The Phosphorylation of Ephb2 Receptor Regulates Migration and Invasion of Human Glioma Cells

[CANCER RESEARCH 64, 3179–3185, May 1, 2004] The Phosphorylation of EphB2 Receptor Regulates Migration and Invasion of Human Glioma Cells

Mitsutoshi Nakada,1,2 Jared A. Niska,2 Hisashi Miyamori,3 Wendy S. McDonough,2 Jie Wu,1 Hiroshi Sato,3 and Michael E. Berens2 1Neuro-Oncology Research, Barrow Neurological Institute, Phoenix, Arizona; 2The Translational Genomics Research Institute, Phoenix, Arizona; and 3Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Ishikawa, Japan

ABSTRACT logical functions have been attributed to Eph receptors and ephrins, including vascular development, tissue-border formation, cell migra- Eph receptor tyrosine kinases and their ligands, ephrins, mediate neu- tion, axon guidance, and synaptic plasticity (4–7). rodevelopmental processes such as boundary formation, axon guidance, There is increasing evidence that the B-family of Eph receptors and vasculogenesis, and cell migration. We determined the expression profiles of the Eph family members in five glioma cell lines under migrating and ephrins are functionally involved in the organization of the central nonmigrating conditions. EphB2 mRNA was overexpressed in all five nervous system during development (3, 4). EphB–ephrin-B signaling during migration (1.2–2.8-fold). We found abundant EphB2 protein as is critical for establishing boundaries between segments of the verte- well as strong phosphorylation of EphB2 in migrating U87 cells. Confocal brate hindbrain (8). Early-migrating EphB2-expressing neural crest imaging showed EphB2 localized in lamellipodia of motile U87 cells. cells are repelled by ephrin-B ligands expressed in the caudal somatic Treatment with ephrin-B1/Fc chimera stimulated migration and invasion compartment (9, 10). Recently it was demonstrated that EphB2 and of U87, whereas treatment with a blocking EphB2 antibody significantly ephrin-B2 signaling mediates glial scarring after spinal cord injury inhibited migration and invasion. Forced expression of EphB2 in U251 (11), suggesting EphB and ephrin-B are also involved in pathological cells stimulated cell migration and invasion and diminished adhesion conditions in the central nervous system. concomitant with the tyrosine phosphorylation of EphB2. U251 stably transfected with EphB2 showed more scattered and more pronounced A characteristic of malignant astrocytic tumors is their ability to invasive growth in an ex vivo rat brain slice. In human brain tumor infiltrate and invade the surrounding normal brain tissue. This phe- specimens, EphB2 expression was higher in glioblastomas than in low- notype makes their complete surgical resection difficult and focal grade astrocytomas or normal brain; patterns of phosphorylated EphB2 therapy ineffective. Thus, understanding the mechanisms of astrocytic matched the expression levels. Laser capture microdissection of invading tumor cell invasion in the brain is essential to developing new strat- glioblastoma cells revealed elevated EphB2 mRNA (1.5–3.5-fold) in 7 of 7 egies to control this malignancy. The process of glioma cell invasion biopsy specimens. Immunohistochemistry demonstrated EphB2 localiza- may include similar mechanisms directing cell movement during tion primarily in glioblastoma cells (56 of 62 cases) and not in normal neural development, suggesting that signaling proteins that contribute brain. This is the first demonstration that migrating glioblastoma cells to migration of neural crest cells may be associated with glioma overexpress EphB2 in vitro and in vivo; glioma migration and invasion are promoted by activation of EphB2 or inhibited by blocking EphB2. Dys- invasion. Among Eph family members, EphA5 expression has been regulation of EphB2 expression or function may underlie glioma invasion. described in glioblastoma cells (12); however, little is known about the role of the other family in this disease. In this study we report a role for EphB receptors in invasive glioma INTRODUCTION cells. Expression of the EphB family was examined in migrating glioma cells. We demonstrate induction of both EphB2 mRNA and Eph kinases constitute the largest family of receptor protein tyro- protein in actively migrating human glioma cells in vitro and in vivo. sine kinases, consisting of 14 distinct members. They are dichoto- Phosphorylation of EphB2 is correlated with migration and invasion, mized into EphA (EphA1 to EphA8) and EphB (EphB1 to EphB6) whereas blocking of EphB2 activation inhibits glioma invasion in according to their sequence homologies and ligand-binding specific- vitro. These results suggest a functional role for EphB2 in the invasive ities (1). Eph ligands are also expressed mainly on cell surfaces and behavior of malignant astrocytic tumors. are termed ephrins. The ephrin-A (ephrin-A1 to ephrin-A5) group are ligands of EphA and are glycosylphosphatidylinositol-anchored proteins. The ephrin-B (ephrin-B1 to ephrin-B3) group are ligands of MATERIALS AND METHODS EphB and are transmembrane proteins. When ligands bind to Eph receptors, the kinase domain is phosphorylated, which leads to phos- Cell Culture Conditions and Extracellular Matrix (ECM) Preparation. phorylation of many cytoplasmic substrate proteins. The transmem- Human embryonic kidney 293T cells and the human astrocytoma cell lines brane ephrin-B ligands can also function as reciprocal receptors for U87, T98G, U251 (American Type Culture Collection, Manassas, VA), EphB molecules and transduce signals into cells (2). One major effect SF767, and G112 (13) were maintained in DMEM supplemented with 10% of this bidirectional activation of Eph receptors and ephrin ligands is fetal bovine serum Astrocytoma-derived ECM was prepared as described cell repulsion. Present understanding of the Eph signaling axis is previously (13). Briefly, a confluent monolayer of SF767 cells was maintained in culture for 3–5 days for ECM deposit. The cells were lysed from the ECM largely derived from studies in the central nervous system, where with 0.5% Triton X-100 for 30 min at 25°C followed by incubation with 0.1 M many of the Eph kinases are abundantly expressed (3). Several bio- NH4OH for 5 min at 25°C, and the residual ECM was thoroughly rinsed with PBS. Received 11/24/03; revised 1/14/04; accepted 2/3/04. Antibodies and Reagents. Antiphosphotyrosine monoclonal antibody was Grant support: NIH Grant NS042262 (to M. E. Berens), Basic Research Fellowship purchased from Cell Signaling Technology (Beverly, MA). Anti-EphB2 poly- Award from the American Brain Tumor Association (to M. Nakada), and a grant from the Naito Foundation for Young Scientists (to M. Nakada). clonal antibody, which recognizes the extracellular domains of EphB2 and The costs of publication of this article were defrayed in part by the payment of page ephrin-B1/Fc chimera, was purchased from R&D systems (Minneapolis, MN). charges. This article must therefore be hereby marked advertisement in accordance with ␣-Tubulin monoclonal antibody was obtained from Oncogene Research 18 U.S.C. Section 1734 solely to indicate this fact. (Boston, MA). Control mouse IgG and Fc fragment of mouse IgG were Requests for reprints: Michael E. Berens, The Translational Genomics Research Institute, 400 North Fifth Street, Suite 1600, Phoenix, AZ 85004. Phone: (602) 850-7007; purchased from Sigma (St. Louis, MO) and Jackson ImmunoResearch (West Fax: (602) 343-8440; E-mail: [email protected]. Grove, PA), respectively. 3179

Migration Assays. Cellular migration was quantified by a microliter scale PCR-amplified using 293T cDNA as a template; the fragment was then radial migration assay as described previously (14). Briefly, astrocytoma- inserted into pEAK plasmid (18). EphB2 cDNA cloned into pEAK, which derived ECM was prepared on 10-well slides (Erie Scientific, Portsmouth, NH) contains a puromycin-resistant gene, was stably transfected into U251 cells by as described above. Approximately 3000 serum-starved glioma cells were the calcium phosphate method, and stable transfectants were selected in the plated in each well by use of a cell sedimentation manifold (CSM Inc., presence of 1.5 ␮g/ml puromycin (Sigma) as described previously (19). As a Phoenix, AZ) to establish a confluent monolayer 1 mm in diameter. The control, cells were transfected with the empty plasmid vector. U251 cells diameter of the circle circumscribing the cells was measured by use of an cotransfected with green fluorescent protein (GFP) and EphB2 or pEAK were inverted microscope (Axiovert; Zeiss, Thornwood, NY) and image analysis obtained by transfecting GFP expression plasmid and EphB2 or pEAK into equipment (Scion Image, Frederick, MD). Cells were then allowed to migrate U251 cells with a selectable neomycin-resistant gene. Cells were then cultured for 24 h in serum-free medium, and the diameter of the circle circumscribing in the presence of 800 ␮g/ml G418 (Sigma) and 1.5 ␮g/ml puromycin to select the population of cells was again measured. The average migration rate of 10 for stable dual transfectants. replicates was calculated from the increase in diameter of the circle circum- Cell Adhesion Assay. The cell adhesion assay was carried out as described scribing the cells as a function of time. previously (20). Briefly, 96-well plastic plates were precoated with astrocyto- To investigate the influence of EphB2 phosphorylation on glioma cell motility, we seeded U87 cells in the migration assay format and allowed them ma-derived ECM. Control dishes were prepared by blocking with BSA alone. ϫ 4 to adhere. The medium was then exchanged for serum-free medium containing Cells were then plated at 1 10 cells/well and allowed to adhere to the dishes 0.2–2 ␮g/ml recombinant ephrin-B1/Fc chimera, anti-EphB2 antibody, or for2hat37°C before staining with 1% crystal violet. After the plate was control mouse IgG, and the migration rate was evaluated for 24 h. washed with PBS, the crystal violet bound to the cells was eluted with 10% Real-Time Quantitative Reverse Transcription-PCR (QRT-PCR). acetic acid and measured by the absorbance at 590 nm (Spectrafluor Plus; QRT-PCR was performed in a LightCycler (Roche Diagnostics, Indianapolis, TECAN, Durham, NC). For ligand stimulation experiments, the EphB2-stably IN) with SYBR green fluorescence signal detection after each cycle of ampli- transfected U251 cells were treated for 15 min with 2 ␮g/ml preclustered fication as described previously (15). PCR was performed with the following ephrin-B1/Fc or control Fc fragment before seeding into the wells. primers: Ex Vivo Invasion Assay on Rat Brain Slices. A brain slice model system EphB1 (NM_004441): sense, 5Ј-AGAGGAGGGAAAAGGACCAGG-3Ј; of rat whole cerebrum was modified according to the organotypic culture antisense, 5Ј-GGTTTCCCACGGCATCTC-3Ј (amplicon size, 183 bp); methods reported previously (21, 22). Brain tissue was retrieved from 4-week- EphB2 (AF025304): sense, 5Ј-AAAAGGGCTTGGGAGATTCAT-3Ј; anti- old male Wister rat [Crl:(WI)BR; Charles River Lab, Wilmington, MA] after sense, 5Ј-GTCCATCTGTCCCGTCCTC-3Ј (amplicon size, 215 bp); halothane anesthesia. The cerebrum was immediately removed and cut verti- EphB3 (NM_004443): sense, 5Ј-GCTGGGCTTGTCTTCGTGGTG-3Ј; cally to the base in 400-␮m-thick sections with a vibratome (1000 Plus; The Ј Ј antisense, 5 -CCTTGGCAAACTCCCGAACA-3 (amplicon size, 184 bp); Vibratome Co., St. Louis, MO). A pinhole was created with a micropipette on Ј Ј EphB4 (NM_004444): sense, 5 -GGCTGCTCGCAACATCCTAGT-3 ; the putamen of the brain slice, and 1 ϫ 105 U251 glioma cells cotransfected Ј Ј antisense, 5 -CCACATCACAATCCCGTAAC-3 (amplicon size, 211 bp); with GFP and EphB2 or pEAK were gently placed in the pinhole (0.5-␮l Ј Ј EphB6 (NM_004445): sense, 5 -GGAGGAAGGGAGGAGAGTAAA-3 ; transfer volume). Typically, six brain slices were used in each experiment. The antisense, 5Ј-CCAGGAGCAGCACCCATAG-3Ј (amplicon size, 241 bp); protocol was approved by the Institutional Animal Care and Use Committee. Histone H3.3 (NM_002107): sense, 5Ј-CCACTGAACTTCTGATTCGC- Fluorescent stereomicroscopic imaging of the specimens was performed at 3Ј; antisense, 5Ј-GCGTGCTAGCTGGATGTCTT-3Ј (amplicon size, 215 bp). ϫ10 magnification using a Macro-Fluorescent Imaging System (SZX12- The nucleotide number and amplicon size for each primer are presented in RFL3; Olympus, Tempe, AZ) with a GFP barrier filter (DP50; Olympus) at 0, parentheses. The PCR data were analyzed with the LightCycler analysis software as described previously (15). 24, and 72 h after seeding of the cells. Immunoprecipitation and Immunoblot Analysis. Immunoprecipitation To quantitate glioma cell invasion into the brain slice, we used an inverted and immunoblot analysis were performed as described previously (16). To laser confocal microscope to observe GFP-labeled cells on a tissue insert with ␮ detect the phosphorylation of EphB2, we stimulated U87 cells with ephrin- the micropore filter membrane, and serial sections were obtained every 20 m B1/Fc for 15 min and treated them with EphB2 antibody for 30 min at 37°C downward from the surface plane to the bottom of the slice. The invasion rate before cell lysate extraction. was calculated as described previously (23). Immunohistochemistry and Immunofluorescent Microscopy. Immuno- Clinical Samples and Histology. Under an Institutional Review Board- histochemistry was performed using avidin-biotin immunoperoxidase tech- approved protocol, fresh human brain tumor tissues were obtained from 26 nique as described previously (16). Anti-EphB2 antibody was used at a dilution patients who underwent therapeutic removal of astrocytic brain tumors. Non- of 1:100. neoplastic control brain tissues were identified from the margins of the tumors For immunofluorescence, after the migration interval, cells were fixed and when possible. Histological diagnosis was made by standard light-microscopic permeabilized. After washing with PBS, cells were blocked with 2% BSA and evaluation of the sections stained with H&E. The classification of human brain 3% goat serum and incubated with anti-EphB2 antiserum (1:100 dilution) for tumors used in this study is based on the revised WHO criteria for tumors of 1 h at 25°C. Negative controls were stained with a 1:50 dilution of preimmu- the central nervous system (24). The 26 astrocytic tumors consisted of 7 nization goat sera. Finally, the cells were incubated for 30 min with a 1:100 low-grade astrocytomas, 8 anaplastic astrocytomas, and 11 glioblastomas. All dilution of Cy3-conjugated antigoat antibody (The Jackson Laboratory). Fluo- of the tumor tissues were obtained at primary resection, and none of the rescence was monitored by inverted confocal laser microscopy (Carl Zeiss, patients had been subjected to chemotherapy or radiation therapy before New York, NY). resection. Cell Invasion Assays. Cell invasion assays were carried out using modi- Laser Capture Microdissection. Cryopreserved glioblastoma specimens fied Boyden chambers consisting of Transwell with precoated Matrigel mem- from seven patients were cut in serial 6–8-␮m sections and mounted on brane filter inserts in 24-well tissue culture plates (BD Biosciences Discovery uncoated slides treated with diethyl pyrocarbonate. We collected 3000 indi- Labware, Bedfold, MA) as described previously (17). Serum-deprived cells vidual cells for QRT-PCR analysis as described previously (15). Laser capture (2 ϫ 105) suspended in 100 ␮l of DMEM containing 1 mg/ml BSA and 0.5% serum were added to each Transwell. After 16 h, noninvading cells were microdissection was performed with a PixCell II Microscope (Arcturus Engi- removed by wiping the upper side of the membrane, and the invading cells neering, Inc., Mountain View, CA). Neoplastic astrocytes in the invasive rim ϳ were fixed with methanol and stained with crystal violet (Sigma). The number 1 cm from the edge of the tumor core were identified according to the criteria of invading cells was quantified by counting of least six random fields (total of nuclear atypia (coarse chromatin, nuclear pleomorphism, and multinucle- magnification, ϫ200) per filter. In certain experiments, ephrin-B1/Fc, EphB2 ation) and, whenever possible, according to nuclear and/or cytoplasmic simi- antibody, or control Fc fragment of mouse IgG was applied to the upper larity with the glioblastoma cells in the core. Reactive astrocytes were iden- chamber. tified by morphology and were avoided. Expression Plasmids and Cell Transfection. The expression plasmid for Statistics. Statistical analyses were performed with the ␹2 test and the EphB2 was constructed as follows. The cDNA fragment encoding EphB2 was two-tailed Mann-Whitney U test. P Ͻ 0.05 was considered significant. 3180

RESULTS

Actively Migrating Glioma Cells Had Increased Expression of EphB2. A microscale radial monolayer migration assay was used to assess the intrinsic migration rate of various glioma cell lines on astrocytoma-derived ECM. Astrocytoma-derived ECM has previously been shown to enhance the motility behavior of glioma cells (13). U87 cells migrated at a significantly higher rate (mean Ϯ SD, 19.25 Ϯ 0.99 ␮m/h; P Ͻ 0.01), whereas U251 migrated more slowly (9.04 Ϯ 1.67 ␮m/h; P Ͻ 0.05) than the other glioma cell lines (Fig. 1A). Because the EphB family has been described to play a role in cell motility (3), we characterized the expression of the various EphB receptors in accordance to cell motility in five glioma cells lines. Glioma cells were deposited on 10-well glass slides precoated with astrocytoma-derived ECM. After 24 h of cell motility, cells actively migrating at the rim and migration-restricted cells at the core were mechanically collected as separate populations. RNA was isolated from the two cell populations, and the expression levels of EphB family members were determined by real-time QRT-PCR. To compare the EphB gene expression level across the different cell lines, the expression level of core U87 mRNA was arbitrarily chosen to nor- malize the data. Among all receptors of the EphB family, only EphB2 was overexpressed in the migrating cells in all cell lines (1.2–2.8-fold) Fig. 2. Production, phosphorylation, and localization of EphB2 in human glioma cell relative to the migration-restricted cells at the core (Fig. 1B). EphB2 lines. A, immunoprecipitation (IP) using total cell lysate from each of the indicated cell expression is highest in U87, which is the most rapidly migrating cell lines. Equal amounts of cell lysates were immunoprecipitated with anti-EphB2 antibody. The immunoprecipitates were probed by immunoblotting (IB) with the antibody indicated. line (Fig. 1, A and B). PY, phosphotyrosine. Whole cell lysates (WCL) were also immunoblotted with EphB2 or ␣-tubulin antibody to control for equal protein loading of the five cell lines. B, U87 migration-restricted cells and migration-activated glioma cells collected after the migration assay and immunoprecipitation and immunoblotting were performed as above. C, U87 cells plated in the migration format on astrocytoma-derived extracellular matrix- coated slides, allowed to migrate overnight, and then fixed. The slide was processed for EphB2 immunocytochemistry. Arrows in panel Rim indicate more intense staining for EphB2 at lamellipodia in migration activating cells. Bar,20␮m.

Actively Migrating Glioma Cells Had Increased Expression of Activated EphB2. Because EphB2 mRNA levels are elevated during glioma migration, we assessed the protein levels of EphB2. Immuno- blot analysis of total cellular lysates detected EphB2 protein expression in three of five glioma cell lines (Fig. 2A, Lanes U87, U251, and T98G). The most motile cell line, U87, had the highest protein expression. To determine whether the EphB2 receptor is active in glioma cells, we immunoblotted the EphB2 immunoprecipitates with a monoclonal antibody recognizing phosphorylated tyrosine residues. Tyrosine-phosphorylated EphB2 receptor was detected only in the highly migratory U87 cells (Fig. 2A). We also assessed whether EphB2 phosphorylation increases in the course of activated cell migration. Consistent with the changes in mRNA expression, we observed a 2-fold increase in EphB2 protein expression in the total lysates of actively migrating U87 cells relative to that from migration-restricted cells (Fig. 2B). As expected, more EphB2 protein was immunoprecipitated in the actively migrating cells and displayed increased tyrosine phosphorylation (Fig. 2B). The sub- cellular localization of EphB2 in migrating glioma cells was determined by immunofluorescent microscopy. Migrating glioma cells displayed increased staining for EphB2 relative to migration-restricted cells (Fig. 2C). In addition, EphB2 staining localized predominantly within lamellipodia at the leading edge of cell migration (Fig. 2C, Rim). These data suggest that EphB2 is active in migrating glioma Fig. 1. Migration of glioma cell lines and profiles of EphB mRNA expression in glioma cells and that it localizes in lamellipodia. cell lines at the core and rim as determined in the migration assay. A, migration rate -P Ͻ 0.01 versus T98G, SF767, and G112. EphB2 Phosphorylation Correlates with Migration and Inva ,ءء ;P Ͻ 0.05 ,ء .obtained after 24 h. Bars, SD B, relative mRNA expression levels of EphB family genes (target mRNA:histone H3.3 sion of U87 Cells in Vitro. To investigate whether the activation of mRNA ratios) in the cells at core (columns C) and rim (columns R) of the migration assay were analyzed by quantitative reverse transcription-PCR. mRNA levels are expressed as EphB2 stimulates glioma cell migration, we used a recombinant a proportion of the U87 core mRNA level, which was given a value of 1. ephrin-B1 ligand to activate endogenous EphB2 in U87. As shown in 3181

Fig. 3A, phosphorylation of EphB2 was induced by addition of ephrin- fected cells. These cells grew with doubling times comparable to those B1/Fc chimera. EphB2 phosphorylation in U87 cells was inhibited in for the parental cells; cells expressing exogenous EphB2 did not a dose-dependent manner by addition of a functional blocking anti- display any cytotoxic effects as determined by changes in trypan blue body to EphB2; treatment with control IgG had no effect. exclusion, propidium iodide uptake, 4Ј,6Ј-diamidino-2-phenylindole Migration assay and invasion studies were performed in the absence or hydrochloride staining, and immunostaining with anti-activated presence of 0.2–2 ␮g/ml recombinant ephrin-B1/Fc chimera, EphB2 caspase 3 antibodies (data not shown). In addition, cells in which antibody, or control mouse IgG. Ephrin-B1/Fc chimera (2.0 ␮g/ml) activation of the EphB2 receptor could be stimulated by treatment effectively stimulated migration of U87 glioma cells (mean Ϯ SD, with an ephrin ligand (Fig. 4C) showed reduced cell substrate adhe- 23.84 Ϯ 0.61 ␮m/h; P Ͻ 0.05) ϳ1.24 fold relative to cells treated with sion on treatment with chimeric ephrin-B1/Fc chimera (mean Ϯ SD, control IgG (Fig. 3B). The migration of U87 was significantly suppressed 0.30 Ϯ 0.025; P Ͻ 0.05; Fig. 4D). There was no difference after by the addition of 2.0 ␮g/ml EphB2 antibody (15.57 Ϯ 1.42 ␮m/h; addition of control Fc relative to EphB2 transfectants (0.68 Ϯ 0.07; P Ͻ 0.05). Similar outcomes were obtained in the migration of U251 and Fig. 4D). T98G, which express EphB2 (data not shown). In addition, overexpression of EphB2 in U251 induced a small but Invasion assay data also indicated that ephrin-B1/Fc chimera stim- measurable increase in migration rate (11.57 Ϯ 1.67 ␮m/h; P Ͻ 0.05) ulated invasion of U87 cells (160 Ϯ 10% of control; P Ͻ 0.01). relative to pEAK-transfected cells (9.6 Ϯ 0.91 ␮m/h). The migration EphB2 antibody inhibited the invasion of U87 cells [87 Ϯ 12% of rate was enhanced by addition of ephrin-B1/Fc chimera in the EphB2 control (P Ͻ 0.05) and 72 Ϯ 10% of control (P Ͻ 0.01)] as concen- transfectants (13.0 Ϯ 2.01 ␮m/h; P Ͻ 0.05), whereas addition of trations of EphB2 antibody increased (Fig. 3C), whereas control IgG control Fc had no measurable effect (11.4 Ϯ 1.34 ␮m/h; Fig. 4E). had no effect on U87 cells invasion. Taken together, these data As shown in Fig. 4F, cell invasion through membranes coated with indicate that activation or interference of EphB2 accelerates or retards Matrigel was increased in cells expressing EphB2 (mean Ϯ SD, glioma cell migration and invasion, respectively. 228 Ϯ 58% of control; P Ͻ 0.05) and in the cells treated with EphB2 Phosphorylation Inhibits Cell Adhesion and Promotes ephrin-B1/Fc chimera (473 Ϯ 53% of control; P Ͻ 0.01), whereas we Cell Migration or Invasion in U251 Cells. To examine the func- observed no significant change between EphB2 and that produced by tional effects of EphB2, we evaluated U251 cells transfected with addition of control Fc (224 Ϯ 46% of control), indicating that the EphB2 or control vector (pEAK) constructs in adhesion, migration, phosphorylation of EphB2 is required for stimulation of cell invasion. and invasion assays. Forced expression of the EphB2 receptor in U251 To evaluate the effects of EphB2 on invasion through a more cells resulted in phosphorylation of the transfected receptor (Fig. 4A). physiologically relevant matrix, we examined U251 stably cotrans- EphB2 activation led to dramatic changes in cell morphology; cells fected with control or EphB2 and GFP expression plasmids for their expressing the activated receptor partially detached from the substrate growth and dispersion within an ex vivo organotypic rat brain slice. and rounded up (Fig. 4B). In contrast, cells transfected with empty Overexpression of EphB2 in U251-GFP-EphB2 cells increased phos- vector remained spread and spindle-shaped (Fig. 4B). Immunocyto- phorylation of the transgene product (data not shown). To compare the chemistry showed that EphB2 localized to the cell membrane (Fig. growth of mock transfectants with that of EphB2 stable transfectants, 4B), suggesting that the transgene functions normally in the trans- we implanted aggregations of each of these engineered cells in the putamen on contralateral sides of the same rat brain slice (Fig. 5A); images were taken at 0, 24, and 72 h after the implantation. The U251-EphB2 cells displayed greater migration and invasion into the organotypic rat brain slice than did the less invasive mock-transfectant cells (Fig. 5B). To quantify cell invasion, serial optical sections were obtained every 20 ␮m downward (Z axis) from the basal plane to the bottom using confocal microscopy. The U251-EphB2 cells penetrated further into the brain slices (mean Ϯ SD, 51.8 Ϯ 14.9 ␮m/72 h) than the mock cells (33.8 Ϯ 10.35 ␮m/72 h; P Ͻ 0.05; Fig. 5C). These data suggest that EphB2 plays a role in invasion both in vitro and in vivo. Overexpression and Phosphorylation of EphB2 in Invading Cells of Glioblastoma. To evaluate a potential role for EphB2 in the malignant behavior of human gliomas, the expression level of EphB2 was evaluated as a function of tumor grade. Levels of EphB2 mRNA in human brain tumors were evaluated by QRT-PCR using histone H3.3 mRNA as an internal reference for normalization. The mRNA levels of the EphB2 gene (EphB2 mRNA:histone H3.3 mRNA ratios) were significantly higher in glioblastoma tissues (mean Ϯ SD, Fig. 3. Influence of EphB2 activation on EphB2 phosphorylation, migration, and 0.391 Ϯ 0.25; n ϭ 11) than those in normal brain tissues invasion of U87 cells. A, serum-starved U87 cells on a plastic substrate were treated with (0.129 Ϯ 0.03; P Ͻ 0.05; n ϭ 3) and low-grade astrocytoma tissues control IgG (IgG), ephrin-B1/Fc chimera (ephrin-B1/Fc), or EphB2-blocking antibody Ϯ Ͻ ϭ (EphB2 Ab) at the indicated concentrations for 15 min, after which immunoprecipitation (0.136 0.082; P 0.01, n 7; Fig. 6A). The expression levels of (IP) of EphB2 was performed. Phosphorylation of EphB2 in U87 was triggered by EphB2 increased significantly as tumor grade increased. To investi- treatment with ephrin-B1/Fc chimera. The EphB2-blocking antibody suppressed the gate the potential role of EphB2 in invasion in vivo, we collected phosphorylation of EphB2. IB, immunoblotting; NA, no addition; PY, phosphotyrosine. B, U87 cells were plated on 10-well glass slides precoated with astrocytoma-derived extra- invading glioblastoma cells and cells in the tumor core by laser cellular matrix and then cultured in serum-free medium in the absence or presence of capture microdissection of seven glioblastoma surgical specimens and control IgG (IgG), ephrin-B1/Fc chimera (ephrin-B1/Fc), or EphB2-blocking antibody performed QRT-PCR of the isolated RNA. EphB2 was overexpressed (EphB2 Ab) at the indicated concentrations after attachment of the cells to the dishes. Cell P Ͻ 0.05 versus no addition (NA) or IgG. in invading glioblastoma cells (1.5–3.5-fold) relative to the cells in the ,ء .migration was assessed over 24 h. Bars, SE C, U87 cells were treated as above and then used in the invasion assay. Cells that invaded tumor core in all seven biopsy specimens (Fig. 6B). to the lower surface of the membrane were fixed and stained. Mean cell counts from at P Ͻ 0.01 Consistent with the QRT-PCR results, protein levels and tyrosine ,ءء ;P Ͻ 0.05 ,ء .least six fields and four experiments are shown. Bars, SD versus no addition (NA) and IgG. phosphorylation of EphB2 were increased in glioblastoma tissue 3182

Fig. 4. Morphological change, cell adhesion, migration, and invasion of U251 cells expressing EphB2. A, activation of EphB2 in stably transfected U251 cells. EphB2 was immunoprecipitated (IP) from cells transfected with EphB2 or pEAK (Mock). The immunoprecipitates were probed by immunoblotting (IB) as indicated. B, morphological changes caused by expression of activated EphB2 in U251 cells. pEAK vector was used as control. Upper panels, phase-contrast microscopy; lower panels, immunofluorescence (IF) for EphB2. EphB2 is localized on the membrane. Bar,50␮m. C, activation of EphB2 by ephrin-B1 ligand. EphB2 was immunoprecipitated (IP) from U251 cells that had been stably transfected with EphB2 and treated with soluble ephrin-B1/Fc chimera at the indicated concentration or left untreated. The immunoprecipitates were probed by immunoblotting (IB) as indicated. D, cells were plated on dishes coated with astrocytoma-derived extracellular matrix (ECM) in the absence (Ϫ) or presence (ϩ) of control Fc (Fc)or2.0␮g/ml ephrin-B1/Fc chimera (ephrin-B1/Fc) and then incubated for 2 h at 37°C. Crystal-violet-stained cells attached to dishes were dissolved in 10% acetic acid, and the absorbance at 590 nm was measured spectropho- tometrically. The mean absorbance value from U251-Mock cells attached to dishes coated with astrocytoma-derived ECM is shown as 1. Bars, P Ͻ 0.01 versus Mock. E, cells were plated ,ءء .SD on 10-well glass slides precoated with astrocytoma-derived ECM and incubated as above after attachment of the cells to the dishes. Cell migration P Ͻ 0.05 ,ء .was assessed over 24 h. Bars, SD versus Mock. F, cells were treated as above and then used in the invasion assay. Mean cell counts from at least six fields and four experiments are P Ͻ 0.01 versus ,ءء ;P Ͻ 0.05 ,ء .shown. Bars, SD Mock. relative to normal brain, low-grade astrocytoma, and anaplastic astro- EphB expression has been reported in various human cancers, cytoma (Fig. 6C). including carcinomas of the lung (25), colon (26), and endometrium EphB2 was immunolocalized predominantly in the neoplastic as- (27), as well as osteosarcoma (28) and neuroblastoma (29). EphB2 trocytes in the majority of all glioblastoma specimens (56 of 62 cases; expression has been confirmed in carcinomas of the colon, stomach, Fig. 6D, panel a). Invading neoplastic cells also contained significant esophagus (30), and neuroblastoma (31); however, its role in the staining for EphB2 (Fig. 6D, panel b). Neoplastic astrocytes were invasion or metastasis processes in human carcinomas has not been identified by nuclear atypia in H&E-stained sections and were con- described. Thus, the present study is the first to demonstrate that firmed by positive glial fibrillary acidic protein staining (data not EphB2 receptor is involved in tumor invasion activity and possibly shown). Reactive astrocytes and occasional endothelial cells of blood malignant progression in human brain tumors. vessels also demonstrated positive immunostaining for EphB2. In EphB2 shares ϳ49–66% amino acid homology with other mem- anaplastic astrocytomas, some atypical cells were weakly immuno- bers of the EphB subgroup, but the homology between human and stained, but no staining was seen in the normal brains (Fig. 6D, panel mouse EphB2 is Ͼ99% (30). This high degree of homology suggests c) or when the primary antibody was substituted for normal serum an important conserved function of EphB2. Kiyokawa et al. (30) (Fig. 6D, panel d). The immunohistochemistry results are consistent reported that EphB2 was expressed in fetal brain but not in adult brain, with those obtained by QRT-PCR and immunoprecipitation analysis. providing evidence that it may be associated with the neurodevelop- mental process. This finding corroborates our QRT-PCR data of its DISCUSSION low expression in the normal brain. Considerable efforts have been The evidence presented here supports a role for EphB2 in glioma made in recent years to elucidate the biological function of EphB cell motility in vitro and in vivo. Expression of EphB2 was elevated family receptors in neural development. To date, EphB2 has been in all five glioma cell lines tested on adoption of a migratory pheno- implicated in proper formation of the anterior commissure (32), reg- type. The fastest migrating glioma cell line, U87, contained the ulation of fluid homeostasis in the semicircular canal (33), and acti- highest level of total EphB2 protein as well as the highest concentra- vation of dendritic spine morphogenesis (34). Unlike other members tion of tyrosine-phosphorylated EphB2 protein; additionally, EphB2 of the Eph family, EphB2 has both kinase-dependent and -independ- was localized to the leading edge at sites of lamellipodia formation. ent effects (4). In the U87 and U251 stable transfectant data, we The activity of migration and invasion in U87 was correlated with the observed a strong correlation between the phosphorylation level of phosphorylation of EphB2. Forced expression of EphB2 in U251, EphB2 and adhesion, migration, and invasion rates. These data indi- which was the least invasive of the five glioma cell lines and showed cate that the kinase activity of EphB2 is important for the invasive low constitutive expression of EphB2, transformed U251 to a more phenotype of glioblastoma cells. highly invasive phenotype as assessed by in vitro and ex vivo assays. In U87 glioma cells, the engineered soluble ligand, ephrin-B1/Fc The expression and phosphorylation of EphB2 were up-regulated in chimera, induced EphB2 tyrosine phosphorylation that correlated with glioblastoma, particularly in the invading cells. These results support increased migration and invasion activity. This is consistent with a role for EphB2 in glioblastoma tumor invasion. previous data obtained with an endothelial cell line (35). Because our 3183

measured with a monolayer radial migration assay, Boyden chambers, and an ex vivo organotypic rat brain slice model. The rat brain slice model is likely to be a more physiologically relevant matrix for invasion than the monolayer assay or Boyden chamber; it may also be subject to artifacts due to interspecies variations in ECM, cell surface molecules, receptor ligands, and unspecific physiological changes accompanying the ex vivo preparation. To date, cross-species com- parisons of three members of the ephrin-B family have been reported, with deduced amino acid homologies of 96, 98, and 100% for human ephrin-B1/rat ephrin-B1, human ephrin-B2/mouse ephrin-B2, and human ephrin-B3/mouse ephrin-B3, respectively (44–48). The ephrin-B1 gene is expressed in rat brain throughout its life (45). It is therefore possible that ephrin-B constitutively present in the rat brain slice provides a ligand for activation of the transfected human EphB2

Fig. 5. Cell migration and invasion in organotypic rat brain slice. A, glioma cells cotransfected with green fluorescent protein and pEAK or EphB2 were transplanted into bilateral putamen on rat organotypic brain slice. B, migrating U251 cells cotransfected with green fluorescent protein and pEAK or EphB2 were observed at the indicated times. Bar, 500 ␮m. C, invasion rates of U251 cells transfected with EphB2 or pEAK were calculated from Z axis images collected by confocal laser scanning microscopy. The mean .P Ͻ 0.05 ,ء .value of invasion rate was obtained from six experiments. Bars, SD

QRT-PCR data showed that EphB1 and EphB4 are also highly expressed in U87 cells, it is possible that endogenous EphB1 and EphB4 are also stimulated by addition of ephrin-B1/Fc. Recent studies have reported that both EphB1 and EphB4 mediate cell migration (36, 37). Although we cannot rule out the possibility that the migration of U87 was partially promoted through EphB1 or EphB4 phosphorylation by addition of ephrin-B1/Fc, an antibody specific for EphB2 prevented phosphorylation of EphB2 and inhibited migration and invasion. Thus, it is reasonable to speculate that the phosphorylation of EphB2 affects these changes in U87 cell behavior. We showed that U251 cells stably transfected with EphB2 manifest dramatic morphological changes, retarded or diminished cell adhesion, and increased migration and invasion activity. It is unknown what molecules are involved in these phenotypic changes downstream of EphB2 signaling. Recent investigations have begun to expand the understanding of EphB2 protein interactions and the downstream biochemical and phenotypic consequences of EphB2 signaling (3). The ability of EphB2 to decrease cell adhesion is reported to be partially mediated by the phosphorylation of R-Ras, a small intracel- lular GTPase (38). Preliminarily, we observed increased R-Ras phosphorylation in U251 cells stably transfected with EphB2 but not in mock-transfected cells (data not shown). R-Ras signals have been Fig. 6. EphB2 expression, phosphorylation, and localization in various human astro- shown to promote migration and invasion through a combination of cytic tumors. A, relative mRNA expression levels of EphB2 gene (EphB2 mRNA:histone phosphatidylinositol 3-kinase and protein kinase C (39). It is possible H3.3 mRNA ratios) in normal brain (NB), low-grade astrocytoma (LGA), anaplastic astrocytoma (AA), and glioblastoma (GB) were analyzed by quantitative reverse transcrip- that R-Ras phosphorylation induced by EphB2 contributes at least in tion-PCR. Each mRNA level is expressed as a proportion of the highest mRNA level of ;P Ͻ 0.05 ,ء .part to behavioral changes of U251 cells. The signaling pathway of EphB2, which was given a value of 1. Horizontal bars indicate mean values -P Ͻ 0.01. B, EphB2 expression was determined by quantitative reverse transcription ,ءء EphB2 also is shown to suppress the extracellular-regulated kinase/ PCR, in tumor core cells (columns C) and invading cells (columns R) in seven glioblas- mitogen-activated protein kinase pathway, resulting in a reduction of toma cases (GB1–GB7) captured by laser capture microdissection. mRNA levels are mitogenic signals and enhancement of cytoskeletal changes (40). expressed as proportions of the mRNA level of EphB2 in the GB1 core, which was given a value of 1. C, results of immunoprecipitation using total cell lysate. Equal amounts of Alternatively, EphB2 demonstrates an association with the exchange cell lysates were immunoprecipitated (IP) with anti-EphB2 antibody. The immunopre- factors intersectin and kalirin, which are involved in the morphogen- cipitates were probed by immunoblotting (IB) with the antibody indicated. PY, phospho- esis and maturation of dendritic spines (41, 42). Lastly, a recent study tyrosine. D, immunolocalization of EphB2 in glioblastoma tissues and normal brain tissues. Paraffin sections were immunostained with polyclonal antibodies against EphB2 indicates that EphB2–ephrin-B1 signaling regulates cell repulsion via (panels a, b, and c) or nonimmune goat IgG (panel d). Note the EphB2 immunostaining modification of RhoA activity (43). Analysis of EphB2 signaling in the glioblastoma cells at the central region in the tumor (panel a, arrows) as well as in invading glioblastoma cells at the tumor border (panel b, arrows) and the reactive pathway in glioma cells is ongoing in our laboratory. astrocytes, whereas no staining is observed in the normal brain with EphB2 (panel c) and Overexpression of EphB2 increased migration and invasion as glioblastoma with nonimmune IgG (panel d). Hematoxylin counterstain. Bar,50␮m. 3184

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Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2004 American Association for Cancer Research. The Phosphorylation of EphB2 Receptor Regulates Migration and Invasion of Human Glioma Cells

Mitsutoshi Nakada, Jared A. Niska, Hisashi Miyamori, et al.

Cancer Res 2004;64:3179-3185.

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