Directly targeting glutathione peroxidase 4 may be more effective than disrupting glutathione on ferroptosis-based cancer therapy

Journal Pre-proof Directly targeting glutathione peroxidase 4 may be more effective than disrupting glutathione on ferroptosis-based cancer therapy

Yunpeng Wei, Huanhuan Lv, Atik Badshah Shaikh, Wei Han, Hongjie Hou, Zhihao Zhang, Shenghang Wang, Peng Shang PII:

S0304-4165(20)30029-5

DOI:

https://doi.org/10.1016/j.bbagen.2020.129539

Reference:

BBAGEN 129539

To appear in:

BBA - General Subjects

Received date:

18 September 2019

Revised date:

11 January 2020

Accepted date:

16 January 2020

Please cite this article as: Y. Wei, H. Lv, A.B. Shaikh, et al., Directly targeting glutathione peroxidase 4 may be more effective than disrupting glutathione on ferroptosis-based cancer therapy, BBA General Subjects(2020), https://doi.org/10.1016/ j.bbagen.2020.129539

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© 2020 Published by Elsevier.

Journal Pre-proof

Directly targeting glutathione peroxidase 4 may be more effective than disrupting glutathione on ferroptosis-based cancer therapy Yunpeng Wei1 , Huanhuan Lv1,2,3 , Atik Bads hah Shaikh1 , Wei Han1 , Hongjie Hou2,3 , Zhihao Zhang2,3 , Shenghang Wang2,3 , Peng Shang1,3* 1

Research & Development Institute of Northwestern Polytechnical University in

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2

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Shenzhen, Shenzhen, 518057, China.

School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072,

Key Laboratory for Space Biosciences and

Biotechnology,

Northwestern

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3

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Shaanxi, China.

Polytechnical University, Xi’an 710072, Shaanxi, China.

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*Corresponding author. [email protected]

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Abstract

Background: Cancer is one of the major threats to human health and current cancer

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therapies have been unsuccessful in eradicating it. Ferroptosis is characterized by

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iron-dependence and lipid hydroperoxides accumulation, and its primary mechanism involves the suppression of system Xc--GSH (glutathione)-GPX4 (glutathione peroxidase 4) axis. Co-incidentally, cancer cells are also metabolically characterized by iron addiction and ROS tolerance, which makes them vulnerable to ferroptosis. This may provide a new tactic for cancer therapy. Scope of review: The general features and mechanisms of ferroptosis, and the basis that makes cancer cells vulnerable to ferroptosis are described. Further, we emphatically discussed that disrupting GSH may not be ideal for triggering ferroptosis of cancer cells in vivo, but directly inhibiting GPX4 and its compensatory members could be more effective. Finally, the various approaches to directly inhibit GPX4 without disturbing GSH were described.

Journal Pre-proof Major conclusions: Targeting system Xc- or GSH may not effectively trigger cancer cells’ ferroptosis in vivo the existence of other compensatory pathways. However, directly targeting GPX4 and its compensatory members without disrupting GSH may be more effective to induce ferroptosis in cancer cells in vivo, as GPX4 is essential in preventing ferroptosis. General significance: Cancer is a severe threat to human health. Ferroptosis-based cancer therapy strategies are promising, but how to effectively induce ferroptosis in

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cancer cells in vivo is still a question without clear answers. Thus, the viewpoints

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raised in this review may provide some references and different perspectives for researchers working on ferroptosis-based cancer therapy.

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Keywords: ferroptosis, GPX4, system Xc-, GSH, cancer therapy

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1. Introduction

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With the development of modern human society and global economy, cancer has become a perilous threat to human health and one of the major causes of death. It is

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estimated that there were about 18.1 million new cancer cases and 9.6 million

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cancer-related deaths in 2018 [1]. In the foreseeable future, cancer may soon become the leading cause of death, replacing cardiovascular disease. Current known

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advancement in anti-cancer drugs, therapeutic strategies, surgical techniques and imaging technology has significantly improved the survival rate of several cancers, immunotherapy could even completely cure some cancer under special conditions [2]. However, there is still no effective therapy to cure most of cancer. Hence, it is urgent to develop newer and effective cancer therapy. Apoptosis is a well-known cell death program. However, in recent years, several types of non-apoptotic cell death pathways have been discovered, including autophagic cell death, necroptosis, ferroptosis and pyroptosis, which extend our understanding about the complexity of cell death. Intensive study on the underlying mechanism of these types of cell deaths may give a glimmer of hope for cancer therapy [3, 4]. Among these non-apoptotic cell death pathways, ferroptosis has

Journal Pre-proof attracted special attention of scientists all over the world. Interestingly, studies have demonstrated that ferroptosis may have the potential to cure quite a few types of cancers, because the unique metabolic characteristics of cancer cells make them vulnerable to ferroptosis [5]. Especially, directly targeting glutathione peroxidase 4 (GPX4), the key regulator of ferroptosis, may be the most efficient way to induce ferroptosis in cancer cells in vivo.

2. Mechanism of ferroptosis: system Xc--GSH-GPX4 axis

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Although the concept of ferroptosis was first proposed by Stockwell in 2012 [6], the pattern of this type of cell death has been noticed for a long time. In 1955,

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cysteine withdrawal mediated cell death was initially characterized by Harry Eagle,

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who characterized the basic principles of what is known today to be the minimal requirement for cell growth in culture [7]. In 2001, it was discovered that excess

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extracellular glutamate could induce nerve cell death in a non-apoptotic manner featured by remarkable oxidative stress, which was named as oxytosis [8]. Then, the

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inhibitory effects of erastin against human foreskin fibroblasts expressing mutant Ras

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oncogene rather than their isogenic primary counterparts were discovered in 2003 [9]. In 2008, RSL3 and RSL5, small- molecule Ras-selective lethal compounds, were also

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found to be cytotoxic to cells expressing mutant Ras oncogene [10]. After Stockwell put forward the term “ferroptosis” and described its characteristic features, it became gradually clear that the similar mechanism actually causes all these cell deaths. As an iron-dependent pattern of regulated cell death, ferroptosis is triggered by the blocking of intracellular lipid hydroperoxides scavenge systems, and executed by overproduced lipid hydroperoxide products [11]. The cellular morphological features of ferroptosis are different from other non-apoptotic cell deaths. In the ferroptosis cells, the mitochondria shrink, crista decreases, and the mitochondrial membrane becomes condensed and fractured [12]. Under general conditions, ferroptosis begins with Fenton reaction catalyzed by iron ion, in which a ferrous iron and a hydrogen peroxide generate a ferric iron, a

Journal Pre-proof hydroxyl and a hydroxyl radical. Then, superoxides reduce ferric iron into ferrous iron, and the cycle continuously produces massive hydroxyl radicals [13]. Hydroxyl radicals are highly unstable and reactive, which could almost oxidize any intracellular macromolecular substances, including lipid, nucleic acid and proteins [14]. In ferroptosis, the oxidation of plasma membrane lipid is critical, because it is the accumulation of membrane lipid hydroperoxide products that leads to cell death [6]. In addition, the generated lipid hydroperoxide products could also produce additional

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lipid peroxide radicals in the presence of iron ion, which further amplifies the toxicity

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of these lipid hydroperoxide products and accelerates cell death [15].

Figure 1 Cascade reactions of lipi d hydroperoxi de products generated in the presence of iron ions.

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Fenton reacti on generates highly reacti ve hydroxyl radical (HO . ), which abstracts hydrogen from

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lipi ds (RH) and generates lipi d radicals (R. ). Li pi d radicals coul d then join oxygen molecules (O2 ) and generate highly reacti ve li pi d hydroperoxi de radicals (ROO .), which c oul d abstract hydrogen

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from other li pi d molecules and generate li pi d hydroperoxi des (ROOH). Li pi d hydroperoxi des then generate other highly reacti ve li pi d hydroperoxi de radicals (ROO. and RO.) under the

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catalysis of iron ions, and the accumulation of these products leads to ferroptosis.

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However, there are intracellular defense mechanisms against ferroptosis, the primary one is described as system Xc--GSH-GPX4 axis [16]. System Xc- ingests cystine into cells by pumping out glutamate, cystine is then reduced to cysteine and utilized in GSH biosynthesis. GPX4, with selenocysteine at its activity site, can reduce lipid hydroperoxide products in the plasma membrane into non-toxic corresponding lipid alcohol using GSH as a cofactor, which eventually prevents ferroptosis. Also, the initialization of ferroptosis needs phospholipids with polyunsaturated fatty acid (PUFA) in the cellular membrane as oxidation substrates, which are mainly biosynthesized by acyl-CoA synthetase long-chain family member 4 (ACSL4) [17]. Thus, disturbing system Xc- (e.g. glutamate, erastin, sulfasalazine and sorafenib), depleting GSH (e.g. buthionine sulfoximine) or inhibiting GPX4 (e.g.

Journal Pre-proof RSL3, ML162 and FIN56) could lead to ferroptosis [18].

Figure 2 System Xc--GS H-GPX4 axis. System Xc- imports cystine (Cys-Cys) into cells by exporting glutamate (Glu) wi th a 1 :1 ratio. Then, cystine is transformed into cysteine (Cys) and partici pates in the bi osynthesis of glutathi one (GS H). GPX4 reduces lipi d hydroperoxi des (ROOH) to harmless lipid alcohol (ROH) using GS H as a cofactor. Disruption of any link of system Xc--GSH-GPX4 axis could lead to ferroptosis.

3.1 Cancer cells are tolerant to iron and ROS

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3. Cancer cells are potentially vulnerable to ferroptosis

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As the second- most abundant metal element in the earth’s crust, iron is an

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essential element in a lot of proteins involved in biological processes, including respiratory chain reaction, oxygen transport, energy metabolism and DNA synthesis &

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repair [13]. However, several studies have demonstrated that the body iron level is positively associated with cancer incidence [19-22]. Iron is vital for cancer cells’

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proliferation, therefore iron metabolism of cancer cells is usually more active than

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normal cells, their iron uptake and intracellular labile iron level could be significantly elevated, while the iron output and storage could be reduced [13]. Likewise, the ROS

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level in cancer cells is also higher than their normal counterparts, as high ROS levels could be involved in signaling for stimulating proliferation [23]. However, high levels of iron and ROS don’t naturally trigger ferroptosis or uncontrolled oxidative stress in cancer cells because they have evolved several defense mechanisms, including excessively activated antioxidative pathways [24]. Interestingly, iron addiction and ROS tolerance may be the archilles heel of cancer cells, and could be used as an advantage to induce ferroptosis in cancer cells by appropriately disturbing their ferroptosis defense systems. 3.2 EMT cancer cells are highly dependent on GPX4 In comparison to normal cells, cancer cells are more dependent on GPX4, especially the epithelial-to- mesenchymal transition (EMT) cancer cells. When cancer

Journal Pre-proof cells derived from epithelial tissues grow to a certain stage, some of these cells could gradually transform into another cell type which have more mesenchymal features, and their original epithelial features decrease or vanish in the meantime [25]. EMT cancer cells are highly invasive and have high metastasis incidences, and they may also be resistant to several types of chemotherapies [26]. In addition, the cancers originated from mesenchymal cells, also known as sarcoma, retain the mesenchymal state from the beginning, they could be more invasive and drug-resistant compared to

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sensitive to GPX4 inhibition induced ferroptosis [27].

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the epithelial originated cancers. Not surprisingly, these cancer cells are usually

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EMT cancer cells, with invasion and metastasis capabilities, require increased membrane fluidity, which needs a higher level of PUFA in the cellular membrane.

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Interestingly, PUFA could be oxidized easier than other kinds of lipids. Thus, that may

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partly explain why EMT cancer cells are more sensitive to GPX4 inhibition and subsequent ferroptosis than non-EMT cancer cells [28].

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4. Disruption of GSH may not effectively inhibit cancer cells in vivo

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Theoretically, disturbing any link of system Xc--GSH-GPX4 axis could trigger ferroptosis in cancer cells. However, it gets more complicated under the practical

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circumstances.

4.1 Blocking system Xc- may not effectively trigger ferroptosis of cancer cells in vivo

A study reported that deletion of SLC7A11 gene (encoding a key component of system Xc-) does not affect the normal development and growth of the mice, but any kind of cells isolated from these mice could not survive in in vitro culture environment. As about 50% of cysteine exists in plasma and extracellular compartment in its reduced form, the cysteine depletion due to the disabled system Xc- could be easily compensated in vivo by other pathway, such as the neutral amino acid transporter ASC, which could also transport extracellular cysteine into to cells [29]. However, all cysteine exists in its oxidized form as cystine in vitro, and its

Journal Pre-proof uptake into cells depends primarily on system Xc-, thus the deletion of SLC7A11 gene blocks the major supply pathway of cysteine and leads to cell death [30]. Besides, some cells could biosynthesize cysteine from methionine via the transsulfuration pathway and bypass the system Xc-. Not surprisingly, these cells are resistant to the inhibition of system Xc- [31]. Thus, these studies demonstrate that blocking system Xc- may be effective to induce ferroptosis in some cancer cells in vitro. However, the effects of this strategy may be mild or insignificant in vivo.

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In addition, there are several reports which claimed that s ystem Xc- inhibitor

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Erastin can inhibit tumor growth in vivo [32, 33]. We consider these reports as several

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special cases due to the metabolic specificities of these cancer cells. Our hypothesis is that the cancer cells in these reports may be lack of fully functional neutral amino acid

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channels and other compensatory pathways like transsulfuration pathway, thus these

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cells are highly dependent on the system Xc- for the uptake of cysteine and Erastin can actually inhibit their growth in vivo. Nevertheless, in other types of cancer cells, the

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fully functional neutral amino acid channels or other compensatory pathways may still be available, which could greatly impair the therapeutic effects based on the

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strategies to target GSH in vivo, but GPX4 targeting therapy may still be effective.

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4.2 Depleting GSH cannot disable GPX4 completely It is well-known that GPX4 requires GSH as a cofactor to reduce lipid hydroperoxide products. However, the cofactors of GPX4 are not limited to GSH. Previous studies have demonstrated that GPX4 could also efficiently utilize low molecular thiols (e.g. cysteine) and protein thiols (e.g. chromatin proteins, sperm mitochondria-associated cysteine-rich proteins and even GPX4 itself) as cofactors to reduce lipid hydroperoxides [34-36], which demanstrates that the depletion of GSH may be less effective in inducing ferroptosis, especially under in vivo conditions.

5. GPX4 is the key enzyme that could effectively prevent ferroptosis Several GPXs family members have been discovered in mammals, including GPX1 to GPX8. Some of the GPXs members are selenoproteins with selenocysteine

Journal Pre-proof at their active sites, including GPX1, GPX2, GPX3, GPX4 and GPX6 (humans). While other GPXs members, including GPX5, GPX6 (mice and rats), GPX7 and GPX8, have cysteine at their active sites instead. However, only GPX4 can effectively scavenge membrane lipid hydroperoxide products, which makes it crucial to induce or prevent ferroptosis [37]. GPX4 is specifically suited to prevent ferroptosis among GPXs family because of its unique ability to reduce large lipid hydroperoxides, which is dependent on its specific amino acid sequence and spatial structure. The active site

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of GPX4 is near the surface of the protein, which is a conserved catalytic triad made

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of selenocysteine (U46), glutamine (Q81) and tryptophan (W136). Mutation any of

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them could greatly impair the normal function of GPX4, especially selenocysteine (U46). The selenocysteine residue is vital for the function of GPX4, because its

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activity could be almost disabled if the selenocysteine residue is replaced by a

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cysteine. It seems that the selenocysteine residue is more suitable than cysteine to perform catalytic function in a deprotonated state at neutral pH [38]. Besides, it is

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reported that genetic deletion of Gpx4 is lethal in mice, while conditional deletion of Gpx4 in brain and fibroblasts leads to neurodegeneration and non-apoptotic neuron

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death in hippocampus featured by massive lipid peroxidation, which further highlights

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the importance of GPX4 in normal development and metabolic process [39-41]. A study found that a widely expressed enzyme named peroxiredoxin 6 (PRDX6) could also reduce phospholipids peroxides in plasma membrane and prevent membrane lipid peroxidation [42]. In comparison to the fatal consequence seen without Gpx4, Prdx6 knockout mice are viable, but their reproductive capacities are degenerated compared to their wild type [43]. Thus, this demonstrates that PRDX6 may not be as crucial as GPX4 and plays a complementary role. In addition, as members of phase II detoxification enzymes, some glutathione S-transferases have non-selenium-dependent peroxidase activity, such as rat hepatic glutathione S-transferase B and rat hepatic microsomal glutathione transferase. The latter could inhibit lipid peroxidation in a glutathione-dependent manner [44, 45]. Thus, these

Journal Pre-proof special glutathione S-transferases may also act as subordinate complementary role to prevent ferroptosis. As one of flavoproteins, apoptosis- inducing factor mitochondria-associated 2 (AIFM2) is very recently reported to act parallel to GPX4 to inhibit ferroptosis, thus it is named as ferroptosis suppressor protein 1 (FSP1). As a member of CoQ 10 oxidoreductase family, FSP1 could effectively inhibit ferroptosis by reducing CoQ 10 using NAD(P)H, and the reduced CoQ 10 could continually scavenge lipid

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hydroperoxides in plasma membranes. Cellular GSH levels and GPX4 activity are not

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related to the anti- ferroptotic function of FSP1. Thus, the expression of FSP1 could

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dramatically increase the resistance of cancer cells against ferroptosis induced by GPX4 inhibition or GSH depletion. In the cancer cells resistant to GPX4 inhibitors,

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blocking FSP1 or depleting CoQ 10 could significantly increase their sensitivities to

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ferroptosis. However, like SLC7A11, FSP1 knockout mice are viable and have no observed anomalies, which demonstrates that FSP1 plays a very powerful

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complementary role to prevent ferroptosis alongside GPX4. The discovery of FSP1 reveals another completely different mechanism of ferroptosis, which partially

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explains the existence of cancer cells that are both resistant to system Xc- and GPX4

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inhibitors, and may also provide potential strategies for the treatment of some resistant cancers [46, 47].

Table 1 Distribution and functions of GPXs family members

GPX type

Distribution

Selenium

Functions

Reference

Reduces hydrogen peroxide

[35]

containing GPX1

Widespread

Yes

and fatty acid hydroperoxides in the cytoplasm, cannot reduce phospholipid hydroperoxides in the cellular membrane.

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GPX2

Gastrointestinal

Yes

Reduces hydrogen peroxide.

[35]

tract GPX3

Plasma

Yes

Reduces hydrogen peroxide.

[35]

GPX4

Widespread

Yes

Effectively reduces

[37, 38]

hydroperoxides of phospholipids, fatty acids, cholesterol and thymine.

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Cannot reduce hydrogen

Epididymis

No

Protects the membranes of

[48]

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GPX5

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peroxide with effect.

spermatozoa from the

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damaging effects of lipid

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peroxidation and prevents premature acrosome reaction.

Embryos and

epithelium

and rats)

(mice

Embryos and

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GPX6

Reduces hydrogen peroxide.

[35]

No

Reduces hydrogen peroxide.

[35]

No

Has mild glutathione

[49]

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adult olfactory

Yes

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GPX6 (humans)

adult olfactory epithelium

GPX7

Endoplasmic reticulum

peroxidase activity, primary function is to sense ROS level and transmit redox signals to other thiol proteins.

GPX8

Endoplasmic reticulum

No

Has mild glutathione peroxidase activity, main function is to prevent

[50]

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endoplasmic reticulum oxidation and stress.

6. The strategies to directly target GPX4 without disturbing GSH Although the disruption of GSH could indirectly disable GPX4, this strategy still seems less effective in vivo because of the existence of other compensatory pathways. In contrast, the strategy to directly target GPX4 may be more efficient to induce ferroptosis in cancer cells in vivo, because GPX4 plays an essential role in preventing

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ferroptosis.

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Figure 3 The strategies to target GPX4 without disturbi ng GS H. (A) RSL3, ML162 and DPIs

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inacti vate GPX4 by directly bi nding to it. (B) FIN56 cripples GPX4 by inducing its degradation. It coul d also deplete CoQ10 by acti vating s qualene synthase (SQS), which may weaken ferroptosis

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resistance medi ated by FSP1. (C) FINO2 inacti vates GPX4 with unknown mechanisms. (D) Statins coul d deplete meval onic aci d by inhi biting HMGCR and lead to obstructi on of IPP

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synthesis, which further inhi bi ts the formation of sec-tRNA and leads to synthesis obstruction of

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selenoproteins, incl uding GPX4. Stains c oul d also inhi bi t the bi osynthesis of CoQ10 , which may

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further block the FSP1 medi ated ferroptosis resistance. (E) GPX4 siRNA coul d s pecifically degrade GPX4 mRNA and block its translation.

The first discovered direct GPX4 inhibitor is RSL3, which contains an electrophilic chloroacetamide and can irreversibly bind to its active site leading to GPX4 inactivation [18]. Other GPX4 inhibitors have also been found later, including ML162, DPI compounds (DPIs, including DPI7, 10, 12, 13, 17, 18 and 19), FIN56 and FINO 2 . Like RSL3, ML162 and DPI compounds also deactivate GPX4 via covalent binding. FIN56 induces the degradation of GPX4 and depletes its abundance. It could also deplete CoQ 10 by activating squalene synthase (SQS), which may suppress ferroptosis resistance mediated by FSP1 in the meantime [46, 47, 51]. FINO 2 indirectly inactivates GPX4 and specifically oxidizes ferrous ions, which triggers ferroptosis in HT-1080 fibrosarcoma cells, however its underlying mechanisms are

Journal Pre-proof still unclear [52]. In addition, it has been reported that statins could also induce ferroptosis. As small- molecule inhibitors of 3-hydroxy-3- methylglutaryl coenzyme A reductase (HMGCR), stains are usually used to treat hyperlipidemia. Like FIN56, it seems that statins could also induce ferroptosis via two pathways. First, statins could block the generation of isopentenyl pyrophosphate (IPP) by inhibiting HMGCR, which further blocks the formation of selenocysteine transporter RNA (sec-tRNA) and inhibits the biosynthesis of selenoproteins, including GPX4 [27]. Second, statins

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could inhibit the biosynthesis of CoQ 10 by blocking mevalonate pathway, which may

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also weaken FSP1 mediated resistance against ferroptosis [46, 47]. Moreover, unlike

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direct GPX4 inhibitors such as RSL3 and ML162, ferroptosis induced by statins cannot be rescued by lipophilic antioxidants such as vitamin E and N-acetylcysteine

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[27]. Also, unlike most of GPX4 inhibitors, the pharmacokinetic properties o f statins

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are quite qualified. Thus, properly using statins may be a promising strategy in ferroptosis based cancer therapy. Besides, GPX4 siRNA could effectively prevent the

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expression of GPX4 in cancer cells as well, and lead to ferroptosis [18]. Despite the uncovering of several ways to inhibit GPX4, it still needs more effective targeting

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strategies for clinical cancer therapy. As GPX4 is widely expressed in tissues and

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essential to prevent membrane lipid peroxidation, disrupting GPX4 in normal tissues could result in unexpected and untoward effects including kidney failure and oxidative brain lesion. In order to avoid side effects, nano carriers for drug delivery (e.g. nanoparticles and nano- liposomes) binding with specific recognition components (e.g. aptamers and antibodies) could be considered [53, 54]. However, the pharmacokinetic properties of most existing GPX4 inhibitors are poor, which limits their clinical application. Thus, improvement of existing GPX4 inhibitors’ pharmacokinetic properties and development of new bioavailable GPX4 inhibitors are also imperative [55]. In addition, as to the cancers resistant to GPX4 inhibition, the supplement of FSP1 or CoQ10 inhibitors may increase their sensitivities to ferroptosis in the presence of suitable GPX4 inhibitors.

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7. Summary and future considerations Various strategies for cancer therapy have been implemented, however, cancer cases and cancer-related deaths are still on the rise over the past few decades. Thus, cancer still remains a serious challenge to the scientific community worldwide [1, 56, 57]. James D. Watson, the father of DNA, had suggested that cancer treatment may need to be focused at the metabolic level rather than genetic level [58, 59]. The genetic anomalies of cancer cells are very complicated and highly unpredictable, but

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their metabolic characteristics are distinct and relatively stable, such as Warburg’s effect, iron addiction and ROS tolerance [13, 23, 60-64]. The discovery of ferroptosis,

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a kind of cell death characterized by iron-dependence and lipid hydroperoxides accumulation, may provide a new perspective for cancer therapy. Actually, several

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studies have demonstrated that some cancers could benefit from ferroptosis based

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therapy, including triple- negative breast cancer, head and neck cancer, glioblastoma, ovarian cancer, non-small cell lung cancer, renal cell carcinomas, diffuse large B cell

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lymphoma, pancreatic ductal adenocarcinoma and acute myeloid leukemia [18,

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65-73]. However, most of the treatments involved the inhibition of system Xc- or GSH rather than GPX4. Compared to GSH targeting strategy, we infer that these and other

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types of cancers may benefit more from GPX4 targeting related strategy. A recent study found that p53 mediated ferroptosis is distinct from the known canonical ferroptosis. This kind of ferroptosis is independent on GSH, GPX4 or ACSL4, but requires the activation of p53 and arachidonate 12- lipoxygenase (ALOX12), and its underlying mechanisms still remain veiled. p53- mediated ferroptosis could be an important complementary role for canonical ferroptosis, because the cancer cells that are lack of ACSL4 expression and resistant to canonical ferroptosis (resistant to erastin and RSL3) could be susceptible to it, such as MFC-7 cells. On the contrary, the cancer cells without ALOX12 expression are resistant to p53 mediated ferroptosis, but sensitive to canonical ferroptosis, such as HCT116 and HT1080 [74]. Besides, it is well-known that cancer cells always alter their metabolic

Journal Pre-proof patterns to escape cell death, such as excessively activating anti-oxidative pathways and inactivating some components in cell death pathways. ALOX12 is necessary for p53-mediated ferroptosis, but in most of human cancer cells, Alox12 gene is deleted, downregulated or mutated, which may be a self-protection mechanism to block the ferroptosis mediated by p53 [75-78]. Thus, most types of human cancer cells may still be sensitive to canonical ferroptosis regulated by GPX4 and its complementary members like FSP1, which means GPX4 could still be the key target spot to induce

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ferroptosis in quite a few human cancers. The discovery of ferroptosis mediated by

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p53 reveals the complexity of ferroptosis itself, and it is inevitable that more

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ferroptosis related pathways will be discovered in the future. GPX4’s role in ferroptosis will be much more evident with the deepening of related research, which

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would further pave the path for the development of GPX4 targeted therapeutic

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strategies for ferroptosis-based cancer therapy.

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Acknowledgements

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This work supported by grants from Science and Technology Planning Project of Shenzhen of China (JCYJ20170412140904406), Shenzhen Virtual University Park

(51777171).

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Special Funds (YFJGJS1.0) and the National Natural Science Foundation of China

Conflict of interest

The authors declare that they have no conflict of interest.

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Highlights 

Iron addiction and ROS tolerance make cancer cells vulnerable to ferroptosis.

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Disrupting of GSH may not effectively trigger cancer cells’ ferroptosis in vivo, because of the existence of compensatory pathway. Directly targeting GPX4 and its compensatory members without disrupting GSH may be more effective to induce ferroptosis in cancer cells in vivo, because GPX4

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is essential in preventing ferroptosis.

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Figure 1

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Figure 3