Control of the apoptosome in epithelial ovarian cancer to enhance chemotherapy

描述(申请人提供)：卵巢癌是一种毁灭性的疾病过程，超过一半的受影响的妇女在确诊后五年内死亡。由于症状微妙，卵巢癌通常不会被发现，直到达到晚期，这一阶段天生就是广泛转移的。外科手术通常会留下大量残留的微小疾病。在这种状态下，唯一的治疗方法是辅助化疗，这通常是由于原发或获得性肿瘤细胞耐药性而失败的。许多常用的化疗方法通过破坏细胞DNA，通过固有的凋亡途径杀死卵巢肿瘤细胞，导致细胞色素c的线粒体释放和活性凋亡体的形成，该凋亡体由细胞色素c、适配蛋白、APAF-1和caspase 9组成。这个活跃的凋亡体然后裂解并激活效应器caspase，进而裂解一系列细胞蛋白，最终导致细胞死亡。耐药肿瘤避免了DNA损伤化疗(如顺铂)诱导的细胞凋亡，部分原因是限制了凋亡体的激活。在卵巢癌中，凋亡体功能的缺乏可以追溯到不能激活APAF-1和/或caspase 9募集缺陷。重要的是，在卵巢癌化疗耐药中控制APAF-1/caspase 9相互作用的机制和凋亡体在体内化疗耐药中的作用都还不清楚。我们的中心假设是，卵巢癌细胞对DNA损伤化疗的敏感性部分取决于凋亡体成分APAF-1和Caspase9的翻译后调节。为了检验这一假设，我们提出了以下目标：(1)确定APAF-1/Caspase9相互作用的调控机制(S)，控制卵巢癌顺铂的敏感性；(2)确定操纵凋亡体是否是体内可行的化疗策略。这项拟议的研究将利用已建立的对顺铂具有差异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伙伴。然后将使用生化工具来监测这些蛋白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，从而验证对凋亡体的差异控制的机制。还将使用分离方法和免疫荧光成像来探索亚细胞定位的差异。然后，我们将使用异种移植模型，通过可诱导的APAF-1基因敲除，作为原则证据来确定凋亡体在体内对化疗敏感性的贡献。随后，我们将在顺铂耐药的异种移植模型中产生第一个目的中确定的差异翻译后调控机制的生化模拟。我们预计，增强顺铂耐药细胞株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易感性，从而导致新的化疗靶点的发现。

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.