Novel Molecular Subtyping Scheme Based on In Silico Analysis of Cuproptosis Regulator Gene Patterns Optimizes Survival Prediction and Treatment of Hepatocellular Carcinoma

Background: The liver plays an important role in maintaining copper homeostasis. Copper ion accumulation was elevated in HCC tissue samples. Copper homeostasis is implicated in cancer cell proliferation and angiogenesis. The potential of copper homeostasis as a new theranostic biomarker for molecular imaging and the targeted therapy of HCC has been demonstrated. Recent studies have reported a novel copper-dependent nonapoptotic form of cell death called cuproptosis, strikingly different from other known forms of cell death. The correlation between cuproptosis and hepatocellular carcinoma (HCC) is not fully understood. Materials and Methods: The transcriptomic data of patients with HCC were retrieved from the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) and were used as a discovery cohort to construct the prognosis model. The gene expression data of patients with HCC retrieved from the International Cancer Genome Consortium (ICGC) and Gene Expression Omnibus (GEO) databases were used as the validation cohort. The Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis was used to construct the prognosis model. A principal component analysis (PCA) was used to evaluate the overall characteristics of cuproptosis regulator genes and obtain the PC1 and PC2 scores. Unsupervised clustering was performed using the ConsensusClusterPlus R package to identify the molecular subtypes of HCC. Cox regression analysis was performed to identify cuproptosis regulator genes that could predict the prognosis of patients with HCC. The receiver operating characteristics curve and Kaplan-Meier survival analysis were used to understand the role of hub genes in predicting the diagnosis and prognosis of patients, as well as the prognosis risk model. A weighted gene co-expression network analysis (WGCNA) was used for screening the cuproptosis subtype-related hub genes. The functional enrichment analysis was performed using Metascape. The 'glmnet' R package was used to perform the LASSO regression analysis, and the randomForest algorithm was performed using the 'randomForest' R package. The 'pRRophetic' R package was used to estimate the anticancer drug sensitivity based on the data retrieved from the Genomics of Drug Sensitivity in Cancer database. The nomogram was constructed using the 'rms' R package. Pearson's correlation analysis was used to analyze the correlations. Results: We constructed a six-gene signature prognosis model and a nomogram to predict the prognosis of patients with HCC. The Kaplan-Meier survival analysis revealed that patients with a high-risk score, which was predicted by the six-gene signature model, had poor prognoses (log-rank test p < 0.001; HR = 1.83). The patients with HCC were grouped into three distinct cuproptosis subtypes (Cu-clusters A, B, and C) based on the expression pattern of cuproptosis regulator genes. The patients in Cu-cluster B had poor prognosis (log-rank test p < 0.001), high genomic instability, and were not sensitive to conventional chemotherapeutic treatment compared to the patients in the other subtypes. Cancer cells in Cu-cluster B exhibited a higher degree of the senescence-associated secretory phenotype (SASP), a marker of cellular senescence. Three representative genes, CDCA8, MCM6, and NCAPG2, were identified in patients in Cu-cluster B using WGCNA and the "randomForest" algorithm. A nomogram was constructed to screen patients in the Cu-cluster B subtype based on three genes: CDCA8, MCM6, and NCAPG2. Conclusion: Publicly available databases and various bioinformatics tools were used to study the heterogeneity of cuproptosis in patients with HCC. Three HCC subtypes were identified, with differences in the survival outcomes, genomic instability, senescence environment, and response to anticancer drugs. Further, three cuproptosis-related genes were identified, which could be used to design personalized therapeutic strategies for HCC.