Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative ...damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsr–double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N-diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner.
Across phyla, reduced nicotinamide adenine dinucleotide phosphate (NADPH) transfers intracellular reducing power to thioredoxin reductase-1 (TrxR1) and glutathione reductase (GR), thereby supporting ...fundamental housekeeping and antioxidant pathways. Here we show that a third, NADPH-independent pathway can bypass the need for TrxR1 and GR in mammalian liver. Most mice genetically engineered to lack both TrxR1 and GR in all hepatocytes ('TR/GR-null livers') remain long-term viable. TR/GR-null livers cannot reduce oxidized glutathione disulfide using NADPH but still require continuous glutathione synthesis. Inhibition of cystathionine γ-lyase causes rapid necrosis of TR/GR-null livers, indicating that methionine-fueled trans-sulfuration supplies the necessary cysteine precursor for glutathione synthesis via an NADPH-independent pathway. We further show that dietary methionine provides the cytosolic disulfide-reducing power and all sulfur amino acids in TR/GR-null livers. Although NADPH is generally considered an essential reducing currency, these results indicate that hepatocytes can adequately sustain cytosolic redox homeostasis pathways using either NADPH or methionine.
Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein. One approach to ...addressing this challenge is to define mutations that frequently co-occur with those in KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function mutations in the KEAP1 gene encoding Kelch-like ECH-associated protein 1 (refs. 2, 3, 4), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR-Cas9-based approach in a mouse model of KRAS-driven LUAD, we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyperactivates NRF2 and promotes KRAS-driven LUAD in mice. Through a combination of CRISPR-Cas9-based genetic screening and metabolomic analyses, we show that Keap1- or Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for stratification of human patients with lung cancer harboring KRAS/KEAP1- or KRAS/NRF2-mutant lung tumors as likely to respond to glutaminase inhibition.
Mutations in the KEAP1-NRF2 (Kelch-like ECH-associated protein 1-nuclear factor-erythroid 2 p45-related factor 2) pathway occur in up to a third of non-small cell lung cancer (NSCLC) cases and often ...confer resistance to therapy and poor outcomes. Here, we developed murine alleles of the KEAP1 and NRF2 mutations found in human NSCLC and comprehensively interrogated their impact on tumor initiation and progression. Chronic NRF2 stabilization by Keap1 or Nrf2 mutation was not sufficient to induce tumorigenesis, even in the absence of tumor suppressors, p53 or LKB1. When combined with KrasG12D/+, constitutive NRF2 activation promoted lung tumor initiation and early progression of hyperplasia to low-grade tumors but impaired their progression to advanced-grade tumors, which was reversed by NRF2 deletion. Finally, NRF2 overexpression in KEAP1 mutant human NSCLC cell lines was detrimental to cell proliferation, viability, and anchorage-independent colony formation. Collectively, these results establish the context-dependence and activity threshold for NRF2 during the lung tumorigenic process.
Stabilization of the transcription factor NRF2 promotes oncogene-driven tumor initiation but blocks tumor progression, indicating distinct, threshold-dependent effects of the KEAP1/NRF2 pathway in different stages of lung tumorigenesis.
Although acquired bone marrow failure (BMF) is considered a T cell-mediated autoimmune disease, few studies have considered contributing roles of innate immune deviations following otherwise ...innocuous infections as a cause underlying the immune defects that lead to BMF. Type I IFN signaling plays an important role in protecting hematopoiesis during systemic stress responses to the opportunistic fungal pathogen Pneumocystis. During Pneumocystis lung infection, mice deficient in both lymphocytes and type I IFN receptor (IFrag(-/-)) develop rapidly progressing BMF associated with accelerated hematopoietic cell apoptosis. However, the communication pathway eliciting the induction of BMF in response to this strictly pulmonary infection has been unclear. We developed a conditional-null allele of Ifnar1 and used tissue-specific induction of the IFrag(-/-) state and found that, following Pneumocystis lung infection, type I IFNs act not only in the lung to prevent systemic immune deviations, but also within the progenitor compartment of the bone marrow to protect hematopoiesis. In addition, transfer of sterile-filtered serum from Pneumocystis-infected mice as well as i.p. injection of Pneumocystis into uninfected IFrag(-/-) mice induced BMF. Although specific cytokine deviations contribute to induction of BMF, immune-suppressive treatment of infected IFrag(-/-) mice ameliorated its progression but did not prevent loss of hematopoietic progenitor functions. This suggested that additional, noncytokine factors also target and impair progenitor functions; and interestingly, fungal β-glucans were also detected in serum. In conclusion, our data demonstrate that type 1 IFN signaling protects hematopoiesis within the bone marrow compartment from the damaging effects of proinflammatory cytokines elicited by Pneumocystis in the lung and possibly at extrapulmonary sites via circulating fungal components.
In this study, we identified Nrf2 as a molecular target of 6-shogaol (6S), a bioactive compound isolated from ginger, in colon epithelial cells in vitro and in vivo. Following 6S treatment of HCT-116 ...cells, the intracellular GSH/GSSG ratio was initially diminished but was then elevated above the basal level. Intracellular reactive oxygen species (ROS) correlated inversely with the GSH/GSSG ratio. Further analysis using gene microarray showed that 6S upregulated the expression of Nrf2 target genes (AKR1B10, FTL, GGTLA4, and HMOX1) in HCT-116 cells. Western blotting confirmed upregulation, phosphorylation, and nuclear translocation of Nrf2 protein followed by Keap1 decrease and upregulation of Nrf2 target genes (AKR1B10, FTL, GGTLA4, HMOX1, and MT1) and glutathione synthesis genes (GCLC and GCLM). Pretreatment of cells with a specific inhibitor of p38 (SB202190), PI3K (LY294002), or MEK1 (PD098059) attenuated these effects of 6S. Using ultra-high-performance liquid chromatography–tandem mass spectrometry, we found that 6S modified multiple cysteine residues of Keap1 protein. In vivo 6S treatment induced Nrf2 nuclear translocation and significantly upregulated the expression of MT1, HMOX1, and GCLC in the colon of wild-type mice but not Nrf2–/– mice. Similar to 6S, a cysteine-conjugated metabolite of 6S (M2), which was previously found to be a carrier of 6S in vitro and in vivo, also activated Nrf2. Our data demonstrated that 6S and its cysteine-conjugated metabolite M2 activate Nrf2 in colon epithelial cells in vitro and in vivo through Keap1-dependent and -independent mechanisms.
Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1) disulfide and glutathione disulfide by thioredoxin reductase-1 ...(TrxR1) and glutathione reductase (Gsr), respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null) causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.
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•Disulfide-reducing power from NADPH or methionine traffic via a diversified network•Reducing power from either Trx1 or Grxs can support mammalian S phase replication•Reductase-insufficient cells accumulate oxidative damage and become hyperproliferative•Hyperproliferation in stressed livers, as in tumors, compensates for cell death
All cells require cytosolic disulfide-reducing power fueled by intracellular NADPH or, in mammals, alternatively generated by catabolism of the essential amino acid methionine. Prigge et al. show that liver homeostasis under cytosolic disulfide reductase paucity involves noncanonical trafficking of reducing power and a hyperproliferation-based organ survival strategy.
Besides helping to maintain a reducing intracellular environment, the thioredoxin (Trx) system impacts bioenergetics and drug metabolism. We show that hepatocyte-specific disruption of Txnrd1, ...encoding Trx reductase-1 (TrxR1), causes a metabolic switch in which lipogenic genes are repressed and periportal hepatocytes become engorged with glycogen. These livers also overexpress machinery for biosynthesis of glutathione and conversion of glycogen into UDP-glucuronate; they stockpile glutathione-S-transferases and UDP-glucuronyl-transferases; and they overexpress xenobiotic exporters. This realigned metabolic profile suggested that the mutant hepatocytes might be preconditioned to more effectively detoxify certain xenobiotic challenges. Hepatocytes convert the pro-toxin acetaminophen (APAP, paracetamol) into cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). APAP defenses include glucuronidation of APAP or glutathionylation of NAPQI, allowing removal by xenobiotic exporters. We found that NAPQI directly inactivates TrxR1, yet Txnrd1-null livers were resistant to APAP-induced hepatotoxicity. Txnrd1-null livers did not have more effective gene expression responses to APAP challenge; however, their constitutive metabolic state supported more robust GSH biosynthesis, glutathionylation, and glucuronidation systems. Following APAP challenge, this effectively sustained the GSH system and attenuated damage.
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Oxidative stress is known to play a pivotal role in the development of oral squamous cell carcinoma (OSCC). We have demonstrated that activation of the nuclear factor erythroid 2-related factor 2 ...(Nrf2) signaling pathway has chemopreventive effects against oxidative stress-associated OSCC. However, Nrf2 have dual roles in cancer development; while it prevents carcinogenesis of normal cells, hyperactive Nrf2 also promotes the survival of cancer cells. This study is aimed to understand the function of Nrf2 in regulating cellular behaviors of OSCC cells, and the potential mechanisms through which Nrf2 facilitates OSCC. We established the Nrf2-overexpressing and Nrf2-knockdown OSCC cell lines, and examined the function of Nrf2 in regulating cell proliferation, migration, invasion, cell cycle and colony formation. Our data showed that Nrf2 overexpression promoted cancer phenotypes in OSCC cells, whereas Nrf2 silencing inhibited these phenotypes. In addition, Nrf2 positively regulated Notch signaling pathway in OSCC cells in vitro. Consistent with this observation, Nrf2 activation in Keap1−/− mice resulted in not only hyperproliferation of squamous epithelial cells in mouse tongue as evidenced by increased expression of PCNA, but also activation of Notch signaling in these cells as evidenced by increased expression of NICD1 and Hes1. In conclusion, Nrf2 regulates cancer behaviors and Notch signaling in OSCC cells.
•Nrf2 overexpression promoted cancer phenotypes in OSCC cells, whereas Nrf2 silencing inhibited cancer phenotypes.•Nrf2 positively regulated Notch signaling pathway in OSCC in vitro and in vivo.•Nrf2 may regulate cellular behaviors of OSCC cells through Notch signaling.
Hydrogen sulfide signaling involves persulfide formation at specific protein Cys residues. However, overcoming current methodological challenges in persulfide detection and elucidation of Cys ...regeneration mechanisms from persulfides are prerequisites for constructing a bona fide signaling model. We here establish a novel, highly specific protein persulfide detection protocol, ProPerDP, with which we quantify 1.52 ± 0.6 and 11.6 ± 6.9 μg/mg protein steady-state protein persulfide concentrations in human embryonic kidney 293 (HEK293) cells and mouse liver, respectively. Upon treatment with polysulfides, HEK293 and A549 cells exhibited increased protein persulfidation. Deletion of the sulfide-producing cystathionine-γ-lyase or cystathionine-β-synthase enzymes in yeast diminished protein persulfide levels, thereby corroborating their involvement in protein persulfidation processes. We here establish that thioredoxin (Trx) and glutathione (GSH) systems can independently catalyze reductions of inorganic polysulfides and protein persulfides. Increased endogenous persulfide levels and protein persulfidation following polysulfide treatment in thioredoxin reductase-1 (TrxR1) or thioredoxin-related protein of 14 kDa (TRP14) knockdown HEK293 cells indicated that these enzymes constitute a potent regeneration system of Cys residues from persulfides in a cellular context. Furthermore, TrxR1-deficient cells were less viable upon treatment with toxic amounts of polysulfides compared to control cells. Emphasizing the dominant role of cytosolic disulfide reduction systems in maintaining sulfane sulfur homeostasis in vivo, protein persulfide levels were markedly elevated in mouse livers where hepatocytes lack both TrxR1 and glutathione reductase (TR/GR-null). The different persulfide patterns observed in wild-type, GR-null, and TR/GR-null livers suggest distinct roles for the Trx and GSH systems in regulating subsets of protein persulfides and thereby fine-tuning sulfide signaling pathways.
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![Ginger Compound [6]-Shogaol...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0893-228X_1520-5010/small "Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo")
