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

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.

口腔和口咽鳞状细胞癌（OOSCC）的生存率较低，主要归因于癌细胞向区域性淋巴结的转移。为了对这些转移进行最佳治疗，需要进行颈部切除术；然而，检测方法的准确性不足导致了治疗不足和治疗过度。新的DNA预后甲基化生物标志物可能有助于提高淋巴结转移的检测。为了确定与淋巴结转移相关的表观遗传调控基因，对6例具有（pN+）淋巴结转移的OOSCC和6例无（pN0）淋巴结转移的OOSCC进行了全基因组甲基化分析，并结合了预测OOSCC中pN+状态的基因表达特征。通过免疫组化和焦磷酸测序验证了所选基因，并使用来自癌症基因组图谱的数据。两步统计分析揭示了14个在pN+ OOSCC中甲基化状态增加和mRNA下调的基因。RAB25，一种已知的肿瘤抑制基因，在发现集中排名最高。在验证集中，RAB25 mRNA（P = 0.015）和蛋白质水平（P = 0.012）在pN+ OOSCC中均较低。RAB25 mRNA水平与RAB25甲基化水平呈负相关（P < 0.001），但RAB25蛋白质表达则不然。我们的研究揭示了启动子甲基化是导致pN+ OOSCC中RAB25表达下调的机制，而表达下调与淋巴结转移相关。RAB25甲基化的检测可能有助于淋巴结转移的诊断，并作为OOSCC中潜在的新型治疗靶点。