ATF5活化转录STAT3的分子机制及其在介导胃癌多药耐药中的作用

Multidrug resistance(MDR) is a major obstacle in chemotherapy of gastric cancer, but its molecular mechanism remains unclear. Activating transcription factor 5(ATF5) is a potent stress-responsive gene and thought to play an important protective role in the cellular stress response. However, whether ATF5 mediate gastric cancer MDR remains unknown. In our previous study, we showed that the protein levels of ATF5 were much higher in the MDR cell lines than in parental cells, and knockdown of ATF5 by siRNA could reverse its MDR phenotype. The protein levels of STAT3 and p-STAT3 was associated with that of ATF5 in MDR cell lines, and knockdown of ATF5 caused the down-regulation of STAT3 and p-STAT3. In addition, two ATF5 putative binding sites were identified within the -650 to -300bp region of the STAT3 promoter with bioinformatics softwares. And the results of luciferase reporter assay showed that the STAT3 promoter activity was markedly activated by ATF5 in a dose-dependent manner. We further confirmed by using STAT3-specific inhibitor JSI-124, inhibition of STAT3 could also re-sensitize MDR cell lines to chemotherapy. Thus we speculate that ATF5 might confer a MDR phenotype to gastric cancer cells through transactivation of STAT3 and subsequent its downstream targets expression. The function and molecular mechanism of ATF5 and the ATF5-STAT3 pathway in gastric cancer MDR will be further investigated in our future work. The results of our study would provide us a more clear understanding of the molecular network in MDR and might reveal a new promising therapeutic target for overcoming MDR in gastric cancer.

胃癌多药耐药(MDR)严重影响化疗效果，但其分子机制仍未阐明。活化转录因子ATF5是一个应激反应基因，在细胞应激保护中具有重要作用。ATF5在胃癌MDR中是否也发挥作用？目前尚无报道。我们前期率先发现：ATF5在胃癌MDR细胞中表达上调，干扰ATF5可逆转其MDR表型；STAT3和p-STAT3的表达在胃癌MDR细胞中和ATF5具有相关性，干扰ATF5可使STAT3、p-STAT3表达下降；生物信息学分析提示STAT3的启动子存在ATF5潜在结合位点，双报告基因实验提示ATF5可激活STAT3启动子活性；抑制STAT3活性可以逆转胃癌细胞MDR表型。据此推测：ATF5转录激活STAT3表达，通过其下游基因的激活导致胃癌多药耐药。本课题拟深入探索ATF5在胃癌MDR中的作用及ATF5-STAT3信号途径的具体作用机制。研究将有助于完善胃癌MDR形成的网络分子机制，进而发现新的潜在干预靶点。

我们前期做了大量的研究证实了ATF4蛋白在食管鳞状细胞癌中的表达及其介导STAT3通路影响食管鳞状细胞癌细胞的多药耐药性（MDR），而对于与ATF4具有相似的分子结构的同家族成员ATF5在胃癌MDR中是否也发挥作用？是本研究的研究重点内容。前期研究发现ATF5在胃癌MDR细胞中具高表达，认为干扰ATF5可逆转其MDR表型；STAT3和p-STAT3的表达在胃癌MDR细胞中和ATF5具有相关性，干扰ATF5可使STAT3、p-STAT3表达下降；生物信息学分析提示STAT3的启动子存在ATF5潜在结合位点，双报告基因实验提示ATF5可激活STAT3启动子活性；抑制STAT3活性可以逆转胃癌细胞MDR表型。本研究在前期研究基础上通过DLR、EMSA、ChIP等实验探索ATF5对STAT3的直接调控作用及具体分子机制，在ATF5过表达的胃癌细胞中阻断STAT3信号途径，明确STAT3是否是ATF5所介导的胃癌多药耐药表型的一个重要途径，还通过裸鼠实验等，综合探讨了探索ATF5在胃癌MDR中的作用及ATF5-STAT3信号途径的具体作用机制。