肿瘤坏死因子受体相关因子4促表皮生长因子受体激酶活性及对肺癌的促进作用

TRAF4 promotes lung cancer development by activating tyrosine kinase of EGFR

目的探讨肿瘤坏死因子受体相关因子4(TRAF4)促进表皮生长因子受体(EGFR)异常激活的作用及其对肺癌细胞增殖、迁移和侵袭的影响。方法收集2015年1月至2017年5月于海军军医大学第一附属医院行肺腺癌切除患者的肿瘤组织,免疫组化检测肺癌组织中TRAF4和Ki-67的表达,实时荧光定量PCR检测Cyclin D和Vimentin mRNA的表达。通过异基因移植模型研究TRAF4对肺癌A549细胞的肿瘤生长能力的影响,采用细胞计数试剂盒8和BrdU实验检测TRAF4或EGFR对肿瘤增殖能力的影响,Transwell实验检测肿瘤细胞转移及侵袭能力。构建不同的TRAF4和EGFR结构域缺失表达载体转染细胞,通过免疫共沉淀实验研究TRAF4和EGFR相互作用方式。结果非小细胞肺癌(NSCLC)组织中TRAF4的表达与Ki-67、Cyclin D和Vimentin的表达呈正相关(r 2分别为0.438、0.695和0.736,均P<0.01)。NSCLC患者肿瘤组织免疫组化检测结果显示,高表达TRAF4的组织同时高表达Ki-67。TRAF4高表达(TRAF4阳性率>30%)患者较TRAF4低表达患者(TRAF4阳性率≤30%)的无进展生存时间缩短(中位无进展生存时间分别为12和19个月,P=0.034)。Traf4^(-/-)细胞较Traf4+/+细胞增殖能力减弱,形成肿瘤体积小(P<0.05)。对形成的肿瘤组织进行Ki-67染色,Cyclin D和Vimentin mRNA表达水平检测,结果显示,Traf4^(-/-)细胞形成的肿瘤组织中Ki-67表达水平[(45.6±8.7)%]低于Traf4+/+细胞形成的肿瘤组织[(62.3±10.3)%,P=0.015],Cyclin D mRNA表达水平(1.01±0.15)和Vimentin mRNA表达水平(1.01±0.12)均低于后者(分别为3.41±0.32和3.12±0.18,均P<0.05)。Western blot结果显示,随着细胞内TRAF4表达水平升高,无论是野生型还是活化突变型细胞,EGFR的磷酸化水平均明显升高。基因敲除EGFR后,A549细胞相对增殖、迁移和侵袭能力减弱(均P<0.01)。免疫共沉淀实验显示,TRAF4通过TRAF结构域与EGFR的近膜区肽段结合,TRAF4过表达条件下,EGFR分子之间的相互结合增强。增加TRAF4表达能促进EGFR分子磷酸化及下游信号的活化。结论TRAF4在NSCLC组织及肿瘤细胞中表达升高,促进肿瘤的增殖、迁移与侵袭。TRAF4直接与EGFR分子相结合,通过促进EGFR分子之间的相互作用增强其自身磷酸化,激活下游信号通路。

ObjectiveTo explore the role of tumor necrosis factor receptor-associated factor 4(TRAF4)in promoting the abnormal activation of epidermal growth factor receptor(EGFR)and its effect on lung cancer cell proliferation,migration and invasion.MethodsTumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University,from January 2015 to May 2017 were collected,and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry,the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR(qRT-PCR).The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model,the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8(CCK8)and BrdU assay,and the migration and invasion abilities of tumor cells were detected by Transwell assay.Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells,and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay.ResultsThe expression of TRAF4 in non-small cell lung cancer(NSCLC)tissues was positively correlated with the expressions of Ki-67,cyclin D,and vimentin(r 2:0.438,0.695,and 0.736,respectively,all P<0.01).Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67.Patients with high TRAF4 expression(TRAF4 positivity>30%)had a shorter progression-free survival(PFS)time than that of patients with low TRAF4 expression(TRAF4 positivity≤30%)(median PFS of 12 and 19 months,respectively;P=0.034).Traf4^(-/-)cells had a weakened proliferative capacity than traf4+/+cells and formed tumors with smaller size(P<0.05).The expression level of Ki-67 in the tumor tissues formed by traf4^(-/-)cells[(45.6±8.7)%]was lower than that in the tumor tissues formed by traf4+/+cells[(62.3±10.3)%,P=0.015],the mRNA levels of cyclin D(1.01±0.15)and vimentin(1.01±0.12)in the traf4^(-/-)cells were lower than those of the traf4+/+cells(3.41±0.32 and 3.12±0.18,respectively,both P<0.05).The western blot results showed that,with the elevated intracellular expression level of TRAF4,phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells.The capacities of proliferation,migration and invasion of A549 cells was weakened after EGFR knockdown(all P<0.01).Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain,and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions.Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling.ConclusionsTRAF4 expression is elevated in NSCLC tissues and tumor cells,which promotes tumor proliferation,migration and invasion.TRAF4 directly binds to EGFR molecules,enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.