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The occurrence of major depressive disorder (MDD) has been linked to an increased vulnerability to stress. The basolateral amygdala (BLA) is one of the critical brain areas that involved in the regulation of pathological reactivity to stress. Increasing evidence indicates that the EphB2 receptor (EphB2) plays a critical role in neuropsychiatric disorders, such as Alzheimer's disease, pain and anxiety. However, whether the EphB2 in the BLA is involved in stress vulnerability is unclear. Here, we identified EphB2 in the BLA as a key regulator contributed to the modulation of stress vulnerability in adult mice. We found that the expression of EphB2 in the BLA was significantly increased in the animal model induced by chronic social stress. Knockdown of EphB2 in the BLA produced antidepressant-like behavioral effects, whereas activation of EphB2 in the BLA increased the susceptibility to subthreshold social defeat stress. Furthermore, we demonstrated that the role of EphB2 in the stress vulnerability was mediated by modulating NMDA receptors, since the knockdown of EphB2 in the BLA prevented not only the increase in the amplitudes of both the miniature and the evoked NMDAR-mediated EPSC, but also the enhancement of surface expression of NMDARs in the defeated mice. Taken together, these results suggest that EphB2 in the BLA is a critical factor contributes to the vulnerability to stress, which may be a potential target for the treatment of depression., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Ephrin receptors (Eph) and ligands are membrane-bound cell-cell communication molecules that regulate the spatial organization of various tissues and organs by repulsive or adhesive signals arising from contact between Eph- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the testis and epididymis are virtually unknown., Objectives: We aimed to investigate the expression of several EphB receptors and ephrin-B ligands in the testis and epididymis of adult mice., Materials and Methods: mRNA and protein expression was detected via reverse transcription-polymerase chain reaction amplification and immunostaining, respectively., Results: Complementary expression patterns were observed in the epithelia along the excurrent duct system in the testis and epididymis; ephrin-B1 was strongly expressed in the epithelia of the rete testis and segment I in the ductus epididymis, whereas EphB2 and/or EphB4 were strongly expressed in the epithelia of the straight tubules and efferent ductules. Moreover, ephrin-B1 was expressed in the spermatogonia, Leydig cells, and peritubular myoid cells in the testis, whereas EphB2 was expressed in elongated spermatids and EphB4 was expressed in the spermatogonia and Leydig cells. Furthermore, these receptors were found to be tyrosine-phosphorylated in the testis and/or epididymis., Discussion: Receptor localization and phosphorylation patterns suggested that EphB/ephrin-B signaling might occur in the seminiferous tubules and epithelial junctions among the straight tubules, rete testis, efferent ductules, and ductus epididymis. Therefore, we propose that EphB/ephrin-B signaling may regulate epithelial boundary formation in the excurrent tubule/ductule/duct system as well as modulate spermatogenesis and spermiation., Conclusion: Overall, this study represents the first analysis of EphB receptor and ephrin-B ligand expression in the normal adult testis and epididymis., (© 2018 American Society of Andrology and European Academy of Andrology.)

MicroRNAs (miRNAs) are small noncoding RNAs that are involved in human carcinogenesis and progression. miR-204 has been reported to be a tumor suppressor in several cancer types. However, the function and underlying molecular mechanism of miR-204 in cervical cancer (CC) are still unclear. In the present study, the expression level of miR-204 was measured using the qRT-PCR method in 30 paired CC clinical samples and in 6 CC cell lines. We found that the expression of miR-204 was significantly downregulated in CC tissues and cell lines compared to normal cervical tissues and cell line. miR-204 was overexpressed by transfection with the miR-204 mimic in HeLa and C33A cell lines in the following experiments. The results showed that overexpression of miR-204 dramatically suppressed cell proliferation, migration, and invasion, caused cell cycle arrest at the G0/G1 phase, promoted cell apoptosis in vitro, and inhibited tumor growth in vivo. Western blot results indicated that overexpressing miR-204 decreased the expressions of CDK2, cyclin E, MMP2, MMP9, Bcl2, whereas it enhanced Bax expression and suppressed the activation of the PI3K/AKT signaling pathways in CC cells. Ephrin type B receptor 2 (EphB2) was identified as a direct target of miR-204 in CC cells according to bioinformatics analysis and luciferase reporter assay. Furthermore, knockdown of EphB2 mimicked the inhibitory effect of miR-204 on the proliferation, invasion, and migration of CC cells. These findings suggested that miR-204 might serve as a tumor suppressor in the development of CC by directly targeting EphB2.

Alzheimer's disease (AD) is a progressive neurodegenerative disease, which affects more and more people. But there is still no effective treatment for preventing or reversing the progression of the disease. Soluble amyloid-beta (Aβ) oligomers, also known as Aβ-derived diffusible ligands (ADDLs) play an important role in AD. Synaptic activity and cognition critically depend on the function of glutamate receptors. Targeting N-methyl-D-aspartic acid (NMDA) receptors trafficking and its regulation is a new strategy for AD early treatment. EphB2 is a key regulator of synaptic localization of NMDA receptors. Aβ oligomers could bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. Here we identified that overexpression of EphB2 with lentiviral vectors in dorsal hippocampus improved impaired memory deficits and anxiety or depression-like behaviors in APPswe/PS1-dE9 (APP/PS1) transgenic mice. Phosphorylation and surface expression of GluN2B-containing NMDA receptors were also improved. Overexpression of EphB2 also rescued the ADDLs-induced depletion of the expression of EphB2 and GluN2B-containing NMDA receptors trafficking in cultured hippocampal neurons. These results suggest that improving the decreased expression of EphB2 and subsequent GluN2B-containing NMDA receptors trafficking in hippocampus may be a promising strategy for AD treatment.

Junctional adhesion molecules are important components of tight junctions, and Eph/ephrin proteins constitute the largest family of receptor tyrosine kinases. Both junctional adhesion molecules and Eph/ephrin are involved in normal tissue development and cancer progression. However, the expression levels and clinical significances of junctional adhesion molecule-A, a member of junctional adhesion molecules, and EphB2, a member of Eph/ephrin family, in lung adenocarcinoma patients are unclear. Therefore, in this study, we aimed to identify the expression and prognostic values of junctional adhesion molecule-A and EphB2 in lung adenocarcinoma patients' cohort. In our study, 70 (55.6%) showed high expression of junctional adhesion molecule-A protein and 51 (40.5%) showed high expression of EphB2 protein in 126 lung adenocarcinoma tissues. Junctional adhesion molecule-A and EphB2 expressions were both significantly increased in tumor tissues compared with noncancerous lung tissues. Kaplan-Meier analysis and log-rank test indicated that low expression of junctional adhesion molecule-A and EphB2 proteins can predict better survival and low mortality rate of lung adenocarcinomas. In univariate analysis, high expression levels of junctional adhesion molecule-A and EphB2 were both found to be significantly correlated with poor overall survival of lung adenocarcinoma patients (hazard ratio = 1.791, 95% confidence interval = 1.041-3.084, p = 0.035; hazard ratio = 1.762, 95% confidence interval = 1.038-2.992, p = 0.036, respectively). The multivariate Cox proportional hazard model demonstrated that EphB2 expression is an independent prognosis parameter in lung adenocarcinoma patients (hazard ratio = 1.738, 95% confidence interval = 1.023-2.952, p = 0.016). Taken together, high expression of junctional adhesion molecule-A and EphB2 can predict poor overall survival and high mortality rate, and EphB2 is an independent prognostic biomarker in lung adenocarcinoma patients.

The Eph receptors are transmembrane proteins that belong to the receptor tyrosine kinases superfamily. Elevated Eph/ephrin expression levels have been associated with angiogenesis and tumor vasculature in many types of human cancers, including breast, lung, and prostate cancers, melanoma, and leukemia. In glioblastoma (GBM), the dysregulated expression of Eph receptors and of corresponding ephrin ligands has been associated with higher tumor grade and poor prognosis making them effective targets for therapeutic drugs. In this study, we describe the GL43.T, an anti-Eph aptamer, able to bind at high-affinity EphB3 and EphB2. Moreover, the GL43.T aptamer inhibits the glioma cell vitality and interferes with ephrine-B1 inhibition of chemotactic serum-stimulated cell migration. GL43.T aptamer represents a promising therapeutic molecule for EphB3-dependent cancers.

EphB2 is an important member of the receptor tyrosine kinases. Recently, EphB2 was shown to facilitate T-cell migration and monocyte activation. However, the effects of EphB2 on B cells remain unknown. In this study, the expression of EphB2 on B cells was tested by Western blot, and the roles of EphB2 in B-cell proliferation, cytokine secretion, and immunoglobulin (Ig) production were evaluated using EphB2 siRNA interference in human B cells from healthy volunteers. Our study revealed that EphB2 was distributed on naive B cells and was up-regulated on activated B cells. Moreover, B-cell proliferation (decreased by 22%, P<0.05), TNF-α secretion (decreased by 40%, P<0.01) and IgG production (decreased by 26%, P < 0.05) were depressed concordantly with the down-regulated EphB2 expression. Subsequently, we screened microRNAs that could regulate EphB2 expression in B cells, and discovered that miR-185 directly targeted to EphB2 mRNA and suppressed its expression. Furthermore, miR-185 overexpression inhibited B-cell activation, and the inhibitor of miR-185 enhanced B-cell activation. Moreover, abatement of EphB2 through miR-185 mimics or EphB2 siRNA attenuated the activation of Src-p65 and Notch1 signaling pathways in human B cells. Our study first suggested that EphB2 was involved in human naive B cell activation through Src-p65 and Notch1 signaling pathways and could be regulated by miR-185., (© FASEB.)

EphB2, a receptor tyrosine kinase for ephrin ligands, is overexpressed in various cancers and plays an important role in tumor progression. However, the expression and functions of EphB2 in cervical cancer remain unknown. In this study, we performed immunohistochemistry in clinical cervical specimens and found that EphB2 was overexpressed in the cervical cancer specimens, and its expression correlated with cancer progression. The percentage of EphB2-positive cells increased gradually from 28% in the normal cervix to 40% in high-grade squamous intraepithelial lesions, and ultimately to 69.8% in squamous cell carcinomas (P < .05). We overexpressed EphB2 in HeLa cells and silenced EphB2 in cervical cancer (C33A) cells, which expressed low and high levels of EphB2, respectively. Exogenous EphB2 promoted cell migration, invasion, and an epithelial-mesenchymal transition (EMT) signature, which is a complex process that occurs during organogenesis and cancer metastasis, whereas EphB2 silencing had the opposite effect (P < .05). Furthermore, HeLa cells with exogenous EphB2 exhibited a stem cell-like state that promoted tumorsphere formation in vitro and exhibited tumorigenesis potential in vivo (P < .05), whereas EphB2 silencing in C33A cells inhibited these stem cell properties (P < .05). In addition, we investigated the intracellular signaling pathways in cervical cancer and found that R-Ras expression correlated positively with EphB2 in clinical samples, and its activity was regulated by EphB2 in cervical cancer. These findings demonstrate that EphB2 plays an important role in cervical cancer progression by orchestrating an EMT program through R-Ras activation., (© 2014.)

Notch is an intercellular signaling pathway related mainly to sprouting neo-angiogenesis. The objective of our study was to evaluate the angiogenic mechanisms involved in the vascular augmentation (sprouting/intussusception) after Notch inhibition within perfused vascular beds using the chick area vasculosa and MxCreNotch1(lox/lox) mice. In vivo monitoring combined with morphological investigations demonstrated that inhibition of Notch signaling within perfused vascular beds remarkably induced intussusceptive angiogenesis (IA) with resultant dense immature capillary plexuses. The latter were characterized by 40 % increase in vascular density, pericyte detachment, enhanced vessel permeability, as well as recruitment and extravasation of mononuclear cells into the incipient transluminal pillars (quintessence of IA). Combination of Notch inhibition with injection of bone marrow-derived mononuclear cells dramatically enhanced IA with 80 % increase in vascular density and pillar number augmentation by 420 %. Additionally, there was down-regulation of ephrinB2 mRNA levels consequent to Notch inhibition. Inhibition of ephrinB2 or EphB4 signaling induced some pericyte detachment and resulted in up-regulation of VEGFRs but with neither an angiogenic response nor recruitment of mononuclear cells. Notably, Tie-2 receptor was down-regulated, and the chemotactic factors SDF-1/CXCR4 were up-regulated only due to the Notch inhibition. Disruption of Notch signaling at the fronts of developing vessels generally results in massive sprouting. On the contrary, in the already existing vascular beds, down-regulation of Notch signaling triggered rapid augmentation of the vasculature predominantly by IA. Notch inhibition disturbed vessel stability and led to pericyte detachment followed by extravasation of mononuclear cells. The mononuclear cells contributed to formation of transluminal pillars with sustained IA resulting in a dense vascular plexus without concomitant vascular remodeling and maturation.

The ability to specifically upregulate genes in vivo holds great therapeutic promise. Here we show that inhibition or degradation of natural antisense transcripts (NATs) by single-stranded oligonucleotides or siRNAs can transiently and reversibly upregulate locus-specific gene expression. Brain-derived neurotrophic factor (BDNF) is normally repressed by a conserved noncoding antisense RNA transcript, BDNF-AS. Inhibition of this transcript upregulates BDNF mRNA by two- to sevenfold, alters chromatin marks at the BDNF locus, leads to increased protein levels and induces neuronal outgrowth and differentiation both in vitro and in vivo. We also show that inhibition of NATs leads to increases in glial-derived neurotrophic factor (GDNF) and ephrin receptor B2 (EPHB2) mRNA. Our data suggest that pharmacological approaches targeting NATs can confer locus-specific gene upregulation effects.

The receptor tyrosine kinase EphB2 is expressed by colon progenitor cells; however, only 39% of colorectal tumors express EphB2 and expression levels decline with disease progression. Conversely, EphB4 is absent in normal colon but is expressed in all 102 colorectal cancer specimens analyzed, and its expression level correlates with higher tumor stage and grade. Both EphB4 and EphB2 are regulated by the Wnt pathway, the activation of which is critically required for the progression of colorectal cancer. Differential usage of transcriptional coactivator cyclic AMP-responsive element binding protein-binding protein (CBP) over p300 by the Wnt/beta-catenin pathway is known to suppress differentiation and increase proliferation. We show that the beta-catenin-CBP complex induces EphB4 and represses EphB2, in contrast to the beta-catenin-p300 complex. Gain of EphB4 provides survival advantage to tumor cells and resistance to innate tumor necrosis factor-related apoptosis-inducing ligand-mediated cell death. Knockdown of EphB4 inhibits tumor growth and metastases. Our work is the first to show that EphB4 is preferentially induced in colorectal cancer, in contrast to EphB2, whereby tumor cells acquire a survival advantage.

The Eph receptors, members of a large family of transmembrane receptor tyrosine kinases, play important roles in a variety of biological functions. Recent studies have suggested that EphA4 and EphB2 participate in the growth and development of various carcinomas. This study examined the relationship of EphA4 and EphB2 gene expression to clinicopathological factors, especially metastasis, in patients with colorectal cancer. We studied surgical specimens of cancer tissue and adjacent normal mucosa obtained from 205 patients with untreated colorectal cancer. The relative expression levels of EphA4 and EphB2 mRNA in the specimens were measured by quantitative real-time, reverse-transcription polymerase chain reaction. The relative expression level of EphA4 mRNA was higher in the presence than in the absence of liver metastasis, whereas the relative expression levels of EphB2 mRNA were similar. Analysis of the relationship between clinicopathological features and gene expression showed that high expression of the EphA4 gene and low expression of the EphB2 gene correlated with liver metastasis. There was no correlation between EphA4 and EphB2 gene expression. Our results suggest that overexpression of the EphA4 gene and reduced expression of the EphB2 gene might promote liver metastasis in colorectal cancer. Overexpression of the EphA4 gene and reduced expression of the EphB2 gene may thus be a useful predictor of liver metastasis in patients with colorectal cancer.

Purpose: To identify genes with upregulated expression at the optic nerve head (ONH) that coincides with retinal ganglion cell (RGC) axon loss in glaucomatous DBA/2J mice. To further demonstrate that the proteins encoded by these genes bind to RGC axons and influence fundamental axon physiology., Methods: In situ hybridization and cell-type-specific immunolabeling were performed on ONH sections from DBA/2J mice (3 to 11 months old) and C57Bl/6NCrl mice (10 months old). EphB2-Fc and ephrin-B2-Fc chimeric proteins were applied to adult RGC axons in vitro and in vivo at the ONH to demonstrate protein binding on axons. EphB2-Fc or control Fc protein was applied in a bath or locally to axons preloaded with the calcium indicator Fluo-4-AM, and changes in intra-axonal calcium were determined., Results: EphB2 and ephrin-B2 were specifically upregulated at the ONH of DBA/2J mice starting at 9 months of age, but not in age-matched C57Bl/6NCrl mice or in DBA/2J animals that did not have axon loss. EphA4 was also present at the ONH, but no difference in expression was detected between unaffected and affected animals. EphB2 was expressed by F4/80(+), MOMA2(+), ED1(-) macrophage-like cells, ephrin-B2 was expressed by Iba-1(+) microglia and GFAP(+) astrocytes, whereas EphA4 was expressed by GFAP(+) astrocytes. EphB2-Fc and ephrin-B2-Fc protein bound to RGC axons in culture and to ONH RGC axons in vivo. Adult RGC axons in vitro elevated intra-axonal calcium in response to EphB2-Fc but not to control Fc protein., Conclusions: The expression of EphB2 and ephrin-B2 is upregulated at the ONH of glaucomatous DBA/2J mice coinciding with RGC axon loss. The direct binding of EphB2 and ephrin-B2 on adult RGC axons at the ONH and the ability of EphB2 to elevate intra-axonal calcium indicate that these proteins may affect RGC axon physiology in the setting of glaucoma and thus affect the development or progression of the disease.

Growth of functional arteries is essential for the restoration of blood flow to ischemic organs. Notch signaling regulates arterial differentiation upstream of ephrin-B2 during embryonic development, but its role during postnatal arteriogenesis is unknown. Here, we identify the Notch ligand Delta-like 1 (Dll1) as an essential regulator of postnatal arteriogenesis. Dll1 expression was specifically detected in arterial endothelial cells, but not in venous endothelial cells or capillaries. During ischemia-induced arteriogenesis endothelial Dll1 expression was strongly induced, Notch signaling activated and ephrin-B2 upregulated, whereas perivascular cells expressed proangiogenic vascular endothelial growth factor, and the ephrin-B2 activator EphB4. In heterozygous Dll1 mutant mice endothelial Notch activation and ephrin-B2 induction after hindlimb ischemia were absent, arterial collateral growth was abrogated and recovery of blood flow was severely impaired, but perivascular vascular endothelial growth factor and EphB4 expression was unaltered. In vitro, angiogenic growth factors synergistically activated Notch signaling by induction of Dll1, which was necessary and sufficient to regulate ephrin-B2 expression and to induce ephrin-B2 and EphB4-dependent branching morphogenesis in human arterial EC. Thus, Dll1-mediated Notch activation regulates ephrin-B2 expression and postnatal arteriogenesis.

Objectives: To analyze expressions of the EphB2 and EphB4 receptors in ovarian carcinomas and explore their clinicopathological correlations and prognostic value., Methods: 115 patients with advanced ovarian carcinoma FIGO IIB to IV were involved. RT-PCR and immunohistochemistry were used to examine the expressions of EphB2/B4 receptor mRNA and protein. Correlations between protein expression and clinicopathological factors were also analyzed., Results: Ovarian carcinoma patients with age elder than 60 years had higher EphB2 expression than younger patients. Expression of EphB2 and EphB4 protein did not significantly correlate with any other clinical variables, including FIGO stage, residual tumor, histological type and differentiation grade. No significant correlation between mRNA and protein expression level for both of these receptors was seen. It was found that patients with strong immunostaining for EphB2 (P = 0.03) or EphB4 (P = 0.003) receptors had poorer survival, and patients with strong immunostaining for EphB4 receptor showed poorer response to chemotherapy (P = 0.036)., Conclusions: These studies suggest that EphB2 and B4 receptors are of prognostic value and EphB4 receptor may be an independent predictor of chemotherapy response in ovarian cancer patients.

More than 10(10) cells are generated every day in the human intestine. Wnt proteins are key regulators of proliferation and are known endogenous mitogens for intestinal progenitor cells. The positioning of cells within the stem cell niche in the intestinal epithelium is controlled by B subclass ephrins through their interaction with EphB receptors. We report that EphB receptors, in addition to directing cell migration, regulate proliferation in the intestine. EphB signaling promotes cell-cycle reentry of progenitor cells and accounts for approximately 50% of the mitogenic activity in the adult mouse small intestine and colon. These data establish EphB receptors as key coordinators of migration and proliferation in the intestinal stem cell niche.

EphB2, a receptor tyrosine kinase regulated by the beta-catenin/Tcf4 complex, is expressed in the proliferative compartment of mouse intestine and regulates bidirectional migration of intestinal precursor cells in the crypt-villus axis through repulsive interaction with Ephrin-B ligands. Recently, it has been shown that reduction of EphB activity accelerates colon tumour progression in the Apc(Min/+) mice. In this study, we examined the expression of EphB2 in normal colon, adenomas, primary colorectal cancers (CRCs), lymph node metastases and liver metastases using immunohistochemistry on tissue microarrays. In addition, EphB2 was overexpressed in SW480 colon cancer cells to study its effect in vitro. We found that EphB2 was expressed in 100% of normal colon crypt base cells, 78% of adenomas, 55.4% of primary CRCs, 37.8% of lymph node metastases and 32.9% of liver metastases (all differences were statistically significant at P < 0.001 compared with primary CRCs). Patients with CRCs that lose EphB2 expression had more advanced tumour stage (P = 0.005), poor differentiation (P < 0.001), poor overall survival (P = 0.005) and disease-free survival (P = 0.001), with the latter being independent of tumour stage. In vitro studies showed that overexpression of EphB2 inhibited colon cancer cell growth in colony formation assay and activation of EphB2 receptor inhibited colon cancer cell adhesion and migration. Our data demonstrated a progressive loss of EphB2 expression in each critical step of colon carcinogenesis, including the onset of invasion, dedifferentiation and metastasis which are paralleled by adverse patient outcome. EphB2 may achieve its tumour suppressor function through regulation of cell survival, adhesion and migration.

Eph receptors and ephrin ligands are membrane-bound cell-cell communication molecules with well-defined functions in development, but their expression patterns and functions in many adult tissues are still largely unknown. We have detected substantial levels of the EphB2 and EphB6 receptors and the ephrin-B1 ligand in the adult mouse kidney by RT-PCR amplification. Immunolocalization experiments revealed that EphB2 is localized in the tubules of the inner and outer medulla and EphB6 is in the tubules of the outer medulla and cortex. By contrast, ephrin-B1 was detected in tubules throughout the whole nephron. Consistent with the overlapping expression of the EphB2 receptor and the ephrin-B1 ligand in the medulla, EphB2 is tyrosine-phosphorylated, and therefore activated, in the kidney. In the outer medulla, however, EphB2 signaling may be attenuated by the co-expressed kinase-inactive EphB6 receptor. Interestingly, we found that EphB signaling induces RhoA activation and Rac1 inactivation as well as cell retraction, enlargement of focal adhesions and prominent stress fibers in primary cultures of medullary tubule cells. These results suggest that EphB receptor signaling through Rho family GTPases regulates the cytoarchitecture and spatial organization of the tubule cells in the adult kidney medulla and, therefore, may affect the reabsorption ability of the kidney.

Gastric cancer is a leading cause of global cancer mortality, but comparatively little is known about the cellular pathways regulating different aspects of the gastric cancer phenotype. To achieve a better understanding of gastric cancer at the levels of systems topology, functional modules, and constituent genes, we assembled and systematically analyzed a consensus gene coexpression meta-network of gastric cancer incorporating >300 tissue samples from four independent patient populations (the "gastrome"). We find that the gastrome exhibits a hierarchical scale-free architecture, with an internal structure comprising multiple deeply embedded modules associated with diverse cellular functions. Individual modules display distinct subtopologies, with some (cellular proliferation) being integrated within the primary network, and others (ribosomal biosynthesis) being relatively isolated. One module associated with intestinal differentiation exhibited a remarkably high degree of autonomy, raising the possibility that its specific topological features may contribute towards the frequent occurrence of intestinal metaplasia in gastric cancer. At the single-gene level, we discovered a novel conserved interaction between the PLA2G2A prognostic marker and the EphB2 receptor, and used tissue microarrays to validate the PLA2G2A/EphB2 association. Finally, because EphB2 is a known target of the Wnt signaling pathway, we tested and provide evidence that the Wnt pathway may also similarly regulate PLA2G2A. Many of these findings were not discernible by studying the single patient populations in isolation. Thus, besides enhancing our knowledge of gastric cancer, our results show the broad utility of applying meta-analytic approaches to genome-wide data for the purposes of biological discovery.

Purpose: To comprehensively evaluate ephrin receptor B2 (EphB2) expression in normal and neoplastic tissues. EphB2 is a tyrosine kinase recently implicated in the deregulation of cell-to-cell communication in many tumors., Experimental Design: EphB2 protein expression was analyzed by immunohistochemistry on tissue microarrays that included 76 different normal tissues, >4,000 samples from 138 different cancer types, and 1,476 samples of colon cancer with clinical follow-up data., Results: We found most prominent EphB2 expression in the intestinal epithelium (colonic crypts) with cancer of the colorectum displaying the highest EphB2 positivity of all tumors. Positivity was found in 100% of 118 colon adenomas but in 33.3% of 45 colon carcinomas. EphB2 expression was also observed in 75 tumor categories, including serous carcinoma of the endometrium (34.8%), adenocarcinoma of the esophagus (33.3%), intestinal adenocarcinoma of the stomach (30.2%), and adenocarcinoma of the small intestine (70%). The occasional finding of strong EphB2 positivity in tumors without EphB2 positivity in the corresponding normal cells [adenocarcinoma of the lung (4%) and pancreas (2.2%)] suggests that deregulation of EphB2 signaling may involve up-regulation of the protein expression. In colon carcinoma, loss of EphB2 expression was associated with advanced stage (P < 0.0001) and was an indicator of poor overall survival (P = 0.0098)., Conclusions: Our results provide an overview on the EphB2 protein expression in normal and neoplastic tissues. Deregulated EphB2 expression may play a role in several cancer types with loss of EphB2 expression serving as an indicator of the possible pathogenetic role of EphB2 signaling in the maintenance of tissue architecture of colon epithelium.

A receptor tyrosine kinase for ephrin ligands, EphB2 is expressed in colorectal cancer and has been proposed as a target for immunoconjugate therapy. The aim of this study was to perform a detailed histologic analysis of EphB2 expression in normal and neoplastic colorectal tissues. In addition, we sought to evaluate EphB2 expression as a prognostic factor in colorectal cancer. Expression of EphB2 was examined in normal colon (n = 28), colorectal cell lines (n = 20), colorectal adenomas (n = 148), primary cancers (n = 28), and metastases (n = 39) using immunohistochemistry. In addition, a series of primary cancers and matched normal (n = 342) with outcome data were profiled in tissue microarrays. The intensity of EphB2 expression was assessed in the entire series by immunohistochemistry, and in a subset by in situ hybridization. Overall survival and recurrence-free survival were correlated with EphB2 protein expression in retrospective subset analyses. Epithelial EphB2 expression was shown at all stages of colorectal tumorigenesis, including the base of all normal crypts, 77% of adenomas, 82% of primary cancers, and 64% of metastases. Although homogeneous expression was observed in adenomas, the pattern of staining was focal (mean 25%) in most malignant lesions. Patients whose tumor stained 2+ for EphB2 expression (versus 0/1+) exhibited significantly prolonged overall survival: mean duration of survival, 2,514 versus 1,044 days; hazard ratio, 0.45; 95% confidence interval, 0.18 to 0.95 (P = 0.035). In summary, EphB2 is expressed in normal crypts, colorectal adenomas, primary cancers, and metastases. High levels of EphB2 expression are associated with a longer mean duration of survival in colorectal cancer.

Eph receptor tyrosine kinases and their ligands, ephrins, mediate neurodevelopmental processes such as boundary formation, axon guidance, vasculogenesis, and cell migration. We determined the expression profiles of the Eph family members in five glioma cell lines under migrating and nonmigrating conditions. EphB2 mRNA was overexpressed in all five during migration (1.2-2.8-fold). We found abundant EphB2 protein as well as strong phosphorylation of EphB2 in migrating U87 cells. Confocal imaging showed EphB2 localized in lamellipodia of motile U87 cells. Treatment with ephrin-B1/Fc chimera stimulated migration and invasion of U87, whereas treatment with a blocking EphB2 antibody significantly inhibited migration and invasion. Forced expression of EphB2 in U251 cells stimulated cell migration and invasion and diminished adhesion concomitant with the tyrosine phosphorylation of EphB2. U251 stably transfected with EphB2 showed more scattered and more pronounced invasive growth in an ex vivo rat brain slice. In human brain tumor specimens, EphB2 expression was higher in glioblastomas than in low-grade astrocytomas or normal brain; patterns of phosphorylated EphB2 matched the expression levels. Laser capture microdissection of invading glioblastoma cells revealed elevated EphB2 mRNA (1.5-3.5-fold) in 7 of 7 biopsy specimens. Immunohistochemistry demonstrated EphB2 localization primarily in glioblastoma cells (56 of 62 cases) and not in normal brain. This is the first demonstration that migrating glioblastoma cells overexpress EphB2 in vitro and in vivo; glioma migration and invasion are promoted by activation of EphB2 or inhibited by blocking EphB2. Dysregulation of EphB2 expression or function may underlie glioma invasion.

Analysis of human colorectal cancer specimens revealed overexpression of the EphB2 receptor tyrosine kinase. Monoclonal antibodies (MAbs) to extracellular sequence of EphB2 were raised and tested for activity against colorectal cancer cells. One of the MAbs, 2H9, effectively blocked the interaction of ephB2 with ephrin ligands and inhibited the resulting autophosphorylation of the receptor. However, this antibody did not affect the proliferation of cancer cells expressing ephB2. Immunocytochemical analysis revealed rapid internalization of the MAb 2H9 on binding ephB2, suggesting that target-dependent cell killing could be achieved with an antibody-drug conjugate. When MAb 2H9 was conjugated to monomethylauristatin E through a cathepsin B-cleavable linker, it specifically killed ephB2-expressing cancer cells in vitro and in vivo. Our results suggest that ephB2 is an attractive target for immunoconjugate cancer therapy.

Eph kinases form the largest family of receptor tyrosine kinases, and their ligands are ephrins (EFNs), which are cell surface proteins. Some Eph kinases and EFNs are expressed on T cells, B cells, and dendritic cells, but their functions in the immune system are largely unknown. In this study, we investigated the effect of EFNB2 on murine T cells. EFNB2 mRNA was expressed in the cortex of the thymus and white pulp of the spleen. At the protein level, it was expressed on T cells and monocytes/macrophages, but not on B cells. EFNB2Rs were expressed mainly on T cells. Solid-phase EFNB2 along with suboptimal anti-CD3 strongly stimulated T cell proliferation, with concomitant augmentation of IFN-gamma but not IL-2 or IL-4 secretion. The activity of cytotoxic T cells was also significantly enhanced in the presence of solid-phase EFNB2. These results indicate that EFNB2R cross-linking results in costimulation of T cells. EFNB2Rs were normally scattered on the T cell surface; after TCR cross-linking, they rapidly congregated to capped TCR complexes and then to patched rafts. This provides a morphological base for EFNB2Rs to participate in T cell costimulation. We also demonstrated that EFNB2R signaling led to augmented p38 and p44/42 mitogen-activated protein kinase activation. Our study shows that EFNB2 plays important roles in immune regulation.