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 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. The construction of a comprehensive cis-regulatory element–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 nondrinkers with the CC or TC genotype. Functional assays confirmed that the variant enhanced FOXP4 transcriptional activity, and upregulated FOXP4 promoted ESCC development in vivo. Chromatin immunoprecipitation sequencing and RNA sequencing 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. Significance: An enhancer variant in FOXP4 mediates the interplay between alcohol consumption, epigenetic alterations, and esophageal carcinogenesis, which could inform development of early detection and prevention strategies.