Characterization of a novel small molecule subtype specific estrogen-related receptor alpha antagonist in MCF-7 breast cancer cells.
Characterization of a novel small molecule subtype specific estrogen-related receptor alpha antagonist in MCF-7 breast cancer cells.
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DOI：
10.1371/journal.pone.0005624
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发表时间：
2009-05-20
期刊：
影响因子：
3.7
通讯作者：
Chen JD
中科院分区：
文献类型：
--
作者：
Chisamore MJ;Cunningham ME;Flores O;Wilkinson HA;Chen JD
孤儿核受体雌激素相关受体α（ERRα）是核受体超家族的成员。它是通过搜索编码雌激素受体α（ERα）相关蛋白质的基因而鉴定的。目前还没有发现内源性配体，但最近报道了一些与ERRα的配体结合域（LBD）高度特异性结合的新型ERRα拮抗剂。研究表明，ERRα可能是治疗乳腺癌和/或代谢紊乱的一个新的药物靶点，这促使人们努力表征新型ERRα特异性拮抗剂N-[（2 Z）-3-（4，5-dihydro-1，3-thiazol-2-yl）-1，3-thiazolidine-2-yl idene]-5 H dibenzo[a，d]annulen-5-amine的作用机制。我们通过荧光素酶试验证明该ERRα配体抑制MCF-7细胞中ERRα的转录活性，但不影响通过实时RT-PCR测量的mRNA水平。此外，经ERRα拮抗剂处理后，ERα（ESR 1）mRNA水平未受影响，但其他ERRα（ESRRA）靶基因，如pS2（TFF 1）、骨桥蛋白（SPP 1）和芳香酶（CYP 19 A1）mRNA水平降低。在体外，ERRα拮抗剂可阻止ERRα与核受体辅激活因子之间的组成性相互作用。此外，我们使用蛋白质印迹证明ERRα亚型特异性拮抗剂增加了ERRα蛋白通过泛素蛋白酶体途径的降解。我们通过染色质免疫沉淀（ChIP）证明，当细胞用配体处理时，ACADM、ESRRA和TFF 1内源性基因启动子与ERRα蛋白之间的相互作用降低。当用我们的ERRα拮抗剂处理MCF-7细胞时，敲低ERRα（shRNA）导致类似的基因组效应。我们报道了一种新的ERRα特异性拮抗剂的作用机制，该拮抗剂抑制ERRα的转录活性，破坏ERRα与核辅激活因子之间的组成性相互作用，并诱导蛋白酶体依赖的ERRα蛋白降解。此外，我们证实，敲低ERRα导致与用ERRα特异性拮抗剂处理时在体外证明的相似的基因组效应。
The orphan nuclear receptor estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily. It was identified through a search for genes encoding proteins related to estrogen receptor α (ERα). An endogenous ligand has not been found. Novel ERRα antagonists that are highly specific for binding to the ligand binding domain (LBD) of ERRα have been recently reported. Research suggests that ERRα may be a novel drug target to treat breast cancer and/or metabolic disorders and this has led to an effort to characterize the mechanisms of action of N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d]annulen-5-amine, a novel ERRα specific antagonist. We demonstrate this ERRα ligand inhibits ERRα transcriptional activity in MCF-7 cells by luciferase assay but does not affect mRNA levels measured by real-time RT-PCR. Also, ERα (ESR1) mRNA levels were not affected upon treatment with the ERRα antagonist, but other ERRα (ESRRA) target genes such as pS2 (TFF1), osteopontin (SPP1), and aromatase (CYP19A1) mRNA levels decreased. In vitro, the ERRα antagonist prevents the constitutive interaction between ERRα and nuclear receptor coactivators. Furthermore, we use Western blots to demonstrate ERRα protein degradation via the ubiquitin proteasome pathway is increased by the ERRα-subtype specific antagonist. We demonstrate by chromatin immunoprecipitation (ChIP) that the interaction between ACADM, ESRRA, and TFF1 endogenous gene promoters and ERRα protein is decreased when cells are treated with the ligand. Knocking-down ERRα (shRNA) led to similar genomic effects seen when MCF-7 cells were treated with our ERRα antagonist. We report the mechanism of action of a novel ERRα specific antagonist that inhibits transcriptional activity of ERRα, disrupts the constitutive interaction between ERRα and nuclear coactivators, and induces proteasome-dependent ERRα protein degradation. Additionally, we confirmed that knocking-down ERRα lead to similar genomic effects demonstrated in vitro when treated with the ERRα specific antagonist.
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