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EPHA5 is a member of the Eph family of tyrosine kinase receptors, which affect carcinogenesis. The expression level of the EPHA5 receptor in a set of lung adenocarcinoma tissue samples was checked using immunohistochemistry. The relationship between EPHA5 expression and clinicopathological parameters, and epidermal growth factor receptor (EGFR) and Braf mutations were analyzed. We also checked the expression level of the EPHA5 receptor in four lung cancer cell lines. High expression of EPHA5 was found in NCI-H460 and H1299 cells, while low expression was observed in A549 and SPC-A1 cells. EPHA5 was knocked down in NCI-H460 and H1299 lung cancer cell lines using siRNAs. The proliferation, clone formation, and invasive ability were analyzed in NCI-H460 and H1299 cells with EPHA5 knockdown. The results show that the EPHA5 receptor is differently expressed in lung adenocarcinoma tissues, in which positive and negative expression of EPHA5 was found in 58.1% and 41.9% of tissues, respectively. Positive expression of EPHA5 was associated with lymph node metastasis (p = 0.002), differentiation (p = 0.020), TNM stage (p = 0.002), and EGFR mutation (p = 0.001). The proliferation, clone formation, and invasive ability were significantly decreased after EPHA5 knockdown in NCI-H460 and H1299 cells. Our data suggest that the EPHA5 receptor plays a role in tumor promotion in lung adenocarcinoma and is a potential target for lung adenocarcinoma treatment., (© 2022 Scandinavian Societies for Medical Microbiology and Pathology.)

Colorectal carcinoma (CRC) is the third most common cancer and a major cause of morbidity and mortality throughout the world. The prognosis of patients has improved markedly over the last 15 years because of the introduction of new therapy including molecular target drugs. To comprehensively understand the molecular process of carcinogenesis of colorectal carcinoma is essential for the diagnosis, prognosis and treatment. EphA5 is a member of the Eph family and plays a critical role in carcinogenesis of lung cancer, prostate cancer, and breast cancer. The expression profile and the role of EphA5 in colorectal carcinoma have not been well investigated till now. In this study, a set of colorectal carcinoma specimens was subjected to immunohistochemical assay using an EphA5 specific antibody. The relationship between the expression of EphA5 and clinicopathological parameters was statistically analyzed. EphA5 was positively expressed in all tested normal mucosa specimens (120/120, 100%) and partly in colorectal carcinoma specimens (70/120, 58.3%). The loss of EphA5 protein was associated with depth of wall invasion (P=0.002), poor tumor differentiation (P<0.001), lymph node metastasis (P<0.001), and advanced TNM stage (P<0.001). The survival analysis showed that patients with reduced expression of EphA5 had a poor overall survival (P=0.017). Our data indicate that EphA5 receptor may be a tumor suppressor in colorectal carcinoma and it may be a new therapeutic target for colorectal carcinoma.

Regenerative medicine holds great promise for the treatment of degenerative retinal disorders. Krüppel-like factors (KLFs) are transcription factors that have recently emerged as key tools in regenerative medicine because some of them can function as epigenetic reprogrammers in stem cell biology. Here, we show that KLF16, one of the least understood members of this family, is a POU4F2 independent transcription factor in retinal ganglion cells (RGCs) as early as embryonic day 15. When overexpressed, KLF16 inhibits RGC neurite outgrowth and enhances RGC growth cone collapse in response to exogenous ephrinA5 ligands. Ephrin/EPH signaling regulates RGC connectivity. The EphA5 promoter contains multiple GC- and GT-rich KLF-binding sites, which, as shown by ChIP-assays, bind KLF16 in vivo In electrophoretic mobility shift assays, KLF16 binds specifically to a single KLF site near the EphA5 transcription start site that is required for KLF16 transactivation. Interestingly, methylation of only six of 98 CpG dinucleotides within the EphA5 promoter blocks its transactivation by KLF16 but enables transactivation by KLF2 and KLF15. These data demonstrate a role for KLF16 in regulation of RGC neurite outgrowth and as a methylation-sensitive transcriptional regulator of EphA5 expression. Together, these data identify differential low level methylation as a novel mechanism for regulating KLF16-mediated EphA5 expression across the retina. Because of the critical role of ephrin/EPH signaling in patterning RGC connectivity, understanding the role of KLFs in regulating neurite outgrowth and Eph receptor expression will be vital for successful restoration of functional vision through optic nerve regenerative therapies., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: EphA5 is a member of the Eph/ephrin family and plays a critical role in the regulation of carcinogenesis. A significant reduction of EphA5 transcripts in high-grade prostate cancer tissue was shown using a transcriptomic analysis, compared to the low-grade prostate cancer tissue. As less is known about the mechanism of EphA5 downregulation and the function of EphA5, here we investigated the expression and an epigenetic change of EphA5 in prostate cancer and determined if these findings were correlated with clinicopathologic characteristics of prostate cancer., Methods: Seven prostate cell lines (RWPE-1, LNCap, LNCap-LN3, CWR22rv-1, PC-3, PC-3M-LN4, and DU145), thirty-nine BPH, twenty-two primary prostate carcinomas, twenty-three paired noncancerous and cancerous prostate tissues were examined via qRT-PCR, methylation-specific PCR, bisulfite sequencing, immunohistochemistry and western blotting. The role of EphA5 in prostate cancer cell migration and invasion was examined by wound healing and transwell assay., Results: Downregulation or loss of EphA5 mRNA or protein expression was detected in 28 of 45 (62.2%) prostate carcinomas, 2 of 39 (5.1%) hyperplasias, and all 6 prostate cancer cell lines. Methylation of the EphA5 promoter region was present in 32 of 45 (71.1%) carcinoma samples, 3 of 39 (7.7%) hyperplasias, and the 6 prostate cancer cell lines. Among 23 paired prostate carcinoma tissues, 16 tumor samples exhibited the hypermethylation of EphA5, and 15 of these 16 specimens (93.8%) shown the downregulation of EphA5 expression than that of their respectively matched noncancerous samples. Immunostaining analysis demonstrated that the EphA5 protein was absent or down-regulated in 10 of 13 (76.9%) available carcinoma samples, and 8 of these 10 samples (80.0%) exhibited hypermethylation. The frequency of EphA5 methylation was higher in cancer patients with an elevated Gleason score or T3-T4 staging. Following the treatment of 6 prostate cancer cell lines with 5-aza-2'-deoxycytidine, the levels of EphA5 mRNA were significantly increased. Prostate cancer cells invasion and migration were significantly suppressed by ectopic expression of EphA5 in vitro., Conclusion: Our study provides evidence that EphA5 is a potential target for epigenetic silencing in primary prostate cancer and is a potentially valuable prognosis predictor and thereapeutic marker for prostate cancer.

Parkin is the most prevalent genetic factor in the onset of autosomal recessive juvenile parkinsonism (AR-JP), and mutations in parkin has been reported to cause motor defects, which result from dopamine deficiency caused by dopaminergic neuronal cell death. Activation of c-Jun N-terminal kinase (JNK) has also been implicated in neuronal cell death in Parkinson's disease (PD). Moreover, Drosophila models for AR-JP, loss of function mutants of Drosophila parkin, also show dopaminergic neural degeneration associated with hyperactivation of JNK, increased apoptosis, and mitochondrial defects. However, the molecular mechanism by which Parkin protects cells from apoptosis remains unclear. In the present study, we tested whether Drosophila Parkin suppressed the JNK signaling pathway in developing tissues. Ectopically expressed parkin strongly suppressed the constitutively active form of Hemipterous (Hep(CA)), a Drosophila JNK kinase that induces an eye degeneration phenotype and apoptosis in the eye imaginal disc. Moreover, parkin also suppressed extra vein formation induced by Basket (Bsk), a Drosophila JNK. Interestingly, the bsk mRNA level was markedly reduced by parkin over-expression, suggesting that the effect of parkin on the phenotype induced by activation of JNK signaling was achieved by transcriptional regulation. Furthermore, we found that the expression level of JNK target genes was reduced by parkin over-expression. Taken together, these results suggest that Drosophila Parkin suppresses JNK signaling by reducing bsk transcription.

Ephrin (Eph) receptors have been reported to be frequently overexpressed in a wide variety of cancer types, being associated with tumor growth, invasion, metastasis and angiogenesis. The aim of the present study was to evaluate the clinical significance of Eph-A1, -A2, -A4, -A5 and -A7 expression in pancreatic ductal adenocarcinoma. Eph-A1, -A2, -A4, -A5 and -A7 expression and staining intensity were assessed immunohistochemically in tumoral samples of 67 pancreatic adenocarcinoma patients and were statistically analyzed in relation to clinicopathological characteristics, tumor proliferative capacity and patients' survival. Eph receptors were abundantly expressed in pancreatic ductal adenocarcinoma cases examined. Eph-A1 staining intensity was significantly associated with tumor size (pT, p = 0.008) and tumor histopathological stage (pStage, p = 0.012). Eph-A2 expression was significantly associated with patients' age (p = 0.007), while Eph-A4 and Eph-A5 with tumor proliferative capacity (p = 0.019 and p = 0.011, respectively). Pancreatic adenocarcinoma patients with moderate/intense Eph-A5 or Eph-A7 staining presented significantly shorter survival times compared to those with negative/mild one (log-rank test, p = 0.024 and p = 0.009, respectively). Multivariate analysis identified Eph-A5 and Eph-A7 staining intensity as independent prognostic factors (p = 0.048 and p = 0.004, respectively). In conclusion, the present study revealed that Eph receptors were associated with pancreatic cancer characteristics, supporting evidence for their potential clinical application in management and prognosis of pancreatic adenocarcinoma patients.

Tumor dormancy has important implications for early detection and treatment of cancer. Lack of experimental models and limited clinical accessibility constitute major obstacles to the molecular characterization of dormant tumors. We have developed models in which human tumors remain dormant for a prolonged period of time (>120 days) until they switch to rapid growth and become strongly angiogenic. These angiogenic tumors retain their ability to grow fast once injected in new mice. We hypothesized that dormant tumors undergo a stable genetic reprogramming during their switch to the fast-growing phenotype. Genome-wide transcriptional analysis was done to dissect the molecular mechanisms underlying the switch of dormant breast carcinoma, glioblastoma, osteosarcoma, and liposarcoma tumors. A consensus expression signature distinguishing all four dormant versus switched fast-growing tumors was generated. In alignment with our phenotypic observation, the angiogenesis process was the most significantly affected functional gene category. The switch of dormant tumors was associated with down-regulation of angiogenesis inhibitor thrombospondin and decreased sensitivity of angiogenic tumors to angiostatin. The conversion of dormant tumors to exponentially growing tumors was also correlated with regulation and activation of pathways not hitherto linked to tumor dormancy process, such as endothelial cell-specific molecule-1, 5'-ecto-nucleotidase, tissue inhibitor of metalloproteinase-3, epidermal growth factor receptor, insulin-like growth factor receptor, and phosphatidylinositol 3-kinase signaling. Further, novel dormancy-specific biomarkers such as H2BK and Eph receptor A5 (EphA5) were discovered. EphA5 plasma levels in mice and mRNA levels in tumor specimens of glioma patients correlated with diseases stage. These data will be instrumental in identifying novel early cancer biomarkers and could provide a rationale for development of dormancy-promoting tumor therapy strategies.

Objective: To investigate the relationship among mossy fiber axon sprouting(MFS), synaptic reorganization, and the alteration of expression of Eph A5 and ephrin A3 in the dentate gyrus in rats with pilocarpine-induced chronic temporal lobe epilepsy., Methods: Mossy fiber sprouting and synaptic formation in rats were observed by Neo-Timm staining, after the acute status epilepticus and chronic spontaneous temporal lobe epilepsy induced by lithium-chloride and pilocarpine. In situ hybridization was used to detect ephrin A3 mRNA and an immunohistochemical staining was applied to determine Eph A5 protein., Results: In entorhinal cortex, only Eph A5 mRNA and protein expressed, which significantly decreased on Day 7 after pilocarpine induced status epilepticus(P<0.01),and resumed to normal levels on Day 30 (P>0.05). In the dentate granule cells, ephrin A3 mRNA reduced obviously on Day 7 after pilocarpine-induced status epilepticus (P<0.01), and returned to normal levels on Day 30 (P>0.05)., Conclusion: The down-regulation of Eph A5 mRNA and protein in entorhinal cortex and dentate gyrus, and ephrin A3 mRNA in dentate gyrus after status epilepticus may be part of the endogenous molecular mechanism of mossy fiber sprouting to the inner molecular layer of dentate gyrus.

Objectives: To study the expression patterns of two Eph family molecules, the receptor EphA5, and the ligand ephrin-A5, during spinal cord development., Methods: The receptor expression was analyzed using beta-galactosidase knockin mice, and affinity ligand probe binding. The ligand expression was assessed using two different affinity probes, and knockout mouse tissues as controls., Results: EphA5 was expressed in the ventral spinal cord, while ephrin-A5 was located in the dorsolateral regions of the spinal cord throughout development., Conclusions: These results show that EphA5 and ephrin-A5 are expressed over broad developmental stages and may play important roles in establishing the dorsoventral organization of the spinal cord.

The Eph family tyrosine kinase receptors and their ligands have been linked to axon guidance and topographic mapping of the developing central nervous system. More specifically, the EphA5 receptor has been shown to play a role in development of hippocamposeptal, retinotectal and thalamocortical projections. Recently, a line of transgenic mice was developed which expresses a truncated EphA5 receptor lacking a functional tyrosine kinase domain. In a previous study, axonal tracing revealed that medial hippocampal axons in this strain projected laterally and ventrally away from their normal target area. In the current study, both transgenic and wild-type controls were evaluated in unconditioned (rotorod and locomotor activity) and conditioned (water maze and active avoidance) behavior tasks which tested hippocampal and striatal functioning. Compared to controls, the transgenic strain did not show differences in rotorod motor activity but did show a transient deficit in spatial navigation ability and a consistent impairment in active avoidance. The dominant-negative mutant receptor also resulted in a decrease in striatal dopamine and serotonin concentrations with no change in hippocampal monoamines. Collectively, these data suggest that animals expressing a truncated EphA5 receptor show deficits related to striatal functioning.

EphA tyrosine kinases are thought to act as topographically specific receptors in the well-characterized projection map from the retina to the tectum. Here, we describe a loss-of-function analysis of EphA receptors in retinotectal mapping. Expressing patches of a cytoplasmically truncated EphA3 receptor in chick retina caused temporal axons to have reduced responsiveness to posterior tectal repellent activity in vitro and to shift more posteriorly within the map in vivo. A gene disruption of mouse EphA5, replacing the intracellular domain with beta-galactosidase, reduced in vitro responsiveness of temporal axons to posterior target membranes. It also caused map abnormalities in vivo, with temporal axons shifted posteriorly and nasal axons anteriorly, but with the entire target still filled by retinal axons. The anterior shift of nasal axons was not accompanied by increased responsiveness to tectal repellent activity, in contrast to the comparable anterior shift in ephrin-A knock-outs, helping to resolve a previous ambiguity in interpreting the ephrin gene knock-outs. The results show the functional requirement for endogenous EphA receptors in retinotectal mapping, show that the receptor intracellular domain is required for a forward signaling response to topographic cues, and provide new evidence for a role of axon competition in topographic mapping.

The Eph family of tyrosine kinase receptors and their ligands, ephrins, are distributed in gradients and serve as molecular guidance cues for axonal patterning during neuronal development. Most of these molecules are also expressed in mature brain. Thus, we examine here the potential roles of such molecules in plasticity and activity-dependent mossy fiber sprouting of adult CNS. We show that the ligand ephrin-A3 and the receptor EphA5 are expressed in complementary gradients in the adult rat mossy fiber system. Using the kindling model, we demonstrate that exogenous immunoadhesins that affect the interaction of endogenous EphA receptors and ephrin-A ligands modulate the development of kindling, one type of long-term plasticity, in mature rat brain. These immunoadhesins, combined with epileptogenic stimulations, alter both the extent and the pattern of collateral axonal sprouting in the mossy fiber pathway. Our results suggest that EphA receptors and ephrin-A ligands modify neuronal plasticity and may serve as spatial cues that modulate the development and pattern of activation-dependent axonal growth in adult CNS.

The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, we provide in vivo evidence that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, we show that the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. Our results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas.

The cerebellum is a modular structure that integrates information in a topographical manner. The membrane receptors of the Eph family and their ligands play important roles in early regionalization, as well as in the formation of topographic connections of the nervous system. Here, we show that the expression of the Eph receptors -A4 and -A7, and of their ligands ephrin-A5 and -A2 correlates with the establishment of territories along the rostro-caudal axis and with the formation of topographically organized connections between the cortex and the cerebellar nuclei. While some sites where co-expression of receptors and ligands are evident, their relative expression mainly define sharp limits along the rostro-caudal axis and, at later stages, complementary gradients in the Purkinje cell layer and the deep cerebellar nuclei.