Reactive oxygen species induce phosphorylation of serine 118 and 167 on estrogen receptor alpha.

Estrogen receptor alpha (ERalpha) is a well-known target for signaling pathways originating from growth factor receptors. Reactive oxygen species (ROS) can induce activation of extracellular response kinase 1/2 (Erk1/2) and protein kinase B (Akt). Both kinases have been implicated in the phosphorylation of serine 118 and serine 167 on ERalpha, respectively. This activity may lead either to ligand-independent activation of ERalpha or down-regulation of ERalpha and may contribute to development of the resistance to endocrine therapy. Treatment of MCF-7 human breast cancer cells with glucose oxidase (GO, 0.1 un/ml) induced transient phosphorylation of serine 118 and serine 167. The increase in expression of p-ser118-ERalpha was 355 +/- 98% (mean +/- SD) and of p-ser167-ERalpha was 632 +/- 355%. These effects were enhanced in Her2 over-expressing MCF7 cells. ERalpha expression declined to 63 +/- 20% within the first 90 min of treatment and was below 10% 24 h later. ROS induced phosphorylation of ERalpha resulted in decreased expression of pS2 and progesterone receptor. Activation of Erk1/2 and Akt was transient with highest levels of Erk1/2 being 595 +/- 143% and p-Akt 311 +/- 125%. Inhibition of Erk1/2 by U0126 (10 microM) decreased p-ser118-ERalpha by 51.7 +/- 8.5% and decreased p-ser167-ERalpha by 41.9 +/- 16.9% whereas inhibition of Akt by LY294002 (20 microM) and wortmannin (500 nM) or by siRNA knock-down, had no effect on p-ser167-ERalpha expression. Our data show for the first time that ROS can induce post-translational modifications of ERalpha at serine 118 and serine 167, and may lead to ERalpha down-regulation in human breast cancer cells. Both the phosphorylation and consequent down-regulation of ERalpha may be a mechanism associated with development of endocrine therapy resistance.

Estrogen receptor-alpha phosphorylated at Ser118 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(118) (P-Ser(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(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(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(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(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(118)-ER-alpha. The lack of effect of HER-2 overexpression on P-Ser(118)-ER-alpha expression in cell models is supported by similar levels of expression of P-Ser(118)-ER-alpha in ER(+)/HER-2-overexpressing and ER(+)/HER-2(-) breast tumors in vivo. Using inhibitors of cyclin-dependent kinase 7 (Cdk7), [(5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole and 2-(R)-1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine], and IkappaB 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(118) in MCF-7 cells. Our data provide direct evidence for a functional role of P-Ser(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(118) by constitutive activation of MAPK pathway.

Assessment of multiple different estrogen receptor-beta antibodies for their ability to immunoprecipitate under chromatin immunoprecipitation conditions.

Several different antibodies to total estrogen receptor (ER)beta, ERbeta1 and ERbeta2/cx have been tested and compared for their ability to immunoprecipitate ERbeta specific isoforms under chromatin immunoprecipitation conditions (ChIP). The rabbit polyclonal antibodies AP-ERbeta1 and AP-ERbeta2/cx, specific for ERbeta1 and ERbeta2/cx isoforms, respectively, were the most efficient for ChIP. The monoclonal antibody MCA1974/PPG5/10 was also able to ChIP ERbeta1, but less efficiently than AP-ERbeta1. All other antibodies tested were not suitable for ChIP analyses although most antibodies tested immunoprecipitated the appropriate ERbeta isoforms under standard conditions. To identify antibodies that can also be used to verify in-vivo expression profiles, a comparison of the antibodies to detect ERbeta isoforms by western blotting and immunohistochemistry was also undertaken. Under the tissue processing and autostaining conditions used at the Manitoba Breast Tumor Bank 385P/GC17, MCA1974/PPG5/10, Ab288/14C8 and MCA2279S/57/3 were found to be the best for IHC of ERbeta isoforms in human breast tissue biopsy sections, while Ab14021, AP-ERbeta1 and AP-ERbeta2/cx were best for western blot detection of ERbeta isoforms.