Unlocking ferroptosis in prostate cancer— the road to novel therapies and imaging markers

Abstract

Ferroptosis is a distinct form of regulated cell death that is predominantly driven by the buildup of intracellular iron and lipid peroxides. Ferroptosis suppression is widely accepted to contribute to the pathogenesis of several tumours including prostate cancer. Results from some studies reported that prostate cancer cells can be highly susceptible to ferroptosis inducers, providing potential for an interesting new avenue of therapeutic intervention for advanced prostate cancer. In this Perspective, we describe novel molecular underpinnings and metabolic drivers of ferroptosis, analyse the functions and mechanisms of ferroptosis in tumours, and highlight prostate cancer-specific susceptibilities to ferroptosis by connecting ferroptosis pathways to the distinctive metabolic reprogramming of prostate cancer cells. Leveraging these novel mechanistic insights could provide innovative therapeutic opportunities in which ferroptosis induction augments the efficacy of currently available prostate cancer treatment regimens, pending the elimination of major bottlenecks for the clinical translation of these treatment combinations such as the development of clinical-grade inhibitors of the anti-ferroptotic enzymes as well as non-invasive biomarkers of ferroptosis, the latter of which could be exploited for diagnostic imaging and treatment decision-making.

Summary

Ferroptosis induction represents a promising new therapeutic strategy for advanced prostate cancer. Here, the authors discuss the interplay between ferroptosis and metabolism, current efforts to target ferroptosis and combination therapies, and emerging methods to monitor this process in patients.

Introduction

The 5-year survival rate for localised prostate cancer is >99% but drops to 30-40% when metastasis is detected and/or with the development of castration-resistant prostate cancer (CRPC)¹. Patients with advanced prostate cancer can be treated with second-generation androgen receptor (AR)-signalling inhibitors (such as enzalutamide, apalutamide, darolutamide, and abiraterone), chemotherapy, radiotherapy, immunotherapy (such as sipuleucel-T), or poly (ADP-ribose) polymerase (PARP) inhibitors (for selected patients harbouring mutations in DNA-damage response genes). However, these current treatment regimens only prolong life expectancy by, on average, 2–3 years². The limited knowledge of the underlying mechanisms that promote the initiation and development of advanced prostate cancer poses a challenge to clinicians in treating these patients.

Major advances in our understanding of the regulated cell death pathway called ferroptosis over the past decade have the potential to transform the therapeutic landscape of cancer. Ferroptosis is an iron-dependent form of cell death that has morphological and mechanistic features distinct from those of other forms of cell death such as necrosis, apoptosis, and autophagy. For instance, different from typical necrotic processes, ferroptosis is not associated with signs of organelle swelling³. Moreover, ferroptosis does not show the usual apoptosis characteristics of chromatin condensation, formation of apoptotic bodies, and cytoskeletal breakdown³. Ferroptosis differs from autophagy by not forming classic double-membraned autophagosomes that enclose cellular cargo. Instead, upon ferroptosis induction, cells undergo a reduction in mitochondrial size and in mitochondrial cristae, accompanied by elevated membrane density³.

A notable hallmark of ferroptosis is the production of peroxides on polyunsaturated fatty acids (PUFAs)⁴ that are part of membrane phospholipids, as well as PUFA cholesterols⁵. PUFAs are particularly susceptible to lipid peroxidation because of multiple double bonds in their carbon chains, which makes this class of lipids uniquely vulnerable to hydrogen abstraction by hydroxyl, alkoxyl, or hydroperoxyl radicals⁶. The sources of oxidants that initiate lipid peroxidation can be the mitochondria, iron overload and iron-dependent generation of reactive oxygen species (ROS), or oxidoreductases (such as NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, and NADPH oxidases)^7–10. The resultant lipid peroxidation leads to a chain reaction that causes a rapid escalation of radical initiation and corresponding radical damage, ultimately causing widespread, irreparable oxidative injury to the cell membrane and eventually, cell death^11,12.

To prevent ferroptosis, cells possess an array of intrinsic systems to avert lipid peroxidation. The classical mechanism of ferroptosis defence involves glutathione peroxidase 4 (GPX4) and the glutathione (GSH) system. GPX4 uses GSH as a cofactor to promote reduction of lethal lipid hydroperoxides to their corresponding non-lethal alcohols, in turn mitigating lipid peroxidation to secure lipid bilayer membrane integrity and prevent ferroptosis¹³. GSH, a tripeptide consisting of glutamate, glycine, and cysteine, is formed by importing cystine into cells through the system x_c-, an antiporter importing cystine in exchange for glutamate¹⁴ . Pharmacologically, ferroptosis can be induced by using a GPX4 inhibitor such as the RAS-selective lethal 3 (RSL3), or by depleting intracellular GSH through the inhibition of system x_c- with small molecules such as erastin, sulfasalazine, and sorafenib ^15,16. Induction of ferroptosis with these agents causes an uncontrolled accumulation of ROS species, which deteriorates the cell membrane and leads to cell death¹⁷.

Beyond the canonical GPX4 system, alternative ferroptosis defence mechanisms exist. Ferroptosis suppressor protein 1 (FSP1; encoded by the AIFM2 gene) and possibly dihydroorotate dehydrogenase (DHODH) can inhibit ferroptosis in a GPX4-independent manner by reducing CoQ10 to CoQ10-H2, a free-radical sequestering antioxidant found in the inner mitochondrial membrane that prevents lipid peroxide propagation^18,19. Reduced CoQ10 is a lipid ubiquinone and acts as an antioxidant that protects lipids and proteins from oxidative damage, therefore helping to maintain the stability and permeability of the cell membrane²⁰. More specifically, CoQ10 acts as a mobile electron carrier within the electron transport chain, shuttling electrons between different enzyme complexes. Upon accepting electrons from NADH, CoQ10 becomes reduced (CoQ10-H2) and can donate these electrons to other molecules, including peroxide species, effectively neutralizing them²¹. Additionally, GTP cyclohydrolase-1 (GCH1) and its metabolic derivatives tetrahydrobiopterin (BH4) and dihydrobiopterin (BH2) constitute another system that antagonizes ferroptosis independently of GPX4. GCH1 specifically shields phosphatidylcholines with two PUFA tails from oxidative damage, consequently preventing ferroptosis. Through its protective effect on CoQ10, GCH1 might also help shield cells from oxidative damage²². GCH1 is the first rate-limiting enzyme in the metabolic pathway that produces BH4. BH4 acts as a cofactor for nitric oxide synthase, enabling the production of nitric oxide- a potent antioxidant. Decreasing levels of BH4 can cause increased levels of superoxides, inducing ferroptosis²².

Cancer cells, which are characterised by a unique metabolic reprogramming and increased levels of ROS, often have intrinsic susceptibility to ferroptosis²³. Hence, disrupting the defence mechanisms against ferroptosis in cancer cells could be a promising therapeutic approach, as these cells heavily rely on these antioxidant systems to survive under stress conditions in the harsh tumour microenvironment. Results from multiple studies in preclinical models showed that targeting major ferroptosis inhibitors, such as the GPX4 enzyme or system x_c-, could preferentially trigger ferroptosis in otherwise drug-resistant cancer cells, in turn enhancing the effectiveness of existing therapies^24–26.

Mounting evidence points to the potential of exploiting ferroptosis for the treatment of advanced prostate cancer. For instance, a commonality across a variety of antiandrogen therapy-resistant, highly mesenchymal prostate cancer cells is the hypersensitivity to ferroptosis and GPX4 dependency, effects that have been linked to substantial lipid remodelling^27,28. Ferroptosis inducers targeting the GPX4—GSH system have also shown anti-cancer activity in xenograft models across multiple prostate cancer variants including adeno-CRPC, neuroendocrine prostate cancer, and double-negative prostate cancer^29,30. Other ferroptosis defence pathways that function independently of GPX4, discovered as recently as 2021, remain relatively understudied in the context of prostate cancer, but might also have important roles in the disease. The therapeutic effectiveness of DHODH, FSP1, and GCH1 inhibitors in prostate cancer has been shown in a few studies. A study using human prostate cancer cell lines treated with the DHODH inhibitor leflunomide demonstrated that the GI50 of the PTEN-mutant cells was lower than that of wildtype PTEN cells, indicating that PTEN-mutant prostate cancers show sensitivity towards DHODH inhibition³¹. Meanwhile, by constructing a ferroptosis-related prognostic gene signature using the Cancer Genome Atlas prostate cancer patient cohort, another study identified FSP1 as an indicator of poor prognosis in prostate cancer patients with high risk of biochemical recurrence³². Notably, controversies exist in the field which often prevent consensus regarding the importance of various pathways in ferroptosis. For example, there are conflicting reports on the role of ferroptosis defence pathways in other cancers such as renal carcinoma and fibrosarcoma^19,33,34. Further, scarce knowledge about the exact mechanisms of action of ferroptosis-inducing drugs and the optimal concentrations to use also confounds data interpretation. For instance, results from a 2023 study showed that DHODH might not be a prevalent ferroptosis defence mechanism³⁴, contrasting what was previously reported¹⁹. In this study, the absence of DHODH barely sensitised cancer cells to ferroptosis compared with FSP1 deletion, whereas reported DHODH inhibitors at high concentrations were shown to sensitise cancer cells to ferroptosis by binding to and inhibiting FSP1 rather than DHODH³⁴. Another important issue in the field is the need to delineate the comparative benefits and pitfalls of targeting distinct ferroptosis defence mechanisms in prostate cancer. For example, results from GPX4 knockout mouse models indicate that GPX4 seems to be essential for survival³⁵, whereas FSP1-knockout mice showed normal development³⁶, indicating that blocking FSP1 might be a less toxic therapeutic strategy than suppressing GPX4. To date, the role of GCH1—BH4 in prostate cancer has not been explored. Together, the growing evidence indicates a causal role for ferroptosis in the reduction of prostate cancer, suggesting that the development and understanding of strategies targeting ferroptosis in prostate cancer are warranted.

In this Perspective, we describe the current understanding of metabolic mechanisms and signalling pathways that govern ferroptosis. We also highlight potential opportunities to integrate ferroptosis inducers into established standard-of-care treatments for prostate cancer, thus establishing a foundation for the development of rational combination therapies to enhance patient outcomes. Last, we describe methods for non-invasively monitoring ferroptosis and their utility in patient selection and as pharmacodynamic biomarkers in the context of ferroptosis-inducing cancer therapies in prostate cancer.

Metabolic processes influencing prostate cancer cells’ vulnerability to ferroptosis

In the past 10 years, progress has been made deciphering the underlying mechanisms of ferroptosis and the intricate links with cancer metabolism regulatory networks. Ferroptosis has been shown to have a crucial role at the intersection of lipid metabolism, iron control, and amino acid metabolism in the context of prostate cancer.

Lipid metabolism and ferroptosis

Ferroptosis is a lipid-dependent form of cell death. Accordingly, ferroptosis susceptibility is affected by the cell’s lipid composition and the processes involved in lipid acquisition, synthesis, storage, and breakdown. Thus, understanding the unique changes in lipid metabolism that define prostate cancer cells is necessary to adequately evaluate ferroptosis sensitivity in prostate cancer. Specifically, malignant cells are often more susceptible to ferroptosis than benign cells owing to an increased demand for iron and an enhanced lipid metabolism^37,38. Prostate cancer cells generate a large proportion of energy through lipid metabolism and have extensively dysregulated fatty acid metabolism^39,40, making this tumour a candidate for metabolism-based therapeutics. Both early-stage and late-stage prostate cancer are characterised by the upregulation of genes linked to lipid metabolism, metabolic rewiring to oxidative phosphorylation, and elevated tricarboxylic acid cycle flux^40–42 , suggesting a resultant increase in intracellular ROS burden that could perturb iron homeostasis and encourage lipid peroxidation⁴³. Results from proteomic studies on primary prostate tumours and bone metastases showed strong positive correlation between the levels of enzymes involved in lipid metabolism and the onset and development of prostate cancer⁴⁴. Major lipid metabolism pathways in prostate cancer that could have potential roles in ferroptosis include production of phospholipids, β-oxidation, and activation of fatty acids.

The intracellular balance between MUFAs and PUFAs

The activation and inclusion of monounsaturated fatty acids (MUFAs), such as oleic acid, in membrane phospholipids is controlled by long-chain acyl-CoA synthetase 3 (ACSL3)⁴⁵. MUFAs can prevent cells from undergoing ferroptosis by replacing oxidizable PUFAs from the membrane lipid bilayer^4,45. Increased PUFA biosynthesis and PUFA incorporation into phospholipids are controlled by long-chain acyl-CoA synthetase 4 (ACSL4) and are essential for ferroptosis induction^45,46. ACSL4 facilitates the accumulation of oxidized cellular membrane phospholipids and subsequent production of lethal lipid peroxides, in turn inducing ferroptosis^47–49 (Fig. 1A). Thus, the balance of MUFAs and PUFAs in phospholipids greatly determines cells’ sensitivity to ferroptosis.

Fig. 1.

Mechanisms of ferroptosis in prostate cancer.

Multiple metabolic pathways contribute to the regulation of ferroptosis in prostate cancer including iron metabolism, lipid metabolism, and glutathione (GSH) synthesis pathways. a) Lipid metabolism. In de novo lipogenesis, stearoyl-CoA desaturase-1 (SCD1) catalyses the production of monounsaturated fatty acids (MUFAs) from the saturated fatty acid (SFA) palmitate. Long-chain acyl-CoA synthetase 3 (ACSL3) subsequently conjugates MUFAs with phospholipids and disrupts the synthesis of phospholipids containing PUFAs. An increased MUFA:PUFA ratio can protect cells from undergoing ferroptosis. Inhibition of fatty acid synthase (FASN), another component of the de novo lipogenesis pathway, could result in ferroptosis. This effect occurs because SFA and MUFA are supplied by FASN to the Lands cycle, which is responsible for remodelling oxidised phospholipids such as phosphatidylcholines. 5’-adenosine monophosphate-activated protein kinase (AMPK)-mediated phosphorylation of acetyl-CoA carboxylase 1 (ACC, an enzyme that catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA), inhibits ACC1 activity, resulting in reduced de novo lipogenesis and decreased ferroptosis in immortalized mouse embryonic fibroblasts (MEFs). Peroxidation of phospholipids containing PUFAs is the main driver of ferroptosis, with oxygenated diacyl arachidonic acid (AA)- and adrenic acid (AdA)-containing species of PE being the most susceptible ferroptosis substrates. The ω-6 de novo PUFA synthesis pathway uses fatty acid elongase 2 and 5 (ELOVL2 and ELOVL5), and fatty acid desaturase 1 and 2 (FADS1 and FADS2) to synthesize AA and AdA from exogenously sourced linoleic acid. Carnitine palmitoyltransferase 1 (CPT-1) imports acylcarnitines into the mitochondria (indicated by dashed arrow) whereas DECR1 catalyses PUFA β-oxidation in the mitochondria (indicated by dashed arrow) to yield acetyl-CoA, which feeds into the TCA cycle to produce hydrogen peroxide (H₂O₂) that reacts with Fe²⁺ to produce ROS and promote lipid peroxidation. Lipid β-oxidation can be a source of ferroptosis suppression, as this pathway diverts PUFAs away from peroxidation towards processes supporting cancer cell survival and drug resistance in castration-resistant prostate cancer (CRPC). Sterol response element-binding protein 1 (SREBP1) is a master transcriptional regulator of genes that encode proteins involved in the de novo lipogenesis, including FASN and ACACA (encoding FAS and ACC1, respectively) and itself is activated by the androgen receptor (AR). The androgen receptor (AR) promotes the transcription of membrane-bound O-acyltransferase 2 (MBOAT2), which inhibits PE-AA and PE-AdA formation, preventing lipid peroxidation and ferroptosis. In addition, AR promotes the transcription of MBOAT2, which inhibits PE-AA and PE-AdA formation. Further, in prostate cancer, AR has been shown to regulate the expression of long-chain acyl-CoA synthetases 3 and 4 (ACSL3 and ACSL4). AR promotes ACSL3 expression to promote MUFA incorporation into membranes, preventing ferroptosis. AR also transcriptionally suppresses ACSL4 expression, which prevents PUFA conjugation to coenzyme A (CoA) and thus, further creation of PUFA-containing phospholipids like PE and subsequent ferroptosis. b) Iron metabolism. Iron is imported into the cell through the transferrin receptor (TfR). Fe³⁺ is converted to Fe²⁺ by the transmembrane ferrireductase six-transmembrane epithelial antigen of the prostate-3 (STEAP3) inside endosomes. Subsequently, the iron chaperone poly(rC)-binding protein 1 (PCBP1) binds and transports Fe²⁺ to ferritin for storage, whereas poly(rC)-binding protein 2 (PCBP2) provides molecular support and transports iron to the iron exporter ferroportin (FPN) for export. Increasing the activity of PCBP1 has been shown to suppress ferroptosis. An excess of iron can increase ROS in large part because iron in its redox-active state (Fe²⁺) participates in the Fenton reaction and creates ferric iron (Fe³⁺) and ROS (such as hydroxyl radicals), which oxidise DNA, proteins, and lipids to induce ferroptosis. In prostate cancer cells, TfR is upregulated, whereas the FPN is downregulated to preserve the labile iron pool. This process helps cancer cells increase proliferation, movement, invasiveness, and the activity of specific proteins that promote metastasis. c) Glutamate, cystine, and GSH metabolism. Ferroptosis was reported to be regulated by extracellular L-glutamine levels and by enzymes involved in glutaminolysis— a glutamine-fuelled intracellular catabolic pathway. Extracellular L-glutamine enters the cell via transporters such as the alanine serine cysteine transporter 2 (ASCT2) and is converted to glutamate. Glutamate can then undergo glutaminolysis and metabolism through the TCA cycle in the mitochondria, producing ROS which can promote ferroptosis. Glutamate also reacts with cysteine in a reaction catalysed by gamma-glutamylcysteine synthetase (γ-GCS) to produce γ-glutamyl-cysteine. Finally, glycine and γ-glutamyl-cysteine form glutathione (GSH) in a reaction catalysed by GSH synthetase (GS). Glutathione peroxidase 4 (GPX4) converts GSH to oxidized GSH (GSSG), using GSH as a reducing agent to reduce toxic hydroperoxides into their respective alcohols. If any step of the process is disrupted, GPX4 activity will be reduced, leading to the accumulation of toxic hydroperoxides that compromises the integrity of membranes and organelles, and thus induces ferroptosis. However, high levels of extracellular glutamine alone cannot cause ferroptosis. Straight arrows represent direct links between proteins. Dashed arrow represents indirect, multiple-steps link between proteins.

In prostate cancer cells, AR synchronously controls the expression of ACSL3 and ACSL4 in opposite directions to render prostate cancer cells reliant on ACSL4-mediated fatty acid metabolism⁵⁰ (Fig. 1A). In prostate cancer patient samples, ACSL3 was shown to be moderately upregulated in early prostate cancer, but ACSL3 expression was reduced during the transition from low-grade to high-grade cancers⁵¹. Conversely, ACSL4’s expression was higher in tumour cells than in surrounding normal epithelial cells, as seen from an immunohistochemical analysis of a human prostate cancer tissue microarray derived from patients across multiple clinicopathological groups and benign prostate tissue⁵². CRPC patient samples also express ACSL4 at higher levels than hormone-naive prostate cancers⁵². The net incorporation of an increased amount of unsaturated fatty acids provides membrane fluidity associated with aggressive cancer cell behaviours such as increased motility and/or proliferation. Interestingly, besides AR, ACSL4 seems to be also a target of E2F. Results from a study in the prostate cancer cell lines LNCaP and PC3 showed that loss of the canonical prostate cancer tumour suppressor RB1 and the resultant E2F activation increased ACSL4 expression and elevated arachidonic acid (AA) and adrenic acid (AdA)–containing phospholipids, which act as primary substrates for lipid peroxidation⁵³. Furthermore, in a PC3 xenograft model and a prostate epithelium-specific Pten and Rb1 double-knockout mouse model, RB1-deficient prostate tumour growth and metastasis were susceptible to ferroptosis induction⁵³. Like in other cancers, high ACSL4 levels were indicated as a mechanism of resistance to drugs such as chemotherapies, tamoxifen, and triacsin C^54–57. Together, these data indicate that in cancers such as prostate cancer, ACSL4 might be upregulated to promote disease progression, but it comes at a cost of increased tumour susceptibility to ferroptosis inducers. This characteristic of prostate cancer can be exploited by combining AR signalling inhibitors with ferroptosis inducers, causing excessive ACSL4-dependent lipid peroxide production and ultimately, ferroptotic cell death.

De novo lipogenesis

Ferroptosis can be regulated by modulating the expression of genes involved in de novo lipogenesis, including essential lipogenic enzymes such as fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC). The irreversible carboxylation of acetyl-CoA to form malonyl-CoA is catalysed by the biotin-dependent ACC, and malonyl-CoA is subsequently converted to long-chain fatty acids by FASN⁵⁸. Stearoyl-CoA desaturase-1 (SCD1) subsequently generates MUFAs from saturated fatty acids (SFAs)(Fig. 1A). Prostate cancer shows high expression of crucial proteins involved in fatty acid synthesis, including ACC and FASN, compared with other cancers. This finding comes from a comparison of 32 cancer types included in The Cancer Genome Atlas PanCan 2018 normalized analysis of primary, treatment-naïve prostate cancers of ~500 men^59,60. Moreover, the expression of ATP citrate lyase (ACLY), ACC, and FASN are higher in prostate cancer patient samples than in the normal prostate healthy controls, indicating an activation of de novo lipogenesis to meet cancer cells’ increased metabolic needs⁵⁹. Androgens are well-known to mediate the expression of these lipogenic enzymes as well as that of SCD1 to fulfil the ever-increasing lipid requirements of prostate cancer cells^40,61,62. An important transcriptional controller of these lipogenic enzymes is the sterol response element-binding protein 1 (SREBP1) transcription factor. Androgens activate the SREBP1 cleavage-activating protein (SCAP), in turn leading to SREBP1 activation^63,64. Active SREBP1 promotes the expression of de novo lipogenesis genes such as FASN⁶⁵. Correspondingly, FASN is a potential biomarker of prostate cancer, with FASN overexpression being associated with a malignant phenotype^66,67. Results from immunohistochemistry analyses showed high expression of FASN in prostate carcinomas, particularly in metastatic cancers^66,67. In fact, FASN expression can be used to predict lymph node metastases regardless of the Gleason score⁶⁸. The effect of FASN and ACC in regulating ferroptosis in the context of prostate cancer is unclear. Studies conducted in other cancer types provide conflicting reports of how FASN and ACC might modulate ferroptosis in cancer. In pancreatic ductal adenocarcinoma cells, system x_c- inhibition (with erastin or sulfasalazine) mediated lipid peroxidation and subsequent ferroptotic death through pyruvate oxidation-dependent fatty acid production. Indeed, the knockdown of FASN or ACC by shRNAs in these cells inhibited erastin-mediated or sulfasalazine-mediated ferroptotic death, whereas the addition of exogenous PUFAs or palmitic acid reversed this phenotype⁶⁹. In another study in mouse embryonic fibroblasts, ferroptosis was inhibited by orlistat, a FASN inhibitor, indicating that ACC1—FASN-mediated lipogenesis was required for ferroptosis in this system⁷⁰. Similarly, adenosine monophosphate-activated protein kinase (AMPK) has been reported to negatively regulate ferroptosis by inhibiting ACC1—FASN-mediated fatty acid synthesis in mouse embryonic fibroblasts and the human renal cancer cell line Caki-1⁷¹. Conversely, in mutant-KRAS lung cancer cell lines, SFAs and MUFAs were shown to be supplied by FASN to the Lands cycle, which is responsible for remodelling oxidized phospholipids such as phosphatidylcholines⁷². Inhibiting the Lands cycle or FASN expression in mutant-KRAS lung cancer cell lines resulted in ferroptosis⁷². Thus, FASN upregulation and the resulting increase in SFA and MUFA can be a defence mechanism against lipid peroxidation. Despite these seemingly contradictory observations, the opposite effects of FASN inhibition on ferroptosis induction could be context-dependent, largely relying on the available pool of peroxidation-prone PUFA or on the ability of cells to take up exogenous PUFAs upon FASN inhibition⁷². Specifically, a potential explanation for FASN-mediated inhibition of ferroptosis⁷⁰ is that FASN inhibition might cause reduction not only in SFA and MUFA lipogenesis but also, potentially, in PUFA biosynthesis, resulting in ferroptosis inhibition. Indeed, FASN inhibition induces adenosine monophosphate buildup, causing AMPK activation,⁷² which could result in the suppression of downstream PUFA biosynthesis⁷¹. Hence, the net effect of these processes on PUFA levels in relation to MUFA and possibly SFA levels is likely to contribute to the regulation of ferroptosis.

AMPK can prevent the production of fatty acids through phosphorylation and the subsequent inhibition of both ACC1 and ACC2 activity under conditions of energy stress⁷³. Inhibition of ACC by small-molecule inhibitors and by AMPK phosphorylation was found to suppress ferroptosis in mouse embryonic fibroblasts⁷¹. Moreover, deletion of AMPK’s alpha catalytic subunits was found to increase the levels of phosphatidylethanolamine (PE) species such as PE 18:0_20:4 and PE 18:0_22:4, which are drivers of ferroptosis⁷¹. Notably, AMPK is frequently activated in human prostate malignancies, and AMPK pharmacological suppression or knockdown was shown to cause prostate cancer cell death and decreased cell growth^74–78. Thus, investigating whether inhibitors of AMPK signalling could synergise with ferroptosis inducers to impede prostate tumour growth will be an interesting area of exploration.

SCD1 produces MUFAs from SFAs by catalysing the formation of a double bond in stearoyl-CoA and palmitoyl-CoA⁷⁹ (Fig. 1A). Prostate cancer patients have a higher MUFA to SFA ratio in the blood compared to healthy controls, suggesting higher SCD1 activity^80–82. SCD1 mRNA and protein levels are higher in human prostate cancer tissue samples than in normal human prostate tissue samples⁸². SCD1 pharmacologic and genetic inhibition suppressed the proliferation of prostate cancer cells both in vitro and in xenograft model⁸². Moreover, SCD1-mediated synthesis of MUFAs has been shown to promote the anti-ferroptosis activity of SREBP1⁸³. This study also demonstrated that PI3K-AKT-mTOR signalling activates downstream SREBP1, which subsequently activates SCD1 to increase ferroptosis-resistant MUFA synthesis and prevent lipid peroxide formation. Thus, through downstream SREBP1-SCD1-facilitated lipogenesis, PI3K-AKT-mTOR signalling could safeguard PTEN (inhibitor of PI3K-AKT signalling and commonly altered tumor suppressor in prostate cancer)-defective prostate cancer cells from oxidative stress and ferroptosis⁸³. Similarly, in ovarian cancer cells, blocking SCD1 induced lipid oxidation and ferroptosis by reducing CoQ10, which increased the anticancer efficacy of ferroptosis inducers⁸⁴.

Collectively, these studies indicate that cancer cells often increase de novo lipogenesis, producing ferroptosis-resistant substrates, such as SFAs and MUFAs, to simultaneously serve their metabolic needs and avoid ROS-induced ferroptotic death. Consequently, inhibitors that target enzymes involved in the de novo lipogenesis pathway could exhibit synergistic effects with ferroptosis inducers, thereby enhancing the efficacy of these inhibitors in inducing cancer cell death.

n-6 de novo PUFA synthesis

The PUFA biosynthesis pathway is another metabolic pathway that contributes to the balance of MUFAs and PUFAs and, ultimately, to ferroptotic sensitivity. Peroxidation of PUFAs is the main driver of ferroptosis, with oxygenated diacyl AA- and AdA-containing species of PE being the most susceptible ferroptosis substrates^4,85. Specifically, exogenous addition of PE-AA-OOH, but not AA-OOH to ACSL4 KO cells increased ferroptosis-mediated cell death⁸⁵, showing an essential role of the oxidative PE pathway in ferroptosis. Thus, these lipid signals are also potential new targets for biomarker and drug discovery. Typically, in the n-6 de novo PUFA synthesis pathway, fatty acid elongase-2 (ELOVL2), fatty acid elongase-5 (ELOVL5), fatty acid desaturase 1 (FADS1), and fatty acid desaturase 2 (FADS2) are used to produce the fatty acids AA and AdA from exogenously sourced linoleic acid^86,87 (Fig. 1A). High expression of ELOVLs and FADS can sensitise gastric cancer cell lines to ferroptotic death, whereas low expression and activity of these proteins are associated with ferroptosis resistance^88,89. In prostate cancer cells, androgen deprivation therapy was shown to amplify PUFA levels, and these levels are even higher in prostate cancer cells expressing neuroendocrine markers^28,90 . The essential PUFA elongation enzyme ELOVL5 is expressed at higher levels in neuroendocrine-like prostate cancer cells compared to adenocarcinoma prostate cells⁹⁰. As a result, PUFAs are upregulated in neuroendocrine-like prostate cancer cells. Increased PUFAs generate greater numbers of lipid raft clusters in the neuroendocrine-like prostate cancer cell membrane, resulting in the induction of AKT-mTOR signalings and subsequent enzalutamide resistance. Despite rendering neuroendocrine-like prostate cancer cells more resistant to enzalutamide, high expression of ELOVL5 can render them more susceptible to ferroptosis inducers due to the rise in PUFAs⁹⁰. These findings are consistent with another study that demonstrated increased AR-mediated activation of ELOVL5 in prostate cancer cells, xenografts, and frozen patients’ radical prostatectomy specimens⁹¹. Here, ELOVL5 was identified as the most commonly overexpressed ELOVL in prostate cancer compared with non-malignant prostate tissues⁹¹. These results suggest that while increased ELOVL5 promotes resistance to enzalutamide, it potentially also creates a therapeutic window for ferroptosis inducers.

β-oxidation

Most studies focus on the role of AR-mediated de novo lipogenesis in prostate cancer, but mounting evidence suggests that fatty acid oxidation (FAO) is also regulated by AR signalling and facilitates the transition to CRPC. Carnitine palmitoyltransferase 1 (CPT-1), specifically the CPT-1A isoform, accelerates the transfer of an acyl group from coenzyme A to L-carnitine, in turn converting acyl-coenzyme As into acyl-carnitines, which can travel across membranes and enter the mitochondria⁹². This reaction is the rate-limiting step of FAO. High-grade neuroendocrine prostate cancer and CRPC tissue samples express higher levels of CPT-1A relative to benign tissues⁹³. The addition of CPT-1A inhibitors such as etomoxir or ranolazine to anti-androgen treatment has been reported to have a synergistic anti-cancer effect in preclinical models of human prostate cancer cell lines⁹³. In a xenograft model using CRPC cell lines, androgen withdrawal increased the susceptibility of CPT-1A knockdown tumours to enzalutamide, while enhancing tumour growth of CPT-1A-overexpressing CRPC cell lines⁹⁴. Another enzyme involved in FAO is DECR1, which catalyses the β-oxidation of PUFAs in the mitochondria. High expression levels of DECR1 were shown to increase prostate cancer cell proliferation as well as resistance to ferroptosis and to antiandrogen therapies⁹⁵. Inhibition of DECR1 in human prostate cancer cell lines induced the accumulation of PUFAs in phospholipids and enhanced mitochondrial ROS, the formation of lipid peroxides, and, ultimately, ferroptosis⁹⁵ (Fig. 1A). Together, enzymes participating in the FAO pathway (such as CPT-1A and DECR1) appear to suppress ferroptosis. One caveat is that the inhibition of β-oxidation might lower ROS production by reducing the amount of acetyl-CoA being channelled to the TCA cycle and the subsequent formation of superoxide anions generated through electron transfer. Nevertheless, ferroptosis inducers might generate enough ROS to overcome this suppression of ROS production. Prostate cancer is often characterised by increased β-oxidation; thus, a combination treatment strategy involving the inhibition of β-oxidation might further enhance the efficacy of ferroptosis inducers in some prostate cancer subtypes.

Iron metabolism and ferroptosis

Cells with elevated free (non-protein bound) iron are particularly susceptible to ferroptosis. Alterations in the transferrin receptor 1 (TfR1), poly(rC)-binding protein 1 and 2 (PCBP1 and PCBP2), and ferroportin systems have been associated with increased levels of iron accumulation within the cell (Fig. 1B). For example, when TfR1 is overexpressed, an increased amount of iron is transported into the cell, indicating that TfR1 is a positive receptor of ferroptosis⁹⁶. After iron is imported through TfR1, PCBP1 and PCBP2 iron chaperones assist the labile iron to bond together and become a stored form of iron called ferritin⁹⁷. PCBP2 also directly interacts with ferroportin, transferring cytoplasmic iron to ferroportin for export outside of cells⁹⁸. Faulty PCBP1 and PCBP2 protein cannot form storage ferritin, leading to an increase in intracellular free iron species. Moreover, a defect in this system halts the removal of iron⁹⁹. Cytosolic iron can also increase through other mechanisms^100–102, but the aberrant expression and activity of these three proteins (PCBP1, PCBP2 and TfR1) are most commonly responsible for iron accumulation.

In prostate cancer, iron is one of the micronutrients essential for proliferation and metastasis. Many enzymes in prostate cancer cells use iron to regulate the transcriptional activity of AR, the central driver of prostate cancer, as well as to modulate other intracellular enzymatic activities that promote the production of energy, DNA synthesis, and heme biosynthesis¹⁰³. Accordingly, iron deficiency slows cancer cell growth, whereas iron-rich environments promote growth^104,105. Consistent with a pro-cancer role for iron, iron-responsive element-binding proteins and TfRs are upregulated not only in various prostate cancer cell lines, but also in the sera of prostate cancer patients, whereas the iron-exporter ferroportin is downregulated to preserve the labile iron pool^106–109. The six-transmembrane epithelial antigen of the prostate-2 (STEAP2), an iron reductase enzyme that has a crucial role in reducing iron within cells has also been shown to be overexpressed in prostate cancer cell lines and enhance cell growth, movement, and invasiveness, as well as activate pro-metastatic genes such as IL1β and MMP7¹¹⁰. However, a surplus of iron can increase ROS, which can trigger mitochondrial dysfunction, excessive lipid peroxidation, cellular damage, and ferroptotic cell death^111,112. Iron in its redox-active state (Fe²⁺) contributes directly to ROS production by participating in the Fenton reaction¹¹², (Fig. 1B) as well as indirectly by modulating iron-dependent enzymes involved in ROS production, such as cytochrome P450 enzymes, NADPH oxidases, and lipoxygenases^113–115. Overall, this dependence on iron for metabolic needs renders prostate cancer highly susceptible to iron overload-mediated ferroptosis.

Iron overload has been identified as a potential promising treatment strategy in multiple disease models. In multiple myeloma cell lines, administering excess iron with the proteasome inhibitor bortezomib- a commonly used therapy for both newly diagnosed and relapsed multiple myeloma patients- increased cell death, whereas lowering ferritin expression through gene silencing mitigated bortezomib resistance, indicating that iron toxicity and the concomitant excess ROS production could be utilized to restrict tumour growth ^116,117. Several prostate cancer cell lines including VCaP, LNCaP, and TRAMP-C2 are particularly susceptible to iron-induced oxidative damage and ferroptosis¹¹⁸. High dosage of iron in the form of iron-dextran and ferric carboxymaltose suppressed tumour growth in mice subcutaneously injected with VCaP and TRAMP-C2 cells, and synergised with bicalutamide to decrease the growth of a castration-resistant PC3 xenograft tumour mouse model, suggesting promise for targeting iron excess even in AR-indifferent prostate cancer¹¹⁸. Results from a study in which the human prostate cancer cell line DU145 and the TRAMP mouse model of prostate cancer were used showed that iron supplementation in the form of ferric ammonium citrate or iron dextran substantially induced RSL3-triggered lipid peroxidation, mitochondrial ROS, and ferroptotic cell death¹¹⁹. Furthermore, enzalutamide enhanced the potency of the RSL3 plus iron combination, resulting in an amplified anti-cancer effect that prevented the onset of CRPC in TRAMP mice, indicating that ferroptosis induction through iron overload can enhance the efficacy of current anti-androgen therapies¹¹⁹.

Iron-based nanoparticles encapsulated in hydrogen peroxide (H₂O₂), which are iron transporters that accumulate in tumours and catalyse Fenton reactions to generate lethal levels of hydroperoxides, are another potential method to induce ferroptosis in cancer cells¹²⁰. For instance, newly developed iron oxide nanoparticles coated with gallic acid and polyacrylic acid (IONP-GA/PAA) were shown to have an inherent cytotoxic effect against numerous glioblastoma, neuroblastoma, and fibrosarcoma cell lines by inducing ferroptosis¹²¹. The ferroptotic effect of IONP-GA/PAA was reversed by conventional ferroptosis inhibitors such as deferoxamine, ciclopirox olamine, and ferrostatin-1, indicating that the ROS-induced ferroptotic cell death is specifically due to the activity of iron derived from IONP-GA/PAA¹²¹. However, little is known about the therapeutic potential of iron oxide nanoparticles in the induction of ferroptosis in prostate cancer. To date, most studies have predominantly focused on the role of these nanoparticles in magnetic resonance imaging (MRI) and chemotherapy delivery to target sites, with little attention paid to combination therapy opportunities that could trigger ferroptosis^122–124.

Glutamate, cystine and GSH metabolism

In addition to promoting lipid metabolism, AR signalling is essential for orchestrating the increased uptake of amino acids that regulate ferroptosis.

Glutamine, the most prevalent amino acid in the human body, is a crucial reservoir of reduced nitrogen for biosynthetic pathways, and also functions as a source of carbon to supply the TCA cycle, synthesize GSH, and produce nucleotides, proteins, and lipids¹²⁵. Glutamine metabolism was shown to affect ferroptotic cell death and influence the vulnerability of prostate malignancies to ferroptosis (Fig. 1C). Mechanistically, glutamine is transported into the cell predominantly through the alanine serine cysteine transporter 2 (ASCT2)— a neutral amino acid transporter belonging to the solute carrier 1 family (SLC1) of proteins —and converted to glutamate, which subsequently reacts with intracellular cysteine and glycine, as mediated by the enzymes y-glutamyl-cysteine (y-GCS) and GSH synthase, to generate GSH and prevent lipid peroxidation¹⁴. Upon inhibition of GSH production through cystine deprivation, excess imported glutamine is channelled into glutaminolysis, a process by which cells convert glutamine into TCA cycle metabolites, instead of into GSH production, creating an additional source of ROS that promotes ferroptosis¹²⁶. High levels of extracellular glutamine alone cannot cause ferroptosis. Only when L-glutamine is available and combined with cystine deprivation can ferroptosis be induced. Similarly, cysteine deprivation or blocking cystine entry alone could not cause ROS buildup, lipid peroxidation, or ferroptosis in conditions of glutamine deficiency or impaired glutaminolysis⁹⁶.

Glutamine metabolism could be a crucial metabolic pathway determining prostate cancer cell fate. For instance, ASCT1 and ASCT2 are upregulated by AR signalling, promoting glutamine absorption (directly and possibly indirectly through altering alternative glutamine uptake mechanisms) and metabolic assimilation¹²⁷. Thus, inhibition of these transporters could slow the growth of tumours in xenograft mouse models of human prostate cancer cell lines by preventing cancer cells from absorbing glutamine, which is an essential source of carbon and nitrogen for macromolecule synthesis and anapleurosis (refilling depleted TCA cycle intermediates)^127,128. Whether these pro-cancer effects of ASCT1 and ASCT2 can be attributed to their impact on ferroptosis is unknown. Moreover, 5α-dihydrotestosterone was found to increase the levels of ASCT2 and also glutaminase in LNCaP and VCaP human prostate cancer cells¹²⁷. Invasive CRPC cell lines such as DU145 and PC3 were shown to express higher basal levels of glutaminase than the less invasive cell line LNCaP cells¹²⁹. This high dependence of prostate cancer cells on glutamine for growth can be exploited to promote ferroptosis by inhibiting GSH production with system x_c- inhibitors or cystine deprivation, in turn channelling an increased amount of glutamate into the TCA cycle to accumulate excess ROS that can induce lipid peroxidation.

Cysteine has various central roles in the metabolic reprogramming of cancer cells. Following import through system x_c-, cystine is converted to cysteine, which serves as a precursor for GSH production through y-GCS (Fig. 1C). Cysteine is also a substrate for one-carbon metabolism, the TCA cycle, and fatty acid synthesis¹³⁰. Cysteine metabolism has been shown to be involved in ferroptosis^131,132 . In this Perspective, we primarily focus on the relevance of cysteine in relation to GSH synthesis concerning prostate cancer. System x_c- expression was shown to be elevated in tissue samples obtained from patients who underwent radical prostatectomy, in patient-derived xenograft models derived from metastatic prostate cancer, as well as in the metastatic stroma of prostate cancer, and is positively correlated with poor prognosis^29,133. In AR-negative DU145 and PC-3 prostate cancer cells, extracellular cystine import is crucial for cell growth¹³⁴. This evidence suggests that prostate cancer cells are highly dependent on cysteine metabolism and GSH production as a ROS scavenging mechanism to cope with metabolic stresses. Hence, targeting the system x_c- and cysteine metabolic pathways to induce ferroptosis might be effective in the treatment of prostate cancer. Indeed, in one study, a human cystathionine-lyase termed Cyst(e)inase that can reduce both cystine and cysteine levels was developed with substantial effectiveness in depleting GSH in human prostate cancer cell lines PC3, DU145 and the murine prostate cancer cell line HMVP2¹³⁵. The use of Cyst(e)inase in such cell lines resulted in G₀/G₁ arrest. This method improved survival in a chronic lymphocytic leukaemia mouse model, and also inhibited the development of prostate (AR-negative) and breast cancer xenografts, highlighting the therapeutic potential for inhibitors of the cysteine metabolic pathway¹³⁵.

In another study, the direct regulation of GSH metabolism by Rb1 loss-induced E2F1 transcriptional activity was shown¹³⁶. This GSH synthesis pathway has been therapeutically exploited by testing isogenic Rb1-depleted models of CRPC cell lines with the system x_c- (encoded by SLC7A11) inhibitor erastin and the y-GCS inhibitor buthionine sulfoximine¹³⁶. These inhibitors were shown to have increased cytotoxicity in Rb1-knockdown as compared to control prostate cancer cells, suggesting a potential novel treatment approach for RB1-deficient tumours¹³⁶. Moreover, in a prostate cancer xenograft model, sulfasalazine suppressed tumour development by causing cystine deficiency and subsequent ferroptotic cell death¹³⁴. These data suggest that further investigation is warranted to determine whether cystine deprivation-induced tumour regression is driven primarily by ferroptosis. Notably, sulfasalazine is a US Food and Drug Administration (FDA)-approved anti-inflammatory medication that is frequently prescribed in clinical settings to manage ulcerative colitis or rheumatoid arthritis^137,138. Thus, sulfasalazine, differently from other ferroptosis inducers that have not entered clinical trials, could be rapidly repurposed for new prostate cancer trials.

Ferroptosis inducers in treatment combinations

The available treatment options for advanced prostate cancer include AR-signalling inhibition, chemotherapy, immunotherapy and radiotherapy (Fig. 2). Due to the synthetic lethality exhibited between PARP inhibition and BRCA mutations, the discovery of alterations in BRCA2 and other homologous recombination repair-related genes in advanced prostate cancer has also led to the approval of PARP inhibitors for the treatment of metastatic CRPC. Crosstalk likely exists between current prostate cancer therapies and ferroptosis. Thus, it is anticipated that combination therapies including ferroptosis inducers could enhance the effectiveness of existing treatments (Table 1).

Fig. 2.

Combination of ferroptosis inducers with standard-of-care therapies in prostate cancer

Combining ferroptosis inducers with current therapies can potentially overcome drug resistance in advanced prostate cancer. a) Ferroptosis inducers such as erastin and RAS-selective lethal 3 (RSL3) have been shown to increase cancer cells’ susceptibility to taxane-based chemotherapies in prostate cancer models^30,139. Suppression of prostate cancer-associated transcript 1 (PCAT1) or upregulation of ChaC glutathione specific gamma-glutamylcyclotransferase 1 (CHAC1) could increase cell sensitivity to ferroptosis. As a result, PCAT1 and CHAC1 could be potential biomarkers used to select for patients for combination therapy with docetaxel plus ferroptosis inducers to overcome docetaxel-resistant prostate cancer. b) Combination of androgen receptor (AR) signalling inhibitors with ferroptosis inducers could prevent the proliferation and metastasis of castration-resistant prostate cancer (CRPC) cell lines and xenograft mouse models^28,29. Treatment with AR antagonists remodels prostate cancer cells’ lipid composition towards higher PUFA accumulation, thus rendering them susceptible to lipid peroxidation and more dependent on lipid peroxide repair pathway to remove toxic lipid peroxides. AR, by itself, also positively regulates ferroptosis inhibitors such as SLC7A11, SLC3A2, GPX4, MBOAT2, ACSL3 while negatively regulating ferroptosis promoters such as DECR1 and ACSL4^{50,95,211–214}. Thus, inhibiting AR can further push prostate cancer cells towards a ferroptosis-prone state, thereby justifying the rationale for combining ferroptosis inducers with AR inhibition. c) Ferroptosis induction can enhance anti-tumour immunity and help overcome immunotherapy resistance. Combining ferroptosis inducers with immune checkpoint blockade has been shown to enhance antitumor immunity. More specifically, in ovarian cancer models, combining the ferroptosis inducer Cyst(e)inase with anti-PDL1 synergistically promoted T cell-mediated antitumor immunity, triggering ferroptosis in cancer cells, and slowing down tumour progression in vivo¹⁴². Moreover, CD8+ T cells activated through immunotherapy can mediate cancer-killing effects through ferroptosis induction. Activated CD8+ T cells release interferon-gamma (IFN-γ) upon PDL1 blockade, which via JAK1-STAT1 signalling, downregulates SLC7A11 expression in tumour cells²⁴⁷. This results in reduced cystine uptake, increased lipid peroxidation, and subsequent ferroptosis. IFN-γ from CD8+ T cells also modifies lipid metabolism in cancer cells by activating the long-chain acyl-CoA synthetase 4 (ACSL4), thus promoting the activation of ferroptosis-prone PUFAs and increasing tumour cell sensitivity to ferroptosis²⁴⁷. Lastly, stimulating ferroptosis also triggers the release of damage-associated molecular patterns (DAMPs) such as HMGB1 and ATP, as well as promotes the expression of cell surface-exposed calreticulin (CRT), subsequently initiating immune cell responses such as the activation of CD8+ T cells and macrophages^249,250,316. Future efforts should be focused on increasing ferroptosis sensitivity only in cancer cells, sparing activated T cells. d) Little is known about radiotherapy and ferroptosis induction in advanced prostate cancer. Results from studies conducted in other cancers showed that ionizing radiation could elevate ROS production and, subsequently, lipid peroxidation. Radiation could also upregulate ACSL4 expression while suppressing solute carrier family 7 member 11 (SLC7A11) and glutathione (GSH) synthesis, causing a decrease in GSH and ultimately promoting ferroptosis induction^192,266. A combination of radiotherapy and the PARP inhibitor niraparib has been shown to synergistically amplify the cytoplasmic accumulation of double-stranded DNA and thus activate cyclic GMP-AMP synthase (cGAS)-mediated ferroptosis in an in vivo model of colorectal cancer²⁶⁸. Ferroptosis inducers, thus, could enhance lipid peroxidation and ROS burden in cancer cells, subsequently re-sensitizing therapy-resistant cancer cells to PARPi + radiation-induced ferroptosis. APC, antigen-presenting cells; cGAS, cyclic GMP-AMP synthase ; CHAC1, ChaC glutathione specific gamma-glutamylcyclotransferase 1; CRT, calreticulin; DAMPs, damage-associated molecular pattern; DECR1, 2,4 Dienoyl-CoA reductase; HMGB1, high mobility group box 1; HnRNP L, heterogeneous nuclear ribonucleoprotein L; IFN-γ, interferon-Gamma; IFNR, interferon receptor; IRF1, interferon regulatory factor 1; JAK, Janus kinase; LRP, LDL-receptor-related protein; MBOAT2, membrane bound O-acyltransferase domain containing 2; MHC II, major histocompatibility complex Class II; MUFA, monounsaturated fatty acids; PD-1, programmed cell death protein 1; PDL1, Programmed death-ligand 1; PE-MUFA, phosphatidylethanolamine containing MUFA; PE-PUFA, phosphatidylethanolamine containing polyunsaturated fatty acids; PARPi, poly (ADP-ribose) polymerase inhibitors; IR, ionizing radiation; PUFA-PLs, phospholipids containing polyunsaturated fatty acids; STAT1, signal transducer and activator of transcription 1; TCR, T cell receptor; TLR, toll-like receptors.

Table 1:

Ferroptosis inducers available for combination treatments with standard-of-care cancer therapies

Ferroptosis inducer	Mechanism and/or targets	Cancer type	Combination therapy	Models	Results	References
Erastin	System xc- inhibitor	Prostate; Other	Chemotherapy Hormone therapy Radiotherapy Immunotherapy	Cell lines, mouse models, patient samples	Combination with chemotherapy inhibited prostate cancer cell growth. Combination with hormone therapy decreased prostate cancer cell growth and migration. Combination with radiotherapy increased cancer cell death and decreased tumour growth. Combination with immunotherapy inhibited tumour growth.	28–30, 69,139–142, 143,144
Imidazole ketone erastin (IKE)	System xc- inhibitor	Other	Radiotherapy	Cell lines	Combination with radiotherapy increased cancer cell death.	145
Sorafenib	System xc- inhibitor	Prostate; Other	Chemotherapy Hormone therapy Radiotherapy	Cell lines, mouse models	Combination with chemotherapy increased cancer cell death and decreased tumour growth. Combination with hormone therapy decreased PTEN-deficient prostate cancer cell growth. Combination with radiotherapy inhibited tumour growth.	145–148
Sulfasalazine	System xc- inhibitor	Prostate; Other	Chemotherapy Radiotherapy	Cell lines	Combination with chemotherapy decreased cancer cell growth. Combination with radiotherapy decreased cancer cell and tumour growth.	134,149–151
Flubenzadole	Induces p53 expression and transcriptional inhibition of SLC7A11	Prostate; Other	Chemotherapy	Cell lines, mouse models	Combination with chemotherapy increased cancer cell death and prostate xenograft tumour growth.	152,153
RSL3	GPX4 inhibitor	Prostate; Other	Chemotherapy Hormone therapy	Cell lines, mouse models	Combination with chemotherapy induced cell cycle arrest and cell death in prostate cancer cells and inhibited tumour progression. Combination with hormone therapy decreased prostate cancer cell growth and migration.	28,29,119,154,155
ML210	GPX4 inhibitor	Prostate; Other	NA	Cell lines	Increased cell death in various cancer cell lines, including prostate cancer cells.	140,156–158
ML162	GPX4 inhibitor	Other	Radiotherapy	Cell lines	Increased lipid peroxidation and cancer cell line sensitivity to radiation therapy.	26,140
JKE-1674	GPX4 inhibitor	Prostate; Other	NA	Cell lines, mouse models	Decreased cancer cell viability, including prostate cancer cells. Inhibited growth of RB-1 deficient prostate tumours and improved survival of mice.	53,140,156
Artesunate	GPX4 inhibitor	Other	Chemotherapy therapy Radiotherapy	Cell lines, mouse models, patient samples	Combination with chemotherapy increased short-term survival in cancer patients. Combination with radiotherapy decreased increased cancer cell death and xenograft tumour formation in mice.	159–162
FIN56	GPX4 degrader and indirectly depletes CoQ10	Other	Radiotherapy	Cell lines	Combination with radiotherapy induced lipid peroxidation and radiosensitivity in cancer cells.	26,163
Withaferin A	GPX4 inhibitor, increases iron levels and induces lipid peroxidation	Prostate; Other	Chemotherapy therapy Radiotherapy Immunotherapy	Cell lines, mouse models	Combination with chemotherapy decreased cancer cell viability, migration, and invasion. Combination with radiotherapy increased cancer cell death. Combination with immunotherapy increased immune cell activation and decreased tumour growth in mice.	164–168
Altretamine	GPX4 inhibitor	Other	Chemotherapy	Cell lines, patient samples	Combination with chemotherapy increased survival in patients.	169–172
Buthionine sulfoximine (BSO)	GCLC inhibitor, GSH depletion	Prostate	Chemotherapy Hormone therapy Radiotherapy	Cell lines, mouse models	Combination with chemotherapy inhibited cancer cell viability. Combination with hormone therapy decreased prostate cancer cell viability. Treatment with BSO increased sensitivity to radiotherapy in mice.	136,173–175
Cisplatin	ROS production, GSH depletion	Other	NA	Cell lines	Decreased cancer cell line viability.	176
Salinomycin (HSB-1216)	Induces iron sequestration in lysosomes and ROS production	Other	NA	Cell lines	Decreased cancer cell line viability.	177–180
Iron oxide nanoparticles (IONP-GA/PAA)	Induces lipid peroxidation	Other	NA	Cell lines	Decreased cancer cell line viability.	121
Artemisinin	The endoperoxide bridge in the compound’s structure reacts with endogenous iron; ROS production	Prostate; Other	Chemotherapy Radiotherapy	Cell lines	Decreased cancer cell line viability, including prostate cancer cells. Combination with chemotherapy decreased cancer cell survival, migration, and invasion. Increases sensitivity of cancer cell lines to radiotherapy.	181–184
Dihydroartemisinin (DHA)	The endoperoxide bridge in the compound’s structure reacts with endogenous iron; ROS production	Prostate; Other	Chemotherapy Radiotherapy Immunotherapy	Cell lines, mouse models	Reduced cancer cell line viability, including prostate cancer cells. Combination with chemotherapy decreased cancer cell line proliferation and tumour formation in mice. Combination with radiotherapy increased cancer cell line death. Combination with immunotherapy increased intratumoral immune cell infiltration and decreased tumour volume in mice.	185–188
FINO2	Oxidizes iron and induces lipid peroxidation	Other	NA	Cell lines	Increased cancer cell death.	189,190
Cyst(e)inase	Depletes extracellular cystine	Prostate; Other	Immunotherapy	Mouse models	Combination with immunotherapy inhibited tumour growth.	135,142,172,191–193

NA, not available.

Ferroptosis inducers and chemotherapy

The most common chemotherapy for CRPC is docetaxel, which is a derivative of paclitaxel and, therefore, is classified as a second-generation member of the taxane family (Fig. 2a). Docetaxel works by binding and disrupting cytoskeletal microtubules during the G2 and M phases of the cell cycle, in turn impeding the normal assembly of the mitotic spindle, and preventing the completion of mitosis and proper cell proliferation, eventually resulting in cell death¹⁹⁴. Docetaxel was also shown to inhibit the expression of the anti-apoptotic protein BCL2, increasing cancer cells vulnerability to apoptosis induction¹⁹⁵. In other studies, docetaxel was shown to prevent nuclear localization of AR in a microtubule-dependent manner¹⁹⁶. Nevertheless, many patients inevitably acquire resistance and develop docetaxel-refractory CRPC. Thus, cabazitaxel, a distinct, third-generation taxane was authorized in 2010 by the FDA as a second-line treatment for metastatic CRPC^197,198. Cabazitaxel exerts anticancer efficacy in docetaxel-resistant malignancies as well as post-docetaxel and chemotherapy-naive settings.

In a study in 22Rv1 and LNCaP95 cells, combination treatment of erastin plus docetaxel consistently outperformed the respective monotherapies in decreasing cancer cell growth³⁰. Similarly, the ferroptosis inducers RSL3 and erastin increased docetaxel susceptibility in previously docetaxel-resistant prostate cancer cell lines¹³⁹. Interestingly, the efficacy of these treatment combinations in subcutaneous xenograft tumour models of docetaxel-resistant prostate cancer cell lines was only maintained with RSL3, and not with erastin¹³⁹. No mechanism for the therapeutic efficacy of this combination treatment approach was provided in either of these studies, and further research is needed to determine the effectiveness and elucidate the mechanisms of this combined approach.

Docetaxel-resistant prostate cancer cells were often shown to be insensitive to ferroptosis; some mechanisms that could explain this observation have been proposed. For instance, prostate cancer-associated transcript 1 (PCAT1), which is substantially elevated in both docetaxel-resistant clinical samples obtained from prostate cancer patients subjected to radical prostatectomy and prostate cancer cell lines¹⁹⁹, was shown to suppress ferroptosis by interacting with c-MYC to maintain c-MYC stability and promote downstream SLC7A11 expression. Deletion of PCAT1 augmented ferroptosis and prostate cancer’s susceptibility to docetaxel treatment, as seen with reduction in cell viability and colony formation upon docetaxel exposure, whereas PCAT1 overexpression reversed these effects¹⁹⁹ . In another study, the expression of ChaC glutathione-specific γ-glutamylcyclotransferase 1 (CHAC1) was shown to be lower in prostate cancer cell lines, DU145 and 22RV1, than in non-transformed human prostate epithelial RWPE-1 cells²⁰⁰. CHAC1’s activity can reduce intracellular GSH levels²⁰¹; thus, overexpressing CHAC1 in DU145 and 22RV1 cells resulted in increased intracellular lipid peroxides due to the lack of the active antioxidant GSH, and sensitised prostate cancer cells not only to ferroptosis induction by erastin, but also to docetaxel²⁰⁰. The study demonstrates that this sensitization to docetaxel was dependent on ferroptosis as the use of a ferroptosis inhibitor negated the docetaxel sensitivity of prostate cancer cells with CHAC1 overexpression²⁰⁰. Thus, PCAT1 and CHAC1 could be used as potential biomarkers to select the right patient population suitable for combined treatment with docetaxel and ferroptosis inducers. Patients with docetaxel-resistant prostate cancer, especially those with increased PCAT1 and suppressed CHAC1, might be candidates for this combined approach. To date, combination treatments with ferroptosis inducers and cabazitaxel have not been reported.

Ferroptosis inducers and hormone therapy

Increased or sustained AR signalling remains the principal mechanism underpinning CRPC progression^202–204. Underlying mechanisms include AR gene or enhancer amplifications, de novo androgen synthesis by cancer cells, and the production of various splice variants missing the ligand-binding domain²⁰⁵. First-line CRPC treatments most often involve second-generation AR signalling inhibitors such as the androgen biosynthesis inhibitor abiraterone or the AR antagonists enzalutamide, apalutamide, and darolutamide^206,207. Co-inhibition of AR and PARP was shown to benefit genomically selected patients with prostate cancer harbouring specific mutations in homologous recombination-repair genes^208,209. PARP inhibitors can downregulate the expression of SLC7A11 in a p53-dependent manner, indicating a potential role of PARP inhibitors in ferroptosis induction²¹⁰. The mechanistic understanding of how these second-generation anti-androgens and PARP inhibitors could enhance the efficacy of ferroptosis inducers or vice versa in treating CRPCs is still preliminary.

AR can strengthen cells’ oxidative resistance mechanisms by directly binding to the promoter regions and promoting the expression of antioxidant-defence genes such as GPX4, SLC7A11, and SLC3A2 ^211–213; thus, suppressing AR activity using antiandrogens has inherent ferroptosis-induction capability and could also synergise with ferroptosis inducers. In LNCaP cells, a substantial lipid remodelling with elevated levels of desaturation and acyl chain lengthening of membrane lipids was shown upon treatment with enzalutamide²⁸. This enzalutamide-induced remodelling raised membrane PUFA content, increasing membrane fluidity and lipid peroxidation, which in turn led to increased susceptibility to GPX4 inhibition and ferroptosis²⁸ (Fig. 2b). In another study in C4-2 adeno-CRPC cells, which express AR and are sensitive to anti-androgens, erastin or RSL3 in combination with either enzalutamide or abiraterone substantially decreased colony formation, migration, and invasion compared with erastin or RSL3 alone²⁹. In this study, erastin or RSL3 in combination with enzalutamide also effectively reduced tumour development in vivo in C4-2 xenografts as compared to single agents without having discernible effects on body weight²⁹. Altogether, the combined approach using second-generation anti-androgens and ferroptosis inducers appears promising for the treatment of adeno-CRPC prostate cancer²⁹. Lastly, compelling evidence supporting the use of ferroptosis inducers with second-generation antiandrogens for the treatment of CRPC was presented in a study in which ferroptosis regulation was shown to occur independently of GPX4 in prostate (and breast) cancers²¹⁴. Specifically, the enzyme membrane bound O-acyltransferase domain containing 2 (MBOAT2) — which selectively transfers MUFAs to the Sn2 position of lysophosphatidylethanolamine (lyso-PE), lysophosphatidylcholine (lyso-PC), and lysophosphatidic acid (lyso-PA)^215,216—was identified as a suppressor of ferroptosis through phospholipid modification independently of GPX4 or FSP1²¹⁴ MBOAT2 was hypothesized to compete for lyso-PE with PUFA-preferred lysophosphatidylethanolamine acyltransferase (such as LPCAT3). The study showed that by overexpressing MBOAT2 in the fibrosarcoma cell line HT-1080, PE-PUFAs, and particularly PE-AAs, were decreased while PE-MUFAs, and particularly PE-OAs (18:1), were simultaneously increased. Thus, in cells or tissues with elevated MBOAT2 activity, PE remodelling could move the cells towards a state of high PE-MUFA but reduced PE-PUFA, resulting in ferroptosis resistance. Importantly, the study also showed that MBOAT2 is transcriptionally upregulated by AR and its pioneering factor FOXA1 in both human prostate cancer samples and in human prostate cancer cell lines²¹⁴ (Fig. 2b). The combination of an AR antagonist (such as enzalutamide) with ferroptosis induction (RSL3) showed a substantial growth inhibition effect in AR-positive prostate cancer cell lines, and in prostate cancer cells resistant to single-agent hormonal therapies²¹⁴. These results indicate that ferroptosis induction, either alone or in conjunction with AR-targeting agents, might constitute a promising treatment approach for advanced prostate cancer.

Ferroptosis inducers and immunotherapy

Immunotherapy has revolutionised cancer treatment since the FDA approval of the first immune checkpoint inhibitor (ICI) in 2011²¹⁷. In prostate cancer, the launch of sipuleucel-T (Fig. 2c), an autologous active cellular immunotherapy approved for men with asymptomatic or minimally symptomatic mCRPC, and ICIs for men with microsatellite instability-high (MSI-H) disease offered additional therapeutic options for this subset of patients. However, to date, immunotherapy has had limited overall success in prostate cancer owing to the existence of a strong immunosuppressive tumour microenvironment (TME) preventing the infiltration of T cells, a substantial heterogeneity in genomic alterations, and low expression of PDL1²¹⁸. Nevertheless, multiple clinical trials have been carried out to investigate the efficacy of immunotherapy as a single-agent therapy and in conjunction with other non-immunotherapies. Current classes of immunotherapies being tested in advanced prostate cancer include: ICIs that target CTLA-4 (such as ipilimumab^219–221) or PD1 and PD-L1 (such as nivolumab^222–225, pembrolizumab^225,226, atezolizumab^227,228, avelumab^229,230, and durvalumab²³¹); vaccine-based therapies including sipuleucel-T²³², DNA vaccines^233,234, PSA-TRICOM vaccine (Prostvac)^235,236, and GVAX^237–239; adoptive cell therapy such as the prostate-specific membrane antigen (PSMA) CAR T cell therapy^240–242 and tumour-infiltrating lymphocyte (TIL) therapy^243,244; and bispecific T cell engager (BiTE) antibodies that target PSMA such as AMG 212 (pasotuxizumab)^245,246.

Interestingly, ferroptosis has been shown to enhance anti-tumour immunity and help overcome immunotherapy resistance in melanoma, fibrosarcoma, and glioma^{142,247–250}. In one study in glioma cells, early ferroptotic cells were shown to secrete danger-associated molecular patterns (DAMPs) (such as HMGB1 and ATP), resulting in the maturation of dendritic cells and the subsequent induction of cytotoxic T cell-mediated adaptive immunity^249,250. In the context of prostate cancer, calreticulin, which is another type of DAMPs , was found to be exposed on transgenic adenocarcinoma of the mouse prostate TRAMP-C1 cancer cells after treatment with vesicles containing ascorbic acid and ferroptosis-inducing iron oxide nanocubes²⁵¹. Prostate cancer is characterised by poor infiltration of CD8+ T cells^141,252; thus, inducing ferroptosis in prostate cancer cells could improve cytotoxic T cell-mediated adaptive immunity and become an attractive approach to improve the efficacy of immunotherapy. Nonetheless, caution should be exerted with the use of ferroptosis inducers, as results from previous studies showed that T cells are also susceptible to ferroptosis²⁵³. For example, CD36-mediated uptake of AA from the TME by CD8+ T cells could trigger T cell ferroptosis²⁵⁴. Thus, efforts aimed at inducing ferroptosis in combination with immunotherapies would necessitate the discovery of novel mechanisms that selectively promote ferroptosis in cancer cells, sparing CD8+ T cells.

In other studies, immune cells were shown to induce ferroptosis in tumour cells. CD8+ T cells can induce ferroptosis in ovarian tumour cells by secreting interferon gamma (IFNγ) into the TME, suppressing the expression of SLC3A2 and SLC7A11 in tumour cells, and limiting cystine import and tumour cells’ ability to remove lipid peroxides¹⁴². Thus, combining ferroptosis inducers such as Cyst(e)inases with anti-PD-L1 antibody could synergistically amplify T cell-mediated anti-tumour immunity¹⁴².

IFNγ and dietary AA was shown to promote ACSL4-dependent tumour ferroptosis through the JAK—STAT1—IRF1 signalling pathway in both mouse and human melanoma cell lines²⁴⁷. Consequently, in syngeneic mouse models bearing murine colon cancer MC38 or murine melanoma Yumm5.2 cells, combination therapy including low-dose AA and anti-PD-L1 yielded maximal tumour growth inhibition compared to single-agent therapies of AA or anti-PD-L1 alone²⁴⁷. Notably, in this study, AA alone could increase the population of IFNγ+, TNFα+, and granzyme B+ CD8+ T cells in the TME, indicating that AA not only reduces tumour cell growth but also increases the recruitment and activities of immune cells²⁴⁷.

In the context of prostate cancer, deletion of the heterogeneous ribonucleoprotein L (HnRNP L) in human CRPC cell lines suppressed the expression of CD274 (encoding PD-L1) by inhibiting the transcription factor YY1²⁵⁵. As a result, in the RM-1 syngeneic mouse model, knockdown of HnRNP L and the concomitant reduction in PD-L1 led to CD4+ and CD8+ T cell tumour infiltration, preventing immune escape. Active T cells can in turn trigger ferroptosis in prostate cancer cells and suppress prostate cancer tumour growth. This study posits the importance of inhibiting HnRNP L to stimulate T cell-mediated ferroptosis, thus improving the efficacy of existing immunotherapy²⁵⁵. Nevertheless, the study did not further explore the potential for combining ferroptosis inducers with anti-PDL1 blockade in prostate cancer. It is possible that the use of ferroptosis inducers could synergistically potentiate the tumour-killing effect of anti-PDL1 blockade. Aside from these studies, our knowledge of the effects of combined ferroptosis inducers with immunotherapy for advanced prostate cancer is scarce. Thus, the potential therapeutic benefits of this combination remain underexplored.

Ferroptosis inducers, radiotherapy, and other DNA-damaging agents

Current systemic radiotherapy treatment regimens for advanced prostate cancer include the use of radium-223 dichloride (radium-223), an α-emitting radionuclide, to specifically target bone metastases, or molecularly targeting agents such as Lutetium-177-prostate-specific membrane antigen-617 (¹⁷⁷Lu-PSMA-617) (Fig. 2d). Radium-223 is an intravenously injected salt that mimics calcium, binding to hydroxyapatite and assimilating into bone areas where active mineralization occurs^256,257. In 2022, the FDA approved ¹⁷⁷Lu-PSMA-617 as the first PSMA-targeted radioligand therapy for the treatment of patients with PSMA-positive metastatic CRPC resistant to anti-androgen drugs and chemotherapy^258–260. Additional PSMA-targeting radioisotopes are currently being intensely investigated for the treatment of CRPCs, such as ²¹³Bi-PSMA-617²⁶¹, ²²⁷Th-PSMA-IgG1²⁶², and ²¹¹At-PSMA-pentanedioic acid²⁶³. Little is known about how radiotherapy interacts with ferroptosis pathways in CRPCs, but insights on the effects of radiotherapy on ferroptosis come from studies in other cancer types.

In other cancers such as uterine sarcoma, fibrosarcoma, glioblastoma, and lung carcinomas ionizing radiations were shown to amplify ROS production, induce lipid peroxidation^26,145, increase the expression of ferroptosis marker genes such as PTGS2 and ACSL4²⁶, and suppress SLC7A11 expression to cause the subsequent depletion of GSH¹⁹², whereas upregulation of the ferroptosis suppressors SLC7A11 and GPX4 was shown to trigger radiation resistance²⁶. A possible explanation for the radiation-induced repression of SLC7A11 could be that, upon exposure to radiation, p53 accumulates intracellularly to induce cell-cycle arrest and apoptosis^264,265. P53 is known to transcriptionally suppress SLC7A11 expression²⁰⁰ by either occupying the SLC7A11 promoter region²⁶⁶ or removing the transcriptionally activating histone H2B on lysine 120 from the SLC7A11 promoter²⁶⁷. Hence, administering ferroptosis inducers in conjunction with radiation can further impair the antioxidant defences of cancer cells and demonstrate synergistic effects on tumour suppression. Moreover, as cancer cells can upregulate SLC7A11 and GPX4 to build resistance against radiation²⁶, they can become heavily reliant on SLC7A11 and GPX4 to cope with excess ROS and toxic hydroperoxides, thus rendering them highly susceptible to ferroptosis inducers.

PARP inhibitors have revolutionised the cancer treatment paradigm and become a mainstay of therapy with radiation in clinical trials. PARP inhibitors were shown to sensitise colon cancer cell lines to radiation-induced ferroptosis through the activation of the transcription factor 3–SLC7A11 axis²⁶⁸. These results suggest that a combination therapy involving radiation, PARP inhibitors and ferroptosis inducers could provide enhanced anti-tumour effects compared with radiation (or ferroptosis induction) alone^269,270. As advanced prostate cancers (both adeno-CRPC and neuroendocrine prostate cancer) express high levels of SLC7A11, GPX4²⁹, and ACSL4⁵², we hypothesize that ferroptosis inducers that suppress either SLC7A11 or GPX4 could enhance lipid peroxidation and re-sensitize therapy-resistant cancer cells to radiation-induced ferroptosis, culminating in radiosensitization.

Ferroptosis inducers and diet

The is increased interest in understanding how dietary changes can enhance patient wellbeing during cancer treatment. Results from several studies showed that dietary levels of various amino acids, MUFAs, PUFAs, and glucose can influence the efficacy of ferroptosis inducers in different cancers. For example, dietary cysteine and methionine deprivation in glioma cell lines and mouse models resulted in enhanced lipid peroxidation and decreased GSH levels²⁷¹. This study found that cysteine and methionine deprivation increased sensitivity to RSL3 treatment, as shown by a decrease in cell viability following treatment. Further, this combination increased lipid peroxidation to levels similar to those obtained with a high dose of RSL3 alone²⁷¹. Another study found that intermittent methionine deprivation followed by imidazole ketone erastin (IKE) treatment or cysteine deprivation enhanced ferroptosis induction because of increased CHAC1 expression and GSH depletion²⁷². These results suggest that a methionine- and cysteine-free diet in combination with ferroptosis inducers might be an efficient therapeutic option for patients with CHAC1-deficient prostate cancer. Furthermore, co-treatment with erastin and exogenous MUFAs, specifically oleic and palmitoleic acid, resulted in suppression of ferroptosis in HT-1080 fibrosarcoma cells, A549 non-small cell lung cancer cells, and T98G glioblastoma cells, highlighting the importance of MUFA:PUFA ratios in the TME⁴⁵. Mechanistically, the anti-ferroptotic effects of oleic acid did not decrease ACSL4 or GPX4 expression but rather reduced the abundance of PUFA-PLs and ROS in the plasma membrane in an ACSL3-dependent manner⁴⁵. Conversely, another study found that an increase in n-3 and n-6 PUFA supplementation decreased cell growth under neutral conditions but induced ferroptosis in cervical, colorectal, and hypopharyngeal cancer cell lines cultured under acidic conditions, indicating that enhanced PUFA uptake by cancer cells in acidic environments increases their susceptibility to ferroptosis as a result of increased lipid peroxidation²⁷³. Further, a PUFA-rich diet in combination with sulfasalazine and erastin treatment increased the anti-tumour effect of ferroptosis inducers in HCT-116 tumour-bearing mice. In the same model, treatment with the ferroptosis inhibitor, ferrostatin-1, in combination with a PUFA-rich diet, resulted in increased tumour growth and substantially reduced survival²⁷³. Together, these studies suggest that cancer patients being treated with ferroptosis inducers might benefit from a PUFA-rich diet compared to a MUFA-rich diet. Further investigation is needed to determine the effects of dietary fatty acid supplementation in conjunction with ferroptosis inducers in patients.

The ketogenic (keto) diet has gained attention as a therapeutic intervention for patients with end-stage cancer suffering from cancer-induced cachexia. Mouse models of colorectal and pancreatic cancers fed a keto diet showed a decrease in the GSH-to-GSSG ratio in the liver, suggesting a decrease in GPX4 activity²⁷⁴. A decrease in cysteine, a critical metabolite necessary for GSH synthesis, was also observed²⁷⁴. Moreover, tumours from keto diet (KD)-fed mice showed increased mutagenic lipid peroxides, accumulation of lipid droplets, and ferrous iron levels. Further, KD-fed mice showed increased accumulation of 4-hydroxynonenal (4-HNE), a product of lipid peroxidation, in the liver, suggesting increased ferroptotic cell death in these tissues²⁷⁴. This increase in lipid peroxidation in KD-fed mice was reduced following treatment with N-acetyl cysteine, an antioxidant that increases GSH synthesis. These results suggest that a decrease in GSH synthesis induced by the keto diet yields decreased GPX4 activity, promoting ferroptosis²⁷⁴. In addition, the amino acid composition of the keto diet may impact ferroptosis independently of lipid peroxidation as it was also demonstrated that the low methionine content in the KD largely contributed to a decrease in expression of genes involved in fatty acid oxidation in the liver²⁷⁵. Combined, these studies suggest that the decrease in GSH synthesis and low methionine content provided by the keto diet might further sensitize cancer cells to ferroptosis inducers. However, there are currently no studies that have investigated treatment with ferroptosis inducers together with the keto diet in cancer patients.

Taken together, these findings imply that diet can alter the susceptibility to ferroptosis. However, in all the currently available studies, dietary changes led to a decrease in weight compared with mice that were fed normal diets. This evidence potentially raises concerns about whether these alternative diets would be beneficial to overall patient health, despite potential therapeutic benefits. These studies are also limited to preclinical cancer models; thus, the influence of special diets on the metabolic reprogramming that regulates ferroptosis and sensitises cancer cells to novel and existing therapies needs to be explored in clinical settings. This research will help optimise diets that could improve the overall health of patients with cancer.

Future of ferroptosis inducers

New ferroptosis inducers such as JKE-1674^140,156 or HSB-1216 have emerged in the past 5 years. JKE-1674, a GPX4 inhibitor, is a derivative of the selective covalent inhibitor of GPX4 called ML210 that is more metabolically stable and demonstrates fewer off-target effects¹⁵⁶. Moreover, the water solubility of JKE-1674 is greatly enhanced by the α-nitroketoxime group, enabling this drug to be detected in the serum of mice¹⁵⁶. This characteristic is an improvement over other ferroptosis inducers such as erastin and RSL3, which have low solubility and undesirable absorption, distribution, metabolism, and excretion characteristics, making them unfavourable for clinical translation^4,276. To date, only one study has assessed the effects of JKE-1674 in prostate cancer. In this study, JKE-1674 suppressed prostate tumour growth and metastasis in prostate epithelium-specific Pten/Rb1 double-knockout mice⁵³ .

The development of FSP1-targeted therapeutics remains an underexplored area. iFSP1, a human FSP1-specific inhibitor, was found to sensitise glioblastoma, lung, breast, and colon cancers to ferroptosis induction through GPX4 inhibition^277,278. Nevertheless, iFSP1 is deemed unsuitable for use in murine models due to its inability to bind to and inhibit mouse FSP1^277,279. Thus, new FSP1 inhibitors with improved pharmacokinetic and pharmacodynamic properties, such as the ferroptosis sensitizer 1²⁸⁰ and NPD4928²⁸¹, have been developed. Many of these inhibitors are yet to be evaluated in preclinical mouse models of cancer.

HSB-1216 is salinomycin encapsulated in polymeric nanoparticles made of PEG-polypropylene glycol (PPG)-PEG-modified PLA tetra-block copolymer. Salinomycin induces the sequestration of iron in lysosomes and the subsequent breakdown of lysosomal ferritin^177,282,283. Lysosomal iron overload then prompts increased production of ROS and drives lysosomal membrane permeabilization, triggering a ferroptosis-like cell death pathway¹⁷⁸. HSB-1216 was found to suppress tumour sphere formation in a dose-dependent manner in a model of late-stage small cell lung cancer¹⁷⁹. In acute myeloid leukemia cell lines, HSB-1216 also reduced their clonogenic survival and increased cancer cell death¹⁸⁰. Future studies are warranted to investigate the therapeutic promise of combining HSB-1216 with antiandrogens for the treatment of prostate cancer.

Advances in ferroptosis imaging

Considering the therapeutic potential of ferroptosis and novel approaches targeting this cell death mechanism, using complementary diagnostic imaging and anticancer approaches will be important for understanding mechanisms of action and then leveraging ferroptosis in the clinic. Ferroptosis imaging has become a major research area of interest in the past decade, both to understand the molecular mechanisms of ferroptosis — to delineate specific biological processes that might not be otherwise visualized directly-- , and to assess the efficacy of ferroptosis-inducing cancer therapy (e.g., use of imaging as a pharmacodynamic biomarker) (Box 1). Unlike other types of cell death, ferroptosis often does not display obvious morphological changes in cell membranes, organelles, nuclei or chromatin structures, presenting a clear challenge for direct detection of ferroptosis²⁸⁴. Further, several markers of ferroptosis including Fe²⁺ and lipid peroxides are usually transient and have relatively low levels beyond detection limits of conventional assays^285,286. These challenges in monitoring ferroptosis could help explain why unique ferroptosis mechanisms were not recognized earlier unlike other types of cell death. Great efforts have been made by several groups to monitor ferroptosis-associated mechanisms both in vitro and in vivo^287–289. Owing to the biological complexity and challenges in the detection of ferroptosis, several different approaches focusing on different aspects of the ferroptotic pathway(s) have been used. For example, iron metabolism and lipid peroxidation are hallmarks of ferroptosis. Direct detection of these processes is difficult due to their short lifespans. Moreover, there are no overt correlations between iron levels and tumour state. Given this, products of lipid peroxidation (levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE)) are currently investigated as stable biomarkers of ferroptosis¹⁸. However, these are static, indirect markers of ferroptosis. Alternatively, it is anticipated that novel, non-invasive imaging modalities (described below) could provide improved longitudinal and spatial insight into ferroptosis activity and tumour biology that could complement existing approaches. Such technologies would thus provide more tractable pharmacodynamic biomarkers of ferroptosis-targeting agents and possibly, predictive biomarkers to select the optimal patient population for such a therapeutic approach. Given that there is no one single consensus ferroptosis pathway for all circumstances, it is likely that multiple biomarkers will be needed to report which aspects of ferroptosis more accurately are being altered.

Box1. Current strategies for detection and clinical monitoring of ferroptosis.

Fluorescent probes

• C11-BODIPY^293,294

• Photochemical activation of membrane lipid peroxidation (PALP)²⁹²

• H₂O₂ fluorescent probes²⁸⁸

Use: fluorescent probes are limited to tissue section analysis and cell culture

Mass spectrometry (MS)

• Matrix-assisted laser desorption/ionization MS (MALDI-MS) to measure peroxidation of long-chain polyunsaturated fatty acids (PUFAs)²⁹⁷

• Liquid chromatography-MS (LC-MS) to measure phosphatidylethanolamines (PEs)⁸⁵

Use: MS-based techniques are useful for monitoring changes in lipid oxidation states, but remain limited to analysis of ex vivo tissue samples

Positron emission tomography (PET) imaging

• ¹⁸F-TRX²⁹⁸

• ⁶⁸Ga-NOTA-Tf and Ga-NOTA-HTF³⁰¹

• ¹⁸F-FSPG^302,304,305

Use: PET imaging approaches are highly sensitive and can provide spatial and longitudinal measures of ferroptosis-linked biology. Several probes have been developed and tested to monitor ferroptosis status in real time in vivo, mainly by monitoring the redox state of cells.

MRI imaging

• Quantitative susceptibility mapping (QSM) using Art-Gd³⁰⁶

• DFeZd³⁰⁷

• ipGdIO-Dox³⁰⁸

• Fe3O4-PLGA-Ce6 nanosystem³⁰⁹

Use: MRI-based approaches to detect ferroptosis are relatively recent and often can be further leveraged to induce ferroptosis for the selective elimination of cancer cells.

Fluorescence probes

Initially, imaging probes and fluorescence-based detection methods were the most widely used methodologies to monitor ferroptosis owing to the ease of detection in in vitro settings. One of the hallmarks of ferroptosis is the peroxidation of PUFAs, as uncontrolled lipid peroxidation and subsequent reactive aldehyde production leads to membrane rupture and induction of ferroptosis (Fig 1). Several chemical approaches have been developed to capture and visualize this step in ferroptosis to identify ferroptosis susceptibility in different physiological and disease conditions^290,291. In one study, a technique was developed to use photochemical activation of membrane lipid peroxidation (PALP) primarily to stratify cells and tissues based on their sensitivity to ferroptosis induction²⁹². In studies in which lipid peroxidation was induced with a laser, fluorescence changes occurred based on the oxidation sensitivity of BODIPY-C11, enabling researchers to monitor lipid peroxidation rates and susceptibility to ferroptosis in different cell types^293,294. A limitation of this technique is the requirement of tissue samples from patients, limiting one’s ability to assess patient sensitivity to ferroptosis induction in real time. Thus, PALP is a powerful method to identify cell and tissue sections that are susceptible to ferroptosis-mediated cancer treatment but is not amenable to high-throughput patient screening. In another study, ferroptosis initiation was indirectly monitored by measuring H₂O₂ levels in live cells using fluorescent probes²⁸⁸. This technique provides a viable approach for measuring the cellular dynamics of redox sensitivity during ferroptosis but is currently limited to in vitro monitoring and only a handful of animal studies, primarily owing to the low signal strength and imprecise methods to specifically target tumour cells. These fluorescence techniques are currently used as an important tool for delineating mechanisms of action. One of the methods to measure levels of Fe²⁺ and lipid peroxides are fluorescent substrates such as RhoNox-1 and BODIPY 581/591 C11 reagent. RhoNox-1 is a cell-permeable compound that can selectively detect basal and endogenous labile Fe²⁺ by enhancement of fluorescence signal in living cells²⁹⁵; thus, offering advantages over conventional ferroptosis biomarkers such as MDA and 4HNE. Similarly, BODIPY 581/591 C11 has been used to detect cellular lipid peroxidation by fluorescence shift from red to green with reaction with lipid peroxides in living cells²⁹⁴.

Mass spectrometry

Mass spectrometry (MS) has been exploited to measure the levels of lipid hydroperoxides during ferroptosis²⁹⁶. Using the mass-to-charge ratio of lipid molecules, MS provides precise information on the molecular mass, elemental structure, and the chemical structure of lipids. MS-based techniques have been used to identify biomarkers of ferroptosis⁸⁵. In one study, liquid chromatography–mass spectrometry (LC-MS)-based redox lipidomics was used to show that oxidation of specific phosphatidylethanolamines acted as death signals during ferroptosis⁸⁵. In another study, Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to measure the peroxidation of long chain PUFAs during ferroptosis in adrenal cell death²⁹⁷. With these MS-based approaches, unique and specific signals that trigger ferroptosis can be quantified with high-precision. Substantial developments using MS-based techniques are underway for the development of reliable clinical biomarkers for ferroptosis.

Positron emission tomography

Positron emission tomography (PET) imaging enables precise, in vivo, non-invasive monitoring of ferroptosis. Some PET tracers do this by tracking labile Fe²⁺ ions. In one study, a novel PET radiotracer termed ¹⁸F-TRX was used to quantify the intracellular labile iron pool in mouse orthotopic human xenograft models. In this study, glioma and renal cell carcinoma were used because of their high expression of the oxidoreductase STEAP3²⁹⁸. This PET imaging modality offers substantial advantages over current methods using fluorescent probes that either act by chelation or reactivity to labile ions due to the high sensitivity of deduction and direct translational ability in clinical setting since PET is a widely established non-invasive modality. The safety and feasibility of this approach are currently being studied in phase II clinical trials in antimalarial studies since ¹⁸F-TRX’s structure is based on artefenomel, an antimalarial compound under investigation. Success of this trial will allow repurposing of ¹⁸F-TRX for ferroptosis detection in cancer²⁹⁹. If successful, ¹⁸F-TRX could also help predict and monitor ferroptosis sensitivity during cancer treatment.

An alternative approach to detect ferroptosis sensitivity is by monitoring transferrin uptake using the cell membrane-localized iron transporter TfR1³⁰⁰. This is useful in determining the iron levels and inferring the ferroptosis sensitivity of cancer cells and might also be useful for determining correlations between specific cancer types and metabolic states in personalized cancer treatment approaches. Exploiting this mechanism, human transferrin (Tf) labelled with gallium-68 (⁶⁸Ga) using 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as a chelator (⁶⁸Ga-NOTA-Tf) was developed to create PET probes that could reflect iron uptake into cancer cells³⁰¹. As hypothesized, it was shown with 68Ga-NOTA-Tf that cells expressing higher TfR1 levels had higher iron levels compared to cells having baseline expression of TfR1. Thus, ⁶⁸Ga-NOTA-Tf could be used clinically as a predictor of susceptibility to iron-dependent processes such as ferroptosis.

Another PET agent to visualise ferroptosis is (4S)-4-(3-[¹⁸F]fluoropropyl)-L-glutamate (¹⁸F-FSPG), an ¹⁸F-labeled glutamate derivative. ¹⁸F-FSPG is transported specifically through the xCT subunit (encoded by SLC7A11) of the x_C⁻ system, which is upregulated in cancer in response to oxidative stress³⁰². Notably, ¹⁸F-FSPG has been successfully used in rodent models to non-invasively monitor x_C⁻ activity in multiple sclerosis (MS) and showed to have high blood clearance and low background activity. This study was primarily focused to identify neuroimmune interactions in MS and found that ¹⁸F-FSPG uptake was significantly higher than control mice with no MS and this was primarily due to xCT subunit activity, providing promise and proof of concept for usage of ¹⁸F-FSPG for monitoring the x_C⁻ system³⁰³. Results from these preclinical studies and ongoing clinical trials have shown ¹⁸F-FSPG to be effective in monitoring patients with not only prostate cancer but also in other relevant tumour models with high x_C⁻ system activity. Specifically, this clinical evaluation study was the first study to use ¹⁸F-FSPG as a radiopharmaceutical for PET imaging of system x_C− activity in recurrent prostate cancer³⁰⁴. Here, it was identified that ¹⁸F-FSPG PET was able to differentiate cancerous prostate lesions from normal tissue and that ¹⁸F-FSPG uptake was also correlated with pathological and IHC markers for x_C⁻ system activity. This landmark study was the first to establish the use of x_C⁻ system activity monitoring with ¹⁸F-FSPG for prostate cancer. Further studies are required for establishing clinical protocols of dosing, diagnostic value for different stages of cancer and its comparison to other clinically established radiotracers³⁰⁴. Furthermore, there are several clinical trials currently underway to diagnose, predict and evaluate a wide variety of tumours using ¹⁸F-FSPG such as liver (NCT02379377), lung (NCT03824535), breast (NCT03144622), and colorectoal cancer (NCT03275974).^302,304,305. Results from these studies suggest that clinically viable methods already exist to evaluate the efficacy of ferroptosis inducers that inhibit system x_C⁻ activity such as Cyst(e)inase, erastin, IKE, or sulfasalazine, which should aid the development of ferroptosis-targeting clinical regimens.

Magnetic resonance imaging

Few methodologies have been developed for imaging ferroptosis using MRI. MRI has been used mostly for resolving soft tissue and anatomical structures in a non-invasive manner, with the use of traditional MRI acquisition being limited in imaging at the molecular scale. In 2023, this notion was challenged by a study in which an emerging MRI-based technique referred to as quantitative susceptibility mapping (QSM) was used to detect ferritin levels in in vivo models³⁰⁶. Here, an artemisinin-based MRI probe was developed by chelating gadolinium ions (Gd (III)) through a 1,4,7,10-tetraazacyclododecane–1,4,7,10-tetraacetic acid (DOTA) group (Art-Gd). Upon interaction with labile Fe²⁺ ions, Art-Gd forms carbon-centred radicals, leading to the formation of the Art-Gd protein complexes as well as the retention of these complexes in cells and tissues and enhanced longitudinal relaxation time (T1) contrast. This technique enables real-time MRI in vivo, although the feasibility and toxicity of this approach have not been confirmed in clinical settings.

Fe-based MRI contrast agents that also act as theranostic agents to induce ferroptosis have been developed. For example, nanoparticles (NPs) containing the pH-responsive contrast agent DFeZd, which has a higher T1 signal in tumour sites compared to normal adjacent tissue, when activated in the acidic tumour microenvironment causes reduction of Fe³⁺ to Fe²⁺, promoting ferroptosis. Hence, this study exploits the concept of 1) detection of tumour tissues and 2) selective induction of ferroptosis in the identified tumor sites³⁰⁷. In another study, a novel magnetic nanocatalyst (iRGD-PEG-ss-PEG-modified gadolinium engineering of magnetic iron oxide-loaded Dox (ipGdIO-Dox)) was developed for MRI-guided chemotherapy and ferroptosis-synergistic cancer therapies³⁰⁸. IpGdIO-Dox has enhanced T1 and T2-weighted MRI signals in the tumour and simultaneously induces ferroptosis because the Fe²⁺ ions catalyse the production of OH radicals from H₂O₂. Further, the ipGdIO-Dox is broken down by high GSH levels often found in advanced tumors, leading to the release of doxorubicin and subsequent synergistic anti-tumour effects with chemotherapy. Similar redox-based ferroptosis-detecting agents have also been developed and may have clinical value. For example, an Fe3O4-PLGA-Ce6 nanosystem was developed to induce similar chemistry for induction of ferroptosis in cancer cells while providing efficient MRI detection properties and synergistic delivery of chemotherapeutic agents³⁰⁹. Many studies using MRI-based approaches for ferroptosis are now focused on dual detection of pH and iron metabolism and complementary anti-cancer theranostic agents. This can be attributed to the inherent challenges of obtaining molecular-scale imaging of ferroptosis mechanisms and thus, need to measure multiple parameters (e.g., pH and iron states) at once to obtain a more accurate measure of ferroptosis. MRI detection strategies are currently limited to measurements of pH, labile Fe ions, and redox levels. Therefore, it is anticipated that the development of novel MRI contrast agents that can detect additional molecules relevant to ferroptosis such as lipid peroxides, iron transporters and GSH targets would enable better real-time imaging of ferroptosis.

Future perspectives

Because of the potential anticancer activity of ferroptosis, there has been great interest in the development of novel methodologies and chemical tools to monitor ferroptosis in the clinical setting. Despite rapid advancements in the detection of ferroptosis, several challenges remain. Ferroptosis is a multifaceted biological process, and many detection techniques only focus on a single phenotype or marker³¹⁰, and still no specific markers of ferroptosis exist²⁸⁴. Thus, identifying specific markers of ferroptosis is of great importance. Moving forward, developing multi-biomarker tracking techniques that enable detection of simultaneous mechanisms involved in ferroptosis such as lipid peroxidation, redox levels, and Fe²⁺ status will be imperative. Fluorescent probes with different chemical compositions and detection strategies can also be of use to observe lipid compositions within plasma membranes to help serve as additional indicators in ferroptosis. However, the scope of these probes is limited by the number of channels of detection than can be used. Additionally, current fluorescent probes are limited in wavelength, as most are in the 400–500 nm range, which has low signal detection strength and autofluorescence. Hence, development of near infra-red probes is warranted, as these probes would enable long-range detection and would probably increase the accuracy of measurements²⁹⁰.

The reliance on one parameter detection of ferroptosis is primarily owed to the lack of clinically reliable and biologically associable ferroptosis biomarkers³. Several previously established biomarkers such as PTGS2 and CHAC1 mRNA do not have direct correlation to molecular events and do not have robust clinical relevance²⁸⁶. Thus, developing reliable clinical biomarkers for ferroptosis and characterizing sensitivity to ferroptosis-based treatments for cancer are of great importance. To address this challenge, advancements in redox-based phospholipidomics and the identification of specific lipids that lead to ferroptosis can enable the characterization of precise biomarkers that could be used to interrogate patient biopsy samples to robustly detect lipid peroxidation³¹¹.

Innovative approaches have been developed for detecting cancer tissues sensitive to ferroptosis-mediated anti-cancer therapy, but the efficacy of these therapeutic approaches and their timelines in clinical settings as well as the toxicity of these therapeutic measures are not fully known^311,312. Predictive biomarkers of sensitivity to ferroptosis induction in tissues are under development and largely link to the underlying biological mechanisms of this process, such as the levels of labile Fe²⁺ and lipid peroxidation³¹³. Strategies such as PALP can be applied to limited sample types, whereas real-time imaging techniques like PET and MRI offer more tractable methods that can be readily translated to the clinic for use as pharmacodynamic biomarkers of ferroptosis-causing agents and possibly, in future, guide patient selection. To date, only single marker-based targeting approaches have been explored in pre-clinical settings so far. Thus, combining two or more markers that are involved in different stages of ferroptosis mechanisms could yield significant breakthroughs in cancer treatment as they would provide a more accurate assessment of ferroptosis, a multi-step process consisting of multiple regulatory nodes.

The current knowledge about alternative ferroptosis regulatory pathways and the role of metabolism in cancer is limited, particularly in prostate cancer³¹⁴. Many open questions remain, for example, about the clear mechanistic crosstalk between ferroptosis, additional cell death pathways and metabolic changes in tumours. An advancement in this area is the use of machine-learning tools to distinguish and classify apoptotic cell death and ferroptosis features through the perturbation of different metabolic pathways and ferroptosis surveillance systems³¹⁵. The use of machine learning is in its infancy and only tissue sections can currently be used; however, this approach has tremendous potential to analyse high-throughput data in clinical settings in combination with the establishment of clear biomarkers and robust phenotypes for ferroptosis-guided therapy.

Conclusions

Since the coining of the term ferroptosis in 2012, the number of publications investigating this mechanism has exponentially grown. In the past ~10 years, most research in the field has been dedicated to uncovering the mechanistic underpinnings and various metabolic pathways regulating lipid peroxidation and ferroptosis. However, additional questions remain. For instance, there is a gap in our understanding of the precise mechanisms through which ferroptosis-related genes impact prostate cancer cells. There is a critical need for reliable ferroptosis markers that can accurately predict and monitor ferroptosis induction and its effects in cancer cells in vivo in real-time. Due to differing sensitivities of various prostate cancer cell lines to ferroptosis, it is important to further develop strategies to overcome cellular tolerance to ferroptosis. Given the significance of AR signalling in prostate cancer progression and the emerging role of ferroptosis in cancer therapy, understanding how these pathways intersect could aid the creation of better treatment strategies. Future clinical studies are warranted to substantiate the role of ferroptosis in prostate cancer treatment. Nevertheless, rapid progress has been made toward leveraging the dependence of cancer cells on ferroptosis-related pathways as a treatment strategy. Specifically, combining ferroptosis inducers with chemotherapy, hormone therapy, radiotherapy, or immunotherapy might each improve anticancer effects and overcome therapy resistance (Fig. 2). However, most of these findings came from preclinical studies in cell and mouse models. Currently available cell line and patient-derived xenograft models are advantageous for studying prostate cancer biology and testing the efficacy of treatments, but the lack of heterogeneity in cell lines and lack of appropriate in vivo models in prostate cancer research limit the translation of preclinical findings to the clinic. Particularly, supraphysiological levels of nutrients in culture media can cause unpredicted effects on the phenotypic behaviour of cells and affect the metabolic fidelity of cancer cell models that makes the results of metabolic drug screening unreliable. Xenograft models do not fully recapitulate the human immune system, tumour microenvironment and the different patient ethnicities, indicating the need for reliable in vivo prostate cancer models with increased fidelity to the disease. Finally, while numerous ferroptosis inducers have been shown to possess potent anti-cancer activity in vitro, issues with their pharmacokinetic properties indicate a need for further development for translation of these ferroptosis-based compounds into clinical use.

Induction of ferroptosis, an iron-dependent form of non-apoptotic cell death, has emerged as a promising therapeutic approach for cancer. Ferroptosis inducers, alone or in combination with existing treatments, represent novel therapeutic approaches for the treatment of aggressive prostate cancer. Advances in ferroptosis detection approaches and biomarker identification for disease monitoring and therapy management hold promise for the future. To that end, new imaging methodologies for monitoring ferroptosis hold promise as biomarkers predictive of ferroptosis-inducing treatment efficacy and could also serve as pharmacodynamic biomarkers of ferroptosis-targeting agents, accelerating drug development efforts. Therefore, a personalized approach integrating multiple biomarker-driven patient selection strategies and subsequent tumour monitoring is anticipated to help advance the development and clinical implementation of ferroptosis-based therapy, and thus unlock the anticancer potential of this promising class of treatments.

Figure 3.