CASC9/HIF-1α正反馈环路调控糖酵解在胰腺癌吉西他滨获得性耐药中的作用及机制研究

Acquired drug-resistance is an important reason for the failure of chemotherapy in pancreatic cancer. Our previous results have demonstrated that acquired gemcitabine chemoresistance is associated with enhanced glycolysis in pancreatic cancer, however, the explicit role and mechanism remain to be further investigated. Recently, it has been reported that CASC9 could stabilize HIF-1α and promote the glycolysis of cancer cells. And the bioinformatics analysis showed that the proximal promoter region of CASC9 contains hypoxia response elements (HREs) sites. Besides, our preliminary experiments have showed gemcitabine could promote the expression of CASC9 and many vital glycolytic enzymes. On the other hand, the induction of HIF-1α by hypoxia also enhanced the expression of CASC9. Therefore, we speculate that gemcitabine may promote profound chemoresistance of pancreatic cancer cells through positive feedback loop of CASC9/HIF-1α and glycolysis-mediated signaling pathway. Thus, we investigate the regulatory relationship of CASC9 and HIF-1α to glycolysis, and explore the mechanism of acquired gemcitabine chemoresistance from tissue level, in vitro and in vivo study, respectively. Altogether, our study will provide new strategies and potential targets for reversing the chemoresistance of pancreatic cancer.

获得性耐药增强是胰腺癌化疗失败的重要原因。申请者前期研究提示，胰腺癌吉西他滨获得性耐药过程伴随着糖酵解途径的增强，然其作用及具体机制仍需深入研究。近来文献报道，CASC9可以通过稳定HIF-1α促进肿瘤的糖酵解，而生物信息学分析表明CASC9的启动子区域又包含缺氧反应元件（HREs）位点。我们的预实验显示，吉西他滨可以促进CASC9及多个糖酵解重要酶的表达，而缺氧诱导HIF-1α的同时又可促进CASC9的上调。我们推测：吉西他滨可能通过诱导并促进CASC9与HIF-1α的正反馈环路，进而介导胰腺癌细胞糖酵解和获得性耐药行为。为此，本课题拟从CASC9、HIF-1α之间的关系对糖酵解的调控来研究吉西他滨作用胰腺癌细胞产生获得性耐药的机制，并分别在组织水平、体外细胞及体内动物水平三个方面进行验证。本课题的研究将为扭转胰腺癌的耐药行为提供新的思路和潜在靶点。

胰腺癌高度恶性，手术切除率低，化疗治疗在其综合治疗中占据重要地位，然其获得性耐药增强往往是化疗失败的重要原因。本课题主要从CASC9与糖酵解以及氧化应激两个层面关系入手，并分别研究AKT、HIF-1α通路以及NRF2抗氧化通路在其介导胰腺癌获得性耐药的作用及机制。首先，通过研究不同吉西他滨浓度对糖酵解重要酶的影响以及对葡萄糖摄取、乳酸释放水平的改变，并通过2-DG抑制糖酵解，检测对吉西他滨化疗敏感性影响，结果发现吉西他滨化疗促进胰腺癌细胞糖酵解，而抑制糖酵解显著增强其化疗敏感性。然后，检测CASC9对糖酵解影响，并分别采用AKT抑制剂LY294002、siRNA敲除HIF-1α来验证其所起的作用，结果证实CASC9促进胰腺癌细胞的糖酵解增强，而AKT及HIF-1α途径介导并促进上述过程。此外，我们研究吉西他滨对CASC9的表达影响及ROS所起的作用，发现ROS介导吉西他滨诱导的CASC9表达升高；进一步检测沉默CASC9对NRF2信号通路及细胞凋亡的影响，结果显示，沉默CASC9抑制吉西他滨诱导NRF2抗氧化系统激活，从而加剧其诱导的氧化应激及细胞凋亡，而通过NAC清除ROS则抑制上述效应。相反，沉默NRF2后则同样抑制吉西他滨诱导的CASC9的表达，说明CASC9与NRF2通路形成正反馈环路，共同减轻吉西他滨诱导的氧化应激及细胞凋亡，从而产生化疗耐药现象。最后，在裸鼠体内通过联合敲除CASC9与吉西他滨化疗，发现敲除CASC9可以显著协同增强吉西他滨对裸鼠移植瘤的化疗杀伤作用。本研究发现吉西他滨耐药的新机制：（1）CASC9通过激活AKT及HIF-1α途径促进糖酵解进而介导胰腺癌细胞吉西他滨化疗耐药；（2）CASC9通过与NRF2抗氧化信号通路形成正反馈环路，进而减轻吉西他滨化疗诱导的氧化应激与凋亡。该研究有望为临床扭转胰腺癌吉西他滨化疗耐药行为提供新的思路和潜在治疗靶点。