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Receptor EphA2 activation with ephrinA1 suppresses growth of malignant mesothelioma (MM)
Available online at www.sciencedirect.com
Cancer Letters 258 (2007) 215–222 www.elsevier.com/locate/canlet
Receptor EphA2 activation with ephrinA1 suppresses growth of malignant mesothelioma (MM) q Najmunnisa Nasreen, Kamal A. Mohammed, Yimu Lai, Veena B. Antony
*
Division of Pulmonary & Critical Care Medicine, Department of Medicine, University of Florida, P.O. Box 100225, Gainesville, FL, USA Received 26 June 2007; received in revised form 6 September 2007; accepted 10 September 2007
Abstract 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. Published by Elsevier Ireland Ltd. Keywords: EphA2; EphrinA1; Malignant mesothelioma; Receptor tyrosine kinase; Proliferation; ERK1/2
1. Introduction Malignant mesothelioma (MM), an asbestos related tumor of the pleura and peritoneum, remains a highly lethal malignancy. Approximately 3000 new cases of MM are diagnosed annually, in United States. Recent evidence suggests that SV40 also a co-factor for the development of MM [1]. The incidence of mesothelioma is expected to increase in Europe and Australia [2,3]. The treatment of MM has been disappointing because most q This work was supported by NIH RO1 AI 45338-02 grant from the National Institute of Health. * Corresponding author. Tel.: +1 352 392 7329; fax: +1 352 271 4559. E-mail address: [email protected]fl.edu (V.B. Antony).
0304-3835/$ - see front matter Published by Elsevier Ireland Ltd. doi:10.1016/j.canlet.2007.09.005
patients die within 12 months after the diagnosis [4]. Although palliative therapy is beneficial to some extent, the response to other treatment modalities such as surgery, radiation and chemotherapy remains poor. Failure of conventional therapeutic approach has led to the interest in identifying novel molecular markers that could be therapeutic targets to restrain tumor growth. Mesothelioma cells exhibit dysregulated growth and produce growth factors [5,6]. We recently reported that receptor EphA2 is overexpressed in MM and blockade of receptor expression decreased MMC proliferation and migration significantly [7]. MM is known to express a variety of receptor tyrosine kinases, such as epidermal growth factor (EGFR), Met, platelet-derived growth factor receptor, vascular endothelial growth factor receptor and
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EphB4 receptor [8–11]. The Eph transmembrane tyrosine kinases constitute the largest family of receptor tyrosine kinases. The Eph receptors are divided into two classes, A and B based on the sequence homology, structure and binding affinity [12]. Currently, 14 Eph receptors and eight ephrin ligands are recognized in humans. EphA receptors predominantly interact with class A ligands and B class receptors interact with B class ligands with few exceptions [13]. EphA2 receptor tyrosine kinase is overexpressed in aggressive cancers [14–18]. While it has been well documented that EphA2 is overexpressed in MM [7], the mechanisms which governs EphA2 expression in this aggressive tumor are not clearly understood. In cancer cells the cell–cell contacts are highly unstable which precludes intercellular contacts. EphA2 binds to the ligands that are anchored to the cell membrane of adjacent cells. In MM this interaction could be critical because the ligand binding of EphA2 regulates cell growth and invasion [19–21]. Ligand and receptor engagement leads to dimerization and internalization of the receptors. Autophosphorylation of the receptor provides recruitment of SH2 domain containing proteins such as PI3 kinase, Src family kinases which are critical signaling intermediates for downstream signaling pathway [22,23]. EphA2 activation with its ligand mediate the downstream signal transduction and modulate malignant behavior [24,25]. The ligation of EphA2 by ephrinA1-Fc inhibit the invasiveness in pancreatic adenocarcinoma via activation of focal adhesion kinase [21]. ERK1/2 MAP kinase pathways have been associated with EphA2 overexpression in malignant epithelial cells [26,27]. However, the molecular mechanisms involved in the activation of EphA2 receptors are not known in MM. In the present study we evaluated the biological outcome of receptor EphA2 activation in MM growth using recombinant ephrinA1-Fc. EphrinA1 ligand and EphA2 receptor engagement negatively regulated the growth of MMC via ERK1/2 MAP kinase dependent signaling. 2. Materials and methods 2.1. Culture of malignant mesothelioma cells MMC line CRL-2081 was obtained from American Type Culture Collection (Manassas, VA). The MM cells were grown in McCoy’s medium (Gibco-BRL, Baltimore, MD) and 10% fetal bovine serum (FBS) from Harlan Bio-
products (Indianapolis, IN) as mentioned earlier [28]. In brief, MMC were resuspended in RPMI-1640 (Gibco Laboratories, Grand Island, NY) containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 lg/ml). The cells were plated in 75 cm2 culture flasks (Corning Costar Corporation, MA) and incubated at 37 C in 5% CO2 and 95% air. The media were changed on alternate days. When the cells were confluent they were trypsinized and seeded into culture flasks/transwell chambers as required for different assays. 2.2. Immuno-precipitation and Western analysis Malignant mesothelioma cells were cultured on a 6well tissue culture plate (Costar) to confluence and the cells were lysed in lysis buffer as reported earlier [29]. Total protein was estimated by BCA method (PIERCE, Rockford, IL) and equal amounts of protein (20 mg/lane) were loaded. Proteins in the samples were separated in denaturing sodium dodecyl sulfate (SDS)–7.5% polyacrylamide gels (Bio-Rad), and transferred electrophoretically onto polyvinylidene difluoride membrane (Immobilon-P, Millipore). The blots were blocked overnight at 4 C with BSA and were incubated with the mouse anti-human EphA2, at 1:500 for 1 h at room temperature (Zymed Laboratories; Carlsbad, CA). After washing, they were incubated with the second antibody (horseradish peroxidase-conjugated anti-mouse IgG Ab) at a dilution of 1:1000 for 1 h. Eph receptors were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). Prestained protein markers were included for molecular mass determination (Bio-Rad). To determine levels of EphA2 phosphorylation, cell lysates prepared using the methods described above. The cell lysates containing 100 lg of total protein were incubated with anti-phosphotyrosine (4G10) obtained from Upstate, Waltham, MA for overnight at 4 C with gentle rotation. Beads were washed extensively with lysis buffer, and immune complexes were eluted with 2· LDS sample buffer (Invitrogen), boiled and centrifuged briefly. Proteins were resolved and detected by Western blotting using anti-EphA2 antibody and ERK1/2 antibody (Cell signaling, Boston, MA). 2.3. Construction of vector containing ephrinA1 (EFN-A1) and transient transfection of MMC The gene transfer vector, pcDNA3.2/V5-DEST was used as an expression vector for the expression of ligand ephrinA1 (EFN-A1) and pcDNA3.2/V5/CAT was used as a control vector (Invitrogen, Carlsbad, CA). The pENTR TOPO vector containing EFN-A1 insert was, expanded in OneShot Top10 cells, and cloned into the destination vector pcDNA3.2/V5-DEST according to the manufacturer’s instructions (Invitrogen, CA). The cloned vector was designated as pcDNA/EFNA1, and
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the control vector or pcDNA/CAT was designated as empty vector. The MMC were transfected with pcDNA/ EFN-A1 and empty vector using lipofectamine-2000 reagent (Invitrogen). The transfected cells were used for further experiments.
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significant effect on MMC. At 3 lg a significant inhibition of EphA2 receptor was noticed compared to control-Fc and unstimulated MMC (Fig. 1). This suggested that activation of receptor EphA2 with its ligand induced rapid internalization and phosphorylation of EphA2 receptor in MMC.
2.4. Brdu cell proliferation assay Cell proliferation was assessed by colorimetric assay kit according to the instructions provided as reported earlier [30] (Calbiochem, San Diego, CA). In brief, MMC were plated in 96-well microplate, at a density of 0.5 · 105 cells/well. The cells activated with ephrinA1-Fc or control-Fc or transfected with plasmid pcDNA-EFNA1 or empty vector by using lipofectamine 2000 and few wells were left untreated. The negative controls received serum free media and some of the wells received inhibitor of ERK1/2 MAP kinase PD98059 (30 lm) prior to treatment. The cell proliferation was assessed in triplicate. The data are presented as a percentage of negative control and proliferation with p < 0.05 were considered being significant. The experiments were repeated four times. 2.5. MMC three-dimensional growth in matrigel Briefly, a 24-well culture plate was coated with 200 ll of matrigel per well and then allowed to polymerize for 30 min at 37 C. MMC at a density of 1 · 103 cells per well were plated in 0.3 ml of 2% FBS containing RPMI1640. The cells were transfected with plasmid containing ephrinA1 construct pcDNA/EFNA1 or control vector or pretreated with ephrinA1-Fc and control-Fc, media were changed every three days. The number of colonies formed was recorded after 12 days of incubation. Four to six randomly chosen fields (10·) from the sample were photographed, and total number of colonies formed were quantitated by the Axio-vision image program. 2.6. Statistical analysis The SigmaStat 3.5 statistical software programme was used to calculate statistical significance. ANOVA was used to compare the experimental groups from the control groups. The differences at p < 0.05 were considered statistically significant.
3.2. EphrinA1-Fc induces phosphorylation of EphA2 in MMC In MMC activation of EphA2 receptor with ephrinA1Fc resulted in an increased phosphorylation of EphA2 as early as 5 min. The phosphorylation of receptor EphA2 was noticed up to 60 min when compared to control-Fc and unstimulated MMC (Fig. 2). However, the phosphorylation of EphA2 was decreased after 1 h of activation with ephrinA1-Fc. This suggests that the phosphorylation of receptor EphA2 is very rapid and remarkable with in 5 min of activation and is sustained for 1 h. 3.3. EphrinA1-Fc activates ERK1/2 MAP kinase in MMC One of the key functions of MAPK activation is the regulation of cell proliferation. In order to determine the mechanism by which ephrinA1 ligand binding regulates malignant behavior of MMC and its growth, MMC were pretreated with ephrinA1-Fc, total and phosphorylated form of ERK1/2 was measured. Increased ERK1/2 activation was noticed as early as 10 min. Maximum activation was noticed at 30 min and decreased there after (Fig. 3). The activation of ERK1/2 by EphA2 was transient and effected the biological function and receptor signaling in MMC. We noticed a significant inhibition of receptor EphA2 expression with ephrin-A1 activation. Since ligand activation negatively regulated receptor EphA2 expression we examined whether expression of receptor is dependent on ERK1/2 signaling. The pretreatment of MMC with inhibitor PD98059 alone did not affect the expression of EphA2 when compared to MMC alone. Moreover, the addition of ephrin-A1 after pretreatment with ERK1/2 inhibitor restored the expres-
3. Results 3.1. EphrinA1-Fc downregulates receptor Eph-A2 expression in MMC EphrinA1-Fc activation downregulated the expression of receptor EphA2 in a concentration dependent manner in MMC. EphrinA1-Fc at lower concentration was ineffective however at higher concentration (>2 lg) showed
Fig. 1. EphrinA1 activation decreases EphA2 receptor expression in MMC in a concentration dependent manner. Resting MMC and MMC activated with ephrinA1-Fc or Control-Fc were subjected to Western blot analysis. The immunoblot presented is a representative of three blots analyzed at different time.
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3.4. Activation of receptor EphA2 suppresses MMC proliferation
Fig. 2. EphrinA1-Fc activation induces EphA2 phosphorylation in MMC. MMC phosphotyrosine (p-Tr) immunoprecipitates derived from ephrinA1-Fc or control-Fc or resting MMC were subjected to Western blot analysis for EphA2 receptor phosphorylation. While control-Fc had no significant effect, ephrinA1-Fc significantly induced phosphorylation of EphA2. The immunoblot presented is a single representative of three blots each analyzed at different times.
Fig. 3. EphrinA1-Fc activation induces ERK1/2 phosphorylation in MMC. MMC were treated with ephrinA1-Fc or control-Fc for indicated period of time were subjected to Western blot analysis for total and phosphorylated ERK1/2 activity. The data presented are a single representative of three similar independent observations noticed at different times.
EphrinA1 ligand stimulation resulted in inhibition of MMC proliferation as determined by Brdu proliferation assay when compared to control MMC. We investigated the mechanism by which ephrinA1 ligand inhibits malignant behavior of MMC. MMC were pretreated with inhibitor for ERK1/2 MAP kinase (PD98059) before activation with ephrinA1-Fc and the proliferation was evaluated. The proliferation of MMC pretreated with inhibitor of ERK1/2 (PD98059) was significantly restored when compared to MMC activated with ephrinA1-Fc alone (Fig. 5). We did not notice any significant affect on proliferation of MMC pretreated with inhibitor PD98059 alone. These results suggest that proliferation of MMC is dependent on activation of ERK1/2 signaling pathway. Whether decrease in MMC proliferation is directly due to decrease in receptor expression was also evaluated by using plasmid containing ephrin-A1 construct called pcDNA/EFNA1. MMC were transfected with pcDNA/ EFNA1 or control-vector or left untransfected. The transfection of MMC by pcDNA/EFNA1 significantly decreased the proliferation of MMC, when compared to control-vector or MMC alone. These results suggest that decreased EphA2 expression is sufficient to decrease MMC proliferation. 3.5. Transfection of MMC with pcDNA/EFNA1 inhibits tumor growth in 3-D matrigels
Fig. 4. Inhibition of ERK1/2 MAP kinase restores the ephrinA1 mediated EphA2 receptor expression inhibition in MMC. The resting cell lysates and MMC pretreated with MAP kinase ERK1/ 2 (PD98059) inhibitor and activated with ephrinA1-Fc were subjected to Western blot analysis. The densitometric values presented are means ± SE of triplicate blots. The statistical significance *P < 0.001 vs control-Fc treated MMC and resting MMC; #P < 0.001 vs ephrinA1-Fc.
sion of receptor EphA2 (Fig. 4). This confirms that ligand mediated activation of receptor EphA2 is dependent on the activation of ERK1/2 in MMC.
We noticed that activation of EphA2 with its ligand ephrinA1-Fc decreased proliferation of MMC. MMC were transfected with a plasmid containing the construct of ephrinA1 (pcDNA/EFNA1), control vector or left untransfected. The MMC transfected with pcDNA/EFNA1 showed high expression of ephrinA1 compared to MMC alone or control vector. Moreover, the expression of receptor EphA2 was remarkably decreased (Fig. 6A). The MMC transfected with pcDNA/EFNA1 or control-vector or untransfected were plated on 3-D matrigel to determine the clonal growth. MMC activated with ephrinA1-Fc or controlFc was also plated simultaneously and the clonal growth was determined (Fig. 6B). The clonal growth was studied for 2 weeks. Interestingly, microscopic examination revealed that untransfected MMC formed tumor like spherical bodies on matrigel (Fig. 6B; panel a). MMC transfected with pcDNA/EFNA1 showed suppressed growth in matrigel compared to control vector or MMC alone (Fig. 6B; panels b–c). MMC activated with ephrinA1-Fc showed remarkable suppression of clonal growth on 3-D matrigel compared to control-Fc (Fig. 6B; panels d–e). These results demonstrate that decrease in EphA2 receptor expression results to decrease of MMC growth.
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Fig. 5. EphrinA1-Fc activation attenuates MMC proliferation. MMC proliferation was estimated by Brdu cell proliferation assay as described in methods. The data represented is mean ± SE of 4 independent experiments carried out at different times in triplicates. The statistical significance *p < 0.001 vs control-Fc treated MMC; #p < 0.001 vs. ephrinA1-Fc treated MMC; **p < 0.001 vs. control-vector treated MMC.
Fig. 6. (A) EphrinA1 transfection with pcDNA/EFNA1 inhibits EphA2 expression in MMC. MMC transfection with either pcDNA/ EFNA1 vector or control vector and EphA2 receptor, ephrinA1 expression were evaluated by Western blot analysis. EphA2 expression was decreased in ephrinA1 transfected MMC. The data presented here is a single representative of three independent experiments carried out at different times. (B) EphrinA1 transfection with pcDNA/EFNA1 inhibits MM growth. MMC transfected with either pcDNA/ EFNA1 or control vector, some cultures were activated either with ephrinA1-Fc or control-Fc and the tumor growth was evaluated in 3-D matrigels. Clonal growth was decreased in vector pcDNA/EFNA1 transfected as well as ephrinA1-Fc activated cultures. The data presented here are a single representative of three similar but independent observations noticed at different times. Magnification = 5 lm.
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4. Discussion Receptor EphA2 is overexpressed in a number of malignancies, including breast, prostrate, colon and melanoma [16,31,32]. We recently reported that EphA2 is overexpressed in MMC and posttranslational silencing of EphA2 significantly suppresses the proliferation and haptotactic migration of MMC [7]. In the present study, we evaluated the mechanisms associated with EphA2 receptor activation in MMC by its ligand ephrinA1 in MM growth. The activation of receptor EphA2 by its ligand induced phosphorylation of EphA2, and consequently decreased the receptor expression. EphrinA1 activation in MMC also resulted in ERK1/2 phosphorylation. Ligand activation or transfection of MMC with pcDNA/EFNA1 significantly inhibited the clonal expansion in 3-D matrigels. In addition activation of EphA2 receptor inhibited the proliferation of MMC. The inhibition of MMC proliferation was dependent on ERK1/2 MAP kinase signaling. EphA2 is an oncoprotein that promotes cell survival, abnormal cell growth and invasion in number of malignancies, including MM [5,14,16,20]. In normal cells the receptor EphA2 is expressed at low levels and binds stably with its ligand ephrinA1 anchored on adjacent cells [4]. However in malignant cells such as MMC, due to dysregulated cell division and abnormal growth the cell–cell contacts are loose which hinders the interaction between the neighboring cells. The loss of contact among the adjacent cells results in accumulation of high levels of intracellular EphA2. The mechanism by which EphA2 overexpression leads to an aggressive cellular phenotype is not clearly understood. In pancreatic adenocarcinoma invasion and metastasis was attenuated by ephrinA1 activation, the ligand activation decreased EphA2 levels due to proteosomal degradation [21]. In addition other studies indicate that activation of EphA2 induces dephosphorylation of focal adhesion kinases and thereby affects cell spreading and migration [24,25]. Earlier we reported that posttranslational silencing of EphA2 significantly attenuated the haptotactic migration of MMC [7]. In the present study, we noticed a significant downregulation of EphA2 receptor expression in MMC upon activation with its ligand, ephrinA1. We also noticed ephrinA1 activation induced rapid internalization, and phosphorylation of EphA2 receptor in MMC. Taken together these results suggest that a decrease in the EphA2 levels
either by activation or silencing attenuates the malignant behavior of tumor cells. Cell surface receptors including EphA2 are known to activate the MAP kinase signaling. ERK1/2 MAP kinase activation and downstream effectors control key cellular functions including proliferation in various tumors [33–35]. We noticed activation of endogenous EphA2 triggered the ERK1/2 MAP kinase signaling in MMC. We also noticed a significant downregulation in MMC proliferation when activated with ephrinA1-Fc. Inclusion of PD98059, a pharmacological inhibitor for ERK1/2 MAP kinase significantly restored the ephrinA1 mediated proliferation inhibition in MMC. When cell growth was measured by clonal growth assay on 3-D matrigels, clonal growth was greatly reduced in MMC activated with ephrinA1-Fc when compared to control-Fc. In addition, transfection of MMC with pcDNA/EFNA1 significantly inhibited the clonal growth in 3-D matrigels. Significantly, less number of colonies were formed in pcDNA/ EFNA1 transfected MMC. The size of the colonies formed was greatly reduced in those cultures. This suggests that the growth of MMC was attenuated when activated with ligand ephrinA1 and ligand mediated activation of EphA2 negatively regulate MM growth. In MMC the activation of EphA2 was sufficient to activate ERK1/2 MAP kinase. The activation of ERK1/2 MAP kinase negatively regulated the proliferation of MMC. EphA2 activation has been linked with the negative regulation of several biological functions, including cell proliferation, migration, survival, invasion and differentiation [15,24,34]. In mammary carcinoma the activation of the EphA2 receptor resulted in activation of ERK1/2 kinase signaling cascade, thereby negatively regulated ECM attachments [27]. Our present finding concur with recent reports that demonstrate ligand-binding of receptor tyrosine kinase activates downstream signaling cascade and negatively regulate tumor cell growth and invasiveness [15,24,26,36]. The novel finding of our present study is that EphA2 mediated induction of the ERK1/2 signaling pathway is necessary for the negative regulation of proliferation. The activation of ERK1/2 was transient, it returned to basal levels within 30– 45 min. The timing of ERK activation is interesting in light of evidence that the duration of ERK1/2 signaling can profoundly impact biological outcomes of receptor signaling [37–39]. The inhibitory action of MAP kinase could be due to induction of MAP
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kinase phosphatase [40]. The degradation of EphA2 could decrease ERK1/2 activity over time by decreasing EphA2 protein levels [19]. However, these findings contradict studies that indicate stimulation of Eph family kinases negatively regulates MAP kinase signaling [26,41]. It is possible that the disparities noticed are cell type specific. It is also possible that cell models presented in various studies respond differently to the ligand engagement. More studies in this context may help understand the signaling pathways responsible for the modulation of biological processes due to EphA2 receptor and its specific ligand ephrinA1 binding in MMC. In conclusion, we present the first evidence that in MMC activation of receptor EphA2 by its ligand ephrinA1 downregulates total EphA2 expression via phosphorylation. The activation of receptor EphA2 by its ligand ephrinA1 suppresses growth of MM via ERK1/2 signaling. This study suggests that cell surface membrane receptor EphA2 could be a potential target and a therapeutic choice to reduce the tumor burden for the treatment of patients with MM.
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Acknowledgements This research was supported by grant NIH RO1 A145338-02 from the National Institute of Health. The technical help of Xiahong Wang and Regev Doron is acknowledged.
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Cancer Letters 258 (2007) 215–222 www.elsevier.com/locate/canlet
Receptor EphA2 activation with ephrinA1 suppresses growth of malignant mesothelioma (MM) q Najmunnisa Nasreen, Kamal A. Mohammed, Yimu Lai, Veena B. Antony
*
Division of Pulmonary & Critical Care Medicine, Department of Medicine, University of Florida, P.O. Box 100225, Gainesville, FL, USA Received 26 June 2007; received in revised form 6 September 2007; accepted 10 September 2007
Abstract 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. Published by Elsevier Ireland Ltd. Keywords: EphA2; EphrinA1; Malignant mesothelioma; Receptor tyrosine kinase; Proliferation; ERK1/2
1. Introduction Malignant mesothelioma (MM), an asbestos related tumor of the pleura and peritoneum, remains a highly lethal malignancy. Approximately 3000 new cases of MM are diagnosed annually, in United States. Recent evidence suggests that SV40 also a co-factor for the development of MM [1]. The incidence of mesothelioma is expected to increase in Europe and Australia [2,3]. The treatment of MM has been disappointing because most q This work was supported by NIH RO1 AI 45338-02 grant from the National Institute of Health. * Corresponding author. Tel.: +1 352 392 7329; fax: +1 352 271 4559. E-mail address: [email protected]fl.edu (V.B. Antony).
0304-3835/$ - see front matter Published by Elsevier Ireland Ltd. doi:10.1016/j.canlet.2007.09.005
patients die within 12 months after the diagnosis [4]. Although palliative therapy is beneficial to some extent, the response to other treatment modalities such as surgery, radiation and chemotherapy remains poor. Failure of conventional therapeutic approach has led to the interest in identifying novel molecular markers that could be therapeutic targets to restrain tumor growth. Mesothelioma cells exhibit dysregulated growth and produce growth factors [5,6]. We recently reported that receptor EphA2 is overexpressed in MM and blockade of receptor expression decreased MMC proliferation and migration significantly [7]. MM is known to express a variety of receptor tyrosine kinases, such as epidermal growth factor (EGFR), Met, platelet-derived growth factor receptor, vascular endothelial growth factor receptor and
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EphB4 receptor [8–11]. The Eph transmembrane tyrosine kinases constitute the largest family of receptor tyrosine kinases. The Eph receptors are divided into two classes, A and B based on the sequence homology, structure and binding affinity [12]. Currently, 14 Eph receptors and eight ephrin ligands are recognized in humans. EphA receptors predominantly interact with class A ligands and B class receptors interact with B class ligands with few exceptions [13]. EphA2 receptor tyrosine kinase is overexpressed in aggressive cancers [14–18]. While it has been well documented that EphA2 is overexpressed in MM [7], the mechanisms which governs EphA2 expression in this aggressive tumor are not clearly understood. In cancer cells the cell–cell contacts are highly unstable which precludes intercellular contacts. EphA2 binds to the ligands that are anchored to the cell membrane of adjacent cells. In MM this interaction could be critical because the ligand binding of EphA2 regulates cell growth and invasion [19–21]. Ligand and receptor engagement leads to dimerization and internalization of the receptors. Autophosphorylation of the receptor provides recruitment of SH2 domain containing proteins such as PI3 kinase, Src family kinases which are critical signaling intermediates for downstream signaling pathway [22,23]. EphA2 activation with its ligand mediate the downstream signal transduction and modulate malignant behavior [24,25]. The ligation of EphA2 by ephrinA1-Fc inhibit the invasiveness in pancreatic adenocarcinoma via activation of focal adhesion kinase [21]. ERK1/2 MAP kinase pathways have been associated with EphA2 overexpression in malignant epithelial cells [26,27]. However, the molecular mechanisms involved in the activation of EphA2 receptors are not known in MM. In the present study we evaluated the biological outcome of receptor EphA2 activation in MM growth using recombinant ephrinA1-Fc. EphrinA1 ligand and EphA2 receptor engagement negatively regulated the growth of MMC via ERK1/2 MAP kinase dependent signaling. 2. Materials and methods 2.1. Culture of malignant mesothelioma cells MMC line CRL-2081 was obtained from American Type Culture Collection (Manassas, VA). The MM cells were grown in McCoy’s medium (Gibco-BRL, Baltimore, MD) and 10% fetal bovine serum (FBS) from Harlan Bio-
products (Indianapolis, IN) as mentioned earlier [28]. In brief, MMC were resuspended in RPMI-1640 (Gibco Laboratories, Grand Island, NY) containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 lg/ml). The cells were plated in 75 cm2 culture flasks (Corning Costar Corporation, MA) and incubated at 37 C in 5% CO2 and 95% air. The media were changed on alternate days. When the cells were confluent they were trypsinized and seeded into culture flasks/transwell chambers as required for different assays. 2.2. Immuno-precipitation and Western analysis Malignant mesothelioma cells were cultured on a 6well tissue culture plate (Costar) to confluence and the cells were lysed in lysis buffer as reported earlier [29]. Total protein was estimated by BCA method (PIERCE, Rockford, IL) and equal amounts of protein (20 mg/lane) were loaded. Proteins in the samples were separated in denaturing sodium dodecyl sulfate (SDS)–7.5% polyacrylamide gels (Bio-Rad), and transferred electrophoretically onto polyvinylidene difluoride membrane (Immobilon-P, Millipore). The blots were blocked overnight at 4 C with BSA and were incubated with the mouse anti-human EphA2, at 1:500 for 1 h at room temperature (Zymed Laboratories; Carlsbad, CA). After washing, they were incubated with the second antibody (horseradish peroxidase-conjugated anti-mouse IgG Ab) at a dilution of 1:1000 for 1 h. Eph receptors were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). Prestained protein markers were included for molecular mass determination (Bio-Rad). To determine levels of EphA2 phosphorylation, cell lysates prepared using the methods described above. The cell lysates containing 100 lg of total protein were incubated with anti-phosphotyrosine (4G10) obtained from Upstate, Waltham, MA for overnight at 4 C with gentle rotation. Beads were washed extensively with lysis buffer, and immune complexes were eluted with 2· LDS sample buffer (Invitrogen), boiled and centrifuged briefly. Proteins were resolved and detected by Western blotting using anti-EphA2 antibody and ERK1/2 antibody (Cell signaling, Boston, MA). 2.3. Construction of vector containing ephrinA1 (EFN-A1) and transient transfection of MMC The gene transfer vector, pcDNA3.2/V5-DEST was used as an expression vector for the expression of ligand ephrinA1 (EFN-A1) and pcDNA3.2/V5/CAT was used as a control vector (Invitrogen, Carlsbad, CA). The pENTR TOPO vector containing EFN-A1 insert was, expanded in OneShot Top10 cells, and cloned into the destination vector pcDNA3.2/V5-DEST according to the manufacturer’s instructions (Invitrogen, CA). The cloned vector was designated as pcDNA/EFNA1, and
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the control vector or pcDNA/CAT was designated as empty vector. The MMC were transfected with pcDNA/ EFN-A1 and empty vector using lipofectamine-2000 reagent (Invitrogen). The transfected cells were used for further experiments.
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significant effect on MMC. At 3 lg a significant inhibition of EphA2 receptor was noticed compared to control-Fc and unstimulated MMC (Fig. 1). This suggested that activation of receptor EphA2 with its ligand induced rapid internalization and phosphorylation of EphA2 receptor in MMC.
2.4. Brdu cell proliferation assay Cell proliferation was assessed by colorimetric assay kit according to the instructions provided as reported earlier [30] (Calbiochem, San Diego, CA). In brief, MMC were plated in 96-well microplate, at a density of 0.5 · 105 cells/well. The cells activated with ephrinA1-Fc or control-Fc or transfected with plasmid pcDNA-EFNA1 or empty vector by using lipofectamine 2000 and few wells were left untreated. The negative controls received serum free media and some of the wells received inhibitor of ERK1/2 MAP kinase PD98059 (30 lm) prior to treatment. The cell proliferation was assessed in triplicate. The data are presented as a percentage of negative control and proliferation with p < 0.05 were considered being significant. The experiments were repeated four times. 2.5. MMC three-dimensional growth in matrigel Briefly, a 24-well culture plate was coated with 200 ll of matrigel per well and then allowed to polymerize for 30 min at 37 C. MMC at a density of 1 · 103 cells per well were plated in 0.3 ml of 2% FBS containing RPMI1640. The cells were transfected with plasmid containing ephrinA1 construct pcDNA/EFNA1 or control vector or pretreated with ephrinA1-Fc and control-Fc, media were changed every three days. The number of colonies formed was recorded after 12 days of incubation. Four to six randomly chosen fields (10·) from the sample were photographed, and total number of colonies formed were quantitated by the Axio-vision image program. 2.6. Statistical analysis The SigmaStat 3.5 statistical software programme was used to calculate statistical significance. ANOVA was used to compare the experimental groups from the control groups. The differences at p < 0.05 were considered statistically significant.
3.2. EphrinA1-Fc induces phosphorylation of EphA2 in MMC In MMC activation of EphA2 receptor with ephrinA1Fc resulted in an increased phosphorylation of EphA2 as early as 5 min. The phosphorylation of receptor EphA2 was noticed up to 60 min when compared to control-Fc and unstimulated MMC (Fig. 2). However, the phosphorylation of EphA2 was decreased after 1 h of activation with ephrinA1-Fc. This suggests that the phosphorylation of receptor EphA2 is very rapid and remarkable with in 5 min of activation and is sustained for 1 h. 3.3. EphrinA1-Fc activates ERK1/2 MAP kinase in MMC One of the key functions of MAPK activation is the regulation of cell proliferation. In order to determine the mechanism by which ephrinA1 ligand binding regulates malignant behavior of MMC and its growth, MMC were pretreated with ephrinA1-Fc, total and phosphorylated form of ERK1/2 was measured. Increased ERK1/2 activation was noticed as early as 10 min. Maximum activation was noticed at 30 min and decreased there after (Fig. 3). The activation of ERK1/2 by EphA2 was transient and effected the biological function and receptor signaling in MMC. We noticed a significant inhibition of receptor EphA2 expression with ephrin-A1 activation. Since ligand activation negatively regulated receptor EphA2 expression we examined whether expression of receptor is dependent on ERK1/2 signaling. The pretreatment of MMC with inhibitor PD98059 alone did not affect the expression of EphA2 when compared to MMC alone. Moreover, the addition of ephrin-A1 after pretreatment with ERK1/2 inhibitor restored the expres-
3. Results 3.1. EphrinA1-Fc downregulates receptor Eph-A2 expression in MMC EphrinA1-Fc activation downregulated the expression of receptor EphA2 in a concentration dependent manner in MMC. EphrinA1-Fc at lower concentration was ineffective however at higher concentration (>2 lg) showed
Fig. 1. EphrinA1 activation decreases EphA2 receptor expression in MMC in a concentration dependent manner. Resting MMC and MMC activated with ephrinA1-Fc or Control-Fc were subjected to Western blot analysis. The immunoblot presented is a representative of three blots analyzed at different time.
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3.4. Activation of receptor EphA2 suppresses MMC proliferation
Fig. 2. EphrinA1-Fc activation induces EphA2 phosphorylation in MMC. MMC phosphotyrosine (p-Tr) immunoprecipitates derived from ephrinA1-Fc or control-Fc or resting MMC were subjected to Western blot analysis for EphA2 receptor phosphorylation. While control-Fc had no significant effect, ephrinA1-Fc significantly induced phosphorylation of EphA2. The immunoblot presented is a single representative of three blots each analyzed at different times.
Fig. 3. EphrinA1-Fc activation induces ERK1/2 phosphorylation in MMC. MMC were treated with ephrinA1-Fc or control-Fc for indicated period of time were subjected to Western blot analysis for total and phosphorylated ERK1/2 activity. The data presented are a single representative of three similar independent observations noticed at different times.
EphrinA1 ligand stimulation resulted in inhibition of MMC proliferation as determined by Brdu proliferation assay when compared to control MMC. We investigated the mechanism by which ephrinA1 ligand inhibits malignant behavior of MMC. MMC were pretreated with inhibitor for ERK1/2 MAP kinase (PD98059) before activation with ephrinA1-Fc and the proliferation was evaluated. The proliferation of MMC pretreated with inhibitor of ERK1/2 (PD98059) was significantly restored when compared to MMC activated with ephrinA1-Fc alone (Fig. 5). We did not notice any significant affect on proliferation of MMC pretreated with inhibitor PD98059 alone. These results suggest that proliferation of MMC is dependent on activation of ERK1/2 signaling pathway. Whether decrease in MMC proliferation is directly due to decrease in receptor expression was also evaluated by using plasmid containing ephrin-A1 construct called pcDNA/EFNA1. MMC were transfected with pcDNA/ EFNA1 or control-vector or left untransfected. The transfection of MMC by pcDNA/EFNA1 significantly decreased the proliferation of MMC, when compared to control-vector or MMC alone. These results suggest that decreased EphA2 expression is sufficient to decrease MMC proliferation. 3.5. Transfection of MMC with pcDNA/EFNA1 inhibits tumor growth in 3-D matrigels
Fig. 4. Inhibition of ERK1/2 MAP kinase restores the ephrinA1 mediated EphA2 receptor expression inhibition in MMC. The resting cell lysates and MMC pretreated with MAP kinase ERK1/ 2 (PD98059) inhibitor and activated with ephrinA1-Fc were subjected to Western blot analysis. The densitometric values presented are means ± SE of triplicate blots. The statistical significance *P < 0.001 vs control-Fc treated MMC and resting MMC; #P < 0.001 vs ephrinA1-Fc.
sion of receptor EphA2 (Fig. 4). This confirms that ligand mediated activation of receptor EphA2 is dependent on the activation of ERK1/2 in MMC.
We noticed that activation of EphA2 with its ligand ephrinA1-Fc decreased proliferation of MMC. MMC were transfected with a plasmid containing the construct of ephrinA1 (pcDNA/EFNA1), control vector or left untransfected. The MMC transfected with pcDNA/EFNA1 showed high expression of ephrinA1 compared to MMC alone or control vector. Moreover, the expression of receptor EphA2 was remarkably decreased (Fig. 6A). The MMC transfected with pcDNA/EFNA1 or control-vector or untransfected were plated on 3-D matrigel to determine the clonal growth. MMC activated with ephrinA1-Fc or controlFc was also plated simultaneously and the clonal growth was determined (Fig. 6B). The clonal growth was studied for 2 weeks. Interestingly, microscopic examination revealed that untransfected MMC formed tumor like spherical bodies on matrigel (Fig. 6B; panel a). MMC transfected with pcDNA/EFNA1 showed suppressed growth in matrigel compared to control vector or MMC alone (Fig. 6B; panels b–c). MMC activated with ephrinA1-Fc showed remarkable suppression of clonal growth on 3-D matrigel compared to control-Fc (Fig. 6B; panels d–e). These results demonstrate that decrease in EphA2 receptor expression results to decrease of MMC growth.
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Fig. 5. EphrinA1-Fc activation attenuates MMC proliferation. MMC proliferation was estimated by Brdu cell proliferation assay as described in methods. The data represented is mean ± SE of 4 independent experiments carried out at different times in triplicates. The statistical significance *p < 0.001 vs control-Fc treated MMC; #p < 0.001 vs. ephrinA1-Fc treated MMC; **p < 0.001 vs. control-vector treated MMC.
Fig. 6. (A) EphrinA1 transfection with pcDNA/EFNA1 inhibits EphA2 expression in MMC. MMC transfection with either pcDNA/ EFNA1 vector or control vector and EphA2 receptor, ephrinA1 expression were evaluated by Western blot analysis. EphA2 expression was decreased in ephrinA1 transfected MMC. The data presented here is a single representative of three independent experiments carried out at different times. (B) EphrinA1 transfection with pcDNA/EFNA1 inhibits MM growth. MMC transfected with either pcDNA/ EFNA1 or control vector, some cultures were activated either with ephrinA1-Fc or control-Fc and the tumor growth was evaluated in 3-D matrigels. Clonal growth was decreased in vector pcDNA/EFNA1 transfected as well as ephrinA1-Fc activated cultures. The data presented here are a single representative of three similar but independent observations noticed at different times. Magnification = 5 lm.
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4. Discussion Receptor EphA2 is overexpressed in a number of malignancies, including breast, prostrate, colon and melanoma [16,31,32]. We recently reported that EphA2 is overexpressed in MMC and posttranslational silencing of EphA2 significantly suppresses the proliferation and haptotactic migration of MMC [7]. In the present study, we evaluated the mechanisms associated with EphA2 receptor activation in MMC by its ligand ephrinA1 in MM growth. The activation of receptor EphA2 by its ligand induced phosphorylation of EphA2, and consequently decreased the receptor expression. EphrinA1 activation in MMC also resulted in ERK1/2 phosphorylation. Ligand activation or transfection of MMC with pcDNA/EFNA1 significantly inhibited the clonal expansion in 3-D matrigels. In addition activation of EphA2 receptor inhibited the proliferation of MMC. The inhibition of MMC proliferation was dependent on ERK1/2 MAP kinase signaling. EphA2 is an oncoprotein that promotes cell survival, abnormal cell growth and invasion in number of malignancies, including MM [5,14,16,20]. In normal cells the receptor EphA2 is expressed at low levels and binds stably with its ligand ephrinA1 anchored on adjacent cells [4]. However in malignant cells such as MMC, due to dysregulated cell division and abnormal growth the cell–cell contacts are loose which hinders the interaction between the neighboring cells. The loss of contact among the adjacent cells results in accumulation of high levels of intracellular EphA2. The mechanism by which EphA2 overexpression leads to an aggressive cellular phenotype is not clearly understood. In pancreatic adenocarcinoma invasion and metastasis was attenuated by ephrinA1 activation, the ligand activation decreased EphA2 levels due to proteosomal degradation [21]. In addition other studies indicate that activation of EphA2 induces dephosphorylation of focal adhesion kinases and thereby affects cell spreading and migration [24,25]. Earlier we reported that posttranslational silencing of EphA2 significantly attenuated the haptotactic migration of MMC [7]. In the present study, we noticed a significant downregulation of EphA2 receptor expression in MMC upon activation with its ligand, ephrinA1. We also noticed ephrinA1 activation induced rapid internalization, and phosphorylation of EphA2 receptor in MMC. Taken together these results suggest that a decrease in the EphA2 levels
either by activation or silencing attenuates the malignant behavior of tumor cells. Cell surface receptors including EphA2 are known to activate the MAP kinase signaling. ERK1/2 MAP kinase activation and downstream effectors control key cellular functions including proliferation in various tumors [33–35]. We noticed activation of endogenous EphA2 triggered the ERK1/2 MAP kinase signaling in MMC. We also noticed a significant downregulation in MMC proliferation when activated with ephrinA1-Fc. Inclusion of PD98059, a pharmacological inhibitor for ERK1/2 MAP kinase significantly restored the ephrinA1 mediated proliferation inhibition in MMC. When cell growth was measured by clonal growth assay on 3-D matrigels, clonal growth was greatly reduced in MMC activated with ephrinA1-Fc when compared to control-Fc. In addition, transfection of MMC with pcDNA/EFNA1 significantly inhibited the clonal growth in 3-D matrigels. Significantly, less number of colonies were formed in pcDNA/ EFNA1 transfected MMC. The size of the colonies formed was greatly reduced in those cultures. This suggests that the growth of MMC was attenuated when activated with ligand ephrinA1 and ligand mediated activation of EphA2 negatively regulate MM growth. In MMC the activation of EphA2 was sufficient to activate ERK1/2 MAP kinase. The activation of ERK1/2 MAP kinase negatively regulated the proliferation of MMC. EphA2 activation has been linked with the negative regulation of several biological functions, including cell proliferation, migration, survival, invasion and differentiation [15,24,34]. In mammary carcinoma the activation of the EphA2 receptor resulted in activation of ERK1/2 kinase signaling cascade, thereby negatively regulated ECM attachments [27]. Our present finding concur with recent reports that demonstrate ligand-binding of receptor tyrosine kinase activates downstream signaling cascade and negatively regulate tumor cell growth and invasiveness [15,24,26,36]. The novel finding of our present study is that EphA2 mediated induction of the ERK1/2 signaling pathway is necessary for the negative regulation of proliferation. The activation of ERK1/2 was transient, it returned to basal levels within 30– 45 min. The timing of ERK activation is interesting in light of evidence that the duration of ERK1/2 signaling can profoundly impact biological outcomes of receptor signaling [37–39]. The inhibitory action of MAP kinase could be due to induction of MAP
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kinase phosphatase [40]. The degradation of EphA2 could decrease ERK1/2 activity over time by decreasing EphA2 protein levels [19]. However, these findings contradict studies that indicate stimulation of Eph family kinases negatively regulates MAP kinase signaling [26,41]. It is possible that the disparities noticed are cell type specific. It is also possible that cell models presented in various studies respond differently to the ligand engagement. More studies in this context may help understand the signaling pathways responsible for the modulation of biological processes due to EphA2 receptor and its specific ligand ephrinA1 binding in MMC. In conclusion, we present the first evidence that in MMC activation of receptor EphA2 by its ligand ephrinA1 downregulates total EphA2 expression via phosphorylation. The activation of receptor EphA2 by its ligand ephrinA1 suppresses growth of MM via ERK1/2 signaling. This study suggests that cell surface membrane receptor EphA2 could be a potential target and a therapeutic choice to reduce the tumor burden for the treatment of patients with MM.
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Acknowledgements This research was supported by grant NIH RO1 A145338-02 from the National Institute of Health. The technical help of Xiahong Wang and Regev Doron is acknowledged.
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