Characterization of mTOR Inhibition and Autophagy Inhibition in Non-Small Cell Lung Cancer




Britt, Rebecca

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Lung cancer continues to be the leading cause of cancer related death in both men and women. Pre-clinical studies of targeted therapies are needed in order to improve upon the chemotherapeutics that are currently in use. The ability to identify subsets of patient tumors which will respond to a particular targeted agent using biomarkers to indicate an acquired vulnerability will improve selection of effective therapeutics and minimize time and money wasted on ineffective drugs. The goal of this dissertation has been to characterize NSCLC response to mTOR inhibition and determine whether there are any molecular biomarkers that can predict response. mTOR is a central regulator of several pro-oncogenic signaling pathways and plays a role in cell growth, proliferation, metabolism, and inhibition of autophagy. Early studies examining mTOR inhibitors were limited by a lack of proper patient selection and the inability of first generation drugs to completely inhibit mTOR signaling. In the present study, we screened a panel of well-characterized NSCLC cell lines with three mTOR inhibitors, classical mTORC1 inhibitor rapamycin, and two novel dual mTORC1/2 inhibitors, Torin1 and AZD8055 in order to identify potential biomarkers that may be used to predict response to these agents. Additionally, in order to further characterize vulnerabilities to mTOR related genes within lung cancer subsets, we performed a genetic knockdown screen individually targeting 55 genes in this important pathway. Because inhibition of mTOR frequently leads to a cytostatic rather than cytotoxic effect, mTOR targeting agents may have greater utility when used in combination with other chemo- and targeted-agents. Therefore, we screened the three mTOR inhibitors in combination with the chemotherapy doublet paclitaxel/carboplatin or the targeted agent erlotinib. Finally, mTOR inhibition and other drug treatments have been shown to lead to autophagy activation. This process of cellular "self-eating" is thought to protect cancer cells from low nutrient availability and therapy induced stress. We screened NSCLC cells for their response to autophagy inhibitors alone and in combination with chemo- and targeted-therapy agents. The studies described in this thesis led us to the following conclusions. A subset of NSCLCs are more responsive to rapamycin than to mTORC1/2 inhibition by Torin1, or AZD8055, and sensitivity to mTOR inhibition is associated with RTK activation such as ERBB2 amplification or EGFR mutation or amplification, while KRAS and/or LKB1 mutations were associated with resistance. RNAi knockdown of various components related to mTOR signaling and autophagy produce a heterogeneous growth effect response in NSCLCs cells, and potentially define subset-specific vulnerabilities. mTOR inhibitors sensitize NSCLC cells to standard targeted- and chemotherapy agents erlotinib and paclitaxel/carboplatin doublet in an additive or synergistic manner, with the greatest level of synergy occurring in cell lines which are resistant to single agent therapies, including those with KRAS mutations. Finally, inhibition of autophagy using chloroquine is not likely to be a successful therapeutic approach in lung cancer as no significant growth effect was seen at physiologically relevant concentrations and no sensitization to standard chemo- or targeted-therapies were observed.

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