木犀草素靶向调控Hippo信号通路抑制三阴性乳腺癌转移的作用及机制研究

Triple negative breast cancer (TNBC) is characterized by strong invasion, distant metastasis and poor prognosis, which has become a difficult part in breast cancer research. Recent studies have confirmed that the Hippo signaling pathway is the core pathway for the regulation of breast cancer metastasis, but the mechanism of YAP/TAZ activities has not been fully elucidated, and fewer drugs are used for intervention. In our previous study, we have established a small molecular screening platform with detection of YAP/TAZ luciferase reporter activity, and find that luteolin can inhibit YAP/TAZ activity. Further studies demonstrate that it can effectively inhibit the transcriptional activity of YAP/TAZ, reduce the expression of its downstream genes and inhibit the migration of TNBC cell. We also found that the expression of miR-4269 was increased after TAZ knockout in TNBC cells. This aim of this project is to further verify the role of luteolin in inhibiting invasion and metastasis of TNBC through a variety of TNBC cell models and mice in situ transplantation, to identify and verify the molecular targets of luteolin regulating Hippo signaling pathway, and to explore the role of miR-4269 in YAP/TAZ regulated breast cancer metastasis. The project will provide a potential target in the downstream pathway of YAP/TAZ, and provide a reference for the drug development based on Hippo pathway.

三阴性乳腺癌（TNBC）具有侵袭力强、远处转移风险大和预后差等特点，成为乳腺癌研究的难点。近期研究证实Hippo-YAP/TAZ通路是调控乳腺癌转移的核心通路，但YAP/TAZ通过何种途径调控乳腺癌的转移尚未完全阐明，以其为干预靶点的药物较少。针对上述问题，我们建立了以YAP/TAZ报告基因检测为核心技术的药物筛选平台，首次发现木犀草素能够抑制YAP/TAZ的活性，降低YAP/TAZ下游基因的表达，抑制TNBC细胞迁移；并且证明TAZ基因敲除能够降低TNBC细胞的迁移能力，增加miR-4269的表达水平。本项目拟进一步观察木犀草素抑制TNBC细胞和小鼠原位移植瘤侵袭转移的作用，阐明木犀草素调控Hippo通路的作用机制，探讨miR-4269在YAP/TAZ调控TNBC细胞转移中的作用。本研究有望发现YAP/TAZ下游的药物作用靶点，为基于Hippo通路抗TNBC转移的药物研发提供参考。

YAP/TAZ在三阴性乳腺癌（TNBC）中存在高表达和过度激活，与TNBC的化疗耐药、肿瘤干细胞样特性的维持、上皮细胞间质转化 (EMT) 和转移密切相关。因此，阐明YAP/TAZ促进TNBC的EMT和转移的分子机制和开发靶向YAP/TAZ的抑制剂对TNBC的治疗具有重要意义。我们前期研究首次发现木犀草素能够有效抑制YAP/TAZ的转录活性。本课题组在此基础上开展相关实验，在细胞和动物水平上验证木犀草素抑制TNBC的增殖、EMT和侵袭转移的作用，发现木犀草素调控Hippo-YAP/TAZ信号通路的靶点，探索TAZ促进TNBC的EMT和转移的分子机制，获得以下结果：（1）木犀草素抑制TNBC细胞的增殖；（2）木犀草素降低TNBC细胞中YAP/TAZ的核内转移和下游基因CTGF和CYR61的表达；（3）木犀草素促进YAP/TAZ的磷酸化和降解，蛋白酶抑制剂MG132能够部分逆转木犀草素诱导的YAP/TAZ降解；（4）通过建立稳定表达TAZ-S89A的TNBC细胞系和利用CRISPR-Cas9方法敲除TAZ的表达，证明TAZ介导木犀草素抑制TNBC细胞增殖的作用；（5）木犀草素抑制TNBC细胞的EMT和迁移；（6）木犀草素能够在体内水平上抑制移植瘤的生长和降低YAP/TAZ的表达；（7）TAZ调控EMT和转移相关的miRNAs，包括miR-200c/141簇和miR-4269；（8）木犀草素在TNBC中增加miR-200c/141簇和miR-4269的表达水平。我们的研究成果提示木犀草素通过调控YAP/TAZ信号轴抑制TNBC的增殖和转移。本项目的研究结果为木犀草素的临床应用提供理论依据，为基于Hippo-YAP/TAZ信号通路的抗肿瘤药物研发提供参考。