In vivo RNAi screen for new tumor suppressor genes in Hepatocellular Carcinoma

The protooncogene c-Myc is a tightly regulated transcription factor in normal cells and deregulated c-Myc plays a significant role in the development of human cancers and is overexpressed in a wide range of human tumors like in human hepatocellular carcinoma (HCC) (50%). Hepatocellular carcinoma constitutes the third most common cause of cancer related death worldwide, a fact that is also largely attributed to the lack of effective treatment options. The multikinase inhibitor Sorafenib represents the first systemic treatment resulting in 2.8 month prolonged survival highlighting the need of a better understanding of the molecular mechanisms of hepatocarcinogenesis that should ultimately lead to the development of new treatment strategies. Short hairpin RNAs capable of stably suppressing gene function by RNA interference (RNAi) can mimic tumor-suppressor-gene loss in mice allowing us to analyze passenger and driver mutations in HCC development. Here we used a novel in vivo RNAi screening platform to directly identify new tumor suppressor genes in a c-Myc driven HCC mouse model. Proof of principle experiments using positive control shRNAs against established tumor suppressor genes such as PTEN showed that our screening setup can identify potent tumor suppressor genes in this model. A focused shRNA library targeting the mouse orthologs of genes deleted in human hepatocellular carcinoma was then co-delivered with c-Myc into p53 heterozygous mouse livers. Genomic DNA was isolated from outgrown tumors and scoring shRNAs were identified via PCR amplification and deep sequencing of the shRNA cassette. Our screen identified several new tumor suppressor candidates that will be presented. Some of these were already functionally validated and mechanistically characterized regarding their role in hepatocarcinogenesis.