The neurotrophin receptor TrkB cooperates with c-Met in enhancing neuroblastoma invasiveness
Monica Hecht 1, *,
Johannes H. Schulte 2,
Angelika Eggert 2,
Joerg Wilting 1 and
Lothar Schweigerer 1
1 Department of Pediatrics, University of Goettingen, Robert-Koch-Strasse 40, D-37075 Goettingen, Germany and 2 University Children's Hospital of Essen, Hufelandstrasse 55, D-45122 Essen, Germany
* To whom correspondence should be addressed at: Department of Oncology and Hematology, University Hospital Charite, Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany. Tel: +49 30 450513303; Fax: +49 30 450513960; Email: monica.hecht{at}charite.de
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Abstract
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Neuroblastoma is the most frequent extracranial solid malignancy of childhood with a high mortality in advanced tumour stages. The hallmark of neuroblastoma is its clinical and biological heterogeneity. The molecular mechanisms leading to favourable or unfavourable tumour behaviour are still speculative. However, amplification of the oncogene MYCN and expression of the neurotrophin receptor TrkB are known to contribute to a highly malignant phenotype. To define the mechanisms through which TrkB may mediate neuroblastoma progression, we stably expressed this receptor in the neuroblastoma cell lines SH-SY5Y and SK-N-AS. The transfectants, but not the controls, had an increased invasive potency both, in vitro and in vivo, as demonstrated by Matrigel-invasion and chorioallantoic membrane assays, respectively. The retinoic acid-induced TrkB expression in parental SH-SY5Y cells was also associated with enhanced cell invasiveness. The TrkB mediated invasiveness involved the upregulation of the hepatocyte growth factor (HGF) and its receptor c-Met, resulting in an autocrine loop. Inhibition of HGF activity by anti-HGF neutralizing antibodies or disabling the function of c-Met by small interfering RNA suppressed the TrkB-induced invasiveness. The enhanced TrkB expression was associated with a significant increase in the secretion of various matrix-degrading proteases. Immunostaining and real-time RTPCR analysis of tumour specimens demonstrated coordinated expression of TrkB and HGF/c-Met in experimental and primary neuroblastomas. We conclude that TrkB expression in neuroblastoma cells results in an increase in their invasive capability via upregulated expression of HGF/c-Met and enhanced activity of proteolytic networks.
Abbreviations: BDNF, brain-derived neurotrophic factor; CAM, chorioallantoic membrane; Ct, threshold cycle; HGF, hepatocyte growth factor; IgG, immunoglobulin G; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; RA, retinoic acid
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Introduction
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Neuroblastoma represents the most common extracranial solid neoplasm found in children and is derived from the neural crest. Although localized and well differentiated tumours can be successfully treated by surgical resection with or without chemotherapy, the prognosis of regionally invasive (stage 3) or metastatic tumours (stage 4) remains poor (1).
Expression of neurotrophin receptors of the Trk family is an important prognostic factor in neuroblastomas. The Trk family is composed of three related proteins, TrkA, TrkB and TrkC, to which the neurotrophins bind differentially (2). Despite the high degree of sequence similarity and common signalling pathways, activation of different Trk receptors in neuroblastomas leads to divergent clinical behaviour (3). One possible explanation is the induction of differential gene and protein expression patterns by TrkA or TrkB receptors leading to favourable or unfavourable prognosis, respectively (4,5). However, the cross-talk between Trk proteins and other receptors on the surface of neuroblastoma cells may determine the specificity of the biological outcome.
TrkB is a 145 kDa cell surface receptor with intrinsic kinase activity: it dimerizes and autophosphorylates cytoplasmic tyrosine residues upon binding of its specific ligand brain-derived neurotrophic factor (BDNF) (6). TrkB is preferentially expressed in neuroblastomas with poor prognosis together with BDNF, conferring invasive and metastatic potency to the tumour cells (7,8). In addition, BDNF/TrkB signalling in neuroblastoma cells has been shown to enhance therapy resistance and to suppress anoikis (911). However, the molecular mechanisms governing the TrkB-induced progression of neuroblastomas are largely unknown.
This study was undertaken to analyse molecules that are regulated by TrkB and to elucidate the molecular pathways contributing to the invasive phenotype of TrkB-expressing neuroblastomas. We have previously shown that highly invasive neuroblastoma cells express c-Met, the receptor of hepatocyte growth factor (HGF). The presence of paracrine HGF/c-Met signalling was associated with increased neuroblastoma invasiveness in vitro and in vivo (12). c-Met, a transmembrane receptor with intrinsic tyrosine kinase activity, is synthesized predominantly by epithelial cells as a 190 kDa precursor protein, which is proteolytically processed into a mature heterodimer consisting of a 50 kDa extracellular
subunit and a 140 kDa catalytic ß subunit (13). HGF, a fibroblast-derived soluble factor, is secreted as an inactive single chain precursor (proHGF) and converted to the heterodimeric active form by serine proteases, such as plasmin (14). c-Met signalling is mediated by autophosphorylation of cytoplasmic tyrosine residues: two of them are a part of the C-terminal multifunctional docking site, which interacts with several cytoplasmic signal transducers. This occurs either directly, such as with the PI 3-kinase (15) and SHIP-1 (16), or indirectly via molecular adapters, such as Grb2 (15) and Shc (17).
Since both c-Met and TrkB are associated with high malignancy of neuroblastomas, we sought to determine if there is any cooperation between the two receptors. The aim was to gain deeper insights into TrkB-mediated cell invasion and consequently neuroblastoma progression.
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Materials and methods
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Cell culture and transfection
Dishes and medium used for cell culture were from Invitrogen (Carlsbad, CA). All cells were grown in RPMI 1640 supplemented with 10% fetal calf serum.
SH-SY5Y and SK-N-AS neuroblastoma cells were obtained from Dr G.Brodeur (The Children's Hospital of Philadelphia, University of Pennsylvania). SH-SY5Y is a neuronal subclone from the neuroblastoma cell line SK-N-SH (18). The full length TrkB cDNA was cloned into the retroviral expression vector pLNCX2 (Clontech, Palo Alto, CA). SH-SY5Y cells were infected with a retrovirus bearing the TrkB-vector construct or the empty vector as described (19). In brief, the TrkB containing vector or the empty vector was transfected into the packaging cell line Bing by electroporation. Virus containing supernatants from these cells added to LipoTaxi (Stratagene, La Jolla, CA) were used to infect SH-SY5Y cells. Transfected cells were selected with geneticin (500 µg/ml; Sigma, St Louis, MO) and subcloned by limiting dilution to obtain clones derived from single cells. SK-N-AS cells were transfected with Superfect reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Transfected cells were selected with geneticin (400 µg/ml; Sigma) and subcloned by limiting dilution to obtain single clonal cell lines. The identity of all transfectants was confirmed by sequencing of the TrkB cDNA after transfection.
c-Met gene silencing was established by transfection of high performance validated siRNA (target region 483533 of human c-Met) from Qiagen into neuroblastoma cells. A human/mouse RNAi control kit (Qiagen) was used in preliminary experiments to optimize the efficiency of transfection and gene silencing. Cells were seeded in 6-well plates and transfection with RNAiFect was performed according to the manufacturer's instructions (Qiagen). Transfection efficiency was monitored using Alexa 488-labelled non-silencing siRNA. Expression levels of c-Met and actin were analysed by real-time RTPCR and western blotting. All transfection experiments were performed in triplicate.
Collection of conditioned medium
Supernatants for HGF determination were collected as described previously (12).
HGF enzyme-linked immunosorbent assay
To determine HGF concentrations in conditioned medium, the Quantikine human HGF immunoassay (R&D Systems, Minneapolis, MN) was used according to the manufacturer's protocol.
Real-time RTPCR
Relative quantification of mRNA expression for TrkB, HGF, c-Met, matrix metalloproteinases MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, uPA and tPA was performed with SYBR Green PCR Master Mix (Eurogentec, Seraing, Belgium) using a thermal cycler ABI PRISM 7700 (Perkin-Elmer-Applied Biosynthesis, Foster City, CA). Total RNA was isolated from neuroblastoma cell lines and primary tumour samples with the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Aliquots of 1 µg of total RNA, random primers and Moloney murine leukaemia virus reverse transcriptase (Invitrogen) were used for cDNA synthesis. The PCR was done with cDNA aliquots and primer sequences specific for human TrkB, HGF, c-Met, MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, uPA, tPA and ß-actin. Primer sequences are available upon request. Relative quantification of target gene expression was calculated by the comparative threshold cycle (Ct) method. The value of target, normalized to an endogenous control (ß-actin) and relative to a calibrator (control transfected cells), is expressed as 2
Ct (fold), where
Ct = Ct of the target Ct of ß-actin, and 
Ct =
Ct of samples for target
Ct of the calibrator for the target.
Immunoprecipitation and western blot analysis
Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed with lysis buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% Triton, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate, 10 µg/ml leupeptin and 10 µg/ml aprotinin). The crude lysate was centrifuged at 16 000 g for 30 min at 4°C to remove cell debris. An aliquot of 500 µg of precleared lysate was incubated with monoclonal antibody against phosphotyrosine (4G10, Upstate Biotechnology Incorporated, Charlottesville, VA), previously conjugated to protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h at 4°C. Immunoprecipitates were washed three times with washing buffer (20 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.1% Triton, 10% glycerol, protease and phosphatase inhibitors as in the lysis buffer) and eluted with Laemmli buffer. Samples were boiled for 5 min at 95°C.
The cell lysates (20 µg protein per lane) or immunoprecipitates were separated on 7.5% SDSpolyacrylamide gels. The fractionated proteins were transferred to a polyvinylidendifluoride membrane (Millipore, Bedford, MA). After blocking, filters were probed with primary antibodies and proteins were visualized with peroxidase-conjugated secondary antibodies using an enhanced luminescence detection system (Amersham Biosciences, Piscata Way, NJ). Anti-c-Met monoclonal antibody, anti-phosphotyrosine monoclonal antibody (4G10) and anti-phospho-c-Met (Tyr 1234/1235) polyclonal antibodies were from Upstate Biotechnology Incorporated. Anti-panTrk, anti-c-Met (C28) and anti-actin polyclonal antibodies were from Santa Cruz Biotechnology. Monoclonal antibodies against MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13 and uPA were purchased from Calbiochem (San Diego, CA). tPA-specific antibody was from Acris Antibodies (Hiddenhausen, Germany). Secondary antibodies were peroxidase-conjugated anti-mouse immunoglobulin G (IgG) or anti-rabbit IgG (Dako, Copenhagen, Denmark).
Zymography
Cells were grown to 70% confluence in 25 cm2 flasks, washed twice with serum-free RPMI and cultured with serum-free medium containing insulin (5 µg/ml), transferrin (5 mg/ml) and selenium (5 ng/ml) (Sigma, MO) for 48 h. Supernatants were collected and centrifuged to remove cellular debris followed by concentration with the Centricon 10 system (Amicon, Bedford, MA). Proteases were detected as described previously by us (12).
Matrigel invasion assay
Assays were performed according to Albini et al. (20). Briefly, transwell chambers with 8 µm-pored polycarbonate filters coated with Matrigel were used (Becton-Dickinson, San Diego, CA). Cells (1.5 x 104) were seeded in serum-free medium in the upper compartment of each chamber. Medium with 10% serum was added to the lower compartment. In some cases, anti-HGF neutralizing antibodies (40 µg/ml), anti-BDNF neutralizing antibodies (20 µg/ml; Oncogene Research, San Diego, CA) or isotype controls were added to the upper compartment at the beginning of the experiment. After 30 h of culture, the cells, which had invaded to lower side of the Matrigel-coated filter, were stained with hematoxylin/eosin and visualized microscopically at 100x magnification. Cells were counted at 400x magnification in 10 representative areas per filter. The experiments were carried out in duplicate and three independent sets of experiments for each type of invasion assay were performed.
Chorioallantoic membrane assay
Cultured neuroblastoma cells were passaged 48 h prior to the experiment. Confluent cultures were harvested, washed once and resuspended in medium without geneticin. 3 x 106 cells were resuspended in 50 µl medium and inoculated inside a Thermanox ring on the chorioallantoic membrane (CAM) of an 8-day-old quail embryo. For this purpose, a window was made into the eggshell of 3-day-old embryos and sealed with Durapore tape. The embryos were reincubated at 37.8°C and 80% relative humidity until day 8, when the inoculation occurred. On 6 days post-inoculation, tumours were harvested and photographed under a dissection microscope.
Immunofluorescence
Cells were cultured at low confluence on plates of 1 cm diameter, washed with cold PBS and fixed with 4% paraformaldehyde. Non-specific binding was blocked by incubation with 1% BSA for 20 min. Polyclonal anti-c-Met antibodies (1:100; Santa Cruz Biotechnology) were added to the cells in a hydrated chamber for 1 h at room temperature. After three washes with PBS for 5 min, secondary Cy3-conjugated goat anti-rabbit IgG (1:200; Molecular Probes, Eugene, OR) was added for 1 h at room temperature. After rinsing, dishes were mounted with fluorescent mounting medium (SouthernBiotech, Birmingham, UK) and analysed with an epifluorescence microscope (Leica, Bensheim, Germany).
Tumours were fixed in Serra's solution [60% absolute ethanol, 30% paraformaldehyde (37%), 10% glacial acetic acid] and embedded in paraplast plus tissue embedding medium (Kendall, Mansfield, MA). Sections of 8 µm thickness were cut, mounted on slides and dried overnight at 45°C. Sections were dewaxed with Roti-Histol (Roth, Karlsruhe, Germany), rehydrated with a graded series of ethanol and finally washed with PBS for 5 min. Non-specific binding was blocked by incubation with 1% BSA for 20 min at room temperature. Sections were then incubated simultaneously with anti-c-Met C28 antibody (1:100; Santa Cruz) and anti-HGF antibody (1:20; R&D Systems) for 1 h. After rinsing, the secondary Alexa Fluor 594-labelled donkey anti-rabbit IgG and Alexa Fluor 488-labelled donkey anti-goat IgG (1:200; Molecular Probes) were applied for 1 h. Slides were rinsed twice, mounted under coverslips and sections were studied with an epifluorescence microscope (Leica).
Immunohistochemistry
Tumours were fixed, cut, mounted, dewaxed and rehydrated as described for immunofluorescence. In order to inhibit endogenous peroxidase activity, sections were incubated for 20 min at room temperature in methanol containing 0.3% hydrogen peroxide. After rinsing with PBS, sections were incubated with the primary antibody solution for 1 h at room temperature. TrkB expression was analysed with polyclonal anti-TrkB 794 antibodies (1:100; Santa Cruz). c-Met immunoreactivity was monitored with polyclonal anti-c-Met C28 antibodies (1:100; Santa Cruz). Endothelial cells were highlighted with QH1 antibody against endothelial and hematopoietic cells of the quail [1:100; (21)]. Slides were rinsed twice with PBS and incubated with appropriate peroxidase-conjugated secondary antibodies (1:200; Sigma) for 1 h at room temperature. After rinsing, immunoreaction was demonstrated by incubating the sections with the chromogene peroxidase substrate diaminobenzidine tetrahydrochloride (DAB; Sigma) for 10 min at room temperature. Finally, sections were counterstained with nuclear fast red for 20 s, dehydrated with a graded series of ethanol and mounted. Negative controls used all reagents except the primary antibodies and did not reveal a signal.
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Results
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TrkB expression and autophosphorylation in TrkB transfected neuroblastoma cells
We stably expressed the full-length construct of TrkB or the empty-vector control (Vec) into the human neuroblastoma cell lines SH-SY5Y and SK-N-AS as described in Materials and methods. Stable transfectants were selected by geneticin and further subcloned to single cell clones. Expression of functional TrkB receptors was examined by real-time RTPCR and immunoblotting. Parental and control-transfected SH-SY5Y and SK-N-AS cells did not express significant amounts of TrkB mRNA or any detectable levels of TrkB protein (Figure 1A and B). In contrast, the TrkB transfectants G7, T6 and T9 demonstrated high-level expression of TrkB (Figure 1A and B). We performed our further studies with the representative TrkB overexpressing cell lines G7 and T6. In addition to the highly TrkB expressing cells, we also used clones with moderate levels of TrkB expression (e.g. G12 and T4; data not shown).

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Fig. 1. Expression and phosphorylation of TrkB in neuroblastoma cell lines as determined by real-time RTPCR analysis and western blots. (A) TrkB mRNA expression in the TrkB-transfected clones G7, G12, T3, T4, T6 and T9. The real-time RTPCR analysis with actin expression as a control was done as described in Materials and methods. The empty-vector transfected cells were designated as a calibrator. (B) Expression of TrkB protein in G7, T3 and T6 cells detected by immunoblotting with anti-pan Trk antibodies. The membranes were stripped and reprobed with antibodies against actin to control equal loading. (C) Basal and ligand-induced autophosphorylation of TrkB in TrkB overexpressing cell lines G7 and T6. The indicated cell lines were serum-starved overnight and treated with (+) or without () BDNF (50 ng/ml) for 15 min. Equal amounts of total protein lysates (500 µg) were immunoprecipitated with anti-phosphotyrosine specific antibodies (4G10) and subjected to SDSPAGE and western blotting with anti-pan Trk antibodies.
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To examine whether the TrkB signalling pathway is active in the transfected cells, we analysed the capability of exogenously applied BDNF to induce rapid autophosphorylation of receptor tyrosine residues. Autophosphorylation of TrkB was examined by immunoprecipitation with anti-phosphotyrosine antibodies followed by western blotting with anti-pan Trk antibodies. In the absence of the exogenous ligand autophosphorylation of TrkB was detected in both clones G7 and T6, suggesting the existence of an autocrine BDNF/TrkB loop. In contrast, there was no detectable TrkB autophosphorylation in empty-vector transfected cells. Treatment with BDNF for 15 min significantly increased the phosphorylation levels of the TrkB receptor in both cell lines G7 and T6 (Figure 1C).
Expression of HGF and its receptor c-Met in TrkB transfectants
In a further set of experiments, we compared the expression profiles of stable TrkB transfectants and control cells (empty-vector transfectants) by oligonucleotide microarray analysis as described (4). The majority of the differentially expressed genes were annotated to the fields of signal transduction, tumourmatrix interactions and cytoskeleton rearrangement. In the field of tumourmatrix interaction, we identified the HGF as an upregulated gene in TrkB-expressing SH-SY5Y cells. We proved the gene expression results on mRNA and protein levels and observed an upregulation of HGF in both TrkB-overexpressing cell lines, G7 and T6 (Figure 2A and B). The HGF receptor c-Met was also strongly expressed in T6 and G7 cells (Figure 2A and C). In contrast, parental SH-SY5Y and SK-N-AS cells as well as control transfectants did not express c-Met, as demonstrated by western blot analysis (Figure 2C).

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Fig. 2. Expression of HGF and its receptor c-Met in TrkB overexpressing neuroblastoma cells. (A) HGF and c-Met mRNA expression in TrkB-transfected SH-SY5Y (clone G7) and SK-N-AS (clone T6) cells detected by real-time RTPCR analysis. Control-transfected cells were designated as a calibrator. (B) HGF immunoreactivity in supernatants of parental, control-transfected and TrkB-transfected SH-SY5Y and SK-N-AS cells. The indicated cell lines were cultured with serum-free medium for 48 h. HGF concentrations in the conditioned media were determined by HGF-ELISA. Values correspond to supernatants of 104 cells and are the means of duplicate determinations, which varied by <10% of the mean. (C) Protein expression and tyrosine phosphorylation of c-Met in TrkB overexpressing neuroblastoma cells. Lysates from the indicated cell lines equalized for total protein were subjected to SDSPAGE and western blotting with antibodies against c-Met and actin. For assaying the phosphorylation status of c-Met, the indicated cell lines were serum starved overnight. Lysates were analysed by western blotting with antibodies against phospho-c-Met (Tyr1234/1235). (D) Expression of c-Met in TrkB overexpressing G7 and T6 cells detected by immunofluorescence staining. Subconfluent monolayers of control- and TrkB-transfected SH-SY5Y and SK-N-AS cells were stained with an antibody against the C-terminus of c-Met and analysed by fluorescence microscopy. The photographs show membrane and cytoplasmic localization of c-Met.
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The co-expression of endogenous HGF and c-Met appeared to represent an autocrine loop in TrkB expressing cells. We therefore investigated whether endogenous HGF was able to activate the c-Met receptor and to initiate c-Met signal transduction in TrkB-transfected cells. Autophosphorylation of c-Met was examined by western blot with anti-phospho-c-Met antibodies (Tyr 1234/1235). The TrkB-expressing cell clones G7 and T6 showed expression of phosphorylated c-Met, suggesting that the autocrine HGF/c-Met loop was constitutively active in these cells (Figure 2C).
In addition, we analysed protein expression and cellular localization of c-Met in TrkB expressing cells by immunostaining. Figure 2D shows representative photomicrographs of c-Met stained neuroblastoma cells. The TrkB transfectants G7 and T6, but not the controls (SH-SY5Y/Vec; SK-N-AS/Vec) displayed strong cytoplasmic and membrane staining of the c-Met receptor.
Effects of TrkB and HGF/c-Met expression on neuroblastoma cell invasiveness
Furthermore, we investigated whether the autocrine HGF/c-Met loop was related to invasion of TrkB-expressing cells into Matrigel. TrkB-transfected SH-SY5Y and SK-N-AS cells demonstrated enhanced invasiveness (5- and 6-fold, respectively) compared with control transfected cells (Figure 3A and D). Interestingly, the invasive potential could be blocked significantly by treatment with neutralizing antibodies against HGF, suggesting that the constitutively activated HGF/c-Met loop in TrkB expressing neuroblastoma cells is the key player of their invasiveness (Figure 3A).

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Fig. 3. Expression of TrkB enhances the invasiveness of neuroblastoma cells. (A) Matrigel invasion assay of the indicated cell lines in the presence or absence of anti-HGF neutralizing antibodies. Cells were seeded in serum-free medium in the upper compartment of the invasion chamber, whereas 10% serum-containing medium was added to the lower compartment. Anti-HGF neutralizing antibodies (40 µg/ml) were applied as indicated. Representative photographs of three independent experiments taken at 100x magnification show cells invading the Matrigel and passing through the porous membrane. Treatment with anti-HGF antibodies inhibited the enhanced invasiveness of TrkB overexpressing cells. (B) Matrigel invasion assay after c-Met gene silencing. Reduction of c-Met expression was associated with impairment of cell invasiveness. Expression of c-Met mRNA and protein was analysed by real-time RTPCR and western blotting. (C) Effect of RA treatment on TrkB, HGF and c-Met expression and on neuroblastoma cell invasion. SH-SY5Y cells were treated with 5 nM RA and with solvent control for 5 days. mRNA expression was analysed by real-time RTPCR. The solvent treated cells were designated as a calibrator. The invasion assays were performed after 4 days of treatment with 5 nM RA. SH-SY5Y cells were placed in the upper compartment of a transwell chamber, BDNF (100 ng/ml) or HGF (100 ng/ml) in complete media were added to the lower wells and cells were incubated for 24 h at 37°C. (D) Graphical illustration of the number of invading cells under the conditions described for Figure 3AC. Columns B1 and B2 represent the number of invading G7 cells after transfection with non-silencing control siRNA and c-Met specific siRNA, respectively. Cells were counted with 400x magnification in 10 representative fields per well. (E) RTPCR analysis for TrkB, HGF and c-Met mRNA in primary neuroblastomas. PCR products (10 µl of each sample) were subjected to agarose gel electrophoresis. A 80 bp TrkB fragment was present in 7 of 25 neuroblastomas. HGF (94 bp) and c-Met (76 bp) products could be detected in all TrkB expressing tumours. Expression of the housekeeping gene ß-actin was used as an internal control.
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Next, we sought to confirm the results obtained with anti-HGF antibodies, and transfected the TrkB expressing G7 cells with a small interfering RNA targeting human c-Met. As a negative control, a non-silencing siRNA was also transfected into the G7 cells. Gene silencing was quantified at 48 h post-transfection, both at mRNA and protein levels. Transfection of c-Met siRNA resulted in a 60% knockdown at the mRNA level (Figure 3B). This was associated with major impairment of the invasive behaviour (30% of the control), as shown in Figure 3B and D.
To validate the results obtained with TrkB transfectants, we performed further experiments with parental neuroblastoma cell lines induced to express TrkB by treatment with all-trans retinoic acid (RA). SH-SY5Y cells were incubated with 5 nM RA for 5 days and analysed for TrkB, HGF and c-Met expression by real-time RTPCR. RA treatment increased the mRNA levels of TrkB (4.5-fold), HGF (4-fold) and c-Met (3.4-fold) (Figure 3C). The expression of endogenous BDNF was not affected by RA treatment (data not shown). Next, we evaluated the effect of TrkB and c-Met stimulation on the invasive capability of RA-treated neuroblastoma cells. Stimulation with BDNF caused a 3-fold increase in the number of invasive cells compared with non-stimulated cells. The addition of HGF also led to an increase in invasiveness (Figure 3C and D). The same set of experiments was also performed with SK-N-SH cells and revealed similar results (data not shown). Thus, activation of the TrkB and c-Met signal transduction pathways by RA in combination with BDNF or HGF stimulates neuroblastoma cell invasiveness.
To extend our observations made on cell lines to patient material we analysed the expression levels of TrkB, HGF and c-Met in tumour specimens from 25 children by real time RTPCR. The ages of patients at diagnosis ranged from 2 days to 174 months (14.5 years) with a median of 31 months. All neuroblastoma patients were diagnosed with advanced stages of disease (stage 3, n = 10 and stage 4, n = 15). The tumour samples were obtained from the competence centre Embryonic Tumours (Children's Hospital, University of Cologne, Germany). We found significant expression of TrkB mRNA in 7 of 25 primary neuroblastomas. Elevated amounts of HGF and c-Met transcripts were also present in TrkB-expressing tumours, indicating coordinated expression of the three molecules in primary neuroblastomas. Figure 3E shows the PCR products for eight tumour samples. Whether the coordinated expression of TrkB and HGF/c-Met in primary neuroblastomas may provide prognostic information remains to be clarified in a larger number of cases.
Cooperation of TrkB with c-Met, both anti-BDNF and anti-HGF neutralizing antibodies inhibit c-Met autophosphorylation and cell invasiveness
We have already shown in Figure 2C that autophosphorylation of c-Met was detected even in the absence of exogenous HGF or BDNF, suggesting that the autocrine HGF/c-Met loop is constitutively active in TrkB-expressing cells. We then monitored c-Met receptor autophosphorylation after treatment with exogenous HGF (100 ng/ml) or BDNF (50 ng/ml) for 15 min: both HGF and BDNF treatment led to an increased expression of phospho-c-Met (Figure 4A). To evaluate the effect of inhibiting one or the other pathway, G7 cells were treated with anti-HGF or anti-BDNF neutralizing antibodies for 24 h and then stimulated with HGF (100 ng/ml) or BDNF (50 ng/ml) for 15 min. Anti-HGF neutralizing antibodies blocked both the endogenous and the HGF-induced phosphorylation of c-Met. Treatment with BDNF increased the amount of phosphorylated c-Met, and this effect could also be blocked by pretreatment of the cells with anti-BDNF neutralizing antibodies. To further address how TrkB overexpression leads to activation of c-Met, we examined whether inhibition of TrkB affected c-Met phosphorylation. The incubation with neutralizing antibodies to BDNF impaired the c-Met phosphorylation (Figure 4A). Thus, activation of c-Met is dependent on TrkB activity. In contrast, TrkB phosphorylation was not affected by inhibition of c-Met with anti-HGF neutralizing antibodies. Interestingly, the decrease in c-Met phosphorylation caused by incubation with anti-HGF neutralizing antibodies could be partially reversed by the addition of exogenous BDNF (Figure 4A). Taken together, the results demonstrate that the cooperation between TrkB and c-Met is dependent on the activation via TrkB.

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Fig. 4. (A) c-Met and TrkB tyrosine phosphorylation and (B) invasion in Matrigel after neutralization of HGF and BDNF. (A) Reduction of basal and ligand-induced c-Met protein phosphorylation after treatment with anti-HGF and anti-BDNF antibodies. Protein expression and phosphorylation were determined after immunoblot analysis of tumour cell lysates. TrkB- or control-transfected SH-SY5Y cells were cultured in 2% serum-containing medium overnight, treated with anti-HGF neutralizing (40 µg/ml) or anti-BDNF neutralizing (20 µg/ml) antibodies for 24 h and stimulated with HGF (100 ng/ml) or BDNF (50 ng/ml) for 15 min. The filters were stripped and reprobed with antibodies against actin to control equal protein loading. Autophosphorylation of TrkB was examined by immunoprecipitation of cell lysates with anti-phosphotyrosine antibodies followed by western blot with anti-Trk antibodies. (B) Reduction of TrkB-induced invasiveness after treatment with anti-HGF and anti-BDNF antibodies, detected by Matrigel invasion assay: graphical illustration of the number of invading cells under the conditions described for Figure 3A. Cells were stained with hematoxylin/eosin and counted at magnification 400x in 10 representative areas per well.
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The activation of the HGF/c-Met signalling pathway in TrkB-overexpressing cells was associated with enhanced invasiveness, as documented in Figures 3 and 4B. The decrease in c-Met autophosphorylation induced by treatment with neutralizing antibodies against HGF or BDNF was correlated with a significant impairment of the invasive behaviour of G7 cells (Figure 4B), suggesting that both BDNF/TrkB and HGF/c-Met signalling are regulating the invasive potential of neuroblastoma cells.
TrkB dependency of protease production and activation in neuroblastoma cells
The invasiveness of tumour cells is dependent on their capability to degrade the extracellular matrix by activating proteases including MMPs as well as the plasminogen activators (PAs) of the urokinase type (uPA) and tissue type (tPA) (22). Although proteases are thought to be produced mainly by stroma cells, we examined whether neuroblastoma cells are able to express matrix degrading enzymes. Using real-time RTPCR, western blotting and zymographic analysis, we monitored changes in expression and activity patterns of various proteases in TrkB expressing SH-SY5Y and SK-N-AS cells compared with parental and control-transfected cells. We observed the presence and upregulation of MMP-2, uPA and tPA transcripts and proteins in TrkB overexpressing SH-SY5Y cells (Figure 5A and B). TrkB-overexpressing SK-N-AS cells displayed production and upregulation of MMP-1, MMP-2, MMP-3 MMP-9, uPA and tPA on both the mRNA and protein levels (Figure 5A and B). Analysis of gelatine zymograms indicated the presence of two increased proteolytic activities in the media from TrkB expressing SH-SY5Y cells, which correspond most likely to the molecular masses of MMP-2 (66 kDa) and MMP-9 (83 kDa). Medium from TrkB expressing SK-N-AS cells displayed one major gelatinolytic activity corresponding to MMP-2. In addition, zymographic analysis performed in casein containing gels showed that both TrkB overexpressing SH-SY5Y and SK-N-AS cell lines produced two lysis zones, which corresponded to the molecular masses of uPA (52 kDa) and tPA (68 kDa) (Figure 5C).

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Fig. 5. Effects of TrkB overexpression on secretion and activation of matrix-degrading enzymes. (A) Determination of the cellular levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, uPA and tPA mRNA in control- and TrkB-transfected SH-SY5Y and SK-N-AS cells by real-time RTPCR. Control-transfected cells were designated as a calibrator. (B) Western blot analysis of conditioned supernatants of the indicated cell lines. Cells at 70% confluence were serum-deprived for 48 h, collected and processed for western blot analysis. Aliquots derived from 5 x 104 cells per lane were studied for expressions of MMP-1, MMP-2, MMP-3, MMP-9, uPA and tPA. (C) Gelatin (upper panel) and casein (lower panel) zymograms from conditioned culture media of the indicated cell lines. Media were collected as described above and subjected to zymographic analysis.
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Invasion of TrkB-expressing neuroblastoma cells into the quail CAM
Having demonstrated that the activation of an autocrine HGF/c-Met loop by the expression of TrkB has substantial effects on neuroblastoma cell invasiveness in vitro, we next sought to determine the effects on their in vivo behaviour. In a further set of experiments, we examined the ability of neuroblastoma cells to invade the CAM of quail embryos. Since the chorion represents an epithelial barrier, only tumour cells with high invasive potential are able to cross it, to get access to the chorionic vessels and to generate solid tumours. CAMs were inoculated with 3 x 106 control- or TrkB-transfected SK-N-AS cells and the embryos were incubated for 7 days. SK-N-AS/Vec cells did not produce any tumours on 6 of 10 CAMs in this time period (Figure 6A). Very small (12 mm) nodules (a total of 4) were observed in 4 of 10 eggs. These appeared macroscopically pale and poorly vascularized (Figure 6B and C). In contrast, TrkB expressing SK-N-AS cells formed multiple large tumours (a total of 34) in 9 of 10 eggs (Figure 6DF). The tumours were well vascularized, grew progressively and reached diameters of up to 15 mm after 1 week of inoculation. Tortuous and dilated arterioles and venules were present at the tumour surface, and, at higher magnification, intratumoural capillaries were seen, in which blood flow could be observed, providing evidence for functional tumour capillaries. CAMs were also inoculated with control- and TrkB-transfected SH-SY5Y neuroblastoma cells (data not shown). The results were similar to those obtained with SK-N-AS cells. The experimental neuroblastomas were further analysed by immunohistochemistry and immunofluorescence. Figure 6GL shows the immunostaining for TrkB, c-Met, HGF and endothelial cells (QH1) in representative tumour sections. Tumours derived from TrkB overexpressing SK-N-AS cells were highly positive for TrkB, c-Met and HGF protein. Staining with QH1 antibodies demarcated numerous quail vessels that had invaded the tumours.

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Fig. 6. Immunohistological detection of TrkB and HGF/c-Met in experimental TrkB overexpressing neuroblastomas in vivo. Control- and TrkB-transfected SK-N-AS neuroblastoma cells were inoculated on the quail CAM. (AF) Macroscopic images: (AC) No or minor tumour formation after inoculation of control cells; (DF) Multiple solid tumours derived from TrkB-transfected SK-N-AS cells were visible after 7 days of incubation. Bar, 4 mm. (GI) Immunohistochemistry of tumours; sections were counterstained with nuclear fast red. Staining with (G) anti-TrkB, (H) anti-c-Met and (I) anti-QH1 antibodies demonstrate TrkB- and c-Met-expression in vivo. The QH1 antibodies demarcate the quail vessels that have invaded the tumours. Bar, 40 µm. (JL) Immunofluorescence studies of experimental neuroblastomas derived from TrkB expressing SK-N-AS cells. Double immunostaining with (J) anti-c-Met and (K) anti-HGF; (L) merged picture. Bar, 40 µm.
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Discussion
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Expression of TrkB and BDNF is usually found in primary neuroblastomas with MYCN-amplification and is associated with an unfavourable outcome (7). One of the hallmarks of TrkB expressing neuroblastomas is their high invasive and metastatic capacity (8,11). In addition, BDNF/TrkB signalling enhances resistance of neuroblastoma cells to chemotherapy (10,23,24). However, the molecular mechanisms underlying these biological processes are as yet incompletely defined. Here, we addressed the effect of TrkB expression on the invasiveness of neuroblastoma cells. We demonstrate that a BDNF/TrkB autocrine pathway is active in TrkB-transfected clones of the SH-SY5Y and SK-N-AS neuroblastoma cell lines. Furthermore, these cell lines display co-expression of HGF and c-Met, setting the stage for an additional autocrine loop, in which endogenously produced HGF binds to c-Met and causes its constitutive activation. We also show that activation of c-Met is dependent on TrkB activity, since inhibition of TrkB by treatment with neutralizing antibodies to BDNF significantly impairs c-Met phosphorylation. Expression of TrkB is also associated with co-expression of HGF and c-Met in retinoic acid-treated neuroblastoma cells, in experimental tumours and in primary neuroblastomas of children. Formation of an autocrine HGF/c-Met loop has been observed in a large number of human tumours of adults, in stringent association with metastatic tendencies (25,26). Here we show that overexpression and activation of c-Met are required to generate TrkB-mediated biological signals relevant for neuroblastoma cell invasiveness: Inhibition of HGF/c-Met signalling with neutralizing antibodies against HGF as well as with siRNA targeting c-Met is sufficient to significantly reduce the invasiveness of TrkB expressing neuroblastoma cells. The interaction between the receptors TrkB and c-Met may be very important for the specificity of the TrkB signalling in neuroblastoma. This idea is strongly supported by previous data showing that c-Met can form complexes with other cell surface molecules, such as
6ß4 integrin, plexin B1 and CD44. These interactions have been reported to diversify the signalling cascades that emanate from the c-Met receptor, thereby determining the specificity of the biological outcome (2729).
Tumour cell invasion into neighbouring tissues is dependent on the degradation of the extracellular matrix by proteases. The proteolytic pathway is regulated by the balance of activators including MMPs and serine proteases, such as uPA/tPA and plasmin and the counteracting proteinase inhibitors (3032). Enhanced invasiveness of retinoic acid-treated neuroblastoma cells has been shown to be dependent on tPA and plasmin activity (33). In the present study, we show that enhanced TrkB expression is associated with a significant increase in the secretion of a subset of MMPs (MMP-1, MMP-2, MMP-9) and the PAs, uPA and tPA. This upregulation contributes to the degradation of the extracellular matrix and supports the role of TrkB in promoting the invasive behaviour of neuroblastoma cells. Our data are in agreement with others (34,35), showing the expression of MMP-2 and MMP-9 in aggressive human neuroblastomas in vivo. MMP-2 is produced by both neoplastic cells and stromal cells, whereas MMP-9 has only been observed in stromal cells (36). We report here that MMP-2 is present in the two engineered TrkB overexpressing SH-SY5Y and SK-N-AS neuroblastoma cell lines, one of them (SK-N-AS) also secretes MMP-9. It has been shown that MMPs and uPA are required for intravasation, a crucial event in the formation of hematogenic and lymphogenic metastases (37). Blocking MMPs with general inhibitors such as batimastat, marimastat or related compounds may be important to reduce the invasiveness, intravasation and consequently the metastatic potential of TrkB expressing neuroblastoma cells. We report in the present study that TrkB expressing cells produce uPA and tPA. Since these PAs are known to convert the inactive proHGF into active HGF (14), the plasminogen/plasmin network may be required not only for matrix degradation but also for activation of the endogenously produced HGF in TrkB expressing neuroblastoma cells.
MMP-2 and MMP-9 play important roles as activators of angiogenesis by promoting the invasion of the extracellular matrix by microvascular endothelial cells and by increasing the bioavailability of VEGF (38). The solid tumours formed by the TrkB expressing SK-N-AS cells on the quail CAM have grown progressively beyond the critical angiogenesis-independent size (23 mm) and reached diameters of up to 15 mm. As demonstrated by immunostaining of vessels within tumour sections, the experimental neuroblastomas are highly vascularized. HGF has been shown to be a potent angiogenic factor both in vitro and in vivo (3941). Thus, the endogenously expressed HGF as well as the upregulated MMP-2 and MMP-9 may stimulate both invasion and angiogenesis of TrkB expressing neuroblastomas.
In summary, we show that TrkB expression mediates activation of an autocrine HGF/c-Met loop in neuroblastoma cells, which contributes in an essential manner to their invasiveness and angiogenesis. Since both processes represent essential steps in tumour progression, abrogation of c-Met kinase activity by specific inhibitors may be an important tool in improving neuroblastoma therapy.
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Acknowledgments
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We thank Mrs M.Czeranski for her technical assistance. We are grateful to Prof. Dr F.Berthold, Dr B.Hero and Dr K.Ernestus for providing neuroblastoma tissue samples. The QH1 antibody was obtained from the Developmental Studies Hybridoma Bank, maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biological Sciences, University of Iowa, Iowa City, IA, under contract N01-HD-6-2915 from the NICHD. This work was supported by Deutsche Krebshilfe.
Conflict of Interest Statement: None declared.
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References
|
---|
- Matthay,K.K., Villablanca,J.G., Seeger,R.C. et al. (1999) Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children's Cancer Group. N. Engl. J. Med., 341, 11651173.[Abstract/Free Full Text]
- Teng,K.K. and Hempstead,B.L. (2003) Neurotrophins and their receptors: signaling trios in complex biological systems. Cell. Mol. Life Sci., 61, 3548.[ISI]
- Brodeur,G.M. (2003) Neuroblastoma: biological insights into a clinical enigma. Nat. Rev. Cancer, 3, 203216.[CrossRef][ISI][Medline]
- Schulte,J.H., Schramm,A., Klein-Hitpass,L. et al. (2005) Microarray analysis reveals differential gene expression patterns and regulation of single target genes contributing to the opposing phenotype of TrkA- and TrkB-expressing neuroblastomas. Oncogene, 24, 165177.[CrossRef][ISI][Medline]
- Sitek,B., Apostolov,O., Stuhler,K., Pfeiffer,K., Meyer,H.E., Eggert,A. and Schramm,A. (2005) Identification of dynamic proteome changes upon ligand-activation of Trk-receptors using two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. Mol. Cell. Proteomics, 4, 291299.[Abstract/Free Full Text]
- Klein,R., Nanduri,V., Jing,S.A., Lamballe,F., Tapley,P., Bryant,S., Cordon-Cardo,C., Jones,K.R., Reichardt,L.F. and Barbacid,M. (1991) The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. Cell, 66, 395403.[CrossRef][ISI][Medline]
- Nakagawara,A., Azar,C.G., Scavarda,N.J. and Brodeur,G.M. (1994) Expression and function of TRK-B and BDNF in human neuroblastomas. Mol. Cell. Biol., 14, 759767.[Abstract]
- Matsumoto,K., Wada,R.K., Yamashiro,J.M., Kaplan,D.R. and Thiele,C.J. (1995) Expression of brain-derived neurotrophic factor and p145TrkB affects survival, differentiation, and invasiveness of human neuroblastoma cells. Cancer Res., 55, 17981806.[Abstract]
- Ho,R., Eggert,A., Hishiki,T., Minturn,J.E., Ikegaki,N., Foster,P., Camoratto,A.M., Evans,A.E. and Brodeur,G.M. (2002) Resistance to chemotherapy mediated by TrkB in neuroblastomas. Cancer Res., 62, 64626466.[Abstract/Free Full Text]
- Jaboin,J., Kim,C.J., Kaplan,D.R. and Thiele,C.J. (2002) Brain-derived neurotrophic factor activation of TrkB protects neuroblastoma cells from chemotherapy-induced apoptosis via phosphatidylinositol 3'-kinase pathway. Cancer Res., 62, 67566763.[Abstract/Free Full Text]
- Douma,S., Van Laar,T., Zevenhoven,J., Meuwissen,R., Van Garderen,E. and Peeper,D.S. (2004) Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB. Nature, 430, 10341039.[CrossRef][ISI][Medline]
- Hecht,M., Papoutsi,M., Tran,H.D., Wilting,J. and Schweigerer,L. (2004) Hepatocyte growth factor/c-Met signaling promotes the progression of experimental human neuroblastomas. Cancer Res., 64, 61096118.[Abstract/Free Full Text]
- Nakamura,T., Nishizawa,T., Hagiya,M., Seki,T., Shimonishi,M., Sugimura,A., Tashiro,K. and Shimizu,S. (1989) Molecular cloning and expression of human hepatocyte growth factor. Nature, 342, 440443.[CrossRef][ISI][Medline]
- Mars,W.M., Zarnegar,R. and Michalopoulos,G.K. (1993) Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA. Am. J. Pathol., 143, 949958.[Abstract]
- Ponzetto,C., Bardelli,A., Zhen,Z., Maina,F., dalla Zonca,P., Giordano,S., Graziani,A., Panayotou,G. and Comoglio,P.M. (1994) A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell, 77, 261271.[CrossRef][ISI][Medline]
- Stefan,M., Koch,A., Mancini,A., Mohr,A., Weidner,K.M., Niemann,H. and Tamura,T. (2001) Src homology 2-containing inositol 5-phosphatase 1 binds to the multifunctional docking site of c-Met and potentiates hepatocyte growth factor-induced branching tubulogenesis. J. Biol. Chem., 276, 30173023.[Abstract/Free Full Text]
- Pelicci,G., Giordano,S., Zhen,Z. et al. (1995) The motogenic and mitogenic responses to HGF are amplified by the Shc adaptor protein. Oncogene, 10, 16311638.[ISI][Medline]
- Biedler,J.L., Helson,L. and Spengler,B.A. (1973) Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res., 33, 26432652.[ISI][Medline]
- Eggert,A., Ikegaki,N., Liu,X.G. and Brodeur,G.M. (2000) Prognostic and biological role of neurotrophin-receptor TrkA and TrkB in neuroblastoma. Klin. Padiatr., 212, 200205.[CrossRef][ISI][Medline]
- Albini,A., Iwamoto,Y., Kleinman,H.K., Martin,G.R., Aaronson,S.A., Kozlowski,J.M. and McEwan,R.N. (1987) A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res., 47, 32393245.[Abstract]
- Pardanaud,L., Altmann,C., Kitos,P., Dieterlen-Lievre,F. and Buck,C.A. (1987) Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells. Development, 100, 339349.[Abstract/Free Full Text]
- Liotta,L.A. and Kohn,E.C. (2003) Cancer's deadly signature. Nat. Genet., 33, 1011.[CrossRef][ISI][Medline]
- Scala,S., Wosikowski,K., Giannakakou,P., Valle,P., Biedler,J.L., Spengler,B.A., Lucarelli,E., Bates,S.E. and Thiele,C.J. (1996) Brain-derived neurotrophic factor protects neuroblastoma cells from vinblastine toxicity. Cancer Res., 56, 37373742.[Abstract]
- Middlemas,D.S., Kihl,B.K., Zhou,J. and Zhu,X. (1999) Brain-derived neurotrophic factor promotes survival and chemoprotection of human neuroblastoma cells. J. Biol. Chem., 274, 1645116460.[Abstract/Free Full Text]
- Trusolino,L. and Comoglio,P.M. (2002) Scatter-factor and semaphorin receptors: cell signalling for invasive growth. Nat. Rev. Cancer, 2, 289300.[CrossRef][ISI][Medline]
- Danilkovitch-Miagkova,A. and Zbar,B. (2002) Dysregulation of Met receptor tyrosine kinase activity in invasive tumors. J. Clin. Invest., 109, 863867.[Free Full Text]
- Trusolino,L., Bertotti,A. and Comoglio,P.M. (2001) A signaling adapter function for alpha6beta4 integrin in the control of HGF-dependent invasive growth. Cell, 107, 643654.[CrossRef][ISI][Medline]
- Giordano,S., Corso,S., Conrotto,P., Artigiani,S., Gilestro,G., Barberis,D., Tamagnone,L. and Comoglio,P.M. (2002) The semaphorin 4D receptor controls invasive growth by coupling with Met. Nat. Cell. Biol., 4, 720724.[CrossRef][ISI][Medline]
- Orian-Rousseau,V., Chen,L., Sleeman,J.P., Herrlich,P. and Ponta,H. (2002) CD44 is required for two consecutive steps in HGF/c-Met signaling. Genes Dev., 16, 30743086.[Abstract/Free Full Text]
- Andreasen,P.A., Kjoller,L., Christensen,L. and Duffy,M.J. (1997) The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer, 72, 122.[CrossRef][ISI][Medline]
- Shapiro,S.D. (1997) Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Curr. Opin. Cell Biol., 10, 602608.[CrossRef][ISI]
- Toi,M., Ishigaki,S. and Tominaga,T. Metalloproteinases and tissue inhibitors of metalloproteinases. (1998) Breast Cancer Res. Treat., 52, 113124.[CrossRef][ISI][Medline]
- Tiberio,A., Farina,A.R., Tacconelli,A., Cappabianca,L., Gulino,A. and Mackay,A.R. (1997) Retinoic acid-enhanced invasion through reconstituted basement membrane by human SK-N-SH neuroblastoma cells involves membrane-associated tissue-type plasminogen activator. Int. J. Cancer, 73, 740748.[CrossRef][ISI][Medline]
- Sugiura,Y., Shimada,H., Seeger,R.C., Laug,W.E. and DeClerck,Y.A. (1998) Matrix metalloproteinases-2 and -9 are expressed in human neuroblastoma: contribution of stromal cells to their production and correlation with metastasis. Cancer Res., 58, 22092216.[Abstract]
- Ara,T., Fukuzawa,M., Kusafuka,T., Komoto,Y., Oue,T., Inoue,M. and Okada,A. (1998) Immunohistochemical expression of MMP-2, MMP-9, and TIMP-2 in neuroblastoma: association with tumor progression and clinical outcome. J. Pediatr. Surg., 33, 12721278.[CrossRef][ISI][Medline]
- Chantrain,C.F., Shimada,H., Jodele,S., Groshen,S., Ye,W., Shalinsky,D.R., Werb,Z., Coussens,L.M. and DeClerck,Y.A. (2004) Stromal matrix metalloproteinase-9 regulates the vascular architecture in neuroblastoma by promoting pericyte recruitment. Cancer Res., 64, 16751686.[Abstract/Free Full Text]
- Kim,J., Yu,W., Kovalski,K. and Ossowski,L. (1998) Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell, 94, 353362.[CrossRef][ISI][Medline]
- Chandrasekar,N., Jasti,S., Alfred-Yung,W.K. et al. (2000) Modulation of endothelial cell morphogenesis in vitro by MMP-9 during glial-endothelial cell interactions. Clin. Exp. Metastasis, 18, 337342.[CrossRef][ISI][Medline]
- Rosen,E.M., Knesel,J. and Goldberg,I.D. (1991) Scatter factor and its relationship to hepatocyte growth factor and met. Cell Growth Differ., 2, 603607.[Free Full Text]
- Bussolino,F., Di Renzo,M.F., Ziche,M., Bocchietto,E., Olivero,M., Naldini,L., Gaudino,G., Tamagnone,L., Coffer,A. and Comoglio,P.M. (1992) Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J. Cell Biol., 119, 629641.[Abstract]
- Lamszus,K., Jin,L., Fuchs,A., Shi,E., Chowdhury,S., Yao,Y., Polverini,P.J., Laterra,J., Goldberg,I.D. and Rosen,E.M. (1997) Scatter factor stimulates tumor growth and tumor angiogenesis in human breast cancers in the mammary fat pads of nude mice. Lab. Invest., 76, 339353.[ISI][Medline]
Received February 15, 2005;
revised July 11, 2005;
accepted July 22, 2005.