Impact of BRCA1-mTORC2 interaction on breast cancer response to DNA damage and ch

描述（由申请人提供）：细胞DNA损伤反应（DDR）是一个高度复杂的系统，它利用蛋白质-蛋白质相互作用和翻译后事件来协调DNA损伤的识别和修复。BRCA 1 C-末端（BRCT）结构域是存在于一组独特的蛋白质中的模块化结构域，例如BRCA 1，其介导DDR和细胞周期检查点。作为串联结构域（tBRCT）出现的BRCT模块可以作为单个结构单元在结合由DDR相关激酶诱导的磷酸化肽中起作用。我们的研究重点是使用7种不同蛋白质的结构域构建tBRCT结构域-蛋白质相互作用网络，该网络由718种蛋白质组成，具有1013种相互作用，其中947种是新的。从这个数据丰富的网络中，发现BRCA 1的tBRCT与Rictor、mSIN 1和PRR 5相互作用，这些是mTORC 2信号传导复合物的核心组分，对增殖和抗凋亡激酶AKT的激活很重要。内源性BRCA 1复合物的免疫沉淀证实了相互作用，并且还发现mTOR在正常培养条件下和DNA损伤后不能结合这些复合物，但在血清饥饿条件下可以结合，这表明了翻译后调节。靶向shRNA抑制BRCA 1表达导致AKT/mTOR通路激活。mTORC 2免疫复合物的分离和随后的体外激酶测定显示，重组BRCA 1 tBRCT能够通过破坏来自Rictor的mTOR来抑制mTORC 2活性。这些结果清楚地表明BRCA 1和mTORC 2信号传导之间的功能联系。因此，我们将检验以下假设：BRCA 1对mTORC 2的负调控是DNA损伤反应的重要组成部分，BRCA 1状态决定了乳腺癌细胞对涉及mTOR抑制剂的治疗的反应。这将通过以下实验完成：1）BRCA 1 tBRCT与Rictor、mSIN 1和PRR 5相互作用的表征; 2）在BRCA 1定位、细胞周期检查点、BRCA 1介导的转录和凋亡方面分析mTORC 2和BRCA 1介导的DNA损伤反应之间的功能联系;和3）确定乳腺细胞系中BRCA 1状态和对掺入mTOR抑制的疗法的应答之间的相关性。这项研究将有助于更好地了解BRCA 1的肿瘤抑制活性以及BRCA 1对DNA损伤的mTORC 2信号传导的调节，以开发利用该信号传导模块的个性化疗法。

DESCRIPTION (provided by applicant): The cellular DNA damage response (DDR) is a highly complex system that utilizes protein-protein interactions and post-translational events to coordinate recognition and repair of DNA lesions. BRCA1 C-Terminal (BRCT) domains are modular domains that exist in an exclusive group of proteins, exemplified by BRCA1, that mediate the DDR and cell cycle checkpoints. BRCT modules that occur as tandem domains (tBRCT) can function as a single structural unit in binding phosphorylated peptides induced by DDR associated kinases. Our research has focused on building a tBRCT domain-protein interaction network using the domains of seven different proteins that identified a network consisting of 718 proteins with 1013 interactions, of which 947 are novel. From this data-rich network, the tBRCT of BRCA1 was found to interact with Rictor, mSIN1, and PRR5, which are core components of the mTORC2-signaling complex, important for activation of the proliferation and anti-apoptotic kinase AKT. Immunoprecipitation of endogenous BRCA1 complexes confirmed the interactions and also found that mTOR is unable to bind these complexes in normal culture conditions and after DNA damage but could bind under serum starvation conditions, suggesting posttranslational regulation. Inhibition of BRCA1 expression by targeted shRNA results in activation of the AKT/mTOR pathway. Isolation of mTORC2 immunocomplexes and subsequent in vitro kinase assays revealed that recombinant BRCA1 tBRCT was able to inhibit mTORC2 activity by disrupting mTOR from Rictor. These results clearly indicate a functional connection between BRCA1 and mTORC2 signaling. Therefore, we will test the hypothesis that negative regulation of mTORC2 by BRCA1 is an important component of the DNA damage response, and BRCA1 status determines the response of breast cancer cells to therapies involving mTOR inhibitors. This will be accomplished with the following experiments: 1) Characterization of the interaction of BRCA1 tBRCT with Rictor, mSIN1, and PRR5; 2) Analysis of the functional connection between mTORC2 and BRCA1-mediated DNA damage response in terms of BRCA1 localization, cell cycle checkpoints, BRCA1-mediated transcription, and apoptosis; and 3) Determine the correlation between BRCA1 status and response to therapies incorporating mTOR inhibition in breast cell lines. This research will lead to a better understanding of the tumor suppressive activity of BRCA1 and the regulation of mTORC2 signaling by BRCA1 in response to DNA damage in order to develop personalized therapies that capitalize on this signaling module.