Oncogenic Activation Of FOXR1 by 11q23 Intrachromosomal Deletion-Fusions In Neuroblastoma. Homo sapiens

Neuroblastoma tumors frequently show loss of heterozygosity of chromosome 11q with a shortest region of overlap in the 11q23 region. These deletions are thought to cause inactivation of tumor suppressor genes leading to haploinsufficiency. Alternatively, micro-deletions could lead to gene fusion products that are tumor-driving. To identify such events we analyzed a series of neuroblastomas by comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) arrays and integrated these data with Affymetrix mRNA profiling data with the bioinformatic tool R2 (http://r2.amc.nl). We identified three neuroblastoma samples with small interstitial deletions at 11q23, upstream of the forkhead-box transcription factor FOXR1. Genes at the proximal side of the deletion were fused to FOXR1, resulting in fusion transcripts of MLL-FOXR1 and PAFAH1B2-FOXR1. FOXR1 expression has only been detected in early embryogenesis. Affymetrix microarray analysis showed high FOXR1 mRNA expression exclusively in the neuroblastomas with micro-deletions and rare cases of other tumor types, including osteosarcoma cell line HOS. RNAi silencing of FOXR1 strongly inhibited proliferation of HOS cells and triggered apoptosis. Expression profiling of these cells and reporter assays suggested that FOXR1 is a negative regulator of forkhead-box factor mediated transcription. The neural crest stem cell line JoMa1 proliferates in culture conditional to activity of a MYC-ER transgene. Over-expression of the wild-type FOXR1 could functionally replace MYC and drive proliferation of JoMa1. We conclude that FOXR1 is recurrently activated in neuroblastoma by intrachromosomal deletion/fusion events, resulting in over-expression of fusion transcripts. Forkhead-box transcription factors have not been previously implicated in neuroblastoma pathogenesis. Furthermore, this is the first identification of intrachromosomal fusion genes in neuroblastoma. Overall design: Time series Affymetrix U133p2 profiling of Osteosarcoma HOS cells transduced with FOXR1 targeted shRNAs or control shRNA

神经母细胞瘤（Neuroblastoma）肿瘤常出现11号染色体长臂（11q）的杂合性缺失（loss of heterozygosity），其最短重叠区域定位于11q23区段。此类缺失被认为会导致抑癌基因失活，进而引发单倍体剂量不足（haploinsufficiency）。此外，微缺失还可能产生驱动肿瘤发生的基因融合产物。为鉴定此类事件，本研究通过比较基因组杂交（comparative genomic hybridization, CGH）与单核苷酸多态性（single nucleotide polymorphism, SNP）芯片分析了一系列神经母细胞瘤样本，并将所得数据与通过生物信息学工具R2（http://r2.amc.nl）获取的Affymetrix mRNA表达谱数据进行整合。我们在3例神经母细胞瘤样本的11q23区段——即叉头框转录因子（forkhead-box transcription factor）FOXR1的上游区域——中鉴定出小型间质缺失。缺失近端侧的基因与FOXR1发生融合，形成MLL-FOXR1与PAFAH1B2-FOXR1融合转录本。此前仅在早期胚胎发生过程中检测到FOXR1的表达。Affymetrix芯片分析显示，FOXR1的高mRNA表达仅见于携带微缺失的神经母细胞瘤样本，以及少数其他肿瘤类型病例，包括骨肉瘤细胞系HOS。通过RNA干扰（RNA interference, RNAi）沉默FOXR1可显著抑制HOS细胞的增殖并诱导细胞凋亡。对这些细胞的表达谱分析与报告基因实验结果表明，FOXR1是叉头框因子介导的转录过程的负调控因子。神经嵴干细胞系JoMa1的体外增殖依赖于MYC-ER转基因的活性。过表达野生型FOXR1可在功能上替代MYC，驱动JoMa1细胞的增殖。本研究得出结论：FOXR1可通过染色体内缺失/融合事件在神经母细胞瘤中反复激活，进而导致融合转录本的过表达。此前尚无叉头框转录因子参与神经母细胞瘤发病机制的相关报道。此外，本研究首次在神经母细胞瘤中鉴定出染色体内融合基因。实验整体设计：对转染FOXR1靶向短发夹RNA（short hairpin RNA, shRNA）或对照短发夹RNA的骨肉瘤HOS细胞进行时序性Affymetrix U133p2芯片表达谱分析。