Ovarian cancer is a devastating disease process, killing more than half of the affected women within five years of diagnosis. Because of subtle symptoms, ovarian cancers often go undetected until they reach an advanced stage which is inherently widely metastatic. Surgical debulking procedures regularly leave significant amounts of residual microscopic disease. In this state, the only recourse for treatment is adjuvant chemotherapy, which often fails due to either primary or acquired tumor cell resistance. Many commonly used chemotherapies kill ovarian tumor cells through the intrinsic apoptotic pathway by damaging cellular DNA, leading to mitochondrial release of cytochrome c and the formation of the active apoptosome, which consists of cytochrome c, the adaptor protein, Apaf-1, and caspase 9. This active apoptosome then cleaves and activates effector caspases, which go on to cleave a host of cellular proteins, ultimately leading to cell death. Resistant cancers avoid apoptosis induced by DNA damaging chemotherapies (e.g., cisplatin) in part by limiting the activation of the apoptosome. In ovarian cancer, lack of apoptosome function can be traced to an inability to activate Apaf-1 and/or a defect in recruitment of caspase 9. Importantly, neither the mechanism controlling Apaf-1/caspase 9 interaction in chemoresistant ovarian cancer, nor the role of apoptosome in chemoresistance in vivo has been clearly defined. Our central hypothesis is that sensitivity to DNA damaging chemotherapy in ovarian cancer cells is determined in part by post-translational regulation of the apoptosome components, Apaf-1 and caspase 9. To test this hypothesis, we propose the following aims: (1) To identify the regulatory mechanism(s) of Apaf-1/ caspase 9 interaction that govern cisplatin sensitivity in ovarian cancer, and (2) To determine whether manipulating the apoptosome is a viable chemotherapeutic strategy in vivo. The proposed research will utilize established ovarian cancer cell lines with differential sensitivities to cisplatin, employing an unbiased proteomics approach to screen for post-translational modifications and binding partners of Apaf-1 and caspase 9. The mechanism of differential control of the apoptosome will then be validated using biochemical tools to monitor changes in binding and activation of these proteins and sensitivity to cytochrome c induced apoptosis. Subcellular localization differences will also be explored using fractionation approaches and immunofluorescent imaging. We will then use a xenograft model, with an inducible knockdown of Apaf-1, as proof of principle to determine the contribution of the apoptosome to chemosensitivity in vivo. Subsequently, we will generate biochemical mimics of differential post-translational regulatory mechanism identified in the first aim in a xenograft model with cisplatin resistant background. We expect that enhancing apoptosome activity in cell lines that are resistant to cisplatin will increase susceptibility of ovarian cancr cells to DNA damage induced apoptosis, leading to the discovery of new chemotherapeutic targets.
Patients with epithelial ovarian cancer often experience resistance to classical chemotherapy agents. To address this problem, we propose to determine how ovarian cancer cells inhibit cell death and potentially identify new therapeutic targets that will sensitize ovarian cancer cells to chemotherapy induced cell death. It is our hope that this research will lead to more effective treatment of epithelial ovarian cancer and increase patient survival.