Abstract 292: Identification of new components of the mTOR signaling pathway in pancreatic cancer cells by a genome-wide RNAi screen

Abstract

Abstract The mammalian Target of Rapamycin (mTOR) is an evolutionary conserved Ser/Thr kinase involved in cell growth regulation and metabolism. Among other factors, mTOR is activated by amino acids. Although the mechanism of mTOR activation in response to growth factors and insulin is well characterized, the pathway of mTOR activation in response to amino acids is not. Previous studies have demonstrated that the latter is dominant over the former and the two pathways act independently of each other. PDAC is the most common form of pancreatic cancer and has a very dismal 5-year survival rate of 3%-5%. The need for discovery of new therapies is clear due to the low survival rates that current treatment options offer. The mTOR/ PI3K pathway is activated in pancreatic cancer and known mTOR inhibitors are currently being tested in clinical trials in pancreatic cancer patients. However, the already existing mTOR inhibitors have yet to demonstrate encouraging clinical responses. To discover druggable targets in pancreatic cancer that will be later tested in vitro and in vivo as potential therapeutics we have completed a genome-wide RNAi screen in pancreatic cancer cells in order to identify gene products whose downregulation inhibits or enhances output from the mTOR pathway. For this purpose, we used the Mia-Paca 2 cells as a representative pancreatic cancer cell line, the Dharmacon SMARTpool siRNA library and phosphorylation levels of the ribosomal S6 protein as a readout of TOR activity, since it is a well-characterized downstream target of TORC1. In initial experiments, we showed that specific RNAis against known regulators of mTORC1 activity alter baseline S6-P levels as expected with a “Z” score of 0.69. We demonstrated the success of the screen by confirming 70% of the potential positive hits from the kinome. The candidate genes found included known or previously identified components of the pathway, and also implicate many additional proteins involved in the mTOR signaling pathway. Bioinformatic analysis that integrates data obtained from this screen with those of previous studies will be presented. Immediate future studies include secondary screens with the goal of mapping where specific hits lie in the mTOR pathway and delineating the underlying mechanism of selected proteins in connection to their a) regulation of mTOR and b) their involvement in cell proliferation and apoptosis. The goal of the former is to understand the mechanism of action of selected targets in relationship to known mTOR regulators which when combined with the latter will aid in the identification of potential druggable targets. In short, we are interested in identifying and characterizing druggable targets which in combination with currently known TOR inhibitors and/ or novel ones identified through this work will sensitize pancreatic cancer cells to cell death and/ or decrease proliferation rates of pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 292.