表皮调节素EREG参与胶质母细胞瘤抵抗双氢青蒿素治疗的作用机制研究

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most aggressive cancer which is characterized by resistance to both radiotherapy and chemotherapy. Nowadays, Dihydroartemisinin (DHA) and some other artemisinin derivatives have shown the therapeutic effects on GBM in vitro and in vivo, becoming a promising candidate and lead compound for the development of innovative drugs against GBM. It is generally accepted that conquering resistances and further improving anti-tumor activity are of the strategic point in the further translational medicine process. In our previous study, the up-regulated expression of Epireglulin (EREG) was detected and confirmed in GBM cells treated with DHA. Interestingly, EREG is a kind of pan-ErbB ligand, and it could bind to ErbB receptors and initiate the downstream signaling pathway more efficiently. In view of the closed relationship between GBM and abnormal activation of ErbB pathway, we hypothesize that EREG might involve in the DHA resistance in GBM. In this study, we systematically combine the in vitro, in vivo and clinical experiments to investigated the effects and mechanisms of DHA resistance in GBM by up-regulating the expression of EREG. Firstly, the resistance effects are analyzed by cell viability assay, cell cycle assay, apoptosis assay, autophagy assay, cell adhesion assay and cell invasion assay. Moreover, the resistance mechanism is studied by ChIP, real-time PCR, Dual-Luciferase reporter assay and Western-blot. Finally, the above-mentioned findings are also confirmed by two-drug combination of DHA and ErbB inhibitor (anti-ErbB Mcabs, TKIs and anti-EREG neutralized Mcab) in treating nude mice xenografts and clinical tissue cultures. We truly hope our research could discover a novel pattern and mechanism of chemo-resistance in GBM, which would like to provide a new target and ideal for further GBM drug modified and development based on artemisinine derivatives. In addition, our findings also would facilitate the clinical application of ErbB inhibitor as an adjuvant in GBM chemo-therapy, redeeming these unlucky drugs in anti-GBM clinical trial.

胶质母细胞瘤（Glioblastoma, GBM）抗药性强，克服抵抗并提高活性是基于双氢青蒿素（Dihydroartemisinin, DHA）开发抗GBM药物的关键。前期发现DHA作用的GBM细胞能上调表达ErbB受体家族的广谱配体表皮调节素（Epiregulin, EREG）。考虑到ErbB的异常活化与GBM相关，课题组推测EREG可能参与GBM对DHA的抵抗。本研究结合体外细胞、整体动物和临床样本研究，采用细胞活力测定、细胞周期分析、细胞凋亡检测、细胞自噬研究、细胞黏附测定、细胞侵袭鉴定、转录因子鉴定、信号途径研究以及抗癌协同效应评价等方法，确认EREG参与GBM抵抗DHA的过程，并探明其分子机制。研究结果将提出一种GBM抗药新机制，为基于DHA的抗GBM药物优化提供新靶点和新思路，为基于DHA的GBM联合治疗提供新组合，也为单独治疗GBM鲜有收效的ErbB抑制剂提供临床应用新方向。

治疗药物匮乏且疗效差是胶质母细胞瘤（Glioblastoma, GBM）的治疗现状。克服抵抗是相关药物开发的瓶颈。近来发现，传统的抗疟药物双氢青蒿素（Dihydroartemisinin, DHA）具有抗癌活性，被视作抗肿瘤创新药物的候选之一。表皮生长因子受体EGFR及其家族成员ErbB的异常活化与GBM的发生、发展和治疗抵抗密切相关。课题组前期证实：DHA具有抗GBM作用，但有效剂量高、治疗活性还需提升。本研究整合了转录组和蛋白组测序、生物信息学分析、细胞活力测定、细胞周期分析、细胞凋亡检测、细胞迁移测定、细胞侵袭鉴定、转录因子分析、信号途径研究以及抗癌协同效应评价等技术对ErbB信号在GBM抵抗DHA治疗中的效应机制进行了探索性研究。结果表明：部分GBM细胞在接受DHA治疗时通过激活Sp1等转录因子而促进ErbB广谱配体表皮调节素（Epiregulin, EREG）的转录、表达和胞外分泌；上调的EREG能够以自分泌和旁分泌的方式激活自身和周围肿瘤细胞的EGFR及其相关JAK/STAT3信号通路，促进细胞因子（IL-6和LIF）、转录因子（SNAI2）、促增殖癌基因（MYC和MKI67）和迁移侵袭相关分子（FN1和MMP-14）的表达，进而以促进细胞增殖、存活、迁移和侵袭等方式推动肿瘤细胞的上皮细胞-间充质转化（Epithelial-Mesenchymal Transition, EMT）和肿瘤微环境（Tumor Microenvironment, TME）改变等恶性进程，进而参与GBM对DHA的治疗抵抗；基于上述机制筛选获得的EGFR酪氨酸激酶抑制剂（EGFR-TKI）阿法替尼能够通过抑制EGFR、STAT3和AKT等信号通路的磷酸化激活而提升GBM细胞对DHA的治疗敏感性，与DHA起到协同抗癌效应，并成功降低两种药物的有效治疗剂量。总而言之，本项目提出了一种依赖EREG/EGFR信号轴的GBM治疗抵抗新机制，为基于DHA的抗GBM药物优化和改造提供了新靶点和新思路（例如：DHA与抗EGFR中和性生物大分子嵌合物），为基于DHA的GBM联合治疗提供了新组合（例如：DHA与EGF-TKIs的联合应用），也为单独治疗GBM鲜有收益的EGFR抑制剂提供了临床应用新方向。