Alcohol, Tobacco, and HPV exposure synergistically regulate promoter methylation of PD-L1 related genes in Head and Neck Squamous Cell Carcinoma

Abstract

Although alcohol, tobacco and human papillomavirus (HPV) are established risk factors for head and neck squamous cell carcinoma (HNSCC), the molecular effects of their co-occurrence remain poorly understood. DNA methylation may link exposure history to regulation of the PD-1/PD-L1 immune checkpoint pathway, whose therapeutic blockade benefits only a subset of patients with recurrent or metastatic HNSCC. We stratified primary tumors from The Cancer Genome Atlas HNSCC cohort by all combinations of these exposures and examined promoter methylation across seven interferon-{gamma}-PD-L1 signaling genes at single-CpG resolution, with region-level validation. Triple-exposure tumors exhibited coordinated methylation changes involving CD274, IRF1, JAK1, PDCD1, MIR33A and MIR34A, with differentially methylated regions overlapping PDCD1, MIR34AHG and JAK1. CD274 promoter methylation is inversely correlated with gene expression. Significant methylation differences occurred only in HPV-positive subgroups, suggesting HPV may be necessary for this phenotype, while concurrent exposures may modify its extent. Transcriptomic analysis and immune deconvolution further suggested greater estimated immune infiltration alongside downregulation of innate and adaptive immune activation pathways, consistent with an inflamed but functionally suppressed tumor microenvironment. These findings suggest combined exposure history may help contextualize checkpoint pathway regulation in HNSCC.