Studies of gene fusions in rhabdomyosarcoma

横纹肌肉瘤基因融合的研究

• 批准号: 8763485
• 负责人: Frederic Barr
• 金额: $ 40.2万
• 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/8763485
• 关键词: Animals; Biological Assay; Candidate Disease Gene; Cell Culture System; Cell Culture Techniques; Cell Death; Cell Line; Cells; Childhood; Chimeric Proteins; Complementary DNA; Data; Detection; Dose; Doxycycline; FGFR4 gene; Fibroblasts; Fusion Protein Expression; Gene Expression; Gene Fusion; Genes; Goals; Growth; Human; IACUC; Laboratory Animals; Laboratory mice; MYCN gene; Malignant Neoplasms; Measures; Medical Oncology; Messenger RNA; Monoclonal Antibodies; Mus; Myoblasts; Oncogene Proteins; Oncogenic; PAX3 gene; PAX7 gene; Pathway interactions; Phenotype; Population; Proteins; Protocols documentation; Publishing; Recurrence; Reverse Transcriptase Polymerase Chain Reaction; Rhabdomyosarcoma; Role; System; Tetracyclines; Toxic effect; Tumorigenicity; Western Blotting; base; cell growth; cell type; cellular transduction; expression vector; genome-wide; in vivo; overexpression; prevent; research study; response; soft tissue; success; translational approach; tumor; tumorigenesis

Our initial goal was to develop cell culture systems in which PAX3-FOXO1 expression can be induced and in which the downstream effects of PAX3-FOXO1 can then be assessed at the gene expression and phenotypic levels. To measure PAX3-FOXO1 expression in the planned studies, a monoclonal antibody to the FOXO1 C-terminus was successfully used in western blot studies of RMS cell lines to detect expression of the wild-type FOXO1 and/or fusion protein. Quantitative RT-PCR assays were similarly validated for detection of the wild-type FOXO1 and PAX3-FOXO1 (or PAX7-FOXO1) mRNA's. To generate inducible expression in our planned studies, a tetracycline-inducible lentiviral expression vector was provided by Dr. Ji Luo of the Medical Oncology Branch. We subcloned the PAX3-FOXO1 cDNA into this expression vector and transduced this PAX3-FOXO1 expression construct into several cell lines. To validate the utility of this inducible construct, we first transduced this construct into murine NIH3T3 fibroblast cells and observed inducible increases in PAX3-FOXO1 mRNA and protein in dose-response studies. The induced level of PAX3-FOXO1 protein reached levels equivalent to and even greater than the high fusion expression level present in fusion-positive RMS cells. Based on our initial success in generating inducible PAX3-FOXO1 expression in NIH3T3 cells, we then introduced this construct into the immortalized human Dbt myoblast cell line. Using our western blot and RT-PCR assays, we found inducible expression of PAX3-FOXO1 in the transduced Dbt cells, from near undetectable levels in the uninduced state to high levels that mirror the high levels in fusion-positive RMS cells. Further use of qRT-PCR assays for FGFR4 and ABAT, two downstream PAX3-FOXO1 targets, revealed inducible increases in expression of both of these genes. We then proceeded with several cell culture assays of the phenotypic effects of PAX3-FOXO1 in these transduced Dbt cells. Similar to our previous published studies of constitutive PAX3-FOXO1 expression constructs, we analyzed phenotypic effects in cells with and without constitutive MYCN expression. The toxic effects of induced PAX3-FOXO1 expression were manifest by the finding of little or no growth of these cells when PAX3-FOXO1 expression was induced by doxycycline in contrast to the brisk growth of these cells in the absence of doxycycline. These toxic effects were seen in the presence or absence of exogenous MYCN expression. Despite these toxic effects, the cells with induced PAX3-FOXO1 expression (as well as exogenous constitutive MYCN expression) demonstrated a high level of focus formation, which is a measure of oncogenic transformation in cell culture. Similar to the cells without doxycycline induction, the cells with induced PAX3-FOXO1 but without exogenous MYCN expression did not demonstrate evidence of oncogenic transformation. To extend these findings to the in vivo setting, an animal protocol has been submitted to the Institutional Animal Care and Use Committee to assess the role of PAX3-FOXO1 (with or without MYCN) in contributing to and maintaining tumorigenicity in laboratory animals.

我们最初的目标是开发可以诱导PAX3-FOXO1表达的细胞培养系统，然后可以在基因表达和表型水平上评估PAX3-FOXO1的下游效应。为了在计划的研究中测量PAX3-FOXO1的表达，FOXO1 c -末端的单克隆抗体成功地用于RMS细胞系的western blot研究，以检测野生型FOXO1和/或融合蛋白的表达。同样，定量RT-PCR检测野生型FOXO1和PAX3-FOXO1（或PAX7-FOXO1） mRNA。为了在我们计划的研究中产生诱导表达，一个四环素诱导的慢病毒表达载体由内科肿瘤科的吉洛博士提供。我们将PAX3-FOXO1 cDNA亚克隆到该表达载体中，并将该PAX3-FOXO1表达构建体转染到多个细胞系中。为了验证该诱导结构的有效性，我们首先将该结构转导到小鼠NIH3T3成纤维细胞中，并在剂量反应研究中观察到PAX3-FOXO1 mRNA和蛋白的诱导增加。诱导的PAX3-FOXO1蛋白表达水平相当于甚至大于融合阳性RMS细胞的高融合表达水平。基于我们在NIH3T3细胞中产生可诱导的PAX3-FOXO1表达的初步成功，我们随后将该构建物引入永生化的人Dbt成肌细胞系。通过western blot和RT-PCR检测，我们发现PAX3-FOXO1在转导的Dbt细胞中可诱导表达，从未诱导状态下接近不可检测的水平到高水平，反映了融合阳性RMS细胞中的高水平。对PAX3-FOXO1的两个下游靶点FGFR4和ABAT的qRT-PCR分析显示，这两个基因的表达均可诱导增加。然后，我们对PAX3-FOXO1在这些转导的Dbt细胞中的表型效应进行了几次细胞培养试验。与我们之前发表的关于组成性PAX3-FOXO1表达结构的研究类似，我们分析了具有和不具有组成性MYCN表达的细胞的表型效应。多西环素诱导PAX3-FOXO1表达时，PAX3-FOXO1细胞的生长很少或不生长，而没有多西环素时，PAX3-FOXO1细胞的生长活跃，这表明了诱导PAX3-FOXO1表达的毒性作用。这些毒性作用在外源性MYCN表达存在或不存在的情况下都可以观察到。尽管存在这些毒性作用，但诱导PAX3-FOXO1表达的细胞（以及外源性构成性MYCN表达）表现出高水平的病灶形成，这是细胞培养中致癌转化的一个指标。与没有强力霉素诱导的细胞类似，诱导PAX3-FOXO1但没有外源MYCN表达的细胞没有显示出致癌转化的证据。为了将这些发现扩展到体内环境，已经向机构动物护理和使用委员会提交了一份动物方案，以评估pax3 - fox01（带或不带MYCN）在实验室动物中促进和维持致瘤性方面的作用。