DCLK1与K-Ras/MAPKs通路相互作用推动胰腺癌发生发展的机制研究

Two of the most important events of molecular mechanism in pancreatic ductal adenocarcinoma (PDAC) formation and progression are tumor stem cell marker Doublecortin-like kinase 1 (DCLK1) expression and K-Ras gene mutation followed by its downstream pathway activation. It’s reported that DCLK1 transcription of PDAC cell was up-regulated by K-Ras mutation and MAPKs pathway activation. Our preliminary study indicates that DCLK1 overexpression promotes PDAC cell functions such as proliferation and migration by K-Ras/MAPKs pathway activation. Structural models of DCLK1 and K-Ras deem a direct physical interaction likely. On the basis of these findings, it’s speculated that directly interaction of DCLK1 and K-Ras forms positive feedback loop between DCLK1 gene expression and K-Ras/MAPKs pathway activation. As a result, persistance of signalling transduction leads to proliferation disorder which will eventually develop into pancreatic cancer. Our hypothesis above will be verified from the following aspects: 1. Assess DCLK1 expression and K-Ras mutation of PDAC patients and correlate with cancer stage and survival. 2. Study direct interaction between DCLK1 and wild type or mutated K-Ras as well as its effect on downstream pathway. Investigate the mechanism of DCLK1 transcriptional up-regulation by K-Ras activating mutation. Evaluate the effects of DCLK1 expression on cell malignant biological behavior through K-Ras/MAPKs using in vitro assays. 3. Explore the effects of interaction between DCLK1 gene expression and K-Ras/MAPKs pathway activation on PDAC initiation in vivo.

肿瘤干细胞标志物DCLK1表达和K-Ras突变激活下游信号通路是驱动胰腺肿瘤形成的关键。业已报道，胰腺癌细胞K-Ras突变激活MAPKs通路，上调DCLK1转录。我们前期研究发现，胰腺癌细胞DCLK1过表达激活K-Ras/MAPKs通路，促进细胞增殖和迁移，蛋白质构象预测DCLK1和K-Ras可能存在结合位点。据此我们推测：DCLK1与K-Ras发生直接相互作用激活其下游信号通路，两者形成正反馈环路，细胞信号持续传导，推动肿瘤发生。本课题拟做如下验证：1.临床样本检测DCLK1表达和K-Ras突变，分析其临床意义；2.体外实验研究DCLK1和（野生/突变）K-Ras蛋白间的直接作用以及对下游通路的影响；明确K-Ras突变上调DCLK1转录的机制；阐明DCLK1对K-Ras/MAPKs通路和细胞功能的影响；3.动物模型探索DCLK1与K-Ras/MAPKs通路正反馈环对胰腺肿瘤发生的影响。

DCLK1作为肿瘤干细胞标志物，与胰腺癌发生发展密切相关，但其具体作用机制尚不清楚。K-Ras突变激活信号通路是胰腺癌发生的重要分子事件，研究报道K-Ras突变激活MAPKs信号通路促进DCLK1的表达。在我们的研究中，DCLK1激活MAPKs通路增加胰腺癌细胞的侵袭迁移能力并促进上皮间质转化，而KRAS-G12D突变也可以激活Ras信号通路促进DCLK1的表达进一步促使胰腺癌细胞发生恶性转化。此外，DCLK1还可以通过Hippo/YAP信号通路促进胰腺癌细胞表达PD-L1逃避细胞毒性T细胞的攻击，体内实验结果也表明DCLK1参与胰腺癌免疫微环境形成表现为增加M2巨噬细胞的浸润和减少T细胞浸润并抑制T细胞功能。综上，本课题揭示了DCLK1在胰腺癌进展中的重要作用及机制，为胰腺癌靶向治疗提供了作用依据。