Pharmacologically Targeting Ferroptosis and Cuproptosis in Neuroblastoma

Abstract

Neuroblastoma is a deadly pediatric cancer that originates from the neural crest and frequently develops in the abdomen or adrenal gland. Although multiple approaches, including chemotherapy, radiotherapy, targeted therapy, and immunotherapy, are recommended for treating neuroblastoma, the tumor will eventually develop resistance, leading to treatment failure and cancer relapse. Therefore, a firm understanding of the molecular mechanisms underlying therapeutic resistance is vital for the development of new effective therapies. Recent research suggests that cancer-specific modifications to multiple subtypes of nonapoptotic regulated cell death (RCD), such as ferroptosis and cuproptosis, contribute to therapeutic resistance in neuroblastoma. Targeting these specific types of RCD may be viable novel targets for future drug discovery in the treatment of neuroblastoma. In this review, we summarize the core mechanisms by which the inability to properly execute ferroptosis and cuproptosis can enhance the pathogenesis of neuroblastoma. Therefore, we focus on emerging therapeutic compounds that can induce ferroptosis or cuproptosis, delineating their beneficial pharmacodynamic effects in neuroblastoma treatment. Cumulatively, we suggest that the pharmacological stimulation of ferroptosis and ferroptosis may be a novel and therapeutically viable strategy to target neuroblastoma.

Introduction

Neuroblastoma (NB), the most common extracranial solid malignancy, is of sympathetic origin and is characterized by a heterogeneous clinical course ranging from a localized tumor to a spontaneous and widely metastatic disease [1–4]. NB accounts for 8–10% of all pediatric tumors and results in approximately 15% of cancer-related deaths in children [5]. Unique features of NB include the high frequency of metastatic disease at diagnosis, the early age of onset, and the tendency for spontaneous regression of tumors in infancy [6].

The main drivers of NB formation are underlying abnormalities in sympathoadrenal cells derived from neural crest cells [7]. Several germline and sporadic genomic rearrangements have been detected in NB, including MYCN [8], anaplastic lymphoma kinase (ALK) [9], encoding lin 28 homolog B (LIN28B) [10], paired-like homeobox 2b (PHOX2B) [11], and polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14) [12]. The amplification of the MYCN oncogene is observed in 20% of all patients with NB, especially in patients who are resistant to therapy and have a poor prognosis [8, 13, 14]. Approximately 2% of NB cases appear to be hereditary, with ALK being the first gene identified to be responsible for familial NB [9, 15]. The specific pathogenesis of NB is still obscure in most cases, leading to limited efficacy in applying specific targeted approaches [16]. NB can be classified into three risk groups (low, intermediate, and high) depending on the extent of disease, age, histology, and presence of cytogenetic abnormalities [17, 18]. The treatment for high-risk NB remains challenging, and the current standard treatment model includes three treatment blocks: induction, consolidation, and maintenance [3, 19]. The consolidation block involves the administration of high-dose chemotherapy followed by autologous stem cell transplantation (ASCT) and radiotherapy [20, 21]. Induction chemotherapy aims to reduce and shrink the tumor, in addition to reducing the risk of metastasis via chemotherapy and surgery [20, 21]. The maintenance block includes immunotherapy via differentiation therapy with 11‐cis retinol, an anti‐disialoganglioside (GD2) monoclonal antibody (mAb), and cytokines [20, 21]. Induction chemotherapy generally uses platinum compounds (cisplatin, carboplatin), cyclophosphamide, vincristine (COJEC), and etoposide [6, 7, 22–25], alongside topotecan (topoisomerase inhibitors) and anthracyclines in North America [26, 27]. These methods of chemotherapeutic induction are preferential inducers of apoptosis [22]. However, this type of pharmacological treatment generates chemotherapeutic drug resistance, hindering the eradication of NB and promoting relapse [4]. Hence, further exploration into the different mechanisms involved in the pathogenesis of NB is needed to identify effective targets for the development of new targeted therapies.

Regulated cell death (RCD) is a ubiquitous process that is essential for restoring biological balance under stress and is essential for maintaining tissue homeostasis [28]. RCD includes apoptosis, programmed cell death (PCD), and regulated necrosis [28]. PCD is a type of RCD that can be activated by external factors and is highly regulated by a number of pathways and intracellular molecules [29]. Ferroptosis [30] and cuproptosis [31] are two newly identified types of PCD. Research has revealed that deficient activation of ferroptosis and cuproptosis is strongly associated with the pathogenesis of many diseases, including NB [32].

Thus, RCD is crucial in NB [33–35]. In this review, we critically discuss the core mechanisms by which nonapoptotic mechanisms of RCD, including ferroptosis and ferroptosis, interact with the pathogenesis and treatment resistance of NB. Next, we focus on therapeutic compounds that modulate RCD activity within NB, delineating their beneficial pharmacological effects. Overall, we suggest that pharmacologically targeting nonapoptotic RCD is a potent therapeutic strategy for treating NB.

Overview of Regulated Cell Death

Ferroptosis

Ferroptosis is a nonapoptotic RCD induced by the iron-mediated oxidative modification of phospholipid membranes [30, 36, 37] (Fig. 1). An imbalance between ferroptosis defense and induction systems dictates the execution of ferroptosis [38]. The cellular antioxidant systems that directly neutralize lipid peroxides constitute the ferroptosis defense systems [39–41]. Five major ferroptosis defense systems with distinctive subcellular localizations have been identified, i.e., the SLC7A11–GPX4 (solute carrier family 7 member 11–glutathione peroxidase 4) axis [38, 42] and the FSP1–CoQH₂ (ferroptosis suppressor protein 1–coenzyme Q₁₀ ubiquinol) [43, 44], GCH1–BH₄ (GTP cyclohydrolase 1–tetrahydrobiopterin) [45, 46], DHODH–CoQH₂ (dihydroorotate dehydrogenase–dihydroubiquione) [47], and MBOAT1/2–MUFA (O-acyltransferase domain containing 1/2–monounsaturated fatty acids) systems [48]. Free iron accumulation and the inhibition of antioxidant systems are the two key initial signals that induce ferroptosis [49]. When iron-dependent reactive oxygen species (ROS) and lipid peroxides (LPOs), the two ferroptosis-promoting factors, substantially override the inhibitory capacity of ferroptosis defense systems, phospholipid polyunsaturated fatty acids (PUFA-PLs) can be converted to peroxidized PUFA-PLs or phospholipid hydroperoxides (PUFA-PLs-OOH) in the membrane via both enzymatic lipid peroxidation (LPO) reactions and nonenzymatic Fenton reactions with the help of bioactive iron and the catalysis of oxidase [50, 51]. PUFA-PLs-OOH accumulate in cellular membranes and eventually cause rupture, resulting in ferroptosis [38].

Fig. 1.

Core mechanisms of ferroptosis

PUFA-PLs are the primary and major substrates for LPO [52]. Acyl-coenzyme A synthetase long chain family member 4 (ACSL4) catalyzes the ligation of CoA with free PUFAs, which include arachidonic acid (AA) and adrenic acid (AdA), to generate PUFA-CoAs in the enzymatic LPO pathway [53, 54]. Lysophosphatidylcholine acyltransferase 3 (LPCAT3) then inserts acyl groups into lysophospholipids and incorporates PUFA-CoAs into PLs to produce PUFA-PLs [53, 55]. Arachidonate lipoxygenases (ALOXs) and cytochrome P450 oxidoreductase (POR), two iron-dependent enzymes, work together with labile iron using O₂ to generate PUFA-PLs-OOH from membranous PUFA-PLs through a peroxidation reaction [52, 56]. Iron functions as an essential cofactor for ALOXs and POR to perform a nonenzymatic Fenton reaction in the LPO pathway. ALOXs and POR promote LPO, the secondary products of which include 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), which induce pore formation at lipid bilayers, leading to ferroptotic cell death [57].

Cuproptosis

Copper functions as an essential micronutrient and trace metal required for life [58]. Redox-active Cu works as an essential key structural or catalytic cofactor for enzymes that are involved in a wide array of biological processes, including OXPHOS, connective tissue cross-linking, biocompound synthesis, iron homeostasis, signal transmission, and ROS detoxification, in almost all organisms [59, 60]. Cu can be cytotoxic, and an overload of Cu promotes Cu-mediated Fenton reactions, producing damaging ROS and disrupting iron‒sulfur cofactor function. Chronic or long-term exposure to copper results in toxicity, and increased intracellular copper leads to many diseases, including cancers. Cu plays vital roles in regulating tumor growth and metastasis [61]. However, the machinery underlying the toxicity and cell death induced by copper remains elusive.

The current goal is to uncover how copper accumulation causes cellular toxicity, inducing copper-mediated cell death, i.e., cuproptosis, and how this process differs from pyroptosis, ferroptosis, necroptosis, and apoptosis [62]. Tsvetkov and colleagues coined the term “cuproptosis” in 2022 (Fig. 2) [31] based on the pioneering works that have shown that copper induces cell death [63] and promotes cancer cell death [64]. Lipoyl synthase (LIAS) and the mitochondrial enzyme ferredoxin (FDX1) were identified as key regulators of Cu toxicity [62], and disulfiram (DSF) was shown to have anticancer activity [65]. Copper-based anticancer compounds induce cell death [65], copper improves the antitumor activity of disulfiram [65], copper induces nonapoptotic programmed cancer cell death [65], elesclomol induces cancer cell apoptosis [65], and copper selectively transported to the mitochondria kills cancer cells [66]. The mechanism underlying how the Cu ionophore elesclomol (ES) exerts anticancer activity was revealed in 2019 [62]; in this context, Tsvetkov and colleagues described Cu-mediated cell death, revealing that ES increases the sensitivity of cancer cells to proteasome inhibitor-induced toxicity in a multiple myeloma mouse model. A mechanistic study revealed that ES-bound Cu²⁺ interacts with FDX1 to reduce Cu⁺² to Cu⁺, leading to increased production of ROS [62, 67]. Gao reported that LPO mediates the lethality of ES [68]. Tsvetkov and colleagues named this unique form of Cu-mediated cell death cuproptosis in 2022 following this discovery, which is characterized by a loss of Fe-S proteins and the aggregation of lipoylated mitochondrial enzymes [31]. Disruption of specific mitochondrial metabolic enzymes plays a role in copper-mediated toxicity, greatly contributing to delineating how copper overload causes mitochondrial dysfunction [31]. Ionophores can transport excess intracellular Cu²⁺ into mitochondria, where it is reduced to Cu⁺ by FDX1. Increased Cu⁺ directly binds to lipoylated DLAT, causing lipoylated protein aggregation and destabilization of Fe-S cluster proteins, resulting in proteotoxic stress and subsequent cuproptosis [31]. Notably, genetic or pharmacological inhibition of ferroptosis, apoptosis, and necroptosis cannot halt ES-Cu complex-induced cell death in multiple cancer cell lines. The antioxidants N-acetylcysteine, ebselene, α-tocopherol, and JP4-039 fail to reverse ES-Cu-induced growth inhibition, indicating that ROS are not required for cuproptosis. However, the hydrophilic antioxidant glutathione (GSH) can inhibit ES-Cu-induced toxicity by chelating intracellular Cu, suggesting that cuproptosis differs from previously regulated cell death mechanisms [31]. Tsvetkov et al. revealed a strong link between mitochondria and copper toxicity. Accordingly, rotenone (an inhibitor of respiratory chain complex I), antimycin (an inhibitor of respiratory chain complex III), UK5099 (an inhibitor of mitochondrial pyruvate uptake), and genetic inhibition of complex I inhibit cuproptosis. Tsvetkov et al. reported that galactose-mediated mitochondrial respiration renders lung cancer cells more sensitive to ES-Cu-induced growth inhibition than cells that rely on glucose-induced glycolysis [31]. Hypoxia (1% O₂) decreases the sensitivity of cancer cells to cuproptosis, whereas basal or adenosine 5′-triphosphate-linked respiration is not affected by ES-Cu in cancer cells. This discriminates cuproptosis from ferroptosis, namely, as a mechanism that requires glucose uptake and pyruvate oxidation. Thus, cuproptosis and ferroptosis are coupled to distinct alterations in mitochondrial function and energy metabolism. Taken together, these findings suggest that cuproptosis is a newly identified oxidative stress-independent, Cu-dependent, and mitochondria-induced cell death mechanism [69].

Fig. 2.

Basic core mechanisms of cuproptosis. The uptake of Cu²⁺ into cells occurs via solute carrier family 31 member 1 (SLC31A1) or copper ionophores. Cu²⁺ then binds selectively to lipoylated tricarboxylic acid cycle proteins and mediates Fe-S cluster protein instability to induce a toxic gain of function through mitochondrial proteotoxic stress, namely, copper-dependent oligomerization of lipoylated proteins, eventually leading to cell death. Elesclomol functions as a copper ionophore to transport copper into cells. The copper importers SLC31A1 and the copper exporter ATPase copper-transporting beta (ATP7B) can also regulate intracellular copper levels. Ferredoxin 1 (FDX1) can reduce Cu²⁺ to Cu⁺ and subsequently lipoylates mitochondrial tricarboxylic acid (TCA) cycle enzymes, especially dihydrolipoamide S-acetyltransferase (DLAT), to promote Fe-S cluster protein loss. Copper-mediated damage to the mitochondrial respiratory chain causes hyperactivation of the energy sensor AMPK, which accelerates cuproptosis and the release of the proinflammatory mediator HMGB1. LA-DLAT, lipoylated DLAT; LIAS, lipoyl synthase

Role of Regulated Cell Death in the Development of Neuroblastoma

Role of Ferroptosis in the Genesis of Neuroblastoma

Oncogenes Dictate the Vulnerability of NB to Ferroptosis

Amplified MYCN is found in 20–25% of cases of NB, and MYCN-amplified NB accounts for a large percentage of pediatric cancer-related deaths. A recent study revealed that amplified MYCN enhances iron influx by increasing the expression of transferrin receptor 1 (TfR1) and lowering the expression of ferroportin, facilitating system/GSH pathway activation by upregulating SLC3A2 and SLC7A11 [70]. This study provides novel insights into how MYCN alters the transcriptome of NB to improve growth and survival. These changes increase the vulnerability of NB to ferroptosis inducers and highlight a potential strategy to treat MYCN-amplified NB by repurposing auranofin. MYCN-amplified NB cells are sensitive to GPX4-targeting ferroptosis inducers through the upregulation of TfR1 expression. TfR1 overexpression selectively increases sensitivity to GPX4 inhibition and ferroptosis. TFRC upregulation confers sensitivity to ferroptosis in NB cells with MYCN amplification, suggesting that GPX4-targeting ferroptosis inducers or TFRC agonists may constitute a new strategy for treating NB harboring MYCN amplification [71]. These observations were corroborated by other studies, which reported that MYCN mediates cysteine adduction and sensitizes NB cells to ferroptosis [72]. Depletion of cysteine promotes MYCN, which induces and sensitizes cells to ferroptosis. The uptake and transsulfuration pathways meet the high cysteine demand in MYCN-amplified childhood NB [72]. When cysteine uptake is limited, protein synthesis mediated by cysteine is maintained, but the loss of GSH triggers ferroptosis, resulting in spontaneous tumor regression in low-risk NB. Pharmacologically inhibiting both cystine uptake and transsulfuration combined with inactivating GPX4 depletes intracellular cysteine and GSH availability and causes tumor remission by triggering ferroptosis in an orthotopic MYCN-amplified NB model. These results suggest that combining multiple ferroptotic targets is a promising therapeutic strategy for aggressive MYCN-amplified NB [72]. Further study revealed that MYCN activates the transsulfuration pathway in NB [73]. Cystathionine beta-synthase (CBS) and methylthioadenosine phosphorylase (MTAP) were increased in MYCN-amplified NB cell lines and tumors. CBS is the rate-limiting enzyme in transsulfuration, and MTAP is an enzyme that helps salvage methionine following polyamine metabolism. MYCN orchestrates both cystine uptake and activation of the transsulfuration pathway to confer ferroptosis vulnerability in MYCN-amplified NB [73]. A recent study revealed that the selenoprotein P receptor LRP8 functions as a key determinant through the inhibition of ferroptosis in MYCN-amplified NB [74]. Loss of LRP8 triggers ferroptosis resulting from an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This study suggested that LRP8 dictates the vulnerability of MYCN-amplified NB to ferroptosis [74]. Taken together, these results suggest that the oncogene MYCN influences the vulnerability of NB to ferroptosis.

Ferroptosis Dictates the Chemosensitivity of Neuroblastoma

A recent study revealed that the E3 ligase tripartite motif (TRIM) 59 protein promotes chemoresistance by suppressing ferroptosis in NB [75]. Silencing TRIM59 enhances the inhibitory effect of doxorubicin hydrochloride (DOX) on the proliferation of neuroblastoma cells. TRIM59 expression was positively linked to cell proliferation in response to DOX. TRIM59 knockdown or overexpression promotes or inhibits ferroptosis, respectively, in neuroblastoma cells [75]. Moreover, TRIM59 inhibits ferroptosis by directly interacting with p53, promoting its ubiquitination and degradation in DOX-exposed neuroblastoma cells. Silencing TRIM59 increases neuroblastoma cell sensitivity to DOX by inducing ferroptosis. The ferroptosis inhibitor ferrostatin-1 (Fer-1) reverses TRIM59 knockdown-induced ferroptosis in neuroblastoma, whereas silencing TRIM59 facilitates the therapeutic efficacy of DOX in xenografted mice [75]. Together, the results of this study suggest that TRIM59 functions as an oncogene to induce chemoresistance to DOX in neuroblastoma through the suppression of ferroptosis through p53 ubiquitination and degradation [75].

Role of Cuproptosis in the Pathogenesis of Neuroblastoma

The role of cuproptosis in the immune landscape and prognosis of NB was first elucidated in 2022 [76]. The authors revealed that the cuproptosis-related gene signature can function as a valuable prognostic biomarker and allows for precise characterization of the tumor immune microenvironment (TIME) in patients with NB. Based on publicly available mRNA expression profile data, the authors initially characterized the specific expression profiles of cuproptosis-related genes (CRGs) in NB samples. The authors classified patients with NB according to the CRGs in the GEO cohort and identified two cuproptosis-related subtypes that were associated with prognosis and the immunophenotype. Then, they constructed a cuproptosis-related prognostic signature, which was validated by LASSO regression. This model can accurately predict patient prognosis, immunotherapy response, and immune infiltration. Silencing the cuproptosis-related gene pyruvate dehydrogenase E1 alpha subunit (PDHA1) significantly inhibited proliferation, migration, and invasion but promoted cell cycle arrest at the S phase and apoptosis in NB cells [76].

A recent study revealed that novel CRG signatures can predict overall survival in pediatric patients with neuroblastoma [77]. Univariate Cox regression analysis revealed that 31 CRGs were associated with overall survival. Patients with NB could be classified into two CRG score groups according to a prognostic model comprising 9 CRGs that was established with LASSO regression analysis [77]. Multivariate analysis suggested that the CRG score was correlated with age, INSS stage, and MYCN status and that COG risk was an independent prognostic indicator. Stratification analysis still revealed a high predictive ability for survival prediction. The higher CRG score group was associated with immune cell infiltration, lower immune scores, and decreased expression of immune checkpoints. The CRG score can predict the drug sensitivity of patients with NB to chemotherapy [77]. These observations were corroborated by other studies, which reported that the established cuproptosis score and prediction model could effectively distinguish between individuals in low- and high-risk groups with high predictive value [78]. In particular, the novel CRG signature PDHA1 plays crucial roles in tumor progression, TIME features, and the long-term prognosis of patients with NB [78]. Taken together, the CRG signature may be used as a prognostic predictor in patients with NB, facilitating the development of effective anticancer therapies for NB.

Therapeutic Strategies for Neuroblastoma That Target Regulated Cell Death

Therapeutic Strategies for Neuroblastoma That Target Ferroptosis

Emerging evidence has shown that the induction of ferroptosis is a novel therapy for NB. Emerging ferroptosis-inducing bioactive compounds (Fig. 3) can kill cancer cells in NB. Table 1 lists some compounds that target ferroptosis to kill NB cells.

Fig. 3.

Chemical structures of small molecules that target ferroptosis to treat neuroblastoma

Table 1.

Emerging compounds that target ferroptosis to inhibit neuroblastoma

Compounds	Experimental model	Targets	Effects	Ref
Erastin (1)	CLP/C57BL/6 J male mice	System xc−	Kills mesenchymal stem cells (BMSCs), mesenchymal stem cells (BMSCs) neuroblastoma (NB)	[66]
RSL3 (2)	LPS/HT22 cells	System xc−	Kills mesenchymal stem cells (BMSCs), mesenchymal stem cells (BMSCs) neuroblastoma (NB)	[66]
Erastin (1) or RSL3 (2)	Neuroblastoma N2A cells	System xc−	Erastin or RSL3 induces ferroptosis in RAS-proficient N2A cells through upregulating HO-1 and downregulating GPX4	[82]
Sulfasalazine (3) or C2-4 (4)	Etoposide-sensitive and resistant neuroblastoma CSCs	System xc−	Etoposide in combination with sulfasalazine or an inhibitor of PKCα (C2-4) sensitizes CSCs to etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, downregulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis	[85]
PTC596 (5)	HTLA-230 and HTLA-ER cells treated with etoposide	System xc−	PTC596, alone or in combination with etoposide, significantly reduces GSH levels, increases peroxide production, stimulates lipid peroxidation, and induces ferroptosis	[86]
Sulfasalazine (3)	PDXs	System xc−	Sulfasalazine blocks growth and induces ferroptosis in MYCN-amplified, patient-derived xenograft models	[70]
Auranofin (6)	PDXs	System xc−	Auranofin blocks growth and induces ferroptosis in MYCN-amplified, patient-derived xenograft models	[70]
Nemorosone (7)	IMR-32 cells	System xc−	↓GSH; ↓system xc− cystine/glutamate antiporter (SLC7A11); ↑intracellular labile Fe2+ pool; ↑heme oxygenase-1 (HMOX1)	[94]
Withaferin A (8)	Neuroblastoma N2A cells	GPX4	Withaferin A induces ferroptosis through activating Nrf2 or inactivating GPX4. Withaferin A boosts the antitumor activity of etoposide or cisplatin in killing a heterogeneous panel of high-risk neuroblastoma cells and in suppressing the growth and relapse rates of neuroblastoma xenografts	[105]
Propargylglycine (9)	IMR5/75, GI-ME-N cells	Cystathionine gamma-lyase	Propargylglycine triggers ferroptosis through blocking cystathionine gamma-lyase, a key enzyme involved in cysteine synthesis in MYCN-driven neuroblastomas	[72]
IONP-GA/PAA (10)	IMR-32 cells		IONP-GA/PAA induces ferroptosis in IMR-32 cells, which is blocked by canonical ferroptosis inhibitors, including deferoxamine and ciclopirox olamine (iron chelators), and ferrostatin-1, the lipophilic radical trap	[106]

4-HNE 4-hydroxy-2-nonenal, BBB blood–brain barrier, BUN blood urea nitrogen, CK-MB creatine kinase MB, CLP cecal ligation and puncture, FPN ferroportin (SLC40A1), FSP1 ferroptosis suppressor protein 1, FTL ferritin light chain, FTH ferritin heavy chain, GSH glutathione, GSSG oxidized glutathione, HFD high-fat diet-fed, HO-1 heme oxygenase-1, IL-1β interleukin-1, IκBα inhibitor of kappa Bα, LDH lactate dehydrogenase, LPS lipopolysaccharide, MCTR1 maresin conjugates in tissue regeneration 1, MDA malondialdehyde, NF-κB nuclear factor kappa B, Nrf2 nuclear factor erythroid-2-related factor 2, PTGS2 prostaglandin endoperoxide synthase 2, SCr serum creatinine, SLC7A11 solute carrier family 7 member 11, SOD superoxide dismutase, TECs renal tubular epithelial cells, TLR4 Toll-like receptor 4, TNF-α tumor necrosis factor-alpha, NCOA4 nuclear receptor coactivator 4

Ferroptosis Inducers Kill NB

Bone marrow mesenchymal stem cells (BMSCs) are central to tumor stroma formation and promote the metastasis of NB to the bone marrow (BM). There is currently no effective method to eradicate these BMSCs affected by NB (NB-BMSCs). A recent study revealed that NB-BMSCs are more sensitive to ferroptosis than normal BMSCs are, as evidenced by how NB-BMSCs synthesize more iron‒sulfur clusters and heme while having lower levels of intracellular free iron and a downregulated xc⁻/GSH/GPX4 system. Erastin (1) is a class I ferroptosis inducer that functions as an inhibitor of system xc⁻, thereby preventing cysteine import and resulting in GSH depletion [79–81]. Accordingly, 1 and RAS-selective lethal 3 (RSL3, 2) could significantly kill NB-BMSCs but not normal BMSCs, providing a potential treatment for tumor-associated BMSCs in patients with NB. This observation has been corroborated by other studies, which showed that 1 and 2 induce ferroptosis in neuroblastoma N2A cells but not in normal neural cells [82]. Small molecules 1 and 2 induce ferroptosis in RAS-proficient neuroblastoma N2A cells. N2A cells are more vulnerable to 1 and 2 than are primary mouse cortical neural stem cells (NSCs) or neurons because of the lower expression of ferritin heavy chain 1 (FTH), a ferroxidase that oxidizes redox-active Fe²⁺ to redox-inactive Fe³⁺, in N2A cells than in NSCs. Overexpression of FTH inhibits ferroptosis by upregulating GPX4 in N2A cells. Decreased expression of FTH was detected in neuroblastoma cell lines. These results suggest that 1 and 2 induce ferroptosis in neuroblastoma N2A cells due to inadequate FTH, highlighting new evidence that inducing ferroptosis is a promising therapeutic target for NB [82].

Ferroptosis Inducers Increase the Antitumor Activity of Chemotherapy

Sulfasalazine (3), an azo bridge-linked anti-inflammatory agent and a class I ferroptosis inducer that was originally synthesized from the antibiotic sulfapyridine, functions as an inhibitor of system xc⁻, thereby preventing cysteine import and resulting in GSH depletion [79–81, 83]. Compound 3 was found to inhibit the absorption of cystine, resulting in the attenuation of GSH and ultimately leading to cell death in certain types of cancer cells [84]. Compound 3 triggers ferroptosis by inhibiting the function of system xc⁻ [30]. MYCN-amplified neuroblastoma accounts for a large percentage of pediatric cancer-related deaths. MYCN confers NB therapeutic vulnerability by rewiring the expression of key receptors, ultimately increasing iron influx by increasing the expression of iron import transferrin receptor 1 (TfR1). This increases NB cell reliance on the cystine/glutamate antiporter (system xc⁻) to detoxify ROS by increasing the transcription of this receptor [70]. Compound 3 and auranofin (6) can block cancer growth by inducing ferroptosis by targeting the system xc⁻/GSH pathway in MYCN-amplified, patient-derived xenograft (PDX) models. The ferroptosis inhibitors ferrostatin-1, N-acetyl-L-cysteine, and deferoxamine (DFO, an iron scavenger) largely reversed this antitumor activity. This study demonstrated how MYCN modulates intracellular iron levels and subsequent GSH pathway activity, revealing the antitumor activity of the FDA-approved auranofin in PDX models of MYCN-amplified NB [70].

C2-4 (4), an inhibitor of PKCα that indirectly targets xCT, increases the antitumor activity of chemotherapy in NB [85]. Compound 4 increases the sensitivity of neuroblastoma stem cells to etoposide by stimulating ferroptosis, as evidenced by decreased intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, downregulating GPX4 activity, and facilitating LPO [85]. Compound 4 enhances the antitumor activity of etoposide by increasing the sensitivity of neuroblastoma stem cells to etoposide through the induction of ferroptosis, highlighting that PKCα inhibition-induced ferroptosis might be a useful strategy to overcome CSC chemoresistance [85].

PTC596 (5), an inhibitor of the oncoprotein BMI-1, has a strong cytotoxic effect on MDR NB cells. PTC596, alone or in combination with etoposide, significantly reduced GSH levels, increased peroxide production, stimulated lipid peroxidation, and induced ferroptosis. These findings indicate that PTC596 could be a promising approach to overcome chemoresistance through triggering ferroptosis by inhibiting BMI-1 in NB [86].

Small Molecules That Induce Ferroptosis

Nemorosone (7) is a bicyclic polyprenylated acylphloroglucinol derivative that was originally isolated from Clusia spp. and can be obtained through chemical synthesis via different synthetic strategies [87]. Compound 6 exerts antitumor effects on several types of malignancies, such as hepatocellular carcinoma, human colorectal cancer, leukemia, pancreatic cancer, and breast cancer [88–93]. Compound 7 functions as a ferroptosis-inducing compound (FIN) in NB; it induces ferroptosis by decreasing glutathione (GSH) levels, blocking SLC7A11 and increasing the intracellular labile Fe²⁺ pool via heme oxygenase-1 (HMOX1) induction [94]. Withaferin A (8), the most active phytocompound extracted from the renowned dietary supplement Withania somnifera (L.) Dunal, has remarkable antitumor efficacy in many cancers [95–103]. Previous studies have shown that 8 exerts antitumor activity by inhibiting activator of transcription 3 (STAT3) in NB [104]. Compound 8 triggers ferroptosis by activating Nrf2 through the targeting of Kelch-like ECH-associated protein 1 (KEAP1) to increase the level of intracellular labile Fe²⁺ upon excessive activation of heme oxygenase-1 (HO-1) and inactivation of GPX4. This dual-edged mechanism enhances the efficacy of WA in combination with etoposide or cisplatin to kill a heterogeneous panel of high-risk BN cells, inhibiting the growth and relapse rates of NB xenografts [105].

Small Molecules Increase the Antitumor Activity of Ferroptosis Inducers

MYCN induces transsulfuration to prevent ferroptosis in NB [72]. Cystathionine gamma-lyase (CTH) and S-adenosyl-L-homocysteine hydrolase (AHCY) are two methyltransferases that feed into Hcy production and show synthetic lethality with high MYCN. CTH converts Cysta to cysteine, and AHCY synthesizes Hcy for transsulfuration. Supplementing either Hcy or Cysta prevents ferroptosis in all cystine-deprived adrenergic neuroblastoma cell lines tested with high or intermediate oncogenic MYC(N) expression but not in the less common mesenchymal NB cell lines. Propargylglycine (PPG), which pharmacologically inhibits CTH, enhances adrenergic cell lines with high MYCN levels to promote either imidazole ketone erastin (IKE)-induced or erastin-induced cell death. Silencing AHCY impaired colony formation in adrenergic high MYCN NB cells, as evidenced by decreased GSH levels and reduced GSH reduced-to-oxidized ratios. These data suggest that the transsulfuration pathway provides an internal cysteine source for GSH biosynthesis, thereby inhibiting ferroptosis in high MYCN adrenergic NB cells.

Therapeutic Strategies for Neuroblastoma That Target Cuproptosis

As a novel unique cell death modality, cuproptosis has sparked great interest in the field of cancer research, providing a novel mechanism to inhibit or kill tumor cells and overcome chemotherapeutic resistance [69, 107, 108]. Interestingly, emerging compounds, such as 2-deoxy-D-glucose, a glucose metabolism inhibitor, which increases the sensitivity of cancer cells to cuproptosis, have been shown to induce cuproptosis in preclinical models [108]. Octyl itaconate (4-OI) increases the sensitivity of cancer cells to cuproptosis by inhibiting aerobic glycolysis mediated by GAPDH [108]. 4-OI also enhances the antitumor activity of elesclomol-Cu in vivo [108]. Importantly, 4-OI promotes cuproptosis by inhibiting aerobic glycolysis, which targets GAPDH [108]. Anisomycin, a p38MAPK signaling pathway agonist, significantly inhibits the proliferation of ovarian cancer stem cells (OCSCs) by promoting cuproptosis and downregulating metallothionein, the PDH complex, the lipoid acid pathway, and FeS cluster protein utilization [109]. Plicamycin may be a cuproptosis inducer that inhibits the progression of HNSCC [110]. Sorafenib, a multityrosine kinase inhibitor, promotes cuproptosis by enhancing copper-mediated lipoylated protein aggregation, suppressing the degradation of FDX1, decreasing intracellular GSH synthesis, and inhibiting cystine import in primary liver cancer cells [111]. Erastin induces ferroptosis by inhibiting system xc⁻, leading to depleted GSH levels [52]. A recent study revealed that erastin and sorafenib could facilitate copper ionophore ES- and ES-Cu-induced cuproptosis by enhancing copper-mediated lipoylated protein aggregation, reducing intracellular GSH synthesis, and decreasing cystine import in HCC cells [111]. This study reveals crosstalk between ferroptosis and cuproptosis, where a combination of ferroptosis inducers and copper ionophores could be a novel therapeutic regimen for HCC. These findings strongly suggest that ferroptosis inducers work as cuproptosis inducers, suggesting that inducers or inhibitors of ferroptosis may also function as inducers or inhibitors of cuproptosis.

Recent research has shown that zinc pyrithione (ZnPT) significantly inhibits TNBC progression by inducing cuproptosis, as evidenced by ZnPT-induced disrupted copper homeostasis, which promotes the oligomerization of dihydrolipoamide S-acetyltransferase, a landmark molecule of cuproptosis [112]. Taken together, these findings indicate that ZnPT can act as a model molecule for future drug discovery toward cuproptosis induction. To our knowledge, no previous studies have determined whether these compounds can induce cuproptosis in NB. Thus, cuproptosis inducers could enable the development of a cuproptosis-based therapy for NB in the future. However, whether cuproptosis can survive as a novel therapeutic regimen for NB remains to be determined.

Conclusions and Perspectives

This review presents recent progress in our understanding of the role of ferroptosis in NB. The oncogene MYCN dictates the vulnerability of NB to ferroptosis. Emerging evidence has confirmed that inducing ferroptosis is generally identified as an effective approach to induce cell death in NB. Ferroptosis inducers can kill NB cells or increase the antitumor activity of chemotherapy. Small molecules induce ferroptosis or increase the antitumor activity of ferroptosis inducers in NB. However, research on the role of ferroptosis is still in its infancy and represents an emerging field. Inevitable challenges remain before its practical application. First, little is known about the role of ferroptosis in NB, along with other cancers such as melanoma [113]. Second, much is still unknown about the role of ferroptosis in overcoming drug resistance to various chemotherapy regimens. Third, although some small-molecule compounds can induce ferroptosis or increase the antitumor activity of chemotherapy, more work is needed to screen and discover novel compounds that induce ferroptosis. Several small molecules can kill cancer cells by inducing ferroptosis. Thus, these drugs may be repurposed for the treatment of NB. Therefore, future exploration of the roles of ferroptosis in NB will promote the discovery of novel therapeutic strategies for NB. Fourth, there remains an urgent unmet need to develop predictive and personal biomarkers for labeling if ferroptosis can be used to treat specific tumors [71, 114, 115]. Fifth, emerging evidence has shown that the Nrf2‒NQO1 pathway regulates ferroptosis-related cell death in brain tumors [116]. However, the role of this pathway in NB is largely unknown and warrants further investigation. Cuproptosis in cancer and NB is still an emerging field and is in its infancy. First, the role of cuproptosis in NB is not yet well understood. Second, the regulatory mechanism underlying cuproptosis in NB needs to be elucidated. Third, the induction of cuproptosis may overcome resistance to conventional chemotherapy in some types of cancer; however, it is not known whether the induction of cuproptosis could overcome drug resistance to chemotherapy in NB. Taken together, ferroptosis and cuproptosis have been identified as novel types of RCD, and the induction of ferroptosis and cuproptosis can kill cancer cells. Thus, inducing ferroptosis and cuproptosis may be a novel potential therapeutic regimen for treating NB.

Author Contributions

YL and LH: writing—original draft preparation; YL and LH: writing—review; HW and JSF editing and visualization; YL and LH: funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Liaoning Province (2022-MS-209), the Liaoning Provincial Department of Education Scientific Research Project (QNZR2020012), the Henan Pediatric Disease Clinical Medical Research Center Foundation (YJZX202207), the CAAE Epilepsy Research Fund (CX-B-2021–02), and 2023 to support China Medical University high-quality development fund (2023JH2/20200119).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics Approval and Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.