Shien-lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, Japan.
Mol Cancer. 2013 Apr 25;12:31. doi: 10.1186/1476-4598-12-31.
Expression of the constitutively activated mutant EGFR variant III (EGFRvIII), the most common mutation in glioblastoma multiforme (GBMs), has been clinically correlated with tumor proliferation, invasion, and angiogenesis. In this study, we examined the role of EGFRvIII on the tumor microenvironment, especially on angiogenesis.
To study the role of EGFRvIII in tumor angiogenesis, we prepared LN229 glioblastoma transfected with enhanced green fluorescent protein (EGFP), wild-type EGFR, or EGFRvIII (LN229-WT or -vIII), and examined tumor growth and microvessel density in the tumors. Additionally, the potential angiogenic factors were identified by real-time PCR analysis, and the functions in LN229-vIII cells were examined.
LN229-vIII cells showed more aggressive tumor growth and higher vascularity as compared to LN229-WT cells in vivo, although there was no significant difference in the cell growth rates in vitro. We next investigated the expression of 60 angiogenesis-related factors to clarify the mechanisms underlying the difference in vascularity between tumor xenografts of LN229-vIII and LN229-WT. We found that the mRNA and protein expressions of angiopoietin-like 4 (Angptl4), a secreted protein involved in angiogenesis and metabolism regulation, were significantly induced by EGFRvIII overexpression, both in vitro and in vivo. Constitutive knockdown of Angptl4 in LN229-vIII using shRNA significantly decreased the microvessel density in the tumor xenografts and suppressed tumor growth. To clarify the regulatory mechanisms of Angptl4 by EGFRvIII, we analyzed the signaling pathways and transcription factors by pharmacological inhibition and RNA interference. U0126, an ERK signal inhibitor dramatically suppressed Angptl4 expression. The transcription factor c-Myc, which is regulated by ERK, was activated in the LN229-vIII cells and knockdown of c-Myc using siRNA also attenuated Angptl4 expression in the LN229-vIII cells. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed increased recruitment of c-Myc to the promoter region of Angptl4 in the LN229-vIII cells.
In summary, we demonstrated that EGFRvIII induces Angptl4 expression through the ERK/c-Myc pathway and promotes tumor angiogenesis in malignant gliomas.
表皮生长因子受体（EGFR）变体 III（EGFRvIII）的组成性激活突变是多形性胶质母细胞瘤（GBM）中最常见的突变，其表达已在临床上与肿瘤增殖、侵袭和血管生成相关联。在这项研究中，我们研究了 EGFRvIII 在肿瘤微环境中的作用，特别是在血管生成方面的作用。
为了研究 EGFRvIII 在肿瘤血管生成中的作用，我们制备了转染增强型绿色荧光蛋白（EGFP）、野生型 EGFR 或 EGFRvIII（LN229-WT 或 -vIII）的 LN229 神经胶质瘤细胞，并在体内检测肿瘤生长和肿瘤中的微血管密度。此外，通过实时 PCR 分析鉴定潜在的血管生成因子，并在 LN229-vIII 细胞中检测其功能。
与 LN229-WT 细胞相比，LN229-vIII 细胞在体内表现出更具侵袭性的肿瘤生长和更高的血管生成，尽管在体外细胞生长速率没有显著差异。我们随后研究了 60 种与血管生成相关的因子的表达，以阐明 LN229-vIII 和 LN229-WT 肿瘤异种移植物血管生成差异的机制。我们发现，血管生成和代谢调节相关分泌蛋白血管生成素样 4（Angptl4）的 mRNA 和蛋白表达在体内和体外均由 EGFRvIII 过表达显著诱导。使用 shRNA 在 LN229-vIII 中持续敲低 Angptl4 可显著降低肿瘤异种移植物中的微血管密度并抑制肿瘤生长。为了阐明 EGFRvIII 对 Angptl4 的调控机制，我们通过药理学抑制和 RNA 干扰分析了信号通路和转录因子。ERK 信号抑制剂 U0126 显著抑制 Angptl4 表达。ERK 调节的转录因子 c-Myc 在 LN229-vIII 细胞中被激活，并且使用 siRNA 敲低 c-Myc 也减弱了 LN229-vIII 细胞中 Angptl4 的表达。此外，染色质免疫沉淀（ChIP）试验显示在 LN229-vIII 细胞中 c-Myc 募集到 Angptl4 启动子区域增加。
总之，我们证明 EGFRvIII 通过 ERK/c-Myc 途径诱导 Angptl4 表达，并促进恶性神经胶质瘤中的肿瘤血管生成。
