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1 James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky;
2 Department of Pathology and Laboratory Medicine and Department of Medicine, University of Alberta and Canadian Blood Services, Edmonton, Alberta, Canada;
3 Department of Transplantology, Polish-American Children’s Hospital CMUJ, Kracow, Poland;
4 Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Why RMS cells metastasize to the bone marrow is still poorly understood. It is likely that bone marrow stroma secretes chemoattractants for RMS cells, and that these cells are attracted into the bone marrow microenvironment where they may find favorable conditions for survival and expansion. In this study we attempted to identify the molecular mechanisms that may be involved in the metastasis of RMS cells to the bone marrow. We recently reported that RMS cells highly express the receptor for the 
-chemokine SDF-1 and that the CXCR4-SDF-1 axis regulates the metastatic behavior of RMS (11)
. RMS cells, however, have also been found to express the tyrosine kinase receptor c-MET (12
, 13)
, known to mediate the multifunctional and potentially oncogenic activities of HGF, also known as scatter factor (14
, 15)
, including cell motility, extracellular matrix degradation, angiogenesis, cell proliferation, and survival (16, 17, 18, 19, 20)
. Perturbation of signaling from the c-MET receptor because of c-MET overexpression/amplification, activating mutations, or autocrine HGF-c-MET loops has been implicated in the pathogenesis of various tumors (1
, 20)
.
Because normal bone marrow stroma cells and osteoblasts highly express and secrete both HGF and SDF-1 (17 , 21, 22, 23, 24, 25, 26) , we hypothesized that the c-MET-HGF axis, like the CXCR4-SDF-1 axis, plays an essential role in directing RMS cells into bone marrow. We focused on the biological responses to stimulation by exogenous HGF of c-MET-positive ARMS and ERMS cell lines, such as phosphorylation of signaling proteins, cell proliferation, survival, adhesion, expression of MMPs, chemotaxis, and chemoinvasion, as well as the survival of RMS cells exposed to radio- and chemotherapy. Our findings indicate that the HGF-c-MET axis, like the CXCR4-SDF-1 axis, regulates the metastatic behavior of RMS cells and their metastasis to the bone marrow and could also contribute to their resistance to conventional treatment modalities.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FACS Analysis.
The expression of c-MET on various RMS cell lines was evaluated by FACS as described previously (11
, 27)
. The c-MET antigen was detected with anti-c-MET MoAb (UPS Biotechnology, Lake Placid, NY), clone DO-24, followed by staining with secondary antibody FITC--murine IgG. Briefly, the cells were stained in PBS (calcium- and magnesium-free) supplemented with 5% BCS (Hyclone, Logan, Utah). After the final wash, cells were fixed in 1% paraformaldehyde, and FACS analysis was performed using the FACscan (Becton Dickinson, San José, CA).
Evaluation of Adhesion Molecules.
The expression of adhesion molecules on RMS cells was evaluated by FACS. Cells were stained with specific anti-PECAM-1, ICAM-1, VCAM-1, E-selectin, and VLA-5 and VLA-4 antibodies detected with phycoerythrin-conjugated secondary phycoerythrin-goat anti-mouse MoAbs as described previously (28)
. The following antibodies were used for this study: 4G6 (IgG2b, mouse anti-human PECAM-1), generously provided by Dr. Steven Albelda; R6.5 (BIRR-1), a murine IgG2a MoAb directed against extracellular domain two of the ICAM-1 molecule, from Boehringer Ingelheim Pharmaceuticals Inc. (Ridgefield, CT); 4B9, an IgG1 MoAb directed against human VCAM-1, from Dr. Roy Lobb, Biogen Inc. (Cambridge, MA); and ES2, (IgG1-
mouse anti-human-E-selectin MoAb), provided by Dr. Rodger McEver, University of Oklahoma (Tulsa, OK). Antibodies against 6ß1 integrin were purchased from PharMingen (San Diego, CA).
Phosphorylation of Intracellular Pathway Proteins.
Western blots were done on extracts prepared from RMS cell lines (1 x 107 cells), which were kept in RPMI 1640 containing low levels of BSA (0.5%) to render the cells quiescent. The cells were then divided and stimulated with optimal doses of HGF (10 ng/ml) for 1 min to 2 h at 37°C before lysing for 10 min on ice in M-Per lysing buffer (Pierce, Rockford, IL) containing protease and phosphatase inhibitors (Sigma). Subsequently, the extracted proteins were separated on either a 12 or 15% SDS-PAGE gel, and the fractionated proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) as described previously (11
, 29
, 30)
. Phosphorylation of the intracellular kinases, 44/42 MAPK (Thr-202/Tyr-204) and AKT, and STAT-1, -3, -5, and -6 proteins was detected using commercial mouse phospho-specific MoAb (p44/42) or rabbit phospho-specific polyclonal antibodies for each of the remainder (all from New England Biolabs, Beverly, MA) with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG as a secondary antibody (Santa Cruz Biotechnology Biotechnology, Santa Cruz Biotechnology, CA) as described (11)
. Equal loading in the lanes was evaluated by stripping the blots and reprobing with appropriate MoAbs: p42/44 anti-MAPK antibody clone #9102, anti-AKT antibody clone #9272, anti-STAT-3 #9132 (New England Biolabs), anti-STAT-1 #sc-464 and anti-STAT-6 #sc-1689 (Santa Cruz Biotechnology), and anti-STAT-5 #89 (Transduction Laboratories, Lexington, KY). The membranes were developed with an ECL reagent (Amersham Life Sciences, Little Chalfont, United Kingdom), dried, and subsequently exposed to film (HyperFilm; Amersham).
Detection of HGF by Western Blot Analysis.
HGF protein was detected in lysates derived from RMS cell lines using specific rabbit polyclonal antibody (from R&D, Minneapolis, MN), which was detected with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG as a secondary antibody (Santa Cruz Biotechnology) as described previously (11)
.
Isolation of mRNA and RT-PCR.
For the analysis of HGF mRNA total mRNA was isolated from RMS cells with the RNeasy Mini Kit (Qiagen Inc., Valencia, CA), mRNA was reverse-transcribed with TaqMan Reverse Transcription Reagents (Applied Biosystems, Branchburg, NJ), and the PCR reaction was carried out with the 5'-GAG GGA CAT AAG AAA AGA AGA-3' sense primer and the 5'-GTG TGG TAT CAT GGA ACT CCA-3' antisense primer. The predicted size of the RT-PCR product for HGF was 383 bp. Amplified products (10 µl) were electrophoresed on a 1.5% agarose gel and transferred to a nylon filter. The specificity of the amplified products was further confirmed by Southern blotting (data not shown).
Real-Time RT-PCR.
To analyze HGF and SDF-1 mRNA levels, total mRNA was isolated from cells that were flushed out from the bone marrow cavities of nonirradiated (control) and irradiated (800 cGy) sacrificed mice with the RNeasy Mini Kit (Qiagen, Inc.) and was reverse-transcribed with TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). Detection of HGF and SDF-1 and ß-actin mRNA levels was performed by real-time RT-PCR assay using an ABI PRISM 7000 Sequence Detection System (ABI, Foster City, CA). A 25-µl reaction mixture contains 12.5 µl of SYBR Green PCR Master Mix, 100 ng of cDNA template, 5'-TGC GTC CAC GAG CTG TTT AC-3' forward and 5'-CCC AAG GGA GTG TCA GGT AGA G-3' reverse primers for SDF-1, and 5'-CTC ACA CCC GCT GGG AGT AC-3' forward and 5'-TCC TTG ACC TTG GAT GCA TTC-3' reverse primers for HGF. The primers were designed with Primer Express software. The threshold cycle (Ct), i.e., the cycle number at which the amount of the amplified gene of interest reaches a fixed threshold, was determined subsequently. Relative quantitation of HGF and SDF-1 mRNA expression was calculated by the comparative Ct method. The relative quantitation value of target, normalized to an endogenous control ß-actin gene and relative to a calibrator, is expressed as 2
Ct (fold), where Ct = Ct of the target gene (HGF and SDF-1) - Ct of endogenous control gene (ß-actin), and Ct = Ct of samples for target gene - Ct of the calibrator for the target gene.
Cell Proliferation and Apoptosis.
Cells were plated in culture flasks at an initial density of 104 cells/cm2 in the presence or absence of HGF (10 ng/ml). Cells were counted at 12, 24, 36, 48, and 60 h after culture initiation (in some experiments also at 72 h). At these time points, cells were harvested from the culture flasks by trypsinization, and the number of cells was determined using a Bürker‘s hemocytometer. Apoptosis was evaluated by the Annexin-V binding assay, and intracellular staining was done for activated caspase-3, as described (28
, 29)
.
Fluorescent Staining of the Actin Cytoskeleton.
To visualize the actin cytoskeleton, cells were cultured for 12 h on glass coverslips in RPMI 1640 supplemented with 10% fetal bovine serum in the absence or presence (10 ng/ml) of HGF. Subsequently, the cells were fixed in 3.7% paraformaldehyde/calcium- and magnesium-free PBS for 15 min, permeabilized by 0.1% Triton X-100 in PBS for 1 min at room temperature, and stained with TRITC-phalloidin at a concentration of 500 ng/ml for 1 h. Cells were examined using a BX51 fluorescence microscope (Olympus America, Melville, NY) equipped in a charge-coupled device camera (Olympus America). Each staining was repeated three times for each cell line.
Recording Cellular Motility.
Two h before starting the motility experiments, CW9019, RH28, RH30, RH18, and SMS-CTR cells were plated in culture dishes at a density of 2 to 2.5 x 104 cells/cm2. Cells were mock-treated or stimulated by HGF (10 ng/ml) for 30 min, and then the tracks of individual RMS cells were recorded with an inverted Hund Wetzlar microscope using phase-contrast optics. In brief, the images of locomoting cells were recorded with a charge-coupled device camera, digitized, and processed. The cell trajectories were constructed from 40 subsequent cell centroid positions recorded for 200 min at time intervals of 5 min. Fifty cell tracks were recorded under each of the experimental conditions tested. To analyze the cellular motility variables, the files containing the tracing data were read into the program Mathematica (Wolfram Research Inc., Champaign, IL; Refs. 31, 32, 33
).
The following parameters characterizing cell movement were computed for each cell: total length of cell trajectory (µm), i.e., the sum of n straight-line segments, each corresponding to cell centroid translocation at a given time interval; the average speed of cell movement (calculated as length of cell path in a given time); total length of cell displacement (µm), i.e., the distance from the starting point directly to the cell’s final position; average rate of cell displacement, i.e., the distance from the starting point directly to the cell’s final position/time of recording; and coefficient of movement efficiency (CME), corresponding to ratio of cell displacement to cell trajectory length. The CME equals 1 for cells moving consistently along a single straight line in a given direction and 0 for random movement.
Transmembrane Chemotaxis.
Cells were seeded in RPMI 1640 containing 10% FBS into 6-well plates. After they had adhered to the dish bottom, the medium was changed, and they were made quiescent as described previously (11)
. Transmembrane chemotaxis across 8-µm pore polycarbonate membranes covered with 50 µl of fibronectin (50 µg/ml) for 2 h at 37°C and overnight at 4°C was examined. Cells were detached (with 0.5 mM EDTA), washed in RPMI 1640, resuspended in RPMI 1640 with 0.5% BSA, and seeded at a density of 105 in 200 µl into the upper chambers of Transwell inserts (Costar Transwell). The lower chambers were filled with HGF (10 ng/ml) or 0.5% BSA RPMI 1640 (control). To determine whether migration was stimulated by the gradient of the chemoattractant, in some experiments HGF was also added to the upper chambers to equalize the difference in concentrations between chambers. After 48 h, the inserts were removed from the Transwells, cells remaining in the upper chambers were scraped off with cotton wool, and cells that had transmigrated were counted either on the lower side of the membranes or on the bottom of the Transwells.
Some of the directional migration experiments to conditioned media harvested from bone marrow-derived fibroblasts were performed on cells preincubated for 30 min at 37°C in the presence of 1 µM T140-truncated polyphemusin analogue (a gift from Dr. Nobutaka Fuji, Kyoto University, Japan) or preincubated in the presence of 1 µM c-Met inhibitor, K-252 (Calbiochem-Novabiochem International). Conditioned media from bone marrow-derived fibroblasts were derived from cells cultured for 24 h in serum-free medium as described previously (11) .
Adhesion of RMS Cells to HUVEC Cells.
RMS cells were labeled with the fluorescent dye calcein-AM and subsequently seeded (for 5 min) onto the 96-well plates covered with HUVEC cells that had been pretreated with HGF (10 ng/ml). After incubation (at 37°C), the plates were washed three times, and cells that adhered to the HUVECs were dissolved using 2% SDS. Fluorescence was measured using the Fusion Universal Microplate Analyzer (Perkin-Elmer, Boston, MA).
MMP Expression.
To evaluate MMP-2 and MMP-9 activities, RMS cells were incubated for 24 h in serum-free media in the absence (control) or presence of HGF (10 ng/ml), and zymography was carried out as described previously by us (11
, 34, 35, 36)
. To evaluate expression of genes for MMP-2 and MMP-9, as well as MT1-MMP and TIMP-2, total RNA was extracted, and the conversion of mRNA to cDNA was carried out using AMVRT (Seigaku America, Ijamsville, MD); polymerase chain reactions were performed following the "primer dropping" method. Sequences for human MMP-2, MMP-9, MT1-MMP, and TIMP-2 were obtained from GenBank (Los Alamos, NM) and used to design primer pairs, as described by us previously (34, 35, 36)
.
Chemoinvasion Assay.
The ability of malignant cells to invade the reconstituted basement membrane Matrigel is regarded as an important measure of their metastatic potential. Three ARMS cell lines (RH28, RH30, and CW9019) and two ERMS cell lines (RH1 and RD) were evaluated in a chemoinvasion assay as described and modified by us (11
, 34, 35, 36)
. Briefly, cells were loaded onto the upper compartments of Boyden chambers (105 cells/chamber) and incubated for 48 h. Cells that invaded the Matrigel barrier toward media alone or toward an HGF gradient (10 ng/ml) were counted on the undersides of filters after fixation and staining with crystal violet. A chemoinvasion index was calculated as the ratio of the number of cells invading the Matrigel toward an HGF gradient to the number of cells invading toward media alone.
Sequencing.
To address whether mutations activating the c-MET receptor in RMS cells could lead to the hyperreactivity of the HGF-c-MET axis in RMS, in addition to overexpression of c-MET, we sequenced c-MET DNA from several RMS cell lines (RH18, RH30, RH28, CW9019, SMS-CTR, and RD) and melanoma SBCL2 (negative control) and searched for potential activating mutations. DNA was extracted by means of the QIAamp DNA Mini Kit (Qiagen). Sequencing PCR was performed on 10 ng of spectrophotometrically quantified DNA with the use of fluorescently labeled nucleotides and exon-specific primers as follows: (a) 5'-TCTTCCTGTTTCAGTCCCCAT-3' and 5'-AGGCCAAAGATAAAATGCTTACTG-3' for exon 15; (b) 5'-CTCATAAAGGGTT-TGATAAATAATTATTT-3' and 5'-GTTTTATTATAAGCTATTTATTAGGTTGCA-3' for exon 16; (c) 5'-GCTCTTCCTATCTAAATTTGACAAAAG-3' and 5'-AGGCCTATTTTGAAGGGATG-3' for exon 17; (d) 5'-TCAGAATTCTAAGGTCAA-AATTAGAACAG-3' and 5'-TTGAACAGTGGGAAACAGATTC-3' for exon 18; (e) 5'-TCTGTAGATATTCAGCATCATTGTAAAT-3' and 5'-AGTGATAAAACTTCAA-AAAAAGTTGG-3' for exon 19. The PCR product was run on Applied Biosystems 310 sequencing apparatus and analyzed by means of Sequencing Analysis software (Applied Biosystems). The sequencing was performed on exons 15–19 encoding the tyrosine kinase domain.
Transplants of RMS Cells into Lethally Irradiated Mice.
To evaluate the in vivo metastatic behavior of RMS cells lines, we injected ARMS or ERMS cells (2 x 106 cells/mouse) i.v. into lethally irradiated (800 cGy) mice. Cells were injected 24 h after irradiation. Then 36 h after injection of these RMS cells, the bone marrow cavities of femora, tibiae, and humeri were flushed to isolate cells present in the bone marrow. The presence of RMS cells in murine bone marrow (i.e., murine-human chimerism) was evaluated by the differences in the level of human -satellite DNA amplified in the extracts isolated from bone marrow-derived cells, using real-time PCR. Briefly, DNA was isolated using the QIAamp DNA Mini Kit (Qiagen Inc.). Detection of -satellite and ß-actin DNA levels was performed by real-time PCR using an ABI PRISM 7000 Sequence Detection System (ABI). A 25-µl reaction mixture contains 12.5 µl of SYBR Green PCR Master Mix, 50 ng of DNA template, 5'-ACACTCTTTTTGCAGGATCTA-3' forward and 5'-AGCAATGTGAAACTCTGGGA-3' reverse primers for -satellite; and 5'-GGA TGC AGA AGG AGA TCA CTG-3' forward and 5'-CGA TCC ACA CGG AGT ACT TG-3' reverse primers for ß-actin. The primers were designed with Primer Express software. The threshold cycle (Ct), i.e., the cycle number at which the amount of amplified gene of interest reaches a fixed threshold, was determined subsequently. Relative quantitation of -satellite DNA was calculated with the comparative Ct method described elsewhere (37)
. The relative quantitation value of target, normalized to a control ß-actin gene and relative to a calibrator, is expressed as 2-Ct (fold difference), where Ct = Ct of target gene (-satellite) - Ct of control gene (ß-actin), and Ct = Ct of samples for target gene - Ct of calibrator for the target gene.
Statistical Analysis.
All results are presented as mean ± SE. Statistical analyses of the data were performed using the nonparametric Mann-Whitney test, with P < 0.05 considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, upper panel). In three ERMS cell lines (SMS-CTR, RD, and RH18), c-MET was also expressed, although at lower levels (Fig. 1
, lower panel). We observed that all eight ARMS cells lines also stained highly positive for CXCR4 (>60% of cells; not shown), which was consistent with our previous findings of high expression of CXCR4 in ARMS cells (11)
. As we reported previously (11)
, CXCR4 is expressed at a much lower level in ERMS cells.
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Because the biology of various tumors may be regulated by autocrine/paracrine axes, we next asked whether these RMS cells express HGF, the c-MET ligand. We found that only 3 (RH1, RH18, and RD) of 11 cell lines investigated in this study expressed a low level of mRNA for HGF (data not shown). The presence of HGF at the protein level was confirmed for RH18 and RD cells. However, blocking the putative c-MET-HGF autocrine regulatory axis by adding anti-HGF blocking MoAb did not affect the proliferation kinetics of these cell lines (data not shown).
HGF Accelerates Locomotion of Individual RMS Cells.
To examine whether HGF influences the locomotion of RMS cells on plastic dishes, we selected three ARMS cell lines (CW9019, RH28, and RH30) and two ERMS cell lines (RH18 and SMS-CTR) and used time-lapse monitoring to record the locomotion of individual cells. The trajectories of RMS cells in the absence or presence of HGF in the culture medium are shown in Fig. 3
. Analysis of these trajectories and mean values and standard errors for the parameters of cell locomotion are summarized in Table 1
.
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. At the same time, the average rate of cell displacement (AvcD) increased much more significantly to 
170% of the control for CW9019 cells, 160% for RH28, 210% for RH30, and 400% for RH18. This indicates that the major increase in cell displacement is achieved mainly because of the change in cell linear movement (Fig. 3)
. Of the cell lines investigated, only SMS-CTR cells did not show a significant acceleration of the average rate of cell displacement (Table 1)
.
HGF Alters the Actin Cytoskeleton.
Next we performed immunofluorescent staining of the actin cytoskeleton to determine its relationship to the results of the migration assays. Immunofluorescent images from Alexa phalloidin, which has a high affinity for the actin cytoskeleton, revealed striking differences between the HGF-treated and untreated RMS cells. In the absence of HGF, RMS cells displayed well-developed filaments of F-actin arranged in parallel to the long axis of the cell (Fig. 4
, A, C, and E), but in its presence they showed motility-related redistribution of the actin cytoskeleton toward the leading edge of the cell (Fig. 4
, B, D, and F).
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We found that HGF significantly increased the chemotactic activity of the ARMS cell lines RH18, RH28, CW9019, and RH30 and the ERMS cell lines SMS-CTR and RD (Fig. 5)
. Interestingly, RH18 cells, which showed the highest increase in locomotion on plastic dishes in the presence of HGF, had the weakest directional chemotaxis to HGF, especially through fibronectin-covered membranes. In contrast, RH 28 cells, which showed weak locomotion in response to HGF, responded much more strongly to directional chemotaxis through fibronectin-covered membranes. RH30 cells that had high spontaneous motility showed the highest directional chemotaxis to HGF.
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. More importantly, we found that inhibition of PI-3K totally inhibits the chemotaxis of RH30 cells (Fig. 6B)
.
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. In a similar set of experiments, we observed that HGF-dependent chemotaxis as well as HGF-mediated phosphorylation of AKT was inhibited in the RH30 ARMS cell line by the ATP analogue K562a, which is a potent inhibitor of the c-MET receptor (38) .
HGF Increases Adhesion to HUVEC.
We found that HGF affected the adhesion to HUVEC cells of three ARMS cell lines investigated (RH28, RH30, and CW9019) and one ERMS cell line (SMS-CTR; Fig. 7
). Generally, the effect on ERMS cells was smaller, and as demonstrated in Fig. 7B
, HGF did not affect adhesion of RD cells. However, when we investigated whether HGF regulates expression/activation of integrins on human RMS using FACS analysis, we did not find any change in the level of expression of VLA-4, VLA-5, PECAM-1, or ICAM-1 on RMS cells after incubation with HGF for 24 h (data not shown).
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, with the exception of MT1-MMP in RH-1 cells. After HGF stimulation, pro-MMP-9 activity increased in RH1, RH28, RH30, and CW9019 cells (as measured by zymography). Pro-MMP-2 activity increased in RH1, RH28, and CW9019 but not in the other cell lines tested (Fig. 8A
and Table 2
). However, we found that HGF stimulation did not influence MT1-MMP and TIMP-2 expression in any of the RMS lines tested (Table 2)
.
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HGF and SDF-1 Increase Chemotaxis of RMS Cells in an Additive Manner.
In our previous work (11)
, we reported that SDF-1, as we have shown here for HGF, regulated the metastatic behavior of RMS cells by increasing cell locomotion, directional chemotaxis, adhesion, and production of MMPs. Hence we decided to investigate whether both factors regulate metastatic behavior in an additive or a synergistic manner. To address this issue, we exposed RH30 ARMS cell lines that show strong chemotaxis to HGF (Fig. 5)
and SDF-1 alone (11)
to increasing doses of HGF (0–10 ng/ml) in the presence of a constant suboptimal dose (50 ng/ml) of SDF-1 (Fig. 9, A and B)
. On the basis of these data, we selected a dose of SDF-1 (50 ng/ml) that alone stimulated 30% of the maximal chemotactic response to SDF-1 (Fig. 9A)
. To this concentration of SDF-1 we added incremental doses of HGF (0–10 ng/ml). As shown in Fig. 9B
, 10 ng/ml of HGF induces almost maximal chemotactic response by these cells.
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summarizes the chemotactic response of RH30 cells to incremental doses of HGF in the absence or presence of 50 ng/ml of SDF-1. We observed that both factors increased the chemotactic activity of RH30 cells in an incremental manner.
RMS Cells Are Chemoattracted into Bone Marrow in Vitro and in Vivo in an HGF- and SDF-1-dependent Manner.
ARMS cells metastasize frequently to the bone marrow, and in fact we found that CM harvested from bone marrow-derived fibroblasts, which are a source for example of HGF (21
, 39)
and SDF-1 (22, 23, 24, 25, 26
, 39)
, chemoattracted the ARMS cell line CW9019 (Fig. 10A)
. Thus, we used K-252a and T140, which are specific blocking agents for c-MET and CXCR4, respectively, to determine whether these molecules inhibit the chemotactic responses of RMS cells to CM harvested from bone marrow-derived fibroblasts. Fig. 10A
also shows that the chemoattraction of CW9019 cells to CM is significantly inhibited by the specific blocking agents K-252a and T140. These experiments suggest that HGF and SDF-1 are major stroma-derived factors that attract RMS cells.
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. Thirty-six h after injection into lethally irradiated/immunosuppressed mice, we found that more ARMS than ERMS cells were present in the bone marrow cavities as evaluated by detection of human specific -satellite DNA using real-time PCR. This assay suggests that both HGF and SDF-1, which are up-regulated in irradiated bone marrow stroma (Fig. 10
A, inset), strongly chemoattracted ARMS cells circulating in the peripheral blood in vivo.
HGF but not SDF-1 Increases Survival of RMS Cells.
Activation of the tyrosine kinase receptor c-MET, in contrast to activation of G-protein-coupled CXCR4, influences cell survival in several cell lines (17
, 19
, 21)
. Thus, to find potential differences between HGF and SDF-1, we evaluated the effects of both factors on the survival of RMS cells. Two ARMS cell lines, RH30 and CW9019, were exposed to HGF or SDF-1 in three experimental models of cell stress that lead to cell apoptosis (Fig. 11)
such as: (a) culture under serum starvation (72 h); (b) 
-irradiation by 1500 cGy; and (c) exposure to vincristine (5 ng/ml) and etoposide (10 µM). We found that HGF, but not SDF-1, enhances the survival of both CW9019 and RH30 cells (P < 0.001). Thus, although both HGF and SDF-1 regulate the metastatic behavior of RMS cells, only HGF increases their survival (Fig. 11)
.
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DISCUSSION |
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In this work we have hypothesized that RMS cells that express both c-MET (1
, 12)
and CXCR4 (11)
could be chemoattracted to the bone marrow by the chemoattractants that are secreted by bone marrow stroma fibroblasts and osteoblasts, such as HGF and SDF-1 (21, 22, 23, 24, 25, 26)
. Supporting this is our finding that CM harvested from human bone marrow derived-fibroblasts chemoattract ARMS cells in an HGF- and SDF-1-dependent manner (Fig. 10A)
. Furthermore, CXCR4 had been implicated recently in the metastasis of several solid tumors (39
, 41, 42, 43, 44, 45)
. The role of the CXCR4-SDF-1 axis in the metastasis of RMS cells was the subject of our previous study (11)
, and here we have focused on the HGF-c-MET axis. The involvement of the HGF-c-MET axis is also known to play an important role in the metastasis/progression of various tumors such as breast, melanoma, and hepatoma (17
, 20
, 21)
. This axis has also been postulated to play a role in the pathogenesis of RMS (12
, 14
, 15) and as shown recently, a synergism between aberrant HGF-c-MET signaling and INK4a/ARF inactivation leads to the induction of RMS with extremely high penetrance and short latency (1)
.
To elucidate the biological effects of HGF in RMS, we selected RMS cell lines that respond to stimulation by HGF by phosphorylation of MAPK p42/44, AKT, or STAT-6. However, despite the fact that these signaling pathways have been shown to be involved in regulating cell proliferation, we did not find that HGF had any effect on the proliferation of RMS cell lines. However, we went on to examine events related to cell metastatic/invasive behavior, such as induction of cell polarity, migration, extracellular matrix degradation, and adhesion. We found that HGF induces motility of RMS cells, their polarity (appearance of the leading edge), and cytoskeletal rearrangements (formation of stress fibers) and as well enhances the production of MMP-2 and MMP-9. All of these processes together may contribute to the egress of RMS cells from the primary tumor and metastasis. Corroborating this, we showed that the majority of RMS cell lines tested express MMP-2, MMP-9, and MT1-MMP, and that HGF stimulated MMP-2 and MMP-9 secretion in some of them. Such up-regulation of MMP-2 and MMP-9, as well as of chemoinvasion, further supports the role of the HGF-c-MET axis in the metastatic behavior of RMS. This HGF-induced migration across the basement membrane and chemotaxis (toward the bone marrow stroma) could direct RMS cells circulating in the peripheral blood into the HGF-rich environment of the bone marrow or lymph nodes. This metastatic response could be further potentiated by SDF-1 secreted in the bone marrow stroma.
Generally, the aberrant HGF-c-MET axis could be the result of: (a) amplification/overexpression of c-MET; (b) activating mutations in the c-MET receptor tyrosine kinase domain; and (c) creation of an autocrine loop by endogenously secreted HGF (1 , 17 , 20 , 21) . In this study we have investigated all three possibilities. Because we did not detect any activating mutations in the tyrosine kinase region of the c-MET receptor in the RMS cell lines studied or the presence of functional autocrine regulatory loops, the only explanation for aberrant HGF signaling in RMS cells must be higher expression of c-MET, modulated by the PAX3-FKHR fusion gene. In fact, the c-MET promoter contains several PAX binding sites, and it had been reported that PAX3 modulates expression of the c-MET receptor during limb development (14) . Moreover, micoarray analysis identified c-MET as a PAX3 downstream target gene (46) .
Supporting this is our observation that, although c-MET was expressed by both ARMS and ERMS cells, its expression was much higher (>80%) on the former. These data support the evidence that c-MET expression may be regulated both by wild-type PAX3 and PAX3-FKHR fusion proteins (4 , 6 , 12) . Moreover, because we recently reported that ARMS cells also expressed more CXCR4 and had a stronger response to SDF-1 (11) , the PAX3-FKHR fusion gene could up-regulate CXCR4 on the surface of ARMS cells as well. Hence our combined data suggest that the PAX3-FKHR fusion gene induces morphological change and invasiveness of human RMS cell lines by up-regulating both c-MET and CXCR4. This, however, requires further confirmation by direct functional analysis of transcriptional regulation of CXCR4 and c-MET promoters, and such studies are currently being performed in our laboratories. As mentioned above, both promoters contain several PAX3 putative binding sites.
Although both ERMS and ARMS cell lines responded to stimulation by HGF, we observed differences in this response. Accordingly, ARMS cells showed stronger directional chemotaxis, adhesion to HUVECs, and Matrigel chemoinvasion. Furthermore, our in vivo experiments support the notion that ARMS cells have a greater "homing/seeding efficiency" to bone marrow in a lethally irradiated/immunocompromised mice model than ERMS cells (Fig. 10B)
. This bone marrow "homing potential" of ARMS cell lines correlates with the expression of the PAX3-FKHR (RH30) and PAX7-FKHR (CW9019) genes as well as the higher expression of c-MET (Table 1)
and CXCR4 (11)
on their surfaces.
Generally, the effect of HGF on the metastatic behavior of RMS cells was similar to those we reported for SDF-1 (11) . Both factors strongly promoted motility and directional chemotaxis of RMS cells in a PI-3K-AKT-dependent manner and increased their adhesion and secretion of MMPs. The most striking and important difference from a therapeutic point of view, however, was that HGF, in contrast to SDF-1, increased the survival of RMS cells after culture in serum-free conditions or after exposure to gamma-irradiation or chemotherapeutic agents such as vincristine and etoposide. Hence we postulate that, although both HGF and SDF-1 primarily direct/attract RMS cells to bone marrow and lymph nodes, HGF alone, or in combination with other factors, stimulates the survival of metastasizing cells and makes them more resistant to radio- and chemotherapy. Thus, targeting the c-MET-HGF axis may be of therapeutic importance both for controlling the metastatic behavior of RMS cells and improving the clinical outcome of radio- and chemotherapy.
On the basis of our observations, we conclude that it is likely that the HGF-c-MET axis plays an important role in RMS dissemination and metastasis to bone marrow, particularly of ARMS. Hence molecular strategies aimed at inhibiting this axis, together with the SDF-1-CXCR4 axis, e.g., the use of small-molecule inhibitors (38 , 47, 48, 49, 50, 51, 52) , could lead to the development of new antimetastatic therapies that would complement conventional radio- or chemotherapy in preventing the dissemination of RMS cells into bone marrow and lymph nodes.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
K. J. and M. K. contributed equally to this work.
Requests for reprints: Mariusz Z. Ratajczak, Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, 529 South Jackson Street, Louisville, KY 40202. Phone: (502) 852-1788; Fax: (502) 852-3032; E-mail: mzrata01{at}louisville.edu
5 The abbreviations used are: RMS, rhabdomyosarcoma; ARMS, alveolar rhabdomyosarcoma; ERMS, embryonal rhabdomyosarcoma; HGF, hepatocyte growth factor; SDF-1, stromal-derived factor 1; MMP, matrix metalloproteinase; MoAb, monoclonal antibody; CXCR4, CXC chemokine receptor 4; PECAM-1, platelet/endothelial cell adhesion molecule 1; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule; HUVEC, human umbilical vein endothelial cell; MAPK, mitogen-activated protein kinase; STAT, signal transducers and activators of transcription; RT-PCR, reverse transcription-PCR; PI-3K, phosphatidylinositol 3-kinase; MT1-MMP, membrane-type 1 MMP; TIMP-2, tissue inhibitor of metalloproteinases-2; CM, conditioned media. 
Received 4/ 7/03. Revised 8/ 4/03. Accepted 9/ 9/03.
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, chemotaxis to control medium (no HGF in lower chamber); 
, chemotaxis to HGF (10 ng/ml). Data from four separate experiments are pooled together. *, P < 0.0001.
) or 300 ng/ml SDF-1 (
). Data from three separate experiments are pooled together. Data from three separate experiments are pooled together, and means are shown; bars, SD. *, P < 0.0001.