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

描述(申请人提供)：细胞DNA损伤反应(DDR)是一个高度复杂的系统，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。BRCA1 C末端(BRCT)结构域是存在于以BRCA1为代表的一组排他性蛋白质中的模块结构域，它们介导DDR和细胞周期检查点。以串联结构域(TBRCT)形式出现的BRCT模块可以作为单个结构单位与DDR相关激酶诱导的磷酸化多肽结合。我们的研究重点是利用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个蛋白质组成，具有1013个相互作用，其中947个是新的。从这个数据丰富的网络中，BRCA1的tBRCT被发现与Rictor、mSIN1和PRR5相互作用，这些是mTORC2-信号复合体的核心成分，对激活增殖和抗凋亡激酶AKT至关重要。免疫沉淀证实了内源性BRCA1复合体的相互作用，并发现mTOR在正常培养条件下和DNA损伤后不能与这些复合体结合，但在血清饥饿条件下可以结合，提示翻译后调节。靶向shRNA抑制BRCA1的表达可激活AKT/mTOR通路。MTORC2免疫复合物的分离和随后的体外激酶检测表明，重组BRCA1 tBRCT能够通过干扰Rictor的mTOR来抑制mTORC2的活性。这些结果清楚地表明BRCA1和mTORC2信号之间存在功能联系。因此，我们将检验这一假设，即BRCA1对mTORC2的负调控是DNA损伤反应的重要组成部分，而BRCA1的状态决定了乳腺癌细胞对涉及mTOR抑制剂的治疗的反应。这将通过以下实验来完成：1)BRCA1 tBRCT与Rictor、mSIN1和PRR5相互作用的表征；2)从BRCA1定位、细胞周期检查点、BRCA1介导的转录和凋亡方面分析mTORC2和BRCA1介导的DNA损伤反应之间的功能联系；以及3)确定BRCA1状态与乳腺细胞系中加入mTOR抑制的治疗反应之间的相关性。这项研究将有助于更好地了解BRCA1的肿瘤抑制活性以及BRCA1在DNA损伤时对mTORC2信号的调节，以便开发利用这一信号模块的个性化治疗方法。

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.