PAK4特异性介导的铁死亡在非小细胞肺癌对Gefitinib耐药过程的作用及机制研究

PAK4 is an important kinase protein that regulates cytoskeleton and migration. Our studies have revealed that PAK4 is an important kinase protein that regulates the metastasis of non-small cell lung cancer (NSCLC). Pretreatment found that PAK4 was highly expressed in Gefitinib-resistant NSCLC. Silencing PAK4 enhanced the sensitivity of drug-resistant cells to gefitinib, and ferroptosis (newly discovered cell death form) was specifically enhanced during this process, whereas apoptosis and autophagy no change. There is no report about ferroptosis involved in Gefitinib resistance. We therefore hypothesized that high expression of PAK4 specifically inhibitted ferroptosis contributed to NSCLC resistance to Gefitinib. In this project, the PAK4 phosphorylated substrate and the phosphorylation site were explored in vitro using PAK4 Tet-on expression system by protein chip technique and in vitro kinase assay. The aim of this study is to elucidate the specific mechanism of PAK4 selectively regulating ferroptosis, and it is expected to clarify the role of PAK4 and its downstream signaling pathway in the resistance of gefitinib, and to explore the mechanism of NSCLC resistance to gefitinib from the new cell death of ferroptosis. All the above will provide new strategies to overcome gefitinib resistance of NSCLC.

PAK4是调节细胞骨架及迁移能力的重要激酶蛋白。我们前期研究揭示PAK4是调控非小细胞肺癌（NSCLC）转移的关键基因；预实验发现PAK4在Gefitinib耐药的NSCLC中表达量更高，沉默PAK4可增强耐药细胞对Gefitinib的敏感性，铁死亡在该过程中特异性增强，而细胞凋亡及自噬功能无变化，目前尚无PAK4及铁死亡参与Gefitinib耐药的报道。因此我们提出假设：高表达PAK4通过特异性抑制铁死亡介导NSCLC对Gefitinib耐药。本项目拟通过蛋白质芯片技术及体外激酶分析等实验，在PAK4 Tet-on表达系统调控下，对PAK4及其下游信号通路在Gefitinib耐药中的作用进行深入探讨，旨在阐明PAK4通过其磷酸化底物特异性调控铁死亡的具体机制，进而从铁死亡这一新的细胞死亡角度探索NSCLC对Gefitinib耐药机制，从而为Gefitinib耐药问题的克服提供新的策略。

本研究拟对PAK4特异性介导的铁死亡在非小细胞肺癌对Gefitinib耐药过程的作用及机制进行探索，遗憾的是我们在动物实验中发现：实验组（PAK4 Knockdown）裸鼠的皮下成瘤与对照组无显著差异，提示PAK4 Knockdown后未能提高皮下种植瘤对Gefitinib的敏感性。幸运的是，我们发现PAK4 Knockdown的裸鼠对化疗药顺铂的敏感性显著增加，随即我们对PAK4与肺癌的化疗敏感性进行探索，发现PAK4在顺铂耐药的非小细胞肺癌细胞株中显著高于顺铂敏感的非小细胞肺癌细胞株，并发现在对顺铂耐药的非小细胞肺癌组织中表达也显著上升。进一步研究发现，PAK4 Knockdown可提高顺铂耐药的非小细胞肺癌细胞株对顺铂的敏感性。.机制研究发现，非小细胞肺癌细胞在使用顺铂化疗过程中会引起内质网应激（ER stress），沉默PAK4 通过调节内质网应激反应，增强非小细胞肺癌化疗耐药细胞的敏感性，.MEK1-ERK1/2-GRP78信号通路在该过程中起重要作用。我们的研究表明PAK4是非小细胞肺癌化疗耐药治疗中一个很有意义的靶点。.另外，我们也发现miR-203a-3p在非小细胞肺癌细胞株及组织中低表达，过表达miR-203a-3p可以显著抑制非小细胞肺癌细胞的恶性生物学行为及调高化疗敏感性。机制研究发现DNMT3B是miR-203a-3p的直接作用靶点，启动子甲基化是miR-203a-3p低表达的重要原因，进一步研究发现miR-203a-3p和 DNMT3B之间存在环状反馈调节。我们的研究表明miR-203a-3p是非小细胞肺癌治疗中一个很有意义的靶点。