Identification of genes preferentially methylated in hepatitis C virus-related hepatocellular carcinoma

Chronic infections by hepatitis B virus (HBV) and hepatitis C virus (HCV) appear to be the most significant causes of hepatocellular carcinoma (HCC). Aberrant promoter methylation is known to be deeply involved in cancer, including in HCC. In this study, we analyzed aberrant promoter methylation by methylated DNA immunoprecipitation-on-chip analysis on a genome-wide scale in six HCCs including three HBV-related and three HCV-related HCCs, six matched noncancerous liver tissues, and three normal liver tissues. Candidate genes with promoter methylation were detected more frequently in HCV-related HCC. Candidate genes methylated preferentially to HBV-related or HCV-related HCCs were detected and selected, and methylation levels of the selected genes were validated by quantitative methylation analysis using MALDI-TOF mass spectrometry using 125 liver tissue samples, including 61 HCCs (28 HBV-related HCCs and 33 HCV-related HCCs) and 59 matched noncancerous livers, and five normal livers. Among analyzed genes, preferential methylation in HBV-related HCC was validated in one gene only. However, 15 genes were found to be methylated preferentially in HCV-related HCC, which was independent from age. Hierarchical clustering of HCC using these genes stratified HCV-related HCC as a cluster of frequently methylated samples. The 15 genes included genes inhibitory to cancer-related signaling such as RAS/RAF/ERK and Wnt/beta-catenin pathways. Methylation of dual specificity phosphatase 4 (DUSP4), cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1), and natriuretic peptide receptor A (NPR1) significantly correlated with recurrence-free survival. It was indicated that genes methylated preferentially in HCV-related HCC exist, and that DNA methylation might play an important role in HCV-related HCC by silencing cancer-related pathway inhibitors, and might perhaps be useful as a prognostic marker.