DERL3 (Derlin 3) is a Derlin family protein involved in endoplasmic reticulum (ER) associated degradation of unfolded or misfolded glycoproteins. Our previous comprehensive genomic and transcriptomic profiling of leukoplakia and oral squamous cell carcinoma (OSCC) revealed amplification at the 22q11.23 locus, which harbours the DERL3 gene. However, DERL3 mRNA expression was found to be downregulated during disease progression, suggesting the role of epigenetic regulation.

We evaluated promoter methylation using the Quantitative real-time methylation-specific PCR technique, copy number alterations using TaqMan assays, and protein expression through immunohistochemistry. The methylation levels between normals, leukoplakia and OSCC were compared using the Kruskal–Wallis test. Correlations between different techniques were computed using Spearman's rank correlation. The indirect and direct effects of DERL3 gene amplification on methylation and protein expression were evaluated using mediation analyses. Recurrence-free survival and disease-specific survival were calculated using a log-rank test.

We observed a sequential, significant increase in promoter methylation of leukoplakia, early-stage, and advanced-stage OSCC (p-value < 0.001), compared to control tissues. The promoter methylation was inversely correlated with DERL3 gene expression. Interestingly, the protein was overexpressed in leukoplakia (p = 0.023), early-stage (p = 0.012), and advanced-stage OSCC (p < 0.001) compared to normal tissue. High protein expression correlated with advanced-stage OSCC, pathological grade and habit profile, while methylation showed a significant association with age, pathological grade and advanced-stage OSCC. However, DERL3 aberrations were not associated with patient survival.

Our findings highlight that while promoter hypermethylation may account for reduced DERL3 mRNA expression, the concomitant increase in DERL3 protein expression suggests that additional regulatory mechanisms contribute to OSCC progression.