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

 描述（由申请人提供）：β-Catenin 是 Wnt 信号通路的主要下游效应子。 Wnt 信号抑制 β-catenin 降解复合物以稳定 β-catenin，然后进行核转位。在细胞核中，β-连环蛋白主要充当转录因子 T 细胞因子 (TCF) 家族的共激活剂。据报道，β-连环蛋白与其他关键信号通路的主要效应子之间存在显着的相互作用，从而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。肝脏中的一种此类相互作用是与 Hippo 信号成分 - Yes Associated Protein-1 (Yap) 的相互作用。 Yap 与 β-catenin 一样，是一种转录辅激活因子，可以调节 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至关重要，但我们对最常见的儿科肝脏肿瘤或肝母细胞瘤 (HB) 中的 β-catenin-Yap 关联进行了有趣的观察。 HB 通常在编码 β-连环蛋白 (CTNNB1) 的基因中存在外显子 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，我们发现人类 HB 样本也同时显示出核 Yap。人类 HB 细胞系的进一步表征确定了这两种分子在细胞增殖和存活方面的协同作用。使用睡美人 (SB) 转座子/转座酶在肝脏中共表达 Yap 和 β-连环蛋白，在注射后 4 周内产生强烈的 HB，并在几周内导致显着的死亡率。这种肿瘤模型为我们提供了一个独特的机会来解决“肝母细胞瘤发生”中 β-连环蛋白和 Yap 蛋白之间复杂的相互作用。我们建议表征两个分子及其转录因子之间的蛋白质-蛋白质相互作用，并阐明 HB 启动和进展所需的下游信号传导。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针对 HB（一种缺乏分子疗法的肿瘤）进行精准治疗。我们将研究这样的假设：Yap 和 β-catenin 以及它们各自的转录因子形成一个功能性相互作用组，进而激活一个独特的转录组，这对 HB 的发育和生长至关重要。我们建议在三个具体目标中全面阐明这些分子机制。目标 1 将确定导致小鼠和患者发生 HB 的 Yap-β-连环蛋白相互作用的生化和遗传基础。该分析还将确定 TCF 和 TEAD 等经典转录因子以及 Tbx5 等最近发现的及时因子的作用。在目标 2 中，我们将确定在小鼠和患者中导致 HB 的 Yap-β-catenin 下游靶标。该分析将再次确定 Yap-β-连环蛋白表达如何强制肝细胞重编程为成肝细胞的分子基础，然后确定已知靶标（如 c-myc）以及通过公正的计算机和基因阵列方法鉴定的靶标的相关性。最终，目标 3 将集中于确定 β-catenin 治疗靶向和/或 Yap 治疗 HB 的效果以及这些观察结果的分子基础。因此，该提议将是一项全面的研究，旨在表征主要儿童肝脏肿瘤的分子基础，并且必将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.