Krizbai István A, Gasparics Ákos, Nagyőszi Péter, Fazakas Csilla, Molnár Judit, Wilhelm Imola, Bencs Rita, Rosivall László, Sebe Attila
PLoS One. 2015 Mar 30;10(3):e0123845. doi: 10.1371/journal.pone.0123845. eCollection 2015.
Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-β, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-β1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, β1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-β signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-β-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-β-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation.
癌症向转移发展遵循一系列被称为转移级联的特定事件顺序。对于外渗和跨内皮迁移，转移细胞首先与内皮细胞相互作用。然而，内皮细胞在转移形成和外渗过程中的作用仍不清楚，并且跨内皮迁移过程中转移细胞与内皮细胞之间的相互作用也了解甚少。由于众所周知肿瘤细胞会表达转化生长因子-β（TGF-β），并且紧密的内皮层在转移性外渗过程中会发生一系列变化（细胞接触破坏、细胞骨架重组、收缩性增强），我们推测内皮-间充质转化（EndMT）可能是转移性外渗所必需的。我们证明，原代培养的大鼠脑内皮细胞（BEC）在TGF-β1处理后会发生EndMT，其特征是紧密连接蛋白和黏附连接蛋白的丢失、纤连蛋白、β1整合素、钙调蛋白和α平滑肌肌动蛋白（SMA）的表达。B16/F10细胞系条件培养基和激活培养基（ACM）具有类似的作用：claudin-5下调、纤连蛋白和SMA表达。在B16/F10 ACM刺激期间使用SB-431542抑制TGF-β信号传导可维持claudin-5水平，并减轻纤连蛋白和SMA表达。B16/F10 ACM对BEC的刺激导致Smad2和Smad3磷酸化。SB-431542在用A2058、MCF-7和MDA-MB231 ACM刺激BEC时也可防止SMA上调。此外，B16/F10 ACM导致跨内皮电阻降低，以TGF-β依赖的方式增加了黑色素瘤细胞黏附并穿过内皮层迁移的数量。这些作用并不局限于BEC：当用乳腺癌细胞系ACM刺激时，人脐静脉内皮细胞（HUVEC）显示出TGF-β依赖的SMA表达。我们的结果表明，EndMT可能是转移性跨内皮迁移所必需的，并且这种转变可能是在被称为转移性外渗的复杂现象中发生的潜在机制之一。
