Down-Regulation of Hedgehog-Interacting Protein through Genetic and Epigenetic Alterations in Human Hepatocellular Carcinoma

Abstract

Purpose: Hedgehog (Hh) signaling is activated in several cancers. However, the mechanisms of Hh signaling activation in hepatocellular carcinoma (HCC) have not been fully elucidated. We analyzed the involvement of Hh-interacting protein (HHIP) gene, a negative regulator of Hh signaling, in HCC. Experimental Design: Glioma-associated oncogene homologue (Gli) reporter assay, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and quantitative real-time reverse transcription^PCR for the target genes of the Hh signals were performed in HHIP stably expressing hepatoma cells. Quantitative real-time PCR for HHIP was performed in hepatoma cells and 36 HCC tissues. The methylation status of hepatoma cells and HCC tissues was also analyzed by sodium bisulfite sequencing, demethylation assay, and quantitative real-time methylation-specific PCR. Loss of heterozygosity (LOH) analysis was also performed in HCC tissues. Results: HHIP overexpression induced significant reductions of Gli reporter activity, cell viability, and transcription of the target genes of the Hh signals. HHIP was hypermethylated and transcriptionally down-regulated in a subset of hepatoma cells. Treatment with a demethylating agent led to the HHIP DNA demethylation and restoration of HHIP transcription. HHIP transcription was also down-regulated in the majority of HCC tissues, and more than half of HCC tissues exhibited HHIP hypermethylation.The HHIP transcription level in HHIP-methylated HCC tissues was significantly lower than in HHIP-unmethylated HCC tissues. More than 30% of HCC tissues showed LOH at the HHIP locus. Conclusions: The down-regulation of HHIP transcription is due to DNA hypermethylation and/ or LOH, and Hh signal activation through the inactivation of HHIP may be implicated in the pathogenesis of human HCC.

Blocking the hedgehog pathway inhibits hepatoblastoma growth

“…[37][38][39] In line with this, loss of heterozygosity at the HHIP locus has been described in one third of HCC cases. 35 Altogether, these data suggest that at least some of the pediatric liver tumors with a constitutively activated Hh signaling pathway could have lost their functional negative regulatory feedback.…”

“…Several lines of evidence have already indicated that HHIP expression could be transcriptionally silenced by CpG island hypermethylation, as shown for pancreatic, 21 gastrointestinal cancers, 34 and just recently HCC. 35 The absence or significant decrease of HHIP mRNA levels found in lung and liver cancers 15,36 additionally suggest a broader role of this epigenetic alteration in tumor development. The aberrant HHIP methylation in 6 of 27 pediatric liver tumors and all liver tumor cell lines with a subsequent low expression of HHIP of our study further strengthen the importance of a mechanism by which the negative regulatory feedback loop of HHIP may be lost or abolished in pediatric liver tumors by promoter methylation.…”

Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms

“…Although Hip-positive staining was observed in PDA lesions [29], another study demonstrated that Hip expression was downregulated in several human cancers of the liver, lung, stomach, and colon [31]. Gene silencing, mediated through methylation, was hypothesized to be responsible for Hip downregulation [32,33], potentially resulting in activation of the ligand-dependent pathway. Our data clearly support the hypothesis that Hip is expressed in benign/ pre-cancerous tumors (PanIN and IPMN) but not in invasive PDA lesions.…”

Hedgehog signaling pathway mediates invasion and metastasis of hepatocellular carcinoma via ERK pathway

“…Previous evidence has demonstrated that the Hh signaling plays an important role in multiple tumor types. This signaling pathway is involved in and participates development, invasion and metastasis of HCC [34][35][36][37][38][39][40] . In this study, we further detected expression of Shh and Gli1 in metastasis and non-metastasis HCC liver tissues, simultaneously we used KAAD-cyc, a specific inhibitor of Hh pathway and Shh, a ligand of the Hh pathway in invasion and metastasis assays of human HCC cell line, and we confirmed the results.…”

“…Recent studies have revealed that the Hh signaling pathway is abnormally activated in human HCC [34][35][36] , and this pathway is thought to participate in the development of HCC [37][38][39] . Moreover, activation of the Hh pathway is correlated closely to invasion and metastasis of HCC [36,40] .…”