Immunohistochemical evaluation of forkhead box A1 and EphA5 markers in serous ovarian carcinomas, and their impact on the clinical outcome of patients.

Ovarian cancer is the most lethal gynaecological neoplasm in females. In ovarian cancer, forkhead box A1 (FOXA1) aids transcription of YAP-associated protein mediated by the cyclic adenosine monophosphate response element-binding protein. As a result, cellular proliferation and migration increased. The roles of erythropoietin-producing human hepatocellular carcinoma cell (Eph) receptors and ephrin ligands in cell adhesion, migration, cell proliferation regulation in various cancers, and angiogenesis are well characterized. This study included formalin-fixed, paraffin-embedded tissue specimens from 41 patients with ovarian serous cystadenocarcinoma, including both low- and high-grade tumours. For each case, a paraffin block with tumour tissue was chosen for an immunohistochemical procedure using primary antibodies against EphA5 and FOXA1. By the end of 2017, patients finished their chemotherapy and were followed for the next 3 years. Positive FOXA1 and EphA5 results were presented in 68.3% and 39% of patients, respectively. A statistically significant correlation was detected between FOXA1 expression and each of CA-125 level, tumour stage, tumour grade, and the presence of lymph node metastasis. In our work, the overall survival was positively correlated with EphA5 expression and inversely correlated to FOXA1 immunoreactivity. The estimated disease-free survival (DFS) and EphA5 immunoreactivity had a significant positive association, whereas DFS and FOXA1 protein expression had a significant inverse link. FOXA1 and EphA5 expression play a role in ovarian cancer progression and prognosis prediction.

Association between EPHA5 methylation status in peripheral blood leukocytes and the risk and prognosis of gastric cancer.

Purpose: Altered DNA methylation, genetic alterations, and environmental factors are involved in tumorigenesis. As a tumor suppressor gene, abnormal EPHA5 methylation was found in gastric cancer (GC) tissues and was linked to the initiation, progression and prognosis of GC. In this study, the EPHA5 methylation level in peripheral blood leukocytes (PBLs) was detected to explore its relationship with GC risk and prognosis. Methods: A total of 366 GC cases and 374 controls were selected as the subjects of this study to collect their environmental factors, and the EPHA5 methylation status was detected through the methylation-sensitive high-resolution melting method. Logistic regression analysis was utilized to evaluate the associations among EPHA5 methylation, environmental factors and GC risk. Meanwhile, the propensity score (PS) was used to adjust the imbalance of some independent variables. Results: After PS adjustment, EPHA5 Pm (positive methylation) was more likely to increase the GC risk than EPHA5 Nm (negative methylation) (ORb = 1.827, 95% CI [1.202-2.777], P = 0.005). EPHA5 Pm had a more significant association with GC risk in the elderly (ORa = 2.785, 95% CI [1.563-4.961], P = 0.001) and H. pylori-negative groups (ORa = 2.758, 95% CI [1.369-5.555], P = 0.005). Moreover, the combined effects of EPHA5 Pm and H. pylori infection (ORc a = 3.543, 95% CI [2.233-5.621], P < 0.001), consumption of alcohol (ORc a = 2.893, 95% CI [1.844-4.539], P < 0.001), and salty food intake (ORc a = 4.018, 95% CI [2.538-6.362], P < 0.001) on increasing the GC risk were observed. In addition, no convincing association was found between EPHA5 Pm and the GC prognosis. Conclusions: EPHA5 methylation in PBLs and its combined effects with environmental risk factors are related to the GC risk.

EPHA5 mutation was associated with adverse outcome of atezolizumab treatment in late-stage non-small cell lung cancers.

BACKGROUND: The aim of the study was to investigate predictive value of gene mutation for atezolizumab treatment response from OAK and POPLAR cohorts. METHODS: Several public databases were used for analyzing gene mutation type of EPHA5 and association with alterations of other genes. Survival analysis was performed for patients receiving atezolizumab from OAK and POPLAR cohorts. RESULTS: EPHA5 mutation have high frequency to harbor TP53 and KEAP1 mutations. The bTMB value has significant difference between EPHA5 mutant and wild-type cases. Patients with EPHA5 mutation got worse survival compared to those without gene mutations receiving atezolizumab (P = 0.0186). CONCLUSIONS: EPHA5 mutant NSCLC may represent a subpopulation which showed worse response after treatment of atezolizumab compared to wild-type ones.

Expression of EPHA5 in lung adenocarcinoma is associated with lymph node metastasis and EGFR mutation.

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.

Long noncoding RNA ZFAS1 suppresses osteogenic differentiation of bone marrow-derived mesenchymal stem cells by upregulating miR-499-EPHA5 axis.

Emerging evidence suggests that the shift between osteogenic and adipogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) determines bone mass. Our study was aimed at testing whether a long noncoding RNA called zinc finger antisense 1 (ZFAS1) participates in the differentiation commitment of BMSCs during osteoporosis. We found that ZFAS1 expression was downregulated during osteogenic differentiation and upregulated during adipogenic differentiation. ZFAS1 knockdown facilitated osteogenic differentiation and suppressed adipogenic differentiation. Furthermore, ZFAS1 knockdown suppressed cell senescence and promoted autophagy. Ovariectomized mice injected with a ZFAS1 knockdown construct showed increased bone mass. Mechanismly, ZFAS1 affected the osteogenic and adipogenic differentiation of BMSCs through sponging miR-499 thereby upregulating ephrin type-A receptor 5 (EPHA5). Taken together, our results revealed that the ZFAS1-miR-499-EPHA5 axis may be important for the osteoporosis-related switch between the osteogenesis and adipogenesis of BMSCs, indicating that ZFAS1 represents a plausible therapeutic target for reversing osteoporotic bone loss.

EphA2 and Ephrin-A5 Guide Eye Lens Suture Alignment and Influence Whole Lens Resilience.

Purpose: Fine focusing of light by the eye lens onto the retina relies on the ability of the lens to change shape during the process of accommodation. Little is known about the cellular structures that regulate elasticity and resilience. We tested whether Eph-ephrin signaling is involved in lens biomechanical properties. Methods: We used confocal microscopy and tissue mechanical testing to examine mouse lenses with genetic disruption of EphA2 or ephrin-A5. Results: Confocal imaging revealed misalignment of the suture between each shell of newly added fiber cells in knockout lenses. Despite having disordered sutures, loss of EphA2 or ephrin-A5 did not affect lens stiffness. Surprisingly, knockout lenses were more resilient and recovered almost completely after load removal. Confocal microscopy and quantitative image analysis from live lenses before, during, and after compression revealed that knockout lenses had misaligned Y-sutures, leading to a change in force distribution during compression. Knockout lenses displayed decreased separation of fiber cell tips at the anterior suture at high loads and had more complete recovery after load removal, which leads to improved whole-lens resiliency. Conclusions: EphA2 and ephrin-A5 are needed for normal patterning of fiber cell tips and the formation of a well-aligned Y-suture with fiber tips stacked on top of previous generations of fiber cells. The misalignment of lens sutures leads to increased resilience after compression. The data suggest that alignment of the Y-suture may constrain the overall elasticity and resilience of the lens.

EPHA5 Enhances the Stemness of Non-small cell lung cancer Cells Through Activating the Wnt Signaling Pathway.

PURPOSE: To explore the effect of erythropoietin-producing hepatocellular receptor A5 (EPHA5) on the stemness of non-small cell lung cancer cells and its molecular mechanism. METHODS: Highly-expressed EPHA5 in NCI-H460 and NCI-H1229 cells was silenced. After EPHA5 silencing, the positive expression level of cluster of differentiation 133 (CD133) in NCI-H460 and NCI-H1229 cells was detected by flow cytometry, and the expression levels of stemness markers sex determining region Y-box 2 (Sox2), Nanog, Kruppel-like factor 4 (KLF4) and octamer-binding transcription factor 4 (Oct4) in cells were detected by Western blotting. RESULTS: The expression level of EPHA5 in non-small cell lung cancer H460 and H1229 cells was higher than that in A549 and SPC-A1 cells. After EPHA5 silencing, the levels of CD133 and stemness markers Sox2, Nanog, KLF4 and Oct4 in H460 and H1229 cells all declined. CCK-8 assay showed that Wnt agonists at a concentration of 2.5 and 5 µm had little effect on the proliferative activity of H460 and H1229 cells. Western blotting revealed that Wnt agonists at a concentration of 5 µm could better enhance the expression of ß-catenin. After treatment with Wnt agonists, the expression of CD133 in H460 and H1229 cells with EPHA5 silencing by siRNA3 was higher than that before treatment, and the expression levels of Sox2, Nanog, KLF4 and Oct4 in the above two cells were also increased compared with those before treatment. However, the levels of the above indexes were all lower after treatment with Wnt agonists than those before silencing. CONCLUSION: Activating the Wnt signaling pathway can induce the increase in EPHA5 expression and enhance the stemness of non-small cell lung cancer cells.

Sarcomatoid hepatocellular carcinoma: From clinical features to cancer genome.

BACKGROUND: Sarcomatoid hepatocellular carcinoma (HCC) is a rare and highly lethal histological subtype of HCC, with completely unknown genetic etiology and therapeutic targets. METHODS: We included 16 patients with sarcomatoid HCC receiving radical resection among 6731 cases of pathological confirmed HCC in year 2008 to 2018 in our hospital. We compared the clinical features, prognosis and cancer genome between 15 sarcomatoid HCC and propensity score-matched 75 non-sarcomatoid HCC patients. The other concurrent case was analyzed using phylogenetic tree to assess the tumor heterogeneity and evolution. RESULTS: Sarcomatoid HCC group showed larger tumor size, more advanced differentiation grade, lower tumor free survival (p = 0.038) and overall survival (p = 0.001), and sarcomatoid type was an independent risk factor for patient death. Integrating sarcomatoid subtype into AJCC staging could increase the diagnostic curve in predicting patient survival. The cancer genome spectrum showed sarcomatoid HCC group had significant higher mutation rates in CDKN2A, EPHA5, FANCM and MAP3K1. Mutations in CDKN2A significantly reduced tumor-free and overall survival in sarcomatoid HCC patients. Moreover, 46.6% sarcomatoid HCC patients had druggable mutations in cell cycle pathway genes, which were targeted by Abemaciclib, et al. We also found sarcomatoid and non-sarcomatoid lesions might originate from a common progenitor but progress differently. CONCLUSION: Our cancer genome analysis showed a specific genomic profile of sarcomatoid HCC, which were characterized by a high mutation rate in cell cycle genes particularly CDKN2A. The results indicate CDK4/6 inhibitors including abemaciclib, ribociclib and palbociclib as potential therapeutic targets and may help for therapeutic decision making.

Deep learning model reveals potential risk genes for ADHD, especially Ephrin receptor gene EPHA5.

Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder. Although genome-wide association studies (GWAS) identify the risk ADHD-associated variants and genes with significant P-values, they may neglect the combined effect of multiple variants with insignificant P-values. Here, we proposed a convolutional neural network (CNN) to classify 1033 individuals diagnosed with ADHD from 950 healthy controls according to their genomic data. The model takes the single nucleotide polymorphism (SNP) loci of P-values $\le{1\times 10^{-3}}$, i.e. 764 loci, as inputs, and achieved an accuracy of 0.9018, AUC of 0.9570, sensitivity of 0.8980 and specificity of 0.9055. By incorporating the saliency analysis for the deep learning network, a total of 96 candidate genes were found, of which 14 genes have been reported in previous ADHD-related studies. Furthermore, joint Gene Ontology enrichment and expression Quantitative Trait Loci analysis identified a potential risk gene for ADHD, EPHA5 with a variant of rs4860671. Overall, our CNN deep learning model exhibited a high accuracy for ADHD classification and demonstrated that the deep learning model could capture variants' combining effect with insignificant P-value, while GWAS fails. To our best knowledge, our model is the first deep learning method for the classification of ADHD with SNPs data.

EPHA5 mutation predicts the durable clinical benefit of immune checkpoint inhibitors in patients with lung adenocarcinoma.

Immune checkpoint inhibitor (ICI) therapy has shown remarkable clinical benefit in lung adenocarcinoma (LUAD) patients. Genomic mutations may be applicable to predict the response to ICIs. Eph receptor A5 (EPHA5) is frequently mutated in breast cancer, lung cancer, and other tumors; however, its association with outcome in patients who receive immunotherapy remains unknown. In this study, we report that EPHA5 mutations were associated with increased tumor mutation burden (TMB), neoantigen load, levels of immune-related gene expression signatures, and enhanced tumor-infiltrating lymphocytes (TILs) in LUAD. LUAD patients with EPHA5 mutations in the immunotherapy cohort achieved a longer progression-free survival (PFS) time than patients with wild-type EPHA5. Immune response pathways were among the top enriched pathways in samples with EPHA5 mutations. In addition, patients with EPHA5 mutations tended to be more sensitive to certain targeted molecular inhibitors, including serdemetan, lox2, and PF1-1. Collectively, our results suggest that identifying mutations in the EPHA5 gene may provide insight into the genome-wide mutational burden and may serve as a biomarker to predict the immune response of patients with LUAD.

PDL1 high expression without TP53, KEAP1 and EPHA5 mutations could better predict survival for patients with NSCLC receiving atezolizumab.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients with high expression of PDL1 are more likely to benefit from atezolizumab. There are no relevant research focusing on the relationship between the PDL1 expression and clinical variables and gene mutation types among NSCLC patients. METHODS: NSCLC patients with confirmed PDL1 expression and gene mutation information from OAK study were included in our study. Logistic regression proportional model was applied to analyze the risk factors on PDL1 high expression. The biomarker evaluable population (BEP) was screened to analyze the gene mutation informaion among these patients. High frequency gene mutations were screened based on different PDL1 expressions. Moreover, the log rank test was applied to analyze the overall survival (OS) difference based on different gene mutation types. RESULTS: A total of 838 patients with NSCLC were included in our study. White patients are more likely to have PDL1 ≥ 1% (P = 0.004). ERBB4, EP300, PREX2, SLIT2, EPHB1 and IGF2R mutations were high frequency mutations in patients with high PDL1 expression, and the patients with EGFR, SMARCA4, EPHA5, FAT1, STK11, TET2 mutations were more likely to be seen in negative PDL1 expression group. Worse survival could be found in patients with KEAP1 (P < 0.001), TP53 (P = 0.004) and EPHA5 (P = 0.013) mutations who received atezolizumab compared with those who had none of these gene mutations. Importantly, for PDL1 high patients without KEAP1, EPHA5, TP53 mutations receiving atezolizumab, they all showed relatively longer median survival with 22.47, 22.18 and 23.33 months, respectively (all, P < 0.01). CONCLUSIONS: Different high frequency gene mutations could be found between the patients with high and negative PDL1. PDL1 expression combined with specific gene mutation may better predict the survival for patients receiving atezolizumab.

EPHA5 mutations predict survival after immunotherapy in lung adenocarcinoma.

Eph receptors constitute the largest family of RTKs, and their associations with antitumor immunity and immunotherapy are largely unknown. By integrating genomic, transcriptomic and clinical data from cohorts in public databases, we identified EPHA5 as the most common mutated gene of Eph receptors in lung adenocarcinoma (LUAD). Moreover, compared with EPHA5 wild-type (WT) patients, EPHA5-mutant (Mut) patients exhibited significantly enhanced infiltration of CD8+ T cells and M1 macrophages, reduced recruitment of immunosuppressive regulatory T cells (Tregs) into the tumor site, as well as the increased level of chemokine, interferon-gamma, inhibitory immune checkpoint signatures, tumor mutation burden (TMB) and tumor neoantigen burden (TNB). Additionally, EPHA5 mutation cooccurred with homologous recombination (HR) or mismatch repair (MMR) gene mutations. These data were validated in the LUAD cell line H1299 and a Chinese LUAD cohort. Most importantly, clinical analysis of a Memorial Sloan Kettering Cancer Center (MSKCC) immunotherapy cohort indicated that LUAD patients with EPHA5 mutations who were treated with immunotherapy had markedly prolonged survival times. Our results revealed the correlation of EPHA5 mutations with tumor immune microenvironment and predictive factors for immunotherapy, implying the potential of EPHA5 mutations as a prognostic marker for the prognosis of LUAD patients to immune checkpoint blockade therapy.

Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor.

Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5'-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.

Alternative proteins are functional regulators in cell reprogramming by PKA activation.

It has been recently shown that many proteins are lacking from reference databases used in mass spectrometry analysis, due to their translation templated on alternative open reading frames. This questions our current understanding of gene annotation and drastically expands the theoretical proteome complexity. The functions of these alternative proteins (AltProts) still remain largely unknown. We have developed a large-scale and unsupervised approach based on cross-linking mass spectrometry (XL-MS) followed by shotgun proteomics to gather information on the functional role of AltProts by mapping them back into known signalling pathways through the identification of their reference protein (RefProt) interactors. We have identified and profiled AltProts in a cancer cell reprogramming system: NCH82 human glioma cells after 0, 16, 24 and 48 h Forskolin stimulation. Forskolin is a protein kinase A activator inducing cell differentiation and epithelial-mesenchymal transition. Our data show that AltMAP2, AltTRNAU1AP and AltEPHA5 interactions with tropomyosin 4 are downregulated under Forskolin treatment. In a wider perspective, Gene Ontology and pathway enrichment analysis (STRING) revealed that RefProts associated with AltProts are enriched in cellular mobility and transfer RNA regulation. This study strongly suggests novel roles of AltProts in multiple essential cellular functions and supports the importance of considering them in future biological studies.

EPHA5 mutation impairs natural killer cell-mediated cytotoxicity against non-small lung cancer cells and promotes cancer cell migration and invasion.

AIM: This study aims to evaluate the role of the EPHA5 mutation in the migration and invasion of non-small cell lung cancer (NSCLC) cells and in modulating the killing effect of natural killer (NK) cells to NSCLC cells. METHODS: EPHA5-wt (wild type) and EPHA5-mut (mutation) plasmids were constructed. EPHA5 was silenced using si-EPHA5. NSCLC cell migration and invasion were determined using Transwell assays. NK cell proliferation and apoptosis were determined using CCK-8 assay and flow cytometry, respectively. The killing effect of NK cells to NSCLC cells was also examined. RESULTS: EPHA5 mutation significantly promoted migration and invasion in NSCLC cells. Furthermore, EPHA5 mutation notably impaired the cytotoxicity of NK cells against NSCLC cells. In contrast, EPHA5-wt overexpression and EPHA5 silencing exerted the opposite effect. CONCLUSION: EPHA5 mutation impairs the NK cell-mediated cytotoxicity against NSCLC cells and promotes migration and invasion in NSCLC cells.

CpG Islands Methylation Analysis of CDH11, EphA5, and HS3ST2 Genes in Gastric Adenocarcinoma Patients.

PURPOSE: Gastric cancer is an aggressive disease which is the fourth prevalent malignancy in the world. Beside the genetic factors, epigenetic alterations such as promoter CpG island hyper methylation are involved in the emergence of gastric cancer. Herein, we investigated the methylation status of CDH11, EphA5, and HS3ST2 genes in patients with and without gastric adenocarcinoma for the first time. METHODS: In the study 40 paraffin-embedded tissue sections from gastric adenocarcinoma patients and 40 specimens from patients with functional dyspepsia were taken. DNA extraction was performed using a modified salting out method. Epizen DNA methylation kit was used to the bisulfite DNA conversion. The methylation status of CDH11, EphA5, and HS3ST2 genes were analyzed by methylation-specific PCR (MSP) technique. RESULTS: Among the 80 specimens, 71 DNA samples were achieved (34 gastric adenocarcinoma patients and 37 control patients). The results showed that CDH11, EphA5, and HS3ST2 genes are methylated in 28 (82.45%), 19 (55.88%), and 26 (76.47%) of 34 DNA samples from gastric adenocarcinoma patients, respectively, whereas, these genes are methylated in 7 (18.91%), 9 (24.32%) and 7 (18.91%) of 37 samples from noncancerous patients, respectively. Statistical analyses using a chi-squared test showed that there is a statistically significant difference in methylation level of CDH11, EphA5, and HS3ST2 genes between gastric cancer and uncancerous patients (p < 0.05). CONCLUSION: To the best of our knowledge, this is the first report on methylation of CDH11, EphA5, and HS3ST2 promoters' in gastric adenocarcinoma patients using MSP. Identification of novel cancer-related molecular mechanisms can be useful in detection of new treatment strategies.

Dynamic Alterations of Retinal EphA5 Expression in Retinocollicular Map Plasticity.

The topographically ordered retinocollicular projection is an excellent system for studying the mechanism of axon guidance. Gradients of EphA receptors in the retina and ephrin-As in the superior colliculus (SC) pattern the anteroposterior axis of the retinocollicular map, but whether they are involved in map plasticity after injury is unknown. Partial damage to the caudal SC at birth creates a compressed, complete retinotopic map in the remaining SC without affecting visual response properties. Previously, we found that the gradient of ephrin-A expression in compressed maps is steeper than normal, suggesting an instructive role in compression. Here we measured EphA5 mRNA and protein levels after caudal SC damage in order to test the hypothesis that changes in retinal EphA5 expression occur that are complementary to the changes in collicular ephrin-A expression. We find that the nasotemporal gradient of EphA5 receptor expression steepens in the retina and overall expression levels change dynamically, especially in temporal retina, supporting the hypothesis. This change in receptor expression occurs after the change in ephrin-A ligand expression. We propose that changes in the retinal EphA5 gradient guide recovery of the retinocollicular projection from early injury. This could occur directly through the change in EphA5 expression instructing retino-SC map compression, or through ephrin-A ligand signaling instructing a change in EphA5 receptor expression that in turn signals the retinocollicular map to compress. Understanding what molecular signals direct compensation for injury is essential to developing rehabilitative strategies and maximizing the potential for recovery.

Autism spectrum disorder in a patient with a genomic rearrangement that only involves the EPHA5 gene.

About one child in 68 is affected by the autism spectrum disorder (ASD), one of the most common neurodevelopmental disorders linked to intellectual disability, especially in males, intellectual disability being diagnosable in about 60-70% of autistic individuals. The biological bases of ASD are not yet fully known, but they are generally considered multifactorial, although many genes and genomic loci have been proposed to be possibly associated with this condition. In this report, we describe the case of a 14-year-old female Italian proband affected by ASD, carrying a novel ~ 270 kb interstitial microduplication, localized at the distal portion of the 4q13.1 region. The rearrangement was inherited from a mild symptomatic father and included a large part of the single EPHA5 gene, a receptor tyrosine kinase involved in the neural development, already indicated to be linked to ASD by previous Genome Wide Association Studies. This imbalance represents, to the best of our knowledge, the smallest duplication identified to date that only impacts the EPHA5 gene. We hypothesize that the duplication of this gene may alter EPHA5 expression and that this may impact the autistic phenotype of the patient.

EPHA5 mediates trastuzumab resistance in HER2-positive breast cancers through regulating cancer stem cell-like properties.

Trastuzumab is a successful, rationally designed therapy that provides significant clinical benefit for human epidermal growth factor receptor-2 (HER2)-positive breast cancer patients. However, about half of individuals with HER2-positive breast cancer do not respond to trastuzumab treatment because of various resistance mechanisms, including but not limited to: 1) shedding of the HER2 extracellular domain, 2) steric hindrance ( e.g., MUC4 and MUC1), 3) parallel pathway activation (this is the general mechanism cited in the quote above), 4) perturbation of downstream signaling events ( e.g., PTEN loss or PIK3CA mutation), and 5) immunologic mechanisms (such as FcR polymorphisms). EPHA5, a receptor tyrosine kinase, has been demonstrated to act as an anticancer agent in several cancer cell types. In this study, deletion of EPHA5 can significantly increase the resistance of HER2-positive breast cancer patients to trastuzumab. To investigate how EPHA5 deficiency induces trastuzumab resistance, clustered regularly interspaced short palindromic repeat technology was used to create EPHA5-deficient variants of breast cancer cells. EPHA5 deficiency effectively increases breast cancer stem cell (BCSC)-like properties, including NANOG, CD133+, E-cadherin expression, and the CD44+/CD24-/low phenotype, concomitantly enhancing mammosphere-forming ability. EPHA5 deficiency also caused significant aggrandized tumor malignancy in trastuzumab-sensitive xenografts, coinciding with the up-regulation of BCSC-related markers and intracellular Notch1 and PTEN/AKT signaling pathway activation. These findings highlight that EPHA5 is a potential prognostic marker for the activity of Notch1 and better sensitivity to trastuzumab in HER2-positive breast cancer. Moreover, patients with HER2-positive breast cancers expressing high Notch1 activation and low EPHA5 expression could be the best candidates for anti-Notch1 therapy.-Li, Y., Chu, J., Feng, W., Yang, M., Zhang, Y., Zhang, Y., Qin, Y., Xu, J., Li, J., Vasilatos, S. N., Fu, Z., Huang, Y., Yin, Y. EPHA5 mediates trastuzumab resistance in HER2-positive breast cancers through regulating cancer stem cell-like properties.

Selection of phage-displayed accessible recombinant targeted antibodies (SPARTA): methodology and applications.

We developed a potentially novel and robust antibody discovery methodology, termed selection of phage-displayed accessible recombinant targeted antibodies (SPARTA). This combines an in vitro screening step of a naive human antibody library against known tumor targets, with in vivo selections based on tumor-homing capabilities of a preenriched antibody pool. This unique approach overcomes several rate-limiting challenges to generate human antibodies amenable to rapid translation into medical applications. As a proof of concept, we evaluated SPARTA on 2 well-established tumor cell surface targets, EphA5 and GRP78. We evaluated antibodies that showed tumor-targeting selectivity as a representative panel of antibody-drug conjugates (ADCs) and were highly efficacious. Our results validate a discovery platform to identify and validate monoclonal antibodies with favorable tumor-targeting attributes. This approach may also extend to other diseases with known cell surface targets and affected tissues easily isolated for in vivo selection.