Identification of ERalpha/ERbeta Heterodimer-specific Ligands by HTS

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DESCRIPTION (provided by applicant): One out of every eight women in the United States will develop breast cancer during her lifetime. The biological actions of estrogens are transduced by its binding to two estrogen receptors (ERs), ER? and ER?, which have a "Ying and Yang" relationship in regulating estrogen action (i.e., ER? promotes while ER? inhibits estrogen-dependent cell growth). ER? is thought to counteract the stimulatory effects of ER? through heterodimerization of the two receptors, and these heterodimers have been proposed to regulate sets of genes distinct from those regulated by either homodimer. However, the mechanism by which ER? functions in concert with ER? as a heterodimer in breast cancer has not been studied in detail due to the lack of specific ER?/??heterodimer ligands. We have established highly robust and reproducible Bioluminescent Resonance Energy Transfer (BRET) assays which can distinguish ligands with ability to induce ER? homodimers, ER? homodimers and ER?/? heterodimers. This proposal outlines three specific aims that focus on high throughput screening (HTS) for identification of ER?/??heterodimer specific ligands. In Aim 1, we take advantage of an ERE-reporter stably integrated cell line that is amenable to HTS. This T47D-KBLuc cell line expresses both ER? and ER?, making it feasible to identify compounds transactivating all forms of ER dimers including ER? and ER? homodimers as well as ER?/??heterodimers. In Aim 2, estrogenic compounds identified from this primary screening will be subjected to the HTS using BRET. In Aim 3, the identified hits will be tested for their abilities to inhibit growth of breast cancer cells co- expressing ER? and ER?. Development of a novel system by which ER?/? heterodimers may be studied will provide a means for direct evidence for heterodimer formation in vivo. Furthermore, these tools will allow preliminary exploration of heterodimer function in breast cancer via cell-based assays. PUBLIC HEALTH RELEVANCE: Estrogen receptors (ERs) play essential function in regulating cell proliferation in breast cancer cells; however the biological functions of ER?/ER? heterodimer remain unknown due to lack of heterodimer-selective compound. Here we propose two-step high throughput screenings for identification of ER?/ER? heterodimer specific ligands.