Studies of amplification in rhabdomyosarcoma

在之前的研究中，我们对CDK4作为融合阳性横纹肌肉瘤（RMS）中12q13-q14扩增事件的重要靶点进行了全面分析。我们假设在这个扩增子中可能存在其他关键靶点。为了进一步研究融合阳性横纹肌肉瘤（RMS）中的12q13-q14扩增，我们检测了44例融合阳性RMS病例的Illumina Omni 2.5M阵列数据。28例PAX3-FOXO1 （P3F）阳性病例中有9例（32%）检测到12q13-q14区扩增，13例pax7 - foxo1阳性病例中有1例（8%）检测到12q13-q14区扩增，证实该扩增子优先出现在P3F阳性病例中。我们以p3f阳性病例为研究对象，采用Bonferroni校正的Mann-Witney U-test将12q13-q14扩增的核心区域定位为0.74 Mb，包含41个基因（包括37个蛋白质编码基因）。为了鉴定12q13-q14扩增病例中表达水平升高的效应基因，我们对5例12q13-q14扩增病例和12例未扩增病例的RNA测序数据进行了类似的统计方法。该分析在核心扩增区鉴定了12个差异表达基因（包括CDK4、OS9和TSPAN31）；这些基因在扩增和未扩增的病例中表现出4倍以上的表达变化。RMS中的这些发现接下来将与12q13-q14在其他癌症类别（如胶质母细胞瘤和肺腺癌）中的扩增和过表达的类似分析进行比较。为了准备潜在扩增靶点的功能研究，我们在一组RMS细胞系中评估了选定靶点的表达。我们特别评估了12q13-q14扩增对这些细胞系中OS9和TSPAN31表达的影响。RT-PCR分析显示，与未扩增12q13-q14的p3f阳性细胞株相比，扩增12q13-q14的Rh30细胞株中OS9和TSPAN31 mRNA的表达量高3-5倍。我们还鉴定了商业抗体，以启动蛋白表达的Western blot研究。与未扩增12q13-q14的p3f阳性细胞系相比，Rh30细胞中OS9蛋白的表达水平更高。相比之下，与未扩增的p3f阳性细胞系相比，TSPAN31蛋白在Rh30中的表达水平相似或更高。因此，12q13-q14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细胞系中可能存在与拷贝数无关的过表达机制。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，然后在这些细胞系中开始选择目标的功能研究。我们之前对融合阳性RMS肿瘤中2p24扩增事件的研究表明，MYCN与该扩增子相关被扩增和过表达。我们在RMS基因融合的研究中证实了MYCN高表达在融合阳性RMS中的功能意义，我们发现MYCN和PAX3-FOXO1的结合刺激了人成肌细胞的致癌转化和快速肿瘤发生，尽管高MYCN和高PAX3-FOXO1表达不足以引起这些致癌作用。在我们的第一组实验中，MYCN在人成肌细胞中组成性表达。为了探索不同水平MYCN表达的影响，并在实验中调节MYCN表达，我们设计了一个可诱导的MYCN表达构建体。特别是，将MYCN cDNA克隆到慢病毒多西环素诱导的表达构建体中，并引入了一个潮霉素耐药基因来代替嘌呤霉素耐药基因。结果表明，强力霉素可诱导高水平MYCN mRNA和蛋白在转导的成肌细胞中表达。然而，在没有强力霉素诱导的情况下，这些细胞的MYCN表达明显高于用空表达结构转导的细胞。这种低水平的泄漏足以在pax3 - fox01存在的情况下允许致癌转化。进一步的研究将用于鉴定MYCN基础表达水平较低的转导细胞。

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.