Studies of amplification in rhabdomyosarcoma

高级研究员于2011财政年度迁往国家癌症研究所，并于2012财政年度上半年聘请人员建立研究实验室。2012年3月开始招募了一名博士后实习生从事RMS放大项目。第一批RMS基因组扩增研究的重点是12 q13-q14扩增子，该扩增子优先发生在PAX 3-FOXO 1阳性RMS的一个子集（25%的病例）和少量PAX 7-FOXO 1阳性和融合阴性病例中。 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。我们随后的微阵列研究表明，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。与儿科肿瘤学分支的Javed Khan博士的合作研究应用高密度阵列分析和下一代测序进一步完善了这些基因组发现。为了确定哪些基因在蛋白质水平上过表达，我们与病理学实验室的Svetlana Pack博士和Stephen休伊特博士以及费城儿童医院（CHOP）的布鲁斯帕维尔博士建立了合作关系。帕维尔博士已经生成了RMS标本的组织微阵列。此外，从儿童肿瘤组（COG）获得了另外两份RMS TMA。我实验室以前的工作已经确定了CHOP和COG TMA上大多数RMS样品的熔合状态。Pack博士正在开发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。然后休伊特博士和帕维尔博士将与我的实验室合作，通过免疫组织化学方法分析这些TMA中选定蛋白质的表达状态。为了开始功能分析，我们首先关注CDK 4基因，然后将考虑其他基因，如我们的其他分析所示。对于这些研究，我们收集了几种具有12 q13-q14扩增子的RMS细胞系-Rh 30（PAX 3-FOXO 1阳性）、30 SJ和18 C（融合阴性）以及HS-RMS-2（多形性）。我们有一系列RMS细胞系，融合阳性和阴性，缺乏12 q13-q14扩增子，并将在我们的研究中使用选定的系。除了在培养中分析这些细胞系之外，我们的目标是从这些细胞系中开发一系列肌内异种移植物。为此，正在对扩增阳性和扩增阴性细胞系的子集进行微生物病原体污染检测，动物研究方案目前正在接受机构动物护理和使用委员会的审查。 正在与儿科肿瘤学分支的Chand卡纳博士合作计划这些动物研究。目前正在对CDK 4实验的试剂进行测试和验证。通过蛋白质印迹检测CDK 4蛋白表达的抗体已经被鉴定并成功地用于几个实验中。使用该抗体的蛋白质印迹分析证实了具有12 q13-q14扩增的细胞系中CDK 4的表达增加。测试了靶向CDK 4基因的初始逆转录病毒shRNA表达构建体。转移到Rh 30细胞中证明了CDK 4蛋白表达降低的证据。此外，细胞培养研究表明表达CDK 4-shRNA的细胞比对照细胞生长更慢。在补充研究中，肿瘤内科分支的Ji Luo博士提供了四环素诱导的慢病毒表达载体。我们已经将CDK 4 cDNA亚克隆到该表达载体中，并将表达构建体转导到Rh 28细胞中，其不具有12 q13-q14区域的扩增。在Rh 28细胞中的初始剂量-反应和时间过程研究证实，与对照相比，在转导的Rh 28细胞中容易诱导CDK 4蛋白表达增加。因此，许多基本的技术现在已经到位，以调查CDK 4的扩增和过度表达在横纹肌肉瘤中的作用。

The Senior Investigator moved to the National Cancer Institute during FY2011 and hired personnel to set up a research laboratory during the first half of FY2012. A postdoctoral trainee was recruited to work on this RMS amplification project starting in March 2012. The first RMS genomic amplification studies are focusing on the 12q13-q14 amplicon, which occurs preferentially in a subset of PAX3-FOXO1-positive RMS (25% of cases) and smaller numbers of PAX7-FOXO1-positivie 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 microarray 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 is further refining these genomic findings.To determine which of these 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, and Dr. Bruce Pawel of the Children's Hospital of Philadelphia (CHOP). Dr. Pawel has generated a tissue microarray of RMS specimens. In addition, two additional RMS TMA's were obtained from the Children's Oncology Group (COG). Previous work in my laboratory has determined the fusion status of most of the RMS specimens on both the CHOP and COG TMA's. Dr. Pack is developing a set of fluorescence in situ hybridization probes to determine the amplification status of the 12q13-q14 region in the cases on these TMA's. Dr. Hewitt and Dr. Pawel will then work with my laboratory to analyze the expression status of selected proteins n these TMA's by immunohistochemistry. To begin the functional analysis, we are initially focusing on the CDK4 gene and then will consider additional genes as indicated by our other analyses. For these studies, we have collected several RMS cell lines with the 12q13-q14 amplicon - Rh30 (PAX3-FOXO1-positive), 30SJ and 18C (fusion-negative) and HS-RMS-2 (pleomorphic). We have a series of RMS cell lines, both fusion-positive and negative, that lack the 12q13-q14 amplicon and will use selected lines in our studies. In addition to analyzing these cell lines in culture, our goal is to also develop a series of intramuscular xenografts from these cell lines. Towards this end, a subset of amplification positive and amplification negative lines is being tested for microbial pathogen contamination, and an animal studies protocol is now under review by the Institutional Animal Care and Use Committee. These animal studies are being planned in collaboration with Dr. Chand Khanna of the Pediatric Oncology Branch. Reagents are now being tested and validated for the CDK4 experiments. An antibody that detects CDK4 protein expression by western blot has been identified and successfully used in several experiments. Western blot analysis with this antibody confirms increased expression of CDK4 in lines with 12q13-q14 amplification. An initial retroviral shRNA expression construct targeted against the CDK4 gene was tested. Transfer into Rh30 cells demonstrated evidence of decreased CDK4 protein expression. Furthermore, cell culture studies indicate that the CDK4-shRNA-expressing cells grow more slowly than control cells. In complementary studies, a tetracycline-inducible lentiviral expression vector was provided by Dr. Ji Luo of the Medical Oncology Branch. We have subcloned the CDK4 cDNA into this expression vector and transduced the expression construct into Rh28 cells, which do not have amplification of the 12q13-q14 region. Initial dose-response and time course studies in Rh28 cells confirm that increased CDK4 protein expression is readily induced in transduced Rh28 cells in comparison to controls. Therefore, many of the basic techniques are now in place to investigate the role of CDK4 amplification and overexpression in rhabdomyosarcoma.