YAP1亚型的鉴定及其在胰腺癌细胞相关上皮-间质转化过程中的作用和机制研究

YAP1 is a key effector protein, which functions downstream of Hippo signaling pathway. It has been reported that YAP1 participates in the process of tumor Epithelial-Mesenchymal Transitions (EMT), but it remains largely unknown as how YAP1 mediate such an effect. Previous research has identified at least 8 YAP1 isoforms as the result of alternative mRNA splicing. Intriguingly, these isoforms differ in two highly conserved regions, the WW motif and the transcription activation domain. To date, the relative expression of these isoforms in pancreatic cancer as well as their role in EMT have not been investigated. In our preliminary study, we have successfully knocked out the endogenous YAP1 in a pancreatic cancer cell line using Crispr/Cas9 and found that the YAP1 knockout cells exhibited markedly reduced cell migration and invasion, unable to undergo EMT upon EMT-induction signal as in the wild type cells. Importantly, we also observed that the expression levels of the YAP1 isoforms in pancreatic cancer cells differ significantly from the normal pancreatic epithelial cells. The result of sequence analysis and preliminary experiments together led us to hypothesize that YAP1 plays an important role in pancreatic cancer EMT process, and that each isoform has unique activity and mechanism toward EMT regulation. The current proposal aims to address the function as well as the molecular mechanism underlying YAP1 regulation of EMT with a focus on cancer-related isoform. We will perform the experiments in both cell culture and animal xenograft models, and use state of art molecular biology as well as proteomics technologies. The successful completion of the project will illuminate the role of YAP1 in pancreatic cancer EMT process, and lay a foundation further therapeutic targeting of this pathway.

YAP1是Hippo信号通路下游最关键的效应蛋白。有研究表明，YAP1参与调节肿瘤细胞上皮-间质转化（EMT）过程，但相关机制仍不明确，在胰腺癌中则未见报道。前期工作发现，经Cas9敲除YAP1基因的胰腺癌细胞侵袭转移能力显著下降，且无法像野生型细胞一样被诱导出EMT。YAP1存在8个亚型，它们在功能结构域上存在显著差异。我们将克隆得到的8个亚型重组到YAP1敲除细胞中构建成YAP1单亚型稳定细胞株后，发现各亚型在调控肿瘤细胞EMT能力上存在差异。基于此，我们推断YAP1在胰腺癌EMT过程中起关键作用，而其各亚型在EMT过程中的作用及调节机制不尽相同。本课题拟通过分子生物学和蛋白组学等技术，结合体外细胞培养和动物实验研究YAP1各亚型的生物学特性及其在胰腺癌EMT过程中的独特作用和机制。本研究将从一个新的角度解析胰腺癌侵袭转移的调节机制，为针对Hippo-YAP1 通道的肿瘤治疗奠定基础。

YAP1是Hippo信号通路下游关键的效应蛋白，参与肿瘤多种恶性化进程。有研究表明，YAP1参与调节肿瘤细胞上皮-间质转化（EMT）过程，但相关机制仍不明确，在胰腺癌中则未见报道。YAP1存在8个亚型，它们在介导蛋白相互作用的WW结构域和介导转录激活的TA结构域上有显著差异。但是没有关于YAP1各亚型功能和调控的深入研究。在本项目中，我们将克隆得到的8个亚型YAP1重组到其内源敲除细胞中构建成YAP1单亚型稳定细胞株，在此基础上研究YAP1各亚型调控和功能的差异。我们结果表明，胰腺癌和肺癌中均存在8个亚型的表达，并且含有两个WW结构域的YAP1-2的mRNA水平高于YAP1-1。但是在TA区域存在差异的α，β，γ，δ亚型之间没有显著规律。我们进一步对各亚型功能和调控研究发现，在TA区域含有VRPQ四个氨基酸插入的β和δ亚型的转录激活活性要低于不含这个片段插入的α和γ亚型。而含有16个氨基酸片段插入的γ和δ亚型的转录激活活性相对于不含这个片段插入的α和β没有显著区别。含有两个WW结构域的YAP1-2由于和LATS1，AMOT，PTPN14等YAP1负调因子的结合活性强于YAP1-1，导致YAP1-2对细胞接触抑制更为敏感，更容易被泛素化降解。因此虽然YAP1-2的mRNA水平较高，但是在实体瘤阶段，YAP1-2的蛋白被大量降解或被磷酸化阻滞在细胞浆中，无法入核发挥其转录辅助因子功能。因此，YAP1-1是实体瘤阶段促进肿瘤恶性化进程的主要YAP1亚型，对胰腺癌细胞增殖、转移和干性的促进作用都强于YAP1-2。但是我们进一步研究发现，在诱导肿瘤发生上皮-间质转化（EMT）过程中，YAP1-2的稳定性和核转移活性提高，并且发挥出比YAP1-1更强的促肿瘤恶性化的作用。我们进一步研究发现YAP1表达密切相关的核转录因子SOX2在促进胰腺癌干性中起着重要作用。并且SOX2的蛋白表达和稳定性受FGF/FGFR信号通路调控。而根据我们的研究结果，该通路也是调控YAP1-2蛋白稳定性，并促进其功能的重要信号通路。这揭示了YAP1和SOX2协同作用，在促胰腺癌恶性化进程中发挥着重要功能。本项目首次阐明了YAP1各亚型分子调控和促肿瘤恶性化进程中的差异，为YAP1的研究开辟了新方向。