Overexpression of the receptor tyrosine kinase EphB4 is common in epithelial cancers and linked to tumor progression by promoting angiogenesis, increasing survival and facilitating invasion and migration. However, other studies have reported loss of EphB4 suggesting a tumor suppressor function in some cancers. These opposing roles may be regulated by (i) the presence of the primary ligand ephrin-B2 that regulates pathways involved in tumor suppression or (ii) the absence of ephrin-B2 that allows EphB4 signaling via ligand-independent pathways that contribute to tumor promotion. To explore this theory, EphB4 was overexpressed in the prostate cancer cell line 22Rv1 and the mammary epithelial cell line MCF-10A. Overexpressed EphB4 localized to lipid-rich regions of the plasma membrane and confirmed to be ligand-responsive as demonstrated by increased phosphorylation of ERK1/2 and internalization. EphB4 overexpressing cells demonstrated enhanced anchorageindependent growth, migration and invasion, all characteristics associated with an aggressive phenotype, and therefore supporting the hypothesis that overexpressed EphB4 facilitates tumor promotion. Importantly, these effects were reversed in the presence of ephrin-B2 which led to a reduction in EphB4 protein levels, demonstrating that ligand-dependent signaling is tumor suppressive. Furthermore, extended ligand stimulation caused a significant decrease in proliferation that correlated with a rise in caspase-3/7 and -8 activities. Together, these results demonstrate that overexpression of EphB4 confers a transformed phenotype in the case of MCF-10A cells and an increased metastatic phenotype in the case of 22Rv1 cancer cells and that both phenotypes can be restrained by stimulation with ephrin-B2, in part by reducing EphB4 levels.EphB4 is a member of the largest subfamily of receptor tyrosine kinases (RTKs) and is commonly overexpressed in cancer cells from several different tissue origins including prostate, 1,2 breast, 3 colon, 4 head and neck 5 and ovary. 6,7 Various studies exploring the role of EphB4 overexpressed in cancer cells have suggested that EphB4 can promote tumor development by stimulating angiogenesis 8,9 increasing cancer cell survival 3,6,10 and facilitating invasion and migration.
11,12Overexpression of EphB4 in the mammary epithelium of MMTV-neuT transgenic mice accelerated the onset of tumor formation and increased metastasis to the lungs. 13 In xenograft models of breast and prostate cancer, knockdown of EphB4 significantly inhibits tumor growth indicating that EphB4 directly contributes to tumor progression.2,3 Similarly, targeting EphB4 using a monoclonal antibody also significantly reduced tumor growth in vivo.14 Collectively, these studies show that in the absence of ligand, EphB4 receptors can contribute to tumor progression.Paradoxically, EphB4 has also been implicated as a tumor suppressor. [15][16][17] This apparent contradiction may be reconciled, at least in part, if distinct EphB4 signaling pathways are differentially regulated by li...