EPHA5 mutation was associated with adverse outcome of atezolizumab treatment in late-stage non-small cell lung cancers.

BACKGROUND: The aim of the study was to investigate predictive value of gene mutation for atezolizumab treatment response from OAK and POPLAR cohorts. METHODS: Several public databases were used for analyzing gene mutation type of EPHA5 and association with alterations of other genes. Survival analysis was performed for patients receiving atezolizumab from OAK and POPLAR cohorts. RESULTS: EPHA5 mutation have high frequency to harbor TP53 and KEAP1 mutations. The bTMB value has significant difference between EPHA5 mutant and wild-type cases. Patients with EPHA5 mutation got worse survival compared to those without gene mutations receiving atezolizumab (P = 0.0186). CONCLUSIONS: EPHA5 mutant NSCLC may represent a subpopulation which showed worse response after treatment of atezolizumab compared to wild-type ones.

EPHA5 mutation predicts the durable clinical benefit of immune checkpoint inhibitors in patients with lung adenocarcinoma.

Immune checkpoint inhibitor (ICI) therapy has shown remarkable clinical benefit in lung adenocarcinoma (LUAD) patients. Genomic mutations may be applicable to predict the response to ICIs. Eph receptor A5 (EPHA5) is frequently mutated in breast cancer, lung cancer, and other tumors; however, its association with outcome in patients who receive immunotherapy remains unknown. In this study, we report that EPHA5 mutations were associated with increased tumor mutation burden (TMB), neoantigen load, levels of immune-related gene expression signatures, and enhanced tumor-infiltrating lymphocytes (TILs) in LUAD. LUAD patients with EPHA5 mutations in the immunotherapy cohort achieved a longer progression-free survival (PFS) time than patients with wild-type EPHA5. Immune response pathways were among the top enriched pathways in samples with EPHA5 mutations. In addition, patients with EPHA5 mutations tended to be more sensitive to certain targeted molecular inhibitors, including serdemetan, lox2, and PF1-1. Collectively, our results suggest that identifying mutations in the EPHA5 gene may provide insight into the genome-wide mutational burden and may serve as a biomarker to predict the immune response of patients with LUAD.

Alternative proteins are functional regulators in cell reprogramming by PKA activation.

It has been recently shown that many proteins are lacking from reference databases used in mass spectrometry analysis, due to their translation templated on alternative open reading frames. This questions our current understanding of gene annotation and drastically expands the theoretical proteome complexity. The functions of these alternative proteins (AltProts) still remain largely unknown. We have developed a large-scale and unsupervised approach based on cross-linking mass spectrometry (XL-MS) followed by shotgun proteomics to gather information on the functional role of AltProts by mapping them back into known signalling pathways through the identification of their reference protein (RefProt) interactors. We have identified and profiled AltProts in a cancer cell reprogramming system: NCH82 human glioma cells after 0, 16, 24 and 48 h Forskolin stimulation. Forskolin is a protein kinase A activator inducing cell differentiation and epithelial-mesenchymal transition. Our data show that AltMAP2, AltTRNAU1AP and AltEPHA5 interactions with tropomyosin 4 are downregulated under Forskolin treatment. In a wider perspective, Gene Ontology and pathway enrichment analysis (STRING) revealed that RefProts associated with AltProts are enriched in cellular mobility and transfer RNA regulation. This study strongly suggests novel roles of AltProts in multiple essential cellular functions and supports the importance of considering them in future biological studies.

CpG Islands Methylation Analysis of CDH11, EphA5, and HS3ST2 Genes in Gastric Adenocarcinoma Patients.

PURPOSE: Gastric cancer is an aggressive disease which is the fourth prevalent malignancy in the world. Beside the genetic factors, epigenetic alterations such as promoter CpG island hyper methylation are involved in the emergence of gastric cancer. Herein, we investigated the methylation status of CDH11, EphA5, and HS3ST2 genes in patients with and without gastric adenocarcinoma for the first time. METHODS: In the study 40 paraffin-embedded tissue sections from gastric adenocarcinoma patients and 40 specimens from patients with functional dyspepsia were taken. DNA extraction was performed using a modified salting out method. Epizen DNA methylation kit was used to the bisulfite DNA conversion. The methylation status of CDH11, EphA5, and HS3ST2 genes were analyzed by methylation-specific PCR (MSP) technique. RESULTS: Among the 80 specimens, 71 DNA samples were achieved (34 gastric adenocarcinoma patients and 37 control patients). The results showed that CDH11, EphA5, and HS3ST2 genes are methylated in 28 (82.45%), 19 (55.88%), and 26 (76.47%) of 34 DNA samples from gastric adenocarcinoma patients, respectively, whereas, these genes are methylated in 7 (18.91%), 9 (24.32%) and 7 (18.91%) of 37 samples from noncancerous patients, respectively. Statistical analyses using a chi-squared test showed that there is a statistically significant difference in methylation level of CDH11, EphA5, and HS3ST2 genes between gastric cancer and uncancerous patients (p < 0.05). CONCLUSION: To the best of our knowledge, this is the first report on methylation of CDH11, EphA5, and HS3ST2 promoters' in gastric adenocarcinoma patients using MSP. Identification of novel cancer-related molecular mechanisms can be useful in detection of new treatment strategies.

Dynamic Alterations of Retinal EphA5 Expression in Retinocollicular Map Plasticity.

The topographically ordered retinocollicular projection is an excellent system for studying the mechanism of axon guidance. Gradients of EphA receptors in the retina and ephrin-As in the superior colliculus (SC) pattern the anteroposterior axis of the retinocollicular map, but whether they are involved in map plasticity after injury is unknown. Partial damage to the caudal SC at birth creates a compressed, complete retinotopic map in the remaining SC without affecting visual response properties. Previously, we found that the gradient of ephrin-A expression in compressed maps is steeper than normal, suggesting an instructive role in compression. Here we measured EphA5 mRNA and protein levels after caudal SC damage in order to test the hypothesis that changes in retinal EphA5 expression occur that are complementary to the changes in collicular ephrin-A expression. We find that the nasotemporal gradient of EphA5 receptor expression steepens in the retina and overall expression levels change dynamically, especially in temporal retina, supporting the hypothesis. This change in receptor expression occurs after the change in ephrin-A ligand expression. We propose that changes in the retinal EphA5 gradient guide recovery of the retinocollicular projection from early injury. This could occur directly through the change in EphA5 expression instructing retino-SC map compression, or through ephrin-A ligand signaling instructing a change in EphA5 receptor expression that in turn signals the retinocollicular map to compress. Understanding what molecular signals direct compensation for injury is essential to developing rehabilitative strategies and maximizing the potential for recovery.

EPHA5 mediates trastuzumab resistance in HER2-positive breast cancers through regulating cancer stem cell-like properties.

Trastuzumab is a successful, rationally designed therapy that provides significant clinical benefit for human epidermal growth factor receptor-2 (HER2)-positive breast cancer patients. However, about half of individuals with HER2-positive breast cancer do not respond to trastuzumab treatment because of various resistance mechanisms, including but not limited to: 1) shedding of the HER2 extracellular domain, 2) steric hindrance ( e.g., MUC4 and MUC1), 3) parallel pathway activation (this is the general mechanism cited in the quote above), 4) perturbation of downstream signaling events ( e.g., PTEN loss or PIK3CA mutation), and 5) immunologic mechanisms (such as FcR polymorphisms). EPHA5, a receptor tyrosine kinase, has been demonstrated to act as an anticancer agent in several cancer cell types. In this study, deletion of EPHA5 can significantly increase the resistance of HER2-positive breast cancer patients to trastuzumab. To investigate how EPHA5 deficiency induces trastuzumab resistance, clustered regularly interspaced short palindromic repeat technology was used to create EPHA5-deficient variants of breast cancer cells. EPHA5 deficiency effectively increases breast cancer stem cell (BCSC)-like properties, including NANOG, CD133+, E-cadherin expression, and the CD44+/CD24-/low phenotype, concomitantly enhancing mammosphere-forming ability. EPHA5 deficiency also caused significant aggrandized tumor malignancy in trastuzumab-sensitive xenografts, coinciding with the up-regulation of BCSC-related markers and intracellular Notch1 and PTEN/AKT signaling pathway activation. These findings highlight that EPHA5 is a potential prognostic marker for the activity of Notch1 and better sensitivity to trastuzumab in HER2-positive breast cancer. Moreover, patients with HER2-positive breast cancers expressing high Notch1 activation and low EPHA5 expression could be the best candidates for anti-Notch1 therapy.-Li, Y., Chu, J., Feng, W., Yang, M., Zhang, Y., Zhang, Y., Qin, Y., Xu, J., Li, J., Vasilatos, S. N., Fu, Z., Huang, Y., Yin, Y. EPHA5 mediates trastuzumab resistance in HER2-positive breast cancers through regulating cancer stem cell-like properties.

c-Fos downregulation positively regulates EphA5 expression in a congenital hypothyroidism rat model.

The EphA5 receptor is well established as an axon guidance molecule during neural system development and plays an important role in dendritic spine formation and synaptogenesis. Our previous study has showed that EphA5 is decreased in the developing brain of congenital hypothyroidism (CH) and the EphA5 promoter methylation modification participates in its decrease. c-Fos, a well-kown transcription factor, has been considered in association with brain development. Bioinformatics analysis showed that the EphA5 promoter region contained five putative c-fos binding sites. The chromatin immunoprecipitation (ChIP) assays were used to assess the direct binding of c-fos to the EphA5 promoter. Furthermore, dual-luciferase assays showed that these three c-fos protein binding sites were positive regulatory elements for EphA5 expression in PC12 cells. Moreover, We verified c-fos positively regulation for EphA5 expression in CH model. Q-PCR and Western blot showed that c-fos overexpression could upregulate EphA5 expression in hippocampal neurons of rats with CH. Our results suggest that c-fos positively regulates EphA5 expression in CH rat model.

Crystal structure of the kinase and UBA domains of SNRK reveals a distinct UBA binding mode in the AMPK family.

Sucrose non-fermenting (Snf1)-related kinase (SNRK) is a novel member of the AMP-activated protein kinase (AMPK) family and is involved in many metabolic processes. Here we report the crystal structure of an N-terminal SNRK fragment containing kinase and adjacent ubiquitin-associated (UBA) domains. This structure shows that the UBA domain binds between the N- and C-lobes of the kinase domain. The mode of UBA binding in SNRK largely resembles that in AMPK and brain specific kinase (BRSK), however, unique interactions play vital roles in stabilizing the KD-UBA interface of SNRK. We further propose a potential role of the UBA domain in the regulation of SNRK kinase activity. This study provides new insights into the structural diversities of the AMPK kinase family.

Decreased expression of EphA5 is associated with Fuhrman nuclear grade and pathological tumour stage in ccRCC.

The incidence of renal cell carcinoma is increasing all over the world. The molecular mechanisms for tumorigenesis, progression and prognosis are still unknown. The erythropoietin-producing hepatoma amplified sequence (Eph) receptors have been reported to be expressed aberrantly in many types of human cancers and in particular EphA5 may play a role in certain human cancers. In this study, a set of clear cell renal cell carcinoma (ccRCC) tissues were subjected to immunohistochemistry. The relationship between EphA5 protein expression and clinicopathological parameters was statistically analysed. Our data show that EphA5 protein was negatively (0) or weakly (1+) expressed in 48 of 78 (61.5%), moderately (2+) expressed in 15 of 78 (19.2%) and strongly (3+) expressed in 15 of 78 (19.2%) tumour samples of ccRCC. Decreased expression of EphA5 was detected more often in females than in males (P = 0.017, rs  = -0.267). Expression of EphA5 was related negatively to Fuhrman grade (P = 0.013, rs  = -0.279) and pathological tumour stage pT (P = 0.003, rs  = -0.334). No relation between the expression of EphA5 and age of patients was found (P = 0.107, rs  = 0.184). Fuhrman grade and pT stage are the most important factors used in prognosis of ccRCC. Hence this study may provide a new and useful prognostic marker in the clinical practice of ccRCC.

EphA5 and EphA7 forward signaling enhances human hematopoietic stem and progenitor cell maintenance, migration, and adhesion via Rac1 activation.

The proliferation, differentiation, adhesion, and migration of hematopoietic stem and progenitor cells (HSPCs) are dependent upon bone marrow stromal cells (BMSCs). In this study, we found that human primitive HSPCs (CD34+CD38-), but not lineage-committed hematopoietic cell populations, express the tyrosine kinase receptors EphA5 and EphA7. Moreover, we found that the ephrinA5 ligand, the high-affinity binding partner of EphA5 and EphA7, is highly expressed by primary human BMSCs. Previous studies have reported that interactions between EphA and ephrinA play important roles in hematopoietic cell trafficking; however, their role in BMSC support of hematopoiesis had not been described previously. Herein, we show that stimulating EphA5 and/or EphA7 forward signaling in HSPCs using soluble ephrinA5-Fc molecules promoted human HSPC-derived colony formation significantly and was associated with increased expression of granulocyte macrophage colony-stimulating factor receptor on HSPCs. Studies using functional blocking peptides to EphA5/7 found that disruption of EphA5/ephrinA5 and/or EphA7/ephrinA5 interactions inhibited HSPC function in BMSC-dependent long-term culture-initiating cell assays. Furthermore, the adhesion and migration of HSPCs was increased significantly in the presence of ephrinA5-Fc molecules compared with human immunoglobulin G-treated controls. Conversely, blocking EphA5 activation led to a reduction of HSPC adhesion, whereas inhibiting EphA5 and/or EphA7 activation hindered HSPC migration. Analysis of HSPC cultured in the presence of ephrinA5-Fc showed that EphA forward signaling stimulated Rac1 gene and protein expression and the Rac1 target molecule WAVE1. Moreover, a significant reduction of ephrinA5-mediated HSPC adhesion and migration was observed in the presence of Rac1 inhibitor. These findings suggest that interactions between EphA and ephrinA5 are important in maintaining the HSPC niche mediated in part by activation of Rac1 signaling.

Axon guidance molecule expression after cell therapy in a mouse model of Parkinson's disease.

BACKGROUND: Cell therapy is a promising approach for Parkinson's disease (PD). Others and we have previously shown that transplantation of ventral mesencephalic fetal cells into substantia nigra (SN) in an animal model of PD enables anatomical and functional repair of the degenerated pathway. However, the molecular basis of this repair is still largely unknown. OBJECTIVE: In this work, we studied the expression of several axon guidance molecules that may be implicated in the repair of the degenerated nigrostriatal pathway. METHODS: The expression of axon guidance molecules was analyzed using qRT-PCR on five specific regions surrounding the nigrostriatal pathway (ventral mesencephalon (VM), thalamus (Thal), medial forebrain bundle (MFB), nucleus accumbens (NAcc) and caudate putamen (CPu)), one and seven days after lesion and transplantation. RESULTS: We showed that mRNA expression of specific axon guidance molecules and their receptors is modified in structures surrounding the nigrostriatal pathway, suggesting their involvement in the axon guidance of grafted neurons. Moreover, we highlight a possible new role for semaphorin 7A in this repair. CONCLUSION: Overall, our data provide a reliable basis to understand how axons of grafted neurons are able to navigate towards their targets and interact with the molecular environment in the adult brain. This should help to improve the efficiency of cell replacement approaches in PD.

EphA5 protein, a potential marker for distinguishing histological grade and prognosis in ovarian serous carcinoma.

BACKGROUND: Ovarian serous carcinoma (OSC) is the most common ovarian epithelial malignancy. Disregulation of Eph/ephrin signaling has been implicated in oncogenesis and tumor progression. EphA5 receptor is one of large families of Eph tyrosine kinase receptor and is documented in the development of nervous system. Till now, there is no published data about the role of EphA5 in ovarian epithelial neoplasmas. METHODS: This study aims to investigate the expression of EphA5 protein in ovarian serous carcinoma, and its relationship to clinical pathological characteristics. Sixty-one cases of ovarian serous carcinoma, 24 cases of benign ovarian serous tumors, 42 cases of serous borderline tumors and 20 cases of normal fallopian tubes were examined using immunohistochemical staining. The relationship between EphA5 expression and pathological parameters was analyzed. Kaplan-Meier survival function was used to analyze prognosis of patients. RESULTS: Immunostaining analysis demonstrated that the EphA5 protein was highly expressed in 100% (20/20) of normal fallopian tube samples, 100% (24/24) of benign epithelial ovarian tumors, 76% (32/42) of ovarian serous borderline tumors, and 31% (19/61) of ovarian serous carcinomas. Loss of EphA5expression was associated with tumor grade (P < 0.001) and FIGO stage (P = 0.005). The survival analysis showed that patients with negative or weak expression of EphA5 protein had a poor outcome than those with positive expression (P = 0.004). CONCLUSIONS: Our results show that EphA5 may be a potential biomarker for distinguishing high-and low-grade ovarian serous carcinoma and a potential prognostic marker.

Altered brain morphology and functional connectivity reflect a vulnerable affective state after cumulative multigenerational stress in rats.

Prenatal stress is a risk factor for abnormal neuroanatomical, cognitive, behavioral and mental health outcomes with potentially transgenerational consequences. Females in general seem more resilient to the effects of prenatal stress than males. Here, we examined if repeated stress across generations may diminish stress resiliency and cumulatively enhance the susceptibility for adverse health outcomes in females. Pregnant female rats of three successive generations were exposed to stress from gestational days 12-18 to generate multigenerational prenatal stress (MPS) in the maternal lineage. Stress response was measured by plasma corticosterone levels and open-field exploration in each generation. Neuromorphological consequences of MPS were investigated in the F3 generation using in vivo manganese-enhanced magnetic resonance imaging (MEMRI), T2-relaxometry, and cytoarchitectonics in relation to candidate gene expression involved in brain plasticity and mental health. Each additional generation of prenatal stress incrementally elevated hypothalamic-pituitary-adrenal axis activation, anxiety-like and aversive behaviors in adult female offspring. Elevated stress responses in the MPS F3 generation were accompanied by reduced neural density in prefrontal cortex, hippocampus and whole brain along with altered brain activation patterns in in vivo MEMRI. MPS increased ephrin receptor A5 (Epha5), neuronal growth regulator (Negr1) and synaptosomal-associated protein 25 (Snap25) gene expression and reduced fibroblast growth factor 12 (Fgf12) in prefrontal cortex. These genes regulate neuronal maturation, arborization and synaptic plasticity and may explain altered brain cytoarchitectonics and connectivity. These findings emphasize that recurrent stress across generations may cumulatively increase stress vulnerability and the risk of adverse health outcomes through perinatal programing in females.

Reconstitution of Fusion Proteins in Supported Lipid Bilayers for the Study of Cell Surface Receptor-Ligand Interactions in Cell-Cell Contact.

Bioactive molecules such as adhesion ligands, growth factors, or enzymes play an important role in modulating cell behavior such as cell adhesion, spreading, and differentiation. Deciphering the mechanism of ligand-mediated cell adhesion and associated signaling is of great interest not only for fundamental biophysical investigations but also for applications in medicine and biotechnology. In the presented work, we developed a new biomimetic platform that enables culturing primary neurons and testing cell surface-receptor ligand interactions in cell-cell contacts as, e.g., in neuronal synapses. This platform consists of a supported lipid bilayer modified with incorporated neuronal adhesion proteins conjugated with the Fc-domain of IgG (ephrin A5 Fc-chimera). We extensively characterized properties of these protein containing bilayers using fluorescence recovery after photobleaching (FRAP), quartz crystal microbalance with dissipation (QCM-D), and immunostaining. We conclude that the Fc-domain is the part responsible for the incorporation of the protein into the bilayer. The biomimetic platform prepared by this new approach was able to promote neuronal cell adhesion and maintain growth as well as facilitate neuronal maturation as shown by electrophysiological measurements. We believe that our approach can be extended to insert other proteins to create a general culture platform for neurons and other cell types.

C-Terminal Auto-Regulatory Motif of Hepatitis C Virus NS5B Interacts with Human VAPB-MSP to Form a Dynamic Replication Complex.

Hepatitis C virus (HCV) is a pathogen of global importance and nearly 200 million people are chronically infected with HCV. HCV is an enveloped single-stranded RNA virus, which is characteristic of the formation of the host membrane associated replication complex. Previous functional studies have already established that the human ER-anchored VAPB protein acts as a host factor to form a complex with HCV NS5A and NS5B, which may be established as a drug target. However, there is lacking of biophysical characterization of the structures and interfaces of the complex, partly due to the dynamic nature of the complex formation and dissociation, which is extensively involved in intrinsically-disordered domains. Here by an integrated use of domain dissection and NMR spectroscopy, for the first time we have successfully deciphered that the HCV NS5B utilizes its auto-regulatory C-linker to bind the VAPB-MSP domain to form a dynamic complex. This finding implies that the NS5B C-linker is capable of playing dual roles by a switch between the folded and disordered states. Interestingly, our previous and present studies together reveal that both HCV NS5A and NS5B bind to the MSP domains of the dimeric VAP with significantly overlapped interfaces and similar affinities. The identification that EphA2 and EphA5 bind to the MSP domain with higher affinity than EphA4 provides a biophysical basis for further exploring whether other than inducing ALS-like syndrome, the HCV infection might also trigger pathogenesis associated with signalling pathways mediated by EphA2 and EphA5.

Developmental Axon Pruning Requires Destabilization of Cell Adhesion by JNK Signaling.

Developmental axon pruning is essential for normal brain wiring in vertebrates and invertebrates. How axon pruning occurs in vivo is not well understood. In a mosaic loss-of-function screen, we found that Bsk, the Drosophila JNK, is required for axon pruning of mushroom body γ neurons, but not their dendrites. By combining in vivo genetics, biochemistry, and high-resolution microscopy, we demonstrate that the mechanism by which Bsk is required for pruning is through reducing the membrane levels of the adhesion molecule Fasciclin II (FasII), the NCAM ortholog. Conversely, overexpression of FasII is sufficient to inhibit axon pruning. Finally, we show that overexpressing other cell adhesion molecules, together with weak attenuation of JNK signaling, strongly inhibits pruning. Taken together, we have uncovered a novel and unexpected interaction between the JNK pathway and cell adhesion and found that destabilization of cell adhesion is necessary for efficient pruning.

DNA Methylation of the EphA5 Promoter Is Associated with Rat Congenital Hypothyroidism.

Thyroid hormones (THs) are essential for normal development of the mammalian central nervous system through regulation of TH-responsive genes. EphA5, an important TH-responsive gene encoding the tyrosine kinase receptor EphA5, regulates synaptogenesis initiation and synaptic remodeling during brain development. Abnormal EphA5 expression is involved in the development of congenital hypothyroidism (CH). To show the regulatory mechanism of EphA5 expression in CH rats, we analyzed the correlation between methylation of the EphA5 promoter and its expression in the hypothyroid hippocampus and hippocampal neurons. Demethylation treatment using 5'-azadeoxycytidine upregulated EphA5 expression and rescued the effects of hypermethylation, suggesting a novel regulatory mechanism of EphA5 expression in CH rats. Our results suggest a potentially new approach for the development of drugs to restore neurocognitive impairments associated with CH.

Polarized activities of AMPK and BRSK in primary hippocampal neurons.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master metabolic regulator that has been shown to inhibit the establishment of neuronal polarity/axogenesis under energy stress conditions, whereas brain-specific kinase (BRSK) promotes the establishment of axon-dendrite polarity and synaptic development. However, little information exists regarding the localized activity and regulation of these kinases in developing neurons. In this study, using a fluorescence resonance energy transfer (FRET)-based activity reporter that responds to both AMPK and BRSK, we found that BRSK activity is elevated in the distal region of axons in polarized hippocampal neurons before any stimulation and does not respond to either Ca(2+) or 2-deoxyglucose (2-DG) stimulation. In contrast, AMPK activity is stimulated by either Ca(2+) or 2-DG in the soma, dendrites, and axons of hippocampal neurons, with maximal stimulated activity observed in the distal region of the axon. Our study shows that the activities of both AMPK and BRSK are polarized in developing hippocampal neurons, with high levels in the distal region of extended axons.

Overexpression of Ephrin A2 receptors in cancer stromal cells is a prognostic factor for the relapse of gastric cancer.

BACKGROUND: Microenvironments control cancer growth and progression. We explored the prognostic impact of stromal reaction and cancer stromal cells on relapse risk and survival after curative gastrectomy in gastric cancer patients. METHODS: Tissue samples were obtained from 107 patients with gastric adenocarcinoma who underwent curative (R0) gastrectomy. Primary stromal cells isolated from gastric cancer tissue (GCSC) and normal gastric tissue (Gastric stromal cell: GSC) in each patient were cultured and subjected to comprehensive proteome (LC-MS/MS) and real-time RT-PCR analysis. Expression of Ephrin A2 receptors (EphA2) in cancers and GCSC was evaluated immunohistochemically. Intermingling of EphA2-positive cancer cells and GCSC (IC/A2+) and overexpression of EphA2 in cancer cells (Ca/A2+) in invasive parts of tumors were assessed, as were relationships of IC/A2+, Ca/A2+, and clinicopathological factors with relapse-free survival and overall survival. RESULTS: Proteome analysis showed that EphA2 expression was significantly higher in GCSC than GSC. Real-time RT-PCR analysis showed that levels of EphA1/A2/A3/A5 and EphB2/B4 were ≥2.0-fold higher in GCSC than GSC. Ca/A2 and IC/A2 were positive in 65 (60.7 %) and 26 (24.3 %) patients, respectively. Relapse was significantly more frequent in IC/A2-positive than in IC/A2-negative (HR, 2.12; 95 % CI, 1.16-5.41; p = 0.0207) patients. Among the 54 patients who received S-1 adjuvant chemotherapy, relapse-free survival (RFS) was significantly shorter in those who were IC/A2-positive than in those who were IC/A2-negative and Ca/A2-negative (HR, 2.83; 95 % CI, 1.12-12.12; p = 0.0339). Multivariable analysis indicated that pathological stage (p = 0.010) and IC/A2+ (p = 0.008) were independent risk factors for recurrence. CONCLUSION: IC/A2+ was predictive of relapse after curative (R0) gastrectomy.