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：
8918262
负责人：
Kimberly Cocce Darlington
金额：
$ 3.35万
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/8918262
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 biochemical tools 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 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）：卵巢癌是一个毁灭性的疾病过程，在诊断后的五年内杀死了一半以上受影响的妇女。由于症状微妙，卵巢癌经常不被发现直到到达固有地转移的高级阶段。手术伪造程序定期留下大量残留微观疾病。在这种状态下，唯一的治疗方法是辅助化学疗法，由于原发性或获得性肿瘤细胞的耐药性通常会失败。许多常用的化学疗法通过损坏细胞DNA通过固有的凋亡途径杀死了卵巢肿瘤细胞，导致细胞色素C的线粒体释放以及活性凋亡组的形成，从而组成细胞色素c，由cytotrome C组成，由旋转蛋白，apaf-1和CASPase 9。许多细胞蛋白，最终导致细胞死亡。耐药性癌症避免了DNA损害化学疗法（例如顺铂）引起的凋亡，部分原因是限制了凋亡组的激活。在卵巢癌中，缺乏可追溯到caspase 9募集中APAF-1和/或缺陷的缺陷。 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确定操纵凋亡组是否是体内可行的化学治疗策略。拟议的研究将利用对顺铂具有不同敏感性的已建立的卵巢癌细胞系，采用一种无偏的蛋白质组学方法来筛选翻译后修饰和APAF-1和Caspase 9的结合伴侣。 C诱导凋亡。还将使用分级方法和免疫荧光成像探索亚细胞定位差异。然后，我们将使用具有APAF-1诱导型敲低的异种移植模型作为原理的证明，以确定凋亡小体对体内化学敏感性的贡献。随后，我们将生成在具有抗顺铂抗性背景的异种移植模型中第一个目标中鉴定出的差异后调节机制的生化模拟。我们预计，增强对顺铂耐药性的细胞系中的凋亡小体活性会增加卵巢cancr细胞对DNA损伤诱导凋亡的敏感性，从而发现新的化学治疗靶标。