Abstract
WNT signaling activation in colorectal cancers (CRCs) occurs through APC inactivation or β-catenin mutations. Both processes promote β-catenin nuclear accumulation, which up-regulates epithelial-to-mesenchymal transition (EMT). We investigated β-catenin localization, transcriptome, and phenotypic differences of HCT116 cells containing a wild-type (HCT116-WT) or mutant β-catenin allele (HCT116-MT), or parental cells with both WT and mutant alleles (HCT116-P). We then analyzed β-catenin expression and associated phenotypes in CRC tissues. Wild-type β-catenin showed membranous localization, whereas mutant showed nuclear localization; both nuclear and non-nuclear localization were observed in HCT116-P. Microarray analysis revealed down-regulation of Claudin-7 and E-cadherin in HCT116-MT vs. HCT116-WT. Claudin-7 was also down-regulated in HCT116-P vs. HCT116-WT without E-cadherin dysregulation. We found that ZEB1 is a critical EMT factor for mutant β-catenin-mediated loss of E-cadherin and Claudin-7 in HCT116-P and HCT116-MT cells. We also demonstrated that E-cadherin binds to both WT and mutant β-catenin, and loss of E-cadherin releases β-catenin from the cell membrane and leads to its degradation. Alteration of Claudin-7, as well as both Claudin-7 and E-cadherin respectively caused tight junction (TJ) impairment in HCT116-P, and dual loss of TJs and adherens junctions (AJs) in HCT116-MT. TJ loss increased cell motility, and subsequent AJ loss further up-regulated that. Immunohistochemistry analysis of 101 CRCs revealed high (14.9%), low (52.5%), and undetectable (32.6%) β-catenin nuclear expression, and high β-catenin nuclear expression was significantly correlated with overall survival of CRC patients (P = 0.009). Our findings suggest that β-catenin activation induces EMT progression by modifying cell-cell junctions, and thereby contributes to CRC aggressiveness.
Highlights
WNT signaling activation in colorectal cancers (CRCs) occurs through adenomatous polyposis coli (APC) inactivation or β-catenin mutations
Loss of cell-cell junctions is critical for epithelialto-mesenchymal transition (EMT) progression; and its clinical significance has been reported in CRC21, relationship between loss of cell-cell junction and β-catenin-mediated EMT is poorly understood
Immunofluorescence microscopy and western blot analysis showed that CRCs with APC mutations more clearly display nuclear β-catenin localization compared www.nature.com/scientificreports to CRC cells with no Wnt pathway relevant mutations and hepatocellular carcinoma (HCC) cells with an AXIN1 mutation
Summary
WNT signaling activation in colorectal cancers (CRCs) occurs through APC inactivation or β-catenin mutations Both processes promote β-catenin nuclear accumulation, which up-regulates epithelialto-mesenchymal transition (EMT). Claudin and Occludin families facilitate tight sealing of cells in the epithelial sheet, whereas zonula occludins (ZO) protein-1, a linker molecule, mediates interaction between Claudins and Occludins and the actin cytoskeleton[11] Of these TJ molecules, abnormal expression of several Claudin proteins (e.g., Claudin-1, -3, -4, and -7) has been associated with tumorigenesis of various cancers, including CRCs. Claudin expression has been correlated with prognosis, invasion, and metastasis in CRCs. Claudin family members show heterogeneous expression patterns and even opposite roles in various types of cancers, and their expressional and functional relationships with β-catenin expression remain unclear[12]. We aimed to investigate the mechanism by which β-catenin activation affects cell-cell junctions during EMT progression using a panel of HCT116 cell lines with differential β-catenin mutation status
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