Down-regulation of hedgehog-interacting protein through genetic and epigenetic alterations in human hepatocellular carcinoma.
OBJECTIVE
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.


METHODS
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.
Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
Motohisa
M
Tada
Fumihiko
F
Kanai
Yasuo
Y
Tanaka
Keisuke
K
Tateishi
Miki
M
Ohta
Yoshinari
Y
Asaoka
Motoko
M
Seto
Ryosuke
R
Muroyama
Kenichi
K
Fukai
Fumio
F
Imazeki
Takao
T
Kawabe
Osamu
O
Yokosuka
Masao
M
Omata
D000368:Aged; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019175:DNA Methylation; D000077209:Decitabine; D015536:Down-Regulation / Q000187:drug effects; D004843:Epistasis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D018197:Hepatoblastoma / Q000235:genetics / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D019656:Loss of Heterozygosity; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D008875:Middle Aged; D014704:Veratrum Alkaloids / Q000494:pharmacology
D002352:Carrier Proteins; C117596:HHIP protein, human; D008562:Membrane Glycoproteins; D014704:Veratrum Alkaloids; D000077209:Decitabine; D001374:Azacitidine; C000541:cyclopamine