EphB4 Phosphorylation Research Articles

A synthetic bivalent peptide ligand of EphB4 with potent agonistic activity

Interaction between ephrin receptor EphB4 and its ligand EFNB2 mediates bidirectional signaling important for cancer: forward EFNB2-to-EphB4 signaling that is tumor suppressive, and reverse EphB4-to-EFNB2 signaling that promotes angiogenesis important for tumor growth and metastasis. Molecular agents targeting these forward and reverse signals of EphB4-EFNB2 interaction can be used to probe the molecular mechanisms of these complex signaling pathways and develop new anticancer therapeutics. In this study, we applied a bivalent ligand design strategy to synthesize a novel dimeric peptide based on an antagonist TNYL-RAW. The dimeric peptide possessed higher EphB4 receptor binding affinity than the monomeric TNYL-RAW peptide. Interestingly, the dimerization of TNYL-RAW peptide converted a monomeric antagonist of EphB4 to a dimeric agonist. This dimeric agonist promoted EphB4 phosphorylation, internalization and degradation, reduced cancer cell motility, and inhibited tube formation of HUVEC. To investigate the mechanism of action of this bivalent dimeric peptide, FRET experiments and molecular dynamic simulation were conducted and suggested that this bivalent ligand recognizes two EphB4 simultaneously which may promote receptor dimerization and oligomerization. This was further supported by the study of this bivalent ligand containing deletion of critical residues on one of its monomers which impaired its simultaneous binding to two EphB4 and ability to cause EphB4 dimerization and phosphorylation. These results demonstrate the value of this novel bivalent agonist ligand of EphB4 as a probe of the bidirectional signaling of EphB4-EFNB2 and lead for cancer drug development.

Abstract 122: Targeting forward and reverse EphB4/EFNB2 signaling by a peptide with dual functions

Although chloroquine administration in vivo following haemorrhage in mice decreases tumour necrosis factor-alpha (TNF-alpha) release by macrophage (M phi), the mechanism remains unknown. To study this, peritoneal M phi (pM phi) from unmanipulated, sham-operated and post-haemorrhage mice were isolated, treated with 0.13 mg/ml chloroquine for 2 hr, and then stimulated with lipopolysaccharide (LPS) for 48 hr. Pretreatment of pM phi from various groups of mice with chloroquine resulted in 75-90% inhibition of TNF-alpha release, determined by bioassay. Total RNA was isolated from pM phi and murine M phi-derived cell lines (P388D1 and RAW 264.7), stimulated with LPS for 0.5 or 1 hr, respectively, and Northern blot analysis for TNF-alpha mRNA performed. Chloroquine inhibited TNF-alpha mRNA expression without interfering with mRNA stability, suggesting that this agent reduces M phi TNF-alpha release by disrupting TNF-alpha gene transcription.

Ligand-dependent EphB4 activation serves as an anchoring signal in glioma cells

Of the erythropoietin-producing human hepatocellular receptors (Ephs), EphB4 has recently emerged as a potential target in several cancers due to its roles in modified cell migration and invasion. As little is known about the roles of EphB4 in glioma, we sought to investigate its function in glioma by in vitro cell migration and invasion assays, immunoblotting and immunostaining. EphB4 was expressed in glioma cell lines and stem-like cell lines. The stimulation of glioma cells with ephrin-B2, the sole ligand of EphB4, conducted EphB4 phosphorylation and suppressed migration and invasion that downregulation of EphB4 using small interfering RNA abrogated. The stimulation also suppressed the phosphorylation of Akt. We confirmed by immunostaining that EphB4-positive cells existing only in the tumor core, whereas ephrin-B2-positive cells widespread in both the tumor core and the invasive area signifying that EphB4-ephrin-B2 reaction occurred only at the tumor core. Taken together, our data suggest that ephrin-B2-dependent EphB4 phosphorylation acts as an anchoring signal to reduce the malignancy by inhibiting Akt phosphorylation in the glioma core, whereas the scarcity of signaling in the tumor periphery promotes invasion into the surrounding brain.

Positively charged polymers modulate the fate of human mesenchymal stromal cells via ephrinB2/EphB4 signaling

Understanding the mechanisms by which mesenchymal stromal cells (MSCs) interact with the physical properties (e.g. topography, charge, ζ-potential, and contact angle) of polymeric surfaces is essential to design new biomaterials capable of regulating stem cell behavior. The present study investigated the ability of two polymers (pHM1 and pHM3) with different positive surface charge densities to modulate the differentiation of MSCs into osteoblast-like phenotype via cell-cell ephrinB2/EphB4 signaling. Although pHM1 promoted the phosphorylation of EphB4, leading to cell differentiation, pHM3, characterized by a high positive surface charge density, had no significant effect on EphB4 activation or MSCs differentiation. When the MSCs were cultured on pHM1 in the presence of a forward signaling blocking peptide, the osteoblast differentiation was compromised. Our results demonstrated that the ephrinB2/EphB4 interaction was required for MSCs differentiation into an osteoblast-like phenotype and that the presence of a high positive surface charge density altered this interaction.

Eph-B4 mediates vein graft adaptation by regulation of endothelial nitric oxide synthase

Vein graft adaptation is characterized by loss of expression of the tyrosine kinase receptor Eph-B4, the embryonic determinant of venous identity, without increased expression of its ligand ephrin-B2, the embryonic determinant of arterial identity. Endothelial nitric oxide synthase (eNOS) is an important mediator of vessel remodeling. We hypothesized that the mechanism of action of Eph-B4 during vein graft adaptation might be through regulation of downstream eNOS activity. Mouse lung endothelial cells were stimulated with ephrin-B2/Fc, without and with preclustering, without and with the eNOS inhibitor Nω-nitro-l-arginine methyl ester hydrochloride or the Eph-B4 inhibitor NVP-BHG712, and assessed by Western blot and immunofluorescence for eNOS and Eph-B4 phosphorylation. Nitric oxide (NO) production was assessed using an NO-specific chemiluminescence analyzer. Cell migration was assessed using a Transwell assay. Human and mouse vein graft specimens were examined for eNOS activity by Western blot, and vessel remodeling was assessed in vein grafts in wild-type or eNOS knockout mice. Ephrin-B2/Fc stimulated both Eph-B4 and eNOS phosphorylation in a bimodal temporal distribution (n= 4; P< .05), with preclustered ephrin-B2/Fc causing prolonged peak Eph-B4 and eNOS phosphorylation as well as altered subcellular localization (n= 4; P< .05). Ephrin-B2/Fc increased NO release (n= 3; P< .01) as well as increased endothelial cell migration (n= 6; P< .05) in an eNOS-dependent fashion. Both human and mouse vein grafts showed increased eNOS phosphorylation compared with normal veins (n= 3; P< .05). Vein grafts from eNOS knockout mice showed less dilation and less wall thickening compared with wild-type vein grafts (n= 7; P< .05). eNOS is a mediator of vein graft adaptation to the arterial environment. Eph-B4 stimulates eNOS phosphorylation in vitro and may mediate vein graft adaptation by regulation of eNOS activity in vivo.

Long term outcomes after carotid endarterectomy in patients with end stage renal disease

INTRODUCTION: Ephrin-B2 is an embryonic determinant of arteries whereas its tyrosine kinase receptor Ephrin (Eph)-B4 is an embryonic determinant of veins. We have previously reported that Eph-B4 expression is reduced, but Ephrin-B2 expression is increased, during vein graft adaptation, and that Eph-B4 inhibition of vein wall thickening is dependent on caveolin-1, a major structural component of caveolae. We hypothesized that Eph-B4 phosphorylation and downstream signaling is differentially activated by arterial and venous magnitudes of shear stress.

Eph-B4 Mediates Vein Graft Adaptation By Regulation of eNOS

Vein graft adaptation is characterized by loss of expression of the tyrosine kinase receptor ephrin type-B receptor 4 (Eph-B4), the embryonic determinant of venous identity, without increased expression of its ligand ephrin-B2, the embryonic determinant of arterial identity. Endothelial nitric oxide synthase (eNOS) is an important mediator of vessel remodeling. We hypothesized that the mechanism of action of Eph-B4 during vein graft adaptation might be via regulation of downstream eNOS activity. Human and mouse vein graft specimens were examined for eNOS activity by Western blot, and vessel remodeling was assessed in vein grafts in wild-type (WT) or eNOS-knockout (KO) mice. Mouse endothelial cells (EC) were stimulated with ephrin-B2/Fc without and with preclustering, without and with L-NAME (1 mM), and assessed by Western blot and immunofluorescence for eNOS and Eph-B4 phosphorylation. NO production was assessed using a NO-specific chemiluminescence analyzer. Cell migration was assessed using a transwell assay. Human and mouse vein grafts both showed increased eNOS phosphorylation compared with normal veins (n = 3; P < .05). Vein grafts from eNOS-KO mice showed less dilation and less wall thickening compared with WT vein grafts (n = 7; P < .05). Ephrin-B2/Fc stimulated both Eph-B4 and eNOS phosphorylation in a bimodal temporal distribution (n = 4; P < .05), with preclustered ephrin-B2/Fc causing prolonged peak Eph-B4 and eNOS phosphorylation as well as altered subcellular localization (n = 4; P < .05). Ephrin-B2/Fc increased NO release from ECs (n = 3; P < .01) as well as a trend towards increased EC migration (n = 6; P = .11) in an eNOS-dependent fashion. eNOS is a mediator of vein graft adaptation to the arterial environment. Eph-B4 stimulates eNOS phosphorylation and mediates vein graft adaptation by regulation of eNOS activity.

EphrinB2/EphB4 Interaction Promotes Myeloid Leukemia Cell Invasion through RhoA-Mediated Mechanism

Background and Objective: Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrinB ligands in a variety of tumors, suggesting a functional relation between EphB/ephrinB signaling and tumor progression. However, how they regulate the invasiveness of myeloid leukemia cells were still unknown. Our previously study suggested that EphB4 were highly expressed in patients with extramedullary leukemia compared with patients without extramedullary leukemia, which indicated that the expression of EphB4 was related with myeloid leukemia cell invasion. To address the molecular mechanism, we aimed to characterize the role of EphB4 and ephrinB2 ligands in the interaction of myeloid leukemia cells.Methods: To clarify the question, myeloid leukemia cell lines (K562 cells and THP-1 cells) treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins were cultured in vitro, then migration and invasion were determined by transwell assay according to different time. Pulldown western immunoblot analysis were used to detect the level of GTP-RhoA and total RhoA; the phosphorylation of EphB4 and MMP9 expression were also determined by immunoblot analysis before and after the treatment of different clustered Fc proteins.Results: The results showed that after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.85-fold, P=0.033), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.46 -fold, P=0.025). However, the numbers of K562 cells migrating through transwell chamber after ephrinA1–Fc stimulation didn’t significantly increase compared to Fc proteins stimulation (P=0.411), and the numbers of K562 cells invading the matrigel also didn’t enhanced (P=0.072) after ephrinA1–Fc stimulation. Moreover, after ephrinB2–Fc stimulation, the numbers of THP-1 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (2.25-fold, P<0.01), meanwhile, the numbers of THP-1 cells invading the matrigel also enhanced (1.66 -fold, P<0.01). However, the numbers of THP-1 cells migrating through transwell chamber and the numbers of THP-1 cells invading the matrigel didn’t significantly enhanced (P>0.05, P>0.05) after ephrinA1–Fc stimulation. Furthermore, EphB4 immunoprecipitation followed by immunoblotting with anti-phosphotyrosine antibody revealed that EphB4 is phosphorylated on tyrosine in K562 cells after ephrinB2–Fc stimulation. Additionally, the level of active RhoA (GTP-RhoA) and MMP9 in K562 cells were both significantly increased in response to EphB4 receptor activation with its ligand ephrin-B2-Fc ( P<0.05).Conclusions: These findings suggested that EphB4/EprinB2 signaling played an important role in myeloid leukemia cells progression by promoting their migratory ability, activating RhoA activity and increasing MMP9 expression. Our findings reveal a novel regulation of this intriguing receptor/ligand family that contributes to the cell invasiveness of myeloid leukemia cells. DisclosuresNo relevant conflicts of interest to declare.

Ephrin type-B receptor 4 activation reduces neointimal hyperplasia in human saphenous vein in vitro

Vein bypass is an essential therapy for patients with advanced peripheral and coronary artery disease despite development of neointimal hyperplasia. We have shown that stimulation of the receptor tyrosine kinase ephrin type-B receptor 4 (Eph-B4) with its ligand ephrin-B2 prevents neointimal hyperplasia in murine vein grafts. This study determines whether Eph-B4 in adult human veins is capable of phosphorylation and activation of downstream signaling pathways, as well as functional to release nitric oxide (NO) and prevent neointimal hyperplasia in vitro. Discarded human saphenous veins were taken from the operating room and placed in organ culture without or with ephrin-B2/Fc (2 μg/mL) for 14 days, and the neointima/media ratio was measured in matched veins. Primary human umbilical vein endothelial cells were treated with ephrin-B2/Fc (2 μg/mL) and examined with quantitative polymerase chain reaction, Western blot, immunoassays, and for release of NO. Ephrin-B2/Fc (2 μg/mL) was placed on the adventitia of saphenous veins treated with arterial shear stress for 24 hours in a bioreactor and activated Eph-B4 examined with immunofluorescence. The baseline intima/media ratio in saphenous vein rings was 0.456 ± 0.097, which increased to 0.726 ± 0.142 in untreated veins after 14 days in organ culture but only to 0.630 ± 0.132 in veins treated with ephrin-B2/Fc (n = 19, P = .017). Ephrin-B2/Fc stimulated Akt, endothelial NO synthase and caveolin-1 phosphorylation, and NO release (P = .007) from human umbilical vein endothelial cells (n = 6). Ephrin-B2/Fc delivered to the adventitia stimulated endothelial Eph-B4 phosphorylation after 24 hours of arterial stress in a bioreactor (n = 3). Eph-B4 is present and functional in adult human saphenous veins, with intact downstream signaling pathways capable of NO release and prevention of neointimal hyperplasia in vitro. Adventitial delivery of ephrin-B2/Fc activates endothelial Eph-B4 in saphenous veins treated with arterial shear stress in vitro. These results suggest that stimulation of Eph-B4 function may be a candidate strategy for translation to human clinical trials designed to inhibit venous neointimal hyperplasia.

Down-regulation of EphB4 phosphorylation is necessary for esophageal squamous cell carcinoma tumorigenecity.

Eph/ephrin signaling system plays a very important role in the tumorigenesis and the formation of blood vessel. However, the function of EphB4 and its ligand ephrin B2 in the carcinogenesis of esophageal squamous cell carcinoma (ESCC) is not fully understood. Here, it was found that the expression of EphB4 was up-regulated in ESCC tissues compared with the paired normal tissues, while ephrin B2 was down-regulated in ESCC samples. Phosphorylation of EphB4 induced by its ligand ephrin B2-Fc inhibited the growth, migration and colony formation of ESCC cells. Moreover, over-expression of EphB4 or EphB4 kinase dead mutant (EphB4 KD) in ESCC cells promoted cell growth and migration, suggesting EphB4 promoted cell growth and migration independent of its kinase activity. Furthermore, we found that EphB4 interacted with the adaptor protein RACK1 and RACK1 decreased the phosphorylation level of EphB4. Taken together, our study revealed the important function and regulation of EphB4 in the progression of ESCC and suggested EphB4 as a novel target for the treatment of ESCC.

Podocyte EphB4 signaling helps recovery from glomerular injury

Eph receptor tyrosine kinases and their ligands (ephrins) have a pivotal role in the homeostasis of many adult organs and are widely expressed in the kidney. Glomerular diseases beginning with mesangiolysis can recover, with podocytes having a critical role in this healing process. We studied here the role of Eph signaling in glomerular disease recovery following mesangiolytic Thy1.1 nephritis in rats. EphB4 and ephrinBs were expressed in healthy glomerular podocytes and were upregulated during Thy1.1 nephritis, with EphB4 strongly phosphorylated around day 9. Treatment with NPV-BHG712, an inhibitor of EphB4 phosphorylation, did not cause glomerular changes in control animals. Nephritic animals treated with vehicle did not have morphological evidence of podocyte injury or loss; however, application of this inhibitor to nephritic rats induced glomerular microaneurysms, podocyte damage, and loss. Prolonged NPV-BHG712 treatment resulted in increased albuminuria and dysregulated mesangial recovery. Additionally, NPV-BHG712 inhibited capillary repair by intussusceptive angiogenesis (an alternative to sprouting angiogenesis), indicating a previously unrecognized role of podocytes in regulating intussusceptive vessel splitting. Thus, our results identify EphB4 signaling as a pathway allowing podocytes to survive transient capillary collapse during glomerular disease.

Cytoplasmic Tyrosines 653 and 774 Are Critical for Eph-B4 Signaling to ERK1/2 and Akt

Introduction: Eph-B4, a receptor tyrosine kinase that determines vascular venous fate during vasculogenesis, is downregulated during vein graft adaptation to the arterial circulation. We have previously shown that stimulation of Eph-B4 with Ephrin-B2/Fc prevents vein graft thickening. However, the mechanisms by which Eph-B4 activation prevents vein graft thickening are unknown. Similarly, we have also shown that Eph-B4 phosphorylation is critical to Eph-B4 action; therefore we examined whether several of the 15 phosphorylation sites in the cytoplasmic portion of Eph-B4 are critical to Eph-B4 stimulation of intracellular signal transduction pathways. Methods: A wild-type Eph-B4 plasmid vector was created, followed by three individual point mutations at tyrosine 581, 653, and 774, with substitution of phenylalanine for tyrosine. Mutations were confirmed by sequencing. Cos-7 cells were transfected with wild-type or mutant Eph-B4 (Y581F-, Y653F-, and Y774F-Eph-B4). Transfected cells were starved for 24 hours and then stimulated with Ephrin-B2/Fc (1 min). Cell lysates were examined via Western blotting for differences in tyrosine-phosphorylation, and phospho-Akt, phospho-ERK1/2, and phospho-FAK activity between wild-type and mutant Eph-B4. Double-labeled immunofluorescence of wild-type and mutant Eph-B4 transfected Cos-7 cells was utilized to examine Eph-B4 tyrosine phosphorylation and colocolization of Eph-B4 with caveolin-1. Results: Stimulation of wild-type Eph-B4 in transfected Cos-7 cells results in phosphorylation of Eph-B4, Akt, and ERK1/2, but not FAK. Tyrosine phosphorylation of Eph-B4 is decreased with the loss of Y774, but not with the loss of Y653 and Y581. Loss of Y653 and Y774 resulted in decreased phospho-AKT activity compared to wild-type Eph-B4 and a loss of Y774 resulted in decreased phospho-ERK1/2. phospho-FAK activity was equivalent between wild-type and all mutant Eph-B4 transfected cells. Loss of Y581 did not influence phosphorylation of Akt, ERK1/2, or FAK. Double-labeled immunofluorescence confirmed the above findings. Wild-type Eph-B4, but not mutant Eph-B4, colocalized with caveolin-1 after Ephrin-B2/Fc stimulation. Conclusions: Eph-B4 downstream cell signaling requires tyrosine residues 653 and 774. Their loss results in decreased phospho-ERK1/2 and phospho-Akt, but not phospho-FAK, activity, and also prevents colocalization of activated Eph-B4 with caveolin-1, showing that tyrosine phosphorylation of these residues is critical to Eph-B4 function. These results suggest novel molecular targets to modulate Eph-B4 activity.

Abstract 1974: EphB2 signaling activity plays a vital role in medulloblastoma cell adhesion and invasion

Abstract Eph/ephrin signaling has been implicated in various types of cancer, enhancing key processes like migration, proliferation and angiogenesis. In medulloblastoma, invading tumor cells characteristically lead to early recurrence and a decreased prognosis. Based on kinase activity profiling data published recently we hypothesized on a key function of the Eph/ephrin signaling system in medulloblastoma invasion. In a panel of medulloblastoma cell lines a limited subset of Eph receptors was found to be expressed of which EphB2 was the most predominant. Furthermore, a significantly higher expression of EphB2 and its ligands ephrin-B1 and ephrin-B3 was observed in medulloblastoma patient tissue compared to normal cerebellum. To determine the relative importance of the EphB and EphA receptor families, 3 medulloblastoma cell lines (Daoy, Uw-402, Res-256) were stimulated with either ephrin-A1 or ephrin-B1 followed by assessment of the migration and adhesion capacity of the cells. Upon stimulation with ephrin-A1 no phenotype could be observed. However, stimulation with ephrin-B1 resulted in a marked increase in migration capacity of Daoy and, to a lesser extent, Uw-402 in concentrations as low as 0.2 ug/mL. The adhesion capacity of these cell lines upon stimulation with ephrin-B1 was determined employing collagen and laminin coated culture dishes. Again, no effects could be observed on Res-256 whereas the adhesive capacity of Daoy and Uw-402 decreased significantly. In addition, stable transfection of Daoy medulloblastoma cells with shRNA against EphB2 significantly reduced the ephrin-B1 induced effects on tumor cell adhesion. The observed differences in the effects of EphB receptor stimulation correspond with the expression levels of EphB2 in the cell lines. Furthermore, a substantial increase of EphB2 and EphB4 phosphorylation could be observed for Daoy and to a lesser extent Uw-402. Res-256 had a low mRNA expression of EphB2 and B4 and also showed no increase in phosphorylation of these receptors upon stimulation. Interestingly, the increase in phosphorylated EphB2 and EphB4 is accompanied by a sharp decrease in EGFR phosphorylation. Possibly this plays a role in the ephrin-B1 induced effects, as has been reported recently for ephrin-A5 in glioblastoma. Recent studies report an epigenetic regulation by hypermethylation of Eph/ephrin family gene expression in acute lymphoblastic leukemia. To assess the presence of epigenetic regulation of EphB receptor expression in medulloblastoma we treated the medulloblastoma cell lines with the demethylating agent 5-aza-2′-deoxycytidine. This resulted in a substantial increase in expression of all EphB type receptors in Daoy. In conclusion, our results indicate a selective Eph expression profile in favor of medulloblastoma invasion. Furthermore, the expression of Eph receptors is regulated, at least in part, through DNA methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1974. doi:10.1158/1538-7445.AM2011-1974

Abstract 3188: Forward and reverse signalings of EphB4/EfnB2 are active in epithelial ovarian cancer and are inhibited by dasatinib

Abstract Ephrin receptor (Eph) family constitutes the largest subfamily of trans-membrane tyrosine kinases. Their ligands, the ephrins (Efn), are membrane-anchored proteins grouped into two classes: class A ligands which are GPI-anchored proteins; class B ligands which contain a trans-membrane and a cytoplasmic tail. Eph-signaling is termed forward, Efn-signaling is termed reverse. Eph/Efn proteins are expressed in virtually all tissues and they are involved in a number of cellular events during embryonic development such as cell migration, repulsion versus adhesion, and cell to cell communication. In cancers, Eph/Efn involment is controversial. The Eph appeared to exert a suppressive role in colon and breast cancers, and the Eph/Efn signaling has been showed to drive or inhibit invasion via a reciprocal regulatory loop in glioblastomas and prostate cancer. In epithelial ovarian cancers (EOCs) the co-expression of EphB4 and EfnB2 in seventy-two EOC samples was associated to worst prognosis, highlighting a possible autocrine/paracrine activation loop in EOCs. We analyzed the signaling through EphB4/EfnB2 pair in EOC cell lines that express both the receptor and the ligand or only the receptor. To analyze the EphB4 activation we evaluated the EphB4 phosphorylation in growing cells and in cells upon binding of a soluble EfnB2-Fc molecule on cell in suspension. We found that EphB4 is phosphorylated in cells growing in presence of fetal calf serum and is further phosphorylated upon EfnB2-Fc stimulation. Accordingly, treatment of growing EOC cells with the soluble EfnB2-Fc induced a decrease in growth capability, meaning that the ligand in soluble form inhibited the binding of the cell membrane ligand to the receptor. In line with this results, silencing of EphB4 by a specific siRNA pool, inhibited cell growth by impairing cell survival. On the other site, silencing of EfnB2 lead to inhibition of EphB4 and src phosphorylations, followed by FAK phosphorylation and remodeling of focal adhesions. Therefore, in EOC cell lines expressing both the receptor and the ligand forward and reverse signaling of EphB4/EfnB2 were active. In addition, we showed that a panel of EOC cells are sensitive (IC50&amp;lt;10 nM) to the kinase inhibitor dasatinib which has been showed to have the same binding capacity on EphB4 and src. Indeed, dasatinib reduced both EphB4 and src phosphorylation in EOC cells. Ongoing experiments of dasatinib treatment in nude mice bearing OVCAR3 xenografts in the peritoneum showed an increase of the survival rate in the treated animals. These in vitro and in vivo experiments demonstrated for the first time that in EOC cells dasatinib targeted both EphB4 and src kinase thus suggesting dasatinib as a valuable therapy in EOC patients. Partially supported by FIRB and AIRC. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3188.

BCR‐ABL‐independent and RAS / MAPK pathway‐dependent form of imatinib resistance in Ph‐positive acute lymphoblastic leukemia cell line with activation of EphB4

We investigated the mechanism responsible for imatinib (IM) resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) cell lines. We established cell lines from a patient with Ph(+) ALL at the time of first diagnosis and relapsed phase and designated as NPhA1 and NPhA2, respectively. We also derived IM-resistant cells, NPhA2/STIR, from NPhA2 under gradually increasing IM concentrations. NPhA1 was sensitive to IM (IC(50) 0.05 microm) and NPhA2 showed mild IM resistance (IC(50) 0.3 microm). NPhA2/STIR could be maintained in the presence of 10 microm IM. Phosphorylation of MEK and ERK was slightly elevated in NPhA2 and significantly elevated in NPhA2/STIR compared to NPhA1 cells. After treatment with IM, phosphorylation of MEK and ERK was not suppressed but rather increased in NPhA2 and NPhA2/STIR. Active RAS was also increased markedly in NPhA2/STIR after IM treatment. The expression of BCL-2 was increased in NPhA2 compared to NPhA1, but no further increase in NPhA2/STIR. Proliferation of NPhA2/STIR was significantly inhibited by a combination of MEK inhibitor and IM. Analysis of tyrosine phosphorylation status with a protein tyrosine kinase array showed increased phosphorylation of EphB4 in NPhA2/STIR after IM treatment. Although transcription of EphB4 was suppressed in NPhA1 and NPhA2 after IM treatment, it was not suppressed and its ligand, ephrinB2, was increased in NPhA2/STIR. Suppression of EphB4 transcripts by introducing short hairpin RNA into NPhA2/STIR partially restored their sensitivity to IM. These results suggest a new mechanism of IM resistance mediated by the activation of RAS/MAPK pathway and EphB4.

The ephrinB2/EphB4 axis is dysregulated in osteoprogenitors from myeloma patients and its activation affects myeloma bone disease and tumor growth

Myeloma bone disease is caused by uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Bidirectional signaling between the cell-surface ligand ephrinB2 and its receptor, EphB4, is involved in the coupling of osteoblastogenesis and osteoclastogenesis and in angiogenesis. EphrinB2 and EphB4 expression in mesenchymal stem cells (MSCs) from myeloma patients and in bone cells in myelomatous bones was lower than in healthy counterparts. Wnt3a induced up-regulation of EphB4 in patient MSCs. Myeloma cells reduced expression of these genes in MSCs, whereas in vivo myeloma cell-conditioned media reduced EphB4 expression in bone. In osteoclast precursors, EphB4-Fc induced ephrinB2 phosphorylation with subsequent inhibition of NFATc1 and differentiation. In MSCs, EphB4-Fc did not induce ephrinB2 phosphorylation, whereas ephrinB2-Fc induced EphB4 phosphorylation and osteogenic differentiation. EphB4-Fc treatment of myelomatous SCID-hu mice inhibited myeloma growth, osteoclastosis, and angiogenesis and stimulated osteoblastogenesis and bone formation, whereas ephrinB2-Fc stimulated angiogenesis, osteoblastogenesis, and bone formation but had no effect on osteoclastogenesis and myeloma growth. These chimeric proteins had similar effects on normal bone. Myeloma cells expressed low to undetectable ephrinB2 and EphB4 and did not respond to the chimeric proteins. The ephrinB2/EphB4 axis is dysregulated in MM, and its activation by EphB4-Fc inhibits myeloma growth and bone disease.

EphB4 controls blood vascular morphogenesis during postnatal angiogenesis

Guidance molecules have attracted interest by demonstration that they regulate patterning of the blood vascular system during development. However, their significance during postnatal angiogenesis has remained unknown. Here, we demonstrate that endothelial cells of human malignant brain tumors also express guidance molecules, such as EphB4 and its ligand ephrinB2. To study their function, EphB4 variants were overexpressed in blood vessels of tumor xenografts. Our studies revealed that EphB4 acts as a negative regulator of blood vessel branching and vascular network formation, switching the vascularization program from sprouting angiogenesis to circumferential vessel growth. In parallel, EphB4 reduces the permeability of the tumor vascular system via activation of the angiopoietin-1/Tie2 system at the endothelium/pericyte interface. Furthermore, overexpression of EphB4 variants in blood vessels during (i) vascularization of non-neoplastic cell grafts and (ii) retinal vascularization revealed that these functions of EphB4 apply to postnatal, non-neoplastic angiogenesis in general. This implies that both neoplastic and non-neoplastic vascularization is driven not only by a vascular initiation program but also by a vascular patterning program mediated by guidance molecules.