FoxM1调控TGF-β1/Smad信号通路促进乳腺癌EMT机制研究

Epithelial mesenchymal transition(EMT) is a key process of cancer metastasis. TGF-β1/Smad plays a critical role in promotion cancer cell acquisition of EMT phenotype. Recently, studies have shown that FoxM1 also relates to the cancer EMT, but the mechanism is unclear. Our previous studies have shown that FoxM1 highly expresses in breast cancer and can promote acquisition of EMT phenotype in breast cancer cells. Further studies showed that FoxM1 can combine with Smad3 in vitro and in vivo, a key element of TGF-β1/Smad signaling pathway. Knock-down of FoxM1 can stop Smad3 nuclear translocation caused by the activation of TGF-β1 pathway. Those results mean that FoxM1 promotes EMT of breast cancer by regulating TGF-β1/Smad signaling pathway. In this program,firstly, we will study that alteration of FoxM1 affects TGF-β1/Smad transcriptional activities with the methods of gene over-expression or knock-down. Secondly, by using Smad3 deletion mutants or FoxM1 deletion mutants, we will test which the domains of FoxM1 or Smad3 are needed for the binding of FoxM1 to Smad3. By using Co-IP, we will test the way of FoxM1 modifying Smad3 nuclear translocation. Finally, we will try to demonstrate the relationship between FoxM1/Smad and Slug, a key factor of EMT. Based on those studies, we will try to understand the mechanism of FoxM1 regulating TGF-β1/Smad signaling pathway and promoting breast cancer cells EMT.

上皮间质转化（EMT）是肿瘤转移的关键。TGF-β1/Smad是目前已知最重要的促EMT信号通路。最近的研究证实FoxM1也可致肿瘤EMT，机制不明。本课题组前期研究证实FoxM1在乳腺癌组织中高表达并可致乳腺癌细胞EMT，进一步观察到FoxM1可与Smad3结合，敲除FoxM1可抑制TGF-β1激活所致的Smad3入核，表明FoxM1可能通过调控TGF-β1/Smad信号通路促进EMT。为此，本课题拟采用基因过表达和敲除技术观察FoxM1对TGF-β1/Smad转录活性影响；构建FoxM1和Smad3不同突变体探讨它们之间的关键结合位点；采用Co-IP、GST pulldown等观察FoxM1对Smad3核转位的作用；采用ChIP等技术对FoxM1/Smad3和EMT调控因子Slug的关联进行探索。通过上述研究，阐明FoxM1调控TGF-β1/Smad机制及促EMT的作用。

TGF-β1/Smad信号通路在多种发育过程和包括肿瘤在内的疾病发生中发挥重要作用。该通路中SMAD蛋白的持续激活是该信号通路的一个主要特征。上皮间质转化（EMT）是肿瘤转移的关键。TGF-β1/Smad 是目前已知最重要的促EMT信号通路。本项目中，我们通过细胞功能实验和分子生物学手段阐述了癌基因FoxM1和TGF-β1/Smad信号通路以及EMT之间的调控关系，揭示了一种崭新的调控网络。FoxM1的高表达和低表达促进乳腺癌细胞侵袭转移，并且FoxM1高表达促进乳腺癌细胞EMT的发生，使E-cadherin降低而Vimentin升高。在TGF刺激下，我们发现FoxM1可与Smad3结合，敲除FoxM1 可抑制TGF-β1 激活所致的Smad3入核，表明FoxM1 可能通过调控TGF-β1/Smad 信号通路促进EMT。接着通过构建FoxM1和Smad3不同突变体探讨它们之间的关键结合位点，实验结果显示SMAD3的C端和FoxM1的N端在它们结合时起关键作用；此外，我们还发现FoxM1通过直接结合Slug启动子调控其转录，进而调控EMT的发生。本项目对TGF-β1/Smad 信号转导通路的研究将有助于我们深入了解该信号通路与肿瘤EMT的关系，为制定和设计针对该信号通路的靶点药物提供理论依据。