RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

ABSTRACT

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC.

KEYWORDS:

• DNA methylation
• epigenetic regulation
• head and neck cancer
• metastasis
• MethylCap-Seq
• oral squamous cell carcinoma

Disclosure of potential conflicts of interest

The CTMM Air Force Consortium is a private/public consortium with involvement of academia, private companies, and the government. It is not a commercial source of funding. M Clausen and M Mastik are funded by CTMM consortium to perform the research project (global methylation screening in HNSCC) that is a part of Clausen's thesis. These funds had no role in study design, data collection and analysis, decision to publish and preparation of the manuscript. No other external funding sources for this study. W van Criekinge is an employee for MDXHealth. The other authors have declared no conflicts of interest.

Funding

This work was funded by the CTMM Air Force consortium (http://www.ctmm.nl). CTMM pays for the salaries of M Clausen and M Mastik (partly), S Denil is supported by an IWT grant (SB101371).