N-cadherin regulates signaling mechanisms required for lens fiber cell elongation and lens morphogenesis.

Tissue development and regeneration involve high-ordered morphogenetic processes that are governed by elements of the cytoskeleton in conjunction with cell adhesion molecules. Such processes are particularly important in the lens whose structure dictates its function. Studies of our lens-specific N-cadherin conditional knockout mouse (N-cadcKO) revealed an essential role for N-cadherin in the migration of the apical tips of differentiating lens fiber cells along the apical surfaces of the epithelium, a region termed the Epithelial Fiber Interface (EFI), that is necessary for normal fiber cell elongation and the morphogenesis. Studies of the N-cadcKO lens suggest that N-cadherin function in fiber cell morphogenesis is linked to the activation of Rac1 and myosin II, both signaling pathways central to the regulation of cell motility including determining the directionality of cellular movement. The absence of N-cadherin did not disrupt lateral contacts between fiber cells during development, and the maintenance of Aquaporin-0 and increased expression of EphA2 at cell-cell interfaces suggests that these molecules may function in this role. E-cadherin was maintained in newly differentiating fiber cells without interfering with expression of lens-specific differentiation proteins but was not able to replace N-cadherin function in these cells. The dependence of migration of the fiber cell apical domains along the EFI for lens morphogenesis on N-cadherin provides new insight into the process of tissue development.

Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies.

The receptor tyrosine kinase EPHA2 is overexpressed in several cancers (breast, head and neck, non-small-cell lung cancer). Small-molecule-based inhibition of the EPHA2 kinase domain (KD) is seen as an important strategy for therapeutic intervention. However, obtaining structural information by crystallography or NMR spectroscopy for drug discovery is severely hampered by the lack of pure, homogeneous protein. Here, different fragments of the EPHA2 KD were expressed and purified from both bacterial (Escherichia coli, BL21(DE3) cells) and insect cells (Spodoptera frugiperda, Sf9 cells).1 H,15 N HSQC was used to determine the proper folding and homogeneity of all the constructs. Protein from E. coli was well-folded but unstable, and it did not crystallize. However, a construct (D596-G900) produced in Sf9 cells yielded homogenous, well-folded protein that crystallized readily, thereby resulting in eleven new EPHA2-ligand crystal structures. We have also established a strategy for selective and uniform 15 N-amino acid labeling of EPHA2 KD in Sf9 cells for investigating dynamics and EPHA2-drug interactions by NMR.

Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling.

Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although the role of Eph receptors and their ligand Ephrins is well studied in developmental processes, their function in tobacco smoke (TS)-induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction, and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly upregulated EphA2 and EphrinA1 expression, disrupted the actin filaments, decreased E-cadherin expression, and increased protein permeability, whereas the focal adhesion protein paxillin was unaffected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS-induced hyperpermeability in BAEpCs. In addition, when BAEpC monolayer was transfected with EphA2-expressing plasmid and treated with recombinant EphrinA1, the transepithelial electrical resistance decreased significantly. Furthermore, TS downregulated E-cadherin expression and induced hyperpermeability across BAEpC monolayer in a Erk1/Erk2, p38, and JNK MAPK-dependent manner. TS induced hyperpermeability in BAEpC monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were unaffected. The present data suggest that TS causes significant damage to the BAEpCs via induction of EphA2 and downregulation of E-cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury.

The role of miR-200a in vasculogenic mimicry and its clinical significance in ovarian cancer.

OBJECTIVE: Vasculogenic mimicry (VM) indicates that aggressive cancer cells can form de novo vascular networks and provide a perfusion pathway for rapidly growing tumors. MiR-200a has been reported significantly deregulated in ovarian cancer. However, miR-200a regulation of VM and its clinical significance in ovarian cancer remain not elucidated. METHODS: In this study, we identified the VM structure by CD34-PAS staining in ovarian cancer tissue. MiR-200a and protein expression was tested by quantitative RT-PCR and western blot. Bioinformatics prediction, luciferase assay and intervention experiments were employed to identify the target of miR-200a. RESULTS: We certified the VM structure in ovarian cancer, and found that the VM positive rate was significantly associated with tumor grade, stage and metastasis. Further study showed that miR-200a expression levels were significantly lower in VM positive ovarian cancer. In addition, our results suggested that miR-200a inhibited VM by negatively regulated EphA2 expression. Consistently, the inverse correlation of miR-200a and EphA2 has also been found in ovarian cancer patients. Moreover, the expression of miR-200a/EphA2 was significantly associated with patient's clinicopathological parameter, such as tumor stage and metastases. Kaplan-Meier curves confirmed that the patients with low miR-200a expression and/or VM positive had a significantly shorter overall survival. CONCLUSIONS: Our research demonstrates that VM, miR-200a and EphA2 play key roles in the progression and prognosis of ovarian cancer, and for the first time suggests that miR-200a inhibits VM by directly regulating EphA2. Therefore, we might have identified a genetic mechanism underlying the involvement of miR-200a in ovarian cancer VM.

EphA2 mutation in lung squamous cell carcinoma promotes increased cell survival, cell invasion, focal adhesions, and mammalian target of rapamycin activation.

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

Smad3 contributes to positioning of proliferating cells in colonic crypts by inducing EphB receptor protein expression.

Deficiency of Smad3, an intracellular mediator of TGF-ß, was shown to significantly accelerate re-epithelialization of the colonic mucosa. This study was performed to investigate the molecular mechanisms by which Smad3 controls colonic epithelial cell proliferation and crypt formation. Smad3(ex8/ex8) C57BL/6 mice were used in this study and wild-type littermates served as controls. The number of proliferating cells in the isolated colonic epithelium of Smad3(-/-) mice was significantly increased compared to that in wild-type littermates. Protein levels of the cell cycle inhibitors p21 and p27 were significantly decreased, while that of c-Myc was increased in the isolated colonic epithelium from Smad3(-/-) mice. In the colonic tissue of wild-type mice, cell proliferation was restricted to the bottom of the crypts in accordance with nuclear ß-catenin staining, whereas proliferating cells were located throughout the crypts in Smad3(-/-) mice in accordance with nuclear ß-catenin staining, suggesting that Smad3 is essential for locating proliferating cells at the bottom of the colonic crypts. Notably, in Smad3(-/-) mice, there was loss of EphB2 and EphB3 receptor protein expression, critical regulators of proliferating cell positioning, while EphB receptor protein expression was confirmed at the bottom of the colonic crypts in wild-type mice. These observations indicated that disturbance of the EphB/ephrin B system brings about mispositioning of proliferating cells in the colonic crypts of Smad3(-/-) mice. In conclusion, Smad3 is essential for controlling number and positioning of proliferating cells in the colonic crypts and contributes to formation of a "proliferative zone" at the bottom of colonic crypts in the normal colon.

Genotype, Age, Genetic Background, and Sex Influence Epha2-Related Cataract Development in Mice.

Purpose: Age-related cataract is the leading cause of blindness worldwide. Variants in the EPHA2 gene increase the disease risk, and its knockout in mice causes cataract. We investigated whether age, sex, and genetic background, risk factors for age-related cataract, and Epha2 genotype influence Epha2-related cataract development in mice. Methods: Cataract development was monitored in Epha2+/+, Epha2+/-, and Epha2-/- mice (Epha2Gt(KST085)Byg) on C57BL/6J and FVB:C57BL/6J (50:50) backgrounds. Cellular architecture of lenses, endoplasmic reticulum (ER) stress, and redox state were determined using histological, molecular, and analytical techniques. Results: Epha2-/- and Epha2+/- mice on C57BL/6J background developed severe cortical cataracts by 18 and 38 weeks of age, respectively, compared to development of similar cataract significantly later in Epha2-/- mice and no cataract in Epha2+/- mice in this strain on FVB background, which was previously reported. On FVB:C57BL/6J background, Epha2-/- mice developed severe cortical cataract by 38 weeks and Epha2+/- mice exhibited mild cortical cataract up to 64 weeks of age. Progression of cataract in Epha2-/- and Epha2+/- female mice on C57BL/6J and mixed background, respectively, was slower than in matched male mice. N-cadherin and ß-catenin immunolabeling showed disorganized lens fiber cells and disruption of lens architecture in Epha2-/- and Epha2+/- lenses, coinciding with development of severe cataracts. EPHA2 immunolabeling showed intracellular accumulation of the mutant EPHA2-ß-galactosidase fusion protein that induced a cytoprotective ER stress response and in Epha2+/- lenses was also accompanied by glutathione redox imbalance. Conclusions: Both, Epha2-/- and Epha2+/- mice develop age-related cortical cataract; age as a function of Epha2 genotype, sex, and genetic background influence Epha2-related cataractogenesis in mice.

CircPAG1 Inhibits the High Glucose-Induced Lens Epithelial Cell Injury by Sponging miR-630 and Upregulating EPHA2.

Background: Circular RNAs (circRNAs) have been considered as vital regulators in the progression of human ocular diseases, including diabetic cataract (DC). This report was designed to research the biological role of circRNA phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (circPAG1) in high glucose (HG)-induced lens epithelial damages.Methods: Lens epithelial damage in DC was investigated by the effects of 25 mM glucose (HG) on human lens epithelial cells (HLE-B3). CircPAG1, microRNA-630 (miR-630), and ephrin type-A receptor 2 (EPHA2) levels were examined by the quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation analysis was performed by 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay and colony formation assay. Cell apoptosis was measured through flow cytometry. Protein levels were detected using western blot. Oxidative stress was determined by malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) levels via the corresponding kits. Dual-luciferase reporter and RNA immunoprecipitation (RIP) and RNA pull-down assays were used for target binding analysis.Results: CircPAG1 expression was downregulated in lens samples of DC patients and HG-treated lens epithelial cells. HG inhibited cell growth but promoted apoptosis and oxidative stress in HLE-B3 cells, while circPAG1 overexpression relieved these damages. Moreover, circPAG1 was identified as a molecular sponge for miR-630. HG-induced cell injury was also attenuated by the inhibition of miR-630, and the function of circPAG1 was related to its sponge effect on miR-630. In addition, miR-630 directly targeted EPHA2 and circPAG1 could regulate the EPHA2 expression via sponging miR-630. Furthermore, we found that the protective role of circPAG1 against the HG-induced cell injury was ascribed to the upregulation of EPHA2.Conclusion: Our evidence suggested that circPAG1 alleviated cell damages in HG-treated human lens epithelial cells by regulating the miR-630/EPHA2 axis.

The prognostic value of ephrin type-A2 receptor and Ki-67 in renal cell carcinoma patients: An Immunohistochemical and Bioinformatical Approach; A STROBE - compliant article.

Patients with renal cell carcinoma (RCC), the most common malignant renal epithelial tumor, usually present with advanced disease and unpredicted clinical behavior. The receptor tyrosine kinase, ephrin type-A receptor 2 (EphA2) was found to be overexpressed in several malignancies and its expression was found to be associated with poor prognostic features.Our study is an observational study with the aim of investigating the prognostic value of EphA2 in RCC patients and its association with clinicopathological parameters as well as Ki-67 expression, which is a well-known proliferative and prognostic marker in RCC.EphA2 and Ki-67 immunohistochemical staining was performed on whole sections representative of 50 patients diagnosed with primary RCC from 2013 to 2018. In addition, the association between EphA2 mRNA expression and clinicopathological parameters as well as the patients' outcome was also evaluated using two large publicly available databases.Our results showed a significant association between EphA2 immunohistochemical expression and tumor size, nuclear grade, tumor stage, patients' outcome and Ki-67 expression (P < .05 for all). The same trend was also observed with EphA2 mRNA expression using larger patients' cohorts in 2 publicly available databases. Notably, EphA2 protein expression showed higher levels of co-expression with the proliferative marker Ki-67.Our results suggested that higher expression of EphA2 and Ki-67 in tumor tissues predicts a locally aggressive behaviour and poor outcome of patients with RCC. Moreover, our results give a rationale for the potential benefits of using novel therapeutic strategies with the aim of targeting EphA2 receptor in RCC patients that might help in improving their outcome.

Interleukin-13 receptor alpha 2, EphA2, and Fos-related antigen 1 as molecular denominators of high-grade astrocytomas and specific targets for combinatorial therapy.

PURPOSE: We investigated the expression of interleukin-13 receptor alpha2 (IL-13R alpha 2), EphA2, and Fos-related antigen 1 (Fra-1) in astrocytomas and normal brain. We sought to document whether the expression of the three factors changed with progression to higher grade malignancy and whether two or three targets in combination might be sufficient to target all patients with high-grade astrocytomas. EXPERIMENTAL DESIGN: Immunohistochemistry was done for IL-13R alpha 2, EphA2, and Fra-1 using human brain tumor tissue microarrays containing 30 specimens of WHO grades II and III astrocytomas, 46 glioblastoma multiformes (GBM), and 9 normal brain samples. Sections were scored based on frequency and intensity of expression. Western blotting was done for all three markers using GBM tumor specimens and xenograft cell lines. Two cytotoxins, IL-13.E13K.PE38QQR and ephrinA1-PE38QQR, which target IL-13R alpha 2 or EphA2, respectively, were tested for cytotoxicity against human GBM primary explant cells and established cells. RESULTS: Expression of all three proteins was significantly higher in GBM compared with normal brain, low-grade, and anaplastic astrocytomas. Greater than 95% of GBM overexpressed at least two of the three markers. Importantly, every GBM overexpressed at least one marker. Human GBM primary explant cells and cell lines were potently killed by IL-13.E13K.PE38QQR and ephrinA1-PE38QQR, in accordance with their level of expression of IL-13R alpha 2 and EphA2, respectively. CONCLUSIONS: IL-13R alpha 2, EphA2, and Fra-1 are attractive therapeutic targets representing molecular denominators of high-grade astrocytomas. One hundred percent of GBM tumors overexpress at least one of these proteins, providing the basis for rational combinatorial targeted therapies/diagnostics suitable for all patients with this disease.

High yield expression of non-phosphorylated protein tyrosine kinases in insect cells.

The key role of kinases in signal transduction and cell growth regulation has been a long standing interest among academics and the pharmaceutical industry. Recombinant enzymes have been used to understand the mechanism of action as well as to screen for chemical inhibitors. The baculo-insect system has been the primary method used to obtain soluble and active kinases, usually producing a mixture of the kinase in various phosphorylation states in different conformations. To obtain a homogenous preparation of non-phosphorylated kinases is critical for biochemical, biophysical and kinetic studies aimed at understanding the mechanism of kinase activation. Taking advantage of the eukaryotic expression property of insect cells, we were able to obtain high yield expression of non-phosphorylated protein tyrosine kinases BTK, JAK3 and Eph2A through coexpression with the tyrosine phosphatase YopH, which suggests that this method can be applied to protein tyrosine kinases in general. We have demonstrated that the fully non-phosphorylated BTK obtained with this method is suitable for various biochemical and kinetic studies.

Involvement of EphA2 in head and neck squamous cell carcinoma: mRNA expression, loss of heterozygosity and immunohistochemical studies.

EphA2 is a 130-kDa transmembrane protein primarily found in adult human epithelial cells and is a member of one of the largest receptor tyrosine kinases. It is located on 1p36.1, a genetic hot spot in cancer. EphA2 overexpression has been observed in aggressive solid tumors and its potential role in tumorigenesis, which includes cell growth, survival, migration and angiogenesis have been reported. However, the role of EphA2 remains unknown in head and neck cancer. In this study, we investigated the genetic profile of EphA2 in primary head and neck squamous cell carcinoma (HNSCC) by determining mRNA level, status of loss of heterozygosity and protein expression. mRNA expression was also correlated with clinicopathological data. Infrequent loss of heterozygosity (20%) was observed, though a 10-fold increase of mRNA expression in tumors compared to normal tissues was noted. A significant number of samples with normal to high mRNA expression was observed among patients with regional metastasis, with T3-T4 tumor size and with moderate to poor differentiation. However, statistical studies did not show any correlation between mRNA expression and any of the clinicopathological parameters. Tumor cells expressed EphA2 protein, but only weakly. These results suggest that EphA2 might be involved in the early development of HNSCC although not directly responsible for its progression.

Increased expression of EphA2 correlates with adverse outcome in primary and recurrent glioblastoma multiforme patients.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor characterized by excessive angiogenesis. The dismal prognosis of patients with GBM warrants the development of new targeting therapies based on novel molecular markers. The EphA2 receptor tyrosine kinase plays a pivotal role in tumor angiogenesis and an increased expression in glioma patients has recently been reported. In this study, we investigated the expression of EphA2 in human normal brain, primary and recurrent GBM and correlated it with clinical pathological parameters and patient's outcome. In addition, intratumor microvascular density was quantified by immunostaining for the endothelial cell marker, von Willebrand factor. A different intensity of the membranous and cytoplastic expression of EphA2 was observed in the 40 primary and recurrent samples of GBM analyzed but not in the normal brain. A high level expression of EphA2 was demonstrated in 24 (60%) of the primary and recurrent GBM analyzed. The increased expression of the EphA2 protein was significantly associated with the adverse outcome of GBM patients (p<0.01 for overall survival). The data presented in this study define the expression pattern of EphA2 in both primary and recurrent glioblastoma and suggest an important role of EphA2 in the pathogenesis of GBM. The EphA2 may be used as a surrogate marker to screen patients for tyrosine kinase inhibitor therapy.

A genome-wide screen reveals functional gene clusters in the cancer genome and identifies EphA2 as a mitogen in glioblastoma.

A novel genome-wide screen that combines patient outcome analysis with array comparative genomic hybridization and mRNA expression profiling was developed to identify genes with copy number alterations, aberrant mRNA expression, and relevance to survival in glioblastoma. The method led to the discovery of physical gene clusters within the cancer genome with boundaries defined by physical proximity, correlated mRNA expression patterns, and survival relatedness. These boundaries delineate a novel genomic interval called the functional common region (FCR). Many FCRs contained genes of high biological relevance to cancer and were used to pinpoint functionally significant DNA alterations that were too small or infrequent to be reliably identified using standard algorithms. One such FCR contained the EphA2 receptor tyrosine kinase. Validation experiments showed that EphA2 mRNA overexpression correlated inversely with patient survival in a panel of 21 glioblastomas, and ligand-mediated EphA2 receptor activation increased glioblastoma proliferation and tumor growth via a mitogen-activated protein kinase-dependent pathway. This novel genome-wide approach greatly expanded the list of target genes in glioblastoma and represents a powerful new strategy to identify the upstream determinants of tumor phenotype in a range of human cancers.

Disruption of EphA2 receptor tyrosine kinase leads to increased susceptibility to carcinogenesis in mouse skin.

EphA2 receptor tyrosine kinase is frequently overexpressed in different human cancers, suggesting that it may promote tumor development and progression. However, evidence also exists that EphA2 may possess antitumorigenic properties, raising a critical question on the role of EphA2 kinase in tumorigenesis in vivo. We report here that deletion of EphA2 in mouse led to markedly enhanced susceptibility to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage skin carcinogenesis. EphA2-null mice developed skin tumors with an increased frequency and shortened latency. Moreover, tumors in homozygous knockout mice grew faster and were twice as likely to show invasive malignant progression. Haploinsufficiency of EphA2 caused an intermediate phenotype in tumor development but had little effects on invasive progression. EphA2 and ephrin-A1 exhibited compartmentalized expression pattern in mouse skin that localized EphA2/ephrin-A1 interactions to the basal layer of epidermis, which was disrupted in tumors. Loss of EphA2 increased tumor cell proliferation, whereas apoptosis was not affected. In vitro, treatment of primary keratinocytes from wild-type mice with ephrin-A1 suppressed cell proliferation and inhibited extracellular signal-regulated kinase 1/2 (ERK1/2) activities. Both effects were abolished in EphA2-null keratinocytes, suggesting that loss of ERK inhibition by EphA2 may be one of the contributing mechanisms for increased tumor susceptibility. Interestingly, despite its tumor suppressive function, EphA2 was overexpressed in skin tumors compared with surrounding normal skin in wild-type mice, similar to the observations in human cancers. EphA2 overexpression may represent a compensatory feedback mechanism during tumorigenesis. Together, these results show that EphA2 is a novel tumor suppressor gene in mammalian skin.

Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery.

Inducing destruction of specific mRNA using small interfering RNA (siRNA) is a powerful tool in analysis of protein function, but its use as a therapeutic modality has been limited by inefficient or impractical delivery systems. We have used siRNA incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo siRNA delivery. In nude mice bearing i.p. ovarian tumors, nonsilencing siRNA tagged with the fluorochrome Alexa 555 was encapsulated into DOPC liposomes and shown to be taken up by the tumor as well as many major organs. Furthermore, DOPC-encapsulated siRNA targeting the oncoprotein EphA2 was highly effective in reducing in vivo EphA2 expression 48 hours after a single dose as measured by both Western blot and immunohistochemistry. Therapy experiments in an orthotopic mouse model of ovarian cancer were initiated 1 week after injection of either HeyA8 or SKOV3ip1 cell lines. Three weeks of treatment with EphA2-targeting siRNA-DOPC (150 microg/kg twice weekly) reduced tumor growth when compared with a nonsilencing siRNA (SKOV3ip1: 0.35 versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16). When EphA2-targeting siRNA-DOPC was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a nonsilencing siRNA (SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus 0.84 g; P = 0.0027). These studies show the feasibility of siRNA as a clinically applicable therapeutic modality.

Functional non-coding polymorphism in an EPHA2 promoter PAX2 binding site modifies expression and alters the MAPK and AKT pathways.

To identify possible genetic variants influencing expression of EPHA2 (Ephrin-receptor Type-A2), a tyrosine kinase receptor that has been shown to be important for lens development and to contribute to both congenital and age related cataract when mutated, the extended promoter region of EPHA2 was screened for variants. SNP rs6603883 lies in a PAX2 binding site in the EPHA2 promoter region. The C (minor) allele decreased EPHA2 transcriptional activity relative to the T allele by reducing the binding affinity of PAX2. Knockdown of PAX2 in human lens epithelial (HLE) cells decreased endogenous expression of EPHA2. Whole RNA sequencing showed that extracellular matrix (ECM), MAPK-AKT signaling pathways and cytoskeleton related genes were dysregulated in EPHA2 knockdown HLE cells. Taken together, these results indicate a functional non-coding SNP in EPHA2 promoter affects PAX2 binding and reduces EPHA2 expression. They further suggest that decreasing EPHA2 levels alters MAPK, AKT signaling pathways and ECM and cytoskeletal genes in lens cells that could contribute to cataract. These results demonstrate a direct role for PAX2 in EPHA2 expression and help delineate the role of EPHA2 in development and homeostasis required for lens transparency.

EphA2 as a novel molecular marker and target in glioblastoma multiforme.

We investigated the presence of EphA2, and its ligand, ephrinA1, in glioblastoma multiforme (GBM), a malignant neoplasm of glial cells, and normal brain. We also initially examined the functional importance of the interaction between EphA2 and ephrinA1 in glioma cells. Expression and localization of EphA2 and ephrinA1 in human GBM and normal brain were examined using Western blotting, immunofluorescence, and immunohistochemistry. A functional role for EphA2 was investigated by assessing the activation status of the receptor and the effect of ephrinA1 on the anchorage-independent growth and invasiveness of GBM cells. We found EphA2 to be elevated in approximately 90% of GBM specimens and cell lines but not in normal brain, whereas ephrinA1 was present at consistently low levels in both GBM and normal brain. EphA2 was activated and phosphorylated by ephrinA1 in GBM cells. Furthermore, ephrinA1 induced a prominent, dose-dependent inhibitory effect on the anchorage-independent growth and invasiveness of GBM cells highly overexpressing EphA2, which was not seen in cells expressing low levels of the receptor. Thus, EphA2 is both specifically overexpressed in GBM and expressed differentially with respect to its ligand, ephrinA1, which may reflect on the oncogenic processes of malignant glioma cells. EphA2 seems to be functionally important in GBM cells and thus may play an important role in GBM pathogenesis. Hence, EphA2 represents a new marker and novel target for the development of molecular therapeutics against GBM.

Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin.

Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.

Expression of EphA2 is prognostic of disease-free interval and overall survival in surgically treated patients with renal cell carcinoma.

Whereas normally expressed at sites of cell-to-cell contact in adult epithelial tissues, recent studies have shown that the receptor tyrosine kinase EphA2 is overexpressed in numerous epithelial-type carcinomas, with the greatest level of EphA2 expression observed in metastatic lesions. In the current study, we have assessed EphA2 expression in archived renal cell carcinoma (RCC) tissues as it relates to patient disease course. Using specific anti-EphA2 monoclonal antibody 208 and immunohistochemistry, we evaluated EphA2 protein expression levels in RCC specimens surgically resected from 34 patients (including 30 conventional clear-cell RCC, 3 papillary, and 1 chromophobic RCC cases) resulting in clinical cures. Regardless of histopathologic subtype, RCC lesions expressing higher levels of EphA2 tended to be of a higher grade (P < 0.05) and larger (P = 0.093), more-highly-vascularized tumors (P = 0.005). Perhaps most notable, the degree of EphA2 overexpression (versus normal matched autologous kidney tissue) seemed predictive of short-term (<1 year) versus longer-term (> or =1 year) disease-free interval (P < 0.001) and of overall survival (P < 0.001) among the RCC patients evaluated. These data suggest that EphA2 expression level may serve as a useful prognostic tool in the clinical management of patients who have been successfully treated with surgery, but who are at greater risk for accelerated disease recurrence and who have a poorer prognosis.