Background/aims: In human hepatocellular carcinoma (HCC) the ras-proto-oncogene is rarely mutated. We therefore studied the possible inactivation of the putative tumor-suppressors and ras-associating proteins, NORE1A, NORE1B, and RASSF1A in HCCs by mutation or epigenetic gene silencing through promoter-CpG hypermethylation.
Methods: SSCP-analyses, sequencing, and methylation-specific PCR were performed in 28 fibrotic/cirrhotic livers and 40 HCCs.
Results: The sequence of NORE1A/B exhibited no deviations and that of the RASSF1A gene a non-silent polymorphism ( approximately 10% of cases) and a missense mutation (one HCC). Both alterations may affect the growth-inhibiting capability of RASSF1A. Epigenetic inactivation of NORE1B was found in 62% of the HCCs and in hepatocarcinoma-cell lines due to considerable promoter-methylation of the gene. Methylation was detected also for RASSF1A in HCCs and hepatocarcinoma cell-lines. As a result, 97% of the HCCs revealed epigenetic silencing of NORE1B, RASSF1A, or both. In contrast every third fibrotic/cirrhotic liver only exhibited silencing of one or both genes.
Conclusions: The candidate tumor suppressor genes NORE1B and RASSF1A are epigenetically down-regulated alone in at least 62%, or in combination in 97% of the HCCs studied. This indicates a frequent and critical event in hepatocarcinogenesis, which may allow HCCs to subverse growth-control in the presence of an unaltered Ras.