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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.

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.

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.

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 β-cells have primary cilia which are implicated in the regulation of glucose metabolism, insulin signaling and secretion. To better understand how β-cell cilia affect glucose handling, we ablate cilia from mature β-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 β-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, a member of the Eph family of receptor tyrosine kinases, has been reported to be overexpressed in some human cancers including glioblastoma. Here, we found that expression of EphA3 is up-regulated in response to epidermal growth factor (EGF) stimulation and promotes formation of cell aggregates in suspension culture of glioblastoma cells. Suppression of EphA3 expression by short hairpin RNA-mediated knockdown or CRISPR/Cas9-mediated gene deletion inhibited EGF-induced promotion of cell aggregate formation, whereas overexpression of EphA3 promoted formation of cell aggregates in suspension culture. EGF-induced EphA3 expression and promotion of cell aggregate formation required Akt activity. Furthermore, N-cadherin, whose expression was regulated by EGF and EphA3, contributed to the formation of cell aggregates in suspension culture. These results suggest that the regulation of EphA3 expression plays a critical role in glioblastoma cell growth in non-adherent conditions.

The Eph and ephrins are involved in the genesis of topographic ordered connections at the visual system. Previously we demonstrated that tectal EphA3 stimulates axon growth of nasal retinal ganglion cells (RGCs) toward the caudal tectum preventing them from branching in the rostral tectum. Now we investigated whether tectal EphA3 plays this role by modulating the axonal EphA4 forward signaling or throughout axonal ephrin-As reverse signaling. For this purpose we used cultures of nasal retinal explants and dissociated retinal neurons from chicken embryos. We treated them with clustered EphA3-Fc, Fc (control), PI-PLC (sheds ephrin-As) or KYL (inhibits ephrin-As-mediated EphA4 activation). We achieved in vitro and in vivo electroporations of chicken embryo retinas with wild type EphA4, Ki-EphA4 (kinase inactive dominant negative EphA4) or EGFP in pMES expression vector. We performed immunocytochemistry, immunoprecipitation and Western blot against Eph/ephrin-As system. Our results showed that: 1) shedding of ephrin-As and the inhibition of ephrin-A-mediated EphA4 activity increase axon length and decrease axonal interstitial filopodia density of nasal RGCs; and 2) a dominant negative form of EphA4 increases axon growth in vitro and induces nasal RGC axons to grow passing throughout their target area in the caudal tectum meanwhile overexpression of EphA4 produces the opposite effects. All together, these results demonstrate that ephrin-A-mediated EphA4 forward signaling decreases the level of axon growth and increases the density of axonal interstitial filopodia of nasal RGCs. Besides, our results showed that: 3) EphA3 ectodomain increases axon growth and decreases the density of axonal interstitial filopodia and branching in vitro and in vivo and 4) EphA3 ectodomain diminishes the ephrin-A2/EphA4 colocalization, and the EphA4 and ephexin1 phosphorylation. All together, these results show that the EphA3 ectodomain produces the opposite effects than the EphA4 forward signaling, by decreasing this signaling pathway throughout competing with EphA4 for ephrin-As binding. Furthermore, it is proposed that tectal EphA3 participates in the establishment of retinotectal mapping throughout this mechanism and that EphAs can regulate axon growth and branching by modulating other EphA receptors forward signaling.

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, 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.

The purpose of this study was to investigate the expression level of EphA3 in nasopharyngeal carcinoma (NPC) and its effect on the proliferative capacity of NPC. Meanwhile, the underlying mechanism by which EphA3 prompts NPC malignant progression was further explored.In this study, the expression of EphA3 in 42 pairs of tumor tissue specimens and paracancerous ones collected from NPC patients was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and the interplay between EphA3 expression and clinical indicators, as well as prognosis of NPC patients was analyzed. Meanwhile, qRT-PCR was also applied to further verify EphA expression in NPC cell lines. In addition, EphA knockdown model was constructed in NPC cell lines, CNE2, and 6-10B, and the impacts of EphA on NPC cell functions was assessed through Cell Counting Kit-8 (CCK-8), cell colony formation, as well as 5-Ethynyl-2'- deoxyuridine (EdU) assays. Finally, a potential interplay between EphA3 and FOG2 was also investigated.In this study, qRT-PCR results revealed that EphA3 expression levels in tumor tissues of patients with NPC were markedly higher than those in adjacent tissues. Compared with patients with low expression of EphA3, those with highly expressed EphA3 had a more advanced pathological stage. In addition, in vitro experiments showed that knocking down EphA3 notably attenuated the proliferation capacity of NPC cells. Subsequently, it was found that the expression of FOG2 in NPC cells was remarkably decreased both in NPC cell lines and tissues, which had a negative correlation with EphA3. Finally, cell recovery experiment revealed a mutual regulation between EphA3 and FOG2, which then together affected the malignant progression of NPC.EphA3 is significantly relevant to pathological staging and poor prognosis of patients with NPC and may enhance the proliferation ability of NPC cells by modulating FOG2.

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.

Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by GBM stem cells (GSC), which have been implicated in resistance to GBM therapy. In this study, we identify several EphRs that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (cytometry by time-of-flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSCs to identify putative rGBM-specific EphR. EPHA2 and EPHA3 coexpression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EPHA2 and EPHA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival in vivo. Treatment of rGBM with a bispecific antibody against EPHA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo via downregulation of AKT and ERK and increased cellular differentiation. In conclusion, we show that EPHA2 and EPHA3 together mark a GSC population in rGBM and that strategic cotargeting of EPHA2 and EPHA3 presents a novel and rational therapeutic approach for rGBM. Significance: Treatment of rGBM with a novel bispecific antibody against EPHA2 and EPHA3 reduces tumor burden, paving the way for the development of therapeutic approaches against biologically relevant targets in rGBM. Cancer Res; 78(17); 5023–37. ©2018 AACR.

Radiotherapy is a well-established therapeutic modality used in the treatment of many cancers. However, radioresistance remains a serious obstacle to successful treatment. Radioresistance can cause local recurrence and distant metastases in some patients after radiation treatment. Thus, many studies have attempted to identify effective radiosensitizers. Eph receptor functions contribute to tumor development, modulating cell-cell adhesion, invasion, neo-angiogenesis, tumor growth and metastasis. However, the role of EphA3 in radioresistance remains unclear. In the current study, we established a stable radioresistant head and neck cancer cell line (AMC HN3R cell line) and found that EphA3 was expressed predominantly in the radioresistant head and neck cancer cell line through DNA microarray, real time PCR and Western blotting. Additionally, we found that EphA3 was overexpressed in recurrent laryngeal cancer specimens after radiation therapy. EphA3 mediated the tumor invasiveness and migration in radioresistant head and neck cancer cell lines and epithelial mesenchymal transition- related protein expression. Inhibition of EphA3 enhanced radiosensitivity in the AMC HN 3R cell line in vitro and in vivo study. In conclusion, our results suggest that EphA3 is overexpressed in radioresistant head and neck cancer and plays a crucial role in the development of radioresistance in head and neck cancers by regulating the epithelial mesenchymal transition pathway.

Nonsyndromic cleft lip with or without palate (NSCL/P) represents a complex condition caused by genetic and environmental factors. The aim of this study was to investigate the relationship between the EPHA3 polymorphisms and NSCL/P.To investigate the relationship between five EPHA3 single nucleotide polymorphisms (SNPs) and NSCL/P, we selected 180 affected patients and 167 normal controls from the Chinese Han Population. EPHA3 SNPs (rs7650466, rs1398197, rs17801309, rs1054750, and rs7632427) were genotyped using the SNaPshot technique; bioinformatic analyses were performed to determine if any of them were potentially functional SNPs.The rs7650466 T allele was associated with the incidence of NSCL/P (OR, 0.211; 95% CI, 0.131-0.338; adjusted p = 4.881 × 10Collectively, our data suggest that the EPHA3 rs7650466 polymorphism confers genetic risk for NSCL/P in the Chinese Han Population. Furthermore, rs7650466 is associated with CL/P incidence in stratified analyses, but not with cleft palate only.

Background Erythropoietin-producing hepatocellular carcinoma (Eph) receptors constitute the largest family of receptor tyrosine kinases. Ephs and their ligands ephrins play an important role in development and carcinogenesis. The expression of EphA3, an Eph family member, has been investigated in a variety of human cancers, with mixed results. High levels of EphA3 protein expression have been reported in colorectal, prostate, and gastric cancers, whereas loss of protein expression has been reported in lung and hematopoietic cancers. EphA3 expression in clear-cell renal cell carcinoma (ccRCC) and its association with clinicopathological parameters has not previously been examined. The aim of this study was to determine the cancerous value of EphA3 protein expression in patients with ccRCC. Materials and Methods This study included 68 patients with ccRCC. EphA3 protein expression was examined in ccRCC tissue samples using immunohistochemistry and a specific polyclonal antibody, and the correlation between EphA3 expression and clinicopathological parameters was subsequently evaluated. Results High EphA3 protein expression was observed in all normal renal tubules. In the 68 ccRCC patient samples examined, EphA3 protein expression was detected in 19 cases (27.9%) and undetectable in 49 cases (72.1%). EphA3 protein expression was significantly associated with tumor diameter ( P = .016) and tumor, node metastases stage ( P = .029). No significant association between protein expression and sex ( P = .387), age ( P = .727), or nuclear grade ( P = .243) was found. Conclusion Ourdata indicate that EphA3 protein expression is reduced in ccRCC, suggesting the possibility that this receptor functions as a tumor suppressor in this disease.

// Antonella Caivano 1 , Francesco La Rocca 1 , Ilaria Laurenzana 1 , Tiziana Annese 2 , Roberto Tamma 2 , Ubaldo Famigliari 3 , Vittorio Simeon 1 , Stefania Trino 1 , Luciana De Luca 1 , Oreste Villani 4 , Simona Berardi 5 , Antonio Basile 5 , Angelo Vacca 5 , Giuseppe Saglio 6 , Luigi Del Vecchio 7, 8 , Pellegrino Musto 9 , Daniela Cilloni 6 1 Laboratory of Pre-clinical and Translational Research, Scientific Institute of Research and Cure (IRCCS), Referral Cancer Center of Basilicata (CROB), Rionero in Vulture, Italy 2 Department of Human Anatomy, Histology and Embryology, University of Bari Medical School, Bari, Italy 3 Division of Pathology, Department of Oncology, St Luigi Hospital, Turin, Italy 4 Department of Onco-Hematology, IRCCS-CROB, Rionero in Vulture, Italy 5 Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy 6 Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy 7 CEINGE-Biotecnologie Avanzate s.c.a r.l and Medical Biotechnologies, Federico II University, Naples, Italy 8 Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy 9 Scientific Direction, IRCCS-CROB, Rionero in Vulture, Italy Correspondence to: Antonella Caivano, email: caivanoa@libero.it Keywords: angiogenesis, bone marrow endothelial cells, receptor tyrosine kinase, EphA3, multiple myeloma Received: September 02, 2016 Accepted: March 01, 2017 Published: March 10, 2017 ABSTRACT This study investigates the role of ephrin receptor A3 (EphA3) in the angiogenesis of Multiple Myeloma (MM) and the effects of a selective target of EphA3 by a specific monoclonal antibody on primary bone marrow endothelial cells (ECs) of MM patients. EphA3 mRNA and protein were evaluated in ECs of MM patients (MMECs), in ECs of patients with monoclonal gammopathies of undetermined significance (MGECs) and in ECs of healthy subjects (control ECs). The effects of EphA3 targeting by mRNA silencing (siRNA) or by the anti EphA3 antibody on the angiogenesis were evaluated. We found that EphA3 is highly expressed in MMECs compared to the other EC types. Loss of function of EphA3 by siRNA significantly inhibited the ability of MMECs to adhere to fibronectin, to migrate and to form tube like structures in vitro , without affecting cell proliferation or viability. In addition, gene expression profiling showed that knockdown of EphA3 down modulated some molecules that regulate adhesion, migration and invasion processes. Interestingly, EphA3 targeting by an anti EphA3 antibody reduced all the MMEC angiogenesis-related functions in vitro . In conclusion, our findings suggest that EphA3 plays an important role in MM angiogenesis.

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.

Background: Src associated with mitosis of 68 kDa (Sam68), is often highly expressed in human cancers. Overexpression of Sam68 has been shown to be correlated with poor survival prognosis in some cancer patients. However, little is known whether Sam68 plays a role in promoting metastasis in breast cancer. Materials and Methods: The expression of Sam68 protein in breast cancer tissue was detected by immunohistochemistry. Trans-well assay, wound-healing, real-time PCR and Western blotting analysis were used to detect the effect of Sam68 on promoting EMT or metastasis of breast cancer. Next-generation RNA sequencing was used to analyze genes that may be regulated by Sam68. Results: Sam68 plays a positive role in promoting breast cancer metastasis. Sam68 was found to be overexpressed in breast cancer along with lymph node metastasis. MMP-9 was also found to be overexpressed in breast cancer tissue and was correlated to the expression of Sam68 (P Conclusion: High expression of Sam68 participates in breast cancer metastasis by up-regulating the EPHA3 gene.

EphA/ephrin signaling regulates axon growth and guidance of neurons, but whether this process occurs also independently of ephrins is unclear. We show that presenilin-1 (PS1)/γ-secretase is required for axon growth in the developing mouse brain. PS1/γ-secretase mediates axon growth by inhibiting RhoA signaling and cleaving EphA3 independently of ligand to generate an intracellular domain (ICD) fragment that reverses axon defects in PS1/γ-secretase- and EphA3-deficient hippocampal neurons. Proteomic analysis revealed that EphA3 ICD binds to non-muscle myosin IIA (NMIIA) and increases its phosphorylation (Ser1943), which promotes NMIIA filament disassembly and cytoskeleton rearrangement. PS1/γ-secretase-deficient neurons show decreased phosphorylated NMIIA and NMIIA/actin colocalization. Moreover, pharmacological NMII inhibition reverses axon retraction in PS-deficient neurons suggesting that NMIIA mediates PS/EphA3-dependent axon elongation. In conclusion, PS/γ-secretase-dependent EphA3 cleavage mediates axon growth by regulating filament assembly through RhoA signaling and NMIIA, suggesting opposite roles of EphA3 on inhibiting (ligand-dependent) and promoting (receptor processing) axon growth in developing neurons. Ministerio de Economia y Competitividad SAF2013-43900-R Carlos A Saura, Ministerio de Economia y Competitividad SAF2016-80027-R Carlos A Saura, Ministerio de Economia y Competitividad CIBERNED CB06/05/0042 Carlos A Saura Generalitat de Catalunya ICREA Academia Carlos A Saura, Generalitat de Catalunya 2017 SGR749 Carlos A Saura.

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.

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.

EphA3 is an Ephrin receptor tyrosine kinase that is overexpressed in most hematologic malignancies. We performed a first-in-human multicenter phase I study of the anti-EphA3 monoclonal antibody KB004 in refractory hematologic malignancies in order to determine safety and tolerability, along with the secondary objectives of pharmacokinetics (PK) and pharmacodynamics (PD) assessments, as well as preliminary assessment of efficacy. Patients were enrolled on a dose escalation phase (DEP) initially, followed by a cohort expansion phase (CEP). KB004 was administered by intravenous infusion on days 1, 8, and 15 of each 21-day cycle in escalating doses. A total of 50 patients (AML 39, MDS/MPN 3, MDS 4, DLBCL 1, MF 3) received KB004 in the DEP; an additional 14 patients were treated on the CEP (AML 8, MDS 6). The most common toxicities were transient grade 1 and grade 2 infusion reactions (IRs) in 79% of patients. IRs were dose limiting above 250mg. Sustained exposure exceeding the predicted effective concentration (1ug/mL) and covering the 7-day interval between doses was achieved above 190mg. Responses were observed in patients with AML, MF, MDS/MPN and MDS. In this study, KB004 was well tolerated and clinically active when given as a weekly infusion.

// Sara Ferluga 1 , Carla Maria Lema Tome 2 , Denise Mazess Herpai 1 , Ralph D’Agostino 3 , Waldemar Debinski 1 1 Department of Cancer Biology, Radiation Oncology and Neurosurgery, Brain Tumor Center of Excellence, Comprehensive Cancer Center of Wake Forest Baptist Medical Center, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA 2 Department of Neurobiology and Anatomy, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA 3 Department of Biostatistical Sciences, Section on Biostatistics, Wake Forest University Health Sciences, Winston-Salem, NC, 27157, USA Correspondence to: Waldemar Debinski, email: debinski@wakehealth.edu Keywords: Eph receptors, ephrin-A5, glioblastoma, cytotoxin, molecular targeting Received: October 12, 2015 Accepted: July 16, 2016 Published: August 1, 2016 ABSTRACT Eph tyrosine kinase receptors are frequently overexpressed and functional in many cancers, and they are attractive candidates for targeted therapy. Here, we analyzed the expression of Eph receptor A3, one of the most up-regulated factors in glioblastoma cells cultured under tumorsphere-forming conditions, together with EphA2 and EphB2 receptors. EphA3 was overexpressed in up to 60% of glioblastoma tumors tested, but not in normal brain. EphA3 was localized in scattered areas of the tumor, the invasive ring, and niches near tumor vessels. EphA3 co-localized with macrophage/leukocyte markers, suggesting EphA3 expression on tumor-infiltrating cells of bone marrow origin. We took advantage of the fact that ephrinA5 (eA5) is a ligand that binds EphA3, EphA2 and EphB2 receptors, and used it to construct a novel targeted anti-glioblastoma cytotoxin. The eA5-based cytotoxin potently and specifically killed glioblastoma cells with an IC 50 of at least 10 -11 M. This and similar cytotoxins will simultaneously target different compartments of glioblastoma tumors while mitigating tumor heterogeneity.

Background This study aimed to preliminarily assess the relationship between erythropoietin‐producing hepatocellular carcinoma receptor A3 (EphA3) and androgen receptor (AR) protein expression levels and prognosis in prostate cancer (PCa) to better understand the role of EphA3 in the prognosis and progression of PCa. Materials We investigated the expression of EphA3 and AR in human PCa by immunohistochemistry. Results EphA3 and AR were both significantly upregulated in PCa, with expression mainly localized to the nucleus. A high level of AR expression was found in 48.4% of 64 tumor samples, which was significantly more than in the adjacent tissue samples (15.6%) (P

Eph receptor tyrosine kinases are critical for cell‑cell communication during normal and oncogenic development. Eph receptor A3 (EphA3) expression is associated with tumor promotion in certain types of cancer; however, it acts as a tumor suppressor in others. The expression levels of EphA3 and its effects on tumor progression in esophageal squamous cell carcinoma (ESCC) cell lines were determined using reverse transcription‑quantitative polymerase chain reaction analysis and a Transwell invasion assay. The present study demonstrated that EphA3 expression was decreased in ESCC tissues and cell lines. Treatment with the DNA methylation inhibitor 5‑aza‑2'‑deoxycytidine increased the mRNA expression levels of EphA3 in the ESCC cell lines KYSE510 and KYSE30. In addition, overexpression of EphA3 in KYSE450 and KYSE510 cells inhibited cell migration and invasion. EphA3 overexpression also decreased RhoA GTPase. Furthermore, EphA3 overexpression induced mesenchymal‑epithelial transition, as demonstrated by epithelial‑like morphological alterations, increased expression of epithelial proteins (E‑cadherin and the tight junction protein 1 zonula occludens‑1) and decreased expression of mesenchymal proteins (Vimentin, N‑cadherin and Snail). Conversely, silencing EphA3 in KYSE410 cells triggered epithelial‑mesenchymal transition, and promoted cell migration and invasion. These results suggested that EphA3 may serve a tumor‑suppressor role in ESCC.

Perineuronal nets (PNNs) are implicated in closure of critical periods of synaptic plasticity in the brain, but the molecular mechanisms by which PNNs regulate synapse development are obscure. A receptor complex of NCAM and EphA3 mediates postnatal remodeling of inhibitory perisomatic synapses of GABAergic interneurons onto pyramidal cells in the mouse frontal cortex necessary for excitatory/inhibitory balance. Here it is shown that enzymatic removal of PNN glycosaminoglycan chains decreased the density of GABAergic perisomatic synapses in mouse organotypic cortical slice cultures. Neurocan, a key component of PNNs, was expressed in postnatal frontal cortex in apposition to perisomatic synapses of parvalbumin-positive interneurons. Polysialylated NCAM (PSA-NCAM), which is required for ephrin-dependent synapse remodeling, bound less efficiently to neurocan than mature, non-PSA-NCAM. Neurocan bound the non-polysialylated form of NCAM at the EphA3 binding site within the immunoglobulin-2 domain. Neurocan inhibited NCAM/EphA3 association, membrane clustering of NCAM/EphA3 in cortical interneuron axons, EphA3 kinase activation, and ephrin-A5-induced growth cone collapse. These studies delineate a novel mechanism wherein neurocan inhibits NCAM/EphA3 signaling and axonal repulsion, which may terminate postnatal remodeling of interneuron axons to stabilize perisomatic synapses in vivo.

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

EphA3 is a member of Eph receptors, which is involved in tumorigenesis. The expression and clinical significance of EphA3 in colorectal cancer (CRC) have not been fully investigated. Four colon cancer cell lines and a set of CRC tissues were examined for EphA3 expression. The methylation status of a CpG island within the EphA3 promoter, the presence of four somatic EPHA3 mutations, and EPHA3 gene copy number variations were also analyzed in colon cancer cell lines. EphA3 expression was lost in all colon cancer cell lines examined. EphA3 expression was lower in tumor tissues when compared with normal intestinal tissues (P

During neuromuscular junction maturation and repair, ephrin-A3 is expressed by all slow (Type I) fibers and only by slow fibers, and inhibits their innervation by fast motor axons via repulsive interactions with the ephrin-A3 receptor EphA8., Each adult mammalian skeletal muscle has a unique complement of fast and slow myofibers, reflecting patterns established during development and reinforced via their innervation by fast and slow motor neurons. Existing data support a model of postnatal "matching" whereby predetermined myofiber type identity promotes pruning of inappropriate motor axons, but no molecular mechanism has yet been identified. We present evidence that fiber type–specific repulsive interactions inhibit innervation of slow myofibers by fast motor axons during both postnatal maturation of the neuromuscular junction and myofiber reinnervation after injury. The repulsive guidance ligand ephrin-A3 is expressed only on slow myofibers, whereas its candidate receptor, EphA8, localizes exclusively to fast motor endplates. Adult mice lacking ephrin-A3 have dramatically fewer slow myofibers in fast and mixed muscles, and misexpression of ephrin-A3 on fast myofibers followed by denervation/reinnervation promotes their respecification to a slow phenotype. We therefore conclude that Eph/ephrin interactions guide the fiber type specificity of neuromuscular interactions during development and adult life.

Eph receptor tyrosine kinases and their ephrin ligands, mediate an important cell communication system both in normal and oncogenic development, and play central roles in a series of processes including angiogenesis, stem cell maintenance and cancer metastasis. Eph receptor A3 (EphA3), commonly overexpressed in a broad range of cancers, including gastric cancer (GC), is related to tumor progression. Our previous study revealed that EphA3 may play important roles in tumorigenesis and angiogenesis in GC. However, its exact role and the mechanisms underlying its function in GC remain unclear. In the present study, lentivirus‑mediated RNA interference was employed to knock down the expression of EphA3 in GC HGC‑27 cells. Functional analyses indicated that depletion of EphA3 expression inhibited the cell growth and tumorigenicity of HGC‑27 cells in vitro and in vivo. Furthermore, knockdown of the expression of EphA3 in HGC‑27 cells inhibited tube formation and migration of HUVEC endothelial cells. Tumor angiogenesis in vivo was also inhibited upon EphA3 knockdown in HGC‑27 cells, with reduced microvessel density (MVD) in xenograft models. We further revealed that EphA3 depletion inhibited tumor angiogenesis and migration through the signal transducer and activator of transcription 3/vascular endothelial growth factor (STAT3/VEGF) signaling pathway. These results indicated that EphA3 may be an effective prognostic indicator and a potential target for GC therapy.

Erythropoietin‑producing hepatocellular carcinoma cell surface type‑A receptor 3 (EPHA3) has been found to promote the proliferation and survival of prostate cancer (PCa) cell lines and prostate tumor development in nude mice. However, the regulation of EPHA3 in PCa remains largely unknown. This study is aimed to investigate the association between EPHA3 expression and androgen receptor (AR) signaling and the potential mechanism. We determined mRNA and protein levels of EPHA3 and AR signaling‑related genes in the PCa cell line 22Rv1 by reverse transcription‑polymerase chain reaction (RT‑PCR) and western blotting, respectively. The EPHA3 mRNA and protein levels were both found to be elevated by dihydrotestosterone (DHT) hormone in a dose‑ and time‑dependent manner, as AR and prostate‑specific antigen (PSA) expression were increased. Similarly, EPHA3 protein levels were also increased in the PCa cell line LNCaP stimulated with DHT or mibolerone (Mib). Overexpression of pEGFP‑AR in 22Rv1 cells significantly increased the EphA3 level, while AR knockdown with small interfering RNA (siRNA) for AR (siAR) markedly decreased the expression of EPHA3. The key EPHA3 promoter region associated with AR regulation was evaluated by co‑transfection of various pGL3‑basic‑luciferase reporter plasmids, containing EPHA3 core promoter fragments differing in length, with the AR plasmid or siAR into 22Rv1 cells. AR overexpression in 22Rvl cells raised the EphA3 promoter transcription activity of pGL3‑EPHA3‑Luc (EPHA3‑Luc)‑789, and vice versa. Similarly, luciferase activity of EPHA3‑Luc‑317 was also clearly affected. However, truncated EPHA3‑Luc‑237 without the transcription factor specific protein 1 (SP1) binding sites or EPHA3‑Luc‑789ΔSP1 with modified SP1 binding sites clearly decreased EPHA3 promoter activity regardless of whether AR was overexpressed or blocked. Treatment of 22Rv1 cells with 10 and 100 nM of the SP1 inhibitor mithramycin A for 24 and 48 h significantly reduced EPHA3 mRNA and protein levels. Additionally, selective inhibition of SP1 with siRNA SP1 (siSP1) at various concentration from 25 to 75 nM, reduced the EPHA3 protein level in PCa LNCaP cells, accordingly. Co‑immunoprecipitation (co‑IP) and chromatin IP (ChIP) assays were performed to determine whether AR forms a transcription factor complex with Sp1 that binds the EPHA3 core promoter region to sense androgen induction. The result suggests that the interaction of AR and SP1 contributes to regulate EPHA3 expression, and the SP1 binding sites (‑295~‑261) in the EPHA3 core promoter region is crucial to the regulation of EPHA3 expression in response to androgen hormone stimuli.

Throughout the visual system, different subtypes of neurons are tuned to distinct aspects of the visual scene, establishing parallel circuits. Defining the mechanisms by which such tuning arises has been a long-standing challenge for neuroscience. To investigate this, we have focused on the retina’s projection to the superior colliculus (SC), where multiple visual neuron subtypes have been described. The SC receives inputs from a variety of retinal ganglion cell (RGC) subtypes; however, which RGCs drive the tuning of different SC neurons remains unclear. Here, we pursued a genetic approach that allowed us to determine the tuning properties of neurons innervated by molecularly defined subpopulations of RGCs. In homozygous Islet2-EphA3 knock-in (Isl2EA3/EA3) mice, Isl2+ and Isl2− RGCs project to non-overlapping sub-regions of the SC. Based on molecular and anatomic data, we show that significantly more Isl2− RGCs are direction-selective (DS) in comparison with Isl2+ RGCs. Targeted recordings of visual responses from each SC sub-region in Isl2EA3/EA3 mice revealed that Isl2− RGC-innervated neurons were significantly more DS than those innervated by Isl2+ RGCs. Axis-selective (AS) neurons were found in both sub-regions, though AS neurons innervated by Isl2+ RGCs were more tightly tuned. Despite this segregation, DS and AS neurons innervated by Isl2+ or Isl2− RGCs did not differ in their spatial summation or spatial frequency (SF) tuning. Further, we did not observe alterations in receptive field (RF) size or structure of SC neurons innervated by Isl2+ or Isl2− RGCs. Together, these data show that innervation by Isl2+ and Isl2− RGCs results in distinct tuning in the SC and set the stage for future studies investigating the mechanisms by which these circuits are built.

MicroRNAs (miRNAs) play key roles in cervical cancer metastasis progression. Accumulated evidences have revealed that miRNAs are related to the pathophysiological process. However, the role of miR-340 in cervical cancer and how it works is still not fully interpreted. Using qRT-PCR to examine the expression of miR-340 in cervical cancer tissues. Transwell migration and invasion experiments were used to detect the role of miR-340 in migration and invasion. Luciferase reporter assay, qRT-PCR, and Western blot were used to detect the relationship between miR-340 and EphA3. Using Transwell migration and invasion experiments to investigate the role of EphA3 on migration and invasion. Restoration expriments were also performed. Western blot was used to assay the influence of miR-340 and EphA3 on EMT. We found that miR-340 was downregulated in cervical cancer tissues compared with the normal tissues. Transwell migration and invasion experiments indicated that overexpression of miR-340 frequently inhibited the migration and invasion of cervical cancer cells. EphA3 is a target of miR-340, and ectopic expression of EphA3 can promote the migration and invasion of cervical cancer cells, whereas restoration of EphA3 in miR-340-overexpressing cervical cancer cells reversed the suppressive effects of miR-340. What's more, the process of migration and invasion which regulated by miR-340/EphA3 was depended on adjusting the EMT way. These findings indicate that miR-340 may act as an anti-tumor factor during the process of tumor metastasis through targeting EphA3, suggesting that miR-340 might be a potential new diagnostic and therapeutic molecule for the treatment of cervical cancer.

Genetic variants are implicated in the development of amyotrophic lateral sclerosis (ALS), but it is unclear whether the burden of rare variants in ALS genes has an effect on survival. We performed whole genome sequencing on 8 familial ALS (FALS) patients with superoxide dismutase 1 (SOD1) mutation and whole exome sequencing on 46 sporadic ALS (SALS) patients living in Hong Kong and found that 67% had at least 1 rare variant in the exons of 40 ALS genes; 22% had 2 or more. Patients with 2 or more rare variants had lower probability of survival than patients with 0 or 1 variant (p = 0.001). After adjusting for other factors, each additional rare variant increased the risk of respiratory failure or death by 60% (p = 0.0098). The presence of the rare variant was associated with the risk of ALS (Odds ratio 1.91, 95% confidence interval 1.03–3.61, p = 0.03), and ALS patients had higher rare variant burden than controls (MB, p = 0.004). Our findings support an oligogenic basis with the burden of rare variants affecting the development and survival of ALS.

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 is expressed in solid tumors and leukemias and is an attractive target for the therapy. We have generated a panel of Humaneered® antibodies to the ligand-binding domain using a Fab epitope-focused library that has the same specificity as monoclonal antibody mIIIA4. A high-affinity antibody was selected that competes with the mIIIA4 antibody for binding to EphA3 and has an improved affinity of ∼1 nM. In order to generate an antibody with potent cell-killing activity the variable regions were assembled with human IgG1k constant regions and expressed in a Chinese hamster ovary (CHO) cell line deficient in fucosyl transferase. Non-fucosylated antibodies have been reported to have enhanced binding affinity for the IgG receptor CD16a (FcγRIIIa). The affinity of the antibody for recombinant CD16a was enhanced approximately 10-fold. This resulted in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity against EphA3-expressing leukemic cells, providing a potent antibody for the evaluation as a therapeutic agent.

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.

The lack of axonal regeneration in the adult central nervous system is in part attributable to the presence of inhibitory molecules present in the environment of injured axons such as the myelin-associated proteins Nogo-A and MAG and the repulsive guidance molecules Ephrins, Netrins and Semaphorins. In the present study, we hypothesized that EphA4 and one of its potential binding partners EphrinA3 may participate in the inhibition of adult axon regeneration in the model of adult mouse optic nerve injury. Axonal regeneration was analysed in three dimensions after tissue clearing of EphA4 knockout (KO), EphrinA3 KO and wild-type (WT) optic nerves. By immunohistochemistry, EphA4 was highly expressed in Müller glia endfeet in the retina and in astrocytes in the retina and the optic nerve, while EphrinA3 was present in retinal ganglion cells and oligodendrocytes. Optic nerve crush did not cause expression changes. Significantly more axons grew in the crushed optic nerve of EphA4 KO mice than in WT or EphrinA3 KO animals. Single axon analysis revealed that EphA4 KO axons were less prone to form aberrant branching than axons in the other mouse groups. The expression of growth-associated proteins Sprr1a and Gap-43 did not vary between EphA4 KO and WT retinae. However, glial fibrillary acidic protein-expressing astrocytes were withdrawn from the perilesional area in EphA4 KO, suggesting that gliosis down-regulation may locally contribute to improve axonal growth at the injury site. In summary, our three-dimensional analysis of injured mouse optic nerves reveals beneficial effects of EphA4 ablation on the intensity and the pattern of optic nerve axon regeneration.

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.

Although EphA3 expression has been associated with progression or prognosis in several types of tumors, the role of EphA3 in hepatocellular carcinoma (HCC) remains unknown. This study sought to investigate the clinicopathological and prognostic relevance of EphA3 expression in HCC as well as the underlying mechanisms responsible. EphA3 protein was mainly localized within the cytoplasm and at the cell membrane. High EphA3 expression was correlated with tumor size, tumor grade, metastasis, venous invasion and AJCC TNM stage (P

Refereed/Peer-reviewed

Ephrin Type-A Receptor 3 (EphA3) belongs to the ephrin receptor subfamily of the protein tyrosine kinase family, and plays an important role in embryogenesis and neurogenesis. This study aimed to investigate the role of EphA3 in promoting malignant transformation of colorectal epithelial cells, and explore underlying molecular mechanisms. Colorectal cancer tissue specimens from 68 patients were analyzed for EphA3 expression. EphA3 expression levels were manipulated in rat colon epithelial cell lines. We found that EphA3 expression level in tumor tissues was associated with patient age (P = 0.015), tumor differentiation (P = 0.001), and lymph node metastasis (P = 0.039). Overexpression of EphA3 and its constitutively active mutants promoted colony formation, migration and invasion, and tumorigenicity of colon epithelial cells in nude mice. The cDNA and lncRNA microarray profiling data revealed that differentially expressed genes and lncRNAs in EphA3 or mutant-transfected cells were associated with cell proliferation, invasion and angiogenesis. Our findings reveal the mechanisms underlying the oncogenic activities of EphA3 in colorectal cells, which could provide novel targets for the prevention, early diagnosis, and treatment of colorectal cancer.

Made available in DSpace on 2018-11-26T15:29:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-02-01 John and Kibble Trust grant Dr William Peter Richards Bequest for Research in Veterinary Pathology 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 hyper plastic 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. (C) 2016 Elsevier Ltd. All rights reserved. Univ Queensland, Sch Vet Sci, Gatton Campus, Gatton, Qld, Australia Univ Estadual Paulista, Sch Vet Med & Anim Sci, Sao Paulo, Brazil Univ Estadual Paulista, Sch Vet Med & Anim Sci, Sao Paulo, Brazil

Cancer initiation and malignant progression are multistep processes that involve loss of growth control, evasion of apoptosis, sustained angiogenesis, tissue invasion, and metastasis (1). Developing cancer cells stochastically acquire and selectively accumulate mutations in the genes that encode oncoproteins, tumor suppressors, and their regulators. Receptor tyrosine kinases (RTKs) are important regulators of signal transduction pathways that promote cell growth, survival, invasion, and motility during malignant progression of solid tumors (2). Dysregulation of RTKs, such as EGF receptor family members, by mutation, amplification, or overexpression, can result in increased kinase activity and ultimately oncogenic transformation. The generality of this paradigm of gain-of-function RTK signaling in cancer has been recently challenged by the discovery of the dual roles of the EPH receptors in both promoting and inhibiting oncogenesis and tumor progression in cell lines and mouse models. To date, there has been insufficient evidence from patient outcome-based studies to verify the findings from mechanistic analyses in tumor models. EPH receptors and their membrane-bound ligands, the ephrins, were originally discovered in the 1990s as axonal guidance molecules, and since that time, the EPH proteins have been found to constitute the largest family of RTKs and to be key regulators of cell–cell communication both in development and disease (3,4). The role of EPH receptors in cancer models is complex; they can either promote or inhibit malignancy, depending on ligand stimulation, signaling cross-talk, and other contextual factors (5–8). For example, EPHA2 overexpression is associated with worsened survival in human breast, prostate, and lung cancers and in glioblastoma multiforme (9–17). Overexpression of EPHA2 can induce ligand-independent signaling, resulting in increased tumor cell malignancy in vitro and accelerated tumor growth and metastasis in vivo (18,19). In keeping with these findings, reduced EPHA2 expression in the presence of short interfering RNA or targeted gene deletion inhibited tumor initiation and metastatic progression (19–21). However, ligand-dependent signaling by EPHA2 in both breast cancer and glioblastoma cell lines inhibited their malignant behavior in vitro and tumor growth in vivo (21,22). The conundrum posed by these findings is not completely resolved, and mechanisms that account for these opposing activities are just beginning to be investigated. Recently, “next-generation” DNA sequencing using large cohorts of human lung cancer samples identified various mutations in EPH receptor genes. Notably, somatic mutations in EPHA3, the gene for EPHA3, were present in 5% to 10% of lung adenocarcinomas (23–26). However, those mutations are associated with amino acid substitutions scattered throughout the receptor, and it is unclear whether these nonrecurrent mutations are “driver” or biologically neutral “passenger” genetic alterations. In addition, because EPH receptor signaling can cause either tumor promotion or tumor suppression, the biological impact of somatically mutated variants of EPHA3 in lung cancer remains unclear. In this report, we sought to characterize the functional effects of EPHA3 mutations identified in primary tumors to distinguish between the oncogenic and tumor-suppressive roles of the protein that EPHA3 encodes. We used a combination of genomic and mutational analyses in cell lines and tumor specimens to investigate the role of EPHA3 and its somatic mutations in non–small cell lung cancer (NSCLC). We performed cell proliferation and apoptosis assays in NSCLC cell lines and used mouse xenograft models to assess the function of EPHA3 in vivo. Finally, we identified molecular mechanisms by which EPHA3 regulates tumorigenicity.

EphA3, a member of the Eph receptor tyrosine kinases, plays important roles in tumor angiogenesis and progression. However, the function of EphA3 in solid tumors has not been widely studied. We aimed to explore EphA3 expression in gastric carcinoma and analyze its role as a potential prognostic factor. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess EphA3 mRNA in a normal gastric mucosa cell line and carcinoma cell lines. Immunohistochemistry for EphA3 and vascular endothelial growth factor (VEGF) was performed in 318 cases of gastric carcinoma. CD34 immunohistochemical staining was used for microvessel density (MVD) counting. Western blotting was used to analyze EphA3 expression in the cell lines and to determine the expression of EphA3 and VEGF in 75 cases of gastric carcinoma and matched normal mucosa. EphA3 mRNA and protein expression was significantly higher in gastric cancer than that in normal mucosa (all P

Background: EphA3 is a component of the Eph receptor family, the largest subgroup of the receptor tyrosine kinase (RTK) family. A recent array-based study implicated the presence of copy-number variations (CNVs) of EphA3 in the genomes of acute myelogenous leukemia. CNVs are present in the general population at varying degrees, and have been found to associate with various types of diseases including hematologic malignancies. However, most of the current studies focused on the genome-wide screening of CNVs, and the functional impact of such regions needs to be extensively investigated in large number of clinical samples. Patients and Methods: In our study, we collected 617 bone marrow samples from multiple types of hematologic malignancies as well as healthy controls. DNA copy numbers and mRNA levels of EphA3 in these samples were examined. Results: We found significant association between the CNVs of EphA3 and these hematologic malignancies including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), and myelodysplastic syndrome (MDS). We also observed a positive correlation between the relative mRNA level and gene dosage of EphA3. Conclusion: The CNVs of EphA3 were associated with multiple types of hematologic malignancies including ALL, AML, CLL, CML, MM, and MDS.

Deficiency of Smad3, an intracellular mediator of TGF-β, was shown to significantly accelerate re-epithelialization of the colonic mucosa. This study was performed to investigate the molecular mechanisms by which Smad3 controls colonic epithelial cell proliferation and crypt formation. Smad3(ex8/ex8) C57BL/6 mice were used in this study and wild-type littermates served as controls. The number of proliferating cells in the isolated colonic epithelium of Smad3(-/-) mice was significantly increased compared to that in wild-type littermates. Protein levels of the cell cycle inhibitors p21 and p27 were significantly decreased, while that of c-Myc was increased in the isolated colonic epithelium from Smad3(-/-) mice. In the colonic tissue of wild-type mice, cell proliferation was restricted to the bottom of the crypts in accordance with nuclear β-catenin staining, whereas proliferating cells were located throughout the crypts in Smad3(-/-) mice in accordance with nuclear β-catenin staining, suggesting that Smad3 is essential for locating proliferating cells at the bottom of the colonic crypts. Notably, in Smad3(-/-) mice, there was loss of EphB2 and EphB3 receptor protein expression, critical regulators of proliferating cell positioning, while EphB receptor protein expression was confirmed at the bottom of the colonic crypts in wild-type mice. These observations indicated that disturbance of the EphB/ephrin B system brings about mispositioning of proliferating cells in the colonic crypts of Smad3(-/-) mice. In conclusion, Smad3 is essential for controlling number and positioning of proliferating cells in the colonic crypts and contributes to formation of a "proliferative zone" at the bottom of colonic crypts in the normal colon.

SummaryIt is a long-standing question how developing motor and sensory neuron projections cooperatively form a common principal grid of peripheral nerve pathways relaying behavioral outputs and somatosensory inputs. Here, we explored this issue through targeted cell lineage and gene manipulation in mouse, combined with in vitro live axon imaging. In the absence of motor projections, dorsal (epaxial) and ventral (hypaxial) sensory projections form in a randomized manner, while removal of EphA3/4 receptor tyrosine kinases expressed by epaxial motor axons triggers selective failure to form epaxial sensory projections. EphA3/4 act non-cell-autonomously by inducing sensory axons to track along preformed epaxial motor projections. This involves cognate ephrin-A proteins on sensory axons but is independent from EphA3/4 signaling in motor axons proper. Assembly of peripheral nerve pathways thus involves motor axon subtype-specific signals that couple sensory projections to discrete motor pathways.