Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Panum Institute, Blegdamsvej 3B, 2200, Copenhagen, Denmark.
Section for Cell Biology and Physiology, Department of Biology, Faculty of Science, University of Copenhagen, Universitetsparken 13, 2100, Copenhagen, Denmark.
BMC Cancer. 2017 Aug 14;17(1):542. doi: 10.1186/s12885-017-3532-x.
Chronic angiogenesis is a hallmark of most tumors and takes place in a hostile tumor microenvironment (TME) characterized by hypoxia, low nutrient and glucose levels, elevated lactate and low pH. Despite this, most studies addressing angiogenic signaling use hypoxia as a proxy for tumor conditions. Here, we compared the effects of hypoxia and TME conditions on regulation of the Na/H exchanger NHE1, Ser/Thr kinases Akt1-3, and downstream effectors in endothelial cells.
Human umbilical vein endothelial cells (HUVEC) and Ea.hy926 endothelial cells were exposed to simulated TME (1% hypoxia, low serum, glucose, pH, high lactate) or 1% hypoxia for 24 or 48 h, with or without NHE1 inhibition or siRNA-mediated knockdown. mRNA and protein levels of NHE1, Akt1-3, and downstream effectors were assessed by qPCR and Western blotting, vascular endothelial growth factor (VEGF) release by ELISA, and motility by scratch assay.
Within 24 h, HIF-1α level and VEGF mRNA level were increased robustly by TME and modestly by hypoxia alone. The NHE1 mRNA level was decreased by both hypoxia and TME, and NHE1 protein was reduced by TME in Ea.hy926 cells. Akt1-3 mRNA was detected in HUVEC and Ea.hy926 cells, Akt1 most abundantly. Akt1 protein expression was reduced by TME yet unaffected by hypoxia, while Akt phosphorylation was increased by TME. The Akt loss was partly reversed by MCF-7 human breast cancer cell conditioned medium, suggesting that in vivo, the cancer cell secretome may compensate for adverse effects of TME on endothelial cells. TME, yet not hypoxia, reduced p70S6 kinase activity and ribosomal protein S6 phosphorylation and increased eIF2α phosphorylation, consistent with inhibition of protein translation. Finally, TME reduced Retinoblastoma protein phosphorylation and induced poly-ADP-ribose polymerase (PARP) cleavage consistent with inhibition of proliferation and induction of apoptosis. NHE1 knockdown, mimicking the effect of TME on NHE1 expression, reduced Ea.hy926 migration. TME effects on HIF-1α, VEGF, Akt, translation, proliferation or apoptosis markers were unaffected by NHE1 knockdown/inhibition.
NHE1 and Akt are downregulated by TME conditions, more potently than by hypoxia alone. This inhibits endothelial cell migration and growth in a manner likely modulated by the cancer cell secretome.
慢性血管生成是大多数肿瘤的标志，发生在以缺氧、低营养和葡萄糖水平、升高的乳酸和低 pH 值为特征的恶劣肿瘤微环境（TME）中。尽管如此，大多数研究血管生成信号的研究都将缺氧作为肿瘤状态的替代物。在这里，我们比较了缺氧和 TME 条件对内皮细胞中 Na/H 交换器 NHE1、丝氨酸/苏氨酸激酶 Akt1-3 及其下游效应物的调节作用。
将人脐静脉内皮细胞（HUVEC）和 Ea.hy926 内皮细胞暴露于模拟的 TME（1%缺氧、低血清、葡萄糖、pH 值、高乳酸）或 1%缺氧 24 或 48 小时，同时或不进行 NHE1 抑制或 siRNA 介导的敲低。通过 qPCR 和 Western blotting 评估 NHE1、Akt1-3 和下游效应物的 mRNA 和蛋白水平，通过 ELISA 评估血管内皮生长因子（VEGF）释放，通过划痕试验评估迁移率。
在 24 小时内，TME 强烈增加了 HIF-1α 水平和 VEGF mRNA 水平，而单独缺氧则适度增加。NHE1 mRNA 水平被缺氧和 TME 降低，并且在 Ea.hy926 细胞中 NHE1 蛋白被 TME 降低。Akt1-3 mRNA 在 HUVEC 和 Ea.hy926 细胞中均有检测到，Akt1 最为丰富。TME 降低了 Akt1 蛋白的表达，而缺氧对其没有影响，而 Akt 磷酸化则被 TME 增加。MCF-7 人乳腺癌细胞条件培养基部分逆转了 Akt 缺失，表明在体内，癌细胞分泌组可能补偿 TME 对内皮细胞的不利影响。TME，而不是缺氧，降低了 p70S6 激酶活性和核糖体蛋白 S6 磷酸化，并增加了 eIF2α 磷酸化，与蛋白质翻译抑制一致。最后，TME 降低了视网膜母细胞瘤蛋白磷酸化并诱导多聚 ADP-核糖聚合酶（PARP）裂解，与增殖抑制和凋亡诱导一致。NHE1 敲低，模拟 TME 对 NHE1 表达的影响，降低了 Ea.hy926 的迁移。TME 对 HIF-1α、VEGF、Akt、翻译、增殖或凋亡标志物的影响不受 NHE1 敲低/抑制的影响。
TME 条件下调 NHE1 和 Akt，比单独缺氧更强烈。这抑制了内皮细胞的迁移和生长，其方式可能受到癌细胞分泌组的调节。
