SIRT4调控线粒体功能影响非小细胞肺癌对EGFR-TKI药物的敏感性

The epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) erlotinib has been approved based on the clinical benefit in non-small cell lung cancer (NSCLC) patients. Unfortunately, cancer cells become resistant to this agent inevitably and limit the improvement in patient outcomes. SIRT4 functions as a tumor suppressor and directs mitochondrial metabolism by inhibiting glutamine metabolism. We founded SIRT4 was low expressed in lung cancer and its low expression was closely correlated with poor overall survival. SIRT4 inhibited mitochondrial fission and induced decreased invasive ability of lung cancer cell. In addition, we found SIRT4 inhibited cell autophagy and promoted lung cancer cells apoptosis induced by erlotinib . Therefore, we hypothesized that SIRT4 increased the effect of erlotinib on lung cancer by hampering mitochondrial function. We will bidirectionally regulate SIRT4 expression and adopt immunofluorescence, flow cytometry assay and microplate reader to detect the effect of SIRT4 on cell mitochondrial function and cell death after erlotinib treatment. Through the detection of glutamine metabolism ,cell autophagy/mitophagy and drug employment, we will clarify SIRT4 inhibit glutamine metabolism and simultaneously cell autophagy/mitophagy ,which aggravates damaged cell mitochondria. Further we intend to test the change of necrosis or mitochondrial apoptosis pathway to explore the underlined molecular mechanisms by which SIRT4 causes cell death as a result of dysfunctional mitochondria . We will also use animal model and clinical samples to explore the clinical and theoretical value of SIRT4 in reversing or inhibiting EGFR - TKI resistance.

解析EGFR-TKI耐药机制及寻找有效的逆转耐药的方法是NSCLC分子靶向治疗中亟需解决的瓶颈问题。最新研究提示细胞/线粒体自噬参与调控EGFR-TKI药物敏感性。我们前期研究证实：线粒体代谢调节器蛋白SIRT4抑制细胞/线粒体自噬，促进EGFR-TKI厄罗替尼诱导的细胞凋亡；抑制谷氨酰胺代谢和肺癌细胞增殖、侵袭能力并与肺癌患者预后密切相关。故提出SIRT4通过抑制线粒体功能而增加肺癌细胞对厄罗替尼的敏感性。我们拟双向调控SIRT4，采用荧光检测、流式细胞、酶标等技术探讨SIRT4对厄罗替尼处理后细胞死亡和线粒体功能的影响；通过检测谷氨酰胺代谢、细胞自噬等证实SIRT4抑制细胞/线粒体自噬进而加重细胞线粒体损伤；坏死性凋亡或线粒体凋亡途径因子变化阐明SIRT4导致线粒体损伤终至细胞死亡的可能分子机制。联合体内和体外实验为SIRT4抑制或逆转EGFR-TKI耐药提供理论补充和临床应用基础。

肺癌已是世界范围内病死率较高的恶性肿瘤之一，多数患者确诊时已属中晚期,常失去外科手术和放疗机会，因此药物治疗的作用就尤为重要，针对EGFR突变的TKI类的分子靶向药物目前已取得临床疗效，但原发性和继发性耐药严重影响EGFR-TKI药物治疗效果，因此探索原发性/继发性EGFR-TKI耐药机制，寻找克服EGFR-TKI耐药的方法是临床提高肺癌患者生存时间和改善生存质量的关键。EGFR-TKI药物耐药细胞的明显特征是细胞在长期药物作用后产生保护作用进而逃离死亡，这种保护作用与真核细胞中高度保守的生命过程----自噬密切相关。SIRT4作为抑癌因子定位于线粒体，具有ADP-核糖基转移酶活性, 主要参与调节能量代谢和线粒体功能。本项目在前期工作基础上，取得创新性成果：1. 体外实验证实线粒体自噬调控因子Sirt4能够提高肺癌细胞对吉非替尼的敏感性；2. 多种形态学及分子生物学实验手段阐明Sirt4抑制吉非替尼诱导的肺癌细胞自噬是通过抑制MEK- ERK信号通路活性来实现；3.吉非替尼诱导的肺癌细胞自噬，在肺癌细胞对抗吉非替尼药物作用中具有保护作用；4.进一步分子机制探讨表明SIRT4通过抑制ATG7-ERK的表达进而抑制肺癌细胞自噬。为全面系统地探索SIRT4 介导耐药的分子机制和临床判定对EGFR-TKI 是否耐药的提供新的思路，本课题初步阐明SIRT4抑制肺癌细胞线粒体功能进而抑制EGFR-TKI耐药，不仅对丰富TKI耐药发生的分子机制具有重要意义，更为寻求提高临床肺癌靶点治疗疗效提供理论补充和临床应用基础。