 days and stored at -20°C. Normal human fibroblasts (NHF) were isolated from healthy neonatal foreskins and cultured in DMEM, 10% FBS, 2 mmol/L l-glutamine and 1% antibiotic-antimycotic. Conditioned media was collected as described for CAFs.

Animal studies

For GEMM studies, Pdx1-Cre, Ink4a fl/fl LSL-Kras G12D (KIC pdx1 ) male mice were obtained by crossing the following strains: Pdx1-Cre, Ink4a fl/fl and LSL-Kras G12D mice provided by D Melton (Harvard Stem Cell Institute, Cambridge, Massachusetts, USA), R Depinho (Dana-Farber Cancer Institute, Boston, Massachusetts, USA) and T Jacks (David H. Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA), respectively. PDACbearing 5-week-old and 8-week-old mice were treated once a week with vehicle or 1(S) (0.1 mg/injection/mouse) for survival follow-up until experimental endpoint: mice reaching sacrificing time or mice reaching physiological endpoints (cachexia criteria, limited movement, drinking or feeding of the mice). No exclusion has to be reported in all experiments as no mice reached a criteria of exclusion such as a fight-related wound or the development of ear inflammation. To study final tumour volume, mice were euthanised at 9 weeks and tumours were weighed and fixed in 4% (wt/vol) formaldehyde for immunochemistry. For survival analyses, male mice were euthanised when they reached ethical clinical end points defined by our institutional guidelines and European animal protection law. For orthotopic tumour studies: the mice were anaesthetised by isoflurane (Vetflurane; Virbac) inhalation in 30% air and 70% O 2 . NMRI nude mice (Janvier Labs) under anaesthesia were injected subcutaneous with 0.2 mg/kg buprenorphine (Vetergesic; Sogeval) and were administered lidocaine (Xylovet; Ceva) at 3.5 mg/kg by infiltration in the abdominal cavity. A first incision was made at the top left of the abdomen and a second at the peritoneum to reach the pancreas. PANC ShRD/PANC ShSig-1R/PANC ShSK2 cells alone (500 000 cells) or plus CAF (1 500 000 cells) contained in 40 µL of PBS were injected into the pancreas. The abdominal musculature was then closed with 4-0 sutures and the external skin with inverted stitches. The mice were euthanised 8 weeks later. The pancreas was removed and weighed and the liver removed and analysed for macroscopic metastases. These organs were fixed in 4% formaldehyde for immunochemistry. Liver metastases were confirmed and counted per mouse using H&E staining.

For all experiments, sample size was predicted following a Mann-Whitney statistical approach involving power calculation of 0.8 with an false discovery rate (FDR) of 0.05. A minimum number of 7 mice per group was predicted as able to reach significance. Bonferroni post-hoc test was used for multiples test comparison (two variance with time and/or treatment). Experimental technicians were proceeding with injections, while personnel related to mice follow-up worked in a blinded approach.

Gene expression analysis of human PDAC samples

We gathered clinicopathological and gene expression data of clinical pancreatic samples from 16 publicly available data sets (online supplemental table S2). Data were collected from Array-Express, EGA, National Center for Biotechnology Information/ Genbank GEO and TCGA databases and had been generated using DNA microarrays (Affymetrix, Agilent and Illumina) and RNA-seq (Illumina). The pooled data set contained 1017 samples, including 925 primary PC samples, and 92 metastases.

Pancreas

Statistical analysis

Each experiment was repeated independently at least three times. Results are presented as median with IQR unless stated otherwise. For comparison between two quantitative variables, we used the Mann-Whitney test. When more than two variables were compared, Kruskal-Wallis tests were used followed by a Dunn's post-test, unless stated otherwise. For comparison between fold change, a one sample t-test was applied. Log-rank (Mantel-Cox) test was used to evaluate mice survival. Analyses were performed using either GraphPad Prism V.8.0.1 or R software. A p value of <0.05 was considered statistically significant.

Additional materials and methods are provided in the online supplemental material.

RESULTS

Secreted cues from CAF stimulate SK2 in pancreatic cancer cells

To determine whether Sig-1R participates in electrical remodelling in PDAC, we recorded ion channel activity in PCC (ie, PANC-1 and MiaPaCa-2), where Sig-1R expression was silenced by short hairpin (sh) RNA. Abrogation of Sig-1R expression led to the inhibition of a K + conductance exhibiting an inward rectification typical of small conductance calcium-dependent K + channels. Further pharmacological and molecular analysis revealed that the current was suppressed by the SK channels inhibitor apamin, the SK2-specific inhibitor leiurotoxin-1 Dab-7 (figure 1A,B), and by molecular silencing of SK2 channel (online supplemental figure S1A, S1B). Interestingly, immunostaining of human PDAC specimens confirmed that Sig-1R and SK2 are effectively expressed in PDAC (figure 1C). To investigate the influence of secreted factors from CAF on SK2 channel in PCC, we have generated a conditioned media using human primary CAF (CAF-CM) obtained from patients with PDAC (CAF-CM; figure 2A). CAF subtyping was investigated by flux cytometry using a panel of markers, including iCAF, myCAF and apCAF markers as well as more transverse markers recently identified such as CD105. As shown in online supplemental figure 2, the CAF cultured for this study were mainly Podoplanin high , CD105 high , PDGFRβ high , FAP high and α-SMA low . We observed that PANC-1 incubation with CAF-CM (overnight) increased the Dab-7-sensitive current (ISK2; figure 2B), an effect abolished by the molecular silencing of SK2 using shRNA or Crispr/Cas9 technology (figure 2B,C). Importantly, CM harvested from NHF cells failed to stimulate the current, indicating that the effect is specific to CAF-secretome (figure 2D). Furthermore, boiled CAF-CM had no impact on the current, ruling out any involvement of secreted metabolites (online supplemental figure S3). The CAF-CM-mediated activation of ISK2 was dependent on

Pancreas

Sig-1R since this effect was abolished by the molecular silencing of Sig-1R and rescued by the expression of C-myc Sig-1R in the shSig-1R cell line (figure 2E,F). Since Sig-1R modulates ion channel function through protein-protein associations, 21 22 we asked whether Sig-1R and SK2 could form complexes on CAF-CM exposure using proximity ligation assay (PLA). In PANC-1 challenged with CAF-CM, the interaction between SK2 and Sig-1R was significantly increased (figure 2G). Altogether, these results show that CAF-CM triggers SK2 in a Sig-1R-dependent manner in PCC.

CAF secretome activates SK2 through an integrin-EGFR-AKT pathway

To understand how CAF-CM stimulates SK2 current (ISK2), we examined the activation state of a subset of signalling kinases in PANC-1 using a phosphokinase array. We observed that the most robustly CAF-CM-activated kinases were associated to two main signalling clusters: one associated to the EGFR-AKT axis (EGFR, Src, AKT, Wnk1, AMPKa1, mTor and CREB) and the other to integrin signalling (Focal Adhesion Kinase (FAK) -Src) (figure 3A). We thus evaluated the effects of treatments altering each of these pathways on SK2 activity. EGF treatment induced a significant increase in ISK2, which was dependent on Sig-1R (figure 3B). Accordingly, the EGFR inhibitor erlotinib and the AKT inhibitor DEBC (10-(4′-(N-diethylamino)butyl)-2chlorophenoxazine . HCl) both suppressed the CAF-CM-induced ISK2 (figure 3C,D). These data indicate that CAF-CM-induced SK2 stimulation depends on EGFR-associated signalling. Given that CAF-CM treatment triggered FAK in the phosphokinase array, we tested the effect of fibronectin (FN), an element of the stroma that activates FAK via the α5β1integrin in PCC. 23 FN mimicked the effects of CAF-CM on ISK2 (figure 3E). Incubation of cells with a functional anti-β1-integrin antibody abolished the CAF-CM-induced increase in ISK2 (figure 3E), further demonstrating that CAF-CM-dependent SK2 activation requires integrin/FAK transduction. To confirm the functional links between the different partners at the molecular level, we performed PLA experiments and observed that SK2 associates with EGFR, the β1integrin subunit and AKT. These associations were promoted by CAF-CM treatment and required Sig-1R, as shown by experiments using ShSig-1R PANC-1 cells (figure 3F). These results reveal that SK2 activity is modulated by a CAF-CM stimulated signalling hub, stabilised by Sig-1R. We, therefore, looked for the presence of EGF and FN in CAF-CM. Surprisingly, we were able to detect EGF neither in a proteomic analysis of the CAF-CM nor using a sensible EGF ELISA test (online supplemental figure S4A). Note that EGF could not be detected in the culture media of PANC-1 cells cultivated with control medium or CAF-CM (online supplemental figure S4B). In support of this, neither EGF nor other EGFR ligands (ie, tumour growth factor alpha (TGFα), heparin binding EGF like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG) and epithelial mitogen (EPGN)) could be detected in the proteomic analysis. By contrast, the analysis revealed that FN is one of the most abundant components of CAF secretome (online supplemental table S1), a result confirmed by an immune-dot blot assay (figure 3G). Interestingly, the treatment of PANC-1 with FN induced the association between AKT and SK2, mimicking the effects of CAF-CM (figure 3H). Since β1integrin can form complex with other α-subunit integrins, we next ask whether other components of the CAF-CM could participate in the stimulation of the current. Proteomic analysis of CAF-CM revealed the presence of collagen I, a ligand of the α2β1integrin, which is expressed in PDAC cells and participates in PDAC cell phenotype. 24 25 Interestingly, the specific α2integrin blocking antibody Gi9 significantly reduced CAF-CM-induced stimulation of SK2 current (online supplemental figure S5), while collagen I stimulated AKT/SK2 association (figure 3H). Finally, these results are globally reinforced by the colocalisation between Sig-1R/ SK2 and p-AKT observed by immunohistochemistry from PDAC patient samples (figure 3I). Altogether, these data indicate that components secreted by CAF-CM that stimulate β1dependent integrins such as FN or collagen I potentially activate SK2 via the same signalling hub.

SK2 is a pivotal regulator of the AKT signalling pathway

Since the activation of ISK2 by the CAF-CM depends on AKT, we hypothesised that SK2 could be a target protein of AKT. Using sequence alignment, we identified three residues (S562, S568 and S569) corresponding to the AKT consensus phosphorylation motif in the SK2 sequence (RxRxxST). Strikingly, this motif was conserved in SK2 orthologs from different species (figure 4A) but was absent in SK1, SK3 and SK4 sequences, suggesting a specificity of AKT for SK2 among the SK family. Co-expression of SK2 and a constitutively active form of AKT (Myr-AKT) in HEK293 cells resulted in a significant increase in SK2 current compared with the control condition. Co-expression of Myr-AKT with SK2 carrying a triple phosphoresistant mutation (S562A, S568A and S569A) failed to increase channel activity, suggesting that AKT stimulates ISK2 by phosphorylating the channel on at least one of the three phosphorylation sites (figure 4B). To further demonstrate that SK2 is a target of AKT, we used a phospho-specific antibody that recognises the AKT phosphorylation consensus motif. Co-immunoprecipitation experiments show that Myr-AKT indeed phosphorylated the WT SK2 channel but failed to phosphorylate the triple phosphoresistant mutant of SK2 (figure 4C). Patch clamp recording in HEK293 transfected with SK2 revealed that S568 and S569 residues were phosphorylated by AKT activation on CAF-CM exposure (figure 4D), demonstrating that SK2 is a specific target of AKT. To further understand how AKT phosphorylation impacts the regulation of the channel, we used the phosphomimetic mutation S568D, which abolished the calcium sensitivity of SK2 (figure 4E). Moreover, on CAF-CM treatment, the activity of SK2 in PANC-1 cells did not change after decreasing the intracellular calcium concentration from 1 µM to 0.1 µM (online supplemental figure S6), indicating that the AKT-dependent phosphorylation of SK2 following CAF-CM exposure renders the channel independent on intracellular calcium concentration. However, at this stage, the functional consequences of AKTdependent SK2 activation on cancer cell behaviour remained an open question. Therefore, we looked for the signalling pathways regulated by CAF-CM-mediated activation of SK2. We measured the phosphorylation levels of the individual kinases monitored with a phosphokinase array in control and SK2-silenced PANC-1 cells challenged with CAF-CM. We observed that the silencing of SK2 blocked CAF-CM-induced phosphorylation of 8 kinases among the 12 that were CAF-CM sensitive (figure 4F). Only kinases showing a significant reduction in their phosphorylation level in both SK2-silenced cell lines (ie, ShSK2 and Crispr-Cas9 KO-SK2 PANC-1 cell lines) were considered and we found that AKT (S473), Yes (Y426), Src (Y419), FAK (Y397), EGFR (Y1086), WnK1 (T60), AMPKa1 (T183) and GSK3β (S21/S9) were activated in an SK2-dependent manner (figure 4G). Since these kinases belong to the EGFR-AKT-β1-integrin-associated signalosomes targeting the channel, these results suggest that

SK2 drives PCC aggressiveness in response to CAF secretome in vitro and in vivo

To test whether SK2 stimulation could favour PCC aggressiveness, we first evaluated the expression of adherens junctions (AJ) markers since loss of AJ coincides with loss of epithelial features and increases invasive phenotypes. 27 Using PCC ShRD as controls, we confirmed that CAF-CM provoked a decrease in E-cadherin, β-catenin and P-120 catenin levels-three AJ markers (figure 5A). The molecular silencing of SK2 (or Sig-1R) abolished the effect of CAF-CM on AJ markers (figure 5A; online supplemental figure S7A, B). To further explore this idea, we performed flow cytometry analyses using an epithelial marker (Epcam/CD326) and a mesenchymal marker, classically activated following epithelial-to-mesenchymal transition (EMT) (CD106). 28 The addition of CAF-CM on control PCC (ShRD) enhances their EMT phenotype, whereas EMT reprogramming is lost in PCC depleted for Sig-1R or SK2 (figure 5B). As a main functional consequence of epithelial markers loss and EMT reprogramming, we then explored invasive potency of PANC-1.

Pancreas

As expected, CAF-CM exposure significantly enhanced PCC invasive abilities (figure 5C). Consistent with epithelial and EMT markers data, the enhanced invasive properties driven by CAF-CM were lost in SK2-deficient or Sig-1R-deficient PCC (figure 5C). Moreover, when grown in 3D condition, CAF-CM increased the formation of protrusions in ShRD control cells, as shown by F-Actin immunofluoresence (figure 5D, left panel) and Filopodia density measurement (figure 5D, right panel), but not in SK2-or Sig-1R-deficient PCC (figure 5D). Interestingly, Sig1R or SK2 silencing altered neither cell growth, nor cell survival, but strongly decreased cell migration (online supplemental figure S8A, B, and C). Moreover, using a public PDAC patient database ((Dataset) oncoR2 platform; http://r2.amc.nl; n=130), 29 we revealed that the expression of both SK2 and Sig-1R are negatively correlated to AJ markers expression (online supplemental figure S9). Reinforcing this result, co-staining between SK2 or Sig-1R and an epithelial marker (pan CK) or a mesenchymal marker (α-SMA) in patient with PDAC samples showed that Sig-1R and SK2 expression patterns are strictly limited to pan cytokeratinexpressing cells and co-localise with α-SMA (figure 5E,F). These results suggest that Sig-1R and SK2 are expressed in epithelial cancer cells that have undergone EMT, a process favoured under CAF crosstalk condition, and reported to impact metastatic burden. 30 To further challenge this hypothesis, we orthotopically transplanted either ShRD, ShSK2 or ShSig-1R PANC-1 cells in the presence, or not, of CAF into the pancreas of NMRI-nude mice (figure 5G-M). As expected, co-injection of ShRD PCC and CAF revealed an increase in tumour weight (figure 5H,L), development of liver metastasis (figure 5I,M) and EMT reprogramming of epithelial cells (figure 5J). Interestingly, silencing of SK2 or Sig-1R in PCC abrogated CAF-induced increase in tumour weight (figure 5H,L), metastasis formation (figure 5I,M) and EMT (pan CK/α-SMA co-labelling; figure 5J) corroborating our hypothesis. Of interest, we controlled the presence of CAFs by the time of euthanasia in co-injected mice by identifying cells only labelled by α-SMA staining as a CAF marker, and could validate their presence, consistently with previous publications using CAF/tumour cell orthotopic co-injections 3 (online supplemental figure S10). Altogether, these results support the idea that SK2 constitutes an essential signal transducer for the intercellular communication between CAF and PCC, bolstering PCC aggressiveness, tumourigenesis and metastasis development.

A Sig-1R ligand inhibits SK2 activity and improves survival in vivo

Considering that the coupling between SK2 and AKT relies on Sig-1R, we sought to test whether targeting SK2 via Sig-1R in PDAC would be a valuable therapeutic strategy. Sig-1R ligands are synthetic small molecules that modify the coupling of Sig-1R with its partners, including ion channels. 10 13 Compound 1(S) is a Sig-1R ligand exhibiting nanomolar affinity for Sig-1R (IC 50 =3.9 nM), high Sig-2R/Sig-1R selectivity (greater than 120) and very low cytotoxicity (selectivity index; ratio: CC 50 /IC 50 , greater than 50 000). 31 We first validated in vitro that 1(S) abolished the CAF-CM-mediated activation of SK2 and prevented the association of SK2 and AKT by patch clamp experiments (figure 6A) and PLA experiments (figure 6B). Proliferation and viability assays, on control Panc-1 cells or cells silenced for Sig1R or SK2, demonstrated that 1(S) treatment does not exhibit any effect on these parameters (online supplemental figure S8A, B). We next evaluated the effect of 1(S) in an endogenous PDAC mice model (KIC pdx1 ). 32 By using a first protocol treating the KIC pdx1 mice at 5 weeks old, before the onset of PDAC (figure 6C), we observed that 1(S) treatment significantly delayed tumour progression and extended the overall survival. The median survival time of KIC pdx1 non-treated animals was 9.5 weeks, whereas median survival of KIC pdx1 -1(S)-treated mice was 11.7 weeks (figure 6D). This data were confirmed on a similar treatment protocol combined to an end-point study at 9 weeks of age (figure 6E), representing the median survival of KIC pdx1 non-treated mice, validating that 1(S) treatment delayed tumour progression. Indeed, as shown in figure 6F, in 1(S)-treated KIC pdx1 mice, the pancreatic tumour/body mass ratio showed a drastic reduction of 69% compared with nontreated ones, decreasing from 0.065 for KIC pdx1 non-treated mice to 0.021 for 1(S)-treated KIC pdx1 mice. Histological examination of pancreas from 1(S)-treated KIC pdx1 mice revealed an almost lack of malignant lesions contrasting with pancreas of non-treated KIC pdx1 mice exhibiting typical PDAC development (figure 6G). In addition, 1(S)-treated mice exhibited a lower number of EMT-reprogramming epithelial cells (pan CK/α-SMA co-staining, figure 6H) and a lower ECM density reflecting a decreased desmoplasia in treated animals (trichrome labelling; figure 6I-K). Furthermore, we have tested the efficiency of 1(S) treatment on PDAC evolution using an alternative protocol starting the treatment of KIC pdx1 mice at 8 weeks of age (figure 6L), when PDAC has already developed. Using this experimental design, we confirmed that 1(S) treatment significantly extended the overall survival. Indeed, the median survival time of KIC pdx1 non-treated animals was 8.8 weeks, whereas median survival of KIC pdx1 -1(S)-treated mice was 11 weeks (figure 6M). Together, these results illustrate that SK2 inhibition via Sig-1R ligand delays PDAC development and significantly extends survival, supporting 1(S) as a valuable therapeutic option to treat PDAC.

SK2 and Sig-1R expression in PDAC correlates with metastatic phenotype

To characterise the expression of Sig-1R in human PDAC, we interrogated publicly available gene expression datasets from 1017 patients with pancreatic carcinoma ((Dataset) online supplemental table S2). First, we found that SIGMAR1 (encoding Sig-1R: figure 7A) and KCNN2 genes (encoding SK2: figure 7B) were more expressed in metastases than in primary tumours, and preferentially in liver metastases. Second, in an analysis limited to the 925 primary cancers, SIGMAR1 expression, assessed as binary value (cut-off equal to median expression level), was not associated with patients' age and gender, pathological tumour type and grade, and American Joint Committee on Cancer stage (online supplemental table S3). Regarding the molecular subtypes (online supplemental table S4), no correlation existed between SIGMAR1 expression (figure 7C) and the (Dataset) Bailey et al's classification 33 (pancreatic progenitor, immunogenic and squamous), the (Dataset) Collisson et al's classification 34 (quasimesenchymal, classical) and the (Dataset) Moffitt et al's classification 35 (basal, classical). By contrast, KCNN2 expression was higher in the squamous Bailey's subtype, the quasi-mesenchymal Collisson's subtype, and the basal Moffitt's tumour subtype (figure 7D). These results revealed higher gene expression of SK2 and Sig-1R in metastases versus primary cancers, and higher expression of SK2 in aggressive molecular subtypes.

DISCUSSION

The stromal compartment participates in PDAC onset, development and aggressiveness. However, recent studies refined the variety of stromal cellular components such as CAF, and

Pancreas

their abilities to potentially support or restrain PDAC progression. 1 5 6 36 37 This knowledge highlighted the necessity to precisely identify and target stromal cues and mechanisms that underlie the protumorous intercellular communication between CAF and PCC. Herein, the potassium channel SK2 was found to mediate intercellular communication between PCC and CAF by amplifying an EGFR-AKT axis on CAF secretome stimulation, promoting metastatic spreading and poor survival in pancreatic cancer. The association between SK2 and its partners is controlled by the ligand-modulated ion channel chaperone Sig-1R, which can be pharmacologically targeted to reduce CAFinduced tumourigenic processes particularly metastasis.

Interestingly, we have shown that exposure to CAF-CM induces an increase in SK2 current in PCC, suggesting that this channel participates in the CAF secretome mediated intercellular communication in PDAC. Several secreted factors from CAF, within PDAC stroma, like growth factors, 38 cytokines 7 and lipids, 8 were shown to mediate paracrine communication that stimulate PCC aggressive features. Our data show that the CAF-CM-mediated activation of SK2 was dependent on EGFR activity. Indeed, this effect was mimicked by EGF treatment and completely abolished using erlotinib. Surprisingly, we have been unable to detect EGF (or other EGFR ligands) in the CAF secretome. Looking deeper, we found that FN and collagen I, two major components of CAF-CM, activate a β1integrin/EGFR/ AKT signalling axis that increases SK2 activity in PCC. The abundant presence of FN and collagen I has been revealed in patients with stromal PDAC and mouse models samples 37 39 as well as

Pancreas

in the secretome obtained from human primary CAF. 40 Several studies have demonstrated that FN and collagen I contribute to PCC aggressiveness, survival and chemoresistance. 24 41 These results strongly suggest a transactivation mechanism of EGFR, that has been previously reported as integrin-related and FAKrelated in PDAC. 26 42 Our data support the idea that secretion of ECM components within PDAC microenvironment would be a key mediator in the CAF-stimulated formation of a signalling hub, including SK2, the β1integrin, EGFR and AKT, that finely tunes the activity level of the downstream intracellular signalling pathway supporting PDAC development and metastasis formation.

SK2 seems to have a pivotal role in this signalling hub. Indeed, we reveal that the channel is a direct target of AKT, since we identified an AKT-specific motif within the channel sequence containing two serine residues that are phosphorylated on CAF-CM treatment (S568 and S569), both being necessary to mediate CAF-CM induced SK2 activity. We also found that the AKT-specific phosphorylation site in SK2 is highly conserved among species but lacks in the other members of the SK channel family (ie, SK1, SK3 and SK4), reinforcing the idea of a functional and specific coupling between SK2 and AKT-dependent pathways. Interestingly, we found that the channel phosphorylation by AKT stimulated the current by increasing its activity at subthreshold intracellular Ca 2+ concentrations. These findings highlight an unexpected direct upregulation of SK2 activity by AKT.

EGFR activation is associated to AKT phosphorylation in PDAC 42 43 and the EGFR-PI3K-AKT signalling axis is one of the main signalling pathways involved in PDAC progression. 44 Therefore, we reasoned that the AKT-dependent activation of SK2 could be crucial in the integrin-EGFR-AKT axis. Our results, showing that SK2 silencing reduced CAF-dependent signalisation, suggest that without a functional channel, PCC remain in a subthreshold state that prevents the metastatic process despite stimulation by CAF. Accordingly, SK2 inhibition reversed the capacity of CAF to induce aggressiveness, and stabilised cells in an epithelial state. Thus, these data provide arguments to propose that SK2 sets an activation threshold that switches PCC from an epithelial state when SK2 activity is low, to a pro-invasive state when channel activity is high, underlined by EMT reprogramming. To support this model, SK2 silencing decreased CAF-induced cell invasiveness, tumour growth and metastasis formation in vivo. These results correlate with our investigation of human PDAC expression datasets revealing that SK2 expression is increased in liver metastasis compared with primary tumours, and that SK2 expression is significantly higher in the more aggressive subtype of PDAC cancer cells. Thus, these results highlighted SK2 as an ion channel playing a pivotal role in transducing extracellular signals from the tumour microenvironment onto intracellular activation of oncogenic signalling pathways in PCC.

Currently, the therapeutic armamentarium to treat PDAC is restricted and poorly efficient. A better understanding of the molecular components that participate in intercellular communication suggests novel strategies for treating PDAC. Here, we have shown that the ligand-modulated chaperone Sig-1R was required for the activation of SK2 current and the assembly of the SK2-AKT-β1-integrin-EGFR signalling hub on PCC stimulation by CAF. This observation is in line with previous studies showing that Sig-1R contributes to electrical remodelling by promoting channel/channel or channel/receptor associations. 13 14 45 Interestingly, Sig-1R silencing recapitulates the effects of SK2 silencing in vitro and in vivo.

On another hand, we have demonstrated that PCC treatment with the selective Sig-1R ligand 1(S) abolished CAF-CM-driven association between SK2 and AKT, underlying current inhibition. More importantly, pharmacological inhibition of Sig-1R was efficient in vivo as mouse models of PDAC treated with 1(S) dramatically delayed tumour development, reduced metastasis onset and extended survival. Although stromal ablation for PDAC treatment remains controversial, our results suggest a potential therapeutic opportunity by targeting an ion channel-dependent signalling hub through its regulator, Sig-1R, a druggable ion channel chaperone, 9 46 to reduce stromal-induced PDAC aggressiveness (figure 8). Because Sig-1R activity is stimulated by stress in diseased tissues, 47 the use of Sig-1R ligands should specifically target SK2 in tumours but not in brain or heart. In support of this, a recent study revealed that Sig-1R ligands modulate ion channels in cardiomyocytes from long QT syndrome patients but not in cardiomyocytes from isogenic controls. 12 Prospective studies combining Sig-1R targeting with other therapeutic approaches, as chemotherapeutics agents, are necessary to evaluate their potential as long-term durable anti-PDAC

  FIGURE1. 1 -THE GASTROINTESTINAL TRACT ....................................................................................................................................... FIGURE1. 2 -LAYERS OF THE GASTROINTESTINAL TRACT ........................................................................................................................... FIGURE1. 3 -THE LARGE INTESTINE ................................................................................................................................................... FIGURE1. 4 -SODIUM ABSORPTION BY COLONOCYTES ............................................................................................................................. FIGURE1. 5 -CHLORIDE SECRETION IN COLONOCYTES ............................................................................................................................. FIGURE1. 6 -THE COLONIC CRYPT...................................................................................................................................................... FIGURE1. 7 -THE COLONIC CRYPT AND ITS CELL TYPES. ........................................................................................................................... FIGURE1. 8 -THE SIGNALLING PATHWAYS AT PLAY IN THE COLONIC CRYPT ................................................................................................... FIGURE2. 1 -OVERVIEW OF WNT NON-CANONICAL SIGNALLING PATHWAYS ................................................................................................ FIGURE2. 2 -MAIN PHOSPHORYLATIONS ON Β-CATENIN, IMPLICATED KINASES, AND EFFECT. ........................................................................... FIGURE2. 3 -OVERVIEW OF ADHERENS JUNCTIONS ............................................................................................................................... FIGURE2. 4 -OVERVIEW OF THE DESTRUCTION COMPLEX ........................................................................................................................ FIGURE2. 5 -WNTS MODIFICATIONS ................................................................................................................................................. FIGURE2. 6 -OVERVIEW OF WNT/Β-CATENIN SIGNALING. ...................................................................................................................... FIGURE2. 7 -MODEL OF Β-CATENIN Y654 PHOSPHORYLATION CONTRIBUTING TO WNT SIGNALLING ................................................................ FIGURE2. 8 -WNT TARGET GENES TRANSCRIPTION ................................................................................................................................ FIGURE2. 9 -WNT PATHWAY REGULATION ......................................................................................................................................... TABLE 1 -MUTATION FREQUENCY OF CANONICAL WNT PATHWAY GENES IN COLORECTAL CANCER.. ................................................................. TABLE 2 -CLASSICAL INTRACELLULAR AND EXTRACELLULAR CONCENTRATION BY IONIC SPECIE. ......................................................................... EQUATION 1 -NERNST EQUATION .................................................................................................................................................... EQUATION 2 -GOLDMAN-HODGKIN-KATZ EQUATION ........................................................................................................................... EQUATION 3 -THE DRIVING FORCE. ................................................................................................................................................... FIGURE 3. 1 -THE POTASSIUM CHANNEL FAMILIES ................................................................................................................................ FIGURE 3. 2 -CANCER HALLMARKS DESCRIBED BY HANAHAN AND WEINBERG ............................................................................................. FIGURE 3. 3 -CANCER HALLMARKS ARE ONCOCHANNELOPATHIES. ............................................................................................................ FIGURE 3. 4 -MAPK ACTIVATION IN K-RASG12VTRANSFORMED BHK CELLS ............................................................................................ FIGURE 3. 5 -MEMBRANE POTENTIAL AND PROLIFERATION. .................................................................................................................. FIGURE 4. 1 -TOPOLOGY AND CHANNEL ARCHITECTURE OF KCNE AND KCNQ1 PROTEINS ............................................................................ FIGURE 4. 2 -BAR DIAGRAM OF KCNE PROTEIN FAMILY ....................................................................................................................... FIGURE 4. 3 -KCNQ1 CURRENTS WITH KCNE1-5 ............................................................................................................................... FIGURE 4. 4 -NEW MODEL OF PIP2 INTERACTION WITH KCNQ1 ............................................................................................................. FIGURE 4. 5 -TIMELINE OF PUBLISHED ARTICLES SHOWING KCNQ1'S ROLE IN CANCERS ................................................................................ FIGURE 4. 6 -KCNQ1 RESTRICTS WNT/Β-CATENIN PATHWAY ACTIVITY AND IS A TRANSCRIPTIONAL TARGET ...................................................... SUPPLEMENTARY FIGURE 1 -PERCENTAGE COLORECTAL CANCER PATIENTS HARBOURING MISSENSE, FRAMESHIFT OR STOP GAIN MUTATIONS ON KCNQ1 ........................................................................................................................................................................................ FIGURE 1 -CANCER PATIENTS HARBOUR MUTATIONS ON KCNQ1. ......................................................................................................... EQUATION 4 -OPEN PROBABILITY .................................................................................................................................................... FIGURE 2 -CANCER-RELATED KCNQ1 MUTATIONS ARE MOSTLY LOSS-OF-FUNCTION .................................................................................. SUPPLEMENTARY FIGURE 2 -KCNQ1 MUTATIONS ............................................................................................................................... FIGURE 3 -LOF MUTATIONS OF KCNQ1 AFFECT WNT/Β-CATENIN PATHWAY .......................................................................................... FIGURE 4 -LOF MUTATIONS OF KCNQ1 INCREASE WNT/Β-CATENIN PATHWAY ACTIVITY ........................................................................... SUPPLEMENTARY FIGURE 3 -MOUSE COLON ORGANOIDS MODEL ............................................................................................................. FIGURE 5 -THE KCNQ1-LOF-INDUCED ACTIVATION OF WNT/Β SIGNALLING IS MEDIATED BY Β-CATENIN INDEPENDENTLY FROM FRIZZLED/LRP ...... FIGURE 6 -RTKS ARE ACTIVATED IN KCNQ1-LOF CELLS.. ................................................................................................................... FIGURE 7 -KCNQ1-LOF INDUCE LOSS OF NEGATIVE REGULATORS OF WNT/Β-CATENIN ............................................................................. FIGURE II-1 -RESTING MEMBRANE POTENTIAL CHANGES IN NON-EXCITABLE CELLS ....................................................................................... FIGURE II-2 -EXPERIMENTAL DEPOLARISATION ACTIVATES WNT/Β-CATENIN SIGNALLING ............................................................................ FIGURE II-3 -DEPOLARISATION INDUCES THE PHOSPHORYLATION OF Β-CATENIN ON ITS TYR654 RESIDUE. ...................................................... FIGURE II-4 -DEPOLARISATION ACTIVATES SERINE/THREONINE KINASES ................................................................................................. FIGURE II-5 -DEPOLARISATION ACTIVATES TYROSINE KINASES .............................................................................................................. FIGURE II-6 -RTKS ARE ACTIVATED UPON DEPOLARISATION ................................................................................................................. FIGURE III-1 -ML277 RESCUES ACTIVITY OF KCNQ1 LOF MUTANT A329T .......................................................................................... FIGURE III-2 -DETACHING Β-CATENIN FROM THE ADHERENS JUNCTIONS INHIBITS KCNQ1:KCNE3 CURRENTS ................................................ FIGURE III-3 -CYSTS ................................................................................................................................................................... FIGURE III-4 -WNT3A. ...............................................................................................................................................................

Figure1. 7 -

 7 Figure1. 7 -The colonic crypt and its cell types. The gradients of signalling pathways are represented by triangles when applicable.

Figure1. 8 -

 8 Figure1. 8 -Schematic and simplified representation of the different signalling pathways at play in the colonic crypt. The Wnt, BMP, Hedgehog, Hippo, Notch and Eph/Ephrin pathways.

Figure2. 4 -

 4 Figure2. 4 -Overview of the destruction complex. The phosphorylated sequence in the amino terminus of β-catenin is shown with CK1 and GSK3 sites circled.(From Stamos and Weis, 2013)

Figure2. 9 -

 9 Figure2. 9 -Wnt pathway regulation. Left: Wnt target gene negative feedback regulators RNF43 and ZNRF3 ubiquitylate Frizzled and mark this receptor for endocytosis and lysosomal degradation (step 1). Right: R-spondins (RSPOs) bind to their cognate receptors leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4), LGR5 or LGR6 and the extracellular domain of RNF43 or ZNRF3. This complex facilitates RNF43 and ZNRF3 clearance from the plasma membrane, thereby potentiating Wnt signalling.(From Bugter et al., 2021)

Figure 3 . 1 -

 31 Figure3. 1 -The Potassium Channel families. Phylogenetic tree of human K + channels. For simplicity, the letter code for K + channels (KCN) have been omitted.(From Heitzmann and Warth, 2008))

Figure 3 . 3 -

 33 Figure 3. 3 -Cancer hallmarks are oncochannelopathies. Cancer hallmarks from first publication (left panel) and added hallmarks (right panel). (From Hanahan and Weinberg, 2000, 2011) -A. Proposed definition of oncochannelopathy. B. Differences between classical channelopathy and oncochannelopathy. (FromPrevarskaya et al., 2018).

Figure 3 . 2 -

 32 Figure 3. 2 -Cancer hallmarks described by Hanahan and Weinberg. Left panel: Original cancer hallmarks[START_REF] Hanahan | The Hallmarks of Cancer[END_REF]. Right panel: Additional cancer hallmarks[START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF].

  The preliminary work in the field was published in 2015 by Zhou and colleagues. They showed that depolarisation induced a reversible rearrangement of lipids, more precisely a rapid nanoclustering of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2) on the inner leaflet of the plasma membrane. The lipid nanoclustering induces a nanoclustering of K-Ras which binds phosphatidylserines. Interestingly, the phosphorylation level (mirroring activation level) of the MAPK effector ERK increases with depolarisation (Figure 3. 4, Figure 3. 5).

Figure 3 . 4 -

 34 Figure 3. 4 -MAPK activation in K-RasG12Vtransformed BHK cells measured by quantitative immunoblotting for phosphorylated ERK (pERK) after plasma membrane depolarization(From Zhou, 2015)

Figure 3 . 5 -

 35 Figure 3. 5 -Membrane Potential and Proliferation. In a normal, nonproliferating cell, the resting membrane potential (Vm ≈ -50 mV) is set by ion channel activity. Phosphatidylserine lipids are in small clusters that localize with K-Ras, which leads to low activation of the RAF-MAPK pathway. Channel overexpression depolarizes the cell (Vm ≈ -10 mV), increasing the clustering of phosphatidylserine and K-Ras. This promotes RAF-MAPK signaling uncontrolled cell proliferation(From Accardi, 2015). -It must be noted that some interpretations of this commentary ofZhou, 2015[START_REF] Accardi | Lipids link ion channels and cancer[END_REF] are inaccurate: overexpression of all ion channels do not induce a depolarisation and depolarisation can be caused by loss of leak potassium channels.

Figure 4 . 1 -

 41 Figure 4. 1 -Topology and channel architecture of KCNE and KCNQ1 proteins A: topology of the KCNE proteins as well as the potassium channel KCNQ1 with indications of some of the domains important for regulation of the channel. All five KCNE proteins hold one transmembrane domain but have varying lengths of NH2 and COOH termini. B: KCNQ1/KCNE channel architecture. Four KCNQ1 α-subunits assemble to form the basic channel. Co-assembly with two to four KCNE β-subunits leads to a β-subunit-specific change in the current characteristics.(From Jespersen et al., 2005).

  KCNE family comprises five members that display a similar structure: they are single-pass transmembrane protein of 103 to 177 residues (Figure4. 2). The N-terminal region is extracellular, and the C-terminal region is intracellular and can carry several post-translational modifications such as glycosylation and a serine phosphorylation site[START_REF] Abbott | Potassium channel subunits encoded by the KCNE gene family: physiology and pathophysiology of the MinK-related peptides (MiRPs)[END_REF].

Figure 4 . 2 -

 42 Figure 4. 2 -Bar diagram of KCNE protein family. Transmembrane domain (TMD) is indicated by a black box.(From Pongs and Schwartz, 2010)

Figure 4 . 3 -

 43 Figure 4. 3 -KCNQ1 currents with KCNE1-5. Whole cell patch clamp recordings from COS-7 cells transfected with DNA encoding (A) WT-KCNQ1 alone or (B-F) with WT-KCNQ1 and each of the 5 KCNE subunits. Cells were held at -80 mV and currents were elicited by potentials ranging from -100 mV to + 40 mV, in 10 mV increment, for KCNE1 to KCNE3, and -100 mV to + 90 mV for KCNE4 and +110mV for KCNE5.(From Bendahhou et al., 2005).

Figure 4 . 4 -

 44 Figure 4. 4 -New model of PIP2 interaction with KCNQ1 (upper panel) compared to the old model (lower panel). (From Mandala and McKinnon, 2023).

  KCNE3 is in direct contact with S4 and blocks it in the up (depolarised/open) conformation. The position of KCNE3 destabilises the downward (hyperpolarised/closed) conformation of S4. The presence of KCNE3 thus locks S4 in the up conformation even at hyperpolarised membrane potentials, thus keeping the channel open under all the physiological range of membrane potentials (Barro-[START_REF] Barro-Soria | KCNE3 acts by promoting voltage sensor activation in KCNQ1[END_REF]

Figure 4 . 5 -

 45 Figure 4. 5 -Timeline of published articles showing KCNQ1's role in cancers. Low KCNQ1 expression is associated with poor survival. Lower left panel: Kaplan-Meier graph depicting Overall Survival (OS) in months, stratified by intensity of KCNQ1 expression in colorectal cancer patients (from Than et al., 2014.) Middle panel: Association of KCNQ1 protein expression levels with Disease Free Survival (DFS) of stage II and III colon cancer patients (From den[START_REF] Den Uil | Loss of KCNQ1 expression in stage II and stage III colon cancer is a strong prognostic factor for disease recurrence[END_REF]. Lower right panel: Kaplan-Meier graph depicting survival stratified by intensity of KCNQ1 protein expression in HepatoCellular Carcinoma patients(From Fan et al., 2018)

Figure 4 . 6 -

 46 Figure 4. 6 -KCNQ1 restricts Wnt/β-catenin pathway activity and is a transcriptional target. Middle panel: normal condition, KCNQ1 is associated with E-cadherin and β-catenin at the plasma membrane. Β-catenin is either sequestered at the adherens junctions or degraded by the Axin degradasome. Wnt transcriptional activity is off. Cells harbour an epithelial phenotype and form spheres with a lumen in 3D. Left panel: pharmacological inhibition of KCNQ1. Right panel: molecular extinction of KCNQ1.In these conditions, the adherens junctions are disrupted. Β-catenin is released, accumulates into the cytosol, is able to translocate into the nucleus where it activates the transcription of Wnt target genes. Among the target genes is KCNQ1 which is repressed by β-catenin transcriptional activity, further enhancing the phenotype observed. Losing the adherens junctions and activating the Wnt/β-catenin pathway has for consequence a loss of epithelial features; mesenchymal cells aggregate but cannot form an organised sphere. In cancer, acquiring mesenchymal features is key in cancer progression and dissemination.(From Rapetti-Mauss, 2018).

FIGURE 1 -

 1 FIGURE 1 -Cancer patients harbour mutations on KCNQ1. A. Bar graph representing the number of mutations on each residue plotted along the sequence of KCNQ1. A colour has been attributed to each cancer type. Under each mutation, the coloured spots correspond to the cancer type in which this particular mutation was found. Transmembrane segments (S1 to S6) of KCNQ1 are represented in red on the x axis. Mutations that are found at least in four patients are represented with red bars. B. Repartition of cancer types for the 221 patients harbouring mutations of KCNQ1. C. Repartition of the types of mutations on KCNQ1: missense, frameshift or stop gain. D. Frequency of each mutation out of the 156 mutations found in databases.

Equation 4 -

 4 Open Probability. From the Ohm law with U being the potential in Volts, R the resistance in Ohm, and I the current in Ampers. In the context of a cell and for a specific channel of interest, U becomes Df -the electrical driving force -which is, as shown above, the membrane potential minus the equilibrium potential of a particular ion, and R is the inverse of G. G being the conductance in Siemens (S = Ω -1 ). These thus gives us the second equation: the current equals the conductance times the electrical driving force. G can be further decomposed into the multiplication of n: the number of channels at the plasma membrane; Po: the open probability of the channel and γ: the unitary conductance of the channel. In a cell being patched, the number of channels at the membrane is constant and the unitary conductance of a channel is also constant. The open probability changes along the input voltage. When plotted, the open probability along the range of potentials draws a Boltzmann sigmoid. Before a potential threshold, Po= 0, then abruptly increases to reach a plateau where Po=1. Po=1 means that all channels are opened. Po=0 means that no channels are opened.

FIGURE 2 -

 2 FIGURE 2 -Cancer-related KCNQ1 mutations are mostly loss-of-function. Patch clamp experiments were performed in the whole cell configuration on CHO cells transfected with KCNQ1 and KCNE3. (A) Currents obtained from the protocol presented on the upper left panel with the WT channel (n=47) or the following mutations: E170K (n=9), V185M (n=32), R243C (n=10), A287T (n=17), A329T (n=10), F339L (n=13), P343S (n=8), R4401Gfs*18 (n=11), K422Sfs*10 (n=10), P448R (n=9), R451Q (n=11), R518* (n=9), G548C (n=8). (B) Current-voltage relationships. (C) Current density (pA/pF) measured at 0mV. (D) Open probability curve and representation of the open probability at -60mV. (E) Current density (pA/pF) measured at 0mV from cotransfection experiments where cDNA coding for a mutation of KCNQ1 was added 1:1 to the WT form. WT-WT (n=27), V185M-WT (n=11), A329T-WT (n=13), K422Sfs*10-WT (n=9), P448R-WT (n=12), R451Q-WT (n=9), R518*-WT (n=13). Data were tested for statistical significance with an ANOVA. * p<0,05, ** p<0,01, *** p<0,001, **** p<0,0001, ns not significant

  mRNA levels are themselves regulated by a great number of processes such as targeted degradation by miRNA, storage in p-bodies, intrinsic stability and translation(Boo and Kim, 0 0; O'Brien et al., 2018;[START_REF] Parker | P Bodies and the Control of mRNA Translation and Degradation[END_REF], we cannot ascertain whether the global activity of Wnt pathway is upor down-regulated.

FIGURE 3 -

 3 FIGURE 3 -LOF mutations of KCNQ1 affect Wnt/β-catenin pathway. Results of RNASeq from four independent biological replicates. (A) Gene Ontology groups that were enriched among the regulated genes in the A329T (left panel), K422Sfs*10 (middle panel) and R518* (right panel) conditions. (B) Statistically differentially expressed genes corresponding to the GO Terms "canonical Wnt signaling pathway" and "Wnt signaling pathway" were clustered based on expression trends. (C) Gene ontology groups enriched among the regulated genes in the TCGA data set of patients suffering from colon adenocarcinoma and harbouring a KCNQ1 mutation. (D) Volcano plot of differentially expressed genes from the colon adenocarcinoma TCGA data set from patients harbouring a KCNQ1 mutation. Thresholds were set at 1.44-fold change and 0.01 p-adjust. Genes corresponding to the GO Term "Wnt signaling pathway" are represented in orange. (E) Top 20 Wnt target genes differentially expressed with the mutations A329T, K422Sfs*10 and R518* clustered by p-adjust.

FIGURE 4 - 3 -

 43 FIGURE 4 -LOF mutations of KCNQ1 increase Wnt/β-catenin pathway activity. (A) Luciferase activity measured in CHO-priHy_KCNE3 cells transiently transfected with WT KCNQ1 or the constructs A329T, F339L, K422Sfs*10, P448R, R451Q, R518* and stimulated with exogenous Wnt3A (n=6). (B) Luciferase activity measured on HCT116 cells stably expressing a vehicle, WT KCNQ1 or the constructs A329T, K422Sfs*10, R518* and stimulated or not (black dots) with exogenous Wnt3A (n=4/5). (C) Schematic representation of the 7TGC plasmid. (D) Representative images acquired on a confocal microscope of the HT29cl19A 7TGC cells overexpressing or not a mutation. 63x magnification, scale bar = 10 µM. Left panel: quantification of GFP+ cells normalised by the number of mCherry+ cells per field (n=9). (E) Western blot and densitometry analysis of active β-catenin normalised by the levels of β-catenin in HT29cl19A (n=8-12). (D) Western blot and densitometry analysis of CD44, Axin2 and CyclinD1 normalised by GAPDH or Tubulin in HT29cl19A (CD44 n=6, Axin2 n=6, CyclinD1 n=6-10). All quantifications were tested for statistical significance using Kruskal-Wallis with post-hoc Dunn's test (A, D), One way Anova with post-hoc Tukey's test (B), Brown-Forsythe and Welch Anova test with post-hoc Dunnett's T3 test (E), One Way Anova with post-hoc Holm-Sidak's test (F -Axin2, CD44), and Mixed-Effect Analysis with post-hoc Holm-Sidak's test (F -CylinD1), * p<0,05, ** p<0,01, *** p<0,001, **** p<0,0001, ns not significant

FIGURE 5 -

 5 FIGURE 5 -The KCNQ1-LOF-induced activation of Wnt/β signalling is mediated by β-catenin independently from Frizzled/Lrp. (A) Protein levels of Axin2 and CD44 normalised by GAPDH treated with vehicle (grey circle) or with 5 µM niclosamide (blue triangles) (n=4). (B) Western blot and densitometry analysis of pLRP6 (S1490) normalised by LRP6 (n=4). (C) Schematic of the targets of pharmacological molecules used. (D) Western Blot and densitometry analysis of Axin2 and CD44 normalised by GAPDH treated with vehicle (grey circles) or 10 µM JW67 (pink diamond) (n=4). (E) Western blot and densitometry analysis of phosphorylated β-catenin on residues Y654 and S33/S37/Thr41 normalised on total β-catenin and GAPDH. (F) Proximity Ligation assays showing interaction between KCNQ1 and β-catenin (n=1) and (G) E-cadherin and β-catenin(n=4). Nuclei are stained with DAPI (blue), interactions are in red. Right side panels: quantification of the number of interactions per cell. 5 to 15 images were acquired and analysed per biological replicate. Each biological replicate is represented by a different symbol on the bar graph. Data were analysed for statistical significance using Two Way Anova with uncorrected Fischer's LSD (A,D),One Way Anova with post-hoc Dunnett's test (B), Kruskal-Wallis with post-hoc Dunn's test (E), and Nested One Way Anova with post-hoc Dunnett's test (F,G). * p<0,05, ** p<0,01, *** p<0,001, **** p<0,0001, ns not significant

  promotes signalling is phosphorylated in the KCNQ1-LOF conditions. Preliminary results seem to indicate that RTK are activated in these conditions and literature has shown that RTKs can directly or indirectly phosphorylate β-catenin on Tyr654 and Ser675 residues and thus promote its transcriptional activity independently from the activation of Frizzled/Lrp. Nonetheless, these results raised an additional question: what happens if Frizzled/Lrp are activated by Wnt ligand?. Would it add up to the activation mediated by RTK ? Interestingly, we noticed that among the Wnt pathway genes that were differentially expressed in our RNASeq and in TCGA patients, negative regulators of the pathway were most often downregulated.

FIGURE 6 -

 6 FIGURE 6 -RTKs are activated in KCNQ1-LOF cells. Chemiluminescence from Proteome Profiler Human Phospho-RTK array kit results of HT19cl19A Ctrl, A329T, K422Sfs*10 and R518* cell lysates after 6 min exposition and with increased contrast (modified B/C) (right panel). Two dots represent technical replicates for each RTK tested in the array. Lower panel: Fold change increase of the phosphorylations of a selection of RTKs in HT29cl19A overexpressing KCNQ1-LOF compared to the control cell line.

1 .

 1 Changing resting membrane potential Potassium leak channels, along with Kir channels are responsible for setting membrane potential at hyperpolarised values, around -60mV (Figure II-1). Upon pharmacological inhibition, molecular extinction, or expression of a LOF mutation of potassium leak channels, the global potassium conductance and by extension the relative permeability of the membrane to potassium decrease. The relative permeability of sodium, chloride and calcium are thus increased. The resting membrane

Figure II- 2 -

 2 Figure II-2 -Experimental depolarisation activates Wnt/β-catenin signalling. Experiments were done in HT29cl19A WT or 7TGC cells. (A) Western Blot and densitometry analysis of Active β catenin normalised on total β-catenin and GAPDH after 0, 10, 30 or 60 min incubation with high K+ solution. (n=4) (B) Western Blot and densitometry analysis of Wnt target genes Slug, Snail and c-Jun normalised on GAPDH after 24h incubation with high K+ solution. (n=3-4) (C) Representative images acquired on a confocal microscope of the HT29cl19A 7TGC cells in a control condition or after treatment with high K+ solution, 100 ng/mL Wnt3A, both, and Wnt3A + 10 µM KCNQ1 inhibitor HMR1556. 63x magnification Lower panel: Mean GFP fluorescence intensity normalised on mCherry fluorescence intensity. 5 to 10 images were acquired per condition. (n=6) All quantifications were tested for statistical significance using Brown Forsythe and Welch with post-hoc Dunnett's T3 test (A), Paired t-tests (for Slug and cJun), Mann-Whitney test (for Snail), Kruskal-Wallis with post-hoc Dunn's test (C). * p<0,05, ** p<0,01, *** p<0,001, **** p<0,0001, ns not significant

Figure II- 3 -

 3 Figure II-3 -Depolarisation induces the phosphorylation of β-catenin on its Tyr654 residue. (A) Western Blot and densitometry analysis of phospho-Y654 β-catenin normalised on total β-catenin and GAPDH after 30-or 60-minutes incubation with high K+ solution of HT29cl91A WT cells (n=5). (B) Western Blot and densitometry analysis of phospho-Y654 β-catenin normalised on total β-catenin and GAPDH after 60 minutes incubation with high K+ solution of isolated rat colonic crypts (n=4). (C)Representative images of immunofluorescence labelling of isolated rat colonic crypt treated or not for 60 minutes with high K+ solution. phospho-Y654 β-catenin is stained in green, nuclei in blue and F-actin in red. Quantifications were tested for statistical significance with Kruskal Wallis with post-hoc Dunn's test (A) or Paired t-test (B). *p<0,05

Figure II- 4 -

 4 Figure II-4 -Depolarisation activates Serine/Threonine Kinases. HT29cl19A WT cells were incubated in the control medium or in high K+ solution for 5, 30 or 60 minutes. (A) Volcano Plot of the bait peptide phosphorylation showing significantly up-and down-regulated peptide phosphorylation after 5, 30 and 60 minutes of high K+ compared to the control condition (n=3). (B) Putative upstream kinases projected on the phylogenetic tree of the human kinome focused on serine threonine kinases. Putative upstream kinases are generated from the peptides that have statistically significant differences in phosphorylation levels. Circle size is correlated with the negative log of the pvalue. (C) Enriched GO process networks following depolarisation. The top five most significantly enriched process networks are sorted by statistical significance based on manually curated objects generated by PamGene fold-change data. Data represent the negative log of the p value determined by String.

Figure II- 5 -

 5 Figure II-5 -Depolarisation activates Tyrosine Kinases. HT29cl19A WT cells were incubated in the control medium or in high K+ solution for 5 minutes (A) Volcano Plot of the bait peptide phosphorylation showing significantly up-and down-regulated peptide phosphorylation after 5 minutes of high K+ compared to the control condition. (n=3) (B) Putative upstream kinases projected on the phylogenetic tree of the human kinome focused on tyrosine kinases. Putative upstream kinases are generated from the peptides that have statistically significant differences in phosphorylation levels. Circle size is correlated with the negative log of the pvalue.

Figure II- 6 -

 6 Figure II-6 -RTKs are activated upon depolarisation Chemiluminescence from Proteome Profiler Human Phospho-RTK array kit results of (A) HT19cl19A WT in the control condition or incubated with high K+ solution for five minutes and (B) HT29cl19A shRNA_scramble (shRD) versus HT29cl19A shRNA_KCNQ1 cell lines. Two dots represent technical replicates for each RTK tested in the array. Right panel: Fold change increase of the phosphorylations of a selection of RTKs in HT29cl19A high K+ compared to the control or in the HT29cl19A shRNA_KCNQ1 compared to HT29cl19A shRD.

  Figure III-1 left panel). More importantly, the resting membrane potential saw a left shift towards hyperpolarised values and even reached -50mV upon ML277 treatment (Figure III-1 right panel). ML277 is thus a tool that could be used to restore the current in some LOF mutations. While we have not tested this molecule on other mutants, its action might be possible in the mutants that are still expressed at the plasma membrane. The specificity of this molecule is increased for KCNQ1:KCNE3 compared to KCNQ1:KCNE1, suggesting that it could be repurposed for cancer patients harbouring a subtype of KCNQ1 mutations.

Figure III- 1 -

 1 Figure III-1 -ML277 rescues activity of KCNQ1 LOF mutant A329T. CHO cells were transfected with WT pEGFP_KCNQ1 and KCNE3 (not shown) or pEGFP_KCNQ1-A329T and KCNE3. Once currents stable, 1 µM of ML277 were added to the extracellular bath via a perfusion system. (A) Representative current-voltage relationship of KCNQ1-A329T:KCNE3 currents before and after addition of ML277 (n= 9). (B) Reversal potential of the current elicited by A329T before and after addition of ML277. Statistical significance was tested by Paired t-test *p<0,05.

  do not know what impact β-catenin has on KCNQ1. To study this question, we used deoxycholic acid, a secondary bile acid present in small quantities in the intestines. It has been shown that deoxycholic acid (DCA) at small concentrations detaches β-catenin from E-cadherin, inducing β-catenin-mediated transcription of pro-tumoral target genes and thus promotes colon cancer cell growth and invasiveness (Pai et al., 2004). Therefore, we used DCA as a tool to detach β-catenin from the adherens junctions without Wnt ligands. DCA at 5 µM induced a large reduction of KCNQ1:KCNE3 current in a few minutes (Figure III-2 A,B). Moreover, some currents showed a tail current indicative of a voltage-dependent channel, suggesting that in a subset of conditions, KCNQ1 was dissociated from the β-subunit KCNE3 as can be seen by the representation of the open probability (Figure III-2 C).

Figure III- 2 -

 2 Figure III-2 -Detaching β-catenin from the adherens junctions inhibits KCNQ1:KCNE3 currents. CHO cells were transiently transfected with KCNQ1:KCNE3. Once currents were stable, 5µM of Deoxycholic acid (DCA) was added to the extracellular bath by a perfusion system. (A) Representative traces of KCNQ1:KCNE3 before and after addition of 5µM DCA. (B) Currentvoltage relationship of KCNQ1:KCNE3 currents before (black) and after addition of DCA (red) (n=5). (C) Open probability of KCNQ1:KCNE3 before (black) and after addition of DCA (red).

Figure III- 3 -

 3 Figure III-3 -HCT116 cells expressing an empty vector (Cneg), WT KCNQ1 or the LOF mutations A329T, K422Sfs*10 or R518* were seeded onto GFR Matrigel. After 10 days in culture, cells were fixed and stained with DAPI (nuclei, in blue), anti-KCNQ1 antibody in green, anti-β-catenin antibody in red, and phalloidin (F-actin, magenta). The number of cysts forming lumen were counted on 5 to 10 images per condition and per biological replicate (n=5). Percentage of cysts forming lumen (white) is represented on the right panel. Contingency analysis was performed for each condition to evaluate statistical significance. ****p<0,0001

Figure III- 4 -

 4 Figure III-4 -CHO cells were transiently transfected with KCNQ1:KCNE3. Currents were recorded in the whole cell configuration. Once current were stable, 100ng/mL of Wnt3A were added to the extracellular bath by a perfusion system. Current voltage relationship of KCNQ1:KCNE3 currents before (black) and after addition of Wnt3A (red) (middle panel). Reversal potential (Prev) of KCNQ1:KCNE3 currents (upper left panel). Current density at 0mV before and after addition of Wnt3A (right panel) (n=7). Statistical significance was tested by Paired t-tests *p<0,05.

  membrane potential, we hypothesized that the mechanisms induced by a LOF of KCNQ1 are mediated by depolarisation. Confirming this hypothesis, we have shown that experimental depolarisation activates Wnt/β-catenin pathway and a set of Receptor Tyrosine Kinases and soluble kinases. While this work requires more work, it answers a rising question in the fields of electrophysiology and cellular biology. Schematic representation of the results. Left panel: Control condition. KCNQ1 is functional and is in the adherens junctions complex with E-cadherin and β-catenin. Β-catenin is either sequestered at the AJ or degraded by the destruction complex. Wnt inhibitory proteins such as WIF1 and DKK1 are present. RTKs and Frizzled/Lrp are not active. Wnt is OFF. Right panel: KCNQ1 carries a non-functional mutation. Membrane is depolarised, RTKs are activated and phosphorylate β-catenin on its Y654 residue. Adherens junctions lose their integrity, β-catenin is in signalling mode and translocates into the nucleus for the transcription of target genes. Wnt inhibitory proteins are downregulated, in the presence of Wnt ligand, Frizzled/Lrp are activated and enhance the shift of β-catenin into signalling mode.

First, these results

  add up to the observations made by Zhou et al. on depolarisation-activated K-Ras signalling, hypotheses made by Levin, and results obtained in Drosophila on depolarisation-induced

  cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heatinactivated Fetal Bovine Serum (FBS) and 50 U/mL Penicillin/Streptomycin. The HEK-293T, CHO-K1 and HCT116 cell lines were purchased from ATCC. HEK-293T were maintained in DMEM supplemented with 10% FBS and 50 U/mL Penicillin/Streptomycin. CHO-K1 cell line was maintained in DMEM/F-12 supplemented with 10% FBS and 50 U/mL Penicillin/Streptomycin. HCT116 cell line was maintained in DMEM supplemented with 5% FBS and 50 U/mL Penicillin/Streptomycin.

FIGURE 1 |

 1 FIGURE 1 | Proposed model of interactions between ion channels and Wnt signaling. (A) KCNQ1 (or hERG) and ClC-2 are associated to E-cadherin and b-catenin at adherent junctions (AJ). Activity of these two channels maintain membrane potential hyperpolarized, which could be a favorable condition for AJ stability, and sequestration of b-catenin at the plasma membrane. CFTR activity maintains intracellular pH, preventing from pHi alkalinisation which induces Dishevelled-dependant inhibition of Destruction Complex (DC). These four channels participate in maintaining Wnt signaling in OFF-state, in which b-catenin is either degraded or sequestrated and cannot translocate into the nucleus to induce Wnt target genes. (B) Ca 2+ entry through TRPC5, TRPM4, TRPV4 and P2X7 channels, induces AKT activation. Thus, AKT phosphorylates GSK3-b which becomes inhibited and subsequently allows the cytosolic accumulation of b-catenin. ASIC1a, by maintaining an acidic pH, decreases b-catenin phosphorylation and ubiquitination, preventing its degradation and inducing a cytosolic accumulation.

  Superieur (ABES). Protected by copyright. on September 2, 2022 at Agence Bibliographique de l Enseignement http://gut.bmj.com/ Gut: first published as 10.1136/gutjnl2021326610 on 1 September 2022. Downloaded from

Figure 1

 1 Figure 1 Sig-1R regulates a potassium current in Pancreatic cancer cells. (A) Patch clamp recordings of current elicited in control (ShRD PANC-1, n=20; MiaPaCa-2, n=15) or Sig-1R-silenced (ShSig-1R PANC-1, n=8; MiaPaCa-2, n=11) PCC, in the absence or presence of SK2 channel inhibitors apamin (apamin:100 nM; PANC-1, n=10) or leiurotoxin-1 Dab-7 (Dab-7: 100 nM; PANC-1, n=10; MiaPaCa-2, n=15). (B) Western blot analysis of shRNA-mediated Sig-1R silencing efficiency in PANC-1 and MiaPaCa-2 cells. (C) Representative immunohistochemistry staining for Sig-1R and SK2 expression in patient with PDAC samples. Scale bar: 50 µm. *p<0.05; **p<0.01; ***p<0.001. ns: not significant; PCC, pancreatic cancer cells; PDAC, pancreatic ductal adenocarcinoma; shRNA, short hairpin RNA.

Figure 3 Figure 4

 34 Figure 3 FN from CAF secretome activates SK2 through an integrin/EGFR-PI3K-AKT pathway. (A) Bar graph showing fold change in phosphorylation levels of a set of signalling proteins on overnight exposure of PANC-1 cells to CAF-CM relative to control (DMEM/F12). Experiments were performed in experimental duplicates (n=3; *p<0,05). (B) ISK2 was recorded in ShRD and ShSig-1R PANC-1 cells, treated overnight with 0% FBS DMEM/F12 (ShRD, n=10; ShSig-1R, n=12) or 0% FBS DMEM/F12 supplemented with 25 ng/mL EGF (ShRD, n=11; ShSig-1R, n=10). (C)-(E) Patch clamp recording of ISK2 in PANC-1 cells exposed overnight to (C) control medium (n=4) or CAF-CM (n=8) pretreated or not treated 1 hour with 10 µM erlotinib before adding CAF-CM (n=12), (D) control medium (n=4) or CAF-CM (n=8) treated or not treated 10 min before recording with 10 µM DEBC (n=11) and (E) control medium (n=9), CAF-CM (n=9), 40 µg/mL FN (n=9) or CAF-CM treated on ice with a functional antibody against the β1integrin 30 min before recording (10 µg/µl; n=9). (F) PLA analysis of the interaction between SK2/EGFR, SK2/AKT and SK2/β1-integrin in ShRD, ShSig-1R or ShSK2 PANC-1 cells treated overnight with control medium or CAF-CM (n=3). Scale bar: 10 µm. (G) Dot blot assay showing the presence of FN in different conditions: DMEM/F12 0% FBS or 1% FBS, and in 2 different batches of CAF-CM. (H) PLA analysis of the interaction between SK2 and AKT in PANC-1 cells, treated overnight with control medium (n=6), 40 µg/mL FN (n=5) or 10 µg/mL collagen I (n=5). Scale bar: 10 µm. (I) Representative images of SK2/Sig-1R and p-AKT-S473 staining in patient with PDAC samples. Arrows highlight cells where SK2/Sig-1R colocalise with p-AKT. Scale bar: 50 µm. *p<0.05; **p<0.01; ***p<0.001. CAF, cancer-associated fibroblasts; FN, fibronectin; ns: not significant; PDAC, pancreatic ductal adenocarcinoma; PLA, proximity ligation assay.

Figure 5

 5 Figure 5 SK2 drives PCC aggressiveness in response to CAF secretome. (A) Western blot analysis showing the protein expression of adherens junction markers in ShRD, ShSig-1R or ShSK2 PANC-1 cells, exposed overnight to control medium (DMEM/F12) or CAF-CM. Densitometric quantifications are shown as scatter plots (E-cadherin, n=9; β-catenin, n=5; p-120 catenin, n=5; Sig-1R, n=4; SK2, n=8). (B) Graphs showing the percentage of cells positive for Epcam (left panel) or CD106 (right panel) markers in Sh-RD, ShSig-1R and ShSK2 challenged with control (DMEM/ F12) or CAF-CM (CAF-CM). Labelling was quantified by flow cytometry (n=3, *p<0,05). (C) ShRD, ShSig-1R and ShSk2 PANC-1 cells were treated with control medium or CAF-CM and subjected to cell invasion assay (n=5; *p<0,05). (D) Confocal immunofluorescence images from ShRD, ShSig-1R and ShSK2 PANC-1 cells, grown in Matrigel (3D cell culture) showing F-actin staining. The scatter plot represents data dispersion and median values of Filopodia density (ShRD DMEM/F12, n=18; ShRD CAF-CM, n=15; ShSig-1R DMEM/F12, n=13; ShSig-1R CAF-CM, n=13; ShSK2, n=11; ShSK2 CAF-CM, n=12). Scale bar: 10 µm. (E) Representative immunofluorescence images of SK2 (magenta), pan CK (green, left panel) or α-SMA (green, right panel) in patient with PDAC samples. Scale bar: 50 µm. (F) Representative immunofluorescence images of Sig-1R (magenta), pan CK (green, left panel) or α-SMA (green, right panel) in patient with PDAC samples. Scale bar: 50 µm. (G) Graphical scheme explaining the experimental workflow for (H)-(J). (H) Graph showing pancreatic tumour weight of PANC ShRD (n=5), PANC ShRD+CAF (n=5), PANC ShSK2 (n=4) and PANC ShSK2+CAF (n=4), 8 weeks after orthotopic injection. Corresponding counted liver macro metastasis per mouse are presented in (I). (J) Corresponding graphs showing the percentage of pan CK labelled cells positive for α-SMA staining (n=3, each). (K) Graphical scheme explaining the experimental workflow for (L) and (M). (L) Graph showing pancreatic tumour weight of PANC-1 ShRD (n=3), PANC-1 ShRD+CAF (n=4), PANC-1 ShSig-1R (n=4) and PANC-1 ShSig-1R+CAF (n=3), 8 weeks after orthotopic injection. Corresponding counted liver macro metastasis per mouse are presented in (M). ANOVA was used for statistical analysis. *p<0.05; **p<0.01. ANOVA, analysis of variance; CAF, cancer-associated fibroblasts; Ctl, control; ns, not significant;.PCC, pancreatic cancer cells; PDAC, pancreatic ductal adenocarcinoma.

  Superieur (ABES). Protected by copyright. on September 2, 2022 at Agence Bibliographique de l Enseignement http://gut.bmj.com/ Gut: first published as 10.1136/gutjnl2021326610 on 1 September 2022. Downloaded from

Figure 6

 6 Figure 6 Sig-1R ligand 1(S) inhibits SK2 activity and reduces CAF secretome-induced aggressiveness. (A) Patch clamp recording of ISK2 in PANC-1 cells exposed to CAF-CM (n=7) or CAF-CM+1(S) ligand (1 µM; n=8) overnight. (B) PLA analysis of SK2/AKT interaction in ShRD PANC-1 cells, treated with control medium, CAF-CM or CAF-CM+1(S) ligand (1 µM; n=3) overnight. Scale bar: 10 µm. (C) Graphical scheme describing the KIC mice treatment workflow. KIC mice were injected at 5 weeks with vehicle or 1(S) ligand treatments, once a week until each animal reached clinical endpoint. (D) Kaplan-Meier survival of KIC mice treated with vehicle (n=8) or 1(S) ligand (n=8) from 5 weeks old. (E) Graphical scheme describing the KIC mice treatment workflow. KIC mice were injected at 5 weeks with vehicle or 1(S) ligand treatment, once a week until the animals were euthanised at 9 weeks old. (F) Graphs showing pancreatic tumour mass over body mass ratio at end point of 9 weeks for KIC mice treated with vehicle (n=7) or 1(S) ligand (n=8). (G) Representative pictures of H&E staining of KIC mice pancreatic tumours treated with vehicle or 1(S) ligand. (H) Graphs showing the percentage of pan CK labelled cells positive for α-SMA staining in control (untreated, n=7) and 1(S) ligand-treated mice (1(S) ligand, n=8). (I) Graphs showing the histological score of trichrome labelling in control (untreated, n=7) and 1(S) ligand-treated mice (1(S) ligand, n=8). (J) Graphs showing the percentage of trichrome staining positive pixels in control (untreated, n=7) and 1(S) ligand-treated mice (1(S) ligand, n=8). (K) Corresponding micrographs showing representative trichrome labelling in control (untreated) and treated animals (1(S) ligand treated). (L) Graphical scheme describing the KIC mice treatment workflow. KIC mice were injected at 8 weeks with vehicle or 1(S) ligand treatment, once a week until each animal reached clinical endpoint. (M) Kaplan-Meier survival of KIC mice treated with vehicle (n=10) or 1(S) ligand (n=8) from 8 weeks old. *p<0.05; **p<0.01; ***p<0.001. CAF, cancer-associated fibroblasts; ns, not significant; PLA, proximity ligation assay.

Figure 8

 8 Figure 8 Schematic diagram showing the mechanism by which CAF secretome activates an SK2-dependent β1integrin-EGFR-AKT signalling hub that promotes PCC aggressiveness. This mechanism can be pharmacologically targeted through Sig1-R to counteract the intercellular communication between CAF and PCC in PDAC. CAF, cancer-associated fibroblasts; EMT, epithelial-to-mesenchymal transition; PCC, pancreatic cancer cells; PDAC, pancreatic ductal adenocarcinoma.

Table des matières

 des Résumé ................................................................................................................................................ Remerciements ................................................................................................................................. Table of Figures .................................................................................................................................

Table of Figures

 of FIGURE1. 1 -THE GASTROINTESTINAL TRACT ....................................................................................................................................... FIGURE1. 2 -LAYERS OF THE GASTROINTESTINAL TRACT ........................................................................................................................... FIGURE1. 3 -THE LARGE INTESTINE ................................................................................................................................................... FIGURE1. 4 -SODIUM ABSORPTION BY COLONOCYTES ............................................................................................................................. FIGURE1. 5 -CHLORIDE SECRETION IN COLONOCYTES ............................................................................................................................. FIGURE1. 6 -THE COLONIC CRYPT...................................................................................................................................................... FIGURE1. 7 -THE COLONIC CRYPT AND ITS CELL TYPES. ........................................................................................................................... FIGURE1. 8 -THE SIGNALLING PATHWAYS AT PLAY IN THE COLONIC CRYPT ................................................................................................... FIGURE2. 1 -OVERVIEW OF WNT NON-CANONICAL SIGNALLING PATHWAYS ................................................................................................ FIGURE2. 2 -MAIN PHOSPHORYLATIONS ON Β-CATENIN, IMPLICATED KINASES, AND EFFECT. ........................................................................... FIGURE2. 3 -OVERVIEW OF ADHERENS JUNCTIONS ............................................................................................................................... FIGURE2. 4 -OVERVIEW OF THE DESTRUCTION COMPLEX ........................................................................................................................ FIGURE2. 5 -WNTS MODIFICATIONS ................................................................................................................................................. FIGURE2. 6 -OVERVIEW OF WNT/Β-CATENIN SIGNALING. ...................................................................................................................... FIGURE2. 7 -MODEL OF Β-CATENIN Y654 PHOSPHORYLATION CONTRIBUTING TO WNT SIGNALLING ................................................................

FIGURE2. 8 -WNT TARGET GENES TRANSCRIPTION ................................................................................................................................ FIGURE2. 9 -WNT PATHWAY REGULATION .........................................................................................................................................

TABLE 1 -

 1 MUTATION FREQUENCY OF CANONICAL WNT PATHWAY GENES IN COLORECTAL CANCER.. .................................................................

TABLE 2 -

 2 CLASSICAL INTRACELLULAR AND EXTRACELLULAR CONCENTRATION BY IONIC SPECIE.

......................................................................... EQUATION 1 -NERNST EQUATION .................................................................................................................................................... EQUATION 2 -GOLDMAN-HODGKIN-KATZ EQUATION ........................................................................................................................... EQUATION 3 -THE DRIVING FORCE.

TABLE 3 -

 3 EXTRACELLULAR AND INTRACELLULAR IONIC CONCENTRATIONS FOR PATCH CLAMP EXPERIMENTS ......................................................

TABLE 4 -

 4 CONCENTRATIONS FOR PREPARATION OF KREBS AND CRYPT ISOLATION BUFFER FOR CRYPT EXTRACTION .............................................

TABLE 5 -

 5 CONCENTRATION FOR CONTROL AND HIGH K+ SOLUTION FOR DEPOLARISATION EXPERIMENTS. ........................................................

		List of abbreviations
	AE	Anionic Exchanger		HGF	Hepatocyte Growth Factor
	AJ	Adherens Junctions		HGFL Hepatocyte Growth Factor-Like
	AKAP A-Kinase Anchoring Protein	Hh	Hedgehog
	APC	Adenomatous Polyposis Coli	IBD	Intestinal Bowel Disease
	BCL9	B Cell CCL/Lymphoma 9 protien	ISC	Intestinal Stem Cells
	BMP	Bone Morphogenetic Protein	JNK	Jun N-terminal Kinase
	bTrCP F-box/WD repeat-containing protein 1A	kDa	kiloDaltons
	CACC Calcium Activated Chloride Channels	Kir	Inwardly rectifying potassium channels
	CaM	Calmodulin			LGR	Leucine-rich repeat-containing G-protein-
					coupled receptor
	CaMKII Calcium/Calmodulin-dependent kinase II
					LOF	Loss-of-Function
	cAMP cyclic Adenosine MonoPhosphate
					LQT	Long QT
	CFTR	Cystic	Fibrosis	Transmembrane
	Conductance Regulator		LRP	Low-density Lipoprotein Receptor-related
					Protein
	CKI	Casein Kinase I
					MAPK Mitogen Activated Protein Kinases
	CM	Conditioned Medium
					mV	milliVolts
	DKK	Dickkopf related protein 1
					NHE	Na+/H+ Exchanger
	Dvl	Dishevelled
					PCP	Planar Cell Polarity
	EGFR Epidermal Growth Factor Receptor
					PIP2	Phosphatidylinositol 4,5 bisphosphate
	ENaC Epithelial Sodium Channels
					PKA	Protein Kinase A
	ERK	Extracellular Signal-regulated Kinase
					PKC	Protein Kinase C
	FGFR	Fibroblast Growth Factor Receptor
					PS	Phosphatidylserine
	Fzd	Frizzled
					pS	pico Siemens
	GFP	Green Fluorescent Protein
					Ptch	Patched
	GIT	GastroIntestinal Tract
					Rbx1	RING Box Protein 1
	Gli	Glioma-associated oncogene
					RNF43 Ring Finger protein 43
	GSK3	Glycogen Synthase Kinase 3

Table 1 -

 1 Mutation frequency of canonical Wnt pathway genes in colorectal cancer. Data were acquired out of 8534 samples from publicly available datasets on cBioPortal. Of note, these data were collected in June 2023 and might differ as new datasets are being added.

Table 2 -Classical intracellular and extracellular concentration by ionic specie for mammalian cells with corresponding theoretical equilibrium potential as calculated by Nernst equation.

 2

  Than and colleagues confirmed that KCNQ1 has a functional role in GIT cancer. KCNQ1 figured in the top 15 genes carrying mutations in intestinal tumours in mice. The APC min mice carrying a targeted deletion mutation in kcnq1 developed more adenomas and adenocarcinomas, mostly in the colon. Furthermore, more colon organoids developed

from these mice, suggesting a role for KCNQ1 in the stem cell compartment. The study identified altered genes including ones involved in immune response, stem cell and goblet cells maintenance and function, and signalling pathways. Lastly, immunohistochemistry in tissues from CRC patients with liver metastases who underwent hepatic resection showed that low expression of KCNQ1 was significantly associated with poor overall survival. All these results point to a tumour-suppressor role for KCNQ1 in GIT cancers

(Figure 4. 5)

. Two years later, another study led by den Uil and colleagues assessed the expression of KCNQ1 among colorectal cancer patients at different stages of disease progression (den

[START_REF] Den Uil | Loss of KCNQ1 expression in stage II and stage III colon cancer is a strong prognostic factor for disease recurrence[END_REF][START_REF] Uil | KCNQ1 and lymphovascular invasion are key features in a prognostic classifier for stage II and III colon cancer[END_REF]

. They found that KCNQ1 mRNA expression was lower in stage II microsatellite stable (MSS) colon cancers and that loss of KCNQ1 protein expression was associated with poor disease-free survival in stage II MSS, stage III MSS and stage III microsatellite instable colon cancer (Figure

  KCNQ1-LOF induce loss of negative regulators of Wnt/β-catenin. (A) Heatmap of significantly differentially expressed genes corresponding to the Gene Ontology Terms "Positive Regulation of Wnt signalling" and "Negative Regulation of Wnt signalling" obtained from HT29Cl19A RNASeq. Genes are clustered by GO Term. (B) Volcano plot of differentially expressed genes from the colon adenocarcinoma TCGA data set from patients harbouring a KCNQ1 mutation. Thresholds were set at 1.44-fold change and 0.01 p-adjust. Genes corresponding to the GO Term "Negative Regulation of Wnt signaling" are represented in blue. Genes corresponding to the GO Term "Positive Regulation of Wnt signaling" are represented in red.(C)

	. WIF1 binds to Representative images acquired on a confocal microscope of the HT29cl19A 7TGC cells overexpressing or not a mutation with
	or without addition of Wnt3A conditioned media (CM). 63x magnification, scale bar = 10 µM. Left panel: quantification of extracellular Wnt ligands to prevent their binding to Frizzled. Protein expression of both Nkd1 and Wif1 GFP+ cells normalised by the number of mCherry+ cells per field (n=5).(D) Western blot and densitometry analysis of pLRP6
	(S1490) normalised by LRP6 (n=3) with or without addition of Wnt3A CM. Western Blot and densitometry analysis of (E) Dkk1, are lowered by 40 to 50% in the KCNQ1-LOF conditions (FIGURE 7 F,G). (F) Nkd1, and (G) Wif1 normalised by GAPDH in HT29cl19A cells overexpressing KCNQ1-LOF mutations. Data were analysed
	for statistical significance using One Way Anova with post-hoc Tukey's (C) or Holm-Sidak's tests (E, F, G). * p<0,05, ** p<0,01,
	*** p<0,001, **** p<0,0001, ns not significant

  that low extracellular pH

TABLE 1 |

 1 Role of ion channels in regulating Wnt pathway activity in cancers.

	References			Rapetti-Mauss et al., 2017		Fan et al., 2018,	Breuer et al., 2019		Strubberg et al., 2018		Jin et al., 2018	Xie et al., 2017		Sagredo et al., 2018	Ashmore et al., 2019		Wang et al., 2015	Zhang et al., 2019	Jin et al., 2017
	Expression in cancers			Down		Down	Up		Down		Down	Up		Up	Up		Up	Up	Up
	Mechanism			Stabilization of b-cat. at the plasma membrane		Stabilization of b-cat at the plasma membrane	Stabilization of b-cat at the plasma membrane		Modulation of the intracellular pH		Stabilization of b-cat at the plasma membrane	Ca 2+ ! AKT ! b-cat accumulation		Ca 2+ ! AKT ! b-cat accumulation	Wnt ligands binding ! calcium		Ca 2+ ! AKT ! b-cat accumulation	AKT !GSK3 b ! b-cat accumulation	Modulation of b-cat phosphorylation and accumulation
	Effect of channels	expression/activity	on Wnt pathway	↓ Wnt activity	Wnt target genes	↓ Wnt activity	↓ Wnt activity	(Channel opened)	↓ Wnt activity	Wnt target genes	↓ Wnt activity	↑ Wnt activity		↑ Wnt activity	↑ Wnt activity		↑ Wnt activity	↑ Wnt activity	↑ Wnt activity
	Channels			KCNQ1		KCNQ1	hERG		CFTR		ClC-2	TRPV4		TRPM4	TRPM8		TRPC5	P2X7	ASIC1a
	Models			Cell lines, Rat colonic crypts		Cell lines	Mouse models,	Cell lines	Mouse intestine		Cell lines; Mouse models	Human sample,	mouse model, Cell lines	Cell lines	Cell lines		Cell lines	Cell lines	Human sample,	Cell lines
	(C) Cancers Type			Colorectal Cancer		Hepatocellular Carcinoma	Breast cancer		Colorectal cancer		Colorectal cancer	Gastric cancer		Prostate cancer	Prostate cancer	Breast cancer	Colorectal cancer	osteosarcoma	Liver cancer

Frontiers in Pharmacology | www.frontiersin.org September 2020 | Volume 11 | Article 525020

Frontiers in Pharmacology | www.frontiersin.org September 2020 | Volume 11 | Article 525020 10

Rapetti-Mauss R, et al. Gut 2022;0:1-14. doi:10.1136/gutjnl-2021-326610

Remerciements

Acknowledgements We thank the Cancer Research Center of Marseille shared resources: Karim Sari and Regis Vitestelle for the PSEA (Platform for Stabling and Animal Experimentation) animal facility, Michel Baccini, Bernard Chetrit and Eric Orlandi for informatical support and Bruno Olivier and Tahagan Titus for technical

Funding The work described in this paper was supported by grants from the Fondation de France (RT: 00087538; OS: 00038330 and 00059283), the French National Institute of Cancer (RT: INCA, PLBio13-134), La Ligue contre le Cancer (RR-M: GB/MA/IQ-10607 and GB/MA/CD/IQ-12187), Foundation ARC (RT: PJA20191209372; OS: PJA 2016 120 4740 and PJA 2019 120 9546; RR-M: PJA 2018 207701) and the Agence Nationale pour la Recherche (HG: ANR19-14-0049-01). JN was supported by Ministère de la Recherche and by Ligue contre le Cancer. CB was supported by the Fondation pour la Recherche Medicale. ST was supported by the French National Institute of Cancer (INCA, Pancreas 2017). BA was supported by the Ministère de la Recherche, de l'Enseignement Supérieur et de l'Innovation.

ORCID iDs

Raphael Rapetti-Mauss http://orcid.org/0000-0002-3518-4845 Jérémy Nigri http://orcid.org/0000-0003-1358-1863 Zainab Hussain http://orcid.org/0000

FUNDING

This work was supported by the Foundation ARC (grant PJA 20181207701), "La Fondation pour la Recherche Medicale"-FRM-(PhD studentship to CB) (n°ECO201906008972) and the Canceŕopole PACA (grant n°2018-13) and Université Côte d'Azur (CSI-2018).

ATCC HCT116-pPriPu-pPriHy_KCNE3

This study This study HCT116-pPriPu_KCNQ1. This study HCT116-pPriPu_KCNQ1.K422Sfs* This study .R518*-pPriHy_KCNE3 This study HT29cl19A-pPriPu_GFP This study HT29cl19A-pPriPu_KCNQ1.A329T GFP This study HT29cl19A-pPriPu_KCNQ1.K422Sfs*10 This study HT29cl19A-pPriPu_KCNQ1.R518* This study RKO-pPriPu This study RKO-pPriPu_KCNQ1 This study RKO-pPriPu_KCNQ1.A329T This study RKO-pPriPu_KCNQ1.K422Sfs*10 This study RKO-pPriPu_KCNQ1.R518* This study CHO-K1-pPriHy_KCNE3 This study M51 Super 8x FOPFlash (TOPFlash mutant) Addgene (Randall Moon) Cat# pRL-SV40P Addgene (Ron Prywes) Cat# pEGFP-N2_KCNQ1 J. Barhanin I. barrau pXOOM_KCNE3 J. Barhanin pEGFP-N2_KCNQ1.E170K This study pEGFP-N2_KCNQ1.V185M This study pEGFP-N2_KCNQ1.R243C This study pEGFP-N2_KCNQ1.A287T This study pEGFP-N2_KCNQ1.A329T This study pEGFP-N2_KCNQ1.F339L This study pEGFP-N2_KCNQ1.P343S This study pEGFP-N2_KCNQ1.R401Gfs*18 This study pEGFP-N2_KCNQ1.K422Sfs*10 This study pEGFP-N2_KCNQ1.P448R This study pEGFP-N2_KCNQ1.R451Q This study pEGFP-N2_KCNQ1.R518* This study pEGFP-N2_KCNQ1.G548C This study pCMV_GAG_pol pCMV_VSVG P81i EVO lab PMD2G EVO lab pPriPu pPriPu _KCNQ1 pPriPu _KCNQ1.A329T This study pPriPu_GFP This study pPriPu_KCNQ1.A329T GFP This study pPriPu_KCNQ1.K422Sfs*10 This study pPriPu_KCNQ1.R518* This study pPriHy_KCNE3 This study 7TGC Addgene (Roel Nusse) Cat# pIRES-GFP Oligonucleotides EcoRI-6His-KCNE3 AATCGAATTCGCCGCCATGGGCCATCAT 6His-KCNE3-MfeI AATCCAATTGTTAGATCATAGACACACC KCNQ1-R451Q-for GACCCCCCAGAAGAGCAGCGGCTGGACCACTTC KCNQ1-R451Q-rev GAAGTGGTCCAGCCGCTGCTCTTCTGGGGGGTC KCNQ1_E170KFor GTGTTCTTCGGGACGAAGTACGTGGTCCGCCTC KCNQ1_E170KRev GAGGCGGACCACGTACTTCGTCCCGAAGAACAC KCNQ1_P448Rfor ATCACGTGCGACCCCCGAGAAGAGCGGCGGCTG KCNQ1_P448RRev CAGCCGCCGCTCTTCTCGGGGGTCGCACGTGAT KCNQ1_R243CFor ATGCTACACGTCGACTGCCAGGGAGGCACCTGG KCNQ1_R243CRev CCAGGTGCCTCCCTGGCAGTCGACGTGTAGCAT KCNQ1_R518StopFor ACCATTAAGGTCATTTGACGCATGCAGTACTTT KCNQ1_R518StopRev AAAGTACTGCATGCGTCAAATGACCTTAATGGT KCNQ1_G548CFor GAGCAGTACTCGCAGTGCCACCTCAACCTCATG KCNQ1_G548CRev CATGAGGTTGAGGTGGCACTGCGAGTACTGCTC KCNQ1_K422SFor GTGGTAAAGAAAAAAAGTTCAAGCTGGACAAAG KCNQ1_K422SRev CTTTGTCCAGCTTGAACTTTTTTTCTTTACCAC KCNQ1_A329TFor GTCGGGAAGACCATCACCTCCTGCTTCTCTGTC KCNQ1_A329TRev GACAGAGAAGCAGGAGGTGATGGTCTTCCCGAC KCNQ1_A287TFor CTGGCTGAGAAGGACACGGTGAACGAGTCAGGC

RESULTS

DISCUSSION

MATERIAL AND METHODS

DuoLink PLA was used to detect interactions between KCNQ1 and β-catenin and β-catenin and Ecadherin. HT29cl19A were seeded on microscope slides at 40 000 cells per well. Cells were washed with PBS, fixed with paraformaldehyde 4% for 10 minutes, permeabilised with triton x100 (0.03%) and blocked with the blocking buffer provided in the kit for 1 hour at 37°C. Cells were immunolabeled with primary antibodies anti β-catenin (Cell Signaling Technology), anti KCNQ1 (Invitrogen) or E-cadherin (BD Biosciences) overnight at 4°C in a humid chamber. Cells were washed with the provided buffer A.

the secondary antibodies with attached PLA probes were supplied in the kit and incubated for 1 hour at 37°C. The ligase provided in the kit was incubated for 30min at 37°C at a 1:40 dilution. The polymerase supplied in the kit was incubated for 100min at 37°C at a 1:80 dilution with Far Red Reagent. Cells were then washed with provided buffer B twice and 0.01x buffer before using the Vectashield DAPI mounting medium. The slides were imaged on the Timelapse: Axiovert 200M -Zeiss (2004) / sCMOS ANDOR Neo camera / MetaMorph software at a 63x magnification and Cy5 / DAPI filters. PLA dots were quantified using a macro on Image J software and divided by the number of cells identified with DAPI staining. 5 to 10 images per condition were acquired and analysed. Negative controls consisted in incubation with one of the primary antibodies alone or none (secondary alone).

The mean number of dots per cell in negative controls was deduced from the number of dots per cell in all experimental conditions.

Cysts formation

HCT116 cells were trypsinised to a single cell suspension of 5x10 3 cells/mL in 0.5% FBS completed with 2% GFR Matrigel. Cells in GFR Matrigel were seeded in 8-well coverglass chambers pre-coated with 100% GFR Matrigel (for 30min at 37°C). Cells were fed every two days and grown for 10 days before fixation and staining.

Cysts immunofluorescence

Cysts were rinsed with PBS, fixed with 4% paraformaldehyde for 30 minutes and permeabilised for 15 minutes in 0.4% triton 100X. Blocking buffer with 10% Bovine Serum Albumin was added for 2 hours at room temperature. Primary antibodies were diluted in 1:1 blocking buffer:wash buffer (0.05% triton) and incubated overnight in a humid chamber at 4°C. Spheroids were then rinsed three times with wash buffer. Secondary antibodies coupled to AlexaFluor647 or AlexaFluor488 and phalloidin-AlexaFluor568 were incubated for 1 hour at room temperature. Spheroids were washed three times in wash buffer and 1:10000 Hoechst was incubated for 10 min at room temperature before mounting the coverglass with Vectashield Vibrance.

Depolarisation experiments

For depolarisation experiments, cells were serum-starved overnight before treatment. Cells were then incubated either for the short kinetic or for 24 hours with a Control Solution or High-K + Solution (Table 5). The potassium equilibrium potential was calculated by Nernst equation, under the assumption that intracellular concentration of potassium is roughly 150mM. The theoretical K + equilibrium potential was -87mV with the Control Solution and -9mV with the High-K + Solution.

PamChip Assays

HT29cl19A cells were treated with control solution or high K+ solution for 5 to 60 minutes. Cells were rinsed with cold PBS and scraped in M-PER Maammalian Extraction Buffer buffer containing Halt protease inhibitor cocktail and phosphatase inhibitor cocktail on ice. Cell lysates were cleared by centrifugation at 12 000 g for 10 minutes at 4°C. Protein concentration was determined by Bradford protein dye assay. Aliquots of 1 mg/mL of protein were snap-frozen in liquid nitrogen. 5 µg of protein extract was used for the PTK array protocol and 1.5 µg for the STK array protocol. Measurements were performed on a Pamstation from PamGene according to the manufacturer's protocol. Briefly, the PTK array was processed in a single-step reaction. Cell extracts, ATP and FITC-labelled pY20 antibody were incubated on the chip and the phosphorylation of individual Tyr-peptides was followed by real-time fluorescent detection. The STK array was processed in two steps. First, the protein extracts, ATP and the primary antibody mixture was incubated with the chip for 110 min. Then, the reaction mix was removed, and the secondary FITC-labelled antibody was added. Development of the fluorescence signal was detected by Alexa488 fluorescence in real-time. Signal intensities were analysed in the Bionavigator software (PamGene) and expressed as log2 fold-ratio versus control condition.

Statistical analysis

Statistical analysis was performed on the software GraphPad Prism (version 9.5.1). When applicable, the Gaussian distribution was tested by the ad-hoc Normality tests of D'Agostino, Anderson-Darling, Shapiro-Wilk, and Kolmogorov-Smirnov, and heteroscedasticity of the data was tested with the ad-hoc Browne-Forsythe and Bartlett's tests. Statistical and post-hoc tests were performed accordingly to the dataset and, if applicable, results of ad-hoc tests. The performed statistical analysis and statistical significance are presented in the legend section of each figure. Statistical analysis of bulk results for RNASeq and PamGene Array were performed by the service provider. Gene Ontology classification and associated statistics were given by the software String.

Antibody

Dilution Increasing evidence point out the important roles of ion channels in the physiopathology of cancers, so that these proteins are now considered as potential new therapeutic targets and biomarkers in this disease. Indeed, ion channels have been largely described to participate in many hallmarks of cancers such as migration, invasion, proliferation, angiogenesis, and resistance to apoptosis. At the molecular level, the development of cancers is characterised by alterations in transduction pathways that control cell behaviors. However, the interactions between ion channels and cancer-related signaling pathways are poorly understood so far. Nevertheless, a limited number of reports have recently addressed this important issue, especially regarding the interaction between ion channels and one of the main driving forces for cancer development: the Wnt/b-catenin signaling pathway. In this review, we propose to explore and discuss the current knowledge regarding the interplay between ion channels and the Wnt/b-catenin signaling pathway in cancers.

AUTHOR CONTRIBUTIONS

RR-M conceived the presented idea and supervised the project. RR-M, CB, BA, and OS wrote the manuscript. All authors contributed to the article and approved the submitted version.

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. S4, these results remained similar and significant after adjustment for the estimated tumour cellularity (ESTIMATE algorithm, R-library, V.1.0.13), for both SIGMAR1 and KCNN2. ns: not significant; PDAC, pancreatic ductal adenocarcinoma. Twitter Jérémy Nigri @jeremynigri, Richard Tomasini tomasini richard@ TomasiniRichard and Olivier Soriani soriani olivier @OlivierSoriani Competing interests None declared.

Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

Patient consent for publication Not applicable.

Ethics approval

The study was approved by our institutional board. All animal care and experimental procedures were performed in agreement with the Animal Ethics Committee of Marseille and the French ministry of research and innovation (reference number: Apafis#16 998-2018100814458519).

Provenance and peer review Not commissioned; externally peer reviewed.

Data availability statement

Supplementary Material

Supplementary Experimental Procedures:

Drug treatment 1(S) compound was synthesized as previously described 1, 2 and solubilized just before administration. standard) for all runs. To do so, a primary CAFhTERT culture was labeled with heavy amino acids (L-arginine and L-lysine) through stable labeling with amino acids in cell culture.

Secretome Liquid Chromatography MS/MS Analysis

Clinical Data Analysis

Patient data were accessed through the R2: genomics analysis and visualization platform (http://r2.amc.nl).

Constructs, Retroviral production and transduction

For stable ShRNA cell lines construction: Control random or Sig-1R-silenced PANC-1 cells were established as previously described 3 . Stable SK2 ShRNA PANC-1 cell line was engineered using the following DNA sequence: Sh SK2 for: 5' TATGGTTCTAATCAGCGTTATCTCGAG ATAACGCTGATTAGAACCATA-3'. The double stranded DNA sequence was inserted in the mammalian expression vector pSuperRT Puro, a derivative of the pPRIG series. Transduction BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Gut doi: 10.1136/gutjnl2021326610 -14.

experiments were performed as previously described 4 . Sh Sig-1R res cell line was obtained by transducing C-myc Sig-1R vector in the Sh Sig-1R PANC-1 cell line.

For KCNN2 (SK2) gene depletion in PANC-1 cells using CRISPR/Cas9 editing technology: guide RNAs targeting human SK2 were designed as described 5 . PANC-1 cells were transfected with pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (obtained from Addgene) carrying gRNAs against human SK2. KCNN2-EX1-pX-FOR : 5'-CACCGCTTAGACACCACGATCTGG -3' and KCNN2-EX1-pX-REV : 5'-AAACCCAGATCGTGGTGTCTAAGC-3'. 48h after transfection (TransIT-LT1, Mirus), cells were selected in 50µg/ml puromycin supplemented culture medium. Resistant cells were seeded to a cell per well in 96 wells plate using a Cell sorter ARIA (BD). Single clones were screened for null SK2 associated channel current by patch clamp analysis and western blotting.

The protocol for obtaining SK2 mutants is adapted from the QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla USA). Forward and reverse primers were obtained from (Sigma-Aldrich). S562A for: 5'-CGG TCC CGG TCC TCG GCC AGG AGG CGG CGG T-3' S568A for: 5'-AGG AGG CGG CGG TCC GCT TCC ACA GCA CCA C-3' S569A for: 5'-GG CGG CGG TCC TCT GCC ACA GCA CCA C-3'. Triple mutation was obtained sequentially from single SK2/S562A and double S568A / S569A mutants. Positive clones were detected by DNA sequencing and then sequenced in their entirety to ensure that no polymerase errors had been introduced. HA-myr-AKT is a kind gift from Dr Julie Guillermet-Guibert (CRCT, Toulouse, France).

EGF quantification

6.10 4 PANC-1 cells were seeded in 12-well plates in DMEM 10% FBS. Two days later, at approximately 80% confluence, cultures were rinsed 3 times in PBS and DMEM without FBS was added for 24 hours. Cultures were again rinsed 3 times in PBS and DMEM/F12 1% FBS or conditioned medium from different batches was added. The culture medium was collected 24 hours later, and cells were counted. EGF quantification in culture media was done using kit BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut doi: 10.1136/gutjnl2021326610 -14. :1 0 2022; Gut , et al.

RapettiMauss R

Quantikine ELISA human EGF (Bio-techne) according to manufacturer instructions. Two different experiments were done with independent cell cultures.

In situ proximity ligation assay

The DuoLink in situ Proximity Ligation Assay (PLA) (Olink Bioscience, Uppsala, Sweden) was used to detect the interaction between SK2 and different candidate partners (C-myc Sig-1R, EGFR, AKT, β1 integrin). PANC-1 cells expressing a stable ShRD, ShSig-1R, or ShSK2 were seeded on poly-Lysine (40μg/ml) coated microscope slides. Cells were fixed with PFA 4% and permeabilized with triton X100 (0.05%). Cells were immunolabeled with primary antibodies: anti SK2 (1:100) and anti C-myc, (1:100) or anti EGFR (1:100), or anti AKT (1:100), or anti β1 integrin (1:100) for 1 h at 37°C. The secondary antibodies with attached PLA probes were supplied in the Duolink kit. Cellular PLA images were captured using an inverted Zeiss Axio Observer Z1 microscope (Zeiss, Jena, Germany). 6 to 10 fields have been imaged and quantified per conditions.

Western blot

Cells were lysed using RIPA lysis buffer containing proteases inhibitor cocktail (cOmplete tablets, mini EDTA-free, EASYpack, Roche) and phosphatases inhibitors (PhosSTOP EASYpack, Roche). Whole-cell lysates were subjected to 8% or 10% SDS/PAGE before proteins being transferred onto a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific). Membranes were probed with the primary antibody overnight at 4°C. Reactive proteins were developed with HRP conjugated secondary antibody and visualized with chemiluminescence according to the manufacturer's protocol (Immobilon® Western, EMD Millipore Corporation) and then exposed to Fusion FX7 Edge (Vilber-Lourmat, France). Details for primary antibodies used are provided in table S5. The protein levels were normalised using GAPDH or α-tubulin when appropriate and quantified by image J software (NIH, MD).

Co-immunoprecipitation

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut doi: 10.1136/gutjnl2021326610 -14. :1 0 2022; Gut , et al.

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HEK 293-T cells were transfected using calcium phosphate with WT SK2, HA-myr-AKT or SK2 S562A/S568A/S569A triple mutant. 24 hours later cells were serum-starved overnight and then proteins were extracted in IP lysis buffer (co-immunoprecipitation kit; Pierce). Anti-Sk2 (Sigma Aldrich) antibody was immobilized on Aminolink plus coupling resin beads using sodium cyanobrohydride as a cross-linking reagent (75 mM). Samples were added to the SK2-beads complex and incubated overnight at 4°C on a rotator. The beads were then washed with IP lysis buffer ten times, and then eluted with elution buffer pH 2.8 (Pierce). Samples were subjected to SDS-PAGE and Western blotting assay.

3D Matrigel cell culture and Filopodia count

For 3D-culture PANC-1 were cultured in Growth Factor Reduced (GFR) Matrigel (BD Biosciences), briefly PANC-1 cells were trypsinized to a single cell suspension of 5×10 3 cells/ml in 1% FBS or CAF-CM completed with 2% GFR Matrigel. Cells in GFR Matrigel were seeded in 8-well coverglass chambers (Nalge Nunc) precoated with 100% GFR Matrigel (for 30min at 37°C). Cells were fed every 2 days and grown for 10 days. Samples were fixed in 4% (wt/vol) PFA for 10 min, washed with PBS, and permeabilized with PBS containing 0.5% (vol/vol) Triton X-100 for 10 min. Cells were then washed with PBS, blocked using a solution containing 10% BSA and 10% normal Goat serum, 0,05 Triton X-100 in PBS, and incubated overnight at 4°C with Alexa Fluor 568 Phalloidin (1/100 in blocking solution). Then samples were exposed to DAPI (1/10 000 in blocking solution) for 10 min, and mounted in moviol mounting medium (Fluka) for further observation. The confocal microscope used to image the samples was a laser scanning confocal microscope LSM710 (Zeiss) with a 63× (NA 1.2 water) objective controlled by ZEN software (2010). Filopodia count were performed using the Image J plugin FiloQuant as described in 6 .

Cell Invasion

Cell invasion assay was performed using CytoSelect™ Cell Invasion Assays from CELL BIOLAB, INC (CBA-11) following the manufacturer's instructions. Briefly, PANC-1 cells stable BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut doi: 10.1136/gutjnl2021326610 -14. :1

transduced with a random ShRNA, a Sig-1R ShRNA or the Sk2 KO construct were pretreated with DMEM/F12 or CAF-CM overnight and then seeded at 1 × 10 5 cells/well on transwell insert pre-filled with Matrigel. The upper chambers were filled with either DMEM/F12 or CAF-CM and the lower chamber with DMEM/F12 10% FBS. 48h later cells were incubated for 20 min with CyQuant® fluorescent dye following manufacturer's instruction. Cell invasion was determined by reading fluorescence at 480 nm using a Fluostar Optima microplate reader (BMG Labtech).

Human phospho-kinase antibody array

Phospho-antibody array analysis was carried out using the Proteome Profiler Human Phospho-Kinase Array (ARY003B; R&D Systems) according to the manufacturer's instructions Cells were cultured in DMEM/F12 containing 1% FBS or CAF-CM and then lysed with Lysis Buffer 6 (R&D Systems) and agitated for 1 hour at 4°C. Cell lysates were clarified by microcentifugation at 12 000 rpm for 15 minutes, and protein concentration was determined using a Bradford protein assay dye (Bio-Rad). Array membranes were blocked with Array Buffer 1 (R&D Systems) and incubated overnight at 4°C with 600 μg of cell lysate. The membranes were washed to remove unbound proteins and then incubated with biotinylated detection antibodies and streptavidin-HRP. Chemiluminescent detection reagents were applied to detect spot densities. Array images were analyzed using Image J software. Array spots were background subtracted and normalized to positive control spots on each membrane to enable comparisons across the different treatment groups. The integrated density of duplicated spots representing each phospho-kinase protein was determined, and data were presented as the fold change compared with the untreated control group. The phosphoantibody array experiment was repeated at least twice, comprising 2 biological and 4 or 6 technical replicates per treatment group.

Fibronectin quantification

For fibronectin detection, the same culture protocol as for EGF quantification was used except that at 80 % confluence, following PBS washing, the culture medium was substituted by DMEM BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut doi: 10.1136/gutjnl2021326610 -14. :1 0 2022; Gut , et al.

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without or with 1% FBS or by conditioned medium from different batches. The culture medium was collected 48 hours later, and fibronectin was quantified by dot blot. Dots of 10 µl serial dilutions of collected culture media were compared to 10 µl DMEM/F12 0% or 1% FBS, conditioned medium and fibronectin concentration ranges from 10 to 0.62 µg/ml. The dot blot was probed with anti-fibronectin (AB1949 Millipore) diluted 1/4000 in TBS 0.1 % tween 1% unfat milk and secondary anti-rabbit Dako 1/2000. 3 different dot blots were done with 3 independent cell cultures seeded in duplicate in each experiment.

Immunochemistry

Formalin-fixed, paraffin-embedded human sections (4 μm) were deparaffinized in xylene and rehydrated through a graded ethanol series. An antigen retrieval step (Dako) was performed before quenching of endogenous peroxidase activity (3%[vol/vol] H2O2). Tissue sections were then incubated with primary antibody (SK2, 1:100, ab9945, Abcam; Sig-1R, 1:100, sc-22948, Santa Cruz Biotechnology) and immunoreactivities were visualized using the Vectastain ABC kit (PK-4005; Vector Laboratories) according to the manufacturer's protocol. Peroxidase activity was revealed using the liquid diaminobenzidine substrate chromogen system (Dako; K3468). Counterstaining with Mayer hematoxylin was followed by a bluing step in 0.1% sodium bicarbonate buffer, before final dehydration, clearance, and mounting of the sections.

Immunofluorescence

Formalin-fixed, paraffin-embedded human sections (4 μm) were deparaffinized in xylene and rehydrated through a graded ethanol series. An antigen retrieval step (10 mM sodium citrate, 0.05% Tween 20, or TRS9 (S236784-2, Agilent Technologies) 95°C) was then performed before tissue sections were preincubated in blocking solution (PBS, 0.1% Triton X-100, 10%[vol/vol] donkey serum) for 1 hour. Tissue sections were incubated in a mixture of 2 primary antibodies against SK2 (1:100, ab99457, Abcam), or Sig-1R (1:100, sc-22948, Santa Cruz Biotechnology), with pAKT (1:25, 9271S, Cell signaling), pan-cytokeratin (panCK, 1:100, ab9377, Abcam), or α-smooth muscle actin (α-SMA, 1:100, ab5694, Abcam) in blocking solution (3% BSA, 0.05% Tween20) overnight at 4°C. After washing in PBS, slides were BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut doi: 10.1136/gutjnl2021326610 -14.

incubated with a mixture of 2 secondary antibodies in blocking solution (Alexa Fluor 568conjugated, A11057, or Alexa Fluor 488-conjugated antibody, A21206, 1:500, Invitrogen).

Stained tissue sections were mounted using Prolong Gold Antifade reagent with DAPI (Life Technologies) before being sequentially scanned at a ×20 magnification under a fluorescent microscope (Zeiss).

For the quantification of PanCK/α-SMA co-staining in mice tissue, the percentage of PanCK stained cells that are positive for α-SMA staining was determined using the JACoP plugin from Fiji software. Data were obtained from 3 independent images per animals.

Proliferation and Cytotoxicity assay

To assess cytotoxicity and proliferation following 1(S) ligand treatment, 25 000 cells were plated in a 12-well plate. 24 hours later cells were washed with PBS 1X and stimulated with 1µM of 1(S) and 200nM of Cytotox Green (Sartorius, #4633). Cells were visualized in an IncuCyte S3 Live-Cell Analysis System (Essen BioScience) at 37 °C, 5% CO2. Phase contrast and green channel (exposure time 300ms) images were taken every 4h, for 72h, with a 10X objective. Images analysis was done using the Cell-by-Cell module of the IncuCyte S3 software (2019B Rev2).

Migration assay

To evaluate cell migration following 1(S) ligand treatment, 200 000 cells were plated on a 24well plate and allowed to adhere. 24 hours later cells were washed with PBS 1X and treated with 1µM of 1(S) ligand overnight. The ligand was subsequently removed by rinsing cells once with PBS 1X. A scratch was performed on the cell monolayer and cell culture medium was added back. Cells were visualized in an IncuCyte S3 Live-Cell Analysis System (Essen BioScience) at 37°C, 5% CO2. Phase contrast images were taken every 4h, for 8h, with a 10X objective. Images were analyzed using ImageJ and recovered area was measured.

Flow cytometry

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut doi: 10.1136/gutjnl2021326610 -14. :1 0 2022; Gut , et al.

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For assessment of epithelial and mesenchymal markers : cells treated or not with conditioned media from CAFs were detached and washed once with PBS then incubated for 15 min on ice with a viability marker (Viobility™ 405/520 Fixable Dye,Miltenyi Biotec) according to manufacturer's instructions. Cells were then centrifuged at 300g for 5 min at 4°C and incubated for 20min on ice with anti-CD326 and anti-CD106 antibodies diluted in FACS buffer (PBS 1X, 0.5% BSA, 2mM EDTA) following the manufacturer's guidelines. Cells were then centrifuged at 300g for 5 min at 4°C and resuspended in FACS buffer. Data were acquired using Fortessa X20 (BD Biosciences, NJ, US) and analyzed with FlowJo version 10.

For CAFs panel : CAFs were washed once with PBS and detached using Stem Pro accutase cell dissociation reagent (Gibco A1110501). Following one wash with PBS, samples were resuspended in FACs buffer (0.5% BSA, 2mM EDTA in PBS) and stained with extracellular fluorochrome-coupled antibodies as listed: anti-FAP, anti-podoplanin, anti-CD29, anti-CD140b, anti-C5L2, anti-CD105, and anti-CD74. Intracellular staining using anti-alpha smooth muscle actin was performed following fixation and permeabilization with BD Cytofix/Cytoperm (BD 554714). Viability staining was performed using a fixable viability dye according to manufacturer's instructions . Samples were rewashed in FACs buffer once before being analyzed using a BD LSR-Fortessa-X20 Cell Analyzer.

Trichrome

Formalin-fixed, paraffin-embedded human sections (4 μm) were deparaffinized in xylene and rehydrated through a graded ethanol series. A Trichrome stain was performed using a kit (ab150686, abcam) following the manufacturer's instructions. Stained tissue sections were mounted using Pertex mounting medium (F/00811, Microm-Microtech, France) before being sequentially scanned at a ×10 magnification under a brightfield microscope (Zeiss).

Histological score

The histological score has been settled as follow. We analyzed 10 pictures per mice and counted the number of images with trichome staining. We scored the number of positive BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut doi: 10.1136/gutjnl2021326610 -14.

images such as 0: is no positive images, 1: less than 2 positive images, 2: less than 4 positive images, 3: less than 8 positive images and 4: between 8 and 10 positive images.

Electrophysiology

Electrophysiological recordings in HEK293-T, PANC-1 and MiaPaCa-2 cells were performed in the whole cell configuration of the patch clamp technique at room temperature. Currents were elicited using a 900 ms-voltage ramp protocol from -120 to 60 mV. Cells were bathed in a solution containing: 5 mM KCl, 145 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, and 10 mM Hepes (pH adjusted to 7.4 with HCl, 300 mosm/l). Soft glass patch electrodes (Brand, Wertheim, Germany) were made on a horizontal pipette puller (P-97, Sutter Instrument Co., Novato, CA) to achieve a final resistance ranging from 3 to 5 MΩ. The internal solution was: 145 mM KCl, 1 mM MgCl2, 0,87 mM CaCl2 (pCa 6) or 0,41 mM CaCl2 (pCa 7), 1 mM EGTA, 10 mM Hepes, 1 mM ATP (pH adjusted to 7.2 with KOH, 308 mosm/l). Recordings were performed using an PC-controlled EPC 9 patch-clamp amplifier (HEKA, Lambrecht/Pfalz, Germany). Currents were acquired and analyzed with Pulse and Pulsefit software (HEKA). Signal were filtered at 10 kHz and digitalized at 20 kHz. Currents were elicited by voltage ramps protocols (from -120 to +60 mV). Current density was measured at 0 mV. For the electrophysiological study of the SK2 mutations, HEK293-T were transfected with the different constructions and the HAmyr-AKT using calcium phosphate and recorded 24h after transfection. Dab-7-sensitive current (ISK2) recorded in PANC-1 cells results from the graphical subtraction of currents recorded before and after Dab-7 perfusion.

Gene expression analysis of human PDAC samples

Data analysis required pre-analytic processing as previously published 7 . SIGMAR1 expression (codes for Sig-R1) in tumors was measured as discrete value by using the median expression level as cut-off, thus defining two tumor classes thereafter designated "SIGMAR1-high" and "SIGMAR1-low". KCNN2 expression (codes for SK2) was similarly analysed. The molecular subtypes of tumors were determined by applying to each sample in each data set separately BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) ADEX subtype and the Collisson's Exocrine-like subtype were not considered as their existence is strongly questioned 11 . Correlations between the tumor classes based on these two genes and clinicopathological features were analysed using the t-test or the Fisher's exact test when appropriate: the variables tested included patients' age, gender, pathological tumor type and grade, pancreatic anatomic location, and American Joint Committee on Cancer (AJCC) stage.

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)