Host-microbe-epigenetic Crosstalk: Dissecting the Role of Microbiome, Immune Dysregulation, and DNA Methylation in Squamous Cell Carcinoma

Abstract Squamous carcinomas of the oral cavity and head and neck share molecular, microbial, and immune features. Dysbiosis and intratumoral microbiota are increasingly implicated in epithelial transformation, with taxa such as Fusobacterium nucleatum linked to inflammation, hypoxia, immune evasion, and metastasis across squamous sites. Defining how host-microbe-immune interactions evolve from oral premalignant lesions to invasive tumors could reveal early biomarkers and modifiable microbial targets for prevention, risk stratification, and intervention. We analyzed samples from 66 oral potentially malignant diseases (OPMD) and 74 head and neck squamous cell carcinoma (HNSCC) using a multi-omics approach. Cohorts were profiled via RNA-seq, shotgun metagenomics, and multiplex cytokine assays. Epigenetic profiling of 20 HNC samples was performed using Nanopore sequencing to assess genome-wide DNA methylation. In OPMDs, a high-risk hypoxic subtype showed an elevated incidence of malignant transformation (p < 0.0001), reduced microbial diversity, and significantly elevated abundance of Fusobacterium nucleatum and Leptotrichia wadei (Log2FC > 1, FDR < 0.05). F. nucleatum activity correlated strongly (R>0.7) with keratinocytes and pericytes. While HNSCC alpha diversity remained stable, community structure shifted (PERMANOVA p=0.001), marked by an enrichment of Lactobacillus spp. driving palmitate metabolism, whereas OPMD favored starch degradation and nitrate reduction. Immune profiling revealed a profound dysregulation within head and neck tumors. While OPMD was characterized by high levels of pro-inflammatory mediators (TNF-α, IL-6), these cytokines were markedly downregulated in HNSCC. This suggests a functional shift from an acute inflammatory response in OPMDs toward a chronic, immune-suppressive state in established carcinoma. Finally, our epigenetic analysis linked oral dysbiosis with host metabolism, revealing significant differential methylation in overweight HNC patients. Our analysis revealed significant hypomethylation affecting key oncogenic and immunomodulatory genes, including NOTCH1, STAT3, and the antigen-presenting gene HLA-DRB1. Our findings demonstrate a conserved microbial trajectory in the progression of head/neck carcinomas, characterized by the enrichment of Fusobacterium and a functional switch from a pro-inflammatory to an immune-evasive tumor microenvironment. These results point to a shared microbial etiology in squamous cancers, and the ongoing epigenetic analysis will provide deeper mechanistic insights into how these host-microbe interactions drive malignant transformation. Citation Format: Armando G. Licata, Cristina Gurizzan, Deborah Lenoci, Marta Lucchetta, Carlo Resteghini, Luigi Lorini, Rosalba Miceli, Paolo Bossi, Loris De Cecco. Host-microbe-epigenetic crosstalk: Dissecting the role of microbiome, immune dysregulation, and DNA methylation in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2866.