The Ras small G proteins are key regulators of many cellular processes downstream of growth factor receptors and adhesion molecules, making them high profile oncogenes. Indeed, RAS-activating mutations are found in ~30% of human cancers. However, cancer is a cooperative process, and RAS activation alone is not sufficient to induce tumour formation. To identify novel cooperative tumour suppressor genes in Ras-driven tumourigenesis, we have undertaken a systematic approach in vivo and in vitro. In vivo, a genome wide shRNA screen identified 947 genes that potentiate RASV12-mediated tumourigenesis and metastasis in the vinegar fly, Drosophila melanogaster (Zoranovic et al., in preparation). In vitro, we will perform a high-throughput shRNA screen in MCF10A RASV12, a breast cell line engineered to express a constitutively active form of human Ha-RAS. MCF10A RASV12 cells will be infected with a pool of shRNA constructs representing a refined list of 234 potential tumour suppressors from the fly screen, and grown in 3D in Matrigel. Invasive colonies will be isolated and next generation sequencing will identify integrated shRNAs providing invasive phenotypes. So far, our analysis in the fly has identified clusters of genes involved in the regulation of diverse biological processes such as cell polarity, chromatin remodelling or autophagy. We are currently characterizing the role of these genes and biological processes in potentiating Ras-mediated tumourigenesis and metastasis. By combining in vivo genetic screen in Drosophila melanogaster, in vitro shRNA screen in mammalian cell lines, and bioinformatics, our integrated, systems level approach will contribute to understanding cooperative tumourigenesis mechanisms and cross-talks involving Ras, and will allow the identification of genes and pathways that could be targeted in cancers in which the Ras signalling module is activated.