A Pan-Cancer Multi-Omics Analysis of CAD: Integrating CRISPR and Metabolomics Data to Unravel the Metabolic-Immune...

Background: CAD (Carbamoyl-Phosphate Synthetase 2, Aspartate Transcarbamylase, and Dihydroorotase) is a pivotal tri-functional enzyme complex associated with the rate-limiting steps of the de novo pyrimidine biosynthetic pathway. Despite its established metabolic role, the multi-dimensional involvement of CAD in the pan-cancer landscape-specifically regarding its regulation of the metabolic-immune axis and its impact on immunotherapy response-remains to be fully elucidated. Methods: We performed a systematic pan-cancer multi-omics analysis integrating TCGA, GTEx, and DepMap datasets to evaluate CAD expression, genomic alterations, and diagnostic potential. In addition, multiple immunotherapy cohorts were integrated for meta-analysis, and metabolomic data were incorporated to explore CAD-associated metabolic features. Results: CAD was significantly upregulated in 17 cancer types, with protein-level evidence supporting this trend. CAD also showed high diagnostic accuracy in several malignancies, particularly LAML and CHOL (AUC approximately 1.0). In immunotherapy-related analyses, CAD expression was positively associated with TMB, MSI, and initial therapeutic response, but was also linked to worse long-term overall survival in pooled cohorts (HR = 1.42, 95% CI: 1.19-1.70). Integrative metabolomic analyses further suggested that high CAD expression was associated with pyrimidine metabolite accumulation and altered amino acid metabolism, indicating a potential link between CAD-related metabolic reprogramming and the tumor immune microenvironment. Conclusions: CAD may represent a promising candidate biomarker across multiple malignancies. Notably, its association with immunotherapy-related features, together with the observed discordance between response-associated signals and long-term survival, warrants further mechanistic and clinical validation.