A case of Epstein-Barr virus-associated lymphoepithelioma-like carcinoma of the colon.

Lymphoepithelioma-like carcinoma is a poorly differentiated carcinoma with prominent lymphoid infiltration occurring in various organs but is exceedingly rare in the colorectal region. This malignancy is frequently associated with Epstein-Barr virus (EBV). Here we report a case of EBV-associated lymphoepithelioma-like carcinoma of the cecum in an 84-year-old male who presented with occult blood. In situ hybridization for EBV-encoded small RNAs (EBER) in an endoscopic submucosal dissection specimen showed that the tumor consisted of EBER-negative well-differentiated tubular adenocarcinoma and EBER-positive lymphoepithelioma-like carcinoma. Real-time PCR detected 7.16 copies of the EBV genome per cell in a sample microdissected from the latter component. Genotyping analysis demonstrated EBV genotype 1, and viral protein/transcript expression in the tumor showed EBV latency I. Expression of Ephrin receptor A2, a recently reported receptor for EBV, was demonstrated in the tumor cells by immunohistochemistry. To our knowledge, this is the first report of lymphoepithelioma-like carcinoma in the colorectal region showing a definite association with EBV infection.

Mass spectrometry-based proteomic platforms for better understanding of SARS-CoV-2 induced pathogenesis and potential diagnostic approaches.

While protein-protein interaction is the first step of the SARS-CoV-2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)-based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS-CoV-2-mediated infections in humans. Comparative analysis of cell-lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS-CoV-2 infection is still incomplete and the tissue-specific response to SARS-CoV-2 infection can probably not be recapitulated efficiently by in vitro experiments. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross-comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K-Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS-CoV-2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS-CoV-2 responsive age-, gender-dependent, tissue-specific protein targets.

Rapid Lipid-Based Approach for Normalization of Quantum-Dot-Detected Biomarker Expression on Extracellular Vesicles in Complex Biological Samples.

Extracellular vesicles (EVs) are of considerable interest as tumor biomarkers because tumor-derived EVs contain a broad array of information about tumor pathophysiology. However, current EV assays cannot distinguish between EV biomarker differences resulting from altered abundance of a target EV population with stable biomarker expression, altered biomarker expression in a stable target EV population, or effects arising from changes in both parameters. We now describe a rapid nanoparticle- and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker levels to EV abundance. In this approach, EVs are captured from complex samples (e.g., serum), stained with a lipophilic dye, and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant and nonmalignant pancreatic cell lines exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its modular design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple expedient of swapping the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker.

Engineering of monobody conjugates for human EphA2-specific optical imaging.

In a previous study, we developed an E1 monobody specific for the tumor biomarker hEphA2 [PLoS ONE (2015) 10(7): e0132976]. E1 showed potential as a molecular probe for in vitro and in vivo targeting of cancers overexpressing hEphA2. In the present study, we constructed expression vectors for E1 conjugated to optical reporters such as Renilla luciferase variant 8 (Rluc8) or enhanced green fluorescent protein (EGFP) and purified such recombinant proteins by affinity chromatography in E. coli. E1-Rluc8 and E1-EGFP specifically bound to hEphA2 in human prostate cancer PC3 cells but not in human cervical cancer HeLa cells, which express hEphA2 at high and low levels, respectively. These recombinant proteins maintained >40% activity in mouse serum at 24 h. In vivo optical imaging for 24 h did not detect E1-EGFP signals, whereas E1-Rluc8 showed tumor-specific luminescence signals in PC3 but not in HeLa xenograft mice. E1-Rluc8 signals were detected at 4 h, peaked at 12 h, and were undetectable at 24 h. These results suggest the potential of E1-Rluc8 as an EphA2-specific optical imaging agent.

Expression of EphA2 protein is positively associated with age, tumor size and Fuhrman nuclear grade in clear cell renal cell carcinomas.

The receptor tyrosine kinase of EphA2 has been shown frequently overexpressed in various types of human carcinomas, which implicated that it plays important roles in carcinogenesis. Although EphA2 protein expression has been investigated in many types of human carcinomas, the relationship between the expression of EphA2 protein in clear cell renal cell carcinoma was not well documented. In the present study, using specific anit-EphA2 polyclonal antibody and immunohistochemistry, we evaluated EphA2 protein expression levels in clear cell RCC specimens surgically resected from 90 patients. Our results shows that EphA2 protein was positively expressed in all normal renal tubes of 90 samples (100%, 3+), which was expressed at low levels in renal cortex but high levels in the collecting ducts of the renal medulla and papilla. EphA2 was negatively or weakly expressed in 30 out of 90 samples (33.3%, 0/1+), moderately expressed in 24 samples (26.7%, 2+) and strongly expressed in 36 samples (40%, 3+). Expression of EphA2 was positively associated with age (P=0.029), tumor diameters (P<0.001) and Fuhrman nuclear grade (P<0.001). Our results indicate that EphA2 variably expressed in clear cell renal cell carcinomas. High expression of EphA2 was more often found in big size and high nuclear grade tumors, which indicated EphA2 protein may be used as a new marker for the prognosis of clear cell renal cell carcinoma.

Surface proteomic analysis of osteosarcoma identifies EPHA2 as receptor for targeted drug delivery.

BACKGROUND: Osteosarcoma (OS) is the most common bone tumour in children and adolescents. Despite aggressive therapy regimens, treatment outcomes are unsatisfactory. Targeted delivery of drugs can provide higher effective doses at the site of the tumour, ultimately improving the efficacy of existing therapy. Identification of suitable receptors for drug targeting is an essential step in the design of targeted therapy for OS. METHODS: We conducted a comparative analysis of the surface proteome of human OS cells and osteoblasts using cell surface biotinylation combined with nano-liquid chromatography - tandem mass spectrometry-based proteomics to identify surface proteins specifically upregulated on OS cells. This approach generated an extensive data set from which we selected a candidate to study for its suitability as receptor for targeted treatment delivery to OS. First, surface expression of the ephrin type-A receptor 2 (EPHA2) receptor was confirmed using FACS analysis. Ephrin type-A receptor 2 expression in human tumour tissue was tested using immunohistochemistry. Receptor targeting and internalisation studies were conducted to assess intracellular uptake of targeted modalities via EPHA2. Finally, tissue micro arrays containing cores of human OS tissue were stained using immunohistochemistry and EPHA2 staining was correlated to clinical outcome measures. RESULTS: Using mass spectrometry, a total of 2841 proteins were identified of which 156 were surface proteins significantly upregulated on OS cells compared with human primary osteoblasts. Ephrin type-A receptor 2 was highly upregulated and the most abundant surface protein on OS cells. In addition, EPHA2 was expressed in a vast majority of human OS samples. Ephrin type-A receptor 2 effectively mediates internalisation of targeted adenoviral vectors into OS cells. Patients with EPHA2-positive tumours showed a trend toward inferior overall survival. CONCLUSION: The results presented here suggest that the EPHA2 receptor can be considered an attractive candidate receptor for targeted delivery of therapeutics to OS.

EphA2/ephrinA1 mRNA expression and protein production in adenoid cystic carcinoma of salivary gland.

PURPOSE: EphA2/ephrinA1 is believed to play a role in tumor growth and metastasis. The purpose of the present study was to determine the presence of EphA2/ephrinA1 in mRNA and protein adenoid cystic carcinoma. MATERIALS AND METHODS: mRNA and protein expression and protein product of EphA2 and ephrinA1 in adenoid cystic carcinoma was investigated using real-time reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemistry. The tyrosine-phosphorylated state of EphA2 in adenoid cystic carcinoma cells was also investigated. RESULTS: Greater expression of EphA2 and ephrinA1 proteins and mRNA was detected in adenoid cystic carcinoma tissues. EphA2/ephrinA1 staining activities in adenoid cystic carcinoma were more significant than those in normal gland tissue (P < .01). EphA2/ephrinA1 expression correlated significantly to the microvessel density (P < .01). EphA2/ephrinA1 expression and microvessel density correlated with the clinical TNM stage, perineural invasion, and vascular invasion (P < .05). In 3 histologic types of adenoid cystic carcinoma, the expression of EphA2/ephrinA1 and microvessel density was significantly greater in the solid type than in the cribriform and tubular types (P < .01). We also noted that EphA2 was present in a nontyrosine-phosphorylated state. CONCLUSIONS: The present study showed a high expression of EphA2/ephrinA1 in adenoid cystic carcinoma. EphA2/ephrinA1 can serve as a novel therapy target for adenoid cystic carcinoma.

Correlation of clinicopathological features and expression of molecular markers with prognosis after ¹³¹I treatment of differentiated thyroid carcinoma.

PURPOSE: The aim of this study was to identify molecular markers associated with differentiated thyroid carcinoma (DTC) patients and to determine whether there was a correlation between clinicopathological features or molecular markers and the outcome of radioiodine therapy. PATIENTS AND METHODS: We retrospectively reviewed the records of 68 patients with papillary thyroid carcinoma (PTC) and 6 patients with follicular thyroid carcinoma (FTC), who underwent iodine treatment. All patients were divided into a cured group (no evidence for disease) and a noncured group (presence of residual or recurrent disease). Immunohistochemical analysis of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER)-2 (C-erbB-2), ephrin receptor (EphA-2), and sodium-iodide symporter (NIS) was performed in all DTC specimens (68 PTCs, 6 FTCs) and in 14 follicular adenoma specimens. RESULTS: Male patients and patients with lymph node and distant metastases had poorer prognosis. Overexpression of ER, PR, HER-2, EphA-2, and NIS was observed in 50.0% (37), 73.0% (54), 52.7% (39), 67.5% (50), and 70.3% (52) of DTC patients, respectively, and was significantly higher than in follicular adenoma patients (0%, 7.14%, 14.29%, 35.7%, and 35.7%, respectively). However, in patients with DTC, no significant difference in the expression of any marker was seen between the cured group and noncured group. CONCLUSIONS: Male gender, and lymph node and distant metastases were found to be poor prognostic factors for patients with DTC. Higher expressions of ER, PR, HER-2, EphA-2, and NIS were associated with DTC, but were not correlated with the effectiveness of radioiodine treatment.

Overexpression of EphA2 correlates with epithelial-mesenchymal transition-related proteins in gastric cancer and their prognostic importance for postoperative patients.

The expression of EphA2 and three epithelial-mesenchymal transition-related proteins (E-cadherin, ß-catenin and vimentin) was detected by immunohistochemistry in human gastric cancer and normal gastric mucosa. The expression of EphA2 and vimentin was significantly higher in gastric cancer tissues than in normal gastric mucosa tissues, and similar results were found for negative E-cadherin expression and ectopic ß-catenin expression. Further analysis showed that the expression of EphA2 was closely correlated with the depth of tumor invasion, tumor-node-metastasis (TNM) stages and lymph node metastasis. Down-regulated expression of the epithelial protein E-cadherin, overexpression of the mesenchymal protein vimentin and ectopic expression of ß-catenin were associated with the depth of tumor invasion, tumor differentiation, TNM stages and lymph node metastasis. The Spearman rank test indicated that the positive expression of EphA2 was negatively associated with E-cadherin expression and was positively correlated with ß-catenin ectopic expression and vimentin expression. In addition, the Kaplan-Meier survival analysis showed that the overexpression of EphA2 and vimentin, ectopic expression of ß-catenin and down-regulation of E-cadherin indicate a poor outcome. Moreover, multivariate Cox analysis showed that TNM stages, lymph node metastasis, EphA2 expression, E-cadherin expression and ß-catenin ectopic expression were independent prognostic factors for postoperative gastric cancer. These findings indicate that the overexpression of EphA2 correlates with the loss of epithelial proteins and the appearance of mesenchymal proteins. Therefore, EphA2 may play a role in epithelial-mesenchymal transition in gastric cancer.

Clinical significance of EphA2 expression in squamous-cell carcinoma of the head and neck.

PURPOSE: EphA2 receptor tyrosine kinase is frequently overexpressed and functionally altered in a variety of human cancers. The study aimed to assess EphA2 expression and to explore its roles in squamous-cell carcinoma of the head and neck (SCCHN). METHODS: EphA2 expression in 98 primary SCCHN tissue specimens was analyzed by immunohistochemistry and correlated with clinicopathological parameters. Additionally, 13 paired SCCHN tissues and 6 SCCHN cell lines were evaluated for EphA2 expression by RT-PCR and immunoblotting. RESULTS: EphA2 overexpressed in SCCHN tissues and SCCHN cell lines. More importantly, high EphA2 expression was significantly associated with tumor site, T classification, clinical stage, recurrence, and lymph node metastasis, respectively. Patients with high EphA2 expression had both poorer disease-free survival and overall survival than patients with low EphA2 expression. Multivariate Cox regression analysis revealed that EphA2 overexpression was an independent prognostic factor for patients with SCCHN. CONCLUSIONS: These findings suggested that EphA2 may contribute to SCCHN progression and represent a novel prognostic indicator for patients with SCCHN.

EphA2 immunoconjugate as molecularly targeted chemotherapy for ovarian carcinoma.

BACKGROUND: EphA2 is overexpressed in many types of human cancer but is absent or expressed at low levels in normal epithelial tissues. We investigated whether a novel immunoconjugate containing an anti-EphA2 monoclonal antibody (1C1) linked to a chemotherapeutic agent (monomethyl auristatin phenylalanine [MMAF]) through a noncleavable linker maleimidocaproyl (mc) had antitumor activity against ovarian cancer cell lines and tumor models. METHODS: Specificity of 1C1-mcMMAF was examined in EphA2-positive HeyA8 and EphA2-negative SKMel28 ovarian cancer cells by antibody binding and internalization assays. Controls were phosphate-buffered saline (PBS), 1C1, or control IgG-mcMMAF. Viability and apoptosis were investigated in ovarian cancer cell lines and tumor models (10 mice per group). Antitumor activities were tested in the HeyA8-luc and SKOV3ip1 orthotopic mouse models of ovarian cancer. Endothelial cells were identified by use of immunohistochemistry and anti-CD31 antibodies. All statistical tests were two-sided. RESULTS: The 1C1-mcMMAF immunoconjugate specifically bound to EphA2-positive HeyA8 cells but not to EphA2-negative cells and was internalized by HeyA8 cells. Treatment with 1C1-mcMMAF decreased the viability of HeyA8-luc cells in an EphA2-specific manner. In orthotopic mouse models, treatment with 1C1-mcMMAF inhibited tumor growth by 85%-98% compared with that in control mice (eg, for weight of HeyA8 tumors, 1C1-mcMMAF = 0.05 g and control = 1.03 g; difference = 0.98 g, 95% confidence interval [CI] = 0.40 to 1.58 g; P = .001). Even in bulkier disease models with HeyA8-luc cells, 1C1-mcMMAF treatment, compared with control treatment, caused regression of established tumors and increased survival of the mice (eg, 1C1-mcMMAF vs control, mean = 60.6 days vs 29.4 days; difference = 31.2 days, 95% CI = 27.6 to 31.2 days; P = .001). The antitumor effects of 1C1-mcMMAF therapy, in SKOV3ip1 tumors, for example, were statistically significantly related to decreased proliferation (eg, 1C1-mcMMAF vs control, mean = 44.1% vs 55.8% proliferating cells; difference = 11.7%, 95% CI = 2.45% to 20.9%; P = .01) and increased apoptosis of tumor cells (eg, 1C1-mcMMAF vs control, mean = 8.6% vs 0.9% apoptotic cells; difference = 7.7%, 95% CI = 3.8% to 11.7%; P < .001) and of mouse endothelial cells (eg, 1C1-mcMMAF vs control, mean 2.8% vs 0.4% apoptotic endothelial cells; difference = 2.4%, 95% CI = 1.4% to 4.6%; P = .034). CONCLUSION: The 1C1-mcMMAF immunoconjugate had antitumor activity in preclinical models of ovarian carcinoma.

Influence of microenvironments on microcirculation patterns and tumor invasion-related protein expression in melanoma.

This study aimed to investigate the influence of different microenvironments on melanoma microcirculation patterns, invasiveness and metastatic behavior. Sixty C57BL/6J mice were randomly divided into two groups with 30 mice per group. Melanoma B16 cells were injected into the subretinal space and groin area of mice synchronously. The number of each type of microcirculation pattern was counted. Invasion and metastasis were observed. Epithelial cell kinase (EphA2), matrix metalloproteinase (MMP)-2 and -9 expression and their mRNA levels were detected by immunohistochemical staining and real-time PCR and compared between the two groups. Five invasions and six lung metastases were found in the subretinal group while no invasion and metastasis were found in the groin group. The number of vasculogenic mimicry (VM) was significantly higher in the subretinal group (P=0.000). However, no significant difference in the numbers of mosaic and endothelium-dependent vessels was observed between the two groups (P=0.076 and 0.146, respectively). EphA2, MMP-2 and MMP-9 expression was significantly higher in the subretinal group. The mRNA levels of EphA2, MMP-2 and MMP-9 were slightly higher in the subretinal tumors (P=0.002, 0.001 and 0.001, respectively). In conclusion, this experimental paradigm can be a powerful one in which to investigate tumor-microenvironment interactions in melanoma. Tumor cells in the intraocular microenvironment had increased EphA2 expression which induced the formation of VM channels. Moreover, expression of MMP-2 and -9 in tumor tissue was increased to enhance the invasiveness and metastatic behavior.

A human antibody-drug conjugate targeting EphA2 inhibits tumor growth in vivo.

The EphA2 receptor tyrosine kinase is selectively expressed on the surface of many different human tumors. We have previously shown that tumor cells can be targeted by EphA2 monoclonal antibodies and that these antibodies function, in part, by inducing EphA2 internalization and degradation. In this report, we describe the isolation and characterization of a fully human monoclonal antibody (1C1) that selectively binds both the human and rodent EphA2 receptor. After cell binding, the antibody induces rapid tyrosine phosphorylation, internalization, and degradation of the EphA2 receptor. Because monoclonal antibodies that selectively bind tumor cells and internalize provide a vehicle for targeted delivery of cytotoxics, 1C1 was conjugated to the microtubule inhibitor monomethylauristatin phenylalanine using a stable maleimidocaproyl linker. The anti-EphA2 antibody-drug conjugate [1C1-maleimidocaproyl-MMAF (mcMMAF)] stimulated the activation of caspase-3/caspase-7 and the death of EphA2-expressing cells with IC(50) values as low as 3 ng/mL. Similarly, the conjugate induced degradation of the EphA2 receptor and inhibited tumor growth in vivo. Administration of 1C1-mcMMAF at doses as low as 1 mg/kg once weekly resulted in significant growth inhibition of EphA2-expressing tumors without any observable adverse effects in mouse xenograft and rat syngeneic tumor models. Our data support the use of an antibody-drug conjugate approach to selectively target and inhibit the growth of EphA2-expressing tumors.

Activation of the EGFR gene target EphA2 inhibits epidermal growth factor-induced cancer cell motility.

EphA2 overexpression has been reported in many cancers and is believed to play an important role in tumor metastasis and angiogenesis. We show that the activated epidermal growth factor receptor (EGFR) and the cancer-specific constitutively active EGFR type III deletion mutant (EGFRvIII) induce the expression of EphA2 in mammalian cell lines, including the human cancer cell lines A431 and HN5. The regulation is partially dependent on downstream activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase and is a direct effect on the EphA2 promoter. Furthermore, EGFR and EphA2 both localize to the plasma membrane and EphA2 coimmunoprecipitates with activated EGFR and EGFRvIII. Ligand activation of EphA2 and EphA2 knockdown by small interfering RNA inhibit EGF-induced cell motility of EGFR-overexpressing human cancer cells, indicating a functional role of EphA2 in EGFR-expressing cancer cells.

EphA2 overexpression is associated with angiogenesis in ovarian cancer.

BACKGROUND: EphA2 is overexpressed in the majority of ovarian cancers and predicts poor clinical outcome. Based on EphA2's emerging role in angiogenesis, we hypothesized that tumors overexpressing EphA2 demonstrate greater microvessel density (MVD) and matrix metalloproteinase (MMP) expression. METHODS: After Institutional Review Board (IRB) approval, 77 invasive epithelial ovarian tumors were analyzed for CD31, EphA2, MMP-2, MMP-9, and MT1-MMP expression. RESULTS: The median age of the patients was 59 years (range, 34-83). EphA2 was overexpressed in 76% of tumors and was associated with advanced-stage disease (P < .001) and high-grade histology (P = .04). MVD was stratified into high (>12.7 vessels/high-power field [HPF]) versus low (

Patterns of EphA2 protein expression in primary and metastatic pancreatic carcinoma and correlation with genetic status.

EphA2 is a transmembrane receptor tyrosine kinase that functions in the regulation of cell growth, survival, angiogenesis, and migration and EphA2 targeting has been proposed as a novel therapeutic strategy for neoplasms that overexpress this protein. EphA2 overexpression has been correlated with increased invasive and metastatic ability in pancreatic cancer cell lines. However, the patterns of EphA2 expression in human pancreatic cancers and associated metastases is unknown, as are the genetics of EphA2 in this tumor type. We collected clinicopathologic data and paraffin-embedded materials from 98 patients with primary and/or metastatic pancreatic cancer and performed immunohistochemical labeling for EphA2 protein. EphA2 protein immunolabeling was found in 207 of 219 samples (95%). The expression was predominantly cytoplasmic, although predominant membranous staining was observed in a minority of cases. When evaluated specifically for labeling intensity, primary and metastatic carcinomas were more strongly positive compared to benign ducts and PanIN lesions (P < 0.00001 and P < 0.01, respectively) and poorly differentiated carcinomas were more strongly positive for EphA2 than well and moderately differentiated tumors (P < 0.005). When primary carcinomas without metastatic disease were specifically compared to carcinomas with associated metastatic disease, the advanced carcinomas showed relatively less strong positive labeling for EphA2 (P < 0.008). Moreover, decreased EphA2 labeling was more commonly found in liver (P < 0.002), lung (P < 0.004) or peritoneal metastases (P < 0.01) as compared to distant lymph node metastases (P < 0.01). Genetic sequencing of the tyrosine kinase domain of EPHA2 in 22 samples of xenograft enriched pancreatic cancer did not reveal any inactivating mutations. However, EPHA2 amplification was found in 1 of 33 pancreatic cancers corresponding to a lymph node metastasis, indicating EPHA2 genomic amplification may underlie EphA2 overexpression in a minority of patients. Our data confirms that EphA2 is overexpressed in pancreatic cancer, but suggests a relative loss of EphA2 in co-existent pancreatic cancer metastases as well as a role for EPHA2 in organ specific metastasis.

VE-cadherin regulates EphA2 in aggressive melanoma cells through a novel signaling pathway: implications for vasculogenic mimicry.

The formation of matrix-rich, vasculogenic-like networks, termed vasculogenic mimicry (VM), is a unique process characteristic of highly aggressive melanoma cells found to express genes previously thought to be exclusively associated with endothelial and epithelial cells. This study contributes new observations demonstrating that VE-cadherin can regulate the expression of EphA2 at the cell membrane by mediating its ability to become phosphorylated through interactions with its membrane bound ligand, ephrin-A1. VE-cadherin and EphA2 were also found to be colocalized in cell-cell adhesion junctions, both in vitro and in vivo. Immunoprecipitation studies revealed that EphA2 and VE-cadherin could interact, directly and/or indirectly, during VM. Furthermore, there was no change in the colocalization of EphA2 and VE-cadherin at cell-cell adhesion sites when EphA2 was phosphorylated on tyrosine residues. Although transient knockout of EphA2 expression did not alter VE-cadherin localization, transient knockout of VE-cadherin expression resulted in the reorganization of EphA2 on the cells' surface, an accumulation of EphA2 in the cytoplasm, and subsequent dephosphorylation of EphA2. Collectively, these results suggest that VE-cadherin and EphA2 act in a coordinated manner as a key regulatory element in the process of melanoma VM and illuminate a novel signaling pathway that could be potentially exploited for therapeutic intervention.

Inhibition of retinal neovascularization by soluble EphA2 receptor.

Eph receptor tyrosine kinases (RTKs) and their ligands, known as ephrins, play an important role in vascular remodeling during embryogenesis, but their functions in adult angiogenesis are just beginning to be investigated. In this report, we investigated the effect of blocking EphA receptor activation on VEGF-induced angiogenic responses of cultured retinal endothelial cells and on retinal neovascularization in a rodent model of retinopathy of prematurity (ROP). Soluble EphA2-Fc receptors inhibited ephrin-A1 ligand or VEGF-induced BRMEC migration and tube formation without affecting proliferation in vitro. Since EphA2-Fc receptors can inhibit activation of multiple EphA receptors, the specific role of EphA2 receptor in angiogenesis was further investigated in EphA2-deficient endothelial cells. Loss of EphA2 in endothelial cells leads to defective cell migration and assembly in response to either ephrin-A1 or VEGF. Finally, a significant reduction in the severity of abnormal retinal neovascularization was observed in the eyes treated with soluble EphA2-Fc receptors, yet the normal total retinal vascular area was not significantly changed. Because soluble Eph receptor significantly inhibited pathologic retinal angiogenesis without affecting normal intraretinal vessels, it may be a promising agent for treatment of retinal angiogenesis in a number of human ocular diseases.