We discuss the molecular mechanisms of this antagonism in colorectal cancer and other cancer types. Additionally, we summarize the available data indicating a reciprocal inhibition of vitamin D action by the activated Wnt/β-catenin pathway. Thus, a complex mutual antagonism between Wnt/β-catenin signaling and the vitamin D system seems to be at the root of many solid cancers.The Wnt/β-catenin signaling pathway is aberrantly activated in most colorectal cancers and less frequently in a variety of other solid neoplasias. Many epidemiological and experimental studies and some clinical trials suggest an anticancer action of vitamin D, mainly against colorectal cancer. The aim of this review was to analyze the literature supporting the interference of Wnt/β-catenin signaling by the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 2D3), the active vitamin D metabolite, usually have opposite effects on cancer cell proliferation and phenotype. In recent years, an increasing number of studies performed in a variety of cancer types have revealed a complex crosstalk between Wnt/β-catenin signaling and 1,25(OH)2D3. Here we review the mechanisms by which 1,25(OH)2D3 inhibits Wnt/β-catenin signaling and, conversely, how the activated Wnt/β-catenin pathway may abrogate vitamin D action. The available data suggest that interaction between Wnt/β-catenin signaling and the vitamin D system is at the crossroads in solid cancers and may have therapeutic applications.Abnormal activation of the Wnt/β-catenin pathway is common in many types of solid cancers. Likewise, a large proportion of cancer patients have vitamin D deficiency. In line with these observations, Wnt/β-catenin signaling and 1α,25-dihydroxyvitamin D Wnt proteins are extracellular signaling molecules that control many key processes during embryonic development and regulate the homeostasis of adult tissues, mainly by modulating the survival, self-renewal, and proliferation of stem cells. They are secreted by a variety of cell types and typically have a short range of action, mediating communication between neighboring cells. Wnt proteins bind to cell surface receptors, of which several classes have been described. Specific Wnt-receptor combinations and cellular contexts determine which of the existing Wnt signaling pathways is engaged .adenomatous polyposis coli) and axin, and the kinases casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β) targets β-catenin for N-terminal phosphorylation and subsequent ubiquitination and proteasome-mediated degradation. Wnt binding to Frizzled and LRP5/6 leads to inhibition of the β-catenin destruction complex and, therefore, to the accumulation of β-catenin in the cytoplasm. A proportion of β-catenin enters the nucleus and binds to transcription factors of the LEF/TCF family acting as a co-activator and regulating the expression of a large variety of genes. Wnt target genes are cell and tissue type-dependent and affect many cellular functions and processes, such as cell proliferation, stemness, migration, and invasion. Some of these targets include the c-MYC and CCND1/cyclin D1 oncogenes, the Wnt inhibitors DKK1 and NKD1/2, and the Wnt effector LEF1. In addition, the Wnt inhibitor AXIN2 is the most ubiquitously regulated β-catenin target gene [The Wnt/β-catenin pathway is triggered by Wnt binding to cell membrane receptors of the Frizzled and low-density lipoprotein receptor-related protein (LRP) families. In the absence of a Wnt ligand, β-catenin protein is mainly located at cell-cell contacts and free cytoplasmic β-catenin is kept low because of a proteolytic destruction machinery. A complex containing the tumor suppressor proteins APC , their extracellular ligands R-spondins (RSPO1-4), and the E3 transmembrane ubiquitin ligases ZNRF3 and RNF43 -6, their. In the APC are present in around 80% of sporadic CRC cases, whereas a small proportion carries mutations in AXIN2 or CTNNB1/β-catenin genes. Moreover, chromosomal rearrangements in R-spondin family members RSPO2 and RSPO3 have been found in 10% of human CRC leading to enhanced Wnt signaling [RNF43 is mutated in a proportion of mismatch repair-deficient colon tumors. Alterations in genes encoding components of the Wnt/β-catenin pathway are frequently mutually exclusive, which confirms that aberrant activation of this pathway is a hallmark of CRC. Mutations in CTNNB1/β-catenin or AXIN2 have been reported in other human tumors, e.g., hepatocellular carcinomas [Dysregulation of Wnt/β-catenin signaling is involved in human diseases including cancer. In many types of cancer, e.g., colorectal, breast, and liver carcinoma, melanoma and leukemia, β-catenin constitutively accumulates within the nucleus of tumor cells ,11,12,13ignaling ,18. In arcinomas ,20,21, wrcinomas ,25,26,273  is a natural seco-steroid whose main source is non-enzymatic production in human skin from UV-B exposed 7-dehydrocholesterol, an abundant cholesterol precursor [3 from skin production and from dietary uptake is hydroxylated in the liver to 25-hydroxyvitamin D3 (25(OH)D3, calcidiol), a stable compound that is used as a biomarker for the vitamin D status of a person [3 at carbon 1, which occurs mainly in the kidneys but also in several types of epithelial and immune cells, renders 1,25-dihydroxyvitamin D3 2D3, calcitriol). This is the most active vitamin D3 metabolite and a high affinity ligand for the vitamin D receptor (VDR) [Vitamin Drecursor ,29. Vitaa person ,31. Subsor (VDR) .2D3 or its synthetic analogues with high specificity [2D3 to VDR promotes the formation of complexes with RXR, the receptor for 9-cis-retinoic acid, and the binding of these VDR/RXR heterodimers to DNA. This leads to epigenetic changes that affect the transcription rate of hundreds of target genes involved in many cellular processes, including proliferation, differentiation and survival [VDR is a member of the nuclear receptor superfamily of transcription factors, which includes receptors for other hormones such as estrogen, progesterone or glucocorticoids, as well as a number of orphan receptors. Nuclear receptors present a highly conserved ligand-binding domain, which in the case of VDR fixes 1,25(OH)cificity . Bindingsurvival ,31. Moresurvival ,35.2D3 and its derivatives modulate signaling pathways that affect cell survival, growth, and differentiation [Current evidence indicates that 1,25(OH)ntiation ,36,37, k3 against CRC by indicating that high UVB exposure or life at lower latitudes, both of which result in higher vitamin D3 synthesis, lead to lower incidence of CRC [3 supplementation human clinical trials have strongly suggested that 1,25(OH)2D3 has anticancer effects, particularly in CRC [Four decades ago, an epidemiological study hinted at the protective effects of vitamin De of CRC . Since ty in CRC ,42,43,442D3 antagonizes the Wnt/β-catenin signaling pathway in colon carcinoma cells [3 observed in epidemiological and animal studies. Previously, other groups had reported a crosstalk between Wnt signaling and other nuclear receptors, such as those for retinoid acid and androgen [Our group was a pioneer in demonstrating that 1,25(OH)ma cells , a mechaandrogen ,47.2D3 interferes with Wnt/β-catenin signaling in human colon carcinoma cells by at least three mechanisms chanisms . Firstlyn of VDR . Interesn of VDR . LithochVDR [2D3 .2D3 induces the expression of E-cadherin protein, which sequesters newly synthesized β-catenin at subcortical cell-cell adherens junctions, thus avoiding its translocation to the nucleus and β-catenin/TCF-mediated transcription [2D3 and concomitant inhibition of the Wnt/β-catenin pathway have also been reported in other cell types [2D3 can antagonize Wnt/β-catenin signaling in colon carcinoma cells that do not express E-cadherin, which implies that this mechanism is not strictly required [Secondly, 1,25(OH)cription . Our datcription ,50,51,52ll types . Howeverrequired .2D3 promotes the expression of Dickkopf 1 (DKK1), a member of a family of extracellular inhibitors of the Wnt/β-catenin pathway [DKK1 is a β-catenin/TCF target gene [DKK1 promoter methylation in a large cohort of CRC patients [Thirdly, 1,25(OH) pathway . DKK1 ca pathway . On the  pathway . In addiget gene ,58,59. Aget gene ,61,62. Sget gene ,64. Moreget gene , and diepatients .2D3 downregulates the expression of DKK4 in both human colon and breast cancer cells. Accordingly, a significant inverse correlation between DKK4 and VDR expression exists in human CRC biopsies [2D3 may be another mechanism for the antitumor action of 1,25(OH)2D3 in CRC.DKK4 is another member of the Dickkopf family of Wnt extracellular inhibitors, although it is a weaker Wnt antagonist than DKK1. We reported that 1,25(OH)biopsies . Unexpecbiopsies . These ebiopsies ,67,68. Tc-MYC oncogene is a well-known β-catenin/TCF target gene that is frequently deregulated in human cancers and activates genetic programs that orchestrate biological processes to promote cell growth and proliferation [c-MYC in CRC [c-MYC and c-FOS oncogenes [loci. In fact, 1,25(OH)2D3 effects on c-MYC gene expression may count as another mechanism of crosstalk between 1,25(OH)2D3 and Wnt/β-catenin signaling pathways. Firstly, ligand-activated VDR represses c-MYC expression by direct interaction with two vitamin D response elements (VDRE) in the promoter region [2D3 on Wnt/β-catenin signaling impairs the transcription of c-MYC mediated by β-catenin/TCF complexes through their binding to several Wnt responsive elements (WRE) at the c-MYC promoter [The feration . Therefoferation . In partC in CRC . A studyncogenes . These dr region ,72. Secopromoter ,73.2D3 crosstalk with Wnt/β-catenin signaling in CRC cells 2D3/VDR induces expression of the negative regulator of the Wnt/β-catenin pathway AXIN1 in CRC cells through a VDRE localized in the regulatory region of the gene [2D3 reduces nuclear β-catenin levels in LT97 colon microadenoma cells and thus downregulates the expression of Wnt target genes such as BCL2, CCND1/cyclin D1, SNAI1, CD44, and LGR5 [Some authors have proposed additional mechanisms of 1,25(OH)RC cells . Beildect on VDR . Tang ant on VDR . Therefothe gene . In addiand LGR5 . Moreoveand LGR5 , which cand LGR5 .2D3 and Wnt/β-catenin signaling pathways but also between carcinoma cells and the tumor microenvironment ironment . They deRC cells . The pos2D3 and Wnt3A (an activator of the Wnt/β-catenin pathway) in human colon fibroblasts. Both agents strongly modulate the gene expression profile and phenotype of these cells. However, in contrast to the antagonism exerted by 1,25(OH)2D3 on the Wnt/β-catenin pathway in colon carcinoma cells, they have a partially overlapping effect. Both compounds inhibit fibroblast proliferation and migration, but while 1,25(OH)2D3 reduces, Wnt3A increases fibroblast capacity to remodel the extracellular matrix [2D3 does not affect the expression of key genes of the Wnt/β-catenin pathway  in human colon tumor or normal organoids derived from CRC patients, where only the DKK4 Wnt/β-catenin target gene is repressed by 1,25(OH)2D3 [2D3 in tumor contexts. Moreover, 1,25(OH)2D3 cooperates with Wnt factors in the differentiation of bone (osteoblasts), skin (keratinocytes) and brain  cells under physiologic conditions [2D3 on overactivation of the Wnt/β-catenin pathway with different effects in particular scenarios.Our group has also studied the interplay between 1,25(OH)r matrix . In addi5(OH)2D3 . This shnditions ,82. Toge2D3 and Wnt/β-catenin signaling in CRC has also been studied in vivo in animal models and patients. Our group showed that the 1,25(OH)2D3 analogue EB1089 reduces the growth of xenografts generated by human CRC cells in immunosuppressed mice. In line with data obtained in cell cultures, this inhibition is associated with an increase of E-cadherin and DKK1 levels, and a decrease of β-catenin nuclear content and of the expression of the β-catenin/TCF target gene ENC1 in the xenografts [2D3 on chemically induced mouse intestinal tumors is concomitant with increased expression of E-cadherin and the inhibition of β-catenin/TCF target genes such as c-Myc and Ccnd1/cyclin D1 in the intestinal crypts of these animals [2D3 and two of its analogues reduce the tumor load in the Apcmin/+ mouse model of intestinal tumorigenesis associated with an increase of E-cadherin protein and a decrease of nuclear β-catenin levels and of the expression of the Wnt target genes c-Myc and Tcf1 [The interplay between 1,25(OH)nografts ,83,84. L animals ,86. Concand Tcf1 .A Western-style diet that is high in fat and low in calcium and vitamin D is a risk factor for gastrointestinal carcinogenesis. This diet increases the frequency of intestinal tumors in normal mice and speeds up tumor formation in mouse models for intestinal cancer . SeveralVdr knockout mice have also been used to study the role of the vitamin D pathway on CRC. Larriba and colleagues [Apcmin/+Vdr−/− mice and discovered that the absence of Vdr results in a higher tumor load and an increased number of premalignant lesions. Interestingly, nuclear staining of β-catenin and expression of Wnt target genes Ccnd1/cyclin D1 and Lef1 are higher in Apcmin/+Vdr−/− than in Apcmin/+Vdr+/+ mice. This suggests that Vdr inactivation facilitates intestinal tumorigenesis fostered by Wnt/β-catenin activation [lleagues  and Zhenlleagues  generatetivation ,92.3 level associates with low promoter methylation of secreted frizzled-related protein 2 (SFRP2) gene that encodes a soluble inhibitor of the Wnt/β-catenin pathway [Remarkably, in a randomized, double-blinded, placebo-controlled clinical trial, Bostick’s group reported that vitamin D supplements increase the expression of APC, E-cadherin and other differentiation markers, and decrease that of β-catenin in the upper part of the crypt of normal rectal mucosa from sporadic colorectal adenoma patients ,94,95,96 pathway . These dAPC, CTNNB1, AXIN2) are rare in breast carcinomas [RSPO2, FZD6) [Although Wnt signaling was first described as inducing breast tumors in mice  and Wnt/rcinomas . The ele2, FZD6) .2D3 downregulates the expression of myoepithelial/basal markers, such as P-cadherin, smooth muscle α-actin, and α6 and β4 integrins in a panel of breast carcinoma cells, and that Vdr−/− mice express higher levels of P-cadherin and smooth muscle α-actin in the mammary gland than wt littermates [2D3/VDR antagonizes the Wnt/β-catenin pathway in breast cancer cells, which might protect against the triple-negative and basal-like phenotype. In line with this, 1,25(OH)2D3 induces DKK1 expression and reduces β-catenin transcriptional activity in R7 murine breast cancer cells, and Vdr deletion and 1,25(OH)2D3 treatment increases and inhibits, respectively, the tumor expression of several Wnt/β-catenin target genes in breast cancer mouse models [2D3 to inhibit spheroid formation by breast cancer stem cells is overcome by β-catenin overexpression, which suggests that inhibition of the Wnt/β-catenin pathway is essential for this action of 1,25(OH)2D3 [VDR expression leads to attenuation of the Wnt/β-catenin pathway in MDA-MB-231 breast cancer cells: cytoplasmic and nuclear levels of β-catenin are reduced with the subsequent downregulation of its target genes AXIN2, CCND1/cyclin D1, IL6, and IL8 [Notably, nuclear β-catenin accumulation in a subset of triple-negative and basal-like breast cancer subtypes has been associated with a poor patient outcome ,99. Our termates . These re models ,102. The5(OH)2D3 . Further5(OH)2D3 . Surpris and IL8 .CCAT2) is upregulated in ovarian cancer cells and promotes epithelial-mesenchymal transition (EMT) at least partially through the Wnt/β-catenin pathway. CCAT2 knockdown represses the expression of β-catenin and the activity of TCF/LEF factors and inhibits EMT by upregulating E-cadherin and downregulating N-cadherin, Snail1, and Twist1 [2D3 inhibits CCAT2 expression in ovarian cancer cells concomitantly with a reduction in cell proliferation, migration, and invasion. This is linked to decreased binding of TCF4 to the c-MYC promoter and, thus, to repression of c-MYC protein expression [2D3. In addition, Srivastava and colleagues have shown that 1,25(OH)2D3/VDR can deplete ovarian cancer stem cells via inhibition of the Wnt/β-catenin pathway [Long non-coding RNA colon cancer-associated transcript 2 2D3. Similarly, 1,25(OH)2D3 inhibits the expression of 73.3% and 77.2% of the Wnt-related genes involved in tissue polarity and cell migration, and in cell cycle, cell growth and proliferation, respectively [2D3 in this cellular context.A recent study has investigated whether 1,25(OH)ectively . These r2D3 and its analogue TX527 increase β-catenin protein levels in the nucleus and at the plasma membrane in a Kaposi’s sarcoma cellular model and potentiate β-catenin/VDR interaction. The net outcome is downregulation of the β-catenin/TCF target genes c-MYC, MMP9 and CCND1/cyclin D1. Moreover, VE-cadherin protein and DKK1 RNA levels are increased [2D3 augments the level of total β-catenin (both cytoplasmic and nuclear pools), while it reduces that of phosphorylated β-catenin in renal cell carcinoma cells [2D3 enhances VDR/β-catenin interaction while attenuating β-catenin/TCF binding. Accordingly, 1,25(OH)2D3 downregulates the expression of CCND1/cyclin D1 and AXIN2 genes. In addition, 1,25(OH)2D3 upregulates E-cadherin expression and blocks TGFβ1-induced nuclear translocation of ZEB1, Snail1 and Twist1, which contributes to the suppression of EMT and the inhibition of cell migration and invasion [2D3 on Kaposi’s sarcoma and renal cell carcinoma cells are largely in agreement with those observed on CRC cells. Distinctly, in pancreatic carcinoma cells, the 1,25(OH)2D3 analogue calcipotriol inhibits Wnt/β-catenin signaling by a different mechanism: the promotion of lysosomal degradation of the Wnt membrane receptor LRP6 [1,25(OH)ncreased . As in Kma cells . More iminvasion . Thus, ttor LRP6 .Vdr deletion predispose mice to either chemical [2D3 enhances β-catenin binding to E-cadherin at the plasma membrane, which promotes epidermal cell differentiation. Moreover, VDR competes with LEF/TCF to recruit β-catenin to gene promoters [2D3 and VDR suppress β-catenin-stimulated LEF1/TCF-driven reporter activity [2D3 analogue EB1089 prevents the development of β-catenin-induced trichofolliculomas, while β-catenin activation in the absence of Vdr results in basal cell carcinomas [VDR expression is associated with upregulation of pathways mediating antitumor immunity and downregulation of proliferative pathways, notably Wnt/β-catenin [2D3 inhibits the expression of Wnt/β-catenin pathway genes. These results suggest that 1,25(OH)2D3/VDR inhibits the pro-proliferative and immunosuppressive Wnt/β-catenin pathway in melanoma and that this is associated with less metastatic disease and stronger host immune responses [Concomitant with an increase in Wnt/β-catenin signaling, global and epidermal-specific chemical  or long-chemical ,114 skinromoters ,116 and activity . The 1,2rcinomas . Recentl-catenin . Functioesponses .VDR knockdown induces, while 1,25(OH)2D3 inhibits, β-catenin binding to and activation of c-MYC promoter in head and neck squamous cell carcinoma [3 and 1,20(OH)2D3, interfere with β-catenin nuclear translocation [2D3 and mitotane, the only chemotherapeutic agent available for adrenocortical carcinoma treatment. These authors reported a reduction in adrenocortical carcinoma cell growth and migration in response to either of the two agents, which is stronger when they are combined [2D3 triggers a decrease in β-catenin RNA and nuclear protein levels, and both 1,25(OH)2D3 and mitotane induce RNA expression of the Wnt inhibitor DKK1, with a more marked effect with the combined treatment, although neither of them can reduce expression of the Wnt target gene c-MYC [Salehi-Tabar and colleagues have reported that arcinoma . In thislocation . In a recombined . 1,25RC cells .2D3 antagonizes Wnt/β-catenin signaling in several neoplasias. However, the interplay between both pathways is a two-way road, that is, activation of the Wnt/β-catenin pathway may also result in 1,25(OH)2D3/VDR inhibition.The abovementioned data indicate that 1,25(OH)2D3 and mediates most if not all 1,25(OH)2D3 effects. Thus, cellular VDR expression is the main determinant of 1,25(OH)2D3 action and its downregulation leads to 1,25(OH)2D3 unresponsiveness. VDR is expressed in most normal human cell types and tissues, but also in cancer cell lines and tumors of diverse origins. In line with the antitumor effects of 1,25(OH)2D3 observed in several neoplasias, high VDR expression in human cancer is usually a hallmark of good prognosis [VDR is the only high affinity receptor for 1,25(OH)rognosis ,37,124. rognosis .SNAI1 transcription via NFκB activation [AXIN2 contributes to Snail1 protein stabilization in breast cancer cells by regulating GSK-3β localization. When levels of AXIN2 increase in response to β-catenin/TCF signaling, GSK-3β is exported from the nuclear compartment leaving Snail1 in its non-phosphorylated transcriptionally active form [SNAI2 RNA levels by Wnt3 has been described in breast cancer cells [Wnt/β-catenin signaling is known to promote EMT through upregulation of the expression and activity of key EMT transcription factors such as Snail1, Snail2, Zeb1 and Twist1 by several mechanisms ,127. Snativation . Furtherive form . In addier cells .VDR gene. Our group demonstrated that Snail1 represses the expression of VDR by two mechanisms [VDR gene transcription by binding to three E-box sequences in its promoter. On the other, Snail1 reduces VDR RNA half-life. As a consequence, Snail1 strongly decreases the level of VDR RNA and protein and the cellular response to 1,25(OH)2D3 [2D3. Therefore, by repressing VDR and CDH1/E-cadherin genes, Snail1 abolishes 1,25(OH)2D3 action and favors the accumulation of β-catenin in the nucleus and the transcription of β-catenin/TCF target genes [VDR gene expression in CRC cells through the same E-boxes in the promoter used by Snail1 2D3 in human osteosarcoma and breast cancer cells [2D3/VDR signaling by inhibiting VDR gene expression.Interestingly, Snail1 and Snail2 are the best-characterized transcriptional repressors of the human chanisms  2D3 ,84. Moreet genes ,84. Latey Snail1 . ActuallVDR gene . SNAI1 agulation ,135,136.I2 genes . VDR expal cells . In addier cells ,139. Knaer cells . Altoget2D3/VDR signaling involves the miR-372/373 cluster  cluster . miR-372r region . Accordir region ,143,144.otential . These ahenotype . These d2D3, a major regulator of the human genome, cooperates with the Wnt/β-catenin pathway in the physiological control of tissues and organs such as bone, skin, and brain. Conversely, 1,25(OH)2D3 attenuates aberrant activation of the Wnt/β-catenin pathway that takes place in most CRC and in a variable proportion of other solid tumors. To do this, 1,25(OH)2D3 modulates a series of genes and mechanisms acting at different levels of the Wnt/β-catenin pathway that vary among cancer types. 1,25(OH)2D3 does not completely block the pathway but rather reduces its overactivation. This probably helps to maintain the physiological effects of Wnt/β-catenin in healthy organs, with few toxic side-effects. As expected from two crucial regulators of the organism and its necessary homeostasis, 1,25(OH)2D3 action is counterbalanced by Wnt factors.The Wnt/β-catenin pathway is frequently overactivated in cancer and promotes tumorigenesis, which makes it an attractive candidate for therapeutic intervention. The active vitamin D metabolite 1,25(OH)2D3 on aberrantly activated Wnt/β-catenin signaling strongly supports the therapeutic utility of vitamin D compounds in cancer prevention and treatment.The multilevel antagonistic action of 1,25(OH)


Wnt signaling is a critical determinant for the maintenance and differentiation of stem/progenitor cells, including trophoblast stem cells during placental development. Hyperactivation of Wnt signaling has been shown to be associated with human trophoblast diseases. However, little is known about the impact and underlying mechanisms of excessive Wnt signaling during placental trophoblast development.In the present work, we observed that two inhibitors of Wnt signaling, secreted frizzled-related proteins 1 and 5 (Sfrp1 and Sfrp5), are highly expressed in the extraembryonic trophoblast suggesting possible roles in early placental development. Sfrp1 and Sfrp5 double knockout mice exhibited disturbed trophoblast differentiation in the placental ectoplacental cone (EPC), which contains the precursors of trophoblast giant cells (TGCs) and spongiotrophoblast cells. In addition, we employed mouse models expressing a truncated β-catenin with exon 3 deletion globally and trophoblast-specifically, as well as trophoblast stem cell lines, and unraveled that hyperactivation of canonical Wnt pathway exhausted the trophoblast precursor cells in the EPC, resulting in the overabundance of giant cells at the expense of spongiotrophoblast cells. Further examination uncovered that hyperactivation of canonical Wnt pathway disturbed trophoblast differentiation in the EPC via repressing Ascl2 expression.Our investigations provide new insights that the homeostasis of canonical Wnt-β-catenin signaling is essential for EPC trophoblast differentiation during placental development, which is of high clinical relevance, since aberrant Wnt signaling is often associated with trophoblast-related diseases. Ascl2, also Mash2), one member of the basic helix–loop–helix (bHLH) family, has been shown to play a critical role during EPC development. Ascl2 is located in the EPC and diminishes as trophoblast cells differentiate into TGCs. Ablation of Ascl2 led to embryonic lethality owing to defective spongiotrophoblast formation [ASCL2 is reported to be expressed in human freshly isolated cytotrophoblasts [Ascl2 remain largely unknown.The placenta, forming the fetal-maternal interface, is essential for the survival and growth of the fetus in eutherian mammals , 2. In mormation . Moreovehoblasts  and proxhoblasts , correspWnt7b null mice died at mid-gestation stage due to defective chorioallantoic fusion [Wnt2 caused impaired labyrinth [Fzd5 mutation was reported to disturb placental labyrinth development [Previous studies have provided evidence that Wnt signaling is a critical player during placental development –15. Wnt7c fusion . Targeteabyrinth . Fzd5 muelopment , and ourelopment . Recentlelopment –18. Althelopment . HoweverAscl2 during EPC trophoblast differentiation, providing direct genetic evidence for the crucial role of suitable canonical Wnt signaling during placental trophoblast differentiation.In the present work, we employed a variety of genetic mouse models and cultured trophoblast cells to demonstrate that hyperactivation of canonical Wnt pathway leads to overabundance of TGCs at the expense of spongiotrophoblast cells, via repressing Sfrp1, Sfrp2, and Sfrp5 during early placental development. Through whole-mount in situ hybridization, we observed that both Sfrp1 and Sfrp5 were expressed in the extraembryonic tissues at E7.5 and E8.5, except Sfrp2 , containing a cysteine-rich domain (CRD) that is 30 to 50% similar in sequence to that of the frizzled protein while lacking the transmembrane domain, serve mainly as extracellular inhibitors of Wnt signaling by directly blocking the interactions between the Wnt and Frizzled receptors . SequencSfrp1 and Sfrp5 during placental development, we examined the pregnancy outcome of Sfrp1 and Sfrp5 double knockout (dKO) females crossed with dKO males, and observed that the average litter size was significantly reduced, compared with that of the wildtype (WT) intercrosses , specifically expressed in TGCs, and trophoblast-specific protein α (Tpbpα), marking EPC and/or spongiotrophoblast cells. While Tpbpα+ trophoblast cells were decreased, Pl1-expressing trophoblast cells were significantly increased in dKO mice , encode two transcription factors of the basic helix-loop-helix (bHLH) family. Ascl2 is required to maintain the EPC progenitor population, determines the specification of Tpbpα-positive trophoblast cells, and inhibits Pl1-expressing giant cell differentiation, while Hand1 has opposing roles and promotes Pl1-expressing TGC differentiation [Ascl2 or Hand1 in mice both lead to embryo lethality owing to defective placental development [Ascl2-positive trophoblast cells were decreased /f(EX3) mouse model, in which the exon 3 of β-catenin gene is flanked by loxp sites, and Cre recombinase-mediated excision would give rise to the expression of a stabilized, constitutively active form of β-catenin resistant to degradation by the GSK3β-mediated proteasome pathway [Ctnnb1f(EX3)/f(EX3) male with Prm-cre (functioning in adult testis) transgenic female, Prm-cre; Ctnnb1f(EX3)/+ male mice were generated. When crossing WT females with Prm-cre; Ctnnb1f(EX3)/+ males, the Ctnnb1Δ/+ conceptus with stabilized β-catenin and hyperactivated canonical Wnt pathway were generated. In this breeding, the average litter size decreased significantly, and Ctnnb1Δ/+ pups failed to be born  m mSfrp1 aCYP19-cre; Ctnnb1f(EX3)/+ mouse model, in which β-catenin protein is dominant-stabilized specifically in extraembryonic trophoblast cells. When CYP19-cre females were crossed with Ctnnb1f(EX3)/+ males, the average litter size was decreased significantly, and the CYP19-cre; Ctnnb1f(EX3)/+ offspring failed to be born /+ conceptus /+ placentas at E9.5 /+ placentas /+ conceptus /+ placentas at E8.5. 209 upregulated genes and 548 downregulated genes were observed in the CYP19-cre; Ctnnb1f(EX3)/+ placentas /+ placentas /+ placentas  cell lines, which undergo differentiation in the absence of FGF4 and mitomycin C-treated mouse embryonic fibroblast-conditioned medium (FCM) [Sfrp1 and Sfrp5 dKO TS cells showed decreased expression of Ascl2 and Tpbpα during differentiation  . Compareion Fig. a, c, conion Fig. b and theion Fig. c were intly Fig. d, f. On cis-acting lincRNA, TCF4/β-catenin complex was recruited to the Ascl2 enhancer immediately downstream of the Ascl2 locus to drive high-level Ascl2 expression [Ascl2 expression, we performed chromatin immunoprecipitation and sequencing (ChIP-Seq) to assess the genome-wide occupancy of β-catenin in differentiated TS cells in the presence of CHIR or not. As expected, β-catenin occupied the promoter regions of the known Wnt target genes, such as Axin2 and Gcm1 /+ global null mice, and CYP19-cre; Ctnnb1f(EX3)/+ trophoblast-specific null mice) that render hyperactivation of canonical Wnt-β-catenin pathway, we found that increased activity of canonical Wnt pathway promoted the excessive differentiation of TGCs via downregulating Ascl2 expression. In addition, since canonical Wnt activity has been reported to promote trophoblast invasion [Wnt signaling has been reported to regulate the proliferation and differentiation of stem and progenitor cells, during the processes of both embryonic development and adult tissue homeostasis , 36, and pathway , exhibit pathway , 39. Exc pathway . As to p pathway . Moreove pathway  and orga pathway , 18. How pathway . Moreove pathway , 41. On invasion , hyperacSfrp1,5 induced hyperactivation of canonical Wnt signaling which impaired trophoblast differentiation, suggesting that the mechanism that keeps the activity of canonical Wnt signaling in a suitable state to maintain the niche for trophoblast differentiation and TGC development, exists in vivo during normal placentation. In order to maintain the niche, a complicated signaling network is needed to preserve the balance of trophoblast differentiation.In addition, as the inhibitors of Wnt pathway, loss of Ascl2 is essential for the maintenance of the EPC during early placental development in mice [Ascl2 knockout and hypomorphic mouse model showed abnormal placentation with decreased EPC layer and/or spongiotrophoblast cell lineage as well as increased number of TGCs [Ascl2 expression was repressed. However, little is known concerning how canonical Wnt signaling regulates Ascl2 expression. In most cases, the canonical Wnt-β-catenin signaling stimulates the transcription of target genes, such as c-Myc [Axin2 [Gcm1 [Ascl2 expression in a similar manner, but to repress Ascl2 expression. Moreover, recent findings have demonstrated that canonical Wnt pathway inhibits the expression of its target genes, including the tumor suppressor 15-prostaglandin dehydrogenase [RANKL [ in mice . Ascl2 k of TGCs , 43. In as c-Myc , Axin2 [c [Axin2  and Gcm1n2 [Gcm1 , 15, thrn2 [Gcm1 , 31, we rogenase , RANKL [e [RANKL  and E-cae [RANKL . HoweverAscl2 expression resulting from hyperactivated canonical Wnt signaling, led to defective chorionic trophoblast differentiation, since the mouse models with Ascl2 deletion [Ascl2 expression [Ascl2-expressing trophoblast cells was not required for labyrinth development [Ascl2-expressing trophoblast cells. In addition, the EPC/spongiotrophoblast layer might functions as structural support for the chorionic/labyrinth development, since Ascl2-expressing trophoblast cells are also present in the chorion and subsequent labyrinth layer.The chorionic development was also impaired, even though the expression of Gcm1, essential for chorionic trophoblast differentiation  and regudeletion  or reducpression  also diselopment , this woSfrp1,5 or the expression of a stabilized β-catenin globally and trophoblast-specifically, resulted in impaired EPC trophoblast differentiation and excessive TGC expansion. Further exploration using the trophoblast stem cell line uncovered that hyper-activated Wnt pathway led to the repression of Ascl2, which is important for trophoblast differentiation. This study provides genetic and molecular evidence that appropriate canonical Wnt pathway is crucial for EPC trophoblast differentiation, which is of high clinical significance since there is a strong correlation between abnormal Wnt signaling and human trophoblast diseases [In summary, hyper-activation of the canonical Wnt signaling, achieved by either the deletion of diseases .Ctnnb1f(EX3)/f(EX3) mice, Sfrp1 and Sfrp5 double mutant mice, and Cyp19-cre transgenic mice were generated as previously described [Prm-cre transgenic mice were obtained from Jackson Laboratory. Ctnnb1f(EX3)/f(EX3) mice were mated with Prm-cre mice to get mice with global hyperactivation of the canonical Wnt pathway. Ctnnb1f(EX3)/f(EX3) mice were mated with Cyp19-cre transgenic mice to get trophoblast-specific hyperactivation of canonical Wnt signaling. Eight-week females were mated with fertile males to induce pregnancy and the day when virginal plugs were seen was considered as embryonic day 0.5 (E0.5). Pregnant females were sacrificed and implantation sites were weighted, frozen, or fixed in 10% neutral buffered formalin. Tail genotyping of the embryos and newborns were determined by PCR.escribed , 52–54. Histological and immunostaining analysis were performed as described previously . In brieIn situ hybridization with isotopes-labeled antisense RNA probes was performed on cryosections 10 μm) as previously described . Whole-m0 μm as pSfrp1 and Sfrp 5 dKO as well as WT TS cells were derived from E3.5 mouse blastocysts as described previously [2. Then, the blastocysts hatched from zona pellucida and attached to the wells to form outgrowth. After disaggregation of the blastocyst outgrowth, feed cells with 70%FCM + 1.5xF4H  for early passages and with 70%FCM + 1xF4H for maintenance. For TS cells differentiation, TS cell medium was used without the supplementation of FGF4, heparin, and FCM. For the treatment of TSC, CHIR99021 , XAV-939 , and iCRT3  were used.eviously . BrieflyRNA extraction and quantitative RT-PCR was performed as described . BrieflyProtein extraction and Western blotting were performed as described previously . AntibodCyp19-cre and Cyp19-cre; Ctnnb1f(EX3)/+ placentas at E8.5 were extracted using RNeasy Micro Kit  according to the manufacturer’s instructions. Purified RNA was prepared using TruSeq® RNA Sample Preparation V2  and subjected to 50-bp single-end sequencing with a BGISEQ-500 sequencer. RNA-seq raw data were initially filtered to obtain clean data after quality control. Clean data were aligned to the mouse genome (mm10) by HISAT2. Raw counts for each gene were calculated by EdgeR. DEGs were defined as genes with P value less than 0.05 and fold change larger than 1.5.RNA from 6 TS cells were cross-linked with 16% formaldehyde  at final concentration of 1% at room temperature for 10 min and quenched with 1/10 volume of 1.25 M glycine for 15 min on ice. Cell lysate in lysis buffer III were sonicated using Bioruptor pico (Diagenode) and then incubated with 4 μg non-phospho (active) β-catenin antibody  overnight at 4°Cwith rotation. Immunoprecipitated complexes were collected with 15 μl Protein A Dynabeads  for 1 h at 4 °C with rotation. Subsequently, beads were washed sequentially once with low-salt buffer, twice with high-salt buffer, once with LiCl buffer, twice with TE, and then eluted in 400 μl of elution buffer for 30 min at 65 °C with shaking. The eluates were incubation at 65 °C for 8 h to reverse the cross-linking. Next, eluates were treated with proteinase K for 1 h at 55 °C and then RNase A for 30 min at 37 °C before DNA was extracted and purified. The ChIP libraries were prepared according to the instruction manual using KAPA DNA HyperPrep Kits  and then run on the Illumina sequencer Hiseq-Xten PE150. Primers for qPCR were listed in Additional file ChIP was performed according to the reported previously . BrieflyThe primary analysis of ChIP-Seq datasets were performed by using Illumina’s Genome Analysis pipeline. The sequencing reads were aligned to the mouse genome (mm10) by HISAT2. Only uniquely aligned reads were kept. MACS2 was applied for peak call using default parameters.t test was performed to determine the significance of a difference between two groups. When comparing the means of more than two groups, a one-way ANOVA was used. Data were presented as means ± SEM in all experiments unless otherwise indicated. *P < 0.05 was considered to indicate a significant result.Statistical analysis was performed with the GraphPad Prism 8 software. In data that was normally distributed, Student’s Additional file 1: : Figure S1. The expression of Sfrp1, Sfrp2 and Sfrp5 during early placental development. A The expression of Sfrp1, Sfrp2, and Sfrp5 was analyzed by whole-mount in situ hybridization at E7.5 and E8.5. B The expression of Sfrp1 and Sfrp5 was detected by in situ hybridization at E7.5-E9.5. The signals were pink. Images in (A) and (B) are representatives of at least two independent experiments. Figure S2. Statistical description of the phenotype of the Sfrp1 and Sfrp5 dKO placenta. A Statistical analysis of spongiotrophoblast thickness and TGCs number in WT and dKO placenta on E9.5. B Quantification of active-β-catenin signal intensity in Fig. P < 0.05. Figure S3. Nuclear localization of active-β-catenin increased in the placenta of Ctnnb1Δ/+ conceptus. A Immunohistochemistry of active-β-catenin in Ctnnb1f(EX3)/+ and Ctnnb1Δ/+ placenta on E9.5. B Quantification of active-β-catenin signal intensity in (A). *, P < 0.05. Figure S4. Global stabilization of β-catenin leads to impaired trophoblast development and embryonic lethality. HE and CK staining of the sections of Ctnnb1f(EX3)/+ and Ctnnb1Δ/+ conceptus on E7.5. Images are representatives of at least three independent experiments. Dec, decidua; EPC, ectoplacental cone; Em, embryo; TGC, trophoblast giant cell. Figure S5. Trophoblast-specific stabilization of β-catenin induces hyperactivation of canonical Wnt pathway. A The localization of active-β-catenin was revealed by immunostaining at E7.5. Cy3-labeled active β-catenin in red, DAPI-labeled nuclei in blue. Images are representatives of at least three independent experiments. B Quantification of active-β-catenin signal intensity in (A). *, P < 0.05. Figure S6. Decreased genes with trophoblast-specific stabilization of β-catenin are related to cell cycles. KEGG analysis of the decreased genes  between Cyp 19-cre and Cyp19-cre; Ctnnb1f(Ex3)/+ placentas . Figure S7.Sfrp1 and Sfrp5 deficiency led to increased activity of Wnt signaling. A TOP-Flash assay in the WT and dKO TS cells. B The expression of Ascl2, Tpbpa and Pl1 in dKO TS cells differentiated for 4 days, in the presence of XAV-939 or not. *, P < 0.05. C QRT-PCR analysis of the expression of Ascl2, Tpbpa and Pl1 in dKO TS cells differentiated for 4 days, with Ascl2 overexpression or not. Figure S8. TCF/LEF might be not required for β-catenin-mediated Ascl2 suppression. A The expression of Ascl2 in WT TS cells treated with indicated conditions. iCRT3, a small molecule that abrogates β-catenin–TCF interaction. *, P < 0.05. ns, not significant. B The top 13 conserved transcription factors binding motif at the β-catenin binding site, 20 kb upstream of the Ascl2 gene. Table S1. Primers Information.Additional file 2: : Table S2. Differentially expressed genes (DEG) between the Cyp19-cre and Cyp19-cre; Ctnnb1f(Ex3)/+ placentas (fold change > 1.5). Table S3. Genes expressed in the Cyp19-cre and Cyp19-cre; Ctnnb1f(Ex3)/+ placentas.


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/β-catenin signaling pathway. In this review, we propose to explore and discuss the current knowledge regarding the interplay between ion channels and the Wnt/β-catenin signaling pathway in cancers. 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  opened n++, Wnt/STOP, influencing planar polarity (2+ homeostasis (Since the first description of Wingless Int 1 (Wnt1) gene in virally induced breast tumors , the Wntpolarity , Ca2+ hoeostasis , or proteostasis , respectIn 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  by the ER-resident acyl-transferase Porcupine . This moHere we will focus on Wnt canonical pathway, which depends on β-catenin stability. In the absence of Wnt ligand (Wnt OFF state), cytosolic β-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 3β (GSK3β) and casein kinase 1α (CK1α). Inside this DC, β-catenin is bound to Axin and phosphorylated by the two constitutively active kinases GSK3β and CK1α at specific Serine and Threonine residues . Then, βInitiation of the Wnt canonical (β-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 CK1α and GSK3β. The phosphorylation of LRP triggers Dishevelled (Dvl) protein polymerisation, thus inactivating the degradation complex . AnotherThe Wnt/β-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 . FurtherAs 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 , and nowWith 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 . HoweverThe potassium channel KCNQ1, also called Kv7.1, belongs to the family of voltage-gated potassium channel. It is constituted by the assembly of four α 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 . Associa+/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  showing hepatic metastases . In lineIt 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 . EpithelAn 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 β-catenin/E-cadherin complex in AJ in colonocytes. Interestingly, the same association between KCNQ1 and β-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 β-catenin in cell lines where KCNQ1 has been silenced by shRNA, indicates that KCNQ1 is necessary to retain β-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/β-catenin signaling pathway. Indeed, silencing of KCNQ1 in CRC induces the re-localisation of β-catenin in the cytosol and increases the expression of Wnt/β-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/β-catenin signaling pathway activity, stabilising β-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 β-catenin to the membrane, repression of the Wnt/β-catenin pathway, and finally reduces the commitment in the EMT process.+ concentration 140mM K+. This protocol causes a cytosolic accumulation of β-catenin, suggesting that the depolarisation induced by the silencing of the channel potentiates Wnt/β-catenin 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 , 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 .By unveiling the bidirectional interaction between Wnt/β-catenin 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 β-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.Another potassium channel has recently been associated to the Wnt/β-catenin pathway, KCNH2 also called hERG or Kv11.1 . This chin vivo by attenuation of the Wnt/β catenin pathway signaling, suggesting that NS1643 mimics the dissociation between hERG and β1-integrin  facilitates Dishevelled’s interaction with the Wnt receptor Frizzled by addressing Dishevelled to the negatively charged phospholipids through its DEP domain (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 ΔF508 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 well-described, but the earliest symptoms concern the digestive system. In addition, CF patients are at high risk of developing gastrointestinal cancers . The stuP domain . StrubbeP domain . All in The main cancer risk for CF patients is gastrointestinal (GI) cancers . Howeveret al. questioned its involvement in adherens junctions (AJ) and its interaction with β-catenin —such as ANO1, also called TMEM16A —CLCA1, i+ and Ca2+ influxes in response to a great variety of stimuli (2+ homeostasis (Transient receptor protein (TRP) channels are cationic channels that generate Na stimuli . Therefoeostasis . Several2+ influx increases AKT activation, which in turn phosphorylates β-catenin on its residue Ser675 and thus activating β-catenin downstream signaling  is overexpressed. Its overexpression contributes to tumor growth, metastasis formation, and poor prognosis. Mechanistically, CasR stimulates TRPV4 channels. The TRPV4-dependent Caignaling .2+ influx stimulates AKT1, which in turn increases the inhibitory Ser9 phosphorylation of GSK3β and the total amount of β-catenin . 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  in 5-Fluorouracil (5-FU), in CRC cells. ABCB1 is a transport protein involved in chemoresistance by mediating efflux of cytotoxic drugs. Enforcing TrpC5 expression increases in a Ca pathway .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 GSK3β phosphorylation and further accumulation of β-catenin in the nucleus. Indeed, Wnt/β-catenin activation by P2X7 stimulation depends on PI3K/AKT stimulation. Interestingly, pharmacological inhibition of P2X7 decreased pro-tumoral behavior of cancer cells .ASIC1a: Acid sensing ion channels (Asic) are proton-gated Na+/Ca2+ channels involved in a wide range of physiological features including pain perception and behavior  leads to the restriction of Wnt activity by a similar mechanism, i.e. the sequestration of β-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 β-catenin at the plasma membrane. Therefore, the remaining question is: how can membrane potential act on β-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 [CaLarge 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.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.This work was supported by the Foundation ARC (grant PJA 20181207701), “La Fondation pour la Recherche Médicale”—FRM—(PhD studentship to CB) (n° ECO201906008972) and the Cancéropole PACA (grant n° 2018-13) and Université Côte d'Azur (CSI-2018).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.


Despite advances in medical treatments, the proportion of the population suffering from alopecia is increasing, thereby creating a need for new treatments to control hair loss and prevent balding. Human hair follicle dermal papilla cells (hDPCs), a type of specialized fibroblast in the hair bulb, play an essential role in controlling hair growth and in conditions like androgenic alopecia. This study aimed to evaluate the intensity-dependent effect of extremely low-frequency electromagnetic fields (ELF-EMFs) on the expression of anagen-related molecules in hDPCs in vitro. We examined the effect of ELF-EMF on hDPCs to determine whether activation of the GSK-3β/ERK/Akt signaling pathway improved hDPC activation and proliferation; hDPCs were exposed to ELF-EMFs at a frequency of 70 Hz and at intensities ranging from 5 to 100 G, over four days. Various PEMF intensities significantly increased the expression of anagen-related molecules, including collagen IV, laminin, ALP, and versican. In particular, an intensity of 10 G is most potent for promoting the proliferation of hDPC and expression of anagen-related molecules. Moreover, 10 G ELF-EMF significantly increased β-catenin and Wnt3α expression and GSK-3β/ERK/Akt phosphorylation. Our results confirmed that ELF-EMFs enhance hDPC activation and proliferation via the GSK-3β/ERK/Akt signaling pathway, suggesting a potential treatment strategy for alopecia. Hair disorders such as hair loss , androgenic alopecia, or excessive hair growth  may impact the social and psychological well-being of an individual ,2. VarioWe focused particularly on EMF in this study. EMF has been approved as a noninvasive treatment by the US Food and Drug Administration (FDA). EMF has been verified for stability and is already clinically used . A largeThe hDPCs are promising cell sources for hair regeneration therapy for alopecia patients . Human hβ-catenin protein levels are regulated by the Wnt signal. In the absence of Wnt3α, β-catenin is ordinarily phosphorylated in the cytoplasm by GSK3β, a serine/threonine protein kinase encoded by GSK3β. When Wnt protein binds to the cell surface receptors of the Frizzled family, β-catenin destruction is prevented via GSK-3β inhibition. Consequently, β-catenin accumulates in the cytoplasm and moves into the nucleus, where it activates gene expression programs. Thus, GSK-3β is a key protein involved in the regulation of Wnt3α/β-catenin signaling, and GSK-3 inhibition may enhance hair growth by inducing an increase in β-catenin levels in DPCs ,24,25,26In this study, we hypothesized that ELF-EMFs would affect the activity of hair cells, so we examined the effect of ELF-EMFs on hDPCs. In particular, we investigated a frequency effect of EMF for inducing hDPCs growth and activating hair growth-related gene and protein expression. First, hDPCs were isolated from patients and exposed to 70 Hz EMF at intensities of 5, 10, 20, 50, and 100 G and compared with control hDPCs, which had not been exposed to EMF. Moreover, to elucidate the mechanisms, we focused on the β-catenin signaling pathway. Our results demonstrated that β-catenin mediated hDPC activation via GSK-3β inhibition by phosphorylation (Ser9). Our findings revealed that ELF-EMF promoted hair growth capacity and could be potentially used as an alternative therapeutic option in hair loss treatment.We exposed hDPCs to ELF-EMF radiations of various intensities, namely 5, 10, 20, 50, and 100 G, at a frequency of 70 Hz, and performed LDH and MTT assays. Following studies on cell morphology, LDH activity assay, and MTT assay, the protein levels of anagen-related proteins  after exposure to ELF-EMFs over four days were quantified via Western blotting. There were significant differences between cells exposed to ELF-EMFs of different intensities . This reThe hDPCs were treated with ELF-EMFs of 10 G intensity, and the expression of β-catenin and Wnt3α was measured via Western blot analysis and immunofluorescence staining. Treatment with an ELF-EMF of 10 G intensity increased the total level of Wnt3α/β-catenin protein expression .To determine whether GSK3β/ERK/AKT signaling was involved in ELF-EMF-induced proliferation of hDPCs and expression of anagen-related molecules. ELF-EMFs induced a significant increase in GSK3β/ERK/AKT phosphorylation . The p-GThe hDPCs regulate the development of human hair follicles, and the Wnt/β-catenin pathway is considered to be essential in maintaining hair-inducing activity of hDPCs . To the This study aimed to determine the impact of ELF-EMF intensity on hDPCs and identify optimal conditions for ELF-EMF-induced expression of anagen-related molecules in hDPCs. We first examine the cytotoxic response of ELF-EMFs. ELF-EMF intensity-dependent cytotoxicity was evaluated by cell morphology and LDH assay. ELF-EMFs do not induce damage to the cell membrane of hDPCs, as verified via lactate dehydrogenase release assay A. AdditiVersican and ALP are key characteristic molecular markers of DPs, which are specifically expressed in DPs during the anagen phase and absent during the telogen phase ,32,33,34Therefore, the study involving investigation of the signaling pathway was conducted at an EMF intensity of 10 G. We hypothesized that ELF-EMFs would modulate Wnt3α/β -catenin signaling in cultured hDPCs because the Wnt3α/β-catenin-mediated signaling pathway plays a pivotal role in the regulation of hair follicle morphogenesis, hair shaft differentiation, and follicular recycling ,35,36. RTo clarify the mechanisms by which ELF-EMF promotes the activity of Wnt3α/β-catenin in hDPCs, we examined changes in the expression of the GSK3β/ERK/AKT signaling pathway. GSK3β is inactivated by phosphorylation at Ser9. Thus, these results suggested that the ELF-EMF-induced increase in β-catenin protein levels occurring in hDPCs may be due to the reduced destruction caused by GSK3β inactivation. Moreover, Wnt3α/β-catenin signaling is involved in regulating the expression of ERK and PI3K/AKT, as well as the proliferation of hDPCs ,28. AKT The hair remaining after hair transplantation was collected after obtaining approval from the Institutional Review Board of the Dongguk University . Hair follicles were also obtained after the hair transplantation surgery, with the patients’ approval. The tissues were washed in Dulbecco’s phosphate balanced solution (DPBS) supplemented with penicillin, streptomycin, and amphotericin B. Hair follicles were delivered to the laboratory in Williams E medium, containing 200 unit/mL penicillin G, 0.2 mg/mL streptomycin, and 0.5 μg/mL amphotericin B.Using a stereoscope at 40× magnification , we sliced off the hair follicle bulb and used a 26 G syringe needle to isolate dermal papilla (DP) and dermal sheath (DS) by softly teasing the papilla. Subsequently, the basal stalk was cut off, and the dermal sheath cells (DSCs) were isolated. Finally, the papilla was transferred to a 35 mm culture dish that had been precoated with 400 µL of FBS, and then Dulbecco’s minimal essential medium  was added, to reach a final concentration of 20% FBS. The media was fixed, while the cells grew from the attached tissue. Once the grown cells could be observed, FBS concentration was reduced to 10%. The media was changed twice a week, and 0.05% trypsin/0.02% EDTA solution was used to subculture confluent cells.2, for 5–7 min. Next, the Accutase was collected and removed from the cells by centrifugation at 800 rpm for 5 min. The supernatant was discarded, and the cell suspension was inoculated into a fresh cell culture medium  and seeded in a 100Φ culture dish.All cell media were replaced every 3 days. DPCs were subcultured when they reached a confluence of 80–90% in a 100Φ culture dish, with subculturing usually performed between 1 and 3 times. The cells were washed with phosphate-buffered saline (PBS), to remove FBS, dissociated using Accutase , and then incubated at 37 °C, in a humid atmosphere of 5% COWe used a Helmholtz coil, which can generate magnetic fields; the apparatus is depicted in 4 cells/well into 24-well plates. After 24 h of incubation, hDPCs irradiated with EMF. After 4 days, the cells were treated with an MTT reagent for 4 h. The samples were assessed by measuring absorbance at 540 nm with an ELISA plate reader. Cytotoxicity was evaluated by the LDH assay; the medium was collected after cell culture, and an LDH-LQ kit was used . The medium (100 μL) was placed in a 96-well plate, with 50 μL of working solution, and incubated at room temperature for 30 min. Then, the reaction was terminated with 50 μL of stop solution (1 N HCl), and the absorbance was measured at 570 nm.Cell proliferation was determined by using an MTT assay. Briefly, hDPCs were seeded at a density of 2 × 10Cells were washed with PBS, extracted with sample buffer (pH 6.8) consisting of 5% 2-mercaptoethanol, 10% glycerol, and 0.1 mg/mL bromophenol blue dissolved in Tris-HCl, and boiled at 100 °C for 5 min. To load the same amount of protein, BCA assay was performed, and the same amount of protein (20–40 μg) was separated via electrophoresis, on 8–10% SDS-PAGE, and transferred onto a nitrocellulose membrane. Subsequently, these separated proteins were incubated with primary antibodies recognizing β-actin , type IV collagen, laminin, β-catenin, Wnt3α, versican, alkaline phosphatase (ALP), p-GSK3β, GSK3β, p-ERK, ERK, p-AKT, and AKT . Protein detection was performed with Lumi Femto  and the Molecular Imager ChemiDoc XRS+ station. ImageJ software  was used to analyze and quantify the Western blot image.Band images were obtained by using the ECL system  and Molecular Imager ChemiDoc XRS+ . ImageJ software was used for the quantitative analysis of Western blot band images.The cells were washed with PBS before being fixed in 4% paraformaldehyde prepared in PBS. Cells were immunostained according to standard protocols, using the following primary antibodies: β-catenin (Abcam) and appropriate fluorescent secondary antibodies . Representative images were captured by using a Nikon Eclipse Ti microscope.p < 0.05 . An ANOVA test was used for multicomponent comparisons, and the Student’s t-test for two-component comparisons after normal distribution was confirmed.All data are presented as the mean ± standard deviation (SD) of three independent experiments. The n-values indicate the number of independent experiments performed or the number of individual experiments. For each independent experiment, at least three technical replicates were used and a minimum number of three independent experiments were performed to ensure adequate statistical power. In all of the analyses, group differences were considered to be statistically significant at Our results suggest that ELF-EMFs have the potential to stimulate anagen-related molecules in hDPCs, as well as hDPC proliferation via the activation of Wnt3α/β-catenin signaling and GSK-3β/ERK/Akt pathway. To the best of our knowledge, this is the first report to demonstrate the effect of various ELF-EMF intensities on anagen-related molecule expression in hDPCs and the underlying mechanisms. Our study also reports the optimal intensity of ELF-EMFs on hDPC proliferation and anagen-related molecule expression. However, considering that the human hair follicle is a complex organ composed of epithelial and mesenchymal tissues and that the interaction of these tissues is essential for hair follicle growth, more studies on human hair follicle organ culture or in vivo studies are warranted. Taken together, these results strongly suggest that an ELF-EMF intensity of 10 G may prolong the anagen phase in hDPCs by increasing type IV collagen, ALP, and versican levels via activation of Wnt3α/β-catenin signaling and phosphorylation of GSK3β/ERK/AKT. Therefore, it is considered that ELF-EMF may be particularly useful for hair loss prevention and hair growth enhancement.