Abstract
Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. We devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, we mechanistically characterized the function of the top hit, the Tetraspanin family member Tsp29Fb, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. Our functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion.
Highlights
Mutations in Ha-RAS, N-RAS, and K-RAS genes are frequent in human tumors and represent oncogenic drivers of cell proliferation and survival
We have undertaken a genome-wide genetic screen using a transgenic RNAi library in the vinegar fly, Drosophila melanogaster, to identify tumor suppressor genes that cooperate with the Ras oncogene (RasV12) in conferring overgrown invasive tumors
We stratified the hits by analyzing the expression of human orthologs of these genes in human epithelial cancers, revealing genes that were strongly downregulated in human cancer
Summary
Mutations in Ha-RAS, N-RAS, and K-RAS genes are frequent in human tumors and represent oncogenic drivers of cell proliferation and survival. Using the Drosophila system, cell polarity genes were identified as suppressors of Ras-driven tumor growth and invasion [14, 15] implicating high inter-species conservation of fundamental genetic networks controlling transformation and metastasis [16]. Given the accessibility of Drosophila to in vivo RNAi screening approaches and conservation of major developmental pathways including Ras, the fly offers a valuable opportunity to genetically dissect cancer-relevant signaling networks in the context of a whole animal, which could lead to the identification of novel biomarkers and new drug targets for a diverse range of epithelial tumors. Ras85D has been shown to be the authentic human ortholog [18, 19], making the fly a suitable model system to genetically dissect Ras-regulated networks
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