Hepatocellular carcinoma (HCC) comprises approximately 90% of primary liver cancers in the world. It is the fifth most prevalent cancer and ranks fourth among cancer-related deaths worldwide [1]. HCC causes approximately 800,000 deaths each year and seems to have a heavy disease burden [2]. In the past 2 or 3 years, there have been some notable advances in the treatment of HCC, such as resection and transplantation [3, 4]. Recently, immunotherapy and molecular targeted therapy for HCC have also been developed and are expected to become new treatment approaches [5,6,7]. However, the survival rate of HCC is still far from satisfactory due to a low rate of early detection, a tendency for recurrence, and chemotherapy resistance [8]. Therefore, it is of great significance for us to identify accurate biomarkers in the diagnosis stage of patients with HCC to evaluate the prognosis of HCC.

Regulated cell death (RCD) is the primary mechanism for eliminating damaged, infected, or redundant cells [9, 10]. Apoptosis was originally thought to be the only RCD mechanism. Nevertheless, as the understanding of the cellular mechanisms that mediate RCD continues to grow, many new forms of non-apoptotic RCD have been discovered, including ferroptosis, pyroptosis, necroptosis, and autophagic cell death [9, 11, 10]. In 2012, Dixon et al. [12] defined the term ferroptosis to describe the form of cell death induced by the small molecule Erastin, which is iron ion-catalyzed necrotic cell death by inhibiting cystine import, resulting in glutathione depletion and phospholipid peroxidase glutathione peroxidase inactivation 4 (GPX4). Ferroptosis was related to the pathophysiological changes of many cancers [13, 14]. Triggering ferroptosis as a novel approach to cancer treatment is highly anticipated and an active area of research. Pyroptosis is an inflammatory RCD that is mainly triggered by inflammatory caspases and gasdermin family proteins and is manifested by the continuous swelling of cells until cell membrane rupture and death [15].

Interestingly, a recent study by Tsvetkov and others revealed that intracellular copper induces a new form of RCD distinct from oxidative stress-related cell death, known as “cuproptosis” [16]. It occurs through the direct binding of copper to fatty acylated components of the tricarboxylic acid cycle, resulting in fatty acylated protein aggregation and iron-sulphur cluster protein loss, leading to proteotoxic stress and ultimately cell death [16]. Studies have shown that an imbalance in copper homeostasis affects tumour growth, causing irreversible damage. Copper can induce multiple forms of cell death through various mechanisms, including reactive oxygen species accumulation, proteasome inhibition, and anti-angiogenesis [17]. For example, blocking SLC31A1-dependent copper uptake increases autophagy in pancreatic cancer cells against cell death [18]. At present, several genes and proteins have been shown to regulate cuproptosis, including FDX1, LIAS, LIPT1, PDHA1, and PDHB [16]. However, the expression patterns and clinical value of cuproptosis-related genes (CRGs) in HCC remain unclear.

In this study, we firstly built a predictive signature based on CRGs as a prognostic biomarker. Next, we created an accurate nomogram to improve the clinical applicability of the risk score. In addition, we analyzed the correlation of CRGs with the prognosis, the TME, immune checkpoint genes, chemotherapy sensitivity, and immunotherapy.

After developing chronic fibrotic liver disease caused by viral or metabolic aetiologies, patients tend to progress to HCC [26]. Nevertheless, the key issue at present is that there is no robust estimation system that can reliably estimate the HCC risk or diagnose HCC during its early stages [27]. As a result, identifying biomarkers is critical for early detection, prognostic analysis, and individualized therapy of HCC.

RCD, or, more specifically, cell suicide, is not only essential in embryonic development but also plays a critical role in the occurrence and development of diseases, especially malignancies [11]. Apoptosis is one of the most classic forms of programmed cell death and is considered the most promising target for tumor therapy [28]. In addition to classical apoptosis, several other forms of RCD have been identified [11]. Copper ionophore-induced cell death is a novel cell death pathway that is distinctly different from traditional death methods [16]. Cuproptosis plays an important role in tumorigenesis and cancer therapy [18, 16, 29]. Dysregulation of CRGs has been shown to be involved in the pathogenesis and development of multiple types of cancer. Li et al. [30] showed that copper chaperone for superoxide dismutase (CCS) promotes breast cancer cell growth and migration by regulating ROS-mediated ERK1/2 activity. Vyas et al. [31] reveal that copper-dependent ATP7B upregulation drives resistance to platinum toxicity in a TMEM16A-overexpressing head and neck cancer model.

Physiologically, enterocytes take up bioavailable copper ions from the diet in a Ctr1-dependent manner, and upon incorporation, cuprous ions are transported to ATP7A, which pumps Cu⁺ from enterocytes into the blood. Copper ions reach the liver through the portal vein and enter hepatocytes through Ctr1 to form membrane pores. Then, Cu⁺ can be secreted into bile or blood through the Atox1/ATP7B/ceruloplasmin pathway. In the blood, this micronutrient can reach peripheral tissues and be reabsorbed by Ctr1 [32]. Wilson’s disease (WD) is an autosomal recessive disorder caused by mutations in the ATP7B gene, which encodes the copper-transporting ATPase, resulting in impaired hepatic copper excretion [33]. Copper metabolism must be tightly controlled in order to achieve homeostasis and avoid disorders. A hereditary or acquired copper unbalance may cause or aggravate many diseases, including cardiovascular diseases, neurodegenerative diseases, Genetic disorders, metabolic diseases, and cancer [34, 35].

The main risk factors for HCC are hepatotropic viruses (HBV and HCV) [36]. In addition, nonalcoholic fatty liver disease (NAFLD) is already the fastest growing cause of HCC in the USA [37]. Evidence reports that inadequate copper intake serum concentration is involved in the pathogenesis of NAFLD. Intrahepatic and serum copper concentrations were lower in subjects with NAFLD compared with other liver diseases [38]. NAFLD-cirrhotic patients were characterized by a statistical significant enhancement of serum copper levels, even more evident in HCC patients [39, 38]. Patients with hepatolenticular degeneration due to impaired copper metabolism have a high incidence of HCC [40], so we hypothesize that cuproptosis has a correlation with the development of HCC. However, the mechanism of action of cuproptosis affecting HCC is not clear.

RCD-based HCC subtype and/or prognostic models are becoming a research hotspot for predicting HCC prognosis. Growing evidence suggests that prognostic models based on next-generation sequencing and public databases provide more comprehensive clinical-genetic prognostic value [41,42,43]. The role including clinical relevance and prognostic significance of CRGs in HCC is unknown. In the present study, TCGA and two external databases (GEO and ICGC) were employed to collect gene expression and clinicopathological information as the training and validation cohorts, respectively. According to an unsupervised consensus clustering analysis of 16 CRGs, HCC patients were categorized into three subtypes. Compared with patients with other subtypes, patients with subtype B had worse OS. The level of immune cell infiltration also differed greatly among the subtypes. The contents of infiltrated immune cells in subtype A and C were significantly higher than those in subtype B, which seemed to contradict its worse survival outcome.

To better evaluate each HCC patient’s prognosis and therapeutic response, it is essential to construct a cuproptosis signature, which could generate a cuproptosis model for individual prediction. Therefore, we identified 81 cuproptosis subtype-related DEGs and developed and tested a novel prognostic signature in HCC patients by using the identified DEGs. The signature contained five cuproptosis-associated genes: PFKFB3, CLEC3B, CFH, HPR, and LAMB1. PFKFB3, a key molecule in glucose metabolism in the cytoplasm, obviously accelerates the rate of glycolysis and is expressed in rapidly proliferating cells and multiple cancers [44]. PFKFB3 can promote cell cycle progression and inhibit apoptosis through Cdk1-mediated phosphorylation of p27, while MAPK increases PFKFB3 transcripts to accelerate cell proliferation [44]. PFKFB3 has been shown to affect the tumorigenesis and progression of HCC through various mechanisms and is a potential target for the treatment of HCC [44–47]. Li et al. [44] found markedly increased OS in individuals with PFKFB3-overexpressing tumors in comparison with the low-expression group. Knockdown of PFKFB3 reduces glucose consumption and disrupts DNA repair function, resulting in G2/M phase arrest and apoptosis in HCC cells. Mechanistically, blockade of PFKFB3 inhibits hepatocellular carcinoma growth by impairing DNA repair via AKT. Matsumoto et al. [46] revealed that inhibition of PFKFB3 suppressed tumor growth and induced tumor vascular normalization in HCC. CLEC3B, a member of the C-type lectin superfamily, has been reported to be downregulated in serum and tumor tissues of HCC [48, 49]. Dai et al. [48] found that downregulation of exosomal CLEC3B in HCC promoted metastasis and angiogenesis through AMPK and VEGF signaling. CFH is a critical regulatory protein of the alternative complement pathway. Mao et al. [50] showed that CFH is enriched in extracellular vesicles (EVs) of metastatic HCC cells and it protects HCC cells by evading complement attack, thereby promoting HCC progression. Therefore, the expression of CRGs was strongly associated with the tumorigenesis, invasion, and outcomes of hepatocellular cancer, corroborating our findings.

The HCC patients were then divided into two risk subgroups based on the calculated cut-off point. Kaplan-Meier curves indicated that the OS rate was markedly higher in the low-risk group than in the high-risk group. The time-dependent ROC curve showed that the risk score presented a good performance for survival prediction. External validation confirmed the value of the predictive signature. Furthermore, multivariate Cox regression analysis confirmed that the risk score was a prognostic factor independent of clinical characteristics. We then developed a nomogram for predicting 3- and 5-year OS in HCC patients, and we also verified the accuracy of the nomogram by calibration. Taken together, the signature may be effective in predicting patient outcomes, thereby facilitating the implementation and evaluation of the model in future clinical practice.

The tumor immune microenvironment is crucial in the initiation and progression of HCC [51]. We calculated 22 TIICs in the two risk subgroups according to the CIBERSORT algorithm. We observed that risk scores were negatively correlated with resting memory CD4 T cells, CD8 T cells, resting and activated NK cells, whereas risk scores were positively correlated M0 and M2 macrophages, indicating that the signature may significantly contribute to modulating immune cell infiltration. Emerging experiments and clinical studies have found that immunotherapy does have advantages that traditional anti-tumor treatments cannot match, which can improve the prognosis of HCC patients [6, 5]. ICI therapy targeting anti-PD-1 or PD-L1 is a crucial step in a combination regimen to improve the prognosis of HCC patients [52]. The combination of anti-CTLA4 and anti-PD-L1 increased tumor-infiltrating lymphocyte function and restored HCC-derived T cell responses to tumor antigens [53]. In the present study, a novel cuproptosis-based signature was built to investigate the relationship between ICIs and the risk score as a predictor of immunotherapy response. The expression of PD-1, PD-L1, and CTLA4 was significantly lower in the high-risk group, suggesting that the signature might be potentially useful for predicting responses to ICI-targeted therapy. We observed lower TIDE scores in low-risk patients compared with high-risk HCC patients. This suggests that low-risk patients are less likely to have tumor immune evasion and are more likely to benefit from immunotherapy, which further explains their better prognosis. We next investigated the correlation between risk score and chemotherapeutic drug sensitivity. And the results indicated patients with high-risk scores seemed to be more responsive to bleomycin, dasatinib, and veliparib, while low-risk patients were more sensitive to sorafenib, lapatinib, metformin, temsirolimus, and palbociclib. The combination of immunotherapy and chemotherapy may provide precise and individualized treatment with different risk scores.

We successfully identified three distinct subtypes of cuproptosis and established a novel prognostic model, providing new insights into the prediction of the outcome of HCC and its response to chemotherapy and immunotherapy.