Signaling cascades in cholangiocarcinoma development

 描述(申请人提供)：肝内胆管细胞癌(ICC)是一种罕见但致命的肝癌。除了只能对少数患者进行手术切除外，对于ICC还没有有效的治疗方法。显然，迫切需要开发新的治疗策略来对抗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Notch1和Notch2，在大多数人ICC样本中都高表达。γ分泌酶抑制剂(GSI)或用分子方法抑制Notch能抑制体外培养的人乳腺癌细胞增殖并诱导其凋亡。从机制上讲，我们证明了Notch对MAPK信号的调节作用。对于河马途径，我们在人和小鼠的ICC样本中发现了河马肿瘤抑制蛋白激酶下游的转录激活因子YAP和TAZ的强烈且普遍存在的激活。在体外，过量表达Lats2或通过显性阴性形式TEAD2(DnTEAD2)阻断YAP/TAZ活性上调河马级联反应强烈地抑制了ICC细胞的生长。当YAP或TAZ在人ICC细胞中的表达被沉默时，也得到了类似的结果。此外，Lats2或dnTEAD2的过表达显著延缓了Pten和TP53肿瘤抑制基因缺失(sgPten/sgP53)诱导的ICC的发生。在体内，我们发现激活形式的YAP(YapS127A)或TAZ(TAZS89A)与激活的AKT信号协同促进ICC的发展。由激活的AKT和YAP/TAZ驱动的ICC依赖于Notch，因为阻断规范的Notch强烈抑制了小鼠ICC的发展。在这些初步研究的基础上，我们假设通过Notch1和/或Notch2调控的典型Notch通路是通过调节MAPK级联而发生ICC所必需的。我们进一步假设，YAP和TAZ是河马肿瘤抑制因子下游的主要转录激活因子，它们在ICC的发展中既有重叠又有不同的作用，阐明这些作用将是利用这一途径的调节来实现翻译含义的关键。两个目标 来检验这些假说。在目标1中，我们将阐明ICC发育过程中典型的Notch信号级联。在目标2中，我们将确定YAP和TAZ在调控ICC发病中的功能贡献。总之，在拟议的应用中，我们将应用体外机制研究与复杂的小鼠遗传学方法相结合，以揭示Notch和河马级联调控ICC肿瘤发生的分子机制。这项研究可能会为开发和应用基于抗Notch和/或YAP/TAZ的疗法治疗ICC提供强有力的证据。基于初步数据的质量和所有必要的体外和体内平衡研究试剂的可用性，我们相信我们将能够解决这些生物学途径在肝脏病理中的重要性，最终设计出更好的理解和治疗ICC的方法。

 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.