Lipid oxidation is strictly regulated by the antioxidant defense system. GPX4 is considered to be a vital antioxidant enzyme, which directly eliminates hydroperoxides in lipid bilayers, thus preventing lethal lipid ROS production (Yang et al., 2014). GSH/GPX4 pathway, FSP1/COQ10/NADPH pathway, DHODH pathway and GCH1/BH4 pathway are identified as defense system in ferroptosis.

Guanosine triphosphate cyclohydrolase 1 (GCH1) is a rate-limiting enzyme for tetrahydrobiopterin BH4 synthesis to inhibit ferroptosis independently of GPX4. BH4 is an effective free radical trapping antioxidant that can prevent lipid peroxidation and can be regenerated by dihydrofolate reductase (DHFR). The inhibition of DHFR cooperates with GPX4 deletion could induce ferroptosis (Kraft et al., 2020). GCH1 promotes the immunosuppression of TNBC by regulating tryptophan metabolism (Wei et al., 2021). Further studies are needed to clarify the molecular mechanism of GCH1/BH4 in ferroptosis defense of breast cancer.

Nuclear factor erythroid2-related factor 2 (Nrf2) can regulate ferroptosis through p62-Keap1-NRF2 pathway. The activation of p62-Keap1-NRF2 signal pathway can also promote the expression of System Xc-, accelerate the transportation of cystine/glutamic acid, and thus eliminate the accumulated lipid peroxidation. The activation of Nrf2 promotes the iron storage, reduces the iron consumption, increasing the expression of target genes NADH Dehydrogenase Quinone 1 (NQO1) and HO-1 which are related to iron and ROS (Xu et al., 2019). Glycogen synthase kinase-3 β (GSK-3 β) promoting erastin-induced ferroptosis by blocking Nrf2 in breast cancer (Wu et al., 2020). Suppressor of fused homolog (SUFU) is a key element of the Hedgehog pathway (Fang et al., 2022). The deletion of SUFU can increase the ferroptosis of breast cancer cells activated by RSL3 by decreasing ACSL4. Vincristine can promote the RSL3 sensitivity of breast cancer cells by downregulating SUFU. Protein kinase C-βII (PKCβII) is activated by lipid peroxidation during ferroptosis, which can directly phosphorylate the Thr328 site of ACSL4 to activate ACSL4, strengthen the biosynthesis of PUFA-containing lipids, and induce lipid peroxidation. PKCβII induces ferroptosis and improves the response of immunotherapy (Zhang et al., 2022).

Recent studies have shown that cancer stem cells (CSC) are relatively enriched in lung metastases due to their high invasive properties, and enriched CSCs can secrete negative Dickkopf1 (DKK1) feedback to inhibit their stem cell properties. Because CSCs are highly sensitive to ferroptosis, DKK1 induces an increase in SLC7A11 expression and regulates inhibition of CSC properties and thus can protect lung metastatic cells from ferroptosis and promote the development of metastases. In multiple models of breast cancer metastasis, small-molecule inhibitors of DKK1 can almost completely block the occurrence of lung metastases (Wu M. et al., 2022). p53 induces ferroptosis by SLC7A11 (Magri et al., 2021). In fact, mutant p53 inhibits Nrf2-dependent SLC7A11 transcription to promote SLC7A11 expression (Clemons et al., 2017). In addition, p53 promotes the nuclear translocation of ubiquitin-specific protease (USP7) and reduces the expression of SLC7A11 by inhibiting H2B monoubiquitination (H2Bub1) (Wang Y. et al., 2019).

DMOCPTL can induce EGR1-mediated apoptosis and ferroptosis and in TNBC by GPX4 ubiquitination. Long-term exposure to the cholesterol metabolite 27-hydroxycholesterol (27HC) causes breast cancer cells to be resistant to 27HC and overexpress GPX4, which inhibits ferroptosis and increases tumorigenicity and tumor metastasis (Liu W. et al., 2021). Up-regulation of the discoidin domain receptor 2 (DDR2) elicits the susceptibility of ferroptosis by GSH consumption of recurrent breast cancer through the Hippo pathway. Epithelial mesenchymal transition-driven upregulation of DDR2 suppresses tumor proliferation in recurrent breast cancer by regulating YAP/TAZ-mediated ferroptosis (Lin et al., 2021). The inhibiting of Fibroblast Growth Factor Receptor 4 (FGFR4) mediated by m⁶A hypomethylation significantly suppress GSH synthesis and efflux of Fe²⁺ through β-catenin/TCF4-SLC7A11/FPN1 signaling pathway, which trigger the excessive production of ROS and abnormal iron pool accumulation in Her-2 positive breast cancer (Zou et al., 2022).

The tumor microenvironment (TME), especially immune cells, has an important impact on ferroptosis in cancer cells. Interferon-γ (IFN-γ) secreted by CD8+ cytotoxic T cells inhibits cystine uptake by cancer cells by downregulating SLC7A11, thus triggering lipid peroxidation and ferroptosis in various tumors (Wang W. et al., 2019). Tumor-associated macrophages (TAM) secrete transforming growth factor beta 2 (TGF-β2) to regulate liver leukemia factor (HLF). HLF inhibits ferroptosis by activating GGT1 and induces TNBC chemoresistance (Li H. et al., 2022). Adipocytes induce ferroptosis resistance in breast cancer by secreting specific fatty acids. Mono-unsaturated Fatty Acid (MUFA) regulates breast cancer cells by binding with acyl-CoA synthetase long chain family member 3 (ACSL3) to phospholipids, and it is confirmed that the regulation of ferroptosis in breast cancer by adipocytes and exogenous MUFA is dependent on ACSL3 (Xie et al., 2022).

Non-coding RNAs (ncRNAs) are regarded as novel regulators of various cellular processes in cancer (Zuo et al., 2022); (Figure 3). Some recent studies have shown that they participate in the molecular regulation of ferroptosis. The ectopic overexpression of SLC7A11 inhibits miR-5096-mediated ferroptosis and diminishes antitumor effects in breast cancer (Yadav et al., 2021). PGM5P3-AS1 regulates MAP1LC3C to confer ferroptosis and thus inhibits the progression of TNBC (Qi et al., 2022). Circular RNA RHOT1 prohibits the occurrence of ferroptosis by targeting the miR-106a-5p/STAT3 signaling pathway and increases breast cancer proliferation (Zhang H. et al., 2021). CircGFRA1 facilitates progression of the malignant phenotype of Her-2 positive breast cancer by sponging with miR-1228 and improving FSP1 expression (Bazhabayi et al., 2021). Circ-BGN binds to OTUB1 and SLC7A11 and inhibits ferroptosis to confer resistance to trastuzumab in HER-2+ breast cancer (Wang S. et al., 2022). A integrity understanding of these regulatory network might facilicate the discovery of vulnerable therapeutic target of ferroptosis in breast cancer.

The demand of tumor cells for iron has increased significantly indicating that iron chelators may be an effective anti-cancer strategy iron chelators exert their anticancer potential by consuming intracellular iron, inhibiting DNA synthesis, inducing apoptosis, and oxidative stress. Furthermore, the reduction of intracellular iron through iron chelation can sensitize breast cancer cells to chemotherapy (Tury et al., 2018). For example, deferoxamine (DFO) and deferoxazole (DFX) show potential antitumor activity in breast cancer by limiting iron bioavailability. DFO promote iron uptake to activate TRF1 and divalent metal transporter 1 (DMT1) via the IL6/PI3K/AKT axis (Chen et al., 2019). The physiologically achievable concentration of DFO monotherapy significantly active the apoptosis of breast cancer cells and the xenotransplantation of mouse breast tumors by activating the tumor necrosis factor-α (TNF-α)/nuclear factor kappa B (NF-κB) axis and transform growth factor β (TGF-β). DFO promoted migration in the ROS-dependent NF-κB and TGF-β signaling pathway by enhancing mitochondrial iron accumulation in TNBC (Liu et al., 2016). Although preclinical studies have confirmed that iron chelating agents inhibit tumor growth and metastasis by certain signaling pathways. However, in clinical studies, iron chelating agents only show moderate therapeutic benefits, and may promote anti-tumor drug resistance due to ferroptosis. It is urgent to improve the bioavailability of iron chelating agents and combination therapy.

Some Chinese medicine and natural compounds have been shown to induce ferroptosis in breast cancer by targeting iron overload. Shuganning induces ferroptosis via Heme oxygenase-1(HO-1) to increase intracellular iron accumulation and suppresses tumor growth in TNBC (Du et al., 2021). Artemisinin is activated by Fe²⁺ to generate free radicals. The iron supplements can improve the anticancer efficacy of artemisinin. Artemisinin can induce ferroptosis by promoting ferritin phagocytosis and thus increasing intracellular iron levels (Chen et al., 2020). A completed phase I clinical trial (NCT00764036) showed that for patients with metastatic breast cancer, oral artemisinin of up to 200 mg per day was deemed safe and well tolerated (von Hagens et al., 2017).

Ferritinophagy serves as a link between ferroptosis and autophagy (Latunde-Dada, 2017). Ferritinophagy transports ferritin to the lysosome for degradation. The release of free iron leads to increased iron, and the Fenton reaction occurs to generate oxidative stress, which is essential for ferroptosis. Autophagic degradation reduces the expression of the ferritin storage protein. Decreased ferritin expression promotes the onset of ferroptosis. The BRD4 inhibitor (+)-JQ1 (JQ1) confer ferroptosis by autophagy in MDA-MB-231 cells. JQ1 was shown to induce ferritin phagocytosis or decreased the expression of GPX4, SLC7A11, and SLC3A2 (Sui et al., 2019). Dihydroartemisinin (DAT) combined with RSL3 was used to treat breast cancer by targeting ferritin. DAT induces lysosomal degradation of ferritin and impact the iron regulatory protein (IRP)/iron response element (IRE) controlled iron homeostasis to promote the level of cellular free iron (Chen et al., 2020).

System Xc–, a glutamate-cystine antiporter, is consists of the light chain subunit SLC7A11 and the heavy chain subunit SLC3A2. SLC3A2 anchors SLC7A11 to the plasma membrane and maintains the stability of SLC7A11 (Liu M. R. et al., 2021). Several compounds-such as erastin, sulfasalazine, and sorafenib—have been utilized as inhibitors of SLC7A11. The erastin is a classic ferroptosis agonist that inhibits the function of System Xc- by irreversibly binding to SLC7A11 and inactivating SLC7A11, resulting in the depletion of GPX4 and GSH. The erastin-induced ferroptosis by increasing cellular lipid ROS in TNBC (Wang H. et al., 2022). Fascin directly interacts with System Xc- and decreases its stability via ubiquitination. Fascin enhances anti-tumor effect to erastin-induced ferroptosis (Chen C. et al., 2022). Sulfasalazine (SAS) can active ROS production to induce the ferroptosis in breast cancer.

Metformin increases Fe²⁺ and lipid ROS, reduces the stability of SLC7A11 by inhibiting the ultrafiltration process of SLC7A11, and metformin combined with SAS can synergistically activate ferroptosis and inhibit the growth of breast cancer cells (Yang et al., 2021). The pH regulator carbonic anhydrase IX (CAIX) induced by hypoxia prevents ferroptosis in cancer cells, and co-targeting of CAIX/XII and xCT induce ferroptosis in TNBC. The novel synthetic lethal interaction will help researchers develop new strategies for solid tumor therapy (Chafe et al., 2021). p53 and BRCA1 Associated Protein 1 (BAP1) inhibit carcinogenesis in part by antagonizing SLC7A11 and induce ferroptosis (Zhang Y. et al., 2019). Eupaformosanin was shown to induce ferroptosis by ubiquitination of the mutant p53 in TNBC (Wei et al., 2022).

GPX4 is an essential enzyme for the elimination of lipid hydrogen peroxide. GPX4 reduces the production of ROS radicals in membrane lipids and protects normal breast cells from ROS DNA damage to inhibit ferroptosis. GPX4 inhibitors include RSL3, the RSL3 analogs DPI7, DPI12, DPI13, DPI17, DPI18 and DPI19, ML210, JKE1647, altretamine, FIN56, FINO2, dihydroartemisinin, and Withaferin A. RSL3 inactivates GPX4, leading to ferroptosis by ROS accumulation (Stockwell, 2022). Erastin also suppresses the expression of GPX4 in breast cancer, especially in TNBC. Vitamin C induces TNBC cells death by suppressing GPX4. Furthermore, sequential treatment with VC and quercetin (Q) significantly increased ROS. A study suggested that vitamin C and Q can be developed as an adjuvant agents for cancer patients by Nrf2 (Mostafavi-Pour et al., 2017). Statins, the mevalonate pathway inhibitors, are identified as ferroptosis agents that target HMGCR. Statins suppress CoQ10 production to induce ferroptosis and decrease GPX4 (Gobel et al., 2019). Lycium barbarum polysaccharide (LBP), a traditional Chinese medicine, promotes ferroptosis in breast cancer by modulating the xCT/GPX4 pathway (Du et al., 2022). Ketamine inhibits GPX4 levels and inhibits histone H3 lysine 27 acetylation (H3K27ac) and enrichment of RNA polymerase II (RNA pol II) by attenuating KAT5 on the GPX4 promoter in breast cancer cells (Li H. et al., 2021). Glycyrrhetinic acid (GA) induces lipid peroxidation to trigger ferroptosis in TNBC cells by reducing the activity of GSH and GPX4 (Wen et al., 2021). DMOCPTL can induce EGR1 mediated apoptosis and ferroptosis and in TNBC by GPX4 ubiquitination (Ding et al., 2021). Researchers screened drug combinations using a synthetic lethal mechanism (simultaneous inhibition of two or more non-lethal genes leading to cell death) and found that low doses of targeted drugs targeting BET and cytoplasmic proteins in vitro could reduce the levels of GPX4 and trigger ferroptosis in TNBC (Verma et al., 2020).

Nanomedicine is a favorable solution for the development of ferroptosis because of its better efficacy and improved targeting ability, low systemic toxicity, and high safety (Zafar et al., 2021). The mechanism of emerging nanomedical therapy is mainly including: 1) triggering Fenton reaction; 2) inhibiting the expression of GPX4; 3) exogenous regulation of lipid peroxidation. The diversified multifunctional nanomaterials can produce H₂O₂, eliminate GSH, and provide Fenton reaction catalyst. In addition, some iron-containing nanomaterials can also induce ferroptosis. Azo-CA4 was loaded into nanocarriers and is converted by near-infrared light to ultraviolet light. After irradiation, Azo-CA4-loaded nanocarriers reduced the viability of TNBC through ferroptosis (Zhu et al., 2020). Sonodynamically amplified ferroptotic red blood cells (SAFEs), a state-of-the-art red blood cell was engineered for simultaneous activation of ferroptosis and oxygen-enriched sonodynamic therapy (SDT). SAFE contains internalized RGD peptide and erythrocyte membrane mixed with camouflaged hemoglobin nanocomplex, perfluorocarbon, sonosensitizer verteporfin, and FINS (dihomo-gamma-linolenic acid, DGLA). SAFE can not only provide abundant oxygen to the hypoxia-related therapeutic resistance of the defect by ultrasound stimulation, but also activate ferroptosis by the combination of ROS production and lipid peroxidation, which selectively accumulates and induces desirable anticancer effects in the tumor (Zhou A. et al., 2022). Heparinase-driven continuous release nanoparticles (HSPE) can enhance the killing power of tumor cells via DOX-induced ferroptosis, regulating the tumor microenvironment, and effectively inhibiting breast cancer metastasis (Zhang J. et al., 2021). The lapatinib and pseudolaric acid B (PAB) were delivered by ferritin-containing nanoparticles, which showed an enhanced tumor suppression ability by increasing ROS (Wu X. et al., 2022).

A tumor-targeted MOF nanosheet system with multienzyme-like catalytic activity can inhibit the antiferroptotic mechanism mediated by GPX4 and FSP1 in tumor cells, thus enhancing ferroptotic damage. After iRGD promoted tumor cells to uptake Au/Cu-TCPP(Fe)@RSL3-PEG-iRGD nanosheets, besides the ability of RSL3 to activate GPX4, the glucose oxidase (Gox) activity of gold nanoparticles could easily comsume intracellular glucose and inhibit the production of NADPH, resulting in reduced expression of GSH and GPX4 (Li K. et al., 2022). Rosuvastatin (RSV) was loaded into silk fibroin (SF) nanoparticles (NPs) (Cu-SF(RSV), to disrupt redox stabilization regulated by the CoQ10/FSP1 axis by interfering with the mevalonate pathway to improve ferroptosis sensitivity in TNBC (Yang J. et al., 2022). In one study, the drug simvastatin (SIM) was loaded into zwitterionic polymer-coated magnetic nanoparticles (Fe₃O₄@PCBMA). PCBMA promoted the accumulation of Fe3O4@PCBMA in tumor lesions, while SIM inhibited the expression of HMGCR, thereby downregulating mevalonate pathway and GPX4 expression, and inducing ferroptosis (Yao et al., 2021). Cinnamaldehyde dimers (CDCs) induce lipid materials to form dimers, which enable them to deplete GSH and deliver therapeutics to enhance ferroptosis and immunotherapeutic effects against breast cancer (Zhou Z. et al., 2022). The nanoparticles consisting of ferritin and a pH-sensitive molecular switch (FPBC@SN) disintegrate in the acidic cytoplasm and release sorafenib and an indoleamine2,3-dioxygenase (IDO) inhibitor NLG919. NLG919 reduces tryptophan metabolism by inhibiting IDO, thus improving tumor immunity and antitumor efficacy. Sorafenib upregulates nuclear receptor coactivator 4 (NCOA4), which produces lipid peroxide (LPO) through ferritin autophagy. Down-regulation of GPX4 and LPO clearance elicit the ferroptosis in tumor cells (Zuo et al., 2021).

The combination of immunotherapy and ferroptosis death nanoparticles can also play an ideal therapeutic effect on breast cancer. A bionic Fe₃O₄ magnetosome (PaM/Ti NC), TGF-β Inhibitor (Ti) is carried in the membrane and PD-1 antibody (Pa) is anchored on the outer surface of the nanoparticles. The level of intracellular H₂O₂ can be increased under immunogenic microenvironment, thus the ferroptosis can be triggered by Fenton reaction. The ideal combined anti-tumor effect is achieved through the mutual promotion of ferroptosis and immunomodulation (Zhang F. et al., 2019).

Lapatinib is a potent double tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) and HER-2 which inhibits the proliferation of breast cancer cells with overexpression of ErbB2 and EGFR (Guarneri et al., 2021). Siramesine is a lysosomotropic agent that releases cathepsin B from lysosomes, leading to increased ROS production and cell death (Petersen et al., 2013). The combination of lapatinib and siramesine could induce ferroptosis in TNBC by boosting intracellular Fe2+ (Ma et al., 2016).

RT is a common cancer treatment that uses the ionizing radiation (IR) to eradicate cancer cells (Delaney et al., 2005). Binding of FINS targeting GPX4 or SLC7A11 to RT can sensitize breast cancer cell lines or xenografts to radiation by enhancing ferroptosis sensitivity. Zhang et al. found that iron-saturated lactoferrin (holo-lactoferrin, Holo-Lf) significantly decreased GPX4 expression in MDA-MB-231 cells and cell viability, and combination therapy with erastin could further inhibit cell viability. The combination of Holo-Lf with RT can significantly increase the production of lipid ROS in MDA-MB-231 cells, indicating that RT can promote cell ferroptosis and improve radiosensitivity (Zhang et al., 2021c). Pre-red laser irradiation combined with gallic acid could enhance anticancer effects by decreasing GPX4 activity (Khorsandi et al., 2020). γ-radiation combined with erastin improves GSH synthesis and controls ROS generation, the therapeutic effectors of radiation therapy (Cobler et al., 2018). Knocking down ESR1 expression may enhance the efficacy of radiation-induced ferroptosis via the NEDD4L/CD71 pathway in breast cancer (Liu et al., 2022). Although the combination of RT and FINS appears to be safe in preclinical studies, some researches have suggested that ferroptosis may also be associated with RT-induced damage of normal tissue (Li et al., 2019). Therefore, it is urgent to clarify whether this combination therapy has negligible toxicity to normal tissues.

Dichloroacetate (DCA) combined with albiziabioside A (AlbA) can inhibit GPX4 and induce ferroptosis to inhibit tumor progression (Wei et al., 2019). DCA can also improve glycolytic lactate immunosuppression and promote the effect of immunotherapy (Ohashi et al., 2013). AlbA has the characteristic of selective antitumor effect without affecting normal cells (Wei et al., 2014). The synergistic effect of the AlbA-DCA conjugate which can affect the polarization of M2-TAM to reprogram the tumor immune microenvironment and ferroptosis will enhance therapeutic efficacy.

Photodynamic therapy (PDT) gather lymphocyte infiltration in tumor lesions and stimulates IFN-γ secretion (Xu et al., 2020). Hemoglobin (Hb) can be used as an oxygen provider in PDT due to its excellent oxygen-carrying properties and can also be used as an iron supplement in the treatment of ferroptosis due to the iron component (Wang et al., 2015). Synergistically induced ferroptosis in cancer cells could be a potential strategy for treating breast cancer (Xu et al., 2020).

The development of various FINS and inhibitors has accelerated research on the ferroptosis and chemosensitivity. ACSL4 in tumor lesions can be used as a predictive factor of complete pathological remission and progression-free survival of neoadjuvant chemotherapy in breast cancer. Importantly, the imbalance between ACSL4/GPX4 can indicate the level of ferroptosis after undergoing neoadjuvant chemotherapy, effectively distinguishing patients with different complete pathological response rates and disease-free survival. With the increase in ACSL4 and the decrease in GPX4, the pathological complete remission rate of patients has gradually increased (Sha et al., 2021).

The iron chelator deferasirox can synergistically enhance the efficacy of doxorubicin and cisplatin in TNBC without increasing side effects (Tury et al., 2018). Saponin formosanin C (FC) is a potent FIN that showed the strongest cytostatic effect among all anticancer drugs, including cisplatin, using the MDA-MB-231 cell line as a model (Chen H. C. et al., 2022). The excessive intake of dietary iron leads to the production of oxidative stress and DNA damage. A collaborative group clinical trial revealed that the use of antioxidant supplements during chemotherapy, such as iron and vitamin B12, increased intracellular iron to serve as a propellant and could increase recurrence and mortality in breast cancer. This finding should be considered when discussing the use of dietary supplements during chemotherapy with breast cancer patients (Ambrosone et al., 2020).