WT1 and beta-catenin targets in Wilms tumor

描述(申请人提供)：主要的努力是确定致癌转录因子的靶标，但来自组织培养实验的数据很少在原发癌症中进行审查。在这里，我们使用Wilms肿瘤作为实验系统来解决这个问题。发生在Denys-Drash和WAGR综合征的Wilms肿瘤携带WT1肿瘤抑制基因的失活突变。相反，WT1突变在散发性肾母细胞瘤中很少见。我们已经证实了先前的数据表明，β-连环蛋白(CTNNB1)的突变仅限于WT1缺失的肿瘤。CTNNB1是Wnt信号通路的一个组成部分，在许多最常见的人类恶性肿瘤中充当癌基因。我们还发现，这些突变在Ser45密码子上显著聚集。通过表达谱分析，我们已经确定了一组区分WT1缺失和WT1阳性肿瘤的基因。这个数据集应该是一个强大的工具来识别WT1和β-catenin/TCF的下游靶点，我们假设它们在这些差异表达的基因中是丰富的。目的1.我们将确定Wnt/β-catenin信号轴在WT1缺失的肾母细胞瘤中是否普遍激活。目的2.为了缩小WT1和β-连环蛋白靶基因的范围，我们将在组织培养中操纵WT1水平和β-连环蛋白途径的成分，并将结果与我们从原发肾母细胞瘤中获得的“金标准”数据进行比较。目的3.为了在体内验证候选的β-连环蛋白靶基因，我们将利用基因敲入的方法将突变的β-连环蛋白表达到发育中的肾脏。通过创建等基因系列，这些小鼠还将允许我们测试Ser45突变与其他磷酸化位置在影响β-连环蛋白的增殖/致癌能力方面的相对效力。我们随后将这些小鼠与Wt1突变杂合子杂交，可能产生Wilms肿瘤的小鼠模型。

DESCRIPTION (provided by applicant): Major efforts have gone into identifying the targets of oncogenic transcription factors, but data from tissue culture experiments have seldom been vetted in primary cancers. Here we address this problem, using Wilms tumor as an experimental system. Wilms tumors occurring in Denys-Drash and WAGR syndromes carry inactivating mutations of the WT1 tumor suppressor gene. In contrast, WT1 mutations are rare in sporadic Wilms tumors. We have confirmed previous data indicating that mutations in beta-catenin (CTNNB1), a component of the Wnt signaling pathway that acts as an oncogene in many of the most common human malignancies, are restricted to the WT1-null tumors. We also find that these mutations are strikingly clustered at codon Ser45. By expression profiling we have identified a panel of genes that distinguish the WT1-null from WT1-positive tumors. This dataset should be a powerful tool to identify downstream targets of WT1 and beta-catenin/TCF, which we hypothesize are enriched among these differentially expressed genes. Aim 1. We will determine whether the Wnt/beta-catenin signaling axis is universally activated in the WT1- null class of Wilms tumors. Aim 2. To narrow the list of WT1 and beta-catenin target genes, we will manipulate WT1 levels, and components of the beta-catenin pathway in tissue culture, and compare the results with our "gold standard" data from the primary Wilms tumors. Aim 3. To validate the candidate beta-catenin target genes in vivo, we will express mutant beta-catenin to the developing kidney, using a gene knock-in approach. By creating an isogenic series, these mice will also allow us to assay for the relative potency of mutation at Ser45 vs. other phosphorylation sites in affecting the proliferative/oncogenic potency of beta-catenin. We will subsequently cross these mice with Wt1-mutant heterozygotes, possibly generating a mouse model for Wilms tumor.