Studies of amplification in rhabdomyosarcoma

横纹肌肉瘤（RMS）中复发性扩增子的第一个研究集中在12 q13-q14扩增子上，该扩增子优先发生在PAX 3-FOXO 1阳性RMS的子集（25%的病例）中，仅发生在PAX 7-FOXO 1阳性和融合阴性病例的较小子集中。我们先前的研究将扩增的最小区域定位到含有28个基因的0.55 Mb区域，包括CDK 4原癌基因。我们随后的表达谱研究表明，这些基因中有7个在扩增的RMS肿瘤中在RNA水平上持续过表达。应用高密度阵列分析和下一代测序与儿科肿瘤学分支的Javed Khan博士进行的合作研究证实了该12 q13-q14扩增子的大小和分布。为了确定28个基因中哪些基因在蛋白质水平过表达，我们与病理学实验室的Svetlana Pack博士和Stephen休伊特博士建立了合作。RMS标本的两个组织微阵列从儿童肿瘤组（COG）获得。使用RT-PCR和FISH检测，我们确定了RMS标本在这些TMA上的融合状态。Pack博士开发了一套荧光原位杂交探针，以确定这些TMA病例中12 q13-q14区域的扩增状态;这些FISH研究的结果与我们先前报告的RMS融合亚群中12 q13-q14扩增频率的结果一致。休伊特博士现在正与我的实验室合作，通过免疫组织化学分析CDK 4蛋白在这些TMA中的表达状态。为了开始开始我们对这种扩增事件的功能后果的分析，我们首先关注CDK 4基因。对于这些研究，我们将Rh 30鉴定为具有12 q13-q14扩增子的融合阳性RMS细胞系。为了比较，我们选择了几个融合阳性RMS细胞系，缺乏12 q13-q14扩增子。除了在培养中分析这些细胞系外，我们还与儿科肿瘤学分支的Chand卡纳博士合作，从这些细胞系中开发了一系列肌内异种移植物。已经鉴定并成功使用了通过蛋白质印迹检测CDK 4蛋白表达的抗体。使用该抗体的蛋白质印迹分析证实了具有12 q13-q14扩增的细胞系和相应异种移植物中CDK 4的表达增加。在一组功能研究中，四环素诱导型慢病毒表达载体由医学肿瘤学分支的Ji Luo博士提供。我们将CDK 4 cDNA亚克隆到该表达载体中，并将表达构建体转导到几种不具有12 q13-q14区域扩增的RMS细胞系中。在这些转导细胞中的初始剂量-反应和时间过程研究证实，与对照相比，在用多西环素处理的转导Rh 28细胞中CDK 4蛋白表达容易增加。在生长和转化的细胞培养研究中评估这些转导细胞的表型。在补充研究中，一组靶向CDK 4基因的慢病毒shRNA表达构建体被转导到Rh 30细胞中，并证明了CDK 4蛋白表达降低的明确证据。此外，这些细胞中CDK 4表达的降低与细胞生长和可能存活的实质性缺陷相关。为了进一步分析这个问题，IPTG诱导的shRNA构建体被开发并在Rh 30中测试。如所预测的，这些诱导型构建体允许Rh 30细胞中CDK 4表达的受控抑制。我们现在正在研究这些细胞中不同水平的CDK 4表达的表型后果。在没有12 q13-q14扩增的融合阳性RMS细胞中，我们选择Rh 28和Rh 41作为具有低和中等水平CDK 4蛋白表达的代表性细胞系。这些细胞系正在用IPTG诱导的shRNA构建体进行类似的测试。最后，动物方案最近已被机构动物护理和使用委员会批准，以扩展我们的细胞培养研究，并评估CDK 4在促进和维持实验室动物致瘤性中的作用。

The first studies of recurrent amplicons in rhabdomyosarcoma (RMS) are focusing on the 12q13-q14 amplicon, which occurs preferentially in a subset of PAX3-FOXO1-positive RMS (25% of cases) and only in a smaller subset of PAX7-FOXO1-positive and fusion-negative cases. Our previous studies localized the minimal region of amplification to a 0.55 Mb region containing 28 genes, including the CDK4 proto-oncogene. Our subsequent expression profiling studies showed that 7 of these genes were consistently overexpressed at the RNA level in amplified RMS tumors. Collaborative studies with Dr. Javed Khan of the Pediatric Oncology branch applying high density array analysis and next generation sequencing has confirmed the size and distribution of this 12q13-q14 amplicon. To determine which of the 28 genes are overexpressed at the protein level, we set up a collaboration with Dr. Svetlana Pack and Dr. Stephen Hewitt of the Laboratory of Pathology. Two tissue microarrays of RMS specimens were obtained from the Children's Oncology Group (COG). Using RT-PCR and FISH assays, we determined the fusion status of the RMS specimens on these TMA's. Dr. Pack developed a set of fluorescence in situ hybridization probes to determine the amplification status of the 12q13-q14 region in cases on these TMA's; the findings from these FISH studies are consistent with our previous reported findings of the frequency of 12q13-q14 amplification in RMS fusion subsets. Dr. Hewitt is now working with my laboratory to analyze the expression status of the CDK4 protein in these TMA's by immunohistochemistry. To begin begin our analysis of the functional consequences of this amplification event, we first focused on the CDK4 gene. For these studies, we identified Rh30 as a fusion-positive RMS cell line with the 12q13-q14 amplicon. For comparison, we selected several fusion-positive RMS cell lines that lack the 12q13-q14 amplicon. In addition to analyzing these cell lines in culture, we developed a series of intramuscular xenografts from these cell lines as part of a collaboration with Dr. Chand Khanna of the Pediatric Oncology Branch. An antibody that detects CDK4 protein expression by western blot has been identified and successfully used. Western blot analysis with this antibody confirms increased expression of CDK4 in lines and corresponding xenografts with 12q13-q14 amplification. In one set of functional studies, a tetracycline-inducible lentiviral expression vector was provided by Dr. Ji Luo of the Medical Oncology Branch. We subcloned the CDK4 cDNA into this expression vector and transduced the expression construct into several RMS cell lines that do not have amplification of the 12q13-q14 region. Initial dose-response and time course studies in these transduced cells confirm that CDK4 protein expression is readily increased in transduced Rh28 cells treated with doxycycline in comparison to controls. The phenotype of these transduced cells is being assessed in cell culture studies of growth and transformation. In complementary studies, a set of lentivral shRNA expression constructs targeted against the CDK4 gene were transduced into Rh30 cells and demonstrated clear evidence of decreased CDK4 protein expression. Furthermore, the decrease in CDK4 expression in these cells is associated with substantial defects in cell growth and possibly survival. To further analyze this issue, IPTG-inducible shRNA constructs were developed and tested in Rh30. As predicted, these inducible constructs permit controlled inhibition of CDK4 expression in the Rh30 cells. We are now investigating the phenotypic consequences of varying levels of CDK4 expression in these cells. Among the fusion-positive RMS cells that do not have 12q13-q14 amplification, we selected Rh28 and Rh41 as representative lines with low and intermediate levels of CDK4 protein expression. These lines are being similarly tested with the IPTG-inducible shRNA constructs. Finally, an animal protocol has recently been approved by the Institutional Animal Care and Use Committee to extend our cell culture studies and assess the role of CDK4 in contributing to and maintaining tumorigenicity in laboratory animals.