Interferon-induced transmembrane protein-1 competitively blocks Ephrin receptor A2-mediated Epstein-Barr virus entry into epithelial cells.

Epstein-Barr virus (EBV) can infect both B cells and epithelial cells (ECs), causing diseases such as mononucleosis and cancer. It enters ECs via Ephrin receptor A2 (EphA2). The function of interferon-induced transmembrane protein-1 (IFITM1) in EBV infection of ECs remains elusive. Here we report that IFITM1 inhibits EphA2-mediated EBV entry into ECs. RNA-sequencing and clinical sample analysis show reduced IFITM1 in EBV-positive ECs and a negative correlation between IFITM1 level and EBV copy number. IFITM1 depletion increases EBV infection and vice versa. Exogenous soluble IFITM1 effectively prevents EBV infection in vitro and in vivo. Furthermore, three-dimensional structure prediction and site-directed mutagenesis demonstrate that IFITM1 interacts with EphA2 via its two specific residues, competitively blocking EphA2 binding to EBV glycoproteins. Finally, YTHDF3, an m6A reader, suppresses IFITM1 via degradation-related DEAD-box protein 5 (DDX5). Thus, this study underscores IFITM1's crucial role in blocking EphA2-mediated EBV entry into ECs, indicating its potential in preventing EBV infection.

Manipulating the Level of Sensorimotor Stimulation during LI-rTMS Can Improve Visual Circuit Reorganisation in Adult Ephrin-A2A5-/- Mice.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that has the potential to treat a variety of neurologic and psychiatric disorders. The extent of rTMS-induced neuroplasticity may be dependent on a subject's brain state at the time of stimulation. Chronic low intensity rTMS (LI-rTMS) has previously been shown to induce beneficial structural and functional reorganisation within the abnormal visual circuits of ephrin-A2A5-/- mice in ambient lighting. Here, we administered chronic LI-rTMS in adult ephrin-A2A5-/- mice either in a dark environment or concurrently with voluntary locomotion. One day after the last stimulation session, optokinetic responses were assessed and fluorescent tracers were injected to map corticotectal and geniculocortical projections. We found that LI-rTMS in either treatment condition refined the geniculocortical map. Corticotectal projections were improved in locomotion+LI-rTMS subjects, but not in dark + LI-rTMS and sham groups. Visuomotor behaviour was not improved in any condition. Our results suggest that the beneficial reorganisation of abnormal visual circuits by rTMS can be significantly influenced by simultaneous, ambient visual input and is enhanced by concomitant physical exercise. Furthermore, the observed pathway-specific effects suggest that regional molecular changes and/or the relative proximity of terminals to the induced electric fields influence the outcomes of LI-rTMS on abnormal circuitry.

EPHA2 Promotes the Invasion and Migration of Human Tongue Squamous Cell Carcinoma Cal-27 Cells by Enhancing AKT/mTOR Signaling Pathway.

EPHA2 is a member of the ephrin receptor tyrosine kinase family and is closely related to the malignant tumor progression. The effect of EPHA2 on OSCC is not clear. This study explored the role of EPHA2 and AKT/mTOR signaling pathways in Cal-27 cell invasion and migration. The expression of EPHA2 and EPHA4 in human OSCC and normal oral tissue was detected by immunohistochemistry. EPHA2-overexpressing and EPHA2-knockdown Cal-27 cells were established, and the cells were treated with an AKT inhibitor (MK2206) and mTOR inhibitor (RAD001). The expression of EPHA2 was detected by qRT-PCR, cell proliferation was evaluated by MTT assay, cell migration and invasion were examined by scratch and Transwell assay, and cell morphology and apoptosis were assessed by Hoechst 33258 staining. Western blot was performed to detect the expression of proteins related to AKT/mTOR signaling, cell cycle, and pseudopod invasion. EPHA2 and EPHA4 were highly expressed in clinical human OSCC. Overexpression of EPHA2 promoted the proliferation, migration, and invasion of Cal-27 cells, inhibited cell cycle blockage and apoptosis, and enhanced the activity of the AKT/mTOR signaling pathway. MK2206 (AKT inhibitor) and RAD001 (mTOR inhibitor) reversed the effect of EPHA2 overexpression on the biological behavior of Cal-27 cells. EPHA2 promotes the invasion and migration of Cal-27 human OSCC cells by enhancing the AKT/mTOR signaling pathway.

EPHA2 Segregates with Microphthalmia and Congenital Cataracts in Two Unrelated Families.

EPHA2 is a transmembrane tyrosine kinase receptor that, when disrupted, causes congenital and age-related cataracts. Cat-Map reports 22 pathogenic EPHA2 variants associated with congenital cataracts, variable microcornea, and lenticonus, but no previous association with microphthalmia (small, underdeveloped eye, ≥2 standard deviations below normal axial length). Microphthalmia arises from ocular maldevelopment with >90 monogenic causes, and can include a complex ocular phenotype. In this paper, we report two pathogenic EPHA2 variants in unrelated families presenting with bilateral microphthalmia and congenital cataracts. Whole genome sequencing through the 100,000 Genomes Project and cataract-related targeted gene panel testing identified autosomal dominant heterozygous mutations segregating with the disease: (i) missense c.1751C>T, p.(Pro584Leu) and (ii) splice site c.2826-9G>A. To functionally validate pathogenicity, morpholino knockdown of epha2a/epha2b in zebrafish resulted in significantly reduced eye size ± cataract formation. Misexpression of N-cadherin and retained fibre cell nuclei were observed in the developing lens of the epha2b knockdown morphant fish by 3 days post-fertilisation, which indicated a putative mechanism for microphthalmia pathogenesis through disruption of cadherin-mediated adherens junctions, preventing lens maturation and the critical signals stimulating eye growth. This study demonstrates a novel association of EPHA2 with microphthalmia, suggesting further analysis of pathogenic variants in unsolved microphthalmia cohorts may increase molecular diagnostic rates.

Ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT.

BACKGROUND: Ephrin-A2, a member of the Eph receptor subgroup, is used in diagnosing and determining the prognosis of prostate cancer. However, the role of ephrin-A2 in prostate cancer is remains elusive. METHODS: We established stable clones overexpressing or silencing ephrin-A2 from prostate cancer cells. Then, CCK-8 was used in analyzing the proliferation ability of cells. CD31 staining was used in evaluating angiogenesis. Migration and invasion assay were conducted in vivo and in vitro. The expression of EMT-related markers was evaluated in prostate cancer cells through Western blotting. RESULTS: We revealed that the ectopic expression of ephrin-A2 in prostate cancer cells facilitated cell migration and invasion in vitro and promoted tumor metastasis and angiogenesis in vivo and that the silencing of ephrin-A2 completely reversed this effect. Although ephrin-A2 did not affect tumor cell proliferation in vitro, ephrin-A2 significantly promoted primary tumor growth in vivo. Furthermore, to determine the biological function of ephrin-A2, we assayed the expression of EMT-related markers in stable-established cell lines. Results showed that the overexpression of ephrin-A2 in prostate cancer cells down-regulated the expression of epithelial markers (ZO-1, E-cadherin, and claudin-1) and up-regulated the expression of mesenchymal markers (N-cadherin, ß-catenin, vimentin, Slug, and Snail), but the knocking out of ephrin-A2 opposed the effects on the expression of EMT markers. CONCLUSIONS: These findings indicate that ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT and may be a potentially therapeutic target in metastatic prostate cancer.

Activation of EphA2-EGFR signaling in oral epithelial cells by Candida albicans virulence factors.

During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of infection. The ephrin type-A receptor 2 (EphA2) detects ß-glucan and plays a central role in stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the Als3 adhesin/invasin and the candidalysin pore-forming toxin. Here, we investigated the interactions among EphA2, EGFR, Als3 and candidalysin during OPC. We found that EGFR and EphA2 constitutively associate with each other as part of a heteromeric physical complex and are mutually dependent for C. albicans-induced activation. Als3-mediated endocytosis of a C. albicans hypha leads to the formation of an endocytic vacuole where candidalysin accumulates at high concentration. Thus, Als3 potentiates targeting of candidalysin, and both Als3 and candidalysin are required for C. albicans to cause maximal damage to oral epithelial cells, sustain activation of EphA2 and EGFR, and stimulate pro-inflammatory cytokine and chemokine secretion. In the mouse model of OPC, C. albicans-induced production of CXCL1/KC and CCL20 is dependent on the presence of candidalysin and EGFR, but independent of Als3. The production of IL-1α and IL-17A also requires candidalysin but is independent of Als3 and EGFR. The production of TNFα requires Als1, Als3, and candidalysin. Collectively, these results delineate the complex interplay among host cell receptors EphA2 and EGFR and C. albicans virulence factors Als1, Als3 and candidalysin during the induction of OPC and the resulting oral inflammatory response.

The Expression of ephrinA1/ephA2 Receptor Increases in Chronic Rhinosinusitis and ephrinA1/ephA2 Signaling Affects Rhinovirus-Induced Innate Immunity in Human Sinonasal Epithelial Cells.

EphA2 receptor and its ephrin ligands are involved in virus infection, epithelial permeability, and chemokine secretion. We hypothesized that ephrinA1/ephA2 signaling participates in rhinovirus (RV)-induced antiviral immune response in sinonasal mucosa of patients with chronic rhinosinusitis (CRS). Therefore, we investigated the expression of ephrinA1/ephA2 in normal and inflamed sinonasal mucosa and evaluated whether they regulate chemokine secretion and the production of antiviral immune mediators including interferons (IFNs) in RV-infected human primary sinonasal epithelial cells. For this purpose, the expression and distribution of ephrinA1/ephA2 in sinonasal mucosa were evaluated with RT-qPCR, immunofluorescence, and western blot. Their roles in chemokine secretion and the production of antiviral immune mediators such as type I and III IFNs, and interferon stimulated genes were evaluated by stimulating ephA2 with ephrinA1 and inactivating ephA2 with ephA2 siRNA or inhibitor in cells exposed to RV and poly(I:C). We found that ephrinA1/ephA2 were expressed in normal mucosa and their levels increased in inflamed sinonasal mucosa of CRS patients. RV infection or poly(I:C) treatment induced chemokine secretion which were attenuated by blocking the action of ephA2 with ephA2 siRNA or inhibitor. The production of antiviral immune mediators enhanced by rhinovirus or poly (I:C) is increased by blocking ephA2 compared with that of cells stimulated by either rhinovirus or poly(I:C) alone. In addition, blocking ephA2 attenuated RV replication in cultured cells. Taken together, these results describe a novel role of ephrinA1/ephA2 signaling in antiviral innate immune response in sinonasal epithelium, suggesting their participation in RV-induced development and exacerbations of CRS.

Application of WES Towards Molecular Investigation of Congenital Cataracts: Identification of Novel Alleles and Genes in a Hospital-Based Cohort of South India.

Congenital cataracts are the prime cause for irreversible blindness in children. The global incidence of congenital cataract is 2.2-13.6 per 10,000 births, with the highest prevalence in Asia. Nearly half of the congenital cataracts are of familial nature, with a predominant autosomal dominant pattern of inheritance. Over 38 of the 45 mapped loci for isolated congenital or infantile cataracts have been associated with a mutation in a specific gene. The clinical and genetic heterogeneity of congenital cataracts makes the molecular diagnosis a bit of a complicated task. Hence, whole exome sequencing (WES) was utilized to concurrently screen all known cataract genes and to examine novel candidate factors for a disease-causing mutation in probands from 11 pedigrees affected with familial congenital cataracts. Analysis of the WES data for known cataract genes identified causative mutations in six pedigrees (55%) in PAX6, FYCO1 (two variants), EPHA2, P3H2,TDRD7 and an additional likely causative mutation in a novel gene NCOA6, which represents the first dominant mutation in this gene. This study identifies a novel cataract gene not yet linked to human disease. NCOA6 is a transcriptional coactivator that interacts with nuclear hormone receptors to enhance their transcriptional activator function.

EPHA2 antisense RNA modulates EPHA2 mRNA levels in basal-like/triple-negative breast cancer cells.

Ephrin type-A receptor 2 (EPHA2) is a receptor tyrosine kinase (RTK), whose over-expression has been observed in a variety of cancers, including breast cancer. EPHA2 expression may be causally related to tumorigenesis; therefore, it is important to understand how EPHA2 gene (EPHA2) expression is regulated. Here, we report that EPHA2 antisense RNA (EPHA2-AS), a natural antisense transcript, is an important modulator of EPHA2 mRNA levels. EPHA2-AS is a ∼1.8 kb long non-coding RNA (lncRNA) with a poly(A) tail that encodes two splice variants, EPHA2-AS1/2. They are constitutively expressed in a concordant manner with EPHA2 mRNA in human breast adenocarcinoma cell lines and in patient samples, with the highest levels detected in the triple-negative breast cancer (TNBC) subtype. The silencing of EPHA2-AS1/2 by a sense oligonucleotide or over-expression of an antisense oligoribonucleotide, which were both designed from the EPHA2 mRNA region (nt 2955-2974) targeted by AS1/2, showed that EPHA2-AS1/2 modulated EPHA2 mRNA levels by interacting with the specific AS1/2-complementary region in the mRNA. The EPHA2-AS1/2 did not prevent microRNAs from acting on the relevant microRNA response elements shared by EPHA2-AS1/2 and EPHA2 mRNA. Our studies demonstrate a crucial role played by EPHA2-AS1/2 in modulating EPHA2 mRNA levels, and hence production of EPHA2 protein, a key oncogenic RTK that contributes to the tumorigenesis of TNBC cells.

EphA2­enriched exosomes promote cell migration and are a potential diagnostic serum marker in pancreatic cancer.

Pancreatic cancer (PC) is the fourth most common cause of cancer­related mortality worldwide and is characterized by high invasiveness and early metastasis. To identify novel diagnostic markers, the present study aimed to understand the mechanism underlying PC progression. The present study demonstrated that exosomes derived from the highly metastatic Panc­1 PC cell line were internalized by a low metastatic cell line, resulting in increased migration of the latter. Proteomics analysis further revealed that the receptor tyrosine kinase Eph receptor A2 (EphA2) was overexpressed in the Panc­1 exosomes, and these Exo_EphA2 had the ability to transfer metastatic potential to recipient cells. Consistent with this, circulating Exo_EphA2 levels were higher in patients with PC compared with healthy controls. Taken together, these results indicated that Exo_EphA2 acts an oncogene in PC and is a potential tumor maker for PC diagnosis.

Epstein-Barr Virus gH/gL and Kaposi's Sarcoma-Associated Herpesvirus gH/gL Bind to Different Sites on EphA2 To Trigger Fusion.

Both Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human gammaherpesviruses and are important in a variety of malignancies. Eph family receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV and EBV. Previous studies identified five conserved residues (ELEFN50-54) in the N-terminal domain of KSHV gH that are critical for Eph binding and KSHV infection. However, the specific domains of EBV gH/gL important for EphA2 binding are not well described. We found that the KSHV gH (ELEFN50-54) motif is important for higher KSHV fusion and that EBV gH/gL does not utilize a similar motif for fusion activity. We previously identified that an EBV gL N-glycosylation mutant (gL-N69L/S71V) was hyperfusogenic in epithelial cells but not in B cells. To determine whether this glycosylation site may be the binding region for EphA2, we compared the EphA2 binding activity of EBV gH/gL and the EBV gH/gL-N69L/S71V mutant. We found that EBV gH/gL-N69L/S71V had higher binding affinity for EphA2, indicating that the EBV gL N-glycosylation site might be responsible for inhibiting the binding of gH/gL to EphA2. Loss of N-glycosylation at this site may remove steric hindrance that reduces EBV gH/gL binding to EphA2. In addition, the mutations located in the large groove of EBV gH/gL (R152A and G49C) also have decreased binding with EphA2. Taken together, our data indicate that the binding site of EphA2 on EBV gH/gL is at least in part proximal to the EBV gL glycosylation site, which in part accounts for differences in EphA2 binding affinity by KSHV.IMPORTANCE Virus entry into target cells is the first step for virus infection. Understanding the overall entry mechanism, including the binding mechanism of specific virus glycoproteins with cellular receptors, can be useful for the design of small molecule inhibitors and vaccine development. Recently, EphA2 was identified as an important entry receptor for both KSHV and EBV. In the present study, we investigated the required binding sites within EphA2 and EBV gH/gL that mediate the interaction of these two proteins allowing entry into epithelial cells and found that it differed in compared to the interaction of KSHV gH/gL with EphA2. Our discoveries may uncover new potential interventional strategies that block EBV and KSHV infection of target epithelial cells.

Y772 phosphorylation of EphA2 is responsible for EphA2-dependent NPC nasopharyngeal carcinoma growth by Shp2/Erk-1/2 signaling pathway.

EphA2 is an important oncogenic protein and emerging drug target, but the oncogenic role and mechanism of ligand-independent phosphorylation of EphA2 at tyrosine 772 (pY772-EphA2) is unclear. In this study, we established nasopharyngeal carcinoma (NPC) cell lines with stable expression of exogenous EphA2 and EphA2-Y772A (phosphorylation inactivation) using endogenous EphA2-knockdown cells, and observed that pY772A EphA2 was responsible for EphA2-promoting NPC cell proliferation and anchorage-independent and in vivo growth in mice. Mechanistically, EphA2-Y772A mediated EphA2-activating Shp2/Erk-1/2 signaling pathway in the NPC cells, and Gab1 (Grb2-associated binder 1) and Grb2 (growth factor receptor-bound protein 2) were involved in pY772-EphA2 activating this signaling pathway. Our results further showed that Shp2/Erk-1/2 signaling mediated pY772-EphA2-promoting NPC cell proliferation and anchorage-independent growth. Moreover, we observed that EphA2 tyrosine kinase inhibitor ALW-II-41-27 inhibited pY772-EphA2 and EphA2-Y772A decreased the inhibitory effect of ALW-II-41-27 on NPC cell proliferation. Collectively, our results demonstrate that pY772-EphA2 is responsible for EphA2-dependent NPC cell growth in vitro and in vivo by activating Shp2/Erk-1/2 signaling pathway, and is a pharmacologic target of ALW-II-41-27, suggesting that pY772-EphA2 can serve as a therapeutic target in NPC and perhaps in other cancers.

ANXA1 Binds and Stabilizes EphA2 to Promote Nasopharyngeal Carcinoma Growth and Metastasis.

Overexpression of ANXA1 and EphA2 has been linked to various cancers and both proteins have attracted considerable attention for the development of new anticancer drugs. Here we report that ANXA1 competes with Cbl for binding EphA2 and increases its stability by inhibiting Cbl-mediated EphA2 ubiquitination and degradation in nasopharyngeal carcinoma (NPC). Binding of ANXA1 to EphA2 promoted NPC cell growth and metastasis in vitro and in vivo by elevating EphA2 levels and increasing activity of EphA2 oncogenic signaling (pS897-EphA2). Expression of ANXA1 and EphA2 was positively correlated and both were significantly higher in NPC tissues than in the normal nasopharyngeal epithelial tissues. Patients with high expression of both proteins presented poorer disease-free survival and overall survival relative to patients with high expression of one protein alone. Furthermore, amino acid residues 20-30aa and 28-30aa of the ANXA1 N-terminus bound EphA2. An 11 amino acid-long ANXA1-derived peptide (EYVQTVKSSKG) was developed on the basis of this N-terminal region, which disrupted the connection of ANXA1 with EphA2, successfully downregulating EphA2 expression and dramatically suppressing NPC cell oncogenicity in vitro and in mice. These findings suggest that ANXA1 promotes NPC growth and metastasis via binding and stabilization of EphA2 and present a strategy for targeting EphA2 degradation and treating NPC with a peptide. This therapeutic strategy may also be extended to other cancers with high expression of both proteins. SIGNIFICANCE: These findings show that EphA2 is a potential target for NPC therapeutics and an ANXA1-derived peptide suppresses NPC growth and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4386/F1.large.jpg.

Baseline Computed Tomography Radiomic and Genomic Assessment of Head and Neck Squamous Cell Carcinoma.

PURPOSE: To determine the relationship between computed tomography (CT) radiomic features and gene expression levels in head and neck squamous cell carcinoma (HNSCC). METHODS: This retrospective study included 66 patients with HNSCC primary lesions (36 oropharyngeal, 6 hypopharyngeal, 10 laryngeal, 14 oral cavity). Gene expression information for 6 targetable genes (fibroblast growth factor receptor [FGFR]1, epidermal growth factor receptor [EGFR], FGFR2, FGFR3, EPHA2, PIK3CA) was obtained via Agilent microarrays from samples collected between 1997 and 2010. Pretreatment contrast-enhanced soft tissue neck CT scans were reviewed, and 142 radiomics features were derived. R was used to calculate Pearson correlation coefficients were calculated between gene expression levels and each radiomic feature. P values were adjusted using the false discovery rate (FDR) method. RESULTS: There were significant correlations between FGFR1 and 5 gray level cooccurrence matrix (GLCM) features with FDR-adjusted P values less than 0.05: inertia (r = 0.366, FDR-adjusted P = 0.006), absolute value (r = 0.31, FDR-adjusted P = 0.024), contrast (r = 0.366, FDR-adjusted P = 0.006), difference average (r = 0.31, FDR-adjusted P = 0.024), and difference variance (r = 0.37, FDR-adjusted P = 0.005). There was 1 correlated feature for FGFR2 with an FDR-adjusted P value less than 0.05: fractal dimension box-coarse (r = 0.33, FDR-adjusted P = 0.018). There was 1 correlated feature for EPHA2 with an FDR-adjusted P value less than 0.05: GLCM entropy (r = -0.28, FDR-adjusted P = 0.049). Six of the 7 features that showed significant correlation belonged to the GLCM class of features. CONCLUSIONS: The CT radiomic features demonstrate correlations with FGFR1 status in HNSCC and should be further investigated for their potential to predict FGFR1 status.

Production of 64Cu-labeled monobody for imaging of human EphA2-expressing tumors.

We previously reported on the monobody E1, which specifically targets the tumor marker hEphA2. In this study, we labeled NOTA-conjugated E1 with 64Cu (64Cu-NOTA-E1) and evaluated biologic characteristics. The uptake of 64Cu-NOTA-E1 in PC3 cells (a human prostate cancer cell line) with high expression of hEphA2 increased in a time-dependent manner. In PC3 xenograft mice, 64Cu-NOTA-E1 injected via the tail vein allowed visualization of tumors on positron emission tomography after 1 h and the highest uptake measured at 24 h post-injection. By contrast, the radioactivity of other tissues either did not increase or decreased over 24 h. This indicates that 64Cu-NOTA-E1 has high tumor uptake and retention, with rapid clearance, and low background values in other tissues. Therefore, 64Cu-NOTA-E1 should be suitable as a novel PET imaging agent for hEphA2-expressing tumors.

Optimization of stereospecific targeting technique for selective production of monoclonal antibodies against native ephrin type-A receptor 2.

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis.

HDAC7 plays a crucial role in cancers, and is the main drug target of several HDAC inhibitors. However, the role and mechanism of HDAC7 in nasopharyngeal carcinoma (NPC) are still unclear. In this study, we observed that HDAC7 was significantly upregulated in the NPC tissues relative to normal nasopharyngeal mucosa (NNM) tissues, HDAC7 expression levels were positively correlated with NPC progression and negatively correlated with patient prognosis, and HDAC7 knockdown dramatically inhibited the in vitro proliferation, migration, and invasion of NPC cells, and the growth of NPC xenografts in mice, indicating the HDAC7 promotes the oncogenicity of NPC. Mechanistically, HDAC7 promoted the in vitro proliferation, migration, and invasion of NPC cells by upregulating EphA2, in which miR-4465 mediated HDAC7-regulating EphA2, a direct target gene of miR-4465. We further showed that miR-4465 was significantly downregulated in the NPC tissues relative to NNM tissues, and inhibited the in vitro proliferation, migration, and invasion of NPC cells by targeting EphA2 expression. Moreover, we observed that the expressions of HDAC7, miR-4465, and EphA2 in NPC tissues were correlated. The results suggest that HDAC7 promotes the oncogenicity of NPC by downregulating miR-4465 and subsequently upregulating EphA2, highlighting HDAC7 as a potential therapeutic target for NPC.

Single nucleotide polymorphism (rs7543472) in EPHA2 gene is associated with age-related cataract in subjects enrolled from Multan in southern Punjab: A case-control study.

OBJECTIVES: To examine whether Ephrin type A receptor 2 gene polymorphisms are associated with susceptibility to age-related cataract. METHODS: The case-control study was conducted from January to May 2014 in Multan, Pakistan, and comprised patients of age-related cataract enrolled from Nishtar Hospital, Multan, and age-matched healthy controls without any type of cataract from the local population. A questionnaire was used to gather clinical and epidemiological data. Deoxyribonucleic acid was extracted from blood samples, and analysis of rs11260867, rs3568293 and rs7543472 single nucleotide polymorphisms was performed by using tetra amplification-refractory mutation system polymerase chain reaction protocol. Data was analysed using SPSS 17. RESULTS: Of the 230 subjects, 129(%) were patients and 101(%) were controls. Among the three polymorphisms analysed, rs7543472 was associated with age-related cataract. Among the epidemiological and clinical factors, age, diabetes, blood pressure, smoking, radiation exposure, steroids usage and use of tranquilisers were associated with age-related cataract (p<0.05 each). CONCLUSIONS: Polymorphism rs7543472 was found to be associated with age-related cataract.

Digenic inheritance of mutations in EPHA2 and SLC26A4 in Pendred syndrome.

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.

A Human Three-Dimensional In Vitro Model of Lens Epithelial Cells as a Model to Study Mechanisms of Drug-Induced Posterior Subcapsular Cataracts.

Purpose: Cataract is a pathological opacification of the lens, which is still one of the leading causes of blindness in the world. Several etiologies are described, among them drug-induced cataract, for example, posterior subcapsular cataract (PSC) after steroid treatment. To investigate different mechanisms of drug-induced cataract a human three-dimensional (3D) lens in vitro model was developed, consisting of immortalized human lens epithelial cells. Methods: These cells were cultivated on 96-well, ultralow attachment plates, where they rapidly form spheroids. By gene expression analysis different markers were observed, which are important to maintain lens transparency, such as ephrin type-A receptor 2 (EphA2) or α-smooth muscle actin (α-SMA). Results: The lens epithelial cells form a spheroid within a few days and show stable expression of important lens marker, and size and viability remain stable up to 26 days in culture. The gene expression of the glucocorticoid-treated spheroids revealed a clear shift in the expression of EphA2, α-SMA, αB-crystallin (CRYAB), and heat shock protein beta-1 (HSPB1). Furthermore, the glucocorticoid treatment did not improve cell survival. Conclusions: This study proposes a useful 3D in vitro model, which expresses important lens markers and is capable of demonstrating features found in drug-induced cataracts. As the viability remains stable over long time, this model can also be used for long-term treatment. The main characteristics are the increased expression of α-SMA, CRYAB, and HSPB1 and the decreased expression of EphA2. The present data provide some first evidence on novel mechanisms involved in glucocorticoid-induced cataracts.