Ferroptosis: Biology and Role in Gastrointestinal Disease

Abstract

Ferroptosis is a form of nonapoptotic cell death that involves iron-dependent phospholipid peroxidation induced by accumulation of reactive oxygen species (ROS), and results in plasma membrane damage and the release of damage-associated molecular patterns. Ferroptosis has been implicated in aging and immunity, as well as disease states including intestinal and liver conditions and cancer. To date, several ferroptosis-associated genes and pathways have been implicated in liver disease. While ferroptotic cell death is associated with dysfunction of the intestinal epithelium, the underlying molecular basis is poorly understood. As the mechanisms regulating ferroptosis become further elucidated, there is clear potential to utilize ferroptosis to achieve therapeutic benefit.

Ferroptosis is an iron-dependent mechanism of non-apoptotic cell death caused by phospholipid peroxidation. As a distinct form of regulated cell death (RCD),^1–3 ferroptosis has gained much attention in recent years due to its involvement in pathological processes and immunity including cancer and its related therapy. Ferroptosis initiators have been broadly classified as extrinsic resulting from altering the activity of cell membrane amino acid transporters and iron transporters, or as intrinsic due to inhibition of intracellular antioxidant enzymes such as glutathione peroxidase GPX4.⁴ Ferroptosis is regulated not by a single universal mechanism, but by diverse and context-dependent pathways that converge at the accumulation of lipid peroxides in the plasma membrane.⁵ Unlike other forms of RCD such as apoptosis (caspases), necroptosis (mixed-lineage kinase domain like pseudokinase, aka MLKL) or pyroptosis (gasdermin D), specific and indispensable mediators for the execution of ferroptosis remain largely unknown, although proposed models exist.^{6, 7} Propagation of ferroptosis through the release of damage-associated molecular patterns (DAMPs) has been shown.^8–10 DAMPs are immuno-modulatory and can connect ferroptosis with the tumor microenvironment (TME) with implications for immunotherapy as cancer treatment. Of much clinical relevance is how to therapeutically target ferroptosis in a disease context. In this review, we discuss mechanisms and regulation of ferroptosis including its relevance in diseases of the gut and cancer. Furthermore, regulatory axes of ferroptosis and their targeting represent a therapeutic opportunity in cancer cells that have developed acquired resistance to other forms of RCD.

Ferroptosis regulation

Key pathways that can trigger ferroptotic cell death converge in the peroxidation of membrane lipids. One such pathway involves the degradation of ferritin and the engagement of transferrin receptor (TFRC)-mediated iron import, both of which serve to increase the intracellular iron pool leading to lipid peroxidation. Through the Fenton reaction, this iron pool and lipid metabolizing enzymes (ACSL4 and LPCAT3) contribute to an increase in phospholipid peroxidation that leads to ferroptosis. Glutathione peroxidase 4 (GPX4) is the canonical ferroptosis-controlling pathway for the regulation of ferroptosis. The NRF2 transcription factor regulates GPX4¹¹ and SCL7A11,¹² as well as other lipid peroxidation and ferroptosis-related genes.¹³ Initiators of ferroptosis that act through the GPX4-dependent pathway include depletion of glutathione (GSH) [the reducing substrate of GPX4 activity], inhibition of the cysteine importer known as system X_c^- using erastin,¹⁴ or GPX4 inhibition via the small molecule RSL3¹⁵. There are also GPX4-independent pathways that serve as alternative antioxidant defenses against ferroptosis, such as the ferroptosis suppressor protein 1 (FSP1), dihydroorotate dehydrogenase (DHODH) and GTP cyclohydrolase 1 (GCH1). These antioxidant enzymes result in generation of metabolites (such as CoQ10, BH4, etc.) with radical-trapping antioxidant activity (RTA) to inhibit lipid peroxidation (Figure 1). Most recently, a novel mechanism independent of both GPX4 and RTA has been discovered to suppress ferroptosis through MBOAT1/2-mediated cellular phospholipid remodeling.¹⁶

Figure 1.

Pathways that can trigger ferroptotic cell death converge in the peroxidation of membrane lipids. End executors of ferroptosis remain to be elucidated. As a receptor in the plasma membrane, cystine-glutamine antiporter (system X_c^-) can be inhibited by erastin or glutamate to extrinsically induce ferroptosis, whereas inhibition of intracellular antioxidant enzymes (GPX4, FSP1, DHODH, GCH1 and DHFR) lead to intrinsically induced ferroptosis.¹⁴ Depletion of GSH by MRP1 export,³² upregulated cys dioxygenase 1 (CDO1),²⁰² or treatment with cisplatin can also sensitize cells to ferroptosis.^{203, 204} The NRF2 pathway is involved in the expression of multiple proteins that can inhibit ferroptosis, including system X_c^-, GPX4 and MT1G. The ferroptosis suppressor protein 1 (FSP1) catalyzes the regeneration of coenzyme Q10 (CoQ10)^{33, 34} whose reduced form ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation and prevents ferroptosis induced by GPX4 depletion. GTP cyclohydroxylase-1 [GCH1] is the rate limiting enzyme of the tetrahydrobiopterin (BH₄) biosynthetic pathway which is another inhibitor of lipid peroxidation.³⁹ The lipid metabolism enzyme ACSL4, which preferentially acylates AA, and LPCAT3 which preferentially inserts acylated AA into membrane phospholipids, have critical roles in GPX4 inhibition-induced ferroptosis.^{86, 87} The first step in GSH synthesis is catalyzed by the glutamate-cysteine ligase catalytic subunit (GCLC) contributing to ferroptosis resistance.²⁰⁵ An increase in the intracellular iron pool through ferritinophagy and TFRC also leads to enhanced lipid peroxidation.⁴³

GPX4 pathway in the regulation of ferroptosis

The intestinal mucosal epithelium is highly dependent on dietary cysteine whose deficiency can lead to impaired cell proliferation, defective barrier function, and cell death due to ferroptosis.¹⁷ A rate limiting step in cellular GSH synthesis is the availability of cysteine that plays an essential role in cellular redox homoeostasis. Cysteine is a key constituent of the GSH tripeptide that is required for GPX4 to inhibit lipid peroxidation and ferroptosis. Cysteine requires specialized transport systems for its import into the cell. System X_c⁻ is a dedicated cystine transporter that imports cystine in exchange for intracellular glutamate. System X_c⁻ is frequently upregulated in cancer cells to counteract elevated levels of ROS, making it an attractive target for anticancer treatment. Furthermore, system X_c^- contains a light chain subunit, SLC7A11, that is targeted by inhibitors such as sulfasalazine or the multikinase inhibitor sorafenib to induce ferroptosis.^18,19 SLC7A11 expression can be repressed by the BRCA1-associated protein 1 (BAP1) that inhibits cystine uptake leading to elevated lipid peroxidation and ferroptosis.²⁰ Several non-coding RNAs^21–23 have also been shown to modulate expression of SLC7A11 resulting in ferroptosis. Ionizing radiation (IR) can inhibit ferroptosis by inducing expression of both SLC7A11 and GPX4 as an adaptive response leading to radioresistance.²⁴ Interestingly, ferroptosis induced by erastin or RSL3 was shown to be enhanced by sodium butyrate, a short-chain fatty acid (SCFA), by inducing lipid reactive oxygen species (ROS) production via downregulation of the expression of SLC7A11 and GPX4²⁵. The nuclear factor erythroid 2-related 2 (NRF2) transcription factor regulates GPX4¹¹ and SCL7A11¹² as well as many other lipid peroxidation- and ferroptosis- related genes such as metallothionein-1 (MT-1G), an important regulator of lipid peroxidation¹³ (Figure 1). Inhibition of NRF2 (using all-trans retinoic acid, or brusatol) induces ferroptosis by mechanisms including sensitizing cells to erastin and sorafenib,¹² reducing MT-1G expression,²⁶ and inducing redox imbalance and apoptosis in cancer cells.²⁷ There is also a synergistic effect of brusatol with lapatinib, an inhibitor of HER2/EGFR.²⁸

Modulation of GPX4 activity regulates ferroptosis whereby inducers of ferroptosis, including statins, can indirectly downregulate GPX4 expression or activity in cancer cells.²⁹ Conversely, upregulation of GPX4 expression by activating transcription factors (TFAP2c and SP1) resulting from selenium supplementation can inhibit ferroptosis.³⁰ MRP1, a multidrug resistance (MDR) gene,³¹ functions to increase the cellular efflux of GSH,³² and high levels of MRP1 expression (MDR phenotype) sensitize cancer cells to pro-ferroptotic agents while conferring resistance to some pro-apoptotic anticancer drugs.³² Accordingly, induction of ferroptosis can be exploited to target treatment-resistant cancer cells.

GPX4-independent pathways

The FSP1-CoQ10-NAD(P)H axis represents a parallel system to the GPX4-GSH axis for inhibition of lipid peroxidation and ferroptosis.^{33, 34} Of note, a combined administration of FSP1 and GPX4 inhibitors showed a more potent induction of ferroptosis compared to individual drugs.³⁵ The ferroptosis suppressor protein 1 (FSP1) can reduce vitamin K to its hydroquinone which acts as an inhibitor of lipid peroxidation, thereby protecting cells from ferroptosis.³⁶ Other parallel systems that inhibit ferroptosis include DHODH,³⁷ and vitamin D3 has been shown to reduce cisplatin-induced intestinal injury by reversing downregulation of both GPX4 and DHODH, explaining its anti-ferroptotic role.³⁸ The tetrahydrobiopterin (BH₄) biosynthesis pathway inhibits ferroptosis independent of cytosolic GPX4 or FSP1 (Figure 1). BH₄, a radical-trapping antioxidant cofactor, prevents membrane lipid peroxidation and its availability alters iron metabolism and mitochondrial function in T cells. Methotrexate inhibition of dihydrofolate reductase, an enzyme that regenerates BH₄, synergizes with GPX4 inhibition in the induction of ferroptosis.³⁹

Iron regulation and iron-induced lipid peroxidation

Phospholipid (PL) peroxidation can be initiated in cells through enzymatic and nonenzymatic processes. If PL hydroperoxide (PLOOH) is formed and not neutralized promptly, it can undergo the iron-catalyzed Fenton reaction, resulting in generation of lipid hydroxyl and lipid peroxyl radicals. As the catalyst of the Fenton reaction, the cellular labile iron pool plays a crucial role in determining cellular susceptibility to ferroptosis. These lipid radicals propagate peroxidation to neighboring PL-containing polyunsaturated fatty acid (PUFA) chains which can then react with oxygen to form lipid peroxyl radicals. This chain reaction amplifies PL peroxidation and ultimately leads to ferroptosis.

Cellular iron homeostasis is exquisitely regulated by iron regulatory protein-1 (IRP1) and IRP2 that control the level and activity of a series of protein factors involved in iron import, export, storage, and release which coordinately maintain cellular iron homeostasis.^{40, 41} As expected, these protein factors can also modulate ferroptosis.^{3, 42} For example, a major mechanism for cellular iron uptake is mediated by transferrin and its receptor, TFRC, which mediates uptake of iron-bound transferrin that is required for ferroptosis induced by cysteine deprivation.⁴³ Plasma membrane expression of TFRC was shown to be significantly increased during ferroptosis and might serve as a specific ferroptosis marker.⁴⁴ Additionally, non-transferrin-bound iron can be imported via encoded transmembrane proteins SLC39A8/ZIP8 and SLC39A14/ZIP14. ZIP14-mediated iron uptake has been reported to promote ferroptotic liver injury.⁴⁵ An excessive amount of cellular iron can be exported by iron exporters such as ferroportin and prominin, and downregulation of these exporters has been shown to promote ferroptosis.^{46, 47} Inside of cells, excess iron is mainly stored in a non-toxic form by the protein complex ferritin. As such, cytosolic ferritin and iron chaperone poly(rC)-binding protein 1 (PCBP1) confer resistance to ferroptosis by limiting iron availability.^{48, 49} Conversely, iron can be released from ferritin through nuclear receptor coactivator 4 (NCOA4)-mediated ferritin-selective autophagy, known as ferritinophagy, which can increase labile iron content and increase the susceptibility of the cell to ferroptosis.^{50, 51}

The maintenance of mitochondrial iron homeostasis is also crucial in regulating ferroptosis. Mitoferrin 1 (SLC25A37) and mitoferrin 2 (SLC25A28) are essential mitochondrial iron importers involved in heme and Fe–S biogenesis. Mitoferrin 2 has been shown to promote ferroptosis likely through increased mitochondria iron content.⁵² Mitochondrial Fe–S proteins are also involved in ferroptosis regulation,⁵³ and can prevent cancer cells from undergoing ferroptosis, probably through limiting mitochondrial iron level.^{54, 55} In contrast, heme oxygenase 1 (HO1), a mitochondrial enzyme that degrades heme to produce ferrous iron, leads to mitochondrial iron overload and sensitizes cancer cells to ferroptosis.^{56, 57} However, it was also reported that mild upregulation of HO1 protected cells from ferroptosis,⁵⁸ suggesting complex and a likely context-dependent role of HO1 in ferroptosis.

Metabolic Pathways

Ferroptosis can be considered as a natural outcome of cellular metabolism. Phospholipid(PL)-PUFA, a product of lipid metabolism and essential component of cell membranes, is the substrate for PL peroxidation, arguably the executing step of ferroptosis. On the other hand, ROS and free radicals, which initiate PL peroxidation and provide the initial trigger for ferroptosis, are inevitable products of cellular metabolism, particularly energy metabolism. Accordingly, various metabolic processes have been demonstrated to be important in ferroptosis of which examples are provided below. Glucose starvation, which mimics an energy stress condition, inhibits lipid peroxidation and ferroptotic cell death of cancer cells. Inactivation of AMPK (AMP-activated protein kinase), serves to inhibit acetyl-CoA carboxylase (ACC) and PUFA biosynthesis, and largely abolishes the protective effects of energy stress on ferroptosis.^{59, 60} Importantly, cancer cells with loss of function mutations of LKB1, a tumor suppressor and upstream AMPK activator, display high basal AMPK activity and show increased resistance to ferroptosis.⁶⁰ Conversely, glucose uptake mediated by SLC2A1 (GLUT1) promotes glycolysis, pyruvate oxidation, tricarboxylic acid cycle (TCA), and fatty acid synthesis which ultimately facilitates ferroptosis in cancer cells.⁶¹ Mammalian target of rapamycin complex 1 (mTORC1) is a nutrient sensor activated by amino acids, energy, and growth factors.⁶² Oncogenic activation of the PI3K-AKT-mTOR signaling axis was shown to suppress ferroptosis in cancer cells by increasing cellular monounsaturated fatty acid (MUFA) content. Conversely, inhibition of PI3K-AKT-mTOR signaling can sensitize cancer cells to ferroptosis induction.⁶³ Specifically, mTORC1 inhibition was shown to sensitize cancer cells to ferroptosis through decreasing GPX4 expression.⁶⁴ However, another study showed that ATP-competitive mTOR inhibitors suppressed ferroptosis triggered by system X_c⁻ inhibition or direct cystine deprivation which may be due to upregulated macropinocytosis of albumin that is an alternative source of cellular cysteine.^{65, 66} Therefore, the effect of mTORC1 on ferroptosis can be context dependent.

PL de novo synthesis and its remodeling process play significant roles in regulating ferroptosis sensitivity by determining the composition of the plasma membrane.⁶⁷ Various factors in these pathways have been shown to increase ferroptosis sensitivity including the uptake of essential PUFA by CD36,^{68, 69} the synthesis of long-chain PUFA by FADS1, FADS2, ELOVL5,^{70, 71} the activation of PUFA by ACSL4,^72–74 as well as the incorporation of PUFA-CoA into lyso-PL by LPCAT3.⁷² Previous studies have demonstrated that exogenous MUFA⁷⁵ or de novo synthesized MUFA inhibits ferroptosis.⁶³ Membrane Bound O-Acyltransferase Domain Containing 1 and 2 (MBOAT1/2) selectively incorporates MUFA-CoA into lyso-phosphatidyl ethanolamine (PE), competitively inhibiting the synthesis of PE-PUFA. MBOAT1/2 suppresses ferroptosis in a GPX4-independent manner. Furthermore, the regulation of MBOAT1 and 2 is mediated by estrogen receptor and androgen receptor signaling, respectively, making them critical therapeutic targets in certain cancer types.¹⁶

NADPH levels play a crucial role in sustaining redox homeostasis and cell survival.⁷⁶ Cellular NADPH abundance serves as a biomarker for predicting sensitivity to ferroptosis across different cancer cell lines.⁷⁷ NADPH can be produced through various metabolic pathways, including the pentose phosphate pathway and NAD kinase (NADK)-mediated phosphorylation of NAD. Suppressing NADK reduces intracellular NADPH levels and increases cell sensitivity to ferroptosis.⁷⁷ Conversely, overexpression of MESH1, a cytosolic NADPH phosphatase that degrades NADPH into NADH, depletes cellular NADPH and sensitizes cells to ferroptosis.⁷⁸

Mitochondrial Metabolism

Mitochondria play a critical role in in cell homeostasis and cellular energy metabolism in eukaryotic cells. While mitochondria provide intracellular ATP via oxidative phosphorylation, they also function as key mediators of various forms of regulated cell death including apoptosis, pyroptosis, necroptosis, and ferroptosis. The involvement of mitochondria in ferroptosis was suggested based on the morphological changes observed in ferroptotic cancer cells and that events associated with mitochondrial energy metabolism promote ferroptosis.^{14, 43, 79} Accumulating evidence suggests that an impaired ferroptotic response is associated with changes in mitochondrial function. Mitochondrial ROS and the release of mitochondrial DAMPs contribute to inflammation in ulcerative colitis (UC).⁸⁰ Furthermore, Paneth cells are highly susceptible to mitochondrial dysfunction in Crohn’s disease (CD).

To date, the role of mitochondria in regulating ferroptosis is poorly understood. Mitochondrial activity is crucial for cysteine-deprivation-induced (CDI) ferroptosis in contrast to RSL3-induced ferroptosis.^{43, 79} CDI ferroptosis fails to occur in the absence of glutamine since glutamine metabolism fuels the mitochondrial TCA cycle to enhance generation of mitochondria-derived ROS, which are necessary for lipid peroxidation during CDI ferroptosis. Metabolite intermediates of the TCA cycle such as α-ketoglutarate, fumarate, succinate, and malate can replace glutamine to induce CDI ferroptosis.⁷⁹ Inhibiting the TCA cycle or the electron transfer chain mitigates CDI ferroptosis.⁷⁹ As mitochondria play a central role in ferroptosis initiation, cells have developed potential ferroptosis suppressing mechanisms localized in the organelle, such as that mediated by DHODH and its product, reduced CoQ10.³⁷

Mitochondria are the center of iron metabolism and energy production, leading to altered lipid peroxidation sensitivity. Mitochondria can modulate ferroptosis through mechanisms including the synthesis of Fe-S clusters. Additionally, mitochondrial Ca²⁺ plays a crucial role in triggering ferroptosis. Reducing Ca²⁺ influx can protect cells from ferroptosis induced by system X_c⁻ inhibitors such as erastin and sulfasalazine.⁸¹ Recent studies have reported that FUNDC2 regulates ferroptosis by interacting with the mitochondrial glutathione transporter SLC25A11 to negatively regulate mitochondrial GSH levels.⁸² It has also been shown that FUNDC2 contributes to cardiomyopathy induced by the anthracycline doxorubicin through ferroptosis.⁸² Betaoxidation is generally believed to have a suppressive effect on ferroptosis by reducing the availability of unesterified PUFAs. The enzyme 2,4-dienoyl-CoA reductase 1 (DECR1), which is involved in PUFA beta-oxidation in the mitochondria, is overexpressed in prostate cancer. Knockout of DECR1 induces endoplasmic reticulum (ER) stress and sensitizes castration-resistant prostate cancer cells to ferroptosis both in vitro and in vivo. Furthermore, inhibiting beta-oxidation enhances ferroptosis in cancer cells.^{83, 84}

As a major regulator of the antioxidant response, NRF2 plays a crucial role in regulating mitochondria respiration, controlling mitochondria-mediated ROS production, as well as the biosynthesis of glutathione and NADPH. It has been shown that NRF2 is a key determinant of the therapeutic response to ferroptosis-targeted therapies in HCC cells.¹² Under oxidative stress, NRF2 is released from KEAP1-mediated ubiquitination. Stabilized NRF2 then accumulates in the nucleus, and transcriptionally activates a panel of genes that are involved in countering oxidative stress, including but not limited to SLC7A11 (cystine transporter); G6PD and PGD (NADPH regeneration); ferritin and ferroportin (iron regulation); FSP1 and DHFR (antioxidant generation), which serves as feedback regulation to maintain cellular redox homeostasis.⁸⁵

Lipid peroxidation

Lipid metabolic enzymes ACSL4 and LPCAT3 have fundamental roles in GPX4-induced ferroptosis (Figure 1).^{86, 87} ACSL4 preferentially activates arachidonic acid (AA) to AA-CoA whereas LPCAT3 preferentially incorporates AA into membrane phospholipids.^{86, 87} Thus, inhibition of these enzymes prevents ferroptosis by reducing the pool of oxidation-sensitive fatty acids in cell membranes.⁸⁶ PKCβII catalyzes the phosphorylation and activation of ACSL4, thus amplifying lipid peroxidation that leads to ferroptosis.⁸⁸ Inhibition of PKCβII-ACSL4 blocks ferroptosis and limits ferroptosis-associated cancer immunotherapy.⁸⁸ Ionizing radiation (IR) induces ferroptosis in cancer cells through increased levels of ROS and upregulation of ACSL4, leading to increased lipid peroxidation. Downregulation of ACSL4 blocks IR-induced ferroptosis and enhances radioresistance.²⁴ In the context of immunotherapy, interferon gamma (IFN-γ) produced by CD8⁺ T cells and AA upregulate ACSL4 leading to lipid peroxidation and immunogenic tumor cell ferroptosis.⁸⁹ Whereas high levels of AA promote tumor ferroptosis, downregulation of ACSL4 enhances tumor progression.⁸⁹ Importantly, low-dose AA administration was shown to enhance the therapeutic efficacy of PD-L1 blockade and improve anti-tumor T cell responses through the IFN-γ signaling pathway.⁸⁹

Ferroptosis in gastrointestinal disease

Common ferroptotic mechanisms in intestinal as well as other diseases include GPX4 inhibition, system X_c⁻ suppression, lipid peroxide accumulation, and iron overload. Key regulators such as GPX4, SLC7A11, ACSL4, and p53 are also important for mediating ferroptosis-associated intestinal diseases. To date, the role of ferroptosis in gastrointestinal (GI) disease remains poorly characterized, and further research is needed to identify disease-specific ferroptotic mechanisms that may enable development of disease context-dependent therapeutic approaches. Studies have found correlations between ferroptosis and other forms of cell death in intestinal diseases that may share common pathways and key regulators, and may guide therapeutic interventions.

Iron overload is an important trigger of ferroptosis that can exacerbate intestinal inflammation. Regulation of iron absorption in enterocytes is dependent on the liver secreted hormone hepcidin that forms a complex with ferroportin. When this complex enters iron-absorptive enterocytes, ferroportin is degraded resulting in iron accumulation with reduced delivery to plasma.90 Excess intracellular iron that is unbound to ferritin, i.e., labile cellular iron, has the potential to increase inflammation and lipid peroxidation resulting in cell death, including ferroptosis. Levels of hepcidin are influenced by gut microbiota, thus affecting iron uptake.91 Iron overload can disrupt the gut microbiome to promote the growth of potentially pathogenic bacteria,⁹⁰ and resultant dysbiosis can lead to enhanced ferroptosis in intestinal epithelial cells. While it has been reported that iron supplementation alters gut microbial homeostasis and exacerbates intestinal inflammation92, the specific mechanisms by which microbiota can affect ferroptosis await further study.

The intestinal epithelium is one of the fastest-renewing tissues in the human body. This rapid turnover requires significant metabolic activity and energy generation leading to increased ROS, iron metabolism,⁹³ and increase susceptibility to ferroptosis. Ferroptosis has been linked to disease generation through intestinal epithelial cell death mediated by ER stress.⁹² Inflammatory responses triggered by ferroptosis can promote immunogenicity via activation of NF-kB by DAMPs.⁹⁴ Furthermore, lipid peroxidation products stimulate phagocytosis (via TLR2 by oxidized phospholipid) and recruit macrophages by secreting chemokines.⁹⁵ While these data suggest that inhibition of ferroptosis may be therapeutically beneficial, acquired resistance to ferroptosis with prevention of cell death has been shown to enhance neoplastic development and progression.⁹⁶ Although beyond the scope of this review, other forms of cell death distinct from ferroptosis occur in GI diseases such as apoptosis, necrosis, necroptosis and pyroptosis. Molecular cross-talk has been described between these forms of cell death and ferroptosis, and multiple forms of cell death can be observed in the context of the same disease.⁹⁷ The relative proportion each mechanism of cell death and its specific contribution to a disease entity awaits further research.^98–100

Inflammatory Bowel Disease

Ferroptosis appears to play a role in inflammation-related conditions such as inflammatory bowel disease (IBD), and has been implicated in both experimental colitis and in human IBD. IBD is characterized by chronic and progressive inflammation of the GI mucosa which has been shown to manifest features of ferroptosis including iron deposition, lipid peroxidation, and GPX4 inactivation.⁹² Ferroptosis was shown to regulate colitis in the dextran sulphate sodium (DSS)-induced model of colitis which showed increased lipid peroxidation.^{92, 101} Furthermore, excess iron can increase mucosal ROS production which was shown to exacerbate intestinal inflammation in the DSS-induced colitis model.⁹² Inhibition of ferroptosis was shown to reduce disease activity scores in DSS-induced colitis.¹⁰²

ER stress signaling can mediate cell death signaling including ferroptosis, that was significantly increased in intestinal epithelial cells (IECs) from patients with UC and in mice with experimentally-induced colitis.^{92, 102} Ferroptosis is recognized as an important contributor to IEC death in both humans and mice with UC.^{14, 92} In IEC-6 cells treated with H₂O_2, downregulation of phosphorylated STAT3 was observed which could be reactivated by the ferroptosis inhibitor, Fer-1.¹⁰³ Phosphorylated NF-κBp65 can inhibit ER stress signaling by binding eukaryotic initiation factor 2α,⁹² and deletion of NF-κBp65 in IECs was shown to upregulate ferroptosis and to exacerbate colitis. Iron overload has also been observed to induce colitis, enhance ferroptosis, and significantly alter the microbiota in mouse models resulting in modulation of the expression of several ferroptosis-related genes such as SLC7A11 and GPX4.¹⁰⁴ GPX4 plays an important role by protecting against lipid peroxidation and ferroptotic cell death¹⁰². Of note, IECs derived from patients with CD were shown to exhibit impaired GPX4 activity and signs of lipid peroxidation.¹⁰⁵ PUFAs, and specifically AA, were shown to trigger a cytokine response in IECs which was restricted by GPX4.¹⁰⁵ Furthermore, a PUFA-enriched Western diet was shown to induce a granuloma-like neutrophilic enteritis in mice that lacked one allele of Gpx4 in IECs, indicating that dietary PUFAs can serve as a trigger of GPX4-restricted mucosal inflammation with a phenotype of human CD. Together, these data indicate that ferroptosis may contribute to IBD via ER stress-mediated IEC cell death, and suggest that ferroptosis is a potential therapeutic target in IBD.⁹²

The purported role of ferroptosis in IBD is believed to be linked to altered short-chain fatty acid (SCFA) metabolism.¹⁰⁶ As previously stated, RSL3- and erastin-induced cell ferroptosis were shown to be enhanced by sodium butyrate. Specifically, sodium butyrate mediates the downregulation of anti-ferroptosis proteins, SLC7A11 and GPX4, thus acting as a pro-ferroptotic SCFA.²⁵ Downregulation of these anti-ferroptotic proteins is mediated by the FFAR2-AKT-NRF2 and the FFAR2-mTORC1 axes.²⁵ Butyrate is a naturally occurring SCFA in the gut produced as a result of the microbial metabolism from dietary fiber. Butyrate is known to play a role in intestinal homeostasis and immune and epithelial barrier function. Butyrate is the primary energy source of IECs¹⁰⁷ and its mitochondrial oxidation accounts for over 70% of the oxygen consumed in IECs.¹⁰⁶ In DSS-induced mice, iron chelation by deferasirox treatment strongly reversed the alterations caused by ferroptosis. Deferasirox was shown to significantly reduce DSS-induced UC in mice in association with a reduction in the level of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and INF-γ)¹⁰⁸. Furthermore, deferasirox treatment reshaped the composition of intestinal microbiota, and metabolomics analysis indicated the SCFAs production was enhanced in deferasirox-treated mice.

Targeting ferroptosis for therapeutic advantage has been demonstrated in animal models of colitis where inhibiting ferroptosis can attenuate intestinal injury in IBD. Suppression of ferroptosis was been shown to ameliorate DSS-induced colitis by blocking Nrf2/HO-1 signaling.¹⁰¹ Inhibition of ferroptosis by ferrostatin-1 was shown to alleviate colitis in the TNBS (trinitrobenzenesulfonic acid)-induced murine model.¹⁰² In addition, a potent ROS scavenger (pH-sensitive molybdenum-based polyoxometalate nanocluster) was shown to attenuate ferroptosis and reduce inflammatory indicators in DSS-induced mouse models of UC.¹⁰⁹ Lipid peroxidation and elevated ferroptotic markers were also detected in the AOM/DSS (azoxymethane/DSS)-induced mouse model of colitis-associated carcinogenesis (CAC). In this model, treatment with ferrostatin-1 was shown to increase the incidence of CAC induced by a high fat diet, while induction of ferroptosis with RSL3 partially reversed this effect.¹¹⁰ It should be noted that while ferrostatin-1 has been shown to inhibit ferroptosis,¹⁴ its mechanism of action relies on preventing the accumulation of lipid peroxides (not a specific protein target) which hinders the potential to further improve the potency of the molecule.¹¹¹ While ferrostatin has been used to inhibit ferroptosis, concerns exists regarding its efficacy as a ferroptosis inhibitor in vivo.

Ferrostatin-1 is unstable in plasma and has low blood-brain barrier permeability which underscores the need for alternative inhibitors with improved pharmacokinetic characteristics.¹¹¹ In fact, there are already ferrostatin-1 analogs like liproxstatin-1 and UAMC-2418, that have better pharmacokinetic properties and outperform other inhibitors of ferroptosis in vivo.¹¹²

Ferroptosis in liver disease

Several forms of programmed cell death including apoptosis, necroptosis, and ferroptosis have been implicated in the pathogenesis of various liver diseases. Ferroptosis appears to be highly context dependent which is further supported by its involvement in conditions affecting the liver. With respect to liver injury and non-neoplastic conditions, normal liver cells are susceptible to ferroptosis whereas liver cancer cells display intrinsic or acquired resistance to ferroptosis. Accumulating evidence suggests that ferroptosis may serve as a promising target for the prevention and treatment of many forms of liver disease.

Ferroptosis has been linked to an expanding number of hepatic metabolic pathways¹¹³ including the NADPH pathway,¹¹⁴ the metabolism of fatty acids,¹¹⁵ and amino acids such as cysteine.¹¹⁶ Excessive iron regulates ferroptosis, and iron metabolism is primarily regulated by the liver which produces transferrin and hepcidin to regulate dietary iron absorption.¹¹⁷ Lower levels of these iron regulators have been found in patients with cirrhosis and liver fibrosis compared to healthy controls.^{45, 118} In animal models, hepatocyte-specific knockout of the transferrin gene Trf coupled with a high iron diet was shown to sensitize to ferroptosis-induced liver fibrosis that could be rescued with ferrostatin-1.⁴⁵ Recently, the antioxidant vitamin E has been proposed to limit ferroptosis in the liver and to decrease hepcidin levels leading to iron depletion.¹¹⁹ However, it remains unknown as to whether abnormal levels of iron regulators such as hepcidin are a cause or a consequence of deregulated iron homoeostasis.¹¹⁷

Acute and chronic liver injury

Acute liver injury frequently results from hepatotoxic agents like drugs, alcohol, ischemic injury, or viral infections.¹²⁰ Acetaminophen is a notable example, with studies indicating that ferroptosis inhibitors can moderately protect against acetaminophen-induced liver damage in mouse hepatocytes.¹²¹ Patients with alcoholic liver disease (ALD) show ethanol-induced upregulation of the transferrin receptor, TFRC, in hepatocytes leading to iron overload.^{122, 123} Chronic alcohol consumption is also associated with downregulation of hepcidin resulting in increased iron transport and absorption in the intestine.¹²⁴ Iron overload leads to oxidative stress and hepatocyte ferroptosis, and both iron chelators and ferroptosis inhibitors can protect against ALD.¹²³ Moreover, ethanol has been shown to inhibit mitochondrial frataxin expression, a key player in iron homeostasis whose downregulation leads to enhanced sensitivity to ethanol-induced ferroptosis, as do increased levels of ACSL4 and reduced system X_c^- and GPX4.¹²⁵ Restoring mitochondrial frataxin expression was shown to decrease sensitivity to ethanol-induced ferroptosis. Alcohol exposure also results in overexpression of fibronectin type III domain-containing protein 3B (FNDC3B) whose deletion in hepatocytes results in ethanol-induced steatosis through the inhibition of the AMPK pathway. Inhibition of AMPK downregulates transferrin expression leading to iron overload and predisposes to increased lipid peroxidation and ferroptosis. ¹²⁶ Other forms of cell death are also involved in alcohol-induced hepatotoxicity, including apoptosis and autophagy.¹²⁵

Lipin-1, a lipid metabolic enzyme expressed in adipose tissue, is overexpressed in animal models of alcoholic steatohepatitis and was shown to enhance hepatic ferroptosis through the inhibition of adiponectin signaling. ¹²⁷ Aberrant liver sirtuin 1 (SIRT1) is also implicated in the pathogenesis of ALD. Animal models of ALD with intestine-specific knock-out of SIRT1 show reduced hepatic inflammation and liver injury, at least partially due to attenuated ferroptosis in the liver.¹²⁸ In mouse models of hepatocyte injury and acute-on-chronic liver failure (ACLF) which has been associated with ferroptosis, Nrf2 activation ameliorates liver injury and protects against hepatotoxicity through inhibition of ferroptosis and inflammation. ¹²⁹ Both ferroptosis and necroptosis have been found to contribute to ACLF through YAP signaling, and inhibition of the YAP pathway results in reduced liver fibrosis partially through inhibition of ferroptosis resulting from upregulation of ACSL4 and downregulation of SLC7A11.¹³⁰ Of note, YAP levels in plasma in patients with ACLF are increased compared to healthy controls.¹³⁰ Given the accumulated evidence linking ethanol consumption with ferroptosis in the liver, therapeutic strategies have the potential to reduce ethanol-related liver injury, although the precise molecular mechanisms await further study.

Viral hepatitis

Infection with hepatitis B and hepatitis C are associated with development of liver fibrosis and in the setting of cirrhosis, can lead to hepatocellular carcinoma (HCC).¹³¹ Exosomes derived from hepatocytes infected with hepatitis B virus were shown to activate hepatic stellate cells and to promote fibrosis through the miR-222/TFRC axis.^{132, 133} In HCC cells, inhibition of miR-142–3p or overexpression of SCL3A2 were shown to inhibit ferroptosis and to reduce cell proliferation, migration and invasion.¹³³ Other studies have revealed that arginine methyltransferase 9 (PRMT9) expression is promoted by the HBV X protein (HBx), and targets the heat shock protein family A member 8 (HSPA8) for arginine methylation resulting in upregulation of CD44 which, in turn, inhibits ferroptosis and promotes HCC progression.^{134, 135} Low doses of selenium have been shown to inhibit the hepatotoxicity of HBx through GPX4-mediated ferroptosis inhibition in cell lines and in mouse models.¹³⁶ The enzyme fatty acid desaturase 2 (FADS2), which promotes lipid peroxidation, has emerged as a rate-limiting factor in HCV replication, indicating a potential role of ferroptosis in hepatitis C infection. In hepatitis C-infected cells, overexpression of FADS2 was found to inhibit viral replication and sensitize cells to ferroptosis.¹³⁷

Metabolic dysfunction-associated steatotic liver disease

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an increasingly prevalent condition that typically begins with steatosis and can progress to fibrosis and ultimately cirrhosis.¹³⁸ Many forms of cell death are involved in MASLD, including ferroptosis.¹³⁹ In patients with metabolic dysfunction-associated steatohepatitis (MASH), elevated end products of lipid peroxidation and iron overload are frequently observed, and iron overload correlates with the histological severity of the liver disease.¹³⁹ Additionally, arachidonate 12-lipoxygenase (ALOX-12), particularly in its interaction with acetyl-CoA carboxylase 1 (ACC1), has been found to promote the progression of MASH.¹⁴⁰ Murine models of MASH show that both a high fat diet and iron overload induce lipophagy and ferritinophagy, respectively, and synergize in promoting ferroptosis that results in lobular inflammation and increased fibrosis.¹⁴¹ Ferroptosis was shown to aggravate MASH progression whereas inhibition of ferroptosis decreased disease severity in a mouse model of MASLD induced by a high-fat diet.¹⁴² Treatment with melatonin, a potent antioxidant, was shown to reduce hepatocyte ferroptosis by inhibition of ER stress through the MT2/cAMP/PKA/IRE1 pathway.¹³⁸ These studies suggest that inhibition of ferroptosis may be a potential therapeutic approach to reduce or prevent liver damage in patients with MASLD.

Hepatic fibrosis

A key step in development of liver fibrosis is the transdifferentiation of hepatic stellate cells (HSCs) into matrix-producing myofibroblasts.¹⁴³ In this context, regulators of ferroptosis in HSCs include RNA-binding proteins ELAV-like protein 1 (ELAVL1)¹⁴⁴ and zinc finger protein 36 (ZFP36).¹⁴⁵ Upon exposure to ferroptosis-inducing compounds, ELAVL1 protein expression was increased through inhibition of the ubiquitin-proteasome pathway. While transfection with an ELAVL1 plasmid induced ferroptosis, ELAVL1 knockdown by siRNA led to ferroptosis resistance. Upregulated ELAVL1 expression also appeared to increase autophagosome formation and autophagic flux, which appeared to be the underlying mechanism for ELAVL1-enhanced ferroptosis.¹⁴⁴ In mice, treatment with sorafenib attenuated murine liver fibrosis by inducing HSC ferroptosis which was impaired by HSC-specific knockdown of ELAVL1. Similarly and upon exposure to ferroptosis-inducing compounds, a ubiquitin ligase (FBXW7/CDC4) decreased expression of the RNA binding protein ZFP36 whereas the ZFP36 plasmid impaired FBXW7 plasmid-induced ferroptosis in HSCs and inhibited induction of autophagy.¹⁴⁵ In mice, HSC-specific overexpression of ZFP36 impaired erastin- or sorafenib-induced ferroptosis. Together, these results identify ELAVL1 and ZFP36 in the induction of autophagy-dependent ferroptosis and as potential targets for the treatment of liver fibrosis, although selective induction of ferroptosis in HSCs is needed with sparing of hepatocytes.

Hemochromatosis

Hereditary hemochromatosis is an iron-overload disease caused by mutations in genes involved in iron absorption.¹⁴⁶ Excessive iron is absorbed by the intestine and deposited in parenchymal cells leading to tissue damage and organ failure. Apart from genetic factors, environmental factors such as alcohol intake and blood loss can also influence iron accumulation. Excessive iron usually produces massive ROS through the Fenton reaction and subsequently leads to DNA damage and tissue injury. Treatment with iron was shown to induce ferroptosis in murine primary hepatocytes, and ferroptosis was also observed in mice fed a high-iron diet or in mouse models of hereditary hemochromatosis with severe iron overload, but not in mice with only mild iron overload.¹¹⁴ Importantly, iron overload–induced liver damage was rescued by inhibition of ferroptosis by ferrostatin-1. Genes found to be significantly up-regulated in iron-treated cells and hemochromatosis include SLC7A11, a known ferroptosis-related gene, whose genetic deletion was insufficient to induce ferroptosis unless SLC7A11^−/− mice were fed a high iron diet. Data indicate that iron can upregulate SLC7A11 expression through the ROS–Nrf2–antioxidant response element axis which may be a potential compensatory mechanism to protect against iron overload-induced ferroptosis in hemochromatosis. In addition, SLC7A11 was shown to confer protection against ferroptosis during iron overload by uptaking cystine and reducing ROS production. In these models, iron-induced ferroptosis was not mediated by ER stress, the mitogen-activated protein kinase (MAPK) pathway, or autophagy.¹¹⁴ These results suggest that ferroptosis may be a target for treating hemochromatosis-related tissue damage.

Ferroptosis and cancer

Ferroptosis has multiple implications in tumor development. First, several cancer signaling pathways are involved in the regulation of ferroptotic cell death.¹⁴⁷ Moreover, cancer cells with dysregulated metabolism, a high accumulation of ROS, and specific mutations render some tumor cells more vulnerable to ferroptosis, and thus, create a therapeutic window for ferroptosis inducers as a therapeutic strategy.¹⁴⁷ Importantly, ferroptosis is triggered by several conventional cancer therapies such as chemotherapy, radiotherapy, immunotherapy, and targeted therapy.¹⁴⁷

Cancer-related pathways in ferroptosis

Several alterations in lipid metabolism have been described in cancer cells¹⁴⁸ including those that contribute to ferroptosis resistance such as activating mutations of PI3K or loss of PTEN function through downstream SREBP1/SCD1-mediated lipogenesis.⁶³ Furthermore, activation of the p62-Keap1-NRF2 pathway confers resistance to ferroptosis in HCC cells.¹² Genes regulated by NRF2, including NQO1, HO1, and FTH1, modify both iron metabolism and lipid peroxidation to confer ferroptosis resistance. Inhibition of the PI3K-AKT-mTOR signaling axis sensitizes cancer cells to ferroptosis induction with therapeutic potential through mTORC1 inhibition.⁶³ SLC47A1 acts as a phospholipid transporter¹⁴⁹ and can block ferroptosis through the inhibition of the ACSL4-SOART1 axis that leads to lipid peroxidation. Targeting SLC47A1 sensitizes tumor cells to ferroptosis induction and may be a strategy for tumor suppression and overcoming drug resistance.¹⁴⁹ Studies indicate that SLC27A5/FATP5, an enzyme involved in the metabolism of fatty acids and bile acids, is downregulated in HCC cells that are resistant to the multikinase inhibitor, sorafenib. SLC27A5 deficiency facilitates this resistance by suppressing ferroptosis. Mechanistically, loss of SLC27A5 enhances glutathione reductase expression in a NRF2dependent manner, and renders HCC cells insensitive to sorafenib-induced ferroptosis in vitro and in vivo.¹⁵⁰

TP53 acts as a metabolic regulator and inhibits cysteine uptake which sensitizes cells to ferroptosis through transcriptional repression of SLC7A11.¹⁵¹ SAT1 expression, which is transcriptionally regulated by p53, can trigger lipid peroxidation and sensitize cells to ROS-induced ferroptosis.¹⁵² Moreover, SAT1 increases expression of ALOX15 which catalyzes peroxidation of AA, and is a key metabolic regulator in turning oxidative stress into lipid peroxidation.¹⁵³ SAT1 expression is downregulated in human tumors possibly contributing to resistance to ferroptosis, and inhibition of ALOX15 abrogates SAT1-induced ferroptosis.¹⁵² On the other hand, p53 has also been shown to promote ferroptosis resistance such as via transcriptionally upregulating p21 and thus, decreasing cellular metabolism¹⁵⁴or via non-transcriptionally blocking the activity of dipeptidyl peptidase-4 (DPP4), which is involved in lipid peroxidation through unknown mechanisms .¹⁵⁵

Intercellular interactions mediated by E-cadherin regulate ferroptosis through the intracellular NF2-Hippo signaling pathway. Inhibition of this axis leads to YAP signaling upregulation of ferroptosis modulators, such as ACSL4 and TFRC.¹⁵⁶ As alterations in the cadherin-NF2-Hippo-YAP signaling axis are common in cancer and can sensitize to ferroptosis, this signaling axis may be therapeutically targeted.¹⁵⁶ Furthermore, because of the role of YAP signaling in epithelial-mesenchymal transition (EMT), the function of YAP to promote ferroptosis provides a mechanism explaining why therapy-resistant mesenchymal tumor cells are often more susceptible to ferroptosis induction and are addicted to GPX4 activity.²⁹ Moreover, dependency on GPX4 is correlated with high level expression of ZEB1 in therapy-resistant tumor cells,²⁹ which links mesenchymal gene expression with lipid metabolism via direct regulation of the transcription of PPARγ and EMT-associated remodeling of the plasma membrane.²⁹ Selective sensitivity of ZEB1 overexpressing cells to lipid-peroxide activity and ferroptosis can be achieved by targeted inhibition of GPX4 or GSH for therapeutic advantage.²⁹ In addition, the RAS family of proto-oncogenes are among the more frequently mutated genes in cancers. Transcription of SLC7A11 is controlled by oncogenic RAS signaling through ETS-1 in a regulatory network that allows RASdriven tumors to evade ferroptosis.¹⁵⁷ This creates an opportunity for targeting cystine uptake and GSH biosynthesis through pharmacological targeting of system X_c^- in RAS-driven tumors, which has been shown to impair tumor growth in vivo.¹⁵⁷

Ferroptosis and CRC

Colorectal cancer (CRC) is the third most common human cancer¹⁵⁸ and ranks fourth as a cause of cancer-related death worldwide.¹⁵⁸ Patients with metastatic disease are generally incurable, and drug resistance underlies treatment failure which underscores the need for new therapies to improve patient survival.

Regulation of ferroptosis in CRC

Tumor cell ferroptosis is a complex and context specific process. In CRC cells with intrinsic resistance to ferroptosis induction, the simultaneous inhibition of the GPX4-GSH axis and the FSP1-CoQ10 axis, still requires additional inhibitors of glycolysis or the Warburg effect to induce ferroptosis.¹⁵⁹ In contrast to TP53 transcription-dependent induction of ferroptosis reported in other tumor cell types, TP53 can block erastin-induced ferroptosis in CRC cells through the inhibition of DPP4 activity in a transcription-independent manner by promoting its localization to the nucleus where it is inactive.¹⁵⁵ Downregulation of TP53 enables DPP4-dependent lipid peroxidation that ultimately leads to ferroptosis.¹⁵⁵ CRC cells can be protected from ferroptosis in a TP53-dependent manner by degradation of the RNA binding protein HNRNPC (heterogeneous nuclear ribonucleoprotein C) mediated by the E3 ubiquitin ligase, cullin-9.¹⁶⁰ Downstream alternative splicing targets of HNRNPC include TP53 and SLC47A1 mRNA.¹⁶⁰ The TP53-induced glycolysis and apoptosis regulator (TIGAR) is overexpressed in CRC, and its activity increases available NADPH to convert oxidized glutathione to GSH and to reduce ROS levels.¹⁶¹ Downregulation of TIGAR sensitizes CRC cells to erastin-induced ferroptosis via increased intracellular ROS, activation of AMPK, and downregulation of SCD1.¹⁶¹ Hypoxia-inducible factor 2α (HIF-2α) is involved in CRC progression, and its activation triggers proinflammatory responses in epithelial cells,¹⁶² and also renders CRC cells vulnerable to ferroptosis through upregulation of lipid and iron regulatory genes.¹⁶²

Butyrate not only has important effects in the induction of ferroptosis but also in preventing the development of CRC through the inhibition of the activation of mTOR1.²⁵ In CRC cells, butyrate induces ferroptosis through both the increased expression of ACSL4, which increases the susceptibility of cells to lipid peroxidation,¹⁶³ and reduction of system X_c^- expression which limits cysteine availability.¹⁶⁴ It should be noted that in CRC, system X_c^- expression is higher compared to tumor-adjacent tissue.¹⁶⁴ Butyrate has been shown to enhance the pro-ferroptotic effects of the cytotoxic chemotherapy drug oxaliplatin in CRC cells in vitro and in vivo.¹⁶⁴ The combined intake of highly fermentable dietary fiber to enhance butyrate production and of omega-3 polyunsaturated fatty acids has been proposed to reduce the risk of CRC. These treatments can promote ferroptosis through GPX4 and mitochondrial metabolism pathways.¹⁰⁷ The “butyrate paradox”, where butyrate enhances proliferation of normal cell but not of cancer cells, also shows the context dependent nature of its metabolism.²⁵

Several genes and RNA molecules with altered expression in CRC have been associated with ferroptosis regulation and tumor development.¹⁵⁸ Furthermore, many ferroptosis-related gene signatures, deposited in public databases, have been proposed to prognosticate patients with CRC,¹⁵⁸ however, limitations exist and there is the need for validation. These signatures have the potential to identify important regulatory pathways of ferroptosis in CRC that may lead to development of new therapeutic strategies.

Therapeutic strategies involving ferroptosis in CRC

Induction of ferroptosis is a potential approach for the treatment of CRC, and several drugs have been associated with this form of RCD.¹⁵⁸ A subset of cancer cells may avoid therapy-induced cell death by switching to a slow, yet reversible proliferation state that is drug tolerant. Drug-tolerant persistent CRC models show increased GPX4 expression making them vulnerable to GPX4 inhibitors,¹⁶⁵ but more potent and bioavailable compounds to achieve GPX4 inhibition are desirable.¹⁶⁵ Overexpression of lipocalin 2 (LCN2), involved in the regulation of iron homeostasis, in CRC cells promotes chemoresistance to 5-fluorouracil (5-FU) through the inhibition of ferroptosis.¹⁶⁶ Conversely, inhibition of LCN2 using monoclonal antibodies was shown to sensitize cells to chemotherapy and to inhibit tumor growth.¹⁶⁶ However, LCN2 was also shown to inhibit the NF-κB/SNAIL pathway and to prevent EMT and metastasis of CRC. Conflicting data exist in that silencing of LCN2 was shown to promote tumor growth¹⁶⁷ and thus, the role of LCN2 in CRC progression remains controversial. It is established that KRAS mutated CRC cell lines are resistant to therapy with EGFR inhibitors, including cetuximab.¹⁶⁸ Inhibition of the Nrf2/HO-1 axis by cetuximab has been shown to promote ferroptosis induced by RSL3 in KRAS-mutated CRC cells.¹⁶⁸

In the context of clinical application for cancer therapy, ferroptosis can be targeted by exploiting cancer cell regulatory alterations that result in enhanced metabolism to sustain increased requirement for energy and metabolite synthesis. These changes can make cancer cells more susceptible to ferroptosis (for example, by being more dependent on GPX4 activity or other ferroptosis surveillance mechanism) creating therapeutic windows for ferroptosis inducers in specific cancers. Cancer cells that have acquired resistance to ferroptosis can be re-sensitized by targeting the specific mechanisms that protect them. Furthermore, ferroptosis inducers can sensitize cancer cells to conventional anti-cancer therapies or overcome acquired resistance to thereby, improve efficacy.¹⁴⁷

The roles of ferroptosis in the tumor microenvironment and immunotherapy

The pros of ferroptosis in immunotherapy

The release of DAMPs is one of the major features of immunogenic cell death which activates the immune system and synergizes with immune checkpoint blockade (ICB). HMGB1 is a DAMP that is released by ferroptotic cells in an autophagy-dependent manner. Of note, the ATG5/ATG7 autophagic axis is necessary for the release of acetylated HMGB1 during ferroptosis¹⁶⁹. Other immunogenic DAMPs, including ATP, CRT, DCN were also confirmed to be released by ferroptotic cells.¹⁷⁰

Moreover, recent studies suggested that CD8+T cells can kill cancer cells via ferroptosis. IFN-γ secreted by activated CD8+ T cells downregulates the expression of SLC7A11 in cancer cells by JAK-STAT1 pathway and sensitizes cancer cells to ferroptosis.¹⁷¹ IFN-γ in combination with AA can further promote ferroptosis in cancer cells. IFN-γ upregulates ACSL4 expression through the STAT1/IRF1 signaling pathway in cancer cells, promotes the incorporation of AA into phospholipids, and sensitizes cancer cells to ferroptosis. Combining AA and ICBs can synergistically inhibit tumor growth through IFN-γ.⁸⁹ These studies have indicated a synergistic effect between ferroptosis induction and ICBs, representing a potential treatment strategy.

The cons of ferroptosis in immunotherapy

Chronic inflammation can lead to tumor initiation, growth and progression by providing a tumor-supportive microenvironment.¹⁷² Tumor cells release proinflammatory molecules that promote immune evasion, angiogenesis, and growth of cancer stem cells.^{8, 172} Several immunosuppressive cell types have been identified in the TME, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAMs), and regulatory T cells (Tregs)¹⁷³ which may participate in ferroptosis-related immunosuppression. Although induction of ferroptosis to target tumors resistant to conventional therapies is promising, the resulting release of proinflammatory signals can have a negative impact on ferroptosis-based therapies for cancer. Cancer cell immune evasion involves mechanisms including macrophage polarization, impaired immune cell cytotoxicity, and upregulation of immunosuppressive cells.^{8, 172} Importantly, ferroptosis plays a role in all of these mechanisms in the TME (Figure 2).

Figure 2.

Involvement of ferroptosis in the tumor microenvironment and modulation of immune responses. Tumor cell ferroptosis promotes the activation of immunosuppressive cells and impairs the activity of anti-tumor immune cells. Immunosuppressive Tregs and MDSCs are protected from ferroptosis through upregulation of GPX4 and ASAH2, respectively. Downregulation of Acyl CoA dehydrogenases (ACADS), and exposure to HMGB1, KRAS^G12D, PGE₂ and 8-OHG resulting from tumor cell ferroptosis in the TME leads to macrophage polarization with a M2 phenotype. TAMs release of proinflammatory TNF is dependent on HMGB1 interaction with the advanced glycosylation end-product specific receptor (AGER) receptor. The inflammatory environment reduces NOS2 expression in M2 macrophages and makes them more vulnerable to ferroptosis. Active CD8⁺ T cells release IFN-γ that impairs tumor cancer cell expression of system X_c^- that results in ferroptotic cell death. Ferroptotic cancer cells increase the expression of COX2 that elaborates PGE₂ which serves to inhibit immune cell (CD8⁺ T cells, DCs and NKs) activities. CD8⁺ T cells are led to ferroptosis through both cysteine depletion as a consequence of their inactive system X_c^- and accumulation inside MDSC and by increased lipids in the TME that are uptaken through the CD36 membrane receptor. In DC cells, downregulation of system X_c^- and increased lipids in the TME (activating the PPAR-γ receptor pathway) enhance ferroptosis and impair their activation of CD8⁺ T cells and their release of proinflammatory IL-6 and TNF. L-KYN release from ferroptotic cancer cells in the TME leads to ferroptosis of NK cells.

The release of HMGB1 has implications for immune responses and cancer, including the proinflammatory TME.^{8, 174} The binding of HMGB1 to toll-like receptor 4 (TLR4) and AGER, mediates immune responses such as the release of the cytokine tumor necrosis factor (TNF).¹⁶⁹ Mutated KRAS^G12D acts as a DAMP during ferroptosis whose release following autophagy-dependent ferroptosis activates AGER and leads to macrophage polarization through STAT3-dependent fatty acid oxidation.¹⁷⁵ Alternatively activated or M2 macrophages is associated with tumor progression due to their angiogenic ¹⁷⁶ and immunosuppressive ¹⁷⁷ activities. In mutated KRAS^G12D pancreatic tumors, tumorigenesis is promoted by depletion of GPX4 or high-iron diets leading to ferroptosis.¹⁷⁸ The release of 8-hydroxyguanosine during ferroptosis activates a DNA-sensing pathway that regulates inflammation and immune responses, causing macrophage migration and activation¹⁷⁸. Polarization of macrophages is also promoted by downregulation of ACADS enzyme which can also enhance CRC tumor growth.¹⁷⁹ Interestingly, M2 macrophages are more susceptible to ferroptotic cell death than M1 macrophages.¹⁸⁰ Upregulation of NOS2 in M1 macrophages protects them from lipid peroxidation, whereas downregulation of NOS2 is promoted in pro-inflammatory conditions including those of the TME.¹⁸⁰

Treg cells are protected from ferroptosis by GPX4, sustaining their activation and anti-tumor immune activity in the TME.¹⁸¹ Treg-specific deletion of GPX4 limits tumor growth and enhances antitumor immune responses, such as production of proinflammatory cytokines including IL-1β.¹⁸¹ MDSCs, which inhibit T-cell mediated anti-tumor immunity, are protected from ferroptosis through N-acylsphingosine amidohydrolase (ASAH2) overexpression. ASAH2 targets and suppresses the p53/Hmox1 axis reducing lipid peroxidation and preventing ferroptosis.¹⁸² One of the mechanisms of MDSCs for blocking T-cell activation is the depletion of cysteine. T-cells lack both cystathionase (that converts methionine to cysteine) and a complete system X_c^- (to import cystine), whereas MDSCs do express a functional system X_c^- but not the ASC neutral amino acid transporter that exports cysteine.¹⁸³ MDSCs block T-cell activation by sequestering cystine and limiting its availability, which leads to T-cell ferroptotic vulnerability in the setting of a TME that is enriched in MDSCs. Mouse models with T cells lacking GPX4 have reduced peripheral CD8⁺ T cells and impaired response to infection, whereas both CD4⁺ and CD8⁺ T-cells lacking GPX4 underwent ferroptotic cell death in vitro.¹⁸⁴ Also, CD8⁺ T-cells are more sensitive to ferroptosis induction using inhibitors of GPX4 activity than are cancer cell lines.¹⁸⁵ Membrane glycoprotein CD36 mediates ferroptosis of CD8⁺ T-cells through its fatty acid uptake activity, impairing CD8⁺ T-cells anti-tumor activity.⁶⁹ Mechanistically, the uptake of oxidized low-density lipoproteins by CD36 induces p38 phosphorylation, and this activation limits secretion of TNF and IFN-γ (effectors of CD8⁺ T-cell immune activity) while also inducing lipid peroxidation and ferroptosis.¹⁸⁶ Knockout of ACSL4 protects these cells from ferroptosis but impairs their antitumor activity, whereas overexpression of GPX4 or FSP1 also protects from ferroptosis while retaining their functionality.¹⁸⁵ In fact, overexpression of GPX4 not only protects CD8⁺ T-cells from ferroptosis, but also enhances their immune activity.¹⁸⁶ IFN-γ released from immunotherapy-activated CD8⁺ T-cells downregulates the expression of system X_c^- in tumor cells, promoting lipid peroxidation and ferroptosis ¹⁷¹ while also sensitizing to radiotherapy.¹⁸⁷ Since IFN-γ induces expression of PD-L1 and promotes tumor progression,¹⁸⁸ the addition of immune checkpoint blockade should be considered as a potential combination therapy. Apolipoprotein L3 (APOL3) promotes ferroptosis of CRC cells and inhibits tumor growth through L-lactate dehydrogenase A (LDHA) ubiquitination and degradation in the proteasome.¹⁸⁹ This pathway enhances the anti-tumor activity of CD8⁺ T-cells increasing the levels of IFN-γ and reducing lactic acid concentration.¹⁸⁹ The combination of overexpression of APOL3, ferroptosis induction with RSL3, and inhibition of PD-L1 has a synergistic effect in CRC models.¹⁸⁹

Other immune cell types have impaired cytotoxic activity in the TME. In this regard, the activity of natural killer (NK) cells is downregulated in the TME¹⁹⁰ whereas activation of the NRF2 signaling pathway restores their anti-tumor activity.¹⁹⁰ Dendritic cells (DCs) are antigen-presenting cells that are important for the activation of cytotoxic T lymphocytes.¹⁹¹ Moreover, DC functions are impaired by lipid peroxidation byproducts that cause ER stress.¹⁹² DC induction of ferroptosis can be achieved with the RSL3 inhibitor of GPX4, but not with the SLC7A11 inhibitor erastin (due to the already low expression of SLC7A11 in DCs).¹⁹³ Mechanistically, RSL3-induced ferroptosis in DCs is dependent on peroxisome proliferator activated receptor gamma (PPAR-γ), acting as a key transcription factor for lipid metabolism.¹⁹³ DCs undergoing ferroptosis lose their immune functions such as production of proinflammatory cytokines (TNF and IL-6) and activation of CD8⁺ T-cells.¹⁹³ Moreover, engulfment of ferroptotic dying cancer cells by DCs not only impedes antigen cross-presentation in DC cells, but also impedes the immunogenicity of chemotherapy-induced apoptotic cells, resulting in decreased proliferative capacity of CTLs.¹⁹⁴

Polymorphonuclear MDSCs (PMN-MDSCs) in the TME are highly sensitive to ferroptosis. Interestingly, spontaneous ferroptosis of PMN-MDSCs does not enhance anti-tumor immunity by reducing the number of PMN-MDSCs. Instead, it confers immunosuppressive activity on PMN-MDSCs. Ferroptotic PMN-MDSCs release PGE2 and oxidized phospholipids, influencing the activity of CD8+ T cells and TAMs and rendering a more immunosuppressive TME. Inhibition of ferroptosis in immunocompetent mice abrogates the immunosuppressive activity of PMN-MDSCs, particularly when combined with ICBs, and reduces tumor progression. This indicates that PMN-MDSC ferroptosis may be a targetable immunosuppressive mechanism for cancer immunotherapy.¹⁹⁵ Overexpression of cyclooxygenase-2 (COX2) in CRC and other cancer cell types¹⁹⁶ has been reported in cancer cells undergoing ferroptosis.¹⁵ COX-2 enzymatic activity is the rate limiting step in prostaglandin E2 (PGE2) synthesis whose membrane receptors can inhibit anti-tumor immune responses.^{197, 198} The COX-2-PGE2-EP axis suppresses activation of myeloid cells¹⁹⁷ and the activity of DCs, NK cells and T-cells, while promoting polarization of TAMs to a M2 phenotype¹⁹⁸ thereby contributing to tumor immune escape. Future studies are needed to clarify the immunomodulatory effects of ferroptosis, including its association with diseases outcomes. In addition, further study of ferroptosis-related signal transduction by different immune cell subpopulations is awaited.

Targeting tumor ferroptosis to improve the efficacy of immunotherapy

Due to the heterogeneity and high complexity of TME, ferroptosis inducing agents (FINs) can cause either immune active or immunosuppressive function in a TME-dependent manner. Therefore, it demands the development of FINs that selectively induce tumor ferroptosis and improve the efficacy of immunotherapy. SLC7A11 had been shown to be dispensable for T cells proliferation in vivo and for their antitumor immune responses. Consequently, tumor cell SLC7A11 loss acts synergistically with the immunotherapeutic agent anti–CTLA4, laying the foundation for utilizing specific SLC7A11 inhibitors to expand the efficacy of existing anticancer immunotherapeutics.¹⁹⁹ Recently, a small molecule compound, N6F11 had been shown to selectively trigger degradation of GPX4 in tumor cells but not immune cells. N6F11 caused ferroptotic cancer cell death, initiated HMGB1-dependent antitumor immunity, and sensitized immune checkpoint blockade in advanced cancer models.²⁰⁰ Another study showed that deubiquitinase inhibitor PR-619 degraded GPX4 and increased the efficacy of anti-PD1 in a colon cancer model.²⁰¹ These findings may establish a safe and efficient strategy to boost ferroptosis-driven antitumor immunity by targeting GPX4 selectively in tumor cells.

Conclusions

Ferroptosis is a form of metabolically regulated cell death. Data continue to emerge regarding ferroptotic regulatory pathways and the relationship between ferroptosis and pathophysiology of diseases including those affecting the gastrointestinal tract. Iron overload, ROS accumulation, lipid peroxidation and impaired antioxidant systems are critical steps in pathways regulating ferroptosis that are involved in GI diseases. While studies have used the accumulation of lipid peroxidation as an indicator of ferroptosis, this process can also be part of other types of RCD including apoptosis, necroptosis, or pyroptosis. Although much progress has been made in understanding pathological roles of ferroptosis, critical questions remain to enable development of ferroptosis-targeted therapies. Ferroptosis appears to have dual roles whereby inhibition of ferroptosis can alleviate intestinal damage whereas induction of ferroptosis has been shown to inhibit CRC cell migration and proliferation. There is an unmet need to identify specific biomarkers of ferroptosis and further research is required to detect ferroptotic mechanisms that are specific for individual disease entities. Therapeutic opportunities related to ferroptosis hold promise for therapy of multiple disease states. Cellular susceptibility to ferroptosis is dependent on the in vivo microenvironment, and vulnerability to ferroptosis among cancer cells, immune cells and within the TME awaits further study. It remains to be determined if we can activate ferroptosis specifically in cancer cells without affecting healthy cells. Studies suggest that ferroptosis plays an important role in tumor suppression as well as resistance to cancer therapy which can provide novel therapeutic opportunities. In conclusion, ferroptosis is a mechanistically and morphologically distinct form of RCD that has provided insights into pathophysiology of multiple disease entities. Elucidating the regulatory pathways related to ferroptosis can provide new opportunities for understanding pathophysiology of disease inlcuding cancer, and identifying novel targets and strategies for exploitation of ferroptosis for therapeutic advantage.

Abbreviations:

ACLF

acute-on-chronic liver failure

ALD

alcoholic liver disease

AOM

azoxymethane

CAC

colitis-associated carcinogenesis

CD

Chron’s disease

CDI

cysteine-deprivation-induced

CRC

colorectal cancer

DAMPs

damage-associated molecular patterns

DCs

dendritic cells

DSS

dextran sulphate sodium

ER

endoplasmic reticulum

FIN

ferroptosis inducing agent

5-FU

5-fluorouracil

GSH

glutathione

HCC

hepatocellular carcinoma

HSCs

hepatic stellate cells

IBD

inflammatory bowel disease

ICB

immune checkpoint blockade

IECs

intestinal epithelial cells

IFN-γ

interferon gamma

IR

ionizing radiation

MDR

multidrug resistant

MDSC

myeloid-derived suppressor cell

MUFA

monounsaturated fatty acid

MASLD

Metabolic dysfunction-associated steatotic liver disease

MASH

metabolic dysfunction-associated steatohepatitis

NK

natural killer

PL

phospholipid

PMN

Polymorphonuclear

PUFA

polyunsaturated fatty acid

RCD

regulated cell death

ROS

reactive oxygen species

RTA

radical-trapping antioxidant

SCFA

short-chain fatty acid

TAMs

tumor associated macrophages

Tregs

regulatory T cells

TME

tumor microenvironment

UC

ulcerative colitis