Anti-Ferroptotic Effects of Nrf2: Beyond the Antioxidant Response
Aryatara Shakya, Nicholas W. McKee, Matthew Dodson, Eli Chapman
Abstract
The transcription factor Nrf2 was originally identified as a master regulator of redox homeostasis, as it governs the expression of a battery of genes involved in mitigating oxidative and electrophilic stress. However, the central role of Nrf2 in dictating multiple facets of the cellular stress response has defined the Nrf2 pathway as a general mediator of cell survival. Recent studies have indicated that Nrf2 regulates the expression of genes controlling ferroptosis, an iron- and lipid peroxidation-dependent form of cell death. While Nrf2 was initially thought to have anti-ferroptotic function primarily through regulation of the antioxidant response, accumulating evidence has indicated that Nrf2 also exerts anti-ferroptotic effects via regulation of key aspects of iron and lipid metabolism. In this review, we will explore the emerging role of Nrf2 in mediating iron homeostasis and lipid peroxidation, where several Nrf2 target genes have been identified that encode critical proteins involved in these pathways. A better understanding of the mechanistic relationship between Nrf2 and ferroptosis, including how genetic and/or pharmacological manipulation of Nrf2 affect the ferroptotic response, should facilitate the development of new therapies that can be used to treat ferroptosis-associated diseases.
INTRODUCTION
The transcription factor nuclear factor erythroid-2 (NF-E2)-related factor 2 (Nrf2, encoded by the NFE2L2 gene), which was originally identified as a master regulator of the cellular antioxidant response, is now recognized as a crucial mediator of a wide range of cellular processes (Dodson et al., 2019). Along with its role in mitigating oxidative stress, Nrf2 has been shown to regulate iron homeostasis, xenobiotic metabolism, proteostasis, DNA repair, metabolism of carbohydrates, amino acids, and lipids, as well as mitochondrial function (Cuadrado et al., 2019; Hayes and Dinkova-Kostova, 2014; Yamamoto et al., 2018). Nrf2 is essential for maintaining proper cell function, and loss of Nrf2 results in enhanced sensitivity to cytotoxic xenobiotics and increased risk of cell death.
Cell death is tightly regulated at the molecular level, and its effectors are often closely integrated with other key cellular processes. Our understanding of how cell death is initiated continues to evolve, with 12 different modes of programmed cell death having now been identified (Galluzzi et al., 2018). Ferroptosis is a recently discovered form of regulated cell death that is genetically, biochemically, and morphologically distinct from other modes of cell death. Ferroptosis is driven by the accumulation of free labile iron, and increased lipid peroxidation, which represent the primary hallmarks of ferroptosis (Forcina and Dixon, 2019; Stockwell et al., 2017). Interestingly, Nrf2 regulates the expression of many genes responsible for preventing these hallmarks from occurring. Since ferroptosis is an oxidative form of cell death, Nrf2 was initially believed to exert its anti-ferroptotic effects primarily through regulation of the antioxidant response; however, recent studies from our group and others have implicated new mechanisms of Nrf2 regulation of ferroptosis that extend beyond antioxidant function into key facets of iron and lipid homeostasis. In this review, we will provide an in-depth exploration of how Nrf2 regulates ferroptosis, with an emphasis on its antioxidant-independent functions, and how modulation of Nrf2-mediated ferroptotic death could affect various disease states.
OVERVIEW OF FERROPTOSIS
Iron homeostasis
Iron exists in two redox states, ferrous (Fe2+) and ferric (Fe3+). While the constant loss or gain of electrons to switch between two redox states makes iron useful for metabolic reactions, the generation of free radicals due to an excess of the highly reactive Fe2+ form is toxic to cells. To prevent iron toxicity, free labile iron in the form of (Fe2+) is controlled by multiple systems at both the systemic and cellular levels to maintain iron homeostasis. Systemic iron homeostasis is regulated by hepcidin, a hormone released from the liver. Iron in the blood in the Fe3+ form is bound by transferrin (Knutson, 2017). As shown in Fig. 1, transferrin-bound Fe3+ is endocytosed into cells by the transferrin receptor (TFR). Once inside the cell, Fe3+ is released from TFR and reduced to Fe2+ by STEAP3. Fe2+ is then transported from the endosomal compartment to the cytoplasm by divalent metal transporter 1 (DMT1), where it is oxidized back to Fe3+ and incorporated into proteins or stored in ferritin cages, with the help of iron chaperones such as poly(RC)-binding protein 1 (PCBP1) (Philpott et al., 2017). Excess Fe2+ can be exported out of the cell by ferroportin (FPN1/SLC40A1) or contribute to the labile iron pool (LIP). Proper iron homeostasis is critical to cell survival, as the presence of excess Fe2+ can lead to iron driven generation of the extremely reactive hydroxyl radical via the Fenton reaction, a key upstream driver of ferroptotic death (Dixon et al., 2012; Kagan et al., 2017; Xie et al., 2016; Yang et al., 2016; Zou et al., 2020). The importance of iron in promoting ferroptosis is further evidenced by the fact that iron chelators block ferroptotic cell death both invitro and invivo, and exogenous iron supplementation sensitizes cells to ferroptosis inducers (i.e., erastin) (Dixon et al., 2012; Hou et al., 2016; Li et al., 2017).

Several Nrf2 target genes have been shown to regulate critical aspects of iron homeostasis, including heme biosynthesis and catabolism, as well as iron uptake, export, storage, and utilization (Fig. 2, Table 1). As a significant portion of functional iron in the body is in the form of heme, dysregulation of heme metabolism could enhance the risk of undergoing ferroptotic death. Many of the Nrf2 target genes involved in heme metabolism were originally identified by Chip-seq analysis of sulforaphane (SF)-treated lymphoblasts, including ATP Binding Cassette Subfamily B Member 6 (ABCB6, porphyrin transport and heme synthesis), ferrochelatase (FECH, heme synthesis), solute carrier family 48 member 1 (SLC48A1, heme transport), alpha-1-microglobulin (AMBP, heme binding/degradation), and thromboxane A synthase 1 (TBXAS1) (Campbell et al., 2013; Chorley et al., 2012). Heme oxygenase-1 (HMOX1), the enzyme that catalyzes the degradation of heme to produce biliverdin, was initially identified as an Nrf2 target gene in curcumin treated renal epithelial cells (Balogun et al., 2003). Treatment of a variety of cancer cell lines with the IκBα inhibitor BAY11-7085 induced ferroptosis in an HMOX1-dependent manner (Chang et al., 2018). Upregulation of HMOX1 was also associated with increased ferroptotic death in the cardiac tissue of sickle cell disease mice (Menon et al., 2022). Conversely, HMOX1 was shown to be anti-ferroptotic in erastin treated renal proximal tubule cells (Adedoyin et al., 2018), indicating a possible tissue and context-dependent role for HMOX1 in mediating ferroptosis. Biliverdin reductases A and B (BLVRA and BLVRB), which convert biliverdin to bilirubin (a strong antioxidant/free radical scavenger) during heme catabolism, were also shown to be Nrf2 target genes, as assessed by ChIP-seq, DNA microarray, and transcriptomic/proteomic analyses (Agyeman et al., 2012; Hirotsu et al., 2012).

Ferritin is responsible for sequestering free iron and preventing it from participating in Fenton reactions. The ferritin cage is comprised of 24 repeating subunits of the ferritin heavy chain (FTH1) and ferritin light chain (FTL), both of which were identified to contain antioxidant response elements (AREs) within their promoter regions (Chorley et al., 2012; Hintze and Theil, 2005; Pietsch et al., 2003; Tsuji et al., 2000; Wasserman and Fahl, 1997). Ferroportin (SLC40A1/FPN1), which as mentioned above mediates the export of excess Fe2+, was also identified to contain an ARE, and can be transcriptionally upregulated by Nrf2 in macrophages (Harada et al., 2011; Marro et al., 2010; Namgaladze et al., 2022), although Nrf2 was also reported to suppress FPN1 in other cell types (Kong et al., 2019). Recently, our group showed that Nrf2 regulates iron homeostasis by controlling both ferritin synthesis and degradation as illustrated in Fig. 3 (Anandhan et al., 2023). First, HERC2, encoding an E3 ubiquitin ligase for NCOA4 (a cargo adapter for ferritin degradation by autophagy [ferritinophagy]) and FBXL5 (an iron-dependent regulator of IRP1/2 that binds to the iron responsive element in the 5’-UTR of FTH1 and FTL mRNA thereby repressing their translation and preventing ferritin synthesis) was identified as a novel Nrf2 target gene. Second, Nrf2 indirectly regulates ferritinophagy via TFEB-dependent transcription of the lysosomal SNARE protein VAMP8 (mediates autophagosome-lysosome fusion). Thus, Nrf2 regulates ferritin protein stability at both the translational and ferritinophagy-dependent turnover levels. Critically, we showed that these two mechanisms are vital to mediating Nrf2-driven resistance to ferroptosis induction in cancer cells, as deletion of Nrf2 resulted in ferritinophagy blockage, NCOA4-dependent recruitment of apoferritin to the autophagosome, an elevated LIP, and enhanced sensitivity to the ferroptosis inducer imidazole ketone erastin (Anandhan et al., 2023). Overall, Nrf2 regulation of iron homeostasis is a critical determinant of a cell’s sensitivity or resistance to ferroptosis, which is independent of its antioxidant function, as discussed below.
Lipid peroxidation
Lipid peroxides are formed when polyunsaturated fatty acids (PUFAs) in the cell membrane, or organellar membranes are oxidized by reactive species, including hydroxyl and hydroperoxyl radicals, reactive nitrogen species (i.e., peroxynitrite), and the actual end products of lipid peroxidation themselves (4-HNE [4-hydroxynonenal] and MDA [malondialdehyde]) (Yin et al., 2011). These highly reactive and electrophilic lipid peroxides, which have a variety of cytotoxic consequences, are now recognized along with free iron as key players in promoting the ferroptotic cascade (Ayala et al., 2014; Conrad et al., 2018). Nrf2 governs the expression of a host of target genes responsible for preventing the formation of lipid peroxides (Fig. 2, Table 1). The CoQ oxidoreductase AIFM2/FSP1 (GSH-independent ferroptosis suppressor), a lipophilic antioxidant that inhibits ferroptosis independently of GPX4, was found to be an Nrf2 target gene (Bersuker et al., 2019; Doll et al., 2019; Emmanuel et al., 2022; Koppula et al., 2022). An earlier study using ChIP seq analysis also identified that Nrf2 could bind to the promoter region of AIFM2 (now called FSP1) in SF-treated lymphoblasts (Chorley et al., 2012). Some members of the aldo-keto-reductase family, i.e., AKR1C1-3, AKR1B1, and AKR1B10, are well-established Nrf2 target genes that have all been associated with ferroptosis resistance, as they convert lipid peroxides into less toxic intermediates (Burchiel et al., 2007; Dixon et al., 2014; Hirotsu et al., 2012; Lou et al., 2006; MacLeod et al., 2009). Similarly, aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which catalyzes the oxidation of reactive aldehydes to their less reactive carboxylic acid form, is an Nrf2 target gene (Hirotsu et al., 2012). Peroxisome proliferator-activated receptor gamma (PPARG) and nuclear receptor subfamily 0, group B, member 2 (NR0B2), which encode PPARγ and small heterodimer partner (SHP), respectively, are nuclear receptors that play essential roles in regulating cholesterol metabolism and thus the structural integrity of lipid membranes. Both were identified as Nrf2 target genes in an invivo comparison of Nrf2 wild type versus KO mice, as well as invitro in Nrf2 induced lymphoblasts (Cho et al., 2010; Chorley et al., 2012; Huang et al., 2010). These studies indicate that Nrf2 directly regulates the expression of several target genes that play a critical role in mitigating the accumulation of lipid peroxides, maintaining membrane integrity, and preventing the subsequent progression of the ferroptotic cascade.
With the production of reactive lipid peroxides that compromise cell membrane integrity and damage DNA, proteins, and organelles, proper processing of these peroxides is critical to prevent their harmful pro-ferroptotic effects. Under ferroptotic conditions, several PUFA metabolizing enzymes, including Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4), Prostaglandin-endoperoxide synthase 2/cyclooxygenase 2 (PTGS2/COX-2), and Arachidonate-5/12/15-lipoxygenase (ALOX5, ALOX12, and ALOX15) are elevated, all of which are regarded as biomarkers of ferroptotic death (Chen et al., 2021; Chu et al., 2019; Dixon et al., 2015; Doll et al., 2017; Yang et al., 2014). In contrast, two critical proteins that prevent excess lipid peroxidation are glutathione peroxidase 4 (GPX4, reduces lipid peroxides to their alcohol form) and system xc- (cystine/glutamate antiporter), which as mentioned above, were identified as the targets of the first ferroptosis inducers RSL3 and erastin, respectively (Dixon et al., 2012; Wolpaw et al., 2011; Yang and Stockwell, 2008). Accordingly, inhibition of these defense mechanisms plays a significant role in promoting ferroptosis. In fact, the ferroptosis inducers identified to date all target these proteins and can be categorized into four classes: (1) Class 1 – system xc- inhibitors (i.e., erastin and its analogs, sulfasalazine, sorafenib), (2) Class 2 – direct inhibitors of GPX4 (i.e., RSL3, RSL5), (3) Class 3 – depleters of GPX4 protein and CoQ10 (i.e., FIN56), and (4) Class 4 (i.e., FINO2) – indirect inhibitors of GPX4 (Abrams et al., 2016; Conrad et al., 2016; Doll et al., 2017; Shimada et al., 2016; Yang and Stockwell, 2016). Intriguingly, both GPX4 and SLC7A11 (subunit of system xc-) have been reported as Nrf2 target genes (Hirotsu et al., 2012; Osburn et al., 2006; Salazar et al., 2006). However, it is important to note that while SLC7A11 is a verified target gene, data supporting GPX4 as an Nrf2 target gene are less convincing, and a functional ARE has not been identified. Along with its regulation of SLC7A11, which provides the cysteine needed to produce GSH, Nrf2 also controls the expression of the catalytic and modulatory subunits of glutamate-cysteine ligase (GCLC/GCLM), glutathione reductase (GSR), and glutathione synthetase (GSS), all of which are vital for GSH metabolism (Fig. 1) (Chan and Kwong, 2000; Chorley et al., 2012; Erickson et al., 2002; Harvey et al., 2009; Hirotsu et al., 2012; Ishii et al., 2000; Kwak et al., 2002; Lee et al., 2005; Sasaki et al., 2002; Wang et al., 2007; Yang et al., 2005). As GSH is a critical cofactor for GPX4, Nrf2 regulation of GSH levels represents a critical aspect of preventing lipid peroxides from accumulating.
THE Nrf2-FERROPTOSIS AXIS IN PATHOLOGY
Cancer
The ferroptosis field emerged during the search for novel classes of small molecules that were able to kill resistant cancer cells. Therefore, the function of ferroptosis in cancer is well reported (Dolma et al., 2003; Stockwell et al., 2017; Yagoda et al., 2007). Over the years, more and more ferroptosis inducers have been identified, and their use to kill resistant cancer cells has been demonstrated in many preclinical cancer models. As we gain more knowledge of the anti-ferroptotic function of Nrf2, it is obvious to predict that inhibition of Nrf2 will significantly enhance the efficacy of ferroptosis inducers. Furthermore, many cancer cells have Nrf2 constitutively activated, which results in the upregulation of cytoprotective genes that promote tumor progression and protect cancer cells from chemotherapeutics, this is known as the dark side of Nrf2 (Wang et al., 2008). Activation of Nrf2 has been reported to protect against ferroptosis in different cancer models. For example, Nrf2 upregulation as a result of autophagy receptor p62-dependent sequestration of Keap1 reduced the sensitivity of hepatocellular carcinoma cells (HCC) to erastin- and sorafenib-induced ferroptosis (Sun et al., 2016). Another study using head and neck cancer (HNC) cells indicated that Nrf2 is essential for these cells to evade RSL3-induced ferroptosis (Shin et al., 2018). Finally, a 3D cell culture model using a CRISPR-Cas9-based screening approach revealed the importance of Nrf2 hyperactivation in promoting the proliferation and survival of lung tumor spheroid cells (Takahashi et al., 2020).
A host of well-established Nrf2 target genes, including SLC7A11, FTH1/FTL, SLC48A1, GPX4, PRDX6, MT1G, and HMOX1, have all been shown to be critical for cancer cell growth and conferring resistance to ferroptosis (Chorley et al., 2012; Chowdhury et al., 2009; Fan et al., 2017; Hassannia et al., 2019; Hirotsu et al., 2012; Houessinon et al., 2016; Na and Surh, 2014; Sun et al., 2016). Additionally, MGST1, a redox-sensitive repressor of ferroptosis in pancreatic cancer cells, was also recently identified as an Nrf2 target gene that dictates pancreatic cancer cell sensitivity to ferroptosis inducers (Kuang et al., 2021). This increased reliance upon Nrf2 and its downstream targets to protect against ferroptosis led to the notion that inhibition of Nrf2 might be needed to synergistically increase the therapeutic efficacy of pro-ferroptotic drugs, particularly against Nrf2 overexpressing cancers. This was demonstrated in a study using the alkaloid trigonelline (a potent, but non-specific Nrf2 inhibitor), which enhanced the anticancer activity of erastin and sorafenib in HCC cells (Sun et al., 2016), as well as artesunate sensitivity in HNC cells (Roh et al., 2017). We have shown that brusatol, the first NRF2 inhibitor developed by our group (Ren et al., 2011), enhanced the efficacy of imidazole ketone erastin in promoting ferroptosis and preventing tumor growth in several pre-clinical models of ovarian cancer (Anandhan et al., 2023). Another study indicated that cotreatment with brusatol and erastin was more effective than either single treatment in killing NSCLC cells, which was partly due to Nrf2 regulation of the FOCAD-FAK signaling pathway (Liu et al., 2020). Overall, cotreatment with pro-ferroptotic therapeutics and an Nrf2 inhibitor represents a promising strategy for inducing ferroptosis to kill resistant cancer cells.
Other diseases
The link between Nrf2 and ferroptosis has also been elucidated in the pathogenesis of diseases other than cancer, indicating the possible relevance of targeting this cascade in other pathological contexts. Studies of Alzheimer’s disease (AD) and Parkinson’s disease (PD) have suggested that ferroptosis could be a main driver of the neuronal cell death that promotes the progression of these neurodegenerative diseases (Morris et al., 2018). For example, our group detected that reduced Nrf2 expression correlated with decline in function of neural stem cells isolated from rats during a critical middle age period (Corenblum et al., 2016). Overall, the gradual loss of Nrf2 with aging is thought to increase susceptibility to ferroptosis, which has been recently demonstrated to play critical roles in the pathogenesis of PD, AD, multiple sclerosis, and other neurodegenerative diseases (Yan et al., 2021). A recent study from our group demonstrated that Nrf2-/- mice exhibit an age-dependent increase in several key markers of ferroptosis in brain regions associated with PD (A et al., 2022). Aside from neurodegenerative diseases, Nrf2-mediated ferroptosis has also been linked to other high-morbidity diseases, such as diabetes, non-alcoholic fatty liver disease (NAFLD), and cardiovascular disease. In an invitro model of diabetic nephropathy, knockdown of Nrf2 increased cell vulnerability to ferroptosis under hyperglycemic conditions, and treatment with an Nrf2 inducer prevented ferroptosis induction (Li et al., 2021). Using invitro hepatocytes and an invivo model of high fat diet-induced NAFLD, our group detected enhanced lipid peroxidation and ferroptosis during the course of NAFLD, in part due to downregulation of ATG7-mediated autophagosome biogenesis and reduced Nrf2, which could be partially rescued by pharmacological induction of Nrf2 (Liu et al., 2022). The occurrence of NAFLD is generally associated with increased lipids and triglyceride accumulation arising from an imbalance between lipid acquisition and removal in the hepatocytes, which in turn could lead to more lipid peroxidation. Overall, through this study we showed that a high-fat diet induced NAFLD causes lipid peroxidation and ferroptosis, both of which can be prevented by upregulating Nrf2 as a potential therapeutic strategy. In a cell model of atherosclerosis, qRT-PCR and western blot data showed a positive correlation between serine protease 22 (PRSS2) and Nrf2, which was correlated with decreased ferroptosis sensitivity (Yang et al., 2021). Therefore, in contrast to ferroptosis induction/Nrf2 inhibition in cancer treatment, ferroptosis inhibition/Nrf2 activation represents a more viable therapeutic option in these non-cancer related diseases.
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
After almost a decade, it has become evident that Nrf2 plays a key role as a ferroptosis suppressor, which is supported by the fact that many Nrf2 target genes have been demonstrated to play important roles in preventing ferroptosis. This notion is also supported by the fact that two critical drivers of ferroptosis, free labile iron and lipid peroxidation, are regulated by Nrf2. While the bulk of the initial studies investigating Nrf2-mediated ferroptosis primarily focused on its antioxidant functions, its anti-ferroptotic role continues to extend beyond just the antioxidant response with the identification of several novel target genes that regulate critical aspects of iron and lipid homeostasis (Fig. 2, Table 1). Further research is needed to dissect the Nrf2-iron-lipid-ferroptosis axis and its role in different pathological contexts. This remains important to the field, as this pathway represents a therapeutic axis to treat ferroptosis-relevant pathologies, either through Nrf2 inhibition in the case of cancer, or through Nrf2 activation in the case of pathological states involving undesirable ferroptotic cell death. Our understanding of Nrf2-mediated ferroptosis has evolved as a result of recent findings but expanding upon this area of research should enhance the therapeutic potential of Nrf2-ferroptosis-based adjuvant therapies.
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