Estrogen-Related Receptor alpha (ERR (alpha))-Coactivator Interactions as Targets for Discovery of New Anti-Breast Cancer Therapeutics

摘要

The steroid nuclear receptor estrogen receptor alpha (ERalpha) is the primary target of current breast cancer therapies, which block ERo activation or estrogen synthesis. Estrogen-related receptor alpha (ERRalpha), a protein with high sequence similarity to ERalpha, has functional similarity to ERalpha in certain breast cancer cell types; though unlike ERalpha, ERRalpha acts independently of steroid ligand. We hypothesize that its activity may be due in part to its interaction with coactivator proteins, and in some instances, ERRalpha may be functionally substituting for ERalpha and playing an important role in the progression of a subset of breast cancers. We found that coactivator recruitment is necessary for full ERRalpha activation of transcription; furthermore, GRIP1 (SRC-2, TIF-2) is cell type- and promoter- specific in its coactivation of transcription with a constitutively active form of ERRalpha. We used this information to develop a cell-based, high- throughput screen for small molecules that inhibit the transcription of an estrogen response element (ERE)-containing luciferase reporter gene in the presence of overexpressed GRIP1 and 'activated' ERRalpha. This screen, performed in MCF-7 cells in 96-well plates, used a 4,160 'known bloactives' compound library. Recently, we were able to improve the cell-based screen by converting to batch transfection of cells and to a 384-well plate format, which allowed us to use 75% less cells and chemical per well, thereby making the assay much more efficient, reproducible, and adaptable to larger chemical libraries. We also have developed four cell-based, high-throughput counter screens to validate 'hits' from primary screens by ruling out compounds that are toxic to cells or that generally inhibit transcription or translation. Lastly, we are generating a targeted, high-throughput screen to determine the effects of remaining 'hits' on inhibition of the GRIP1-ERRalpha interaction in vitro.