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[Cancer Research 61, 4229-4237, May 15, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Enhanced Invasion and Tumor Growth of Fibroblast Growth Factor 8b-overexpressing MCF-7 Human Breast Cancer Cells1

Johanna K. Ruohola, Tiina P. Viitanen, Eeva M. Valve, Jani A. Seppänen, Niina T. Loponen, Jaakko J. Keskitalo, Päivi T. Lakkakorpi and Pirkko L. Härkönen2

Department of Anatomy and MediCity Research Laboratory, Institute of Biomedicine, University of Turku, Turku, Finland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fibroblast growth factor 8 (FGF-8) is a secreted heparin-binding protein, which has mitogenic and transforming activity. Increased expression of FGF-8 has been found in human breast cancer, and it has a potential autocrine role in its progression. Human FGF-8 is alternatively spliced to generate four protein isoforms (a, b, e, and f). Isoform b has been shown to be the most transforming. In this work, we studied the role of FGF-8b in the growth (in vitro and in vivo) of MCF-7 human breast cancer cells, which proliferate in an estrogen-dependent manner. Constitutive overexpression of FGF-8b in MCF-7 cells down-regulated FGF-8b-binding receptors FGF receptor (FGFR) 1IIIc, FGFR2IIIc, and FGFR4 found to be expressed in these cells. FGF-8b overexpression led to an increase in the anchorage-independent proliferation rate in suspension culture and colony formation in soft agar, when MCF-7 cells were cultured with or without estradiol. FGF-8b also provided an additional growth advantage for cells stimulated with estradiol. In addition, FGF-8b-transfected cells invaded more actively through Matrigel than did control cells. This was possibly due to the increased secretion of matrix metalloproteinase 9. In vivo, FGF-8b-transfected MCF-7 cells formed faster growing tumors than vector-only-transfected cells when xenografted into nude mice. The tumors formed by FGF-8b-transfected cells were more vascular than the tumors formed by vector-only-transfected cells. In conclusion, FGF-8b expression confers a growth advantage to MCF-7 breast carcinoma cells, both in vitro and in vivo. In addition to stimulation of proliferation, this growth advantage probably arises from increased invasion and tumor vascularization induced by FGF-8b. The results suggest that FGF-8b signaling may be an important factor in the regulation of tumorigenesis and progression of human breast cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The FGFs3 form a family of at least 23 growth-regulatory proteins. They share 35–50% amino acid sequence identity and induce proliferation and differentiation in a wide range of cells of epithelial, mesodermal, and neuroectodermal origin (1 , 2) . FGF-8 was originally isolated from the conditioned medium of an androgen-dependent mouse mammary tumor cell line (SC-3) as an androgen-induced growth factor and was later classified as a member of the FGF family on the basis of structural similarity (3) . FGF-8 has been found to have an important role in embryogenesis and morphogenesis (4) . It is expressed during gastrulation, in brain development, and in the process of limb and facial morphogenesis of the developing mouse (5, 6, 7, 8, 9, 10) . It may thus play a critical role in the development of the face, limbs, and brain. In adult mice, FGF-8 expression has been detected only in the ovaries and testes (11, 12, 13) . FGF-8 has been identified as an oncogene product on the basis of its overexpression in NIH3T3 cells (14) . In addition, the generation of FGF-8-transgenic mice, using the mouse mammary tumor virus long terminal repeat promoter, produced mammary lobular adenocarcinomas, salivary gland tumors, and ovarian stromal hyperplasia (15) .

The structure of the FGF-8 gene is more complicated than that of the other members of the FGF family. Alternative splicing of the FGF-8 gene potentially gives rise to eight different protein isoforms (a–h) in the mouse and four (a, b, e, and f) in humans (6 , 16 , 17) . The isoforms differ in the NH2 terminus. The biological function of these forms is not exactly known, but at least they differ in transforming potential. FGF-8b has been found to have the highest NIH3T3 cell-transforming capacity (18 , 19) . FGF-8b has also been shown to be the first member of the FGF family having an increased expression level in breast cancer and a potential autocrine role in its progression (20) . We have recently found that besides FGF-8b, FGF-8 isoforms a and e may also be involved in the development and progression of human prostate cancer (21) .

FGF signaling is transduced through the formation of a complex of a growth factor, a proteoglycan, and a high-affinity FGFR, which is a transmembrane tyrosine kinase receptor (22) . Four different high-affinity receptors (FGFR 1, FGFR 2, FGFR 3, and FGFR 4) bind FGF ligands and display varying patterns of expression (reviewed in Refs. 1 and 22 ). Alternative mRNA splicing generates isoforms of receptors 1–3 that exhibit unique ligand-binding properties (1 , 23) . FGF-8 preferentially activates FGFR2IIIc and FGFR3IIIc splice forms and FGFR 4 (17 , 24) , although there are differences between the activation potential of various FGF-8 isoforms. FGF-8b also activates FGFR1IIIc, but only at a very high concentration (17) .

FGF-8 protein has been shown to be expressed in both ductal and epithelial cells of normal breast tissue and also in various breast diseases, such as fibroadenoma, intraductal papilloma, ductal hyperplasia, and breast cancer (25) . In one study, analysis of FGF-8 mRNA showed that it was present at a significantly higher level in breast cancer than in nonmalignant breast tissues (20) . FGF-8 mRNA has also been found in various human breast cancer cell lines (26, 27, 28) . Previous studies on the effects of FGFs in MCF-7 breast cancer cells have produced conflicting results. Transfection of FGF-1 or FGF-4 conferred a hormone-independent, antiestrogen-resistant, and metastatic growth phenotype to MCF-7 breast cancer cells in vivo (29 , 30) , whereas the results of other studies have indicated a growth-inhibitory role of exogenous FGF-4 on estrogen-dependent MCF-7 cells (27) . These conflicting data suggest that FGFs are pleiotropic biological activators that are capable of inducing mutually exclusive cellular functions (i.e., growth inhibition and growth stimulation) under different conditions.

The aim of the present study was to examine the effect of FGF-8 on the in vitro and in vivo growth of human breast cancer. Estrogen-dependent MCF-7 breast cancer cells were used as a model, since FGF-8 is endogenously expressed in MCF-7 cells only at a low level (20 , 27) . Since FGF-8 isoform b has been shown to have the most transforming potential among the different isoforms, we stably transfected a plasmid containing the coding sequence for FGF-8b into human MCF-7 breast carcinoma cells. These cells normally produce slow-growing, nonmetastatic, and hormone-dependent tumors when xenografted s.c. into nude mice. Stable transfectants were investigated for the expression of FGF-8-binding receptors, growth and invasion in vitro, and angiogenic and tumorigenic potential in vivo.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The human breast cancer cell line MCF-7 dependent on estrogen for growth was a kind gift from Dr. C. K. Osborne (University of Texas Health Science Center, San Antonio, TX) (31) . The cells were maintained in RPMI 1640 culture medium supplemented with 10% heat-inactivated FBS, 1 nM 17-ß-estradiol (E2), 2 mM L-glutamine, and 4 µg insulin/ml. RPMI 1640, E2 and insulin were purchased from Sigma (St. Louis, MO). FBS was purchased from Life Technologies, Inc. (Paisley, Scotland, United Kingdom) and L-glutamine from Fluka (Riedel de-Haën, Germany). Tam was obtained from the Orion Corporation, Medipolar (Oulu, Finland). E2 was dissolved in 94% ethanol and Tam in 70% ethanol and they were added to the culture medium as indicated in "Results."

Proliferation Assays with Recombinant FGF-8b.
To determine the effect of FGF-8b on the proliferation of MCF-7 cells, [3H]thymidine incorporation was used. MCF-7 cells were plated on 96-well plates at a density of 5000/well in phenol red-free RPMI 1640 with 5% DC-FBS. Next day, the medium was changed and 1 nM E2, FGF-8b (recombinant murine FGF-8b; R&D Systems, Minneapolis, MN), and FGF-2 (recombinant human FGF-2; Sigma) were added. After a 48-h stimulation, the cells were pulse labeled with [3H]thymidine (0.2 µCi/well; Amersham Life Science, Buckinghamshire, England) for 2 h. In the experiment using a reduced serum concentration, the cells were made quiescent by reducing the serum level to 1% for 18 h before E2 or growth factors were added and stimulated for 24 h. The radioactivity incorporated into the cells was determined by trypsinizing the cells and harvesting them on glass fiber filters (Printed Filtermat A; Wallac Oy, Turku, Finland). The radioactivity was measured in a MicroBeta counter (Wallac Oy). For determination of the cell population doubling times, 2 x 104 MCF-7 cells were plated on 6-well plates and cultured with E2 or growth factors for 5 days. Cell numbers were counted with a Coulter Counter (Coulter, Harpenden, United Kingdom) in isotone solution (Coulter) after washing the cells with PBS and lysing them in 0.01 M HEPES and 1.5 mM MgCl2 containing Zap-Oglobin (Coulter) as a detergent. Population doubling times were calculated using the formula: , where N2 is the number of cells at time t2 and N1 is the number of cells at time t1.

Transfection.
The expression vector pcDNA3 (Invitrogen, Groningen, The Netherlands), containing human FGF-8 isoform b cDNA in an EcoRI site under the control of cytomegalovirus promoter, was a kind gift from Dr. P. Roy-Burman (University of Southern California School of Medicine, Los Angeles, CA) (19) . As a control, we used an empty pcDNA3 vector (mock). Plasmids (5 µg) were transfected into MCF-7 cells by using SuperFect (Qiagen, Germany) according to the manufacturer’s instructions. Forty-eight hours after transfection, the cells were transferred to selection medium containing 500 µg G418/ml (Calbiochem, Germany) for 14 days. Surviving colonies from four to five dishes were pooled together and characterized. In addition, 10 different cell clones were subcloned and compared with pooled population cell lines.

RNA Isolation and Northern Blot Analysis.
Total RNA was extracted using the guanidinium isothiocyanate method (32) . Samples of total RNA (20 µg) were separated by electrophoresis, stained with ethidium bromide, and blotted on GeneScreen Plus nylon membrane (NEN Research Products, Boston, MA) using standard conditions. The PCR clone for FGF-8 (33) and glyceraldehyde-3-phosphate dehydrogenase cDNA (33) were [32P]dCTP labeled by the random priming method (Ready-To-Go DNA Labeling Beads; Amersham Pharmacia Biotech, Piscataway, NJ). Hybridization was carried out using standard conditions and exposed to X-ray film. Intensities of hybridization signals were quantified by densitometry using an MCID M4 Image Analyzer (Imaging Research, St. Catherines, Ontario, Canada).

Western Blot Analysis.
Serum-free RPMI 1640 medium with 1 nM E2 was conditioned by FGF-8b- or vector-only (mock)-transfected MCF-7 cells for 24 h. Cells from conditioned dishes were lysed with lysis buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, and 2 mM phenylmethylsulfonyl fluoride. The amount of proteins was measured by using a micro-bicinchoninic acid protein assay (Pierce, Rockford, IL). Heparin-binding proteins were isolated from the conditioned medium as previously described (35) . Proteins from cell lysate (20 µg) and corresponding amounts of heparin-bound proteins from conditioned medium were separated by SDS-PAGE electrophoresis. FGF-8 was detected by using a 0.2-µg/ml concentration of goat anti-FGF-8b antibody (R&D Systems) and a secondary antibody, horseradish peroxidase-labeled antigoat IgG, at a 1:2000 dilution (Dako A/S, Copenhagen, Denmark). Protein bands were visualized by using an enhanced chemiluminescence detection system (Amersham Life Science). As a negative control, normal goat IgG (R&D Systems) was used instead of anti-FGF-8b, at the same concentration.

RT-PCR and Southern Blotting for FGFRs.
RT-PCR for FGFR1IIIc, FGFR3IIIc, and FGFR4 was performed according to Ittman and Mansukhani (36) . To verify the negative result of FGFR3IIIc expression, RT-PCR was also performed with another set of primers, a 5' primer: 5'-ACCCTACGTTACCGTGCTCAA-3' and a 3' primer: 5'-CCGCCAGGCAGGTGTACT-3' for 30 cycles: (94°C for 1 min, 60°C for 1 min, and 72°C for 3 min). RT-PCR for FGFR2IIIc was carried out according to Miki et al. (37) . RT-PCR for ß-actin was performed to serve as an internal control, as previously described (11) . Samples (20 µl) of the RT-PCR reaction mixtures for FGFRs and ß-actin were run on 1.5% NuSieve 3:1 agarose gels (FMC BioProducts, Rockland, ME) and transferred to GeneScreen plus nylon membranes. The RT-PCR product for FGFR1IIIc was confirmed with an oligonucleotide: 5'-TGTGTAAGGTGTACAGTGA-3'. The oligonucleotides were 3' end labeled with digoxigenin-ddUTP (Boehringer Mannheim, Mannheim, Germany) according to the protocol of the manufacturer. Washes and detection of the signal were carried out as described in the digoxigenin luminescent detection protocol (Boehringer Mannheim). The RT-PCR products of FGFR2IIIc, FGFR4, and ß-actin were confirmed by hybridization with 32P-labeled FGFR2 cDNA (38) , FGFR4 cDNA (39) , or ß-actin control probe (Oncor Inc., Gaithersburg, MD) in standard conditions. The intensities of hybridization signals were quantified by densitometry using an MCID M4 Image Analyzer (Imaging Research).

Determination of Anchorage-dependent and -independent Growth of Transfected Cells.
Cells (5 x 104) were seeded into 6-well plates in monolayers in phenol red-free RPMI 1640 with 5% DC-FBS without any hormones. Next day, the medium was changed and 1 nM E2 and/or 5 µM Tam was added. Cells were fed with fresh media every other day. On the days indicated, cell numbers were determined by counting cell nuclei in a Coulter Counter. Cells (5 x 104) in suspension culture were grown in 30-mm plastic bacteriological dishes (Sterilin, Teddington, United Kingdom), to which <2% of the cells attach. On the days indicated, cells were harvested and counted as for monolayers except that lysis took about 1 h with gentle rocking of the colonies. Cells in soft agar colony assay were cultured in 6-well plates first covered with an agar layer (phenol red-free RPMI 1640 with 0.5% agar and 5% DC-FBS). The middle layer contained 5 x 104 cells in phenol red-free RPMI 1640 with 0.33% agar and 5% DC-FBS with or without 1 nM E2. The top layer, consisting of medium, was added to prevent drying of the agarose gels. The plates were incubated for 18 days, after which the cultures were inspected and photographed.

Invasion Assay.
The potential of FGF-8b to trigger invasion was tested on Falcon Cell Culture Inserts of 8-µm pore size (Becton Dickinson Labware Europe, France) coated with Matrigel (100 µg/cm2; Becton Dickinson Labware, Bedford, MA). FGF-8b-transfected, mock-transfected, and wild-type MCF-7 cells (1 x 105) were seeded into inserts in 24-well plates and incubated for 2–3 days in RPMI 1640 with 10% inactivated FBS, 10 nM E2, and 4 µg insulin/ml in triplicate or quadruplicate. After the indicated time, the inserts were fixed in 4% paraformaldehyde for 10 min, washed with PBS, and stained with Mayer’s hematoxylin for at least 1 h, washed once with H2O, dipped in 70% ethanol + 5% HCl, and washed once again with H2O. The cells on the upper side of the membrane were wiped off with cotton wool, and the cells fixed on the lower side of the membrane were counted by microscopy (x100 magnification) from 15 fields, representing almost the whole membrane area.

Zymography.
Serum-free RPMI 1640 medium with 1 nM E2 was conditioned by FGF-8b- and Mock-transfected MCF-7 cells for 24 h. Samples of concentrated conditioned media, referred to same number of cells in culture, were applied to nondenaturating 10% SDS-PAGE containing gelatin (1 mg/ml). After electrophoresis, gels were washed in 50 mM Tris-HCl ( pH 7.5) containing 2.5% Triton X-100 to remove SDS and developed by incubation in 50 mM Tris-HCl (pH 7.5) containing 5 mM CaCl2 and 1 µM ZnCl2 for 48 h at +37°C. Gels were fixed with 7% acetic acid containing 50% methanol for 1 h followed by staining with 0.2% Coomassie Blue G250. No destaining was needed. Gelatinolytic enzymes were detected as transparent bands on the blue background of Coomassie Blue-stained gel. Conditioned medium from the HT-1080 human fibrosarcoma cells was used as a positive control, because they secrete the high amounts of MMP-2 and MMP-9 (40) .

In Vivo Studies.
Tumorigenicity was examined by inoculation of cells s.c. into the back of the neck (2 x 106) or into the mammary fat pad (5 x 106) of athymic female mice (BALB/cABom-nu/nu; Bomholtgård, Denmark), which simultaneously received a 60-day release pellet containing 0.72 mg of E2 (Innovative Research of America, Toledo, OH). Tumor measurements were performed twice a week. After 4 weeks the mice bearing s.c. tumors formed from FGF-8b-transfected cells were divided into two groups, and the pellet was removed from one group (n = 4). The tumors were measured in two dimensions, and the volumes were calculated according to the formula: , where d1 and d2 are two perpendicular tumor diameters (41) . Tumors obtained were divided into two parts. One was Formalin fixed for histological examination and the other was frozen for RNA and protein analysis.

Determination of Tumor Angiogenesis.
The three most vascular nonoverlapping fields of each H&E-stained section were chosen under a microscope. The maximal areas of neovascularization were identified by scanning at low magnification (x40). Three separate fields (magnification x100–200) were captured and stored in a microcomputer by using a Kappa CF8/1 FMC digital camera. The relative areas of existing vessels (in pixels) in the three separate fields were measured by using Adobe Photoshop version 5.

Immunohistochemistry.
After dehydration, 5-µm sections of paraffin-embedded tumor samples on silane-coated glass slides were incubated in 0.1% H2O2 in PBS to block endogenous peroxidase activity. After trypsin treatment, nonspecific binding of the IgG was reduced by a 1-h preincubation of the slides in 10% normal goat serum at +4°C. The primary antibody (rabbit antihuman von Willebrand factor; Dako A/S) was applied at a dilution of 1:250 in PBS/3% BSA, and the sections were incubated at +4°C overnight. Normal rabbit serum instead of primary antibody was used as a negative control. After washes in PBS, the samples were incubated with biotinylated goat antirabbit IgG secondary antibody (Vector Laboratories, Burlingame, CA) at a 1:200 dilution for 1 h at room temperature. Detection was performed with an avidin-biotin complex kit (Vector Laboratories). The slides were then stained with diaminobenzidine, washed, counterstained with Delafield’s hematoxylin, dehydrated, treated with xylene, and mounted.

Statistical Analysis.
One-way ANOVA or the corresponding nonparametric tests (Kruskal-Wallis or Mann-Whitney U tests) were used to test for differences in the vessel area or in the cell proliferation and invasion assays. To avoid the multiplicity problem, Tukey least-significant difference tests or Duncan’s multiple range comparison tests were applied. Sizes of s.c. tumors formed by FGF-8b- and mock-transfected cells at day 28 (before E2 pellet was taken away) were analyzed with the Mann-Whitney U test. The growth curves of individual tumors were also analyzed with the Kaplan-Meier estimation method and tumor latencies were compared with the nonparametric log rank test. The two-sample t test was used to test for differences in tumor size grown in mammary fat pad at the end of the experiment. Statistical significance in all tests is based on two-sided P < 5%.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Exogenous FGF-8b and FGF-2 on the Proliferation of MCF-7 Cells.
To mimic paracrine regulation and to check whether exogenous FGF-8b has a mitogenic effect on MCF-7 cells, a proliferation study at different concentrations of recombinant FGF-8b was performed by means of the [3H]thymidine incorporation assay. Fig. 1Citation shows that FGF-8b increased thymidine uptake of MCF-7 cells in a dose-dependent manner. Stimulation of DNA synthesis was maximal (5- to 10-fold) at a concentration of 50 ng/ml and it slightly declined with higher concentrations. The experiment was performed with both normal (5%) and reduced (1%) serum levels. More marked stimulation was observed with less serum although the cells seemed to slightly suffer in these conditions, since a smaller response to E2 was observed. The effect of FGF-8b on DNA synthesis was compared with that of E2 and FGF-2. In 1% serum, the effect of FGF-8b was stronger than that of E2 (10-fold versus 7-fold) but in 5% serum E2 was more potent than FGF-8b (8-fold versus 5-fold). FGF-2 gave a maximal response at a concentration of 10 ng/ml (4- to 7-fold).



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  		Fig. 1. [3H]Thymidine incorporation by MCF-7 cells grown with and without different concentrations of FGF-8b (5, 10, 25, 50, and 100 ng/ml), FGF-2 (1, 5, 10, and 50 ng/ml), and 1 nM E2. Columns (cpm) show mean ± SD of eight replicate wells cultured with 1% serum or 5% serum on 96-well plates. The experiment was repeated four times with similar results. Statistical analysis showed a significant difference between control (no additions) versus others (***, P < 0.001).

 
To estimate cell population doubling times, we also counted the cell numbers in a 5-day culture. MCF-7 cells without E2 or growth factors had the mean cell population doubling time of 46 ± 3 h, but it was decreased to 35 ± 3 h (P = 0.01) when cultured in the presence of E2. The doubling time of cells treated with FGF-8b (25 ng/ml) or FGF-2 (10 ng/ml) was decreased to 38 ± 3 h (P = 0.03; data not shown). These results suggested that FGF-8b is a mitogen for MCF-7 breast cancer cells when added exogenously to the culture medium.

Transfection of FGF-8b into MCF-7 Cells.
The expression plasmid pcDNA3 containing the coding sequence for FGF-8b under the transcriptional control of the human cytomegalovirus promoter was transfected into MCF-7 breast carcinoma cells. Plasmid without cDNA (mock) was used as a negative control. G418-resistant clones of FGF-8b-transfected and mock-transfected MCF-7 cells were isolated and expression of the FGF-8b mRNA was confirmed by Northern blot analysis. Two cell lines derived from pooled colonies, named FGF-8b and mock, were chosen for further study. Estradiol had no effect on the constitutive expression of FGF-8b (Fig. 2A)Citation . We did not detect any endogenous expression of FGF-8b mRNA in wild-type MCF-7 cells by Northern blot analysis. In addition, 10 different clonal cell populations were obtained by subcloning the pooled population cell line. They all expressed comparable levels of mRNA for FGF-8 than did pooled population cells (data not shown).



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  		Fig. 2. The expression of FGF-8 mRNA and protein in transfected MCF-7 cells. A, Northern blot analysis of FGF-8b- and mock-transfected cells cultured with and without 10 nM E2 for 6 days. For Northern blot analysis, 20 µg RNA of FGF-8b- transfected, mock-transfected, and wild-type MCF-7 cells were loaded on the gel. Columns show the relative level of FGF-8 mRNA (FGF-8/glyceraldehyde-3-phosphate dehydrogenase mRNA; scanning units) in each sample. B, Western blot analysis of FGF-8b- and mock-transfected cell lysates and conditioned media. Proteins from cell lysate (20 µg; Lane 1, FGF-8b; Lane 2, mock) and corresponding amounts of heparin-bound proteins from conditioned media (Lane 3, FGF-8b; Lane 4, mock) were separated by 12% SDS-PAGE. Secreted FGF-8b protein (Mr {approx}34,000) was found only in the conditioned medium of FGF-8b-transfected cells. Lane 5, recombinant FGF-8b protein (20 ng), which served as a positive control (Mr {approx}32,000).

 
Western blot analysis revealed the presence of FGF-8b protein ({approx}34 kD) in the media conditioned by FGF-8b-transfected cells and partially purified on heparin-Sepharose (Fig. 2B)Citation . As expected, FGF-8b protein secreted from cells differed in size from recombinant FGF-8b protein used as a positive control because the protein produced by cells is glycosylated but recombinant FGF-8b is not. We did not detect FGF-8b in extracts of FGF-8b-transfected cells, indicating that it was actively secreted from the cells. Neither did we detect FGF-8b protein in the conditioned media or cell extracts of mock cells.

Expression of FGF-8b-binding FGFRs in Transfected MCF-7 Cells.
To study the FGFRs mediating FGF-8b signaling in MCF-7 cells, we performed semiquantitative RT-PCR analysis of FGFR1IIIc, FGFR2IIIc, FGFR3IIIc, and FGFR4, which have previously been shown to mediate a mitogenic effect of FGF-8 in BaF3 cells (17 , 24) . We found that MCF-7 cells expressed FGFR1IIIc, FGFR2IIIc, and FGFR4, but not FGFR3IIIc. Overexpression of FGF-8b in MCF-7 cells was found to down-regulate all FGFRs expressed in the cells (Fig. 3)Citation . FGFR1IIIc and FGFR2IIIc were found to be up-regulated by E2 in mock-transfected cells, whereas no effect was found on the expression of FGFR4.



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  		Fig. 3. Expression of FGFR1IIIc, FGFR2IIIc, and FGFR4 in transfected MCF-7 cells studied by RT-PCR. A, detection of FGFR1IIIc, FGFR2IIIc, FGFR4, and ß-actin mRNA expression in MCF-7 cells by RT-PCR analysis. Southern blot hybridization of FGFR RT-PCR products using a specific oligonucleotide probe for FGFR1IIIc (673 and 940 bp), a cDNA probe for FGFR2IIIc (150 bp), a cDNA probe for FGFR4 (637 bp), and a cDNA probe for ß-actin (770 bp) is shown. PCR products from FGF-8b-expressing cells cultured with E2 (Lane 1), from FGF-8b-expressing cells cultured without E2 (Lane 2), from mock cells cultured without E2 (Lane 3), and from mock cells cultured with E2 (Lane 4) are shown. B, densitometric analysis of Southern blots. The intensity values of FGFRs were corrected by the corresponding intensity values of ß-actin. Relative levels of FGFR mRNAs (FGFR/ß-actin mRNA; scanning units) are shown.

 
Effect of FGF-8b Transfection on the Growth and Morphology of MCF-7 Cells.
The growth characteristics and hormone dependence of the transfected cells were studied next. We found that in monolayer cultures FGF-8b was not able to increase the proliferation rate of MCF-7 cells. They actually grew more slowly than mock or wild-type MCF-7 cells when cultured with E2 (Fig. 4)Citation and did not reach corresponding cell densities even in very confluent cultures. Estradiol induction of proliferation of FGF-8b transfectants was {approx}50% lower than in control cells, but there was no difference in the number of cells grown without any hormones. Tam was able to inhibit the growth of FGF-8b-expressing cells, thus indicating that the cells had retained their responsiveness to antihormones.



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  		Fig. 4. Growth of FGF-8b- and mock-transfected and wild-type MCF-7 cells in monolayer culture. Cells (5 x 104/well) were seeded into 6-well plates in phenol red-free RPMI 1640 with 5% DC-FBS without any hormones. The next day, the medium was changed and 1 nM E2 and/or 5 µM Tam was added. Treatments: a, control, no hormones added; b, Tam; c, E2; and d, E2+Tam. Cells were fed with fresh media every other day. The cultures were incubated for the time periods shown. Each bar represents the mean ± SD of triplicate wells. The experiment was repeated twice with similar results.

 
The effect of FGF-8b transfection on the morphology of MCF-7 cells was studied in the presence or absence of estradiol. FGF-8b-expressing MCF-7 cells were rounded and more loosely attached to the culture dishes than mock-transfected or wild-type MCF-7 cells. There were also more detached cells in the culture media of FGF-8b-expressing cells when cultured with E2 (data not shown).

Anchorage-independent Growth of FGF-8b-Expressing MCF-7 Cells.
The acquisition of anchorage-independent growth is generally considered to be one of the in vitro properties associated with the malignancy of cells. The transfected cells were examined for their ability to grow in suspension culture or in soft agar. FGF-8b-expressing cells grew faster than mock-transfected or wild-type MCF-7 cells in suspension culture (Fig. 5A)Citation . At day 10, the number of FGF-8b-expressing MCF-7 cells was 2-fold, both in E2-containing cultures and in cultures without E2 when compared with the controls. FGF-8b-expressing cells also formed more colonies, which were looser and larger in size than those in control cultures, in which tight round cell aggregates were formed. When the cells were grown in soft agar for 18 days, FGF-8b-expressing cells were able to form colonies even without E2, whereas mock-transfected cells formed colonies only when grown in the presence of E2 (Fig. 5B)Citation .



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  		Fig. 5. Anchorage-independent growth of FGF-8b- and mock-transfected and wild-type (wt) MCF-7 cells. A, cells (5 x 104/well) were plated into 30-mm bacteriological dishes in phenol red-free RPMI 1640 containing 5% DC-FBS with and without 1 nM E2. The cultures were incubated for the time periods shown. Each point represents the mean ± SD of triplicate wells. The experiment was repeated twice with similar results. The statistical analysis of differences in the growth between FGF-8b-transfected and mock-transfected or wild-type MCF-7 cells were analyzed by one-way ANOVA (***, P < 0.001; **, P < 0.01). B, colony formation of FGF-8b- or mock-transfected cells grown in soft agar. FGF-8b (left) and mock (right) cells were cultured with and without 1 nM E2 for 18 days, when the colonies were evaluated and photographed. Bar, 100 µm.

 
FGF-8b Overexpression Increases the Invasiveness of MCF-7 Cells.
The effect of FGF-8b on the invasiveness of MCF-7 cells was measured by in vitro invasion assays. FGF-8b-transfected, mock, or wild-type MCF-7 cells were seeded on the upper surface of cell culture insert membranes covered with Matrigel. Fig. 6ACitation shows the results of a representative sample of invasion experiments. The invasive capacity of parental MCF-7 and mock-transfected cells was poor, but in FGF-8b-overexpressing cells it was clearly increased (2-fold, P = 0.021).



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  		Fig. 6. A, invasion capacity of FGF-8b- or mock-transfected and MCF-7 wild-type (wt) cells. Cells were seeded using normal medium in the upper compartment of invasion chambers to test their ability to invade through Matrigel-coated semipermeable filters inserted into the wells. After a 72-h incubation, the number of the migrated cells per membrane was determined. Bars show the mean number ± SD of invaded cells per membrane in a representative experiment performed in quadruplicate and repeated four times with similar results. The number of invaded FGF-8b-expressing cells was significantly higher than that of mock or wild-type MCF-7 cells (*, P = 0.021). B, gelatinolytic activity of MMP-2 and MMP-9 in the conditioned media of wild-type, FGF-8b-transfected, and Mock-transfected MCF-7 cells (2 x 105; Lanes 1, 2, and 3, respectively) or conditioned medium of HT-1080 cells, which was used as a positive control (Lane 4). The experiment was repeated three times with similar results.

 
As one of the possible mechanisms, the increased invasiveness of FGF-8b-expressing cells may be due to the increased level or activity of matrix-degrading proteases, such as matrix MMPs (42) . Therefore, the expression levels of MMP-2 and MMP-9 were studied by zymography. We found that MCF-7 cells produced both MMPs, but the activity of MMP-9 was clearly increased in FGF-8b-transfected cells when compared to mock-transfected or wild-type MCF-7 cells (Fig. 6B)Citation . We did not find any difference between the cells in the activity of MMP-2.

FGF-8b Overexpression Increases Tumor Growth.
To investigate the tumorigenicity of the transfectants in vivo, the cells were transplanted both s.c. to the back of the neck (ectopic location) and into mammary fat pads (orthotopic location) of BALB/c nude mice bearing s.c. E2 pellets. Table 1Citation shows a summary of the growth characteristics of the FGF-8b-expressing cells versus mock-transfected cells. Tumor formation was very effective in both cell lines, being 100% in mammary fat pads and 100% and 67% in s.c. tumors formed by FGF-8b- and mock-transfected cells, respectively. In the ectopic location, the latency time for tumor formation was delayed (2–7 weeks) in tumors formed by mock-transfected cells when compared with tumors formed by FGF-8b-expressing cells (2–4 weeks; P = 0.0405), but when injected into mammary fat pad both cell lines had a latency time of only 1 week.


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  		Table 1 Tumorigenicity of FGF-8b- or mock-transfected MCF-7 cells in nude mice bearing E2 pelletsa

 
Fig. 7Citation shows the growth of all individual s.c. tumors over a 1-month period. Although the growth rates of different tumors varied greatly, it was obvious that the cells expressing FGF-8b grew faster as tumors than the controls. Mock-transfected cells were able to produce only small tumors (mean ± SE, 36 ± 33 mm3) in a 1-month period (Fig. 7BCitation ; Table 1Citation ), whereas most of the FGF-8b-expressing cells produced large tumors (mean ± SE, 204 ± 164 mm3; Fig. 7ACitation ; Table 1Citation ). The difference was found to be statistically significant (P = 0.0032). However, the tumors were still dependent on E2 for growth and started to decrease in size if the E2 pellets were removed (Fig. 7C)Citation . In addition, FGF-8b-transfected MCF-7 cells were not able to produce tumors without E2 supplementation. In fact, the effect of FGF-8b on the growth of MCF-7 cells as tumors seemed to be dependent on factor(s) produced by E2. We did not find any signs of invasion or macrometastases to distant organs (lungs, liver).



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  		Fig. 7. Growth of FGF-8b- or mock-transfected MCF-7 cells in vivo. FGF-8b-expressing, (A) and mock cells (B, 2 x 106) were implanted s.c. into nude mice (FGF-8b, n = 11; mock, n = 12) bearing E2 pellets. Tumor measurements were performed twice a week. The tumors were measured in two dimensions, and the volumes were calculated according to the formula V = ({pi}/6)(d1 x d2)3/2, where d1 and d2 are two perpendicular tumor diameters. The growth of individual tumors formed by FGF-8b-expressing cells (a–j) and mock cells (a–e) are shown. The difference between the tumor growth of FGF-8b- and mock-transfected cells was found to be statistically significant (P = 0.0032). C, mean volumes of tumors derived from FGF-8b-expressing and mock cells. After 4 weeks (marked *), the mice bearing tumors formed from FGF-8b-transfected cells were divided into two groups, were anesthetized again, and the pellet was removed for 2 weeks from one group (n = 4). During the 2-week follow-up period, the tumors growing larger than 200 mm3 in mice retaining E2 pellets were removed at the time points marked with an arrow.

 
As in the ectopic location, orthotopically inoculated FGF-8b-expressing MCF-7 cells produced larger tumors in a 1-month experiment (mean ± SE, 379 ± 181 mm3) when compared to mock cells (mean ± SE, 112 ± 34 mm3). The difference was found to be statistically significant (P = 0.046; Table 1Citation ). No obvious signs of invasion or metastasis were found to lymph nodes, lungs, liver, spleen, and bone either. In addition, three clonal FGF-8b-expressing cell lines were injected s.c. into nude mice and compared to the growth of two mock-transfected cell lines. These experiments revealed similar results, thus suggesting that FGF-8b expression provided a growth advantage for MCF-7 cells (data not shown).

All tumors produced by FGF-8b transfectants were found to express FGF-8 mRNA at a high level, but nothing was seen in mock controls. The expression of FGF-8 mRNA was also high after the E2 pellets were removed (data not shown).

Increased Angiogenesis of FGF-8b-expressing Tumors.
FGF-8b tumors contained more large sinusoid-like vessels and showed an overall angiogenic morphology when examined from H&E-stained sections. In addition, there was very little necrosis in these tumors. Mock cells produced small tumors in which only a few vessels were seen (data not shown). Vascularization was quantified by measuring the relative area of vessels in three fields of the most vascularized areas in each tumor. The area covered by vessels in FGF-8b tumors grown in E2 pellet-carrying mice was significantly greater than in mock tumors (P = 0.0018; Fig. 8ACitation ). Although the tumor volume of FGF-8b tumors grown for 4 weeks rapidly decreased when the E2 pellet was removed, the relative vessel area was not decreased correspondingly, suggesting a possible additive effect of E2 and FGF-8b. FGF-8b expression thus seemed to maintain the angiogenic morphology, which differed from mock controls even in the absence of an estrogen effect (vessel area of FGF-8b tumors was about 3-fold when compared with mock controls, P = 0.04; Fig. 8ACitation ). Tumor sections were also stained with an anti-von Willebrand factor antibody staining endothelial cells. FGF-8b tumors were more intensively stained than control tumors, which further confirmed the increased vascularization of FGF-8b-expressing tumors (Fig. 8B)Citation .



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  		Fig. 8. Determination of tumor vascularization. A, relative areas of vessels (in pixels) in three fields of H&E-stained sections are shown (mean ± SE). Differences in vessel area were found to be statistically significant (**, P = 0.0018 when compared with mock tumors; *, P = 0.04 when compared with mock tumors). There was no statistical difference between the FGF-8b tumors grown in the presence and absence of an E2 pellet. B, von Willebrand factor immunohistochemical staining of endothelial cells in nude mouse tumors. Deparaffinized sections (5 µm) of FGF-8b and mock tumors grown in the presence of E2 pellet, were incubated with rabbit antihuman von Willebrand factor antibody at +4°C overnight, followed by biotinylated goat antirabbit IgG secondary antibody. Detection was carried out with an avidin-biotin complex kit, followed by use of diaminobenzidine. Counterstain is Delafield’s hematoxylin. Bar, 100 µm.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
FGF-8 is the only member of the FGF family known to be expressed at an elevated level in human breast cancer (20) . In addition, it is known to have transforming potential in experimental models (19) . Because endogenous expression of FGF-8 in MCF-7 cells is low (20) , we used them to examine the effect of FGF-8 on the growth properties of human breast cancer cells in vivo and in vitro. We demonstrate here that overexpression of FGF-8 in MCF-7 cells by transfection leads to increased tumor growth and an invasive and angiogenic capacity.

FGF-8b protein added to the culture medium of estrogen-dependent MCF-7 cells was able to increase the rate of proliferation similarly to E2, suggesting that FGF-8 is able to function as a mitogen for MCF-7 cells. In our experiments, the effect was comparable to that of FGF-2, which is a classic mitogen for fibroblasts and endothelial cells and also for tumor cells (43) . An autocrine action of FGF-2 in the growth of immortal human breast cancer cells suggested that FGF-2 is important in the early events leading to neoplastic transformation of human mammary epithelial cells (44) . In addition, FGF-2 is one of the primary angiogenic factors in breast cancer (43) . The proliferative response of various breast cancer cell lines to FGF-2 has, however, been variable: either inhibitory (45 , 46) or stimulatory (47 , 48) . This is probably caused by the multifunctional properties of FGF-2, varying from one cell type and growth condition to another.

Previous data concerning the expression of FGFRs in MCF-7 cells have also been very conflicting and different expression profiles of FGFRs in these cells have been reported (27 , 49) . In the present study, we demonstrated expression of FGFR1IIIc, FGFR2IIIc, and FGFR4 but not FGFR3IIIc in MCF-7 cells. We used two different pairs of primers for FGFR3IIIc but did not obtain any specific message. The expression of FGFR1IIIc and FGFR2IIIc were found to be up-regulated by E2, whereas E2 had no effect on FGFR4 expression. Previously, we have reported that androgens can modulate the expression of FGFRs in an androgen-sensitive S115 mouse mammary tumor cell line, possibly via the production of FGF-8 (33) . The mechanism of the estrogen-induced increase of FGFRs was not studied here, but we speculate that estrogen indirectly increases the expression of FGFRs by regulating the production of FGFs, as previously shown in different tissues (50 , 51) . Transfection of FGF-8b to MCF-7 cells down-regulated the expression of all three receptors. This suggested that ectopically expressed FGF-8 was functional and probably interacted with the FGFRs located on the cell membrane of MCF-7 cells. A similar down-regulation of FGFRs was found when FGF-2 was overexpressed in HEC-1-B endometrial cells (52) . In these cells FGF-2 induced growth and vascularization in vivo, which responses are in accordance with our results concerning the effects of FGF-8b on breast cancer cells.

We demonstrate here that MCF-7 cells stably transfected with FGF-8 acquire the ability to proliferate in the absence of E2 in suspension and in semi-solid medium, thus displaying anchorage-independent growth. In suspension culture, colonies of FGF-8b-expressing cells were loose, with empty spaces inside the colony. In addition, when cultured as a monolayer, the cells had rounded morphology and poorer attachment to culture dishes than vector-only-transfected cells. Their ability to invade was also clearly increased. These changes in morphology, attachment, anchorage-independent growth, and invasion potential may be a result, for example, of changes in the expression of cell-cell adhesion molecules such as cadherins. Several investigators have shown that decreased expression of E-cadherin is correlated with increased invasiveness of breast cancer and the expression of N-cadherin promotes motility and invasion of breast cancer cells (53) . It has also been suggested that N-cadherin-dependent motility is mediated by FGFR signaling (53) . Data from FGF-8-/- mouse embryos also suggest that FGF-8 is an important factor in regulation of cellular migration (54) .

Other molecules which affect matrix degradation, such as matrix MMPs, tissue inhibitors of metalloproteinases, plasminogen activators (urokinase-type plasminogen activator and tissue plasminogen), and plasminogen activator inhibitors, may also be involved in the increased invasion capacity of FGF-8b-transfected MCF-7 cells. We studied the activity of the matrix metalloproteinases MMP-2 and MMP-9 in FGF-8b-transfected, mock- transfected, or wild-type MCF-7 cells. These MMPs, known as gelatinases A and B, mediate invasion and metastasis by degrading the components of basement membrane, such as type IV collagen (55) . MMP-2 and MMP-9 activities are increased in malignant breast carcinoma when compared with benign breast tumors (56) . Both MMP-2 and MMP-9 were expressed in FGF-8b- and mock- transfected or wild-type MCF-7 cells, but the activity of MMP-9 was increased in FGF-8b-expressing cells. Previously, nontransformed mammary myoepithelial cells transfected with FGF-3 have been found to be highly invasive and metastatic and to produce an increased level of MMP-9 (57) . It is thus probable that increased invasiveness of FGF-8b-expressing MCF-7 cells observed in vitro is associated with increased MMP-9 activity. It is not known whether FGF-8b directly regulates MMP-9 expression but the promoter region of the MMP-9 gene contains activator protein 1 and nuclear factor {kappa}B sites (58) known to be targets for FGF signaling (59 , 60) . We could not, however, detect increased invasion in vivo in any of the tumors formed by FGF-8b-transfected cells. It is possible that additional factors or capacities are required to facilitate invasion in vivo. The role of other possible factors, such as cadherins, promoting cell migration and invasion in FGF-8b-transfected cells presently remains to be studied.

Tumor growth of FGF-8b-expressing cells in nude mice was fast and evaluation of tumor morphology showed a rich vasculature in FGF-8b-expressing tumors. Staining with an endothelial cell marker (anti-von Willebrand factor) demonstrated increased size and number of vessels in FGF-8b-expressing tumors. It is now well established that tumor growth (and metastasis) requires new blood vessel growth for the tumor to grow beyond 1–2 mm3 (reviewed in Ref. 61 ). Evidence from numerous studies has revealed that angiogenesis is a discrete component of the tumor phenotype which is often activated during the early preneoplastic stages of development of a tumor. This is thus an effective phase for antiangiogenic therapy to block tumor growth. Our recent studies have shown that FGF-8 is also able to stimulate proliferation, migration, and tube formation of endothelial cells (62) , which suggests that FGF-8 has a direct angiogenic effect in tumors.

The FGF-8b provided growth advantage of MCF-7 cells in our study was dependent on the presence of E2 in vivo. Also, when the E2 pellet was removed from the mice, the tumor growth ceased and tumor size decreased to the level of mock tumors grown with the E2 pellet. We have previously shown that E2 is able to induce the production of VEGF by MCF-7 cells (35) , which may be one of the estrogen-regulated factors required for the full effect of FGF-8b on growth and angiogenesis in MCF-7 tumors. VEGF transfection of MCF-7 cells similarly led to increased vascular density and enhanced tumor growth, but did not affect hormone or antihormone dependence of the cells (63) . Previous studies have suggested that the effect of FGF-4 on angiogenesis is possibly mediated by increased levels of VEGF (64) . To study possible mediation of FGF-8 action by VEGF, we measured the level of VEGF mRNA and protein in FGF-8b-expressing cells, tumors, and serum of the mice bearing tumors formed by FGF-8b-expressing cells, but the levels were not different from those in controls (data not shown). Previous studies have also shown that FGF-2 and VEGF may have synergistic effect on angiogenesis in vitro (65) and in vivo (66) . It is thus conceivable that FGF-8b also has a synergistic effect with E2-induced VEGF in MCF-7 cells but additional experiments are needed to study the possible mechanisms.

To our knowledge, this is the first report demonstrating stimulation of tumor growth and angiogenesis by FGF-8 in human breast cancer cells. The in vitro data show that FGF-8 is able to stimulate proliferation and anchorage-independent growth of breast cancer cells and to increase their invasion via the elevated production of MMP-9. In addition, the in vivo experiments demonstrate that FGF-8 also increases vessel formation, which most probably contributes to accelerated tumorigenesis. These data suggest that FGF-8 represents an important component of FGF signaling pathways regulating invasion and tumor growth of breast cancer cells.


   ACKNOWLEDGMENTS
 
We thank Dr. Pradip Roy-Burman for giving the FGF-8b-pcDNA3 expression plasmid. Susanna Hinkka is acknowledged for helping in the statistical analysis. Soili Jussila, Anneli Kurkela, Pirkko Rauhamäki, and Leena Simola are warmly thanked for excellent technical assistance. Johanna Ruohola is a graduate student of the Turku Graduate School in Biomedical Sciences.


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

1 Supported by grants from the Ida Mountin Foundation and the Finnish Medical Foundation Duodecim (to J. K. R.) and the Academy of Finland (to P. L. H. and P. T. L.). Back

2 To whom requests for reprints should be addressed, at Department of Anatomy, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. Phone: 358-2-3337379; Fax: +358-2-3337352. E-mail: pirkko.harkonen{at}utu.fi Back

3 The abbreviations used are: FGF, fibroblast growth factor; FGF-8, fibroblast growth factor 8; FGFR, fibroblast growth factor receptor; RT, reverse transcription; E2, 17ß-estradiol; FBS, fetal bovine serum; Tam, tamoxifen; DC, dextran-coated charcoal treated; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor. Back

Received 5/30/00. Accepted 3/16/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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