Mukherjee Joydeep, DeSouza Leroi V, Micallef Johann, Karim Zia, Croul Sid, Siu K W Michael, Guha Abhijit
Arthur and Sonia Labatts Brain Tumor Research Center, Hospital for Sick Children's Research Institute, University of Toronto, Toronto, Canada.
Cancer Res. 2009 Nov 15;69(22):8545-54. doi: 10.1158/0008-5472.CAN-09-1778. Epub 2009 Nov 10.
Glioblastoma multiforme (GBM) is the most common and lethal primary human brain tumor. GBMs are characterized by a variety of genetic alterations, among which oncogenic mutations of epidermal growth factor receptor (EGFRvIII) is most common. GBMs harboring EGFRvIII have increased proliferation and invasive characteristics versus those expressing wild-type (wt) EGFR. To identify the molecular basis of this increased tumorgenic phenotype, we used iTRAQ-labeling differential proteomic analysis. Among several differentially expressed proteins, we selected CRMP1, a protein implicated in cellular invasion that was markedly decreased in GBMs expressing EGFRvIII, for further study. The differential expression of CRMP1 was confirmed in a panel of human GBM cell lines and operative specimens that express wtEGFR or mutant EGFRvIII by quantitative real-time PCR, Western blot, and immunohistochemical analysis. In human GBM samples, decreased expression of CRMP1 correlated with EGFRvIII positivity. Knockdown of CRMP1 by siRNA resulted in increased invasion of wtEGFR expressing human GBM cells (U87 and U373) to those found in isogenic GBM cells. Exogenous expression of EGFRvIII in these wtEGFR-expressing GBM cells promoted their ability to invade and was accompanied by decreased expression of CRMP1. Rescuing CRMP1 expression decreased invasion of the EGFRvIII-expressing GBM cells by tilting the balance between Rac and Rho. Collectively, these results show that the loss of CRMP1 contribute to the increased invasive phenotype of human GBMs expressing mutant EGFRvIII.
多形性胶质母细胞瘤（GBM）是最常见且致命的原发性人脑肿瘤。GBM具有多种基因改变，其中表皮生长因子受体（EGFRvIII）的致癌突变最为常见。与表达野生型（wt）EGFR的GBM相比，携带EGFRvIII的GBM具有更强的增殖和侵袭特性。为了确定这种肿瘤发生表型增加的分子基础，我们使用了iTRAQ标记差异蛋白质组学分析。在几种差异表达的蛋白质中，我们选择了CRMP1，一种与细胞侵袭有关的蛋白质，它在表达EGFRvIII的GBM中显著降低，以便进一步研究。通过定量实时PCR、蛋白质印迹和免疫组织化学分析，在一组表达wtEGFR或突变型EGFRvIII的人GBM细胞系和手术标本中证实了CRMP1的差异表达。在人GBM样本中，CRMP1表达降低与EGFRvIII阳性相关。通过小干扰RNA敲低CRMP1导致表达wtEGFR的人GBM细胞（U87和U373）的侵袭能力高于同基因GBM细胞。在这些表达wtEGFR的GBM细胞中外源性表达EGFRvIII促进了它们的侵袭能力，并伴随着CRMP1表达的降低。挽救CRMP1表达通过改变Rac和Rho之间的平衡降低了表达EGFRvIII的GBM细胞的侵袭能力。总体而言，这些结果表明CRMP1的缺失导致了表达突变型EGFRvIII的人GBM侵袭表型的增加。
