Studies of amplification in rhabdomyosarcoma

在以前的研究中，我们对CDK4作为融合阳性横纹肌肉瘤(RMS)12q13-q14扩增事件的重要靶点进行了全面的分析。我们假设在这个扩增子中可能有额外的关键靶点。为了扩展我们对融合阳性横纹肌肉瘤(RMS)12q13-Q14扩增的研究，我们检查了来自44例融合阳性横纹肌肉瘤(RMS)新队列的Illumina Omni2.5M阵列数据。在28例PAX3-FOXO1(P3F)阳性病例中有9例(32%)和13例PAX7-FOXO1阳性病例中1例(8%)检测到12q13-Q14区扩增，证实该扩增片段在P3F阳性病例中优先出现。我们以P3F阳性病例为研究对象，采用带Bonferroni校正的Mann-Witney U检验，将12q13-Q14扩增的核心区定位在0.74Mb区域，包含41个基因(包括37个蛋白编码基因)。为了确定12q13-q14扩增病例中表达水平升高的效应基因，我们对5例有12q13-q14扩增的病例和12例没有12q13-q14扩增的病例的RNA测序数据进行了类似的统计方法。这一分析确定了核心扩增区域内的12个差异表达基因(包括CDK4、OS9和TSPAN31)；这些基因在扩增和非扩增病例中的表达变化是4倍或更高。RMS的这些发现接下来将与12q13-q14扩增和在其他癌症类别中过度表达的类似分析进行比较，例如胶质母细胞瘤和肺腺癌。为了准备潜在扩增靶点的功能研究，我们评估了选定靶点在一组RMS细胞系中的表达。特别是，我们评估了12q13-q14扩增对这些细胞系中OS9和TSPAN31表达的影响。RT-PCR分析表明，在12q13-Q14扩增的Rh30细胞系中，OS9和TSPAN31的mRNA表达水平是未扩增的P3F阳性细胞系的3-5倍。我们还鉴定了商用抗体，以启动蛋白质表达的蛋白质印迹研究。与未扩增12q13-Q14的P3F阳性株相比，Rh30细胞中OS9蛋白的表达水平更高。相反，TSPAN31蛋白在Rh30中的表达水平与未扩增的P3F阳性品系相似或更高。因此，12q13-Q14的扩增与这两种蛋白的高表达有关，尽管在一些没有该扩增子的品系中可能存在拷贝数非依赖的过表达机制。来自12q13-q14扩增子的其他基因将在RMS细胞系中进行类似的评估，然后开始对这些细胞系中选定的靶点进行功能研究。我们先前对融合阳性RMS肿瘤中2p24扩增事件的研究表明，MYCN被扩增并与该扩增子相关地过度表达。在我们对融合阳性RMS中的基因融合的研究中，我们证明了MYCN的高表达在RMS中的功能意义，其中我们发现MYCN和PAX3-FOXO1的结合促进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作用。在我们的第一组实验中，MYCN在人成肌细胞中有结构性表达。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的表达，我们设计了一个可诱导的MYCN表达载体。特别是将MYCN基因克隆到了慢病毒多西环素诱导表达载体中，并引入了潮霉素抗性基因来代替嘌呤霉素抗性基因。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。将进行进一步的研究，以确定基础水平较低的MYCN表达的转导细胞。

In previous studies, we performed a comprehensive analysis of CDK4 as an important target of the 12q13-q14 amplification event in fusion-positive rhabdomyosarcoma (RMS). We hypothesize that there may be additional critical targets within this amplicon. To extend our studies of 12q13-q14 amplification in fusion-positive rhabdomyosarcoma (RMS), we examined Illumina Omni 2.5M array data from a new cohort of 44 fusion-positive RMS cases. Amplification of the 12q13-q14 region was detected in 9 of 28 (32%) PAX3-FOXO1 (P3F)-positive cases, and 1 of 13 (8%) PAX7-FOXO1-positive cases, confirming the preferential occurrence of this amplicon in P3F-positive cases. We focused on the P3F-positive cases and used the Mann-Witney U-test with Bonferroni correction to localize the core region of 12q13-q14 amplification to a 0.74 Mb region containing 41 genes (including 37 protein-coding genes). To identify effector genes expressed at increased levels in 12q13-q14-amplified cases, we performed a similar statistical approach on RNA sequencing data from 5 cases with and 12 cases without 12q13-q14 amplification. This analysis identified 12 differentially expressed genes (including CDK4, OS9 and TSPAN31) within the core-amplified region; these genes showed 4-fold or higher change in expression between amplified and non-amplified cases. These findings in RMS will next be compared with similar analyses of 12q13-q14 amplification and over-expression in other cancer categories, such as glioblastoma and lung adenocarcinoma. In preparation for functional studies of potential amplification targets, we assessed the expression of selected targets in a panel of RMS cell lines. In particular, we assessed the effect of 12q13-q14 amplification on OS9 and TSPAN31 expression in these cell lines. RT-PCR analysis showed that OS9 and TSPAN31 mRNA are expressed at 3-5-fold higher levels in the 12q13-q14-amplified Rh30 cell line compared to P3F-positive lines without 12q13-q14 amplification. We also identified commercial antibodies to initiate Western blot studies of protein expression. OS9 protein expression was detected at higher levels in Rh30 cells compared to P3F-positive lines without 12q13-q14 amplification. In contrast, TSPAN31 protein was expressed at similar or higher levels in Rh30 compared to non-amplified P3F-positive lines. Therefore, 12q13-q14 amplification is associated with high expression of both proteins though there may be copy number-independent overexpression mechanisms in some lines without this amplicon. Additional genes from the 12q13-q14 amplicon will be similarly assessed in RMS cell lines, before commencing functional studies of selected targets in these cell lines. Our previous studies of the 2p24 amplification event in fusion-positive RMS tumors 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 our studies of the gene fusions in RMS in which we show that the combination of MYCN and PAX3-FOXO1 stimulated oncogenic transformation and rapid tumorigenesis of human myoblasts, though neither high MYCN nor high PAX3-FOXO1 expression were sufficient to cause these oncogenic effects. In our first set of experiments, MYCN was constitutively expressed in the human myoblasts. To explore the effect of different levels of MYCN expression and to modulate MYCN expression during experiments, we designed an inducible MYCN expression construct. In particular, the MYCN cDNA was cloned into a lentiviral doxycycline-inducible expression construct, and a hygromycin resistance gene was also introduced in place of the puromycin resistance gene. 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. Additional studies will be performed to identify transduced cells with lower basal levels of MYCN expression.