Abstract B33: Understanding resistance mechanisms to PI3K/mTOR inhibitors in ovarian cancer model systems

Abstract

Abstract The combined use of computational models and experimental data is an emerging tool in systems biology to help explain the mechanism of resistance to various protein inhibitors in oncological medicine. Improved understanding of the signaling network dynamics may offer novel approaches to overcoming drug resistance. The aim of this study was to analyze the signaling dynamics of the PI3K/Akt/mTOR pathway in ovarian cancer cells after treatment with different PI3K/mTOR inhibitors. Systems computational approach was applied to unwind the role of multiple feedbacks in PI3K/Akt/mTOR pathway in the responses of cancer cells bearing mutations in PTEN, PIK3CA and HER2 overexpression. In this study, a set of PI3K (LY294002), mTOR (Rapamycin) and dual PI3K/mTOR (BEZ235) inhibitors were applied to a panel of ovarian cancer cell lines under heregulin driven conditions over 10 point time-course treatment, in order to assess the association of growth inhibition with pathway activation. The evaluation of expression levels of various proteins and phospho-proteins was assessed using reverse phase protein array (RPPA) as a high throughput proteomic analysis to investigate the dynamic changes in response to the inhibitors. In part, PTEN expression status was analyzed as biomarker of the feedback-dependent pAkt activation in response to mTOR inhibition by rapamycin in different cell lines. The three inhibitors displayed a significant inhibition in cell proliferation on all the ovarian cancer cell lines following a 5-day treatment. Our results have shown a significant positive correlation between the signaling dynamics of phosphorylated Src (Tyr527) and phosphorylated PTEN (Ser380/Thr382/383) upon treatment with the drugs, consistent with the role of Src in the inactivation of PTEN. Additionally, we demonstrate that PI3K plays a role in the activation of RAS pathway, as phosphorylated cRaf (Ser259 and Ser338) and phosphorylated mTOR (Ser2481) were upregulated and showed a significant positive correlation in their signaling dynamics. Although, all the cell lines have shown a significant inhibition to cell proliferation following a 5-day treatment with the inhibitors, the signaling expression of 12 different phospho-proteins within the PI3K/mTOR and MAPK pathways differ. Rapamycin has been shown to downregulate the expression of phospho-mTOR (Ser2448) and its substrates, phospho-4E-BP1 and phospho-S6K1 but has shown an upregulation in phospho-Akt (Ser473), confirming the existence of the negative feedback loop, S6K1-Akt, upon treatment. BEZ235 presented the highest efficacy in inhibiting cell proliferation in all the ovarian cancer cell lines but failed to downregulate the phosphorylation of ERK. Due to the high overexpression of erbB2 that may cause HER2 homodimerization, the signaling expression of the ovarian cancer cell line, SKOV3, showed upregulation in all the measured phospho-proteins. We propose an extended model of PI3K/Akt/mTOR pathway in ovarian cancer, based on theoretical modeling and experimental data, highlighting possible resistance compensatory mechanisms within the pathway and with its parallel cross-talk pathway MAPK. The model highlights potential novel protein interactions and feedback loops, offering strategies for combinatorial anti-cancer therapy. Citation Format: Ghassan Tashkandi, Alexey Goltsov, Peter Mullen, Jim Bown, David Harrison, Simon Langdon. Understanding resistance mechanisms to PI3K/mTOR inhibitors in ovarian cancer model systems. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B33.