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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 induce phosphorylation of Tyr731VE-cadherin and Ser33/Ser37/Thr41β-catenin in HUVECs in vitroA & B, αAM and αAMR induce Tyr731VE-cadherin phosphorylation in HUVECs in a time-dependent manner. C & D, αAM and αAMR induce Ser33/Ser37/Thr41 β-catenin phosphorylation in HUVECs in a time-dependent manner. No changes were observed in the total amount of the VE-cadherin (A, B) and β-catenin (C, D) suggesting that the increase in the phosphorylation patterns is independent of the changes in the overall protein expression. β-actin was used as a loading control. (E) Phospho-β-catenin is localized in the cytoplasm. The phospho-β-catenin Ser33/Ser37/Thr41 in confluent HUVECs monolayers was determined by fluorescent microscopy at different time points upon treatment with IgG-control (70 μg/ml), AM (10−7 M), αAM (70 μg/ml), and αAMR (70 μg/ml). DAPI-stained nuclei are in blue.
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Figure 5: αAM and αAMR induce phosphorylation of Tyr731VE-cadherin and Ser33/Ser37/Thr41β-catenin in HUVECs in vitroA & B, αAM and αAMR induce Tyr731VE-cadherin phosphorylation in HUVECs in a time-dependent manner. C & D, αAM and αAMR induce Ser33/Ser37/Thr41 β-catenin phosphorylation in HUVECs in a time-dependent manner. No changes were observed in the total amount of the VE-cadherin (A, B) and β-catenin (C, D) suggesting that the increase in the phosphorylation patterns is independent of the changes in the overall protein expression. β-actin was used as a loading control. (E) Phospho-β-catenin is localized in the cytoplasm. The phospho-β-catenin Ser33/Ser37/Thr41 in confluent HUVECs monolayers was determined by fluorescent microscopy at different time points upon treatment with IgG-control (70 μg/ml), AM (10−7 M), αAM (70 μg/ml), and αAMR (70 μg/ml). DAPI-stained nuclei are in blue.

Mentions: Previous studies have demonstrated that disruption of cell-cell junctions depends on intracellular kinases and/or phosphatases that regulate the phosphorylation state of cadherins and the cytosolic binding partners [31–34]. Accordingly, we sought to determine whether αAM or αAMR could interfere with VE-cadherin function, possibly through modulating VE-cadherin/β-catenin phosphorylation status. The mechanism by which αAM and αAMR modulates VE-cadherin/β-catenin function was further dissected in the presence or absence of 70 μg/ml of αAM or αAMR. The endothelial cell lysates were examined using anti-phosphoTyr731-VE-cadherin antibody, which recognizes VE-cadherin when it is phosphorylated on Tyr731, and anti-phospho-β-catenin Ser33/Ser37/Thr41 antibody, which recognizes β-catenin when it is phosphorylated at 1 or more of 3 specific sites, namely Ser33, Ser37 or Thr41. αAM and αAMR induced the phosphorylation of VE-cadherin on Tyr731 (Figures 5A, 5B) and β-catenin on Ser33, Ser37 or Thr41 (Figures 5C, 5D). No changes in the total amount of the VE-cadherin and β-catenin could be observed, suggesting that the increase of phosphorylation patterns are independent of the changes in the overall protein expression (Figures 5A–5D). The increase in phospho-β-catenin Ser33/Ser37/Thr41 was confirmed by immunostaining of αAM-treated endothelial cells and starts as early as 1 h (Figure 5E) and 16 h post-treatment demonstrates that the phospho-β-catenin reside specifically in the cytoplasm and is not translocated into the nucleus (Figure 5E, inset). These findings suggest that αAM and αAMR may interfere with β-catenin function, since phosphorylation of serine/threonine residues in the N-terminal region of β-catenin targets the protein for ubiquitination and subsequent proteasomal degradation [35, 36]. These data indicate that αAM or αAMR destabilizes the endothelial cell-cell junctions by promoting the phosphorylation of VE-cadherin and β-catenin which results in the loss of anchorage of endothelial cells and subsequently the disruption with its near cell partners.


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 induce phosphorylation of Tyr731VE-cadherin and Ser33/Ser37/Thr41β-catenin in HUVECs in vitroA & B, αAM and αAMR induce Tyr731VE-cadherin phosphorylation in HUVECs in a time-dependent manner. C & D, αAM and αAMR induce Ser33/Ser37/Thr41 β-catenin phosphorylation in HUVECs in a time-dependent manner. No changes were observed in the total amount of the VE-cadherin (A, B) and β-catenin (C, D) suggesting that the increase in the phosphorylation patterns is independent of the changes in the overall protein expression. β-actin was used as a loading control. (E) Phospho-β-catenin is localized in the cytoplasm. The phospho-β-catenin Ser33/Ser37/Thr41 in confluent HUVECs monolayers was determined by fluorescent microscopy at different time points upon treatment with IgG-control (70 μg/ml), AM (10−7 M), αAM (70 μg/ml), and αAMR (70 μg/ml). DAPI-stained nuclei are in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: αAM and αAMR induce phosphorylation of Tyr731VE-cadherin and Ser33/Ser37/Thr41β-catenin in HUVECs in vitroA & B, αAM and αAMR induce Tyr731VE-cadherin phosphorylation in HUVECs in a time-dependent manner. C & D, αAM and αAMR induce Ser33/Ser37/Thr41 β-catenin phosphorylation in HUVECs in a time-dependent manner. No changes were observed in the total amount of the VE-cadherin (A, B) and β-catenin (C, D) suggesting that the increase in the phosphorylation patterns is independent of the changes in the overall protein expression. β-actin was used as a loading control. (E) Phospho-β-catenin is localized in the cytoplasm. The phospho-β-catenin Ser33/Ser37/Thr41 in confluent HUVECs monolayers was determined by fluorescent microscopy at different time points upon treatment with IgG-control (70 μg/ml), AM (10−7 M), αAM (70 μg/ml), and αAMR (70 μg/ml). DAPI-stained nuclei are in blue.
Mentions: Previous studies have demonstrated that disruption of cell-cell junctions depends on intracellular kinases and/or phosphatases that regulate the phosphorylation state of cadherins and the cytosolic binding partners [31–34]. Accordingly, we sought to determine whether αAM or αAMR could interfere with VE-cadherin function, possibly through modulating VE-cadherin/β-catenin phosphorylation status. The mechanism by which αAM and αAMR modulates VE-cadherin/β-catenin function was further dissected in the presence or absence of 70 μg/ml of αAM or αAMR. The endothelial cell lysates were examined using anti-phosphoTyr731-VE-cadherin antibody, which recognizes VE-cadherin when it is phosphorylated on Tyr731, and anti-phospho-β-catenin Ser33/Ser37/Thr41 antibody, which recognizes β-catenin when it is phosphorylated at 1 or more of 3 specific sites, namely Ser33, Ser37 or Thr41. αAM and αAMR induced the phosphorylation of VE-cadherin on Tyr731 (Figures 5A, 5B) and β-catenin on Ser33, Ser37 or Thr41 (Figures 5C, 5D). No changes in the total amount of the VE-cadherin and β-catenin could be observed, suggesting that the increase of phosphorylation patterns are independent of the changes in the overall protein expression (Figures 5A–5D). The increase in phospho-β-catenin Ser33/Ser37/Thr41 was confirmed by immunostaining of αAM-treated endothelial cells and starts as early as 1 h (Figure 5E) and 16 h post-treatment demonstrates that the phospho-β-catenin reside specifically in the cytoplasm and is not translocated into the nucleus (Figure 5E, inset). These findings suggest that αAM and αAMR may interfere with β-catenin function, since phosphorylation of serine/threonine residues in the N-terminal region of β-catenin targets the protein for ubiquitination and subsequent proteasomal degradation [35, 36]. These data indicate that αAM or αAMR destabilizes the endothelial cell-cell junctions by promoting the phosphorylation of VE-cadherin and β-catenin which results in the loss of anchorage of endothelial cells and subsequently the disruption with its near cell partners.

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