Estrogen Receptor- alpha Phosphorylated at Ser super(118) Is Present at the Promoters of Estrogen-Regulated Genes and Is Not Altered Due to HER-2 Overexpression

Detection of estrogen receptor (ER)- alpha phosphorylated at Ser super(118) (P-Ser super(118)-ER- alpha ) may be an indicator of an intact ligand-dependent ER- alpha in breast tumors in vivo and may predict responsiveness to endocrine therapy. The current study addresses whether P-Ser super(118)-ER- alpha is functionally involved in ER target gene transcription and if this is modulated by HER-2 overexpression. Using chromatin immunoprecipitation analysis, P-Ser super(118)-ER- alpha was found associated with the promoters of several estrogen-regulated genes in MCF-7 breast cancer cells 30 minutes following estrogen treatment. Coactivators AIB1 and p300 were coimmunoprecipitated with P-Ser super(118)-ER- alpha following estrogen treatment. The overexpression of HER-2 protein in MCF-7 cells did not affect estrogen induction of phosphorylation of Ser super(118) or its presence at the promoters of several estrogen-regulated genes. U0126, an inhibitor of mitogen-activated protein kinase (MAPK) pathway, had no effect on P-Ser super(118)-ER- alpha . The lack of effect of HER-2 overexpression on P-Ser super(118)-ER- alpha expression in cell models is supported by similar levels of expression of P-Ser super(118)-ER- alpha in ER super(+)/HER-2-overexpressing and ER super(+)/HER-2 super(-) breast tumors in vivo. Using inhibitors of cyclin-dependent kinase 7 (Cdk7), [(5,6-dichloro-1-{szligbeta}-D-ribofuranosylbenzimidazole and 2-(R)-1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine ] , and I Kappa B kinase- alpha (IKK- alpha ; BAY-11-7082), we show that IKK- alpha , but not Cdk7, is at least in part involved in estrogen-mediated phosphorylation at Ser super(118) in MCF-7 cells. Our data provide direct evidence for a functional role of P-Ser super(118)-ER- alpha in estrogen-regulated signaling and do not support the hypothesis that resistance of breast tumors to tamoxifen therapy involves ligand independent activation of ER- alpha due to constitutive phosphorylation of Ser super(118) by constitutive activation of MAPK pathway. (Cancer Res 2006; 66(20): 10162-70)