Undoubtedly ovarian cancer is a vexing, incurable disease for patients with recurrent cancer and has been declared one of four untreatable cancers by NIH. Effective therapeutic options are quite limited; and, as a result, morbidity worsens and survival shortens dramatically. In view of this unfortunate dilemma, our goal in this proposal is to explore the therapeutic potential of Bmi-1 in ovarian carcinogenesis. In this context, determining the mechanism by which loss of Bmi-1 sensitizes the cancer cells to cisplatin would be important for development of new therapeutic strategies to combat ovarian cancer. Bmi-1 (B lymphoma mouse Moloney leukemia virus insertion region) regulates the self-renewal of normal stem and progenitor cells. In addition, overexpression of Bmi-1 and its correlation with clinical grade/stage has been reported in a variety of cancers including ovarian cancer. We demonstrated that, downregulation of Bmi-1 decreased clonogenicity, proliferation and sensitized ovarian cancer cells to cisplatin-induced apoptosis. Bmi- 1 causes neoplastic transformation of lymphocytes and co-operates with H-Ras giving rise to metastatic breast cancer in mice, all strongly suggesting an oncogenic role in epithelial malignancies. Hence the development of new therapeutic strategies to inactivate Bmi-1 to combat ovarian cancer most likely will have a strong impact on clinical outcome and patient management. Here we postulate that Bmi-1 plays an important role in ovarian cancer growth and can be manipulated to increase sensitivity to cisplatin. Furthermore, we will investigate the molecular mechanism by which downregulation of Bmi-1 sensitizes the ovarian cancer cells to cisplatin to develop new therapeutic strategies. To address our hypothesis, we propose the following aims: Aim1: To determine the mechanism by which loss of Bmi-1 enhances drug-sensitivity. Rationale and hypothesis: We recently demonstrated that downregulation of Bmi-1 by siRNA or miR significantly enhanced cisplatin-induced apoptosis in ovarian cancer cells including the cisplatin resistant CP- 70. Importantly use of the oxygen scavenger N-Acetylcysteine (NAC) abolished cisplatin induced apoptosis suggesting involvement of reactive oxygen species (ROS) and/or NFkappaB (NF:B) in this process. Furthermore, cisplatin treatment in Bmi-1 knockdown cells led to significant engagement of the DNA damage response (DDR) pathway leading to apoptosis. Hence, we hypothesize that silencing Bmi-1 enhances cisplatin-induced apoptosis through NF:B and/or ROS mediated activation of the DDR pathway. Approach: Therefore (i) NF:B activation status, (ii) total and mitochondrial ROS generation in Bmi-1 silenced cisplatin treated cells wil be determined. Mitochondrial parameters such as oxygen consumption, ATP production, gene products involved in redox homeostasis will be studied. (iii) DDR engagement leading to apoptosis will be studied by changes in phosphorylation or localization of different markers including ATM1, ATR, Chk1 and Chk2. Cleavage of Caspase-3, 8, 9 and PARP will also be determined. Aim2: To determine the therapeutic potential of Bmi-1 knockdown as a modulator of chemosensitivity to cisplatin in ovarian cancer in vivo. Rationale and hypothesis: We recently demonstrated that ovarian tumor growth and nodule formation were significantly reduced upon delivery of Bmi-1 siRNA by nanoliposome in vivo in an A-2780/CP-20 intraperitoneal model. Downregulation of Bmi-1 by siRNA or microRNA-15a/16 that directly target Bmi-1 mRNA resulted in inhibition of proliferation, clonogenicity and sensitized cells to cisplatin-induced apoptosis. Hence, we hypothesize that downregulation of Bmi-1 will inhibit ovarian tumor growth and sensitize the tumor cells to cisplatin in a preclinical mouse model. Approach: We will test the therapeutic efficacy in orthotopic intraperitoneal ovarian tumor mouse models generated by injecting A-2780 or CP-70 (cisplatin resistant) cels. To simulate treatment of advanced smal- volume disease, cisplatin and or nanoliposomal conjugates of siRNA/miR will be administered intraperitoneally 1 week after tumor inoculation and continued for 4 weeks. We will determine (i) if knockdown of Bmi-1 by siRNA, (ii) if knockdown of Bmi-1 by miR-15a/16 inhibits tumor growth and sensitizes cells to cisplatin in vivo; (iii) Alternatively, doxycycline inducible conditional knockdown of Bmi-1 by shRNA in A-2780 and CP-70 cells will be utilized; (iv) Validate the molecular targets of the DDR and apoptosis pathway as in Aim 1, in tumor cells in vivo by Western blot analysis, RT-PCR and immunohistochemistry (IHC). The proposed studies will validate Bmi-1 as an important new target using readily translatable nanoliposomal delivery methods with potential for therapy even in chemoresistant ovarian cancer.

R01CA157481

2012-09-07

2018-06-30