Extensive Methylation Is Associated with β-Catenin Mutations in Hepatocellular Carcinoma: Evidence for Two Distinct Pathways of Human Hepatocarcinogenesis

Naoshi Nishida,Goel Ajay,C Richard Boland,Takafumi Nishimura,Takeshi Nagasaka,Iwao Ikai

doi:10.1158/0008-5472.can-06-3464

Abstract

Hepatocellular carcinoma (HCC) with p53 mutations is usually characterized by extensive chromosomal instability (CIN), whereas those with beta-catenin mutations have relatively less CIN and the molecular pathogenesis of these tumors is unknown. Methylation of CpG dinucleotides in the promoters of cancer-related genes is another characteristic feature of HCCs. The aim of this study was to determine the contribution of the methylator phenotype to HCC and its relationship to genomic instability. Fractional allelic loss (FAL) was determined using 400 microsatellite markers in 81 HCCs and 77 corresponding noncancerous livers as a measure of CIN. Methylation of 21 genetic loci was quantitated using combined bisulfite restriction analysis. Using hierarchical clustering analysis based upon the quantification of methylation levels, all HCCs were segregated into two groups characterized by either limited or extensive methylation. Mutations in the beta-catenin and p53 genes were determined by DNA sequencing. We found that the methylation levels were significantly higher in the HCCs than in noncancerous livers in 18 of the 21 loci (P values ranged from 0.035 to <0.0001). Among 18 loci, elevated levels of methylation at nine loci were significantly associated with beta-catenin mutations (P values ranged from 0.02 to <0.0001). In addition, the presence of beta-catenin mutations was associated with HCCs in the extensive methylation group (P < 0.0001), whereas p53 mutations correlated with high FAL scores (P = 0.0036). These data suggest that HCCs can be classified into two distinct categories based upon promoter methylation, CIN, and mutations of cancer-related genes. HCCs with extensive methylation harbor frequent beta-catenin mutations, whereas HCCs with high levels of CIN are associated with p53 mutations, suggesting the presence of two independent pathways for the pathogenesis of HCC.

Highlights

Hepatocellular carcinoma (HCC) is a common malignancy worldwide, and the overall prevalence of the at-risk population is expected to grow with time
For characterization of HCCs according to the methylation status, quantitative methylation profiling is necessary, as methylation densities are not uniform in all tumors and these have biological implications for gene silencing
We have analyzed a large subset of HCCs using quantitative methylation analysis for several gene promoters and provide evidence that a significant majority of HCC is characterized by an extensive methylation phenotype

Summary

Introduction

Hepatocellular carcinoma (HCC) is a common malignancy worldwide, and the overall prevalence of the at-risk population is expected to grow with time. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Doi:10.1158/0008-5472.CAN-06-3464 several distinct risk factors [1], and different forms of genetic and epigenetic alterations have been described in HCC [2]. Previous reports suggest that human HCC with p53 mutations tend to have extensive chromosomal instability (CIN), whereas others have mutations in b-catenin and relatively little CIN [3]. These findings are consistent with animals studies in which liver tumors in transgenic mice have been classified based upon CIN and b-catenin mutations [4]

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