Bmi-1, a potential therapeutic target in ovarian cancer

描述（由申请人提供）：毫无疑问，卵巢癌是一种令人烦恼的，无法治愈的疾病，复发性癌症患者，并已宣布由美国国立卫生研究院的四个无法治愈的癌症之一。有效的治疗选择相当有限;因此，发病率和生存率大大缩短。鉴于这一不幸的困境，我们本提案的目标是探索Bmi-1在卵巢癌发生中的治疗潜力。在这种情况下，确定Bmi-1的缺失使癌细胞对顺铂敏感的机制对于开发新的治疗策略以对抗卵巢癌将是重要的。Bmi-1（B淋巴瘤小鼠莫洛尼白血病病毒插入区）调节正常干细胞和祖细胞的自我更新。此外，Bmi-1的过表达及其与临床分级/分期的相关性已在包括卵巢癌在内的多种癌症中报道。我们证明，Bmi-1的下调降低了卵巢癌细胞的克隆形成，增殖和敏感的顺铂诱导的凋亡。Bmi-1导致淋巴细胞的肿瘤转化，并与H-Ras合作，引起小鼠转移性乳腺癌，所有这些都强烈表明在上皮恶性肿瘤中的致癌作用。因此，开发新的治疗策略来抑制Bmi-1以对抗卵巢癌很可能会对临床结果和患者管理产生强烈影响。在这里，我们假设Bmi-1在卵巢癌的生长中起着重要的作用，并且可以被操纵以增加对顺铂的敏感性。此外，我们将研究Bmi-1的下调使卵巢癌细胞对顺铂敏感的分子机制，以开发新的治疗策略。为了解决我们的假设，我们提出了以下目标：目的1：确定Bmi-1的丢失增强药物敏感性的机制。原理和假设：我们最近证明，通过siRNA或miR下调Bmi-1显著增强顺铂诱导的卵巢癌细胞凋亡，包括顺铂耐药的CP-70。重要的是，氧清除剂N-乙酰半胱氨酸（NAC）的使用消除了顺铂诱导的细胞凋亡，表明活性氧（ROS）和/或NF κ B（NF：B）参与了该过程。此外，顺铂治疗Bmi-1敲低细胞导致DNA损伤反应（DDR）途径的显著参与，导致细胞凋亡。因此，我们假设沉默Bmi-1通过NF：B和/或ROS介导的DDR途径的激活增强顺铂诱导的细胞凋亡。方法：因此，将确定Bmi-1沉默的顺铂处理的细胞中的（i）NF：B活化状态，（ii）总的和线粒体ROS产生。线粒体参数，如耗氧量，ATP的生产，参与氧化还原稳态的基因产物将被研究。(iii)将通过不同标志物（包括ATM 1、ATR、Chk 1和Chk 2）的磷酸化或定位的变化来研究导致细胞凋亡的DDR接合。还将测定Caspase-3、8、9和PARP的裂解。目的2：确定Bmi-1基因敲低作为卵巢癌体内顺铂化疗敏感性调节剂的治疗潜力。原理和假设：我们最近证明，在A-2780/CP-20腹腔内模型中，通过纳米脂质体体内递送Bmi-1 siRNA后，卵巢肿瘤生长和结节形成显著减少。通过直接靶向Bmi-1 mRNA的siRNA或microRNA-15a/16下调Bmi-1导致增殖抑制、克隆形成和对顺铂诱导的细胞凋亡敏感的细胞。因此，我们假设Bmi-1的下调将抑制卵巢肿瘤的生长，并在临床前小鼠模型中使肿瘤细胞对顺铂敏感。方法：我们将在通过注射A-2780或CP-70（顺铂耐药）顺铂产生的原位腹膜内卵巢肿瘤小鼠模型中检测治疗效果。为了模拟晚期小体积疾病的治疗，顺铂和/或siRNA/miR的纳米脂质体缀合物将在肿瘤接种后1周腹膜内施用并持续4周。我们将确定（i）是否通过siRNA敲低Bmi-1，（ii）是否通过miR-15 a/16敲低Bmi-1抑制肿瘤生长并使细胞对顺铂体内敏感;（iii）或者，将利用A-2780和CP-70细胞中通过shRNA的多西环素诱导的Bmi-1条件性敲低;（iv）通过Western印迹分析、RT-PCR和免疫组织化学（IHC）在体内肿瘤细胞中鉴定如目标1中的DDR和凋亡途径的分子靶标。拟议的研究将验证Bmi-1作为一个重要的新靶点，使用易于翻译的纳米脂质体递送方法，即使在化疗耐药的卵巢癌中也具有治疗潜力。

DESCRIPTION (provided by applicant): 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.