Biologic Significance of 5-Hydroxymethylcytosine Expression in Oral Mucosal Epithelial Dysplasia and Oral Squamous Cell Carcinoma

Abstract

Current modeling postulates that the development of cancer is driven by the accumulation of genetic mutations and epigenetic modifications, resulting in a clonal population of cells with deregulated growth characteristics. Three epigenetic mechanisms have been well documented, including DNA methylation, histone modifications and non-coding RNAs. Of these, DNA methylation is the best studied epigenetic alteration in cancer. Characterization of the expression of the epigenetic marker 5-hydroxymethylcytosine (5-hmC) in human and experimental melanoma and its precursors has established it as an important functionally relevant epigenetic biomarker in cancer. We therefore posited that 5-hmC in oral premalignant lesions and oral squamous cell carcinomas represents an opportunity to identify a novel epigenetic molecular pathway that may serve as a biomarker for early diagnosis of oral neoplasms and subsequent improvements in the prognosis of oral cancer. A total of sixty-six histologic samples (N=66) were obtained from the Harvard School of Dental Medicine affiliated bio-archives. Immunohistochemistry for 5-hmC was performed on nine cases diagnosed as fibromas (F, nF=9) with uninvolved margins used to evaluate non-inflamed/non-cancerous mucosa; nine cases diagnosed as frictional keratosis (FK, nFK=9); ten cases diagnosed as lichen planus (LP, nLP=10); fifteen cases diagnosed as moderate-to-severe oral epithelial dysplasia (OED, nOED=15) and twenty-three cases diagnosed as oral squamous cell carcinoma (oSCC, noSCC=23). Human cell lines of oral keratinocytes from benign oral mucosa, oral epithelial dysplasia and four types of oral squamous cell carcinoma were also evaluated to detect the expression of 5-hmC genomic DNA. Finally, immunohistochemistry for 5-hmC was also performed in histologic samples of a murine model where oral squamous cell carcinoma was induced with the carcinogen 4-nitroquinoline-1-oxide (N=10). Five samples consisted of non-inflamed/non-neoplastic normal dorsal tongue mucosa (T, nT=5) and five consisted of oral squamous cell carcinoma (mSCC, nmSCC=5). Progressive loss from benign and reactive/inflammatory oral mucosal lesions to oral epithelial dysplasia and oral squamous cell carcinoma was observed in patient samples. Likewise, distinct patterns with regard to the distribution of staining was noted in benign non-inflamed lesions versus reactive inflammatory lesions such as frictional keratosis and lichen planus, providing further explanation germane for the understanding of the responses of the oral mucosa to tissue injury. The finding in human tissue of loss of 5-hmC immunoreactivity within dysplasia and carcinoma were substantiated in human cell lines at a DNA level, and further validated in an in vivo experimental model of murine oral SCC. In aggregate, our results confirm the potential importance of loss of 5-hmC as a novel epigenetic mark in oral SCC at both diagnostic and biological levels, and establish a murine model of oral SCC induction as a potentially informative approach to understanding the role of epigenetic regulation in oral carcinogenesis.