The acquisition of a mesenchymal phenotype is a critical step in the metastatic progression of epithelial carcinomas. Adherens junctions (AJs) are required for suppressing this epithelial-mesenchymal transition (EMT) but less is known about the role of tight junctions (TJs) in this process. Here, we investigated the functions of blood vessel epicardial substance (BVES, also known as POPDC1 and POP1), an integral membrane protein that regulates TJ formation. BVES was found to be underexpressed in all stages of human colorectal carcinoma (CRC) and in adenomatous polyps, indicating its suppression occurs early in transformation. Similarly, the majority of CRC cell lines tested exhibited decreased BVES expression and promoter DNA hypermethylation, a modification associated with transcriptional silencing. Treatment with a DNA-demethylating agent restored BVES expression in CRC cell lines, indicating that methylation represses BVES expression. Reexpression of BVES in CRC cell lines promoted an epithelial phenotype, featuring decreased proliferation, migration, invasion, and anchorage-independent growth; impaired growth of an orthotopic xenograft; and blocked metastasis. Conversely, interfering with BVES function by expressing a dominant-negative mutant in human corneal epithelial cells induced mesenchymal features. These biological outcomes were associated with changes in AJ and TJ composition and related signaling. Therefore, BVES prevents EMT, and its epigenetic silencing may be an important step in promoting EMT programs during colon carcinogenesis.

Notch signaling largely determines intestinal epithelial cell fate. High Notch activity drives progenitors toward absorptive enterocytes by repressing secretory differentiation programs, whereas low Notch permits secretory cell assignment. Myeloid translocation gene-related 1 (MTGR1) is a transcriptional corepressor in the myeloid translocation gene/Eight-Twenty-One family. Given that Mtgr1(-/-) mice have a dramatic reduction of intestinal epithelial secretory cells, we hypothesized that MTGR1 is a key repressor of Notch signaling. In support of this, transcriptome analysis of laser capture microdissected Mtgr1(-/-) intestinal crypts revealed Notch activation, and secretory markers Mucin2, Chromogranin A, and Growth factor-independent 1 (Gfi1) were down-regulated in Mtgr1(-/-) whole intestines and Mtgr1(-/-) enteroids. We demonstrate that MTGR1 is in a complex with Suppressor of Hairless Homolog, a key Notch effector, and represses Notch-induced Hairy/Enhancer of Split 1 activity. Moreover, pharmacologic Notch inhibition using a γ-secretase inhibitor (GSI) rescued the hyperproliferative baseline phenotype in the Mtgr1(-/-) intestine and increased production of goblet and enteroendocrine lineages in Mtgr1(-/-) mice. GSI increased Paneth cell production in wild-type mice but failed to do so in Mtgr1(-/-) mice. We determined that MTGR1 can interact with GFI1, a transcriptional corepressor required for Paneth cell differentiation, and repress GFI1 targets. Overall, the data suggest that MTGR1, a transcriptional corepressor well characterized in hematopoiesis, plays a critical role in intestinal lineage allocation.

BVES is novel adhesion protein that functions as a modulator of tight junction (TJ) formation with TJs level directly correlating with BVES expression. TJs, in addition to regulating paracellular passage of molecules, can also directly regulate Rho activity and transcriptional activation of cell cycle regulatory genes (ERBB2, PCNA, CDK4, and Cyclin-D1). Hence, alteration in BVES expression also modulates TJ associated signaling. Interestingly, overexpressing a dominant negative BVES induced human corneal epithelial cells to exhibit increased mesenchymal characteristics. Together, these observations led us to hypothesize that increasing BVES expression in carcinoma cells would induce an epithelial-like phenotype. To evaluate the function of BVES in carcinoma, a semi-adherent LIM2405 human colorectal cancer cell line was stably transfected with wild type BVES (WT-LIM2405) or empty vector (V-LIM2405) as control. Multiple stably transfected clonal isolates of LIM2405 overexpressing BVES exhibited increased epithelial features as evidenced by: i) uniform monolayer formation, ii) decreased vimentin and increased cytokeratin, Zo-1, and occludin expression, iii) decreased cellular motility (66% reduction, p<0.001), and iv) decreased colony number in soft-agar assays (53.5%, p<0.01). In addition, WT-LIM2405 cells progressively displayed reduced cellular proliferation, prolonged cell-doubling time (35%), and decreased S-phase and increased G1 fractions. This was more apparent with increasing confluence, suggesting that contact inhibition is restored with BVES expression. To evaluate the affect of BVES on tumor growth in vivo, V-LIM2405 and WT-LIM2405 cells were grown as orthotopic xenografts in athymic mice. WT-LIM2405 tumor growth was significantly attenuated (86%, p<0.001). Intra-tumoral proliferation rates were equivalent, however apoptosis was increased in WT-LIM2405 tumors (TUNEL positive cells/HPF, p=0.04). We next surveyed BVES expression in colorectal carcinoma. 80% of human CRCs samples exhibited at least a 40% reduction in BVES protein by immunoblotting. In addition, immunolocalization studies showed disorganized or absent BVES and ZO-1 staining only in carcinomatous regions. BVES expression is decrease not only in colorectal cancer (p=0.004), but also in breast (p=0.018) and thyroid carcinoma (p=0.20). Global expression analysis of 250 staged CRC samples compared with normal and adenoma colon specimens showed a decrease in BVES expression as early as carcinoma in situ (p<0.05). These findings indicate endogenous BVES expression is decrease in carcinoma and restoration of its’ expression induces an epithelial-like phenotype in colorectal cancer cells, thus implicating BVES as a tumor suppressor. Further investigation to determine the role of BVES in epithelial cancer biology is warranted and may lead to novel insight into targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3076.

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