Identification of ERalpha/ERbeta Heterodimer-specific Ligands by HTS

描述（由申请人提供）：在美国，每八个女性中就有一个会在她的一生中患上乳腺癌。雌激素的生物学作用是通过与两种雌激素受体（ER）结合来传导的。呃?，它们在调节雌激素的作用中具有“阴阳”关系(即ER？促进，而ER？抑制雌激素依赖性细胞生长)。呃?能抵消雌激素受体的刺激作用？通过这两种受体的异源二聚体，这些异源二聚体被认为可以调节不同于任何一种同型二聚体调节的基因。然而，ER的机制是什么？与急诊室协调的功能？作为异源二聚体在乳腺癌中的作用，由于缺乏特异性ER，尚未得到详细研究。异质二聚体配体。我们已经建立了高度稳健和可重复的生物发光共振能量转移（BRET）检测方法，可以区分具有诱导ER？为,呃?同型二聚体和ER?/？形成。本提案概述了三个具体目标，重点是高通量筛选（HTS）用于鉴定ER？异二聚体特异性配体。在Aim 1中，我们利用了一种适合HTS的e -报告细胞稳定整合细胞系。T47D-KBLuc细胞系同时表达ER？呃?，使得鉴定可激活所有形式的ER二聚体的化合物(包括ER？呃?同型二聚体以及ER /异源二聚体。在Aim 2中，从该初步筛选中鉴定的雌激素化合物将使用BRET进行HTS。在Aim 3中，鉴定出的靶点将被测试其抑制乳腺癌细胞共表达ER？呃?。开发一种新的系统，使ER?/？研究异二聚体将为体内异二聚体的形成提供直接证据。此外，这些工具将允许通过基于细胞的检测来初步探索异二聚体在乳腺癌中的功能。

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.