The catalytically defective receptor protein tyrosine kinase EphA10 promotes tumorigenesis in pancreatic cancer cells.

EphA10 (erythropoietin-producing hepatocellular carcinoma receptor A10) is a catalytically defective receptor protein tyrosine kinase in the ephrin receptor family. Although EphA10 is involved in the malignancy of some types of cancer, its role as an oncogene has not been extensively studied. Here, we investigated the influence of EphA10 on the tumorigenic potential of pancreatic cancer cells. Analysis of expression profiles from The Cancer Genome Atlas confirmed that EphA10 was elevated and higher in tumor tissues than in normal tissues in some cancer types, including pancreatic cancer. EphA10 silencing reduced the proliferation, migration, and adhesion of MIA PaCa-2 and AsPC-1 pancreatic cancer cells. These effects were reversed by overexpression of EphA10 in MIA PaCa-2 cells. Importantly, overexpression and silencing of EphA10 respectively increased and decreased the weight, volume, and number of Ki-67-positive proliferating cells in MIA PaCa-2 xenograft tumors. Further, EphA10 expression was positively correlated with invasion and gelatin degradation in MIA PaCa-2 cells. Moreover, overexpression of EphA10 enhanced the expression and secretion of MMP-9 in MIA PaCa-2 cells and increased the expression of MMP-9 and the vascular density in xenograft tumors. Finally, expression of EphA10 increased the phosphorylation of ERK, JNK, AKT, FAK, and NF-κB, which are important for cell proliferation, survival, adhesion, migration, and invasion. Therefore, we suggest that EphA10 plays a pivotal role in the tumorigenesis of pancreatic epithelial cells and is a novel therapeutic target for pancreatic cancer.

Generation and characterization of a bispecific diabody targeting both EPH receptor A10 and CD3.

The EPH receptor A10 (EphA10) is up-regulated in breast cancer but is not normally expressed in healthy tissue, thus it has been suggested that EphA10 may be a useful target for cancer therapy. This study reports a diabody, an antibody derivative binding two different target molecules, EphA10 expressed in tumor cells and CD3 expressed in T cells, which showed T cell dependent-cytotoxicity. The diabody, which has His-tagged and FLAG-tagged chains, was expressed in Escherichia coli and purified in both heterodimer (Db-1) and homodimer (Db-2) formulations by liquid chromatography. Flow cytometry analysis using EphA10-expressing cells showed that binding activity of heterodimers was stronger than that of homodimers. Addition of diabodies to PBMC cultures resulted in T-cell mediated redirected lysis, and the bioactivity was consistent with the stronger binding activity of heterodimeric diabody formulations. Our results indicate that diabodies recognizing both EphA10 and CD3 could have a range of potential applications in cancer therapy, such as breast cancers that express the EPH receptor A10, especially triple negative breast cancer.

EphA10 and EphB3 are prognostic markers in gallbladder squamous cell/adenosquamous carcinomas and adenocarcinomas.

AIMS: Squamous cell/adenosquamous carcinomas (SC/ASC) are rare subtypes of gallbladder cancers (GBCs). Clinical characteristics of SC/ASC have not been well documented, and no biological markers of GBC carcinogenesis, progression and prognosis are available. METHODS: We detected EphA10 and EphB3 expression in 69 SC/ASCs and 146 adenocarcinomas (ACs) with EnVision immunohistochemistry. RESULTS: The percentage of cases with a patient age of > 45 years, lymph node metastasis and invasion was significantly higher in the SCs/ASCs compared with the ACs (P < 0.05). The positive expression of EphA10 and negative expression of EphB3 were significantly higher in the cases of SC/ASC and AC than in chronic cholecystitis (P < 0.01). The positive expressions of EphA10 and negative expression of EphB3 were significantly higher in the cases of poorly differentiation, large tumor size, high TNM stage, lymph node metastasis, invasion and no resection (only biopsy) of SC/ASC and AC. The negative correlation was found between EphA10 and EphB3 expression in SC/ASC and AC (P < 0.01). The univariate Kaplan-Meier analysis showed that positive EphA10 and negative EphB3, differentiation, tumor size, TNM stage, lymph node metastasis, invasion and surgical curability, is closely associated with a decreased overall survival in SC/ASC and AC patients (P < 0.05 or P < 0.01). The multivariate Cox regression analysis identified that positive EphA10 and negative EphB3 expression are independent factors for a poor-prognosis in SC/ASC and AC patients. The AUC for EphA10 and EphB3 showed might have role for carcinogenesis and progression of SC/ASC and AC. CONCLUSIONS: The present study indicates that positive EphA10 and negative EphB3 expression are closely associated with the pathogenesis, clinical, pathological and biological behaviors, and poor prognosis in gallbladder cancer.

Isoform expression patterns of EPHA10 protein mediate breast cancer progression by regulating the E-Cadherin and ß-catenin complex.

Overexpression of EPHA10 protein was reported in concomitance with clinical severity of breast cancer. In this study, we annotate overexpression of EPHA10 protein with changes of isoform expression as EphA10s (EPHA10 isoform 2) and EphA10 (EPHA10 isoform 3). In the process of malignant transformation, secretory protein EphA10s is in low expression, and pseudo-kinase EphA10 is overexpressed and cytoplasmically enriched. Down-regulated EphA10s blunts stabilization of membrane-associate ß-catenin via the interaction with ephrin A5. Cytoplasmic EphA10 maintains phosphorylation of E-cadherin. Restoring isoform expression pattern by up-regulated EphA10s and down-regulated cytoplasmic EphA10 inhibits cell invasion and lymph node metastasis by strengthening the stability of the complex of E-cadherin and ß-catenin in membrane. Taken together, we defined the novel interaction via expression patterns of EphA10s and EphA10 that promote malignant transformation of breast cancer, and demonstrated the potential benefit in clinical usage.

EPHA7 and EPHA10 Physically Interact and Differentially Co-localize in Normal Breast and Breast Carcinoma Cell Lines, and the Co-localization Pattern Is Altered in EPHB6-expressing MDA-MB-231 Cells.

Erythropoietin-producing hepatocellular carcinoma cell (EPH) receptors comprise the most abundant receptor tyrosine kinase family characterized to date in mammals including humans. These proteins are involved in axon guidance, tissue organization, vascular development and the intricate process of various diseases including cancer. These diverse functions of EPH receptors are attributed, in part, to their abilities for heterodimerization. While the interacting partners of kinase-deficient EPHB6 receptor have been characterized, the interaction of the kinase-dead EPHA10 with any other receptor has not been identified. By using co-immunoprecipitation, we demonstrated physical interaction between kinase-deficient EPHA10 with kinase-sufficient EPHA7 receptor. Immunocytochemical analyses have revealed that these two receptors co-localize on the cell surface, and soluble portions of the receptors exist as a complex in the cytoplasm as well as the nuclei. While EPHA7 and EPHA10 co-localize similarly on the membrane in MCF10A and MCF7 cells, they were differentially co-localized in MDA-MB-231 cells stably transfected with empty pcDNA vector (MDA-MB-231-PC) or an expression construct of EPHB6 (MDA-MB-231-B6). The full-length isoforms of these receptors were co-localized on the cell surface, and the soluble forms were present as a complex in the cytoplasm as well as the nucleus in MDA-MB-231-PC cells. MDA-MB-231-B6 cells, on the other hand, were distinguished by the absence of any signal in the nuclei. Our results represent the first demonstration of physical interaction between EPHA10 and EPHA7 and their cellular co-localization. Furthermore, these observations also suggest gene-regulatory functions of the complex of the soluble forms of these receptors in breast carcinoma cells of differential invasiveness.

Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA.

Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol-Schiff base-polyethylene glycol (Chol-SIB-PEG)-modified cationic liposome-siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol-SIB-PEG was successfully synthesized and confirmed with FTIR and (1)H-NMR. An Eph-PEG-SIB-Chol-modified liposome-siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application.