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Adrenomedullin blockade induces regression of tumor neovessels through interference with vascular endothelial-cadherin signalling.

Khalfaoui-Bendriss G, Dussault N, Fernandez-Sauze S, Berenguer-Daizé C, Sigaud R, Delfino C, Cayol M, Metellus P, Chinot O, Mabrouk K, Martin PM, Ouafik L - Oncotarget (2015)

Bottom Line: At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function.Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin.In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, CRO2, UMR_S 911, Faculté de Médecine, Marseille, France.

ABSTRACT
The cellular and molecular mechanisms by which adrenomedullin (AM) blockade suppresses tumor neovessels are not well defined. Herein, we show that AM blockade using anti-AM and anti-AM receptors antibodies targets vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and induces regression of unstable nascent tumor neovessels. The underlying mechanism involved, and shown in vitro and in vivo in mice, is the disruption of the molecular engagement of the endothelial cell-specific junctional molecules vascular endothelial-cadherin (VE-cadherin)/β-catenin complex. AM blockade increases endothelial cell permeability by inhibiting cell-cell contacts predominantly through disruption of VE-cadherin/β-catenin/Akt signalling pathway, thereby leading to vascular collapse and regression of tumor neovessels. At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function. Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin. In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation. We found that AM blockade induces β-catenin phosphorylation on Ser33/Ser37/Thr41 sites in both ECs and VSMCs both in vitro and in vivo in mice. These data suggest that AM blockade selectively induces regression of unstable tumor neovessels, through disruption of VE-cadherin signalling. Targeting AM system may present a novel therapeutic target to selectively disrupt assembly and induce regression of nascent tumor neovessels, without affecting normal stabilized vasculature.

No MeSH data available.


Related in: MedlinePlus

αAM and αAMR decrease phosphorylation of Ser473Akt in vitro(A & B) αAM (70 μg/ml) and αAMR (70 μg/ml) decrease pSer473Akt in HUVECs in a time-dependent manner. No changes were observed in the total amount of the Akt protein. (C) incubation of HUVECs for 16 h with PI3K inhibitor LY 294002 induces phosphorylation of Ser33/Ser37/Thr41 β-catenin in HUVECs. β-actin was used as a loading control.
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Figure 7: αAM and αAMR decrease phosphorylation of Ser473Akt in vitro(A & B) αAM (70 μg/ml) and αAMR (70 μg/ml) decrease pSer473Akt in HUVECs in a time-dependent manner. No changes were observed in the total amount of the Akt protein. (C) incubation of HUVECs for 16 h with PI3K inhibitor LY 294002 induces phosphorylation of Ser33/Ser37/Thr41 β-catenin in HUVECs. β-actin was used as a loading control.

Mentions: Several studies have shown that AM acts through PI3K, Akt, MAPK, FAK and other components of the cell-cell adhesion machinery [38–40], suggesting that the PI3K/Akt signalling pathway might be inhibited by AM blockade to produce phospho-Ser33/Ser37/Thr41 β-catenin. Accordingly, we further examined whether αAM or αAMR inhibits 2% FBS-mediated Akt phosphorylation. The incubation of 2% FBS-stimulated endothelial cells with αAM or αAMR did not affect the phosphorylation of Akt at Ser473 during the first hours of treatment (Figures 7A, 7B). However, αAM or αAMR decreased the pAkt-Ser473 levels after a 10-hour incubation and reached low levels upon a 24-hour incubation (Figures 7A, 7B). This reduction was clearly related to a decrease in phosphorylation, since the total amount of Akt was not altered by αAM nor αAMR treatment (Figures 7A, 7B). We showed that treatment of HUVECs for 16 hours, with PI3K inhibitor LY294002 induced a phosphorylation of Ser33/Ser37/Thr41 β-catenin (Figure 7C). Thus, signalling via a PI3K-Akt-dependent pathway could be involved in AM-CLR-RAMP2/RAMP3 mediated angiogenesis and vascular stability.


Adrenomedullin blockade induces regression of tumor neovessels through interference with vascular endothelial-cadherin signalling.

Khalfaoui-Bendriss G, Dussault N, Fernandez-Sauze S, Berenguer-Daizé C, Sigaud R, Delfino C, Cayol M, Metellus P, Chinot O, Mabrouk K, Martin PM, Ouafik L - Oncotarget (2015)

αAM and αAMR decrease phosphorylation of Ser473Akt in vitro(A & B) αAM (70 μg/ml) and αAMR (70 μg/ml) decrease pSer473Akt in HUVECs in a time-dependent manner. No changes were observed in the total amount of the Akt protein. (C) incubation of HUVECs for 16 h with PI3K inhibitor LY 294002 induces phosphorylation of Ser33/Ser37/Thr41 β-catenin in HUVECs. β-actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480698&req=5

Figure 7: αAM and αAMR decrease phosphorylation of Ser473Akt in vitro(A & B) αAM (70 μg/ml) and αAMR (70 μg/ml) decrease pSer473Akt in HUVECs in a time-dependent manner. No changes were observed in the total amount of the Akt protein. (C) incubation of HUVECs for 16 h with PI3K inhibitor LY 294002 induces phosphorylation of Ser33/Ser37/Thr41 β-catenin in HUVECs. β-actin was used as a loading control.
Mentions: Several studies have shown that AM acts through PI3K, Akt, MAPK, FAK and other components of the cell-cell adhesion machinery [38–40], suggesting that the PI3K/Akt signalling pathway might be inhibited by AM blockade to produce phospho-Ser33/Ser37/Thr41 β-catenin. Accordingly, we further examined whether αAM or αAMR inhibits 2% FBS-mediated Akt phosphorylation. The incubation of 2% FBS-stimulated endothelial cells with αAM or αAMR did not affect the phosphorylation of Akt at Ser473 during the first hours of treatment (Figures 7A, 7B). However, αAM or αAMR decreased the pAkt-Ser473 levels after a 10-hour incubation and reached low levels upon a 24-hour incubation (Figures 7A, 7B). This reduction was clearly related to a decrease in phosphorylation, since the total amount of Akt was not altered by αAM nor αAMR treatment (Figures 7A, 7B). We showed that treatment of HUVECs for 16 hours, with PI3K inhibitor LY294002 induced a phosphorylation of Ser33/Ser37/Thr41 β-catenin (Figure 7C). Thus, signalling via a PI3K-Akt-dependent pathway could be involved in AM-CLR-RAMP2/RAMP3 mediated angiogenesis and vascular stability.

Bottom Line: At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function.Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin.In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, CRO2, UMR_S 911, Faculté de Médecine, Marseille, France.

ABSTRACT
The cellular and molecular mechanisms by which adrenomedullin (AM) blockade suppresses tumor neovessels are not well defined. Herein, we show that AM blockade using anti-AM and anti-AM receptors antibodies targets vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and induces regression of unstable nascent tumor neovessels. The underlying mechanism involved, and shown in vitro and in vivo in mice, is the disruption of the molecular engagement of the endothelial cell-specific junctional molecules vascular endothelial-cadherin (VE-cadherin)/β-catenin complex. AM blockade increases endothelial cell permeability by inhibiting cell-cell contacts predominantly through disruption of VE-cadherin/β-catenin/Akt signalling pathway, thereby leading to vascular collapse and regression of tumor neovessels. At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function. Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin. In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation. We found that AM blockade induces β-catenin phosphorylation on Ser33/Ser37/Thr41 sites in both ECs and VSMCs both in vitro and in vivo in mice. These data suggest that AM blockade selectively induces regression of unstable tumor neovessels, through disruption of VE-cadherin signalling. Targeting AM system may present a novel therapeutic target to selectively disrupt assembly and induce regression of nascent tumor neovessels, without affecting normal stabilized vasculature.

No MeSH data available.


Related in: MedlinePlus