With the huge advancement in the area of bioinformatics algorithms and multi-omics research methodologies22,23, the researchers could get a better understanding of colorectal cancer. The studies of the characteristics and heterogeneity of the tumor immune microenvironment(TIME) which was illustrated as a complex and dynamic community, could help get a more precise classification and novel clinical strategies for patients with colorectal cancer24–26. Copper is a critical factor for the metabolism of all cells, however, when the organism has more copper than its homeostasis, the excess copper would directly bind to the lipoylated proteins of the tricarboxylic acid (TCA) cycle and further induce the dysfunction of the mitochondria which could finally lead to the death of cells11. In tumor cells, three pathways containing reactive oxygen species(ROS) accumulation, proteasome inhibition, and antiangiogenesis were illustrated as the mechanisms correlated with the process of copper-induced cell death, also termed cuproptosis13. The exploration of the correlation between copper-induced cell death and TIME could help find the vulnerabilities and novel biomarkers for the immunotherapies of colorectal cancer.
In this study, all these ten cuproptosis-related genes(CRGs) significantly showed different expressions in the colorectal cancer tissues, and the functional enrichment analysis suggested that these CRGs were mainly involved in the function and signaling pathways of tricarboxylic acid(TCA) cycle, mitochondrial metabolism, and the metabolism-related protein complex activities which were related with the disorders of energy metabolism and dysfunction of mitochondria of colorectal cancer27,28. Five CRGs containing PDHB, MTF1, DLD, DLAT, and CDKN2A were demonstrated with prognostic value in colorectal cancer, meanwhile, the CDKN2A and DLD were demonstrated with a significant correlation with the pathological characteristics in patients with colorectal cancer by the univariate analysis. Moreover, based on the prognostic value of these five CRGs, a gene signature containing these genes was developed by the LASSO Cox regression analysis, and according to the calculated results of the signature, the high-risk score group was associated with shorter overall survival(OS) rate. The above results revealed that these CRGs not only have a significant expression in colorectal cancer tissues but also could have a correlation with the prognosis and pathological characteristics of the patients with colorectal cancer.
To clarify whether these five prognostic CRGs(PDHB, MTF1, DLD, DLAT, and CDKN2A) were associated with the TIME which was the immune parts of TME in colorectal cancer, the TIMER algorithm was first selected for estimating the immune cell infiltration of these five CRGs, meanwhile, the genes of immune suppressor, MHC, Chemokines, and Chemokines receptor were selected to detect the immune roles of CRGs in colorectal cancer. The results of the TIMER analysis revealed the correlation of these CRGs with the infiltration of the immune cells and four CRGs were illustrated the significant correlation with the B cell, CD8 + T cell, as well as the macrophage which was illustrated as the critical roles in the oncogenesis, metastasis and cell death such as apoptosis and pyroptosis of the tumor cells29,30. The immune suppressor and immune checkpoints have been demonstrated with a significant impact on the escape from the immune surveillance of the tumor cells and immunotherapies in colorectal cancer31,32, MHC genes have shown the correlation with the cell death and function of the immune cells such as CD4 + T cells and CD8 + T cells33,34, chemokines and chemokines receptor genes were associated with the energy metabolism and the macrophage polarization in tumor cells35,36. The above immune-related estimation illustrated the significant correlation between CRGs and TIME in colorectal cancer, especially the MTF1. Moreover, the TMB and MSI analysis results of these five CRGs in colorectal cancer demonstrated that MTF1 and DMAT could serve as the independent biomarkers for the immunotherapies of colorectal cancer37,38. All the above analysis results suggested that these five prognostic CRGs have a significant correlation between the TIME and immune infiltration cells in colorectal cancer, more interestingly, the correlation between the CRGs and the immune genes suggested the CRGs could have an impact on the polarization of macrophages, function of B cell and CD8 + T cell in colorectal cancer and the metastasis of the tumor cells.
For further screening of the immune and functional landscape of these CRGs in colorectal cancer, consensus clustering analysis was selected to explore the novel patterns of these CRGs in TCGA and two clusters were finally identified. The different expression of mRNAs between these two clusters was first detected, and the results revealed the significant characteristics for the classification of CRGs in colorectal cancer. The comparison of the function between these two clusters was subsequently performed by KEGG and GO databases, and the results were summarized with up-regulated and down-regulated. The up-regulated mRNAs were mainly associated with the process of the cell cycle and the metabolism by GO analysis, meanwhile, the pathway patterns by the KEGG database revealed the up clusters mainly participate in the pathways of colorectal cancer, p53 signaling pathway, and the cell cycle pathway which were related with the oncogenesis and metabolism of tumor cells39–41. While the down-regulated mRNAs were mainly related to protein targeting like targeting to membrane, the process of TCA cycle and mitochondrion metabolism such as oxidative phosphorylation and process of ATP metabolism by GO analysis, and the pathways analysis revealed the down-regulated mRNAs mainly participated in the glutathione metabolism and oxidative phosphorylation signaling pathways which were associated with the cell death as well as the dysfunction of the mitochondrion in tumor cells42–44. For screening the difference in immune infiltration landscape between these two clusters, the TIMER algorithm was selected and the results showed a significant difference in the immune infiltration cells, moreover, five immune-checkpoints genes expression between these two clusters also showed a statistically significant difference. The difference of mRNAsi calculated by the OCLR algorithm illustrated the significant difference in stem cell-like characteristics between these two clusters which were associated with the behavior, growth, communication, and immune response of tumor cells in colorectal cancer45–47.
The above results revealed that the CRGs were mainly characterized by the process of the TCA cycle and energy metabolism of tumor cells in colorectal cancer. The results of immune infiltration patterns and mRNA stemness index could provide the clues that the immune checkpoints and stem cell-like characteristics might play key roles in the precise classification of the copper-induced cell death in colorectal cancer, respectively. Meanwhile, the relationship between CRGs and immune cell infiltration also suggested the directions for anti-cancer research such as immunotherapies in colorectal cancer.
There are also some limitations to this research. All studies were carried out with the TCGA-COADREAD cohort, and more data from in vivo, in vitro research, and clinical studies could be used to corroborate the results.