Yap and beta-catenin interactions in liver: Implications in Pathophysiology

 描述（由申请人提供）：β-连环蛋白是Wnt信号通路的主要下游效应物。Wnt信号抑制β-catenin降解复合物以稳定β-catenin，随后使其核转位。在细胞核中，β-catenin主要作为T细胞因子（TCF）家族转录因子的共激活因子起作用。现在已经报道了β-连环蛋白与其他关键信号通路的主要效应物的显著相互作用，其揭示了复杂的相互作用组，这可能对人类健康和疾病产生重大影响。肝脏中的一种这样的相互作用是与Hippo信号传导组分- Yes Associated Protein-1（雅普）的相互作用。雅普与β-catenin一样，是一种转录辅激活因子，可调节TE结构域（TEAD）转录因子的活性。虽然这两种途径对于调节细胞命运和增殖都是至关重要的，但我们在最常见的儿科肝脏肿瘤或肝母细胞瘤（HB）中对β-连环蛋白-Yap的相关性进行了有趣的观察。HB典型地在编码β-连环蛋白的基因（CTNNB 1）中具有外显子3缺失，从而呈现截短的稳定蛋白，并且在大多数HB中观察到β-连环蛋白的核易位。有趣的是，我们鉴定出人HB样品也同时显示核雅普。在人HB细胞系中的进一步表征鉴定了两种分子在细胞增殖和存活中的协同作用。使用睡美人（SB）转座子/转座酶在肝脏中共表达雅普和β-连环蛋白，在注射后4周内导致稳健的HB，其在几周内导致显著死亡。这种肿瘤模型为我们提供了一个独特的机会来解决β-连环蛋白和雅普蛋白在“肝母细胞瘤发生”中的复杂相互作用。我们建议表征两个分子之间的蛋白质-蛋白质相互作用沿着其转录因子以及阐明下游信号传导，这是HB启动和进展所必需的。同时，这些研究将使我们能够利用这些相互作用和靶基因来精确治疗HB，这是一种缺乏分子疗法的肿瘤。我们将研究以下假设：雅普和β-catenin沿着与它们各自的转录因子一起形成功能性相互作用组，进而激活独特的转录组，这对HB的发育和生长至关重要。我们建议全面阐明这些分子机制的3个具体目标。目的1将确定雅普-β-连环蛋白相互作用的生化和遗传基础，导致小鼠和患者的HB发展。该分析还将确定典型转录因子如TCF和TEAD以及最近鉴定的及时因子如Tbx 5的作用。在目标2中，我们将鉴定导致小鼠和患者中HB的雅普-β-连环蛋白的下游靶标。该分析将再次确定雅普-β-连环蛋白表达如何迫使肝细胞重编程为肝母细胞的分子基础，然后确定已知靶点如c-myc和通过无偏的计算机和基因阵列方法鉴定的靶点的相关性。最终，目标3将集中于确定β-连环蛋白和/或雅普治疗HB的治疗靶向效果沿着这些观察结果的分子基础。因此，这一建议将是一个全面的调查，以表征领先的儿科肝脏肿瘤的分子基础，必将有重大的生物学和治疗意义。

 DESCRIPTION (provided by applicant): β-Catenin is a chief downstream effector of the Wnt signaling pathway. Wnt signals inhibit β-catenin degradation complex to stabilize β-catenin followed by its nuclear translocation. In the nucleus, β-catenin acts chiefly as a co-activator fo T cell factor (TCF) family of transcription factors. Notable interactions of β- catenin have now bee reported with major effectors of other key signaling pathways that divulges a complex interactome, which could have significant repercussions in human health and disease. One such interaction in the liver is that with the Hippo signaling component- Yes Associated Protein-1 (Yap). Yap, like β-catenin, is a transcriptional co-activator, which can regulate activity of TE domain (TEAD) transcription factors. While both pathways are critical for regulating cell fate and proliferation, we made an intriguing observation of β-catenin-Yap association in the commonest pediatric liver tumor or hepatoblastoma (HB). HB classically harbors exon-3 deletions in gene encoding β-catenin (CTNNB1) rendering a truncated stable protein and is observed as nuclear translocation of β-catenin in majority of HB. Interestingly, we identified human HB samples to also simultaneously show nuclear Yap as well. Further characterization in human HB cell lines identified a synergy between the two molecules in cell proliferation and survival. Co- expression of Yap and β-catenin in liver using sleeping beauty (SB) transposon/transposase, led to robust HB within 4 weeks after injection that led to significant mortality in few weeks. This tumor model allows us a unique opportunity to address the complex interplay between β-catenin and Yap proteins in `hepatoblastomagenesis'. We propose to characterize protein-protein interactions between the two molecules along with their transcription factors as well as elucidate downstream signaling, which is required for HB initiation and progression. Simultaneously, these studies will allow us to exploit these interactions and target genes for precision therapies against HB, a tumor which lacks molecular therapies. We will investigate the hypothesize that Yap and β-catenin along with their respective transcription factors form a functional interactome to in turn activate a unique transcriptome, critical to development and growth of HB. We propose to comprehensively elucidate these molecular mechanisms in the 3 specific aims. Aim 1 will determine the biochemical and genetic basis of Yap-β-catenin interactions that lead to HB development in mice and patients. The analysis will also determine role of canonical transcription factors such as TCF and TEAD as well as recently identified and timely factors such as Tbx5. In Aim 2 we will identify downstream targets of Yap-β-catenin that lead to HB in mice and patients. This analysis will again determine the molecular basis of how Yap-β-catenin expression coerces hepatocyte reprogramming to hepatoblast and then determine the relevance of known targets like c-myc and those identified through unbiased in silico and genearray approaches. Eventually, Aim 3 will be focused at determining the effect of therapeutic targeting of β-catenin and/or Yap treatment of HB along with the molecular basis of these observations. Thus, this proposal will be a comprehensive investigation to characterize the molecular basis of the leading pediatric liver tumor, and is bound to have significant biological and therapeutic implications.