Novel Estrogen-Response-Element Binding Proteins That Compete with Estrogen Receptor-α Predict Clinical Outcome of Human Breast Carcinoma.

Background: Low molecular weight ERE-binding proteins (ERE-BP) were detected while investigating estrogen response element (ERE) binding properties of estrogen receptor-a (ERa) in breast cancer. Properties of ERE-BP were evaluated by electrophoretic mobility shift (EMSA) and super-shift assays (SSA) with ERE sequences present in 59-flanking regions of estrogen responsive genes. Materials & Methods: Cytosols, prepared from de-identified tissue biopsies in 50 mM K 2 HPO 4 /KH 2 PO 4 buffer, pH 7.4 with 1.5 mM EDTA, 10 mM Na 2 MoO 4 , 1 mM PMSF & 10 μM monothioglycerol, were incubated 16 hr, 4 ° C with [ 32 P]ERE and separated by EMSA. ERE-BP level was measured from band intensity of an EMSA profile, expressed in digital light units (DLU)/mg protein (Cyclone Phosphor System with OptiQuant®). ERα, ERβ and ERE-BP exhibited distinct EMSA migration, allowing quantification. Specificity of ERE-BP association was determined with ERE sequences as labeled ligands and as unlabeled competitors. SSA were performed with AER 314, 320, 611, 1D5, 5D11 (NeoMarkers) or a polyclonal antibody (PanVera/Invitrogen). Results: ERE-BP recognized [ 32 P]ERE sequences (e.g., VitA2, h-fos, jun, cath D & pS2) present in promoter regions of responsive genes. Addition of unlabeled EREs to EMSA with [ 32 P]VitA2-ERE diminished intensities of ERE-BP bands, indicating specific interaction. ERE-BP did not super-shift when incubated with ERα or ERβ antibodies suggesting no identity with either receptor isoform. Competition with cytosol and constant [ERα] and [ERE], revealed extracts inhibited ERα binding with simultaneous ERE-BP appearance. No correlation was observed between ERE-binding and either patient age, race, nodal status, cancer grade, stage or tumor maker levels. When disease-free (DFS) or overall survival (OS) was analyzed as a function of above or below median ERE-BP expression using Kaplan-Meier plots, patients with biopsies in the above median group exhibited lower DFS and OS than women in the below median group. Addition of ERE-BP expression within a single ERα status (i.e., either positive or negative) identified subsets exhibiting decreased DFS and OS. Patients with ERα negative cancer and high ERE-BP expression exhibited the poorest OS of any group, including those with ER negative/ERE-BP negative cancer. Conclusion: Collectively, ERE-binding specificity, antibody recognition and molecular weights suggest ERE-BP are not ERα or ERβ fragments. Results support the hypothesis that presence of these novel ERE-BP in breast carcinoma is an indicator of poor prognosis. Over-expression of ERE-BP may lead to suppression of ER-mediated transcription by competing with ERα for ERE-binding. Thus patients with biopsies exhibiting elevated ERE-BP levels have poor DFS and OS because they are effectively estrogen (and thus tamoxifen) resistant, i.e., ERα signal transduction is inhibited. ERE-BP expression may provide a more clinically relevant pathologic classification compared to conventional criteria while correlating with clinical presentation, prognosis and disease course. Supported in part by NIH/NCI 1 R43 CA106059-01A1 & Phi Beta Psi Charity Trust. TLK is a recipient of a DOD BCRP Pre-doctoral Traineeship Award W81WXWH-08-0775. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3040.