Overexpression of Vascular Endothelial Growth Factor 189 in Breast Cancer Cells Leads to Delayed Tumor Uptake with Dilated Intratumoral Vessels
2007; Elsevier BV; Volume: 172; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2008.070181
ISSN1525-2191
AutoresMarie-Astrid Hervé, Hélène Buteau-Lozano, Roger Vassy, Ivan Bièche, Guillaume Velasco, Marika Pla, G. Perret, Samia Mourah, Martine Perrot‐Applanat,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoVascular endothelial growth factor (VEGF) is essential for breast cancer progression and is a relevant target in anti-angiogenesis. Although VEGF121 and VEGF165, the fully or partially secreted isoforms, respectively, have been the focus of intense studies, the role of the cell-associated VEGF189 isoform is not understood. To clarify the contribution of VEGF189 to human mammary carcinogenesis, we established several clones of MDA-MB-231 cells stably overexpressing VEGF189 (V189) and VEGF165 (V165). V189 and V165 clones increased tumor growth and angiogenesis in vivo. Remarkably, V165 induced the most rapid tumor uptake, whereas V189 increased vasodilation. In vitro overexpression of VEGF165 and VEGF189 increases the proliferation and chemokinesis of these cancer cells. Interestingly, overexpression of VEGF189 increased cell adhesion on fibronectin (1.9-fold) and vitronectin (1.6-fold), as compared to VEGF165, through α5β1 and αvβ5 integrins. Using the BIACore system we demonstrated for the first time that VEGF189 binds directly to neuropilin-1, which is strongly expressed in MDA-MB-231 cells. In contrast, VEGF-R2 was not significantly expressed and VEGF-R1 was expressed at low level. Our in vitro results suggest an autocrine effect of VEGF189 on breast cancer cells, probably through neuropilin-1. In conclusion, our data indicate that VEGF189 participates in mammary tumor growth through both angiogenesis and nonangiogenic functions. Whether VEGF189 overexpression is correlated to prognosis in human breast tumors remains to be established. Vascular endothelial growth factor (VEGF) is essential for breast cancer progression and is a relevant target in anti-angiogenesis. Although VEGF121 and VEGF165, the fully or partially secreted isoforms, respectively, have been the focus of intense studies, the role of the cell-associated VEGF189 isoform is not understood. To clarify the contribution of VEGF189 to human mammary carcinogenesis, we established several clones of MDA-MB-231 cells stably overexpressing VEGF189 (V189) and VEGF165 (V165). V189 and V165 clones increased tumor growth and angiogenesis in vivo. Remarkably, V165 induced the most rapid tumor uptake, whereas V189 increased vasodilation. In vitro overexpression of VEGF165 and VEGF189 increases the proliferation and chemokinesis of these cancer cells. Interestingly, overexpression of VEGF189 increased cell adhesion on fibronectin (1.9-fold) and vitronectin (1.6-fold), as compared to VEGF165, through α5β1 and αvβ5 integrins. Using the BIACore system we demonstrated for the first time that VEGF189 binds directly to neuropilin-1, which is strongly expressed in MDA-MB-231 cells. In contrast, VEGF-R2 was not significantly expressed and VEGF-R1 was expressed at low level. Our in vitro results suggest an autocrine effect of VEGF189 on breast cancer cells, probably through neuropilin-1. In conclusion, our data indicate that VEGF189 participates in mammary tumor growth through both angiogenesis and nonangiogenic functions. Whether VEGF189 overexpression is correlated to prognosis in human breast tumors remains to be established. 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In certain cancers, the expression of the VEGF165 or VEGF189 isoform has been associated with differences in tumor growth.24Guo P Xu L Pan S Brekken RA Yang ST Whitaker GB Nagane M Thorpe PE Rosenbaum JS Su Huang HJ Cavenee WK Cheng SY Vascular endothelial growth factor isoforms display distinct activities in promoting tumor angiogenesis at different anatomic sites.Cancer Res. 2001; 61: 8569-8577PubMed Google Scholar, 25Yu JL Rak JW Klement G Kerbel RS Vascular endothelial growth factor isoform expression as a determinant of blood vessel patterning in human melanoma xenografts.Cancer Res. 2002; 62: 1838-1846PubMed Google Scholar, 26Yuan A Yu CJ Kuo SH Chen WJ Lin FY Luh KT Yang PC Lee YC Vascular endothelial growth factor 189 mRNA isoform expression specifically correlates with tumor angiogenesis, patient survival, and postoperative relapse in non-small-cell lung cancer.J Clin Oncol. 2001; 19: 432-441Crossref PubMed Scopus (198) Google Scholar An increase of cell-associated VEGF189 expression has been observed in lung and colon cancers and in glioblastomas.16Cheung N Wong MP Yuen ST Leung SY Chung LP Tissue-specific expression pattern of vascular endothelial growth factor isoforms in the malignant transformation of lung and colon.Hum Pathol. 1998; 29: 910-914Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 24Guo P Xu L Pan S Brekken RA Yang ST Whitaker GB Nagane M Thorpe PE Rosenbaum JS Su Huang HJ Cavenee WK Cheng SY Vascular endothelial growth factor isoforms display distinct activities in promoting tumor angiogenesis at different anatomic sites.Cancer Res. 2001; 61: 8569-8577PubMed Google Scholar, 25Yu JL Rak JW Klement G Kerbel RS Vascular endothelial growth factor isoform expression as a determinant of blood vessel patterning in human melanoma xenografts.Cancer Res. 2002; 62: 1838-1846PubMed Google Scholar, 26Yuan A Yu CJ Kuo SH Chen WJ Lin FY Luh KT Yang PC Lee YC Vascular endothelial growth factor 189 mRNA isoform expression specifically correlates with tumor angiogenesis, patient survival, and postoperative relapse in non-small-cell lung cancer.J Clin Oncol. 2001; 19: 432-441Crossref PubMed Scopus (198) Google Scholar VEGF189 is related to poorer prognosis in lung cancer and osteosarcoma.26Yuan A Yu CJ Kuo SH Chen WJ Lin FY Luh KT Yang PC Lee YC Vascular endothelial growth factor 189 mRNA isoform expression specifically correlates with tumor angiogenesis, patient survival, and postoperative relapse in non-small-cell lung cancer.J Clin Oncol. 2001; 19: 432-441Crossref PubMed Scopus (198) Google Scholar, 27Lee YH Tokunaga T Oshika Y Suto R Yanagisawa K Tomisawa M Fukuda H Nakano H Abe S Tateishi A Kijima H Yamazaki H Tamaoki N Ueyama Y Nakamura M Cell-retained isoforms of vascular endothelial growth factor (VEGF) are correlated with poor prognosis in osteosarcoma.Eur J Cancer. 1999; 35: 1089-1093Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 28Nishi M Abe Y Tomii Y Tsukamoto H Kijima H Yamazaki H Ohnishi Y Iwasaki M Inoue H Ueyama Y Nakamura M Cell binding isoforms of vascular endothelial growth factor-A (VEGF189) contribute to blood flow-distant metastasis of pulmonary adenocarcinoma.Int J Oncol. 2005; 26: 1517-1524PubMed Google Scholar, 29Oshika Y Nakamura M Tokunaga T Ozeki Y Fukushima Y Hatanaka H Abe Y Yamazaki H Kijima H Tamaoki N Ueyama Y Expression of cell-associated isoform of vascular endothelial growth factor 189 and its prognostic relevance in non-small cell lung cancer.Int J Oncol. 1998; 12: 541-544PubMed Google Scholar Xenografts of VEGF189-overexpressing colon cancer cells grew more slowly than those of VEGF165-overexpressing cells.30Tomii Y Yamazaki H Sawa N Ohnishi Y Kamochi J Tokunaga T Osamura Y Sadahiro S Kijima H Abe Y Ueyama Y Tamaoki N Nakamura M Unique properties of 189 amino acid isoform of vascular endothelial growth factor in tumorigenesis.Int J Oncol. 2002; 21: 1251-1257PubMed Google Scholar VEGF188-expressing mouse fibrosarcoma, although hypervascularized, was not associated with tumor growth,31Grunstein J Masbad JJ Hickey R Giordano F Johnson RS Isoforms of vascular endothelial growth factor act in a coordinate fashion to recruit and expand tumor vasculature.Mol Cell Biol. 2000; 20: 7282-7291Crossref PubMed Scopus (214) Google Scholar and melanoma cells transfected with VEGF189 remained nontumorigenic and dormant.25Yu JL Rak JW Klement G Kerbel RS Vascular endothelial growth factor isoform expression as a determinant of blood vessel patterning in human melanoma xenografts.Cancer Res. 2002; 62: 1838-1846PubMed Google Scholar, 31Grunstein J Masbad JJ Hickey R Giordano F Johnson RS Isoforms of vascular endothelial growth factor act in a coordinate fashion to recruit and expand tumor vasculature.Mol Cell Biol. 2000; 20: 7282-7291Crossref PubMed Scopus (214) Google Scholar These results emphasize a complex role of VEGF189 in tumoral development. In this study, we evaluated the role of VEGF189 in the progression of human breast cell carcinoma. For this purpose, we generated stable human breast carcinoma cells (MDA-MB-231) overexpressing VEGF165 or VEGF189 isoforms. The effect of VEGF189 expression on angiogenic potential and tumor cell behavior were determined both in vitro and in vivo. Interestingly, we demonstrate that VEGF189 binds to NRP-1, a specific property demonstrated so far for VEGF165. For the first time, our findings show that VEGF189, as well as VEGF165, contributes to breast cancer progression and angiogenesis. Furthermore, we provide evidence that their effects are mediated through different paracrine and autocrine mechanisms. Human breast cancer cells, MDA-MB-231 (American Type Culture Collection, Molsheim, France), were routinely maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 100 μg/μl sodium pyruvate. The anti-α5β1, anti-αvβ3, anti-αvβ5 antibodies were purchased from AbCys (Paris, France). Human umbilical vein endothelial cells (HUVECs) were isolated with collagenase I.32Jaffe EA Nachman RL Becker CG Minick CR Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.J Clin Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (5968) Google Scholar Cells were cultured on 0.2% gelatin-coated dishes in Medium 199 (M199) supplemented with 20% FBS, 2 mmol/L glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 15 mmol/L of HEPES, and 0.4% endothelial cell growth supplement (Promocell, Heidelberg, Germany). Second passage cells and a mixture of three cords were used for all experiments. All cell culture reagents were from Life Technologies Inc. (Cergy Pontoise, France). The clones pUC-VEGF165 and pUC-VEGF189 were initially obtained from Dr. J. Abraham (Scios-Nova, Mountain View, CA). The cDNA fragment corresponding to VEGF165 or VEGF189 gene was subcloned into a bicistronic eukaryotic expression vector, pRCEN, containing the neomycin resistance gene as a selective marker, as previously described.7Plouët J Moro F Bertagnolli S Coldeboeuf N Mazarguil H Clamens S Bayard F Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic effect.J Biol Chem. 1997; 272: 13390-13396Crossref PubMed Scopus (212) Google Scholar Stable transfections of MDA-MB-231 cells were performed using the Fugene 6 reagent according to manufacturer's instructions (Roche Diagnostics, Meylan, France).33Buteau-Lozano H Ancelin M Lardeux B Milanini J Perrot-Applanat M Transcriptional regulation of vascular endothelial growth factor by estradiol and tamoxifen in breast cancer cells: a complex interplay between estrogen receptors alpha and beta.Cancer Res. 2002; 62: 4977-4984PubMed Google Scholar Parental cells were also transfected with pRCEN vector (control plasmid). The transfected cells were selected with G418 antibiotic (1 mg/ml, Life Technologies) in DMEM-10% FBS for 6 to 8 weeks. Stable transfected clones were isolated and maintained in DMEM-10% FBS in the presence of 500 μg/ml of G418. Seventy percent confluent MDA-MB-231 cells were cultured in DMEM-10% FBS. RNA was isolated using TRIzol reagent according to the manufacturer's instructions (Invitrogen, Cergy Pontoise, France). Reverse transcription of 1 μg of RNA was performed using 200 U of Superscript II RNase H reverse transcriptase with random hexamers (Invitrogen). Transcript quantification for VEGF was performed using TaqMan technology (LightCycler 2.0, Roche) and standard curve quantification method using the LightCycler software 3-1, according to described techniques. Briefly, cDNAs for VEGF189 and VEGF165 were prepared from total RNA, amplified by RT-PCR, and cloned using TOPO II TA cloning kit (Invitrogen). A standard curve for each transcript was generated using serial dilutions of cloned products ranging from 1 to 109 molecules/μl. The copy number of unknown samples was calculated by setting their PCR cycle number to the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of interest transcripts were normalized to the housekeeping β2-microglobulin gene transcripts. The following primers and probes (Eurogentec, Angers, France) were used: VEGF189-antisense, 5′-CCACAGGGAACGCTTAGGAC-3′; VEGF165-antisense, 5′-GCTTTCTCCGCTCTGAGCA-3′; VEGF sense 5′-AGCAAGACAAGAAAAAAAATCAGTTCGAGGAAA-3′. Transcript quantification of VEGF receptors, VEGF-R1, VEGF-R2, and NRP-1 was performed as previously described34Bieche I Tozlu S Girault I Onody P Driouch K Vidaud M Lidereau R Expression of PEA3/E1AF/ETV4, an Ets-related transcription factor, in breast tumors: positive links to MMP2, NRG1 and CGB expression.Carcinogenesis. 2004; 25: 405-411Crossref PubMed Scopus (50) Google Scholar using the TATA box-binding protein (TBP) as the endogenous RNA control, and each sample was normalized on the basis of its TBP content. Results are expressed as N-fold differences in target gene expression relative to TBP gene expression. Cells (5 × 105) were plated and grown in DMEM in Petri dishes until subconfluence was reached. Medium was changed, and cells were incubated in DMEM medium supplemented with heparin (50 μg/ml; Sigma, Steinheim, Germany) for 24 hours to release cell-associated VEGF. Conditioned media (CM) were collected and concentrated using Centriprep (Amicon, Bedford, MA). Total cell extracts were obtained after cell lysis. Protein concentration was determined using the BCA protein assay (Pierce, Rockford, IL). The concentration of cell culture medium was performed before Western blot and analysis of biological activity (endothelial cell proliferation assay). VEGF concentrations were measured in cell culture supernatant from heparin-treated cells using a standard enzyme-linked immunosorbent assay (ELISA) protocol (Quantikine human VEGF; R&D Systems, Minneapolis, MN). All data were normalized to cell protein content obtained after cell lysis. Results were expressed as ng VEGF/mg cell protein. Each sample was measured in duplicate. Protein samples (40 μg from CM) in Laemmli buffer were loaded onto a 12.5% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis under reducing conditions and then transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). VEGF was revealed after incubation with rabbit anti-human VEGF-A antibody (1:200; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) at room temperature for 1 hour, followed by a 1-hour incubation with horseradish peroxidase-conjugated mouse anti-rabbit antibody (1:10,000; DAKO, Glostrup, Denmark). Visualization was performed with ECL reagent (Amersham Biosciences, Orsay, France).19Ancelin M Buteau-Lozano H Meduri G Osborne-Pellegrin M Sordello S Plouet J Perrot-Applanat M A dynamic shift of VEGF isoforms with a transient and selective progesterone-induced expression of VEGF189 regulates angiogenesis and vascular permeability in human uterus.Proc Natl Acad Sci USA. 2002; 99: 6023-6028Crossref PubMed Scopus (108) Google Scholar HUVECs were seeded into gelatin-coated 96-well plates (5 × 103/well) and maintained in M199 containing 1% FBS for 24 hours. VEGF-induced proliferation was measured by adding CM from heparin-treated clones to HUVECs for 72 hours. Cell proliferation was measured using the XTT assay (Roche Diagnostics), as previously described.12Hervé MA Buteau-Lozano H Mourah S Calvo F Perrot-Applanat M VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA.Exp Cell Res. 2005; 309: 24-31Crossref PubMed Scopus (51) Google Scholar Results were normalized to data obtained with HUVECs incubated with CM from control-vector transfected cells. Recombinant human VEGF165 (R&D Systems) and VEGF1897Plouët J Moro F Bertagnolli S Coldeboeuf N Mazarguil H Clamens S Bayard F Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic effect.J Biol Chem. 1997; 272: 13390-13396Crossref PubMed Scopus (212) Google Scholar were used as controls. The experiments were performed three times in triplicate. Cells were plated (105 cells into 6-cm Petri dishes) in DMEM containing 10% FBS. Cell counts were performed every 24 hours in duplicate. The doubling time of the various clones was calculated using the formula Tc = 0.3T/logA/A0, where A is the cell number at time T of proliferation, and A0 is the cell number at an initial time point Tc.35Korah RM Sysounthone V Golowa Y Wieder R Basic fibroblast growth factor confers a less malignant phenotype in MDA-MB-231 human breast cancer cells.Cancer Res. 2000; 60: 733-740PubMed Google Scholar Each clone was tested in triplicate in three different experiments. Cells were plated into 96-well plates (5 × 103/well) in DMEM without serum at 37°C, 5% CO2. Metabolic activity was analyzed using the XTT assay for 72 hours. In some experiments, cells were incubated with anti-human VEGF-A neutralizing antibody at 10 μg/ml (R&D Systems). Ninety-six-well plates were coated for 2 hours at 37°C with vitronectin [10 μg/ml in phosphate-buffered saline (PBS)] or fibronectin (30 μg/ml in PBS) (Sigma-Aldrich). Negative controls consisted of 2% BSA-coated wells. Cell adhesion assay was performed as described.36Fernandis AZ Prasad A Band H Klosel R Ganju RK Regulation of CXCR4-mediated chemotaxis and chemoinvasion of breast cancer cells.Oncogene. 2004; 23: 157-167Crossref PubMed Scopus (240) Google Scholar Cells (30,000 cells/well) were allowed to adhere to the substrate for 1 hour at 37°C. Unattached cells were removed by gently washing with PBS and 1% BSA, then adherent cells were rinsed, fixed with paraformaldehyde (4%), and quantified using crystal violet staining (0.1%). The optical density was measured at 540 nm. To determine the role of the integrin in cell adhesion to fibronectin or vitronectin, the cells were pretreated with anti-α5β1 (MAB1969, 10 μg/ml), anti-αvβ3 (VMA1976, 10 μg/ml), anti-αvβ5 antibody (VMA1961, 10 μg/ml), or a control IgG for 30 minutes at 4°C before adhesion to fibronectin- or vitronectin-coated dishes. Confluent cell cultures were grown on 24-well plates in DMEM-10% FBS. Scratches were ma
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