Transmembrane helix interactions regulate oligomerization of the receptor tyrosine kinase EphA2.

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.

EphA2 is a functional entry receptor for HCMV infection of glioblastoma cells.

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.

EphA2 promotes the transcription of KLF4 to facilitate stemness in oral squamous cell carcinoma.

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.

LDHA-mediated M2-type macrophage polarization via tumor-derived exosomal EPHA2 promotes renal cell carcinoma progression.

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.

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication.

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.

Targeting the EphA2 pathway: could it be the way for bone sarcomas?

Bone sarcomas are malignant tumors of mesenchymal origin. Complete surgical resection is the cornerstone of multidisciplinary treatment. However, advanced, unresectable forms remain incurable. A crucial step towards addressing this challenge involves comprehending the molecular mechanisms underpinning tumor progression and metastasis, laying the groundwork for innovative precision medicine-based interventions. We previously showed that tyrosine kinase receptor Ephrin Type-A Receptor 2 (EphA2) is overexpressed in bone sarcomas. EphA2 is a key oncofetal protein implicated in metastasis, self-renewal, and chemoresistance. Molecular, genetic, biochemical, and pharmacological approaches have been developed to target EphA2 and its signaling pathway aiming to interfere with its tumor-promoting effects or as a carrier for drug delivery. This review synthesizes the main functions of EphA2 and their relevance in bone sarcomas, providing strategies devised to leverage this receptor for diagnostic and therapeutic purposes, with a focus on its applicability in the three most common bone sarcoma histotypes: osteosarcoma, chondrosarcoma, and Ewing sarcoma.

Exploring the potential of EphA2 receptor signaling pathway: a comprehensive review in cancer treatment.

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.

Sesamol-mediated targeting of EPHA2 sensitises cervical cancer for cisplatin treatment by regulating mitochondrial dynamics, autophagy, and mitophagy.

BACKGROUND: Cervical cancer ranks as the fourth most prevalent cancer among women globally, presenting a significant therapeutic challenge due to its resistance to cisplatin. Ephrin type-A receptor 2 (EPHA2) is prominently overexpressed in cervical cancer and plays a vital role in cisplatin resistance, although the underlying mechanisms remain incompletely elucidated. Mitochondrial dynamics, autophagy, and mitophagy are critical in mediating cisplatin resistance. Sesamol, a phytochemical compound, has exhibited promising anticancer properties. This study aims to investigate the regulatory role of EPHA2 in these pathways underlying cisplatin resistance and to investigate the potential of sesamol in overcoming this resistance and inhibiting cervical cancer progression. METHODS AND RESULT: In this study, we knocked down EPHA2 in the SiHa cell line and evaluated the resulting changes in molecular markers associated with mitochondrial dynamics, mitophagy, and autophagy. Our results indicated that EPHA2 knockdown (EPHA2-KD) led to enhanced mitochondrial fusion and reduced mitochondrial fission, mitophagy, and autophagy. Furthermore, we investigated the effect of EPHA2-KD and sesamol treatment on sensitising cervical cancer to cisplatin treatment. Our data revealed that EPHA2-KD and sesamol treatment significantly increases cellular sensitivity to cisplatin-induced cytotoxicity. Additionally, we demonstrated that sesamol effectively targets EPHA2, as evidenced by decreased EPHA2 expression levels following sesamol treatment. CONCLUSION: In summary, targeting EPHA2 through knockdown or sesamol treatment enhances cisplatin sensitivity in cervical cancer by modulating mitochondrial dynamics, autophagy and mitophagy, suggesting promising therapeutic strategies to overcome chemoresistance.

TYRO3 and EPHA2 Expression Are Dysregulated in Breast Cancer.

Receptor tyrosine kinases (RTKs) are involved in cell growth, motility, and differentiation. Deregulation of RTKs signaling is associated with tumor development and therapy resistance. Potential RTKs like TAM (TYRO3, AXL, MERTK), RON, EPH, and MET have been evaluated in many cancers like lung, prostate, and colorectal, but little is known in breast tumors. In this study, 51 luminal breast cancer tissue and 8 triple negative breast cancer (TNBC) subtypes were evaluated by qPCR for the expression of TAM, RON, EPHA2, and MET genes. Statistical analysis was performed to determine the correlation to clinical data. TYRO3 is related to tumor subtype and stage, patient's age, smoking habits, and obesity. MET expression is correlated to EPHA2 and TAM gene expression. EPHA2 expression is also related to aging and smoking habits. The expression levels of the TAM and EPHA2 genes seem to play an important role in breast cancer, being also influenced by the patient's lifestyle.

Comprehensive analysis of EphA2 in pan-cancer: A prognostic biomarker associated with cancer immunity.

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.

Integrative multi-omic analysis reveals neurodevelopmental gene dysregulation in CIC-knockout and IDH1-mutant cells.

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.

mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition.

Meningiomas (MNs), arising from the arachnoid/meningeal layer, are nonresponsive to chemotherapies, with ∼50% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously, we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mechanistic target of rapamycin complex 2 (mTORC2) signaling, leading to clinical trials for NF2 and MN. Recently our omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly neuregulin-1/heregulin (NRG1), and confirm increased NRG1 secretion and activation of V-ERB-B avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3) receptor kinase. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2, and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a mechanism independent of NRG1-ERBB3 but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of insulin-like growth factor receptor 1 (IGF1R). Drug treatment co-targeting mTORC1/2 and IGF1R/insulin receptor attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion/activation of NRG1-ERBB3 signaling. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/insulin receptor and co-targeting these pathways may prove effective for treatment of NF2-deficient MN.

Restored microRNA-519a enhances the radiosensitivity of non-small cell lung cancer via suppressing EphA2.

Accumulating evidence has demonstrated that microRNA-519a (miR-519a) acts as the tumor suppressor in various cancers, but little is known regarding its intrinsic regulatory mechanisms in non-small cell lung cancer (NSCLC). Here, we aimed to investigate the role of miR-519a-targeted ephrinA2 receptor (EphA2) in radiosensitivity of NSCLC. MiR-519a and EphA2 expression in NSCLC and paracancerous tissues were detected using RT-qPCR and western blot analysis. A549 cell line was cultured and radiation-resistant cell line A549R was constructed using fractionated X-ray irradiation of these cells at 60 Gy. Colony formation ability and radioresistance of parent strain A549 and resistant strain A549R were detected with restored miR-519a and depleted EphA2. MTT assay was used to measure cell proliferation, flow cytometry was performed for determination of cell cycle distribution and apoptosis. The migration and invasion abilities were assessed by Transwell assay. The target relationship between miR-519a and EphA2 was verified. Results suggested that miR-519a was downregulated and EphA2 was upregulated in NSCLC tissues and cells, and miR-519a targeted EphA2. MiR-519a expression declined, while EphA2 expression elevated in A549R cells versus A549 cells. Upregulated miR-519a and downregulated EphA2 suppressed D0, Dq, survival fraction (SF2) and N-value, arrested cells at G0/G1 phase, advanced the apoptosis and attenuated migration, proliferation, and invasion of A549 and A549R cells. Overexpression of EphA2 reversed the promotion of upregulated miR-519a on radiosensitivity of NSCLC cells. Our results revealed that miR-519a enhances radiosensitivity of NSCLC by inhibiting EphA2 expression. Moreover, miR-519a serves as a target for NSCLC treatment.

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

BACKGROUND: The 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. METHODS: The 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). RESULTS: Non-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. CONCLUSION: EphA2 MAT-related signalling confers transendothelial invasion. This is associated with a substantially worse prognosis in PTEN-deficient PCa.

Ephrin-A3/EphA2 axis regulates cellular metabolic plasticity to enhance cancer stemness in hypoxic hepatocellular carcinoma.

BACKGROUND & AIMS: The highly proliferative nature of hepatocellular carcinoma (HCC) frequently results in a hypoxic intratumoural microenvironment, which creates a therapeutic challenge owing to a lack of mechanistic understanding of the phenomenon. We aimed to identify critical drivers of HCC development and progression in the hypoxic microenvironment. METHODS: We performed integrative analysis of multiple transcriptomic and genomic profiles specific for HCC and hypoxia and identified the Ephrin-A3/Eph receptor A2 (EphA2) axis as a clinically relevant and hypoxia-inducible signalling axis in HCC. The functional significance and mechanistic consequences of the Ephrin-A3/EphA2 axis were examined in EFNA3- and EPHA2- knockdown/overexpressing HCC cells. The potential downstream pathways were investigated by transcriptome sequencing, quantitative reverse-transcription PCR, western blotting analysis and metabolomics. RESULTS: EFNA3 was frequently upregulated in HCC and its overexpression was associated with more aggressive tumour behaviours. HIF-1α directly and positively regulated EFNA3 expression under hypoxia. EFNA3 functionally contributed to self-renewal, proliferation and migration in HCC cells. EphA2 was identified as a key functional downstream mediator of EFNA3. Functional characterisation of the Ephrin-A3/EphA2 forward-signalling axis demonstrated a promotion of self-renewal ability and tumour initiation. Mechanistically, the Ephrin-A3/EphA2 axis promoted the maturation of SREBP1 and expression of its transcriptional target, ACLY, was significantly associated with the expression of EFNA3 and hypoxia markers in clinical cohorts. The metabolic signature of EPHA2 and ACLY stable knockdown HCC cells demonstrated significant overlap in fatty acid, cholesterol and tricarboxylic acid cycle metabolite profiles. ACLY was confirmed to mediate the self-renewal function of the Ephrin-A3/EphA2 axis. CONCLUSIONS: Our findings revealed the novel role of the Ephrin-A3/EphA2 axis as a hypoxia-sensitive modulator of HCC cell metabolism and a key contributor to HCC initiation and progression. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a fast-growing tumour; hence, areas of the tumour often have insufficient vasculature and become hypoxic. The presence of hypoxia within tumours has been shown to negatively impact on the survival of patients with tumours, including HCC. Herein, we identified the Ephrin-A3/EphA2 axis as a key functional driver of tumour initiation and progression in response to hypoxia. Additionally, we showed that SREBP1-ACLY-mediated metabolic rewiring was an important downstream effector that induced cancer stemness in response to Ephrin-A3/EphA2 forward-signalling.

EphA2 contributes to disruption of the blood-brain barrier in cerebral malaria.

Disruption of blood-brain barrier (BBB) function is a key feature of cerebral malaria. Increased barrier permeability occurs due to disassembly of tight and adherens junctions between endothelial cells, yet the mechanisms governing junction disassembly and vascular permeability during cerebral malaria remain poorly characterized. We found that EphA2 is a principal receptor tyrosine kinase mediating BBB breakdown during Plasmodium infection. Upregulated on brain microvascular endothelial cells in response to inflammatory cytokines, EphA2 is required for the loss of junction proteins on mouse and human brain microvascular endothelial cells. Furthermore, EphA2 is necessary for CD8+ T cell brain infiltration and subsequent BBB breakdown in a mouse model of cerebral malaria. Blocking EphA2 protects against BBB breakdown highlighting EphA2 as a potential therapeutic target for cerebral malaria.

RSK-Mediated Non-canonical Activation of EphA2 by Tamoxifen.

The long-term administration of tamoxifen to estrogen receptor α (ERα)-positive breast cancer patients is an established treatment that reduces mortality and recurrence. However, resistance to tamoxifen and an increased risk of endometrial cancer may occur; therefore, the mechanisms by which tamoxifen causes these adverse effects warrant further study. Tamoxifen has been shown to activate mitogen-activated protein kinase (MAPK) in an ERα-independent manner; therefore, we investigated its effects on the MAPK-mediated non-canonical activation of EphA2, a critical event regulating cell migration. Tamoxifen at slightly higher concentrations induced the rapid phosphorylation of EphA2 at Ser-897 via the MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK-ribosomal S6 kinases (RSK) pathway in HeLa cells. In addition, tamoxifen significantly enhanced the migration ability of ERα-negative MDA-MB-231 breast cancer cells in RSK- and EphA2-dependent manners. Phosphorylated EphA2 was internalized and re-localized to the plasma membrane, including lamellipodia, in an RSK-dependent manner. Collectively, the present results provide novel insights into the tumor-promoting activity of tamoxifen.

EphA2 promotes tumorigenicity of cervical cancer by up-regulating CDK6.

Erythropoietin-producing hepatocellular receptor A2 (EphA2) receptor tyrosine kinase plays an important role in tissue organization and homeostasis in normal organs. EphA2 is overexpressed in a variety of types of solid tumours with oncogenic functions. However, the role of EphA2 in cervical cancer (CC) is still needed to be further explored. Here, we examined the role of EphA2 by establishing a stable EphA2 knock-down CC cell lines or a stable EphA2-overexpressed CC cells lines. Overexpression of EphA2 increased cell proliferation and migration of CC while EphA2 knock-down decreased the CC tumorigenicity. In addition, EphA2 knock-down suppressed CC tumour development in the xenograft mouse model. Inhibition of EphA2 by AWL-II-41-27, EphA2-specific tyrosine kinase inhibitor, or knock-down of EphA2 decreased mRNA and protein expression of cyclin-dependent kinase (CDK) 6 in CC cells, which increased cellular susceptibility to epirubicin (EPI), an anti-cancer chemotherapy drug. A clinicopathological study of EphA2 was conducted on a cohort of 158 human CC patients. EphA2 protein expression was positively correlated with CDK6 protein expression, invasion depth, lymph node metastasis and clinicopathological stage (P < .05). This study demonstrates the oncogenic activity of EphA2 in vitro and in vivo, which provides insights into the relevant mechanisms that might lead to novel treatments for CC.

Gastric epithelial attachment of Helicobacter pylori induces EphA2 and NMHC-IIA receptors for Epstein-Barr virus.

Epstein-Barr virus (EBV)-associated gastric cancer belongs to 1 of the 4 subtypes of gastric cancer and accounts for 10% of total gastric cancers. However, most cases of gastric cancer have a history of Helicobacter pylori infection. Therefore, we investigated the possibility that H. pylori infection promotes the development of EBV-associated gastric cancer. H. pylori was exposed to principal EBV receptor, CD21, negative gastric epithelial cells, and then infected with EBV recombinant expressing enhanced green fluorescent protein. Changes in EBV infectivity due to prior H. pylori exposure were analyzed using flow cytometry. The treatment of gastric epithelial cells with H. pylori increased the efficiency of EBV infection. An increase was also observed when CagA-deficient, VacA-deficient, and FlaA-deficient H. pylori strains were used, but not when cag pathogenicity island-deficient H. pylori was used. The treatment of epithelial cells with H. pylori induced the expression of accessory EBV receptors, EphA2 and NMHC-IIA, and increased the efficiency of EBV infection depending on their expression levels. When gastric epithelial cells were treated with EPHA2 or NMHC-IIA siRNA, EBV infection via H. pylori attachment was decreased. The adhesion of H. pylori induced the expression of accessory EBV receptors in gastric epithelial cells and increased the efficiency of EBV infection.

Functional analysis of deleterious EPHA2 SNPs in lens epithelial cells.

Purpose: Ephrin (Eph) receptor A2 (EPHA2) polymorphism has been associated with age-related cataract (ARC) in different populations worldwide, but the mechanisms by which this polymorphism results in the development of ARC are unclear. Here, we chose four EPHA2 single nucleotide polymorphisms (SNPs; rs35903225, rs145592908, rs137853199, and rs116506614) and studied their function in human lens epithelial cells (LECs). Methods: The four EPHA2 mutants were overexpressed using lentiviral transduction in human LECs. Cells expressing wild-type (WT) and mutated EPHA2 were subjected to quantitative PCR (qPCR), western blot, immunoprecipitation (IP), and transwell migration assay. MG132 and chloroquine were used to inhibit the degradation of the WT and mutated EPHA2. The structural changes induced by rs137853199 were predicted and optimized using Schrödinger software. IP-mass spectrometry (IP-MS) was performed to examine the proteins that directly interact with WT and rs137853199 EPHA2. Sanger sequencing was performed to determine the frequency of rs137853199 in 184 patients with ARC (73 cortical cataracts, 56 nuclear cataracts, and 55 posterior subcapsular cataracts) and 49 normal controls. Results: Compared with the WT and the other three mutations, the rs137853199 mutation specifically resulted in a significant decrease in the expression of EPHA2. We identified that EPHA2 rs137853199 is degraded via the ubiquitin-proteasomal pathway through a lysine-48 (K48) residue linkage. Furthermore, the knockdown of EPHA2 reduced cell migration; while the overexpression of WT EPHA2 rescued this defect, the overexpression of rs137853199 EPHA2 did not. In addition, in cells overexpressing rs137853199 EPHA2, the expression of ß-catenin, a key protein that regulates cell migration, significantly decreased. We predicted that rs137853199 would induce a conformational change at a linker position in the carboxyl terminal of EPHA2. The IP-MS results showed that the main molecular functions of the proteins that specifically bind WT or rs137853199 EPHA2 are binding and catalysis, while the main protein class is the protein-modifying enzyme. Finally, we discovered that the minor allele frequency of rs137853199 was significantly higher in cortical cataract patients than it was in normal controls. Conclusions: In summary, these findings suggest a mechanism by which a point mutation in EPHA2 disrupts protein stability, expedites protein degradation, and decreases cell mobility. Importantly, this mutant is associated with cortical cataracts.