The matricellular protein CYR61 interferes with normal pancreatic islets architecture and promotes pancreatic neuroendocrine tumor progression

The significance of matricellular proteins during development and cancer progression is widely recognized. However, how these proteins actively contribute to physiological development and pathological cancer progression is only partially elucidated. In this study, we investigated the role of the matricellular protein Cysteine-rich 61 (CYR61) in pancreatic islet development and carcinogenesis. Transgenic expression of CYR61 in β cells (Rip1CYR mice) caused irregular islets morphology and distorted sorting of α cells, but did not alter islets size, number or vascularization. To investigate the function of CYR61 during carcinogenesis, we crossed Rip1CYR mice with Rip1Tag2 mice, a well-established model of β cell carcinogenesis. Beta tumors in Rip1Tag2CYR mice were larger, more invasive and more vascularized compared to tumors in Rip1Tag2 mice. The effect of CYR61 on angiogenesis was fully abrogated by treating mice with the anti-VEGFR2 mAb DC101. Results from in vitro assays demonstrated that CYR61 modulated integrin α₆β₁-dependent invasion and adhesion without altering its expression. Taken together, these results show that CYR61 expression in pancreatic β cells interferes with normal islet architecture, promotes islet tumor growth, invasion and VEGF/VERGFR-2-dependent tumor angiogenesis. Taken together, these observations demonstrate that CYR61 acts as a tumor-promoting gene in pancreatic neuroendocrine tumors

The Integrin Antagonist Cilengitide Activates αVβ3, Disrupts VE-Cadherin Localization at Cell Junctions and Enhances Permeability in Endothelial Cells

Cilengitide is a high-affinity cyclic pentapeptdic αV integrin antagonist previously reported to suppress angiogenesis by inducing anoikis of endothelial cells adhering through αVβ3/αVβ5 integrins. Angiogenic endothelial cells express multiple integrins, in particular those of the β1 family, and little is known on the effect of cilengitide on endothelial cells expressing αVβ3 but adhering through β1 integrins. Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on αVβ3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the β1 ligands fibronectin and collagen I. We show that cilengitide activated cell surface αVβ3, stimulated phosphorylation of FAK (Y397 and Y576/577), Src (S418) and VE-cadherin (Y658 and Y731), redistributed αVβ3 at the cell periphery, caused disappearance of VE-cadherin from cellular junctions, increased the permeability of HUVEC monolayers and detached HUVEC adhering on low-density β1 integrin ligands. Pharmacological inhibition of Src kinase activity fully prevented cilengitide-induced phosphorylation of Src, FAK and VE-cadherin, and redistribution of αVβ3 and VE-cadherin and partially prevented increased permeability, but did not prevent HUVEC detachment from low-density matrices. Taken together, these observations reveal a previously unreported effect of cilengitide on endothelial cells namely its ability to elicit signaling events disrupting VE-cadherin localization at cellular contacts and to increase endothelial monolayer permeability. These effects are potentially relevant to the clinical use of cilengitide as anticancer agent

Relative cell death of HUVEC cultures exposed to cilengitide or EMD135981.

<p>HUVEC were cultured for 18 hours on vitronectin (αVβ3 ligand), fibronectin (α5β1>αVβ3 ligand) and collagen I (α1β1 and α2β1 ligand) before they were exposed for 4 hours to EMD135981 or cilengitide. Cells were collected and Sub-G1 DNA content determined by propidium iodide staining and flow cytometry analysis. Results are expressed as percent increase of cell death relative to untreated conditions. Cilengitide increased HUVEC cell death on vitronectin at high and low protein coating concentrations, while on fibronectin and collagen it only did it at low protein coating concentrations. (n = 3).</p

Cilengitide interferes with HUVEC adhesion on low-density β1 integrin substrates.

<p>(a) HUVEC short-term adhesion assays performed on vitronectin (αVβ3 ligand), fibronectin (α5β1>αVβ3 ligand) and collagen I (α1β1/α2β1 ligand) coated at the indicated concentrations, in medium only (black bars) or in the presence of EMD135981 (gray bars) or cilengitide (white bars). On vitronectin, cilengitide inhibited adhesion at all coating concentrations while on fibronectin and collagen I it blocked adhesion only at low coating concentrations. (n = 5). (b) HUVEC detachment assays. HUVEC were cultured for 18 hours on vitronectin, fibronectin or collagen I coated at the indicated concentrations, to allow for full attachment, before exposure for 4 hours to medium only (black bars), EMD135981 (gray bars) or cilengitide (white bars). Cilengitide detached HUVEC cultured on vitronectin at all coating concentrations, while it induced HUVEC detachment from fibronectin and collagen only at low coating concentrations (Triplicate wells/condition, n = 3). (c) HUVEC short-term adhesion assays performed on fibronectin and collagen I coated at the indicated concentrations, in medium only (black bars), in the presence of CGP77675 (white bars), cilengitide (gray bars), cilengitide+CGP77675 (hatched bars). Src inhibition did not prevent cilengitide-induced inhibition of cell adhesion on low matrix concentrations. (Triplicate wells/condition, n = 2). Attached cells were quantified by Crystal Violet staining and OD determination at 540 nm wavelength. Asterisks indicate statistical significant differences of the values relative to untreated controls (p<0.05).</p

Cilengitide induces VE-cadherin loss form cellular junctions.

<p>(a) Confluent HUVEC plated on fibronectin, were exposed to cilengitide or EMD135981 (10 µM each) or VEGF (100 ng/ml) for 20 minutes and stained for VE-cadherin. Cilengitide and VEGF treatments disrupted VE-cadherin localization at cellular junctions, while EMD135981 showed no effect (n = 3). Optical magnification: 400×; Bar: 10 µm. (b) Confluent HUVEC plated on fibronectin or collagen I were exposed to cilengitide (10 µM each) for the indicated time and double stained for VE-cadherin and β3 integrin. Cilengitide disrupted VE-cadherin staining and promoted appearance of β3 at VE-cadherin-depleted cell-cell borders (arrows). Paralleling loss of VE-cadherin from cell-cell junctions, ‘gaps’ appeared in the monolayer (asterisks). (n = 4). Optical magnification: 400×; Bar: 10 µm. (c) Higher magnification (2× zooming in) of HUVEC cultures of the experiment shown in panel b to demonstrate rare co-localization of VE-cadherin and β3 integrin at cellular junctions upon cilengitide stimulation (arrowheads). (n = 4). Bars: 10 µm.</p

Cilengitide augments the permeability of HUVEC monolayers.

<p>(a). HUVEC were grown on fibronectin- or collagen I-coated PET filter inserts for 20 hours to ensure confluence and treated with cilengitide (10 µM), CGP77675 (2.5 µM) or a combination thereof. Permeability was measured using the tracer molecule FITC-dextran. Cilengitide increased HUVEC monolayer permeability on both matrices and CGP77675 only partially prevented this increase. Results represent the increase in permeability of treated cultures relative to untreated controls at t = 0 and is given in arbitrary fluorescence units (AU). (b) Crystal violet staining of control and treated filters at the end of the assay (240 minutes) revealed that cilengitide did not cause extensive detachment of HUVEC but induced the appearance of retraced, dendritic-like cells (arrows). (Triplicate filters/condition, n = 3).</p

Cilengitide activates αVβ3 on HUVEC.

<p>(a) HUVEC in suspension were exposed to 10 µM of cilengitide or EMD135981 for 10 minutes, stained by immunofluorescence for β3 LIBS and total αVβ3 expression (with LIBS-1 and LM609 mAbs, respectively) and analyzed by flow cytometry. Cilengitide, but not EMD135981, induced LIBS expression (left histograms, thick lines), without affecting total αVβ3 expression (right histograms, thick lines). Dotted lines: cellular fluorescence in the absence of primary antibody. (n = 3). (b) Fibronectin-adherent HUVEC were exposed to 10 µM cilengitide, EMD135981, or 1 mM MnCl₂ for 10 minutes, stained for β3 LIBS and total αVβ3 expression (with CRC54 or LM609 mAbs, respectively) and analyzed by immunofluorescence microscopy. Total αVβ3 and β3 LIBS were present at focal adhesions in unstimulated HUVEC and at tiny patches at the cell edge in cilengitide-exposed HUVEC, thus confirming that αVβ3-positive patches contain active αVβ3. MnCl₂ stimulated recruitment and activation of αVβ3 at focal adhesions. (n = 2). Optical magnification: 400×; Bars: 10 µm.</p

Proposed model of cilengitide effects on endothelial cells.

<p>Cilengitide acts on αVβ3-expressing endothelial cells in three ways: 1, it suppresses αVβ3-dependent adhesion by directly inhibiting αVβ3-ligand-binding function; 2, it interferes with β1 integrin-mediated cell adhesion through a transdominant negative effect induced by activated αVβ3; 3, it stimulates phosphorylation of VE-cadherin cytoplasmic domain and disrupts VE-cadherin localization at cell-cell contacts through activation of αVβ3 and Src-dependent signaling. Abbreviations: ECM, extracellular matrix; RGDfV, cilengitide; TDNE, transdominant negative effect; pp, phosphorylation.</p

Cilengitide induces Src and FAK phosphorylation.

<p>(a) Western blotting analysis of Src phosphorylation at Y⁵²⁹ and Y⁴¹⁹ and total Src in HUVEC grown on fibronectin and exposed for 10 minutes to EMD135981, cilengitide (10 µM each) and CGP77675 (2.5 µM) as indicated. Cilengitide increased Src phosphorylation at Y⁴¹⁹ but did not alter Y⁵²⁹ phosphorylation. CGP77675 prevented Y⁴¹⁹ phosphorylation. (b) Western blotting analysis of the same cells as in panel a, but for phosphorylation of FAK at Y³⁹⁷ and Y⁵⁷⁶ and total FAK. Cilengitide increased FAK phosphorylation at both tyrosine residues and this was inhibited by CGP77675. EMD135981 had no effect on Src or FAK phosphorylation. Actin was detected to demonstrate equal loading of the lanes. The bar graph represents the relative level of phospho Src/FAK over total Src/FAK as determined by band density analysis. (n = 3).</p