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.
In this proposal we aim to determine the effects on DNA damage signaling regulated by the novel protein- protein interaction between the breast and ovarian cancer associated protein BRCA1 and components of the oncogenic kinase mTORC2. This study is noteworthy as it has the potential to be immediately translatable to breast cancer patients by advancing personalized therapies tailored to individual gene profiles.
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