Background and Aims: The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenomic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data of global methylation changes are scarce.
Methods: We performed a genome-wide analysis for aberrant promoter methylation in human CCs. We profiled ten intrahepatic (ICC) and eight extrahepatic (ECC) CCs, as well as corresponding and non-corresponding non-tumorous biliary tissue specimens using the methyl-CpG immunoprecipitation (MCIp) technology combined with whole-genome CpG island arrays. DNA methylation was confirmed by quantitative MassARRAY® analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments, using 5-aza-2'deoxycytidine treatment of two CC cell lines (EGI-1 and TFK-1). Immunohistochemistry and Tissue Microarrays (TMAs) were used to analyze candidate gene expression at the protein level.
Results: Biostatistical evaluation of the data revealed non-random promoter hypermethylation and identified overrepresentation of genes involved in cancer related pathways including Wnt/β-catenin, BMP and TGF-β signaling pathways. Silencing of genes in the Wnt/β-catenin pathway (e.g. SOX17, WNT3A, DKK2, SFRP1, SFRP2 and WNT5A), is consistent with previous reports describing an activation of this pathway in CCs. 5-aza-2'deoxycytidine treatment leads to demethylation and gene reexpression in cell lines showing methylated promoters for SFRP1, SFRP2, WIF1, SFRP4 or Sox17. Analysis of DNA methylation and gene expression in matched normal/tumor pairs demonstrated epigenetic gene silencing in the primary tumor samples. For the promising candidate gene SFRP2, immunohistochemical expression analysis was performed using TMAs comprising 223 biliary tract carcinomas (BTC) including 98 ECCs, 56 ICCs, and 69 adenocarcinomas of the gallbladder. This revealed a substantial downregulation of SFRP2 in neoplastic tissues of all BTC subtypes.
Conclusions: Here, we provide a comprehensive approach to define the genome-wide methylation landscape of CC. Herein, we could detect that hypermethylation of promoter CpG islands of tumor suppressor genes is a common epigenetic event in CC. Several candidate genes of cancer relevant signaling pathways were identified, and among these a cluster of genes acting in the Wnt/β-catenin-signaling pathway. This study may provide a solid basis for future attempts of detecting epigenetic changes in cholangiocarcinogenesis.
