cancers

Review
Ferroptosis in Cancer Cell Biology
Christina M. Bebber 1,2,3 , Fabienne Müller 1,2 , Laura Prieto Clemente 1,2 , Josephine Weber 1,2
and Silvia von Karstedt 1,2, *
1 Department of Translational Genomics, Medical Faculty, University of Cologne, Weyertal 155b,
50931 Cologne, Germany; christina.bebber@uk-koeln.de (C.M.B.); fabienne.mueller@uk-koeln.de (F.M.);
lprietoc@uni-koeln.de (L.P.C.); jweber38@smail.uni-koeln.de (J.W.)
2 Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases (CECAD),
Medical Faculty, University of Cologne, Joseph-Stelzmann-Straße 26, 50931 Cologne, Germany
3 Department I of Internal Medicine, University Hospital of Cologne, Kerpener Straße 62,
50937 Cologne, Germany
* Correspondence: s.vonkarstedt@uni-koeln.de; Tel.: +49-(0)221-4788-4340




Received: 29 November 2019; Accepted: 7 January 2020; Published: 9 January 2020


Abstract: A major hallmark of cancer is successful evasion of regulated forms of cell death.
Ferroptosis is a recently discovered type of regulated necrosis which, unlike apoptosis or necroptosis,
is independent of caspase activity and receptor-interacting protein 1 (RIPK1) kinase activity. Instead,
ferroptotic cells die following iron-dependent lipid peroxidation, a process which is antagonised
by glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1). Importantly,
tumour cells escaping other forms of cell death have been suggested to maintain or acquire sensitivity
to ferroptosis. Therefore, therapeutic exploitation of ferroptosis in cancer has received increasing
attention. Here, we systematically review current literature on ferroptosis signalling, cross-signalling
to cellular metabolism in cancer and a potential role for ferroptosis in tumour suppression and
tumour immunology. By summarising current findings on cell biology relevant to ferroptosis in
cancer, we aim to point out new conceptual avenues for utilising ferroptosis in systemic treatment
approaches for cancer.

Keywords: ferroptosis; cancer; cell death; GPX4; inflammation

1. Introduction
The emergence of electron transport chains as a means to generate a chemical gradient for
the generation of adenosine triphosphate (ATP) is as ancient as the first single-celled organism
undergoing photosynthesis to produce oxygen. Billions of years of evolution later, multicellular aerobic
organisms generate ATP mainly by oxidative phosphorylation (OXPHOS) at the expense of atmospheric
oxygen [1]. Herein, protein complexes belonging to the electron transport chain are located in the
inner mitochondrial membrane where they transport electrons derived from nicotinamide adenine
dinucleotide hydrogen (NADH) via redox reduction to the terminal electron acceptor oxygen (O2 ),
which is thereby reduced to water (H2 O). NADH is one of the major products of the tricarboxylic
acid (TCA) cycle within the mitochondrial lumen driven by metabolites such as Acetyl-CoA derived
from glycolysis and catabolic fatty acid oxidation (β-oxidation) [1]. During electron transport, a small
proportion of electrons leak out and react with oxygen molecules to generate highly reactive superoxide
(O·−2 ) [2]. Superoxides can be transported into the cytosol using the mitochondrial permeability
transition pore (mPTP) in the outer mitochondrial membrane. Thereby, OXPHOS is a major source
of reactive oxygen species (ROS) in aerobic cells which can cause aberrant oxidation of proteins,
lipids and DNA.

Cancers 2020, 12, 164; doi:10.3390/cancers12010164 www.mdpi.com/journal/cancers

Cancers 2020, 12, 164 2 of 24

Therefore, cells have evolved to develop a complex cellular antioxidant defence system to ensure
cellular survival. As such, superoxide dismutases (SOD1), localised either in the cytoplasm or in
the mitochondrial matrix (SOD2), catalyse the dismutation of superoxides (O·−2 ) generated during
OXPHOS into slightly less reactive hydrogen peroxide (H2 O2 ) and water (H2 O) [2]. H2 O2 is then further
reduced by catalases, glutathione peroxidases (GPXs) or peroxiredoxins (PRDXs) [3]. Many antioxidant
defence proteins including SOD1, catalase and glutathione peroxidase 4 (GPX4) [4] are transcriptionally
induced by the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (NRF2)
which is activated upon oxidative stress-induced degradation of its negative regulator kelch-associated
protein 1 (KEAP1) [5]. Yet, in the presence of redox-active metals such as divalent iron (Fe2+ ) catalysing
the Fenton reaction, hydroxyl radicals (HO·) are generated from hydrogen peroxide (H2 O2 ) [6]. Hence,
limiting the availability of free divalent redox-active metals via sequestration within metal-binding
proteins is an additional integral part of the cellular antioxidant defence machinery [7].
Paradoxically, despite OXPHOS being the most efficient way to generate ATP, many cancer cells
have undergone metabolic reprogramming, wherein they mainly generate ATP from cytosolic aerobic
glycolysis coupled to lactate fermentation. This metabolic re-programming in cancer was famously
discovered by Warburg and Cori in the 1920s [8,9] and has been suggested as a cancer cell means to
evade toxic levels of ROS production. However, maintenance of this Warburg effect requires higher
glucose uptake and elevated metabolic activity making tumour cells nevertheless heavily reliant on
the antioxidant machinery and maybe even more susceptible to oxidative stress [10,11]. Therefore,
highly proliferative cancer cells are known to require handling of elevated cellular ROS levels in order
to successfully establish tumours [12–14].
One of the most important hallmarks of cancer is the efficient evasion of regulated cell death.
Recently, ferroptosis, a new, yet potentially evolutionary ancient type of regulated necrosis which is
triggered upon collapse of a lipid radical-specific antioxidant defence system was described [15] (recently
also reviewed [16–20]). Whereas apoptosis, necroptosis and pyroptosis are all either directly dependent
on caspases or inhibited by their activity (reviewed in-depth elsewhere [21]), ferroptosis seems to have
evolved separately with, as of yet, very little known direct molecular cross-talk to other pathways of
regulated cell death [21]. As the study of ferroptosis is a relatively young, yet rapidly growing field,
here, we will provide an updated systemic overview on processes regulating ferroptosis and potential
outcomes in cancer.

2. Ferroptosis Pathway Regulation

One hallmark of ferroptosis is the requirement for iron, as demonstrated by the fact that chelation
of iron by deferoxamine (DFO) rescues experimental induction of ferroptosis [15]. Consequently,
transferrin, which binds free ferric iron (Fe3+ ) and shuttles it into cells, was shown to regulate
ferroptosis [22]. Once Fe3+ is imported, endosomal six-transmembrane epithelial antigen of prostate
3 (STEAP3) catalyses the reduction to divalent iron (Fe2+ ) and releases it to the cellular labile iron
pool through the divalent metal transporter 1 (DMT1) [23]. Interestingly, DMT1 has been shown to be
up-regulated upon ferroptosis-induction [24].
Another hallmark is the characteristic accumulation of membrane lipid peroxides preceding
cell death [15]. Lipid peroxides were recently modelled to destabilise the lipid bilayer resulting in
disintegration of cellular membranes in silico [25]. Through the use of lipidomics, arachidonic acid
(AA)- and adrenic acid (AdA)-containing phosphatidylethanolamine (PE) species were identified as
in vivo lipid products of ferroptosis [26]. These lipids can undergo spontaneous peroxidation in the
presence of hydroxyl radicals (HO·) generated from Fenton reactions of redox active divalent iron (Fe2+ )
and hydroperoxide (H2 O2 ). Hydroxyl radicals (HO·) can react directly with polyunsaturated fatty
acids (PUFAs) in membrane phospholipids which can trigger a chain reaction of lipid ROS attacking
proximal PUFAs. Alternatively, divalent iron can serve as a cofactor for lipoxygenase (LOX) to catalyse
PUFA peroxidation enzymatically [27]. PUFAs are especially sensitive to lipid peroxidation due
to the presence of highly reactive hydrogen atoms within methylene bridges [28]. Interestingly,
Cancers 2020, 12, 164 3 of 24

4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) are fairly specific lipid peroxidation
by-products, which have frequently been used as general markers of oxidative stress in tissue
sections. Acyl-CoA synthetase long-chain family member 4 (ACSL4) mediates esterification of AA
and AdA with coenzyme A (CoA) forming Acyl-CoA which can then undergo either ß-oxidation or
anabolic PUFA biosynthesis [29–31]. Importantly, ACSL4 was identified to be required for cells to
undergo ferroptosis by generating the lipid target pool for peroxidation [20,29]. In a similar manner,
lysophosphatidylcholine acyltransferase 3 (LPCAT3) contributes to ferroptosis by incorporation of
AA into phospholipids of cellular membranes thereby contributing to substrate generation for lipid
peroxidation [29,32,33]. Together, these findings demonstrate that PUFA synthesis and peroxidation is
an essential prerequisite for cells to die via ferroptosis.
Vice versa, GPX4 was shown to constitutively hydrolyse lipid hydroperoxides and thereby
serve cellular protection from ferroptosis [34]. Antagonising GPX4 with the small molecule inhibitor
rat sarcoma viral oncogene homolog (RAS)-selective lethal 3 (RSL3) led to efficient induction of
ferroptosis [15]. GPX4 requires glutathione (GSH) as an electron donor to reduce lipid hydroperoxides.
GSH is an abundant cellular tripeptide consisting of glycine, glutamate and cysteine and is utilised as
one of the major cellular non-protein antioxidants [35]. GSH synthesis depends on the availability
of intracellular cysteine which can be generated from cystine imported from the extracellular space
via the sodium-independent cystine/glutamate antiporter System xc-. System xc- is a heterodimer
consisting of a heavy chain (4F2, gene name SLC3A2) and a light chain (xCT, gene name SLC7A11) [36].
Interestingly, xCT, the subunit decisive for specific amino acid antiport was shown to be a molecular
target of the small molecule eradicator of RAS and ST-expressing cells (erastin) and the resulting
cystine depletion triggered ferroptosis [37,38].
Recently, ferroptosis suppressor protein 1 (FSP1), formerly called apoptosis-inducing factor
mitochondria associated 2 (AIFM2), was identified as another ferroptosis protective factor in a
CRISPR-Cas9 knockout screen for synthetic lethality with the GPX4 small molecule inhibitor RSL3 [39]
and a cDNA overexpression screen complementing for GPX4 loss [40]. Both studies reported that FSP1
is recruited to the plasma membrane by N-terminal myristoylation, where it acts as an oxidoreductase,
reducing ubiquinone (=Coenzyme Q10) to the lipophilic radical scavenger ubiquinol which limits
accumulation of lipid ROS within membranes in the absence of GPX4. Hence, ubiquinol generated by
FSP1 acts as an endogenous functional equivalent of the described small-molecule lipophilic radical
scavengers ferrostatin-1 (Fer-1) and liproxstatin-1 inhibiting ferroptosis [15]. Interestingly, in hundreds
of cancer cell lines, FSP1 expression correlated with ferroptosis resistance in non-haematopoietic cancer
cell lines, yet most significantly in lung cancer cells, suggesting upregulation of FSP1 to be a strategy of
ferroptosis escape in cancer [40,41].

3. Ferroptosis and Mitochondria

Mitochondria are indispensable for most normal cell types due to their role in generating ATP
through OXPHOS [22,42]. However, this process comes at a cost of ROS production as a byproduct
of OXPHOS [43]. Mitochondria are involved in the execution of various types of regulated cell
death such as extrinsic and intrinsic apoptosis and autophagy, thereby playing a central role in tissue
homeostasis [44,45]. Interestingly, experimental induction of ferroptosis through pharmacological
inhibition of xCT was shown to induce mitochondrial fragmentation, mitochondrial ROS production,
loss of the mitochondrial membrane potential (MMP) and ATP depletion [18,42,46–49]. Supporting a
requirement for mitochondrial metabolism in the execution of ferroptosis [47], depletion of mitochondria
via Parkin-mediated mitophagy in vitro or inhibition of OXPHOS rescued cells from ferroptosis
induced by cystine deprivation or erastin [42]. Yet, in the initial characterisation of ferroptosis,
mitochondrial DNA (mtDNA)-depleted ρ0 cells remained sensitive to oxidative stress and ferroptosis
induction [15]. Hence, whether or not mitochondria are involved in ferroptosis across all cell types is
still controversial and there may be cell-specific differences similar to a type I and type II cell scenario
as described for extrinsic apoptosis [50]. Of note, the Bcl-2 family member BH3-interacting domain
Cancers 2020, 12, 164 4 of 24

death agonist (BID), whose cleavage into truncated Bid (tBID) is known to be essential during extrinsic
apoptosis in type II cells, was shown to be required for erastin-induced ferroptosis and oxytosis in
neurons [46].

3.1. The Upstream and Downstream of Mitochondria in Ferroptosis

Interestingly, there seems to be a marked difference in the requirement for mitochondrial
metabolism in the execution of ferroptosis depending on the strategy by which ferroptosis is triggered.
When triggered by cystine starvation or by erastin, resulting in GSH depletion, activity of the
mitochondrial TCA cycle was necessary for ferroptosis induction [22]. In fact, cancer cells deficient
for the mitochondrial tumour suppressor fumarate hydratase (FH), a metabolic enzyme of the TCA
cycle, were unable to undergo ferroptosis upon cystine deprivation [42]. Yet, when GPX4 was
pharmacologically inhibited or deleted, cells underwent ferroptosis regardless of the TCA cycle,
functional OXPHOS or mitochondria suggesting GPX4 activity required for ferroptosis prevention to lie
downstream of mitochondria [42]. Supporting this idea, mitochondrial damage and mitochondrial ROS
production are events taking place during the execution of ferroptosis upon inhibition of xCT or cystine
starvation but are not necessary for GPX4 inhibition-induced ferroptosis [42,46]. Yet, arguing against
an exclusive role of GPX4 downstream of mitochondria, apoptosis-inducing factor (AIF), which is
associated with the inner mitochondrial membrane, translocates from mitochondria to the nucleus
contributing to ferroptotic death upon GPX4 deletion suggesting a certain level of mitochondrial
permeability during ferroptosis [51]. This permeability, however, is Bcl-2 associated X protein (BAX)
and Bcl-2 homologous antagonist/killer (BAK1) independent, as cells deficient for both proteins were
able to undergo ferroptosis [15].
Interestingly, GPX4 is expressed as three different splice variants before processing, which leads
to initial sorting to different cellular compartments [52], whilst, after processing, these isoforms are
identical. A signal peptide within the long isoform (lGPX4) leads to mitochondrial import, the short
form (sGPX4) is found in the cytosol, mitochondria and microsomes and the nuclear isoform (nGPX4) in
the nucleus [52]. Thereby, through these different localisations, GPX4 may fulfil compartment-specific
functions which may account for the conflicting results obtained regarding mitochondrial involvement
in ferroptosis after GPX4 deletion. Importantly, apart from its role in ferroptosis which has gained much
attention recently, GPX4 was previously shown to preserve mitochondrial ATP generation by protecting
mitochondria from ROS build-up during tert-Butyl hydroperoxide (TBHP)-induced cell death [53].
Taken together, GPX4, or at least a particular isoform thereof, seems to exert an important function in
the prevention of ferroptosis independently of mitochondria, and its role in protecting mitochondrial
metabolism from ROS-induced membrane damage may also feed into its ferroptosis-protective activity
(summarised in Figure 1).
Cancers 2020, 12, 164 5 of 24
Cancers 2020, 12, x 5 of 23

Figure 1. Schematic view of the ferroptosis pathway. Ferroptosis pursues upon aberrant build-up of
Figure 1. Schematic view of the ferroptosis pathway. Ferroptosis pursues upon aberrant build-up of lipid
lipid reactive oxygen species (ROS) leading to peroxidation (-OOH) of polyunsaturated fatty acids
reactive oxygen species (ROS) leading to peroxidation (-OOH) of polyunsaturated fatty acids (PUFAs).
(PUFAs). Main peroxidation target PUFAs are arachidonic acid (AA) phosphatidylethanolamine (PE)
Main peroxidation target PUFAs are arachidonic acid (AA) phosphatidylethanolamine (PE) lipid species
lipid species within cellular membranes leading to membrane destabilisation and rupture. Lipid
within cellular membranes
peroxidation can be triggeredleading bytocytosolic
membrane destabilisation
redox active iron (Fe and2+) rupture.
shuttled Lipid peroxidation
into cells bound to can
be triggered by cytosolic redox active iron (Fe 2+ ) shuttled into cells bound to transferrin via transferrin
transferrin via transferrin receptor (TFRC) endocytosis and endosomal release mediated by divalent
receptor
metal(TFRC) endocytosis
transporter 1 (DMT1). and endosomal
In the presence release
of H2O2,mediated by divalent
Fe2+ catalyses hydroxylmetalradicaltransporter 1 (DMT1).
(HO∙) generation
In the presence of H O , Fe 2+ catalyses hydroxyl radical (HO·) generation in a Fenton reaction, which
in a Fenton reaction,2 2 which sets of a radical lipid peroxidation chain reaction. Lipoxygenase (LOX)
sets can
of aequally
radicalcatalyse
lipid peroxidation
lipid peroxidation chainusing
reaction. Lipoxygenase
Fe2+. As (LOX) canforequally
a required prerequisite catalyse
ferroptosis, Acyl-lipid
peroxidation
CoA synthetase 2+
usinglong-chain
Fe . As afamily required prerequisite
member 4 (ACSL4)for andferroptosis, Acyl-CoA synthetase
lysophosphatidylcholine long-chain
acyltransferase 3
(LPCAT3)
family member generate
4 (ACSL4)the pool
andoflysophosphatidylcholine
AA-containing target lipids. Glutathione peroxidase
acyltransferase 3 (LPCAT3) 4 (GPX4), in turn,
generate the pool
hydrolyses lipidtarget
of AA-containing peroxides converting
lipids. them peroxidase
Glutathione into their respective
4 (GPX4),non-toxic
in turn, lipid alcohols (-OH).
hydrolyses GPX4
lipid peroxides
requiresthem
converting glutathione (GSH)
into their as a cofactor
respective which lipid
non-toxic upon alcohols
its oxidation (GSSG)
(-OH). GPX4by requires
GPX4 is reduced to GSH
glutathione (GSH)
by glutathione reductase (GR). GSH synthesis depends on glutamate cysteine
as a cofactor which upon its oxidation (GSSG) by GPX4 is reduced to GSH by glutathione reductase ligase (GCL) and
glutathione synthetase (GSS) as well as on intracellular cystine shuttled into the cell in exchange for
(GR). GSH synthesis depends on glutamate cysteine ligase (GCL) and glutathione synthetase (GSS) as
glutamate mediated by system xc- (SLC3A2 and SCL7A11/xCT). Independently of GSH, ferroptosis
well as on intracellular cystine shuttled into the cell in exchange for glutamate mediated by system xc-
suppressor protein 1 (FSP1) generates ubiquinol from ubiquinone which acts as a lipophilic radical
(SLC3A2 and SCL7A11/xCT). Independently of GSH, ferroptosis suppressor protein 1 (FSP1) generates
trapping agent within membranes thereby protecting from ferroptosis. Oxidative phosphorylation
ubiquinol from ubiquinone which acts as a lipophilic radical trapping agent within membranes thereby
(OXPHOS) and the tricarboxylic acid (TCA) cycle have both been described to be required for
protecting from
ferroptosis ferroptosis.
triggered Oxidative phosphorylation
by cystine-depletion or system xc- but (OXPHOS) and the tricarboxylic acid (TCA)
not GPX4 inhibition.
cycle have both been described to be required for ferroptosis triggered by cystine-depletion or system
xc- but not GPX4 inhibition.
Cancers 2020, 12, 164 6 of 24

3.2. Lipid Peroxidation in Mitochondria

GPX4 is one of the major intracellular enzymes involved in hydrolysing lipid peroxides,
thereby ensuring repair of lipid peroxide-perturbed cellular membranes. In a counter regulation
step, GPX4 inhibition and GSH depletion induces the activation of 12/15 lipoxygenases (12/15-LOX),
which are involved in lipid peroxidation [51,54]. Once 12/15-LOX are activated they can directly
attack the mitochondrial membrane inducing local lipid peroxidation in neurons [54–56]. Therefore,
although the current view of ferroptosis favours the idea of lipid peroxide accumulation in the plasma
membrane leading to cellular rupture [25], lipid peroxides have also been shown to accumulate in
the mitochondrial membrane during ferroptosis. In addition to 12/15-LOX regulating this process,
CDGSH iron sulfur domain 1 (CISD1) is an iron-containing protein whose N-terminus is inserted into
the outer mitochondrial membrane where it regulates mitochondrial iron uptake. Upon CISD1 deletion,
iron accumulation inside the mitochondria facilitates the generation of mitochondrial lipid peroxides
contributing to ferroptosis [31]. On the other hand, cholesterol hydroperoxide (ChOOH) species
(5alpha-OOH, 6alpha/6beta-OOH, 7alpha/7beta-OOH) and phospholipid hydroperoxide (PLOOH)
families (PCOOH, PEOOH, PSOOH) which are peroxidised in the cytosol can be transported to
mitochondria via the sterol carrier protein 2 (SCP-2) upon cystine deprivation [55,57]. This suggests a
potential role for these lipid peroxides in mitochondria during ferroptosis triggered via this route.
Taken together, mitochondrial metabolism is a main source of cellular ROS and contributes to
ferroptosis in many cellular systems. However, mechanisms of how ferroptosis and mitochondria
cross signal and whether xCT and GPX4 always signal in a single hierarchical pathway is poorly
understood. Therefore, additional studies on the mechanistic contribution of mitochondria in ferroptosis
are warranted.

4. Ferroptosis Discovery as an Oncogene-Selective Death

Although not named at the time, ferroptosis was first discovered as part of a synthetic
lethality screen for small molecules selectively targeting Harvey rat sarcoma viral oncogene homolog
(HRAS)G12V -mutant human foreskin fibroblasts (BJeLR) [58]. Next, the small molecules erastin [48] and
RSL3 [59] were described to induce an oncogenic RAS-specific oxidative type of cell death independently
of caspases. The observed HRASG12V selectivity was proposed to stem from an increased expression of
transferrin receptor TFRC and thereby increased intracellular iron levels in HRAS-mutant cells [59].
Moreover, Kirsten sarcoma viral oncogene homolog (KRAS)-mutant Calu-1 lung cancer cells exhibited
higher sensitivity to erastin and silencing of KRAS by small hairpin (sh) RNA reduced erastin’s efficacy
in these cells. Additionally, A-673 cells, which harbour an activating BRAFV600E mutation, became more
resistant to erastin treatment upon shRNA-mediated suppression of BRAF [48]. Supporting an increased
ferroptosis sensitivity of oncogene-expressing cells, human mammary epithelial (HME) cells expressing
mutant epithelial growth factor receptor (EGFR) were more sensitive to cystine deprivation-induced
ferroptosis through increased mitogen-activated protein kinase (MAPK) pathway activation [60].
Additionally, in non-small-cell lung cancer (NSCLC) cell lines, it was shown that the level of MAPK
pathway activity correlates with sensitivity to ferroptosis induced by cystine deprivation [60].
Apart from their potential influence on ferroptosis execution, oncogenic RAS isoforms and
induction of cellular ROS have been extensively studied. In this regards, oncogenic NRASG12D and
HRASG12V expression was shown to activate ROS production and to trigger a p38 mitogen-activated
protein kinase (MAPK)-mediated oxidative stress response [61]. Moreover, RAS-stimulated ROS
production was shown to be mediated via the activation of nicotinamide adenine dinucleotide
phosphate hydrogen (NADPH)-oxidases (NOX) regulated by the PI3K/Rac1 and RAF/MEK/ERK
RAS-effector pathways [62,63]. KRAS also induced translocation of p47phox , a subunit of NADPH
oxidase 1 (NOX1), to the plasma membrane thereby promoting its activation and aiding cellular
transformation [64]. Additionally, in the context of inactivation of the tumour suppressor p16, KRASG12V
expression also up-regulated NADPH oxidase 4 (NOX4) [65]. Thereby, oncogenic RAS isoforms may
Cancers 2020, 12, 164 7 of 24

also influence lipid peroxidation and ferroptosis through enhanced feeding into the general cellular
ROS pool.
Whilst these and other studies have demonstrated an induction of ROS under acute oncogenic
RAS overexpression in vitro, which may promote cellular transformation at the cost of elevated ROS,
the question remained how tumours expressing oncogenic KRAS from the endogenous locus would
handle ROS in vivo. Interestingly, expression of KRASG12D , BRAFV619E and MYCERT2 oncogenes
from their respective endogenous loci activated nuclear NRF2, a major transcription factor inducing
antioxidant defence genes [66,67]. Importantly, NRF2 is responsible for the positive regulation of
different genes involved in GSH synthesis including xCT, glutamate-cysteine ligase catalytic subunit
(GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) [68–70]. In addition, NRF2 promotes
expression of Ferritin (FTH) [71] which may act as a scavenger for redox active iron suggesting a
protective function of NRF2 in ferroptosis. In line with this suggestion, hepatocellular carcinoma
cells became more sensitive to ferroptosis inducers upon deletion or pharmacological inhibition
of NRF2 [71,72]. NRF2 interacts with KEAP1, a tumour suppressor protein, which also regulates
the expression of the ATP binding cassette (ABC)-family transporter multidrug resistance protein 1
(MRP1). Interestingly, MRP1 was shown to sensitise to ferroptosis by mediating glutathione efflux [73].
Additionally, it was shown that there is a high co-occurrence of KEAP1 and KRAS mutations in human
lung cancers which elevates cellular rates of glutaminolysis [74]. Increased glutamate shuttling into
the TCA cycle, namely glutaminolysis, may compete with glutamate requirement for cystine antiport
which might affect GSH levels. Yet, NRF2 was also shown to be activated by withaferin A leading to
induction of its bona-fide target gene heme oxygenase 1 (HMOX1) which causes an excess of cytosolic
labile iron through catalysing its release from haeme promoting ferroptosis in neuroblastoma [75].
Hence, NRF2 activation and target gene expression can lead to opposing outcomes for ferroptosis and
its actual effect on ferroptosis is possibly cell type specific.
Also arguing against mutant KRAS as a marker of ferroptosis sensitivity, artesunate (ART)
was shown to induce ferroptosis in an iron- and ROS-dependent manner in pancreatic ductal
adenocarcinoma (PDAC) cell lines irrespective of KRAS status [76]. Furthermore, erastin treatment
induced growth inhibition in acute myeloid leukaemia (AML) cells with an NRASQ61L mutation
(HL-60), but not in other cell lines harbouring RAS mutations (e.g., NRASG12D or KRASA18D ) or RAS
wild type (WT) suggesting other genetic or non-genetic factors influencing ferroptosis sensitivity [77].
Moreover, there is evidence suggesting that oncogenic KRAS mutations may in fact protect cells from
ferroptosis. In this regards it was shown that rhabdomyosarcoma cells (RMS13) expressing either
NRASG12V , HRASG12V or KRASG12V were more resistant to ferroptosis than respective empty vector
control cells [78]. Interestingly it was recently shown that fibroblasts, expressing oncogenic KRASG12V ,
are protected from hydrogen peroxide-induced cell death by up-regulating xCT which allows for
KRAS-induced tumourigenicity in vivo [79].
It was demonstrated that the mitochondrial TCA cycle is required for ferroptosis [42]. Interestingly,
a shift in the metabolic pathway in KRAS-driven cancers can enhance different characteristics like
glutaminolysis, glycolysis or nutrient uptake and thereby affect the TCA cycle [80]. Moreover, NADPH
is needed to maintain the TCA cycle, fatty acid synthesis and glutamine metabolism and is used by
glutathione reductase (GR) to reduce oxidised GSSG to GSH. Of note, KRAS was shown to elevate
NADPH levels through metabolic reprogramming which may enable an improved rate of GSH
regeneration and ferroptosis protection [81,82].
Intriguingly, cells expressing homozygous KRASG12D/G12D demonstrated upregulation of genes
related to glycolysis, enhanced glucose consumption and significantly increased levels of TCA cycle
enzymes. Moreover, it was shown that homozygous KRAS mutant cells shuttle glucose towards
glutathione synthesis, again suggesting that these cells may be more protected from ROS and thereby
more resistant to ferroptosis [83].
Apart from oncogene status, in a panel of 117 cancer cell lines from different tissues, sensitivity to
erastin-induced cell death was examined to define possible additional determinants of erastin sensitivity.
Cancers 2020, 12, 164 8 of 24

Interestingly, tissue origin was a much stronger predictor of ferroptosis sensitivity in cancer cell lines
than oncogene mutational status [34]. Within this study, diffuse large B-cell lymphoma (DLBCL) cell
lines were identified as particularly sensitive to ferroptosis [34]. Although, it was described that
suspension cells are more sensitive to small molecules which induce growth inhibition, this was
not the underlying mechanism for increased ferroptosis sensitivity in DLBCL which remains to be
addressed [34,84]. Apart from tissue origin, another important factor determining ferroptosis sensitivity
may be cellular differentiation. Interestingly, in melanoma, ferroptosis sensitivity did not correlate
with MAPK pathway activity but instead depended on the dedifferentiation status [34]. Moreover,
cells with an expression signature indicative of an epithelial-to-mesenchymal transition (EMT) were
more sensitive to GPX4 inhibitors [85]. Interestingly, high cell confluence inhibits ferroptosis via loss
of YAP-mediated transcriptional upregulation of TFRC and ACSL4, an experimental variable which
therefore needs to be tightly controlled in studies aiming for the identification of factors influencing
ferroptosis sensitivity [86].

5. In Vivo Relevance of Ferroptosis: Lessons Learned from Knockout Mice

One of the key bottlenecks for protection from ferroptosis is the availability of GSH, which serves as
a redox equivalent for GPXs including GPX4. As mentioned above, one important source of intracellular
cysteine for GSH synthesis is cystine imported via xCT (=Slc7a11). xCT is highly expressed in neurons
and brush border membranes of the kidney and duodenum [37]. Furthermore, it has been reported to
be expressed in the thyroid gland [87]. Expression can be induced by oxidative stress stimuli such as
hydrogen peroxide, oxygen and sodium arsenite leading to elevated GSH production [87,88]. In line
with a role in cystine import, xCT-deficient mice display increased cystine concentrations in blood
plasma and decreased intracellular GSH levels in comparison to WT mice. However, cellular cysteine
levels are comparable between knockout (KO) and WT littermates, suggesting compensatory cysteine
synthesis via the transsulfuration pathway [87]. Mouse embryonic fibroblasts (MEFs) isolated from
Slc7a11 KO mice fail to survive in cell culture except when supplemented with 2-mercaptoethanol or
N-acetyl cysteine (NAC) both described to serve as alternative cystine sources for cells. These data
suggest that cells grown in 2D culture in vitro may lack compensation via the transsulfuration pathway.
Interestingly, Slc7a11 KO mice are protected from neurotoxic insults induced by 6-hydroxydopamine
(6-OHDA), which can trigger Parkinson’s Disease (PD) via decreasing extracellular glutamate levels in
the brain [89].
During GSH synthesis, glutamate and cysteine are ligated by the glutamate-cysteine ligase (GCL)
forming the GSH precursor γ-glutamylcysteine (see Figure 1). GCL is a heterodimer consisting of
the catalytic subunit GCLC and the regulatory subunit GCLM [90]. Deletion of Gclc is embryonically
lethal before E8.5 as mice are unable to synthesise GSH [91,92]. Moreover, Gclc knockout embryos
fail to gastrulate and instead present with increased cell death assayed by TUNEL staining [92].
In contrast, Gclm-deficient mice develop normally but synthesise approximately 75–90% less GSH in
comparison to WT littermates, suggesting that either 10–25% of cellular GSH are sufficient for healthy
embryonic development or Gclc may fulfil additional functions during embryonic development [93].
Glutathione synthetase (GSS) catalyses condensation of glycine and γ-glutamylcysteine to generate
GSH. Mice deficient in Gss die at E7.5, whilst heterozygous littermates survive and do not display
an overt phenotype [94]. These data underline the important role of GSH synthesis for normal
embryogenesis and development and may in the case of Gclc-deficiency hint at a connection with cell
death prevention during embryonic development.
The transsulfuration pathway represents an alternative strategy for cysteine generation
from methionine, in which the cystathionine beta-synthase (CBS) processes homocysteine [95,96].
Cbs deletion in mice results in severe growth retardation and postnatal death 5 weeks after birth. This is
accompanied by elevated levels of the cysteine precursor homocysteine in plasma, generating a mouse
model for severe homocystinuria [97,98]. Interestingly, a recently developed CBS inhibitor has been
Cancers 2020, 12, 164 9 of 24

identified to induce ferroptosis in different cancer cell lines [99]. Yet, whether any aspect of the Cbs KO
phenotype is related to overt induction of ferroptosis is unknown.
Downstream of GSH synthesis, GPX4 is a GSH-dependent key regulator of the ferroptosis
pathway. Human GPX4 contains a selenocysteine encoded by a “Stop” codon within its catalytic
domain. GPX4 has been shown to be indispensable for embryogenesis as Gpx4 KO mice die in utero
at E7.5 and display abnormal organ compartmentalisation [100,101]. Interestingly, mice with an
inactivating serine exchange mutation in the enzymatically active selenocysteine of Gpx4 die at the
same stage of embryonic development whereas mice with heterozygous loss of selenocysteine within
Gpx4 are born but display defects in spermatogenesis [52,102]. Of note, mice containing a cysteine
instead of a selenocysteine in the catalytically active site of Gpx4 are viable but display seizures and
cell death in interneurons when on a mixed genetic background [103,104]. Whole-body inducible
knockout of Gpx4 increased oxidative stress and mitochondrial dysfunction in Gpx4-depleted organs
with decreased activity of electron transport chain members complex I and IV demonstrating that
Gpx4 is an important protector of mitochondrial integrity [105]. Adult mice die within two weeks
after systemic induction of Gpx4 KO caused by acute renal failure demonstrating its essential role
also for adult tissue homeostasis [55,105]. Interestingly, Gpx4 deletion in hematopoietic cells causes
receptor-interacting protein 3 (Rip3)-dependent cell death in erythroid precursor cells resulting in
anaemia in mice [106]. Mechanistically, the authors show that GPX4 deletion leads to glutathionylation
and inactivation of caspase 8, which triggers necroptosis independently of tumor necrosis factor
(TNF). Moreover, T-cell-specific deletion of Gpx4 resulted in T cell ferroptosis which was dependent on
receptor-interacting protein 1 (Rip1) and Rip3, suggesting that Gpx4 may be essential for T-cell mediated
immune responses [107]. Thereby, these two studies strongly suggest an interaction between ferroptosis
and necroptosis takes place in vivo. This has also recently been discussed in Florean et al. [19].
Mice harbouring a x-chromosomal deletion of the important PUFA-regulator Acsl4 are viable but
adipocyte-specific deletion results in decreased levels of PUFA-derived fatty acyl-CoAs which fuels
lipid peroxidation [108,109].
Thereby, protection from ferroptosis plays a crucial role for tissue homeostasis, whilst many
proteins involved in ferroptosis also fulfil metabolic functions unrelated to regulated cell death which
may be causative for some of the phenotypes seen in KO mouse models.

Cancer Mouse Models and Genetic Evidence for Ferroptosis in Cancer

In mouse models of mammary tumours, lymphomas and sarcomas, Gclm deficiency
leading to a significant reduction of cellular GSH caused impaired tumour initiation and
progression [110]. Moreover, murine MC38 colon cancer and Pan02 pancreatic cancer cells, harbouring
a CRISPR-Cas9-mediated Slc7a11 knockout displayed impaired in vivo tumour growth accompanied
by increased ROS levels and decreased GSH levels [111]. Interestingly, Arensman et al. showed
that while cystine uptake by Slc7a11 is essential for T cell viability and proliferation in vitro, it is
dispensable for T cells in vivo and does not impact T cell infiltration in tumours in immunocompetent
mice. These results show that targeting xCT unlike GPX4 [107] may represent an effective cancer
treatment strategy without compromising anti-tumour immunity. Recently, it has been demonstrated
that Gpx4 KO significantly impairs tumour growth upon Fer-1 withdrawal, whilst additional Fsp1 KO
enhances this effect [39].

6. Ferroptosis as a p53-Mediated Tumour-Suppressive Mechanism

Cell cycle arrest, cell death and senescence can, in many cases, serve tumour suppression. Therefore,
p53, a master regulator of cell cycle arrest and transcription of intrinsic apoptosis genes, has long been
assumed to exclusively suppress tumours via these mechanisms [112–114]. Surprisingly, mutating three
lysine residues within the DNA-binding domain of p53 (p533KR ), which prevents their acetylation, was
sufficient to suppress tumour growth in mice despite impaired functionality of this mutant in inducing
cell cycle arrest, senescence and apoptosis [115]. Strikingly, it was discovered that the p533KR mutant
Cancers 2020, 12, 164 10 of 24

was still capable of suppressing xCT transcription via direct promotor binding, thereby sensitising
cells to ferroptosis upon ROS-induced stress [116]. Hence, ferroptosis induction has been identified to
belong to the arsenal of tumour-suppressive activities of p53. Importantly, mutating a fourth identified
acetylation site in p53 (K98) in addition to the three previously described ones led to a complete loss of
its tumour suppressor activity. Importantly, this mutant had also lost the capacity to induce ferroptosis
sensitisation through xCT suppression, which indicates that the acetylation of the fourth acetylated
lysine residue K98 in p53 in addition to the other three sites is vital for tumour suppression and
ferroptosis induction by p53 [117]. Interestingly, cytosolic accumulation of p53 mutants typically found
in cancer mutants was shown to bind and thereby sequester NRF2, preventing nuclear translocation of
NRF2 and induction of its target genes including xCT [118]. These data might offer an explanation as to
how p53 mutants may indirectly promote suppression of xCT expression irrespective of DNA binding.
In addition, cytoplasmic accumulation preceding nuclear translocation may contribute to wild type
p53-mediated xCT suppression. Another study demonstrated that the INK4 locus alternative reading
frame (ARF) protein functions as a p53-independent tumour suppressor by limiting NRF2-mediated
xCT induction resulting in tumour growth suppression [68]. Thereby, ferroptosis sensitivity is also
linked to ARF status and its interaction with NRF2. Interestingly, APR-246, a clinical reactivator of
mutant p53 was shown to decrease intracellular glutathione levels through direct binding of cysteines
within GSH, leading to an increase of cellular ROS [119]. Yet, it remains to be established whether
APR-246-mediated decrease in cellular GSH also stems from p53-mediated xCT suppression and
resulting decrease of cystine import. Interestingly, a p53 Ser47 single nucleotide polymorphism (SNP)
was identified in a population of African descent which rendered cells more resistant to RSL3 treatment
and generated knock-in mice more prone to spontaneous tumour development [120]. This Ser47 variant
impaired phosphorylation on adjacent Ser46, which was important for p53 to induce spontaneous
cell death in different cell lines [121]. Moreover, a lack of this phosphorylation in Ser46 decreased
the ability of Ser47 to bind to p53 target genes which might explain decreased RSL3 sensitivity by
de-repression of xCT [120].
In contrast to the above-mentioned studies showing that p53 can induce ferroptosis through
suppression of xCT expression [116,118], in human colorectal cancer (CRC) cells, p53 was shown
to promote SLC7A11 expression [122]. Furthermore, the same study showed that loss of p53
inhibits accumulation of dipeptidyl-peptidase-4 (DPP4) in the nucleus, which results in enhanced
plasma-membrane associated DPP4-dependent lipid peroxidation via ROS-generating NOX enzymes
resulting in ferroptosis [122]. Thereby, loss of p53 may equally sensitise cells to ferroptosis in certain
contexts. In addition, treatment with the MDM2 inhibitor nutlin-3 stabilised WT p53, reduced cellular
ferroptosis sensitivity and induced p21 [123]. p21 upregulation in turn promoted intracellular
glutathione storage leading to reduced accumulation of lipid ROS also in the presence of p53
transcriptional activity [123].
Although many of these studies propose that there is a relationship between tumour suppression
and ferroptosis sensitivity, so far, there is no genetic evidence that p53 expression directly induces
ferroptosis and thereby mediates tumour suppression in vivo. Moreover, results obtained with loss of
p53 do not account for the NRF2 sequestering function seen for mutated p53 in the cytosol. Therefore,
future studies will have to unravel whether ferroptosis can suppress tumour growth and to what extent
it is part of the constitutive p53-mediated tumour-suppressive machinery.

7. Ferroptosis-Inducing Therapy (FIT) for Cancer Treatment

A breakthrough discovery for a potential use of ferroptosis-inducing therapy (FIT) for cancer
treatment came with the finding that cells with acquired resistance to the human epidermal growth
factor receptor (HER1/HER2) tyrosinkinase inhibitor Lapatinib, so called persister cells with a high
mesenchymal state, were selectively sensitised to the induction of ferroptosis [85,124]. These studies
for the first time pointed out that cells which had escaped other means of killing may be selectively
sensitised to ferroptosis. Moreover, immunotherapy was recently shown to sensitise to tumour cell
Cancers 2020, 12, 164 11 of 24

ferroptosis (see Section 9). Considering that xCT is often aberrantly expressed in many cancers [125,126],
ferroptosis induction may just prove to be a weak spot of cancer.
However, the two main ferroptosis inducers used in vitro, RSL3 and erastin, do not meet
pharmacokinetic standards for in vivo application yet due to poor water solubility and metabolic
instability [34,127,128]. To circumvent this problem, several efforts have been undertaken to render
erastin more suitable for in vivo application. In one approach, triple-negative breast cancer (TNBC)
cells, which highly express folate receptor, were treated with erastin packaged in exosomes covered
with folate to specifically target folate receptor-overexpressing TNBC [127]. Another metabolically
more stable form of erastin is piperazine-coupled erastin which has demonstrated anti-tumour activity
in a xenograft model using human fibrosarcoma HT-1080 cells [15,34]. In addition, imidazole-ketone
erastin, a metabolically stable variant of erastin, was shown to reduce tumour growth in a SU-DHL-6
DLBCL xenograft model [128].
Whilst small molecule backbones for RSL3 and erastin will have to be further optimised for clinical
application, interestingly, a number of food and drug administration (FDA)-approved drugs have been
identified to function via the induction of ferroptosis in different cancer entities:
Sorafenib, is an FDA-approved multi-kinase inhibitor for treatment of advanced renal cell
carcinoma (RCC) and advanced hepatocellular carcinoma (HCC). Molecularly, Sorafenib was shown
to inhibit system xc- [38]. Moreover, cell death induced in HCC by sorafenib was suppressed by
ferropstatin-1 and iron-chelators [129]. It has further been reported that the tumour suppressor
retinoblastoma protein (RB1) suppresses ferroptosis induced by sorafenib treatment [130] suggesting a
possible biomarker for sorafenib treatment-induced ferroptosis.
Sulfasalazine (SAS), an FDA-approved drug for the treatment of rheumatoid arthritis and
inflammatory bowel diseases (Crohn, ulcerative colitis), is thought to act as anti-inflammatory
drug [131]. Described targets of SAS are arachidonat-5-lipoxygenase (ALOX-5) [132], cyclooxygenase
2 (COX-2) [133] and nuclear factor ’kappa-light-chain-enhancer’ (NF-κB) [134]. It has also been shown
to inhibit the system xc- subunit xCT and effectively induce ferroptosis in non-Hodgkin lymphoma
cells [131].
Altretamine (hexamethylmelamine), an FDA-approved alkylating antineoplastic drug, is used
for the treatment of ovarian cancer [135]. It has also been shown to inhibit GPX4 and effectively kill
U-2932 DLBCL cells in vitro [136].
Statins, such as cerivastatin and simvastatin, have been shown to reduce the synthesis of Coenzyme
Q10 via blocking the mevalonate pathway and thereby induce ferroptosis in the human fibrosarcoma
cell line HT-1080 [85].
Thereby, the induction of ferroptosis may underlie treatment efficacies of several already approved
cancer drugs which may shorten clinical development for the concept of therapeutic induction of
ferroptosis in human cancers [17,19]. Which ones these are apart from the already identified ones
above will have to be discovered in the future. Current compounds known to induce ferroptosis are
summarised in Table 1.
Cancers 2020, 12, 164 12 of 24

Table 1. Ferroptosis-inducing drugs.

Reagent Mechanism of Action FDA Approved/Clinical Use Reference

(1S,3R)-RSL3 GPX4 inhibitor No [15,34,124,137]
Altretamine GPX4 inhibitor Yes/Ovarian cancer treatment [135,136]
Artesunate Glutathione S transferase No/Malaria treatment [76]
Mitochondrial complex I
BAY 87-2243 inhibitor/hypoxia-inducible factor-1 No [138,139]
(HIF-1) inhibitor
Buthionine No/Clinical trial for
γ-GCS inhibitor [34,110,137]
sulfoximine (BSO) neuroblastoma treatment
Cyst(e)inase [Cys] depletion No [140]
erastin System xc- inhibitor No
Gpx4 degradation/squalene synthase
FIN56 No [141,142]
activator
Imidazole-ketone
System xc-inhibitor No [128]
erastin
Piperazine erastin System xc-inhibitor No [15,34]
Yes/Renal cell, thyroid, and hepatocellular
Sorafenib System xc-inhibitor [38,129,130]
carcinoma treatment
Statins Block biosynthesis of CoQ10 Yes [85]
Yes/Rheumatoid arthritis and
Sulfasalazine System xc-inhibitor [24,131–134]
inflammatory bowel diseases treatment
Withaferin A Gpx4 inactivation/Keap1 inactivation No/Clinical trial for schizophrenia [75,142]

8. Ferroptosis and Inflammation in Pathology

A hallmark of regulated necrosis pathways is the loss of plasma membrane integrity [143].
This distinct feature of regulated necrosis allows for the release of cytosolic content and exposure
of this content to the surrounding tissue. Many of these released cytosolic factors function as
immunogenic signals, so called damage-associated molecular patterns (DAMPs) [144]. Not only
DAMPs but also alarmins that are released upon cell death or injury have the ability to induce
inflammatory immune responses [145]. Mechanistically, DAMPs and alarmins operate as ligands that
stimulate pattern recognition receptor (PRR)-expressing immune cells resulting in an inflammatory
process, termed necroinflammation if regulated necrosis was the initiator of immune activation [144].
Although precise mechanisms remain to be established, accumulating evidence suggests that ferroptosis
may be inflammatory through lipid peroxidation-mediated plasma membrane rupture [146] leading
to sterile inflammation [36]. As such, the small molecule inhibitor Fer-1 reduced immune cell
infiltrations into diseased tissues in models of acute kidney injury (AKI) [147,148]. Fer-1 treatment also
decreased cytokine and chemokine expression levels (C-X-C-motif chemokine 2 (CXCL-2), interleukin 6
(IL-6), p65 subunit of NF-κB [147], interleukin 33 (IL-33), TNF-α, monocyte chemotactic protein 1
(MCP-1) [148]), suggesting that ferroptotic DAMPs may have the capacity to elicit secondary immune
cell activation and cytokine production or that production of these alarmins is directly blocked by Fer-1.
Interestingly, the application of the caspase inhibitor zVAD-fmk and the RIPK1 inhibitor necrostatin-1
appeared to be futile for folic acid-induced AKI amelioration [148]. Mononuclear interstitial infiltrations
were also observed to occur upon GPX4 deletion in kidneys [55]. These data suggest that inflammatory
processes and activation of the immune system in acute kidney injury are induced through ferroptotic
cell death.
Fer-1 treatment further prevented neutrophil recruitment and their interferon (IFN)-dependent
adhesion to coronary veins following ischemia/reperfusion injury (IRI) through a Toll-like receptor 4
(TLR4)-mediated signalling pathway [149]. Ferroptotic cell death was also shown to contribute
to human diseases affecting the brain such as intracerebral haemorrhage [150] as well as
neurodegeneration [151,152]. Interestingly, conditional neuron-specific Gpx4 knockout mouse models
Cancers 2020, 12, 164 13 of 24

revealed up-regulated proinflammatory cytokine levels (TNF-α and IL-6) associated with neuronal
degeneration and cognitive impairment [151].
Furthermore, Trolox, another radical scavenger known to block ferroptosis, was equally shown to
decrease expression levels of proinflammatory cytokines (TNF-α, IL-6, interleukin 1 beta (IL-1β)) in
steatohepatitis [153]. This metabolic liver disease is characterised by the progression from steatosis
into fibrosis through inflammatory processes [154]. Overall, it is strongly suggested that ferroptosis is
closely associated with overt inflammatory signatures exhibiting elevated levels of proinflammatory
cytokines in damaged tissues [147,148,151,153]. Collectively, these results imply that ferroptosis exerts
a critical role in early inflammatory processes and that the prevention of necroinflammation provides a
novel therapeutic strategy in combating inflammatory tissue damage and disease.
Although other regulated necrosis pathways such as necroptosis and pyroptosis were shown
to mitigate inflammation in several diseases [155], a growing body of literature suggests that
ferroptosis may be present from the outset of inflammatory [20,148,149,153] potentially triggering
or sensitising to other inflammatory events. Importantly, immunogenic cell death results in the
recruitment and activation of immune cells through alarmins which in turn has been hypothesised to
induce further necrotic cell death pathways, culminating in a necroinflammatory auto-amplification
loop [156]. However, the exact molecular pathways/machineries driving primary and secondary
necroinflammation and the underlying hierarchical sequence of events remains yet to be disentangled.

M1 versus M2-Type Immunity in Cancer: Potential Implications for Ferroptosis

Necroptotic cancer cells can release DAMPs, triggering inflammation in normal tissues [157].
Recently, it was shown that ferroptosis can equally allow for the release of the known DAMP
high-mobility group protein B1 (HMGB1) in a manner dependent on the cellular autophagy
machinery [158]. Apart from this, it is conceivable that the induction of ferroptosis in cancer cells
might elicit innate immune responses which can influence tumours via various ways. The M1/M2
polarisation axis proposed by Mills et al. in 2000 provides a simplistic approach to distinguish between
two types of macrophages with different metabolic programmes [159]. Whereas the M1 macrophage
phenotype was reported to result in tumour regression [160] and inhibition of tumour growth [161],
M2 macrophages have been shown to exhibit tumour-promoting activity as they produce angiogenesis
factors [162] and inhibit M1 macrophages, thereby suppressing anti-tumour immunity. The importance
of macrophage polarisation in the tumour microenvironment was further demonstrated by showing
a chemokine-mediated decrease in M2-like macrophages in the vicinity of the tumour leading to
reduced tumour growth [163]. In view of the intricate dynamics between innate and adaptive immune
responses, it is important to highlight that M1/M2 macrophages promote adaptive Th1/Th2 lymphocyte
responses, respectively [159]. M1/Th1 responses are associated with the release of proinflammatory
cytokines, such as TNF [164] as well as IFN-γ, a cytokine that is in turn able to stimulate M1 polarisation
in a feed-forward loop [165] as well as downregulate xCT [137]. In contrast, macrophage-induced
Th2 responses are closely linked to anti-inflammatory mediators, such as IL-4 and IL-10 [164,166].
Interestingly, cytokines released from Th2 T-cell clones have been shown to avert cytokine production
by Th1 cells, essentially strengthening the suppression of M1/kill type activity [159,167]. Furthermore,
tumour cells have developed mechanisms to stimulate tumour cell migration and to maintain a high
M2 to M1 ratio in the tumour microenvironment, thereby working in their best interest [168–170].
Of note, the majority of tumour-associated macrophages (TAM) in the tumour microenvironment
is predominantly composed of M2 macrophages [171]. As a result, anti-cancer macrophage innate
conversion therapies have emerged in recent years, directly targeting intracellular signalling pathways
to increase the M1/M2 ratio [172,173]. Beatty et al. showed that the combination therapy of gemcitabine
and agonist CD40 antibody obtained macrophage-dependent tumour regression in vivo which was
suggested to be attributable to M1 activity [174].
As the M1/M2 dichotomy influences inflammatory events in opposite directions, it is of
great interest to elucidate the quality of a ferroptotic secretome released from dying cancer cells
Cancers 2020, 12, 164 14 of 24

as it may either trigger M2 tumour-promoting activity or initiate an M1 anti-tumour immune

response. Given that ferroptosis in tissues has been causatively linked to the presence of TNF,
this may suggest the potential for the promotion of an M1-type immune microenvironment.
Supporting the idea of ferroptosis to be inflammatory in the context of cancer, ferroptosis triggered
in neuroblastoma by withaferin A led to an influx of immune cells into the tumour [75].
Moreover, recent research has proposed that sensitising tumour cells to ferroptosis enhances
the efficacy of combinatorial cancer immune-activatory therapies [175]. Interestingly, xCT was
reported to be down-regulated through both immunotherapy-activated IFNγ [137,175] as well as
radiotherapy-induced ataxia-telangiectasia mutated gene (ATM), resulting in increased tumour
cell lipid peroxidation and tumoural ferroptosis [175]. Furthermore, immunotherapy-induced
cytokines, namely IFNγ and TNF-α, were shown to provoke dedifferentiation in melanoma cells
which was accompanied by resistance development. Intriguingly, this transition by tumour
cells resulted in an increased susceptibility to ferroptosis induction and affected their overall
immunosuppressive abilities [176]. Therefore, not only the induction but the sensitisation of tumour
cells to ferroptosis may supply a novel tool for anti-tumour immunity and may serve the optimisation
of established immunotherapies.

9. Conclusions
Many cancers highly express xCT, suggesting a selective dependency on either cystine or GSH,
which may be exploited therapeutically by targeting these signalling nodes. Moreover, cancer cells
appear to acquire ferroptosis sensitivity as part of an escape strategy against other targeted therapies,
posing an opportunity for FIT in therapeutic management of relapse. Whilst the pathway will require
additional in-depth characterisation and the validity of genetic induction of ferroptosis remains to
be tested in genetically engineered mouse models of cancer, intriguingly, immunotherapy seems to
positively interact with ferroptosis in vivo. These and other data strongly suggest ferroptosis to either
be directly immunogenic or to prime an inflammatory response to other forms of regulated necrosis in
the tumour microenvironment. Collectively, we propose that tumour cell ferroptosis may promote
four possible outcomes in cancer: (A) if immuno-silent and completely killing, tumour cell ferroptosis
should suppress tumours, (B) if immuno-silent but fractional killing causes selection, ferroptosis may
result in tumour promotion through selection of the fittest cancer cell clone, (C) if an M1-type immunity
is triggered, ferroptosis should be tumour-suppressive via activation of anti-tumour immunity and (D)
if an M2-type immunity is triggered, ferroptosis would be overall tumour-protective, as it would aid
shielding tumours against anti-tumour immune attack (proposed concepts summarised in Figure 2).
Importantly, mixed responses within these four response types are likely to occur due to tumour
heterogeneity. Thus, it will be important in future work to determine not only cellular determinants of
ferroptosis sensitivity and resistance but also systemic responses and mechanisms of how these are
interlinked with other types of regulated cell death in order to fully harness the potential of FIT for
cancer treatment.
protective, as it would aid shielding tumours against anti-tumour immune attack (proposed concepts
summarised in Figure 2). Importantly, mixed responses within these four response types are likely
to occur due to tumour heterogeneity. Thus, it will be important in future work to determine not only
cellular determinants of ferroptosis sensitivity and resistance but also systemic responses and
Cancers 2020, 12,of
mechanisms 164how these are interlinked with other types of regulated cell death in order to15fully
of 24
harness the potential of FIT for cancer treatment.

Figure
Figure 2.
2. Proposed
Proposed concepts
concepts for for the
the influence
influence ofof ferroptosis
ferroptosis onon tumour
tumour outcome.
outcome. (A) (A) without
without raising
raising
an immune response, ferroptosis may result in selective and complete killing
an immune response, ferroptosis may result in selective and complete killing of tumour cells of tumour cells leading
leading to
to tumour
tumour eradication.
eradication. (B)(B) if immune-silent,
if immune-silent, ferroptosis
ferroptosis maymayalsoalso merely
merely result
result in fractional
in fractional killing
killing of
of cells
cells
withinwithin a heterogeneous
a heterogeneous tumour. tumour.
Over Over
time, time, this would
this would lead tolead to selection
selection of ferroptosis
of ferroptosis resistant
resistant clones
clones and their outgrowth and overall promotion of tumours. (C) if ferroptosis were
and their outgrowth and overall promotion of tumours. (C) if ferroptosis were able to raise an M1-type able to raise an
M1-type immune response,
immune response, M1 macrophagesM1 macrophages would
would aid T-cell aid T-cell
activation andactivation
maintain an and maintain an
anti-tumour anti-
immune
tumour
responseimmune
resultingresponse
in tumour resulting in tumour
eradication. (D) iferadication.
ferroptosis (D) if ferroptosis
instead instead
were to raise were to immune
an M2-type raise an
M2-type immune response, M2-macrophages would protect tumour cells
response, M2-macrophages would protect tumour cells from T-cell-mediated anti-tumour immune from T-cell-mediated anti-
tumour immune attack, leading to ferroptosis-initiated tumour immune
attack, leading to ferroptosis-initiated tumour immune protection and immune escape. Thereby, protection and immune
escape.
concept Thereby, concept
A and C offer a modelA and C offeran
explaining a anti-tumour
model explaining
effect ofan anti-tumour
ferroptosis, effectB of
whereas andferroptosis,
D propose
whereas B and D propose
model mechanisms model mechanisms
for ferroptosis-induced for ferroptosis-induced
tumour promotion. tumour promotion.

Author Contributions: Writing-original draft preparation, C.M.B., F.M., L.P.C., J.W. and S.v.K.; writing-review
and editing, C.M.B. and S.v.K.; visualisation, F.M.; funding acquisition, S.v.K. All authors have read and agreed to
the published version of the manuscript.
Funding: This work was funded by a Max-Eder-Junior Research Group grant (701125509) by the German cancer
aid (Deutsche Krebshilfe (DKH), awarded to S.v.K.) and supported by the Koeln Fortune Program of the Faculty
of Medicine, University of Cologne (awarded to J.W.).
Conflicts of Interest: The authors declare no conflict of interest.
Cancers 2020, 12, 164 16 of 24

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