YAP Ser403特异磷酸酶的鉴定、功能解析及化学干预

Activation of YAP/TAZ is critical to cancer cell growth, stemness maintenance, metastasis and drug-resistance. Our previous study found that IKKε could phosphorylate YAP at Ser403 and target it for lysosomal degradation. Further investigation revealed that YAP Ser403 phosphorylation is undetectable in aggressive breast cancer cells but becomes obvious when the cells are treated with broad-spectrum phosphatase inhibitor, suggesting that there should be endogenous phosphatase that could specifically remove YAP S403 phosphorylation thus block lysosome-mediated degradation of YAP. Given this hypothesis, we aim to identify such phosphatase and examine its tumor promoting function and the underlying mechanisms. Moreover, we also aim to develop high throughput screening system by reading phosphotase activity in vitro. Our preliminary works have identified the phosphates and the potent lead compound that could suppress its activity. We will further explore whether it could be a good strategy to control YAP expression and YAP-mediated tumor development. Upon successful conclusion of this work, we will have provided new directions and references for tumor therapy.

Hippo信号通路中YAP的激活是促进肿瘤细胞生长和增殖、维持肿瘤细胞干性、增强肿瘤细胞转移和抗药能力的关键因素。我们前期的研究发现YAP的Ser403位点能被IKKε磷酸化，该磷酸化能不依赖Hippo/Lats促进YAP经溶酶体降解。进一步我们发现，YAP Ser403位磷酸化修饰在恶性乳腺癌细胞中被抑制在极低水平，而在经过广谱磷酸酶处理后则得到了显著增加。由此我们推断恶性肿瘤细胞中存在YAP Ser403特异性磷酸酶阻断了YAP的溶酶体降解。因此本课题旨在：鉴定特异去除YAP Ser403位磷酸化的磷酸酶；在细胞及动物水平阐述其在肿瘤发生发展中的功能及相应的分子机制；以该磷酸酶为靶标搭建小分子药物筛选平台，筛选靶向该磷酸酶的小分子抑制剂、获取先导化合物，以期找到能控制YAP表达水平及肿瘤发展的药物，为肿瘤靶向治疗提供新的方向和理论依据。

Hippo信号通路中YAP的激活是促进肿瘤细胞生长和增殖、维持肿瘤细胞干性、增强肿瘤细胞转移和抗药能力的关键因素。我们前期的研究发现YAP的Ser403位点能被IKKε磷酸化，该磷酸化能不依赖Hippo/Lats促进YAP经溶酶体降解。进一步我们发现，YAP Ser403位磷酸化修饰在恶性乳腺癌细胞中被抑制在极低水平，而在经过广谱磷酸酶处理后则得到了显著增加。由此我们推断恶性肿瘤细胞中存在YAP Ser403特异性磷酸酶阻断了YAP的溶酶体降解。本项目在此基础上鉴定了特异去除YAP Ser403位磷酸化的磷酸酶PPM1B；临床乳腺癌数据库的分析表明PPM1B的mRNA表达水平与乳腺癌病人的存活率呈负相关。进一步我们构建了PPM1B条件敲除小鼠，在乳腺中特异敲除PPM1B并与乳腺癌自发肿的MMTV-PyMT转基因小鼠杂交，证实在乳腺中特异敲除PPM1B基因能抑制自发乳腺肿瘤的生成。同时我们也构建了PPM1B髓系敲除小鼠，证明该小鼠细胞中YAP表达减少，固有免疫响应增强并且小鼠的抗病毒能力增加。针对PPM1B与YAP相互作用的结构域分析，我们设计了靶向PPM1B的短肽，筛选得到有效的能抑制PPM1B与YAP相互作用，并增加YAP S403位磷酸化的短肽，用该短肽注射小鼠，可以有效抑制小鼠模型中肿瘤的生长。这些研究为乳腺癌的研究提供了新的视角，为乳腺癌治疗药物的开发提供了新的方向。