EphA8 inhibits cell apoptosis via AKT signaling and is associated with poor prognosis in breast cancer.

Erythropoietin­producing hepatocellular receptors (Ephs) comprise the largest subfamily of receptor tyrosine kinases and have been reported to be involved in a variety of biological cellular processes, including tumorigenesis and cancer progression. The present study aimed to determine the expression levels and clinicopathological significance of EphA8 in breast cancer (BC) using immunohistochemistry analysis of tissue microarrays. The results of the present study revealed that EphA8 expression levels were upregulated in BC tissue and were associated with tumor size and TNM stage. In addition, upregulated expression levels of EphA8 were identified to be a poor prognostic biomarker for patients with BC. The knockdown of EphA8 expression using short hairpin RNA resulted in increased levels of apoptosis as well as decreased proliferation, migration and invasion of BC cells both in vivo and in vitro. The knockdown of EphA8 also decreased the phosphorylation of AKT, which was accompanied by downregulation of Bcl­2 expression levels and upregulation of p53, Caspase­3 and Bax expression levels. Moreover, knockdown of EphA8 expression increased the chemosensitivity of BC cells to paclitaxel. In conclusion, the results of the present study indicated that EphA8 may be a useful prognostic marker in BC and that knockdown of EphA8 may represent a novel strategy in adjuvant chemotherapy for the treatment of BC.

Bone marrow stromal cells derived exosomal miR-10a and miR-16 may be involved in progression of patients with multiple myeloma by regulating EPHA8 or IGF1R/CCND1.

ABSTRACT: Interaction with bone marrow stromal cells (BMSCs) has been suggested as an important mechanism for the progression of multiple myeloma (MM) cells, while exosomes are crucial mediators for cell-to-cell communication. The study was to investigate the miRNA profile changes in exosomes released by BMSCs of MM patients and explore their possible function roles.The microarray datasets of exosomal miRNAs in BMSCs were downloaded from the Gene Expression Omnibus database (GSE110271: 6 MM patients, 2 healthy donors; GSE78865: 4 donors and 2 MM patients; GSE39571: 7 MM patients and 4 controls). The differentially expressed miRNAs (DEMs) were identified using the LIMMA method. The target genes of DEMs were predicted by the miRwalk 2.0 database and the hub genes were screened by constructing the protein-protein interaction (PPI) network, module analysis and overlapping with the differentially expressed genes (DEGs) after overexpression or knockout of miRNAs.Three downregulated DEMs were found to distinguish MM from normal and MM-MGUS controls in the GSE39571 dataset; one downregulated and one upregulated DEMs (hsa-miR-10a) could differentiate MM from normal and MM-MGUS controls in the GSE110271-GSE78865 merged dataset. Furthermore, 11 downregulated (hsa-miR-16) and 1 upregulated DEMs were shared between GSE39571 and merged dataset when comparing MM with normal samples. The target genes were predicted for these 17 DEMs. PPI with module analysis showed IGF1R and CCND1 were hub genes and regulated by hsa-miR-16. Furthermore, EPHA8 was identified as a DEG that was downregulated in MM cells when the use of has-miR-10a mimics; while IGF1R, CCND1, CUL3, and ELAVL1 were also screened as DEGs that were upregulated in MM cells when silencing of hsa-miR-16.BMSCs-derived exosomal miR-10a and miR-16 may be involved in MM progression by regulating EPHA8 or IGF1R/CCND1/CUL3/ELAVL1, respectively. These exosomal miRNAs or genes may represent potential biomarkers for diagnosis of MM and prediction of progression and targets for developing therapeutic drugs.

EphA8 acts as an oncogene and contributes to poor prognosis in gastric cancer via regulation of ADAM10.

EphA8 is a member of the erythropoietin-producing hepatocellular receptor (Eph) family of receptor tyrosine kinases. Ephs and their ephrins ligands play crucial roles in many cellular processed by mediating intracellular signaling resulting from cell-cell interactions. But the underlying mechanisms of EphA8 in gastric cancer (GC) remains unclearly. 298 clinical specimens in tissues microarray, and was found to be significantly higher in GC tissues compared with nontumor tissues (p < 0.001). EphA8 expression was also strongly associated with differentiation level (p = 0.025), tumor-node-metastasis stage (p = 0.019), and poor 5 years survival (p < 0.001). A panel of GC cell lines showed reduced proliferation, invasion, and migration capacities after RNA-mediated knockdown of EphA8, concomitant with downregulation of the proliferation-related proteins (cyclin A, cyclin D1, and cyclin-dependent kinase 4) and the metastasis-related (matrix metalloproteinases MMP2, and MMP9). EphA8 knockdown also decreased expression of the protease ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) and ADAM10-related protein AKT, suggesting an interaction between EphA8 and ADAM10. In conclusion, we found that EphA8, which is highly expressed in GC tissues, stimulates proliferation, invasion, and migration of cancer cells, and is an independent risk factor for poor prognosis of GC. These dates suggest that EphA8 could be new diagnostic and/or therapeutic targets for GC.

EphA8 is a Prognostic Factor for Oral Tongue Squamous Cell Carcinoma.

BACKGROUND Oral tongue squamous cell carcinoma (OTSCC) is the most common malignancy of the oral cavity. Here we explore the potential effects of EphA8, which is one of the receptors in Ephs subfamily of RTKs (receptor tyrosine kinases), in the progression and prognosis of OTSCC. MATERIAL AND METHODS A total of 119 OTSCC patients were enrolled in this retrospective study. Immunohistochemistry (IHC) staining and quantitative polymerase chain reaction (Q-PCR) were utilized to examine the expression of EphA8 in OTSSC tissues and adjacent non-tumor tissues. The relationship between EphA8 expression and the clinicopathological features of OTSCC patients were analyzed by chi-square. Survival analysis was carried out with Kaplan-Meier curve and the related log-rank test. Multivariate analysis was then undertaken to assess the prognosis factor by utilizing the Cox proportional hazard regression model. In addition, MTT assay and Matrigel invasion assay were performed to examine the effects of EphA8 on the proliferation and invasion capacities of human oral squamous carcinoma cells (SCC-25) and human tongue squamous cell carcinoma cells (H357). RESULTS Q-PCR and IHC staining revealed that EphA8 was highly expressed in OTSCC tissues, especially in advanced stage OTSCC tissues. Kaplan-Meier curve showed that high EphA8 expression was significantly associated with poor prognosis, similar to age, smoking habit, drinking habit, tumor size, and TNM stage. Multivariate analysis indicated that EphA8 expression could serve as an independent prognostic factor in OTSCC. In vitro experiments revealed that overexpression of EphA8 might promote the progression of OTSCC via enhancing the invasion capacity but not proliferation capacity of tumor cells. CONCLUSIONS EphA8 was highly expressed in OTSCC tissues and was significantly associated with poor prognosis of OTSCC.

Foxp2 Regulates Identities and Projection Patterns of Thalamic Nuclei During Development.

The molecular mechanisms underlying the formation of the thalamus during development have been investigated intensively. Although transcription factors distinguishing the thalamic primordium from adjacent brain structures have been uncovered, those involved in patterning inside the thalamus are largely unclear. Here, we show that Foxp2, a member of the forkhead transcription factor family, regulates thalamic patterning during development. We found a graded expression pattern of Foxp2 in the thalamic primordium of the mouse embryo. The expression levels of Foxp2 were high in the posterior region and low in the anterior region of the thalamic primordium. In Foxp2 (R552H) knockin mice, which have a missense loss-of-function mutation in the forkhead domain of Foxp2, thalamic nuclei of the posterior region of the thalamus were shrunken, while those of the intermediate region were expanded. Consistently, Foxp2 (R552H) knockin mice showed changes in thalamocortical projection patterns. Our results uncovered important roles of Foxp2 in thalamic patterning and thalamocortical projections during development.

EphA8 is a prognostic marker for epithelial ovarian cancer.

EphA8 is one of the Eph receptors in the Eph/ephrin receptor tyrosine kinase (RTK) subfamily. During tumorigenesis, EphA8 is involved in angiogenesis, cell adhesion and migration. In this study, we determined the mRNA and protein expression levels of EphA8 in cancerous and normal ovarian tissue samples by quantitative reverse transcription PCR (qRT-PCR) (N = 60) and tissue microarray immunohistochemistry analysis (TMA-IHC) (N = 223) respectively. EphA8 protein levels in cancer tissues were correlated with epithelial ovarian cancer (EOC) patients' clinical characteristics and overall survival. Both EphA8 mRNA and protein levels were significantly higher in EOC tissues than in normal or benign ovarian tissues (all P < 0.05). High EphA8 protein level was associated older age at diagnosis, higher FIGO stage, positive lymph nodes, presence of metastasis, positive ascitic fluid, and higher serum CA-125 level. High EphA8 protein level is an independent prognostic marker in EOC. We conclude that EphA8 acts as an oncogene in EOC development and progression. Detection of EphA8 expression could be a useful prognosis marker and targeting EphA8 represents a novel strategy for EOC treatment.

Ephrin-A3 promotes and maintains slow muscle fiber identity during postnatal development and reinnervation.

Each adult mammalian skeletal muscle has a unique complement of fast and slow myofibers, reflecting patterns established during development and reinforced via their innervation by fast and slow motor neurons. Existing data support a model of postnatal "matching" whereby predetermined myofiber type identity promotes pruning of inappropriate motor axons, but no molecular mechanism has yet been identified. We present evidence that fiber type-specific repulsive interactions inhibit innervation of slow myofibers by fast motor axons during both postnatal maturation of the neuromuscular junction and myofiber reinnervation after injury. The repulsive guidance ligand ephrin-A3 is expressed only on slow myofibers, whereas its candidate receptor, EphA8, localizes exclusively to fast motor endplates. Adult mice lacking ephrin-A3 have dramatically fewer slow myofibers in fast and mixed muscles, and misexpression of ephrin-A3 on fast myofibers followed by denervation/reinnervation promotes their respecification to a slow phenotype. We therefore conclude that Eph/ephrin interactions guide the fiber type specificity of neuromuscular interactions during development and adult life.

miR-10a controls glioma migration and invasion through regulating epithelial-mesenchymal transition via EphA8.

MicroRNAs (miRNAs) play a critical role in the development of cancers. However, the role of miRNAs in glioma is still poorly understood. In this study, we demonstrate that microRNA-10a (miR-10a) promotes cell migration and invasion by negatively regulating the expression of Eph tyrosine kinase receptor A8 (EphA8). Ectopic expression of EphA8 counteracts the promotion of migration and invasion induced by miR-10a. We further demonstrate that miR-10a and EphA8 regulate epithelial-mesenchymal transition (EMT) to affect cell migration and invasion. Collectively, we unveil a branch of the miR-10a/EphA8 pathway that regulates the progression of glioma.

EphrinB1 expression is dysregulated and promotes oncogenic signaling in medulloblastoma.

Eph receptors and ephrin ligands are master regulators of oncogenic signaling required for proliferation, migration, and metastasis. Yet, Eph/ephrin expression and activity in medulloblastoma (MB), the most common malignant brain tumor of childhood, remains poorly defined. We hypothesized that Eph/ephrins are differentially expressed by sonic hedgehog (SHH) and non-SHH MB and that specific members contribute to the aggressive phenotype. Affymetrix gene expression profiling of 29 childhood MB, separated into SHH (N = 11) and non-SHH (N = 18), was performed followed by protein validation of selected Eph/ephrins in another 60 MB and two MB cell lines (DAOY, D556). Functional assays were performed using MB cells overexpressing or deleted for selected ephrins. We found EPHB4 and EFNA4 almost exclusively expressed by SHH MB, whereas EPHA2, EPHA8, EFNA1 and EFNA3 are predominantly expressed by non-SHH MB. The remaining family members, except EFNB1, are ubiquitously expressed by over 70-90 % MB, irrespective of subgroup. EFNB1 is the only member differentially expressed by 28 % of SHH and non-SHH MB. Corresponding protein expression for EphB/ephrinB1 and B2 was validated in MB. Only ephrinB2 was also detected in fetal cerebellum, indicating that EphB/ephrinB1 expression is MB-specific. EphrinB1 immunopositivity localizes to tumor cells within MB with the highest proliferative index. EphrinB1 overexpression promotes EphB activation, alters F-actin distribution and morphology, decreases adhesion, and significantly promotes proliferation. Either silencing or overexpression of ephrinB1 impairs migration. These results indicate that EphrinB1 is uniquely dysregulated in MB and promotes oncogenic responses in MB cells, implicating ephrinB1 as a potential target.

Aberrant hypomethylated STAT3 was identified as a biomarker of chronic benzene poisoning through integrating DNA methylation and mRNA expression data.

Chronic occupational benzene exposure is associated with an increased risk of hematological malignancies such as aplastic anemia and leukemia. The new biomarker and action mechanisms of chronic benzene poisoning are still required to be explored. Aberrant DNA methylation, which may lead to genomic instability and the altered gene expression, is frequently observed in hematological cancers. To gain an insight into the new biomarkers and molecular mechanisms of chronic benzene poisoning, DNA methylation profiles and mRNA expression pattern from the peripheral blood mononuclear cells of four chronic benzene poisoning patients and four health controls that matched age and gender without benzene exposure were performed using the high resolution Infinium 450K methylation array and Gene Chip Human Gene 2.0ST Arrays, respectively. By integrating DNA methylation and mRNA expression data, we identified 3 hypermethylated genes showing concurrent down-regulation (PRKG1, PARD3, EPHA8) and 2 hypomethylated genes showing increased expression (STAT3, IFNGR1). Signal net analysis of differential methylation genes associated with chronic benzene poisoning showed that two key hypomethylated STAT3 and hypermethylated GNAI1 were identified. Further GO analysis and pathway analysis indicated that hypomethylated STAT3 played central roles through regulation of transcription, DNA-dependent, positive regulation of transcription from RNA polymerase II promoter, JAK-STAT cascade and adipocytokine signaling pathway, Acute myeloid leukemia, and JAK-STAT signaling pathway. In conclusion, the aberrant hypomethylated STAT3 might be a potential biomarker of chronic benzene poisoning.

Expression of EphA8-Fc in transgenic mouse embryos induces apoptosis of neural epithelial cells during brain development.

EphAs and ephrin-As are expressed in multiple regions of the developing brain and have been implicated in regulating brain size. Here, we report the identification of a novel mechanism in which reverse signaling through ephrin-As controls neural epithelial cell number in the developing brain. Ectopic expression of EphA8-Fc in transgenic embryos induced apoptosis of neural epithelial cells, which was accompanied by a dramatic decrease in brain size. The number of ephrin-A5-expressing cells was significantly reduced in the brain region where EphA8-Fc was ectopically expressed. Furthermore, in vitro culture of the dissociated neuroepithelial cells revealed that EphA8-Fc enhanced apoptotic cell death of the ephrinA5-expressing cells in a caspase-dependent manner. Thus, our results suggest that reverse signaling through ephrin-As is biochemically linked with caspase-dependent proapoptotic signaling during early brain development.

RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin.

The Rab family of small guanosine triphosphatases (GTPases) plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs). Ras and Rab interactor (or Ras interaction/interference)-like (RINL), which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM) domain-containing (Anks) protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.

EphA8-ephrinA5 signaling and clathrin-mediated endocytosis is regulated by Tiam-1, a Rac-specific guanine nucleotide exchange factor.

Recent studies indicate that endocytosis of Eph-ephrin complexes may be one of the mechanisms by which a high affinity cell-cell adhesion is converted to a repulsive interaction. In this study, we show that EphA8 undergoes clathrin-mediated endocytosis upon treatment with ephrin-A5, and that EphA8 is associated tightly with Tiam-1, a Rac-specific guanine nucleotide exchange factor. Analysis of EphA8 deletion mutants revealed that a juxtamembrane region in EphA8 is critically involved in endocytosis of EphA8-ephrinA5 complexes. An EphA8 mutant lacking this juxtamembrane portion was defective for endocytosis with ephrinA5, and also displayed a weak association with Tiam-1. Expression of an endocytosis-defective version of EphA8 resulted in a low level of Rac activity in response to ephrin-A5 stimulation. More importantly, down-regulation of Tiam-1 resulted in inefficient endocytosis of EphA8-ephrinA5 complexes. These results suggest that Tiam-1 plays a role in clathrin-dependent endocytosis of EphA8-ephrinA5 complexes.

The SAM domains of Anks family proteins are critically involved in modulating the degradation of EphA receptors.

We recently reported that the phosphotyrosine-binding (PTB) domain of Anks family proteins binds to EphA8, thereby positively regulating EphA8-mediated signaling pathways. In the current study, we identified a potential role for the SAM domains of Anks family proteins in EphA signaling. We found that SAM domains of Anks family proteins directly bind to ubiquitin, suggesting that Anks proteins regulate the degradation of ubiquitinated EphA receptors. Consistent with the role of Cbl ubiquitin ligases in the degradation of Eph receptors, our results revealed that the ubiquitin ligase c-Cbl induced the ubiquitination and degradation of EphA8 upon ligand binding. Ubiquitinated EphA8 also bound to the SAM domains of Odin, a member of the Anks family proteins. More importantly, the overexpression of wild-type Odin protected EphA8 and EphA2 from undergoing degradation following ligand stimulation and promoted EphA-mediated inhibition of cell migration. In contrast, a SAM domain deletion mutant of Odin strongly impaired the function of endogenous Odin, suggesting that the mutant functions in a dominant-negative manner. An analysis of Odin-deficient primary embryonic fibroblasts indicated that Odin levels play a critical role in regulating the stability of EphA2 in response to ligand stimulation. Taken together, our studies suggest that the SAM domains of Anks family proteins play a pivotal role in enhancing the stability of EphA receptors by modulating the ubiquitination process.

Identification of phosphotyrosine binding domain-containing proteins as novel downstream targets of the EphA8 signaling function.

Eph receptors and ephrins have been implicated in a variety of cellular processes, including morphology and motility, because of their ability to modulate intricate signaling networks. Here we show that the phosphotyrosine binding (PTB) domain-containing proteins AIDA-1b and Odin are tightly associated with the EphA8 receptor in response to ligand stimulation. Both AIDA-1b and Odin belong to the ankyrin repeat and sterile alpha motif domain-containing (Anks) protein family. The PTB domain of Anks family proteins is crucial for their association with the juxtamembrane domain of EphA8, whereas EphA8 tyrosine kinase activity is not required for this protein-protein interaction. In addition, we found that Odin is a more physiologically relevant partner of EphA8 in mammalian cells. Interestingly, overexpression of the Odin PTB domain alone attenuated EphA8-mediated inhibition of cell migration in HEK293 cells, suggesting that it acts as a dominant-negative mutant of the endogenous Odin protein. More importantly, small interfering RNA-mediated Odin silencing significantly diminished ephrinA5-induced EphA8 signaling effects, which inhibit cell migration in HEK293 cells and retract growing neurites of Neuro2a cells. Taken together, our findings support a possible function for Anks family proteins as scaffolding proteins of the EphA8 signaling pathway.

H3 histamine receptor agonist inhibits biliary growth of BDL rats by downregulation of the cAMP-dependent PKA/ERK1/2/ELK-1 pathway.

Histamine regulates many functions by binding to four histamine G-coupled receptor proteins (H1R, H2R, H3R and H4R). As H3R exerts their effects by coupling to Galpha(i/o) proteins reducing adenosine 3', 5'-monophosphate (cAMP) levels (a key player in the modulation of cholangiocyte hyperplasia/damage), we evaluated the role of H3R in the regulation of biliary growth. We posed the following questions: (1) Do cholangiocytes express H3R? (2) Does in vivo administration of (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH) (H3R agonist), thioperamide maleate (H3R antagonist) or histamine, in the absence/presence of thioperamide maleate, to bile duct ligated (BDL) rats regulate cholangiocyte proliferation? and (3) Does RAMH inhibit cholangiocyte proliferation by downregulation of cAMP-dependent phosphorylation of protein kinase A (PKA)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ets-like gene-1 (Elk-1)? The expression of H3R was evaluated in liver sections by immunohistochemistry and immunofluorescence, and by real-time PCR in cholangiocyte RNA from normal and BDL rats. BDL rats (immediately after BDL) were treated daily with RAMH, thioperamide maleate or histamine in the absence/presence of thioperamide maleate for 1 week. Following in vivo treatment of BDL rats with RAMH for 1 week, and in vitro stimulation of BDL cholangiocytes with RAMH, we evaluated cholangiocyte proliferation, cAMP levels and PKA, ERK1/2 and Elk-1 phosphorylation. Cholangiocytes from normal and BDL rats express H3R. The expression of H3R mRNA increased in BDL compared to normal cholangiocytes. Histamine decreased cholangiocyte growth of BDL rats to a lower extent than that observed in BDL RAMH-treated rats; histamine-induced inhibition of cholangiocyte growth was partly blocked by thioperamide maleate. In BDL rats treated with thioperamide maleate, cholangiocyte hyperplasia was slightly higher than that of BDL rats. In vitro, RAMH inhibited the proliferation of BDL cholangiocytes. RAMH inhibition of cholangiocyte growth was associated with decreased cAMP levels and PKA/ERK1/2/Elk-1 phosphorylation. Downregulation of cAMP-dependent PKA/ERK1/2/Elk-1 phosphorylation (by activation of H3R) is important in the inhibition of cholangiocyte growth in liver diseases.

Rapid electrical and delayed molecular signals regulate the serum response element after nerve injury: convergence of injury and learning signals.

Axotomy elicits changes in gene expression, but little is known about how information from the site of injury is communicated to the cell nucleus. We crushed nerves in Aplysia californica and the sciatic nerve in the mouse and found short- and long-term activation of an Elk1-SRF transcription complex that binds to the serum response element (SRE). The enhanced short-term binding appeared rapidly and was attributed to the injury-induced activation of an Elk1 kinase that phosphorylates Elk1 at ser383. This kinase is the previously described Aplysia (ap) ERK2 homologue, apMAPK. Nerve crush evoked action potentials that propagated along the axon to the cell soma. Exposing axons to medium containing high K(+), which evoked a similar burst of spikes, or bathing the ganglia in 20 microM serotonin (5HT) for 20 min, activated the apMAPK and enhanced SRE binding. Since 5HT is released in response to electrical activity, our data indicate that the short-term process is initiated by an injury-induced electrical discharge that causes the release of 5HT which activates apMAPK. 5HT is also released in response to noxious stimuli for aversive learning. Hence, apMAPK is a point of convergence for injury signals and learning signals. The delay before the onset of the long-term SRE binding was reduced when the crush was closer to the ganglion and was attributed to an Elk1 kinase that is activated by injury in the axon and retrogradely transported to the cell body. Although this Elk1 kinase phosphorylates mammalian rElk1 at ser383, it is distinct from apMAPK.

The EphA8 receptor phosphorylates and activates low molecular weight phosphotyrosine protein phosphatase in vitro.

Low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) has been implicated in modulating the EphB1-mediated signaling pathway. In this study, we demonstrated that the EphA8 receptor phosphorylates LMW-PTP in vitro. In addition, we discovered that mixing these two proteins leads to EphA8 dephosphorylation in the absence of phosphatase inhibitors. Finally, we demonstrated that LMW-PTP, modified by the EphA8 autokinase activity, possesses enhanced catalytic activity in vitro. These results suggest that LMW-PTP may also participate in a feedback-control mechanism of the EphA8 receptor autokinase activity in vivo.