Inhibition of Tumor Growth and Angiogenesis by Soluble EphB4

Abstract

EphB receptors and their ephrinB ligands play a key role in the formation of a regular vascular system. Recent studies have also shown the involvement of Eph/ephrin interactions in malignant tumor progression and angiogenesis. We have generated soluble monomeric EphB4 (sEphB4)-expressing A375 melanoma cells to study the effect of dominant negatively acting sEphB4 on tumor growth and angiogenesis. Soluble EphB4-expressing A375 tumors grown subcutaneously in nude mice show dramatically reduced tumor growth compared to control tumors. The proliferative capacity of sEphB4-expressing cells in monolayer culture is not altered. Yet, sEphB4-expressing A375 cells cannot establish proper cell-cell contacts in three-dimensional spheroids. However, sEphB4 transfectants have reduced proliferation and apoptosis rates when grown in three-dimensional culture in vitro or in subcutaneous tumors in vivo. Analysis of the vascular phenotype of the tumors revealed a reduction of intratumoral microvessel density in sEphB4-expressing tumors. Corresponding to these mouse experiments, a matched pair analysis of EphB4 and ephrinB2 expression in human colon carcinomas revealed significantly upregulated levels of EphB4 expression compared to adjacent normal tissue. Taken together, the data identify dual effects of sEphB4 on the tumor and the vascular compartment that collectively inhibit tumor growth.

EphB Receptor-binding Peptides Identified by Phage Display Enable Design of an Antagonist with Ephrin-like Affinity

“…Remarkably, this value is 10,000 times lower than the IC 50 of ϳ150 M for the non-biotinylated TNYL (Fig. 6A) and comparable with the IC 50 of ϳ9 nM for the dimeric ephrin-B2 Fc (Fig. 6B).…”

“…3, left panels and data not shown). The concentration of biotinylated peptides necessary to inhibit binding of the dimeric ephrin-B2 AP by 50% (IC 50 ) is ϳ10 M for EWLS, 150 M for AHTF, 15 M for SNEW, 50 M for TNYL, and 200 M for DHNH (Fig. 3, left panels, and Table I).…”

“…Thus, the TNYL-RAW peptide could be developed to inhibit tumor progression and other forms of pathological angiogenesis that may similarly depend on EphB4 and ephrin-B2. It would also be interesting to test combinations of peptides binding to A-and B-class Eph receptors involved in a particular pathological process, such as EphB4 and EphA2 in cancer (5,8,49,50). Finally, the affinity of the EphB receptorbinding peptides may be greatly increased by dimerization through a flexible linker (36,42,43).…”

Structural and Biophysical Characterization of the EphB4·EphrinB2 Protein-Protein Interaction and Receptor Specificity

“…Several groups have more recently demonstrated that the full extracellular domain of EphB4 is indeed a viable therapeutic target. First, the soluble extracellular domain of EphB4 was described to block both forward and reverse signaling, resulting in an inhibition of tumor growth in vivo (28,29). Second, phage display studies have identified a peptide (TNYL-RAW) that antagonizes the EphB4⅐ephrinB2 interaction in the high nanomolar range (30).…”

The EphB4 Receptor-tyrosine Kinase Promotes the Migration of Melanoma Cells through Rho-mediated Actin Cytoskeleton Reorganization

“…EphA receptors have also been reported to be up-regulated during the development of breast and prostate carcinomas (9 -11). Besides the EphA/ephrin-A system, several studies have also reported the expression of B class Eph receptors and ephrins in different tumors, including melanomas, and suggested a functional relation between EphB/ ephrin-B expression and tumor progression (12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”