The tumor suppressive role of HGF activator inhibitor 2(HAI-2) in hepatocellular carcinoma

Abstract

Epigenetic alteration is one of the important mechanisms in inactivation of tumor suppressor genes in different types of cancer including hepatocellular carcinoma (HCC). In this study, we aimed to identify epigenetically silenced candidate tumor suppressor genes in human HCC. Following treatment with 5-aza-2'-deoxycytidine (5-aza-dC) in high-throughput cDNA microarray methylation study, the expression of HGF activator inhibitors (HAIs), HAI-1 and HAI-2, was found to be significantly upregulated in two of the three HCC cell lines (Hep3B, SMMC7721, and BEL7402). HAIs belong to Kunitz-type serine protease inhibitor family that are synthesized as integral transmembrane proteins and subsequently released from the cell surface by proteolysis. They inhibit the generation of biologically active hepatocyte growth factor (HGF) through their interaction with HGF activator (HGFA). In addition, HAIs inhibit a wide variety of serine proteases including matriptase, hepsin, trypsin, plasmin, tissue and plasma kallikreins and factor XIa. The mRNA expression of HAI-2 was either absent or low in 14 HCC cell lines, but 5-Aza-dC treatment significantly upregulated HAI-2 expression in 11 (79%) of these cell lines. The mRNA levels of HAI-2 were found to be significantly under-expressed in ~80% of human HCCs as compared with their corresponding nontumorous livers. HAI-2 promoter methylation was detected in 71% of human HCCs. Using bisulfite DNA sequencing, we confirmed that the promoter of HAI-2 gene was frequently hypermethylated in the HCC cell lines and human HCCs, and HAI-2 promoter hypermethylation was associated with the low mRNA expression levels of HAI-2. These results showed that HAI-2 were frequently hypermethylated and under-expressed in human HCCs. Ectopic expression of HAI-2 in SMMC cells significantly suppressed the growth of subcutaneous tumors in nude mice. To further elucidate the effect of HAI proteins, HAI-2 mutants were constructed. Changes of the P1 amino acids in the functional Kunitz domains (KD1 and KD2) of HAI-2 were introduced by site-directed mutagenesis. We demonstrated that, in contrast, the HAI-2 with mutated KD1 domain, which is the major functional domain of human HAI-2 protein, failed to suppress the growth of the subcutaneous tumors in nude mice. However, the mutated KD2 domain did not affect the suppressive function of HAI-2 in the mice injection assay. Our findings suggest that HAI-2 inactivation may play a role in the development of HCC and KD1 domain of HAI-2 is implicated to be the key region responsible for the tumor suppressive function.

(This study was funded in part by the Michael Kadoorie Cancer Genetic Research Program of the Kadoorie Charitable Foundation)