C1GALT1 is associated with poor survival and promotes soluble Ephrin A1-mediated cell migration through activation of EPHA2 in gastric cancer.
ABSTRACT: C1GALT1 controls the crucial step of GalNAc-type O-glycosylation and is associated with both physiologic and pathologic conditions, including cancers. EPH receptors comprise the largest family of receptor tyrosine kinases (RTKs) and modulate a diverse range of developmental processes and human diseases. However, the role of C1GALT1 in the signaling of EPH receptors remains largely overlooked. Here, we showed that C1GALT1 high expression in gastric adenocarcinomas correlated with adverse clinicopathologic features and is an independent prognostic factor for poor overall survival. Silencing or loss of C1GALT1 inhibited cell viability, migration, invasion, tumor growth and metastasis, as well as increased apoptosis and cytotoxicity of 5-fluorouracil in AGS and MKN45 cells. Phospho-RTK array and western blot analysis showed that C1GALT1 depletion suppressed tyrosine phosphorylation of EPHA2 induced by soluble Ephrin A1-Fc. O-glycans on EPHA2 were modified by C1GALT1 and both S277A and T429A mutants, which are O-glycosites on EPHA2, dramatically enhanced phosphorylation of Y588, suggesting that not only overall O-glycan structures but also site-specific O-glycosylation can regulate EPHA2 activity. Furthermore, depletion of C1GALT1 decreased Ephrin A1-Fc induced migration and reduced Ephrin A1 binding to cell surfaces. The effects of C1GALT1 knockdown or knockout on cell invasiveness in vitro and in vivo were phenocopied by EPHA2 knockdown in gastric cancer cells. These results suggest that C1GALT1 promotes phosphorylation of EPHA2 and enhances soluble Ephrin A1-mediated migration primarily by modifying EPHA2 O-glycosylation. Our study highlights the importance of GalNAc-type O-glycosylation in EPH receptor-regulated diseases and identifies C1GALT1 as a potential therapeutic target for gastric cancer.
Project description:The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand's carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.
Project description:Eph/ephrin signaling proteins are present in the corneal epithelium, where their function remains unknown. The authors examined the role of the EphA2 receptor and ephrin-A1 ligand in human corneal epithelial cell migration.Immunohistochemical analysis of EphA2 and ephrin-A1 in healthy and diabetic corneas was performed in concert with linear scratch wound healing studies in primary and telomerase-immortalized human corneal epithelial cells. Corneal epithelial cells were exposed to a soluble ephrin-A1-Fc peptide mimetic that targets EphA2 to trigger receptor phosphorylation and subsequent downregulation. Genetic modulation of EphA2 and ephrin-A1 levels was combined with manipulation of Erk1/2 or Akt signaling during wound healing.EphA2 was immunolocalized to human corneal epithelial cells in vivo and in vitro. Ephrin-A1 ligand targeting of EphA2 restricted the ability of corneal epithelial cells to seal linear scratch wounds in a manner that was associated with a transient reduction in Erk1/2 and Akt activation state. Ephrin-A1-Fc treatment delayed wound healing independently of Mek-Erk1/2 signaling but was no longer capable of restricting migration after pharmacologic blockade of the PI3K-Akt pathway. Interestingly, ephrin-A1 immunoreactivity was increased in the corneal epithelia of diabetic individuals, mice maintained on a high-fat diet, or cultured corneal epithelial cells exposed to high glucose, which exhibit impaired Akt signaling and slower wound healing responses.EphA2 attenuates corneal epithelial cell migration when stimulated by ephrin-A1 ligand in a manner that involves the suppression of Akt. Elevated levels of ephrin-A1 may contribute to diabetic keratopathies by persistently engaging EphA2 and prohibiting Akt-dependent corneal epithelial repair processes.
Project description:Eph receptor tyrosine kinases and ephrin ligands control many physiological and pathological processes, and molecules interfering with their interaction are useful probes to elucidate their complex biological functions. Moreover, targeting Eph receptors might enable new strategies to inhibit cancer progression and pathological angiogenesis as well as promote nerve regeneration. Because our previous work suggested the importance of the salicylic acid group in antagonistic small molecules targeting Eph receptors, we screened a series of salicylic acid derivatives to identify novel Eph receptor antagonists. This identified a disalicylic acid-furanyl derivative that inhibits ephrin-A5 binding to EphA4 with an IC(50) of 3 ?m in ELISAs. This compound, which appears to bind to the ephrin-binding pocket of EphA4, also targets several other Eph receptors. Furthermore, it inhibits EphA2 and EphA4 tyrosine phosphorylation in cells stimulated with ephrin while not affecting phosphorylation of EphB2, which is not a target receptor. In endothelial cells, the disalicylic acid-furanyl derivative inhibits EphA2 phosphorylation in response to TNF? and capillary-like tube formation on Matrigel, two effects that depend on EphA2 interaction with endogenous ephrin-A1. These findings suggest that salicylic acid derivatives could be used as starting points to design new small molecule antagonists of Eph receptors.
Project description:Ephrin receptors (Ephs) are reported to control metastatic signaling of non-small cell lung cancer (NSCLC) and other tumors. Here we show for the first time that blocking expression of the Eph ligand Ephrin B3 inhibits NSCLC cell migration and invasion. We demonstrate that Ephrin B3 directly binds the EphAs EphA2, EphA3, EphA4, and EphA5. EphA2 Ser897 was previously shown to drive migration propensity of tumor cells and our study reveals that EphA2 stays phosphorylated on Ser897 in the Ephrin B3/EphA2 complex in NSCLC cells of different histology. Moreover, we report that within such Ephrin B3/EphA2 complex both Akt Ser 129 and p38MAPK are found indicating a potential to drive migration/proliferation. We also found the EMT marker E-cadherin expression to be maintained or increased upon Ephrin B3 blockade in NSCLC cells. Expression of Ephrin B3 was furthermore analyzed in a cohort of NSCLC stage IA-IB cases (n=200) alongside EphA2 and Ephrin A1. We found that Ephrin B3 was concomitantly expressed with EphA2 and Ephrin A1 with higher Ephrin B3 levels found in non-squamous than in squamous tumors, whereas EphA2 was higher expressed in well-differentiated than in low-differentiated tumors. In the entire NSCLC cohort, Ephrin B3 expression was not linked to patient survival, whereas a high EphA2 expression was associated with improved survival (p=0.03). In conclusion, we show that blocking Ephrin B3 expression inhibits NSCLC proliferation-, migration- and invasion capacity which calls for further studies on interference with Ephrin B3 as a possible therapeutic avenue in this tumor malignancy.
Project description:RATIONALE:Understanding the mechanisms that regulate trafficking of human cardiac stem cells (hCSCs) may lead to development of new therapeutic approaches for the failing heart. OBJECTIVE:We tested whether the motility of hCSCs in immunosuppressed infarcted animals is controlled by the guidance system that involves the interaction of Eph receptors with ephrin ligands. METHODS AND RESULTS:Within the cardiac niches, cardiomyocytes expressed preferentially the ephrin A1 ligand, whereas hCSCs possessed the EphA2 receptor. Treatment of hCSCs with ephrin A1 resulted in the rapid internalization of the ephrin A1-EphA2 complex, posttranslational modifications of Src kinases, and morphological changes consistent with the acquisition of a motile cell phenotype. Ephrin A1 enhanced the motility of hCSCs in vitro, and their migration in vivo following acute myocardial infarction. At 2 weeks after infarction, the volume of the regenerated myocardium was 2-fold larger in animals injected with ephrin A1-activated hCSCs than in animals receiving control hCSCs; this difference was dictated by a greater number of newly formed cardiomyocytes and coronary vessels. The increased recovery in myocardial mass with ephrin A1-treated hCSCs was characterized by further restoration of cardiac function and by a reduction in arrhythmic events. CONCLUSIONS:Ephrin A1 promotes the motility of EphA2-positive hCSCs, facilitates their migration to the area of damage, and enhances cardiac repair. Thus, in situ stimulation of resident hCSCs with ephrin A1 or their ex vivo activation before myocardial delivery improves cell targeting to sites of injury, possibly providing a novel strategy for the management of the diseased heart.
Project description: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.
Project description:BACKGROUND: Increased expression of Eph receptor tyrosine kinases and their ephrin ligands has been implicated in tumor progression in a number of malignancies. This report describes aberrant expression of these genes in ovarian cancer, the commonest cause of death amongst gynaecological malignancies. METHODS: Eph and ephrin expression was determined using quantitative real time RT-PCR. Correlation of gene expression was measured using Spearman's rho statistic. Survival was analysed using log-rank analysis and (was visualised by) Kaplan-Meier survival curves. RESULTS: Greater than 10 fold over-expression of EphA1 and a more modest over-expression of EphA2 were observed in partially overlapping subsets of tumors. Over-expression of EphA1 strongly correlated (r = 0.801; p < 0.01) with the high affinity ligand ephrin A1. A similar trend was observed between EphA2 and ephrin A1 (r = 0.387; p = 0.06). A striking correlation of both ephrin A1 and ephrin A5 expression with poor survival (r = -0.470; p = 0.02 and r = -0.562; p < 0.01) was observed. Intriguingly, there was no correlation between survival and other clinical parameters or Eph expression. CONCLUSION: These data imply that increased levels of ephrins A1 and A5 in the presence of high expression of Ephs A1 and A2 lead to a more aggressive tumor phenotype. The known functions of Eph/ephrin signalling in cell de-adhesion and movement may explain the observed correlation of ephrin expression with poor prognosis.
Project description:Both pro- and antioncogenic properties have been attributed to EphA2 kinase. We report that a possible cause for this apparent paradox is diametrically opposite roles of EphA2 in regulating cell migration and invasion. While activation of EphA2 with its ligand ephrin-A1 inhibited chemotactic migration of glioma and prostate cancer cells, EphA2 overexpression promoted migration in a ligand-independent manner. Surprisingly, the latter effects required phosphorylation of EphA2 on serine 897 by Akt, and S897A mutation abolished ligand-independent promotion of cell motility. Ephrin-A1 stimulation of EphA2 negated Akt activation by growth factors and caused EphA2 dephosphorylation on S897. In human astrocytoma, S897 phosphorylation was correlated with tumor grades and Akt activation, suggesting that the Akt-EphA2 crosstalk may contribute to brain tumor progression.
Project description:Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-transformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki =? 49 µM). Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC(50)? = 48 and 66 µM, respectively) without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1?, IRK?) activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells. In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis.
Project description:Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B-class receptors. Here, we present the crystal structures of an A-class complex between EphA2 and ephrin-A1 and of unbound EphA2. Although these structures are similar overall to their B-class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A-class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a 'lock-and-key'-type binding mechanism, in contrast to the 'induced fit' mechanism defining the B-class molecules. This model is supported by structure-based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell-based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2-specific homotypic cell adhesion interactions.