Promoter hypermethylation of Wnt pathway inhibitors in hepatitis C virus - induced multistep hepatocarcinogenesis

Background: Aberrant DNA methylation profiles are a characteristic feature of almost all types of cancers including hepatocellular carcinoma (HCC) and play an important role in carcinogenesis. In spite of the accumulating evidence that suggests appearance of such aberrations at precancerous stages, very little effort has been invested to investigate such possible methylation events in patients at risk of developing HCC i.e. those suffering from chronic hepatitis C virus (HCV) infection and liver cirrhosis (LC). We reasoned that such an analysis could lead to the identification of novel predictive biomarkers as well as potential drug targets. Methods: Promoter methylation status of two Wnt inhibitors SFRP2 and DKK1 was quantitatively analyzed by bisulfite pyrosequencing in a series of liver biopsy samples. These biopsies were collected from HCV-infected individuals suffering from chronic hepatitis (CH; n = 15), liver cirrhosis (LC; n = 13) and hepatocellular carcinoma (HCC; n = 41). DNA isolated from infection free normal livers (N; n = 10) was used as control. Results: Our analysis revealed that both of the genomic loci were significantly hypermethylated in CH patients' livers as compared to normal controls (p = 0.0136 & 0.0084 for SFRP2 and DKK1, respectively; Mann-Whitney U test). DNA methylation levels for both loci were also significantly higher in all the diseased cohorts as compared to normal controls (p < 0.0001 and = 0.0011 for SFRP2 and DKK1, respectively; Kruskal-Wallis test). However, a comparison between three disease cohorts (CH, LC & HCC) revealed no significant difference in levels of DNA methylation at DKK1 promoter. In contrast, a progressive increase in DNA methylation levels was observed at the SFRP2 promoter (i.e. N < CH & LC < HCC). Conclusion: This study demonstrated that in HCV infected liver tissues hypermethylation at promoter regions of key cancer related genes SFRP2 and DKK1, appears early at CH and LC stages, long before the appearance of HCC.