Abstract 3068: Proteotoxic stress associated with mTORC1 activation in ovarian carcinoma: proteasome inhibition as a therapeutic strategy

Abstract

Genetic profiling studies of high grade serous ovarian carcinoma have revealed recurrent alterations in the mTORC1 signalling network (e.g. mutations/copy number alterations in PTEN, TSC1, TSC2, and PIK3CA) and pathway activation, detected by phospho-4E-BP1, has been associated with poor prognosis. We sought to characterize functionally the role of mTORC1 signalling and its therapeutic implications in ovarian cancer. Treatment of ovarian cancer cell lines with rapamycin resulted in inhibition of mTORC1 signalling and decreased rate of protein synthesis. However, irrespective of PTEN mutation status, only mild cytostatic effects were achieved even with high concentrations of rapamycin. We next examined the phenotypic consequences of mTOR activation, using siRNA directed against TSC2. Surprisingly, we observed striking growth inhibition in the majority of ovarian cancer cell lines, whether grown under adherent monolayer culture or in 3-dimensional spheroid culture conditions. While mTORC1 pathway activation was confirmed biochemically, knockdown of TSC2 also resulted in activation of the unfolded protein response (UPR), with elevated levels of phospho-EIF2α and ATF4, consistent with the accumulation of misfolded proteins in the endoplasmic reticulum.

From a therapeutic standpoint, the resulting burden on the ubiquitin-proteasome system should render these cells particularly sensitive to proteasome inhibition. We show that treatment with the proteasome inhibitor, bortezomib, causes increased accumulation of detergent-insoluble poly-ubiquinated proteins and formation of larger and more abundant cytoplasmic protein aggregates in siTSC2-transfected compared to scrambled siRNA-transfected ovarian carcinoma cells. This was accompanied by a more pronounced UPR stress response, including induction of pro-apoptotic CHOP, and suppression of autophagy, resulting in marked cytotoxicity. Conversely, we show that inhibition of protein synthesis by cycloheximide renders tumor cells resistant to bortezomib. Increased resistance to bortezomib was also noted when cells were grown as spheroids, a condition associated with suppression of mTORC1 signalling and decreased protein synthesis. This resistant phenotype of tumour spheroids however was ameliorated with TSC2 knockdown. Our findings demonstrate that protein homeostasis is finely-tuned in ovarian cancer and that mutations in mTORC1 pathway components do not necessarily imply “oncogene addiction”. In early-stage clinical trials, bortezomib has achieved notable responses in a few patients. Ovarian carcinomas with genetic alterations causing increased mTORC1 signalling may be particularly amenable to treatment with proteasome inhibitors.

Citation Format: M. Herman Chui, Patricia Shaw, Robert Rottapel. Proteotoxic stress associated with mTORC1 activation in ovarian carcinoma: proteasome inhibition as a therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3068. doi:10.1158/1538-7445.AM2017-3068