Biochemical mechanism of ferroptosis-mediated cancer cell death in triple-negative breast cancer: An insight

Anitha Chidamabaram; Malarvili Thekkumalai; B Prabasheela; Tripta S Bhagat; Jyoti Batra; Sivanesan Dhandayuthapani

doi:10.4103/sujhs.sujhs_37_22

REVIEW ARTICLE

Year : 2022 | Volume : 8 | Issue : 2 | Page : 90-94

Biochemical mechanism of ferroptosis-mediated cancer cell death in triple-negative breast cancer: An insight

Anitha Chidamabaram¹, Malarvili Thekkumalai², B Prabasheela³, Tripta S Bhagat⁴, Jyoti Batra⁵, Sivanesan Dhandayuthapani⁶
¹ Department of Biochemistry, Government Arts and Science College, Orathanadu, Thanjavur, Tamil Nadu, India
² Department of Biochemistry, Government Arts and Science College, Srirangam, Tiruchirappalli, Tamil Nadu, India
³ Department of Pharmaceutical Engineering, Aarupadai Veedu Institute of Technology, Paiyanoor, Kancheepuram District, Tamil Nadu, India
⁴ Department of General Surgery, Santosh Deemed to be University, Ghaziabad, Uttar Pradesh, India
⁵ Department of Biochemistry, Santosh Deemed to be University, Ghaziabad, Uttar Pradesh, India
⁶ Department of Central Research Facility, Santosh Deemed to be University, Ghaziabad, Uttar Pradesh, India

Date of Submission	23-Nov-2022
Date of Acceptance	24-Nov-2022
Date of Web Publication	11-Jan-2023

Correspondence Address:
Sivanesan Dhandayuthapani
Central Research Facility Santosh, Deemed to be University, Ghaziabad, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/sujhs.sujhs_37_22

Abstract

Ferroptosis is a form of programmed cell death (PCD), distinct from apoptosis, that was identified in 2012. The process is driven by the iron-dependent oxidative degeneration of lipids. Ferroptosis causes cell death through the accumulation of iron-dependent lipid reactive oxygen species. Free radicals cause degradation of lipid molecules by the removal of electrons through oxidation. The process is dependent on intracellular iron as the accumulation of iron acts as a catalyst for converting peroxides into free radicals. The oxidative degradation of lipids occurs when there is depletion of the antioxidant glutathione and a loss of activity of the lipid repair enzyme glutathione peroxidase 4. The lipid peroxidation then leads to cell membrane denaturation. The biochemical mechanism behind the unique iron-dependent programmed cell death with reference to the triple negative breast cancer have been reviewed in this article.

Keywords: Ferroptosis, iron-dependent cell death, regulated cell death, triple-negative breast cancer

How to cite this article:
Chidamabaram A, Thekkumalai M, Prabasheela B, Bhagat TS, Batra J, Dhandayuthapani S. Biochemical mechanism of ferroptosis-mediated cancer cell death in triple-negative breast cancer: An insight. Santosh Univ J Health Sci 2022;8:90-4

How to cite this URL:
Chidamabaram A, Thekkumalai M, Prabasheela B, Bhagat TS, Batra J, Dhandayuthapani S. Biochemical mechanism of ferroptosis-mediated cancer cell death in triple-negative breast cancer: An insight. Santosh Univ J Health Sci [serial online] 2022 [cited 2023 May 30];8:90-4. Available from: http://www.sujhs.org/text.asp?2022/8/2/90/367572

Introduction

The International Agency for Research on Cancer of the World Health Organization in the biennial report (2020-2021) stated the reality of increasing incidence of cancer and its rising trend makes cancer as one of the main reasons that threaten human life. Although cancer is becoming one of the most common diseases globally, it has its own unique epidemiological characteristics and patient types as well.^[1] For example, the epidermal growth factor receptor mutation rate of lung adenocarcinoma patients in China is 61%, while that in the United States is only 11%, whereas it is reported as 23% out of 907 nonsmall cell lung cancer patients in India ethnicity.^[2] In the face of the high incidence of cancer and the rising trend, there is an urgent need to clarify its pathogenic mechanism in depth and to carry out more focused and targeted treatment.

Cell Death

Processes of cell death mechanism can be categorized based on several criteria such as the morphological measures, cellular context, and the main triggering stimulus. A pool of experts those who are pioneers in the field of studying the cell death mechanisms have classified the cell death into two major types namely: (i) accidental cell death (ACD); and (ii) regulated cell death (RCD) and have published their article entitled “Molecular Mechanisms of Cell Death: Recommendations of the Nomenclature Committee on Cell Death” in the year 2018.^[3] A process that leads to uncontrolled cell death is termed as ACD, which occurs under the trigger of any stimulus that is caused by any accidental injuries those exceed the cells' adjustability that results in cell death are called as ACD. On the other hand, autonomous and orderly cell death that is normally being controlled by genes and where without any disturbances to the stability of internal environment of the cells are termed as RCDs. The process of RCD that occurs under normal physiological conditions is also called as programmed cell death (PCD).^[3] In general, the induction and execution RCDs are based on the formation of signals and its amplification complexes by the responsible set of genes and been vital for the development of immune responses.^[4]

Reactive Oxygen SpecieS

Abundant production of reactive oxygen species (ROS) and its accumulation triggers PCD of senescence through cellular signaling in wide variety of cellular responses that includes the DNA damage and other signal transduction responses.^[4] It is well known that ROS-mediated DNA damage could normally activates the ATM/ATR-p53 pathway, thereby resulting in cellular senescence of cell death. Further, it is also most common that accumulation ROS would cause cell death by activating the apoptosis signal-regulating kinase 1-p38/c-Jun N-terminal kinase pathway. Hence, it is quite obvious that the cell survival/cell death is primarily regulated by the stress responses.

Even though several initiatives have been reported to play roles in producing ROS, iron is also identified as one of the major components that plays vital role in producing ROS, thereby contributing to ROS-mediated senescence or cell death pathways including apoptosis and necrosis. Many types of RCD, which are often characterized by plasma membrane rupture, cellular swelling, and inflammation induction, have been reported recently.^[5]

Importance of Oxygen and its Role in the Generation of Reactive Oxygen Species

Oxygen is one of the most important molecules for the survival of living organisms. However, it is not only important as a fuel provider for energy production but also generates the by-products such as ROS and thereby acts as a double-edged sword. Oxidative stress-mediated ROS generation and accumulation due to metabolic and signaling aberrations is one of the most common features of cancers.^[6],[7] Overload of ROS could damage the cellular components such as the DNA, proteins, and lipids quite easily.

Ferroptosis

Abandoned peroxidation of the lipid bilayers of the cellular membrane is termed as the hallmarks of a unique form of RCD call as “ferroptosis,” which was coined by Brent Stockwell and Scott J. Dixon, a decade ago in 2012.^[8],[9] Ferroptosis has its own uniqueness by utilizing the iron molecule for the induction of lipid peroxidation of the lipid bilayers, which is distinct from other forms of RDCs such as apoptosis and pyroptosis.^{[10],[11],[12]}

Ferroptosis Mechanism

Ferroptosis is a modality of RCD driven by iron-dependent lipid peroxidation [Figure 1]. Three key hallmarks of ferroptosis have been deciphered: the peroxidation of membrane lipids, the availability of intracellular iron, and the loss of antioxidant defense.^[8] The sensitivity of cancer cells to ferroptosis is determined by intracellular metabolic processes including lipid metabolism, iron metabolism, and amino acid metabolism.^[13],[14]

Figure 1: Cellular mechanism of ferroptosis

Click here to view

Lipid Peroxidation

Lipid peroxidation causes the destruction of the lipid bilayer and the damage of membranes, subsequently leading to cell death.^[15] The cellular membranes are rich in phospholipids (PLs) containing polyunsaturated fatty acids (PUFAs), which are highly vulnerable to ROS-induced peroxidation. The availability of membrane PUFAs competent to undergo per-oxidation is essential for the execution of ferroptosis.^[16] PUFAs need to be synthesized, activated, and incorporated into membrane PLs to participate in this lethal process, which requires two key enzymes, acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3. ACSL4 is able to catalyze the ligation of long-chain PUFAs with coenzyme A and LPCAT3 promotes the esterification and incorporation of these products into membrane PLs.^[17],[18]

Certain lipoxygenases (LOXs) are considered the major enzymes that can directly oxygenate PUFA-containing lipids in membrane bilayers.^[19] However, the mechanisms underlying LOX-mediated ferroptosis induction remain to be further dissected.^[20] Another enzyme, cytochrome P450 oxidoreductase (POR), has recently been implicated to be involved in initiating the peroxidation of lipid.^[21]

Iron Overload

As the name “ferroptosis” implies, iron is essential for the execution of ferroptotic cell death. Iron is indispensable for Fenton reaction, which generates free radicals and mediates lipid peroxidation.^[22] In addition, iron is required for the activation of iron-containing enzymes LOXs and POR, which are responsible for oxidizing membrane PUFAs. Moreover, iron is important for redox-based metabolic processes involved in the production of cellular ROS.

Due to the key role of iron in the execution of ferroptosis, cellular iron pool is intricately controlled via the regulation of genes involved in intracellular iron storage, release, import, and export.^[23],[24] Change in cellular labile iron affects the sensitivity of cells to ferroptosis. For example, the increase of iron importer transferrin^[25] or the degradation of iron storage protein ferritin^[26] has been reported to increase cellular iron availability and sensitizes cells to ferroptosis.

Vulnerability of Antioxidants

Under normal conditions, iron-mediated lipid oxidation is tightly controlled by cellular anti-antioxidant defense systems. Glutathione peroxidase 4 (GPX4) is thought to be the key antioxidant enzyme directly acting to eliminate the hydroperoxides in lipid bilayers and prevent the accumulation of lethal lipid ROS.^[27] GPX4 uses glutathione (GSH) as a substrate and reduces the membrane PL hydroperoxide to harmless lipid alcohols. The synthesis of GSH, which is essential for the activity of GPX4, requires three amino acids: cysteine, glycine, and glutamic acid. Cysteine, as an essential cellular building block of GSH, is the rate-limiting substrate of GSH synthesis. The abundance of cysteine within mammalian cells is mainly regulated by system xc, which consists of two subunits, solute carrier family 7 member 11 (SLC7A11) and SLC3A2.^[28] System xc plays a major role in importing cystine (the oxidized form of cysteine) into cells, and its expression and activity is exquisitely regulated by oncogenes and tumor suppressors in cancer cells through a variety of mechanisms.^{[29],[30],[31]} Small molecule inhibitors such as erastin,^[32] which suppresses SLC7A11-mediated cystine import, can induce ferroptosis in multiple cancers.

An alternative GXP4-independent ferroptosis-suppressing mechanism has been recently uncovered.^[33],[34] Two independent genetic screens revealed that the ferroptosis suppressor protein 1 (FSP1)-CoQ system is capable of protecting cells against ferroptosis induced by GPX4 inhibition.^[33] FSP1 could prevent lipid peroxidation via reduction of lipid radicals. Therefore, cells utilize two pathways, cyst(e)ine-GSH-GPX4 and FSP1-CoQ axis, to suppress lipid peroxidation and prevent ferroptosis. Ferroptosis occurs when these antioxidant defense systems are overwhelmed by iron-dependent lipid ROS accumulation.

Ferroptosis in Triple-Negative Breast Cancer

Ferroptosis sensitivity varies widely among different cancers. Since ferroptosis is linked to tumor suppression, a key question in cancer therapy is which tumor types would be more likely to benefit from the ferroptosis inducers (FINs). Recent evidence suggests that the expression of genes involved in ferroptosis-associated metabolic pathways,^[14] such as lipid, iron, and amino acid metabolism, is altered in TNBC, rendering this difficult-to-treat tumor intrinsically susceptible to ferroptosis. The particular sensitivity of TNBC to ferroptosis highlights this nonapoptotic death pathway as an attractive TNBC druggable target. Herein, we use TNBC as a model to summarize the regulation of ferroptosis in cancer, and similar mechanisms may also exist in other cancer types.

Role of Metabolism of Lipid

Deregulated lipid metabolism can lead to lipid peroxidation and induce ferroptosis. In an effort to identify the key factors that determine ferroptosis sensitivity, two independent screening approaches were applied and uncovered ACSL4 as an essential component for ferroptosis execution.^[35] Interestingly, it was found that ACSL4 was preferentially expressed in TNBC compared with other types of breast cancer, and its expression predicted their sensitivity to ferroptosis. Significant high expression of ACSL4 in TNBC tumors and cell lines was also observed in a recent study.^[36] Given that ACSL4 is responsible for enriching cellular membranes with long PUFAs, these findings suggest that TNBC is prone to be PUFA rich and thus particularly sensitive to ferroptosis.

Role of Iron Metabolism

Sufficient intracellular iron is essential for the execution of ferroptosis. Compared with normal cells, cancer cells exhibit higher reliance on iron to enable growth.^[37] By analysis of clinical datasets and breast cancer specimens, a recent study revealed that the genes that regulate intracellular iron levels were distinctly expressed in TNBC versus non-TNBC tumors and cell lines.^[36] In particular, a substantial low level of the iron exporter ferroportin was observed in TNBC, concomitant with the high expression level of the iron importer transferrin receptor.^[36],[38] These alterations in the expression of genes involved in iron metabolism regulation may contribute to an increase in cellular labile iron pool and facilitate iron-dependent lipid peroxidation, rendering TNBC to be iron-rich tumor and susceptible to ferroptosis.

Role of Amino Acid Metabolism

Amino acid metabolism is essential for the antioxidation defense system composed by SLC-7A11-mediated cystine uptake, GSH biosynthesis, and GPX4 activity. Cancer cells may exhibit altered dependency on specific amino acid metabolic pathways, and targeting these dependencies will be a promising therapeutic strategy. An earlier study applying functional metabolic analyses found that compared with other types of breast cancer, TNBC exhibited a marked reliance on glutamine metabolism necessary to fuel SLC7A11,^[39] hinting a potential link of TNBC to ferroptosis and unveiling SLC7A11 as a promising therapeutic target for TNBC. As a nonenzymatic antioxidant molecule, GSH plays an important role in maintaining redox homeostasis. The expression of GSH synthetase (GSS), one of the critical enzymes for GSH synthesis, was found to be decreased in TNBC versus non-TNBC tumors.^[36] Indeed, an earlier study used mass spectrometry-based metabolomics analysis and revealed that levels of the cellular GSH were lower in TNBC cell lines compared to controls.^[40] Moreover, the expression of GPX4 was also lower in TNBC compared with other types of breast cancer.^[36] Low intracellular GSH and reduced GPX4 expression may weaken the antioxidation defense capacity and increase the probability of lipid peroxidation, rendering TNBC particularly responsive to agents that promote ferroptosis.

Conclusion

Ferroptosis is driven by the oxidation of PUFA containing lipids, the accumulation of intracellular iron, and the loss of antioxidant defense. The regulation of ferroptosis has been characterized in a variety of cancer types including TNBC. TNBC exhibits a unique pattern of expression of the ferroptosis-related genes, rendering it prone to have sufficient PUFAs and iron, and decreased antioxidation capacity, thus particularly vulnerable to ferroptosis inducers. Therefore, targeting ferroptosis is likely to be a promising therapeutic strategy for this difficult-to-treat tumor.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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