WT1 and beta-catenin targets in Wilms tumor

描述（由申请人提供）：主要的努力已经进入确定致癌转录因子的目标，但组织培养实验的数据很少在原发性癌症中进行审查。在这里，我们解决这个问题，使用肾母细胞瘤作为一个实验系统。Denys-Drash综合征和WAGR综合征中发生的肾母细胞瘤携带WT 1肿瘤抑制基因的失活突变。相反，WT 1突变在散发性肾母细胞瘤中是罕见的。我们已经证实了以前的数据表明，β-连环蛋白（CTNNB 1），作为一个致癌基因在许多最常见的人类恶性肿瘤的Wnt信号通路的组成部分，突变仅限于WT 1-null肿瘤。我们还发现，这些突变是惊人的聚集在密码子Ser 45。通过表达谱分析，我们已经确定了一组区分WT 1-null和WT 1-阳性肿瘤的基因。这个数据集应该是一个强大的工具，以确定WT 1和β-catenin/TCF，我们假设这些差异表达的基因中富集的下游目标。目标1。我们将确定Wnt/β-catenin信号轴是否在WT 1- null类Wilms肿瘤中普遍激活。目标2.为了缩小WT 1和β-连环蛋白靶基因的范围，我们将在组织培养中操纵WT 1水平和β-连环蛋白通路的组分，并将结果与我们的原发性Wilms肿瘤的“金标准”数据进行比较。目标3.为了在体内验证候选的β-连环蛋白靶基因，我们将使用基因敲入方法将突变的β-连环蛋白表达到发育中的肾脏。通过建立一个同基因系列，这些小鼠也将允许我们测定Ser 45突变相对于其他磷酸化位点在影响β-连环蛋白增殖/致癌效力方面的相对效力。我们随后将这些小鼠与Wt 1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.