Ferroptosis: a dual-edged sword in tumour growth

Ferroptosis, a recently identified form of non-apoptotic cell death, is distinguished by its dependence on iron-triggered lipid peroxidation and accumulation of iron. It has been linked to various disorders, including the development of tumours. Interestingly, ferroptosis appears to exhibit a dual role in the context of tumour growth. This article provides a thorough exploration of the inherent ambivalence within ferroptosis, encompassing both its facilitation and inhibition of tumorous proliferation. It examines potential therapeutic targets associated with ferroptosis, the susceptibility of cancerous cells to ferroptosis, strategies to enhance the efficacy of existing cancer treatments, the interaction between ferroptosis and the immune response to tumours, and the fundamental mechanisms governing ferroptosis-induced tumour progression. A comprehensive understanding of how ferroptosis contributes to tumour biology and the strategic management of its dual nature are crucial for maximizing its therapeutic potential.


Introduction
Cell death plays a crucial role in maintaining tissue balance and controlling the unregulated growth of tumour cells (Fuchs and Steller, 2011).However, tumour cells have evolved mechanisms to evade cell death regulation, promoting unchecked cell replication.Ferroptosis, a unique form of non-apoptotic cell death characterised by lipid peroxidation and unstable iron buildup, differs in morphology, physiology, and biochemistry from classical programmed cell death (Dixon et al., 2012;Friedmann Angeli et al., 2014;Stockwell et al., 2017;Hassannia et al., 2019).An increasing body of evidence implicates ferroptosis in the development of various diseases, including the onset and progression of tumours (Tang et al., 2021).
Currently, ferroptosis has emerged as a significant focus in oncology research.Most studies suggest a beneficial role in restraining tumour growth through interactions between ferroptosis and tumours (Table 1), highlighting its potential as a therapeutic target in oncology.Tumour cells can bypass ferroptosis to promote their own growth by employing defense mechanisms, such as activating System Xc-, boosting glutathione peroxidase 4 (GPX4) activity, and altering glutathione (GSH) metabolism (Dixon et al., 2012).Disrupting or eliminating these mechanisms can trigger ferroptosis and hinder tumour expansion.Additionally, regulating lipid metabolism and iron metabolism pathways can induce ferroptosis, thereby inhibiting tumour growth (Martinez-Outschoorn et al., 2017;Wolpaw and Dang, 2018;Sang et al., 2019;Zou et al., 2020;Lei et al., 2022).Ferroptosis inducers presents a promising approach to curbing tumour growth.Furthermore, combining ferroptosis with chemotherapy, radiotherapy, targeted therapy, or immunotherapy shows potential to enhance antitumour effectiveness and overcome drug resistance (Yamaguchi et al., 2013;Yu et al., 2015;Wang et al., 2019a;Ye et al., 2020) (Table 3).
Consequently, ferroptosis holds the potential to reshape tumour treatment strategies and improve clinical outcomes.
However, it's important to note that ferroptosis can also have a negative impact on promoting tumour growth (Table 2).Through various pathways, such as ferroptosis metabolic pathways (Dai et al., 2020), inflammation-related pathways (Tang et al., 2021;Li and Li, 2020), antigen presentation process (Legrand et al., 2019), and the modulation of immune cell function (Wang et al., 2020;Luo et al., 2021), ferroptosis has been identified as a promoter of tumour growth.This article offers a comprehensive review of ferroptosis's dual role in both promoting and inhibiting tumours, laying a theoretical foundation for further research into ferroptosis in tumour treatment.A thorough understanding of this duality allows for maximizing the clinical effectiveness of ferroptosis-based treatments while minimizing potential adverse effects.

Inhibition of ferroptosis through regulating lipid metabolism thereby promoting tumour growth
Lipid metabolism is closely related to ferroptosis.Lipid peroxidation is a free radical-driven reaction that primarily affects unsaturated fatty acids in cell membranes (Tang et al., 2021).Acyl-coenzyme A synthetase long chain family member 4 (ACSL4) and Lysophosphatidylcholine acyltransferase 3 (LPCAT3) are key regulators of PUFA-PLs synthesis.Phospholipase A2 (PLA2) cleaves PUFAs into free PUFAs and lysophospholipids (Tang et al., 2021).ACSL4 catalyzes the attachment of free PUFAs to coenzyme A to generate PUFA-CoAs, which are re-esterified and incorporated into phospholipids (PLs) by LPCAT3 to form PUFA-containing phospholipids (PUFA-PLs) (Lei et al., 2022;Doll et al., 2017;Dixon et al., 2015).Due to the presence of a bis-allylic moieties of PUFAs, PUFA-PLs are especially susceptible to peroxidation (Conrad and Pratt, 2019).
The downregulation of PUFAs in tumour cells is associated with ferroptosis evasion and the promotion of tumour growth (Lei et al., 2022) (Figure 1; Table 1).For instance, in renal cell carcinoma (RCC), reducing peroxidized PUFAs through the adipokine chemerin allows tumour cells to avoid ferroptosis and supports RCC growth (Tan et al., 2021).KRAS mutations in lung cancer also increase the expression of Acyl-coenzyme A synthetase long chain family member 3 (ACSL3) to reprogram lipid metabolism, promote Monounsaturated fatty acidsphospholipids (MUFA-PL) biosynthesis and ferroptosis resistance, and facilitate lung cancer progression (Friedmann Angeli et al., 2014;Padanad et al., 2016).
In human tumour cell lines, cells in a mesenchymal-like state show selective susceptibility to ferroptosis (Sang et al., 2019).Research indicates that mesenchymal tumour cells exhibit higher enzyme activity, promoting PUFAs synthesis and lipid peroxide production, ultimately leading to ferroptosis occurrence (Viswanathan et al., 2017;Xu et al., 2019) (Figure 1).Specific overexpression of elongation of very long-chain fatty acid protein 5 (ELOVL5) and fatty acid desaturase 1 (FADS1) in mesenchymal gastric cancer cells, both involved in PUFAs synthesis, makes cancer cells particularly susceptible to ferroptosis (Lee et al., 2020) (Table 1).

Escaping ferroptosis by interfering with the antioxidant system and affecting amino acid metabolism contributes to tumour growth
Ferroptosis is associated with disruption of the antioxidant system and amino acid metabolism (Figure 1).GSH-GPX4 is involved in the intracellular antioxidant system and is a key factor influencing the onset of ferroptosis.GPX4, the only member of the GPX protein family capable of converting phospholipid hydroperoxides into phosphatidyl alcohols, prevents lipid peroxidation, thus restraining ferroptosis and supporting tumour growth (Ursini et al., 1982;Brigelius-Flohé and Maiorino, 2013;Seibt et al., 2019;Brigelius-Flohé and Flohé, 2020).GSH, a co-factor for GPX4, is synthesized from glycine, glutamate, and cysteine, with cysteine being the rate-limiting precursor (Forman et al., 2009;Koppula et al., 2018;Friedmann Angeli et al., 2019).
Cysteine/glutathione antiporter, also known as System Xc-, is an important intracellular antioxidant element.System Xc-is a transmembrane protein, consisting of SLC7A11 and SLC3A2, responsible for the exchange of extracellular cystine with intracellular glutamate (Bannai, 1986;Conrad and Sato, 2012).SLC7A11 mediates cystine/glutamate antotransporter protein activity and SLC3A2 maintains SLC7A11 protein stability (Bannai, 1986;Sato et al., 1999;Conrad and Sato, 2012;Koppula et al., 2018).Therefore, inhibition of System Xc-leads to an imbalance of the antioxidant system thereby causing ferroptosis.The SLC7A11-GSH-GPX4 system plays a crucial role as the main defense against ferroptosis in tumours (Dixon et al., 2012;Friedmann Angeli et al., 2014;Stockwell et al., 2017;Hassannia et al., 2019).GPX4 is a central control factor of ferroptosis, and intracellular GSH content directly affects GPX4 activity (Maiorino et al., 2018).Ferroptosis inducers have demonstrated efficacy in tumour cells by directly binding to and inhibiting GPX4 (Table 1).The ferroptosis activator RSL3, an inhibitor of the antioxidant system, directly inactivates GPX4 and inhibits tumour growth in a xenograft mouse model of BJeLR cell origin (Fuchs and Steller, 2011).FIN56, induces ferroptosis in HT1080 cells by depleting GPX4 protein as well as activating farnesyldiphosphate farnesyltransferase 1 (FDFT1/SQS) to block coenzyme Q10 production (Shimada et al., 2016).Kras/TP53-driven pancreatic tumours induce ferroptosis and inhibit tumour growth by depleting cystine or cysteine through cyst (e) inase (Badgley et al., 2020).Sulfasalazine inhibits System Xc-and diminishes cellular glutathione, leading to the excessive buildup of lipid peroxides in tumour cells, inducing ferroptosis.This demonstrates an antitumour effect in pancreatic cancer (Lo et al., 2010).

Inhibiting tumour growth by affecting iron metabolism pathways to induce ferroptosis
Iron metabolism is a necessary process for ferroptosis.Iron overload induces ferroptosis through the Fenton reaction, which generates a large number of hydroxyl radicals and triggers a strong oxidative stress response that produces a large number of ROS (Conrad and Pratt, 2019).Transferrin (TFR) and divalent metal ion transporter protein-1 (DMT1) take up extracellular iron, and ferroportin (FPN) transfers intracellular iron to the outside of the cell (Figure 1).These proteins collaborate to maintain intracellular iron homeostasis (Seiler et al., 2008;Mandal et al., 2010).Iron is also essential for participation in lipid peroxidation, and lipoxygenase (LOXs) and cytochrome P450 oxidoreductase (PORs) require iron for catalysis (Jiang et al., 2021a).
Tumour cells show an increased demand for iron and display heightened oxidative metabolic processes compared to nonmalignant cells (Martinez-Outschoorn et al., 2017;Wolpaw and  Dang, 2018; Zou et al., 2020).The level of intracellular iron impacts sensitivity to ferroptosis.Elevated intracellular iron in tumour cells leads to higher production of ROS and lipid metabolites, aiding ferroptosis development (Table 1; Figure 1).Tumours abundant in iron, like hepatocellular carcinoma (HCC) and breast cancer, or those rich in ROS like lung cancer, along with tumours with increased iron use and overload, demonstrate heightened sensitivity to ferroptosis (Ma et al., 2016).Iron oxide nanoparticles, breaking down within the acidic tumour cell environment, release intracellular iron, leading to increased iron and ROS production, ultimately inducing ferroptosis and hindering tumour growth (Ma et al., 2017).Artemisinin prompts lung cancer cells to absorb and release substantial iron amounts, heightening their susceptibility to ferroptosis (Chen et al., 2020).In high-grade serous ovarian cancer (HGSOC), elevated iron intake and reduced expression of the iron efflux pump FPN result in excessive intracellular iron, further promoting ferroptosis onset (Basuli et al., 2017).
Thus, adjusting iron levels-enhancing iron intake, reducing storage, and restricting iron release-holds potential to promote ferroptosis and impede tumour growth.

Promotional effect of ferroptosis on tumour growth
Ferroptosis promotes tumour growth and progression by regulating metabolic pathways, triggering inflammationassociated immunosuppression, and cancer cells dying from ferroptosis to compromise antitumour immune responses (Figure 2; Table 2).

Cancer cells dying from ferroptosis affects the antigen presentation process and produces immunosuppressive effects, thus compromising antitumour immune responses
Immunogenicity refers to antigens' ability to provoke an immune response, involving immune effector molecule production, activation, proliferation, and differentiation (Aaes and Vandenabeele, 2021).Immunogenic cell death occurs when tumour-associated antigens (TAAs) are processed and presented on the surfaces of tumour cells and dendritic cells (DCs) (Legrand et al., 2019;Yatim et al., 2017).Research suggests that ferroptosis may possess immunomodulatory traits influencing neighbouring tumour cells' response to immunogenic death (Blüml et al., 2005;Blüml et al., 2009).Observations indicate that cancer cells undergoing ferroptosis impede TAA processing and presentation.Co-culturing ferroptotic cancer cells with DCs revealed that these "initial" iron-depleted cells hinder DCs maturation, phagocytosis, and antigen-cross-presentation (Wiernicki et al., 2022) (Figure 2; Table 2).Consequently, ferroptosis negatively impacts antigen-presenting cells, influencing adaptive immune responses and antitumour immunity.

The role of ferroptosis in different cancer therapies, such as chemotherapy, immunotherapy, radiotherapy and reversal of tumour resistance
Currently, numerous studies corroborate the synergistic effect of ferroptosis combination therapy in bolstering the efficacy of antitumour treatment.However, evidence supporting the inhibitory effect of combination regimens remains relatively scarce.We've compiled studies showcasing the potential of combined ferroptosis therapy to heighten antitumour efficacy and surmount drug resistance (Table 3).Further pertinent research is warranted to build upon this foundation.

Ferroptosis combination with immunotherapy
The immune system wields significant influence over both tumour development and treatment.Recent research highlights ferroptosis as a factor impeding tumour growth by modulating the immune response (Wang et al., 2019a).Combining immune checkpoint inhibitors with ferroptosis inducers enhances immunotherapy efficacy (Jiang et al., 2021b).Ferroptosis, by recruiting and activating immune cells within the tumour environment, serves as a foundation for using ferroptosis inducers to augment immunotherapy.Ferroptotic tumour cells release DAMPs and trigger Major Histocompatibility Complex (MHC) class I molecule expression, activating T cells and macrophages (Wen et al., 2019).In the tumour microenvironment, CD8 (+) T cells produce IFN-γ, downregulating SLC7A11 expression, reducing cystine uptake, fostering lipid peroxide accumulation, and inducing ferroptosis in Melanoma and ovarian cancer cells (Wang et al., 2019a).The ferroptosis inducer BEBT-908 triggers ferroptosis, elevates MHC class I molecule expression, and activates the IFN-γ signalling pathway in Colorectal cancer (CRC), human diffuse large B-cell lymphoma, and lung cancer, bolstering the body's immune response and exerting antitumour effects (Fan et al., 2021) (Figure 1; Table 3).

Future perspective and conclusion
We have summarized multiple studies delving into the interplay between ferroptosis and tumour growth, aiming to grasp their relationship comprehensively and provide insights for targeted therapeutic strategies.It's vital to decipher how to counteract ferroptosis' role in promoting tumour growth while harnessing its therapeutic potential.One study proposed RCH NPs, a self-amplifying nanomedicine, aiming to optimize therapeutic efficacy in tumours by addressing ferroptosis' dual nature.RCH NPs displayed robust ferroptotic damage and bolstered the immune response, enhancing ferroptosis' positive effects in inhibiting tumour growth.They also mitigated inflammation-linked immunosuppression and IFNγ-associated adaptive immune resistance, countering ferroptosis' negative impact on immunotherapy (Zhang et al., 2022).More research is anticipated to design effective therapies that balance ferroptosis' dual effects on tumour growth.
Yet, ongoing research on ferroptosis remains in its early stages, leaving unanswered queries.Most studies investigating ferroptosis and its tumour association have relied on cellular and animal models, lacking validated clinical evidence.For instance, though targeting GPX4, a crucial component of the ferroptosis defense system, might theoretically restrain tumour growth, GPX4 is essential for life, with studies suggesting its loss heightens mortality rates in mice (Yant et al., 2003).Hence, it's crucial to ascertain the potential harm to normal tissue due to GPX4 inhibitors.Despite numerous potential targets linked to tumours and ferroptosis, it's unclear which of these findings can transition into clinical investigations.
Furthermore, there's a lack of research examining the effectiveness and safety of drugs intended to target ferroptosis.For instance, inhibiting SLC7A11 has shown potential in triggering tumour ferroptosis and reversing tumour resistance without observable impact on the development and survival of mice (Sato et al., 2005).However, this treatment may not be effective for tumours not reliant on the System Xc-.Ferroptosis is linked to the onset and progression of various diseases, extending beyond cancer to include degenerative conditions (Stockwell et al., 2017).Therefore, it's crucial to develop tailored therapies inducing ferroptosis in tumours while avoiding systemic adverse reactions.Combination therapies involving ferroptosis inducers and RT have shown safety in preclinical studies, but there are indications that ferroptosis might also contribute to radiation-induced damage in normal tissues (Su et al., 2022).Hence, further research is necessary to understand the impact of ferroptosis inducers on normal tissues and identify the patient population most likely to benefit from these treatments.
Finally, there's a notable absence of biomarkers available for assessing ferroptosis within the human body.Identifying suitable biomarkers would significantly aid in in vivo studies and clinical monitoring.Discovering predictive biomarkers capable of forecasting a tumour's response to ferroptosis-inducing therapies is crucial for categorizing tumour patients and guiding subsequent antitumour interventions involving ferroptosis induction.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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TABLE 1
Inhibitory effect of ferroptosis on tumour growth by regulating metabolisms.

TABLE 2
Promotional effect of ferroptosis on tumour growth.