Studies of amplification in rhabdomyosarcoma

为了扩展我们对融合阳性横纹肌肉瘤(RMS)中12q13-Q14扩增的研究，我们使用了来自癌症基因组图谱(TCGA)的数据来比较我们在RMS中的发现与在其他癌症类别中该染色体区域的扩增。特别是，我们在多形性胶质母细胞瘤(GBM)、去分化脂肪肉瘤(DLPS)和肺腺癌(LAC)中发现了多个12q13-q14扩增的病例。我们之前的研究发现，在25%的PAX3-FOXO1阳性的RMS中存在该扩增片段，而对TCGA数据的分析发现，12q13-Q14扩增在7%的LAC、20%的GBM和86%的DLPS中存在。然后，我们使用统计方法来描绘每个癌症类别中持续放大的区域。在这种分析中，围绕扩增区域的一大片基因组区域被分成多个区间，然后在扩增阳性和扩增阴性的样本中评估每个区间中的拷贝数。然后用带Bonferroni校正的Mann-Whitney U检验来评估每个区间的显著拷贝数差异。这项分析确定了四种癌症类别中共同的0.24Mb扩增区域(包含13个基因，包括CDK4)。除了常见的扩增区域外，在融合阳性的RMS中，扩增区域沿着丝粒方向延伸，在GBM、DLPS和LAC中，扩增区域沿端粒方向延伸。为了评估这些扩增事件的表达后果，分析了RNA-Seq数据在每个癌症类别中扩增阳性和扩增阴性样本之间的差异表达，并使用如上所述的类似统计方法来确定显著的表达差异。这一分析表明，在所有四个类别中，与该扩增事件相关的共同重叠区中的七个基因过度表达。此外，在融合阳性的RMS中有4个与扩增延伸区域特异相关的过表达基因，在GBM、LAD和DLPS中有3个与扩增延伸区域特异相关的过表达基因。这些发现表明，细胞遗传学上相似的扩增事件实际上由共同的核心扩增区域和特定癌症类别特有的附加扩增区域组成。我们先前对融合阳性RMS的2p24扩增事件的研究表明，MYCN被扩增和过度表达与该扩增子相关。在融合阳性的RMS中，MYCN的高表达具有功能意义，我们的研究表明，外源MYCN和PAX3-FOXO1的结合刺激了人成肌细胞的致癌转化和快速成瘤，尽管MYCN和PAX3-FOXO1的高表达都不足以引起这些致癌效应。为了探索不同水平的MYCN表达对MYCN表达的影响，并在实验过程中对MYCN的表达进行调控，我们设计了一种MYCN可诱导表达的载体，方法是将MYCN基因和潮霉素抗性基因克隆到慢病毒多西环素诱导表达载体中。结果表明，多西环素在转导的成肌细胞中诱导了高水平的MYCN mRNA和蛋白。然而，在没有多西环素诱导的情况下，这些细胞表现出显著高于转导空表达载体的细胞的MYCN表达。这种低水平的泄漏足以在PAX3-FOXO1存在的情况下进行致癌转化。为了获得基础MYCN表达水平较低的转导细胞，我们进行了几个独立的转导实验，并分析了转导复制以找到基础MYCN表达最低的群体，然后从这个群体中获得了多个亚克隆。虽然在特定的亚克隆中发现了非常低的基础MYCN表达，但随着细胞的传代，这一基础水平上升，这表明存在对高基础MYCN表达的强烈选择，这使得这些计划中的研究变得复杂。

To extend our studies of the 12q13-q14 amplification in fusion-positive rhabdomyosarcoma (RMS), we used data from The Cancer Genome Atlas (TCGA) to compare our findings in RMS with amplification of this chromosomal region in other cancer categories. In particular, we identified multiple cases with 12q13-q14 amplification in glioblastoma multiforme (GBM), dedifferentiated liposarcoma (dLPS), and lung adenocarcinoma (LAC). Whereas our previous studies identified this amplicon in 25% of cases of PAX3-FOXO1-positive RMS, analysis of the TCGA data found 12q13-q14 amplification in 7% of LAC, 20% of GBM, and 86% of dLPS. We then used a statistical approach to delineate the consistently amplified region in each cancer category. In this analysis, a large genomic region surrounding the amplified region was split into numerous intervals, and the copy number in each interval was then assessed in amplicon-positive and amplicon-negative samples. Significant copy number differences in each interval were then evaluated by Mann-Whitney U tests with Bonferroni corrections. This analysis identified a common 0.24 Mb region of amplification (containing 13 genes, including CDK4) across the four cancer categories. In addition to the commonly amplified regions, the region of amplification extended in the centromeric direction in fusion-positive RMS and extended in the telomeric direction in GBM, dLPS, and LAC. To evaluate the expression consequences of these amplification events, RNA-Seq data was analyzed for differential expression between amplicon-positive and amplicon-negative samples in each of the cancer categories, and the similar statistical methods as described above were used to determine significant expression differences. This analysis revealed that seven genes within the common overlap region were overexpressed in association with this amplification event in all four categories. In addition, there were four overexpressed genes specifically associated with the extended region of amplification in fusion-positive RMS and three overexpressed genes specifically associated with the extended region of amplification in GBM, LAD, and dLPS. These findings indicate that cytogenetically similar amplification events actually consist of a common core amplified region and additional extended regions of amplification that are specific to certain cancer categories. Our previous studies of the 2p24 amplification event in fusion-positive RMS indicated that MYCN was amplified and over-expressed in association with this amplicon. The functional significance of high MYCN expression in fusion-positive RMS was demonstrated in studies in which we show that the combination of exogenous MYCN and PAX3-FOXO1 stimulated oncogenic transformation and rapid tumorigenesis of human myoblasts, though neither high MYCN nor high PAX3-FOXO1 expression was sufficient to cause these oncogenic effects. To explore the effect of different levels of MYCN expression and to modulate MYCN expression during experiments, we designed an inducible MYCN expression construct by cloning the MYCN cDNA and a hygromycin resistance gene into a lentiviral doxycycline-inducible expression construct. The resulting construct showed doxycycline induction of high levels of MYCN mRNA and protein in transduced myoblasts. However, in the absence of doxycycline induction, these cells showed significantly higher MYCN expression than cells transduced with the empty expression construct. This low level of leakiness was sufficient to permit oncogenic transformation in the presence of PAX3-FOXO1. To derive transduced cells with lower basal levels of MYCN expression, we performed several independent transduction experiments and analyzed the transduced replicates to find the population with the lowest basal MYCN expression and then obtained multiple subclones from this population. Though very low basal MYCN expression was found in specific subclones, this basal level increased as the cells were passed,indicating that there is a strong selection for high basal MYCN expression that complicated these planned studies.