Wnt/β-catenin信号转导通路的激活诱导细胞死亡的机制研究

Wnt/β-catenin signaling transduction pathway regulates a multitude of cellular processes including cell proliferation, differentiation, migration and apoptosis through mediating the activity of β-catenin. The pathway is one of the most fundamental pathways that can direct embryonic development as well as maintenance of tissue homeostasis. Aberrant activation of Wnt/β-catenin signaling can promote stem cell proliferation and self-renewal, which is associated with the formation and development of many types of cancer. Wnt/β-catenin signaling pathway is generally considered as oncogenic. However, our preliminary studies found out that activation of Wnt/β-catenin signaling in glioma stem cell (GSC) isolated from mouse malignant glioma could lead to cellular death, the underlying mechanisms of which is unclear. In this project, we will use in vitro cultured glioma stem cells to identify the possible downstream factors and signaling pathways involved in Wnt/β-catenin signaling induced GSC death through a serious of biochemistry and molecular biology methods such as lentivirus mediated gene manipulation, gene expression data analysis, real-time qPCR (RT-qPCR). The completion of this project will provide us with new insights into the comprehensive understanding of the complex roles of Wnt/β-catenin signaling pathway played in cancer and the development of new strategies targeting Wnt/β-catenin signaling pathway in the treatment of cancer. ..

Wnt/β-catenin信号转导通路通过调节β-catenin的活性来调控细胞增殖、分化、迁移、凋亡等生命过程，是生物体正常发育和组织稳定维持的重要调节机制之一。Wnt/β-catenin信号的异常激活能促进干细胞的增殖与更新，与多种癌症的发生、发展有关，因而被认为是一条促癌的信号转导通路。然而，我们前期的研究工作却发现，在小鼠神经胶质瘤中分离得到的胶质瘤干细胞（GSC）中，激活Wnt/β-catenin信号却能诱导GSC的死亡，其作用机制有待进一步研究。本项目将以体外培养的胶质瘤干细胞为研究对象，通过慢病毒载体介导的基因操作、基因表达芯片分析、荧光定量PCR等一系列生化与分子生物学实验方法，系统探索Wnt/β-catenin信号激活诱导GSC死亡的下游调控因子与可能的信号通路，为全面认识Wnt/β-catenin信号通路在癌症中复杂的作用机制和以此为靶点的抗癌药物的研发提供新的策略。

胶质瘤是中枢神经系统中最常见并且死亡率最高的恶性肿瘤。具有自我更新能力的胶质瘤干细胞的存在被认为是胶质瘤对化疗和放疗产生抗性和复发的根源。因而有针对性的消除胶质瘤干细胞成为治疗胶质瘤的有效途径。本研究发现一种GSK3抑制剂CHIR可以抑制胶质瘤干细胞的生长， EdU细胞增殖实验和AnnexinV-PI染色结合流式分析证明CHIR可以抑制胶质瘤干细胞的增殖和诱导胶质瘤干细胞的凋亡。过表达constitutively active β-catenin的胶质瘤干细胞对CHIR更加敏感，而过表达dominant negative TCF的胶质瘤干细胞对CHIR不敏感，证明CHIR是通过激活Wnt/β-catenin信号通路来抑制胶质瘤干细胞的生长。进一步通过RNA-seq分析发现Wnt/β-catenin信号通路的激活可以激活Hippo-Yap信号通路。Western blot分析发现 Wnt/β-catenin信号通路可以磷酸化Hippo-Yap信号通路的核心激酶Lats，磷酸化的Lats可以进一步磷酸化下游的Yap并导致Yap蛋白水平的下降。沉默了Yap的胶质瘤干细胞生长明显变慢，并且增加了对化疗药物carboplatin和5FU的敏感性。同时，沉默Lats1或者过表达激活形式的YapS127A可以使得胶质瘤干细胞对CHIR的敏感度降低，说明抑制Hippo-Yap信号通路可以减慢胶质瘤干细胞的生长并且减弱CHIR对胶质瘤干细胞生长的抑制作用。本研究的发现将为治疗胶质瘤提供新的可能的药物开发靶点。