Protein kinase D (PKD) 1 influences cell migration by mediating both trans-Golgi vesicle fission and integrin recycling to the cell surface. Using restriction landmark genomic scanning methods, we found that the promoter region of PKD1 was aberrantly methylated in gastric cancer cell lines. Silencing of PKD1 expression was detected in 72.7% of gastric cancer cell lines examined, and the silencing was associated with CpG hypermethylation in the promoter region of PKD1. Treatment with 5-aza-2#-deoxycytidine and trichostatin A partially reversed PKD1 methylation and restored gene expression in PKD1-silenced cell lines. Real-time reverse transcription-polymerase chain reaction analysis of 96 paired clinical primary gastric cancer samples revealed that 59% of the analyzed tumors had a >2-fold decrease in PKD1 expression compared with each normal-appearing tissue and that this downregulation of PKD1 expression was significantly correlated with increased methylation. We also observed a gradual increase in the level of promoter methylation of PKD1 in aging, normal-appearing mucosal tissues, suggesting that PKD1 methylation may be one of the earliest events that predispose an individual to gastric cancer. PKD1 expression was required for directional migration of gastric cancer cells. Furthermore, knock down of PKD1 by RNA interference promoted the invasiveness of cell lines that expressed PKD1 at relatively high levels. Based on these results, we propose that PKD1 is frequently silenced by epigenetic regulation, which plays a role in cell migration and metastasis in gastric cancer.
In this study, the promoter of the gene coiled-coil domain-containing 67 (CCDC67) was found to be frequently methylated in gastric cancer cell lines and in primary gastric tumors, as examined by restriction landmark genomic scanning. In addition, CCDC67 expression was down-regulated in 72.7% of gastric cancer cell lines tested. In most cases, gene down-regulation was associated with CpG hypermethylation in the CCDC67 promoter. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin A restored CCDC67 expression in down-regulated cell lines. Pyrosequencing analysis of 150 paired primary gastric cancer samples revealed that promoter CpG methylation was increased in 74% of tested tumors compared with paired adjacent normal tissues, and this hypermethylation correlated significantly with down-regulation of CCDC67. CCDC67 protein was localized to the cell membrane by immunocytochemistry. Stable transfection of a CCDC67 gene in one gastric cancer cell line inhibited adhesion-dependent and -independent colony formation, and CCDC67 expression suppressed tumorigenesis in nude mice. We suggest that CCDC67 is a putative tumor suppressor gene that is silenced in gastric cancers by promoter CpG methylation and that it may play an important role in cell signaling and migration related to tumorigenesis.
Transcriptional factor 4 (TCF4), encoding a basic helix-loop-helix transcriptional factor, has recently been demonstrated as a causative gene for Pitt-Hopkins syndrome, a neurodevelopmental disease. Examination of gastric cancers using the restriction landmark genomic scanning technique revealed methylation at a NotI enzyme site in TCF4 intron 8 and further identified CpG dinucleotide hypermethylation in TCF4 exon 1, strongly associated with gene silencing in gastric cancer cell lines. Treatment with 5-aza-2′-deoxycytidine and/or trichostatin A restored TCF4 expression in TCF4-silenced gastric cancer cell lines. Real-time reverse transcription–polymerase chain reaction analysis of 77 paired primary gastric tumor samples revealed that 38% of analyzed tumors had a >2-fold decrease in TCF4 expression compared with adjacent normal-appearing tissue, and the decrease significantly correlated with increased CpG methylation in TCF4 exon 1. Clinicopathologic data showed that decreased TCF4 expression occurred significantly more frequently in intestinal-type (22/37, 59%) than in diffuse-type (7/37, 19%) gastric cancers (P = 0.0004) and likewise more frequently in early (12/18, 67%) than in advanced (17/59, 29%) gastric cancers (P = 0.004). CpG methylation markedly increased with patient age among normal-appearing tissues, suggesting that CpG methylation in gastric mucosa may be one of the earliest events in carcinogenesis of intestinal-type gastric cancers. Furthermore, ectopic expression of TCF4 decreased cell growth in a gastric cancer cell line, and the knock down of TCF4 using small interfering RNA increased cell migration. Based on these results, we propose that the observed frequent epigenetic-mediated TCF4 silencing plays a role in tumor formation and progression.
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