以APC基因突变为背景的肠癌双靶点模型的建立及机制研究

The mutation of APC gene affect Wnt/β-catenin signal pathway. The processes of dysregulation of Wnt/β-catenin in intestinal polyps might response to various pro-inflammatory stresses for development of intestinal cancer via the formation of new signal pathways. In this study, we aim to establish the mice models with double specific targets in intestinal cancer. These specific models will be used in studies of medicine and pharmacology. ApcMin/+ mice will cross with the mice of Fas deficiency, Vegfr2-KI(knock in) and COX transgenes, respectively, to generate mice of APCMin+/Fas deficiency, APCMin+/Vegfr2-KI(knock in) and APCMin+/COX transgenes. Among them, the APCMin+/COX transgenes will established by using the transgenic technology in our laboratory. We then investigate the mechanisms of these models. The mechanisms of APCMin+/Fas/CD95 deficiency and APCMin+/COX transgenes might be associated with the stimulation of COX-2/PGE2-MMPs-Fas/CD95 signaling pathway and therefore the intestinal polyps in these models show continuously growth and resistance to the induction of apoptosis. Also, in the model of APCMin+/COX transgenes, we will investigate the mechanism of development of intestinal cancer from polyps. This development might due to the activation of signal pathway of NF-κB-COX/2/PGE2-c-Met-JAK1-STAT3. In the model of APCMin+/Vegfr2-KI(knock in), we suggest that the invasion and metastasis of intestinal cancer is associate with the stimulation of angiogenesis and epithelial-mesenchymal transition (EMT). The mechanism of this cancer development might due to the activation of NF-κB-COX-2/PGE2-KDR-PLC-γ-PKC-Raf-MEK-MAPK signaling pathway . In addition, we will examine the inhibitory effects of some new compounds and drugs on the growth of polyps and cancers in intestines using these specific models. These assays will show the good value of these models in study of cancers and drug discovery. After that, these models will provide to scientists for study of intestinal cancers and also for the drug screening in medicine.

APC突变致Wnt/β-catenin信号异常激活可能影响炎症信号参入结肠癌的发病过程并可能形成新信号通路。拟以杂交和转基因方法建立具有APCMin+背景的小鼠肠癌双靶点模型，为医学和药理学研究服务。拟证明APCMin+/Fas deficiency和APCMin+/COX transgenes模型所表现出的肠癌增殖和凋亡抵抗与COX-2/PGE2-MMPs-Fas/CD95信号异常传导有关；APCMin+/COX transgenes模型肠腺瘤癌变机制可能与激活NF-κB-COX/2/PGE2-c-Met-JAK1-STAT3信号有关；在APCMin+/Vegfr2-KI(knock in)模型，我们拟证明其微血管形成和肠上皮-间质化EMT与激活NF-κB-COX-2/PGE2-KDR-PLC-γ-PKC-Raf-MEK-MAPK有关。拟实例验证上述模型的正确，评价其理论意义和使用价值。

趋化因子受体CXCR7和CXCR4表达水平与结肠癌炎症微环境形成密切相关。本课题研究发现，人结肠癌组织明显高表达CXCR7和CXCR4并形成异二聚体CXCR7/CXCR4 heterodimer，临床资料显示有65%患者结肠癌组织高表达CXCR7/CXCR4 heterodimer。我们构建了villin-CXCR7和villin-CXCR4转基因小鼠，并通过杂合方式建立了villin-CXCR7-CXCR4小鼠以及与APC突变小鼠杂合APCMin/+/villin-CXCR7-CXCR4小鼠，发现CXCR7/CXCR4 heterodimer表达与结肠组织M-MDSCs细胞和M2型巨噬细胞浸润有关，并产生大量炎症因子M-CSF、GM-CSF、IL-6和 TNF-α，形成典型炎癌转化微环境，促进结肠癌产生与恶性发展。进一步分析发现，CXCR7/CXCR4 heterodimer刺激β-arrestin 1向细胞核内聚集，激活核内组蛋白去甲基化酶JMJD2A。组蛋白H3K9me3和H3K36m3去甲基后转变成为H3K9和H3K36，导致多种炎症因子和增殖因子转录，刺激癌细胞增殖。.CXCR2也是重要炎症因子，我们通过杂合方式构建APCmin/+/CXCR2+/-双靶点小鼠模型，发现敲除CXCR2后肠腺瘤生长受到明显抑制，其肠粘膜及外周血TNF-α、IL-1β、COX-2、p-NFκB、PGE2和IL-6水平明显低于APCmin/+小鼠。与APCmin/+比较，APCmin/+/ CXCR2+/-小鼠肠上皮腺瘤细胞p-PDK，p-AKT，AKT 和p-P38，P38，p-Erk，Erk等表达水平降低。APCmin/+小鼠肠上皮细胞核β-catenin水平升高，而敲除CXCR2的APCmin/+/CXCR2+/-小鼠其细胞核内β-catenin水平明显降低。提示：CXCR2在维持炎症状态、刺激腺瘤增殖方面具有重要作用。.本项目还发现，天然产物杨梅素对APCmin/+小鼠肠腺瘤具有明显抑制作用,可明显抑制Wnt/β-catenin信号激活，抑制炎症因子IL-1β, IL-6, TNF-α和PGE2等表达，其机制可能通过调控GSK-3β活性和p38 MAPK/Akt/mTOR信号表达水平，进而阻止β-catenin入核有关。该化合物对于进一步开发防治结直肠癌药物具有重要意义。