HAI‐2 is epigenetically downregulated in human hepatocellular carcinoma, and its Kunitz domain type 1 is critical for anti‐invasive functions

Abstract

Pharmacological demethylation-based gene expression profile analysis is a useful tool to identify epigenetically silenced tumour suppressor genes. HGF activator inhibitor 2 (HAI-2), a serine protease inhibitor, has been identified as one of the candidate tumour suppressor genes in human hepatocellular carcinoma (HCC) with this technique. In this study, we aimed to characterise the epigenetic status and tumour suppressive function of HAI-2 in HCC. We validated that HAI-2 expression was either absent or low in most of the HCC cell lines tested, and 5-Aza-2 0 -deoxycytidine treatment significantly restored its expression in 9 (75%) of these 12 cell lines. HAI-2 was found to be frequently underexpressed in human HCCs (p < 0.001). With bisulphite DNA sequencing and methylation-specific PCR, we found that the promoter of the HAI-2 gene was frequently hypermethylated in both HCC cell lines and human HCCs. Ectopic expression of HAI-2 significantly inhibited cell migration and invasiveness of HCC cells in vitro and suppressed tumourigenicity in vivo. In addition, we also provided the first evidence that HAI-2 mediated its tumour suppressor function via the Kunitz domain 1 (KD-1), as KD-1 but not KD-2 inactivating mutant abolished its anti-tumour invasiveness in vitro. Our findings suggest that HAI-2 is a candidate tumour suppressor gene that is frequently hypermethylated and underexpressed in human HCCs, and the KD-1 domain of HAI-2 is the key region responsible for its anti-invasive function. '

Methylation Profiles Reveal Distinct Subgroup of Hepatocellular Carcinoma Patients with Poor Prognosis

“…Two of these, GSTP1 [13], [40] and SPINT2 [41], [42] were previously reported tumor suppressor genes which were hypermethylated in tumors with corresponding down-regulated gene expression, while the other 2 (CYB5R2 and SH3YL1) represent potential novel tumor suppressor genes. Quantitative real-time PCR and pyrosequencing successfully validated all four genes (Figure 4C).…”

Aberrant Methylation of Promoter Region of SPINT2/HAI-2 Gene: An Epigenetic Mechanism in Hepatitis C Virus-Induced Hepatocarcinogenesis

“…Most of the studies dealing with the methylation profile in HCCs did not tackle the role of HCV in epigenetic changes during hepatocarcinogenesis. Some had less than five HCV-induced HCC cases (Deng et al, 2010;Ammerpohl et al, 2012), while others lacked normal controls and only included adjacent noncancerous liver tissues (Fukai et al, 2003;Su et al, 2008;Hernandez-Vargas et al, 2010;Shen et al, 2012;Shin et al, 2010;Shitani et al, 2012) as well as HCC cell lines (Tung et al, 2009). Moribe et al (2009) reported that the SPINT2/HAI-2 gene promoter was highly unmethylated in the normal liver from healthy individuals and in the liver from HCV carriers.…”

“…The inhibitory effect of SPINT2/HAI-2 promoter hypermethylation on its gene expression was proved by Tung et al (2009), using bisulfite sequencing and MSP; they found that most HCC cases had underexpression of the SPINT2/ HAI-2 gene when the promoter region was hypermethylated. It is noteworthy to mention that such a low expression noted in HCC can be hardly explained by somatic mutations of SPINT2/HAI-2, which are quite rare (Morris et al, 2005), thus favoring the role of epigenetics.…”

“…SPINT2/HAI-2 is an endogenous inhibitor of the HGF activator enzyme responsible for HGF activation and hence limits the signaling by the HGF/c-Met receptor pathway (Morris et al, 2005). The significantly underexpressed SPINT2/HAI-2 gene demonstrated in human HCC and HCC cell lines (Tung et al, 2009) and overexpressed c-Met in HCC correlated with poor prognosis (Ueki et al, 1997). Furthermore, HGF peptide levels were reported to be higher in the liver and serum of cirrhotic cases and correlated with occurrence of HCC (Yamagamim et al, 2002).…”

“…In addition, it inhibits the formation of active HGF by inhibiting the action of the HGF activator (Tung et al, 2009). It is recognized as a tumor suppressor gene and its underexpression has been reported in cancers, such as breast cancer (Parr et al, 2004) and glioblastoma (Hamasuna et al, 2001;Schuster et al, 2003).…”