Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis

Abstract

To determine the methylation profile of multiple tumor-related genes during multistep hepatocarcinogenesis, we investigated the methylation status of CpG islands of 9 genes, using methylation-specific polymerase chain reaction for 60 paired hepatocellular carcinoma (HCC) and non-HCC liver tissue samples, 22 dysplastic nodule (DN), 30 liver cirrhosis (LC), 34 chronic hepatitis (CH) and 20 normal liver samples. The methylation status of 9 genes was correlated to the clinicopathological findings of HCC patients. All HCC samples showed methylation of at least one gene, whereas it was shown in 72.7% of DN and 40% of LC, but was not shown in CH and normal liver samples (P < 0.001). The number of genes methylated showed a stepwise increase with the progression of stages (0 for normal liver and CH, 0.5 for LC, 1.5 for DN, and 3.7 for HCC (P < 0.001)). The genes frequently methylated in HCC were APC (81.7%), GSTP1 (76.7%), RASSF1A (66.7%), p16 (48.3%), COX-2 (35%), and E-cadherin (33.3%). COX-2, p16, RASSF1A, and TIMP-3 were not methylated in LC and CH from patients without concurrent HCC. Chronic liver diseases with concurrent HCC showed higher methylation frequencies of the tested genes, and a higher number of methylated genes than those without concurrent HCC. HCC patients with methylation of E-cadherin or GSTP1 showed poorer survival than those without (P = 0.034 and 0.043, respectively). In conclusion, our results indicated that CpG island methylation of tumor-related genes is an early and frequent event, and accumulates step-by-step during a multistep hepatocarcinogenesis. CpG island methylation of E-cadherin or GSTP1 might serve as a potential biomarker for prognostication of HCC patients.

Figure 1.

Representative examples of MSP analysis of APC, COX-2, E-cadherin, DAP-kinase, GSTP1, p16, RASSF1A, TIMP3, and hMLH1 in HCC, corresponding non-cancerous liver tissue, DN, LC, and CH samples. DNA extracted from 226 liver tissues was amplified with primers specific to the unmethylated (U) or the methylated (M) CpG islands of each gene after modification with sodium bisulfite.

Figure 2.

Summary of methylation analysis of APC, COX-2, E-cadherin, DAP-kinase, GSTP1, p16, RASSF1A, TIMP3, and hMLH1 in 226 liver samples. Filled boxes indicates the presence of methylation and open boxes indicates the absence of methylation. DX, diagnosis; T, tumor; N, paired non-tumorous liver tissue; No*, case number; No^†, number of genes methylated; No^‡, number of MINT loci methylated; CIMP-P, CpG island methylator phenotype-positive cases; CIMP-N, CIMP-negative cases; DAP-k, DAP-kinase; E-cad, E-cadherin.

Figure 3.

Frequencies of CpG island methylation of APC, COX-2, DAP-kinase, E-cadherin, GSTP1, hMLH1, p16, RASSF1A, and TIMP3 in normal liver tissue, preneoplastic lesions, and hepatocellular carcinoma samples.

Figure 4.

Overall survival of hepatocellular carcinoma patients according to the methylation status of GSTP1 (A) and E-cadherin (B). Solid line, patients without methylation of examined gene; dotted line, patients with methylation of examined gene.