Interferon-induced transmembrane protein-1 competitively blocks Ephrin receptor A2-mediated Epstein-Barr virus entry into epithelial cells.

Epstein-Barr virus (EBV) can infect both B cells and epithelial cells (ECs), causing diseases such as mononucleosis and cancer. It enters ECs via Ephrin receptor A2 (EphA2). The function of interferon-induced transmembrane protein-1 (IFITM1) in EBV infection of ECs remains elusive. Here we report that IFITM1 inhibits EphA2-mediated EBV entry into ECs. RNA-sequencing and clinical sample analysis show reduced IFITM1 in EBV-positive ECs and a negative correlation between IFITM1 level and EBV copy number. IFITM1 depletion increases EBV infection and vice versa. Exogenous soluble IFITM1 effectively prevents EBV infection in vitro and in vivo. Furthermore, three-dimensional structure prediction and site-directed mutagenesis demonstrate that IFITM1 interacts with EphA2 via its two specific residues, competitively blocking EphA2 binding to EBV glycoproteins. Finally, YTHDF3, an m6A reader, suppresses IFITM1 via degradation-related DEAD-box protein 5 (DDX5). Thus, this study underscores IFITM1's crucial role in blocking EphA2-mediated EBV entry into ECs, indicating its potential in preventing EBV infection.

A phase 1 safety and bioimaging trial of antibody DS-8895a against EphA2 in patients with advanced or metastatic EphA2 positive cancers.

Ephrin type-A 2 (EphA2) is a transmembrane receptor expressed in epithelial cancers. We report on a phase I dose escalation and biodistribution study of DS-8895a, an anti-EphA2 antibody, in patients with advanced EphA2 positive cancers. DS-8895a was administered at 1, 3, 10 or 20 mg/kg every 2 weeks to determine safety, pharmacokinetics and anti-tumor efficacy. All patients underwent 89Zr trace-labelled infusion of DS-8895a (89Zr-DS-8995a) positron emission tomography imaging to determine the biodistribution of DS-8895a, and correlate findings with EphA2 expression, receptor saturation and response. Nine patients were enrolled on study. Of patients enrolled, seven patients received at least one infusion of DS-8895a: four patients received 1 mg/kg dose (Cohort 1) and three patients received 3 mg/kg dose (Cohort 2). Median age was 67.0 years (range 52-81), majority male (71%), and median number of prior systemic therapies was three (range 0-8). The primary cancer diagnosis was colorectal cancer (two patients) and one patient each had gastric, head and neck, high-grade serous adenocarcinoma, lung, and pancreatic cancers. No dose-limiting toxicities or treatment-related adverse events reported. The best response for the patients in Cohort 1 was stable disease and in Cohort 2 was progressive disease. 89Zr-DS-8895a demonstrated no normal tissue uptake and specific low-grade uptake in most tumours. DS-8895a had limited therapeutic efficacy at doses evaluated and 89Zr-DS-8895a demonstrated low tumour uptake. The biodistribution data from this study were key in halting further development of DS-8895a, highlighting the importance of biodistribution studies in drug development. (Trial registration: ClinicalTrials.gov Identifier NCT02252211).

Manipulating the Level of Sensorimotor Stimulation during LI-rTMS Can Improve Visual Circuit Reorganisation in Adult Ephrin-A2A5-/- Mice.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that has the potential to treat a variety of neurologic and psychiatric disorders. The extent of rTMS-induced neuroplasticity may be dependent on a subject's brain state at the time of stimulation. Chronic low intensity rTMS (LI-rTMS) has previously been shown to induce beneficial structural and functional reorganisation within the abnormal visual circuits of ephrin-A2A5-/- mice in ambient lighting. Here, we administered chronic LI-rTMS in adult ephrin-A2A5-/- mice either in a dark environment or concurrently with voluntary locomotion. One day after the last stimulation session, optokinetic responses were assessed and fluorescent tracers were injected to map corticotectal and geniculocortical projections. We found that LI-rTMS in either treatment condition refined the geniculocortical map. Corticotectal projections were improved in locomotion+LI-rTMS subjects, but not in dark + LI-rTMS and sham groups. Visuomotor behaviour was not improved in any condition. Our results suggest that the beneficial reorganisation of abnormal visual circuits by rTMS can be significantly influenced by simultaneous, ambient visual input and is enhanced by concomitant physical exercise. Furthermore, the observed pathway-specific effects suggest that regional molecular changes and/or the relative proximity of terminals to the induced electric fields influence the outcomes of LI-rTMS on abnormal circuitry.

May EPH/Ephrin Targeting Revolutionize Lung Cancer Treatment?

Lung cancer (LC) is the leading cause of cancer death in the United States. Erythropoietin-producing hepatocellular receptors (EPHs) comprise the largest receptor tyrosine kinases (RTKs) family in mammals. EPHs along with their ligands, EPH-family receptor-interacting proteins (ephrins), have been found to be either up- or downregulated in LC cells, hence exhibiting a defining role in LC carcinogenesis and tumor progression. In their capacity as membrane-bound molecules, EPHs/ephrins may represent feasible targets in the context of precision cancer treatment. In order to investigate available therapeutics targeting the EPH/ephrin system in LC, a literature review was conducted, using the MEDLINE, LIVIVO, and Google Scholar databases. EPHA2 is the most well-studied EPH/ephrin target in LC treatment. The targeting of EPHA2, EPHA3, EPHA5, EPHA7, EPHB4, EPHB6, ephrin-A1, ephrin-A2, ephrin-B2, and ephrin-B3 in LC cells or xenograft models not only directly correlates with a profound LC suppression but also enriches the effects of well-established therapeutic regimens. However, the sole clinical trial incorporating a NSCLC patient could not describe objective anti-cancer effects after anti-EPHA2 antibody administration. Collectively, EPHs/ephrins seem to represent promising treatment targets in LC. However, large clinical trials still need to be performed, with a view to examining the effects of EPH/ephrin targeting in the clinical setting.

Scleraxis upregulated by transforming growth factor-ß1 signaling inhibits tension-induced osteoblast differentiation of priodontal ligament cells via ephrin A2.

During tooth movement in orthodontic treatment, bone formation and resorption occur on the tension and compression sides of the alveolar bone, respectively. Although the bone formation activity increases in the periodontal ligament (PDL) on the tension side, the PDL itself is not ossified and maintains its homeostasis, indicating that there are negative regulators of bone formation in the PDL. Our previous report suggested that scleraxis (Scx) has an inhibitory effect on ossification of the PDL on the tension side through the suppression of calcified extracellular matrix formation. However, the molecular biological mechanisms of Scx-modulated inhibition of ossification in the tensioned PDL are not fully understood. The aim of the present study is to clarify the inhibitory role of Scx in osteoblast differentiation of PDL cells and its underlying mechanism. Our in vivo experiment using a mouse experimental tooth movement model showed that Scx expression was increased during early response of the PDL to tensile force. Scx knockdown upregulated expression of alkaline phosphatase, an early osteoblast differentiation marker, in the tensile force-loaded PDL cells in vitro. Transforming growth factor (TGF)-ß1-Smad3 signaling in the PDL was activated by tensile force and inhibitors of TGF-ß receptor and Smad3 suppressed the tensile force-induced Scx expression in PDL cells. Tensile force induced ephrin A2 (Efna2) expression in the PDL and Efna2 knockdown upregulated alkaline phosphatase expression in PDL cells under tensile force loading. Scx knockdown eliminated the tensile force-induced Efna2 expression in PDL cells. These findings suggest that the TGF-ß1-Scx-Efna2 axis is a novel molecular mechanism that negatively regulates the tensile force-induced osteoblast differentiation of PDL cells.

EPHA2 Promotes the Invasion and Migration of Human Tongue Squamous Cell Carcinoma Cal-27 Cells by Enhancing AKT/mTOR Signaling Pathway.

EPHA2 is a member of the ephrin receptor tyrosine kinase family and is closely related to the malignant tumor progression. The effect of EPHA2 on OSCC is not clear. This study explored the role of EPHA2 and AKT/mTOR signaling pathways in Cal-27 cell invasion and migration. The expression of EPHA2 and EPHA4 in human OSCC and normal oral tissue was detected by immunohistochemistry. EPHA2-overexpressing and EPHA2-knockdown Cal-27 cells were established, and the cells were treated with an AKT inhibitor (MK2206) and mTOR inhibitor (RAD001). The expression of EPHA2 was detected by qRT-PCR, cell proliferation was evaluated by MTT assay, cell migration and invasion were examined by scratch and Transwell assay, and cell morphology and apoptosis were assessed by Hoechst 33258 staining. Western blot was performed to detect the expression of proteins related to AKT/mTOR signaling, cell cycle, and pseudopod invasion. EPHA2 and EPHA4 were highly expressed in clinical human OSCC. Overexpression of EPHA2 promoted the proliferation, migration, and invasion of Cal-27 cells, inhibited cell cycle blockage and apoptosis, and enhanced the activity of the AKT/mTOR signaling pathway. MK2206 (AKT inhibitor) and RAD001 (mTOR inhibitor) reversed the effect of EPHA2 overexpression on the biological behavior of Cal-27 cells. EPHA2 promotes the invasion and migration of Cal-27 human OSCC cells by enhancing the AKT/mTOR signaling pathway.

EPHA2 Segregates with Microphthalmia and Congenital Cataracts in Two Unrelated Families.

EPHA2 is a transmembrane tyrosine kinase receptor that, when disrupted, causes congenital and age-related cataracts. Cat-Map reports 22 pathogenic EPHA2 variants associated with congenital cataracts, variable microcornea, and lenticonus, but no previous association with microphthalmia (small, underdeveloped eye, ≥2 standard deviations below normal axial length). Microphthalmia arises from ocular maldevelopment with >90 monogenic causes, and can include a complex ocular phenotype. In this paper, we report two pathogenic EPHA2 variants in unrelated families presenting with bilateral microphthalmia and congenital cataracts. Whole genome sequencing through the 100,000 Genomes Project and cataract-related targeted gene panel testing identified autosomal dominant heterozygous mutations segregating with the disease: (i) missense c.1751C>T, p.(Pro584Leu) and (ii) splice site c.2826-9G>A. To functionally validate pathogenicity, morpholino knockdown of epha2a/epha2b in zebrafish resulted in significantly reduced eye size ± cataract formation. Misexpression of N-cadherin and retained fibre cell nuclei were observed in the developing lens of the epha2b knockdown morphant fish by 3 days post-fertilisation, which indicated a putative mechanism for microphthalmia pathogenesis through disruption of cadherin-mediated adherens junctions, preventing lens maturation and the critical signals stimulating eye growth. This study demonstrates a novel association of EPHA2 with microphthalmia, suggesting further analysis of pathogenic variants in unsolved microphthalmia cohorts may increase molecular diagnostic rates.

Ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT.

BACKGROUND: Ephrin-A2, a member of the Eph receptor subgroup, is used in diagnosing and determining the prognosis of prostate cancer. However, the role of ephrin-A2 in prostate cancer is remains elusive. METHODS: We established stable clones overexpressing or silencing ephrin-A2 from prostate cancer cells. Then, CCK-8 was used in analyzing the proliferation ability of cells. CD31 staining was used in evaluating angiogenesis. Migration and invasion assay were conducted in vivo and in vitro. The expression of EMT-related markers was evaluated in prostate cancer cells through Western blotting. RESULTS: We revealed that the ectopic expression of ephrin-A2 in prostate cancer cells facilitated cell migration and invasion in vitro and promoted tumor metastasis and angiogenesis in vivo and that the silencing of ephrin-A2 completely reversed this effect. Although ephrin-A2 did not affect tumor cell proliferation in vitro, ephrin-A2 significantly promoted primary tumor growth in vivo. Furthermore, to determine the biological function of ephrin-A2, we assayed the expression of EMT-related markers in stable-established cell lines. Results showed that the overexpression of ephrin-A2 in prostate cancer cells down-regulated the expression of epithelial markers (ZO-1, E-cadherin, and claudin-1) and up-regulated the expression of mesenchymal markers (N-cadherin, ß-catenin, vimentin, Slug, and Snail), but the knocking out of ephrin-A2 opposed the effects on the expression of EMT markers. CONCLUSIONS: These findings indicate that ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT and may be a potentially therapeutic target in metastatic prostate cancer.

EphA2, vascular endothelial growth factor, and vascular endothelial growth factor correlate with adverse outcomes and poor survival in patients with glioma.

PURPOSE: To assess expression levels of Ephrin type-A receptor 2 (EphA2), vascular endothelial growth factor (VEGF), and von Willebrand factor (vWF), and assess their potentials as prognostic biomarkers to predict the risk of poor survival in patients with primary lower grade glioma. METHOD: The study included75 patients with histopathologically confirmed primary glioma (World Health Organization Grade IV). All patients underwent combined surgery and postoperative radiotherapy for the management of primary glioma. Immuno-histochemical analysis was performed to evaluate expression levels ofEphA2 and VEGF. Evaluation of tumor microvessel density was also performed at angiogenesis hot spots due to tumor growth. Main outcomes of the study were the prognostic efficiencies of EphA2, VEGF, and vWF in primary low-grade glioma, as well as whether their expression levels were associated with cancer progression. RESULTS: Of the patients with glioma, 67% had very strong expression of EphA2. Overall survival was inversely correlated with the expression of EphA2. Regarding VEGF expression, 38 patients (51%) had strong expression, 29 patients (39%) had weak expression, and 8 patients (11%) had no expression. Strong VEGF expression was associated with poor prognosis and poor survival. CONCLUSION: EphA2, VEGF, and vWF could be considered prognostic markers for assessment of primary glioma.

Activation of EphA2-EGFR signaling in oral epithelial cells by Candida albicans virulence factors.

During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of infection. The ephrin type-A receptor 2 (EphA2) detects ß-glucan and plays a central role in stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the Als3 adhesin/invasin and the candidalysin pore-forming toxin. Here, we investigated the interactions among EphA2, EGFR, Als3 and candidalysin during OPC. We found that EGFR and EphA2 constitutively associate with each other as part of a heteromeric physical complex and are mutually dependent for C. albicans-induced activation. Als3-mediated endocytosis of a C. albicans hypha leads to the formation of an endocytic vacuole where candidalysin accumulates at high concentration. Thus, Als3 potentiates targeting of candidalysin, and both Als3 and candidalysin are required for C. albicans to cause maximal damage to oral epithelial cells, sustain activation of EphA2 and EGFR, and stimulate pro-inflammatory cytokine and chemokine secretion. In the mouse model of OPC, C. albicans-induced production of CXCL1/KC and CCL20 is dependent on the presence of candidalysin and EGFR, but independent of Als3. The production of IL-1α and IL-17A also requires candidalysin but is independent of Als3 and EGFR. The production of TNFα requires Als1, Als3, and candidalysin. Collectively, these results delineate the complex interplay among host cell receptors EphA2 and EGFR and C. albicans virulence factors Als1, Als3 and candidalysin during the induction of OPC and the resulting oral inflammatory response.

The Expression of ephrinA1/ephA2 Receptor Increases in Chronic Rhinosinusitis and ephrinA1/ephA2 Signaling Affects Rhinovirus-Induced Innate Immunity in Human Sinonasal Epithelial Cells.

EphA2 receptor and its ephrin ligands are involved in virus infection, epithelial permeability, and chemokine secretion. We hypothesized that ephrinA1/ephA2 signaling participates in rhinovirus (RV)-induced antiviral immune response in sinonasal mucosa of patients with chronic rhinosinusitis (CRS). Therefore, we investigated the expression of ephrinA1/ephA2 in normal and inflamed sinonasal mucosa and evaluated whether they regulate chemokine secretion and the production of antiviral immune mediators including interferons (IFNs) in RV-infected human primary sinonasal epithelial cells. For this purpose, the expression and distribution of ephrinA1/ephA2 in sinonasal mucosa were evaluated with RT-qPCR, immunofluorescence, and western blot. Their roles in chemokine secretion and the production of antiviral immune mediators such as type I and III IFNs, and interferon stimulated genes were evaluated by stimulating ephA2 with ephrinA1 and inactivating ephA2 with ephA2 siRNA or inhibitor in cells exposed to RV and poly(I:C). We found that ephrinA1/ephA2 were expressed in normal mucosa and their levels increased in inflamed sinonasal mucosa of CRS patients. RV infection or poly(I:C) treatment induced chemokine secretion which were attenuated by blocking the action of ephA2 with ephA2 siRNA or inhibitor. The production of antiviral immune mediators enhanced by rhinovirus or poly (I:C) is increased by blocking ephA2 compared with that of cells stimulated by either rhinovirus or poly(I:C) alone. In addition, blocking ephA2 attenuated RV replication in cultured cells. Taken together, these results describe a novel role of ephrinA1/ephA2 signaling in antiviral innate immune response in sinonasal epithelium, suggesting their participation in RV-induced development and exacerbations of CRS.

Concurrent LI-rTMS induces changes in c-Fos expression but not behavior during a progressive ratio task with adult ephrin-A2A5-/- mice.

Changes within the dopaminergic system induced by repetitive transcranial magnetic stimulation (rTMS) may contribute to its therapeutic effects; however, dopamine-related behavioral effects of rTMS have not been widely investigated. We recently showed that ephrin-A2A5-/- mice completed significantly fewer trials in a visual task than wildtype mice, and that concurrent low-intensity (LI-) rTMS during the task could partially rescue the abnormal behavior [Poh et al. 2018, eNeuro, vol. 5]. Here, we investigated whether the behavioral differences in ephrin-A2A5-/- mice are due to abnormal motivation, primarily a dopamine-modulated behavior, and whether LI-rTMS would increase motivation. Ephrin-A2A5-/- and wildtype mice underwent 14 daily sessions of progressive ratio (PR) tasks and received either sham or LI-rTMS during the first 10 min. Ephrin-A2A5-/- mice responded more than wildtype comparisons, and LI-rTMS did not influence task performance for either strain. Therefore concurrent stimulation does not influence motivation in a PR task. However, ephrin-A2A5-/- mice did have abnormal performance in the PR tasks after a change in the PR schedule which suggests perseverative behavior. We stained for c-Fos in the prelimbic area (PrL), ventral tegmental area and nucleus accumbens (NAc) core and shell to examine neuronal activity from the final PR session. Sham ephrin-A2A5-/- mice had lower c-Fos expression in the PrL and NAc vs. wildtype mice. Ephrin-A2A5-/- mice that received LI-rTMS showed c-Fos expression closer to wildtype levels in the NAc. Combined with high PR performance, ephrin-A2A5-/- mice show an abnormal shift to habitual responding and LI-rTMS may attenuate this shift.

Application of WES Towards Molecular Investigation of Congenital Cataracts: Identification of Novel Alleles and Genes in a Hospital-Based Cohort of South India.

Congenital cataracts are the prime cause for irreversible blindness in children. The global incidence of congenital cataract is 2.2-13.6 per 10,000 births, with the highest prevalence in Asia. Nearly half of the congenital cataracts are of familial nature, with a predominant autosomal dominant pattern of inheritance. Over 38 of the 45 mapped loci for isolated congenital or infantile cataracts have been associated with a mutation in a specific gene. The clinical and genetic heterogeneity of congenital cataracts makes the molecular diagnosis a bit of a complicated task. Hence, whole exome sequencing (WES) was utilized to concurrently screen all known cataract genes and to examine novel candidate factors for a disease-causing mutation in probands from 11 pedigrees affected with familial congenital cataracts. Analysis of the WES data for known cataract genes identified causative mutations in six pedigrees (55%) in PAX6, FYCO1 (two variants), EPHA2, P3H2,TDRD7 and an additional likely causative mutation in a novel gene NCOA6, which represents the first dominant mutation in this gene. This study identifies a novel cataract gene not yet linked to human disease. NCOA6 is a transcriptional coactivator that interacts with nuclear hormone receptors to enhance their transcriptional activator function.

EPHA2 antisense RNA modulates EPHA2 mRNA levels in basal-like/triple-negative breast cancer cells.

Ephrin type-A receptor 2 (EPHA2) is a receptor tyrosine kinase (RTK), whose over-expression has been observed in a variety of cancers, including breast cancer. EPHA2 expression may be causally related to tumorigenesis; therefore, it is important to understand how EPHA2 gene (EPHA2) expression is regulated. Here, we report that EPHA2 antisense RNA (EPHA2-AS), a natural antisense transcript, is an important modulator of EPHA2 mRNA levels. EPHA2-AS is a ∼1.8 kb long non-coding RNA (lncRNA) with a poly(A) tail that encodes two splice variants, EPHA2-AS1/2. They are constitutively expressed in a concordant manner with EPHA2 mRNA in human breast adenocarcinoma cell lines and in patient samples, with the highest levels detected in the triple-negative breast cancer (TNBC) subtype. The silencing of EPHA2-AS1/2 by a sense oligonucleotide or over-expression of an antisense oligoribonucleotide, which were both designed from the EPHA2 mRNA region (nt 2955-2974) targeted by AS1/2, showed that EPHA2-AS1/2 modulated EPHA2 mRNA levels by interacting with the specific AS1/2-complementary region in the mRNA. The EPHA2-AS1/2 did not prevent microRNAs from acting on the relevant microRNA response elements shared by EPHA2-AS1/2 and EPHA2 mRNA. Our studies demonstrate a crucial role played by EPHA2-AS1/2 in modulating EPHA2 mRNA levels, and hence production of EPHA2 protein, a key oncogenic RTK that contributes to the tumorigenesis of TNBC cells.

microRNA-451a promoter methylation regulated by DNMT3B expedites bladder cancer development via the EPHA2/PI3K/AKT axis.

BACKGROUND: The downregulation of microRNA (miR)-451a has been reported in bladder cancer (BCa) tissues. Herein, we elucidated the role of miR-451a in BCa with the involvement of DNA methyltransferase 3B (DNMT3B). METHODS: We first screened the differentially expressed miRNAs from the serum of 12 BCa patients and 10 healthy controls in the BCa database GSE113486. Subsequently, we detected miR-451a expression and CpG island methylation of the promoter in BCa cells T24 and 5637 with DNMT3B knockdown. The downstream mRNAs of miR-451a were predicted by bioinformatics and KEGG enrichment analysis. Afterwards, the expression patterns of DNMT3B, miR-451a and erythropoietin-producing hepatocellular receptor tyrosine kinase class A2 (EPHA2) were altered in BCa cells to test the ability of cell proliferation, apoptosis, migration as well as invasion. Finally, the effect of miR-451a and DNMT3B was evaluated in vivo. RESULTS: miR-451a was significantly reduced in serum of BCa patients and cell lines. Moreover, the expression of DNMT3B in BCa cells was significantly increased, thus promoting methylation of the miR-451a promoter, resulting in miR-451a inhibition. Additionally, we found that miR-451a targeted and negatively regulated EPHA2, while EPHA2 could activate the PI3K/AKT signaling, driving BCa cell growth and metastasis. CONCLUSIONS: Our study proposed and demonstrated that miR-451a downregulation mediated by DNMT3B is critical for proliferation, migration, and invasion of BCa, which may be beneficial for developing more effective therapies against BCa.

MiR-26a targets EphA2 to resist intracellular Listeria monocytogenes in macrophages.

At infection sites, macrophages are sentinels that resist and destroy various pathogens, through direct phagocytosis. In macrophages, microRNAs play a variety of crucial roles, the most striking of which is the regulation of the ability of the host cell to resist infection. However, the underlying mechanisms associated with the anti-infection effects mediated by microRNAs remain largely unknown. Here, we demonstrated that miR-26a is downregulated during infection by Listeria monocytogenes (Lm). In miR-26a overexpressing mice, the Lm bacterial burden of liver and spleen decreased significantly within 72 h of infection, compared with that in control mice. Subsequently, RNA sequencing (RNA-seq) data suggested that miR-26a may attenuate the survival of Lm by targeting the Ephrin receptor tyrosine kinase A2 (EphA2). The knockdown of EphA2 in RAW264.7 macrophage cells resulted in decreased intracellular Lm burden. Taken together, these findings validate EphA2 as a target of miR-26a and provide a mechanism through which Lm may survive within macrophages by altering host miRNA expression.

The EphA2 and cancer connection: potential for immune-based interventions.

The Eph (erythropoietin-producing human hepatocellular) receptors form the largest known subfamily of receptor tyrosine kinases. These receptors interact with membrane-bound ephrin ligands via direct cell-cell interactions resulting in bi-directional activation of signal pathways. Importantly, the Eph receptors play critical roles in embryonic tissue organization and homeostasis, and in the maintenance of adult processes such as long-term potentiation, angiogenesis, and stem cell differentiation. The Eph receptors also display properties of both tumor promoters and suppressors depending on the cellular context. Characterization of EphA2 receptor in regard to EphA2 dysregulation has revealed associations with various pathological processes, especially cancer. The analysis of various tumor types generally identify EphA2 receptor as overexpressed and/or mutated, and for certain types of cancers EphA2 is linked with poor prognosis and decreased patient survival. Thus, here we highlight the role of EphA2 in malignant tissues that are specific to cancer; these include glioblastoma multiforme, prostate cancer, ovarian and uterine cancers, gastric carcinoma, melanoma, and breast cancer. Due to its large extracellular domain, therapeutic targeting of EphA2 with monoclonal antibodies (mAbs), which may function as inhibitors of ligand activation or as molecular agonists, has been an oft-attempted strategy. Therefore, we review the most current mAb-based therapies against EphA2 expressing cancers currently in pre-clinical and/or clinical stages. Finally, we discuss the latest peptides and cyclical-peptides that function as selective agonists for EphA2 receptor.

EphA2­enriched exosomes promote cell migration and are a potential diagnostic serum marker in pancreatic cancer.

Pancreatic cancer (PC) is the fourth most common cause of cancer­related mortality worldwide and is characterized by high invasiveness and early metastasis. To identify novel diagnostic markers, the present study aimed to understand the mechanism underlying PC progression. The present study demonstrated that exosomes derived from the highly metastatic Panc­1 PC cell line were internalized by a low metastatic cell line, resulting in increased migration of the latter. Proteomics analysis further revealed that the receptor tyrosine kinase Eph receptor A2 (EphA2) was overexpressed in the Panc­1 exosomes, and these Exo_EphA2 had the ability to transfer metastatic potential to recipient cells. Consistent with this, circulating Exo_EphA2 levels were higher in patients with PC compared with healthy controls. Taken together, these results indicated that Exo_EphA2 acts an oncogene in PC and is a potential tumor maker for PC diagnosis.

Biochemical and NMR characterization of the interactions of Vav2-SH2 domain with lipids and the EphA2 juxtamembrane region on membrane.

Vav2 is a ubiquitous guanine nucleotide exchange factor (GEF) for Rho family GTPases that is involved in regulating a wide range of biological processes. It interacts with several tyrosine-phosphorylated cell surface receptors, including the Eph family receptors, through its SH2 domain. The interaction of Vav2 with EphA2 is crucial for EphA2-mediated tumor angiogenesis. Here we show that Vav2-SH2 domain is a lipid-binding module that can recognize PI(4,5)P2 and PI(3,4,5)P3 lipids weakly but specifically. The specific lipid-binding site in Vav2-SH2 domain was identified by NMR chemical shift perturbation experiments using the head groups of PI(4,5)P2 and PI(3,4,5)P3, both of which bind to Vav2-SH2 with millimolar binding affinities. In addition, the interaction between Vav2-SH2 and the phosphorylated juxtamembrane region (JM) of EphA2 (Y594 phosphorylated) was investigated using NMR techniques. Furthermore, by using a nickel-lipid containing peptide-based nanodiscs system, we studied the binding of Vav2-SH2 to the phosphorylated JM region of EphA2 on lipid membrane and uncovered a role of membrane environment in modulating this protein-protein recognition.

Comparative Proteomic Analysis Identifies EphA2 as a Specific Cell Surface Marker for Wharton's Jelly-Derived Mesenchymal Stem Cells.

Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are a valuable tool in stem cell research due to their high proliferation rate, multi-lineage differentiation potential, and immunotolerance properties. However, fibroblast impurity during WJ-MSCs isolation is unavoidable because of morphological similarities and shared surface markers. Here, a proteomic approach was employed to identify specific proteins differentially expressed by WJ-MSCs in comparison to those by neonatal foreskin and adult skin fibroblasts (NFFs and ASFs, respectively). Mass spectrometry analysis identified 454 proteins with a transmembrane domain. These proteins were then compared across the different cell-lines and categorized based on their cellular localizations, biological processes, and molecular functions. The expression patterns of a selected set of proteins were further confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorescence assays. As anticipated, most of the studied proteins had common expression patterns. However, EphA2, SLC25A4, and SOD2 were predominantly expressed by WJ-MSCs, while CDH2 and Talin2 were specific to NFFs and ASFs, respectively. Here, EphA2 was established as a potential surface-specific marker to distinguish WJ-MSCs from fibroblasts and for prospective use to prepare pure primary cultures of WJ-MSCs. Additionally, CDH2 could be used for a negative-selection isolation/depletion method to remove neonatal fibroblasts contaminating preparations of WJ-MSCs.