Low Nrf2 Research Articles

Lipocalin-2 as a prognostic marker in patients with acute exacerbation of idiopathic pulmonary fibrosis

BackgroundLipocalin-2 (LCN2) is a secretory glycoprotein upregulated by oxidative stress; moreover, patients with idiopathic pulmonary fibrosis (IPF) have shown increased LCN2 levels in bronchoalveolar lavage fluid (BALF). This study aimed to determine whether circulatory LCN2 could be a systemic biomarker in patients with IPF and to investigate the role of LCN2 in a bleomycin-induced lung injury mouse model.MethodsWe measured serum LCN2 levels in 99 patients with stable IPF, 27 patients with acute exacerbation (AE) of IPF, 51 patients with chronic hypersensitivity pneumonitis, and 67 healthy controls. Further, LCN2 expression in lung tissue was evaluated in a bleomycin-induced lung injury mouse model, and the role of LCN2 was investigated using LCN2-knockout (LCN2 -/-) mice.ResultsSerum levels of LCN2 were significantly higher in patients with AE-IPF than in the other groups. The multivariate Cox proportional hazards model showed that elevated serum LCN2 level was an independent predictor of poor survival in patients with AE-IPF. In the bleomycin-induced lung injury mouse model, a higher dose of bleomycin resulted in higher LCN2 levels and shorter survival. Bleomycin-treated LCN2 -/- mice exhibited increased BALF cell and protein levels as well as hydroxyproline content. Moreover, compared with wild-type mice, LCN2-/- mice showed higher levels of circulatory 8-isoprostane as well as lower Nrf-2, GCLC, and NQO1 expression levels in lung tissue following bleomycin administration.ConclusionsOur findings demonstrate that serum LCN2 might be a potential prognostic marker of AE-IPF. Moreover, LCN2 expression levels may reflect the severity of lung injury, and LCN2 may be a protective factor against bleomycin-induced acute lung injury and oxidative stress.

Abstract 1212: Impact of KEAP1/STK11 co-mutations and NRF2 signaling on resistance to adagrasib in advanced NSCLC

Abstract Background: KRAS G12C inhibitors (G12Ci) are revolutionizing the therapeutic landscape of advanced NSCLC, but mechanisms of limited clinical efficacy observed in some patients (pts) merit continued exploration. In the largest cohort of advanced KRAS G12C-mutant (KG12C) NSCLC pts treated with G12Ci, we demonstrated KEAP1 and STK11 co-mutations are enriched in early progression to G12Ci. Yet the independent predictive role of KEAP1 and STK11 co-mutations and the impact of enhanced NRF2 signaling in clinical outcomes to G12Ci remains incompletely characterized. Methods: Clinico-genomic data were collected from 129 pts with advanced KG12C NSCLC treated with adagrasib (ada) (KRYSTAL-1 - NCT03785249). NRF2 signaling was estimated by NRF2 score (HTG transcriptome panel). Kaplan-Meier estimates of PFS and OS were assessed with log-rank test. Hazard ratios (HR) were estimated using Cox regression model. KG12C NSCLC cell lines and CDX/PDX models were used for pre-clinical experiments. Results KEAP1 and STK11 co-mutations were each associated with significantly shorter survival upon ada treatment (KEAP1: PFS 4.1 vs 9.9 mo, HR 2.7, p<0.01; OS 5.4 vs 19.0 mo, HR 3.6, p<0.01; STK11: PFS 4.2 vs 11.0 mo, HR 2.2, p<0.01; OS 9.8 mo vs not reached [NR], HR 2.6, p<0.01). KEAP1WT/STK11WT status identified a group of pts with significantly longer PFS (16.85 mo) and OS (NR) upon treatment with ada. In cell viability and colony formation assays, KEAP1 re-expression sensitized all KEAP1MUT/STK11MUT KG12C NSCLC cell lines to ada treatment. Similar results were observed with STK11/LKB1 re-expression. KEAP1MUT was linked to limited response to ada across a broad panel of NSCLC PDX/CDX. In vivo drug anchored CRISPR screens in the ada-sensitive LU99 cell line identified KEAP1 as the most significant gene promoting resistance to ada. KEAP1 KO in the LU99 model determined early onset of ada resistance. In vitro drug screens showed high synergy scores and higher anti-tumor activity for mTOR inhibitor (mTORi) and ada combinations in KG12C KEAP1MUT/STK11MUT NSCLC. High NRF2 score was associated with significantly shorter survival with ada (PFS: 4.2 vs 8.4 mo, HR 2.0, p=0.02; OS: 6.5 vs 19.0 mo, HR 2.8, p<0.01) even in KEAP1WT NSCLC pts. Integration of KEAP1WT, STK11WT and low NRF2 score status identified pts - 32% - with significant and meaningfully longer survival when treated with ada (PFS 12.0 vs 4.2 mo, HR 0.2, P<0.01; OS NR vs 8.0 mo, HR 0.1, p<0.01). Conclusions: Co-mutations in KEAP1 and STK11 and NRF2 signaling define a subgroup of KG12C NSCLC pts with markedly distinct outcomes upon treatment with ada. The mTORi and ada combination shows high efficacy for targeting KG12C NSCLC harboring KEAP1 and STK11 co-mutations. The clinical safety and efficacy of mTORi nab-sirolimus and ada will be determined in the ongoing KRYSTAL-19 trial (NCT05840510). Citation Format: Marcelo V. Negrao, David Molkentine, Alvaro G. Paula, Laura Hover, Monique Nilsson, Natalie Vokes, Lars Engstrom, Andrew Calinisan, Laura Waters, David M. Briere, Jill Hallin, George R. Blumenschein, Ferdinandos Skoulidis, Scott E. Kopetz, David S. Hong, Don L. Gibbons, Peter Olson, James Christensen, John V. Heymach. Impact of KEAP1/STK11 co-mutations and NRF2 signaling on resistance to adagrasib in advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1212.

Plasma Concentrations of High Mobility Group Box 1 Proteins and Soluble Receptors for Advanced Glycation End-Products Are Relevant Biomarkers of Cognitive Impairment in Alcohol Use Disorder: A Pilot Study.

Alcohol use disorder (AUD) is a major component in the etiology of cognitive decline and dementia. Underlying mechanisms by which long-term alcohol abuse causes cognitive dysfunction include excessive oxidative stress and inflammation in the brain, activated by increased reactive oxygen/nitrogen species (ROS/RNS), advanced glycation end-products (AGEs) and high-mobility group box 1 protein (HMGB1). In a pilot study, we examine the potential clinical value of circulating biomarkers of oxidative stress including ROS/RNS, HMGB1, the soluble receptor for AGE (sRAGE), the brain biomarker of aging apolipoprotein D (ApoD), and the antioxidant regulator nuclear factor erythroid 2-related factor 2 (NRF2) as predictive indices for cognitive impairment (CI) in abstinent patients with AUD (n = 25) compared to patients with established Alzheimer's disease (AD, n = 26) and control subjects (n = 25). Plasma concentrations of sRAGE were evaluated with immunoblotting; ROS/RNS with a fluorometric kit; and HMGB1, ApoD, and NRF2 by ELISA. Abstinent AUD patients had higher sRAGE, ROS/RNS (p < 0.05), and ApoD (p < 0.01) concentrations, similar to those of AD patients, and lower NRF2 (p < 0.01) concentrations, compared to controls. These changes were remarkable in AUD patients with CI. HMGB1, and sRAGE correlated positively with duration of alcohol use (rho = 0.398, p = 0.022; rho = 0.404, p = 0.018), whereas sRAGE correlated negatively with periods of alcohol abstinence (rho = -0.340, p = 0.045). A predictive model including ROS/RNS, HMGB1, sRAGE, alcohol use duration, and alcohol abstinence periods was able to differentiate AUD patients with CI (92.3% of correct predictions, ROC-AUC= 0.90) from those without CI. In conclusion, we propose ROS/RNS, HMGB1, and sRAGE as stress biomarkers capable of predicting cognitive impairment in AUD patients.

Abstract B004: Molecular crosstalk between NF-κB and NRF2 signaling affects prognosis in HPV-associated head and neck cancer

Abstract The incidence of head and neck squamous cell carcinoma (HNSCC) is on the rise and is expected to increase by 50% by 2030. Around half of the patient population affected are diagnosed when the tumor is at a locally advanced stage. These patients are usually treated with intense radiotherapy, leading to severe side-effects. Precision medicine would revolutionize the treatment of head and neck cancer by accurately identifying patients that are at low risk of recurrence, reduce treatment related side-effects and aid in overall improvement in quality of life of patients after treatment. This generates the need to develop biomarkers to identify patients with less aggressive tumors as candidates for treatment de-escalation. We applied an unbiased approach (weighted gene correlation network analysis (WGCNA)) that allows detection of autocorrelated gene sets on 3 independent cohorts of HPV+ HNSCC and created 22 consensus transcriptional modules by selecting genes that grouped together in WGCNA analyses from all 3 cohorts. Interestingly, only 1 module intrinsically divided HPV+ HNSCC into 2 subtypes, displaying different mutational profiles, mutational signatures, HPV gene expression patterns, HPV integration status, and patient survival. Gene set enrichment analysis revealed that this module was enriched in NF-kB genes and strongly associated with NF-κB signaling, confirming our prior findings that defined HPV+ HNSCC subtypes by presence or absence of NF-kB regulators, TRAF3 or CYLD defects and by the NF-κB activity classifier. We hypothesized that NF-kB-driven intrinsic tumor characteristics contribute to increased sensitivity of NF-kB active HPV+ head and neck tumors to radiation, providing patients survival benefits. Indeed, TRAF3 or CYLD deletion dramatically increased radiation sensitivity of HPV+ head and neck cancer cells. As we began to investigate mechanisms of radiation sensitization associated with TRAF3 and CYLD deletion, we found that activation of NF-κB significantly correlated with marked downregulation of nuclear factor erythroid 2-related factor 2 (NRF2) activity in tumors from 3 independent cohorts, as well as in HPV+ HNSCC cells that harbor constitutively active NF-kB due to deletion of TRAF3 or CYLD. Interestingly, TRAF3 CRISPR KO cells had lower NRF2 protein levels that were restored by MG132 treatment, indicating an involvement of KEAP1/CUL3 mediated proteasomal degradation of NRF2. In summary, our data showcases an inverse correlation between NF-kB and NRF2 pathways in HPV+ HNSCC. Currently, there are no prognostic biomarkers to distinguish patients who require aggressive therapy versus those that are appropriate for reduced intensity treatment. Identification of markers distinguishing the 2 subtypes of HPV+ HNSCC (high NF-kB and low NRF2 activity) may serve as prognostic biomarkers to help clinicians with therapeutic decisions. Citation Format: Aditi Kothari, Travis Schrank, Wendell Yarbrough, Natalia Isaeva. Molecular crosstalk between NF-κB and NRF2 signaling affects prognosis in HPV-associated head and neck cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B004.

Human papillomavirus-driven repression of NRF2 signalling confers chemo-radio sensitivity and predicts prognosis in head and neck squamous cell carcinoma

PurposeTo investigate the role of NRF2 signalling in conferring superior prognosis in patients with HPV positive (HPV+ve) head & neck squamous cell carcinomas (HNSCC) compared to HPV negative (HPV−ve) HNSCC and develop molecular markers for selection of HPV+ve HNSCC patients for treatment de-escalation trials. MethodsNRF2 activity (NRF2, KEAP1, and NRF2-transcriptional targets), p16, and p53 levels between HPV+ve HNSCC and HPV−ve HNSCC in prospective and retrospective tumor samples as well as from TCGA database were compared. Cancer cells were transfected with HPV-E6/E7 plasmid to elucidate if HPV infection represses NRF2 activity and sensitizes to chemo-radiotherapy. ResultsProspective analysis revealed a marked reduction in expression of NRF2, and its downstream genes in HPV+ve tumors compared to HPV−ve tumors. A retrospective analysis by IHC revealed significantly lower NQO1 in p16high tumors compared to p16low tumors and the NQO1 expression correlated negatively with p16 and positively with p53. Analysis of the TCGA database confirmed low constitutive NRF2 activity in HPV+ve HNSCC compared to HPV−ve HNSCC and revealed that HPV+ve HNSCC patients with ‘low NQO1’ expression showed better overall survival compared to HPV+ve HNSCC patients with ‘high NQO1’ expression. Ectopic expression of HPV-E6/E7 plasmid in various cancer cells repressed constitutive NRF2 activity, reduced total GSH, increased ROS levels, and sensitized the cancer cells to cisplatin and ionizing radiation. ConclusionLow constitutive NRF2 activity contributes to better prognosis of HPV+ve HNSCC patients. Co-expression of p16high, NQO1low, and p53low could serve as a predictive biomarker for the selection of HPV + ve HNSCC patients for de-escalation trials.

Prognostic Worth of Nrf2/BACH1/HO-1 Protein Expression in the Development of Breast Cancer

Objectives: Nrf2/BACH1/HO-1 proteins have been implicated in the development and progression of tumors. However, their clinical relevance in breast cancer remains unclear and understudied. This study evaluated Nrf2/BACH1/HO-1 protein expression and its relationship with age, tumor grade, tumor stage, TNM, ER, PR, HER2, and histologic type. Methods: 114 female breast cancer and 30 noncancerous tissues were evaluated for Nrf2/BACH1/HO-1 protein expression using immunohistochemistry and Western blot. The relationships between the expression and clinicopathologic factors were assessed using the χ² test. Results: 74% of the cancerous samples had high Nrf2 protein expression, and 26% of them had low Nrf2 protein expression. Regarding the non-cancer samples, 43% had high Nrf2 protein expression and 57% had low Nrf2 protein expression (p < 0.002). 39% of the cancerous samples had high BACH1 protein expression, and 61% had low BACH1 protein expression. For the non-cancer samples, 80% had high BACH1 protein expression and 20% had low BACH1 protein expression (p < 0.031). 67% of the cancerous samples had high HO-1 protein expression, and 33% had low HO-1 protein expression. However, for the non-cancer samples, 17% of them had high HO-1 protein expression and 83% had low HO-1 protein expression (p < 0.001). The expression of Nrf2 and HO-1 significantly correlated with tumor grade, while BACH1 was significantly associated with tumor stage (p < 0.05). Conclusion: Nrf2, BACH1, and HO-1 could be explored as a biomarker for cancer stage, progression, and prognosis.

In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls-Part 2: Adaptive Response.

Oxidized in vitro genomic DNA (gDNA) is known to launch an adaptive response in human cell cultures. The cfDNA extracted from the plasma of schizophrenic patients (sz-cfDNA) and healthy controls (hc-cfDNA) contains increased amounts of 8-oxodG, a DNA-oxidation marker. The aim of the research was answering a question: can the human cfDNA isolated from blood plasma stimulate the adaptive response in human cells? In vitro responses of ten human skin fibroblasts (HSFs) and four peripheral blood mononuclear cell (PBMC) lines after 1-24 h of incubation with sz-cfDNA, gDNA and hc-cfDNA containing different amounts of 8-oxodG were examined. Expressions of RNA of eight genes (NOX4, NFE2L2, SOD1, HIF1A, BRCA1, BRCA2, BAX and BCL2), six proteins (NOX4, NRF2, SOD1, HIF1A, γH2AX and BRCA1) and DNA-oxidation marker 8-oxodG were analyzed by RT-qPCR and flow cytometry (when analyzing the data, a subpopulation of lymphocytes (PBL) was identified). Adding hc-cfDNA or sz-cfDNA to HSFs or PBMC media in equal amounts (50 ng/mL, 1-3 h) stimulated transient synthesis of free radicals (ROS), which correlated with an increase in the expressions of NOX4 and SOD1 genes and with an increase in the levels of the markers of DNA damage γH2AX and 8-oxodG. ROS and DNA damage induced an antioxidant response (expression of NFE2L2 and HIF1A), DNA damage response (BRCA1 and BRCA2 gene expression) and anti-apoptotic response (changes in BAX and BCL2 genes expression). Heterogeneity of cells of the same HSFs or PBL population was found with respect to the type of response to (sz,hc)-cfDNA. Most cells responded to oxidative stress with an increase in the amount of NRF2 and BRCA1 proteins along with a moderate increase in the amount of NOX4 protein and a low amount of 8-oxodG oxidation marker. However, upon the exposure to (sz,hc)-cfDNA, the size of the subpopulation with apoptosis signs (high DNA damage degree, high NOX4 and low NRF2 and BRCA1 levels) also increased. No significant difference between the responses to sz-cfDNA and hc-cfDNA was observed. Sz-cfDNA and hc-cfDNA showed similarly high bioactivity towards fibroblasts and lymphocytes. Conclusion: In cultured human cells, hc-cfDNA and sz-cfDNA equally stimulated an adaptive response aimed at launching the antioxidant, repair, and anti-apoptotic processes. The mediator of the development of the adaptive response are ROS produced by, among others, NOX4 and SOD1 enzymes.

The nuclear factor erythroid 2-related factor 2 agonist tert-butylhydroquinone improves bone marrow mesenchymal stromal cell function in prolonged isolated thrombocytopenia after allogeneic haematopoietic stem cell transplantation.

Prolonged isolated thrombocytopenia (PT) is a life-threatening comorbidity associated with allogeneic haematopoietic stem cell transplantation (allo-HSCT). Our previous study indicated that dysfunctional bone marrow mesenchymal stromal cells (BM MSCs) played a role in PT pathogenesis and that reactive oxygen species (ROS) accumulation was related to BM MSC senescence and apoptosis. However, the mechanism of the increase in ROS levels in the BM MSCs of PT patients is unknown. In the current case-control study, we investigated whether nuclear factor erythroid 2-related factor 2 (NRF2), which is a central regulator of the cellular anti-oxidant response that can clear ROS in human BM MSCs, was associated with PT after allo-HSCT. We evaluated whether an NRF2 agonist (tert-butylhydroquinone, TBHQ) could enhance BM MSCs from PT patients in vitro. We found that BM MSCs from PT patients exhibited increased ROS levels and reduced NRF2 expression. Multivariate analysis showed that low NRF2 expression was an independent risk factor for primary PT [p=0.032, Odds ratio (OR) 0.868, 95% confidence interval (CI) 0.764-0.988]. In-vitro treatment with TBHQ improved the quantity and function of BM MSCs from PT patients by downregulating ROS levels and rescued the impaired BM MSC support of megakaryocytopoiesis. In conclusion, these results suggested that NRF2 downregulation in human BM MSCs might be involved in the pathogenesis of PT after allo-HSCT and that BM MSC impairment could be improved by NRF2 agonist in vitro. Although further validation is needed, our data indicate that NRF2 agonists might be a potential therapeutic approach for PT patients after allo-HSCT.

Oxidative Stress Suppresses Nrf2 Protein Synthesis through Global Protein Synthesis Inhibition

Oxidative and electrophilic stresses are common aggressors in chronic diseases ranging from arthritis to cancer to neurodegenerative disease. The transcription factor Nrf2 is an essential mediator of the cell’s cytoprotective response to these stressors. Nrf2 is constantly synthesized, ubiquitinated, and degraded with a short half‐life of 20 minutes that maintains low basal Nrf2 protein levels. Electrophiles and oxidative stress cause Nrf2 to accumulate and subsequently to activate cytoprotective genes by modifying cysteines of the Keap1 protein, the repressor that targets Nrf2 for ubiquitination. We previously found that paradoxically, reactive oxygen species (ROS) stimulate the production of cytoprotective proteins, but inhibit Nrf2 protein synthesis, revealing a “Dr. Jekyll and Mr. Hyde” effect of ROS on Nrf2 accumulation. We tested the hypothesis that oxidative conditions that activate the Nrf2 pathway simultaneously inhibit global protein synthesis, thereby suppressing Nrf2 protein synthesis. We investigated both ROS generated by redox cycling of the oxidizable phenol, di‐tert‐butylhydroquinone (dtBHQ), and direct addition of H2O2 to HaCaT keratinocyte cells. We found that 100 μM to 400 μM H2O2 and 50 μM dtBHQ inhibit both Nrf2 protein synthesis and global protein synthesis. Addition of antioxidants, such as the manganese porphyrin MnTMPyP, rescued both Nrf2‐specific and global protein synthesis. Therefore, antioxidants such as MnTMPyP that mitigate the suppressive effects of ROS on global protein synthesis during Nrf2 activation could enhance the Nrf2/cytoprotective response under conditions of electrophilic and oxidative stress.

Brusatol inhibits the invasion and migration of pancreatic cancer cells by suppressing the NRF2/NF-κB/STAT3 signal cascade

• Pancreatic cancer-and immune-related pathways are enriched in tumors with high NFE2L2 expression. • Brusatol could inhibit invasion and migration of pancreatic cancer cells. • Reduced NRF2 expression results in suppressed invasion and migration of pancreatic cancer cells via the NF-κB/STAT3 pathway. • Brusatol could reverse gemcitabine-induced activation of the NF-κB/STAT3 pathway. The previous study has established that brusatol exhibits growth inhibitory and pro-apoptotic effects. However, whether brusatol can suppress the invasion and migration of pancreatic cancer cells remains unclear. Information in The Cancer Genome Atlas database indicates that NRF2 expression is tightly associated with the status of lymphatic metastasis and overall survival. Gene set enrichment analysis has also revealed that the JAK-STAT pathway could be suppressed by brusatol and is significantly enriched for phenotypes associated with high expression of the NRF2 gene. Therefore, we speculated and confirmed that brusatol can inhibit cell invasion and migration via the NRF2 pathway. Additionally, low NRF2 gene expression results in a function similar to that of brusatol as well as decreased expression of NF-κB and p-STAT3. Furthermore, brusatol reversed gemcitabine-induced overexpression of NF-κB and p-STAT3. Together, our results suggest that brusatol can inhibit the invasion and migration of pancreatic cancer cells through NRF2/NF-κB/STAT3 signaling cascade.

The Age-Specific Features and Clinical Significance of NRF2 and MAPK10 Expression in HCC Patients

BackgroundNuclear factor (erythroid-derived 2)-like 2 (NRF2) functions decline with age; however, cancer cells can hijack its pathways to ensure survival and aggressiveness. Yet, the role of NRF2 in hepatocellular carcinoma (HCC) is rarely investigated in an age-specific manner. This study investigates the expression of NRF2 and its activator (MAPK10) in different age groups of HCC patients, in addition to the age-specific features of NRF2 and MAPK10 interaction and their clinical significance.MethodsTumor and near-tumor tissue samples of 181 HCC patients were used to complete a protein expression analysis of NRF2 and MAPK10. Patients’ survival and clinical data were collected for clinical analysis. Global databases (TCGA, ICGC) were used to collect MAPK10 genetic mutation and mRNA expression data in patients with HCC, colorectal, stomach, and pancreatic cancers.ResultsOur findings revealed an increase in NRF2 protein expression but only in younger HCC patients, along with a decline in MAPK10 ability to activate NRF2 in older patients. We also found an increased MAPK10 genetic mutation rate and decreased mRNA expression in older patients. Low MAPK10 and NRF2 expression levels were associated with shorter survival and poorer prognosis due to positive correlation with microvascular invasion, tumor thrombus, elevated AFP levels, and larger tumor size.ConclusionNRF2 expression and oxidative stress mechanism in HCC patients are influenced by age. This magnifies the need to consider patients’ age in treatment strategies and guidelines and re-evaluates the application of studies’ age-standardized findings in older patients who are usually excluded from relevant research.

Removal of oxidative stress and genotoxic activities during drinking water production by ozonation and granular activated carbon filtration

BackgroundBioanalytical tools have been shown to be useful in drinking water quality assessments. Here, we applied a panel of in vitro bioassays to assess the treatment efficiency of two pilot-scale treatments: ozonation and granular activated carbon (GAC) filtration at a drinking water treatment plant (DWTP). The pilot-scale systems were studied alongside a full-scale treatment process consisting of biological activated carbon (BAC) filtration, UV disinfection, and monochloramine dosing. Both systems were fed the same raw water treated with coagulation/flocculation/sedimentation and sand filtration. The endpoints studied were oxidative stress (Nrf2 activity), genotoxicity (micronuclei formations), aryl hydrocarbon receptor (AhR) activation, as well as estrogen receptor (ER) and androgen receptor (AR) activity.ResultsNrf2, AhR, and ER activities and genotoxic effects were detected in the incoming raw water and variability was observed between the sampling events. Compared to most of the samples taken from the full-scale treatment system, lower Nrf2, AhR, and ER bioactivities as well as genotoxicity were observed in all samples from the pilot-scale systems across all sampling events. The most pronounced treatment effect was a 12-fold reduction in Nrf2 activity and a sixfold decrease in micronuclei formations following ozonation alone. GAC filtration alone resulted in sevenfold and fivefold reductions in Nrf2 activity and genotoxicity, respectively, in the same sampling event. Higher bioactivities were detected in most samples from the full-scale system suggesting a lack of treatment effect. No androgenic nor anti-androgenic activities were observed in any sample across all sampling events.ConclusionsUsing effect-based methods, we have shown the presence of bioactive chemicals in the raw water used for drinking water production, including oxidative stress, AhR and ER activities as well as genotoxicity. The currently used treatment technologies were unable to fully remove the observed bioactivities. Ozonation and GAC filtration showed a high treatment efficiency and were able to consistently remove the bioactivities observed in the incoming water. This is important knowledge for the optimization of existing drinking water treatment designs and the utilization of alternative treatment technologies.

Perspectives in liver redox imbalance: Toxicological and pharmacological aspects underlying iron overloading, nonalcoholic fatty liver disease, and thyroid hormone action.

Oxidative stress is an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control, and/or molecular damage altering cellular functions. This redox imbalance may trigger different responses depending on the antioxidant potential of a given cell, the level of reactive oxygen/nitrogen species (ROS/RNS) attained and the time of exposure, with protective effects being induced at low ROS/RNS levels in acute or short-term conditions, and harmful effects after high ROS/RNS exposure in prolonged situations. Relevant conditions underlying liver redox imbalance include iron overload associated with ROS production via Fenton chemistry and the magnitude of the iron labile pool achieved, with low iron exposure inducing protective effects related to nuclear factor-κB, signal transducer and activation of transcription 3, and nuclear factor erythroid-related factor 2 (Nrf2) activation and upregulation of ferritin, hepcidin, acute-phase response and antioxidant components, whereas high iron exposure causes drastic oxidation of biomolecules, mitochondrial dysfunction, and cell death due to necrosis, apoptosis and/or ferroptosis. Redox imbalance in nonalcoholic fatty liver disease (NAFLD) is related to polyunsaturated fatty acid depletion, lipogenic factor sterol regulatory element-binding protein-1c upregulation, fatty acid oxidation-dependent peroxisome proliferator-activated receptor-α downregulation, low antioxidant factor Nrf2 and insulin resistance, a phenomenon that is exacerbated in nonalcoholic steatohepatitis triggering an inflammatory response. Thyroid hormone (T3 ) administration determines liver preconditioning against ischemia-reperfusion injury due to the redox activation of several transcription factors, AMP-activated protein kinase, unfolded protein response and autophagy. High grade liver redox imbalance occurring in severe iron overload is adequately handled by iron chelation, however, that underlying NAFLD/NASH is currently under study in several Phase II and Phase III trials.

NRF2 Activation Promotes Aggressive Lung Cancer and Associates with Poor Clinical Outcomes.

Stabilization of the transcription factor NRF2 through genomic alterations in KEAP1 and NFE2L2 occurs in a quarter of patients with lung adenocarcinoma and a third of patients with lung squamous cell carcinoma. In lung adenocarcinoma, KEAP1 loss often co-occurs with STK11 loss and KRAS-activating alterations. Despite its prevalence, the impact of NRF2 activation on tumor progression and patient outcomes is not fully defined. We model NRF2 activation, STK11 loss, and KRAS activation in vivo using novel genetically engineered mouse models. Furthermore, we derive a NRF2 activation signature from human non-small cell lung tumors that we use to dissect how these genomic events impact outcomes and immune contexture of participants in the OAK and IMpower131 immunotherapy trials. Our in vivo data reveal roles for NRF2 activation in (i) promoting rapid-onset, multifocal intrabronchiolar carcinomas, leading to lethal pulmonary dysfunction, and (ii) decreasing elevated redox stress in KRAS-mutant, STK11-null tumors. In patients with nonsquamous tumors, the NRF2 signature is negatively prognostic independently of STK11 loss. Patients with lung squamous cell carcinoma with low NRF2 signature survive longer when receiving anti-PD-L1 treatment. Our in vivo modeling establishes NRF2 activation as a critical oncogenic driver, cooperating with STK11 loss and KRAS activation to promote aggressive lung adenocarcinoma. In patients, oncogenic events alter the tumor immune contexture, possibly having an impact on treatment responses. Importantly, patients with NRF2-activated nonsquamous or squamous tumors have poor prognosis and show limited response to anti-PD-L1 treatment.

NRF2 mRNA Expression Signature Associated with Regional Nodal Recurrence in Early-stage Non-small Cell Lung Cancer (NSCLC)

Lung stereotactic body radiation therapy (SBRT) achieves high local control in the treatment of early-stage non-small cell lung cancer (NSCLC). Nevertheless, up to 15-20% of patients may have regional nodal or distant recurrence after treatment. Molecular profiling may allow for personalized identification of patients at highest risk for recurrence. To this end, we conducted a transcriptomic analysis of lung SBRT samples with focus on the NRF2 pathway, which is involved in antioxidant regulation and has been associated with recurrence and survival in a variety of cancers. We studied the association of a NRF2 pathway mRNA expression profile with recurrence and survival. We identified patients with T1-3N0 NSCLC treated with SBRT at our institution. We isolated total RNA from formalin fixed paraffin embedded pretreatment tumor biopsy samples using an RNA Purification kit. Expression of mRNAs was analyzed using an assay. We used a published 20-mRNA risk score based on NRF2 pathway genes and dichotomized patients into high- and low-NRF2 groups using the median risk score value of the dataset. Kaplan-Meier analysis was used to study the association of the high NRF2 score with recurrence and survival. Cox proportional hazards multivariable analysis was used to account for histology, age, T-stage, and ECOG performance status. We identified 92 patients treated with lung SBRT with available mRNA data. There were no significant differences in age, sex, race, performance status, T-stage, histology, biologically effective dose, or central vs peripheral tumor location between high NRF2 (n = 46) and low NRF2 (n = 46) cohorts. There was significantly worse regional nodal recurrence in high- vs low-NRF2 patients (HR = 1.69; 95% CI 10.13 – 25.8; p = 0.035). There was no statistically significant difference in local recurrence (HR = 1.78; 95% CI 0.42 – 7.56; p = 0.43), distant recurrence (HR = 1.19; 95% CI 0.74 – 1.93; p = 0.48), or overall survival (HR = 1.67; 95% CI -.88 – 3.15; p = 0.12). On multivariable analysis, the association of high NRF2 score with regional nodal recurrence persisted (HR = 4.5; 95% CI 1.02 – 26.6; p = 0.046). There was no significant association of histology, age, T-stage, or ECOG performance status with regional nodal recurrence on multivariable analysis. We validated a published NRF2 mRNA expression signature, and found a high NRF2 signature was associated with regional nodal recurrence in patients with early-stage NSCLC treated with lung SBRT. This risk score may help to identify patients who would benefit from more aggressive mediastinal staging. These findings should be validated in an independent cohort.

Role of PDK4 in insulin signaling pathway in periadrenal adipose tissue of pheochromocytoma patients.

Studies have shown that pheochromocytoma (PHEO) is associated with glucose intolerance and decreased insulin sensitivity. In adipocytes, pyruvate dehydrogenase kinase 4 (PDK4) is involved in glucose uptake. However, very little is known about the role of PDK4 in the insulin signaling pathway in the adipose tissue of PHEO patients. We analyzed the expression of adipokines, oxidative stress-related genes, PDK4, phosphorylated AMPK (pAMPK) and phosphorylated IRS1 (pIRS1) in the periadrenal adipose tissue (peri-A) of patients with PHEO and non-functioning adrenal adenoma (NFA). We also investigated the effects of epinephrine on PDK4, pAMPK and pIRS1 in human stromal vascular fraction (SVF) cells, mouse 3T3-L1 preadipocytes and brown preadipocytes. PHEO patients had higher mRNA levels of PGC1α, C/EBPα, C/EBPβ, COXII and AP2 and lower mRNA levels of PPARγ in their peri-A than NFA patients. Decreased pAMPK and increased PDK4 and pIRS1 were observed in the peri-A of PHEO patients. PHEO patients also had significantly higher NOX4 protein expression and lower Nrf2 and HO-1 protein expression in their peri-A than NFA patients. In vitro, epinephrine treatment upregulated PDK4 expression, inhibited AMPK phosphorylation and enhanced IRS1 phosphorylation. The knockdown of PDK4 by siRNA upregulated pAMPK and downregulated pIRS1. In conclusion, PDK4 may play an essential role in hypercatecholamine-induced insulin resistance in the periadrenal adipose tissues of PHEO patients.

Association of single nucleotide polymorphisms in the NRF2 promoter with vascular stiffness with aging

Pulse wave velocity (PWV), an indicator of vascular stiffness, increases with age and is increasingly recognized as an independent risk factor for cardiovascular disease (CVD). Although many mechanical and chemical factors underlie the stiffness of the elastic artery, genetic risk factors related to age-dependent increases in PWV in apparently healthy people are largely unknown. The transcription factor nuclear factor E2 (NF-E2)-related factor 2 (Nrf2), which is activated by unidirectional vascular pulsatile shear stress or oxidative stress, regulates vascular redox homeostasis. Previous reports have shown that a SNP in the NRF2 gene regulatory region (-617C>A; hereafter called SNP-617) affects NRF2 gene expression such that the minor A allele confers lower gene expression compared to the C allele, and it is associated with various diseases, including CVD. We aimed to investigate whether SNP-617 affects vascular stiffness with aging in apparently healthy people. Analyzing wide-ranging data obtained from a public health survey performed in Japan, we evaluated whether SNP-617 affected brachial-ankle PWV (baPWV) in never-smoking healthy subjects (n = 642). We also evaluated the effects of SNP-617 on other cardiovascular and blood test measurements. We have shown that not only AA carriers (n = 55) but also CA carriers (n = 247) show arterial stiffness compared to CC carriers (n = 340). Furthermore, SNP-617 also affected blood pressure indexes such as systolic blood pressure and mean arterial pressure but not the ankle brachial pressure index, an indicator of atherosclerosis. Multivariate analysis showed that SNP-617 accelerates the incremental ratio of baPWV with age. This study is the first to show that SNP-617 affects the age-dependent increase in vascular stiffness. Our results indicate that low NRF2 activity induces premature vascular aging and could be targeted for the prevention of cardiovascular diseases associated with aging.

NF-E2-Related Factor 2 Regulates Interferon Receptor Expression and Alters Macrophage Polarization in Lupus.

Pristane-induced lupus is associated with nonresolving inflammation and deficiency of proresolving macrophages. Proresolving nonclassic macrophages (NCMs) are less responsive to type I interferon (IFN) than classic macrophages (CMs; which are proinflammatory), reflecting their relative expression levels of the type I IFN receptor (IFNAR). This study was undertaken to investigate the regulation of IFNAR expression in macrophages. We carried out gene expression profiling of purified CMs and NCMs from mice treated with pristane (which develop lupus) or mineral oil (non-lupus controls). Macrophage differentiation and IFNAR expression were examined in mice treated with NF-E2-related factor 2 (Nrf2) activators and inhibitors and in Nrf2-deficient mice. Nrf2 activity was also assessed in blood cells from patients with systemic lupus erythematosus (SLE). Significant differences were determined by Student's t-test. RNA sequencing revealed increased expression of genes regulated by the transcription factor Nrf2 in NCMs from mineral oil-treated versus pristane-treated mice and in NCMs versus CMs. The Nrf2 activator CDDO-imidazole (CDDO-Im) decreased CMs (P < 0.0001) and promoted the development of proresolving NCMs (P = 0.06), whereas the Nrf2 inhibitor brusatol increased CMs (P < 0.05) and decreased NCMs (P < 0.001). CDDO-Im decreased Ifnar1 (P < 0.001) and IFN-stimulated gene (ISG) expression in macrophages and alleviated oxidative stress (P < 0.05), whereas brusatol had the opposite effect (P < 0.01). Moreover, Ifnar1 and ISG expression levels were higher in Nrf2-knockout mice than controls (P < 0.05). As seen in mice with lupus, SLE patients showed evidence of low Nrf2 activity. Our findings indicate that Nrf2 activation favors the resolution of chronic inflammation in lupus. Since autoantibody production and lupus nephritis depend on IFNAR signaling, the ability of Nrf2 activators to repolarize macrophages and reduce the INF signature suggests that these agents may warrant consideration for treating lupus.

Impact of Gene Expression Associated with Glucocorticoid-Induced Transcript 1 (GLCCI1) on Severe Asthma and Future Exacerbation.

Genetic variations in glucocorticoid-induced transcript 1 (GLCCI1) have been associated with the response to corticosteroid treatment. However, the associations of GLCCI1 polymorphisms or gene expression with the prognosis of asthma and pathophysiological factors related to steroid insensitivity remain unclear. We sought to investigate the associations of GLCCI1, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and histone deacetylase 2 (HDAC2) mRNA expression levels and the GLCCI1 rs37973 polymorphism with asthma severity and future exacerbation in patients with asthma. Subjects included 25 patients with severe asthma and 127 patients with nonsevere asthma. mRNA expression levels in peripheral blood mononuclear cells were measured and evaluated as predictors of severe asthma using receiver operating characteristic (ROC) analysis. The hazard ratios of the mRNA expression levels for time to first exacerbation in the 1-year follow-up period were calculated. GLCCI1, Nrf2, and HDAC2 mRNA expression levels were significantly lower in patients with severe asthma than in patients with nonsevere asthma and could predict severe asthma with an area under the ROC curve of 0.68, 0.71, and 0.65, respectively. In contrast, no relationship was found between the GLCCI1 rs37973 polymorphism and severe asthma. The hazard ratios for asthma exacerbation in patients with low GLCCI1, Nrf2, and HDAC2 mRNA expression levels were 3.24 (95% confidence interval, 1.42-7.40), 3.13 (1.37-7.16), and 2.98 (1.22-7.25), respectively. Patients with severe asthma could be distinguished by lower GLCCI1, Nrf2, and HDAC2 mRNA levels in peripheral blood cells, and all of these gene signatures could predict future asthma exacerbations.

Activation of Nrf2/HO-1 Pathway and Human Atherosclerotic Plaque Vulnerability:an In Vitro and In Vivo Study.

Reactive oxygen species (ROS) induce nuclear factor erythroid 2–related factor 2 (Nrf2) activation as an adaptive defense mechanism, determining the synthesis of antioxidant molecules, including heme-oxygenase-1 (HO-1). HO-1 protects cells against oxidative injury, degrading free heme and inhibiting ROS production. HO-1 is highly expressed in macrophages during plaque growth. Macrophages are morpho-functionally heterogeneous, and the prevalence of a specific phenotype may influence the plaque fate. This heterogeneity has also been observed in monocyte-derived macrophages (MDMs), a model of macrophages infiltrating tissue. The study aims to assess oxidative stress status and Nrf2/HO-1 axis in MDM morphotypes obtained from healthy subjects and coronary artery disease (CAD) patients, in relation to coronary plaque features evaluated in vivo by optical coherence tomography (OCT). We found that MDMs of healthy subjects exhibited a lower oxidative stress status, lower Nrf2 and HO-1 levels as compared to CAD patients. High HO-1 levels in MDMs were associated with the presence of a higher macrophage content, a thinner fibrous cap, and a ruptured plaque with thrombus formation, detected by OCT analysis. These findings suggest the presence of a relationship between in vivo plaque characteristics and in vitro MDM profile, and may help to identify patients with rupture-prone coronary plaque.