Increased Non-Homologous End Joining Makes DNA-PK a Promising Target for Therapeutic Intervention in Uveal Melanoma
Increased Non-Homologous End Joining Makes DNA-PK a Promising Target for Therapeutic Intervention in Uveal Melanoma
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DOI：
10.3390/cancers11091278
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发表时间：
2019-09-01
期刊：
影响因子：
5.2
通讯作者：
Sisley, Karen
中科院分区：
文献类型：
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作者：
Doherty, Rachel E.;Bryant, Helen E.;Sisley, Karen
葡萄膜黑色素瘤（UM）是成人中最常见的原发性眼内肿瘤，转移后的平均生存期为6个月。30多年来，生存率没有提高。这项研究表明，姐妹染色单体交换（SCE）是低UM这可能是由于FANCD 2的表达减少。由于FANCD 2可以起到抑制非同源末端连接（NHEJ）的作用，因此本研究研究了UM中的NHEJ。NHEJ途径蛋白DNA依赖性蛋白激酶（DNA-PK）的催化亚基的活化通过分析焦点形成和使用基于质粒的末端连接测定确定的NHEJ连接效率来测量。使用小干扰RNA（siRNA）敲低和DNA-PK的化学抑制剂，测定原代UM培养物和两种细胞系的存活率。为了评估响应于DNA-PK抑制的同源重组能力，进行了SCE分析。此外，为了支持这些发现，使用癌症基因组图谱（TCGA）-UM RNAseq数据（n = 79）分析了与NHEJ相关的基因的信使RNA（mRNA）表达。UM中NHEJ活性和DNA-PKcs活化上调，并且DNA-PK的抑制选择性地诱导细胞凋亡，并且对电离辐射和链间交联剂敏感。NHEJ蛋白DNA-PK的抑制对UM是致命的，表明单独或作为其他治疗的敏化剂是潜在有效的治疗选择。
Uveal melanoma (UM) is the most common primary intraocular tumour in adults, with a mean survival of six months following metastasis. The survival rates have not improved in over 30 years. This study has shown that sister chromatid exchange (SCE) is low in UM which is likely due to a reduced expression of FANCD2. As FANCD2 can function to suppress non-homologous end joining (NHEJ), this study therefore investigated NHEJ in UM. The activation of the catalytic subunit of the NHEJ pathway protein DNA-dependent protein kinase (DNA-PK) was measured by analysing the foci formation and the ligation efficiency by NHEJ determined using a plasmid-based end-joining assay. Using small-interfering RNA (siRNA) knock-down, and chemical inhibitors of DNA-PK, the survival of primary UM cultures and two cell lines were determined. To assess the homologous recombination capacity in response to the inhibition of DNA-PK, a SCE analysis was performed. In addition, to support the findings, the messenger RNA (mRNA) expression of genes associated with NHEJ was analysed using the Cancer Genome Atlas (TCGA)-UM RNAseq data (n = 79). The NHEJ activity and DNA-PKcs activation was upregulated in UM and the inhibition of DNA-PK selectively induced apoptosis and sensitized to ionising radiation and inter-strand cross-linking agents. The inhibition of the NHEJ protein DNA-PK is lethal to UM, indicating a potentially effective therapeutic option, either alone or as a sensitizer for other treatments.