抑癌基因缺失下EGFR活化通过调控微环境适变介导胶质母细胞瘤恶性发展

Formation of murine primary glioblastoma multiforme (GBM) induced by overexpression of the epidermal growth factor receptor (EGFR) accompanied with loss of tumor suppressor PTEN and INK4a/ARF was recently demonstrated. The tumor was highly proliferating and infiltrative, however the mechanisms are yet unknown. In this study, U87MG GBM cells which intrinsically carry mutated PTEN and deleted INK4a/ARF locus was used as the primary GBM cell model. Different phenotypes of stable transfected U87MG cells were constructed including U87MG- EGFRvIII (mutated EGFR), U87MG-EGFR (wild type), U87MG-PTEN, U87MG-INK4a/ARF, U87MG-PTEN-INK4a/ARF and U87MG-vector as the vehicle control. Based on these cell models, in vitro, in vivo and ex vivo GBM models was further set up and compared. Combined methodology with Bioinformatics methods and laboratory techniques was employed to investigate mechanisms of the combined effect induced by the three gene variations in GBM cells on key intracellular signaling networks, functional modules with key molecule signatures and the tumor adaptation with the microenvironment through in vitro, in vivo and ex vivo transitions. Furthermore, the role of EGFR siganing activation on tumor microenvironmental adaptation and shaping up was the most investigated. We aim to find key molecular targets, multiple kinase target combination for effective suppression on tumor growth and infiltration in vivo, and in vitro methodology for monitoring efficacy of targeted therapy in GBM in vivo. Moreover, bioinformatics tools was used to analyze and compare high-throughout data of the present biological models in this study, those of clinical GBM of the same phenotype from relevant public databases such as TCGA, and those of Immune and inflammatory related diseases and chronic metabolism diseases obtained from public resources, in order to reveal common features on the regulation network and key nodes among different diseases. Moreover, combinations of anti-inflammation drugs, or antidiabetic drugs with targeted agents were tested on our GBM models to acquire the most effective treatment strategy on the GBM models. In summary, the methodology and results of our project may not only illustrate key mechanisms involved in the formation and progression of human GBM, but also provide interdisciplinary technical platform on relevant cancer research studies.

表皮生长因子EGFR过表达以及抑癌基因PTEN、INK4a/ARF突变缺失是一部分人胶质母细胞瘤（GBM）的特征性分子变异。这些基因变异可联合促发鼠原发性GBM发生，其肿瘤呈高侵袭性及恶性进展；而仅有抑癌基因缺失的肿瘤则较局限并呈慢性进展。本研究通过已构建的三基因联合变异的GBM模型包括体外细胞、裸鼠原位以及活体外组织培养模型等，结合生物学实验与生物信息学分析，剖析三基因变异组合所致肿瘤信号通路网络、功能模块和效应分子标签、可控性分子靶点以及携带基因变异细胞所介导的体内外微环境适变及其分子机制。通过比较抑癌基因缺失背景下EGFR活化通路对肿瘤体系建立和微环境的影响，寻找最佳靶控分子点，揭示肿瘤发生发展的机制。进一步通过在生物学模型所获得的高通量数据与临床肿瘤及免疫炎性疾病数据的比对分析，揭示不同疾病发展的规律，通过联合应用抗炎药及靶向抑制剂摧毁肿瘤微环境及其体系，有效控制GBM侵袭及进展。

本研究基于经典型胶质母细胞瘤（GBM）中三基因变异是致病的重要基因变异产生致病表型而展开研究工作。这些变异为抑癌基因PTEN、INK4a/ARF缺失，癌基因EGFR基因扩增、突变致使其蛋白过表达及高活性。EGFR及其突变体EGFRvIII是GBM靶向治疗方案的重要靶点，但极易产生耐药性。通过构建三基因变异的细胞学模型，以及裸鼠原位模型和收集相关临床数据及对比分析等策略，从体外、体内裸鼠原位GBM、以及临床标本活体等不同侧面收集研究数据。其中，1）体外GBM细胞模型实验中明确，PTEN缺失可引起炎症通路STAT3活化并促进细胞形态改变，快速侵袭与迁移；抑癌基因确实背景下，EGFRvIII对炎性微环境产生应答效应，表现为EGFRvIII/NF-kB/STAT3形成三分子复合体，促进GBM细胞侵袭与迁移；EGFRvIII在裸鼠内显著促进血管生成，表现为血管密度增加，并存在血管生成拟态现象（vasculogenic mimicry, VM）;在体外可形成管网状结构，并与内皮细胞可通过NOTCH途径促进血管生成，其中STAT3可为重要靶点。2）在裸鼠颅内原位模型中，在抑癌基因缺失下，EGFRvIII表达可显著促进肿瘤恶性进展。通过对裸鼠颅内肿瘤微环境中不同部位的分析发现，肿瘤免疫微环境中单核-巨噬细胞是最多浸润的免疫细胞，肿瘤中心与边缘呈现不同的免疫调控特征。对临床标本的整理与分析发现，3）GBM患者颅内肿瘤也存在大量的单核-巨噬细胞和CD4+Th细胞等。综上所述，本研究明确STAT3信号通路作为抑癌基因缺失下，EGFRvIII+肿瘤及微环境中STAT3可能为重要靶点，联合应用可有效抑制EGFRvIII介导的微环境适变及肿瘤恶性进展。相关临床数据在进一步分析中。本研究研究体系及分析可为相关治疗提供重要线索与依据，为进一步申请专利、申报成果奖及转化应用打下坚实基础。

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