EphA2 Activation Research Articles

EphA2 blockade enhances the anti-endothelial effect of radiation and inhibits irradiated tumor cell-induced migration of endothelial cells.

EphA2 tyrosine kinase plays an important role in tumor angiogenesis, but whether targeting this pathway can affect response to ionizing radiation (IR) remains unknown. We investigated, using a soluble EphA2-Fc chimera, whether EphA2 inhibition could sensitize A549 and MCF-7 tumor cells, as well as human umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (HDMEC), to IR. EphA2-Fc resulted in a greater response of endothelial cells (EC) to IR than either treatment alone. EphA2-Fc significantly increased apoptosis and decreased clonogenic survival, tube formation and migration in irradiated EC after stimulation with vascular endothelial growth factor (VEGF), without an affecting their proliferation. No difference in proliferation or survival was found in A549 and MCF-7 tumor cells. In a co-culture model, EphA2-Fc inhibited an irradiated A549 cell-induced increase in EC migration. VEGF supplementation, as well as condiotioned medium from irradiated A549 cells, phosphorylated EphA2 in EC. The latter was abrogated by EphA2-Fc. EC were most sensitive to a combination of EphA2 inhibition and radiotherapy. The induction of paracrine growth factors and activation of EphA2 in EC suggest a protective mechanism that tumors probably use to attenuate IR-induced antivascular effects. Our data justify further investigation to explore targeting EphA2 in tumor radiosensitivity in vivo.

The effect of alphastatin peptide suppressing the hypoxia-induced vasculogenic mimicry formation of glioma

Objective To investigate the vasculogenic mimicry formation induced by hypoxia in II-III human glioma cell and the effect of alphastatin peptide suppressing the hypoxia-induced vasculogenic mimicry formation and the mechanism. Methods MTT, Transwell and three- dimentional culture were used to detect the proliferation, migration and tubule formation of SHG44. The expression of vascular endothelial growth factor-a(VEGF-α), erythropoietin-producing hepatocellular carcinoma-A2 (EphA2) and matrix metalloproteinases 2 (MMP2) was detected by RT-PCR and Western blotting analysis. Results The OD 490 in hypoxia group was 0.60±0.06 and in control group was 0.46±0.05. The number of cell migration was 178.71±18.81 in hypoxia group and 85.86±17.92 in control group. The tubule formation was 56.80±12.21 in hypoxia group and 4.20±2.62 in control group. The proliferation, migration and tubule formation in hypoxia group were significantly higher than that in control group. The expression of VEGF-α, EphA2 and MMP2 was upregulated in hypoxia. When various concentrations of alphastatin (100, 1 000, 10 000 nmol/L) were added to hypoxia group, the numbers of cell migration were 142.57±12.12, 92.71±17.68, 30.00±7.72 and the tubule formation were 47.71±10.58, 18.86±8.40, 8.43±5.62. The cell migration and tubule formation were significantly suppressed by alphastatin in a dose-dependent manner. In alphastatin group, the phosphorylation of EphA2 protein ( P=0.037, F=4.629) and activation of MMP2 protein ( P=0.005, F=9.331) were significantly suppressed but there was no change in VEGF-α protein. Conclusion II-III human glioma cell is able to form vasculogenic mimicry induced by hypoxia and alphastatin peptide can suppress the hypoxia-induced vasculogenic mimicry. VEGF-α induced EphA2 phospharilation and MMP2 activation maybe the key pathway to form vasculogenic mimicry.

Ligand Targeting of EphA2 Enhances Keratinocyte Adhesion and Differentiation via Desmoglein 1

EphA2 is a receptor tyrosine kinase that is engaged and activated by membrane-linked ephrin-A ligands residing on adjacent cell surfaces. Ligand targeting of EphA2 has been implicated in epithelial growth regulation by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2)-mitogen activated protein kinase (MAPK) pathway. Although contact-dependent EphA2 activation was required for dampening Erk1/2-MAPK signaling after a calcium switch in primary human epidermal keratinocytes, the loss of this receptor did not prevent exit from the cell cycle. Incubating keratinocytes with a soluble ephrin-A1-Fc peptide mimetic to target EphA2 further increased receptor activation leading to its down-regulation. Moreover, soluble ligand targeting of EphA2 restricted the lateral expansion of epidermal cell colonies without limiting proliferation in these primary cultures. Rather, ephrin-A1-Fc peptide treatment promoted epidermal cell colony compaction and stratification in a manner that was associated with increased keratinocyte differentiation. The ligand-dependent increase in keratinocyte adhesion and differentiation relied largely upon the up-regulation of desmoglein 1, a desmosomal cadherin that maintains the integrity and differentiated state of suprabasal keratinocytes in the epidermis. These data suggest that keratinocytes expressing EphA2 in the basal layer may respond to ephrin-A1-based cues from their neighbors to facilitate entry into a terminal differentiation pathway.

Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.

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.

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

Abstract 5216: An Akt-EphA2 reciprocal regulatory loop controls tumor cell migration and invasion

Abstract EphA2 is a member of Eph family receptor tyrosine kinases that has been implicated in many types of human cancers. Perplexingly, both pro- and anti-oncogenic properties have been attributed to EphA2 kinase. We report here that a possible cause for this apparent paradox is diametrically opposite roles of EphA2 in regulating cell migration and invasion. While activation of EphA2 with its ligand ephrin-A1 inhibited chemotactic migration of glioma and prostate cancer cells, EphA2 overexpression promoted migration in a ligand-independent manner. Surprisingly, the latter effects required phosphorylation of EphA2 on serine 897 by Akt, and S897A mutation abolished ligand-independent promotion of cell motility. Ephrin-A1 stimulation of EphA2 negated Akt activation by growth factors and caused EphA2 dephosphorylation on S897. In human astrocytoma, S897 phosphorylation was correlated with tumor grades and Akt activation, suggesting that the Akt-EphA2 crosstalk may contribute to brain tumor progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5216.

Receptor Cluster Size Affects Signaling in Breast Epithelial Cancer Cells

Our purpose is to study a potentially novel mechanical regulatory mechanism in the EphA2 signaling pathway. EphA2 is a receptor tyrosine kinase that is known to be up-regulated in 40% of human breast cancers and plays an active role in metastasis. Activation of EphA2 occurs after binding to its ligand, presented on an opposing cell membrane. Monomer binding is followed by dimerization of the receptor-ligand complex and then oligomerization. Past research, through predominantly biochemical methods, have concluded that EphA2 signaling depends on the degrees of multimerization of the proteins and the topology of the ligand presentation. However, clustering mechanisms of EphA2 proteins are not well understood because these signaling molecules function in the cell membrane, which is an environment that is difficult to characterize and manipulate. Our hypothesis is that the multi-scale organization of EphA2 in the cell membrane regulates its biochemical function. To mimic the cell-cell junction, we use a supported lipid bilayer - cell membrane hybrid system. Breast cancer cells presenting EphA2 are cultured on a fluid lipid bilayer consisting of ligand fusion proteins, which can stably interact with a subset of capturing lipids within the bilayer. This interaction allows us to control the protein density, precisely image it, and maintain molecular mobility so ligand-induced receptor clustering can occur. Receptor cluster size is varied by changing the cluster size and degrees of oligomerization of its ligand. On the nanometer length scale, antibodies are used to cross link monomeric forms of ligand fusion proteins and thereby vary the degrees of oligomerization. On the micrometer scale, patterned chromium substrates are used to segregate ligands into corrals of variable cluster sizes. Our results suggest that the spatial organization of receptor plays a role in orchestrating the cascade of signaling switches.

EphA2 Mediates Ligand-Dependent Inhibition and Ligand-Independent Promotion of Cell Migration and Invasion via a Reciprocal Regulatory Loop with Akt

Both pro- and antioncogenic properties have been attributed to EphA2 kinase. We report that a possible cause for this apparent paradox is diametrically opposite roles of EphA2 in regulating cell migration and invasion. While activation of EphA2 with its ligand ephrin-A1 inhibited chemotactic migration of glioma and prostate cancer cells, EphA2 overexpression promoted migration in a ligand-independent manner. Surprisingly, the latter effects required phosphorylation of EphA2 on serine 897 by Akt, and S897A mutation abolished ligand-independent promotion of cell motility. Ephrin-A1 stimulation of EphA2 negated Akt activation by growth factors and caused EphA2 dephosphorylation on S897. In human astrocytoma, S897 phosphorylation was correlated with tumor grades and Akt activation, suggesting that the Akt-EphA2 crosstalk may contribute to brain tumor progression.

Expression of the receptor tyrosine kinase EphA2 is increased in smokers and predicts poor survival in non-small cell lung cancer.

Up-regulation of the receptor tyrosine kinase EphA2 has been shown in several epithelial cancers. Epidermal growth factor receptor (EGFR) and K-Ras have been reported to regulate EphA2 in several in vitro models, but this regulation has never been examined in tumors from patients. Because of the established importance of EGFR and K-Ras mutations in non-small cell lung cancer (NSCLC), we investigated the relationship between these mutations and EphA2 in this cancer type. The significance of EphA2 expression was further examined by testing for correlation with other clinical parameters. EphA2 expression was analyzed by immunohistochemistry in tissue microarray format using surgically resected NSCLC specimens (n = 279). EGFR and K-Ras mutation status was determined for most specimens. The correlation between EphA2 expression and EGFR or K-Ras mutation status was examined, along with several clinicopathologic variables of the tumors. The effects of increasing EGFR and K-Ras activity on EphA2 expression and activity were examined in two cell lines. EphA2 expression was detected in >90% of tumor samples. Expression of EphA2 was positively correlated with activated EGFR but not with EGFR mutations. EphA2 expression was increased in patients harboring K-Ras mutations. EphA2 expression was positively correlated with a history of smoking, and high EphA2 scores predicted poorer progression-free and overall survivals. EphA2 expression in NSCLC is associated with K-Ras mutations, EGFR activation, smoking history, and poor prognosis. EphA2 expression is up-regulated in the context of EGFR or K-Ras activation. The potential of EphA2 as a therapeutic target for NSCLC should be further investigated.

Hsp90 Is an Essential Regulator of EphA2 Receptor Stability and Signaling: Implications for Cancer Cell Migration and Metastasis

A subset of Eph receptors and their corresponding ligands are commonly expressed in tumor cells where they mediate biological processes such as cell migration and adhesion, whereas their expression in endothelial cells promotes angiogenesis. In particular, the tumor-specific up-regulation of EphA2 confers properties of increased cellular motility, invasiveness, tumor angiogenesis, and tumor progression, and its overexpression correlates with poor prognosis in several cancer types. The cellular chaperone Hsp90 also plays a significant role in regulating cell migration and angiogenesis, although the full repertoire of motility driving proteins dependent on Hsp90 function remain poorly defined. We explored the hypothesis that Hsp90 may regulate the activity of EphA2 and examined the potential relationship between EphA2 receptor signaling and chaperone function. We show that geldanamycin, an Hsp90 antagonist, dramatically destabilizes newly synthesized EphA2 protein and diminishes receptor levels in a proteasome-dependent pathway. In addition, geldanamycin treatment impairs EphA2 signaling, as evidenced by a decrease in ligand-dependent receptor phosphorylation and subsequent cell rounding. Therefore, Hsp90 exerts a dual role in regulating the stability of nascent EphA2 protein and maintaining the signaling capacity of the mature receptor. Our findings also suggest that the geldanamycin-dependent mitigation of EphA2 signaling in receptor-overexpressing cancer cells may be sufficient to recapitulate the antimotility effects of this drug. Finally, the identification of a pharmacologic approach to suppress EphA2 expression and signaling highlights the attractive possibility that Hsp90 inhibitors may have clinical utility in antagonizing EphA2-dependent tumorigenic progression.

Kinase-Dependent and -Independent Roles of EphA2 in the Regulation of Prostate Cancer Invasion and Metastasis

Ligand-activated Eph tyrosine kinases regulate cellular repulsion, morphology, adhesion, and motility. EphA2 kinase is frequently up-regulated in several different types of cancers, including prostate, breast, colon, and lung carcinomas, as well as in melanoma. The existing data do not clarify whether EphA2 receptor phosphorylation or its simple overexpression, which likely leads to Eph kinase-independent responses, plays a role in the progression of malignant prostate cancer. In this study, we address the role of EphA2 tyrosine phosphorylation in prostate carcinoma cell adhesion, motility, invasion, and formation of metastases. Tumor cells expressing kinase-deficient EphA2 mutants, as well as an EphA2 variant lacking the cytoplasmic domain, are defective in ephrinA1-mediated cell rounding, retraction fiber formation, de-adhesion from the extracellular matrix, RhoA and Rac1 GTPase regulation, three-dimensional matrix invasion, and in vivo metastasis, suggesting a key role for EphA2 kinase activity. Nevertheless, EphA2 regulation of cell motility and invasion, as well as the formation of bone and visceral tumor colonies, reveals a component of both EphA2 kinase-dependent and -independent features. These results uncover a differential requirement for EphA2 kinase activity in the regulation of prostate carcinoma metastasis outcome, suggesting that although the kinase activity of EphA2 is required for the regulation of cell adhesion and cytoskeletal rearrangement, some distinct kinase-dependent and -independent pathways likely cooperate to drive cancer cell migration, invasion, and metastasis outcome.

EphA2 Reexpression Prompts Invasion of Melanoma Cells Shifting from Mesenchymal to Amoeboid-like Motility Style

Eph tyrosine kinases instruct cell for a repulsive behavior, regulating cell shape, adhesion, and motility. Beside its role during embryogenesis, neurogenesis, and angiogenesis, EphA2 kinase is frequently up-regulated in tumor cells of different histotypes, including prostate, breast, colon, and lung carcinoma, as well as melanoma. Although a function in both tumor onset and metastasis has been proposed, the role played by EphA2 is still debated. Here, we showed that EphA2 reexpression in B16 murine melanoma cells, which use a defined mesenchymal invasion strategy, converts their migration style from mesenchymal to amoeboid-like, conferring a plasticity in tumor cell invasiveness. Indeed, in response to reexpression and activation of EphA2, melanoma cells activate a nonproteolytic invasive program that proceeds through the activation of cytoskeleton motility, the retraction of cell protrusions, a Rho-mediated rounding of the cell body, and squeezing among three-dimensional matrix, giving rise to successful lung and peritoneal lymph node metastases. Our results suggest that, among the redundant mechanisms operating in tumor cells to penetrate the anatomic barriers of host tissues, EphA2 plays a pivotal role in the adaptive switch in migration pattern and mechanism, defining and distinguishing tumor cell invasion strategies. Thus, targeting EphA2 might represent a future approach for the therapy of cancer dissemination.

Loss of EphA2 receptor tyrosine kinase reduces Apcmin/+ tumorigenesis

The Eph receptor A2 (EphA2) is overexpressed in a range of human epithelial cancers, a phenotype that is associated with cancer cell proliferation, progression and angiogenesis. Mouse models of mammary neoplasia have confirmed the role of EphA2 as mice carrying a knockout allele of EphA2 were resistant to breast cancer, a phenotype that was associated with interactions between EphA2 and ErbB2. We investigated in vivo the role of EphA2 in GI cancer. To determine whether EphA2 influences intestinal tumorigenesis, we used qRT-PCR to examine the mRNA expression levels of EphA2 in tumors from the small intestine and colon of Apc(Min/+) mice. We found that EphA2 was significantly up-regulated in tumors from both regions when compared with normal control tissues. We then evaluated the spatial expression patterns of EphA2 protein using immunohistochemistry in both the small intestine and colon and found that in normal tissues EphA2 was robustly expressed in highly differentiated cells, such as cells of the villi, but that EphA2 expression was largely absent from the stem cell niche and proliferative zones of intestinal crypts. In contrast, in tumors EphA2 was broadly expressed. Finally, we created a strain of Apc(Min/+) mice carrying a genetic knockout of the EphA2 gene. These mice developed significantly fewer and smaller tumors in both the small and large intestine. Overall, our results indicate that EphA2 plays an oncogenic role in the mammalian intestine suggesting that strategies to target EphA2 activity may offer new therapeutic modalities for colorectal cancer.

179 POSTER Phase I study of the novel anti-cancer drug PM00104 as a 1-hour weekly infusion resting every fourth week in patients with advanced solid tumors or lymphoma

The Eph receptor/ephrin system is a recently discovered regulator of vascular development during embryogenesis. Activation of EphA2, one of the Eph receptors, reportedly suppresses cell proliferation and adhesion in a wide range of cell types, including vascular endothelial cells. Vascular endothelial growth factor (VEGF) plays a primary role in both pathological angiogenesis and abnormal vascular leakage in diabetic retinopathy. In the study described herein, we demonstrated that EphA2 stimulation by ephrinA1 in cultured bovine retinal endothelial cells inhibits VEGF-induced VEGFR2 receptor phosphorylation and its downstream signaling cascades, including PKC (protein kinase C)-ERK (extracellular signal-regulated kinase) 1/2 and Akt. This inhibition resulted in the reduction of VEGF-induced angiogenic cell activity, including migration, tube formation, and cellular proliferation. These inhibitory effects were further confirmed in animal models. Intraocular injection of ephrinA1 suppressed ischemic retinal neovascularization in a dose-dependent manner in a mouse model. At a dose of 125 ng/eye, the inhibition was 36.0 ± 14.9% (P < 0.001). EphrinA1 also inhibited VEGF-induced retinal vascular permeability in a rat model by 46.0 ± 10.0% (P < 0.05). These findings suggest a novel therapeutic potential for EphA2/ephrinA1 in the treatment of neovascularization and vasopermeability abnormalities in diabetic retinopathy.

Preclinical evaluation of dasatinib, a potent Src kinase inhibitor, in melanoma cell lines

BackgroundMetastatic melanoma is a highly chemotherapy resistant tumour. The use of newer targeted therapies alone and in combination with chemotherapy may offer new hope of improving response to treatment. Dasatinib, a multi-target kinase inhibitor, is currently approved for the treatment of chronic myeloid leukaemia and has shown promising results in preclinical studies in a number of solid tumours.MethodsWe examined the effects of dasatinib on proliferation, chemo-sensitivity, cell cycle arrest, apoptosis, migration and invasion in human melanoma cell lines. Expression and activation of Src kinase, FAK and EphA2 were also examined in the melanoma cells.ResultsDasatinib inhibited growth of three of the five melanoma cell lines. Comparison with sorafenib showed that in these three cell lines dasatinib inhibited growth at lower concentrations than sorafenib. Dasatinib in combination with the chemotherapy drug temozolomide showed greater efficacy than either drug alone. Dasatinib induced cell cycle arrest and apoptosis and significantly inhibited cell migration and invasion of melanoma cells. Dasatinib inhibition of proliferation was associated with reduced phosphorylation of Src kinase, while decreased phosphorylation of FAK was implicated in dasatinib-mediated inhibition of migration and invasion in melanoma cells.ConclusionDasatinib has both anti-proliferative and anti-invasive effects in melanoma cells and combined with chemotherapy may have clinical benefit in the treatment of malignant melanoma.

Overexpression of EPHA2 receptor destabilizes adherens junctions via a RhoA-dependent mechanism

EPHA2 receptor tyrosine kinase is overexpressed in several human cancer types and promotes malignancy. However, the mechanisms by which EPHA2 promotes tumor progression are not completely understood. Here we report that overexpression of a wild-type EPHA2, but not a signaling-defective cytoplasmic truncation mutant (DeltaC), in human mammary epithelial cells weakens E-cadherin-mediated cell-cell adhesion. Interestingly, the total level of cadherins and the composition of the adherens junction complexes were not affected, nor was the tyrosine phosphorylation of the cadherin complex components changed. By contrast, RhoA GTPase activity was significantly affected by modulating the EPHA2 activity in MCF-10A cells. Treatment with a ROCK kinase inhibitor rescued cell-cell adhesion defects in EPHA2-overexpressing cells, whereas expression of constitutively activated Rho disrupted adherens junctions in DeltaC-expressing cells. EPHA2-dependent Rho activation and destabilization of adherens junctions appeared to be regulated via a signaling pathway involving Src kinase, low molecular weight phosphotyrosine phosphatase (LMW-PTP) and p190 RhoGAP. EPHA2 interacted with both Src and LMW-PTP, and the interactions increased in EPHA2-overexpressing cells. In addition, LMW-PTP phosphatase activity was elevated, and this elevation was accompanied by a decrease in tyrosine phosphorylation of p190 RhoGAP and destabilization of cell-cell adhesion. Expression of either a dominant negative LMW-PTP mutant, C12S, or a wild-type p190 RhoGAP rescued adhesion defects in EPHA2-overexpressing cells. Together, these data suggest that EPHA2 promotes tumor malignancy through a mechanism involving RhoA-dependent destabilization of adherens junctions.

The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling

Overexpression of the receptor tyrosine kinase EPH receptor A2 (EphA2) is commonly observed in aggressive breast cancer and correlates with a poor prognosis. However, while EphA2 has been reported to enhance tumorigenesis, proliferation, and MAPK activation in several model systems, other studies suggest that EphA2 activation diminishes these processes and inhibits the activity of MAPK upon ligand stimulation. In this study, we eliminated EphA2 expression in 2 transgenic mouse models of mammary carcinoma. EphA2 deficiency impaired tumor initiation and metastatic progression in mice overexpressing ErbB2 (also known as Neu) in the mammary epithelium (MMTV-Neu mice), but not in mice overexpressing the polyomavirus middle T antigen in mammary epithelium (MMTV-PyV-mT mice). Histologic and ex vivo analyses of MMTV-Neu mouse mammary epithelium indicated that EphA2 enhanced tumor proliferation and motility. Biochemical analyses revealed that EphA2 formed a complex with ErbB2 in human and murine breast carcinoma cells, resulting in enhanced activation of Ras-MAPK signaling and RhoA GTPase. Additionally, MMTV-Neu, but not MMTV-PyV-mT, tumors were sensitive to therapeutic inhibition of EphA2. These data suggest that EphA2 cooperates with ErbB2 to promote tumor progression in mice and may provide a novel therapeutic target for ErbB2-dependent tumors in humans. Moreover, EphA2 function in tumor progression appeared to depend on oncogene context, an important consideration for the application of therapies targeting EphA2.

Receptor EphA2 activation with ephrinA1 suppresses growth of malignant mesothelioma (MM)

The objective of this study was to understand the possible mechanisms of activation of receptor EphA2 by its ligand ephrinA1 in malignant mesothelioma cell (MMC) growth. Activation of receptor EphA2 by its ligand ephrinA1 triggered the phosphorylation of EphA2. Ligand activation of EphA2 also induced phosphorylation of ERK1/ 2 and significantly decreased MMC proliferation. Ligand activated and ephrinA1 vector (pcDNA/EFNA1) transfected MMC demonstrated decreased clonal growth in 3-D matrigels when compared to resting MMC. These studies suggest that EphA2 activation by its ligand ephrinA1 transmits intracellular signals from cell membrane to nucleus via ERK1/ 2 signaling cascade and inhibits MM growth.

Ephrin-A1 is a negative regulator in glioma through down-reguation of EphA2 and FAK

Eph receptors, the largest receptor tyrosine kinases, and their ephrin ligands play important roles in axon guidance and cell migration during development of the nervous system. Recently, these molecules are also found involved in tumorigenesis of different kinds of cancers. In this study, we demonstrated that expression of ephrin-A1 was dramatically down-regulated in glioma cell lines and in primary gliomas compared to the matched normal tissues. Forced expression of ephrin-A1 attenuated cell migration, cell proliferation, and adhesion-independent growth in human glioma U251 cells. EphA2, a receptor for ephrin-A1 and an oncoprotein, was greatly decreased in ephrin-A1-transfected glioma cells. Overexpression of ephrin-A1 stimulated activation of EphA2 by phosphorylation and led to its degradation. Furthermore, focal adhesion kinase (FAK), a known downstream molecule of EphA2, was also down-regulated in the ephrin-A1 transfected cells. These results suggested that ephrin-A1 serves as a critical negative regulator in the tumorigenesis of gliomas by down-regulating EphA2 and FAK, which may provide potential valuable targets for therapeutic intervention.