EphB4: A promising target for upper aerodigestive malignancies

Abstract

The erythropoietin-producing hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that include two major subclasses, EphA and EphB. They form an important cell communication system with critical and diverse roles in a variety of biological processes during embryonic development. However, dysregulation of the Eph/ephrin interactions is implicated in cancer contributing to tumour growth, metastasis, and angiogenesis. Here, we focus on EphB4 and review recent developments in elucidating its role in upper aerodigestive malignancies to include lung cancer, head and neck cancer, and mesothelioma. In particular, we summarize information regarding EphB4 structure/function and role in disease pathobiology. We also review the data supporting EphB4 as a potential pharmacological and immunotherapy target and finally, progress in the development of new therapeutic strategies including small molecule inhibitors of its activity is discussed. The emerging picture suggests that EphB4 is a valuable and attractive therapeutic target for upper aerodigestive malignancies.

Introduction

The erythropoietin-producing hepatoma (Eph) receptors represent the largest class of receptor tyrosine kinases (RTKs). They are type I transmembrane proteins that interact with their membrane-bound ligands the ephrins and facilitate cell-to-cell contacts resulting in bidirectional intracellular signaling [1,2]. Ephrin stimulation causes receptor dimerization and autophosphorylation of the Eph receptor on the juxtamembrane regions and cytoplasmic tails driving the subsequent recruitment of down-stream signaling molecules [3]. These include SH2 and SH3 adapter proteins, Src family kinases, PI3K, MAP kinases, small GTPases, guanine nucleotide exchange factors, and phosphatases, each of which contribute to the complex cell repulsion and adhesion pathways that modulate cell shape, motility and attachment [4].

Based on sequence homologies and their binding affinities and characteristics, the Eph receptors are grouped into subclasses namely, Eph A and Eph B receptors, although there is significant redundancy and cross talk between subclasses [[5], [6], [7], [8]]. Furthermore, while the members of the ephrin-A subclass (ephrin-A1 to -A5) are tethered to the membrane via a glycosylphosphatidylinositol (GPI) anchor, the members of the ephrin-B subclass (ephrin-B1 to -B3) are transmembrane proteins and have a highly conserved cytoplasmic domain [9].

The EphB subfamily, the largest of receptor tyrosine kinases, comprises of six members (EphB1 to 6). Five of them are catalytically competent, while the more divergent EphB6 is catalytically dead [10]. Unlike many of the other members in this family that exhibit promiscuity [4], the EphB4 receptor has distinctive specificity for a single ligand, ephrinB2; however, EphB4 also binds to ephrinB1 and ephrinB3 albeit weakly [11]. Physiologically, EphB4 plays important roles in vascular remodeling of primitive capillary networks into distinct arteries and veins [12,13]. On the other hand, EphB4 is also known to regulate vascularization in malignant tumours [14], and is up-regulated in numerous cancer types including upper aerodigestive cancers (reviewed, [8,15,16]). Nonetheless, since Eph-ephrin interactions have been shown to either promote or inhibit tumour growth [8,17], the role of these receptors in cancer remains a matter for debate. However, crosstalk between elevated Eph receptors and other oncogenes, such as the ErbB family of receptor tyrosine kinases is thought to result in enhanced cell proliferation and tumorigenesis, presumably independent of ephrin stimulation (reviewed, [18]) (Fig. 1). Here, we review the function of EphB4, its potential as a therapeutic target, the developments to date on small molecule inhibitors, and EphB4 immunotherapy in upper aerodigestive malignancies.

EphB4, like all other Eph receptors, is a type I transmembrane protein that has a prototypical RTK topology. Thus, the protein is characterized by an N-terminal multidomain extracellular region, a single transmembrane segment, and a cytoplasmic region that contains the kinase domain in the C-terminus [19]. The extracellular domain includes a globular ligand-binding domain, a cysteine-rich region, composed of an epidermal growth factor (EGF)-like motif, and two fibronectin type III repeats [11]. The ephrin-binding domain has a high- affinity binding site that mediates the biochemical communication between EphB4 and the ephrinB2 in adjacent cells. The cytoplasmic region contains a tyrosine kinase domain and protein-protein interacting modules, including a sterile-α-motif (SAM) and a PDZ-binding motif [1]. The SAM domain is a protein-interacting domain that promotes homo-dimerization and the oligomerization of receptors. The PDZ-binding motif mediates the organization of protein complexes at the plasma membrane [20] (Fig. 2).

The crystal structures of the EphB4 receptor in complex with the extracellular domain of ephrinB2 complex [21] and the EphB4 receptor in complex with antagonistic peptide TNYL-RAW (TNYLFSPNGPIARAW) that binds with high-affinity (15 nM) [22] have been solved. The EphB4-ephrinB2 structural analysis suggested that L95 plays a particularly important role in defining ligand selectivity of EphB4. Indeed, all other members of this receptor family that promiscuously bind many ephrins have a conserved R at the position corresponding to L95 of EphB4. Furthermore, amino acid changes in the EphB4 ligand binding cavity when designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yielded EphB4 variants with altered binding affinity for ephrinB2 and TNYL-RAW. Finally, binding studies employing isothermal titration calorimetry with the EphB4 L95R mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and TNYL-RAW [22].

More recently, Dai et al. [23] by simulating conformational ensembles and recognition energy landscapes starting from separated Eph and ephrin molecules and proceeding up to the formation of receptor-ligand complexes, demonstrated that two pairs of dynamic salt bridges are unique to EphB4, resulting in ephrinB2 recognition and conformational selection. Together these structural biology and computational studies provide tremendous insight on the specificities for the interaction between EphB4 and ephrinB2 and how this knowledge could be applied to drug discovery in designing novel compounds that recapitulate the critical contacts of the peptide and EphB4 with good pharmacokinetic properties.

Since the receptor and the ligand typically reside on different cells, a rather unique feature of the Eph/ephrin axis is that the complexes emanate signals bidirectionally. Thus, forward signals that depend on Eph kinase activity propagate in the receptor-expressing cell, and reverse signals that depend on Src family kinases propagate in the ligand (ephrin)-expressing cell (Fig. 3). Eph signaling controls cell morphology, adhesion, migration and invasion by modifying the organization of the actin cytoskeleton and influencing the activities of integrins and intercellular adhesion molecules [1,4]. Additionally, recent work has also uncovered Eph effects on cell proliferation and survival as well as specialized cellular functions such as synaptic plasticity, insulin secretion, bone remodeling and immune function [1].

With regard to forward signaling by EphB4 in cancer cells, while the receptor is upregulated in many cancers, its response to ephrin is muted and, in some cases, ephrin-dependent Eph forward signaling may even be detrimental to tumour progression. For example, EphB4 receptors that are activated by ephrins acquire the remarkable ability to inhibit oncogenic signaling pathways, including Abl-Crk, PI3K-Akt and HRAS-Erk [24]. In contrast, some cancer cells are stimulated by activation [25] and in others, overexpression of the active EphB4 enhanced anchorage-independent growth, migration and invasion, all characteristics associated with an aggressive phenotype [26] suggesting that the overexpression of EphB4 facilitates tumour progression by forward signaling. However, overexpression of EphB4 or that of an inactive mutant EphB4 kinase in esophageal squamous cell carcinoma cells promoted cell growth and migration, suggesting EphB4 promoted cell growth and migration independently of its kinase activity and that repressed tumourigenesis [27].

Insofar as reverse signaling is concerned, since B ephrins do not possess intrinsic catalytic activity, they rely on the recruitment of signaling molecules such as Src family kinases, which phosphorylate specific tyrosine residues in the intra-cytoplasmic domain of B ephrins, resulting in receptor engagement and clustering [28]. Furthermore, similar to the situation with forward signaling, reverse signaling has also been observed to result in both tumour suppression as well as progression. For example, tumour cells expressing dominant negative EphB4 are incapable of forward signaling, but remain able to stimulate ephrinB2 reverse signaling, that in turn promotes cell invasion and proliferation. The overexpression of EphrinB2 in cultured cancer cells enhances integrin mediated attachment, migration and invasion [29,30]. Considered together, these observations on bidirectional signaling suggest that the role of EphB4/ephrinB2 is context-dependent and can vary from one cancer type to the other. Furthermore, mutations dysregulating Eph function likely also play a role in cancer progression.

Many receptor tyrosine kinases (RTKs), including several members of the Eph family, are frequently altered in lung cancer where they are believed to play important roles [31]. For example, a mutation in EphA2 (G391R) causes constitutive kinase activation in lung squamous cell carcinoma (SCC) where it promotes increased cell survival, cell invasion, focal adhesions, and activation of mammalian target of rapamycin [32]. Three potentially relevant EphB6 mutations were identified in NSCLC patients (3.8%) that included two point mutations and an in-frame deletion mutation (del915–917). Although catalytically inactive, the point mutations in EphB6 lead to instable proteins while the in-frame deletion enhanced migration and accelerated wound healing in vitro. Furthermore, the del915–917 mutation also increased the metastatic capability of NSCLC cells in an in vivo mouse model suggesting that EphB6 mutations promote metastasis in a subset of patients with non-small cell lung cancer (NSCLC) cells [33]. Similarly, it has been reported that EphA3 and EphA5 are frequently altered in NSCLC. While mutations in EphA3 appear to have pro-tumourigenic effects thereby transforming tumour suppressor function of the WT protein in the lung [34], the functional significance of these alterations in EphA5 is unknown [35].

Comparatively, there is a lot more information on EphB4, particularly in lung cancer. For example, it has been reported that the gene is overexpressed and amplified in several lung cancer subtypes and is necessary for the growth of lung adenocarcinoma xenografts in mice [36]. Several non-synonymous mutations in EphB4 have also been identified, in human tumour tissues and cell lines. Thus, a mutation resulting in an R564K substitution occurring in the intracellular juxtamembrane (JM) domain was detected in one multiple myeloma cell line [37], and an R889W substitution was detected in one gastric carcinoma tissue sample [38]. A detailed genetic analysis of small cell lung cancer (SCLC) also identified several mutations in the ephrin receptor family including EphB4 [86].

Another comprehensive study by Ferguson et al. [39] revealed EphB4 can be mutated in lung cancer and that they lead to putative structural alterations as well as increased cellular proliferation and motility. Notably, the authors detected eight NS EphB4 mutations with one (A230V) in an extracellular linker region, two (A371V and P381S) in the first extracellular fibronectin III repeat, two (W534* and E536K) in the extracellular juxtamembrane domain, two (G723S and A742V) in the tyrosine kinase domain, and one (P881S) in an intracellular linker region just C-terminal to the tyrosine kinase domain. Three of these (A230V, A371V, and P381S) occurred in adenocarcinoma, one (A742V) occurred in SCC, and four (W534*, E536K, G723S, and P881S) occurred in SCLC. Seven of these eight mutations (all except A371V) had not been previously detected (Fig. 4). Overall, the non-synonymous mutations occurred in 7% of samples, with non-synonymous mutation rates of 9% in adenocarcinoma, 9% in SCLC, and 3% in SCC. These observations together with several others [[40], [41], [42]] provide a panoramic view of the EphB4 mutational landscape in lung cancer. Interestingly, of the 16 sites with EphB4 mutations in adenocarcinoma tissues identified by Ferguson et al. [39], 12.5% were located in the kinase domain underscoring the functional implications. Furthermore, a bioinformatics analysis of these mutations revealed that a) they are mutually exclusive from other common RTK variants in lung cancer, b) they correspond to analogous sites of other RTK's variations in cancers, c) they are predicted to be oncogenic based on biochemical, evolutionary, and domain-function constraints, and d) EPHB4 mutations can induce broad changes in the kinome signature of lung cancer cells [39]. Taken together, these data illuminate the role of EphB4 in lung cancer and further identify EphB4 as a potentially important therapeutic target. However, thus far, no non-synonymous EphB4 mutations have been reported in HNSCC or pleural mesothelioma tissues.