STAT3反馈激活介导胃癌细胞对PI3K-AKT-MTOR抑制剂的耐药性研究

发布时间：2018-03-27 19:31

本文选题：胃癌　切入点：STAT3　出处：《中国人民解放军医学院》2017年硕士论文

【摘要】：背景:胃癌是全球发病率第四位,死亡率第二位的常见恶性肿瘤。我国每年有40多万例新发病例,约占世界新发病例数42%,每年约35万人死于胃癌,在我国城市地区,该病是导致病人死亡的第二位原因。PI3K/AKT/mTOR信号通路在肿瘤细胞中被异常激活,因此一系列针对该信号通路分子的抑制剂被开发并进入临床实验。靶向治疗药物的出现大大促进了胃癌治疗的进展,但是肿瘤细胞不可避免地产生耐药性限制了针对该信号通路抑制剂的治疗效果,且分子机制也不清楚。STAT3反馈激活在肿瘤细胞对广泛的肿瘤靶向治疗药物产生耐药性过程中发挥主导作用。因此研究针对这两种靶点联合用药的策略,对于改善胃癌靶向性治疗疗效具有重要的意义。目的:分析和揭示STAT3激酶反馈激活是PTEN缺失的胃癌细胞对PI3K/AKT/mTOR抑制剂产生耐药性的重要因素;阐明STAT3激酶反馈激活的分子机制;探索以STAT3激酶和PI3K/AKT/mTOR为胃癌治疗靶点,联合用药的策略以及对胃癌细胞的协同抑制功能。方法:利用 PI3K/AKT/mTOR 抑制剂(LY294002、BEZ235、MK2206、RAD001)单一或联合STAT3激酶抑制剂(STATTIC)处理一系列胃癌细胞系,利用Western-blot检测AKT激酶和STAT3激酶的表达及磷酸化水平;应用流式细胞技术等观察单一或联合用药后对细胞周期、凋亡等细胞现象的影响;通过siRNA建立了 HGC27敲低STAT3表达的稳定细胞系,并建立相应的荷瘤小鼠模型,然后通过CCK8实验、平板克隆实验等检测细胞生长;利用荷瘤小鼠模型,检测STAT3表达前后以及BEZ235处理后胃癌细胞生长速度。结果:(1)在PTEN阴性的胃癌细胞中,PI3K/AKT/mTOR信号通路抑制剂反馈激活STAT3激酶活性;(2)抑制STAT3激酶活性能够增加胃癌细胞对PI3K/AKT/mTOR抑制剂的敏感性;(3)抑制PI3K/AKT/mTOR激酶和STAT3激酶活性协同抑制胃癌细胞生长;(4) SDF-1/CXCR4轴参与了 STAT3反馈激活,STAT3的反馈激活与细胞分泌的可溶性细胞因子以及遗传背景等有关。结论:在PTEN阴性的胃癌细胞中,P13K/AKT/mTOR抑制剂反馈激活STAT3激酶活性,并与某些可溶性细胞因子有关,这对于理解肿瘤耐药性提供了一定的基础。联合抑制STAT3激酶和PI3K/AKT/mTOR激酶活性协同抑制胃癌细胞生长。该研究结果为PTEN阴性胃癌患者的治疗策略提供了指导。
[Abstract]:Background: gastric cancer is the fourth most common malignant tumor in the world. There are more than 400,000 new cases every year in China, accounting for 42 million new cases in the world. About 350000 people die of gastric cancer every year. The disease is the second leading cause of death. The PI3K / AKT / mTOR signaling pathway is unusually activated in tumor cells. Therefore, a series of inhibitors targeting the signaling pathway molecules have been developed and entered clinical trials. The emergence of targeted therapeutic drugs has greatly promoted the progress of gastric cancer treatment. But the inevitable drug resistance of tumor cells limits the therapeutic effect of the signaling pathway inhibitor. And the molecular mechanism is not clear. STAT3 feedback activation plays a leading role in the development of drug resistance of tumor cells to a wide range of tumor targeted treatment drugs. Objective: to analyze and reveal that the activation of STAT3 kinase feedback is an important factor in the drug resistance of gastric cancer cells with PTEN deficiency to PI3K/AKT/mTOR inhibitors, and to elucidate the molecular mechanism of STAT3 kinase feedback activation. Methods: a series of gastric cancer cell lines were treated by PI3K/AKT/mTOR inhibitor LY294002BEZ235MK2206 RAD001 (single or combined with STAT3 kinase inhibitor STATTIC). Western-blot was used to detect the expression and phosphorylation of AKT kinase and STAT3 kinase, and flow cytometry was used to observe the effect of single or combined administration on cell cycle and apoptosis. A stable cell line with low STAT3 expression of HGC27 knockout was established by siRNA, and the corresponding tumor-bearing mouse model was established. Then, the cell growth was detected by CCK8 experiment, plate cloning assay and so on, and the tumor-bearing mouse model was used. The growth rate of gastric cancer cells was measured before and after STAT3 expression and after BEZ235 treatment. Results the inhibition of STAT3 kinase activity by PI3K / AKT / mTOR signaling pathway inhibitor feedback activated STAT3 kinase activity in PTEN negative gastric cancer cells could increase the activity of STAT3 kinase in gastric cancer cells with PI3K/AKT/mTOR. The inhibitory activity of PI3K/AKT/mTOR kinase and STAT3 kinase combined with inhibition of growth of gastric cancer cells by SDF-1/CXCR4 axis is involved in the feedback activation of STAT3 feedback activator STAT3, which is related to soluble cytokines secreted by the cells and genetic background. Conclusion: P13K / AKT / mTOR inhibitor feedback activates the activity of STAT3 kinase in PTEN negative gastric cancer cells. And related to some soluble cytokines, This study provides a basis for understanding tumor resistance. The combination of inhibition of STAT3 kinase and PI3K/AKT/mTOR kinase activity in combination with the inhibition of gastric cancer cell growth. The results of this study provide guidance for the treatment strategy of PTEN negative gastric cancer patients.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

【参考文献】