EphA2 Signaling Research Articles

Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

Disruption of Eph-ephrin bidirectional signaling leads to human congenital and age-related cataracts, but the mechanisms for these opacities in the eye lens remain unclear. Eph receptors bind to ephrin ligands on neighboring cells to induce canonical ligand-mediated signaling. The EphA2 receptor also signals non-canonically without ligand binding in cancerous cells, leading to epithelial-to-mesenchymal transition (EMT). We have previously shown that the receptor EphA2 and the ligand ephrin-A5 have diverse functions in maintaining lens transparency in mice. Loss of ephrin-A5 leads to anterior cataracts due to EMT. Surprisingly, both canonical and non-canonical EphA2 activation are present in normal wild-type lenses and in the ephrin-A5 knockout lenses. Canonical EphA2 signaling is localized exclusively to lens epithelial cells and does not change with age. Non-canonical EphA2 signaling is in both epithelial and fiber cells and increases significantly with age. We hypothesize that canonical ligand-dependent EphA2 signaling is required for the morphogenesis and organization of hexagonal equatorial epithelial cells while non-canonical ligand-independent EphA2 signaling is needed for complex membrane interdigitations that change during fiber cell differentiation and maturation. This is the first demonstration of non-canonical EphA2 activation in a non-cancerous tissue or cell and suggests a possible physiological function for ligand-independent EphA2 signaling.

A biocompatible nanoformulation of curcumin analogue and curd exosomes targeting EphA2 signalling cascade in head and neck cancer

BackgroundMajor therapeutic developments have been made in the prevention of head and neck cancer (HNC), and crucial measures have been implemented for the survival of patients. The advent of cancer nano-theranostic as an effective approach targets cancer by allowing drug aggregation at the tumour site, its proper bioaccessibility, and tumour cell death. Curd exosomes are the cellular interactive nanovesicles, considered a convenient conveyance medium for cargoes still unexplored. Curcumin analogue alanine is primarily recognised for its superior radical scavenging activity and anti-mutagen properties compared with curcumin.MethodsThe current study focussed on the isolation and characterisation of curd exosomes, followed by their interaction with cancer cells to deliver their content conveniently. Herein, we developed a nanoformulation of curd exosomes loaded with curcumin alanine to determine its bioaccessibility and anti-proliferative effect compared with curcumin alanine free drug. In addition, the influence of curcumin alanine and its nanoformulation on cell morphology, nucleus structures, colony formation potential, and tumour cell death was observed. The expression of EphA2 and its associated molecules was determined using western blot and PCR to explore the mechanism at the cellular level.ResultsThe recent investigation revealed the encapsulation of curcumin analogue alanine in curd exosomes enhanced the bioaccessibility in contrast with curcumin alanine. Then, we focussed on the curcumin alanine effect on HNC cells to monitor morphological alterations, a reduction in cell multiplication, and triggering apoptosis. Particularly, we found considerable suppression of EphA2 influencing mitochondrial dynamics with the strengthening of mitochondrial fusion MFN1 and MFN2, whereas fission-associated protein DRP1 was down-regulated by the treatment of curcumin alanine nanoformulation. Furthermore, curcumin alanine nanoformulation activates the apoptotic marker caspase-7 and suppresses the anti-apoptotic marker Bcl-xL.ConclusionHence, these findings have drawn attention to curd exosomes loaded curcumin alanine nanoformulation impairing cell multiplication and mitochondrial fission, leading to apoptotic cell death, as one of the effective approaches for the treatment of HNC.Graphical

Inhibition of the EphA2-Sam/Ship2-Sam Association through Peptide Ligands: Studying the Combined Effect of Charge and Aromatic Character.

The Sam (sterile alpha motif) domain from the lipid phosphatase Ship2 binds the Sam domain from the EphA2 receptor to negatively regulate receptor endocytosis and degradation. This interaction is primarily linked to pro-oncogenic effects. We report on the design and evaluation of EphA2-Sam/Ship2-Sam peptide inhibitors provided with positive charges and different aromatic characters. Starting from the sequence of previously identified Ship2-Sam targeting peptides, an in silico approach was set up to predict higher affinity peptide ligands. A few peptides were experimentally tested through an interdisciplinary approach. Interaction studies were performed by nuclear magnetic resonance spectroscopy and biolayer interferometry. 3D models of Ship2-Sam/peptide complexes were predicted by AlphaFold2. Cell-based assays were carried out to investigate whether such peptide sequences might have an influence on EphA2 signaling. The approach led to the identification of novel Ship2-Sam ligands and shed further light on original approaches to design inhibitors of the Ship2-Sam/EphA2-Sam interaction.

Abstract 6866: Investigating the role of Epha2 in breast cancer-mediated myeloid cell expansion and function

Abstract Bone metastases are present in over 70% of metastatic breast cancer patients, and current therapy reduces osteolysis in tumor-induced bone disease (TIBD) but does not improve overall survival. Frequently, TIBD is characterized by an increase in osteoclast proliferation and bone-resorbing activity, and patients often experience pain and pathologic fractures. Increased bone resorption releases growth factors that aid in the progression of tumor growth causing the ‘vicious cycle’. Additionally, bone metastatic patients are often resistant to first- and second-line therapies highlighting an importance for research in this area. The receptor tyrosine kinase, Ephrin-type-A 2 receptor (EphA2), participates in both forward and reverse signaling during receptor/ligand engagement and participates in many physiological and pathological processes, including bone homeostasis, immunity, and cancer. Interestingly, EphA2 is highly expressed in bone metastases, and previous evidence suggests that EphA2 can promote TIBD and reduce anti-tumor immunity. We therefore hypothesize that increased expression of EphA2 in breast cancer cells at the site of bone metastasis promotes expansion of myeloid cells, leading to TIBD. Previous data shows a reduction in osteoclast number and bone lesions following EphA2 knockout. However, the mechanism by which EphA2 mediates the expansion of osteoclasts is not well understood. In agreement with previous work, our data shows that EphA2 deletion in murine breast cancer (BC) cell line, 4T1, reduces bone lesion area and number and the presence of bone metastasis-associated macrophages. Additionally, we observed a significant reduction in Ly6Chi cells. To assess the effects of EphA2 deletion on the behavior of 4T1 cells, we conducted a wound healing assay and observed a decrease in wound healing capacity in 4T1 cells following EphA2 deletion. This suggests that EphA2 expression in 4T1 cells may increase their metastatic potential. We will repeat these studies and incorporate factors from the bone marrow. Our future studies will investigate the role of EphA2 signaling and reverse signaling in BC cell lines, myeloid progenitor cells, osteoclasts, and bone metastasis-associated macrophages. We will utilize murine and human BC cell lines to elucidate the effects of EphA2 deletion in BC on immune cell populations, bone metastasis, and tumor progression. Additionally, we will investigate how EphA2 reverse signaling impacts osteoclasts’ ability to modulate immune cells. We aim to reduce TIBD and tumor burden by reducing the expansion of osteoclasts and shifting the immune cells to an anti-tumorigenic phenotype. Our studies will provide additional insight into the underlying mechanisms of the vicious cycle which will offer new treatment strategies for patients with osteolytic breast cancer. Citation Format: Dominique Parker, Verra Ngwa, Jin Chen, Julie Rhoades. Investigating the role of Epha2 in breast cancer-mediated myeloid cell expansion and function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6866.

Targeting host tyrosine kinase receptor EphA2 signaling via small-molecule ALW-II-41-27 inhibits macrophage pro-inflammatory signaling responses to Pneumocystis carinii β-glucans.

Pneumocystis jirovecii, the fungus that causes Pneumocystis jirovecii pneumonia (PJP), is a leading cause of morbidity and mortality in immunocompromised individuals. We have previously shown that lung epithelial cells can bind Pneumocystis spp. β-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. β-glucans activation of the inflammatory signaling cascade in macrophages can be pharmacodynamically inhibited with the EphA2 receptor small-molecule inhibitor ALW-II-41-27. In vitro, when ALW-II-41-27 is administrated via intraperitoneal to mice prior to the administration of highly proinflammatory Saccharomyces cerevisiae β-glucans in the lung, a significant reduction in TNF-alpha release was noted in the ALW-II-41-27 pre-treated group. Taken together, our data suggest that targeting host lung macrophage activation via EphA2 receptor-fungal β-glucans interactions with ALW-II-41-27 or other EphA2 receptor kinase targeting inhibitors might be an attractive and viable strategy to reduce detrimental lung inflammation associated with PJP.

Effectiveness of anti-erythropoietin producing Hepatocellular receptor Type-A2 antibody in pancreatic cancer treatment

Erythropoietin-producing hepatocyte receptor type A2 (EphA2) is a tyrosine kinase that binds to ephrins (e.g., ephrin-A1) to initiate bidirectional signaling between cells. The binding of EphA2 and ephrin-A1 leads to the inhibition of Ras-MAPK activity and tumor growth. During tumorigenesis, the normal interaction between EphA2 and ephrin-A1 is hindered, which leads to the overexpression of EphA2 and induces cancer. The overexpression of EphA2 has been identified as a notable tumor marker in diagnosing and treating pancreatic cancer. In this study, we used phage display to isolate specific antibodies against the active site of EphA2 by using a discontinuous recombinant epitope for immunization. The therapeutic efficacy and inhibition mechanism of the generated antibody against pancreatic cancer was validated and clarified. The generated antibodies were bound to the conformational epitope of endogenous EphA2 on cancer cells, thus inducing cellular endocytosis and causing EphA2 degradation. Molecule signals pAKT, pERK, pFAK, and pSTAT3 were weakened, inhibiting the proliferation and migration of pancreatic cancer cells. The humanized antibody hSD5 could effectively inhibit the growth of the xenograft pancreatic cancer tumor cells BxPc-3 and Mia PaCa-2 in mice, respectively. When antibody hSD5 was administered with gemcitabine, significantly improved effects on tumor growth inhibition were observed. Based on the efficacy of the IgG hSD5 antibodies, clinical administration of the hSD5 antibodies is likely to suppress tumors in patients with pancreatic cancer and abnormal activation or overexpression of EphA2 signaling.

Abstract 321: Altered Epha2 Signaling In Non-alcoholic Fatty Liver Disease Progression

Background: Nonalcoholic fatty liver disease (NAFLD), the leading cause of chronic liver disease worldwide, results from diet-induced hepatic steatosis that can result in hepatocyte ballooning, inflammation and fibrosis driving nonalcoholic steatohepatitis (NASH). The Eph receptors, the largest family of receptor tyrosine kinases in the mammalian genome, affect inflammation and fibrosis in other model systems, and we have shown that EphA2 deletion reduces atherosclerosis despite increasing plasma cholesterol levels. While a GWAS study has linked EphA2 to NAFLD, the potential role of EphA2 in NAFLD has never been investigated. Methods: EphA2 expression and signaling was assessed in mice fed a high fat diet (HFD) for 8 and 24 weeks, in human liver samples from NASH patients, and in HuH7 cells treated with palmitic acid (FA), low density lipoprotein (LDL), and/or fructose. Global EphA2 KO mice were fed a HFD for 12 weeks followed by histological and molecular analyses. Results: Mice with early-stage non-alcoholic fatty liver disease (NAFLD) showed elevated EphA2 expression along with markers of EphA2 ligand-dependent signaling (EphA2 tyrosine phosphorylation). Consistently, HuH7 cells treated with FA, LDL, and/or fructose displayed a similar enhancement of EphA2 expression upon treatment. However, a mouse model of late-stage NASH exhibited reduced expression of the EphA2 ligand ephrinA1 and elevated markers of ligand-independent signaling (Ser897 phosphorylation). Similarly, human liver samples from NASH patients show reduced ephrinA1 expression and elevated markers of EphA2 ligand independent signaling. Consistent with a role for EphA2 in NAFLD/NASH, global EphA2 KO mice show significantly lower indices of NAFLD following high fat diet feeding, including reduced liver-to-body weight ratios, hepatic steatosis, and inflammation. Conclusion: Taken together, our data demonstrate that EphA2 expression and signaling are altered during NAFLD/NASH pathogenesis and suggest that EphA2 inhibition may reduce NAFLD/NASH disease progression.

The role of EphA2 in ADAM17- and ionizing radiation-enhanced lung cancer cell migration.

Ionizing radiation (IR) enhances the migratory capacity of cancer cells. Here we investigate in non-small-cell-lung-cancer (NSCLC) cells a novel link between IR-enhanced ADAM17 activity and the non-canonical pathway of EphA2 in the cellular stress response to irradiation. Cancer cell migration in dependence of IR, EphA2, and paracrine signaling mediated by ADAM17 was determined using transwell migration assays. Changes of EphA2 pS897 and mRNA expression levels upon different ADAM17-directed treatment strategies, including the small molecular inhibitor TMI-005, the monoclonal antibody MEDI3622, and shRNAs, were mechanistically investigated. ADAM17-mediated release and cleavage of the EphA2 ligand ephrin-A1 was measured using ELISA and an acellular cleavage assay. Irradiation with 5 Gy enhanced tumor cell migration of NSCLC NCI-H358 cells in dependence of EphA2. At the same time, IR increased growth factor-induced EphA2 S897 phosphorylation via auto- and paracrine signaling. Genetic and pharmaceutical downregulation of ADAM17 activity abrogated growth factor (e.g. amphiregulin) release, which reduced MAPK pathway-mediated EphA2 S897 phosphorylation in an auto- and paracrine way (non-canonical EphA2-pathway) in NCI-H358 and A549 cells. These signaling processes were associated with reduced cell migration towards conditioned media derived from ADAM17-deficient cells. Interestingly, ADAM17 inhibition with the small molecular inhibitor TMI-005 led to the internalization and proteasomal degradation of EphA2, which was rescued by amphiregulin or MG-132 treatment. In addition, ADAM17 inhibition also abrogated ephrin-A1 cleavage and thereby interfered with the canonical EphA2-pathway. We identified ADAM17 and the receptor tyrosine kinase EphA2 as two important drivers for (IR-) induced NSCLC cell migration and described a unique interrelation between ADAM17 and EphA2. We demonstrated that ADAM17 influences both, EphA2 (pS897) and its GPI-anchored ligand ephrin-A1. Using different cellular and molecular readouts, we generated a comprehensive picture of how ADAM17 and IR influence the EphA2 canonical and non-canonical pathway in NSCLC cells.

Targeting EphA2 and DDR signaling can overcome the BRAF and MEK inhibitors acquired resistance in melanoma cell lines

The BRAF and MEK inhibitors combined strategies have dramatically changed the outcome of BRAF-mutated metastatic melanoma patients. However, despite the initial promising results, the onset of primary or acquired resistance occurs in nearly half of the patients at about one year from the diagnosis. Understanding the mechanisms of resistance to these inhibitors is therefore critical for planning more effective therapeutic strategies able to improve patient outcomes. To this aim we generated BRAF and MEK inhibitors resistant melanoma cells starting from the SAN and A375 lines, both harboring the most common BRAF-V600 mutation and sensitive to these drugs. The obtained double-resistant cell lines were characterized by MTT cell proliferation, migration, invasion assays, phosphoarray and western blot analysis. Here we report that the overexpression of several Tyrosine Kinase Receptors (TKRs), such as EphA2 and DDRs, drives the resistance to these drugs and that this resistance can be overcome by treatment with ALW‑II‑41‑27 multikinase inhibitor. ALW‑II‑41‑27 blocks not only TKRs expression, but also the related downstream AKT and MAPK signaling pathways and its efficacy is documented by decreased cell viability and reduced cell invasion/migration of the resistant cells. Our results can delineate a novel promising therapeutic approach to overcoming the drug resistance occurring in BRAF-mutated metastatic melanoma.

Abstract B003: Hypoxia-induced secretion of fucosylated PVR/CD155 from brain met-associated fibroblasts drives breast cancer invasive capacity by altering cell-cell contacts & focal adhesion

Abstract Background: Brain metastasis (BM) develops in ~10-30% of breast cancer (BC) patients; triple negative breast cancer (TNBC), the most aggressive subtype of BC, exhibits the highest incidence of BM. Treatment options for BCBM are extremely limited due to the poor biological understanding. Hence, there is an urgent need to elucidate molecular mechanisms driving BCBM. We aim to study BCBM pathogenesis by dissecting the role of tumorigenic fucosylated proteins secreted by BM-associated fibroblasts (bmCAFs). Methods: We assessed global fucosylation (post-translational protein modification by L-fucose) patterns by fucose-binding lectin pulldown and immunoblot (IB) analysis. Conditioned media (CM) derived from CAFs that were depleted or not of fucosylated proteins were used to treat BC cells to assess motility and invasion. Fuco-proteomics and phosphoproteomic profiling identified bmCAF-secreted fucosylated (sf) proteins in bmCAF-derived CM and associated global signaling changes induced in BC cells, respectively. qRT-PCR was performed to analyze FUT11 levels under normoxia and hypoxia. Immunofluorescence and IB analyses of BC cells treated ± with bmCAF-derived CM was performed to validate PVR downstream signaling changes. Stereotactic intracranial implantation of BC cells alone or with ctrl/PVR knocked-down- bmCAFs was used for the BCBM mouse model. Results: We discovered that fucosylated proteins secreted uniquely by bmCAFs but not by normal-breast fibroblasts (NBF) or primary tumor-CAFs (tCAF), potently drives BC proliferation and invasiveness. Fucosylated proteomic profiling of the bmCAF secretome identified a soluble Polio Virus Receptor (PVR) isoform as uniquely upregulated, fucosylated, and secreted by bmCAFs. Of the 13 fucosyltransferases (FUTs), we found that bmCAFs upregulate FUT11, a key hypoxia-related gene. FUT11 and secreted fucosylated PVR (sfPVR) are significantly increased in bmCAFs exposed to hypoxia, consistent with the hypoxic environment of the brain. PVR can exist as transmembrane and secreted isoforms. Whereas transmembrane PVR is known to contribute to poor prognosis in a number of cancers by facilitating tumorigenic cell:matrix interactions and attenuating anti-tumor immunity, roles of secreted fucosylated PVR (sfPVR) in cancer are unknown. Our phosphoproteomics analyses of BC cells identified cellular adhesion/cytoskeletal and EPHA2 signaling as significantly modulated by bmCAF sfPVR to drive BC cell motility and invasiveness. Indeed, PVR knockdown abrogates the ability of bmCAFs to enhance BC growth/spread in the brain in a mouse model. Conclusion: Our data demonstrate that pathological hypoxia drives FUT11-mediated fucosylation and secretion of PVR by bmCAFs, which potently drives BC cells motility and invasiveness, representing a mechanism by which bmCAFs can promote BCBM. We expect our ongoing and further studies to advance our understanding of how sfPVR from bmCAFs promotes BCBM and to establish a basis for therapeutic targeting of PVR and/or use of fucosylated PVR and its signaling effectors as biomarkers. Citation Format: Emma Adhikari, Qian Liu, Viktoriya Marusyk, Victoria Lzumi, John M. Koomen, Andriy Marusyk, Eric Lau. Hypoxia-induced secretion of fucosylated PVR/CD155 from brain met-associated fibroblasts drives breast cancer invasive capacity by altering cell-cell contacts & focal adhesion [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B003.

Targeting Host Tyrosine Kinase Receptor EPHA2 Signaling Affects Uropathogen Infection in Human Bladder Epithelial Cells.

Urinary tract infections (UTIs) affect a major proportion of the world population but have limited non-antibiotic-based therapeutic and preventative strategies against UTIs. Facultative intracellular uropathogens such as strains of uropathogenic E. coli, K. pneumoniae, E. faecalis, E. cloacae are well-known uropathogens causing UTIs. These pathogens manipulate several host-signaling pathways during infection, which contributes to recurrent UTIs and inappropriate antibiotic application. Since host cell receptor tyrosine kinases (RTKs) are critical for the entry, survival and replication of intracellular pathogens, we investigated whether different uropathogens require host EPHA2 receptors for their intracellular survival using a cell culture model of intracellular infection in human bladder epithelial cells (BECs). Infection of BECs with seven different uropathogens enhanced the expression levels and activation of EPHA2. The significance of EPHA2 signaling for uropathogen infection was investigated by silencing EPHA2 expression using RNA interference or by inhibiting the kinase activity of EPHA2 using small-molecule compounds such as dasatinib or ALW-II-41-27. Both preventive and therapeutic tyrosine kinase inhibition significantly reduced the intracellular bacterial load. Thus, our results demonstrate the involvement of host cell EPHA2 receptor during intracellular uropathogen infection of BECs, and targeting RTK activity is a viable non-antibiotic therapeutic strategy for managing recurrent UTIs.

The efficacy of receptor tyrosine kinase EphA2 autophosphorylation increases with EphA2 oligomer size

The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell–cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms high-order oligomers upon ligand binding. Here, we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used fluorescence intensity fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Our comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, an autophosphorylation response contributing to EphA2 activation, correlates with EphA2 mean oligomer size. However, we found that other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. Taken together, this work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features.

Non-canonical EphA2 activation underpins PTEN-mediated metastatic migration and poor clinical outcome in prostate cancer

BackgroundThe key process of mesenchymal to amoeboid transition (MAT), which enables prostate cancer (PCa) transendothelial migration and subsequent development of metastases in red bone marrow stroma, is driven by phosphorylation of EphA2S897 by pAkt, which is induced by the omega-6 polyunsaturated fatty acid arachidonic acid. Here we investigate the influence of EphA2 signalling in PCa progression and long-term survival.MethodsThe mechanisms underpinning metastatic biopotential of altered EphA2 signalling in relation to PTEN status were assessed in vitro using canonical (EphA2D739N) and non-canonical (EphA2S897G) PC3-M mutants, interrogation of publicly available PTEN-stratified databases and clinical validation using a PCa TMA (n = 177) with long-term follow-up data. Spatial heterogeneity of EphA2 was assessed using a radical prostatectomy cohort (n = 67).ResultsNon-canonical EphA2 signalling via pEphA2S897 is required for PCa transendothelial invasion of bone marrow endothelium. High expression of EphA2 or pEphA2S897 in a PTENlow background is associated with poor overall survival. Expression of EphA2, pEphA2S897 and the associated MAT marker pMLC2 are spatially regulated with the highest levels found within lesion areas within 500 µm of the prostate margin.ConclusionEphA2 MAT-related signalling confers transendothelial invasion. This is associated with a substantially worse prognosis in PTEN-deficient PCa.

Direct quantification of ligand-induced lipid and protein microdomains with distinctive signaling properties.

Lipid rafts are ordered lipid domains that are enriched in saturated lipids, such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have not been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here, we report that microscopic GM1-enriched domains can form, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains. By measuring dissociation constants, we demonstrate that the propensity of EphA2 to oligomerize is similar in EphA2-enriched and -depleted domains. However, surprisingly, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biophysical chemistry of lipid rafts.

Roles of Eph-Ephrin Signaling in the Eye Lens Cataractogenesis, Biomechanics, and Homeostasis.

The eye lens is responsible for fine focusing of light onto the retina, and its function relies on tissue transparency and biomechanical properties. Recent studies have demonstrated the importance of Eph-ephrin signaling for the maintenance of life-long lens homeostasis. The binding of Eph receptor tyrosine kinases to ephrin ligands leads to a bidirectional signaling pathway that controls many cellular processes. In particular, dysfunction of the receptor EphA2 or the ligand ephrin-A5 lead to a variety of congenital and age-related cataracts, defined as any opacity in the lens, in human patients. In addition, a wealth of animal studies reveal the unique and overlapping functions of EphA2 and ephrin-A5 in lens cell shape, cell organization and patterning, and overall tissue optical and biomechanical properties. Significant differences in lens phenotypes of mouse models with disrupted EphA2 or ephrin-A5 signaling indicate that genetic modifiers likely affect cataract phenotypes and progression, suggesting a possible reason for the variability of human cataracts due to Eph-ephrin dysfunction. This review summarizes the roles of EphA2 and ephrin-A5 in the lens and suggests future avenues of study.

Yeast β-glucan Increases Etoposide Sensitivity in Lung Cancer Cell Line A549 by Suppressing Nuclear Factor Erythroid 2-Related Factor 2 via the Noncanonical Nuclear Factor Kappa B Pathway.

Etoposide is regarded as one of the main standard cytotoxic drugs for lung cancer. However, mutations in Kelch-like ECH-associated protein 1 (Keap1), the main regulator of nuclear factor erythroid 2-related factor 2 (Nrf2), are often detected in lung cancer and lead to chemoresistance. Since the aberrant activation of Nrf2 enhances drug resistance, the suppression of the Nrf2 pathway is a promising therapeutic strategy for lung cancer. We herein used the human lung adenocarcinoma cell line A549 because it harbors a Keap1 loss-of-function mutation. A treatment with β-glucan, a major component of the fungal cell wall, reduced Nrf2 protein levels; downregulated the expression of cytochrome P450 3A5, UDP glucuronosyltransferase 1A1, and multidrug resistance protein 1; and increased etoposide sensitivity in A549 cells. Furthermore, the ephrin type-A receptor 2 (EphA2) receptor was important for the recognition and biologic activity of β-glucan in A549 cells. EphA2 signaling includes nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), and p38 mitogen-activated protein kinase (MAPK). However, treatment of cells with stattic (STAT3 inhibitor) or SB203580 (p38 MAPK inhibitor) did not diminish the effects of β-glucan. In contrast, knockdown of v-rel reticuloendotheliosis viral oncogene homolog B (RelB) abolished the effects of β-glucan, suggesting the involvement of the noncanonical NF-κB pathway. The β-glucan effects were also attenuated by the knockdown of WD40 Repeat protein 23 (WDR23). The β-glucan treatment and RelB overexpression induced the expression of Cullin-4A (CUL4A), which increased WDR23 ligase activity and promoted the subsequent depletion of Nrf2. These results revealed a novel property of β-glucan as a resistance-modifying agent in addition to its widely reported immunomodulatory effects for lung cancer therapy via the EphA2-RelB-CUL4A-Nrf2 axis. SIGNIFICANCE STATEMENT: Chemotherapeutic resistance remains a major obstacle in cancer therapy despite extensive efforts to elucidate the underlying molecular mechanisms and overcome multidrug resistance. The present study revealed a novel resistance-modifying property of β-glucan, thereby expanding our knowledge on the beneficial roles of β-glucan and providing an alternative strategy to prevent drug resistance by cancer. The present results provide evidence for the involvement of a novel mode of NF-κB and Nrf2 crosstalk in the drug resistance phenotype.

Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling

SummaryThe EphA2 receptor tyrosine kinase activates signaling pathways with different, and sometimes opposite, effects in cancer and other pathologies. Thus, highly specific and potent biased ligands that differentially control EphA2 signaling responses could be therapeutically valuable. Here, we use EphA2-specific monomeric peptides to engineer dimeric ligands with three different geometric configurations to combine a potential ability to differentially modulate EphA2 signaling responses with the high potency and prolonged receptor residence time characteristic of dimeric ligands. The different dimeric peptides readily induce EphA2 clustering, autophosphorylation and signaling, the best with sub-nanomolar potency. Yet, there are differences in two EphA2 signaling responses induced by peptides with different configurations, which exhibit distinct potency and efficacy. The peptides bias signaling when compared with the ephrinA1-Fc ligand and do so via different mechanisms. These findings provide insights into Eph receptor signaling, and proof-of-principle that different Eph signaling responses can be distinctly modulated.

Patterned Substrate of Mobile and Immobile Ligands to Probe EphA2 Receptor Clustering.

A multitude of membrane-localized receptors are utilized by cells to integrate both biochemical and physical signals from their microenvironment. The clustering of membrane receptors is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we describe a method to fabricate multicomponent, ligand-functionalized microarrays, for spatially segregated and simultaneous monitoring of receptor activation and signaling in individual living cells. While existing micropatterning techniques allow for the display of fixed ligands, this protocol uniquely allows for functionalization of both mobile membrane corrals and immobile polymers with selective ligands, as well as microscopic monitoring of cognate receptor activation at the cell membrane interface. This protocol has been developed to study the effects of clustering on EphA2 signaling transduction. It is potentially applicable to multiple cell signaling systems, or microbe/host interactions. Graphical abstract: A side-by-side comparison of clustered or non-clustered EphA2 receptor signaling in a single cell.

Regulation of the EphA2 receptor intracellular region by phosphomimetic negative charges in the kinase-SAM linker

Eph receptor tyrosine kinases play a key role in cell-cell communication. Lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered understanding of their signaling mechanisms. Here, we use integrative structural biology to investigate the structure and dynamics of the EphA2 intracellular region. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling involving serine/threonine phosphorylation of the linker connecting its kinase and SAM domains. We show that accumulation of multiple linker negative charges, mimicking phosphorylation, induces cooperative changes in the EphA2 intracellular region from more closed to more extended conformations and perturbs the EphA2 juxtamembrane segment and kinase domain. In cells, linker negative charges promote EphA2 oligomerization. We also identify multiple kinases catalyzing linker phosphorylation. Our findings suggest multiple effects of linker phosphorylation on EphA2 signaling and imply that coordination of different kinases is necessary to promote EphA2 non-canonical signaling.

EphA2 signaling within integrin adhesions regulates fibrillar adhesion elongation and fibronectin deposition

The multifunctional glycoprotein fibronectin influences several crucial cellular processes and contributes to multiple pathologies. While a link exists between fibronectin-associated pathologies and the receptor tyrosine kinase EphA2, the mechanism by which EphA2 promotes fibronectin matrix remodeling remains unknown. We previously demonstrated that EphA2 deletion reduces smooth muscle fibronectin deposition and blunts fibronectin deposition in atherosclerosis without influencing fibronectin expression. We now show that EphA2 expression is required for contractility-dependent elongation of tensin- and α5β1 integrin-rich fibrillar adhesions that drive fibronectin fibrillogenesis. Mechanistically, EphA2 localizes to integrin adhesions where focal adhesion kinase mediates ligand-independent Y772 phosphorylation, and mutation of this site significantly blunts fibrillar adhesion length. EphA2 deficiency decreases smooth muscle cell contractility by enhancing p190RhoGAP activation and reducing RhoA activity, whereas stimulating RhoA signaling in EphA2 deficient cells rescues fibrillar adhesion elongation. Together, these data identify EphA2 as a novel regulator of fibrillar adhesion elongation and provide the first data identifying a role for EphA2 signaling in integrin adhesions.