Studies of gene fusions in rhabdomyosarcoma

在以前的研究中，我们将一个可被多西环素诱导的PAX3-FOXO1表达载体引入永生化的人成肌细胞中(有和没有MYCN的结构性表达载体)，以建立一个细胞培养系统，在这个细胞培养系统中，PAX3-FOXO1的表达可以被多西环素处理上调，然后被多西环素停用而下调。在细胞培养研究中，在PAX3-FOXO1和MYCN表达的细胞中检测到了致癌转化，而在仅表达PAX3-FOXO1的细胞中没有检测到致癌转化。在接受肌肉注射的动物中，当用多西环素处理时，PAX3-FOXO1诱导的成肌细胞形成渐进性生长的肿瘤；同时表达PAX3-FOXO1和MYCN的细胞比表达PAX3-FOXO1的细胞早几周形成肿瘤。当停用多西环素时，肿瘤形成停止并退化，然后在持续缺乏多西环素的情况下，几周后逐渐生长的肿瘤重新形成。在过去的一年里，我们分析了动物研究中形成的原发和复发肿瘤的组织病理学和特定蛋白的表达。所有肿瘤的组织学形态与人类手臂相似，MyoD和Mygenin高表达。频繁的有丝分裂和高Ki67染色指数表明高增殖率。蛋白质印迹分析显示，退行性肿瘤的PAX3-FOXO1蛋白表达迅速下降。退行性病变的显微镜检查显示多核巨细胞显著增加，提示肌源性分化。在许多巨细胞中，固缩细胞核的存在和caspase-3染色的大量瞬时增加提供了叠加细胞死亡的证据。Ki67染色也有一过性下降，表明细胞增殖受阻。复发肿瘤的Western印迹分析显示，其中一半的肿瘤没有检测到PAX3-FOXO1蛋白的表达。1例复发的肿瘤有一个大的显性结节，结节内有致密排列的未分化小细胞，周围是松散排列的分化肌管。该结节具有较高的Ki67染色指数，与增殖活性一致。在其他复发肿瘤中，小未分化细胞和高Ki67染色的区域呈斑块状分布。这些发现强调了PAX3-FOXO1在体内刺激生长、抑制分化和细胞死亡的作用，并提示复发肿瘤的形成可能有多种机制。在原发和复发肿瘤来源的细胞系中，多西环素诱导PAX3-FOXO1表达，停药导致PAX3-FOXO1表达缺失。这些发现表明，在肿瘤发生和复发过程中，诱导系统是完整的。在病灶形成试验中，多西环素在原发和复发肿瘤的细胞系中诱导显著的病灶形成。在没有多西环素的情况下，一个复发的肿瘤来源株显示PAX3-FOXO1非依赖的病灶形成。对于转导PAX3-FOXO1而不转导MYCN的细胞，肿瘤来源的细胞发生了转化，而亲本细胞没有转化，提示在肿瘤发生过程中选择了替代MYCN的细胞。

In previous studies, we introduced a doxycycline-inducible PAX3-FOXO1 expression construct into immortalized human myoblasts (with and without a constitutive MYCN expression construct) to generate a cell culture system in which PAX3-FOXO1 expression can be up-regulated by doxycycline treatment and then down-regulated by doxycycline withdrawal. In cell culture studies, oncogenic transformation was detected in cells with PAX3-FOXO1 and MYCN expression, but not in cells only expressing PAX3-FOXO1 alone. In animals receiving an intramuscular injection, progressively growing tumors formed from PAX3-FOXO1-inducible myoblasts when treated with doxycycline; cells expressing both PAX3-FOXO1 and MYCN formed tumors several weeks earlier than cells expressing PAX3-FOXO1 without exogenous MYCN. When doxycycline was withdrawn, tumor formation stopped and regressed, and then progressively growing tumors re-formed several weeks later in the continued absence of doxycycline. During the past year, we analyzed the histopathology and expression of specific proteins in the primary and recurrent tumors formed in the animal studies. 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. Western blot analysis of the regressing tumors showed a rapid decrease in PAX3-FOXO1 protein expression. Microscopic examination of regressing lesions showed a striking increase in multinucleated giant cells, indicative of myogenic differentiation. In many giant cells, the presence of pyknotic nuclei and a large transient increase in caspase-3 staining provided evidence of superimposed cell death. There was also a transient decrease in Ki67 staining indicative of a block in proliferation. Western blot analysis of recurrent tumors showed that half of these tumors have no detectable PAX3-FOXO1 protein expression. One recurrent tumor contained a large dominant nodule of densely packed undifferentiated small cells surrounded by loosely packed differentiated myotubes. This nodule had a high Ki67 staining index consistent with proliferative activity. In other recurrent tumors, there was a patchy distribution of regions with small undifferentiated cells and high Ki67 staining. These findings emphasize the in vivo role of PAX3-FOXO1 in stimulating growth and inhibiting differentiation and cell death, and suggest there may multiple mechanisms underlying formation of recurrent tumors. In cell lines derived from primary and recurrent tumors, doxycycline addition induced PAX3-FOXO1 expression and withdrawal resulted in loss of PAX3-FOXO1 expression. These findings indicate that the inducible system is intact during tumorigenesis and recurrence. In focus formation assays, doxycycline induced significant focus formation in cell lines from primary and recurrent tumors. In the absence of doxycycline, one of the recurrent tumor-derived lines showed PAX3-FOXO1-independent focus formation. For the cells transduced with PAX3-FOXO1 but not MYCN, the tumor-derived cells were transformed whereas the parental cells were not transformed, suggesting selection of cells with a change that substitutes for MYCN during tumorigenesis.