Ariazi Eric A, Kraus Richard J, Farrell Michael L, Jordan V Craig, Mertz Janet E
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706, USA.
Mol Cancer Res. 2007 Jan;5(1):71-85. doi: 10.1158/1541-7786.MCR-06-0227.
We previously showed that (a) estrogen-related receptor alpha1 (ERRalpha1) down-modulates estrogen receptor (ER)-stimulated transcription in low ErbB2-expressing MCF-7 mammary carcinoma cells, and (b) ERRalpha and ErbB2 mRNA levels positively correlate in clinical breast tumors. We show here that ERRalpha1 represses ERalpha-mediated activation in MCF-7 cells because it failed to recruit the coactivator glucocorticoid receptor interacting protein 1 (GRIP1) when bound to an estrogen response element. In contrast, ERRalpha1 activated estrogen response element- and ERR response element-mediated transcription in ERalpha-positive, high ErbB2-expressing BT-474 mammary carcinoma cells, activation that was enhanced by overexpression of GRIP1. Likewise, regulation of the endogenous genes pS2, progesterone receptor, and ErbB2 by ERRalpha1 reflected the cell type-specific differences observed with our reporter plasmids. Importantly, overexpression of activated ErbB2 in MCF-7 cells led to transcriptional activation, rather than repression, by ERRalpha1. Two-dimensional PAGE of radiophosphate-labeled ERRalpha1 indicated that it was hyperphosphorylated in BT-474 relative to MCF-7 cells; incubation of these cells with anti-ErbB2 antibody led to reduction in the extent of ERRalpha1 phosphorylation. Additionally, mitogen-activated protein kinases (MAPK) and Akts, components of the ErbB2 pathway, phosphorylated ERRalpha1 in vitro. ERRalpha1-activated transcription in BT-474 cells was inhibited by disruption of ErbB2/epidermal growth factor receptor signaling with trastuzumab or gefitinib or inactivation of downstream components of this signaling, MAPK kinase/MAPK, and phosphatidylinositol-3-OH kinase/Akt, with U0126 or LY294002, respectively. Thus, ERRalpha1 activities are regulated, in part, via ErbB2 signaling, with ERRalpha1 likely positively feedback-regulating ErbB2 expression. Taken together, we conclude that ERRalpha1 phosphorylation status shows potential as a biomarker of clinical course and antihormonal- and ErbB2-based treatment options, with ERRalpha1 serving as a novel target for drug development.
（a）在低表达ErbB2的MCF-7乳腺癌细胞中，雌激素相关受体α1（ERRα1）下调雌激素受体（ER）刺激的转录；（b）在临床乳腺肿瘤中，ERRα和ErbB2的mRNA水平呈正相关。我们在此表明，ERRα1在MCF-7细胞中抑制ERα介导的激活，因为当它与雌激素反应元件结合时，无法募集共激活因子糖皮质激素受体相互作用蛋白1（GRIP1）。相反，ERRα1在ERα阳性、高表达ErbB2的BT-474乳腺癌细胞中激活雌激素反应元件和ERR反应元件介导的转录，GRIP1的过表达增强了这种激活。同样，ERRα1对内源性基因pS2、孕激素受体和ErbB2的调控反映了我们用报告质粒观察到的细胞类型特异性差异。重要的是，在MCF-7细胞中过表达活化的ErbB2导致ERRα1的转录激活，而非抑制。对放射性磷酸标记的ERRα1进行二维电泳表明，相对于MCF-7细胞，它在BT-474细胞中发生了过度磷酸化；用抗ErbB2抗体孵育这些细胞导致ERRα1磷酸化程度降低。此外，丝裂原活化蛋白激酶（MAPK）和Akt（ErbB2信号通路的组成部分）在体外使ERRα1磷酸化。用曲妥珠单抗或吉非替尼破坏ErbB2/表皮生长因子受体信号传导，或分别用U0126或LY294002使该信号传导的下游成分MAPK激酶/MAPK和磷脂酰肌醇-3-OH激酶/Akt失活，均可抑制ERRα1在BT-474细胞中激活的转录。因此，ERRα1的活性部分通过ErbB2信号传导进行调控，ERRα1可能对ErbB2表达起正反馈调节作用。综上所述，我们得出结论，ERRα1的磷酸化状态具有作为临床病程以及基于抗激素和ErbB2治疗方案生物标志物的潜力，ERRα1可作为药物开发的新靶点。
