Due to the lack of treatment options hepatocellular carcinoma (HCC) represents the third most frequent cause of cancer related death. The major obstacle in HCC treatment is resistance to conventional chemotherapy and it becomes clear that new treatment strategies are urgently needed. We devised a system to conduct negative selection in vivo RNAi screens to identify new therapeutic target genes in Ras driven murine liver carcinomas. Multilocular invasive hepatocellular carcinomas develop after short latency when oncogenic Nras(G12V) is stably delivered into p19-/- mouse livers via transposable elements. As a proof of principle that RNAi mediated knockdown of target genes can cause lethality of Nras(G12V) driven liver tumors, we showed that stable expression of an shRNA targeting a cell essential DNA replication factor (replication protein 3, Rpa3) completely blocks Nras(G12V) driven tumorigenesis and that shRNA against Ras downstream target B-Raf reduced the tumor burden of injected mice. To identify vulnerabilities in Nras(G12V);p19-/- tumors that may be exploited as new therapeutic targets; we next performed a negative selection in vivo RNAi screen. Transposable elements co-expressing Nras(G12V) and a focused shRNA library were stably delivered into p19-/- mouse livers. The frequency of each shRNA in the library before delivery into hepatocytes and after liver tumor development (in tumor nodules) was quantified via deep sequencing and a comparison of obtained profiles identified hairpins against a ribosomal protein as top depleted. Functional validation experiments with different hairpins show that knockdown of our candidate gene strongly suppresses Nras(G12V) driven tumorigenesis in p19-/- mice whereas the observed tumor suppressive effects correlated with the knockdown efficiency. Suppression of the identified ribosomal candidate gene via RNAi causes a ribosomal stress response that drives murine p19-/- hepatoma cells into an irreversible cell cycle arrest. Interestingly, we show that the cells undergo a full senescence arrest and upregulate cytokines involved in activation of immune cells that mediate their clearance in vivo.
