Exploring Ferroptosis-Inducing Therapies for Cancer Treatment: Challenges and Opportunities

Abstract

Conventional cancer therapies typically aim to eliminate tumor cells by inducing cell death. The emergence of resistance to these standard treatments has spurred a shift in focus towards exploring alternative cell death pathways beyond apoptosis. Ferroptosis—an iron-dependent regulated cell death triggered by lipid peroxide accumulation—has gained prominence in cancer research in recent years. Ferroptosis-inducing therapies hold promise for overcoming resistance encountered with conventional treatments. However, challenges, including the lack of distinctive ferroptosis markers and the intricate role of ferroptosis within the tumor microenvironment, currently hinder the clinical translation of these therapies. This perspective article critically outlines these hurdles and highlights unexplored opportunities in ferroptosis research, aiming to refine its therapeutic utilization in combating cancer.

Introduction

Evading cell death represents a core hallmark of cancer. While initial studies primarily focused on apoptosis in cancer, the discoveries of alternative non-apoptotic cell death pathways, coupled with clinical observations revealing tumor resistance to conventional therapies that involve apoptosis induction, has ignited a compelling exploration into diverse cell death modalities in cancer. Among these modalities, ferroptosis has captured significant attention within the cancer research community in recent years (1,2). This surge in ferroptosis-related studies stems from several factors, contributing to an exponential growth in research understanding the role of ferroptosis in cancer.

1. Ferroptosis—an iron-dependent form of regulated cell death induced by the excessive accumulation of lipid peroxides in cellular membranes—operates distinctly from apoptosis and other cell death pathways (2,3). Therefore, cancer therapies aimed at inducing ferroptosis hold the potential in circumventing resistance encountered with traditional apoptosis-inducing treatments (1).

2. Cells have evolved antioxidant defense mechanisms, most notably the solute carrier family 7 member 11 (SLC7A11)-glutathione peroxidase 4 (GPX4) signaling axis (Fig. 1), to counteract lipid peroxides generated as byproducts of various cellular metabolic activities, thereby suppressing ferroptosis (2). Ferroptosis ensues when these defense systems are significantly compromised. The initial discovery of diverse ferroptosis-inducing compounds, such as erastin targeting SLC7A11 and RSL3 inhibiting GPX4 (Fig. 1), has sparked extensive research aimed at developing more potent ferroptosis inducers (FINs) and exploring these FINs in translational cancer studies (1,2).

3. An expanding body of research strongly corroborates the concept that, much like apoptosis, ferroptosis operates as a natural antitumor mechanism. Its tumor-suppressive effects involve interactions with a spectrum of tumor suppressor genes (e.g., p53 and BAP1) and critical oncogenic pathways (e.g., KRAS and PI3K), laying the groundwork for investigating ferroptosis-inducing strategies in cancer therapy (1,2).

4. It has been increasingly recognized that standard-of-care therapies—chemotherapy, radiotherapy, targeted therapy, and immunotherapy—can induce lipid peroxidation and ferroptosis in tumors (1,2). This realization has led to a surge in studies exploring the combination of FINs with these conventional therapies in cancer treatment (1).

Figure 1. Overview of ferroptosis interplay in cancer cells and immune cells, and potential biomarkers for ferroptosis detection.

The SLC7A11 transporter facilitates the uptake of extracellular cystine, aiding in the intracellular biosynthesis of glutathione (GSH), which is utilized by GPX4 for detoxifying lipid peroxides. Inactivation of the SLC7A11-GSH-GPX4 axis by ferroptosis inducers (FINs), such as erastin targeting SLC7A11 or RSL3 inhibiting GPX4, leads to the lethal accumulation of lipid peroxides, triggering ferroptosis.

Several markers such as C11 BODIPY 581/591, LiperFluo, 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), cyclooxygenase-2 (COX-2 or PTGS2), and peroxiredoxin 3 (PRDX3) have been used to monitor lipid peroxidation and ferroptosis. However, each marker has its specific limitations and undiscovered features, necessitating further exploration.

Ferroptotic cancer cells can modulate immune cells within the tumor microenvironment, reciprocally influencing or being influenced by these immune cells. The behavior of immune cells can either promote or inhibit ferroptosis in cancer cells, depending on the functionality and cellular context of these immune cells. Notably, various immune cell types exhibit differing susceptibilities to ferroptosis induction; for instance, M1-like macrophages demonstrate resistance, while M2-like macrophages are sensitive to ferroptosis induction. This adds more complexities for systemic ferroptosis-inducing therapy in cancer treatment.

However, several obstacles stand in the way of translating ferroptosis research into clinical applications. In this Perspective article, we will delve into two such challenges, the absence of specific ferroptosis markers and the complex role of ferroptosis in the tumor microenvironment. Additionally, we will highlight new opportunities that warrant further investigation in ferroptosis translational research.

Ferroptosis detection and markers

The potential of pharmacologically inducing ferroptosis holds great promise in treating therapy-resistant cancers (1,2). Translating ferroptosis-based treatments into clinical practice necessitates a comprehensive evaluation of ferroptosis occurrence during cancer therapy. Hence, there is considerable value in discovering specialized probes or proteins designed to specifically detect cancer cells undergoing ferroptosis. Preclinical studies in animal models have indicated the occurrence of ferroptosis across various stages of tumor development, including initiation, progression, and metastasis (1). Nonetheless, the absence of established biomarkers, similar to cleaved caspase-3 used for apoptosis detection, poses a significant obstacle in accurately identifying ferroptosis within tumor tissues.

For instance, fluorescent probes such as C11 BODIPY 581/591 and LiperFluo are widely used as indicators of lipid peroxidation in live cell imaging but face challenges in their application to fixed tumor tissue sections (nonetheless, they present potential candidates for ferroptosis biomarkers in blood or biofluids). Assessing oxidized phospholipids through lipidomic analyses serves as a compelling marker for ferroptosis; however, its technical complexity presents a significant limitation, restraining its routine use in clinical settings.

In preclinical studies, aldehyde secondary products of lipid peroxidation, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), have been commonly used as ferroptosis biomarkers via immunohistochemical analyses on tumor tissue sections (2). Nevertheless, these lipid peroxidation byproducts can arise from other unrelated oxidative stress conditions. Similarly, PTGS2 (cyclooxygenase-2 or COX-2) has been proposed as a ferroptosis biomarker due to its substantial upregulation following ferroptosis-inducing treatment in cancer cells (2). Yet, PTGS2 is also known to be upregulated in response to various inflammatory stimuli and stress conditions, not all of which are linked to ferroptosis.

Furthermore, transferrin receptor has been identified as a specific marker for ferroptosis (4). However, it is important to note that transferrin receptor is also upregulated when cells experience iron depletion, which serves as part of the cellular response to bolster its ability to acquire iron under low iron conditions. Given that iron depletion often correlates with resistance to ferroptosis in cancer cells, this complicates using transferrin receptor as a universal marker for identifying ferroptosis. In summary, the primary constraints of current ferroptosis biomarkers include their unsuitability for routine clinical application and their detection in contexts not associated with ferroptosis.

In our view, this challenge in pinpointing reliable ferroptosis biomarkers relates to a key mechanistic difference between ferroptosis and apoptosis. Apoptosis, along with other well-known regulated cell death forms like necroptosis and pyroptosis, involve distinct cell signaling cascades culminating in the activation of cell death executioner proteins (5). These pathways usually entail specific protein cleavage or modifications unique to each cell death process—such as caspase cleavage in apoptosis, mixed-lineage kinase domain-like protein (MLKL) phosphorylation in necroptosis, and gasdermin D cleavage in pyroptosis— making these modifications reliable markers for identifying respective cell death mechanisms (5).

However, ferroptosis is induced by lipid peroxide accumulation rather than defined protein modifications. During ferroptosis, significant changes occur in diverse metabolic pathways related to antioxidants, lipid, and iron metabolism (2). These alterations, intricately intertwined with numerous metabolic cues and downstream effects, pose a challenge in harnessing them as specific biomarkers to accurately detect ferroptosis.

Despite these challenges, we envision that a more in-depth understanding of ferroptosis regulatory mechanisms will continue to offer hope for uncovering new ferroptosis biomarkers. In particular, a recent study identified a role of peroxiredoxin 3 (PRDX3) in regulating ferroptosis; importantly, it revealed that PRDX3 undergoes a process called hyperoxidation—a type of posttranslational modification using peroxides as substrates—specifically triggered under ferroptosis-inducing conditions but not in other forms of cell death (6). Given that hyperoxidation involves a protein modification, hyperoxidized PRDX3 holds the potential as a unique ferroptosis marker. Additionally, the availability of antibodies targeting hyperoxidized PRDX3 should facilitate its detection in tumor sample sections, although it will be critical to validate the anti-hyperoxidized PRDX3 monoclonal antibody in future analyses, including an assessment of its potential cross-reaction with other members of the PRDX family. Furthermore, it is crucial to analyze this potential ferroptosis marker under various conditions, including non-ferroptosis-related oxidative stress scenarios, and therapeutic contexts involving ferroptosis-inducing treatments like immunotherapy or radiotherapy. This study will also spark further investigation into identifying other protein modifications involved in ferroptosis regulation as potential ferroptosis markers.

The intricate role of ferroptosis within the tumor microenvironment

Ferroptosis represents a pivotal natural mechanism in tumor suppression, offering promising avenues through therapies that trigger ferroptosis within tumor cells to combat tumor progression (1). However, systemic ferroptosis-inducing treatments in vivo extend beyond the targeted tumor cells, potentially impacting the functionality and vitality of various immune cells cohabiting within the tumor microenvironment (1). The effects of inducing ferroptosis in specific immune cells can either fortify or undermine the overall effectiveness of ferroptosis-based cancer therapies, depending upon the influence of these immune cells on tumor cells (Fig. 1). Navigating these complexities highlights the challenge of balancing the eradication of tumor cells with the imperative preservation of essential immune responses in ferroptosis-inducing therapies. As discussed below, this dilemma underscores the diverse and intricate roles that ferroptosis plays across various immune cell types in the context of cancer treatment.

Ferroptosis-inducing therapeutic strategies have shown potential in selectively eliminating certain immunosuppressive cells within the tumor microenvironment. For example, M2-like macrophages, known for their immunosuppressive roles, display heightened susceptibility to ferroptosis induction; in contrast, the immunostimulatory M1-like macrophages exhibit a notable resistance to ferroptosis (1). This susceptibility opens avenues for leveraging ferroptosis-inducing therapies to selectively eliminate M2 macrophages while preserving the M1 macrophage population; additionally, these therapies hold potential for reprogramming M2 macrophages into the more immunostimulatory M1 phenotype, thereby reshaping the immune landscape within tumors. Extending beyond macrophages, recent investigations have highlighted the significance of targeting specific molecular pathways to enhance the susceptibility of immunosuppressive myeloid-derived suppressor cells and tumor-infiltrating neutrophils to ferroptosis. Inhibition of n-acylsphingosine amidohydrolase 2 (ASAH2) or aconitate decarboxylase 1 (ACOD1), for instance, has been identified as an effective strategy to heighten the vulnerability of these immunosuppressive cells to ferroptosis induction, potentially paving the way for more precise and impactful therapeutic interventions (1,7). Additional studies revealed the susceptibility of regulatory T cells, another critical subset of immunosuppressive cells, to ferroptosis induced by GPX4 inhibition (1). Collectively, these findings advocate for the strategic use of ferroptosis-based cancer therapies to selectively trigger ferroptosis in various immunosuppressive cell populations. Such targeted interventions hold the promise of not only eliminating specific immunosuppressive cells but also potentially bolstering the antitumor immune response. This dual effect may significantly enhance the efficacy of these therapies in cancer treatment.

The dynamic interplay between cancer cells and immune cells may further amplify the antitumor immune response during ferroptosis-inducing cancer therapies. Ferroptosis in cancer cells triggers the release of a diverse array of immunostimulatory signals, capable of activating antigen-presenting cells (such as dendritic cells and M1-like macrophages) and T cells, resembling a potential mechanism similar to a tumor vaccine (1). This intriguing aspect positions ferroptosis as a form of immunogenic cell death, enhancing the immune system’s response against cancer. On the other hand, activated CD8⁺ T cells contribute to the promotion of ferroptosis within cancer cells by releasing interferon. This process involves modulating key ferroptosis regulatory pathways within cancer cells, notably upregulating acyl-coenzyme A synthetase long chain family member 4 (ACSL4) to synthesize polyunsaturated fatty acid-containing phospholipids (PUFA-PLs), pivotal substrates for lipid peroxidation, and downregulating SLC7A11-mediated ferroptosis defense mechanisms (8,9). This regulation highlights the bidirectional relationship between ferroptosis and immune activation, amplifying the therapeutic potential of this interaction in cancer treatment.

Significantly, interventions targeting ferroptosis pathways exhibit remarkable efficacy in suppressing tumor growth in immunocompetent animal models. For instance, dietary supplementation with arachidonic acid, a type of PUFA, or treatment with cyst(e)inase to degrade extracellular cystine and cysteine, triggers ferroptosis and effectively inhibits tumor growth; furthermore, combining immunotherapy with arachidonic acid or cyst(e)inase demonstrates a striking synergistic effect, further repressing tumor growth by augmenting ferroptosis induction (8,9). These studies highlight the potential of harnessing ferroptosis within the immunotherapeutic framework for cancer treatment.

However, other recent studies have revealed an intricate relationship between ferroptosis and various immunostimulatory cells, introducing complexities that impact antitumor immunity. Certain immune cells known for their immunostimulatory roles, such as B cells and natural killer cells, have shown susceptibility to ferroptosis (1). This susceptibility raises concerns that systemic ferroptosis-inducing therapies, by inadvertently targeting and eliminating these immune cells, might compromise the crucial antitumor immune response. The effects of ferroptosis on CD8⁺ T cells, pivotal players in adaptive immunity, also remain a subject of debate. Some evidence suggests that ferroptosis might not adversely affect CD8⁺ T cells and could potentially even augment their numbers; conversely, conflicting perspectives argue that CD8⁺ T cells exhibit heightened sensitivity to ferroptosis, possibly due to their elevated reliance on GPX4 and increased fatty acid uptake facilitated by CD36 (1).

Notably, recent findings have highlighted the vulnerability of immunosuppressive polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) to ferroptosis induction (10). However, the ferroptosis vulnerability in PMN-MDSCs presents a paradoxical twist in the context of antitumor immunity: ferroptotic PMN-MDSCs release specific oxidized PLs, which in turn hinder T cell infiltration and function, thereby suppressing the antitumor immune response. Consequently, inhibiting ferroptosis unexpectedly resulted in an antitumor effect in an immunocompetent animal model (10). It is noteworthy that this study diverges from the majority of other studies, which have shown that inducing ferroptosis in immunosuppressive cells typically enhances the antitumor immune response; in addition, in immunocompetent animal models, treatment with ferroptosis inducers, rather than inhibitors, usually leads to antitumor effects (1,7–9). The reasons for these disparities, whether they stem from the heterogeneity of PMN-MDSCs/neutrophils within tumors or variations in experimental design or animal models, remain to be clarified in future investigations.

In summary, the interplay between ferroptosis and diverse immune cells (Fig. 1) underscores the intricate balance required to harness ferroptosis-inducing therapies for cancer treatment. While ferroptosis is considered a potential form of immunogenic cell death, which selectively eliminates immunosuppressive cells, potentially augments antitumor immunity, and mediates the efficacy of immunotherapy, its impact on other immune components, such as CD8⁺ T cells, remains controversial and warrants careful investigation. These revelations pose both a challenge and an opportunity: effectively navigating the fine line between exploiting ferroptosis for cancer therapy and preserving essential immune responses, while also identifying new avenues for enhancing antitumor immunity by modulating ferroptosis in specific immune cell populations.

It is also important to note that the majority of prior preclinical studies investigating ferroptosis-inducing cancer therapies have relied on immunocompromised animal models, limiting the exploration of ferroptosis’s role in the immune system. These observations discussed above (which were conducted in immunocompetent animal models) therefore underscore the critical need in future studies for utilizing preclinical models that better mirror the complexities of the immune system and the tumor microenvironment, such as syngeneic models and genetically engineered mouse models. Such models are crucial in advancing ferroptosis-based translational research, offering a more comprehensive understanding of the intricate interactions between ferroptosis and the immune system in the future.

Conclusion

The exploitation of ferroptosis in cancer therapy has gained significant traction in the cancer research community, driven by its potential to overcome resistance linked to apoptosis-inducing treatments and exploit distinctive metabolic vulnerabilities in cancer cells. The evolving landscape of ferroptosis research within cancer therapy reveals both significant challenges and promising avenues. Within this article, we spotlight two primary challenges and discuss corresponding opportunities: the identification of ferroptosis biomarkers and understanding the intricate dynamics of ferroptosis within the complex tumor microenvironment (Fig. 1). Owing to space limitation, several other critical challenges in ferroptosis translational research remain unexplored in this article. These include exploration of optimal therapeutic windows that selectively eliminate tumor cells via ferroptosis while preserving normal tissues, the development of more potent and specific FINs (such as GPX4 inhibitors) suitable for clinical application, identification of patient cohorts suitable for selective combination therapies involving ferroptosis-inducing treatments and conventional therapies, and the formulation of strategies to counter potential ferroptosis resistance in patients. Addressing the first challenge, existing studies have shown that certain cancer cells display specific vulnerabilities to ferroptosis due to metabolic reprogramming, distinct genetic alterations, or imbalances in ferroptosis defense mechanisms (1). Exploiting these ferroptosis vulnerabilities in cancer cells has the potential to selectively target tumors while sparing normal tissues. However, further clinical investigations are warranted to validate these concepts derived from preclinical studies.

Moving forward, it is critical to attain a comprehensive fundamental understanding of the multifaceted role of ferroptosis in cancer. Concurrently, refining treatment approaches through translational research is crucial to maximize the therapeutic effectiveness of ferroptosis-inducing strategies while minimizing unintended side effects. This balanced approach holds the key to fully harnessing the potential of ferroptosis-targeted therapies in the ongoing fight against cancer.