Ephexin4 and EphA2 mediate resistance to anoikis through RhoG and phosphatidylinositol 3-kinase

Disruption of cell–extracellular matrix interaction causes epithelial cells to undergo apoptosis called anoikis, and resistance to anoikis has been suggested to be a critical step for cancer cells to metastasize. EphA2 is frequently overexpressed in a variety of human cancers, and recent studies have found that overexpression of EphA2 contributes to malignant cellular behavior, including resistance to anoikis, in several different types of cancer cells. Here we show that Ephexin4, a guanine nucleotide exchange factor for the small GTPase RhoG that interacts with EphA2, plays an important role in the regulation of anoikis. Knockdown of Ephexin4 promoted anoikis in HeLa cells, and experiments using a knockdown-rescue approach showed that activation of RhoG, phosphatidylinositol 3-kinase (PI3K), and Akt was required for the Ephexin4-mediated suppression of anoikis. Indeed, Ephexin4 knockdown caused a decrease in RhoG activity and Akt phosphorylation in HeLa cells cultured in suspension. In addition, Ephexin4 was involved in the EphA2-mediated suppression of anoikis. Taken together, these results suggest that Ephexin4 mediates resistance to anoikis through activation of RhoG and PI3K downstream of EphA2.     

Ephexin4 and EphA2 mediate cell migration through a RhoG-dependent mechanism

EphA2, a member of the Eph receptor family, is frequently overexpressed in a variety of human cancers, including breast cancers, and promotes cancer cell motility and invasion independently of its ligand ephrin stimulation. In this study, we identify Ephexin4 as a guanine nucleotide exchange factor (GEF) for RhoG that interacts with EphA2 in breast cancer cells, and knockdown and rescue experiments show that Ephexin4 acts downstream of EphA2 to promote ligand-independent breast cancer cell migration and invasion toward epidermal growth factor through activation of RhoG. The activation of RhoG recruits its effector ELMO2 and a Rac GEF Dock4 to form a complex with EphA2 at the tips of cortactin-rich protrusions in migrating breast cancer cells. In addition, the Dock4-mediated Rac activation is required for breast cancer cell migration. Our findings reveal a novel link between EphA2 and Rac activation that contributes to the cell motility and invasiveness of breast cancer cells.     

Interactions between EGFR and EphA2 promote tumorigenesis through the action of Ephexin1

The cell signaling factors EGFR, EphA2, and Ephexin1 are associated with lung and colorectal cancer and play an important role in tumorigenesis. Although the respective functional roles of EGFR and EphA2 are well known, interactions between these proteins and a functional role for the complex is not understood. Here, we showed that Ephexin1, EphA2, and EGFR are each expressed at higher levels in lung and colorectal cancer patient tissues, and binding of EGFR to EphA2 was associated with both increased tumor grade and metastatic cases in both cancer types. Treatment with Epidermal Growth Factor (EGF) induced binding of the RR domain of EGFR to the kinase domain of EphA2, and this binding was promoted by Ephexin1. Additionally, the AKT-mediated phosphorylation of EphA2 (at Ser897) promoted interactions with EGFR, pointing to the importance of this pathway. Two mutations in EGFR, L858R and T790M, that are frequently observed in lung cancer patients, promoted binding to EphA2, and this binding was dependent on Ephexin1. Our results indicate that the formation of a complex between EGFR, EphA2, and Ephexin1 plays an important role in lung and colorectal cancers, and that inhibition of this complex may be an effective target for cancer therapy.Subject terms: Oncogenes, Cancer models     

RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85alpha and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.     

The cystine/glutamate antiporter xCT is a key regulator of EphA2 S897 phosphorylation under glucose-limited conditions

EphA2, which belongs to the Eph family of receptor tyrosine kinases, is overexpressed in a variety of human cancers. Serine 897 (S897) phosphorylation of EphA2 is known to promote cancer cell migration and proliferation in a ligand-independent manner. In this study, we show that glucose deprivation induces S897 phosphorylation of EphA2 in glioblastoma cells. The phosphorylation requires the activity of the cystine/glutamate antiporter xCT and reactive oxygen species (ROS)-dependent ERK and RSK activation. Furthermore, depletion of EphA2 in glioblastoma cells leads to decreased cell viability under glucose starvation. Our results suggest a role of EphA2 in glioblastoma cell viability under glucose-limited conditions.     

Protein kinase C phosphorylates the EphA2 receptor on serine 892 in the regulatory linker connecting the kinase and SAM domains

The EphA2 receptor tyrosine kinase signals through two distinct mechanisms, one regulated by tyrosine phosphorylation and the other by serine/threonine phosphorylation. Serine 892 (S892) is one of the major serine/threonine phosphorylation sites in EphA2, but little is known about its regulation and function. S892 is located in the linker connecting the EphA2 kinase and SAM domains, and is part of a cluster of five phosphorylated residues that includes the well characterized S897. EphA2 can be phosphorylated on S897 by the RSK, AKT and PKA kinases to promote a non-canonical form of signaling that plays an important role in cancer malignancy. Here we show that the Protein Kinase C (PKC) family phosphorylates the EphA2 S892 motif in vitro and in cells. By using a newly developed phosphospecific antibody, we detected EphA2 S892 phosphorylation in a variety of cell lines. As expected for a PKC target site, the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) increases S892 phosphorylation whereas the broad-spectrum PKC inhibitor Go 6983 inhibits both basal and TPA-induced S892 phosphorylation. Besides phosphorylating S892, PKC can also increase EphA2 phosphorylation on S897 through the MEK kinase, which regulates the ERK-RSK signaling axis. We also found that S892 and S897 phosphorylation induced by PKC activation can be downregulated by ephrin ligand-induced EphA2 canonical signaling. Our data reveal that the PKC family contributes to the phosphorylation cluster in the EphA2 kinase-SAM linker, which regulates EphA2 non-canonical signaling and cancer malignancy.     

EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling

The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression.     

EphA2 induces metastatic growth regulating amoeboid motility and clonogenic potential in prostate carcinoma cells

EphA2 kinase regulates cell shape, adhesion, and motility and is frequently overexpressed in several cancers, including melanoma, prostate, breast, and colon cancers and lung carcinoma. Although a function in both tumor onset and metastasis has been proposed, the role played by EphA2 in tumor progression is still debated. In melanoma, EphA2 has been reported to affect cell migration and invasiveness allowing cells to move by a proteolysis-independent strategy, commonly referred as amoeboid motility. With the aim to understand the role of EphA2 in prostate cancer metastatic spreading, we stably silenced EphA2 expression in a model of aggressive metastatic prostate carcinoma. Our results show that EphA2 drives the metastatic program of prostate carcinoma, although its involvement greatly differs among metastatic steps. Indeed, EphA2 expression (i) greatly affects prostate carcinoma cell motility style, guiding an amoeboid movement based on Rho-mediated cell rounding and independent from metalloprotases, (ii) is ineffective on transendothelial migration, adhesion onto extracellular matrix proteins, and on resistance to anoikis, (iii) regulates clonogenic potential of prostate carcinoma, thereby increasing anchorage-independent growth and self-renewal, prostasphere formation, tumor onset, dissemination to bone, and growth of metastatic colonies. Our finding indicate that EphA2-overexpressing prostate carcinoma cells gain an invasive benefit from their amoeboid motility style to escape from primary tumors and then, enhancing their clonogenic potential successfully target bone and grow metastases, thereby acknowledging EphA2 as a target for antimetastatic therapy of aggressive prostate cancers.     

Distinctive Structure of the EphA3/Ephrin-A5 Complex Reveals a Dual Mode of Eph Receptor Interaction for Ephrin-A5

The Eph receptor tyrosine kinase/ephrin ligand system regulates a wide spectrum of physiological processes, while its dysregulation has been implicated in cancer progression. The human EphA3 receptor is widely upregulated in the tumor microenvironment and is highly expressed in some types of cancer cells. Furthermore, EphA3 is among the most highly mutated genes in lung cancer and it is also frequently mutated in other cancers. We report the structure of the ligand-binding domain of the EphA3 receptor in complex with its preferred ligand, ephrin-A5. The structure of the complex reveals a pronounced tilt of the ephrin-A5 ligand compared to its orientation when bound to the EphA2 and EphB2 receptors and similar to its orientation when bound to EphA4. This tilt brings an additional area of ephrin-A5 into contact with regions of EphA3 outside the ephrin-binding pocket thereby enlarging the size of the interface, which is consistent with the high binding affinity of ephrin-A5 for EphA3. This large variation in the tilt of ephrin-A5 bound to different Eph receptors has not been previously observed for other ephrins.     

Claudin-4 controls the receptor tyrosine kinase EphA2 pro-oncogenic switch through ?-catenin

Background

The EphA2 receptor, which is expressed in many types of cancer, is activated by two different mechanisms. Activation by engagement with one of its ephrin ligands is anti-oncogenic whereas phosphorylation of S897 by AKT increases migration, invasion and metastasis. Down-regulation of claudin-4 (CLDN4) produces a loss of E-cadherin and increased ?-catenin signaling and a phenotype similar to that produced by oncogenic activation of EphA2, suggesting that CLDN4 may serve to restrain the pro-oncogenic signaling of EphA2.

Results

We found that constitutive knockdown of CLDN4 was associated with a 4.5-fold increase in EphA2 mRNA and a 2.5-fold increase in EphA2 protein which was reversible by re-expression of CLDN4. Knockdown of EphA2 blocked the migratory phenotype induced by loss of CLDN4. Knockdown of CLDN4 resulted in a 5.8-fold increase in pEphA(S897), the oncogenic form of the receptor, as well as partial mislocalization of the excess EphA2 to the interior of the cell. Forced expression of E-cadherin did not reduce total EphA2 or pEphA(S897) whereas re-expression of CLDN4 restored localization and reduced EphA2 and pEphA(S897) even in cells not expressing E-cadherin. Transient siRNA-mediated knockdown of EphA2 and ?-catenin, and inhibition of PI3K by LY294002, demonstrated that increased pEphA(S897) in the CLDN4 knockdown cells was attributable to an increase in the level of active dephospho-?-catenin upstream of PI3K and AKT.

Conclusions

We conclude that CLDN4 serves to restrain pro-oncogenic signaling from EphA2 by limiting the activity of ?-catenin and PI3K and preventing phosphorylation of EphA2 on S897 by AKT. This suggests that interventions directed at enhancing the level or functional activity of CLDN4 may be of therapeutic interest.

     

Crystal structure and NMR binding reveal that two small molecule antagonists target the high affinity ephrin-binding channel of the EphA4 receptor

The Eph receptor tyrosine kinases regulate a variety of physiological and pathological processes not only during development but also in adult organs, and therefore they represent a promising class of drug targets. The EphA4 receptor plays important roles in the inhibition of the regeneration of injured axons, synaptic plasticity, platelet aggregation, and likely in certain types of cancer. Here we report the first crystal structure of the EphA4 ligand-binding domain, which adopts the same jellyroll beta-sandwich architecture as shown previously for EphB2 and EphB4. The similarity with EphB receptors is high in the core beta-stranded regions, whereas large variations exist in the loops, particularly the D-E and J-K loops, which form the high affinity ephrin binding channel. We also used isothermal titration calorimetry, NMR spectroscopy, and computational docking to characterize the binding to EphA4 of two small molecules, 4- and 5-(2,5 dimethyl-pyrrol-1-yl)-2-hydroxybenzoic acid which antagonize ephrin-induced effects in EphA4-expressing cells. We show that the two molecules bind to the EphA4 ligand-binding domain with K(d) values of 20.4 and 26.4 microm, respectively. NMR heteronuclear single quantum coherence titrations revealed that upon binding, both molecules significantly perturb EphA4 residues Ile(31)-Met(32) in the D-E loop, Gln(43) in the E beta-strand, and Ile(131)-Gly(132) in the J-K loop. Molecular docking shows that they can occupy a cavity in the high affinity ephrin binding channel of EphA4 in a similar manner, by interacting mainly with the EphA4 residues in the E strand and D-E and J-K loops. However, many of the interactions observed in Eph receptor-ephrin complexes are absent, which is consistent with the small size of the two molecules and may account for their relatively weak binding affinity. Thus, our studies provide the first published structure of the ligand-binding domain of an EphA receptor of the A subclass. Furthermore, the results demonstrate that the high affinity ephrin binding channel of the Eph receptors is amenable to targeting with small molecule antagonists and suggest avenues for further optimization.     

Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in adult mouse forebrain

The ephrin receptors EphA4 and EphB2 have been implicated in synaptogenesis and long-term potentiation in the cerebral cortex and hippocampus, where they are generally viewed as post-synaptic receptors. To determine the precise distribution of EphA4 and EphB2 in mature brain synapses, we used subcellular fractionation and electron microscopy to examine the adult mouse forebrain/midbrain. EphA4 and EphB2 were both enriched in microsomes and synaptosomes. In synaptosomes, they were present in the membrane and the synaptic vesicle fractions. While EphA4 was tightly associated with PSD-95-enriched post-synaptic density fractions, EphB2 was easily extracted with detergents. In contrast, both receptors were found in the pre-synaptic active zone fraction. By electron microscopy, EphA4 was mainly detected in axon terminals, whereas EphB2 was more frequently detected in large dendritic shafts, in the hippocampus and cerebral cortex. However, in the ventrobasal thalamus, EphB2 was detected most frequently in axon terminals and thin dendritic shafts. The localization of EphA4 and EphB2 in multiple compartments of neurons and synaptic junctions suggests that they interact with several distinct scaffolding proteins and play diverse roles at synapses.     

MicroRNA-335 suppresses the proliferation,migration, and invasion of breast cancer cells by targeting EphA4

MicroRNAs (miRNAs) are small noncoding RNAs that exert their functions by targeting specific mRNA sequences. Many studies have demonstrated that miRNAs are crucial for cancer progression, during which they can act as either oncogenes or tumor suppressors. Previous research has shown that miR-335 is downregulated in breast cancer, and it has been shown to be a breast cancer suppressor. In addition, emerging evidence indicates that erythropoietin-producing hepatocellular A4 (EphA4) is implicated in cancer cell proliferation, migration, and invasion. However, little is known about the relationship between miR-335 and EphA4 in breast cancer. In the present study, we used bioinformatic and biochemical analyses to demonstrate that EphA4 is a direct downstream target of miR-335 in human breast cancer MCF-7 and MDA-MB-23 cells and revealed that miR-335 negatively regulates the expression of EphA4 in these cells. Further investigation revealed that miR-335 overexpression inhibits MCF-7 and MDA-MB-231 cell proliferation and that this inhibition is attenuated by EphA4 coexpression. Similarly, miR-335 overexpression also inhibited growth and downregulated EphA4 expression in tumors in nude mice. Moreover, our results demonstrated that miR-335 overexpression suppresses migration and invasion in MCF-7 and MDA-MB-231 cells, an effect that was reversed by EphA4 overexpression. These findings confirmed that EphA4 is a direct target gene of miR-335 and that miR-335 suppresses breast cancer cell proliferation and motility in part by directly inhibiting EphA4 expression.     

A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers

BackgroundThe EphA2 receptor tyrosine kinase is known to promote cancer cell malignancy in the absence of activation by ephrin ligands. This behavior depends on high EphA2 phosphorylation on Ser897 and low tyrosine phosphorylation, resulting in increased cell migration and invasiveness. We have previously shown that EphA2 forms dimers in the absence of ephrin ligand binding, and that dimerization of unliganded EphA2 can decrease EphA2 Ser897 phosphorylation. We have also identified a small peptide called YSA, which binds EphA2 and competes with the naturally occurring ephrin ligands.MethodsHere, we investigate the effect of YSA on EphA2 dimer stability and EphA2 function using quantitative FRET techniques, Western blotting, and cell motility assays.ResultsWe find that the YSA peptide stabilizes the EphA2 dimer, increases EphA2 Tyr phosphorylation, and decreases both Ser897 phosphorylation and cell migration.ConclusionsThe experiments demonstrate that the small peptide ligand YSA reduces EphA2 Ser897 pro-tumorigenic signaling by stabilizing the EphA2 dimer.General significanceThis work is a proof-of-principle demonstration that EphA2 homointeractions in the plasma membrane can be pharmacologically modulated to decrease the pro-tumorigenic signaling of the receptor.     

EphB-mediated degradation of the RhoA GEF Ephexin5 relieves a developmental brake on excitatory synapse formation

The mechanisms that promote excitatory synapse formation and maturation have been extensively studied. However, the molecular events that limit excitatory synapse development so that synapses form at the right time and place and in the correct numbers are less well understood. We have identified a RhoA guanine nucleotide exchange factor, Ephexin5, which negatively regulates excitatory synapse development until EphrinB binding to the EphB receptor tyrosine kinase triggers Ephexin5 phosphorylation, ubiquitination, and degradation. The degradation of Ephexin5 promotes EphB-dependent excitatory synapse development and is mediated by Ube3A, a ubiquitin ligase that is mutated in the human cognitive disorder Angelman syndrome and duplicated in some forms of Autism Spectrum Disorders (ASDs). These findings suggest that aberrant EphB/Ephexin5 signaling during the development of synapses may contribute to the abnormal cognitive function that occurs in Angelman syndrome and, possibly, ASDs.