Targeting EphA2 Research Articles

EphA2 blockade enhances the anti-endothelial effect of radiation and inhibits irradiated tumor cell-induced migration of endothelial cells.

EphA2 tyrosine kinase plays an important role in tumor angiogenesis, but whether targeting this pathway can affect response to ionizing radiation (IR) remains unknown. We investigated, using a soluble EphA2-Fc chimera, whether EphA2 inhibition could sensitize A549 and MCF-7 tumor cells, as well as human umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (HDMEC), to IR. EphA2-Fc resulted in a greater response of endothelial cells (EC) to IR than either treatment alone. EphA2-Fc significantly increased apoptosis and decreased clonogenic survival, tube formation and migration in irradiated EC after stimulation with vascular endothelial growth factor (VEGF), without an affecting their proliferation. No difference in proliferation or survival was found in A549 and MCF-7 tumor cells. In a co-culture model, EphA2-Fc inhibited an irradiated A549 cell-induced increase in EC migration. VEGF supplementation, as well as condiotioned medium from irradiated A549 cells, phosphorylated EphA2 in EC. The latter was abrogated by EphA2-Fc. EC were most sensitive to a combination of EphA2 inhibition and radiotherapy. The induction of paracrine growth factors and activation of EphA2 in EC suggest a protective mechanism that tumors probably use to attenuate IR-induced antivascular effects. Our data justify further investigation to explore targeting EphA2 in tumor radiosensitivity in vivo.

Abstract 826: Expression levels of circulating soluble-EphA2 receptor in cancer patients

Abstract EphA2 is a receptor tyrosine kinase that critically controls many aspects of cell behavior. It is expressed in a manner that is consistent with the regulation of cell growth, migration and invasion. Analysis by IHC has shown that elevated expression of EphA2 exhibits predictive value in lung cancer survival and recurrence. Importantly, current data suggests that targeting EphA2 could be beneficial and is currently being tested in an ovarian cancer setting. We developed a prototype ELISA for circulating EphA2 receptor in order to investigate expression levels of the soluble form of EphA2 in certain cancer types. We have developed a prototype ELISA which measures circulating human EphA2 receptor. 96 well Nunc plates were coated with an affinity-purified goat polyclonal antibody specific for the extracellular domain (ECD) of human EphA2. Human serum and plasma samples were diluted 5-fold in a sample diluent with blocking reagents. The standard was a mammalian cell-derived form of the ECD for the human EphA2 receptor. Standards and samples were incubated on the plates for 2 Hours, 37C. The detector antibody was a biotinylated affinity-purified goat polyclonal antibody and was added to the plates for 1 Hour, 37C. Plates were washed and a streptavidin-HRP conjugate was added to the plates for 30 minutes, 22C. A final wash was performed and a TMB substrate was added to the plates for color development. The reaction was stopped with sulfuric acid and plates were read at an absorbance of 450nm. Data generated to date using this prototype ELISA indicates that over-expression of soluble-EphA2 occurs in many cancers versus normal samples tested. The 95% CI of total normal plasma and serum was 672_1306 and 894_1429pg/mL, respectively. Capture Inhibition studies revealed specificity of the assay in human samples (100% inhibition). Parallelism studies revealed acceptable performance in serially diluted samples. Significant over-expression of soluble-EphA2 was observed in benign lung cancers versus normals (100%, 10/10) while staged lung cancers exhibited significantly lower levels of circulating EphA2 (p<0.001) compared to benign (96%, 148/154). Elevations of circulating EphA2 were found in glioblastoma (73%, 22/30), ovarian (48%, 28/59), pancreatic (46%, 19/41), and late stage breast (44%, 15/34) cancer patients. Immunoprecipitation experiments are ongoing to visualize soluble EphA2 by western blot, with initial data showing a specific band at ∼60kD. This study describes the discovery of circulating EphA2 receptor in human samples using a prototype ELISA. Levels of soluble-EphA2 are over-expressed in glioblastoma, ovarian, pancreatic, and late stage breast cancer samples. Decreased expression of the circulating form of EphA2 is observed in staged lung cancer versus benign. Measuring the levels of the soluble-EphA2 in circulation may have potential utility for cancer patients who may benefit from EphA2 targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 826.

Abstract B201: Selective peptides for targeting EphA2 in cancer cells

Abstract EphA2 receptor tyrosine kinase is highly overexpressed in tumors and is rapidly emerging as a promising new therapeutic target in cancer. Agents that target EphA2 have been shown to deliver cytotoxins/drugs selectively to cells expressing EphA2 and to decrease tumor growth and metastasis in animal models. We have recently isolated two peptides (YSA and SWL) by phage display that selectively target EphA2, and show the ability to deliver quantum dots and phage particles to cancer cells overexpressing EphA2. Furthermore, we elucidated the structure-activity relationship of one of the EphA2 agonistic peptides (YSA) by alanine scanning mutagenesis, characterized the SWL peptide and a series of related peptides, all of which retain their selectivity for EphA2. The selectivity of the peptides was determined by measuring the ability of the Eph receptors to bind immobilized peptide, and their potency was measured using an ELISA assay that monitors the inhibition of ephrin-A1 binding to EphA2. Our analysis revealed that only 4 of the 12 amino acids are critical for binding of YSA, and a 5-mer peptide derived from YSA retains selectivity for EphA2. All these peptides are reasonably stable in conditioned cell culture medium and rat liver microsomal preparations. In addition, at least some of these peptides show the ability to activate the EphA2 receptor inducing its phosphorylation and inhibit serum-induced phosphorylation of Akt and Erk1/Erk2 MAP kinases, which are key mediators in major oncogenic pathways. Therefore, our results indicate that in addition to selectively targeting cancer cells, these peptides can also lead to inhibition of oncogenic pathways. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B201.

RNA interference targeting EphA2 inhibits proliferation, induces apoptosis, and cooperates with cytotoxic drugs in human glioma cells

Background Overexpression of EphA2 was detected in low- and high-grade glioma. To examine the role of EphA2 in human glioma cells, we studied its effects on proliferation and apoptosis using gene silencing through RNA interference. Methods One siRNA targeting EphA2 gene was synthesized in vitro and was transfected into the glioma U251n cells. Expression of EphA2 proteins was detected by Western blots and immunofluorescence. Cell apoptosis and mitochondrial membrane potential were analyzed by flow cytometry and annexin-V/fluorescein isothiocyanate/propidium iodide, respectively. Caspase-3 activity was measured by a spectrofluorometer. MTT assay was used to examine changes in cell proliferation. Results After treatment with sequence-specific siRNA targeting EphA2, the protein level of the transfected group decreased significantly. As compared to non-siRNA transfected cells, the transfected group showed lower proliferation, higher apoptosis, and loss of mitochondrial membrane potential. Caspase-3 activity increased in cells treated with siRNA and downregulated when treated with caspase-3 inhibitor. And the effects were clearly additive when siRNA transfected cells treated with the anticancer agents. Conclusions The results suggest that EphA2-siRNA inhibit U251n cell proliferation and induce their apoptosis. It is possible that EphA2 via mitochondrial and caspase-3 inhibits U251n cell apoptosis. And EphA2-siRNA transfection enhances U251n cells' sensitivity to chemotherapy. EphA2 may be an effective therapeutic target in patients with glioma. Silencing the receptor EphA2 gene is a novel approach for the containment of growth and migration of tumor in patients with malignant glioma.

EphA2 as a target for ovarian cancer therapy

EphA2 is a receptor tyrosine kinase that is overexpressed by many human cancers, and is often associated with poor prognostic features. It is involved in many processes crucial to malignant progression, such as migration, invasion, metastasis, proliferation, survival and angiogenesis. Inducing EphA2 downregulation by any one of several mechanisms (antibody-mediated inhibition of signalling, antibody-mediated downregulation of total EphA2 expression and siRNA-mediated inhibition of expression) has been shown to decrease tumour growth, prolong survival and inhibit angiogenesis in multiple preclinical models of ovarian, breast and pancreatic cancer. Targeting EphA2 is especially attractive in ovarian cancer, in which overexpression is present in > 75% of cases. This disease is highly responsive to chemotherapy, and EphA2 inhibition is especially effective in combination with taxanes. This demonstrated efficacy, along with the low expression of EphA2 by normal adult tissues and lack of demonstrable toxicities in preclinical models, suggest that long-term treatment with EphA2-targeting agents is an attractive approach for ovarian cancer therapy.

EphA2 as a Glioma-Associated Antigen: A Novel Target for Glioma Vaccines

EphA2 is a receptor tyrosine kinase, is frequently overexpressed in a wide array of advanced cancers. We demonstrate in the current study that the EphA2 protein is restrictedly expressed in primary glioblastoma multiforme, anaplastic astrocytoma tissues in comparison to normal brain tissues. To evaluate the possibility of targeting EphA2 in glioma vaccine strategies, we stimulated human leukocyte antigen (HLA) A2+ peripheral blood mononuclear cells (PBMCs) obtained from healthy donors, glioma patients with autologous dendritic cells (DCs) loaded with synthetic EphA2$$3_$91 peptide (TLADFDPRV), which has previously been reported to induce interferon-δ in HLAA2+ PBMCs. Stimulated PBMCs demonstrated antigenspecific cytotoxic T lymphocyte (CTL) responses as detected by specific lysis of T2 cells loaded with the EphA2883 peptide as well as HLA-A2+ glioma cells, SNB19, U251, that express EphA2. Furthermore, in vivo immunization of HLA-A2 transgenic HHD mice with the EphA2883-891 peptide resulted in the development of an epitope-specific CTL response in splenocytes, despite the fact that EphA2883-891 is an autoantigen in these mice. Taken together, these data suggest that EphA2883-891 may be an attractive antigen epitope for molecularly targeted glioma vaccines.