ABSTRACT: Background: Potassium voltage-gated channel subfamily Q member 3 (KCNQ3) is primarily recognized for its function in neuronal excitability; however, its expression patterns, genetic landscape, and functional significance across human malignancies remain largely unexplored. This study aimed to systematically investigate the pan-cancer expression profile, prognostic value, immune regulatory functions, and molecular mechanisms of KCNQ3, with a focused functional validation in cholangiocarcinoma (CHOL). Methods: We integrated multi-omics data from public databases including GTEx, HPA, TIMER2, TISCH2, cBioPortal, SMART, and TIMER3 to comprehensively analyze KCNQ3 expression, genetic alterations, DNA methylation, clinical prognosis, immune infiltration, and pathway enrichment across 33 cancer types. Single-cell RNA sequencing and spatial transcriptomics were employed to dissect KCNQ3 expression at the cellular level. In vitro and in vivo functional experiments, including CCK-8, colony formation, EdU, Transwell, wound healing, flow cytometry, and xenograft models, were conducted in CHOL cell lines (RBE and 9810). RNA sequencing, Western blotting, and pharmacological inhibition were utilized to elucidate the underlying molecular mechanisms. Results: KCNQ3 exhibited tissue-specific expression in normal tissues and was significantly dysregulated across multiple tumor types, with notable upregulation in breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), lung adenocarcinoma (LUAD), and thyroid carcinoma (THCA). Single-cell analysis revealed predominant KCNQ3 expression in malignant cells, mono/macrophages, endothelial cells, and myofibroblasts, with spatial transcriptomics confirming elevated expression in malignant regions. Pan-cancer genomic analysis identified copy number amplifications as the predominant alteration, which correlated with worse progression-free survival (PFS, P = 1.709 × 10⁻³) and disease-free survival (DFS, P = 7.350 × 10⁻⁶). Widespread DNA hypomethylation of KCNQ3 was observed across tumors, potentially contributing to transcriptional activation. Elevated KCNQ3 expression was significantly associated with unfavorable overall survival (OS), PFS, disease-specific survival (DSS), and disease-free interval (DFI) in multiple malignancies. Gene set enrichment analysis demonstrated robust positive enrichment of epithelial-mesenchymal transition (EMT), inflammatory response, angiogenesis, and KRAS signaling, coupled with suppression of oxidative phosphorylation. Immune landscape analysis revealed extensive correlations between KCNQ3 and diverse immune cell subsets, immune stimulators, inhibitors, and chemokines, suggesting a role in tumor immune microenvironment modulation. Notably, KCNQ3-high malignant cells exhibited enhanced cell-cell communication with surrounding stromal and immune populations. Functional studies in CHOL demonstrated that KCNQ3 promoted cell proliferation, suppressed apoptosis, and enhanced migration, invasion, and EMT in vitro, while stable knockdown significantly inhibited tumor growth and pulmonary metastasis in vivo. Mechanistically, transcriptomic profiling and pathway analysis identified ERK1/2-MAPK cascade activation as a central mediator of KCNQ3-driven oncogenesis, and pharmacological ERK inhibition substantially reversed the pro-tumorigenic effects of KCNQ3 overexpression. Conclusion: Our pan-cancer analysis establishes KCNQ3 as a prognostic biomarker and functional oncogene across multiple human cancers. KCNQ3 orchestrates tumor progression, immune microenvironment remodeling, and metabolic reprogramming, with its oncogenic effects in cholangiocarcinoma mediated through activation of the ERK1/2 signaling pathway. These findings highlight KCNQ3 as a potential therapeutic target and prognostic indicator in precision oncology.
