title: Characterization of estrogen response element binding proteins as biomarkers of breast cancer behavior.
abstract: BACKGROUND
  While investigating estrogen response element (ERE) binding properties of human estrogen receptor-α (hERα) in breast cancer cytosols, other ERE-binding proteins (ERE-BP) were observed.
  
  
  METHODS
  Recognition properties of ERE-BP were evaluated by electrophoretic mobility shift assays (EMSA) with ERE sequences of the 5'-flanking region of the estrogen responsive gene vitellogenin A2 (VitA2). Cytosols were incubated 16 h, 4 °C with [32P]ERE sequences and separated by EMSA. A method of estimating ERE-BP levels was developed by measuring band intensity from EMSA profiles, expressed in digital light units (DLU)/μg protein and normalized to total DLU. ERE-BP were purified by affinity chromatography and EMSA, and then identified by mass spectrometry.
  
  
  RESULTS
  ERE-BP in cytosols did not supershift in the presence of anti-hERα or anti-hERβ antibodies recognizing different ER epitopes suggesting that they are not fragments of either receptor isoform. ERE-BP competed with hERα for binding to VitA2-ERE. Increased levels of ERE-BP DNA-binding activities measured in 310 cytosols prepared from breast cancer biopsies correlated with decreased patient survival. Strikingly, breast cancer patients with ER negative status and high ERE-BP expression exhibited the poorest disease-free and overall survival. After purification, ERE-BP were identified as Ku70 (XRCC6) and Ku80 (XRCC5) using mass spectrometry. ERE-BP were confirmed to be Ku70/80 by supershift assay.
  
  
  CONCLUSIONS
  Presence of these novel ERE-binding proteins in a breast carcinoma is a strong predictor of poor prognosis. Our results suggest that ERE-BP, identified as Ku70/Ku80, in cytosols prepared from breast carcinoma biopsies are useful biomarkers for assessing risk of breast cancer recurrence.
authors.affiliation: Hormone Receptor Laboratory, Department of Biochemistry & Molecular Biology, Brown Cancer Center, University of Louisville, Louisville, KY 40292, USA; Institute for Molecular Diversity & Drug Design, University of Louisville, Louisville, KY 40292, USA.
authors.forename: Traci L
authors.initials: TL
authors.lastname: Kruer
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Cummins
authors.forename: David W
authors.initials: DW
authors.lastname: Powell
authors.forename: James L
authors.initials: JL
authors.lastname: Wittliff
mesh_terms: D000912:Antibodies, Neoplasm / Q000276:immunology; D034961:Antigens, Nuclear / Q000378:metabolism; D001667:Binding, Competitive; D014408:Biomarkers, Tumor / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D018572:Disease-Free Survival; D047628:Estrogen Receptor alpha / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D006801:Humans; D000072200:Ku Autoantigen; D013058:Mass Spectrometry; D009363:Neoplasm Proteins / Q000378:metabolism*; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics*; D014020:Tissue Extracts
chemical_list: D000912:Antibodies, Neoplasm; D034961:Antigens, Nuclear; D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D004967:Estrogens; D009363:Neoplasm Proteins; D014020:Tissue Extracts; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen
keywords: Biomarker; Breast cancer; Estrogen response element