Ephrin Receptor A2
Erythropoietin-producing human hepatocellular carcinoma receptors (EPH) have been discovered through a screening aimed at identifying novel kinases involved in cancer (Holland et al. 1997). They form the largest subfamily of receptor tyrosine kinases (RTKs) and are among the oldest evolutionarily conserved receptor/ligand pairs (Pasquale 2008). EPH receptors and their ephrin (EFNs) ligands are expressed in all embryonic germ layers of vertebrates and mediate cell migration and positioning, boundary formation, and segmentation during important developmental processes such as gastrulation (Pasquale 2008). Axon guidance is one of the main functions of EPHs/EFNs; however, this system is nowadays regarded as a universal cell-to-cell communication pathway that allows cellular moving to a specific position and maintains cellular organization through chemotactic/chemorepulsive forces (Pasquale 2008; Genander 2012). Recent studies are also focusing on the role of EPHs/EFNs in the regulation of adult stem niche in various organs (Genander 2012; Perez White and Getsios 2014) as well as on their function in regulating specialized functions such as synaptic plasticity, memory formation, epithelial and vascular homeostasis and integrity, bone remodeling, insulin secretion, and inflammatory and immune response. EPHA2 is an active modulator of the cellular adhesion process and it appears to be closely connected with adhesion molecules like E-cadherin, mediating EPHA2 localization at cell-cell contact, or like claudin-4 that is phosphorylated by EPHA2 at the tight junctions, decreasing cell-cell adhesion and enhancing paracellular permeability (Tanaka et al. 2005). Interestingly, rare genetic variants of EPHA2 (on chromosome 1p36) have been linked with cataract occurrence in Caucasians. One of these variants, Arg721Gln increases basal kinase activity of EPHA2.
Structure and Activation
Like all the other RTKs, upon ligand binding, EPHA2 undergoes dimerization/oligomerization with conformational changes triggering the autophosphorylation of the receptor on specific tyrosine residues, whereas at the same time the phosphorylation of tyrosines located in the inhibitory juxtamembrane domain facilitates receptor activation and phosphorylation of cytoplasmic downstream signal proteins (Davis et al. 2009). EPHA2 contains also an autophosphorylated tyrosine residue within the activation loop; however, differently from other RTKs, phosphorylation of this residue is not essential for kinase activity (Pasquale 2008). Similarly to other RTKs, receptor-ligand binding induces conformational reorganization of EPHA2 LBD that facilitates the interaction with other EPHs, resulting in rapid oligomers as well as heterodimers formation, allowing joining of different dimer pairs into tetramers and formation of high-order cluster complexes. Interestingly, binding of different EPHs to EPHA2 is able to modulate signaling in the absence of EFN binding and to suppress EPH protumorigenic signaling. Examples are EPHB6 suppression of EPHA2 ligand-independent signaling in breast cancer cell or EPHA7-mediated inhibition of EPHA2 oncogenic signaling in lymphoma.
Because EFNA1 ligand is bound to surface of neighboring cells, the interaction with EPHA2 elicits a bidirectional signaling. In ligand-expressing cells, the binding through the GPI anchor stimulates a “reverse signaling” involving juxtamembrane proteins such as Src or, in turn, interacting with other membrane-bound receptors such as RET or NTRK1 (Pasquale 2008).
Several evidences suggest that the EPHA2 ligand-dependent signaling is modulating the effect of other RTKs, such as FGFR, TRKB, and IGF1R (Miao et al. 2001; Pasquale 2008). This inhibitory function involves recruitment of p120RAS-GAP that promotes GTP hydrolysis and therefore switches off p21 RAS (Minami et al. 2011; Pasquale 2008). Similarly, EPHA2 forward signaling reduces AKT phosphorylation on both T308 and S473 sites (Miao et al. 2009). However, it is worth mentioning that RTKs activation leads to MAPK activity and EPHA2 overexpression, activating a positive regulatory feedback loop (Macrae et al. 2005). An interesting crosstalk between EPHA2/EFNA1 and VEGFR has been described in cancer, where high expression of EFNA1 in endothelial cells is recognized as chemoattractant in tissue angiogenesis and tumor neovasculature, supporting the first discovery of EFNA1 as a TNFα-induced gene in human umbilical vein endothelial cells (HUVECs). Of note, EFNA1 and EPHA2 expressions are upregulated by VEGFR and HIF-1α in hypoxic conditions, leading to EPHA2 phosphorylation and increased expression of VEGFR that subsequently activates distant endothelial cells (Beauchamp and Debinski 2012). This crosstalk suggests a role for EPHA2-EFNA1 in regulating tumor-stroma interaction and tumor progression. Similarly, EPHA2-EFNA1 binding is decreasing MET activation and inhibiting EGF-induced motility (Macrae et al. 2005). In conclusion, although a consistent number of studies have identified several molecular players in EPHA2 “ligand-dependent” signaling, the precise cellular effects are still far from being understood. Moreover, numerous evidences suggest the existence of a “ligand-independent” activation mechanism with a prominent role in tumor promotion, adding more complexity to the EPHA2-EFNA1 system.
Role of EPHA2 in Cancer
Considering the multiplicity and the versatility played in adult and embryonic cellular activities, it is not surprising that EPHs/EFNs play a key role in tumors (Pasquale 2008). As previously discussed, most of the reports point to a tumor-suppressive effect of the “ligand-dependent” EPH stimulation compared to an oncogenic effect of “ligand-independent” stimulation (Pasquale 2008). Accordingly, in several cancer types, it is often documented a reduced expression of the EFN ligands and at the same time an increased expression of the EPH receptors.
EPHA2 represents the family member most upregulated in human cancer, through a deregulation that most takes place at the transcriptional and posttranslational level. It is overexpressed in a variety of human malignancies, and it is often associated to increased tumor grade and poor prognosis in a number of malignancies such as breast, lung, prostate, skin, esophageal, gastric, and renal carcinoma, as well as in thyroid carcinomas (Amato et al. 2014). Despite its high-expression levels, tyrosine phosphorylation of EPHA2 is often downregulated in malignant cells compared to normal cells, and EFNA1 is frequently downregulated in tumor cells contributing to the loss of cell-cell contact among cancer cells (Pasquale 2008). The main phosphotyrosine involved in ligand-independent activation of EPHA2 has been identified as serine 897 (Ser897), mapping near the EPHA2 SAM domain. This phosphorylation has been reported to mediate the switch from the antioncogenic to the prooncogenic function of EPHA2. Miao and coworkers recently demonstrated that phosphorylation of EPHA2 on Ser897 is mediated by AKT and it is crucial for glioma and prostate cancer cell invasiveness and stem cell maintenance. S897A mutation abolished the ligand-independent promotion of cell motility. In contrast, EFNA1 stimulation of EPHA2 blunted AKT and caused EPHA2 dephosphorylation on Ser897 (Miao et al. 2009). Moreover, overexpression of EPHA2 in glioblastoma cell lines promoted cell invasion. These effects were ligand independent and required the AKT-mediated phosphorylation on Ser897. Finally, overexpression of EPHA2 promoted stem cell properties in a kinase-independent manner (Miao et al. 2015a).
EPHA2 Therapeutic Targeting
EPHA2 is rarely mutated but it is frequently overexpressed in carcinomas of the breast, lung, and several other cancer types. In non-small cell lung carcinomas (NSCLC), EPHA2 is associated to poor outcome and its genetic disruption in KRAS-induced NSCLC impairs tumor growth. In vitro EPHA2 knockdown reduces growth and survival of NSCLC cell lines and EPHA2 ATP-competitive kinase inhibitor ALW-II-41-27 reduces tumorigenesis in a NSCLC xenograft model (Amato et al. 2014). More recently, EPHA2 inhibition by ALW-II-41-27 has been demonstrated to overcome resistance of NSCLC cell lines to the EGFR TKIs erlotinib or AZD9291 (Amato et al. 2016). In mammary carcinomas, EPHA2 has been reported to be overexpressed in aggressive subtypes featuring poor prognosis. Moreover, EPHA2 ablation reduced initiation and metastatic progression of mammary carcinomas in MMTV-ERBB2 transgenic mice. In melanomas, EPHA2 was recently reported to be involved in resistance and adaptation to BRAF/MEK kinase inhibitors. Chronic exposure to BRAF inhibitors caused upregulation of EPHA2 phosphorylated on Ser897, accompanied by increased cell invasion and reduced expression of EFNA1 ligand. Similarly, in melanoma cell lines that developed resistance to BRAF chemical inhibition as well as in tumor specimens from patients who relapsed upon BRAF inhibitor treatment, EPHA2 was found overexpressed and hyperphosphorylated on Ser897 and treatment with ALW-II-41-27 EPHA2 TKI was able to suppress BRAF inhibitor-resistant melanoma cells (Miao et al. 2015b). Interestingly, knockout mice showed that EPHA2 was dispensable for normal hematopoiesis as well as for acute myeloid leukemias initiated by the MLL-AF9 oncogene, while radiolabeled anti-EPHA2 monoclonal antibodies blocked leukemogenesis in experimental mice.
According to these evidences, EPHA2 has been considered as a promising molecular target for cancer treatment and various tools are being explored to hit EPHA2 in different cancer models, including EPHA2 monoclonal antibody, polyspecific antibodies designed to target different EPH simultaneously, soluble or endotoxin-conjugated EFNA1, small molecule tyrosine kinase inhibitors (such as ALW-II-41-27), gold- or polyethylene glycol (PEG)-coated EFNA1 nanoshells, human adenoviruses engineered with EFA1 extracellular domain or with EFNA1 mimetic homing peptide (YSAYPDSVPMMSK named YSA), and functionalized YSA-nanocarriers for drug delivery (www.clinicaltrials.gov). Finally, some studies have tested the combination of EPHA2-targeting therapies with chemotherapeutics or other targeted therapies, such as combination of EPHA2-antibody and paclitaxel in ovarian tumors, EPHA2 siRNA with FAK or Src siRNAs and EPHA2 antibody with tamoxifen, EPHA2 inhibitors, and gemcitabine in pancreatic cancer (Amato et al. 2016).
EPHA2/EFNA1 system acts as a main player in several physiological cellular processes such as cell-cell contact, cellular adhesion, and connection to extracellular matrix, and therefore these molecules have been investigated in different pathological phenomena as well as in cancer. Among the different EPHs, EPHA2 is the most overexpressed in human cancers, although the precise mechanism of action has not been clarified yet. Many evidences point to an antioncogenic effect of the ligand-dependent EPHA2 activation and to a prooncogenic function of the ligand-independent receptor activation, and therefore several blocking strategies are being tested in different cancer models to evaluate their therapeutic application.
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