The Intricacies of NRF2 Regulation in Cancer

Abstract

The complex role of NRF2 in the context of cancer continues to evolve. As a transcription factor, NRF2 regulates various genes involved in redox homeostasis, protein degradation, DNA repair, and xenobiotic metabolism. As such, NRF2 is critical in preserving cell function and viability, particularly during stress. Importantly, NRF2 itself is regulated via a variety of mechanisms, and the mode of NRF2 activation often dictates the duration of NRF2 signaling and its role in either preventing cancer initiation or promoting cancer progression. Herein, different modes of NRF2 regulation, including oxidative stress, autophagy dysfunction, protein-protein interactions, and epigenetics, as well as pharmacological modulators targeting this cascade in cancer, are explored. Specifically, how the timing and duration of these different mechanisms of NRF2 induction affect tumor initiation, progression, and metastasis are discussed. Additionally, progress in the discovery and development of NRF2 inhibitors for the treatment of NRF2-addicted cancers is highlighted, including modulators that inhibit specific NRF2 downstream targets. Overall, a better understanding of the intricate nature of NRF2 regulation in specific cancer contexts should facilitate the generation of novel therapeutics designed to not only prevent tumor initiation, but also halt progression and ultimately improve patient wellbeing and survival.

1. Introduction

Cellular stress plays a critical role in not only promoting tumor formation, but also ensuring the successful treatment of cancer post-initiation [1]. For example, oxidative stress has been shown to damage nucleic acids, proteins, and lipids, increasing the chance of DNA mutation and disrupting basic cellular processes, which in turn, leads to an increased risk of malignant transformation and tumorigenesis [2, 3]. In this context, increased antioxidant capacity and enhanced detoxification play a vital role in combatting oxidative stress and preventing malignant adaptations prior to tumor initiation [4, 5]. However, once a tumor is formed, malignantly transformed cells can also utilize these same defense systems to maintain the reductive environment needed to navigate the stresses of rapid proliferation and a hostile microenvironment [6]. Confoundingly, this also affords cancer cells the ability to combat therapeutics that cause cytotoxicity by generating oxidative stress (i.e. ionizing radiation, doxorubicin, etc.) [7, 8]. Therefore, while cellular stress response pathways are crucial to prevent cancer initiation, these same protective systems also allow cancer cells to survive and thrive.

Nuclear factor (erythroid derived-2)-like 2 (NRF2) is a transcription factor that has been deemed the master regulator of redox and metabolic homeostasis [9]. Structurally, NRF2 consists of seven defined domains (Neh1–7) that mediate its interaction with DNA, transcriptional coactivators, and the E3 ligase complexes responsible for its degradation [10, 11]. Under normal conditions, NRF2 is bound in the cytosol by Kelch-like ECH-associated protein 1 (KEAP1), a substrate adaptor protein of the cullin-3/ring-box 1 (CUL3/RBX1) E3 ubiquitin ligase complex that facilitates its ubiquitylation. Following ubiquitylation, valosin-containing protein (p97) extracts NRF2 from this complex and transports it to the 26S proteasome for degradation [12–15]. However, during stress, NRF2 degradation is disrupted, allowing newly translated NRF2 to translocate to the nucleus, bind to small musculoaponeurotic fibrosarcoma (sMAF), and transcribe genes containing antioxidant response elements (AREs). Despite its initial discovery as a regulator of the antioxidant response, NRF2 also controls the transcription of genes involved in almost every aspect of cellular metabolism. This includes heme and iron metabolism (heme oxygenase-1 [HMOX1], ferritin heavy chain [FTH1]) [16, 17], phase I, II, and III metabolism (aldo-keto reductase 1C1 [AKR1C1], NAD(P)H dehydrogenase (quinone) 1 [NQO1], glutathione S-transferase alpha 1 [GSTA1], multidrug resistance-associated protein 1 [ABCC1]) [18–20], DNA repair (DNA repair protein RAD51 homolog 1 [RAD51]) [21], proteasomal assembly (proteasome subunit alpha type-1 [PSMA1], proteasome maturation protein [POMP]) [22, 23], other transcription factors (aryl hydrocarbon receptor [AHR], neurogenic locus notch homolog protein 1 [NOTCH1]) [24, 25], glutathione metabolism (glutamate-cysteine ligase catalytic subunit [GCLC], cystine/glutamate transporter [xCT/SLC7A11]) [26, 27], anti-apoptosis (B-cell lymphoma 2 [BCL2], Bcl-2-like Protein 1 [BCL2L1]) [28, 29], and autophagy (sequestosome 1 [p62/SQSTM1], autophagy related 5/autophagy related 7 [ATG5/ATG7]) [19, 30–32].

Critically, over two decades of NRF2-based research has revealed a wide variety of NRF2 regulatory mechanisms, including canonical alteration of the KEAP1-NRF2 complex via cysteine oxidation or reversible ETGE disruption (one of the two motifs that mediate KEAP1-NRF2 binding), non-canonical p62-dependent dysregulation of the NRF2-KEAP1 interaction, and transcriptional/translational regulation of NRF2 mRNA and protein expression [33]. Importantly, the mode of NRF2 activation dictates its role in cancer, as induction of NRF2 in healthy cells can be utilized for chemoprevention; whereas, somatic mutations in genes involved in NRF2 regulation give rise to NRF2-addicted cancers that rely on persistent NRF2 activation for their survival [34]. In the context of NRF2-addiction, recent evidence has highlighted various modes of NRF2 inhibition that may serve as targetable cascades to treat this subset of cancers. This is of particular importance as constitutive activation of NRF2 has been shown to enhance resistance to cancer therapies and promote survival of metastasized cancer cells in a continually increasing number of cancer types, making inhibiting NRF2 in cancer an ongoing area of interest [35–37].

Based on its dual role in cancer, a more detailed understanding of the mechanisms by which NRF2 is regulated could offer insight into possible genetic and pharmacological manipulations that could be utilized for chemoprevention and chemotherapy [38]. Along these lines, targeting the different regulatory mechanisms that govern prolonged NRF2 activation, whether it be transcriptional, epigenetic, or protein-protein interaction-based, all represent viable strategies to directly affect NRF2 in a cancer setting [39–41]. It is also important to consider not only the mechanism of activation, but also the temporal nature of NRF2 induction, as acute versus prolonged activation plays a critical role in determining its beneficial versus detrimental effects. For example, in the context of an acute xenobiotic exposure, NRF2 can be rapidly induced to initiate key stress response pathways, and upon resolution of said stress, return to a basally low state. This temporal attenuation of the NRF2 signal when the stress is resolved is utterly important in determining the fate of the cell; for example, prolonged NRF2 activation in a chronic low-dose environmental exposure setting (i.e. metals/metalloids) can result in malignant transformation of the cell, thus resulting in tumor initiation [42]. This is somewhat similar to cancers driven by somatic mutations in NRF2, KEAP1 and CUL3, where NRF2 is constitutively activated and aids in cancer progression and resistance to therapies. Overall, an increased understanding of contextual and temporal control of NRF2-mediated signaling cascades, modes of regulation, and specific targets affected by pharmacological modulators of this pathway will enhance the future development of NRF2-based cancer therapeutics. Here, we review the different means by which NRF2 and its downstream targets can be activated or inhibited, with a particular emphasis on how modulation of the NRF2 cascade affects cancer initiation, progression, and metastasis.

2. Chemoprevention

Mentioned briefly above, chemopreventive induction of NRF2 to prevent tumor initiation has long been an active area of interest. The wide array of cytoprotective target genes controlled by NRF2 are critical in preventing the oxidative, proteotoxic, and metabolic stresses that contribute to malignant transformation. Thus, it is not surprising that targeting the NRF2 signaling pathway for chemoprevention has been shown to prevent tumor initiation in numerous cancer types [43]. Interestingly, despite the increasing number of defined modes of NRF2 activation, canonical NRF2 activation still represents the most common mechanism of attaining NRF2-dependent chemoprevention (Figure 1). Canonical activation of NRF2, including chemopreventive compounds that mediate their anti-cancer effects via this mechanism, will be discussed in detail below.

Figure 1: NRF2 activation mechanisms.

NRF2 transcriptional regulation of ARE-containing genes is controlled via a variety of mechanisms, including 1) canonical, 2) autophagy dysfunction, 3) transcriptional/translational/epigenetics, 4) post-translational modifications, 5) protein-protein interactions, and 6) mutations.

2.1. Canonical NRF2 activation

Traditionally, canonical NRF2 activation refers to NRF2 upregulation as a result of oxidative or electrophilic modification of KEAP1 cysteines (i.e. Cys151). This modification then results in a conformational change in the NRF2-KEAP1 complex that prevents degradation of NRF2 [44, 45]. As a result, newly synthesized NRF2 is able to evade KEAP1-dependent degradation and translocate to the nucleus, where it activates transcription [46]. Oxidative/electrophilic modification of KEAP1 can occur due to a variety of factors, including exposure to environmental toxicants and free radical generating compounds, or pharmacological interventions that either directly adduct to KEAP1 or trigger the endogenous generation of reactive oxygen species (ROS). In each of these cases, the cell responds to an oxidizing environment by upregulating NRF2 and its cytoprotective target genes to restore redox homeostasis [47]. Upon restoration of homeostasis, KEAP1 then shuttles to the nucleus and returns NRF2 to the cytosol for degradation, which in turn prevents prolonged NRF2 activation [48]. In the context of chemoprevention, NRF2 has been shown to be induced in a canonical manner by a wide variety of dietary compounds [49]. Thus, the canonical mode of NRF2 induction is essential in initiating the detoxification of harmful carcinogens and preventing excessive oxidative stress to prevent malignant transformation.

2.2. NRF2 Inducers

Pharmacological induction of NRF2 has long been studied as a mechanism of chemoprevention [50]. Mechanistically, as discussed above, several inducers of NRF2 have been shown to affect the KEAP1-NRF2 interaction by directly modifying or increasing the oxidation of KEAP1 cysteines [44]. Early work in the NRF2 field indicated that treatment of bladder cells or keratinocytes with oltipraz or 3H-1,2-Dithiole-3-thione, respectively, could serve as chemopreventive agents by inducing NRF2-dependent upregulation of phase II detoxifying enzymes [51–53]. Interestingly, a number of natural compounds obtained from everyday components of the diet, including sulforaphane (broccoli sprouts), bixin (achiote), and cinnamaldehyde (cinnamon), can also serve as NRF2-based chemopreventive compounds [54–60]. For instance, a number of studies have indicated that consumption of cinnamaldehyde decreases colon tumorigenesis in an NRF2-dependent manner [61]. Similarly, sulforaphane has shown antitumorigenic properties and is currently in clinical trials as a means of lung cancer chemoprevention [62, 63]. Topical bixin application induced NRF2 in skin which mitigated nonionizing and ionizing radiation damage, indicating that use of this pharmacological approach could protect against off-target skin toxicities and potentially skin cancer initiation [57, 64]. Although not via canonical NRF2 induction, curcumin and ursolic acid were found to induce NRF2 activity epigenetically by reversing the methylated state of the NFE2L2 promoter, which in the case of ursolic acid prevented skin cancer [65]. A number of synthetic canonical inducers of NRF2 have also been shown to have chemopreventive properties. For example, bardoxolone methyl (CDDO-Me), a synthetic triterpenoid, as well as its derivative CDDO-Im, both activate NRF2-dependent cytoprotection, and thus could be used as chemopreventive agents [66]. Additional classes of chemicals have been identified to induce drug-metabolizing and antioxidant enzymes as a means of NRF2-dependent chemoprevention, including carotenoids, dithiolethiones, flavonoids, isothiocyanates, and phenols [67]. Recently, new strategies to pharmacologically induce NRF2 utilize non-covalent binding of compounds, like Geopyxin F, to KEAP1, which induces NRF2 independently from Cys151. This approach is important as non-covalent binding is reversible, thus NRF2 will not be upregulated in a prolonged manner, and presumably this will have less toxicity due to the lack of ROS generated [33, 68]. Despite the promising nature of NRF2 induction as a chemoprevention strategy, and vast number of NRF2-inducing chemical compounds, no NRF2 inducers have made it past clinical trials for chemoprevention. As many NRF2 inducing compounds are electrophiles, and thus carry the risk of numerous off-target effects, the development of approvable NRF2 inducers for disease intervention continues to represent a difficult challenge. For example, in the case of bardoxolone, its variant CDDO-Im was shown in a proteomic analysis to interact with 577 proteins. This promiscuity indicates that while NRF2 is activated, several other proteins, including additional transcription factors, are also altered. Thus, while electrophilic inducers like bardoxolone have shown pre-clinical promise, the search for safe and efficacious NRF2 activators that can be used in clinic continues [69, 70]. To date, dimethyl fumarate (DMF) remains the only FDA approved compound that directly activates NRF2 for the treatment of multiple sclerosis [71]; however, whether or not it could be used for chemoprevention has yet to be explored. Therefore, the progression of canonical NRF2 inducers for the purposes of chemoprevention past the clinical trial phase is still needed.

3. Cancer Initiation and Progression

While NRF2 activation plays a key role in chemoprevention, a great deal of evidence in the literature also indicates that prolonged activation of NRF2 promotes cancer initiation and progression. In the context of initiation, it is important to note that while NRF2 may be activated during the earliest stages of tumor formation, this could just be a secondary effect of the increased stress associated with malignant transformation. Thus, whether or not NRF2 actually drives initiation across multiple cancer contexts remains unclear. Despite this fact, it stands to reason that while transformation itself may not involve NRF2, upregulation of this pathway is critical for early tumorigenesis, as stress generated by replication errors or exogeneous toxicants can be negated by NRF2 transcriptional programs. Furthermore, prolonged NRF2 activation could suppress apoptosis of newly transformed cells by continually upregulating detoxification and DNA repair processes [38]. However, while evidence supporting NRF2-promotion of cancer initiation is less clear, its role in mediating tumor growth and survival has been thoroughly demonstrated, particularly in the context of chronic toxicant exposure. Regardless, expanding our current understanding of what role NRF2 and its various modes of activation play during cancer initiation and progression is important to consider.

3.1. Canonical/Toxicants

Early studies in the NRF2 field noted that toxicants play a significant role in canonical NRF2 activation due to the generation of oxidative stress. Specifically, 2,3,7,8-tetrachloroibenzo-P-dioxin (TCDD) or dioxin, was shown to induce cytoprotective enzymes (i.e. NQO1) in an NRF2-dependent manner in mouse hepatoma cells [72]. Also, exposure to metals/metalloids, including As, Cd, Co, Cu, Fe, Hg, and Pb, has been shown to increase NRF2 expression, as acute metal exposure leads to an increase in ROS and a depletion of glutathione, ultimately resulting in oxidative tissue damage [73]. While exposure to toxic metals and other compounds generate high ROS levels and induce NRF2, it remains unclear if NRF2 is critical for cancer initiation in this context. Additionally, it has been proposed that DNA damage as a result of toxic metal exposure in combination with continuous redox cycling could contribute to tumor initiation [74]. High levels of ROS could cause DNA mutations and other damage in cells, which would normally trigger apoptosis, but in a prolonged NRF2 activated setting, NRF2 could combat the stress to prevent apoptosis, allowing the damaged cell to survive and potentially transform. Overall, this highlights that ROS-dependent NRF2 induction could promote cancer initiation, but more evidence supporting this notion is needed.

3.2. Non-Canonical (Autophagy Dysfunction)

While the major role of NRF2 following acute toxicant exposure is the immediate neutralization of oxidative stress and removal of the xenobiotic insult, prolonged NRF2 activation during chronic low-dose toxicant exposure has actually been linked to NRF2-dependent malignant transformation. For example, only NRF2^+/+ but not NRF2^−/− immortalized bronchial epithelial cells can form colonies in soft agar following chronic exposure to low environmentally-relevant doses of arsenic [75]. Unlike treatment with higher concentrations, which have been shown to increase ROS, exposure to lower levels of arsenic causes proteotoxic stress and non-canonical p62-dependent activation of NRF2 [76]. Non-canonical activation of the NRF2 signaling pathway refers to KEAP1 sequestration by p62 during autophagic dysfunction that results in NRF2 protein accumulation and prolonged NRF2 signaling [77]. As p62 shares a similar amino acid motif (STGE) to NRF2 (ETGE) that dictates KEAP1 interaction, p62 can compete with NRF2 for KEAP1 binding [78]. Specifically, when the serine residue of the STGE motif of p62 is phosphorylated, its affinity for KEAP1 is increased. This allows p62 to outcompete NRF2 for KEAP1 binding, and the p62-KEAP1 complex is then recruited to the autophagosome for autophagic degradation. However, upon arsenic exposure, fusion between the autophagosome and lysosome is disrupted, resulting in the accumulation of p62-bound KEAP1 in autophagosomes [79]. Thus, in a chronic arsenic exposure setting, p62-dependent sequestration of KEAP1 could lead to prolonged activation of NRF2 and tumor formation. Accordingly, loss of p62 prevents NRF2 activation and malignant transformation of human keratinocytes in a chronic arsenic exposure model [80]. Additionally, p62 accumulation as a result of liver-specific loss of ATG5 or ATG7, two key autophagy initiation proteins, results in increased NRF2 activity and the development of hepatocellular carcinoma; however, knockout of NRF2 mitigated these tumorigenic pathological changes [81–83]. Interestingly, recent evidence demonstrates that both ATG5 and ATG7, as well as p62, are putative target genes of NRF2, indicating that NRF2 and autophagy are intimately linked [31]. Exploration of the relationship between non-canonical NRF2 induction and cancer initiation warrants further investigation and offers a potentially exploitable therapeutic niche for the manipulation of NRF2, particularly in cancers resulting from environmental exposure to carcinogens, including metals.

3.3. Genetic and Epigenetic Regulation

Several aspects of transcriptional and translational regulation of NRF2 have been shown to alter NRF2 expression. At the DNA level, it has been demonstrated that NRF2 can actually regulate transcription of itself via a putative ARE sequence detected in its promoter region, creating the possibility of a positive feedback loop [84]. Other transcription factors, such as AhR, have also been shown to regulate NRF2 expression. Interestingly, AhR has been shown to shape the tumor microenvironment of BRCA1-associated breast cancer via modulation of redox homeostasis, which could involve its regulation of NRF2, although this has not been shown [85, 86]. Additionally, Kirsten rat sarcoma viral oncogene homolog (KRAS) was shown to transcriptionally induce NRF2 expression by binding to a TPA response element (TRE) in the promoter region of NFE2L2; in turn, this results in increased drug resistance in NSCLC [87]. More recently, several microRNAs have been shown to regulate KEAP1 and NRF2 mRNA levels in the context of cancer. Specifically, miR-200 decreases KEAP1 mRNA levels and leads to NRF2 upregulation in breast cancer, while miR-101 targets CUL3 mRNA to increase NRF2 expression [88, 89]. NRF2 signaling is also influenced by epigenetic changes that affect its expression, including DNA methylation and chromatin modifications. For example, oxidative stress can induce DNA demethylases, which results in increased NRF2 expression and leads to chemoresistance [90, 91]. Similarly, methylation of H3K4 is mediated by oxidative stress-induced recruitment of O-linked N-acetylglucosamine transferase (OGT) to the NFE2L2 promoter, resulting in increased NRF2 transcriptional activity [92]. Conversely, methylation of the KEAP1 promoter has been reported to suppress KEAP1 levels in lung cancer, which is correlated with increased expression of NRF2 [93]. Overall, transcriptional, translational, and epigenetic modifications play an important role in upregulating NRF2 in cancer and promoting chemoresistance, but the exact mechanisms that induce these changes across multiple cancer types needs further investigation.

3.4. Post-Translational Modifications

The role of post-translational modifications in regulating NRF2 function in cancer has also been identified. Early evidence that post-translational modification could affect NRF2 signaling was shown by Sun et al., who demonstrated that acetylation of NRF2 by p300/CREB-binding protein (CBP) in the Neh1 domain facilitated ARE-binding [94]. In the context of lung cancer, reduced sumoylation of NRF2 results in increased oxidative stress levels and activation of the c-Jun N-terminal kinase (JNK) pathway, which in turn promoted lung cancer cell invasion [95]. In papillary renal cell carcinoma type II, mutations in the fumarate hydratase gene (FH) result in fumarate hydratase deficiency, which causes the accumulation and subsequent fumarate-dependent succination of KEAP1. Succination of KEAP1 results in stabilized NRF2 protein levels that increase resistance to cancer therapeutics in renal carcinoma patients [96, 97]. While current evidence shows that post-translational modifications of NRF2 and KEAP1 could upregulate NRF2 in cancer, the exact function, relevance, and abundance of each modification in different cancer types needs further elucidation.

3.5. Protein-Protein Interactions

As the DLG and ETGE motifs of NRF2 each bind to a homodimer of KEAP1 to ensure its ubiquitylation and degradation, disruption of this interaction can result in NRF2 activation. Specifically, the DLG motif serves as a weak binding latch that can be easily disrupted, while the ETGE motif acts as a strong hinge; therefore, ETGE binding dictates KEAP1 interactions. Several proteins that also contain ETGE motifs have been shown to bind to KEAP1 and result in an increase in NRF2 protein levels. As mentioned earlier, p62 can outcompete NRF2 for binding with KEAP1, as both proteins bind to KEAP1 via similar motifs, resulting in NRF2 protein accumulation [77]. Another example is nuclear factor (erythroid-derived 2)-like 1 (NRF1), another CNC leucine zipper transcription factor, which can bind to KEAP1 and thus prolong the half-life of NRF2. Intriguingly, despite being bound by KEAP1 in an ETGE-dependent manner, NRF1 is not regulated in the same manner as NRF2 and is not ubiquitylated by the KEAP1-CUL3-RBX1 complex [98]. Thus, it is possible that high levels of NRF1 could drive prolonged activation of NRF2 in certain cancer settings. Other proteins, such as cell-cell junction associated protein family with sequence similarity 129, member B (FAM129B) have also been shown to bind to KEAP1 via an ETGE motif and increase chemoresistance, with cellular localization changes caused by phosphorylation of FAM129B leading to constitutive activation of NRF2 and higher metastatic potential in BRAF mutant melanoma cells [99, 100]. Other KEAP1-interacting proteins that have been shown to increase cancer cell survival are dipeptidyl peptidase 3 (DPP3), Wilms tumor gene on X chromosome (WTX), and partner and localizer of BRCA1 (PALB2), due to their shared ability to bind to KEAP1 and prevent NRF2 degradation [101–103]. Similarly, cyclin-dependent kinase inhibitor 1 (p21) results in NRF2 activation via disruption of the KEAP1-NRF2 complex; however, p21 is unique in that it binds to NRF2 to disrupt the interaction [104]. In each of these contexts, disrupting the NRF2-KEAP1 interaction increases cancer progression and chemoresistance by activating the NRF2 signaling pathway.

3.6. Mutations

In the context of cancer, somatic mutations in any of the genes encoding members of the NRF2-KEAP1-CUL3-RBX1 complex can result in increased NRF2 protein levels and constitutive upregulation of cytoprotective target genes. As a result, unmitigated NRF2 activation results in resistance to cancer therapies and an overall more aggressive phenotype. For example, ~30% of non-small cell lung cancer (NSCLC) cases exhibit KEAP1 mutations [105]. Similarly, mutations in CUL3 have been shown in renal cell carcinoma [106]. Additionally, mutations in NFE2L2 itself have been detected in esophageal squamous cell carcinoma, hepatocellular carcinoma, and NSCLC [107–109]. In each of these cases, constitutive activation of the NRF2 signaling pathway enhances cancer progression, resulting in a worse prognosis for patients. This is important as NRF2 is ubiquitously expressed in all mammalian cell types, and as such any mutations in these proteins that result in constitutive activation of NRF2 could contribute to progression and therapeutic resistance in a variety of cancers. The exact cause of mutations in KEAP1, NFE2L2, and CUL3 remain random in nature, but evidence suggests that toxicant exposure and aging both play a significant role in the initiation and maintenance of NRF2-addicted tumors [110].

4. Metastasis

Without therapeutic intervention, continual expansion of the primary tumor often results in metastasis, which significantly decreases patient prognosis and survival. While the role of NRF2 in metastasis is still emerging, mounting evidence has linked upregulation of NRF2, either via genetic or pharmacological manipulation, to an increased risk of metastasis [37]. NRF2 was shown to contribute to NSCLC migration and invasion via transcriptional upregulation of pro-metastatic target genes [75]. Also, upregulation of HO-1 increased NSCLC metastasis by inhibiting the degradation of BACH1 (BACH1) [111]. Recent evidence has also shown that deletion of NRF2 resulted in decreased breast cancer migration and invasion due to suppression of focal adhesion formation downstream of Ras homolog family member A/Rho-associated protein kinase (RhoA/ROCK) signaling [112]. Additionally, NRF2 was essential for mitochondrial calcium uniporter regulator 1 (MCUR1)-induced epithelial-mesenchymal transition (EMT) via its upregulation of Notch1, which in turn promoted pro-metastatic proteins, and increased hepatocellular carcinoma metastasis [113]. While the mechanisms of NRF2-driven metastasis are just being investigated, it is clear that inhibitors of NRF2 are needed in order to not only halt primary tumor progression, but prevent metastasis as well.

5. NRF2 Inhibition in Cancer (Figure 2)

Figure 2: Modes of NRF2 inhibition decrease cancer cell viability.

Prolonged or constitutive NRF2 activation increases cancer progression, but NRF2 inhibition via 1) E3-ligase dependent proteasomal degradation, 2) DNA binding disruption, and 3) translational or epigenetic regulation can decrease overall NRF2 expression and activity to decrease cancer cell viability.

As NRF2 aids in tumor initiation, progression, and metastasis, the NRF2 signaling pathway represents a targetable cascade at every stage of cancer treatment. Currently, several endogenous mechanisms of NRF2 inhibition have been shown to increase chemosensitivity and decrease cancer cell survival. Additionally, recent developments in the implementation of pharmacological inhibitors of NRF2 to treat different cancer types continues to show promise. Overall, understanding mechanisms of NRF2 inhibition that could be utilized to treat cancer could have a profound impact on improving patient wellbeing and survival.

5.1. NRF2 Protein Degradation

Under normal conditions, NRF2 protein levels are kept low by several E3 ubiquitin ligase complexes that facilitate the ubiquitylation and degradation of NRF2. As mentioned above, KEAP1 binds to NRF2 via its Neh2 domain and recruits the RBX1-CUL3 E3 ligase complex to target NRF2 for proteasomal degradation [12]. The ability of KEAP1 to facilitate NRF2 degradation has also been shown to be enhanced via O-GlnNAcylation of KEAP1 at S104; thus, indicating that NRF2 levels can also be modulated by post-translational modification of KEAP1 [114]. Additionally, beta-transducin repeat containing E3 ubiquitin protein ligase (β-TrCP) was shown to facilitate ubiquitylation and degradation of NRF2 by binding to the Neh6 domain and then acting as a substrate adaptor for S-phase kinase-associated protein 1-cullin-1-ring-box1/regulator of cullins-1 (SCF) E3 complex [115]. Upon the receipt of certain stimuli, activation of glycogen synthase kinase-3 (GSK-3) leads to the phosphorylation of two separate degrons in the Neh6 domain, which recruit the β-TrCP complex and target NRF2 for degradation [116]. This offers a potentially targetable alternative mode of NRF2 degradation in KEAP1-mutant/NRF2-addicted cancers. Similarly, ERAD-associated E3 ubiquitin-protein ligase (HRD1), an important endoplasmic reticulum (ER) protein in the inositol-requiring enzyme 1 (IRE1) arm of the ER stress pathway, was shown to increase NRF2 ubiquitylation and degradation by directly interacting with NRF2 via its Neh4–5 domains [117]. As various pathological conditions could differentiate the role and predominance of individual E3 complexes in NRF2 degradation, further exploration into the specific modulators of NRF2 protein degradation machinery could offer insight into NRF2 inhibition in cancer.

5.2. Genetic and Epigenetic Regulation

In NRF2-addicted cancers, protein degradation machinery may be defective, such as with KEAP1 mutations, therefore, targeting NRF2-DNA interactions to block NRF2-mediated transcription may be a viable approach. For instance, retinoid X receptor alpha (RXRα) was shown to suppress NRF2 signaling by binding to the Neh7 domain, which in turn sensitized NSCLC cells to drug intervention [118]. Similarly, nuclear receptor estrogen-related receptor beta (ERRβ) was shown to repress NRF2 transcriptional activation via physically interacting with NRF2 and repressing its transactivation [119]. Additionally, NF-κB and NRF2 have an inverse relationship in reference to target gene expression, as both compete for CBP transcriptional coactivators [120]. Independent of direct binding to NRF2, nuclear factor (erythroid-derived 2)-like 3 (NRF3) was shown to compete with NRF2 for binding to the ARE sequence in the promoter region of the NQO1 gene. When NRF3 was bound to the ARE, it repressed transcriptional activity and caused a decrease in overall NQO1 expression [121]. Transcriptional regulator protein BACH1 has also been linked to the negative regulation of ARE-containing NRF2 target genes, as BACH1 can also bind to AREs resulting in decreased expression [122]. As the target genes themselves are what drive resistance to therapies and enhance tumor progression, suppression of NRF2 transcriptional activity could serve as a viable method to decrease NRF2 target gene expression, especially when NRF2 protein degradation machinery is non-functional.

While the role of epigenetic upregulation of NRF2 has been widely explored, epigenetic mechanisms that result in inhibition of NRF2 signaling remain relatively unknown. For example, several CpG islands have been identified in the promoter region of the NFE2L2 gene, which upon hypermethylation decrease NRF2 expression and promote prostate tumorigenesis [123]. This indicates that hypermethylation could result in carcinogenesis in some cells, but the role of DNA methylation in mediating NRF2 expression in an NRF2-addicted cancer setting has not yet been explored. In addition to the disruption of DNA binding as mentioned above, NF-κB p65 recruits HDAC3, a corepressor of NRF2 target genes, to bind with CBP; this results in histone hypoacetylation that affects NRF2 recruitment to the E1 enhancer region of its target gene HO-1, resulting in decreased ARE-dependent gene expression [120]. Also, miR-144 and miR-28 both directly target NRF2 mRNA and ultimately result in decreased NRF2 expression [124, 125]. By decreasing NRF2 protein synthesis, miR-144 increased the chemosensitivity of several hepatocellular carcinoma cell lines [126]. While little evidence currently exists to argue that epigenetic targeting of NRF2 could help kill cancer cells, the data presented here demonstrates the ability to inhibit the expression of NRF2 and its target genes epigenetically, which warrants further investigation as a strategy for treating NRF2-addicted cancer.

5.3. Pharmacological inhibitors

While one of the main functions of NRF2 upregulation is to combat oxidative stress, this is an unwanted response in cancer treatment as it could enhance tumor progression and chemoresistance. In fact, the mechanism of action of several established chemotherapies is the production of high levels of ROS that in turn cause cellular damage and induce cell death [127]. Therefore, if NRF2 is induced in this context, negation of the damage caused by chemotherapies in cancer cells can undermine treatment and ultimately decrease efficacy of these therapeutic drugs. Thus, the use of ROS-inducing chemotherapies (i.e., doxorubicin, arsenic trioxide, etc.) has a narrow window in which the dose must be high enough to overcome NRF2-based protection in the tumor without inducing major off-target toxicities in healthy tissues. Together, these findings demonstrate that NRF2 activation represents a significant hurdle in the utilization of ROS/DNA adduct-based chemotherapeutics, and strongly supports the need for adjuvant NRF2-targeted therapeutics in order to suppress NRF2-addicted mechanisms of resistance. This is especially true for NRF2-addicted tumors, where a pharmacological inhibitor of NRF2 would be needed in order to enhance the efficacy of current oxidative stress-inducing chemotherapeutics.

Clearly, there is an urgent need to develop NRF2 inhibitors as an adjuvant drug to conventional chemotherapy to overcome resistance in cancers where NRF2 levels are high. In the context of NRF2-addicted cancers, knockout of NRF2 sensitizes NSCLC cells to ionizing radiation by increasing ROS levels, ultimately resulting in increased radiation-induced apoptosis [128]. Similarly, downregulation of NRF2 in NSCLC cells with a KEAP1 mutation resulted in increased sensitivity to cisplatin treatment both in vitro and in vivo [129]. In 2011, brusatol was identified as the first compound to inhibit NRF2 and sensitize NRF2-addicted NSCLC cells to many platinum-based chemotherapeutic drugs [129]. Mechanistically, brusatol inhibits general protein translation, thus inhibiting short-lived NRF2 more effectively than long-lived proteins; however, it still remains one of the most potent and useful tools for studying NRF2 inhibition [130]. Halofuginone was shown to inhibit NRF2 in NRF2-addicted cancer cells, which in turn sensitized cancer cells to radio- and chemotherapies; however, halofuginone induced amino acid starvation which, like brusatol, also repressed global protein translation [131]. Another compound trigonelline can induce apoptosis in pancreatic cancer cells via suppression of NRF2 nuclear translocation and target gene expression [132, 133]. Ochratoxin A (OTA) also inhibited NRF2-dependent glutathione synthesis, sensitizing cells to chemical-induced injury; however, the specific mode of NRF2 inhibition by OTA remains unclear. Additionally, chronic OTA exposure could also induce somatic mutations in NFE2L2 and KEAP1, indicating a potential limitation in its use [134]. Although no NRF2-specific inhibitors have been validated, the small molecule ML385 was claimed to bind to the Neh1 domain of NRF2 and block its interaction with MAFG, thus inhibiting its ability to activate transcription of target genes [135]. Currently, there is no FDA approved drug to inhibit NRF2; therefore, developing NRF2 inhibitors with heightened specificity and limited off-target toxicity is needed in order to improve the treatment of NRF2-addicted cancers.

In addition to NRF2 suppression, inhibition of specific NRF2 target genes has been shown to decrease cancer cell proliferation, induce cell death, and alleviate tumor burden. For example, use of the compounds erastin or sulfasalazine, which inhibit SLC7A11 (xCT), and RSL-3, which inhibits glutathione peroxidase 4 (GPX4), increased cisplatin sensitivity of NRF2-addicted NSCLC cells and ultimately induced iron-dependent programmed cell death, also known as ferroptosis [136, 137]. Treatment of cells with polydatin inhibited glucose-6-phosphate dehydrogenase (G6PD), which ultimately repressed the pentose phosphate pathway (PPP) and reduced tongue cancer growth and metastasis [138]. Additionally, knockdown of HO-1 increased the chemosensitivity of pancreatic cancer cells, and while pharmacological inhibitors for HO-1 exist and have been shown to suppress Mycobacterium tuberculosis infection, they have not been characterized in the context of cancer [139, 140]. Overall, NRF2 and its target genes play a significant role in resistance to cancer therapies; therefore, identification, characterization, and development of NRF2 inhibitors in cancer treatment regimens remains an important area of research.

As discussed above, there are numerous upstream regulatory mechanisms that govern NRF2 activation, thus pharmacologically targeting upstream effectors of NRF2 could serve as an effective means to reduce NRF2 expression in certain cancers. Mentioned earlier, KRAS has been shown to upregulate NRF2 expression via a TPA response element in the promoter region of NRF2 [87]. This suggests that pharmacological inhibitors that target kinases upstream of KRAS (i.e. gefitinib, cetuximab, etc.), could lessen NRF2 transcription. However, as these kinases regulate numerous downstream effectors, the relevance of NRF2 inhibition in this context would need to be validated. Prior work has also indicated that the mitogen-activated protein kinase (MAPK) pathway enhanced ARE-containing gene expression in an NRF2-dependent manner, indicating that crosstalk occurs between these two pathways [141]. Recent evidence also indicated that elevated ERK activity leads to increased NRF2 protein levels in melanoma, and that inhibition of MEK decreases tumor aggressiveness in an NRF2-dependent manner [100]. Therefore, the MAPK pathway could serve as a target for NRF2-addicted cancers, particularly in a non-mutated KEAP1 context. While no NRF2 inhibitors are currently FDA approved, deeper mechanistic insight into modes of NRF2 inhibition, as well as the continued discovery of novel NRF2 inhibitors with heightened specificity but less toxicity, will help provide new reagents to treat NRF2-addicted cancers.

6. Conclusion

The number of mechanisms by which NRF2 is regulated continues to expand at an exponential rate. Overexpression of NRF2 is a major factor that drives tumor aggressiveness and resistance to therapies; yet, controlled NRF2 activation in normal tissues remains a useful strategy for chemoprevention. Therefore, induction of NRF2 is context-dependent in cancer. Transient NRF2 induction via canonical inducers that target cysteine residues of KEAP1 or non-covalently bind to KEAP1 and disrupt ETGE interactions provide cytoprotection for chemopreventive purposes; meanwhile, chronic toxicant exposure, protein-protein interactions, epigenetic modifiers, transcriptional/translational regulators, post-translational modification, and mutations have all been shown to induce NRF2 in a prolonged manner in cancer and promote cancer initiation, progression, and metastasis. In these cases, NRF2 inhibition is needed. However, due to the known difficulty in targeting transcription factors and the complexity of the NRF2 signaling cascade, inhibition of NRF2 has remained elusive. Despite this fact, numerous research efforts have outlined a variety of endogenous modes of NRF2 inhibition including E3-ligase-dependent degradation, disruption of DNA binding, and epigenetic modifications which decrease NRF2 levels and/or its transcriptional activation; all of which could serve as viable targets in the development of novel therapeutics targeting NRF2 in cancer. Regardless of the complex and difficult nature of targeting NRF2 in cancer, finding ways to manipulate each individual mode of NRF2 activation continues to represent a crucial strategy for preventing or slowing tumorigenesis, decreasing the aggressiveness of NRF2-addicted cancers, and ultimately improving patient prognosis. Overall, the temporal and contextual nature of NRF2 modulation in cancer remains a complicated balancing act, further highlighting the importance of considering how therapeutics affect this pathway when developing NRF2-targeted drugs to combat cancer.