Heterozygous germline deletion in Hif3a exacerbates esophageal squamous cell carcinoma development

Abstract Germline variations contribute to esophageal squamous cell carcinoma (ESCC) susceptibility. We identified a germline deletion (exons 7–8) in HIF3A in an ESCC family and investigated its functional impact using CRISPR/Cas9‐engineered cells and Hif3a ‐eKO1 mice (heterozygous for exons 7–8 deletion). Multi‐omics analysis of Hif3a ‐eKO1 and WT mice revealed dysregulated pathways in normal esophagus and during 4NQO‐induced carcinogenesis, with key biomarkers validated by immunohistochemistry. Hif3a deficiency enhanced ESCC cell proliferation and invasion in vitro and accelerated 4NQO‐induced tumorigenesis in vivo , with Hif3a ‐eKO1 mice developing more and larger neoplastic lesions. Multi‐omics analysis revealed downregulation of cytokeratin‐related genes (notably Krt17) and γδ T cells in normal esophagus of Hif3a ‐eKO1 compared with WT. Consistently reduced Krt17 expression in Hif3a ‐eKO1 was confirmed by both esophageal immunohistochemistry and cellular Western blot analyses. During 4NQO‐induced carcinogenesis, Hif3a deficiency upregulated DNA damage response markers, including Krüppel‐like factor 4 (Klf4) and ATR serine/threonine kinase (Atr). Notably, epithelial cells with abundant γH 2 AX foci lacked Krt17 expression, while Krt17‐positive cells showed minimal γH 2 AX foci. Heterozygous germline Hif3a deletion (exons 7–8) may promote ESCC by disrupting esophageal barrier function—impairing Krt17‐mediated epithelial integrity and reducing γδ T cells—while exacerbating genomic instability. These findings reveal ESCC predisposition mechanisms and therapeutic targets. © 2026 The Pathological Society of Great Britain and Ireland.