Methylation profiling of tumor suppressor genes and oncogenes in hepatitis virus-related hepatocellular carcinoma in northern India Manjula Kiran a , Yogesh K. Chawla b , Jyotdeep Kaur a, * a Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160 012, India b Department of Hepatology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160 012, India Received 14 May 2009; received in revised form 17 June 2009; accepted 20 June 2009 Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer in India, and hepatitis B virus and hepatitis C virus infections are major risk factors. DNA methylation alterations have been linked to various carcinomas in different populations. Aberrant CpG island methylation of genes has been recognized in HCC, information is limited for hepatitis viruserelated hepatocarcinogen- esis. HCC risk has not previously been associated with gene-specific DNA methylation in India. Promoter region methylation of a panel of six tumor suppressor genes (CDKN2A, CDKN2B, CDH1, GSTP1, SOCS1, and APC ) and three oncogenes (MYC, HRAS, and KRAS) was determined by methylation-specific PCR among 23 HCC samples and 20 control hepatitis samples. CDKN2B methylation frequency in HCC was double that for hepatitis, and methylation allele density of APC, GSTP1, and CDKN2B increased 2.2-, 2.3-, and 7.6-fold, respectively. Epigenetic silencing of tumor suppressor genes starts during viral infection and progresses toward HCC with the chronicity of the disease. Findings of altered methylation status support involvement of these tumor suppressor genes in HCC. MYC showed decreased methylation in HCC, relative to hepatitis. These observations on DNA methylation suggest the involvement of CDKN2B, SOCS1, CDH1, GSTP1, and MYC in path- ogenesis of HCC in India and implicate altered DNA methylation in the molecular pathogen- esis. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Hepatocellular carcinoma (HCC) is the third most common cancer worldwide, with annual incidence of more than 600,000 [1]. Asia has the highest number of cases (~76% globally) followed by Africa, Europe, the United States, and Australia. India, however, has been classified as a low-incidence zone for area, with !5 cases/100,000 population. Various epidemiological studies have shown the major risk factors for HCC to be infection with hepatitis B virus (HBV) and hepatitis C virus (HCV), which together account for three-quarters of all HCC cases worldwide. Hepatitis B virus is known to become integrated into the host genome and undergo epigenetic changes along with the host DNA [2]. Hepatitis C virus, however, indirectly induces hepatocarcinogenesis by modulating apoptosis and inflammatory responses. There are studies in which chronic hepatitis patients were monitored for years until HCC development, and not all patients with HBV or HCV infection develop HCC. This implies that, in addition to virus-related changes, there may be various genetic or epigenetic changes playing an important role in modifying cancer risk [2]. The local demethylation of promoter CpG islands has been linked to activation of the otherwise transcriptionally silenced oncogenes [3]. Also, the hypermethylation in the promoter region has been reported to inactivate the tumor suppressor genes [4]. Profiling of the DNA methylation in cancer is thus very much desirable. In HCC, a growing number of genes have been recog- nized as undergoing aberrant methylation at CpG islands, suggesting it to be an important molecular mechanism for the development of HCC [5,6]. Hypermethylation has been found to be associated with the inactivation of virtually all pathways involved with the cancer development, including cell cycle regulation (CDKN2A, alias p16; CDKN2B, alias p15), metabolism and energy pathway (GSTP1), cell adhe- sion (CDH1, or E-cadherin; APC ), and the JAK/STAT pathway (SOCS1). Also, in HBV- or HCV-related chronic * Corresponding author. Tel.: þ91-172-2755181; fax: þ91-172- 2744401/2745078. E-mail address: jyotdeep2001@yahoo.co.in (J. Kaur). 0165-4608/09/$ e see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2009.06.021 Cancer Genetics and Cytogenetics 195 (2009) 112e119