靶向抑制eIF4E磷酸化增效RAD001治疗非小细胞肺癌的分子机制研究

mTOR （mammalian target of rapamycin，mTOR）inhibitors increase Mnk (MAP kinase integrating kinase, Mnk)-dependent phosphorylation of eIF4E (eukaryotic translation initiation factor 4E, eIF4E) through mTOR inhibition-induced feedback mechanism and decrease mTOR inhibitor's anticancer activity. Phosphorylation of eIF4E is specifically catalyzed by mitogen-activated protein kinase Mnk1. Phosphorylated eIF4E plays a crucial role in inhibiting cell apoptosis and causing cell proliferation. Our findings indicate increase the expression of phosphorylated eIF4E in the non small cell lung cancer (NSCLC) xenografts and cell lines with RAD001-treated. Our data also demonstrated Mnk inhibitor CGP57380 is capable of inhibiting the phosphorylation of eIF4E and inducing high expression of DR5 (death receptor 5) and down-regulation of c-FLIP(cellular FLICE-like inhibitory protein) in NSCLC cell lines. Combination CGP57380 and RAD001 enhance the antitumor activity by inducing apoptosis and suppressing the proliferation of cultured human NSCLC cell lines. The most important part of the research project is to investigate the molecular mechanism of enhancing the anticancer effect of NSCLC with RAD001 treatment and CGP 57380 selectively inhibited the phosphorylation of eIF4E and inducing NSCLC cell apoptosis through induction of DR5 and down-regulation of c-FLIP protein. The achievement of the novel research project may be first to elucidate the molecular mechanism of the synergistic anticancer activity of CGP57380 combined with RAD001 by inducing apoptosis of NSCLC cells through the death receptor pathway. Therefore, the project might have theoretical significance and important implications for clinical applications.

mTOR 抑制剂反馈激活Mnk (MAP kinase integrating kinase, Mnk)依赖性eIF4E(eukaryotic translation initiation factor 4E, eIF4E)的磷酸化，降低其抗肿瘤疗效。Mnk 1是eIF4E磷酸化的唯一激酶，磷酸化eIF4E抑制细胞凋亡, 促进肿瘤增殖。我们前期研究发现mTOR抑制剂RAD001（RAD）增加肺癌的磷酸化eIF4E。Mnk1抑制剂CGP57380（CGP）抑制eIF4E磷酸化，增加DR5和下调c-FLIP的表达。CGP联合 RAD诱导肺癌细胞凋亡，增加抗肿瘤效应。本项目主要研究CGP抑制eIF4E磷酸化，经何种机制调控DR5和c-FLIP的表达，诱导细胞凋亡，增加RAD治疗非小细胞肺癌的疗效。项目的完成将首次阐明CGP可能通过死亡受体通路诱导细胞凋亡增效RAD的分子机制，具有重要理论意义。

项目背景：.靶向治疗耐药影响非小细胞肺癌（NSCLC）患者疗效。eIF4E的磷酸化激活上调肿瘤相关蛋白表达，促进肿瘤的发生、发展。eIF4E磷酸化的唯一激酶磷酸化Mnk1通过Mnk1-eIF4G 相互作用致eIF4E Ser-209位点磷酸化。mTOR抑制剂反馈激活Akt和eIF4E磷酸化，降低其抗肿瘤效应。Mnk抑制剂CGP57380靶向抑制mTOR抑制剂Rapamycin的衍生物RAD001诱导的eIF4E磷酸化，促进凋亡、抑制肿瘤细胞增殖，生长和转移。本项目旨在阐明CGP57380 靶向抑制eIF4E 磷酸化，经何种途径诱导细胞凋亡增加RAD001 治疗NSCLC 疗效的分子机制。.本项目主要包括三个方面的研究：.①研究CGP57380靶向抑制eIF4E磷酸化，是否增加RAD001治疗NSCLC的疗效。.②研究CGP57380靶向抑制eIF4E磷酸化，经何途径诱导细胞凋亡，增效RAD001治疗NSCLC分子机制。.③ miRNA芯片检测筛选CGP57380和 RAD001处理的A549细胞中调控Akt/mTOR/Mnk通路关键分子的miRNA。.本项目取得四个方面的重要研究成果：.① p-Mnk1和p-eIF4E在NSCLC的发生、发展和侵袭转移中起重要作用，p-Mnk1可作为NSCLC患者预后差的独立分子标志。.② NSCLC尤其肺腺癌存在EGFR基因高突变、外显子19、21突变、高表达EGFR 和Flot-2 有作为NSCLC靶向疗效评估的分子标志。.③本课题首次阐明RAD001和CGP57380联合靶向抑制Akt/mTOR和Mnk/eIF4E通路，抑制NSCLC细胞增殖，逆转RAD001诱导的eIF4E磷酸化和抑制AKT的活性，经由死亡受体通路和线粒体途径诱导凋亡，增效RAD001治疗NSCLC的分子机制。.④文献回顾发现存在NSCLC靶向治疗耐药性miRNA，抑制耐药性miRNA表达能逆转耐药。生物信息学分析CGP57380和 RAD001处理后的A549细胞miRNA芯片，筛选获得Akt/mTOR/Mnk通路多个调控Mnk和eIF4E磷酸化的 miRNA。为深入探讨CGP57380抑制RAD001诱导eIF4E磷酸化，增效其抗NSCLC作用的分子调控机制提供实验基础。.本项目的完成为NSCLC个体化联合靶向治疗提供实验依据，具有重要临床转化应用前景。