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Item Apurinic/Apyrimidinic Endonuclease/Redox Factor-1 (APE1/Ref-1) redox function negatively regulates NRF2(2015-01) Fishel, Melissa L.; Wu, Xue; Devlin, Cecilia M.; Logsdon, Derek P.; Jiang, Yanlin; Luo, Meihua; He, Ying; Yu, Zhangsheng; Tong, Yan; Lipking, Kelsey P.; Maitra, Anirban; Rajeshkumar, N. V.; Scandura, Glenda; Kelley, Mark R.; Ivan, Mircea; Department of Pediatrics, Indiana University School of MedicineApurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) (henceforth referred to as Ref-1) is a multifunctional protein that in addition to its base excision DNA repair activity exerts redox control of multiple transcription factors, including nuclear factor κ-light chain enhancer of activated B cells (NF-κB), STAT3, activator protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), and tumor protein 53 (p53). In recent years, Ref-1 has emerged as a promising therapeutic target in cancer, particularly in pancreatic ductal carcinoma. Although a significant amount of research has centered on Ref-1, no wide-ranging approach had been performed on the effects of Ref-1 inhibition and transcription factor activity perturbation. Starting with a broader approach, we identified a previously unsuspected effect on the nuclear factor erythroid-related factor 2 (NRF2), a critical regulator of cellular defenses against oxidative stress. Based on genetic and small molecule inhibitor-based methodologies, we demonstrated that repression of Ref-1 potently activates NRF2 and its downstream targets in a dose-dependent fashion, and that the redox, rather than the DNA repair function of Ref-1 is critical for this effect. Intriguingly, our results also indicate that this pathway does not involve reactive oxygen species. The link between Ref-1 and NRF2 appears to be present in all cells tested in vitro, noncancerous and cancerous, including patient-derived tumor samples. In particular, we focused on understanding the implications of the novel interaction between these two pathways in primary pancreatic ductal adenocarcinoma tumor cells and provide the first evidence that this mechanism has implications for overcoming the resistance against experimental drugs targeting Ref-1 activity, with clear translational implications.Item Inhibition of TXNRD or SOD1 overcomes NRF2-mediated resistance to β-lapachone(Elsevier, 2020-02) Torrente, Laura; Prieto-Farigua, Nicolas; Falzone, Aimee; Elkins, Cody M.; Boothman, David A.; Haura, Eric B.; DeNicola, Gina M.; Biochemistry and Molecular Biology, School of MedicineAlterations in the NRF2/KEAP1 pathway result in the constitutive activation of NRF2, leading to the aberrant induction of antioxidant and detoxification enzymes, including NQO1. The NQO1 bioactivatable agent β-lapachone can target cells with high NQO1 expression but relies in the generation of reactive oxygen species (ROS), which are actively scavenged in cells with NRF2/KEAP1 mutations. However, whether NRF2/KEAP1 mutations influence the response to β-lapachone treatment remains unknown. To address this question, we assessed the cytotoxicity of β-lapachone in a panel of NSCLC cell lines bearing either wild-type or mutant KEAP1. We found that, despite overexpression of NQO1, KEAP1 mutant cells were resistant to β-lapachone due to enhanced detoxification of ROS, which prevented DNA damage and cell death. To evaluate whether specific inhibition of the NRF2-regulated antioxidant enzymes could abrogate resistance to β-lapachone, we systematically inhibited the four major antioxidant cellular systems using genetic and/or pharmacologic approaches. We demonstrated that inhibition of the thioredoxin-dependent system or copper-zinc superoxide dismutase (SOD1) could abrogate NRF2-mediated resistance to β-lapachone, while depletion of catalase or glutathione was ineffective. Interestingly, inhibition of SOD1 selectively sensitized KEAP1 mutant cells to β-lapachone exposure. Our results suggest that NRF2/KEAP1 mutational status might serve as a predictive biomarker for response to NQO1-bioactivatable quinones in patients. Further, our results suggest SOD1 inhibition may have potential utility in combination with other ROS inducers in patients with KEAP1/NRF2 mutations.Item The Role of Lipoxygenase and Interleukin-6 on Islet β-cell Oxidative Stress and Dysfunction(2019-06) Conteh, Abass M.; Mirmira, Raghavendra G.; Linnemann, Amelia K.; Anderson, Ryan M.; Considine, Robert V.; Harrington, Maureen A.Type 1 and Type 2 diabetes (T1D/T2D) share a common etiology that involves an increase in oxidative stress that leads to dysfunction and subsequent β cell death. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids to form lipid metabolites involved in a variety of biological functions including cellular oxidative stress response. On the other hand, Interleukin 6 (IL-6) signaling has been demonstrated to be protective in islets. In this study, we explored the effect of lipoxygenase enzymes 12-Lipoxygenase, 12/15 Lipoxygenase and IL-6 on β cell function and survival in mice using both STZ and high-fat diet (HFD) models of diabetes. Alox12-/- mice showed greater impairment in glucose tolerance following STZ and HFD compared to wild-type mice (WT), whereas Alox15-/- were protected against dysglycemia. These findings were accompanied by evidence of islet oxidative stress in Alox12-/- mice and reduced oxidative stress in Alox15-/- mice, consistent with alterations in the expression of antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12-/- mice showed a compensatory increase in Alox15 gene expression and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. IL-6 was able to significantly attenuate the generation of reactive oxygen species by proinflammatory cytokines in human pancreatic islets. Furthermore, we find that IL-6 regulates the master antioxidant response protein NRF2. Collectively these results show that loss of Alox12 activates a compensatory increase in Alox15 that sensitizes β cells to oxidative stress and signaling by IL-6 is required for maximal antioxidant response under conditions of increased ROS formation, such as obesity.