# Cancer-associated loss-of-function mutations in KCNQ1 enhance Wnt/β-catenin signalling disrupting epithelial homeostasis

Camille Berenguier, Xingyu Chen, Benoit Allegrini, Hélène Guizouarn, Franck Borgese, Catherine Etchebest, Olivier Soriani, Raphael Rapetti-Mauss

<figure><img src="image_1.png" /></figure>

► To cite this version:

Camille Berenguier, Xingyu Chen, Benoit Allegrini, Hélène Guizouarn, Franck Borgese, et al. Cancer-associated loss-of-function mutations in KCNQ1 enhance Wnt/β-catenin signalling disrupting epithelial homeostasis. Oncogene, In press, 10.1038/s41388-025-03447-4. hal-05087788

HAL Id: hal-05087788

https://hal.science/hal-05087788v1

Submitted on 28 May 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 Cancer-Associated Loss-of-Function Mutations in KCNQ1 Enhance Wnt/β-catenin

2 Signalling Disrupting Epithelial Homeostasis

3 **Authors:** Camille Berenguier¹, Xingyu Chen², Benoit Allegrini¹, Hélène Guizouarn¹, Franck Borgese¹, Catherine Etchebest², Olivier Soriani¹, Raphael Rapetti-Mauss¹*

5 ¹Université Côte d'azur, CNRS, Inserm, iBV, Nice, France

6 ²Université Paris Cité and Université de la Réunion, INSERM, BIGR, U1134, DSIMB

7 Bioinformatics Team, F-75015 Paris, France

8 *Corresponding author. Email: Raphael Rapetti-Mauss, [Raphael.Rapetti-Mauss@univ-cotedazur.fr](mailto:Raphael.Rapetti-Mauss@univ-cotedazur.fr)

9 [cotedazur.fr](http://cotedazur.fr)

10 **Conflict of interests:** The authors declare that they have no conflict of interest.
11 Abstract:

12 Ion channels are emerging as regulators of intracellular signalling pathway, yet the molecular
13 mechanisms underlying this role remain poorly understood. KCNQ1, a potassium channel with
14 tumour suppressor functions, restricts Wnt/β-catenin signalling, a pathway whose
15 dysregulation, often driven by protein-altering mutations, is a hallmark of several epithelial
16 cancers. Here, we identify loss-of-function (LOF) mutations in KCNQ1 across multiple epithelial
17 cancers and elucidate their impact on Wnt/β-catenin signalling. Our findings reveal that
18 cancer-associated KCNQ1-LOF mutations regulate the β-catenin pathway through a dual
19 mechanism. First, they drive β-catenin transcriptional activity through triggering MET
20 receptor, bypassing Frizzled/LRP6 receptor complex activation. Second, these mutations
21 suppress the expression of key negative regulators of Wnt signalling, such as DKK-1, Wif-1 and
22 NKD-1, leading to amplified pathway activation in response to Wnt ligand stimulation. This
23 dysregulation disrupts epithelial homeostasis, as demonstrated by impaired crypt
24 organization and increased proliferation in mouse colon-derived organoids. Together, these
25 findings uncover an original mechanism linking KCNQ1 dysfunction to aberrant Wnt/β-catenin
26 signalling, highlighting the role of ion channel in regulating epithelial signalling networks and
27 tissue homeostasis.
28 INTRODUCTION

The function of ion channels in cancers has been extensively studied, first by demonstrating that ion channels participate in cellular processes associated to cancer physiopathology such as cell migration, invasion, resistance to apoptosis, or gain of stem cell features[1]. More recently, progress has been made regarding the mechanistic events that associate ion channels to cancer biology. Particularly, it has been shown that ion channels can participate in the regulation of several cancer-related signalling pathways, including MEK/ERK[2], PI3K/AKT[3], STAT3-p53[4], β1-integrin/FAK[5] MAPK/AP-1 axis[6] and Wnt/β-catenin[7-9]. Nevertheless, the molecular mechanisms that are linking ion channels to the regulation of these signalling pathways are far from being understood. Especially the mutational landscape of these proteins in cancer biology and their impact on cell signalling pathway activity remains unexplored[10].

The Wnt/β-catenin pathway is a major developmental signalling pathway crucial for adult tissue homeostasis but a major driver in several cancers when hyperactivated[11]. This pathway is therefore tightly regulated by a number of different mechanisms, especially by negative regulators that prevent deleterious hyperactivation of the pathway. Several secreted factors directly interact with Wnt ligand such as Wif-1, sFRPs, and Notum, reducing the ability of Wnt ligands to interact with and activate Frizzled receptor[11]. Other secreted factors such as DKK-1 prevent Wnt-induced Fizzled/LRP6 complex formation[12]. Another regulatory system is constituted by the receptor LGR5, which when activated by its ligand R-spondin, inhibits the RNF43-ZNRF3 ubiquitinase system that regulates the membrane abundance of Frizzled[13, 14]. An additional example of this wide system of negative regulation is NKD-1
50 which inhibits the pathway via its interaction with Dishevelled and β-catenin and prevents β-catenin nuclear shuttling[15].
51

52 KCNQ1 is a voltage-dependent potassium channel, functional as a tetramer, the function of
53 which varies depending on the associated KCNE accessory subunits[16]. Paired with KCNE1, it
54 forms the IKs current crucial for cardiac repolarization[17]. With KCNE2, it recycles potassium
55 in stomach parietal cells to support H+ secretion[18]. In association with KCNE3 in epithelial
56 cells, it becomes a voltage-independent "leak" current, aiding potassium recycling and
57 membrane hyperpolarization[19-21]. Besides its functions in cardiac and epithelial physiology,
58 KCNQ1, more recently emerged as a tumour suppressor in several epithelial cancers including
59 colorectal cancer (CRC)[7, 22, 23], hepatocellular cancer[24], oesophageal cancer[25], renal
60 cell carcinoma[26] and uterine cancer[27]. Further studies in CRC report that a low expression
61 of KCNQ1 is associated with poor disease-free survival in stage II and stage III colorectal
cancer[22, 28]. Interestingly, There is an interplay between KCNQ1 and the Wnt/β-catenin
62 pathway[7]. Indeed, KCNQ1 restricts the activity of the Wnt/β-catenin pathway by retaining
63 β-catenin at the adherens junction, while the activation of the Wnt/β-catenin pathway leads
64 to the downregulation of the channel expression. Therefore, KCNQ1 functions as a negative
65 regulator of the Wnt pathway, thereby providing an additional layer of regulation.
66

67 In this study, we identified KCNQ1 cancer-associated mutation across several epithelial
cancers. We analysed 13 selected KCNQ1 cancer-associated mutations, most of which were
68 loss-of-function (LOF). Interestingly, we have shown that KCNQ1 LOF mutations promote
69 Wnt/β-catenin pathway activity independently of Wnt ligands and Fizzled/LRP6 receptor
70 complex activation; instead, mediating ligand-independent MET activation. This Wnt/β-
catenin overactivation impaired the ability of mouse colon-derived organoids to differentiate
71
72
73 into crypt domains. Additionally, KCNQ1 LOF mutations reduced Wnt negative regulators such
74 as DKK-1, Wif-1, and NKD-1, amplifying Wnt ligand stimulated pathway activation. These
75 findings provide further mechanistic insights into how an ion channel interacts with a major
76 intracellular pathway and point out the impact of ion channel mutations on intracellular
77 signalling pathway.

## 78 RESULTS

79 KCNQ1 mutations occur in several epithelial cancer types

80 We analysed publicly available mutational databases from patients with different types of
cancers, using cBioPortal, the International Cancer Genome Consortium (ICGC), and the
81 Catalogue of Somatic Mutations in Cancer (COSMIC). We identified 256 KCNQ1 mutations in
82 243 patients, predominantly in skin (26.2%) and colorectal cancers (24.2%), with stomach
83 (9%), liver (9.4%), lung (12.1%), uterine (12.5%), breast (5%), and ovarian (1.6%) cancers also
84 exhibiting mutations (Fig. 1B). Most KCNQ1 mutations were missense (82%), with frameshift
85 (12.5%) and stop-gain (4.7%) less common (Fig. 1C). The frameshift mutation K422Sfs*10 was
86 most frequent (Fig. 1D). Protein-altering KCNQ1 mutations occurred in colon (2.65%,
87 58/2183), gastric (1.7%, 23/1318), liver (1.4%, 22/1529), lung (1.8%, 31/1719), uterine (4.7%,
88 29/615), skin (4.2%, 63/1511), breast (0.8%, 13/1602), and ovarian cancers (0.7%, 4/585) (Sup.
89 Table1). To better understand the mutations, we mapped them onto the KCNQ1 structure[29],
90 showing their distribution across channel domains (Fig.1E). Among the most frequent
91 mutations A329T is located at helix S6 near the selectivity filter, in the pore-forming domain,
92 possibly destabilizing it, while A287T is at the S5-extracellular loop junction close to the filter.
93 Six mutations (R243C, V185M/A, E170K, G219E/M) appear in the voltage-sensing domain
94 (VSD), with R243C and E170K potentially disrupting VSD function via altered activation or
95
96 charge repulsion. F339L and P343S are near gating residues in S6, possibly affecting pore
97 opening. Cytoplasmic mutations include R518* (calmodulin-binding HB helix), G548C (HC-HD),
98 and nine mutations in the HA-HB loop (R401Gfs18/L/W/Pfs62, R451Q, P448R, K420fs,
99 K422Sfs*10/R), which remains unresolved due to its high flexibility. Mutations E170K, V185M,
100 R243C, A287T, A329T, F339L, P343S, P448R, R451Q, R401Gfs18, K422Sfs10, R518*, and G548C
101 were selected for electrophysiological studies.

102 Most cancer-associated mutations of KCNQ1 cause loss-of-function phenotype

103 Electrophysiological properties of selected KCNQ1 mutations were recorded in CHO-K1 cells
104 using patch-clamp in whole-cell configuration with KCNE3, an important KCNQ1 accessory
105 subunit in epithelial cells (Fig. 2A, B, C), or alone (Fig. S1A, B, C). Tested mutants expressed
106 with KCNE3 exhibited little to no current (Fig. 2A, B, C), except from A287T and G548C, which
107 matched wild-type currents. Mutations E170K, R243C, A329T, R401Gfs18, K422Sfs10, and
108 R518* showed no current, while V185M, F339L, P343S, P448R, and R451Q produced reduced
109 currents, indicating channel loss-of-function (LOF). Protein expression was confirmed by
110 Western blot (Fig. S1D). We selected the mutations with a low-amplitude current (threshold:
111 5 pA/pF at 0 mV) to study the apparent open probability, evaluated by measuring the tail
112 current amplitude at -120 mV[30]. Some mutations showed differences in the open
113 probability curve. The V1/2 of wild-type KCNQ1:KCNE3 is at -150 mV, where the channel is
114 nearly fully open (Po = 0.8-1) [31]. However, at -60 mV, which is close to the resting membrane
115 potential of colonic epithelial cells[32, 33], some mutations, including V185M, F339L, P448R,
116 and R451Q, exhibited a lower open probability (Fig. 2D). In patients, these mutations are likely
117 heterozygous, so we questioned whether co-expression with the wild-type channel showed a
118 dominant negative effect. We found that tested LOF mutations prevailed over the wild-type
119 channel, resulting in low or no current (Fig. 2E). Overall, out of the 13 most frequent KCNQ1
120 mutations, only two (A287T and G548C) were silent, while the other 11 caused LOF with
121 KCNE3. We selected the frequent A329T mutation to study its impact on channel structure
122 using molecular dynamics simulations. No major structural changes or significant differences
123 in pore profiles were observed compared to the wild type (Figs. S2, S3). However, differences
124 in residue pair behaviour in the cross-correlation matrix (DCCM) suggested altered channel
125 dynamics, particularly involving residues in the voltage sensor domain (S4) and distant regions
126 (Fig. S4). Ion transport analysis showed that ions in the cavity reached the selectivity filter
127 more often in wild-type channels than in A329T mutants (Fig. S5). These findings indicate that
128 A329T impairs channel dynamics and potassium ion transport, contributing to the channel's
129 loss-of-function.

130
131 **LOF cancer-associated mutations in KCNQ1 promote Wnt pathway activity and impaired**
**epithelium homeostasis**

132 Given the dominant negative effect of the mutations on the wild-type channel, we expressed
133 the three types of mutations—missense (A329T), frameshift (K422Sfs*10), and stop-gain
134 (R518*)—in HT29cl19A, a colorectal cell line derived from HT29 with endogenous KCNQ1
135 expression and epithelial features[34]. Expression of mutated channels was confirmed by
136 western blot (Fig. S6), and activity by patch clamping (Figs. 3A, B, C). To explore the broader
137 effects of LOF mutations, we performed RNA sequencing (RNA-seq) and analysed differentially
138 expressed genes for functional module enrichment using Amine[35] and String software[36].
139 Consistent with our hypothesis, the Wnt/β-catenin pathway was among the ten most
140 dysregulated processes with all three LOF mutations (Fig. 3D), illustrated by the heatmap of
141 the top 15 differentially expressed Wnt-related genes significantly up- or down-regulated in
142 KCNQ1-LOF mutants (Fig. 3E). Using the muTarget[37] tool and TCGA datasets, we analysed
gene expression in colon adenocarcinoma samples (396 patients) to identify altered biological
processes. Comparing patients with KCNQ1 mutations (n=10) to those without (n=386), we
found the Wnt/β-catenin signalling pathway among the most modulated processes (Fig. 3F,
G), confirming our findings in HT29cl19A. A volcano plot highlights the differentially expressed
genes between these groups (Fig. 3H). These results suggest that KCNQ1 loss-of-function
mutations disrupt the Wnt/β-catenin pathway.

149 To further analyse the effect of KCNQ1-LOF mutations on the Wnt/β-catenin pathway we used
a 7 repeat TCF/LEF-eGFP[38] reporter (Fig.4A) to monitor the activity of the pathway in
150 HT29cl19A. Cells expressing the A329T, K422Sfs*10, or R518* mutations exhibited a three-
151 fold increase in mean fluorescence of eGFP compared to WT control (Fig.4B). The levels of
152 active β-catenin—meaning β-catenin that is not phosphorylated by CK1 and GSK3 for
153 degradation—were also elevated by at least two-fold in HT29cl19A expressing KCNQ1
154 mutants (Fig. 4C). We then quantified the protein expression of a set of canonical Wnt target
155 genes: Axin2, CD44, cyclin D1 and Claudin-1, that was increased in the KCNQ1 mutation-
156 expressing cells, providing further evidence of an activated Wnt/β-catenin signalling pathway
157 (Fig. 4D). Consistently, results from the RNA-seq showed that several Wnt target genes were
158 increased in the KCNQ1-LOF mutant cells compared to control conditions including MYC,
159 CD44, CLDN-1, LGR5, and MET (Fig. 4E). Using the 7TGC reporter in a colon organoid model
160 established from isolated mouse colonic crypts, we confirmed that the A329T mutation
161 increased TCF activity (Fig. 4F), demonstrating heightened Wnt pathway activation.
162 Additionally, a Top/Fop assay in CHO-K1 cells treated with Wnt3A-conditioned medium
163
showed that KCNQ1-LOF mutations enhanced Wnt/β-catenin signalling in a simplified system (Fig. S7).

Using colon organoid, we assessed the impact of KCNQ1-LOF mutations on colonic epithelial homeostasis. Mutant organoids were significantly larger than controls, with more exceeding 100 µm in diameter (Fig. 4G) and an increase in Ki67+ cells (Fig. 4H), indicating enhanced proliferation. This aligns with findings by Cui et al., who demonstrated that increased Wnt/β-catenin activity drives larger organoid size through enhanced proliferation[39]. Notably, in Wnt3A-deprived conditions (ENR), KCNQ1-LOF organoids showed reduced crypt formation and fewer crypts per organoid (Figs. 4I, J), suggesting impaired differentiation. These findings suggest that KCNQ1-LOF mutations disrupt the balance between proliferation and differentiation in colonic epithelial cells, contributing to altered tissue homeostasis.

**LOF cancer-associated mutations of KCNQ1 activate β-catenin pathway independently of FZD/LRP6 activation.**

To explore the mechanism underlying KCNQ1-LOF-induced Wnt/β-catenin pathway activation, we examined the role of the Frizzled/LRP6 complex. Cells expressing WT or LOF-mutated KCNQ1 were treated with niclosamide (5 µM), a Frizzled inhibitor, which reduced Frizzled membrane density[40]. CD44 and Axin2 protein levels remained elevated in KCNQ1-mutated cells compared to WT, regardless of treatment (Fig. 5A). Additionally, KCNQ1 mutations did not affect LRP6 phosphorylation (Fig. 5B), indicating that the Wnt signalosome is not overactivated by KCNQ1-LOF mutations. Since active β-catenin levels and Wnt target gene expression increased with the mutations, we investigated β-catenin involvement. HT29cl19A were treated with JW67, an activator of the Axin degradasome[41] (Fig. 5C). If the
186 increased pathway activity were mediated by β-catenin, enhancing its degradation would
187 negate the effects. Indeed, JW67 treatment reduced CD44 and Axin2 expression to WT-like
188 levels in KCNQ1-LOF cells (Fig. 5D), showing that target gene transcription depends primarily
189 on β-catenin. In control cells, JW67 had little effect, suggesting β-catenin shifts from
190 degradation in controls to active signalling in the KCNQ1-LOF context. We thus assessed β-
191 catenin phosphorylation and found decreased levels at Ser33, Ser37, and Thr41, which signal
192 proteasomal degradation, consistent with JW67 results. Conversely, phosphorylation
193 increased at Tyr654 (detaching β-catenin from E-cadherin) [42], Ser675 (promoting nuclear
194 translocation) [43], and Tyr142 (dissociating β-catenin from α-catenin) [44] (Fig. 5E). This
195 suggests reduced degradation and dissociation of β-catenin from adherens junctions in the
196 KCNQ1-LOF context. Proximity ligation assays confirmed reduced interaction between β-
197 catenin and E-cadherin when LOF mutations were present (Fig. 5F). These findings show that
198 KCNQ1-LOF mutations activate the Wnt/β-catenin pathway independently of Frizzled/LRP6
199 but requires β-catenin signalling.

200 LOF cancer-associated mutations of KCNQ1 activate β-catenin pathway through MET
201 receptor activity

202 Receptor Tyrosine Kinases (RTKs) can phosphorylate β-catenin at Tyr654 and Tyr142 residues
203 directly or via the activation of soluble kinases SRC and AKT[42, 45, 46] enhancing pathway
204 activity independently of Wnt/Frizzled/LRP6. Using a phospho-proteome kit, we found
205 increased phosphorylation of MET and ErbB3 in KCNQ1-LOF mutations (Fig. 6A), confirmed by
206 western blot for MET (Fig. 6B) across all three mutations. Interestingly MET has been found to
207 interact with the Wnt/β-catenin pathway[47-49], therefore we have focused on this RTK. To
208 test MET role in Wnt/β-catenin activation, we used the MET inhibitor Crizotinib, which
209 abolished the effects of KCNQ1-LOF mutations on CD44 expression (Fig. 6C), Tyr654
210 phosphorylation (Fig. 6D), TCF activity (Fig. 6E), and organoid size (Fig. 6F). Bapta-AM, a Ca²⁺
211 chelator, did not counteract these effects on the expression of CD44 and the activity of TCF
212 (Figs. S8A, S8B), ruling out the involvement of an increase in intracellular calcium
213 concentration in this process. These data confirm that MET mediates KCNQ1-LOF-induced
214 Wnt/β-catenin pathway activation.

215 LOF mutations in KCNQ1 diminish negative regulation of Wnt signalling and enhance ligand-activated Wnt signalling
216

217 In RNA-seq analyses of HT29cl19A cells and TCGA patient data, negative regulators of the Wnt
218 pathway were mainly downregulated, while most positive regulators were upregulated (Fig.
219 7A, 7B). Protein-level validation showed that the Wnt negative regulators, DKK-1, Wif-1, and
220 NKD-1, were decreased in KCNQ1-LOF mutants compared to controls (Fig. 7C). Thus, we
221 hypothesized that KCNQ1 may reduce ligand-activated Wnt signalling via the expression of
222 negative regulators. Testing this, Wnt3A-conditioned medium (Wnt3A-CM) treatment of wild-
223 type HT29cl19A cells caused no change in Wnt pathway activity, as assessed by a TCF-eGFP
224 reporter assay. In contrast, KCNQ1-LOF mutants showed increased Wnt activity under the
225 same treatment conditions (Fig. 7D). This increase, compared to control condition, was
226 confirmed by enhanced phosphorylation of LRP6, in mutant cells treated with Wnt3A-CM (Fig.
227 7E). Further, adding recombinant DKK-1 and Wif-1 to Wnt3A-CM-treated cells restored the
228 WT phenotype, demonstrating that the loss of negative regulators underlies the
229 overactivation of the Wnt/β-catenin pathway in KCNQ1-LOF mutants (Fig. 7F). These findings
230 suggest that KCNQ1-LOF mutations promote ligand-dependent Wnt signalling by reducing
231 negative regulator expression, driving pathway hyperactivation.
232

DISCUSSION

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

Ion channels play an important role in the biology of cancers and recent work have demonstrated that these transmembrane proteins participate in regulating cancer-related intracellular signalling pathways. However, an important open question is whether mutations carried by ion channels are present in patients with cancer and more importantly whether these mutations can impact the activity of cancer-related signalling pathway. Our data show that mutations of KCNQ1 are present in patients suffering from different types of cancers and that these mutations are mostly loss of function. Interestingly the expression of LOF mutations of KCNQ1 favours the activity of the Wnt/β-catenin pathway. We have chosen to focus our study on colonic tissue, where KCNQ1 is associated with KCNE3, as the interplay between KCNQ1, Wnt/β-catenin pathway and Colon cancer has been reported. However, KCNQ1 in association with KCNE3 or KCNE2 have been reported in other epithelia such as lung[50], mammary[51], thyroid[52] and choroid plexus[53] tissue. Interestingly KCNE2 have been associated with neoplasia in stomach[54] and we have identified mutation in KCNQ1 in this type of cancer suggesting that KCNQ1/KCNE2 complex could have a role in stomach cancer physiopathology. Therefore, the function of the KCNQ1 mutations depicted in this study might be extrapolated to other epithelia. We identified several mutations on KCNQ1 in colorectal cancer, with a low frequency of about 3% of patients that was similar in other types of cancer. In CRC, 80% of patients carry mutations on APC, but other components of the pathway are also mutated with a lower frequency such as β-catenin (5%), Axin1 (6%) or RNF43 (10%) approaching the rate of KCNQ1 mutations[55].

Even if driver mutations have been largely identified and studied in cancers, the functional role of passenger mutations has emerged in the last years. Driver mutations are defined as
those, among all the genetic alterations accumulated in cancer cells, that promote initiation
and progression of the oncogenic process and are generally high-frequency mutations.
Passenger mutations have been initially defined as mutations occurring along with driver
mutations but without functional effect and present in cancer patients with a low
frequency [56]. However, several recent studies have shown that low-frequency passenger
mutations can actually have an important role in oncogenesis and cancer physiopathology.
This is the case for DOT1L which Gain-of-function mutations are found in about 3% of lung
adenocarcinomas and have been shown to promote malignancy through the activation of
MAPK/ERK pathway [57]. Gain of function mutations in G9a have been found in melanoma and
contribute to cancer progression through the activation of the Wnt pathway [58]. Finally,
CDK13 is mutated in about 4% of melanomas and contribute to oncogenesis by inducing a
deficient RNA surveillance [59]. This redefines the role of low-frequency mutations in cancer
biology. Here we show that KCNQ1-LOF mutations promote Wnt/β-catenin activity, one of the
main cancer-driver pathways, and impair epithelial homeostasis, suggesting that KCNQ1-LOF
mutations may participate in the oncogenic processes. From a diagnostic and prognostic point
of view, low-frequency mutations can be used to finely classify subtypes of cancer
patients [60]. Expression of KCNQ1 has been shown to be a prognostic marker for CRC
recurrence [22, 28]. KCNQ1 mutational status could also be used as a prognostic marker for
patients with CRC or other cancers.

Only few studies report cancer patients harbouring mutations on ion channels[61-64], and
there is no study that functionally link these mutations to a change in cancer-related signalling
pathway or an impact on cancer physiopathology. Here we have found that most tested
somatic mutations of KCNQ1 identified in cancer lead to a loss-of-function and result in an
increased in Wnt/β-catenin pathway activity. This is consistent with our previously published
279 data showing the interplay between KCNQ1 and the Wnt pathway, where KCNQ1 acts as a
280 negative regulator of the pathway [7].

281 Our findings highlight the broader implications of KCNQ1-LOF mutations for epithelial tissue
282 integrity. Using organoid models derived from mouse colonic crypts, we demonstrate that
283 KCNQ1-LOF mutations promote hyperproliferation, as evidenced by larger organoid sizes and
284 increased Ki67+ staining. Moreover, these mutations also impair differentiation and crypt
285 domain formation, in Wnt3A-deprived conditions, similarly to what can be observed with APC
286 loss-of-function [65]. This phenotype aligns with the consequences of dysregulated Wnt/β-
287 catenin signalling. These observations underscore the importance of KCNQ1 in maintaining
288 the delicate balance between proliferation and differentiation in epithelial tissues, a balance
289 that is disrupted when KCNQ1 function is lost.

290 Another unanswered question in the field is the molecular mechanisms involved in ion
291 channel-mediated regulation of intracellular signalling pathway. Interestingly, here we have
292 revealed that KCNQ1-LOF-mediated activation of β-catenin activity is independent on the
293 classical Frizzled/LRP6 receptors and occurs in the absence of Wnt ligands; but does involve
294 β-catenin. β-catenin is released from the adherens junctions, its phosphorylation on Ser33
295 residue -which promotes degradation- is decreased, and its phosphorylation on Tyr654,
296 Tyr142 and Ser675 residue—which promote signalling—are increased in the KCNQ1-LOF
297 conditions. An alternative path to induce the transcriptional activation of the Wnt/β-catenin
298 pathway is to target β-catenin phosphorylation through the activation of RTKs [66]. Among the
299 RTKs, MET has been described to activate this pathway by directly phosphorylating β-catenin
300 on both Tyr654 [67], Tyr142 [44, 68] residues. Our data demonstrate that the inhibition of MET
301 completely reverses the phenotype induced by the expression of KCNQ1-LOF mutations on
302 Wnt/β-catenin activation, thus demonstrating that these mutations trigger the activation of
303 MET. These results raise the complex question of how an ion channel loss-of-function could
304 lead to the activation of a membrane associated receptor and this, independently of
305 intracellular calcium increase. Zhou et al. have demonstrated that membrane depolarization
306 induced rearrangement of membrane phospholipids supporting the clustering of K-Ras which
307 leads to the activation of downstream signalling[69]. We can speculate that a similar
308 mechanism could link the loss-of-function of KCNQ1 to the activation of MET, but further
309 investigations are needed to conclude.

310 Interestingly, our results also show that in cells expressing WT KCNQ1, Wnt3A treatment failed
311 to activate the Wnt/β-catenin pathway after 30 hours. However, in the same condition cell
312 expressing LOF mutation of KCNQ1 showed an activation of the pathway. We have shown that
313 this difference in the ability of Wnt3A to activate the pathway resides in the levels of Wnt
314 negative regulators including DKK-1, NKD-1, and WIF-1 which are decreased in LOF mutants.
315 These data suggest a complex role for KCNQ1, which may influence the negative feedback
316 loop of the Wnt pathway or regulate other pathways that control the expression of negative
317 regulators. For instance, WIF-1 has been described as a Smad-1 target gene in lung epithelium,
318 retaining Wnt/β-catenin signalling when expressed[70], illustrating the intricate regulation of
319 Wnt negative regulator expression.

320 These results suggest that KCNQ1-LOF mutations have a dual effect. They directly activate the
321 β-catenin pathway through MET activation while also decreasing the expression of negative
322 regulators, creating a permissive state for further activation by Wnt ligands. KCNQ1 thus
323 prevents against Wnt/β-catenin overactivation through both a direct effect via restricting MET
324 activity and an indirect effect by maintaining high levels of negative regulators (Fig. 8).
In conclusion, this study provides key mechanistic insights into how ion channels regulate Wnt/β-catenin signalling and highlights a new avenue of research exploring the connection between cancer-related signalling pathways and ion channel mutations. These proteins, along with their cancer-associated mutations, hold promise as valuable tools for cancer diagnosis and prognosis, emphasizing the importance of further investigation.

## MATERIALS AND METHODS

For a more detailed description of the materials and methods used in this study, please refer to the supplementary information file.

### Cell lines culture

HT29CI19A cells were obtained from Dr. Christian Laboisse[34], cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated Fetal Bovine Serum and 50 U/mL Penicillin/Streptomycin (Gibco). The HEK-293T, and CHO-K1 cell lines were purchased from ATCC (RRID: CVCL_0214). HEK-293T (RRID: CVCL_0063) were maintained in DMEM supplemented with 10% FBS and 50 U/mL Penicillin/Streptomycin. CHO-K1 cell line was maintained in DMEM/F-12 (Gibco) supplemented with 10% FBS and 50 U/mL Penicillin/Streptomycin. Cells were grown in a humidified atmosphere containing 5% CO2 at 37°C and the medium was replaced every 2 to 3 days. Cells were checked for mycoplasma contamination every 2 months.

### Organoid medium preparation

Wnt3A conditioned medium was harvested as follows: L-Cells producing Wnt3A 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. R>spondin conditioned medium was harvested in the same conditions from R-spondin-producing HEK-293T cells, except that the selection of cells was done with 3 µg/mL Zeocin. R-spondin-producing HEK-293T cells have been purchased from Sigma-Aldrich and L-Cells producing Wnt3A were a kind gift from Dr. Hans Clevers.

### Organoid Culture

Colonic crypts from 6-8-week-old male C57BL/6J wild-type mice were obtained post-mortem from animals used in prior ethically approved studies at our institute. All work complied with the guidelines of the local animal ethics committee (Comité Institutionnel d'Éthique Pour l'Animal de Laboratoire - CIEPAL-Azur) for the reuse of animal materials in research. Only male was used as KCNQ1 expression is variable over oestrus cycle in female[71]. Freshly dissected male mouse colon was everted on a glass rod, washed in PBS supplemented with 10mM glucose and 50 U/mL Penicillin/Streptomycin (PBS++) and then placed in 10 mL crypt isolation buffer (NaCl 96mM; KCl 1.5mM; EDTA 27 mM; Sorbitol 55mM; HEPES 10mM; Tris 10mM; Sucrose 44mM; pH7.4) 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 PBS++. After three washes in PBS++ (1000 x g; 10min), isolated crypts were resuspended in Advanced-DMEM/F-12 (Gibco) supplemented with Hepes 10mM, Glutamax 1x (Gibco) and 50 U/mL Penicillin/Streptomycin. Intact crypts were counted and resuspended in the proper amount of Growth Factor Reduced Matrigel (Corning 356231) to seed 1000 crypts per 40 µL GFR-Matrigel drop. The matrigel drops containing the crypts in a 24-well plate were left to polymerise upside down for 20 min
at 37°C. 500 µL of WENR medium containing Primocin (100 µg/mL Invivogene) 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. WENR medium was composed as follow: Wnt3a-CM 50%, EGF 50 ng/ml (PreproTech AF-100-15), Noggin 5ng/ml (PreproTech 250-38), Rspondin-CM 20%, N-acetyl-l-cysteine 1 mM (Sigma-Aldrich A9165), B27 supplement 1X (Life technologies 17504-044 50X), N2 supplement 1X (Life technologies 17502-048 100X). Colon organoids were passaged every 7 to 10 days. For each subsequent passages Martigel drops were dissociated with cool Advanced-DMEM/F-12, then organoids were harvested into single cells using TrypLE (ThermoFisher, 12605036). 1 µM Y27632- dihydrochloride (TargetMol; T1725) was added to prevent anoïkis. To reach organoid crypts formation WENR medium was replaced after 4 days by ENR medium (same composition as WENR but Wnt3a-CM was omitted).

## Whole-Exome sequencing Datasets

Cancer-associated mutation of KCNQ1 were obtained from publicly available Whole-Exome sequencing datasets. Data were obtained from TCGA, ICGC, and several publications listed in Sup table1. Only nonsynonymous mutations have been taken into account.

## 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 10x magnification (RRID:SCR_002368). Diameters were measured using Image J software (RRID:SCR_003070).

## Wnt reporter assay 7TGC

7TGC was a gift from Roel Nusse (Addgene plasmid # 24304 ; http://n2t.net/addgene:24304 ; RRID:Addgene_24304)[38]. 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 µg of 7TGC plasmid, 9 µg P89.1 and 2 µg pMD2G plasmids. P891 and pMD2G were kind gifts from Dr. Zeinab Rekad[72]. Viral supernatant was collected after 48 hours, filtered through sterile 0.45 µm filters, then added directly to HT29cl19A cells or Organoids. 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 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 (vector Laboratories. Inc. H-1800). 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.

## Immunofluorescence

Immunofluorescence staining protocol for organoids has been adapted from Bergdorf et al[73]. Briefly, organoids have been fixed in 4% PFA for 1h at RT and then after 3 washes in TBS 0.1% triton 20x, permeabilized for 30 min at RT in TBS 0.5% triton 20x in suspension. Organoids have been then blocked in 3% BSA, TBS 0.1% triton 20x solution for 1h at RT. Organoids were then incubated with primary antibody (Ki67 SC-D3B5 1:500) overnight at 4°C. Secondary antibody (Alexa 488 1:3000) has been added after 3 washes (5min, 2500 g, RT) for 1h at RT. After 3 final washes Organoids were mounted in Vectashield DAPI Vibrance® and then observed under a confocal microscope (Zeiss 710 or 880).

## Statistical analysis
Statistical analysis was performed on the software GraphPad Prism (version 10; RRID:SCR_002798). 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. Outlier values were excluded according to the outlier identification test (GraphPad Prism). Quantitative data were presented as median with interquartile range. A formal power analysis was not conducted. Sample sizes were chosen based on previous studies and preliminary results obtained from triplicate experiments. Statistical analysis of bulk results for RNA-Seq were performed by the service provider. Gene Ontology classification and associated statistics were given by the software String.

## Acknowledgments:

We thank the Prism imaging facility of iBV: Baptiste Monterroso and Sameh Ben Aicha for technical support. We thank Dr. Stephan Clavel (IPMC-Valbonne, France) and Dr Victor Tiroille (C3M-Nice, France) for their help with Organoids culture. This work was granted access to the HPC resources of IDRIS under the allocation 2024-A0160714918 made by GENCI

## Funding: The work described in this paper was supported by research fundings from:

* National Institute of Cancer – INCA grant PLBIO INCA_16710 (RRM)

* Fondation ARC grant PJA 2018 207701 (RRM),

* Canceropole PACA, INCA, Region Sud grant 2018-13 (RRM)

* Université Côte d'Azur CSI: Crédits Scientifiques Incitatifs (RRM)

* Agence Nationale pour la Recherche ANR grant ANR19-14-0049-01 (HG)

* Fondation pour la Recherche Medicale FRM PhD grant (CB)
439 • Fondation ARC Fourth year PhD grant (CB)

440 Author contributions:

441 • Conceptualization: RRM, CB

442 • Data Curation: RRM, CB, XC, CE

443 • Formal analysis: RRM, CB, XE, CE

444 • Funding acquisition: RRM, HG

445 • Projet administration: RRM

446 • Validation: RRM

447 • Methodology: RRM, CB, HG, FB, XE, CE, OS

448 • Investigation: RRM, CB, HG, FB, BA, XE, CE,

449 • Visualization: RRM, CB

450 • Supervision: RRM

451 • Writing—original draft: RRM, CB

452 • Writing—review & editing: RRM, CB, HG, FB, BA, XE, CE, OS

453 **Conflict of interests:** The authors declare that they have no conflict of interest.

454 **Data and materials availability:** The datasets generated during and/or analysed during the
current study are available from the corresponding author on reasonable request. The RNA
sequencing data have been deposited to Figshare under the accession number :

457 10.6084/m9.fishare.28420820

458

459 References

460

461 1 Prevarskaya N, Skryma R, Shuba Y. Ion Channels in Cancer: Are Cancer Hallmarks

462 Oncochannelopathies? *Physiol Rev* 2018; 98: 559-621.
<table>
  <tr>
    <td>463</td>
    <td>2</td>
    <td>Ji CD, Wang YX, Xiang DF, Liu Q, Zhou ZH, Qian F et al. Kir2.1 Interaction with Stk38 Promotes Invasion and Metastasis of Human Gastric Cancer by Enhancing MEKK2-MEK1/2-ERK1/2 Signaling. Cancer Res 2018; 78: 3041-3053.</td>
  </tr>
  <tr>
    <td>464</td>
    <td>465</td>
    <td></td>
  </tr>
  <tr>
    <td>466</td>
    <td>467</td>
    <td></td>
  </tr>
  <tr>
    <td>468</td>
    <td>469</td>
    <td>Raphael Rapetti-Mauss, Jérémy Nigri, Camille Berenguier, Pascal Finetti, Sarah Simha Tubiana, Bonnie Labrum et al. SK2 channels set a signalling hub bolstering CAF-triggered tumourigenic processes in pancreatic cancer. Gut 2022.</td>
  </tr>
  <tr>
    <td>470</td>
    <td>471</td>
    <td></td>
  </tr>
  <tr>
    <td>472</td>
    <td>473</td>
    <td>Prosdocimi E, Carpanese V, Todesca LM, Varanita T, Bachmann M, Festa M et al. BioID-based intact cell interactome of the Kv1.3 potassium channel identifies a Kv1.3-STAT3-p53 cellular signaling pathway. Sci Adv 2024; 10: eadn9361.</td>
  </tr>
  <tr>
    <td>474</td>
    <td>475</td>
    <td></td>
  </tr>
  <tr>
    <td>476</td>
    <td>477</td>
    <td>Becchetti A, Crescioli S, Zanieri F, Petroni G, Mercatelli R, Coppola S et al. The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression. Sci Signal 2017; 10.</td>
  </tr>
  <tr>
    <td>478</td>
    <td>479</td>
    <td></td>
  </tr>
  <tr>
    <td>480</td>
    <td>481</td>
    <td>Scarth JA, Wasson CW, Patterson MR, Evans D, Barba-Moreno D, Carden H et al. Exploitation of ATP-sensitive potassium ion (K. Oncogene 2023; 42: 2558-2577.</td>
  </tr>
  <tr>
    <td>482</td>
    <td>483</td>
    <td></td>
  </tr>
  <tr>
    <td>483</td>
    <td>484</td>
    <td>Rapetti-Mauss R, Bustos V, Thomas W, McBryan J, Harvey H, Lajczak N et al. Bidirectional KCNQ1:β-catenin interaction drives colorectal cancer cell differentiation. Proc Natl Acad Sci U S A 2017; 114: 4159-4164.</td>
  </tr>
  <tr>
    <td>485</td>
    <td>486</td>
    <td></td>
  </tr>
  <tr>
    <td>486</td>
    <td>487</td>
    <td>Rapetti-Mauss R, Berenguier C, Allegrini B, Soriani O. Interplay Between Ion Channels and the Wnt/β-Catenin Signaling Pathway in Cancers. Front Pharmacol 2020; 11: 525020.</td>
  </tr>
  <tr>
    <td>487</td>
    <td>488</td>
    <td></td>
  </tr>
  <tr>
    <td>488</td>
    <td>489</td>
    <td></td>
  </tr>
  <tr>
    <td>490</td>
    <td>491</td>
    <td>Breuer EK, Fukushiro-Lopes D, Dalheim A, Burnette M, Zartman J, Kaja S et al. Potassium channel activity controls breast cancer metastasis by affecting β-catenin signaling. Cell Death Dis 2019; 10: 180.</td>
  </tr>
  <tr>
    <td>492</td>
    <td>493</td>
    <td></td>
  </tr>
  <tr>
    <td>493</td>
    <td>494</td>
    <td>Huang X, Jan LY. Targeting potassium channels in cancer. J Cell Biol 2014; 206: 151-162.</td>
  </tr>
  <tr>
    <td>495</td>
    <td>496</td>
    <td></td>
  </tr>
  <tr>
    <td>496</td>
    <td>497</td>
    <td>Nusse R, Clevers H. Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell 2017; 169: 985-999.</td>
  </tr>
  <tr>
    <td>498</td>
    <td>499</td>
    <td></td>
  </tr>
  <tr>
    <td>499</td>
    <td>500</td>
    <td>Semënov MV, Tamai K, Brott BK, Kühl M, Sokol S, He X. Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr Biol 2001; 11: 951-961.</td>
  </tr>
  <tr>
    <td>501</td>
    <td>502</td>
    <td></td>
  </tr>
  <tr>
    <td>502</td>
    <td>503</td>
    <td>Koo BK, Spit M, Jordens I, Low TY, Stange DE, van de Wetering M et al. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 2012; 488: 665-669.</td>
  </tr>
  <tr>
    <td>504</td>
    <td>505</td>
    <td></td>
  </tr>
  <tr>
    <td>505</td>
    <td>506</td>
    <td>Hao HX, Xie Y, Zhang Y, Charlat O, Oster E, Avello M et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 2012; 485: 195-200.</td>
  </tr>
  <tr>
    <td>507</td>
    <td>508</td>
    <td></td>
  </tr>
  <tr>
    <td>508</td>
    <td>509</td>
    <td>Van Raay TJ, Fortino NJ, Miller BW, Ma H, Lau G, Li C et al. Naked1 antagonizes Wnt signaling by preventing nuclear accumulation of β-catenin. PLoS One 2011; 6: e18650.</td>
  </tr>
  <tr>
    <td>510</td>
    <td>511</td>
    <td></td>
  </tr>
  <tr>
    <td>511</td>
    <td>512</td>
    <td>Abbott GW. Biology of the KCNQ1 Potassium Channel. New Journal of Science 2014; 2014(Article ID 237431): 26.</td>
  </tr>
  <tr>
    <td>513</td>
    <td>514</td>
    <td></td>
  </tr>
  <tr>
    <td>514</td>
    <td>515</td>
    <td>Barhanin J, Lesage F, Guillemare E, Fink M, Lazdunski M, Romey G. K(V)LQT1 and IsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature 1996; 384: 78-80.</td>
  </tr>
  <tr>
    <td>516</td>
    <td>517</td>
    <td></td>
  </tr>
  <tr>
    <td>517</td>
    <td>518</td>
    <td>Grahammer F, Herling AW, Lang HJ, Schmitt-Gräff A, Wittekindt OH, Nitschke R et al. The cardiac K+ channel KCNQ1 is essential for gastric acid secretion. Gastroenterology 2001; 120: 1363-1371.</td>
  </tr>
  <tr>
    <td>519</td>
    <td>520</td>
    <td></td>
  </tr>
  <tr>
    <td>520</td>
    <td>521</td>
    <td>Schroeder BC, Waldegger S, Fehr S, Bleich M, Warth R, Greger R et al. A constitutively open potassium channel formed by KCNQ1 and KCNE3. Nature 2000; 403: 196-199.</td>
  </tr>
  <tr>
    <td>522</td>
    <td>522</td>
    <td></td>
  </tr>
</table>
523 20 Preston P, Wartosch L, Günzel D, Fromm M, Kongsuphol P, Ousingsawat *J et al.* Disruption of the K+ channel beta-subunit KCNE3 reveals an important role in intestinal and tracheal Cl- transport. *J Biol Chem* 2010; 285: 7165-7175.

526
527 Julio-Kalajzić F, Villanueva S, Burgos J, Ojeda M, Cid LP, Jentsch TJ *et al.* K. *J Physiol* 2018; 596: 393-407.

529
530 den Uil SH, Coupé VM, Linnekamp JF, van den Broek E, Goos JA, Delis-van Diemen PM *et al.* Loss of KCNQ1 expression in stage II and stage III colon cancer is a strong prognostic factor for disease recurrence. *Br J Cancer* 2016; 115: 1565-1574.

533
534 Than BL, Goos JA, Sarver AL, O'Sullivan MG, Rod A, Starr TK *et al.* The role of KCNQ1 in mouse and human gastrointestinal cancers. *Oncogene* 2014; 33: 3861-3868.

536
537 Fan H, Zhang M, Liu W. Hypermethylated KCNQ1 acts as a tumor suppressor in hepatocellular carcinoma. *Biochem Biophys Res Commun* 2018; 503: 3100-3107.

539
540 Chen Y, Wang H, Zhu S, Lan X. miR-483-5p promotes esophageal cancer progression by targeting KCNQ1. *Biochem Biophys Res Commun* 2020; 531: 615-621.

542
543 Huang C, Li J, Zhang X, Xiong T, Ye J, Yu J *et al.* The miR-140-5p/KLF9/KCNQ1 axis promotes the progression of renal cell carcinoma. *FASEB J* 2020.

545
546 Gu C, Yang H, Chang K, Zhang B, Xie F, Ye J *et al.* Melatonin alleviates progression of uterine endometrial cancer by suppressing estrogen/ubiquitin C/SDHB-mediated succinate accumulation. *Cancer Lett* 2020; 476: 34-47.

549
550 Uil SH, Coupé VMH, Bril H, Meijer GA, Fijneman RJA, Stockmann HBAC. KCNQ1 and lymphovascular invasion are key features in a prognostic classifier for stage II and III colon cancer. *BMC Cancer* 2022; 22: 372.

553
554 Sun J, MacKinnon R. Structural Basis of Human KCNQ1 Modulation and Gating. *Cell* 2020; 180: 340-347.e349.

556
557 Pusch M, Steinmeyer K, Koch MC, Jentsch TJ. Mutations in dominant human myotonia congenita drastically alter the voltage dependence of the CIC-1 chloride channel. *Neuron* 1995; 15: 1455-1463.

559
560 Barro-Soria R, Perez ME, Larsson HP. KCNE3 acts by promoting voltage sensor activation in KCNQ1. *Proc Natl Acad Sci U S A* 2015; 112: E7286-7292.

562
563 Klaerke DA, Wiener H, Zeuthen T, Jørgensen PL. Ca2+ activation and pH dependence of a maxi K+ channel from rabbit distal colon epithelium. *J Membr Biol* 1993; 136: 9-21.

565
566 Burckhardt BC, Gögelein H. Small and maxi K+ channels in the basolateral membrane of isolated crypts from rat distal colon: single-channel and slow whole-cell recordings. *Pflugers Arch* 1992; 420: 54-60.

569
570 Augeron C, Laboisse CL. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. *Cancer Res* 1984; 44: 3961-3969.

572
573 Pasquier C, Guerlais V, Pallez D, Rapetti-Mauss R, Soriani O. A network embedding approach to identify active modules in biological interaction networks. *Life Sci Alliance* 2023; 6.

575
576 Szklarczyk D, Kirsch R, Koutrouli M, Nastou K, Mehryary F, Hachilif R *et al.* The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. *Nucleic Acids Res* 2023; 51: D638-D646.

579
580 Nagy Á, Győrffy B. muTarget: A platform linking gene expression changes and mutation status in solid tumors. *Int J Cancer* 2021; 148: 502-511.

582
<table>
  <tr>
    <td>583</td>
    <td>38</td>
    <td>Fuerer C, Nusse R. Lentiviral vectors to probe and manipulate the Wnt signaling pathway. PLoS One 2010; 5: e9370.</td>
  </tr>
  <tr>
    <td>584</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>585</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>586</td>
    <td>39</td>
    <td>Cui H, Tang D, Garside GB, Zeng T, Wang Y, Tao Z et al. Wnt Signaling Mediates the Aging-Induced Differentiation Impairment of Intestinal Stem Cells. Stem Cell Rev Rep 2019; 15: 448-455.</td>
  </tr>
  <tr>
    <td>587</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>588</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>589</td>
    <td>40</td>
    <td>Lu W, Lin C, Roberts MJ, Waud WR, Piazza GA, Li Y. Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/β-catenin pathway. PLoS One 2011; 6: e29290.</td>
  </tr>
  <tr>
    <td>590</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>591</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>592</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>593</td>
    <td>41</td>
    <td>Waaler J, Machon O, von Kries JP, Wilson SR, Lundenes E, Wedlich D et al. Novel synthetic antagonists of canonical Wnt signaling inhibit colorectal cancer cell growth. Cancer Res 2011; 71: 197-205.</td>
  </tr>
  <tr>
    <td>594</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>595</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>596</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>597</td>
    <td>42</td>
    <td>van Veelen W, Le NH, Helvensteijn W, Blonden L, Theeuwes M, Bakker ER et al. β-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis. Gut 2011; 60: 1204-1212.</td>
  </tr>
  <tr>
    <td>598</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>599</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>600</td>
    <td>43</td>
    <td>Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase. J Biol Chem 2006; 281: 9971-9976.</td>
  </tr>
  <tr>
    <td>601</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>602</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>603</td>
    <td>44</td>
    <td>Brembeck FH, Schwarz-Romond T, Bakkers J, Wilhelm S, Hammerschmidt M, Birchmeier W. Essential role of BCL9-2 in the switch between beta-catenin's adhesive and transcriptional functions. Genes Dev 2004; 18: 2225-2230.</td>
  </tr>
  <tr>
    <td>604</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>605</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>606</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>607</td>
    <td>45</td>
    <td>Fernández-Sánchez ME, Barbier S, Whitehead J, Béalle G, Michel A, Latorre-Ossa H et al. Mechanical induction of the tumorigenic β-catenin pathway by tumour growth pressure. Nature 2015; 523: 92-95.</td>
  </tr>
  <tr>
    <td>608</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>609</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>610</td>
    <td>46</td>
    <td>Gujral TS, van Veelen W, Richardson DS, Myers SM, Meens JA, Acton DS et al. A novel RET kinase-beta-catenin signaling pathway contributes to tumorigenesis in thyroid carcinoma. Cancer Res 2008; 68: 1338-1346.</td>
  </tr>
  <tr>
    <td>611</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>612</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>613</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>614</td>
    <td>47</td>
    <td>Monga SP, Mars WM, Pediaditakis P, Bell A, Mulé K, Bowen WC et al. Hepatocyte growth factor induces Wnt-independent nuclear translocation of beta-catenin after Met-beta-catenin dissociation in hepatocytes. Cancer Res 2002; 62: 2064-2071.</td>
  </tr>
  <tr>
    <td>615</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>616</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>617</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>618</td>
    <td>48</td>
    <td>Huang FI, Chen YL, Chang CN, Yuan RH, Jeng YM. Hepatocyte growth factor activates Wnt pathway by transcriptional activation of LEF1 to facilitate tumor invasion. Carcinogenesis 2012; 33: 1142-1148.</td>
  </tr>
  <tr>
    <td>619</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>620</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>621</td>
    <td>49</td>
    <td>Rasola A, Fassetta M, De Bacco F, D'Alessandro L, Gramaglia D, Di Renzo MF et al. A positive feedback loop between hepatocyte growth factor receptor and beta-catenin sustains colorectal cancer cell invasive growth. Oncogene 2007; 26: 1078-1087.</td>
  </tr>
  <tr>
    <td>622</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>623</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>624</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>625</td>
    <td>50</td>
    <td>Cowley EA, Linsdell P. Characterization of basolateral K+ channels underlying anion secretion in the human airway cell line Calu-3. J Physiol 2002; 538: 747-757.</td>
  </tr>
  <tr>
    <td>626</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>627</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>628</td>
    <td>51</td>
    <td>van Tol BL, Missan S, Crack J, Moser S, Baldridge WH, Linsdell P et al. Contribution of KCNQ1 to the regulatory volume decrease in the human mammary epithelial cell line MCF-7. Am J Physiol Cell Physiol 2007; 293: C1010-1019.</td>
  </tr>
  <tr>
    <td>629</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>630</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>631</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>632</td>
    <td>52</td>
    <td>Roepke TK, King EC, Reyna-Neyra A, Paroder M, Purtell K, Koba W et al. Kcne2 deletion uncovers its crucial role in thyroid hormone biosynthesis. Nat Med 2009; 15: 1186-1194.</td>
  </tr>
  <tr>
    <td>633</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>634</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>635</td>
    <td>53</td>
    <td>Roepke TK, Kanda VA, Purtell K, King EC, Lerner DJ, Abbott GW. KCNE2 forms potassium channels with KCNA3 and KCNQ1 in the choroid plexus epithelium. FASEB J 2011; 25: 4264-4273.</td>
  </tr>
  <tr>
    <td>636</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>637</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>638</td>
    <td>54</td>
    <td>Roepke TK, Purtell K, King EC, La Perle KM, Lerner DJ, Abbott GW. Targeted deletion of Kcne2 causes gastritis cystica profunda and gastric neoplasia. PLoS One 2010; 5: e11451.</td>
  </tr>
  <tr>
    <td>639</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>640</td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>641</td>
    <td>55</td>
    <td>Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene 2017; 36: 1461-1473.</td>
  </tr>
  <tr>
    <td>642</td>
    <td></td>
    <td></td>
  </tr>
</table>
643 56 McFarland CD, Yaglom JA, Wojtkowiak JW, Scott JG, Morse DL, Sherman MY *et al.* The Damaging Effect of Passenger Mutations on Cancer Progression. *Cancer Res* 2017; 77: 4763-4772.

644 57 Zhang J, Yang T, Han M, Wang X, Yang W, Guo N *et al.* Gain-of-function mutations in the catalytic domain of. *Sci Adv* 2023; 9: eadc9273.

648 58 Kato S, Weng QY, Insco ML, Chen KY, Muralidhar S, Pozniak J *et al.* Gain-of-Function Genetic Alterations of G9a Drive Oncogenesis. *Cancer Discov* 2020; 10: 980-997.

651 59 Insco ML, Abraham BJ, Dubbury SJ, Kaltheuner IH, Dust S, Wu C *et al.* Oncogenic. *Science* 2023; 380: eabn7625.

654 60 Salvadores M, Mas-Ponte D, Supek F. Passenger mutations accurately classify human tumors. *PLoS Comput Biol* 2019; 15: e1006953.

657 61 Rahrmann EP, Shorthouse D, Jassim A, Hu LP, Ortiz M, Mahler-Araujo B *et al.* The NALCN channel regulates metastasis and nonmalignant cell dissemination. *Nat Genet* 2022; 54: 1827-1838.

660 62 Pelzmann B, Hatab A, Scheruebel S, Langthaler S, Rienmueller T, Sokolowski A *et al.* Consequences of somatic mutations of GIRK1 detected in primary malign tumors on expression and function of G-protein activated, inwardly rectifying, K. *Front Oncol* 2022; 12: 998907.

664 63 Joshi AD, Parsons DW, Velculescu VE, Riggins GJ. Sodium ion channel mutations in glioblastoma patients correlate with shorter survival. *Mol Cancer* 2011; 10: 17.

667 64 Shorthouse D, Zhuang L, Rahrmann EP, Kosmidou C, Wickham Rahrmann K, Hall M *et al.* KCNQ potassium channels modulate Wnt activity in gastro-oesophageal adenocarcinomas. *Life Sci Alliance* 2023; 6.

671 65 Novellasdemunt L, Foglizzo V, Cuadrado L, Antas P, Kucharska A, Encheva V *et al.* USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating β-Catenin Deubiquitination. *Cell Rep* 2017; 21: 612-627.

675 66 Shah K, Kazi JU. Phosphorylation-Dependent Regulation of WNT/Beta-Catenin Signaling. *Front Oncol* 2022; 12: 858782.

678 67 Zeng G, Apte U, Micsenyi A, Bell A, Monga SP. Tyrosine residues 654 and 670 in beta-catenin are crucial in regulation of Met-beta-catenin interactions. *Exp Cell Res* 2006; 312: 3620-3630.

681 68 David MD, Yeramian A, Duñach M, Llovera M, Cantí C, de Herreros AG *et al.* Signalling by neurotrophins and hepatocyte growth factor regulates axon morphogenesis by differential beta-catenin phosphorylation. *J Cell Sci* 2008; 121: 2718-2730.

685 69 Zhou Y, Wong CO, Cho KJ, van der Hoeven D, Liang H, Thakur DP *et al.* SIGNAL TRANSDUCTION. Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling. *Science* 2015; 349: 873-876.

689 70 Xu B, Chen C, Chen H, Zheng SG, Bringas P, Xu M *et al.* Smad1 and its target gene Wif1 coordinate BMP and Wnt signaling activities to regulate fetal lung development. *Development* 2011; 138: 925-935.

692 71 Alzamora R, O'Mahony F, Bustos V, Rapetti-Mauss R, Urbach V, Cid LP *et al.* Sexual dimorphism and oestrogen regulation of KCNE3 expression modulates the functional properties of KCNQ1 K⁺ channels. *J Physiol* 2011; 589: 5091-5107.

696 72 Rekad Z, Ruff M, Radwanska A, Grall D, Ciais D, Van Obberghen-Schilling E. Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly. *Elife* 2023; 12.

700
701 73 Bergdorf KN, Phifer CJ, Bechard ME, Lee MA, McDonald OG, Lee E *et al*. Immunofluorescent staining of cancer spheroids and fine-needle aspiration-derived organoids. *STAR Protoc* 2021; 2: 100578.

704
705

706 **Figure Legends:**
707

708

<caption>FIGURE 1: KCNQ1 somatic mutations identified in cancer patient datasets.</caption>

709 A KCNQ1 nonsynonymous somatic mutations are mapped to the protein sequence, showing
710 mutation counts at each position across cancer types (Table S1). Coloured spots indicate
711 cancer types for each mutation. Transmembrane segments (S1–S6) are marked in red, and
712 mutations found in at least four patients are highlighted with red bars. B. Repartition of cancer
713 types for the 243 patients harbouring mutations of KCNQ1. C. Repartition of the types of
714 mutations on KCNQ1: missense, frameshift or stop gain. D. Frequency of each mutation out
715 of the 256 mutations found in databases. E CRC-associated mutation distributions in KCNQ1
716 model complex. Proteins are present in cartoon mode: KCNQ1 tetramer in light green, KCNE3
717 protein light pink and Calmodulin in yellow. PIP2 lipid is represented in dynamical bond mode
718 and coloured in orange. The location of the various mutations is indicated in space-filling
719 mode.

720 FIGURE 2 – Multiple cancer-associated KCNQ1 mutations are loss-of-function.

721 A Representative currents traces obtained from the protocol shown in the upper left panel
722 with the WT channel (n=47) or the following mutations: E170K (n=9), V185M (n=28), R243C
723 (n=10), A287T (n=17), A329T (n=10), F339L (n=13), P343S (n=8), R4401Gfs*18 (n=11),
724 K422Sfs*10 (n=10), P448R (n=9), R451Q (n=11), R518* (n=9), G548C (n=8) recorded in the
725 whole cell configuration of the patch clamp technique in CHO-K1 cells transfected with KCNQ1
726 and KCNE3. B Current-voltage relationships plotted from traces obtained in A normalised for
727 cell size. C Current density (pA/pF) measured at 0mV. Statistical significance has been tested
using Kruskal-Wallis test followed by Dunn's post-test. * p<0.05, ** p<0.01, *** p<0.001, ****
p<0.0001, ns not significant. D Normalized activation curves (apparent open probability -
G/Gmax) were measured after steeping to -120mV, as a function of pre-pulse potential. In the
insert, apparent open probability at -60mV are given. Statistical significance has been tested
using Kruskal-Wallis test followed by Dunn's post-test. * p<0.05, ** p<0.01, *** p<0.001, ****
p<0.0001, ns not significant. E Current density (pA/pF) measured at 0mV from co-transfection
experiments where cDNA encoding a KCNQ1 mutation was added in a 1:1 ratio to the WT form
in CHO-K1 cells. WT-WT (n=27), V185M-WT (n=9), A329T-WT (n=13), K422Sfs*10-WT (n=9),
P448R-WT (n=12), R451Q-WT (n=9), R518*-WT (n=13). Statistical significance has been tested
using Kruskal-Wallis test followed by Dunn's post-test. * p<0.05, ** p<0.01, *** p<0.001, ****
p<0.0001, ns not significant.

739 FIGURE 3 - KCNQ1 loss-of-function (LOF) mutations affect the Wnt/β-catenin pathway.

740 A Representative whole cell configuration patch clamp traces recorded of wild type KCNQ1
741 and LOF mutated KCNQ1 in HT29CI19A (WT n=10, A329T n=9, K422Sfs*10 n=8, R518* n=7). B
742 I/V curve obtained from traces in b. C Current density (pA/pF) measured at -20mV. Statistical
743 significance has been tested using Kruskal and Wallis test followed by a Dunn's post-test. *
744 p<0,05, ** p<0,01, *** p<0,001, **** p<0,0001, ns not significant. D Gene ontology terms
745 enriched among the regulated genes in the A329T (left panel), K422Sfs*10 (middle panel), and
746 R518* (right panel) conditions obtained from RNA sequencing. The online software AMINE
747 has been used to analyse the data. E Heat maps showing the top 30 significantly differentially
748 expressed genes corresponding to the Wnt signalling pathway. n= 4 per groups. F Schematic
749 representing the TCGA dataset where CRC patients (carrying a set of mutations but not on
750 KCNQ1) are compared to CRC patients with KCNQ1 mutations. G Gene ontology groups
enriched among the regulated genes in the TCGA dataset of colon adenocarcinoma patients carrying a KCNQ1 mutation obtained with AMINE. H Volcano plot representation of differentially expressed genes from the TCGA colon adenocarcinoma dataset from patients with a KCNQ1 mutation. Thresholds were set at a 1.44-fold change and a p-adjust of 0.01. Genes corresponding to the GO term "Wnt signalling pathway" are shown in orange.

**FIGURE 4 - KCNQ1 loss-of-function (LOF) mutations increase Wnt/β-catenin pathway activity in colon cell line and organoids.**

**A** Schematic representation of the 7xTCF-Sv40-m-cherry (7TGC) construct. **B** Representative images of HT29cl19A-7TGC cells acquired on a confocal microscope, expressing or not LOF KCNQ1 mutations. eGFP in green and mCherry in red. Magnification 63x, scale bar = 10 µm.
Left panel: quantification of mean fluorescence intensity of eGFP+ cells normalized by the number of mCherry+ cells per field (n=10). Statistical significance has been tested using ANOVA test followed by Holm-Sidak post-test.
**C** Western blot analysis and densitometry of active β-catenin normalized by β-catenin levels in HT29cl19A cells (n=7-11). Statistical significance has been tested using ANOVA test followed by Holm-Sidak post-test.
**D** Western blot analysis and densitometry of CD44, Axin2, Cyclin D1, and Claudin-1 normalized by GAPDH or Tubulin in HT29cl19A cells (CD44 n=6, Axin2 n=5-6, Cyclin D1 n=7-10, Claudin-1 n=8). Statistical significance has been tested using ANOVA test followed by Holm-Sidak post-test.
**E** Differentially expressed Wnt/β-catenin target genes in HT29cl19A with A329T, K422Sfs*10, and R518* mutations obtained from RNA sequencing.
**F** Representative confocal images of organoid-7TGC carrying or not a LOF KCNQ1 mutation. Green eGFP, m-Cherry in red. scale bar = 100 µm. On the right panel: quantification of the mean fluorescence intensity of GFP+ organoid normalized by organoid size (N=5, WT n=58, A329T n=40 organoids). Statistical
significance has been tested using Mann and Whitney test. G Representative images showing organoids derived from C57BL/6J mouse colon crypts cultured for 6 days in WENR medium carrying or not LOF KCNQ1 mutation. Organoid diameter was measured from brightfield images (10x magnification), scale bar = 100 µm. Violin plot shows the distribution of organoid diameters (µm) for each condition (N=5-4, WT: n=600 organoids, A329T: n=585 organoids, K422Sfs*10 n=427 organoids). Statistical significance has been tested using Kruskal-Wallis test followed by Dunn's post-test. H Representative confocal images of Ki 67 staining (green) in organoids, (nucleus in blue, F-actine in red) at 40x magnification, scale bar = 100 µm. The graph represents the ratio of Ki67+ cells to the total number of cells (N=4, WT n=28, A329T n=32, K422Sfs*10 n=32 organoids). Statistical significance has been tested using ANOVA followed by a Holm-Sidak post-test. I Workflow for the experiments on mouse colon organoids. Organoids were first cultured in WENR medium for proliferation, on day 4, medium were switch to ENR medium for differentiation until D8. Number of organoids with crypts and number of crypts per organoid were assess on D8. J Representative brightfield images of organoids in ENR taken at 10x magnification, scale bar = 100µm. White stars highlight crypts. Upper right panel shows ratio of the organoids with crypts over the total number of organoids, and the lower right panel shows the number of crypts by organoids (N=5, WT n=131, A329T n=181, K422Sfs*10 n=184 organoids). Statistical significance has been tested using ANOVA followed by a Holm-Sidak post-test (Organoids with crypts) or Kruskal and Wallis followed by Dunn's test (Crypts/organoids). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns not significant.

**FIGURE 5 – Activation of the Wnt/β-catenin signalling pathway induced by KCNQ1 mutations is mediated by β-catenin, independently of Frizzled/Lrp.**
A Protein levels of Axin2 and CD44 normalized by GAPDH treated with vehicle (grey circles) or with 5 µM niclosamide overnight (blue diamonds) (n=4). Statistical significance has been tested using two-way ANOVA followed by a Holm-Sidak post-test. B Western blot analysis and densitometry of pLRP6 (S1490) normalized by LRP6 (n=4). Statistical significance has been tested using ANOVA followed by a Holm-Sidak post-test. C Cartoon showing how the pharmacological molecules used target the Wnt pathway. D Western blot analysis and densitometry of Axin2 and CD44 normalized by GAPDH treated with vehicle (grey circles) or 10 µM JW67 overnight (pink diamonds) (n=4). Statistical significance has been tested using two-way ANOVA followed by a Holm-Sidak post-test. E Western blot analysis and densitometry of β-catenin phosphorylated on residues Y654 (n=8), Y142 (n=8), S675 (n=6), and S33/S37/Thr41 (n=5) normalized by total β-catenin. Statistical significance has been tested using ANOVA followed by a Holm-Sidak post-test or a Kruskal and Wallis test followed by a Dunns' test (Y654). F Proximity ligation assay (PLA) showing the interaction between E-cadherin and β-catenin (n=3-4). Nuclei are stained with DAPI (blue), interactions are in red (scale bar = 100 µm, images on the right are zoom from the main images). Right 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. Statistical significance has been tested using Nested one-ANOVA followed by a Holm-Sidak post-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns not significant.

**FIGURE 6 – The effect of KCNQ1 LOF mutants on Wnt/β-catenin pathway activation is mediated through MET activation.**

A Images showing the phosphorylation status of 49 RTKs (human Proteome Profiler), in cell lysates from HT29cl19A Ctrl, A329T, K422Sfs*10, and R518*. Right panel: quantification of the
phosphorylations of the two most increased RTKs in KCNQ1 LOF mutants (Fold increase from the control). B Western blot showing the phosphorylation of MET at residues Y1234/1235 in KCNQ1 LOF mutants (n=8). Statistical significance has been tested using ANOVA followed by a Holm-Sidak post-test. C Western blot showing the effect of Crizotinib treatment (1 µM overnight) on CD44 expression (n=8). Statistical significance has been tested using Two-way ANOVA followed by a Holm-Sidak post-test. D Western blot showing the effect of Crizotinib treatment (1 µM overnight) on β-catenin phosphorylation at Y654 (n=6). Statistical significance has been tested using Two-way ANOVA followed by a Holm-Sidak post-test. E Representative images acquired on a confocal microscope of HT29cl19A 7TGC cells expressing the K422Sfs*10 mutation or in control condition treated with Crizotinib (1 µM overnight). Magnification 63x, scale bar = 10 µm. Left panel: quantification of GFP+ cells normalized by the number of mCherry+ cells per field (n=4). Statistical significance has been tested using Two-way ANOVA followed by a Holm-Sidak post-test. F Effect of Crizotinib (1 µM) on organoids derived from mouse colon crypts cultured for 6 days in WENR medium. Crizotinib treatment was done from day 3 to day 6. Organoid diameter was measured from brightfield images at 10x magnification, scale bar = 100 µm (Vehicle: WT n=62, A329T n=138, K422Sfs*10 n=119; Crizotinib: WT n= 101, A329T n=151, K422Sfs*10 n=130). Statistical significance has been tested using Two-way ANOVA followed by a Holm-Sidak post-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns not significant.

FIGURE 7 - LOF mutations of KCNQ1 induce a loss of negative regulators of the Wnt/β-catenin pathway.

A Top 20 significantly differentially expressed genes corresponding to the Gene Ontology terms "Positive regulation of Wnt signalling" in red and "Negative regulation of Wnt signalling"
in blue, obtained from HT29cl19A RNA sequencing data (n=4). B Volcano plot of differentially expressed genes from the TCGA dataset on colon adenocarcinoma patients with KCNQ1 mutations. Thresholds were set at a 1.44-fold change and an adjusted p-value of 0.01. Genes corresponding to the Gene Ontology term "Negative regulation of Wnt signalling" are shown in blue. Genes corresponding to the Gene Ontology term "Positive regulation of Wnt signalling" are shown in red. C Western blot analysis and densitometry of Dkk1 (n=7), Nkd1 (n=4), and Wif1 (n=5) normalized by GAPDH in HT29cl19A cells overexpressing KCNQ1-LOF mutations. Statistical significance has been tested using ANOVA followed by a Holm-Sidak post-test. D Representative images acquired on a confocal microscope of HT29cl19A 7TGC cells overexpressing or not a mutation, with or without the addition of Wnt3A conditioned medium (CM) for 30 hours. Magnification 63x, scale bar = 10 µM. Left panel: quantification of GFP+ cells normalized by the number of mCherry+ cells per field (n=10-8). Statistical significance has been tested using Two-way ANOVA followed by a Holm-Sidak post-test. E Western blot analysis and densitometry of pLRP6 (S1490) normalized by LRP6 with or without the addition of Wnt3A CM treated for 2 hours (n=5). Statistical significance has been tested by condition using a Mann and Whitney test. F Representative confocal microscope images of HT29cl19A 7TGC cells expressing the K422Sfs*10 mutation, treated with Wnt3A-CM with or without, DKK-1 150 nM and Wif-1 500 nM for 30h, or left untreated. Magnification 63x, scale bar = 10 µM. Left panel: quantification of GFP+ cells normalized by the number of mCherry+ cells per field (n=4). Statistical significance has been tested using ANOVA followed by a Holm-Sidak post-test. * p<0,05, ** p<0,01, *** p<0,001, **** p<0,0001, ns not significant.

**Figure 8** –Schematic diagram showing how cancer-associated KCNQ1 mutations activate the Wnt/β-catenin signalling pathway. Wild type KCNQ1 favour a low activity of the Wnt pathway by retaining β-catenin at the membrane and promoting the expression of Wnt negative
867 regulators (i.e. DKK-1 and Wif-1). Cancer-associated KCNQ1 mutation favour a high activity of
868 the Wnt/β-catenin pathway by promoting β-catenin Y654 and Y142 phosphorylation through
869 the activation of MET. Loss of KCNQ1 function also lower Wnt negative regulators expression.
870 This results in maintaining cystic colon derived organoid when grown in ENR medium
871 contrarily to organoid expressing the wild type channel which develop crypt domain in this
872 condition.
<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_12.png" /></figure>
<figure><img src="image_13.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>D</figcaption></figure>

A329T

K422S sf10*

R518*

GO terms Biological processes

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>E</figcaption></figure>

Wnt signaling pathway related genes

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_21.png" /></figure>

CRC Patients
(n=386)

Vs

<figure><img src="image_22.png" /></figure>

CRC Patients
With KCNQ1
mutations (n=10)

<caption>G</caption>

TCGA

GO terms Biological processes

<figure><img src="image_23.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_24.png" /></figure>

Log2 FC
<figure><img src="image_25.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>HT29 CI.19A</figcaption></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>
A

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

B

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

C

<figure><img src="image_47.png" /></figure>

D

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

E

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

F

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>
<figure><img src="image_61.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_65.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>
<figure><img src="image_71.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_72.png" /></figure>

<figure><img src="image_73.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_74.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>

<figure><img src="image_77.png" /></figure>

<figure><img src="image_78.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_79.png" /></figure>

<figure><img src="image_80.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_81.png" /></figure>

<figure><img src="image_82.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_83.png" /></figure>

<figure><img src="image_84.png" /></figure>
<figure><img src="image_85.png" /></figure>

<figure><img src="image_86.png" /></figure>