Studies of gene fusions in rhabdomyosarcoma

横纹肌肉瘤基因融合的研究

基本信息

  • 批准号:
    9343899
  • 负责人:
  • 金额:
    $ 59.23万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
  • 资助国家:
    美国
  • 起止时间:
  • 项目状态:
    未结题

项目摘要

In previous studies, we constructed a doxycycline-inducible PAX3-FOXO1 expression construct and introduced this construct into immortalized human myoblasts (with and without a constitutive MYCN expression construct). In the resulting cell culture system, PAX3-FOXO1 expression can be up-regulated by doxycycline treatment and then down-regulated by doxycycline withdrawal. In cell culture studies, doxycycline-treated myoblasts transduced with MYCN and PAX3-FOXO1 showed a high level of oncogenic transformation in a focus formation assay whereas there was no transformation detected without doxycycline treatment or in cells transduced only with PAX3-FOXO1 or MYCN. When doxycycline was removed during the course of the focus formation assay of cells transduced with MYCN and inducible PAX3-FOXO1 constructs, smaller foci formed with prominent myogenic differentiation and cell death. To study these oncogenic events in vivo, mice were injected intramuscularly with these cells, and progressively growing tumors formed from PAX3-FOXO1-inducible myoblasts when the mice were fed a doxycycline-supplemented diet; cells expressing both PAX3-FOXO1 and MYCN formed rapidly growing tumors whereas cells expressing PAX3-FOXO1 without exogenous MYCN formed tumors several weeks later. All tumors showed histologic resemblance to human ARMS with high expression of MyoD and myogenin. Frequent mitoses and a high Ki67 staining index were indicative of a high proliferative rate. To determine the requirement for continued PAX3-FOXO1 expression in these tumors, doxycycline was withdrawn after small palpable tumors formed, resulting in a cessation of tumor growth and regression to undetectable levels. Expression studies confirmed that PAX3-FOXO1 mRNA and protein levels drastically decreased, and microscopic examination of regressing tumors revealed a transient decrease in cellular proliferation in association with widespread myogenic differentiation and cell death. Following tumor regression in these animal studies, tumors generally recurred several weeks later despite the continued absence of inducing agent. Analysis of recurrent tumor samples revealed that there was no detectable PAX3-FOXO1 expression in more than half of these recurrent tumors. Cell lines were next generated from primary and recurrent tumors to further investigate these oncogenic effects. Primary tumor-derived lines expressing exogenous PAX3-FOXO1 and MYCN demonstrated PAX3-FOXO1-dependent transformation and tumorigenesis similar to the original transduced parental population. In contrast, primary tumor-derived lines expressing only exogenous PAX3-FOXO1 were transformed in culture and formed tumors at a much faster rate than the original transduced parental population, consistent with the selection of PAX3-FOXO1-dependent transformed variants during tumorigenesis. Studies of cell lines derived from recurrent tumors that did not express the fusion protein revealed transformation and tumorigenesis, both in the absence of doxycycline, consistent with the acquisition of additional oncogenic events that render these cells independent of the fusion protein. The formation of similar recurrences with the primary tumor-derived cell lines or subclones of the original transduced parental cells provided evidence of the generality of this recurrence phenomenon. Finally, though cell lines derived from primary tumors were dependent on PAX3-FOXO1 and differentiated when doxycycline was removed, the recurrent tumor-derived cells did not differentiate under these conditions and instead proliferated continuously, consistent with a PAX3-FOXO1-independent block in differentiation.
在以前的研究中,我们构建了一个强力霉素诱导的PAX 3-FOXO 1表达构建体,并将该构建体引入永生化的人成肌细胞(有和没有组成性MYCN表达构建体)。在所得细胞培养系统中,PAX 3-FOXO 1表达可通过多西环素处理上调,然后通过多西环素撤除下调。在细胞培养研究中,用MYCN和PAX 3-FOXO 1转导的多西环素处理的成肌细胞在病灶形成测定中显示出高水平的致癌转化,而在没有多西环素处理或仅用PAX 3-FOXO 1或MYCN转导的细胞中没有检测到转化。当在用MYCN和诱导型PAX 3-FOXO 1构建体转导的细胞的病灶形成试验过程中去除强力霉素时,形成较小的病灶,并伴有明显的肌源性分化和细胞死亡。为了在体内研究这些致癌事件,小鼠肌内注射这些细胞,并且当小鼠被喂食多西环素补充的饮食时,由PAX 3-FOXO 1诱导的成肌细胞形成逐渐生长的肿瘤;表达PAX 3-FOXO 1和MYCN的细胞形成快速生长的肿瘤,而表达PAX 3-FOXO 1而没有外源性MYCN的细胞在几周后形成肿瘤。所有肿瘤显示与人ARMS的组织学相似性,具有高表达MyoD和肌细胞生成素。频繁的有丝分裂和高Ki 67染色指数指示高增殖率。为了确定在这些肿瘤中持续PAX 3-FOXO 1表达的需要,在小的可触知肿瘤形成后撤回多西环素,导致肿瘤生长停止并消退至不可检测的水平。表达研究证实,PAX 3-FOXO 1 mRNA和蛋白水平急剧下降,和显微镜检查的消退肿瘤显示与广泛的肌原性分化和细胞死亡的细胞增殖的短暂减少。在这些动物研究中,肿瘤消退后,尽管继续缺乏诱导剂,但肿瘤通常在几周后复发。对复发性肿瘤样本的分析显示,在这些复发性肿瘤的一半以上中没有可检测到的PAX 3-FOXO 1表达。接下来从原发性和复发性肿瘤产生细胞系以进一步研究这些致癌作用。表达外源性PAX 3-FOXO 1和MYCN的原代肿瘤衍生系表现出与原始转导亲本群体相似的PAX 3-FOXO 1依赖性转化和肿瘤发生。相比之下,仅表达外源PAX 3-FOXO 1的原代肿瘤衍生系在培养物中转化,并以比原始转导的亲本群体快得多的速率形成肿瘤,这与肿瘤发生期间PAX 3-FOXO 1依赖性转化变体的选择一致。对不表达融合蛋白的复发性肿瘤来源的细胞系的研究显示,在不存在强力霉素的情况下,转化和肿瘤发生与使这些细胞独立于融合蛋白的额外致癌事件的获得一致。与原代肿瘤衍生细胞系或原始转导亲本细胞的亚克隆形成类似的复发提供了这种复发现象的普遍性的证据。最后,虽然来自原发性肿瘤的细胞系依赖于PAX 3-FOXO 1,并且在去除多西环素时分化,但复发性肿瘤衍生的细胞在这些条件下不分化,而是连续增殖,这与PAX 3-FOXO 1独立的分化阻滞一致。

项目成果

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Frederic Barr其他文献

Frederic Barr的其他文献

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{{ truncateString('Frederic Barr', 18)}}的其他基金

Studies of gene fusions in rhabdomyosarcoma
横纹肌肉瘤基因融合的研究
  • 批准号:
    10486830
  • 财政年份:
  • 资助金额:
    $ 59.23万
  • 项目类别:
Studies of amplification in rhabdomyosarcoma
横纹肌肉瘤扩增的研究
  • 批准号:
    8763479
  • 财政年份:
  • 资助金额:
    $ 59.23万
  • 项目类别:
Studies of gene fusions in rhabdomyosarcoma
横纹肌肉瘤基因融合的研究
  • 批准号:
    8763485
  • 财政年份:
  • 资助金额:
    $ 59.23万
  • 项目类别:
Studies of gene fusions in rhabdomyosarcoma
横纹肌肉瘤基因融合的研究
  • 批准号:
    9153887
  • 财政年份:
  • 资助金额:
    $ 59.23万
  • 项目类别:
Clinical Operations for Laboratory of Pathology
病理实验室临床操作
  • 批准号:
    9556867
  • 财政年份:
  • 资助金额:
    $ 59.23万
  • 项目类别:
Studies of amplification in rhabdomyosarcoma
横纹肌肉瘤扩增的研究
  • 批准号:
    8349507
  • 财政年份:
  • 资助金额:
    $ 59.23万
  • 项目类别:
Studies of amplification in rhabdomyosarcoma
横纹肌肉瘤扩增的研究
  • 批准号:
    9556544
  • 财政年份:
  • 资助金额:
    $ 59.23万
  • 项目类别:
Epigenetic studies in rhabdomyosarcoma
横纹肌肉瘤的表观遗传学研究
  • 批准号:
    9153908
  • 财政年份:
  • 资助金额:
    $ 59.23万
  • 项目类别:
Epigenetic studies in rhabdomyosarcoma
横纹肌肉瘤的表观遗传学研究
  • 批准号:
    8553174
  • 财政年份:
  • 资助金额:
    $ 59.23万
  • 项目类别:
Epigenetic studies in rhabdomyosarcoma
横纹肌肉瘤的表观遗传学研究
  • 批准号:
    8938110
  • 财政年份:
  • 资助金额:
    $ 59.23万
  • 项目类别:

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