高表达CXCR4可通过乳酸脱氢酶A磷酸化诱导肺腺癌细胞产生吉非替尼耐药  

Overexpression of CXCR4 induces resistance to gefitinib in lung adenocarcinoma cells via enhancing lactate dehydrogenase A phosphorylation

作　　者：祝巧良 卢春来 古杰 葛棣 ZHU Qiao-liang;LU Chun-lai;GU Jie;GE Di(Department of Thoracic Surgery,Zhongshan Hospital,Fudan University,Shanghai 200032,China)

机构地区：[1]复旦大学附属中山医院胸外科,上海200032

出　　处：《中国临床医学》2021年第3期408-415,共8页Chinese Journal of Clinical Medicine

基　　金：国家自然科学基金(81872291)。

摘　　要：目的:探讨CXC趋化因子受体4(CXCR4)高表达诱导肺腺癌细胞产生吉非替尼耐药的潜在机制。方法:检测含EGFR-19del点突变的肺腺癌细胞中CXCR4的表达情况,以siRNA干扰或慢病毒感染调控细胞CXCR4的表达,通过克隆形成、CCK-8、流式细胞凋亡术、细胞凋亡相关蛋白测定、裸鼠皮下成瘤等实验鉴定细胞的吉非替尼敏感性。采用免疫共沉淀结合质谱分析法筛选CXCR4的相互作用蛋白。检测细胞的有氧糖酵解水平,并检测细胞在糖酵解抑制剂处理下的吉非替尼敏感性。结果:PC9/GR细胞存在显著的吉非替尼耐药性,PC9/GR细胞的吉非替尼IC50是PC9细胞的近100倍(19.15μmol/L vs 0.195μmol/L)。CXCR4在PC9/GR细胞中高表达(>4倍高于PC9细胞),抑制CXCR4可显著逆转PC9/GR细胞的耐药性。过表达CXCR4可诱导PC9细胞产生耐药,使吉非替尼IC50由0.89μmol/L升高至10.10μmol/L。乳酸脱氢酶A(lactate dehydrogenase A,LDHA)是新型CXCR4底物:CXCR4过表达通过促进LDHA磷酸化,从而增强细胞的有氧糖酵解功能;糖酵解抑制剂可显著逆转CXCR4高表达所诱导的吉非替尼耐药。结论:CXCR4高表达通过促进LDHA磷酸化使有氧糖酵解功能增强,从而诱导肺腺癌细胞产生吉非替尼耐药,是潜在的吉非替尼耐药治疗靶点。Objective:To explore the underlying mechanism of CXC chemokine receptor 4(CXCR4)-induced resistance to gefitinib in lung adenocarcinoma.Methods:CXCR4 expression of lung adenocarcinoma cells bearing EGFR-19del mutation was detected and modulated with siRNA interference or lentiviral infection.Assays of colony-formation,CCK-8,flow cytometric apoptosis,detection of apoptotic proteins and EGFR downstream signaling molecules,and tumor formation in vivo were conducted to identify gefitinib sensitivity.Co-immunoprecipitation combined with mass spectrometric analysis was applied to screen CXCR4-interacting proteins.Glycolysis levels of cells were detected and glycolysis inhibitors were applied to detect cell functions under gefitinib.Results:PC9/GR cells exhibited significant resistance to gefitinib.The IC50 for gefitinib of PC9/GR cells is nearly 100 times higher than that of PC9 cells(19.15μmol/L vs 0.195μmol/L).CXCR4 was overexpressed in PC9/GR cells(more than 4 times higher than that of PC9 cells),and inhibition of CXCR4 sensitized PC9/GR cells to gefitinib.Conversely,overexpressing CXCR4 conferred resistance to PC9 cells,by which the IC50 for gefitinib was increased from 0.89μmol/L to 10.10μmol/L.Furthermore,we identified lactate dehydrogenase A as a novel CXCR4 substrate,finding that overexpression of CXCR4 promoted LDHA phosphorylation by which aerobic glycolysis was enhanced.Importantly,CXCR4-induced resistance to gefitinib in lung adenocarcinoma cells could be reversed by glycolysis inhibitors both in vitro and in vivo.Conclusions:CXCR4 overexpression induced resistance to gefitinib in lung adenocarcinoma cells through enhancing glycolysis by promoting LDHA phosphorylation.Combination of CXCR4 antagonist and glycolysis inhibitors might provide an alternative strategy to overcome the progression of lung cancer after gefitinib treatment.

关 键 词：CXC趋化因子受体4 乳酸脱氢酶A 吉非替尼耐药 肺腺癌 

分 类 号：R734.2[医药卫生—肿瘤]