Wingless-type MMTV Integration Site Family Research Articles

Mutation analysis of WNT10B in obese children, adolescents and adults

Wingless-type MMTV integration site family, member 10B (WNT10B) is an activator of the Wnt pathway. The Wnt pathway is known to play an important role in maintenance and differentiation of stem cells and has been implicated in the origination of obesity. To evaluate the role of genetic variation in WNT10B in obesity further, we performed a mutation analysis on Belgian obese patients and control subjects. A mutation analysis of WNT10B by means of high-resolution melting curve analysis and direct sequencing was performed on 546 obese children and adolescents (mean Z-score of 2.6 ± 0.6 and 2.5 ± 0.4 respectively), 86 morbidly obese adults (mean BMI of 48.0 ± 0.4 kg/m(2)) and 447 lean, healthy controls (mean BMI of 22.1 ± 1.7 kg/m(2)). A total of five novel non-synonymous variants were identified. R228Q was the only coding, non-synonymous variant that was exclusively found in patients, but the variant did not co-segregate with obesity in the three investigated siblings. The remaining four variants were either found both in cases and in control samples (G181D) or only in control samples (A108P, S187R and P315S). The frequency of non-synonymous variants in lean individuals (0.9 %) was higher than in obese individuals (0.3 %) and familial co-segregation of the most promising variant in patients could not be demonstrated. Therefore, we conclude that variations in WNT10B do not contribute to human monogenic obesity in our population.

Excessive Wnt/β‐catenin signaling disturbs tooth‐root formation

Wingless-type MMTV integration site family (Wnt)/β-catenin signaling plays an essential role in cellular differentiation and matrix formation during skeletal development. However, little is known about its role in tooth-root formation. In a previous study, we found excessive formation of dentin and cementum in mice with constitutive β-catenin stabilization in the dental mesenchyme. In the present study we analyzed the molar roots of these mice to investigate the role of Wnt/β-catenin signaling in root formation in more detail. We generated OC-Cre:Catnb(+/lox(ex3)) mice by intercrossing Catnb(+/lox(ex3)) and OC-Cre mice, and we analyzed their mandibular molars using radiography, histomorphometry and immunohistochemistry. OC-Cre:Catnb(+/lox(ex3)) mice showed impaired root formation. At the beginning of root formation in mutant molars, dental papilla cells did not show normal differentiation into odontoblasts; rather, they were prematurely differentiated and had a disorganized arrangement. Interestingly, SMAD family member 4 was upregulated in premature odontoblasts. In 4-wk-old mutant mice, molar roots were about half the length of those in their wild-type littermates. In contrast to excessively formed dentin in crown, root dentin was thin and hypomineralized in mutant mice. Biglycan and dentin sialophosphoprotein were downregulated in root dentin of mutant mice, whereas dentin matrix protein 1 and Dickkopf-related protein 1 were upregulated. Additionally, ectonucleotide pyrophosphatase/phosphodiesterase 1 was significantly downregulated in the cementoblasts of mutant molars. Finally, in the cementum of mutant mice, bone sialoprotein was downregulated but Dickkopf-related protein 2 was upregulated. These results suggest that temporospatial regulation of Wnt/β-catenin signaling plays an important role in cell differentiation and matrix formation during root and cementum formation.

Alterations of the WNT7A gene in clear cell renal cell carcinomas.

WNT7A (wingless-type MMTV integration site family, member 7A) is a known tumor suppressor gene of non-small cell lung carcinomas (NSCLC) and is frequently inactivated due to CpG-island hypermethylation in human cancers. The members of WNT family are involved in cell signaling and play crucial roles in cancer development. In the present work hypermethylation of the WNT7A gene was detected in 66% (29/44) of analyzed clear cell renal cell carcinomas (RCCs) using methyl-specific PCR (MSP). Moreover, bisulfite sequencing confirmed intensive hypermethylation of the 5′-CpG island of the WNT7A gene. Methylation analysis revealed positive correlations between tumor stage, Fuhrman nuclear grade and WNT7A hypermethylation. Additionally, restoration of WNT7A gene expression in the A498 cell line by 5-aza-2′-deoxycytidine treatment confirmed a direct contribution of hypermethylation in silencing of the WNT7A gene. High frequency of loss of heterozygosity (LOH) was demonstrated on chromosome 3p25 in regions surrounding the WNT7A gene. The frequent down-regulation of WNT7A gene expression was detected in 88% (15/17) of clear cell RCCs. We have also shown that the WNT7A gene possesses tumor suppression function by colony-formation and cell proliferation assays in RCC cell lines. In summary, the WNT7A gene is inactivated by genetic/epigenetic alterations in clear cell RCC and demonstrates tumor suppressor properties.

Glucocorticoids induce CCN5/WISP-2 expression and attenuate invasion in oestrogen receptor-negative human breast cancer cells

CCN5 (cysteine-rich 61/connective tissue growth factor/nephroblastoma overexpressed 5)/WISP-2 [WNT1 (wingless-type MMTV integration site family, member 1)-inducible signalling pathway protein 2] is an oestrogen-regulated member of the CCN family. CCN5 is a transcriptional repressor of genes associated with the EMT (epithelial-mesenchymal transition) and plays an important role in maintenance of the differentiated phenotype in ER (oestrogen receptor)-positive breast cancer cells. In contrast, CCN5 is undetectable in more aggressive ER-negative breast cancer cells. We now report that CCN5 is induced in ER-negative breast cancer cells such as MDA-MB-231 following glucocorticoid exposure, due to interaction of the endogenous glucocorticoid receptor with a functional glucocorticoid-response element in the CCN5 gene promoter. Glucocorticoid treatment of MDA-MB-231 cells is accompanied by morphological alterations, decreased invasiveness and attenuated expression of mesenchymal markers, including vimentin, cadherin 11 and ZEB1 (zinc finger E-box binding homeobox 1). Interestingly, glucocorticoid exposure did not increase CCN5 expression in ER-positive breast cancer cells, but rather down-regulated ER expression, thereby attenuating oestrogen pathway signalling. Taken together, our results indicate that glucocorticoid treatment of ER-negative breast cancer cells induces high levels of CCN5 expression and is accompanied by the appearance of a more differentiated and less invasive epithelial phenotype. These findings propose a novel therapeutic strategy for high-risk breast cancer patients.

Involvement of the WNT and FGF signaling pathways in non-isolated anorectal malformations: Sequencing analysis of WNT3A, WNT5A, WNT11, DACT1, FGF10, FGFR2 and the T gene

Anorectal malformations (ARMs) comprise a broad spectrum of anomalies, including anal atresia, congenital anal fistula and persistence of the cloaca. Research suggests that genetic factors play an important role in ARM development. However, few genetic variants have been identified. Embryogenesis is orchestrated by crosstalk of the wingless-type MMTV integration site family (WNT) and fibroblast growth factor (FGF) signaling pathways in a process that involves several intracellular cascades. Studies in mice have implicated several genes from these pathways in the etiology of ARMs. We performed sequencing analysis of seven of these previously reported genes in 78 patients with ARMs occurring within the context of at least one additional congenital anomaly. No associations were identified with variants in WNT3A, WNT5A, WNT11, DACT1, FGF10 or the T gene. In the FGFR2 gene, three novel heterozygous nucleotide substitutions were identified. Further investigations, including the study of family members, revealed that these variants were not causally related to the phenotype in the present ARM cohort. Mutations in the seven investigated genes may nonetheless be a cause of ARMs in rare cases. However, further studies should consider genes encoding other proteins in the WNT/FGF signaling pathways as possible candidates.

Different levels of testicular organization during gonadal differentiation in B6.YTir mice manifesting sex reversal

B6.Y(Tir) (mice with Y chromosome from a strain in Tirano, Italy, and autosomes and X-chromosomes from the B6 strain) mice provide an excellent model for analysing sex development that occurs during gonadal differentiation; however, the molecular mechanisms that contribute to sex reversal are unclear. Our aim has been to establish which molecular events participate in this sex reversal. The pattern of gene expression related to testicular [Sry (sex-determining region of the Y chromosome), Sox9 (Sry-related high-mobility group box gene 9) and Mis (Müllerian-inhibiting substance)] and ovarian [Wnt4 (Wingless-type MMTV (murine-mammary-tumour virus) integration site family, member 4), Rspo1 (cysteine-rich secretory protein containing a thrombospondin type 1 repeat) and Stra8 (stimulated by retinoic acid gene 8)] differentiation was analysed by applying immunofluorescence and real-time RT-PCR (reverse transcription-PCR), focusing on XY gonads from the B6.Y(Tir) mouse, but also analysing the normal strains CD-1 and C57BL/6J (B6). The expression of genes related to the process of sexual differentiation was altered in the case of the B6.Y(Tir) strain, both at the transcript and protein level, inducing differentiation of ovaries and ovotestes, but not the formation of the testes, which were normal. Our results indicate that the expression of testicular genes is inhibited at various levels, permitting the expression of ovarian genes such as Wnt4, Stra8 and Rspo1. However, their activity was not clear when the data were averaged. Correlation analysis indicated that an ovary differentiation pathway is activated when the testicular differentiation pathway is inhibited.

RTTN Mutations Link Primary Cilia Function to Organization of the Human Cerebral Cortex

Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals.

Circulation Research Thematic Synopsis

<i>Circulation Research</i> Thematic Synopsis

Treatment-induced damage to the tumor microenvironment promotes prostate cancer therapy resistance through WNT16B

Acquired resistance to anticancer treatments is a substantial barrier to reducing the morbidity and mortality that is attributable to malignant tumors. Components of tissue microenvironments are recognized to profoundly influence cellular phenotypes, including susceptibilities to toxic insults. Using a genome-wide analysis of transcriptional responses to genotoxic stress induced by cancer therapeutics, we identified a spectrum of secreted proteins derived from the tumor microenvironment that includes the Wnt family member wingless-type MMTV integration site family member 16B (WNT16B). We determined that WNT16B expression is regulated by nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB) after DNA damage and subsequently signals in a paracrine manner to activate the canonical Wnt program in tumor cells. The expression of WNT16B in the prostate tumor microenvironment attenuated the effects of cytotoxic chemotherapy in vivo, promoting tumor cell survival and disease progression. These results delineate a mechanism by which genotoxic therapies given in a cyclical manner can enhance subsequent treatment resistance through cell nonautonomous effects that are contributed by the tumor microenvironment.

Wingless-type MMTV integration site family member 16B (WNT16B)

Wingless-type MMTV integration site family member 16B (WNT16B)

Genetic deletion of low-density lipoprotein receptor-related protein 5 increases cartilage degradation in instability-induced osteoarthritis

The wingless-type MMTV integration site family (WNT) signalling pathway plays an important role in embryonic joint and bone development and has been associated with osteoporosis and osteoarthritis (OA). Loss-of-function mutations in low-density lipoprotein receptor-related protein 5 (LRP5), a WNT co-receptor, result in low bone mass. Lrp5(-)(/)(-) mice also have low bone mass phenotypes. Recently an OA-susceptibility locus containing the LRP5 gene was suggested. We investigated the effects of loss of Lrp5 in joint biology in three different mouse models of OA. Total body bone mineral parameters were measured by dual-energy X-ray absorptiometry. Trabecular and cortical bone parameters of tibia and femur were assessed ex vivo by peripheral quantitative CT. Osteoarthritic changes were induced in Lrp5(-)(/)(-) and wild-type C57Bl/6J mice using the surgically induced destabilization of the medial meniscus model and the chemically induced papain and collagenase model. The severity of joint disease was investigated by histological analysis of the knee joints. Bone mineral density and weight were significantly decreased in Lrp5(-)(/)(-) C57Bl/6J mice compared with their wild-type littermates. Surgically induced destabilization of the knee joint resulted in significantly increased cartilage degradation in the medial tibia of Lrp5(-)(/)(-) mice compared with wild-type control mice. In the medial femur, a similar trend was found but did not reach statistical significance. In the papain- and collagenase-induced models, these differences were not observed. Inflammation scores were comparable between wild-type and Lrp5(-)(/)(-) mice. These data show that loss of function of Lrp5 increases cartilage degradation in mild instability-induced OA models in mice. Low bone mass density could have contributed to this effect.

Inhibition of Wnt1 expression reduces the enrichment of cancer stem cells in a mouse model of breast cancer

Breast cancer is the leading cause of deaths from cancer in women. Cancer recurrence is the most common cause of mortality in breast cancer patients. The cancer stem cell (CSC) hypothesis proposes that CSCs are the center of cancer development and recurrence. Targeting CSCs, in combination with standard chemotherapy, may prevent cancer recurrence and improve long-term survival. Stem cells can be enriched in non-adherent sphere cultures. To identify molecular targets in breast CSCs, we evaluated the transcription levels of stem cell-related genes in 4T1 mouse mammary cancer cells grown as spheres or in a monolayer culture. The most differentially expressed gene was found to be wingless-type MMTV integration site family member 1 (Wnt1) in the 4T1 sphere culture. Functionally, knockdown of Wnt1 in breast cancer cell lines suppressed the in vitro properties of the stem-like cells, including their sphere-forming ability and ALDH activity, whereas the addition of recombinant Wnt1 to breast cancer cell lines enhanced the in vitro properties of these stem-like cells. In addition, knockdown of Wnt1 in 4T1 cells affected the properties of the stem-like cells in vivo, including their tumorigenic potential and tumor initiation ability. Collectively, these results suggest that Wnt1 expression may give rise to the properties of CSCs in breast tumors. Therefore, targeting Wnt1-associated signaling proteins may provide an effective therapeutic approach for the treatment of advanced breast cancer.

Polymorphisms of the bovine WNT10B gene and their associations with growth traits

Wingless-type MMTV integration site family, member 10B (WNT10B) may play an important role in inhibiting differentiation of preadipocytes in vitro and impairing adipose development in vivo. In this study, based on DNA sequencing and PCR–RFLP methods, we attempted to characterize the associations between common genetic polymorphisms in WNT10B and growth traits of 435 female cattle from three breeds (Jiaxian, Qinchuan and Luxi cattle). The results indicated that g.220A>G was in the intron 1, and g.1617C>T, g.3980G>T, g.4711G>C were in the coding region. At the g.3980G>T locus, Jiaxian cattle individuals with genotype TT had greater body length than those with genotypes GG and GT (P<0.05). At the g.220A>G locus, Qinchuan cattle individuals with genotype GG had greater growth traits than those with genotype AA and AG (P<0.05 or P<0.01). These statistical results showed that the WNT10B gene might be a potential candidate gene for marker-assisted selection (MAS).

Canonical Wnt suppressor, Axin2, promotes colon carcinoma oncogenic activity

Aberrant activation of canonical Wingless-type MMTV integration site family (Wnt) signaling is pathognomonic of colorectal cancers (CRC) harboring functional mutations in either adenomatous polyposis coli or β-catenin. Coincident with Wnt cascade activation, CRCs also up-regulate the expression of Wnt pathway feedback inhibitors, particularly the putative tumor suppressor, Axin2. Because Axin2 serves as a negative regulator of canonical Wnt signaling in normal cells, recent attention has focused on the utility of increasing Axin2 levels in CRCs as a means to slow tumor progression. However, rather than functioning as a tumor suppressor, we demonstrate that Axin2 acts as a potent promoter of carcinoma behavior by up-regulating the activity of the transcriptional repressor, Snail1, inducing a functional epithelial-mesenchymal transition (EMT) program and driving metastatic activity. Silencing Axin2 expression decreases Snail1 activity, reverses EMT, and inhibits CRC invasive and metastatic activities in concert with global effects on the Wnt-regulated cancer cell transcriptome. The further identification of Axin2 and nuclear Snail1 proteins at the invasive front of human CRCs supports a revised model wherein Axin2 acts as a potent tumor promoter in vivo.

Mesenchymal Stem Cells Provide an Advantageous Tumor Microenvironment for the Restoration of Cancer Stem Cells

Objective: Accumulating evidences suggest that cancer-associated fibroblasts are provided from bone-marrow-derived mesenchymal stem cells (BM-MSCs); however, little is known about the mechanism(s) by which BM-MSCs accelerate cancer aggressiveness. Methods: Gastric carcinoma (GC)-derived MKN-7 cells were cocultured with UE6E7T-12 BM-MSCs. The gene expression profile in MKN-7 cells was investigated by microarray analysis. Between two major types of GCs (intestinal- and diffuse-type), the expression of genes was detected by immunohistochemistry. Results: We found that direct attachment to UE6E7T-12 induced proliferation and cluster formation of MKN-7 cells. Coculture with UE6E7T-12 increased the population of CD133+ MKN-7 cells in vitro and coimplantation of these in mice resulted in subcutaneous tumors in vivo. The wingless-type MMTV integration site (WNT) family member 5A (WNT5A) and transforming growth factor-β (TGF-β)-induced (TGFBI) genes were found to be upregulated in MKN-7 cells directly attached to UE6E7T-12. Recruitment of CD271+ BM-MSC was detected preferentially in the stroma of the diffuse-type GC and this type of GC cell also showed frequent expression of WNT5A, TGF-β type I receptor and CD133. Conclusion: BM-MSC-mediated activations of the WNT and TGF-β signaling pathways were thought to provide advantageous microenvironments for cancer progression by supporting the reacquisition and maintenance of cancer stem cells.

WNT2 Locus Is Involved in Genetic Susceptibility of Peyronie's Disease

Peyronie's disease (PD) is a fibromatosis of the penis, with a pathology very similar to what is seen in the hand (palmar fascia) in Dupuytren's disease (DD). Recently, we performed a genome-wide association study and identified nine genetic loci containing common variants associated with DD. Seven of these loci mapped within or near genes of the canonical WNT pathway and each locus yielded relatively large odds ratios (ORs) for DD disease status. Given the clinical overlap between PD and DD, we examined whether the nine DD susceptibility loci are also involved in PD. An association study was performed using a case/control design. From 2007 to 2010, we prospectively included 111 men who had been clinically diagnosed with PD. Control subjects (N = 490 males) were randomly drawn from a population-based cohort from the same region of the Netherlands. Allele frequencies in the 111 PD cases and 490 controls were compared using a 1-degree-of-freedom basic chi-square test. A P value < 0.05 after Bonferroni correction for the nine tested single nucleotide polymorphisms (SNPs) was considered statistically significant (i.e., P < 0.0056). Association of genetic markers (SNPs) with PD. We observed significant association with SNP rs4730775 at the wingless-type MMTV integration site family member 2 (WNT2) locus on chromosome 7 (P = 0.0015, OR 0.61), but found no evidence for the other eight loci being involved with PD despite the large effect size seen for some of these variants in DD. The WNT2 association was even more significant after we removed 15 patients with comorbid DD. WNT2 is a susceptibility locus for PD and our finding provides evidence for a partly shared genetic susceptibility between PD and DD.

Abstract 5201: Suppression of WNT1 expression inhibits the maintenance and expansion of cancer stem cells in a mouse model of breast cancer

Abstract Cancer relapse is the most common cause of mortality in breast cancer patients. The cancer stem cell (CSC) hypothesis proposes that CSCs are the root of cancer. CSC-targeted therapies may prevent cancer relapse and provide more effective treatment. It has been known that stem cells can be enriched in non-adherent sphere culture. To identify molecular targets in breast CSC, we compared transcription levels of stem cell-related genes between tumorspheres established from Balb/c mice-derived 4T1 mammary carcinoma and mammospheres from the mammary glands of Balb/c mice. The most differentially expressed gene was found to be Wingless-type MMTV integration site family, member 1 (WNT1). To determine whether WNT1 has any relevance in breast carcinoma, we analyzed the expression of WNT1 in the human matched normal breast and breast cancer samples. Immunofluorescent staining showed that WNT1 expression was elevated ∼17-63-fold in cancerous tissues compared with non-cancerous tissues from the same breast cancer patients. Functionally, knockdown of WNT1 in 4T1 cells reduced the Aldefluorpos cell compartment enriched in CSCs and suppressed the properties of CSCs, including anti-apoptosis, sphere-forming ability, and tumor initiation ability in vivo. Collectively, these results show that WNT1 expression gives rise to properties of CSC in breast cancer. Therefore, the targeting of WNT1-associated signaling may provide an effective therapeutic approach in the treatment of advanced breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5201. doi:1538-7445.AM2012-5201

Receptor tyrosine kinase–like orphan receptor 2 (ROR2); wingless-type MMTV integration site family member 5A (WNT5A)

Receptor tyrosine kinase–like orphan receptor 2 (ROR2); wingless-type MMTV integration site family member 5A (WNT5A)

MED12 mutations in uterine fibroids—their relationship to cytogenetic subgroups

Recurrent chromosomal alterations are found in roughly 20% of all uterine fibroids but in the majority cytogenetic changes are lacking. Recently, mutations of the gene mediator subcomplex 12 (MED12) have been detected in a majority of fibroids but no information is available whether or not they co-occur with cytogenetic subtypes as, e.g., rearrangements of the genes encoding high mobility group AT-hook (HMGA) proteins. In a total of 80 cytogenetically characterized fibroids from 50 patients, we were not only able to confirm the frequent occurrence of MED12 mutations but also to stratify two mutually exclusive pathways of leiomyomagenesis with either rearrangements of HMGA2 reflected by clonal chromosome abnormalities affecting 12q14~15 or by mutations affecting exon 2 of MED12. On average the latter mutations were associated with a significantly smaller tumor size. However, G>A transitions of nucleotides c.130 or c.131 correlate with a significantly larger size of the fibroids compared to other MED12 mutations thus explaining the high prevalence of the former mutations among clinically detectable fibroids. Interestingly, fibroids with MED12 mutations expressed significantly higher levels of the gene encoding wingless-type MMTV integration site family, member 4 (WNT4). Based on these findings and data from the literature, we hypothesize that estrogen and the mutated MED12 cooperate in activating the Wnt pathway which in turn activates β-catenin known to cause leiomyoma-like lesions in a mouse model. The occurrence of a "fibroid-type mutation" in a rare histologic subtype of endometrial polyps suggests that this mechanism is not confined to uterine leiomyomas.

WNT6 is a novel target gene of caveolin-1 promoting chemoresistance to epirubicin in human gastric cancer cells

Resistance to chemotherapy is a major obstacle for curative treatment of human gastric cancer (GC). However, the underlying molecular mechanisms are largely unknown. Wingless-type MMTV integration site family members (WNTs) are secreted glycoproteins involved in embryogenesis and, on inappropriate expression in the adult, in cancer. Here, we show expression of WNT6 in GC patient specimens, human GC cell lines and in a mouse model of GC. In human GC cells, WNT6 expression was enhanced by caveolin-1 (Cav1), a scaffold protein of plasma membrane caveolae. WNT6 knock-down and overexpression experiments demonstrated that WNT6 increased the resistance to apoptotic cell death induced by the anthracycline chemotherapeutics epirubicin (Epi) and doxorubicin (Dox). Epi increased the activity of the human WNT6 promoter through Cav1-dependent binding of β-catenin to the proximal WNT6 promoter. Epi increased both WNT6/Wnt6 and Cav1 expression in human GC cells and within the tumor area of a murine model of GC (CEA424-SV40 TAg). In GC patients, WNT6 expression was positively associated with the tumor stage and the nodal status, and inversely correlated with the response to ECF (Epi, cisplatin, 5-fluorouracil) chemotherapy. These results showed that WNT6 and Cav1 are upregulated by chemotherapeutics and enhance the resistance of GC cells to anthracycline drugs. Understanding the molecular mechanisms driving WNT6/Cav1-induced drug resistance will provide benefits in developing new therapies for GC.