Downregulation of FIP200 Induces Apoptosis of Glioblastoma Cells and Microvascular Endothelial Cells by Enhancing Pyk2 Activity

The expression of focal adhesion kinase family interacting protein of 200-kDa (FIP200) in normal brain is limited to some neurons and glial cells. On immunohistochemical analysis of biopsies of glioblastoma tumors, we detected FIP200 in the tumor cells, tumor-associated endothelial cells, and occasional glial cells. Human glioblastoma tumor cell lines and immortalized human astrocytes cultured in complete media also expressed FIP200 as did primary human brain microvessel endothelial cells (MvEC), which proliferate in culture and resemble reactive endothelial cells. Downregulation of endogenous expression of FIP200 using small interfering RNA resulted in induction of apoptosis in the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC. It has been shown by other investigators using cells from other tissues that FIP200 can interact directly with, and inhibit, proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK). In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK. Coimmunoprecipitation and colocalization studies indicated that the endogenous FIP200 was largely associated with Pyk2, rather than FAK, in the glioblastoma tumor cells and brain MvEC. Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells. In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors.

200kDa黏着斑激酶家族相互作用蛋白（FIP200）在正常脑组织中的表达局限于部分神经元与胶质细胞。针对胶质母细胞瘤（glioblastoma）活检样本（biopsies）的免疫组化分析（immunohistochemical analysis）显示，我们在肿瘤细胞、肿瘤相关内皮细胞以及偶见的胶质细胞中检测到了FIP200的表达。在完全培养基中培养的人胶质母细胞瘤细胞系、永生化人脑星形胶质细胞（immortalized human astrocytes），以及可在体外增殖并模拟反应性内皮细胞的原代人脑微血管内皮细胞（MvEC），同样表达FIP200。通过小干扰RNA（small interfering RNA）下调内源性FIP200的表达，可诱导人胶质母细胞瘤细胞、永生化人脑星形胶质细胞以及原代人脑MvEC发生细胞凋亡（apoptosis）。既往已有其他研究团队利用其他组织来源的细胞证实，FIP200可直接与富脯氨酸酪氨酸激酶2（Pyk2）和黏着斑激酶（FAK）结合并抑制其活性。在人胶质母细胞瘤细胞、永生化人脑星形胶质细胞以及原代人脑MvEC中，我们发现下调FIP200的表达可提升Pyk2的活性，但不会改变其蛋白表达水平，却对FAK的活性与表达均无影响。免疫共沉淀（coimmunoprecipitation）与共定位（colocalization）实验结果表明，在胶质母细胞瘤细胞及人脑MvEC中，内源性FIP200主要与Pyk2结合，而非FAK。此外，在上述细胞中，包含Pyk2自磷酸化位点（autophosphorylation site）的TAT-Pyk2融合蛋白（TAT-Pyk2-fusion protein）可显著抑制FIP200下调所引发的促凋亡效应。综上，在胶质母细胞瘤相关的肿瘤细胞、星形胶质细胞以及人脑MvEC中，下调内源性FIP200蛋白可促进细胞凋亡，这一效应极有可能源于FIP200与Pyk2的直接相互作用被解除，进而解除了其对Pyk2激活的抑制作用，提示FIP200的表达对于胶质母细胞瘤肿瘤中三类细胞的存活均不可或缺。