ARTICLE

Role of Human Cripto-1 in Tumor Angiogenesis

Caterina Bianco, Luigi Strizzi, Andreas Ebert, Cindy Chang, Aasia Rehman, Nicola Normanno, Liliana Guedez, Rita Salloum, Erika Ginsburg, Youping Sun, Nadia Khan, Morihisa Hirota, Brenda Wallace-Jones, Christian Wechselberger, Barbara K. Vonderhaar, Giovanna Tosato, William G. Stetler-Stevenson, Michele Sanicola, David S. Salomon

Affiliations of authors: Tumor Growth Factor Section, Mammary Biology and Tumorigenesis Laboratory (CB, LS, CC, YS, NK, MH, BW-J), Molecular and Cellular Endocrinology Section, Mammary Biology and Tumorigenesis Laboratory (EG, BKV), Extracellular Matrix Section, Laboratory of Pathology (LG, RS, WGS-S), Experimental Transplantation and Immunology Branch (GT), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD; Department of Gynecology, Charite Campus Benjamin Franklin, Berlin, Germany (AE); Department of Molecular and Cellular Biology, University of Michigan, Ann Arbor, MI (AR); Division of Haematological Oncology and Department of Experimental Oncology, ITN-Fondazione Pascale, Naples, Italy (NN); Upper Austrian Research GmbH Zentrum, Linz, Austria (CW); Biogen-Idec Inc., Cambridge, MA (MS)

Correspondence to: David S. Salomon, PhD, Tumor Growth Factor Section, Mammary Biology and Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg. 10, Rm. 5B39, Bethesda, MD 20892 (e-mail: salomond{at}mail.nih.gov)


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Human cripto-1 (CR-1) promotes cell transformation and increases migration and invasion of various mouse and human epithelial cell lines. We investigated whether CR-1 also stimulates angiogenesis. Methods: We used human umbilical vein endothelial cells (HUVECs) to measure in vitro migration with fibronectin-coated Boyden chambers, invasion with Matrigel-coated Boyden chambers, proliferation with a tetrazolium salt, and differentiation with an in vitro Matrigel assay. We investigated new blood vessel formation in vivo by use of Matrigel-filled silicone cylinders implanted under the skin of nude mice and by use of a breast cancer xenograft model with CR-1-transfected or control Neo-transfected MCF-7 human breast cancer cells. We also used a blocking anti-CR-1 monoclonal antibody to investigate the role of CR-1 in angiogenesis in vivo and in vitro. All statistical tests were two-sided. Results: CR-1 stimulated HUVEC proliferation, migration, and invasion and induced HUVEC differentiation into vascular-like structures on Matrigel. In vivo, recombinant CR-1 protein induced microvessel formation in Matrigel-filled silicone cylinders, and microvessel formation was statistically significantly inhibited with a blocking anti-CR-1 monoclonal antibody (CR-1 and antibody = 127% of microvessel formation compared with that in untreated control cylinders and CR-1 alone = 259%; difference = 132%, 95% confidence interval [CI] = 123% to 140%; P<.001). Tumors formed by CR-1-transfected MCF-7 cells in the cleared mammary fat pad of nude mice had higher microvessel density than tumors formed by control Neo-transfected MCF-7 cells (CR-1-transfected cells = 4.66 vessels per field and Neo-transfected cells = 2.33 vessels per field; difference = 2.33 vessels per field, 95% CI = 1.2 to 2.8; P = .004). Conclusion: CR-1 appears to have an important role in the multistep process of angiogenesis.



    INTRODUCTION
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human cripto-1 (CR-1) is the founding member of the epidermal growth factor (EGF)-CFC (named for the founding members of the family: cripto in humans, FRL1 in Xenopus, and cryptic in mice) family of proteins (1). CR-1 is overexpressed in 50%–80% of different types of primary human carcinomas, such as breast, cervix, colon, stomach, pancreas, lung, ovary, and testis carcinomas (1). Proteins in the EGF-CFC family contain a modified EGF-like domain, a cysteine-rich CFC domain, and a short hydrophobic carboxyl terminus that is essential for membrane attachment by a glycosylphosphatidylinositol moiety (1). CR-1 can apparently function as an oncogene by inducing in vitro cell transformation, migration, invasion, epithelial-to-mesenchimal transition, and branching morphogenesis in mouse and human mammary epithelial cells and in human breast and cervical carcinoma cells (25). CR-1 overexpression can induce tumor formation, as shown by the development of mammary hyperplasias and papillary adenocarcinomas in transgenic mice that stably overexpress a human CR-1 transgene in the mammary gland (5).

Besides performing a crucial role in cellular transformation, members of the EGF-CFC family have been shown to be essential for early embryonic development (6). During embryogenesis, CR-1 functions as an essential coreceptor for nodal, a member of the transforming growth factor {beta}(TGF-{beta}) family (7). CR-1 apparently recruits nodal to the serine-threonine kinase activin type I (ALK4)/activin type II receptor complex by interacting with nodal through its EGF-like domain and with ALK4 through its CFC domain and allows nodal to induce Smad-2 phosphorylation and activation (8,9).

Despite the clear association between CR-1 overexpression and human tumors, the mechanism used by CR-1 to promote cellular transformation is not clear. CR-1 can activate two major signaling pathways—a nodal/ALK4/Smad-2 signaling pathway and a nodal- and ALK4-independent signaling pathway that leads to activation of ras/raf/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways (7,915). Activation of MAPK and PI3-K/AKT pathways requires CR-1 to bind a cell-surface heparan sulfate proteoglycan, glypican-1, and then to activate the cytoplasmic tyrosine kinase c-Src (13).

Several approaches have been developed to inhibit CR-1 expression in tumor cells, including the use of monoclonal antibodies and antisense technology. We have previously generated a panel of mouse anti-CR-1 monoclonal antibodies (mAbs) that bind to different functional epitopes in the EGF-like and CFC domains of CR-1 protein. Anti-CR-1 mAb A8.G3.5, directed against the CFC domain, blocks the binding of CR-1 to ALK4 and to activin B and inhibits up to 70% of the in vivo growth of human testicular and colon cancer xenografts in nude mice (16). CR-1 antisense oligonucleotides inhibit the in vitro and in vivo growth of human breast, colon, ovarian, and testicular carcinoma cells (17,18). In fact, treatment of nude mice bearing human GEO colon carcinoma xenografts with CR-1 antisense oligonucleotides reduces the microvessel density in GEO tumors, suggesting that CR-1 might participate in neovascularization during tumor formation (18).

We investigated whether CR-1 is involved in tumor angiogenesis by evaluating the effect of CR-1 at different stages of neovascularization of human vascular endothelial cells, including proliferation, migration, invasion, and differentiation into vascular-like structures. We also investigated whether the neutralizing anti-CR-1 mAb A8.G3.5 can block angiogenesis in vitro and in vivo.


    MATERIALS AND METHODS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell Culture, Growth Factors, Inhibitors, and Antibodies

Human umbilical vein endothelial cells (HUVECs, purchased from Clonetics Cambrex, Rockland, ME) were grown in the endothelial cell growth medium system EBM-2 (Clonetics Cambrex) supplemented with endothelial growth supplements (EGM-2 single quotes; Clonetics Cambrex) and 2% fetal bovine serum (Invitrogen, Carlsbad, CA). For all the experiments, HUVECs were used at passage 4 or less. Human MCF-7 breast cancer cells were cultured and transfected with a human CR-1 expression vector or with an empty control Neo vector, as previously described (10). A human glycosylphosphatidylinositol-truncated recombinant CR-1 protein containing an Fc tag was expressed in Chinese hamster ovary cells and purified, as previously described (19). Recombinant vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) proteins were purchased from R&D Systems (Minneapolis, MN). The MAPK inhibitor PD98059, the PI3-K inhibitor LY294002, the c-Src inhibitor PP2, and the VEGF receptor tyrosine kinase inhibitor (VEGFRI) 4',4'-chloro-2'-fluorophenylamino-6,7-dimethoxyquinazoline were purchased from Calbiochem (San Diego, CA). The ALK4 inhibitor SB-431542 was kindly provided by GlaxoSmithKline (20). The blocking anti-CR-1 mAb A8.G3.5, specific for the CFC domain, and the nonblocking anti-CR-1 mAb B3.F6.17, specific for the amino-terminal domain, were gifts from Biogen-Idec (16).

Reverse Transcription-Polymerase Chain Reaction (PCR) Analysis for ALK4 and Nodal Expression in HUVECs

Total RNA (2 µg) was prepared from HUVECs and reverse transcribed to cDNA with Superscript II (Invitrogen) and with random primers in a reaction volume of 20 µL; 2 µL of this reaction mixture was used for PCR amplification with Platinum PCR Supermix (Invitrogen). For nodal, PCR was performed for 30 cycles for 30 seconds at 94 °C, 45 seconds at 62 °C, and 45 seconds at 72 °C. For ALK4, PCR was performed for 30 cycles as follows: 1 minute at 94 °C, 1 minute at 55 °C, and 1 minute at 72 °C. Primers for amplification of human ALK4 and nodal have been previously described (10,16).

Cell Proliferation Assay

HUVECs were cultured at 3 x 104 cells per well on 96-well microtiter plates in EBM-2 medium containing 2% fetal bovine serum and endothelial growth supplements (Clonetics Cambrex). After 24 hours, the cells were washed twice with phosphate-buffered saline (PBS), and then EBM-2 medium (Clonetics Cambrex) without supplements and serum but containing recombinant CR-1 (1, 10, 50, or 100 ng/mL), VEGF (10 ng/mL), or bFGF (10 ng/mL) proteins was added. These concentrations of CR-1 and bFGF or VEGF have been previously shown to stimulate proliferation of epithelial or endothelial cells (915,21). To measure cell proliferation, we used a colorimetric assay that is based on the cleavage of the tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1; Roche, Indianapolis, IN) to formazan by mitochondrial dehydrogenase in viable cells. Cultures were incubated for 48 hours at 37 °C, WST-1 (10 µL per well, dilution 1:10) was added, and cultures were incubated for 4 hours at 37 °C. The reaction product was quantified by measuring the optical density of the formazan product at 450 nm. The experiment was repeated three times, and the samples were tested in triplicate.

Migration and Invasion Assays

Cell migration and invasion assays, respectively, were performed in fibronectin-coated Boyden chambers and Matrigel-coated Boyden chambers (QCM-FN Quantitative Cell Migration Assay and Cell Invasion Assay Kit, Chemicon, Temecula, CA). For both migration and invasion assays, Dulbecco’s modified Eagle medium (DMEM) containing 5% fetal bovine serum was used in the lower Boyden chamber as the chemoattractant. HUVECs were cultured in EBM-2 medium (Clonetics Cambrex) without serum and supplements for 24 hours, harvested by trypsinization, and resuspended in DMEM containing 5% bovine serum albumin at 4 x 105 cells per milliliter. We placed 0.5 mL of this cell suspension in the upper chamber with the components indicated at the following concentrations: recombinant VEGF at 10 ng/mL, recombinant CR-1 protein at 200 ng/mL, anti-CR-1 mAb A8.G3.5 at 25 µg/mL, anti-CR-1 mAb B3.F6.17 at 25 µg/mL, VEGFRI at 1 or 10 mM, the MAPK inhibitor PD98059 at 10 µM, the PI3-K inhibitor LY294002 at 10 µM, the c-Src inhibitor PP2 at 10 µM, and the ALK4 inhibitor SB-431542 at 10 µM. The Boyden chambers were incubated overnight at 37 °C. Cells on the top side of the filter were removed, and cells that had migrated and invaded the Matrigel through the filter and attached to the bottom of the membrane were stained with crystal violet stain solution (Chemicon). The crystal violet stain solution was eluted with 10% acetic acid extraction buffer (Chemicon) and transferred to wells of a 96-multiwell plate, and the absorbance was read at 595 nm in each well. Experiments were repeated three times with duplicate samples.

Protein Phosphorylation and Western Blot Analysis

HUVECs were cultured in 60-mm-diameter plates (1.5 x 106 cells per plate) and were serum starved in complete serum-free medium (Cellgro, Mediatech, Herndon, VA) for 24 hours. We have previously shown that CR-1-mediated phosphorylation of MAPK is detected within 5 minutes and that the CR-1-mediated phosphorylation of c-Src, AKT, and Smad-2 is detected within 15–30 minutes (9,13). Cells were stimulated with CR-1 protein at either 200 or 400 ng/mL for 5 minutes to assess MAPK phosphorylation or for 30 minutes to assess Smad-2, AKT, and c-Src phosphorylation. HUVECs were also pretreated with the inhibitors 10 µM PD98059 (for MAPK), 10 µM LY294002 (for PI3-K), 10 µM PP2 (for c-Src), or 10 µM SB-431542 (for ALK4) for 30 minutes at 37 °C and then stimulated with CR-1 at 400 ng/mL as a control for the specificity of the various inhibitors (13,20). Levels of phosphorylated and total MAPK, AKT, Smad-2, and c-Src were measured by western blot analysis as previously described (9). Densitometric analysis of the bands on the western blots was performed with the NIH Image program (http://rsb.info.nih.gov/nih-image/). Density of the bands was normalized to total protein expression of the nonphosphorylated forms and expressed as relative band intensity units (U).

In Vitro Capillary Tube Formation on Matrigel

HUVECs that had been cultured in EBM-2 medium containing serum and endothelial cell supplements were washed twice with PBS and trypsinized, and then 5 x 104 HUVECs per well were cultured in 96-well microtiter plates coated with Matrigel (In Vitro Angiogenesis Assay Kit, Chemicon) in the presence of the following components, as indicated: recombinant VEGF at 10 ng/mL, the anti-CR-1 mAb A8.G3.5 at 25 µg/mL, the anti-CR-1 mAb B3.F6.17 at 25 µg/mL, recombinant CR-1 protein at 200 ng/mL, VEGFRI at 1 or 10 mM, 10 µM PD98059, 10 µM LY294002, 10 µM PP2, and 10 µM SB-431542. After 18 hours, tube formation was assessed under an inverted light microscope, and cultures were photographed. The experiment was repeated three times, with duplicate samples.

VEGF and bFGF Immunoassays

To assess secreted VEGF and bFGF levels in cell culture medium, we used a quantitative sandwich enzyme immunoassay (human bFGF and human VEGF Quantikine, R&D Systems). In this assay, we compared Neo-transfected MCF-7 cells, which have low levels of endogenous CR-1 expression, with CR-1-transfected MCF-7 cells (10). Cell culture supernatant, from 80% confluent control Neo-transfected MCF-7 cells or CR-1-transfected MCF-7 cells cultured in DMEM containing 10% fetal bovine serum, was added to 96-well microtiter plates (200 µL per well) that were precoated with a monoclonal antibody specific for either VEGF or bFGF (R&D Systems) and incubated for 2 hours at room temperature. After several washings with PBS to remove any unbound protein, a horseradish peroxidase-conjugated polyclonal antibody against human VEGF or bFGF (R&D Systems) was added to the wells and the reaction mixture was incubated for 2 hours at room temperature. The reaction was developed by adding substrate solution containing hydrogen peroxide and the chromogen tetramethylbenzidine at 200 µL per well (R&D Systems). After color development, the reaction was stopped by adding 50 µL of 2 N sulfuric acid to each well. The intensity of the color in each well was read at 450 nm. VEGF and bFGF standard curves ranging from 15.6 to 1000 pg/mL were used to determine VEGF and bFGF concentrations in the culture medium.

Directed In Vivo Angiogenic Assay (DIVAA)

Surgical-grade silicone tubes (1 cm long, with an internal diameter of 0.15 cm; New Age Industries, Southampton, PA) were closed at one end with metal plugs. For each angioreactor implant, the lumen of each tube was filled with 18 µL of Matrigel (Collaborative Research, Becton Dickinson) containing the following components as indicated: recombinant VEGF at 50 ng/mL, recombinant CR-1 at 100 ng/mL, recombinant bFGF at 50 ng/mL, anti-CR-1 mAb A8.G3.5 at 25 µg/mL, and 1 x 104 Neo-transfected MCF-7 cells or 1 x 104 CR-1-transfected MCF-7 cells (22). The tubes were held at 37 °C to allow the Matrigel to solidify. Athymic nude mice (females, 6–8 weeks of age; National Cancer Institute, Frederick, MD) were anesthetized by intraperitoneal injection with 0.015 mL of 2.5% Avertin (Aldrich, St. Louis, MO; 0.02 mg/g of body weight). Animal care was in accordance with institutional guidelines, and all experiments were performed under an approved protocol by the National Institutes of Health. Two tubes were inserted into a skin pocket in the flank of each anesthetized nude mouse, and the pocket was sealed with surgical staples. After 9 days, the mice were injected intravenously with fluorescein isothiocyanate (FITC)-dextran, to quantify the vascular volume within the angioreactors (25 mg/mL; 100 µL per mouse; Sigma, St. Louis, MO). After 20 minutes, the tubes were removed from the skin pockets and photographed with an inverted light microscope. Matrigel was removed from angioreactors and digested in 200 µL of dispase solution (Collaborative Research). The FITC fluorescence that was trapped in the implant was measured with an HP Spectrophotometer (Perkin-Elmer, Foster City, CA) reflects the volume of blood circulating through the newly formed capillary vessels (i.e., the vascular volume). The experiment was repeated three times, with at least three mice per treatment condition.

MCF-7 Cell Xenograft Tumors in Nude Mice

The mammary fat pad of female nude mice (4–6 weeks of age) was cleared of epithelium by the method of DeOme (23). After the mice were anesthetized by intraperitoneal injection with 0.015 mL of 2.5% Avertin (Aldrich; 0.02 mg/g of body weight), we made a single incision laterally across the abdomen, revealing the fourth mammary gland. We removed the nipple-associated epithelial portion of the fourth mammary gland with an electrical cauterizing scalpel, leaving a mammary fat pad cleared of its epithelium. Neo-transfected or CR-1-transfected MCF-7 cells were grown until they were 80% confluent, trypsinized, and resuspended in DMEM to 5 x 106 cells per 100 µL; all 100 µL was injected into the cleared mammary fat pad (five animals per group). At the same time, the mice also were implanted subcutaneously in the intrascapular region with a 10-mg cholesterol-based pellet containing 0.72 mg of 17{beta}-estradiol (Innovative Research of America, Sarasota, FL) to allow the growth of estrogen-dependent MCF-7 cells (24). Tumor volumes were measured with a caliber twice a week. After 3 weeks, mice were killed and tumors were excised. Results shown are representative of two independent experiments with similar results.

Immunohistochemistry and Assessment of Vessel Density

To evaluate tumor vessel density, MCF-7 tumor xenografts were surgically removed from the mice after 3 weeks, fixed in PBS-buffered formalin, and embedded in paraffin. Five-micrometer-thick sections of the paraffin-embedded tissue were deparaffinized in xylene, rehydrated in a series of graded ethanols, and predigested with a ready-to-use pepsin solution (Digest-All3, Zymed, San Francisico, CA) for 10 minutes at 37 °C. Endogenous peroxidase activity was blocked by a 5-minute incubation in 3% H2O2. To identify intratumoral blood vessels, the sections were then incubated for 1 hour at room temperature with rabbit anti-CD31 polyclonal antibody (sc-8306; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100. This antibody is used to measure CD31, which is expressed on endothelial cells and, therefore, identifies blood vessels. In negative controls, the primary antibody was replaced with an irrelevant control isotype IgG. Immunostaining was carried out with the Vectastain ABC kit (Vector, Burlingame, CA) by the manufacturer’s instruction. Color was developed with AEC peroxidase substrate (Vector) by following the manufacturer’s instructions, and sections were counterstained with hematoxylin. Average tumor vessel counts were obtained as described (25). Briefly, areas with relatively high vascular density, commonly referred to as hot spots, were identified microscopically at a magnification of x100. CD31-positive vessels in these areas were counted in three separate fields at a magnification of x400. The mean value was calculated, and tissue vessel density was expressed as the number of vessels per field.

Statistical Analysis

Student’s t test was used to assess the statistical significance of the differences between various groups in the western blot analysis. For all other experiments, the statistical significance of differences between groups was evaluated by using the nonparametric Wilcoxon rank sum test. Results were expressed as the mean values and 95% confidence intervals (CIs). Statistical calculations were performed with the use of Statistical Package for Social Sciences software package, version 11.0 (SPSS, Chicago, IL). All statistical tests were two-sided, and data were considered statistically significant at P<.05.


    RESULTS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CR-1 and the Nodal/ALK4/Smad-2 and Glypican-1/c-Src/MAPK/AKT Signaling Pathways in HUVECs

HUVECs express the signaling pathway components ALK1, ALK5, glypican 1, c-Src, MAPK, and AKT (2629). Because CR-1 can signal through the activation of the glypican-1/c-Src/MAPK/AKT or nodal/ALK4/Smad-2 signaling pathway, we investigated whether HUVECs also express nodal and ALK4. By use of reverse transcription-PCR and primers specific for human nodal and ALK4, we detected the expression of both nodal and ALK4 in HUVECs (Fig.1, A). We next determined whether treatment of HUVECs with recombinant CR-1 enhances the activation (i.e., phosphorylation) of intracellular signaling molecules that function downstream of glypican-1 or nodal and ALK4. The level of MAPK phosphorylation was threefold higher in CR-1-treated HUVECs than in untreated HUVECs (Fig. 1, B). The level of AKT phosphorylation was twofold higher and the level of c-Src phosphorylation was threefold higher in CR-1-treated HUVECs than in untreated HUVECs (Fig. 1, C and D). In addition, CR-1 treatment activated a nodal/ALK4/Smad-2 pathway in HUVECs, as shown by a twofold increase in the level of Smad-2 phosphorylation (Fig. 1, E). Pretreatment of HUVECs with the specific inhibitors of MAPK (PD98059), AKT (LY294002), c-Src (PP2), or ALK4 (SB-431542) statistically significantly interfered with the CR-1-induced phosphorylation of the corresponding signaling component in HUVECs (e.g., for phosphorylated AKT, CR-1 at 400 ng/mL and LY294002 = 55 U versus CR-1 = 159 U; difference = 104 U, 95% CI = 93 to 114 U; P = .005; and, for phosphorylated c-Src, CR-1 at 400 ng/mL and PP2 = 65.5 U versus CR-1 = 169 U; difference = 103.5 U, 95% CI = 94 to 108 U; P = .003) (Fig. 1, B–E). Thus, all components of both nodal/ALK4/Smad-2 and glypican-1/c-Src/MAPK/AKT signaling pathways are expressed in HUVECs, appear to be activated by CR-1, and may contribute to the function of CR-1.



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Fig. 1. Expression of signaling pathway components in human umbilical vein endothelial cells (HUVECs). A) Reverse transcription–polymerase chain reaction using specific primers for ALK4 (435 base pairs [bp]) and nodal (251 bp) in HUVECs. In the lane to the left is shown HaeIII fragments of {phi}X174 replicative form DNA, shown as DNA markers. BE) Mitogen-activated protein kinase (MAPK), AKT, c-Src, and Smad-2 activation by cripto-1 (CR-1) in HUVECs. Serum-starved HUVECs were stimulated with recombinant CR-1 protein at 200 ng/mL or 400 ng/mL for 5 minutes for MAPK phosphorylation or for 30 minutes for AKT, c-Src, and Smad-2 phosphorylation, as indicated. Phosphorylation was analyzed by western blot analysis with antibodies against phosphorylated and nonphosphorylated forms of MAPK (B), AKT (C), c-Src (D), and Smad-2 (E). Cells were also pretreated with the MAPK inhibitor PD98059 at 10 µM (B), the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 at 10 µM (C), the c-Src inhibitor PP2 at 10 µM (D), or the ALK4 inhibitor SB-431542 at 10 µM (E) and then stimulated with recombinant CR-1 protein (400 ng/mL).

 
CR-1 and Proliferation of Human Endothelial Cells

Proliferation of endothelial cells (HUVECs) is an important step during early angiogenic events. We evaluated the ability of purified recombinant CR-1, VEGF, and bFGF proteins to stimulate HUVEC proliferation. All three proteins stimulated HUVEC proliferation under serum-free conditions (Fig. 2). VEGF treatment increased proliferation of HUVECs by 53% (95% CI = 42% to 63%; P = .004), and bFGF treatment increased proliferation of HUVECs by 218% (95% CI = 204% to 231%; P = .004), both compared with the proliferation of untreated HUVECs. CR-1 also induced a statistically significant dose-dependent increase in the proliferation of HUVECs. The activity of CR-1 at 10 ng/mL was comparable to that of VEGF (57% increase, 95% CI = 47% to 67%; P = .039), and the activity of CR-1 at 100 ng/mL was comparable to that of bFGF (212% increase, 95% CI = 200% to 223%; P = .004). Thus, CR-1 could induce the proliferation of HUVECs as well as the potent angiogenic molecules VEGF and bFGF.



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Fig. 2. Cripto-1 (CR-1) and the proliferation of human umbilical vein endothelial cells (HUVECs). Serum-starved HUVECs were stimulated with vascular endothelial growth factor (VEGF, 10 ng/mL), basic fibroblast growth factor (bFGF, 10 ng/mL), or CR-1 (1, 10, 50, or 100 ng/mL). After 48 hours, 10 µL per well of the reagent 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) was added, and the assay was quantified by measuring the optical density (OD) of the dye solution at 450 nm. Data are expressed as percentage of increase over control. Error bars = upper 95% confidence interval. *P = .004, compared with control. All statistical tests were two-sided.

 
CR-1 and HUVEC Migration and Invasion

To form new blood vessels in a tumor, endothelial cells must invade the tumor’s extracellular matrix and migrate through its basement membrane into the remodeled perivascular space. To determine whether CR-1 affects endothelial cell migration and invasion, HUVECs were incubated in Boyden chambers with recombinant CR-1 protein or recombinant VEGF protein alone or in combination for 18 hours. For the migration assay, we used membranes coated with the extracellular matrix protein fibronectin, which promotes cell adhesion and allows cell migration. For the invasion assay, we used membranes coated with Matrigel, which cells must degrade to reach the underside of the membrane. Treatment with CR-1 or VEGF individually increased the number of migrating and invading cells approximately twofold (for the migration assay, CR-1 = 265% versus control = 100%, difference = 165%, 95% CI = 158% to 172%, P = .031; for the invasion assay, CR-1 = 195% versus control = 100%, difference = 95%, 95% CI = 93% to 97%, P = .031) (Fig. 3). The combination of VEGF and CR-1 proteins did not enhance HUVEC migration or invasion further (Fig. 3). In addition, treatment of HUVECs with a specific VEGF receptor tyrosine kinase inhibitor (VEGFRI) induced a dose-dependent inhibition of VEGF-enhanced cell migration or invasion but did not affect the level of CR-1-induced migration or invasion, suggesting that CR-1 is not acting through the autocrine production of VEGF.



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Fig. 3. Cripto-1 (CR-1) and migration and invasion of human umbilical vein endothelial cells (HUVECs). HUVECs were seeded on fibronectin-coated filters for the migration assay (A) and Matrigel-coated filters for the invasion assay (B). Endothelial cells were incubated with vascular endothelial growth factor (VEGF, 10 ng/mL) alone or in combination with monoclonal antibody (mAb) A8.G3.5 (25 µg/mL) or with VEGF receptor tyrosine kinase inhibitor (VEGFRI, 1 or 10 µM), or CR-1 (200 ng/mL) alone or in combination with VEGF (10 ng/mL), the mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10 µM), phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (10 µM), c-Src inhibitor PP2 (10 µM), ALK4 inhibitor SB-431542 (10 µM), mAb A8.G3.5 (25 µg/mL), mAb B3.F6.17 (25 µg/mL), or VEGFRI (1 or 10 µM). After an overnight incubation, cells that migrated or invaded were stained with crystal violet stain solution (Chemicon). Stain was eluted with 10% acetic acid extraction buffer (Chemicon) and transferred to a 96-multiwell plate, and absorbance was read at 595 nm in each well. Error bars = upper 95% confidence interval. *, P = .031, compared with control; **, P = .031, compared with VEGF-treated cells; and ***, P = .031, compared with CR-1-treated cells. All statistical tests were two-sided.

 
To elucidate the type of signaling pathways involved in CR-1-induced HUVEC migration and invasion, we used specific inhibitors of signaling molecules and the neutralizing anti-CR-1 mAb A8.G3.5, which has been shown to inhibit tumor growth in vivo (16), and the control nonblocking anti-CR-1 mAb B3.F6.17 (also of the IgG1 isotype) (16). CR-1-induced enhanced migration and invasion of HUVECs was statistically significantly inhibited by adding the blocking mAb A8.G3.5 with CR-1 (55% and 42% inhibition in migration and invasion assays, respectively, P = .031 for both sets of results), the c-Src inhibitor PP2 (approximately 50% inhibition in both assays), or the PI3-K inhibitor LY2940002 (83% and 48% inhibition in migration and invasion assays, respectively) (Fig. 3). In contrast, no statistically significant decrease in the endothelial cell migration and invasion in response to CR-1 was observed in the presence of the MAPK inhibitor PD98059 or the control nonblocking anti-CR-1 mAb B3.F6.17. The ALK4 inhibitor SB-431542 induced a nonstatistically significant reduction in endothelial cell migration, whereas it had no effect on CR-1-induced endothelial cell invasion (Fig. 3). Finally, the blocking anti-CR-1 mAb A8.G3.5 had no effect on VEGF-enhanced migration and invasion in HUVECs. Thus, c-Src and PI3-K/AKT appear to be directly involved in mediating CR-1 chemotactic function in HUVECs, and the neutralizing anti-CR-1 mAb A8.G3.5, which recognizes an epitope in the CFC domain of CR-1, appears to block the in vitro proangiogenic function of CR-1 in HUVECs by inhibiting CR-1-induced endothelial cell migration and invasion.

CR-1 and In Vitro Morphologic Differentiation of HUVECs

To explore the ability of CR-1 to induce angiogenesis, we evaluated the formation of vascular tube-like structures by CR-1-treated cells in an in vitro Matrigel assay. In the absence of growth factors, HUVECs did not form tube-like structures and remained round and isolated on the Matrigel bed (Fig. 4). Addition of recombinant CR-1 protein induced formation of distinct rings and cords of cells (Fig. 4) that were visible by 18 hours. VEGF induced similar structures. Coadministration of CR-1 and VEGF did not further increase the formation of vascular structures over that observed with CR-1 or VEGF alone. VEGFRI strongly blocked the formation of VEGF-induced vascular structures by HUVECs but did not affect CR-1-induced capillary-like tube formation. The combination of CR-1 and anti-CR-1 mAb A8.G3.5, PP2, or LY294002 markedly decreased formation of vascular structures, but the combination of CR-1 and MAPK inhibitor PD98059, the ALK4 inhibitor SB-431542, or the nonblocking mAb B3.F6.17 did not affect the CR-1-induced formation of vascular structures. Finally, the blocking anti-CR-1 mAb A8.G3.5 did not interfere with VEGF-induced formation of vascular structures. Thus, CR-1 appears to stimulate differentiation of endothelial cells through a c-Src/PI3-K/AKT signaling pathway.



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Fig. 4. Cripto-1 (CR-1) and human umbilical vein endothelial cell (HUVEC) differentiation into vascular-like structures. HUVECs were cultured in Matrigel-precoated wells in absence (control) or in presence of vascular endothelial growth factor (VEGF), VEGF plus monoclonal antibody (mAb) A8.G3.5 (25 µg/mL), VEGF plus VEGF receptor tyrosine kinase inhibitor (VEGFRI, 1 or 10 µM), CR-1 plus VEGF, CR-1, CR-1 plus mAb A8.G3.5 (25 µg/mL), CR-1 plus mAb B3.F6.17 (25 µg/mL), CR-1 plus PP2 (10 µM), CR-1 plus LY294002 (10 µM), CR-1 plus PD98059 (10 µM), CR-1 plus SB-431542 (10 µM), or CR-1 plus VEGFRI (1 or 10 µM). VEGF and CR-1 proteins were used at 10 ng/mL or 200 ng/mL, respectively.

 
CR-1 and Neovascularization In Vivo

We had shown that CR-1 stimulates endothelial cell proliferation, migration, invasion, and differentiation in vitro. However, agents that affect angiogenesis in vitro do not always affect angiogenesis the same way in vivo. Therefore, we used a recently described quantitative in vivo angiogenesis assay—the directed in vivo angiogenesis assay—to determine the angiogenic potential of CR-1-overexpressing MCF-7 human breast cancer cells or of purified recombinant CR-1 protein (22). We have previously shown (10) that CR-1 overexpression in MCF-7 cells is associated with enhanced levels of invasion and a more aggressive phenotype in vitro. VEGF and bFGF are secreted in the supernatant of Neo-transfected MCF-7 cells and CR-1-transfected MCF-7 cells, but expression of CR-1 did not appear to increase the levels of VEGF and bFGF, as determined by immunoassay (data not shown). Neo-transfected MCF-7 cells or CR-1-transfected MCF-7 cells and recombinant CR-1, bFGF, or VEGF proteins were embedded in Matrigel inside semiclosed silicone cylinders (i.e., angioreactors) and implanted subcutaneously into nude mice. We also combined CR-1 and VEGF proteins to evaluate whether they cooperate to enhance new vessel formation in vivo. After 9 days, the implants were removed, and the degree of vascularization within these angio-reactors was quantified after the intravenous injection of FITC-dextran into the nude mice as an indirect measure of the volume of blood circulating through the newly formed vessels within the silicone cylinders (22).

Implants containing either CR-1 protein or CR-1-overexpressing MCF-7 cells contained visible capillaries within the angioreactors, whereas the cells transfected with the empty vector had very few new vessels (Fig. 5, A–C). Implants containing either purified recombinant CR-1 protein or CR-1-overexpressing MCF-7 cells had a statistically significantly greater volume of blood in newly formed vessels than implants containing Matrigel alone or control Neo-transfected MCF-7 cells (recombinant CR-1 protein alone = 259% versus control = 100%, difference = 159%, 95% CI = 147% to 171%, P<.001; and CR-1-transfected MCF-7 cells = 243% versus Neo-transfected MCF-7 cells = 129%, difference = 114%, 95% CI = 109% to 118%, P<.001) (Fig. 5, D). Combined treatment with VEGF and CR-1 resulted only in a slight increase of new vessel formation in vivo, compared with CR-1 or VEGF alone. As expected, a statistically significant angiogenic response was also detected in angioreactors containing VEGF or bFGF (P<.001); these responses were similar to but less than that induced by CR-1. Finally, we investigated the ability of anti-CR-1 mAb A8.G3.5 to inhibit CR-1-induced angiogenesis in vivo. Addition of anti-CR-1 mAb A8.G3.5 to CR-1 statistically significantly inhibited new vessel formation in angioreactors (CR-1 and A8.G3.5 = 127% versus CR-1 alone = 259%, difference = 132%, 95% CI = 123% to 140%, P<.001), suggesting that this neutralizing anti-CR-1 antibody has substantial antiangiogenic activity in vivo (Fig. 5, D).



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Fig. 5. Cripto-1 (CR-1) and angiogenesis. Angiogenesis was assessed in vivo by using the directed in vivo giogenesis assay. Paired angioreactors recovered at 9 days after implantation with Neo-transfected MCF-7 cells (A), CR-1-transfected MCF-7 cells (B), or recombinant CR-1 protein (100 ng/mL) (C). In the figure, angioreactors are oriented with the open end to the left and with the sealed end to the right. Arrows = vascular structures within the lumen. D) Quantification of directed in vivo angiogenesis assay by fluorescein isothiocyanate (FITC)–dextran injection. Results are expressed as relative fluorescence units (percentage of control) of FITC-dextran. Recombinant proteins and monoclonal antibody (mAb) were used at the following concentration: basic fibroblast growth factor (bFGF, 50 ng/mL), vascular endothelial growth factor (VEGF, 50 ng/mL), CR-1 (100 ng/mL) alone or in combination with mAb A8.G3.5 (25 µg/mL) or VEGF (50 ng/mL). MCF-7 Neo = Neo-transfected MCF-7 cells; MCF-7 CR-1 = CR-1-transfected MCF-7 cells. Error bars = upper 95% confidence interval. *, P<.001, compared with control; **, P<.001, compared with CR-1-containing angioreactors; ***, P<.001, compared with no-transfected MCF-7 cells. All statistical tests were two-sided.

 
CR-1 and New Vessel Formation in CR-1-Expressing MCF-7 Xenografts

We next determined whether the CR-1 expressed by CR-1-transfected MCF-7 cells would enhance tumorigenesis of these cells in nude mice and/or neovascularization of the tumors. 17{beta}-Estradiol-dependent Neo-transfected MCF-7 cells or CR-1-transfected MCF-7 cells were injected into the cleared mammary fat pad of nude mice implanted subcutaneously with 17{beta}-estradiol pellets (five mice per group). If mice were not implanted subcutaneously with 17{beta}-estradiol pellets, MCF-7 cells did not grow (10). After 10 days, mice that received CR-1-transfected MCF-7 cells started to develop tumors, whereas no tumors were detected in the control group, which received Neo-transfected MCF-7 cells (CR-1-transfected MCF-7 tumors = 0.11 mm2 [mean] versus Neo-transfected MCF-7 tumors = 0 mm2 [mean]; difference = 0.11 mm2, 95% CI = 0.07 to 0.14 mm2; P = .032; Fig. 6, A). After 20 days, however, all mice in both groups had tumors, with no statistically significant difference in tumor volume between them (Fig. 6, A). Finally, the endothelial cell marker CD31 identified a statistically significantly higher microvessel density in sections of CR-1-transfected MCF-7 tumors than Neo-transfected MCF-7 tumors (CR-1-transfected MCF-7 tumors = 4.66 vessels per field versus Neo-transfected MCF-7 = 2.33 vessels per field; difference = 2.33 vessels per field, 95% CI = 1.2 to 2.8 vessels per field; P = .004; Fig. 6, B and C).



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Fig. 6. Overexpression of cripto-1 (CR-1) in MCF-7 cells and microvessel density. A) Neo-transfected MCF-7 cells (MCF-7 Neo) or CR-1-transfected MCF-7 cells (MCF-7 CR-1) were inoculated in the cleared fat pad of nude mice implanted with 17{beta}-estradiol pellets (five mice per group). Tumor size at 10 and 20 days after injection of the cells was determined with a caliper. B) Immunohistochemistry for the endothelial cell marker CD31 in CR-1-transfected MCF-7 cell tumor sections. Brown color and arrowheads indicate endothelial cells and representative blood vessels, respectively. C) Microvessel quantification. Tissue vessel density was expressed as the number of vessels per high-power magnification field. Error bars = upper 95% confidence interval. *P = .039. All statistical tests were two-sided.

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We show, to our knowledge for the first time, that CR-1 has a strong angiogenic activity in cultured endothelial cells (i.e., HUVECs). CR-1 enhanced the proliferation of HUVECs in a dose-dependent manner, suggesting that it can function as a mitogenic factor for endothelial cells, as do VEGF and bFGF (30). CR-1 also induced migration and invasion of HUVECs, as does VEGF. Moreover, CR-1-treated HUVECs aligned to form vascular-like structures on Matrigel, indicating that CR-1 can stimulate differentiation in cultured endothelial cells. Because the specific inhibitor VEGFRI did not block CR-1-induced migration, invasion, and capillary-tube formation in HUVECs, CR-1 does not appear to function through the autocrine production of VEGF.

We also demonstrated that CR-1 can stimulate endothelial cells by activation of a c-Src/MAPK/PI3-K/AKT or a nodal/ALK4/Smad-2 signaling pathway. Specific inhibitors of c-Src or PI3-K strongly inhibited CR-1-induced migration, invasion, and differentiation of HUVECs. We have previously shown that activation of c-Src and PI3-K/AKT signaling pathways in mammary epithelial cells or in human cervical and breast cancer cell lines is required for CR-1-dependent cell migration, invasion, and transformation (2,4,10,12,13). The PI3-K/AKT signaling pathway has been identified as a major regulator of VEGF-induced endothelial cell motility (27,28). However, the inability of a specific MAPK inhibitor to block CR-1-induced HUVEC migration, invasion, and morphologic differentiation in our study demonstrates that these CR-1-induced responses do not require activation of a MAPK signaling cascade. Similarly, the MAPK inhibitor PD98059 had no effect on VEGF-induced endothelial cell migration (27).

The inhibition of CR-1-induced migration, invasion, and capillary-like tube formation induced by treatment with a neutralizing anti-CR-1 mAb, but not by the nonblocking anti-CR-1 mAb B3.F6.17, strongly indicates that CR-1 directly modulates angiogenesis in HUVECs. The mAb A8.G3.5 binds to an epitope in the CFC domain of CR-1, thereby preventing CR-1 binding to ALK4 and perturbing nodal signaling (16). Surprisingly, blockade of ALK4/Smad-2 signaling pathway with the ALK4 inhibitor SB-431542 had a marginal effect on the CR-1-induced proangiogenic phenotype in HUVECs in vitro. However, the CFC domain may also bind to other proteins that act through an ALK4-independent signaling pathway. For example, CR-1 can bind to glypican-1, Vg1/GDF1, Lefty, activin, and tomoregulin 1 (13,16,3134). Therefore, mAb A8.G3.5 may interfere with the binding of CR-1 to one or more of these signaling molecules in addition to blocking a nodal/ALK4 signaling pathway. Alternatively, CR-1 may function as an activin antagonist (16,34). Because activin has been recently shown to inhibit proliferation and differentiation of vascular endothelial cells in vitro, mAb A8.G3.5 might disrupt the binding of CR-1 to activin B and thereby restore activin B growth suppression in endothelial cells (35). In agreement with our results, blocking the function of activin with SB-431542 does not inhibit proliferation and/or differentiation of endothelial cells (35). Interestingly, treatment of HUVECs with the combination of CR-1 and SB-431542 actually produced a higher level of differentiation than treatment with CR-1 alone (Fig. 4), suggesting that CR-1 and SB-431542 cooperate in blocking activin signaling in endothelial cells.

The strong CR-1-induced angiogenic effects in endothelial cells observed in vitro were then extended by in vivo experiments. CR-1, either as a purified recombinant protein or when overexpressed in MCF-7 breast cancer cells, stimulated various endothelial cell responses that are associated with microvessel formation in vivo. Anti-CR-1 mAb A8.G3.5 inhibited 50% of the CR-1-induced neovessel formation, suggesting that this mAb has potent antiangiogenic activity and that the strong antitumor activity exerted by mAb A8.G3.5 in testicular and colon cancer xenografts in nude mice may be mediated by its direct anti-angiogenic activity and by its inhibition of tumor cell proliferation (16).

Interestingly, coadministration of CR-1 and VEGF did not produce a greater effect than that observed with administration of CR-1 or VEGF alone, suggesting that these two angiogenic molecules may act through the same signaling pathways. In this regard, PI3-K and AKT have been strongly implicated in mediating different VEGF functions and, therefore, may be common effector molecules in the process of angiogenesis induced by VEGF and/or CR-1 (28).

In accord with the involvement of CR-1 in regulating new blood vessel formation, overexpression of CR-1 in MCF-7 breast cancer cells enhanced tumor neovascularization in a xenograft model. Because CR-1 overexpression in MCF-7 cells did not increase the secretion of VEGF or bFGF, CR-1 may not be acting indirectly in MCF-7 cells by inducing VEGF and/or bFGF expression. Although CR-1-transfected MCF-7 cells produced tumors more quickly than control Neo-transfected MCF-7 cells, after 20 days the size of both types of MCF-7 mammary tumor xenografts were comparable. We have previously shown (10) that CR-1 overexpression fails to induce an estrogen-independent phenotype in estrogen-responsive MCF-7 cells. However, in serum-free conditions, CR-1 overexpression in MCF-7 cells could enhance proliferation, survival, and invasion (10). Therefore, in our xenograft model, estrogen supplementation, which is normally required for the proper growth of estrogen-dependent MCF-7 cells in vivo, may account for some of the reduction in CR-1-induced tumor growth observed.

In conclusion, we have demonstrated a critical role for CR-1 signaling in angiogenesis and have shown that a blocking anti-CR-1 mAb inhibits microvessel formation in vivo. We believe that further experiments are warranted to determine whether the blocking anti-CR-1 mAb is a good therapeutic candidate to inhibit angiogenesis.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported in part by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC) to Nicola Normanno.


    REFERENCES
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received April 30, 2004; revised October 25, 2004; accepted November 22, 2004.



             
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