Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide, with human papillomavirus (HPV)-related HNSCC rising to concerning levels. Extensive clinical, genetic and epigenetic differences exist between HPV-associated HNSCC and HPV-negative HNSCC, which is often linked to tobacco use. However, 5-hydroxymethylation (5hmC), an oxidative derivative of DNA methylation and its heterogeneity among HNSCC subtypes, has not been studied. We characterized genome-wide 5hmC profiles in HNSCC by HPV status and subtype in 18 HPV(+) and 18 HPV(−) well-characterized tumors. Results showed significant genome-wide hyper-5hmC in HPV(−) tumors, with both promoter and enhancer 5hmC able to distinguish meaningful tumor subgroups. We identified specific genes whose differential expression by HPV status is driven by differential hydroxymethylation. CDKN2A (p16), used as a key biomarker for HPV status, exhibited the most extensive hyper-5hmC in HPV(+) tumors, while HPV(−) tumors showed hyper-5hmC in CDH13, TIMP2, MMP2 and other cancer-related genes. Among the previously reported two HPV(+) subtypes, IMU (stronger immune response) and KRT (more keratinization), the IMU subtype revealed hyper-5hmC and up-regulation of genes in cell migration, and hypo-5hmC with down-regulation in keratinization and cell junctions. We experimentally validated our key prediction of higher secreted and intracellular protein levels of the invasion gene MMP2 in HPV(−) oral cavity cell lines. Our results implicate 5hmC in driving differences in keratinization, cell junctions and other cancer-related processes among tumor subtypes. We conclude that 5hmC levels are critical for defining tumor characteristics and potentially used to define clinically meaningful cancer patient subgroups.

中文翻译：

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5-羟甲基化突出了头颈癌角化和细胞连接的异质性

头颈部鳞状细胞癌 (HNSCC) 是全球第六大流行的癌症，与人乳头瘤病毒 (HPV) 相关的 HNSCC 上升到令人担忧的水平。HPV 相关 HNSCC 和 HPV 阴性 HNSCC 之间存在广泛的临床、遗传和表观遗传差异，这通常与烟草使用有关。然而，尚未研究 5-羟甲基化 (5hmC)，一种 DNA 甲基化的氧化衍生物及其在 HNSCC 亚型中的异质性。我们通过 18 种 HPV(+) 和 18 种 HPV(-) 特征明确的肿瘤中的 HPV 状态和亚型来表征 HNSCC 中的全基因组 5hmC 谱。结果显示 HPV(-) 肿瘤中显着的全基因组 hyper-5hmC，启动子和增强子 5hmC 都能够区分有意义的肿瘤亚组。我们确定了特定基因，其由 HPV 状态引起的差异表达是由差异羟甲基化驱动的。CDKN2A (p16)，用作 HPV 状态的关键生物标志物，在 HPV(+) 肿瘤中表现出最广泛的 hyper-5hmC，而 HPV(-) 肿瘤在 CDH13、TIMP2、MMP2 和其他癌症相关基因中表现出 hyper-5hmC . 在之前报道的两种 HPV(+) 亚型中，IMU（更强的免疫反应）和 KRT（更多角化），IMU 亚型显示高 5hmC 和细胞迁移基因的上调，以及低 5hmC 和下调角化和细胞连接。我们通过实验验证了我们对 HPV(-) 口腔细胞系中侵袭基因 MMP2 的更高分泌和细胞内蛋白质水平的关键预测。我们的结果表明 5hmC 导致角化差异，肿瘤亚型之间的细胞连接和其他癌症相关过程。我们得出结论，5hmC 水平对于定义肿瘤特征至关重要，并可能用于定义具有临床意义的癌症患者亚组。