Transcriptomic Analyses of MYCN-Regulated Genes in Anaplastic Wilms' Tumour Cell Lines Reveals Oncogenic Pathways and Potential Therapeutic Vulnerabilities.
Transcriptomic Analyses of MYCN-Regulated Genes in Anaplastic Wilms' Tumour Cell Lines Reveals Oncogenic Pathways and Potential Therapeutic Vulnerabilities.
复制标题
DOI：
10.3390/cancers13040656
复制
发表时间：
2021-02-06
期刊：
影响因子：
5.2
通讯作者：
Malik K
中科院分区：
文献类型：
--
作者：
Szemes M;Melegh Z;Bellamy J;Park JH;Chen B;Greenhough A;Catchpoole D;Malik K
肾母细胞瘤 (WT) 是最常见的儿科实体恶性肿瘤之一，产生于发育中的肾脏。大多数 WT 的治疗相对成功，但具有间变组织学的肿瘤除外。间变性 WT 存活率可低至 30-50%，这强调需要更好地了解其分子病因学，以促进针对不良预后 WT 的新疗法的开发。先前的研究表明，在发育不全的野生型中，MYCN 在 DNA 和 RNA 水平上增加，尽管 MYCN 蛋白的生物学功能尚未报道。在本研究中，我们定义了 WT 中 MYCN 蛋白的表达模式，并调节间变性 WT 细胞系中的 MYCN 蛋白水平，以确定 MYCN 在 WT 中的功能。我们发现 MYCN 耗竭会导致细胞生长减少，并确定了 MYCN 调节的基因和通路，这些基因和通路可能代表了预后不良的 WT 的治疗脆弱性。 MYCN 原癌基因在许多癌症中失调，尤其是在神经母细胞瘤中，其中 MYCN 基因扩增识别出预后非常差的临床子集。基因表达和 DNA 分析还表明，在预后较差的维尔姆斯肿瘤（包括弥漫性退行性变的肿瘤）中，MYCN mRNA 过度表达，以及 MYCN 的局灶性扩增、拷贝数增加和功能突变的推定变化。然而，令人惊讶的是，MYCN 蛋白在肾母细胞瘤中的表达和功能仍然不清楚。在这项研究中，我们使用组织微阵列的免疫组织化学评估了原发性肾母细胞瘤中 MYCN 蛋白的表达。我们发现 MYCN 蛋白在肿瘤胚细胞中表达，而在基质和上皮成分中不表达。对于功能研究，我们使用两种间变性肾母细胞瘤细胞系 WiT49 和 17.94 来研究 MYCN 耗竭的生物学和转录组效应。我们发现 MYCN 敲低始终导致生长抑制，但不会导致细胞死亡。 RNA 测序鉴定出 WiT49 和 17.94 细胞系共有的 561 个 MYCN 调控基因。正如预期的那样，许多细胞过程都位于 MYCN 的下游。 MYCN 正向调节 miRNA 调节因子和已知的肾母细胞瘤癌基因 LIN28B，编码甲基小体蛋白 PRMT1、PRMT5 和 WDR77 的基因，以及线粒体转位酶基因 TOMM20 和 TIMM50。 MYCN 抑制的基因包括发育信号受体 ROBO1 和基质标记物 COL1A1。重要的是，我们发现 MYCN 还抑制了假定的 Wilms 肿瘤抑制基因 REST，MYCN 敲低导致 REST 蛋白增加，并同时抑制 RE1 沉默转录因子 (REST) 靶基因。总之，我们的研究确定了与 MYCN 相互作用的调控轴，为预后不良的维尔姆斯肿瘤的潜在靶向治疗提供了新的途径。
Wilms’ tumour (WT) is one of the most common solid paediatric malignancies and arises in the developing kidney. Treatment of most WTs is relatively successful, with the notable exception of tumours with anaplastic histology. Anaplastic WT survival rates can be as low as 30–50%, emphasising the need for a better understanding of their molecular aetiology, in order to facilitate the development of novel therapeutics for poor-prognosis WT. Previous studies have implicated increases in MYCN at the DNA and RNA level in WTs with anaplasia, although the biological functions of MYCN protein have not been reported. In this study, we define the expression pattern of MYCN protein in WTs and also modulate MYCN protein levels in anaplastic WT cell-lines to define the functions of MYCN in WT. We show that MYCN depletion leads to decreased cell growth and identify MYCN-regulated genes and pathways that may represent therapeutic vulnerabilities in poor-prognosis WT. The MYCN proto-oncogene is deregulated in many cancers, most notably in neuroblastoma, where MYCN gene amplification identifies a clinical subset with very poor prognosis. Gene expression and DNA analyses have also demonstrated overexpression of MYCN mRNA, as well as focal amplifications, copy number gains and presumptive change of function mutations of MYCN in Wilms’ tumours with poorer outcomes, including tumours with diffuse anaplasia. Surprisingly, however, the expression and functions of the MYCN protein in Wilms’ tumours still remain obscure. In this study, we assessed MYCN protein expression in primary Wilms’ tumours using immunohistochemistry of tissue microarrays. We found MYCN protein to be expressed in tumour blastemal cells, and absent in stromal and epithelial components. For functional studies, we used two anaplastic Wilms’ tumour cell-lines, WiT49 and 17.94, to study the biological and transcriptomic effects of MYCN depletion. We found that MYCN knockdown consistently led to growth suppression but not cell death. RNA sequencing identified 561 MYCN-regulated genes shared by WiT49 and 17.94 cell-lines. As expected, numerous cellular processes were downstream of MYCN. MYCN positively regulated the miRNA regulator and known Wilms’ tumour oncogene LIN28B, the genes encoding methylosome proteins PRMT1, PRMT5 and WDR77, and the mitochondrial translocase genes TOMM20 and TIMM50. MYCN repressed genes including the developmental signalling receptor ROBO1 and the stromal marker COL1A1. Importantly, we found that MYCN also repressed the presumptive Wilms’ tumour suppressor gene REST, with MYCN knockdown resulting in increased REST protein and concomitant repression of RE1-Silencing Transcription factor (REST) target genes. Together, our study identifies regulatory axes that interact with MYCN, providing novel pathways for potential targeted therapeutics for poor-prognosis Wilms’ tumour.
登录
查看更多内容
影响因子：
8.8
作者：
通讯作者：
--
影响因子：
1.9
作者：
Brown, Keith W.;Charles, Adrian;Malik, Karim
通讯作者：
Malik, Karim
影响因子：
5.4
作者：
Huang M;Weiss WA
通讯作者：
Weiss WA
影响因子：
30.8
作者：
BARDEESY, N;FALKOFF, D;PELLETIER, J
通讯作者：
PELLETIER, J
影响因子：
11.8
作者：
Grieshammer, U;Ma, L;Martin, GR
通讯作者：
Martin, GR