Epigenetic inactivation of serine protease inhibitor TFPI-2 in human HCC

Abstract

Liver cancer (hepatocellular carcinoma, HCC) is a prevalent cancer worldwide and is the second most common fatal cancer in Southeast Asia and Hong Kong. In liver cancer, tumor suppressor genes are known to play very important roles in carcinogenesis. Apart from deletions, epigenetic mechanisms such as aberrant promoter methylation and histone modification also significantly contribute to the inactivation of tumor suppressor genes. In this study, we used a high-throughput microarray technology to compare the changes of gene expression profiles of three HCC cell lines (Hep3B, SMMC-7721 and BEL-7402) after treatment by a global demethylating agent (5-aza-2’-deoxycytidine, 5-aza-2’-dC). By screening more than 15,000 genes, we identified a substantial number of genes with mRNA expression significantly restored after 5-aza-2’-dC treatment. About 300 transcripts were found to be upregulated in at least two of these three HCC cell lines. One of the candidate genes we identified with this approach is TFPI-2 (tissue-factor pathway inhibitor-2), a newly identified Kunitz-type serine protease inhibitor. TFPI-2 strongly inhibited the activity of trypsin, plasmin, and factor VIIa-tissue factor complex and was found to be suppresses cancer invasion both in vivo and in vitro. In this study, we aim at investigate the role of TFPI-2 in human HCC. We found that TFPI-2 mRNA expression was lower in HCC cell lines and was significantly upregulated with 5-Aza-dC treatment. Bisulfite DNA sequencing confirmed that the TFPI-2 promoter was hypermethylated in these HCC cell lines. In human HCC samples, with bisulfite DNA sequencing, we found that TFPI-2 promoter was hypermethylated in a subset of human HCCs. In addition, TFPI-2 mRNA expression was frequently downregulated in HCC by ≥2 folds, as compared with the corresponding non-tumorous livers. Our findings suggest that TFPI-2 was frequently inactivated in human HCC and epigenetic silencing of the TFPI-2 gene may contribute to human hepatocarcinogenesis. (This study was funded in part by Michael Kadoorie Cancer Genetic Research Program of the Kadoorie Charitable Foundation)