VEZT, a novel putative tumor suppressor, suppresses the growth and tumorigenicity of gastric cancer

Abstract

Vezatin (VEZT), an adherens junctions transmembrane protein, was identified as a putative tumor suppressor in our previous study. However, the role of VEZT in tumorigenesis remains elusive. We aimed to clarify its epigenetic regulation and biological functions in gastric cancer. In this study, we show that the expression level of VEZT is involved in lymphatic metastasis, depth of cancer invasion and TNM stage in 104 gastric cancer patients. Bisulfate sequencing polymerase chain reaction (BSP) methods showed that VEZT was hypermethylated in tissues and corresponding blood of gastric cancer patients compared with healthy controls. Helicobacter pylori (H. pylori) infection induces the methylation and silencing of VEZT in GES-1 cells. Restoring VEZT expression in MKN-45 and NCI-N87 gastric cancer cells inhibited growth, invasion and tumorigenesis in vitro and in vivo. Global microarray analysis was applied to analyze the molecular basis of the biological functions of VEZT after VEZT transfection combined with real-time PCR and chromatin immunoprecipitation assay. G protein-coupled receptor 56(GPR56), cell growth, cell division cycle 42(CDC42), migration/invasion and transcription factor 19(TCF19), cell cycle progression, were identified as direct VEZT target genes. TCF19, a novel target of VEZT, was functionally validated. Overexpression of TCF19 in MKN-45 cells increased cell cycle progress and growth ability. This study provides novel insight into the regulation of the VEZT gene, which could represent a potential target for therapeutic anti-cancer strategies.

Figure 1. Methylation analysis of normal gastric tissues, primary gastric cancer tissues and peripheral blood from gastric carcinoma patients and healthy controls.

(A) Online bioinformatics software was used to analyze the promoter region and the methylation status of the VEZT gene. (B) Methylation status of 34 CpG sites of the VEZT promoter from 30 normal gastric tissues and 30 primary gastric cancer tissues. Primary tumor tissues showed higher methylation levels in the VEZT promoter region when compared with normal gastric tissues. (C) Methylation status of 34 CpG sites of the VEZT promoter from peripheral blood plasma DNA of 30 gastric carcinoma patients and 30 healthy controls. Peripheral blood from gastric carcinoma patients showed higher methylation levels in VEZT promoter region when compared with healthy controls. Each row of circles represents an integrated methylation ratio from three clones, and each circle represents a single CpG site. Open circle represents unmethylated cytosine, whereas filled circles or partially filled circles represent the methylated ratio of CpG sites.

Figure 2. Helicobacter pylori infection promotes the methylation and silencing of VEZT.

(A) H. pylori-positive gastritis patients showed higher methylation levels in the VEZT promoter region when compared with H. pylori-negative gastritis patients. Each row of circles represents an integrated methylation ratio from three clones, and each circle represents a single CpG site. Open circle represents unmethylated cytosine, whereas filled circles or partially filled circles represent the methylated ratio of CpG sites. (B) After 24-h infection with H. pylori, the attachment of H. pylori was observed by transmission electron microscopy on the surface of GES-1 cells in the experimental cells relative to the control cells. (C) VEZT expression level in GES-1 cells was reduced after a 24-h H. pylori infection relative to negative control cells; however, H. pylori infection induced the IL-6, AKT, TNF-α, IL-8, ATF3 and IRX5 expression by western blot analysis. (D) The methylation of the VEZT promoter was detected by MSP after a 24-h H. pylori infection in GES-1 cells (marked as M), whereas methylation of the VEZT promoter in GES-1 cells that were not infected with H. pylori was not observed (marked as U). (E) Schematic summary of 34 CpG sites in the promoter region of the VEZT gene from -171 to -428 by BSP analysis. GES-1 cells showed a higher level of methylation after H. pylori infection relative to the control samples. The bar represents 5000 nm.

Figure 3. Function of VEZT in gastric cancer cells.

(A) The different expression levels of VEZT in five gastric cancer cell lines and one immortalized gastric mucosal cell line. (B) Expression of VEZT was upregulated in MKN-45 and NCI-N87 cells upon VEZT vector transfection relative to N1-controls. (C) Invasive, migratory and tubular formation capacities of VEZT-transfected MKN-45 and NCI-N87 cells were suppressed as determined by the transwell and tubular formation assays. (D) Overexpression of VEZT leads to cell growth arrest as determined by the CCK-8 assay. (E) Colony formation rates were significantly different between VEZT-transfected cells and N1-controls in MKN-45 and NCI-N87 cells. (F) The overexpression of VEZT in MKN-45 and NCI-N87 cells inhibited tumorigenesis in nude mice. Tumor nodules resected from the VEZT-transfected group were smaller than those from N1-controls. (G) Tumor growth curves for the VEZT/N1 group and N1-controls show rapid tumor growth in the MKN-45 or NCI-N87/N1control groups. *P < 0.05. Each bar represents the mean value ± standard deviation from three independent experiments.

Figure 4. Target gene identification by global microarray analysis.

(A) Clustering map of differentially expressed genes overlapped with cancer-associated genes set in the Molecular Signatures Database. Row represents gene, column represents experimental cells. Upregulated genes are shown in red (left) and downregulated genes in green (right). (B) Upregulation of HMGN5, ITGA5, PGM3, MXD1, ATF3, CDIPT, FOXP1 and GPR56 were confirmed (up). Downregulation of URI1, HOXD3, PLCD1, RAB4A, PXN, LTBP4, TCF19, IL-8, CDC42 and DSTN were confirmed (down). (c) Chromatin immunoprecipitation analysis was done with VEZT antibody using lysates from MKN-45 cells. Promoter pulldowns were assessed through quantitative PCR, which revealed amplified GPR56, TCF19 and CDC42 compared with the negative IgG control.

Figure 5. Molecular mechanism of the inhibitory activity of VEZT on gastric cancer growth.

(A) TCF19-transfected MKN-45 showed increased percentage of G2/M phase cells and decreased percentage of S and G0/ G1 phase cells. (B) Over-expression of TCF19 promoted cell growth by CCK-8 assay. (C) Schematic summary of the VEZT tumor suppressor gene on carcinogenesis of gastric cancer. VEZT is inactivated via hypermethylation, which may be induced by H. pylori infection. Restoring VEZT expression inhibits cell proliferation, migration, invasion and tumorigenesis both in vitro and in vivo, which could be explained by the downregulation of specific target genes identified by global microarray analysis. *P < 0.05. Each bar represents the mean value ± standard deviation from three independent experiments.