Characterization of estrogen response element binding proteins as biomarkers of breast cancer behavior

• Published in: Clinical biochemistry 2013-11, Vol.46 (16-17), p.1739-1746
• Main Authors: Kruer, Traci L., Cummins, Timothy D., Powell, David W., Wittliff, James L.
• Format: Article
• Language: English
• Subjects: Antibodies, Neoplasm - immunology; Antigens, Nuclear - metabolism; Binding, Competitive; Biomarker; Biomarkers, Tumor - metabolism; Breast cancer; Cytosol - metabolism; Disease-Free Survival; DNA, Neoplasm - metabolism; DNA-Binding Proteins - metabolism; Estrogen Receptor alpha - metabolism; Estrogen Receptor beta - metabolism; Estrogen response element; Estrogens - metabolism; Female; Humans; Ku Autoantigen; Mass Spectrometry; Neoplasm Proteins - metabolism; Protein Binding; Response Elements - genetics; Tissue Extracts
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2013.07.005

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. 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 16h, 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. 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. 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. •ERE-BP were identified with properties differing from those of ER in breast cancer.•Patients with ER- and high ERE-BP cancers exhibited poor DFS and overall survival.•ERE-BP were identified as Ku70/Ku80 proteins which was confirmed by supershift assay.