Studies of amplification in rhabdomyosarcoma

横纹肌肉瘤扩增的研究

• 批准号: 9556544
• 负责人: Frederic Barr
• 金额: $ 30.47万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9556544
• 关键词: 12q13; 13q14; 1p36; Affect; Behavior; Biological; Biological Assay; CDK4 gene; Candidate Disease Gene; Categories; Cell Culture System; Cell Line; Cells; Chemicals; Chromosomal Duplication; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Common Core; Complement; Complementary DNA; Cytogenetics; Data; Dedifferentiated Liposarcomas; Doxycycline; Event; FOXO1A gene; Family; Gene Amplification; Genes; Genomic Segment; Genomics; Glioblastoma; Goals; Growth; Heterogeneity; Human; Laboratory mice; Lung Adenocarcinoma; MYCN gene; Malignant Neoplasms; Mann-Whitney U Test; Messenger RNA; Molecular; Myoblasts; Oncogenic; Oncoproteins; PAX3 gene; PAX7 gene; Pathogenesis; Pathway interactions; Phenotype; Population; Proteins; RNA Interference; Reagent; Recurrence; Rhabdomyosarcoma; Role; Sampling; Statistical Methods; The Cancer Genome Atlas; Therapeutic Intervention; Tissue Microarray; Transcript; base; cellular transduction; design; differential expression; directed attention; experimental study; fusion gene; genome-wide; hygromycin A; inducible gene expression; new therapeutic target; overexpression; resistance gene; therapeutic target; transcriptome sequencing; tumor; tumorigenesis

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.

为了扩展我们对融合阳性横纹肌肉瘤（RMS）中12 q13-q14扩增的研究，我们使用来自癌症基因组图谱（TCGA）的数据将我们在RMS中的发现与其他癌症类别中该染色体区域的扩增进行比较。特别是，我们确定了多例12 q13-q14扩增的多形性胶质母细胞瘤（GBM），去分化脂肪肉瘤（dLPS）和肺腺癌（LAC）。尽管我们先前的研究在25%的PAX 3-FOXO 1阳性RMS病例中鉴定出这种扩增子，但TCGA数据分析发现12 q13-q14扩增在7%的LAC、20%的GBM和86%的dLPS中。然后，我们使用统计方法来描绘每个癌症类别中的一致扩增区域。在该分析中，将扩增区域周围的大基因组区域分成多个区间，然后在扩增子阳性和扩增子阴性样品中评估每个区间中的拷贝数。然后通过具有Bonferroni校正的Mann-Whitney U检验评价每个间隔中的显著拷贝数差异。该分析在四种癌症类别中鉴定了共同的0.24 Mb扩增区域（含有13个基因，包括CDK 4）。除了通常扩增的区域，扩增区域在融合阳性RMS中在着丝粒方向上延伸，并且在GBM、dLPS和LAC中在端粒方向上延伸。为了评估这些扩增事件的表达结果，分析RNA-Seq数据以获得每种癌症类别中扩增子阳性和扩增子阴性样品之间的差异表达，并使用如上所述的类似统计方法来确定显著的表达差异。该分析显示，在所有四个类别中，共同重叠区域内的七个基因与该扩增事件相关地过表达。此外，有4个过度表达的基因与融合阳性RMS的扩增扩展区域特异性相关，3个过度表达的基因与GBM、LAD和dLPS的扩增扩展区域特异性相关。这些发现表明，细胞遗传学上类似的扩增事件实际上由共同的核心扩增区域和特定于某些癌症类别的额外的扩增延伸区域组成。我们先前对融合阳性RMS中2 p24扩增事件的研究表明，MYCN与该扩增子相关扩增并过表达。MYCN高表达在融合阳性RMS中的功能意义在研究中得到了证实，在这些研究中，我们表明外源性MYCN和PAX 3-FOXO 1的组合刺激了人成肌细胞的致癌转化和快速肿瘤发生，尽管MYCN高表达和PAX 3-FOXO 1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表达的影响，并在实验过程中调节MYCN表达，我们设计了一个诱导型MYCN表达构建体，通过克隆MYCN cDNA和潮霉素抗性基因到慢病毒多西环素诱导型表达构建体中。所得到的构建体显示在转导的成肌细胞中多西环素诱导高水平的MYCN mRNA和蛋白。然而，在没有强力霉素诱导的情况下，这些细胞显示出比用空表达构建体转导的细胞显著更高的MYCN表达。这种低水平的泄漏足以允许在PAX 3-FOXO 1存在下的致癌转化。为了获得具有较低基础MYCN表达水平的转导细胞，我们进行了几次独立的转导实验，并分析了转导的重复以找到具有最低基础MYCN表达的群体，然后从该群体获得多个亚克隆。虽然在特定亚克隆中发现了非常低的基础MYCN表达，但随着细胞传代，该基础水平增加，表明存在对高基础MYCN表达的强选择，这使这些计划的研究复杂化。