Gene Summary

Gene:EPHB4; EPH receptor B4
Summary:Ephrin receptors and their ligands, the ephrins, mediate numerous developmental processes, particularly in the nervous system. Based on their structures and sequence relationships, ephrins are divided into the ephrin-A (EFNA) class, which are anchored to the membrane by a glycosylphosphatidylinositol linkage, and the ephrin-B (EFNB) class, which are transmembrane proteins. The Eph family of receptors are divided into 2 groups based on the similarity of their extracellular domain sequences and their affinities for binding ephrin-A and ephrin-B ligands. Ephrin receptors make up the largest subgroup of the receptor tyrosine kinase (RTK) family. The protein encoded by this gene binds to ephrin-B2 and plays an essential role in vascular development. [provided by RefSeq, Jul 2008]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:ephrin type-B receptor 4
Source:NCBIAccessed: 31 August, 2019


What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 31 August 2019 using data from PubMed using criteria.

Literature Analysis

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Tag cloud generated 31 August, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: EPHB4 (cancer-related)

Hanna DL, Loupakis F, Yang D, et al.
Prognostic Value of ACVRL1 Expression in Metastatic Colorectal Cancer Patients Receiving First-line Chemotherapy With Bevacizumab: Results From the Triplet Plus Bevacizumab (TRIBE) Study.
Clin Colorectal Cancer. 2018; 17(3):e471-e488 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
BACKGROUND: No biomarkers exist to predict benefit from antiangiogenic therapy in metastatic colorectal cancer patients. ACVRL1 (activin receptor like-protein 1) encodes for ALK1, a member of the transforming growth factor-β receptor family, which directs pathologic angiogenesis. We examined the intratumoral expression of ACVRL1 and other angiogenesis pathway-related genes to identify molecular markers in the TRIBE study.
MATERIALS AND METHODS: Of 503 randomized patients, 228 had sufficient tissue for analysis. Formalin-fixed paraffin-embedded specimens were examined for expression of VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR2, ACVRL1, EphB4, and EGFL7 using reverse transcription polymerase chain reaction. A maximal χ
RESULTS: High ACVRL1 expression was associated with superior OS in both treatment arms (FOLFOXIRI [5-fluorouracil, leucovorin, oxaliplatin, irinotecan]-bevacizumab, 32.7 vs. 13.5 months, hazard ratio [HR], 0.38, P = .023; FOLFIRI [5-fluorouracil, leucovorin, irinotecan]-bevacizumab, 35.1 vs. 22.0 months, HR, 0.36, P = .006) and prolonged PFS (11.7 vs. 5.9 months, multivariate HR, 0.17; P = .001) for patients receiving FOLFOXIRI-bevacizumab on univariate and multivariate analyses. In recursive partitioning analysis, ACVRL1 was the strongest discriminator of the response rate, PFS, and OS in patients receiving FOLFOXIRI-bevacizumab and of OS in patients receiving FOLFIRI-bevacizumab. In silico validation revealed significant associations between ACVRL1 expression, disease recurrence, and 1-year survival (P < .05) among all colorectal cancer stages.
CONCLUSION: ACVRL1 expression could serve as a prognostic biomarker in metastatic colorectal cancer patients receiving chemotherapy and bevacizumab and warrants further evaluation in prospective studies.

Liu X, Wang Y, Sun L, et al.
Long noncoding RNA BC005927 upregulates EPHB4 and promotes gastric cancer metastasis under hypoxia.
Cancer Sci. 2018; 109(4):988-1000 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Hypoxia plays a critical role in the metastasis of gastric cancer (GC), yet the underlying mechanism remains largely unclear. It is also not known whether long, noncoding RNAs (lncRNAs) are involved in the contribution of hypoxia to GC metastasis. In the present study, we found that lncRNA BC005927 can be induced by hypoxia in GC cells and mediates hypoxia-induced GC cell metastasis. Furthermore, BC005927 is frequently upregulated in GC samples and increased BC005927 expression was correlated with a higher tumor-node-metastasis stage. GC patients with higher BC005927 expression had poorer prognoses than those with lower expression. Additional experiments showed that BC005927 expression is induced by hypoxia inducible factor-1 alpha (HIF-1α); ChIP assay and luciferase reporter assays confirmed that this lncRNA is a direct transcriptional target of HIF-1α. Next, we found that EPHB4, a metastasis-related gene, is regulated by BC005927 and that the expression of EPHB4 was positively correlated with that of BC005927 in the clinical GC samples assessed. Intriguingly, EPHB4 expression was also increased under hypoxia, and its upregulation by BC005927 resulted in hypoxia-induced GC cell metastasis. These results advance the current understanding of the role of BC005927 in the regulation of hypoxia signaling and offer new avenues for the development of therapeutic interventions against cancer progression.

Salgia R, Kulkarni P, Gill PS
EphB4: A promising target for upper aerodigestive malignancies.
Biochim Biophys Acta Rev Cancer. 2018; 1869(2):128-137 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
The erythropoietin-producing hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that include two major subclasses, EphA and EphB. They form an important cell communication system with critical and diverse roles in a variety of biological processes during embryonic development. However, dysregulation of the Eph/ephrin interactions is implicated in cancer contributing to tumour growth, metastasis, and angiogenesis. Here, we focus on EphB4 and review recent developments in elucidating its role in upper aerodigestive malignancies to include lung cancer, head and neck cancer, and mesothelioma. In particular, we summarize information regarding EphB4 structure/function and role in disease pathobiology. We also review the data supporting EphB4 as a potential pharmacological and immunotherapy target and finally, progress in the development of new therapeutic strategies including small molecule inhibitors of its activity is discussed. The emerging picture suggests that EphB4 is a valuable and attractive therapeutic target for upper aerodigestive malignancies.

Yin J, Cui Y, Li L, et al.
Overexpression of EPHB4 Is Associated with Poor Survival of Patients with Gastric Cancer.
Anticancer Res. 2017; 37(8):4489-4497 [PubMed] Related Publications
BACKGROUND: Increased expression of erythropoietin-producing human hepatoma (EPHB4) leads to enhanced cell migration, growth and adhesion in tumor cells. However, little is known regarding the effects of EPHB4 in gastric cancer. The present study aimed to examine the clinical relevance of EPHB4 and its association with the prognosis of gastric cancer.
MATERIALS AND METHODS: EPHB4 transcript expression in 324 gastric cancer samples with paired adjacent normal gastric tissues was determined using quantitative polymerase chain reaction and the results were statistically analyzed against patient clinicopathological data. AGS and HGC27 cell lines were transfected with EPHB4 siRNA and the effects examined by functional analysis.
RESULTS: EPHB4 mRNA levels in gastric cancer tissues were significantly elevated when compared to non-cancerous tissues (p=0.0110). Tissue samples from male patients exhibited lower expression than those from female patients (p=0.0110). Non-cardiac gastric tumors (fundus, corpus and pylorus) expressed a higher number of EPHB4 transcripts in comparison to cardiac gastric tumors (p<0.001). Increased expression of EPHB4 was significantly associated with poorer overall (p=0.0051) and progression-free (p=0.0262) survival. EPHB4 knockdown appeared to reduce post-wound migration of AGS cells (p=0.0057) and increase migration of HGC27 cells (p=0.0337). EPHB4 knockdown significantly increased adhesive ability in HGC27 (p<0.0001).
CONCLUSION: The expression of EPHB4 was increased in gastric cancer and increased EPHB4 expression was correlated with poor survival. Knockdown of EPHB4 promoted adhesion and exerted diverse effects on migration of gastric cancer cells. Further investigations may highlight its predictive and therapeutic potential in gastric cancer.

Ulivi P, Scarpi E, Chiadini E, et al.
Right- vs. Left-Sided Metastatic Colorectal Cancer: Differences in Tumor Biology and Bevacizumab Efficacy.
Int J Mol Sci. 2017; 18(6) [PubMed] Article available free on PMC after 01/09/2019 Related Publications
There is evidence of a different response to treatment with regard to the primary tumor localization (right-sided or left-sided) in patients with metastatic colorectal cancer (mCRC). We analyzed the different outcomes and biomolecular characteristics in relation to tumor localization in 122 of the 370 patients with metastatic colorectal cancer enrolled onto the phase III prospective multicenter "Italian Trial in Advanced Colorectal Cancer (ITACa)", randomized to receive first-line chemotherapy (CT) or CT plus bevacizumab (CT + B).

Marisi G, Scarpi E, Passardi A, et al.
Circulating VEGF and eNOS variations as predictors of outcome in metastatic colorectal cancer patients receiving bevacizumab.
Sci Rep. 2017; 7(1):1293 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Novel predictive biomarkers are needed to improve patient selection and optimize the use of bevacizumab (B) in metastatic colorectal cancer. We analyzed the potential of five circulating biomarkers to predict B efficacy and monitor response. Peripheral blood samples collected at baseline, at the first clinical evaluation and at progression were available for 129 patients enrolled in the prospective multicentric ITACa trial and randomized to receive FOLFOX4/FOLFIRI (CT) with (64 patients) or without B (65 patients). VEGF-A, eNOS, EPHB4, COX2 and HIF-1α mRNA levels were measured by qRT-PCR. Baseline marker expression levels and their modulation during therapy were analyzed in relation to objective response, progression-free survival and overall survival (OS). VEGF and eNOS expression was significantly correlated in both groups (Spearman's correlation coefficient = 0.80; P < 0.0001 and 0.75; P < 0.0001, respectively). B-treated patients with >30% reduction in eNOS and VEGF levels from baseline to the first clinical evaluation showed better OS than the others (median OS 31.6 months, 95% CI 21.3-49.5 months and median OS 14.4 months, 95% CI 9.0-22.7 months, respectively, HR 0.38, 95% CI 0.19-0.78, P = 0.008). A reduction in eNOS and VEGF expression from baseline to the first clinical evaluation may indicate a response to B.

Wang W, Jia WD, Hu B, Pan YY
RAB10 overexpression promotes tumor growth and indicates poor prognosis of hepatocellular carcinoma.
Oncotarget. 2017; 8(16):26434-26447 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Hepatocellular carcinoma (HCC), one of the most common and lethal cancers worldwide, has a high recurrence rate with current treatment modalities. Identifying biomarkers for early diagnosis and discovering new sufficient molecular targets for the development of targeted therapies are urgently needed. RAB10, a member of the RAS family, has been shown to be highly expressed in HCC. However, the function of RAB10 in HCC is less studied. Here we report that RAB10 acts as an oncogene in HCC. The shRNA-mediated knockdown of RAB10 significantly reduced the proliferation of HCC cells and colony formation, induced cell cycle arrest at G0/G1 phase and increased apoptosis in vitro. In addition, RAB10 knockdown suppressed HCC growth in nude mice. Moreover, RAB10 silencing decreased the phosphorylation of InsR, Met/HGFR, Ron/MST1R, Ret, c-Kit/SCFR, EphA3, EphB4, Tyro3/Dtk, Axl, Tie2/TEK, VEGFR2/KDR, Akt/PKB/Rac, S6 Ribosomal Protein and c-Abl, while the phosphorylation of HSP27, p38 MAPK, Chk2 and TAK1 increased significantly. These results suggest that RAB10 regulates cell survival and proliferation through multiple oncogenic, cell stress and apoptosis pathways. More importantly, high RAB10 expression levels in HCC cells correlated with a poor prognosis in HCC patients. Therefore, our findings revealed an oncogenic role for RAB10 in the pathogenesis of HCC and that RAB10 is a potential molecular target or a biomarker for HCC.

Takada S, Hojo M, Tanigaki K, Miyamoto S
Contribution of Endothelial-to-Mesenchymal Transition to the Pathogenesis of Human Cerebral and Orbital Cavernous Malformations.
Neurosurgery. 2017; 81(1):176-183 [PubMed] Related Publications
BACKGROUND: The analysis of gene-targeted mouse mutants has demonstrated that endothelial-to-mesenchymal transition (EndMT) is crucial to the onset and progression of cerebral cavernous malformations (CMs). It has also been shown that Notch and ephrin/Eph signaling are involved in EndMT. However, their roles in the pathogenesis of human intracranial CMs remain unclear.
OBJECTIVE: To elucidate the contribution of EndMT, the Notch pathway, and ephrin-B2/EphB4 signaling to the pathogenesis of human intracranial CMs.
METHODS: Eight human intracranial CMs (5 cerebral and 3 orbital CMs) were immunohistochemically investigated.
RESULTS: CD31 (an endothelial marker) and EndMT markers, such as α-smooth muscle actin (a mesenchymal marker) and CD44 (a mesenchymal stem cell marker), were expressed in the endothelial layer of vascular sinusoids in all cases, suggesting that endothelial cells (ECs) have acquired mesenchymal and stem-cell-like characteristics and undergone EndMT in all cerebral and orbital CMs. EndMT was observed in about 70% and 35% of ECs in cerebral and orbital CMs, respectively. In all cases, Notch3 was expressed in the endothelial layer, indicating that ECs of vascular sinusoids have acquired mesenchymal features. In all cases, both ephrin-B2 and EphB4 were detected in the endothelial layer, suggesting that ECs of vascular sinusoids are immature or malformed cells and have both arterial and venous characteristics.
CONCLUSION: EndMT plays a critical role in the pathogenesis of human cerebral and orbital CMs. Modulating EndMT is expected to be a new therapeutic strategy for cerebral and orbital CMs.

Bhatia S, Hirsch K, Sharma J, et al.
Enhancing radiosensitization in EphB4 receptor-expressing Head and Neck Squamous Cell Carcinomas.
Sci Rep. 2016; 6:38792 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas.

Yu XM, Wu YC, Liu X, et al.
Cell-Free RNA Content in Peripheral Blood as Potential Biomarkers for Detecting Circulating Tumor Cells in Non-Small Cell Lung Carcinoma.
Int J Mol Sci. 2016; 17(11) [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Circulating tumor cells (CTCs) have been implicated in tumor progression and prognosis. Techniques detecting CTCs in the peripheral blood of patients with non-small cell lung carcinoma (NSCLC) may help to identify individuals likely to benefit from early systemic treatment. However, the detection of CTCs with a single marker is challenging, owing to low specificity and sensitivity and due to the heterogeneity and rareness of CTCs. Herein, the probability of cell-free RNA content in the peripheral blood as a potential biomarker for detecting CTCs in cancer patients was investigated. An immunomagnetic enrichment of real-time reverse-transcription PCR (RT-PCR) technology for analysis of CTCs in NSCLC patients was also developed. The mRNA levels of four candidate genes, cytokeratin 7 (

Stremitzer S, Zhang W, Yang D, et al.
Expression of Genes Involved in Vascular Morphogenesis and Maturation Predicts Efficacy of Bevacizumab-Based Chemotherapy in Patients Undergoing Liver Resection.
Mol Cancer Ther. 2016; 15(11):2814-2821 [PubMed] Related Publications
Angiogenesis-related gene expression is associated with the efficacy of anti-VEGF therapy. We tested whether intratumoral mRNA expression levels of genes involved in vascular morphogenesis and early vessel maturation predict response, recurrence-free survival (RFS), and overall survival (OS) in a unique cohort of patients with colorectal liver metastases (CLM) treated with bevacizumab-based chemotherapy followed by curative liver resection. Intratumoral mRNA was isolated from resected bevacizumab-pretreated CLM from 125 patients. In 42 patients, a matching primary tumor sample collected before bevacizumab treatment was available. Relative mRNA levels of 9 genes (ACVRL1, EGFL7, EPHB4, HIF1A, VEGFA, VEGFB, VEGFC, FLT1, and KDR) were analyzed by RT-PCR and evaluated for associations with response, RFS, and OS. P values for the associations between the individual dichotomized expression level and RFS were adjusted for choosing the optimal cut-off value. In CLM, high expression of VEGFB, VEGFC, HIF1A, and KDR and low expression of EGFL7 were associated with favorable RFS in multivariable analysis (P < 0.05). High ACVRL1 levels predicted favorable 3-year OS (P = 0.041) and radiologic response (PR = 1.093, SD = 0.539, P = 0.002). In primary tumors, low VEGFA and high EGFL7 were associated with radiologic and histologic response (P < 0.05). High VEGFA expression predicted shorter RFS (10.1 vs. 22.6 months; HR = 2.83, P = 0.038). High VEGFB (46% vs. 85%; HR = 5.75, P = 0.009) and low FLT1 (55% vs. 100%; P = 0.031) predicted lower 3-year OS rates. Our data suggest that intratumoral mRNA expression of genes involved in vascular morphogenesis and early vessel maturation may be promising predictive and/or prognostic biomarkers. Mol Cancer Ther; 15(11); 2814-21. ©2016 AACR.

Pierscianek D, Wolf S, Keyvani K, et al.
Study of angiogenic signaling pathways in hemangioblastoma.
Neuropathology. 2017; 37(1):3-11 [PubMed] Related Publications
Hemangioblastoma (HB) is mainly located in the brain and the spinal cord. The tumor is composed of two major components, namely neoplastic stromal cells and abundant microvessels. Thus, hyper-vascularization is the hallmark of this tumor. Despite the identification of germline and/or epigenetic mutations of Von Hippel Lindau (VHL) gene as an important pathogenic mechanism of HB, little is known about the molecular signaling involved in this highly vascularized tumor. The present study investigated the key players of multiple angiogenic signaling pathways including VEGF/VEGFR2, EphB4/EphrinB2, SDF1α/CXCR4 and Notch/Dll4 pathways in surgical specimens of 22 HB. The expression of key angiogenic factors was detected by RT

Zhang W, Lv Y, Xue Y, et al.
Co-expression modules of NF1, PTEN and sprouty enable distinction of adult diffuse gliomas according to pathway activities of receptor tyrosine kinases.
Oncotarget. 2016; 7(37):59098-59114 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Inter-individual variability causing elevated signaling of receptor tyrosine kinases (RTK) may have hampered the efficacy of targeted therapies. We developed a molecular signature for clustering adult diffuse gliomas based on the extent of RTK pathway activities. Glioma gene modules co-expressed with NF1 (NF1-M), Sprouty (SPRY-M) and PTEN (PTEN-M) were identified, their signatures enabled robust clustering of adult diffuse gliomas of WHO grades II-IV from five independent data sets into two subtypes with distinct activities of RAS-RAF-MEK-MAPK cascade and PI3K-AKT pathway (named RMPAhigh and RMPAlow subtypes) in a morphology-independent manner. The RMPAhigh gliomas were associated with poor prognosis compared to the RMPAlow gliomas. The RMPAhigh and RMPAlow glioma subtypes harbored unique sets of genomic alterations in the RTK signaling-related genes. The RMPAhigh gliomas were enriched in immature vessel cells and tumor associated macrophages, and both cell types expressed high levels of pro-angiogenic RTKs including MET, VEGFR1, KDR, EPHB4 and NRP1. In gliomas with major genomic lesions unrelated to RTK pathway, high RMPA signature was associated with short survival. Thus, the RMPA signatures capture RTK activities in both glioma cells and glioma microenvironment, and RTK signaling in the glioma microenvironment contributes to glioma progression.

McCall JL, Gehring D, Clymer BK, et al.
KSR1 and EPHB4 Regulate Myc and PGC1β To Promote Survival of Human Colon Tumors.
Mol Cell Biol. 2016; 36(17):2246-61 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Identification and characterization of survival pathways active in tumor cells but absent in normal tissues provide opportunities to develop effective anticancer therapies with reduced toxicity to the patient. We show here that, like kinase suppressor of Ras 1 (KSR1), EPH (erythropoietin-producing hepatocellular carcinoma) receptor B4 (EPHB4) is aberrantly overexpressed in human colon tumor cell lines and selectively required for their survival. KSR1 and EPHB4 support tumor cell survival by promoting the expression of downstream targets, Myc and the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1β (PGC1β). While KSR1 promotes the aberrant expression of Myc and the PGC1β protein via a posttranscriptional mechanism, EPHB4 has a greater effect on Myc and PGC1β expression via its ability to elevate mRNA levels. Subsequent analysis of the posttranscriptional regulation demonstrated that KSR1 promotes the translation of Myc protein. These findings reveal novel KSR1- and EPHB4-dependent signaling pathways supporting the survival of colorectal cancer cells through regulation of Myc and PGC1β, suggesting that inhibition of KSR1 or EPHB4 effectors may lead to selective toxicity in colorectal tumors.

Moshayedi M, Barneh F, Haghjooy-Javanmard S, et al.
A rapid and sensitive method for EphB4 identification as a diagnostic and therapeutic biomarker in invasive breast cancer.
J Cancer Res Ther. 2016 Jan-Mar; 12(1):188-92 [PubMed] Related Publications
BACKGROUND: In the roadmap to design diagnostic and therapeutic markers for breast cancer, EphB4 is of special interest due to its multiple roles in tumor initiation, progression and invasion. The aim of present study was to characterize a rapid and sensitive ELISA-based method to measure EphB4 level and its phosphorylation status following stimulation with its ligand, ephrinB2, in an invasive breast cancer cell line.
MATERIALS AND METHODS: MDA-MB-231 breast cancer cells were lysed and EphB4 level was measured using ELISA. EphB4 level was measured in sub- and post-confluent states in culture dishes. Receptor phosphorylation was also detected by ELISA assay, using various concentrations of pre-clustered ephrinB2 for 20 minutes.
RESULTS: Expression of EphB4 receptor was detected by ELISA in all samples. EphB4 level was significantly higher in post.confluent than sub.confluent cells. Phosphorylated receptor was also detectable with this method when cells were exogenously stimulated.
CONCLUSIONS: Quantitative data from ELISA manifested a difference between levels of EphB4 in two states of different invasive properties. Moreover, ELISA method may be considered rapid and sensitive enough to detect even low levels of total and phosphorylated EphB4 Cost-effectiveness of this method for the detection of differential expression of EphB4 proteins in clinics is also noticeable.

Pradeep S, Huang J, Mora EM, et al.
Erythropoietin Stimulates Tumor Growth via EphB4.
Cancer Cell. 2015; 28(5):610-622 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
While recombinant human erythropoietin (rhEpo) has been widely used to treat anemia in cancer patients, concerns about its adverse effects on patient survival have emerged. A lack of correlation between expression of the canonical EpoR and rhEpo's effects on cancer cells prompted us to consider the existence of an alternative Epo receptor. Here, we identified EphB4 as an Epo receptor that triggers downstream signaling via STAT3 and promotes rhEpo-induced tumor growth and progression. In human ovarian and breast cancer samples, expression of EphB4 rather than the canonical EpoR correlated with decreased disease-specific survival in rhEpo-treated patients. These results identify EphB4 as a critical mediator of erythropoietin-induced tumor progression and further provide clinically significant dimension to the biology of erythropoietin.

Liersch-Löhn B, Slavova N, Buhr HJ, Bennani-Baiti IM
Differential protein expression and oncogenic gene network link tyrosine kinase ephrin B4 receptor to aggressive gastric and gastroesophageal junction cancers.
Int J Cancer. 2016; 138(5):1220-31 [PubMed] Related Publications
Transmembrane tyrosine-kinase Ephrin receptors promote tumor progression and/or metastasis of several malignancies including leukemia, follicular lymphoma, glioma, malignant pleural mesothelioma, papillary thyroid carcinoma, sarcomas and ovarian, breast, bladder and non-small cell lung cancers. They also drive intestinal stem cell proliferation and positioning, control intestinal tissue boundaries and are involved in liver, pancreatic and colorectal cancers, indicating involvement in additional digestive system malignancies. We investigated the role of Ephrin-B4 receptor (EPHB4), and its ligand EFNB2, in gastric and gastroesophageal junction cancers in patient cohorts through computational, mathematical, molecular and immunohistochemical analyses. We show that EPHB4 is upregulated in preneoplastic gastroesophageal lesions and its expression further increased in gastroesophageal cancers in several independent cohorts. The closely related EPHB6 receptor, which also binds EFNB2, was downregulated in all tested cohorts, consistent with its tumor-suppressive properties in other cancers. EFNB2 expression is induced in esophageal cells by acidity, suggesting that gastroesophageal reflux disease (GERD) may constitute an early triggering event in activating EFNB2-EPHB4 signaling. Association of EPHB4 to both Barrett's esophagus and to advanced tumor stages, and its overexpression at the tumor invasion front and vascular endothelial cells intimate the notion that EPHB4 may be associated with multiple steps of gastroesophageal tumorigenesis. Analysis of oncogenomic signatures uncovered the first EPHB4-associated gene network (false discovery rate: 7 × 10(-90) ) composed of a five-transcription factor interconnected gene network that drives proliferation, angiogenesis and invasiveness. The EPHB4 oncogenomic network provides a molecular basis for its role in tumor progression and points to EPHB4 as a potential tumor aggressiveness biomarker and drug target in gastroesophageal cancers.

Yang X, Yang Y, Tang S, et al.
EphB4 inhibitor overcome the acquired resistance to cisplatin in melanomas xenograft model.
J Pharmacol Sci. 2015; 129(1):65-71 [PubMed] Related Publications
The purpose of this paper is to investigate the possible mechanisms of resistance to chemotherapy in melanoma from the perspective of molecular biology and to discuss the strategies to overcome them. Cisplatin, a DNA-damaging compound that triggers apoptotic cell death, is commonly used in the treatment of malignant melanoma. However, most patients develop mechanisms of acquired resistance and about 25% of them do not achieve tumor regression at all, due to intrinsic resistance to therapy. In the current study, we reported the tumor xenografts of the human A375 melanoma, after 40-weeks' consecutive therapy with cisplatin that developed resistance as a result of EphB4 overexpression. Moreover, the expression of phospho-AKT and phospho-ERK were significantly increased in cisplatin-resistant tumors. In addition, combined of cisplatin with EphB4 selective inhibitor could abrogate this acquired mechanism of drug resistance due to an enhanced apoptotic effect in cisplatin-resistant xenografts. In summary, these results help to understand the mechanisms of acquired resistance to chemotherapy and provide important information for clinical treatment strategies.

Huang G, Li M
The role of EphB4 and IGF-IR expression in breast cancer cells.
Int J Clin Exp Pathol. 2015; 8(5):5997-6004 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
OBJECTIVE: to investigate the role of EphB4 and IGF-1R in the proliferation and migration of breast cancer.
METHODS: The relative mRNA levels of EphB4 were measured by RT-PCR. The proliferation of the cells was determined by MTT assay, and cells migration and invasive ability was analyzed using the scratch migration assay.
RESULTS: The expression of EphB4 in control group was significantly decreased when compared with IGF-I group (P<0.001). The expression of EphB4 in IGF-I+LY and LY group were lower than that of the control group (P<0.001).The cell proliferation and migration ability of the cells in IGF-I group increased significantly compared to the cells in the control group (P<0.001), while the cells in IGF-1+LY group and LY group showed a decreased proliferation and migration ability compared to the control group (P<0.001).
CONCLUSION: IGF-IR might be a upstream gene of EphB4. Besides, higher expression of EphB4 shows increased tumor proliferation and migration in breast cells. The study of EphB4 upstream gene and signaling pathway can provide more targeted anti-tumor point selection for targeted therapy.

Dimasi N, Fleming R, Hay C, et al.
Development of a Trispecific Antibody Designed to Simultaneously and Efficiently Target Three Different Antigens on Tumor Cells.
Mol Pharm. 2015; 12(9):3490-501 [PubMed] Related Publications
Targeting Eph (erythropoietin producing hepatoma) receptors with monoclonal antibodies is being explored as therapy for several types of cancer. To test whether simultaneous targeting of EphA2, EphA4, and EphB4 would be an effective approach to cancer therapy, we generated a recombinant trispecific antibody using the variable domain genes of anti-EphA2, anti-EphA4, and anti-EphB4 monoclonal antibodies. A multidisciplinary approach combining biochemical, biophysical, and cellular-based assays was used to characterize the trispecific antibody in vitro and in vivo. Here we demonstrate that the trispecific antibody is expressed at high levels by mammalian cells, monodispersed in solution, thermostable, capable of simultaneously binding the three receptors, and able to activate the three targets effectively as evidenced by receptor internalization and degradation both in vitro and in vivo. Furthermore, pharmacokinetic analysis using tumor-bearing nude mice showed that the trispecific antibody remains in the circulation similarly to its respective parental antibodies. These results indicate that simultaneous blockade of EphA2, EphA4, and EphB4 could be an attractive approach to cancer therapy.

Ferguson BD, Tan YH, Kanteti RS, et al.
Novel EPHB4 Receptor Tyrosine Kinase Mutations and Kinomic Pathway Analysis in Lung Cancer.
Sci Rep. 2015; 5:10641 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Lung cancer outcomes remain poor despite the identification of several potential therapeutic targets. The EPHB4 receptor tyrosine kinase (RTK) has recently emerged as an oncogenic factor in many cancers, including lung cancer. Mutations of EPHB4 in lung cancers have previously been identified, though their significance remains unknown. Here, we report the identification of novel EPHB4 mutations that lead to putative structural alterations as well as increased cellular proliferation and motility. We also conducted a bioinformatic analysis of these mutations to demonstrate that they are mutually exclusive from other common RTK variants in lung cancer, that they correspond to analogous sites of other RTKs' variations in cancers, and that they are predicted to be oncogenic based on biochemical, evolutionary, and domain-function constraints. Finally, we show that EPHB4 mutations can induce broad changes in the kinome signature of lung cancer cells. Taken together, these data illuminate the role of EPHB4 in lung cancer and further identify EPHB4 as a potentially important therapeutic target.

Mertens-Walker I, Fernandini BC, Maharaj MS, et al.
The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of integrin-β8 in prostate cancer cells.
BMC Cancer. 2015; 15:164 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
BACKGROUND: The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways.
METHODS: We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion.
RESULTS: We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin β8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion.
CONCLUSIONS: These results reveal that EphB4 regulates integrin β8 expression and that integrin β8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin β8 may be a new treatment strategy for prostate cancer.

Olmez I, Shen W, McDonald H, Ozpolat B
Dedifferentiation of patient-derived glioblastoma multiforme cell lines results in a cancer stem cell-like state with mitogen-independent growth.
J Cell Mol Med. 2015; 19(6):1262-72 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Emerging evidence shows that glioblastoma multiforme (GBM) originates from cancer stem cells (CSCs). Characterization of CSC-specific signalling pathways would help identify new therapeutic targets and perhaps lead to the development of more efficient therapies selectively targeting CSCs. Here; we successfully dedifferentiated two patient-derived GBM cell lines into CSC-like cells (induced glioma stem cells, iGSCs) through expression of Oct4, Sox2 and Nanog transcription factors. Transformed cells exhibited significant suppression of epidermal growth factor receptor and its downstream pathways. Compared with parental GBM cells, iGSCs formed large neurospheres even in the absence of exogenous mitogens; they exhibited significant sensitivity to salinomycin and chemoresistance to temozolomide. Further characterization of iGSCs revealed induction of NOTCH1 and Wnt/β-catenin signalling and expression of CD133, CD44 and ALDH1A1. Our results indicate that iGSCs may help us understand CSC physiology and lead to development of potential therapeutic interventions aimed at differentiating tumour cells to render them more sensitive to chemotherapy or other standard agents.

Mertens-Walker I, Lisle JE, Nyberg WA, et al.
EphB4 localises to the nucleus of prostate cancer cells.
Exp Cell Res. 2015; 333(1):105-15 [PubMed] Related Publications
The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.

Becerikli M, Merwart B, Lam MC, et al.
EPHB4 tyrosine-kinase receptor expression and biological significance in soft tissue sarcoma.
Int J Cancer. 2015; 136(8):1781-91 [PubMed] Related Publications
Soft tissue sarcomas (STS) are heterogeneous malignant tumors of mesenchymal origin. Due to low incidence and high number of different histological subtypes, their pathogenesis and thus potential targets for their therapy remain barely investigated. Several studies revealed significant higher EPHB4 expression in malignancies such as prostate and colorectal cancer showing survival advantages for these tumor cells. Therefore we studied the expression of EPHB4 in a total of 46 clinical human specimens of different STS and human fibroblasts. EPHB4 mRNA and protein expression were significantly increased in synovial sarcoma. After targeting EPHB4 in fibrosarcoma, synovial sarcoma, liposarcoma and MFH sarcoma cell lines by siRNA or by inhibition of autophosphorylation using the specific EPHB4 kinase inhibitor NVP-BHG712 a decreased proliferation rate/vitality of synovial- and fibrosarcoma cells was observed. Silencing of EPHB4 significantly reduced the transmigration of synovial sarcoma cells towards fibroblasts and endothelial cells. In addition, we assessed the anti-metastatic effect of EPHB4 inhibition in vivo by intraperitoneal administration of the EPHB4 inhibitor in an appropriate sarcoma lung metastasis xenograft model. As result 43% of NVP-BHG712 treated mice (n = 3/7) developed pulmonary metastases whereas all control mice (n = 5) revealed lung metastases. The residual 57% of mice (n = 4/7) showed only small local tumor cell spots. Size measurements of the Vimentin positive area explained significant decrease in lung metastasis formation (p < 0.05) after EPHB4 kinase inhibition. In summary, these data provide first evidence of the importance of EPHB4 in the tumorigenesis of synovial sarcoma and present EPHB4 as a potential target in the therapy of this malignancy.

McKinney N, Yuan L, Zhang H, et al.
EphrinB1 expression is dysregulated and promotes oncogenic signaling in medulloblastoma.
J Neurooncol. 2015; 121(1):109-18 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Eph receptors and ephrin ligands are master regulators of oncogenic signaling required for proliferation, migration, and metastasis. Yet, Eph/ephrin expression and activity in medulloblastoma (MB), the most common malignant brain tumor of childhood, remains poorly defined. We hypothesized that Eph/ephrins are differentially expressed by sonic hedgehog (SHH) and non-SHH MB and that specific members contribute to the aggressive phenotype. Affymetrix gene expression profiling of 29 childhood MB, separated into SHH (N = 11) and non-SHH (N = 18), was performed followed by protein validation of selected Eph/ephrins in another 60 MB and two MB cell lines (DAOY, D556). Functional assays were performed using MB cells overexpressing or deleted for selected ephrins. We found EPHB4 and EFNA4 almost exclusively expressed by SHH MB, whereas EPHA2, EPHA8, EFNA1 and EFNA3 are predominantly expressed by non-SHH MB. The remaining family members, except EFNB1, are ubiquitously expressed by over 70-90 % MB, irrespective of subgroup. EFNB1 is the only member differentially expressed by 28 % of SHH and non-SHH MB. Corresponding protein expression for EphB/ephrinB1 and B2 was validated in MB. Only ephrinB2 was also detected in fetal cerebellum, indicating that EphB/ephrinB1 expression is MB-specific. EphrinB1 immunopositivity localizes to tumor cells within MB with the highest proliferative index. EphrinB1 overexpression promotes EphB activation, alters F-actin distribution and morphology, decreases adhesion, and significantly promotes proliferation. Either silencing or overexpression of ephrinB1 impairs migration. These results indicate that EphrinB1 is uniquely dysregulated in MB and promotes oncogenic responses in MB cells, implicating ephrinB1 as a potential target.

Li X, Choi WW, Yan R, et al.
The differential expression of EphB2 and EphB4 receptor kinases in normal bladder and in transitional cell carcinoma of the bladder.
PLoS One. 2014; 9(8):e105326 [PubMed] Article available free on PMC after 01/09/2019 Related Publications
Effective treatment of transitional cell carcinoma (TCC) of the bladder requires early diagnosis. Identifying novel molecular markers in TCC would guide the development of diagnostic and therapeutic targets. Ephrins mediate signals via tyrosine kinase activity that modulates diverse physiologic and developmental processes, and ephrins are increasingly implicated in carcinogenesis. The aim of our study was to examine the differential regulation of EphB4 and EphB2 in normal bladder and in TCC of the bladder in 40 patients undergoing radical cystectomy for curative intent. Immunostaining and Western blotting revealed that normal urothelium expresses EphB2 (20 of 24 cases, 83% of the time) not EphB4 (0 of 24 cases, 0%). In sharp contrast, TCC specimens show loss of EphB2 expression (0 of 34 cases, 0%) and gain of EphB4 expression (32 of 34, 94%). Furthermore, EphB4 signal strength statistically correlated with higher tumor stage, and trended toward the presence of carcinoma in situ (CIS). These results are confirmed by analysis of normal urothelial and tumor cell lines. EphB2 is not a survival factor in normal urothelium, while EphB4 is a survival factor in TCC. Treatment of bladder tumor xenograft with an EphB4 inhibitor sEphB4-HSA leads to 62% tumor regression and complete remission when combined with Bevacizumab. Furthermore, tissue analysis revealed that sEphB4-HSA led to increased apoptosis, decreased proliferation, and reduced vessel density, implicating direct tumor cell targeting as well as anti-angiogenesis effect. In summary loss of EphB2 and gain of EphB4 expression represents an inflection point in the development, growth and possibly progression of TCC. Therapeutic compounds targeting EphB4 have potential for diagnosing and treating TCC.

Li M, Zhao J, Qiao J, et al.
EphB4 regulates the growth and migration of pancreatic cancer cells.
Tumour Biol. 2014; 35(7):6855-9 [PubMed] Related Publications
Pancreatic cancer is a serious threat to human life. Moreover, its treatment is complicated and its prognosis is very poor. Therefore, a new method for the diagnosis and treatment of pancreatic cancer is very essential. In this study, a eukaryotic expression plasmid targeting EphB4 was constructed and transfected into PANC-1 pancreatic cancer cells to investigate the inhibition of cell growth and the progression of iRNA against EphB4. This study provides the basis for the gene therapy of pancreatic cancer. The recombinant eukaryotic EphB4 expression plasmid, pSIREN-RetroQ-ZsGreen-EphB4 and a negative control plasmid, pSIREN-RetroQ-ZsGreen-N, were constructed. At 48 h after transfection, the relative messenger RNA (mRNA) and protein levels of EphB4 were measured by RT-PCR and western blot. The proliferation of the transfected cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while cell migration ability was analyzed using the scratch migration assay. At 48 h after transient transfection, EphB4 mRNA expression was significantly decreased in transfected PANC-1 cells as compared to the control group (P < 0.01). In vitro, inhibition of EphB4 expression weakened the proliferation and cell migration ability of PANC-1 cells compared to the control group. The small interfering RNA (siRNA) eukaryotic expression plasmid vector targeting EphB4 was successfully constructed and effectively transfected into PANC-1 cells. The recombinant plasmid can inhibit the expression of EphB4 mRNA and protein in PANC-1 cells, as well as cell growth and migration.

Takahashi Y, Itoh M, Nara N, Tohda S
Effect of EPH-ephrin signaling on the growth of human leukemia cells.
Anticancer Res. 2014; 34(6):2913-8 [PubMed] Related Publications
BACKGROUND: Signaling induced by binding of erythropoietin-producing hepatoma-amplified sequence (EPH) receptors to their cell-surface ephrin ligands is implicated in hematopoiesis and growth of various cancer cells. However, the roles of EPH-ephrin signaling in leukemia have not been elucidated. We investigated the effects of EPHB4 and ephrin B2 on the growth of leukemia cells.
MATERIALS AND METHODS: Seven human leukemia cell lines were used to examine the effects of recombinant ephrin B2 and EPHB4 on cell proliferation by colorimetric WST-1 assay and colony assays; on protein tyrosine phosphorylation; and on mRNA expression by reverse transcription-polymerase chain reaction and microarray analysis.
RESULTS: In an erythroid leukemia-derived cell line AA, exogenous ephrin B2 induced proliferation and colony formation; in addition, it up-regulated protein tyrosine phosphorylation and the expression of growth-related genes such as FBJ murine osteosarcoma viral oncogene homolog B and v-src avian sarcoma viral oncogene homolog.
CONCLUSION: Growth-promoting effects of ephrin B2 were observed in an erythroid leukemia cell line, suggesting that the EPH-ephrin signaling may be involved in the pathology of leukemia.

Hu F, Tao Z, Shen Z, et al.
Down-regulation of EphB4 phosphorylation is necessary for esophageal squamous cell carcinoma tumorigenecity.
Tumour Biol. 2014; 35(7):7225-32 [PubMed] Related Publications
Eph/ephrin signaling system plays a very important role in the tumorigenesis and the formation of blood vessel. However, the function of EphB4 and its ligand ephrin B2 in the carcinogenesis of esophageal squamous cell carcinoma (ESCC) is not fully understood. Here, it was found that the expression of EphB4 was up-regulated in ESCC tissues compared with the paired normal tissues, while ephrin B2 was down-regulated in ESCC samples. Phosphorylation of EphB4 induced by its ligand ephrin B2-Fc inhibited the growth, migration and colony formation of ESCC cells. Moreover, over-expression of EphB4 or EphB4 kinase dead mutant (EphB4 KD) in ESCC cells promoted cell growth and migration, suggesting EphB4 promoted cell growth and migration independent of its kinase activity. Furthermore, we found that EphB4 interacted with the adaptor protein RACK1 and RACK1 decreased the phosphorylation level of EphB4. Taken together, our study revealed the important function and regulation of EphB4 in the progression of ESCC and suggested EphB4 as a novel target for the treatment of ESCC.

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