Engineered Recombinant EGFP-Azurin Theranostic Nanosystem for Targeted Therapy of Prostate Cancer.

Erythropoietin-producing hepatocellular (Eph) receptors and their ligands, ephrins, are the largest subfamily of receptor tyrosine kinases (RTKs) that have emerged as a new class of cancer biomarkers due to their aberrant expression in cancer progression. The activation of Eph receptors either due to their hyperexpression or via high affinity binding with their respective ephrin ligands initiates a cascade of signals that impacts cancer development and progression. In prostate cancer, the overexpression of the EphA6 receptor has been correlated with increased metastatic potential. Azurin, a small redox protein, is known to prevent tumor progression by binding to cell surface Eph receptors, inhibiting its autophosphorylation in the kinase domain and thereby disrupting Eph-ephrin signaling. Hence, a self-assembled, theranostic nanosystem of recombinant fusion protein his6EGFP-azu (80-128) was designed by conjugating enhanced green fluorescent protein (EGFP) with the C-terminal region of azurin. This design was inspired by the in silico binding study, where the analogue of ephrinA, his6EGFP-azu (80-128) showed higher binding affinity for the EphA6 receptor than the ephrinA ligands. The his6EGFP-azu (80-128) nanosystem which assembled as nanoparticles was tested for its ability to simultaneously detect and kill the prostate cancer cells, LNCaP. This was achieved by specifically targeting EphA6 receptors overexpressed on the cancer cell surface via C-terminal peptide, azu (80-128). Herein, we report antiproliferative, apoptotic, antimigratory, and anti-invasive effects of this nanosystem on LNCaP cells, while having no similar effects on EphA6 negative human normal lung cells, WI-38.

Molecular Dynamics Simulation Investigation of the Binding and Interaction of the EphA6-Odin Protein Complex.

Protein-protein interaction plays an important role in the development of almost all cells. Elucidating the dynamic binding and affinity of a protein-protein complex is essential for understanding the biological functions of proteins. EphA6 and Odin proteins are members of the Eph (erythropoietin-producing hepatocyte) receptor family and the Anks (ankyrin repeat and sterile α motif domain-containing) family, respectively. Odin significantly functions in regulating endocytosis, degradation, and stability of EphA receptors. In this work, the key residues of the interaction interface were determined through a hydrogen bond, root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and dynamic correlation analysis of the conventional molecular dynamics (MD) simulations. The calculated standard binding free energy, -7.92 kcal/mol, between EphA6 and Odin is quite consistent with the experimental measurement value, -8.73 kcal/mol. By the combination of several MD simulation techniques, our investigation of the binding process reveals the detailed representative characteristics of the entire binding pathway at the molecular level. Based on the obtained potential of the mean force (PMF) curve, the analysis of the simulation trajectories shows that the residue Arg1013 in the receptor EphA6 is responsible for capturing Asp739 and Asp740 in the ligand Odin during the initial stage of binding. In the later stage of binding, the hydrogen bonds and salt bridges between a series of residues Lys973, Leu1007, Gly1009, His1010, and Arg1012 in the receptor and residues Leu735, Asn736, Asp739, Asp740, and Asp753 in the ligand mainly contribute to the stability of the protein complex. In addition, the specific change process of the receptor-ligand-binding mode is also clarified during the binding process. Our present simulation will promote a deep understanding of the protein-protein interaction, and the identified key interresidue interaction will be theoretical guidance for the design of protein drugs.

A genome-wide association study for prolificacy in three Polish sheep breeds.

Reproductive traits (especially litter size) are usually characterized by low heritability, and thus, phenotypic selection is often ineffective and slow. In order to improve fertility characteristics such as ovulation rate and litter size, it seems more effective to select breeding animals based on their genotype. The aim of the study was to use genome-wide association study (GWAS) in three sheep breeds to identify the genetic variants affecting the litter size in sheep. The study allowed us to identify one genome-wide significant SNP (rs402032081-located in ephrin type-A receptor 6, EPHA6) showing an association with litter size in Polish Mountain Sheep. We suggest that the EPHA6 gene can be a candidate gene for prolificacy trait in selected breeds of sheep; however, it needs further functional data for validation.

Functional genomic analysis identifies drug targetable pathways in invasive and metastatic cutaneous squamous cell carcinoma.

Although cutaneous squamous cell carcinoma (cSCC) is treatable in the majority of cases, deadly invasive and metastatic cases do occur. To date there are neither reliable predictive biomarkers of disease progression nor FDA-approved targeted therapies as standard of care. To address these issues, we screened patient-derived primary cultured cells from invasive/metastatic cSCC with 107 small-molecule inhibitors. In-house bioinformatics tools were used to cross-analyze drug responses and DNA mutations in tumors detected by whole-exome sequencing (WES). Aberrations in molecular pathways with evidence of potential drug targets were identified, including the Eph-ephrin and neutrophil degranulation signaling pathways. Using a screening panel of siRNAs, we identified EPHA6 and EPHA7 as targets within the Eph-ephrin pathway responsible for mitigating decreased cell viability. These studies form a plausible foundation for detecting biomarkers of high-risk progressive disease applicable in dermatopathology and for patient-specific therapeutic options for invasive/metastatic cSCC.

IFNß-induced exosomal linc-EPHA6-1 promotes cytotoxicity of NK cells by acting as a ceRNA for hsa-miR-4485-5p to up-regulate NKp46 expression.

AIMS: Exosomes contain functional molecules from their cells of origin and can enter recipient cells for intercellular communication. Interferon ß (IFNß) has been shown to induce some lncRNAs to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (NK) cells. We aim to clarify whether IFNß induced exosomes can regulate the cytotoxicity of NK cells by transferring specific lncRNAs into NK cells. MAIN METHODS: Exosomes were isolated from the supernatants of A549 cells with or without IFNß treatment. Co-culture and ELISA assay were used to analyze the effect of exosomes on the cytotoxicity of NK cells. Human transcriptome array (HTA) was performed to analyze the profiling of RNAs wrapped in exosomes. Then subcellular location, qPCR, western blotting, dual-luciferase reporter assay and ELISA were used to determine long noncoding RNAs (lcnRNAs) location, sponge absorb effects, the expression of NKp46 and cytotoxicity of NK cells. KEY FINDINGS: ELISA assay showed IFNß induced exosomes can strengthen the cytotoxicity of NK cells. Through HTA we found the expression levels of 69 lncRNAs were significantly changed within IFNß induced exosomes. Additionally, we found a specific exosomal cargo, linc-EPHA6-1, acted as a competing endogenous RNA (ceRNA) for hsa-miR-4485-5p which subsequently up-regulate one of the natural cytotoxicity receptors (NKp46) expression. Furthermore, we verified over-expression of linc-EPHA6-1 significantly enhances the cytotoxicity of NK cells against A549 cells and Zika virus infected A549 cells. SIGNIFICANCE: Our results demonstrated that IFNß-induced exosomal linc-EPHA6-1 can regulate the cytotoxicity of NK cells.

Podocyte RNA sequencing reveals Wnt- and ECM-associated genes as central in FSGS.

Loss of podocyte differentiation can cause nephrotic-range proteinuria and Focal and Segmental Glomerulosclerosis (FSGS). As specific therapy is still lacking, FSGS frequently progresses to end-stage renal disease. The exact molecular mechanisms of FSGS and gene expression changes in podocytes are complex and widely unknown as marker changes have mostly been assessed on the glomerular level. To gain a better insight, we isolated podocytes of miR-193a overexpressing mice, which suffer from FSGS due to suppression of the podocyte master regulator Wt1. We characterised the podocytic gene expression changes by RNAseq and identified many novel candidate genes not linked to FSGS so far. This included strong upregulation of the receptor tyrosine kinase EphA6 and a massive dysregulation of circadian genes including the loss of the transcriptional activator Arntl. By comparison with podocyte-specific changes in other FSGS models we found a shared dysregulation of genes associated with the Wnt signaling cascade, while classical podocyte-specific genes appeared widely unaltered. An overlap with gene expression screens from human FSGS patients revealed a strong enrichment in genes associated with extra-cellular matrix (ECM) and metabolism. Our data suggest that FSGS progression might frequently depend on pathways that are often overlooked when considering podocyte homeostasis.

Epigenetic age signatures in bones.

Age prediction can help identify skeletal remains by limiting the search range for a missing person. Although age prediction methods based on odontology and anthropology are frequently used in the forensic field, DNA methylation is particularly promising age-predictive biomarker. In this study, we generated genome-wide DNA methylation profiles of bone samples from 32 identified skeletal remains with an age at death ranging from 31 to 96 years. Only 12 provided more than 800 K quality-filtered CpG methylation values using Illumina's Infinium MethylationEPIC BeadChip array. Methylation ages of the bone samples calculated using a recently developed skin & blood clock composed of 391 CpG sites were found to be very similar to their actual ages (MAD = 6.4 years). However, the low success rate in methylation profiling of bone DNA samples may prevent researchers from applying the array to this type of samples. Therefore, we selected a set of CpG sites that would enable age prediction based on only a few CpG sites in bone DNA samples. Nineteen age-associated CpG marker candidates were selected from 720 K quality-filtered CpG values of 21 male skeletal remain samples. Because age signatures for blood, such as markers on the ELOVL2, FHL2, KLF14 and TRIM59 genes, had showed strong age associations in 12 bone samples, we further tested the age association of the 5 well-known markers in a blood-based model and the 13 out of 19 CpG markers from the array of 21 bone samples with an independent set of 30 skeletal remain samples using SNaPshot multiplex based on single nucleotide primer extension. Four CpG sites on TMEM51, TRIM59, ELOVL2, and EPHA6 genes showed moderate or weak correlations between methylation and age, which suggests further investigation of these markers to predict the age of bones.

Tyrosine kinase Eph receptor A6 sensitizes glioma-initiating cells towards bone morphogenetic protein-induced apoptosis.

Bone morphogenetic protein (BMP) signaling plays important roles in glioblastoma multiforme (GBM), a lethal form of brain tumor. BMP reduces GBM tumorigenicity through its differentiation- and apoptosis-inducing effects on glioma-initiating cells (GIC). However, some GIC do not respond to the tumor suppressive effects of BMP. Using a phosphoreceptor tyrosine kinase array, we found that EPHA6 (erythropoietin-producing hepatocellular carcinoma receptor A6) phosphorylation was regulated by BMP-2 signaling in some GIC. Analysis of The Cancer Genome Atlas showed that EPHA6 expression was lower in patients with GBM than in the normal brain, and that high EPHA6 expression was correlated with better prognosis. EPHA6 receptor increased the susceptibility of both sensitive and resistant GIC to BMP-2-induced apoptosis. The cooperative effect on apoptosis induction depended on the kinase activity of BMP type I receptor but was independent of EPHA6 kinase function. Overexpression of the EPHA6 receptor in GIC resulted in the formation of a protein complex of EPHA6 receptor and the BMP type I receptor ALK-2, which was associated with BMP-induced apoptosis in GIC. Intracranial injection of GIC into nude mice showed that gain-of-function of EPHA6 together with BMP-2 pretreatment slowed GBM tumor progression in the mouse brain and promoted mouse survival. In summary, EPHA6 together with BMP-2 signaling led to apoptotic cell death in GIC, and thus is a putative tumor suppressor in GBM.

EPHA6 rs4857055 C > T polymorphism associates with hypertension through triglyceride and LDL particle size in the Korean population.

BACKGROUND: Erythropoietin-producing human hepatocellular (Eph) receptors might contribute to the development of atherosclerosis. A genome-wide association study indicated that the Eph receptor A6 gene (EPHA6) associated with at least 1 blood pressure (BP) phenotype. The objective of the present study was to determine whether EPHA6 is a novel candidate gene for hypertension in a Korean population. METHODS: A total 2146 study participants with normotension and hypertension were included. Genotype data were obtained using a Korean Chip. To assess the association between single-nucleotide polymorphisms (SNPs) and BP, we performed a linear regression analysis, which showed that rs4850755 in the EPHA6 gene was the SNP most highly associated with both systolic and diastolic BP. RESULTS: The presence of the TT genotype of the EPHA6 rs4857055 C > T SNP was associated with a higher risk of hypertension after adjusting for age, sex, body mass index (BMI), smoking, and drinking [odds ratio 1.533, P = 0.001]. In the control group, significant associations were observed between systolic BP and the rs4857055 polymorphism and between diastolic BP and the rs4857055 polymorphism. In the hypertension group, a significant association was observed between systolic BP and the rs4857055 polymorphism. In the hypertension group, subjects with the TT genotype showed significantly higher systolic BP than CC subjects. Additionally, in the hypertension group, TT carriers showed a higher tendency of serum triglyceride (P = 0.069) and significantly higher apolipoprotein B (P = 0.015) and smaller low-density lipoprotein (LDL) particle size (P < 0.001) than either TC or CC subjects. CONCLUSIONS: These results could suggest that the EPHA6 rs4857055 C > T SNP is a novel candidate gene for hypertension in the Korean population. Additionally, the TT genotype could be associated with hypertriglyceridemia and small LDL particle size in hypertension.

Targeted Sequencing Reveals Low-Frequency Variants in EPHA Genes as Markers of Paclitaxel-Induced Peripheral Neuropathy.

Purpose: Neuropathy is the dose-limiting toxicity of paclitaxel and a major cause for decreased quality of life. Genetic factors have been shown to contribute to paclitaxel neuropathy susceptibility; however, the major causes for interindividual differences remain unexplained. In this study, we identified genetic markers associated with paclitaxel-induced neuropathy through massive sequencing of candidate genes.Experimental Design: We sequenced the coding region of 4 EPHA genes, 5 genes involved in paclitaxel pharmacokinetics, and 30 Charcot-Marie-Tooth genes, in 228 cancer patients with no/low neuropathy or high-grade neuropathy during paclitaxel treatment. An independent validation series included 202 paclitaxel-treated patients. Variation-/gene-based analyses were used to compare variant frequencies among neuropathy groups, and Cox regression models were used to analyze neuropathy along treatment.Results: Gene-based analysis identified EPHA6 as the gene most significantly associated with paclitaxel-induced neuropathy. Low-frequency nonsynonymous variants in EPHA6 were present exclusively in patients with high neuropathy, and all affected the ligand-binding domain of the protein. Accumulated dose analysis in the discovery series showed a significantly higher neuropathy risk for EPHA5/6/8 low-frequency nonsynonymous variant carriers [HR, 14.60; 95% confidence interval (CI), 2.33-91.62; P = 0.0042], and an independent cohort confirmed an increased neuropathy risk (HR, 2.07; 95% CI, 1.14-3.77; P = 0.017). Combining the series gave an estimated 2.5-fold higher risk of neuropathy (95% CI, 1.46-4.31; P = 9.1 × 10-4).Conclusions: This first study sequencing EPHA genes revealed that low-frequency variants in EPHA6, EPHA5, and EPHA8 contribute to the susceptibility to paclitaxel-induced neuropathy. Furthermore, EPHA's neuronal injury repair function suggests that these genes might constitute important neuropathy markers for many neurotoxic drugs. Clin Cancer Res; 23(5); 1227-35. ©2016 AACR.

Genetic polymorphisms and paclitaxel- or docetaxel-induced toxicities: A systematic review.

BACKGROUND: Taxanes, including paclitaxel and docetaxel, are indispensable for treatment of cancer. Development of toxicity frequently necessitates dose reduction or discontinuation of therapy, despite clinical response. OBJECTIVE: Pharmacogenetic studies were reviewed for identification of genetic variants possibly underlying individual susceptibility to adverse events. METHOD: We conducted a systematic search in Pubmed and Embase for pharmacogenetic reports with focus on commonly reported taxane-related gastrointestinal, hematological and neurological toxicities in adult patients with solid tumors. The findings from a total of 51 eligible studies are presented in a comprehensive way. RESULTS: Most frequently investigated single nucleotide polymorphisms (SNPs) were located in genes encoding proteins affecting pharmacokinetics, such as drug transporters and genes of the cytochrome P450 family. Inconclusive data for risk of toxicity as well as for effects on drug exposure were reported on variants in ABCB1, CYP3A4, CYP3A5 and, for paclitaxel, CYP2C8. Interest is also dedicated towards genes involved in pharmacodynamics, such as detoxification of reactive oxygen species, DNA repair, neuronal processes and microtubule function. Recent studies include variants in TUBB2A, EPHA5 and EPHA6 for a possible association with neurotoxicity. Variations in methodological approach, sample size, study design, treatment schedule and end-point of toxicity affect consistency of results. CONCLUSION: This review illustrates the complexity to well design pharmacogenetic studies for validation of SNPs that may clarify differences in taxane-induced toxicities among individuals. Novel genes encoding cellular targets of taxanes deserve further analysis by means of robust patient cohorts and definition of objective end-points.

Identification of disrupted AUTS2 and EPHA6 genes by array painting in a patient carrying a de novo balanced translocation t(3;7) with intellectual disability and neurodevelopment disorder.

Intellectual disability (ID) is a frequent feature but is highly clinically and genetically heterogeneous. The establishment of the precise diagnosis in patients with ID is challenging due to this heterogeneity but crucial for genetic counseling and appropriate care for the patients. Among the etiologies of patients with ID, apparently balanced de novo rearrangements represent 0.6%. Several mechanisms explain the ID in patients with apparently balanced de novo rearrangement. Among them, disruption of a disease gene at the breakpoint, is frequently evoked. In this context, technologies recently developed are used to characterize precisely such chromosomal rearrangements. Here, we report the case of a boy with ID, facial features and autistic behavior who is carrying a de novo balanced reciprocal translocation t(3;7)(q11.2;q11.22)dn. Using microarray analysis, array painting (AP) technology combined with molecular study, we have identified the interruption of the autism susceptibility candidate 2 gene (AUTS2) and EPH receptor A6 gene (EPHA6). We consider that the disruption of AUTS2 explains the phenotype of the patient; the exact role of EPHA6 in human pathology is not well defined. Based on the observation of recurrent germinal and somatic translocations involving AUTS2 and the molecular environment content, we put forward the hypothesis that the likely chromosomal mechanism responsible for the translocation could be due either to replicative stress or to recombination-based mechanisms.

EphA6 promotes angiogenesis and prostate cancer metastasis and is associated with human prostate cancer progression.

Metastasis is the primary cause of prostate cancer (CaP)-related death. We investigate the molecular, pathologic and clinical outcome associations of EphA6 expression and CaP metastasis. The expression profiling of Eph receptors (Ephs) and their ephrin ligands was performed in parental and metastatic CaP cell lines. Among Ephs and ephrins, only EphA6 is consistently overexpressed in metastatic CaP cells. Metastatic potential of EphA6 is assessed by RNAi in a CaP spontaneous metastasis mouse model. EphA6 knock-down in human PC-3M cells causes decreased invasion in vitro and reduced lung and lymph node metastasis in vivo. In addition, knock-down of EphA6 decreases tube formation in vitro and angiogenesis in vivo. EphA6 mRNA expression is higher in 112 CaP tumor samples compared with benign tissues from 58 benign prostate hyperplasia patients. Positive correlation was identified between EphA6 expression and vascular invasion, neural invasion, PSA level, and TNM staging in CaP cases. Further, genome-wide gene expression analysis in EphA6 knock-down cells identified a panel of differentially regulated genes including PIK3IPA, AKT1, and EIF5A2, which could contribute to EphA6-regulated cancer progression. These findings identify EphA6 as a potentially novel metastasis gene which positively correlates with CaP progression. EphA6 may be a therapeutic target in metastatic CaP.

Novel recurrently mutated genes in African American colon cancers.

We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors.

A dimerized HMX1 inhibits EPHA6/epha4b in mouse and zebrafish retinas.

HMX1 is a homeobox-containing transcription factor implicated in eye development and responsible for the oculo-auricular syndrome of Schorderet-Munier-Franceschetti. HMX1 is composed of two exons with three conserved domains in exon 2, a homeobox and two domains called SD1 and SD2. The function of the latter two domains remains unknown. During retinal development, HMX1 is expressed in a polarized manner and thus seems to play a role in the establishment of retinal polarity although its exact role and mode of action in eye development are unknown. Here, we demonstrated that HMX1 dimerized and that the SD1 and homeodomains are required for this function. In addition, we showed that proper nuclear localization requires the presence of the homeodomain. We also identified that EPHA6, a gene implicated in retinal axon guidance, is one of its targets in eye development and showed that a dimerized HMX1 is needed to inhibit EPHA6 expression.

Transcription factor Foxd1 is required for the specification of the temporal retina in mammals.

The organization of the visual system is different in birds and mammals. In both, retinal axons project topographically to the visual targets in the brain; but whereas in birds visual fibers from the entire retina decussate at the optic chiasm, in mammals, a number of axons from the temporal retina diverge at the midline to project ipsilaterally. Gain-of-function experiments in chick raised the hypothesis that the transcription factor Foxd1 specifies retinal temporal identity. However, it remains unknown whether Foxd1 is necessary for this function. In mammals, the crucial role of Foxd1 in the patterning of the optic chiasm region has complicated the interpretation of its cell-autonomous function in the retina. Furthermore, target molecules identified for Foxd1 are different in chicks and mice, leading to question the function of Foxd1 in mammals. Here we show that in the mouse, Foxd1 imprints temporal features in the retina such as axonal ipsilaterality and rostral targeting in collicular areas and that EphA6 is a Foxd1 downstream effector that sends temporal axons to the rostral colliculus. In addition, our data support a model in which the desensitization of EphA6 by ephrinA5 in cis is not necessary for the proper functioning of EphA6. Overall, these results indicate that Foxd1 functions as a conserved determinant of temporal identity but reveal that the downstream effectors, and likely their mechanisms of action, are different in mammals and birds.

Learning and memory impairment in Eph receptor A6 knockout mice.

Genetic inhibition of the ephrin receptor (EphA6) in mice produced behavioral deficits specifically in tests of learning and memory. Using a fear conditioning training paradigm, mice deficient in EphA6 did not acquire the task as strongly as did wild type (WT) mice. When tested in the same context 24h later, knockout (KO) mice did not freeze as much as WT mice indicating reduced memory of the consequences of the training context. The KO mice also displayed less freezing when presented with the conditioning stimulus (CS) in a separate context. In the hidden platform phase of the Morris water maze (MWM) task, KO mice did not reach the same level of proficiency as did WT mice. KO mice also exhibited less preference for the target quadrant during a probe trial and were significantly impaired on an initial reversal of the platform. These findings suggest that EphA6, in line with a number of other Eph receptors and their ephrin ligands, is involved in neural circuits underlying aspects of learning and memory.

HOXA13 directly regulates EphA6 and EphA7 expression in the genital tubercle vascular endothelia.

Hypospadias, a common defect affecting the growth and closure of the external genitalia, is often accompanied by gross enlargements of the genital tubercle (GT) vasculature. Because Hoxa13 homozygous mutant mice also exhibit hypospadias and GT vessel expansion, we examined whether genes playing a role in angiogenesis exhibit reduced expression in the GT. From this analysis, reductions in EphA6 and EphA7 were detected. Characterization of EphA6 and EphA7 expression in the GT confirmed colocalization with HOXA13 in the GT vascular endothelia. Analysis of the EphA6 and EphA7 promoter regions revealed a series of highly conserved cis-regulatory elements bound by HOXA13 with high affinity. GT chromatin immunoprecipitation confirmed that HOXA13 binds these gene-regulatory elements in vivo. In vitro, HOXA13 activates gene expression through the EphA6 and EphA7 gene-regulatory elements. Together these findings indicate that HOXA13 directly regulates EphA6 and EphA7 in the developing GT and identifies the GT vascular endothelia as a novel site for HOXA13-dependent expression of EphA6 and EphA7.