Integrative Analysis of Epigenomic and Transcriptomic Alterations in Tissue-Engineered Models of Oral Cancer Progression

Abstract

Oral potentially malignant disorders (OPMDs) clinically present as white and/or red lesions with histological changes of oral epithelial dysplasia (OED) that can progress to oral squamous cell carcinoma (OSCC). However, no molecular or epigenetic markers currently exist that reliably predict progression to OSCC. DNA methylation has emerged as a critical hallmark of cancer, although its role in OPMDs remains poorly understood. Most studies rely on monolayer cell cultures, which fail to replicate the physiological in vivo environment. This study developed tissue-engineered oral mucosa constructs to model OSCC progression and investigate DNA methylation associated with OPMDs. Full-thickness, 3D tissue-engineered oral mucosa models were generated using normal (FNB6), dysplastic (DOK, D19), and OSCC (H357) keratinocyte cell lines grown on collagen hydrogels populated with primary fibroblasts. Histological and immunohistochemical comparisons with normal, OED and OSCC human tissues were performed to validate the models. Next-generation RNA sequencing and DNA-methylation profiling were used to identify differentially expressed and methylated genes. RNA sequencing revealed distinct gene expression patterns in 3D models resembling in vivo conditions more closely than traditional 2D cultures. DNA methylation profiling of OED identified early promoter methylation disturbances. PODXL emerged as a novel epithelial marker for oral cancer progression based on RNA sequencing across normal, OED, and OSCC samples. Furthermore, integrative analysis highlighted 59 hypermethylated and downregulated genes implicated in p53 signalling, tumour suppression, and choline metabolism. The hypomethylating agent decitabine restored the expression of several target genes in mild OED but showed limited efficacy in severe OED, likely due to chromosomal abnormalities. These findings demonstrate the value of 3D tissue-engineered models for studying oral cancer progression and highlight the importance of epigenetic alterations in OED. Decitabine therapy may reverse early dysplastic changes, though its impact decreases at advanced stages. Future research should investigate interventions aimed at these early disruptions.