Estrogen receptor-β: why may it influence clinical outcome in estrogen receptor-α positive breast cancer?

In the previous issue of the journal, Lin and coworkers present data demonstrate that increased expression of estrogen receptor (ER)-β in ER-α-positive breast cancer cells antagonizes a defined group of ER-α/estrogen stimulated genes that are involved in cell cycle regulation and DNA replication. Similar expression patterns for these genes were found human ER-α positive breast tumors expressing higher levels or ER-β, and this correlated with better clinical outcome. The implications for these data, which suggest that ER-β is a positive actor and diagnostic marker for therapeutic outcome, are discussed

Differential responses of LHβ promoter mutants to WT(+KTS).

<p>LHβ promoter mutants were transfected into LβT2 cells with or without 1μg of WT1 (+KTS) plasmid or control plasmid to normalize DNA. Data were analyzed and expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116825#pone.0116825.g007" target="_blank">Fig. 7</a>.</p

Model of WT1 variant actions on the LHβ promoter.

<p>WT1 (-KTS) may associate directly with the 3’Egr1 site of the LHβ promoter to stimulate basal promoter activity, but in the presence of GnRH stimulates Egr1 expression. Egr1 then binds to both the 3’- and 5’-Egr1 sites on the promoter and further increases LHβ transcription. WT1 (+KTS) may associate with the 3’-Egr1 site, but also requires the SF1 site for biological activity. Decreased expression of WT1 (+KTS) would stimulate basal LHβ expression as the suppressor is reduced. The isoforms likely compete for association to the LHβ promoter at the same sites.</p

WT1 (-KTS and +KTS) expression and chromatin association in LβT2 cells.

<p>A. WT1 mRNA (+KTS and –KTS) splice variant PCR products expressed in LβT2 cells. Whole cell RNA was extracted from LβT2 cells, reverse transcribed to cDNA and quantified by real-time PCR, using specific primers to detect +KTS and –KTS splice variants, then displayed on a 1% agarose gel. Bands corresponding to the products for +KTS (301bp) and –KTS (292 bp) WT1 were detected in 4 independent samples of mRNA. B. WT1 and Egr1 protein expression in LβT2 cells. Cell proteins (30μg) were separated on 10% polyacrylamide-SDS gels, then analyzed by immunoblotting with specific antibodies for WT1, Egr1 and β-actin. Specific proteins were detected in the same samples of untreated LβT2 cells. C. Chromatin association of WT1 with the endogenous LHβ promoter in LβT2 cells. The association of WT1 and RNA Polymerase II with the LHβ promoter in untreated LβT2 cells was measured by Chromatin immunoprecipitation assays with antibodies against WT1 and phosphorylated RNApol II, as well as control (no Antibody). LHβ promoter occupancy was measured by quantitative real time PCR using primers specific for the LHβ promoter, and normalized for chromatin input in each sample. In this study, background binding (no Antibody) was set at 100% and association of RNA Polymerase II and WT1 are expressed relative to background values. Association was measured in 3 independent experiments with duplicate samples.</p

WT1 mRNA and protein levels are decreased by GnRH.

<p>A. WT1 (-KTS) and (+ KTS) mRNA variant levels in LβT2 cells treated with 50nM GnRH for 90min. RNA was extracted and mRNAs levels measured by RT-PCR and normalized against GAPDH mRNA. Values for WT1 mRNAs are shown for both Reverse Transcriptase (RT) and no RT (negative control) conditions. Note that for no RT, the Y axis is interrupted and expanded to show the low values. Data is the mean ± SEM from 5–7 experiments. * = P<0.05, -GnRH vs +GnRH; ** = P<0.001, -GnRH vs +GnRH B. WT1 and Egr1 protein levels in LβT2 cells treated with 50nM GnRH for 0 to 3.5 h. The experiment was performed three times. Upper panel: Cells were lysed after GnRH treatment and proteins (30μg) were separated by 10% SDS-PAGE, then detected with antibodies against WT1 or Egr1. Immunoblotting for β-actin was performed on the same blots as WT1 and Egr1, and used for normalization of these proteins quantified by densitometry analysis. A representative blot is shown. Lower panel: Quantification of protein bands was performed with densitometry, and normalized protein levels are shown from combined experiments. Bands for Egr1 proteins were not detected (ND) in any blot at time zero without GnRH.</p

WT1 and Egr1 occupancy of the endogenous LHβ promoter in response to GnRH.

<p>LβT2 cells were incubated with or without 50 nM GnRH and collected every 10 min for 120 min. ChIP assays were performed using antibody against WT1, Egr1 and phosphorylated pRNA Pol II. LHβ promoter occupancy was measured by quantitative real time PCR using primers for the LHβ promoter. The experiment was performed three times with duplicate samples and replicate PCR measurements in each sample. Data are presented as the mean + SEM and expressed as LHβ promoter occupancy relative to basal (no GnRH at time zero) binding.</p

WT1 (+KTS) decreases basal and GnRH-stimulated LHβ promoter activity.

<p>LβT2 cells were transfected with either <b>A)</b> A luciferase reporter construct driven by the rat LHβ promoter (-617 to +41 bp) including both distal and proximal GnRH responsive promoter regions, or <b>B)</b> A luciferase reporter construct driven by the rat LHβ promoter (-245 to +44 bp), including only the proximal GnRH response region of the promoter. Constructs were cotransfected with or without 0.5, 0.7,1 μg of WT1 (+KTS) plasmid or control plasmid to normalize DNA. At 48 h post-transfection, cells were treated with 50nM GnRH for 6 h and collected in lysis buffer. Luciferase activity was measured, and data expressed as average ± SE for 6 samples; the experiment was performed 3 times each. Statistical significance was determined using ANOVA (confidence interval determined by the Bonferroni multiple comparison test). * p<.05 -GnRH vs +GnRH, # = p<.05 control, -GnRH vs –GnRH+WT1, a = p<.05 control, +GnRH vs +GnRH +WT1.</p