Researchers find that reinstating BVES expression in CRC reverses EMT and slows metastasis in mice.
Scientists have identified a potential new target for preventing metastasis in epithelial tumors such as colorectal cancer (CRC). Studies showed that BVES (blood vessel epicardial substance), a protein involved in regulating the formation of tight junctions (TJs) between epithelial cells, is downregulated in colorectal cancer, allowing the cancer cells to undergo epithelial-mesenchymal transition (EMT), the first stage in cancer metastasis.
The Vanderbilt University School of Medicine team’s work found that re-expressing BVES in CRC cell lines effectively blocked EMT, resulted in cells converting to a more nonmalignant phenotype, and impaired the growth and metastasis of tumors in experimental mice. The scientists claim downregulation of BVES expression occurs early in the course of cancer development and is caused by hypermethylation of the gene’s promoter.
The studies are reported in The Journal of Clinical Investigation, in a paper titled “BVES regulates EMT in human corneal and colon cancer cells and is silenced via promoter methylation in human colorectal carcinoma.”
The Vanderbilt team’s research built on previous studies demonstrating a role for BVES in modulating the transition between epithelial and mesenchymal phenotypes during embryogenesis. These studies also showed that ectopic expression of BVES attenuates CRC tumor growth and metastasis in orthotopic xenografts. They hypothesized that if BVES controls epithelial-mesenchymal phenotypes in epithelial cancers by regulating junctional complex formation, then its expression in these cancers might be reduced.
Screening a multitissue cancer panel for BVES mRNA levels confirmed that expression of the gene was, indeed, markedly reduced in colon and breast cancers when compared with respective normal tissues. The findings were verified through analysis of the Moffit Cancer Center/Vanderbilt Medical Center colon cancer expression array data set, which comprises another 250 tumor samples organized by tumor stage and includes samples from adjacent normal tissue.
Tumor tissue from these samples demonstrated far lower BVES transcript levels than the normal adjacent mucosal specimens. BVES transcription was also depressed in premalignant lesions (adenomas), suggesting BVES loss occurs early in the tumorigenic process, the authors note. The findings for BVES mRNA were mirrored at the protein level: immunofluorescence studies showed that in CRC tissue the BVES protein was virtually lacking, whereas it was clearly evident and correctly localized in the normal colonic mucosa.
Interestingly, there was an inverse relationship between BVES and ZEB1 transcription levels in the tumor dataset. ZEB1 promotes EMT by repressing junction-related proteins such as E-cadherin, and increased expression of this gene is associated with mesenchymal phenotype. The inverse relationship between BVES and ZEB1 thus reinforces the hypothesis that BVES loss favors mesenchymal transcriptional programs in CRC, the researchers suggest.
They moved on to evaluate BVES mRNA in a panel of CRC cell lines. Levels of the transcript were either low or undetectable in 12/13 of the cancer lines evaluated. Relatively high levels of BVES mRNA were only expressed in the Caco-2 cancer cell line displayed, which, significantly, is also capable of forming a differentiated, polarized epithelium, they add.
Quantitative bisulfite pyrosequencing analysis of 18 matched CRC and normal tissues identified hypermethylation of the BVES promoter as the probable cause of the gene’s downregulation in CRC. While the BVES promoter demonstrated low methylation levels (averaging at 4.9%) in normal samples, in cancerous tissue methylation was significantly increased at each individual CpG site examined and reached an average of 26.6%.
“Analysis of our expression array data indicated that for these 18 samples, BVES transcript levels were inversely related to promoter methylation status, consistent with increased methylation silencing gene expression,” the researchers state.
Dramatically increased BVES promoter methylation was similarly found in 12 of the 13 cancer cell lines previously assessed for mRNA levels. The exception, again, was in the Caco-2 cells. While the other 12 cell lines demonstrated BVES promoter methylation levels of 80–90%, which correlated with the general absence of transcripts, BVES promoter methylation in Caco-2 cells was much lower, at about 30%, correlating with higher levels of the transcript in these cells.
The role of promoter methylation in transcriptional regulation of BVES was confirmed by treating cancer cells with the demethylation agent 5-aza-2′-deoxycytidine. In these cells BVES promoter DNA methylation was decreased, and BVES gene expression restored.
“These results indicate that promoter hypermethylation is one mechanism mediating transcriptional silencing of BVES in colorectal adenocarcinoma,” the authors remark. “It is possible that DNA methylation represents a common mechanism by which BVES is silenced in multiple solid tumors.”
The findings thus far indicated linked loss of BVES expression with a mesenchymal phenotype in CRC cells. What wasn’t yet clear was whether this plays an active role in modulating epithelial-mesenchymal phenotypes or is merely a bystander in the malignant transformation process.
The researchers answered this question through the engineering of LIM2405 colorectal cancer cells, which normally exhibit prominent mesenchymal features and express little or no detectable BVES. They used stable transfection techniques to generate a mixed population of LIM2405 cells that stably express a wild-type BVES and multiple BVES-expressing LIM2405 clones.
Immunofluorescence microscopy and immunoblot analyses confirmed the re-establishment of BVES expression in the cells and that the resulting BVES protein displayed polarized localization similar to TJ protein expression patterns in normal, nonmalignant epithelium. The LIM2405 colorectal cancer cell line is characterized by prominent mesenchymal features including anchorage- and cell contact-independent growth.
Restoration of BVES expression in the transfected cells, however, resulted in the re-establishment of epithelial features. The cells grew in flat, adherent sheets. Pooled or clonal BVES-expressing LIM2405 cells exhibited increased transepithelial resistance (TER), a marker for epithelial polarization. Proliferation rate in the BVES-expressing cells was also reduced.
“Consistent with epithelial characteristics, BVES expression attenuated LIM2405 cell migration by 66%, and reduced anchorage-independent growth by 53.5%,” the researchers add.
Epithelial traits were separately found to be reinstated following BVES re-expression in both an ocular melanoma cell line and a murine breast cancer cell line, both of which would normally show low BVES levels. “Collectively, these experiments demonstrate that BVES can reverse mesenchymal growth characteristics of carcinoma cells, resulting in restoration of a more epithelial phenotype.”
Previous work by the team had suggested that transient manipulation of BVES could regulate EMT: In these studies they showed that transient disruption of either BVES expression or its cell membrane localization effectively attenuated cell-cell contact and resulted in epithelial cells assuming a fibroblast morphology.
To test whether loss of BVES function does actually promote EMT, the investigators stably transfected HCE cells with carboxyterminal truncated DN-BVES, which results in abnormal BVES trafficking and has a dominant-negative effect. The resulting HCE-DN cells demonstrated a very different morphology from control HCE cells and HCE-WT cells (that stably overexpress a wild-type HCE).
HCE and HCE-WT cell proliferation was associated with the formation of organized cell clusters and normal-looking epithelial monolayers that displayed cell membrane adhesion junction proteins in an expected pattern. In contrast, the HCE-DN cells demonstrated attenuated cell-cell contact and proliferated excessively, forming irregular foci that exhibited little or no junctioanl proteins at the cell membrane. HCE-DN cells in addition displayed increased motility in comparison with both HCE and HCE-WT cells.
Significantly, there was a direct relationship between BVES expression in transfected cancer cell lines and the expression of junction proteins including E-cadherin. Further analyses suggested that BVES may play a role in regulating TJ and adherent junction (AJ) signaling programs and, indeed, cross-talk between the two.
The team finally evaluated the effects of BVES expression on tumor growth in mice. BVES nonexpressing LIM2405 cells and three different transfected BVES-expressing clones were transplanted into nude mice. While the nonexpressing cancer cells formed large tumors within 20 days, the three BVES-expressing LIM2405 clones showed a roughly 8-fold reduction in tumor growth. Evaluation of one of the BVES-expressing tumors indicated an increase in apoptosis, without any change in cell proliferation, indicating that at least a partial effect of introducing BVES expression is restoration of cell death programs, the researchers state.
“This study provides direct evidence that BVES regulates EMT/MET in a reversible manner in normal and cancer epithelial cells,” they conclude. “BVES transcriptional suppression occurs during early tumorigenesis, raising the possibility that BVES could be a therapeutic or preventative target in CRC and perhaps other solid tumor malignancies.”