Abstract
Background: Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies, with a rising global incidence. Despite advances in diagnosis and treatment, effective biomarkers for early detection and targeted therapy remain limited. β-Klotho (KLB), a co-receptor in the FGF signaling pathway, has been implicated in tumor progression and poor prognosis in several cancers. However, its role in CRC is still poorly understood. Methods: To investigate the function of KLB in CRC, we performed a comprehensive pan-cancer analysis using datasets from TCGA, GTEx, Human Protein Atlas, cBioPortal, UALCAN, and TIMER2.0. Analyses included KLB expression profiles, promoter methylation, genetic alterations, immune checkpoint associations, and immune cell infiltration. Functional enrichment was conducted via GO/KEGG and GSEA. A prognostic model was constructed using biostatistical methods. In vitro and in vivo assays-including CCK-8, colony formation, wound healing, and subcutaneous tumor formation in mice-were conducted to evaluate the biological effects of KLB on CRC cells. Additionally, single-cell RNA sequencing data were analyzed to explore the cell-type-specific expression of KLB within the tumor microenvironment. Results: KLB expression was significantly downregulated in CRC tissues compared to normal tissues and negatively correlated with TNM stage and overall prognosis. Promoter methylation and somatic mutations were observed in CRC, suggesting epigenetic and genetic regulation of KLB. Functional enrichment revealed that KLB is involved in regulating the cell cycle, chromatin remodeling, and immune responses. The prognostic nomogram based on KLB expression showed improved predictive performance for progression-free interval (C-index = 0.778). Experimentally, KLB overexpression inhibited CRC cell proliferation and migration. Single-cell RNA-seq analysis demonstrated that KLB is primarily expressed in fibroblasts and stromal cells within the CRC tumor microenvironment, implicating its role in intercellular communication and immune modulation through pathways such as MIF and MK signaling. Conclusion: KLB functions as a potential tumor suppressor in CRC, with diagnostic, prognostic, and therapeutic implications. Its expression in stromal components of the tumor microenvironment suggests a regulatory role in immune response and tumor progression. These findings support KLB as a promising biomarker and therapeutic target for CRC.