Eguchi R, Nakano T, Wakabayashi I
Department of Environmental and Preventive Medicine, Hyogo College of Medicine, Hyogo, Japan.
Division of Respiratory Medicine, Department of Internal Medicine, Hyogo College of Medicine, Hyogo, Japan.
Oncogene. 2017 Feb 2;36(5):714-722. doi: 10.1038/onc.2016.226. Epub 2016 Jun 27.
Malignant mesothelioma is an aggressive tumor arising from the mesothelial cells of serous membranes and is associated with tumor angiogenesis, which is a prerequisite for tumor progression. Vascular endothelial growth factors (VEGFs) including VEGF-A have a crucial role in tumor angiogenesis. However, bevacizumab, a monoclonal antibody to VEGF-A, has recently been reported not to improve the progression-free survival of patients with malignant mesothelioma. Cell culture supernatant contains extracellular components such as serum, which can mask the existence of unknown cell-derived factors in the supernatant and make it difficult to detect the factors by subsequent protein analysis. We tried using serum-free culture for human mesothelioma cell lines, NCI-H28, NCI-H2452 and NCI-H2052, and only NCI-H2052 cells adapted to serum-free culture. We found that serum-free culture supernatant derived from NCI-H2052 cells induces the formation of capillary-like tube structures (tube formation) in three-dimensional culture, in which endothelial cells sandwiched between two layers of collagen or embedded in collagen are incubated with various angiogenic inducers. However, neither neutralization of VEGF-A nor RNA interference of VEGF receptor 2 (VEGFR2) suppressed the supernatant-induced tube formation. Using mass spectrometry, we identified a total of 399 proteins in the supernatant, among which interleukin-8 (IL-8), growth-regulated α-protein, midkine, IL-18, IL-6, hepatoma-derived growth factor, clusterin and granulin (GRN), also known as progranulin (PGRN), were included as a candidate protein inducing angiogenesis. Neutralizing assays and RNA interference showed that PGRN, but not the above seven candidate proteins, caused the supernatant-induced tube formation. We also found that NCI-H28 and NCI-H2452 cells express PGRN. Furthermore, we demonstrate that not only PGRN but also GRN-like protein have an important role in the supernatant-induced tube formation. Thus, mesothelioma-derived GRNs induce VEGF-independent angiogenesis.
恶性间皮瘤是一种起源于浆膜间皮细胞的侵袭性肿瘤，与肿瘤血管生成相关，而肿瘤血管生成是肿瘤进展的一个先决条件。包括血管内皮生长因子A（VEGF-A）在内的血管内皮生长因子在肿瘤血管生成中起关键作用。然而，最近有报道称，VEGF-A单克隆抗体贝伐单抗并不能改善恶性间皮瘤患者的无进展生存期。细胞培养上清液含有血清等细胞外成分，这可能会掩盖上清液中未知细胞衍生因子的存在，并且通过后续蛋白质分析难以检测到这些因子。我们尝试对人恶性间皮瘤细胞系NCI-H28、NCI-H2452和NCI-H2052进行无血清培养，只有NCI-H2052细胞适应了无血清培养。我们发现，源自NCI-H2052细胞的无血清培养上清液在三维培养中可诱导形成毛细血管样管状结构（管形成），即在两层胶原蛋白之间夹有或嵌入胶原蛋白中的内皮细胞与各种血管生成诱导剂一起孵育。然而，VEGF-A的中和或血管内皮生长因子受体2（VEGFR2）的RNA干扰均不能抑制上清液诱导的管形成。通过质谱分析，我们在上清液中总共鉴定出399种蛋白质，其中白细胞介素-8（IL-8）、生长调节α蛋白、中期因子、IL-18、IL-6、肝癌衍生生长因子、簇集蛋白和颗粒蛋白（GRN，也称为前颗粒蛋白（PGRN））被列为诱导血管生成的候选蛋白。中和试验和RNA干扰表明，是PGRN而非上述七种候选蛋白导致了上清液诱导的管形成。我们还发现NCI-H28和NCI-H2452细胞表达PGRN。此外，我们证明不仅PGRN而且类GRN蛋白在上清液诱导的管形成中都起重要作用。因此，间皮瘤衍生的GRN诱导不依赖VEGF的血管生成。
