IDH1突变经EGFR介导的自噬信号通路调控胶质母细胞瘤增殖及侵袭的作用

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. Isocitrate dehydrogenase-1 (IDH1) is a newly discovered TCA enzymes mutated in GBM. The mutation of IDH1 is associated to the clinical classification and prognosis of GBM. IDH1 mutation might play an important role in GBM but its role and mechanisms remains unclear. Our preliminary results show that IDH1 mutation binds direactly to EGFR, the binding between IDH1 mutation and EGFRvIII become stronger under OGD condition, overexpression of IDH1 mutation up-regulates autophagy protein Beclin and LC3, indicating that there is “Energy metabolism-IDH1 mutation-EGFR/PI3K/AKT/mTOR-autophagy” signaling in GBM cells. The purpose of this study is to: 1) verify the binding between IDH1 mutation and EGFR under glucose deprivation、hypoxia and free amino acids by GST pull down、Co-IP and FRET, and investigate the effect of autophagy on GBM cell proliferation and invasion; 2) screen the shortest peptide of IDH1 mutation binding with EGFR by GST pull down. The study will provide new mechanism between abnormal energy metabolism and the development of tumor, and the results may provide potential therapeutic targets for GBM.

胶质母细胞瘤(GBM)是成人最多见、恶性程度最高的脑肿瘤。异柠檬酸脱氢酶1(IDH1)是GBM特异的三羧酸循环代谢酶, 与GBM分型及预后密切相关。IDH1突变在GBM发生发展中的确切作用和机制还不清楚。前期实验发现IDH1突变与癌基因EGFR蛋白结合，缺糖缺氧条件下IDH1突变与EGFRvIII蛋白结合增强。过表达IDH1突变上调自噬蛋白Beclin和LC3表达，提示GBM细胞中存在“能量代谢-IDH1突变-EGFR/PI3K/AKT/mTOR-自噬”信号通路。本项目拟1）无糖、无氧、无氨基酸条件下GST pull down、Co-IP和FRET进一步确证IDH1突变与EGFR的结合，并观察自噬激活对GBM细胞增殖和侵袭的影响；2）GST pull down筛选IDH1突变与EGFR结合的最短功能肽段。本研究将为揭示能量代谢异常与肿瘤发生发展的机制提出新观点，并为GBM药物研发提供基础。

胶质母细胞瘤（Glioblastoma, GBM）是脑内胶质瘤中恶性程度极高的肿瘤，治疗后平均存活时间14个月。GBM高侵袭性、组织坏死和微血管过度增生的病理特征均与肿瘤组织的异常能量代谢相关。异柠檬酸脱氢酶（isocitrate dehydrogenase, IDH）是糖代谢中的三羧酸循环代谢酶，其中IDH1在GBM突变率极高，是GBM临床分型和预后的重要标志。本项目主要研究IDH1突变在GBM发生发展中的作用和机制。首先，Western blot观察GBM细胞系LN229和U87MG中内源性IDH1和突变的IDH1R132H都有表达，其中U87MG中两者表达量较LN229高。慢病毒感染过表达突变IDH1能抑制U87MG和LN229细胞的增殖和迁移；同时CRISPR/Cas9建立稳定敲除IDH1后GBM细胞增殖和迁移明显抑制。裸鼠皮下接种稳定转染IDH1、IDH1R132H、CRISPR敲除IDH1的U87MG细胞，突变组以及敲除组肿瘤的生长均变缓慢，说明突变IDH1R132H具有抑制胶质瘤生长的作用，正常的IDH1是促进胶质瘤生长的必要条件。其次在慢病毒感染过表达IDH1R132H以及CRISPR/Cas9敲除内源性IDH1的GBM细胞中，正常培养条件下，R132H突变组以及敲除组细胞容易产生自噬；EBSS和无糖条件处理3小时后两者上调p-AKT Ser473，p-mTOR Ser2448水平, 抑制细胞自噬，说明在正常条件下，IDH1敲除或R132H突变使得异柠檬酸脱氢酶活力下降，降低三羧酸循环为基础的能量代谢，引起细胞自噬；在无糖培养和EBSS无氨基酸培养中，IDH1敲除或R132H突变不能影响三羧酸循环中以非糖物质为底物的其他酶的活性，细胞机器可以调整维持基本的能量代谢，细胞自噬情况不明显。进一步在GBM细胞和293T细胞采用时间分辨能量共转移和GST pull down方法证明IDH1 R132H与EGFR有结合，且两者相互作用依赖于IDH1的完整结构，会影响PI3K-AKT-mTOR通路而引起细胞自噬。综上所述，本研究证明了IDH1突变通过与EGFR相结合后调控PI3K/AKT/mTOR信号通路，引起细胞自噬，影响GBM增殖和迁移。本研究将为揭示能量代谢异常与肿瘤发生发展的机制提出新观点。