Development and validation of a cell death gene signature for overall survival in clear cell renal cell carcinoma

Kidney renal clear carcinoma (KIRC) is a common genitourinary malignancy with high morbidity and mortality rates despite multimodal therapies. Programmed cell death (PCD) is an active death process that occurs not only in the normal development of individuals but also in abnormal physiological states or diseases. PCD pathways dysfunction is associated with the formation and progression of cancers. Expression profiles of KIRC specimens and corresponding clinical information were obtained from the Cancer Genome Atlas (TCGA) and ArrayExpress databases. Single-cell transcriptome were collected from GEO database. Eight cell death pathways genes were downloaded from Kyoto Encyclopedia of Genes and Genomes database, Gene Set Enrichment Analysis website, Human Autophagy Database website and FerrDb website and the literature, respectively. Consensus clustering analysis, differential gene analysis, survival analysis, correlation analysis, immune cell infiltration analysis, least absolute shrinkage and selection operator regression analysis, univariate and multivariate Cox analysis were conducted using R software. Experimental validation was performed using RT-qPCR, Western blotting, immunohistochemistry, siRNA-mediated gene knockdown, and flow cytometry. We identified three cell death-relevant subtypes of KIRC based on 709 genes of eight PCD-related pathways. Each cluster displayed distinct clinical, cellular, and immunological characteristics. Moreover, a novel cell death related prognostic model was constructed in this research. Survival analysis and time-dependent receiver operating characteristic analysis demonstrated that the model was reliable and robust not only in TCGA cohort, but also in an external validation dataset (E-MTAB-1980 cohort). In addition, RT-qPCR results confirmed the differential expressions of DEGs in the tissue samples, which was in line with the results of TCGA. Western blotting and immunohistochemistry further confirmed the elevated protein expression of IRF9, CXCL2, and EIF4EBP1 in KIRC tissues. In addition, siRNA-mediated IRF9 knockdown increased apoptosis in 786-O and A-498 cells. This study identified three cell death-relevant subtypes of KIRC based on the genes of eight PCD-related pathways, which were associated with OS and DSS of KIRC. We also defined a novel prognostic model of seven cell death-related genes, which was proven to be independently associated with the OS of KIRC, and the prognostic model was validated in an external dataset. The experimental findings further suggest that IRF9 may contribute to apoptosis resistance in ccRCC cells.