Ramsawhook Ashley, Lewis Lara, Coyle Beth, Ruzov Alexey
Division of Cancer and Stem Cells, School of Medicine, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD UK.
Children's Brain Tumour Research Centre, School of Medicine, QMC, University of Nottingham, Nottingham, NG7 2UH UK.
Clin Epigenetics. 2017 Feb 13;9:18. doi: 10.1186/s13148-016-0306-2. eCollection 2017.
Alteration of DNA methylation (5-methylcytosine, 5mC) patterns represents one of the causes of tumorigenesis and cancer progression. Tet proteins can oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine (5caC). Although the roles of these oxidised forms of 5mC (oxi-mCs) in cancer pathogenesis are still largely unknown, there are indications that they may be involved in the mechanisms of malignant transformation. Thus, reduction of 5hmC content represents an epigenetic hallmark of human tumours, and according to our recent report, 5caC is enriched in a proportion of breast cancers and gliomas. Nevertheless, the distribution of oxi-mCs in paediatric brain tumours has not been assessed.
Here, we analyse the global levels and spatial distribution of 5hmC and 5caC in four brain tumour cell lines derived from paediatric sonic hedgehog (SHH) pathway-activated medulloblastomas (Daoy and UW228-3) and ependymomas (BXD-1425EPN and DKFZ-EP1NS). We show that, unlike HeLa cells, the paediatric tumour cell lines possess both 5hmC and 5caC at immunochemically detectable levels and demonstrate that both modifications display high degrees of spatial overlap in the nuclei of medulloblastomas and ependymomas. Moreover, although 5hmC levels are comparable in the four brain tumour cell lines, 5caC staining intensities differ dramatically between them with highest levels of this mark in a subpopulation of DKFZ-EP1NS cells. Remarkably, the 5caC enrichment does not correlate with 5hmC levels and is not associated with alterations in thymine DNA glycosylase () expression in SHH medulloblastoma and ependymoma cell lines but corresponds to elevated levels of transcript in UW228-3 and DKFZ-EP1NS cells.
We demonstrate that both 5caC enrichment and elevated expression are observed in SHH medulloblastomas and ependymomas. Our results suggest that increased Tet-dependent 5mC oxidation may represent one of the epigenetic signatures of cancers with neural stem cell origin and, thus, may contribute to development of novel approaches for diagnosis and therapy of the brain tumours.
DNA甲基化（5-甲基胞嘧啶，5mC）模式的改变是肿瘤发生和癌症进展的原因之一。Tet蛋白可将5mC氧化为5-羟甲基胞嘧啶（5hmC）、5-甲酰基胞嘧啶和5-羧基胞嘧啶（5caC）。尽管这些5mC氧化形式（oxi-mCs）在癌症发病机制中的作用仍大多未知，但有迹象表明它们可能参与恶性转化机制。因此，5hmC含量的降低是人类肿瘤的一种表观遗传特征，并且根据我们最近的报告，5caC在一部分乳腺癌和神经胶质瘤中富集。然而，oxi-mCs在儿童脑肿瘤中的分布尚未得到评估。
在这里，我们分析了来自儿童音猬因子（SHH）通路激活的髓母细胞瘤（Dao和UW228-3）和室管膜瘤（BXD-1425EPN和DKFZ-EP1NS）的四种脑肿瘤细胞系中5hmC和5caC的整体水平和空间分布。我们表明，与HeLa细胞不同，儿童肿瘤细胞系在免疫化学可检测水平上同时具有5hmC和5caC，并证明这两种修饰在髓母细胞瘤和室管膜瘤细胞核中显示出高度的空间重叠。此外，尽管四种脑肿瘤细胞系中的5hmC水平相当，但它们之间的5caC染色强度差异很大，在DKFZ-EP1NS细胞亚群中该标记水平最高。值得注意的是，5caC的富集与5hmC水平无关，并且与SHH髓母细胞瘤和室管膜瘤细胞系中胸腺嘧啶DNA糖基化酶（）表达的改变无关，但与UW228-3和DKFZ-EP1NS细胞中转录本水平的升高相对应。
我们证明在SHH髓母细胞瘤和室管膜瘤中观察到5caC富集和转录本表达升高。我们的结果表明，增加的Tet依赖性5mC氧化可能代表具有神经干细胞起源的癌症的表观遗传特征之一，因此可能有助于开发脑肿瘤诊断和治疗的新方法。
