The Ferroptosis Pathway

Death is part of the natural process of all living things, including individual cells. This process can occur in a variety of ways, and new pathways of cell death continue to be discovered. One of them, named ferroptosis, was first described in 2012 as a nonapoptotic, iron-dependent form of cell death.¹ Years earlier, in the search for compounds that are selectively lethal to RAS-mutated tumor cells, researchers already identified two structurally independent small molecules named erastin and RSL3 that were able to induce a unique form of cell death.² Further investigation revealed that this type of cell death does not share classic features of apoptosis such as caspase activation and chromatin fragmentation and is characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. In contrast, cells that undergo ferroptosis seem to exhibit distinct morphological characteristics such as shrunken and damaged mitochondria.³

While several proteins have been shown to regulate ferroptosis, glutathione peroxidase 4 (GPX4) is the central enzyme of this pathway. GPX4 effectively inhibits ferroptosis by reducing and thus limiting lipid peroxides and reactive oxygen species (ROS).⁴ This process requires the substrate glutathione (GSH), which is provided by the enzyme xCT via an intermediate step.

Figure: Simplified ferroptotic cascade. Free iron accumulation is an initiator of ferroptosis. GPX4 normally inhibits ferroptosis by limiting lipid peroxides generated by ROS. This process requires the substrate GSH, which is provided by the enzyme xCT. (Adapted from Dodson et al., 2019, created with BioRender.com)

Since its initial discovery, ferroptosis has attracted great interest in its process and function. According to PubGrade  , the number of publications has increased exponentially in past years, from 405 in 2019, 849 in 2020, to 1670 in 2021. Rockland's portfolio contains numerous antibodies against key components of the ferroptosis pathway that can help to further unravel the underlying mechanisms and thereby pave the way for new applications in cancer therapy, the treatment of neurodegenerative diseases, and in inflammation.

Antibodies Against Modulators of Ferroptosis

Numerous signaling pathways and their associated proteins have an impact on ferroptosis and regulate this process. (Table adapted from Tang et al., 2021)

Calcium Pathway

Dysregulated ORAI1-mediated Ca²⁺ influx contributes to ferroptosis induced by GSH depletion.⁷

Cell Adhesion

Cadherin-mediated intercellular interactions suppress ferroptosis by activating intracellular NF2.⁸

Cysteine Metabolism

The availability of free cysteine determines the extent of GSH synthesis and protection against ferroptosis.⁹

DNA Damage Pathway

ATM has been identified as a target for tumor cell ferroptosis, as it can be activated by radiotherapy and increases lipid oxidative damage.¹⁰

Epithelial–Mesenchymal Transition Pathway 

ZEB1 provides a bridge between mesenchymal gene expression and lipid peroxide susceptibility.¹¹

ER Stress

Ferroptosis is associated with increased ER stress. The chaperone GRP78 (through activation of ATF4) inhibits GPX4 degradation and promotes oxidative stress resistance.¹²

Glutamine Metabolism

GLS2-mediated glutamate production is required for erastin-induced ferroptosis.¹³

Iron Metabolism

Iron is required for the accumulation of lipid peroxides. In this context, the iron carrier protein transferrin plays a key role in the import of iron into the cell.²

KRAS Pathway

Mutations in the oncogene B-raf render cells more susceptible to erastin-induced ferroptosis.¹⁴

Lipid Metabolism

Glutathione peroxidase 4 (GPX4) is the central enzyme of the ferroptosis pathway. GPX4 effectively inhibits ferroptosis by reducing and thus limiting lipid peroxides and reactive oxygen species.⁴

Lysosome & Autophagy

Several autophagy-related genes modulate ferroptosis by autophagic degradation of cellular iron storage proteins.¹⁵

Mitochondrial Function

The ferroptotic small molecules, erastin and artesunate, induce pro-apoptotic PUMA expression.¹⁶

NRF2 Pathway

NRF2 is an important transcriptional regulator of anti-ferroptotic genes and is itself regulated by enzymes such as KEAP1.⁵

NOX Pathway

The NOX family of proteins promote lipid peroxidation in ferroptosis via ROS production.⁶

RNS Pathway

Scaffolding protein Cav-1 is involved in erastin-induced ferroptosis and links reactive nitrogen species (RNS) to ferroptosis.¹⁷

References

1. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B 3rd, Stockwell BR. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012 May 25;149(5):1060-72.
2. Yang WS, Stockwell BR. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol. 2008 Mar;15(3):234-45.
3. Stockwell BR, Friedmann Angeli JP, Bayir H, et al. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell. 2017;171(2):273-285.
4. Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, Cheah JH, Clemons PA, Shamji AF, Clish CB, Brown LM, Girotti AW, Cornish VW, Schreiber SL, Stockwell BR. Regulation of ferroptotic cancer cell death by GPX4. Cell. 2014 Jan 16;156(1-2):317-331.
5. Dodson M, Castro-Portuguez R, Zhang DD. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol. 2019 May;23:101107.
6. Tang D, Chen X, Kang R, Kroemer G. Ferroptosis: molecular mechanisms and health implications. Cell Res. 2021 Feb;31(2):107-125.
7. Henke N, Albrecht P, Bouchachia I, Ryazantseva M, Knoll K, Lewerenz J, Kaznacheyeva E, Maher P, Methner A. The plasma membrane channel ORAI1 mediates detrimental calcium influx caused by endogenous oxidative stress. Cell Death Dis. 2013 Jan 24;4(1):e470.
8. Wu J, Minikes AM, Gao M, Bian H, Li Y, Stockwell BR, Chen ZN, Jiang X. Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signalling. Nature. 2019 Aug;572(7769):402-406.
9. Fujii J, Homma T, Kobayashi S. Ferroptosis caused by cysteine insufficiency and oxidative insult. Free Radic Res. 2020 Dec;54(11-12):969-980.
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11. Viswanathan, V., Ryan, M., Dhruv, H. et al. Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway. Nature 547, 453–457 (2017).
12. Zhu S, Zhang Q, Sun X, Zeh HJ 3rd, Lotze MT, Kang R, Tang D. HSPA5 Regulates Ferroptotic Cell Death in Cancer Cells. Cancer Res. 2017 Apr 15;77(8):2064-2077.
13. Gao M, Monian P, Quadri N, Ramasamy R, Jiang X. Glutaminolysis and Transferrin Regulate Ferroptosis. Mol Cell. 2015 Jul 16;59(2):298-308.
14. Yagoda N, von Rechenberg M, Zaganjor E, Bauer AJ, Yang WS, Fridman DJ, Wolpaw AJ, Smukste I, Peltier JM, Boniface JJ, Smith R, Lessnick SL, Sahasrabudhe S, Stockwell BR. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature. 2007 Jun 14;447(7146):864-8.
15. Gao M, Monian P, Pan Q, Zhang W, Xiang J, Jiang X. Ferroptosis is an autophagic cell death process. Cell Res. 2016 Sep;26(9):1021-32.
16. Hong SH, Lee DH, Lee YS, Jo MJ, Jeong YA, Kwon WT, Choudry HA, Bartlett DL, Lee YJ. Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression. Oncotarget. 2017 Dec 8;8(70):115164-115178.
17. Deng G, Li Y, Ma S, Gao Z, Zeng T, Chen L, Ye H, Yang M, Shi H, Yao X, Zeng Z, Chen Y, Song Y, Liu B, Gao L. Caveolin-1 dictates ferroptosis in the execution of acute immune-mediated hepatic damage by attenuating nitrogen stress. Free Radic Biol Med. 2020 Feb 20;148:151-161.