Abstract 1175: Lossof claudin-3 expression induces de-differentiation in colonic epithelial cellsto promote colon cancer malignancy and associates with poor patient survival

Abstract

Abstract Dysregulation of colonocyte differentiation, imposing a crypt progenitor phenotype, characterize colorectal cancer (CRC) progression. Thus, an improved understanding of the molecular processes that regulate colonocyte differentiation can help identify novel therapeutic targets and biomarkers. In this regard, we found claudin-3, a tight junction integral protein, to be the highest expressed cell-cell adhesion moiety in the normal colonic epithelium and particularly concentrated amongst terminally differentiated colonocytes at the crypt top. We therefore postulated a key role for claudin-3 in maintaining colonocyte differentiation and negative association with colon tumorigenesis. In accordance, claudin-3 expression was low in 10 out of 14 CRC cells lines tested and undetectable in poorly differentiated and highly tumorigenic HCT116 and SW620 cell lines. In further analysis, using mRNA and protein expression, and utilizing samples from a large patient cohort (<250 CRC specimen), we found claudin-3 expression to be significantly suppressed (p<0.001 versus normal) in cancer tissues versus normal mucosa. The colon tissues from the established mouse models of colon cancer (APCmin mice and Azoxymethane (AOM)-DSS-induced colon cancer) demonstrated similar tumor specific decrease in claudin-3 expression. Interestingly, claudin-3 negative tumors retained E-cadherin expression. Most notably, we found a significant and positive correlation (p<0.001) of the greater levels of claudin-3 expression with patient survival. Genetic manipulation studies using colon cancer cells further supported a causal role of claudin-3 in upholding colonocyte differentiation, by modulating ZEB-1 protein synthesis and Wnt-signaling activation (Topflash promoter activity, p<0.05; claudin-3 siRNA versus control cells), and inhibiting cancer cell mobility and invasive ability. Importantly, homozygous deletion of claudin-3 expression in mice similarly inhibited colonocyte differentiation (down-regulated P-27 expression and up-regulated Vimentin expression), and promoted colon cancer growth and invasion when subjected to the AOM-DSS-induced mouse model of colorectal cancer (p<0.001 versus WT littermates). Pharmacological manipulations further revealed epigenetic regulation as potential mechanism for the inhibition of caludin-3 expression in CRC. Taken together, our data reveal a tissue-specific and inverse causal association of claudin-3 expression with CRC progression and patient survival, and highlights key importance of claudin-3 in maintaining colonocyte differentiation in Wnt-ZEB1-dependent manner. Citation Format: Rizwan Ahmad, Zhimin Chen, Balawant Kumar, Xi Chen, Mary kay Washington, Punita Dhawan, Amar B. Singh. Lossof claudin-3 expression induces de-differentiation in colonic epithelial cellsto promote colon cancer malignancy and associates with poor patient survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1175.