RESUMEN
EPH receptors (EPHs), the largest family of tyrosine kinases, phosphorylate downstream substrates upon binding of ephrin cell surface-associated ligands. In a large cohort of endometriotic lesions from individuals with endometriosis, we found that EPHA2 and EPHA4 expressions are increased in endometriotic lesions relative to normal eutopic endometrium. Because signaling through EPHs is associated with increased cell migration and invasion, we hypothesized that chemical inhibition of EPHA2/4 could have therapeutic value. We screened DNA-encoded chemical libraries (DECL) to rapidly identify EPHA2/4 kinase inhibitors. Hit compound, CDD-2693, exhibited picomolar/nanomolar kinase activity against EPHA2 (Ki: 4.0 nM) and EPHA4 (Ki: 0.81 nM). Kinome profiling revealed that CDD-2693 bound to most EPH family and SRC family kinases. Using NanoBRET target engagement assays, CDD-2693 had nanomolar activity versus EPHA2 (IC50: 461 nM) and EPHA4 (IC50: 40 nM) but was a micromolar inhibitor of SRC, YES, and FGR. Chemical optimization produced CDD-3167, having picomolar biochemical activity toward EPHA2 (Ki: 0.13 nM) and EPHA4 (Ki: 0.38 nM) with excellent cell-based potency EPHA2 (IC50: 8.0 nM) and EPHA4 (IC50: 2.3 nM). Moreover, CDD-3167 maintained superior off-target cellular selectivity. In 12Z endometriotic epithelial cells, CDD-2693 and CDD-3167 significantly decreased EFNA5 (ligand) induced phosphorylation of EPHA2/4, decreased 12Z cell viability, and decreased IL-1ß-mediated expression of prostaglandin synthase 2 (PTGS2). CDD-2693 and CDD-3167 decreased expansion of primary endometrial epithelial organoids from patients with endometriosis and decreased Ewing's sarcoma viability. Thus, using DECL, we identified potent pan-EPH inhibitors that show specificity and activity in cellular models of endometriosis and cancer.
Asunto(s)
Inhibidores de Proteínas Quinasas , Humanos , Femenino , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Endometriosis/tratamiento farmacológico , Endometriosis/metabolismo , Endometriosis/patología , ADN/metabolismo , Receptores de la Familia Eph/metabolismo , Receptores de la Familia Eph/antagonistas & inhibidores , Receptor EphA2/metabolismo , Receptor EphA2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Movimiento Celular/efectos de los fármacosRESUMEN
The transmembrane helices of receptor tyrosine kinases (RTKs) have been proposed to switch between two different dimeric conformations, one associated with the inactive RTK and the other with the active RTK. Furthermore, recent work has demonstrated that some full-length RTKs are associated into oligomers that are larger than dimers, raising questions about the roles of the TM helices in the assembly and function of these oligomers. Here we probe the roles of the TM helices in the assembly of EphA2 RTK oligomers in the plasma membrane. We employ mutagenesis to evaluate the relevance of a published NMR dimeric structure of the isolated EphA2 TM helix in the context of the full-length EphA2 in the plasma membrane. We use two fluorescence methods, Förster Resonance Energy Transfer and Fluorescence Intensity Fluctuations spectrometry, which yield complementary information about the EphA2 oligomerization process. These studies reveal that the TM helix mutations affect the stability, structure, and size of EphA2 oligomers. However, the effects are multifaceted and point to a more complex role of the TM helix than the one expected from the "TM dimer switch" model.
Asunto(s)
Multimerización de Proteína , Receptor EphA2 , Receptor EphA2/metabolismo , Receptor EphA2/química , Receptor EphA2/genética , Humanos , Transferencia Resonante de Energía de Fluorescencia , Membrana Celular/metabolismo , Conformación Proteica en Hélice alfa , MutaciónRESUMEN
Human cytomegalovirus (HCMV) infection is associated with human glioblastoma, the most common and aggressive primary brain tumor, but the underlying infection mechanism has not been fully demonstrated. Here, we show that EphA2 was upregulated in glioblastoma and correlated with the poor prognosis of the patients. EphA2 silencing inhibits, whereas overexpression promotes HCMV infection, establishing EphA2 as a crucial cell factor for HCMV infection of glioblastoma cells. Mechanistically, EphA2 binds to HCMV gH/gL complex to mediate membrane fusion. Importantly, the HCMV infection was inhibited by the treatment of inhibitor or antibody targeting EphA2 in glioblastoma cells. Furthermore, HCMV infection was also impaired in optimal glioblastoma organoids by EphA2 inhibitor. Taken together, we propose EphA2 as a crucial cell factor for HCMV infection in glioblastoma cells and a potential target for intervention.
Asunto(s)
Infecciones por Citomegalovirus , Glioblastoma , Receptor EphA2 , Humanos , Proteínas del Envoltorio Viral/metabolismo , Glioblastoma/genética , Citomegalovirus/fisiología , Receptor EphA2/genéticaRESUMEN
Ephrin receptor A2 (EphA2), a member of the Ephrin receptor family, is closely related to the progression of oral squamous cell carcinoma (OSCC). Cancer stem cells (CSCs) play essential roles in OSCC development and occurrence. The underlying mechanisms between EphA2 and CSCs, however, are not yet fully understood. Here, we found that EphA2 was overexpressed in OSCC tissues and was associated with poor prognosis. Knockdown of EphA2 dampened the CSC phenotype and the tumour-initiating frequency of OSCC cells. Crucially, the effects of EphA2 on the CSC phenotype relied on KLF4, a key transcription factor for CSCs. Mechanistically, EphA2 activated the ERK signalling pathway, promoting the nuclear translocation of YAP. Subsequently, YAP was bound to TEAD3, leading to the transcription of KLF4. Overall, our findings revealed that EphA2 can enhance the stemness of OSCC cells, and this study identified the EphA2/KLF4 axis as a potential target for treating OSCC.
Asunto(s)
Carcinoma de Células Escamosas , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Neoplasias de la Boca , Células Madre Neoplásicas , Receptor EphA2 , Factor 4 Similar a Kruppel/metabolismo , Humanos , Receptor EphA2/metabolismo , Receptor EphA2/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Animales , Ratones , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Femenino , Ratones Desnudos , Masculino , Pronóstico , Sistema de Señalización de MAP Quinasas/genética , Transcripción GenéticaRESUMEN
The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) is overexpressed in malignant tumors. We previously reported that non-canonical EphA2 phosphorylation at Ser-897 was catalyzed by p90 ribosomal S6 kinase (RSK) via the MEK-ERK pathway in ligand- and tyrosine kinase-independent manners. Non-canonical EphA2 activation plays a key role in tumor progression; however, its activation mechanism remains unclear. In the present study, we focused on cellular stress signaling as a novel inducer of non-canonical EphA2 activation. p38, instead of ERK in the case of epidermal growth factor signaling, activated RSK-EphA2 under cellular stress conditions, including anisomycin, cisplatin, and high osmotic stress. Notably, p38 activated the RSK-EphA2 axis via downstream MAPK-activated protein kinase 2 (MK2). Furthermore, MK2 directly phosphorylated both RSK1 Ser-380 and RSK2 Ser-386, critical residues for the activation of their N-terminal kinases, which is consistent with the result showing that the C-terminal kinase domain of RSK1 was dispensable for MK2-mediated EphA2 phosphorylation. Moreover, the p38-MK2-RSK-EphA2 axis promoted glioblastoma cell migration induced by temozolomide, a chemotherapeutic agent for the treatment of glioblastoma patients. Collectively, the present results reveal a novel molecular mechanism for non-canonical EphA2 activation under stress conditions in the tumor microenvironment.
Asunto(s)
Glioblastoma , Receptor EphA2 , Transducción de Señal , Humanos , Anisomicina/farmacología , Movimiento Celular , Cisplatino/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Presión Osmótica , Fosforilación , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor EphA2/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Microambiente TumoralRESUMEN
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. ß-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. ß-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 ß-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 ß-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.
Asunto(s)
Benzamidas , Niacinamida/análogos & derivados , Pneumocystis carinii , Pneumocystis , Neumonía por Pneumocystis , Receptor EphA2 , beta-Glucanos , Ratones , Animales , beta-Glucanos/metabolismo , Proteínas Tirosina Quinasas Receptoras , Neumonía por Pneumocystis/microbiología , Macrófagos/microbiología , Huésped InmunocomprometidoRESUMEN
Lactate dehydrogenase A (LDHA) is known to promote the growth and invasion of various types of tumors, affects tumor resistance, and is associated with tumor immune escape. But how LDHA reshapes the tumor microenvironment and promotes the progression of renal cell carcinoma (RCC) remains unclear. In this study, we found that LDHA was highly expressed in clear cell RCC (ccRCC), and this high expression was associated with macrophage infiltration, while macrophages were highly infiltrated in ccRCC, affecting patient prognosis via M2-type polarization. Our in vivo and in vitro experiments demonstrated that LDHA and M2-type macrophages could enhance the proliferation, invasion, and migration abilities of ccRCC cells. Mechanistically, high expression of LDHA in ccRCC cells upregulated the expression of EPHA2 in exosomes derived from renal cancer. Exosomal EPHA2 promoted M2-type polarization of macrophages by promoting activation of the PI3K/AKT/mTOR pathway in macrophages, thereby promoting the progression of ccRCC. All these findings suggest that EPHA2 may prove to be a potential therapeutic target for advanced RCC.
Asunto(s)
Carcinoma de Células Renales , Progresión de la Enfermedad , Exosomas , Neoplasias Renales , Macrófagos , Receptor EphA2 , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/genética , Receptor EphA2/metabolismo , Receptor EphA2/genética , Humanos , Neoplasias Renales/patología , Neoplasias Renales/metabolismo , Neoplasias Renales/genética , Exosomas/metabolismo , Animales , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Línea Celular Tumoral , Proliferación Celular , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/genética , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Masculino , Microambiente Tumoral , Pronóstico , Serina-Treonina Quinasas TOR/metabolismo , Femenino , Transducción de Señal , Ratones Desnudos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.
Asunto(s)
Proteasas Virales 3C , Autofagia , Picornaviridae , Receptor EphA2 , Transducción de Señal , Serina-Treonina Quinasas TOR , Proteínas Virales , Replicación Viral , Animales , Receptor EphA2/metabolismo , Receptor EphA2/genética , Serina-Treonina Quinasas TOR/metabolismo , Línea Celular , Porcinos , Picornaviridae/fisiología , Picornaviridae/genética , Proteasas Virales 3C/metabolismo , Proteínas Virales/metabolismo , Proteínas Virales/genética , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/genética , Proteolisis , Cricetinae , Interacciones Huésped-Patógeno , Carga ViralRESUMEN
The protein encoded by the ephrin type-A receptor 2 (EphA2) gene is a member of the ephrin receptor subfamily of the receptor tyrosine kinase family (RTKs). Eph receptors play a significant role in various biological processes, particularly cancer progression, development, and pathogenesis. They have been observed to regulate cancer cell growth, migration, invasion, tumor development, invasiveness, angiogenesis, and metastasis. To target EphA2 activity, various molecular, genetic, biochemical, and pharmacological strategies have been extensively tested in laboratory cultures and animal models. Notably, drugs, such as dasatinib, initially designed to target the kinase family, have demonstrated an additional capability to target EphA2 activity. Additionally, a novel monoclonal antibody named EA5 has emerged as a promising option to counteract the effects of EphA2 overexpression and restore tamoxifen sensitivity in EphA2-transfected MCF-7 cells during in vitro experiments. This antibody mimicked the binding of Ephrin A to EphA2. These methods offer potential avenues for inhibiting EphA2 activity, which could significantly decelerate breast cancer progression and restore sensitivity to certain drugs. This review article comprehensively covers EphA2's involvement in multiple malignancies, including ovarian, colorectal, breast, lung, glioma, and melanoma. Furthermore, we discuss the structure of EphA2, the Eph-Ephrin signaling pathway, various EphA2 inhibitors, and the mechanisms of EphA2 degradation. This article provides an extensive overview of EphA2's vital role in different types of cancers and outlines potential therapeutic approaches to target EphA2, shedding light on the underlying molecular mechanisms that make it an attractive target for cancer treatment.
Asunto(s)
Neoplasias , Receptor EphA2 , Animales , Receptor EphA2/genética , Receptor EphA2/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Efrinas/farmacología , Línea Celular TumoralRESUMEN
BACKGROUND: Several studies have reported a significant relationship between Ephrin receptor A2 (EphA2) and malignant progression in numerous cancers. However, there is a lack of comprehensive pan-cancer analysis on the prognostic value, mutation status, methylation landscape, and potential immunological function of EphA2. METHOD: Using The Cancer Genome Atlas, Genotype Tissue Expression Database and GEO data, we analysed the differences in EphA2 expression between normal and tumour tissues and the effects of EphA2 on the prognosis of different tumours. Furthermore, using GSCALite, cBioPortal, TISDB, ULCLAN and TIMER 2.0 databases or platforms, we comprehensively analysed the potential oncogenic mechanisms or manifestations of EphA2 in 33 different tumour types, including tumour mutation status, DNA methylation status and immune cell infiltration. The correlation of EphA2 with immune checkpoints, tumour mutational burden, DNA microsatellite instability and DNA repair genes was also calculated. Finally, the effects of EphA2 inhibitors on the proliferation of human glioma and lung cancer cells were verified in cellular experiments. RESULTS: EphA2 is differentially expressed in different tumours, and patients with overexpression have poorer overall survival. In addition, gene mutations, gene copy number variation and DNA/RNA methylation of EphA2 have been identified in various tumours. Moreover, EphA2 is positively associated with immune infiltration involving macrophages and CD8+ T cells. Further, EphA2 mRNA expression is significantly associated with immune checkpoint in various cancers, especially programmed death-ligand 1. Finally, the EphA2 inhibitor ALW-II-41-27 shows potent anti-tumour activity. CONCLUSION: Our first pan-cancer study of EphA2 provides insight into the prognostic and immunological roles of EphA2 in different tumours, suggesting that EphA2 might be a potential biomarker for poor prognosis and immune infiltration in cancer.
Asunto(s)
Biomarcadores de Tumor , Neoplasias , Receptor EphA2 , Humanos , Receptor EphA2/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Pronóstico , Neoplasias/inmunología , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Mutación , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Proliferación Celular/genéticaRESUMEN
Endometrial cancer is the most frequent malignant tumor of the female reproductive tract but lacks effective therapy. EphA2, a receptor tyrosine kinase, is overexpressed by various cancers including endometrial cancer and is associated with poor clinical outcomes. In preclinical models, EphA2-targeted drugs had modest efficacy. To discover potential synergistic partners for EphA2-targeted drugs, we performed a high-throughput drug screen and identified panobinostat, a histone deacetylase inhibitor, as a candidate. We hypothesized that combination therapy with an EphA2 inhibitor and panobinostat leads to synergistic cell death. Indeed, we found that the combination enhanced DNA damage, increased apoptosis, and decreased clonogenic survival in Ishikawa and Hec1A endometrial cancer cells and significantly reduced tumor burden in mouse models of endometrial carcinoma. Upon RNA sequencing, the combination was associated with downregulation of cell survival pathways, including senescence, cyclins, and cell cycle regulators. The Axl-PI3K-Akt-mTOR pathway was also decreased by combination therapy. Together, our results highlight EphA2 and histone deacetylase as promising therapeutic targets for endometrial cancer.
Asunto(s)
Neoplasias Endometriales , Inhibidores de Histona Desacetilasas , Receptor EphA2 , Animales , Femenino , Humanos , Ratones , Apoptosis , Línea Celular Tumoral , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Inhibidores de Histona Desacetilasas/uso terapéutico , Panobinostat/farmacología , Panobinostat/uso terapéutico , Fosfatidilinositol 3-Quinasas , Terapia Molecular Dirigida , Receptor EphA2/antagonistas & inhibidoresRESUMEN
The lipid phosphatase Ship2 interacts with the EphA2 receptor by forming a heterotypic Sam (sterile alpha motif)-Sam complex. Ship2 works as a negative regulator of receptor endocytosis and consequent degradation, and anti-oncogenic effects in cancer cells should be induced by hindering its association with EphA2. Herein, a computational approach is presented to investigate the relationship between Ship2-Sam/EphA2-Sam interaction and cancer onset and further progression. A search was first conducted through the COSMIC (Catalogue of Somatic Mutations in Cancer) database to identify cancer-related missense mutations positioned inside or close to the EphA2-Sam and Ship2-Sam reciprocal binding interfaces. Next, potential differences in the chemical-physical properties of mutant and wild-type Sam domains were evaluated by bioinformatics tools based on analyses of primary sequences. Three-dimensional (3D) structural models of mutated EphA2-Sam and Ship2-Sam domains were built as well and deeply analysed with diverse computational instruments, including molecular dynamics, to classify potentially stabilizing and destabilizing mutations. In the end, the influence of mutations on the EphA2-Sam/Ship2-Sam interaction was studied through docking techniques. This in silico approach contributes to understanding, at the molecular level, the mutation/cancer relationship by predicting if amino acid substitutions could modulate EphA2 receptor endocytosis.
Asunto(s)
Neoplasias , Receptor EphA2 , Motivo alfa Estéril , Receptor EphA2/química , Unión Proteica , Mutación , Monoéster Fosfórico Hidrolasas/metabolismo , LípidosRESUMEN
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.
Asunto(s)
Efrina-A1 , Receptor EphA2 , Células Endoteliales/metabolismo , Efrina-A1/química , Ligandos , Fosforilación , Receptor EphA2/metabolismo , HumanosRESUMEN
Epstein-Barr virus (EBV) is best known for infection of B cells, in which it usually establishes an asymptomatic lifelong infection, but is also associated with the development of multiple B cell lymphomas. EBV also infects epithelial cells and is associated with all cases of undifferentiated nasopharyngeal carcinoma (NPC). EBV is etiologically linked with at least 8% of gastric cancer (EBVaGC) that comprises a genetically and epigenetically distinct subset of GC. Although we have a very good understanding of B cell entry and lymphomagenesis, the sequence of events leading to EBVaGC remains poorly understood. Recently, ephrin receptor A2 (EPHA2) was proposed as the epithelial cell receptor on human cancer cell lines. Although we confirm some of these results, we demonstrate that EBV does not infect healthy adult stem cell-derived gastric organoids. In matched pairs of normal and cancer-derived organoids from the same patient, EBV only reproducibly infected the cancer organoids. While there was no clear pattern of differential expression between normal and cancer organoids for EPHA2 at the RNA and protein level, the subcellular location of the protein differed markedly. Confocal microscopy showed EPHA2 localization at the cell-cell junctions in primary cells, but not in cancer cell lines. Furthermore, histologic analysis of patient tissue revealed the absence of EBV in healthy epithelium and presence of EBV in epithelial cells from inflamed tissue. These data suggest that the EPHA2 receptor is not accessible to EBV on healthy gastric epithelial cells with intact cell-cell contacts, but either this or another, yet to be identified receptor may become accessible following cellular changes induced by inflammation or transformation, rendering changes in the cellular architecture an essential prerequisite to EBV infection.
Asunto(s)
Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Organoides/virología , Receptor EphA2/metabolismo , Estómago/virología , Internalización del Virus , Células Epiteliales/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Humanos , Organoides/metabolismo , Estómago/fisiología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/virologíaRESUMEN
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.
Asunto(s)
Candida albicans/fisiología , Candidiasis Bucal/patología , Efrina-A2/metabolismo , Células Epiteliales/patología , Orofaringe/patología , Factores de Virulencia/metabolismo , Animales , Candidiasis Bucal/genética , Candidiasis Bucal/metabolismo , Candidiasis Bucal/microbiología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Efrina-A2/genética , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Orofaringe/metabolismo , Orofaringe/microbiología , Receptor EphA2 , Factores de Virulencia/genéticaRESUMEN
With the aim of identifying novel antagonists selective for the EphA receptor family, a combined experimental and computational approach was taken to investigate the molecular basis of the recognition between a prototypical Eph-ephrin antagonist (UniPR1447) and two representative receptors of the EphA and EphB subfamilies, namely, EphA2 and EphB2 receptors. The conformational free-energy surface (FES) of the binding state of UniPR1447 within the ligand binding domain of EphA2 and EphB2, reconstructed from molecular dynamics (MD) simulations performed on the microsecond time scale, was exploited to drive the design and synthesis of a novel antagonist selective for EphA2 over the EphB2 receptor. The availability of compounds with this pharmacological profile will help discriminate the importance of these two receptors in the insurgence and progression of cancer.
Asunto(s)
Receptor EphA2 , Receptor EphB2 , Humanos , Ligandos , Simulación de Dinámica Molecular , Unión Proteica , Receptor EphA2/antagonistas & inhibidores , Receptor EphB2/antagonistas & inhibidoresRESUMEN
Capicua (CIC)'s transcriptional repressor function is implicated in neurodevelopment and in oligodendroglioma (ODG) aetiology. However, CIC's role in these contexts remains obscure, primarily from our currently limited knowledge regarding its biological functions. Moreover, CIC mutations in ODG invariably co-occur with a neomorphic IDH1/2 mutation, yet the functional relationship between these two genetic events is unknown. Here, we analysed models derived from an E6/E7/hTERT-immortalized (i.e. p53- and RB-deficient) normal human astrocyte cell line. To examine the consequences of CIC loss, we compared transcriptomic and epigenomic profiles between CIC wild-type and knockout cell lines, with and without mutant IDH1 expression. Our analyses revealed dysregulation of neurodevelopmental genes in association with CIC loss. CIC ChIP-seq was also performed to expand upon the currently limited ensemble of known CIC target genes. Among the newly identified direct CIC target genes were EPHA2 and ID1, whose functions are linked to neurodevelopment and the tumourigenicity of in vivo glioma tumour models. NFIA, a known mediator of gliogenesis, was discovered to be uniquely overexpressed in CIC-knockout cells expressing mutant IDH1-R132H protein. These results identify neurodevelopment and specific genes within this context as candidate targets through which CIC alterations may contribute to the progression of IDH-mutant gliomas. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Astrocitos/enzimología , Epigenoma , Epigenómica , Perfilación de la Expresión Génica , Isocitrato Deshidrogenasa/genética , Mutación , Proteínas Represoras/genética , Transcriptoma , Astrocitos/patología , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Metilación de ADN , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Isocitrato Deshidrogenasa/metabolismo , Factores de Transcripción NFI/genética , Factores de Transcripción NFI/metabolismo , Oligodendroglioma/enzimología , Oligodendroglioma/genética , Oligodendroglioma/patología , Receptor EphA2/genética , Receptor EphA2/metabolismo , Proteínas Represoras/deficienciaRESUMEN
Deficiencies in epithelial barrier integrity are involved in the pathogenesis of chronic rhinosinusitis (CRS). This study aimed to investigate the role of ephrinA1/ephA2 signaling on sinonasal epithelial permeability and rhinovirus-induced epithelial permeability. This role in the process of epithelial permeability was evaluated by stimulating ephA2 with ephrinA1 and inactivating ephA2 with ephA2 siRNA or inhibitor in cells exposed to rhinovirus infection. EphrinA1 treatment increased epithelial permeability, which was associated with decreased expression of ZO-1, ZO-2, and occludin. These effects of ephrinA1 were attenuated by blocking the action of ephA2 with ephA2 siRNA or inhibitor. Furthermore, rhinovirus infection upregulated the expression levels of ephrinA1 and ephA2, increasing epithelial permeability, which was suppressed in ephA2-deficient cells. These results suggest a novel role of ephrinA1/ephA2 signaling in epithelial barrier integrity in the sinonasal epithelium, suggesting their participation in rhinovirus-induced epithelial dysfunction.
Asunto(s)
Permeabilidad de la Membrana Celular , Células Epiteliales , Receptor EphA1 , Receptor EphA2 , Humanos , Permeabilidad de la Membrana Celular/genética , Permeabilidad de la Membrana Celular/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Infecciones por Picornaviridae/metabolismo , Receptor EphA2/metabolismo , Rhinovirus/patogenicidad , ARN Bicatenario , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiologíaRESUMEN
EphA2 tyrosine kinase is upregulated in many cancers and correlated with poor survival of patients, including those with endometrial cancer. EphA2-targeted drugs have shown modest clinical benefit. To improve the therapeutic response to such drugs, we performed a high-throughput chemical screen to discover novel synergistic partners for EphA2-targeted therapeutics. Our screen identified the Wee1 kinase inhibitor, MK1775, as a synergistic partner to EphA2, and this finding was confirmed using both in vitro and in vivo experiments. We hypothesized that Wee1 inhibition would sensitize cells to EphA2-targeted therapy. Combination treatment decreased cell viability, induced apoptosis, and reduced clonogenic potential in endometrial cancer cell lines. In vivo Hec1A and Ishikawa-Luc orthotopic mouse models of endometrial cancer showed greater anti-tumor responses to combination treatment than to either monotherapy. RNASeq analysis highlighted reduced cell proliferation and defective DNA damage response pathways as potential mediators of the combination's effects. In conclusion, our preclinical findings indicate that Wee1 inhibition can enhance the response to EphA2-targeted therapeutics in endometrial cancer; this strategy thus warrants further development.
Asunto(s)
Antineoplásicos , Neoplasias Endometriales , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas , Proteínas Tirosina Quinasas , Receptor EphA2 , Animales , Femenino , Humanos , Ratones , Antineoplásicos/uso terapéutico , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Neoplasias Endometriales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptor EphA2/antagonistas & inhibidoresRESUMEN
Pneumocystis species interaction with myeloid cells is well known, especially in macrophages; however, how the organism binds to lung epithelial cells is incompletely understood. Ephrin type-A receptor 2 (EphA2) has been previously identified as a lung epithelial pattern recognition receptor that binds to fungal ß-glucans. Herein, we also report that EphA2 can also bind Pneumocystis ß-glucans, both in isolated forms and also on exposed surfaces of the organism. Furthermore, binding of Pneumocystis ß-glucans resulted in phosphorylation of the EphA2 receptor, which has been shown to be important for downstream proinflammatory response. Indeed, we also show that interleukin 6 cytokine is significantly increased when lung epithelial cells are exposed to Pneumocystis ß-glucans, and that this response could be blocked by preincubation with a specific antibody to EphA2. Our study presents another Pneumocystis lung epithelial cell receptor with implications for initial colonization and possible therapeutic intervention.