TGF-β-Notch信号通路cross-talk对胶质瘤干细胞的功能调控作用及机制研究

Emerging data suggest that glioblastoma stem cells (GSCs) are responsible for tumor growth and maintenance and may account for tumor recurrence despite conventional therapies. The identification of these cells has spurred intense interest in understanding molecular mechanisms that regulate their biological behavior. Such knowledge promises to open entirely new avenues of treatment for gliblastoma patients by targeting these cell...Our recently published data show that TGF-β signaling pathway plays a critical role in the regulation of tumorigenic properties of GSCs. Blockade of TGF-β pathway inhibited the proliferation, self-renewal and radioresistance of GSCs. Moreover, Notch1 and Hey1 genes were found to be down-regulated by TGF-β singnaling inhibition,stongly indicating that cross-talk between TGF-β and Notch may be involved in the TGF-β mediated regulation of GSCs.Interaction between TGF-β and Notch signaling has been reported in multiple cell types,however the relationship of Notch and TGF-β signaling in GSCs is unknown.. .The present project is to address the mechanisms of cross-talk between TGF-β and Notch and its role in the regulation of GSCc at the molecular and functional level. To examine molecular cross-talk between TGF-β and Notch signaling in GSCs, some key molecules of TGF-β and Notch signaling pathways will be evaluated when one or the other pathway is inhibited or activated in cultured GSCs. To determine the role of aforementioned cross-talk in GSCs, proliferation, self-renewal,differentiation, apoptosis and radiation response of GSCs under various intervention of TGF-β and/or Notch signaling will be investigated.In addition, orthotopic mouse model will be used to examine the cross-talk between TGF-β and Notch in vivo. GSCs transfected with lentivirus encoding NICD or Notch1-shRNA or control vector will be transplanted into the brains of SCID mouse, then treated with or without TGF-β signaling inhibitor. Tumor size, animal survival, CD133 positive cells fraction, apoptosis, microvessel density,and some molecules of both signaling pathways will be measured. Furthermore, gene expression profiling will be performed using transcriptome microarray and pathway network will be analyzed using Ingenuity Pathway Analysis software to define the relevant molecular mechanisms. The data generated from this novel study are highly expected to provide new molecular targets and new concepts for GSCs-targeted treatment in glioblastoma.

胶质瘤干细胞（GSCs）被认为是胶质母细胞瘤恶性表型的根源和关键治疗靶点。深入探讨GSCs的调控机制是针对这些细胞进行分子干预的前提。申请者最新发表的研究显示，TGF-β信号通路对GSCs的功能调控发挥关键作用，并提示Notch通路可能参与TGF-β对GSCs的调控作用，两者之间存在cross-talk机制。基于此研究背景，本项目以GSCs和小鼠颅内移植胶质瘤模型为实验对象，研究TGF-β和/或Notch信号通路干预对上述通路关键节点分子的表达及GSCs生物学行为的影响，同时采用基因微阵列技术检测GSCs基因表达谱的变化，并运用生物信息软件分析差异表达基因的功能和信号通路网络。通过本项目的实施，将揭示TGF-β-Notch信号通路cross-talk对GSCs的功能调控作用及机制，为设计以GSCs关键信号通路为靶点的治疗策略提供理论基础和实验依据。

申请者前期研究显示，TGF-β信号通路对胶质瘤细胞（包括胶质瘤干细胞）的功能调控发挥关键作用，并提示Notch通路可能参与介导TGF-β通路对胶质瘤细胞恶性表型的调控作用。本课题探讨TGF-β对Notch1信号通路的调控作用及其对胶质瘤生物学行为以及放射敏感性的影响。69例GBM手术标本免疫组化染色显示TGF-β和Notch1在胶质瘤中高表达，其表达水平与患者预后相关。荧光素酶报告基因检测发现转染TGF-β受体特异性siRNA抑制Notch1基因的转录活性，TGF-β信号通路抑制剂显著下调Notch1及其下游基因的表达，证实TGF-β在胶质瘤细胞中转录调控Notch1信号通路。进一步实验证实TGF-β通过Notch1通路调控胶质瘤的生长、放射敏感性和胶质瘤干细胞的自我更新能力。CRISPR/CAS9介导的Notch1基因敲除抑制胶质瘤干细胞和胶质瘤建系细胞的增殖，抑制肿瘤相关的血管生成，促进凋亡，增加放射诱导的DNA损伤和抑制DNA损伤的修复，从而增加胶质瘤细胞的放射敏感性。裸鼠皮下移植肿瘤模型和小鼠颅内原位移植肿瘤模型均证实Notch1基因敲除抑制肿瘤生长，联合放疗可以产生协同作用，增加肿瘤的放射敏感性。免疫组化结果显示Notch1基因敲除联合放疗可以显著抑制裸鼠皮下移植肿瘤的增殖，血管生成和乏氧。为进一步研究Notch1基因对胶质瘤生物学行为调控的分子机制，我们采用microarray技术检测Notch1基因敲除前后基因表达谱的变化，并进行相关生物信息学分析。结果证实Notch1在胶质瘤细胞上通过调控CCND1、CDC25C、CDK1和CHEK1基因的表达对胶质瘤的生物学行为发挥作用，值得下一步更深入研究。总之，我们的结果表明TGF-β和Notch1信号通路对胶质瘤（包括胶质瘤干细胞）的功能具有重要调控作用，为设计以TGF-β和Notch1信号通路为靶点的治疗策略提供理论基础和实验依据。