Hyperactivation Of Nrf2 Research Articles

Persistent activation of Nrf2 promotes a vicious cycle of oxidative stress and autophagy inhibition in cadmium-induced kidney injury

Nuclear factor erythroid 2-related factor 2 (Nrf2) serves as the master regulator of antioxidant signaling and inhibition or hyperactivation of Nrf2 pathway will result in the redox imbalance to induce tissue injury. Herein, we established cadmium (Cd)-exposed rat kidney injury model by intraperitoneal injection with CdCl2 (1.5 mg/kg body weight) and cytotoxicity model of NRK-52E cells by CdCl2 (5 μM) exposure to reveal the role of Nrf2 hyperactivation in Cd-induced nephrotoxicity. Data from the in vitro and in vivo study showed that Cd caused Nrf2 nuclear retention due to nuclear-cytoplasmic depletion of Kelch-like ECH-associated protein 1 (Keap1) and Sequestosome-1(SQSTM1/p62) accumulation, leading to the persistent activation of Nrf2. Moreover, we established inhibited models of Cd-induced prolonged Nrf2 activation using siRNA-mediated gene silencing in vitro and pharmacological inhibition in vivo for subsequent assays. First, Cd-induced cytotoxicity, renal injury and concomitant oxidative stress were markedly alleviated by Nrf2 inhibition. Second, Cd-induced autophagy inhibition was notably alleviated by Nrf2 inhibition. Further, we revealed underlying molecular mechanisms of the crosstalk between persistent activation of Nrf2 and autophagy inhibition in Cd-induced nephrotoxicity. Data showed that Cd-induced lysosomal dysfunction evidenced by impaired lysosomal biogenesis and degradation capacity was markedly recovered by Nrf2 inhibition. Meanwhile, Cd-impaired autophagosome-lysosome fusion was obviously restored by Nrf2 inhibition. In conclusion, our findings revealed that persistent activation of Nrf2 promoted a vicious cycle of oxidative stress and autophagy inhibition in Cd-induced nephrotoxicity.

Thioredoxin reductase-1 levels are associated with NRF2 pathway activation and tumor recurrence in non-small cell lung cancer

Activating mutations in the KEAP1/NRF2 pathway characterize a subset of non-small cell lung cancer (NSCLC) associated with chemoresistance and poor prognosis. We herein evaluated the relationship between 64 oxidative stress-related genes and overall survival data from 35 lung cancer datasets. Thioredoxin reductase-1 (TXNRD1) stood out as the most significant predictor of poor outcome. In a cohort of NSCLC patients, high TXNRD1 protein levels correlated with shorter disease-free survival and distal metastasis-free survival post-surgery, including a subset of individuals treated with platinum-based adjuvant chemotherapy. Bioinformatics analysis revealed that NSCLC tumors harboring genetic alterations in the NRF2 pathway (KEAP1, NFE2L2 and CUL3 mutations, and NFE2L2 amplification) overexpress TXNRD1, while no association with EGFR, KRAS, TP53 and PIK3CA mutations was found. In addition, nuclear accumulation of NRF2 overlapped with upregulated TXNRD1 protein in NSCLC tumors. Functional cell assays and gene dependency analysis revealed that NRF2, but not TXNRD1, has a pivotal role in KEAP1 mutant cells’ survival. KEAP1 mutants overexpress TXNRD1 and are less susceptible to the cytotoxic effects of the TXNRD1 inhibitor auranofin when compared to wild-type cell lines. Inhibition of NRF2 with siRNA or ML-385, and glutathione depletion with buthionine-sulfoximine, sensitized KEAP1 mutant A549 cells to auranofin. NRF2 knockdown and GSH depletion also augmented cisplatin cytotoxicity in A549 cells, whereas auranofin had no effect. In summary, these findings suggest that TXNRD1 is not a key determinant of malignant phenotypes in KEAP1 mutant cells, although this protein can be a surrogate marker of NRF2 pathway activation, predicting tumor recurrence and possibly other aggressive phenotypes associated with NRF2 hyperactivation in NSCLC.

Abstract 2507: Does a threshold exist for NRF2 hyperactivation to block tumor progression in KRAS mutant, TP53-deficient NSCLC

Abstract Lung cancer is responsible for the most cancer-related deaths worldwide. Within the most prominent histological subtype, non-small cell lung cancer (NSCLC), there is an unmet clinical need: lung adenocarcinomas (ADCs) driven by mutant KRAS. Within this subset of tumors, KRAS mutations co-occur with mutations in tumor suppressor genes including TP53 and the redox regulator KEAP1. KEAP1 is the negative regulator of transcription factor NRF2, which directs the antioxidant response and multiple facets of metabolism. In NSCLC, alterations in the KEAP1-NRF2 circuit result in constitutive NRF2 activation and are often associated with resistance to therapy and poor outcomes in patients. While NRF2 hyperactivation has been associated with tumor progression, our lab's recent findings suggest that this may be context-dependent, and that too much NRF2 activation may be detrimental. To study the role of NRF2 hyperactivation on tumor progression, we have utilized KRAS mutant genetically engineered mouse models of NSCLC harboring TP53 deletion. These studies are based on our lab's finding that the homozygous KEAP1R554Q loss-of-function mutation decreases tumor size in a Kras mutant, Trp53-deficient (KP) lung ADC model (Kang et al. 2019 eLife). In parallel to these studies, we have also developed a conditional murine allele of the NRF2D29H mutation found in human NSCLC to serve as a secondary model of NRF2 hyperactivation in the KP mouse (KPN). Consistent with our homozygous KEAP1 mutant model (KPKK), we found that KPN mice demonstrated constitutive NRF2 activation, as observed by increased immunohistochemical staining of canonical NRF2 target, NQO1. This degree of NRF2 activation in KPN mice was slightly lower than that of KPKK mice, suggesting that the KPN mouse is an intermediate model of NRF2 activation. Supportingly, we also found that KPN mice had decreased tumor burden, although not to the same extent as KPKK mice. Interestingly, our heterozygous KEAP1 mutant model (KPK) demonstrates only modest NRF2 activation but did not exhibit decreased tumor burden. Importantly, analyses of tumor number suggested that KPKK and KPN tumors are impaired in tumor progression, rather than initiation. KPKK and KPN tumors also exhibited lower proliferative indices when compared to KP mice, in correspondence with their reduced tumor burden. Collectively, these results suggest that there may be a threshold for NRF2 activation to block tumor progression in the KP model. Current studies are focused on determining whether this impediment to tumor burden is NRF2-dependent, and what NRF2-dependent mechanisms may impair tumor progression. Importantly, these studies may help identify whether a threshold for NRF2 hyperactivation to promote or block tumor progression exists, and if this can be therapeutically exploited in patients with KRAS mutant, TP53-deficient lung tumors. Citation Format: Janine M. DeBlasi, Aimee Falzone, Gina M. DeNicola. Does a threshold exist for NRF2 hyperactivation to block tumor progression in KRAS mutant, TP53-deficient NSCLC [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 2507.

Long non‑coding RNA XIST inhibits osteoblast differentiation and promotes osteoporosis via Nrf2 hyperactivation by targeting CUL3.

Osteoporosis (OP) is a common skeletal disorder characterized by a low bone mass and the deterioration of bone structure. Long non-coding (lnc)RNA X inactive-specific transcript (XIST) is highly expressed in the serum and monocytes of patients with OP. Thus, the purpose of the present study was to explore the mechanisms underlying the role of XIST in the progression of OP. To establish animal models of OP, female rats underwent a bilateral ovariectomy. The bone mineral density of individual rats was measured using dual-energy X-ray absorptiometry. The combination of XIST and cullin-3 (CUL3) was analyzed using a dual-luciferase reporter assay. Bone histopathological changes were assessed by hematoxylin and eosin staining. Alkaline phosphatase activity was examined by ALP staining. Finally, a series of functional experiments were performed to examine the effects of XIST on cellular behaviors. In the present study, XIST promoted OP and inhibited bone formation by regulating the expression levels of CUL3 and nuclear factor erythroid 2-related factor 2 (Nrf2) in the rats with OP. Moreover, XIST directly targeted CUL3 and negatively regulated its expression. Of note, CUL3 downregulation reversed the effects of XIST silencing on cell viability, differentiation and mineralization, as well as the expression of Nrf2 and CUL3 in MC3T3-E1 cells. Collectively, XIST was demonstrated to inhibit the differentiation of osteoblasts and promote OP by inhibiting the degradation of Nrf2 via targeting CUL3.

Identification of Beilschmiedia tsangii Root Extract as a Liver Cancer Cell-Normal Keratinocyte Dual-Selective NRF2 Regulator.

Transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) plays a crucial role in regulating the expression of genes participating in cellular defense mechanisms against oxidative or xenobiotic insults. However, there is increasing evidence showing that hyperactivation of NRF2 is associated with chemoresistance in several cancers, including hepatocellular carcinoma (HCC), thus making NRF2 an attractive target for cancer therapy. Another important issue in cancer medication is the adverse effects of these substances on normal cells. Here, we attempted to identify a dual-selective NRF2 regulator that exerts opposite effects on NRF2-hyperactivated HCC cells and normal keratinocytes. An antioxidant response element driven luciferase reporter assay was established in Huh7 and HaCaT cells as high-throughput screening platforms. Screening of 3,000 crude extracts from the Taiwanese Indigenous Plant Extract Library resulted in the identification of Beilschmiedia tsangii (BT) root extract as a dual-selective NRF2 regulator. Multiple compounds were found to contribute to the dual-selective effects of BT extract on NRF2 signaling in two cell lines. BT extract reduced NRF2 protein level and target gene expression levels in Huh7 cells but increased them in HaCaT cells. Furthermore, notable combinatory cytotoxic effects of BT extract and sorafenib on Huh7 cells were observed. On the contrary, sorafenib-induced inflammatory reactions in HaCaT cells were reduced by BT extract. In conclusion, our results suggest that the combination of a selective NRF2 activator and inhibitor could be a practical strategy for fine-tuning NRF2 activity for better cancer treatment and that plant extracts or partially purified fractions could be a promising source for the discovery of dual-selective NRF2 regulators.

Modeling paraquat-induced lung fibrosis in C. elegans reveals KRIT1 as a key regulator of collagen gene transcription.

Paraquat poisoning causes lung fibrosis, which often results in long-term pulmonary dysfunction. Lung fibrosis has been attributed to collagens accumulation, but the underlying regulatory pathway remains unclear. Here we use the genetically tractable C. elegans as a model to study collagen gene transcription in response to paraquat. We find that paraquat robustly up-regulates collagen gene transcription, which is dependent on KRI-1, a poorly studied protein homologous to human KRIT1/CCM1. KRI-1 knockdown prevents paraquat from activating the oxidative stress response transcription factor SKN-1/Nrf2, resulting in reduced collagen transcription and increased paraquat sensitivity. Using human lung fibroblasts (MRC-5), we confirm that both KRIT1 and Nrf2 are required for collagen transcription in response to paraquat. Nrf2 hyper-activation by KEAP1 knockdown bypasses KRIT1 to up-regulate collagen transcription. Our findings on the regulation of collagen gene transcription by paraquat could suggest potential strategies to treat pulmonary fibrosis caused by paraquat poisoning.

P62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.

Effect of Nrf2 in tumor progression and its inhibitors in cancer therapy

Nrf2 is the key transcription factor mainly for regulating oxidative homeostasis and cytoprotective responses against oxidative stress. Nrf2/Keap1 pathway is one of the most important cellular defense mechanisms against endogenous or exogenous oxidative stress. With its activation, a wide range of stress-related genes is transactivated to restore the cellular homeostasis. Recent studies revealed that the aberrant activation of Nrf2 is related to the malignant progression, chemotherapeutic drug resistance and poor prognosis. Nrf2 plays a crucial role in cancer malignancy and chemotherapeutic resistance by controlling the intracellular redox homeostasis through the activation of cytoprotective antioxidant genes. Nrf2 inhibitor containing many natural products has been deemed as a novel therapeutic strategy for human malignancies. This article reviews the progress of studies of the Nrf2/Keap1 pathway, and its biological impact in solid malignancies and molecular mechanisms for causing Nrf2 hyperactivation in cancer cells. In conclusion, we summarized the deve-lopment of Nrf2 inhibitors in recent years, in the expectation of providing reference for further drug development and clinical studies.

Evaluation of Nrf2, Keap1 and Apoptotic Pathway Genes Expression in Acute Myeloid Leukemia Patients

The aim of this study was to evaluate the expression Nrf2 (Nuclear factor-erythroid 2-p45 derived factor 2) and Keap1 (Kelch-like ECH-associated protein 1) genes and Bcl-2 (B-cell lymphoma 2), Bcl-XL (B-cell lymphoma-extra large), Bax (Bcl2-associated X protein) apoptotic pathway genes in acute myeloid leukemia patients. In this case-control study, the expression of genes encoding Nrf2, Keap1, Bcl2, Bcl- XL and Bax in 40 acute myeloid leukemia (AML) patients were compared with 40 normal individuals in the Iranian population. We evaluated the mRNA expression of genes by using the real-time quantitative polymerase chain reaction. The expression of Nrf2, Bcl2 and Bcl- XL genes in new AML patients were increased (p < 0.05). The patients treated with chemotherapy had a significantly more than four times higher expression level of Nrf2 than new case patients (P < 0.05), while there was a decrease in the expression level of Bcl2 and Bcl-XL, which was not statistically significant. In other hands in relapsed patients, the expressions of Nrf2, Bcl2 and Bcl- XL were higher level than new case patients (p < 0.05) but this was less than patients treated with chemotherapy (p > 0.05). The high levels of mentioned genes may be associated with poor treatment response, chemoresistance and disease recurrence. Because of hyperactivation and overexpression of Nrf2 in leukemia, suggest that Nrf2 inhibitors could be used as a pharmacological target in combination with classical chemotherapeutic agents to increase the efficacy of anticancer therapy.

Enhancer-bound Nrf2 licenses HIF-1α transcription under hypoxia to promote cisplatin resistance in hepatocellular carcinoma cells.

Tumor microenvironment is hypoxic, which can cause resistance to chemotherapy, but the detailed mechanisms remain elusive. Here we find that mild hypoxia (5% O2) further increases cisplatin resistance in the already resistant HepG2/DDP but not the sensitive HepG2 cells. We find that Nrf2 is responsible for cisplatin resistance under hypoxia, as Nrf2 knockdown sensitizes HepG2/DDP cells while Nrf2 hyper-activation (though KEAP1 knockdown) increases resistance of HepG2 cells to cisplatin. Nrf2 binds to an enhancer element in the upstream of HIF-1α gene independently of hypoxia, promoting HIF-1α mRNA synthesis under hypoxic condition. As a result, Nrf2-dependent transcription counteracts HIF-1α degradation under mild hypoxia condition, leading to preferential cisplatin-resistance in HepG2/DDP cells. Our data suggest that Nrf2 regulation of HIF-1α could be an important mechanism for chemotherapy resistance in vivo.

Role of the KEAP1-NRF2 Axis in Renal Cell Carcinoma.

Simple SummaryRenal cancers are common types of tumors affecting humans. While many different genes have been found to be mutated and to drive the initiation and progression of these lethal cancers, a fine molecular understanding of the process is still lacking. One important pathway that emerges central in many different types of renal cancers is one called KEAP-NRF2. This axis is very important in normal kidneys as a defense against oxidative stress. Here, we summarize a large body of literature suggesting that this axis is exploited by tumor cells to escape control and to transform, and thus it could represent a good target for therapy.NRF2 is a transcription factor that coordinates the antioxidant response in many different tissues, ensuring cytoprotection from endogenous and exogenous stress stimuli. In the kidney, its function is essential in appropriate cellular response to oxidative stress, however its aberrant activation supports progression, metastasis, and resistance to therapies in renal cell carcinoma, similarly to what happens in other nonrenal cancers. While at the moment direct inhibitors of NRF2 are not available, understanding the molecular mechanisms that regulate its hyperactivation in specific tumor types is crucial as it may open new therapeutic perspectives. Here, we focus our attention on renal cell carcinoma, describing how NRF2 hyperactivation can contribute to tumor progression and chemoresistance. Furthermore, we highlight the mechanism whereby the many pathways that are generally altered in these tumors converge to dysregulation of the KEAP1-NRF2 axis.

3D Culture Models with CRISPR Screens Reveal Hyperactive NRF2 as a Prerequisite for Spheroid Formation via Regulation of Proliferation and Ferroptosis

Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.

Phase 1 Trial of MLN0128 (Sapanisertib) and CB-839 HCl (Telaglenastat) in Patients With Advanced NSCLC (NCI 10327): Rationale and Study Design

IntroductionThere are currently no approved targeted therapies for lung squamous-cell carcinoma (LSCC) and KRAS-mutant lung adenocarcinoma (LUAD). About 30% of LSCC and 25% of KRAS-mutant LUAD exhibit hyperactive NRF2 pathway activation through mutations in NFE2L2 (the gene encoding NRF2) or its negative regulator, KEAP1. Preclinical data demonstrate that these tumors are uniquely sensitive to dual inhibition of glycolysis and glutaminolysis via mammalian target of rapamycin (mTOR) and glutaminase inhibitors. This phase 1 study was designed to assess safety and preliminary activity of the mTOR inhibitor MLN0128 (sapanisertib) in combination with the glutaminase inhibitor CB-839 HCl. MethodsPhase 1 dose finding will use the queue-based variation of the 3 + 3 dose escalation scheme with the primary endpoint of identifying the recommended expansion dose. To confirm the acceptable tolerability of the recommended expansion dose, patients will subsequently enroll onto 1 of 4 expansion cohorts (n = 14 per cohort): (1) LSCC harboring NFE2L2 or (2) KEAP1 mutations, or (3) LUAD harboring KRAS/(KEAP1 or NFE2L2) coalterations, or (4) LSCC wild type for NFE2L2 and KEAP1. The primary endpoint of the dose expansion is to determine the preliminary efficacy of MLN0128/CB-839 combination therapy. ConclusionThis phase 1 study will determine the recommended expansion dose and preliminary efficacy of MLN0128 and CB-839 in advanced non–small-cell lung cancer with a focus on subsets of LSCC and KRAS-mutant LUAD harboring NFE2L2 or KEAP1 mutations.

A conditional mouse expressing an activating mutation in NRF2 displays hyperplasia of the upper gastrointestinal tract and decreased white adipose tissue.

Activation of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co-complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL-Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2-activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3-dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)-positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA-seq profiling and follow-up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2-regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14-restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

NRF2/ACSS2 axis mediates the metabolic effect of alcohol drinking on esophageal squamous cell carcinoma.

Alcohol drinking is a leading risk factor for the development of esophageal squamous cell carcinoma (ESCC). However, the molecular mechanisms of alcohol-associated ESCC remain poorly understood. One of the most commonly mutated genes in ESCC is nuclear factor erythroid 2 like 2 (NFE2L2 or NRF2), which is a critical transcription factor regulating oxidative stress response and drug detoxification. When NRF2 is hyperactive in cancer cells, however, it leads to metabolic reprogramming, cell proliferation, chemoradioresistance, and poor prognosis. In this study, hyperactive NRF2 was found to up-regulate acetyl-CoA synthetase short-chain family members 2 (ACSS2), an enzyme that converts acetate to acetyl-CoA, in ESCC cells and mouse esophagus. We also showed that knockdown of NRF2 or ACSS2 led to decreased ACSS2 expression, which in turn reduced the levels of acetyl-CoA and ATP with or without ethanol exposure. In addition, ethanol exposure enhanced lipid synthesis in ESCC cells. Moreover, we observed a change in the metabolic profile of ESCC cells exposed to ethanol as a result of their NRF2 or ACSS2 status. We further showed that ACSS2 contributed to the invasive capability of NRF2high ESCC cells exposed to ethanol. In conclusion, the NRF2/ACSS2 axis mediates the metabolic effect of alcohol drinking on ESCC.

Chronic arsenic exposure enhances metastatic potential via NRF2-mediated upregulation of SOX9

Chronic low dose arsenic exposure continues to be a worldwide health concern because of its prevalence and link to increased cancer risk, including non-small cell lung cancer (NSCLC). Mortality of NSCLC patients increases with the development of a metastatic lesion compared to when the tumor is localized; however, the exact mechanism for what causes NSCLC cells to metastasize in the context of environmental toxicant exposure has yet to be fully elucidated. One proposed contributor to metastasis in NSCLC is nuclear factor (erythroid-derived 2)-like 2 (NRF2), a transcription factor with known oncogenic properties that has proved to be critical for arsenic carcinogenesis. Here, we demonstrate that chronic arsenic exposure enhances the invasive and migratory capacity of immortalized lung epithelial cells via NRF2-dependent upregulation of SRY-box 9 (SOX9), another transcription factor linked with cell proliferation, epithelial-mesenchymal transition, and metastasis. We identified a functional antioxidant response element (ARE) in the promoter region of SOX9, suggesting that it is an NRF2 target gene, with mutation of the ARE preventing NRF2 binding. Pharmacological induction or inhibition of NRF2 increased or decreased SOX9 expression, respectively. Furthermore, we demonstrate that hyperactivation of NRF2 via knockout of Kelch-like ECH-associated protein 1 (KEAP1), its negative regulator, contributes to proliferation; while, inhibition of NRF2 or direct knockdown of SOX9 slowed the ability of NSCLC cells to proliferate, migrate, and invade. Overall, this study suggests that NRF2-mediated SOX9 upregulation can contribute to the metastatic potential of both environmentally and genetically driven lung tumors.

Small molecular Nrf2 inhibitors as chemosensitizers for cancer therapy.

The basic leucine zipper transcription factor Nrf2is the primary regulator of cellular oxidative stress. Activation of Nrf2 is regarded as a potential preventive and therapeutic strategy. However, aberrant hyperactivation of Nrf2 is found in a variety of cancers and promotes cancer progression and metastasis. Moreover, constitutive activation of Nrf2 confers cancer cells resistance to chemo- and radio-therapy. Thus, inhibiting Nrf2 could be a new therapeutic strategy for cancer. With the aim of accelerating the discovery and development of novel Nrf2 inhibitors, we summarize the biological and pathological functions of Nrf2 in cancer. Furthermore, the recent studies of small molecular Nrf2 inhibitors and potential Nrf2 inhibitory mechanisms are also summarized in this review.

Moderate Nrf2 Activation by Genetic Disruption of Keap1 Has Sex-Specific Effects on Bone Mass in Mice

Keap1 is a negative controller of the transcription factor Nrf2 for its activity. The Keap1/Nrf2 signaling pathway has been considered as a master regulator of cytoprotective genes, and exists in many cell types including osteoblasts and osteoclasts. Our previous study shows Nrf2 deletion decreases bone formation. Recent studies show hyperactivation of Nrf2 causes osteopenia in Keap1−/− mice, and Keap1−/− osteoblasts have significantly less proliferative potential than Keap1+/− osteoblasts. We aimed to examine if moderate Nrf2 activation by disruption of Keap1 impacts bone metabolism. We examined bone phenotype of Keap1 heterozygotic mice (Ht) in comparison with Keap1 wild type (WT) mice. Deletion or knockdown of Keap1 enhanced the gene expression of Nrf2, ALP and wnt5a in cultured primary osteoblasts compared to WT control. In male mice, compared with their age-matched littermate WT controls, Keap1 Ht mice showed significant increase in bone formation rate (+30.7%, P = 0.0029), but did not change the ultimate force (P < 0.01). The osteoclast cell numbers (−32.45%, P = 0.01) and surface (−32.58%, P = 0.03) were significantly reduced by Keap1 deficiency in male mice. Compared to male WT mice, serum bone resorption marker in male Keap1 Ht mice was significantly decreased. Our data suggest that moderate Nrf2 activation by disruption of Keap1 improved bone mass by regulating bone remodeling in male mice.

Dietary Supplementation with Fish Oil or Conjugated Linoleic Acid Relieves Depression Markers in Mice by Modulation of the Nrf2 Pathway.

Inflammation and oxidative stress play an important role in the pathogenesis of depressive disorders and nuclear erythroid related factor 2 (Nrf2), a regulator of RedOx homeostasis and inflammation, is a promising target for depression prevention/treatment. As fish oil (FO) and conjugated linoleic acid (CLA) are known Nrf2 inducers, their protective ability is comparatively evaluated in a murine model of depression (MRL/MpJ-Faslpr ). Oxidative stress, fatty acids content, and critical factors reflecting brain functioning-namely brain-derived neurotrophic factor (BDNF), synaptic markers, and cholinergic signaling-are preliminarily evaluated in the frontal cortex of 8-week (Young) and in 22-week old animals (Old), which are used as model of depression. These markers are measured in Old mice at the end of a 5-week pretreatment with FO or CLA (728 or 650mg kg-1 , respectively). Old mice exhibit disrupted Redox homeostasis, compensatory Nrf2 hyperactivation, lower docosaheaxaenoic acid (DHA), and lower BDNF and synaptic function proteins compared to Young mice. FO and CLA treatment relieves almost all the pathophysiological hallmarks at a level comparable to Young mice. Presented data provide the first evidence for the comparable efficacy of FO or CLA supplementation in preventing depression signs in Old MRL/lpr mice, likely through their ability of improving Nrf2-mediated antioxidant defenses.

Abstract 884: Interplay between NRF2 and GPX4 is critical for cancer cell survival in 3D spheroids

Abstract Antioxidants have historically been thought of as cancer preventative agents; however, increasing evidence in recent years indicate that antioxidants promote cancer cell survival. Using 3D spheroid models, our lab previously demonstrated that normal cells die in the inner layers of spheroids, whereas cancer cells survive. Multiple lines of evidence indicate that survival of inner cells within tumor spheroids is dependent on escape from apoptosis and reactive oxygen species (ROS)-induced cell death through upregulation of anti-apoptotic and antioxidant programs. While the mechanisms associated with apoptosis are well-established, there is relatively little understood about the cellular processes involved in ROS-induced cell death (origins of ROS, types of oxidative stress experienced, mechanisms of cell death, antioxidants involved etc.). Tumor cells with mutations in the nuclear factor erythroid 2-related factor 2 (NRF2) pathway that lead to hyperactivation of this transcription factor serve as an excellent model to study the role of antioxidant programs in the regulation of survival processes. NRF2 is the master regulator of the antioxidant response directly regulating expression of antioxidant enzymes as well as proteins required for the production of non-enzymatic antioxidants. Furthermore, mutations in the NRF2 pathway are frequent in tumor types that are associated with oxidative stress such as lung cancer. We found that knock-down of NRF2 affected cell survival and growth in 3D by regulating (i) cell proliferation and (ii) inner cell survival. To better understand the specific processes responsible for ROS-induced cell death and the antioxidant program(s) utilized by cancer cells to bypass these death programs, we performed CRISPR screens in 3D using lung cancer cell lines with NRF2 hyperactivation to identify downstream targets of NRF2 and/or antioxidants that collaborate with NRF2 to regulate cell survival and growth in 3D. As predicted, we observed drop-out of sgRNAs targeting NRF2. Notably, a top drop-out hit was glutathione peroxidase 4 (GPX4), an essential antioxidant enzyme that reduces membrane phospholipid hydroperoxides to prevent a form of non-apoptotic cell death known as ferroptosis. We found that inhibition of GPX4 resulted in elevated levels of lipid peroxidation and death in the inner cells of 3D spheroids which were rescuable by the ferroptosis inhibitor Ferrostatin-1, implicating ferroptotic cell death in 3D biology. Surprisingly, knock-down of NRF2 resulted in stabilization of GPX4 at the post-transcriptional level. Finally, knock-down of NRF2 combined with GPX4 inhibition induced cell death in both inner and outer cells of 3D spheroids. These data suggest that cancer cells exploit the functional interplay between NRF2 and GPX4 to survive in high oxidative stress environments. This work was supported by funding from the ROADS Program funded by F. Hoffmann-La Roche Ltd.. Note: This abstract was not presented at the meeting. Citation Format: Patricia Cho, Nobuaki Takahashi, Laura M. Selfors, Hendrik J. Kuiken, Joan S. Brugge. Interplay between NRF2 and GPX4 is critical for cancer cell survival in 3D spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 884.