In a recent study published in Science, Tsvetkov et al
1
. shed a light on a new form of cell death, copper-dependent cell death (termed cuproptosis).
They defined “cuproptosis” as a nonapoptotic cell death pathway. They have demonstrated
that copper directly binds to lipoylated components of the tricarboxylic acid (TCA)
cycle. Then aggregation of these copper-bound, lipoylated mitochondrial proteins and
subsequent Fe-S cluster protein loss triggered proteotoxic stress and a distinct form
of cell death.
As an essential cofactor, copper homeostasis is critical for various physiological
processes. Dysregulation of intracellular copper bioavailability can induce oxidative
stress and cytotoxicity
2
. In the animal kingdom, from prokaryotes to eukaryotes, copper homeostasis is finely
regulated mainly by preventing excessive accumulation of copper ions in cells which
threats cell survival. Pioneering studies have explored several cell death forms,
such as apoptosis, necroptosis, pyroptosis, and ferroptosis, however, the phenomenon
that copper overload causes cellular toxicity has been less elucidated. In terms of
the exact mechanism by which copper ions cause cell death, several hypotheses have
been proposed, including the induction of apoptosis, caspase-independent cell death,
the induction of reactive oxygen species (ROS), and inhibition of ubiquitin-proteasome
system
1
, etc. However, a well-acknowledged theory has not been clearly defined yet. Thus,
precise mechanisms of copper-induced cell death still need to be further elucidated.
In this study, Tsvetkov et al. firstly proposed “cuproptosis”, which depended on the
accumulation of copper in cells and was a unique cell death pathway distinct from
all other known ones. Copper ionophores (like elesclomol) are small molecular transporters
of copper ions into cells, which can be excellent tools to explore the mechanisms
of cytotoxicity of copper ions. This work illuminated that copper toxicity was highly
correlated with mitochondrial activity. Cells with active mitochondrial respiration
were more sensitive to elesclomol treatment than cells that rely on anaerobic glycolysis,
and the copper accumulation induced cell death with the participation of pivotal components
of the TCA cycle.
To determine the specific metabolic pathway of copper-mediated cytotoxicity, genome-wide
CRISPR-Cas9 loss-of-function screens were performed, followed by individual gene knockout
studies for further identification of key genes responsible for copper-induced cell
death. Authors found that the knockout of FDX1 (Ferredoxin 1, a direct target of copper
ionophores) or lipoylated enzymes could rescue cells from copper toxicity. Protein
lipoylation is a type of posttranslational modification, which is known to occur only
in metabolism-related molecules that involve the initiation process of the TCA cycle.
Furthermore, to explore the regulatory relationship among the screened genes, the
bioinformatics analysis, human tumor samples immunological analysis and gene knockdown
technology were carried out. Results suggested that as an upstream molecule, FDX1
regulated protein lipoylation. FDX1 knockout experiment suggested that FDX1 depletion
reduced protein lipoylation of DLAT and DLST (TCA cycle components). Then, the authors
demonstrated that DLAT and DLST no longer bound copper after the depletion of FDX1.
Naturally, the authors concluded that the lipoyl moiety was necessary for copper to
bind. The work also illuminated that copper bound directly to DLAT and led to subsequent
oligomerization of DLAT dependent on lipoylation. In addition, the mass spectrometric
analysis revealed that the cellular loss of Fe-S cluster proteins after elesclomol
treatment was dependent on FDX1 and facilitated by proteotoxic stress. Furthermore,
human embryonic kidney 293 T and ABC1 cells overexpressing SLC31A1 (a copper importer)
showed a higher cellular sensitivity to copper ions at physiological concentrations.
Finally, for in vivo verification of the mechanism of cuproptosis, a mouse model of
Wilson’s disease in which ATP7B (a copper exporter) was deleted was used, and the
result showed the same cytotoxicity as treatment of copper ionophores. A schematic
illustration of the cuproptosis mechanism has been presented at the end of this article
(Fig. 1).
Fig. 1
Schematic illustration of cuproptosis mechanism. The effect of intercellular copper
concentration on cell activity shows that cells maintain highly bioactive only within
a suitable range of extremely narrow copper ion concentrations (a). Elesclomol, a
copper ionophore, shuttles copper into the cells. FDX1 encodes a reductase to reduce
Cu2+ to Cu+ and is a direct target of elesclomol. DLAT, a protein target of lipoylation,
involves with mediating carbon entry to the TCA cycle. DLAT lipoylation was promoted
by FDX1, and Cu+ enhanced lipoylated protein aggregation and iron-sulfur cluster protein
reduction, which triggered proteotoxic stress and cell death (b).
Pioneering studies have reported that various metal ions can trigger cell death in
independent-apoptosis manners, such as phospholipid peroxidation-triggered ferroptosis
3
. However, the exact mechanism by which excessive copper induces cell death has rarely
been reported. The work by Tsvetkov et al. provided an in-depth analysis of the mechanisms
of cuproptosis, coupled with its potential pathophysiological functions. Copper, platinum,
and iron were widely studied in drug delivery systems development, which showed bright
prospects in an array of anticancer purposes
4
. Metal, like copper, and iron, were reported to be cytotoxic, thus care must be taken
to rigorously test the effects of metal ions themselves on biological processes. Beyond
that, there is a pressing need to explore if excessive accumulation leading to cell
damage is a common feature of all metals, especially for some metals commonly used
as drug carriers (gold, silicon, etc.).
Additionally, in this work, the authors indicated that mitochondrial glutathione (GSH,
a natural intracellular copper chaperone) decelerated copper-dependent cell death
via suppressing lipoylation and promoting DLAT oligomerization. Evidence suggested
that mitochondrial GSH, as a reductase, reduced oxidative phosphorylation, and depleted
mitochondrial GSH increased ROS generation precedes procaspase 3 activation (a critical
process of apoptosis)
5
. Therefore, whether a connection can be established between cuproptosis inhibition
mediated by GSH and apoptosis-related mechanisms remain to be clarified.
The field of cuproptosis is nonetheless nascent in many ways, thus further studies
are urgently needed to be carried out in the following aspects. (1) The molecular
mechanisms of copper toxicity in cancers need to be fully fleshed out. Additionally,
if cuproptosis evolves towards well-established cell death patterns remains to be
further illuminated. (2) Clinical trials of copper-induced cytotoxicity should be
based on either the discovery of biomarkers from appropriate patient populations or
in-depth knowledge of the molecule’s mechanism of action. (3) Given the differences
in the abundance of lipoylated proteins and respiratory patterns in various human
tumors, authors suggested that the precise range of concentrations that cause cytotoxicity
needs to be determined in various diseases model and even cell types, and then personalized
copper-based treatment strategies are reasonable and well-founded.
Taken together, this groundbreaking study unlocked a refreshing cell death pathway.
The discovery of “cuproptosis” provides a new avenue for anticancer treatments by
fully exploiting the pathophysiological role of copper. Additionally, this study inspires
us to further explore the cytotoxicity of other metals. Promisingly, cuproptosis translational
medicine might be a potential candidate in clinical applications after a series of
safety and efficacy tests, not only for genetic disorders, more importantly, for a
variety of human cancers.