mTORC1/2激酶抑制剂通过上调EGFR表达介导胰腺癌耐药的新机制研究

Pancreatic cancer is the fourth leading cause of cancer death and the high mortality rate of pancreatic cancers is in part due to the cancer resistance to treatments. There is therefore an urgent need for development of new effective targeted therapies that target cancer signaling pathways. Mammalian target of rapamycin (mTOR) drives cancer formation and progress, thus emerging as a cancer therapeutic target. There are two mTOR complexes: mTOR complex 1 (mTOR1) and complex 2 (mTORC2). In earlier studies, we have shown pancreatic cancers are resistant to the treatment of rapamycin due to the feedback activation of AKT cell growth pathway. Recently, mTORC1/2 dual inhibitor (AZD8055) has been developed for cancer therapies. Our preliminary studies have shown that the treatment of AZD8055 activates AKT pathway. A phosphor-receptor tyrosine kinase array has revealed the phosphorylation and activation of epidermal growth factor receptor (EGFR). In this proposal, we will therefore test the hypothesis that the AZD8055-induced activation of Akt occurs through the feedback EGFR activation. First, we will employ SRB assay,RT-qPCR, western blot and report gene approach in analysis of AZD8055 treated pancreatic cells, thus determining whether the EGFR activation occurs through transcriptional mechanisms and/or EGFR phosphorylation. We will also take small interfering RNA (siRNA) approach and thus determine whether knockdown of EGFR eliminates the feedback Akt activation.Finally, we will generate pancreatic xenografts in mice, treat the mice with the combination of AZD8055 and EGFR inhibitor(Erlotinib) and assess whether the combination therapy can inhibit the xenograft growth. This proposal will therefore provide new therapeutic strategy in treating human pancreatic cancers.

胰腺癌手术切除率低，传统放化疗治疗效果差，因此寻求有效的靶向药物是提高胰腺癌治疗的关键。胰腺癌细胞内mTOR信号通路异常激活,但临床应用mTOR抑制剂（雷帕霉素）治疗效果不显著。为解决其由于AKT负反馈激活而引起的肿瘤耐药，第二代mTORC1/2抑制剂（AZD8055）被研制出来。本课题组发现，胰腺癌仍对其耐药，应用RTK array显示AZD8055诱导EGFR表达明显上调，但EGFR是否参与胰腺癌对AZD8055的耐药以及具体的耐药机制尚不清楚。本课题拟在前期工作基础上，通过流式细胞术、Western、RT-qPCR、免疫共沉淀、SiRNA干涉、报告基因等技术，明确以EGFR为靶点改善胰腺癌对AZD8055耐药的作用，并从不同层面阐明AZD8055引起EGFR表达升高的机制，从而为以EGFR为靶点逆转胰腺癌对AZD8055的耐药，为开发切实有效的胰腺癌靶向药物联合治疗方案提供依据。

mTOR通路在肿瘤发生发展中发挥着重要的作用，第一代mTOR抑制剂（Rapamycin及其类似物）由于诱导存在IGF-1R/AKT的负反馈激活而导致细胞耐药，第二代mTOR抑制剂（AZD8055）可以抑制AKT/mTORC2的激活而被寄予厚望，但在胰腺癌中的治疗效果尚不完全清楚。我们的研究发现，与第一代抑制剂Everolimus相比较，AZD8055对胰腺癌细胞的增殖抑制没有显著提高，AZD8055可短暂的抑制AKT的磷酸化，但随后AKT被重新磷酸化激活。同时，我们发现AZD8055诱导的AKT短暂抑制通过释放转录因子FoxO1/3a的活性而介导EGFR的表达激活上调，进而进一步导致AKT的继续激活和胰腺癌细胞的耐药。最后，通过体内体外实验我们证实联合EGFR抑制剂（Erlotinib）可以有效逆转胰腺癌对AZD8055的耐药，抑制细胞增殖和移植瘤的生长，以及下调EGFR/AKT通路相关蛋白的表达。该研究为有效逆转胰腺癌对AZD8055的耐药提供新的靶点和思路。