Control of the apoptosome in epithelial ovarian cancer to enhance chemotherapy

控制上皮性卵巢癌中的凋亡体以增强化疗

基本信息

批准号：
8649372
负责人：
Kimberly Cocce Darlington
金额：
$ 3.31万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8649372
关键词：
Adaptor Signaling Protein Address Adjuvant Chemotherapy Affect Apoptosis Apoptotic Binding Biochemical Bypass Cancer cell line Caspase Cell Death Cell Line Cells Cessation of life Cisplatin Cleaved cell Complex DNA DNA Damage Defect Development Diagnosis Disease Epithelial ovarian cancer Experimental Designs Fractionation Functional disorder Human Image Immunohistochemistry In Vitro Inhibition of Apoptosis Lead Left Malignant Neoplasms Malignant neoplasm of ovary Mass Spectrum Analysis Mediating Mediator of activation protein Microscopic Mitochondria Molecular Molecular Target Monitor Mus Operative Surgical Procedures Ovarian Pathway interactions Patients Platinum Play Post-Translational Protein Processing Post-Translational Regulation Predisposition Procedures Process Proliferating Proteins Proteomics Regimen Regulation Research Residual state Resistance Role Sampling Staging Survival Rate Symptoms Taxane Compound Testing Tumor Debulking Woman Xenograft Model apoptotic protease-activating factor 1 base cancer cell cancer therapy caspase-3 caspase-9 chemotherapeutic agent chemotherapy cytochrome c effective therapy experience in vivo killings neoplastic cell new therapeutic target ovarian neoplasm prevent pro-caspase-9 protein activation public health relevance response small hairpin RNA taxane therapeutic target tool tumor

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：卵巢癌是一种毁灭性的疾病，超过一半的受影响女性在诊断后五年内死亡。由于症状微妙，卵巢癌常常未被发现，直到达到晚期阶段，而晚期阶段本质上已经广泛转移。外科减瘤手术通常会留下大量残留的微观疾病。在这种情况下，唯一的治疗手段是辅助化疗，但由于原发性或获得性肿瘤细胞耐药性，辅助化疗常常失败。许多常用的化疗药物通过破坏细胞 DNA 的内在凋亡途径杀死卵巢肿瘤细胞，导致线粒体释放细胞色素 c 并形成活性凋亡体，该凋亡体由细胞色素 c、衔接蛋白、Apaf-1 和 caspase 9 组成然后，这种活性凋亡体会裂解并激活效应半胱天冬酶，进而裂解大量细胞蛋白，最终导致细胞死亡。耐药性癌症部分通过限制凋亡体的激活来避免 DNA 损伤性化疗（例如顺铂）诱导的细胞凋亡。在卵巢癌中，凋亡体功能的缺乏可归因于无法激活 Apaf-1 和/或 caspase 9 募集缺陷。重要的是，化疗耐药性卵巢癌中控制 Apaf-1/caspase 9 相互作用的机制和凋亡体在体内化学抗性中的作用已被明确定义。我们的中心假设是，卵巢癌细胞对 DNA 损伤性化疗的敏感性部分取决于凋亡体成分 Apaf-1 和 caspase 9 的翻译后调节。为了检验这一假设，我们提出以下目标：(1)确定控制卵巢癌顺铂敏感性的 Apaf-1/ caspase 9 相互作用的调节机制，以及 (2) 确定操纵凋亡体是否是一种可行的体内化疗策略。拟议的研究将利用已建立的对顺铂具有不同敏感性的卵巢癌细胞系，采用无偏见的蛋白质组学方法来筛选 Apaf-1 和 caspase 9 的翻译后修饰和结合伴侣。然后，将研究凋亡体的差异控制机制。使用生化工具进行验证，以监测这些蛋白质的结合和激活的变化以及对细胞色素 c 诱导的细胞凋亡的敏感性。还将使用分级方法和免疫荧光成像来探索亚细胞定位差异。然后，我们将使用可诱导敲低 Apaf-1 的异种移植模型作为原理证明，以确定凋亡体对体内化学敏感性的贡献。随后，我们将在具有顺铂耐药背景的异种移植模型中生成第一个目标中确定的差异翻译后调节机制的生化模拟物。我们预计，增强对顺铂耐药的细胞系中的凋亡体活性将增加卵巢癌细胞对DNA损伤诱导的细胞凋亡的敏感性，从而导致新的化疗靶点的发现。