APE1/Ref-1 Regulates STAT3 Transcriptional Activity and APE1/Ref-1–STAT3 Dual-Targeting Effectively Inhibits Pancreatic Cancer Cell Survival

Abstract

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 redox-selective inhibitor E3330 abrogates STAT3 DNA binding. Since APE1/Ref-1 also exerts redox control on other cancer-associated 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.

Novel Thiosemicarbazones Regulate the Signal Transducer and Activator of Transcription 3 (STAT3) Pathway: Inhibition of Constitutive and Interleukin 6–Induced Activation by Iron Depletion

“…Interestingly, ROS can inhibit STAT3 signaling by oxidation of conserved cysteines on JAK and STAT proteins that decrease their activation, implicating STAT3 as a mediator of redox homeostasis (Mamoon et al, 2007;Li et al, 2010). More recently, STAT3 transcriptional activity has been shown to be directly regulated by the redox function of the APE1/Ref-1 endonuclease (Cardoso et al, 2012). , Src, p-cAbl, cAbl, p-JAK2, and JAK2 were measured by Western blot analysis in (A) PANC-1, (B) MIAPaCa-2, and (C) DU145 cells after a 24-hour/37°C incubation with control medium, DFO (250 mM), Dp44mT (5 mM), DpC (5 mM), DFO:Fe (1:1; 250 mM), Dp44mT:Fe (2:1; 5 mM), DpC:Fe (2:1; 5 mM), FeCl 3 (250 mM), or Dp2mT (5 mM).…”

Identification and Characterization of New Chemical Entities Targeting Apurinic/Apyrimidinic Endonuclease 1 for the Prevention of Chemotherapy-Induced Peripheral Neuropathy

“…Reporter assays were performed as previously described Kelley et al, 2011;Cardoso et al, 2012;Luo et al, 2012). Cells were transfected with a nuclear factor-kB (NF-kB)-luciferase construct containing an NF-kB-response promoter and driving the expression of a luciferase gene and a Renilla luciferase control reporter vector pRL-CMV.…”

APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cellRNAsequencing

“…Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1; henceforth referred to as APE1) is a multifunctional protein that is involved in repairing DNA damage via its endonuclease activity in base excision repair (Fung and Demple, 2005; Izumi et al ., 2005; Jiang et al ., 2009; Kelley et al ., 2014), and using its redox protein–protein signaling function to control the activity of numerous transcription factors such as STAT3, NFκB, AP‐1, p53, and hypoxia‐inducible factor 1α (HIF1α), among others (Cardoso et al ., 2012; Fishel et al ., 2015; Gaiddon et al ., 1999; Jiang et al ., 2010; Kelley et al ., 2012; Lando et al ., 2000; Logsdon et al ., 2016). It also contributes to the removal of damaged bases within RNA (Poletto et al ., 2016; Vascotto et al ., 2014).…”

“…While a number of studies have investigated genes regulated by APE1, and specifically its redox signaling function (Cardoso et al ., 2012; Fishel et al ., 2015; Gaiddon et al ., 1999; Jiang et al ., 2010; Kelley et al ., 2012; Lando et al ., 2000; Logsdon et al ., 2016; Nishi et al ., 2002; Xanthoudakis et al ., 1992), it has been difficult to compile a comprehensive list of genes regulated by APE1 as it is essential for cell viability. APE1‐knockout in mice results in embryonic lethality, postimplantation, between days E5‐E9 (Ludwig et al ., 1998; Xanthoudakis et al ., 1996).…”

“…Approaches to circumvent this dilemma have utilized conditional knockouts and siRNA knockdowns (Fung and Demple, 2005; Izumi et al ., 2005; Jiang et al ., 2010). For example, using siRNA knockdowns, our laboratory has previously identified APE1 directly regulating STAT3 transcriptional activity (Cardoso et al ., 2012), suppressing Nrf2‐induced gene expression (Fishel et al ., 2015) and, most recently, regulating carbonic anhydrase 9 (CA9) via HIF‐1α under hypoxic conditions (Logsdon et al ., 2016). …”