Identification and characteriation of novel tumor suppressors using in vivo tumor model in Drosophila melanogaster

Abstract

Cancer is a disease that hitch hikes growth regulatory network and causes uncontrollable cell proliferation and metastasis which is usually fatal. Genetic alterations in cancer cells enable such hitch hiking of regulatory networks. These alterations allow cancer cells to acquire abilities to grow and metastasize, which together with other enabling characteristics of cancer form the hallmarks of cancer. Cancer cells gradually acquire multiple hallmark characteristics defining cancer progression as a multi-step process. Disease progression and accumulating genetic alterations suggest cooperative mechanisms underlying progression of disease which is also supported by experimental evidence. Thus, it is of fundamental importance to identify causal genetic alterations in different cancer types. This has motivated a large number of genome wide omics studies, which have revealed a plethora of genomic, transcriptomic changes in cancer tissues. Resultant large data has presented a bigger challenge of identifying relevant causal factors. To understand the cooperative mechanisms underlying tumorigenesis we carried out a genome-wide screen in Drosophila tumor model for epithelial cancers. We used epithelial growth factor (EGFR) and Yorkie (Yki) as oncogenic drivers and depleted one gene at a time using RNAi mediated knock down in each of these contexts. We have identified several novel putative tumor suppressors, depletion of which enhance effects of EGFR and Yki resulting in massive overgrowth of wing imaginal discs. Interestingly, we identified components of the Negative Elongation Factor Complex (NELF) as tumor suppressors specifically in context of Yki. These findings were particularly intriguing because, Yki is a co-activator of transcription, while the NELF complex is required for promoter proximal pausing (PPP). PPP, as the name suggests, occurs in region proximal to transcription start site and has been shown to be a critical regulatory mechanism in transcription of genes in response to stimuli before elongation phase and after promoter escape by RNA Pol II. Characterization of the overgrown wing disc tissue formed by combination of depletion of NELF complex components and Yki overexpression showed neoplastic transformation. Additionally, we observed that CDK9, the kinase component of Positive transcription elongation factor (P-TEFb) is necessary as well as cooperative with Yki in tumorigenesis. Our work shows that PPP functions to limit tumorigenic potential of Yki activity. I will also present evidence from RNA-seq of tumor discs which indicate possible mechanisms that underlie tumorigenesis observed in combination of NELF depleted and Yki overexpressing wing discs. These findings shed light upon mechanisms that might regulate oncogenic outcome of Yes associated protein 1 (YAP1), mammalian ortholog of well conserved Hippo pathway.