Hepatocyte growth factor activator inhibitor 2/placental bikunin (HAI-2/PB) gene is frequently hypermethylated in human hepatocellular carcinoma

We previously identified apolipoprotein D (Apo D) as a novel tumor suppressor gene based on the pharmacological unmasking of epigenetic silencing. We analyzed Apo D expression using realtime reverse transcription-PCR and evaluated its expression status based on the clinicopathological parameters of 70 patients with hepatocellular carcinoma (HCC). Immunohistochemical staining was also performed. We determined the methylation status of Apo D gene promoter by methylation-specific PCR (MSP). The Apo D gene-expression in tumor tissue was significantly lower than that in nontumor tissue (p = 0.011). A low Apo D expression significantly correlated with less-differentiated HCC ( p = 0.019). Immunohistochemical studies confirmed a decreased Apo D expression in poorly differentiated tumors. The prognosis of patients with a lower Apo D gene-expression was significantly worse than that in those with a higher expression (p = 0.028). The Apo D gene-expression was an independent prognostic factor (relative risk: 2.36, p = 0.018). An MSP assay showed a low-level of methylation in well differentiated HCC and a high-level of methylation in less differentiated tumors. Apo D may be a novel tumor suppressor gene of HCC, and its expression status may be a useful biomarker for predicting the patient outcome. ' 2005 Wiley-Liss, Inc.Key words: apolipoprotein D; hepatocellular carcinoma; histological grade; prognostic factor; methylationWe recently performed a comprehensive survey of commonly inactivated tumor suppressor genes in esophageal cancer cell lines based on functional reactivation by a demethylating agent. 1 A total of 58 silenced genes were thus identified using this approach, and one of those genes was Apolipoprotein D (Apo D). In our report, we detected the promoter hypermethylation of Apo D gene using bisulfite DNA sequencing. The methylation status of Apo D correlated closely with the Apo D expression status. Moreover, the growth suppressive activity of Apo D was also demonstrated by a colony focus assay. 1 These findings thus prompted us to analyze the clinical significance of the Apo D expression status in human cancers.Apo D, belonging to the lipocalin superfamily, is a glycoprotein of about 30 kDa found in the high-density lipoprotein fraction of human plasma. 2 The functional role of Apo D remains unclear, but the observation that this protein forms complexes with lecithin-cholesterol acyltransferase suggests that Apo D may be involved in cholesterol esterification. 3 More recently, the Apo D gene-expression was reported to be induced by retinoic acid in breast cancer cell lines. The induction of the Apo D expression was accompanied by an inhibition of cell proliferation and a progression through a more differentiated phenotype. These finding suggest that the mechanisms controlling retinoic acid-induced growth arrest, cell differentiation and Apo D synthesis may be directly coordinated in human breast cancer cells. 4 Furthermore, a low expression of Apo D determined by an immunohistochemical study was significantly as...

Section: Discussionmentioning

confidence: 99%

Clinicopathologic and prognostic values of apolipoprotein D alterations in hepatocellular carcinoma

Utsunomiya

Ogawa

Yoshinaga

et al. 2005

“…[5][6][7] Clinical studies investigating associations between carcinogenesis and abnormal methylation on CpG islands in tumor suppressor genes or genes involved in cell proliferation and death have been conducted in various cancers. [8][9][10][11][12] For HCC, it has been reported that some genes undergo hypermethylation in liver tissues, [13][14][15][16][17][18][19][20][21] supporting the hypothesis that determination of methylation in specific genes may be useful for HCC diagnosis. However, to the best of our knowledge, the diagnosis of HCC, in particular early HCC, based on the methylation levels of a single gene has not been clinically investigated to date.…”

mentioning

confidence: 91%

“…However, to the best of our knowledge, the diagnosis of HCC, in particular early HCC, based on the methylation levels of a single gene has not been clinically investigated to date. [13][14][15][16][17][18][19][20][21] The aim of the current study was to identify a combination of methylated marker genes suitable for use in the diagnosis of a small HCCs less than 3 cm HCC or a well-differentiated HCC as an early HCC, and to establish a user-friendly methylation-specific PCR (MSP) system to measure methylation status of these HCC gene markers. To achieve the aim, we selected 12 genes (APC, CASP8, CCND2, CFTR, CDKN2A, GSTP1, HIC1, POU3F1, RASSF1A, RUNX3, SPINT2 and TP73) with a clear methylation in >50% (actually 68-100%) of HCC cases examined in studies reported previously as genetic marker candidates for diagnosis of early HCC.…”

mentioning

confidence: 99%

Methylation of multiple genes as molecular markers for diagnosis of a small, well‐differentiated hepatocellular carcinoma

Moribe

Iizuka

Miura

et al. 2009

101

The current study was conducted to identify robust methylation markers and their combinations that may prove useful for the diagnosis of early hepatocellular carcinoma (HCC). To achieve this, we performed in silico CpG mapping, direct sequencing and pyrosequencing after bisulfite treatment, and quantitative methylation-specific PCR (MSP) in HCC and non-HCC liver tissues. In the filtering group (25 HCCs), our direct sequencing analysis showed that, among the 12 methylation genes listed by in silico CpG mapping, 7 genes (RASSF1A, CCND2, SPINT2, RUNX3, GSTP1, APC and CFTR) were aberrantly methylated in stages I and II HCCs. In the validation group (20 pairs of HCCs and the corresponding non-tumor liver tissues), pyrosequencing analysis confirmed that the 7 genes were aberrantly and strongly methylated in early HCCs, but not in any of the corresponding nontumor liver tissues (p < 0.00001). The results obtained using our novel quantitative MSP assay correlated well with those observed using the pyrosequencing analysis. Notably, in MSP assay, RASSF1A showed the most robust performance for the discrimination of HCC and non-HCC liver tissues. Furthermore, a combination of RASSF1A, CCND2 and SPINT2 showed 89-95% sensitivity, 91-100% specificity and 89-97% accuracy in discriminating between HCC and non-HCC tissues, and correctly diagnosed all early HCCs. These results indicate that the combination of these 3 genes may aid in the accurate diagnosis of early HCC. ' 2009 UICC

“…16 Therefore, somatic mutation of HAI-2 may not be a common cause of HAI-2 inactivation in human cancers. Although one recent study by Fukai et al indicated that HAI-2 was frequently hypermethylated in human HCC, 25 the functional implications of HAI-2 inactivation in HCC have not been explored. This prompted us to further characterise the potential tumour suppressive role of HAI-2 in HCC.…”

Section: Discussionmentioning

confidence: 99%

“…[15][16][17]19,25,39 Previous study has shown that KD-1 was the major functional domain in HAI-2 in inhibiting the activity of its target proteases in vitro. 6 However, the roles of the 2 KDs of HAI-2 in tumour suppressive functions have not been clarified.…”

Section: Discussionmentioning

confidence: 99%

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

Tung

Wong

Yau

et al. 2009

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. '