ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis.

Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis.

eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity.

The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

Targeting Axl favors an antitumorigenic microenvironment that enhances immunotherapy responses by decreasing Hif-1α levels.

Hypoxia is an important phenomenon in solid tumors that contributes to metastasis, tumor microenvironment (TME) deregulation, and resistance to therapies. The receptor tyrosine kinase AXL is an HIF target, but its roles during hypoxic stress leading to the TME deregulation are not well defined. We report here that the mammary gland-specific deletion of Axl in a HER2+ mouse model of breast cancer leads to a normalization of the blood vessels, a proinflammatory TME, and a reduction of lung metastases by dampening the hypoxic response in tumor cells. During hypoxia, interfering with AXL reduces HIF-1α levels altering the hypoxic response leading to a reduction of hypoxia-induced epithelial-to-mesenchymal transition (EMT), invasion, and production of key cytokines for macrophages behaviors. These observations suggest that inhibition of Axl generates a suitable setting to increase immunotherapy. Accordingly, combining pharmacological inhibition of Axl with anti-PD-1 in a preclinical model of HER2+ breast cancer reduces the primary tumor and metastatic burdens, suggesting a potential therapeutic approach to manage HER2+ patients whose tumors present high hypoxic features.

AXL confers cell migration and invasion by hijacking a PEAK1-regulated focal adhesion protein network.

Aberrant expression of receptor tyrosine kinase AXL is linked to metastasis. AXL can be activated by its ligand GAS6 or by other kinases, but the signaling pathways conferring its metastatic activity are unknown. Here, we define the AXL-regulated phosphoproteome in breast cancer cells. We reveal that AXL stimulates the phosphorylation of a network of focal adhesion (FA) proteins, culminating in faster FA disassembly. Mechanistically, AXL phosphorylates NEDD9, leading to its binding to CRKII which in turn associates with and orchestrates the phosphorylation of the pseudo-kinase PEAK1. We find that PEAK1 is in complex with the tyrosine kinase CSK to mediate the phosphorylation of PAXILLIN. Uncoupling of PEAK1 from AXL signaling decreases metastasis in vivo, but not tumor growth. Our results uncover a contribution of AXL signaling to FA dynamics, reveal a long sought-after mechanism underlying AXL metastatic activity, and identify PEAK1 as a therapeutic target in AXL positive tumors.

Rap1 is Involved in Angiopoietin-1-Induced Cell-Cell Junction Stabilization and Endothelial Cell Sprouting.

Angiopoietin-1 (Ang-1) is an important proangiogenic factor also involved in the maintenance of endothelial-barrier integrity. The small GTPase Rap1 is involved in the regulation of adherens junctions through VE-cadherin-mediated adhesion, and in endothelial permeability. While many studies established that Rap1 activation is critical for endothelial cell-cell adhesions, its roles in the antipermeability effects of Ang-1 are ill-defined. Thus, we determined the contribution of Rap1 to Ang-1-stimulated angiogenic effects on endothelial cells (ECs). We found that Rap1 is activated following Ang-1 stimulation and is required for the antipermeability effects of Ang-1 on EC monolayers. Our results also revealed that Rap1 is necessary for EC sprouting stimulated by Ang-1 but had no significant effect on Ang-1-induced EC migration and adhesion. In contrast, downregulation of VE-cadherin markedly increased the adhesiveness of ECs to the substratum, which resulted in inhibition of Ang-1-stimulated migration. These results revealed that Rap1 is central to the effects of Ang-1 at intercellular junctions of ECs, whereas VE-cadherin is also involved in the adhesion of ECs to the extracellular matrix.

Polarization and sprouting of endothelial cells by angiopoietin-1 require PAK2 and paxillin-dependent Cdc42 activation.

Binding of angiopoietin-1 (Ang-1) to its receptor Tie2 on endothelial cells (ECs) promotes vessel barrier integrity and angiogenesis. Here, we identify PAK2 and paxillin as critical targets of Ang-1 responsible for EC migration, polarization, and sprouting. We found that Ang-1 increases PAK2-dependent paxillin phosphorylation and remodeling of focal adhesions and that PAK2 and paxillin are required for EC polarization, migration, and angiogenic sprouting in response to Ang-1. Our findings show that Ang-1 triggers Cdc42 activation at the leading edges of migrating ECs, which is dependent on PAK2 and paxillin expression. We also established that the polarity protein Par3 interacts with Cdc42 in response to Ang-1 in a PAK2- and paxillin-dependent manner. Par3 is recruited at the leading edges of migrating cells and in focal adhesion, where it forms a signaling complex with PAK2 and paxillin in response to Ang-1. These results show that Ang-1 triggers EC polarization and angiogenic sprouting through PAK2-dependent paxillin activation and remodeling of focal adhesions, which are necessary for local activation of Cdc42 and the associated polarity complex. We have shown that PAK2 controls a signaling pathway important for angiogenic sprouting that links focal adhesions to polarity signaling in ECs.

AXL knockdown gene signature reveals a drug repurposing opportunity for a class of antipsychotics to reduce growth and metastasis of triple-negative breast cancer.

Triple-Negative Breast Cancer (TNBC) is an aggressive cancer subtype that is associated with a poor prognosis due to its propensity to form metastases. The receptor tyrosine kinase AXL plays a role in tumor cell dissemination and its expression in breast cancers correlates with poor patient survival. Here, we explored whether already used drugs might elicit a gene signature similar to that seen with AXL knockdown in TNBC cells and which could, therefore, offer an opportunity for drug repurposing. To this end, we queried the Connectivity Map with an AXL gene signature which revealed a class of dopamine receptors antagonists named phenothiazines (Thioridazine, Fluphenazine and Trifluoperazine) typically used as anti-psychotics. We next tested if these drugs, similarly to AXL depletion, were able to limit growth and metastatic progression of TNBC cells and found that phenothiazines are able to reduce cell invasion, proliferation, viability and increase apoptosis of TNBC cells in vitro. Mechanistically, these drugs did not affect AXL activity but instead reduced PI3K/AKT/mTOR and ERK signaling. When administered to mice bearing TNBC xenografts, phenothiazines were able to reduce tumor growth and metastatic burden. Collectively, these results suggest that these antipsychotics display anti-tumor and anti-metastatic activity and that they could potentially be repurposed, in combination with standard chemotherapy, for the treatment of TNBC.

NOTCH1 signaling induces pathological vascular permeability in diabetic retinopathy.

Diabetic macular edema is a major complication of diabetes resulting in loss of central vision. Although heightened vessel leakiness has been linked to glial and neuronal-derived factors, relatively little is known on the mechanisms by which mature endothelial cells exit from a quiescent state and compromise barrier function. Here we report that endothelial NOTCH1 signaling in mature diabetic retinas contributes to increased vascular permeability. By providing both human and mouse data, we show that NOTCH1 ligands JAGGED1 and DELTA LIKE-4 are up-regulated secondary to hyperglycemia and activate both canonical and rapid noncanonical NOTCH1 pathways that ultimately disrupt endothelial adherens junctions in diabetic retinas by causing dissociation of vascular endothelial-cadherin from ß-catenin. We further demonstrate that neutralization of NOTCH1 ligands prevents diabetes-induced retinal edema. Collectively, these results identify a fundamental process in diabetes-mediated vascular permeability and provide translational rational for targeting the NOTCH pathway (primarily JAGGED1) in conditions characterized by compromised vascular barrier function.

The Receptor Tyrosine Kinase AXL Is Required at Multiple Steps of the Metastatic Cascade during HER2-Positive Breast Cancer Progression.

AXL is activated by its ligand GAS6 and is expressed in triple-negative breast cancer cells. In the current study, we report AXL expression in HER2-positive (HER2+) breast cancers where it correlates with poor patient survival. Using murine models of HER2+ breast cancer, Axl, but not its ligand Gas6, was found to be essential for metastasis. We determined that AXL is required for intravasation, extravasation, and growth at the metastatic site. We found that AXL is expressed in HER2+ cancers displaying epithelial-to-mesenchymal transition (EMT) signatures where it contributes to sustain EMT. Interfering with AXL in a patient-derived xenograft (PDX) impaired transforming growth factor ß (TGF-ß)-induced cell invasion. Last, pharmacological inhibition of AXL specifically decreased the metastatic burden of mice developing HER2+ breast cancer. Our data identify AXL as a potential anti-metastatic co-therapeutic target for the treatment of HER2+ breast cancers.

CdGAP/ARHGAP31 is regulated by RSK phosphorylation and binding to 14-3-3ß adaptor protein.

Cdc42 GTPase-activating protein (CdGAP, also named ARHGAP31) is a negative regulator of the GTPases Rac1 and Cdc42. Associated with the rare developmental disorder Adams-Oliver Syndrome (AOS), CdGAP is critical for embryonic vascular development and VEGF-mediated angiogenesis. Moreover, CdGAP is an essential component in the synergistic interaction between TGFß and ErbB-2 signaling pathways during breast cancer cell migration and invasion, and is a novel E-cadherin transcriptional co-repressor with Zeb2 in breast cancer. CdGAP is highly phosphorylated on serine and threonine residues in response to growth factors and is a substrate of ERK1/2 and GSK-3. Here, we identified Ser1093 and Ser1163 in the C-terminal region of CdGAP, which are phosphorylated by RSK in response to phorbol ester. These phospho-residues create docking sites for binding to 14-3-3 adaptor proteins. The interaction between CdGAP and 14-3-3 proteins inhibits the GAP activity of CdGAP and sequesters CdGAP into the cytoplasm. Consequently, the nucleocytoplasmic shuttling of CdGAP is inhibited and CdGAP-induced cell rounding is abolished. In addition, 14-3-3ß inhibits the ability of CdGAP to repress the E-cadherin promoter and to induce cell migration. Finally, we show that 14-3-3ß is unable to regulate the activity and subcellular localization of the AOS-related mutant proteins lacking these phospho-residues. Altogether, we provide a novel mechanism of regulation of CdGAP activity and localization, which impacts directly on a better understanding of the role of CdGAP as a promoter of breast cancer and in the molecular causes of AOS.

Cell adhesion controlled by adhesion G protein-coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo-Dock.

Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gßγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses.

Endothelial NO Synthase-Dependent S-Nitrosylation of ß-Catenin Prevents Its Association with TCF4 and Inhibits Proliferation of Endothelial Cells Stimulated by Wnt3a.

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) modulates many functions in endothelial cells. S-nitrosylation (SNO) of cysteine residues on ß-catenin by eNOS-derived NO has been shown to influence intercellular contacts between endothelial cells. However, the implication of SNO in the regulation of ß-catenin transcriptional activity is ill defined. Here, we report that NO inhibits the transcriptional activity of ß-catenin and endothelial cell proliferation induced by activation of Wnt/ß-catenin signaling. Interestingly, induction by Wnt3a of ß-catenin target genes, such as the axin2 gene, is repressed in an eNOS-dependent manner by vascular endothelial growth factor (VEGF). We identified Cys466 of ß-catenin as a target for SNO by eNOS-derived NO and as the critical residue for the repressive effects of NO on ß-catenin transcriptional activity. Furthermore, we observed that Cys466 of ß-catenin, located at the binding interface of the ß-catenin-TCF4 transcriptional complex, is essential for disruption of this complex by NO. Importantly, Cys466 of ß-catenin is necessary for the inhibitory effects of NO on Wnt3a-stimulated proliferation of endothelial cells. Thus, our data define the mechanism responsible for the repressive effects of NO on the transcriptional activity of ß-catenin and link eNOS-derived NO to the modulation by VEGF of Wnt/ß-catenin-induced endothelial cell proliferation.

Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation.

VEGF and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through regulation of a plethora of signaling events in endothelial cells (ECs). Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes conferring specific responses to each factor remain poorly defined. Thus, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in ECs using mass spectrometry. Analysis of VEGF and Ang-1 networks of regulated phosphoproteins revealed that the junctional proteins ZO-1, ZO-2, JUP and p120-catenin are part of a cluster of proteins phosphorylated following VEGF stimulation that are linked to MAPK1 activation. Down-regulation of these junctional proteins led to MAPK1 activation and accordingly, increased proliferation of ECs stimulated specifically by VEGF, but not by Ang-1. We identified ZO-1 as the central regulator of this effect and showed that modulation of cellular ZO-1 levels is necessary for EC proliferation during vascular development of the mouse postnatal retina. In conclusion, we uncovered ZO-1 as part of a signaling node activated by VEGF, but not Ang-1, that specifically modulates EC proliferation during angiogenesis.

Axl phosphorylates Elmo scaffold proteins to promote Rac activation and cell invasion.

The receptor tyrosine kinase Axl contributes to cell migration and invasion. Expression of Axl correlates with metastatic progression in cancer patients, yet the specific signaling events promoting invasion downstream of Axl are poorly defined. Herein, we report Elmo scaffolds to be direct substrates and binding partners of Axl. Elmo proteins are established to interact with Dock family guanine nucleotide exchange factors to control Rac-mediated cytoskeletal dynamics. Proteomics and mutagenesis studies reveal that Axl phosphorylates Elmo1/2 on a conserved carboxyl-terminal tyrosine residue. Upon Gas6-dependent activation of Axl, endogenous Elmo2 becomes phosphorylated on Tyr-713 and enters into a physical complex with Axl in breast cancer cells. Interfering with Elmo2 expression prevented Gas6-induced Rac1 activation in breast cancer cells. Similarly to blocking of Axl, Elmo2 knockdown or pharmacological inhibition of Dock1 abolishes breast cancer cell invasion. Interestingly, Axl or Elmo2 knockdown diminishes breast cancer cell proliferation. Rescue of Elmo2 knockdown cells with the wild-type protein but not with Elmo2 harboring Tyr-713-Phe mutations restores cell invasion and cell proliferation. These results define a new mechanism by which Axl promotes cell proliferation and invasion and identifies inhibition of the Elmo-Dock pathway as a potential therapeutic target to stop Axl-induced metastases.

Acyclic tethers mimicking subunits of polysaccharide ligands: selectin antagonists.

We report on the design and synthesis of molecules having E- and P-selectins blocking activity both in vitro and in vivo. The GlcNAc component of the selectin ligand sialyl Lewis(X) was replaced by an acyclic tether that links two saccharide units. The minimization of intramolecular dipole-dipole interactions and the gauche effect would be at the origin of the conformational bias imposed by this acyclic tether. The stereoselective synthesis of these molecules, their biochemical and biological evaluations using surface plasmon resonance spectroscopy (SPR), and in vivo assays are described. Because the structure of our analogues differs from the most potent E-selectin antagonists reported, our acyclic analogues offer new opportunities for chemical diversity.

The phosphatase PTP-PEST/PTPN12 regulates endothelial cell migration and adhesion, but not permeability, and controls vascular development and embryonic viability.

Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability.

Formation of a PKCζ/ß-catenin complex in endothelial cells promotes angiopoietin-1-induced collective directional migration and angiogenic sprouting.

Angiogenic sprouting requires that cell-cell contacts be maintained during migration of endothelial cells. Angiopoietin-1 (Ang-1) and vascular endothelial growth factor act oppositely on endothelial cell junctions. We found that Ang-1 promotes collective and directional migration and, in contrast to VEGF, induces the formation of a complex formed of atypical protein kinase C (PKC)-ζ and ß-catenin at cell-cell junctions and at the leading edge of migrating endothelial cells. This complex brings Par3, Par6, and adherens junction proteins at the front of migrating cells to locally activate Rac1 in response to Ang-1. The colocalization of PKCζ and ß-catenin at leading edge along with PKCζ-dependent stabilization of cell-cell contacts promotes directed and collective endothelial cell migration. Consistent with these results, down-regulation of PKCζ in endothelial cells alters Ang-1-induced sprouting in vitro and knockdown in developing zebrafish results in intersegmental vessel defects caused by a perturbed directionality of tip cells and by loss of cell contacts between tip and stalk cells. These results reveal that PKCζ and ß-catenin function in a complex at adherens junctions and at the leading edge of migrating endothelial cells to modulate collective and directional migration during angiogenesis.

Modulation of the cochaperone AHA1 regulates heat-shock protein 90 and endothelial NO synthase activation by vascular endothelial growth factor.

OBJECTIVE: Vascular endothelial growth factor (VEGF) signaling to endothelial NO synthase (eNOS) plays a central role in angiogenesis. In endothelial cells (ECs), heat-shock protein 90 (Hsp90) is also a regulator of eNOS activity. Our study is designed to determine whether modulation of the activator of Hsp90 ATPase 1 (AHA1) regulates the function of Hsp90 in ECs. METHODS AND RESULTS: We show that eNOS phosphorylation on Ser-1179 after VEGF stimulation is significantly reduced in ECs transfected with a small interfering RNA against AHA1. Accordingly, VEGF-stimulated NO production, endothelial permeability, cell migration, and EC invasion in Matrigel implants in mice are reduced in small interfering RNA against AHA1-treated conditions. Furthermore, the induction of eNOS association with Hsp90 after VEGF stimulation is decreased in AHA1-downregulated cells. We also demonstrate that modulation of Hsp90 activity by AHA1 regulates phosphorylation of Hsp90 on Tyr-300. Interestingly, the association of AHA1 with Hsp90 is increased after c-Src-mediated phosphorylation of Hsp90 on Tyr-300. Finally, we show that overexpression of AHA1 in ECs promotes association of eNOS and Hsp90, phosphorylation of Ser-1179 of eNOS, increases NO production, and cell migration. CONCLUSIONS: These results reveal that modulation of Hsp90 activity by AHA1 regulates VEGF signaling to eNOS and angiogenesis.