EphA2 Signaling Research Articles

Tumor cell-intrinsic EPHA2 suppresses anti-tumor immunity by regulating PTGS2 (COX-2).

Resistance to immunotherapy is one of the biggest problems of current oncotherapeutics. WhileT cell abundance is essential for tumor responsiveness to immunotherapy, factors that define the T cell inflamed tumor microenvironment are not fully understood. We conducted an unbiased approach to identify tumor-intrinsic mechanisms shaping the immune tumor microenvironment(TME), focusing on pancreatic adenocarcinoma because it is refractory to immunotherapy and excludes T cells from the TME. From human tumors, we identified EPHA2 as a candidate tumor intrinsic driver of immunosuppression. Epha2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy. We found that PTGS2, the gene encoding cyclooxygenase-2, lies downstream of EPHA2 signaling through TGFβ and is associated with poor patient survival. Ptgs2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy; pharmacological inhibition of PTGS2 was similarly effective. Thus, EPHA2-PTGS2 signaling in tumor cells regulates tumor immune phenotypes; blockade may represent a novel therapeutic avenue for immunotherapy-refractory cancers. Our findings warrant clinical trials testing the effectiveness of therapies combining EPHA2-TGFβ-PTGS2 pathway inhibitors with anti-tumor immunotherapy, and may change the treatment of notoriously therapy-resistant pancreatic adenocarcinoma.

Role of EphA2-PI3K signaling in vasculogenic mimicry induced by cancer-associated fibroblasts in gastric cancer cells.

Although erythropoietin-producing human hepatocellular receptor A2 (EphA2) signaling serves an important role in the tumor microenvironment, its contribution to vasculogenic mimicry (VM) formation in gastric cancer cells remains unclear. The aim of the present study was to investigate the role of EphA2 in VM formation induced by cancer-associated fibroblasts (CAFs). The conditioned medium of CAFs (CAF-CM) was prepared from 12 patients with gastric adenocarcinoma. VM was evaluated by the number of tubules and intersections in gastric cancer cells following CAF-CM treatment. The role of EphA2-phosphoinositide 3-kinase (PI3K) in VM was investigated using EphA2-targeted small interfering (si)RNAs (siEphA2), EphA2 inhibitors and PI3K-inhibitors. CAF-CM-induced VM formation was significantly associated with high protein expression levels of EphA2. EphA2 inhibitor and siEphA2 manipulation significantly decreased VM formation by CAF-CM. In siEphA2 cells, decreased expression levels of VM-associated proteins were observed. CAF-CM-induced VM formation was blocked by the PI3K-inhibitor. In conclusion, CAFs facilitate VM formation via EphA2-PI3K signaling in gastric cancer cells. Thus, EphA2-PI3K signaling may be required for CAF-promoted VM formation during gastric tumorigenesis.

Dasatinib, paclitaxel and carboplatin in women with advanced-stage and recurrent endometrial cancer: A pilot clinical and translational study

Abstract Purpose To evaluate the effect of dasatinib therapy on EphA2 signaling in cancers of women with measurable (biopsy amenable) advanced-stage, chemo-naive primary or recurrent endometrial cancer. Preliminary efficacy was also assessed. Patients and methods We performed a pilot study of single-agent dasatinib lead-in, followed by triplet dasatinib, paclitaxel, and carboplatin. We measured the downstream effectors of EphA2 signaling in pre- and post-dasatinib treatment biopsy tissue samples; we also determined the severity of adverse events and patients' progression-free survival and overall survival durations. Results Eighteen patients were recruited and given dasatinib (150 mg orally daily for 14 days), followed by paclitaxel, carboplatin and dasatinib (daily) for six cycles (21-day cycles). Seventeen patients were evaluable for toxicity and 11 patients for response. A reverse phase protein array and proximity ligation assay revealed that CRAF/BRAF dimerization, caveolin-1 level, and Notch pathway signaling were predictive of response and resistance to dasatinib. Overall, the objective response rate was 45% (95% CI: 17%–77%), with median progression-free survival duration of 10.5 months and median overall survival duration of 30.4 months. The most common grade 3 or 4 adverse events were neutropenia (76%), thrombocytopenia (53%), anemia (53%), and fatigue (12%). Conclusions Caveolin-1 expression, in combination with CRAF/BRAF heterodimerization, is associated with resistance to EphA2 targeting by dasatinib. The triplet combination showed interesting clinical activity in endometrial cancer with acceptable toxicity. Pretreatment with dasatinib may accentuate combination therapy toxicity.

Engineering nanomolar peptide ligands that differentially modulate EphA2 receptor signaling

The EPH receptor A2 (EphA2) tyrosine kinase plays an important role in a plethora of biological and disease processes, ranging from angiogenesis and cancer to inflammation and parasitic infections. EphA2 is therefore considered an important drug target. Two short peptides previously identified by phage display, named YSA and SWL, are widely used as EphA2-targeting agents owing to their high specificity for this receptor. However, these peptides have only modest (micromolar) potency. Lack of structural information on the binding interactions of YSA and SWL with the extracellular EphA2 ligand-binding domain (LBD) has for many years precluded structure-guided improvements. We now report the high-resolution (1.53-2.20 Å) crystal structures of the YSA peptide and several of its improved derivatives in complex with the EphA2 LBD, disclosing that YSA targets the ephrin-binding pocket of EphA2 and mimics binding features of the ephrin-A ligands. The structural information obtained enabled iterative peptide modifications conferring low nanomolar potency. Furthermore, contacts observed in the crystal structures shed light on how C-terminal features can convert YSA derivatives from antagonists to agonists that likely bivalently interact with two EphA2 molecules to promote receptor oligomerization, autophosphorylation, and downstream signaling. Consistent with this model, quantitative FRET measurements in live cells revealed that the peptide agonists promote the formation of EphA2 oligomeric assemblies. Our findings now enable rational strategies to differentially modify EphA2 signaling toward desired outcomes by using appropriately engineered peptides. Such peptides could be used as research tools to interrogate EphA2 function and to develop pharmacological leads.

β-glucan priming enhances neutrophil Fcγ receptor-mediated antifungal activity during oral mucosal infection

Abstract Neutrophils are key to control fungal proliferation in the oral cavity. Recently we identified that the ephrin type-A receptor 2 (EphA2) functions as an oral epithelial cell β-glucan receptor that triggers epithelial cell production of proinflammatory mediators in response to Candida albicans infection. Because EphA2 is also expressed by neutrophils, we investigated the role of EphA2 in neutrophil candidacidal activity during OPC. We found that when OPC was induced in EphA2−/− mice, there was delayed accumulation of phagocytes into the oral tissues and more severe disease relative to wild-type mice. Depletion of neutrophils in EphA2−/− mice did not further increase oral fungal burden, nor did adoptive transfer of EphA2−/− neutrophils into CD18−/− mice decrease oral fungal burden, indicating that EphA2 on neutrophils plays a central role in the host defense against OPC. Mice lacking the Fcγ receptor I (CD64) were also highly susceptible to OPC. In vitro studies showed that EphA2 signaling augments neutrophil Fcγ receptor-mediated antifungal activity. The interaction of serum-opsonized C. albicansyeast with neutrophil EphA2 activated the MEK-ERK signaling pathway, leading to NADPH subunit p47phox site specific phospho-priming. EphA2-induced priming of neutrophil p47phox increased intracellular ROS production and enhanced killing of opsonized C. albicans yeast. Thus, in neutrophils, EphA2 serves as a receptor for β-glucans that enhances Fcγ receptor-mediated antifungal activity and is crucial to control early fungal proliferation during OPC.

361 Ephrin-A1 ligand strengthens tight junction function and prevents Th2 cytokine-induced barrier collapse

Atopic dermatitis (AD) is a chronic skin disease affecting all ages at a worldwide prevalence rate up to 20%. AD is largely driven by immune signals initiated by Th2 cytokines including interleukin (IL)-4 and IL-13. However, mounting evidence points to a central role for epidermal barrier dysfunction in the pathogenesis of AD. We have shown that the epidermal tight junction (TJ) network, a critical component of the barrier, depends on the receptor tyrosine kinase EphA2. Therefore, we hypothesize that EphA2 is a positive regulator of TJs in epidermis and can be targeted to improve epidermal barrier function. In 2D keratinocyte cultures, engaging EphA2 with its ligand ephrin-A1 enhances TJ barrier function compared to control (P=0.0001), as measured by trans-epithelial electrical resistance (TEER). This enhancement is accompanied by the immunofluorescent accumulation of TJ proteins, but only small changes in protein and transcript expression. Increased TEER (P<0.05) and TJ formation are also seen in 3D human epidermal equivalents (3D HEE) after ephrin-A1 treatment. These results led us to ask if ephrin-A1 can prevent epidermal barrier defects in in vitro models of AD. Concurrent treatment with IL-4 and IL-13 induces an AD-like phenotype apparent by changes in epidermal morphology. We show that ephrin-A1 treatment in 3D human skin equivalents (3D HSE) restores keratohyalin granules and the stratum corneum which are lost in 3D HSE after cytokine treatment alone. In human explant cultures of truncal skin, IL-4+IL-13 cause epidermal spongiosis and increased epidermal thickness, effects that are abrogated by ephrin-A1. In addition to altering epidermal morphology, we find that IL-4+IL-13 reduce TEER in 3D HEE compared to controls (P<0.05) and obliterate TJs, shown by loss of occludin immunofluorescence. This cytokine-induced catastrophe of TJ function and formation was rescued by co-treatment with ephrin-A1. Taken together, these results suggest that targeting the EphA2 signaling axis not only enhances TJs, but can also restore epidermal barrier function in the face of Th2 cytokine-driven TJ disassembly.

Morphological characteristics of vasculogenic mimicry and its correlation with EphA2 expression in gastric adenocarcinoma

Genetically deregulated tumor cells generate vascular channels by vasculogenic mimicry (VM) that is independent of endothelial blood vessels. The morphological characteristics of VM and the role of EphA2 in the formation of VM were evaluated in 144 clinical samples of gastric adenocarcinoma and AGS gastric cancer cell line. It has long been believed that VM consists of PAS-positive basement membrane and CD31/CD34-negative cells. Interestingly, we found that the luminal surface of gastric tumor cells that form VM channels showed PAS-positive reaction, and that the involvement of CD31/CD34-positive tumor cells in the formation of VM channels. Highly aggressive tumor cells that formed VM were found to express CD31 or CD34, implicating the angiogenic and vasculogenic potential of the genetically deregulated tumor cells. VM occurrence was positively correlated with high expression of EphA2 in our patient cohort, and the indispensable role of EphA2 in VM formation was identified by gene silencing in AGS cells. We also report that Epstein–Barr virus (EBV)-positive tumor cells were involved in the formation of VM channels in EBV-associated gastric cancer samples. Overall, our results suggest that EphA2 signaling promotes tumor metastasis by inducing VM formation during gastric tumorigenesis.

Retraction of: Age-Associated Defects in EphA2 Signaling Impair the Migration of Human Cardiac Progenitor Cells.

Retraction of: Age-Associated Defects in EphA2 Signaling Impair the Migration of Human Cardiac Progenitor Cells.

Investigating the Role of the Transmembrane Helix of EphA2 in Signal Transduction across the Plasma Membrane

Receptor tyrosine kinases (RTKs), the second largest class of membrane receptors, transduce biochemical signals across the cell membrane via lateral dimerization or oligomerization. RTKs control cell growth, differentiation and motility, and are involved in many diseases such as cancer. The single transmembrane domains of RTKs have been hypothesized to play important roles in signal transduction. First, sequence-specific interactions between the transmembrane domains are believed to contribute to the overall stability of RTK dimers and oligomers. Second, it has been proposed that the transmembrane domain dimer controls the spatial separation and orientation of the catalytic domains in the RTK oligomers. In this study, we focus on the transmembrane domain of EphA2, an RTK involved in many critical physiological processes, including tissue homeostasis, angiogenesis and tumorigenesis. Previous studies in our lab have shown that EphA2 can associate into two types of dimers, as well as clusters, depending on the type of ligand (dimeric EphrinA1-Fc, Ephrin A1 and the engineered peptide YSA), with distinct interaction interfaces in the extracellular region. By mutating key interaction motifs in the transmembrane domain and utilizing a quantitative FRET methodology, we seek to determine if the interaction between the transmembrane domains is affected by the type of bound ligands. These experiments will give new insights into the role of the transmembrane domain in EphA2 signaling.

EphA2 is a Neutrophil Receptor for Candida Albicans that Stimulates Antifungal Activity During Oropharyngeal Infection

During oropharyngeal candidiasis (OPC), Candida albicans proliferates and invades the superficial oral epithelium. The ephrin type-A receptor 2 (EphA2) functions as an oral epithelial cell β-glucan receptor that triggers the production of proinflammatory mediators in response to fungal infection. Because EphA2 is also expressed by neutrophils, we investigated the role of EphA2 in neutrophil candidacidal activity during OPC. We found that when OPC was induced in EphA2–/– mice, there was delayed accumulation of phagocytes in the oral tissues and more severe disease relative to wild-type mice. Depletion of neutrophils in EphA2–/– mice did not further increase oral fungal burden, nor did adoptive transfer of EphA2–/– neutrophils into CD18−/− mice decrease oral fungal burden, indicating that EphA2 on neutrophils plays a central role in the host defense against OPC. Mice lacking the Fcγ receptor I were also highly susceptible to OPC. In vitro studies showed that EphA2 signaling augments neutrophil Fcγ receptor-mediated antifungal activity. The interaction of serum-opsonized C. albicans yeast with neutrophil EphA2 activated the MEK-ERK signaling pathway, leading to NADPH subunit p47phox site specific phospho-priming. EphA2-induced priming of neutrophil p47phox increased intracellular ROS production and enhanced killing of opsonized C. albicans yeast. Thus, in neutrophils, EphA2 serves as a receptor for β-glucans that enhances Fcγ receptor-mediated antifungal activity and is crucial to control early fungal proliferation during OPC.

Abstract 3025: Ligand-independent EphA2 signaling drives an amoeboid phenotype that promotes melanoma brain metastasis development

Abstract The majority of melanoma patients treated with BRAF inhibitors (BRAFi) ultimately develop resistance and fail on therapy. It has previously been shown that BRAFi resistance is highly correlated with the adoption of a strongly invasive phenotype through ligand-independent EphA2 signaling. In this study, we used comprehensive mass spectrometry-based proteomic approaches to delineate the global signaling changes associated with the aggressive phenotype driven by ligand-independent EphA2 signaling. Preliminary data have identified new signaling adaptations associated with a mesenchymal-to-amoeboid transition (MAT) phenotype, which was confirmed in 3D collagen cultures. Functional experiments demonstrated that the amoeboid phenotype promoted cell migration and invasion, which was mediated through the interaction of EphA2 and CDC42. These findings were confirmed by expressing a constitutively activated form of CDC42. BRAFi resistant cell lines also exhibited the MAT phenotype and were more invasive compared to their treatment-naïve counterparts, in line with their dependence upon ligand-independent EphA2 signaling following BRAFi selection pressure. To further demonstrate the metastatic potential of these amoeboid cells driven by ligand-independent EpHA2 signaling in vivo, we performed intracardiac injections in mice with cell lines expressing either EpHA2 S897A (inactive) or S897E (constitutively active phosphomimetic). Interestingly, we found a preferential homing of EphA2 S897E cells to the brain, but no difference in metastasis to other organs including the lung and liver. An analysis of brain metastasis specimens from patients failing BRAF and BRAF/MEK inhibitor therapy showed strong staining for the amoeboid phenotype marker EphA2. To investigate whether the amoeboid phenotype may confer a survival advantage in circulation, we carried out shear stress assays. These experiments demonstrated that the amoeboid phenotype driven by ligand-independent EphA2 signaling promoted the survival of melanoma cells under shear stress. Cell attachment assays, trans-endothelial invasion assays and vascular permeability assays show that these cells are better suited to attach to and permeate an endothelial monolayer. We further show that inhibiting PI3K reversed the amoeboid phenotype and limited the EphA2-driven invasive capacity. In summary, we show for the first time that BRAFi resistance is associated with the adoption of an MAT phenotype that increases the metastatic seeding of melanoma cells to the brain, which can be reversed through inhibition of PI3K signaling. Citation Format: Chao Zhang, Inna Smalley, Ritin Sharma, Michael Emmons, Jane Messina, John Koomen, Keiran Smalley. Ligand-independent EphA2 signaling drives an amoeboid phenotype that promotes melanoma brain metastasis development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3025.

Eph/ephrin signalling serves a bidirectional role in lipopolysaccharide‑induced intestinal injury

A growing body of evidence has demonstrated that Eph/ephrin signalling may serve a central role in intestinal diseases. However, whether erythropoietin-producing hepatocellular (Eph)/ephrin signalling is associated with the development of post-infectious irritable bowel syndrome (PI-IBS) is still unknown. In the present study, the role of Eph/Ephrin signalling in lipopolysaccharide (LPS)-induced intestinal injury was evaluated in vivo and in vitro. LPS treatment significantly increased the levels of proinflammatory mediators [monocyte chemoattractant protein-1, tumour necrosis factor α, interleukin (IL)-1β, IL-6, intercellular adhesion molecule 1 and vascular cell adhesion molecule-1], activated the EphA2-Ephrin A1, protein kinase B (Akt)-nuclear factor (NF)-κB, Src-NF-κB and Wnt/β-catenin signalling pathways, and inhibited EphB1-Ephrin B3 signalling in colon tissues, and primary cultured enteric neuronal and glial cells. Notably, EphA2 monoclonal antibody (mAb) treatment or Ephrin B3 overexpression could partially alleviate the LPS-induced upregulation of proinflammatory mediators, and Akt-NF-κB, Src-NF-κB and Wnt/β-catenin signalling pathways. In addition, EphA2 mAb treatment could partially inhibit LPS-induced inactivation of EphB-Ephrin B3 signalling, while Ephrin B3 overexpression could abrogate LPS-induced activation of EphA2-Ephrin A1 signalling. EphB1/Ephrin B3 signalling may antagonise the EphA2/Ephrin A1-dependent pathway following LPS treatment. The results associated with the EphA2 signaling pathway, indicated that Eph/ephrin signalling may serve a bidirectional role in LPS-induced intestinal injury. Eph/ephrin signalling may be a novel therapeutic target for LPS-induced intestinal injury and potentially PI-IBS.

Cancer-associated fibroblasts promote gastric tumorigenesis through EphA2 activation in a ligand-independent manner.

Under physiologic conditions, the binding of erythropoietin-producing hepatocellular (Eph) A2 receptor and its ligand ephrinA1 results in decreased EphA2 level and tumor suppression. However, EphA2 and ephrinA1 are highly expressed in human cancers including gastric adenocarcinoma. In this study, we tested our hypothesis that cancer-associated fibroblasts (CAFs) promote gastric tumorigenesis through EphA2 signaling in a ligand-independent manner. Expression of EphA2 protein in primary tumor tissues of 91 patients who underwent curative surgery for gastric adenocarcinoma was evaluated by immunohistochemistry and western blotting. Conditioned medium of cancer-associated fibroblasts (CAF-CM) was used to evaluate the tumorigenic effect of CAFs on gastric cancer cell lines. Epithelial-mesenchymal transition (EMT), cell proliferation, migration, and invasion were assessed. EphrinA1-Fc ligand was used to determine the suppressor role of EphA2 receptor-ligand binding. CAF-CM-induced EMT and promoted cancer cell motility even without cell-cell interaction. Treatment with a selective EphA2 inhibitor (ALW-II-41-27) or EphA2-targeted siRNA markedly reduced CAF-CM-induced gastric tumorigenesis. EphrinA1-Fc ligand treatment showing ligand-dependent tumor suppression diminished the EphA2 expression and EMT progression. In contrast, ephrinA1-targeted siRNA did not significantly affect CAF-CM-mediated increases in EphA2 expression and EMT progression. Treatment with VEGF showed effects like CAF-CM in terms of EphA2 activation and EMT progression. CAFs may contribute to gastric tumorigenesis by activating EphA2 signaling pathway in a ligand-independent manner. Our results suggest that ligand-independent activation of EphA2 was triggered by VEGF released from CAF-CM. Our result may partially explain why ligand-dependent tumor suppressor roles of EphA2 are not evident in gastric cancer despite the prominent level of ephrinA1.

Spatially modulated ephrinA1:EphA2 signaling increases local contractility and global focal adhesion dynamics to promote cell motility

Recent studies have revealed pronounced effects of the spatial distribution of EphA2 receptors on cellular response to receptor activation. However, little is known about molecular mechanisms underlying this spatial sensitivity, in part due to lack of experimental systems. Here, we introduce a hybrid live-cell patterned supported lipid bilayer experimental platform in which the sites of EphA2 activation and integrin adhesion are spatially controlled. Using a series of live-cell imaging and single-molecule tracking experiments, we map the transmission of signals from ephrinA1:EphA2 complexes. Results show that ligand-dependent EphA2 activation induces localized myosin-dependent contractions while simultaneously increasing focal adhesion dynamics throughout the cell. Mechanistically, Src kinase is activated at sites of ephrinA1:EphA2 clustering and subsequently diffuses on the membrane to focal adhesions, where it up-regulates FAK and paxillin tyrosine phosphorylation. EphrinA1:EphA2 signaling triggers multiple cellular responses with differing spatial dependencies to enable a directed migratory response to spatially resolved contact with ephrinA1 ligands.

687 Autophagy and ciliogenesis are coordinately regulated by EphA2 in epidermis

Autophagy and ciliogenesis are required for proper epidermal differentiation; however, the link between epidermal autophagy and ciliogenesis remains unclear. Knockdown of the receptor tyrosine kinase EphA2 in a human keratinocyte cell line (hTCEpi) resulted in an increase in: (i) large vacuoles; (ii) LC3II, a mature form of LC3 protein; and (iii) p62, a negative marker of autophagy flux. Loss of EphA2 in human epidermal keratinocytes (HEKs) decreased LC3 turnover, another measure of autophagy flux. This establishes a positive role for EphA2 in autophagy. BioID assays combined with unbiased proteomic analyses reveal that in HEK 3-D raft cultures, 10% of proteins interacting with EphA2 involved autophagy. Among them was phospholipase D1 (PLD1), a critical regulator of end-stage autophagy. Pulldown and co-immunoprecipitation assays confirmed that EphA2 binds to PLD1. Pharmacologic and genetic inhibition of PLD1 signaling reversed vacuole accumulation and increased LC3II in hTCEpi cells lacking EphA2. This is compelling evidence that EphA2 regulates autophagy via PLD1. Impaired autophagy also impacted ciliogenesis in epithelial cells lacking EphA2, as the percentage of ciliated cells were decreased in hTCEpi cells and HEKs threated with siEphA2. Such a decrease was reversed by knockdown of PLD1. Embryonic skin of Xenopus, a well-established in vivo model of ciliogenesis, was treated with a Morpholino oligo targeting Epha2, which impaired ciliogenesis, clearly establishing a role for EphA2 in this process. We show that EphA2, in part, coordinates autophagy and ciliogenesis, processes essential for proper epidermal differentiation. An abnormality in EphA2 signaling has been demonstrated in psoriasis; a disease characterized by impaired epidermal differentiation. Thus, understanding the relationships between EphA2 signaling, autophagy and ciliogenesis on the background of psoriasis is warranted.

EphA2 receptor is a key player in the metastatic onset of Ewing sarcoma.

Ewing sarcoma (ES) is the second most common bone malignancy affecting children and young adults with poor prognosis due to high metastasis incidence. Our group previously described that EphA2, a tyrosine kinase receptor, promotes angiogenesis in Ewing sarcoma (ES) cells via ligand-dependent signaling. Now we wanted to explore EphA2 ligand-independent activity, controlled upon phosphorylation at S897 (p-EphA2S897 ), as it has been linked to metastasis in several malignancies. By reverse genetic engineering we explored the phenotypic changes after EphA2 removal or reintroduction. Gene expression microarray was used to identify key players in EphA2 signaling. Mice were employed to reproduce metastatic processes from orthotopically implanted engineered cells. We established a correlation between ES cells aggressiveness and p-EphA2S897 . Moreover, stable overexpression of EphA2 in low EphA2 expression ES cells enhanced proliferation and migration, but not a non-phosphorylable mutant (S987A). Consistently, silencing of EphA2 reduced tumorigenicity, migration and invasion in vitro, and lung metastasis incidence in experimental and spontaneous metastasis assays in vivo. A gene expression microarray revealed the implication of EphA2 in cell signaling, cellular movement and survival. ADAM19 knockdown by siRNA technology strongly reproduced the negative effects on cell migration observed after EphA2 silencing. Altogether, our results suggest that p-EphA2S897 correlates with aggressiveness in ES, so blocking its function may be a promising treatment.

EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling.

PurposeProgenitor cells of the limbal epithelium reside in a discrete area peripheral to the more differentiated corneal epithelium and maintain tissue homeostasis. What regulates the limbal–corneal epithelial boundary is a major unanswered question. Ephrin-A1 ligand is enriched in the limbal epithelium, whereas EphA2 receptor is concentrated in the corneal epithelium. This reciprocal pattern led us to assess the role of ephrin-A1 and EphA2 in limbal–corneal epithelial boundary organization.MethodsEphA2-expressing corneal epithelial cells engineered to express ephrin-A1 were used to study boundary formation in vitro in a manner that mimicked the relative abundance of these juxtamembrane signaling proteins in the limbal and corneal epithelium in vivo. Interaction of these two distinct cell populations following initial seeding into discrete culture compartments was assessed by live cell imaging. Immunofluoresence and immunoblotting was used to evaluate the contribution of downstream growth factor signaling and cell–cell adhesion systems to boundary formation at sites of heterotypic contact between ephrin-A1 and EphA2 expressing cells.ResultsEphrin-A1–expressing cells impeded and reversed the migration of EphA2-expressing corneal epithelial cells upon heterotypic contact formation leading to coordinated migration of the two cell populations in the direction of an ephrin-A1–expressing leading front. Genetic silencing and pharmacologic inhibitor studies demonstrated that the ability of ephrin-A1 to direct migration of EphA2-expressing cells depended on an a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and epidermal growth factor receptor (EGFR) signaling pathway that limited E-cadherin–mediated adhesion at heterotypic boundaries.ConclusionsEphrin-A1/EphA2 signaling complexes play a key role in limbal–corneal epithelial compartmentalization and the response of these tissues to injury.

EphA2 signaling is impacted by carcinoembryonic antigen cell adhesion molecule 1-L expression in colorectal cancer liver metastasis in a cell context-dependent manner.

We have shown that carcinoembryonic antigen cell adhesion molecule 1 long isoform (CEACAM1-L) expression in MC38 metastatic colorectal cancer (CRC) cells results in liver metastasis inhibition via CCL2 and STAT3 signaling. But other molecular mechanisms orchestrating CEACAM1-L-mediated metastasis inhibition remain to be defined. We screened a panel of mouse and human CRC cells and evaluated their metastatic outcome after CEACAM1 overexpression or downregulation. An unbiased transcript profiling and a phospho-receptor tyrosine kinase screen comparing MC38 CEACAM1-L-expressing and non-expressing (CT) CRC cells revealed reduced ephrin type-A receptor 2 (EPHA2) expression and activity. An EPHA2-specific inhibitor reduced EPHA2 downstream signaling in CT cells similar to that in CEACAM1-L cells with decreased proliferation and migration. Human CRC patients exhibiting high CEACAM1 in combination with low EPHA2 expression benefited from longer time to first recurrence/metastasis compared to those with high EPHA2 expression. With the added interaction of CEACAM6, we denoted that CEACAM1 high- and EPHA2 low-expressing patient samples with lower CEACAM6 expression also exhibited a longer time to first recurrence/metastasis. In HT29 human CRC cells, down-regulation of CEACAM1 along with CEA and CEACAM6 up-regulation led to higher metastatic burden. Overall, CEACAM1-L expression in poorly differentiated CRC can inhibit liver metastasis through cell context-dependent EPHA2-mediated signaling. However, CEACAM1’s role should be considered in the presence of other CEACAM family members.

Abstract 213: Deletion of Epha2 Reduces Fibroproliferative Remodeling in Atherosclerosis and Vascular Smooth Muscle

Advanced atherosclerosis involves the recruitment of vascular smooth muscle cells, leading to smooth muscle-mediated fibroproliferative remodeling and plaque stiffening. We previously demonstrated the neuronal guidance receptor EphA2 shows enhanced expression in murine and human atherosclerosis, and EphA2 deletion in atherosclerosis-prone ApoE knockout mice attenuates lesion formation and endothelial cell activation in models of atherosclerosis. We now demonstrate deletion of EphA2 attenuates plaque progression to advanced stages associated with diminished smooth muscle content. While contractile smooth muscle cells show minimal EphA2 expression in vitro and in vivo , transition to the synthetic phenotype results in enhanced EphA2 expression and luciferase reporter activity in vitro and in atherosclerotic lesions. Furthermore, forced expression of EphA2 in quiescent smooth muscle cells is sufficient to suppress contractile smooth muscle markers, suggesting a role for EphA2 in smooth muscle phenotypic switch. Depletion of EphA2 results in significant reductions in proliferation within atherosclerotic plaques and in smooth muscle cells with associated reductions in serum-induced ERK and Akt signaling. Conversely, overexpression of EphA2 enhances smooth muscle proliferation in vitro , suggesting EphA2 is sufficient to drive proliferation. EphA2-deficient mice show reductions in plaque fibrosis and matrix remodeling. Consistent with this, depletion of EphA2 in vascular smooth muscle cells exhibit a significant reduction in matrix deposition. Together these data suggest a role for EphA2 in smooth muscle-mediated fibroproliferative remodeling, representing the first link between EphA2 signaling and smooth muscle function in atherosclerosis.

Shear stress inhibits IL-17A-mediated induction of osteoclastogenesis via osteocyte pathways

Interleukin (IL)-17 is crucial to osteoclast differentiation and activation. Osteocytes support osteoclast formation and are thought to orchestrate bone remodeling in response to fluid flow. The contribution of IL-17 to osteocyte-related bone resorption remains unclear. Here, we used the osteocyte-like MLO-Y4 cell line to examine the role of IL-17 and fluid flow in osteoclastogenesis. It was the first time to demonstrate that IL-17A promoted MLO-Y4 cell proliferation, enhanced expression of receptor activator of nuclear factor κ-B ligand (RANKL) and tumor necrosis factor-α (TNF-α), and induced osteoclastogenesis when MLO-Y4 cells were co-cultured with bone marrow-derived macrophage (BMM) cells. Additionally, shear stress upregulated osteoprotegerin expression in osteocytes, downregulated the effect of IL-17A on RANKL and TNF-α expression, and attenuated IL-17A-activated osteoclastic differentiation in the co-culture system of MLO-Y4 and BMM cells. Furthermore, we explored the signaling pathways that potentially mediate these effects in osteocytes, and found that the extracellular signal-regulated kinase (ERK)1/2 and signal transducer and activator of transcription (STAT3) pathways were suppressed by IL-17A but induced by fluid flow. EphA2 signaling enhances osteoclastogenesis in osteocytes, and the intercellular reversed EphA2-ephrinA2 signaling from osteocytes to BMM play an important role in IL-17A-dependent osteoclastic differentiation. EphB4 signaling inhibits osteoclastogenesis in osteocytes, and the intercellular reversed EphB4-ephrinB2 signaling from osteocytes to BMM could inhibit IL-17A-dependent osteoclastic differentiation. The current findings suggest that IL-17A as a promoter of bone resorption and fluid shear stress critically regulate bone remodeling via osteocyte-specific signaling pathways. IL-17 modulation-based approaches may be developed as a novel therapeutic strategy for enhancing bone remodeling efficiency and stability.