基于功能激酶组学的新型EMT调节分子的筛选验证及调节机制研究

The epithelial-mesenchymal transition (EMT) is closely associated with cancer metastasis. However, few EMT regulators can be used as cancer drug targets. Since kinases have been recognized as ideal drug targets, we carried out human kinome cDNA screens that surveyed 500 human kinases to identify novel EMT regulators. In this screen, we used vimentin promoter luciferase assay to identify potential EMT regulators, and then validated their EMT-inducing function by establishing stable cell lines. Furthermore, NCBI GO analysis uncovered that most effective EMT-inducing kinases were associated cell cycle regulation, cell metabolism, autophagy, or histione modification. According these results, we came up with a hypothesis explaining EMT of cancer cells: when cancer cells feel "hungry", they will fulfill their exuberant growth requirement by enhancing glycolysis, autophagy or promoting G0/G1 arrest, through which they get raw material, intermediate products, and best opportunity for anabolic metabolism. At the same time, the histone, the sensor of abnormal metabloites, could transfer the changes of cell metabolism into changes of some genes expression through reprogramming cell epigenetic modification. Therefore, cancer cells undergo EMT and spread into other organs with enough energy to furfill their strong requirement for energy. Through identifying novel EMT regulators from human kinome cDNA screens and uncovering their regulation mechanism, this study will open a new window to see EMT and provide new potential targets for anticancer drug development.

EMT与肿瘤转移密切相关。已知的EMT调节分子中，可作为药物靶标者为数甚少。激酶是理想的药物作用靶点，我们前期利用EMT标记物Vimentin的报告载体，从激酶组cDNA文库中筛选出新型EMT调节分子，之后构建稳定表达这些激酶的细胞株，验证其表型和功能，得到有效调节EMT激酶。通过NCBI GO数据库分析有效激酶的已知功能，发现大多与细胞周期、代谢、自噬和组蛋白修饰相关。据此，我们提出"能源"诉求驱动肿瘤细胞发生EMT的作用机制：当肿瘤细胞感知能源匮乏时，会通过增强糖酵解、增强自噬和使细胞阻滞于G0/G1期，为其合成代谢提供更多原料、中间产物和创造最佳时机。同时，组蛋白修饰酶作为传感器，将代谢产物的异常变化通过表观遗传重塑转化为基因表达的变化，使肿瘤细胞发生EMT，转移到能源丰富的器官。本课题将为全面理解肿瘤细胞EMT提供全新的视角和可靠的实验依据，为新型EMT调节激酶的药物研发奠定基础。

EMT与肿瘤转移、耐药，以及干细胞样特性都密切相关。已知的EMT调节分子中，可作为药物靶标者为数甚少。激酶是理想的药物作用靶点。为了寻找新型药物靶表型EMT调节分子，我们利用EMT标记物Vimentin的报告载体，从激酶组cDNA文库中筛选出新型EMT调节分子，进行一系列的表型和功能验证，得到有效调节EMT激酶CDKL2和ZAK。CDKL2和ZAK能够明显诱导EMT的表型及功能，增强细胞的“耐饥恶”和“耐药”的特性，并增强乳腺上皮细胞中干细胞亚群的比例和功能，使其表现出自我更新和多向分化的特性；另一方面，敲低ZAK，能够逆转EMT的表型及功能，削弱和延迟乳腺癌骨转移的发生发展。值得关注的是，临床数据分析均显示激酶ZAK高表达与乳腺癌患者的生存期缩短密切相关；ZAK可以作为一个独立的诊断指标预测患者的生存期，其预测准确性与ER、PR、HER2联合预测的准确性相当。ZAK有望作为三阴性乳腺癌的全新治疗靶点。.阐释“能源”诉求驱动肿瘤细胞发生EMT的作用机制：“能源”供应告急时，肿瘤细胞会穷尽一切手段，维持其生存：（1）增强糖酵解，为肿瘤细胞的合成代谢直接提供原料。（2）增强自噬，通过降解蛋白质和细胞器，为肿瘤细胞的合成代谢补给中间产物。（3）细胞周期G0/G1阻滞，为细胞合成代谢创造了最佳时机。而组蛋白作为细胞代谢的传感器，将细胞代谢变化通过表观遗传重塑转化为基因表达的变化，最终引起细胞表型和功能的重塑，包括耐饥饿、耐缺氧、获得迁移活性、获得干细胞样表型。因此，肿瘤细胞的EMT可以看作是为了应对“能源”诉求的一种全方位的细胞重塑过程。.针对激酶在体活性检测局限性的问题，设计构建特定EMT调节激酶活性的小动物成像系统，并将其应用于新药筛选。筛选到了有效的EMT抑制剂HYD-PEP06，经过系统和完整的药效和机理研究，HYD-PEP06已于2019年1月获CFDA批准正式进入临床研究。