Epigenetic Regulation of Tumor Suppressor AKAP12 in Human Hepatocellular Carcinoma

Abstract

Molecular defects leading to hepatocellular carcinoma (HCC), one of the more frequent cancers worldwide, are poorly understood. We have previously shown a downregulation of the tumor suppressor A-kinase Anchoring Protein 12 (AKAP12) in HCC and premalignant lesions. Here, we concentrated on epigenetic gene silencing that may cause the downregulation of AKAP12 in hepatocarcinogenesis. BioCOBRA was used to achieve semi-quantitative DNA methylation data of the AKAP12 gene promoter in a panel of representative specimens (n=39). This analysis revealed a mean DNA methylation of 25.1% for HCC, 2.4% for CL, and 6.6% for NL, thus indicating a hypermethylation of the AKAP12 promoter in human HCC specimens. Additionally, human HCC cell lines (HepG2, Hep3B, HuH7, PLC, AKN-1) as well as the nontumorigenic liver epithelial cell line HACL-1 were tested. Elevated methylation levels were observed in 50% (3/6) of the tested cell lines. The analysis revealed 91% (AKN-1), 34.9% (HepG2), 28.5% (Hep3B), 2.1% (HUH7), 1.5% (PLC) and 0% (HACL-1). Consecutively, restoration experiments using 5-aza-2’-deoxycytidine (5-aza-dC) as demethylating agent were performed. The two cell lines with the highest DNA methylation levels, AKN-1 and HepG2 were tested and the 5-aza-dC treatment resulted in both cell lines in a decrease in promoter methylation paralleled by an increase in AKAP12 mRNA expression indicating a correlation between gene expression and promoter methylation for AKAP12. Hence, our findings show that there is a distinctive downregulation of AKAP12 in human hepatocarcinogenesis, and downregulation of AKAP12 in HCC may be due to epigenetic silencing of the AKAP12 promoter.