Studies of amplification in rhabdomyosarcoma

我们最近对横纹肌肉瘤（RMS）复发性扩增子的研究集中在12 q13-q14扩增子上，该扩增子优先发生在PAX 3-FOXO 1阳性RMS的一个子集（25%的病例）中，仅发生在PAX 7-FOXO 1阳性和融合阴性病例的一个较小子集中。我们先前的研究将扩增的最小区域定位到含有28个基因的0.55 Mb区域，包括CDK 4原癌基因。在有和没有这种扩增子的RMS肿瘤中的表达谱研究表明，这些基因中的7个（包括CDK 4）在扩增的RMS肿瘤中在RNA水平上一致地过表达。然后，我们专注于CDK 4基因，并通过与病理学实验室的Svetlana Pack和Stephen休伊特博士合作，评估CDK 4蛋白表达和扩增状态之间的关系。RMS标本的两个组织微阵列（TMA）从儿童肿瘤组获得。Pack博士使用荧光原位杂交探针确定每个病例TMA上的12 q13-q14扩增状态，休伊特博士使用免疫组织化学方法评估这些病例中CDK 4蛋白的表达。这些TMA研究的综合结果表明，12 q13-q14扩增的病例表达高水平的CDK 4蛋白，12号染色体额外拷贝的病例表达中等水平的蛋白，12 q13-q14拷贝数正常的病例表达低水平的蛋白。为了分析CDK 4扩增的功能后果，将针对CDK 4基因的IPTG诱导的shRNA构建体转导到Rh 30细胞中，Rh 30细胞是具有12 q13-q14扩增和相关的CDK 4 mRNA和蛋白质高水平表达的融合阳性RMS系。在Rh 30细胞中诱导CDK 4-shRNA构建体导致CDK 4表达的抑制。此外，CDK 4表达的这种抑制与细胞生长缺陷相关，其特征在于通过流式细胞术的G1期阻滞。在实验室小鼠的肿瘤发生研究中，Rh 30细胞中的CDK 4 shRNA诱导导致异种移植物形成延迟。用诱导型CDK 4 shRNA对Rh 30细胞的表达微阵列分析表明，CDK 4-RB通路中的基因表达降低，如E2 F转录靶点和其他细胞周期相关基因。在使用IPTG诱导的CDK 4 shRNA构建体的进一步实验中，在没有12 q13-q14扩增的Rh 41细胞系中诱导CDK 4 shRNA也显示出细胞生长的类似降低。这一发现提供了初步证据，表明12 q13-q14扩增或过表达与对CDK 4抑制的敏感性增加无关。为了进一步分析这个问题，我们比较了5种融合阳性RMS细胞系对LEE 011（Novartis）（CDK 4/CDK 6的选择性抑制剂）生长抑制的敏感性。用一系列LEE 011浓度处理这五个细胞系导致细胞数量的剂量依赖性减少和CDK 4-RB途径靶点表达的相应减少。在这些细胞系中，对生长抑制的敏感性与CDK 4或CDK 6表达水平之间没有关系。特别是，CDK 4在Rh 30中的高表达与对生长抑制的更高易感性无关。

Our recent studies of recurrent amplicons in rhabdomyosarcoma (RMS) focused 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. Expression profiling studies in RMS tumors with and without this amplicon showed that 7 of these genes (including CDK4) were consistently overexpressed at the RNA level in amplified RMS tumors. We then focused on the CDK4 gene and assessed the relationship between CDK4 protein expression and amplification status through a collaboration with Drs. Svetlana Pack and Stephen Hewitt of the Laboratory of Pathology. Two tissue microarrays (TMA's) of RMS specimens were obtained from the Children's Oncology Group. Dr. Pack used fluorescence in situ hybridization probes to determine 12q13-q14 amplification status in each case on the TMA, and Dr. Hewitt used an immunohistochemical approach to assess CDK4 protein expression in these cases. The combined results from these TMA studies indicated that cases with 12q13-q14 amplification expressed high levels of CDK4 protein, cases with extra copies of chromosome 12 expressed intermediate protein levels and cases with normal 12q13-q14 copy number expressed low protein levels. To analyze the functional consequences of CDK4 amplification, IPTG-inducible shRNA constructs targeted against the CDK4 gene were transduced into Rh30 cells, a fusion-positive RMS line with 12q13-q14 amplification and associated high level expression of CDK4 mRNA and protein. Induction of the CDK4-shRNA constructs in Rh30 cells resulted in inhibition of CDK4 expression. In addition, this inhibition of CDK4 expression was associated with defects in cell growth, characterized as a G1 arrest by flow cytometry. In tumorigenesis studies in laboratory mice, CDK4 shRNA induction in Rh30 cells resulted in a delay in xenograft formation. Expression microarray analysis of Rh30 cells with inducible CDK4 shRNA demonstrated decreased expression of genes in the CDK4-RB pathway, such as E2F transcriptional targets and other cell cycle-related genes. In further experiments with the IPTG-inducible CDK4 shRNA constructs, induction of CDK4 shRNA in the Rh41 cell line, which does not have 12q13-q14 amplification, also demonstrated similar decreases in cell growth. This finding provides initial evidence that 12q13-q14 amplification or overexpression is not associated with increased sensitivity to CDK4 inhibition. To further analyze this issue, we compared the sensitivity of five fusion-positive RMS cell lines to growth inhibition by LEE011 (Novartis), a selective inhibitor of CDK4/CDK6. Treatment of these five lines with a range of LEE011 concentrations resulted in a dose-dependent decrease in cell numbers and a corresponding decrease in expression of CDK4-RB pathway targets. There was no relationship between sensitivity to growth inhibition and the CDK4 or CDK6 expression levels in these cell lines. In particular, the high CDK4 expression in Rh30 was not associated with higher susceptibility to growth inhibition.