The erythropoietin-producing hepatocellular carcinoma (Eph) receptors constitute the largest sub-family of receptor tyrosine kinases (RTKs) identified until now (Eph Nomenclature Committee, 1997). The Eph receptors have diverse activities, including effects on the actin cytoskeleton, cell attachment, cell shape, and cell mobility. Moreover, recent work has also found that these receptors also influence cell proliferation, survival, secretion, and differentiation. These activities depend on the interaction between the Eph receptors and the ephrins (Eph receptor interacting proteins) (Pasquale, 2005; Pasquale, 2008). Based on sequence identity, structure, and their binding affinity for ligands, the Eph receptors are grouped into two subclasses, EphA receptor (EphA1-10) and EphB receptor (EphB1–6). The ligands for Eph receptors, ephrins, are cell-surface bound proteins that are divided into two subclasses ephrin-A (ephrin-A1 to -A6) and ephrin-B (ephrin-B1 to -B3) according to how they bind to the plasma membrane (Pasquale, 2004; Pasquale, 2005). Ephrin-A ligands are glycosylphosphatidylinositol (GPI) anchored, and ephrin-A ligands can transmit signals despite the lack of a cytoplasmic domain. The reverse signaling mechanisms of ephrin-A ligands are considered to be related to ephrin-A clustering and recruitment of regulatory proteins (Davy et al., 1999). Ephrin-A ligands are anchored to the membrane via covalent linkage to GPI, and rely on transmembrane coreceptors to transmit signals intracellularly (Bonanomi et al., 2012). Ephrin-B ligands are similar to Eph receptors in that they contain a cytoplasmic region, a single transmembrane domain, and a PDZ-binding motif. Ephrin-B reverse signaling also involves the Src family kinases, which are responsible for ephrin-B phosphorylation after the binding of Eph receptor (Pasquale, 2005; Arvanitis and Davy, 2008). Phosphorylated ephrin-B can initiate reverse signaling via SH2 or PDZ domain-containing proteins (Cowan and Henkemeyer, 2001; Lu et al., 2001). In general, EphA receptors are promiscuously activated by glycosylphosphatidylinositol (GPI)-linked ephrin-A ligands, and EphB receptors are promiscuously activated by transmembrane ephrin-B ligands. However, there are some exceptions, cross interactions have been observed between EphA4 and ephrin-B2/B3 as well as between EphB2 and ephrin-A5 (Himanen et al., 2004; Kania and Klein, 2016; Royet et al., 2017). Furthermore, the membrane attachment of both Eph receptors and ephrin ligands provides a mechanism whereby Eph-ephrin receptors signaling activation requires cell-cell contacts. Eph receptors interact with their membrane-bound ligands the ephrins and promote cell-cell contacts, leading to bidirectional intracellular signaling and downstream signaling cascades that induce autophosphorylation of tyrosine residues in the juxtamembrane region and kinase domain, which further drive the recruitment of downstream signaling molecules (Kullander and Klein, 2002). These include Src family kinases, mitogen-activated protein (MAP) kinases, Src homology 2 and 3 adapter proteins, guanine nucleotide exchange factors, phosphatidylinositol 3-kinase (PI3K), small GTPases, and phosphatases. They are all involved in complex cell-cell repulsion and adhesion pathways, which modulate cell morphology, motility and attachment (Pasquale, 2005).

It is widely known that Eph receptors have essential roles in embryonic development, and in the past decade their critical roles in the occurrence and progression of human disease, especially in tumorigenesis, have become more and more clear (Pasquale, 2008; Xi et al., 2012; Husa et al., 2016). Undoubtedly, enriching our understanding of the roles Eph receptors play in the potential biomarkers, stemness, and drug resistance of cancer will provide new opportunities for tumor therapy (Pasquale, 2010; Leung et al., 2021). Eph receptor B2 (EphB2) has been demonstrated to play a crucial modulatory role in tumor progression (Guo et al., 2006; Lam et al., 2014; Buckens et al., 2020; Morales et al., 2021). Perplexingly, EphB2 can function as both tumor promoters and suppressors in different cellular contexts. In many different human tumors, such as breast cancer, cervical cancer, and medulloblastoma (Wu et al., 2004; Sikkema et al., 2012; Duan et al., 2018), EphB2 acts as a tumor promoter that promotes migration and invasion of tumor cells, and its expression is upregulated. On the contrary, in colorectal cancer and bladder cancer (Xi et al., 2012; Lee et al., 2021), EphB2 functions as a tumor suppressor and its expression level is reduced. However, there are no reviews focusing on the dual roles of EphB2 in cancer. Hence, in the subsequent chapters we first review the main biological features and the related signaling regulatory mechanisms of EphB2, and then we summarize the roles of EphB2 in cancer through current studies, in order to provide some fundamental knowledge for following studies. Finally, we provide our viewpoint on the future prospects of cancer research focusing on EphB2, especially with regard to the effects of EphB2 on tumoral immunity.

The EphB2 receptor is a 117-kDa transmembrane protein consisting of 1,055 amino acids, which is encoded on chromosome 1p36.12 in humans. It was cloned from chicken cDNA in 1991 (Pasquale, 1991). EphB2 has a prototypical RTK topology including an N-terminal multidomain extracellular region, a membrane spanning region, and an intracellular region (Figure 1) (Beckmann et al., 1994; Lisabeth et al., 2013). The extracellular region includes two fibronectin type-III repeats, a cysteine-rich domain (containing an epidermal growth factor (EGF)-like motif), and an ephrin-binding region (Himanen et al., 2001; Pasquale, 2005). The ephrin-binding region of EphB2 is a spherical ligand-binding region containing a cavity that accommodates a hydrophobic loop protruding from the ephrin (Himanen et al., 2001). Interestingly, so as to accommodate to ephrin-A5 or ephrin-B2, different conformational changes occur in the ligand-binding cavity of EphB2 (Toth et al., 2001; Himanen et al., 2004). Moreover, EphB2 can also bind ephrin-B1 and ephrin-B3 (Tanaka et al., 2007; McClelland et al., 2010).

The intracellular region encompasses a juxtamembrane region, a tyrosine kinase domain, a sterile-α-motif (SAM) domain, and a PDZ-binding motif (Pasquale, 2008). The SAM domain is a protein interaction domain that facilitates receptors homo-dimerization and the oligomerization (Thanos et al., 1999). Moreover, biophysical and structural studies have revealed that isolated extracellular Eph and ephrin regions initially form high-affinity heterodimers around the hydrophobic loop of the ligand. Then, these dimers may form ring-like heterotetramers with lower affinity. On cell surface, Eph-ephrin complexes are further arranged into higher-order aggregates or clusters, and begin a bidirectional signaling (Himanen et al., 2001; Himanen and Nikolov, 2002; Himanen, 2012). EphB2 signals may also be propagated through the Ras binding protein AF6 and other proteins containing the SH2 domain, which bind to the C-terminus of the Eph receptors (Hock et al., 1998; Torres et al., 1998; Buchert et al., 1999). Furthermore, there are two conserved autophosphorylation sites (tyrosines 605 and 611) in the EphB2 juxtamembrane region (Zisch et al., 2000). In order to investigate their role in Src binding, Zisch et al. have mutated tyrosines 605 and 611 of EphB2 to the amino acid phenylalanine that cannot be phosphorylated (Zisch et al., 1998). However, replacing tyrosines 605 and 611 with phenylalanine reduce EphB2 kinase activity, which complicates the analysis of their role in EphB2-mediated signaling and their function as Src homology 2 (SH2) domain binding sites. In contrast, replacing them with glutamic acid, which like phosphotyrosine is negatively charged, will disrupt SH2 domain binding without impairing EphB2 kinase activity (Zisch et al., 2000). Functionally, Ephb2 is related to monocyte adhesion to endothelial cells (Vreeken et al., 2020), endothelial cell chemotaxis and branching remodeling (Salvucci et al., 2006), T-cell and B-cell activation (Nguyen et al., 2013; Yu et al., 2014; Mimche et al., 2015a), autophagic cell death (Kandouz et al., 2010; Tanabe et al., 2011), cell repulsive responses (Lin et al., 2008; Poliakov et al., 2008; Schaupp et al., 2014; Gaitanos et al., 2016; Okumura et al., 2017; Evergren et al., 2018), platelet function (Vaiyapuri et al., 2015; Berndt and Andrews, 2018; Berrou et al., 2018), angiogenesis (Sato et al., 2019), and liver fibrosis (Mimche et al., 2015b; Butler and Schmidt, 2015; Mimche et al., 2018; Chen et al., 2020; Huang et al., 2021).

A distinctive characteristic of Eph-ephrin complexes is that they can generate bidirectional signals: Eph/ephrin forward signaling is triggered by activating tyrosine kinase domain after the binding of ephrin ligand, and propagates in the receptor-expressing cells, whereas Eph/ephrin reverse signaling is initiated by activating Src family kinase domain after the binding of Eph receptor, and propagates in the ligand (ephrin)-expressing cells (Surawska et al., 2004; Pasquale, 2010). Eph signaling modifies the actin cytoskeleton organization and affects the activities of intercellular adhesion molecules and integrins, thereby controlling cell morphology, adhesion, invasion, migration, and the epithelial phenotype (Pasquale, 2005; Pasquale, 2008). In addition, recent work has also discovered Eph influences on cell proliferation, survival, and special cellular functions such as insulin secretion, immune function, synaptic plasticity and bone remodeling (Pasquale, 2005).

Regarding forward signaling by EphB2 in cancer cells, although the receptor is upregulated in most cancers, its response to ephrin is silent. In some cases, Eph forward signaling that relies on ephrin may even be harmful to tumor progression. For example, the medulloblastoma cell lines with high expression of EphB2 were stimulated by ephrin-B1, the cell adhesion ability in vitro was significantly decreased, and the invasion ability was increased (Sikkema et al., 2012). At the same time, overexpression of EphB2 in glioma tissues and cells inhibited cell adhesion and promoted cell invasion, indicating that the overexpression of EphB2 promotes tumor progression via forward signaling (Nakada et al., 2004; Nakada et al., 2005). However, EphB2 inactivation promoted cell proliferation, motility, and invasion of bladder cancer (Lee et al., 2021). Silencing EphB2 accelerated pancreatic cancer growth by facilitating cell proliferation through triggering G1/S phase transition, indicating EphB2 forward signaling has a tumor suppressing function (Hua et al., 2011).

Unlike Eph receptors, since ephrin-Bs do not have intrinsic catalytic activity, they depend on the recruitment of signaling molecules (such as Src family kinases) to signal, which phosphorylate specific tyrosine residues in the cytoplasmic region of ephrin-Bs, leading to receptor engagement and clustering (Salvucci and Tosato, 2012; Salgia et al., 2018). Moreover, similar to the case of forward signaling, reverse signaling was also found to lead to tumor progression and suppression. For example, EphB2-ephrin-B1 promoted the invasion of pancreatic cancer cells (Tanaka et al., 2007). However, EphB2/ephrin signaling was able to suppress colorectal cancer expansion and invasion via repulsive mechanisms (Okumura et al., 2017; Evergren et al., 2018). In summary, these results on bidirectional signaling indicated that EphB2/ephrin has diverse and complex functions in different cancer types and surrounding environment. In addition, mutational inactivation of EPHB2 may also play an important role in cancer progression (Huusko et al., 2004).

Many studies have verified that EphB2 is abnormally expressed in many cancer types Table 1. EphB2 is overexpressed in most tumors, such as hepatocellular carcinoma, breast cancer, glioma, and malignant mesothelioma (Leung et al., 2021; Wu et al., 2004; Nakada et al., 2004; Goparaju et al., 2013), and it functions as a tumor promoter. However, the expression of EphB2 is low in other tumors, such as colorectal cancer and bladder cancer (Xi et al., 2012; Lee et al., 2021), indicating that it exerts a tumor-suppression effect. These studies show that EphB2 expression is dynamically regulated in different tumor progression, and that EphB2 exerts its regulatory functions in multiple ways (Figure 2). Moreover, EphB2 expression is related to elevated metastatic potential, poor prognosis, and decreased survival of tumor patients (Wu et al., 2006; Yu et al., 2011; Husa et al., 2016; Koh et al., 2020). Consequently, EphB2’s functional relevance and expression patterns in malignancies make this protein a potential prognostic biomarker and therapeutic target in cancer.

EphB2 is a significant member of the Eph receptor family, which was thought to be distributed on tumor cells and endothelial cells in previous researches (Salvucci et al., 2006; Wang et al., 2012). Recently, as EphB2 was found to be expressed on some immunocytes such as monocytes, T cells, and B cells, increasing researches have reported on the roles of EphB2 in immunity (Alfaro et al., 2008; Braun et al., 2011; Yu et al., 2014). Forward EphB2 signaling induced by the specific binding of ephrin-B1/B2 and EphB2 could promote monocyte activation and T-cell migration (Alfaro et al., 2008; Braun et al., 2011). In addition, transdifferentiation of human monocytes into macrophages was correlated with increased expression of EphB2, and exposure of monocytes to immobilized ephrinB2 led to phosphorylation of receptors, followed by increased expression of proinflammatory chemokines such as monocyte chemotactic protein-1/CCL2 and interleukin-8 (Braun et al., 2011). Yu et al. reported that EphB2 was involved in the activation of human naive B-cell via Notch1 and Src-p65 signaling pathways and was regulated by miR-185 (Yu et al., 2014). In this study, they used Western blot to test the expression of EphB2 on B cells, and used EphB2 siRNA interference in human B cells from healthy volunteers to evaluate the roles of EphB2 in immunoglobulin (Ig) production, cytokine secretion, and B-cell proliferation. Their demonstrated that EphB2 was scattered on naive B cells and its expression was up-regulated on activated B cells. IgG production (decreased by 26%, p < 0.05), TNF-α secretion (decreased by 40%, p < 0.01), and B-cell proliferation (decreased by 22%, p < 0.05) were decreased concordantly with the down-regulated EphB2 expression. Subsequently, they found that miR-185 directly targeted EphB2 mRNA and inhibited its expression. Moreover, miR-185 overexpression suppressed B-cell activation and miR-185 inhibitor promoted B-cell activation. Furthermore, abatement of EphB2 via EphB2 siRNA or miR-185 mimics attenuated the activation of Notch1 and Src-p65 signaling pathways in human B cells (Yu et al., 2014). In conclusion, it can be speculated that EphB2 might be involved in tumor immunity and this issue certainly worthy further investigation in future research. In this review, we first systematically summarized and discussed the roles of EphB2 in cancer, as well as listed researches that may deepen our understanding of how it regulates cancer progression. Overall, EphB2 serves as a tumor promoter in most cases, facilitating tumor cell proliferation, invasion, and migration through different signaling pathways. However, EphB2 expression and its specific functions in gastric cancer and prostate cancer are controversial and need to be further studied. Moreover, the relationship between EphB2 expression and clinicopathological features was summarized. In detail, the abnormal expression of EphB2 was remarkably associated with clinicopathological features, including overall survival, disease-free survival, lymph node metastasis, histological differentiation, tumor grade and stage, reflecting its potential value as a sensitive and effective biomarker for cancer diagnosis, prognosis, and therapy. Furthermore, The Eph receptor family is an attractive tumor therapeutic target. Previous studies have developed a peptide, monoclonal antibody or small molecule against EphB2 in an attempt to prevent its activation (Mao et al., 2004; Koolpe et al., 2005; Toledo-Sherman et al., 2005), which may be used as a potential treatment for cancer. Accordingly, deepening the understanding of the structure, biogenesis, and molecular mechanisms of EphB2 will provide valuable information for functional research and improving the efficiency of rational drug design.