EGFR过表达协同PTEN缺失介导的免疫炎性网络促进胶质母细胞瘤恶性进展的分子机制

As key features for human glioblastoma multiforme (GBM), tumor rapid proliferation and infiltration to normal brain are related to specific tumor microenvironment. In our previous work, we found that constitutive RTK signaling induced by over-expression of wild-type or mutant EGFR cooperated with loss of tumor suppressor PTEN function in promoting significant alteration of immune and inflammation-related molecules and thereof functions. Therefore, such gene alterations may promote tumor interaction with microenvironment and further malignant progression of the tumor. .In this study, we investigated the related inflammatory network in GBM cells which either over expressed wild type EGFR or mutant EGFR accompanied with loss of PTEN, in comparison with glioma cells with only low level of EGFR with/out intact PTEN. Tumor samples of such phenotype and the controls were also applied. We selected the best fitted 3D- or 2D- in vitro cultural condition for such phenotypic GBM cells to infiltrate and proliferate in vitro, on which cDNA microarray analysis was applied, with tumor cells without EGFR/PTEN alteration as the controls. Bioinformatic tools such as gene set enrichment array (GSEA), GO, KEGG pathway database and IPA（Ingenuity Pathways Analysis）was used to compare and extract data related to immune-inflammatory network and key nodes in public databases, array data from clinical samples and related serum cytokine array, in order to reveal key inflammatory network and nodes relevant to such phenotypic GBM. Targeted agents, siRNA, antibody, cytokine and anti-inflammation drug were used to verify the final results. .In summary, our results may provide useful information on key mechanisms linking tumor progression and immune regulatory network and molecular targets for targeted therapy. Our 3-D or 2-D cell culture system may be useful tools as microenvironment to test the efficacy of targeted therapy with tumor specimens from human GBM.

人类胶质母细胞瘤(GBM)快速增殖、侵袭的特性与炎性微环境存在复杂关联。前期工作发现经典型GBM的分子特征，EGFR突变/过表达和抑癌基因PTEN突变性缺失可协同促进肿瘤细胞免疫炎性因子及相关功能的显著变化，提示基因变异可能促进肿瘤与微环境相互作用乃至恶性进展。本研究以经典型GBM和对照细胞为对象，比较缺氧条件下其与细胞外基质和/或黏附分子的相互作用以及单核细胞对肿瘤/纤维细胞球的浸润等，筛选出最适于经典型GBM细胞增殖与侵袭的二维和三维细胞培养体系；结合其表达谱数据与生物信息学工具，提取特征性免疫炎性调控网络和关键分子节点；应用靶向抑制剂、细胞因子、抗炎药等进行实验验证。进一步将在体外模型上识别的免疫炎性网络在相关临床GBM标本、肿瘤相关炎性细胞及其血清免疫因子表达谱等数据中进行证实，最终确定临床上影响经典型GBM恶性进展的免疫炎性网络、关键节点并建立模拟肿瘤炎性微环境的体外培养体系。

人类恶性肿瘤的发生发展是一系列癌基因活化及抑癌基因失活的程序性结果。其中肿瘤与其局部微环境直接接触和相互作用，以及与此相关的系统性表征在恶性肿瘤演变过程中发挥关键作用。本项目基于“非可控性炎症恶性转化的调控网络及其分子机制”重大研究计划指南中的科学问题“非可控性炎症恶性转化的调控网络与关键节点”为中心而展开研究，主要分为两部分内容。.第一部分以恶性胶质母细胞瘤（glioblastoma, GBM）为研究对象，基于炎症与肿瘤相关的内源性途径，即起源于基因变异所引起炎症反应促进肿瘤恶性进展为线索展开研究。聚焦GBM中以1/4频率发生的特征性基因变异包括表皮生长因子受体（EGFR）活化和抑癌基因PTEN突变性缺失等。通过对具有该基因变异特征的细胞模型、动物模型以及所收集的临床标本和公共数据库中病人标本进行生物学实验和生物信息学分析，解析基于基因变异介导的内源性炎症反应驱动疾病恶性进展的调控网络和分子机制。特别是在体外细胞模型系列性研究中通过模拟活体内微环境找寻线索，进而在动物模型和临床标本中验证重要调控网络和分子机制，对于明确有效靶点和耐药机制具有重要的临床应用意义。研究首次发现，EGFR活化（活化型突变体）和PTEN缺失协同引起炎症相关JAK2/STAT3通路激活并介导肿瘤侵袭；该通路的激活依赖于EGFR参与的黏着斑复合体集聚重要细胞内活化途径如PI3K、Ras等。靶向JAK2/STAT3抑制剂AG490、WP1066等，而非STAT3抑制剂可有效抑制该表型肿瘤的侵袭。通过模拟缺氧、细胞外基质微环境，证实EGFR突变体、PTEN突变型缺失通过促进黏着斑复合体重组装以及激活相关通路发挥作用，并在动物模型及临床标本中证实。本研究通过收集系列性临床标本和构建体外模拟微环境，已初步建立研究体系以利于下一步转化应用与产品研发。.第二部分研究以结直肠癌（colorectal cancer, CRC）为研究对象揭示炎症微环境促进结直肠癌恶性进展的调控网络与分子机制。研究首次发现转录抑制子GFI1是炎症微环境促进结直肠癌细胞转移的关键调控因子，并在动物实验和临床标本进一步确证。.在研期间以通讯作者发表论文16篇，其中影响因子大于5两篇，拟投Cancer Cell 1篇（通讯作者）。总之，本项目取得的系列性研究结果，揭示了炎-癌转化新机制，为下一步转化医学研发与应用奠定了良好基础。