Frequent epigenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma.

Methylation associated inactivation of RASSF1, a putative tumor suppressor identified at 3p21.3, has been frequently observed in several human malignancies, including lung and breast cancers. To explore the penetrance of RASSF1 in gastric carcinogenesis, we performed expression and mutation analyses of 3 isotypes of RASSF1 (A, B, and C) in 150 gastric specimens, including 15 carcinoma cell lines. RASSF1A and RASSF1B transcripts were not expressed in 60% (9 of 15) and 33% (5 of 15) of gastric carcinoma cell lines, respectively, whereas RASSF1C was detectable in all cell lines. Bisulfite DNA sequencing analysis revealed that the CpG island in the RASSF1A promoter is hypermethylated in all RASSF1A-nonexpressing cell lines. In addition, both RASSF1A and RASSF1B were re-expressed by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Among 90 primary gastric adenocarcinomas examined, 41 (46%) and 19 (21%) expressed no or abnormally low levels of RASSF1A and RASSF1B, respectively, and 12 (13%) tumors showed no expression of both isoforms. Loss or abnormal down-regulation of RASSF1A correlated with tumor stage and grade but not with histological types of tumors. Methylation-specific PCR analysis demonstrated that 95% (39 of 41) of RASSF1A-nonexpressing primary tumors are methylated at the CpG sites in the promoter, whereas none of the adjacent noncancerous or normal tissues are methylated. No somatic mutations were detected in RASSF1 transcripts expressed in unmethylated tumors. However, 10 methylated tumors, including 4 cell lines, showed low genomic levels of RASSF1 and expressed no RASSF1A transcripts, suggesting that RASSF1A inactivation might be caused by both epigenetic and genetic mechanisms in a subset of gastric adenocarcinomas. In conclusion, our data indicate that epigenetic transcriptional silencing of RASSF1, especially RASSF1A isoform, is a frequent event in gastric tumorigenesis and might play an important role in the malignant progression of gastric adenocarcinomas.