Kinase-Dependent and -Independent Roles of EphA2 in the Regulation of Prostate Cancer Invasion and Metastasis
2009; Elsevier BV; Volume: 174; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.080473
ISSN1525-2191
AutoresMaria Letizia Taddei, Matteo Parri, Adriano Angelucci, Barbara Onnis, Francesca Bianchini, Elisa Giannoni, Giovanni Raugei, Lido Calorini, Nadia Rucci, Anna Teti, Mauro Bologna, Paola Chiarugi,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoLigand-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. 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. Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs), are involved in many biological processes including angiogenesis, tissue-border formation, cell migration, axon guidance, and synaptic plasticity. Ephs\ephrins are important mediators of cell-cell communication regulating cell attachment to extracellular matrix, cell shape, and motility.1Pasquale EB Eph receptor signalling casts a wide net on cell behaviour.Nat Rev Mol Cell Biol. 2005; 6: 462-475Crossref PubMed Scopus (866) Google Scholar Their frequent overexpression in human cancers and the correlation with poor prognosis and high vascularity in cancer tissues emphasize emerging roles in tumor progression.2Kiyokawa E Takai S Tanaka M Iwase T Suzuki M Xiang YY Naito Y Yamada K Sugimura H Kino I Overexpression of ERK, an EPH family receptor protein tyrosine kinase, in various human tumors.Cancer Res. 1994; 54: 3645-3650PubMed Google Scholar EphA2 has been implicated in carcinogenesis of several cancers including melanomas and prostate, breast, colon, lung, and esophageal carcinomas.3Kinch MS Carles-Kinch K Overexpression and functional alterations of the EphA2 tyrosine kinase in cancer.Clin Exp Metastasis. 2003; 20: 59-68Crossref PubMed Scopus (120) Google Scholar These studies showed high levels of EphA2 in both tissue and cell explants of these diseases and especially in the more aggressive stages of progression.4Dodelet VC Pasquale EB Eph receptors and ephrin ligands: embryogenesis to tumorigenesis.Oncogene. 2000; 19: 5614-5619Crossref PubMed Scopus (263) Google Scholar In particular, ectopic overexpression of EphA2 gives untransformed epithelial cells both tumorigenic and metastatic potential.5Zelinski DP Zantek ND Stewart JC Irizarry AR Kinch MS EphA2 overexpression causes tumorigenesis of mammary epithelial cells.Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar Certain Ephs and their ligands are expressed and up-regulated at sites of dynamic neovascularization, eg, in endothelial cells during tumor invasion.6Shin D Garcia-Cardena G Hayashi S Gerety S Asahara T Stavrakis G Isner J Folkman J Gimbrone Jr, MA Anderson DJ Expression of ephrinB2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization.Dev Biol. 2001; 230: 139-150Crossref PubMed Scopus (278) Google Scholar EphA2-deficient mice displayed decreased tumor volume, microvasculature density, and lung metastasis.7Brantley-Sieders DM Fang WB Hicks DJ Zhuang G Shyr Y Chen J Impaired tumor microenvironment in EphA2-deficient mice inhibits tumor angiogenesis and metastatic progression.FASEB J. 2005; 19: 1884-1886Crossref PubMed Scopus (115) Google Scholar Actually, the role of EphA2 in the regulation of malignant transformation/progression is far from clear. Indeed recent results unveil some antitumorigenic functions of EphA2: EphA2 is localized on chromosome 1p36.13, a region frequently deleted in a number of human cancers, including prostate and brain tumor and disruption of EphA2 kinase in mice leads to increased susceptibility to skin carcinogenesis, suggesting EphA2 as a potential tumor suppressor gene in mammalian skin.8Guo H Miao H Gerber L Singh J Denning MF Gilliam AC Wang B Disruption of EphA2 receptor tyrosine kinase leads to increased susceptibility to carcinogenesis in mouse skin.Cancer Res. 2006; 66: 7050-7058Crossref PubMed Scopus (115) Google Scholar Beside being implicated in tumor aggressiveness and vasculogenesis, class A ephrin/Eph interactions have recently been implicated in the organization of cell migration during several physiological and pathophysiological processes, including development, tissue morphogenesis, and cancer cell migration.1Pasquale EB Eph receptor signalling casts a wide net on cell behaviour.Nat Rev Mol Cell Biol. 2005; 6: 462-475Crossref PubMed Scopus (866) Google Scholar As for other receptor tyrosine kinases, ligand binding of EphA receptors induces receptor clustering, activation of kinase activity, and subsequent trans-phosphorylation of the cytoplasmic domains, creating docking sites for a number of signaling proteins.9Hubbard SR Juxtamembrane autoinhibition in receptor tyrosine kinases.Nat Rev Mol Cell Biol. 2004; 5: 464-471Crossref PubMed Scopus (242) Google Scholar, 10Kullander K Klein R Mechanisms and functions of Eph and ephrin signalling.Nat Rev Mol Cell Biol. 2002; 3: 475-486Crossref PubMed Scopus (972) Google Scholar The role of class A Eph receptors in regulating endothelial cell migration and assembly is strongly supported by several studies in angiogenic remodeling.11Ogawa K Pasqualini R Lindberg RA Kain R Freeman AL Pasquale EB The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization.Oncogene. 2000; 19: 6043-6052Crossref PubMed Scopus (338) Google Scholar On the contrary, a clear role of EphA2 kinase in the regulation of cancer cell motility has not been delineated. We recently reported that in prostate carcinoma cells, ephrinA1 elicits a motility response by activating a Rho- and focal adhesion kinase (FAK)-dependent cytoskeleton rearrangement, finally driving the retraction of the cell body and the inhibition of directional cell migration.12Parri M Buricchi F Giannoni E Grimaldi G Mello T Raugei G Ramponi G Chiarugi P EphrinA1 activates a Src/focal adhesion kinase-mediated motility response leading to rho-dependent actino/myosin contractility.J Biol Chem. 2007; 282: 19619-19628Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar In addition, activation of EphA2 is able to redirect motility and inhibit invasion of adenocarcinoma cells, again through a FAK-mediated pathway.13Duxbury MS Ito H Zinner MJ Ashley SW Whang EE Ligation of EphA2 by ephrin A1-Fc inhibits pancreatic adenocarcinoma cellular invasiveness.Biochem Biophys Res Commun. 2004; 320: 1096-1102Crossref PubMed Scopus (99) Google Scholar Ephrin/Eph interaction gives rise to complex cell-cell signaling culminating in a bidirectional pathway. Cells bearing the ephrin ligand engage in reverse signaling, and cells carrying the Eph receptors undergo forward signaling.10Kullander K Klein R Mechanisms and functions of Eph and ephrin signalling.Nat Rev Mol Cell Biol. 2002; 3: 475-486Crossref PubMed Scopus (972) Google Scholar, 14Holland SJ Gale NW Mbamalu G Yancopoulos GD Henkemeyer M Pawson T Bidirectional signalling through the EPH-family receptor Nuk and its transmembrane ligands.Nature. 1996; 383: 722-725Crossref PubMed Scopus (464) Google Scholar, 15Henkemeyer M Orioli D Henderson JT Saxton TM Roder J Pawson T Klein R Nuk controls pathfinding of commissural axons in the mammalian central nervous system.Cell. 1996; 86: 35-46Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar Although the reverse signaling of ephrin As is recognized as kinase-independent, attributable to their lack of enzymatic activity, the forward response elicited by the Eph kinase receptors is puzzling, because both kinase-dependent and -independent components have been reported. Indeed, several lines of evidence describe EphA2 signaling as mainly kinase-dependent. First, mutations of the kinase domain of EphA2 affect vascular endothelial cell growth and vascular endothelial growth factor-dependent angiogenesis.16Cheng N Brantley DM Liu H Lin Q Enriquez M Gale N Yancopoulos G Cerretti DP Daniel TO Chen J Blockade of EphA receptor tyrosine kinase activation inhibits vascular endothelial cell growth factor-induced angiogenesis.Mol Cancer Res. 2002; 1: 2-11Crossref PubMed Scopus (38) Google Scholar Second, recent data showed that EphA2 receptor phosphorylation may be vital in granting oncogenic potential.17Fang WB Brantley-Sieders DM Parker MA Reith AD Chen J A kinase-dependent role for EphA2 receptor in promoting tumor growth and metastasis.Oncogene. 2005; 24: 7859-7868Crossref PubMed Scopus (112) Google Scholar In agreement, the block of EphA2 receptor activation through EphA2-Fc results in a decrease in phosphorylation that was concurrent with decreased tumor volume.18Dobrzanski P Hunter K Jones-Bolin S Chang H Robinson C Pritchard S Zhao H Ruggeri B Antiangiogenic and antitumor efficacy of EphA2 receptor antagonist.Cancer Res. 2004; 64: 910-919Crossref PubMed Scopus (144) Google Scholar In keeping with these data, emerging evidence suggests that protein tyrosine phosphatases (PTPs) are involved in regulating Eph-mediated responses,19Miao H Burnett E Kinch M Simon E Wang B Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation.Nat Cell Biol. 2000; 2: 62-69Crossref PubMed Scopus (475) Google Scholar, 20Shintani T Ihara M Sakuta H Takahashi H Watakabe I Noda M Eph receptors are negatively controlled by protein tyrosine phosphatase receptor type O.Nat Neurosci. 2006; 9: 761-769Crossref PubMed Scopus (89) Google Scholar strongly supporting a role for Eph kinase activity. Nevertheless Eph receptors are nonclassical receptor tyrosine kinases because, beside kinase-dependent signaling, ligation of certain members of the Eph family can also trigger kinase-independent responses.21Birgbauer E Cowan CA Sretavan DW Henkemeyer M Kinase independent function of EphB receptors in retinal axon pathfinding to the optic disc from dorsal but not ventral retina.Development. 2000; 127: 1231-1241Crossref PubMed Google Scholar, 22Dalva MB Takasu MA Lin MZ Shamah SM Hu L Gale NW Greenberg ME EphB receptors interact with NMDA receptors and regulate excitatory synapse formation.Cell. 2000; 103: 945-956Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 23Grunwald IC Korte M Wolfer D Wilkinson GA Unsicker K Lipp HP Bonhoeffer T Klein R Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity.Neuron. 2001; 32: 1027-1040Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 24Kullander K Mather NK Diella F Dottori M Boyd AW Klein R Kinase-dependent and kinase-independent functions of EphA4 receptors in major axon tract formation in vivo.Neuron. 2001; 29: 73-84Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar First, the presence of kinase-inactive together with wild-type EphA7 within the same cell changes its ligand-induced response from repulsion to adhesion,25Holmberg J Clarke DL Frisen J Regulation of repulsion versus adhesion by different splice forms of an Eph receptor.Nature. 2000; 408: 203-206Crossref PubMed Scopus (291) Google Scholar indicating different functions of Ephs owing to their phosphorylation and degree of clustering. Second, EphA8 receptor localizes p110γ phosphatidylinositol 3-kinase to the plasma membrane in a tyrosine kinase-independent manner, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion.26Gu C Park S The p110 gamma PI-3 kinase is required for EphA8-stimulated cell migration.FEBS Lett. 2003; 540: 65-70Crossref PubMed Scopus (12) Google Scholar Third, the simple removal of membrane-associated EphA2 through ligand-independent endocytosis reduces malignant behavior of the cells and tumor growth.13Duxbury MS Ito H Zinner MJ Ashley SW Whang EE Ligation of EphA2 by ephrin A1-Fc inhibits pancreatic adenocarcinoma cellular invasiveness.Biochem Biophys Res Commun. 2004; 320: 1096-1102Crossref PubMed Scopus (99) Google Scholar Finally, Miao and colleagues27Miao H Strebhardt K Pasquale EB Shen TL Guan JL Wang B Inhibition of integrin-mediated cell adhesion but not directional cell migration requires catalytic activity of EphB3 receptor tyrosine kinase. Role of Rho family small GTPases.J Biol Chem. 2005; 280: 923-932Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar recently reported that EphB3 catalytic activity is required for inhibition of integrin-mediated cell adhesion but is dispensable for directional cell migration. In the context of this controversial literature, we investigated the role of tyrosine phosphorylation of EphA2 kinase in the regulation of prostate carcinoma cell motility and invasion. On the whole, our findings point to a kinase-dependent role of EphA2 receptor for the regulation of cell motility, adhesion, cytoskeleton rearrangements, as well as for invasion and metastasis formation in nude mice, although the invasive and prometastatic effect of EphA2 show a kinase-independent component. Unless specified all reagents were obtained from Sigma (St. Louis, MO). PC3, DU145, LNCaP, and HEK293T cells were from ATCC, Rockville, MD; PNT1A cells were a generous gift of Rosario Notaro (the Department of Pharmacology, University of Florence, Italy), recombinant mouse Fc and ephrinA1-Fc chimera were from R&D Systems (Minneapolis, MN), antiphosphotyrosine (clone 4G10) and anti-EphA2 antibodies (clone D7) were from Upstate Biotechnology Inc. (Charlottesville, VA), anti-RhoA antibodies were from BD (New Jersey, USA). Primers for EphA2 reverse transcriptase-polymerase chain reaction (RT-PCR) were 5′-ATGGAGCTCCAGGCAGCCCG-3′ and 5′-TCAGATGGGGATCCCCACAGT-3′. Total RNA was isolated from PC3 cells with TRIzol reagent, and cDNA obtained with SuperScript one-step RT-PCR. EphA2 was subcloned into pTargetT vector (Promega, Madison, WI). EphA2 mutants were obtained using a QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). Phenylalanine replaced Tyr587 and Tyr593 in the double-mutant (DM) and arginine replaced Lys645 in the kinase dead (KD) mutant. EphA2 cytoplasmic domain truncation mutant (ΔCyto), was generated by PCR amplification of the extracellular and transmembrane domain of EphA2 (from N-ter to Lys 562). PC3 human prostate carcinoma were cultured in Ham's F12. HEK293T human embryonic kidney cells and DU-145 human prostate carcinoma cells (brain metastasis) were cultured in Dulbecco's modified Eagle's medium. PNT1A human postpubertal prostate normal cells and LNCaP human prostate carcinoma cells (lymph node metastasis) were cultured in RPMI 1640. All media are supplemented with 10% fetal calf serum in a 5% CO2 humidified atmosphere. HEK293T cells were transiently transfected using Lipofectamine 2000 (Invitrogen Milano, IT) using 4 μg of plasmid DNA. Forty-eight hours after transfection the cells were recovered for analysis. PC3 and DU-145 cells were stably transfected with the same procedure, except that 48 hours after transfection cells were selected with 400 mg/L G418 for neomycin resistance. For studies using soluble ephrinA1, cells were stimulated with 1 μg ml−1 Fc or ephrinA1-Fc for the indicated times. After washing with phosphate-buffered saline (PBS), the cells were fixed with 3.7% formaldehyde solution in PBS for 20 minutes at 4°C. Then, cells were permeabilized with 0.1% Triton X-100 in PBS and stained with 50 μg/ml of phalloidin-tetramethyl-rhodamine isothiocyanate for 1 hour at room temperature, mounted with glycerol plastine, and observed under a laser-scanning confocal microscope (Leica SP5, Mannheim, Germany). For anti-EphA2 immunoprecipitation, we used either anti-EphA2 antibodies or 1 μg ml−1 ephrinA1-Fc fusion protein with similar results. Immune complexes were collected on protein A Sepharose, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose. For chemiluminescence detection we used a Gel Logic 2200 Kodak Imaging System (Eastman-Kodak, Rochester, NY), equipped with a charge-coupled device camera, which guarantees a high linearity, and Quantity-One software (Bio-Rad, Hercules, CA) was used to obtain quantitative analyses. Cells were serum-starved for 24 hours before detaching with 0.25% trypsin for 1 minute. Trypsin was blocked with 0.2 mg/ml soybean trypsin inhibitor, and cells were resuspended in fresh medium, maintained in suspension for 30 minutes at 37°C, and then directly seeded onto precoated dishes treated overnight with 10 μg/ml of human fibronectin for 4 hours in the presence of either 1 μg ml−1 Fc or ephrinA1-Fc. Cells were cultured in 6-cm plates until confluence. The monolayer of PC3 cells was serum-starved for 24 hours and then was scratched using a thin sterile pipette tip. Pictures were taken before and 24 hours after the addition of complete medium with either 1 μg/ml of ephrinA1-Fc or Fc using an inverted Leica microscope equipped with a Nikon digital camera (Tokyo, Japan). The transwell system of Costar (Lowell, MA), equipped with 8-μm-pore polyvinylpyrrolidone-free polycarbonate filters was used. Cells were loaded into the upper compartment (5 × 105 cells in 500 μl) in serum-free growth medium. The upper sides of the porous polycarbonate filters were coated with 50 μg/cm2 of reconstituted Matrigel basement membrane and placed into six-well culture dishes containing 1 ml of complete growth medium together with either 1 μg/ml Fc or ephrinA1-Fc. After 24 hours of incubation at 37°C, noninvading cells were removed mechanically using cotton swabs, and the microporous membrane was stained with DiffQuick solution. Chemotaxis was evaluated by counting the cells migrated to the lower surface of the polycarbonate filters (six randomly chosen fields, mean ± SD). Aliquots from media conditioned by PC3 cells and human fibrosarcoma HT1080 cells, used as positive control, were electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gels co-polymerized with 0.1% (w/v) type A gelatin. After electrophoresis, the gels were washed in 2.5% v/v Triton X-100 for 30 minutes to remove sodium dodecyl sulfate. Gelatin substrate gels were then incubated in 50 mmol/L Tris-HCl, pH 7.4, 200 mmol/L NaCl, and 5 mmol/L CaCl2 for 24 hours at 37°C. After incubation, the gels were stained with 0.1% Coomassie brilliant blue in acetic acid, methanol, and distilled water at a volume ratio of 1:2:3, respectively, for 60 minutes at room temperature. After destaining, the gels were immersed in distilled water and scanned immediately with QuantityOne Image Analysis software (Bio-Rad). PC3 cells were directly lysed in RIPA buffer, the lysates were clarified by centrifugation, and RhoA-GTP or Rac-GTP levels were quantified. Briefly, lysates were incubated with 10 μg of Rhotekin-GST fusion protein (Becton Dickinson, Franklin Lakes, New Jersey) or p21-activated kinase (PAK)-GST fusion protein, both absorbed on glutathione Sepharose beads for 1 hour at 4°C. Immunoreactive RhoA or Rac1 were then quantified by Western blot analysis. Lysates were normalized for RhoA or Rac1 content by immunoblot. Male CD1 nude mice, used for in vivo bone metastasis experiments, were purchased from Charles River (Milan, Italy). Mice were maintained under the guidelines established by our institution (University of L'Aquila, Medical School and Science and Technology School Board Regulations, complying with the Italian Government Regulation n.116, 27/1/1992). The procedure of heart injection of prostate cancer cells in nude mice has been previously described.28Angelucci A Gravina GL Rucci N Festuccia C Muzi P Vicentini C Teti A Bologna M Evaluation of metastatic potential in prostate carcinoma: an in vivo model.Int J Oncol. 2004; 25: 1713-1720PubMed Google Scholar Briefly, 1 × 105 cells in 0.1 ml of saline solution, were injected in the left ventricle of 4-week-old nude mice previously anesthetized with a mixture of ketamine (25 mg/ml)/xylazine (5 mg/ml). The development of tumor colonies in the whole skeletal apparatus was monitored at times by radiography using a Faxitron cabinet X-ray system (Faxitron X-ray Corp., Wheeling, IL). All animals were subjected to accurate necroscopy for the evaluation of the presence of tumor colonies in other anatomical sites. The quantification of human Alu sequences was previously described.29Schneider T Osl F Friess T Stockinger H Scheuer WV Quantification of human Alu sequences by real-time PCR—an improved method to measure therapeutic efficacy of anti-metastatic drugs in human xenotransplants.Clin Exp Metastasis. 2002; 19: 571-582Crossref PubMed Scopus (65) Google Scholar Briefly, mice were sacrificed by carbon dioxide inhalation and the rear limbs were accurately deprived of surrounding soft tissues and opened with a surgical blade to expose the medulla. Bone fragments were incubated with 100 mmol/L Tris-HCl, pH 8.5, 0.5 mol/L ethylenediaminetetraacetic acid, 10% sodium dodecyl sulfate, 5 mol/L NaCl, 20 mg/ml proteinase K at 37°C for 12 hours. DNA was isolated by phenol/chloroform extraction, precipitated with ethanol, and suspended in 0.1 mol/L Tris-HCl, pH 8.0, 0.5 mol/L ethylenediaminetetraacetic acid. After spectrophotometric quantification, 2 ng of genomic DNA was analyzed by real-time PCR amplification using Stratagene MX3000P personal Q-PCR (M-Medical, Milano, IT) in the presence of SYBR Green. Quantification of human DNA was based on standard curve using genomic DNA extracted from PC3 cells. Cells (105) were plated in triplicate directly onto 24-well cell culture dishes in the presence of complete medium. Cellular growth was stopped after 4 hours (To) and after 7 days in culture, by removing the medium and 0.5% crystal violet solution in 20% methanol was added. After staining for 5 minutes the fixed cells were washed with PBS and solubilized with 200 μl/well 0.1 mol/L sodium citrate, pH 4.2. The absorbance at 595 nm was evaluated using a microplate reader. All experiments were done at least in triplicate. Statistical analysis of the data were performed by Student's t-test, P values ≤0.05 were considered statistically significant. Statistical analysis of in vivo experiments was performed using SPSS 11.0 software (SPSS, Inc., Chicago, IL). All statistical tests were two-tailed. Differences in the success rate between treatments were compared with χ2 test for 2 × 2 tables or Fisher's exact test when the tables were too sparse. EphA2 has already been reported to be overexpressed in prostate cancers30Walker-Daniels J Coffman K Azimi M Rhim JS Bostwick DG Snyder P Kerns BJ Waters DJ Kinch MS Overexpression of the EphA2 tyrosine kinase in prostate cancer.Prostate. 1999; 41: 275-280Crossref PubMed Scopus (233) Google Scholar, 31Wang XD Reeves K Luo FR Xu LA Lee F Clark E Huang F Identification of candidate predictive and surrogate molecular markers for dasatinib in prostate cancer: rationale for patient selection and efficacy monitoring.Genome Biol. 2007; 8: R255Crossref PubMed Scopus (70) Google Scholar and the expression of the kinase has been correlated with tumor aggressiveness.32Zeng G Hu Z Kinch MS Pan CX Flockhart DA Kao C Gardner TA Zhang S Li L Baldridge LA Koch MO Ulbright TM Eble JN Cheng L High-level expression of EphA2 receptor tyrosine kinase in prostatic intraepithelial neoplasia.Am J Pathol. 2003; 163: 2271-2276Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar The strong correlation between EphA2 expression and prostate carcinoma aggressiveness prompted us to select an experimental model to study the role of EphA2 in the regulation of cell motility and invasiveness of prostate carcinoma cells. We therefore selected PC3 prostate tumor cells as a model because of their expression of both EphA2 and ephrinA1 ligand (Figure 1A). Prior studies have shown that co-expressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains. These results raise the possibility that cis- and trans-configurations of ligand/receptor proteins allow cells to use both Ephs and ephrins as functional guidance molecules within the same moving cell, raising both reverse and forward signaling.33Marquardt T Shirasaki R Ghosh S Andrews SE Carter N Hunter T Pfaff SL Coexpressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains.Cell. 2005; 121: 127-139Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar We observed that the expression of endogenous ephrinA1 ligand is increased by cell density (100% or 30% confluence, respectively, dense or sparse), whereas the amount of EphA2 kinase is unaffected (Figure 1, B and C). We therefore determined whether in PC3 cells EphA2 receptors are tyrosine phosphorylated by engaging endogenous ephrinAs in a cell-cell contact-dependent manner, and whether the level of EphA2 tyrosine phosphorylation can be further elevated by exogenous ephrinA1-Fc ligand. Cells were again plated with 100% or 30% confluence and stimulated with 1 μg/ml of exogenous ephrinA1-Fc ligand for 15 minutes. As shown in Figure 1D, the basal level of EphA2 receptor phosphorylation is not influenced by increased cell concentration, suggesting that the trans-stimulation via cell-cell contacts is ineffective in PC3 cells. On the contrary, the phosphorylation of EphA2 is strongly induced by exogenous ephrinA1-Fc ligand. In particular, high cell density reduces the exogenously-induced EphA2 phosphorylation, suggesting that endogenous ephrinA1 ligands may behave as a trans-acting and cell-cell contact-dependent factor, leading to desensitize EphA2. To focus our studies on forward trans-phosphorylation of EphA2 by the exogenous ligand, thus avoiding any contamination of the forward signaling with the reverse signaling, all of the following experiments were performed in low confluence cultures (≤30%). To test directly the role of EphA2 receptor phosphorylation/kinase activity, we cloned the human EphA2 cDNA and by site-specific mutagenesis we generated the following mutants: Y587F/Y593F in the juxtamembrane domain (DM); K645R a kinase-dead (KD) mutant in the ATP binding site; and the ΔCyto mutant, deleted of the entire cytoplasmic region (Figure 2A). Our assumption is that all these mutants may act as dominant-negative factors by contrasting and/or eliminating the trans-phosphorylation of receptor dimers, whereas the ΔCytoEphA2 leads to abrogation to both kinase-dependent and -independent function of EphA2. Preliminary studies on the EphA2-negative HEK293T cell line as recipient (Figure 1A), demonstrated that the ΔCytoEphA2, the DM, and the KD mutants exert a dominant-negative activity on both EphA2 and FAK phosphorylation (data not shown). Nevertheless EphA2 has been implied in prostate carcinogenesis, a role for both ligand-dependent tyrosine phosphorylation of EphA2 or for its simple overexpression have been entailed.16Cheng N Brantley DM Liu H Lin Q Enriquez M Gale N Yancopoulos G Cerretti DP Daniel TO Chen J Blockade of EphA receptor tyrosine kinase activation inhibits vascular endothelial cell growth factor-induced angiogenesis.Mol Cancer Res. 2002; 1: 2-11Crossref PubMed Scopus (38) Google Scholar, 49Zantek ND Azimi M Fedor-Chaiken M Wang B Brackenbury R Kinch MS E-cadherin regulates the
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