Identification of novel Ras pathway genes using an inducible Drosophila tumor cell line
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Date
2016-05
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The Ohio State University
Abstract
The oncogenic form of Ras signaling is associated with 30% of human cancers, including 90% of pancreatic tumors, was first genetically characterized in Drosophila. Subsequent genetic screens have identified many additional genes in the pathway. To identify novel candidates, we have conducted an in vitro screen using an inducible Ras oncogene in Drosophila tissue culture cells developed in our lab. This conditional cell line requires the control of oncogenic RasV12 expression induced by GeneSwitch-Gal4 (GSR) for proliferation. Using RNAseq analysis, 363 gene transcripts were identified that were upregulated two-fold or greater by oncogenic RasV12 expression. Of these, 91 candidate genes were tested further for functional analysis using RNAi in flies. Ubiquitous knock-down of 38 target genes caused death or decreased viability of the organism, suggesting their essential role, and depletion of another gene caused a wing phenotype. As the Ras pathway is known to play an important role in wing-vein patterning, I carried out tissue-specific knock-downs in the wing. I am characterizing further three genes whose depletion results in the most robust wing venation defects. Knock-down of G protein gamma 30A (Gγ30A), a G-protein subunit, in the wing causes extra vein phenotypes, consistent with a negative Ras pathway regulator. Knock-down of CG12009, a chitin-binding protein, and CG31689, an ABC transporter, in the wing causes loss of vein phenotypes, consistent with positive Ras pathway regulators.
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Keywords
Drosophila, Cancer, Ras, Genetic Screen