Bmi-1, a potential therapeutic target in ovarian cancer

Bmi-1，卵巢癌的潜在治疗靶点

• 批准号: 9067819
• 负责人: Resham Bhattacharya
• 金额: $ 31.15万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 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 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9067819
• 关键词: Acetylcysteine; Address; Apoptosis; B-Cell Lymphomas; CASP3 gene; 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 breast cancer; Methods; MicroRNAs; Mitochondria; Modeling; Molecular; Molecular Target; Moloney Leukemia Virus; Morbidity - disease rate; Mus; Neoplastic Cell Transformation; Nodule; Oncogenic; Operative Surgical Procedures; Ovarian; Oxidation-Reduction; Oxygen; Oxygen Consumption; Paclitaxel; Pathway interactions; Patient-Focused Outcomes; Patients; Phosphorylation; Play; Polycomb; Process; Production; Reactive Oxygen Species; Recurrent Malignant Neoplasm; Relapse; Reporting; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Small Interfering RNA; Staging; Stem cells; Testing; Therapeutic; Tissues; Treatment Efficacy; Tumor Debulking; United States National Institutes of Health; Western Blotting; cancer cell; carcinogenesis; chemotherapy; combat; drug sensitivity; gene product; in vivo; innovation; intraperitoneal; knock-down; mortality; mouse model; nanoliposome; neoplastic cell; novel therapeutic intervention; 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.

描述（由申请人提供）：毫无疑问，卵巢癌对复发性癌症患者来说是一种令人烦恼、无法治愈的疾病，已被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；因此，发病率恶化，生存期大大缩短。鉴于这一不幸的困境，我们的目标是探索Bmi-1在卵巢癌发生中的治疗潜力。在这种情况下，确定Bmi-1缺失使癌细胞对顺铂敏感的机制对于开发对抗卵巢癌的新治疗策略非常重要。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，在包括卵巢癌在内的多种癌症中，Bmi-1的过表达及其与临床分级/分期的相关性已被报道。我们证明，下调Bmi-1可降低卵巢癌细胞的克隆性、增殖，并使卵巢癌细胞对顺铂诱导的凋亡敏感。Bmi- 1引起淋巴细胞的肿瘤转化，并与H-Ras合作，在小鼠中引起转移性乳腺癌，这些都强烈提示在上皮恶性肿瘤中起致瘤作用。因此，开发新的治疗策略，使Bmi-1失活，以对抗卵巢癌，很可能对临床结果和患者管理产生重大影响。在这里，我们假设Bmi-1在卵巢癌的生长中起重要作用，并且可以通过操纵来增加对顺铂的敏感性。此外，我们将研究下调Bmi-1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。为了验证我们的假设，我们提出以下目标：目的1：确定Bmi-1缺失增强药物敏感性的机制。基本原理和假设：我们最近证明，通过siRNA或miR下调Bmi-1可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，这表明活性氧（ROS）和/或NFkappaB （NF:B）参与了这一过程。此外，顺铂治疗Bmi-1敲低的细胞导致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可以通过NF:B和/或ROS介导的DDR通路激活来增强顺铂诱导的细胞凋亡。方法：因此(i) NF:B激活状态，（ii） Bmi-1沉默顺铂处理细胞的总ROS和线粒体ROS生成将被确定。线粒体参数，如氧气消耗，ATP生产，基因产品参与氧化还原稳态将被研究。（iii）通过ATM1、ATR、Chk1和Chk2等不同标记物磷酸化或定位的变化来研究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2：确定体内Bmi-1敲低作为卵巢癌顺铂化疗敏感性调节剂的治疗潜力。基本原理和假设：我们最近在A-2780/CP-20腹腔模型中证明，纳米脂质体在体内递送Bmi-1 siRNA后，卵巢肿瘤的生长和结节形成显著减少。直接靶向Bmi-1 mRNA的siRNA或microRNA-15a/16下调Bmi-1可抑制细胞增殖、克隆原性并使细胞对顺铂诱导的凋亡敏感。因此，我们假设在临床前小鼠模型中，下调Bmi-1会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）细胞制备原位卵巢肿瘤小鼠模型，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siRNA/miR纳米脂质体偶联物将在肿瘤接种后1周腹腔注射，并持续4周。我们将确定(i)是否通过siRNA敲低Bmi-1， （ii） miR-15a/16敲低Bmi-1是否抑制肿瘤生长并使细胞对体内顺铂敏感；（iii）或者利用强力霉素诱导的shRNA在A-2780和CP-70细胞中有条件地敲低Bmi-1；（iv）通过Western blot分析、RT-PCR和免疫组化（IHC）验证活体肿瘤细胞中DDR和凋亡通路的分子靶点，如Aim 1所示。这些研究将验证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，甚至在化疗耐药卵巢癌中也有潜在的治疗潜力。

项目成果

Role of TGF-β signaling in uterine carcinosarcoma.

• DOI: 10.18632/oncotarget.3711
• 发表时间: 2015-06-10
• 期刊: Oncotarget
• 作者: Dwivedi SK;McMeekin SD;Slaughter K;Bhattacharya R
• 通讯作者: Bhattacharya R

MDR1 mediated chemoresistance: BMI1 and TIP60 in action.

• DOI: 10.1016/j.bbagrm.2016.06.002
• 发表时间: 2016-08
• 期刊: Biochimica et biophysica acta
• 作者: Banerjee Mustafi S;Chakraborty PK;Naz S;Dwivedi SK;Street M;Basak R;Yang D;Ding K;Mukherjee P;Bhattacharya R
• 通讯作者: Bhattacharya R

Bmi-1: At the crossroads of physiological and pathological biology.

• DOI: 10.1016/j.gendis.2015.04.001
• 发表时间: 2015-09
• 期刊: Genes & diseases
• 影响因子: 6.8
• 作者: Bhattacharya R;Mustafi SB;Street M;Dey A;Dwivedi SK
• 通讯作者: Dwivedi SK

Therapeutic evaluation of microRNA-15a and microRNA-16 in ovarian cancer.

• DOI: 10.18632/oncotarget.7618
• 发表时间: 2016-03-22
• 期刊: Oncotarget
• 作者: Dwivedi SK;Mustafi SB;Mangala LS;Jiang D;Pradeep S;Rodriguez-Aguayo C;Ling H;Ivan C;Mukherjee P;Calin GA;Lopez-Berestein G;Sood AK;Bhattacharya R
• 通讯作者: Bhattacharya R