NRF2-mediated Stress Response Research Articles

Differential genomic effects of four nano-sized and one micro-sized CeO2 particles on HepG2 cells.

The objective of this research was to perform a genomics study of five cerium oxide particles, 4 nano and one micrometer-sized particles which have been studied previously by our group with respect to cytotoxicity, biochemistry and metabolomics. Human liver carcinoma HepG2 cells were exposed to between 0.3 to 300 ug/ml of CeO2 particles for 72 hours and then total RNA was harvested. Fatty acid accumulation was observed with W4, X5, Z7 and less with Q but not Y6. The gene expression changes in the fatty acid metabolism genes correlated the fatty acid accumulation we detected in the prior metabolomics study for the CeO2 particles named W4, Y6, Z7 and Q, but not for X5. In particular, the observed genomics effects on fatty acid uptake and fatty acid oxidation offer a possible explanation of why many CeO2 particles increase cellular free fatty acid concentrations in HepG2 cells. The major genomic changes observed in this study were sirtuin, ubiquitination signaling pathways, NRF2-mediated stress response and mitochondrial dysfunction. The sirtuin pathway was affected by many CeO2 particle treatments. Sirtuin signaling itself is sensitive to oxidative stress state of the cells and may be an important contributor in CeO2 particle induced fatty acid accumulation. Ubiquitination pathway regulates many protein functions in the cells, including sirtuin signaling, NRF2 mediated stress, and mitochondrial dysfunction pathways. NRF2-mediated stress response and mitochondrial were reported to be altered in many nanoparticles treated cells. All these pathways may contribute to the fatty acid accumulation in the CeO2 particle treated cells.

Dichotomous roles of RIPK3 in regulating the IFN response and NLRP3 inflammasome in human monocytes.

Regulation of the profile and magnitude of toll-like receptor (TLR) responses is important for effective host defense against infections while minimizing inflammatory toxicity. The chemokine CXCL4 regulates the TLR8 response to amplify inflammatory gene and inflammasome activation while attenuating the interferon (IFN) response in primary monocytes. In this study, we describe an unexpected role for the kinase RIPK3 in suppressing the CXCL4 + TLR8-induced IFN response and providing signal 2 to activate the NLRP3 inflammasome and interleukin (IL)-1 production in primary human monocytes. RIPK3 also amplifies induction of inflammatory genes such as TNF, IL6, and IL1B while suppressing IL12B. Mechanistically, RIPK3 inhibits STAT1 activation and activates PI3K-Akt-dependent and XBP1- and NRF2-mediated stress responses to regulate downstream genes in a dichotomous manner. These findings identify new functions for RIPK3 in modulating TLR responses and provide potential mechanisms by which RIPK3 plays roles in inflammatory diseases and suggest targeting RIPK3 and XBP1- and NRF2-mediated stress responses as therapeutic strategies to suppress inflammation while preserving the IFN response for host defense.

Abstract 306: Alternatively activated macrophages induce bystander effects via arginase 1

Abstract Colorectal cancer is a leading cause of cancer mortality and arises from genetic and epigenetic changes in colon epithelial cells. One of the mechanisms driving colon epithelial cell transformation is the bystander effect (BSE). BSE primarily arises from polarized macrophages (MØs) in stromal tissue. Herein, we report that alternatively activated (M2) MØs, as generated by interleukin (IL)-4, induce double-strand DNA breaks in target cells through BSE. Unlike M1 MØs that are activated by intestinal microbiota, BSE and DNA damage due to M2-like MØs occur through a distinct mechanism that differs from M1 MØs and does not involve pro-inflammatory mediators (e.g., cyclooxygenase-2 or tumor necrosis factor-α). Murine MØs (RAW264.7 cells) treated with IL-4 were polarized to an M2-like phenotype and found to strongly express arginase 1 (ARG1), a prototypical enzyme that converts L-arginine to L-ornithine and urea. L-Ornithine is a precursor in polyamine synthesis with H2O2 being a key oxidizing byproduct of polyamine catabolism. In a dual-chamber system using Young Adult Mouse Colon (YAMC) cells as targets for BSE generated by M2-like MØs, inhibition of ARG1 by N-hydroxy-nor-L-arginine abolished DNA damage (as measured by γH2AX). Direct treatment of YAMC cells with L-ornithine resulted in similar levels of DNA damage as M2-like MØs. YAMC cells exposed to M2-like MØs had increased intracellular H2O2. MDL-72527, an inhibitor polyamine oxidase and polyamine catabolism, reduced intracellular H2O2 to background levels and decreased DNA damage. YAMC cells sustaining DNA damage due to BSE from M2-like MØs showed specific phosphorylation of p53(ser15) and NF-κB(ser536) with no evidence for active caspase-3. These data along with an increased expression of cyclinD1 in YAMC cells indicated that DNA damage caused by M2-like MØs occurred via polyamine catabolism and was not due to cellular toxicity or apoptosis. Pathway analysis of RNAseq data for YAMC cells exposed to M2-like MØs identified multiple activated pathways including DNA repair, ubiquitination, Nrf2-mediated stress responses, endoplasmic reticulum overload responses, and lipid biosynthesis. These results provide additional evidence for global cellular responses to BSE generated by M2-like MØs. In conclusion, our findings show that M2-like MØs can generate BSE via a novel mechanism involving ARG1. Induction of BSE by M2-like MØs results in acute DNA damage in colon epithelial cells and activates multiple signaling pathways in target epithelial cells. These data imply a role for alternatively activated M2-like MØs in the malignant transformation of epithelial cells. Citation Format: Ram B. Undi, Hunter L. Porter, Jonathan D. Wren, Naushad Ali, Mark M. Huycke. Alternatively activated macrophages induce bystander effects via arginase 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 306.

Upregulation of Nox4 induces a pro-survival Nrf2 response in cancer-associated fibroblasts that promotes tumorigenesis and metastasis, in part via Birc5 induction

BackgroundA pro-oxidant enzyme, NADPH oxidase 4 (Nox4) has been reported to be a critical downstream effector of TGFβ-induced myofibroblast transformation during fibrosis. While there are a small number of studies suggesting an oncogenic role of Nox4 derived from activated fibroblasts, direct evidence linking this pro-oxidant to the tumor-supporting CAF phenotype and the mechanisms involved are lacking, particularly in breast cancer.MethodsWe targeted Nox4 in breast patient-derived CAFs via siRNA-mediated knockdown or administration of a pharmaceutical inhibitor (GKT137831). We also determine primary tumor growth and metastasis of implanted tumor cells using a stable Nox4-/- syngeneic mouse model. Autophagic flux of CAFs was assessed using a tandem fluorescent-tagged ptfl-LC3 plasmid via confocal microscopy analysis and determination of the expression level of autophagy markers (beclin-1 and LC3B). Nox4 overexpressing CAFs depend on the Nrf2 (nuclear factor-erythroid factor 2-related factor 2) pathway for survival. We then determined the dependency of Nox4-overexpressing CAFs on the Nrf2-mediated adaptive stress response pathway for survival. Furthermore, we investigated the involvement of Birc5 on CAF phenotype (viability and collagen contraction activity) as well as the expression level of CAF markers, FAP and αSMA.ConclusionsWe found that deletion of stroma Nox4 and pharmaceutically targeting its activity with GKT137831 significantly inhibited orthotopic tumor growth and metastasis of implanted E0771 and 4T1 murine mammary carcinoma cell lines in mice. More importantly, we found a significant upregulation of Nox4 expression in CAFs isolated from human breast tumors versus normal mammary fibroblasts (RMFs). Our in situ RNA hybridization analysis for Nox4 transcription on a human breast tumor microarray further support a role of this pro-oxidant in the stroma of breast carcinomas. In addition, we found that Nox4 promotes autophagy in CAFs. Moreover, we found that Nox4 promoted survival of CAFs via activation of Nrf2, a master regulator of oxidative stress response. We have further shown Birc5 is involved as a downstream modulator of Nrf2-mediated pro-survival phenotype. Together these studies indicate a role of redox signaling via the Nox4-Nrf2 pathway in tumorigenesis and metastasis of breast cancer cells by promoting autophagy and survival of CAFs.

Rationally Designed Molecules Synergistically Modulate Multifaceted Aβ Toxicity, Microglial Activation, and Neuroinflammation.

Synergistic modulation of multifaceted toxicity is the key to tackle multifactorial Alzheimer's disease (AD). The etiology of AD includes amyloid β (Aβ) amyloidosis, metal ion dyshomeostasis, reactive oxygen species (ROS), oxidative stress, mitochondrial damage, and neuroinflammation. We rationally designed multifunctional modulators by integrating pharmacophores for metal chelation, antioxidant and anti-inflammatory properties, and modulation of Aβ42 aggregation on the naphthalene monoimide (NMI) scaffold. The in vitro and cellular studies of NMIs revealed that M3 synergistically modulates metal-independent and -dependent amyloid toxicity, scavenges ROS, alleviates oxidative stress, and emulates Nrf2-mediated stress response in neuronal cells. M3 effectively reduced structural and functional damage of mitochondria, reduced Cyt c levels, and rescued cells from apoptosis. The biological atomic force microscopy and Western blot analysis revealed the ability of M3 to suppress microglial activation and neuroinflammation through inhibition of the NF-κβ pathway. The synergistic action of M3 is in agreement with our design strategy to develop a multifunctional therapeutic candidate by integrating multiple pharmacophores with distinct structural and functional elements to ameliorate the multifaceted toxicity of AD.

Tetramethylpyrazine nitrone TBN extends the lifespan of C. elegans by activating the Nrf2/SKN-1 signaling pathway

SKN-1, the ortholog of mammalian Nrf2 proteins, is a transcription factor that plays an important role in oxidative stress resistance and longevity. Similar to other defense systems, the Nrf2-mediated stress response is compromised in aging and neurodegenerative diseases. Our previous studies demonstrated that tetramethylpyrazine nitrone (TBN), a derivative of tetramethylpyrazine armed with a potent free radical-scavenging nitrone moiety, exerted multifunctional neuroprotection in neurological and other diseases. However, the ability of TBN to extend a healthy lifespan and its underlying mechanisms of action are not yet clear. C. elegans have become a popular animal model in aging research. Herein, we demonstrate that TBN can extend the lifespan, promote age-associated health indicators, and restore mitochondrial function in C. elegans. TBN also significantly reduced ROS levels and superoxide accumulation in C. elegans. We show that TBN-mediated lifespan extension is SKN-1dependent. The present study provides valuable insights into the mechanisms by which TBN inhibits aging via the Nrf2/SKN-1 pathway in C. elegans.

Human intestinal bitter taste receptors regulate innate immune responses and metabolic regulators in obesity.

Bitter taste receptors (taste 2 receptors, TAS2Rs) serve as warning sensors in the lingual system against the ingestion of potentially poisonous food. Here, we investigated the functional role of TAS2Rs in the human gut and focused on their potential to trigger an additional host defense pathway in the intestine. Human jejunal crypts, especially those from individuals with obesity, responded to bitter agonists by inducing the release of antimicrobial peptides (α-defensin 5 and regenerating islet–derived protein 3 α [REG3A]) but also regulated the expression of other innate immune factors (mucins, chemokines) that affected E. coli growth. We found that the effect of aloin on E. coli growth and on the release of the mucus glycoprotein CLCA1, identified via proteomics, was affected by TAS2R43 deletion polymorphisms and thus confirmed a role for TAS2R43. RNA-Seq revealed that denatonium benzoate induced an NRF2-mediated nutrient stress response and an unfolded protein response that increased the expression of the mitokine GDF15 but also ADM2 and LDLR, genes that are involved in anorectic signaling and lipid homeostasis. In conclusion, TAS2Rs in the intestine constitute a promising target for treating diseases that involve disturbances in the innate immune system and body weight control. TAS2R polymorphisms may be valuable genetic markers to predict therapeutic responses.

High intensity muscle stimulation activates a systemic Nrf2-mediated redox stress response

High intensity exercise is a popular mode of exercise to elicit similar or greater adaptive responses compared to traditional moderate intensity continuous exercise. However, the molecular mechanisms underlying these adaptive responses are still unclear. The purpose of this pilot study was to compare high and low intensity contractile stimulus on the Nrf2-mediated redox stress response in mouse skeletal muscle. An intra-animal design was used to control for variations in individual responses to muscle stimulation by comparing a stimulated limb (STIM) to the contralateral unstimulated control limb (CON). High Intensity (HI – 100Hz), Low Intensity (LI – 50Hz), and Naïve Control (NC – Mock stimulation vs CON) groups were used to compare these effects on Nrf2-ARE binding, Keap1 protein, and downstream gene and protein expression of Nrf2 target genes. Muscle stimulation significantly increased Nrf2-ARE binding in LI-STIM compared to LI-CON (p = 0.0098), while Nrf2-ARE binding was elevated in both HI-CON and HI-STIM compared to NC (p = 0.0007). The Nrf2-ARE results were mirrored in the downregulation of Keap1, where Keap1 expression in HI-CON and HI-STIM were both significantly lower than NC (p = 0.008) and decreased in LI-STIM compared to LI-CON (p = 0.015). In addition, stimulation increased NQO1 protein compared to contralateral control regardless of stimulation intensity (p = 0.019), and HO1 protein was significantly higher in high intensity compared to the Naïve control group (p = 0.002). Taken together, these data suggest a systemic redox signaling exerkine is activating Nrf2-ARE binding and is intensity gated, where Nrf2-ARE activation in contralateral control limbs were only seen in the HI group. Other research in exercise induced Nrf2 signaling support the general finding that Nrf2 is activated in peripheral tissues in response to exercise, however the specific exerkine responsible for the systemic signaling effects is not known. Future work should aim to delineate these redox sensitive systemic signaling mechanisms.

ATRT-02. THE DUAL MTORC1/2 INHIBITOR, TAK-228 COMBINES SYNERGISTICALLY WITH THE BH3 MIMETIC, OBATOCLAX TO IMPROVE SURVIVAL IN MICE BEARING ORTHOTOPIC XENOGRAFTS OF AT/RT

mTOR activation drives tumorigenicity by regulating transcription factor expression and downstream growth and survival pathways. We have previously shown that mTORC1 and mTORC2 are highly activated in AT/RT and the dual mTORC1/2 inhibitor, TAK-228 (Sapanisertib) improves survival in mice bearing orthotopic xenografts of AT/RT. To design a rational combination therapy that enhances TAK-228’s efficacy and durability, we performed RNASeq 4 hours after TAK-228 treatment of AT/RT cell models. Pathway analysis revealed disruption of the NRF2-mediated stress response. NRF2 is a cap’n’collar leucine zipper transcription factor that regulates expression of genes involved in redox homeostasis, energy metabolism, cell proliferation, and survival. Analysis of publicly available RNASeq data on 32 human tumors identified elevated expression of NRF2 in AT/RT (median expression 40.78, normal brain 18.81). Short-hairpin knockdown of NRF2 decreased the expression of NRF2 as well as the anti-apoptotic proteins MCL-1, BCL-xL, and BCL-2 (western blot), and intracellular concentrations of reduced glutathione (p<0.005, t-test). TAK-228 similarly decreased expression of NRF2, MCL-1, and glutathione (p<0.005, t-test) demonstrating that TAK-228 compromises AT/RT defenses against oxidative stress and cell death. The brain-penetrant BH3 mimetic, Obatoclax increases oxidative stress and induces apoptosis in AT/RT (MUSE oxidative stress, cPARP western blot, t-test p<0.05). These complementary mechanisms of action synergize to slow AT/RT cell growth (MUSE Cell viability assay, ANOVA p<0.05) and induce high rates of cell death (MUSE ANNEXIN V assay, ANOVA p<0.05, Western blot for cPARP, Compusyn Synergy analysis CI<1.0). Once-weekly treatments of TAK-228 combined with Obatoclax in orthotopic mouse models of AT/RT is well tolerated, slows tumor growth (bioluminescence imaging, ANOVA p<0.05) and significantly extends median survival from 35 to 55 days (Log-rank p<0.05). These findings support a new clinical trial aimed at improving AT/RT survival.

ATRT-23. THE DUAL mTORC1/2 INHIBITOR SAPANISERTIB DISRUPTS THE NRF2-MEDIATED STRESS RESPONSE AND COMBINES SYNERGISTICALLY WITH THE BH3 MIMETIC OBATOCLAX TO EXTEND AT/RT SURVIVAL

Atypical teratoid/rhabdoid tumors are aggressive infantile tumors highly resistant to intensive therapies. We aim to identify and target critical factors driving this therapy resistance to improve AT/RT survival. Analysis of publically available RNASeq on 32 AT/RT identified elevated expression of NRF2 (median expression 40.78, normal brain 18.81). NRF2 is a master regulator of cell’s stress response whose expression is correlated with therapy resistance and poor survival. NRF2 activation is sensitive to mTOR activity and is a biomarker predicting response to the dual mTORC1/2 inhibitor, Sapanisertib (TAK228, INK128). We performed RNASeq on 4 human-derived AT/RT cell models after Sapanisertib treatment. Pathway analysis reveals disruption of the NRF2-mediated stress response (-log p value 0.39, Z-score 1.0). As a result, Sapanisertib decreases ROS scavengers like glutathione (Metabolite analysis UHPLC-MS, t-test p<0.05) and induces a pro-death phenotype (decreased MCL-1 expression, western blot; gene-expression analysis, RNASeq). The brain-penetrant BH3 mimetic Obatoclax increases ROS generation and induces apoptosis (MUSE oxidative stress and ANNEXIN V assays, t-test p<0.05). These complementary mechanisms of action synergize to induce high rates of cell death (MUSE ANNEXIN V assay, ANOVA p<0.05, C-PARP western blot, Compusyn Synergy analysis CI<1.0) and slow cell growth (MUSE Cell viability, ANOVA p<0.05). Once-weekly treatments of Sapanisertib combined with Obatoclax in orthotopic mouse models of AT/RT are well tolerated, slow tumor growth (bioluminescence imaging) and significantly extend median survival from 35 to 55 days (Log-rank p<0.05). These findings support a new clinical trial aimed at improving AT/RT survival.

Abstract 6226: Sapanisertib interferes with the NRF2-mediated stress response to synergize with Obatoclax and extend AT/RT survival

Abstract Atypical teratoid rhabdoid tumors (AT/RT) are the most common malignant brain tumors of infancy, highly resistant to all therapies, and responsible for one of the greatest numbers of life years lost due to cancer. We have previously demonstrated that Sapanisertib combines synergistically with cisplatin to extend survival in AT/RT. To understand the mechanisms underlying this synergy and to identify further novel combination therapies to improve AT/RT survival, we performed RNASeq on AT/RT cell models 4 hours after Sapanisertib treatment. Pathway analysis revealed that Sapanisertib disrupts the NRF2-mediated stress response and apoptotic signaling pathway leading to a pro-death phenotype. Activating mutations in the NRF2 coding gene, NFE2L2, have been identified as a possible biomarker predicting Sapanisertib efficacy in lung cancer clinical trials. Short hairpin knockdown of NRF2 led to decreased expression of the anti-apoptotic proteins MCL-1, BCL-2, and BCL-xL suggesting that interfering with NRF2 may drive this pro-death phenotype in AT/RT. Obatoclax is a synthetic small molecule inhibitor of the BCL-2 family of proteins that was well tolerated in phase II clinical trials and readily crosses the blood brain barrier. Obatoclax treatment of NRF2 knockdown AT/RT cell models slows cell growth, induces high rates of apoptosis, and leads to increased oxidative stress compared to short hairpin scramble controls (cell viability assay, t-test p&amp;lt;0.05; ANNEXIN V cell sorting assay t-test p&amp;lt;0.05, MUSE oxidative stress assay t-test p&amp;lt;0.05). We treated AT/RT cell models with Sapanisertib and showed that treatment decreases expression of NRF2 and MCL-1 (western blot). Sapanisertib combines synergistically with Obatoclax to increase oxidative stress, slow cell growth, and increase apoptosis (MUSE oxidative stress assay ANOVA p&amp;lt;0.05; cell viability assay, ANOVA p&amp;lt;0.05; ANNEXIN V cell sorting assay ANOVA p&amp;lt;0.05, Compusyn model of synergy, CI&amp;lt;1.0). We next developed orthotopic mouse models of AT/RT using our luciferase-labeled CHLA06 and BT37 AT/RT cell models. We administered Sapinisertib and Obatoclax using mouse equivalent dosing to human clinical trials (Sapinisertib 6mg/kg oral weekly, Obatoclax 10mg/kg oral twice a week). Combination treatment was well tolerated and more than doubled median survival. Our data supports a new clinical trial combining Sapinisertib with Obatoclax to extend survival in AT/RT. Citation Format: Ashlyn Parkhurst, Sabrina Wang, Harpreet Kaur, Antje Arnold, Charles Eberhart, Eric Raabe, Jeffrey Rubens. Sapanisertib interferes with the NRF2-mediated stress response to synergize with Obatoclax and extend AT/RT survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6226.

Cytoplasmic DAXX drives SQSTM1/p62 phase condensation to activate Nrf2-mediated stress response

Autophagy cargo recognition and clearance are essential for intracellular protein quality control. SQSTM1/p62 sequesters intracellular aberrant proteins and mediates cargo delivery for their selective autophagic degradation. The formation of p62 non-membrane-bound liquid compartments is critical for its function as a cargo receptor. The regulation of p62 phase separation/condensation has yet been poorly characterised. Using an unbiased yeast two-hybrid screening and complementary approaches, we found that DAXX physically interacts with p62. Cytoplasmic DAXX promotes p62 puncta formation. We further elucidate that DAXX drives p62 liquid phase condensation by inducing p62 oligomerisation. This effect promotes p62 recruitment of Keap1 and subsequent Nrf2-mediated stress response. The present study suggests a mechanism of p62 phase condensation by a protein interaction, and indicates that DAXX regulates redox homoeostasis, providing a mechanistic insight into the prosurvival function of DAXX.

Excess homocysteine upregulates the NRF2-antioxidant pathway in retinal Müller glial cells

This study evaluated the effects of elevated homocysteine (Hcy) on the oxidative stress response in retinal Müller glial cells. Elevated Hcy has been implicated in retinal diseases including glaucoma and optic neuropathy, which are characterized by retinal ganglion cell (RGC) loss. To understand the mechanisms of Hcy-induced RGC loss, in vitro and in vivo models have been utilized. In vitro isolated RGCs are quite sensitive to elevated Hcy levels, while in vivo murine models of hyperhomocysteinemia (HHcy) demonstrate a more modest RGC loss (∼20%) over a period of many months. This differential response to Hcy between isolated cells and the intact retina suggests that the retinal milieu invokes mechanisms that buffer excess Hcy. Oxidative stress has been implicated as a mechanism of Hcy-induced neuron loss and NRF2 is a transcription factor that plays a major role in regulating cytoprotective responses to oxidative stress. In the present study we investigated whether HHcy upregulates NRF2-mediated stress responses in Müller cells, the chief retinal glial cell responsible for providing trophic support to retinal neurons. Primary Müller cells were exposed to L-Hcy-thiolactone [50μM–10mM] and assessed for viability, reactive oxygen species (ROS), and glutathione (GSH) levels. Gene/protein levels of Nrf2 and levels of NRF2-regulated antioxidants (NQO1, CAT, SOD2, HMOX1, GPX1) were assessed in Hcy-exposed Müller cells. Unlike isolated RGCs, isolated Müller cells are viable over a wide range of Hcy concentrations [50 μM – 1 mM]. Moreover, when exposed to elevated Hcy, Müller cells demonstrate decreased oxidative stress and decreased ROS levels. GSH levels increased by ∼20% within 24 h exposure to Hcy. Molecular analyses revealed 2-fold increase in Nrf2 expression. Expression of antioxidant genes Nqo1, Cat, Sod2, Hmox1, Gpx1 increased significantly. The consequences of Hcy exposure were evaluated also in Müller cells harvested from Nrf2−/− mice. In contrast to WT Müller cells, in which oxidative stress decreased upon exposure to Hcy, the Nrf2−/− Müller cells showed a significant increase in oxidative stress. Our data suggest that at least during early stages of Hhcy, a cytoprotective response may be in place, mediated in part by NRF2 in Müller cells.

Hydralazine induces stress resistance and extends C. elegans lifespan by activating the NRF2/SKN-1 signalling pathway

Nuclear factor (erythroid-derived 2)-like 2 and its Caenorhabditiselegans ortholog, SKN-1, are transcription factors that have a pivotal role in the oxidative stress response, cellular homeostasis, and organismal lifespan. Similar to other defense systems, the NRF2-mediated stress response is compromised in aging and neurodegenerative diseases. Here, we report that the FDA approved drug hydralazine is a bona fide activator of the NRF2/SKN-1 signaling pathway. We demonstrate that hydralazine extends healthy lifespan (~25%) in wild type and tauopathy model C. elegans at least as effectively as other anti-aging compounds, such as curcumin and metformin. We show that hydralazine-mediated lifespan extension is SKN-1 dependent, with a mechanism most likely mimicking calorie restriction. Using both in vitro and in vivo models, we go on to demonstrate that hydralazine has neuroprotective properties against endogenous and exogenous stressors. Our data suggest that hydralazine may be a viable candidate for the treatment of age-related disorders.

Abstract 5457: OGT restrains the NRF2 antioxidant pathway via O-GlcNAcylation of KEAP1

Abstract O-GlcNAcylation is a reversible post-translational modification that adds an O-linked β-N-acetylglucosamine (O-GlcNAc) moiety onto serine/threonine residues of target proteins. This modification is regulated by only two enzymes: O-GlcNAc transferase (OGT, the writer) and O-GlcNAcase (OGA, the eraser) in mammals. Recent studies have revealed that OGT expression and O-GlcNAc modifications are elevated in several cancers, but specific O-GlcNAc targets are not well defined. We conducted a global transcriptome profiling in MDA-MB-231 breast cancer cells to search for signaling events that respond to O-GlcNAc fluctuation. We found significant up-regulation of genes involved in the NRF2-dependent stress response when OGT activity is inhibited in different tumor types. We also discovered a strong positive correlation of gene signatures between low OGT activity and NRF2 activation in multiple human tumor gene expression datasets. NRF2, the primary regulator of redox balance, is usually activated by oxidative stress and repressed under basal conditions by the KEAP1-CUL3 ubiquitin ligase complex. However, we found that OGT inhibition increases NRF2 protein level through reducing poly-ubiquitination of NRF2 in the absence of oxidative stress. By chemical sugar labeling and mass spectrometry assays, we identified that KEAP1 is directly O-GlcNAcylated by OGT especially within the BTB and Kelch motifs. Of all 11 putative O-GlcNAc sites on KEAP1, we found serine 104 is responsible for regulating NRF2 activity through affecting KEAP1-CUL3 interaction. Interestingly, we found the amount of intracellular glucose co-vary with KEAP1 O-GlcNAcylation and NRF2 protein, suggesting that glucose metabolites may utilize the nutrient sensing O-GlcNAcylation to fine-tune the antioxidant response. We propose that cancer cells could utilize O-GlcNAcylation, specifically on KEAP1, to regulate NRF2-mediated stress responses in response to the dynamics of intracellular glucose level. Since the NRF2 pathway is inappropriately activated in certain tumor types due to dysregulated function of KEAP1, such as non-small cell lung cancer, where it provides stress resistance and a growth advantage. Our study could provide new insight into redox cancer biology and provide novel strategy to modulate NRF2 activity during tumor development. Citation Format: Po-Han Chen, Timothy J. Smith, Jianli Wu, Michael Boyce, Jen-Tsan Ashley Chi. OGT restrains the NRF2 antioxidant pathway via O-GlcNAcylation of KEAP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5457. doi:10.1158/1538-7445.AM2017-5457

Characterization of chemical-induced sterile inflammation in vitro: application of the model compound ketoconazole in a human hepatic co-culture system.

Liver injury as a result of a sterile inflammation is closely linked to the activation of immune cells, including macrophages, by damaged hepatocytes. This interaction between immune cells and hepatocytes is as yet not considered in any of the in vitro test systems applied during the generation of new drugs. Here, we established and characterized a novel in vitro co-culture model with two human cell lines, HepG2 and differentiated THP-1. Ketoconazole, an antifungal drug known for its hepatotoxicity, was used as a model compound in the testing of the co-culture. Single cultures of HepG2 and THP-1 cells were studied as controls. Different metabolism patterns of ketoconazole were observed for the single and co-culture incubations as well as for the different cell types. The main metabolite N-deacetyl ketoconazole was found in cell pellets, but not in supernatants of cell cultures. Global proteome analysis showed that the NRF2-mediated stress response and the CXCL8 (IL-8) pathway were induced by ketoconazole treatment under co-culture conditions. The upregulation and ketoconazole-induced secretion of several pro-inflammatory cytokines, including CXCL8, TNF-α and CCL3, was observed in the co-culture system only, but not in single cell cultures. Taking together, we provide evidence that the co-culture model applied might be suitable to serve as tool for the prediction of chemical-induced sterile inflammation in liver tissue in vivo.

Differential Genomic Effects of Six Different TiO2 Nanomaterials on Human Liver HepG2 Cells.

Human HepG2 cells were exposed to six TiO2 nanomaterials (with dry primary particle sizes ranging from 22 to 214 nm, either 0.3, 3, or 30 μg/mL) for 3 days. Some of these canonical pathways changed by nano-TiO2 in vitro treatments have been already reported in the literature, such as NRF2-mediated stress response, fatty acid metabolism, cell cycle and apoptosis, immune response, cholesterol biosynthesis, and glycolysis. But this genomic study also revealed some novel effects such as protein synthesis, protein ubiquitination, hepatic fibrosis, and cancer-related signaling pathways. More importantly, this genomic analysis of nano-TiO2 treated HepG2 cells linked some of the in vitro canonical pathways to in vivo adverse outcomes: NRF2-mediated response pathways to oxidative stress, acute phase response to inflammation, cholesterol biosynthesis to steroid hormones alteration, fatty acid metabolism changes to lipid homeostasis alteration, G2/M cell checkpoint regulation to apoptosis, and hepatic fibrosis/stellate cell activation to liver fibrosis.

Differential Genomic Effects on Signaling Pathways by Two Different CeO2 Nanoparticles in HepG2 Cells.

To investigate genomic effects, human liver hepatocellular carcinoma (HepG2) cells were exposed for three days to two different forms of nanoparticles both composed of CeO2 (0.3, 3 and 30 μg/mL). The two CeO2 nanoparticles had dry primary particle sizes of 8 nanometers {(M) made by NanoAmor} and 58 nanometers {(L) made by Alfa Aesar} and differ in various other physical-chemical properties as well. The smaller particle has stronger antioxidant properties, probably because it has higher Ce3+ levels on the particle surface, as well as more surface area per unit weight. Nanoparticle M showed a normal dose-response pattern with 363, 633 and 1273 differentially expressed genes (DEGs) at 0.3, 3 and 30 μg/mL, respectively. In contrast, nanoparticle L showed a puzzling dose-response pattern with the most DEGs found in the lowest exposure group with 1049, 303 and 323 DEGs at 0.3, 3 and 30 μg/mL, respectively. This systems biological genomic study showed that the major altered pathways by these two nano cerium oxides were protein synthesis, stress response, proliferation/cell cycle, cytoskeleton remodeling/actin polymerization and cellular metabolism. Some of the canonical pathways affected were mTOR signaling, EIF2 signaling, fatty acid activation, G2/M DNA damage checkpoint regulation, glycolysis and protein ubiquitination. These two CeO2 nanoparticles differed considerably in their genomic effects. M is more active than L in respect to altering the pathways of mitochondrial dysfunction, acute phase response, apoptosis, 14-3-3 mediated signaling, remodeling of epithelial adherens junction signaling, actin nucleation by ARP-WASP complex, altered TCA cycle and elevated fatty acid concentrations by metabolomics. However, L is more active than M in respect to the pathways of NRF2-mediated stress response and hepatic fibrosis/hepatic stellate cell activation. One major difference in the cell response to nano M and L is that nano M caused the Warburg effect while nano L did not.

LAPTM4B is associated with poor prognosis in NSCLC and promotes the NRF2-mediated stress response pathway in lung cancer cells.

We recently demonstrated that lysosomal protein transmembrane 4 beta (LAPTM4B) is elevated in non-small cell lung cancers (NSCLCs) and in the surrounding premalignant airway field of cancerization. In the present study, we sought to begin to understand the relevance of LAPTM4B expression and signaling to NSCLC pathogenesis. In situ hybridization analysis of LAPTM4B transcript in tissue microarrays comprised of 368 NSCLCs demonstrated that LAPTM4B expression was significantly increased in smoker compared to non-smoker lung adenocarcinoma tumors (P < 0.001) and was significantly associated with poor overall survival (P < 0.05) in adenocarcinoma patients. Knockdown of LAPTM4B expression inhibited cell growth, induced cellular apoptosis and decreased cellular autophagy in serum starved lung cancer cells. Expression profiling coupled with pathways analysis revealed decreased activation of the nuclear factor erythroid 2-like 2 (NRF2) stress response pathway following LAPTM4B knockdown. Further analysis demonstrated that LAPTM4B augmented the expression and nuclear translocation of the NRF2 transcription factor following serum deprivation as well as increased the expression of NRF2 target genes such as heme oxygenase 1/HMOX1). Our study points to the relevance of LAPTM4B expression to NSCLC pathogenesis as well as to the probable role of LAPTM4B/NRF2 signaling in promoting lung cancer cell survival.

Preoperative fasting protects against renal ischemia-reperfusion injury in aged and overweight mice.

Ischemia-reperfusion injury (IRI) is inevitable during kidney transplantation leading to oxidative stress and inflammation. We previously reported that preoperative fasting in young-lean male mice protects against IRI. Since patients are generally of older age with morbidities possibly leading to a different response to fasting, we investigated the effects of preoperative fasting on renal IRI in aged-overweight male and female mice. Male and female F1-FVB/C57BL6-hybrid mice, average age 73 weeks weighing 47.2 grams, were randomized to preoperative ad libitum feeding or 3 days fasting, followed by renal IRI. Body weight, kidney function and survival of the animals were monitored until day 28 postoperatively. Kidney histopathology was scored for all animals and gene expression profiles after fasting were analyzed in kidneys of young and aged male mice. Preoperative fasting significantly improved survival after renal IRI in both sexes compared with normal fed mice. Fasted groups had a better kidney function shown by lower serum urea levels after renal IRI. Histopathology showed less acute tubular necrosis and more regeneration in kidneys from fasted mice. A mRNA analysis indicated the involvement of metabolic processes including fatty acid oxidation and retinol metabolism, and the NRF2-mediated stress response. Similar to young-lean, healthy male mice, preoperative fasting protects against renal IRI in aged-overweight mice of both genders. These findings suggest a general protective response of fasting against renal IRI regardless of age, gender, body weight and genetic background. Therefore, fasting could be a non-invasive intervention inducing increased oxidative stress resistance in older and overweight patients as well.