Cuproplasia and cuproptosis in hepatocellular carcinoma: mechanisms, relationship and potential role in tumor microenvironment and treatment

Abstract

Background

Hepatocellular carcinoma (HCC) is the main phenotype of liver cancer with a poor prognosis. Copper is vital in liver function, and HCC cells rely on it for growth and metastasis, leading to cuproplasia. Excessive copper can induce cell death, termed cuproptosis. Tumor microenvironment (TME) is pivotal in HCC, especially in immunotherapy, and copper is closely related to the TME pathogenesis. However, how these two mechanisms contribute to the TME is intriguing.

Main body

We conducted the latest progress literature on cuproplasia and cuproptosis in HCC, and summarized their specific roles in TME and treatment strategies. The mechanisms of cuproplasia and cuproptosis and their relationship and role in TME have been deeply summarized. Cuproplasia fosters TME formation, angiogenesis, and metastasis, whereas cuproptosis may alleviate mitochondrial dysfunction and hypoxic conditions in the TME. Inhibiting cuproplasia and enhancing cuproptosis in HCC are essential for achieving therapeutic efficacy in HCC.

Conclusion

An in-depth analysis of cuproplasia and cuproptosis mechanisms within the TME of HCC unveils their opposing nature and their impact on copper regulation. Grasping the equilibrium between these two factors is crucial for a deeper understanding of HCC mechanisms to shed light on novel directions in treating HCC.

Introduction

Liver cancer poses a serious global health challenge and exhibits the most rapid rise in mortality in decades [1]. According to the latest cancer statistics in the United States, liver cancer ranks fourth in mortality among men and seventh among women [1]. Hepatocellular carcinoma (HCC) accounts for approximately 90% of cases, making it the predominant subtype of liver cancer [2]. Hepatitis B virus, hepatitis C virus infection, nonalcoholic steatohepatitis, aflatoxin exposure, liver cirrhosis, autoimmune liver disease, and metabolic syndrome are prominent risk factors for HCC, [3–6] especially Hepatitis B virus infection in China and nonalcoholic steatohepatitis in the West [7]. Despite advancements in drug development and treatment protocols, the prognosis of HCC patients has improved but remains relatively poor [8]. Furthermore, the specific molecular mechanisms underlying HCC are not well established, highlighting the pressing need for predictive biomarkers and novel targeted therapies for the diagnosis and treatment of HCC.

Copper is an essential trace element for the human body. Copper, serving as an indispensable coenzyme for metabolic enzymes, participates in a series of physiological processes such as respiration, lipolysis, cell growth, and proliferation [9]. Cancer, especially HCC, exhibits an increased demand for copper attributed to tumor growth and metastasis [9, 10]. Serum copper levels are notably higher in HCC patients compared to healthy individuals [11]. Elevated copper levels directly correlate with HCC progression, leading to cuproplasia [12]. Cuproplasia, characterized by copper-dependent cell growth and proliferation, manifests itself as copper-dependent tumor formation and development [9]. Nevertheless, cellular copper levels demonstrate a bidirectional, U-shaped dose–response relationship [13]. Copper's inherent redox properties render it beneficial yet potentially toxic to cells. Excessive extracellular and intracellular copper can lead to kidney disease, liver disease, and brain damage [14, 15]. Consequently, increased copper levels in HCC tissues also induce cuproptosis, potentially eliminating cancerous cells. Cuproptosis was first discovered by Tsvetkov and his colleagues, who identified a novel form of regulated cell death induced by copper, distinct from the known cell death modes such as necroptosis, apoptosis, and ferroptosis. They coined the term ‘cuproptosis’ [16]. This discovery has been published in science and has garnered considerable attention in scientific research. The prevalent hypoxic conditions within the tumor microenvironment (TME) influence copper metabolism in HCC tissue [17].

This review concentrates on elucidating the mechanisms of cuproplasia and cuproptosis in HCC, along with their implications in HCC prognosis, hoping this insight will steer novel avenues for the diagnosis and treatment of HCC.

Copper metabolism and role in HCC

Copper metabolism is primarily regulated by the liver [18]. Humans typically contain approximately 80 mg of copper, predominantly distributed among vital organs such as the liver, brain, and eyes [19]. Daily dietary intake provides 1.3 mg of copper, with 0.8 mg directed to hepatic circulation [19]. Copper is an indispensable trace metal, acting as a cofactor for various copper-dependent enzymes, notably respiratory enzymes in mitochondria (cytochrome c oxidase(COX)). Its involvement extends to crucial physiological processes such as lipolysis ( phosphodiesterase 3B), [9] crosslinks of elastin and collagen (lysyl oxidase (LOX) and LOXL2), [20, 21] cell growth and proliferation (mitogen-activated protein kinase kinase 1 (MEK1) and MEK2), [9] autophagy (the kinases Unc-51 Like Autophagy Activating Kinase 1 (ULK1) and ULK2), [22] iron absorption and transport (ceruloplasmin), [23] signal transduction (dopamine β-hydroxylase), [24] reactive oxygen species (superoxide dismutase (SOD), glutathione (GSH)), epigenetic modification (LOXL2), leukocyte trafficking (amine oxidase copper containing 3), [25] as well as playing a role in cardiovascular, nervous, and immune systems [14]. Dynamic fluctuations in copper levels within the body orchestrate the changes in external stimuli and biological states to control and regulate biological functions, underscoring the significance of copper homeostasis in sustaining normal physiological processes.

However, copper ion homeostasis can be easily disrupted. Copper deficiency impairs the activity of copper-dependent enzymes, affecting energy metabolism, glucose tolerance, immune responses, and the antioxidant defense system, culminating in oxidative stress-induced damage. Conversely, excess copper can cause cell damage mainly by enhancing free radicals to exacerbate oxidative stress and DNA damage, ultimately fostering malignant transformations [26]. Notably, perturbations in copper homeostasis correlate with chemotherapy resistance and immune checkpoint dysregulation [27, 28]. Abnormal copper homeostasis may lead to neurodegenerative diseases [29], metabolic diseases [30], cardiovascular diseases [31], tumors [32] and other multi-system diseases. Studies have demonstrated the association between elevated serum copper levels and various tumors, [32] elucidating the multifaceted involvement of copper in tumorigenesis. For HCC fields, Caroline I. Davis et al. have demonstrated the vulnerability of copper homeostasis in HCC [33]. Increased serum copper content may promote the progression from cirrhosis to HCC [34]. P. Dongiovanni has shown that increased copper concentration in HCC is positively correlated with oxidative stress, impacting the occurrence and development of tumors [35]. C. Porcu's study has shown that high copper concentrations regulate the Copper Transport Protein 1 axis, promoting the growth, migration, and invasion of liver cancer cells [34]. Xianglong Liu et al. studied the differences between high copper and low copper phenotypes in HCC, showing that compared with low copper subtype, patients with high copper subtype had significantly abnormal immune function, a higher probability of gene mutation, and significantly weaker sensitivity and reactivity to chemotherapy drugs [36]. The specific mechanisms of copper in HCC are described as follows.

Cuproplasia and HCC

The mechanism of cuproplasia in HCC

Elevated copper levels pose a heightened risk of HCC, [34, 37] with HCC necessitating higher concentrations of copper than normal cells for tumor growth and proliferation [9]. Consequently, the concept of cuproplasia emerged. Cuproplasia is characterized by copper-dependent cell growth and proliferation, [9] encompassing neoplasia and hyperplasia, as well as the primary and secondary effects of copper [38]. The mechanism of cuproplasia has been described in Fig. 1. Mechanistically, existing studies have shown that cuproplasia drives tumor progression via the following mechanisms: (1) Elevated copper levels affect glycolysis, lipid metabolism, gluconeogenesis, collagen crosslinking, autophagy and other biological processes (2) Copper assists mitochondrial COX activity, promoting reactive oxygen species (ROS) production and tumor cell proliferation; [39, 40] (3) Copper regulation of signaling pathways like the Antioxidant protein 1 (ATOX)- Adenosine 5'-triphosphatase copper transporting alpha (ATP7A)-LOX pathway enhances metastasis and expansion; [41] (4) Copper activation of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and tumor necrosis factor (TNF) promotes tumor angiogenesis; [9] (5) Copper-induced immune checkpoint expression, such as programmed death-ligand 1 (PD-L1), [42] aids in evading immune injury, ultimately leading to tumor development and chemotherapy resistance [36].

Fig. 1

The mechanism of cuproplasia in HCC. The cuproplasia is defined as the copper-dependent cell growth and proliferation, containing both neoplasia and hyperplasia. Copper is translated by the SLC31A1, and ATP7B and forms into the labile pool. (1) Copper could decrease the level of G6PDH and GRD, leading to the production of ROS and decreased antioxidant function, thus increasing the risk of genetic mutations in hepatocytes. Besides, through the BRAF-RAS-RAF-MEK-ERK and PI3K-PDK1-PKB signaling pathways, the risk of genetic mutations is increasing collectively in order to contribute to neoplasia. (2) Copper could promote hyperplasia through the mitochondrial pathways and non-mitochondrial pathways. Copper is the cofactor of various respiratory enzymes, the elevated copper could increase the number and the function of the respiratory enzymes in mitochondria such as the CCS, SOD1, COX, and ATOX1, thus the mitochondrial respiration is enhanced. Besides, copper could inhibit the PDE, which could degrade the cAMP. Thus, more triglycerides are transformed into glycerol and fatty acids through cAMP pathway, increasing the lipolysis in HCC, and consequently promoting tumor proliferation. (3) In addition, copper alleviates the ULK1 and ULK2 pathways to enhance autophagic flux, providing more copper-dependent targets for tumor proliferation by controlling protein quality. (4) Furthermore, copper is involved in the COMMD family and LOX family through HIF1α/VEGF/NF-κB pathway and ATOX–ATP7A–LOX pathways, then the angiogenesis is promoted. All of those above lead to hyperplasia. ATOX1 antioxidant protein 1, cAMP 3',5'-cyclic AMP, cdc25 cell division cyclin25, CCS copper chaperone for superoxide dismutase, COMMD copper metabolism MURR1 domain, COX cytochrome c oxidase, eNOS endothelial nitric oxide synthase, G6PDH glucose 6-phosphate dehydrogenase, LOX lysyl oxidase, MEK1/2 mitogen-activated protein kinase kinase 1/2, HIF-1α hypoxia-inducible factor-1α, mTOR mammalian target of rapamycin, Nf-kB Nuclear factor kappa-B, NO nitric oxide, p53 transformation-related protein 53, PDE phosphodiesterase, PDK1 3-Phosphoinositide-dependent protein kinase 1, PDGF platelet-derived growth factor, PI3K phosphatidylinositol 3-kinase, PKB protein kinase B, ROS reactive oxygen species, SCO1 synthesis of cytochrome c oxidase 1, SOD recombinant superoxide dismutase, STEAPs Six-transmembrane epithelial antigen of the prostate, ULK Unc-51 Like Autophagy Activating Kinase, VEGF vascular endothelial growth factor

Copper is strongly implicated in the development of cirrhosis and HCC, contributing to neoplasia [10, 37, 43]. Patients with Wilson disease, an autosomal recessive disorder of copper metabolism, exhibit a significantly elevated incidence of HCC. Copper accumulation may promote malignant transformation and cell death of liver cells [33]. Prolonged exposure to high-soluble copper induces significant morphological changes in the liver, increased cell cycle arrest, and suppressed lymphocytes, [44] thereby elevating the frequency of gene mutation [43, 45]. Excessive copper exposure of liver cells could lead to the decrease of glucose 6-phosphate dehydrogenase (G6PDH) and GSH reductase, [46] excessive production of ROS, and decreased antioxidant function, which increases the probability of gene damage. Moreover, it may induce mitochondria-mediated liver cell death and apoptosis [18, 47]. Additionally, copper directly binds to dual-specificity protein kinases1/2 (DSPK1/2) with high affinity, and is closely related to the classical oncogenic pathways such as BRAF-RAS-RAF-DSPK1/2-extracellular-regulated kinase1(ERK1) and phosphatidylinositol 3-kinase (PI3K)-3-Phosphoinositide-dependent protein kinase1-protein kinase B (PKB) signaling pathways [48]. Consequently, the collective increase in the risk of genetic mutations contributes to neoplasia. In addition, studies have shown that blocking copper transporter-1(also known as copper importer solute carrier family 31 member 1 (SLC31A1)) or reducing intracellular copper levels could inhibit PI3K-PKB and mitogen-activated protein kinase (MAPK) signaling pathway, thereby suppressing neoplasia [49–51]. SLC31A1 represents an important potential therapeutic target in HCC.

Clinical research has demonstrated the increased tissue and intracellular copper accumulation in human HCC tissue samples, suggesting the necessity of copper for tumor proliferation [52]. Elevated serum copper levels correlate with poorer HCC survival [53]. Isotopic copper studies suggested that the increased copper burden in the TME is not sourced from dietary intake, but rather from the redistribution of copper to cysteine-rich proteins in the body [17]. Due to the substantial proliferation demands, HCC cells necessitate increased energy, supported by copper acting as a cofactor for various mitochondrial respiratory enzymes (e.g., COX, SOD1, ATOX1). Consequently, HCC cells require higher copper levels compared to normal cells for proliferation [9, 10, 43, 45]. Elevated copper levels enhance the function of the respiratory enzymes in mitochondria, thereby augmenting mitochondrial respiration.

Copper also promotes hyperplasia via non-mitochondrial pathways. For instance, copper degrades phosphodiesterase, altering the activity of 3',5'-cyclic AMP to stimulate lipolysis [54]. Dysregulated lipid metabolism is a hallmark of HCC cells [55]. The cAMP pathway facilitates the conversion of more triglycerides into glycerol and fatty acids, which can subsequently promote tumor proliferation. Additionally, copper alleviates the ULK1 and ULK2 pathways to enhance autophagic flux, [22] thus providing more copper-dependent targets for tumor proliferation by regulating protein quality. The copper metabolism MURR1 domain (COMMD) family plays crucial roles in either promoting or inhibiting HCC hyperplasia [56]. Increased COMMD 7 expressions and the reduction of COMMD1 and COMMD10 expressions in HCC tissues could promote hyperplasia via Nuclear factor kappa-B signal pathways [56, 57]. Elevated COMMD 3 expression in HCC tissues could stimulate the angiogenesis through hypoxia-inducible factor-1α/ VEGF/nuclear factor kappa-B pathway [58].

Cuproplasia in TME of HCC

TME, the microenvironment surrounding the tumor cells, significantly influences the pathogenesis of HCC [59]. Apart from malignant hepatocytes, the TME of HCC encompasses surrounding extracellular matrix (ECM), innate and adaptive immune cells, inflammatory cells, Tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), tumor-associated neutrophils, myeloid-derived suppressor cells (MDSCs), endothelial cells, surrounding micro-vessels, and various cytokines and chemokines [60]. These cells interact with HCC, forming an immunosuppressive microenvironment [61, 62]. In addition, modified enzymes such as proteases, metabolic feedstocks, metabolites, exosomes, microparticles, and biophysical properties including adhesion and viscoelasticity are also important parts of TME.

The TME in HCC is marked by aberrant angiogenesis, immunosuppression, dysregulated ECM remodeling, and chronic inflammation, initiating tumorigenesis, growth, self-renewal, metastasis, and immune escape, while hindering various anti-tumor treatments [63, 64]. For instance, malignant hepatocytes secrete VEGF to create a tumor-promoting microenvironment, [65] while innate and acquired immune cells exhibit dual effects of tumor promotion and anti-tumor activities [8]. Tumor-infiltrating lymphocytes, comprising T cells, B cells, and others, are frontline defenders combating tumors with relentless attacks until pathogens are eradicated. However, antitumor responses of Tumor-infiltrating lymphocytes are hindered in tumor patients, impeding effective tumor eradication due to factors like cuproplasia induced by mild copper elevation, as discussed below.

T cells, pivotal in adaptive immunity and antitumor responses, consist of helper, cytotoxic, and memory subsets. Nonetheless, within the tumor microenvironment, these cells endure prolonged ischemia, hypoxia, and arginine deficiency, impairing their normal functions. For instance, microenvironmental lactate accumulation substantially hampers T cell proliferation, cytokines secretion, and cytotoxic activity [66]. Furthermore, studies have indicated that increased microenvironmental copper levels correlate positively with PD-L1 expression. PD-L1, a well-studied immune checkpoint, binds to PD1, inducing T cell apoptosis and exhaustion by inactivating downstream pathways like Ras/MAPK, PI3K/AKT, etc [67]. Zhou et al. reported similar findings, suggesting DSF/Cu upregulates PD-L1 expression by inhibiting poly (ADP-ribose) polymerase 1 (PARP1) activity and inactivating glycogen synthase kinase-3β (GSK3β). This concurrent upregulation of ligands and receptors for immune checkpoints likely enables HCC to evade T cell-mediated killing [68]. Although upregulation of ligands and receptors for immune checkpoints, such as anti-PD1 therapy, has been approved for HCC and generally have manageable side effects, some patients may struggle with tolerability, leading to potential treatment discontinuation or reduced efficacy [69]. Understanding individual tolerance levels and side-effect profiles is thus critical for optimizing therapy in HCC patients and enhancing responses to immune checkpoint inhibitors. In order to enhance the tolerability and efficacy of treatments, future research directions may include gaining a deeper understanding of the biology of immune checkpoints to improve current checkpoint blockade therapies and inform the development of the next generation of immunotherapies [70].

Macrophages, crucial TME components, form an immune barrier, engaging in antitumor activities through antigen phagocytosis and cytokine secretion like TNF-α. Moreover, macrophages present antigens to lymphocytes, bolstering adaptive immunity [71]. TAMs, a subtype of macrophages in TME, are abnormally activated, including the naïve macrophages (also known as M0 macrophages), M1 macrophages activated by interferon, and the M2 macrophages activated by anti-inflammatory factors [72]. M1 macrophages produce anti-tumor factors like TNF-α, whereas M2 macrophages with lower antigen-presenting ability produce tumor growth factors and angiogenic factors (such as IL-6, IL-10, VEGF) that promote tumor growth [72]. Excess copper could lead to significant suppression of macrophage function [73]. Given significantly elevated copper levels in both tumor tissue and serum of HCC patients compared to normal individuals, copper likely influences macrophage differentiation akin to other metabolites and cytokines, which promote M2 macrophage polarization [74].

CAFs are inhibitory intermediates in the TME that correlate with poor prognosis in HCC [75]. CAFs participate in ECM remodeling, [76] and release IL-6, FGFs, VEGF, hepatocyte growth factors, and other cytokines to recruit inflammatory and immune cells to affect the immune response process [77, 78]. Tumor-infiltrating natural killer (NK) cells, whose abundance correlates positively with overall survival, exhibit cytotoxicity through perforin, granzyme, Factor-related Apoptosis ligand, etc., while modulating immune responses via cytokines and chemokines secretion, along with antigen presentation [79]. Copper chelators have been observed to augment NK cell infiltration, suggesting elevated copper levels may impede NK cell survival, proliferation, recruitment, and cytotoxic activity. Thus, employing copper chelators could offer a novel and promising therapeutic strategy [80]. Additionally, diverse immune cells (e.g., B cells, neutrophils, MDSCs) represent crucial immunosuppressive elements in the TME [3]. These cells interact with HCC, shaping an immunosuppressive microenvironment [61, 62].

The anti-tumor activity of the immune cells relies on intact mitochondrial respiration [81]. Imbalance of copper homeostasis can impair immune response of immune cells to tumor cells [81]. Due to the significance of copper in mitochondrial metabolism, cuproplasia enhances the mitochondrial respiration of HCC cells, rendering them more resistant to immune cell elimination. Besides, in the COMMD family, the expression of COMMD2/3/10 is strongly associated with immune infiltration in HCC, [82] especially M0 macrophages, and neutrophils, [83] promoting the occurrence and development of TME. The LOX family, as the copper-dependent enzyme, acts on the remodeling of structural ECM crosslinks, promoting TME formation in HCC [84]. Research showed that the LOX-like2 (LOXL2) and LOXL4 is highly expressed in HCC tissues [85, 86]. ECM induces the upregulation of hypoxia-inducible factor-1α through DSPK1/2-ERK1/2 pathway that further stimulates LOXL2 expression in TAMs [87]. CAFs could up-regulate the expressions of LOXL2 in HCC cells, while HCC could also up-regulate the expression of LOXL2 in CAFs. This forms a positive cycle to significantly promote TME and HCC invasion [86].

Furthermore, LOXL4 induces an immunosuppressive phenotype of macrophages, leading to upregulation of PD-L1 expression and further inhibiting CD8 + T cell function [88]. Voli and his colleagues first indicated that copper regulates PD-L1 expression, serving as a downstream target of intratumoral copper [28]. The significant function of PD-L1 in the suppressive TME has been fully discussed, and enormous drugs targeting PD-L1 have been developed [89]. Beyond being highly expressed on the surface of T lymphocytes, B lymphocytes, macrophages, and dendritic cells, PD-L1 is highly expressed in HCC cells [60]. PD-1/PD-L1 pathway induces T lymphocytes apoptosis and exhaustion, suppresses B lymphocytes activation, negatively affects the differentiation of T lymphocytes, and inhibits tumor-specific T cell proliferation, [90] thus promoting immune tolerance and suppressive TME. The research found that copper chelators reducing the copper in tumor tissues could significantly decrease the expression of PD-L1, thereby increasing the tumor-infiltrating NK cells and CD8⁺ T cells to promote new immune cell clones and enhance the anti-tumor immune responses, indicating the inhibitory effect of copper on TME [28].

Moreover, cuproplasia in TME activates HCC cells to secret more proangiogenic factors such as basic fibroblast growth factors, VEGF, and fibroblast growth factor 2, 3-phosphoinositide dependent protein kinase 1, enhancing migration and invasion of endothelial cells, thereby promoting angiogenesis [49, 62, 91]. Angiogenesis is an important factor in tumor progression. LOX family also promotes peritumoral angiogenesis by upregulating the expression of VEGF and platelet-derived growth factors [88, 92]. Through the ATOX1–ATP7A–LOX pathways, the LOX family promotes the distant metastasis of HCC [41]. Copper in TME also activates copper-related pathways such as the MAPK pathway, [48] the apyrimidinic endonuclease-1/redox effector factor 1 to promote HCC tumorigenesis and metastasis [93]. Studies have shown that Cu²⁺ could bind with CD147 to activate the PI3K-PKB pathway, stimulating surrounding fibroblasts to highly express angiogenic activators like matrix metalloproteinase 2, thus increasing HCC invasiveness [12].

Cuproptosis and HCC

The mechanism of cuproptosis

Cuproptosis is a currently discovered form of programmed cell death, distinct from known mechanisms triggered by copper and dependent on mitochondrial respiration [16]. The current understanding of the cuproptosis mechanism is depicted in Fig. 2. However, the more precise mechanisms require further investigation.

Fig. 2

Cuproplasia in TME of HCC. The TME in HCC is marked by aberrant angiogenesis, immunosuppression, dysregulated ECM remodeling, hypoxia, reprogramming, and chronic inflammation, initiating tumorigenesis, growth, self-renewal, metastasis, and immune escape. Cuproplasia induces high expression of LOX2 or LOX4 in HCC cells, CAFs, and TAMs, leading to the release of factors such as VEGF, PDGF, HGF, and FGF. These factors promote endothelial cell survival, proliferation, and angiogenesis, while also upregulating MMP2 expression by CAFs, thereby enhancing HCC invasiveness. Furthermore, cuproplasia upregulates PDL1 expression in HCC cells, CAFs, TAMs, and NK cells, resulting in apoptosis and exhaustion of CD8 + T cells. Alongside other cytokines, chemokines, metabolic substances, ROS, exosomes, etc., an immunosuppressive TME ultimately develops. CAFs cancer-associated fibroblasts, ECM extracellular matrix, FAK focal adhesion kinase, FGF fibroblast growth factor, HCC hepatocellular carcinoma, HGF hepatocyte growth factor, LOX2 lysyl oxidase-like2, LOX4 lysyl oxidase-like4, MAPK Mitogen-Activated Protein Kinase, MMP2 Matrix metalloproteinase 2, NK cells natural killer cells, PI3K phosphatidylinositol 3-kinase, PDGF platelet-derived growth factor, PDL1 programmed death-ligand 1, RAS rat sarcoma, ROS reactive oxygen species, TAMs Tumor-associated macrophages, TAN tumor-associated neutrophils, TME tumor microenvironment, VEGF vascular endothelial growth factor

Copper, a key element in mitochondria, is involved in the assembly of copper enzymes such as COX, antioxidant enzyme superoxide dismutase 1, and respiratory complex IV [43]. The onset of cuproptosis relies on mitochondrial respiration and is closely related to the tricarboxylic acid (TCA) cycle [16]. Normal cells regulate intracellular copper through copper importer SLC31A1 and exporter ATP7B [94]. Both transporters are strongly related to cuproptosis [95]. When the level of copper in ECM is elevated, elesclomol, a copper ionophore, transports copper into the intracellular matrix [96]. Elesclomol enhances the ferredoxin 1 (FDX1), a reductase capable of reducing Cu²⁺ to more toxic Cu⁺ [97]. Additionally, FDX1 disrupts Fe-S protein balance and promotes lipidation and aggregation of enzymes involved in the regulation of the TCA cycle, especially dihydrolipoamide S-acetyltransferase (DLAT) [98]. Excess Cu⁺ induces proteotoxic stress rather than copper-induced mitochondrial oxidative stress by causing mitochondrial lipidated protein oligomerization [16]. Moreover, cuproptosis induction impairs mitochondrial respiration, resulting in reduced ATP synthesis. ATP depletion activates Adenosine 5'-monophosphate(AMP)-activated protein kinase (AMPK), triggering the phosphorylation of high-mobility group box 1 (HMGB1) [99]. Under normal circumstances, HMGB1 primarily resides in the nucleus, tightly bound to nucleosomes [100]. Upon AMPK-induced phosphorylation, HMGB1 dissociates from histones, leading to increased extracellular release, inflammation, [99] exacerbation of cell death, and tissue damage (Fig. 3).

Fig. 3

Cuproptosis and its mechanism in HCC. The cuproptosis is closely related to mitochondrial respiration and the TCA cycle and is triggered by the elesclomol, which could translate elevated extracellular copper to intracellular matrix not only the cell membrane importer SLC31A1 and exporter ATP7B. Besides, elesclomol could enhance the FDX1 to reduce Cu²⁺ to become the more toxic Cu⁺. FDX1 is the key enzyme of cuproptosis, causing Fe-S protein imbalance thus leading to the Fe-S cluster, and promoting the lipoylation of DLAT. The Cu + binds to the lipoylated DLAT aggregation to participate in the formation of the PDH complex, which could affect the mitochondrial TCA cycle, leading to ATP depletion. Depletion of ATP will lead to intracellular inflammation through the AMPK pathway. In addition, the lipoylated DLAT aggregation and excess Cu.⁺ could induce proteotoxic stress through mitochondrial lipidated protein oligomerization. Eventually, these serious consequences lead to the cuproptosis. AMPK Adenosine 5'-monophosphate(AMP)-activated protein kinase, ATP Adenosine 5'-triphosphate, ATP7B Adenosine 5'-triphosphatase copper transporting beta, DLAT dihydrolipoamide S-acetyltransferase, ECT electron transport chain, FDX1 ferredoxin 1, LIAS lipoic acid synthetase, TCA tricarboxylic acid, PDH Pyruvate dehydrogenase, SLC31A1 solute carrier family 31 member 1

Cuproptosis in HCC

Cuproptosis is triggered by abnormal expression of cuproptosis-related genes (CRGs) [101]. Numerous CRGs have been identified in HCC involving in copper ion metabolism and mitochondrial function (Table 1). Sun et al. found that FDX1 downregulation activates mitophagy and the PI3K/AKT signaling pathway, promoting HCC progression through elevated ROS production [102]. DLAT was overexpressed in HCC tissues and promotes HCC cell proliferation [103]. Lipoic acid significantly inhibited cell migration and invasion in HCC cells [104]. These genes contribute to regulating copper-dependent cell death mechanisms, which could influence HCC progression. Additionally, anti-cuproptosis genes (e.g., GLS, MTF1, CDKN2A) may be linked to antioxidant functions, stemness formation, angiogenesis, DNA repair, and methylation [105–107]. Copper transporters (e.g., SLC31A1, ATP7B) enable HCC cells to counteract the cytotoxic effects of copper buildup, allowing tumor cells to evade apoptosis [108]. For additional information on CRGs' roles and mechanisms in HCC development, refer to Table 1. These findings indicate that targeting CRGs and copper homeostasis may offer new therapeutic strategies for HCC. Further research is needed to clarify these pathways and their effects on HCC treatment outcomes.

Table 1 The CRGs and their developed scoring methods in HCC

Among the CRGs, 7 pro-cuproptosis genes (FDX1, DLAT, lipoic acid synthetase gene, lipoyltransferase 1, dihydrolipoamide dehydrogenase gene, pyruvate dehydrogenase E1 subunit beta gene, and pyruvate dehydrogenase E1 subunit alpha 1 gene), 3 anti-cuproptosis genes (glutaminase gene, metal regulatory transcription factor 1 gene, and cyclin-dependent kinase inhibitor 2A gene), and 2 transporters (SLC31A1 and ATP3B) are involved in the process of cuproptosis [95]. Unlike apoptosis, mitochondrial ROS, B-cell lymphoma-2-associated X protein, B-cell lymphoma-2-antagonist/killer 1, and caspase activation required for apoptosis are not required for cuproptosis, [16] despite the decrease in mitochondrial membrane potential [16, 99]. Oxidative stress inhibitors (e.g., N-acetylcysteine), ferroptosis inhibitors (e.g., ferrostatin-1), or cell necrosis inhibitors (e.g., necrostatin-1) cannot inhibit cuproptosis. However, copper chelators, inhibitors of respiratory chain complex I (Rotenone), II (Rustin), and III (antimycin A), and inhibitors of mitochondrial pyruvate uptake (UK5099) can inhibit cuproptosis [16]. Cuproptosis is unaffected by mitochondrial uncouplers like Synonyms of Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, suggesting that mitochondrial respiration rather than ATP production is required for cuproptosis [16]. The mitochondrial quality control systems like mitophagy, and AMPK-mediated autophagy pathway could promote mitochondrial health and homeostasis. Mitophagy could self-repair mitochondria to enhance respiratory function under stressful situations, [109] indicating that activating those mitochondrial quality control systems may limit cuproptosis [98, 109].

The dysregulation of CRGs is closely related to the occurrence and development of HCC, [101, 110–113] and CRGs expressions strongly correlate with the immune-suppressive TME in HCC [111, 114]. CRGs enable the identification of HCC subtypes (Table 1). Altered expression of CRGs such as SLC31A1 and ATP7B is significantly associated with elevated copper levels in HCC tissues [33]. Multi-omics analysis reveals that CRGs could identify HCC patients with suppressed immune TME, increased expression of immune checkpoint molecules, and poor prognosis [111]. CRG scores inversely correlate with the expression of immune-related genes and positively correlate with the expression of DNA repair-related genes [115]. A bioinformatics analysis results showed that CRG score was significantly correlated with the infiltration of M2 macrophages and neutrophils in TME [116]. M2 macrophages induce hypoxia and suppress the immune system, [117] while neutrophils secrete chemokine ligands that mediate the infiltration of M2 macrophages and regulatory T cells, reshaping the TME to suppress the immune response [118].

CRGs were expressed to varying degrees in all cell types of TME [111]. For instance, SLC31A1 is positively correlated with the abundance of M1 macrophages and neutrophils, while FDX1 positively correlates with the abundance of activated memory CD4⁺ T cells [111, 114]. Some cuproptosis-activating genes (e.g., lipoic acid synthetase gene, lipoyltransferase 1, FDX1, and pyruvate dehydrogenase E1 subunit alpha 1 gene) exhibit resistance to drugs with broad anti-inflammatory activity, speculating the similar anti-inflammatory biological activities of those genes, consistent with the suppressed immune infiltration [115]. High expression of DLAT in cancer tissues enables HCC cells to evade the immune system and foster an inhibitory immune microenvironment [119]. The proportion of Tregs among HCC patients with long overall survival is higher than that among those with shorter survival, [110] partly due to Tregs' involvement in regulating cuproptosis-induced inflammatory responses and oxidative stress in the TME [110].

Cuproptosis is speculated to shape the antitumor immune environment, but its inhibitory effect on immunotherapy remains uncertain. [120] Previous research showed that various immune checkpoint genes (ICGs) such as butyrophilin subfamily 2 member A1 gene, Butyrophilin-like protein 9, CD40 antigen ligand, signal regulatory protein alpha gene, and TNF receptor superfamily member 4 gene are co-expressed with CRGs [114]. High expression of CRGs is positively correlated with the immune checkpoint molecules [111]. ICGs are closely related to the incidence and development of HCC, which forms the basis of immunotherapy and immune checkpoint inhibitor (ICI) therapy [121]. Additionally, the CRGs are closely associated with the immunosuppressive TME, which is also an essential indicator of ICI therapy. Therefore, CRGs affect the immune microenvironment and are throughout the development of HCC, which is closely related to immunotherapy.

HCC cells exhibit both aerobic respiration (oxidative phosphorylation) and glycolysis, with a preference for glycolysis, a phenomenon known as the Warburg effect [122]. This metabolic characteristic suggests that HCC proliferation is characterized by increased glycolysis even under the presence of O₂ [123]. Besides, HCC thrives in a hypoxic TME, further promoting glycolysis [61]. The increased glycolysis is closely related to the proliferation, angiogenesis, and metastasis of HCC [124, 125]. Thus, inhibiting glycolysis or reversing the hypoxic TME can potentially delay HCC progression [126]. Cuproptosis is closely related to mitochondrial respiration, mainly involving oxidative phosphorylation [16]. The occurrence of cuproptosis requires enhanced oxidative phosphorylation level and improved aerobic TME. Therefore, promoting cuproptosis in HCC may alleviate mitochondrial dysfunction and hypoxic TME, thus reducing the glycolysis in HCC cells, offering a fascinating perspective for clinical treatment [127].

The relationship between cuproplasia and cuproptosis

The occurrence of cuproplasia or cuproptosis in a cell is contingent upon the concentration of copper ions. Slightly increased copper concentrations not only sustain cellular functions and fulfill physiological and metabolic needs but also contribute to tumorigenesis, as previously elucidated in detail, resulting in cuproplasia [9]. Conversely, surpassing a specific threshold of copper leads to cuproptosis, apoptosis, ferroptosis, and other cellular responses [27]. Nonetheless, cells exhibit varying degrees of copper tolerance. Studies have revealed notably heightened copper levels in diverse tumor tissues, indicating enhanced capacity of tumor cells in copper utilization and tolerance [80]. This could be attributed to the maximal utilization of cuproplasia by tumor cells to proliferate via the aforementioned mechanisms, along with heightened autophagy aimed at clearing ROS produced by the elevated copper-induced Fenton reaction, thereby shielding tumor cells from cytotoxic effects of uncontrolled peroxidation. However, genes linked to these mechanisms could be either downregulated or upregulated in anti-tumor immune cells. For instance, autophagy plays a crucial role in sustaining T cell proliferation and function. However, tumor-infiltrating T cells exhibited notable downregulation of autophagy-related genes with decreased autophagic flux, leading to inhibition of T cell proliferation and oxidative stress [128].

The autophagy pathway plays a crucial role in connecting the mechanisms of both cuproplasia and cuproptosis. Lower copper levels can alleviate the ULK1 and ULK2 pathways, thereby enhancing autophagic flux, regulating protein quality, and ultimately promoting copper-dependent tumor proliferation. Additionally, autophagy could be induced by metabolic stress, hypoxia, redox stress, and immune signaling such as damage-associated molecular patterns [129]. Upregulated autophagy in tumor cells can facilitate tumorigenesis and progression by reducing ROS accumulation and providing essential nutrients for survival. Autophagy also contributes to the degradation of granzyme B released by NK cells and cytotoxic T lymphocytes, as well as major histocompatibility complex class I in dendritic cells, promoting tumor immune evasion [130]. Furthermore, chemoresistance and distant metastasis in HCC could be attributed to enhanced autophagy, which decreases the sensitivity to chemotherapy and inhibits anoikis [130].

However, the disruption of the mitochondrial TCA cycle by excess copper leads to ATP depletion, triggering intracellular inflammation and increased autophagy via the AMPK pathway, exacerbating the process of cuproptosis. Therefore, autophagy acts as a double-edged sword in tumors, with its specific role determined by the specific tumor type, differentiation degree, pathological stage, and the TME context. Elesclomol–CuCl2 can mitigate resistance to docetaxel by inhibiting autophagy in prostate cancer cells [131]. In colorectal cancer models, tretinoin demonstrates antitumor effects by inhibiting both proliferation and autophagy [132]. Although direct evidence is lacking for the involvement of autophagy inhibition in cuproplasia or cuproptosis in HCC, the role played by inhibition of autophagy in other tumors presents suggestive testimony. ROS mediates the processes of cuproplasia and cuproptosis. Copper may reduce G6PDH and GRD levels, resulting in ROS production and decreased antioxidant function. This increases the risk of genetic mutations in hepatocytes, thereby promoting neoplasia [133]. Copper also enhances the activity of copper chaperone for superoxide dismutase and SOD1, which convert superoxide, with high activity, into hydrogen peroxide. This reduces ROS production in the mitochondrial membrane and mitigates ROS-induced damage to tumor cell proteins and lipid membranes [134]. Moreover, ROS increases autophagic flux, thereby offering additional copper-dependent targets for tumor proliferation by regulating protein quality [22]. MDSCs, TAMs, and neutrophils exert immunosuppressive functions through various pathways such as Treg induction, production of high levels of arginase-1 and ROS. Copper chelator usage substantially decreased MDSCs infiltration, indicating a proportional relationship between copper levels and MDSCs recruitment [80]. Elevated copper levels promote increased MDSCs and ROS levels, creating an immunosuppressive microenvironment that mediates immune evasion. Mitochondrial Fe-S cluster proteins, involved in the synthesis of heme, lipoic acid, and biotin in mitochondria, are crucial constituents of the TCA cycle and the electron transport chain complexes [135]. Copper cytotoxicity results in Fe-S cluster loss, leading to reduced mitochondrial membrane potential, inhibition of the electron transport chain complexes and TCA cycle, and initiation of the Fenton reaction, resulting in substantial ROS production. ROS burst exacerbates cuproptosis by inducing multifaceted cellular damage, including DNA damage, mitochondrial dysfunction, and membrane integrity disruption.

The clinical significance and prospect of cuproplasia and cuproptosis in HCC

Copper compounds and nano-copper

Copper induces both cuproplasia and cuproptosis in HCC. Tumor cells need more copper than normal cells for proliferation, angiogenesis, and metastasis, [43] while excessive copper also triggers cuproptosis. Thus, inhibiting cuproplasia and enhancing cuproptosis in HCC could be prospective therapeutic strategies. Cellular fate, whether cuproplasia or cuproptosis, is determined by the level of copper concentration. Consequently, targeting copper via copper chelators to mitigate cuproplasia or copper ionophores to enhance cuproptosis has emerged as a highly promising therapeutic avenue for various copper-associated diseases, including HCC. Identifying the multifaceted roles of crucial molecules involved in autophagy and ROS regulation in cuproplasia and cuproptosis, along with potential targets, agents, and combined therapeutic interventions utilizing copper compounds, may yield more efficacious therapeutic strategies. Copper compounds, appropriately complexed, hold promise as potential drugs for HCC treatment with minimal side effects [27].

Clinically used copper compounds include copper chelators and copper ionophores, both exhibiting outstanding anticancer activity and promising prospects in cancer therapy (Table 2) [91]. Copper chelators inhibit cuproplasia in tumor cells, contributing to therapeutic efficacy [32]. Numerous copper chelators have been developed at present, [136] and studies have demonstrated their ability to inhibit the formation of new blood vessels, thereby preventing angiogenesis [52, 137]. Tetrathiomolybdate (TTM), a copper chelator capable of inhibiting copper absorption, has demonstrated efficacy in reducing the tumorigenicity of HCC cell lines. It also inhibits glycolysis, reducing the energy supply to tumor cells, and thereby impeding tumor initiation and progression [33]. Given that the occurrence and development of HCC are closely linked to angiogenesis, there has been a surge in research aiming to inhibit this process, leading to advancements in anti-HCC therapies focused on antiangiogenesis. Penicillamine, known for its role as an antidote for heavy metal poisoning and in treating Wilson's disease, has emerged as a potential anti-HCC drug due to its strong copper chelating properties and anti-angiogenic effects [138]. Trientine, an alternative for patients with Wilson's disease who cannot tolerate penicillamine, is another effective copper chelator that inhibits angiogenesis. Sone K et al. reported that trientine not only inhibits the proliferation of vascular endothelial cells but also promotes tumor cell apoptosis, exhibiting a remarkable and promising anti-tumor activity [139]. Besides, copper chelators could reduce PD-L1 expression in tumor tissue, stimulate anticancer immune responses, and inhibit immune checkpoints [28]. In addition, pro-chelators are developed to enhance selectivity against cancer cells [140]. By utilizing stimuli primarily present in the TME, pro-chelators enhance targeting activity and therapeutic effects with little off-target toxicity [136, 141]. Regarding copper ionophores, research found that in some cases there may be a critical copper solubility and a narrow window that enables more copper to accumulate in HCC tumor cells leading to cuproptosis and thus selectively killing tumor cells [15]. Disulfiram, a widely studied copper ionophore, can inhibit the activity of PARP1, promote the phosphorylation of GSK-3β at the Ser9 site, and ultimately lead to the increase of PD-L1 expression and stimulate cell apoptosis [68]. Elesclomol, a highly lipophilic and potent copper-binding molecule, transports excess copper into mitochondria, causing loss of lipoylated mitochondrial proteins and Fe-S cluster protein, triggering intense oxidative stress, ultimately leading to cuproptosis in HCC cells [142].

Table 2 Drugs or medicines for HCC according to the copper mechanism

To enhance the selectivity of copper ionophores, pro-ionophores and nano-drug delivery systems can be used as Valentina Oliveri and her colleagues detailed in their review [32]. Nano-copper is a popular potential anti-tumor drug for chemodynamic therapy recently, [143] which regulates the immunosuppressive TME by activating ROS to kill tumor cells and alleviate the hypoxic microenvironment to trigger immunogenic cell death [144]. GSH is overexpressed in the TME and severely depletes ROS to limit the chemodynamic therapy [145]. Studies have found that Cu²⁺ released by nano-copper can effectively consume the overexpressed GSH in TME, thereby reducing the elimination of hydroxyl radicals and amplifying cascade oxidative stress [143]. In addition, the generated Cu⁺ utilizes the characteristics of TME to trigger nano-catalysis reactions via Fenton-like reactions, [146] producing highly toxic hydroxyl radicals and hydrogen peroxide, leading to the outbreak of ROS in the TME [147]. High concentrations of ROS induce severe oxidative stress thus triggering immunogenic cell death. During the immunogenic cell death, the antigen-presenting cells and cytotoxic T lymphocytes are activated and the systemic anti-tumor immune responses are triggered [144]. In addition, to enhance the therapeutic effect, enormous materials such as copper nanocrystalline-doped folic acid-based super carbon dots, [145] copper-encapsulating magnetic nanoassemblies, [148] thermosensitive hydrogel systems, and copper-coordinated nanogenerators were developed [143, 147]. Although success has been achieved in preclinical research, clinical applicability still needs further research.

DC_AC50, another copper-based anticancer compound, acts as a copper-trafficking protein inhibitor, inhibiting ATOX1/copper chaperone for superoxide dismutase to enhance ROS-mediated cell death in lung cancer H1299 cells, head and neck cancer 212LN cells, and breast cancer cells [134]. Preclinical studies in melanoma patients have revealed the significant therapeutic potential of DC_AC50, offering suggestive evidence for copper-targeted therapy in HCC [149].

Combination therapy

Combination with targeted drugs. Lenvatinib, a receptor tyrosine kinase inhibitor that suppresses vascular endothelial growth factor receptors, has already widely been put into clinical applications in advanced HCC. However, the therapeutic effects of Lenvatinib are still subject to certain limitations such as the rapid development of drug resistance and side effects. In 2021, Qi Xu and co-workers constructed a nano platform where Lenvatinib and copper sulfide nanocrystals (Cu2-xS NCs) were co-encapsulated [150]. The evidence from this study indicated that the combinatorial treatment enhanced tumoricidal efficacy and provided additional therapeutic benefits. Similarly, surveys conducted by Li Nan et al. in 2023 conclusively suggested that the application of a combination of TTM with Lenvatinib presented markedly decreased angiogenesis and showed synergistic antitumor responses [151]. Sorafenib, another widely studied receptor tyrosine kinase inhibitor, exhibits a significant advantage in suppressing angiogenesis and tumor cell proliferation, extending the survival time of advanced patients [152]. Mechanistically, sorafenib inhibits mitochondrial matrix-related proteases-mediated FDX1 degradation and directs tumor cells to cuproptosis [153]. Wang and colleagues found that disulfiram (a copper ionophore mentioned above) combined with sorafenib has significant synergistic cytotoxicity against neoplastic cells of the liver and shows extremely encouraging anti-cancer and anti-metastasis efficacy. Collectively, these results provide novel insights into combination treatment strategies in HCC [154].

Combination with chemotherapy and radiotherapy. Li et al. published the findings of the prediction response of 10 drugs in hepatocellular carcinoma. Among them, they found that patients with a high CRGs score subtype were more sensitive to 5-fluorouracil, sunitinib, gemcitabine, and bleomycin than patients with a low CRGs score subtype [155]. Therefore, it is conceivable that the combinative treatment of copper compounds and chemotherapy drugs may have clinical benefits. This speculation was further exemplified in studies conducted by Hassan and coworkers. Their evidence suggested that 5-fluorouracil in combination with Cu and disulfiram presented a more markedly decreased proliferation of tumor cells and considerably ameliorated tumor burden with a remarkably decreased level of damage to cellular structures such as lipids, proteins, and DNA [156]. Likewise, Wang et al. found that disulfiram combined with 5-fluorouracil showed a remarkably antineoplastic activity with reduced metastatic and recurrence risk [154]. In the same vein, such results have also been consistently verified in other tumor models, such as colorectal cancer, pancreatic cancer, etc [157]. Clinical studies have reported significant elevated copper after radiotherapy in tumor patients, suggesting that serum copper levels may provide partial evidence for the efficacy of radiation therapy [158]. Copper treatment downregulated the expression of copper metabolism MURR1 domain 10 in animal models endowing cancer cells with increased resistance to radiation. Tetravinylpentylamine, a copper chelator, significantly decreased the serum level of copper and resensitized the tumor cells to radiation in mice fed copper, indicating that Tetravinylpentylamine is a copper-dependent selective radiosensitizer [159]. At the same time, targeting COMMD10 and related signaling may provide novel directions for discovering potential biomarkers and therapeutic strategies to alleviate and overcome radioresistance.

Combination with other agents. Both cuproptosis and ferroptosis are novel types of programmed necrosis. Whether there exists a connection between the two has aroused the curiosity of many researchers. Wang et al. explored the potential interaction between ferroptosis and cuproptosis in HCC. They found that two ferroptosis inducers, sorafenib and erastin, inhibited mitochondrial matrix-associated protease-mediated degradation of FDX1, causing protein lipoylation and subsequent cuproptosis in hepatocellular carcinoma cells. Greater degrees of cell death could be observed when ferroptosis inducers and cuproptosis inducers were used simultaneously [160]. This discovery provides new insights into our further understanding of the role of cuproptosis and ferroptosis in the pathophysiological process and potential therapeutic targets of HCC.

Scores, models, and biomarkers

Based on the CRGs, the cuproptosis signature, [101] cuproptosis-related risk score, [10, 111] cuproptosis-related gene prognostic index have been developed to predict the prognosis and the TME of HCC [112]. The risk scores are positively correlated with M0 and M2 macrophages, while negatively correlated with CD4⁺ T cells, CD8⁺ T cells, and NK cells, indicating the suppressed immune TME [127]. The higher the CRG score, the worse the effect of immunotherapy [161]. Thus, those CRG scores can help judge the immune infiltration and the effect of immunotherapy. Furthermore, since CRGs are closely related to ICGs, targeting these CRGs can reverse the suppressive immune microenvironment, and improve the efficacy of ICIs and prognosis in HCC treatment [121].

Furthermore, various long non-coding RNAs (lncRNAs) can be used as biomarkers in HCC treatment (Table 3). The selected lncRNAs are associated with the prognosis of HCC, [162] the TME conditions, and expressions of key immune checkpoints [163]. Besides, the cuproptosis-related lncRNA signature, [164] the cuproptosis-related lncRNAs risk-scoring model, [162] the lncRNA profile [165] were built to predict the immune cell activity and prognosis of immunotherapy in HCC, as well as the targeted therapy evaluation.

Table 3 The summary of research on cuproptosis-associated RNA biomarkers in HCC

FDX1 may have promise as a biomarker and therapeutic target based on preliminary studies, which is closely related to cancer mutation, immunity, and prognosis, [166] and is expected to become a new therapeutic target for HCC. FDX1 is down-expressed in various cancer cells, [166] including HCC, and loss of FDX1 renders cancer cells resistant to cuproptosis [16]. Clinical studies have shown that HCC patients with high FDX1 expression exhibited longer survival times [10]. Besides, FDX1 is highly associated with and directly targeted by elesclomol [167]. DLAT can also serve as a new predictive biomarker for HCC prognosis and is closely related to the TME and immune system of HCC patients [119]. HCC patients with reduced DLAT expression have a better prognosis with better OS and disease-specific survival [119]. Elesclomol is an anticancer drug that heavily relies on its transport of extracellular copper [96]. Through elesclomol administration, the FDX1 is activated, thereby inducing cuproptosis. Both preclinical and clinical trials have confirmed the safety and the cytotoxicity of elesclomol to treat cancer, [168–170] which deserves special attention in HCC therapy.

Challenges targeting cuproplasia and cuproptosis in HCC

Although copper compounds, nano-copper, and combination therapies seem to be poised to become part of standard medical practice, certain clinical trials have not yet released their findings, leading to uncertainties and inquiries regarding the efficacy of this approach to treating cancer [NCT00006332]. The lack of detailed experimental information hinders our ability to accurately determine the reasons for potential failures. Challenges such as inadequate clinical efficacy, flawed experimental design and methodologies, safety concerns regarding toxicity and side effects, poor drug-like properties, and limited commercial viability, collectively cast doubt on the future prospects of these treatments. The robust stability and prevalent presence of lncRNAs, as indicated in Table 3, suggest their potential utility as dependable cancer biomarkers. However, some non-coding RNAs lack specificity, as elevated levels may not always indicate a specific cancer type but could potentially be attributed to other malignancies or inflammatory responses triggered by bacterial and viral infections. Furthermore, discrepancies in findings across various studies investigating the same non-coding RNA may stem from factors such as sample size, statistical approaches, specificity of detection techniques, and other methodological considerations. Given the above shortcomings, non-coding RNA still faces great challenges in entering the clinical translation stage. Establishing standard operating procedures for evaluating non-coding RNA, including sample selection, RNA extraction, detection, and standardization methods, would facilitate the clinical application of non-coding RNA. Researchers have combined genomics, proteomics, metabolomics, etc. with clinical big data, artificial intelligence, and machine learning to develop various risk scores and prognostic models. These models help doctors make better clinical decisions about HCC and improve patient prognosis. However, due to the complexity and diversity in the pathogenesis, clinical behaviors, biology, pathology, and molecular characteristics of HCC subtypes, as well as variations in chemotherapeutic sensitivities and prognosis, the ability of prediction models established for a single subtype is subject to certain limitations, so that these models still face the dilemma of being difficult for further generalization. Building more comprehensive, more sensitive, and algorithmically better models remains a key challenge. Establishing closer connections between these models and immune infiltration may provide some inspiration for clinical treatment. Furthermore, there is an urgent need for a better understanding of the cross-talk between cuproplasia, cuproptosis, and other forms of cell death, such as ferroptosis. This insight might reveal a potential correlation not only for related cell and animal experiments assessing the candidate contribution of crosstalk between different types of cell death to certain diseases, but, more importantly, also in the coming time clinical research evaluating the efficacy of potential drugs attempting to provide more practical guidance for the combination therapy and clinical decision-making of diseases linked with cuproplasia and cuproptosis (e.g., cancer, neurodegenerative diseases, and obesity, as well as Wilson's disease).

Concluding remarks

HCC presents a significant global health challenge, exerting considerable strain on healthcare systems worldwide. An in-depth analysis of cuproplasia and cuproptosis mechanisms within the TME of HCC unveils their opposing nature and their impact on copper regulation within the TME. Cuproplasia fosters TME formation, angiogenesis, and metastasis, whereas cuproptosis may alleviate mitochondrial dysfunction and hypoxic conditions in the TME. Therefore, inhibiting cuproplasia and enhancing cuproptosis in HCC are essential for achieving therapeutic efficacy in HCC. These areas necessitate further investigation and offer promising research prospects.