Abstract MIP-051: EFFICACY OF MTORC1/2 INHIBITION ON OVARIAN CANCER STEM CELLS
Authors: Jing-Yi Chern, Melissa Frey, Fernanda Musa, Amandine Alard, Stephanie V. Blank, Robert Schneider
Journal: Clinical Cancer Research (2017)

Abstract INTRODUCTION: Ovarian cancer is the most lethal gynecologic malignancy. Initial treatment with platinum and taxane drugs are effective, however inevitably these patients will recur and become resistant to cytotoxic therapies. Thus, new alternatives for durable treatments need to be identified. One approach is to explore the heterogeneity of epithelial ovarian cancer and identify the subpopulation of cells that are resistant to treatment. It has been hypothesized, that cancer stem cells (CSCs) can survive chemotherapy and have enhanced tumor-initiating capabilities. The AKT-PI3K-mTOR pathway is well studied and has been shown to be critical for tumor cell survival. This pathway closely regulates both mTOR complexes (mTORC1 and mTORC2). mTORC1 regulates protein synthesis and autophagy whereas mTORC2 regulates cell motility. mTORC1 and 2 ultimately affect cell proliferation, survival and angiogenesis, all inherently important in tumorgenesis. OBJECTIVES: Rapalogs are specific and potent inhibitors of mTORC1 but have no effect of mTORC2. We investigate the efficacy of dual mTORC1/2 inhibitors and its effect on the CSC population. METHODS: Carboplatin resistant ovarian cancer cell line OVCAR3 were cultured and treated with dimethyl sulfoxide (DMSO), carboplatin, or INK128, a potent dual inhibitor of mTORC1/2. Using cell surface markers CD133 and CD44, CSCs were quantified by flow cytometry. OVCAR3 spheroids were cultured as a surrogate for CSCs. Colony formation-survival assays were performed with spheroids treated with the above mentioned conditions. Analysis was performed using ArrayScan technology. Doxycyline inducible silencing cell lines derived from OVCAR3 were generated; Non-silenced (Nsi), Raptor (Sh-mTORC1) and Rictor (Sh-mTORC2). Colony formation assays using spheroids from silenced lineages were performed to evaluate growth and survival. Western blot analysis was performed to confirm silencing of targets, and to evaluate protein expression in CSCs. RESULTS: 4.87% of the untreated OVCAR3 population were CSCs. INK128 treatment of the cells confered a 2 fold increase of CD44+/CD133+ cells, presumably CSCs, P&amp;lt;0.005 and no difference between DMSO and carboplatin treated cells (NS). INK128 treated cells formed fewer colonies in comparison to DMSO (mean number of colonies 36.3 + 23.8 vs. 83.3 + 32.1, P&amp;lt;0.001) and smaller colonies (mean number of cells in a colony 5260 + 835.8 vs 6907 + 702.4 P &amp;lt;0.0002). Similar populations of CD44+/CD133+ were isolated in Nsi and Raptor (NS), and there were no difference in the number or size of colonies. In comparison to Nsi, Rictor resulted in 50% decrease in CD44+/CD133+ cells, (P &amp;lt;0.002) and fewer colonies (1.6+1.3 vs 10+7.2, p&amp;lt; 0.03). There was no difference in colony size. CSCs expressed Oct4, Sox2 and phosphorylated 4E-BP1 confirms activation of AKT. CONCLUSIONS: The PI3K/AKT/mTOR pathway appears to have a role in the biogenesis of platinum resistant ovarian cancer cells. The inhibition of mTORC1/2 delays growth of OVCAR3 cells, and may have a role in activating in stem like CSCs. However, it does not appear to selectively inhibit CD133+/CD44+ cells. These findings suggest that targeting CSCs may lead to advances in ovarian cancer treatment. Citation Format: Jing-Yi Chern MD, Melissa Frey MD, Fernanda Musa MD, Amandine Alard PhD, Stephanie V. Blank, MD, Robert Schneider, PhD. EFFICACY OF MTORC1/2 INHIBITION ON OVARIAN CANCER STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-051.