PHOX2B-Mediated Regulation of ALK Expression: In Vitro Identification of a Functional Relationship between Two Genes Involved in Neuroblastoma
PHOX2B-Mediated Regulation of ALK Expression: In Vitro Identification of a Functional Relationship between Two Genes Involved in Neuroblastoma
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DOI：
10.1371/journal.pone.0013108
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发表时间：
2010-10-01
期刊：
影响因子：
3.7
通讯作者：
Perri, Patrizia
中科院分区：
文献类型：
--
作者：
Bachetti, Tiziana;Di Paolo, Daniela;Perri, Patrizia
背景资料：神经母细胞瘤（NB）是一种严重的儿科肿瘤，起源于神经嵴衍生物，占儿童癌症死亡率的15%。在有限比例的NB患者中，通过鉴定编码受体酪氨酸激酶间变性淋巴瘤激酶（ALK）和转录因子配对样同源异型盒2B（PHOX 2B）的两种基因突变，证实了异质性和复杂的遗传病因。有趣的是，这两个基因在绝大多数原代NB样品和细胞系中过表达。这些观察结果使我们测试的假设ALK和PHOX 2B之间的监管或功能关系NB pathogenicity.Methodology/Principal Findings：根据这种可能性，我们首先证实了一个惊人的相关性之间的转录水平的ALK，PHOX 2B和它的直接目标PHOX 2A在一个小组的NB细胞系。然后，我们通过siRNA介导的敲低来操纵它们在NB细胞系中的表达，并迫使分析中的每个基因过表达。令人惊讶的是，PHOX 2B和PHOX 2A定向的siRNA有效地下调彼此以及ALK基因，并且一致地，NB细胞中PHOX 2B的表达增强产生ALK蛋白的增加。我们最终证明，PHOX 2B驱动ALK基因转录直接结合其启动子，因此代表了一种新的PHOX 2B target.Conclusions/Significance：这些研究结果提供了一个令人信服的解释同时参与这两个基因在NB发病机制，并将促进更好地了解分子相互作用的基础上的疾病。此外，这项工作为传统疗法难治的NB开辟了新的前景，这些疗法可能受益于针对多个基因靶点的新型治疗性RNAi方法的设计。
Background: Neuroblastoma (NB) is a severe pediatric tumor originating from neural crest derivatives and accounting for 15% of childhood cancer mortality. The heterogeneous and complex genetic etiology has been confirmed with the identification of mutations in two genes, encoding for the receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) and the transcription factor Paired-like Homeobox 2B (PHOX2B), in a limited proportion of NB patients. Interestingly, these two genes are overexpressed in the great majority of primary NB samples and cell lines. These observations led us to test the hypothesis of a regulatory or functional relationship between ALK and PHOX2B underlying NB pathogenesis.Methodology/Principal Findings: Following this possibility, we first confirmed a striking correlation between the transcription levels of ALK, PHOX2B and its direct target PHOX2A in a panel of NB cell lines. Then, we manipulated their expression in NB cell lines by siRNA-mediated knock-down and forced over-expression of each gene under analysis. Surprisingly, PHOX2B- and PHOX2A-directed siRNAs efficiently downregulated each other as well as ALK gene and, consistently, the enhanced expression of PHOX2B in NB cells yielded an increment of ALK protein. We finally demonstrated that PHOX2B drives ALK gene transcription by directly binding its promoter, which therefore represents a novel PHOX2B target.Conclusions/Significance: These findings provide a compelling explanation of the concurrent involvement of these two genes in NB pathogenesis and are going to foster a better understanding of molecular interactions at the base of the disease. Moreover, this work opens new perspectives for NBs refractory to conventional therapies that may benefit from the design of novel therapeutic RNAi-based approaches for multiple gene targets.