Department of Neurological Surgery, University of California, Irvine, Irvine, CA 92697, USA.
J Neurooncol. 2010 Jan;96(2):191-200. doi: 10.1007/s11060-009-9963-8. Epub 2009 Jul 19.
We reported that PAX6 suppresses glioblastoma cell growth in vivo and anchorage-independent growth without significant alteration of cell proliferation in vitro, suggesting that PAX6 may alter the tumor microenvironment. Because we found that PAX6 downregulates expression of the gene encoding vascular endothelial growth factor A (VEGFA) in glioma cells, we used a subcutaneous xenograft model to verify PAX6 suppression of VEGFA-induced angiogenesis based on CD31-immunostaining of endothelial cells. The results showed a significant reduction of VEGFA at the transcription level in PAX6-transfected cells in xenografts and PAX6 has a suppressive effect on the microvascular amplification typically seen in glioblastoma. We showed that PAX6 suppression of VEGFA expression requires its DNA binding-domain. The C-terminal truncation mutant of PAX6, however, did not show the dominant negative function in regulating VEGFA expression that it showed previously in regulating MMP2 expression. In the glioma cell line U251HF, we further determined that blocking the PI3K/Akt signaling pathway with either adenoviral-mediated PTEN expression or LY294002 enhanced PAX6-mediated suppression of VEGFA in an additive manner; thus, PAX6-mediated suppression of VEGFA is not via the canonical pathway through HIF1A. These two VEGFA-regulatory pathways can also be similarly modulated in another malignant glioma cell line, U87, but not in LN229 where the basal VEGFA level is low and PTEN is wild-type. PAX6 suppression of VEGFA appears to be blocked in LN229. In conclusion, our data showed that PAX6 can initiate in glioma cells a new signaling pathway independent of PI3K/Akt-HIF1A signaling to suppress VEGFA expression.
我们曾报道 PAX6 在体内抑制胶质母细胞瘤细胞生长和体外无锚定依赖性生长，而对细胞增殖无明显改变，这表明 PAX6 可能改变肿瘤微环境。由于我们发现 PAX6 下调了编码血管内皮生长因子 A（VEGFA）的基因在神经胶质瘤细胞中的表达，我们使用皮下异种移植模型，基于内皮细胞 CD31 免疫染色来验证 PAX6 对 VEGFA 诱导的血管生成的抑制作用。结果表明，在异种移植物中 PAX6 转染细胞中 VEGFA 的转录水平显著降低，并且 PAX6 对胶质母细胞瘤中常见的微血管扩增具有抑制作用。我们表明 PAX6 对 VEGFA 表达的抑制需要其 DNA 结合域。然而，PAX6 的 C 末端截断突变体在调节 VEGFA 表达方面没有表现出先前在调节 MMP2 表达方面的显性负作用。在神经胶质瘤细胞系 U251HF 中，我们进一步确定通过腺病毒介导的 PTEN 表达或 LY294002 阻断 PI3K/Akt 信号通路以相加的方式增强了 PAX6 介导的 VEGFA 抑制；因此，PAX6 介导的 VEGFA 抑制不是通过 HIF1A 的经典途径。这两个 VEGFA 调节途径也可以在另一种恶性神经胶质瘤细胞系 U87 中类似地调节，但在 LN229 中不行，因为 LN229 的基础 VEGFA 水平较低且 PTEN 为野生型。PAX6 对 VEGFA 的抑制似乎在 LN229 中受阻。总之，我们的数据表明 PAX6 可以在神经胶质瘤细胞中启动独立于 PI3K/Akt-HIF1A 信号的新信号通路，以抑制 VEGFA 表达。
