DEPDC1通过CDC20信号通路调控胶质母细胞瘤的恶性表型和放射敏感性及机制研究

Glioblastoma multiforme (GBM) is the most common primary brain tumor with extremely dismal prognosis despite multimodal conventional therapies. Thus, it’s very urgent to seek alternate treatments through a better understanding of the cell biology and through some new molecular targets. . .DEPDC1 is a highly-conserved protein containing the DEP (Dishevelled, EGL-10, Pleckstrin) domain which has been reported to regulate a broad range of cellular functions. Emerging data suggests that DEPDC1 plays an important role in tumorgenesis, in particular, bladder cancer, breast cancer and multiple myeloma. However, the exact role of DEPDC1 in GBM has not been reported. Our DNA microarrays and/or qRT-PCR analysis revealed an evident up-regulation of DEPDC1 mRNA level in GBM cell lines and glioblastoma stem cells (GSC), which was confirmed in clinical samples of GBM using immunohistochemical staining. Kaplan-Meier analysis showed that high expression of DEPDC1 conferred a poor prognosis. Knockdown of DEPDC1 in GBM cells inhibited cell proliferation, increased cell apoptosis and radiosensitivity, as well as retarded xenograft tumor growth in vivo. More importantly, DEPDC1 knockdown significantly attenuated the self-renewal capability of GSCs by neurosphere formation and limiting dilution assay. These data suggested that DEPDC1 may be a key regulator of the malignant phenotype and radiosensitivity of GBM. To further explore the potential molecular mechanism, we searched correlated gene with DEPDC1 in TCGA cohort using R2: Genomics Analysis and Visualization Platform and found that CDC20 was significantly positively correlated with DEPDC1, which was then validated in GBM tissues. Microarrays and Western blot analysis showed that knockdown of DEPDC1 in GBM cells decreased the expression of CDC20. Luciferase reporter assay revealed that the transcription activity of CDC20 was inhibited by DEPDC1 shRNA transfection. Furthermore, the cell growth inhibitory effect of DEPDC1 knockdown was completely abrogated by additional ectopic over-expression of CDC20. All of the above results suggest that DEPDC1 may regulate the malignant phenotype and radiosensitivity of GBM through CDC20 signaling pathway. But the underlying molecular and cellular mechanism is still elusive and need to be further explored...This project intends to test the hypothesis that DEPDC1 regulates malignant phenotype and radiosensitivity of GBM through CDC20 signaling pathway. To this end, the correlation between DEPDC1 and CDC20, as well as other related markers and clinicopathologic features in GBM patients will be evaluated. Some key molecules in the CDC20 signaling pathway will be measured when DEPDC1 is down or up-regulated in GBM cells and GSCs. Proliferation, invasion, apoptosis, cell cycle and radiosensitivity of GBM cell lines as well as self-renewal and differentiation of GSCs under various intervention of DEPDC1 and/or CDC20 signaling will be investigated. In addition, orthotopic intracranial model will be utilized to evaluate the effects of DEPDC1-CDC20 signaling network on the malignant phenotype of GBM in vivo. Tumor size, animal survival, apoptosis and some valuable molecules 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. Through the implementation of this project, we will reveal the role of DEPDC1-CDC20 network in the regulation of malignant phenotype and radiosensitivity in GBM, as well as the possible underlying mechanisms, providing a theoretic and experimental basis for development of novel targeted therapies in GBM.

胶质母细胞瘤（GBM）是成人颅内最常见的原发恶性肿瘤。申请者前期研究发现DEPDC1在 GBM和胶质瘤干细胞（GSC）中表达增加，下调DEPDC1显著抑制GBM细胞增殖，促进凋亡，提高放射敏感性，抑制裸鼠皮下移植瘤的生长和GSC的自我更新能力。TCGA数据库分析和GBM组织标本检测显示DEPDC1与CDC20表达正相关；敲减DEPDC1明显降低CDC20表达水平，过表达CDC20逆转DEPDC1下调对GBM的增殖抑制作用。据此，我们推测DEPDC1可能通过CDC20通路调控GBM的恶性表型和放射敏感性。本项目以GBM细胞系、GSC和小鼠颅内移植瘤模型为对象，通过分子生物学、细胞生物学、CRISPR/CAS9基因编辑和基因微阵列等技术和方法，深入研究DEPDC1通过CDC20通路对GBM恶性表型和放射敏感性的调控作用，并阐明其中可能的分子和细胞学机制，为GBM治疗提供新的靶点和和实验依据。

胶质母细胞瘤（GBM）是成人颅内最常见的原发恶性肿瘤。申请者前期研究发现DEPDC1在GBM和胶质瘤干细胞（GSC）中表达增加，下调DEPDC1显著抑制GBM细胞增殖，促进凋亡，提高放射敏感性，敲减DEPDC1明显降低CDK1表达水平，我们推测DEPDC1可能通过CDK1通路调控GBM的恶性表型和放射敏感性。本课题旨在探讨DEPDC1对CDK1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。脑胶质瘤组织芯片以及72例GBM手术标本免疫组化染色显示DEPDC1在胶质瘤中高表达，其表达水平与患者预后相关。DEPDC1基因敲减明显抑制胶质瘤细胞增殖、迁移和胶质瘤干细胞的自我更新，促进肿瘤细胞凋亡，增加放射线诱导的DNA断裂，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证实DEPDC1基因敲减抑制胶质瘤生长。为进一步研究DEPDC1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DEPDC1基因敲减前后基因表达谱的变化，并进行相关生物信息学分析。荧光素酶报告基因检测结果证实DEPDC1对CDK1有转录调控作用，免疫共沉淀实验（CO-IP）结果进一步证实DEPDC1和CDK1有蛋白互作关系，结果提示DEPDC1通过调控CDK1基因的表达对胶质瘤的生物学行为发挥作用。通过功能回复实验，过表达CDK1可以逆转DEPDC1敲减对胶质瘤细胞生物学行为的影响。实验结果验证了我们的假设：DEPDC1通过CDK1信号通路调控胶质瘤恶性表型，针对DEPDC1-CDK1信号通路的靶向治疗可以有效抑制胶质瘤生长，提高放疗效果，值得下一步深入研究。本课题通过细胞、动物和分子水平的系统研究，已经明确DEPDC1通过对CDK1的表达进行转录调控，对胶质瘤细胞的生物学行为具有重要的调控作用，为设计以 DEPDC1-CDK1 信号通路为靶点的治疗策略和预后判断提供新的理论基础和实验依据。