|Home | About | Journals | Submit | Contact Us | Français|
The Eph receptor tyrosine kinases and their ephrin ligands have intriguing expression patterns in cancer cells and tumor blood vessels, which suggest important roles for their bidirectional signals in multiple aspects of cancer development and progression. Eph gene mutations likely also contribute to cancer pathogenesis. Eph receptors and ephrins have been shown to affect the growth and migration/invasion of cancer cells in culture as well as tumor growth, invasiveness, angiogenesis, and metastasis in vivo. However, Eph signaling activities in cancer appear to be complex, and are characterized by puzzling dichotomies. The Eph receptors nevertheless represent promising new therapeutic targets in cancer.
Eph receptors and their ephrin ligands together form an important cell communication system with widespread roles in normal physiology and disease pathogenesis1. Links between Eph receptors and cancer date back to the first identified Eph family member2. EphA1 was cloned from a carcinoma cell line in a screen for novel oncogenic tyrosine kinases. The novel receptor was found to be upregulated in tumor versus normal tissues and its overexpression caused the oncogenic transformation of NIH3T3 fibroblasts2,3. The first Eph receptor-interacting (ephrin) ligand, EPHRIN-A1, was also identified from cancer cells a few years later4. The evidence implicating Eph receptors and ephrins in cancer is now extensive, and continues to grow.
The activities of the Eph system in cancer are complex, and intriguing in their paradoxical effects. For example, multiple Eph receptors and/or ephrins are present in essentially all types of cancer cells. However, not only increased but also decreased Eph expression has been linked to cancer progression. Consistent with this dichotomy, there is good evidence that Eph receptors and ephrins can both promote and inhibit tumorigenicity. The factors responsible for these divergent activities are only beginning to be uncovered.
Following a brief overview of the Eph and ephrin families and their bidirectional signaling mechanisms, the factors that regulate their expression and the remarkable multiplicity of their roles in cancer will be discussed, and the strategies under evaluation to target the Eph system for cancer therapy outlined. Other reviews provide more in depth information on Eph signaling mechanisms in development and adult physiology1,5–8.
In the human genome there are 9 EphA receptors, which promiscuously bind 5 glycosylphosphatidylinositol (GPI)-linked ephrin-A ligands, and 5 EphB receptors, which promiscuously bind 3 transmembrane ephrin-B ligands5. Exceptions are the EPHA4 and EPHB2 receptors, which can also bind ephrin-Bs and EPHRIN-A5, respectively, and EPHB4, which preferentially binds only EPHRIN-B2. Eph receptors typically interact with the cell surface-associated ephrins at sites of cell-cell contact (Fig. 1). In addition, soluble ephrin-As released from the cell surface retain the ability to activate EPHA24,9,10.
Eph-ephrin complexes emanate bidirectional signals: 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 ephrin-expressing cell. Ephrin-dependent but kinase-independent Eph signals can also occur11–13. 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 molecules1,5. 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 function1.
Bidirectional signals can lead to removal of the adhesive Eph-ephrin complexes from cell contact sites through an unusual endocytic mechanism that involves their internalization, together with patches of the surrounding plasma membranes, into the receptor- or the ephrin-expressing cell5. This enables separation of the engaged cell surfaces to produce the characteristic Eph repulsive responses. Another mechanism allowing cell separation involves protease-mediated cleavage of the Eph or ephrin extracellular domains14–18. Internalization and cleavage result in degradation, which can profoundly downregulate Eph levels. However, in certain cellular contexts Eph-ephrin complexes persist at intercellular junctions and emanate prolonged bidirectional signals that favor adhesiveness. For example, the cell adhesion molecule E-CADHERIN promotes EPHA2/EPHRIN-A1 localization at epithelial cell junctions and the metalloprotease ADAM19 stabilizes EPHA4/EPHRIN-A5 at neuromuscular junctions independently of its proteolytic activity19–21. A combination of Eph-dependent repulsive and adhesive forces can drive the segregation of cell populations expressing different repertoires of Eph receptors and ephrins, which may include transformed and normal cells or divergent subpopulations of tumor cells5,22,23.
There is also increasing evidence that other signaling modalities beyond “conventional” bidirectional signaling contribute to the multiple activities of the Eph system in cancer. For example, an initial extracellular Eph or ephrin cleavage by metalloproteases followed by γ-secretase-mediated cleavage within the transmembrane segment releases intracellular domains that can generate distinctive signals14,16,24,25. Eph receptors and ephrins can also signal independently of each other, through crosstalk with other signaling systems, which produces yet more distinctive outcomes. In addition, they participate in feedback loops that may switch between different outputs depending on the state of other cellular signaling networks (Fig. 2).
The Eph and ephrin families have grown in complexity during evolution, keeping pace with the increasingly more sophisticated tissue organization of higher organisms. Finely coordinated spatial and temporal regulation of Eph receptor and ephrin expression controls many processes that are critical for development and tissue homeostasis, including the formation of tissue boundaries, assembly of intricate neuronal circuits, remodeling of blood vessels, and organ size1,5. Multiple Eph receptors and/or ephrins are also expressed in both cancer cells and the tumor microenvironment, where they influence tumor properties by enabling aberrant cell-cell communication within and between tumor compartments26–32. Mutations dysregulating Eph function likely also play a role in cancer progression.
Many studies have correlated Eph and ephrin expression levels with cancer progression, metastatic spread and patient survival (Table 1). EPHA2, for example, is upregulated in a wide variety of cancers and its expression has been linked to increased malignancy and a poor clinical prognosis27,28,31,33. Furthermore, EPHA2 seems to be preferentially expressed in the malignant breast and prostate cancers with a basal phenotype34,35. EPHB4 is also widely expressed in cancer cells and its increased abundance has been correlated with cancer progression29,36,37. However, decreased Eph or ephrin levels in malignant cancer cell lines and tumor specimens have been reported as well. For example, EPHA1 is downregulated in advanced human skin and colorectal cancers38,39, EphB receptors in colorectal cancer23,40–42 and EPHRIN-A5 in glioblastomas43. Furthermore, EPHB6 expression is lower in metastatic than non-metastatic lung cancers44. Reconciling these discrepancies, recent studies show that an initial Eph receptor upregulation (due to activated oncogenic signaling pathways and other factors) can be followed by epigenetic silencing in more advanced stages due to promoter hypermethylation, as shown for several EphB receptors and EPHA1 in colorectal cancer23,39–41. Transcriptional repression, such as repression of EPHB2 by C-REL (a member of the nuclear factor-κB family) in colorectal cancer, may also play a role in Eph silencing45. Intriguingly, differential transcriptional regulation has been reported for EPHB2 and EPHB4 expression during colorectal cancer progression37. This was attributed to a switch in the association of β-catenin from the p300 coactivator (which induces EphB2) to the CBP coactivator (which induces EphB4). An inverse expression pattern has also been observed for EPHA2 versus ephrin-A expression in breast cancer cell lines, owing at least in part to feedback loops (Fig. 2A), and for several EphB receptors versus ephrin-Bs in early colorectal tumors and breast cancer cell lines23,46,47.
Chromosomal alterations and changes in mRNA stability also regulate Eph and ephrin expression in cancer cells (Table 1). A number of Eph receptor and ephrin genes are located in chromosomal regions frequently lost in cancer cells. For example, EPHA2, EPHA8 and EPHB2 are clustered at chromosomal region 1p36, which undergoes loss of heterozygosity in many cancers48,49. Some Eph genes, however, are in amplified regions50. Nonsense-mediated mRNA decay and interaction with mRNA-binding proteins can also regulate Eph mRNA stability in cancer cells49,51. These complex mechanisms of regulation parallel the multiplicity of Eph activities in cancer cells.
Several Eph receptors and ephrins are upregulated in vascular cells by tumor-derived factors and hypoxia. For example, tumor necrosis factor α (TNFα), vascular endothelial growth factor-A (VEGF-A) and the hypoxia-inducible factor HIF-2α have been shown to upregulate EPHRIN-A1 in cultured endothelial cells52–54. Endothelial EPHRIN-B2 is upregulated by VEGF through the Notch pathway, by cyclic stretch, hypoxic stress and contact with smooth muscle cells, whereas shear stress seems to decrease EPHRIN-B2 expression in endothelial cells but increase it in endothelial precursors by inducing their differentiation55–59. Moreover, EPHRIN-B2 is expressed in pericytes and vascular smooth muscle cells57,60. Expression of EPHA2/EPHRIN-A1 and EPHB4/EPHRIN-B2 in tumor blood vessels has been most extensively characterized, but other Eph receptors and ephrins are also present in the tumor vasculature54–57,61,62. In contrast, little is known about Eph and ephrin expression in other tumor compartments, such as activated fibroblasts and infiltrating immune and inflammatory cells. Nevertheless, Eph-dependent communication between these cells and tumor cells likely plays an important role in tumor homeostasis.
Screens of tumor specimens and cell lines have recently identified mutations in the genes encoding all of the Eph receptors, whereas cancer-related ephrin mutations have not been reported so far, perhaps in part because many of the screens have focused on the kinome63–67 (http://www.sanger.ac.uk/genetics/CGP/cosmic). Mutations of at least some Eph receptors are predicted to play a role in cancer pathogenesis. For example, EPHB2 mutations have been identified in human prostate, gastric, colorectal and melanoma tumors40,49,67–69. Some of these mutations may impair kinase function, and some are accompanied by loss of heterozygosity, suggesting a tumor suppressor role for EPHB2 forward signaling. Furthermore, a number of Eph receptors –particularly EPHA3 and EPHA5 – are frequently mutated in lung cancer63,70. The mutations are typically scattered throughout the Eph domains, including the ephrin-binding domain and other extracellular regions67,70. Elucidating the effects of the mutations will provide important insight into the functional roles of the Eph system in cancer.
In many cancer cell lines, Eph receptors appear to be highly expressed but poorly activated by ephrins, as judged by their low level of tyrosine phosphorylation1,29,37,47,71,72. This was one of the first clues that ephrin-dependent Eph forward signaling may be detrimental to tumor progression. Furthermore, recent expression profiling of ApcMin/+ intestinal tumors from wild-type and Ephb4+/− mice has revealed an extensive transcriptional reprogramming that suggests anti-proliferative and anti-invasive activities of EPHB4 in colorectal cancer73.
Forcing Eph receptor activation with soluble Fc fusion proteins of ephrin ligands can inhibit proliferation, survival, migration and invasion of many types of cancer cells in culture as well as tumor growth in several mouse models5,29,42,47,74. Conversely, a dominant negative form of EPHB4 has been shown to promote colorectal cancer proliferation and invasion73. These studies demonstrate that Eph forward signaling pathways can lead to tumor suppression (Fig. 3). Indeed, Eph receptors activated by ephrins acquire the remarkable ability to inhibit oncogenic signaling pathways, such as the HRAS-Erk, PI3 kinase-Akt and Abl-Crk pathways. Interestingly, this may reflect a physiological function of the Eph system in epithelial homeostasis by promoting contact-dependent growth inhibition and decreasing motility and invasiveness. These changes are reminiscent of mesenchymal-to-epithelial transition (Box 1).
Cancer cells appear to use a variety of mechanisms to minimize the tumor suppressor effects of Eph forward signaling. For example, the high EPHA2 or EphB expression and low ephrin expression observed in some cancers result in low bidirectional signaling23,46,47. Furthermore, co-expressed Eph receptors and ephrins often do not interact effectively in cancer cells20. This may be because they engage in lateral interactions that silence their signaling function, as has been shown in neurons and transfected cells5. Alternatively, loss of E- or VE-cadherin impairs endogenous EPHA2-EPHRIN-A1 interaction in malignant breast cancer and melanoma cells, respectively20,75. The two cadherins appear to promote EPHA2-EPHRIN-A1 interaction by stabilizing intercellular contacts and promoting the localization of EPHA2 at cell-cell junctions. Phosphotyrosine phosphatases also negatively regulate Eph receptor forward signaling in some cancer cells76. For example, the low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) has been implicated in cell transformation through its ability to dephosphorylate EPHA2, thus counteracting ephrin-dependent activation77. The receptor-type phosphatases PTPRO and PTPRF, and PTEN in C. elegans, also dephosphorylate Eph receptors78–80. However, it is not known whether this plays a role in cancer. Eph mutations may also contribute to disrupting forward signaling by impairing ephrin binding or kinase activity. For example, the EPHA3 E53K mutation in the MeWo melanoma cell line abrogates ephrin binding66,81 and the EPHB2 G787R mutation found in colorectal cancer impairs kinase activity69. It will also be interesting to investigate whether soluble Eph ectodomains generated by alternative splicing82,83 or proteolysis14–18,24 and proteins containing a major sperm protein (MSP) domain84 (Fig. 1) may decrease Eph signaling in cancer cells by acting as naturally occurring antagonists.
The tumor suppressor effects of Eph forward signaling may be active at the tumor periphery if the surrounding tissues express ephrins. In mouse tumor models, ephrins present in normal tissues have been proposed to inhibit expansion and invasiveness of incipient colorectal and skin tumors expressing Eph receptors23,85,86. In addition, recent experiments in the developing zebrafish hindbrain raise the possibility that elevated Eph or ephrin levels may drive segregation of tumor cells from surrounding normal tissues, thereby decreasing invasiveness, not only through repulsive mechanisms but also by promoting adhesiveness between tumor cells22. Eph receptors may further decrease tumor invasiveness by promoting the formation of tight junctions in neighboring epithelial cells through stimulation of ephrin-B reverse signaling87 (see next section). Indeed, recent systems-level studies have implicated complex, asymmetric signaling networks in the sorting of EPHRIN-B1-expressing HEK293 cells from EPHB2-expressing cells88. It is tempting to speculate that Eph receptors may contribute to tumor dormancy through these types of bidirectional signaling mechanisms that restrict tumor expansion. Accordingly, high EPHA5 levels have been detected in various dormant but not fast-growing tumor xenograft models89.
Ephrin reverse signaling in cancer cells may in some cases also contribute to tumor suppression (Fig. 3). In the Xenopus system and HT29 colon cancer cells, EPHRIN-B1 tyrosine phosphorylation (which can be induced by interaction with EphB receptors or by activated growth factor receptors and Src) disrupts binding of the ephrin to the scaffolding protein PAR6, promoting the formation of tight junctions between cells87,90. Similar to its role in neurons, ephrin-B reverse signaling may also inhibit the migratory and invasive effects of the CXCR4 G protein-coupled chemokine receptor in cancer cells5,6. EPHRIN-A5 can downregulate epidermal growth factor receptor (EGFR) levels in glioblastoma cells43.
Conversely, forward and/or reverse Eph-ephrin signals can enhance malignant transformation in some cases. There is also increasing evidence that the Eph receptors are capable of unconventional signaling activities that do not depend on activation by ephrin ligands and that support cancer progression. Moreover, it is well established that the Eph system promotes tumor angiogenesis.
In certain cellular contexts, Eph receptors activated by ephrins may have lost the ability to suppress tumorigenicity, and even acquired oncogenic ability. For example, activating mutations may render oncogenic signaling pathways resistant to inhibition by Eph forward signaling. Furthermore, EPHB2 can promote proliferation in mouse intestinal progenitor cells and ApcMin/+ adenomas through Abl-mediated increase in CYCLIN-D1 levels even though it inhibits invasiveness through other pathways91 (Fig. 4). Activation of RHOA downstream of EPHA2 and EPHB4 promotes ameboid-type migration of cancer cells and destabilizes epithelial adherens junctions in various cancer cell lines (Fig. 4), even though RHOA inhibits mesenchymal-type migration92–94 (Fig. 3). EPHA2 forward signaling in malignant melanoma and ovarian cancer cells can also promote vasculogenic mimicry75,95.
RRAS phosphorylation downstream of EPHB2 (Fig. 4) can enhance glioma cell invasiveness, possibly by decreasing cell substrate adhesion96, even though in other cell types Eph forward signals decrease cell adhesion and migration5,47,97. Instead of inhibiting the HRAS-Erk MAP kinase pathway, depending on the circumstances, EPHB2 can sometimes activate it5,79. In turn, activation of the Erk MAP kinase pathway enhances ephrin-dependent activation of overexpressed EPHB2 in cultured cells79. This may result in different EPHB2/MAP kinase feedback loops (Fig. 2B) that could either enhance or diminish cancer cell malignancy. Indeed, activation of an engineered membrane-anchored cytoplasmic domain of fibroblast growth factor receptor 1 (FGFR1) inhibits ephrin-dependent repulsive signaling by overexpressed EPHB2 through a mechanism involving downregulation of the HRAS-Erk pathway, suggesting that FGFR1 activation could neutralize the anti-invasive effects of EPHB2 in cancer cells79. In contrast, overexpressed EPHA4 and FGFR1 associate and potentiate each other's oncogenic activities in cultured glioma and other cell types98,99. It will also be interesting to determine whether Eph receptors can downregulate PTEN levels and perhaps activity in cancer cells, as suggested by recent studies in C. elegans80.
Downregulation of EPHA2 or EPHB4 by small interfering RNAs (siRNAs) or antisense oligonucleotides decreases cancer cell malignancy in culture and inhibits tumor growth in a number of mouse cancer models36,37,100–102. Furthermore, EPHA2 overexpression causes oncogenic transformation of mammary epithelial cells in culture as well as in vivo71,103. These experiments demonstrate positive effects of Eph receptors on cancer progression. Given the low levels of Eph forward signaling observed in many cancer cells, these tumor promoting activities are likely to be independent of ephrin stimulation and possibly also of kinase activity. Recent evidence indeed shows that oncogenic signaling pathways can co-opt Eph receptors to increase cancer cell malignancy.
Notable examples of how the altered signaling networks of cancer cells can subvert Eph function involve EPHA2. This receptor has been found to mediate some of the oncogenic activities of EGFR family members, including cancer cell migration in culture and tumor growth and metastasis in a transgenic mouse breast cancer model104,105 (Fig. 4). EPHA2 also appears to be required for Src-dependent invasiveness of colorectal cancer cells in culture90. These effects may be ligand-independent and explained at least in part by the recently discovered crosstalk between EPHA2 and Akt, a serine/threonine kinase frequently activated in cancer cells72 (Figs. 2C and and4).4). Phosphorylation by Akt of a single serine in EPHA2 appears to promote cancer cell migration and invasion, an effect that interestingly does not require EPHA2 kinase activity and is reversed by EPHRIN-A1 stimulation72. It will be important to investigate the details of the Akt/EPHA2 crosstalk and whether other Eph receptors may contribute to cancer progression through analogous mechanisms. EPHA2 has also been recently shown to promote epithelial proliferation and branching morphogenesis in the developing mouse mammary gland by mediating hepatocyte growth factor (HGF)-dependent inhibition of RHOA activity106, which is in contrast to the RHOA activation induced by EPHA2 overexpression, ephrin stimulation, or crosstalk with the ERBB2 receptor103,105,107. It is not yet known if an ephrin-independent EPHA2/HGF receptor crosstalk may play a role in cancer. Ephrin-independent activities of Eph receptors may also include modulation of the subcellular localization of signaling partners that are constitutively associated with an Eph receptor (Fig. 1) or become associated as a result of Eph phosphorylation by other kinases.
With regard to other receptors, the recently discovered ephrin-independent downregulation of β1-integrin levels and cell substrate adhesion by endogenous EPHB4 may promote migration and invasiveness in some cancer cell types although it is inhibitory in others96,97. Additionally, distinctive signaling activities of Eph intracellular domain fragments generated by metalloprotease and γ-secretase cleavage may promote cancer cell malignancy. For example, the EPHA4 cytoplasmic domain released by γ-secretase can enhance RAC1 activity in cultured cells independently of ephrin stimulation and kinase activity24. Furthermore, EPHRIN-B3 stimulation can block apoptosis caused by caspase-dependent cleavage of overexpressed EPHA4 in cultured cells, which interestingly suggests a role for EPHA4 as a “dependence” receptor108.
Little is known about the effects of ephrin-A reverse signaling in epithelial cells. One study has shown that ephrin-A1 is highly upregulated in hepatocellular carcinoma and promotes the proliferation and expression of genes associated with proliferation and invasion in human liver cancer cells109. In fibroblasts, EphA-dependent stimulation of EPHRIN-A5 activates the FYN Src family kinase, integrin-mediated adhesion and Erk MAP kinases5,6 (Fig. 4). Accordingly, EPHRIN-A5 overexpression can increase fibroblast growth in soft agar, invasion and morphological transformation110. Ephrin-B reverse signaling also involves Src family kinases, which phosphorylate the ephrin-B cytoplasmic domain thus regulating its interaction with signaling molecules5,6. Src activation has been proposed to require release of the ephrin-B intracellular domain by metalloprotease and γ-secretase cleavage following EphB binding, which decreases Src association with its inhibitory kinase CSK14. Furthermore, homophilic engagement of claudins, which are tight junction proteins, causes Src-mediated EPHRIN-B1 phosphorylation that slows down the formation of epithelial cell junctions and may thus enhance invasiveness111. This is in contrast to the promotion of tight junction formation due to EPHRIN-B1 phosphorylation discussed above. Whether phosphorylation of different tyrosines, different levels of phosphorylation, or the cellular context may lead to positive versus negative effects of ephrin-Bs on intercellular adhesion remains to be determined.
Other recurring themes in ephrin-B reverse signaling are a localization in lipid rafts and RAC1 activation, which can occur through multiple mechanisms and increase cancer cell migration and invasion112–114 (Fig. 4). For example, EPHRIN-B3 is upregulated in invading cells of glioma biopsies and promotes RAC1-dependent invasion of glioma cell lines112 while EPHRIN-B2 is upregulated in invading cells of glioma and melanoma biopsies and its forced overexpression in the cultured cancer cells enhances integrin-mediated attachment, migration and invasion115,116. Furthermore, EPHRIN-B1 reverse signaling has been reported to induce secretion of matrix metalloprotease 8 (MMP8) and promote invasion of glioma, pancreatic, gastric and leukemic cancer cells in vitro and in mouse tumor models17,113,117.
Ephrin-B reverse signaling may also modulate gene transcription in cancer cells. Ephrin-B1 binds and activates STAT3, a transcription factor involved in cancer progression118 (Fig. 4). Furthermore, in neural progenitors EPHRIN-B1 intracellular domain fragments can localize to the nucleus and bind the ZHX2 transcriptional repressor, potentiating its activity, although it is not known whether this regulation also plays a role in cancer25.
Blood vessels are critical for tumor growth and represent an important venue for metastatic dissemination. Several Eph receptors and ephrins promote angiogenesis by mediating communication of vascular cells with other vascular cells as well as tumor cells. The latter interactions may occur especially during blood vessel growth and in tumor vessels with discontinuous endothelial lining. Furthermore, they may affect not only the endothelial cells but also, reciprocally, tumor cell behavior119.
Analysis of tumors grown in Epha2 mutant mice or mice treated with inhibitory EphA-Fc fusion proteins suggests that EPHA2 forward signaling promotes tumor angiogenesis27,31,56. In contrast, EPHA2 does not seem to play a major role in developmental angiogenesis, and only recently abnormalities in capillary development that may be due to defective pericyte coverage have been revealed in Epha2-deficient mice120. In vitro and in vivo data also show that EPHA2 forward signaling can increase blood vessel permeability, perhaps in part through phosphorylation of claudins8,121. A major ligand for endothelial EPHA2 is EPHRIN-A1, whose upregulation in endothelial cells and consequent activation of EPHA2 have been reported to play an important role in the angiogenic effects of VEGF-A and TNFα52,53. In tumors, EPHRIN-A1 can be expressed by both endothelial and tumor cells52,122,123. Interestingly, the upregulation of EPHA2 and EPHRIN-A1 observed in pancreatic tumors of mice treated with VEGF inhibitors suggests that EPHA2-dependent angiogenesis may contribute to the development of resistance to anti-VEGF therapies, perhaps by promoting endothelial coverage by pericytes and smooth muscle cells120,124. Curiously EPHRIN-A3, another ephrin ligand for EPHA2, is downregulated in hypoxic endothelial cells in culture by the microRNA miR-210 and appears to inhibit angiogenic responses in hypoxic human umbilical vein endothelial cells62. It will be important in future studies to evaluate the combined activities of all relevant EphA receptors and ephrin-A ligands in the regulation of capillary sprouting, vessel permeability and pericyte coverage, as well as their possible redundancies and opposing functions in tumor blood vessels.
EPHB4 and EPHRIN-B2 also play a role in tumor angiogenesis. During development, they are characteristically expressed in the endothelial cells of veins and arteries, respectively, and enable arterial-venous vessel segregation and vascular remodeling55–57,125. The information available so far highlights the importance of EPHRIN-B2 reverse signaling in tumor angiogenesis, while little is known about the role of EPHB4 forward signaling56,126–128. Reverse signaling by EPHRIN-B2, and possibly other ephrin-Bs, in tumor endothelial cells, pericytes and smooth muscle cells likely depends on interaction with several EphB receptors expressed by vascular and/or tumor cells and has been shown to be important for blood vessel assembly, enlargement and decreased permeability both in cell culture and in vivo57,126,127. EPHRIN-B2 signaling also promotes the interaction between endothelial cells and pericytes or vascular smooth muscle cells60,128, suggesting that upregulation of this ephrin may stabilize the vessels of tumors recurring after anti-VEGF therapy129. EPHRIN-B2 in the tumor endothelium may also play additional roles. For example, it may enhance the recruitment of bone marrow-derived endothelial progenitor cells that could participate in tumor vascularization, through a mechanism involving EPHB4-dependent upregulation of selectin ligands130. It will be interesting to determine whether EPHRIN-B2 may also promote extravasation of EphB-positive metastatic tumor cells through the vascular endothelium, similar to its in vitro effect on monocytes58,131.
Eph receptors and ephrins represent promising new therapeutic targets in cancer. A variety of strategies are under evaluation to interfere with their tumor-promoting effects or enhance their tumor-suppressing effects, although our limited mechanistic understanding of the dichotomous Eph activities represents a challenge in the design of therapeutic agents. Other approaches that do not rely on interfering with Eph function involve using Eph receptor-targeting molecules for the selective delivery of drugs, toxins or imaging agents to tumors, and the use Eph-derived antigenic peptides to stimulate anti-tumor immune responses.
Inhibiting the Eph system may be particularly useful for anti-angiogenic therapies, and possibly to overcome resistance to anti-VEGF therapies27,29,55,124,129. Efforts to identify small molecules that target the Eph kinase domain have begun to yield some high affinity inhibitors132–136 (Table 2). Furthermore, a number of inhibitors designed to target other kinases also inhibit Eph receptors. For example dasatinib, a multi-targeted kinase inhibitor already used in the treatment of chronic myelogenous leukemia and under clinical evaluation to treat solid tumors, potently inhibits EPHA2 and other Eph receptors besides its primary targets Abl and Src34,137,138 (http://clinicaltrials.gov/ct2/results?term=epha2). Interestingly, EPHA2 has also been identified as a biomarker for dasatinib sensitivity of cancer cells34,35. Moreover XL647, an orally bioavailable EGF and VEGF receptor inhibitor being evaluated in clinical trials for lung cancer, also targets EPHB4 (http://clinicaltrials.gov/ct2/results?term=EphB4).
Downregulation of EPHA2 or EPHB4 expression with siRNAs or antisense oligonucleotides has been shown to inhibit malignant cell behavior in culture and tumor growth in vivo36,37,100–102 (Table 2). For example, delivery of EPHA2 siRNA to tumors using neutral liposomes inhibits tumor growth and metastasis in mouse models of ovarian cancer, particularly when combined with delivery of siRNA silencing focal adhesion kinase (FAK) or with paclitaxel chemotherapy102,139. Eph receptor levels and function might also be reduced in vivo, as they are in vitro, by drugs that target the chaperone protein HSP90140,141, although other proteins will also be concomitantly downregulated.
Another strategy that shows promise for cancer anti-angiogenic therapy is to inhibit Eph-ephrin interactions. A variety of molecules can be used for this purpose (Table 2). The dimeric EPHA2 ectodomain fused to Fc (which inhibits EPHA forward signaling but promotes reverse signaling) and the monomeric soluble EPHB4 ectodomain (which inhibits both forward and reverse signaling) can both reduce tumor growth in mouse cancer models, at least in part by inhibiting tumor angiogenesis27,31,57,142. Antagonistic antibodies143,144 and peptides that inhibit ephrin binding to individual Eph receptors or subsets of receptors145–147 could be useful for inhibiting Eph-ephrin interactions and bidirectional signaling with greater selectivity than the promiscuous Eph ectodomains. At least two of these peptides bind to the high-affinity ephrin-binding channel of their target receptor148,149. This Eph channel also appears suitable for targeting with chemical compounds, and two isomeric small molecules that preferentially inhibit ephrin binding to EPHA2 and EPHA4, albeit with low affinity, have been identified150,151. Structural characterization of additional small molecules and peptides in complex with Eph receptors may reveal general rules enabling the rational design of chemical compounds capable of selectively targeting Eph receptors with high affinity.
Intriguingly, ephrin ligands and agonistic antibodies have also been successfully used to inhibit tumor progression in mouse cancer models despite being activators rather than inhibitors of Eph-ephrin signaling (Table 2). These agonists have been proposed to act by stimulating Eph forward signaling pathways with tumor suppressor activity and/or receptor degradation in the cancer cells47,152–154. Antibody-dependent cell-mediated cytotoxicity may also contribute to the anti-cancer effects of some of the antibodies155, perhaps explaining the discrepancies in the effectiveness of different EPHA2 antibodies with similar agonistic properties155,156. Eph agonistic antibodies may also be useful in combination with chemotherapy33,153.
Eph-targeting agents likely act through a combination of multiple effects on cancer cells and the tumor microenvironment, which may explain the efficacy of agents with opposite mechanisms of action. For example, EPHA2 agonists would be expected to enhance tumor suppressor signaling pathways and receptor degradation in the cancer cells but promote tumor angiogenesis31. On the other hand, some Eph kinase inhibitors with anti-angiogenic activity might also block possible Eph tumor suppressor activities. Such inhibitors may therefore be particularly effective for the treatment of tumors where Eph forward signaling pathways with tumor suppressor activity are not activated. EPHB4 agonists that also antagonize ephrin binding may be particularly beneficial by both enhancing EPHB4-dependent tumor suppression in cancer cells and inhibiting EPHRIN-B2-dependent angiogenesis47,127. Ultimately, how a tumor will respond to a particular Eph-targeted strategy will likely depend on the tumor type, stage and microenvironment. Selecting optimal strategies to interfere with Eph function for cancer therapy will therefore require a better understanding of Eph signaling mechanisms in the different cellular compartments of tumors. Eph-dependent oncogenic signaling networks may also represent suitable therapeutic targets. Newly developed targeting molecules, in particular those with selectivity for individual Eph receptors or ephrins, in turn represent useful research tools to further our knowledge of Eph cancer biology.
Because of their elevated expression in many tumors compared to normal tissues, Eph receptors also represent attractive targets for the delivery of drugs or toxins and imaging agents to cancer tissue. Several chemotherapeutic drugs and toxins conjugated to Eph antibodies or an ephrin, which cause receptor-mediated drug internalization, appear promising in initial studies (Table 2). EPHA2- or EPHB2-targeting antibodies coupled to derivatives of the peptide drug auristatin, which disrupts microtubule dynamics, inhibit the growth of several cancers in rodent models143,157,158. Another potential application is the targeted delivery of gold-coated nanoshells conjugated to ephrins for photothermal destruction of Eph-positive cancer cells159. Importantly, systemic toxic effects have not been apparent so far and the EPHA2 antibody coupled to an auristatin derivative is under clinical evaluation (http://clinicaltrials.gov/ct2/show/NCT00924235). Notably, targeting Eph surfaces that are preferentially exposed on tumor cells, which may include the ephrin-binding channel, could further improve the therapeutic index152.
Antibodies, ephrins and peptides can also be used to deliver imaging agents for diagnostic purposes. EPHA2 is a particularly attractive target for this application given its widespread expression in both cancer cells and tumor vasculature and low expression in most adult tissues27,56,160. Promising results have been obtained in animal models by using an EPHA2 antibody labeled with 64Cu through the chelating agent 1,4,7,10-tetraazacyclododecane N,N′,N″,N″′-tetraacetic acid (DOTA) for radioimmunoPET imaging and an EPHA3 antibody coupled to 111Indium for gamma camera imaging160,161.
In addition to the immune cell-mediated cytotoxicity that can be elicited by Eph-targeted antibodies, a bispecific single-chain antibody that simultaneously binds both EPHA2 and the T-cell receptor/CD3 complex causes T-cell-mediated destruction of EPHA2-positive tumor cells in vitro and decreases tumor growth in vivo162 (Table 2). Eph receptors that are preferentially expressed in tumors compared to normal tissues are also attractive targets for cancer vaccines. EPHA2, EPHA3 and an EPHB6 isoform have been identified as sources of tumor-associated peptide antigens that are recognized by cancer-specific cytotoxic T-cells163–166. Interestingly, agonists and drugs that stimulate Eph receptor degradation may inhibit tumor growth at least in part by enhancing the presentation of Eph-derived peptides that can be recognized by effector T-cells141,154. Vaccination with Eph-derived epitopes also shows promise as a strategy to elicit tumor rejection167,168.
Accumulating evidence implicates deregulation of the Eph cell communication system in cancer pathogenesis. The Eph receptors are emerging as master regulators capable of either potentiating the activities of oncogenic signaling networks or repressing them, depending on ephrin stimulation and other contextual factors. Remarkably, Eph receptors and ephrins can switch between contrasting activities by using bidirectional signaling as well as other signaling modalities to influence cancer cell behavior. We still know relatively little about how the Eph system regulates tumorigenesis at the molecular level, but clearly there is extensive cell context dependency for many Eph pathways. For example, many of the differences observed in Eph/ephrin signaling outcomes may relate to differences in spatial and temporal coordination of imput signals and relays, and thus vary between cell types and in vitro versus in vivo environments169. An important step forward will be to understand in detail the Eph activities beyond bidirectional signaling and the crosstalk with oncogenic pathways. Furthermore, systems-level studies will be instrumental for: providing a comprehensive overview of the effects of Eph bidirectional and unconventional signaling mechanisms in cancer and stromal cells; comparing the signaling activities of different Eph/ephrin family members; examining the consequences of changes in Eph/ephrin expression, for example to compare the effects of Eph receptor downregulation by agonists and by transcriptional silencing; and elucidating the effects of cancer-relevant Eph/ephrin mutations. Indeed, a recent proteomic analysis combined with siRNA screening and data-driven network modeling has provided a wealth of tantalizing new information on the asymmetric bidirectional signaling networks initiated by ephrin-B1 and EphB2 at sites of cell-cell contact88. Another area of great interest is how the Eph system influences the metastatic process, including tissue invasion, dissemination through the vascular system, possible reversal of epithelial-to-mesenchymal transition at distant sites, and dormancy of Eph-expressing micrometastases seeded in ephrin-rich tissues.
To advance our understanding of Eph cancer biology, it will also be important to examine the effects of Eph or ephrin loss, increased expression, and cancer relevant mutations in genetically engineered mouse models that mimic the progression of human cancers. Such in vivo models are key for studying the Eph system, given its penchant for regulating communication between different cell types, which is difficult to accurately recapitulate in vitro. The mouse models will also be useful for preclinical evaluation of new Eph-based therapies.
Eph and ephrin expression promises to be a powerful predictor of prognosis and perhaps drug sensitivity. For example, increased EPHA2 expression can confer sensitivity to dasatinib but resistance to the ERBB2-targeting antibody trastuzumab33–35. Therefore, there is a need for a comprehensive assessment of Eph and ephrin protein expression in large cohorts of human tumors in correlation with stages of malignancy and clinical outcome. Carefully validated antibodies and quantitative proteomics approaches are needed to ensure the reliability of such studies. Understanding the complexities of the Eph system will contribute to clarify the mechanisms of cancer development, progression and metastasis as well as aid development of novel anti-cancer therapies.
Forward signaling by EPHA2 and several EphB receptors in epithelial and cancer cells can induce morphological changes reminiscent of mesenchymal-to-epithelial transition. For example, stimulation of EPHA2 forward signaling with EPHRIN-A1-Fc in sparse MDCK epithelial cells enhances maturation of cell-cell junctions and cell compaction170,171 (see figure) In a positive feedback loop, E-CADHERIN can promote EPHA2 expression and surface localization in epithelial and cancer cells that have reached high density, thereby prolonging EPHA2 interaction with co-expressed EPHRIN-A1 and forward signaling19,20,170 (Fig. 2D). Stimulation of EPHB2 forward signaling with EPHRIN-B1-Fc can also couple increased intercellular adhesion with cell contraction and apico-basal polarization in colorectal cancer cells by promoting the membrane localization of E-CADHERIN86. Interestingly, the consequences are dramatically different in colorectal cancer cells expressing EPHB2 but lacking E-CADHERIN, where EPHRIN-B1-Fc stimulation causes cell contraction and separation instead of promoting cell-cell adhesion86. Stable transfection of EPHB3 in HT29 colon cancer cells, which endogenously express ephrin-Bs and E-CADHERIN, also causes changes consistent with mesenchymal-to-epithelial transition42. Furthermore, moderate ephrin-B expression and phosphorylation can promote the integrity of adherens and tight junctions in Xenopus and HT29 cells87. Conversely, EPHB4-EPHRIN-B2 antagonists have been shown to disturb intercellular junctions in MCF-10A mammary epithelial cells47. Thus, interplay with E-CADHERIN can convert Eph repulsive signals into signals that promote cell-cell adhesion. It is not known whether a similar interplay may occur with N-CADHERIN, which often replaces E-CADHERIN in malignant cancer cells that have undergone epithelial-to-mesenchymal transition. Studies in normal tissues suggest that Eph receptors can promote N-CADHERIN-dependent adhesion. For example, EPHA4 forward signaling is critical for the N-CADHERIN-dependent mesenchymal-to-epithelial transition that occurs at the borders of developing zebrafish somites172. Interestingly, EPHA2 mutations in humans and EPHA2 or EPHRIN-A5 loss in mice disrupt the N-CADHERIN-dependent intercellular junctions in the lens epithelium, causing cataracts173,174.
The author thanks members of her laboratory for helpful comments on the manuscript. Work in the author's laboratory is supported by grants from the National Institutes of Health, the Department of Defense, the Tobacco-Related Disease Research Program, and Sanford Children's Health.