Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Gabriel E. DiMattia

Abstract

The most prevalent subtype of ovarian cancer – high-grade serous (HGS) carcinoma – is also the most lethal, since the majority of cases are characterized by advanced-stage (metastatic) presentation. Metastasis of this cancer proceeds by an intra-peritoneal route, involving detachment of cells from the primary tumour and dissemination throughout the peritoneal cavity as multicellular aggregates, or spheroids. Herein, we demonstrate that HGS patient-derived tumour cells cultured to form in vitro spheroids exhibit features of cancer dormancy, a cellular state known to promote therapeutic resistance and disease recurrence. We discovered that upon spheroid formation, cells became non-proliferative, exhibiting a cell cycle profile and protein expression pattern (elevated p27Kip1 and RBL2/p130) that was consistent with quiescence. This was accompanied by decreased AKT kinase activity, which may be important in mediating cell cycle exit via the SCF ubiquitin-ligase complex member p45/SKP2. Moreover, when spheroids were re-attached to an adherent substratum, quiescence was rapidly reversed in an AKT-dependent manner. Aside from quiescence, we also discovered that the cellular self-digestion mechanism autophagy was upregulated during spheroid formation. Induction of this process was also observed in adherent cells (and augmented in spheroids) by pharmacologic AKT inhibition. To determine autophagy’s effect on cell viability, we attempted to block it using siRNAs targeting critical autophagy-related (ATG) genes. Interestingly, depletion of Beclin1/ATG6 had no effect, despite its role as a canonical inducer of the process. Conversely, depletion of ATG5 and ATG7 led to efficient autophagy blockade, as did treatment with the classical autophagy inhibitor Chloroquine and the novel agent Spautin-1. These approaches caused a loss of viability in both adherent and spheroid cultures. Moreover, combining autophagy blockade with AKT inhibition synergistically reduced viability, thus implying that autophagy upregulation functions as a survival mechanism. Taken together, these data reveal that two cellular processes, quiescence and autophagy, are readily induced by metastatic ovarian cancer cells as features of a dormant phenotype. We therefore propose that therapeutically targeting these dormant cells will prove highly effective in combating metastasis, resistance, and recurrence in patients with metastatic HGS ovarian cancer.

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