Cysteine Depletion, a Key Action to Challenge Cancer Cells to Ferroptotic Cell Death

Abstract

Cancer cells are characterized as highly proliferative at the expense of enhancement of metabolic rate. Consequently, cancer cells rely on antioxidant defenses to overcome the associated increased production of reactive oxygen species (ROS). The reliance of tumor metabolism on amino acids, especially amino acid transport systems, has been extensively studied over the past decade. Although cysteine is the least abundant amino acid in the cell, evidences described it as one of the most important amino acid for cell survival and growth. Regarding its multi-functionality as a nutrient, protein folding, and major component for redox balance due to its involvement in glutathione synthesis, disruption of cysteine homeostasis appears to be promising strategy for induction of cancer cell death. Ten years ago, ferroptosis, a new form of non-apoptotic cell death, has been described as a result of cysteine insufficiency leading to a collapse of intracellular glutathione level. In the present review, we summarized the metabolic networks involving the amino acid cysteine in cancer and ferroptosis and we focused on describing the recently discovered glutathione-independent pathway, a potential player in cancer ferroptosis resistance. Then, we discuss the implication of cysteine as key player in ferroptosis as a precursor for glutathione first, but also as metabolic precursor in glutathione-independent ferroptosis axis.

Full text

MINI REVIEW
published: 07 May 2020
doi: 10.3389/fonc.2020.00723
Frontiers in Oncology | www.frontiersin.org 1May 2020 | Volume 10 | Article 723
Edited by:
Ana Preto,
University of Minho, Portugal
Reviewed by:
Cinzia Domenicotti,
University of Genoa, Italy
Barbara Marengo,
University of Genoa, Italy
*Correspondence:
Boutaina Daher
bdaher@centrescientifique.mc
Jacques Pouysségur
pouysseg@unice.fr
Specialty section:
This article was submitted to
Cancer Metabolism,
a section of the journal
Frontiers in Oncology
Received: 21 February 2020
Accepted: 16 April 2020
Published: 07 May 2020
Citation:
Daher B, Vu ˇ
ceti ´
c M and Pouysségur J
(2020) Cysteine Depletion, a Key
Action to Challenge Cancer Cells to
Ferroptotic Cell Death.
Front. Oncol. 10:723.
doi: 10.3389/fonc.2020.00723
Cysteine Depletion, a Key Action to
Challenge Cancer Cells to
Ferroptotic Cell Death
Boutaina Daher 1
*, Milica Vu ˇ
ceti ´
c1and Jacques Pouysségur 1,2
*
1Medical Biology Department, Centre Scientifique de Monaco (CSM), Monaco, Monaco, 2Institute for Research on Cancer
and Aging (IRCAN), CNRS, INSERM, Centre A. Lacassagne, Université Côte d’Azur, Nice, France
Cancer cells are characterized as highly proliferative at the expense of enhancement of
metabolic rate. Consequently, cancer cells rely on antioxidant defenses to overcome
the associated increased production of reactive oxygen species (ROS). The reliance
of tumor metabolism on amino acids, especially amino acid transport systems, has
been extensively studied over the past decade. Although cysteine is the least abundant
amino acid in the cell, evidences described it as one of the most important amino
acid for cell survival and growth. Regarding its multi-functionality as a nutrient, protein
folding, and major component for redox balance due to its involvement in glutathione
synthesis, disruption of cysteine homeostasis appears to be promising strategy for
induction of cancer cell death. Ten years ago, ferroptosis, a new form of non-apoptotic
cell death, has been described as a result of cysteine insufficiency leading to a collapse
of intracellular glutathione level. In the present review, we summarized the metabolic
networks involving the amino acid cysteine in cancer and ferroptosis and we focused on
describing the recently discovered glutathione-independent pathway, a potential player
in cancer ferroptosis resistance. Then, we discuss the implication of cysteine as key
player in ferroptosis as a precursor for glutathione first, but also as metabolic precursor
in glutathione-independent ferroptosis axis.
Keywords: xCT transporter, cysteine, lipid peroxides, glutathione, ferroptosis, tumor-resistance
INTRODUCTION
Since the early 20th century, the reprogramming of cellular metabolism has been recognized as
one of the major hallmarks of oncogenesis (1) that has great potential for anti-cancer treatment.
Key signatures of cancer cells, due to their highly proliferative nature, are intensified metabolic
rate, increased oxidative pressure, and consequently, reliance on antioxidant defense in term
of redox homeostasis maintenance. The central role of amino acids in antioxidant defense is
mainly due to the involvement of serine, glutamine/glutamate and cysteine, in glutathione (GSH)
and nicotinamide adenine dinucleotide phosphate (NADPH) production (2,3). One particularity
of cysteine is its semi-essential nature and its implication in GSH homeostasis. GSH is a
key non-enzymatic player in cellular antioxidant defense and is broadly implicated in tumor
initiation, progression, metastasis, and resistance. Nutrient supply and redox balance are therefore
intertwined and of great importance for anti-cancer treatment. In the present review, we will

Daher et al. Cysteine, Key Player in Ferroptosi
describe in more details cysteine implication in cancer
(patho)physiology from two aspects; cysteine as a proteinogenic
amino acid and cysteine as an amino acid involved in the GSH-
and thus redox- homeostasis.
CYSTEINE, A KEY PLAYER IN TUMOR
METABOLISM
Cysteine is a sulfur-containing amino acid. Even though it is
described to be a “non-essential” amino acid, in conditions
of high nutrient demands, it becomes essential. In the liver, a
particular metabolic pathway called transsulfuration permits the
supply of cysteine by conversion of an essential amino acid:
methionine. Yet, this amino acid interconversion is insufficient
to provide the cysteine requirements of rapidly dividing cancer
cells (4). As mentioned previously, cysteine is a thiol-containing
amino acid, which nucleophilicity makes it highly susceptible to
redox changes. Notably, impressive complexity of the cysteinome
dynamic reflects its important role in the cell. Indeed cysteine
has a crucial role in many processes such as assembly, protein
folding stability and trafficking, biosynthesis of coenzyme A and
taurine, iron-sulfur (Fe-S) cluster biogenesis, detoxification of
heavy metals and redox balance (5). A number of pathologies
have been characterized by an unbalanced cysteinome profile,
including cystinuria, renal calculi, Huntington’s disease, and
Alzheimer’s disease (6–8). In cancer, the implication of cysteine
in tumor formation, propagation and resistance has been widely
described (5).
The building block for three essential nutrients
(carbohydrates, lipids, and proteins): simple sugars, fatty
and amino acids, are provided from diet. Contrary to for
example fatty acids, amino acids due to their lipophobicity
require transporters for the import/export. Up to now, more
than 30 different amino acid transporters have been described
in mammalian cells, however, a small, co-called “minimal set”
among them is consistently overexpressed in many different
tumor types (9–11). These transporters are LAT1, ASCT2
and the Xc−system. According to our previous study, LAT1
(standing for L-type Amino acid Transporter 1) is indispensable
for transport of essential amino acids, general amino acid
homeostasis, and consequently, tumor growth (12). ASCT2
or Alanine-Serine-Cysteine Transporter 2 is a transporter that
exchanges small neutral amino acids and plays a crucial role
in glutamine uptake and the promotion of tumor growth,
independently of LAT1 activity (13). The third overexpressed
transporter in cancer is the X−
csystem, an exchanger that
imports cystine, the oxidized form of cysteine, and exports
glutamate. This sodium-independent antiporter is composed of
two subunits: xCT (gene name SLC7A11), a subunit responsible
for the amino acid exchange, and a chaperone CD98 (gene
name SLC3A2). In 2011, the transmembrane glycoprotein CD44,
a cancer stem-like cell marker, and more precisely the CD44
variant (CD44v) capable to bind hyaluronan has also been
described to interacts and stabilizes X−
csystem (14) (Figure 1).
Although the role of CD44 in the transport activity of xCT
has not been validated so far, an interesting implication in
iron endocytosis via CD44-bound hyaluronates is proposed
(15) (Figure 1). Our group recently described that a genetic
disruption of the xCT subunit using CRISPR-Cas9 inhibits
protein synthesis and proliferation in vitro (16) and leads to a
specific non-apoptotic cell death named ferroptosis, that will
be described later in this review. A 14C-cystine transport assay
in xCT knockout (xCT-KO) cells revealed this transporter as
unique and indispensible for cystine uptake, as a complete
abolishment of cystine transport has been observed. In contrast,
in in vivo assay, xCT-KO pancreatic ductal adenocarcinoma
(PDAC) cells injected subcutaneously managed to form a
tumor, although with a short delay. This indicates that other
mechanisms are involved in the maintenance of intracellular
cysteine pool in vivo allowing tumor growth. Indeed, one of
the poorly discussed limits of cystine transport study in vitro
is the fact that the commonly used culture media contains
exclusively oxidized form of cysteine. Consistent with this, use
of a reducing source such as β-mercaptoethanol allows reversal
of xCT-KO phenotype, as it has been reported couple decades
ago by Bannai’s group (17,18). Therefore, highly dynamic ratio
of cystine/cysteine couple in vivo can explain the discrepancy
with in vitro phenotype. Transport of reduced form of cysteine
has been assigned to the transporters form ASCT family.
However, in case of the ASCT2, studies showed that cysteine is
actually a competitive inhibitor and not a substrate for ASCT2
(19,20). Similarly, preliminary results in our group indicate
that ASCT2 is not involved in cysteine uptake in surviving
xCT-ASCT2 double knockout PDAC cells in presence of β-
mercaptoethanol. Our laboratory at the moment is focused on the
examination of this highly elusive transport system for the import
of cysteine.
The highly conserved mechanistic target of rapamycin
(mTOR) regulates protein synthesis, metabolism and growth.
Activation of the mTOR complex 1 (mTORC1) relies not only
on insulin and growth factors activating, respectively, PI3K
and ERK1/2, but also on amino acids. Indeed translocation
of mTORC1 from the cytoplasm to the lysosome, a rich
compartment in amino acids, is critical for mTORC1 activation
(21). In addition the specific activation of mTORC1 by the
amino acids glutamine, arginine and leucine is well-described
(21,22). Interestingly, recent report suggested that cysteine per
se is also able to regulate mTORC1 activity (23). In line with
this, disruption of cystine uptake inhibits mTORC1 activation,
leading to an inhibition of protein synthesis (16,24). It is
interesting to note that the capacity of sensing amino acids
has been achieved by different mechanisms so that intracellular
protein synthesis homeostasis is ensured with high fidelity.
Except mTORC1, another important pathway in this regard
is amino acid-starvation pathway. Namely, the protein kinase
General Control Nonderepressible (GCN2) is a sensor of amino
acids that is activated by the intracellular accumulation of
uncharged tRNA (25,26). GCN2 represses general protein
synthesis and activates the transcription of genes involved in
the synthesis and transport of amino acids via activation of
ATF4 transcriptional factor. This GCN2-ATF4 pathway is crucial
for tumor cell survival during nutrient deprivation (27). Our
data from PDAC cell lines showed that genetic ablation of
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Daher et al. Cysteine, Key Player in Ferroptosi
FIGURE 1 | Intracellular cysteine pool supply. Extracellular oxidized cystine is imported at the expense of one glutamate molecule via Xc−system composed of two
subunits: xCT transporter and the chaperone CD98. This complex xCT is also associated with the stem-like cancer cell marker CD44v. Imported cystine is then
reduced to cysteine by cystine reductase (CR) (1). Methionine conversion leads to cysteine synthesis via the transsulfuration pathway (2). Two important steps in this
synthesis are conversion from homocysteine to cystathionine by cystathionine β-synthase (CBS) and synthesis of cysteine from cystathionine by cystathionase (CTH).
Degradation of glutathione (GSH) via CHAC1 intracellularly provides cysteine supply (3). GSH, either from exogenous sources or exported from cells via Multidrug
Resistance Protein 1 exporter (MRP1), is cleaved extracellularly by γ-Glutamyl transferase (GGT) forming γ-Glutamyl-X substrate and Cysteinyl-Glycine. This
Cysteinyl-Glycine dipeptide can either be potentially transported via PEPT2 or cleave by dipeptidase releasing cysteine and glycine (5). γ-Glutamyl moiety can be
complexed to available extracellular cyst(e)ine forming γ-Glutamyl-cysteine. Cysteine supply from GSH is one of the main function of γ-Glutamyl-cycle (4). Available
extracellular cysteine is then transported via ASCT family members but can also be oxidized and imported via xCT.
xCT transporter leads to intracellular cysteine deficiency, and
therefore GCN2-ATF4 pathway activation (16). The results
described in PDAC cells have also been confirmed in human
breast cancer after genetic or pharmacologic inhibition of this
transporter (28).
In brief, activation of the GCN2-ATF4 amino acid stress
pathway and inhibition of protein synthesis through inhibition
of mTORC1 demonstrates the strong proteogenic role played
by cysteine in tumor cells. Nevertheless, the role of building
the protein molecules is not the only function of cysteine.
The particularity of this amino acid is its bifunctionality,
and its other essential role is the building up of cellular
antioxidant defenses via the biosynthesis of the most
conserved and abundant non-enzymatic antioxidant in the
cell: glutathione.
GLUTATHIONE: HOMEOSTASIS AND
FUNCTIONS
Glutathione (GSH) is the most abundant non-protein thiol
in mammalian cells, reaching an intracellular concentration in
mM range, whereas its plasma concentration does not exceed
micromolar range. In the cell, 90% of GSH is located in the
cytoplasm, 10–12% in the mitochondria, and a small percentage
in the endoplasmic reticulum (ER) (29). This small tripeptide is
composed of glutamate, cysteine, and glycine. Its biosynthesis is
a two-step enzymatic cascade, including first a specific γ-ligation
of glutamate and cysteine by γ-glutamate-cysteine ligase (GCL)
and then the formation of peptide bond between this dipeptide
and glycine by glutathione synthetase (GS). The GCL enzyme
consists of two subunits, a heavy catalytic subunit, GCLc, and
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Daher et al. Cysteine, Key Player in Ferroptosi
FIGURE 2 | Glutathione dependent and independent ferroptosis axis. Ferroptosis-cell death is dependent of accumulation of lipid peroxides in the membrane leading
to its disruption and cell bubbling (photography representing xCT-KO cells dying by ferroptosis). GSH-dependent axis follows cysteine-dependent import via xCT and
GSH synthesis. Some of up-to-date known inhibitors of xCT are erastin, imidazole ketone erastin (IKE), high extracellular glutamate but also sorafenib or sulfasalazine
(SSZ). Cysteine is the rate limiting component of GSH synthesis via glutamate-cysteine ligase (GCL). This GSH biosynthesis can be inhibited by buthionine sulfoximine
(BSO). GSH can be reduced via GSH reductase (GR) and then used as a cofactor by GSH peroxidase 4 (GPX4) to detoxify lipids peroxides. GPX4 can be inhibited by
different inhibitors such as RSL3, ML210, and FIN56 to induce ferroptosis. GSH-independent axis follows detoxification of lipid peroxides by ubiquinol leading to its
oxidation to ubiquinone. Ferroptosis suppressor protein 1 (FSP1) is responsible of the regeneration of ubiquinone to ubiquinol and can be interrupted by “inhibitor of
FSP1” (iFSP1). Acetyl-CoA is a precursor of ubiquinol and mevalonate pathway. Cysteine is potentially implicated in ubiquinol synthesis via pantothenate pathway
which uses cysteine for acetyl-coA synthesis. Pantothenate synthesis is inhibited by pantothenate kinase inhibitors (PanKi).
a light regulatory subunit, GCLm. Cysteine availability is the
limiting factor of GSH synthesis due to the fact that GCLc Km
for cysteine, around 270 µM, is roughly equal to its intracellular
concentration. GSH is involved in many important cellular
functions via its key role in antioxidant defense, protecting the
cell against free radicals produced as metabolic by-products,
either directly or indirectly. Numerous studies demonstrated that
this small molecule is crucial in many different human diseases
such as aging, diabetes, acquired immune deficiency syndrome
(AIDS), as well as neurodegenerative and liver diseases (30). The
importance of glutathione in tumor metabolism and particularly
in resistance mechanisms has been widely studied during the last
decades. One of the well-described roles played by GSH is the
detoxification of xenobiotics such as different drugs, and thus it is
fundamental for the resistance to chemo-, but also radiotherapy.
Indeed, multidrug and radiation resistance in tumor cells have
been associated with higher intracellular levels of GSH, and
increased level of GSH is a poor prognostic factor in many types
of cancer (31).
The γ-glutamatyl cycle or Meister cycle initially proposed in
the 60s described the synthesis and breakdown of GSH, making
it a strong cysteine donor in physiological and pathological
conditions (32). Cysteine synthesis via the transsulfuration
pathway and GSH biosynthesis occurs primarily in the liver
in physiological conditions, while in the pathology other cells
can take over the same role or contribute to it (developed
later in next paragraph). GSH excreted in the blood is cleaved,
to its constituents; and de novo synthesis of GSH by cancer
cells occurs as follows: GSH is first exported from the cell of
origin via transporters known as Multiresistance Drug Proteins
(MRPs), which belongs to the ATP binding cassette (ABC)s
transporter family and is well-known player in cancer resistance
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Daher et al. Cysteine, Key Player in Ferroptosi
mechanisms (33). Then, once in the extracellular space, GSH
is cleaved by γ-Glutamyl-Transferase (GGT), which is also
known as a poor prognostic factor for cancer patients (34).
With an active site at the external surface of the plasma
membrane, GGT catalyzes the transfer of γ-glutamatyl moiety
from GSH to free amino acid, and thereby, released cysteine-
glycine dipeptide that can be transported in its intact form
via the proton-coupled oligopeptide transporter family member
PEPT2 (35,36) or further cleaved by dipeptidases to cysteine
and glycine. On the other side, the γ-glutamyl-amino acids
are converted into 5-oxoproline and the corresponding amino
acid by γ-glutamyl cyclotransferase. One interesting possibility
is that γ-glutamyl can be complexed to extracellular available
free cyst(e)ine, imported into the cell and as such can serve
as a substrate for GS during GSH synthesis (bypassing GCL
reaction). In physiological conditions, oxidized GSH can be
recycled intracellularly by GSH reductase (GR) using NADPH as
a reducing power. However, in stressful, oxidative-compromising
conditions, this reductase seems not to be sufficient, underlying
the importance of this γ-glutamyl cycle (37). As described by
Bannai, this recycling cycle provides very reliable source of
cysteine, and thus GSH, to the cells. Therefore, GGT localization
across the membrane permits direct uptake of the extracellular
reduced form of cysteine before its oxidation (38). However,
whether intact GSH can cross the cellular membrane via a specific
transporter remains unclear (39). Further investigation of the
pathways involving GSH is expected to bring important insights
into cancer (patho)physiology understanding (2).
Besides ATF4, another transcription factor that regulates
xCT expression is the nuclear factor erythroid 2-related
factor 2 (NRF2) via the antioxidant response element (ARE)
present in the promoter region of xCT gene (40,41). GSH
levels directly correlate with cysteine availability; therefore, a
disruption of cysteine uptake, either genetically or chemically,
efficiently depletes GSH intracellular levels (16,42). As described
previously, GSH has multiple roles in the cell and one of
them is functioning as cofactor for the enzyme glutathione
peroxidase 4 (GPx4). This peroxidase, with a selenocysteine at
its active site, converts lipid hydroperoxides to lipid alcohols
using reducing power of GSH. Lipid peroxides can be produced
either spontaneously or by enzyme-catalyzed processes. Free-
radical chain reaction occurs in an oxidatively-compromised
environment where ROS production overcomes their removal.
In the specific case of cysteine-deprived cell death, lipid
peroxidation acquires a character of chain-reaction due to
the Fenton reaction with ferrous iron (Fe2+). Namely, redox-
active metals, like Fe2+, react with peroxides generating highly
active hydroxyl radicals (R-HO•) that further propagate the
peroxidation reaction (43). Ferroptosis, coined by Stockwell’s
group in 2012, designates a specific non-apoptotic cell death
caused by such accumulation of lipid peroxides following cystine
deprivation (42). Disruption of cysteine uptake and collapse of
intracellular GSH pool induces an inhibition of the detoxifying
activity of GPx4 and excessive accumulation of oxidatively
damaged lipids at the membrane, although it remains unclear
if this solely affects the cell plasma membrane, or the effect
also extends to organelle membranes. All in all, the different
pathways involved in GSH homeostasis are an indication of
the complex dynamic and quick turn over of this tripeptide,
and provide clues for potential targets for a GSH-depleting,
ferroptosis-inducing strategy.
CYSTEINE, LIPID PEROXIDES AND
FERROPTOSIS
Glutathione-Dependent Ferroptosis
Since characterization of ferroptosis in 2012, the cysteine-GSH-
GPx4 axis is described as essential pathway for its regulation,
and thus seen as potential therapeutic target. Up to now,
powerful genetic tools allowed clarification of the significance,
dispensability and potential of cystine transporters for ferroptotic
cell death in PDAC and breast cancer (16,28). On the other
side, now there is growing interest in the development of specific
pharmacological inhibitors of xCT that will prove fundamental
research in the clinical settings. Almost 20 years ago, a high
extracellular level of glutamate was described as inhibitor for
cystine uptake and inductor of a specific cell death termed
oxytosis (44). Indeed, in 1988 Bannai reported glutamine import
via ASCT2 and conversion into glutamate that is exported
in exchange of cystine import (1:1) via xCT (45). Yet, more
recently, a metabolomic analysis reveals the importance of
glutamine uptake by ASCT2 and its conversion into ROS-
producing intermediate metabolite α-Ketoglutarate as marker
of sensitivity during sulfasalazine xCT-inhibition cell death in
head and neck squamous cells carcinoma (46). Glutaminolysis
was also reported to sensitize melanoma cells to ferroptosis (47).
Therefore, those studies suggest that ASCT2 expression can be a
marker of ferroptosis sensitivity.
A high extracellular glutamate concentration was one of the
first xCT inhibitors described but a few years later, erastin
(standing for eradicator of RAS and small T antigen-expressing
cells) was identified by Stockwell’s group in RAS-mutated cancer
cell lines as inhibitor of xCT transport (42). Erastin is widely
used in in vitro studies as efficient inducer of ferroptosis in
many different cancer types, including breast, PDAC, lymphoma,
renal, brain and ovarian cancers (48). A recent study has
described a new form of erastin, imidazole ketone erastin
(IKE), characterized as more metabolically stable than the
previous form (49). Other small compounds are also described
as ferroptosis inducers, including two FDA-approved drugs,
sulfasalazine, prescribed for lung carcinoma and fibrosarcoma
(50,51) and sorafenib (52). However, our team found that
the specificity of those two compounds to xCT was relatively
low, as addition of cysteine analog N-Acetyl-Cysteine (NAC),
could not rescue cells from dying (16). Another possible way to
induce depletion of intracellular cysteine has been proposed by
Cramer’s group using systemic depletion of cysteine in the plasma
of leukemic mice. This was done by employing an engineered
cyst(e)inase enzyme resulting in the suppression of tumor growth
in breast and prostate cancer xenografts (53). Nevertheless, it
is crucial to note that recently conversion of methionine to
cysteine via transsulfuration pathway is a potential resistance
mechanism under cysteine depletion conditions. The conversion
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Daher et al. Cysteine, Key Player in Ferroptosi
from homocysteine to cystathionine by cystathionine β-synthase
(CBS) is described to be a key player in restoring intracellular
cysteine pool as decreasing its expression increases sensitivity to
ferroptosis in ovarian cancer cells upon erastin treatment (54).
Downstream reaction that forms cysteine from cystathionine by
cystathionase (CTH) has also been recently described to play
a key role in adaptation mechanisms during cysteine-deprived
induced stress in a wide variety of cancer cells lines (55).
Investigating the molecular mechanisms involved in restoring
the intracellular redox-buffer cysteine is of a great interest in
the understanding of ferroptosis resistance mechanisms. The
importance of this pathway is not only crucial for ferroptosis
as cystathionase is also described to be involved in senescence
evasion in melanocytes and melanoma cells (56).
However, other systems downstream of cystine uptake
can also play an important role. For instance, GSH can
be depleted, through inhibition of its synthesis using an
inhibitor of GCL (buthionine sulfoximine, BSO) (57) under
conditions that maintain the intracellular cysteine pool intact,.
Another strategy to deplete GSH is increasing its efflux. Dixon’s
group demonstrated that the GSH exporter MRP1 sensitizes
HPA1 erastin-treated cells to ferroptosis and concordantly,
MRP1 inhibition leads to a retention of GSH and leads
to ferroptosis resistance (58). Furthermore, another recently
described player involved in GSH catabolism is the specific
cytoplasmic GSH-degrading enzyme CHAC1 (59). Interestingly,
this enzyme enhances cysteine starvation-induced ferroptosis
through activation of the GCN2-eIF2α-ATF4 pathway in human
triple negative breast cancer cells (60). The implication of CHAC1
in increasing sensitivity to ferroptosis was recently confirmed in
Burkitt’s lymphoma during artesunate-induced ferroptosis (61).
Those two mechanisms involved in efflux and intracellular GSH
degradation can be of great interest to challenge ferroptosis-
resistant cells.
The third major target is the selenoenzyme GPx4. Different
inhibitors of GPx4 induce ferroptosis such as RLS3, FIN56,
and ML210 (24) and more recently, resibufugenin (62). Post-
chemotherapy-“persister” cells resistant to lapatinib treatment
in breast, melanoma, lung, and ovarian cancer have been
characterized as GPx4-dependent (63). Therefore, making
ferroptosis inducers druggable for cancer therapy sounds like
a promising strategy to challenge and overcome acquired
resistance to other drugs. In theory, targeting any node of
cysteine-GSH-GPx4 axis seems to be sufficient to induce
ferroptosis. Importantly, results obtained from knockout mice
suggests that xCT inhibition could have the least off-side effect in
comparison with the GPx4 and GCL (18,64,65). Indeed, xCT−/−
mice are healthy and fertile despite an increase in cystine plasma
concentration and a decrease in GSH plasma level. Therefore,
developing an efficient and specific xCT inhibitor is a promise
of great advance in cancer therapy.
Glutathione-Independent Ferroptosis
Although described since the beginning to be the main actor
of ferroptosis inhibition, it has been recently described that the
cysteine-GSH-GPx4 axis can be, at least in part, dispensable.
A recent genetic screen of genes complementing the loss
of GPx4 in resistant cell lines uncovered new players for
ferroptosis inhibition. A specific oxidoreductase, previously
known as apoptosis-inducing-factor mitochondrial-2 (AIFM2),
capable of recycling reduced ubiquinol (Co-enzymeQ10H2)
from ubiquinone at the expense of NAD(P)H, has been
presented as a potential ferroptosis inhibitor due to the fact
that its overexpression complements the loss of GPx4 in
PFA1 and human fibrosarcoma (66,67). Therefore, since then,
this AIFM2 oxidoreductase has been re-named to Ferroptosis
Suppressor Protein-1 (FSP1) (Figure 2). Those compensatory
mechanisms depend on the NAD(P)H-mevalonate pathway that
synthesize ubiquinol. Ubiquinol traps radicals undergoing lipid
peroxidation in the membrane. Therefore, the discovery of
this parallel GSH-independent mechanism for lipid peroxide
scavenging is of great interest for development of ferroptosis-
based potential chemotherapeutics. Finally, membrane lipid
composition and more importantly the long polyunsaturated
fatty acid (PUFA) is playing a key role in ferroptosis sensitivity.
This PUFA membrane enrichment is triggered by the specific
enzyme acyl-CoA synthetase long-chain family member 4
(ACSL4). Interestingly ACSL4 was preferentially expressed in a
panel of basal-like breast cancer cell lines and predicted their
sensitivity to ferroptosis (68).
CONCLUSION AND REMAINING
QUESTIONS
During past decade, investigation of ferroptosis from both
aspects: induction and prevention, has become a topic of
interests for numerous different pathologies. Nevertheless, a
remaining undiscussed point is the role of cysteine in ferroptosis
independently from GSH synthesis. In other words, how similar
are the phenotypes of cysteine-depleted vs. GSH-depleted cells?
Is ferroptosis caused exclusively by an excessive lipid peroxides
accumulation due to GSH depletion and oxidative damage,
or also by cysteine insufficiency itself? To investigate the role
played by this amino acid, labeled cystine was used to follow
its incorporation in PDAC cells before and after treatment with
IKE. As described previously, the major part of exogenous cystine
is incorporated in GSH, yet unexpectedly, the remaining part
is incorporated in co-enzyme A synthesis via the pantothenate
pathway (69). Co-enzyme A is a precursor of cholesterol and co-
enzyme Q10 (ubiquinol) a product of the mevalonate pathway,
but also a key player in fatty acid biosynthesis and β-oxidation.
Notably, lipid metabolism plays a crucial role in ferroptosis (70).
We therefore suggest an additional role of cysteine, independent
of GSH synthesis, in the prevention of ferroptosis. On the other
hand, inhibition of GSH synthesis with BSO, independently
of the cysteine pool, has been repeatedly described to induce
ferroptosis (42). In contrast, many recent studies explored and
demonstrated the implication of GSH depletion in induction of
apoptosis via depletion of mitochondrial GSH pool leading to
the release of cytochrome c, when combined with chemotherapy
in breast cancer and leukemia, respectively (71,72). Moreover,
GSH has also been involved in other types of cell death such
as necroptosis and for more details, refer to Lv’s review (73).
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Daher et al. Cysteine, Key Player in Ferroptosi
In line with this, one other mechanism recently described to be
involved in the resistance to GSH depletion is the overexpression
of deubiquitinases that inhibit protein degradation following
ER-stress (74). This system, independent of lipid peroxide and
apoptotic-mitochondrial defect, reveals the complexity of GSH-
depletion-induced cell death pathways. Our team is currently
validating this hypothesis using knockout cell lines specifically
deficient for GSH synthesis. All in all, despite recent progress in
the field, the detailed mechanisms of ferroptosis are still largely
unknown and a significant amount of research remains to be
developed in this new exciting area of research.
AUTHOR CONTRIBUTIONS
BD wrote this review and MV and JP revised it.
ACKNOWLEDGMENTS
BD and MV are founded by the Center Scientifique de
Monaco (CSM) and the Monegasque association GEMLUC
(Groupement des Entreprises Monégasques dans la Lutte contre
le Cancer).
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Conflict of Interest: The authors declare that the research was conducted in the
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