Cells and copper have a finicky relationship. While the trace metal is essential for many cellular processes, too much copper is toxic to the cell. Copper deficiency is the hallmark of the rare disorder Menkes syndrome, while Wilson’s disease is characterized by excess copper. In cancer cells, some copper is needed as a nutrient to support proliferation (a phenomenon known as cuproplasia), while surplus copper can cause cell death (1).

Drugs that manipulate copper levels are used to treat copper regulation disorders, but they also enable researchers to exploit the cell’s finely-tuned copper requirements for other therapeutic applications. For example, copper ionophores that bind to copper and release it inside the cell may be used to promote copper toxicity and kill cancer cells. While these drugs have been explored for cancer treatment, they have shown inconsistent results, motivating efforts to better understand how copper causes cell death. 

In a recent study in Science, researchers mapped the series of events from copper’s entry into a cell to the cell’s death (2). They discovered that copper triggers a unique form of cell death, which they term cuproptosis, by binding to a discrete set of proteins involved in the tricarboxylic acid (TCA) cycle. By identifying the cellular targets of copper toxicity, this work can guide the use of copper-transporting drugs for treating various diseases. 

The researchers first established that cell death depends on the copper metal itself rather than the copper ionophore drug. When they removed copper from the cell culture media, a potent copper ionophore molecule could not induce cell death on its own, demonstrating that “it's all about bringing the copper into the cell and not some other activity of the molecule,” said Peter Tsvetkov, a biochemist and cell and molecular biologist at the Broad Institute of MIT and Harvard and coauthor of the study. 

The researchers treated cells with the copper ionophore in the nanomolar range for a few hours, which resulted in cell death more than one day later. Bombarding the cells with high concentrations of copper “is just like spilling bleach; it hits everything, and it's not specific,” Tsvetkov said. “If you throw bleach on the cells, they will die, but that doesn't mean that there is a biological cascade of events that causes this cell death.” In contrast, the drawn-out cell death at low concentrations of drug indicates that copper induces cytotoxicity through a regulated biological pathway. 

This sequence of cellular events is distinct from known forms of cell death such as apoptosis, necroptosis, and ferroptosis. “If we chemically or genetically inhibit all of these pathways, we still cannot inhibit this copper-induced cell death, suggesting that it's something different,” Tsvetkov said. 

This mysterious pathway began to unravel as the team observed that when cells rely more heavily on mitochondrial respiration than glycolysis to produce energy, they are nearly 1,000 times more susceptible to copper toxicity. 

To pinpoint the exact proteins involved, the team used CRISPR-Cas9 to individually delete every gene in an ovarian cancer cell’s genome, then screened the library of cells against two copper-loaded ionophores. They identified seven genes where deletion rescued cells from death upon treatment with either ionophore, indicating that they play a role in the mechanism of copper toxicity. All of the genes encoded proteins that are either regulators or targets of lipoylation, a post-translational modification of lysine with an organosulfur lipoyl group. Lipoylation is known to occur on only four proteins in the entire human proteome, all of which belong to metabolic complexes that regulate entry into the TCA cycle (3). 

Given that copper binds tightly to free lipoic acid, the researchers measured the affinity of copper for proteins that undergo lipoylation and found that binding occurred only when proteins were modified with a lipoyl group. When copper bound to lipoylated dihydrolipoamide S-acetyltransferase (DLAT), a component of the TCA cycle, the protein formed clumps, leading the researchers to propose that copper directly binds to lipoylated TCA cycle proteins and causes them to aggregate, placing stress on the cell that ultimately culminates in death. Proteomic analysis revealed that copper ionophore treatment leads to a loss of iron-sulfur cluster proteins across the cell, which could be a separate or related manifestation of copper toxicity.       

Finally, the researchers examined protein levels resulting from excess intracellular copper in a mouse model of Wilson’s disease. They observed a decrease in lipoylated and iron-sulfur cluster proteins, suggesting that natural and drug-based forms of copper overload have a similar effect on the cell.   

“It’s really an integrative study using high-throughput-omics approaches to pinpoint this one pathway in mitochondria that is responsible for copper-mediated death,” said Vishal Gohil, a biochemist at Texas A&M University who was not involved in the study. “In a bigger picture, the advancement is that this paper identified this new target of copper toxicity.” While the study does convincingly demonstrate that copper causes lipoylated proteins to aggregate, the question of how exactly that leads to cell death remains unanswered, Gohil said.

 “I hope that similar work in the future by us and others will elaborate more on what the downstream processes are,” Tsvetkov said. 

According to Gohil, the possibility that copper toxicity in Wilson’s disease occurs through this mechanism should be investigated in greater detail. “We do not know fully how physiologically relevant cuproptosis is in settings of cell development and differentiation,” he said. 

By identifying aggregated lipoylated proteins as a sign of copper toxicity, this work can help inform safe ionophore treatment of copper deficiency in Menkes syndrome. “You want to ensure that you don't give too much copper, triggering this cuproptosis,” Gohil said. “So really, the biggest contribution of this study is that we now have a new biomarker of copper toxicity.” 

Understanding the mechanism of copper-induced cytotoxicity may also help identify the types of tumors most sensitive to copper ionophore treatment and guide clinical selection criteria. A clinical trial testing a copper ionophore as part of a combination therapy for melanoma showed poor efficacy overall, but revealed some evidence of activity in tumors with specific metabolic profiles (4). These tumors had shifted toward mitochondrial respiration to grow, making them more responsive to TCA cycle-targeting cuproptosis. “If you know how a drug works, you can potentially find the patient populations that are particularly dependent on this mechanism,” Tsvetkov said. 

For Tsvetkov, uncovering cuproptosis revealed a wealth of shiny new research directions. “I find it super exciting that we did this unbiased genomic screen and we stumbled upon a pathway that I've never heard about before,” he said. “There's a lot of room for new discovery in this interface of metal homeostasis, in particular copper, and cellular metabolism.”

References

1. Ge, E.J. et al. Connecting copper and cancer: From transition metal signalling to metalloplasia. Nat Rev Cancer  22, 102-113 (2022). 
2. Tsvetkov, P. et al. Copper induces cell death by targeting lipoylated TCA cycle proteins. Science  375, 1254-1261 (2022). 
3. Rowland, E.A., Snowden, C.K., & Cristea, I.M. Protein lipoylation: An evolutionarily conserved metabolic regulator of health and disease. Curr Opin Chem Biol  42, 76-85 (2018). 
4. O’Day, S.J. et al. Final results of phase III SYMMETRY study: Randomized, double-blind trial of elesclomol plus paclitaxel versus paclitaxel alone as treatment for chemotherapy-naive patients with advanced melanoma. J Clin Oncol  31, 1211-1218 (2013).