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[Cancer Research 66, 775-783, January 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Angiopoietin 2 Induces Glioma Cell Invasion by Stimulating Matrix Metalloprotease 2 Expression through the {alpha}vß1 Integrin and Focal Adhesion Kinase Signaling Pathway

Bo Hu1,2, Michael J. Jarzynka1,3, Ping Guo1,3,4, Yorihisha Imanishi1,3, David D. Schlaepfer5 and Shi-Yuan Cheng1,3

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

Requests for reprints: Shi-Yuan Cheng, University of Pittsburgh Cancer Institute and Department of Pathology, Research Pavilion at Hillman Cancer Center, Suite 2.26, 5117 Centre Avenue, Pittsburgh, PA 15213-1863. Phone: 412-623-3261; Fax: 412-623-4840; E-mail: chengs{at}upmc.edu or Bo Hu, Department of Medicine, Research Pavilion at Hillman Cancer Center, Suite 2.26, 5117 Centre Avenue, Pittsburgh, PA 15213-1863. Phone: 412-623-3261; Fax: 412-623-4840; E-mail: hub{at}upmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Accumulating evidence reveals a significant correlation between angiopoietin 2 (Ang2) expression and tumor invasion and metastasis in various human cancers, but the major focus of recent studies has been on the angiogenic effects of Ang2. We recently reported that Ang2-stimulated glioma cell invasion results from the up-regulation and activation of matrix metalloprotease 2 (MMP-2) in tumor cells. In this study, we identify a novel mechanism by which Ang2 stimulates MMP-2 expression leading to glioma cell invasion. We show that Ang2 interacts with {alpha}vß1 integrin in Tie2-deficient human glioma cells, activating focal adhesion kinase (FAK), p130Cas, extracellular signal–regulated protein kinase (ERK) 1/2, and c-jun NH2-terminal kinase (JNK) and substantially enhancing MMP-2 expression and secretion. The Ang2/{alpha}vß1 integrin signaling pathway was attenuated by functional inhibition of ß1 and {alpha}v integrins, FAK, p130Cas, ERK1/2, and JNK. Furthermore, expression of a negative regulator of FAK, FAK-related nonkinase, by U87MG/Ang2–expressing glioma xenografts suppressed Ang2-induced MMP-2 expression and glioma cell infiltration in the murine brain. These data establish a functional link between Ang2 interaction with {alpha}vß1 integrin and glioma cell invasion through the FAK/p130Cas/ERK1/2 and JNK-mediated signaling pathway. (Cancer Res 2006; 66(2): 775-83)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Angiopoietin 2 (Ang2), a known angiogenic regulator, plays important roles in angiogenesis and tumor progression (1). Ang2, expressed in both endothelial cells and tumor cells, has been significantly correlated with tumor metastasis and invasion (2–6), but the mechanism by which Ang2 acts on tumor cells remains obscure. The highly conserved COOH-terminal fibrinogen-like domain of the angiopoietins implies a functional association with the integrin receptor family (7, 8). Integrins are crucial for migration and invasion of tumor cells, not only for mediating adhesion of tumor cells to the extracellular matrix but also for regulating the processes of cell migration and invasion (9). Integrins are heterodimeric glycoproteins composed of one {alpha} chain and one ß chain. The ß1 family of integrins associates with at least one of the 12 {alpha}-chains ({alpha}1-{alpha}11 and {alpha}v) representing the major group of cellular extracellular matrix receptors. ß1-Integrin promotes metastasis of ß1-null tumor cells (9). Mutation of the intracellular domain of ß1 integrin differentially affects cell adhesion, invasion, and metastasis (10). Ang2, as well as other members of the angiopoietin family, is a potential substrate for the integrin receptor family in endothelial cells, fibroblasts, and myocytes (11–13). Ang2 not only enhances cell adhesion in both endothelial cells and Tie2-deficient fibroblasts but also triggers integrin-mediated intracellular signal transduction pathways in these cells.

Malignant human gliomas are pathophysiologically characterized by their insidious infiltration of the brain (14). Invasion of glioma cells into adjacent brain structures occurs through the activation of multigenic programs, including matrix metalloproteases (MMP) such as MMP-2, which degrade extracellular matrix to overcome the extracellular matrix barrier at the invasive fronts of tumors (15). Several reports have recently shown that up-regulation of MMP-2 correlates with the invasiveness of human gliomas (16–18). Interestingly, integrin-mediated signaling pathways are involved in regulation of MMP-2 expression and cell invasion in tumor cells (19). Expression patterns of several integrins are correlated with invasive and migratory behaviors in glioma cells in vitro and in vivo (17). MMP-2 expression and tumor cell invasion have also been associated with the activation of downstream regulators of integrins such as focal adhesion kinase (FAK), p130Cas, extracellular signal–regulated protein kinase (ERK)/mitogen-activated protein kinase, and c-jun NH2-terminal kinase (JNK)/stress-activated protein kinase (20, 21). We recently reported that coexpression of Ang2 with MMP-2 was found in the invasive areas, but not in the central regions, of primary human glioma specimens (16, 18). Stable expression of Ang2 by glioma cells leads to an increase in glioma cell invasiveness in vitro as well as in murine intracranial xenografts accomplished through up-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 secretion in glioma cells. We show that Ang2 interacts with {alpha}vß1 integrin in U87MG glioma cells, resulting in the activation of FAK, p130Cas, ERK1/2, and JNK, thereby enhancing MMP-2 expression and secretion. As a functional consequence of increased MMP-2 secretion, glioma cell invasion is promoted. Furthermore, stable expression of dominant-negative forms of FAK or p130Cas in glioma cells inhibits Ang2-stimulated MMP-2 expression and secretion by blocking activation of JNK and ERK1/2. Our data suggest that elevation of Ang2 expression in the gliomas leads to increased MMP-2 secretion along with increased cell invasion via {alpha}vß1 integrin/FAK–mediated activation of ERK1/2 and JNK.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and reagents. Human U87MG and T98G glioma cells were obtained from American Type Culture Collection (Manassas, VA), U251MG and U373MG glioma cells were from our collection, and their culture was previously described (16). The following reagents were used for this study: anti-Ang2 (C-19), anti-p130Cas (C-20), anti-JNK1 (C-17), anti-FAK (C-20), and anti-ß3 integrin (H-96) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); neutralizing anti-ß3 integrin (B3A), anti-ß1 integrin (JB1A), and anti-{alpha}3 integrin antibodies (P1B5); a ß1 integrin polyclonal investigator kit; an {alpha} integrin blocking investigator kit that includes anti-ß1, anti-{alpha}1, anti-{alpha}2, anti-{alpha}3, anti-{alpha}4, anti-{alpha}5, anti-{alpha}6, anti-{alpha}v, anti-{alpha}vß3, and anti-{alpha}vß5 antibodies; FITC-conjugated anti-{alpha}vß3 (clone LM609) and anti-{alpha}vß5 antibodies (clone P1F6; both for fluorescence-activated cell sorting analyses) and anti-MMP-2 antibodies (Chemicon International, Inc., Temecula, CA); FITC-conjugated anti-ß1 antibody (clone 4B4-FITC, for FACS analyses, Coulter Corp., Miami, FL); anti-Ang2 antibody and human recombinant Ang2 protein (R&D Systems, Minneapolis, MN); phosphospecific antibodies against activated ERK1/2 (E10 to pT202/pY204) and activated JNK (G9 to pT183/pY185, Cell Signaling, Beverly, MA); anti-phosphotyrosine (pY) antibody (4G10, Upstate Biotechnology, Lake Placid, NY); anti-FAK (pY397 and pY861) antibodies (Biosource, Camarillo, CA); anti-hemagglutinin (HA) antibody (12CA5, Covance, Berkeley, CA) and anti–Myc tag antibody (Medical & Biological Laboratory International, Woburn, MA). Fibronectin, RGD, RAD, U0126, SP600125, and MMPi-III were from Calbiochem (San Diego, CA). Protein G-plus and Protein G-plus/Protein A agarose bead slurry were from Oncogene Research (San Diego, CA). Ni+-NTA beads were from Qiagen (Valencia, CA). Alexa Fluor 594–conjugated goat anti-mouse immunoglobulin G (IgG) and Alexa Fluor 594–conjugated donkey anti-goat IgG antibodies were from Molecular Probes, Inc. (Carlsbad, CA). Other reagents were from Invitrogen (Carlsbad, CA), Sigma (St. Louis, MO), Fisher Scientific (Hampton, NH), or BD Biosciences (San Diego, CA).

Cell adhesion assay. Microtiter plates were coated with 10 µg/mL Ang2, fibronectin, or bovine serum albumin (BSA). Cells were harvested, preincubated in serum-free DMEM with or without various treatments for 30 minutes on ice, seeded into the plate, and incubated for another 30 minutes at 37°C. Nonattached cells were removed by vigorous agitation and aspiration. Attached cells were fixed and stained with crystal violet (0.2% in H2O). Cell-associated crystal violet was eluted by addition of 100 µL of 10% acetic acid. Cell adhesion was quantified by measuring the absorbance of the eluted crystal violet at a wavelength of 600 nm using a microplate reader (22).

Pull-down assay and coimmunoprecipitation. Pull-down assay and coimmunoprecipitation for integrin and ligand interaction were done as previously described (23). Briefly, cultured U87MG cells were incubated with conditioned medium (CM)/Ang2 or CM/control at 37°C/5% CO2 for 2 hours and washed thrice with TBS containing CaCl2 and MnCl2 (1 mmol/L each). In the pull-down assays, the cells were lysed with a buffer containing 20 mmol/L Tris-HCl (pH 8.0), 300 mmol/L NaCl, 1 mmol/L CaCl2, 1 mmol/L MnCl2, 30 mmol/L imidazole, 1.5% Triton X-100, and 2 mmol/L phenylmethylsulfonyl fluoride (PMSF) and centrifuged. The supernatant was rotated overnight with Ni+-NTA beads at 4°C. The incubated beads were extensively washed with a wash buffer containing 25 mmol/L NaHPO4, 150 mmol/L NaCl, 2 mmol/L CaCl, and 45 mmol/L imidazole, eluted with the wash buffer containing 500 mmol/L imidazole, and precipitated with trichloroacetic acid. In coimmunoprecipitation assays, the cells were lysed with a lysis buffer containing 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L CaCl2, 2 mmol/L MnCl2, 1.5% Triton X-100, 2 mmol/L PMSF, and 10 µg/mL leupeptin and precleared with various nonspecific IgG and either Protein G-plus or Protein G-plus/Protein A agarose beads. The precleared cell lysate containing 1 mg of total protein was separately incubated overnight at 4°C with 5 µg different antibodies [anti-ß1 (JB1A), anti-ß3 (B3A), anti-{alpha}3, anti-{alpha}5, and anti-{alpha}v integrin antibodies that were included in the {alpha} integrin blocking investigator kit, anti-Myc (9B11), and anti-Ang2 antibodies], and then collected with 50 µL of either a Protein 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 conditions and detected with an anti-ß1 integrin (JB1A) or an anti-Ang2 antibody.

Gelatin zymography and in vitro invasion assays. Zymographic analyses for MMP-2 and in vitro invasion assays were done as previously described (16).

Intracranial brain tumor xenografts and immunohistochemistry. Stereotactic implantation of various types of U87MG cells, sacrifice of glioma-bearing mice, tumor processing, and analyses of intracranial U87MG glioma invasiveness were done as previously described (16).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ang2-enhanced adhesion of Tie2-deficient glioma cells is mediated by integrins. We first used FACS, immunoblot, and reverse transcription-PCR (RT-PCR) analyses (see Supplementary materials) to assess the expression of 11 integrin subunits and complexes that have been implicated in cancer cell migration and invasion (19). RT-PCR analyses were done to further confirm the absence of the particular integrin subunits in U87MG cells that had shown negative results by immunoblot analysis. We found that ß1, ß3, {alpha}2, {alpha}3, {alpha}5, {alpha}v, {alpha}6, {alpha}vß3, and {alpha}vß5 are expressed in U87MG cells whereas the expression of ß4, {alpha}1, and {alpha}4 in U87MG cells was undetectable (Supplementary Table S1). Then, we purified recombinant Ang2 from the CM of U87MG/Ang2–expressing (U87MG/Ang2) cells using Ni+-NTA affinity chromatography, achieving an ~60-fold increase in Ang2 protein concentration with 90% purity (data not shown). The biological activity of the purified Ang2 was verified by competitive inhibition of Ang1-stimulated tyrosine phosphorylation of Tie2 in endothelial cells (16).

Next, we assessed whether Ang2 stimulates U87MG cell adhesion and which integrin subunit is involved in Ang2-stimulated cell adhesion and motility. As shown in Supplementary Fig. S1, U87MG cells tightly adhered to the Ang2-coated surface compared with the BSA-coated surface. A general integrin blocking reagent, EDTA caused complete inhibition of U87MG cell adherence to Ang2, and a RGD peptide showed more potent inhibitory effect when compared with a RAD peptide. We also assessed whether the integrin receptors expressed in U87MG glioma cells (Supplementary Table S1) are involved in Ang2-stimulated cell adhesion by preincubating the cells with functional blocking integrin antibodies.

Among these functional blocking antibodies, only an anti-ß1 antibody caused complete inhibition of U87MG cells adhering to Ang2 compared with mouse IgG control. Furthermore, anti-ß3, anti-{alpha}2, anti-{alpha}3, anti-{alpha}5, anti-{alpha}6, and anti-{alpha}v antibodies blocked Ang2-stimulated U87MG cell adhesion to a lesser degree compared with the anti-ß1 antibody. These data suggest that interaction of Ang2 with integrins is involved in Ang2-stimulated U87MG cell adhesion and ß1, ß3, {alpha}3, {alpha}5, and {alpha}v integrins are likely involved in this adhesion process.

Ang2 associates with ß1, {alpha}5, and {alpha}v integrins in Tie2-deficient human glioma cells. To evaluate whether Ang2 is associated with ß1, ß3, {alpha}3, {alpha}5, and {alpha}v integrins in Tie2-deficient glioma cells, we did pull-down and coimmunoprecipitation assays that have been used to identify integrin-associated proteins (23). Integrin subunits ß1, {alpha}5, {alpha}v, but not ß3, {alpha}3, and Ang2, were detected in the pull-down portion by immunoblot analyses of U87MG cells that had been incubated with serum-free CM of U87MG/Ang2–expressing cells (CM/Ang2; Fig. 1A). In contrast, these integrins and Ang2 were undetectable in the pull-down portion from the cells that were incubated with the serum-free CM of U87MG cells (CM/control) whereas these integrins were found in the total U87MG cell lysates without exposure to CM. In the immunoprecipitation assays, equal amounts of ß1 integrin proteins in the immunoprecipitated complexes from both groups were seen by immunoblot analysis, but Ang2 was only present in the immunoprecipitated 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 immunoprecipitates from U87MG cells exposed to CM/Ang2 reacted strongly with an anti-ß1 antibody (Fig. 1B, b and c).


Figure 1
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  		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 lysed. A, pull-down/immunoblotting (IB) assays. The eluted proteins from Ni+-NTA beads were analyzed by immunoblot using anti-ß1, anti-ß3, anti-{alpha}3, anti-{alpha}5, and anti-{alpha}v antibodies (top) or an anti-Ang2 antibody (bottom). B, cell lysates were immunoprecipitated (IP) separately with anti-ß1 (a), anti-c-Myc (b), or anti-Ang2 (c) antibodies followed by immunoblot separately using the anti-ß1 and anti-Ang2 antibodies. Representative of three independent experiments.

 
{alpha}vß1 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 adherent to Ang2. Anti-ß1 and anti-{alpha}v antibodies attenuated the Ang2-enhanced tyrosine phosphorylation of proteins in U87MG cells whereas the anti-ß3, anti-{alpha}3, and anti-{alpha}5 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 {alpha}vß1 integrin may be the principal integrin receptor that is involved in Ang2-stimulated tyrosine phosphorylation of multiple proteins in U87MG cells.


Figure 2
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  		Figure 2. {alpha}vß1 integrin mediates Ang2-stimulated tyrosine phosphorylation of FAK and p130Cas. 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-ß1, anti-ß3, anti-{alpha}3, anti-{alpha}5, and anti-{alpha}v 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-ß1, anti-ß3, anti-{alpha}5, and anti-{alpha}v 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.

 
We then assessed whether Ang2 stimulates tyrosine phosphorylation of FAK and p130Cas through the association with {alpha}vß1 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-ß1 or anti-{alpha}v antibodies inhibited phosphorylation of both proteins enhanced by Ang2 compared with the control (mouse IgG) whereas inhibition of ß3 and {alpha}5 had little or no effect. These results indicate that Ang2 stimulates tyrosine phosphorylation of FAK and p130Cas through {alpha}vß1 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 Ang2-S10) did not affect Ang2-induced tyrosine phosphorylation of Tyr861 of FAK and p130Cas. These results show that adhesion to Ang2 increases tyrosine phosphorylation of FAK and p130Cas in U87MG cells and Ang2-induced tyrosine phosphorylation of p130Cas is mediated through FAK activation.

Ang2 activates JNK1 and ERK1/2 through the {alpha}vß1 integrin/FAK/p130Cas signaling pathway. We examined the activation of JNK1 and ERK1/2 in U87MG cells adhering to Ang2. As shown in Fig. 3A, increased phosphorylation of both JNK1 and ERK1/2 in U87MG cells was observed as early as 20 minutes and peaked between 40 and 60 minutes after the cells adhered to Ang2. The Ang2-stimulated activation of JNK1 and ERK1/2 in U87MG cells was mediated by {alpha}vß1 integrin because inhibition of {alpha}v or ß1, but not {alpha}5 or ß3, attenuated the phosphorylation of both JNK1 and ERK1/2 compared with control mouse IgG (Fig. 3C and D). The Ang2 stimulation of JNK1 and ERK1/2 in U87MG cells was also specific because a JNK inhibitor, SP600125 (Fig. 3C; ref. 25), and an ERK1/2 inhibitor, U0126 (Fig. 3D; refs. 26, 27), completely blocked the protein phosphorylation induced by Ang2 compared with the controls.


Figure 3
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  		Figure 3. Ang2 activates JNK1 and ERK1/2 via ß1 integrin/FAK/p130Cas signaling pathway. A, time course activation of JNK1 and ERK1/2 by Ang2. U87MG cells were plated onto 10 µg/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 µg/mL anti-ß1, anti-ß3, anti-{alpha}5, and anti-{alpha}v antibodies, mouse IgG, 25 µmol/L SP600125, and 25 µmol/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.

 
Next, we determined whether p130Cas is an important mediator of the Ang2/{alpha}vß1/FAK signaling pathway by expressing the Src homology 3 (SH3) domain of p130Cas (CasSH3) in U87MG cells. CasSH3 binds to FAK and inhibits endogenous p130Cas binding, thus acting in a dominant-negative manner (28). Expression of CasSH3 in U87MG cells suppressed Ang2-stimulated tyrosine phosphorylation of p130Cas (Fig. 3B) but did not cause any morphologic changes or alteration in cell growth rate compared with U87MG parental cells (data not shown).

Because JNK1 and ERK1/2 are potential downstream effectors of FAK and p130Cas in mediating cell motility and invasion (21), we tested whether expression of CasSH3 or FRNK in U87MG cells blocks Ang2-stimulated activation of JNK1 and ERK1/2. Expression of FRNK (F19) or CasSH3 (C8) in U87MG cells inhibited Ang2-stimulated activation of both JNK1 (Fig. 3C) and ERK1/2 (Fig. 3D) compared with controls (mouse IgG, DMSO, and a FRNK S-1034–expressing cell clone, S11). In parallel, expression of FRNK in U87MG/Ang2 cell clones (Ang2-F1 and Ang2-F7) also attenuated Ang2-stimulated activation of JNK1 and ERK1/2 when compared with parental U87MG/Ang2 cells or U87MG/Ang2/FRNK S-1034 cell clones, Ang2-S2 and Ang2-S10 (data not shown). These data suggest that Ang2 activates JNK1 and ERK1/2 in U87MG cells and the activation is mediated by the Ang2/{alpha}vß1 integrin/FAK/p130Cas signaling pathway.

Ang2 stimulates MMP-2 expression and secretion via {alpha}vß1 integrin/FAK/p130Cas–mediated activation of ERK1/2 and JNK1. We examined whether stimulation of MMP-2 by Ang2 is mediated by the {alpha}vß1 integrin/FAK/p130Cas signaling pathway through activation of JNK1 and ERK1/2 in glioma cells. As shown in Fig. 4A, an increased amount of pro-MMP-2 (72 kDa) was detected in the serum-free CM of U87MG cells plated onto Ang2-coated plates by gelatin zymography and immunoblot analyses in a concentration-dependent manner compared with the cells seeded onto BSA-coated plates. The active form of MMP-2 (64 kDa) was almost undetectable in the CM of U87MG cells plated either on Ang2 or BSA, probably due to insufficient MMP-2 activation resulting from a short period of cell culture (18 hours). Inhibition of ß1 or {alpha}v, but not {alpha}5, by neutralizing antibodies significantly suppressed Ang2-stimulated MMP-2 secretion (Fig. 4B). Both SP600125 and U0126 also effectively inhibited Ang2-stimulated MMP-2 secretion in U87MG cells (Fig. 4B).


Figure 4
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  		Figure 4. Ang2 stimulates MMP-2 expression and secretion via {alpha}vß1 integrin/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 preincubated in the absence or presence of 25 µg/mL anti-ß1, anti-{alpha}5, and anti-{alpha}v antibodies, mouse IgG, 25 µmol/L SP600125, and 25 µmol/L U0126 or DMSO and seeded onto plates coated with 10 µg/mL BSA or Ang2. MMP-2 secretion in the CM of 18-hour cultures was analyzed by zymographic assays. Whole-cell lysates were analyzed by immunoblot assays using an anti-MMP-2 antibody. Immunoblots for ß-actin were used as a protein loading control. The experiments were done at least three independent times with similar results.

 
We next assessed whether inhibition of FAK and p130Cas by FRNK and CasSH3 blocks Ang2-stimulated MMP-2 expression and secretion (Fig. 4C). We found that MMP-2 expression and secretion were decreased in U87MG/FRNK–expressing cell clones (F13 and F19) and U87MG/CasSH3–expressing cell clones (C3 and C8) compared with parental U87MG cells or U87MG/FRNK S-1034–expressing cell clones (S11 and S21) exposed to Ang2. Lastly, we determined whether Ang2 stimulates MMP-2 secretion via ß1 integrin in other Tie2-deficient human glioma cell lines. As shown in Fig. 4D, preincubation of T98G, U251MG, and U373MG glioma cells with an anti-ß1 antibody significantly inhibited Ang2-stimulated MMP-2 secretion. Additionally, expression of MMP-2 in U87MG cells exposed to various treatments was also assessed with immunoblot analyses using an anti-MMP-2 antibody. The detected levels of expression of MMP-2 were correlated to the amounts of secretion of MMP-2 in the CM by the gelatin zymographic analyses (Fig. 4A-D). Taken together, our results show that Ang2 is capable of stimulating the expression and secretion of MMP-2 in Tie2-deficient glioma cells through {alpha}vß1 integrin/FAK/p130Cas–mediated activation of ERK1/2 and JNK1. Interestingly, we could not detect MMP-9 by gelatin zymography from CM of the various cells after an 18-hour cell culture with the various treatments, suggesting that MMP-9 is not involved in Ang2-stimulated human glioma invasion nor regulated by the Ang2/{alpha}vß1 integrin/FAK/p130Cas/ERK1/2 and JNK1 signaling pathway (data not shown).

Ang2 stimulates cell invasion via the {alpha}vß1 integrin/FAK/p130Cas signaling pathway through ERK1/2 and JNK1 activation. To determine whether Ang2 stimulation of the {alpha}vß1 integrin/FAK/p130Cas signaling pathway leads to increased U87MG glioma cell invasion, we did in vitro cell invasion assays. As shown in Fig. 5A, exposure to exogenous Ang2 promoted cell invasion whereas Ang2-stimulated cell invasion was blocked by a MMP inhibitor (MMPi-III, 50 µmol/L) that preferentially attenuates MMP-2 activity (Calbiochem; ref. 16), anti-ß1 and anti-{alpha}v antibodies (25 µg/mL), SP600125 (25 µmol/L), and U0126 (25 µmol/L) compared with cells treated with mouse IgG or DMSO. Expressing FRNK (F13 and F19) and CasSH3 (C3 and C8) in U87MG cells also suppressed Ang2-stimulated cell invasion compared with parental U87MG cells or U87MG/FRNK S-1034–expressing cells (S11 and S21).


Figure 5
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  		Figure 5. Ang2 stimulates glioma cell invasion via {alpha}vß1 integrin/FAK/p130Cas 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 µmol/L MMPi-III, 25 µg/mL anti-ß1 and anti-{alpha}v antibodies, mouse IgG, 25 µmol/L SP600125, and 25 µmol/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.

 
Next, we assessed whether inhibition of ß1, {alpha}v integrin, FAK, JNK1, ERK1/2, and MMP-2 in U87MG/Ang2–expressing cells (Ang2) suppresses Ang2-induced cell invasion. As shown in Fig. 5B, FRNK expression in U87MG/Ang2 expressing cells (Ang2-F1 and Ang2-F7) effectively suppressed Ang2-stimulated cell invasion compared with U87MG/Ang2–expressing cells (Ang2) and U87MG/Ang2/FRNK S-1034–expressing clones (Ang2-S2 and Ang2-S10). Similar to the results with parental U87MG cells seeded on an Ang2-coated surface described in Fig. 4A, pretreatment of U87MG/Ang2–expressing cells with MMPi-III (50 µmol/L), anti-ß1 and anti-{alpha}v antibodies (25 µg/mL), SP600125 (25 µmol/L), or U0126 (25 µmol/L) also inhibited the cell invasion enhanced by expressing Ang2 in U87MG cells (Fig. 5B). These data suggest that both the {alpha}vß1 integrin/FAK/p130Cas/ERK1/2 and JNK1 signaling pathway and MMP-2 are involved in Ang2-stimulated glioma cell invasion.

Inhibition of FAK suppresses Ang2-stimulated MMP-2 expression and glioma invasion in vivo. Lastly, a glioma xenograft model in the murine brain was employed to determine whether inhibition of FAK by FRNK expression could inhibit Ang2-stimulated glioma cell infiltration in vivo. As shown in Fig. 6, mice that received U87MG/Ang2-expressing cells developed invasive gliomas (Fig. 6B). The U87MG/Ang2–expressing 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 with that of U87MG/Ang2 expressing tumors (Fig. 6J) whereas there was no inhibition of MMP-2 expression by FRNK S-1034 (Fig. 6L). Immunoblot analyses confirmed that expression of FRNK or FRNK S-1034 by these cell clones did not alter expression levels of Ang2 but FRNK expression did inhibit Ang2-stimulated MMP-2 expression compared with that in U87MG/Ang2–expressing cells (data not shown). The sensitivity of detecting MMP-2 expression in established U87MG gliomas by immunohistochemical staining (Fig. 6I to L) is lower than that in cultured U87MG cells by zymographic and Western blot assays (Fig. 4A-C). Expression of HA-tagged FRNK and FRNK S-1034 by U87MG/Ang2–expressing cells in vivo was detected by immunohistochemistry using an anti-HA tag antibody (Fig. 6M and N). Furthermore, although expression of FRNK, but not FRNK S-1034, in U87MG/Ang2–expressing cells inhibited Ang2-stimulated glioma cell invasion in vitro and in vivo, no alteration of tumor formation of intracranial gliomas formed by either U87MG/Ang2/FRNK–expressing or U87MG/Ang2/FRNK S-1034–expressing cells was observed. Together, these results show that inhibition of FAK by FRNK in U87MG/Ang2–expressing cells suppressed Ang2-stimulated MMP-2 expression and glioma cell infiltration in vivo.


Figure 6
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  		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 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Growth factors and angiopoietin family members recently have emerged as novel ligands for integrins eliciting specific biological effects. For example, latent forms of transforming growth factor ß directly bind to integrin {alpha}vß6 (29) and {alpha}vß1 (30) in regulating pulmonary inflammation and fibrosis. VEGF165 and VEGF189 promote endothelial cell adhesion, migration, and survival through interaction with several integrins independent of VEGF receptors (31). Fibrinogen has been shown as a ligand for several integrins including the {alpha}5ß1 integrin receptor (32). Members of the angiopoietin family contain a fibrinogen-like domain at the COOH terminus of their proteins (8, 33), suggesting integrins as potential candidate receptors (7). Adhesion of Tie2-deficient cells, such as fibroblasts and skeletal myocytes, to Ang2-coated as well Ang1-coated surfaces was recently shown (11, 12). Lacking Tie2 receptors, fibroblasts and myocytes adhere to Ang2- or Ang1-coated surfaces and the adhesion is sensitive to the inhibition of ß1, ß3, {alpha}6, and {alpha}v integrins. Interactions of Ang1 and/or Ang2 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 {alpha}5ß1 integrin outside-in FAK activation, leading to a cross-talk of Tie2 and {alpha}5ß1, and fine-tuning modulation of the vascular effect (13). In this study, we report that Ang2 associates with {alpha}vß1 integrin in Tie2-deficient glioma cells. We observed that Ang2 induces extracellular adhesion and activates intracellular signal transduction pathways in glioma cells. Pull-down and immunoprecipitation assays showed that {alpha}vß1 integrin could be copurified with Ang2. The association of Ang2 with {alpha}vß1 integrin was highly calcium and manganese dependent in both pull-down and coimmunoprecipitation assays, suggesting the association of Ang2 with {alpha}vß1 integrin is similar to that of Ang1, Ang2 to various integrins (12), and PG-MV/vesican with ß1 integrin (23). Our data further show that Ang2 stimulates {alpha}vß1 integrin/FAK–mediated signaling, leading to an increase in MMP-2 expression and glioma cell invasion.

FAK and p130Cas are involved in early integrin receptor signaling. Up-regulated FAK expression and activation have been found in various types of human cancers. An increase of FAK phosphorylation on Tyr397 and Tyr861 strongly correlates with the acquisition of an invasive phenotype of tumor cells (21, 34). We found that Ang2 stimulates FAK tyrosine phosphorylation on these two tyrosine residues in U87MG cells. Expression of FRNK, but not its mutant form FRNK S-1034, suppressed Ang2-induced activation of FAK/p130Cas/ERK1/2 and JNK1, MMP-2 expression and secretion, and cell invasion in vitro and reversed the phenotype of Ang2-stimulated glioma cell infiltration and MMP-2 expression in vivo. These results are consistent with previous reports that FAK Tyr397 phosphorylation is required to promote JNK activation and cell invasion by v-Src (35) and that FRNK expression suppressed Src-stimulated Try861 phosphorylation, inhibiting Src activation of FAK downstream signaling, MMP-2 expression, and tumor metastasis (25). Moreover, FRNK overexpression disrupts focal adhesion and induces anoikis in ventricular myocytes by inhibiting endothelin-1-induced phosphorylation of proline-rich tyrosine kinase 2 (PyK2; ref. 36). In glioma cells, PyK2 plays critical roles in cell migration. Overexpression of PyK2 stimulates glioma cell migration whereas inhibition of PyK2 by FRNK expression or siRNA for PyK2 significantly inhibits glioma cell migration (37, 38). Therefore, it is plausible that in addition to suppression of FAK, FRNK expression in U87MG/Ang2–expressing cells also inhibits PyK2 activation, leading to decreased Ang2-induced glioma cell invasion in vitro and in vivo.

p130Cas is an adaptor protein that associates with FAK directly through its SH3 domain to a proline-rich region of FAK (amino acids 712-718; ref. 21). p130Cas also interacts with FAK indirectly through binding to Src family kinase via its SH3 domain. Src stimulates FAK Tyr397 phosphorylation, resulting in binding of FAK to an SH2 domain of the Src protein (39). Our data show that Ang2-induced p130Cas tyrosine phosphorylation was completely abolished by neutralizing anti-ß1 and anti-{alpha}v antibodies or FRNK, but not FRNK S-1034, suggesting that the interaction of phosphorylated FAK at Tyr397 with Src family kinase is also involved in Ang2/{alpha}vß1/FAK/p130Cas signaling (21). Additionally, expression of a dominant-negative regulator for p130Cas, CasSH3 (28), significantly attenuated Ang2-induced phosphorylation of p130Cas and downstream signaling activities in U87MG cells, showing that the interaction between FAK and p130Cas mediates Ang2/{alpha}vß1 integrin stimulation of glioma cell invasion.

Previous studies have shown that integrin-mediated stimulation of FAK/p130Cas signaling pathways activates ERK1/2 and JNK1 (19), MMP-2 secretion, and cell invasion (25, 35, 40, 41). Our results identify an {alpha}vß1 integrin/FAK/p130Cas/ERK1/2 and JNK1 signaling pathway that mediates Ang2-stimulated MMP-2 expression, secretion, and cell invasion. Functional blocking antibodies for Ang2 and {alpha}v and ß1 integrins inhibited Ang2 stimulation of ERK1/2 and JNK. FRNK and CasSH3 or inhibitors for ERK1/2 (U0126) and JNK1 (SP600125) suppressed Ang2 activation of ERK1/2 and JNK1. Furthermore, Ang2-stimulated MMP-2 expression and cell invasion were attenuated by {alpha}v and ß1 inhibition, FRNK, CasSH3, U0126, and SP600125. These results are consistent with previous findings that both ERK1/2 and JNK mediate Src-stimulated v-Src3T3 cell invasion and FRNK inhibits v-Src-stimulated phosphorylation of ERK1/2 and JNK (25). Moreover, our results differ from a study showing that only JNK1 activation, and not ERK1/2 phosphorylation, is required in v-Src-stimulated FAK-Src-p130Cas signaling in FAK-null fibroblasts (35).

ß1-Integrin has been shown to activate FAK-mediated signaling pathways leading to promotion of invasion of various types of human carcinoma cells (10, 42, 43). Suppression of ß1 expression in rat C6 glioma cells prevented diffuse glioma cell invasion in the brain (44). On the other hand, several {alpha} integrin subunits that associate with the ß1 integrin subunit, such as {alpha}v, {alpha}5, and {alpha}6 integrins, have been implicated in mediating tumor cell invasion (19, 42, 43). In addition, {alpha}vß3 integrin is also associated with invasion of human gliomas (17, 45). In our study, we show that {alpha}vß1, but not other integrin receptors, is associated with Ang2-stimulated glioma cell invasion. Moreover, we cannot rule out other molecules involved in Ang2-stimulated glioma cell invasion. For example, Ang2 may associate with {alpha}vß5 integrin because functional blocking of {alpha}v, {alpha}5, ß1, ß3, and {alpha}vß5 inhibited fibroblast or myocyte adhesion to Ang2 (11, 12). ANGPTL3 and tenascin C interact with {alpha}vß3 integrin through the fibrinogen-like domain that is also present in Ang2 (7, 8). We found that {alpha}3 and ß3 are not associated with Ang2 whereas {alpha}5 is weakly associated with Ang2. However, inhibition of {alpha}3, {alpha}5, and ß3 only moderately suppressed Ang2-mediated cell adhesion (Supplementary Table S1 and Fig. S1), but not phosphorylation of FAK and p130Cas (Fig. 2). Thus, {alpha}vß3 and {alpha}5ß1 integrins are probably not involved in Ang2-stimulated glioma cell invasion in our model. Additionally, we have recently reported that MT1-MMP, laminin-5 {gamma}2, and Ang2 are coexpressed in the invasive regions, but not in the central areas, of primary glioma specimens and exogenous Ang2 stimulates expression and activation of both MT1-MMP and laminin-5 {gamma}2 in U87MG/Ang2–expressing glioma xenografts in the murine brain and in vitro (18). Taken together, these results warrant further investigation to determine other signaling pathways involved in Ang2-induced glioma cell invasion.

In summary, this study identifies a novel molecular mechanism by which Ang2 stimulates human glioma cell invasion via the {alpha}vß1 integrin/FAK/p130Cas/ERK1/2 and JNK1 signaling pathway and MMP-2 expression (Supplementary Fig. S2). Considering the fact that Ang2 expression has significant links to tumor invasion and metastases in various types of human cancers (2–6), our studies provide a basis for further characterization of the molecular cascades involved in Ang2-stimulated glioma invasion, which may yield promising insight for the development of new diagnostic and therapeutic applications for invasive tumors.


   Acknowledgments
 
Grant support: James S. McDonnell Foundation grant and NIH grants CA102011 and CA102310 (D.D. Schlaepfer) and an ACS grant RSG CSM107144 (S-Y. Cheng).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Kari Alitalo for an expression vector containing human Ang2 cDNA and Dr. Jun-Lin Gun for an expression vector containing human CasSH3 cDNA.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 4/ 6/05. Revised 10/11/05. Accepted 10/28/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth factors and blood vessel formation. Nature 2000;407:242–8.[CrossRef][Medline]
  2. Sfiligoi C, de Luca A, Cascone I, et al. Angiopoietin-2 expression in breast cancer correlates with lymph node invasion and short survival. Int J Cancer 2003;103:466–74.[CrossRef][Medline]
  3. Etoh T, Inoue H, Tanaka S, Barnard GF, Kitano S, Mori M. Angiopoietin-2 is related to tumor angiogenesis in gastric carcinoma: possible in vivo regulation via induction of proteases. Cancer Res 2001;61:2145–53.[Abstract/Free Full Text]
  4. Ochiumi T, Tanaka S, Oka S, et al. Clinical significance of angiopoietin-2 expression at the deepest invasive tumor site of advanced colorectal carcinoma. Int J Oncol 2004;24:539–47.[Medline]
  5. Koga K, Todaka T, Morioka M, et al. Expression of angiopoietin-2 in human glioma cells and its role for angiogenesis. Cancer Res 2001;61:6248–54.[Abstract/Free Full Text]
  6. Lind AJ, Wikstrom P, Granfors T, Egevad L, Stattin P, Bergh A. Angiopoietin 2 expression is related to histological grade, vascular density, metastases, and outcome in prostate cancer. Prostate 2005;62:394–9.[CrossRef][Medline]
  7. Yokoyama K, Erickson HP, Ikeda Y, Takada Y. Identification of amino acid sequences in fibrinogen {gamma}-chain and tenascin C C-terminal domains critical for binding to integrin {alpha}vß3. J Biol Chem 2000;275:16891–8.[Abstract/Free Full Text]
  8. Camenisch G, Pisabarro MT, Sherman D, et al. ANGPTL3 stimulates endothelial cell adhesion and migration via integrin {alpha}vß3 and induces blood vessel formation in vivo. J Biol Chem 2002;277:17281–90.[Abstract/Free Full Text]
  9. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002;110:673–87.[CrossRef][Medline]
  10. Brakebusch C, Bouvard D, Stanchi F, Sakai T, Fassler R. Integrins in invasive growth. J Clin Invest 2002;109:999–1006.[CrossRef][Medline]
  11. Carlson TR, Feng Y, Maisonpierre PC, Mrksich M, Morla AO. Direct cell adhesion to the angiopoietins mediated by integrins. J Biol Chem 2001;276:26516–25.[Abstract/Free Full Text]
  12. Dallabrida SM, Ismail N, Oberle JR, Himes BE, Rupnick MA. Angiopoietin-1 promotes cardiac and skeletal myocyte survival through integrins. Circ Res 2005;96:e8–24.[Abstract/Free Full Text]
  13. Cascone I, Napione L, Maniero F, Serini G, Bussolino F. Stable interaction between {alpha}5ß1 integrin and Tie2 tyrosine kinase receptor regulates endothelial cell response to Ang-1. J Cell Biol 2005;170:993–1004.[Abstract/Free Full Text]
  14. Kleihues P, Cavenee WK, editors. Pathology and genetics: tumours of the nervous system. Lyon: IARC Press; 2000.
  15. Giese A, Westphal M. Glioma invasion in the central nervous system. Neurosurgery 1996;39:235–50; discussion 50–2.[CrossRef][Medline]
  16. Hu B, Guo P, Fang Q, et al. Angiopoietin-2 induces human glioma invasion through the activation of matrix metalloprotease-2. Proc Natl Acad Sci U S A 2003;100:8904–9.[Abstract/Free Full Text]
  17. Bellail AC, Hunter SB, Brat DJ, Tan C, Van Meir EG. Microregional extracellular matrix heterogeneity in brain modulates glioma cell invasion. Int J Biochem Cell Biol 2004;36:1046–69.[CrossRef][Medline]
  18. Guo P, Imanishi Y, Cackowski FC, et al. Up-regulation of angiopoietin-2, matrix metalloprotease-2, membrane type 1 metalloprotease, and laminin 5 {gamma}2 correlates with the invasiveness of human glioma. Am J Pathol 2005;166:877–90.[Abstract/Free Full Text]
  19. Hood JD, Cheresh DA. Role of Integrins in Cell Invasion and Migration. Nat Rev Cancer 2002;2:91–100.[CrossRef][Medline]
  20. Hanks SK, Ryzhova L, Shin NY, Brabek J. Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility. Front Biosci 2003;8:d982–96.[Medline]
  21. Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 2005;6:56–68.[CrossRef][Medline]
  22. Kramer RH, McDonald KA, Crowley E, Ramos DM, Damsky CH. Melanoma cell adhesion to basement membrane mediated by integrin-related complexes. Cancer Res 1989;49:393–402.[Abstract/Free Full Text]
  23. Wu Y, Chen L, Zheng PS, Yang BB. ß1-Integrin-mediated glioma cell adhesion and free radical-induced apoptosis are regulated by binding to a C-terminal domain of PG-M/versican. J Biol Chem 2002;277:12294–301.[Abstract/Free Full Text]
  24. Richardson A, Parsons T. A mechanism for regulation of the adhesion-associated proteintyrosine kinase pp125FAK. Nature 1996;380:538–40.[CrossRef][Medline]
  25. Hauck CR, Hsia DA, Puente XS, Cheresh DA, Schlaepfer DD. FRNK blocks v-Src-stimulated invasion and experimental metastases without effects on cell motility or growth. EMBO J 2002;21:6289–302.[CrossRef][Medline]
  26. Rice PL, Goldberg RJ, Ray EC, Driggers LJ, Ahnen DJ. Inhibition of extracellular signal-regulated kinase 1/2 phosphorylation and induction of apoptosis by sulindac metabolites. Cancer Res 2001;61:1541–7.[Abstract/Free Full Text]
  27. Thottassery JV, Sun Y, Westbrook L, et al. Prolonged extracellular signal-regulated kinase 1/2 activation during fibroblast growth factor 1-or heregulin ß1-induced antiestrogen-resistant growth of breast cancer cells is resistant to mitogen-activated protein/extracellular regulated kinase kinase inhibitors. Cancer Res 2004;64:4637–47.[Abstract/Free Full Text]
  28. Cary LA, Han DC, Polte TR, Hanks SK, Guan JL. Identification of p130Cas as a mediator of focal adhesion kinase-promoted cell migration. J Cell Biol 1998;140:211–21.[Abstract/Free Full Text]
  29. Munger JS, Harpel JG, Giancotti FG, Rifkin DB. Interactions between growth factors and integrins: latent forms of transforming growth factor-ß are ligands for the integrin {alpha}vß1. Mol Biol Cell 1998;9:2627–38.[Abstract/Free Full Text]
  30. Munger JS, Huang X, Kawakatsu H, et al. The integrin {alpha}vß6 binds and activates latent TGF ß1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 1999;96:319–28.[CrossRef][Medline]
  31. Hutchings H, Ortega N, Plouet J. Extracellular matrix-bound vascular endothelial growth factor promotes endothelial cell adhesion, migration, and survival through integrin ligation. FASEB J 2003;17:1520–2.[Abstract/Free Full Text]
  32. Suehiro K, Gailit J, Plow EF. Fibrinogen is a ligand for integrin {alpha}5ß1 on endothelial cells. J Biol Chem 1997;272:5360–6.[Abstract/Free Full Text]
  33. Davis S, Papadopoulos N, Aldrich TH, et al. Angiopoietins have distinct modular domains essential for receptor binding, dimerization and superclustering. Nat Struct Biol 2003;10:38–44.[CrossRef][Medline]
  34. Carragher NO, Frame MC. Focal adhesion and actin dynamics: a place where kinases and proteases meet to promote invasion. Trends Cell Biol 2004;14:241–9.[CrossRef][Medline]
  35. Hsia DA, Mitra SK, Hauck CR, et al. Differential regulation of cell motility and invasion by FAK. J Cell Biol 2003;160:753–67.[Abstract/Free Full Text]
  36. Heidkamp MC, Bayer AL, Kalina JA, Eble DM, Samarel AM. GFP-FRNK disrupts focal adhesions and induces anoikis in neonatal rat ventricular myocytes. Circ Res 2002;90:1282–9.[Abstract/Free Full Text]
  37. Lipinski CA, Tran NL, Bay C, et al. Differential role of proline-rich tyrosine kinase 2 and focal adhesion kinase in determining glioblastoma migration and proliferation. Mol Cancer Res 2003;1:323–32.[Abstract/Free Full Text]
  38. Lipinski CA, Tran NL, Menashi E, et al. The tyrosine kinase pyk2 promotes migration and invasionof glioma cells. Neoplasia 2005;7:435–45.[CrossRef][Medline]
  39. Salazar EP, Rozengurt E. Src family kinases are required for integrinmediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397. J Biol Chem 2001;276:17788–95.[Abstract/Free Full Text]
  40. Kurata H, Thant AA, Matsuo S, et al. Constitutive activation of MAP kinase kinase (MEK1) is critical and sufficient for the activation of MMP-2. Exp Cell Res 2000;254:180–8.[CrossRef][Medline]
  41. Zhang Y, Thant AA, Hiraiwa Y, et al. A role for focal adhesion kinase in hyluronan-dependent MMP-2 secretion in a human small-cell lung carcinoma cell line, QG90. Biochem Biophys Res Commun 2002;290:1123–7.[CrossRef][Medline]
  42. Uhm JH, Gladson CL, Rao JS. The role of integrins in the malignant phenotype of gliomas. Front Biosci 1999;4:D188–99.[Medline]
  43. Giannelli G, Astigiano S, Antonaci S, et al. Role of the {alpha}3ß1 and {alpha}6ß4 integrins in tumor invasion. Clin Exp Metastasis 2002;19:217–23.[CrossRef][Medline]
  44. Paulus W, Baur I, Beutler AS, Reeves SA. Diffuse brain invasion of glioma cells requires ß1 integrins. Lab Invest 1996;75:819–26.[Medline]
  45. Kanamori M, Vanden Berg SR, Bergers G, Berger MS, Pieper RO. Integrin ß3 overexpression suppresses tumor growth in a human model of gliomagenesis: implications for the role of ß3 overexpression in glioblastoma multiforme. Cancer Res 2004;64:2751–8.[Abstract/Free Full Text]



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