Metformin协同HDACi调控BIM信号通路克服EGFR-TKI耐药的作用和机制

Drug resistance is the key problem that restricts the clinical use of Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs). Recent study shows that BIM deletion polymorphism and low expression play important roles in TKI resistance. Histone deacetylase inhibitors (HDACi) could overcome TKI resistance through BIM upregulation in lung cancer cells with BIM deletion polymorphism, while it has little effect in BIM-wild type lung cancer cells, or cells with high expression of anti-apoptotic proteins including BCL-2 and MCL-1. Our reseach indicated that a standard anti-diabetic drug, metformin, could significantly overcome TKI resistance. Surprisingly, metformin was found to increase BIM expression in a number of lung cancer cells, including TKI sensitive cells and TKI resistant cells due to T790M mutation, K-RAS mutation, C-Met amplification, or EMT. Moveover, metformin can inhibit expression of MCL-1 and BCL-2. Thus, metformin and HDACi may cooperately target BIM signaling pathway, and the combination may overcome TKI resistance of multiple mechanisms. The current research aims to study the effect and mechanisms of metformin in combination with HDACi on TKI resistance. Hopefully we will prove the complementary synergy of co-treatment with metformin and HDACi on TKI sensitivity, thus providing a new approach which may be generally adoptted to treat NSCLC patients with EGFR mutations.

EGFR-TKI耐药是限制其疗效和临床应用的瓶颈。近来研究表明，BIM多态性和低表达是TKI耐药的重要原因，HDACi可以克服BIM多态性逆转耐药，但对BIM野生型及MCL-1等抗凋亡蛋白高表达的肺癌作用有限。我们前期工作发现，经典降糖药Metformin可逆转TKI耐药，并能显著上调BIM野生型TKI耐药细胞中的BIM的表达，抑制MCL-1等抗凋亡蛋白。因此，Metformin和HDACi可能具有协同调控BIM通路及凋亡的作用，二者协同可能克服多种耐药机制所致TKI耐药。本项目拟研究Metformin协同HDACi对多种机制所致TKI耐药肺癌细胞的作用，进一步探讨TKI耐药的分子机制及逆转策略，有望证实Metformin和HDACi通过调控BIM信号通路克服原发及继发TKI耐药的协同作用，为临床提供较广泛适用的克服TKI耐药的联合用药方案，具有重要科学意义。

EGFR-TKI耐药是限制其疗效和临床应用的瓶颈。BIM多态性和低表达是TKI耐药的重要原因，HDACi可以克服BIM多态性逆转耐药，但对BIM野生型及MCL-1等抗凋亡蛋白高表达的肺癌作用有限。我们前期工作发现，Metformin可逆转TKI耐药，并能显著上调BIM野生型TKI耐药细胞中的BIM的表达，抑制MCL-1等抗凋亡蛋白。因此，Metformin和HDACi可能具有协同调控BIM通路及凋亡的作用，协同克服多种耐药机制所致TKI耐药。本项目以EGFR-TKI耐药肺癌细胞株H1975、PC-9GR为研究对象，通过系列研究评估细胞侵袭、细胞凋亡，验证Metformin联合泛HDACi vorinostat逆转TKI耐药性的效应。通过western blot、siRNA技术检测凋亡信号通路相关蛋白表达变化并探索Metformin联合Vorinostat逆转TKI耐药性的关键机制。结果发现Metformin与Vorinostat均可一定程度增加EGFR-TKI耐药细胞对Gefitinib的敏感性，两药联用可协同克服Gefitinib耐药，诱导EGFR-TKI耐药细胞凋亡；在H1975、PC-9GR细胞株中，Metformin与Vorinostat共同作用于凋亡信号通路，表现为促凋亡蛋白BIM、BAX表达水平显著增加，抗凋亡蛋白Bcl-2、Bcl-xL和p-Mcl-1表达水平显著下调。且协同抑制EGFR、AKT、ERK的磷酸化。此外，Metformin可抑制被Vorinostat和/或Gefitinib诱导的保护性自噬，进一步促进凋亡。BIM是Metformin与Vorinostat协同诱导凋亡，克服EGFR-TKI耐药的关键靶点。上述发现有望为临床提供较广泛适用的克服TKI耐药的联合用药方案，具有重要科学意义。