Hagemann Carsten, Gloger Jens, Anacker Jelena, Said Harun M, Gerngras Stefanie, Kühnel Siglinde, Meyer Christoph, Rapp Ulf Rüdiger, Kämmerer Ulrike, Vordermark Dirk, Flentje Michael, Roosen Klaus, Vince Giles Hamilton
Department of Neurosurgery, Tumorbiology Laboratory, University of Würzburg, D-97080 Würzburg, Germany.
Int J Mol Med. 2009 Jan;23(1):17-31.
RAF proteins are well known oncoproteins. The B-RAF has been shown to be activated by mutations in a multitude of human cancers. Alterations of C-RAF expression are discussed to play a role in lung cancer. Only for A-RAF no link to tumorigenesis has been published so far. Malignant gliomas are the most prevalent primary brain tumors of adults. They are highly invasive and very difficult to treat, despite of surgery, gamma-irradiation and chemotherapy. Although a role of the mitogenic Ras-RAF-MEK-ERK signalling cascade in brain tumor development is well established, there are only few reports available addressing alterations in RAF sequence or protein expression and function in human gliomas. We analysed the mutational status of A-RAF and B-RAF in human glioblastomas (GBM) by sequencing. Then we checked for RAF gene amplification by dot blot hybridization and examined RAF mRNA and protein expression patterns in human astrocytic gliomas of WHO grade II (LGA) and IV (GBM) by semiquantitative RT-PCR and Western blotting, respectively. The results were correlated with patients prognosis. Finally, we performed functional assays to address a putative function of A-RAF in glioma cell proliferation and migration. We showed that RAF mutations are a rare event in glioblastoma multiforme. A-raf gene amplification was more often detected and overexpression of all three RAF proteins on mRNA and protein level was regularly found in human malignant gliomas. Whereas A-RAF and C-RAF expression was negatively correlated with the patients prognosis, B-RAF expression had a positive effect. Since neither A-RAF, nor C-RAF expression had any influence on proliferation and migration of GBM cells, putative functions of C-RAF in angiogenesis and of A-RAF in regulation of metabolism are discussed. Our data indicate that RAF proteins might be valuable targets for small molecule therapies. However, initially specific functions of RAF during tumorigenesis have to be elucidated.
RAF蛋白是众所周知的癌蛋白。已证明B-RAF在多种人类癌症中因突变而被激活。有讨论认为C-RAF表达的改变在肺癌中起作用。迄今为止，仅A-RAF尚未发表与肿瘤发生相关的报道。恶性胶质瘤是成人中最常见的原发性脑肿瘤。它们具有高度侵袭性，尽管进行了手术、伽马射线照射和化疗，仍很难治疗。虽然有丝分裂原性的Ras-RAF-MEK-ERK信号级联在脑肿瘤发生中的作用已得到充分证实，但关于人类胶质瘤中RAF序列、蛋白表达及功能改变的报道却很少。我们通过测序分析了人类胶质母细胞瘤（GBM）中A-RAF和B-RAF的突变状态。然后我们通过斑点印迹杂交检测RAF基因扩增，并分别通过半定量逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法检测WHO二级（LGA）和四级（GBM）人类星形胶质细胞瘤中RAF mRNA和蛋白表达模式。结果与患者预后相关。最后，我们进行了功能试验以探讨A-RAF在胶质瘤细胞增殖和迁移中的假定功能。我们发现RAF突变在多形性胶质母细胞瘤中是罕见事件。在人类恶性胶质瘤中更常检测到A-raf基因扩增，并且在mRNA和蛋白水平上经常发现所有三种RAF蛋白的过表达。虽然A-RAF和C-RAF表达与患者预后呈负相关，但B-RAF表达具有积极作用。由于A-RAF和C-RAF表达对GBM细胞的增殖和迁移均无任何影响，因此讨论了C-RAF在血管生成中的假定功能以及A-RAF在代谢调节中的假定功能。我们的数据表明RAF蛋白可能是小分子疗法的有价值靶点。然而，首先必须阐明RAF在肿瘤发生过程中的特定功能。
