Signaling cascades in cholangiocarcinoma development

 描述（由申请人提供）：肝内胆管细胞癌（ICC）是一种罕见但致命的肝癌。除了只能应用于少数患者的手术切除外，没有有效的治疗ICC的方法。有一个明确的迫切需要开发新的治疗策略对这种致命的恶性肿瘤。最近，我们和其他人证明了Notch和Hippo信号级联在ICC发病机制中的关键作用。对于Notch通路，我们发现Notch受体，包括Notch 1和Notch 2在大多数人ICC样品中高度表达。γ-分泌酶抑制剂（GSI）或Notch抑制剂在体外可抑制人ICC细胞增殖并诱导细胞凋亡。从机制上讲，我们表明Notch的功能是调节MAPK信号传导。对于Hippo通路，我们发现了人ICC和小鼠ICC样品中Hippo肿瘤抑制激酶下游的转录激活因子雅普和TAZ的强烈且普遍的激活。在体外，通过过表达Lats 2或通过显性负性形式的TEAD 2（dnTEAD 2）阻断雅普/TAZ活性来上调Hippo级联反应强烈抑制ICC细胞生长。当在人ICC细胞中沉默雅普或TAZ表达时，获得了类似的结果。此外，Lats 2或dnTEAD 2的过表达强烈延迟了由Pten和TP 53肿瘤抑制因子（sgPten/sgP 53）的缺失诱导的ICC发展。在体内，使用流体动力学转染，我们发现活化形式的雅普（YapS 127 A）或TAZ（TAZ 89 A）与活化的AKT信号传导协同促进ICC发育。由激活的AKT和雅普/TAZ驱动的ICC依赖于Notch，因为阻断经典Notch强烈抑制小鼠中的ICC发育。基于这些初步研究，我们假设经典的Notch途径介导的Notch 1和/或Notch 2是需要ICC的发展，通过调节MAPK级联。我们进一步假设，雅普和TAZ，主要的转录激活剂下游的河马肿瘤抑制激酶，有重叠，但不同的作用，在ICC的发展和阐明这些作用将是至关重要的，利用调制这一途径的翻译影响。两个目标 来检验这些假设。在目标1中，我们将阐明ICC发展中的经典Notch信号级联。在目标2中，我们将确定雅普和TAZ在调节ICC发病机制中的功能贡献。总之，在拟议的申请中，我们将应用体外机制研究结合复杂的小鼠遗传方法，以揭示Notch和Hippo级联在调节ICC肿瘤发生中的分子机制。该研究将可能提供强有力的证据来支持基于抗Notch和/或雅普/TAZ的治疗剂用于ICC治疗的开发和应用。基于初步数据的质量和平衡的体外和体内研究的所有必要试剂的可用性，我们相信我们将能够解决这些生物学途径在肝脏病理学中的重要性，最终设计出更好的理解和ICC的治疗方法。

 DESCRIPTION (provided by applicant): Intrahepatic cholangiocarcinoma (ICC) is a rare yet deadly form of liver cancer. Beyond surgical resection which can only be applied to a minority of the patients, there is no effective treatment for ICC. There is a clear urgent need to develop novel treatment strategies against this deadly malignancy. Recently, we and others demonstrated the critical role of Notch and Hippo signaling cascades in ICC pathogenesis. For the Notch pathway, we found that Notch receptors, including Notch1 and Notch2 are highly expressed in majorities of human ICC samples. Treatment of γ-secreatase inhibitors (GSI) or inhibition of Notch using molecular approaches is able to repress human ICC cell proliferation and induce apoptosis in vitro. Mechanistically, we showed that Notch functions to regulate MAPK signaling. For the Hippo pathway, we uncovered a strong and ubiquitous activation of Yap and TAZ, the transcriptional activators downstream of Hippo tumor suppressor kinases, in human ICC as well as mouse ICC samples. In vitro, upregulation of Hippo cascade by overexpressing Lats2 or blocking Yap/TAZ activity via dominant negative form of TEAD2 (dnTEAD2) strongly inhibited ICC cell growth. Similar results were obtained when Yap or TAZ expression was silenced in human ICC cells. Furthermore, overexpression of Lats2 or dnTEAD2 strongly delayed ICC development induced by loss of Pten and TP53 tumor suppressors (sgPten/sgP53). In vivo, using hydrodynamic transfection, we discovered that activated forms of Yap (YapS127A) or TAZ (TAZS89A) synergized with activated AKT signaling to promote ICC development. ICC driven by activated AKT and Yap/TAZ depends on Notch as blocking canonical Notch strongly inhibited ICC development in mice. Based on these preliminary studies, we hypothesize that the canonical Notch pathway medicated via Notch1 and/or Notch2 is required for ICC development via modulating MAPK cascade. We further hypothesize that Yap and TAZ, the major transcriptional activators downstream of Hippo tumor suppressor kinases, have overlapping yet distinct roles in ICC development and elucidation of these roles will be critical to exploit the modulation of this pathway for translational implications. Two aims are proposed to test these hypotheses. In Aim 1, we will elucidate the canonical Notch signaling cascade in ICC development. In Aim 2, we will define the functional contribution of Yap and TAZ in regulating ICC pathogenesis. Altogether, in the proposed application, we will apply in vitro mechanistic studies in combination with sophisticated mouse genetic approaches to uncover the molecular mechanisms underlying Notch and Hippo cascades in regulating ICC tumorigenesis. The study will likely provide strong evidence to support the development and application of anti-Notch and/or Yap/TAZ based therapeutics for ICC treatment. Based on the quality of preliminary data and availability of all necessary reagents for balanced in vitro and in vivo studies, we feel confident that we will be able to address the significance of these biological pathways in liver pathology to eventually devise better understanding and hence therapies for ICC.