Cis-regulatory Alterations in FOXP4 Modulate Esophageal Cancer Susceptibility Induced by Chronic Alcohol Exposure

Abstract Chronic alcohol exposure is a risk factor for developing esophageal squamous cell carcinoma (ESCC). To identify alcohol-responsive genes involved in esophageal carcinogenesis, we employed mouse models to systematically investigate alterations in cis-regulatory elements (CREs) in the esophageal epithelium across different ethanol exposure durations. A key exposure duration, 16 weeks of exposure to 20% ethanol, corresponded with increased expression of 222 genes that correlated with ESCC progression and were enriched in pathways related to epithelial proliferation and oncogenesis. Construction of a comprehensive CRE-gene map in human ESCC enables further evaluation of the role of the alcohol-responsive genes in ESCC susceptibility, identifying promoter and enhancer variants. A three-stage case-control study involving 9,033 ESCC cases and 10,801 controls revealed an enhancer variant, rs10223516, in FOXP4 that was associated with ESCC susceptibility through gene-alcohol interaction. The rs10223516 variant modulated FOXP4 expression through a long-range interaction, with the T allele exhibiting higher enhancer activity. Alcohol drinkers with the TT genotype exhibited a 76% higher risk of developing ESCC than non-drinkers with CC or TC genotype. Functional assays confirmed that the variant enhanced FOXP4 transcriptional activity, and upregulated FOXP4 promoted ESCC development in vivo. ChIP-seq and RNA-seq analyses further demonstrated that FOXP4 enhanced ESCC susceptibility and tumor growth by transcriptionally activating CYP26B1 and MYC. These findings highlight the complex gene-environment interactions between alcohol consumption and epigenetic alterations in esophageal tumorigenesis, offering potential targets for ESCC detection and prevention.