Department of Pathology, University of Virginia, Charlottesville, VA 22908, USA.
BMC Cancer. 2011 May 20;11:184. doi: 10.1186/1471-2407-11-184.
Glioblastoma (GBM) is a highly aggressive malignant primary brain tumor, characterized by rapid growth, diffuse infiltration of cells into both adjacent and remote brain regions, and a generalized resistance to currently available treatment modalities. Recent reports in the literature suggest that Signal Transducers and Activators of Transcription (STATs) play important roles in the regulation of GBM pathophysiology.
STAT6 protein expression was analyzed by Western blotting in GBM cell lines and by immunohistochemistry in a tissue microarray (TMA) of glioma patient tissues. We utilized shRNA against STAT6 to investigate the effects of prolonged STAT6 depletion on the growth and invasion of two STAT6-positive GBM cell lines. Cell proliferation was assessed by measuring (3)H-Thymidine uptake over time. Invasion was measured using an in vitro transwell assay in which cells invade through a type IV collagen matrix toward a chemoattractant (Fetal Bovine Serum). Cells were then stained and counted. Kaplan-Meyer survival curves were generated to show the correlation between STAT6 gene expression and patient survival in 343 glioma patients and in a subset of patients with only GBM. Gene expression microarray and clinical data were acquired from the Rembrandt 1 public data depository (https://caintegrator.nci.nih.gov/rembrandt/). Lastly, a genome-wide expression microarray analysis was performed to compare gene expression in wild-type GBM cells to expression in stable STAT6 knockdown clones.
STAT6 was expressed in 2 GBM cell lines, U-1242MG and U-87MG, and in normal astrocytes (NHA) but not in the U-251MG GBM cell line. In our TMA study, STAT6 immunostaining was visible in the majority of astrocytomas of all grades (I-IV) but not in normal brain tissue. In positive cells, STAT6 was localized exclusively in the nuclei over 95% of the time. STAT6-deficient GBM cells showed a reduction in (3)H-Thymidine uptake compared to the wild-type. There was some variation among the different shRNA- silenced clones, but all had a reduction in (3)H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1) and urokinase Plasminogen activator (uPA) as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt 1 gene expression microarray and clinical data, there was a significant difference in survival (P < 0.05) between glioma patients with up- and down-regulated STAT6. Decreased STAT6 expression correlated with longer survival times. In two subsets of patients with either grade IV tumors (GBM) or Grade II/III astrocytomas, there was a similar trend that however did not reach statistical significance.
Taken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target.
胶质母细胞瘤（GBM）是一种高度侵袭性的恶性原发性脑肿瘤，其特征为快速生长、细胞弥漫性浸润到相邻和远处的脑区，以及对目前可用的治疗方法普遍耐药。最近的文献报道表明，信号转导和转录激活因子（STATs）在调节 GBM 病理生理学方面发挥着重要作用。
通过 Western blot 在 GBM 细胞系中分析 STAT6 蛋白表达，并通过组织微阵列（TMA）对神经胶质瘤患者组织进行免疫组织化学分析。我们利用针对 STAT6 的 shRNA 来研究长期 STAT6 耗竭对两种 STAT6 阳性 GBM 细胞系生长和侵袭的影响。通过测量（3）H-胸苷摄取随时间的变化来评估细胞增殖。通过体外 Transwell 侵袭实验测量侵袭，其中细胞穿过 IV 型胶原基质向趋化因子（胎牛血清）侵袭。然后对细胞进行染色和计数。Kaplan-Meier 生存曲线用于显示 343 名神经胶质瘤患者和仅 GBM 患者亚组中 STAT6 基因表达与患者生存之间的相关性。从 Rembrandt 1 公共数据存储库（https://caintegrator.nci.nih.gov/rembrandt/）获取基因表达微阵列和临床数据。最后，进行全基因组表达微阵列分析，比较野生型 GBM 细胞与稳定 STAT6 敲低克隆的基因表达。
STAT6 在 2 种 GBM 细胞系 U-1242MG 和 U-87MG 以及正常星形胶质细胞（NHA）中表达，但在 U-251MG GBM 细胞系中不表达。在我们的 TMA 研究中，STAT6 免疫染色可见于所有级别（I-IV）的星形细胞瘤中，但在正常脑组织中不可见。在阳性细胞中，STAT6 主要定位于细胞核，核内定位占 95%以上。与野生型相比，STAT6 缺陷型 GBM 细胞的（3）H-胸苷摄取减少。不同的 shRNA 沉默克隆之间存在一些差异，但在 U-1242MG 和 U-87MG 细胞中，（3）H-胸苷摄取均减少了 35%-70%，减少了 40%-50%。此外，在我们的体外跨膜侵袭实验中，STAT6 耗竭的细胞比对照细胞的侵袭性降低。U-1242MG 和 U-87MG 细胞的侵袭性分别降低了 25%-40%和 30%-75%。微阵列分析鉴定出基质金属蛋白酶 1（MMP-1）和尿激酶纤溶酶原激活物（uPA）作为可能参与促进 GBM 细胞侵袭的 STAT6 靶基因。基于 Rembrandt 1 基因表达微阵列和临床数据的 Kaplan-Meier 生存曲线显示，STAT6 上调和下调的神经胶质瘤患者的生存存在显著差异（P<0.05）。STAT6 表达降低与较长的生存时间相关。在具有 IV 级肿瘤（GBM）或 II/III 级星形细胞瘤的两个亚组患者中，存在相似的趋势，但未达到统计学意义。
综上所述，这些发现表明 STAT6 在增强 GBM 中的细胞增殖和侵袭中发挥作用，这可能解释了为什么 STAT6 的上调与神经胶质瘤患者的生存时间较短有关。本报告因此确定 STAT6 为新的潜在有前途的治疗靶点。
