Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK
1 Division of Biochemistry, Department of Oncology, Biomedical Research Centre, Osaka University Medical School, Suita, Osaka, Japan
2 To whom correspondence should be addressed. Tel: +44 29 20744711; Fax: +44 20 20761723; Email: martinta1{at}cf.ac.uk
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Abstract |
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Abbreviations: HGF, hepatocyte growth factor; SF, scatter factor
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Introduction |
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HGF/SF is secreted by fibroblasts in vivo (2) and plays a role in a number of important physiological processes, including cell motility and migration, proliferation and cell invasion. HGF/SF is able to regulate the angiogenic process and to hasten tissue repair after insult (35), where activation of the inactive molecule occurs by proteases responding to tissue damage.
HGF/SF is strongly implicated as a regulator of tumour progression and metastasis (6,7). HGF/SF is implicated as a powerful angiogenic factor due to its ability to stimulate endothelial cell proliferation and motility in vitro and has been shown to induce blood vessel growth (810). HGF/SF has also been shown to act as a paracrine factor in regulating the expression of other angiogenic factors by tumour cells (11).
Cancer cells (including breast cancer) over-express c-Met (7). Stromal fibroblasts from breast cancer tissues have been shown to express and produce large amounts of bioactive HGF/SF compared with those from normal tissue stroma. Clinical studies indicate that HGF/SF and this receptor are influential factors associated with progression and prognosis of patients with breast cancer (1214). Edakuni et al. (15) have shown that the HGF/SF and c-Met proteins are expressed in cancer and stromal cells, with autocrine and paracrine patterns. Co-expression of HGF and c-Met at the leading cancer front of cancer tissues was correlated with histological grade, reduced patient survival and a high Ki-67 labelling index. HGF/SF and c-Met are over-expressed in breast carcinoma when compared with benign breast tissue, and can be co-expressed in cancer cells (16). In multivariate analysis (17) immunoreactive HGF/SF levels were found to be the most important independent factor in predicting relapse-free and overall survival. It was also found to be of greater import than lymph node involvement. Tsarfaty et al. (18) suggest that in a subpopulation of node-negative breast cancer patients, a high tumor c-Met level relative to normal tissue is an indicator of poor overall survival. Detection of occult metastatic breast cancer cells in blood by a multimolecular marker assay, including a marker for c-Met, has potential clinical utility in monitoring tumor progression by a blood test; c-Met signal correlated with tumour size (19). In addition, high levels of c-Met expression were associated with death due to metastatic disease in patients with axillary lymph node-negative breast carcinoma. Expression of c-Met may be a useful prognostic indicator of more aggressive disease in patients whose prognosis is not easily stratified by current histopathological markers (20).
Agents able to suppress the activity of HGF/SF may be of great importance clinically. A number of such agents are known to exhibit the inhibitory effects of HGF/SF on cancer cells. One such compound is the HGF/SF antagonist NK4.
NK4 is an HGF/SF variant that contains the receptor domain and all four kringle domains, but no ß chain. This variant is generated by enzymatic cleavage of mature human HGF/SF (21). Due to the loss of the ß chain, NK4 is able to bind to the HGF/SF receptor, but is unable to activate it. NK4 therefore competes with HGF/SF molecules in receptor binding. It has already been found to deactivate HGF/SF-induced receptor binding in MDCK cells (22), but has no mitogenic or motogenic effects (21,24). We have previously reported that NK4 has no agonistic effect on human vascular endothelial cells and that NK4 is able to inhibit HGF/SF-promoted tubule formation and endothelial cell migration (7). Proliferation and migration of human microvascular endothelial cells has also been inhibited by NK4 (24). NK4 is also able to suppress HGF/SF-induced invasion of prostate cancer cell lines (25) and ovarian cancer cells (26).
We now report that NK4 is able to inhibit the HGF/SF-induced growth of human breast cancer in the female nude mouse tumour model. We propose that this inhibition is due to inhibition of the angiogenic effect of HGF/SF, shown by decreased tumour vascularity in those mice treated with NK4.
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Materials and methods |
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Cell lines
Human breast cancer cell line MDA MB 231 and human fibroblast cell line MRC5 (both from ECACC, Wiltshire, UK) were maintained in DMEM with 10% foetal calf serum.
SDSPAGE and western blotting
For immunoprecipitation, cells were first subject to serum starvation for 2 h, before treatment with HGF/SF, NK4, fibroblast-conditioned medium (serum-free) or combinations thereof. Cells were pelleted and lysed in HCMF buffer, 0.5% Triton X-100, 2 mM CaCl2, 100 µg/ml phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin and 10 mM sodium orthovanadate for 40 min. Cells treated with orthovanadate and hydrogen peroxide were used as a positive control. Cell debris was removed by centrifugation (13 000 g for 10 min). Anti-paxillin or anti-cMet antibody was added to each sample of equal protein concentration (a small portion of the lysate sample was first collected for use as a loading control). Protein A/Gagarose was added after 1 h and incubated at 4°C overnight. The precipitates were washed three times and lysed in sample buffer. After western blotting, the membranes were probed with anti-phosphotyrosine antibody (PY99). Equal amounts of lysate from each cell sample were placed on an 8% polyacrylamide gel. Following electrophoresis, proteins were blotted onto nitrocellulose sheets and blocked in 10% skimmed milk for 60 min before probing with anti-actin antibody, followed by peroxidase-conjugated secondary antibody (1:2000). Protein bands were visualized with an enhanced chemiluminescence (ECL) system (Insight Biotechnology, Wembley, Middlesex, UK). The band intensities on photographic film were analysed with densitometry software (Optimas 6.0; Optimas UK Ltd) and are shown as relative values.
Cell migration assay
MDA MB 231 cells were seeded into 96-well plates and allowed to reach confluence. An area clear of cells was made using a 0.5 mm needle, followed by addition of HGF/SF (10 ng/ml) or fibroblast-conditioned medium and/or NK4 (200 ng/ml) (molar ratio NK4:HGF/SF 40:1). Mineral oil was used to overlay the medium and the cells were viewed microscopically on a heated (37°C) stage for 2 h whilst being recorded with a time-lapse video recorder. Decreases in distance between the two leading fronts of cells were assessed at 5 min intervals using Optimas.
Tumour cell invasion assay
Invasiveness of MDA MB 231 breast cancer cell line was assessed using this in vitro assay. Transwell chambers equipped with 6.5 mm diameter polycarbonate filter (pore size 8 µm) (Becton Dickinson Labware, Oxford, UK) were pre-coated with 50 µg/membrane of solubilized basement membrane in the form of Matrigel (Collaborative Research Products, Bedford, MA). After membrane re-hydration, 15 000 cells were aliquoted into each insert with HGF/SF (10 ng/ml) or fibroblast-conditioned medium and/or NK4 (200 ng/ml) (molar ratio NK4:HGF/SF 40:1). After 96 h co-culture non-invasive cells were removed with cotton swabs. Invasive cells on the underside of the insert were fixed and stained with crystal violet, followed by microscopic counting (20 fields/insert).
HGF/SF bioassay
A MDCK cell scatter assay was used to determine the bioactivity of active HGF/SF secreted from MRC5 fibroblast cells. Fibroblast cells were seeded into 25 cm2 tissue culture flasks at semi-confluence and allowed to reach full confluency. After washing the flasks with BSS buffer, 4 ml of either complete medium (with 10% foetal calf serum, for cell migration, invasion and scatter assay) or serum-free medium (for HGF/SF receptor activation assay) was added to the flasks for a further 24 h. Following centrifugation to remove cell debris, the conditioned medium was stored at -20°C until used. MDCK cells were seeded on a 96-well plate and the serially diluted fibroblast-conditioned medium added, with recombinant HGF/SF as an internal standard. After 24 h, cells were fixed, stained with crystal violet and scattering assessed. HGF/SF concentration is taken from the highest dilution of fibroblast-conditioned medium that causes scattering, which is compared with the HGF/SF standard.
Tumour study in nude mice
For the nude mice tumour model, MDA MB 231 breast cancer cells (1 x 106 cells) in Matrigel (0.5 mg/ml) were s.c. injected into the interscapular region of 7-week-old female athymic nude mice (CD-1; Charles River Laboratories, UK). Human fibroblasts (fibroblast cell line MRC5) (5 x 105) were co-injected (with MDA MB 231 cells) as a source of bioactive HGF/SF. Following injection of cells, HGF/SF and/or NK4 were delivered by implantation of osmotic mini-pumps having a constant release rate over a period of 4 weeks (experimental end-point) or until termination was required (tumours >1 cm in diameter or weight loss >25% of total body weight, in accordance with UK Home Office Regulations). For implantation of the osmotic pumps, the nude mice were anaesthetized (halothane) and Alzet minipumps (model 2004) containing BSA (1 mg/ml as a protein control), HGF/SF (40 µg/kg/day), NK4 (400 µg/kg/day) or HGF/SF and NK4 were implanted s.c. in the right flank after a single lateral incision. The combination allows 400 µg/kg/day of NK4 and 40 µg/kg/day of HGF/SF, giving a molar ratio of 20:1 (NK4:HGF/SF), to allow maximum inhibition in vitro (2426). The nude mice were kept in sterilized, filtered cages and under 12 h dark/light standardized environmental conditions throughout the experiments. The mice were weighed twice weekly according to the Home Office guidelines. Tumour size was measured using digital callipers, twice weekly. The tumour volume was calculated as 0.512 x width2 x length (mm3). Weight of the tumour was determined after dissection at the end of the experiment and was found to correlate with the final volume (Spearman correlation coefficient 0.93).
Immunohistochemical staining of primary tumours
On excision, primary tumours were immediately immersed in a fixation solution of neutral buffered formalin for 48 h, followed by processing on a Reichert-Jung automatic tissue processor (24 h cycle) before embedding in pastillated paraffin wax (congealing point 5760°C). Sections (7 µm) were cut and placed on Super Frost Plus slides and incubated overnight at 60°C. Sections were stained using anti-VE-cadherin, an endothelium-specific cell adhesion molecule (28) or vimentin (to enable visualization of fibroblasts in the sections). The sections were prepared for immunohistochemical staining by treating with xylene and absolute ethanol, with endogenous peroxidase activity removed by subsequent submersion in H2O2/ethanol mix and washing with H2O. Antigen enhancement of the sections was carried out using a solution of EDTA (pH 8.0) in a microwave for 20 min. Sections were then washed and treated with horse serum for 20 min as a blocking agent to non-specific binding. VE-cadherin primary antibody was used (1:400) for 45 min and then the sections were washed in buffer. The secondary antibody (biotinylated) was added (in horse serum/buffer solution) for 30 min, followed by numerous washings. Avidin/biotin complex was added for 30 min, again followed by washes. Diaminobenzadine was used as a chromogen to visualize the antibodyantigen complex. Sections were counterstained in Mayer's haematoxylin for 1 min, dehydrated, cleared and mounted in DPX and screened using a x40 objective.
Angiogenesis screening criteria for VE-cadherin stained primary tumours
Four researchers carried out screening independently. Dark brown granules in interstitial spaces demonstrated VE-cadherin positive staining: staining was discounted if it was only found on the cell surface; lumen staining was not necessary for positive identification of a blood vessel. Five non-overlapping fields were screened after locating and identifying a definite blood vessel within the stromal tissue with which to compare staining intensity.
Percentage of fibroblast component in primary tumours
Sections were screened independently by four researchers. Dark brown coloured cells denoted positive staining. Five non-overlapping fields were chosen at random. The number of positively stained cells (fibroblasts) and total number of cells were counted under a high power field using a bright field microscope. The percentage of fibroblasts in the tumour was calculated as (no. of positive cells)/(total no. cells) x 100.
Statistical analysis
Statistical analysis was performed with MINITAB version 9.2 (Minitab Inc., State College, PA) using a two sample Student's t-test and Microsoft Excel for 2 tests and Microsoft Excel (97) for correlation coefficients and
2 tests.
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Results |
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NK4 decreased HGF/SF-induced breast tumour growth in nude mice
Over the 4 week period, none of the mice displayed significant weight loss (>10% of body weight). No noticeable side effects were observed. However, in HGF/SF only and fibroblasts/MDA MB 231 groups, tumour size in two of the mice exceeded 1 cm in diameter. These mice were eliminated from the final analysis. In the animal model, tumour weight (g) in the control group (cancer cells only, n = 6) was 0.508 ± 0.18 (SE) g, in the HGF/SF group 0.734 ± 0.17 g (n = 8), and in the NK4-treated group this weight was reduced to 0.258 ± 0.08 g (P = 0.02 versus control) (Figure 4A). NK4 significantly reduced HGF/SF-induced tumour growth [NK4 + HGF/SF, 0.225 ± 0.052 g (n = 6), P = 0.01 versus HGF/SF]. The antagonist markedly retarded fibroblast-induced tumour growth [NK4 + fibroblasts 0.261 ± 0.12 g (n = 6), P = 0.01 versus fibroblasts alone, 0.771 ± 0.16 g (n = 6)].
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In the group in which mice were co-injected with MDA MB 231 cell and MRC5 fibroblasts, mice generally bore bigger tumours, compared with those who received MDA MB 231 cell injection only (1201 ± 180 versus 721 ± 172 mm3, respectively) (Figure 4B). The increased tumour volume in the fibroblast only group could be partly attributed to the co-injected fibroblast cells, as shown by immunohistochemical staining of vimentin in these tissues. There was no significant difference in the percentage of fibroblasts in those tumours in the control group (cancer cells alone) compared with those groups where fibroblasts were co-injected with cancer cells: control group (cancer cells alone) 8.14 ± 7.22% stained fibroblasts; cancer cells plus fibroblasts 14.40 ± 11.75% stained fibroblasts; cancer cells plus fibroblasts treated with NK4 13.25 ± 8.56% stained fibroblasts (P = 0.88). Again, the NK4 antagonist markedly retarded fibroblast-induced tumour growth to 256 ± 64 mm3 (P = 0.007 versus fibroblasts alone, n = 6).
NK4 decreased HGF/SF-induced tumour angiogenesis in nude mice
Primary tumour sections were stained with anti-VE-cadherin in order to observe tumour angiogenesis by counting the number of vessels. VE-cadherin staining was decreased in those mice that had been administered NK4 (Figure 5). In the control group (cancer cells only, Figure 5A) staining was found in 10.50 ± 2.9 (SE) vessels, in the HGF/SF group in 18.60 ± 2.5 (Figure 5C) and in the group treated with NK4 alone (Figure 5B) 4.17 ± 1.2 (P = 0.05 versus control). NK4 suppressed HGF/SF-induced angiogenesis (Figure 5D) to 2.5 ± 0.87 vessels (P = 0.001 versus HGF/SF alone, n = 5). Human fibroblasts, acting as a natural source of HGF/SF, enhanced angiogenesis (to 18.00 ± 1.8 vessels) (Figure 5E). Again, the NK4 antagonist markedly retarded fibroblast-induced angiogenesis (Figure 5F) to 4.75 ± 1.4 vessels (P = 0.001 versus fibroblasts alone, n = 4), shown graphically in Figure 6.
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Discussion |
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One of the most interesting discoveries in the current study is that NK4 reduced fibroblast-induced breast cancer growth (in both weight and volume) in the in vivo nude mouse model. In our tumour model, MRC5 fibroblasts, when co-injected with breast cancer cells, markedly increased the growth of breast tumour tissue. We view this increase in tumour growth as largely due to the tumour stimulating factors, including HGF/SF, produced by the MRC5 fibroblast cells, as co-injected fibroblasts only contributed to a marginal increase in vimentin-positive cells (fibroblasts) in the tumours. Thus, we have demonstrated an important role of NK4 in the control of stromal fibroblast-induced tumour growth.
Some reduction in tumour growth was observed in the NK4 only group: the murine met receptor is very similar to human c-Met (>85% homology) and human and murine HGF/SF share homology of >90% (31). We can thus infer that NK4 was also acting by inhibiting HGF/SF produced by mouse stromal cells themselves. This is the first study in which human fibroblasts have been used as a source of HGF/SF. As most tumour cells are surrounded by stroma, interaction between the stroma and the malignant cells is extremely important in the development of tumour angiogenesis (32). As most cancers, especially breast cancers, are surrounded by stromal cells that have a significant effect on cancer cells, NK4 inhibition of fibroblast-induced angiogenesis and tumour growth is of particular relevance. Indeed, as breast cancer cells express a higher level of the HGF/SF receptor (c-Met) at the protein and mRNA levels, an agent such as NK4 that can block the activation of this important proto-oncogene is a valuable tool in the prevention of tumour growth (33). Stromal fibroblasts surrounding tumours in breast cancer patients express higher than normal amounts of bioactive HGF/SF, thus, the levels of HGF/SF and its receptor are among the most relevant factors in the prognosis of patients with breast cancer (17,34,35).
NK4 affected a decrease in tumour growth by reducing tumour angiogenesis, as indicated by immunochemical staining of the endothelium-specific adhesion molecule VE-cadherin. Metastasis of cancer cells proceeds via a number of steps: tumour cells must first invade the surrounding stroma and initialize angiogenesis and then the tumour must develop, which requires transport of nutrients to and removal of waste products from the tumour site (37). For tumours to continue to grow, a connection must be made to the blood supply. The blood vessels within the tumour can then provide a route for detached tumour cells to enter the circulatory system and metastasize to distant locations (37,38). To metastasize, the detached tumour cells must enter the blood circulation, survive the immune system and arrive at a distant site. HGF/SF is a known angiogenic factor and this aspect of HGF/SF has particular relevance in cancer, in which an increased level of this cytokine is proposed to be related to the invasiveness and metastatic ability of such cancers (33). HGF/SF has been shown to regulate motility, proliferation, morphogenesis and cancer cell adhesion to the endothelium within tumours. It has been shown to be a powerful inducer of angiogenesis in cancer cells (7) and a stimulator of endothelial cell motility and migration in vivo. HGF/SF has been shown to modulate the expression of the endothelial cellcell adhesion molecule VE-cadherin (39,40). HGF/SF-induced tumour angiogenesis is therefore an important target for anticancer therapies.
In vitro studies have shown that NK4 inhibits HGF/SF-enhanced human microvascular endothelial cell proliferation and migration (24). Angiogenesis of human vascular endothelial cells has also been shown to be inhibited by NK4 (33). More recently, in vivo animal studies have shown that NK4 is effective against HGF/SF-enhanced invasion of gall bladder cancer cells and increased apoptotic death of these cells, resulting in retarded tumour growth (21). NK4 has also been effective against HGF/SF-induced growth, invasion and metastasis of human pancreatic cancer cells in nude mice (41) and against HGF/SF-enhanced tumour growth and metastasis of both Lewis lung carcinoma cells and Jug-MC(A) mammary cancer cells in nude mice (24). Our data, together with others, indicates that NK4 is strongly anti-angiogenic and that this effect contributes to the anti-tumour-growth effect of NK4 in vivo.
This study has shown that the HGF/SF antagonist NK4 inhibits HGF/SF-induced increases in breast cancer cell migration and invasion, as assessed by in vitro methods. NK4 has been shown to inhibit the matrix adhesion and invasion of numerous prostate cell lines by preventing phosphorylation of paxillin (25). Spreading and invasion of human pancreatic cancer cells is also inhibited by this molecule (42). NK4 inhibits HGF/SF-induced breast cancer cell motility (43) and colon cancer cell, gall bladder cancer cell, cholangiocarcinoma cell and human uterus cervical carcinoma cell motility and invasion (21,44).
The concentrations of HGF/SF and NK4 used in this study were devised to maximize the inhibitory effect of NK4. This molecule is effective as it exhibits competitive inhibition with HGF/SF with regard to binding the Met receptor. Thus, higher levels of NK4 are required in order to successfully prevent binding and activation of the c-Met receptor by HGF/SF. Previous in vitro studies have shown that up to 10 times as much NK4 may be required to fully inhibit the effect of HGF/SF (31,43,44). Here, we used NK4 at a 20-fold higher concentration than the HGF/SF administered to the mice.
Levels of HGF/SF in human breast cancer tissues have been measured by Yamashita et al. (17,35) and were found to be between 1.4 and 306.5 ng/100 mg protein, with a median level of 11.2 ng. Blood and serum levels of HGF/SF in normal volunteers ranged from 0.46 ± 0.04 to 1.18 ± 0.12 ng/ml in whole blood (45) and from 0.1999 ± 0.073 to 0.847 ng/ml in serum (46,47). In patients with gastric cancer, serum levels were raised to 0.578 ± 0.258 ng/ml (45). Previous in vivo studies used NK4 administered at 1.5 mg/kg/day (40) and 25 µg/day, or 1 mg/kg/day (24).
Finally, this study has further confirmed that NK4 is a complete antagonist to HGF/SF. NK4 at very high concentrations has no effect on activation of the HGF/SF receptor, c-Met, but can inhibit HGF/SF-induced receptor activation. The molecular events downstream of the HGF/SF receptor include the activation of a number of signalling complexes in the cells, such as the mitogen-activated protein kinase, phospholipase C- and phosphoinositide kinase-3 pathways (48). We and others have also shown that HGF/SF can increase tumour matrix adhesion by activating the FAKpaxillin pathway (48). The current study has further demonstrated that NK4 can inhibit HGF/SF-induced phosphorylation of paxillin in human breast cancer cells.
Preventing tumour angiogenesis is an attractive target for cancer therapies. Although anti-angiogenesis therapies would be most effective against cancer cells of a more invasive, metastatic nature, they may also be effective against angiogenesis-dependent tumour invasion (49). The results presented here indicate that NK4 acts by reducing the migration and potential invasiveness of cancer cells and by decreasing tumour angiogenesis, thereby reducing the size of primary tumours. This study, for the first time, shows that NK4 can inhibit the effect of HGF/SF both as a human recombinant form and as a bioactive form from human fibroblasts, also showing that fibroblast production of HGF/SF is a major contributory factor on the growth of breast cancers. We conclude that NK4 has a significant effect on the growth of human breast tumours in nude mice, particularly when stimulated by HGF/SF or fibroblasts. This gives a clear indication of the therapeutic value of NK4.
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Acknowledgments |
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References |
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