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 3

BVES expression is silenced via promoter hypermethylation.

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BVES expression is silenced via promoter hypermethylation. (A) Schematic...
(A) Schematic representation of the human BVES promoter. The CpG island of BVES extends from –997 to +394 from TSS. Each red tick mark represents 1 CpG site. The arrows indicate the TSS. Pyosequencing assay to determine methylation level (%) was carried out on 7 CpG sites, as underlined by the black bar. TSS, transcriptional start site. (B) BVES promoter methylation status in colon adenocarcinoma compared with that in adjacent normal mucosa samples. Methylation level (%) at each of the 7 CpG dinucleotides was determined (left). For normal versus tumor comparisons, a horizontal bar represents the mean. The average level of the 7 CpG sites is shaded in gray. Individual symbols represent individual samples, and horizontal bars represent the mean. Before/after plot of matched samples, demonstrating 100% concordant increase in BVES methylation in cancers (right) (n = 18). P < 0.001, 2-tailed unpaired t test. N = normal; T = tumor tissue. (C) Pyrosequencing of BVES promoter DNA in the CRC panel. Data are presented as the average methylation percentage of 7 CpG pairs. (D) Pyrosequencing results of LIM2405 cells treated with either vehicle or 5-aza-2′-deoxycytidine for 72 hours (left). Data are presented as the average of triplicate measurements from duplicate experiments. qRT-PCR in LIM2405 cells showed detection of BVES mRNA following treatment with 5-aza-2′-deoxycytidine. RFU, relative fluorescence units. This demonstrates that BVES transcription in both clinical samples and CRC lines is silenced via promoter hypermethylation.