Integrative Mendelian Randomization and Single‐Cell Pseudotime Analysis Reveal DKK3 as a PI3K–AKT‐Modulated Driver of Esophageal Squamous Cell Carcinoma

Background Esophageal squamous cell carcinoma (ESCC) remains a highly lethal malignancy, and the molecular drivers of its progression are not fully defined. Dickkopf‐3 (DKK3), a context‐dependent modulator of oncogenic signaling, has been implicated in several solid tumors, but its role in ESCC is unclear. Methods We integrated Mendelian randomization based on cis‐expression quantitative trait loci (cis‐eQTLs) from 31,684 individuals with The Cancer Genome Atlas (TCGA) ESCC transcriptomic data (|log 2 FC| > 1 and p < 0.05) , single‐cell RNA sequencing, and in vitro/in vivo functional assays to identify causal ESCC‐associated genes. DKK3 expression was validated by Western blotting, its effects on ESCC cell proliferation, migration, and clonogenicity were tested experimentally, and DKK3‐related pathways were explored by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Results Integrative differential expression and MR analyses identified 22 ESCC‐associated genes. Single‐cell epithelial profiling further prioritized three candidates (DKK3, NINJ2, and SAPCD2), among which only DKK3 showed high and progressively increasing expression along the malignant epithelial trajectory and was therefore selected for mechanistic validation. DKK3 knockdown significantly inhibited ESCC cell proliferation, migration, and clonogenic capacity in vitro and suppressed tumor growth in nude mice. DKK3 silencing reduced p‐PI3K and p‐AKT levels, and enrichment analyses supported that DKK3 promotes ESCC progression at least partly through activation of the PI3K–AKT signaling pathway. Conclusion By linking germline regulatory variation with single‐cell tumor profiling and functional validation, this study identifies DKK3 as a causally relevant oncogenic regulator in ESCC that drives epithelial tumor growth and migration via PI3K–AKT pathway activation, supporting DKK3 as a potential biomarker and therapeutic targe