The Hippo effector YAP1/TEAD1 regulates EPHA3 expression to control cell contact and motility.

The EPHA3 protein tyrosine kinase, a member of the ephrin receptor family, regulates cell fate, cell motility, and cell-cell interaction. These cellular events are critical for tissue development, immunological responses, and the processes of tumorigenesis. Earlier studies revealed that signaling via the STK4-encoded MST1 serine-threonine protein kinase, a core component of the Hippo pathway, attenuated EPHA3 expression. Here, we investigated the mechanism by which MST1 regulates EPHA3. Our findings have revealed that the transcriptional regulators YAP1 and TEAD1 are crucial activators of EPHA3 transcription. Silencing YAP1 and TEAD1 suppressed the EPHA3 protein and mRNA levels. In addition, we identified putative TEAD enhancers in the distal EPHA3 promoter, where YAP1 and TEAD1 bind and promote EPHA3 expression. Furthermore, EPHA3 knockout by CRISPR/Cas9 technology reduced cell-cell interaction and cell motility. These findings demonstrate that EPHA3 is transcriptionally regulated by YAP1/TEAD1 of the Hippo pathway, suggesting that it is sensitive to cell contact-dependent interactions.

Antibody-mediated depletion of CCR10+EphA3+ cells ameliorates fibrosis in IPF.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant repair that diminishes lung function via mechanisms that remain poorly understood. CC chemokine receptor (CCR10) and its ligand CCL28 were both elevated in IPF compared with normal donors. CCR10 was highly expressed by various cells from IPF lungs, most notably stage-specific embryonic antigen-4-positive mesenchymal progenitor cells (MPCs). In vitro, CCL28 promoted the proliferation of CCR10+ MPCs while CRISPR/Cas9-mediated targeting of CCR10 resulted in the death of MPCs. Following the intravenous injection of various cells from IPF lungs into immunodeficient (NOD/SCID-γ, NSG) mice, human CCR10+ cells initiated and maintained fibrosis in NSG mice. Eph receptor A3 (EphA3) was among the highest expressed receptor tyrosine kinases detected on IPF CCR10+ cells. Ifabotuzumab-targeted killing of EphA3+ cells significantly reduced the numbers of CCR10+ cells and ameliorated pulmonary fibrosis in humanized NSG mice. Thus, human CCR10+ cells promote pulmonary fibrosis, and EphA3 mAb-directed elimination of these cells inhibits lung fibrosis.

EPHA3 Contributes to Epigenetic Suppression of PTEN in Radioresistant Head and Neck Cancer.

EPHA3, a member of the EPH family, is overexpressed in various cancers. We demonstrated previously that EPHA3 is associated with radiation resistance in head and neck cancer via the PTEN/Akt/EMT pathway; the inhibition of EPHA3 significantly enhances the efficacy of radiotherapy in vitro and in vivo. In this study, we investigated the mechanisms of PTEN regulation through EPHA3-related signaling. Increased DNA methyltransferase 1 (DNMT1) and enhancer of zeste homolog 2 (EZH2) levels, along with increased histone H3 lysine 27 trimethylation (H3K27me3) levels, correlated with decreased levels of PTEN in radioresistant head and neck cancer cells. Furthermore, PTEN is regulated in two ways: DNMT1-mediated DNA methylation, and EZH2-mediated histone methylation through EPHA3/C-myc signaling. Our results suggest that EPHA3 could display a novel regulatory mechanism for the epigenetic regulation of PTEN in radioresistant head and neck cancer cells.

Intranasal Delivery of Temozolomide-Conjugated Gold Nanoparticles Functionalized with Anti-EphA3 for Glioblastoma Targeting.

Glioblastoma multiforme (GBM) is a highly lethal and aggressive tumor of the brain that carries a poor prognosis. Temozolomide (TMZ) has been widely used as a first-line treatment for GBM. However, poor brain targeting, side effects, and drug resistance limit its application for the treatment of GBM. We designed a Temozolomide-conjugated gold nanoparticle functionalized with an antibody against the ephrin type-A receptor 3 (anti-EphA3-TMZ@GNPs) for targeted GBM therapy via intranasal administration. The system can bypass the blood-brain barrier and target active glioma cells to improve the glioma targeting of TMZ and enhance the treatment efficacy, while reducing the peripheral toxicity and drug resistance. The prepared anti-EphA3-TMZ@GNPs were 46.12 ± 2.0 nm and suitable for intranasal administration, which demonstrated high safety to the nasal mucosa in a toxicity assay. In vitro studies showed that anti-EphA3-TMZ@GNPs exhibited significantly enhanced cellular uptake and toxicity, and a higher cell apoptosis ratio has been seen compared with that of TMZ (54.9 and 14.1%, respectively) toward glioma cells (C6). The results from experiments on TMZ-resistant glioma cells (T98G) demonstrated that the IC50 of anti-EphA3-TMZ@GNPs (64.06 ± 0.16 µM) was 18.5-fold lower than that of TMZ. In addition, Western blot analysis also revealed that anti-EphA3-TMZ@GNPs effectively down-modulated expression of O6-methylguanine-DNA methyltransferase and increased chemosensitivity of T98G to TMZ. The antiglioma efficacy in vivo was investigated in orthotopic glioma-bearing rats, and the results demonstrated that the anti-EphA3-TMZ@GNPs prolonged the median survival time to 42 days and increased tumor-cell apoptosis dramatically compared with TMZ. In conclusion, anti-EphA3-TMZ@GNPs could serve as an intranasal drug delivery system for efficacious treatment of GBM.

Critical role of EphA3 in cancer and current state of EphA3 drug therapeutics.

The erythropoietin-producing human hepatocellular (Eph) receptors are transmembrane glycoprotein members of the tyrosine kinase receptors family. The Ephs may bind to various ephrin ligands resulting in the phosphorylation of their tyrosine kinase domain and the activation of the Eph receptor. In this review we focus on EphA3, one receptor of the 14 different Ephs, as it carries out both redundant and restricted functions in the germline development of mammals and in the maintenance of various adult tissues. The loss of EphA3 regulation is correlated with various human malignancies, the most notable being cancer. This receptor is overexpressed and/or mutated in multiple tumors, and is also associated with poor prognosis and decreased survival in patients. Here we highlight the role of EphA3 in normal and malignant tissues that are specific to cancer; these include hematologic disorders, gastric cancer, glioblastoma multiforme, colorectal cancer, lung cancer, renal cell carcinoma, and prostate cancer. Moreover, various anticancer agents against EphA3 have been developed to either inhibit its kinase domain activity or to function as agonists. Thus, we examine the most potent small molecule drugs and mAb-based therapeutics against EphA3 that are currently in pre-clinical or clinical stages.

A panel of protein kinase high expression is associated with postoperative recurrence in cholangiocarcinoma.

BACKGROUND: Cancer recurrence is one of the most concerning clinical problems of cholangiocarcinoma (CCA) patients after treatment. However, an identification of predictive factor on Opisthorchis viverrini (OV)-associated CCA recurrence is not well elucidated. In the present study, we aimed to investigate the correlation of twelve targeted protein kinases with CCA recurrence. METHODS: Twelve protein kinases, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2, 3, 4 (HER2, HER3, HER4), vascular endothelial growth factor receptor 3 (VEGFR3), vascular endothelial growth factor-C (VEGF-C), erythropoietin-producing hepatocellular carcinoma receptor type-A3 (EphA3), EphrinA1, phosphor-serine/threonine kinase 1 (p-Akt1), serine/threonine kinase 1 (Akt1), beta-catenin and protein Wnt5a (Wnt5a) were examined using immunohistochemistry. Pre-operative serum tumor markers, CA19-9 and CEA were also investigated. RESULTS: Among twelve protein kinases, EGFR, HER4, and EphA3 were associated with tumor recurrence status, recurrence-free survival (RFS) and overall survival (OS). Multivariate cox regression demonstrated that EGFR, HER4, EphA3 or the panel of high expression of these proteins was an independent prognostic factor for tumor recurrence. The combination of high expression of these proteins with a high level of CA19-9 could improve the predictive ability on tumor recurrence. Moreover, the patients were stratified more accurately when analyzed using the combination of high expression of these proteins with primary tumor (T) or lymph node metastasis (N) status. CONCLUSION: EGFR, HER4, EphA3 or the panel of high expression of these proteins is an independent prognostic factor for post-operative CCA recurrence.

Glucose homeostasis is regulated by pancreatic ß-cell cilia via endosomal EphA-processing.

Diabetes mellitus affects one in eleven adults worldwide. Most suffer from Type 2 Diabetes which features elevated blood glucose levels and an inability to adequately secrete or respond to insulin. Insulin producing ß-cells have primary cilia which are implicated in the regulation of glucose metabolism, insulin signaling and secretion. To better understand how ß-cell cilia affect glucose handling, we ablate cilia from mature ß-cells by deleting key cilia component Ift88. Here we report that glucose homeostasis and insulin secretion deteriorate over 12 weeks post-induction. Cilia/basal body components are required to suppress spontaneous auto-activation of EphA3 and hyper-phosphorylation of EphA receptors inhibits insulin secretion. In ß-cells, loss of cilia/basal body function leads to polarity defects and epithelial-to-mesenchymal transition. Defective insulin secretion from IFT88-depleted human islets and elevated pEPHA3 in islets from diabetic donors both point to a role for cilia/basal body proteins in human glucose homeostasis.

EPHA3 enhances macrophage autophagy and apoptosis by disrupting the mTOR signaling pathway in mice with endometriosis.

Background: Endometriosis is a chronic fibrotic disease characterized by agonizing pelvic pain and low fertility, mainly affecting middle-aged women. The aim of the present study is to assess the potential effects of erythropoietin-producing hepatocellular carcinoma A3 (EPHA3) on endometriosis, with emphasis on the autophagy and apoptosis of macrophages via inhibition of the mammalian target of rapamycin (mTOR) signaling pathway.Methods: The mouse models of endometriosis were established followed by culturing the macrophages and macrophage transfection via the EPHA3 vector, siRNA EPHA3, and RAPA (an inhibitor of the mTOR signaling pathway). The expression of EPHA3, related factors in the mTOR signaling pathway, macrophage autophagy (autophagy-related gene 3 (Atg3), light chain 3-I (LC3-I), light chain 3-II (LC3-II) and Beclin1) and apoptosis (B-cell lymphoma-2 (bcl-2), bax and fas) were all detected and documented, respectively. The changes of autophagic lysosomes and the apoptosis of macrophages in each group following transfection were also inspected and detected.Results: The results of the in silico analysis ascertained EPHA3 to be a candidate gene of endometriosis. After successful modeling, the uterine tissues of endometriosis mice presented with a low expression of EPHA3 and activated mTOR signaling pathway. Overexpression of EPHA3 inhibited the activation of the mTOR signaling pathway, down-regulated bcl-2 expression, up-regulated the expression of Atg3, LC3-II/LC3-I, Beclin1, bax and fas, and also promoted the autophagy and apoptosis of macrophages in endometriosis mice.Conclusion: Altogether, EPHA3 could potentially promote the autophagy and apoptosis of macrophages in endometriosis via inhibition of the mTOR signaling pathway, highlighting the potential of EPHA3 as the target to treat endometriosis.

Prognostic Significance of High EphA1-4 Expression Levels in Locally Advanced Gastric Cancer.

BACKGROUND/AIM: Erythropoietin-producing hepatocellular carcinoma receptor A (EphA) is associated with angiogenesis and invasive tumor progression. In this study, we evaluated the EphA1-4 expression levels in advanced gastric cancer. PATIENTS AND METHODS: Tumor tissues obtained from 114 patients with advanced gastric adenocarcinoma who underwent gastrectomy were analyzed. In addition, the impact of EPHA 1-4 mRNA expression on survival was analyzed using the Kaplan-Meier plotter database on the website. RESULTS: High EphA 1, 2, and 4 expression levels were significantly related to recurrence (p<0.01, p=0.04, and p<0.01). Both high EphA 1 and 4 expression levels were independent predictors of relapse-free interval (hazard ratio [HR]=2.0, p=0.03; HR=2.4, p=0.03) and disease-specific survival (HR=2.0, 95% p=0.03; HR=2.5, p=0.02) on multivariate analysis. In the Kaplan-Meier plotter database, high EPHA2 mRNA expression was significantly associated with poor survival in patients with gastric cancer (p=0.0098), and high expression levels of EPHA1 and 4 tended to be associated with poor survival (p=0.050, p=0.052). CONCLUSION: EphA 1, 2, and 4 may play key roles in recurrence and survival in patients with advanced gastric cancer.

EphA3 targeting reduces in vitro adhesion and invasion and in vivo growth and angiogenesis of multiple myeloma cells.

PURPOSE: Multiple myeloma (MM) is a hematologic malignancy characterized by a clonal expansion of plasma cells (PCs) in the bone marrow (BM). Since MM has so far remained incurable, further insights into its pathogenesis and the concomitant identification of new therapeutic targets are urgently needed. The tyrosine kinase receptor EphA3 is known to be involved in various cellular processes including cell viability, cell movement and cell-cell interactions. Recently, EphA3 has emerged as a potential therapeutic target in several hematologic and solid tumors. Here, we aimed to uncover the role of EphA3 in MM. METHODS: EphA3 mRNA and protein expression in primary MM bone marrow plasma cells (BMPCs), in MM-derived cell lines and in healthy controls (HCs) was assessed using qRT-PCR, Western blotting and flow cytometry. The effects of siRNA-mediated EphA3 silencing and anti EphA3 antibody (EphA3mAb) treatment on MM PC trafficking and viability were evaluated using in vitro assays. The effects of EphA3mAb treatment were also assessed in two MM-derived mouse xenograft models. RESULTS: We found that EphA3 was overexpressed in primary MM BMPCs and MM-derived cell lines compared to HCs. We also found that siRNA-mediated EphA3 silencing and EphA3mAb treatment significantly inhibited the ability of MM PCs to adhere to fibronectin and stromal cells and to invade in vitro, without affecting cell proliferation and viability. Gene expression profiling showed that EphA3 silencing resulted in expression modulation of several molecules that regulate adhesion, migration and invasion processes. Importantly, we found that EphA3mAb treatment significantly inhibited in vivo tumor growth and angiogenesis in two MM-derived mouse xenograft models. CONCLUSIONS: Our findings suggest that EphA3 plays an important role in the pathogenesis of MM and provide support for the notion that its targeting may represent a novel therapeutic opportunity for MM.

EphA3 as a target for antibody immunotherapy in acute lymphoblastic leukemia.

The human EphA3 gene was discovered in a pre-B acute lymphoblastic leukemia (pre-B-ALL) using the EphA3-specific monoclonal antibody (mAb), IIIA4, which binds and activates both human and mouse EphA3. We use two models of human pre-B-ALL to examine EphA3 function, demonstrating effects on pre-B-cell receptor signaling. In therapeutic targeting studies, we demonstrated antitumor effects of the IIIA4 mAb in EphA3-expressing leukemic xenografts and no antitumor effect in the xenografts with no EphA3 expression providing evidence that EphA3 is a functional therapeutic target in pre-B-ALL. Here we show that the therapeutic effect of the anti-EphA3 antibody was greatly enhanced by adding an α-particle-emitting 213Bismuth payload.

The Neural Cell Adhesion Molecule (NCAM) Promotes Clustering and Activation of EphA3 Receptors in GABAergic Interneurons to Induce Ras Homolog Gene Family, Member A (RhoA)/Rho-associated protein kinase (ROCK)-mediated Growth Cone Collapse.

Establishment of a proper balance of excitatory and inhibitory connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity. The neural cell adhesion molecule (NCAM) and the receptor tyrosine kinase EphA3 regulate the perisomatic synapse density of inhibitory GABAergic interneurons in the mouse frontal cortex through ephrin-A5-induced growth cone collapse. In this study, it was demonstrated that binding of NCAM and EphA3 occurred between the NCAM Ig2 domain and EphA3 cysteine-rich domain (CRD). The binding interface was further refined through molecular modeling and mutagenesis and shown to be comprised of complementary charged residues in the NCAM Ig2 domain (Arg-156 and Lys-162) and the EphA3 CRD (Glu-248 and Glu-264). Ephrin-A5 induced co-clustering of surface-bound NCAM and EphA3 in GABAergic cortical interneurons in culture. Receptor clustering was impaired by a charge reversal mutation that disrupted NCAM/EphA3 association, emphasizing the importance of the NCAM/EphA3 binding interface for cluster formation. NCAM enhanced ephrin-A5-induced EphA3 autophosphorylation and activation of RhoA GTPase, indicating a role for NCAM in activating EphA3 signaling through clustering. NCAM-mediated clustering of EphA3 was essential for ephrin-A5-induced growth cone collapse in cortical GABAergic interneurons, and RhoA and a principal effector, Rho-associated protein kinase, mediated the collapse response. This study delineates a mechanism in which NCAM promotes ephrin-A5-dependent clustering of EphA3 through interaction of the NCAM Ig2 domain and the EphA3 CRD, stimulating EphA3 autophosphorylation and RhoA signaling necessary for growth cone repulsion in GABAergic interneurons in vitro, which may extend to remodeling of axonal terminals of interneurons in vivo.

Overexpression of Ephrin A3 Receptor in Canine Prostatic Carcinoma.

Ephrin A3 (EphA3), a member of the ephrin receptor tyrosine kinase family, is involved in a variety of functions in normal cells, especially during embryonic development, and alterations in its expression profile have been observed in several human cancers. However, there are no reports of the expression of EphA3 in normal, hyperplastic or neoplastic canine prostate tissue or in other types of canine tumours. Six normal, 15 hyperplastic and 21 neoplastic canine prostates were examined immunohistochemically with a polyclonal antibody specific for human EphA3. The percentage of positive cells in all prostatic carcinomas was increased, with a mean of 89.28 ± 5.18% compared with normal (9.17 ± 6.72%) and hyperplastic prostates (20.00 ± 8.28%). EphA3 expression was not correlated with the histological subtypes of prostate cancer or with the Gleason score. The increase in EphA3 expression in canine prostatic carcinomas suggests the involvement of this receptor in prostatic carcinogenesis and its potential use as a target for new therapeutic strategies.

Distinctive Structure of the EphA3/Ephrin-A5 Complex Reveals a Dual Mode of Eph Receptor Interaction for Ephrin-A5.

The Eph receptor tyrosine kinase/ephrin ligand system regulates a wide spectrum of physiological processes, while its dysregulation has been implicated in cancer progression. The human EphA3 receptor is widely upregulated in the tumor microenvironment and is highly expressed in some types of cancer cells. Furthermore, EphA3 is among the most highly mutated genes in lung cancer and it is also frequently mutated in other cancers. We report the structure of the ligand-binding domain of the EphA3 receptor in complex with its preferred ligand, ephrin-A5. The structure of the complex reveals a pronounced tilt of the ephrin-A5 ligand compared to its orientation when bound to the EphA2 and EphB2 receptors and similar to its orientation when bound to EphA4. This tilt brings an additional area of ephrin-A5 into contact with regions of EphA3 outside the ephrin-binding pocket thereby enlarging the size of the interface, which is consistent with the high binding affinity of ephrin-A5 for EphA3. This large variation in the tilt of ephrin-A5 bound to different Eph receptors has not been previously observed for other ephrins.

The putative tumor suppressor gene EphA3 fails to demonstrate a crucial role in murine lung tumorigenesis or morphogenesis.

Treatment of non-small cell lung cancer (NSCLC) is based on histological analysis and molecular profiling of targetable driver oncogenes. Therapeutic responses are further defined by the landscape of passenger mutations, or loss of tumor suppressor genes. We report here a thorough study to address the physiological role of the putative lung cancer tumor suppressor EPH receptor A3 (EPHA3), a gene that is frequently mutated in human lung adenocarcinomas. Our data shows that homozygous or heterozygous loss of EphA3 does not alter the progression of murine adenocarcinomas that result from Kras mutation or loss of Trp53, and we detected negligible postnatal expression of EphA3 in adult wild-type lungs. Yet, EphA3 was expressed in the distal mesenchyme of developing mouse lungs, neighboring the epithelial expression of its Efna1 ligand; this is consistent with the known roles of EPH receptors in embryonic development. However, the partial loss of EphA3 leads only to subtle changes in epithelial Nkx2-1, endothelial Cd31 and mesenchymal Fgf10 RNA expression levels, and no macroscopic phenotypic effects on lung epithelial branching, mesenchymal cell proliferation, or abundance and localization of CD31-positive endothelia. The lack of a discernible lung phenotype in EphA3-null mice might indicate lack of an overt role for EPHA3 in the murine lung, or imply functional redundancy between EPHA receptors. Our study shows how biological complexity can challenge in vivo functional validation of mutations identified in sequencing efforts, and provides an incentive for the design of knock-in or conditional models to assign the role of EPHA3 mutation during lung tumorigenesis.

Hypoxia-controlled EphA3 marks a human endometrium-derived multipotent mesenchymal stromal cell that supports vascular growth.

Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF)-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs), but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29+/CD73+/CD90+/CD146+ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.

Targeting EphA3 inhibits cancer growth by disrupting the tumor stromal microenvironment.

Eph receptor tyrosine kinases are critical for cell-cell communication during normal and oncogenic tissue patterning and tumor growth. Somatic mutation profiles of several cancer genomes suggest EphA3 as a tumor suppressor, but its oncogenic expression pattern and role in tumorigenesis remain largely undefined. Here, we report unexpected EphA3 overexpression within the microenvironment of a range of human cancers and mouse tumor xenografts where its activation inhibits tumor growth. EphA3 is found on mouse bone marrow-derived cells with mesenchymal and myeloid phenotypes, and activation of EphA3(+)/CD90(+)/Sca1(+) mesenchymal/stromal cells with an EphA3 agonist leads to cell contraction, cell-cell segregation, and apoptosis. Treatment of mice with an agonistic α-EphA3 antibody inhibits tumor growth by severely disrupting the integrity and function of newly formed tumor stroma and microvasculature. Our data define EphA3 as a novel target for selective ablation of the tumor microenvironment and demonstrate the potential of EphA3 agonists for anticancer therapy.

Pyrrolo[3,2-b]quinoxaline derivatives as types I1/2 and II Eph tyrosine kinase inhibitors: structure-based design, synthesis, and in vivo validation.

The X-ray crystal structures of the catalytic domain of the EphA3 tyrosine kinase in complex with two type I inhibitors previously discovered in silico (compounds A and B) were used to design type I1/2 and II inhibitors. Chemical synthesis of about 25 derivatives culminated in the discovery of compounds 11d (type I1/2), 7b, and 7g (both of type II), which have low-nanomolar affinity for Eph kinases in vitro and a good selectivity profile on a panel of 453 human kinases (395 nonmutant). Surface plasmon resonance measurements show a very slow unbinding rate (1/115 min) for inhibitor 7m. Slow dissociation is consistent with a type II binding mode in which the hydrophobic moiety (trifluoromethyl-benzene) of the inhibitor is deeply buried in a cavity originating from the displacement of the Phe side chain of the so-called DFG motif as observed in the crystal structure of compound 7m. The inhibitor 11d displayed good in vivo efficacy in a human breast cancer xenograft.

EphrinA/EphA-induced ectodomain shedding of neural cell adhesion molecule regulates growth cone repulsion through ADAM10 metalloprotease.

EphrinA/EphA-dependent axon repulsion is crucial for synaptic targeting in developing neurons but downstream molecular mechanisms remain obscure. Here, it is shown that ephrinA5/EphA3 triggers proteolysis of the neural cell adhesion molecule (NCAM) by the metalloprotease a disintegrin and metalloprotease (ADAM)10 to promote growth cone collapse in neurons from mouse neocortex. EphrinA5 induced ADAM10 activity to promote ectodomain shedding of polysialic acid-NCAM in cortical neuron cultures, releasing a ~ 250 kDa soluble fragment consisting of most of its extracellular region. NCAM shedding was dependent on ADAM10 and EphA3 kinase activity as shown in HEK293T cells transfected with dominant negative ADAM10 and kinase-inactive EphA3 (K653R) mutants. Purified ADAM10 cleaved NCAM at a sequence within the E-F loop of the second fibronectin type III domain (Leu(671) -Lys(672) /Ser(673) -Leu(674) ) identified by mass spectrometry. Mutations of NCAM within the ADAM10 cleavage sequence prevented EphA3-induced shedding of NCAM in HEK293T cells. EphrinA5-induced growth cone collapse was dependent on ADAM10 activity, was inhibited in cortical cultures from NCAM null mice, and was rescued by WT but not ADAM10 cleavage site mutants of NCAM. Regulated proteolysis of NCAM through the ephrin5/EphA3/ADAM10 mechanism likely impacts synapse development, and may lead to excess NCAM shedding when disrupted, as implicated in neurodevelopmental disorders such as schizophrenia. PSA-NCAM and ephrinA/EphA3 coordinately regulate inhibitory synapse development. Here, we have found that ephrinA5 stimulates EphA3 kinase and ADAM10 activity to promote PSA-NCAM cleavage at a site in its second FNIII repeat, which regulates ephrinA5-induced growth cone collapse in GABAergic and non-GABAergic neurons. These findings identify a new regulatory mechanism which may contribute to inhibitory connectivity.

Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands.

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.