APE 1 / Ref-1 Regulates STAT 3 Transcriptional Activity and APE 1 / Ref-1 – STAT 3 Dual-Targeting Effectively Inhibits Pancreatic Cancer Cell Survival

Pancreatic cancer is a largely incurable disease, and increasing evidence supports strategies targeting multiple molecular mediators of critical functions of pancreatic ductal adenocarcinoma cells. Intracellular redox state modulates the activity of various signal transduction pathways and biological processes, including cell survival, drug resistance and responsiveness to microenvironmental factors. Recently, it has been shown that the transcription factor STAT3 is under redox control, but the mechanisms involved in its regulation are unknown. Here, we demonstrate for the first time that STAT3 DNA binding and transcriptional activity is directly regulated by the redox function of the APE1/Ref-1 endonuclease, using overexpression and redox-specific mutational strategies, and gene knockdown. Also, pharmacological blockade of APE1/Ref-1 by the redoxselective inhibitor E3330 abrogates STAT3 DNA binding. Since APE1/Ref-1 also exerts redox control on other cancerassociated transcription factors, we assessed the impact of dual-targeting of STAT3 signaling and APE1/Ref-1 redox on pancreatic cancer cell functions. We observed that disruption of APE1/Ref-1 redox activity synergizes with STAT3 blockade to potently inhibit the proliferation and viability of human PDAC cells. Mechanistically, we show that STAT3–APE1/Ref-1 dual targeting promotes marked tumor cell apoptosis, with engagement of caspase-3 signaling, which are significantly increased in comparison to the effects triggered by single target blockade. Also, we show that STAT3–APE1/Ref-1 dual blockade results in significant inhibition of tumor cell migration. Overall, this work demonstrates that the transcriptional activity of STAT3 is directly regulated by the redox function of APE1/Ref-1, and that concurrent blockade of STAT3 and APE1/Ref-1 redox synergize effectively inhibit critical PDAC cell functions. Citation: Cardoso AA, Jiang Y, Luo M, Reed AM, Shahda S, et al. (2012) APE1/Ref-1 Regulates STAT3 Transcriptional Activity and APE1/Ref-1–STAT3 Dual-Targeting Effectively Inhibits Pancreatic Cancer Cell Survival. PLoS ONE 7(10): e47462. doi:10.1371/journal.pone. 0047462 Editor: Nils Cordes, Dresden University of Technology, Germany Received April 27, 2012; Accepted September 17, 2012; Published October 19, 2012 Copyright: 2012 Cardoso et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support for this work was provided by the National Institutes of Health National Cancer Institute (NCI) CA121168, CA114571, and CA121168S1 (MRK), CA122298 (MLF), CA113669, CA134292 and CA134767 (AM) the Riley Children’s Foundation (MRK) www.rileykids. org/and the Ralph W. and Grace M. Showalter Research Trust Fund (MLF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and declare that they have no conflict of interest except for Dr. Mark R. Kelley who is the Chief Scientific Founder and consultant for ApeX Therapeutics, a company that has licensed IP from his work. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: mfishel@iupui.edu