Bmi-1, a potential therapeutic target in ovarian cancer

Bmi-1，卵巢癌的潜在治疗靶点

• 批准号: 8233863
• 负责人: Resham Bhattacharya
• 金额: $ 34.45万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-07 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8233863
• 关键词: Acetylcysteine; Address; Apoptosis; B-Cell Lymphomas; Cancer Patient; Carboplatin; Cell Line; Cells; Cisplatin; Clinical; DNA Damage; Data; Development; Disease; Down-Regulation; Doxycycline; Drug resistance; Epithelial; Generations; Genes; Goals; Growth; Gynecologic; HRAS gene; Homeostasis; Immunohistochemistry; In Vitro; Lymphocyte; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Messenger RNA; Metastatic Neoplasm to the Breast; Methods; MicroRNAs; Mitochondria; Modeling; Molecular; Molecular Target; Moloney Leukemia Virus; Morbidity - disease rate; Mus; Neoplastic Cell Transformation; Nodule; Oncogenic; Operative Surgical Procedures; Outcome; Ovarian; Oxidation-Reduction; Oxygen; Oxygen Consumption; Paclitaxel; Pathway interactions; Patients; Phosphorylation; Play; Polycomb; Process; Production; Proteins; Reactive Oxygen Species; Recurrent Malignant Neoplasm; Relapse; Reporting; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Simulate; Small Interfering RNA; Staging; Stem cells; Testing; Therapeutic; Tissues; Treatment Efficacy; Tumor Debulking; United States National Institutes of Health; Western Blotting; cancer cell; carcinogenesis; caspase-3; chemotherapy; combat; drug sensitivity; in vivo; innovation; intraperitoneal; mortality; mouse model; nanoliposome; neoplastic cell; novel therapeutics; ovarian neoplasm; overexpression; pre-clinical; response; self-renewal; small hairpin RNA; stem; therapeutic target; tumor; tumor growth; tumor progression

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. PUBLIC HEALTH RELEVANCE: 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 and therefore most likely will have a strong impact on clinical outcome and patient management.

描述(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，对于复发的癌症患者来说，它已经被NIH宣布为四种无法治疗的癌症之一。有效的治疗选择相当有限；其结果是，发病率恶化，存活率大幅缩短。鉴于这一不幸的两难境地，我们在这项建议中的目标是探索BMI-1在卵巢癌发生中的治疗潜力。在这种背景下，确定BMI-1缺失使癌细胞对顺铂敏感的机制对于开发抗击卵巢癌的新治疗策略将是重要的。BMI-1(B淋巴瘤小鼠Moloney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的过度表达及其与临床分级/分期的相关性已被报道在包括卵巢癌在内的多种癌症中。我们证明，BMI-1的下调降低了卵巢癌细胞的克隆性、增殖性和对顺铂诱导的凋亡的敏感性。Bmi-1导致淋巴细胞的肿瘤性转化，并与H-RAS协同作用导致小鼠转移性乳腺癌，所有这些都强烈表明在上皮性恶性肿瘤中具有致癌作用。因此，开发新的治疗策略来灭活BMI-1来对抗卵巢癌，很可能会对临床结果和患者管理产生强烈的影响。在这里，我们假设BMI-1在卵巢癌的生长中起着重要的作用，并且可以被操纵来增加对顺铂的敏感性。此外，我们将研究BMI-1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：确定BMI-1缺失增强药物敏感性的机制。理论基础和假设：我们最近证明，siRNA或miR下调Bmi-1可显著增强顺铂诱导的卵巢癌细胞的凋亡，包括顺铂耐药的CP-70。重要的是，使用氧清除剂N-乙酰半胱氨酸(NAC)可阻断顺铂诱导的细胞凋亡，提示ROS和/或NFkappaB(NF：B)参与了这一过程。此外，顺铂在BMI-1基因敲除细胞中的处理导致DNA损伤反应(DDR)通路的显著参与，从而导致细胞凋亡。因此，我们假设沉默BMI-1通过核因子：B和/或ROS介导的DDR通路的激活来增强顺铂诱导的细胞凋亡。方法：测定BMI-1沉默的顺铂处理细胞的核因子：B的活化状态，(Ii)总ROS和线粒体ROS的产生。线粒体参数，如耗氧量、ATP产量、参与氧化还原动态平衡的基因产物等将被研究。(Iii)通过ATM1、ATR、Chk1和Chk2等不同标志物的磷酸化或定位变化来研究DDR参与导致细胞凋亡。Caspase-3、8、9和PARP的切割也将被确定。目的：探讨BMI-1基因敲除作为顺铂化疗敏感性调节剂对卵巢癌的体内治疗作用。理论基础和假设：我们最近在A-2780/CP-20腹膜内模型中证明，通过体内纳米脂质体传递Bmi-1 siRNA后，卵巢肿瘤的生长和结节形成显著减少。通过siRNA或microRNA-15a/16下调Bmi-1的表达，可抑制细胞的增殖和克隆形成，并使细胞对顺铂诱导的细胞凋亡敏感。因此，我们假设，在临床前小鼠模型中，BMI-1的下调将抑制卵巢肿瘤的生长，并使肿瘤细胞对顺铂敏感。方法：我们将通过注射A-2780或CP-70(顺铂耐药)细胞建立的小鼠原位腹膜内卵巢肿瘤模型来测试其治疗效果。为了模拟晚期小体积疾病的治疗，顺铂和或纳米脂质体的siRNA/miR偶联物在肿瘤接种后1周内给予，持续4周。我们将确定(I)是否通过siRNA下调Bmi-1，(Ii)是否通过miR-15a/16下调Bmi-1在体内是否抑制肿瘤生长并增强细胞对顺铂的敏感性；(Iii)或者，将利用多西环素在A-2780和CP-70细胞中通过shRNA诱导的条件下调Bmi-1的分子靶点；(Iv)通过Western印迹分析、RT-PCR和免疫组织化学(IHC)验证目的1中的DDR和凋亡途径的分子靶点。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。 公共卫生相关性：拟议的研究将验证BMI-1作为一种重要的新靶点，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，因此最有可能对临床结果和患者管理产生重大影响。