Abstract. Aberrant methylation of CpG islands in the promoter region of genes is a common epigenetic phenomenon found in early cancers. Therefore conducting genome-scale methylation studies will enhance our understanding of the epigenetic etiology behind carcinogenesis by providing reliable biomarkers for early detection of cancer. To discover novel hypermethylated genes in colorectal cancer by genome-wide search, we first defined a subset of genes epigenetically reactivated in colon cancer cells after treatment with a demethylating agent. Next, we identified another subset of genes with relatively down-regulated expression patterns in colorectal primary tumors when compared with normal appearing-adjacent regions. Among 29 genes obtained by cross-comparison of the two gene-sets, we subsequently selected, through stepwise subtraction processes, two novel genes, GABRA1 and LAMA2, as methylation targets in colorectal cancer. For clinical validation pyrosequencing was used to assess methylation in 134 matched tissue samples from CRC patients. Aberrant methylation at target CpG sites in GABRA1 and LAMA2 was observed with high frequency in tumor tissues (92.5% and 80.6%, respectively), while less frequently in matched tumor-adjacent normal tissues (33.6% for GABRA1 and 13.4% for LAMA2). Methylation levels in primary tumors were not significantly correlated with clinicopathological features including age, sex, survival and TNM stage. Additionally, we found that ectopic overexpression of GABRA1 in colon cancer cell lines resulted in strong inhibition of cell growth. These results suggest that two novel hypermethylated genes in colorectal cancer, GABRA1 and LAMA2, may have roles in colorectal tumorigenesis and could be potential biomarkers for the screening and the detection of colorectal cancer in clinical practice.
IntroductionColorectal cancer (CRC) is one of the most common types of neoplasia in developed countries and is the second leading cause of cancer related death (1,2). The mean 5-year survival rate for CRC is estimated to be <10% once metastasis occurs, but it would be greater than 90% if the cancer is found in an early stage (3). So far, digital rectal exams, fecal occult blood tests, sigmoidoscopy and colonoscopy have been used as acceptable options for early detection of CRC. But these methods lack the needed sensitivity and could be supplemented with a more sensitive and efficient screening assays using novel biomarkers. During carcinogenesis, genetic and epigenetic alterations of tumor suppressor genes are believed to play a pathogenic role (4,5). Epigenetic gene silencing of tumor suppressor genes, due to hypermethylation of CpG islands in their promoter region, is known to frequently occur in the early stages of cancer development. Such aberrant events can be utilized as moleculardiagnostic biomarkers for early detection and risk identification in cancer patients (6).Methylation status of p16, p14, hMLH1, TSP1, GSTP1 and MGMT genes have been intensively studied in CRC (7-12). However, detection of such...
