Molecular studies of the gene CG2943 in the development of normal structures and tumors induced by genetic manipulation in Drosophila melanogaster

• Grant number: 13/21242-2
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: March 01, 2014
• End date: April 30, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Enilza Maria Espreafico
• Grantee: Carlos Antonio Couto Lima
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The development of cancer is a multistep process, which involves the cooperation of mutations in components of signaling pathways, cell cycle and cell death, as well as the interactions between the tumor and the tumor microenvironment. Recent studies demonstrate the power of Drosophila melanogaster to evaluate these processes, allowing a better understanding of several molecular signaling pathways essential for developmental processes and cell growth control. Recent data from our laboratory show that knocking-down KIAA0090/EMC1 in melanoma cells reduces the rate of proliferation, migration and cell survival and attenuates tumor growth in vivo. The overexpression of EMC1 in tumor breast cells increases the cell proliferation and migration. During my master project (FAPESP 2011/16666-2), we knocked-down the KIAA0090/EMC1 orthologous (CG2943), in D. melanogaster muscle, by using an RNAi driven to this tissue, and produced a phenotype of severe motor disability, in adult flies, associated with ultrastructural defects in the mitochondria of muscle fibers. Driving the RNAi expression to the precursor tissue (the imaginal discs) of wings generated animals unable to fly and with variable degrees of defects in this structure, resembling the phenotypes described for genes already characterized, such as Notch. In this project, aiming at elucidating the functional pathways and mechanisms of action of the CG2943 in the formation of normal structures during the development, we will focus on: i) generating antibodies to characterize the tissue expression pattern and subcellular localization of the CG2943 gene product; ii) getting new alleles of CG2943 gene using the CRISPR RNA/ Cas9 recently developed method to induce the deletions of the entire gene or of specific regions; iii) generation of transgenic lineages carrying CG2943 under the control of UAS-GAL4 for tissue-specific overexpression of the protein, intact or fused to fluorescent tags; iv) characterize the phenotypic defects at cellular and molecular levels, by combining genetic and confocal/multiphoton fluorescence microscopy approaches, consisting in creating lineages carrying interference on the CG2943 gene combined with single or multiple fluorescent markers for in vivo imaging acquisition; v) studying genetic interactions based on phenotypic complementation or modification. To explore the role of this gene in cancer, we will induce tumors in the imaginal disc compartments through genetic manipulation, by mosaicism, of oncogenes and tumor suppressor genes, as previously described, and by genetic crosses with lineages carrying alterations in CG2943, we will be able to evaluate how the CG2943 depletion or its overexpression influences tumorigenesis and the processes of tumor growth and invasion. (AU)