Assistant Professor, Eppley Institute
Director of the Proteomics & Systems Biology
Tel: 402-559-2248 (Office)
Protein-Protein Interactions: Dysregulation of the DNA damage response in cancer
Summary of Research:
The major focus of our laboratory is to discover novel regulatory mechanisms in the DNA damage response (DDR) network through proteomic profiling of protein-protein interactions. Our lab uses an integrated pipeline of proteomics discovery, bioinformatic analysis, and functional profiling of interactions using standard biochemical techniques. This approach is focused on discovering novel mechanisms in the DDR that can be exploited to develop and refine precision therapies in cancer.
The DDR network 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 a small specialized 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 BRCT domain-protein interaction network using all of the BRCT domains in the human proteome in a comprehensive screening process that identified a network consisting of 1682 proteins with 3185 interactions. From this data-rich network, the tandem BRCT domain of BRCA1 was found to interact with Rictor, mSIN1, and PRR5 which are core components of the oncogenic 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 the recombinant BRCA1 tBRCT was able to inhibit mTORC2 activity by disrupting mTOR from Rictor. Currently, we are examining the correlation between BRCA1 expression or mutation status in breast cancer and their sensitivity to mTOR inhibition. This study will hopefully lead to the identification of breast cancer patient subpopulations that are sensitive to mTOR inhibitor based therapies.
The BRCT domain interactome has also identified several functionally significant interactions with Fanconi Anemia (FA) genes. In addition, young patients with DDR disruptions in FA genes are significantly more susceptible to endocrinopathies. Therefore, ongoing projects in the lab are exploring the intersection of BRCT domain-mediated DNA damage recognition and repair with the regulation of the FA pathway in human cancer, as well as defining the cell-specific protein interactome of all FA proteins with a focus on freshly isolated human primary pancreas endocrine cells. These studies are also designed to determine the disruption of FA protein interactions caused by cancer-associated mutations. A deeper understanding of the mechanisms regulating the DDR may identify opportunities to sensitize cancer cells to DNA damaging radiation and chemotherapies to improve cancer patient survival and outcome.