Expression Pattern and Prognostic Value of EPHA/EFNA in Breast Cancer by Bioinformatics Analysis: Revealing Its Importance in Chemotherapy.

The activities of the ephrin family in breast cancer (BrCa) are complex. Family A receptors (EPHA) and ligands (EFNA) can act as oncogenes or tumor suppressors and are implicated in chemoresistance. Here, we examined the expression pattern and prognostic value of the EPHA/EFNA family in patients with breast cancer, including patients with different subtypes or different chemotherapy cohorts. In the UALCAN database, the mRNA expression of EPHA1, EPHA10, EFNA1, EFNA3, and EFNA4 was significantly higher, whereas that of EPHA2, EPHA4, EPHA5, and EFNA5 was significantly lower in breast cancer tissues than in paracancerous tissues. The transcriptional levels of EPHA/EFNA family members were correlated with intrinsic subclasses of breast cancer. The relationship between EPHA/EFNA and the clinicopathological parameters of BrCa was analyzed using bc-GenExMiner V4.5. EPHA1, EPHA2, EPHA4, EPHA7, EFNA3, EFNA4, and EFNA5 were upregulated in estrogen receptor- (ER-) and progesterone receptor- (PR-) negative tumors, whereas EPHA3, EPHA6, and EFNA1 were upregulated in ER- and PR-positive tumors. EPHA1, EPHA2, EFNA3, and EFNA4 mRNA expression was significantly higher in human epidermal growth factor receptor 2- (HER2-) positive tumors than in HER2-negative tumors. Triple-negative status was positively correlated with EPHA1, EPHA2, EPHA4, EPHA7, EFNA3, EFNA4, and EFNA5 and negatively correlated with EPHA3 and EPHA10 mRNA expression. Genetic alterations of EPHA/EFNA in breast cancer varied from 1.1% to 10% for individual genes, as determined by the cBioPortal database. The Kaplan-Meier plotter indicated that high EphA7 mRNA expression was associated with poor overall survival (OS) and recurrence-free survival (RFS), especially in the HER2 and luminal A subtypes. EFNA4 was predicted to have poor OS and RFS in breast cancers, especially in luminal B, basal-like subtype, and patients treated with adjuvant chemotherapy. High EPHA3 expression was significantly associated with better OS and RFS, especially in the luminal A subtype, but with poor RFS in BrCa patients receiving chemotherapy. Our findings systematically elucidate the expression pattern and prognostic value of the EPHA/EFNA family in BrCa, which might provide potential prognostic factors and novel targets in BrCa patients, including those with different subtypes or treated with chemotherapy.

Synthesis, radiolabelling and initial biological characterisation of 18F-labelled xanthine derivatives for PET imaging of Eph receptors.

Eph receptor tyrosine kinases, particularly EphA2 and EphB4, represent promising candidates for molecular imaging due to their essential role in cancer progression and therapy resistance. Xanthine derivatives were identified to be potent Eph receptor inhibitors with IC50 values in the low nanomolar range (1-40 nm). These compounds occupy the hydrophobic pocket of the ATP-binding site in the kinase domain. Based on lead compound 1, we designed two fluorine-18-labelled receptor tyrosine kinase inhibitors ([18F]2/3) as potential tracers for positron emission tomography (PET). Docking into the ATP-binding site allowed us to find the best position for radiolabelling. The replacement of the methyl group at the uracil residue ([18F]3) rather than the methyl group of the phenoxy moiety ([18F]2) by a fluoropropyl group was predicted to preserve the affinity of the lead compound 1. Herein, we point out a synthesis route to [18F]2 and [18F]3 and the respective tosylate precursors as well as a labelling procedure to insert fluorine-18. After radiolabelling, both radiotracers were obtained in approximately 5% radiochemical yield with high radiochemical purity (>98%) and a molar activity of >10 GBq µmol-1. In line with the docking studies, first cell experiments revealed specific, time-dependent binding and uptake of [18F]3 to EphA2 and EphB4-overexpressing A375 human melanoma cells, whereas [18F]2 did not accumulate at these cells. Since both tracers [18F]3 and [18F]2 are stable in rat blood, the novel radiotracers might be suitable for in vivo molecular imaging of Eph receptors with PET.

Expression of RNA-binding protein LIN28 in classic gastric hepatoid carcinomas, gastric fetal type gastrointestinal adenocarcinomas, and hepatocellular carcinomas: An immunohistochemical study with comparison to SALL4, alpha-fetoprotein, glypican-3, and Hep Par1.

INTRODUCTION: Gastric hepatoid carcinomas (GHCs) include type I (classic) and type II (fetal type gastrointestinal adenocarcinoma). The classic type shows overlapping morphologic features with those of hepatocellular carcinoma (HCC). The aim of this study is to investigate expression of LIN28 in GHCs and explore its utility to distinguish classic GHC from HCC. METHODS: We investigated immunohistochemical expression of LIN28 in 93 primary GHCs (47 type I, 46 type II) and 60 HCCs with comparison to SALL4, AFP, glypican-3, Hep Par1, p-CEA and CK7. We also stained LIN28 and SALL4 in 52 conventional gastric adenocarcinomas to assess their specificity in gastric carcinomas. RESULTS: Classic GHCs and fetal type gastrointestinal adenocarcinomas showed positive LIN28 in 21/47 (45%) and 10/46 (22%), SALL4 in 41/47 (87%) and 36/46 (78%), AFP in 30/46 (65%) and 33/46 (72%), glypican-3 in 31/41 (76%) and 24/38 (63%), Hep Par1 in 27/41 (66%) and 28/37 (76%), and CK7 in 15/40 (38%) and 25/38 (66%), respectively. p-CEA staining was seen in 19/44 (43%) classic GHCs. Among HCCs, LIN28, SALL4, AFP, glypican-3, Hep Par1, p-CEA and CK7 was seen in 1/60 (2%), 0/60 (0%), 6/30 (20%), 23/30 (77%), 29/30 (97%), 28/30 (93%) and 21/30 (70%) cases, respectively. LIN28 and SALL4 staining was seen in 2/52 (4%) and 14/52 (27%) gastric conventional adenocarcinomas, respectively. The sensitivity and specificity of distinguishing classic GHCs from HCCs was 45% and 98% for LIN28, 87% and 100% for SALL4, 65% and 80% for AFP, 76% and 30% for glypican-3, 66% and 3% for Hep Par1, 43% and 7% for p-CEA, and 38% and 30% for CK7, respectively. Combining LIN28 and SALL4 increased the sensitivity to 96% with 98% specificity to distinguish classic GHCs from HCCs. CONCLUSIONS: LIN28 is a very specific marker (98% specificity) for distinguishing classic GHCs from HCCs though it is not as sensitive as SALL4. AFP, glypican-3, Hep Par1 and p-CEA are not useful in distinguishing classic GHCs from HCCs. Combining LIN28 and SALL4 increased the sensitivity to distinguish classic PHCs from HCCs.

Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland.

Sox2-expressing stem/progenitor cells in the anterior lobe of the pituitary gland form two types of micro-environments (niches): the marginal cell layer and dense cell clusters in the parenchyma. In relation to the mechanism of regulation of niches, juxtacrine signaling via ephrin and its receptor Eph is known to play important roles in various niches. The ephrin and Eph families are divided into two subclasses to create ephrin/Eph signaling in co-operation with confined partners. Recently, we reported that ephrin-B2 localizes specifically to both pituitary niches. However, the Ephs interacting with ephrin-B2 in these pituitary niches have not yet been identified. Therefore, the present study aims to identify the Ephs interacting with ephrin-B2 and the cells that produce them in the rat pituitary gland. In situ hybridization and immunohistochemistry demonstrated cell type-specific localization of candidate interacting partners for ephrin-B2, including EphA4 in cells located in the posterior lobe, EphB1 in gonadotropes, EphB2 in corticotropes, EphB3 in stem/progenitor cells and EphB4 in endothelial cells in the adult pituitary gland. In particular, double-immunohistochemistry showed cis-interactions between EphB3 and ephrin-B2 in the apical cell membranes of stem/progenitor cell niches throughout life and trans-interactions between EphB2 produced by corticotropes and ephrin-B2 located in the basolateral cell membranes of stem/progenitor cells in the early postnatal pituitary gland. These data indicate that ephrin-B2 plays a role in pituitary stem/progenitor cell niches by selective interaction with EphB3 in cis and EphB2 in trans.

High expression of EphB6 protein in tongue squamous cell carcinoma is associated with a poor outcome.

EphB6 is a member in the receptor tyrosine kinase Eph family in that its kinase domain contains several alterations in conserved amino acids and is catalytically inactive. Although EphB6 is expressed both in a variety of embryonic and adult tissues, biological functions of this receptor are largely unknown. In this study, we examined the expression of EphB6 protein in 54 of tissue specimens of tongue squamous cell carcinoma by using a specific polyclonal anti-EphB6 antibody. The relationship between expression of EphB6 and clinical pathologic parameters was analyzed. The expression level of EphB6 in carcinoma cells from 34 out of 54 (63%) specimens was no alterative compared with normal squamous cells in same patient. The level of EphB6 protein staining was increased in carcinoma cells in 20 out of 54 (37%) specimens compared with normal squamous cells in same patient. The high-expression of EphB6 was significantly associated with age (P=0.021), tumor TNM stage (P=0.026) and lymph node metastasis (P=0.046). Patients with high expressed EphB6 protein had a high mortality (P=0.057). No significant relationship between expression of EphB6 and sex, tumor grade, HPV infection, relapse and smoke was found. We showed that patients with high expression of EphB6 had a significantly poor overall survival (OS) compared to patients with negative or weak expression (P=0.042). Our results indicated that EphB6 protein may be used as a new marker for prognosis for tongue squamous cell carcinoma.

Ephrin A/EphA controls the rostral turning polarity of a lateral commissural tract in chick hindbrain.

Most post-crossing commissural axons turn into longitudinal paths to make synaptic connections with their targets. Mechanisms that control their rostrocaudal turning polarity are still poorly understood. We used the hindbrain as a model system to investigate the rostral turning of a laterally located commissural tract, identified as the caudal group of contralateral cerebellar-projecting second-order vestibular neurons (cC-VC). We found that the caudal hindbrain possessed a graded non-permissive/repulsive activity for growing cC-VC axons. This non-permissiveness/repulsion was in part mediated by glycosyl-phosphatidylinositol (GPI)-anchored ephrin A. We further demonstrated that ephrin A2 was distributed in a caudal-high/rostral-low gradient in the caudolateral hindbrain and cC-VC axons expressed EphA receptors. Finally, perturbing ephrin A/EphA signalling both in vitro and in vivo led to rostrocaudal pathfinding errors of post-crossing cC-VC axons. These results suggest that ephrin A/EphA interactions play a key role in regulating the polarity of post-crossing cC-VC axons as they turn into the longitudinal axis.

Characterization of a novel Eph receptor tyrosine kinase, EphA10, expressed in testis.

In mammals, 14 members of the Eph receptor tyrosine kinase family have been described so far. Here we present a not yet described member of this family denoted EphA10. We report the identification of three putative EphA10 isoforms: one soluble and two transmembrane isoforms. One of the latter isoforms lacked the sterile alpha motif commonly found in Eph receptors. The gene encoding EphA10 is located on chromosome 1p34 and expression studies show that EphA10 mRNA is mainly expressed in testis. Binding studies to ephrin ligands suggests that this receptor belongs to the EphA subclass of Eph receptors binding mainly to ephrin-A ligands.

Mediolateral compartmentalization of the cerebellum is determined on the "birth date" of Purkinje cells.

The adult cerebellum is functionally compartmentalized into clusters along the mediolateral axis (M-L clusters), and a variety of molecular makers are expressed in specific subsets of M-L clusters. These M-L clusters appear to be the basic structure in which cerebellar functions are performed, but the mechanisms by which cerebellar mediolateral compartmentalization is established are still unclear. To address these questions, we examined the development of M-L clusters using replication-defective adenoviral vectors. The adenoviral vectors effectively introduced foreign genes into the neuronal progenitor cells of the cerebellum in a birth date-specific manner, allowing us to observe the native behavior of each cohort of birth date-related progenitor cells. When the adenoviral vectors were injected into the midbrain ventricle of mouse embryos on embryonic days 10.5 (E10.5), E11.5, and E12.5, the virally infected cerebellar progenitor cells developed into Purkinje cells. Notably, the Purkinje cells that shared the same birth date formed specific subsets of M-L clusters in the cerebellum. Each subset of M-L clusters displayed nested and, in part, mutually complementary patterns, and these patterns were unchanged from the late embryonic stage to adulthood, suggesting that Purkinje cell progenitors are fated to form specific subsets of M-L clusters after their birth between E10.5 and E12.5. This study represents the first such direct observation of Purkinje cell development. Moreover, we also show that there is a correlation between the M-L clusters established by the birth date-related Purkinje cells and the domains of engrailed-2, Wnt-7B, L7/pcp2, and EphA4 receptor tyrosine kinase expression.