Pygo2在TGF-β信号刺激的乳腺癌上皮-间质转化（EMT）形成中的作用机制研究

Pygo2 is a newly found β-catenin associated protein, which is essential for Wnt signaling transduction in both drosophila and some of mammalian tissues. Our published study demonstrated that Pygo2 plays a pivotal role in expansion of breast cancer stem cells; however, weather Pygo2 regulates the invasion and metastasis of breast cancer is still obscure. Our previous study of this proposal showed that Pygo2 expression is positively correlated with the cancer progression. Moreover, the cancer/pericancerous tissue ratio for Pygo2 protein expression is much higher than that of mRNA. Pygo2 protein but not mRNA level presented time and dose dependent increase upon TGF-β stimulation. Further studies indicated that LY294002, the PI3K-Akt specific inhibitor could effectively inhibit the TGF-β function in Pygo2 expression improvement and phosphorylation, and impair the association between Pygo2 and MLL2-HMT or GCN5-HAT. These results suggest that TGF-β signaling may improve the Pygo2 expression as well as the binding ability between Pygo2 and the downstream proteins via the PI3K-Akt phosphorylation. Knockdown of Pygo2 expression in breast cancer cells resulted in impaired the changes in cell morphology, migration, invasion and EMT markers stimulated by TGF-β, which suggests that Pygo2 is involved in TGF-β signaling mediated EMT process. In this proposal, we will at first demonstrate the upstream and downstream mechanisms for Pygo2 in regulating EMT of breast cancer cells in both molecular and cellular levels. Then, we will perform the in vivo study of Pygo2 in regulating EMT process by virtue of transgenic /knockout mouse models. Finally, we will analyze the clinical samples to establish the correlation between Pygo2 and cancer progression.

Pygo2是新近发现的β-catenin下游结合蛋白，在果蝇和部分哺乳动物组织的Wnt信号转导中不可缺少。我们先前的研究证明Pygo2在扩增乳腺癌干细胞中发挥重要作用，然而Pygo2是否调节乳腺癌侵袭、转移仍然不清楚。本项目前期实验发现， Pygo2表达与肿瘤进展正相关，TGF-β信号可以通过PI3K-Akt磷酸化增强Pygo2的蛋白表达以及与甲基转移酶MLL2-HMT、乙酰基转移酶GCN5-HAT的结合能力。在乳腺癌细胞中knockdown Pygo2的表达，能明显削弱TGF-β信号刺激引起的细胞形态、侵袭迁移能力以及EMT标志物的变化，表明Pygo2参与了TGF-β信号介导的EMT过程。本课题将首先在分子和细胞水平证明Pygo2调节乳腺癌 EMT的作用，然后应用转基因/基因敲除小鼠体内研究Pygo2调节乳腺癌 EMT的作用，最后分析临床标本建立Pygo2与肿瘤进展的相关性。

肿瘤的侵袭转移是最终引起病人死亡的主要原因，而EMT在肿瘤的侵袭过程中发挥了重要作用。TGFβ信号通路和Wnt信号通路是体内诱导EMT的重要元素。Pygo2 蛋白是Wnt信号通路的重要成员，通过Bcl9蛋白与β-Catenin蛋白结合，在扩增乳腺癌干细胞中发挥重要作用。本课题研究发现在乳腺癌侵袭与转移过程中Pygo2同样发挥重要作用。（1）在临床乳腺癌样本中，Pygo2表达水平与肿瘤进展正相关。Pygo2表达水平高的ER-PR-乳腺癌患者预后比Pygo2表达水平低的ER-PR-患者差，并且Pygo2表达水平与乳腺癌分级有相关性，在III、IV级乳腺癌样本中Pygo2表达高于癌旁的比例高于I、II级乳腺癌。（2）分子水平的研究发现，TGFβ对pygo 2蛋白水平的调控具有时间与剂量依赖性。Pygo2在上游接受TGFβ信号调节，下游介导Wnt信号通路，初步阐明了Pygo2的作用机理。TGFβ刺激细胞能够引起Pygo2蛋白N端、C端Akt底物序列磷酸化，蛋白稳定性增加，表达水平升高，磷酸化的Pygo2与GCN5、MLL2等蛋白复合体的结合能力增强，上调Wnt信号通路，增强Snail、Slug、Axin2等下游基因转录表达，促进细胞发生EMT，间质细胞标志蛋白Vimentin、Cox2表达增强，上皮细胞标志蛋白E-cadherin成熟体表达下降。（3）细胞水平的研究发现，TGFβ刺激细胞能够引起细胞发生上皮间质转化（EMT），细胞形态发生改变，敲减Pygo2后细胞形态改变消失。通过划痕愈合实验与Transwell小室迁移实验，证实TGFβ刺激能够促进乳腺癌细胞侵袭转移，磷酸化的Pygo2促进细胞侵袭转移的能力增强。通过本课题研究，确定pygo2在乳腺癌侵袭转移中的作用，对于寻找新的有效药物或干预靶点、提高乳腺癌治疗效果、改善病人生活质量、以及提高我国在肿瘤治疗领域的水平都有着重要的社会意义和经济意义。