DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection

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Abstract

Hepatocellular carcinoma (HCC) is known to be associated with both HBV and HCV. While epigenetic changes have been previously reported to be associated with hepatocellular carcinoma (HCC), whether the epigenetic profile of HBC associated HCC differs from that of HCV-associated HCC is unclear. We analyzed DNA methylation of ten genes (APC, CCND2, CDKN2A, GSTP1, HOXA9, RARB, RASSF1, RUNX, SFRP1, and TWIST1) using MethyLight assays on 65 archived liver tissue blocks. Three genes (APC, CCND2, and GSTP1) were frequently methylated in normal liver tissues. Five genes (APC, CDKN2A, HOXA9, RASSF1, and RUNX) were significantly more frequently methylated in malignant liver tissues than normal liver tissues. Among HCC cases, HOXA9, RASSF1 and SFRP1 were methylated more frequently in HBV-positive HCC cases, while CDKN2A were significantly more frequently methylated in HCV-positive HCC cases. Our data support the hypothesis that HCC resulting from different viral etiologies is associated with different epigenetic changes.

Introduction

Hepatocellular carcinoma (HCC), the predominant form of human liver cancer, is the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death, accounting for approximately 600,000 deaths per year (Bosch et al., 2005, Thomas and Zhu, 2005). Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the major etiological factors for HCC, accounting for 80% of all HCC cases. It is hypothesized that HBV and HCV infection lead to hepatocarcinogenesis through increased hepatocyte regeneration and turnover leading to inflammation, oxidative DNA damage and chronic hepatitis. This microenvironment sets the stage for malignant transformation of hepatocytes through accumulation of both genetic and epigenetic changes (Levrero, 2006). Alternatively, specific viral proteins can directly lead to cell transformation, including X-protein encoded by HBV (HBx), core, NS3, NS4B and NS5A proteins encoded by HCV (Koike, 2002, Koike, 2005, Levrero, 2006).

Since HBV is a DNA virus, which integrates into the genome, while HCV is an RNA virus, which does not involve a DNA intermediate, it is likely that different mechanisms are involved in hepatocarcinogenesis caused by these two viruses (Farazi and DePinho, 2006, Kirk et al., 2006, Levrero, 2006). Epidemiologically, HBV infection in childhood is more likely to cause HCC in young adults, while HCV infection rarely causes HCC in adults younger than 50 years old (Kirk et al., 2006). The male-to-female ratio is higher in HBV-caused HCC than HCV-caused HCC (Shiratori et al., 1995). Patients co-infected with HBV and HCV are at increased risk for developing HCC compared to singly infected patients, further supporting the hypothesis of different pathways between the two viruses. Morphologically, HBV-caused HCCs tend to be infiltrative and multinodular, while HCV-caused HCCs tend to be solitary, smaller in size and encapsulated (But et al., 2008). However, at the molecular level, both loss of heterozygosity (LOH) and microarray analyses have failed to consistently identify specific genetic alterations and gene expression changes associated with specific viral infection (Thorgeirsson et al., 2006).

Epigenetic alterations, such as DNA methylation, play an important role in tumorigenesis (Jones and Baylin, 2002, Laird, 2003). DNA methylation, referring here to the addition of a methyl group to the cytosine in the CpG dinucleotides, when occurring at the promoter region of a gene, leads to gene silencing. Consequently, DNA methylation of a tumor suppressor gene has the equivalent effect as genetic mutations to inactivate the gene. Recent studies have reported the detection of DNA methylation of various panels of genes during the stepwise progression of HCC. For example, DNA methylation of four genes (Col1A2, IGFBP2, CTGF, and fibronectin 1) has been shown to progressively increase from normal liver, chronic hepatitis, liver cirrhosis to hepatoma (Chiba et al., 2005, Kwon et al., 2005). Similarly, methylation of p16 (CDKN2A), p15 and SFRP1 is present not only in HCC, but is present also at low frequencies in chronic hepatitis and liver cirrhosis samples (Fukai et al., 2005, Shih et al., 2006), all supporting the hypothesis that CpG island methylation of tumor-related genes is an early and frequent event, and methylation changes accumulate during a multistep hepatocarcinogenesis (Lee et al., 2003). In addition, several studies have investigated the methylation patterns associated with specific viral infection without definitive conclusion. Jicai reported finding that p16 methylation preferentially occurred in liver cancerous tissues with HBV infections, as compared to those without HBV infection (Jicai et al., 2006), while others have reported higher frequency of p16 methylation in HCV-HCCs than HBV-HCCs (Katoh et al., 2006, Li et al., 2004, Narimatsu et al., 2004). These studies were confounded by other risk factors such as age, ethnic background and geographic locations, which might have lead to differential methylation patterns.

In the present study we compared the epigenetic profile of normal liver tissue, HBV-associated HCC, and HCV-associated HCC using archived liver tissues biopsies, after adjusting for relevant co-factors.

Section snippets

Collection of clinical tissue specimens

Archived formalin-fixed paraffin-embedded tissue blocks were obtained from University of Washington Medical Center and Harborview Medical Center. A total of 62 cancerous liver tissue blocks from 40 HCC patients had sufficient material for the study, with 30 subjects having a single block, and 10 subjects having 2–8 blocks each. In addition, 25 normal liver biopsy samples from 25 liver transplant donors were used as normal control tissue for this study. Age, gender, race, and HBV, HCV serology

Study population

The baseline characteristics of the 25 normal and 40 HCC subjects are summarized in Table 1. Subjects with HCC were older than normal subjects (53.7 vs. 40.6; p = 0.0005), but both were approximately one-quarter female. Approximately half of both cases and controls were Caucasian; however 30% of subjects with HCC were Asian, while 40% of normal subjects were either Black or Hispanic. Only 16% of normal subjects were seropositive for HBV or HCV, while 30% of HCC subjects were seropositive for HBV

Discussion

We determined the methylation status by the MethyLight assay of ten genes in both normal and malignant liver tissues. We have identified three genes that were frequently methylated (in approximately 50% of patients) in normal liver tissues. Five additional genes were significantly more frequently methylated in HCC tissues compared to normal liver tissues. Finally, we observed that methylation of CDKN2A was frequent in HCV, but not HBV-associated HCC. These data suggest that epigenetic

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    Current address: Institute of Dermatology, Chinese Academy of Medical Sciences, Nanjing, P.R.China.

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