Ferroptosis Overview - Signaling Pathway. Diagnostics Marker and Targeted Therapy

1 Major Signaling Pathways in Ferroptosis

The regulation of ferroptosis is orchestrated by a complex network of interconnected metabolic pathways, including iron metabolism, lipid metabolism, and antioxidant defense systems. Understanding these pathways is crucial for designing effective therapeutic strategies.

1.1 Iron Metabolism

Iron is the central catalyst for ferroptosis. The intracellular labile iron pool (LIP), primarily consisting of ferrous iron (Fe2+), fuels the Fenton reaction, which generates highly destructive reactive oxygen species (ROS). Cellular iron homeostasis is tightly controlled by proteins responsible for its uptake, storage, and export. For instance, the transferrin receptor 1 (TFR1) mediates iron uptake, while the iron storage protein ferritin sequesters excess iron to prevent toxicity. Ferroptosis can be triggered by iron overload or by the release of iron from ferritin through a specific autophagic process called ferritinophagy, which is mediated by the cargo receptor NCOA4.

1.2 Antioxidant Defense Systems

Cells possess robust antioxidant systems to counteract oxidative stress and protect against ferroptosis. The most well-known defense is the System Xc⁻/GSH/GPX4 axis. System Xc⁻, a cystine-glutamate antiporter, imports cystine, which is then converted into cysteine, the rate-limiting precursor for glutathione (GSH). GSH serves as a critical cofactor for glutathione peroxidase 4 (GPX4), an enzyme that functions as the primary "ferroptosis gatekeeper" by reducing toxic lipid hydroperoxides to non-toxic lipid alcohols. A second, independent pathway involves ferroptosis suppressor protein 1 (FSP1), which reduces coenzyme Q10 (also called CoQ10) to ubiquinol, a potent antioxidant that directly scavenges lipid peroxyl radicals. The p62-KEAP1-NRF2 pathway also plays a critical role by upregulating the transcription of genes encoding antioxidant and iron-metabolism proteins.

1.3 Lipid Peroxidation

The accumulation of lipid peroxides is the direct cause of ferroptotic cell death. Polyunsaturated fatty acids (PUFAs) integrated into cell membranes are particularly susceptible to this process. Enzymes such as acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) are instrumental in generating phospholipid-PUFAs (PL-PUFAs), which are the essential substrates for lipid peroxidation. This process is further catalyzed by iron-dependent lipoxygenases (LOXs). Blocking these enzymes or providing alternative, peroxidation-resistant fatty acids can effectively inhibit ferroptosis.

2 Diagnosis of Ferroptosis

The accurate diagnosis of ferroptosis is essential for both research and clinical applications. Specific biomarkers and detection methods allow for the precise identification and quantification of ferroptosis in different contexts.

2.1 Ferroptosis Detection Methods

• Measurement of Lipid Peroxidation: Quantifying lipid ROS using fluorescent probes.
• Total ROS Measurement: Using probes to assess the overall level of reactive oxygen species within the cell.
• Ferrous Iron Quantification: Employing specific probes to directly detect the key catalytic ion (Fe²⁺).
• Protein and Gene Expression Analysis: Using techniques such as Western Blot, immunohistochemistry (IHC), and real-time PCR to measure the levels of key regulatory proteins like GPX4 and SLC7A11.
• Morphological Analysis: Observing distinct mitochondrial changes via transmission electron microscopy (TEM), including shrunken mitochondria with increased membrane density and a reduction in cristae.

2.2 Ferroptosis Biomarkers

• GPX4: A central negative regulator of ferroptosis. Low expression or inactivation is a definitive indicator of ferroptosis induction.
• SLC7A11 (xCT): A subunit of System Xc⁻. Its reduced expression or inhibition leads to GSH depletion and is a classic biomarker for a specific type of ferroptosis.
• ACSL4: An enzyme that promotes the synthesis of substrates for lipid peroxidation. Its high expression is associated with sensitivity to ferroptosis.
• Malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE): These are stable end-products of lipid peroxidation and serve as direct, quantifiable biomarkers of ferroptosis in tissues and plasma.
• NCOA4: The cargo receptor for ferritinophagy. Increased NCOA4 activity or expression indicates the breakdown of iron stores, promoting ferroptosis.
• TFRC (Transferrin Receptor): An increase in TFRC content is a biomarker for the activation of ferroptosis.
• PTGS2 (Prostaglandin-Endoperoxide Synthase 2) / COX2: Elevated expression of this enzyme is a widely recognized biomarker for ferroptosis, often associated with a pro-inflammatory state.
• CHAC1: As a downstream effector of the GCN2-eIF2α-ATF4 pathway, increased expression of CHAC1 is a biomarker associated with ferroptosis induction.
• NRF2 (The nuclear factor erythroid 2-related factor 2): NRF2 acts as a master transcriptional regulator that can both promote and restrict ferroptosis depending on cellular context.

3 Targeted Therapy for Ferroptosis

3.1 Targeting Iron Homeostasis

Therapies focused on iron metabolism aim to modulate the availability of ferrous iron. For diseases driven by ferroptosis, such as neurodegeneration, iron chelators are used to sequester free iron, preventing the Fenton reaction. The most common iron chelators include deferoxamine (DFO), deferiprone (DFP), and deferasirox (DFX). For cancer therapy, strategies are designed to increase intracellular iron levels. This can be achieved using nanoparticles engineered to deliver iron directly to tumor cells. Another approach is to target iron receptors and transporters. For example, a TFR1-specific antibody can inhibit cellular iron uptake, thereby suppressing ferroptosis. This strategy is particularly relevant for cancers that rely on high iron uptake for rapid proliferation.

3.2 Targeting Antioxidant Defenses

Targeting antioxidant defenses is a primary focus for therapeutic development, particularly for inducing ferroptosis in cancer. The main strategy involves inhibiting System Xc⁻, which disrupts cystine uptake and depletes glutathione (GSH), using compounds such as erastin and its derivatives, as well as clinically approved drugs like sulfasalazine and sorafenib. Another approach is to directly inhibit GPX4 using compounds like RSL3, ML162, and ML210. Newer strategies include the use of protein degraders to specifically degrade the GPX4 protein and FSP1 pathway inhibitors to inhibit the GPX4-independent defense against ferroptosis. Other agents, such as the gold-containing compound auranofin and Buthionine sulfoximine (BSO), are also used to induce ferroptosis by inhibiting thioredoxin reductase or GSH synthesis. Conversely, to protect healthy tissues, antioxidants like N-acetylcysteine (NAC) and its derivative NACA are used to restore GSH levels. The NRF2 pathway is a key regulator of ferroptosis, and its modulators, like trigonelline and sitagliptin, provide different therapeutic avenues by either inhibiting or activating NRF2.

3.3 Targeting Lipid Metabolism

Modulating lipid metabolism is a powerful way to control ferroptosis by altering the supply of substrates for lipid peroxidation. Inhibitors of enzymes such as ACSL4 (e.g., thiazolidinediones like rosiglitazone and pioglitazone) and lipoxygenases (LOXs) like ALOX5 and ALOX12 (e.g., zileuton and NDGA) can prevent the synthesis and peroxidation of pro-ferroptotic lipids, thereby suppressing ferroptosis. A class of lipid chain-breaking antioxidants called radical-trapping antioxidants (RTAs), including ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1), directly scavenge lipid hydroperoxyl radicals, offering potent cytoprotection, while other RTAs like edaravone have been shown to protect against ferroptosis under various pathological conditions. Finally, inhibiting the mevalonate pathway with statins can make cancer cells more susceptible to ferroptosis by altering cellular lipid composition.

Cancer Immunotherapy Targeting Ferroptosis