Transposon-based insertional mutagenesis screens have been used successfully in numerous studies to induce mutations in Drosophila. Recently, transposon and retrovirus-based insertional mutagenesis screens have been used in the mouse to identify new candidate tumor suppressors and oncogenes present in somatic tumors. This approach is especially important today as it is now clear that the spectrum of mutated genes in a tumor is complex and varies from tissues to tissues. Despite its promises, the limitation of the genetic tools available in the mouse together with the expense associated with mammalian experiments present significant obstacles to the large-scale application of this approach. During my postdoctoral studies, I propose to combine the recent ingenuity of the mammalian screens with the genetic tools available in Drosophila to develop a general method for large-scale F1 somatic screens to identify new oncogenes and tumor suppressors. Specifically, I propose to use conditionally modified piggyBac transposons to create insertional mutations that will result in either gene inactivation or ectopic expression in specific tissues (eye and gut) in F1 animals. Mobilization of the piggyBac elements in specific tissues will be accomplished by expressing the piggyBac transposase using the Gal4-UAS system. Depending on their integration sites the piggybac elements in some cases will inactivate some genes. In addition, because I will use piggyBac elements that contain a UAS sequence, in some cases the inserted elements will lead to expression of nearby genes in the presence of Gal4. Further, to increase the frequency of events, an ammunition chromosome that carries multiple piggyBac transposable elements carrying a UAS will be used. Finally, to allow for detection of recessive tumor suppressor genes FLP-FRT chromosomes will be used to create homozygous clones of the chromosomes that are targeted by the piggyBac elements. These screens will be performed in two established models to study tumor phenotypes, the eye and in the adult gut, to compare the spectrum of mutations leading to tumor phenotypes in different tissues. Animals will be screened for the presence of tumors that will be analyzed using Next Generation Sequencing (NGS) to identify the genes affected. Finally, validation of the genes identified in these experiments will identify in which pathways they are acting. Altogether, these studies will not only identify tumor suppressors and oncogenes but also provide important information on the contextual differences between tumor phenotypes in different tissues.
This project is seeking support for the identification of novel tumor suppressors and oncogenes in Drosophila. The result of this study will greatly increase our understanding of which genes, out of the many that are mutated in cancerous tissues, are responsible for causing tumor phenotypes. This work is relevant to human cancer because of the amazing gene conservation between fly and humans.
| Tipping, Marla; Perrimon, Norbert (2014) Drosophila as a model for context-dependent tumorigenesis. J Cell Physiol 229:27-33 |
