N-MYC regulates focal adhesion kinase expression in human neuroblastoma
N-MYC regulates focal adhesion kinase expression in human neuroblastoma
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DOI：
10.1074/jbc.m701450200
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发表时间：
2007-04-27
影响因子：
4.8
通讯作者：
Golubovskaya, Vita M.
中科院分区：
文献类型：
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作者：
Beierle, Elizabeth A.;Trujillo, Angelica;Golubovskaya, Vita M.
N-MYC 是一种转录因子，在神经母细胞瘤的细胞存活中发挥重要作用，N-MYC 癌基因的扩增是神经母细胞瘤的主要不良预后指标。粘着斑激酶 (FAK) 是一种生存因子，已被证明在多种人类癌症中过度表达。在这项研究中，我们研究了神经母细胞瘤中 N-MYC 对 FAK 表达的调节作用。我们首先发现神经母细胞瘤中 N-MYC 和 FAK 表达之间的相关性。实时定量 PCR 表明，与未扩增的 SK-N-AS 神经母细胞瘤细胞系相比，N-MYC 扩增的 IMR-32 中的 FAK mRNA 丰度有所增加。与未扩增的 SK-N-AS 神经母细胞瘤细胞系相比，在 N-MYC 扩增的 IMR-32 细胞系中，FAK 蛋白表达也与 N-MYC 表达呈正相关。在同基因 N-MYC+ (Tet(-)) 和 N-MYC+ (Tet(-)) 神经母细胞瘤细胞系中观察到相同的结果。启动子报告基因检测显示，与非扩增和 N-MYC 非表达细胞系相比，在 N-MYC 扩增的 IMR-32 细胞系、N-MYC 转染的 SK-N-AS 非扩增细胞系和等基因 N-MYC- ( Tet(+)) 神经母细胞瘤细胞系中，FAK 启动子的活性增加。我们还鉴定了 FAK 启动子序列中的两个 N-MYC 结合位点，并通过电泳迁移率变动、染色质免疫沉淀和双荧光素酶测定显示 N-MYC 转录因子与 FAK 启动子的结合。最后，与同基因 N-MYC- (Tet(+)) 细胞相比，用 FAK 小干扰 RNA 或显性失活 FAK 抑制剂 (AdFAK-CD) 下调 N-MYC 诱导神经母细胞瘤细胞系中的 FAK 表达，显着降低 N-MYC+ (Tet(-)) 细胞的活力并增加细胞凋亡，这证明了 N-MYC 表达细胞系中 FAK 过表达的生物学意义。这是第一份在神经母细胞瘤中将 N-MYC 和 FAK 联系起来的报告，它清楚地表明 N-MYC 诱导 FAK 表达。结果表明，N-MYC 对 FAK 表达的调节可以控制同基因 N-MYC-/+ (Tet(+/-)) 神经母细胞瘤细胞系的细胞功能。
N-MYC is a transcription factor that plays an important role in cellular survival in neuroblastoma, and amplification of the N-MYC oncogene is the primary adverse prognostic indicator for neuroblastoma. Focal adhesion kinase ( FAK) is a survival factor that has been shown to be overexpressed in many types of human cancers. In this study, we investigated the role of N-MYC regulation of FAK expression in neuroblastoma. We first found a correlation between N-MYC and FAK expression in neuroblastoma. Real time quantitative PCR demonstrated an increase in FAK mRNA abundance in the N-MYC-amplified IMR-32 compared with the nonamplified SK-N-AS neuroblastoma cell lines. FAK protein expression also correlated positively with N-MYC expression in the N-MYC-amplified IMR-32 versus nonamplified SK-N-AS neuroblastoma cell lines. The same results were seen with the isogenic N-MYC+ ( Tet(-)) and N-MYC+ ( Tet(-)) neuroblastoma cell lines. Promoter-reporter assays showed that activity of the FAK promoter was increased in the N-MYC-amplified IMR-32 cell line, in the N-MYC-transfected SK-N-AS nonamplified cell line, and in the isogenic N-MYC- ( Tet(+)) neuroblastoma cell lines compared with the nonamplified and N-MYC-nonexpressing cell lines. We also identified two N-MYC binding sites in the FAK promoter sequence and showed binding of N-MYC transcription factor to the FAK promoter through electrophoretic mobility shift, chromatin immunoprecipitation, and dual luciferase assays. Finally down-regulation of FAK expression in N-MYC-inducible neuroblastoma cell lines with FAK small interfering RNA or a dominant-negative FAK inhibitor ( AdFAK-CD) significantly decreased viability and increased apoptosis in the N-MYC+ ( Tet(-)) cells compared with the isogenic N-MYC- ( Tet(+)) cells, demonstrating the biological significance of FAK overexpression in the N-MYC-expressing cell lines. This is the first report linking N-MYC and FAK in neuroblastoma, and it clearly demonstrates that N-MYC induces FAK expression. The results indicate that N-MYC regulation of FAK expression can control cellular functions in isogenic N-MYC-/+ ( Tet(+/-)) neuroblastoma cell lines.