Allele loss and epigenetic inactivation of 3p21.3 in malignant liver tumors.

Previously, the RASSF1A, BLU and SEMAPHORIN 3B (SEMA3B) candidate tumor suppressor genes on chromosome 3p21.3 were found to be inactivated and downregulated by genetic and epigenetic changes in lung cancer. We analyzed the methylation status of RASSF1A, BLU and SEMA3B in 35 hepatocellular carcinomas (HCCs) and 15 cholangiocarcinomas (CCs) by methylation-specific PCR and loss of heterozygosity (LOH) at 3p21.3 after microdissection. The presence of mRNA transcripts was confirmed by semiquantitative PCR. SEMA3B hypermethylation was found in 29/35 HCCs (83%) and in all (15/15) patients with CC. BLU promoter hypermethylation was detected in 7/35 (20%) HCCs and 3/15 (20%) CCs. In 2 corresponding specimens of hepatitis B virus-related liver cirrhosis, BLU methylation was also observed, but not in uninvolved normal liver tissue. RASSF1A was methylated in 21/35 HCCs (60%) and in 10/15 CCs (67%). LOH at 3p21.3 occurred in 8/35 (23%) HCCs and 3/15 (20%) CCs. The presence of hypermethylation was statistically associated with LOH of SEMA3B and correlated with downregulation of mRNA transcripts. SEMA3B transcripts increased upon treatment of HCC cell lines with the demethylation compound 5-aza-2-deoxycytidine. In conclusion, our data indicate that 2-hit gene silencing of SEMA3B through epigenetic changes and allele loss is a common and important event in the carcinogenesis of malignant liver tumors.