Minimal Active Domain and Mechanism of Action of the Angiogenesis Inhibitor Histidine-Rich Glycoprotein

Research Article

Johan Dixelius, Anna-Karin Olsson, A˚sa Thulin, Chunsik Lee, Irja Johansson, and Lena Claesson-Welsh

Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden

Abstract by the receptor tyrosine kinase VEGF receptor 2 (VEGFR-2; also Histidine-rich glycoprotein (HRGP) is an abundant heparin- known as KDR in the human and Flk-1 in the mouse). Most, if not all, binding plasma protein that efficiently arrests growth and tumors express VEGF, which induces angiogenesis in neighboring VEGFR-2-expressing vessels and which promotes endothelial cell vascularization of mouse tumor models. We have shown that the antiangiogenic effect of HRGP is dependent on its permeability, leading to extravasation of provisional matrix compo- histidine/proline–rich domain, which needs to be released nents such as fibrin, fibronectin, and vitronectin. Binding of from the mother protein to exert its effects. Here we identify a endothelial cell integrins to these matrix components further 35-amino-acid peptide, HRGP330, derived from the histidine/ modulates the angiogenic response by inducing downstream proline–rich domain as endowed with antiangiogenic proper- signaling pathways converging with those induced by activation ties in vitro and in vivo. The mechanism of action of HRGP330 of VEGFR-2. Targeting the vasculature by neutralizing the action of involves subversion of focal adhesion function by disruption of VEGF with antibodies (Avastin/bevacizumab) prolonged the lives of integrin-linked kinase (ILK) and focal adhesion kinase (FAK) patients with metastatic renal or colorectal cancer in phase III functions, inhibition of vascular endothelial growth factor clinical trials (5, 6). This provides evidence that targeting the (VEGF)–induced tyrosine phosphorylation of the FAK sub- vasculature is a valid approach in cancer treatment. A number of strate A-actinin, and, as a consequence, an arrest in endo- endogenous antiangiogenic factors, derived from naturally occur- thelial cell motility. The disturbed focal adhesion function is ring proteins, have been identified and shown to inhibit growth of mouse tumors (see ref. 7 for a review). reflected in the ability of HRGP as well as of HRGP330 to prevent endothelial cell adhesion to vitronectin in a manner A common theme for inhibitors such as endostatin, tumstatin, and kininostatin, fragments of collagen XVIII, collagen IV, and involving AvB3 integrin. In conclusion, HRGP330, which we define as the minimal antiangiogenic domain of HRGP, exerts kininogen, respectively, is their interference with integrin function its effects through signal transduction targeting focal adhe- either by using integrins as receptors (8, 9) or by specifically binding sions, thereby interrupting VEGF-induced endothelial cell to different matrix components (10). Endostatin is reported to act via a5h1 integrins and tumstatin via a h motility. (Cancer Res 2006; 66(4): 2089-97) v 3. Binding of endostatin to endothelial cells also involves heparan sulfates (11, 12) and the leukocyte adhesion antigen E-selectin (13), indicating that the Introduction receptor mechanism may be complex and dependent on several Angiogenesis, formation of new blood vessels from preexisting components. ones, is a physiologic process that occurs during development, as a We and others have identified histidine-rich glycoprotein (HRGP; result of exercise, in wound healing and during the menstrual cycle. also known as HRG/HPRG, for review see ref. 14) as a potent Angiogenesis is tightly regulated by a number of proangiogenic and inhibitor of angiogenesis, an effect mediated via the histidine/ antiangiogenic factors acting in concert to adjust the distribution proline–rich (His/Pro–rich) domain of the protein (15, 16). We have of oxygen and nutrients to meet the demands of the tissue. A shown that HRGP reduces endothelial cell migration and tumor number of pathologic conditions including rheumatoid arthritis, vascularization and growth in mice. HRGP is a member of the diabetic retinopathy, tumor growth, atherosclerosis, and diabetic cystatin superfamily together with a2-HS glycoprotein/fetuin, loss of peripheral circulation are characterized by deregulated fetuin-B, cystatin, and kininogen (17, 18). The central domain of angiogenesis (1, 2). The consequences range from inadequate tissue HRGP is rich in histidine and proline residues and contains multiple growth to ischemia. more or less conserved tandem repeats of the pentapeptide GHHPH. Vascular endothelial growth factor (VEGF; first identified as We have shown that this domain can be proteolytically released from vascular permeability factor; refs. 3, 4) acts as a proangiogenic the full-length protein, which is required for the antiangiogenic factor by promoting endothelial migration, proliferation, differen- activity (16). HRGP preparations always contain proteolytic frag- tiation, and tissue degradation. These effects are primarily mediated ments of distinct masses (16, 19) and one of these fragments corresponds to the His/Pro–rich domain (16). In the present study, we further define the minimal active domain within the His/Pro– Note: Supplementary data for this article are available at Cancer Research Online rich fragment of HRGP and describe its mechanism of action. (http://cancerres.aacrjournals.org/). J. Dixelius and A-K. Olsson contributed equally to this work. J. Dixelius is presently at Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles Materials and Methods va¨g 2, SE-17177 Stockholm, Sweden. Requests for reprints: Lena Claesson-Welsh, Rudbeck Laboratory, Department of Antibodies. The antibodies used were neutralizing anti-avh3 (MAB Genetics and Pathology, Uppsala University, Dag Hammarskjo¨lds va¨g 20, SE-751 85 1976z, Chemicon, Temecula, CA); anti–focal adhesion kinase (FAK; Santa Uppsala, Sweden. E-mail: [email protected]. I2006 American Association for Cancer Research. Cruz Biotechnology, Santa Cruz, CA); anti-paxillin (Transduction Lab, doi:10.1158/0008-5472.CAN-05-2217 Franklin Lakes, NJ); anti-a-actinin; horseradish peroxidase (HRP) anti-goat www.aacrjournals.org 2089 Cancer Res 2006; 66: (4). February 15, 2006

(Sigma-Aldrich, Stockholm, Sweden); anti–integrin-linked kinase (ILK; Cell edges’’ function. Coloring was done by manually outlining the cells using the Signaling, Danvers, MA); anti-phosphotyrosine (4G10, Upstate Biotechnol- ‘‘pen tool.’’ ogy, Charlottesville, VA); Alexa 488 antimouse; Alexa 555 antimouse; Alexa Adhesion assay. Ninety-six-well cell culture plates were incubated 488 antirabbit and Alexa 568 antirabbit (Molecular Probes, Eugene, OR); overnight with vitronectin (1 Ag/mL in PBS) or BSA (2% BSA in PBS), HRP antirabbit and antimouse (Amersham Biosciences, Uppsala, Sweden); blocked for 2 hours with BSA in all wells, and washed thrice with PBS. Cells antimouse CD31 (Becton Dickinson, Franklin Lakes, NJ); and biotinylated plated the previous day in complete medium were washed once and antirat antibody (Vector Laboratories, Burlingame, CA). incubated for 60 minutes in medium [0.2% BSA, 20 mmol/L HEPES (pH 7.2), HRGP and HRGP-derived synthetic peptides. Recombinant His-tagged 2 ng FGF-2/mL; assay medium]. Cells were dissociated using nonenzymatic HRGP was produced and purified as previously described (16). In ‘‘Cell dissociation solution’’ (Sigma), washed once, and resuspended in assay preparations of purified HRGP, there are always proteolytic fragments of medium. Reagents were added to the cell suspension at the following distinct masses present (16, 19). One of these fragments corresponds to the concentrations: HRGP, 100 ng/mL; HRGP330, 100 ng/mL; LM609, 10 Ag/mL, His/Pro–rich domain of HRGP (16). The designation ‘‘HRGP’’ in this study and incubated for 15 or 5 minutes before seeding to allow complete or only refers to the purified preparation consisting of the mix of full-length HRGP partial inhibition of adhesion, respectively. After 45 minutes in 37jC, cells and proteolytic fragments thereof. HRGP-derived synthetic peptides were washed thrice in TBS and fixed in Zn-fix for 10 minutes. Finally, HRGP330, HRGP365, and HRGP398 were purchased from Innovagen AB cells were stained with Hoechst 33342 (1 Ag/mL) washed once in TBS, (Lund, Sweden). The peptides were analyzed by reverse-phase high- photographed (2Â objective), and cell number was quantified using the Easy performance liquid chromatography and mass spectral analysis and were Image software (Tekno Optik AB, Ska¨rholmen, Sweden). The experiments dissolved in TBS [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl] containing were repeated at least thrice and the mean value of at least four wells per

1 mmol/L ZnCl2. The peptides have the following sequences: HRGP330 condition was used. Error bars represent 1 SD. Statistical significance was (amino acids 330-364), DLHPHKHHSHEQHPHGHHPHAHHPHEHDTHRQ- calculated using Students t test. Differences with P < 0.05 were considered HPH-COOH; HRGP365 (amino acids 365-397), GHHPHGHHPHGHHPH- significant. GHHPHGHHPHCHDFQDYG-COOH; and HRGP398 (amino acid 398-439), Chemotaxis assay. The chemotaxis assay was done using a modified PCDPPPHNQGHCCHGHGPPPGHLRRRGPGKGPRPFHCRQIGS-COOH. Boyden chamber, as described earlier (16), with 8-Am micropore polycar- Cell culture. Bovine capillary endothelial cells, a kind gift from Dr. R. bonate filters (PFB8-50; Neuro Probe, Inc., Gaithersburg, MD) coated Christofferson (Department of Medical Cell Biology, Uppsala University), with type 1 collagen solution at 100 Ag/mL (Vitrogen 100; Collagen Corp., were cultured on gelatin-coated dishes in DMEM (Life Technologies, Inc., Palo Alto, CA). Bovine capillary endothelial cells that had been starved Rockville, MD), 10% newborn calf serum, and 2 ng fibroblast growth factor overnight in 0.5% NCS were trypsinized and resuspended at 7.5 Â 105/mL 2 (FGF-2)/mL (complete medium). Telomerase immortalized endothelial in DMEM, 0.25% BSA, and trasylol (aprotinin) at 1,000 KIE/mL. The cell cells, derived from human dermal microvascular endothelial cells, were a suspension was added in the upper chamber and VEGF-A (Peprotech, Rocky kind gift from Dr. Martin McMahon (Cancer Research Institute, University Hill, NJ) at 10 ng/mL in the lower chamber. HRGP peptides (100 ng/mL) of California San Francisco, San Francisco, CA; ref. 20). Telomerase and avh3 neutralizing antibody (LM609; 10 Ag/mL) were added both in the immortalized endothelial cells were cultured in complete endothelial cell upper and lower chambers. After 5 hours at 37jC, cells that had migrated basal medium (EBM MV2, PromoCell, Heidelberg, Germany) without through the filter were stained with Giemsa and counted using the Easy antibiotics on gelatin-coated cell culture plastic. Image Analysis software (Tekno Optik). All samples were analyzed in at least Conditions for microscopy and immunoblotting. For all microscopy six wells for each treatment and at several separate occasions. Error bars and immunoblotting experiments, cells were seeded on vitronectin-coated represent 1 SD. Statistical significance was calculated using Students t test. (V8379, Sigma-Aldrich) Petri dishes or cell culture slides in complete Differences with P < 0.05 were considered significant. medium, incubated for 24 hours, washed, and incubated in DMEM/1% Animal studies. Animal work was approved by the Uppsala University newborn calf serum (starvation medium) for 20 to 24 hours. To prevent board of animal experimentation and thus was done according to the United contribution from serum-derived HRGP, the medium was replaced with Kingdom Coordinating Committee on Cancer Research guidelines for fresh EBM/0.1% bovine serum albumin (BSA), 20 mmol/L HEPES (pH 7.2), the welfare of animals in experimental neoplasia (21). The mice were and incubated for 90 minutes. The cells were then stimulated as indicated. anesthetized with isoflurane (Forene; Abbott, Abbott Park, IL) during all Microphotography. The slides were put on ice, washed in ice-cold TBS manipulations. Six-week-old female Fox Chase severe combined immunode- [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl], and fixed in Zn-fix [0.05% ficient mice were injected s.c. with 2.5 Â 106 BxPC3 pancreatic carcinoma f 3 Ca-acetate, 0.5% Zn-acetate, 0.5% ZnCl2 in TBS (pH 6.6), and 0.2% Triton cells on their left flank. When the tumors had reached a size of 200 mm , X-100] for 15 minutes at room temperature. The preparations were washed the animals were randomized into two groups (n = 7 per group) and in TBS, blocked in TBS/10% FCS, and stained using standard protocols. The treatment was initiated with daily s.c injections on the right flank with 60Â/NA 1.4 or 10Â/NA 0.45 objectives of a Nikon E1000 microscope and a either 5 mg/kg/d HRGP330 (dissolved in 1 mmol/L ZnCl2, 68 mmol/L NaCl, Nikon DXM1200 camera were used. 1.4 mmol/L KCl, 2.1 mmol/L Na2HPO4Á7H2O, 0.7 mmol/L KH2PO4) or vehicle. Immunoblotting. Cells were put on ice and quickly washed once in The tumors were measured with a caliper, in a blind procedure, and volumes 2 ice-cold TBS/100 Amol/L Na3VO4, then lysed in NP40 lysis buffer [1% NP40, were calculated by the formula p /6Â width Â length. Tumor material was 20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 10% glycerol, 2.5 mmol/L snap-frozen in dry ice/isopentane and sectioned at 3 Am. EDTA, 10 kallikrein inhibitory units (KIE) aprotinin/mL, 1 mmol/L phenyl- CD31 staining and quantification of vascular variables. Frozen methylsulfonyl fluoride, and 100 Amol/L Na3VO4]. Lysates were clarified sections of the tumor tissue were fixed in acetone and blocked in 2% rabbit by centrifugation. Immunoprecipitation, SDS-PAGE, and immunoblotting serum before incubation with a rat anti-mouse CD31 antibody (Becton were done using standard procedures. Immunoreactive sites on the filters Dickinson), diluted 1:500, and incubated at 4jC overnight. Detection of were detected by enhanced chemiluminescence. the primary antibody binding sites was done using a biotinylated antirat Time lapse. Telomerase immortalized endothelial cells were seeded on antibody (BA-4001; Vector Laboratories), diluted 1:300, and streptavidin- vitronectin-coated coverslips at a subconfluent density in complete EBM HRP (SA-5004; Vector Laboratories) diluted 1:200. The enzymatic signal was medium and incubated for 24 hours, washed, and starved in EBM/1% FCS visualized using AEC peroxidase substrate kit (SK-4200; Vector Laborato- for 20 hours. The coverslips were mounted in observation dishes in a 1:1 ries). Hematoxylin staining was done to visualize all cells. Stereological mixture of conditioned EBM/1% FCS and EBM/20 mmol/L HEPES (pH 7.2) quantification of vascular variables was done as described earlier (16) on at in a temperature-controlled chamber set to 37jC on a Zeiss LSM 510 Meta least three different tumors from each treatment group. This method of confocal microscope. The cells were allowed to stabilize for 60 minutes; quantifying tumor angiogenesis relates the length, volume, and surface area after which, one differential interference contrast image per minute was of the vessels to tumor volume (22). Error bars represent 1 SD. Statistical collected for 2 hours using the 40Â/NA 1.3 objective. The image in Fig. 4A significance was calculated using Students t test. Differences with P < 0.05 was processed in Adobe Photoshop software (San Jose, CA) using the ‘‘find were considered significant.

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Results (HRGP330, amino acids 330-364; HRGP365, amino acids 365-397; Identification of the minimal active domain of HRGP. We and HRGP398, amino acids 398-439) derived sequentially from have shown that inhibition of endothelial cell chemotaxis by the the His/Pro–rich region were screened for their ability to inhibit angiogenesis inhibitor HRGP is mediated by the His/Pro–rich endothelial cell chemotaxis (Fig. 1A-C). Of these, only HRGP330 domain (16). HRGP is a 75-kDa multidomain molecule which was able to inhibit VEGF-induced chemotaxis of primary endo- engages in interactions with a broad spectrum of molecules (14). thelial cells (Fig. 1A). As seen in Fig. 1D, HRGP330 attenuated To avoid complications in our studies from such interactive chemotaxis in a dose-dependent manner. partners (e.g., thrombospondins 1 and 2), we wished to identify the HRGP330 is antiangiogenic in vivo. We next tested whether minimal active domain of HRGP. A series of synthetic peptides HRGP330 retained the antiangiogenic properties of HRGP in vivo.

Figure 1. Identification of the minimal active domain of HRGP. A to C, chemotaxis of primary endothelial cells induced by 10 ng/mL VEGF was inhibited by 100 ng/mL HRGP330 (H330; A) but not by HRGP365 (B) or HRGP398 (C). D, the 35-amino-acid residue peptide HRGP330 inhibited VEGF-induced endothelial cell chemotaxis in a dose-dependent manner. E, growth curves of BxPC3 tumors in HRGP330-treated (5 mg/kg/d; w ) and control-treated (5) mice (n = 7 per group). F, visualization of the tumor vasculature by immunohistochemical staining against CD31 revealed a striking reduction in CD31-positive vessels in tumors from HRGP330-treated animals. Bar, 100 Am. G, stereological quantification of vascular parameters in tumors from control- or HRGP330-treated mice showed a significant reduction in vessel length, volume, and surface area. Bars, 1 SD. Significance at the P < 0.01 or P < 0.05 level is indicated.

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Figure 2. HRGP330 disrupts ILK-paxillin complex formation. A, endothelial cells on vitronectin were treated with HRGP330 (100 ng/mL) and VEGF (50 ng/mL), individually or in combination, for 10 minutes. VEGF induced increased complex formation between paxillin and ILK as shown by immunoprecipitation (IP) of paxillin and immunoblotting (IB) for ILK (top; arrow, ILK). Cotreatment with HRGP330 and VEGF disrupted ILK-paxillin complex formation. Control immunoblotting for paxillin on the paxillin immunoprecipitates (middle) and immunoblotting for actin on total cell lysates (bottom), show equal protein amounts in cell lysates used for immunoprecipitation. B, the reciprocal experiment (IP:ILK, IB:paxillin) gave the same result. The broad paxillin band is indicated. Immunoblotting for ILK and actin showed equal amount of protein in total cell lysates. Control immunoblotting for the amount of immunoprecipitated ILK was not feasible due to comigration with the immunoglobulin heavy chain. C, immunocytochemical staining for paxillin (red) and ILK (green), using the same conditions as in (A), revealed colocalization of paxillin and ILK after VEGF stimulation (Merge; yellow, indicated by arrowheads), which was disrupted by cotreatment with VEGF and HRGP330. Bar, 4 Am.

Female severe combined immunodeficient mice (n = 7 per group) showed a striking reduction primarily in length and volume but were injected with BxPC3 human pancreatic carcinoma cells on also in surface area of the blood vessels in tumors from HRGP330- their left flank. The tumors were allowed to become well established treated mice as compared with control-treated mice (Fig. 1G). and reach f200 mm3 before treatment was initiated with daily These data show that HRGP330 possesses potent antiangiogenic s.c. injections on their right flank with HRGP330 (5 mg/kg/d in properties in an in vivo setting. 1 mmol/L ZnCl2) or vehicle. The tumor growth curves (Fig. 1E) HRGP330 inhibits VEGF-induced complex formation of ILK showed no significant difference in tumor size between the groups and paxillin. Migration requires coordinated signaling from although there was a tendency for reduced growth in the treatment growth factor receptors and integrins. The focal adhesion group. After 31 days, the study had to be interrupted due to weight components ILK and FAK are instrumental integrators of these loss of animals in both groups. To determine whether HRGP330 signals. ILK is a serine/threonine kinase; however, its contribution exerted antiangiogenic effect in vivo, the degree of tumor to focal adhesion function seems to be dependent on its role as an vascularization was analyzed by immunohistochemical staining adapter for a large number of molecules (e.g., paxillin). Complex for expression of the vascular marker CD31. As seen in Fig. 1F, tumor formation between ILK and paxillin is needed for subcellular vascularization of HRGP330-treated mice was dramatically reduced. localization of ILK to focal adhesions and for cellular migration The control mice tumor vasculature was composed of larger vessels (23). We have previously shown that HRGP induces rearrangement and an even, dense capillary network. In contrast, tumors from the of focal adhesions in endothelial cells after 10 minutes of treatment. HRGP330-treated mice retained the large vessels whereas the small To seek the mechanism of action of HRGP330, we subjected capillaries were essentially absent (Fig. 1F). Quantification of tumor endothelial cells to treatment with the peptide for 10 minutes in vascularization according to the method by Gunderssen et al. (22) the presence and absence of VEGF. Immunoprecipitation of either

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Angiogenesis Inhibition by HRGP paxillin (Fig. 2A) or ILK (Fig. 2B) followed by immunoblotting for HRGP330 inhibits VEGF-induced tyrosine phosphorylation the partner showed that ILK-paxillin complex formation was of FAK and A-actinin. The cytoplasmic tyrosine kinase FAK is enhanced by VEGF whereas cotreatment with HRGP330 blocked crucial for focal adhesion function. Several angiogenesis inhibitors the VEGF-induced increase. The reduced ILK-paxillin complex such as angiostatin and endostatin have been shown to activate formation was not due to decreased protein expression levels; in FAK in endothelial cells (24, 25). Both VEGF and HRGP330 contrast, there was a tendency for increased recovery of paxillin in individually stimulated tyrosine phosphorylation (reflecting acti- the biochemical analysis of cells treated with HRGP330 (Fig. 2A). vation) of FAK in endothelial cells, with a slight increase after 10 Because ILK migrates at the same position of the immunoglobulin minutes and a more pronounced effect after 2 hours of treatment heavy chain, immunoblotting for ILK was done on total cell (Fig. 3A). In contrast, in cells cotreated with VEGF and HRGP330, lysates from stimulated cells (Fig. 2B, middle), which revealed FAK tyrosine phosphorylation was attenuated. One important similar recovery of ILK independent of treatment. Immunostain- substrate for the FAK kinase is a-actinin, of which binding to actin ing of endothelial cells (Fig. 2C), using the same experimental is negatively regulated by its tyrosine phosphorylation (26, 27). conditions as above, confirmed increased colocalization of ILK Tyrosine phosphorylation of a-actinin was studied in cells treated and paxillin in focal adhesions in response to treatment with with HRGP330 and VEGF for different time periods under the same VEGF. The colocalization was blocked by inclusion of HRGP330 conditions as above (Fig. 3B). Immunoblotting analyses showed together with VEGF. Disruption of VEGF-induced complex that VEGF as well as HRGP330 induced a robust phosphorylation formation between ILK and paxillin by HRGP330, visualized by of a-actinin both at 10 minutes and 2 hours. However, cotreatment immunoblotting or immunostaining of intact cells, provides a of the cells with VEGF and HRGP330 for 2 hours blocked a-actinin mechanistic explanation for the inhibition of migration by the phosphorylation, indicating persistent binding to actin and angiogenesis inhibitor. reduced cellular motility.

Figure 3. HRGP330 inhibits VEGF-induced activation of FAK and tyrosine phosphorylation of a-actinin and induces redistribution of a-actinin. Endothelial cells on vitronectin were treated with HRGP330 (100 ng/mL) and VEGF (50 ng/mL in A and B, 10 ng/ml in C), individually or in combination, for 10 minutes and 2 hours. A, both VEGF and HRGP330 added individually induced tyrosine phosphorylation of FAK at 10 minutes and more pronounced after 2 hours, as shown by immunoprecipitation of FAK and subsequent immunoblotting for phosphotyrosine (PY). Cotreatment of the cells with VEGF and HRGP330, both at 10 minutes and 2 hours, abolished FAK phosphorylation. Bottom, control immunoblotting for FAK. Levels of phosphorylated FAK normalized to total FAK levels are depicted graphically below the blot (PY/FAK). B, VEGF and HRGP330 added individually induced a pronounced increase in tyrosine phosphorylation of a-actinin (a-act), both at 10 minutes and 2 hours, as shown by immunoprecipitation for a-actinin, followed by immunoblotting for phosphotyrosine or a-actinin. Cotreatment of cells with VEGF and HRGP330 for 10 minutes allowed a-actinin phosphorylation whereas, at 2 hours, the level of phosphorylation had returned to basal, in contrast to treatment with the individual factors. Bottom, control immunoblotting for a-actinin. Levels of phosphorylated a-actinin normalized to total a-actinin levels are depicted graphically below the blot (PY/a-act). C, immunocytochemical staining for a-actinin (red) was done in endothelial cells on vitronectin treated with VEGF and HRGP330, individually or in combination, for 10 minutes and 2 hours (i.e., the same experimental setup as in A and B). FAK staining (green) was used to visualize focal contacts. No significant change in a-actinin staining pattern compared with control cells was detected after individual treatment with VEGF or HRGP330 or after the combined treatment for 10 minutes. Cotreatment with VEGF and HRGP330 for 2 hours resulted in accumulation of a-actinin along actinlike fiber structures. Bar, 8 Am. www.aacrjournals.org 2093 Cancer Res 2006; 66: (4). February 15, 2006

HRGP330 and VEGF cotreatment induces redistribution of A-actinin. Migrating cells have a rapid turnover of focal adhesions and a dynamic actin cytoskeleton whereas nonmigrating cells are characterized by localization of a-actinin to their stable focal adhesions (27). Further, a-actinin incorporation into actin fibers is likely to promote a less dynamic cytoskeleton (28). The distribution of a-actinin was investigated by immunofluorescent staining of endothelial cells treated with VEGF and HRGP330 as indicated (Fig. 3C). Untreated cells or cells treated with VEGF or HRGP330 alone, for 10 minutes or 2 hours, showed low a-actinin immuno- reactivity. However, in cells cotreated with VEGF and HRGP330 for 2 hours, a-actinin-positive fibers were clearly visible, merging with focal adhesions visualized by FAK staining. These data reinforce the notion that HRGP330 acts to inhibit endothelial cell motility responses by subverting the function of focal adhesions. HRGP330 interferes with VEGF-induced polarization and lamellopodia formation but does not inhibit VEGFR-2 activation. Cell migration requires the concerted regulation of growth factor–induced lamellopodia formation and cell polarization. Endo- thelial cells on coverslips were monitored by time-lapse microscopy to study the effect of HRGP330 on these cellular responses (Fig. 4A; Supplementary data). Untreated cells were nonpolarized with a basal lamellopodia activity. Treatment with HRGP330 alone (Supplementary data) did not affect the cell morphology whereas inclusion of VEGF resulted in a transient burst of increased formation of lamellopodia at 30 minutes and, within two hours, a shift towards a polarized phenotype with elongated cells, consistent with a migratory phenotype. Cotreatment of the cells with VEGF and HRGP330 abrogated the VEGF-induced changes, indicating that HRGP330 directly or indirectly interferes with signaling events induced by VEGF (Fig. 4A; Supplementary data). To show that HRGP330 did not simply exert its effects by direct inhibition of activation of VEGFR-2, the main transducer of endothelial cell responses to VEGF, cells were treated with VEGF in the presence and absence of HRGP330 (i.e., similar conditions as Figure 4. HRGP330 reverts VEGF-induced polarization and lamellopodia formation. A, time-lapse images of endothelial cells on vitronectin treated those used above to show HRGP330 activation of FAK). As shown with VEGF alone (10 ng/mL) or in combination with HRGP330 (100 ng/mL). in Fig. 4B, VEGFR-2 tyrosine phosphorylation, indicative of its VEGF stimulation resulted in increased lamellopodia formation (arrows)at30 activation, was induced by VEGF irrespective of whether HRGP330 minutes and a shift towards a polarized phenotype with elongated cells within 2 hours, consistent with a migratory phenotype. In the presence of HRGP330, was included in the treatment or not. these VEGF-induced morphologic changes were abrogated. B, VEGFR-2 A Involvement of vB3 integrin in HRGP action. We have tyrosine phosphorylation induced by VEGF (50 ng/mL) was unaffected by the previously shown that HRGP interferes with the ability of endothelial presence of HRGP330 (100 ng/mL) as shown by immunoprecipitation of VEGFR-2 and immunoblotting for phosphotyrosine. The expression level of cells to adhere to vitronectin (16). As shown in Fig. 5A, HRGP330 VEGFR-2 is low in primary endothelial cells and immunoblotting for Erk was also inhibited adhesion of endothelial cells to vitronectin, possibly therefore used as a control for protein amount in total cell lysates. h implicating the av 3 integrin, the main vitronectin receptor on endothelial cells, as a target molecule for HRGP330. To address because vitronectin coating of the polycarbonate filters used in the h the role of av 3 in the adhesion assay, we compared the effect of chemotaxis assay did not support endothelial cell adhesion. It is h treatment with the neutralizing anti-av 3 antibodies and HRGP/ conceivable that avh3 integrins used cryptic sites exposed in the HRGP330 on endothelial cell adhesion to vitronectin. As shown in partially denatured collagen I coat for movement and that these h Fig. 5A, treatment with HRGP/HRGP330 and anti-av 3 antibodies interactions were obliterated by the neutralizing antibody, thereby inhibited the vitronectin-induced adhesion. Under conditions where attenuating the migratory response (Fig. 5C). This result shows that adhesion was only partially inhibited by individual treatment with migration of the endothelial cells in the present experimental setup HRGP or by the neutralizing antibody LM609, we noted that the is dependent on avh3 integrins and supports the notion that combined treatment did not further reduce adhesion of the HRGP330 may act by interfering with integrin signaling. endothelial cells (Fig. 5B). These data indicate an overlapping target for HRGP and the anti-avh3 antibody, suggesting that HRGP/ Discussion HRGP330 may act via interference with avh3 function. h That av 3 integrin function was important for endothelial cell HRGP330 exerts its antiangiogenic effect by arresting the actin motility under the conditions used in the chemotaxis assay was cytoskeleton in an immobile state and preventing growth factor– shown by inclusion of a neutralizing anti-avh3 antibody, which induced promigratory events, such as lamellopodia formation and reduced VEGF-induced chemotaxis to basal levels (Fig. 5C). The cell polarization. The molecular mechanism involves disruption of chemotaxis experiment was done using collagen I–coated filters VEGF-induced complex formation between paxillin and ILK, as well

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Angiogenesis Inhibition by HRGP as prevention of VEGF-induced phosphorylation of FAK and its downstream substrate a-actinin. VEGFR-2 activation is not affected by the presence of HRGP330. Instead, our data implicate avh3, the main vitronectin receptor on endothelial cells, in the mechanism of action of HRGP330. However, we do not exclude that the receptor mechanism involves one or more additional cell-surface components. A summary and possible model for the mechanism of action of HRGP330, based on the above findings, is depicted in Fig. 6. HRGP interacts with a number of plasma proteins (14). Complex formation with thrombospondins 1 and 2, involving a COOH- terminal CLESH-1 domain of HRGP (29, 30), has received attention as it apparently leads to neutralization of the antiangiogenic effect of thrombospondin. Therefore, the identification of a 35-amino-acid residue peptide, HRGP330, which lacks the thrombospondin binding domain but retains the antiangiogenic properties, is important in further development of HRGP as an angiogenesis inhibitor (15, 16, 31). Although it is likely that the stability of HRGP330 is low in vivo, we achieved a significant reduction in BxPC3 vascularization with a virtual disappearance of small vessels in tumors in the treated animals. Donate et al. (31) recently reported on a multimerized pentapeptide based on the repetitive sequence GHHPH from the His/Pro–rich domain of HRGP, which was used to inhibit

Figure 6. Mechanism of action of HRGP330. The antiangiogenic effect of HRGP330 is exerted through an arrest of the endothelial cell actin cytoskeleton and a consequent inhibition of VEGF-induced effects such as lamellopodia formation and polarization, events needed for migration, and an angiogenic response. HRGP330 disrupts VEGF-induced complex formation between paxillin and ILK in focal adhesions, as well as VEGF-induced phosphorylation/activation of FAK and its downstream substrate a-actinin. VEGFR-2 activation is not affected by the presence of HRGP330. The main vitronectin receptor on endothelial cells, avh3 integrin, is implicated in the mechanism of action of HRGP330. However, it is likely that the receptor mechanism involves one or several cell-surface molecules, such as heparan sulfates, or other thus far unidentified components (?). Vn, vitronectin; HSPG, heparan sulfate proteoglycan; pax, paxillin; a-act, a-actinin.

angiogenesis in s.c. Matrigel plugs and tumor growth in mice at 80 mg/kg/d. Five repeats of the sequence GHHPH are contained within the peptide HRGP365 that did not show any antichemotactic effect on endothelial cells in our hands (Fig. 1B) whereas HRGP330 contains one GHHPH repeat. Another difference between HRGP330 and HRGP365 and HRGP398 is that HRGP330 binds to heparin/ heparan sulfate, showing that HRGP330 is dependent on sequences distinct from GHHPH for its biological properties.1 How does HRGP330 interact with the endothelial cell surface h Figure 5. Involvement of av 3 integrin in HRGP action. A, adhesion of and transduce its effects? Considering the effect of HRGP/ endothelial cells to vitronectin was significantly reduced in the presence of HRGP330 on endothelial cell adhesion, migration, and focal HRGP, HRGP330, or neutralizing avh3 antibody (LM609). B, under conditions where adhesion was only partially decreased (compare extent of HRGP and adhesions, integrins are obvious receptor candidates. The major HRGP330 block in relation to BSA between A and B), no further reduction vitronectin receptor on endothelial cells is avh3 integrin, which is was seen as a result of combined treatment with HRGP and LM609. C, chemotaxis of endothelial cells towards VEGF in a modified Boyden chamber was reduced to similar extents by inclusion of HRGP, HRGP330, or neutralizing avh3 antibody (LM609). Bars, 1 SD. All changes induced by HRGP, HRGP330, or LM609, compared with control in (A and B), or by the combination of 1 M. Vanwildemeersch, A.K. Olsson, E. Gottfridsson, L. Claesson-Welsh, U. Lindahl, VEGF and HRGP, HRGP330, or LM609, compared with VEGF in (C), were D. Spillmann. Angiogenesis inhibition by HRGP involves Zn2+-dependent binding of significant (P < 0.01; P < 0.05). heparan sulfate to the His/Pro–rich domain. 2005, submitted for publication. www.aacrjournals.org 2095 Cancer Res 2006; 66: (4). February 15, 2006

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research expressed on angiogenic endothelial cells in conjunction with role in linking integrins to the actin cytoskeleton (41). Endothelial vessel remodeling (32). We show that endothelial cell chemotaxis is cell–specific ablation of the serine/threonine kinase ILK leads to dependent on avh3; moreover, neutralizing anti-avh3 antibodies severe vascular defects and embryonic death at E11.5 (42) and block endothelial cell adhesion to vitronectin to the same extent as inhibition of ILK kinase activity inhibits tumor angiogenesis in HRGP330. These data are compatible with the notion that the mice (43). The importance of ILK serine/threonine kinase activity, mechanism of action of HRGP330 involves interference with avh3 however, has been questioned as genetic data from C. elegans and D. function. Moreover, ongoing studies implicate heparan sulfate in melanogaster suggest that the kinase activity is dispensable in vivo 1 h the mechanism of action of HRGP330. It is of interest that av 3 (41, 44). Of particular relevance for interpretation of data in this has been shown to occur in complex with VEGFR-2 and to be study, ovexpression of a mutant form of ILK that lacks the ability to required for VEGFR-2 responsiveness (33). In this context, it is engage in complex formation with paxillin leads to severely reduced important to note that HRGP330 does not simply interfere with motility (23). Moreover, cells overexpressing wild-type ILK fail to VEGFR-2 activation by binding either to heparin/heparan sulfate or polarize and display a poorly spread phenotype similar to that to avh3 (see Fig. 4B). Thus, we suggest that there may be more than of cells overexpressing RhoA. Forced expression of FAK, which one interactive partner for HRGP330 on the endothelial cell negatively regulates RhoA (45), rescues this phenotype in wild-type surface. The His/Pro–rich region of HRGP is able to bind Zn2+, ILK–transfected cells, suggesting that a balanced activation of FAK which is also required for HRGP330 to exert its antiangiogenic and ILK is required for polarization and migration. Our results on the function.1 Spectra obtained using far-UV technique indicate that effect of HRGP330 on FAK and ILK are in excellent agreement with HRGP belongs to the category of intrinsically unstructured proteins the well-established and central role of these cytoplasmic kinases in (34). A probable effect of Zn2+ binding is stabilization of the cell migration. conformation of the His/Pro–rich domain, which may be required Cancer is a major cause of premature death. Proof of the concept for its interaction with a receptor. that angiogiogenesis inhibition has clinical benefits in tumor Other potential mechanisms of action of HRGP330 include the treatment is given by the VEGF-blocking antibody Avastin possibility of interaction with circulating vitronectin, which may (bevacizumab). Recent reports indicate that VEGFR-2 expression promote homing of HRGP330 to sites of active angiogenesis. This and function is not restricted to angiogenic endothelium and effects type of mechanism has recently been proposed for the antiangio- on neuronal survival cannot be excluded in long-term anti-VEGF genic molecules anginex and anastellin (10). Moreover, two recent treatments (46). We have, at this point, no indications of adverse reports from the same group (31, 35) suggest membrane-bound effects on normal vasculature by systemic HRGP/HRGP330 treat- tropomyosin as a receptor for HRGP. Tropomyosins constitute a ment but these studies will continue. It seems clear that members of large family of widely expressed intracellular actin-binding mole- the cystatin superfamily, to which HRGP belongs, present a number cules lacking transmembrane domain (36). It is noteworthy that of interesting features that support our focus on HRGP as an tropomyosin has been suggested to also serve as a receptor for angiogenesis inhibitor. Thus, fetuin-B has recently been implicated kininogen (37) as well as for endostatin (38). The current consensus as a tumor suppressor (47) and kininogen possesses antiangiogenic is that endostatin requires a5h1 integrins and heparan sulfate to properties, also mediated via a histidine-rich domain (37). Deletion exert its effects on endothelial cells. of the proximal region of mouse chromosome 16, containing fetuin, Our in vitro data show that HRGP330 potently inhibits chemo- fetuin-B, HRGP, and kininogen of the cystatin superfamily, as well as taxis of endothelial cells in the nanogram range. Cell motility is a other genes, has been associated with the ‘‘angiogenic switch’’ in a complex process, which requires cells to polarize and concentrate transgenic model of multistage tumorigenesis (48). A recent report lamellopodia and filopodia activity to the leading edge, where small on HRGP gene inactivation implicates HRGP in regulation of blood focal adhesions are formed de novo. Actin stress fibers, microtubuli, coagulation (49). The consequence of loss of HRGP expression and RhoA activity are confined to and necessary for the trailing end for pathologic angiogenesis awaits further characterization. In where focal adhesions are larger and need to be remodeled or conclusion, antiangiogenic drugs have a large potential, perhaps in disrupted. The chemoattractant signal relayed by receptor tyrosine particular when combined with cytotoxic drugs or surgery, and their kinases, such as VEGFR-2, converge with signals from integrin development will be aided by mechanistic insights. receptors to orchestrate the motility response. This integration may be achieved by direct interaction (e.g., between VEGFR-2 and avh3) serving to amplify reciprocal signals (see ref. 39 for review). Acknowledgments Furthermore, downstream signaling pathways integrate to transmit Received 6/23/2005; revised 11/27/2005; accepted 12/16/2005. a permissive migratory signal. Such pathways include the cytoplas- Grant support: Swedish Cancer Society (project no. 3820-B04-09XAC), Innoventus Project AB, and the Sixth European Union Framework Programme (Integrated Project mic kinases FAK and ILK, both of which interact directly or indirectly ‘‘Angiotargeting’’; contract no. 504743) in the area of Life Sciences, Genomics, and with integrins as well as growth factor receptors. FAK has been Biotechnology for Health (L. Claesson-Welsh); Magnus Bergvalls Foundation and Erik, Karin, and Go¨sta Selanders Foundation (A-K. Olsson); and Swedish Society of Medical ascribed a critical role in focal adhesion turnover (40) and FAK- Research (J. Dixelius). dependent tyrosine phosphorylation of a-actinin blocks its associ- The costs of publication of this article were defrayed in part by the payment of page ation with actin, allowing increased actin dynamics (26). In FAKÀ/À charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cells, a-actinin fails to become tyrosine phosphorylated, indicating a We thank Dr. Martin McMahon for the kind gift of telomerase immortalized key role for FAK in this signaling pathway (26). ILK has a structural endothelial cells.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Minimal Active Domain and Mechanism of Action of the Angiogenesis Inhibitor Histidine-Rich Glycoprotein

Johan Dixelius, Anna-Karin Olsson, Åsa Thulin, et al.

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