A hub for an herb: is STAT3 blockade at the epicenter of toosendanin-induced NCOA4-dependent ferritinophagy and ferroptosis in cancer cells?

Ferroptosis is an iron-dependent, but tightly regulated, cell death that is mechanistically different from other forms of cell death such as apoptosis or necroptosis (1,2). Even though iron is obligatory for several important functions in a cell, excess free iron in the form of its divalent Fe²⁺ can be detrimental because it generates via the Fenton reaction cycle potent reactive oxygen species (ROS) such as hydroxyl (OH^l) and hydroperoxyl (HOO^l) radicals that oxidize double bonds in polyunsaturated fatty acids present in phospholipids in the lipid bilayer of biological membranes. This phenomenon is called lipid peroxidation and generates lipid peroxides as the end products. As phospholipids are the most predominant lipids that are fundamental for the bilayer structure of cellular membranes, their destruction via peroxidation causes damage to membrane integrity and barrier function, thus leading to cell death. Ferroptosis is highly regulated by intricate mechanisms, all ultimately coalescing towards controlling either the cellular levels of free Fe²⁺ (called labile iron pool) or the ability to scavenge the damage-causing free radicals. Understandably, if this process occurs in normal tissues, it compromises organ function, thus contributing to tissue pathology and clinical manifestations in various diseases such as diabetes, inflammatory bowel disease, ischemia-reperfusion injury, neurodegeneration, polycystic ovary syndrome, etc. The scenario is quite different in tumor tissues. Cancer cells need excess iron to support biological functions pertinent to increased proliferation and growth and accordingly regulate iron acquisition (upregulation of transferrin receptor 1 and iron importer SLC11A2) and iron efflux mechanisms (downregulation of iron exporter SLC40A1) to meet their increased demands for iron (3,4). At the same time, these cells need to make sure that the cellular levels of the labile iron pool are maintained low and the capacity of the cells to prevent lipid peroxidation is sustained high to avoid cell death by ferroptosis. This goal is accomplished by inducing the production of the iron-sequestering protein ferritin and by upregulating their antioxidant machinery (e.g., increased glutathione levels, induction of glutathione peroxidase GPX4, induction of the cystine importer SLC7A11 to support glutathione synthesis, activation of the antioxidant transcription factor NRF2). Through these regulatory pathways, cancer cells resist ferroptosis even though these cells have the machinery to acquire more iron than normal cells (5-7). Given this unique scenario, induction of ferroptotic death could be the Achilles’ heel in cancer cells and offers a potentially effective strategy for cancer treatment.

There are several pharmacological options to exploit the vulnerability of cancer cells to ferroptosis: (I) suppression of GPX4; (II) suppression of SLC7A11; (III) blockade of NRF2 signaling; and (IV) degradation of ferritin via ferritinophagy to load the cells with excess free iron. Each of these options is being targeted for cancer therapy with currently ongoing intense investigations in many laboratories. Several small molecules have been identified as potent inhibitors of GPX4 (e.g., RSL3), SLC7A11 (e.g., erastin, niclosamide), and NRF2 (e.g., ML385, pyrimethamine) and as potent inducers of ferritin degradation (e.g., emodin). There are also numerous examples of natural compounds (e.g., herbal constituents) that exhibit potency to induce ferroptosis in cancer cells via one or more of the mechanisms listed above and hence hold promise as therapeutic agents in cancer treatment. A recently reported study by Feng et al. in the Journal of Gastrointestinal Oncology (8) describes the ability of toosendanin, a triterpenoid constituent of a Chinese medicinal herb (9), to promote ferroptotic cell death in gastrointestinal stromal tumor (GIST) cells. The most significant finding in this study is that the mechanisms of toosendanin as a ferroptosis inducer in these cells are multi-faceted, involving decreased GPX4 and SLC7A11 as well as increased ferritinophagy, thus leading to increase in the labile iron (Fe²⁺) pool, decrease in glutathione and increase in ROS. This in vitro study was carried out with a cell line model for GIST. The use of a single cell line (GIST-T1 cells) is a significant weakness in this investigation. Several cell lines are available from commercial sources as suitable models for GIST; it is therefore not readily apparent why the authors chose to conduct the study with a single cell line without validating their findings in additional cell lines. Notwithstanding this notable weakness, the experiments reported in the study are thorough and the data and their interpretations appear reliable and logical. GISTs are rare, but often malignant, tumors of the gastrointestinal tract; these tumors arise from a specialized cell type, known as the interstitial cells of Cajal (ICCs). ICCs are the pacemaker cells that are responsible for the slow waves in the gastrointestinal tract, thus obligatory for gut motility. Morphologically these cells are neither smooth muscle type nor neuronal type, but they express a specific cell-surface marker known as KIT (CD117), a tyrosine kinase-associated receptor that is activated by stem cell factor as a specific ligand. KIT is mutated in ~80% of GISTs leading to its constitutive overactivation which is the key driver of tumor initiation and growth (10). The GIST-T1 cell line used in the study by Feng et al. (8) does have an activating mutation in this receptor, thus making it a suitable cell-line model to represent the most predominant molecular type of GIST. Imatinib is a highly selective inhibitor of receptor-associated tyrosine kinases and targets KIT as well as other receptor tyrosine kinases such as PDGF receptor. This drug constitutes the first-line therapy in the treatment of GIST with KIT mutations. However, 60–75% of patients develop resistance to this drug within 2 years of treatment, highlighting the need for newer therapeutic strategies. In this context lies the significance of the study by Feng et al. (8) which identifies toosendanin as a potential agent for the treatment of GIST with a pharmacological mechanism distinct from that of imatinib and hence holding promise to circumvent resistance to imatinib.

Let us first summarize the findings of the study. Treatment of GIST-T1 cells at submicromolar concentrations decreases cell viability with an IC₅₀ value of 0.13 µM. This effect is prevented most effectively by ferrostatin-1, a selective inhibitor of ferroptosis. Inhibitors of other forms of cell death (apoptosis, necroptosis or pyroptosis) are less effective. The decreased cell viability is associated with decreased levels of the antioxidant glutathione and increased levels of the lipid peroxidation end product malondialdehyde (in the figure panel relating to this finding, glutathione and MDA are mislabeled as cytokines in the manuscript). In addition, the protein levels of GPX4 and SLC7A11 are decreased consistent with the changes in glutathione and malondialdehyde. There is also evidence for increased ferritinophagy, supported by the increase in NCOA4 and the decrease in ferritin (most likely it is the ferritin heavy chain that was measured in the study even though it is not specifically mentioned) along with an increase in free Fe²⁺ levels. NCOA4 is a selective cargo receptor for autophagic degradation of ferritin and release of free iron; this protein binds to ferritin heavy chain (FTH1) and channels it into autophagosomes for lysosomal degradation (11,12). Treatment with toosendanin suppresses not only cell viability but also the colony-formation ability of the cells as well as their migration and invasion capabilities. NCOA4-mediated ferritinophagy appears to be at the center of these effects because siRNA-mediated downregulation of NCOA4 significantly reverses these effects. Similarly, when ferroptosis is blocked with ferrostatin-1 in toosendanin-treated cells, the protein levels of GPX4 and SLC7A11 increase and the levels of malondialdehyde decrease. Taken collectively, these findings lead to the conclusion that toosendanin induces NCOA4-mediated ferritinophagy and leads to ferroptotic cell death in GIST-T1 cells.

The ability of toosendanin to induce ferroptosis in normal cells as well as in cancer cells has been described in several reports, both prior to (13,14) and after (15-17) the publication of the study by Feng et al. (8). But what is new in the Feng et al. study is that it is the first report on the involvement of NCOA4-mediated ferritinophagy in toosendanin-mediated ferroptosis induction. However, this could not be the sole mechanism underlying the pharmacological action of toosendanin as a ferroptosis inducer. This is evident from other published reports which describe additional mechanisms such as the activation of PERK-eIF2α-ATF4-ATF3-TFRC pathway to increase the cellular uptake of iron (13), activation of WWOX leading to increased transcriptional activity of p53 and suppressed transcriptional activity of NRF2, thus leading to decreased expression of GPX4 and SLC7A11 and increased expression of the iron exporter SLC40A1 (14), activation of PLK1 (15), activation of 5-lipoxygenase (ALOX5) to directly induce lipid peroxidation (16), and inhibition of O-GlcNAcylation of NRF2, thus leading to suppression of its transcriptional activity (17).

Let us now critically analyze the experimental data presented in the study by Feng et al. (8), specifically focusing on potential missing pieces of the puzzle. Toosendanin reduces cell proliferation, colony formation, migration and invasion of GIST-T1 cells. The ferroptotic cell death caused by toosendanin most likely underlies the reduction in cell proliferation and colony formation. Since lipid peroxidation is induced, it may disrupt the integrity of cellular membranes and cytoskeleton network, thereby leading to decreased cell migration and invasion. The increased levels of NCOA4 protein in toosendanin-treated cells explain the decreased levels of ferritin as one would expect from enhanced NCOA4-dependent ferritinophagy. This process is accompanied with an increase in Fe²⁺ levels that is necessary for the induction of ferroptosis. There are however some important steps between ferritinophagy and free Fe²⁺ that are not addressed in the study. Ferritin has the capacity to store only the trivalent form of iron Fe³⁺; therefore, ferritin degradation would release only Fe³⁺ within the autophagosomes. Iron is known to be exported out of the autophagosomes via the H⁺-coupled transporter DMT1 (divalent metal ion transporter, SLC11A2) which accepts Fe²⁺, not Fe³⁺, as the substrate. This necessitates the conversion of Fe³⁺ to Fe²⁺ in autophagosomes prior to its release into cytoplasm; this conversion is catalyzed by the endosomal metalloreductase STEAP3. At present, it is not known if toosendanin has any effect on the expression and/or activity of either of these components (i.e., SLC11A2 and STEAP3). Furthermore, the expression of GPX4 and SLC7A11 is suppressed by toosendanin. It is not known whether the induction of ferritinophagy is directly related to these expression changes. The release of excess iron by ferritinophagy most likely causes stress in cells that could lead to suppression of GPX4 and SLC7A11, but this has not been investigated. It is tempting to speculate that the observed increase in cellular levels of NCOA4 protein lies upstream of all other findings observed in toosendanin-treated cells. This then leaves the following questions: (I) Is the increase in NCOA4 protein levels due to changes in gene expression or in stability of mRNA or protein? (II) Does toosendanin elicit these effects by direct interaction with NCOA4 protein or by some upstream signaling?

There is no evidence in published literature for direct binding of toosendanin to NCOA4 protein. However, several molecular targets have been identified for this compound in mammalian cells, the direct binding confirmed in most cases by multiple experimental approaches such as surface plasmon resonance, cellular thermal shift and in silico molecular docking. Among these targets are STAT3, vacuolar type H⁺-ATPase, Src family protein kinases, STING (stimulator of interferon genes), eukaryotic translational elongation factor 2, sonic hedgehog Shh, and κ-opioid receptor (18-23). After a careful review of the pertinent literature, we posit that STAT3 as the upstream pharmacological target for toosendanin may convincingly explain the changes in NCOA4, FTH1, labile free Fe²⁺, GPX4 and SLC7A11 observed in toosendanin-treated GIST-T1 cells. Toosendanin directly binds to the SH2 domain of STAT3 with a K_D value of ~0.24 µM (17). This interaction inhibits the activity of receptor-associated tyrosine kinase and consequently impairs STAT3 dimerization and nuclear translocation, and hence its transcriptional activity. STAT3 is overactive in multiple types of cancer and it is a negative regulator of ferroptosis, promoting resistance to this form of cell death in cancer cells (24). Therefore, blockade of STAT3 signaling via its direct binding to toosendanin may induce ferroptosis. In corroboration with this possibility are the findings that STAT3 is a positive transcriptional regulator of the genes coding for FTH1, GPX4 and SLC7A11 (25). Based on these published reports, one can expect to see a decrease in the expression and protein levels of these three proteins in GIST-T1 cells in response to toosendanin treatment. This is indeed the case in the study by Feng et al. (8). This mechanism by itself may be sufficient to explain the observed increase in free labile Fe²⁺ and decrease in glutathione, together causing an increase in ROS and lipid peroxidation. ROS and oxidative stress are known to induce the expression of NCOA4 (26,27) and activate the ATF3-STEAP3 axis (28,29). As such, toosendanin is expected to increase the protein levels of NCOA4 and STEAP3 in GIST-T1 cells, which could promote ferritinophagy and the conversion of released Fe³⁺ into Fe²⁺ within the autophagosomes. The data reported by Feng et al. (8) support this line of logic. Interestingly, the K_D value (~0.24 µM) is in the same order of magnitude as the IC₅₀ value (0.13 µM) for the ability of toosendanin to decrease cell proliferation in GIST-T1 cells. The most likely biochemical/signaling pathways that might underlie the pharmacological effects of toosendanin in GIST-T1 cells are schematically represented in Figure 1. This scheme is still hypothetical but is easily tenable for direct experimental validation with pharmacological and/or genetic approaches. However, it has to be borne in mind that STAT3 is not the only pharmacological target for toosendanin; therefore, potential involvement of targets other than STAT3 in toosendanin-induced ferroptosis should not be ignored.

Figure 1 A possible mechanistic scheme for the induction of NCOA4-dependent ferritinophagy and ferroptosis in GIST-T1 cells by toosendanin with inhibition of STAT3 signaling as the upstream epicenter. The abbreviations for all the components of the signaling pathway proposed in this pathway are expanded at the beginning of the article. ATF, activating transcription factor; FTH1, ferritin heavy chain; GIST, gastrointestinal stromal tumor; GPX4, glutathione peroxidase 4; GSH, glutathione; NCOA4, nuclear receptor coactivator 4; ROS, reactive oxygen species; SLC7A11, 11th member of the solute carrier gene family 7, subfamily A; STAT3, signal transducer and activator of transcription 3; STEAP3, six-transmembrane epithelial antigen of prostate 3.

As a final point, the study by Feng et al. (8) represents an in vitro experimentation in its entirety. It would be necessary to demonstrate the ability of toosendanin to induce NCOA4-dependent ferritinophagy and ferroptosis and hence to suppress tumor growth using in vivo models of GIST. Another point of significance is that the mechanism of toosendanin described in GIST-T1 cells is not likely to be an isolated phenomenon seen only in this cancer cell because the general pathways leading to the development of resistance to ferroptotic cell death are quite similar across cancer cells of various tissue origin. As such, toosendanin may represent a promising therapeutic agent for the treatment of not only GIST but also other cancer types. This is evident from published literature demonstrating the promising therapeutic efficacy of toosendanin in models of multiple cancers, including colon cancer, pancreatic cancer, breast cancer, liver cancer, gastric cancer, lung cancer, and ovarian cancer [reviewed in (30-32)]. Notwithstanding this encouraging experimental evidence for toosendanin as a promising anti-cancer agent, there is a need for detailed in vivo studies to validate its therapeutic potential because of the unresolved caveats relating to safety and toxicity concerns and possible undesirable off-target side effects of the agent.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Journal of Gastrointestinal Oncology. The article has undergone external peer review.

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Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0151/coif). The authors have no conflicts of interest to declare.

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