The uptake and expulsion of copper by CRC cells is a complex process involving multiple mechanisms and proteins. In nature, copper mainly exists as Cu²⁺, but the human body primarily absorbs Cu⁺. Metal reductases like six-transmembrane epithelial antigen of the prostate (STEAP) and duodenal cytochrome b convert bivalent copper to monovalent copper, enhancing its uptake into cells through specific transport proteins at the cell membrane (18, 19). These transporters comprise copper transport proteins (CTRs) and copper ion transport ATPases (ATP7A and ATP7B) (20). After copper enters the cells, a portion is transported through ATP7A and ATP7B, then enriched and stored in the Golgi apparatus and vesicles (21, 22). Copper is primarily excreted from the body through the bile and then eliminated via the intestine (23). At the cellular level, the copper transporter ATP7A/B plays a crucial role in facilitating copper excretion by removing excess copper from the cells. Under normal conditions, these transporters are located in the trans-Golgi network (TGN), where they move copper ions from the cytoplasm into the TGN lumen. With increased copper exposure, ATP7A and ATP7B relocate to either the plasma membrane or the intracellular vesicle compartment. Meanwhile, ATP7A and ATP7B shuttle copper from the trans-Golgi network to the post-Golgi vesicles. These vesicles containing copper can merge with the cell membrane, releasing copper into the surrounding environment (24, 25). Furthermore, in the colorectal tract, copper is reabsorbed through specific transport proteins such as CTR1 to maintain the body’s copper balance.

Under physiological conditions, copper enters the cell membrane and is transported to various cellular substructures, including the Golgi apparatus, cytoplasm, mitochondria, and nucleus (26). The cytoplasm contains high concentrations of glutathione (GSH) and metallothionein (MT), which are natural chelators of copper ions (27, 28). These proteins are more numerous than copper ions, so most of the copper in the cytoplasm is in a chelated state. In addition, chelated copper is not free in the cytosol, but is mediated by ATP7B for vesicle sequestration (29). First, sequestrated copper can maintain a negative concentration gradient of the cytoplasmic membrane, thereby promoting copper uptake by CTR1 along the concentration gradient (30). Second, sequestrated copper can stabilize the concentration of free copper ions, prevent cytotoxicity, and produce reactive oxygen species (ROS) (31, 32). MT and GSH form an intrinsic defense mechanism against copper-induced cytotoxicity. In addition, copper ions can bind to chaperone proteins, specifically the copper chaperone of superoxide dismutase (CCS), which interacts with copper ions and transports them to superoxide dismutase 1 (SOD1), promoting the formation of disulfide bonds, which are essential for their proper structure and enzymatic activity (33, 34). In addition, CCS regulates the distribution of SOD1 in the intermembrane space and cytoplasm in an oxygen-dependent manner. This regulatory mechanism is essential to maintain ROS levels in vivo and reduce ROS production by electron transport chains, thereby preventing oxidative damage caused by copper overload (35, 36). Notably, copper sequestration can affect copper concentrations in the tumor microenvironment, thereby affecting the aggressiveness and metastasis of tumor cells (37). The role of copper in angiogenesis and inflammatory response may also be influenced by sequestration, which is the key factor in tumor progression (38, 39).

Copper plays a complex and important role in the proliferation of CRC, and this copper-dependent growth phenomenon is known as cuproplasia, which promotes CRC cell proliferation by promoting a series of enzymatic or non-enzymatic reactions in CRC cells (40). Moreover, copper is also closely associated with the spread of CRC (41).

Copper is a cofactor for many enzymes and is involved in key processes such as cellular respiration and antioxidant defense. Excessive accumulation of copper causes oxidative stress that can lead to the degradation of key cancer-suppressor proteins, including p53, affecting cell cycle regulation and inhibiting programmed cell death, thereby improving the viability of CRC cells (42). In addition, Copper can allosterically activate the E2 binding enzyme UBE2D1-UBE2D4, leading to the binding of numerous proteins (including p53) for ubiquitination and degradation (43). Otherwise, copper enzymes activate mammalian kinases such as ULK1 and ULK2, which are downstream targets of the nutrient-sensing kinase mTOR (target of rapamycin) and targets of copper-dependent kinases (44). Notably, disinhibition of ULK1 and ULK2 induces autophagy, allowing CRC cells to recycle intracellular components to support biosynthesis and bioenergy supply, particularly important in the TME (45, 46).

Epithelial-mesenchymal transition (EMT) play a crucial role in the metastasis of CRC, and both processes are closely linked to copper and copper-binding proteins. Copper is essential for the lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) proteins involved in the crosslinking of collagen and elastin. Cancer cells secrete LOX that remodels the extracellular matrix to form pre-metastatic niches, thereby recruiting bone marrow-derived cells that promote EMT in CRC (47, 48). Moreover, the interaction between the copper-mediated hypoxia response element and HIF-1α promotes EMT in CRC through CCS activating the transcription factors ZEB 1, ZEB 2, and Snail (49). Additionally, antioxidant 1 copper chaperone (ATOX1), a copper homeostasis factor, enters the nucleus to activate transcription. This promotes the expression of Cyclin D1 and NADPH oxidase subunit p47phox, critical for cell proliferation and response to oxidative stress. ATOX1 is also a vital copper chaperone protein in human cells that helps maintain copper balance. It carries copper ions from the cytoplasm to the secretory pathway, which is essential for activating copper-dependent enzymes involved in neurotransmitter biosynthesis, iron efflux, neoangiogenesis, wound healing, and blood pressure regulation (50–52). ATOX1 plays a role in regulating the distribution of copper ions within cells, ensuring they are properly allocated to the organelles and molecules that need them. This process helps prevent the improper accumulation and toxicity of copper (53). In addition, ATOX1 protects cells from oxidative stress damage, potentially related to or independent of its copper chaperone role (54). ATOX1 is involved in the regulation and elimination of cellular copper load by promoting the transfer of copper ions to the secretory pathway through its interaction with copper-transporting ATPases such as ATP7A and ATP7B (55). The activity of ATOX1 is affected by the cell’s redox state and GSH balance, as cysteine residues can form disulfide bonds regulated by GSH and glutathione reductase (Grx1) (56). ATOX1 may also be involved in the transfer of copper ions from the copper transporter CTR1 on the cell membrane to intracellular copper chaperone, which is essential for intracellular transport of copper ions (57).

Angiogenesis is crucial for tumor growth and metastasis, with vascular endothelial growth factor regulating vascular growth. Copper ions play a key role in the early stages of tumor vascular formation (38, 58). The activation of angiogenic factors includes basic fibroblast growth factor, vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha, and interleukin-1 (IL-1). These factors, when combined with endothelial cells, promote their transition from G0 to G1 and induce proliferation (59). Moreover, copper ions can increase the production of vasodilator nitric oxide and promote angiogenesis by altering the activity of endothelial nitric oxide synthase (60).

From the perspective of tumor proliferation and metastasis, CRC seems to maintain the abnormal state of copper metabolism deliberately. CRC maintains high concentrations of copper ions in CRC cells through a series of copper transporters and copper chaperones (20, 53). This is due to the extensive involvement of copper in the maintenance of the microenvironment, angiogenesis, and rapid proliferation in CRC progression (6). This kind of copper enrichment to promote self-proliferation is also called copper proliferation. However, the over-enrichment of copper puts CRC in a very dangerous situation due to the presence of cuproptosis, as excess copper can lead to protein aggregation by binding to the lipid acylated components of the tricarboxylic acid cycle (TCA), which in turn leads to the loss of iron-sulfur protein clusters and ultimately to protein toxic stress and cell death (11).

Copper is a heavy metal. Like other heavy metals, copper has biological activity that leads to protein denaturation. However, this does not fully explain the biological toxicity caused by excess copper in cells. Excessive copper-promoting cuproptosis is a complex process. Specifically, Copper enters cells via transporters like SLC31A1/CTR1 and SLC31A2/CTR2 (61), causing copper overload. In that case, copper can enter mitochondria and directly bind to the TCA FDX1-modified TCAT in mitochondrial respiration, to induce the oligomerization of lipoylated dihydro thioamide S-acetyltransferase (DLAT). Oligomerization of lipoylated DLAT causes cytotoxicity and induced cell death. In addition, the reduced ferredoxin 1 (FDX1) reduces the bivalent copper to the monovalent copper, which has greater cytotoxicity and can induce conformational changes in the Fe-S cluster protein, which is more unstable (62). Under the double action, the cells undergo proteotoxic stress, which eventually leads to cell death ( Figure 2 ).

Sixteen regulators of cuproptosis were identified, including FDX1, MTF1, DBT, CDKN2A, DLST, DLAT, LIPT1, LIAS, GLS, DLD, PDHA1, PDHB, GCSH, SLC31A1, ATP7A, and ATP7B (11). All of these proteins play important roles in the proliferation and metastasis of CRC cells. At the same time, these known molecules associated with copper death can play an important role in predicting CRC progression. For example, low expression of FDX1 in CRC was associated with a poor prognosis. Analysis of the immune microenvironment showed a significantly lower proportion of CD8+ T cells than in adjacent normal tissues, and the opposite proportion of CD4+ T cells (63).

Non-coding RNAs (ncRNAs) are RNA molecules that do not code for proteins but instead play various regulatory roles in gene expression and cellular processes (64–67). Based on their sizes, tructure, and function, ncRNAs can be classified into different categories based on their size, such as miRNAs, lncRNAs, small siRNAs, and circRNAs. Non-coding RNAs (ncRNAs) related to cuproptosis play crucial roles in the regulation process of CRC cells ( Table 1 ). These RNAs include microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), which participate in cuproptosis regulation through different mechanisms and play crucial role in the occurrence of cuproptosis in CRC cells and their prognosis prediction (68–70).

Compared to normal cells, CRC cells have significant differences in biochemical metabolism. Due to the Warburg effect, tumor cells continue to use glycolysis for energy even in the presence of oxygen. This phenomenon is also known as aerobic glycolysis or biochemical reprogramming (76). This effect often leads to the accumulation of lactic acid. The change in biochemical reactions decreases oxygen demand, which further reduces oxygen content in the TME. CRC cells require copper for their growth and spread. These cells often have high levels of copper ionophores, such as ATP7A/B, to absorb and retain copper (77). In addition, the intestine, as the human body absorbs copper, has a higher copper content than other tissues (78). This dual action results in a high concentration of copper in the TME of CRC.

In the unique biochemical environment surrounding tumor cells, oxygen levels are often low, leading to oxidative stress and disruption of the redox balance in CRC cells (79, 80). Under normal circumstances, cells maintain internal stability through a series of redox reactions to adapt to environmental changes and sustain vital energy processes. However, CRC cells may experience redox imbalance and this imbalance can manifest as increased oxidative stress, where the generation of oxidative species exceeds the capacity of antioxidant defense systems, consequently elevating intracellular oxidative stress levels. Of particular concern is the disruption of the GSH system, which is crucial for the transition of copper valence in CRC cells (81). This heightened oxidative stress can have diverse effects on CRC cells, including increased sensitivity to copper (82, 83). In conditions of oxidative stress, copper’s reactivity may be enhanced, facilitating its interaction with cuproptosis-targeting proteins within the cell, thereby augmenting sensitivity to copper.

Elevated levels of copper in both serum and tissue samples from patients with CRC cell lines indicate a necessity for copper in tumor proliferation. Dysregulation of copper homeostasis and the resultant excess cuproplasia emerge as pivotal factors in CRC development (40, 84, 85). Copper homeostasis is a negative feedback regulation mode that actively regulates the intracellular copper concentration. In general, copper homeostasis is very stable and not easy to destroy, but around CRC cells, due to their copper tropism, the copper content is often an order of magnitude higher than in normal cells. At such high copper concentrations, CRC cells are in fact on the verge of undergoing cuproptosis. Therefore, in an environment with more foreign copper, CRC cells will quickly uptake excessive copper, triggering the process of cuproptosis, and leading to cell death (86). Consequently, in environments abundant with exogenous copper, CRC cells swiftly internalize excessive copper, triggering the process of cuproptosis and eventual cell demise.

Cuproptosis is a completely new mode of cell death, and treatment with other inhibitors of known cell death mechanisms — including ferroptosis, necroptosis, and oxidative stress (87–89) — has failed to eliminate copper ionophore-induced cell death. Due to drug resistance, current treatments like chemotherapy, radiotherapy, immunotherapy, and targeted therapy often do not effectively combat colorectal cancer (2). However, a cuproptosis-based treatment could effectively prevent this. First, the cause of cuproptosis is an overload of copper ions in cells. However, CRC cells require high levels of copper to maintain their proliferation and metastasis, making it difficult to eliminate this cause. Secondly, the sites of cuproptosis, such as FDX1 and DLAT, are relatively conserved. This suggests that CRC cells may struggle to evade cuproptosis through mutations (11). The cuproptosis pathway offers advantages such as a brief duration, rapid progression, widespread occurrence, and easy pharmaceutical targeting, demonstrating its potential for treating colorectal cancer.

Glyceraldehyde-3-phosphate dehydrogena (GAPDH) is a key enzyme in the sixth step of glycolysis, catalyzing the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate (90). In the process of glycolysis, glucose enters cells via GLUT and is converted to 6-phosphogluconate under the action of hexokinase (91). Subsequently, it is isomerized to fructose-6-phosphate by phosphoglucose isomerase. Then, glyceraldehyde-3-phosphate (GAP) is formed under the catalysis of phosphofructokinase (PFK) (92). However, for glycolysis products to enter the mitochondria and participate in the TCA cycle, they must be converted to pyruvate by GAPDH before further conversion to acetyl-CoA to enter the mitochondria. This highlights the significance of GAPDH in converting glucose into the universal reactant acetyl-CoA in cells ( Figure 3 ). Numerous studies have confirmed that it is a potential therapeutic approach to inhibit aerobic glycolysis in tumor cells (93–95).

GAPDH is an important enzyme in aerobic glycolysis. If the activity of GAPDH is inhibited, it will directly inhibit the whole aerobic glycolysis, making the energy produced by yeast greatly reduced (96–98). In CRC, due to their special biochemical environment (99, 100), tumor cell cells mainly rely on glycolysis for energy, so inhibiting the activity of GAPDH will directly lead to the reduction in energy gain leading to CRC. In the case of energy shortage, the metabolic speed in CRC is weakened, and the copper in cells will not be excluded by ATP7A/B, which will cause copper retention in cells, leading to the destruction of copper homeostasis, and eventually excessive copper accumulation and cuproptosis. In addition, the inhibition of GAPDH will also reduce acetyl-CoA in CRC. In such cells, both protein synthesis and DNA replication will slow down, leading to the decreased proliferation, diffusion and stress resistance of CRC. When cuproptosis occurs in CRC, cells will be unable to remove the lipoylated proteins that are aggregation and allowing them to exert toxic effects. Overall, the CRC showed an increased sensitivity to cuproptosis.

In fact, under normal circumstances, tumor cells produce energy through glycolysis, while mitochondrial respiration is strictly inhibite (101). However, this does not mean that tumor cells cannot undergo mitochondrial respiration. In the case of GAPDH inhibition, CRC compensates for the lack of ATP and acetyl-CoA by engaging in aerobic metabolism such as aerobic respiration and lipid metabolism (102). Under this circumstance, due to the opening of aerobic respiration, increased permeability of the mitochondrial membrane, and changes in the redox environment in cells, Cu (II) ions are more easily reduced to Cu (I) ions and enter mitochondria to trigger cuproptosis.

In the first step of cuproptosis, the copper ionophore mediate the entry of copper ions into cells, playing an extremely important role in the development of cell cuproptosis. Among them, SLC31A1, responsible for transporting Cu (I) ions, plays a pivotal role (61). On the surface of tumor cells, Cu (II) ions are reduced to Cu (I) ions by STEAP, after which SLC31A1 facilitates their entry into the cell, triggering subsequent reactions (103). Experimental evidence has demonstrated that the use of CTR inhibitors can significantly alleviate the extent of cuproptosis, underscoring the regulatory role of CTR in cuproptosis (104).

Mitochondria are the main targets of cuproptosis, characterized by oxidatively damaging mitochondrial membranes and impaired enzyme function in the TCA cycle (105–107). Metabolomic analysis of cells treated with copper ions reveals a time-dependent increase in the dysregulation of several TCA cycle-related metabolites. Additionally, significant attenuation of cuproptosis is observed upon inhibition of electron transport chain complexes I and II. Furthermore, copper ions alter a range of metabolic enzymes by lipoylation (11), a highly conserved posttranslational modification, and all lipidated proteins participate in the TCA cycle, although relatively few proteins are lipidated in mammalian cells. One such lipidated protein is DLAT, a subunit of the pyruvate dehydrogenase complex, and copper can directly bind to DLAT to promote the disulfide bond-dependent aggregation of lipidated DLAT (108, 109) ( Figure 4 ). When tumor cells are in hypoxia, mitochondrial respiration is inhibited, and copper is difficult to enter mitochondria, and cuproptosis is greatly inhibited.

FDX1 is a very small iron-sulfur protein that transfers electrons from NADPH to mitochondrial cytochrome P450 by ferredoxin reductase, involved in the aerobic metabolism of steroids, vitamin D, and bile acids (110, 111). It can not only play the role of electron transport but also can reduce the Cu (II) to the more toxic form (112, 113). The researchers found that if the FDX1 gene is knocked down with CRISPR-Cas9, not only will mitochondrial respiration be inhibited, but copper will be more difficult to enter mitochondria, but copper will also weaken its toxicity due to its inability, thus greatly alleviating the effect of cuproptosis on cells (114).

In addition, protein lipoylation also plays a key role in the cuproptosis process (9, 11, 115, 116). Lipoylation is not common in the biochemical environment of the human body. The proteins that can undergo lipoylation are concentrated in mitochondrial respiration, mainly TCA (117), among which the main roles are LIPT1, DLD, LIAS, DLAT, PDHA1, and PDHB (118, 119). In the cuproptosis process, TCA related proteins are lipoylated and aggregated, resulting in the stalling of whole mitochondrial respiration and great biotoxicity (120). Knockout of the above protein genes can rescue the cell toxicity of copper ions.

It should be noted that FDX1 is an upstream regulator of protein lipoylation throughout, and in fact that lipoylation of proteins is inaccessible in the absence of FDX1. FDX1, as an important component of the electron transport chain, does not work without mitochondrial respiration (111, 121), which also shows that the necessary condition for cuproptosis is mitochondrial respiration.

Immunotherapy of PD-1 with PD-L1 is an immunotherapy strategy used to enhance the aggression of the immune system against cancer. Programmed cell death protein 1(PD-1) is a cell-surface receptor, whereas programmed death-ligand 1(PD-L1) is its ligand (122, 123). Under normal circumstances, the binding of PD-1 to PD-L1 can inhibit the activity of T cells, thus preventing the excessive activation of the immune system and causing its own tissue damage (124). However, in some cancers, tumor cells or other cells will overexpress PD-L1 to inhibit the surrounding T cells by binding to PD-1 to evade immune surveillance (125). The rationale of PD-1 and PD-L1 immunotherapy is to use antibodies against anti-PD-1 or anti-PD-L1 to block the binding between PD-1 and PD-L1, thus releasing the inhibitory effect on T cells and enhancing the tumor attack of the immune system (122, 123). This therapeutic strategy has been shown to be effective in a variety of cancers, including melanoma, non-small cell lung cancer, CRC, etc. However, in the actual treatment, the effect of PD-1 and PD-L1 immunotherapy is not effective, because the tumor cells will continuously make PD-1 to the cell surface.

For PD-1/PD-L1 immunotherapy to continue to work, it needs to inhibit tumor cells to continuously make PD-1. This effect can be achieved by drugs that induce cuproptosis, which reduces PD-1 expression by promoting tumor cell death or interfering with material synthesis in the cells. In addition, the strong specificity between PD-1 and PD-L1 makes it possible to make relevant targeted drugs, which will greatly improve the anticancer properties of drugs and reduce the damage to normal cells in the human body. In the latest study, researchers developed a sodium alginate hydrogel composed of sodium dichloroacetate copper and galactose to induce sustained atrophy, resulting in a reduction of PD-L1 on the surface of CRC cells. When implanted into the tumor, the preformed hydrogel can further crosslink in the presence of physiological calcium ions (Ca²⁺), forming a hydrogel that controls the release of elesclomol-Cu²⁺ (ES-Cu) and galactose. This hydrogel effectively induces DLAT oligomerization and copper-induced cell death in CRC cells. Additionally, the radiation-induced upregulation of PD-L1 is abrogated in the presence of the hydrogel, which releases ES-Cu and galactose. Consequently, the tumor’s sensitivity to radiotherapy and immunotherapy is significantly enhanced, further prolonging the survival of tumor-bearing mice with both local and metastatic tumors (126).

Chemotherapy is the cornerstone of treatment for CRC. After years of development, several effective drugs, including capecitabine, 5-fluorouracil, and oxaliplatin, are widely used in clinical practice (127). In many clinical treatments, drug resistance of CRC often occurs, leading to treatment failure (128). This is due to the unique microenvironment and metabolic pattern of CRC. Treatment targeting cuproptosis can avoid and even utilize the microenvironment characteristics of CRC. While conventional chemotherapeutic agents typically work by blocking or interfering with DNA replication, cuproptosis operates through a different mechanism, that is, excess copper can lead to protein aggregation by binding to the lipid-acylated components of the TCA, which in turn leads to the loss of iron-sulfur protein clusters and ultimately leads to protein toxic stress and cell death (129). Additionally, there is a type of drug that can enhance cuproptosis by targeting GAPDH, and these drugs can block glycolysis, reduce the energy and raw material source needed for DNA replication, and inhibit the proliferation of CRC cells (130). Based on this, the combination of traditional chemotherapy and targeted cuproptosis drugs will greatly improve the effect of traditional therapy.