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

 描述(申请人提供)：β-catenin是Wnt信号通路的主要下游效应器。WNT信号抑制β-连环蛋白降解复合体稳定β-连环蛋白，进而抑制其核转位。在细胞核中，β-连环蛋白主要作为T细胞因子家族转录因子的共激活因子。据报道，β-连环蛋白与其他关键信号通路的主要效应者之间存在显著的相互作用，这些信号通路揭示了一个复杂的相互作用组，这可能会对人类健康和疾病产生重大影响。肝脏中的一个这样的相互作用是与河马信号组件-YAP(YAP)的相互作用。YAP和β-连环蛋白一样，是一种转录共激活因子，可以调节TE结构域(TEAD)转录因子的活性。虽然这两条途径对调节细胞命运和增殖都很关键，但我们在最常见的儿科肝肿瘤或肝母细胞瘤(HB)中发现了β-连环蛋白-YAP的关联。Hb编码β-连环蛋白(CTNNB1)基因的外显子-3缺失，是一种截短的稳定蛋白，在大多数Hb中被观察到为β-连环蛋白的核易位。有趣的是，我们鉴定了人类Hb样本，同时也显示了核YAP。在人类HB细胞系中的进一步鉴定证实了这两个分子在细胞增殖和存活方面的协同作用。利用睡美人(SB)转座子/转座酶在肝脏中共表达YAP和β-catenin，在注射后4周内产生强劲的Hb，导致在几周内显著死亡。这个肿瘤模型给我们提供了一个独特的机会来解决β-连环蛋白和YAP蛋白在‘肝母细胞吻合’中的复杂相互作用。我们建议表征这两个分子之间的蛋白质-蛋白质相互作用及其转录因子，并阐明下游信号，这是Hb启动和发展所必需的。同时，这些研究将使我们能够利用这些相互作用并针对基因进行精确治疗，以对抗HB，一种缺乏分子治疗的肿瘤。我们将研究这样的假设，即YAP和β-catenin与它们各自的转录因子形成一个功能相互作用的组，进而激活一个独特的转录组，对Hb的发育和生长至关重要。我们建议从三个特定的目的来全面阐明这些分子机制。目的1将确定YAP-β-连环蛋白相互作用的生化和遗传基础，该相互作用导致小鼠和患者发生乙肝。分析还将确定Tcf和TEAD等规范转录因子的作用，以及最近发现的和及时的因子，如Tbx5。在目标2中，我们将确定在小鼠和患者中导致Hb的YAP-β-Catenin的下游靶点。这一分析将再次确定yap-β-catenin的表达如何迫使肝细胞重新编程为肝母细胞的分子基础，然后确定c-myc等已知靶点与那些通过电子显微镜和基因扫描方法无偏倚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YAP治疗HB的治疗靶向的效果以及这些观察的分子基础。因此，这项建议将是一项全面的调查，以确定主要的儿科肝脏肿瘤的分子基础，并必然具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.