In vivo RNAi screening identifies new mediators of p53 independent tumor suppressive functions of p19Arf in the liver

The sources of hepatocellular carcinoma are liver progenitor cells or hepatocytes. Interestingly, we observed differences between progenitor cell- and hepatocyte- derived hepatocellular carcinomas in mice. Oncogenic Nras (NrasG12V) efficiently triggers hepatocellular carcinomas derived from p53-/- liver progenitor cells, however almost no tumor growth is observed, when NrasG12V is delivered into p53-/- hepatocytes. Since aggressive hepatocellular carcinomas develop after short latency upon delivery of oncogenic Nras into p19Arf-/- hepatocytes, we suggest that p19Arf mediates a p53-independent tumor suppressive function in hepatocytes. To identify mediators of this p53-independent tumor suppressive function of p19Arf in the mouse liver, we set up an in vivo RNAi screen. As a proof of principle we could show that co-delivery of NrasG12V and p19Arf-shRNAs into p53-/- hepatocytes via transposable elements was able to phenocopy NrasG12V-driven hepatocarcinogenesis in p19Arf-/- livers. A focused shRNAmir library was compiled consisting of shRNAs targeting genes differenzially expressed in NrasG12V; p53-/- mouse livers compared to NrasG12V; p19Arf-/- livers. This shRNA library was divided into several low complexity pools and subjected to a positive selection screen in a p53-/- background. Via deep sequencing of gDNA derived from NrasG12V; p53-/- tumors, we identified several shRNAs knocking down new candidate genes mediating p53-independent tumor suppressive functions of p19Arf in the mouse liver. Among them are genes involved in the mitotic spindle assembly and the spindle checkpoint. Functional validation experiments using single hairpins have already been pursued for several candidates. ShRNA-mediated knockdown of those candidate genes allows for Ras driven tumorigenesis in a p53-deficient background.