Characterization of DNA-binding proteins as clinically relevant biomarkers of breast cancer behavior.

Abstract

While investigating estrogen response element (ERE) binding properties of Era in de-identified human breast cancer extracts, additional proteins were observed that recognized ERE sequences (ERE-BP). In order to unravel the apparent role of these proteins, our goal was to compare properties of these novel ERE-BP with those of ERa, determine their identity and evaluate their clinical relevance in breast cancer behavior. ERE-BP were present in various tissue types including breast, ovarian, uterine and colon cancers and normal tissues. These proteins were present in both cytoplasm and nuclei although higher binding activities were detected in nuclear extracts. ERE-BP did not supershift with numerous anti-ERa or ERß antibodies recognizing different ER epitopes suggesting that they are not fragments of either ERa or ERß. ERE-BP competed with rhERa for binding to the VitA2-ERE yet overall exhibited significantly different sequence specificity compared to that of human ERa. The ERE-BP we observed in breast cancer extracts were not specific for ERE sequences. To further support this conclusion, various estrogens had no effect on the ERE-binding of these proteins in contrast to rhERa. Furthermore, ERE-BP activities were not correlated with levels of expression of in either ERa- or ERE-mediated transcription. An immune-based method was established for purifying ERE-BP from tissue extracts and proteins were identified by mass spectrometry. Ku70 (XRCC6) and Ku80 (XRCC5) were determined to be the most likely candidates for the identity of ERE-BP. Supershift assays confirmed that ERE-BP/ERE complexes observed by EMSA were specifically recognized by antibodies to the Ku70/Ku80 heterodimer (Ku). Western blotting with Ku70/Ku80 antibodies confirmed their presence in breast cancer extracts. Increased Ku DNA-binding activities in cytosols of breast biopsies correlated with higher grade tumors, positive lymph node status and decreased patient survival. Also increased Ku DNA-binding activities in cancers from patients receiving adjuvant chemotherapy correlated with decreased survival, suggesting Ku DNA-binding activities may be used to predict response to treatment. Collectively, our results suggest that Ku DNA-binding activities in cytosols prepared from carcinoma biopsies are useful biomarkers for assessing breast cancer recurrence and response to therapy