Zeb1 mediates EMT/plasticity-associated ferroptosis sensitivity in cancer cells by regulating lipogenic enzyme expression and phospholipid composition.

Therapy resistance and metastasis, the most fatal steps in cancer, are often triggered by a (partial) activation of the epithelial-mesenchymal transition (EMT) programme. A mesenchymal phenotype predisposes to ferroptosis, a cell death pathway exerted by an iron and oxygen-radical-mediated peroxidation of phospholipids containing polyunsaturated fatty acids. We here show that various forms of EMT activation, including TGFß stimulation and acquired therapy resistance, increase ferroptosis susceptibility in cancer cells, which depends on the EMT transcription factor Zeb1. We demonstrate that Zeb1 increases the ratio of phospholipids containing pro-ferroptotic polyunsaturated fatty acids over cyto-protective monounsaturated fatty acids by modulating the differential expression of the underlying crucial enzymes stearoyl-Co-A desaturase 1 (SCD), fatty acid synthase (FASN), fatty acid desaturase 2 (FADS2), elongation of very long-chain fatty acid 5 (ELOVL5) and long-chain acyl-CoA synthetase 4 (ACSL4). Pharmacological inhibition of selected lipogenic enzymes (SCD and FADS2) allows the manipulation of ferroptosis sensitivity preferentially in high-Zeb1-expressing cancer cells. Our data are of potential translational relevance and suggest a combination of ferroptosis activators and SCD inhibitors for the treatment of aggressive cancers expressing high Zeb1.

Enhancing 7-dehydrocholesterol suppresses brain ferroptosis and tissue injury after neonatal hypoxia-ischemia.

Neonatal hypoxic-ischemic brain injury (HIBI) results in part from excess reactive oxygen species and iron-dependent lipid peroxidation (i.e. ferroptosis). The vitamin D precursor 7-dehydrocholesterol (7-DHC) may inhibit iron-dependent lipid peroxidation. Primary neurons underwent oxygen and glucose deprivation (OGD) injury and treatment with 7-DHC-elevating medications such as cariprazine (CAR) or vehicle. Postnatal day 9 mice underwent sham surgery or carotid artery ligation and hypoxia and received intraperitoneal CAR. In neurons, CAR administration resulted in significantly increased cell survival compared to vehicle controls, whether administered 48 h prior to or 30 min after OGD, and was associated with increased 7-DHC. In the mouse model, malondialdehyde and infarct area significantly increased after HIBI in the vehicle group, which were attenuated by post-treatment with CAR and were negatively correlated with tissue 7-DHC concentrations. Elevating 7-DHC concentrations with CAR was associated with improved cellular and tissue viability after hypoxic-ischemic injury, suggesting a novel therapeutic avenue.

Treatment with siRNAs is commonly associated with GPX4 up-regulation and target knockdown-independent sensitization to ferroptosis.

Small interfering RNAs (siRNAs) are widely used in biomedical research and in clinical trials. Here, we demonstrate that siRNA treatment is commonly associated with significant sensitization to ferroptosis, independently of the target protein knockdown. Genetically targeting mitochondrial antiviral-signaling protein (MAVS) reversed the siRNA-mediated sensitizing effect, but no activation of canonical MAVS signaling, which involves phosphorylation of IkBα and interferon regulatory transcription factor 3 (IRF3), was observed. In contrast, MAVS mediated a noncanonical signal resulting in a prominent increase in mitochondrial ROS levels, and increase in the BACH1/pNRF2 transcription factor ratio and GPX4 up-regulation, which was associated with a 50% decrease in intracellular glutathione levels. We conclude that siRNAs commonly sensitize to ferroptosis and may severely compromise the conclusions drawn from silencing approaches in biomedical research. Finally, as ferroptosis contributes to a variety of pathophysiological processes, we cannot exclude side effects in human siRNA-based therapeutical concepts that should be clinically tested.

Integrated chemical and genetic screens unveil FSP1 mechanisms of ferroptosis regulation.

Ferroptosis, marked by iron-dependent lipid peroxidation, may present an Achilles heel for the treatment of cancers. Ferroptosis suppressor protein-1 (FSP1), as the second ferroptosis mainstay, efficiently prevents lipid peroxidation via NAD(P)H-dependent reduction of quinones. Because its molecular mechanisms have remained obscure, we studied numerous FSP1 mutations present in cancer or identified by untargeted random mutagenesis. This mutational analysis elucidates the FAD/NAD(P)H-binding site and proton-transfer function of FSP1, which emerged to be evolutionarily conserved among different NADH quinone reductases. Using random mutagenesis screens, we uncover the mechanism of action of next-generation FSP1 inhibitors. Our studies identify the binding pocket of the first FSP1 inhibitor, iFSP1, and introduce the first species-independent FSP1 inhibitor, targeting the NAD(P)H-binding pocket. Conclusively, our study provides new insights into the molecular functions of FSP1 and enables the rational design of FSP1 inhibitors targeting cancer cells.

Loss of the cystine/glutamate antiporter in melanoma abrogates tumor metastasis and markedly increases survival rates of mice.

The cystine/glutamate antiporter, system xc- , is essential for the efficient uptake of cystine into cells. Interest in the mechanisms of system xc- function soared with the recognition that system xc- presents the most upstream node of ferroptosis, a recently described form of regulated necrosis relevant for degenerative diseases and cancer. Since targeting system xc- hold the great potential to efficiently combat tumor growth and metastasis of certain tumors, we disrupted the substrate-specific subunit of system xc- , xCT (SLC7A11) in the highly metastatic mouse B16F10 melanoma cell line and assessed the impact on tumor growth and metastasis. Subcutaneous injection of tumor cells into the syngeneic B16F10 mouse melanoma model uncovered a marked decrease in the tumor-forming ability and growth of KO cells compared to control cell lines. Strikingly, the metastatic potential of KO cells was markedly reduced as shown in several in vivo models of experimental and spontaneous metastasis. Accordingly, survival rates of KO tumor-bearing mice were significantly prolonged in contrast to those transplanted with control cells. Analyzing the in vitro ability of KO and control B16F10 cells in terms of endothelial cell adhesion and spheroid formation revealed that xCT expression indeed plays an important role during metastasis. Hence, system xc- emerges to be essential for tumor metastasis in mice, thus qualifying as a highly attractive anticancer drug target, particularly in light of its dispensable role for normal life in mice.

Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction.

Conditions of impaired adrenal function and tissue destruction, such as in Addison's disease, and treatment resistance of adrenocortical carcinoma (ACC) necessitate improved understanding of the pathophysiology of adrenal cell death. Due to relevant oxidative processes in the adrenal cortex, our study investigated the role of ferroptosis, an iron-dependent cell death mechanism and found high adrenocortical expression of glutathione peroxidase 4 (GPX4) and long-chain-fatty-acid CoA ligase 4 (ACSL4) genes, key factors in the initiation of ferroptosis. By applying MALDI mass spectrometry imaging to normal and neoplastic adrenocortical tissue, we detected high abundance of arachidonic and adrenic acid, two long chain polyunsaturated fatty acids which undergo peroxidation during ferroptosis. In three available adrenal cortex cell models (H295R, CU-ACC1 and CU-ACC-2) a high susceptibility to GPX4 inhibition with RSL3 was documented with EC50 values of 5.7 × 10-8, 8.1 × 10-7 and 2.1 × 10-8 M, respectively, while all non-steroidogenic cells were significantly less sensitive. Complete block of GPX4 activity by RSL3 led to ferroptosis which was completely reversed in adrenal cortex cells by inhibition of steroidogenesis with ketoconazole but not by blocking the final step of cortisol synthesis with metyrapone. Mitotane, the only approved drug for ACC did not induce ferroptosis, despite strong induction of lipid peroxidation in ACC cells. Together, this report is the first to demonstrate extraordinary sensitivity of adrenal cortex cells to ferroptosis dependent on their active steroid synthetic pathways. Mitotane does not induce this form of cell death in ACC cells.

FSP1 is a glutathione-independent ferroptosis suppressor.

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

Novel Allosteric Activators for Ferroptosis Regulator Glutathione Peroxidase 4.

Glutathione peroxidase 4 (GPX4) is essential for cell membrane repair, inflammation suppression, and ferroptosis inhibition. GPX4 upregulation provides unique drug discovery opportunities for inflammation and ferroptosis-related diseases. However, rational design of protein activators is challenging. Until now, no compound has been reported to activate the enzyme activity of GPX4. Here, we identified a potential allosteric site in GPX4 and successfully found eight GPX4 activators using a novel computational strategy and experimental studies. Compound 1 from the virtual screen increased GPX4 activity, suppressed ferroptosis, reduced pro-inflammatory lipid mediator production, and inhibited NF-κB pathway activation. Further chemical synthesis and structure-activity relationship studies revealed seven more activators. The strongest compound, 1d4, increased GPX4 activity to 150% at 20 µM in the cell-free assay and 61 µM in cell extracts. Therefore, we demonstrated that GPX4 can be directly activated using chemical compounds to suppress ferroptosis and inflammation. Meanwhile, the discovery of GPX4 activators verified the possibility of rational design of allosteric activators.

Nano-targeted induction of dual ferroptotic mechanisms eradicates high-risk neuroblastoma.

High-risk neuroblastoma is a devastating malignancy with very limited therapeutic options. Here, we identify withaferin A (WA) as a natural ferroptosis-inducing agent in neuroblastoma, which acts through a novel double-edged mechanism. WA dose-dependently either activates the nuclear factor-like 2 pathway through targeting of Kelch-like ECH-associated protein 1 (noncanonical ferroptosis induction) or inactivates glutathione peroxidase 4 (canonical ferroptosis induction). Noncanonical ferroptosis induction is characterized by an increase in intracellular labile Fe(II) upon excessive activation of heme oxygenase-1, which is sufficient to induce ferroptosis. This double-edged mechanism might explain the superior efficacy of WA as compared with etoposide or cisplatin in killing a heterogeneous panel of high-risk neuroblastoma cells, and in suppressing the growth and relapse rate of neuroblastoma xenografts. Nano-targeting of WA allows systemic application and suppressed tumor growth due to an enhanced accumulation at the tumor site. Collectively, our data propose a novel therapeutic strategy to efficiently kill cancer cells by ferroptosis.

Ferroptosis Inhibition: Mechanisms and Opportunities.

The past decade has yielded tremendous insights into how cells die. This has come with our understanding that several distinct forms of cell death are encompassed under the umbrella term necrosis. Among these distinct forms of regulated necrotic cell death, ferroptosis has attracted considerable attention owing to its putative involvement in diverse pathophysiological processes. A key feature of the ferroptosis process is the requirement of phospholipid peroxidation, a process that has been linked with several human pathologies. Now with the establishment of a connection between lipid peroxidation and a distinctive cell death pathway, the search for new small molecules able to suppress lipid peroxidation has gained momentum and may yield novel cytoprotective strategies. We review here advances in our understanding of the ferroptotic process and summarize the development of lipid peroxidation inhibitors with the ultimate goal of suppressing ferroptosis-relevant cell death and related pathologies.

Direct participation of DNA in the formation of singlet oxygen and base damage under UVA irradiation.

UVA light is hardly absorbed by the DNA molecule, but recent works point to a direct mechanism of DNA lesion by these wavelengths. UVA light also excite endogenous chromophores, which causes DNA damage through ROS. In this study, DNA samples were irradiated with UVA light in different conditions to investigate possible mechanisms involved in the induction of DNA damage. The different types of DNA lesions formed after irradiation were determined through the use of endonucleases, which recognize and cleave sites containing oxidized bases and cyclobutane pyrimidine dimers (CPDs), as well as through antibody recognition. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG) was also studied in more detail using electrochemical detection. The results show that high NaCl concentration and concentrated DNA are capable of reducing the induction of CPDs. Moreover, concerning damage caused by oxidative stress, the presence of sodium azide and metal chelators reduce their induction, while deuterated water increases the amounts of oxidized bases, confirming the involvement of singlet oxygen in the generation of these lesions. Curiously, however, high concentrations of DNA also enhanced the formation of oxidized bases, in a reaction that paralleled the increase in the formation of singlet oxygen in the solution. This was interpreted as being due to an intrinsic photosensitization mechanism, depending directly on the DNA molecule to absorb UVA and generate singlet oxygen. Therefore, the DNA molecule itself may act as a chromophore for UVA light, locally producing a damaging agent, which may lead to even greater concerns about the deleterious impact of sunlight.

Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis.

Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis-a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls-arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.

ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.

Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate ferroptosis are needed. We applied two independent approaches-a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines-to uncover acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential component for ferroptosis execution. Specifically, Gpx4-Acsl4 double-knockout cells showed marked resistance to ferroptosis. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 was preferentially expressed in a panel of basal-like breast cancer cell lines and predicted their sensitivity to ferroptosis. Pharmacological targeting of ACSL4 with thiazolidinediones, a class of antidiabetic compound, ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases.

Regulated necrosis: disease relevance and therapeutic opportunities.

The discovery of regulated cell death presents tantalizing possibilities for gaining control over the life-death decisions made by cells in disease. Although apoptosis has been the focus of drug discovery for many years, recent research has identified regulatory mechanisms and signalling pathways for previously unrecognized, regulated necrotic cell death routines. Distinct critical nodes have been characterized for some of these alternative cell death routines, whereas other cell death routines are just beginning to be unravelled. In this Review, we describe forms of regulated necrotic cell death, including necroptosis, the emerging cell death modality of ferroptosis (and the related oxytosis) and the less well comprehended parthanatos and cyclophilin D-mediated necrosis. We focus on small molecules, proteins and pathways that can induce and inhibit these non-apoptotic forms of cell death, and discuss strategies for translating this understanding into new therapeutics for certain disease contexts.

The antioxidant requirement for plasma membrane repair in skeletal muscle.

Vitamin E (VE) deficiency results in pronounced muscle weakness and atrophy but the cell biological mechanism of the pathology is unknown. We previously showed that VE supplementation promotes membrane repair in cultured cells and that oxidants potently inhibit repair. Here we provide three independent lines of evidence that VE is required for skeletal muscle myocyte plasma membrane repair in vivo. We also show that when another lipid-directed antioxidant, glutathione peroxidase 4 (Gpx4), is genetically deleted in mouse embryonic fibroblasts, repair fails catastrophically, unless cells are supplemented with VE. We conclude that lipid-directed antioxidant activity provided by VE, and possibly also Gpx4, is an essential component of the membrane repair mechanism in skeletal muscle. This work explains why VE is essential to muscle health and identifies VE as a requisite component of the plasma membrane repair mechanism in vivo.

Knockout of mitochondrial thioredoxin reductase stabilizes prolyl hydroxylase 2 and inhibits tumor growth and tumor-derived angiogenesis.

AIMS: Mitochondrial thioredoxin reductase (Txnrd2) is a central player in the control of mitochondrial hydrogen peroxide (H2O2) abundance by serving as a direct electron donor to the thioredoxin-peroxiredoxin axis. In this study, we investigated the impact of targeted disruption of Txnrd2 on tumor growth. RESULTS: Tumor cells with a Txnrd2 deficiency failed to activate hypoxia-inducible factor-1α (Hif-1α) signaling; it rather caused PHD2 accumulation, Hif-1α degradation and decreased vascular endothelial growth factor (VEGF) levels, ultimately leading to reduced tumor growth and tumor vascularization. Increased c-Jun NH2-terminal Kinase (JNK) activation proved to be the molecular link between the loss of Txnrd2, an altered mitochondrial redox balance with compensatory upregulation of glutaredoxin-2, and elevated PHD2 expression. INNOVATION: Our data provide compelling evidence for a yet-unrecognized mitochondrial Txnrd-driven, regulatory mechanism that ultimately prevents cellular Hif-1α accumulation. In addition, simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems was used as an efficient therapeutic approach in hindering tumor growth. CONCLUSION: This work demonstrates an unexpected regulatory link between mitochondrial Txnrd and the JNK-PHD2-Hif-1α axis, which highlights how the loss of Txnrd2 and the resulting altered mitochondrial redox balance impairs tumor growth as well as tumor-related angiogenesis. Furthermore, it opens a new avenue for a therapeutic approach to hinder tumor growth by the simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems.

Cardiolipin signaling mechanisms: collapse of asymmetry and oxidation.

SIGNIFICANCE: An ancient anionic phospholipid, cardiolipin (CL), ubiquitously present in prokaryotic and eukaryotic membranes, is essential for several structural and functional purposes. RECENT ADVANCES: The emerging role of CLs in signaling has become the focus of many studies. CRITICAL ISSUES: In this work, we describe two major pathways through which mitochondrial CLs may fulfill the signaling functions via utilization of their (i) asymmetric distribution across membranes and translocations, leading to the surface externalization and (ii) ability to undergo oxidation reactions to yield the signature products recognizable by the executionary machinery of cells. FUTURE DIRECTIONS: We present a concept that CLs and their oxidation/hydrolysis products constitute a rich communication language utilized by mitochondria of eukaryotic cells for diversified regulation of cell physiology and metabolism as well as for inter-cellular interactions.

ROS, thiols and thiol-regulating systems in male gametogenesis.

BACKGROUND: During maturation and storage, spermatozoa generate substantial amounts of reactive oxygen species (ROS) and are thus forced to cope with an increasingly oxidative environment that is both needed and detrimental to their biology. Such a janus-faceted intermediate needs to be tightly controlled and this is done by a wide array of redox enzymes. These enzymes not only have to prevent unspecific modifications of essential cellular biomolecules by quenching undesired ROS, but they are also required and often directly involved in critical protein modifications. SCOPE OF REVIEW: The present review is conceived to present an update on what is known about critical roles of redox enzymes, whereby special emphasis is put on the family of glutathione peroxidases, which for the time being presents the best characterized tasks during gametogenesis. MAJOR CONCLUSIONS: We therefore demonstrate that understanding the function of (seleno)thiol-based oxidases/reductases is not a trivial task and relevant knowledge will be mainly gained by using robust systems, as exemplified by several (conditional) knockout studies. We thus stress the importance of using such models for providing unequivocal evidence in the molecular understanding of redox regulatory mechanisms in sperm maturation. GENERAL SIGNIFICANCE: ROS are not merely detrimental by-products of metabolism and their proper generation and usage by specific enzymes is essential for vital functions as beautifully exemplified during male gametogenesis. As such, lessons learnt from thiol-based oxidases/reductases in male gametogenesis could be used as a general principle for other organs as it is most likely not only restricted to this developmental phase. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.

Antigenotoxic properties of chlorophyll b against cisplatin-induced DNA damage and its relationship with distribution of platinum and magnesium in vivo.

The chemotherapeutic agent cisplatin (cDDP) is widely used to treat a variety of solid and hematological tumors. However, cDDP exerts severe side effects, such as nephrotoxicity, neurotoxicity, and bone-marrow suppression. The use of some dietary compounds to protect organs that are not targets in association with chemotherapy has been encouraged. This study evaluated the protective effects of chlorophyll b (CLb) on DNA damage induced by cDDP by use of single-cell gel electrophoresis (SCGE) assays. Further, this investigation also determined platinum (Pt) and magnesium (Mg) bioaccumulation in mice tissues after treatment with CLb alone and/or in association of cDDP (simultaneous treatment) by inductively coupled plasma-mass spectroscopy (ICP-MS). All parameters were studied in peripheral blood cells (PBC), kidneys, and liver of mice after administration of CLb (0.2 or 0.5 mg/kg of body weight [b.w.]), cDDP (6 mg/kg b.w.), and the combination CLb 0.2 plus cDDP or CLb 0.5 plus cDDP. Pt accumulation in liver and kidneys was higher than that found in PBC, while DNA damage was higher in kidneys and liver than in PBC. Further, treatment with CLb alone did not induce DNA damage. Evidence indicates that genotoxic effects produced by cDDP may not be related to Pt accumulation and distribution. In combined treatments, CLb decreased DNA damage in tissues, but the PT contents did not change and these treatments also showed that CLb may be an important source of Mg. Thus, our results indicate that consumption of CLb-rich foods may diminish the adverse health effects induced by cDDP exposure.