Identification of ERalpha/ERbeta Heterodimer-specific Ligands by HTS

描述(申请人提供)：在美国，每八名女性中就有一名会在有生之年患上乳腺癌。雌激素的生物学作用是通过与两种雌激素受体(ER、ER、ER)结合而实现的。和ER？，在调节雌激素作用方面具有“阴阳”关系(即ER？促销的同时急诊室？抑制雌激素依赖的细胞生长)。呃？被认为可以抵消内质网的刺激作用？通过两个受体的异源二聚体，这些异源二聚体被认为调节不同于任何一个同源二聚体调控的基因集。然而，ER是通过什么机制实现的？与急诊室协同工作？作为一种异源二聚体在乳腺癌中的作用，由于缺乏特异性的内质网/异源二聚体配体，目前还没有详细的研究。我们已经建立了高度可靠和重复性好的生物发光共振能量转移(BRET)分析方法，可以区分具有诱导ER？同源二聚体，ER？同源二聚体和ER？/？杂二聚体。这项建议概述了三个特定的目标，重点是高通量筛选(HTS)以鉴定内质网/异二聚体特异性配体。在目标1中，我们利用了一种稳定整合的细胞系，该细胞系对HTS具有适应性。T47D-KBLuc细胞表达ER？和ER？，使鉴定反式激活所有形式的ER二聚体包括ER？的化合物成为可能。急诊室呢？同二聚体和内质网？/？异二聚体。在目标2中，从这一初步筛选中鉴定出的雌激素化合物将使用BRET进行HTS。在目标3中，将测试识别的HITS是否具有抑制共表达ER？的乳腺癌细胞生长的能力。那急诊室呢？开发一种新的系统，通过哪个ER？/？杂二聚体的研究将为体内杂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分析初步探索异二聚体在乳腺癌中的功能。 公共卫生相关性：雌激素受体(ER)在调节乳腺癌细胞增殖中起着重要作用，但ER？/ER？由于缺乏对杂二聚体具有选择性的化合物，杂二聚体仍然未知。在这里，我们提出了两步高通量筛选来鉴定ER？/ER？杂二聚体特异性配体。

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.