Studies of gene fusions in rhabdomyosarcoma

在之前的研究中，我们构建了强力霉素诱导的PAX3-FOXO1表达构建体，并将该构建体引入永生化的人成肌细胞（有或没有MYCN表达构建体）。在所得到的细胞培养体系中，PAX3-FOXO1的表达可以在强力霉素处理下上调，然后在强力霉素停用后下调。在细胞培养研究中，多西环素处理的MYCN和PAX3-FOXO1转导的成肌细胞在病灶形成实验中显示出高水平的致癌转化，而未经多西环素处理或仅用PAX3-FOXO1或MYCN转导的细胞没有检测到转化。当在MYCN和诱导pax3 - fox01构建体转导的细胞形成病灶的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，在小鼠肌肉内注射这些细胞，当小鼠被喂食强力霉素补充的饮食时，pax3 - fox01诱导的成肌细胞逐渐形成肿瘤；同时表达PAX3-FOXO1和MYCN的细胞形成了快速生长的肿瘤，而不表达外源MYCN的PAX3-FOXO1细胞在几周后形成肿瘤。所有肿瘤在组织学上与人类ARMS相似，MyoD和myogenin高表达。频繁的有丝分裂和高Ki67染色指数表明高增殖率。为了确定PAX3-FOXO1在这些肿瘤中持续表达的需求，在可触及的小肿瘤形成后停用强力霉素，导致肿瘤停止生长并退回到不可检测的水平。表达研究证实，PAX3-FOXO1 mRNA和蛋白水平急剧下降，显微镜检查显示，退化肿瘤的细胞增殖短暂减少，与广泛的肌源性分化和细胞死亡有关。在这些动物实验中，肿瘤消退后，尽管持续缺乏诱导剂，肿瘤通常在几周后复发。对复发肿瘤样本的分析显示，超过一半的复发肿瘤中没有检测到PAX3-FOXO1的表达。然后从原发性和复发性肿瘤中产生细胞系，以进一步研究这些致癌作用。表达外源性PAX3-FOXO1和MYCN的原发肿瘤来源系表现出与原始转导亲本群体相似的PAX3-FOXO1依赖性转化和肿瘤发生。相比之下，仅表达外源性PAX3-FOXO1的原发肿瘤衍生系在培养中转化并形成肿瘤的速度比原始转导的亲代群体快得多，这与肿瘤发生过程中PAX3-FOXO1依赖性转化变体的选择一致。对不表达融合蛋白的复发肿瘤细胞系的研究显示，在缺乏强力霉素的情况下，转化和肿瘤发生与获得额外的致癌事件一致，这些事件使这些细胞独立于融合蛋白。原发肿瘤衍生细胞系或原始转导亲代细胞的亚克隆形成类似的复发，为这种复发现象的普遍性提供了证据。最后，虽然原发肿瘤来源的细胞系依赖于PAX3-FOXO1，并在去除多西环素后分化，但在这些条件下，复发肿瘤来源的细胞不分化，而是持续增殖，这与PAX3-FOXO1不依赖于分化的阻滞一致。

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.