Eph Receptor Tyrosine Kinases Research Articles

Abstract LB-12: EphA2 as a diagnostic imaging target in glioblastoma: A PET/MRI study

Abstract Neuroimaging has evolved from being a purely anatomical tool to a discipline that can offer anatomical, functional, hemodynamic, metabolic and molecular information. From the perspective of patients with glioblastoma multiforme (GBM), the use of neuroimaging techniques is an essential stage in diagnosis and classification of the tumour and to assess patient response following treatment.[1] The Eph receptor tyrosine kinases (RTKs) and their Eph-interacting ligands (ephrins) are the largest family of RTKs consisting of 16 Eph receptors and 9 ephrin ligands.[2] From the discovery of the first Eph RTK evidence has been presented to implicate the Eph family in tumour development and progression, a body of work which continues to grow. The complex bidirectional signaling mechanisms between Eph RTKs and ephrin ligands have been shown to influence cell morphology, adhesion, migration, invasion, cell proliferation and survival.[2-4] Recently, many studies have linked Eph and ephrin expression levels with tumour progression and metastatic spread and, as a consequence, patient survival. In the specific case of EphA2, an increasing number of studies have shown a correlation between EphA2 overexpression and poor patient prognosis.[5,6] As such, EphA2 represents an attractive target for highly specific PET imaging using a labeled monoclonal antibody (mAb). In this study we present simultaneous PET/MRI of a 64Cu labeled mAb specific to the human form of EphA2. We show efficient targeting of the tumour in three intracranial mouse models of GBM. In addition, we show that non-specific binding can be significantly reduced by blocking removal of the mAb from the blood pool via Fc receptor binding. In all cases we compare the ability of the mAb to delineate tumour boundaries with the current clinical imaging techniques of 18F-FDOPA PET imaging and gadolinium (Gd) contrast enhanced MRI. [1] Henson, J. W.; Gonzalez, R. G. Expert review of neurotherapeutics 2004, 4. [2] Pasquale, E. B. Nature Reviews Cancer 2010, 10, 165. [3] Pasquale, E. B. Cell 2008, 133, 38. [4] Pasquale, E. B. Nat Rev Mol Cell Bio 2005, 6, 462. [5] Lackmann, M.; Boyd, A. W. Science Signaling 2008, 1. [6] Zhuang, G. L. et al. Cancer Res 2010, 70, 299. Citation Format: Simon Puttick, Brett W. Stringer, Bryan W. Day, Karine Mardon, Gary J. Cowin, Michael Fay, Kristofer J. Thurecht, Andrew K. Whittaker, Andrew W. Boyd, Stephen Rose. EphA2 as a diagnostic imaging target in glioblastoma: A PET/MRI study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-12. doi:10.1158/1538-7445.AM2014-LB-12

Abstract 3819: Clinical potential of the Eph/ephrin profile in breast cancer

Abstract The Eph receptors belong to a large family of receptors tyrosine kinase. The Ephs and their ligands ephrins are aberrantly expressed in cancer. However, due their complex signaling and promiscuous interactions, a wide screening of the entire family will be important to determine their clinical value. Purpose: The Eph receptor tyrosine kinase family has been implicated in breast cancer but their role in tumor progression remains elusive. Therefore we aimed to profile the Eph receptors and their ligands ephrins in breast cancer samples to define them as potential clinical markers or therapeutic targets. Experimental design: Gene expression of Eph/ephrins was quantified by RT-PCR using a TaqMan® Micro Fluidics Cards Microarray custom designed for 21 Eph/ephrin family members: EphA1-A8, EphB1-B4 and EphB6, ephrinA1-A5, ephrinB1-B3 and 2 endogenous controls: GAPDH and HPRT1 in 70 breast cancer tumors. Results: We found that high expression of EphBs, and particularly EphB2, was associated to worse outcome among postmenopausal patients with lymph node infiltration. Additionally high EphB2 expression correlated with HER2 positive status. Conclusions: Our results suggest that the EphB receptor family may have prognostic value for postmenopausal breast cancer patients with lymph nodal infiltration. We will continue this study in a larger patient material in order to better evaluate the therapeutic impact of this family. Citation Format: Gizeh Perez-Tenorio, Anna-Maria Husa, Olle Stål. Clinical potential of the Eph/ephrin profile in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3819. doi:10.1158/1538-7445.AM2014-3819

UniPR129 is a competitive small molecule Eph‐ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations

The Eph receptor tyrosine kinases and their ephrin ligands are key players in tumorigenesis and many reports have correlated changes in their expression with a poor clinical prognosis in many solid tumours. Agents targeting the Eph-ephrin system might emerge as new tools useful for the inhibition of different components of cancer progression. Even if different classes of small molecules targeting Eph-ephrin interactions have been reported, their use is hampered by poor chemical stability and low potency. Stable and potent ligands are crucial to achieve robust pharmacological performance. UniPR129 (the L-homo-Trp conjugate of lithocholic acid) was designed by means of computational methods, synthetized and tested for its ability to inhibit the interaction between the EphA2 receptor and the ephrin-A1 ligand in an elisa binding study. The ability of UniPR129 to disrupt EphA2-ephrin-A1 interaction was functionally evaluated in a prostate adenocarcinoma cell line and its anti-angiogenic effect was tested in vitro using cultures of HUVECs. UniPR129 disrupted EphA2-ephrin-A1 interaction with Ki = 370 nM in an elisa binding assay and with low micromolar potency in cellular functional assays, including inhibition of EphA2 activation, inhibition of PC3 cell rounding and disruption of in vitro angiogenesis, without cytotoxic effects. The discovery of UniPR129 represents not only a major advance in potency compared with the existing Eph-ephrin antagonists but also an improvement in terms of cytotoxicity, making this molecule a useful pharmacological tool and a promising lead compound.

Effects of RNA interference targeting Eph receptor tyrosine kinase A7 gene on the biological behavior of SMMC-7721 hepatoma cell line in vitro

Objective To investigate the effects of RNA interference targeting Eph receptor tyrosine kinase A7 (EphA7) gene on the biological behavior of SMMC-7721 hepatoma cell line.Methods Recombinant plasmid of EphA7 gene-targeting small interfering RNA (siRNA) was transfected into liver cancer SMMC-7721 cells by LipofectamineTM 2000.The expression of EphA7 gene mRNA and protein in SMMC-7721 cells transfected with pRNA-siEphA7 was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting.Methyl thiazol tetrazolium (MTT) assay was used to detect the proliferative activity of the transfected SMMC-7721 cells.The changes of cell cycle after transfection were detected by flow cytometry.Transwell chamber invasion assay was used to measure the changes of invasion and migration abilities of SMMC-7721 cells after transfection.Results As compared with empty vector transfected group (0.4293 ± 0.0533,0.5822 ± 0.0291) and untransfected group (0.431 1 ± 0.041 0,0.583 2 ±0.015 4),the expression of EphA7 mRNA (0.191 1 ±0.015 3,0.148 8 ±0.019 9,0.087 4 ± 0.012 3) and protein (0.278 6 ± 0.039 2,0.301 1 ± 0.037 2,0.214 2 ± 0.014 7) in transfected group was remarkably suppressed (P ＜ 0.05).MTT assay showed that the proliferation rate in transfected group was lower than in empty vector transfected group and untransfected group (P ＜ 0.05).Flow cytometry results revealed that among the transfected group,empty vector transfected group and untransfected group,there was no significant difference in the proportion of cells in G1,G2 and S phases (P ＞ 0.05).Transwell chamber invasion assay demonstrated that the amount of cells penetrating through the membrane in transfected group (27.00 ±5.57,13.40 ±3.43,23.60 ±3.65) was less than that in the empty vector transfected group (68.6 ± 6.50) and untransfected group (72.00 ± 9.30,P ＜ 0.05).Conclusion The down-regulation of EphA7 gene can reduce the proliferation activity,and weaken the invasive ability in vitro of SMMC-7721 cell.EphA7 may play an important role in the invasion and metastasis progression of hepatocellular carcinoma. Key words: Carcinoma, hepatocellular; Transfection; RNA interference; Eph receptor tyrosine kinase A7; Gene threapy

4-Substituted quinazoline derivatives as novel EphA2 receptor tyrosine kinase inhibitors

Erythropoietin-producing hepatocellular receptor tyrosine kinase subtype A2 (EphA2) is an attractive therapeutic target for suppressing tumor progression. In our efforts to discover novel small molecules to inhibit EphA2, a class of compound based on 4-substituted quinazoline containing 7-(morpholin-2-ylmethoxy) group was identified as a novel hit by high throughput screening campaign. Structural modification of parent quinazoline scaffolds by introducing substituents on aniline displayed potent inhibitory activities toward EphA2.

Altered distribution of the EphA4 kinase in hippocampal brain tissue of patients with Alzheimer’s disease correlates with pathology

Synaptic dysfunction occurs early in the progression of Alzheimer’s disease (AD) and correlates with memory decline. There is emerging evidence that deregulation of Erythropoietin-producing hepatocellular (Eph) receptor tyrosine kinases (RTK) signaling contributes to the aberrant synaptic functions associated with neurodegeneration. The Eph receptor A4 is highly expressed in human adult hippocampal brain tissue and was previously linked to cognitive impairment in a transgenic mouse model for AD. Whether EphA4 levels are altered in AD brain remains elusive. Therefore we investigated the protein levels and localization of EphA4 in human hippocampus derived from AD (n = 29) as well as non-demented control cases (n = 19).The total EphA4 protein levels were not changed in AD patients compared to control cases. However, immunohistochemical localization of EphA4 revealed an altered distribution in AD compared to control hippocampus. EphA4 immunoreactivity was observed in plaque-like structures in AD cases. Double-labelling with phosphorylated tau and amyloid beta indicates that EphA4 co-localizes with neuritic plaques in AD. This altered distribution pattern was observed at early stages (Braak stage II) and correlates with the hallmarks of AD pathology suggesting a reduced availability of EphA4 that is likely to contribute to synaptic dysfunction that occurs early in AD.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0079-9) contains supplementary material, which is available to authorized users.

Lack of ephrin receptor A1 is a favorable independent prognostic factor in clear cell renal cell carcinoma.

The EPH receptor tyrosine kinases and their cell-bound ligands, the ephrins, have been shown to be associated with cancer development and progression. In this study, mRNA and protein expression of the receptors EPHA1 and EPHA2 as well as of their ligand EFNA1 and their prognostic relevance in clear cell renal cell carcinoma was evaluated. Gene expression was measured in 75 cryo-preserved primary tumors and matched non-malignant renal specimens by quantitative PCR. Protein expression was analyzed by immunohistochemistry on tissue microarrays comprising non-malignant, primary tumors and metastatic renal tissues of 241 patients. Gene and protein expression of all three factors was altered in tumor specimens with EPHA1 and EPHA2 being generally diminished in tumors compared to normal renal tissue, whereas EFNA1 was commonly elevated. A positive EPHA1 and EPHA2 protein staining as well as a low EFNA1 protein level were significantly linked to more aggressive tumor features, but only a positive EPHA1 immunoreactivity was significantly associated with poor survival. In subgroup analyses, EPHA1 and EPHA2 protein levels were significantly higher in metastatic than in primary lesions. Patients with EPHA1/EPHA2-positive tumors or with tumors with positive EPHA1 and low EFNA1 immunoreactivity had the shortest survival rates compared to the respective other combinations. In a multivariate model, EPHA1 was an independent prognostic marker for different survival endpoints. In conclusion, an impaired EPH-ephrin signaling could contribute to the pathogenesis and progression of clear cell renal cell carcinoma.

Corticofugal projections from medial primary somatosensory cortex avoid EphA7-expressing neurons in striatum and thalamus

Within the first two postnatal weeks, corticostriatal axons from the primary somatosensory cortex (S1) form topographic projections that organize into characteristic bands of axon terminals in the dorsolateral striatum. Molecules regulating the development of these topographically organized projections are currently unknown. Thus, the present study investigated whether EphA receptor tyrosine kinases, which regulate axonal guidance in the visual system via axon repulsion, could participate in the formation of corticostriatal connections during development. Prior studies indicate that EphA7-expressing striatal neurons are organized into banded compartments resembling the matrisome innervation pattern formed by cortical afferents from the S1 cortex and that ephrin-A5, a known EphA7 ligand, is expressed in a medial (high) to lateral (low) gradient in S1. Thus, we hypothesized that the organization of EphA7-expressing striatal neurons in banded domains provides a repulsive barrier preventing corticostriatal axons containing EphA7-ligands from innervating inappropriate regions of the striatum. To evaluate this, we injected the anterograde tracer, biotinylated dextran amine (BDA), into two locations in medial areas of S1 (the anterior and posterior whisker fields), which are reported to express high levels of ephrin-A5 during development. Injections were made in mouse pups on postnatal day 9 (P9) and the animals were processed for immunohistochemistry on P12. Our data demonstrate that projections from both the forelimb/anterior whisker field and the posterior whisker field avoid EphA7-expressing neurons and terminate in a banded pattern in regions with very low EphA7-expression. We also determined that corticothalamic projections from medial S1 also exhibit a restricted distribution in the thalamus and avoid neurons expressing EphA7. Thus, our results support the hypothesis that the anatomical organization of striatal and thalamic neurons expressing EphA7 receptors restricts the topographic distribution of cortical afferents from medial regions of S1 which express high levels of ephrin-A5.

Binding of the Kaposi's sarcoma-associated herpesvirus to the ephrin binding surface of the EphA2 receptor and its inhibition by a small molecule.

The ephrin receptor tyrosine kinase A2 (EphA2) is an entry receptor for Kaposi's sarcoma-associated herpesvirus (KSHV) that is engaged by the virus through its gH/gL glycoprotein complex. We describe here that natural ephrin ligands inhibit the gH/gL-EphA2 interaction. The effects of point mutations within EphA2 demonstrated that KSHV gH/gL interacts with EphA2 through a restricted set of the same residues that mediate binding of A-type ephrins. Two previously described inhibitors of the EphA2 interaction with ephrin A5 also inhibited binding of KSHV gH/gL to EphA2. The more potent of the two compounds inhibited KSHV infection of blood vessel and lymphatic endothelial cells in the micromolar concentration range. Our results demonstrate that interaction of KSHV with EphA2 occurs in a fashion similar to that of the natural ephrin ligands. Our data further indicate a new avenue for drug development against KSHV. Our study reports two important findings. First, we show that KSHV engages its receptor, the receptor tyrosine kinase EphA2, at a site that overlaps the binding site of the natural ephrin ligands. Second, we demonstrate that KSHV infection of target cells can be blocked by a small-molecule inhibitor of the viral glycoprotein-EphA2 interaction. These findings represent a novel avenue for the development of strategies to treat KSHV-associated diseases.

Eph receptor tyrosine kinases in cancer stem cells

Eph receptor tyrosine kinases (RTKs) and their ligands, ephrins, play critical roles in development, tissue homeostasis, and cancer. Because Eph receptors are expressed in most adult stem cell niches and in many types of cancers, it has been long suspected that this family of RTKs may also regulate the function of cancer stem-like cells (CSCs). This review will focus on recent studies to elucidate the contribution of Eph/ephrin molecules in CSC self-renewal and tumorigenicity, as well as describe efforts to target these molecules in cancer. Because CSCs are often resistant to therapeutic intervention and have been shown to depend on Eph RTKs for self-renewal, targeting Eph receptors may hold promise for the treatment of drug-resistant cancers.

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L(535)X3G(539)X2A(542)X3V(546)X2L(549) rather than through the alternative glycine zipper motif A(536)X3G(540)X3G(544) (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr(588) and/or Tyr(594)) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.

JNK Signaling Mediates EPHA2-Dependent Tumor Cell Proliferation, Motility, and Cancer Stem Cell–like Properties in Non–Small Cell Lung Cancer

Recent genome-wide analyses in human lung cancer revealed that EPHA2 receptor tyrosine kinase is overexpressed in non-small cell lung cancer (NSCLC), and high levels of EPHA2 correlate with poor clinical outcome. However, the mechanistic basis for EPHA2-mediated tumor promotion in lung cancer remains poorly understood. Here, we show that the JNK/c-JUN signaling mediates EPHA2-dependent tumor cell proliferation and motility. A screen of phospho-kinase arrays revealed a decrease in phospho-c-JUN levels in EPHA2 knockdown cells. Knockdown of EPHA2 inhibited p-JNK and p-c-JUN levels in approximately 50% of NSCLC lines tested. Treatment of parental cells with SP600125, a c-JUN-NH2-kinase (JNK) inhibitor, recapitulated defects in EPHA2-deficient tumor cells, whereas constitutively activated JNK mutants were sufficient to rescue phenotypes. Knockdown of EPHA2 also inhibited tumor formation and progression in xenograft animal models in vivo. Furthermore, we investigated the role of EPHA2 in cancer stem-like cells (CSC). RNA interference-mediated depletion of EPHA2 in multiple NSCLC lines decreased the ALDH(+) cancer stem-like population and tumor spheroid formation in suspension. Depletion of EPHA2 in sorted ALDH(+) populations markedly inhibited tumorigenicity in nude mice. Furthermore, analysis of a human lung cancer tissue microarray revealed a significant, positive association between EPHA2 and ALDH expression, indicating an important role for EPHA2 in human lung CSCs. Collectively, these studies revealed a critical role of JNK signaling in EPHA2-dependent lung cancer cell proliferation and motility and a role for EPHA2 in CSC function, providing evidence for EPHA2 as a potential therapeutic target in NSCLC. Cancer Res; 74(9); 2444-54. ©2014 AACR.

Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC

Genome-wide analyses determined previously that the receptor tyrosine kinase (RTK) EPHA2 is commonly overexpressed in non-small cell lung cancers (NSCLCs). EPHA2 overexpression is associated with poor clinical outcomes; therefore, EPHA2 may represent a promising therapeutic target for patients with NSCLC. In support of this hypothesis, here we have shown that targeted disruption of EphA2 in a murine model of aggressive Kras-mutant NSCLC impairs tumor growth. Knockdown of EPHA2 in human NSCLC cell lines reduced cell growth and viability, confirming the epithelial cell autonomous requirements for EPHA2 in NSCLCs. Targeting EPHA2 in NSCLCs decreased S6K1-mediated phosphorylation of cell death agonist BAD and induced apoptosis. Induction of EPHA2 knockdown within established NSCLC tumors in a subcutaneous murine model reduced tumor volume and induced tumor cell death. Furthermore, an ATP-competitive EPHA2 RTK inhibitor, ALW-II-41-27, reduced the number of viable NSCLC cells in a time-dependent and dose-dependent manner in vitro and induced tumor regression in human NSCLC xenografts in vivo. Collectively, these data demonstrate a role for EPHA2 in the maintenance and progression of NSCLCs and provide evidence that ALW-II-41-27 effectively inhibits EPHA2-mediated tumor growth in preclinical models of NSCLC.

Ephrin reverse signaling induces mouse palatal fusion without TGFβ3 signaling (921.4)

Orofacial clefts are the most prevalent craniofacial birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium which adheres to form the midline epithelial seam (MES). MES cells then proceed through epithelial to mesenchymal transition (EMT) forming a fused palate. TGF‐β3 is essential for the disintegration of the palatal MES. Eph receptor tyrosine kinases and their ephrin ligands are responsible for many developmental processes. Binding of ephrins causes receptor activation in Eph‐bearing cells (forward signaling), and intracellular signaling inside ephrin‐bearing cells (reverse signaling). We asked if activation of ephrin reverse signaling is sufficient to fuse mouse palates if we blocked TGFβ receptors. We cultured E13.5 mouse palatal shelves in different conditions: Control IgG Fc; anti‐TGF‐β3 with or without clustered recombinant EphB2/Fc protein; TGF‐β RI kinase inhibitor (SB 431542) with or without EphB2/Fc. Palatal fusion was quantified and analyzed with a mean fusion score (MFS) in anterior, medial and posterior regions. Palates treated with anti‐TGF‐β3 or (SB 431542) did not fuse while those treated with SB 431542 and EphB2/Fc and those treated with anti‐TGF‐β3 and EphB2/Fc were partially fused. Palates from the control groups fused. These results demonstrate that ephrin reverse signaling induces fusion of the mammalian palate without TGF‐β3 signaling.Grant Funding Source: Biomedical Sciences Department, baylor College of Dentistry, Baylor Oral Health Foundation

EphA4 activation of c-Abl mediates synaptic loss and LTP blockade caused by amyloid-β oligomers.

The early stages of Alzheimer's disease are characterised by impaired synaptic plasticity and synapse loss. Here, we show that amyloid-β oligomers (AβOs) activate the c-Abl kinase in dendritic spines of cultured hippocampal neurons and that c-Abl kinase activity is required for AβOs-induced synaptic loss. We also show that the EphA4 receptor tyrosine kinase is upstream of c-Abl activation by AβOs. EphA4 tyrosine phosphorylation (activation) is increased in cultured neurons and synaptoneurosomes exposed to AβOs, and in Alzheimer-transgenic mice brain. We do not detect c-Abl activation in EphA4-knockout neurons exposed to AβOs. More interestingly, we demonstrate EphA4/c-Abl activation is a key-signalling event that mediates the synaptic damage induced by AβOs. According to this results, the EphA4 antagonistic peptide KYL and c-Abl inhibitor STI prevented i) dendritic spine reduction, ii) the blocking of LTP induction and iii) neuronal apoptosis caused by AβOs. Moreover, EphA4-/- neurons or sh-EphA4-transfected neurons showed reduced synaptotoxicity by AβOs. Our results are consistent with EphA4 being a novel receptor that mediates synaptic damage induced by AβOs. EphA4/c-Abl signalling could be a relevant pathway involved in the early cognitive decline observed in Alzheimer's disease patients.

SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2

The adaptor SLAP is a negative regulator of receptor signalling in immune cells but its role in human cancer is ill defined. Here we report that SLAP is abundantly expressed in healthy epithelial intestine but strongly downregulated in 50% of colorectal cancer. SLAP overexpression suppresses cell tumorigenicity and invasiveness while SLAP silencing enhances these transforming properties. Mechanistically, SLAP controls SRC/EPHA2/AKT signalling via destabilization of the SRC substrate and receptor tyrosine kinase EPHA2. This activity is independent from CBL but requires SLAP SH3 interaction with the ubiquitination factor UBE4A and SLAP SH2 interaction with pTyr594-EPHA2. SRC phosphorylates EPHA2 on Tyr594, thus creating a feedback loop that promotes EPHA2 destruction and thereby self-regulates its transforming potential. SLAP silencing enhances SRC oncogenicity and sensitizes colorectal tumour cells to SRC inhibitors. Collectively, these data establish a tumour-suppressive role for SLAP in colorectal cancer and a mechanism of SRC oncogenic induction through stabilization of its cognate substrates.

Altered distribution of the EphA4 kinase in hippocampal brain tissue of patients with Alzheimer¿s disease correlates with pathology

Synaptic dysfunction occurs early in the progression of Alzheimer’s disease (AD) and correlates with memory decline. There is emerging evidence that deregulation of Erythropoietin-producing hepatocellular (Eph) receptor tyrosine kinases (RTK) signaling contributes to the aberrant synaptic functions associated with neurodegeneration. The Eph receptor A4 is highly expressed in human adult hippocampal brain tissue and was previously linked to cognitive impairment in a transgenic mouse model for AD. Whether EphA4 levels are altered in AD brain remains elusive. Therefore we investigated the protein levels and localization of EphA4 in human hippocampus derived from AD (n = 29) as well as non-demented control cases (n = 19).The total EphA4 protein levels were not changed in AD patients compared to control cases. However, immunohistochemical localization of EphA4 revealed an altered distribution in AD compared to control hippocampus. EphA4 immunoreactivity was observed in plaque-like structures in AD cases. Double-labelling with phosphorylated tau and amyloid beta indicates that EphA4 co-localizes with neuritic plaques in AD. This altered distribution pattern was observed at early stages (Braak stage II) and correlates with the hallmarks of AD pathology suggesting a reduced availability of EphA4 that is likely to contribute to synaptic dysfunction that occurs early in AD.

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.

EphA3 As a Target For Monoclonal Antibody Therapy For Acute Leukemia

Eph receptor tyrosine kinases interact with cell-surface ephrin ligands to direct cell growth, migration and cell positioning. High expression of EphA3 in a number of cancers has been linked to progression through facilitation of invasiveness and metastatic spread. EphA3 protein, which was originally described in leukemia, has also reported to be expressed in sarcomas, lung cancer, melanoma and glioblastoma. Using a Humaneered® derivative of the IIIA4 anti-EphA3 monoclonal antibody, KB004 (KaloBios Pharmaceuticals Inc), a clinical trial has commenced which targets the EphA3 protein in leukemia. The EphA3-specific monoclonal antibody, IIIA4, binds and activates human and mouse EphA3 with similar affinities, binding is followed by internalization of receptor-antibody complexes. We have shown high expression of EphA3 in the LK63 pre-B ALL and no expression of EphA3 in the Reh, a similar pre-B ALL cell line. We have examined the effect of the IIIA4 antibody in LK63 and Reh NOD/SCID xenograft models. In the LK63 xenograft model, administration of the IIIA4 antibody led to inhibition of tumour growth and decrease spread from bone marrow to the spleen and other organs and increased the latency of the disease, however no reduction in engraftment was observed in Reh xenograft model, suggesting that the effect was directed against the leukemic cells rather than the stromal and vascular elements. To further analyse the function of EphA3 expression, LK63 EphA3 knock down and EphA3-transfected Reh cells were tested in the xengraft model. Similar to LK63, antibody-treated Reh EphA3 xenografts showed reduction in the bone marrow engraftment and increased the latency of the disease. In contrast, LK63 EphA3 knock down xenografts showed minimal difference between treated and control group but notably showed a significant reduction in the splenic engraftment compared to the normal LK63 model. These results are consistent with IIIA4-mediated effects on the growth of EphA3-positive leukemic cells in the bone marrow and a secondary effect on the subsequent spread of the leukemic cells to extra-medullary sites and provides evidence that EphA3 is a functional therapeutic target in acute leukemias. Disclosures:No relevant conflicts of interest to declare.

Discovery and Characterization of a Novel Cyclic Peptide That Effectively Inhibits Ephrin Binding to the EphA4 Receptor and Displays Anti-Angiogenesis Activity

The EphA4 receptor tyrosine kinase regulates a variety of physiological and pathological processes during neural development and the formation of tumor blood vessels; thus, it represents a new and promising therapeutic target. We used a combination of phage peptide display and computer modeling/docking approaches and discovered a novel cyclic nonapeptide, now designated TYY. This peptide selectively inhibits the binding of the ephrinA5 ligand with EphA4 and significantly blocks angiogenesis in a 3D matrigel culture system. Molecular docking reveals that TYY recognizes the same binding pocket on EphA4 that the natural ephrin ligand binds to and that the Tyr3 and Tyr4 side chains of TYY are both critical for the TYY/EphA4 interaction. The discovery of TYY introduces a valuable probe of EphA4 function and a new lead for EphA4-targeted therapeutic development.