Abstract
ABSTRACTCholangiocarcinoma (CC) is a rare malignancy of the extrahepatic or intrahepatic biliary tract with an outstanding poor prognosis. Non-surgical therapeutic regimens result in minimally improved survival of CC patients. Global genomic analyses identified a few recurrently mutated genes, some of them in genes involved in epigenetic patterning. In a previous study, we demonstrated global DNA methylation changes in CC, indicating major contribution of epigenetic alterations to cholangiocarcinogenesis. Here, we aimed at the identification and characterization of CC-related, differentially methylated regions (DMRs) in potential microRNA promoters and of genes targeted by identified microRNAs. Twenty-seven hypermethylated and 13 hypomethylated potential promoter regions of microRNAs, known to be associated with cancer-related pathways like Wnt, ErbB, and PI3K-Akt signaling, were identified. Selected DMRs were confirmed in 2 independent patient cohorts. Inverse correlation between promoter methylation and expression suggested miR-129-2 and members of the miR-200 family (miR-200a, miR-200b, and miR-429) as novel tumor suppressors and oncomiRs, respectively, in CC. Tumor suppressor genes deleted in liver cancer 1 (DLC1), F-box/WD-repeat-containing protein 7 (FBXW7), and cadherin-6 (CDH6) were identified as presumed targets in CC. Tissue microarrays of a representative and well-characterized cohort of biliary tract cancers (n=212) displayed stepwise downregulation of CDH6 and association with poor patient outcome. Ectopic expression of CDH6 on the other hand, delayed growth in the CC cell lines EGI-1 and TFK-1, together suggesting a tumor suppressive function of CDH6. Our work represents a valuable repository for the study of epigenetically altered miRNAs in cholangiocarcinogenesis and novel putative, CC-related tumor suppressive miRNAs and oncomiRs.
Highlights
Cholangiocarcinomas (CCs) are heterogeneous hepatobiliary cancers with features of cholangiocyte differentiation.[1]
We considered 1099 hypermethylated and 565 hypomethylated regions, common to more than 50%, i.e., more than 9 of 18 CC cases examined in a previous study, and found overlaps with 27 and 13 miRNA promoters, respectively (Fig. 1; Supplementary Table S2 and S3)
Screening miRCancer,[13] a literature-based database covering 454 cancer-related miRNAs, we found that 20 miRNAs or miRNA families with CC-related promoter differentially methylated regions (DMRs) had been already described as dysregulated in cancer (Supplementary Table S4); yet, only one of them, miR-200b, in CC.[14]
Summary
Cholangiocarcinomas (CCs) are heterogeneous hepatobiliary cancers with features of cholangiocyte differentiation.[1] From a clinical point of view, CC is an orphan cancer with poor patient outcome. Therapeutic options for CC are limited, and surgical resection remains the only option with curative intent. Current clinical trials often employ mixed cohorts of biliary tract cancers (BTCs) including intrahepatic CC (ICC), extrahepatic CC (ECC), and gallbladder carcinoma (GBC) and, so far, promising chemotherapeutic treatment strategies have not been identified.[2] Recurrent mutations in CC have been found in TP53, KRAS, and SMAD4.3 Profiling for additional mutations using exome sequencing identified mutations in genes associated with functions in epigenetic programming (e.g., BAP1, MLL3, ARID1A, IDH1, and IDH2) suggesting alterations in epigenetic patterns.[4,5]. In a recent global DNA methylation screen in ECC and ICC patients, we identified many aberrantly methylated candidate genes related to cancer-relevant signaling pathways, thereby demonstrating the substantial contribution of DNA methylation changes in the pathogenesis of CC.[7]
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