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Original Article |
Laboratory of Endocrine Cell Biology (Ju.H.H., Ji.H.H., H.K.C., D.W.K., E.S.H., J.M.S., H.K., O.-Y.K., H.K.R., D.Y.J., M.S.), National Research Laboratory Program, Department of Internal Medicine, Chungnam National University College of Medicine; and Department of Biology (K.-H.Y.), College of Natural Sciences, Chungnam National University, Daejeon 301-721, Korea
Address all correspondence and requests for reprints to: Minho Shong and Deog Yeon Jo, Laboratory of Endocrine Cell Biology, Department of Internal Medicine, Chungnam National University College of Medicine, 640 Daesadong Chungku Daejeon, Taejon 301-721, Korea. E-mail: minhos{at}cnu.ac.kr.
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Abstract |
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transcripts, detected by RT-PCR. Fluorescence activated cell sorting analysis of CXCR4 expression in normal and ATC cells showed that ARO cells expressed significant levels of CXCR4. FRO, NPA, and normal thyroid cells did not express membrane CXCR4, as determined by fluorescence activated cell sorting analysis. To identify the functional role of CXCR4 in ARO cells, we treated ARO cells with SDF-1 and analyzed the signaling pathways, cellular migration, and proliferation. SDF-1 enhanced the migration but did not affect the proliferation of ARO cells or activate the Janus kinase/signal transducer and activator of transcription signaling pathways. However, SDF-1/CXCR4 activation resulted in phosphorylation of the p70S6 kinase and its target protein, ribosomal S6 protein, and also activation of the ERK1/ERK2 signaling pathways. Furthermore, SDF-1/CXCR4- mediated activation of the p70S6 kinase and phosphorylation of the S6 protein were inhibited by treatment with an mTOR/FRAP inhibitor.
The specificity of the CXCR4-mediated migration of ARO cells was demonstrated by the dose-dependent inhibition of migration by neutralizing anti-CXCR4. The ATC cells, FRO and NPA, which do not express CXCR4, did not demonstrate significant SDF-1-mediated migration in vitro. In addition, the CXCR4-mediated migration of ARO cells was inhibited by treatment with pertussis toxin (a Gi-protein inhibitor) and PD 98059 (a mitogen-activated ERK kinase inhibitor) but not by LY294002 and wortmanin, phosphatidylinositol 3-kinase inhibitors.
These findings suggest that a subset of ATC cells expresses functional CXCR4, which may be important in tumor cell migration and local tumor invasion.
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Introduction |
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Chemokines are a superfamily of small, cytokine-like proteins (8) that induce, through their interaction with G protein-coupled receptors, cytoskeletal rearrangement, firm adhesion to endothelial cells, and directional migration. The chemokine stromal cell-derived factor 1 (SDF-1/CXCL12) and its unique receptor CXC chemokine receptor 4 (CXCR4) are expressed in epithelial cancer cells and have a critical role in migration and metastasis (9, 10). Muller et al. (11) screened human breast cancer cells for 17 different chemokine receptor genes and found that the expression levels of CXCR4 and CC chemokine receptor 7 were the only genes consistently increased relative to the levels in normal mammary epithelial cells. Activation of breast cancer cells with either SDF-1/CXCL12 or CCL21 triggered chemotaxis and tissue invasion in vitro. In addition, Muller et al. (11) showed that extracts of organs targeted by breast cancer had chemotactic activity for breast cancer cells that could be specifically neutralized by antibodies against CXCR4, suggesting that CXCR4 determines the site of metastasis.
Thyrocytes have been shown to produce SDF-1/CXCL12 in vitro and in vivo (12, 13); however, the functional expression of CXCR4, the receptor for SDF-1/CXCL12, is controversial. Aust et al. (13) examined the expression of SDF-1/CXCL12 and CXCR4 in normal and pathological thyroid tissues. SDF1/CXCL12 mRNA was detectable, and CXCR4 mRNA was present in low levels in normal thyrocytes. However, the significance of SDF1/CXCL12 and CXCR4 in thyroid cells is largely unknown. In this study, we found that CXCR4 is expressed in human ATC cells and is important in migration through the activation of diverse signaling pathways involving ERK and the phosphatidylinositol 3-kinase (PI3K)-dependent p70S6 kinase. The inhibition of CXCR4-mediated activation of ERK and p70S6 kinase decreases the migration of ATC cells.
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Materials and Methods |
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Media and cell culture reagents and materials were purchased from Life Technologies, Inc. (Gaithersburg, MD), Sigma (St. Louis, MO), Fisher Scientific (Fairlawn, NJ), Corning, Inc. (Corning, NY), and Hyclone Laboratories, Inc. (Logan, UT). Rapamycin and wortmannin were obtained from Calbiochem (La Jolla, CA). LY294002 and PD98059 were obtained from Sigma and New England Biolabs, Inc. (Beverly, MA), respectively. Recombinant human (rh) SDF-1/pre-B-cell growth-stimulating factor (PBSF; no. 350-NS) was obtained from R&D Systems (Abingdon, UK). The following antibodies were used in this study: human anti-CXCR4 monoclonal antibody (R&D Systems), anti-phospho-ERK polyclonal antibody, anti-phospho-Stat1 polyclonal antibody (pS727), anti-phospho-Stat3 polyclonal antibody (pS727, pY705), and anti-phospho-p70S6K1 polyclonal antibody (pT 389; New England Biolabs, Inc.). [3H]Thymidine and [
-32P]ATP (specific activity, 3000 Ci/mmol) were from NEN Life Science Products (Boston, MA).
Cell culture and treatments
Three human thyroid carcinoma cell lines (ARO, FRO, and NPA) were cultured in RPMI 1640 (Life Technologies, Inc., Cergy Pontoise, France) supplemented with 7% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD) at 37 C with 5% CO2 (13).
Thyroid tissue samples were obtained by total thyroidectomy from patients with unilobar papillary thyroid cancer, and normal contralateral human thyroid follicular cells in primary culture were prepared as previously described (14, 15) and maintained in Ham F12 medium with 5% FBS and ampicillin/streptomycin. The use of human tissues in this study was approved by the Clinical Research Committee at Chungnam National University School of Medicine. Written informed consent was also obtained from each patient.
rhSDF-1/PBSF, purchased from R&D Systems, was dissolved in PBS containing at least 0.1% BSA. After culture in RPMI 1640 without serum for 12 h, the cells used in this study were treated with 10 ng/ml rhSDF-1/PBSF for the indicated time periods.
RT-PCR
Total cellular RNA from ARO, NPA, FRO, bone marrow stromal, and bone marrow endothelial cells (BMEC) was isolated by standard procedures, and the total cellular RNA from papillary thyroid and normal thyroid cells was prepared from primary cultured cells. For RT-PCR, first-strand cDNA was synthesized using reverse transcriptase (Life Technologies, Inc., Grand Island, NY); PCR was performed using ampliTaq DNA polymerase (Perkin-Elmer Corp., Norwalk, CT) in a Perkin-Elmer 9700 thermocycler. PCR conditions for SDF-1 were: predenaturation at 94 C for 5 min, followed by 30 cycles of denaturation at 94 C for 1 min, annealing at 65 C for 1 min, and elongation at 72 C for 1 min. PCR conditions for CXCR4 were: predenaturation at 94 C for 5 min, followed by 30 cycles of denaturation at 94 C for 1 min, annealing at 62 C for 90 sec, and elongation at 72 C for 2 min. The primers used for SDF-1 were: 5'-TGATCGTCTGACTGGTGTTA-3' (sense) and 5'-CTTAGGGGATTTGGAAGTTT-3' (antisense); and primers for CXCR4 were: 5'-AATCTTCCTGCCCACCATCTAC-3' (sense) and 5'-GGCAGATGACAGATATATCTGTGACCGC-3' (antisense).
Flow cytometric analysis
ARO, NPA, FRO, and normal thyroid cells were cultured in RPMI 1640 media with 10% FBS at 37 C with 5% CO2. Cells were washed three times in an isotonic cold PBS buffer (supplemented with 0.5% BSA) after trypsin/EDTA treatment and incubated for 30 min at 4 C with fluorescein isothiocyanate-conjugated CXCR4 monoclonal antibodies. Isotype mouse IgG1 (Becton Dickinson and Co., Heidelberg, Germany) was used as a control. After this incubation, unbound anti-CXCR4 antibodies were removed by washing, and cells were resuspended in 200 µl PBS buffer for the final flow cytometric analysis. Cells were analyzed using a Becton Dickinson and Co. FACScan.
Migration assay
Migration of each cell type used in this experiment was assessed in 24-well chemotaxis chambers (Corning, Inc.). Briefly, the upper compartment of the chamber was loaded with 5 x 104 cells (resuspended in serum-free RPMI 1640 media), and the lower compartment of the chamber was loaded with different concentrations of rhSDF-1 or without rhSDF-1 (resuspended in serum-free RPMI 1640 media). The two compartments were separated by an 8-µm pore size polycarbonate filter (BD PharMingen). To determine whether rhSDF-1/CXCR4 signals were involved in ARO cell migration, NPA and FRO cells were used as a negative control in media conditions containing 100 ng/ml rhSDF-1. To investigate whether ARO cell migration involves G protein, mTOR, Erk1/2, and PI3K pathways, the lower compartments of the chamber were treated with 100 ng/ml pertussis toxin (PTX; Sigma), 10 ng/ml rapamycin (Sigma), 50 µM PD98059, and 1 µM LY294002 in serum-free RPMI 1640 media throughout this experiment at 37 C with 5% CO2. After a 24-hr incubation at 37 C, the filter was removed, and cells that migrated were counted by light microscopy.
Western blot analysis
Immunoblot analysis was performed using anti-STAT1 (pY701, total), anti-STAT3 (pY 705, pS 727, total), anti-p70S6K1 (pT 389, total), anti-nuclear factor-
B (NF-B), anti-inhibitor B (IB), anti-AKT (pT 308, pS 473), and anti-MAPK p44/p42 (pT 202/Y204, total) antibodies (Cell Signaling Technology, Inc., Beverly, MA). For Western blot analysis, cells used in this experiment were starved in serum-free RPMI 1640 for 12 h and then stimulated with 10 ng/ml rhSDF-1 for the indicated period of time at 37 C. The treated cells were scraped, lysed by addition of SDS sample buffer [62.5 mM Tris-HCl (pH 6.8), 6% (wt/vol), 30% glycerol, 125 mM DTT, and 0.03% (wt/vol) bromophenol blue], and separated by 10% SDS-PAGE along with biotinylated molecular weight standards. The proteins were transferred to a nitrocellulose membrane by electrotransfer for 1 h. After soaking in blocking buffer [1x TBS, 0.1% Tween 20 with blocking reagent (5% milk)], the membrane was incubated with primary antibody overnight at 4 C. Blots were developed using the horseradish peroxidase (HRP)-linked secondary antibody and a chemiluminescent detection system (Phototope-HRP Western Blot Detection Kit, New England Biolabs, Inc.).
Immune complex kinase assay
ARO ATC cells were transiently transfected with hemagglutinin antigen (HA)-tagged p70S6K and immunoprecipitated by anti-HA monoclonal antibody coupled to protein G-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). The samples were washed twice with Buffer A [containing 20 mM Tris-HCl (pH 7.5), 0.1% Nonidet p40, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 2 mM dithiothreitol, 40 µg/ml phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin] and then twice with Buffer A containing 500 mM NaCl. Finally, the immune complexes were washed with Buffer C [containing 20 mM HEPES (pH 7.2), 10 mM MgCl2, 0.1 mg/ml BSA, and 3 mM ß-mercaptoethanol] and Buffer D [containing 20 mM HEPES (pH 7.2), 10 mM MgCl2, 10 mM MnCl2, 1 mM dithiothreitol, and 10% glycerol]. S6 phosphotransferase activities were assayed in a reaction mixture consisting of 1x Buffer C, 1 µg S6 peptide, 20 µM ATP, and 5 µCi [-32P]ATP at 30 C for 20 min. Samples were counted by liquid scintillation (Hewlett-Packard Co., Palo Alto, CA).
Cell proliferation assay
ARO ATC cells (1 x 104 cells per well) were plated in a 96-well plate for the indicated times in 100 µl RPMI 1640 with 7% FBS or without serum; each well was then stimulated with 10 ng/ml rhSDF-1 for the indicated periods of time or not stimulated. To determine the number of viable cells, we used the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI) according to the manufacturer’s protocol. Briefly, 20 µl methylthiazoletetrazolium (MTT) solution was added to each well, and, after a 2-h incubation period at 37 C, absorbance at 490 nm was measured.
SDF-1 immunoassay
To measure SDF-1 secretion from ATC cells, ARO, FRO, and NPA cells were cultured in RPMI 1640 with 10% FBS. The cells were shifted into serum-free RPMI 1640 and into RPMI 1640 with cytokines (IL-1ß, 10 ng/ml; IFN-, 10 ng/ml; TGB-ß, 10 ng/ml; and TNF-, 10 ng/ml) for 72 h. The culture supernatant was collected and measured by ELISA (human SDF-1 immunoassay; Quantikine, R&D Systems).
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and CXCR4 in normal and ATC cells
We were interested in determining whether thyrocytes express SDF-1 and CXCR4. To initiate this study, primary cultured normal human thyrocytes and the papillary cancer ATC cell lines, FRO, NPA, and ARO, were evaluated for SDF-1 and CXCR4 expression by RT-PCR and fluorescence activated cell sorting analysis. Bone marrow-derived stromal cells and BMEC were used as negative and positive control cells for SDF-1 expression, respectively. The cultured cells obtained primarily from papillary thyroid cancer and surrounding normal thyroid gland tissue expressed SDF-1 and CXCR4 mRNA (Fig. 1
, lanes 1 and 2). SDF-1 and CXCR4 transcripts were detectable by RT-PCR in ARO, FRO, and NPA ATC cells (Fig. 1, lanes 3, 4, and 5).
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, A, C, and D). However, ARO cells expressed CXCR4 at significant levels (Fig. 2B). These findings suggest that functional CXCR4 receptor expression is not always correlated with CXCR4 transcripts in thyroid cells. Because the SDF-1 transcript was detected in ATC cells, we measured soluble SDF-1 levels in culture medium. The supernatant medium from FRO, NPA, and ARO cells produced similar levels (400 pg/ml) of SDF-1. SDF-1 production was not increased by the cytokines IL-1ß, IFN-, TGF-ß, and TNF- in FRO and ARO cells. However, TNF- increased SDF-1 production in NPA cells (Fig. 1C
). These findings suggest that SDF-1 is constitutively produced by ATC cells and its production is enhanced in a specific cell type by TNF-.
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To evaluate the SDF-1-induced migration of CXCR4-expressing ARO cells, we employed modified Boyden chamber analysis. Because FRO and NPA cancer cells did not express CXCR4, as measured by flow cytometry, we used them to directly evaluate the specific role of CXCR4 in the migration of ARO cells to SDF-1. ARO cells migrate to rhSDF-1 in a dose-dependent manner (P < 0.001 for cell migration to all rhSDF-1 concentrations vs. migration to the control medium; Fig. 3A). Optimal migration of ARO cells was induced by 5–10 ng/ml rhSDF-1. In contrast, FRO and NPA cells did not migrate toward 100 ng/ml rhSDF-1 (Fig. 3B). These results suggest that CXCR4 expression in ATC cells is functional and important in SDF-1-dependent chemotaxis and migration.
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secretion from thyroid cells is due to autocrine or paracrine effects, we performed a migration assay with the culture-supernatant from NPA cells treated with TNF- (Fig. 1C). The addition of culture supernatant (SDF-1 concentration equal to 800 pg/ml) resulted in significant migration of ARO cells, but not of FRO and NPA cells (Fig. 3C). These observations suggest that SDF-1 secreted from thyroid cells is functionally active and may cause migration of a CXCR4-positive thyroid cancer cell line.
SDF-1-induced activation of chemokine receptors has been shown to be mediated primarily by members of the Gi subclass of G proteins, but also by other members of the G class. SDF-1/CXCR4 induces activation of multiple downstream signaling pathways. To determine the signaling pathways responsible for the migration of ARO ATC cells, we measured chemotaxis in the presence of several inhibitors: PTX, LY294002, PD98059, and rapamycin. PTX, a specific inhibitor of heterotrimeric G protein coupling to G protein-coupled receptors, is a potent signaling inhibitor of Gi proteins. An immediate, complete inhibition of rhSDF-1- induced migratory response was induced in ARO cells by PTX (Fig. 3D). In a simultaneous experiment, the specific mitogen-activated ERK inhibitor PD98059 also inhibited rhSDF-1-induced chemotactic activity in ARO cells. LY294002 and rapamycin, the specific chemical inhibitors of PI3K and mTOR/FRAP, respectively, did not suppress the rhSDF-1-induced migratory response in ARO cells. All of these findings suggest that the rhSDF-1-induced chemotactic activity of ARO cells requires activation of Gi and MAPK pathways.
SDF-1/CXCR4 stimulates ERK and S6 kinases but does not activate STAT1, STAT3, and NF-B
Because the above results suggest that ARO cell CXCR4 functions in migratory responses, we analyzed the signaling pathways activated by CXCR4 in response to SDF-1 treatment. The ATC ARO, FRO, and NPA cells had phosphorylated MAPK p44/p42 forms without SDF-1 treatment. Phosphorylation of p44 and p42 MAPKs increased in ARO cells in response to SDF-1 treatment (Fig. 4A). However, SDF-1 did not affect the level of total and phosphorylated p44 and p42 MAPKs in FRO and NPA cells.
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on PI3K pathways in ARO cells, we treated ARO cells with SDF-1 and measured the phosphorylated forms of AKT/PKB and S6K1. The phosphorylation of the T308 and S473 residues of AKT are closely related to the kinase activity of the protein. The phosphorylated forms of AKT were not detected by phosphospecific antibodies after treatment with SDF-1 in ARO cells (Fig. 5A). AKT/PKB activity in immunoprecipitates from ARO cells was not changed by SDF-1 (data not shown). S6K1 is activated upon phosphorylation by several kinases: PDK-1, mTOR/FRAP, and unidentified hydrophobic motif kinases. The T389 residue of S6K1 has been shown to be phosphorylated by mTOR/FRAP. SDF-1 treatment induced T389 phosphorylation in S6K1 (Fig. 5B), which correlated with increased S6K1 activity (Fig. 5D). In addition, phosphorylation of the ribosomal S6 protein, a substrate of S6K1, was blocked by rapamycin (Fig. 5C, lane 4).
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activates Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways in CXCR4-positive ARO cells, we measured levels of phosphorylated STAT1 and STAT3 after SDF-1 treatment. SDF-1 did not induce S727 and Y705 STAT3 phosphorylation or Y701 STAT1 phosphorylation in ARO cells. The basal levels of STAT1 S727 phosphorylation measured in ARO cells were not increased by SDF-1 treatment (Fig. 6A). In addition, the total levels of IB and p65 in ARO cells were not changed by SDF-1 treatment (Fig. 6B).
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/CXCR4 did not affect ATC cellular proliferation and survival
SDF-1 was initially designated PBSF because rhSDF-1 supports the proliferation of a stromal cell-dependent B-cell line (DW34; Ref. 16). However, SDF-1 stimulates neuronal apoptosis in vitro, indicating a direct cytotoxic effect on neuronal cells (17). To determine whether SDF-1 promotes cellular proliferation in ARO cells, we cultured ARO cells in the presence or absence of rhSDF-1. rhSDF-1 did not enhance proliferation of ARO cells cultured in RPMI 1640 (including 7% FBS; Fig. 7A). In a parallel experiment, serum deprivation for 24 h caused a 50% reduction in ARO cells cultured in RPMI 1640 (with 7% FBS). The addition of rhSDF-1 did not protect against cell death during serum deprivation. These findings suggest that SDF-1 is not a potent enough growth factor in CXCR4-expressing ATC cells to stimulate cellular proliferation and prevent cellular apoptosis.
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-mediated tumor cell migration. The expression of SDF-1, a unique ligand of CXCR4 expressed in all tissues including normal thyroid gland, is reduced in autonomous thyroid adenomas (13). We found that normal thyroid cells and ATC cells also expressed the SDF-1 transcript (Fig. 1A). In addition, ATC cells secrete SDF-1 into the culture medium. However, ATC cells did not secrete SDF-1 in response to IL-1ß, IFN-, TGF-ß, and TNF-. Cultured human thyroid cells also secrete SDF-1 and SDF-1ß, but SDF-1 secretion is not stimulated by lipopolysaccharide, IL-1, and TNF (13). Secreted SDF-1 is functionally active for the initiation of ARO cell migration (Fig. 3C). These results suggest that autocrine SDF-1 activity may be involved in the local invasion of ATC.
We found CXCR4 RT-PCR products in cultured human thyroid cells and the ARO, FRO, and NPA ATC cells. The RT-PCR product sequences matched the human CXCR4 sequence (data not shown). However, we detected membrane CXCR4 only in ARO cells and not in normal thyroid, FRO, and NPA cells. The discrepancy between the presence of CXCR4 transcripts and the lack of CXCR4 membrane expression may be due to several reasons. Instability of membrane CXCR4 expression has been noted in other cells, such as neuroblastoma cells (21). Because SDF-1 treatment results in the down-regulation of CXCR4 expression, autocrine secretion of SDF-1 may result in endocytosis (22, 23) and ubiquitin-mediated degradation of CXCR4 (24).
CXCR4 may be important in the infiltration of macrophages and lymphocytes in such cancers as melanoma, carcinomas of the ovary, breast, and cervix, and sarcomas and gliomas (25, 26, 27). In addition, CXCR4 has been shown to mediate metastasis by SDF-1 gradients. These findings may be important in the development of new biological cancer therapies. Among the ATC cells evaluated, in vitro migration was SDF-1-dependent in CXCR4-positive ARO cells. CXCR4-mediated migration of ARO cells was specific, as shown by the dose-dependent inhibition of migration by neutralizing anti-CXCR4. The ATC cells, FRO and NPA, which did not express CXCR4, did not demonstrate significant SDF-1-mediated migration in vitro. These observations suggest that CXCR4 expression in ATC cells may affect biological behaviors, such as local invasion and distant metastasis of ATC. Several adhesion molecules are involved in the migration of tumor cells; we phenotyped, with respect to the expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin, the ARO cells that responded to SDF-1 stimulation. Surprisingly, SDF-1 stimulation in ARO cells did not affect the expression levels (data not shown). In vitro SDF-1-induced-migration of ARO cells was inhibited by PTX (100 ng/ml) and PD98059, but not by the PI3K inhibitor LY294002 or the mTOR/FRAP inhibitor rapamycin. These observations suggest that CXCR4 expressed in ATC cells functions through Gi-coupled MAPK signaling pathways in SDF-1-dependent migration. The signaling molecules associated with the activation of CXCR4 by SDF-1 have recently been clarified and include Erk1/2 MAPK (28, 29), Pyk2 (30), paxillin, Crk (31), AKT/PKB, PI3K (32, 33), and NF-B (34). The JAK and STAT system has also been implicated in the CXCR4 signaling pathway (35, 36). However, these signaling pathways are strictly regulated by cell type. We found that CXCR4-expressing ARO cells rapidly phosphorylated the T202 and Y204 residues of Erk1 and Erk2 in response to SDF-1 treatment. When pretreated with PTX and PD98059, SDF-1-induced phosphorylation of Erk1 and Erk2 was blocked in ARO cells. These findings suggest that SDF-1-induced Erk1 and Erk2 activation are downstream effectors of a Gi protein coupled to CXCR4. These observations are compatible with the PTX- and PD98059-induced inhibition of the in vitro migration of ARO cells in response to SDF-1.
One of the major downstream kinases of PI3K is AKT/PKB. AKT/PKB activation is associated with the phosphorylation of T308 and S473, residues in an activation loop and hydrophobic motif, respectively. Phosphorylation of these residues was not detected by phosphospecific antibodies after SDF-1 treatment in ARO cells. However, p70S6 kinase (Fig. 5B) and its substrate ribosomal S6 protein were rapidly phosphorylated by SDF-1 in ARO cells. p70S6 Kinase activity was closely related to phosphorylation of its T389 residue. Activation of p70S6 kinase is associated with ribogenesis, cellular proliferation, and apoptosis inhibition by phosphorylating the proapoptotic molecule, BAD (37, 38, 39). However, SDF-1 treatment in ARO cells did not affect cellular proliferation or cellular death induced by serum deprivation (Fig. 7). These findings suggest that SDF-1 alone does not regulate cellular proliferation and survival in ATC cells.
Zhang et al. (36) have identified that SDF-1 stimulates tyrosine phosphorylation of JAK2, JAK1, TYK2, STAT2, and STAT4 in the human progenitor cell line, CTS. SDF-1 did not induce tyrosine phosphorylation of JAK1 and JAK2 (data not shown) or of STAT1 and STAT3 in ARO cells. These findings suggest that SDF-1/CXCR4 signaling is cell type-specific. SDF-1 activates NF-B through direct pre-B lymphoma cell line L1.2 and in primary astrocytes (34), respectively. We monitored the IB level in ARO cells after short and long time periods of SDF-1 treatment to trace the activation of NF-B (Fig. 6B). However, the IB level did not decrease with SDF-1 treatment, suggesting that SDF-1 does not activate the NF-B pathway in CXCR4-expressing ARO cells.
In summary, migration of CXCR4-expressing ARO cells was SDF-1-dependent in vitro and required mainly the Gi-coupled Erk1 and Erk2 MAPK pathway. Although SDF-1 activates MAPKs and p70S6 kinase, it alone is not competent to stimulate proliferation and enhance the survival of CXCR4-expressing ARO cells. In addition, CXCR4-expressing ARO cells did not activate several known signaling pathways; SDF-1 activates MAPK and p70S6 kinase in CXCR4-expressing ARO cells but not JAK/STAT, AKT, and NF-B, which are known to be activated by SDF-1 in other cell types. We postulate that the acquisition of functional CXCR4 expression during the process of carcinogenesis in ATC may be involved in local invasion by autocrine and paracrine SDF-1 stimulation.
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Acknowledgments |
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Footnotes |
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Abbreviations: ATC, Anaplastic thyroid cancer; CXCR4, CXC chemokine receptor 4; FBS, fetal bovine serum; IB, inhibitor B; HA, hemagglutinin antigen; JAK, Janus kinase; MTT, methylthiazoletetrazolium; NF-B, nuclear factor-B; PBSF, pre-B-cell growth-stimulating factor; PI3K, phosphatidylinositol 3-kinase; PTX, pertussis toxin; rh, recombinant human; SDF, stromal cell derived factor; STAT, signal transducer and activator of transcription.
Received August 30, 2002.
Accepted October 2, 2002.
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 I. Airoldi, L. Raffaghello, E. Piovan, C. Cocco, B. Carlini, A. Amadori, M. V. Corrias, and V. Pistoia CXCL12 Does Not Attract CXCR4+ Human Metastatic Neuroblastoma Cells: Clinical Implications Clin. Cancer Res., January 1, 2006; 12(1): 77 - 82. [Abstract] [Full Text] [PDF]
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 J. T. Kaifi, E. F. Yekebas, P. Schurr, D. Obonyo, R. Wachowiak, P. Busch, A. Heinecke, K. Pantel, and J. R. Izbicki Tumor-Cell Homing to Lymph Nodes and Bone Marrow and CXCR4 Expression in Esophageal Cancer J Natl Cancer Inst, December 21, 2005; 97(24): 1840 - 1847. [Abstract] [Full Text] [PDF]
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A. Katayama, T. Ogino, N. Bandoh, S. Nonaka, and Y. Harabuchi Expression of CXCR4 and Its Down-Regulation by IFN-{gamma} in Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., April 15, 2005; 11(8): 2937 - 2946. [Abstract] [Full Text] [PDF]
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C. Laverdiere, B. H. Hoang, R. Yang, R. Sowers, J. Qin, P. A. Meyers, A. G. Huvos, J. H. Healey, and R. Gorlick Messenger RNA Expression Levels of CXCR4 Correlate with Metastatic Behavior and Outcome in Patients with Osteosarcoma Clin. Cancer Res., April 1, 2005; 11(7): 2561 - 2567. [Abstract] [Full Text] [PDF]
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 F. Pacifico, C. Mauro, C. Barone, E. Crescenzi, S. Mellone, M. Monaco, G. Chiappetta, G. Terrazzano, D. Liguoro, P. Vito, et al. Oncogenic and Anti-apoptotic Activity of NF-{kappa}B in Human Thyroid Carcinomas J. Biol. Chem., December 24, 2004; 279(52): 54610 - 54619. [Abstract] [Full Text] [PDF]
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G. G. Vaday, S.-B. Hua, D. M. Peehl, M. H. Pauling, Y.-H. Lin, L. Zhu, D. M. Lawrence, H. D. Foda, and S. Zucker CXCR4 and CXCL12 (SDF-1) in Prostate Cancer: Inhibitory Effects of Human Single Chain Fv Antibodies Clin. Cancer Res., August 15, 2004; 10(16): 5630 - 5639. [Abstract] [Full Text] [PDF]
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 Y. S. Haviv, W. J. van Houdt, B. Lu, D. T. Curiel, and Z. B. Zhu Transcriptional targeting in renal cancer cell lines via the human CXCR4 promoter Mol. Cancer Ther., June 1, 2004; 3(6): 687 - 691. [Abstract] [Full Text] [PDF]
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 X. Wu, D.-J. Li, M.-M. Yuan, Y. Zhu, and M.-Y. Wang The Expression of CXCR4/CXCL12 in First-Trimester Human Trophoblast Cells Biol Reprod, June 1, 2004; 70(6): 1877 - 1885. [Abstract] [Full Text] [PDF]
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 J.-P. Spano, F. Andre, L. Morat, L. Sabatier, B. Besse, C. Combadiere, P. Deterre, A. Martin, J. Azorin, D. Valeyre, et al. Chemokine receptor CXCR4 and early-stage non-small cell lung cancer: pattern of expression and correlation with outcome Ann. Onc., April 1, 2004; 15(4): 613 - 617. [Abstract] [Full Text] [PDF]
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