Angiopoietin 2 Induces Glioma Cell Invasion by Stimulating Matrix Metalloprotease 2 Expression Through the Αvβ1 Integrin and Focal Adhesion Kinase Signaling Pathway

Research Article

Angiopoietin 2 Induces Glioma Cell Invasion by Stimulating

Matrix Metalloprotease 2 Expression through the AvB1 Integrin and Focal Adhesion Kinase Signaling Pathway

Bo Hu,1,2 Michael J. Jarzynka,1,3 Ping Guo,1,3,4 Yorihisha Imanishi,1,3 David D. Schlaepfer,5 and Shi-Yuan Cheng1,3

1University of Pittsburgh Cancer Institute and Departments of 2Medicine and 3Pathology, Research Pavilion at the Hillman Cancer Center, Pittsburgh, Pennsylvania; 4North China Coal Medical College, Tangshan, Hebei, P.R. China; and 5Department of Immunology, The Scripps Research Institute, La Jolla, California

Abstract regulating the processes of cell migration and invasion (9). a Accumulating evidence reveals a significant correlation Integrins are heterodimeric glycoproteins composed of one h h between angiopoietin 2 (Ang2) expression and tumor invasion chain and one chain. The 1 family of integrins associates with at and metastasis in various human cancers, but the major focus least one of the 12 a-chains (a1-a11 and av) representing the major h of recent studies has been on the angiogenic effects of Ang2. group of cellular extracellular matrix receptors. 1-Integrin h We recently reported that Ang2-stimulated glioma cell promotes metastasis of 1-null tumor cells (9). Mutation of the h invasion results from the up-regulation and activation of intracellular domain of 1 integrin differentially affects cell matrix metalloprotease 2 (MMP-2) in tumor cells. In this adhesion, invasion, and metastasis (10). Ang2, as well as other study, we identify a novel mechanism by which Ang2 members of the angiopoietin family, is a potential substrate for the stimulates MMP-2 expression leading to glioma cell invasion. integrin receptor family in endothelial cells, fibroblasts, and myocytes (11–13). Ang2 not only enhances cell adhesion in both We show that Ang2 interacts with AvB1 integrin in Tie2- deficient human glioma cells, activating focal adhesion kinase endothelial cells and Tie2-deficient fibroblasts but also triggers (FAK), p130Cas, extracellular signal–regulated protein kinase integrin-mediated intracellular signal transduction pathways in these cells. (ERK) 1/2, and c-jun NH2-terminal kinase (JNK) and substan- tially enhancing MMP-2 expression and secretion. The Ang2/ Malignant human gliomas are pathophysiologically characterized by their insidious infiltration of the brain (14). Invasion of AvB1 integrin signaling pathway was attenuated by functional Cas inhibition of B1 and Av integrins, FAK, p130 , ERK1/2, and glioma cells into adjacent brain structures occurs through the JNK. Furthermore, expression of a negative regulator of FAK, activation of multigenic programs, including matrix metallopro- FAK-related nonkinase, by U87MG/Ang2–expressing glioma teases (MMP) such as MMP-2, which degrade extracellular matrix xenografts suppressed Ang2-induced MMP-2 expression and to overcome the extracellular matrix barrier at the invasive fronts glioma cell infiltration in the murine brain. These data of tumors (15). Several reports have recently shown that up- establish a functional link between Ang2 interaction with regulation of MMP-2 correlates with the invasiveness of human AvB1 integrin and glioma cell invasion through the FAK/ gliomas (16–18). Interestingly, integrin-mediated signaling path- p130Cas/ERK1/2 and JNK-mediated signaling pathway. (Cancer ways are involved in regulation of MMP-2 expression and cell Res 2006; 66(2): 775-83) invasion in tumor cells (19). Expression patterns of several integrins are correlated with invasive and migratory behaviors in Introduction glioma cells in vitro and in vivo (17). MMP-2 expression and tumor cell invasion have also been associated with the activation of Angiopoietin 2 (Ang2), a known angiogenic regulator, plays downstream regulators of integrins such as focal adhesion kinase important roles in angiogenesis and tumor progression (1). Ang2, (FAK), p130Cas, extracellular signal–regulated protein kinase (ERK)/ expressed in both endothelial cells and tumor cells, has been mitogen-activated protein kinase, and c-jun NH -terminal kinase significantly correlated with tumor metastasis and invasion (2–6), 2 (JNK)/stress-activated protein kinase (20, 21). We recently reported but the mechanism by which Ang2 acts on tumor cells remains that coexpression of Ang2 with MMP-2 was found in the invasive obscure. The highly conserved COOH-terminal fibrinogen-like areas, but not in the central regions, of primary human glioma domain of the angiopoietins implies a functional association with specimens (16, 18). Stable expression of Ang2 by glioma cells leads the integrin receptor family (7, 8). Integrins are crucial for to an increase in glioma cell invasiveness in vitro as well as migration and invasion of tumor cells, not only for mediating in murine intracranial xenografts accomplished through up- adhesion of tumor cells to the extracellular matrix but also for regulation and activation of MMP-2 (16). In this study, we characterize the mechanism by which Ang2 induces glioma cell invasion by stimulating MMP-2 expression and Note: Supplementary data for this article are available at Cancer Research Online h (http://cancerres.aacrjournals.org/). secretion in glioma cells. We show that Ang2 interacts with av 1 Requests for reprints: Shi-Yuan Cheng, University of Pittsburgh Cancer Institute integrin in U87MG glioma cells, resulting in the activation of FAK, and Department of Pathology, Research Pavilion at Hillman Cancer Center, Suite 2.26, Cas 5117 Centre Avenue, Pittsburgh, PA 15213-1863. Phone: 412-623-3261; Fax: 412-623- p130 , ERK1/2, and JNK, thereby enhancing MMP-2 expression 4840; E-mail: [email protected] or Bo Hu, Department of Medicine, Research Pavilion and secretion. As a functional consequence of increased MMP-2 at Hillman Cancer Center, Suite 2.26, 5117 Centre Avenue, Pittsburgh, PA 15213-1863. secretion, glioma cell invasion is promoted. Furthermore, stable Phone: 412-623-3261; Fax: 412-623-4840; E-mail: [email protected]. Cas I2006 American Association for Cancer Research. expression of dominant-negative forms of FAK or p130 in glioma doi:10.1158/0008-5472.CAN-05-1149 cells inhibits Ang2-stimulated MMP-2 expression and secretion by www.aacrjournals.org 775 Cancer Res 2006; 66: (2). January 15, 2006

h blocking activation of JNK and ERK1/2. Our data suggest that anti- 3 (B3A), anti-a3, anti-a5, and anti-av integrin antibodies that were elevation of Ang2 expression in the gliomas leads to increased included in the a integrin blocking investigator kit, anti-Myc (9B11), and anti-Ang2 antibodies], and then collected with 50 AL of either a Protein MMP-2 secretion along with increased cell invasion via avh1 integrin/FAK–mediated activation of ERK1/2 and JNK. G-plus or a Protein G-plus/Protein A agarose bead slurry. The eluted proteins from Ni+-NTA beads or the antibody-complexed proteins from coimmunoprecipitation were subjected to SDS-PAGE under reducing Materials and Methods h conditions and detected with an anti- 1 integrin (JB1A) or an anti-Ang2 Antibodies and reagents. Human U87MG and T98G glioma cells were antibody. obtained from American Type Culture Collection (Manassas, VA), U251MG Gelatin zymography and in vitro invasion assays. Zymographic and U373MG glioma cells were from our collection, and their culture was analyses for MMP-2 and in vitro invasion assays were done as previously previously described (16). The following reagents were used for this study: described (16). anti-Ang2 (C-19), anti-p130Cas (C-20), anti-JNK1 (C-17), anti-FAK (C-20), and Intracranial brain tumor xenografts and immunohistochemistry. anti-h3 integrin (H-96) antibodies (Santa Cruz Biotechnology, Santa Cruz, Stereotactic implantation of various types of U87MG cells, sacrifice of CA); neutralizing anti-h3 integrin (B3A), anti-h1 integrin (JB1A), and anti-a3 glioma-bearing mice, tumor processing, and analyses of intracranial integrin antibodies (P1B5); a h1 integrin polyclonal investigator kit; an a U87MG glioma invasiveness were done as previously described (16). h integrin blocking investigator kit that includes anti- 1, anti-a1, anti-a2, h h anti-a3, anti-a4, anti-a5, anti-a6, anti-av, anti-av 3, and anti-av 5 antibodies; Results FITC-conjugated anti-avh3 (clone LM609) and anti-avh5 antibodies (clone P1F6; both for fluorescence-activated cell sorting analyses) and anti-MMP-2 Ang2-enhanced adhesion of Tie2-deficient glioma cells is antibodies (Chemicon International, Inc., Temecula, CA); FITC-conjugated mediated by integrins. We first used FACS, immunoblot, and anti-h1 antibody (clone 4B4-FITC, for FACS analyses, Coulter Corp., Miami, reverse transcription-PCR (RT-PCR) analyses (see Supplementary FL); anti-Ang2 antibody and human recombinant Ang2 protein (R&D materials) to assess the expression of 11 integrin subunits and Systems, Minneapolis, MN); phosphospecific antibodies against activated complexes that have been implicated in cancer cell migration and ERK1/2 (E10 to pT202/pY204) and activated JNK (G9 to pT183/pY185, Cell invasion (19). RT-PCR analyses were done to further confirm the Signaling, Beverly, MA); anti-phosphotyrosine (pY) antibody (4G10, Upstate absence of the particular integrin subunits in U87MG cells that had Biotechnology, Lake Placid, NY); anti-FAK (pY397 and pY861) antibodies h shown negative results by immunoblot analysis. We found that 1, (Biosource, Camarillo, CA); anti-hemagglutinin (HA) antibody (12CA5, h , a , a , a , a , a , a h , and a h are expressed in U87MG cells Covance, Berkeley, CA) and anti–Myc tag antibody (Medical & Biological 3 2 3 5 v 6 v 3 v 5 whereas the expression of h , a , and a in U87MG cells was Laboratory International, Woburn, MA). Fibronectin, RGD, RAD, U0126, 4 1 4 SP600125, and MMPi-III were from Calbiochem (San Diego, CA). Protein G- undetectable (Supplementary Table S1). Then, we purified recom- plus and Protein G-plus/Protein A agarose bead slurry were from Oncogene binant Ang2 from the CM of U87MG/Ang2–expressing (U87MG/ + Research (San Diego, CA). Ni+-NTA beads were from Qiagen (Valencia, CA). Ang2) cells using Ni -NTA affinity chromatography, achieving an Alexa Fluor 594–conjugated goat anti-mouse immunoglobulin G (IgG) and f60-fold increase in Ang2 protein concentration with 90% purity Alexa Fluor 594–conjugated donkey anti-goat IgG antibodies were from (data not shown). The biological activity of the purified Ang2 was Molecular Probes, Inc. (Carlsbad, CA). Other reagents were from Invitrogen verified by competitive inhibition of Ang1-stimulated tyrosine (Carlsbad, CA), Sigma (St. Louis, MO), Fisher Scientific (Hampton, NH), or phosphorylation of Tie2 in endothelial cells (16). BD Biosciences (San Diego, CA). Next, we assessed whether Ang2 stimulates U87MG cell adhesion A Cell adhesion assay. Microtiter plates were coated with 10 g/mL Ang2, and which integrin subunit is involved in Ang2-stimulated cell fibronectin, or bovine serum albumin (BSA). Cells were harvested, adhesion and motility. As shown in Supplementary Fig. S1, U87MG preincubated in serum-free DMEM with or without various treatments for 30 minutes on ice, seeded into the plate, and incubated for another 30 cells tightly adhered to the Ang2-coated surface compared with the minutes at 37jC. Nonattached cells were removed by vigorous agitation and BSA-coated surface. A general integrin blocking reagent, EDTA aspiration. Attached cells were fixed and stained with crystal violet (0.2% in caused complete inhibition of U87MG cell adherence to Ang2,

H2O). Cell-associated crystal violet was eluted by addition of 100 AL of 10% and a RGD peptide showed more potent inhibitory effect when acetic acid. Cell adhesion was quantified by measuring the absorbance compared with a RAD peptide. We also assessed whether the of the eluted crystal violet at a wavelength of 600 nm using a microplate integrin receptors expressed in U87MG glioma cells (Supplemen- reader (22). tary Table S1) are involved in Ang2-stimulated cell adhesion by Pull-down assay and coimmunoprecipitation. Pull-down assay and preincubating the cells with functional blocking integrin anti- coimmunoprecipitation for integrin and ligand interaction were done as bodies. previously described (23). Briefly, cultured U87MG cells were incubated with Among these functional blocking antibodies, only an anti-h1 conditioned medium (CM)/Ang2 or CM/control at 37jC/5% CO2 for 2 hours and washed thrice with TBS containing CaCl and MnCl (1 mmol/L each). antibody caused complete inhibition of U87MG cells adhering to 2 2 h In the pull-down assays, the cells were lysed with a buffer containing Ang2 compared with mouse IgG control. Furthermore, anti- 3, a a a a a 20 mmol/L Tris-HCl (pH 8.0), 300 mmol/L NaCl, 1 mmol/L CaCl2, 1 mmol/L anti- 2, anti- 3, anti- 5, anti- 6, and anti- v antibodies blocked MnCl2, 30 mmol/L imidazole, 1.5% Triton X-100, and 2 mmol/L phenyl- Ang2-stimulated U87MG cell adhesion to a lesser degree compared methylsulfonyl fluoride (PMSF) and centrifuged. The supernatant was with the anti-h1 antibody. These data suggest that interaction of rotated overnight with Ni+-NTA beads at 4jC. The incubated beads were Ang2 with integrins is involved in Ang2-stimulated U87MG cell extensively washed with a wash buffer containing 25 mmol/L NaHPO4, adhesion and h1, h3, a3, a5, and av integrins are likely involved in 150 mmol/L NaCl, 2 mmol/L CaCl, and 45 mmol/L imidazole, eluted with this adhesion process. the wash buffer containing 500 mmol/L imidazole, and precipitated with Ang2 associates with B1, A5, and Av integrins in Tie2- trichloroacetic acid. In coimmunoprecipitation assays, the cells were lysed deficient human glioma cells. To evaluate whether Ang2 is with a lysis buffer containing 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L h h associated with 1, 3, a3, a5, and av integrins in Tie2-deficient NaCl, 1 mmol/L CaCl2, 2 mmol/L MnCl2, 1.5% Triton X-100, 2 mmol/L PMSF, and 10 Ag/mL leupeptin and precleared with various nonspecific IgG glioma cells, we did pull-down and coimmunoprecipitation assays and either Protein G-plus or Protein G-plus/Protein A agarose beads. The that have been used to identify integrin-associated proteins (23). h h precleared cell lysate containing 1 mg of total protein was separately Integrin subunits 1, a5, av, but not 3, a3, and Ang2, were detected j h incubated overnight at 4 C with 5 Ag different antibodies [anti- 1 (JB1A), in the pull-down portion by immunoblot analyses of U87MG cells

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that had been incubated with serum-free CM of U87MG/Ang2– Ang2 activates JNK1 and ERK1/2 through the AvB1 integrin/ expressing cells (CM/Ang2; Fig. 1A). In contrast, these integrins and FAK/p130Cas signaling pathway. We examined the activation of Ang2 were undetectable in the pull-down portion from the cells JNK1 and ERK1/2 in U87MG cells adhering to Ang2. As shown in that were incubated with the serum-free CM of U87MG cells (CM/ Fig. 3A, increased phosphorylation of both JNK1 and ERK1/2 in control) whereas these integrins were found in the total U87MG U87MG cells was observed as early as 20 minutes and peaked cell lysates without exposure to CM. In the immunoprecipitation between 40 and 60 minutes after the cells adhered to Ang2. The assays, equal amounts of h1 integrin proteins in the immunopre- Ang2-stimulated activation of JNK1 and ERK1/2 in U87MG cells h h cipitated complexes from both groups were seen by immunoblot was mediated by av 1 integrin because inhibition of av or 1, but analysis, but Ang2 was only present in the immunoprecipitated not a5 or h3, attenuated the phosphorylation of both JNK1 and complexes from the U87MG cells incubated with CM/Ang2 (Fig. 1B, a). In parallel immunoprecipitation experiments using an anti-Myc antibody or an anti-Ang2 antibody, only the immu- noprecipitates from U87MG cells exposed to CM/Ang2 reacted h strongly with an anti- 1 antibody (Fig. 1B, b and c). AvB1 integrin mediates Ang2-stimulated tyrosine phosphorylation of FAK and p130Cas. We analyzed whole-cell lysates by immunoblot analyses with an anti-phosphotyrosine antibody. As shown in Fig. 2A, increased tyrosine phosphorylation (pTyr) of proteins with molecular weights in the range of 105 to 160 kDa and 70 kDa was observed in the U87MG cells h adherent to Ang2. Anti- 1 and anti-av antibodies attenuated the Ang2-enhanced tyrosine phosphorylation of proteins in U87MG cells whereas the anti-h3, anti-a3, and anti-a5 antibodies had no effect on tyrosine phosphorylation. We also found that tyrosine phosphorylation of proteins in Ang2 stably transfected U87MG cells was constitutively up-regulated, which may explain why these cells adhere to cell culture plates in a more robust fashion than parental U87MG cells. These results suggest that the avh1 integrin may be the principal integrin receptor that is involved in Ang2-stimulated tyrosine phosphorylation of multiple proteins in U87MG cells. We then assessed whether Ang2 stimulates tyrosine phosphor- Cas ylation of FAK and p130 through the association with avh1 integrin in U87MG cells. As shown in Fig. 2B, the extent of phosphorylation of FAK was enhanced in U87MG cells seeded onto Ang2 as compared with BSA, whereas phosphorylation of p130Cas was dramatically induced in the cells plated onto Ang2. Pretreatment of U87MG cells with neutralizing anti-h1 or anti-av antibodies inhibited phosphorylation of both proteins enhanced by Ang2 compared with the control (mouse IgG) whereas inhibition of h3 and a5 had little or no effect. These results indicate that Ang2 stimulates tyrosine phosphorylation of FAK and p130Cas through avh1 integrin in U87MG cells. Next, we established U87MG cell clones that stably expressed FAK-related nonkinase (FRNK, an endogenous FAK inhibitor; ref. 24) or its mutant form, FRNK S-1034, in U87MG cells or U87MG/Ang2 cells (Fig. 2C). Expression of FRNK or FRNK S-1034 in U87MG or U87MG/Ang2 cells did not result in any changes in cell morphology, cell growth rate, or expression levels of Ang2 compared with that of parental U87MG or U87MG/Ang2 cells (data not shown). Compared with Ang2-treated U87MG cells, decreased tyrosine phosphorylation of FAK and p130Cas was seen in FRNK- expressing cell clones (F19, Ang2-F1; Fig. 2D). Interestingly, expression of FRNK in U87MG and U87MG/Ang2 cells suppressed Ang2-stimulated tyrosine phosphorylation of FAK at amino acids Tyr397 and Tyr861. As expected, FRNK S-1034 expression (S11 or Figure 1. Ang2 associates with integrins in Tie2-deficient U87MG glioma cells. U87MG cells were incubated with CM/Ang2, CM/control, or without CM and Ang2-S10) did not affect Ang2-induced tyrosine phosphorylation of lysed. A, pull-down/immunoblotting (IB) assays. The eluted proteins from Cas + Tyr861 of FAK and p130 . These results show that adhesion to Ni -NTA beads were analyzed by immunoblot using anti-h1, anti-h3, anti-a3, Cas Ang2 increases tyrosine phosphorylation of FAK and p130 in anti-a5, and anti-av antibodies (top) or an anti-Ang2 antibody (bottom). B, cell lysates were immunoprecipitated (IP) separately with anti-h1 (a), anti-c-Myc (b), U87MG cells and Ang2-induced tyrosine phosphorylation of or anti-Ang2 (c) antibodies followed by immunoblot separately using the anti-h Cas 1 p130 is mediated through FAK activation. and anti-Ang2 antibodies. Representative of three independent experiments. www.aacrjournals.org 777 Cancer Res 2006; 66: (2). January 15, 2006

Cas Figure 2. avh1 integrin mediates Ang2-stimulated tyrosine phosphorylation of FAK and p130 . A, whole-cell lysates from U87MG cells, U87MG/Ang2–expressing cells, and the U87MG cells seeded onto Ang2-coated plates with preincubation using mouse IgG, anti-h1, anti-h3, anti-a3, anti-a5, and anti-av antibodies were analyzed by immunoblot using anti-pTyr or anti-FAK antibodies. These anti-integrin antibodies were used to block Ang2-induced cell adhesion (Supplementary Fig. S1) and examine the involvement of these integrins in Ang2-stimulated glioma cell invasion. Arrows, top, enhanced tyrosine phosphorylation of cellular proteins. Arrow, bottom, FAK protein as a loading control. B, U87MG cells with or without pretreatment using anti-h1, anti-h3, anti-a5, and anti-av integrin antibodies or mouse IgG were seeded onto BSA- or Ang2-coated plates. FAK or p130Cas was immunoprecipitated with anti-FAK and anti-p130Cas antibodies and analyzed by immunoblot using an anti-pTyr antibody. Equal protein expression was verified by reprobing the blots with anti-FAK or anti-p130Cas antibodies. C, expression of HA-tagged FRNK and FRNK S-1034 in U87MG/FRNK–expressing (F19), U87MG/FRNK S-1034–expressing (S11), U87MG/Ang2 and FRNK–expressing (Ang2-F1), and U87MG/ Ang2 and FRNK S-1034–expressing (Ang2-S10) cell clones were characterized by immunoblot using an anti-HA antibody. D, tyrosine phosphorylation of FAK, pY397-FAK, pY861-FAK, and p130Cas in U87MG, F19, Ang2-F1, S11, and Ang2-S10 cells that were plated onto BSA- or Ang2-coated surfaces were immunoprecipitated with anti-FAK and anti-p130Cas antibodies and analyzed by immunoblot using anti-pTyr, anti–pY397-FAK, and anti–pY861-FAK antibodies. Equal protein expression was verified by reprobing the blots with anti-FAK or anti-p130Cas antibodies. Representative of three independent experiments.

ERK1/2 compared with control mouse IgG (Fig. 3C and D). The cell clones, Ang2-S2 and Ang2-S10 (data not shown). These data Ang2 stimulation of JNK1 and ERK1/2 in U87MG cells was also suggest that Ang2 activates JNK1 and ERK1/2 in U87MG cells and Cas specific because a JNK inhibitor, SP600125 (Fig. 3C; ref. 25), and an the activation is mediated by the Ang2/avh1 integrin/FAK/p130 ERK1/2 inhibitor, U0126 (Fig. 3D; refs. 26, 27), completely blocked signaling pathway. the protein phosphorylation induced by Ang2 compared with the Ang2 stimulates MMP-2 expression and secretion via AvB1 controls. integrin/FAK/p130Cas–mediated activation of ERK1/2 and Next, we determined whether p130Cas is an important mediator JNK1. We examined whether stimulation of MMP-2 by Ang2 is Cas of the Ang2/avh1/FAK signaling pathway by expressing the Src mediated by the avh1 integrin/FAK/p130 signaling pathway homology 3 (SH3) domain of p130Cas (CasSH3) in U87MG cells. through activation of JNK1 and ERK1/2 in glioma cells. As CasSH3 binds to FAK and inhibits endogenous p130Cas binding, showninFig.4A, an increased amount of pro-MMP-2 (72 kDa) thus acting in a dominant-negative manner (28). Expression of was detected in the serum-free CM of U87MG cells plated onto CasSH3 in U87MG cells suppressed Ang2-stimulated tyrosine Ang2-coated plates by gelatin zymography and immunoblot phosphorylation of p130Cas (Fig. 3B) but did not cause any analyses in a concentration-dependent manner compared with morphologic changes or alteration in cell growth rate compared the cells seeded onto BSA-coated plates. The active form of with U87MG parental cells (data not shown). MMP-2 (64 kDa) was almost undetectable in the CM of U87MG Because JNK1 and ERK1/2 are potential downstream effectors of cells plated either on Ang2 or BSA, probably due to insufficient FAK and p130Cas in mediating cell motility and invasion (21), we MMP-2 activation resulting from a short period of cell culture tested whether expression of CasSH3 or FRNK in U87MG cells (18 hours). Inhibition of h1 or av,butnota5, by neutralizing blocks Ang2-stimulated activation of JNK1 and ERK1/2. Expression antibodies significantly suppressed Ang2-stimulated MMP-2 of FRNK (F19) or CasSH3 (C8) in U87MG cells inhibited Ang2- secretion (Fig. 4B). Both SP600125 and U0126 also effectively stimulated activation of both JNK1 (Fig. 3C) and ERK1/2 (Fig. 3D) inhibited Ang2-stimulated MMP-2 secretion in U87MG cells compared with controls (mouse IgG, DMSO, and a FRNK S-1034– (Fig. 4B). expressing cell clone, S11). In parallel, expression of FRNK in We next assessed whether inhibition of FAK and p130Cas U87MG/Ang2 cell clones (Ang2-F1 and Ang2-F7) also attenuated by FRNK and CasSH3 blocks Ang2-stimulated MMP-2 expression Ang2-stimulated activation of JNK1 and ERK1/2 when compared and secretion (Fig. 4C). We found that MMP-2 expression and with parental U87MG/Ang2 cells or U87MG/Ang2/FRNK S-1034 secretion were decreased in U87MG/FRNK–expressing cell clones

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h (F13 and F19) and U87MG/CasSH3–expressing cell clones (C3 MMP-2 activity (Calbiochem; ref. 16), anti- 1 and anti-av anti- and C8) compared with parental U87MG cells or U87MG/FRNK bodies (25 Ag/mL), SP600125 (25 Amol/L), and U0126 (25 Amol/L) S-1034–expressing cell clones (S11 and S21) exposed to Ang2. compared with cells treated with mouse IgG or DMSO. Expressing Lastly, we determined whether Ang2 stimulates MMP-2 secretion FRNK (F13 and F19) and CasSH3 (C3 and C8) in U87MG cells via h1 integrin in other Tie2-deficient human glioma cell lines. As also suppressed Ang2-stimulated cell invasion compared with shown in Fig. 4D, preincubation of T98G, U251MG, and U373MG parental U87MG cells or U87MG/FRNK S-1034–expressing cells glioma cells with an anti-h1 antibody significantly inhibited Ang2- (S11 and S21). h stimulated MMP-2 secretion. Additionally, expression of MMP-2 in Next, we assessed whether inhibition of 1, av integrin, FAK, U87MG cells exposed to various treatments was also assessed JNK1, ERK1/2, and MMP-2 in U87MG/Ang2–expressing cells with immunoblot analyses using an anti-MMP-2 antibody. The (Ang2) suppresses Ang2-induced cell invasion. As shown in detected levels of expression of MMP-2 were correlated to the Fig. 5B, FRNK expression in U87MG/Ang2 expressing cells (Ang2- amounts of secretion of MMP-2 in the CM by the gelatin F1 and Ang2-F7) effectively suppressed Ang2-stimulated cell zymographic analyses (Fig. 4A-D). Taken together, our results invasion compared with U87MG/Ang2–expressing cells (Ang2) show that Ang2 is capable of stimulating the expression and and U87MG/Ang2/FRNK S-1034–expressing clones (Ang2-S2 and secretion of MMP-2 in Tie2-deficient glioma cells through avh1 Ang2-S10). Similar to the results with parental U87MG cells seeded integrin/FAK/p130Cas–mediated activation of ERK1/2 and JNK1. on an Ang2-coated surface described in Fig. 4A, pretreatment Interestingly, we could not detect MMP-9 by gelatin zymography of U87MG/Ang2–expressing cells with MMPi-III (50 Amol/L), anti- from CM of the various cells after an 18-hour cell culture with the h1 and anti-av antibodies (25 Ag/mL), SP600125 (25 Amol/L), or various treatments, suggesting that MMP-9 is not involved in U0126 (25 Amol/L) also inhibited the cell invasion enhanced by Ang2-stimulated human glioma invasion nor regulated by the expressing Ang2 in U87MG cells (Fig. 5B). These data suggest that h Cas h Cas Ang2/av 1 integrin/FAK/p130 /ERK1/2 and JNK1 signaling both the av 1 integrin/FAK/p130 /ERK1/2 and JNK1 signaling pathway (data not shown). pathway and MMP-2 are involved in Ang2-stimulated glioma cell Ang2 stimulates cell invasion via the AvB1 integrin/ invasion. FAK/p130Cas signaling pathway through ERK1/2 and JNK1 Inhibition of FAK suppresses Ang2-stimulated MMP-2 activation. To determine whether Ang2 stimulation of the avh1 expression and glioma invasion in vivo. Lastly, a glioma integrin/FAK/p130Cas signaling pathway leads to increased U87MG xenograft model in the murine brain was employed to determine glioma cell invasion, we did in vitro cell invasion assays. As shown whether inhibition of FAK by FRNK expression could inhibit in Fig. 5A, exposure to exogenous Ang2 promoted cell invasion Ang2-stimulated glioma cell infiltration in vivo. As shown in whereas Ang2-stimulated cell invasion was blocked by a MMP Fig. 6, mice that received U87MG/Ang2-expressing cells devel- inhibitor (MMPi-III, 50 Amol/L) that preferentially attenuates oped invasive gliomas (Fig. 6B). The U87MG/Ang2–expressing

Cas Figure 3. Ang2 activates JNK1 and ERK1/2 via h1 integrin/FAK/p130 signaling pathway. A, time course activation of JNK1 and ERK1/2 by Ang2. U87MG cells were plated onto 10 Ag/mL of Ang2-coated (A) or BSA-coated (B) plates for 20, 40, or 60 minutes. Whole-cell lysates were analyzed with anti–phospho-JNK and anti–phospho-ERK1/2 antibodies. Equal protein expression was verified by reprobing the blots with anti-JNK1 and anti-ERK1/2 antibodies. B, cell lysates from U87MG and U87MG/CasSH3–expressing cell clones (C3 and C8) were immunoprecipitated with an anti-p130Cas antibody and analyzed by immunoblot using an anti-pTyr antibody. Equal protein expression was verified by reprobing the blots with an anti-p130Cas antibody. C and D, whole-cell lysates from parental U87MG cells pretreated with 25 Ag/mL anti-h1, anti-h3, anti-a5, and anti-av antibodies, mouse IgG, 25 Amol/L SP600125, and 25 Amol/L U0126 or DMSO, and U87MG/CasSH3–expressing (C3), U87MG/FRNK–expressing (F19), and U87MG/FRNK S-1034–expressing (S11) cell clones were analyzed with anti–phospho-JNK (recognizes p-JNK1 in double band; refs. 25, 35) and anti–phospho-ERK1/2 antibodies. Equal protein expression was verified by reprobing the blots with anti-JNK1 (recognizes JNK1 as a single band; refs. 25, 35) and anti-ERK1/2 antibodies. Representative of three independent experiments. www.aacrjournals.org 779 Cancer Res 2006; 66: (2). January 15, 2006

Cas Figure 5. Ang2 stimulates glioma cell invasion via avh1 integrin/FAK/p130 signaling. Parental U87MG and U87MG clone cells (C3, C8, F13, F19, S11, and S21; A) or U87MG/Ang2–expressing cells and U87MG/Ang2–expressing clone cells (Ang2-F1, Ang2-F7, Ang2-S2, and Ang2-S10; B) were preincubated in the absence or presence of 50 Amol/L MMPi-III, 25 Ag/mL anti-h1 and anti-av antibodies, mouse IgG, 25 Amol/L SP600125, and 25 Amol/L U0126 or DMSO. The cells were then seeded onto transwell inserts with (A) or without (B) Ang2 coating on the Matrigel membrane. Cell invasion was allowed for 24 hours. Columns, mean fold increase in the number of invading cells; bars, SD. The experiments were done in triplicate at least three independent times with similar results.

cells that additionally express FRNK did not exhibit an invasive phenotype and formed well-circumscribed gliomas (Fig. 6C), which are analogous to U87MG/LacZ–expressing tumors (Fig. 6A). In contrast, the expression of FRNK S-1034 in U87MG/Ang2– expressing cells (Ang2-S2 and Ang2-S10) was unable to inhibit Ang2-stimulated invasion (Fig. 6D). Immunohistochemical and immunoblot analyses using anti-Ang2 and anti-MMP-2 antibodies show that expression of FRNK or FRNK S-1034 had no affect on Ang2 expression (Fig. 6G and H) compared with that of U87MG/ Ang2-expressing tumors (Fig. 6F). However, FRNK expression did noticeably reduce the MMP-2 expression (Fig. 6K) compared h Figure 4. Ang2 stimulates MMP-2 expression and secretion via av 1 integrin/ with that of U87MG/Ang2 expressing tumors (Fig. 6J) whereas FAK/p130Cas signaling in glioma cells. U87MG (A and B), U87MG, C3, C8, F13, F19, S11, S21 (C), T98G, U251MG, and U373MG cells (D) were there was no inhibition of MMP-2 expression by FRNK S-1034 A h preincubated in the absence or presence of 25 g/mL anti- 1, anti-a5, and anti-av (Fig. 6L). Immunoblot analyses confirmed that expression of antibodies, mouse IgG, 25 Amol/L SP600125, and 25 Amol/L U0126 or DMSO and seeded onto plates coated with 10 Ag/mL BSA or Ang2. MMP-2 secretion in FRNK or FRNK S-1034 by these cell clones did not alter the CM of 18-hour cultures was analyzed by zymographic assays. Whole-cell expression levels of Ang2 but FRNK expression did inhibit Ang2- lysates were analyzed by immunoblot assays using an anti-MMP-2 antibody. Immunoblots for h-actin were used as a protein loading control. The experiments stimulated MMP-2 expression compared with that in U87MG/ were done at least three independent times with similar results. Ang2–expressing cells (data not shown). The sensitivity of

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h h detecting MMP-2 expression in established U87MG gliomas by directly bind to integrin av 6 (29) and av 1 (30) in regulating immunohistochemical staining (Fig. 6I to L) is lower than that pulmonary inflammation and fibrosis. VEGF165 and VEGF189 in cultured U87MG cells by zymographic and Western blot promote endothelial cell adhesion, migration, and survival assays (Fig. 4A-C). Expression of HA-tagged FRNK and FRNK through interaction with several integrins independent of VEGF S-1034 by U87MG/Ang2–expressing cells in vivo was detected by receptors (31). Fibrinogen has been shown as a ligand for several immunohistochemistry using an anti-HA tag antibody (Fig. 6M integrins including the a5h1 integrin receptor (32). Members of and N). Furthermore, although expression of FRNK, but not the angiopoietin family contain a fibrinogen-like domain at the FRNK S-1034, in U87MG/Ang2–expressing cells inhibited Ang2- COOH terminus of their proteins (8, 33), suggesting integrins as stimulated glioma cell invasion in vitro and in vivo, no alteration potential candidate receptors (7). Adhesion of Tie2-deficient cells, of tumor formation of intracranial gliomas formed by either such as fibroblasts and skeletal myocytes, to Ang2-coated as well U87MG/Ang2/FRNK–expressing or U87MG/Ang2/FRNK S-1034– Ang1-coated surfaces was recently shown (11, 12). Lacking Tie2 expressing cells was observed. Together, these results show that receptors, fibroblasts and myocytes adhere to Ang2- or Ang1- inhibition of FAK by FRNK in U87MG/Ang2–expressing cells coated surfaces and the adhesion is sensitive to the inhibition of h h suppressed Ang2-stimulated MMP-2 expression and glioma cell 1, 3, a6, and av integrins. Interactions of Ang1 and/or Ang2 infiltration in vivo. with these integrins activate FAK and ERK1/2, increasing cell adhesion (11), and Akt, FAK, and ERK1/2, promoting cell survival (12). Even in endothelial cells, Ang1 is able to mediate selectively Discussion a5h1 integrin outside-in FAK activation, leading to a cross-talk Growth factors and angiopoietin family members recently have of Tie2 and a5h1, and fine-tuning modulation of the vascular emerged as novel ligands for integrins eliciting specific biological effect (13). In this study, we report that Ang2 associates with h h effects. For example, latent forms of transforming growth factor av 1 integrin in Tie2-deficient glioma cells. We observed that

Figure 6. FRNK expression blocks Ang2-induced glioma invasion in the murine brain. Gliomas established by control U87MG LacZ (A, E, and I), Ang2 (B, F, and J), Ang2/FRNK (C, G, K, and M), and Ang2/FRNK S-1034 (D, H, L, and N) cells in the murine brain were analyzed by H&E staining (A-D) and immunohistochemistry of serial sections of the brains using anti-Ang2 (E-H), anti-MMP-2 (I-L), and anti-HA (M and N) antibodies. Insets, isotype-matched IgG (negative) controls. Arrowheads, clean edge of the tumor. Arrows, invasive extensions as well as disseminated tumor clusters. The animal experiments were done two independent times with six mice per group using separate U87MG cell clones: LacZ, Ang2-20, Ang2-35 (16), Ang2-F1, Ang2-F7, Ang2-S1, and Ang2-S10 with similar results. Bar, 50 Am. www.aacrjournals.org 781 Cancer Res 2006; 66: (2). January 15, 2006

Ang2 induces extracellular adhesion and activates intracellular and JNK. FRNK and CasSH3 or inhibitors for ERK1/2 (U0126) and signal transduction pathways in glioma cells. Pull-down and JNK1 (SP600125) suppressed Ang2 activation of ERK1/2 and JNK1. immunoprecipitation assays showed that avh1 integrin could be Furthermore, Ang2-stimulated MMP-2 expression and cell invasion h h copu-rified with Ang2. The association of Ang2 with av 1 integrin were attenuated by av and 1 inhibition, FRNK, CasSH3, U0126, was highly calcium and manganese dependent in both pull-down and SP600125. These results are consistent with previous findings and coimmunoprecipitation assays, suggesting the association of that both ERK1/2 and JNK mediate Src-stimulated v-Src3T3 cell Ang2 with avh1 integrin is similar to that of Ang1, Ang2 to invasion and FRNK inhibits v-Src-stimulated phosphorylation of h various integrins (12), and PG-MV/vesican with 1 integrin (23). ERK1/2 and JNK (25). Moreover, our results differ from a study Our data further show that Ang2 stimulates avh1 integrin/FAK– showing that only JNK1 activation, and not ERK1/2 phosphoryla- mediated signaling, leading to an increase in MMP-2 expression tion, is required in v-Src-stimulated FAK-Src-p130Cas signaling in and glioma cell invasion. FAK-null fibroblasts (35). Cas FAK and p130 are involved in early integrin receptor signaling. h1-Integrin has been shown to activate FAK-mediated signaling Up-regulated FAK expression and activation have been found in pathways leading to promotion of invasion of various types of h various types of human cancers. An increase of FAK phosphory- human carcinoma cells (10, 42, 43). Suppression of 1 expression in lation on Tyr397 and Tyr861 strongly correlates with the rat C6 glioma cells prevented diffuse glioma cell invasion in the brain acquisition of an invasive phenotype of tumor cells (21, 34). We (44). On the other hand, several a integrin subunits that associate h found that Ang2 stimulates FAK tyrosine phosphorylation on these with the 1 integrin subunit, such as av, a5, and a6 integrins, have two tyrosine residues in U87MG cells. Expression of FRNK, but not been implicated in mediating tumor cell invasion (19, 42, 43). In its mutant form FRNK S-1034, suppressed Ang2-induced activation addition, avh3 integrin is also associated with invasion of human Cas of FAK/p130 /ERK1/2 and JNK1, MMP-2 expression and gliomas (17, 45). In our study, we show that avh1, but not other secretion, and cell invasion in vitro and reversed the phenotype integrin receptors, is associated with Ang2-stimulated glioma cell of Ang2-stimulated glioma cell infiltration and MMP-2 expression invasion. Moreover, we cannot rule out other molecules involved in in vivo. These results are consistent with previous reports that FAK Ang2-stimulated glioma cell invasion. For example, Ang2 may h Tyr397 phosphorylation is required to promote JNK activation and associate with av 5 integrin because functional blocking of av, a5, cell invasion by v-Src (35) and that FRNK expression suppressed h1, h3, and avh5 inhibited fibroblast or myocyte adhesion to Ang2 Src-stimulated Try861 phosphorylation, inhibiting Src activation of (11, 12). ANGPTL3 and tenascin C interact with avh3 integrin FAK downstream signaling, MMP-2 expression, and tumor through the fibrinogen-like domain that is also present in Ang2 (7, 8). metastasis (25). Moreover, FRNK overexpression disrupts focal We found that a3 and h3 are not associated with Ang2 whereas a5 adhesion and induces anoikis in ventricular myocytes by inhibiting is weakly associated with Ang2. However, inhibition of a3, a5, and h endothelin-1-induced phosphorylation of proline-rich tyrosine 3 only moderately suppressed Ang2-mediated cell adhesion kinase 2 (PyK2; ref. 36). In glioma cells, PyK2 plays critical roles (Supplementary Table S1 and Fig. S1), but not phosphorylation of Cas in cell migration. Overexpression of PyK2 stimulates glioma cell FAK and p130 (Fig. 2). Thus, avh3 and a5h1 integrins are probably migration whereas inhibition of PyK2 by FRNK expression or siRNA not involved in Ang2-stimulated glioma cell invasion in our model. for PyK2 significantly inhibits glioma cell migration (37, 38). Additionally, we have recently reported that MT1-MMP, laminin-5 Therefore, it is plausible that in addition to suppression of FAK, g2, and Ang2 are coexpressed in the invasive regions, but not in the FRNK expression in U87MG/Ang2–expressing cells also inhibits central areas, of primary glioma specimens and exogenous Ang2 PyK2 activation, leading to decreased Ang2-induced glioma cell stimulates expression and activation of both MT1-MMP and invasion in vitro and in vivo. laminin-5 g2 in U87MG/Ang2–expressing glioma xenografts in the p130Cas is an adaptor protein that associates with FAK directly murine brain and in vitro (18). Taken together, these results warrant through its SH3 domain to a proline-rich region of FAK (amino further investigation to determine other signaling pathways acids 712-718; ref. 21). p130Cas also interacts with FAK indirectly involved in Ang2-induced glioma cell invasion. through binding to Src family kinase via its SH3 domain. Src In summary, this study identifies a novel molecular mecha- stimulates FAK Tyr397 phosphorylation, resulting in binding of nism by which Ang2 stimulates human glioma cell invasion via h Cas FAK to an SH2 domain of the Src protein (39). Our data show the av 1 integrin/FAK/p130 /ERK1/2 and JNK1 signaling that Ang2-induced p130Cas tyrosine phosphorylation was com- pathway and MMP-2 expression (Supplementary Fig. S2). pletely abolished by neutralizing anti-h1 and anti-av antibodies or Considering the fact that Ang2 expression has significant links FRNK, but not FRNK S-1034, suggesting that the interaction of to tumor invasion and metastases in various types of human phosphorylated FAK at Tyr397 with Src family kinase is also cancers (2–6), our studies provide a basis for further character- Cas involved in Ang2/avh1/FAK/p130 signaling (21). Additionally, ization of the molecular cascades involved in Ang2-stimulated expression of a dominant-negative regulator for p130Cas,CasSH3 glioma invasion, which may yield promising insight for the (28), significantly attenuated Ang2-induced phosphorylation of development of new diagnostic and therapeutic applications for p130Cas and downstream signaling activities in U87MG cells, invasive tumors. showing that the interaction between FAK and p130Cas mediates h Ang2/av 1 integrin stimulation of glioma cell invasion. Previous studies have shown that integrin-mediated stimulation Acknowledgments of FAK/p130Cas signaling pathways activates ERK1/2 and JNK1 (19), Received 4/6/2005; revised 10/11/2005; accepted 10/28/2005. Grant support: James S. McDonnell Foundation grant and NIH grants CA102011 MMP-2 secretion, and cell invasion (25, 35, 40, 41). Our results and CA102310 (D.D. Schlaepfer) and an ACS grant RSG CSM107144 (S-Y. Cheng) . Cas identify an avh1 integrin/FAK/p130 /ERK1/2 and JNK1 signaling The costs of publication of this article were defrayed in part by the payment of page pathway that mediates Ang2-stimulated MMP-2 expression, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. secretion, and cell invasion. Functional blocking antibodies for We thank Dr. Kari Alitalo for an expression vector containing human Ang2 cDNA Ang2 and av and h1 integrins inhibited Ang2 stimulation of ERK1/2 and Dr. Jun-Lin Gun for an expression vector containing human CasSH3 cDNA.

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