Frequent epigenetic inactivation of the RASSF1A gene in hepatocellular carcinoma.

Undraga Schagdarsurengin,Doris Steinemann,Gerd P Pfeifer,Brigitte Schlegelberger,Reinhard Dammann,Hans H Kreipe,Ludwig Wilkens,Peer Flemming

doi:10.1038/sj.onc.1206338

Abstract

Aberrant promoter methylation is a fundamental mechanism 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, hepatocirrhosis. Out of 15, 14 (93%) HCC were methylated at the RASSF1A CpG island and hypermethylation was independent 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 methylation 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 RARbeta2. 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. Therefore, 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.

Highlights

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies worldwide, with Eastern Asia and sub-Saharan Africa being the most prevalent regions (Schafer and Sorrell, 1999; Flemming et al, 2001)
Aberrant promoter methylation and loss of heterozygosity (LOH) were detected in noncancerous hepatitis and cirrhosis from patients with HCC (Kondo et al, 2000; Roncalli et al, 2002)
We analysed the expression of Ras association domain family 1A gene (RASSF1A) in HCC cell lines and we used bisulfite methylationspecific PCR (MSP) to investigate the aberrant hypermethylation of RASSF1A and other tumor suppressor genes (p16INK4A, TIMP3, PTEN, CDH1, and RARb2) in primary HCC, hepatocellular adenoma and in HCC cancer cell lines

Summary

Introduction

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies worldwide, with Eastern Asia and sub-Saharan Africa being the most prevalent regions (Schafer and Sorrell, 1999; Flemming et al, 2001). Aberrant promoter methylation and LOH were detected in noncancerous hepatitis and cirrhosis from patients with HCC (Kondo et al, 2000; Roncalli et al, 2002). We analysed the expression of RASSF1A in HCC cell lines and we used bisulfite methylationspecific PCR (MSP) to investigate the aberrant hypermethylation of RASSF1A and other tumor suppressor genes (p16INK4A, TIMP3, PTEN, CDH1, and RARb2) in primary HCC, hepatocellular adenoma and in HCC cancer cell lines.

Results

Conclusion