Redox Effector Factor-1 Research Articles

APE-1/Ref-1 Inhibition Blocks Malignant Pleural Mesothelioma Cell Proliferation and Migration: Crosstalk between Oxidative Stress and Epithelial Mesenchymal Transition (EMT) in Driving Carcinogenesis and Metastasis.

Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure. MPM pathogenesis has been related both to oxidative stress, evoked by and in response to asbestos fibers exposure, and epithelial mesenchymal transition (EMT), an event induced by oxidative stress itself and related to cancer proliferation and metastasis. Asbestos-related primary oxidative damage is counteracted in the lungs by various redox-sensitive factors, often hyperactivated in some cancers. Among these redox-sensitive factors, Apurinic-apyrimidinic endonuclease 1 (APE-1)/Redox effector factor 1 (Ref-1) has been demonstrated to be overexpressed in MPM and lung cancer, but the molecular mechanism has not yet been fully understood. Moreover, asbestos exposure has been associated with induced EMT events, via some EMT transcription factors, such as Twist, Zeb-1 and Snail-1, in possible crosstalk with oxidative stress and inflammation events. To demonstrate this hypothesis, we inhibited/silenced Ref-1 in MPM cells; as a consequence, both EMT (Twist, Zeb-1 and Snail-1) markers and cellular migration/proliferation were significantly inhibited. Taken as a whole, these results show, for the first time, crosstalk between oxidative stress and EMT in MPM carcinogenesis and invasiveness, thus improving the knowledge to better address a preventive and therapeutic approach against this aggressive cancer.

Schwann cell-derived CXCL2 contributes to cancer pain by modulating macrophage infiltration in a mouse breast cancer model

Pain is one of the most severe complications affecting the quality of life of cancer patients. Although substantial progress has been made in the diagnosis and treatment of cancer, the neurobiological mechanism of cancer pain is still unclear. In the present study, we identified the critical role of CXC chemokine 2 (CXCL2), released by Schwann cells after being activated by cancer cells, in maintaining cancer-induced macrophage infiltration and the resulting mechanical hypersensitivity and persistent spontaneous nociception. In vitro, Schwann cells cocultured with breast cancer cells exhibited a significant increase in CXCL2 expression; in addition, conditioned medium from Schwann cells activated by breast cancer cells had a similar effect to recombinant CXCL2 in terms of inducing macrophage migration. Targeting CXCL2 signaling by both CXC chemokine receptor 2 (CXCR2) antagonist pharmacological blockade and anti-CXCL2 mAb immunological blockade robustly prevented conditioned medium-induced macrophage migration. In vivo, both application of recombinant CXCL2 and perineural breast cancer cell implantation resulted in mechanical hypersensitivity and persistent spontaneous nociception in mice, along with increased macrophage infiltration into the sciatic nerves. Similar to the in vitro results, inhibition of CXCL2/CXCR2 signaling or conditional knockdown of CXCL2 in sciatic nerve Schwann cells effectively attenuated breast cancer cell-induced mechanical hypersensitivity, persistent spontaneous nociception, and macrophage recruitment in the sciatic nerve. Mechanistically, we found that redox effector factor-1 (Ref-1) secreted by breast cancer cells activated hypoxia inducible factor-1α (HIF-1α) expression and inhibited reactive oxygen species (ROS) production in Schwann cells, ultimately inducing CXCL2 expression in Schwann cells. In brief, the present study expands new insights into cancer pain mechanisms from promising animal models to provide new strategies for the control of cancer pain.

Abstract 2475: Differential sensitivity of mouse PDAC KrasG12D cells to Ref-1/APE1 redox signalling inhibitors: Role of NFkB as a primary target of Ref-1/APE1 in Kras driven pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, with a five-year survival rate of less than ten percent. Kras is a common driver mutation of PDAC, contributing to progression and chemotherapy resistance. Additionally, inflammation is implicated in the development of PDAC and NFκB is recognized as a key mediator of inflammation and has been frequently observed to be upregulated in PDAC. Ref-1/APE1 (redox effector factor 1/apurinic endonuclease 1), a multifunctional protein involved in redox signaling of selected transcription factors (TF), is an upstream activator of NFκB and a therapeutic target. We have developed inhibitors to prevent Ref-1/APE1's redox signaling function, including activation of NFkB. We screened eight murine PDAC KC cell lines, which contain the KrasG12D mutation, to determine if there are subsets of these tumor cells that would respond differentially to Ref-1/APE1 redox signaling which recently completed phase I clinical trials for solid tumors. Additionally, we tested a series of second-generation inhibitors, including APX2009, APX2014 and others. The cell lines split into two distinct groups of sensitive and resistant to the APX compounds. In RNAseq analysis, the sensitive cell lines displayed enriched pathway genes and increased expression of TNFα-NFkB, epithelial to mesenchymal transition, inflammatory response and JAK/STAT3 signaling pathways as well as others, while the resistant lines were enriched for mTORC1, MYC and estrogen response pathways. Previous studies by our group have demonstrated the regulation of the JAK/STAT3 and NFkB pathways by Ref-1/APE1 and inhibition by APX compounds. To further tease out which of the downstream TFs and pathways were most dominant in the sensitivity response, we used KC3590 cells containing the KrasG12D mutation and expressing only a truncated, inactive form of NFκB. Rescue cells were created which have a full-length NFκB. When challenged with Ref-1/APE1 inhibitors APX3330, APX2009, and APX2014, cells containing the full-length, active NFκB were more sensitive to the APX compounds. To ascertain whether NFkB is the dominant TF reflecting APX inhibitor sensitivity, we studied whether knocking down RelA and STAT3, using siRNA, would alter the response of the cells to the APX compounds. STAT3 is known to be regulated by Ref-1/APE1 redox signaling. In these studies, the cellular response to APX inhibitors was unaffected following siRNA knock-down of RelA or STAT3. Overall, these experiments reveal suppression of NFκB as a primary determinant of Ref-1/APE1 cytotoxicity in KrasG12D cells. These findings allow us to begin to determine whether a specific gene signature within KRas driven PDAC tumors will inform which tumors would be more sensitive to Ref-1/APE1 redox signaling inhibitors. Citation Format: Rachel A. Caston, Randy Wireman, Lee Armstrong, Silpa Gampala, Olivia Babb, Nayela Chowdhury, Zonera Hassan, Christian Schneeweis, Gunter Schneider, Melissa L. Fishel, Mark R. Kelley. Differential sensitivity of mouse PDAC KrasG12D cells to Ref-1/APE1 redox signalling inhibitors: Role of NFkB as a primary target of Ref-1/APE1 in Kras driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2475.

Identification of Redox-Sensitive Transcription Factors as Markers of Malignant Pleural Mesothelioma.

Simple SummaryMalignant pleural mesothelioma is a lung tumor associated with asbestos exposure, with a poor prognosis, and a difficult pharmacological approach. Asbestos exposure is very toxic for the lungs, which counteract this toxic effect by activating some antioxidant defense proteins. When these proteins are more active that in normal conditions, as in several cancers, these tumors become able to survive and resist to stress or chemotherapy. In our laboratory, we collected cellular samples of mesothelioma and non-transformed mesothelium from Hospital’s Biobank and we evaluated these proteins. Our results demonstrated these proteins are upregulated in mesothelioma cells and not in non-transformed mesothelium. This event could be associated to toxic effects evoked by asbestos exposure, highlighting the need in the future to monitor asbestos-exposed people by measuring biomarkers identified, in the attempt to identify them as possible predictive markers and potential pharmacological targets addressed to improve mesothelioma prognosis.Although asbestos has been banned in most countries around the world, malignant pleural mesothelioma (MPM) is a current problem. MPM is an aggressive tumor with a poor prognosis, so it is crucial to identify new markers in the preventive field. Asbestos exposure induces oxidative stress and its carcinogenesis has been linked to a strong oxidative damage, event counteracted by antioxidant systems at the pulmonary level. The present study has been focused on some redox-sensitive transcription factors that regulate cellular antioxidant defense and are overexpressed in many tumors, such as Nrf2 (Nuclear factor erythroid 2-related factor 2), Ref-1 (Redox effector factor 1), and FOXM1 (Forkhead box protein M1). The research was performed in human mesothelial and MPM cells. Our results have clearly demonstrated an overexpression of Nrf2, Ref-1, and FOXM1 in mesothelioma towards mesothelium, and a consequent activation of downstream genes controlled by these factors, which in turn regulates antioxidant defense. This event is mediated by oxidative free radicals produced when mesothelial cells are exposed to asbestos fibers. We observed an increased expression of Nrf2, Ref-1, and FOXM1 towards untreated cells, confirming asbestos as the mediator of oxidative stress evoked at the mesothelium level. These factors can therefore be considered predictive biomarkers of MPM and potential pharmacological targets in the treatment of this aggressive cancer.

Unbiased High-Throughput Drug Combination Pilot Screening Identifies Synergistic Drug Combinations Effective against Patient-Derived and Drug-Resistant Melanoma Cell Lines.

We describe the development, optimization, and validation of 384-well growth inhibition assays for six patient-derived melanoma cell lines (PDMCLs), three wild type (WT) for BRAF and three with V600E-BRAF mutations. We conducted a pilot drug combination (DC) high-throughput screening (HTS) of 45 pairwise 4×4 DC matrices prepared from 10 drugs in the PDMCL assays: two B-Raf inhibitors (BRAFi), a MEK inhibitor (MEKi), and a methylation agent approved for melanoma; cytotoxic topoisomerase II and DNA methyltransferase chemotherapies; and drugs targeting the base excision DNA repair enzyme APE1 (apurinic/apyrimidinic endonuclease-1/redox effector factor-1), SRC family tyrosine kinases, the heat shock protein 90 (HSP90) molecular chaperone, and histone deacetylases.Pairwise DCs between dasatinib and three drugs approved for melanoma therapy-dabrafenib, vemurafenib, or trametinib-were flagged as synergistic in PDMCLs. Exposure to fixed DC ratios of the SRC inhibitor dasatinib with the BRAFis or MEKis interacted synergistically to increase PDMCL sensitivity to growth inhibition and enhance cytotoxicity independently of PDMCL BRAF status. These DCs synergistically inhibited the growth of mouse melanoma cell lines that either were dabrafenib-sensitive or had acquired resistance to dabrafenib with cross resistance to vemurafenib, trametinib, and dasatinib. Dasatinib DCs with dabrafenib, vemurafenib, or trametinib activated apoptosis and increased cell death in melanoma cells independently of their BRAF status or their drug resistance phenotypes. These preclinical in vitro studies provide a data-driven rationale for the further investigation of DCs between dasatinib and BRAFis or MEKis as candidates for melanoma combination therapies with the potential to improve outcomes and/or prevent or delay the emergence of disease resistance.

Abstract PR01: A phase I study targeting the APE1/Ref-1 DNA repair-redox signaling protein with the APX3330 inhibitor

Abstract APX3330 is an orally administered anti-cancer small molecule that targets the APE1/Ref-1 protein (apurinic endonuclease 1 / redox effector factor 1). APE1/Ref-1 is both a DNA repair and reduction-oxidation (redox) signaling protein that participates in the DNA base excision repair (BER) pathway. Additionally, APE1/Ref-1 regulates the redox status of a number of important transcription factors (TFs) in tumor cells. These include NFkB, STAT3, and HIF-1a. APE1/ref-1 converts these TFs from an off (oxidized) to an on (reduced) state allowing them to bind to the promoter sequence of genes regulating tumor growth, metastasis, metabolism and survival of tumor cells. APX3330 selectively binds to APE1/Ref-1, specifically inhibiting its redox signaling activity. APE1/Ref-1 also plays a key role in a variety of inflammatory disorders beyond cancers including diabetic macular edema, inflammatory bowel disorders and others. Data will be presented for study NCT03375086 evaluating APX3330 in patients with incurable malignancies. Eligibility required adequate organ function, PS 0-2 and tumors not amenable to curative therapy. Primary and secondary objectives included determining the recommended phase 2 dose (RP2D), the safety and PK/PD profiles of APX3330 and reporting any RECIST anti-tumor activity. Patients received APX3330 b.i.d., in 21-day cycles. APX3330 was well tolerated at dose levels from 240-600 mg/d. The most frequent treatment-related adverse events (all grades) included G1 nausea (16%) and fatigue (16%). Six subjects had disease stabilization for > 4 cycles, and of these, four subjects with the following diagnosis, RECIST response and days on study included: colorectal cancer, PR (partial response), 357 days; endometrial, SD (stable disease), 421days; melanoma, SD, 337 days; prostate, SD, 252 days. All study objectives were completed and determined that APX3330 is safe for chronic dosing from 240 up to and including 600 mg/day. It provides clinical benefit to patients with a variety of tumor types (e.g., endometrial, colorectal, prostate and melanoma cancer). Pharmacodynamic (PD) studies from patient biopsy evaluation indicates APX3330-mediated effect upon cancer cells, including decrease in transcription factors activity for those regulated by the APE1/Ref-1 protein. Differentially expressed proteins (DEPs) were analyzed comparing pre-treatment and on-treatment tumor biopsies. Specifically studied were NFkB, HIFa and STAT3 genes downstream of these three TFs. Circulating tumor cell (CTC) analysis was performed on sixteen patients who had baseline and on-treatment samples that were evaluated longitudinally. Of these, 7/16 (44%) of patients showed a reduction in CTCs after initiation of treatment with APX3330. APE1/Ref-1 serum levels were determined to be elevated in aggressive tumors and were reduced following treatment with APX3330 in SD patients (past four treatment cycles). Pharmacokinetic (PK) data indicate confirmation of pre-clinical data. Conclusions: APX3330 is an orally administered inhibitor of APE1/Ref-1 specifically targeting the redox signaling function of this protein. This phase I study identified 600 mg PO daily as the RP2D for further development. RECIST evaluation identified signs of clinical activity in this un-selected population of patients with advanced cancer. PD analyses (proteomics, CTC, serum) indicate APX3330 mediated targeting of the APE1/Ref-1 protein. Citation Format: Mark R. Kelley, Safi Shahda, Nehal J. Lakhani, Bert O'Neil, Lincy Chu, Amanda K. Anderson, Jun Wan, Amber L. Mosley, Hao Liu, Richard A. Messmann. A phase I study targeting the APE1/Ref-1 DNA repair-redox signaling protein with the APX3330 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR01. doi:10.1158/1535-7163.TARG-19-PR01

Blocking HIF signaling via novel inhibitors of CA9 and APE1/Ref-1 dramatically affects pancreatic cancer cell survival

Pancreatic ductal adenocarcinoma (PDAC) has reactive stroma that promotes tumor signaling, fibrosis, inflammation, and hypoxia, which activates HIF-1α to increase tumor cell metastasis and therapeutic resistance. Carbonic anhydrase IX (CA9) stabilizes intracellular pH following induction by HIF-1α. Redox effector factor-1 (APE1/Ref-1) is a multifunctional protein with redox signaling activity that converts certain oxidized transcription factors to a reduced state, enabling them to upregulate tumor-promoting genes. Our studies evaluate PDAC hypoxia responses and APE1/Ref-1 redox signaling contributions to HIF-1α-mediated CA9 transcription. Our previous studies implicated this pathway in PDAC cell survival under hypoxia. We expand those studies, comparing drug responses using patient-derived PDAC cells displaying differential hypoxic responses in 3D spheroid tumor-stroma models to characterize second generation APE1/Ref-1 redox signaling and CA9 inhibitors. Our data demonstrates that HIF-1α-mediated CA9 induction differs between patient-derived PDAC cells and that APE1/Ref-1 redox inhibition attenuates this induction by decreasing hypoxia-induced HIF-1 DNA binding. Dual-targeting of APE1/Ref-1 and CA9 in 3D spheroids demonstrated that this combination effectively kills PDAC tumor cells displaying drastically different levels of CA9. New APE1/Ref-1 and CA9 inhibitors were significantly more potent alone and in combination, highlighting the potential of combination therapy targeting the APE1-Ref-1 signaling axis with significant clinical potential.

Apurinic/apyrimidinic endonuclease 1 performs multiple roles in controlling the outcome of cancer cells toward radiation and chemotherapeutic agents

Many endogenous and exogenous sources produce reactive oxygen species such as superoxide radical anions and hydrogen peroxide that are converted to the highly reactive form, hydroxyl radical. It is this latter species that can damage several macromolecules in the cells, in particular, the DNA to produce a variety of DNA lesions. These DNA lesions include oxidatively damaged purine and pyrimidine bases, as well as single-strand and double-strand breaks. These unrepaired DNA lesions lead to base substitutions, deletions, insertions, and rearrangements of the chromosome, ultimately altering the stability of the genome. Maintaining the integrity of the genome is essential to prevent various diseases such as several types of cancers. There are several DNA repair pathways including base-excision repair (BER), nucleotide-excision repair, mismatch repair, homologous recombination, and nonhomologous end joining that operate in the human cells to prevent genomic instability. Each of these DNA repair pathways consists of multiple enzymes that execute specific function (s). This review focuses on a key enzyme apurinic/apyrimidinic endonuclease 1 (APE1) that belongs to the BER pathway that plays a pivotal role in the removal of modified DNA bases. We provide an overview of the multifaceted roles performed by APE1, which also serves as a redox factor and referred to as redox effector factor 1 (Ref-1) or APE1/Ref-1. In addition, we discuss more recent findings whereby (i) peroxiredoxin 1 controls the redox activity of APE1 and (ii) CUT-like homeobox 1 protein, a transcription factor that binds to DNA and stimulates the DNA repair activities of APE1 to confer resistance to radio- and chemotherapy.

The anti-inflammatory role of extranuclear apurinic/apyrimidinic endonuclease 1/redox effector factor-1 in reactive astrocytes.

Apurinic/apyrimidinic endonuclease 1 (APE1), a ubiquitous multipurpose protein, is also known as redox effector factor-1 (Ref-1). It is involved in DNA repair and redox signaling and, in turn, oxidative stress-induced neurodegeneration. Although previous studies have demonstrated that APE1/Ref-1 functions as a negative regulator of inflammatory response via several mechanisms in neuronal cells, little is known about the roles of APE1/Ref-1 in glial cells. In this study, we found that cytoplasmic APE1/Ref-1 expression was upregulated in reactive astrocytes of the kainic acid- or lipopolysaccharide (LPS)-injected hippocampus. Analysis of the inflammatory response induced by extranuclear APE1/Ref-1 (ΔNLS-Ref-1) in cultured primary astrocytes revealed that it markedly suppressed inducible nitric oxide synthase (iNOS) expression and tumor necrosis factor-α (TNF-α) secretion induced by LPS to a similar extent as did wild type APE1/Ref-1 (WT-Ref-1), supporting the concept an anti-inflammatory role of extranuclear APE1/Ref-1 in astrocytes. Additionally, overexpression of WT- and ΔNLS-Ref-1 suppressed the transcriptional activity of nuclear factor-κB (NF-κB), although it effectively enhanced activator protein 1 (AP-1) activity. The blunting effect of APE1/Ref-1 on LPS-induced NF-κB activation was not mediated by IκB kinase (IKK) activity. Instead, APE1/Ref-1 inhibited p300-mediated acetylation of p65 by suppressing intracellular reactive oxygen species (ROS) levels following LPS treatment. Taken together, our results showed that altered expression and/or subcellular distribution of APE1/Ref-1 in activated astrocytes regulated the neuroinflammatory response to excitotoxin and endotoxin insults used in model of neurodegenerative brain diseases.

Regulation of HIF1α under Hypoxia by APE1/Ref-1 Impacts CA9 Expression: Dual Targeting in Patient-Derived 3D Pancreatic Cancer Models.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality in the United States. Aggressive treatment regimens have not changed the disease course, and the median survival has just recently reached a year. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including hypoxia, which creates a more aggressive phenotype with increased metastatic potential and impaired therapeutic efficacy. AP Endonuclease-1/Redox Effector Factor 1 (APE1/Ref-1) is a multifunctional protein possessing a DNA repair function in base excision repair and the ability to reduce oxidized transcription factors, enabling them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in survival mechanisms, tumor growth, and hypoxia signaling. Here, we explore the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling activity, which regulates the transcriptional activation of hypoxia-inducible factor 1 alpha (HIF1α). Carbonic anhydrase IX (CA9) is regulated by HIF1α and functions as a part of the cellular response to hypoxia to regulate intracellular pH, thereby promoting cell survival. We hypothesized that modulating APE1/Ref-1 function will block activation of downstream transcription factors, STAT3 and HIF1α, interfering with the hypoxia-induced gene expression. We demonstrate APE1/Ref-1 inhibition in patient-derived and established PDAC cells results in decreased HIF1α-mediated induction of CA9. Furthermore, an ex vivo three-dimensional tumor coculture model demonstrates dramatic enhancement of APE1/Ref-1-induced cell killing upon dual targeting of APE1/Ref-1 and CA9. Both APE1/Ref-1 and CA9 are under clinical development; therefore, these studies have the potential to direct novel PDAC therapeutic treatment. Mol Cancer Ther; 15(11); 2722-32. ©2016 AACR.

APE1/Ref-1 facilitates recovery of gray and white matter and neurological function after mild stroke injury

A major hallmark of oxidative DNA damage after stroke is the induction of apurinic/apyrimidinic (AP) sites and strand breaks. To mitigate cell loss after oxidative DNA damage, ischemic cells rapidly engage the base excision-repair proteins, such as the AP site-repairing enzyme AP endonuclease-1 (APE1), also named redox effector factor-1 (Ref-1). Although forced overexpression of APE1 is known to protect against oxidative stress-induced neurodegeneration, there is no concrete evidence demonstrating a role for endogenous APE1 in the long-term recovery of gray and white matter following ischemic injury. To address this gap, we generated, to our knowledge, the first APE1 conditional knockout (cKO) mouse line under control of tamoxifen-dependent Cre recombinase. Using a well-established model of transient focal cerebral ischemia (tFCI), we show that induced deletion of APE1 dramatically enlarged infarct volume and impaired the recovery of sensorimotor and cognitive deficits. APE1 cKO markedly increased postischemic neuronal and oligodendrocyte degeneration, demonstrating that endogenous APE1 preserves both gray and white matter after tFCI. Because white matter repair is instrumental in behavioral recovery after stroke, we also examined the impact of APE1 cKO on demyelination and axonal conduction and discovered that APE1 cKO aggravated myelin loss and impaired neuronal communication following tFCI. Furthermore, APE1 cKO increased AP sites and activated the prodeath signaling proteins, PUMA and PARP1, after tFCI in topographically distinct manners. Our findings provide evidence that endogenous APE1 protects against ischemic infarction in both gray and white matter and facilitates the functional recovery of the central nervous system after mild stroke injury.

Acute exercise stress promotes Ref1/Nrf2 signalling and increases mitochondrial antioxidant activity in skeletal muscle.

What is the central question of this study? How does acute exercise affect the redox effector factor-1 (Ref1) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling and its association with mitochondrial H(2)O(2) production and antioxidant capacity? What is the main finding and its importance? Ref1/Nrf2 signalling in skeletal muscles was activated by acute exercise, and this activation was correlated with increased mitochondrial H(2)O(2) content and antioxidant capacity (reduced glutathione and manganese superoxide dismutase). The finding indicates that the oxidative stress induced by acute exercise in skeletal muscle mitochondria is associated with the upregulation of Ref1/Nrf2 signalling and enhancement of antioxidant defense pathways. This mechanism may play a role in preventing cellular oxidative stress resistance during acute exercise. The molecular mechanism of exercise-induced oxidative stress and adaptive activation of antioxidant responses in skeletal muscle has not been fully elucidated. This study aimed to investigate the effect of acute exercise on redox effector factor-1 (Ref1) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling and associations with mitochondrial H(2)O(2) production and antioxidant mechanisms in skeletal muscles. Groups of male ICR/CD-1 mice were subjected to an acute exercise bout of different durations (45, 90, 120 or 150 min). Muscle tissues (gastrocnemius and quadriceps femoris) were harvested after exercise to measure mitochondrial manganese superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase (CuZnSOD) activities, reduced glutathione (GSH) content and expression of Ref1/Nrf2 genes and Ref1/Nrf2 proteins. The acute exercise increased oxidative stress and activated Ref1/Nrf2 signalling in a time-dependent manner, with a linear correlation between the mitochondrial H(2)O(2) content and Ref1/Nrf2 expressions. The GSH content and MnSOD activity were also significantly increased, but CuZnSOD activity was not significantly affected. The findings indicate that the H(2)O(2) production induced by acute exercise in skeletal muscle mitochondria in the mouse is closely associated with upregulation of the Ref1/Nrf2 signalling pathway and enhancement of antioxidant defense components, including GSH and MnSOD. Activation of Ref1/Nrf2/antioxidant defense pathways may play a role in preventing cellular oxidative stress resistance during acute exercise.

Glial plasticity after hexahydrobenzene exposure.

Glial plasticity after hexahydrobenzene exposure.

Abstract B158: Targeting APE1/Ref-1 results in inhibition of hypoxia signaling genes

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related mortality in the US. Most patients present with advanced disease, and ∼95% die within five years. Treatment with chemotherapy has not changed the natural course of this disease, and just recently, with combination of chemotherapeutic agents, the median survival reached a year. Several mechanisms are proposed to play a role in the aggressive, treatment-resistant phenotype of PDAC, including adaptation to hypoxia, which leads to increased potential for metastasis and impairs the efficacy of chemotherapy and radiotherapy. Hypoxia-Inducible Factor-1α (HIF1α), a major oxygen sensor in cells, is a transcription factor that is rapidly degraded under normoxic conditions but upregulates a number of genes under hypoxic conditions that contribute to survival, metastasis, and angiogenic signaling in the tumor microenvironment. One of the most notable HIF targets is Carbonic Anhydrase IX (CA9), which promotes tumor cell survival and metastasis by maintaining a steady intracellular pH while acidifying the microenvironment, thereby encouraging epithelial-mesenchymal transition and contributing to extracellular matrix degradation. AP Endonuclease1/ Redox Effector Factor 1 (APE1/Ref-1) is a dual function protein that possesses a DNA repair function as well as the ability to reduce transcription factors and enable them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in preventing apoptosis, survival mechanisms, and hypoxia signaling, including HIF-1α. Therefore, we hypothesized that APE1/Ref-1 inhibition impairs HIF-1α-mediated signaling, and this leads to decreased survival and invasion of tumor cells exposed to hypoxic conditions. Methods: We performed co-immunoprecipitation (co-IP) studies to look at the interaction of APE1/Ref-1 with transcriptional targets, HIF-1α, STAT3, and NFκB along with RT-PCR and Western blotting to confirm expression of hypoxia signaling genes. Luciferase reporter assays were used to quantitate transcriptional activation under hypoxia. Boyden chamber was used to look at migration and invasion as well as proliferation based assays following manipulation of APE1/Ref-1 and hypoxia. Results: HIF-1α and STAT3, but not NFκB associate with APE1/Ref-1 under hypoxia. Moreover, we found that knockdown of APE1/Ref-1 protein diminishes HIF-mediated transcription in hypoxia, as shown by analysis of luciferase reporter assays. Next, we showed that, in hypoxia, APE1/Ref-1 inhibition diminishes HIF-1α-induced downstream targets including CA9 and ANGPTL4 further indicating that APE1/Ref-1 redox activity regulates HIF signaling. Importantly, we found that hypoxia, in the presence or absence of APE1/Ref-1, no longer induced CA9 mRNA levels in HIF-deficient MEFs, proving that hypoxia-dependent regulation of CA9 expression is fully mediated by HIF-1α. A blockade of both CA9 activity via small molecule and CA9 transcription via APE1/Ref-1 leads to decreased PDAC cell proliferation under hypoxia. These data indicate that APE1/Ref-1 inhibition interferes with ηψπoξια-mediated signaling and can further sensitize PDAC cells to CA9/12 inhibition even under the conditions of extreme oxygen deprivation. Ongoing experiments will determine the role APE1/Ref-1 plays in the survival and invasion of tumor cells exposed to hypoxia. Citation Format: Derek P. Logsdon, Huiwen Cheng, Meihua Luo, Safi Shahda, Yangyang Hao, Yan Tong, Zhangsheng Yu, Nicholas Zyromski, Ernestina Schipani, Yunlong Liu, Claudiu T. Supuran, Mircea Ivan, Mark R. Kelley, Melissa L. Fishel. Targeting APE1/Ref-1 results in inhibition of hypoxia signaling genes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B158.

An oxidative DNA "damage" and repair mechanism localized in the VEGF promoter is important for hypoxia-induced VEGF mRNA expression.

In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation.

Abstract 5097: APE1/Ref-1 promotes cell adhesion and migration in cervical cancer cells

Abstract Apurinic and apyrimidinic endonuclease 1 (APE1)/redox effector factor-1 (Ref-1) is known to be involved in DNA base excision repair pathway and redox-dependent regulation of a number of transcription factors, such as AP1, p53, and Pax-8. It is expressed in numerous human cancers such as pancreatic cancer, prostate cancer, breast cancer, cervical cancer, and colon cancer. Our previous studies suggested APE1/Ref-1 can promote cancer progression through its involvement in cell proliferation, metastasis and angiogenesis; however, the molecular mechanism by which APE1/Ref-1 functions in these processes has not been characterized. In this study, we are the first to report that APE1/Ref-1 activates cell adhesion-related proteins to trigger cell migration. To investigate whether APE1/Ref-1 is involved in cell adhesion in normal fibroblasts, an adhesion assay was performed using GM00637 human fibroblast cell lines stably overexpressing APE1/Ref-1. Cell adhesion to vitronectin, a known ligand of the adhesion molecules integrin ανβ3 and integrin ανβ5, was significantly increased in APE1/Ref-1-overexpressing GM00637 cells compared to control fibroblast cells. FACS analysis showed APE1/Ref-1 induces the expression and activation of adhesion molecules including integrin αν and β3 in APE/Ref-1-overexpressing GM00637 cells. To evaluate whether APE1/Ref-1-mediated regulation of integrin ανβ3 is involved in cell migration in cervical cancer cells, a migration assay was performed using APE1/Ref-1-specific siRNA and an integrin ανβ3-specific antibody. Cell migration was significantly decreased in the absence of APE1/Ref-1 expression in cervical cancer cells, which was comparable with inhibition of integrin ανβ3. Consistent with these results, APE1/Ref-1 increased the activation and expression of integrin ανβ3 in both cervical cancer cells and cervical cancer patient tissue. Combined, these studies suggest that APE/Ref-1 can promote cell adhesion and migration through integrin ανβ3 signaling and thereby this signaling mechanism could contribute to cervical cancer progression. Citation Format: Mihwa Kim, Ho Jin You, Dae Joon Kim. APE1/Ref-1 promotes cell adhesion and migration in cervical cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5097. doi:10.1158/1538-7445.AM2015-5097

Design and activity of AP endonuclease-1 inhibitors.

Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1) is a critical component of base excision repair that excises abasic lesions created enzymatically by the action of DNA glycosylases on modified bases and non-enzymatically by hydrolytic depurination/depyrimidination of nucleobases. Many anticancer drugs generate DNA adducts that are processed by base excision repair, and tumor resistance is frequently associated with enhanced APE-1 expression. Accordingly, APE-1 is a potential therapeutic target to treat cancer. Using computational approaches and the high resolution structure of APE-1, we developed a 5-point pharmacophore model for APE-1 small molecule inhibitors. One of the nM APE-1 inhibitors (AJAY-4) that was identified based on this model exhibited an overall median growth inhibition (GI50) of 4.19μM in the NCI-60 cell line panel. The mechanism of action is shown to be related to the buildup of abasic sites that cause PARP activation and PARP cleavage, and the activation of caspase-3 and caspase-7, which is consistent with cell death by apoptosis. In a drug combination growth inhibition screen conducted in 10 randomly selected NCI-60 cell lines and with 20 clinically used non-genotoxic anticancer drugs, a synergy was flagged in the SK-MEL-5 melanoma cell line exposed to combinations of vemurafenib, which targets melanoma cells with V600E mutated BRAF, and AJAY-4, our most potent APE-1 inhibitor. The synergy between AJAY-4 and vemurafenib was not observed in cell lines expressing wild-type B-Raf protein. This synergistic combination may provide a solution to the resistance that develops in tumors treated with B-Raf-targeting drugs.

A review on protein-protein interaction network of APE1/Ref-1 and its associated biological functions.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a classic example of functionally variable protein. Besides its well-known role in (i) DNA repair of oxidative base damage, APE1 also plays a critical role in (ii) redox regulation of transcription factors controlling gene expression for cell survival pathways, for which it is also known as redox effector factor 1 (Ref-1), and recent evidences advocates for (iii) coordinated control of other non-canonical protein-protein interaction(s) responsible for significant biological functions in mammalian cells. The diverse functions of APE1 can be ascribed to its ability to interact with different protein partners, owing to the attainment of unfolded domains during evolution. Association of dysregulation of APE1 with various human pathologies, such as cancer, cardiovascular diseases and neurodegeneration, is attributable to its multifunctional nature, and this makes APE1 a potential therapeutic target. This review covers the important aspects of APE1 in terms of its significant protein-protein interaction(s), and this knowledge is required to understand the onset and development of human pathologies and to design or improve the strategies to target such interactions for treatment and management of various human diseases.

The role of APE/Ref-1 signaling pathway in hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is responsible for a third of the estimated cancer-caused deaths worldwide. To deeply understand the mechanisms controlling HCC progression is of primary importance to develop new approaches for treatment. Apurinic/apyrimidinic endonuclease-1/redox effector factor 1 (APE/Ref-1) has been uncovered elevated in various types of cancer, including HCC. Additionally, HCC progression is always correlated with elevated copper (Cu). Our previous data demonstrated that Cu treatment initiated APE/Ref-1 expression and its downstream targets. Therefore, we hypothesized that APE/Ref-1 may be involved in HCC progression through mediating the effect of Cu to its signaling cascades. Following different treatments, human HCC cell line (Hep3B) and immortalized non-malignant hepatocyte cell line (THLE3) were analyzed to explore the role of APE/Ref-1 signaling pathway. Unstained human tissue microarrays (TMA) were subjected to IHC analysis to study the relationship between APE/Ref-1 expression and clinic features. APE/Ref-1 was upregulated in HCC cells consistent with the strong expression of APE/Ref-1 in HCC tissue microarray. Greater cytoplasmic accumulation of APE/Ref-1 was found in poorly differentiated and more aggressive tumors. Also we provide evidence to show that APE/Ref-1 signaling pathway stimulates cellular proliferation, enhances antiapoptosis, and facilitates metastasis through experimental knockdown of APE/Ref-1 using siRNA in Hep3B cells or overexpressing APE/Ref-1 in THLE3 cells. These results define a novel role of APE/Ref-1 in HCC progression as being an important mediating and potentiating molecule, and also provide a basis for further investigations utilizing appropriate APE/Ref-1 inhibitors in combination with chemo-drugs for HCC treatment.

APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1α, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a ‘hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed.