This proposal aims to identify a chemical probe that will serve as an activator of the tumor suppressor gene, breast cancer susceptibility gene 1 (BRCA1). In the absence of somatic mutations, BRCA1 has been found to be down regulated in 30-40% of sporadic breast cancer cases. Decreased expression of BRCA1 accelerates growth of mammary tumor cells, while increased expression leads to growth arrest and apoptosis. Furthermore, over expression of BRCA1 in the murine mammary gland provides protection against mutagen- induced mammary neoplasia. Through this grant mechanism, we aim to gain access to the largest academic screening collection provided by the NIH Molecular Libraries Roadmap Initiative and the Molecular Libraries Probe Production Centers Network (MLPCN). Therefore, this proposal describes a high throughput-ready BRCA1 promoter- driven-Firefly luciferase reporter assay to serve as a primary tool to screen the MLPCN small molecule repository for small molecule activators of BRCA1. Primary screen hits that increase BRCA1 promoter-driven- Firefly luciferase activity greater than two standard deviations above the vehicle control will then be screened in three different secondary assays: 1) A BRCA1 promoter-driven-Renilla luciferase assay and 2) a biochemical luciferase inhibitor assay will serve as counter-screens to determine any false positives, while 3) quantitative real-time PCR will be used as an orthogonal screen to determine if primary screen hits (that are not luciferase inhibitors) increase endogenous BRCA1 mRNA expression. Finally, we propose Western blotting as a tertiary assay to assess validated hits for their ability to increase BRCA1 protein expression. Compounds that increase BRCA1 protein will be further developed in follow-up medicinal chemistry and cheminformatics studies. In 2009, the American Cancer Society estimated that 254,650 new cases of invasive and in situ breast cancer were diagnosed among American women. Based on estimates of BRCA1 dysfunction in sporadic breast cancers, over 80,000 newly diagnosed malignancies would have decreased BRCA1 expression. Therefore, identification and characterization of a plausible active compound that enhances BRCA1 expression could potentially translate into a novel prevention or therapeutic option for breast cancer patients with significant impact on incidence and/or survival.
In 2009, the American Cancer Society estimated that 254,650 new cases of invasive and in situ breast cancer were diagnosed among American women. Based on estimates of breast cancer susceptibility gene 1 (BRCA1) dysfunction in sporadic breast cancers, over 80,000 of these cases would have decreased BRCA1 expression. Loss of BRCA1 has been shown to contribute to the development of an aggressive type of breast cancer for which there is currently no targeted therapies. Therefore, our goal is to identify a drug that restores BRCA1 expression and tumor suppressor function, ultimately resulting in a novel BRCA1-targeted therapy providing breast cancer patients extended progression-free survival rates, more treatable breast cancers, or an alternative to cytotoxic chemotherapy.
