Mol. Cells 2023; 46(3): 165-175
Published online March 24, 2023
https://doi.org/10.14348/molcells.2023.0005
© The Korean Society for Molecular and Cellular Biology
Correspondence to : zhangd@arizona.edu
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
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 ironand 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.
Keywords cancer, ferroptosis, Nrf2
The transcription factor nuclear factor erythroid-2 (NF-E2)-related factor 2 (Nrf2, encoded by the
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.
Coined by Brent Stockwell’s group in 2012, the term ferroptosis, as the name indicates, is an iron-dependent form of cell death. The actual origins of ferroptosis date back to 2003, with the discovery of two molecules, erastin and RAS Selective Lethal 3 (RSL3), which caused a non-apoptotic form of cell death in mutant HRAS-expressing foreskin fibroblasts that could ultimately be rescued by lipophilic antioxidants or iron chelating agents (Dolma et al., 2003; Stockwell et al., 2017; Yagoda et al., 2007; Yang and Stockwell, 2008). While erastin inhibits the system xCT cystine/glutamate antiporter, causing depletion of intracellular glutathione (GSH) and inactivation of GPX4 due to loss of this critical cofactor, RSL3 directly binds to and inhibits GPX4 function, preventing cells from effectively reducing lipid peroxides and causing eventual cell death (Dixon et al., 2012; Wolpaw et al., 2011). Consistent with these original observations, many other studies over the years have provided evidence indicating that the core ferroptotic cascade includes two salient features: (1) iron accumulation and (2) increased lipid peroxidation as shown in Fig. 1. Nrf2 regulates the major defense pathways responsible for ensuring that these pro-ferroptotic changes are kept in check, indicating its central role in preventing the initiation of the ferroptosis cascade. In the following sections, we will briefly discuss key aspects of each of these critical drivers of ferroptosis, including how they are directly and indirectly influenced by Nrf2 (Fig. 2, Table 1).
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/
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 (
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 (
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
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
The pathological role of ferroptosis in human diseases, including cancer, neurodegeneration, liver injury, kidney failure, and diabetes, is an emerging area of research. In this section, we will briefly discuss the studies that have implicated Nrf2 regulation of ferroptosis in mediating disease progression and treatment.
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
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
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.
D.D.Z. is supported by the following grants from the National Institutes of Health: R35ES031575 and P42ES004940.
A.S., N.W.M., and M.D. wrote the manuscript. A.S. and N.W.M. made the figures and table. D.D.Z. and E.C. edited the final manuscript.
The authors have no potential conflicts of interest to disclose.
Putative Nrf2 target genes involved in regulating ferroptosis
Gene symbol | Gene name | ARE verification | Function | Reference |
---|---|---|---|---|
ATP binding cassette subfamily B member 6 | ChIP-seq | Mitochondrial uptake of porphyrins. | (Campbell et al., 2013; Chorley et al., 2012) | |
Aldo-keto reductase family 1 member C1/C2/C3 | EMSA RGA ChIP-seq | Belongs to aldo-keto reductase family, reduces aldehydes, ketones, and quinones to corresponding alcohol. | (Burchiel et al., 2007; Hirotsu et al., 2012; Lou et al., 2006; MacLeod et al., 2009) | |
Aldehyde dehydrogenase 1 family member A1 | ChIP-seq | Belongs to aldehyde dehydrogenase family, oxidizes aldehydes to carboxylic acid. | (Hirotsu et al., 2012) | |
Alpha-1-microglobulin/bikunin precursor | ChIP-seq | Encodes A1M and bikunin proteins which have roles in heme catabolism and structural incorporation of extracellular matrix. | (Campbell et al., 2013) | |
Biliverdin reductase A/B | ChIP-seq | NADPH or NADH-dependent catalysis of the conversion of biliverdin to bilirubin. | (Agyeman et al., 2012; Hirotsu et al., 2012) | |
Ferrochelatase | ChIP-seq | Catalyzes the installation of ferrous iron into protoporphyrin IX. | (Campbell et al., 2013; Chorley et al., 2012) | |
Apoptosis inducing factor mitochondria associated 2 | RGA ChIP-seq | CoQ oxidoreductase that generates the reduced form of coenzyme Q10, which traps lipid peroxide. | (Chorley et al., 2012; Koppula et al., 2022) | |
Ferritin heavy chain 1 | EMSA RGA ChIP-seq | Heavy chain of ferritin which is a major intracellular iron storage protein; FTH1 is involved in the oxidation of Fe2+ to Fe3+. | (Chorley et al., 2012; Pietsch et al., 2003) | |
Ferritin light chain | RGA ChIP-seq | Light chain of ferritin which is a major intracellular iron storage protein. | (Chorley et al., 2012; Hintze and Theil, 2005) | |
Glutamate-cysteine ligase catalytic/modifier subunit | EMSA RGA ChIP-seq | GCLC is the catalytic subunit of glutamate-cysteine ligase (GCS) while GCLM is the modifier subunit of GCS. GCS is the first rate-limiting enzyme of glutathione synthesis. | (Chorley et al., 2012; Erickson et al., 2002; Hirotsu et al., 2012; Yang et al., 2005) | |
Glutathione peroxidase 4 | ChIP-seq | Catalyzes the reduction of hydrogen peroxide, organic hydroperoxides, and lipid hydroperoxides. | (Hirotsu et al., 2012) | |
Glutathione synthetase | EMSA RGA | Forms homodimer which catalyzes the ATP-dependent conversion of gamma-L-glutamyl-L-cysteine to glutathione. | (Lee et al., 2005) | |
Glutathione-disulfide reductase | RGA ChIP-seq | Reduces sulfur atoms in a glutathione disulfide bond (GS-SG) into their sulfhydryl form (GSH). | (Chorley et al., 2012; Harvey et al., 2009; Wang et al., 2007) | |
Heme oxygenase 1 | EMSA RGA ChIP-seq | Cleaves heme to form biliverdin. | (Balogun et al., 2003; Campbell et al., 2013; Chorley et al., 2012; Hirotsu et al., 2012) | |
Metallothionein 1 | RGA ChIP-seq | Cysteine rich proteins involved in metal detoxification and homeostasis as well as protection against oxidative stress. | (Hirotsu et al., 2012; Houessinon et al., 2016) | |
Nuclear receptor subfamily 0 group B member 2 | RGA ChIP-seq | An orphan nuclear receptor that interacts with retinoid receptors, thyroid hormone receptors, and estrogen receptors, preventing the respective receptor's ligand-dependent transcriptional activation. | (Huang et al., 2010) | |
Peroxisome proliferator activated receptor gamma | EMSA ChIP-seq | Nuclear receptor, functioning as a transcription factor that regulates fatty acid storage and cholesterol metabolism. | (Cho et al., 2010; Chorley et al., 2012) | |
Peroxiredoxin 6 | RGA ChIP-seq | Reduces various peroxides such as short chain organic, phospholipid, and fatty acid hydroperoxides. | (Chorley et al., 2012; Chowdhury et al., 2009; Hirotsu et al., 2012) | |
Solute carrier family 40 member 1 | RGA | Cell membrane protein involved in export of iron from cells. | (Campbell et al., 2013; Marro et al., 2010) | |
Solute carrier family 48 member 1 | ChIP-seq | Cell membrane protein involved in heme transport through endosome membrane, lysosomal membrane, and plasma membrane. | ||
Solute carrier family 7 member 11 | RGA ChIP-seq | Cell membrane protein that mediates the transport of anionic cysteine in exchange for glutamate. | (Hirotsu et al., 2012; Sasaki et al., 2002) | |
Thromboxane A synthase 1 | ChIP-seq | Monooxygenase that catalyzes the conversion of prostaglandin H2 to thromboxane A2. | (Campbell et al., 2013) |
These genes listed were identified by ChIP-Seq analysis and subsequently validated by the indicated experimental method used to identify a functional antioxidant response elements (AREs), including electrophoretic mobility shift assay (EMSA), reporter gene assay (RGA), or computational search (CS).
Mol. Cells 2023; 46(3): 165-175
Published online March 31, 2023 https://doi.org/10.14348/molcells.2023.0005
Copyright © The Korean Society for Molecular and Cellular Biology.
Aryatara Shakya1 , Nicholas W. McKee1
, Matthew Dodson1
, Eli Chapman1
, and Donna D. Zhang1,2,*
1Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721, USA, 2The University of Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA
Correspondence to:zhangd@arizona.edu
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
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 ironand 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.
Keywords: cancer, ferroptosis, Nrf2
The transcription factor nuclear factor erythroid-2 (NF-E2)-related factor 2 (Nrf2, encoded by the
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.
Coined by Brent Stockwell’s group in 2012, the term ferroptosis, as the name indicates, is an iron-dependent form of cell death. The actual origins of ferroptosis date back to 2003, with the discovery of two molecules, erastin and RAS Selective Lethal 3 (RSL3), which caused a non-apoptotic form of cell death in mutant HRAS-expressing foreskin fibroblasts that could ultimately be rescued by lipophilic antioxidants or iron chelating agents (Dolma et al., 2003; Stockwell et al., 2017; Yagoda et al., 2007; Yang and Stockwell, 2008). While erastin inhibits the system xCT cystine/glutamate antiporter, causing depletion of intracellular glutathione (GSH) and inactivation of GPX4 due to loss of this critical cofactor, RSL3 directly binds to and inhibits GPX4 function, preventing cells from effectively reducing lipid peroxides and causing eventual cell death (Dixon et al., 2012; Wolpaw et al., 2011). Consistent with these original observations, many other studies over the years have provided evidence indicating that the core ferroptotic cascade includes two salient features: (1) iron accumulation and (2) increased lipid peroxidation as shown in Fig. 1. Nrf2 regulates the major defense pathways responsible for ensuring that these pro-ferroptotic changes are kept in check, indicating its central role in preventing the initiation of the ferroptosis cascade. In the following sections, we will briefly discuss key aspects of each of these critical drivers of ferroptosis, including how they are directly and indirectly influenced by Nrf2 (Fig. 2, Table 1).
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/
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 (
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 (
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
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
The pathological role of ferroptosis in human diseases, including cancer, neurodegeneration, liver injury, kidney failure, and diabetes, is an emerging area of research. In this section, we will briefly discuss the studies that have implicated Nrf2 regulation of ferroptosis in mediating disease progression and treatment.
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
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
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.
D.D.Z. is supported by the following grants from the National Institutes of Health: R35ES031575 and P42ES004940.
A.S., N.W.M., and M.D. wrote the manuscript. A.S. and N.W.M. made the figures and table. D.D.Z. and E.C. edited the final manuscript.
The authors have no potential conflicts of interest to disclose.
Putative Nrf2 target genes involved in regulating ferroptosis
Gene symbol |
Gene name | ARE verification |
Function | Reference |
---|---|---|---|---|
ATP binding cassette subfamily B member 6 | ChIP-seq | Mitochondrial uptake of porphyrins. | (Campbell et al., 2013; Chorley et al., 2012) | |
Aldo-keto reductase family 1 member C1/C2/C3 | EMSA RGA ChIP-seq |
Belongs to aldo-keto reductase family, reduces aldehydes, ketones, and quinones to corresponding alcohol. | (Burchiel et al., 2007; Hirotsu et al., 2012; Lou et al., 2006; MacLeod et al., 2009) | |
Aldehyde dehydrogenase 1 family member A1 | ChIP-seq | Belongs to aldehyde dehydrogenase family, oxidizes aldehydes to carboxylic acid. | (Hirotsu et al., 2012) | |
Alpha-1-microglobulin/bikunin precursor | ChIP-seq | Encodes A1M and bikunin proteins which have roles in heme catabolism and structural incorporation of extracellular matrix. | (Campbell et al., 2013) | |
Biliverdin reductase A/B | ChIP-seq | NADPH or NADH-dependent catalysis of the conversion of biliverdin to bilirubin. | (Agyeman et al., 2012; Hirotsu et al., 2012) | |
Ferrochelatase | ChIP-seq | Catalyzes the installation of ferrous iron into protoporphyrin IX. | (Campbell et al., 2013; Chorley et al., 2012) | |
Apoptosis inducing factor mitochondria associated 2 | RGA ChIP-seq |
CoQ oxidoreductase that generates the reduced form of coenzyme Q10, which traps lipid peroxide. | (Chorley et al., 2012; Koppula et al., 2022) | |
Ferritin heavy chain 1 | EMSA RGA ChIP-seq |
Heavy chain of ferritin which is a major intracellular iron storage protein; FTH1 is involved in the oxidation of Fe2+ to Fe3+. | (Chorley et al., 2012; Pietsch et al., 2003) | |
Ferritin light chain | RGA ChIP-seq |
Light chain of ferritin which is a major intracellular iron storage protein. | (Chorley et al., 2012; Hintze and Theil, 2005) | |
Glutamate-cysteine ligase catalytic/modifier subunit | EMSA RGA ChIP-seq |
GCLC is the catalytic subunit of glutamate-cysteine ligase (GCS) while GCLM is the modifier subunit of GCS. GCS is the first rate-limiting enzyme of glutathione synthesis. | (Chorley et al., 2012; Erickson et al., 2002; Hirotsu et al., 2012; Yang et al., 2005) | |
Glutathione peroxidase 4 | ChIP-seq | Catalyzes the reduction of hydrogen peroxide, organic hydroperoxides, and lipid hydroperoxides. | (Hirotsu et al., 2012) | |
Glutathione synthetase | EMSA RGA |
Forms homodimer which catalyzes the ATP-dependent conversion of gamma-L-glutamyl-L-cysteine to glutathione. | (Lee et al., 2005) | |
Glutathione-disulfide reductase | RGA ChIP-seq |
Reduces sulfur atoms in a glutathione disulfide bond (GS-SG) into their sulfhydryl form (GSH). | (Chorley et al., 2012; Harvey et al., 2009; Wang et al., 2007) | |
Heme oxygenase 1 | EMSA RGA ChIP-seq |
Cleaves heme to form biliverdin. | (Balogun et al., 2003; Campbell et al., 2013; Chorley et al., 2012; Hirotsu et al., 2012) | |
Metallothionein 1 | RGA ChIP-seq |
Cysteine rich proteins involved in metal detoxification and homeostasis as well as protection against oxidative stress. | (Hirotsu et al., 2012; Houessinon et al., 2016) | |
Nuclear receptor subfamily 0 group B member 2 | RGA ChIP-seq |
An orphan nuclear receptor that interacts with retinoid receptors, thyroid hormone receptors, and estrogen receptors, preventing the respective receptor's ligand-dependent transcriptional activation. | (Huang et al., 2010) | |
Peroxisome proliferator activated receptor gamma | EMSA ChIP-seq |
Nuclear receptor, functioning as a transcription factor that regulates fatty acid storage and cholesterol metabolism. | (Cho et al., 2010; Chorley et al., 2012) | |
Peroxiredoxin 6 | RGA ChIP-seq |
Reduces various peroxides such as short chain organic, phospholipid, and fatty acid hydroperoxides. | (Chorley et al., 2012; Chowdhury et al., 2009; Hirotsu et al., 2012) | |
Solute carrier family 40 member 1 | RGA | Cell membrane protein involved in export of iron from cells. | (Campbell et al., 2013; Marro et al., 2010) | |
Solute carrier family 48 member 1 | ChIP-seq | Cell membrane protein involved in heme transport through endosome membrane, lysosomal membrane, and plasma membrane. | ||
Solute carrier family 7 member 11 | RGA ChIP-seq |
Cell membrane protein that mediates the transport of anionic cysteine in exchange for glutamate. | (Hirotsu et al., 2012; Sasaki et al., 2002) | |
Thromboxane A synthase 1 | ChIP-seq | Monooxygenase that catalyzes the conversion of prostaglandin H2 to thromboxane A2. | (Campbell et al., 2013) |
These genes listed were identified by ChIP-Seq analysis and subsequently validated by the indicated experimental method used to identify a functional antioxidant response elements (AREs), including electrophoretic mobility shift assay (EMSA), reporter gene assay (RGA), or computational search (CS).
. Putative Nrf2 target genes involved in regulating ferroptosis.
Gene symbol | Gene name | ARE verification | Function | Reference |
---|---|---|---|---|
ATP binding cassette subfamily B member 6 | ChIP-seq | Mitochondrial uptake of porphyrins. | (Campbell et al., 2013; Chorley et al., 2012) | |
Aldo-keto reductase family 1 member C1/C2/C3 | EMSA RGA ChIP-seq | Belongs to aldo-keto reductase family, reduces aldehydes, ketones, and quinones to corresponding alcohol. | (Burchiel et al., 2007; Hirotsu et al., 2012; Lou et al., 2006; MacLeod et al., 2009) | |
Aldehyde dehydrogenase 1 family member A1 | ChIP-seq | Belongs to aldehyde dehydrogenase family, oxidizes aldehydes to carboxylic acid. | (Hirotsu et al., 2012) | |
Alpha-1-microglobulin/bikunin precursor | ChIP-seq | Encodes A1M and bikunin proteins which have roles in heme catabolism and structural incorporation of extracellular matrix. | (Campbell et al., 2013) | |
Biliverdin reductase A/B | ChIP-seq | NADPH or NADH-dependent catalysis of the conversion of biliverdin to bilirubin. | (Agyeman et al., 2012; Hirotsu et al., 2012) | |
Ferrochelatase | ChIP-seq | Catalyzes the installation of ferrous iron into protoporphyrin IX. | (Campbell et al., 2013; Chorley et al., 2012) | |
Apoptosis inducing factor mitochondria associated 2 | RGA ChIP-seq | CoQ oxidoreductase that generates the reduced form of coenzyme Q10, which traps lipid peroxide. | (Chorley et al., 2012; Koppula et al., 2022) | |
Ferritin heavy chain 1 | EMSA RGA ChIP-seq | Heavy chain of ferritin which is a major intracellular iron storage protein; FTH1 is involved in the oxidation of Fe2+ to Fe3+. | (Chorley et al., 2012; Pietsch et al., 2003) | |
Ferritin light chain | RGA ChIP-seq | Light chain of ferritin which is a major intracellular iron storage protein. | (Chorley et al., 2012; Hintze and Theil, 2005) | |
Glutamate-cysteine ligase catalytic/modifier subunit | EMSA RGA ChIP-seq | GCLC is the catalytic subunit of glutamate-cysteine ligase (GCS) while GCLM is the modifier subunit of GCS. GCS is the first rate-limiting enzyme of glutathione synthesis. | (Chorley et al., 2012; Erickson et al., 2002; Hirotsu et al., 2012; Yang et al., 2005) | |
Glutathione peroxidase 4 | ChIP-seq | Catalyzes the reduction of hydrogen peroxide, organic hydroperoxides, and lipid hydroperoxides. | (Hirotsu et al., 2012) | |
Glutathione synthetase | EMSA RGA | Forms homodimer which catalyzes the ATP-dependent conversion of gamma-L-glutamyl-L-cysteine to glutathione. | (Lee et al., 2005) | |
Glutathione-disulfide reductase | RGA ChIP-seq | Reduces sulfur atoms in a glutathione disulfide bond (GS-SG) into their sulfhydryl form (GSH). | (Chorley et al., 2012; Harvey et al., 2009; Wang et al., 2007) | |
Heme oxygenase 1 | EMSA RGA ChIP-seq | Cleaves heme to form biliverdin. | (Balogun et al., 2003; Campbell et al., 2013; Chorley et al., 2012; Hirotsu et al., 2012) | |
Metallothionein 1 | RGA ChIP-seq | Cysteine rich proteins involved in metal detoxification and homeostasis as well as protection against oxidative stress. | (Hirotsu et al., 2012; Houessinon et al., 2016) | |
Nuclear receptor subfamily 0 group B member 2 | RGA ChIP-seq | An orphan nuclear receptor that interacts with retinoid receptors, thyroid hormone receptors, and estrogen receptors, preventing the respective receptor's ligand-dependent transcriptional activation. | (Huang et al., 2010) | |
Peroxisome proliferator activated receptor gamma | EMSA ChIP-seq | Nuclear receptor, functioning as a transcription factor that regulates fatty acid storage and cholesterol metabolism. | (Cho et al., 2010; Chorley et al., 2012) | |
Peroxiredoxin 6 | RGA ChIP-seq | Reduces various peroxides such as short chain organic, phospholipid, and fatty acid hydroperoxides. | (Chorley et al., 2012; Chowdhury et al., 2009; Hirotsu et al., 2012) | |
Solute carrier family 40 member 1 | RGA | Cell membrane protein involved in export of iron from cells. | (Campbell et al., 2013; Marro et al., 2010) | |
Solute carrier family 48 member 1 | ChIP-seq | Cell membrane protein involved in heme transport through endosome membrane, lysosomal membrane, and plasma membrane. | ||
Solute carrier family 7 member 11 | RGA ChIP-seq | Cell membrane protein that mediates the transport of anionic cysteine in exchange for glutamate. | (Hirotsu et al., 2012; Sasaki et al., 2002) | |
Thromboxane A synthase 1 | ChIP-seq | Monooxygenase that catalyzes the conversion of prostaglandin H2 to thromboxane A2. | (Campbell et al., 2013) |
These genes listed were identified by ChIP-Seq analysis and subsequently validated by the indicated experimental method used to identify a functional antioxidant response elements (AREs), including electrophoretic mobility shift assay (EMSA), reporter gene assay (RGA), or computational search (CS)..
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