| dc.description.abstract | Ovarian cancer is the fifth leading cause of cancer-related death among women and the deadliest of all gynecological cancers. Treatment failure is a major contributing factor in ovarian cancer-related mortality. Advances in the development of new agents that target critical pathways in cancer may provide better options to overcome treatment failure. One of the molecular mechanisms that can be targeted for ovarian cancer therapy is the protein quality control. Components of the protein quality control, such as heat shock proteins, the ubiquitin-proteasome system, the unfolded protein response and autophagy, regulate protein homeostasis. Protein quality control also provides phenotypic stability by buffering cryptic genetic variations that could potentially reduce the fitness of cancer cells. This concept was best illustrated by studies that genetically or pharmacologically inhibited heat-shock protein 90, and reported variations in the phenotype following the disruption of heat shock protein 90. This dissertation investigates the therapeutic efficacy of targeting valosin-containing protein (VCP), an important component of the protein quality control, in ovarian cancer. VCP or p97, a member of the ATPase Associated with diverse cellular Activities-ATPase (AAA-ATPase) protein family, has been associated with various cellular functions including endoplasmic reticulum associated degradation, the ubiquitin proteasome system, golgi membrane reassembly, autophagy, DNA repair and cell division making it an important regulator of the protein quality control. Recent studies identified VCP and the ubiquitin proteasome system as synthetic lethal targets in ovarian cancer. This dissertation describes the preclinical activity of VCP inhibitors in ovarian cancer. Results presented in this dissertation show that quinazoline-based VCP inhibitors initiate Gap 1 (G1) cell cycle arrest, attenuate cap-dependent protein translation and induce programmed cell death via the intrinsic and the extrinsic modes of apoptosis. Mechanistic studies point to the unresolved unfolded protein response as a mechanism by which VCP inhibitors contribute to cytotoxicity. These results support an emerging concept that the unfolded protein response pathway may be targeted in ovarian cancer as a source of vulnerability. Since prolonged induction of the unfolded protein response results in CCAAT/enhancer binding protein homologous protein (CHOP) mediated cell death, we tested the hypothesis that VCP inhibitors act synergistically with compounds that enhance CHOP expression. Here, we show that VCP inhibitors act synergistically with salubrinal, an inhibitor of growth arrest and DNA-damage-inducible 34 (GADD34), by enhancing CHOP expression in ovarian cancer cell lines. Our results provide a proof-of-concept that VCP inhibitors can be used as a single agent and can be synergized with compounds that enhance CHOP expression to induce cell death. While the synergistic effects observed between VCP inhibitors and GADD34 inhibitor provides an in vitro proof-of-concept, these results have little clinical relevance because salubrinal is not a clinical candidate. Therefore, we identified mifepristone, an FDA approved steroidal progesterone antagonist, to act synergistic with VCP inhibitors across several ovarian cancer cell lines. Combination treatment between clinically achievable doses of mifepristone and oral VCP inhibitor- CB-5083 show an atypical increase in the unfolded protein response, a significant decrease in colony formation as well as an increase in caspase activity. Mechanistic studies upon mifepristone treatment point towards the inhibition of activating transcription factor 6 (ATF6) branch and the activation of heme- regulated inhibitor (HRI) kinase pathway as two plausible mechanisms contributing towards the synergistic effect. Oral VCP inhibitor- CB-5083 is currently in Phase I clinical trials because it shows therapeutic effects in multiple tumor xenograft models; however, the mechanism of resistance to CB-5083 is unknown. Here, we characterize the molecular mechanism of resistance to CB-5083. Using incremental exposure to CB-5083, we established CB-5083- resistant ovarian cancer cells that show 5 to 6-fold resistance in vitro compared to parental cells. Genomic and complementary DNA sequencing of the VCP coding region revealed a pattern of co-selected mutations: (1) missense mutations at codon 470 in one copy resulting in increased ATPase activity; and (2) nonsense or frameshift mutations at codon 606 or codon 616 in another copy causing the loss of allele-specific expression. Unbiased molecular docking studies show codon 470 as a putative binding site for CB-5083. Furthermore, the analysis of somatic mutations in cancer genomes from the cancer genome atlas (TCGA) indicate that codon 616 is frequently mutated in human cancers. Thus, identification of these mutations associated with in vitro resistance to VCP inhibitors may be useful as potential theranostic markers while screening for patients to enroll in clinical trials. VCP has emerged as a viable therapeutic target for several cancer types, and therefore targeting such hyperactive VCP mutants should aid in improving the therapeutic outcome in cancer patients. Overall, this dissertation 1) provides a proof-of-concept that VCP can be targeted in ovarian cancer, 2) proves that VCP inhibitors can act synergistically with other compounds that modulate the protein quality control, 3) identified a clinically relevant drug combination (CB-5083 and mifepristone) that should be investigated further, 4) unearths a novel mechanism of resistance towards the oral VCP inhibitor, CB-5083, in ovarian cancer cells. | |
