The role of NRF2 in human cancers: Pre-clinical insights paving the way for clinical trials

Abstract

Tumorigenesis is considered as a complex, multistep process in which genetic mutations play a crucial role. The genetic mutations cause notable changes, not only in the biological behavior of tumor cells but also in their reactions to treatment. Nuclear factor erythroid 2-related factor 2 (NRF2) is one of the most disrupted molecular pathways in human cancers, and during cancer development, the expression of this factor rises to enhance survival rates. The NRF2 seems crucial for shielding tumor cells from apoptosis and oxidative damage, while promoting pro-survival autophagy to increase the survival rate. Crucially, NRF2 plays a dual role in enhancing both the growth and metastasis of cancer cells, and the upregulation of this factor boosts the stemness and cancer-stem cell characteristics of tumor cells, while also promoting drug resistance and radioresistance. The elevation of glycolysis, activation of epithelial-mesenchymal transition (EMT), and inhibition of ferroptosis are additional features of NRF2 upregulation in human cancers. Among the different pathways that control NRF2, non-coding RNA transcripts play a significant role, and by altering NRF2 expression, they influence tumor development. The pharmacological modulation of NRF2 can occur through both direct and indirect methods; in the direct method, NRF2 is inhibited, whereas in the indirect method, the regulators and associated pathways of NRF2, such as Kelch-like ECH-associated protein 1 (KEAP1), are influenced. The nanoparticles have been engineered to inhibit NRF2 in decreasing tumorigenesis. Consequently, the clinical application of current discoveries can enhance cancer treatment capabilities for patients in the near future.

Graphical abstract

Highlights

• •NRF2 upregulation drives cancer progression by enhancing stemness, drug resistance, glycolysis, and metastasis while preventing apoptosis and ferroptosis.
• •MiRNAs, lncRNAs, and circRNAs play crucial roles in modulating NRF2 expression, influencing tumorigenesis and therapy response.
• •Pharmacological and nanoparticle-based strategies targeting NRF2 and its regulatory pathways, such as Keap1, offer promising avenues for cancer therapy.

1. Introduction

The idea of oxidative stress was initially documented in the 1970s, although its true beginnings trace back to the 1950s, when scientists investigated the side effects of ionizing radiation, free radicals, and various aspects of molecular oxygen [1,2]. Harman [3] then concentrated on the aging phenomenon and the influence of these factors in 1956. Nonetheless, the idea of free radical biology was slowly embraced by the scientific community, potentially because of the theoretical aspects of the concepts and the absence of adequate experiments to support the hypothesis. Consequently, additional research was conducted in the following years to validate the hypothesis. The endeavors to comprehend the biological system's capacity to produce superoxide free radicals through typical metabolic routes [4], along with the role of superoxide dismutase (SOD) as an enzyme that assists aerobic organisms in mitigating the toxicity of free radicals [5,6], have heightened interest in this area. With the notable advancements in medicine, it is now clear that oxidative damage plays a crucial role in the development of various pathological events, and thus, its regulation can lead to substantial enhancements in disease treatment. For example, in the situation of retinal neovascularization, nanoenzymes have been created for the precise targeting of mitochondria that can avert oxidative damage [7]. Oxidative damage poses a significant risk to the brain and may lead to neurological disorders [8]; notably, decreasing both oxidative damage and inflammation can lessen the pathogenesis of Parkinson's disease [9]. The increase in the intake of green foods such as fruits and vegetables can reduce oxidative damage [10]. While oxidative damage can lead to various human diseases, the situation with cancer is unique. Due to the detrimental effects of oxidative stress, the promotion of this damage is a focus in cancer treatment. The increase in reactive oxygen species (ROS) generation and oxidative damage can induce apoptosis in cancer cells by causing mitochondrial dysfunction [11]. It is proposed that the loading of MutT homolog 1 (MTH1) inhibitor onto nanocomposites can facilitate tumor suppression caused by oxidative damage [12]. Therefore, enhancing oxidative damage through nanostructures or pharmaceutical agents can reduce tumorigenesis [13,14]. The nuclear factor erythroid 2-related factor 2 (NRF2) acts as a regulator of ROS with a crucial role in various diseases such as periodontitis [15], nerve damage repair [16], liver diseases [17] and most notably, cancer [18,19]. Fig. 1 clearly illustrates the multifaceted role of NRF2 in cancer.

Fig. 1.

Nuclear factor erythroid 2-related factor 2 (NRF2) drives cancer progression by promoting growth, metastasis, and therapy resistance via epithelial-mesenchymal transition (EMT), cell death inhibition, and metabolic changes. It is regulated by non-coding RNAs, and nanoparticle-based therapies are being developed to target its effects.

This review aims to offer an extensive examination of the molecular context of NRF2 in human cancers, emphasizing its pre-clinical assessment and possible effects on upcoming clinical trials. NRF2, an important regulator of the oxidative stress response, has a dual function in cancer development, aiding in both tumor formation and chemoresistance. This analysis explores the complex processes by which NRF2 affects the growth, survival, spread, and blood vessel formation in cancer cells, along with its relationships with non-coding RNAs and drug compounds. This review also aims to clarify the complex roles of NRF2, emphasizing its promise as a therapeutic target and providing insights on how its regulation might be utilized to enhance cancer treatments and address issues of treatment resistance. In recent years, several review articles have emerged focusing on NRF2, addressing specialized topics such as NRF2's interaction with hypoxia-inducible factors (HIFs) in cancer [20], the interplay between autophagy and NRF2 in cancer [21], the role of NRF2 in ferroptosis-driven cancer therapy [22], NRF2's involvement in amino acid metabolism within cancer [23], and its significance in pancreatic cancer [24], among various others. Nonetheless, this review offers an extensive examination of NRF2's role in interaction with various molecular pathways, regulation of biological processes in cancers, and its link to therapy resistance, encompassing both drug resistance and radioresistance.

2. The structure and signaling of NRF2

The NRF2 is a transcription factor that is part of Cap ‘n’ Collar (CNC) family, consisting of 605 amino acids and seven specific domains with individual roles, including NRF2-ECH homology domains 1 to 7 (Neh1 to Neh7) [25]. The localization of NRF2 in the cytoplasm is facilitated by the action of Neh5. Additionally, the regulation of NRF2 stability along with its ubiquitination and degradation by Kelch-like ECH-associated protein 1 (KEAP1) is controlled by the N-terminal domain [26]. Following its transfer to the nucleus, NRF2 must attach to DNA to control gene expression, and this function is enabled by the Neh1 domain, which supports DNA-binding via a CNC basic leucine zipper (Bzip) configuration in the Neh1 domain [27]. Additionally, the nuclear transfer of NRF2 is facilitated by the Neh1 domain via a nuclear localization signal (NLS) [28]. In carrying out its function, NRF2 needs to collaborate and engage with coactivators, which is facilitated by Neh3, Neh4, and Neh5 acting as transactivation domains [29,30]. The Neh6 domain is abundant in serine residues and is viewed as a regulatory domain with a negative role that binds to beta-transducin repeat containing protein (β-TrCP) to initiate NRF2 ubiquitination [31]. Neh7 domain facilitates the binding of NRF2 to retinoid X receptor alpha (RXRα), inhibiting the NRF2/antioxidant response element (ARE) pathway [32].

The canonical pathway of NRF2 activation is the most thoroughly researched mechanism and mainly concerns the regulation of NRF2 by the KEAP1-cullin 3 (CUL3) ubiquitin ligase complex, which functions as the main defense against oxidative stress [26,33]. In typical circumstances, NRF2 undergoes continuous degradation through the ubiquitin-proteasome pathway, with KEAP1 serving as a substrate adaptor for the CUL3-ring box protein 1 (CUL3-RBX1) E3 ubiquitin ligase complex, which ubiquitinates NRF2 and signals it for proteasomal degradation, thus ensuring that NRF2 levels remain low in the cytoplasm. Nevertheless, under conditions of oxidative stress or when exposed to electrophiles, ROS or electrophiles alter particular cysteine residues on KEAP1, impairing its interaction with NRF2 and inhibiting NRF2 ubiquitination, thereby enabling the accumulation of newly synthesized NRF2 in the cytoplasm and its translocation to the nucleus. Within the nucleus, NRF2 forms heterodimers with small musculoaponeurotic fibrosarcoma (Maf) proteins and attaches to AREs in the promoter areas of target genes, which promotes the transcription of genes responsible for detoxification, antioxidant protection, and anti-inflammatory actions, including heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), glutathione (GSH) S-transferases (GSTs), and glutamate-cysteine ligase catalytic subunit/modifier subunit (GCLC/GCLM). This process is vital for sustaining redox balance and safeguarding cells from harm due to ROS and electrophiles, with feedback regulation maintaining stringent oversight once the stressor is managed.

Conversely, the non-canonical NRF2 activation pathway functions without the regulation of KEAP1 and engages different signaling mechanisms that are frequently triggered by particular stimuli, including growth factors, cytokines, or disease states such as cancer [33,34]. This pathway depends on post-translational modifications of NRF2 and its interactions with signaling pathways, including phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK). Kinases such as Akt, PKC, and MAPKs can phosphorylate NRF2, thereby increasing its stability, nuclear translocation, or transcriptional activity. In addition, sequestosome 1 (p62/SQSTM1) can bind to KEAP1, release NRF2 from KEAP1-mediated repression, and promote NRF2 accumulation and nuclear translocation, a process frequently observed under conditions of dysfunctional autophagy, such as cancer and neurodegenerative diseases. Moreover, interactions with nuclear factor kappaB cells (NF-κB) signaling and epigenetic control through histone modifications and DNA methylation further influence NRF2 activity, adding an extra layer of regulation that permits cells to adjust their responses to various stimuli (Fig. 2).

Fig. 2.

Nuclear factor erythroid 2-related factor 2 (NRF2) is activated through canonical and non-canonical pathways, leading to its stabilization, nuclear translocation, and activation of antioxidant and cytoprotective genes via binding to the antioxidant response element (ARE). In the canonical pathway, oxidative stress disrupts the NRF2-Kelch-like ECH-associated protein 1 (KEAP1) interaction, while in the non-canonical pathway, autophagy inhibition and sequestosome 1 (p62/SQSTM1) accumulation sequester KEAP1, promoting NRF2 activation. ROS: reactive oxygen species; NQO1: NAD(P)H quinone dehydrogenase 1; HO-1: heme oxygenase-1; mTORC1: mechanistic target of rapamycin complex 1.

The function of NRF2 in cancer is complex, featuring both general and specific mechanisms that influence its role in tumor development, resistance to treatment, and control of oxidative stress (Table S1). Typically, NRF2 functions as a principal regulator of the antioxidant response, preserving redox equilibrium by enhancing the expression of genes such as HO-1, NQO1, and GCLM, thereby shielding cancer cells from oxidative damage. For example, in bladder cancer, the KEAP1/NRF2/GCLM pathway facilitates GSH production, improving cell survival [35], whereas in hepatocellular carcinoma, reduced NRF2 levels heighten chemotherapy sensitivity by inhibiting HIF-1α/heat shock protein 70 (HSP70) [36]. NRF2 is crucial in drug resistance, frequently enhancing drug efflux pumps such as P-glycoprotein and detoxification mechanisms, as observed in colorectal cancer (CRC), where exosomes from oxaliplatin-resistant cells convey NRF2 to boost P-glycoprotein levels and resistance to drugs [37]. Moreover, NRF2 facilitates tumor growth by boosting anti-apoptotic and pro-survival mechanisms, exemplified in prostate cancer, where the interaction between estrogen receptor alpha (ERα) and NRF2 increases the expression of resistance-related genes, aiding in castration-resistant prostate cancer (CRPC) progression [38]. Certain mechanisms, nonetheless, emphasize distinct pathways or interactions in specific types of cancer. In CRC, the stabilization of arrest-defective 1/NRF2 (ARD1/NRF2) via acetylation enhances NRF2 function [39], whereas in lung cancer, the sirtuin 1 (SIRT1)/NRF2 pathway facilitates mitophagy and resistance to oxidative stress, contributing to tumor persistence [40]. In pancreatic cancer, exosomal HSP beta-1 (HSPB1) works with fused in sarcoma (FUS) to reduce NRF2 messenger RNA (mRNA) stability, inhibiting ferroptosis [41], while in gastric cancer, GST Mu 3 (GSTM3) enhances NRF2 stability by interfering with its ubiquitination, functioning as a tumor suppressor [42]. Cervical cancer shows distinct pathways such as the NRF2/GSH peroxidase 4 (GPX4) axis, wherepeptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) influences ferroptosis and cisplatin responsiveness [43], and the neurogenic locus Notch homolog protein 1/recombination signal binding protein for immunoglobulin kappa J region/NRF2/ferredoxin-1 (Notch1/RBP-J/NRF2/FDX1) pathway, where [1-propyl-3,5-bis(2-bromobenzylidene)-4-piperidinone] (PBPD) triggers cuproptosis [44]. These results emphasize NRF2's dual function in cancer, showcasing its shared mechanisms like oxidative stress control and drug resistance, along with its unique interactions that differ among cancer types, presenting possible therapeutic targets for precision oncology.

3. NRF2 and cancer proliferation and survival rate

A defining feature of human cancer is the elevated proliferation of tumor cells compared to normal cells, and this heightened growth rate can lead to acidity in tumor microenvironment (TME) due to the induction of glycolysis as a method of energy production. Due to the oncogenic role of NRF2, it can lead to an increased proliferation rate of tumor cells, which may in turn result in the inhibition of ferroptosis [45]. At times, the rise in the proliferation rate of tumor cells by NRF2 is attributed to the boosting of enzyme activity related to metabolism. The activation of NRF2 can be stimulated by cellular myelocytomatosis oncogene (c-Myc) in head and neck cancer, leading to the upregulation of glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) to promote proliferation [46]. Subsequently, NRF2 boosts GSH metabolism levels in cancer cells to promote tumor formation [47]. Indeed, the potential of NRF2 in cancer pertains to both the growth and survival of tumor cells, and if its stability is increased by ubiquitin specific peptidase 11 (USP11), this enhancement leads to tumor cell progression [48]. Consequently, when NRF2 expression decreases, the ultimate effect is a reduction in tumorigenesis. The role of propofol as an anti-cancer agent in gastric cancer is linked to the downregulation of NRF2, which disrupts the polyol pathway and decreases the growth rate of cancer cells [49]. Nonetheless, the functions of anti-tumor agents are occasionally debated because of the dual role they play during tumorigenesis, with propofol being one of them. While prior research emphasizes propofol's role in NRF2 downregulation, a separate experiment shows that propofol activates the NRF2 pathway, promoting tumor cell proliferation and progression while inhibiting apoptosis [50]. Following a decrease in NRF2 expression, oxidative damage rises, hindering the advancement of cancer cells [51]. Nonetheless, when p62 upregulates NRF2, the levels of ROS decrease, leading to a reduction in the growth of prostate tumor cells [52]. Thus, inhibiting the p62/NRF2 pathway hinders tumor cell proliferation and triggers ferroptosis [53].

4. NRF2 and cancer metastasis and angiogenesis

The spread of tumor cells poses a significant issue, with nearly 90% of cancer fatalities resulting from invasion and metastasis. Tumor cells commonly exhibit invasion, which is essential for the spread of cancer cells to remote tissues and organs. The function of polygalacin D hinders the metastasis and advancement of esophageal cancer cells. When employed as an anti-cancer agent, polygalacin D boosts miR-142-5p expression to lower NRF2 levels, aiding in the suppression of esophageal cancer progression and the prevention of lung metastasis [54]. The KEAP1 mutation and NRF2 upregulation may lead to increased cancer invasion and the development of resistance to cisplatin [55]. Therefore, if the function of NRF2 enhances the metastasis of tumor cells, it may lead to therapy resistance. The role of NRF2 can also boost the invasion in gastric tumor cells. Notably, an increase in nestin levels in gastric cancer may accelerate the advancement of gastric tumor cells. Nestin can elevate NRF2 levels by downregulating KEAP1, thereby increasing the invasion of gastric cancer [56]. At times, NRF2's role is connected to its effect on the pathways associated with tumor invasion. Notably, the enhancement of Akt and ERK is crucial for the increase in invasion of gastric tumor cells. The checkpoint with forkhead and ring finger domains (CHFR), as an oncogenic factor, can enhance NRF2 expression, leading to decreased ROS levels. Subsequently, the upregulation of Akt and ERK takes place to enhance invasion and metastasis of gastric tumor cells [57]. Because of unusual alterations in the genomic framework of tumor cells, they can elevate NRF2 expression. Notably, the absence of mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) in melanoma leads to the upregulation of NRF2, which enhances the invasion and metastasis of tumor cells [58]. These investigations emphasize the role of NRF2 in cancer invasion and angiogenesis, as outlined in Table S2 and Fig. 3.

Fig. 3.

Nuclear factor erythroid 2-related factor 2 (NRF2) contributes to cancer development. Elevated NRF2 levels stimulate pathways including vascular endothelial growth factor A (VEGF-A), Ras-related protein Rap1b (Rap1b), and protein kinase B/extracellular signal-regulated kinase (Akt/ERK), glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT). In addition, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), genes associated with retinoid-interferon-induced mortality 19 (GRIM-19), checkpoint with forkhead and ring finger (CHFR), Kelch-like ECH-associated protein 1 (KEAP1), and hypoxia-inducible factor 1-alpha (HIF-1α) further participate in NRF2-mediated cancer progression.

5. NRF2 in cancer drug resistance

Chemoresistance is regarded as one of the most prevalent and difficult issues in the area of cancer treatment. The fundamental processes involved in tumor formation must be thoroughly recognized and tackled. The rationale is that gene therapy is emerging as a novel strategy for cancer, and by emphasizing NRF2 and associated pathways in tumorigenesis, genes aimed at these pathways can be developed to combat drug resistance. Due to the role of NRF2 in enhancing tumorigenesis and safeguarding cancer cells, the elevation of NRF2 can lead to drug resistance. The suppression of NRF2 by brucein D has led to an increase in gemcitabine sensitivity in pancreatic tumor cells, which has also been validated in vivo [59]. The main cause of NRF2-driven drug resistance is the significant increase in NRF2 expression in cancer cells, protecting them from the ROS produced by chemotherapy drugs [[60], [61], [62]]. It is significant that tripterygium glycosides inhibit NRF2, which leads to a downregulation of NQO1, GPX4, and HO-1, thereby inducing ferroptosis and overcoming resistance to cisplatin [19]. NRF2 can enhance the self-renewal and survival of cancer stem cells (CSCs), leading to carcinogenesis and resistance to drugs [63].

The link between N⁶-methyladenosine (m⁶A) changes and ferroptosis in intrahepatic cholangiocarcinoma (ICC) and its effect on cisplatin resistance was examined. Cells resistant to cisplatin were created. The effect of methyltransferase like 3 (METTL3) on drug resistance, migration, and invasion was evaluated. The target gene's expression and the impacts of overexpression and repression were evaluated. The connection between METTL3 and NRF2 was investigated. The role of the METTL3/NRF2 axis in resistance to tumor treatment was confirmed through rescue experiments. METTL3 showed increased expression in cisplatin-resistant cells, enhancing m⁶A modification levels, stabilizing NRF2 mRNA, and boosting NRF2 protein expression to inhibit ferroptosis [64]. The expression of carnitine palmitoyltransferase 1B (CPT1B) is linked to lower survival rates in individuals with pancreatic cancer. Blocking CPT1B significantly decreases the growth and dissemination of pancreatic cancer cells. Additionally, it was demonstrated that CPT1B associates with KEAP1. Inhibition of CPT1B results in reduced NRF2 expression and initiates ferroptosis. Furthermore, CPT1B expression increases following gemcitabine treatment and is notably present in gemcitabine-resistant pancreatic ductal adenocarcinoma cells. CPT1B knockdown-induced ferroptosis enhances the cytotoxicity of gemcitabine in pancreatic ductal carcinoma [65]. Patients with newly diagnosed acute myeloid leukemia (AML) and FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) exhibit poorer treatment outcomes. Arsenic trioxide (ATO), effective in treating acute promyelocytic leukemia (APL), has shown the capability to break down FLT3 protein in AML cell lines and to enhance sensitivity in non-APL AML patient samples in vitro. The pharmacological or molecular blockage of NRF2, a key regulator of the antioxidant response, enhances chemosensitivity to ATO and daunorubicin in FLT3-ITD mutant AML cells, along with non-FLT3-ITD mutated cell lines and original samples. In the FLT3-ITD mutant AML cell line MV4-11-ATO-R, known for developing ATO resistance, an increase in NRF2 expression, its nuclear localization, and the overexpression of NRF2 target genes has been reported. The suppression of NRF2 in these resistant cells increases ATO sensitivity in vitro. Digoxin treatment decreases p-Akt levels and prevents NRF2 from entering the nucleus, thereby increasing cellular susceptibility to ATO. However, digoxin and ATO do not make the non-ITD AML cell line THP1 more sensitive, which shows increased NRF2 expression. In MV4-11 ATO-R xenograft mice, digoxin lowers leukemic load and prolongs lifespan, indicating that altering NRF2 expression could mitigate acquired ATO resistance in FLT3-ITD AML [66].

Growth differentiation factor 15 (GDF15) decreases oxidative stress, leading to resistance against oxiplatin (L-OHP) chemotherapy in CRC. GDF15 posttranscriptionally influences the stability of NRF2 protein via the PI3K/Akt/glycogen synthase kinase 3 beta (GSK3β) signaling pathway. NRF2 acts as a transcription factor regulating GDF15 expression, creating a positive feedback loop that maintains redox homeostasis in CRC. A notable correlation between GDF15 and NRF2 was found in clinical CRC samples, and their simultaneous overexpression was linked to poorer outcomes in CRC patients receiving L-OHP treatment. In a mouse xenograft model derived from L-OHP-resistant CRC cells, simultaneous inhibition of GDF15 and NRF2 significantly enhanced the sensitivity to L-OHP [67]. Chemoresistance plays a major role in the poorer prognosis of patients with PC. Recent studies suggest that ferroptosis might increase chemoresistance, yet the mechanisms involved are still unclear. This research discovered a significant increase in HSP90α in the peripheral blood and tissue samples of patients displaying chemoresistant PC. HSP90α boosts the growth, movement, and invasion of chemoresistant pancreatic cancer cells by suppressing ferroptosis. It rivals KEAP1 for attachment, thus releasing NRF2 from KEAP1 confinement. NRF2 then moves to the nucleus and activated GPX4 pathway, which prevents ferroptosis and worsens chemoresistance in pancreatic cancer cells [68]. Consequently, NRF2 acts as a promoter of chemoresistance in human malignancies, as outlined in Table S3.

6. NRF2 in radioresistance

Another standard cancer treatment is radiotherapy, which targets irradiation to cause DNA damage and elevate cell death in human cancer cells. Nonetheless, this type of therapy has also encountered issues, and despite side effects, the emergence of chemoresistance can lead to treatment failure in patients. Due to the antioxidant role of NRF2, it can enhance radiotherapy resistance in human cancers. Both CRC cells and xenograft models exhibit an increase in NRF2 expression. The antioxidant activity and metabolic reprogramming are enhanced through NRF2's function to promote radioresistance in CRC [69]. Inhibition of NRF2 boosts ROS production, leads to oxidative injury, and triggers apoptosis in glioma, thereby heightening radio-sensitivity [70]. Indeed, when irradiation is applied, the goal is to enhance ROS production. Nonetheless, the upregulation of NRF2 decreases ROS production, which negatively affects radio-sensitivity in tumor cells [71]. The decrease in ROS production is connected to the increased expression of antioxidant enzymes through the NRF2 pathway. Reduced levels of Raf kinase inhibitor protein (RKIP) lead to decreased expression of miR-450b-5p. Subsequently, the overproduction of NRF2 takes place to increase NQO1 levels, boosting the antioxidant defense mechanism and safeguarding against radiotherapy [72]. In this scenario, an elevation in KEAP1 levels as a negative regulator of NRF2 can enhance sensitivity to radiotherapy. Elevated KEAP1 levels lead to the downregulation of NRF2, which increases radio-sensitivity in nasopharyngeal cancer [73], as outlined in Table S4 and Fig. 4.

Fig. 4.

Nuclear factor erythroid 2-related factor 2 (NRF2) facilitates chemoresistance and radioresistance. Additionally, NRF2 suppresses ferroptosis and boosts permeability glycoprotein (P-glycoprotein) function. TUG1: taurine upregulated gene 1; MMP-2/9: matrix metalloproteinase-2 and -9; CPT1B: carnitine palmitoyltransferase 1B; METTL3: methyltransferase-like 3; LC3-II: microtubule-associated protein 1A/1B-light chain 3-II; ROS: reactive oxygen species; NQO1: NAD(P)H quinone dehydrogenase 1.

7. NRF2 and cancer stemness

CSCs represent a specific population that is relatively small, making up 1%–2% of the entire tumor population. With the rise in stemness of cancer cells, their progression accelerates, allowing them to acquire the capability of developing resistance to therapy. Thus, the initial action is to pinpoint the essential mechanisms that lead to carcinogenesis and stemness, in order to address them in the future. NRF2 serves as a powerful regulator of stemness in human cancers, and its increased expression fosters CSC-like characteristics that enhance tumor development and contribute to chemoresistance. When the CD133 receptor is activated, it triggers the PI3K/Akt pathway, which increases NRF2 levels to support CSC characteristics [74]. Nonetheless, CD33 is not the sole contributor to CSC characteristics in tumor cells. Increased expression of CD44 leads to the p62/NRF2 pathway, influencing CSC-like characteristics in breast cancer (BC) [75]. In chronic hypoxia, there is an increase in tumor cell progression, which is also linked to stemness. The beneficial link between NRF2 and HIF-2α is crucial for enhancing stemness in chronic hypoxic conditions. Notably, NRF2 lowers levels of miR-181a-2-3p to enhance HIF-2α expression. Subsequently, it increases the expression of Krüppel-like factor 4 (KLF4), octamer-binding transcription factor (OCT4), and β-catenin, which enhances CSC-like traits and fosters tumorigenesis [76].

8. NRF2 and biological mechanisms

8.1. Apoptosis

Apoptosis is a mechanism of cell death that is inhibited in tumor cells to guarantee their survival rate. Photodynamic therapy (PDT) is utilized to diminish the survival of cancer cells. Nonetheless, when NRF2 is upregulated, it diminishes the effectiveness of PDT. Luteolin, acting as a NRF2 inhibitor, enhances ROS production and boosts PDT efficacy by as much as 45% [77]. In addition, hyperthermia can trigger apoptosis in lung cancer, while NRF2 reduces ROS production to inhibit apoptosis and encourage tumor development [78]. Therefore, the increased expression of NRF2 inhibits apoptosis in cancer cells. Notably, p62 is regarded as a trigger for NRF2. Neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) can inhibit the p62/NRF2 pathway to promote apoptosis in bladder cancer and hinder growth and metastasis [79]. When NRF2 inhibits apoptosis, it leads to tumor cells becoming resistant to chemotherapy. Inhibiting NRF2 promotes apoptosis and ferroptosis, while increasing the sensitivity of esophageal cancer cells to cisplatin [80]. This results from NRF2 decreasing oxidative damage and apoptosis to promote chemoresistance in cancer cells [81].

8.2. Autophagy

An autophagy mechanism, which serves as a programmed cell death process, may play a role in regulating tumorigenesis. Autophagy refers to the creation of autophagosomes, where cargoes are encased and subsequently degraded in the cytoplasm with the aid of lysosomes. The fundamental levels of autophagy are preserved in cells, whereas stressful situations like starvation can trigger the induction of autophagy. While apoptosis may decrease the survival of tumor cells, it has been noted that the role of autophagy does not consistently parallel that of apoptosis, and it can either promote or inhibit cancer progression. The function of autophagy in cancer biology has been well-investigated. There is a link between NRF2 and autophagy in human cancers [[82], [83], [84]]. To enhance tumorigenesis, NRF2 promotes the autophagy process [85]. To initiate autophagy, following nuclear transfer, NRF2 enhances p62 expression to facilitate autophagy-mediated survival of cancer cells [86]. The presence of Helicobacter pylori (H. pylori) infection contributes to the accelerated development of gastric cancer. Remarkably, this infectious agent boosts NRF2/HO-1 expression to trigger autophagy, aiding the advancement of gastric cancer [87]. Nevertheless, the relationship is not always such that NRF2 regulates autophagy, and at times, autophagy may influence the nuclear translocation of NRF2. The phosphorylation of the JNK/ERK axis in gastric tumors leads to the induction of autophagy by increasing the levels of beclin-1 and p62. Subsequently, p62 promotes NRF2 expression by downregulating KEAP1, which facilitates the nuclear translocation of NRF2, thereby improving the survival rate of tumor cells [88]. Due to the oncogenic role of autophagy, research has concentrated on inhibiting it to stop tumorigenesis in human cancers. Nevertheless, it has been demonstrated that inhibiting autophagy does not always inhibit tumor formation. It seems that cancer cells can evade the suppression of autophagy, and to achieve this, they promote NRF2-mediated macropinocytosis [89]. Additionally, when autophagy is inhibited, tumor cells enhance NRF2 expression to facilitate their growth [90]. Consequently, the inhibition of both autophagy and NRF2 leads to a reduction in tumorigenesis [91]. In addition, the relationship between NRF2 and autophagy can enhance the survival of tumor cells. Notably, apatinib as an anti-cancer drug can enhance ROS production in lung cancer, leading to reduced NRF2 and p62 expression which triggers apoptosis and autophagic cell death [92]. As NRF2 regulates ROS in human cancers and considering the role of ROS in autophagy regulation, there likely exists an interaction among these elements and autophagy during tumorigenesis. BDH2 acts as a tumorigenesis suppressor by interacting with KEAP1, which facilitates the degradation and ubiquitination of NRF2. Subsequently, there is an increase in ROS production that inhibits the PI3K/Akt/mTOR pathway in initiating apoptosis and autophagic cell death [93]. Interestingly, the molecular modulators of autophagy can influence the NRF2 pathway. p62/SQSTM1 acts as an autophagy regulator, while modulator of apoptosis 1 (MOAP-1) can facilitate the separation of p62. Subsequently, the release of KEAP1 takes place to inhibit the NRF2 pathway [94].

8.3. Necroptosis

The B2 protein of betanodavirus attaches to mitochondria and triggers mitochondrion-mediated apoptotic signaling in lung cancer cells, although its molecular mechanism remains unclear. This study demonstrates that B2 enhances hydrogen peroxide/NRF2-related stress signals, dynamically regulating programmed cell death in non-small cell lung cancer (NSCLC). The B2 protein serves as a necrosis inducer, promoting the death of lung cancer cells through p53 overexpression and RIP3 expression, offering an innovative method for treating lung cancer. The B2 protein was used to focus on A549 lung cancer cells and solid tumors in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Tumors were collected, processed with hematoxylin and eosin staining on tissue and cellular sections, and serum samples were analyzed for blood biochemistry. The findings showed that B2 effectively eliminated A549 cell-induced solid tumors in NOD/SCID mice, whereas the mutant ΔB2 was not effective. In these animals, B2 (but not ΔB2) activated both p53/B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)-induced apoptosis and RIPK3-mediated necroptosis. Immunochemical research showed that hydrogen peroxide/p38/NRF2 stress significantly reduced the production of tumor markers CD133, Thy-1 cell surface antigen (Thy1), and napsin, associated with the mobility and invasion of cancer cells. The B2-induced, ROS/NRF2-driven stress signal initiated numerous pathways, leading to the death of A549 lung cancer tumor cells [95].

8.4. Pyroptosis

Pyroptosis, a type of programmed cell death, is marked by cell swelling along with vesicle formation and the release of inflammatory cytokines. Piperlongumine (PL), a naturally occurring bioactive compound from Piper longum L., has demonstrated significant anti-cancer effects in oncology. However, its exact impacts and molecular mechanisms in ESCC are still insufficiently clarified. Previous article aimed to explore the role and mechanisms of PL in ESCC through both in vitro and in vivo approaches [96]. In vitro experiments demonstrated that the half maximal inhibitory concentration (IC₅₀) of PL in ESCC cells was 28.55 μM. PL significantly hindered malignant behaviors by inducing pyroptosis in ESCC cells, thereby suppressing proliferation, migration, invasion, and colony formation in KYSE-30 cells. It additionally increased the expression of ASC, cleaved-caspase-1, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and gasdermin D (GSDMD), while promoting ROS generation. NRF2 knockdown increased thioredoxin interacting protein (TXNIP) expression, whereas NRF2 overexpression decreased TXNIP expression. However, PL therapy reduced these impacts. Moreover, the suppressive effects of PL on esophageal squamous cell carcinoma (ESCC) cell malignancy were alleviated by dimethyl fumarate (DMF) (an NRF2 stimulant) or N-acetylcysteine (NAC) (a ROS neutralizer). Additionally, PL significantly boosted the levels of ASC, cleaved-caspase-1, NLRP3, GSDMD, and ROS generation; however, these impacts were reduced by TXNIP knockdown or resveratrol (RUS), which is a TXNIP inhibitor. The KYSE-30 xenograft model showed that PL obstructed the growth of ESCC tumors by promoting pyroptosis [96]. Near-infrared fluorescence (NiRF) induce cell pyroptosis and oxidative stress through golgi phosphoprotein 3 (GOLPH3) activation, while metformin mitigates these consequences. NRF2 plays a crucial role in metformin's protective effect, operating downstream of the adenosine monophosphate (AMP)-activated protein kinase/cyclic AMP (cAMP) response element-binding protein (AMPK/CREB) signaling pathway. A lack of NRF2 diminishes the protective benefits of metformin. In vivo experiments on male C57BL/6 mice exposed to NiRF (2 mg/kg) and given metformin (100, 200, and 300 mg/kg) for four weeks demonstrated that NiRF promotes pyroptosis via the upregulation of NLRP3, caspase-1, N-GSDMD, interleukin-18 (IL-18), and IL-1β. Metformin treatment reduces the changes caused by NiRF [97].

NRF2, a transcription factor, regulates the activation of antioxidant genes and plays a role in carcinogenesis when redox-sensitive signaling is disrupted. In BC, increased NRF2 activity is linked to enhanced metastatic capability. The relationship between peroxisome proliferator-activated receptor alpha (PPARα) and NRF2 pathways in cancer has been studied, leading to the assessment of novel PPARα antagonists (IB42, IB44, and IB66) alongside the control compound GW6471 in MCF7 breast cancer cells. The strongest antagonist, IB66, inhibited MCF7 growth by inducing G2/M cell cycle arrest. This effect was facilitated through a caspase 3/Akt-dependent apoptotic/pyroptotic route triggered by increased oxidative stress. IB66 also controlled the NRF2 and cyclooxygenase-2 (COX2) pathways in the cells that were treated. The results suggest that the NRF2/PPARα axis could be targeted to combat BC chemoresistance and promote different cell death pathways like pyroptosis [98]. Oxaliplatin is commonly used in CRC chemotherapy, though its efficacy is often limited by chemoresistance. A previous experimental study investigated how NRF2 inhibition enhances oxaliplatin sensitivity in CRC and the underlying mechanisms involved [99]. In vitro studies used 4-octyl itaconate (4-OI) for NRF2 activation and lentivirus to reduce NRF2 levels in CRC cell lines. The findings showed that both oxaliplatin and lobaplatin reduced the proliferation of HCT-116 and LoVo human colorectal cancer cell line (LOVO) cells in a dose-dependent manner while increasing NRF2 expression. The activation of NRF2 using 4-OI diminished the sensitivity of CRC cells to oxaliplatin and lobaplatin, while knocking down NRF2 heightened sensitivity. Analysis of public databases revealed higher GPX4 expression in CRC tissues compared to normal tissues, with increased GPX4 levels linked to poorer prognosis. Oxaliplatin reduced GPX4 expression in vitro, and NRF2 knockdown additionally decreased GPX4 and GSH levels while increasing malondialdehyde (MDA) levels, thus promoting oxaliplatin-induced ferroptosis. Additionally, oxaliplatin increased gasdermin E N-terminal fragment (GSDME-N) expression and induced the release of lactate dehydrogenase (LDH), IL-1β, and tumor necrosis factor alpha (TNF-α), while NRF2 knockdown augmented GSDME-mediated pyroptosis. In vivo knockdown of NRF2 enhanced the growth-inhibitory impact of oxaliplatin on the development of HCT116 xenograft tumors. The results suggest that inhibiting NRF2 increases sensitivity to oxaliplatin in CRC cells by promoting ferroptosis and pyroptosis, highlighting a possible treatment target for overcoming chemoresistance in CRC [99]. Ginsenoside Rh3 (GRh3) exhibits anticancer effects in CRC by inducing GSDMD-dependent pyroptosis and solute carrier family 7 member 11 (SLC7A11)-mediated ferroptosis through the signal transducer and activator of transcription 3 (STAT3)/p53/NRF2 pathway. GRh3 prevents NRF2 from moving to the nucleus, reducing HO-1 levels to promote pyroptosis and inhibiting SLC7A11 to lower GSH and raise iron, lipid ROS, and MDA, leading to ferroptosis, while demonstrating notable anticancer effectiveness with minimal harm to normal cells [100].

8.5. Ferroptosis

Ferroptosis is another mechanism of cell death that relies on iron and the generation of ROS, exhibiting notable morphological, biochemical, and genetic distinctions from apoptosis, autophagy, and necrosis [101]. Ferroptosis is regarded as a novel treatment approach to combat chemoresistance [102,103]. The enhancement of NRF2 inhibits ferroptosis in colorectal tumors. In Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant CRC, cetuximab-induced downregulation of the NRF2/HO-1 axis may lead to ferroptosis [104]. When cancer cells exhibit drug resistance, research concentrates on the use of alternative anti-cancer agents. Nonetheless, when there is an upregulation of NRF2, it leads to the suppression of artesunate-induced ferroptosis; in this scenario, silencing NRF2 can improve the sensitivity of head and neck cancer cells to artesunate-induced ferroptosis [105]. Table S5 provides a summary of the interaction between NRF2 and ferroptosis in human cancers. Fig. 5 emphasizes the interaction between NRF2 and cell death mechanisms in cancer.

Fig. 5.

Nuclear factor erythroid 2-related factor 2 (NRF2) regulates multiple cell death pathways by inhibiting apoptosis. In contrast, NRF2 inhibition by the NRF2 inhibitor IB66 can induce pyroptosis, contributing to breast cancer (BC) suppression. Bcl-2: B-cell lymphoma 2; IB66: a phenylsulfonimide PPARα antagonist; H. pylori: Helicobacter pylori; p62/SQSTM1: sequestosome 1; HO-1:heme oxygenase-1.

8.6. Epithelial-mesenchymal transition (EMT)

EMT is viewed as a process that promotes tumorigenesis and cancer spread because it can convert epithelial cells into mesenchymal cells. By elevating N-cadherin and vimentin levels while decreasing E-cadherin levels, EMT facilitates cancer metastasis, potentially leading to the spread and invasion of tumor cell [106,107]. There is a strong link between NRF2 and EMT in controlling the invasion and metastasis of cancer cells. The enhancement of NRF2 supports tumor development, which occurs by influencing macrophage polarization; when tumor cells interact with macrophages, they can trigger EMT [108]. Bacterial infections can also enhance the invasion and spread of tumor cells. Fusobacterium nucleatum can activate the Toll-like receptor 4 (TLR4)/Akt pathway to boost NRF2 expression by downregulating KEAP1 during EMT induction and increasing metastasis in CRC [109]. Therefore, pharmacological inhibition of NRF2 can significantly aid in decreasing the invasion of tumor cells. Interestingly, the upregulation of MAPK and NRF2 in lung cancer can not only promote metastasis but also contribute to gefitinib resistance in tumor cells [110]. Consequently, the role of NRF2 extends beyond mere regulation of the antioxidant defense system in human cancers [111] and the downregulation of NRF2 by diallyl disulfide can hinder tumorigenesis and the invasion of lung cancer [112].

8.7. Glycolysis

The stimulation of glycolysis substantially enhances the proliferation of tumor cells. Glycolysis, or glucose metabolism, represents a novel metabolic pathway in tumor cells, where rapidly proliferating cancer cells favor glycolysis over oxidative phosphorylation. The wealth of evidence has emphasized the role of glycolysis in promoting tumorigenesis. AlkB homolog 5 (ALKBH5) can boost m⁶A demethylation of glucose transporter type 4 (GLUT4) in BC and encourages tumorigenesis by activating glycolysis [113]. When glycolysis is inhibited, the growth rate of tumor cells decreases [114]. The mutation of DNMT3A R882H can enhance the survival rate of acute leukemia cells. When the DNMT3A R882H mutation takes place, it triggers NRF2/NQO1 activation, promoting glycolysis and facilitating carcinogenesis [115]. Nonetheless, glycolysis is not solely linked to the proliferation rate of cancer cells; at times, it can also promote stemness. The occurrence of endoplasmic reticulum (ER) stress can elevate NRF2 levels, initiating the Warburg effect and enhancing the stemness of cancer cells [116]. As noted earlier, infection presence may lead to cancer development in certain instances, and thus, the role of infections in enhancing glucose metabolism should be assessed. The hepatitis B virus is capable of leading to liver cancer and by boosting NRF2 expression, and it promotes G6PD expression to increase glycolysis in hepatocytes, which is regarded as one of the fundamental mechanisms in hepatocarcinoma progression [117]. Fig. 6 illustrates the role of NRF2 in the regulation of EMT and glycolysis.

Fig. 6.

Nuclear factor erythroid 2-related factor 2 (NRF2) promotes epithelial-mesenchymal transition (EMT) and cancer metastasis. Additionally, factors such as mitogen-activated protein kinase (MAPK), DNA methyltransferase 3 alpha (DNMT3A), and endoplasmic reticulum (ER) stress enhance NRF2 activity. ATP: adenosine triphosphate; ADP: adenosine diphosphate; NAD: nicotinamide adenine dinucleotide; HIF-1: hypoxia-inducible factor 1.

9. Non-coding RNA-mediated regulation of NRF2

9.1. microRNAs (miRNAs)

miRNAs are brief, natural non-coding RNAs that show dysregulation in human cancers and play a role in regulating tumor development. The function of miRNAs involves attaching to the 3′-untranslated region (UTR) of target mRNA to inhibit translation or facilitate degradation. The NRF2 can be controlled by miRNAs in human cancers. In hypoxia, levels of NRF2 rise to promote tumorigenesis. Nonetheless, miR-140-5p can inhibit the NRF2/HO-1 pathway, thereby hindering tumorigenesis in BC [118]. In a fascinating manner, NRF2 has the capacity to regulate the expression levels of miRNAs, as this protein can attach to the promoters of miRNAs. NRF2 binds to the 5′-UTR of miR-125B and miR-29B, enhancing their expression and leading to tumorigenesis [119]. Additionally, a connection can be established between NRF2 and miRNAs in human cancers. miR-29b-1-5p can lower the levels of Akt and NRF2, whereas NRF2 decreases the expression of miR-29b-1-5p in the context of ROS reduction and promoting proliferation in triple-negative BC [120].

9.2. Long non-coding RNAs (lncRNAs)

The lncRNAs represent another category of non-coding RNA that can influence proteins, genes, and miRNAs. The cytoplasmic interaction between lncRNA and miRNAs involves lncRNAs sponging miRNAs to decrease their expression levels. Unlike miRNAs, which exist solely in their mature form within the cytoplasm, lncRNAs are present in the mitochondria, nucleus, and cytoplasm, and their functions range from chromatin remodeling to influencing proteins and various epigenetic factors. The interaction between lncRNA and NRF2 can influence the advancement of cancer cells. The role of lncRNA SLC7A11-antisense RNA 1 (AS1) in CRC can promote tumorigenesis by enhancing the nuclear translocation of NRF2, which decreases ROS production and boosts growth and invasion [121]. This interaction can likewise influence how tumor cells respond to chemotherapy. The lncRNA MIR4435-2HG has been demonstrated to boost the NRF2/HO-1 pathway, aiding in the prevention of apoptosis and the promotion of tumorigenesis, which leads to cisplatin resistance [122].

9.3. Circular RNAs (circRNAs)

The negative aspect of NRF2 regulation in human cancers may be seen in its interaction with circRNAs, given that significant focus has been placed on miRNAs and lncRNAs. Just a single study has assessed the relationship between circRNAs and NRF2. The capacity of circular RNA derived from the KEAP1 gene (circKEAP1) to inhibit tumor formation in lung cancer has been assessed, and this circRNA exhibits low expression levels. The circKEAP1 sequesters miR-141-3p to inhibit NRF2 by enhancing KEAP1 levels, thereby contributing to lung cancer progression [123]. Nevertheless, there remains significant potential for comprehending the interaction between circRNAs and NRF2 in human cancers (Table S6 and Fig. 7).

Fig. 7.

Non-coding RNAs regulate nuclear factor erythroid 2-related factor 2 (NRF2) activity in cancer. HIF-1α: hypoxia-inducible factor 1; VEGF: vascular endothelial growth factor; SLC7A11: solute carrier family 7 member 11; CRC: colorectal cancer; nEDD4: neural precursor cell expressed, developmentally downregulated 4; KLF8: Krüppel-like factor 8; BC: breast cancer; CircKEAP1: KEAP1-derived circular RNA.

10. Pharmacological targeting of NRF2 in cancer therapy

Focusing on NRF2 is advantageous in cancer treatment due to its role in oncogenesis; thus, its downregulation facilitates the suppression of tumor formation. Decreased expression levels of NRF2 may contribute to diminished tumorigenesis, and through the action of ailanthone, NRF2 downregulation takes place in bladder cancer treatment, which lowers proliferation and metastasis [124]. Combination therapy is proposed to be advantageous in cancer treatment, and a blend of cyaniding-3-O-glucoside and cisplatin can lower NRF2 expression, which plays a role in oxidative damage and apoptosis [125]. Nonetheless, debates exist regarding the regulation of NRF2 by pharmaceutical agents, and while several studies support NRF2 inhibition in cancer treatment, evidence suggests that NRF2 activation may also hinder tumor development. 2-(7-(Diethylamino)-2-oxo-2H-chromen-3-yl)cyclohexa-2,5-diene-1,4-dione (PBQC) is a small molecule that targets GSH, and by inducing the overexpression of NRF2 and KEAP1 S-glutathionylation, it promotes apoptosis and hinders proliferation [126]. Additionally, the elevation of NRF2 expression by cyanidin chloride inhibits the NF-κB pathway, thereby decreasing tumor development in CRC [127]. In certain situations, the quandary in NRF2 regulation and function is associated with antioxidant activity. Sulforaphane is regarded as a powerful antioxidant that can enhance NRF2 expression. In bladder cancer, sulforaphane increases ROS production and NRF2 expression, leading to apoptosis. TNF-related apoptosis-inducing ligand (TRAIL) can inhibit the upregulation of NRF2 by sulforaphane, which speeds up the generation of ROS in cancer treatment [128]. Nonetheless, the action of anti-cancer agents is occasionally influenced by the regulation of several pathways. The induction of apoptosis and the inhibition of metastasis in BC can be achieved by the action of alantolactone, which lowers the levels of NRF2 and NF-κB while enhancing the p38 MAPK pathway [129]. Indeed, one method to promote apoptosis in human cancer cells is to increase the production of ROS. Apatinib functions as a tyrosine kinase inhibitor, capable of inhibiting the NRF2/HO-1 pathway to promote ROS-induced apoptosis and autophagy in ovarian cancer [130].

The KEAP1-NRF2 pathway is crucial for protecting cells against oxidative and xenobiotic stress by regulating the expression of cytoprotective genes. Generally, KEAP1 suppresses NRF2 by promoting its degradation. In stressful situations, NRF2 gathers and stimulates protective genes. In various cancers, NRF2 builds up continuously because of failures in KEAP1-driven degradation, granting drug and radiation resistance to tumor cells. Investigators examined NRF2 inhibitors and found febrifugine derivatives, notably halofuginone. Halofuginone, even at low doses, reduces NRF2 levels by initiating an amino acid scarcity response, which hinders overall protein synthesis and diminishes NRF2. This intervention markedly reduced drug resistance in neoplastic cells reliant on NRF2 [131]. Our team identified brusatol, the initial NRF2 inhibitor, to address chemoresistance. However, the method of inhibition has not been determined up to this moment. Brusatol's mode of action does not involve the direct inhibition of the NRF2 pathway. Brusatol suppresses both cap-dependent and cap-independent protein translation, affecting multiple short-lived proteins, such as NRF2 [132]. Brusatol, a distinctive NRF2 pathway inhibitor, boosts the sensitivity of various cancer cells and xenografts to chemotherapy drugs like cisplatin by reducing NRF2 protein levels through enhanced ubiquitination and degradation, thus obstructing the NRF2-driven protective mechanism. This leads to increased apoptosis, diminished cell proliferation, and greater tumor growth inhibition in A549 xenografts, with actions that are dependent on NRF2, suggesting its potential as a supplementary chemotherapeutic agent [133]. ML385, a novel NRF2 inhibitor identified through high-throughput screening, targets the Neh1 domain of NRF2, disrupts the DNA binding of the v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog G (MAFG)-NRF2 complex, and enhances cytotoxic effects when used in combination with chemotherapy drugs in NSCLC cells, particularly those with KEAP1 mutations or showing NRF2 gain-of-function. In preclinical models, the combination of ML385 and carboplatin demonstrated significant antitumor effectiveness, highlighting its potential as a targeted therapy for NSCLC with increased NRF2 activation [134]. Trigonelline (TRG) showed scolicidal effectiveness against Echinococcus granulosus protoscoleces by inhibiting the NRF2 signaling pathway, reducing the activities of NQO-1 and HO-1, increasing ROS levels, and triggering caspase-3, leading to significant structural damage and reduced viability of protoscoleces. The NRF2 protein, primarily located in protoscolex cells, was significantly downregulated by TRG, highlighting its potential as a treatment targeting the NRF2 pathway in Echinococcus granulosus [135]. TRG greatly reduces NRF2 activity and lowers NRF2-driven proteasome activity in pancreatic cancer cells, leading to increased vulnerability to anticancer drugs and TRAIL-induced apoptosis by inhibiting cytoprotective gene expression and proteasomal activity. TRG acts as a potent NRF2 inhibitor, enhancing the efficacy of anticancer medications in both cell lines and tumor-bearing animals, suggesting its promise as a therapeutic agent to improve cancer treatment outcomes [136]. NRF2 is controlled through various mechanisms, including the KEAP1-CUL3-RBX1 E3-ligase system, KEAP1-independent E3 ligases such as β-TrCP and HRD1 E3 ubiquitin-protein ligase (Hrd1), autophagy-associated pathways, and transcriptional regulation by oncogenic Ras, leading to its dual role in cellular protection and cancer progression. Understanding these regulatory mechanisms has aided the creation of NRF2 modulators, which encompass inducers for chemoprevention and inhibitors to combat chemoresistance in tumors with persistent NRF2 activation [137]. Consequently, both synthetic and natural products possess the capability to modulate NRF2 in cancer treatment, as outlined in Table S7.

11. Nanostructures regulating NRF2

The biological role of NRF2 in different human cancers has been thoroughly recorded. Based on the conversations, the role of NRF2 is multifaceted, allowing it to enhance tumor cell advancement and contribute to the characteristics of cancer. Consequently, inhibiting NRF2 hinders tumor formation. This is the reason that pharmacological agents for regulating NRF2 have been created. Nevertheless, drugs and anti-cancer agents face low bioavailability; hence, nanostructures are utilized for their delivery to enhance the therapeutic index. Lately, nanoparticles have been developed for the modulation of NRF2 in cancer treatment. Administering oridonin via poly(lactic-co-glycolic acid)/polyethylene glycol (PLGA/PEG) nanostructures into mice may lead to its uptake by cancer cells, thereby inhibiting the NRF2 axis, decreasing NQO-1 and HO-1 levels, and promoting ROS production, ultimately causing apoptosis [138]. More significantly, the sensitivity of cancer cells to chemotherapy can increase through the use of nanoparticles. The lipidic nanostructures containing quinacrine can suppress the NRF2 pathway, contributing to cell death while enhancing cisplatin sensitivity in lung tumors [139]. Luteolin serves as an additional anti-cancer medication for cancer treatment. Luteolin can be incorporated into phytosomes, which subsequently inhibit the NRF2 pathway, increasing BC sensitivity to doxorubicin [140]. The notable aspect is that despite a substantial rise in the anti-cancer effectiveness of drugs, nanocarriers exhibit high biocompatibility. The micellar nanostructures can encapsulate halofuginone, and despite their ability to inhibit tumorigenesis, they exhibit low systemic toxicity and are biocompatible [141]. The phytochemical attachment to the nanostructures can significantly enhance their potential in cancer treatment. EGCG serves as a modulator for NRF2, and when it is attached to magnetic nanostructures, these can activate the NRF2/HO-1 pathway and trigger apoptosis [142]. One of the complications is the existence of several research studies that demonstrate the role of NRF2 upregulation in cancer treatment, akin to the earlier experiment. Lately, gene therapy and the regulation of NRF2 through genetic techniques have been implemented, and to enhance this capability, gene delivery via nanostructures has been pursued. Nanostructures can achieve an encapsulation efficiency of 90% for NRF2-small interfering RNA (siRNA), and upon exposure to ultrasound, nanobubbles are capable of releasing NRF2 for melanoma treatment [143]. Additionally, the downregulation of NRF2 via siRNA-loaded carbosilane dendrimers can reduce cisplatin resistance in bladder cancer [144].

12. A summary of targets

NRF2 is a vital transcription factor that regulates numerous target genes, many of which play important roles in cancer biology. The NRF2 target genes that have been studied most extensively are HO-1, NQO1, and GSTs. HO-1 is elevated in various cancers, such as bladder and liver cancer, where it promotes tumor cell survival by diminishing oxidative stress and enhancing resistance to therapy. In bladder cancer, the KEAP1/NRF2/GCLM pathway stimulates GSH synthesis, improving cellular defense against oxidative damage and chemotherapy agents. NQO1 is often overexpressed in CRC, where it detoxifies harmful quinones and aids treatment resistance by metabolizing anticancer drugs like mitomycin C. GSTs, a class of enzymes regulated by NRF2, are typically elevated in cancers such as melanoma and glioblastoma, enabling cancer cells to evade chemotherapy-induced apoptosis by attaching GSH to reactive agents, thus promoting their removal. These instances underscore the dual role of NRF2 in protecting normal cells from oxidative damage while offering survival advantages to cancerous cells.

The intricate relationship between NRF2 and its target genes goes beyond mere detoxification and antioxidant protection mechanisms. The overexpression of glycolytic enzymes such as G6PD and TKT, driven by NRF2, aids in the metabolic reprogramming of cancer cells, enhancing rapid growth and endurance in challenging environments. The metabolic change is particularly evident in head and neck cancers, where c-Myc-driven NRF2 activation boosts G6PD and TKT activity, promoting tumor development and progression. Furthermore, the influence of NRF2 on EMT and metastasis is enhanced by the overproduction of Snail and additional transcription factors that trigger EMT, thereby encouraging cancer cell invasion and dissemination. The SIRT1/NRF2 pathway in lung cancer promotes mitophagy and improves oxidative stress resistance, thereby aiding tumor survival and growth. Additionally, NRF2's role in regulating ferroptosis, a form of regulated cell death triggered by iron-dependent lipid peroxidation, is becoming increasingly acknowledged. NRF2 can prevent ferroptosis by enhancing the expression of genes linked to GSH metabolism and lipid repair, like GPX4, thus supporting cancer cell survival and resistance to therapies that induce this type of cell death.

Non-coding RNAs, including lncRNAs and miRNAs, add another level of complexity to NRF2 signaling in cancer. LncRNAs such as SLC7A11-AS1 and MIR4435-2HG have shown the ability to influence NRF2 function, impacting cancer development and treatment resistance. SLC7A11-AS1 aids the movement of NRF2 into the nucleus, reducing ROS levels and promoting growth and invasion in CRC. Conversely, inhibiting lncRNA TUG1 has been demonstrated to increase sensitivity to cisplatin by reducing NRF2 levels in bladder cancer. miRNAs, such as miR-155, can promote tolerance to ATO in lung cancer cells by activating NRF2 and preventing apoptosis. The interaction between NRF2 and these non-coding RNAs highlights the intricate regulation of NRF2 signaling, which could be utilized for therapeutic benefits. Understanding the precise mechanisms through which non-coding RNAs influence NRF2 activity could lead to novel strategies for targeting NRF2 in cancer therapy.

Strategies involving pharmacological approaches and nanoparticles to modulate NRF2 offer promising avenues for cancer treatment. Pharmacological inhibitors like brusatol and ML385 have shown effectiveness in increasing cancer cells' sensitivity to chemotherapy and radiation by disrupting redox balance and triggering cell death pathways, including apoptosis, ferroptosis, and autophagy. Compounds such as sulforaphane and curcumin might offer benefits in early-stage cancers or healthy tissues by protecting against damage caused by oxidative stress and preventing cancerous transformation. However, incorrect activation of NRF2 in advanced cancers might inadvertently enhance tumor growth, spread, and resistance to treatment. Methods of delivery based on nanotechnology have surfaced as efficient means to improve the accuracy and safety of NRF2-targeted therapies. Nanoparticles can be engineered to selectively deliver NRF2 modulators to tumor cells by exploiting the unique characteristics of the TME, such as enhanced permeability and retention (EPR) effects, low pH levels, and overexpressed surface receptors. Stimuli-responsive nanomaterials, such as pH-sensitive and redox-sensitive nanoparticles, enable the controlled release of NRF2 modulators in response to specific tumor-related stimuli, thus improving tumor selectivity and minimizing off-target effects. These advancements greatly improve the therapeutic index of NRF2-targeted therapies, thereby benefiting outcomes for cancer patients.

The transcription factor NRF2 interacts with various signaling pathways, influencing and being influenced by other transcriptional regulators. NRF2 can influence the expression or stability of various transcription factors, including Notch1, aryl hydrocarbon receptor (AhR), and NF-κB. Studies on cells lacking NFE2L2 reveal lower expression levels of Notch1 and AhR target genes, along with increased NF-κB activity due to enhanced IκB kinase β activity [[145], [146], [147]]. Additionally, some substances that trigger NRF2 can simultaneously activate various transcriptional regulators, creating complex relationships, like the connection between NRF2 and AhR when exposed to specific chemicals. A functional relationship is present between the NRF2 and Notch1 pathways, as demonstrated by studies on liver regeneration [[148], [149], [150]]. The activity of NRF2 may be affected by its interactions with mutant p53, which influences the regulation of proteasome genes and contributes to drug resistance [151]. To improve understanding of these connections, tools such as NRF2-ome have been developed to outline NRF2 interactions and regulatory networks, highlighting the need for integrated approaches to fully comprehend NRF2-driven gene regulation [145].

13. Conclusion

The disruption of molecular pathways facilitates the advancement of human cancers, and the atypical expression of NRF2 has been thoroughly recorded in early, localized, advanced, and metastatic cancer stages. The role of NRF2 is believed to be multifaceted, influencing various cancer hallmarks, including growth, metastasis, and response to therapy. A key advantage of research is comprehending the biological behavior of cancer cells in relation to the role of NRF2. This role is to enhance proliferation and metastasis while also promoting angiogenesis. Nonetheless, several studies indicate that the role of NRF2 is not consistently oncogenic and may act as a tumor-suppressor factor; however, these studies are few, and the primary emphasis remains on its oncogenic role. The stemness of cancer cells is increased by NRF2, which can promote chemo-resistance and radioresistance. Consequently, targeting NRF2 therapeutically may be utilized to decrease tumor formation and enhance treatment response. A key aspect is the connection between NRF2 and biological processes in tumor cells. NRF2 can inhibit apoptosis and ferroptosis during tumor development, while it promotes pro-survival autophagy to boost carcinogenesis and avoid apoptosis in human cancers. NRF2 shows a positive correlation with the metastasis of cancer cells due to its stimulation of EMT and increased motility of cancer cells. Notably, NRF2 enhances glycolysis and promotes proliferation and metabolic reprogramming in cancerous cells. The control of NRF2 can be influenced by non-coding RNA transcripts such as miRNAs, lncRNAs, and circRNAs, impacting tumorigenesis. The pharmacological modulation of NRF2 has been observed in cancer treatment, where both upregulation and down-regulation of NRF2 can be seen in cancer therapy. Notably, the regulation of NRF2 through nanoparticles has been conducted to hinder tumor formation. Regarding the dual role of NRF2 in human cancers, the authors suggest that it is connected to the role of ROS, as NRF2 regulates the production of ROS. According to the research, the generation of ROS does not consistently ensure a reduction in tumorigenesis and the induction of apoptosis via oxidative damage; at times, it can even encourage tumorigenesis. Consequently, it is accurate to say that a decrease in NRF2 expression can enhance ROS production during apoptosis; in instances where ROS has a cancer-promoting role, increasing NRF2 can inhibit tumor development by lowering ROS levels. Nonetheless, additional targeted research needs to be conducted to clarify the precise reason for the dual role of NRF2 in human cancers and to show whether this function is context-dependent or not.

The deregulation of molecular pathways, particularly the NRF2 signaling axis, is essential in the progression of human cancers. This article highlights that NRF2 functions as a dual entity in cancer biology, exhibiting both oncogenic and tumor-suppressive functions depending on the cellular context, type of tumor, and stage of cancer development. NRF2 is acknowledged for promoting cancer cell survival, growth, and treatment resistance through its antioxidant and cytoprotective roles; nevertheless, it can also suppress tumor development in certain situations, especially when oxidative stress is employed to induce cancer cell death. This duality emphasizes the complexity of targeting NRF2 in cancer therapy and raises important issues regarding its clinical application. The potential of using NRF2 as a therapeutic target in cancer therapy is highly influenced by the specific context. The inhibition of NRF2 has been shown to make cancer cells more receptive to chemotherapy, radiation, and targeted therapies by disrupting redox balance, enhancing oxidative damage, and promoting cell death mechanisms like apoptosis, ferroptosis, and autophagy. This approach is particularly relevant in cancers where NRF2 remains continuously active because of mutations in KEAP1, NRF2, or other regulatory factors, leading to heightened antioxidant defenses and treatment resistance. Conversely, the activation of NRF2 could be beneficial in particular situations, such as in early-stage cancers or in healthy tissues, where it may protect against damage caused by oxidative stress and hinder malignant transformation. The timing and method of NRF2 modulation are crucial. Unintentional activation of NRF2 in advanced cancers may inadvertently promote tumor growth, spread, and drug resistance by increasing the survival of CSCs and protecting tumor cells from oxidative damage. Conversely, the early suppression of NRF2 in healthy tissues or initial-stage lesions might amplify oxidative stress, leading to genomic instability and an increased risk of cancer. Therefore, the development of NRF2-focused treatments must be carefully tailored to the specific type of cancer, its stage, and the tumor's genetic traits. Pharmacological agents that modulate NRF2 activity, such as NRF2 inhibitors (brusatol and luteolin) and activators (sulforaphane and curcumin), should be used in a context-dependent manner. Therapeutic regimens that simultaneously address NRF2 alongside other cancer-related pathways (such as PI3K/Akt, MAPK, or NF-κB) could offer a more successful strategy to overcome resistance and enhance treatment efficacy. Utilizing nanotechnology-driven delivery techniques to precisely aim at NRF2 within tumor cells, while safeguarding healthy tissues, could minimize off-target effects and improve the therapeutic index. While NRF2 presents a promising therapeutic target for cancer, its dual role in tumor progression and suppression necessitates a complex strategy for its regulation. Upcoming preclinical and clinical studies should aim to identify biomarkers that indicate responsiveness to NRF2-targeted therapies, enhance the timing and dosage of NRF2 modulators, and develop combination strategies that take advantage of the weaknesses of NRF2-dependent tumors. By addressing these challenges, we can effectively utilize NRF2 as a therapeutic target and improve results for cancer patients.

A key challenge in NRF2-focused therapy is achieving precise targeting of tumors while minimizing unintended effects on healthy tissues. Traditional pharmacological agents that impact NRF2 activity, including small molecule inhibitors like brusatol and ML385 or activators such as sulforaphane and bardoxolone methyl, often show a lack of selectivity, leading to potential toxicity and harmful effects on healthy tissues. To address this limitation, the progress of nanotechnology-driven delivery systems has emerged as a promising method to enhance the precision and safety of NRF2-targeted therapies. Nanomaterials such as liposomes, polymeric nanoparticles, and dendrimers can be engineered to specifically deliver NRF2 modulators to cancer cells by utilizing the unique properties of the TME, which include EPR effects, acidic pH, and elevated surface receptor expression. Nanoparticles loaded with NRF2-siRNA have shown effectiveness in inhibiting NRF2 expression in cancer cells, thereby enhancing their vulnerability to chemotherapy and radiation while protecting normal tissues. Similarly, NRF2 inhibitors enclosed in tumor-targeting nanocarriers, like folate-modified liposomes or antibody-linked nanoparticles, may preferentially accumulate at tumor sites, thus reducing systemic toxicity and improving therapeutic effectiveness. Moreover, employing stimuli-responsive nanomaterials like pH-sensitive or redox-sensitive nanoparticles enables the controlled release of NRF2 modulators in response to specific tumor-related stimuli, thereby enhancing tumor selectivity and minimizing off-target effects. In addition to improving tumor targeting, nanomaterials also enhance medication stability, bioavailability, and pharmacokinetics. Encapsulating NRF2 inhibitors within polymeric nanoparticles, including PLGA or PEG-based carriers, could protect them from degradation, prolong their circulation time, and improve their accumulation in tumor tissues. Furthermore, the combination of NRF2-targeted nanomaterials with other treatment methods, including PDT or immunotherapy, could synergistically enhance anticancer efficacy by simultaneously targeting multiple pathways involved in tumor progression and treatment resistance. While nanomaterials hold great promise for improving the precision and efficacy of NRF2-targeted treatments, their safety profile needs additional evaluation. Issues may include toxicity caused by nanoparticles, immune responses, and prolonged biodistribution. Comprehensive preclinical studies are necessary to enhance the design, composition, and surface properties of nanomaterials, thus reducing risks and ensuring their biocompatibility and biodegradability. Additionally, the progress of biomarker-based strategies to identify patients who will benefit from NRF2-targeted treatments can enhance both the safety and effectiveness of therapies.

Funding

This research was supported by Liaoning Province Science and Technology Plan Joint Program (Natural Science Foundation - General Project) (2024-MSLH-559& 2024-MSLH-579), the Medical Engineering Interdisciplinary Research Funds of Liaoning Cancer Hospital & Institute-Dalian University of Technology under Grant No.LD202218, Collaborative Research Project of China Medical University, as well as the 345 Talent Project of Shengjing Hospital (30C&30D).

CRediT authorship contribution statement

Yi Pei: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Jianqiao Yin: Writing – review & editing, Writing – original draft, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization. Jiamei Liu: Writing – original draft, Project administration, Methodology, Investigation, Data curation, Conceptualization. Dongze Liu: Writing – review & editing, Writing – original draft, Software, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization. Qianlong Wu: Writing – review & editing, Writing – original draft, Visualization, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization. Xue Cai: Writing – review & editing, Writing – original draft, Visualization, Software, Resources, Methodology, Investigation, Data curation. Mingming Han: Writing – review & editing, Writing – original draft, Software, Resources, Methodology, Investigation. Yu Tian: Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Liyu Yang: Writing – review & editing, Writing – original draft, Supervision, Software, Project administration, Methodology, Investigation, Conceptualization. Shengye Liu: Writing – review & editing, Writing – original draft, Visualization, Supervision, Software, Resources, Methodology, Investigation, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.