Altered DNA methylation is associated with aberrant stemness gene expression in early­stage HNSCC.

ABSTRACT

Head and neck squamous cell carcinoma (HNSCC) is characterized by morphological and functional cellular heterogeneity, which are properties of progenitor cells, as opposed to cell alterations caused by accidental expression of stem cell­related molecules. The expression levels of stemness molecules and their distribution in HNSCC are unclear. As regards sporadic cellular heterogeneity, methylation is an important factor for transcriptional regulation in tumors. Integrative screening analysis of mRNA expression and altered methylation status was performed with original microarrays in 12 tumor and non­tumor pairs of oral squamous cell carcinoma (SCC) cases. From this data set, genes regulated via aberrant DNA methylation and classified proteins were validated by function clustering. Olfactomedin 4 (OLFM4), known as an intestinal stemness molecule and cell­cell adhesion factor, was found to be highly expressed in tumors, with an mRNA expression ratio [tumor/normal (T/N)] of 40.7686 and low methylation (­18.02%) in the promoter region. In addition, the OLFM4 expression levels increased following treatment with the demethylating agent 5­azacytidine in two HNSCC cell lines. Furthermore, the expression levels of OLFM4 in 59 cases of early­stage tongue SCC were analyzed using immunohistochemistry to examine protein expression corresponding to the histopathological definition of tumors and to evaluate prognosis. The aberrant stemness gene expression caused by altered DNA methylation appeared to regulate early­stage HNSCC characteristics. The results of the present study indicated a correlation between OLFM4 expression and promoter methylation, and suggest that it plays an important role in tumor cell heterogeneity in HNSCC.