Cancer Cell

Review

Targeting Ferroptosis to Iron Out Cancer

Behrouz Hassannia,1,2 Peter Vandenabeele,1,2,3 and Tom Vanden Berghe1,2,4,5,*
1VIB Center for Inflammation Research (IRC), Ghent, Belgium
2Department of Biomedical Molecular Biology (DBMB), Ghent University, Ghent, Belgium
3Methusalem Program, Ghent University, Ghent, Belgium
4Laboratory of Pathophysiology, Department of Biomedical Sciences, University of Antwerp, 2000 Antwerp, Belgium
5Ferroptosis And Inflammation Research (FAIR), VIB-Ghent University and University of Antwerp, Belgium

*Correspondence: [email protected]
https://doi.org/10.1016/j.ccell.2019.04.002

One of the key challenges in cancer research is how to effectively kill cancer cells while leaving the healthy
cells intact. Cancer cells often have defects in cell death executioner mechanisms, which is one of the
main reasons for therapy resistance. To enable growth, cancer cells exhibit an increased iron demand
compared with normal, non-cancer cells. This iron dependency can make cancer cells more vulnerable to
iron-catalyzed necrosis, referred to as ferroptosis. The identification of FDA-approved drugs as ferroptosis
inducers creates high expectations for the potential of ferroptosis to be a new promising way to kill ther-
apy-resistant cancers.

Introduction for their selective cytotoxicity in engineered cells overexpressing

Despite considerable advances in treatment, cancer remains the the oncogenic mutant HRAS (Dolma et al., 2003; Weiwer et al.,
second leading cause of death worldwide (WHO, 2018). Trig- 2012; Yang and Stockwell, 2008). However, further studies did
gering apoptotic cell death with anti-cancer drugs is one of the not corroborate the selective lethality of erastin in RAS-mutated
principal approaches for killing cancer cells. However, the effec- cancer cell lines (Dixon et al., 2012; Yang et al., 2014). Morpho-
tiveness of apoptosis induction in tumors is limited, due to the logically, cells undergoing ferroptosis have a typical necrotic
acquired or intrinsic resistance of cancer cells to apoptosis (Hol- morphology, along with dysmorphic small mitochondria with
ohan et al., 2013; Okada and Mak, 2004; Su et al., 2016). There- decreased crista, a condensed membrane, and a ruptured outer
fore, exploiting other forms of non-apoptotic cell death opens membrane (Dixon et al., 2012; Friedmann Angeli et al., 2014).
new therapeutic avenues for eliminating cancer cells and limiting Furthermore, ferroptotic cells do not display any hallmarks of
the survival of drug-resistant clones. Two decades of intensive apoptosis, and knockdown of necroptosis mediators RIPK1
cell death research has revealed the existence of several modes and RIPK3 do not protect cells from ferroptosis (Dixon et al.,
of regulated necrosis (Vanden Berghe et al., 2014). Two types of 2012; Friedmann Angeli et al., 2014). Instead, ferroptosis execu-
regulated necrosis, necroptosis and ferroptosis, are being tion is characterized by an iron-catalyzed excessive peroxidation
explored as alternative ways to eradicate apoptosis-resistant of PUFA-containing phospholipids (PLs), present in excess in
cancer cells. A growing list of compounds and anti-cancer drugs mammalian cell membranes. Supplementing cells with PUFAs
has been reported to initiate necroptosis (Fulda, 2014) as well as promotes ferroptosis, while exchanging PUFAs with deuterated
ferroptosis (Conrad et al., 2016; Yang and Stockwell, 2016). Nec- PUFAs that are less susceptible to oxidation or deletion of genes
roptosis, the best-characterized form of regulated necrosis, is required for the activation and incorporation of PUFAs into PLs
mediated by the concerted action of receptor interacting inhibits ferroptosis (Dixon et al., 2015; Doll et al., 2017; Kagan
protein kinase 3 (RIPK3) and mixed-lineage kinase domain-like et al., 2017; Yang et al., 2016; Yuan et al., 2016b). Mechanisti-
(Pasparakis and Vandenabeele, 2015; Vanden Berghe et al., cally, double bonds adjacent to methylene groups in PUFAs
2016). Ferroptosis is defined as an iron-catalyzed form of regu- weaken the hydrogen bond energy of the bis-allylic methylene
lated necrosis that occurs through excessive peroxidation groups, resulting in increased susceptibility of hydrogen
of polyunsaturated fatty acids (PUFAs) (Dixon et al., 2012). abstraction and consequent oxygenation, which can be moni-
Recently ferroptosis has gained a lot of interest, especially in tored through oxidative lipidomics approaches (Else, 2017;
view of the downregulation and silencing of genes involved in Friedmann Angeli et al., 2014; Gaschler and Stockwell, 2017; Re-
the initiation and execution of necroptosis in cancers (Chen petto et al., 2012; Yang et al., 2016). The deleterious effects of
et al., 2016). In this review, we first briefly describe the current lipid peroxidation in ferroptosis can be neutralized by iron chela-
molecular understanding of ferroptosis induction and execution, tors such as deferoxamine (DFO), and a wide variety of lipophilic
and how it is modulated. Next, we elaborate on strategies of can- radical traps such as vitamin E, ferrostatin-1 (Fer1), and liprox-
cer therapy that are based on ferroptosis induction, and finally statin-1 (Lip1) (Dixon et al., 2012; Friedmann Angeli et al., 2014).
we provide a future perspective on this emerging field. Non-enzymatic Lipid Peroxidation
Non-enzymatic lipid peroxidation or auto-oxidation of lipids is a
Ferroptosis Execution: Biological Rust of Unsaturated free radical-driven chain reaction in which reactive oxygen spe-
Membranes cies (ROS) initiate the oxidation of PUFAs (Figure 1). The most
Ferroptosis was originally recognized as a unique form of cell chemically reactive species of activated oxygen is hydroxyl
death induced by small molecules, erastin and RSL3, in screens radical (OH$), which is a highly mobile, water-soluble form of

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Figure 1. Lipid Peroxidation Process and Toxicity

Ferrous iron (Fe2+) is oxidized to ferric iron (Fe3+) through reaction with hydrogen peroxide (H2O2), resulting in formation of highly reactive hydroxyl radicals (OH$),
referred to as the Fenton reaction. Fe3+ is reduced back to Fe2+ through reaction with superoxide radicals (O2$–). This redox cycle is referred to as the Harber-
Weiss reaction. In non-enzymatic lipid peroxidation, free radicals, such as hydroxyl radicals (OH$), abstract a hydrogen from a polyunsaturated fatty acid (PUFA)
forming a carbon-centered phospholipid (PL) radical (PL$). PL$ reacts with molecular oxygen (O2), forming a phospholipid peroxyl radical (PLOO$). PLOO$ ab-
stracts hydrogen from another PUFA, forming a phospholipid hydroperoxide (PLOOH) and a new PL$, which can react again with O2. In enzymatic lipid per-
oxidation, lipoxygenases (LOX) catalyze the dioxygenation of PUFAs and generate PLOOH. On the one hand, PLOOH in the presence of ferrous iron (Fe2+) can be
decomposed to alkoxyl phospholipid radical (PLO$), which by attacking another PUFA promotes further propagation of lipid peroxidation. On the other hand,
PLOOH may decompose to 4-hydroxynonenal (4-HNE) or malondialdehyde (MDA), which by crosslinking may inactivate proteins. Peroxidation of PLs and
generation of 4-HNE or MDA cause membrane instability and permeabilization, leading to cell death. Glutathione peroxidase 4 (GPX4) possesses a unique
capability to reduce reactive PL hydroperoxides to unreactive PL-alcohol (PL-OH), which interrupts the free radical chain reaction and suppresses lipid per-
oxidation.

ROS that can initiate lipid peroxidation. Fenton and Fenton-like iron (Fe2+), are the main sources of hydroxyl radical formation
reactions, which mainly involve the reaction between hydrogen (Ayala et al., 2014; Fenton, 1894). As the first step of non-enzy-
peroxide (H2O2) and a transition metal such as free ferrous labile matic lipid peroxidation, a hydroxyl radical abstracts a hydrogen

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from PUFA to form carbon-centered lipid radical (L$). The rapid Canonical Ferroptosis Induction
reaction of molecular oxygen (O2) with a lipid radical yields a lipid The canonical pathway induces ferroptosis by inactivating the
peroxyl radical (LOO$). Subsequently, the lipid peroxyl radical major protective mechanism of membranes against peroxidation
can abstract hydrogen from an adjacent PUFA, leading to the damage. A major detoxifying mechanism is achieved through the
formation of a lipid hydroperoxide (LOOH) and a new lipid activity of glutathione peroxidases (GPXs) (Bochkov et al., 2010).
radical, which can initiate another lipid radical chain reaction. Among GPXs, GPX4 possesses a unique capability to detoxify
Lipid hydroperoxide is converted to an alkoxyl radical (LO$) in hydroperoxides in complex lipids such as PL and cholesterol,
the presence of ferrous iron, which subsequently reacts with even when they are inserted into membranes or lipoproteins (Bri-
an adjacent PUFA to initiate another lipid radical chain reaction. gelius-Flohe and Maiorino, 2013). Therefore, GPX4 is considered
This auto-amplifying process, which is catalyzed by iron and the only GPX that protects biomembranes from peroxidation
oxygen, can result in membrane destruction and cell death damage (Brigelius-Flohe and Maiorino, 2013). GPX4 can be inac-
when the molecular guardians that keep lipid peroxidation in tivated through direct or indirect targeting mechanisms such as
check fail. As a primary non-enzymatic defense mechanism, depletion of intracellular glutathione (GSH), as an essential
chain-breaking lipophilic anti-oxidants can neutralize lipid radi- cofactor of GPX4 (Table 1) (Dixon et al., 2012; Yang et al.,
cals by donating electrons. Alternatively, under conditions of 2014, 2016).
high concentrations of radicals, two radicals can react with Depletion of Intracellular GSH
each other and form stable non-radical molecules (Reis and The system XC
is composed of disulfide-linked heterodimers
Spickett, 2012; Repetto et al., 2012; Valko et al., 2005). SLC7A11 (xCT) and SLC3A2 (4F2hc) that imports the extracel-
Enzymatic Lipid Peroxidation lular oxidized form of cysteine, cystine, in exchange for intracel-
Enzymatic lipid peroxidation is mediated in a controlled manner lular glutamate (Cao and Dixon, 2016; Sato et al., 1999). Inhibit-
by the activity of the lipoxygenase (LOX) family. LOXs are non- ing the import of cystine, which is required for GSH synthesis,
heme iron-containing enzymes that catalyze the dioxygenation culminates in depletion of intracellular GSH levels (Dixon et al.,
of free and esterified PUFAs to generate various lipid hydroper- 2012, 2014; Lu, 2009). GSH is a tripeptide anti-oxidant
oxides (Figure 1). In mammalian cells, linoleic acid (LA) and that serves as a cofactor for selenium-dependent GPX4 to
arachidonic acid (AA) are the most abundant PUFAs serving as reduce lipid hydroperoxides (Lu, 2009; Yant et al., 2003). There-
substrates for LOXs (Kuhn et al., 2015). Lipoxygenase 5 (LOX5) fore, depletion of GSH by erastin indirectly inactivates GPX4
synthesizes 5-hydroperoxyeicosatetraenoic acid (5-HPETE) (Figure 2A), leading to accumulation of toxic lipid ROS and
from oxygenation of AA at carbon 5. LOX12 and LOX15 synthe- ensuing lipid peroxidation (Dixon et al., 2012, 2014). Of note,
size 12-HPETE and 15-HPETE from AA and 9-hydroperoxyocta- early observations trace back to the 1950s and 1970s showing
decadienoic acid (9-HPODE) and 13-HPODE from LA (Gaschler that deprivation of cystine inhibited the growth of cell cultures
and Stockwell, 2017). In contrast to LOX5, which needs prior hy- (Eagle, 1955; Hirschhorn and Stockwell, 2019), and this type of
drolysis of esterified AA from membrane PLs by cytosolic phos- cell death was inhibited by lipophilic anti-oxidants and iron che-
pholipase A2 (cPLA2), LOX12 and LOX15 can directly oxygenate lators (Bannai et al., 1977; Hirschhorn and Stockwell, 2019; Mur-
AA containing PLs (Jung et al., 1985; Takahashi et al., 1993). In phy et al., 1989, 1990). Direct inhibition of GSH synthesis also
support of the role of lipoxygenases in ferroptosis, inhibition or suffices to induce ferroptosis in some cells. For instance, inhibi-
knockdown of lipoxygenases inhibits ferroptosis in some cell tion of glutamate-cysteine ligase (GCL), an enzyme involved in
types (Seiler et al., 2008; Yang et al., 2016). Recently, it was sug- de novo synthesis of GSH, using buthionine sulfoximine (BSO),
gested that vitamin E also inhibits LOX activity, in addition to its can induce ferroptosis in some cellular contexts (Figure 2A)
lipophilic radical trap activity, which might explain its potent (Yang et al., 2014).
inhibitory action on ferroptosis (Kagan et al., 2017). Inactivation/Depletion of GPX4
Toxicity of Lipid Peroxidation A chemoproteomic assay identified that the ferroptosis inducer
The exact mechanisms leading to ferroptotic cell death down- RSL3 covalently binds to selenocysteine (Sec) at the active site
stream of lipid peroxidation remain elusive. The mechanism of GPX4, thereby directly inhibiting PL-peroxidase activity of
may involve the formation of a structured lipid pore, similar to GPX4 (Figure 2B) (Yang et al., 2014, 2016). Overexpression of
the proteinaceous pores observed in necroptosis and pyroptosis GPX4 in cells confers resistance to RSL3-induced ferroptosis,
(Kayagaki et al., 2015; Wang et al., 2017b). Alternatively, while knockdown of GPX4 promotes ferroptosis (Yang et al.,
continued extensive oxidation and depletion of PUFAs might 2014). Other compounds such as ML162, withaferin A (WA),
modify membrane fluidity and structure, and increase mem- and the Food and Drug Administration (FDA)-approved anti-
brane permeability, eventually leading to a loss of membrane cancer agent altretamine can also induce ferroptosis by inacti-
integrity. Using molecular dynamics models, it is hypothesized vating GPX4 (Figure 2B) (Cao and Dixon, 2016; Gaschler et al.,
that during ferroptosis membrane thinning and increased curva- 2018a; Hassannia et al., 2018; Weiwer et al., 2012; Woo et al.,
ture drive a vicious cycle of access by oxidants, which ultimately 2015). FIN56 promotes ferroptosis by binding and activating
destabilizes the membrane, leading to pore and micelle forma- squalene synthase (SQS), an enzyme involved in cholesterol
tion (Agmon et al., 2018; Cheng et al., 2018). Additionally, lipid synthesis (Shimada et al., 2016b). This might result in suppres-
hydroperoxides may decompose to reactive toxic aldehydes sion of some metabolites such as lipid-soluble anti-oxidant
such as 4-hydroxy-2-nonenals (4-HNEs) or malondialdehydes coenzyme Q10 (coQ10) and Sec-tRNA, and account for the
(MDAs), which by crosslinking may inactivate proteins involved observed depletion/inactivation of GPX4 (Figure 2C). Similarly,
in essential cellular processes to promote ferroptosis (Zhong genetic depletion of GPX4 leads to the rapid accumulation of
and Yin, 2015) (Figure 1). lipid ROS, which can be suppressed by lipophilic radical traps

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Table 1. Ferroptosis Stimuli

Mechanism Target Compound/Drug Phenotype and Readout References
GPX4 inactivation system XC
erastin lipid ROSa Dixon et al., 2012; Dolma
due to GSH depletion death inhibited by DFO, et al., 2003
(class I FINs) Fer1, and Trolox
piperazine erastin increase in PTGS2 mRNA Yang et al., 2014
regression HT1080
xenografts
imidazole ketone lipid ROSa Larraufie et al., 2015;
erastin (IKE) Yang et al., 2016
sulfasalazine death inhibited by DFO, Dixon et al., 2012; Gout
Fer1, and Trolox et al., 2001
sorafenib lipid ROSa Louandre et al., 2013
death inhibited by DFO
and Fer1
glutamate inhibited by CPX Dixon et al., 2012
and Fer1
glutamate-cysteine buthionine sulfoximine lipid ROSa Griffith, 1982; Yang
ligase (BSO) et al., 2014
glutathione S-transferase artesunate lipid ROSa Eling et al., 2015;
death inhibited by DFO, Hamacher-Brady et al.,
Fer1, and Trolox 2011; Lisewski
et al., 2014
unknown DPI2 death inhibited by DFO Yang et al., 2014
and a-Toc
[Cys] depletion cyst(e)inase reduced GSH levels Cramer et al., 2017
regression of MDA-MB-
361, DU145 and PC3
xenografts
ND dehydrogenase BAY 87-2243 lipid ROSa Basit et al., 2017;
death inhibited by Fer1 Schockel et al., 2015
and a-Toc
GPX4 inactivation/ GPX4 1S,3R-RSL3b,c lipid ROSa Dixon et al., 2012; Yang
depletion (class II, death inhibited by DFO et al., 2014; Yang and
III FINs) and Fer1 Stockwell, 2008
increase in PTGS2 mRNA
HT1080 tumor regression
DPI7/ML162b,DPI10/ lipid ROSa Weiwer et al., 2012; Yang
ML210b, DPI12-13b, et al., 2014
DPI17-19b
altretamineb lipid ROSa Woo et al., 2015
b
FIN56 lipid ROSa Gaschler et al., 2018a;
death inhibited by DFO Shimada et al., 2016b
and a-Toc
withaferin Ac lipid ROSa in Hassannia et al., 2018
neuroblastoma cells
death inhibited by DFO,
Fer1 and a-Toc
Squalene synthase FIN56b lipid ROSa Gaschler et al., 2018a;
death inhibited by DFO Shimada et al., 2016b
and a-Toc
HMG-CoA reductase fluvastatin, lovastatin lipid ROSa Shimada et al., 2016b;
acid, simvastatin Viswanathan et al., 2017
(Continued on next page)

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Table 1. Continued
Mechanism Target Compound/Drug Phenotype and Readout References
a
Iron loading (class hemoglobin lipid ROS Li et al., 2017
IV FINs) death inhibited by Fer1 in
organotypic
hippocampal slice
cultures
FeCl2 death inhibited by Fer1 in Li et al., 2017
organotypic
hippocampal slice
cultures
hemin death inhibited by DFO Hassannia et al., 2018;
and Fer1 Imoto et al., 2018;
NaveenKumar
et al., 2018
(NH4)2Fe(SO4)2 death inhibited by Fer1 Hassannia et al., 2018
non-thermal plasma lipid ROSa Furuta et al., 2018; Shi
death inhibited by DFO et al., 2017
salinomycin lipid ROSa Mai et al., 2017
death inhibited by Fer1
amino acid depletion + lipid ROSa Kim et al., 2016
Cornell dots death inhibited by Fer1
and Trolox
amino acid depletion, death inhibited by Fer1 Gao et al., 2015
cystine deprivation +
holo-transferrin
transferrin [ lapatinib + siramesine death inhibited by DFO Ma et al., 2016
ferroportin-1 Y and Fer1
Iron oxidation (class FINO2 lipid ROSa Gaschler et al., 2018a
IV FINs) death inhibited by Fer1
Increase in LIP by KEAP1 inactivation withaferin A lipid ROSa in Hassannia et al., 2018
HMOX1[ (class IV neuroblastoma
FINs) death inhibited by DFO,
Fer1 and a-Toc
IkBa BAY 11-7085 death inhibited by Fer1, Chang et al., 2018
Lip1 and NAC
Unknown lanperisone death inhibited by DFO Shaw et al., 2011
and Trolox
artemisinin derivatives death inhibited by DFO Lin et al., 2016; Ooko
and Fer1 et al., 2015
CIL41, CIL56, CIL69, lipid ROSa for CIL56 Shimada et al., 2016b
CIL70, CIL75, and CIL79 death inhibited by DFO
and a-Toc
a-Toc, a-tocopherol; CIL, caspase-3/7-independent lethal; CPX, ciclopirox olamine; DFO, deferoxamine; Fer1, ferrostatin-1; FIN, ferroptosis-inducing
compound, GPX4, glutathione peroxidase 4; GSH, glutathione; HMOX1, heme oxygenase-1; IkBa, nuclear factor of k light-chain polypeptide gene
enhancer in B cell inhibitor a; KEAP1, kelch-like ECH-associated protein 1; LIP, labile iron pool; Lip1, liproxstatin-1; PTGS2, prostaglandin-endoper-
oxide synthase 2, ROS, reactive oxygen species; VDAC, voltage-dependent anion channel.
a
Lipid ROS shown by C11-BODIPY staining.
b
GPX4 inactivation shown using LC-MS-based GPX4 assay (PCOOH).
c
Direct GPX4 binding shown through pull-down.

and iron chelators (Friedmann Angeli et al., 2014; Yang and can further propagate lipid peroxidation. Moreover, iron
et al., 2014). and iron derivatives, such as heme or iron-sulfur [Fe-S] clusters,
are crucial for the activity of ROS-producing enzymes such as
Non-canonical Ferroptosis Induction nicotinamide adenine dinucleotide phosphate hydride (NADPH)
Focusing on the lipid peroxidation mechanism, it is clear that iron oxidases (NOXs), LOXs, and mitochondrial electron transport
plays an important role in ferroptosis. Small pools of iron mostly complexes, which can fuel ROS production (Dixon and Stock-
in the form of Fe2+, referred to as the labile iron pool (LIP), can well, 2014). Non-canonical ferroptosis induction refers to ferrop-
directly catalyze free radical formation via Fenton reactions tosis that is initiated by increasing the LIP (Hassannia et al.,

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Figure 2. Mechanism of Ferroptosis Induction

(A) Class I ferroptosis-inducing compounds (FINs) trigger ferroptosis by depleting intracellular glutathione (GSH) through inhibition of system XC
or through
inhibition of glutamate-cysteine ligase (GCL).
(legend continued on next page)

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2018), for example due to excessive activation of heme oxygen- while a moderate upregulation could be cytoprotective (Suttner
ase 1 (HMOX1), decreased ferroportin expression, or increased and Dennery, 1999).
transferrin expression (Chang et al., 2018; Hassannia et al., Lipid Metabolism
2018; Ma et al., 2016). Addition of holo-transferrin as an iron car- Incorporation of de novo synthesized fatty acids into PLs requires
rier protein also unleashes ferroptotic cell death upon amino acid the conversion of long-chain fatty acids to acyl-coenzyme A (CoA)
deprivation in Bax/Bak double-knockout cells (Gao et al., 2015) catalyzed by acyl-CoA synthetase (ACS) and subsequent reacy-
(Table 1). Although the mechanism of action is still unclear, over- lation catalyzed by lysophospholipid acyltransferases (LPLATs)
loading cells with iron using for example iron chloride, hemoglo- (Hishikawa et al., 2014; Mashima et al., 2009). In this regard,
bin, hemin, or ferrous ammonium sulfate is sufficient to induce knockdown of acyl-CoA synthetase long-chain family member
ferroptosis (Hassannia et al., 2018; Imoto et al., 2018; Li et al., 4 (ACSL4), which preferably converts AA to acylated AA, or loss
2017; NaveenKumar et al., 2018) (Figure 2D). of lysophosphatidylcholine acyltransferase 3 (LPCAT3), which
catalyzes the insertion of acylated AA into PLs, makes cells resis-
Modulators of Ferroptosis tant to ferroptosis (Dixon et al., 2015; Doll et al., 2017; Kagan
Considering the fact that ferroptosis is a result of metabolic et al., 2017; Yuan et al., 2016b) (Table 2 and Figure 3H). Recently,
dysfunction involving ROS, iron, and PUFAs, various genes a scaffold protein inhibitor of protein kinase cascades coined
and pathways related to metabolism in iron or energy, lipid syn- phosphatidylethanolamine-binding protein 1 (PEBP1) was shown
thesis, and oxidative stress have been identified, which poten- to bind and direct LOX15 toward PUFAs in the cell membrane to
tially modulate the sensitivity to ferroptosis (Table 2 and Figure 3). promote ferroptosis (Wenzel et al., 2017). Upon studying the
mechanism of action of FIN56-induced ferroptosis, the mevalo-
Iron Metabolism nate pathway was identified (Figure 3F) (Shimada et al., 2016b).
Iron is a redox-active metal that can be engaged in free radical A direct metabolite of mevalonate, isopentenyl pyrophosphate
formation and propagation of lipid peroxidation (Figure 3C). (IPP), is crucial for cholesterol biosynthesis, isopentenylation of
Therefore, elevated levels of iron can increase the vulnerability Sec-tRNA, and CoQ10 production (Moosmann and Behl, 2004).
to ferroptosis. Various genes or proteins involved in iron homeo- Repressing the activity of squalene synthase (SQS) or squalene
stasis including its import, export, and storage have been shown monooxygenase, which are downstream of isopentenyl pyro-
to modulate sensitivity to ferroptosis (Table 2; Figures 3A and phosphate involved in cholesterol synthesis, impedes ferroptosis.
3B). Suppression of nitrogen fixation 1 (NFS1), a cysteine desul- Conversely, inhibition of HMG-CoA reductase by statins, up-
furase that supplies sulfur from cysteine for the synthesis of iron- stream of isopentenyl pyrophosphate synthesis, enhances
sulfur clusters, sensitizes cells to ferroptosis by activating the ferroptosis (Shimada et al., 2016b). Putatively, the mevalonate
iron-starvation response through concurrent increased levels pathway can affect ferroptosis through two mechanisms. Inhibi-
of transferrin receptor (TFRC) and decreased levels of ferritin tion of isopentenyl pyrophosphate synthesis interferes with matu-
(FTH) (Alvarez et al., 2017). Lysosomes, through degradation of ration of Sec-tRNA, which is required for incorporation of Sec to
FTH (coined ferritinophagy), can accumulate large amounts of the selenoprotein GPX4 (Yang and Stockwell, 2016). Additionally,
iron (Terman and Kurz, 2013) (Figure 3D). Inhibition of lysosomal suppression of coenzyme Q10 production can cause abnormal
activity (Torii et al., 2016) or silencing of nuclear receptor coacti- respiratory function and oxidative damage in the mitochondria.
vator 4 (NCOA4), a cargo receptor recruiting FTH to autophago- In this regard, supplementation of cells with CoQ10 effectively
somes for lysosomal degradation and iron release, suppresses suppresses ferroptosis (Shimada et al., 2016b).
ferroptosis (Gao et al., 2016; Hou et al., 2016). Excessive activa- (Anti-)Oxidant Metabolism
tion of HMOX1, which catalyzes the degradation of heme to GSH metabolism and anti-oxidant capacity regulates sensitivity
ferrous iron, biliverdin, and carbon monoxide, enhances ferrop- to ferroptosis (Table 2). Analysis of sensitivity of ferroptosis stim-
tosis by increasing LIP (Chang et al., 2018; Hassannia et al., uli across 60 different human cell lines, the NCI-60 cell line panel,
2018). Pharmacological inhibition or silencing of HMOX1 confers using transcriptome data revealed that NADP(H) abundance as a
resistance to ferroptosis induced by withaferin A, erastin, and biomarker, which is inversely correlated with sensitivity to ferrop-
BAY 11-7085 (Chang et al., 2018; Hassannia et al., 2018; Kwon tosis inducers (Shimada et al., 2016a). The transsulfuration
et al., 2015). However, HMOX1 can also act in a cytoprotective pathway, through which methionine supplies cysteine from cys-
way (Sun et al., 2016), probably depending on the level of activa- tathionine for glutathione synthesis, modulates ferroptosis in
tion. The protective effect of HMOX1 is attributed to its anti- some cells when system XC
is inhibited (Figure 3G) (Belalcazar
oxidant activity while its toxic effect is due to enhanced genera- et al., 2014; Hayano et al., 2016). Upregulation of the transsulfu-
tion of ferrous iron that boosts Fenton-mediated decomposition ration pathway in response to silencing of certain tRNA synthe-
of peroxides in case of insufficient buffering capacity by ferritin. tases (CARS, HARS, and EPRS) renders cells insensitive to fer-
Thus excessive upregulation of HMOX1 could be cytotoxic, roptosis induced by erastin (Hayano et al., 2016). tRNA

(B) Class II FINs trigger ferroptosis by direct inhibition and inactivation of GPX4.
(C) Class III FINs trigger ferroptosis by indirect inhibition and inactivation of GPX4. FIN56, for example, binds and activates squalene synthase (SQS), an enzyme
involved in cholesterol synthesis. This might result in suppression of some metabolites such as CoQ10 and Sec-tRNA, and account for the observed depletion/
inactivation of GPX4.
(D) Class IV FINs trigger ferroptosis by increasing the labile iron pool (LIP) through direct iron overload or excessive activity of heme oxygenase 1 (HMOX1). Glu,
glutamate; BSO, buthionine sulfoximine; GR, glutathione reductase; GS, glutathione synthase; Gly, glycine, GSSG, glutathione disulfide; HMGCR, 3-hydroxy-3-
methylglutaryl CoA reductase; WA, withaferin A; SAS, sulfasalazine; FPP, farnesyl pyrophosphate; KEAP1, kelch-like ECH-associated protein 1; NRF2, nuclear
factor erythroid 2-related factor 2; DMT1, divalent metal transporter 1.

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Table 2. Modulators of Ferroptosis

Gene Protein Function Modulatory Effect References
Iron Metabolism
TFRC transferrin receptor cellular transferrin-iron uptake knockdown suppresses Yang and Stockwell,
erastin-induced ferroptosis 2008
knockdown suppresses Gao et al., 2015,
ferroptosis induced by amino 2016
acid/cystine deprivation
PHKG2 phosphorylase kinase, g2 activates glycogen iron regulatory function? Yang et al., 2016
phosphorylase to release knockdown suppresses
glucose-1-phosphate from erastin-induced ferroptosis
glycogen
IREB2 iron response element-binding RNA-binding protein that knockdown suppresses Dixon et al., 2012;
protein 2 regulates iron levels in the cells ferroptosis induced by erastin Gao et al., 2016
by regulating the translation or amino acid/cystine
and stability of mRNAs that deprivation
affect iron homeostasis upon
iron depletion
HSBP1 heat-shock 27-kDa protein 1 activated in heat stress iron regulatory function? Sun et al., 2015
response by heat-shock knockdown enhances erastin-
factor 1 (HSF1) induced ferroptosis in vitro and
in vivo
HMOX1 heme oxygenase 1 catalyzes the degradation of inhibition or knockdown Hassannia et al.,
heme to biliverdin, CO, suppresses withaferin 2018
and Fe2+ A-induced ferroptosis
inhibition or knockout Kwon et al., 2015
suppresses erastin-induced
ferroptosis
inhibition or knockdown Chang et al., 2018
suppresses BAY-induced
ferroptosis
CISD1/mitoNEET CDGSH iron-sulfur domain 1 inhibits mitochondrial iron knockdown enhances erastin- Yuan et al., 2016a
transport into the matrix induced ferroptosis
NCOA4 nuclear receptor coactivator 4 cargo receptor mediating knockdown suppresses Gao et al., 2016
ferritinophagy ferroptosis induced by amino
acid/cystine deprivation
knockdown suppresses Hou et al., 2016
erastin-induced ferroptosis
ACO1 aconitase 1 iron-sulfur protein that knockdown suppresses Gao et al., 2016
converses citrate to isocitrate, ferroptosis induced by amino
controls iron inside cell acid/cystine deprivation
FTH1 ferritin heavy chain 1 subunit of major intracellular expression level controls Yang and Stockwell,
iron storage protein ferroptosis sensitivity 2008
knockdown enhances erastin- Sun et al., 2016
or sorafenib-induced
ferroptosis in hepatocellular
carcinoma
STEAP3 six-transmembrane epithelial metalloreductase converting upregulated in response to Song et al., 2016
antigen of prostate 3 Fe3+ to Fe2+ erastin in bone marrow
stromal cells
FANCD2 Fanconi anemia nuclear protein involved in iron regulatory function? Song et al., 2016
complementation group D2 DNA damage repair knockout enhances erastin-
induced ferroptosis in bone
marrow stromal cells
NFS1 cysteine desulfurase enzyme involved in knockdown activates the iron- Alvarez et al., 2017
synthesizing iron-sulfur starvation response promoting
clusters using sulfur from erastin-induced ferroptosis
cysteine
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Table 2. Continued
Gene Protein Function Modulatory Effect References
Lipid Metabolism
ACSF2 acyl-CoA synthetase family regulation of mitochondrial knockdown suppresses Dixon et al., 2012
member 2 fatty acid metabolism erastin-induced ferroptosis
CS citrate synthase regulation of mitochondrial knockdown suppresses Dixon et al., 2012
fatty acid metabolism erastin-induced ferroptosis
LPCAT3 lysophosphatidylcholine incorporation of acylated fatty identified in haploid cell Dixon et al., 2015
acyltransferase 3 acids into membranes by genetic screen
catalyzing the reacylation of knockdown suppresses RSL3- Kagan et al., 2017
lysophospholipids to induced ferroptosis
phospholipids
ACSL4 acyl-CoA synthetase long- converts free fatty acids identified in haploid cell Dixon et al., 2015
chain family member 4 (preferentially AA) into fatty genetic screen
acyl-CoAs knockdown suppresses Yuan et al., 2016b
erastin-induced ferroptosis
inhibition or knockout Kagan et al., 2017
suppresses RSL3-induced
ferroptosis
inhibition or knockout Doll et al., 2017
suppresses erastin-, RSL3-, or
GPX4-depletion-induced
ferroptosis
inhibition suppresses GPX4-
depletion-induced damage
in vivo
ACSL3 acyl-CoA synthetase long- converts exogenous required for MUFA-induced Magtanong et al.,
chain family member 3 monounsaturated fatty acids protection from erastin2- 2018
(MUFAs) into fatty acyl-CoAs induced ferroptosis
knockout attenuates MUFA-
induced resistance to
ferroptosis
ACACA Acetyl-CoA carboxylase alpha Converts acetyl-CoA to identified in haploid cell Dixon et al., 2015
malonyl-CoA, the rate-limiting genetic screen
step in fatty acid synthesis inhibition suppresses FIN56-, Dixon et al., 2015;
but not erastin- or RSL3- Shimada et al.,
induced ferroptosis 2016b
GPX4 glutathione peroxidase 4 lipid repair enzyme inhibition or knockout induces Yang et al., 2014
ferroptosis
AKR1C aldo-keto reductase family 1 regulate the detoxification of upregulation confers Dixon et al., 2014
member C1 oxidative lipid breakdown protection against erastin-
products induced ferroptosis
LOX lipoxygenase-12/15 catalyzes the dioxygenation of inhibition or knockout Seiler et al., 2008
polyunsaturated fatty acids in suppresses GPX4-depletion-
lipids induced ferroptosis
lipoxygenases knockout protects against Yang et al., 2016
imidazole keto erastin (IKE)-,
but not RSL3-induced
ferroptosis
inhibition protects against
erastin-induced ferroptosis
PEBP1 phosphatidylethanolamine- protein scaffold controls substrate specificity Wenzel et al., 2017
binding protein 1 of LOX15
knockdown suppresses RSL3-
induced ferroptosis
ZEB1 zinc finger E-box-binding EMT regulator and lipogenic knockout suppresses GPX4- Viswanathan et al.,
homeobox 1 factor depletion-induced ferroptosis 2017
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Table 2. Continued
Gene Protein Function Modulatory Effect References
SQS/FDFT1 squalene synthase/farnesyl- responsible for synthesis of knockdown or inhibition Shimada et al.,
diphosphate squalene and involved in suppresses FIN-56-induced 2016b
farnesyltransferase 1 cholesterol synthesis ferroptosis
knockout or inhibition Garcia-Bermudez
sensitizes SQLE-deficient et al., 2019
anaplastic large cell lymphoma
(ALCL) cells to ML162-induced
ferroptosis
SQLE squalene monooxygenase catalyzes the conversion of overexpression sensitizes Garcia-Bermudez
squalene to squalene-2,3- SQLE-deficient ALCL cells to et al., 2019
epoxide and involved in ML162-induced ferroptosis
cholesterol synthesis
HMGCR 3-hydroxy-3-methyl-glutaryl- synthesis of mevalonic acid inhibition enhances FIN-56- Shimada et al.,
coenzyme A reductase induced ferroptosis 2016b
FADS2 fatty acid desaturase 2/acyl- involved in biosynthesis of knockdown decreases Vriens et al., 2019
CoA 6-desaturase highly unsaturated fatty acids, sapienate production and
desaturates palmitate to suppresses RSL3- induced
produce the monounsaturated lipid peroxidation in HUH7 liver
fatty acid sapienate cancer cells
(Anti)oxidant Metabolism
NRF2 nuclear factor erythroid key regulator of anti-oxidant inhibition or knockdown Sun et al., 2016
2-related factor 2 response including the enhances erastin- or
expression of system XC
sorafenib-induced ferroptosis
in hepatocellular carcinoma
in vitro and in vivo
knockdown enhances Roh et al., 2017
artesunate-induced
ferroptosis in head and neck
cancer in vitro and in vivo
overexpression confers Fan et al., 2017
resistance to erastin- and
RSL3-induced ferroptosis in
glioma cells, while knockdown
enhances ferroptosis
KEAP1 kelch-like ECH- associated binds to and regulates NRF2 overexpression enhances Fan et al., 2017
protein 1 by keeping its levels at control erastin- and RSL3-induced
ferroptosis in glioma cells,
while knockdown confers
resistance to ferroptosis
knockdown confers resistance Roh et al., 2017
to artesunate-induced
ferroptosis in head and neck
cancer
HMOX1 heme oxygenase 1 catalyzes the degradation of knockout enhances erastin- Adedoyin et al.,
heme to biliverdin, CO, induced ferroptosis in proximal 2018
and Fe2+ tubular cells
knockout enhances erastin- Sun et al., 2016
and sorafenib-induced
ferroptosis in hepatocellular
carcinoma cells
NQO1 quinone oxidoreductase-1 reduces quinones to knockdown enhances erastin- Sun et al., 2016
hydroquinones and sorafenib-induced
ferroptosis in hepatocellular
carcinoma cells
SLC7A11 solute carrier family 7 subunit of system Xc
to inhibition induces ferroptosis Dixon et al., 2012
member 11 import cystine in the cell
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Table 2. Continued
Gene Protein Function Modulatory Effect References
GCLC glutamate-cysteine ligase enzyme involved in GSH inhibition induces ferroptosis Yang et al., 2014
catalytic subunit synthesis
CARS cysteinyl-tRNA synthetase protein translation knockdown suppresses Hayano et al., 2016
erastin-induced ferroptosis
through compensatory
activation of transsulfuration
pathway [Cys][
CBS cystathionine-b-synthase converts homocysteine to inhibition or knockdown Hayano et al., 2016
cystathionine resensitizes erastin-induced
ferroptosis in CARS
knockdown cells
NOX NADPH oxidase superoxide-producing inhibition suppresses erastin- Dixon et al., 2012
enzymes induced ferroptosis in non-
small cell lung cancer cells
ABCC1/MRP1 multidrug-resistance protein 1 mediates GSH and overexpression sensitizes to Cao et al., 2019
chemotherapeutics efflux ferroptosis induced by erastin2
from cells and cyst(e)inase, RSL3
and ML162
Energy Metabolism
SLC1A5 solute carrier family 1 amino acid transporter feeding inhibition or knockdown Gao et al., 2015
member 5 glutaminolysis suppresses ferroptosis
induced by amino acid/cystine
deprivation
GLS2 glutaminase 2 converts glutamine to inhibition or knockdown Gao et al., 2015
glutamate suppresses ferroptosis
induced by erastin or amino
acid/cystine deprivation
GOT1 glutamic-oxaloacetic involved in synthesis of inhibition suppresses Gao et al., 2015
transaminase 1 a-ketoglutarate from ferroptosis induced by erastin
glutamate or amino acid/cystine
deprivation
microRNA 137 targets and regulates SLC1A5 overexpression suppresses Luo et al., 2018
levels SLC1A5 and confers
resistance to erastin- and
RSL3-induced ferroptosis,
while inhibition sensitizes to
ferroptosis
G6PD glucose-6-phosphate involved in pentose phosphate knockdown suppresses Dixon et al., 2012
dehydrogenase pathway erastin-induced ferroptosis in
non-small cell lung cancer
cells Calu-1
knockdown suppresses Gao et al., 2016
ferroptosis induced by amino
acid/cystine deprivation
PGD phosphoglycerate involved in pentose phosphate knockdown suppresses Dixon et al., 2012
dehydrogenase pathway erastin-induced ferroptosis in
non-small cell lung cancer
cells Calu-1

synthetase inhibition is suggested to induce an integrated stress protein after treating cells with erastin and sorafenib render
response, leading to enhanced synthesis of cysteine, which ulti- cells resistant to ferroptosis. This protection is attributed to upre-
mately feeds GSH synthesis (Hayano et al., 2016). gulation of NRF2 target genes including NQO1, HMOX1, and
Importantly, pharmacological inhibition of nuclear factor FTH1. Mitochondria are important sources of ROS formation in
erythroid 2-related factor 2 (NRF2) and silencing of NRF2-related mammalian cells through the electron transport chain (ETC)
genes increases sensitivity of cells to ferroptosis (Sun et al., that were suggested to contribute to ferroptosis, though strictly
2016), emphasizing its crucial role in inducing anti-oxidant not required. Cells depleted of mitochondria were initially found
mechanisms (Nguyen et al., 2009). Increased levels of NRF2 to remain sensitive to ferroptosis (Dixon et al., 2012; Gaschler

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Figure 3. Overview of Ferroptosis Modulation

(A–D) Iron metabolism and its regulators including transferrin, TFRC, and ferritinophagy modulate lipid peroxidation and ferroptosis by increasing the levels of
intracellular labile iron pool (LIP).

(legend continued on next page)

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et al., 2018b). However, inhibiting the activity of ETC complexes cannot repress SLC7A11 expression and fail to promote ferrop-
or depleting mitochondria, in a similar but more extended tosis, which highlights BAP1-mediated tumor suppression by
approach, could rescue ferroptosis induced by cystine depriva- ferroptosis (Zhang et al., 2018).
tion or erastin, but not by GPX4 inhibition (Gao et al., 2019). p53-Mediated Regulation of Ferroptosis
Energy Metabolism The guardian of the genome p53, encoded by the TP53 gene, is
Glutamate metabolism, known as glutaminolysis, is required for a crucial tumor suppressor that is mutated or inactivated in
ferroptosis triggered by cystine deprivation (Gao et al., 2015). more than half of all human cancers. The tumor-suppressor
Inhibition of glutamine uptake through the SLC1A5 transporter, activity of p53 has been primarily linked to its canonical func-
inhibition of glutamine metabolism to glutamate through mito- tions including induction of cell-cycle arrest, senescence, or
chondrial glutaminase (GLS2), as well as blocking the synthesis apoptosis. However, non-canonical functions of p53 such as
of a-ketoglutarate from glutamate through glutamic-oxaloacetic controlling metabolism and redox state can also contribute to
transaminase 1 (GOT1), counteract ferroptosis (Table 2, tumor suppression by regulating ferroptosis (Bieging et al.,
Figure 3E) (Gao et al., 2015). Glutaminolysis is supposed to modu- 2014; Kaiser and Attardi, 2018). Depending on the p53 muta-
late ferroptosis by supplying a-ketoglutaric acid (aKG) in tion status and cellular context, p53 can have either pro- or
the mitochondrial tricarboxylic acid cycle (Gao et al., 2019). Inhibi- anti-ferroptotic functions in response to oxidative stress (Gna-
tion of the pentose phosphate pathway (PPP) and silencing of two napradeepan et al., 2018) (Table 3). p53 might act as a rheo-
PPP enzymes, glucose-6-phosphate dehydrogenase (G6PD) stat, preventing ferroptosis under basal or low ROS stress,
and phosphoglycerate dehydrogenase (PGD), hinders erastin- while promoting ferroptosis under high oxidative stress (Kruis-
induced ferroptosis in human lung cancer cells (Dixon et al., wijk et al., 2015).
2012). The PPP generates NADPH, which is essential for preser- Under cellular stress, p53 sensitizes cells to ferroptosis by
ving the cellular levels of GSH and resistance to ferroptosis. How- transcriptional suppression of SLC7A11 and thereby repression
ever, it can also supply NADPH to NOXs and thereby contributes to of cystine uptake. For example, activation of p53 by nutlin-3
ROS production and ferroptosis in some cellular contexts. triggers ferroptosis in osteosarcoma U2OS cells in response
to ROS stress (Jiang et al., 2015). Regulation of SLC7A11
Ferroptosis and Cancer expression requires acetylation of the DNA-binding domain of
Tumor suppressors, such as BAP1 and p53, control the activa- p53 (Wang et al., 2016). Remarkably, an acetylation-defective
tion of ferroptosis (Table 3). Furthermore, the activation of ferrop- mutant p53 with three mutated lysines (K117/161/162R,
tosis by several small molecules and FDA-approved clinical referred to as p533KR) that fails to induce apoptosis, senes-
drugs in cancer cells, and the efficacy of tumor suppression by cence, and cell-cycle arrest (Li et al., 2012), sensitizes cells to
ferroptosis inducers in various experimental cancer models, un- ferroptosis (Jiang et al., 2015). The p533KR knockin mice do
derline the potential of ferroptosis as a novel anti-cancer therapy. not form spontaneous tumors, suggesting that p53 suppresses
This is further strengthened by the observed efficacy of ferropto- tumor formation through induction of ferroptosis (Jiang et al.,
sis to kill therapy-resistant cancer cells. 2015). Ectopic expression of the quadruple acetylation-defec-
BAP1-Mediated Regulation of Ferroptosis tive mutant p534KR in p53-null cells, in contrast to p533KR, fails
The tumor suppressor BAP1 encodes a nuclear deubiquitinat- to repress SLC7A11 and does not suppress tumor formation
ing enzyme, which in the form of a polycomb-repressive deubi- in xenograft models (Wang et al., 2016). p53 can also regulate
quitinase (PR-DUB) complex epigenetically regulates gene ferroptosis through its metabolic target gene spermidine/sper-
expression by decreasing histone 2A ubiquitination in nucleo- mine N1-acetyltransferase (SAT1), which encodes a protein
somes (Carbone et al., 2013; Ventii et al., 2008). BAP1 muta- involved in the polyamine metabolism pathway that is often
tions are found in a variety of sporadic human cancers whose downregulated in human tumors. Knockout of SAT1 markedly
germline mutations are considered to be an important predis- abrogates p53-mediated ferroptosis, while elevated levels of
posing factor for hereditary cancers (Abdel-Rahman et al., SAT1 expression sensitizes cells to ferroptosis upon ROS
2011; Jiao et al., 2013; Murali et al., 2013; Wiesner et al., stress (Ou et al., 2016). The African-restricted polymorphism
2011). The tumor-suppressor activity of BAP1 is partly medi- S47 in the p53 gene (TP53S47) also impairs the ability of p53
ated by ferroptosis through deubiquitination of H2A on the to suppress the transcription of SLC7A11 and makes cells
SLC7A11 promoter, resulting in repression of SLC7A11 expres- markedly resistant to ferroptosis (Gao et al., 2015; Jennis
sion (Zhang et al., 2018). Also in vivo, restoring BAP1 expres- et al., 2016). Although p53S47 could induce apoptosis, senes-
sion in BAP1-deficient cells suppresses xenograft tumor cence, and cell-cycle arrest, p53S47 knockin mice are vulner-
development. Human cancer-associated BAP1 mutations able to spontaneous tumor development (Jennis et al., 2016).

(E) Glutamine and glutaminolysis promote ferroptosis by supplying precursors, such as citrate, for fatty acid synthesis.
(F) Mevalonate pathway metabolite isopentenyl pyrophosphate (IPP) is crucial for cholesterol biosynthesis, isopentenylation, and Sec-tRNA and CoQ10 pro-
duction, and regulates ferroptosis induced by FIN56.
(G) Transsulfuration pathway affects sensitivity to ferroptosis by supplying cysteine from cystathionine for glutathione synthesis.
(H) GPX4 is the main negative regulator of ferroptosis by detoxifying lipid hydroperoxides. GSH and selenium are required for the activity of GPX4. The amount of
arachidonoyl (AA)/adrenoyl (AdA) esterified in PLs, which is regulated by the activity of acyl-CoA synthetase long-chain family member 4 (ACSL4) and lyso-
phosphatidylcholine acyltransferase 3 (LPCAT3), define sensitivity to ferroptosis. LOXs by direct oxygenation of PUFAs produce toxic PL hydroperoxides. TF,
transferrin; TFRC, transferrin receptor; STEAP3, six-transmembrane epithelial antigen of prostate 3; NCOA4, nuclear receptor coactivator 4; GLS2, glutaminase
2; GOT1, glutamic-oxaloacetic transaminase 1; FPP, farnesyl pyrophosphate; DMT1, divalent metal transporter 1; SQS, squalene synthase; Met, methionine;
Cys, cysteine.

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Table 3. Regulation of Ferroptosis by Cancer Metabolism

Gene Protein Function Modulatory Effect References
TP53 wild-type p53 tumor suppressor knockout/knockdown increases Jiang et al., 2015;
cystine uptake and suppresses Xie et al., 2017
ferroptosis in osteosarcoma U2OS
and breast cancer MCF7 cells
knockdown sensitizes colorectal Xie et al., 2017
cancer HCT116 and SW48 cell to
ferroptosis
stabilization with nutlin-3 in mouse Tarangelo et al.,
embryonic fibroblasts, HT-1080 2018
fibrosarcoma, renal cancer Caki-1,
and osteosarcoma U2OS cells
suppresses ferroptosis
mutated p533KR triple acetylation-defective mutant retains the ability to regulate Jiang et al., 2015
(K117/161/162) that fails to induce SLC7A11 expression and induce
cell-cycle arrest, senescence, and ferroptosis
apoptosis
Mutated p53S47 nonsynonymous single-nucleotide impaired ferroptosis induction in Jennis et al., 2016
polymorphism at codon 47 in p53S47 knockin MEF cells
African-descent populations intact ferroptosis induction in E1A/ Basu et al., 2016
Ras-transformed p53S47
knockin cells
mutated p534KR quadruple acetylation-defective impaired ferroptosis induction and Wang et al., 2016
mutant (K98/117/161/162) that fails loss of tumor-suppressor activity
to induce cell-cycle arrest,
senescence, and apoptosis
mutated p53 missense mutations (R273H, accumulated mutant-p53 protein Liu et al., 2017
R175H) that impair p53 sequence- sensitizes esophageal and lung
specific binding to DNA cancer cells to ferroptosis
BAP1 BRCA1-associated nuclear deubiquitinating promotes ferroptosis by repression Zhang et al., 2018
protein 1 epigenetically regulates gene of SLC7A11 expression
expression
CDKN1A cyclin-dependent inhibits CDK causing cell-cycle overexpression confers resistance Tarangelo et al.,
kinase (CDK) inhibitor arrest to erastin2-induced ferroptosis (by 2018
1A (p21) retarding GSH depletion?)
SAT1 spermidine/spermine acetylates spermidine and spermine transcription target of p53 Ou et al., 2016
N1-acetyltransferase 1 overexpression leads to ferroptosis
upon ROS stress
knockout suppresses p53-induced
ferroptosis
SOCS1 suppressor of cytokine cytokine-induced negative regulates p53 expression and Saint-Germain et al.,
signaling 1 regulators of cytokine signaling sensitizes cells to ferroptosis 2017
TP63 DN tumor protein 63a oncogene orchestrates GSH metabolism Wang et al., 2017a
overexpression confers resistance
to erastin- or RSL3-induced
ferroptosis, while knockdown
enhances ferroptosis
OTUB1 ovarian tumor (OTU) directly interacts with SLC7A11 and suppresses ferroptosis by Liu et al., 2019
family member regulates SLC7A11 stability stabilizing SLC7A11
deubiquitinase knockout sensitizes to erastin-
induced ferroptosis

Other mutant forms of p53 such as p53R273H and p53R175H, impedes tumor growth in a patient-derived xenograft model
which result from missense mutations in TP53, can suppress (Liu et al., 2017).
SLC7A11 expression by hindering NRF2-mediated SLC7A11 Apparently, p53 can also negatively regulate ferroptosis in
upregulation (Habib et al., 2015; Liu et al., 2017; Sasaki et al., some cellular contexts. Stabilizing and restoring the activity of
2002). Interestingly, reactivation of p53G266R using its activator wild-type p53 by nutlin-3 protects fibrosarcoma, renal cancer,
APR-246 restores system XC
inhibition by sulfasalazine, which and osteosarcoma cells against ferroptosis induced by system

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XC
inhibition through sustaining intracellular GSH in a p53-21- ovarian tumor growth (Basuli et al., 2017). Two erastin analogs,
dependent axis (Tarangelo et al., 2018). This might give cancer piperazine erastin and imidazole ketone erastin (IKE), effectively
cells a survival benefit under metabolic stress conditions suppress tumor growth in experimental models for fibrosarcoma
such as cystine deprivation. Another mechanism was proposed and diffuse large B cell lymphoma (Larraufie et al., 2015; Yang
through blocking dipeptidyl-peptidase-4 (DPP4) activity by p53, et al., 2014; Zhang et al., 2019). The system XC
inhibitor sulfa-
which blocks erastin-induced ferroptosis in colorectal cancer salazine, an FDA-approved immunosuppressant used for the
cells (Xie et al., 2017). In the absence of p53, the DPP4 interacts treatment of some types of arthritis, inhibits the growth of lym-
with NOX1, forming a NOX1-DPP4 complex that mediates phomas, pancreatic tumors, and lung tumors in experimental
plasma membrane lipid peroxidation and ferroptosis. cancer models (Gout et al., 2001; Guan et al., 2009; Lo et al.,
Sensitivity of Therapy-Resistant Cancer Cells to 2010). However, the poor pharmacokinetics, low potency, and
Ferroptosis metabolic stability of sulfasalazine limit its efficacy of system
Triggering ferroptosis is one of the best ways to circumvent XC
targeting and its therapeutic application (Azadkhan et al.,
therapy-resistant cancer cells. For example, therapy-resistant 1982; Dixon et al., 2014; Robe et al., 2009). Sorafenib, an FDA-
mesenchymal cells, typically characterized by high zinc finger approved anti-cancer drug for treatment of hepatocellular and
E-box-binding homeobox 1 (ZEB1) expression, seem highly renal cell carcinoma and thyroid cancer, was also identified as
sensitive for ferroptosis induced by GPX4 inhibition or statin a class I FIN (Dixon et al., 2014; Sun et al., 2017). Early inhibition
treatment (Viswanathan et al., 2017). ZEB1 is hypothesized to of GSH synthesis by BSO upon tumor onset also reduces the
act as a lipogenic factor and to regulate lipid metabolism, mammary tumor burden in mice (Harris et al., 2015) and sensi-
providing a bridge between mesenchymal gene expression tizes melanoma and neuroblastoma cells to the chemothera-
and lipid peroxide vulnerability. Similarly, the survival of drug- peutic agent melphalan (Anderson and Reynolds, 2002; Ongaro
tolerant persister cells, which are a major challenge in cancer et al., 2015). Finally, as a genetic approach to increase the effi-
treatment, also relies on GPX4 for survival, which makes cacy of GSH depletion, an optimized human cystathionine g-
GPX4 an ideal target to induce ferroptosis (Hangauer et al., lyase (CGL), coined cyst(e)inase, was engineered to degrade
2017). GPX4 is also shown to be required for tumor relapse cysteine and cystine with a higher kinetic rate. This approach im-
in a melanoma xenograft model (Hangauer et al., 2017). The pedes the growth of prostate and breast cancer xenografts and
BRAF kinase inhibitor vemurafenib also sensitizes persistent increases mouse survival in a chronic lymphocytic leukemia
melanoma cells to ferroptosis induced by several triggers model (Cramer et al., 2017).
(Tsoi et al., 2018). In therapy-resistant high-risk neuroblastoma, Class II and III FINs
ferroptosis also seems to be effective. We identified that simul- Higher levels of SLC7A11 have been detected in several types of
taneous induction of canonical and non-canonical ferroptosis human cancers, which suggests system XC
inhibitors as prom-
by withaferin A efficiently kills a heterogeneous panel of high- ising anti-cancer agents. However, it can also represent a hurdle
risk neuroblastoma cells, suppresses tumor growth, and shows limiting the potency of system XC
inhibitor in some cancer con-
superior activity in relation to the relapse rate of neuroblastoma texts (Huang et al., 2005; Jiang et al., 2015). Upregulation of
xenografts compared with etoposide or cisplatin (Hassannia heat-shock protein HSPB1 in cancer cells upon erastin treat-
et al., 2018). ment confers resistance to ferroptosis induced by class I FINs
(Sun et al., 2015). Inhibition of GSH synthesis downstream of
Anti-cancer Therapeutic Potential of Ferroptosis- system XC
fails to induce ferroptosis in some cells, presumably
Inducing Compounds due to upregulation of GSH-independent thioredoxin (Txn) anti-
Four ways of initiating ferroptosis have been discovered (Feng oxidant pathway (Harris et al., 2015; Mandal et al., 2010). The
and Stockwell, 2018). Class I ferroptosis inducers (FINs) act thioredoxin system has been suggested to preserve the endog-
by depleting GSH, class II FINs directly target and inactivate enous vitamin E in a reduced state by preventing it from oxidative
GPX4, class III FINs deplete GPX4 and CoQ10 via the SQS-me- loss (May et al., 2002). Thus, because some cancer cells adapt to
valonate pathway, and class IV FINs induce lipid peroxidation resist the activation of ferroptosis by class I FINs, targeting of
by increasing the LIP or oxidizing iron. GPX4 by class II or III FINs (Table 1; Figures 2B and 2C) is gaining
Class I FINs interest for triggering ferroptosis as an anti-cancer strategy.
It is conceivable that abrogation of anti-oxidant systems of can- Among class II FINs, RSL3 has been shown to inhibit the growth
cer cells makes them more vulnerable to oxidative damage and of fibrosarcoma in a mouse model (Yang et al., 2014). Withaferin
cell death (Trachootham et al., 2009). The anti-oxidant GSH has A suppresses neuroblastoma xenograft growth and relapse rate
been considered as cancer’s Achilles’ heel (Balendiran et al., (Hassannia et al., 2018). Importantly, altretamine, an FDA-
2004; Traverso et al., 2013). System XC
and the transsulfuration approved anti-neoplastic drug that is used for ovarian cancer
pathway are the two main sources of cysteine for GSH synthesis treatment, induces ferroptosis through GPX4 inhibition (Woo
and maintenance of intracellular thiol redox potential. In some et al., 2015).
cancers, epigenetic silencing or defects in the transsulfuration Class IV FINs
enzymes has been reported, which makes them exclusively Cancer cells have been shown to have higher iron requirements
dependent on system XC
for their cystine uptake (Jeschke than normal, non-cancer cells, often referred to as ‘‘iron addic-
et al., 2013; Yang et al., 2014; Yang and Stockwell, 2016; Zhao tion.’’ Essentially this makes cancer cells more vulnerable for
et al., 2012). Thus, system XC
might be a good therapeutic ferroptosis induction upon boosting the LIP, and might provide
anti-cancer target. Among class I FINs, erastin regresses tumors new opportunities in cancer therapy. These class IV FINs induce
in cervical carcinoma xenografts (Sun et al., 2015) and inhibits lipid peroxidation through increasing the LIP or oxidizing iron.

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Interestingly, the sensitivity of renal carcinoma to ferroptosis is Perspectives

proposed to be related to its iron-enriched tumor environment Since the discovery of ferroptosis, compelling evidence
(Yang and Stockwell, 2016). Upregulation of the iron-starvation has shown anti-cancer features from targeting ferroptosis in
response in combination with treatment with sulfasalazine or experimental cancer models. Vulnerabilities of therapy-resistant
BSO reduces the growth of lung tumors in a mouse xenograft cancers to ferroptosis and the recognition of the FDA-approved
model (Alvarez et al., 2017). In addition to inactivation of GPX4, drugs altretamine, sorafenib, and silica nanoparticles as ferropto-
withaferin A induces ferroptosis in neuroblastoma by increasing sis inducers in cancer create high expectations for the therapeutic
the LIP through HMOX1-mediated degradation of heme (Has- potential of ferroptosis. However, many questions still remain that
sannia et al., 2018). Hemin and ferrous ammonium sulfate induce need further clarification. What is the specificity and how can po-
iron accumulation in cells, which causes ferroptosis in neuro- tential adverse effects of ferroptosis induction in preclinical and
blastoma cells (Hassannia et al., 2018). BAY 87-2243, a well- clinical cancer settings be controlled? To what extent does the
known IkBa inhibitor, induces ferroptotic death in cancer cells mutation profile affect the sensitivity to ferroptosis? How can tu-
in a nuclear factor kB-independent manner through upregulation mors be sensitized to ferroptosis using epigenome editing? How
of HMOX1 and iron accumulation (Chang et al., 2018). FINO2, an immunogenic is ferroptosis? How effective is ferroptosis induction
endoperoxide-containing 1,2-dioxolane, triggers ferroptosis in compared with immunotherapy? What is the plasticity of the lipid
cancer cells through GPX4 inactivation and iron oxidation metabolism of cancer cells for the control of ferroptosis sensitivity,
(Gaschler et al., 2018a). Combination of a kinase tyrosine inhib- as recently suggested (Vriens et al., 2019)? Considering the effec-
itor lapatinib, used for breast cancer treatment, and the lysoso- tiveness of GPX4 inhibitors in therapy-resistant cancers, it will be a
motropic drug siramesine, which destabilizes lysosomes, syner- challenge to develop and optimize GPX4-targeting compounds
gistically induces ferroptosis through iron transport disruption in with improved pharmacokinetics and targeting. In this regard,
breast cancer cells (Ma et al., 2016). Pushing iron overload, using nanomedicine approaches could provide opportunities for devel-
iron oxide nanoparticles, has been shown to induce ferroptosis oping ferroptosis therapeutics with higher efficacy and improved
in nutrient-deprived cancer cells and suppresses tumor growth targeting and, hence, lower overall systemic toxicity and
(Kim et al., 2016). increased safety. In addition to the anti-cancer context, what is
Nanomedicine the biological function of ferroptosis and why is it evolutionarily
An emerging anti-cancer strategy to induce ferroptosis is the conserved? To what extent is lipid peroxidation functionally
use of nanomedicines (Shen et al., 2018). The increasing num- linked to ferroptosis? Is lipid peroxidation the cause of mem-
ber of FDA-approved anti-cancer nanomedicines underscores brane permeabilization or are lipid adduct-induced membrane
this emerging field with the goal of developing therapeutics protein modifications required to induce functional changes in
with higher efficacy and improved safety and toxicological pro- channels, pores, or receptors? Do current fluorescence-based
files (Bobo et al., 2016). The iron oxide nanoparticles kill cancer techniques for lipid peroxide detection, on which most mech-
cells through elevation of iron levels and ROS. Hydrolysis of anistic studies rely, accurately and precisely measure cellular
iron oxide nanoparticles following uptake and degradation in lipid peroxides? What is the therapeutic potential of lipophilic
the acidic environment of cancer cells can release intracellular radical traps for the control of lipid ROS, lipid peroxidation,
iron that enhances Fenton-like reactions and ROS generation. and ferroptosis-induced damage? More detailed mechanistic
In this regard, sensitivity of cancer cells to cisplatin is increased insights are needed as well as real-time diagnostic tools to
by loading cisplatin prodrug onto iron oxide nanoparticles detect ferroptosis in response to improved strategies to com-
(FePt-NP2) (Ma et al., 2017). The singlet oxygen (1O2) kills can- bat the ‘‘rust’’ of therapy-resistant cancers as a stepping
cer cells after internalization of iron-based nanoparticles teth- stone toward translation into the clinic.
ered with LA hydroperoxide (Zhou et al., 2017). Metal organic
network nanoparticles, encapsulated with p53 plasmid by
inducing Fenton reactions combined with p53-mediated inhibi- ACKNOWLEDGMENTS
tion of GSH synthesis, produce lipid peroxides, which leads to
We thank Martin Bronwen for editing. We apologize for not referencing all
ferroptosis and inhibition of tumor growth (Zheng et al., 2017). relevant reports due to limitations on the number of references. B.H. is post-
The FDA-approved ultra-small poly(ethylene glycol)-coated sil- doctoral researcher at Ghent University and the IRC. T.V.B. is assistant profes-
ica nanoparticles, coined Cornel dots (C0 dots), induce ferropto- sor at the University of Antwerp, guest professor at Ghent University, and team
leader at the VIB Center for Inflammation Research (IRC). His Ferroptosis
sis in starved cancer cells and suppress tumor growth. C0 dots
and Inflammation Research (FAIR) lab at Ghent University and at the IRC
were found to absorb and incorporate iron from the extracel- is supported by Excellence of Science (EOS 30826052 MODEL-IDI),
lular environment into their structure (Kim et al., 2016). Nano- Research Foundation Flanders (FWO G0B7118N), VLIR-UOS (grant number:
medicine-based strategies can also be used to deliver FINs TEAM2018-SEL018), Charcot Foundation, Stichting Tegen Kanker (FAF-
C/2018/1250), Ghent University, and VIB. His pathophysiology lab at the
to cancer cells and circumvent systemic toxicity. For example, University of Antwerp is part of a consortium of excellence focusing on inflam-
a nanoparticle formulation of withaferin A promoted its tumor mation (INFLA-MED), has frequent partnerships with international pharma and
accumulation due to an increased permeability and retention is part of national and European research programs such as TOP-BOF (32254)
and FWO (G0C0119N). P.V. is senior full professor at Ghent University and
(EPR) effect and suppressed tumor growth in neuroblastoma senior PI at the VIB IRC. His research is supported by Belgian grants
(Hassannia et al., 2018). Similarly, the employment of IKE (EOS 30826052 MODEL-IDI), Flemish grants (Research Foundation
nanoparticles resulted in increased accumulation of IKE in Flanders: FWO G.0C31.14N, G.0C31.14N, G.0C37.14N, FWO G0E04.16N,
diffuse large B cell lymphoma tumors and suppressed the tu- G.0C76.18N, G.0B71.18N), grants from Ghent University (BOF16/
MET_V/007 Methusalem grant), Foundation against Cancer (FAF-F/2016/
mor growth in mice with reduced toxicity compared with free 865), and VIB. Some illustration templates used in the figures were derived
IKE (Zhang et al., 2019). from www.somersault1824.com.

Cancer Cell 35, June 10, 2019 845

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