Methylation profiling combined with genomic and transcriptomic data points out to new tumor suppressor genes in human hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer and the third leading cause of cancer mortality worldwide. Its etiologies are well-established, most prominently chronic hepatitis B (HBV) and C (HCV) virus infections and chronic alcohol abuse. About 10% of HCCs are classified as cryptogenic, but according to recent epidemiological data likely derive from nonalcoholic steatohepatitis (NASH). In contrast, the underlying molecular mechanisms are largely unknown. In order to identify host changes related to tumor etiology as well as novel driver genes of human hepatocarcinogenesis in general, we explored the genome-wide methylation of 27578 CpG-sites covering more than 14000 CpG islands throughout the whole genome in 64 human HCC and 10 normal liver controls. 4104 CpG sites showed methylation differences between tumor and normal tissue, out of them 1348 were hypermethylated and 2756 CpG were hypomethylated in tumors. Hypermethylated CpGs were particularly frequent in genes known to be silenced by the Polycomb repressor complex 2 (PRC2), while hypomethylated CpGs were enriched in genes known to be imprinted. After defining genes with tumor-specific methylation alterations, we focused on the identification of new tumor suppressor candidates. Therefore we combined genome-wide methylation data with already existing genomic data. After confirming the aberrant methylaton of our candidate genes in HCC by pyrosequencing, expression analysis was performed, and 5-Aza-dC treatment was used to validate the methylation-dependent changes in gene expression. Selected candidates underwent further functional analyses following re-expession in suitable cell lines in vitro. In conclusion, we ware able to identify new tumor suppressor gene candidates of human hepatocarcinogenesis through the vertical integration of genomic and epigenomic data sets.

HCC - genomic - methylation - profiling