Frequent epigenetic inactivation of the RASSF1A gene in hepatocellular carcinoma Undraga Schagdarsurengin 1 , Ludwig Wilkens 2 , Doris Steinemann 3 , Peer Flemming 2 , Hans H Kreipe 2 , Gerd P Pfeifer 4 , Brigitte Schlegelberger 3 and Reinhard Dammann* , 1 1 AG Tumorgenetik der Medizinischen Fakulta¨t, Martin-Luther-Universita¨t Halle-Wittenberg, 06097 Halle/Saale, Germany; 2 Institut fu¨r Pathologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany; 3 Institut fu¨r Zell- und Molekularpathologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany; 4 Department of Biology, Beckman Research Institute, City of Hope Cancer Center, Duarte, CA 91010, USA Aberrant promoter methylation is a fundamental mechan- ism of inactivation of tumor suppressor genes in cancer. The Ras association domain family 1A gene (RASSF1A) is frequently epigenetically silenced in several types of human solid tumors. In this study, we have investigated the expression and methylation status of the RASSF1A gene in hepatocellular carcinoma (HCC). In two HCC cell lines (HepG2 and Hep3B) RASSF1A was inactivated and treatment of these cell lines with a DNA methylation inhibitor reactivated the transcription of RASSF1A. The methylation status of the RASSF1A promoter region was analysed in 26 primary liver tissues including HCC, hepatocellular adenoma (HCA), liver fibrosis, hepatocir- rhosis. Out of 15, 14 (93%) HCC were methylated at the RASSF1A CpG island and hypermethylation was inde- pendent of hepatitis virus infection. RASSF1A was also methylated in two out of two fibrosis and in three (75%) out of four cirrhosis; the latter carries an increased risk of developing HCC. Additionally, we analysed the methyla- tion status of p16 INK4a and other cancer-related genes in the same liver tumors. Aberrant methylation in the HCC samples was detected in 71% of samples for p16, 25% for TIMP3, 17% for PTEN, 13% for CDH1, and 7% for RARb2. In conclusion, our results demonstrate that RASSF1A and p16 INK4a inactivation by methylation are frequent events in hepatocellular carcinoma, but not in HCA, which is in contrast to HCC without cirrhosis, viral hepatitis, storage diseases, or genetic background. There- fore, this study gives additional evidence against a progression of adenoma to carcinoma in the liver. Thus, RASSF1A hypermethylation could be useful as a marker of malignancy and to distinguish between the distinct forms of highly differentiated liver neoplasm. Oncogene (2003) 22, 1866–1871. doi:10.1038/sj.onc.1206338 Keywords: hepatocellular carcinoma; hepatocellular adenoma;methylation; RASSF1;tumorsuppressorgene Hepatocellular carcinoma (HCC) is one of the most frequentmalignanciesworldwide,withEasternAsiaand sub-Saharan Africa being the most prevalent regions (Schafer and Sorrell, 1999; Flemming et al., 2001). Chronic exposure to aflatoxin B1 is thought to be a major etiological mechanism in these geographic re- gions. Epidemiologic data indicate that hepatitis B (HBV) and C (HCV) virus are major risk factors for thedevelopmentofHCC(SchaferandSorrell,1999).In Europe and the United States chronic alcoholism may contribute to hepatocellular carcinogenesis. The devel- opment of HCC is a multistep process, which is also suggestedbyhistologicalstudies(Feitelson etal.,2002). HCC appears within the context of chronic viral hepatitis, cirrhosis, and distinct storage diseases as tyrosinemia, glycogenoses, and genetic hemochro- matosis. Inactivation of tumor suppressor genes occurs by mutations, loss of heterozygosity (LOH), and/or epige- netic silencing (Esteller and Herman, 2002; Jones and Baylin,2002).InHCC,inactivationof p16 (Wong etal., 1999; Shen etal., 2002) and GSTP1 gene (Zhong etal., 2002) is common. Aberrant promoter methylation and LOH were detected in noncancerous hepatitis and cirrhosis from patients with HCC (Kondo et al., 2000; Roncalli et al., 2002). In HCC, LOH on several chromosomal arms including 1p, 4q, 5q, 6q, 8p, 10q, 11p, 13q, 16q, and 17p has been reported (Boige etal., 1997; Marchio etal., 1997; Wilkens etal.,2001)andin recentstudies,LOHonchromosome3pwasidentifiedin cirrhosisandHCC(Roncalli etal.,2000;Li etal.,2001). Inseveraltypesofhumancarcinoma,LOHof3pisone of the most frequent and earliest events in the pathogenesis of cancer (Kok et al., 1997; Wistuba et al., 2000). Recently, we and others have cloned and character- ized the Ras association domain family 1A gene (RASSF1)fromthelungcancertumorsuppressorlocus 3p21.3 (Dammann et al., 2000; Lerman and Minna, 2000; Burbee et al., 2001). The two main forms (RASSF1A and RASSF1C)aretranscribedfromdistinct CpG island promoters. The RASSF1A promoter is intensively hypermethylated in a variety of primary human cancers, for example, in lung, breast, ovarian, Received 28 August 2002; revised 19 December 2002; accepted 19 December 2002 *Correspondence: R Dammann, Institut fu¨r Humangenetik und Medizinische Biologie, Martin-Luther-Universita¨t Halle-Wittenberg, Magdeburger Strasse 2, 06097 Halle/Saale, Germany; E-mail: reinhard.dammann@medizin.uni-halle.de Oncogene (2003) 22, 1866–1871 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc