Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma

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Abstract
One of the main regulatory pathways reported to be altered in hepatocellular carcinoma (HCC) is that of cell cycle control involving RB1 gene-related cell inhibitors. We investigated p14ARF , p15INK4B , p16INK4A , p18INK4C , and RB1 genes in a series of HCCs and associated cirrhosis with the goal of ascertaining their pattern of inactivation by gene methylation. Thirty-three HCCs, adjacent nonneoplastic cirrhotic tissues, and 6 HCC cell lines were studied. Cirrhoses (25 of 33, 76%), HCCs (31 of 33, 94%), and 3 of 6 (50%) cell lines showed 1 or more methylated genes. Cirrhoses (17 of 33, 51%) had more frequently than HCCs (11 of 33, 33%, P = .01) only 1 methylated gene. With the exception of p18INK4C the genes under study showed promoter methylation with frequency ranging from 82% (p16INK4A in HCC) to 33% and 39% (p15INK4B and p16INK4A in cirrhoses). In cases with only 1 methylated gene, p15INK4B in cirrhosis (8 of 17, 47%) and p16INK4A in HCC (10 of 11, 91%) were the more frequently altered. An optimal correlation was found between p15 and p16 gene methylation and complete protein loss in HCC detected by immunocytochemistry, whereas a partial loss of the same proteins was a feature of methylated cirrhoses. Inactivation by DNA methylation of several genes of the RB1 pathway is common to cirrhosis and HCC. An early pattern of methylatory events (1 methylated gene) is a feature of cirrhosis rather than HCC, whereas an advanced one (≥3 methylated genes) is characteristic of malignancy. Early methylation changes seem to involve p15INK4B and p16INK4A in cirrhosis and p16INK4A in HCC. In conclusion, a stepwise progression of methylating events is a feature of the sequence cirrhosis-HCC and contributes to the process of hepatic carcinogenesis with potential clinical implications.