Cancer remains a public health threat worldwide, with the International Agency for Research on Cancer reporting a staggering 19.3 million new cases and nearly 10 million deaths in 2020 (1). Despite significant advances in its treatment, the battle against cancer is hampered by the significant side effects of traditional therapies and the inevitable emergence of resistance associated with such treatments. Therefore, the identification of more effective and better-tolerated anticancer therapies is critical to meet clinical requirements. As first described by Dixon et al (2) in 2012, ferroptosis is a distinct form of cell death characterized by the accumulation of LPOs (3,4). Recently, ferroptosis has garnered attention owing to its involvement in numerous diseases, ranging from neurodegeneration to lethal malignancies, such as liver, breast, and lung cancers (5–8). Over the past two decades, comprehensive studies have investigated the intricacies of ferroptosis in diverse cancer types, elucidating its pivotal role in governing cancer progression via diverse signaling cascades. Cancer cells that exhibit resistance to conventional therapies or possess a high propensity for metastasis are more susceptible to ferroptosis (9–11). Therefore, the regulation of ferroptosis and its target proteins represents a novel and promising strategy for cancer treatment (12,13). The various signaling pathways involved in ferroptosis have been extensively summarized those studies. Ferroptosis primarily occurs through the L-cystine/glutamate antiporter (system Xc-)/glutathione (GSH)/GSH peroxidase 4 (GPX4) pathway, the GTP cyclohydroxylase-1 (GCH1)/tetrahydrobiopterin (BH4) pathway, the dihydroorotate dehydrogenase (DHODH) pathway, the O-acyltransferase 1/2 (MBOAT1/2)-monounsaturated fatty acid (MUFA) pathway, the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathways, the mevalonate pathway and the apoptosis-inducing factor mitochondria-associated 2 (AIFM2, also known as FSP1 and referred to as FSP1 throughout) pathway-mainly the FSP1-coenzyme Q10 (CoQ10)-NAD(P)H, FSP1-vitamin K (VK)-NAD(P)H and FSP1-endosomal sorting complex, required for transport (ESCRT)-III pathways (Fig. 1). Among these systems, the Xc-/GSH/GPX4 pathway has garnered significant attention as a crucial regulatory mechanism of ferroptosis, sparking extensive research on its ability to induce ferroptosis in tumor cells. However, the clinical application of inhibitors targeting this system has been hindered by their poor selectivity, toxicity and the presence of FSP1, which runs parallel to GSH-GPX4. Furthermore, certain cancer cells exhibit resistance to ferroptosis induction (14–16).

FSP1, a crucial modulator of ferroptosis, has emerged as a key player in ferroptosis resistance, operating independently of the GSH-GPX4 pathway. This protein primarily blocks ferroptosis in tumor cells by regulating the oxidation of NADPH, thereby hampering the efficacy of cancer treatment strategies (17). Furthermore, FSP1 is highly expressed in various cancer cells and its expression has been linked to poor prognosis (18,19). Therefore, targeting FSP1 to inhibit its activity, thus promoting ferroptosis, may be a promising approach for cancer therapy, particularly in the case of difficult-to-treat tumors. This article provides a comprehensive review of the ferroptosis pathway, the mechanism by which FSP1 modulates ferroptosis and the potential of FSP1-related molecular inhibitors in cancer treatment.

Ferroptosis is a non-apoptotic form of iron-dependent cell death. Key hallmarks of ferroptosis include elevated iron levels, lipid peroxidation and disruption of the mitochondrial architecture (20–22). Ferroptosis has been associated with various factors, including iron metabolism level, GPX4, GCH1/BH4, DHODH, FSP1 and MBOAT1/2 (Fig. 1). There is a significant association between intracellular iron metabolism and ferroptosis. Excessive iron loading can lead to ferroptosis. Transferrin receptor 1, a marker of ferroptosis, has a pivotal role in facilitating the transport of Fe³⁺ into cells (23). Within the cellular milieu, divalent metal transporter 1 converts Fe³⁺ into Fe²⁺, which is subsequently stored in ferritin. However, when intracellular levels of labile Fe²⁺ become excessively high, it reacts with hydroxyl radicals via the Fenton reaction, triggering a surge in reactive oxygen species (ROS) production and the accumulation of lipid peroxidation. These processes ultimately induce ferroptosis (24). Furthermore, a phospholipase present in the cytoplasm, namely platelet-activating factor (PAF)-acetylhydrolase (II), specifically inhibits short-chain fatty acid oxidation, interfering with the cell's redox capacity and blocking the aggregation of oxidized phospholipids such as PAF; this leads to membrane rupture, inhibiting cell ferroptosis (25). Acetyl-coenzyme A (CoA) of the mevalonate pathway inhibits ferroptosis by triggering the NADPH-FSP1-CoQ10 pathway via the regulation of CoQ10 synthesis. Furthermore, various types of fatty acids have different degrees of regulatory roles in ferroptosis. Among these, polyunsaturated fatty acids (PUFAs) are particularly prone to peroxidation during ferroptosis. This process involves the esterification of membrane-forming phospholipids with the assistance of acyl-CoA synthetase long-chain family member 4, which is a fatty acid-activating enzyme found in the endoplasmic reticulum and outer mitochondrial membrane. In addition, lysophosphatidylcholine acyltransferase (LPLAT) contributes to the destruction of the lipid bilayer structure, leading to enhanced membrane permeability and ultimately triggering ferroptosis (26,27). Conversely, the inhibition of lipid peroxidation has a pivotal role in preventing ferroptosis by suppressing the formation of LPOs and free radicals. This involves crucial pathways discussed in the following paragraphs.

Multiple studies have shown that inhibition of FSP1 enhances the antitumor efficacy of GPX4 inhibitors (95–97). For instance, FSP1-mediated ferroptosis can be effectively targeted for the treatment of challenging cancers, including triple-negative breast cancer (TNBC), hepatocellular carcinoma (HCC), colon cancer, acute lymphocyte leukemia and NSCLC. Laboratory studies have further demonstrated that FSP1 inhibitors can increase cancer cells' susceptibility to ferroptosis. Thus, FSP1 holds significant potential as a target for tumor therapy, offering hope for more effective treatment options.

Lee et al (99) proposed a strong connection between FSP1 overexpression and HCC, indicating that elevated FSP1 levels are associated with shorter overall survival among patients. iFSP1, a specific inhibitor of FSP1, effectively triggers ferroptosis in hepatoma cells by attracting immune cells. This approach has been shown to reduce HCC cell counts and enhance immune infiltration of dendritic cells, macrophages and T cells (Table I). When tested on MHCC97L cells at various concentrations, iFSP1 was shown to effectively suppress tumor-cell proliferation without causing significant changes in body weight. Treatment with iFSP1 also resulted in modifications to the immune landscape of HCC tumor cells. Therefore, iFSP1, as an FSP1 inhibitor, holds immense potential as a superior therapeutic agent for the treatment of HCC (99,100).

Hendricks et al (101) discovered that FSEN1 is an inhibitor of FSP1 and exerts synergistic effects with multiple ferroptosis inducers (Table I). Their findings demonstrated that FSEN1 acts specifically through FSP1. To induce ferroptosis in H460^C Cas9 cells, an optimal dosage of FSEN1 at 0.55 mM and RSL3 at 0.55 mM was identified. This dosage achieved EC₅₀ values of 69.363 nM in H460^C GPX4 knockdown cells. Pharmacokinetics analysis conducted in mice revealed a plasma half-life of 8 h and an intrinsic clearance rate of 11.54 ml/min/mg in mouse liver microsomes. These findings suggest improved metabolic stability of FSEN1 in vivo (102). Evaluation of the impact of FSEN1 on cell ferroptosis triggered by GPX4 inhibitors across diverse cancer cell lines, including lung, liver and breast cancer cells, revealed that FSEN1 enhances the sensitivity of cancer cells, to varying degrees, towards RSL3-induced ferroptosis. Furthermore, preclinical studies have demonstrated synergistic effects when FSEN1 was used alongside endoperoxide-derived ferroptosis inducers, such as dihydroartemisinic acid (101). These findings provide further evidence supporting the potential of FSEN1 as an FSP1 inhibitor in cancer therapy.

Nakamura et al (103) found that icFSP1 exhibits specificity towards ferroptosis and does not stimulate the myristylation of FSP1 in vitro (Table I). Their findings suggest that icFSP1 may regulate FSP1-membrane interactions, inducing the production of condensates that lead to reduced membrane-binding affinity of FSP1. Furthermore, the induction of FSP1 condensate formation within tumor cells triggers the phase separation of FSP1, ultimately inducing ferroptosis in cancer cells. the EC₅₀ value of icFSP1, measured in Pfa1 cells, was found to be 0.21 µM. Treatment with the FSP1 inhibitor icFSP1 impaired tumor-cell growth and significantly reduced tumor weight. However, at higher concentrations, there was no significant impact on tumor weight, indicating no off-target effects. Furthermore, icFSP1 significantly improved microsomal stability and maximum tolerated dose in mouse plasma compared with iFSP1. The stronger pharmacokinetic and metabolic stability of icFSP1 renders it suitable for use in vivo (103), making it a promising candidate for targeting FSP1-dependent phase separation in anticancer therapy. These findings provide the basis for targeting FSP1-dependent phase separation as an effective anticancer therapy.

Nakamura et al (104) proposed that the treatment of Pfa1 cells expressing mouse FSP1 or human FSP1 with viFSP1, a multifunctional inhibitor of FSP1, leads to ferroptosis, which can be reversed by administration of the ferroptosis inhibitor liproxstatin-1 (Table I), confirming that the dependence of viFSP1 on FSP1 causes ferroptosis. Specifically, viFSP1 targets the NADH binding pocket around residues A328, F294, M327 and T1 in FSP1. In addition, both human and mouse FSP1 cells exhibited similar sensitivity to viFSP1 with no significant difference in the calculated IC₅₀ values. Thus, viFSP1 emerges as a species-independent direct inhibitor of FSP1, with an EC₅₀ value of 170 nM in Pfa1 cells. In multiple human and mouse cancer cell lines, as well as in rat fibroblasts and Pfa1 cells overexpressing FSP1, the concurrent use of viFSP1 and the GPX4 inhibitor RSL3 synergistically enhanced ferroptosis induction, suggesting its potential therapeutic utility in tumor treatment (104).

Ubiquitination, a form of posttranslational modification, has a crucial role in regulating cellular processes. Sorafenib, a multi-target kinase inhibitor, inhibits cell proliferation by interfering with serine-threonine kinase-related signaling pathways and hinders tumor angiogenesis by targeting specific tyrosine kinases (Table I). In HCC cells, sorafenib has been demonstrated to induce ferroptosis (105). Lai et al (106) revealed that sorafenib regulates the interaction between tripartite motif containing 54 and FSP1 through the MAPK/ERK kinase pathway, promoting the ubiquitination of FSP1. Elevated FSP1 expression partially reverses sorafenib-induced ferroptosis, whereas FSP1 inhibition enhances HCC cell sensitivity to sorafenib-induced ferroptosis (106). Thus, high levels of FSP1 inhibit sorafenib-induced ferroptosis in HCC cells (95). This suggests that downregulation of FSP1 in HCC cells may promote sorafenib-induced ferroptosis to facilitate therapeutic effects against HCC.

Mishima et al (107), proposed that the DHODH inhibitor brequinar suppresses FSP1 activity at elevated dosages, thereby mitigating resistance to ferroptosis (Table I). Even in the absence of DHODH, high concentrations of brequinar were shown to effectively induce ferroptosis in Pfa1 cells derived from GPX4-deficient mouse fibroblasts. However, in FSP1-knockdown cells, ferroptosis was not induced, which was in contrast to the effects observed with BAY-2402234, another DHODH inhibitor that lacks inhibitory activity against FSP1 (108). These findings suggest that the ferroptosis-inducing effect of brequinar is mediated by the inhibition of FSP1 rather than DHODH.

Zhou et al (109) demonstrated that curcumin has the capacity to induce ferroptosis in highly tumorigenic A549 CD133+ cells via the GSH-GPX4 and FSP1-COQ10-NADH signaling pathways (Table I). Treatment with curcumin resulted in a dose-dependent decrease in the activity of CD133⁺ cells and the expression levels of GPX4 and FSP1 protein. The levels of ROS, GSH, CoQ10 and NAD+/NADH in the curcumin-treated group were significantly lower than in the control group; this effect was attenuated by ferroptosis inhibitor Fer-1. Furthermore, the IC₅₀ value of curcumin was determined to be 36 µM, indicating its high potency. In addition, curcumin demonstrated robust stability in vivo. Upon curcumin treatment, tumor growth was effectively suppressed, tumor pathology improved and the expression of Ki-67, a marker of tumor proliferation, was notably downregulated. Furthermore, the inhibitory effect of curcumin was higher than that of RSL3 and iFSP1. However, Fer-1 significantly inhibited the antitumor effects of curcumin (109). Thus, curcumin can inhibit GPX4 and FSP1 and promote ferroptosis to treat tumors.

Miyazaki et al (110) demonstrated that Andrographis exhibited comparable pharmacological effects to curcumin, effectively suppressing the activities of GPX4 and FSP1, thereby inducing ferroptosis in colorectal cancer cells (Table I). Treatment of SW480 and HCT116 cells with Andrographis significantly reduced cell viability as well as GPX4 and FSP1 expression; these effects were comparable to those elicited by RSL3 or iFSP1. In addition, the IC₅₀ of Andrographis in both cell lines was 40 µg/ml, indicating its stability and reliability in vivo. Treatment with Andrographis was shown to inhibit the growth of cancer cells, with a reduction in invasiveness. Furthermore, the drug had no adverse effect on body weight. Fer-1 was found to mitigate the inhibitory action of Andrographis on colorectal cancer. The combination of Andrographis and curcumin exhibited a superior inhibitory effect against GPX4 and FSP1 to either drug alone.

Li et al (112) employed [Fe (III)] Cu-tetra (4-carboxyphenyl) porphyrin chloride [Cu-TCPP (Fe)] metal organic framework (MOF) nanosheets as substrates for the deposition of Au NPs through in situ nucleation. Subsequently, they successfully deposited the ferroptosis inducer RSL3 onto the surface of these NPs via π-π stacking interactions to obtain NPs capable of targeting ferroptosis for TNBC therapy. Furthermore, they enhanced the system by integrating a long-circulating polyvinyl sugar segment and a tumor-targeting iRGD ligand (112). Liu et al (113) demonstrated that Au NPs possess similar activity to glucose oxidase, which oxidized glucose into gluconic acid, effectively depleting glucose levels, disrupting the pentose phosphate pathway and inhibiting GSH biosynthesis. This prevents the conversion of CoQ10 to CoQ10H2, leading to an elevation in the NADP+/NADPH ratio and effectively suppressing the FSP1-CoQ10H2 pathway. At the same time, increased NADP⁺/NADPH ratios further inhibit the conversion of cystine to cysteine, reducing GSH and GPX4 biosynthesis. Furthermore, Yuan et al (114) showed that Cu²⁺ immobilized within the TCPP MOF nanoplates exhibited the ability to oxidize GSH into GSH oxide, depleting the cofactor essential for GPX4 activity. This process attenuated GPX4′s inhibitory function against ferroptosis, effectively blocking the GPX4/GSH pathway. In addition, this nanosystem possessed the ability to release Cu²⁺, further enhancing its inhibitory effect against the GPX4/GSH pathway. Studies have shown that Au/Cu-TCPP-Fe-polyethylene glycol (PEG) exhibits a prolonged blood circulation time and an enhanced tumor-targeting effect in vivo. Mice treated with Au/Cu-TCPP (Fe)@RSL3-PEG-iRGD had the longest survival with a median survival time of 60 days, and only a small number of tumor cells retaining proliferative activity. In addition, these nano-tablets demonstrate excellent tolerability in vivo, even at very high dosages. Thus, multienzyme-like reactions of NPs can simultaneously inhibit GPX4 and the FSP1-CoQ10H2 pathway in TNBC cells to promote ferroptosis, providing a promising avenue for the clinical treatment of drug-resistant tumors.

Yang et al (115) utilized photodynamic therapy to target FSP1-mediated ferroptosis. They employed a photoresponsive nanocomposite composed of boron-dipyrromethene (BODIPY)-modified polyamide (amidoamine) (BMP), which was used to encapsulate iFSP1 and chlorin e6 (Ce6); this was facilitated by π-π stacking and hydrophobic interactions between Ce6 and BODIPY. Thus, BMP and ferroptosis inducers were integrated into NPs. Under light irradiation, these NPs effectively triggered ferroptosis both in vitro and in vivo, leading to a significant slowing of tumor growth. Ferroptosis directly promotes immunogenic cell death, a process in which cells release ATP and high mobility group box 1 proteins, while exposing calreticulin on their surface. During this process damage-associated molecular patterns mediate the recruitment and activation of dendritic cells, thereby facilitating T-cell infiltration and cytotoxicity. It was suggested that IFN-γ secreted by T cells can trigger LPO of tumor cells; however, in the absence of NPs, cell death is not elicited, particularly when GPX4 and FSP1 are inhibited. In addition, pharmacokinetics studies have shown that the half-life of Ce6 is prolonged when it is encapsulated within NPs, and furthermore, during treatment, the drug did not accumulate in normal organs, with stable body weights maintained across groups. Of note, no significant histological damage or morphological alterations were observed in any of the organs. Therefore, light-responsive nanocomposites have the potential to synergistically induce ferroptosis in cancer immunotherapy.

The exploitation of ferroptosis, a type of programmed cell death, holds great promise for various cancer therapies. Ferroptosis inducers heighten tumor cell sensitivity towards chemotherapeutic agents and facilitate the elimination of tumor cells that are refractory to other programmed cell death modalities. Consequently, the induction of ferroptosis has emerged as a promising approach for treating refractory tumors. Although numerous ferroptosis inducers have been identified, the primary focus has been limited to targeting the system xc-/GSH/GPX4 pathway. This limited perspective has somewhat hindered the exploration of the ferroptosis regulatory system as a target for cancer therapy.

FSP1, a linchpin anti-ferroptosis factor, enhances cancer-cell resistance to ferroptosis, making it an attractive target for cancer therapy. The current review reported on FSP1 inhibitors that boost cancer-cell sensitivity to ferroptosis. These inhibitors exhibit pharmacokinetic and metabolic stability, suitable for in vivo studies, and have been shown to effectively block the FSP1-mediated cellular defense mechanism against ferroptosis. Among them, the FSP1 inhibitors mentioned above have demonstrated remarkable potency in overcoming ferroptosis resistance in several cancer cell lines, potentially enabling the development of more effective cancer therapies.

Although FSP1 inhibitors may potentially have a significant role in cancer treatment, several challenges remain to be tackled. First, the specific mechanism by which FSP1 regulates ferroptosis remains unclear, necessitating further research. Although FSP1 is highly expressed in a variety of tumors, it is also expressed in normal tissues, making target selection a challenge. Furthermore, the association between FSP1 expression levels and tumor malignancy as well as poor prognosis further complicates treatment strategies. Third, research pertaining to FSP1 inhibitors is currently in its early stages. Although preliminary experimental studies have yielded encouraging outcomes, extensive further exploration is imperative to establish their clinical utility. Fourth, the number of reported FSP1 inhibitors is limited and it is crucial to develop more drug molecular structures through natural drug screening and AI technology. The goal is to develop inhibitors that are highly selective, exhibit low toxicity and possess desirable pharmaceutical characteristics. In addition, further research is required to investigate the potential clinical application of combinations of FSP1 inhibitors with other antineoplastic agents and immune checkpoint inhibitors. Moreover, nanomaterials hold immense promise in this domain, offering novel avenues for research.

In conclusion, although FSP1 inhibitors demonstrated tremendous potential in tumor therapy, further research is necessary to address existing challenges and optimize the application of these compounds in clinical practice.