Mutations in the breast cancer susceptibility protein, BRCA1, are heavily implicated in familial breast and ovarian cancers that are classified as triple negative. Triple negative tumors lack estrogen receptors, progesterone receptors and Her2 expression that are commonly used drug targets to enhance treatment options for other forms of breast cancer. Thus, patients afflicted with triple negative cancers have limited treatment options and succumb to recurrence in less time following conventional therapy. Under normal conditions, the BRCA1 protein acts as a tumor suppressor, helping correct breaks in genomic DNA and ensure fidelity in newly synthesized mRNA. Defects in these regulatory processes lead to genomic instability and to tumor initiation. Understanding the molecular basis for triple negative breast cancer induction related to BRCA1 mutations could significantly contribute to the development of new treatment options for patients afflicted with this aggressive disease. Our overall goal is to develop a new tunable microchip-based strategy to study the structural attributes of BRCA1 protein assemblies involved in nuclear protective processes -- and to examine how defects in the BRCA1 protein can impact the formation of these essential protein assemblies. Our proposed research will provide a multi-disciplinary opportunity to bridge technologies used in cancer biology, material science, and structural biology to address long-standing questions involving the role BRCA1 in gene regulatory events. Tunable Affinity Capture microchip devices will be used recruit functionally distinct BRCA1- associated protein complexes from human breast cancer cells. We will establish an on-chip molecular sorting technique that employs adaptor molecules specific for transcription-related complexes bound to DNA. Isolated complexes will be characterized using biochemical methodologies. We will then utilize cryo-Electron Microscopy (EM) to determine the 3D architecture of the microchip-sorted BRCA1 protein assemblies. Combined techniques of antibody labeling and molecular modeling will permit us to interpret EM density maps that will reveal the molecular interactions among the BRCA1-associated assemblies. Long-term, our findings could provide a structural template for the design of new therapies aimed at tuning protein-protein interactions in basal-like subtypes of breast and ovarian cancers.

Public Health Relevance

Treatment options are limited for patients with triple negative breast and ovarian cancers, and there is no known cure. We will investigate the actions of a prime culprit implicated in causing the disease, the protein factor, BRCA1. We will determine in 3D how BRCA1 operates during the genetic readout of DNA, with the expectation that this information can lead to new therapies to better manage BRCA1-related cancers.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-EBIT-J (09))
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Knowlton, John R
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Virginia Polytechnic Institute and State University
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United States
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Gilmore, Brian L; Varano, A Cameron; Dearnaley, William et al. (2018) Preparation of Tunable Microchips to Visualize Native Protein Complexes for Single-Particle Electron Microscopy. Methods Mol Biol 1764:45-58
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