EphB2, EphB4, and ephrin-B1 expression and localization in postnatal developing epididymis in mice.

BACKGROUND: Eph receptors and ephrin ligands, the transmembrane proteins, function as a mechanism of communication between cells. Therefore, we intended to explore the expression array of EphB2 and EphB4 receptors and ephrin-B1 ligand in postnatal developing mouse epididymis during 1 day to 8 weeks using RT-PCR amplification and immunofluorescence staining. RESULTS: RT-PCR analysis indicated that the expression levels of EphB2, EphB4, and ephrin-B1 in the epididymis declined with the advancement of age during the initial phases of postnatal development and stayed relatively near to adult levels until 4 weeks. We discovered that the predominant compartments expressing EphB2/B4 and ephrin-B1 emerged in the excurrent duct epithelia of postnatal developing epididymis until 3 weeks. Consequently, even before spermatozoa reach the excurrent duct in epididymis, at the age of 3 weeks, the epididymal excurrent duct system exhibits characteristics similar to those of an adult in terms of expression of EphB2/B4 and ephrin-B1. Moreover, ephrin-B1 was expressed in epididymal epithelial cells throughout the development and EphB4 was expressed only in early postnatal stages while basal cells expressed EphB4 throughout the postnatal development. CONCLUSION: The study represents the first expression analysis of ephrin-B1, EphB2, and EphB4 in the normal mouse epididymis during the postnatal development.

EGR1 modulates EPHB4-induced trophoblast dysfunction in recurrent spontaneous abortion†.

Recurrent spontaneous abortion, defined as at least three unexplained abortions occurring before the 20-24 week of pregnancy, has a great impact on women's quality of life. Ephrin receptor B4 has been associated with trophoblast function in preeclampsia. The present study aimed to verify the hypothesis that ephrin receptor B4 regulates the biological functions of trophoblasts in recurrent spontaneous abortion and to explore the upstream mechanism. Ephrin receptor B4 was overexpressed in mice with recurrent spontaneous abortion. Moreover, ephrin receptor B4 inhibited trophoblast proliferation, migration, and invasion while promoting apoptosis. Downregulation of early growth response protein 1 expression in mice with recurrent spontaneous abortion led to ephrin receptor B4 overexpression. Poor expression of WT1-associated protein in mice with recurrent spontaneous abortion reduced the modification of early growth response protein 1 mRNA methylation, resulting in decreased early growth response protein 1 mRNA stability and expression. Overexpression of WT1-associated protein reduced the incidence of recurrent spontaneous abortion in mice by controlling the phenotype of trophoblasts, which was reversed by early growth response protein 1 knockdown. All in all, our findings demonstrate that dysregulation of WT1-associated protein contributes to the instability of early growth response protein 1, thereby activating ephrin receptor B4-induced trophoblast dysfunction in recurrent spontaneous abortion. Our study provides novel insights into understanding the molecular pathogenesis of recurrent spontaneous abortion.

SAM domain variants of EPHB4 associated with aberrant signaling are linked to lymphatic-related fetal hydrops and facial dysmorphology.

Variants in EPHB4 (Ephrin type B receptor 4), a transmembrane tyrosine kinase receptor, have been identified in individuals with various vascular anomalies including Capillary Malformation-Arteriovenous Malformation syndrome 2 and lymphatic-related (non-immune) fetal hydrops (LRHF). Here, we identify two novel variants in EPHB4 that disrupt the SAM domain in two unrelated individuals. Proband 1 presented within the LRHF phenotypic spectrum with hydrops, and proband 2 presented with large nuchal translucency prenatally that spontaneously resolved in addition to dysmorphic features on exam postnatally. These are the first disease associated variants identified that do not disrupt EPHB4 protein expression or tyrosine-kinase activity. We identify that EPHB4 SAM domain disruptions can lead to aberrant downstream signaling, with a loss of the SAM domain resulting in elevated MAPK signaling in proband 1, and a missense variant within the SAM domain resulting in increased cell proliferation in proband 2. This data highlights that a functional SAM domain is required for proper EPHB4 function and vascular development.

Unilateral segmental presentation and a novel EPHB4 gene variant in capillary malformation-arteriovenous malformation type 2.

Capillary malformation-arteriovenous malformation is a rare autosomal dominant disorder associated with EPHB4 loss-of-function mutations. We report the unique presentation of a 6-year-old girl with multiple capillary malformations in a unilateral segmental distribution affecting the right hemiface, right upper chest, and right arm associated with overgrowth. Targeted next-generation sequencing on a tissue sample revealed a novel heterozygotic variant in the EPHB4 gene (NM_004444.5 (EPHB4): c.715T>A, p.[Cys239Ser]). This case highlights a distinct presentation of CM-AVM type 2 and showcases a new variant in EPHB4 not previously reported in the literature.

Discovery and Characterization of Ephrin B2 and EphB4 Dysregulation and Novel Mutations in Cerebral Cavernous Malformations: In Vitro and Patient-Derived Evidence of Ephrin-Mediated Endothelial Cell Pathophysiology.

Intracranial vascular malformations manifest on a continuum ranging from predominantly arterial to predominantly venous in pathology. Cerebral cavernous malformations (CCMs) are capillary malformations that exist at the midpoint of this continuum. The axon guidance factor Ephrin B2 and its receptor EphB4 are critical regulators of vasculogenesis in the developing central nervous system. Ephrin B2/EphB4 dysregulation has been implicated in the pathogenesis of arterial-derived arteriovenous malformations and vein-based vein of Galen malformations. Increasing evidence supports the hypothesis that aberrant Ephrin B2/EphB4 signaling may contribute to developing vascular malformations, but their role in CCMs remains largely uncharacterized. Evidence of Ephrin dysregulation in CCMs would be important to establish a common link in the pathogenic spectrum of EphrinB2/Ephb4 dysregulation. By studying patient-derived primary CCM endothelial cells (CCMECs), we established that CCMECs are functionally distinct from healthy endothelial cell controls; CCMECs demonstrated altered patterns of migration, motility, and impaired tube formation. In addition to the altered phenotype, the CCMECs also displayed an increased ratio of EphrinB2/EphB4 compared to the healthy endothelial control cells. Furthermore, whole exome sequencing identified mutations in both EphrinB2 and EphB4 in the CCMECs. These findings identify functional alterations in the EphrinB2/EphB4 ratio as a feature linking pathophysiology across the spectrum of arterial, capillary, and venous structural malformations in the central nervous system while revealing a putative therapeutic target.

Erythropoietin induces odontoblastic differentiation of human-derived pulp stem cells via EphB4-Mediated MAPK signaling pathway.

OBJECTIVES: Human-derived pulp stem cells play key roles during dentinogenesis. Erythropoietin is reportedly involved in osteoblastogenesis and facilitates bone formation. However, the mechanism is still unknown. This research was to study the potential of erythropoietin in enhancing odontoblastic differentiation of human-derived pulp stem cells and to determine the underlying mechanism. METHODS: The human-derived pulp stem cells were treated with erythropoietin, EphB4 inhibitor, and MAPK inhibitors, and the odontoblastic differentiation was measured by ALP staining, ALP activity assay, alizarin red S staining, and their quantitative analysis, and RT-qPCR of DSPP, DMP1, OCN, and RUNX2. The direct pulp capping model was established to evaluate the formation of tertiary dentin after treatment with erythropoietin. Western blot assay was conducted to assess relevant protein expressions in the phosphorylated EphB4 and MAPK pathway. RESULTS: The results showed that erythropoietin promoted odontoblastic differentiation of human-derived pulp stem cells at 20 U/ml. Erythropoietin induced tertiary dentin formation in vivo. The potential mechanism of this was upregulating phosphorylated EphB4 and phosphorylated MAPK; furthermore, this effect could be decreased by EphB4 inhibitors, which inhibited MAPK phosphorylation. Blockage of MAPK pathways attenuated human-derived pulp stem cells' odontoblastic differentiation, suggesting that MAPK pathways are involved. CONCLUSION: Erythropoietin induced tertiary dentin formation in vivo. And erythropoietin enhanced human-derived pulp stem cells' odontoblastic differentiation via the EphB4-mediated MAPK signaling pathway.

Osteoblast Jmjd3 regulates osteoclastogenesis via EphB4 and RANKL signalling.

BACKGROUND: Osteoblasts suppress osteoclastogenesis during the reversal phase of bone remodelling and the mechanism needs to be further investigated. Here, we investigated the role of histone demethylase Jumonji domain-containing 3 (Jmjd3) in osteoblasts on regulating osteoclastogenesis. METHODS: Jmjd3 expression was silenced in osteoblasts. Osteoblasts and osteoclasts were co-cultured in direct or indirect contact ways, and osteoclastogenesis was determined by tartrate-resistant acid phosphatase (TRAP) staining and Western blotting. Additionally, Ephrin receptor B4 (EphB4) and receptor activator of nuclear factor-kappa Β ligand (RANKL) expression were quantified in osteoblasts via real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. Subsequently, EphB4 was overexpressed in osteoblasts and RANKL expression and osteoclastogenesis was quantified. RESULTS: Osteoclastogenesis and marker protein expression levels was promoted when osteoclasts were co-cultured with Jmjd3-silenced osteoblasts. Silencing of Jmjd3 expression in osteoblasts decreased EphB4 expression, owing to suppression of demethylation of H3K27me3 on the promoter region of EphB4. Whereas RANKL expression was upregulated in Jmjd3-silenced osteoblasts. Overexpression of EphB4 in osteoblasts inhibited osteoclastogenesis and RANKL expression. CONCLUSION: Jmjd3 in osteoblasts is a crucial regulator of osteoblast-to-osteoclast communication through EphB4-EphrinB2, RANKL-RANK and EphB4-RANKL signalling axes, suggesting the pivotal role of Jmjd3 in bone remodelling process in bone destruction disease such as chronic apical periodontitis.

De novo cerebral cavernous malformations with PIK3CA somatic mutation and EPHB4 germline mutation in a child with multiple developmental venous anomalies and cutaneous vascular malformations.

Cavernous malformations (CM) that arise in the central nervous system have long been considered congenital, while there are many reports of de novo non-familial-type CM adjacent to developmental venous anomalies (DVA) or after radiation. The mechanisms that cause de novo formations of sporadic cavernous malformation (CM) still remain unknown and purely speculative. We report a case of de novo cerebral CM in a child with multiple developmental venous anomalies and cutaneous vascular malformations. Histological examination and whole-exome sequencing (WES) was performed on a fresh-frozen tissue sample of the CM. WES revealed 2 missense non-synonymous variants in two genes, EPHB4 and PIK3CA. The mutant allele of EPHB4 (NM_004444.4: c.1840 T > C, p.Y614H) appeared in 248/469 WES reads (allele frequency, 52.88%), which suggested the mutation a germline one. PIK3CA (NM_006218.2) somatic mutations were found in exon 9: c.1624G > A (p.Glu542Lys) with variant frequency of 2.2% (2/89 WES reads). We did not find any non-synonymous mutations of the three CCM genes (KRIT1, CCM2, and PDCD10) in this patient. Our findings suggested that the combination of gain of function in PIK3CA and loss of function in EPHB4 may play an important role in the pathogenesis of CM, which can develop in acquired form like tumorigenesis.

The role of ephrinB2-EphB4 signalling in bone remodelling during orthodontic tooth movement.

OBJECTIVE: The aim of this study was to investigate the role of ephrinB2-EphB4 signalling in alveolar bone remodelling on the tension side during orthodontic tooth movement (OTM). MATERIALS AND METHODS: An OTM model was established on sixty 8-week-old male Wistar rats. They were randomly divided into the experimental group and the control group. The animals in the experimental group were administrated with subcutaneous injection of EphB4 inhibitor NVP-BHG712 every other day, whereas the control group received only the vehicle. Samples containing the maxillary first molar and the surrounding bone were collected after 0, 3, 7, 14 and 21 days of tooth movement. RESULTS: EphrinB2-EphB4 signalling was actively expressed on the tension side during tooth movement. Micro-CT analysis showed the distance of tooth movement in the experimental group was significantly greater than that of the control group (P < .05) with significantly increased trabecular separation (Tb. Sp) and decreased trabecular number (Tb. N) from day 14 to day 21. The number of osteoclasts significantly increased in the experimental group compared with the control group after 3 and 7 days of tooth movement (P < .05). The expressions of alkaline phosphatase (ALP) and osteopontin (OPN) were significantly reduced by inhibition of EphB4 (P < .05). CONCLUSION: The inhibition of EphB4 suppressed bone formation and enhanced bone resorption activities on the tension side of tooth movement. The ephrinB2-EphB4 signalling might play an important role in alveolar bone remodelling during OTM.

Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IRA Signal in Malignant Mesothelioma.

EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein-protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as "DTX3c"), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway.

The protective role of Ephrin-B2/EphB4 signaling in osteogenic differentiation under inflammatory environment.

Inflammation and alveolar bone destruction constitute the main pathological process of periodontitis. However, the molecular mechanisms of bone destruction under the inflammation environment remain unclear. This study aims to explore the role of Ephrin-B2/EphB4 signaling in osteogenic differentiation under the inflammation environment. Mouse pre-osteoblasts MC3T3-E1 were pretreated with lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS). The Ephrin-B2/EphB4 signaling was activated, and the osteogenic differentiation of cells was examined. The results showed that activation of Ephrin-B2/EphB4 signaling promoted the expression levels of osteogenic differentiation-related genes, and also relieved the inhibitory effect of Pg-LPS on osteogenesis. Noticeably, the effect of Ephrin-B2/EphB4 signaling might be related to the mitogen-activated protein kinase (MAPK) pathway. While applying Ephrin-B2-Fc and EphB4-Fc to periodontitis mice, we observed the reduction of alveolar crest destruction. The current study revealed the possible role of Ephrin-B2/EphB4 signaling in reducing bone destruction in periodontitis and suggested its potential values for further research.

Inulin coated Mn3O4 nanocuboids coupled with RNA interference reverse intestinal tumorigenesis in Apc knockout murine colon cancer models.

This study reports the development and pre-clinical evaluation of biodrug using RNA interference and nanotechnology. The major challenges in achieving targeted gene silencing in vivo include the stability of RNA molecules, accumulation into pharmacological levels, and site-specific targeting of the tumor. We report the use of Inulin for coating the arginine stabilized manganese oxide nanocuboids (MNCs) for oral delivery of shRNA to the gut. Furthermore, bio-distribution analysis exhibited site-specific targeting in the intestines, improved pharmacokinetic properties, and faster elimination from the system without cytotoxicity. To evaluate the therapeutic possibility and effectiveness of this multimodal bio-drug, it was orally delivered to Apc knockout colon cancer mice models. Persistent and efficient delivery of bio-drug was demonstrated by the knockdown of target genes and increased median survival in the treated cohorts. This promising utility of RNAi-Nanotechnology approach advocates the use of bio-drug in an effort to replace chemo-drugs as the future of cancer therapeutics.

Extracellular signal-regulated kinase inhibition prevents venous adaptive remodeling via regulation of Eph-B4.

OBJECTIVES: Vein graft adaptation (VGA) is a process that vein as a vascular graft conduits in arterial reconstructive surgery; VGA can lead to postoperative vein graft stenosis (VGS) and complications after coronary artery bypass graft and other peripheral artery bypass surgeries. VGA is characterized by vein graft loss the venous features without exhibiting arterial features; furthermore, the activation of ERK inhibited the maintenance of venous properties of the vein graft. We hypothesized that ERK inhibition can affect vein VGS through regulating the expression of EphB4. METHODS: Rat vein transplantation model was established using wild-type and EphB4+/- Sprague-Dawley rats. Hematoxylin-eosin, Masson, Verhoeff, actin staining, and immunohistochemistry were applied to observe the structure of the vein grafts. Vascular smooth muscle cells (VSMCs) were isolated from the vein and vein grafts. Western blotting was used to determine the expression of p-ERK1/2 and EphB4, and immunofluorescence was applied to detect the expression and location of EphB4. Cell wound scratch assay and CCK8 assay were used to determine the migration and proliferation of VSMCs. Real-time polymerase chain reaction was used to determine the mRNA expression of EphB4. RESULTS: Western blotting in vein sample and vein graft sample detected p-ERK1/2 and ERK1/2 expression in both EphB4+/+ and EphB4+/- rats. The expression of p-ERK was increased in vein graft compared to vein. Immunofluorescence in VSMCs form EphB4+/+ and EphB4+/- rats detected EphB4 expression in both cells, and the expression of EphB4 was increased in VSMCs form EphB4+/+ rats. SCH772984 reduces the proliferation and migration of VSMCs. Inhibition of ERK suppressed the increase of vein graft wall thickness, and the expression of collagen fibers, elastic fibers, and α-actin was decreased. Vein graft from EphB4+/- rats reduces the expression of EphB4, and SCH772984 suppressed the decrease of EphB4 in vivo. Vein graft from EphB4+/- rats increased the expression of EphB4, and SCH772984 suppressed the increase of EphB4 in vivo. CONCLUSIONS: The inhibition of ERK1/2 suppressed the process of VGS by decreasing the proliferation of VSMCs. The ERK-inhibitor SCH772984 suppressed the level of VGS by extending the time of EphB4 expression during the process of VGA, thus maintaining the venousization of vein graft. The mechanism may be that the inhibitor SCH772984 suppresses the level of VGS by extending the time of EphB4 expression during the process of VGA. Therefore, our research provides a new target of VGS treatment by inhibiting the expression of ERK1/2 through the process of VGA.

Quercetin regulates vascular endothelium function in chronic renal failure via modulation of Eph/Cav-1 signaling.

Arteriovenous fistula (AVF) is frequently believed to be the best vascular access for chronic renal failure (CRF) patients. Vascular endothelial cell dysfunction has been implicated in AVF maturation. Quercetin (Quer) is a natural polyphenolic compound widely used in traditional Chinese medicine. We aimed to uncover the impacts of Quer on vascular endothelial cells in a CRF rat model and human umbilical vein endothelial cells (HUVECs) stimulated by lipopolysaccharide (LPS) and serum from rat with CRF. Blood urea nitrogen and serum creatinine levels were tested in CRF rat model after administration of Quer. H&E staining was used to estimate endothelial damage. Nitric oxide (NO), endothelial NO synthase (eNOS), EPH receptor B4 (EphB4), EphrinB2, and p-caveolin-1 (p-Cav-1) levels in the serum were examined by enzyme-linked immunosorbent assay. Western blot was employed to analyze the expressions of eNOS, phosphorylated (p)-eNOS, EphB4, and Cav-1 in arterial tissues and HUVECs. Cell counting kit-8 was applied for assessing cell proliferation. TUNEL (terminal-deoxynucleotidyl transferase-mediated nick end labeling) assay was employed to estimate cell apoptosis. Results showed that Quer ameliorated renal function impairment and endothelial injury in vivo. Meanwhile, Quer boosted the proliferation and suppressed the apoptosis of HUVECs stimulated by LPS and serum from rat with CRF. Additionally, Quer elevated NO and eNOS levels, upregulated p-eNOS expression but downregulated EphB4, EphrinB2, and p-Cav-1 expressions. Moreover, EphB4 inhibitor had the similar effect as Quer treatment in HUVECs stimulated by LPS and serum from rat with CRF. Collectively, Quer might effectively regulate vascular function to prevent AVF failure in CRF via modulation of Eph/Cav-1 signaling.

Short intronic repeat sequences facilitate circular RNA production.

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery "backsplices" and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼ 30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3' end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA.

EphrinB2-EphB4 Signaling in Neurooncological Disease.

EphrinB2-EphB4 signaling is critical during embryogenesis for cardiovascular formation and neuronal guidance. Intriguingly, critical expression patterns have been discovered in cancer pathologies over the last two decades. Multiple connections to tumor migration, growth, angiogenesis, apoptosis, and metastasis have been identified in vitro and in vivo. However, the molecular signaling pathways are manifold and signaling of the EphB4 receptor or the ephrinB2 ligand is cancer type specific. Here we explore the impact of these signaling pathways in neurooncological disease, including glioma, brain metastasis, and spinal bone metastasis. We identify potential downstream pathways that mediate cancer suppression or progression and seek to understand it´s role in antiangiogenic therapy resistance in glioma. Despite the Janus-faced functions of ephrinB2-EphB4 signaling in cancer Eph signaling remains a promising clinical target.

Inhibition of the erythropoietin-producing receptor EPHB4 antagonizes androgen receptor overexpression and reduces enzalutamide resistance.

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.

EphB4: A promising target for upper aerodigestive malignancies.

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.

Activation of EphrinB2 Signaling Promotes Adaptive Venous Remodeling in Murine Arteriovenous Fistulae.

BACKGROUND: Arteriovenous fistulae (AVF) are the preferred mode of vascular access for hemodialysis. Before use, AVF remodel by thickening and dilating to achieve a functional conduit via an adaptive process characterized by expression of molecular markers characteristic of both venous and arterial identity. Although signaling via EphB4, a determinant of venous identity, mediates AVF maturation, the role of its counterpart EphrinB2, a determinant of arterial identity, remains unclear. We hypothesize that EphrinB2 signaling is active during AVF maturation and may be a mechanism of venous remodeling. METHODS: Aortocaval fistulae were created or sham laparotomy was performed in C57Bl/6 mice, and specimens were examined on Days 7 or 21. EphrinB2 reverse signaling was activated with EphB4-Fc applied periadventitially in vivo and in endothelial cell culture medium in vitro. Downstream signaling was assessed using immunoblotting and immunofluorescence. RESULTS: Venous remodeling during AVF maturation was characterized by increased expression of EphrinB2 as well as Akt1, extracellular signal-regulated kinases 1/2 (ERK1/2), and p38. Activation of EphrinB2 with EphB4-Fc increased phosphorylation of EphrinB2, endothelial nitric oxide synthase, Akt1, ERK1/2, and p38 and was associated with increased diameter and wall thickness in the AVF. Both mouse and human endothelial cells treated with EphB4-Fc increased phosphorylation of EphrinB2, endothelial nitric oxide synthase, Akt1, ERK1/2, and p38 and increased endothelial cell tube formation and migration. CONCLUSIONS: Activation of EphrinB2 signaling by EphB4-Fc was associated with adaptive venous remodeling in vivo while activating endothelial cell function in vitro. Regulation of EphrinB2 signaling may be a new strategy to improve AVF maturation and patency.

EphB4 as a Novel Target for the EGFR-Independent Suppressive Effects of Osimertinib on Cell Cycle Progression in Non-Small Cell Lung Cancer.

Osimertinib is the latest generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor used for patients with EGFR-mutated non-small cell lung cancer (NSCLC). We aimed to explore the novel mechanisms of osimertinib by particularly focusing on EGFR-independent effects, which have not been well characterized. We explored the EGFR-independent effects of osimertinib on cell proliferation using NSCLC cell lines, an antibody array analysis, and the association between the action of osimertinib and the ephrin receptor B4 (EphB4). We also studied the clinicopathological significance of EphB4 in 84 lung adenocarcinoma patients. Osimertinib exerted significant inhibitory effects on cell growth and cell cycle progression by promoting the phosphorylation of p53 and p21 and decreasing cyclin D1 expression independently of EGFR. EphB4 was significantly suppressed by osimertinib and promoted cell growth and sensitivity to osimertinib. The EphB4 status in carcinoma cells was positively correlated with tumor size, T factor, and Ki-67 labeling index in all patients and was associated with poor relapse-free survival in EGFR mutation-positive patients. EphB4 is associated with the EGFR-independent suppressive effects of osimertinib on cell cycle and with a poor clinical outcome. Osimertinib can exert significant growth inhibitory effects in EGFR-mutated NSCLC patients with a high EphB4 status.