Ferroptosis Suppressor Protein 1 (FSP1)-CoQ10-NADPH-Axis Is Responsible for Erastin Resistance in MCF-7 Breast Cancer Cells.

1. Introduction
Breast cancer is a serious global threat to women’s health, and emergence of multidrug resistance following chemotherapy leads to poor prognosis and decreased survival rates [1,2,3]. Erastin (ER) and its analogs are promising anticancer agents due to their ability to sensitize resistant tumor cells to chemotherapeutic agents [4]. It has been found that triggering ferroptosis in breast cancer cells can significantly inhibit their proliferation and invasion, as well as improving sensitivity to radiotherapy and chemotherapy [4]. Accordingly, drugs that induce ferroptotic tumor cell death have attracted attention as a potential therapeutic strategy, particularly for cancers refractory to conventional therapies [5].
Ferroptosis plays a critical role in cancer and chemical toxicology due to its unique cell death mechanism that is driven by iron-dependent lipid peroxidation [6,7,8,9]. Ferroptotic cell death has been shown to be morphologically, biochemically, and genetically distinct from other forms of cell death such as apoptosis, necrosis, and autophagy [6,8,10]. Ferroptosis is characterized by the accumulation of lethal reactive oxygen species (ROSs) and lipid radicals, resulting in damage to cellular membranes and causing ferroptosis [6,8,10]. Human normal as well as tumor cells have an enhanced antioxidant system and protect cells from this ROS-induced toxicity. This system includes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPXs), and reduced glutathione to protect cells [11]. This mechanism has become extremely relevant to cancer treatment and cancer-related diseases because exposure to certain anticancer drugs can intensify oxidative stress, leading to ferroptotic cell death [9] as well as damage in organs like the liver, kidneys, and lungs [12].
At the molecular level, ER induces ferroptosis by suppressing the glutamate/cystine antiporter (system xCT), leading to inhibition of cellular cystine uptake and inhibiting glutathione synthesis [13]. System xCT, composed of the light-chain SLC7A11 and the heavy-chain SLC3A2, has been reported to be a key regulator of cellular redox homeostasis. It mediates the uptake of extracellular cystine in exchange for intracellular glutamate, supplying cystine for its intracellular reduction to cysteine, the rate-limiting precursor for glutathione (GSH) synthesis. By maintaining GSH levels, system xCT supports the activity of antioxidant enzymes such as GPX4 and thereby protects cells from lipid peroxidation and oxidative stress. Inhibition of system xCT causes a rapid drop in intracellular glutathione levels, resulting in cell death from the accumulation of lipid-derived reactive oxygen species (ROSs). Glutathione is a primary cellular antioxidant that plays critical roles in maintaining the redox balance and defending against oxidative stress, including ROS. Not only ER has been shown to induce ferroptosis in several types of cancer cells, and it is selectively more cytotoxic to tumors harboring RAS mutations [14]. During ferroptosis, glutathione peroxidase 4 (GPX4) is inhibited, and intracellular lipid hydroperoxides (LOOHs) accumulate, causing damage to cellular membranes (lipid peroxidation) in the presence of iron [15].
Ferroptosis suppressor protein 1 (FSP1; also called AIFM2 or AMID) utilizes NAD(P)H to reduce oxidized coenzyme Q10 (ubiquinone) to its active, reduced form, ubiquinol. Ubiquinol (CoQH2) then acts as an antioxidant that neutralizes lipid radicals, preventing them from propagating and initiating ferroptosis [16,17]. FSP1 can also reduce vitamin K to its hydroquinone form (VKH) to inhibit ferroptosis. Ferroptosis inducers (FIN) inactivate of these ferroptosis surveillance systems, leading to a rapid buildup of lipid peroxides, causing ferroptosis in many cancer cell lines and in vivo tumor models [6,10].
Recently, we as well as others have shown that ER is extremely effective in reversing and sensitizing various chemotherapeutic agents in several ABC transporter-expressing cell lines. We found that ER effectively reverses resistance to Adriamycin and topotecan in ABCB1- and ABCG2-expressing tumor cell lines by enhancing cellular uptake of Adriamycin and topotecan, respectively [18,19,20]. More importantly, our studies showed that both ER and RSL3 (Ras selective ligand3) were significantly more cytotoxic to MCF-7/MXR cells, a BCRP-expressing cell line [20], than the parent MCF-7 cells. Furthermore, both ER and RSL3 were effective in reversing topotecan resistance in the MCF-7/MXR breast cancer cell line [20].
Our previous studies demonstrating ER enhanced cytotoxicity of topotecan in MCF-7/MXR breast cancer cells suggested that ferroptosis inducers may be clinically valuable in combination with existing chemotherapeutics for the treatment of resistant breast cancers [20]. However, the mechanisms of ER-induced cell death and resistance in breast cancer cells are poorly understood. Understanding the mechanisms and pathways of how cancer cells acquire resistance to ferroptosis are extremely important for the development of newer strategies to specifically target breast cancer cells.
In this study, we investigated mechanisms underlying the differential sensitivity of parental MCF-7 breast cancer cells and their BCRP-overexpressing derivative, MCF-7/MXR, to ferroptosis-inducing agents, with a particular focus on ER. Our findings indicate that an enhanced antioxidant system centered on the FSP1–CoQ10–NADPH axis plays a key role in mediating ER resistance in MCF-7 cells, whereas reduced GSH levels together with ER-dependent inhibition of GPX4 activity enhances ER sensitivity to MCF-7/MXR cells. Ultimately, this knowledge could aid the development of highly effective ferroptosis-based treatments towards advancing cancer therapies.

2. Materials and Methods
Erastin, RSL3, Ferrostatin-1, DFO, N-acetyl cysteine (NAC), FSEN1 and iFSP1 were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and were dissolved in DMSO. Stock solutions were stored at −80 °C. Fresh drug solutions, prepared from the stock solutions, were used in all experiments.

2.1. Cell Culture
Human MCF-7 breast tumor cells were purchased from ATCC (Manassas, VA, USA). MCF-7/MXR breast tumor cells (MXR) were a gift from Dr. Erasmus Schneider (NCI/NIH [21]. Cells were cultured in Phenol Red-free RPMI media supplemented with 10% fetal bovine serum and antibiotics. Cells were only used for 20–25 passages, after which a new cell culture was started from fresh, frozen stock.

2.2. Cytotoxicity Studies
For cytotoxicity studies, both CellTiter-Glo (Promega, Madison, WI, USA) and Trypan Exclusion methods were utilized. We seeded about 2500–3000 cells/well for CellTiter-Glo assay in opaque white, 96-well plates that were allowed to attach overnight. Cells were then treated with various concentrations of drugs for 72 h, and cytotoxicity was determined according to the manufacturer’s instructions. For the Trypan Blue assay, about 30,000–40,000 cells/well were seeded onto a 12-well plate, and were allowed to attach for 24 h and treated with various drugs for 24 h. Following trypsinization, surviving cells were collected and 15 L of cell mixtures were combined with 15 µL of Trypan Blue and counted in T20 automatic cell counter (Bio-Rad, Hercules, CA, USA). Effects of various inhibitors (DFO, NAC, FES, FESN1, and iFSP1) were examined on the cytotoxicity of ER by preincubating with inhibitors for 1–2 h followed by the addition of various concentrations of ER and incubated for 24 h in complete medium.

2.3. Lipid Peroxidation Assay
Formation of malondialdehyde (MDA) using 2-thiobarbituric acid was used to assess cellular lipid peroxidation as previously described [22]. Briefly, approximately 2.5–3.0 × 10^6 cells/mL (2 mL) were incubated with different concentrations of ER for 4 h at 37 °C. The reactions were terminated by adding 2 mL of 10% trichloroacetic acid, followed by centrifugation at 1000× g for 5 min. Aliquots (1.5 mL) of the supernatants were mixed with 1.5 mL of 2% 2-thiobarbituric acid, and the mixture was heated to 90 °C for 10 min to develop the chromophore. After cooling, absorbance was measured at 532 nm.

2.4. RT-PCR
The expression levels of selected transcripts were confirmed by reverse transcription polymerase chain reaction (RT-PCR) using Absolute SYBR Green ROX Mix (Thermo Fisher Scientific, Rochester, NY, USA) as previously described [23]. Relative gene expression was analyzed using the ΔΔCt method, with cycle threshold values normalized to β-actin in time-matched samples. Primers for the selected genes were obtained from OriGene (Gaithersburg, MD, USA; Table 1). Real-time fluorescence detection was performed using an iCycler system (Bio-Rad, Hercules, CA). Data are presented as mean ± SEM from three independent experiments and were analyzed using an unpaired Student’s t-test, with p ≤ 0.05 considered statistically significant.

2.5. Detection of ROS
Invitrogen Molecular Probes fluorogenic reagents—CellROX Deep Red—was utilized for the detection and quantitation of ROS in MCF-7 and MCF-7/MXR cells following treatment with ER. The CellRox reagent is cell-permeable and is nonfluorescent in the reduced state. Upon oxidation by ROS, the reagents exhibit a strong fluorogenic signal that has an absorption/emission maxima of 644/665 nm and remains localized in the cytoplasm [24,25]. Cells were incubated with different concentrations of ER for 2–4 h in RPMI 1640 (no phenol red RPMI1640 media supplemented with 2.0% FBS) media. Following treatment, media was removed and washed twice with media followed by ice-cold PBS (4 x). Cells were then placed on ice in 1 mL PBS and fluorescence was imaged on a Zeiss inverted epifluorescent microscope.
Statistical Analysis: The results are expressed as mean ± SEM of minimum of 3 independent experiments (n = 3). One-way analysis of variance (ANOVA) or Student’s t-test was used for statistical analysis using Graph Pad Prism (GraphPad Software, Inc., La Jolla, CA, USA) and the results were considered statistically significant when p < 0.05.

3. Results

3.1. Cytotoxicity of ER and RSL3 in MCF-7 and MXR Cells
As previously reported, ER and RSL3 were significantly cytotoxic to MCF-7/MXR cells compared to parent MCF-7 cells (Figure 1) at 24 h and 72 h. MCF-7 cells were almost 10-fold resistant to both ER (Figure 1A–C) and RSL3 (Figure 1D,E).

3.2. RSL3 Enhances ER Cytotoxicity in MCF-7 and MXR Cells
RSL3, a direct inhibitor of GPX4, significantly enhanced cytotoxicity of ER in both cell lines (Figure 2A,B), suggesting that GPX4 was involved in the cytotoxicity of ER in these cells. This increased ER cytotoxicity was significantly attenuated by NAC (Figure 2C,D), indicating ROS-mediated cytotoxicity.

3.3. Ferrostatin-1, DFO and NAC Decrease Erasatin Cytotoxicity in MCF-7 and MXR Cells
Ferrostatin-1 (FES) and DFO, an iron chelator, are known inhibitors of ferroptosis. NAC is a known inhibitor of ROS. We utilized these agents to evaluate ER cytotoxicity in MCF-7 and MXR cells. As shown in Figure 3, these agents (at minimally cytotoxic doses) were highly effective in inhibiting ER cytotoxicity in MCF-7 and MXR cells, suggesting that ER cytotoxicity is ROS driven, is dependent upon presence of iron and formation of the lipid peroxides, which is a key factor in ferroptotic cell death.

3.4. ER Induces Lipid Peroxidation in MXR Cells
Formation of lipid peroxides by ER in MCF-7 and MXR cells was investigated. As shown in Figure 4A, significant amounts of peroxidation (measured as MDA) were detected only in MXR cells in a dose-dependent manner. In contrast, no MDA formation was detected in MCF-7 cells (Figure 4A). The effect of RSL3 on MDA formation was also examined since GPX4 is a known quencher of peroxide formation. As shown in Figure 4B, RSL3 significantly induced MDA formation in MCF-7 cells without markedly affecting MDA formation in MXR cells. However, more MDA was formed in MXR cells than MCF-7 cells when treated with RSL3.

3.5. Erastin Forms ROS in MCF-7 and MXR Cells
ER is known to generate ROS, causing oxidative damage in cells and leading to cell death [24,25]. Formation of ROS was therefore examined in MCF-7 and MXR cells using CellRox Deep Red [26,27], producing brightly deep red fluorescent. As shown in Figure 5, ER generated significant amounts of ROS in both cells in a dose-dependent manner. However, significantly more ROS was formed in MXR cells compared to MCF-7 cells, especially at lower concentrations of ER (Figure 5, ER at 5µM and 10 µM).

3.6. Modulations of Gene Expression Changes by ER in MCF-7 and MXR Cells
RT-PCR studies were carried out to monitor changes in gene expressions by ER. As shown in Figure 6, ER significantly modulated activity of several oxidative stress- and ferroptosis-related genes. Heme oxygenase-1 gene (HMOX1) expression was significantly induced in both cells by ER, indicating an enhanced labile iron pool and oxidative stress in both cells. CHAC1, responsible for hydrolysis (degradation) of cellular GSH, was markedly increased in MCF-7 cells while ER decreased CHAC1 expression in MXR cells. Although ER treatment significantly inhibited GPX4 expression in MXR cells, it was induced in MCF-7 cells. SLC7A11, the Xc- transporter, was induced in MCF-7 by ER, but was significantly inhibited in MXR cells.

3.7. Effects of Inhibitors of FSP1 on Erastin Cytotoxicity in MCF-7 and MXR Cells
To elucidate mechanism of differential cytotoxicity of ER in these cells, effects of various known inhibitors of FSP1, a protein involved in the inhibition of ferroptosis in cells and causing drug resistance [28,29,30,31], was examined. Treatment of MCF-7 and MXR with FSEN1, a strong non-competitive inhibitor of substrate-binding pocket of FSP1 [32,33], showed similar cytotoxicity in both cell lines. Treatment of MCF-7 and MXR cells with a non-toxic dose of FSEN1 in the presence of ER showed markedly different outcomes; FESN1 significantly enhanced ER cytotoxicity in MCF-7 while it had minimal effects on ER cytotoxicity in MXR cells (Figure 7). These observations suggest that MCF-7 cells have either higher expression of FSP1 or are dependent upon the FSP1/CoQ10 pathway for their antioxidant defense, based on their decreased sensitivity to ER-induced ferroptosis as observed here. In contrast, MXR cells must have lower expression of FSP1 or other antioxidants as FSEN1 does not affect the cytotoxicity of ER on MXR cells.
To further confirm these observations, we utilized another inhibitor of FSP1. iFSP1 is a known inhibitor of FSP1 but differs from FSEN1 in that it binds to the FSP1 protein and deactivates its oxidoreductase function, preventing FSP1 from using NAD(P)H to reduce ubiquinone [16]. iFSP1 was not significantly toxic to MCF-7 cells at concentrations utilized here (Figure 7E), but was cytotoxic to MXR cells at higher concentrations (Figure 7F). iFSP1, when used at minimally cytotoxic doses (10 µM), significantly enhanced cytotoxicity of ER in MCF-7 cells (Figure 7G), but it had minimal effects on ER cytotoxicity in MXR cells (Figure 7H). These results further suggest that MCF-7 cells depend upon the FSP1-coenzyme Q10 (CoQ10)-NAD(P)H axis for its antioxidant properties to prevent ferroptosis and reduce cytotoxicity of ER in MCF-7 cells.

3.8. Erastin Decreases FSP1 and NQO1 Expressions in MCF-7 Cells
To further elucidate the mechanism underlying the differential enhancement of ER cytotoxicity by FSP1 inhibitors in MCF-7 cells, we performed RT-PCR analyses to examine the effects of ER on the expression of FSP1, NQO1, and other antioxidant genes in MCF-7 and MXR cells. ER significantly suppressed the expression of both FSP1 and NQO1 in MCF-7 cells, whereas no significant changes were observed in MXR cells (Figure 6B). In addition, ER markedly modulated the expression of multiple antioxidant response genes in MCF-7 cells, including NRF2, GSR, SOD1, and SOD2 (Figure 6B). Although ER reduced NRF2 expression in MXR cells, this effect was significantly weaker than that observed in MCF-7 cells, and ER had no measurable impact on GSR, SOD1, or SOD2 expression in MXR cells.
Basal gene expression levels of GPX4, SCL7A11, FSP1 and NQO1 were also compared which showed significant differences between MCF-7 and MXR cells (Figure 6C). GPX4 and FSP1 expression levels were comparable between MCF-7 and MXR cells; however, MXR cells exhibited significantly reduced expression of both SLC7A11 and NQO1, indicating that differences in ferroptosis sensitivity may arise from impaired cystine uptake and redox cofactor regeneration rather than from altered expression of core ferroptosis suppressor proteins.

4. Discussion
While significant achievements have been made in the treatment of breast cancer, it is still a leading cause of cancer death in women worldwide, accounting for 23% of cancer diagnoses and 14% of cancer deaths each year [1,3,34]. In breast cancer, drug resistance is responsible for up to 90% of deaths and multidrug resistance (MDR) hinders the efficacy of chemotherapeutic drugs, often leading to relapse and metastasis [35]. A major cause of treatment failure is due to the development of therapy resistance and new therapeutic agents are urgently needed. Recently, ferroptosis has attracted significant attention in the treatment of human tumors, including breast cancers, as ferroptosis-inducing agents can sensitize resistant tumor cells to chemotherapeutic agents. Studies have shown that ferroptosis inducers such as ER can reverse drug resistance and sensitize tumor cells to various chemotherapeutic agents in multiple ABC transporter-expressing cell lines. Zhou et al. [18] reported that docetaxel, an ABCB1 substrate, is highly synergistic with ER in an ABCB1-expressing ovarian cancer cell line. Erastin significantly increased intracellular docetaxel accumulation by inhibiting the drug-efflux activity of ABCB1 without altering its expression, suggesting that the combination of ER and docetaxel may represent an effective strategy for treating chemo-resistant ovarian cancer. Our previous studies [19,20] also demonstrated strong synergy between ER and doxorubicin (Adriamycin) in an ABCB1-overexpressing ovarian cancer cell line. In addition, ER markedly enhanced the cytotoxicity of topotecan in an ABCG2-overexpressing breast cancer cell line. In all cases, the reversal of drug resistance was associated with ER-mediated inhibition of transporter function, leading to increased intracellular drug accumulation.
Consistent with these findings, Chen et al. [36] showed that ER synergized with docetaxel in docetaxel-resistant prostate cancer cells, overexpressing the ABCB1 protein. Erastin inhibited ABCB1 activity without affecting its protein expression or subcellular localization, indicating that ER reverses docetaxel resistance by suppressing the function of this multidrug-resistance transporter.
Furthermore, ER has been reported to sensitize ovarian cancer cells to cisplatin [37], while PRLX939, an ER analog, synergized with cisplatin in non-small-cell lung cancer cells [38]. RSL3 has also been shown to act synergistically with several anticancer drugs and with radiotherapy [39]. Sorafenib, a tyrosine kinase inhibitor and ferroptosis inducer, enhances sensitivity to cisplatin [40] and doxorubicin [41]. Similarly, sulfasalazine, a system xCT inhibitor and potent ferroptosis inducer, has demonstrated synergy with cisplatin [42] and doxorubicin [43] in bladder and breast cancer cells, respectively. Collectively, these studies highlight the potential of ferroptosis inducers as promising agents for overcoming chemotherapy resistance in clinical settings.
Our studies show that both ER and RSL3 are markedly more cytotoxic to MCF-7/MXR cells than the parent MCF-7 breast cancer cells. The mechanisms of ER resistance to tumor cells remain understudied and there is an urgent need to understand reasons for this in breast and other cancers for better treatment options. In this study, we sought to understand both the mechanism (s) of ER-dependent cell death and how resistance to ferroptosis-inducing agents develops in breast cancer cells. Studies showed that while MCF-7 cells are resistant to ER, RSL3, a direct inhibitor of GPX4, sensitized both MCF-7 and MXR breast tumor cells to ER. However, RSL3 was less effective in MCF-7 cells than MXR cells, indicating that GPX4 is responsible, in part, for the decreased toxicity of ER in MCF-7 cells. MCF-7 cells have been shown to have high GSH and GPX4 activity [44]. Additionally, we found that ER did not induce MDA formation in MCF-7 cells while MXR cells were highly sensitive to ER, generating significant amounts of MDA. Presence of RSL3 again significantly enhanced MDA formation in MCF-7 without affecting MXR cells, indicating that GPX4 is involved in decreased MDA formation in MCF-7 cells. Cytotoxicity of ER was inhibited by FES (inhibitor of lipid radicals) and DFO (an iron chelator) in both cell lines, indicating ferroptotic cell death by ER. Furthermore, NAC, an inhibitor of ROS also significantly inhibited the cytotoxicity of ER and RSL3 in both cell lines, indicating ROS-mediated cell death. Formation of ROS was confirmed in both cells.
We found that ER significantly induced the expression of HMOX1, a biomarker of oxidative stress, in both cell lines. However, ER-dependent induction of HMOX1 was more pronounced in the MXR cells than in the MCF-7 cells, consistent with the increased ROS production we observed in MXR cells. However, effects of ER on GPX4 and SLC7A11, two genes responsible for either use or import of cellular GSH respectively, were markedly different in these cell lines. Expression of GPX4 and SLC7A11 were significantly decreased by ER in MXR cells while expression of these genes was increased in MCF-7 cells. This suggests that the GSH-GPX4 pathway may be more active in MXR cells than in MCF-7 cells, potentially contributing to increased cell death. Still, that MCF-7 cells showed minimal lipid peroxidation and markedly reduced cytotoxicity in response to ER suggests that additional ferroptosis-inhibiting pathways may be involved.
To investigate additional pathways responsible for ER-resistance in MCF-7 cells, we utilized inhibitors of FSP1, a protein that confers resistance to both ferroptosis and apoptosis in tumor cells [17,28,30,45,46,47]. Two different inhibitors of FSP1, FSEN1 and iFSP1, were utilized to examine their effects on ER-induced cell death in these cell lines. Minimally cytotoxic doses of these inhibitors were significantly effective in enhancing ER-induced cytotoxicity in MCF-7 cells without modulating ER cytotoxicity in MXR cells. This differential enhancement of ER cytotoxicity in MCF-7 cells suggest that FSP1 plays a significant role in protecting MCF-7 cells from ferroptotic death, resulting in decreased cytotoxicity of ER. These conclusions are further highlighted by our finding that there are significant differences in expression levels of ferroptosis-related genes, SCL7A11 and NQO1 between MCF-7 and MXR cells despite comparable GPX4 and FSP1 expression. MCF-7 cells exhibited significantly higher levels of SLC7A11 and NQO1, whereas MXR cells showed marked downregulation of both genes. Since SLC7A11 plays critical roles in cystine uptake and glutathione synthesis and NQO1 in coenzyme Q redox cycling, these findings indicate that MXR cells possess an impaired upstream redox and metabolic capacity to support ferroptosis defense. This molecular context provides a mechanistic explanation for our drug response data, in which combined inhibition of system xCT (ER) and FSP1 produced strong synergistic cytotoxicity in MCF-7 cells but not in MXR cells. In MCF-7 cells, elevated SLC7A11 and NQO1 sustain parallel GPX4- and FSP1-dependent protective pathways, rendering dual pathway blockade highly effective. In contrast, reduced SLC7A11 and NQO1 expression in MXR cells limit glutathione availability and CoQ redox buffering upstream of GPX4 and FSP1, thereby diminishing the added benefit of FSP1 inhibition.
These results highlight a previously uncharacterized mechanistic divergence in ferroptosis regulation between MCF-7 and MXR breast cancer cells. MXR cells exhibit classical GPX4-dependent ferroptosis sensitivity, whereas MCF-7 cells are protected by a robust FSP1-driven defense system, rendering them less responsive to ER alone. This mechanistic distinction has important implications for designing targeted therapies aimed at overcoming ferroptosis resistance in breast cancer. Unfortunately, FSP1 inhibitors such as iFSP1 and FSEN1 are not in the clinic due to poor solubility, limited potency, and unfavorable chemical scaffolds that restrict further medicinal chemistry optimization into viable therapeutic agents. Moreover, FSP1 is expressed in both normal and tumor tissues, posing a significant challenge for achieving cancer-selective inhibition without inducing toxicity in healthy cells. Nevertheless, these compounds serve as valuable experimental tools for elucidating the role of FSP1 in regulating ferroptosis in preclinical models.

5. Conclusions
This study demonstrates that parental MCF-7 breast cancer cells and their MXR derivative exhibit fundamentally different mechanisms governing their sensitivity to ferroptosis induction. MCF-7 resistance to ER arises primarily from activation of the FSP1–CoQ10–NADPH pathway and upregulation of the GSH–GPX4 antioxidant system, which together suppress lipid peroxidation and ferroptotic death. In contrast, MXR cells rely heavily on the xCT–GSH–GPX4 axis, making them highly susceptible to ER-induced ferroptosis. Our findings identify FSP1 as a critical regulator of ferroptosis resistance in MCF-7 cells and suggest that co-targeting GPX4 and FSP1 may provide a powerful strategy to sensitize resistant breast cancer cells to ferroptosis-inducing agents. These insights improve our understanding of ferroptosis regulation in breast cancer and support further investigation into combination therapies involving ER, RSL3, and FSP1 inhibitors as potential approaches to overcome drug resistance.