Endothelial-mesenchymal transition of brain endothelial cells: possible role during metastatic extravasation

PLoS One. 2015 Mar 5;10(3):e0119655. doi: 10.1371/journal.pone.0119655. eCollection 2015.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzamides / pharmacology
  • Brain / cytology*
  • Brain / drug effects
  • Cell Line, Tumor
  • Cells, Cultured
  • Culture Media, Conditioned / pharmacology
  • Dioxoles / pharmacology
  • Endothelial Cells / drug effects
  • Endothelial Cells / physiology*
  • Epithelial-Mesenchymal Transition*
  • Gene Expression Regulation / drug effects*
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • MCF-7 Cells
  • Mice
  • Neoplasm Metastasis
  • Rats
  • Transforming Growth Factor beta1 / antagonists & inhibitors
  • Transforming Growth Factor beta1 / pharmacology*

Substances

  • 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide
  • Benzamides
  • Culture Media, Conditioned
  • Dioxoles
  • Transforming Growth Factor beta1

Grants and funding

This work was supported by grants from the Hungarian Research Fund (OTKA PD-100958, K-100807), the National Development Agency (Hungary-Romania Cross-Border Co-operation Programme 2007-2013: HURO/1101/173/2.2.1; and the TÁMOP-4.2.2.A-11/1/KONV-2012-0052 project) and the Hungarian Kidney Foundation. I. Wilhelm was supported by the János Bolyai Research Fellowship of the Hungarian Academy of Sciences. The research of C. Fazakas was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/ 2-11/1-2012-0001 ‘National Excellence Program’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.