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

  • University of Nice-Sophia Antipolis and Centre scientifique de Monaco (CSM)

Abstract and Figures

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.
This content is subject to copyright.
published: 07 May 2020
doi: 10.3389/fonc.2020.00723
Frontiers in Oncology | 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
Boutaina Daher
Jacques Pouysségur
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
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
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 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 (68). 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 (911). These transporters are LAT1, ASCT2
and the Xcsystem. 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 Xcsystem 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 (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.
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).
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.
BD wrote this review and MV and JP revised it.
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
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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c and Pouysségur. This is an open-access article
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Frontiers in Oncology | 9May 2020 | Volume 10 | Article 723
... 25 Depletion of extracellular cystine leads to a loss of intracellular glutathione supply and the accumulation of oxidative damage to membrane lipids. [22][23][24]26 Uncontrolled lipid peroxidation propagates by reacting with ferrous iron and producing hydroxyl radicals leading to a form of cell death known as ferroptosis. 26,27 Ferroptosis is fundamentally a product of aberrant metabolic behavior including changes in central carbon metabolism such as increased mitochondrial glutaminolysis and increased dependence on glucose flux through the pentose phosphate pathway to generate NADPH. ...
... [22][23][24]26 Uncontrolled lipid peroxidation propagates by reacting with ferrous iron and producing hydroxyl radicals leading to a form of cell death known as ferroptosis. 26,27 Ferroptosis is fundamentally a product of aberrant metabolic behavior including changes in central carbon metabolism such as increased mitochondrial glutaminolysis and increased dependence on glucose flux through the pentose phosphate pathway to generate NADPH. [28][29][30] The impact of PKM2, which also plays a critical role in metabolic reprograming and the management of oxidative stress, on ferroptosis in pancreatic cancer remains poorly characterized. ...
Full-text available
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with high mortality and limited efficacious therapeutic options. PDAC cells undergo metabolic alterations to survive within a nutrient-depleted tumor microenvironment. One critical metabolic shift in PDAC cells occurs through altered isoform expression of the glycolytic enzyme, pyruvate kinase (PK). Pancreatic cancer cells preferentially upregulate pyruvate kinase muscle isoform 2 isoform (PKM2). PKM2 expression reprograms many metabolic pathways, but little is known about its impact on cystine metabolism. Cystine metabolism is critical for supporting survival through its role in defense against ferroptosis, a non-apoptotic iron-dependent form of cell death characterized by unchecked lipid peroxidation. To improve our understanding of the role of PKM2 in cystine metabolism and ferroptosis in PDAC, we generated PKM2 knockout (KO) human PDAC cells. Fascinatingly, PKM2KO cells demonstrate a remarkable resistance to cystine starvation mediated ferroptosis. This resistance to ferroptosis is caused by decreased PK activity, rather than an isoform-specific effect. We further utilized stable isotope tracing to evaluate the impact of glucose and glutamine reprogramming in PKM2KO cells. PKM2KO cells depend on glutamine metabolism to support antioxidant defenses against lipid peroxidation, primarily by increased glutamine flux through the malate aspartate shuttle and utilization of ME1 to produce NADPH. Ferroptosis can be synergistically induced by the combination of PKM2 activation and inhibition of the cystine/glutamate antiporter in vitro. Proof-of-concept in vivo experiments demonstrate the efficacy of this mechanism as a novel treatment strategy for PDAC.
... However, excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells [13]. L-Cysteine deprivation induces apoptosis in respiring by limiting iron-sulfur (Fe-S) cluster synthesis in the mitochondria [14,15]. Hence detection of excess Lcysteine is of great importance (Scheme 1). ...
... Third, our unbiased metabolic CRISPR knockout screen identified five candidate genes whose necessity for GBM invasion was validated in clonal KD cell lines, with each of these genes having demonstrated roles in the cellular response to oxidative stress. Prior studies have suggested that cancer cells have an increased cysteine demand that exceeds the amino acid's availability in the microenvironment (47,48). This activates the transsulfuration pathway to meet the metabolic requirements of the cancer cells (48). ...
Full-text available
While the poor prognosis of glioblastoma arises from the invasion of a subset of tumor cells, little is known of the metabolic alterations within these cells that fuel invasion. We integrated spatially addressable hydrogel biomaterial platforms, patient site-directed biopsies, and multi-omics analyses to define metabolic drivers of invasive glioblastoma cells. Metabolomics and lipidomics revealed elevations in the redox buffers cystathionine, hexosylceramides, and glucosyl ceramides in the invasive front of both hydrogel-cultured tumors and patient site-directed biopsies, with immunofluorescence indicating elevated reactive oxygen species (ROS) markers in invasive cells. Transcriptomics confirmed upregulation of ROS-producing and response genes at the invasive front in both hydrogel models and patient tumors. Amongst oncologic ROS, H2O2 specifically promoted glioblastoma invasion in 3D hydrogel spheroid cultures. A CRISPR metabolic gene screen revealed cystathionine gamma-lyase (CTH), which converts cystathionine to the non-essential amino acid cysteine in the transsulfuration pathway, to be essential for glioblastoma invasion. Correspondingly, supplementing CTH knockdown cells with exogenous cysteine rescued invasion. Pharmacologic CTH inhibition suppressed glioblastoma invasion, while CTH knockdown slowed glioblastoma invasion in vivo. Our studies highlight the importance of ROS metabolism in invasive glioblastoma cells and support further exploration of the transsulfuration pathway as a mechanistic and therapeutic target.
... It is one of the most abundant intracellular thiols, with concentrations reaching the millimolar range within cells. 89 GSH plays a pivotal role as a small molecule antioxidant that maintains intracellular redox balance. The biosynthesis of GSH involves a two-step enzymatic cascade process. ...
Full-text available
Ferroptosis is a novel form of programmed cell death morphologically, genetically, and biochemically distinct from other cell death pathways and characterized by the accumulation of iron‐dependent lipid peroxides and oxidative damage. It is now understood that ferroptosis plays an essential role in various biological processes, especially in the metabolism of iron, lipids, and amino acids. Gastric cancer (GC) is a prevalent malignant tumor worldwide with low early diagnosis rates and high metastasis rates, accounting for its relatively poor prognosis. Although chemotherapy is commonly used to treat GC, drug resistance often leads to poor therapeutic outcomes. In the last several years, extensive research on ferroptosis has highlighted its significant potential in GC therapy, providing a promising strategy to address drug resistance associated with standard cancer therapies. In this review, we offer an extensive summary of the key regulatory factors related to the mechanisms underlying ferroptosis. Various inducers and inhibitors specifically targeting ferroptosis are uncovered. Additionally, we explore the prospective applications and outcomes of these agents in the field of GC therapy, emphasizing their capacity to improve the outcomes of this patient population.
... 42 It plays an important role in many processes, such as protein folding stability and trafficking, assembly, biosynthesis of coenzyme A, detoxification of heavy metals, and redox balance. 43,44 Inside cells, L-cysteine is the prevailing form due to the highly reducing conditions. 45 The thiol group present in the structure of cysteine makes it a unique amino acid, which can undergo a variety of different nucleophilic reactions and increase its affinity to enzyme binding sites of metals, such as zinc or iron. ...
Full-text available
Xanthene and thioxanthene analogues have been investigated for their potential as anticancer and anti-inflammatory agents. Additionally, cysteine analogues have been found to possess antioxidant, anti-inflammatory, and anticancer activities due to their role in cellular redox balance, scavenging of free radicals, and involvement in nucleophilic reactions and enzyme binding sites. In this study, we synthesized a library of tertiary alcohols derived from xanthene and thioxanthene, and further, some of these compounds were coupled with cysteine. The objective of this research was to explore the potential anticancer, antioxidant, and anti-inflammatory activities of the synthesized compounds. The synthesized compounds were subjected to test for anticancer, antioxidant, and anti-inflammatory activities. Results indicated that compound 3 exhibited excellent inhibition activity (IC50 = 9.6 ± 1.1 nM) against colon cancer cells (Caco-2), while compound 2 showed good inhibition activity (IC50 = 161.3 ± 41 nM) against hepatocellular carcinoma (Hep G2) cells. Compound 4 demonstrated potent antioxidant inhibition activity (IC50 = 15.44 ± 6 nM), and compound 7 exhibited potent anti-inflammatory activity with cyclooxygenase-2 (COX-2) inhibition IC50 (4.37 ± 0.78 nM) and high selectivity for COX-2 (3.83). In conclusion, certain synthesized compounds displayed promising anticancer activity and anti-inflammatory effects. Nevertheless, additional research is necessary to create more analogues, develop a more distinct comprehension of the structure–activity relationship (SAR), and perform in vivo experiments to evaluate the pharmacokinetic and pharmacodynamic characteristics of the compounds under examination. Such research may pave the way for the development of novel therapeutic agents with potential applications in cancer and inflammatory diseases.
... Among them, iron metabolism, lipid metabolism, and amino acid metabolism are the three important pathways. Specifically, the sulfur-containing proteinogenic amino acid, cysteine, is the key precursor and rate-limiting substrate of GSH [12]. Cysteine metabolism, especially cysteine uptake (cells absorb cysteine in its oxidized form, cystine, through the cystine-glutamate antiporter System Xc-, which is the exact target of erastin) as well as de novo cysteine synthesis (mainly referring to the transsulfuration pathway, which channels methionine to cysteine and other metabolites), determines the intracellular cysteine for the biosynthesis of cysteinebased metabolites including GSH, CoQ, and other ferroptosis-suppressive molecules [13]. ...
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Ferroptosis is a newly characterized form of programmed cell death. The fundamental biochemical feature of ferroptosis is the lethal accumulation of iron-catalyzed lipid peroxidation. It has gradually been recognized that ferroptosis is implicated in the pathogenesis of a variety of human diseases. Increasing evidence has shed light on ferroptosis regulation by amino acid metabolism. Herein, we report that arginine deprivation potently inhibits erastin-induced ferroptosis, but not RSL3-induced ferroptosis, in several types of mammalian cells. Arginine presence reduces the intracellular glutathione (GSH) level by sustaining the biosynthesis of fumarate, which functions as a reactive α,β-unsaturated electrophilic metabolite and covalently binds to GSH to generate succinicGSH. siRNA-mediated knockdown of argininosuccinate lyase, the critical urea cycle enzyme directly catalyzing the biosynthesis of fumarate, significantly decreases cellular fumarate and thus relieves erastin-induced ferroptosis in the presence of arginine. Furthermore, fumarate is decreased during erastin exposure, suggesting that a protective mechanism exists to decelerate GSH depletion in response to pro-ferroptotic insult. Collectively, this study reveals the ferroptosis regulation by the arginine metabolism and expands the biochemical functionalities of arginine.
... In 2012, Stockwell lab formally defined ferroptosis as a unique iron-dependent cell death due to the accumulation of peroxidized lipids, which cannot be suppressed by apoptosis and necrosis inhibitors [9]. Different from apoptosis and necrosis, ferroptotic cells are characterized by a "ballooning morphology" of clear cytosol without cell swelling or shrinkage [27]. Mitochondria seems to be a key organelle affected by ferroptosis, since mitochondrial shrinkage, disrupted mitochondrial cristea and an increase of mitochondrial membrane density were reported upon ferroptosis induction [9,26,28]. ...
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Ferroptosis has been conceptualized as a novel cell death modality distinct from apoptosis, necroptosis, pyroptosis and autophagic cell death. The sensitivity of cellular ferroptosis is regulated at multiple layers, including polyunsaturated fatty acid metabolism, glutathione-GPX4 axis, iron homeostasis, mitochondria and other parallel pathways. In addition, microRNAs (miRNAs) have been implicated in modulating ferroptosis susceptibility through targeting different players involved in the execution or avoidance of ferroptosis. A growing body of evidence pinpoints the deregulation of miRNA-regulated ferroptosis as a critical factor in the development and progression of various pathophysiological conditions related to iron overload. The revelation of mechanisms of miRNA-dependent ferroptosis provides novel insights into the etiology of diseases and offers opportunities for therapeutic intervention. In this review, we discuss the interplay of emerging miRNA regulators and ferroptosis players under different pathological conditions, such as cancers, ischemia/reperfusion, neurodegenerative diseases, acute kidney injury and cardiomyopathy. We emphasize on the relevance of miRNA-regulated ferroptosis to disease progression and the targetability for therapeutic interventions.
... Исследования роли основного канцерогенного фактора -эстрогена и его рецепторов (ER, существует два вида рецепторов -α и β) -в развитии и прогрессировании рака молочной железы демонстрируют низкий уровень общих тиолов в сыворотке пациенток с ER-положительным в сравнении с ER-отрицательным РМЖ [39,40], что отражается на уровне риска рецидива и выживаемости [41]. В данном случае предполагается, что активация ERα увеличивает пролиферацию клеток РМЖ (вследствие усиленной транскрипции гена CCND1, который кодирует белок циклин D1, специфически регулирующий фазовый переход G1/S-фаза в клеточном цикле), а, следовательно, и рост окислительного стресса, влияющего на уровень сывороточных тиолов. ...
Background . Imbalance of aminothiol metabolism is a potential risk factor for malignant transformation of cells and caner development, including breast cancer, which is the most commonly diagnosed cancer in the world. The purpose of the study was to summarize the available data on the characteristics of thiol metabolism as one of the factors contributing to the progression of breast cancer. Material and Methods . Data were searched from 1999 to 2022 using the Web of Science, Scopus, MedLine, The Cochrane Library, PubMed databases, which made it possible to assess the role of thiol-dependent metabolic disturbances in the regulation of tissue redox balance in breast cancer genesis. Results . The review considers the results of both our own data and international studies on breast cancer, which suggest that an imbalance of thiol compounds necessary to maintain a moderately reducing cellular environment that counteracts oxidative stress during cellular metabolism and detoxifcation under conditions of tumor progression can provoke reprogramming of the leading links of antiblastoma resistance, contributing to cancer progression. Conclusion . A more detailed study of the mechanisms of aminothiol metabolism in breast cancer emphasizes their particular importance for stabilizing the cellular genome and providing antitoxic protection of the cell, as well as understanding the important role of thiols as a coordination center in redox signaling. Disturbances at any stage of thiol metabolism can play an etiological role in oncogenetic pathologies, while the role of thiols as signaling molecules and the regulation of their metabolism should not be generalized for the entire group of diseases. Determination of serum markers of the redox state in patients with breast cancer, especially during antitumor therapy, can serve for an objective assessment of the effectiveness of treatment and the adaptive capabilities of the body, as well as predicting tumor growth and optimizing the program for screening and preventing cancer.
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In cases of cellular injury, there is an observed increase in the production of reactive oxygen species (ROS). When this production becomes excessive, it can result in various conditions, including cancerogenesis. Glutathione (GSH), the most abundant thiol-containing antioxidant, is fundamental to re-establishing redox homeostasis. In order to evaluate the role of GSH and its antioxi-dant effects in patients affected by cancer, we performed a thorough search on Medline and EMBASE databases for relevant clinical and/or preclinical studies, with particular regard to diet, toxicities, and pharmacological processes. The conjugation of GSH with xenobiotics, including anti-cancer drugs, can result in either of two effects: xenobiotics may lose their harmful effects, or GSH conjugation may enhance their toxicity by inducing bioactivation. While being an interesting weapon against chemotherapy-induced toxicities, GSH may also have a potential protective role for cancer cells. New studies are necessary to better explain the relationship between GSH and cancer. Although self-prescribed glutathione (GSH) implementation is prevalent among cancer patients with the intention of reducing the toxic effects of anticancer treatments and potentially preventing damage to normal tissues, this belief lacks substantial scientific evidence for its efficacy in reducing toxicity, except in the case of cisplatin-related neurotoxicity. Therefore, the use of GSH should only be considered under medical supervision, taking into account the appropriate timing and setting.
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Ferroptotic cell death and cancer Cell death can occur through different mechanisms, several of which are being explored as potential targets for cancer treatment. One form of cell death that has attracted recent interest is ferroptosis, which is triggered by high intracellular levels of lipid reactive oxygen species. Pancreatic cancer cells have high levels of reactive oxygen species but manage to avoid ferroptosis by importing extracellular cysteine. Studying mice bearing pancreatic tumors, Badgley et al. found that administration of a drug inhibiting cysteine import induced tumor-selective ferroptosis and inhibited tumor growth. Further work will be required to determine whether this therapeutic strategy will be effective in human pancreatic cancer, a tumor type for which new treatments are urgently needed. Science , this issue p. 85
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Cystinuria, a genetic disorder of cystine transport, is characterized by excessive excretion of cystine in the urine and recurrent cystine stones in the kidneys and, to a lesser extent, in the bladder. Males generally are more severely affected than females. The disorder may lead to chronic kidney disease in many patients. The cystine transporter (b0,+) is a heterodimer consisting of the rBAT (encoded by SLC3A1) and b0,+AT (encoded by SLC7A9) subunits joined by a disulfide bridge. The molecular basis of cystinuria is known in great detail, and this information is now being used to define genotype-phenotype correlations. Current treatments for cystinuria include increased fluid intake to increase cystine solubility and the administration of thiol drugs for more severe cases. These drugs, however, have poor patient compliance due to adverse effects. Thus, there is a need to reduce or eliminate the risks associated with therapy for cystinuria. Four mouse models for cystinuria have been described and these models provide a resource for evaluating the safety and efficacy of new therapies for cystinuria. We are evaluating a new approach for the treatment of cystine stones based on the inhibition of cystine crystal growth by cystine analogs. Our ongoing studies indicate that cystine diamides are effective in preventing cystine stone formation in the Slc3a1 knockout mouse model for cystinuria. In addition to crystal growth, crystal aggregation is required for stone formation. Male and female mice with cystinuria have comparable levels of crystalluria, but very few female mice form stones. The identification of factors that inhibit cystine crystal aggregation in female mice may provide insight into the gender difference in disease severity in patients with cystinuria.
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Ferroptosis is an iron-dependent, lipid peroxide-mediated cell death that may be exploited to selective elimination of damaged and malignant cells. Recent studies have identified that small-molecule erastin specifically inhibits transmembrane cystine–glutamate antiporter system xc⁻, prevents extracellular cystine import and ultimately causes ferroptosis in certain cancer cells. In this study, we aimed to investigate the molecular mechanism underlying erastin-induced ferroptosis resistance in ovarian cancer cells. We treated ovarian cancer cells with erastin and examined cell viability, cellular ROS and metabolites of the transsulfuration pathway. We also depleted cystathionine β-synthase (CBS) and NRF2 to investigate the CBS and NRF2 dependency in erastin-resistant cells. We found that prolonged erastin treatment induced ferroptosis resistance. Upon exposure to erastin, cells gradually adapted to cystine deprivation via sustained activation of the reverse transsulfuration pathway, allowing the cells to bypass erastin insult. CBS, the biosynthetic enzyme for cysteine, was constantly upregulated and was critical for the resistance. Knockdown of CBS by RNAi in erastin-resistant cells caused ferroptotic cell death, while CBS overexpression conferred ferroptosis resistance. We determined that the antioxidant transcriptional factor, NRF2 was constitutively activated in erastin-resistant cells and NRF2 transcriptionally upregulated CBS. Genetically repression of NRF2 enhanced ferroptosis susceptibility. Based on these results, we concluded that constitutive activation of NRF2/CBS signalling confers erastin-induced ferroptosis resistance. This study demonstrates a new mechanism underlying ferroptosis resistance, and has implications for the therapeutic response to erastin-induced ferroptosis.
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Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been believed to be restrained only by the phospholipid hydroperoxide (PLOOH)-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants (RTAs)5,6. The factors which underlie a given cell type’s sensitivity to ferroptosis⁷ are, however, critical to understand the pathophysiological role of ferroptosis and how it may be exploited for cancer treatment. Although metabolic constraints⁸ and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been yet been identified that account for ferroptosis resistance. We undertook an expression cloning approach to identify genes able to complement GPX4 loss. These efforts uncovered the flavoprotein “apoptosis inducing factor mitochondria-associated 2 (AIFM2)” as a previously unrecognized anti-ferroptotic gene. AIFM2, hereafter renamed “ferroptosis-suppressor-protein 1” (FSP1), initially described as a pro-apoptotic gene¹¹, confers an unprecedented protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that ferroptosis suppression by FSP1 is mediated via ubiquinone (CoQ10): its reduced form ubiquinol traps lipid peroxyl radicals that mediate lipid peroxidation, while FSP1 catalyses its regeneration by using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, FSP1/CoQ10/NAD(P)H exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione (GSH) to suppress phospholipid peroxidation (pLPO) and ferroptosis.
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Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has been implicated in the cell death that underlies several degenerative conditions², and induction of ferroptosis by inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death⁵. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines⁶, suggesting that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR–Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ), generating a lipophilic radical-trapping antioxidant (RTA) that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumor xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a new ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutics.
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Ferroptosis is a newly discovered form of regulated cell death that is the nexus between metabolism, redox biology, and human health. Emerging evidence shows the potential of triggering ferroptosis for cancer therapy, particularly for eradicating aggressive malignancies that are resistant to traditional therapies. Recently, there has been a great deal of effort to design and develop anticancer drugs based on ferroptosis induction. Recent advances of ferroptosis-inducing agents at the intersection of chemistry, materials science, and cancer biology are presented. The basis of ferroptosis is summarized first to highlight the feasibility and characteristics of triggering ferroptosis for cancer therapy. A literature review of ferroptosis inducers (including small molecules and nanomaterials) is then presented to delineate their design, action mechanisms, and anticancer applications. Finally, some considerations for research on ferroptosis inducers are spotlighted, followed by a discussion on the challenges and future development directions of this burgeoning field.
Although chemoresistance remains a primary challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC), exploiting oxidative stress might offer novel therapeutic clues. Here we explored the potential of targeting cystine/glutamate exchanger (SLC7A11/xCT), which contributes to the maintenance of intracellular glutathione (GSH). Genomic disruption of xCT via CRISPR-Cas9 was achieved in two PDAC cell lines, MiaPaCa-2 and Capan-2, and xCT-KO clones were cultivated in the presence of N-acetylcysteine. Although several cystine/cysteine transporters have been identified, our findings demonstrate that, in vitro, xCT plays the major role in intracellular cysteine balance and GSH biosynthesis. As a consequence, both xCT-KO cell lines exhibited amino acid stress with activation of GCN2 and subsequent induction of ATF4, inhibition of mTORC1, proliferation arrest, and cell death. Tumor xenograft growth was delayed but not suppressed in xCT-KO cells, which indicated both the key role of xCT and also the presence of additional mechanisms for cysteine homeostasis in vivo. Moreover, rapid depletion of intracellular GSH in xCT-KO cells led to accumulation of lipid peroxides and cell swelling. These two hallmarks of ferroptotic cell death were prevented by vitamin E or iron chelation. Finally, in vitro pharmacologic inhibition of xCT by low concentrations of erastin phenocopied xCT-KO and potentiated the cytotoxic effects of both gemcitabine and cisplatin in PDAC cell lines. In conclusion, our findings strongly support that inhibition of xCT, by its dual induction of nutritional and oxidative cellular stresses, has great potential as an anticancer strategy. Significance The cystine/glutamate exchanger xCT is essential for amino acid and redox homeostasis and its inhibition has potential for anticancer therapy by inducing ferroptosis.
Resibufogenin (RB) has been used for cancer treatment, but the underlying mechanisms are still unclear. This study aimed to investigate the effects of RB treatment on colorectal cancer (CRC) cells, and to determine the underlying mechanisms. The cell counting kit-8 assay was used to determine cell viability. Cell morphology was observed under light microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was employed to detect cell apoptosis. Intracellular ferrous iron (Fe2+ ), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species levels were detected by using commercial iron assay kit, MDA assay kit, GSH assay kit, and 2,7-dichlorodihydrofluorescein diacetate probes, respectively. The protein expressions were determined by Western blot and immunohistochemistry. RB inhibited cell viability in the CRC cell lines (HT29 and SW480) in a dose- and time-dependent manner, and caused cytotoxicity to the normal colonic epithelial cell line (NCM460) at high dose. Similarly, RB induced morphological changes in CRC cells from normal to round shape, and promoted cell death. Of note, RB triggered oxidative stress and ferroptotic cell death in CRC cells, and only ferroptosis inhibitors (deferoxamine and ferrostatin-1), instead of inhibitors for other types of cell death (apoptosis, autophagy, and necroptosis), reversed the inhibitory effects of RB on CRC cell proliferation. Furthermore, glutathione peroxidase 4 (GPX4) was inactivated by RB treatment, and overexpression of GPX4 alleviated RB-induced oxidative cell death in CRC cells. Consistently, the in vivo experiments validated that RB also triggered oxidative stress, and inhibited CRC cells growth and tumorigenicity in mice models. RB can inhibit CRC cells growth and tumorigenesis by triggering ferroptotic cell death in a GPX4 inactivation-dependent manner.
The mechanistic target of rapamycin (mTOR) signaling pathway coordinates environmental and intracellular cues to control eukaryotic cell growth. As a pivot point between anabolic and catabolic processes, mTOR complex 1 (mTORC1) signaling has established roles in regulating metabolism, translation and autophagy. Hyperactivity of the mTOR pathway is associated with numerous human diseases, including diabetes, cancer and epilepsy. Pharmacological inhibition of the mTOR pathway can extend lifespan in a variety of model organisms. Given its broad control of essential cellular processes and clear relevance to human health, there is extensive interest in elucidating how upstream inputs regulate mTORC1 activation. In this Cell Science at a Glance article and accompanying poster, we summarize our understanding of how extracellular and intracellular signals feed into the mTOR pathway, how the lysosome acts as an mTOR signaling hub, and how downstream signaling controls autophagy and lysosome biogenesis.
Cysteine acts both as a building unit for protein translation and as the limiting substrate for glutathione synthesis to support the cellular antioxidant system. In addition to transporter-mediated uptake, cellular cysteine can also be synthesized from methionine through the transsulfuration pathway. Here, we investigate the regulation of transsulfuration and its role in sustaining cell proliferation upon extracellular cysteine limitation, a condition reported to occur in human tumors as they grow in size. We observed constitutive expression of transsulfuration enzymes in a subset of cancer cell lines, while in other cells, these enzymes are induced following cysteine deprivation. We show that both constitutive and inducible transsulfuration activities contribute to the cellular cysteine pool and redox homeostasis. The rate of transsulfuration is determined by the cellular capacity to conduct methylation reactions that convert S-adenosylmethionine to S-adenosylhomocysteine. Finally, our results demonstrate that transsulfuration-mediated cysteine synthesis is critical in promoting tumor growth in vivo.