Silencing of caspase-8 in murine hepatocellular carcinomas is mediated via methylation of an essential promoter element.
OBJECTIVE
Caspase-8 is the apical caspase essential for triggering Fas-induced apoptosis. In this study, we investigated caspase-8 expression in hepatocellular carcinomas (HCCs) using recently described HCC mouse models (c-myc and IgEGF transgenes).


METHODS
HCCs were isolated from c-myc and IgEGF transgenic animals. Expression of caspase-8 was monitored by reverse-transcription polymerase chain reaction. The murine caspase-8 promoter was characterized by luciferase-reporter analysis and the analysis of promoter methylation was performed by bisulfite genomic sequencing.


RESULTS
In HCCs investigated, we frequently found a lack of caspase-8 messenger RNA expression. Genomic deletions at the caspase-8 locus did not contribute to caspase-8 silencing. We examined tumor-derived promoter sequences and found significant hypermethylation at distinct CpG sites. In parallel, we characterized the murine caspase-8 promoter and identified a 30-bp promoter element that is indispensable for basal promoter activity. This minimal promoter element contained SP1 binding motifs that are colocalized with CpG sites and were methylated in tumor-derived promoter sequences. Electrophoretic mobility shift assay analysis showed that methylation of these SP1 sites is sufficient to prevent SP1 complex formation. To support our data, we mimicked the methylation pattern of a tumor-derived caspase-8 promoter in vitro using CpG methylase and found a strong reduction of promoter activity.


CONCLUSIONS
We show that HCCs are correlated frequently with silencing of caspase-8 expression and provide data suggesting that caspase-8 silencing is a direct consequence of inhibiting SP1-dependent transactivation caused by CpG methylation at its essential binding sites in the promoter region. Our data support the hypothesis that inhibition of apoptosis triggers hepatocarcinogenesis.
Liedtke
Christian
C
Department of Medicine III, University Hospital Aachen, Aachen University, Aachen, Germany.
Zschemisch
Nils-Holger
NH
Cohrs
Anne
A
Roskams
Tania
T
Borlak
Jürgen
J
Manns
Michael P
MP
Trautwein
Christian
C
D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism; D053181:Caspase 8; D020169:Caspases / Q000235:genetics*; D045744:Cell Line, Tumor; D016384:Consensus Sequence; D018899:CpG Islands / Q000502:physiology; D019175:DNA Methylation; D004815:Epidermal Growth Factor / Q000235:genetics; D020868:Gene Silencing; D017930:Genes, Reporter; D016259:Genes, myc; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism
D016329:Sp1 Transcription Factor; D004815:Epidermal Growth Factor; D008156:Luciferases; C505530:CASP8 protein, human; C505526:Casp8 protein, mouse; D053181:Caspase 8; D020169:Caspases