BVES regulates EMT in human corneal and colon cancer cells and is silenced via promoter methylation in human colorectal carcinoma
Christopher S. Williams, … , R. Daniel Beauchamp, Min S. Chang
Christopher S. Williams, … , R. Daniel Beauchamp, Min S. Chang
Published September 12, 2011
Citation Information: J Clin Invest. 2011;121(10):4056-4069. https://doi.org/10.1172/JCI44228.
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Research Article Oncology

BVES regulates EMT in human corneal and colon cancer cells and is silenced via promoter methylation in human colorectal carcinoma

Abstract

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.

Authors

Christopher S. Williams, Baolin Zhang, J. Joshua Smith, Ashwath Jayagopal, Caitlyn W. Barrett, Christopher Pino, Patricia Russ, Sai H. Presley, DunFa Peng, Daniel O. Rosenblatt, Frederick R. Haselton, Jin-Long Yang, M. Kay Washington, Xi Chen, Steven Eschrich, Timothy J. Yeatman, Wael El-Rifai, R. Daniel Beauchamp, Min S. Chang

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Figure 7

BVES regulates TJ-associated RhoA signaling.

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BVES regulates TJ-associated RhoA signaling. (A) Immunofluorescent local...
(A) Immunofluorescent localization studies for ZO-1 (green) and GEF-H1 (red) in LIM2405-P (LIM-P), LIM2405-V (LIM-V), and LIM-32 clones overexpressing BVES. LIM-32 cells exhibit corresponding cell membrane localization of ZO-1 and GEF-H1 as indicated on the merged image (yellow, cell border). Both LIM2405-P and LIM2405-V exhibit only scattered regions of cell membrane distribution of either ZO-1 or GEF-H1, with little corresponding cell membrane localization (original magnification, ×400). (B) Immuno­purification of BVES and GEF-H1. Lysates of cells transiently overexpressing Flag-tagged BVES revealed co-immunopurification of BVES and GEF-H1. Either FLAG or BVES antibodies were used to precipitate proteins. (L= Molelecular Weight Marker). (C) RhoA activity assay measured on LIM2405 and pooled BVES LIM2405 (LIM2405 pBVES) cells after GEF-H1 transfection. Immunoblot of RhoA total protein levels. ***P < 0.005. (D) Ratios of phosphorylated MLC (p-myosin) over total MLC (myosin) were obtained through densitometric analysis of immunoblots (n = 4) of LIM2405 (P), WT BVES (LIM-32, LIM-15, LIM-9) clones, and dominant-negative LIM (DN). WT-BVES clones exhibit a significant decrease in phosphorylated myosin compared the LIM2405-P, while the DN LIM cells exhibited a significant increase in p-myosin. (E) Boyden chamber invasion assay of LIM2405 or pooled BVES LIM2405 cells, comparing effects of ROCK inhibition via treatment with Y-27362 or vehicle (control) (n = 3). *P < 0.05, **P < 0.005, Student’s t test. (F) Schematic of BVES modulating TJ and AJ.