NRF2 was discovered in 1994 and belongs to the Cap and Collar (CNC) basic-region leucine zipper transcription factor family [
20]. NRF2 has seven conserved NRF2-ECH homology domains comprising Neh1 to Neh7 (A). Neh1, Neh3, Neh4, and Neh5 domains are involved in the transcriptional activation of NRF2 by binding its co-activators. Neh2, Neh6, and Neh7 control the stability of NRF2 through responding as a negative regulatory domain [
16]. Neh1 domain is known as a CNC-bZIP domain that allows NRF2 to bind antioxidant response element (ARE), also known as the electrophile response element (EpRE) through interaction with other factors like small musculoaponeurotic fibrosarcoma (sMAF) [
21]. Neh2 domain functions as a major regulatory domain of NRF2 containing ETGE and DLG regions that are required for the interaction with KEAP1. In addition, Neh2 domain has lysine rich residues responsible for the ubiquitination and subsequent proteasomal degradation of NRF2 [
22]. Neh3 is the transactivation domain recruiting co-activators that are necessary for the transactivation of NRF2 [
23]. NRF2 also possesses Neh4 and Neh5 domains containing acid-rich residues that interact with CREB-binding protein with histone acetyltransferase activity (CBP) [
24]. The Neh6 domain contains serine-rich residues that can be phosphorylated by Glycogen Synthase Kinase 3b (GSK-3β) and leads to proteasomal degradation of NRF2 through cullin 1 (Cul1)-dependent ubiquitination [
25]. The Neh7 domain mediates the binding of RXRα (retinoid X receptor α) that inhibits the NRF2 transcriptional activity [
26].
KEAP1 was identified as a negative regulator of NRF2 that consists of five functional domains, namely, the N-terminal domain, Broad complex/Tramtrack, Bric-a-Brac domain (BTB), a cysteine-rich intervening region (IVR), Kelch domain, or double glycine repeat (DGR), and carboxyterminal domain (B) [
27]. The BTB domain is required for homo dimerization of KEAP1 and plays a critical role in ubiquitination of NRF2 through the interaction with the CUL3-based E3 ubiquitin ligase complex [
28]. The IVR domain has highly reactive cysteine residues that are responsible for sensing reactive oxygen species (ROS), reactive nitrogen species (RNS), and hydrogen sulfide (H
2S) [
29,
30,
31]. The Kelch/DGR domain functions as an NRF2 repressor, and it contains of six Kelch motif repeats, which are required for interaction with Neh2 domain of NRF2 [
32].
KEAP1-NRF2 pathway is one of the major signaling cascades that promote antioxidant defense in normal cells, which is a crucial mechanism in the prevention of cancer development. Many studies have shown that KEAP1 and NRF2 proteins function as tumor suppressors, as their absence leads to tumorigenesis while other work indicates that NRF2 can also promote tumor progression. In the following sections, we will briefly discuss past and present studies focused on this seemingly paradoxical aspect.
2.3. The Carcinogenic Role of NRF2
While many studies show that activation of NRF2 protects normal cells against various toxic substances and diseases, it has been shown that the overactivation of NRF2 also supports cancer progression and protects cancer cells from oxidative damage leading to chemoresistance and radioresistance (). Elevated levels of NRF2 in cancer induce the upregulation of glucose 6-phosphate dehydrogenase (G6PD), transketolase (TKT), 6-phosphogluconate dehydrogenase (PGD), and other metabolic enzymes [
40]. The augmented activation of these metabolic enzymes increases the synthesis of purine and amino acids and refills the NADPH pool via the pentose phosphate pathway (PPP) leading to metabolic reprogramming for cell proliferation and enhanced antioxidant capacity. Moreover, NRF2 regulates the basal expression of Mdm2, a direct inhibitor of p53 [
41]. Therefore, increased Nrf2 expression indirectly downregulates p53 and contributes to tumor survival by suppressing p53-related apoptotic signals.
Figure 2. NRF2 activation in healthy and cancer cells. (A) In healthy cell, under normal conditions NRF2 level is inhibited by KEAP1-mediated proteasomal degradation; under stress conditions, NRF2 dissociates from KEAP1, accumulates in nucleus and activates cytoprotective gene expression. (B) In cancer cells, different molecular mechanisms cause constitutive NRF2 activation that results in drug resistance, stress adaptation, cells proliferation, and activation of metabolic reprogramming and induces expression of genes related to tumor progression. NRF2, nuclear factor erythroid 2-related factor 2; KEAP1, Kelch-like ECH-associated protein 1; MAF, small musculoaponeurotic fibrosarcoma protein; Pol II; RNA polymerase II; ARE: antioxidant response element.
Recent studies have demonstrated that NRF2 plays a critical role in promoting intrinsic and acquired chemoresistance of cancer cells to common chemotherapeutics by activating drug resistance proteins and drug transporters such as UDP-glucuronosyl-transferase 1A1 (UGT1A) and multidrug-resistance-associated protein-1 (MRP1). In a study conducted in human doxorubicin-resistant ovarian cancer cells, NRF2 level was found to be elevated compared to the control cell line, and silencing of NRF2 expression via siRNA restored drug sensitivity [
42]. In another study, chemical activation of NRF2 provided a survival advantage to neuroblastoma cells in response to cancer drugs such as cisplatin, doxorubicin, and etoposide [
43]. Based on these findings, Cho et al. demonstrated that depletion of NRF2 expression via siRNA knockdown increased the effectiveness of cisplatin in ovarian cancer cells [
14].
Moreover, persistent activation of NRF2 was reported to attenuate the toxicity of ionizing radiation and drug treatment in human lung cancer cells, while NRF2 knockdown enhanced cellular response to ionizing radiation and chemotherapeutic drugs. These findings suggest that targeting NRF2 activity alone or in combination with other drugs could be an effective strategy to improve the sensitivity of malignant cells to anticancer therapies [
44,
45].
Furthermore, recent studies demonstrate the role of NRF2 in tumor metastasis. Constitutively active NRF2 promotes lung cancer via inhibiting degradation of a pro-metastatic transcription factor Bach1 [
46]. Overexpression of NRF2 in breast cancer leads to cell proliferation and metastasis by activating the RhoA gene and its downstream signal proteins [
47]. The critical role of NRF2 in tumor metastasis and proliferation has been shown in human hepatocellular carcinoma via regulating expression of Bcl-xL and Metalloproteinase-9 (MMP-9) genes [
48]. NRF2 also activates epithelial mesenchymal transition (EMT) and invasion in pancreatic adenosquamous carcinoma cells by decreasing E-cadherin gene expression [
49]. In addition, depletion of NRF2 decreases radiation-induced NSCLC invasion through promoting E-cadherin expression and reducing N-cadherin and MMP2/9 expression [
50].
3. NRF2 Activation Mechanisms in Cancer
Comprehensive studies validated that NRF2-KEAP1 signaling pathway is activated in several cancers such as skin, lung, bladder, hepatocellular carcinoma, esophagus, ovarian, prostate, pancreatic, and breast cancer [6,52,53,55,57]. The molecular mechanisms responsible for the activation of NRF2 in cancer are schematized in and further details are discussed below:
Figure 3. Different molecular mechanisms are responsible for activation of NRF2-KEAP1 pathway in cancer; somatic mutations in KEAP1 or NRF2; epigenetic modifications in KEAP1 and NRF2 promoter; post-transcriptional activation of NRF2; oncogenic signals; hormonal activation.
3.1. The Somatic Mutations in KEAP1 or NRF2
In cancer, somatic loss of function mutations in
KEAP1 or
NFE2L2 genes are the most known mechanisms that reduce NRF2-KEAP1 binding and prevent degradation of NRF2 through KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex [
45,
51]. Increasing evidence has established that the inhibition of NRF2-KEAP1 interaction leads to the overexpression of NRF2 in cancer cells that, in turn, enhances the activation of antioxidant defense system, and proteins involved in chemoresistance and radioresistance system via activating ARE-containing gene expression. Most of the inactivating mutations in the
NFE2L2 gene were detected within ETGE and DLG motifs in various cancers such as lung, head, neck, and esophageal carcinoma [
53]. It was also reported that the exon2 loss of the
NF2EL2 pre-mRNA abolishes the KEAP1–NRF2 protein–protein interaction, thereby inducing NRF2 accumulation and transcriptional activation of its target genes in lung, head, and neck cancers [
58].
Inactivating mutations in
KEAP1 gene occur frequently in many cancer types and largely affect the NRF2-KEAP1 interaction. Unlike
NFE2L2,
KEAP1 mutations can be missense or nonsense mutations and observed on the entire gene [
45,
55]. Some of the mutations in
KEAP1 gene lead to deregulation of apoptosis, autophagy, and inflammation by accumulation of BCL2 and p62 proteins [
59,
60]. The first loss-of-function mutations in Kelch/DGR domain of KEAP1 were reported in human lung adenocarcinoma cell lines [
54]. Then, somatic mutations in Kelch/IVR domain of KEAP1 were detected in both human NSCLC cell lines and clinical NSCLC patients’ tumor samples [
45,
55]. Recently, different research groups also reported that
KEAP1 genetic alterations could be novel molecular hallmarks in high neuroendocrine gene expressing lung cancers [
61,
62].
3.2. Epigenetic Modifications in KEAP1 and NRF2 Promoters
Besides somatic mutations, epigenetic changes at
KEAP1 and
NFE2L2 promoters may promote to the accumulation of NRF2 and depletion of KEAP1 in cancer cells. Several studies indicate that epigenetic mechanisms play a role in the regulation of KEAP1/NRF2 signaling. In particular, silencing of
KEAP1 by different epigenetic mechanisms in many tumors causes NRF2 accumulation. In lung, colon, and prostate cancers,
KEAP1 promoter was found to be significantly hypermethylated [
43,
63,
64,
65]. Moreover, hypermethylation within the promoter region of
KEAP1 was associated with poor clinical prognoses in patients with glioma [
66]. On the other hand, it has been shown that
NFE2L2 promoter demethylation resulted in NRF2 accumulation and chemoresistance in colon cancer cells [
67]. Therefore, from a therapeutic perspective,
KEAP1 methylation or
NFE2L2 demethylation can be targeted to inhibit abnormal NRF2 expression in different cancers.
3.3. Post-Transcriptional Regulation of NRF2 Activation
MicroRNAs (miRNAs) are small, 19–25 nucleotides in length, non-coding RNA molecules that play roles in regulating gene expression by sequence-specific binding to mRNA sequences [
68]. Several studies concluded that KEAP1 and NRF2 levels can be regulated at the post-transcriptional level in different cancers by abnormal expression of miRNAs targeting these genes. For example, miR-507, miR-634, miR-450a, and miR-129-5p directly target and suppress NRF2 activity. Studies have shown that these miRNAs are downregulated in esophageal squamous cell carcinoma (ESCC) and lead to upregulation of NRF2 mRNA [
69].
Furthermore, miR-27a, miR-141, miR-144, miR-153, miR-200a, miR-432, and miR-23a modulate
KEAP1 mRNA expression and induce NRF2 activation [
70]. It was reported that miR-141 is overexpressed in breast and ovarian cancer, and additionally, overexpression of this miRNA increased chemoresistance of HCC cells to 5-fluorouracil through the activation of NRF2-driven antioxidant pathways [
71,
72].
3.4. Disruptor Proteins
Several disrupting proteins are involved in the activation of NRF2 in cancer. Moreover, p62, also known as sequestosome 1 (SQSTM1), is an autophagy receptor protein that contains the STGE motif, which is similar to the ETGE motif of NRF2. This protein competes with NRF2 for KEAP1 binding and promotes autophagic degradation of KEAP1 [
73,
74,
75,
76]. Studies proved that when p62 expression was decreased by siRNA-mediated knockdown, NRF2 and its target genes were downregulated, while the half-life of KEAP1 increased by twofold [
73,
76]. In addition, elevated p62 contributed to renal cancer progression and hepatocellular carcinoma through the activation of NRF2 [
77,
78,
79]. These studies emphasize the critical role of p62 and NRF2 axis in the regulation of tumor development.
Besides, p21, which is a direct target of p53, associates with ETGE and/or DLG motifs in NRF2 and disrupts NRF2-KEAP1 binding causing NRF2 accumulation [
80]. Furthermore, Wilms tumor gene on the X chromosome (WTX) and partner and localizer of BRCA2, also known as PALB2 proteins have been shown to bind KEAP1 and suppress NRF2 ubiquitination [
81,
82]. Similarly, the protein dipeptidyl peptidase 3 (DPP3) was shown to inhibit NRF2 ubiquitination through binding to KEAP1, thus activating NRF2-dependent gene transcription in breast cancer [
83].
3.5. Oncogenic Signals
Oncogenic signals contribute to abnormal NRF2 activation in cancer through transcriptional upregulation of NRF2. DeNicola et al. reported that NRF2 level can be increased by the activation of oncogenic alleles of
KRAS,
BRAF, and
C-MYC (KRASG12D, BRAFV619E, and C-MYCERT12) [
84]. In addition, they also demonstrated that K-RAS and B-RAF activated Jun and Myc transcription factors, which, in turn, promoted cancer cell survival and chemoresistance [
84]. Similarly, disruption of tumor suppressor phosphatase and tensin homologue (PTEN) protein-activated NRF2 in human cancers [
85]. Moreover, KRAS-ERK signaling pathway plays a critical role in elevation of NRF2 transcription via 12-O Tetradecanoylphorbol-13-acetate (TPA) response element that localizes in a regulator site in NRF2 exon1 [
86]. Furthermore, the phosphatidylinositol-4,5-bisphosphate 3-Kinase (PI3K) serine/threonine kinase (AKT) signaling pathway also upregulated NRF2 transcription through the inhibition of GSK3-β-TrCP-induced proteasomal degradation of NRF2 [
87].
3.6. Hormonal Activation
Several studies validated the effects of hormonal activation of NRF2 on cancer progression. Gonadotropins and sex steroid hormones, including follicle-stimulating hormone (FSH), estrogen (E2), and luteinizing hormone (LH), have been reported to be critical in activation of NRF2 through the induction of ROS that inhibit KEAP1 by oxidation of its cysteine residues [88]. In addition, follicle-stimulating hormone (FSH) is known to induce expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1α (HIF1α). Thus, FSH contributes to tumor angiogenesis through ROS-mediated NRF2 signaling [89].