Studies of amplification in rhabdomyosarcoma

横纹肌肉瘤扩增的研究

• 批准号: 8763479
• 负责人: Frederic Barr
• 金额: $ 33.5万
• 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/8763479
• 关键词: 12q13-q14; 13q14; 2p24; Animals; Antibodies; Behavior; Biological; Biological Assay; CDK4 gene; Candidate Disease Gene; Cell Culture Techniques; Cell Line; Cells; Chemicals; Children' s Oncology Group; Clinical; Collaborations; Complementary DNA; Cyclin-Dependent Kinase 4; Data; Defect; Dose; Doxycycline; Event; Family; Fluorescent in Situ Hybridization; Frequencies; Genes; Genetic; Genetic Transcription; Genomics; Goals; Growth; Heterogeneity; IACUC; Immunohistochemistry; Intramuscular; Isopropyl Thiogalactoside; Laboratories; Laboratory Animals; Laboratory mice; Maps; Medical Oncology; Microarray Analysis; Molecular; Molecular Profiling; Oncogene Proteins; Oncogenic; Other Genetics; PAX3 gene; PAX7 gene; Pathogenesis; Pathology; Pathway interactions; Pediatric Oncology; Phenotype; Proteins; Proto-Oncogenes; Protocols documentation; RNA; RNA Interference; Reaction Time; Reagent; Recurrence; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Rhabdomyosarcoma; Role; Series; Simulate; Specimen; Testing; Tetracyclines; Therapeutic Intervention; Tissue Microarray; Tumorigenicity; Western Blotting; Work; Xenograft procedure; base; cell growth; cellular transduction; density; directed attention; expression vector; next generation sequencing; overexpression; protein expression; research study; small hairpin RNA; therapeutic target; tumor; tumorigenesis

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.

对横纹肌肉瘤(RMS)复发扩增的最早研究集中在12q13-Q14扩增，它优先出现在PAX3-FOXO1阳性横纹肌肉瘤的一部分(25%)，仅出现在PAX7-FOXO1阳性和融合阴性的一小部分横纹肌肉瘤病例中。我们以前的研究将最小扩增区域定位到0.55Mb区域，包含28个基因，包括CDK4原癌基因。我们随后的表达谱研究表明，在扩增的RMS肿瘤中，这些基因中有7个在RNA水平上持续过表达。与儿科肿瘤科的贾维德·汗博士应用高密度阵列分析和下一代测序进行的合作研究证实了12q13-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上过度表达，我们与斯维特拉娜·帕克博士和病理学实验室的斯蒂芬·休伊特博士建立了合作关系。两个RMS标本的组织微阵列来自儿童肿瘤组(COG)。利用RT-PCR和FISH分析，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来确定这些TMA上的12q13-Q14区域的扩增状态；这些FISH研究的结果与我们之前报道的RMS融合亚群中12q13-Q14扩增频率的发现一致。休伊特博士现在正在与我的实验室合作，通过免疫组织化学方法分析CDK4蛋白在这些TMA中的表达状态。为了开始我们对这个扩增事件的功能后果的分析，我们首先关注CDK4基因。在这些研究中，我们确定Rh30是一个带有12q13-q14扩增子的融合阳性RMS细胞株。为了进行比较，我们选择了几个缺乏12q13-q14扩增子的融合阳性RMS细胞株。除了在培养中分析这些细胞系外，作为与儿科肿瘤学分会的Chand Khanna博士合作的一部分，我们还从这些细胞系中开发了一系列肌肉内异种移植。一种通过蛋白质印迹检测CDK4蛋白表达的抗体已经被鉴定并成功应用。Western印迹分析证实CDK4在12q13-q14扩增的细胞系和相应的异种移植瘤中表达增加。在一组功能研究中，四环素诱导的慢病毒表达载体由医学肿瘤科的纪罗博士提供。我们将CDK4基因亚克隆到该表达载体中，并将其转化到几个未扩增12q13-q14区域的RMS细胞系中。在这些转导细胞中的初步剂量-反应和时间过程研究证实，与对照组相比，经多西环素处理的转导Rh28细胞中CDK4蛋白的表达很容易增加。这些转导细胞的表型正在生长和转化的细胞培养研究中进行评估。在补充性研究中，一组针对CDK4基因的慢病毒shRNA表达载体被导入Rh30细胞，并显示CDK4蛋白表达降低的明显证据。此外，CDK4在这些细胞中的表达减少与细胞生长和可能的生存能力的严重缺陷有关。为了进一步分析这个问题，我们开发了IPTG诱导的shRNA结构，并在Rh30中进行了测试。正如预测的那样，这些可诱导的构建体允许可控地抑制Rh30细胞中CDK4的表达。我们现在正在研究这些细胞中不同水平的CDK4表达的表型后果。在没有12q13-Q14扩增的融合阳性RMS细胞中，我们选择了低水平和中等水平CDK4蛋白表达的代表性品系Rh28和Rh41。这些品系正在用IPTG可诱导的shRNA构建物进行类似的测试。最后，机构动物保护和使用委员会最近批准了一项动物方案，以延长我们的细胞培养研究，并评估CDK4在促进和维持实验动物致瘤性方面的作用。