Targeting ferroptosis to overcome drug resistance in lung cancer.

Introduction
Ferroptosis is a unique form of cell death that has been increasingly recognized in recent years as a key factor in numerous physiological and pathological conditions, which is dependent on elemental iron and accompanied by lipid peroxidation processes (1). Since the concept of ferroptosis was coined in 2012, research in the realm of ferroptosis has climbed at an exponential pace. This form of cell death is characterized by an iron-dependent accumulation of lipid peroxidation products, leading to membrane damage and ultimately cell death. This process is under the regulation of multiple enzymes and metabolic pathways, particularly glutathione peroxidase 4 (GPX4) and lipoxygenase (LOXs) (2). LOXs promotes ferroptosis through generating lipid peroxides, and in contrast, GPX4 inhibits ferroptosis using the reductive capacity of glutathione (GSH) (1). Recent studies found that ferroptosis can inhibit tumorigenesis and eliminate the cancer cells with high proliferative capacity (3,4).
Lung cancer stands as one of the most prevalent malignant tumors and constitutes the primary cause of mortality resulting from neoplastic diseases worldwide (5). Although there have been considerable advances in lung cancer treatment, the emergence of drug resistance in lung cancer continues to be a major challenge. Extensive preclinical and clinical studies focus on surmounting drug resistance (6). Recent studies have shown that ferroptosis is linked to cancer treatment resistance, and inducing immunogenic ferroptosis can be beneficial in cancer patients who are resistant to apoptosis-inducing drugs (7). High mesenchymal stem cell (MSC) status in lung cancer has consistently been recognized as being associated with resistance to multiple lung cancer treatment modalities. Drug-resistant MSC status is highly dependent on the lipid peroxidase pathway regulated by GPX4, which renders them especially susceptible to ferroptosis (8). Furthermore, numerous studies have shown that regulation of ferroptosis affects the effectiveness of tumor therapy and even reverses tumor treatment resistance (9,10).
Here, we describe in detail the mechanisms underlying targeting ferroptosis and the therapeutic role of modulating ferroptosis in counteracting lung cancer resistance to common therapies, such as chemotherapy, targeted therapy, immunotherapy, and photodynamic therapy (PDT). Furthermore, we discuss the prospects and challenges of targeted ferroptosis as a therapeutic strategy for reversing treatment resistance in lung cancer.

Regulatory mechanisms of ferroptosis
Ferroptosis represents a newly identified form of regulatory cell death (RCD), which is characterized by phospholipid membrane damage due to iron-dependent oxidation (11). Ever since the GPX4-centered mechanism of ferroptosis was elucidated in 2014 (12), an increasing number of studies have been engaged to identify other GPX4-independent regulators [e.g., ferroptosis suppressor protein 1 (FSP1) (13,14) and guanosine triphosphate cyclohydrolase 1 (GCH1) (15,16)] controlling ferroptosis. The current studies provide a strong theoretical underpinning for initiating the process of ferroptosis, which are mainly categorized into the following pathways: the classical GPX4-regulated pathway, the iron metabolic pathway, and the lipid metabolic pathway (Figure 1) (17). In addition, we have also noticed some emerging regulatory mechanisms of ferroptosis, regulated by non-coding RNAs. This is related or different from the three traditional pathways, which we will discuss separately here.

GPX4-regulated pathway
The solute carrier family 7 member 11 (SLC7A11)-GSH-GPX4 axis constitutes the key ferroptosis defense system (4,12). Therefore, ferroptosis-inducing compounds (FINs) were identified to either indirectly inactivate GPX4 by depleting GSH or directly inhibit GPX4 activity.
GPX4, which belongs to the glutathione peroxidase family, is especially crucial in catalyzing the conversion of phospholipid hydroperoxides (PLOOH) into the relevant phospholipid alcohols (18). GSH serves as a reducing agent, and its presence is essential for the activity of GPX4 (13). The first rate-limiting step of GSH synthesis in the cell is mediated by the glutamate cysteine ligase (GCL), which catalyzes the synthesis of gamma-glutamylcysteine from glutamate and cysteine (19). The synthesis of GSH is dependent on the uptake of cysteine by the cystine/glutamate antiporter SLC7A11 (also known as xCT system), and thus the downregulation of SLC7A11 causes indirect inhibition of GPX4 (20). As a result, the deactivationof GPX4 leads to the accumulation of PLOOH, which triggers cell membrane damage and ferroptosis. Studies have consistently shown that deactivationof GPX4 triggers oxidative cell death (21-23). The genetic inhibition of GPX4 can induce ferroptosis of tumor cells and effectively inhibit tumor growth in vivo, suggesting that the GPX4 regulatory pathway plays a significant role in tumor regulation.
The p53-p21 axis is a key regulator of ferroptosis sensitivity. p53 significantly promotes ferroptosis through its transcriptional activity, especially by downregulating the expression of SLC7A11 and affecting the GSH/GPX4 axis (24). Interestingly, p53 inhibits lung cancer cell growth via the ferroptosis pathway. p53 induces ferroptosis in lung cancer cells by downregulating SLC7A11 expression and upregulating Reactive oxygen species (ROS) production. In contrast, p53 inhibits dipeptidyl peptidase-4 (DPP4) activity to downregulate erastin-induced ferroptosis in a transcription-independent manner (25). At the same time, p21, as a downstream target of p53, also indirectly participates in this process (26,27). The dual functions of p53-p21 axis verify that the regulation of ferroptosis highly depends on the microenvironment.
However, some cancer cell lines remained resistant to ferroptosis after GPX4 inactivation, likely due to the presence of additional ferroptosis defense mechanisms, such as FSP1 (13,14). FSP1 reduces coenzyme Q10 (CoQ10) to ubiquinol (CoQ10H2) via NAD(P)H. Ubiquinol, as a lipophilic antioxidant, directly scavenges membrane lipid peroxides, thereby inhibiting ferroptosis.

Iron metabolic pathway
Iron is an essential dietary element in human. In general, intracellular iron balance is regulated in a balance between iron absorption, output, utilization, and storage (28). Ferroptosis is featured by an elevation in Fe2+ within a labile iron pool (LIP) (29). Ferritinophagy promotes ferroptosis by breaking down ferritin in cancer cells (30,31). When ferroptosis occurs, large amounts of free Fe2+ accumulate in the cells. The augmented intracellular Fe2+ can produce hydroxyl radicals via the Fenton reaction [a chemical reaction between Fe2+ and hydrogen peroxide (H2O2)] (32), and participate in phospholipid peroxidation to form PLOOH (33). Peroxidation of membrane lipids can alter the relationship between receptors and ligands, substance transport, and the symmetry of lipid bilayer, all of which can lead to cell dysfunction and eventually cell death (34). The overexpression of mitochondrial ferritin (FtMt), an iron storage protein, decreases free Fe2+, thus inhibiting ferroptotic cell death (29). Cancer cells exhibit an enhanced dependence on iron for their survival compared to normal cells (35). Rapidly proliferating cancer cells enhance iron absorption, leading to elevated intracellular iron levels, which highlights the potential of targeting ferroptosis as a therapeutic strategy in cancer treatment. The inhibition of iron-sulfur cluster biosynthetase NFS1 increases intracellular LIP, reduces the growth of lung cancer, and enhances lung cancer cells sensitivity to ferroptosis (21). Lipid transporter 2 (LCN2) and divalent metal transporter 1 (DMT1) are two key proteins in iron homeostasis regulation (36,37). The overexpression of LCN2 is correlated with resistance to 5-fluorouracil (5-FU) in colorectal cancer. Overcoming 5-FU resistance by targeting LCN2 leads to ferroptosis of tumor cells by increasing intracellular iron levels (38). An increase in intracellular lipid peroxides due to DMT1 inhibition triggeres ferroptosis, thereby reversing multidrug resistance and killing breast cancer stem cells (39). In summary, iron metabolic pathways may serve as key therapeutic targets to interfere ferroptosis in cancer cells.

Lipid metabolic pathway
The distinct feature of ferroptosis is the enhancement of lipid peroxidation. Consequently, lipid metabolism is regarded as a crucial aspect in the ferroptosis process (40). Lipid peroxidation occurs through peroxidation of membrane phospholipids to yield PLOOH, and the decomposition of PLOOH to produce malondialdehyde or 4-hydroxynonenal (41). This leads to membrane instability and increased permeability, ultimately causing cell death (34). Lipid peroxidation also change the molecular structure of polyunsaturated fatty acids (PUFAs), disrupts the fluidity and stability of cell membrane structure, increase the permeability of cell membrane, makes it prone to rupture and death (40).
The acyl-CoA synthase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) are significant promoters of ferroptosis. PUFAs are bound to coenzyme A (CoA) through the action of ACSL4 to form acyl-CoA. Subsequently, acyl-CoA can be re-esterified into phospholipids by LPCAT3 to generate PUFA-PL (42,43). Therefore, the regulation of ACSL4 and LPCAT3 may determine the sensitivity of ferroptosis. Meanwhile, LOXs, especially the LOX-15 subtype, play an important role during ferroptosis in cancer cells. Overexpression of LOXs will elevate the susceptibility of cells to ferroptosis, and the direct oxidation of PUFAs catalyzed by LOXs will contribute to the occurrence of ferroptosis (44). In contrast, LOX inhibitors, such as baicalein, safeguard cells against ferroptosis and avoid damage from lipid peroxidation (45,46). As a member of the lipoxygenase (LOX) family, arachidonate 15-lipoxygenase-1 (ALOX15) catalyzes the oxygenation of PUFAs to generate bioactive lipid mediators, and its expression, promoted by miR-522-3p up-regulation, can induce gefitinib resistance in lung cancer PC9 cells. Accordingly, down-regulating miR-522-3p can reverse this resistance, but its application in chemotherapy drugs for lung cancer remains to be studied (47). We elaborate on targeting ferroptosis to counteract drug resistance in lung cancer in the next section.

Regulation of ferroptosis by non-coding RNAs
Recent evidence indicates that microRNAs (miRNAs), long non-coding RNAs (lncRNAs) play important regulatory roles in ferroptosis and lung cancer resistance. MicroRNA-101-3p (miR-101-3p) restores ferroptosis in tumor cells by directly targeting TBLR1 (48). MicroRNA-302a-3p (miR-302a-3p) directly binds to the 3’-untranslated region of iron transporter protein to reduce its protein expression, and functions as a tumor inhibitor, at least partly, via targeting ferroportin to induce ferroptosis of non-small cell lung cancer (NSCLC) (49). The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1) drives drug resistance by suppressing miR-145 and activating MUC1, thereby inhibiting ferroptosis (50). As a lncRNA, LINC02266 inhibits erastin-induced ferroptosis by suppressing the level of ACSL4, which may be related to the AKT pathway (51). Targeting these non-coding RNAs or their downstream pathways may become a new strategy to overcome resistance.

Targeting ferroptosis for overcoming drug resistance in lung cancer

Reversing resistance to traditional chemotherapy by targeting ferroptosis
Chemotherapy is one of the primary means of lung cancer treatment, but drug resistance poses a significant challenge. For example, patients with NSCLC may develop resistance to platinum-based chemotherapy regimens. Cancer stem cells (CSCs) play a crucial role in driving tumor drug resistance. In recent years, certain small molecule substances (e.g., ironomycin AM5 and TRPML1 inhibitors) have been found to induce ferroptosis, thereby reducing the tumorigenicity and chemotherapy resistance of cancer stem cells (52,53). lncFERO inhibits ferroptosis by regulating the expression of stearoyl-CoA desaturase 1 (SCD1), a protein related to iron metabolism, thereby enhancing tumorgenicity and chemotherapy resistance of gastric cancer stem cells (54). Correspondingly, targeting ferroptosis can reverse tumor chemotherapy resistance of tumors to a certain extent. Some recent strategies have been found to target ferroptosis in lung cancer cells (Table 1).

Cisplatin resistance
Cisplatin stands as one of the most promising and extensively applied drugs in the treatment of multiple solid cancers, including lung cancer, cervical cancer, melanoma, lymphomas, etc. Cisplatin exerts its anticancer activity via multiple mechanisms, but its most widely accepted mechanism is that it generates of DNA lesions by interacting with purine bases on DNA, ultimately resulting in apoptosis (68). Nevertheless, side effects and drug resistance are the two intrinsic challenges associated with cisplatin, which constrain its application and effectiveness. Cisplatin resistance is attributed to reduced drug accumulation within cancer cells, detoxification through reactions with GSH and metallothioneins, and accelerated repair of DNA lesions (69-71).
GPX4 works synergistically with GSH to inhibit ferroptosis, which plays a crucial role in promoting cisplatin resistance (18). Recently, ent-kaurane diterpenes are able to overcome cisplatin resistance by inducing ferroptosis through targeting peroxidase I/II and depletion of GSH (55). Isoorientin serves as a crucial mediator by modulating the SIRT6/Nrf2/GPX4 signaling pathway, particularly through the downregulation of GPX4 expression, to induce cellular ferroptosis and reverse drug resistance in lung cancer cells (58). Isoorientin treatment led to a marked reduction in the viability of drug-resistant cells, a significant elevation in intracellular iron, malondialdehyde (MDA), and ROS levels, and a substantial decrement in glutathione concentration in vitro and in vivo. These effects culminated in the induction of ferroptosis in lung cancer cells, ultimately counteracting chemotherapeutic resistance in lung cancer treatment (56).
Falnidamol, an anticancer pyrimidine compound, boosts cisplatin’s toxicity against NSCLC cells. Falnidamol regulates ROS by inhibiting DUSP26, thereby promoting ferroptosis and reversing chemotherapeutic resistance (58). Combined therapy with Falnidamol and cisplatin triggers ferroptosis, iron buildup, lipid peroxidation, and suppresses EMT and EGFR phosphorylation by decreasing DUSP26 expression in NSCLC cells. Inhibiting xCT systems (e.g., by erastin and sorafenib) has also been shown to be able to reverse cisplatin resistance in NSCLC cells (72). In addition, α-hederin treatment increases the chemical sensitivity of NSCLC cells to cisplatin through downregulating glutathione peroxidase 2 (GPX2) and glutathione synthase (GSS) expression to suppress the synthesis of GSH (57). There is reason to believe that more exploration of targeting ferroptosis could significantly remove the application and efficacy limitations of cisplatin.

5-FU resistance
5-FU is a commonly used antitumor drug, belonging to the pyrimidine analogs, which inhibits tumor cell proliferation by interfering with the synthesis of DNA and RNA (73).
Uracil-tegafur (UFT) is an oral medication, a derivative of 5-FU, which contains a dihydropyrimidine dehydrogenase (DPD) inhibitor. Actually, UFT has been demonstrated to be effective in a postoperative adjuvant context for early-stage NSCLC in multiple randomized controlled trials (RCTs) (74). Although 5-FU has shown significant efficacy in the treatment of various cancers, many patients develop resistance during the treatment process, limiting its clinical application.
Certain 5-FU-resistant cells exhibit an accumulation of lipid droplets, which function as organelles that suppress ferroptosis (60). Precise regulation of the cell cycle is crucial for comprehending the pathophysiological and molecular mechanisms underlying lung cancer and for developing effective therapeutic agents (60,75). Mechanically, cell cycle arrest triggers diacylglycerol acyltransferase (DGAT)-dependent lipid droplet formation, which sequesters the excessive PUFAs accumulated in arrested cells into triacylglycerols (TAGs), thereby suppressing ferroptosis. This sequestration is similar to an antioxidant effect, which protects cells from lipid peroxidation. Therefore, DGAT inhibition coordinates a redistribution of PUFAs from TAGs to phospholipids and re-sensitizes arrested cells to ferroptosis (76,77). The combined use of ferroptosis inducers and DGAT inhibitors (e.g., T863 and PF06427878) effectively suppresses the growth of 5-FU-resistant tumors by targeting ferroptosis (59,60). Together, these findings indicate a function of cell cycle arrest in promoting ferroptosis resistance and propose a ferroptosis-inducing therapeutic approach for targeting slow-cycling and therapy-resistant cancers.

Gemcitabine resistance
Gemcitabine, belonging to the deoxycytidine analogs, inhibits the growth and proliferation of tumor cells by interfering with DNA synthesis and is widely used in the treatment of NSCLC (78). The acquisition of therapeutic resistance by tumors is mediated through the lipid peroxidase pathway. This phenomenon is especially prominent in cancer cells that exhibit a high mesenchymal phenotype, subsequently leading to resistance against a variety of therapeutic modalities (8). Research has confirmed that gemcitabine resistance can be reversed by affecting ferroptosis through regulating tumor fatty acid metabolism and GPX4 (79). Triacsin C, a PUFA analogue with diverse biological activities, acts as an ACSL inhibitor, blocking intracellular lipid accumulation by suppressing ACSLs (e.g., ACSL1, ACSL4, and ACSL5) activity (80). Combining the ACSL inhibitor triacsin C with Gemcitabine significantly enhances the growth-inhibitory effect and may help lung cancer patients overcome drug resistance (61).

Reversing resistance to targeted therapy by targeting ferroptosis
Targeted therapeutics, including epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs), are of great significance in the treatment of NSCLC, but drug resistance remains a major challenge. Strategies to combat drug resistance are therefore urgently needed. Newly emerging evidence indicates that ferroptosis plays a crucial role in cancer treatment by targeting relevant molecules and is also associated with cancer progression (Figure 2). Moreover, it has been reported that targeting ferroptosis can be employed to surmount resistance to targeted therapies (Table 1).

Intrinsic resistance to EGFR-TKIs
EGFR is actively engaged in the modulation of multiple mechanisms related to cancer progression, such as angiogenesis, differentiation and migration (81). Therefore, targeting EGFR has emerged as a prominent approach for the treatment of various types of cancers, including NSCLC, pancreatic cancer, glioblastoma (82). Advanced EGFR-TKIs generations exhibit anti-neoplastic potency; however, drug resistance poses a significant obstacle. The EGFR T790M mutation is the main mechanism underlying resistance to 1st and 2nd generation EGFR TKIs (83). Meanwhile, beyond the mainstream EGFR T790M mutation mechanism, studies have proven that lung cancer cells with EGFR mutations are more sensitive to ferroptosis, making the targeting ferroptosis a potential strategy for treating EGFR-TKIs resistant lung cancer (84). Overcoming the intrinsic and acquired resistance due to EGFR mutations makes it necessary to further explore alternative strategies and discover new inhibitors.
The United States Food and Drug Administration (FDA) approved gefitinib, erlotinib, and lapatinib as anti-EGFR therapeutic agents for the treatment of NSCLC patients. Consequently, they became the first-ever clinically approved EGFR inhibitors (85-87). However, due to their reversible characteristics, first generation EGFR-TKIs quickly falls into the drug-resistant category within a very short period of time (88,89). Therefore, patients undergone the treatment with gefitinib frequently developed resistance to anti-cancer drugs after 9 to 15 months of progression-free survival (90).
Targeting ferroptosis, a form of iron-dependent cell death, presents a potential strategy to combat intrinsic resistance in EGFR-mutant lung cancer. Although the direct link between ferroptosis and de novoresistance is an ongoing research area, modulating key players in this pathway can influence cell survival upon treatment pressure. For instance, the upregulation of miR-522-3p, associated with LOX-15, has been implicated in reducing the sensitivity of lung cancer PC9 cells (a cell model with EGFR mutation) to gefitinib, potentially by altering the cellular redox state and counteracting drug-induced death signals; conversely, inhibiting miR-522-3p can restore sensitivity (43). Furthermore, since the anti-apoptotic pathways often contribute to intrinsic resistance, inducing ferroptosis by inhibiting regulators like GPX4 could theoretically help overcome the initial survival advantage of some treatment-resistant cancer cells, as seen in studies involving other cancer types (79). The exploration of agents like the ACSL inhibitor triacsin C, which has shown growth-inhibitory effects in combination with gemcitabine in EGFR wild-type lung cancer models, underscores the broader relevance of targeting lipid metabolism to enhance cytotoxicity (68), although its specific role in ferroptosis induction in EGFR-mutant contexts requires further validation.
β-elemene, a sesquiterpene compound extracted from turmeric, acts as a ferroptosis inducer with antitumor properties. It enhances erlotinib’s cytotoxicity against EGFR-mutated, EGFR-TKI-resistant NSCLC cells, with ferroptosis contributing to the therapeutic effect (91). In addition, research indicates that β-elemene binds to TFEB in EGFR-wild-type NSCLC cells, enhancing lysosomal function. This binding increases GPX4 ubiquitination and lysosomal degradation, thus promoting ferroptosis (92). These findings suggest that β-elemene-induced ferroptosis acts as a potential new strategy for reversing targeted therapy resistance in lung cancer through GPX4 degradation. Additionally, diacetylfuran diacylfuroxans-induced ferroptosis shows promise in overcoming cancer resistance (93-95). Consistently, GPX4 and mTORC1 pathways are upregulated in lapatinib-resistant NSCLC cells. Targeting GPX4 and mTORC1 pathways may reverse lapatinib resistance and improve its therapeutic efficacy (96).
Erianin and its derivatives have shown potential in reversing resistance to EGFR-TKIs. Both exert their effects through a dual mechanism of “signaling pathway regulation and ferroptosis induction”: Erianin monomers directly trigger ferroptosis via the Ca2+/CaM signaling axis as a core switch, while its derivatives act through dual-target inhibition of EGFR and tubulin (blocking both EGFR-mediated resistance signaling and microtubule dysfunction-associated resistance), coupled with autophagy-dependent amplification of ferroptosis, thereby optimizing resistance reversal efficiency. In terms of resistance types, both primarily target acquired resistance (such as secondary EGFR mutations or compensatory signaling activation), while also addressing certain cases of intrinsic resistance (e.g., EGFR wild-type) through non-EGFR-dependent pathways (62,63). This provides a new candidate drug direction for the treatment of EGFR-TKI-resistant NSCLC.

Intrinsic resistance to ALK-TKIs
Olaparib has been approved by the U.S. Food and Drug Administration for the treatment and maintenance of patients with breast cancer gene 1/2 (BRCA1/2) mutations in a small number of tumor types and is a well-known poly (ADP-ribose) polymerase (PARP) inhibitor. However, patients who have functional BRCA (without germline BRCA mutations) did not obtain benefits from olaparib. It has been reported that PARP inhibition can promote ferroptosis by suppressing SLC7A11-mediated glutathione (GSH) synthesis, which is why olaparib can induce ferroptosis. In addition, ferroptosis enhanced by ferroptosis inducer synergistically enhances sensitivity to the PARP inhibitor olaparib, thereby sensitizing ovarian cancer with a BRCA1/2 mutation (66). Based on PARP’s success in ovarian cancer, researchers and clinicians are also exploring the potential use of PARP inhibitors in the treatment of lung cancer. Targeting ferroptosis based on PARP inhibitors may be a novel strategy to reverse resistance in lung cancer patients with BRCA1/2 mutation.

Acquired resistance to EGFR-TKIs
Acquired resistance inevitably emerges, which becomes a clinical challenge. Acquired resistance occurs when tumor cells develop resistance to initial effective treatments, leading to treatment failure. The main mechanisms of acquired resistance involve activation of the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways, tertiary mutations and amplification of EGFR, as well as histological/phenotypic transformations [small cell lung cancer (SCLC) transformation and epithelial mesenchymal transition] (83). The use of GPX4 inhibitors alone or in combination with the mTOR inhibitor, everolimus (RAD001), can inhibit the proliferation of EGFR-resistant mutant cells (97). This indicates that the interaction between ferroptosis-related proteins and the mTOR pathway plays an important role in EGFR-TKIs resistant cells, and inducing ferroptosis may be an effective therapeutic strategy to overcome EGFR-TKIs resistance. Besides, the histone deacetylase inhibitor vorinostat promotes ferroptosis in EGFR-mutated lung adenocarcinoma cells through inhibiting the expression of SLC7A11 and enhancing the efficacy of the ferroptosis inducers. It may serve as a potential therapeutic strategy for overcoming EGFR-TKIs resistance (64). All these studies indicate that targeting ferroptosis may be an effective therapeutic strategy to overcome EGFR-TKIs resistance.
The aberrant activation of the PDZ-binding motif-containing transcriptional coactivator TAZ (Transcriptional Coactivator with PDZ-binding Motif, WWTR1) is a key mechanism of resistance to targeted therapies like EGFR-TKIs in NSCLC. TAZ is linked to ferroptosis regulation via controlling the gene expression of ANGPTL4 (98). Artesunate effectively induces the proteasomal degradation of TAZ, inhibits tumor cell growth, and shows efficacy against EGFR inhibitor-resistant NSCLC tumors (65). Gossypol, a YAP (Yes-associated protein) /TAZ inhibitor, shows potential in tackling EGFR-TKI resistance in NSCLC, particularly in cases with the EGFR L858R/T790M mutation. It inhibits growth, promotes apoptosis, and sensitizes resistant cells to EGFR-TKIs (85). As such, gossypol emerges as a promising candidate for overcoming acquired resistance to EGFR-TKI, offering new hope for improved therapeutic outcomes in NSCLC patients.

Acquired resistance to ALK-TKIs
For ALK-positive NSCLC patients who have received extensive treatment and usually possess resistance mechanisms to ALK-TKIs, the treatment options are generally restricted to chemotherapy. However, chemotherapy only brings about limited clinical benefits and may cause severe toxicity. Olaparib, a well-known poly (ADP-ribose) polymerase (PARP) inhibitor, has been approved by the United States Food and Drug Administration for the treatment and maintenance of patients with breast cancer gene 1/2 (BRCA1/2) mutations in a limited number of tumor types. The combined using of ALK-TKIs crizotinib and olaparib increased the level of ROS, induced DNA damage, and reduced the phosphorylation of AKT, mTOR, and ULK-1, thereby enhancing olaparib-induced cell death in drug-resistant cell lines (99). Although the involvement of ferroptosis remains largely unexplored, we believe that therapeutic strategies for targeting ferroptosis may be developed to overcome resistance to targeted therapies in lung cancer.

Reversing resistance to immunotherapy by targeting ferroptosis
Immune checkpoint inhibitors (ICIs) have changed lung cancer treatment, but intrinsic and secondary resistance remains a problem. Dysregulated immune metabolism significantly affects the tumor microenvironment (TME) and the host. T cell immune deficiency may lead to T cell dysfunction, including decreased proliferation capacity, reduced effector function, and abnormal signaling. These dysfunctions directly affect the ability of T cells to recognize and clear tumor cells, resulting in poor efficacy of immunotherapy. T cell immunodeficiency helps explain the underlying mechanism of primary resistance to therapy and provides new insights into immunotherapy (100).
Recent studies have indicated that ferroptosis is involved in T-cell immunity and cancer immunotherapy (101). Inhibiting ferroptosis is conducive to overcoming resistance to anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) therapy (102). These findings suggest that targeting ferroptosis may be a strategy to overcome immunotherapy resistance (Figure 3). Two methods to overcome immunotherapy resistance by targeting ferroptosis include: an intrinsic method that targets tumor cells to stimulate anti-tumor immunity via a vaccine-like effect; an extrinsic method that affects immune cell sensitivity in the TME, particularly by reducing immunosuppressive cells (103).

Tumor-cell-intrinsic mechanisms
The primary mechanism of ICIs treatment is to block specific immune checkpoints, enabling immune cells to regain the ability to recognize tumors and avoid immune evasion. However, during immunotherapy, due to the compensatory effect, the expression of other immune checkpoint pathways will increase, leading to treatment resistance (104). They are highly expressed in animal T cells that have developed resistance to anti-PD-1 or anti-CTLA-4 therapies, and the use of inhibitors targeting these alternative immune checkpoints in combination therapy can reverse the occurrence of resistance to immunotherapy.
Additionally, the reasons for immune therapy resistance caused by intrinsic factors of tumors also include the loss of tumor immunogenicity. The factors causing immunogenicity loss have been determined. These factors comprise activation of the mitogen-activated protein kinase (MAPK) signaling pathway, loss of PTEN expression, activation of the WNT/β-catenin signaling pathway, impairment of the interferon-γ signaling pathway, and decreased expression of tumor antigens (103). Tumors downregulate the expression of tumor-specific antigens or tumor-associated antigens, leading to the inability of T cell receptors (TCR) to specifically recognize and bind to MHC molecules. These changes result in the failure of the body to produce an effective anti-tumor immune response.
Cancer stem cells (CSCs) contribute to immunotherapy resistance. Combining targeting ferroptosis with immunotherapy shows promise. CSCs’ metabolic changes lead to resistance, but how they resist ferroptosis during immune evasion remains elusive. CPT1A, a key enzyme of fatty acid oxidation, interacts with macrophage L-carnitine to make lung cancer cells resistant to ferroptosis, weakening CD8+ T cells. This creates a positive feedback loop formed by CPT1A/c-Myc significantly enhances the antioxidant capacity and reduces PUFAs by down-regulating ACSL4, thus protecting CSCs from ferroptosis. Essentially, therapeutic strategies targeting ferroptosis via CPT1A can reverse drug resistance in lung cancer, improve the anti-tumor effect of immune checkpoint blockers (105). This leads us to believe strategies targeting ferroptosis in CSCs hold promise for improving the anti-tumor effect of immune checkpoint blockers, thereby overcoming resistance to immunotherapy.
Immunogenic cell death can activate the adaptive immune system, potentially transforming tumors from an immunologically “cold” to a “hot” state responsive to immunotherapy. Interestingly, ferroptosis has been shown to be immunogenic (106,107). This presents a novel immunotherapy approach, particularly valuable in instances of immunotherapy resistance involving the adaptive immune system. Early ferroptosis cells releases injury-associated molecular patterns (DAMPs) [like ATP and high mobility group box 1 (HMGB1)] and enhancing the maturation of bone marrow-derived dendritic cells in vitro (106,107). In addition, steaoryl-2-15-HpETE-sn-glycero-3-phosphatidylethanolamine (SAPE-OOH) on the surface of ferroptotic tumor cells acts as an eat-me signal to guide phagocytosis by binding with TLR2 on macrophages (108). Furthermore, a transcriptional coactivator with a PDZ-binding motif (TAZ, also known as WWTR1) promotes the process of tumor immune escape by up-regulating the expression of PD-L1. Ferroptosis inducers, such as artesunate, decrease the expression of TAZ and PD-L1, reversed immune escape in vivo, enhanced anti-tumor immunity, and reversed lung cancer immunotherapy resistance (65). In summary, targeting ferroptosis in cancer cells may activate immunogenicity to stimulate antitumor immunity, thereby overcoming resistance to immunotherapy.
Overall, targeting ferroptosis in lung cancer cells may trigger an internally generated, vaccination-like effect through improve the anti-tumor effect of immune checkpoint blockers and activate immunogenicity to stimulate anti-tumor immunity to overcome immunotherapy resistance.

Tumor-cell-extrinsic mechanisms
Immunotherapy resistance may be related to the increased expression of immunosuppressive cells and immunosuppressive molecules in the TME, or the escape mechanism of tumor cells to immune attack. The TME is primarily composed of tumor cells, immune and inflammatory response cells surrounding them, tumor-associated fibroblasts, adjacent stromal tissue, microvessels, as well as various cytokines and chemokines. It is a complex and integrated system, which can be classified into an immune microenvironment dominated by immune cells and a non-immune microenvironment dominated by fibroblasts. When the number of immunosuppressive cells in the TME increases, or immunotoxic cells are absent or dysfunctional, it will lead to resistance to immunotherapy.

Infiltration of inhibitory immune cells in TME
Immunosuppressive TAMs and Tregs within the TME play a role in combating immunotherapy resistance in lung cancer (109). The anti-tumor TAM1, which causes elevated levels of induced nitric oxide synthase (iNOS)/NO·, is more resistant to ferroptosis than TAM2. Regulation of ferroptosis by iNOS/NO· inhibits the survival of TAM2 without affecting TAM1, thereby enhancing anti-tumor immunity in the TME (110). In addition, high tyrosine kinase receptor tyrosine 3 (TYRO3) expression is associated with anti-PD-1/PD-L1 immunotherapy resistance in preclinical mouse models and patients. Inhibition of TYRO3 promotes ferroptosis of tumor cells and promoted TAM1 to pro-tumor TAM2 polarization (111). Inhibition of TYRO3 also triggers ferroptosis and reprograms TAMs, thereby restoring the sensitivity of resistant tumor cells to immunotherapy.
Additionally, Tregs are a subset of T cells with immunosuppressive functions that promote tumor progression by suppressing anti-tumor immune responses. Tregs can produce IL-10 or IL-35, and through their interaction, promote the exhaustion of CD8+ tumor-infiltrating lymphocytes, thereby limiting the anti-tumor immune effect (112). GPX4 prevents lipid peroxidation and ferroptosis in Treg cells in the regulation of immune homeostasis and anti-tumor immunity. The infiltration of Tregs is associated with anti-PD-L1 immunotherapy resistance, while the depletion of Tregs restores anti-tumor immunity (113). GPX4-deficient Tregs produce interleukin-1β (IL-1β) and enhance mitochondrial superoxide production to promote the T helper cell 17 (Th17) response, which enhances anti-tumor immunity (114). In summary, targeting ferroptosis by inhibiting GPX4 in Tregs may reverse immunotherapy resistance.

T cell exhaustion and T cell dysfunction
Cancer immunotherapy restores or enhances effector function of CD8+ T cells in the TME (115-117). Cancer immunotherapy-activated CD8+ T cells clear tumors primarily by inducing cell death through the perforin-granzyme and Fas-Fas ligand pathways (118,119). Immunotherapy-activated CD8+ T cells intensify tumor-specific lipid peroxidation during ferroptosis, improving immunotherapy’s antitumor effects. Mechanistically, IFN-γ from these T cells lowers SLC3A2 and SLC7A11 levels, reducing cystine uptake in tumor cells and fostering lipid peroxidation and ferroptosis (101). Therefore, T-cell-promoted tumor ferroptosis is an anti-tumor mechanism, and targeting this pathway in combination with checkpoint blockade is a potential therapeutic strategy. In addition, inhibition of CD36 can protect CD8+ T cells from ferroptosis and improve the effectiveness of ICI immunotherapy (120). The TME can induce ferroptosis in CD36+ T cells, but ferroptosis inhibitors (e.g., ferrostatin-1) can protect T cells and block immunosuppressive signals. Combining ferrostatin-1 with an immune checkpoint inhibitor shows potential to overcome tumor resistance (121).

Novel nanomaterials
Furthermore, a novel kind of nanoassembly was engineered to combine ultra-small iron nanoparticles (USINPs) with radioactive iodine-labeled anti-PD-L1 antibodies (131I-aPD-L1). The nanoassembly is stable in the bloodstream, effectively targets tumors, and breaks down in the presence of ATP within the TME, releasing iron ions and inducing ferroptosis. Immunogenic cell death induced by radiopharmaceutical therapy (RPT) and ferroptosis combined with PD-L1 immune checkpoint blocking therapy showed strong anti-tumor immunity (122). This study provides a novel approach to improve tumor resistance to ferroptosis inducers and radiopharmaceutics, demonstrating the potential of effective single photon emission computed tomography (SPECT) guided targeting ferroptosis to reverse immunotherapy resistance.

Reversing resistance to PDT by targeting ferroptosis
PDT is a clinically approved, minimally invasive therapeutic procedure that can exert a selective cytotoxic activity toward malignant cells. It is based on the local or systemic application of a photosensitive compound-the photosensitizer. Cancerous cells can develop resistance to PDT through diverse mechanisms, such as utilizing molecular pumps on their cell membranes to expel photosensitizers (123), producing specific proteins that break down the drugs (124), or modulating the cellular redox balance to neutralize the produced ROS (125). It is significant to point out that among all lung cancer cases treated with PDT, 70–85% exhibit resistance, which consequently results in the failure of the PDT treatment (126).
A novel mechanism of PDT resistance has been reported, where PDT induces DNA damage response (DDR) and upregulates GPX4 to gradually degrade the generated ROS. Dihydroartemisinin (DHA) can activate Fe and produce abundant ROS (127). Importantly, DHA showed a significant inhibitory effect on GPX4 expression, thereby triggering ferroptosis and enhancing the anti-lung cancer efficacy of PDT (128). A new bionic nanoplatform has been developed for targeted delivery of the photosensitizer Ce6, heme, and PEP20 (a CD47 inhibitor). This platform enhances PDT with oxygen, activates ferroptosis, and blocks CD47-SIRPα signaling. Heme chloride reduces hypoxia and boosts PDT via catalase-like activity. The nano-platform induces ferroptosis through both GPX4 downregulation and Fe2+ overload. Combining PEP20 with PDT improves anti-cancer immunity and overcomes tumor immune resistance (129).
The exploration of targeting ferroptosis with nanocarriers and corresponding drugs is anticipated to enhance the promise of PDT, an emerging minimally invasive treatment approach.

Perspectives
Currently, research on reversing lung cancer drug resistance by targeting ferroptosis has achieved significant breakthroughs at the basic mechanistic level. However, translating these findings into clinically applicable therapeutic strategies still faces multiple challenges, while also presenting vast opportunities for further exploration. Compared to other malignancies such as breast cancer, studies on the relationship between ferroptosis and drug resistance in lung cancer remain relatively limited, with many key scientific questions awaiting answers. For instance, the mechanisms underlying the differential sensitivity of various pathological subtypes (e.g., NSCLC vs. SCLC) to ferroptosis inducers, as well as the interactions between tumor microenvironment components (such as cancer-associated fibroblasts) and ferroptosis regulation in lung cancer cells, require deeper mechanistic investigation to fill existing research gaps.
In clinical translation, the applicability of ferroptosis inducers remains unclear. There is still insufficient evidence to determine whether their resistance-reversing effects are limited to lung cancers with specific molecular features (e.g., EGFR mutations or ALK fusions) or can be broadly applied to most lung cancer types. Therefore, precise molecular subtyping studies are needed to identify potential beneficiary populations and provide a basis for personalized treatment strategies. Additionally, the lack of specific biomarkers for ferroptosis in vivo severely hampers treatment efficacy evaluation. The inability to clinically detect the occurrence and extent of ferroptosis in tumor cells in real time makes it difficult to dynamically adjust treatment approaches. Hence, identifying and validating reliable biomarkers (such as specific lipid peroxides or iron metabolism-related protein expression changes) is a critical step toward advancing clinical translation in this field.
Moreover, the non-specific nature of ferroptosis regulation may pose safety risks-given its involvement in cell death pathways in degenerative diseases, ischemic conditions, and other pathological processes, excessive activation of ferroptosis could lead to damage in normal tissues of iron metabolism-sensitive organs such as the liver and kidneys. Nanoparticle-based ferroptosis inducers, which can accumulate in tumor sites via passive targeting (e.g., enhanced permeability and retention effect) or active targeting (e.g., modification with tumor-specific antibodies) (122,130), offer a potential solution to reduce normal tissue toxicity and improve treatment safety, warranting further exploration.

Conclusions
Ferroptosis is a type of programmed cell death, which is featured by the accumulation of ROS and the excessive production of lipid peroxides, in addition to apoptosis that is driven by iron-dependent phospholipid peroxidation. Targeting cell death processes is a common approach to cancer treatment, and ferroptosis has recently been recognized as playing an important role in anti-cancer therapies. Intrinsic and acquired drug resistance is a major obstacle to cancer treatment. This study provides implications for reversing cancer drug resistance by inducing ferroptosis. As indicated by numerous studies, modulating ferroptosis may overcome resistance to conventional chemotherapy, targeted therapy, and immunotherapy. However, this field still faces multiple challenges, including insufficient lung cancer-specific research, the lack of reliable biomarkers, and issues regarding treatment specificity. Future studies should focus on elucidating the underlying mechanisms, identifying specific biomarkers, developing targeted delivery systems, and utilizing advanced strategies such as nanotechnology to improve treatment specificity and safety. Although the path toward clinical translation remains long, research on novel cell death mechanisms-represented by targeting ferroptosis-undoubtedly provides a valuable direction and hope for overcoming drug resistance in lung cancer. The findings summarized in this review may offer hope for the development of new therapies to overcome cancer resistance by inducing ferroptosis.

Supplementary
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