Vascular endothelial growth factor-C in activating vascular endothelial growth factor receptor-3 and chemokine receptor-4 in melanoma adhesion

Abstract

Vascular endothelial growth factor-C (VEGF-C) binds to receptor vascular endothelial growth factor receptor-3 (VEGFR-3) expressed on lymphatic endothelial and melanoma cells. Binding of VEGF-C to VEGFR-3 enhances receptor phosphorylation that activates mitogen-activated protein kinase (MAP-K) and phosphatidylinositol-3-kinase (PI3K). These signalling pathways regulate cell migration and adhesion in response to internal or external changes. In addition, the overexpression of VEGF-C upregulates chemokine receptor CXCR-4 in tumours (melanoma). CXCR-4 is expressed on cells of the immune system (natural killer cells) and facilitates the migration of leukocytes in response to the CXCL12 ligand. The latter is expressed by lymphatic endothelial cells and by stromal cells in the tumour microenvironment (TME). The gradient established between CXCR-4 expressed on tumour cells and CXCL12 produced by stromal and lymphatic endothelial cells enhances tumour cell metastasis. 3-(4-Dimethylamino-naphthalen-1-ylmethylene)-1, 3-dihydroindol-2-one, MAZ-51, is an indolinone-based synthetic molecule that inhibits the phosphorylation of the tyrosine kinase receptor VEGFR-3. CTCE-9908, a CXCR-4 antagonist derived from human CXCL12, hinders receptor phosphorylation and the subsequent signalling pathways that would be activated. VEGF-C is stimulated by transforming growth factor-beta 1 (TGF-β1), which facilitates cell-cell and cell-matrix adhesion by regulating cadherins through the activation of focal adhesion kinase (FAK) and mediates paxillin upregulation. Increased VEGF-C protein levels stimulated by TGF-β bound to VEGFR-3 impact on intracellular pathways that promote tumour cell adhesion. In addition, increased VEGF-C protein levels lead to enhanced CXCR-4 protein expression. Therefore, effective blocking of VEGR-3 and CXCR-4 may inhibit tumour cell metastasis by hampering intracellular proteins promoting adhesion.

FIGURE 1

TGF‐β ligand binding the two pair serine/threonine receptor activating receptor phosphorylation and triggering the signalling pathways JNK/p38, Cdc42/Rho (family of GTPases) and Par6 affecting migration and adhesion, cell shape and cell‐to‐cell contact. Additional TGF‐β signalling may occur through the Smad pathway where TGF‐β gene transcription enhances tumour suppressive responses that enhance apoptosis. This indicates the dual role of TGF‐β to enhance tumour cell metastasis or to induce apoptosis depending on the intracellular pathway activated. (Image was designed by Y.N Hlophe using Microsoft PowerPoint 2013; 2013 Microsoft Corporation).

FIGURE 2

Chemical structure of MAZ‐51. An indoline at the oxindole core binds the adenine ring of the vascular endothelial growth factor receptor‐3. The aldehyde section determines the tyrosine kinase receptor that MAZ‐51 can bind to. (Image was designed by Y.N Hlophe using Microsoft PowerPoint 2013; 2013 Microsoft Corporation).

FIGURE 3

CXCR‐4 signalling pathway: MAP‐K and PI3K. A diagram indicating CXCR‐4 activation by the heterotrimeric G‐proteins (Gβ, Gα and Gγ) located on the intracellular section of the plasma membrane. Gα activates adenylate cyclase, which leads to MAP‐K activation. Gγ and Gβ activate the PI3K pathway, which leads to Akt stimulation resulting in tumour cell proliferation. Paxillin is a focal adhesion adapter protein. When paxillin is phosphorylated on tyrosine‐ and serine residue sites it recruits signalling molecules involved in cell migration. (Image was designed by Y.N Hlophe using Microsoft PowerPoint 2013; 2013 Microsoft Corporation).

FIGURE 4

Focal adhesion kinase linear structure. The linear structure of FAK and tyrosine phosphorylation sites. The focal adhesion targeting (FAT) domain is important for adhesion‐dependent tyrosine phosphorylation and to contain integrin adhesion sites. The proline‐rich motifs (PR1 and PR2) facilitate interaction with the Src‐homology 3 domain. The majority of the N‐terminal comprises the JEF domain, although the function of the JEF domain is not well‐understood. (Image was designed by Y.N Hlophe using Microsoft PowerPoint 2013; 2013 Microsoft Corporation).

FIGURE 5

Paxillin linear structure. Linear paxillin structure indicating domains and tyrosine binding sites. Paxillin is a nonenzymatic docking protein with multiple binding domains. The 5 LD motifs are found at the N‐terminal and comprise eight residue leucine‐rich sequences. The LIM domains are double zinc‐finger motifs that form the C‐terminal. LIM 2 + 3 are important to recruit paxillin to focal adhesions. FAK relates with paxillin via LD motifs 2 + 4. (Image was designed by Y.N Hlophe using Microsoft PowerPoint 2013; 2013 Microsoft Corporation).

FIGURE 6

VEGFR‐3 signalling pathways: MAP‐K and PI3K. VEGF‐C binds VEGFR‐3 and activates JNK 1/2, MAP‐K/p38, extracellular signal‐regulated kinase (ERK) 1/2, PI3K/Akt pathways promoting migration, proliferation and survival of melanoma cells, ^. Focal adhesion kinase (FAK) and paxillin are activated by growth factor signalling and facilitate tumour cell adhesion. (Image was designed by Y.N Hlophe and modified by Carolyn Nadasen using Microsoft PowerPoint 2013; 2013 Microsoft Corporation).

FIGURE 7

Ligand‐activated intracellular pathways promoting tumour cell adhesion. Activation of intracellular signalling pathways (MAP‐K and PI3K) promotes tumour cell adhesion once growth factor receptors (VEGFR‐2/3) and a chemokine receptor (CXCR‐4) are phosphorylated. TGF‐β1 ligand binding the two pair serine/threonine receptor activating receptor phosphorylation facilitates cell–cell and cell‐matrix adhesion by regulating cadherins through activation of focal adhesion kinase (FAK) and mediates paxillin upregulation. The activation of intracellular adhesion proteins (FAK, paxillin and cadherin) when ligands (VEGF‐C, TGF‐β1 and CXCL12) bind their specific receptors. Reduced expression of adhesion proteins will contribute to limiting the activity of the intracellular pathways that promote tumour cell adhesion. (Image was designed by Y.N Hlophe using Microsoft PowerPoint 2013; 2013 Microsoft CorporationUnited States of America).