Estrogen Receptors (hERs) are ligand-inducible transcription factors critical for the progression of breast cancers. The estrogen antagonist tamoxifen is used to treat breast carcinomas by blocking hormone binding to hERs. Although tamoxifen chemotherapy is beneficial, this drug causes endometrial (uterine) cancer. This serious side effect results from the inability of tamoxifen to fully inhibit hER function in the cell nucleus. The long-term objective of this research focuses on the development of a novel paradigm for total blockage of hER function. This paradigm is based on utilizing synthetic ligands to prohibit hER nuclear localization. The proposed research will test the following hypothesis: Since hERs affect transcription in the cell nucleus, novel synthetic ligands that recruit hERs to the plasma membrane will fully inhibit hER-mediated transcriptional activation. This hypothesis will be tested through evaluation and chemical synthesis of chimeric ligands with distinct hER-binding and membrane-binding domains. Surface plasmon resonance measurements of membrane anchoring functionality, chimeric hER ligands and hER-ligand complexes will be utilized to investigate membrane affinities in vitro. Furthermore, chimeric ligands will be analyzed in vivo through transcriptional reporter gene assays and green fluorescent protein assays of subcellular localization. Preliminary work has already demonstrated that (a) 7-alpha-substituted beta-estradiol hER ligands can be efficiently synthesized and (b) that hER-promoted reporter gene expression in vivo is abolished by genetically fusing the Ras membrane targeting sequence to hERs. Thus, Ras membrane targeting provides a much stronger directing force than hER nuclear localization signals. These data suggest that synthetic ligands that target hERs to the plasma membrane may confer total hormonal blockage, thereby providing more effective antiestrogen therapeutics. The strategy proposed herein has the potential to provide a novel approach to nuclear hormone receptor antagonism.
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