Actinomyosin Contraction, Phosphorylation of VE-Cadherin, and Actin Remodeling Enable Melanoma-Induced Endothelial Cell-Cell Junction Disassembly

<div><p>During melanoma cell extravasation through the vascular endothelium, melanoma cells interact with endothelial cells through secretion of cytokines and by adhesion between proteins displayed on opposing cell surfaces. How these tumor cell associated signals together regulate the dynamics of intracellular signaling pathways within endothelial cells leading to endothelial cell-cell junction disruption is not well understood. Here, we used a combination of experimental and computational approaches to examine the individual and combined effects of activation of the vascular cell adhesion molecule (VCAM)-1, interleukin (IL)-8, and IL-1β signaling pathways on the integrity of vascular junctions. Our simulations predict a multifaceted interplay of signaling resulting from individual activation of VCAM-1, IL-8 and IL-1β pathways that is neither synergistic nor additive compared to all inputs turned on simultaneously. Furthermore, we show that the levels of phosphorylated proteins associated with actinomyosin contractility and junction disassembly peak prior to those related to actin remodeling. The results of this work provide insight into the dynamics of tumor-mediated endothelial junction disassembly and suggest that targeting proteins downstream of several interaction pathways may be the most effective therapeutic approach to reduce melanoma extravasation.</p></div

Computational simulations show that melanoma signals differentially affect endothelial contractility, actin remodeling, and junction disassembly.

<p>Phosphorylation dynamics as a function of time for (A) MLC, (B) HSP-27, and (C) VE-cadherin following activation of VCAM-1, IL-8, and IL-1β individually or simultaneous activation of all inputs together.</p

Simulations reveal that knockout of p38 MAPK influences MLC and VE-cadherin phosphorylation dynamics and completely inhibits HSP-27 phosphorylation.

<p>Phosphorylation dynamics as a function of time for p38 on and knockout of p38 for (A) MLC, (B) HSP-27, and (C) VE-cadherin.</p

Schematic of network model connections.

<p>Pathways are distinguished by color with IL-8 in blue, VCAM-1 in orange, IL-1β in green, c-Src in magenta, and model outputs in black. Grey indicates locations where all pathways converge. Phosphorylated proteins are indicated with the letter P and protein activation is designated with an asterisk.</p

Co-culture of endothelial monolayers with A2058 melanoma cells induces endothelial cell-cell junction disruption local to melanoma cell adhesion sites.

<p>(A) Phase contrast images of HUVEC monolayers with corresponding immunofluorescence staining of VE-cadherin junctions. VE-cadherin staining indicates existence of cell-cell junction integrity in the endothelial monolayer. With A2058 co-culture, VE-cadherin staining shows dissolution of cell-cell junctions. White arrows indicate the location of A2058 melanoma cells. (B) Quantification of the percentage gap formation between endothelial cells with A2058 melanoma cell co-culture with and without inhibitors of cellular contractility, actin remodeling, and Src kinase signaling. *p<0.001 in comparison to HUVEC only controls. #p<0.001 in comparison to HUVEC/A2058 co-culture sample incubated with DMSO. Scale bars, 25 µm.</p

Simulations show that knockout of MLCK reduces MLC phosphorylation, whereas HSP-27 and VE-cadherin phosphorylation is minimally affected.

<p>Phosphorylation dynamics as a function of time for MLCK on and knockout of MLCK for (A) MLC, (B) HSP-27, and (C) VE-cadherin.</p

Knockout of PKC affects the simulated dynamics of MLC and VE-cadherin phosphorylation.

<p>Phosphorylation dynamics as a function of time for MLCK on and knockout of MLCK for (A) MLC, (B) HSP-27, and (C) VE-cadherin.</p