Control of the apoptosome in epithelial ovarian cancer to enhance chemotherapy

描述（由申请人提供）：卵巢癌是一种毁灭性的疾病过程，在诊断后五年内杀死一半以上的受影响妇女。由于微妙的症状，卵巢癌往往不被发现，直到他们达到一个先进的阶段，这是固有的广泛转移。外科减积手术经常会留下大量的残余显微病变。在这种情况下，唯一的治疗手段是辅助化疗，这往往是由于原发性或获得性肿瘤细胞耐药而失败的。许多常用的化疗药物通过损伤细胞DNA的内在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核糖体的形成，其由细胞色素c、衔接蛋白、Apaf-1和半胱天冬酶9组成。然后，这种活跃的核糖体切割并激活效应物半胱天冬酶，后者继续切割宿主细胞蛋白，最终导致细胞死亡。 耐药癌症避免了DNA损伤化疗诱导的细胞凋亡（例如，顺铂），部分通过限制脂质体的活化。在卵巢癌中，溶酶体功能的缺乏可以追溯到无法激活Apaf-1和/或半胱天冬酶9的募集缺陷。重要的是，在化疗耐药的卵巢癌中，控制Apaf-1/caspase 9相互作用的机制，以及体内化疗耐药中Apaf-1/caspase 9的作用都没有明确的定义。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转录后调节的转录体成分，Apaf-1和半胱天冬酶9。为了验证这一假设，我们提出了以下目标：（1）确定Apaf-1/ caspase 9相互作用的调节机制，这些机制控制卵巢癌顺铂敏感性，（2）确定操纵肿瘤小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筛选Apaf-1和caspase 9的翻译后修饰和结合伴侣。然后将使用生物化学工具来验证微囊体的差异控制机制，以监测这些蛋白质的结合和活化的变化以及对细胞色素c诱导的细胞凋亡的敏感性。亚细胞定位差异也将探讨使用分馏方法和免疫荧光成像。然后，我们将使用异种移植模型，与诱导敲低的Apaf-1，作为原则的证据，以确定在体内的化疗敏感性的贡献。随后，我们将在具有顺铂耐药背景的异种移植模型中产生在第一个目标中鉴定的差异翻译后调节机制的生化模拟物。我们期望增强对顺铂耐药的细胞系中的溶酶体活性将增加卵巢癌细胞对DNA损伤诱导的凋亡的易感性，从而发现新的化疗靶点。

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.