Degradation Of Nrf2 Research Articles

Tiliroside targets TBK1 to induce ferroptosis and sensitize hepatocellular carcinoma to sorafenib

BackgroundCombination therapy with other antineoplastic agent is a favorable approach for targeting the molecules involved in sorafenib resistance. PurposeIn the present study, we determined whether tiliroside, a natural flavonoid glycoside isolated from oriental paperbush flower, could improve the sensitivity of hepatocellular carcinoma (HCC) cells to sorafenib. Furthermore, we investigated the mechanisms and identified the potential drug targets of tiliroside. MethodsSynergy was performed using CalcuSyn. Transcriptomic studies were adopted to investigate whether tiliroside could induce ferroptosis and inhibit the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in HCC cells. Ferroptosis was analyzed using western blotting, flow cytometry, and transmission electron microscopy. Immunofluorescence, co-immunoprecipitation, and Nrf2 knockdown or overexpression were performed to confirm the involvement of Nrf2 in tiliroside-induced ferroptosis. Additionally, molecular docking and biolayer interferometry-based measurements were used to confirm the direct target of tiliroside. Finally, subcutaneous xenograft and orthotopic xenograft tumors in nude mice were used to assess the effects of tiliroside in vivo. ResultsTiliroside significantly enhanced the anti-HCC activity of sorafenib without any discernible side effects. Moreover, the combination of tiliroside and sorafenib induced synergistic effects against HCC in vitro. The inhibitory effects of tiliroside on HCC were antagonized by N-acetylcysteine and the ferroptosis inhibitor liproxstatin-1. Studies on the mechanism of action revealed that tiliroside could directly bind to TANK-binding kinase 1 (TBK1) and inhibit its enzymatic activity. Inhibition of TBK1 by tiliroside decreased the phosphorylation of serine 349 on sequestosome-1 (p62) and the affinity of p62 for kelch like ECH-associated protein 1 (Keap1) and promoted Keap1-mediated Nrf2 ubiquitination and degradation. The downstream target proteins of Nrf2, including glutathione peroxidase 4, ferritin heavy chain 1, and glucose-6-phosphate dehydrogenase, demonstrated similar results to that of Nrf2 protein, inducing ferroptosis in tiliroside-treated HCC cells. We extended these findings in vivo and found that tiliroside inhibited the growth of HepG2 tumors in both subcutaneous xenograft and orthotopic xenograft tumor models of HCC. ConclusionOur findings imply that tiliroside is a potent TBK1 inhibitor and a candidate natural anti-cancer product that could function as a sensitizer of sorafenib in HCC treatment by targeting TBK1 to induce ferroptosis.

Bazedoxifene-induced ROS promote mitochondrial dysfunction and enhance osimertinib sensitivity by inhibiting the p-STAT3/SOCS3 and KEAP1/NRF2 pathways in non-small cell lung cancer

Although the advent of osimertinib has brought revolutionary changes to the treatment landscape of non-small cell lung cancer (NSCLC) patients, acquired resistance remains a major obstacle limiting long-term survival benefits for the treatment of cancer. The purpose of this study was to examine the mechanisms involved in the ability of bazedoxifene to synergistically enhance osimertinib sensitivity, which will aid in delaying and overcoming osimertinib resistance to improve patient outcomes. Here, we found that osimertinib increased the production of reactive oxygen species (ROS), promoted mitochondrial fission, diminished mitochondrial membrane potential, and activated cell apoptosis. Moreover, the p-STAT3/suppressor of cytokine signaling 3 (SOCS3) and KEAP1/NRF2 signaling pathways were activated to scavenge ROS and promote osimertinib resistance. Mechanistically, SOCS3 can directly bind to KEAP1 to prevent the degradation of NRF2, resulting in the activation of an NRF2-dependent transcriptional program. Furthermore, the osimertinib-induced mitochondrial dysfunction and apoptosis were enhanced by bazedoxifene, thereby delaying and overcoming osimertinib resistance by inhibiting these pathways in vitro and in vivo. These findings identified a new critical link in the p-STAT3/SOCS3 pathway, KEAP1/NRF2 pathway, mitochondrial dysfunction, and osimertinib resistance. The present study demonstrated that bazedoxifene can be used for delaying or overcoming osimertinib resistance in NSCLC.

Celastrol targeting Nedd4 reduces Nrf2-mediated oxidative stress in astrocytes after ischemic stroke

Stroke is the second leading cause of death worldwide, and oxidative stress plays a crucial role. Celastrol exhibits strong antioxidant properties in several diseases; however, whether it can affect oxidation in cerebral ischemic-reperfusion injury (CIRI) remains unclear. This study aimed to determine whether celastrol could reduce oxidative damage during CIRI and to elucidate the underlying mechanisms. Here, we found that celastrol attenuated oxidative injury in CIRI by upregulating nuclear factor E2-related factor 2 (Nrf2). Using alkynyl-tagged celastrol and liquid chromatography-tandem mass spectrometry, we showed that celastrol directly bound to neuronally expressed developmentally downregulated 4 (Nedd4) and then released Nrf2 from Nedd4 in astrocytes. Nedd4 promoted the degradation of Nrf2 through K48-linked ubiquitination and thus contributed to astrocytic reactive oxygen species production in CIRI, which was significantly blocked by celastrol. Furthermore, by inhibiting oxidative stress and astrocyte activation, celastrol effectively rescued neurons from axon damage and apoptosis. Our study uncovered Nedd4 as a direct target of celastrol, and that celastrol exerts an antioxidative effect on astrocytes by inhibiting the interaction between Nedd4 and Nrf2 and reducing Nrf2 degradation in CIRI.

Serine/threonine kinase 3 promotes oxidative stress and mitochondrial damage in septic cardiomyopathy through inducing Kelch-like ECH-associated protein 1 phosphorylation and nuclear factor erythroid 2-related factor 2 degradation.

Serine/threonine kinases (STK3) is a core component of the Hippo pathway and modulates oxidative stress and inflammatory responses in cardiovascular diseases. However, its potential role in septic cardiomyopathy remains undefined. STK3-mediated phosphorylation of Kelch-like ECH-associated protein 1 (KEAP1) was shown to suppress antioxidant gene transcription controlled by nuclear factor erythroid 2-related factor 2 (Nrf2) in macrophages. To explore whether STK3 induces KEAP1-mediated suppression of Nrf2 in septic cardiomyopathy, wild-type and global STK3 knockout (STK3 -/- ) mice were treated with LPS. LPS treatment upregulated cardiac STK3 expression. STK3 deletion attenuated myocardial inflammation and cardiomyocyte death, and improved myocardial structure and function. In LPS-challenged HL-1 cardiomyocytes, shRNA-mediated STK3 knockdown normalized mitochondrial membrane potential and ATP production, attenuated apoptosis, and rescued antioxidant gene expression by preventing Nrf2 downregulation. Co-IP, docking analysis, western blotting, and immunofluorescence assays further showed that STK3 binds and phosphorylates KEAP1, promoting Nrf2 downregulation. Accordingly, transfection of phosphodefective KEAP1 mutant protein in cardiomyocyte restored Nrf2 expression and mitochondrial performance upon LPS, while expression of a phosphomimetic KEAP1 mutant abolished the mitochondria-protective and pro-survival effects of STK3 deletion. These findings suggest that STK3 upregulation contributes to septic cardiomyopathy by phosphorylating KEAP1 to promote Nrf2 degradation and suppression of the antioxidant response.

CSIG-26. PROTEIN KINASE B (PKB/AKT) PROTECTS IDH-MUTATED GLIOMA FROM FERROPTOSIS VIA NRF2

Abstract BACKGROUND Mutations of the isocitrate dehydrogenase (IDH) gene are highly prevalent in WHO II/III glioma. IDH-mutated glioma frequently exhibits strong molecular signatures of PI3K/AKT/mTOR signaling, whereas the precise roles of this oncogenic pathway in IDH-mutated glioma remain elusive. METHODS In this study, we performed unbiased RNA sequencing and gene set enrichment analysis (GSEA) to investigate the relevant molecular pathways governed by the Phosphoinositide 3-kinase (PI3K)/AKT in IDH-mutated cancer cells. The role of nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione de novo synthesis were further analyzed through biochemistry assays. Moreover, the role of AKT in glioma disease outcomes was evaluated in a preclinical animal model bearing IDH-mutated orthoptic intracranial xenograft. RESULTS RNA sequencing and GSEA analysis indicated that the AKT pathway significantly correlated with Nrf2-guided gene expression and ferroptosis-related pathways. Mechanistically, AKT activation compromised the phosphorylation of Nrf2 at its SDS domain, which is a critical post-translational modification that determines Nrf2 degradation through an E3 ligase β-transducin repeat-containing protein (β-TrCP). Thus, the activation of the AKT pathway limited the proteolysis of Nrf2 and a remarkably enhanced Nrf2 transcriptional activity, which enables cytoprotective pathways such as antioxidant, tumor microenvironment remodeling, and ferroptosis protection. Moreover, a combination of AKT inhibitor ipatasertib with genotoxic agent temozolomide resulted in potent synthetic lethality in IDH-mutated cancer cells, highlighted with enhanced ferroptotic cell death. Further, the AKT inhibitor and TMZ combination regimen improved disease outcomes in a preclinical animal model with IDH-mutated orthotopic intracranial xenograft, with significantly prolonged overall survival. CONCLUSION Overall, our findings revealed an uncharted oncogenic mechanism in IDH-mutated glioma, highlighted with AKT/Nrf2-guided cytoprotective pathway. Our study also provides critical preclinical evidence for a synthetic lethality approach for IDH mutated cancers.

In silico and docking studies on the binding activities of Keap1 of antioxidant compounds in non-oilseed legumes

We used in silico methods to predict the physiochemical and pharmacological characteristics, toxicity, and biological activities of the screened compounds. All compounds showed positive results while calculating their physiochemical and pharmacokinetic descriptors. Using the Prediction of Activity Spectra for Substances (PASS) software on compounds form non-oilseed legumes, we identified compounds (mainly 4 polyphenol compounds) with anti-infective, anti-eczematic, antimutagenic, muco-membranous protector, fibrinolytic, anticarcinogenic, hepato-protectant, cardio-protectant, antioxidant, and astringent effect. PASS predicted HO-1 expression enhancing and free radical scavenging properties for gallic acid, coumaric acid, catechin, and epicatechin. Data about validation protocols for molecular docking of ligand IVV to Keap1 was performed by root mean square deviation (RMSD) value is used to validate docking protocol and representation mainly for analyzing stability of protein and predicting conformational changes of protein. Molecular docking is a powerful technique for studies of receptor-ligand interaction and has led to the discovery of Keap1-Nrf2 small molecule inhibitors. Keap1 inhibits the degradation of Nrf2. Our results suggest that screened compounds from non-oilseed legumes can effectively interact with the Keap1 binding site and dissociate Keap1 and Nrf2. The screened compounds from non-oilseed legumes that displayed high binding affinities with Keap1 are promising new Nrf2 activators. We performed molecular docking to identify the molecular interactions of gallic acid, catechin, and epicatechin with Keap1. Non-oilseed legumes plant is a natural source of potent antioxidant that may prevent diseases and could be potentially used as functional food, nutraceuticals, and drugs.

FTO inhibits oxidative stress by mediating m6A demethylation of Nrf2 to alleviate cerebral ischemia/reperfusion injury.

Current therapies are of limited efficacy in cerebral ischemia/reperfusion (I/R) injury. Based on the important role of oxidative stress in cerebral I/R injury, this study aimed to explore how the N6-adenosine methylation (m6A) demethylase FTO affects oxidative stress. Middle cerebral artery occlusion/reperfusion (MCAO/R)-induced rat model and oxygen and glucose deprivation/re-oxygenation (OGD/R)-induced SH-SY5Y cells were established as in vivo and in vitro model, respectively. The neurological score of rats was measured, and the volume of cerebral infarction was measured by TTC staining. The levels of FTO, nuclear factor-erythroid 2-related factor (Nrf2), and the activity of m6A demethylase FTO were detected. The m6A methylation level of Nrf2 mRNA was detected by MeRIP experiment. Flow cytometry and MTT assay were used to detect apoptosis and proliferation in vitro. TUNEL assay was used to detect apoptosis in brain tissues. FTO and Nrf2 expressions were decreased in the MCAO/R rat brain tissues and OGD/R SH-SY5Y cells, while the m6A methylation level of Nrf2 mRNA was significantly increased. Overexpression of FTO upregulated Nrf2 expression by decreasing the m6A methylation level of Nrf2 mRNA. m6A binding protein YT521-B homology (YTH) domain family protein 2 (YTHDF2) promoted the degradation of Nrf2 by promoting the m6A methylation level of Nrf2 mRNA. Furthermore, SH-SY5Y cell apoptosis was increased and cell viability was decreased after the addition of methyltransferases METTL 3/14, thus blocking FTO to protect SH-SY5Y cells from oxidative stress injury. In vivo, overexpression of FTO decreased the area of cerebral ischemia infarction and the extent of cell apoptosis. In conclusion, FTO increases Nrf2 expression by mediating m6A demethylation of Nrf2 mRNA, thereby inhibiting oxidative stress response and ultimately alleviating cerebral I/R injury.

The RSL3 Induction of KLK Lung Adenocarcinoma Cell Ferroptosis by Inhibition of USP11 Activity and the NRF2-GSH Axis.

Antioxidant transcription factor NRF2 plays a pivotal role in cell ferroptosis. KLK lung adenocarcinoma (LUAD) is a specific molecular subtype of Kras-mutant LUAD. The activation of mutant Kras in combination with the inactivation of Lkb1 and Keap1 abnormally increases NRF2 expression, while high NRF2 confers KLK LUAD cell resistance to ferroptosis. This study assessed the inhibition of NRF2-GSH axis to sensitize a small molecule RSL3 to induce KLK LUAD cell ferroptosis and then explored the underlying molecular mechanisms. The data showed that the NRF2-GSH inhibition sensitized RSL3 induction of KLK LUAD cell ferroptosis in vitro, while RSL3 treatment reduced level of NRF2 protein in KLK LUAD during ferroptosis. Moreover, RSL3 treatment inhibited activity of the NRF2-GSH signaling during in KLK LUAD cell ferroptosis in vitro and in vivo. Mechanistically, the RSL3 reduction of NRF2 expression was through the promotion of NRF2 ubiquitination in KLK LUAD cells. In addition, RSL3 was able to directly bind to USP11, a recently identified de-ubiquitinase of NRF2, and inactivate USP11 protein to induce NRF2 protein ubiquitination and degradation in KLK LUAD cells. These data revealed a novel mechanism of RSL3 induction in KLK LUAD cell ferroptosis by suppression of the USP11-NRF2-GSH signaling. Future study will confirm RSL3 as a novel therapeutic approach in control of KLK lung adenocarcinoma.

Cullin 3 mutant causing familial hyperkalemic hypertension lacks normal activity in the kidney.

Mutations in the ubiquitin ligase scaffold protein cullin 3 (CUL3) cause the disease familial hyperkalemic hypertension (FHHt). We recently reported that in the kidney, aberrant mutant CUL3 (CUL3-Δ9) activity lowers the abundance of CUL3-Δ9 and Kelch-like 3, the CUL3 substrate adaptor for with-no-lysine kinase 4 (WNK4) and that this is mechanistically important. However, whether CUL3-Δ9 exerts additional effects on other targets that may alter renal function is unclear. Here, we sought to determine 1) whether CUL3-Δ9 expression can rescue the phenotype of renal tubule-specific Cul3 knockout mice, and 2) whether CUL3-Δ9 expression affects other CUL3 substrates. Using an inducible renal tubule-specific system, we studied two CUL3-Δ9-expressing mouse models: Cul3 knockout (Cul3-/-/Δ9) and Cul3 heterozygous background (Cul3+/-/Δ9, FHHt model). The effects of CUL3-Δ9 in these mice were compared with Cul3-/- and Cul3+/- mice. Similar to Cul3-/- mice, Cul3-/-/Δ9 mice displayed polyuria with loss of aquaporin 2 and collecting duct injury; proximal tubule injury also occurred. CUL3-Δ9 did not promote degradation of two CUL3 targets that accumulate in the Cul3-/- kidney: high-molecular-weight (HMW) cyclin E and NAD(P)H:quinone oxidoreductase 1 (NQO1) [a surrogate for the CUL3-Kelch-like ECH-associated protein 1 (KEAP1) substrate nuclear factor erythroid-2-related factor 2]. Since CUL3-Δ9 expression cannot rescue the Cul3-/- phenotype, our data suggest that CUL3-Δ9 cannot normally function in ubiquitin ligase complexes. In Cul3+/-/Δ9 mice, KEAP1 abundance did not differ but NQO1 abundance was higher, suggesting adaptor sequestration by CUL3-Δ9 in vivo. Together, our results provide evidence that in the kidney, CUL3-Δ9 completely lacks normal activity and can trap CUL3 substrate adaptors in inactive complexes.NEW & NOTEWORTHY CUL3 mutation (CUL3-Δ9) causes familial hyperkalemic hypertension (FHHt) by reducing adaptor KLHL3, impairing substrate WNK4 degradation. Whether CUL3-Δ9 affects other targets in kidneys remains unclear. We found that CUL3-Δ9 cannot degrade two CUL3 targets, cyclin E and nuclear factor erythroid-2-related factor 2 (NRF2; using a surrogate marker NQO1), or rescue injury or polyuria caused by Cul3 disruption. In an FHHt model, CUL3-Δ9 impaired NRF2 degradation without reduction of its adaptor KEAP1. Our data provide additional insights into CUL3-Δ9 function in the kidney.

Proanthocyanidins Activate Nrf2/ARE Signaling Pathway in Intestinal Epithelial Cells by Inhibiting the Ubiquitinated Degradation of Nrf2.

Nrf2 plays a key role in the antioxidant system, and many antioxidants can activate the Nrf2/ARE signaling pathway and alleviate oxidative stress. However, the underlying mechanisms of antioxidants, such as proanthocyanidin- (PC-) induced Nrf2 activation, remain poorly understood. In this study, PC was used on MODE-K cells at different concentrations (0, 1, 2.5, and 5 μg/mL) and different times (0, 3, 6, 12, and 24 h); then, immunoprecipitation, immunofluorescence, and Western blotting were performed to test Nrf2, Bach1, Keap1, HO-1, and NQO1 protein expressions in MODE-K cells. Results showed that PC increased Nrf2, HO-1, and NQO1 protein expressions, decreased Keap1 and Bach1 protein expressions, and enhanced ARE gene activity. PC also decreased the ubiquitinated degradation of the Nrf2 protein, increased Nrf2 protein stability, and increased Nrf2 protein expression by inhibiting Keap1-dependent Nrf2 protein degradation, promoted Nrf2 entry into the nucleus, competed with Bach1, and activated ARE elements, which in turn initiated the Nrf2/ARE signaling pathway. Thus, we conclude that PC activates the Nrf2/ARE signaling pathway in intestinal epithelial cells by inhibiting the ubiquitinated degradation of Nrf2, increasing Nrf2 protein stability and expression, and then regulating key antioxidant enzymes such as HO-1 and NQO1 to initiate cytoprotective effects.

NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

Cystathionine-y-lyase (CSE) is a critical enzyme for hydrogen sulfide (H2S) biosynthesis and plays a key role in respiratory syncytial virus (RSV) pathogenesis. The transcription factor NRF2 is the master regulator of cytoprotective and antioxidant gene expression, and is degraded during RSV infection. While some evidence supports the role of NRF2 in CSE gene transcription, its role in CSE expression in airway epithelial cells is not known. Here, we show that RSV infection decreased CSE expression and activity in primary small airway epithelial (SAE) cells, while treatment with tert-butylhydroquinone (tBHQ), an NRF2 inducer, led to an increase of both. Using reporter gene assays, we identified an NRF2 response element required for the NRF2 inducible expression of the CSE promoter. Electrophoretic mobility shift assays demonstrated inducible specific NRF2 binding to the DNA probe corresponding to the putative CSE promoter NRF2 binding sequence. Using chromatin immunoprecipitation assays, we found a 50% reduction in NRF2 binding to the endogenous CSE proximal promoter in SAE cells infected with RSV, and increased binding in cells stimulated with tBHQ. Our results support the hypothesis that NRF2 regulates CSE gene transcription in airway epithelial cells, and that RSV-induced NRF2 degradation likely accounts for the observed reduced CSE expression and activity.

Lysionotin Induces Ferroptosis to Suppress Development of Colorectal Cancer via Promoting Nrf2 Degradation.

Extensive use of substances derived from natural sources has been documented in the treatment of colorectal cancer (CRC). Lysionotin (Lys) is a flavonoid present in the flowers and leaves of Gesneriaceae family plants. Despite its various pharmacological properties, which include neuroprotective, pro, antimalarial, and anticancer effects, the therapeutic advantages of Lys for CRC remain uncertain. In this present study, we demonstrated that Lys treatment successfully inhibited cell proliferation, migration, and invasion in HCT116 and SW480 CRC cells in vitro. Intriguingly, significant ferroptosis and reactive oxygen species (ROS) accumulation in CRC cells were induced by Lys treatment, whereas antagonism of ferroptosis by Liproxstatin-1 (Lip1) pretreatment retarded the anti-CRC effects of Lys. In addition, Lys reduced the amount of Nrf2 protein in CRC cells by increasing the rate at which it is degraded. Overexpression of Nrf2 rescued Lys reduced ferroptosis, suggesting the Nrf2 signaling is a crucial determinant of whether Lys induces ferroptosis in CRC cells. We also revealed that Lys suppressed tumor growth in vivo without obvious adverse effects on the main organs of mice. In conclusion, our results discovered that Lys treatment induced ferroptosis to exert antitumor effects in HCT116 and SW480 CRC cells by modulating Nrf2 signaling, providing a potential therapeutic approach for the prevention of colorectal cancer.

Methyl 3,4-dihydroxybenzoate inhibits RANKL-induced osteoclastogenesis via Nrf2 signaling in vitro and suppresses LPS-induced osteolysis and ovariectomy-induced osteoporosis in vivo.

Osteoporosis deteriorates bone mass and biomechanical strength and is life-threatening to the elderly. In this study, we show that methyl 3,4-dihydroxybenzoate (MDHB), an antioxidant small-molecule compound extracted from natural plants, inhibits receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in vitro. Furthermore, MDHB attenuates the activation of mitogen-activated protein kinase (MAPK) and NF-κB pathways by reducing the levels of reactive oxygen species (ROS), which leads to downregulated protein expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1). We also confirm that MDHB upregulates the protein expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), an important transcription factor involved in ROS regulation, by inhibiting the ubiquitination-mediated proteasomal degradation of Nrf2. Next, animal experiments show that MDHB has an effective therapeutic effect on lipopolysaccharide (LPS)- and ovariectomized (OVX)-induced bone loss in mice. Our study demonstrates that MDHB can upregulate Nrf2 and suppress excessive osteoclast activity in mice to treat osteoporosis.

Hydrogen Sulfide Donor GYY4137 Rescues NRF2 Activation in Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) can cause severe respiratory illness in infants, immunocompromised, and older adults. Despite its burden, no vaccine or specific treatment is available. RSV infection is associated with increased reactive oxygen species (ROS) production, degradation of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), and decreased antioxidant enzymes (AOEs), leading to oxidative damage and lung injury. Hydrogen sulfide (H2S) is an endogenous gaseous molecule that plays a physiological role in numerous cellular processes and a protective role in multiple pathological conditions, displaying vasoactive, cytoprotective, anti-inflammatory, and antioxidant activities. H2S can promote NRF2 activation through the sulfhydration of Kelch-like ECH-associated protein 1, the cytoplasmic repressor of NRF2. Here we investigated whether increasing cellular H2S levels could rescue NRF2 and NRF2-dependent gene expression in RSV-infected primary airway epithelial cells. We found that treatment with the H2S donor GYY4137 significantly increased NRF2 levels and AOEs gene expression by decreasing KEAP1 levels, and by modulating pathways involved in RSV-induced NRF2 degradation, such as NRF2 ubiquitination, and promyelocytic leukemia (PML) protein levels. These results suggest that the administration of exogenous H2S can positively impact the altered redox balance associated with RSV infection, which represents an important determinant of RSV-induced lung disease.

The β-TrCP-Mediated Pathway Cooperates with the Keap1-Mediated Pathway in Nrf2 Degradation In Vivo.

Nrf2 activates cytoprotective gene expression, and Nrf2 activity is regulated through at least two protein degradation pathways: the Keap1-mediated and β-TrCP-mediated pathways. To address the relative contributions of these pathways, we generated knock-in mouse lines expressing an Nrf2SA mutant that harbored two substitution mutations of serine residues interacting with β-TrCP. The homozygous (Nrf2SA/SA) mice grew normally, with Nrf2 levels comparable to those of wild-type (WT) mice under unstressed conditions. However, when Keap1 activity was suppressed, high levels of Nrf2 accumulated in Nrf2SA/SA macrophages compared with that in WT macrophages. We crossed Nrf2SA/SA mice with mice in which Keap1 was knocked down to two different levels. We found that the Nrf2SA/SA mutation induced higher Nrf2 activity when the Keap1 level was strongly reduced, and these mice showed severe growth retardation. However, activation and growth retardation were not evident when Keap1 was moderately suppressed. These increases in Nrf2 activity induced by the Nrf2SA mutation caused severe hyperplasia and hyperkeratosis in the esophageal epithelium but did not cause abnormalities in the other tissues/organs examined. These results indicate that the β-TrCP-mediated pathway cooperates with the Keap1-mediated pathway to regulate Nrf2 activity, which is apparent when the Keap1-mediated pathway is profoundly suppressed.

Kynurenine monooxygenase inhibition and associated reduced quinolinic acid reverses depression-like behaviour by upregulating Nrf2/ARE pathway in mouse model of depression: In-vivo and In-silico studies

Kynurenine pathway, a neuroimmunological pathway plays a substantial role in depression. Consistently, increased levels of neurotoxic metabolite of kynurenine pathway; quinolinic acid (QA) found in the suicidal patients and remitted major depressive patients. QA, an endogenous modulator of N-methyl-d-aspartate receptor is produced by microglial cells, may serve as a potential candidate for a link between antioxidant defence system and immune changes in depression. Further, nuclear factor (erythroid-derived 2) like 2 (Nrf2), an endogenous antioxidant transcription factor plays a significant role in maintaining antioxidant homeostasis during basal and stress conditions. The present study was designed to explore the effects of KMO-inhibition (Kynurenine monooxygenase) and association of reduced QA on Keap1/Nrf2/ARE pathway activity in olfactory bulbectomized mice (OBX-mice). KMO catalysis the neurotoxic branch of kynurenine pathway directing the synthesis of QA. KMO inhibitionshowed significant reversal of depressive-like behaviour, restored Keap-1 and Nrf2 mRNA expression, and associated antioxidant levels in cortex and hippocampus of OBX-mice. KMO inhibition also increased PI3K/AKT mRNA expression in OBX-mice. KMO inhibition and associated reduced QA significantly decreased inflammatory markers, kynurenine and increased the 5-HT, 5-HIAA and tryptophan levels in OBX-mice. Furthermore, molecular docking studies has shown good binding affinity of QA towards ubiquitin proteasome complex and PI3K protein involved in Keap-1 dependent and independent proteasome degradation of Nrf2 respectively supporting our in-vivo findings. Hence, QA might act as pro-oxidant through downregulating Nrf2/ARE pathway along with modulating other pathways and KMO inhibition could be a potential therapeutic target for depression treatment.

Abstract 1472: Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer

Abstract Nuclear factor E2-related factor 2 (NRF2) is a master regulator of cellular redox homeostasis. The sustained overactivation of NRF2 has been observed in many different types of human malignancy, conferring an advantage for growth and survival of cancer cells. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) recognizes phosphorylated serine or threonine of a target protein and isomerizes the adjacent proline residue, thereby altering the structure, stability, and function of target proteins. We identified the three specific serine residues (Ser215, 408, and 577) present in NRF2, each of which precedes proline and undergoes phosphorylation. This prompted us to investigate whether PIN1 could bind to NRF2 and thereby influence its stability. For this purpose, we initially co-transfected HEK293T cells with expression vectors for GST-PIN1 and HA-tagged wild-type Nrf2 or non-phosphorylatable mutant constructs (NRF2S215A, NRF2S408A, and NRF2S577A). Notably, there was the most pronounced ubiquitination with concomitant degradation of NRF2 in cells harbouring NRF2S215A which was associated with abrogation of its interaction with PIN1. We then investigated the clinical relevance of NRF2 and PIN1 interaction to breast cancer progression. Immunofluorescence analysis of tissue microarrays (90 breast cancer tissues and 10 adjacent normal ones) revealed much higher (P < 0.001) expression of both NRF2 and PIN1 in the invasive ductal carcinomas than in normal tissues, which correlated with disease stages. The overexpression of NRF2 and PIN1 in breast cancer patients was also confirmed by immunohistochemical and immunoblot analyses. Next, we compared the co-localization as well as expression of NRF2 and PIN1 in the two molecular subtype tissues (140 each). Both expression and co-localization of NRF2 and PIN1 were significantly higher (P < 0.001) in more aggressive TNBC than in the luminal type tissues. We also detected the significantly elevated levels of the Pin1-Nrf2 complex formed in human breast cancer specimens, compared with those in adjacent normal tissues. Silencing of Pin1 reduced the nuclear accumulation of NRF2 which was attributable to enhanced ubiquitination. The cycloheximide pulse-chase assay unravelled a significantly faster degradation of pre-existing NRF2 in PIN1-silenced MDA-MB-231 cells, corroborating the stabilization of NRF2 protein by PIN1. To explore the regulation of NRF2 by PIN1 and their interaction in vivo, we inoculated MDA-MB-231 cells into male BALB/c nude mice. Tumors derived from PIN1 knockdown cells displayed markedly reduced levels of NRF2. In conclusion, stabilization of NRF2 through direct interaction with PIN1 may provide a novel mechanism underlying their cooperative role in breast cancer growth and progression. Citation Format: Soma Saeidi, Su-Jung Kim, Yanymee N Guillen-Quispe, Achanta Sri Venkata Jagadeesh, Young-Joon Surh. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1472.

Abstract 2113: Suppression of macrophage migration inhibitory factor (MIF) impairs tumor growth and overcomes immunotherapy resistance in KEAP1-deficient NSCLC tumors

Abstract Background: KEAP1, which regulates the degradation of the antioxidant transcription factor NRF2, is the third most commonly mutated tumor suppressor in lung adenocarcinoma (LUAD). Recent reports have provided clinical evidence that mutations in STK11/LKB1 and KEAP1 are strongly associated with immune checkpoint blockade resistance in LUAD, particularly those harboring KRAS mutations. Nevertheless, the specific mechanisms by which loss of KEAP1 impacts anti-tumor immunity in KRAS mutant tumors remains to be determined. Methods: KRAS-mutant (K) and LKB1 (KL), and/or KEAP1-deficient (KK and KLK) murine tumor models were profiled using single-cell RNA sequencing (scRNA-seq) and multiplex staining, and response to anti-PD1 treatment was assessed. Clinical samples from the MD Anderson ICON study (a cohort of 148 resected tumors from early-stage lung cancer patients) and TCGA lung cohorts were used to validate pre-clinical findings. Results: While K tumors were sensitive to anti-PD1 treatment, KEAP1-deficient isogenic tumors (KK; KLK) were refractory. KEAP1-deficient tumors were found to exhibit low immune cell infiltration and an enrichment of cancer associated fibroblasts (CAFs) and endothelial cells. scRNA-seq analysis indicated that KEAP1-deficient tumors had reduced T cell infiltration, in particular, CD8 and NK T cells, decreased B cell populations, and a marked change in M2 macrophage polarization as compared to KEAP1-proficient tumors. Multiplex analysis of CD3 and F4/80 markers confirmed these findings. In the TCGA lung cancer cohort, CD8B expression was dramatically decreased while MIF (macrophage migration inhibitory factor) was upregulated in KK tumors as compared to K LUAD tumors, and expression of KEAP1 inversely correlated with CD163, ARG2 and IL10, which are mainly secreted by macrophages. KEAP1-deficient pre-clinical tumor models showed a significant upregulation of MIF expression and secretion. CRISPR-Cas9 deletion of MIF dramatically impaired in vivo tumor growth, and enhanced T cell cytotoxic effects, anti-tumor immune response and anti-PD1 treatment in KK and KLK tumor models. Conclusions: These findings indicate that loss of KEAP1, alone or in combination with STK11/LKB1 alterations, contributes to an immunosuppressed tumor immune microenvironment. These changes appear to be mediated at least in part through MIF upregulation, providing a potential therapeutic strategy for overcoming KEAP1-dependent resistance to immunotherapy. Citation Format: Ana Galan-Cobo, Yu Qian, Fuduan Peng, Daniel James McGrail, Sonia Patel, Fahao Zhang, Xiang Zhang, Ferdinandos Skoulidis, Edwin Parra, Minghao Dang, Saxon Rodriguez, Alexandre Reuben, Ignacio Wistuba, Linghua Wang, John Victor Heymach. Suppression of macrophage migration inhibitory factor (MIF) impairs tumor growth and overcomes immunotherapy resistance in KEAP1-deficient NSCLC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2113.

Abstract 2369: The NRF2 and dimeric STAT3 complex coordinately regulates IL-23 expression: Implications for breast cancer progression

Abstract Basal-like breast cancer (BLBC) is an aggressive malignancy with a poor prognosis. Persistent activation of NRF2 and STAT3 is considered to stimulate the aggressive behavior of BLBC. An online database, NRF2-ome predicted the possibility of interplay between NRF2 and STAT3 based on domain motif interactions. However, cooperation between NRF2 and STAT3 and its implications for breast cancer progression remain elusive. Immunoprecipitation and in situ proximity analyses revealed that NRF2 and STAT3 directly interact with each other, which predominantly occurred in the nucleus of human breast cancer cells. After confirming the physical interaction between STAT3 and NRF2, we examined whether this could influence the expression or stability of each transcription factor. While silencing of NRF2 failed to alter the STAT3 expression, STAT3 knockdown facilitated degradation of NRF2. Of note, silencing of STAT3 did not affect the mRNA expression of NRF2, suggesting that STAT3-induced accumulation of NRF2 is attributable to stabilization of the preexisting protein. As the NRF2-STAT3 complex was found to accumulate predominantly in the nucleus, we speculate that NRF2 could bind to a functionally active phosphorylated form of STAT3 (P-STAT3Y705). The substitution of Y705 of STAT3 with the non-phosphorylatable phenylalanine (STAT3Y705F) abolished interaction with NRF2. Notably, the dimeric form, but not a monomeric form, of STAT3 interacts directly with the Neh1 and Neh3 domains of NRF2. Next, we investigated the functional significance of STAT3- NRF2 interaction in BLBC growth in vivo. STAT3 knockdown significantly reduced expression of NRF2 in the xenograft tumor tissues derived from MDA-MB-231 human breast cancer cells. Tumors derived from cells transfected with the non-specific control siRNA showed a pronounced interaction between STAT3 and NRF2, whereas such interaction was abolished in the STAT3-silenced xenograft tumor. Analysis of the TCGA multi-omics data showed that high levels of STAT3 and NRF2 mRNA transcripts were correlated with elevated expression of P-STAT3Y705 and NRF2 target proteins in breast cancer patients. Next generation RNA sequencing analysis identified the gene encoding interleukin-23A (IL-23A) upregulated by concurrent binding of STAT3 and NRF2 to its promoter. IL-23A is markedly overexpressed in BLBC, compared with other subtypes of breast cancer. Combined silencing of NRF2 and STAT3 inhibited BLBC growth to a greater extent than that achieved with single siRNA transfection. IL-23A depletion by siRNA also showed the similar phenotypic changes to those caused by double knockdown of both transcription factors. In conclusion, these findings suggest that the STAT3-NRF2 interaction accelerates BLBC growth and progression by augmenting IL-23A expression, which underscores the importance of subtype-specific molecular pathways in human breast cancer. Citation Format: Su-Jung Kim, Soma Saeidi, Nam-Chul Cho, Seung Hyeon Kim, Han-Byoel Lee, Wonshik Han, Hye-Kyung Na, Young-Joon Surh. The NRF2 and dimeric STAT3 complex coordinately regulates IL-23 expression: Implications for breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2369.

Reciprocal regulation of NRF2 by autophagy and ubiquitin–proteasome modulates vascular endothelial injury induced by copper oxide nanoparticles

NRF2 is the key antioxidant molecule to maintain redox homeostasis, however the intrinsic mechanisms of NRF2 activation in the context of nanoparticles (NPs) exposure remain unclear. In this study, we revealed that copper oxide NPs (CuONPs) exposure activated NRF2 pathway in vascular endothelial cells. NRF2 knockout remarkably aggravated oxidative stress, which were remarkably mitigated by ROS scavenger. We also demonstrated that KEAP1 (the negative regulator of NRF2) was not primarily involved in NRF2 activation in that KEAP1 knockdown did not significantly affect CuONPs-induced NRF2 activation. Notably, we demonstrated that autophagy promoted NRF2 activation as evidenced by that ATG5 knockout or autophagy inhibitors significantly blocked NRF2 pathway. Mechanically, CuONPs disturbed ubiquitin–proteasome pathway and consequently inhibited the proteasome-dependent degradation of NRF2. However, autophagy deficiency reciprocally promoted proteasome activity, leading to the acceleration of degradation of NRF2 via ubiquitin–proteasome pathway. In addition, the notion that the reciprocal regulation of NRF2 by autophagy and ubiquitin–proteasome was further proven in a CuONPs pulmonary exposure mice model. Together, this study uncovers a novel regulatory mechanism of NRF2 activation by protein degradation machineries in response to CuONPs exposure, which opens a novel intriguing scenario to uncover therapeutic strategies against NPs-induced vascular injury and disease.Graphical