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Dipartimento di Medicina Interna e di Medicina Specialistica (R.M., A.C., F.F., L.S., S.R., R.V.), Cattedra di Endocrinologia, University of Catania, Ospedale Garibaldi, 95123 Catania, Italy; and Dipartimento di Medicina Sperimentale e Clinica (A.B.), Cattedra di Endocrinologia, Policlinico Mater Domini, University of Catanzaro "Magna Graecia," 88100 Catanzaro, Italy
Address all correspondence and requests for reprints to: Antonino Belfiore, M.D., Dipartimento di Medicina Sperimentale e Clinica, Policlinico Mater Domini, University of Catanzaro "Magna Graecia," via Tommaso Campanella 115, 88100 Catanzaro, Italy. E-mail: belfiore{at}unicz.it.
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Introduction |
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We recently observed that approximately 70% of papillary thyroid cancers overexpress Met (3), the tyrosine kinase receptor for the hepatocyte growth factor (HGF)/scatter factor. HGF is a cytokine with several biological activities, including stimulation of cell proliferation, motility, and morphogenesis. HGF is secreted as an inactive precursor that is converted to the active heterodimeric form by secreted proteases, including plasminogen activators (4, 5, 6, 7). Because Met is not expressed or only focally expressed at low level in the normal thyroid, its aberrant expression in papillary cancer suggests a role for the HGF-Met system in the development or the progression of these tumors. When we investigated the possible prognostic significance of Met immunostaining in a large series of thyroid carcinomas, we found that Met overexpression was a specific and early event in papillary cancer development (8). Surprisingly, Met overexpression was inversely correlated with the occurrence of distant metastases. In fact, negative or low Met expression was the most effective predictive factor for the occurrence of distant metastases and patient survival (8).
To better understand the biological role of the Met overexpression in papillary thyroid carcinomas, we investigated whether Met tyrosine kinase is activated by locally produced HGF in these tumors. We also examined the biological effects of HGF in primary cultures of papillary cancer cells expressing Met at different levels.
In papillary thyroid cancer specimens, we found that Met is activated, and HGF is locally produced by the tumor stromal fibroblasts. In cultured thyroid cancer cells, HGF was a potent mitogenic and survival factor. HGF, however, elicited different biological effects, depending on the Met expression level. In cells with high Met expression (high-Met cells), the major effect of HGF was branching morphogenesis, with no appreciable stimulation of cell migration or invasion of reconstituted basal membranes. In contrast, in cells with low Met expression (low-Met cells), HGF caused little branching morphogenesis but effectively stimulated cell migration and invasion of reconstituted basal membranes.
Therefore, these data suggest that, in papillary thyroid cancer cells, a high Met expression favors growth and morphogenetic epithelial-mesenchymal interactions but not cell migration and invasiveness. These findings may help explain why a high Met expression is associated with a low occurrence of distant metastases in papillary thyroid cancer.
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Materials and Methods |
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Cell cultures obtained from six different papillary carcinomas were used to carry out functional studies. Two cultures had low Met levels, and four cultures had high Met levels (see Results). All cultures were obtained from well-differentiated cancers. Extrathyroid invasion and locoregional lymph node metastases were present in two of two low-Met tumors and in one of four high-Met tumors at diagnosis. Patient follow-up was available for both low-Met patients but only two of four high-Met patients (33 ± 4 months, mean ± SD). No patient developed distant metastases. To exclude the presence of met mutations in the papillary cell culture studied, sequences corresponding to exons 16–19 were amplified and screened for possible mutations by automatic sequencing (10). None of these tumors had met mutations.
Fresh tissue specimens (used for Western blot studies) were collected at surgery, immediately frozen, and stored in liquid nitrogen until processing.
Established cell lines
Established thyroid cell lines from papillary (TPC-1, BC-PAP, and NPA), follicular (WRO), or anaplastic thyroid cancer (ARO and FF-1) were grown in RPMI 1640 supplemented with 2 mM glutamine, 10% FCS, and 50 µg/ml gentamicin. Simian virus 40-transformed human thyrocytes (4A1), which were obtained as previously described (11), were cultured in DMEM supplemented with 2 mM glutamine and 5% FCS.
Met and HGF mRNA evaluation
Both Met and HGF mRNAs were evaluated in thyroid cells and tissue specimens by RT-PCR. Total RNA was prepared using a commercial kit (Ultraspec II RNA; Biotecx Laboratories, Inc, Houston, TX). cDNA was synthesized by adding 0.3 µg of total RNA to 15 U/ml murine leukemia virus reverse transcriptase (Life Technologies, Inc., Paisley, UK), 2.5 mM random hexamers, 0.5 mM of each deoxynucleotide triphosphate, 3 mM MgCl2, 75 mM KCl, 10 mM dithiothreitol, and 50 mM Tris-HCl in a total volume of 20 µl and incubated at 37 C for 70 min. PCR amplification was performed with 5 µl cDNA, adding 1.25 U Taq polymerase (Applied Biosystems, Foster City, CA), 2.5 mM specific primers, 2.5 mM MgCl2, and 0.5 mM of each deoxynucleotide triphosphate. The following primer sequences were used: met, sense primer, nucleotides 3936–3960, 5'-TACTTGTTGCAAGGGAGAAGACTCCTA-3'; met, antisense primer, nucleotides 4391–4416, 5'-GGGACCAAGCCTCTGGTTCTGATGC-3'; HGF, sense primer, nucleotides 945–970, 5'-GGAATGGAATTCCATGTCAGCGTT-3'; and HGF, antisense primer, nucleotides 1587–1609, 5'-TCAAGTCTCGAGAAGGGAAACA-3'. Conditions for met PCR were as follows: 94 C x 60 sec, 77 C x 60 sec, and 72 C x 60 sec for 30 cycles. Conditions for HGF PCR were as follows: 94 C x 60 sec, 64 C x 45 sec, and 72 C x 90 sec for 35 cycles. PCR products were analyzed by 2% agarose gel electrophoresis, followed by ethidium bromide staining (12).
Met and HGF protein measurement by Western blot
Frozen tissues were pulverized in the presence of liquid nitrogen (Mikro-Dismembrator; B. Braun Biotech International, Melsungen, Germany). Powdered tissues or subconfluent cell monolayers were solubilized with 50 mM HEPES (pH 7.4), 1% Triton X-100, and 2 mM phenylmethylsulfonylfluoride. For Met protein measurement, solubilized extracts were first immunoprecipitated with anti-Met antibody sc161 (Santa Cruz Biotechnology, Santa Cruz, CA) raised against the carboxy terminal of human Met (13).
After centrifugation at 10,000 x g for 5 min, the pellets were washed three times at high stringency (0.5 M NaCl; 10 mM sodium phosphate, pH 7.4; 0.5% Nonidet P-40; 2 mM EDTA; and 0.04% BSA), boiled in Laemmli buffer, and centrifuged at 10,000 x g for 5 min at 4 C. The supernatants were then subjected to 7.5% PAGE under reducing conditions and transferred to nitrocellulose membranes. Filters were then incubated with either 0.5 µg/ml anti-Met DQ-13 monoclonal antibody (14) (an antibody directed against a peptide corresponding to the last 19 amino acids of human Met carboxy terminal; UBI, Lake Placid, NY) or 1 µg/ml 
PY antibody (Transduction Laboratories, Lexington, KY).
To measure HGF protein content, tissue extracts (100 µg/lane) were subjected to PAGE under nonreducing conditions, transferred to nitrocellulose membranes, and incubated with an anti-HGF antibody (R&D Systems Ltd., Abingdon, UK). Filters were then incubated with a second antibody conjugated with horseradish peroxidase, and the reaction was developed by an enhanced chemiluminescence detection system (Amersham International, Amersham, UK). The signal was subjected to densitometric analysis.
Small Interfering RNA (siRNA) Met knock-down
TPC-1 cells (2 x 105/well) were plated in 60-mm tissue culture plates and grown in regular medium without antibiotics for 24 h. Cells, at 90–95% confluency, were then transfected with Met-specific siRNA oligonucleotides (Met siRNA smart pool, catalog no. M-003156-01-05; Dharmacon, Lafayette, CO) and with scramble RNA oligonucleotides as control (nonspecific control duplex-XIII, catalog no. D-001206-13-05; Dharmacon) using Lipofectamine 2000 (Invitrogen Laboratories, Paisley, UK) following the manufacturer’s instructions. The final concentration of siRNA was 100 nM. The lipid/siRNA complex was added drop-wise to the cultures and was replaced with fresh medium after 6 h. Experiments to evaluate the biological effects of HGF were then carried out 24 h after siRNA transfection. Met down-regulation in transfected cells was confirmed by Western blot analysis, as described earlier.
ERK1/2, Akt, and Stat-3 phosphorylation in response to HGF
TPC-1 cells, which were transfected with Met siRNAs or with scramble RNA oligonucleotides, were washed twice with PBS (pH 7.4) and serum starved for 48 h. The cells were then treated with HGF (20 ng/ml) for 10 min. Ligand stimulation was terminated by two washes with ice-cold PBS (pH 7.4), buffer removal by aspiration, and addition of ice-cold sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris, pH 6.8; 10% glycerol; 2% SDS; 50 mM dithiothreitol; and 0.1% bromophenol blue). After scraping, samples were sonicated for 10 sec and heated to 95–100 C for 5 min. Whole-cell lysates were then subjected to reducing SDS-PAGE on 10% polyacrylamide gel. After electrophoresis, the resolved proteins were transferred to nitrocellulose membranes and subjected to immunoblot analysis.
To study the principal Met signaling pathways, the blots were probed with polyclonal antibodies against the phosphorylated form of either ERK1/2 or Akt. Both phosphospecific antibodies were from New England Biolabs (Beverly, MA).
To control for protein content, the membranes were stripped with stripping buffer Restore (Pierce, Rockford, IL) for 15 min at room temperature and subsequently reprobed with specific polyclonal antibodies, either anti-ERK or anti-Akt polyclonal antibody (all from New England Biolabs). All immunoblots were revealed by enhanced chemiluminescence method, autoradiographed, and subjected to densitometric analysis.
Immunofluorescence studies
TPC-1 cells treated with siRNA or with scramble RNA oligonucleotides were cultured onto coverslips and either exposed or not to HGF (20 ng/ml) for 10 min. Cells were then fixed in 3.7% formaldehyde, permeabilized with PBS/0.3% Triton X-100, blocked with PBS/10% normal goat serum, and incubated with primary antibodies for 1 h. Cells were then incubated with Alexa-conjugated (Alexa Fluor 594 or 488) secondary antibodies (Molecular Probes, Leiden, The Netherlands) for 1 h. To visualize the cytoplasm, the cells were also incubated with Alexa-conjugated phalloidin (Molecular Probes) for an additional 30 min. The cells were finally counterstained with Hoechst 33258 (Sigma Chemical Co., St. Louis, MO) to color the nuclei. Epifluorescence microscopy was performed with an Olympus microscope (Olympus, Tokyo, Japan). The images were digitally acquired with an Orca CCD Camera (Hamamatsu, Hamamatsu City, Japan) and processed with the Image-Pro Plus 4.0 software (Media Cybernetics, Silver Spring, MD).
Cell growth studies
The growth response of thyroid cells to the mitogenic effect of HGF was evaluated both by measuring [3H]thymidine incorporation and cellular DNA.
For [3H]thymidine incorporation experiments, cells (3 x 104/well) were plated in 24-well tissue culture plates and grown in their regular medium for 24 h. The medium was then replaced with fresh medium containing 0.3% charcoal-stripped FCS. Twenty-four hours later, HGF was added to each well at the indicated concentrations. After 24 h, 18.5 kBq/well of [3H]thymidine was added for 4 h. At the end of the incubation, cell monolayers were washed twice with cold buffer, incubated with cold 10% trichloroacetic acid solution for 30 min, solubilized with 0.1 N NaOH, and counted by liquid scintillation.
In parallel experiments, cells were seeded in 24-well tissue culture plates at a density of 3 x 104 cells/well and incubated with HGF for 4 d with a medium change on the d 3. Cells were detached with a 0.2% EDTA solution and counted in a hemochromocytometer. The cellular suspension was then centrifuged, the pellet was solubilized with 0.03% SDS, and the cellular DNA content was determined by the fluorometric method of Labarca and Paigen (15).
Cell cycle analysis
TPC-1 cells treated with siRNA or with scramble RNA oligonucleotides were synchronized for 24 h in serum-free medium and then incubated in the presence or the absence of HGF (20 ng/ml) for another 24 h. Bromodeoxyuridine was added directly to the culture medium to the final concentration of 10 µM for 30 min. Cells were harvested and resuspended in 70% ethanol and stored at –20 C. Staining with antibromodeoxyuridine antibody was performed as suggested by the manufacturer’s instructions. Cells were resuspended in PBS containing 20 mg/ml propidium iodide plus 40 mg/ml RNase (Sigma) for 30 min in the dark. Cells were then subjected to FACS analysis (FACScalibur; BD Bioscience, Bedford, MA).
Invasion, migration, and adhesion assays
Invasion assays were performed with the Boyden’s chamber technique. Approximately 105 cells, resuspended in 200 µl of media, were placed on 6.5-mm diameter polycarbonate filters (8-µm pore size; Corning Costar Corp., Cambridge, MA) coated at the lower and the upper side (chemotaxis assay) with 1.2 mg/ml of Matrigel (BD Biosciences Labware, Bedford, MA). Various doses of HGF (0–50 ng/ml) in 1 ml of medium were added to the lower compartment. The plates were incubated at 37 C with 5% CO2 for 6 h (chemotaxis assay) or 48 h (invasion assay). At the end of incubation, the cells or the Matrigel at the upper side of the filter were removed with a cotton swab. Cells that had migrated to the lower side of the filter were fixed with 11% glutaraldehyde for 15 min at room temperature and stained with 0.1% crystal violet in 20% methanol for 20 min. After three washes with water and complete drying, the crystal violet was solubilized by immersion of the filters in 10% acetic acid. The concentration of the solubilized crystal violet was evaluated as absorbance at 590 nm.
Migration assay in TPC-1-transfected cells was performed in Boyden chambers with modifications. Briefly, cells were seeded onto six-well plates (5 x 104 cells/well) and maintained in complete medium for 24 h. Cells were then transfected by FuGene6 method (Roche Applied Science, Indianapolis, IN) with either an empty vector (2 µg/well) or Met (0.5, 2, or 4 µg/well) along with histone 2B (H2B)-green fluorescent protein (GFP) (0.4 µg/well) to mark transfected cells (DNA to Fugene6 ratio, 1:3). Forty-eight hours after transfection, cells were harvested by trypsinization and allowed to migrate for 6 h in 250 µg/ml collagen IV-coated translucent transwells (1 x 105 cells/transwell; Becton Dickinson, Franklin Lakes, NJ). To measure haptotaxis (migration toward the matrix: basal and HGF stimulated), transwells were coated with collagen IV on the lower side. To measure HGF-induced chemotaxis, transwells were coated with collagen IV on both sides (lower and upper), and HGF was added in the lower chamber. To evaluate HGF-induced random migration, HGF was added in both chambers (lower and upper). Chambers were then put in 3.7% formaldehyde, washed with PBS, and incubated for 10 min with PBS plus 5 µg/ml Hoechst to stain cell nuclei. Chambers were then examined under a fluorescence invertoscope (Olympus) at x20 magnification. Hoechst-stained cells represented the total migrated population, whereas GFP-positive cells represented the transfected migrated population. This procedure was allowed by the characteristics of transwells, which are translucent to the florescent light and allow the visualization of cells present on the lower side only. In parallel experiments, migrated cells were stained with crystal violet. Numbers obtained with migrated GFP-positive cells were normalized for numbers obtained with Hoechst- and crystal violet-stained cells and expressed as a percentage of basal. Results are mean of three separate experiments performed in triplicates.
Adhesion assay was performed with TPC-1 thyroid cancer cells transfected as reported earlier. Cells were plated onto collagen-coated six-well plates (2 x 105 cells/plate) and allowed to adhere in complete medium for 10, 30, 45, 60, and 120 min at 37 C with 5% CO2. At each time point, cells were rinsed three times with PBS and fixed with 3.7% formaldehyde, washed with PBS, and incubated for 10 min with PBS plus 5 µg/ml Hoechst to stain cell nuclei. Plates were then examined under a fluorescence invertoscope (Olympus) at x20 magnification. Hoechst-stained cells represented the total adherent population, whereas GFP-positive cells represented the transfected adherent population. In parallel experiments, adherent cells were stained with crystal violet. Numbers obtained with adherent GFP-positive cells were normalized for numbers obtained with Hoechst- and crystal violet-stained cells and expressed as a percentage of adherent cells at time 10 min. Results are the mean of three separate experiments performed in triplicates.
Chemoinvasiveness in response to HGF was also evaluated in TPC-1 cells treated with siRNA or with scramble RNA oligonucleotides.
Morphogenic effect
Approximately 5 x 103 cells were seeded into 48-well plastic plates, which had been previously coated with 150 µl of Matrigel (10 mg/ml) and allowed to set for 30 min at 37 C. The following day, various concentrations of HGF (0–50 ng/ml) were added in fresh medium. The cells were incubated with daily feeding of HGF and then photographed after 7 d (16). To evaluate cell growth, Matrigel was dissolved with dispase. The cellular suspension was then centrifuged, the pellet was solubilized with 0.03% SDS, and the cellular DNA content was determined by the fluorometric method of Labarca and Paigen (15).
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Results |
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). We then evaluated HGF expression by RT-PCR and Western blot analysis. RT-PCR analysis demonstrated the presence of the HGF transcript in all cancer specimens (data not shown), and Western blot analysis revealed the presence of HGF protein in all specimens (Fig. 1B). HGF concentration ranged between 15 and 110 ng/100 µg protein, as evaluated by Western blot using recombinant HGF as a standard (data not shown).
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To identify the source of HGF production, separate cultures of either thyrocytes or fibroblasts were obtained from both neoplastic and normal thyroid tissue specimens (n = 3 for each condition). Western blot analysis revealed that fibroblasts, but not thyrocytes, express the HGF protein (Fig. 2A). The same results were obtained by RT-PCR (Fig. 2B).
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). Taken together, these data indicate that Met is activated in papillary thyroid carcinomas and that HGF is produced by stromal fibroblasts.
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).
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). Very similar results were obtained with DNA measurements, as described in Materials and Methods (results not shown).
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, A and B). In the presence of HGF, low-Met cells formed only rare tubule-like structures, and cells predominantly aggregated in spheroids (Fig. 4C), whereas high-Met cells developed an extensive tubule network in a dose-dependent manner starting at a HGF concentration of 5–10 ng/ml (Fig. 4D).
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Motogenic effect.
HGF typically stimulates chemotaxis and chemoinvasion in a variety of epithelial cells. To evaluate the ability of papillary cancer thyrocytes to respond to HGF-induced chemotaxis, we measured HGF-stimulated migration through filters in a Boyden chamber. Furthermore, we evaluated HGF-stimulated chemoinvasion in terms of the cancer thyrocyte ability to cross a Matrigel barrier obtained by coating filters of the Boyden chamber with a thin layer of Matrigel. This property is considered a measure of the cell invasive potential.
In low-Met cells, both chemotaxis and chemoinvasion were actively stimulated by HGF, with the maximal effect reached at 20 ng/ml HGF (Fig. 5, A and B). In high-Met cells, chemotaxis was only modestly stimulated by HGF, and chemoinvasion was only marginally responsive to HGF (Fig. 5, A and B).
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). The difference in Met expression between low- or high-Met cell clones was similar to that observed when using different cell lines. Also, in this model, only high-Met thyroid cancer cells underwent branching morphogenesis when cultured in Matrigel in the presence of 20 ng/ml HGF (Fig. 6B). In contrast, HGF-stimulated invasiveness in Boyden chambers was higher in low-Met cell clones than in high-Met cell clones (+150–188% vs. +102–130%, respectively; P < 0.002; range of values obtained in four different experiments). The maximal growth response to HGF was similar in both groups (165 ± 19% and 161 ± 6% in high- or low-Met clones, respectively).
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). In Met siRNA-treated cells, HGF-stimulated chemoinvasion was increased compared with cells transfected with scramble RNA oligonucleotides (Fig. 7B). Moreover, cells with silenced Met were unable to form tubule-like structures in Matrigel in contrast with cells transfected with scramble-siRNA (Fig. 7C). We then evaluated whether siRNA treatment affected cell cycle progression in response to HGF. TPC-1 cells that were treated with either Met siRNAs or with scramble RNA oligonucleotides were exposed to 20 ng/ml of HGF for 24 h, and the cell cycle was, thereafter, analyzed by FACS analysis. In control cells, HGF induced an increase in cell progression to S phase (from 40–51%), with a reduction of cells in the G1 and G2 phases. Met siRNA-treated cells showed a smaller progression to S phase and no reduction of G2 phase after exposure to HGF (Fig. 7D).
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. Cells were cotransfected with H2B-GFP to mark the transfected population. Chemotaxis in response to HGF was then evaluated in transwells coated with collagen IV on both sides and HGF added to the lower chamber. Met transfection caused a dose-dependent reduced chemotaxis both in basal conditions and, more evidently, in response to HGF (Fig. 8B). We also evaluated random motility (HGF in both chambers), which resulted to be even more impaired than HGF-induced chemotaxis (data not shown). Very similar results were obtained when haptotaxis (migration through transwells coated with collagen IV only on the lower side) was evaluated. Similarly to what was observed with chemotaxis, random haptotaxis (HGF in both chambers) was more impaired than HGF-driven haptotaxis (HGF in the lower chamber; data not shown).
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) showed that Met overexpression significantly reduced thyroid cancer cell adhesion. These data may help explain the restraining effect of Met on thyroid cancer cell migration.
Met down-regulation leads to a relocalization of phosphorylated focal adhesion kinase (FAK) in response to HGF stimulation but not to a preferential Akt or ERK pathway activation
To explore the mechanisms underlying the enhanced cell migration in cells with low Met, we used TPC-1 papillary thyroid cancer cells treated with Met siRNA. TPC-1 cells were treated with either siRNA Met or scramble oligonucleotides, as described in Materials and Methods, and stimulated with 20 ng/ml HGF for 10 min. Cells were then solubilized and subjected to Western blot analysis with phosphospecific antibodies to Akt and ERK1/2, two major signaling pathways stimulated by HGF. In parallel experiments, siRNA Met-treated TPC-1 were seeded onto coverslips, stimulated with 20 ng/ml HGF for 10 min, and then processed for immunofluorescence studies. Western blot analysis showed that siRNA-induced Met down-regulation completely blocked both Akt and ERK1/2 phosphorylation in response to HGF stimulation (Fig. 9).
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). By contrast, HGF stimulation in cells treated with siRNA led to a pFAK relocalization at the cell edge (Fig. 10). Experiments performed with anti-FAK antibody were unable to show any significant difference in FAK localization between high and low Met-expressing cells (data not shown). These data indicate that, despite the attenuation in Met signaling observed under Met protein down-regulation, HGF stimulation in low-Met cells results in a different pFAK localization, suggesting that FAK may be responsible for the enhanced HGF-induced migration.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study, undertaken to clarify the biological role of the aberrant Met expression in papillary thyroid cancer, has yielded two major findings.
First, we observed that HGF, the Met ligand, is locally produced at a relatively high level in papillary thyroid cancer tissue and may cause ligand-induced activation of its receptor that is overexpressed by the malignant thyrocytes. We found that HGF is produced by stromal fibroblasts, whereas no HGF production could be detected in primary cultures from either normal thyroid or papillary thyroid cancers. Moreover, no autocrine HGF production was observed in a panel of established thyroid cancer cell lines. Cultured fibroblasts from either normal or neoplastic thyroid tissue expressed similar amounts of HGF. It is possible, therefore, that the increased tumor HGF production originates from fibroblasts stimulated by unidentified mediators produced by the malignant cells. The possibility also exists that stromal cells, other than fibroblasts, substantially contribute to the increased HGF content in thyroid cancer specimens. Macrophages may be such cells because they produce HGF and are a major component of the lymphoreticular infiltrate commonly present in papillary thyroid cancer (17). Therefore, we can conclude that Met overexpression in epithelial thyroid cancer cells and increased paracrine HGF production by tumor stromal cells result in abnormal Met activation in papillary thyroid cancer. Paracrine HGF secretion by stromal cells has been previously described in nonneoplastic tissues as well as in a variety of malignancies (18, 19). Trovato et al. (20) reported both paracrine and autocrine HGF production in papillary thyroid carcinomas. However, the use of immunohistochemistry as the sole method to detect HGF is a limitation of that study. Further studies are required to assess whether a subset of thyroid carcinomas indeed produces HGF.
A second novel observation in the present study is that HGF induces different biological effects in papillary cancer malignant thyrocytes depending on Met expression levels. High-Met cells were highly sensitive to HGF-induced growth and branching morphogenesis. In contrast, low-Met cells were less sensitive to HGF-induced growth and hardly underwent branching morphogenesis in response to HGF. Interestingly, both basal and HGF-stimulated cell migration was reduced in high-Met cells compared with low-Met cells. These results were confirmed in cell clones with a high or low Met expression derived from TPC-1 papillary thyroid cancer cell line.
Met down-regulation obtained by specific siRNAs confirmed that Met reduction inhibits branching morphogenesis but not cell migration in response to HGF. Conversely, Met-transfected cells allowed us to further confirm that Met overexpression causes an overall reduced migration in thyroid cancer cells. This model also allowed us to find that Met overexpression affects not only HGF-driven migration (chemotaxis) but also matrix-driven migration (haptotaxis). These data may suggest that the effect of Met overexpression on thyroid cancer cell invasiveness may depend on HGF concentrations and the extracellular matrix surrounding thyroid cancer cells.
These results are in agreement with previous studies demonstrating that the different biological responses elicited by the HGF-Met interaction (mitogenesis, branching morphogenesis, and cell motility) have different molecular bases. Mitogenesis appears to be mediated by the recruitment of the Grb2-SoS complex that, in turn, stimulates Ras, whereas cell motility is mediated by the phosphatidylinositol-3-OH kinase pathway. Branching morphogenesis requires a sustained recruitment of phospholipase C-
to Gab-1 (21). Interestingly, the expression of a Gab-1 mutant unable to bind phospholipase C- does not affect cell motility; it only partially reduces cell growth stimulation but completely inhibits branching morphogenesis (21). Activation of the Stat-3 pathway is also required for morphogenesis (22); this pathway is associated with cell differentiation but not with cell motility and growth. Recently, we observed that c-abl may be a negative regulator of HGF-induced thyroid cancer cell motility (23), a mechanism that may be linked to a high Met expression because it is not present in normal thyroid cells.
Our present finding of a different localization of pFAK in low Met-expressing cells in response to HGF may suggest different hypotheses. First, Met down-regulation in thyroid cancer cells may cause a reduction in the focal contacts formation in response to HGF and, as a consequence, a reduced cell adhesion. These observations may explain the higher responsiveness of low Met-expressing cells to HGF in terms of migration and are consistent with the results obtained with adhesion curves (Fig. 8). Second, because pFAK is able to recruit several docking proteins (including paxillin and p130Cas) involved in multiple signal transduction pathways (24), it is reasonable to suppose that pFAK relocalization in low Met-expressing cells may be responsible for a signaling partitioning at cell migrating edge, with a consequent enhancement in cell migration mechanisms.
Our data in differentiated thyroid cancer cells are in concert with the recent observations that Met activation confers an invasive phenotype only in undifferentiated breast cancer cells, whereas it has a predominant morphogenetic effect in the same cells transfected with and expressing E-cadherins (21).
The present findings may also help explain our previous clinical data indicating that papillary thyroid cancers with Met overexpression have a reduced risk of developing hematogenous metastases (8) and underline the complexity of the effect of Met overexpression and the interplay between Met and the extracellular matrix.
Other different mechanisms may also play a role in determining a good prognosis in papillary tumors that overexpress Met. Recently, it has been shown that HGF stimulation of Met-positive thyroid cancer cells is one of the mechanisms involved in the recruitment of dendritic cells (25). We and others have found that dendritic cell infiltration is strongly associated with a low risk of developing distant metastases and with a more favorable prognosis in papillary cancer patients (16, 26).
Interestingly, a high Met expression may be also associated with a more favorable outcome in patients with either pancreatic (27) or breast cancer (28).
Taken together, these data indicate that abnormal Met activation in papillary thyroid cancer may play an important role in promoting growth and survival of neoplastic cells from the initial stages of thyroid tumorigenesis. At the same time the relative expression of Met and its ligand HGF may affect papillary thyroid cancer characteristics such as growth, structure, invasiveness, and distant metastases, which are all factors that will determine the tumor progression rate, its ability to spread to adjacent and distant sites, and eventually, the patient outcome.
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Footnotes |
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A.B. and R.V. contributed equally to this work.
Abbreviations: FAK, Focal adhesion kinases; FCS, fetal calf serum; GFP, green fluorescent protein; H2B, histone 2B; HGF, hepatocyte growth factor; pFAK, phosphorylated FAK; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA.
Received December 31, 2003.
Accepted for publication June 2, 2004.
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, Empty-transfected cells; 
, Met-transfected cells.
