EphrinB2-EphB4 Signaling in Neurooncological Disease

Abstract

EphrinB2-EphB4 signaling is critical during embryogenesis for cardiovascular formation and neuronal guidance. Intriguingly, critical expression patterns have been discovered in cancer pathologies over the last two decades. Multiple connections to tumor migration, growth, angiogenesis, apoptosis, and metastasis have been identified in vitro and in vivo. However, the molecular signaling pathways are manifold and signaling of the EphB4 receptor or the ephrinB2 ligand is cancer type specific. Here we explore the impact of these signaling pathways in neurooncological disease, including glioma, brain metastasis, and spinal bone metastasis. We identify potential downstream pathways that mediate cancer suppression or progression and seek to understand it´s role in antiangiogenic therapy resistance in glioma. Despite the Janus-faced functions of ephrinB2-EphB4 signaling in cancer Eph signaling remains a promising clinical target.

Keywords: EphB4; bone; bone metastasis; brain metastasis; ephrinB2; glioblastoma; glioma; metastasis; neurooncology; spinal metastasis.

Figure 1

Molecular compartmentalization of ephrinB2 and EphB4. EphB4 forward signaling involves dimerization, Src homology 2 (SH2), sterile alpha motif (SAM), and PSD95, DLG, ZO1 (PDZ) autophosphorylation (orange P). Kinase independent signaling involves guanine nucleotide exchange factor (GEF) binding followed by autophosphorylation. Phosphotyrosine phosphatases are known to reduce EphB4 activity. EphrinB2 reverse signaling involves Src-mediated phosphorylation (orange P) in combination with SH2 and PDZ protein assembly. Serine residue phosphorylation was discovered in both directions (yellow P). Small molecule inhibitors (SMI) for the EphB4 kinase domain are available, the extracellular domain of ephrinB2 and EphB4 have been fused to Fragment crystallizable (ephrinB2-FC & EphB4-FC) regions to generate antibody therapies, and small interfering peptides (TNYL) are available that bind to the ligand pocket and block signaling.

Figure 2

Tumor promoting and suppressing pathways identified after ephrinB2–EphB4 binding. (a) Tumor promoting pathways. EphrinB reverse signaling promotes invasiveness, epithelial–mesenchymal transition (EMT) and migration through STAT3, Src, RAC1 and MMP8 [47,48]. In endothelial cells, ephrinB2 reverse signaling regulates VEGFR2 internalization critical for angiogenesis [49]. Non phosphorylated ephrinB will block tight junction formation via PAR6 [50]. EphB forward signaling in tumors promotes survival via Myc and proliferation via Cyclin-D1 through multiple pathways [51,52,53]. Migration can be enhanced through the Akt/PI3K NFkB pathway [31]. Other migratory pathways of EphBs include phosphorylation of RRAS and activation of RHOA [54,55]. (b) Tumor suppressing pathways. Endothelial ephrinB disturbs focal adhesion via GRB4 during reverse signaling [56,57]. Tight junctions are formed after PAR6 binding and trimerization with atypical PKC (aPKC) and CDC42 [50]. Constant regulatory feedback is imposed by Src [56]. Cytokine inhibition was found trough modulation of the CXCR4R pathway [56]. Forward singling in tumors involves the upregulation of the PI3K subunit p110 [58]. Abl activation was found to inhibit RAP1 and RAC1 [20,59,60]. RRAS and HRAS are blocked after p120RASGAP activation [61]. RRAS and HRAS are also reduced by direct phosphorylation [62]. Internal calcium can be regulated by S100G which activates SOX17 and reduces proliferation [46]. Different MMPs are significantly upregulated when EphB4 is depleted [46]. Pathways identified in other celltypes but potentially active in endothelial and/or glioma cells are indicated with a dashed line.

Figure 3

Effects of antiangiogenic therapy in glioma. Antiangiogenic treatment leads to elimination of small blood vessels followed by an increase in hypoxia and a larger necrotic tumor core. Consequently, the tumor will undergo therapy evasion and increase resistance. Phenotypically, this is manifested by increased tumor cell invasion and vascular maturation mediated by, but not limited to ephrinB2–EphB4 signaling.

Figure 4

EphB4 resistance mechanisms towards antiangiogenic treatment in glioma. EphrinB2 expression in tumors is reduced after antiangiogenic treatment, arbitrated by means of elevated levels of hypoxia-inducible factor (HIF-1α) and zinc finger E-box-binding homeobox 2 (ZEB2) [107]. Among other effects, this pathway enhances tumor invasiveness. In forward signaling, EphB4 controls the activation of the EGF receptor via the Wnt/beta-catenin signaling pathway. The EGF receptor phosphorylates phosphoinositide phospholipase C-gamma-1 (PLC-γ1), which was found to drive gene expression changes in Sunitinib resistant glioma [105]. PLC-γ1 phosphorylation increases tumor invasiveness in other tumor entities. In the endothe-lium, antiangiogenic therapy blocks VEGFR–EphrinB2 dimerization and Delta-Like Canonical Notch Ligand 4 (DLL4) activa-tion [106]. EphB4 is progressively expressed in Bevacizumab resistant blood vessels and regulates DLL4/Notch proportions via cerebral cavernous malformation 3 (CCM3) [103,106]. Endothelial EphB4 overexpression forms large, treatment insensitive vessels expressing increased amounts of TIE2 and ANG1 and decreased amounts of ANG2 [94]. Additionally, it protects cells from apoptosis possibly through the PI3K-AKT-ERK pathway [106].

Figure 5

Effects of ephrin-B2–EphB4 interaction on extravasation of circulating melanoma cells in the spine. (a) Physi-ological interaction between EphB4 expressing melanoma cells and ephrin-B2 expressing spinal endothelial cells leads to re-pulsion (green arrows) of CTCs and hampers extravasation. (b) Conditional KO of ephrin-B2 on spinal endothelium decreases repulsive forces and enables CTC extravasation (red arrows). (c) Increased extravasation is also observed under small mole-cule inhibition (NVP-BHG 712) of EphB4 forward signaling. (d) No significant effects (grey arrows) on CTC extravasation in absence of endothelial ephrin-B2. (e) Binding of soluble ephrin-B2-Fc to EphB4 in presence of endothelial Ephrin-B2 decreases CTC repulsion and enables extravasation. (f) In the absence of endothelial ephrin-B2, soluble ephrin-B2-Fc partially restores physiologic barrier functions and decreases CTC extravasation.