Wnt signalling drives context-dependent differentiation or proliferation in neuroblastoma
Wnt signalling drives context-dependent differentiation or proliferation in neuroblastoma
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DOI：
10.1101/236745
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发表时间：
2018-01
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通讯作者：
M. Szemes;A. Greenhough;Z. Melegh;Sally Malik;A. Yuksel;D. Catchpoole;Kelli Gallacher;M. Kollareddy;J. H. Park;K. Malik
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作者：
M. Szemes;A. Greenhough;Z. Melegh;Sally Malik;A. Yuksel;D. Catchpoole;Kelli Gallacher;M. Kollareddy;J. H. Park;K. Malik
神经母细胞瘤是儿童时期最常见和最致命的实体瘤之一，被认为是由神经嵴发育中的交感肾上腺素能谱系分化破坏引起的。神经母细胞瘤表现出肿瘤内和肿瘤间的异质性，高风险肿瘤的特点是分化差，这可归因于 MYCN 介导的神经元分化相关基因的抑制。已知 MYCN 与 Alk、Akt 和 MEK/ERK 信号等致癌信号传导途径协同作用，并且与 c-MYC 一起已被证明在各种组织中被 Wnt 信号传导激活。然而，我们之前的工作表明，一些神经母细胞瘤细胞系的 Wnt3a/Rspo2 处理可以矛盾地减少 c-MYC 和 MYCN 蛋白。这促使我们使用 RNA 测序来定义神经母细胞瘤特异性 Wnt3a/Rspo2 驱动的转录组，并描述细胞生物学中伴随的变化。在这里，我们报告了 90 个 Wnt 靶基因的鉴定，并表明 Wnt 信号传导是神经母细胞瘤中众多转录因子和信号传导途径的上游。使用活细胞成像，我们表明 Wnt 信号传导可以驱动 SK-N-BE(2)-C 和 SH-SY5Y 细胞系的分化，但相反，可以驱动 SK-N-AS 细胞的增殖。我们发现，分化的细胞系会诱导促分化 BMP4 和 EPAS1 蛋白，这在 SK-N-AS 细胞中并不明显。相反，SK-N-AS细胞响应Wnt3a/Rspo2而表现出CCND1、磷酸化RB和E2F1的增加，这与它们的增殖反应一致，并且这些蛋白质在分化系中没有增加。通过对原发肿瘤基因表达数据库中 90 个基因的表达进行荟萃分析，我们证明了 Wnt 基因在具有不同预后的患者群体中的离散表达模式。此外，我们的分析揭示了 Wnt 基因子集内的互连性，其中一个子集由 MYCN 抑制的神经元分化的新推定驱动因素组成。 β-连环蛋白免疫组织化学评估显示肿瘤中的 β-连环蛋白水平较高且分化较好，进一步支持经典 Wnt 信号在神经母细胞瘤分化中的作用。
Neuroblastoma is one of the commonest and deadliest solid tumours of childhood, and is thought to result from disrupted differentiation of the developing sympathoadrenergic lineage of the neural crest. Neuroblastoma exhibits intra-and intertumoural heterogeneity, with high risk tumours characterised by poor differentiation, which can be attributable to MYCN-mediated repression of genes involved in neuronal differentiation. MYCN is known to co-operate with oncogenic signalling pathways such as Alk, Akt and MEK/ERK signalling, and, together with c-MYC has been shown to be activated by Wnt signalling in various tissues. However, our previous work demonstrated that Wnt3a/Rspo2 treatment of some neuroblastoma cell lines can, paradoxically, decrease c-MYC and MYCN proteins. This prompted us to define the neuroblastoma-specific Wnt3a/Rspo2-driven transcriptome using RNA sequencing, and characterise the accompanying changes in cell biology. Here we report the identification of ninety Wnt target genes, and show that Wnt signalling is upstream of numerous transcription factors and signalling pathways in neuroblastoma. Using live-cell imaging, we show that Wnt signalling can drive differentiation of SK-N-BE(2)-C and SH-SY5Y cell-lines, but, conversely, proliferation of SK-N-AS cells. We show that cell-lines that differentiate show induction of pro-differentiation BMP4 and EPAS1 proteins, which is not apparent in the SK-N-AS cells. In contrast, SK-N-AS cells show increased CCND1, phosphorylated RB and E2F1 in response to Wnt3a/Rspo2, consistent with their proliferative response, and these proteins are not increased in differentiating lines. By meta-analysis of the expression of our 90 genes in primary tumour gene expression databases, we demonstrate discrete expression patterns of our Wnt genes in patient cohorts with different prognosis. Furthermore our analysis reveals interconnectivity within subsets of our Wnt genes, with one subset comprised of novel putative drivers of neuronal differentiation repressed by MYCN. Assessment of β-catenin immunohistochemistry shows high levels of β-catenin in tumours with better differentiation, further supporting a role for canonical Wnt signalling in neuroblastoma differentiation.