Nuclear factor erythroid 2–related factor 2 (Nrf2) serves as a “main regulator” in response to internal or external cell stressors through coordinated induction of a wide range of cytoprotective genes. In cancer cells, Nrf2 increases expression of cytoprotective genes and, as a result, promotes proliferation through inhibition of apoptosis and metabolic reprogramming. Therefore, the activation of Nrf2 is an important regulator for prevention of cancer triggered by stresses and toxins. Defense system is activated by cellular pathways to ensure that response to stresses and toxins is sufficient for needs of the body. Nrf2 is a regulator of genes mediated by antioxidant response elements. Nrf2 is a pleiotropic gene that represents highly researched strategy in cancers. During recent decades, emerging evidence shows that Nrf2 is generally activated in many types of cancer by many mechanisms. Nrf2 has been showed to contribute to chemoresistance of cancer cells, as well as carcinogenesis due to inflammation, in recent studies. This review provides an overview of current mechanisms of regulation of Nrf2 in normal cells and its dual effects in cancer. This chapter aims to rationalize these double roles by criticizing dependence of Nrf2 functions and methods behind these contradictory data.
- oxidative stress
Nrf2 is a central regulator of antioxidant response element (ARE)-related gene expression and immune response. This gene encodes a transcription factor that is a member of basic leucine zipper (bZIP) protein family. The encoded transcription factor regulates genes containing antioxidant response elements (ARE) that many of these genes involve the generation of free radicals. Nrf2 is expressed in the kidney, liver, and intestine where detoxification occurs routinely. Nrf2 is located in cytoskeleton attached to Keap1. Nrf2, encoded by the nuclear factor (erythroid derived 2)-like 2 (Nfe2l2) gene, is a leucine zipper protein and a polypeptide. It has a molecular weight of 66 kDa and is widely expressed [1, 2]. Nfe2l2 gene contains a xenobiotic responsive element (XRE) at −712(XREL1) position of promoter region and two additional XRE-like elements found in +755(XREL2) and + 850(XREL3) positions, which are directly modulated by aryl hydrocarbon receptor (AHR) activation . Nrf2, which is found in the cytoplasm in normal or stress-free conditions, migrates to the nucleus in case of oxidative stress and attaches to DNA. Mutations and changes in Nrf2 expression have been described in many cancer types [4, 5, 6, 7, 8, 9]. Upregulation of Nrf2 is linked with many types of cancer, including the lung, skin, prostate, breast, and head–neck [6, 8, 10, 11]. Many mechanisms have been reported for the increased activity of Nrf2 in cancer cells. Some of them, (1) somatic mutations in Kelch-like ECH-related protein 1 (Keap1), Cullin 3 (CUL3) or Nrf2 ; (2) epigenetic silencing of Keap1 ; (3) abnormal protein accumulation that disrupts the interaction between Nrf2 and Keap1 ; (4) transcriptional upregulation of Nrf2 through oncogene-dependent signaling ; and modification of Keap1 by metabolic programming . Increasing studies show that Nrf2 activation may not be beneficial in all types and stages of cancer over the past few years. In fact, Nrf2 can ensure survival of not only normal cells, but also cancer cells, and supports the process by which Nrf2 activation in malignant cells can sustain development of the disease. The roles of the bad or good side of Nrf2 [7, 8] have caused debates because it is still not clear whether Nrf2 acts as a tumor suppressor or oncogene [7, 9]. Nrf2 hyperactivity in tumors creates a protective environment that promotes survival by protecting cancer cells from radiotherapy, oxidative stress, and chemotherapeutic agents. Therefore, there is a growing range of research aimed at identifying the boundaries between Nrf2 positive and negative responses in cancer and targeting Nrf2 therapeutically [17, 18, 19]. It is clear that Nrf2 exactly plays its protective role without distinguishing for cancer and normal cells. Current studies acknowledge the double roles of Nrf2 in carcinogenesis: protective in the early stages and harmful in the later stages.
2. Basal state induction and cellular positions
Nrf2 is inactivated transcriptionally by binding to its regulator Keap1, which targets Nrf2 for proteasomal degradation in basal conditions. CUL3 ubiquitin ligase, which governs the degradation, is a third protein. Under stable conditions, ubiquitous Nrf2 is rapidly disrupted by 26S proteasome (Figure 1). Nrf2 has a very short half-life of less than 30 minutes. Therefore, Nrf2 is not abundant under basal conditions and, in light of current studies, supports the claim that Nrf2 is found at a relatively low level in most organs or tissues [20, 21, 22]. Nrf2-Keap1 is of first importance in balancing homeostatic environment since cells need to respond by adapting to different stresses. Cells can use highly toxic molecules to be used in the physiological signal. These molecules contain reactive oxygen species (ROS) and reactive nitrogen types (RNSs) such as hydrogen peroxide (H2O2) and nitric oxide (NO). Low concentrations of these molecules are used for adaptive intracellular signaling, and high concentrations are used for defense mechanisms against microorganisms [23, 24]. But, physiological concentrations of these molecules need to be precisely regulated, and Nrf2-Keap1 play important role in this signaling.
Nrf2 is a transcription factor that regulates cellular stress signals and reacts by directing different transcriptional programs. Limited number of researchers were working only on inhibiting its protective role and carcinogenesis in suppressing oxidative or electrophilic stress till a little more than a decade ago [25, 26], but recently, Nrf2 has become a topic of widespread interest and research area. Kelch-like ECH-related protein 1 (Keap1) is a negative regulator, has encouraged many publications, and has become an important topic of discussion. The debate focuses on whether Nrf2 is tumor suppressor or reverse oncogenic, leading to the question of whether Nrf2 should be targeted for anticancer therapeutic agents .
Genetic analysis has shown mutations in Nrf2 and Keap1 in some cancers. These mutations increase Nrf2 expression and are related with resistance to chemotherapy and poor survival rate from cancer [18, 28]. Sequence analyses of Nrf2 and Keap1 have identified many mutations within Kelch domain and in the Neh2 domain of Keap1-Nrf2; this causes Nrf2 to unstable due to Keap1’s inability to target Nrf2 for degradation and ubiquitination (Figure 1a). Whether activation or inhibition of Nrf2 is a good strategy for treatment or prevention of cancer is still unclear. In vivo studies have shown that basal Nrf2 protein levels decrease with age and associate with lower expression target genes of Nrf2 [29, 30]. Therefore, it is more probable that Nrf2 acts as a defense against the aging process caused by free radicals, which gradually decreases over time, leading to accumulation of free radicals that can cause cancer progression [31, 32].
3. Keap1-Nrf2-ARE signaling
Keap1 has more than 20 groups of free sulfhydryl (-SH) in the cysteine residues. These highly reactive molecules for stress act as sensitive sensors. Reactive cysteine thiols are present as (S-) under physiological pH and are more reactive to ROS/RNS than sulfhydryl groups . Keap1 alters cysteine residues, giving a response to oxidative or electrophilic stresses [34, 35]. Tert-butylhydroquinone (tBHQ), an electrophile, reacts with reactive cysteine residues in Keap1 to activate Nrf2 . Binding of tBHQ to Keap1 does not impair binding of Nrf2 to Keap1; this indicates that sequestration of Nrf2 from Keap1 homodimer cannot explain electrophilic mediated induction of Nrf2 accumulation within cells . These modifications result in conformational changes of Keap1 and reverse degradation of Nrf2, which is then transcriptionally activated.
Different types of stressors react differently with the cysteine residues in Keap1, suggesting that the residues of cysteine in some way contribute to activity of Keap1 individually or in combination (23,24). This indicates that Nrf2-Keap1 mechanism is not a simple “on” or “off” button mechanism but can instead respond with different mechanisms by various stress factors [23, 35].
Some of the promising Nrf2 activator or inhibitor agents are currently in different phases of clinical trials [38, 39, 40]. Human clinical trials were kept assessing the effects of inducers [41, 42]. These include: (1) approved and other purpose agents such as dimethyl fumarate (DMF) and Oltipraz; (2) compounds purified from natural sources such as broccoli sprouts, curcumin, resveratrol, and sulforaphane; and (3) highly potent triterpenoid derivatives, e.g., RTA 402-408 and CXA-10 .
Nrf2 protein, which forms a heterodimer structure with MA or Jun protein in the nucleus (Figure 1b), binds to ARE (Antioxidant Response Element) sequence on DNA and provides regulation of related gene expressions in favor of activation of antioxidant mechanisms . PI3-kinase is responsible for nuclear translocation of Nrf2 and binding of Nrf2 to ARE to induce enzymes such as GST, HO-1, and NQO1. It is essentially a common DNA sequence called an antioxidant response element (ARE) similar to Nrf2-binding motif for induction .
Cancer cells have higher ROS levels than normal cells. Nevertheless, they can adapt to high ROS levels with the activating of certain ways that allow them to proliferate and survive. These ways include the activation of antioxidants to reduce ROS, as well as metabolic reprogramming pathways that can produce more ROS and make cancer cells more vulnerable to future stress [46, 47]. Keap1-Nrf2 pathway is one of the most important signaling pathways that play a role in the survival and defense of cell against xenobiotics and oxidative stress.
4. Keap1-dependent regulation of Nrf2
Keap1 contains 27 cysteines, which account for 4.33% of all amino acids, whereas the average cysteines content in human proteins is 2.26% . Proteomic analyses have found that several of the 27 cysteines in Keap1 have been modified to respond to different electrophiles . However, only three of the Keap1 cysteines, Cys151, Cys273, and Cys288, were found to be functionally important for Keap1-Nrf2 regulation . Cys273 and Cys288 target Cys151, which is a subset of Nrf2 activators, although they are essentials for Keap1 to inhibit Nrf2 under basal conditions. During oxidative stress, ROS reacts with cysteine residues of Keap1, including C151, C273, and C288, allowing Nrf2 to escape Keap1-mediated degradation .
Human Nrf2 protein has 605 amino acids and seven highly preserved Neh (Nrf2-ECH homology) domains from Neh1 to Neh7 (Figure 2). Neh1 domain contains small musculoaponeurotic fibrosarcoma homologous proteins (MafF, MafG or MafK) and a basic leucine zipper (bZIP) motif that is heterodimerized for DNA binding and transcriptional activation . Neh2 regulatory domain of N-terminal contains DLG and ETGE motifs critical to Keap1 binding and resulting in Nrf2 degradation .
Neh3 domain is located in C-terminal of Nrf2 contains VFLVPK motif, which is critical for binding to CHD6 helicase . Nrf2’s N-terminal includes two transactivation domains, Neh4 and Neh5, and both domains were found to be necessary for Nrf2’s maximum transactivation activity . Neh6 domain contains a degron containing a DSG motif embedded in a set of serine-rich residues. This binding region is a docking site for adaptor protein β-TrCP, which mediates ubiquitin ligase of Nrf2 by a Cullin1-Rbx1 complex . Neh7 domain interacts with the retinoic acid receptor a (RARa) and suppresses Nrf2 transcriptional activity in the nucleus .
Nrf2 degradation can be stopped when exposed to electrophiles and ROS. Reactive cysteines are a small set of protein cysteines with pKa values relatively low around 4 and 5 due to the effect of surrounding amino acid microenvironment, unlike most protein cysteine thiols with pKa values of about 8.5. Reactive thiols are perfectly targets for electrophiles, and indeed, several electrophilic reagents have been shown to directly alter thiols. The modification of Keap1 is thought to impair structural integrity of Keap1-Cul3 E3 ligase complex, causing a decrease in ubiquitination activity, thereby facilitating accumulation of Nrf2 [58, 59]. In recent studies, presence of unrestricted Keap1 has recognized deleterious effects to cellular homeostasis and highlighted Nrf2’s role as Keap1’s suppressor that implies that Nrf2 and Keap1 are mutually blocking each other . Under normal conditions, Keap1 plays an important role in limiting Nrf2 activity by binding to DLG/ETGE motifs in the Neh2 domain and inducing ubiquitination and proteasomal degradation of Nrf2 (Figure 1a) .
Permanent activation of Nrf2 in tumor cells is activation of p62, that is, a multifunctional protein involved in selective autophagy that is often overexpressed in tumors . p62 in phosphoryl form can bind with Keap1 in the same binding domain for Nrf2, thereby competitively inhibiting Keap1/Nrf2 interaction resulting in Nrf2 stabilization and translocated into the nucleus. Nrf2 can consecutively upregulate p62 gene expression, thereby upregulation of a pro-survival circuit that can support tumor formation. The accumulation of proteins and metabolites that disrupt Keap1-Nrf2 can activate Nrf2 in cancer. p62 is the best-known disruptor that competes with Nrf2 for direct attachment to Keap1 through an SQSTM1 motif similar to ETGE motif in Nrf2 . After p62 is bound to Keap1, it causes Keap1 to go into autophagic degradation . Recent studies have shown that p62 gene expression is upregulated in hepatocellular carcinoma and that the activation of Nrf2 induced by p62 is critical for HCC development [64, 65].
Kelch domain of Keap1 interacts with two different sequences of amino acids found in the N-terminal of Nrf2: ETGE and DLG . Based on a series of critical observations, “Hinge-Latch model” (Figure 3) that is Keap1-Nrf2 interactive two-site binding model was proposed . ETGE motif has a higher affinity for Kelch-repetition domain than DLG motif. Therefore, Keap1 captures Nrf2 through ETGE motif before DLG motif is attached to adjacent unoccupied Kelch-repeat domains; this is called the “hinge and latch” mechanism [67, 68]. The modes of binding DLG and ETGE to Keap1 are quite different . Keap1-DLG binding is characterized as kinetically a “fast-on-fast-off,” which is thermodynamically guided by both enthalpy and entropy. In contrast, ETGE-Keap1 binding is characterized by completely enthalpy guided and involves a two-state reaction that leads to more stable conformation [70, 71]. These findings support the claim that DLG motif serves as a converter that transmits environmental stress to Nrf2 induction as a latch (Figure 3).
5. Nrf2-regulated cytoprotective genes
Nrf2 plays a major role in the protective mechanism against xenobiotics, which can initiate carcinogenesis by damaging DNA . Nrf2 increases expression of antioxidant enzymes. Gene transcription profiles showed that not all genes around Nrf2 are transcriptionally regulated by Nrf2 binding. These genes require transcription factors, cofactors, and intermediaries for complete activation . Antioxidant molecules such as glutathione (GSH), vitamin C and E, bilirubin, and antioxidant proteins such as thioredoxin (Trx), Superoxide Dismutase (SOD), catalase, peroxiredoxin, glutathione peroxidase (GPx) are major antioxidant molecules that play a role in balancing oxidative stress. Nrf2 and its downregulatory effectors have been shown to be critically important regulators in the regulation of intracellular redox state and in protecting cells from oxidative stress and chemical damage in the lungs and liver [74, 75]. Nrf2 loss has been associated with advanced metastasis. For example, loss of Nrf2 initiates a harmful cascading of decreased GST expression and raises ROS level, ultimately leading to DNA damage and tumor formation . The role of Nrf2 signaling as a tumor suppressor is due to a lot of in vivo studies comparing susceptibility to carcinogenicity chemically induced in Nrf2-knockout mice (Nfe2l2−/−) and wild-type mice. In this context, Nrf2-null mice were found to be more prone to developing bladder, stomach, and skin cancer when exposed to carcinogen substances compared with wild-type mice. This gene has a deficiency and susceptibility to oxidative damage, and chemical carcinogenesis increases in Nrf2-knockout mice . Expression of antioxidant and phase II enzymes was found to be eliminated in mice with Nrf2 deficiency. Heavy quinone-induced made mice with Nrf2 deficiency more prone to skin cancer, while NQO1 and GST expression regulated by Nrf2 decreased compared with wild mice . In addition, expression levels of ARE-mediated genes such as glutathione-
Nrf2 downstream targets are separated into three main groups: phase I and phase II drug metabolizing enzymes (DMEs) and phase III drug carriers. Phase I enzymes oxidize drugs or xenobiotics such as aldo-ketoreductases (AKRs) and cytochrome P450s (CYPs) encoded by genes regulated by Nrf2; Phase II enzymes conjugate products of phase I reactions, while phase III enzymes carry final metabolites out of cells in collaboration to implement a cytoprotective function. Several phase I enzymes also play important roles in removal of xenobiotics through hydrolysis, reduction, and oxidation. Phase I, cytochrome P450 (CYP) family, aldehyde oxidase (ACO) contain modification of xenobiotics by enzymes such as aldehyde dehydrogenases (ALDHs), aldo-ketoreductases (AKRs), alcohol dehydrogenases (ADHs), esterase, flavins-containing monooxygenases (FMOs), and cyclooxygenases (COXs) ; (ii) phase II enzymes such as GST, UGT, Sulfotransferases (SULTs), N-acetyltransferases (NATs), and methyltransferases (MTs) add polar groups to phase I products to prepare them from excretion ; and (iii) carriers, ATP-binding cassette (ABC), and dissolved solute-carrier (SLC) export metabolites (modified) out of the cell .
Phase II drug metabolism or conjugation reactions involve a different group of enzymes, which often lead to water-soluble products that can be excreted with bile or urine. Conjugation reactions include: glucuronidation, acetylation, sulfation, methylation, amino acid and glutathione conjugation.
The protective effects of upregulation of Nrf2 signaling can be in various forms. Protection can be instantaneous by stimulation of genes that are directly regulated through Nrf2 binding to AREs in the target genes [83, 84]. Protective effects can be secondary through stimulation of macromolecular damage removal/repair mechanisms [84, 85]. Protective effects can be tertial through induction of tissue repair/regeneration pathways . p53, which is a tumor suppressor, reduces Nrf2 activity, stopping cell growth and inducing apoptosis .
Regulation of Nrf2 can be unstable against the loss of inducible nature of Nrf2 signaling and acquisition of a structurally active phenotype. The constructive signal for expression of cytoprotective enzymes would give cells surviving chance under stress conditions. This seemingly positive condition would become a serious disadvantage in progression of cancer pathogenesis and treatment. Therefore, structural activation or increased signaling of Nrf2 pathway can be determined for destiny of cell during tumorigenesis and affect response to radiotherapy and chemotherapy. Under these circumstances, Nrf2 can be described as an oncogene [32, 88].
6. Tumor suppressor and oncogenic roles of Nrf2
Nrf2 protects cells from oxidative stress, in vivo studies with rats have shown that basal Nrf2 protein levels decrease with age and correlate with lower expression of Nrf2 target genes . Increasing Nrf2 activity, which facilitates tumor formation and proliferation of K-Ras, B-Raf, and Myc in cancer cells, helps reduce intracellular ROS levels. Overexpression of Nrf2 also regulates cell proliferation by directing glucose and glutamine to anabolic pathways, increasing purine synthesis and affecting pentose phosphate pathway to promote cell proliferation . In addition, under hypoxia/reoxygenation, Keap1 reduced expression and increased expression of Prx1, Nrf2, and peroxiredoxin-1 (Prx1) proteins, which reduce ROS levels and ultimately protect cancer cell . In a study of Nrf2+/− mice exposed to diesel exhaust and N-nitroso butyl (4-hydroxybutyl) amine, increased pulmonary DNA adducts and bladder tumors were shown [92, 93].
Mutations that cause Keap1 loss of function and Nrf2 function gain and gene mutations that lead to electrophilic metabolite accumulation can also trigger continuous Nrf2 activity in cancer . Nrf2 and its target gene expression levels can serve as biomarkers for diagnosis of lung cancers. Large-scale multi-tumor sequencing efforts by The Cancer Genome Atlas (TCGA) project found that CUL3 and Keap1 function loss and Nrf2 functional gain mutations were significantly enriched in lung adenocarcinoma, pulmonary squamous cell carcinoma and lung squamous cell carcinoma, and bladder cancer [95, 96, 97].
Oncogene activation, including oncogenic mutants of K-Ras, B-Raf, and c-Myc, may cause upregulated expression of Nfe2l2 gene . K-Ras and B-Raf activations induce transcription of Nrf2 through activation of Jun and Myc transcription factors. This activation of Nrf2 has been shown to be critical for increased chemoresistance and tumor growth of Ras mutant cancer cells [15, 99].
mRNA and protein levels of AKRs have been shown to be biomarkers for diagnosis of cancers activated by Nrf2 . AKRs are detected with greater precision than Nrf2. Several studies have shown that cancer cells with high levels of Nrf2 are less susceptible to common chemotherapeutic agents such as etoposide, carboplatin, cisplatin, 5-fluorouracil, and doxorubicin [10, 11, 101, 102].
Nrf2-targeting agents with advanced specificity are needed to increase effectiveness of cancer treatment. In addition, controversial roles of Nrf2 in cancer prevention and progression suggest that more issues need to be addressed to determine optimal use of Nrf2 activators or inhibitors in the clinic . Nrf2 may have both tumor-suppressive and -promoting effects (Figure 4). Nrf2 target genes regulate autophagy, mitochondrial physiology, redox homeostasis, proteasomal degradation, energy metabolism, iron metabolism, amino acid metabolism, survival, reproduction, DNA repair, and drug metabolism and excretion [104, 105, 106].
Overexpression of sMaf proteins results in a decrease in transcriptional activity of Nrf2. However, there are no studies that identify different gene expressions or mutations of sMaf family proteins in tumors .
Dysregulation of epigenetic mechanism is hallmark of cancer. It is shown that acetylation conditions resulted in promoting nuclear localization of Nrf2, while deacetylation promoting cytoplasmic rather than nuclear localization of Nrf2. Hypermethylation of Keap1 promoter has been detected in the breast, lung, brain, and colorectal [108, 109, 110] and causes a decrease in Keap1 mRNA production and therefore Nrf2 activation [111, 112, 113].
In general, the question of whether Nrf2 activation is bilateral role is explained via several contexts, levels, and mechanisms. The above data strongly suggest that inhibition or activation of Nrf2 alone or in combination can be a promising therapeutic strategy for cancer treatment. In numerous in vitro and in vivo studies, multiple genomic, transcriptional, and proteomic mechanisms related to Nrf2 activation in cancer have been explained in detail. Targeting one of redox signaling factors, such as Nrf2, seems like a crucial challenge for designing efficient cancer therapeutic strategies. Nrf2 is a well-known regulator that regulates antioxidant system and mediates tumorigenesis and suppression. Nrf2 should be considered an important therapeutic target. Nrf2 is in the focus of worldwide research, and we are expected to continue to see more research outputs in the future.
Conflict of interest
There is no conflict of interest.
Chanas SA, Jiang Q, McMahon M, McWalter GK, McLellan LI, Elcombe CR, et al. Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. The Biochemical Journal. 2002; 365(Pt 2):405-416
Kwak MK, Wakabayashi N, Itoh K, Motohashi H, Yamamoto M, Kensler TW. Modulation of gene expression by cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway. Identification of novel gene clusters for cell survival. The Journal of Biological Chemistry. 2003; 278(10):8135-8145
Miao W, Hu L, Scrivens PJ, Batist G. Transcriptional regulation of NF-E2 p45-related factor (NRF2) expression by the aryl hydrocarbon receptor-xenobiotic response element signaling pathway: Direct cross-talk between phase I and II drug-metabolizing enzymes. The Journal of Biological Chemistry. 2005; 280(21):20340-20348
Nioi P, Nguyen T. A mutation of Keap1 found in breast cancer impairs its ability to repress Nrf2 activity. Biochemical and Biophysical Research Communications. 2007; 362(4):816-821
Jiang T, Chen N, Zhao F, Wang XJ, Kong B, Zheng W, et al. High levels of Nrf2 determine chemoresistance in type II endometrial cancer. Cancer Research. 2010; 70(13):5486-5496
Padmanabhan B, Tong KI, Ohta T, Nakamura Y, Scharlock M, Ohtsuji M, et al. Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer. Molecular Cell. 2006; 21(5):689-700
Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, et al. Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Medicine. 2006; 3(10):1865-1876
Kim YR, Oh JE, Kim MS, Kang MR, Park SW, Han JY, et al. Oncogenic NRF2 mutations in squamous cell carcinomas of oesophagus and skin. The Journal of Pathology. 2010; 220(4):446-451
He F, Ru X, Wen T. NRF2, a transcription factor for stress response and beyond. International Journal of Molecular Sciences. 2020; 21(13):4777
Ohta T, Iijima K, Miyamoto M, Nakahara I, Tanaka H, Ohtsuji M, et al. Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer cell growth. Cancer Research. 2008; 68(5):1303-1309
Shibata T, Kokubu A, Gotoh M, Ojima H, Ohta T, Yamamoto M, et al. Genetic alteration of Keap1 confers constitutive Nrf2 activation and resistance to chemotherapy in gallbladder cancer. Gastroenterology. 2008; 135(4):1358-1368. 1368 e1-4
Yoo NJ, Kim HR, Kim YR, An CH, Lee SH. Somatic mutations of the KEAP1 gene in common solid cancers. Histopathology. 2012; 60(6):943-952
Cheng D, Wu R, Guo Y, Kong AN. Regulation of Keap1-Nrf2 signaling: The role of epigenetics. Curr Opin Toxicol. 2016; 1:134-138
Motohashi H, Yamamoto M. Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends in Molecular Medicine. 2004; 10(11):549-557
DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K, et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature. 2011; 475(7354):106-109
Leinonen HM, Kansanen E, Polonen P, Heinaniemi M, Levonen AL. Dysregulation of the Keap1-Nrf2 pathway in cancer. Biochemical Society Transactions. 2015; 43(4):645-649
Lau A, Villeneuve NF, Sun Z, Wong PK, Zhang DD. Dual roles of Nrf2 in cancer. Pharmacological Research. 2008; 58(5-6):262-270
Wang XJ, Sun Z, Villeneuve NF, Zhang S, Zhao F, Li Y, et al. Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2. Carcinogenesis. 2008; 29(6):1235-1243
Sporn MB, Liby KT. NRF2 and cancer: The good, the bad and the importance of context. Nature Reviews. Cancer. 2012; 12(8):564-571
Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T, et al. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Molecular and Cellular Biology. 2004; 24(16):7130-7139
Taguchi K, Yamamoto M. The KEAP1-NRF2 system as a molecular target of cancer treatment. Cancers (Basel). 2020; 13(1):46
Itoh K, Wakabayashi N, Katoh Y, Ishii T, O'Connor T, Yamamoto M. Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electrophiles. Genes to Cells. 2003; 8(4):379-391
Yamamoto T, Suzuki T, Kobayashi A, Wakabayashi J, Maher J, Motohashi H, et al. Physiological significance of reactive cysteine residues of Keap1 in determining Nrf2 activity. Molecular and Cellular Biology. 2008; 28(8):2758-2770
Budak M. Radiation and DNA Methylation Mechanisms, DNA Methylation Mechanism. Rijeka: IntechOpen; 2020. pp. 3-17
Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, et al. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochemical and Biophysical Research Communications. 1997; 236(2):313-322
Ramos-Gomez M, Kwak MK, Dolan PM, Itoh K, Yamamoto M, Talalay P, et al. Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proceedings of the National Academy of Sciences of the United States of America. 2001; 98(6):3410-3415
Kensler TW, Wakabayashi N. Nrf2: Friend or foe for chemoprevention? Carcinogenesis. 2010; 31(1):90-99
Taguchi K, Motohashi H, Yamamoto M. Molecular mechanisms of the Keap1-Nrf2 pathway in stress response and cancer evolution. Genes to Cells. 2011; 16(2):123-140
Zhang H, Davies KJA, Forman HJ. Oxidative stress response and Nrf2 signaling in aging. Free Radical Biology & Medicine. 2015; 88(Pt B):314-336
Schmidlin CJ, Dodson MB, Madhavan L, Zhang DD. Redox regulation by NRF2 in aging and disease. Free Radical Biology & Medicine. 2019; 134:702-707
Sykiotis GP, Bohmann D. Stress-activated cap'n'collar transcription factors in aging and human disease. Science Signaling. 2010; 3(112):re3
Shelton P, Jaiswal AK. The transcription factor NF-E2-related factor 2 (Nrf2): A protooncogene? The FASEB Journal. 2013; 27(2):414-423
Janssen-Heininger YM, Mossman BT, Heintz NH, Forman HJ, Kalyanaraman B, Finkel T, et al. Redox-based regulation of signal transduction: Principles, pitfalls, and promises. Free Radical Biology & Medicine. 2008; 45(1):1-17
Hayes JD, McMahon M, Chowdhry S, Dinkova-Kostova AT. Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway. Antioxidants & Redox Signaling. 2010; 13(11):1713-1748
Kobayashi M, Li L, Iwamoto N, Nakajima-Takagi Y, Kaneko H, Nakayama Y, et al. The antioxidant defense system Keap1-Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds. Molecular and Cellular Biology. 2009; 29(2):493-502
Abiko Y, Miura T, Phuc BH, Shinkai Y, Kumagai Y. Participation of covalent modification of Keap1 in the activation of Nrf2 by tert-butylbenzoquinone, an electrophilic metabolite of butylated hydroxyanisole. Toxicol Appl Pharm. 2011; 255(1):32-39
Taguchi K, Yamamoto M. The KEAP1-NRF2 system in cancer. Frontiers in Oncology. 2017; 7:85
Magesh S, Chen Y, Hu L. Small molecule modulators of K eap1-N rf2-ARE pathway as potential preventive and therapeutic agents. Medicinal Research Reviews. 2012; 32(4):687-726
Jimenez-Osorio AS, Gonzalez-Reyes S, Pedraza-Chaverri J. Natural Nrf2 activators in diabetes. Clinica Chimica Acta. 2015; 448:182-192
Houghton CA, Fassett RG, Coombes JS. Sulforaphane and other nutrigenomic Nrf2 activators: Can the Clinician’s expectation Be matched by the reality? Oxidative Medicine and Cellular Longevity. 2016; 2016:7857186
Kensler TW, Chen JG, Egner PA, Fahey JW, Jacobson LP, Stephenson KK, et al. Effects of glucosinolate-rich broccoli sprouts on urinary levels of aflatoxin-DNA adducts and phenanthrene tetraols in a randomized clinical trial in He Zuo township, Qidong, People's Republic of China. Cancer Epidemiology, Biomarkers & Prevention. 2005; 14(11 Pt 1):2605-2613
Wang JS, Shen X, He X, Zhu YR, Zhang BC, Wang JB, et al. Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People's Republic of China. Journal of the National Cancer Institute. 1999; 91(4):347-354
Tebay LE, Robertson H, Durant ST, Vitale SR, Penning TM, Dinkova-Kostova AT, et al. Mechanisms of activation of the transcription factor Nrf2 by redox stressors, nutrient cues, and energy status and the pathways through which it attenuates degenerative disease. Free Radical Biology & Medicine. 2015; 88(Pt B):108-146
Nguyen T, Sherratt PJ, Pickett CB. Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annual Review of Pharmacology and Toxicology. 2003; 43(1):233-260
Ma Q. Role of nrf2 in oxidative stress and toxicity. Annual Review of Pharmacology and Toxicology. 2013; 53:401-426
Harris IS, Treloar AE, Inoue S, Sasaki M, Gorrini C, Lee KC, et al. Glutathione and thioredoxin antioxidant pathways synergize to drive cancer initiation and progression. Cancer Cell. 2015; 27(2):211-222
Schumacker PT. Reactive oxygen species in cancer: A dance with the devil. Cancer Cell. 2015; 27(2):156-157
Kopacz A, Kloska D, Forman HJ, Jozkowicz A, Grochot-Przeczek A. Beyond repression of Nrf2: An update on Keap1. Free Radical Biology & Medicine. 2020; 157:63-74
Kansanen E, Jyrkkanen HK, Levonen AL. Activation of stress signaling pathways by electrophilic oxidized and nitrated lipids. Free Radical Biology & Medicine. 2012; 52(6):973-982
Shibata T, Ohta T, Tong KI, Kokubu A, Odogawa R, Tsuta K, et al. Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy. Proceedings of the National Academy of Sciences. 2008; 105(36):13568-13573
Baird L, Lleres D, Swift S, Dinkova-Kostova AT. Regulatory flexibility in the Nrf2-mediated stress response is conferred by conformational cycling of the Keap1-Nrf2 protein complex. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110(38):15259-15264
Chen QM. Nrf2 for cardiac protection: Pharmacological options against oxidative stress. Trends in Pharmacological Sciences. 2021; 42(9):729-744
Tong KI, Katoh Y, Kusunoki H, Itoh K, Tanaka T, Yamamoto M. Keap1 recruits Neh2 through binding to ETGE and DLG motifs: Characterization of the two-site molecular recognition model. Molecular and Cellular Biology. 2006; 26(8):2887-2900
Nam LB, Keum YS. Binding partners of NRF2: Functions and regulatory mechanisms. Archives of Biochemistry and Biophysics. 2019; 678:108184
Katoh Y, Itoh K, Yoshida E, Miyagishi M, Fukamizu A, Yamamoto M. Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription. Genes to Cells. 2001; 6(10):857-868
Rojo AI, Medina-Campos ON, Rada P, Zuniga-Toala A, Lopez-Gazcon A, Espada S, et al. Signaling pathways activated by the phytochemical nordihydroguaiaretic acid contribute to a Keap1-independent regulation of Nrf2 stability: Role of glycogen synthase kinase-3. Free Radical Biology & Medicine. 2012; 52(2):473-487
Keum YS, Choi BY. Molecular and chemical regulation of the Keap1-Nrf2 signaling pathway. Molecules. 2014; 19(7):10074-10089
Holland R, Hawkins AE, Eggler AL, Mesecar AD, Fabris D, Fishbein JC. Prospective type 1 and type 2 disulfides of Keap1 protein. Chemical Research in Toxicology. 2008; 21(10):2051-2060
Sekhar KR, Rachakonda G, Freeman ML. Cysteine-based regulation of the CUL3 adaptor protein Keap1. Toxicol Appl Pharm. 2010; 244(1):21-26
Bono S, Feligioni M, Corbo M. Impaired antioxidant KEAP1-NRF2 system in amyotrophic lateral sclerosis: NRF2 activation as a potential therapeutic strategy. Molecular Neurodegeneration. 2021; 16(1):71
Itoh K, Mimura J, Yamamoto M. Discovery of the negative regulator of Nrf2, Keap1: A historical overview. Antioxidants & Redox Signaling. 2010; 13(11):1665-1678
Copple IM, Lister A, Obeng AD, Kitteringham NR, Jenkins RE, Layfield R, et al. Physical and functional interaction of sequestosome 1 with Keap1 regulates the Keap1-Nrf2 cell defense pathway. The Journal of Biological Chemistry. 2010; 285(22):16782-16788
Jung BJ, Yoo HS, Shin S, Park YJ, Jeon SM. Dysregulation of NRF2 in cancer: From molecular mechanisms to therapeutic opportunities. Biomol Ther (Seoul). 2018; 26(1):57-68
Komatsu M, Kurokawa H, Waguri S, Taguchi K, Kobayashi A, Ichimura Y, et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nature Cell Biology. 2010; 12(3):213-223
Umemura A, He F, Taniguchi K, Nakagawa H, Yamachika S, Font-Burgada J, et al. p62, upregulated during Preneoplasia, induces hepatocellular carcinogenesis by maintaining survival of stressed HCC-initiating cells. Cancer Cell. 2016; 29(6):935-948
Tong KI, Kobayashi A, Katsuoka F, Yamamoto M. Two-site substrate recognition model for the Keap1-Nrf2 system: A hinge and latch mechanism. Biological Chemistry. 2006; 387(10-11):1311-1320
Hayes JD, McMahon M. NRF2 and KEAP1 mutations: Permanent activation of an adaptive response in cancer. Trends in Biochemical Sciences. 2009; 34(4):176-188
Martin-Montalvo A, Villalba JM, Navas P, de Cabo R. NRF2, cancer and calorie restriction. Oncogene. 2011; 30(5):505-520
Suzuki T, Yamamoto M. Molecular basis of the Keap1-Nrf2 system. Free Radical Biology & Medicine. 2015; 88(Pt B):93-100
Fukutomi T, Takagi K, Mizushima T, Ohuchi N, Yamamoto M. Kinetic, thermodynamic, and structural characterizations of the association between Nrf2-DLGex degron and Keap1. Molecular and Cellular Biology. 2014; 34(5):832-846
Baird L, Swift S, Lleres D, Dinkova-Kostova AT. Monitoring Keap1-Nrf2 interactions in single live cells. Biotechnology Advances. 2014; 32(6):1133-1144
Giudice A, Montella M. Activation of the Nrf2-ARE signaling pathway: A promising strategy in cancer prevention. BioEssays. 2006; 28(2):169-181
Chorley BN, Campbell MR, Wang X, Karaca M, Sambandan D, Bangura F, et al. Identification of novel NRF2-regulated genes by ChIP-Seq: Influence on retinoid X receptor alpha. Nucleic Acids Research. 2012; 40(15):7416-7429
Chan K, Han XD, Kan YW. An important function of Nrf2 in combating oxidative stress: Detoxification of acetaminophen. Proceedings of the National Academy of Sciences of the United States of America. 2001; 98(8):4611-4616
Chan K, Kan YW. Nrf2 is essential for protection against acute pulmonary injury in mice. Proceedings of the National Academy of Sciences of the United States of America. 1999; 96(22):12731-12736
Frohlich DA, McCabe MT, Arnold RS, Day ML. The role of Nrf2 in increased reactive oxygen species and DNA damage in prostate tumorigenesis. Oncogene. 2008; 27(31):4353-4362
Long DJ 2nd, Waikel RL, Wang XJ, Perlaky L, Roop DR, Jaiswal AK. NAD(P)H:Quinone oxidoreductase 1 deficiency increases susceptibility to benzo(a)pyrene-induced mouse skin carcinogenesis. Cancer Research. 2000; 60(21):5913-5915
Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang MI, Kobayashi A, Yamamoto M, et al. Protection against electrophile and oxidant stress by induction of the phase 2 response: Fate of cysteines of the Keap1 sensor modified by inducers. Proceedings of the National Academy of Sciences of the United States of America. 2004; 101(7):2040-2045
Telkoparan-Akillilar P, Suzen S, Saso L. Pharmacological applications of Nrf2 inhibitors as potential antineoplastic drugs. International Journal of Molecular Sciences. 2019; 20(8):2025
Fan PW, Zhang D, Halladay JS, Driscoll JP, Khojasteh SC. Going beyond common drug metabolizing enzymes: Case studies of biotransformation involving aldehyde oxidase, gamma-Glutamyl Transpeptidase, Cathepsin B, Flavin-containing monooxygenase, and ADP-Ribosyltransferase. Drug Metabolism and Disposition. 2016; 44(8):1253-1261
Stanley LA. Drug metabolism. In: Badal S, Delgoda R, editors. Pharmacognosy. London, UK: Elsevier; 2017. pp. 527-545. DOI: 10.1016/B978-0-12-802104-0.00027-5
I.T. Consortium. Membrane transporters in drug development, Nature reviews. Drug discovery. 2010; 9(3):215
Kim YC, Masutani H, Yamaguchi Y, Itoh K, Yamamoto M, Yodoi J. Hemin-induced activation of the thioredoxin gene by Nrf2. A differential regulation of the antioxidant responsive element by a switch of its binding factors. The Journal of Biological Chemistry. 2001; 276(21):18399-18406
Wakabayashi N, Slocum SL, Skoko JJ, Shin S, Kensler TW. When NRF2 talks, who's listening? Antioxidants & Redox Signaling. 2010; 13(11):1649-1663
Kwak MK, Wakabayashi N, Greenlaw JL, Yamamoto M, Kensler TW. Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway. Molecular and Cellular Biology. 2003; 23(23):8786-8794
Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as an anticancer strategy. Nature Reviews. Drug Discovery. 2013; 12(12):931-947
Wild AC, Moinova HR, Mulcahy RT. Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. The Journal of Biological Chemistry. 1999; 274(47):33627-33636
Ganan-Gomez I, Wei Y, Yang H, Boyano-Adanez MC, Garcia-Manero G. Oncogenic functions of the transcription factor Nrf2. Free Radical Biology & Medicine. 2013; 65:750-764
Wang R, Liu L, Liu H, Wu K, Liu Y, Bai L, et al. Reduced NRF2 expression suppresses endothelial progenitor cell function and induces senescence during aging. Aging (Albany NY). 2019; 11(17):7021
Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H, et al. Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell. 2012; 22(1):66-79
Kim YJ, Ahn JY, Liang P, Ip C, Zhang Y, Park YM. Human prx1 gene is a target of Nrf2 and is up-regulated by hypoxia/reoxygenation: Implication to tumor biology. Cancer Research. 2007; 67(2):546-554
Aoki Y, Sato H, Nishimura N, Takahashi S, Itoh K, Yamamoto M. Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust. Toxicol Appl Pharm. 2001; 173(3):154-160
Iida K, Itoh K, Kumagai Y, Oyasu R, Hattori K, Kawai K, et al. Nrf2 is essential for the chemopreventive efficacy of oltipraz against urinary bladder carcinogenesis. Cancer Research. 2004; 64(18):6424-6431
Ooi A, Dykema K, Ansari A, Petillo D, Snider J, Kahnoski R, et al. CUL3 and NRF2 mutations confer an NRF2 activation phenotype in a sporadic form of papillary renal cell carcinoma. Cancer Research. 2013; 73(7):2044-2051
Collisson E, Campbell J, Brooks A, Berger A, Lee W, Chmielecki J, et al. Comprehensive molecular profiling of lung adenocarcinoma: The cancer genome atlas research network. Nature. 2014; 511(7511):543-550
Network CGAR. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature. 2014; 507(7492):315
Network CGAR. Comprehensive genomic characterization of squamous cell lung cancers. Nature. 2012; 489(7417):519
Praslicka BJ, Kerins MJ, Ooi A. The complex role of NRF2 in cancer: A genomic view. Current Opinion in Toxicology. 2016; 1:37-45
Tao S, Wang S, Moghaddam SJ, Ooi A, Chapman E, Wong PK, et al. Oncogenic KRAS confers chemoresistance by upregulating NRF2. Cancer Research. 2014; 74(24):7430-7441
MacLeod AK, Acosta-Jimenez L, Coates PJ, McMahon M, Carey FA, Honda T, et al. Aldo-keto reductases are biomarkers of NRF2 activity and are co-ordinately overexpressed in non-small cell lung cancer. British Journal of Cancer. 2016; 115(12):1530-1539
Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, et al. Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Medicine. 2006; 3(10):e420
Jaramillo MC, Zhang DD. The emerging role of the Nrf2-Keap1 signaling pathway in cancer. Genes & Development. 2013; 27(20):2179-2191
Moon EJ, Giaccia A. Dual roles of NRF2 in tumor prevention and progression: Possible implications in cancer treatment. Free Radical Biology & Medicine. 2015; 79:292-299
Hayes JD, Dinkova-Kostova AT. The Nrf2 regulatory network provides an interface between redox and intermediary metabolism. Trends in Biochemical Sciences. 2014; 39(4):199-218
Lee SB, Sellers BN, DeNicola GM. The regulation of NRF2 by nutrient-responsive Signaling and its role in anabolic cancer metabolism. Antioxidants & Redox Signaling. 2018; 29(17):1774-1791
de la Vega MR, Chapman E, Zhang DD. NRF2 and the hallmarks of cancer. Cancer Cell. 2018; 34(1):21-43
Katsuoka F, Yamamoto M. Small Maf proteins (MafF, MafG, MafK): History, structure and function. Gene. 2016; 586(2):197-205
Muscarella LA, Barbano R, D'Angelo V, Copetti M, Coco M, Balsamo T, et al. Regulation of KEAP1 expression by promoter methylation in malignant gliomas and association with patient's outcome. Epigenetics. 2011; 6(3):317-325
Kovac M, Navas C, Horswell S, Salm M, Bardella C, Rowan A, et al. Recurrent chromosomal gains and heterogeneous driver mutations characterise papillary renal cancer evolution. Nature Communications. 2015; 6(1):6336
Budak M, Yildiz M. Epigenetic Modifications and Potential Treatment Approaches in Lung Cancers, Lung Cancer-Strategies for Diagnosis and Treatment. Rijeka: IntechOpen; 2018. pp. 115-135
Wang R, An J, Ji F, Jiao H, Sun H, Zhou D. Hypermethylation of the Keap1 gene in human lung cancer cell lines and lung cancer tissues. Biochemical and Biophysical Research Communications. 2008; 373(1):151-154
Hanada N, Takahata T, Zhou Q, Ye X, Sun R, Itoh J, et al. Methylation of the KEAP1 gene promoter region in human colorectal cancer. BMC Cancer. 2012; 12(1):66
Barbano R, Muscarella LA, Pasculli B, Valori VM, Fontana A, Coco M, et al. Aberrant Keap1 methylation in breast cancer and association with clinicopathological features. Epigenetics. 2013; 8(1):105-112