Affiliation of authors: M. S. Merchant, C.-W. Woo, C. L. Mackall, C. J. Thiele, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD.
Correspondence to: Melinda S. Merchant, M.D., Ph.D., 10 Center Dr., MSC 1298, Bldg. 10/13N240, Bethesda, MD 20892 (e-mail: merchanm{at}mail.nih.gov).
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ABSTRACT |
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INTRODUCTION |
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Ewings sarcoma is a small, round, blue-cell tumor of bone and soft tissue, with peak incidence during childhood and adolescence. Despite frequent responses to initial multimodality therapy, the majority of patients with metastatic Ewings sarcoma succumb to recurrent disease, which highlights the need for effective alternative therapies directed at minimal residual Ewings sarcoma. Interaction of c-kit with its ligand, SCF, has been implicated in the growth, survival, and metastatic potential of Ewings sarcoma (6,8). Cell lines and fresh isolates from the Ewings sarcoma family of tumors express cell surface c-kit (8) and SCF in both soluble and membrane-bound forms (6). Expression of both receptor and ligand suggests an autocrine or juxtacrine loop that may contribute to the unregulated growth of Ewings sarcoma cells. Blockade of c-kit signaling by monoclonal antibodies or antisense oligonucleotides inhibits cell growth and leads to apoptosis in cultures of Ewings cell lines (8).
The c-abl, platelet-derived growth factor receptor (PDGFR), and c-kit tyrosine kinases are specifically inhibited by imatinib mesylate (hereafter imatinib, formerly STI571, Gleevec; Novartis Pharmaceuticals, East Hanover, NJ), approved by the U.S. Food and Drug Administration (9,10). Both preclinical and clinical studies have shown that imatinib induces apoptosis and is associated with remission in patients with bcr-abl-positive chronic myelogenous leukemia as well as tumor shrinkage in patients with c-kit-positive gastrointestinal stromal tumors (1113). Imatinib can inhibit the ligand-dependent growth of small-cell lung cancer cells in vitro by blocking SCF-mediated c-kit phosphorylation (7). In this study, we examined the effects of imatinib on the in vitro proliferation of Ewings sarcoma cell lines, the induction of apoptosis in these lines, and the in vivo growth of Ewings sarcoma xenografts.
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MATERIALS AND METHODS |
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The following Ewings sarcoma cell lines were used: TC71, TC32, RD-ES, 5838, A4573, EWS-925, NCI-EWS-94, and NCI-EWS-95 (14). NCI-EWS-011 and NCI-EWS-021 cell lines were generated at the National Cancer Institute from tumor tissue obtained from recurrent Ewings sarcomas. Both resected tumors and the recently generated cell lines were positive for the t(11;22) EWS/FLI-1 translocation. The rhabdomyosarcoma line RD4A (15) and the neuroblastoma cell lines CHP-212 and KCNR (16) were used where indicated as negative controls. Cell lines were grown in RPMI-1640 medium supplemented with 2 mM L-glutamine and 0.1% or 10% fetal calf serum (Life Technologies, Gaithersburg, MD). Cells were plated 24 hours before treatment and cultured in medium containing SCF at 100 ng/mL (Intergen Co., Purchase, NY) or medium alone in the presence or absence of imatinib. Cell proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay (Sigma Chemical Co., St. Louis, MO) 2472 hours after treatment.
Detection of Surface Receptor Expression and Apoptosis
Adherent cells were removed from culture dishes by treatment with 0.05% trypsin/0.53 mM EDTA4Na (Life Technologies) and washed in flow cytometry buffer (phosphate-buffered saline [PBS] containing 2% bovine serum albumin and 0.1% NaN3). Approximately 15 x 105 cells were stained with phycoerythrin-conjugated anti-CD117 (c-kit), anti-CD140a (PDGFR), or anti-CD140b (PDGFR
) (BD Biosciences Pharmingen, San Diego, CA) or with Annexin V-conjugated fluorescein isothiocyanate and propidium iodide (PI) in calcium-containing buffer. One- or two-color immunofluorescence was detected with a FACSCalibur (BD Biosciences, Burlington, MA). Data from a minimum of 10 000 cells were acquired and analyzed with CellQuest software (BD Biosciences Immunocytometry Systems, Franklin Lakes, NJ). For the morphologic assessment of apoptosis by fluorescence microscopy, 10 µL of Hoechst 33342 (50 µg/mL; Sigma Chemical Co.) was added to each culture well and incubated for 10 minutes at 37 °C. PI was then added (50 µg/mL), and samples were analyzed for nuclear condensation and fragmentation by fluorescence microscopy.
Immunoprecipitation and Immunoblotting
Two million cells were cultured on 150-mm tissue culture plates for 3 days at 37 °C in an atmosphere of 5% CO2/95% air. Cells were preincubated for 30 minutes with imatinib as indicated before stimulation with SCF at 100 ng/mL (Pierce, Rockford, IL) for 7 minutes at 37 °C. After two washes with ice-cold PBS, total cell lysates were prepared in 1 mL of 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM TrisHCl [pH 8.0], 500 µM sodium orthovanadate [pH 9.0], 140 mM NaCl, 10% glycerol, aprotinin at 10 µg/mL, leupeptin at 1 µg/mL, and 1 mM phenylmethylsulfonyl fluoride; Sigma Chemical Co.) at 4 °C for 30 minutes. Insoluble material was removed by centrifugation at 4 °C for 15 minutes at 10 000g. Protein concentrations were determined with the Bradford protein assay. The protein lysates (1 mg) were immunoprecipitated with 1 µg of monoclonal anti-Kit antibody (K45; NeoMarkers, Fremont, CA) for 2 hours at 4 °C. The immune complexes were then collected by rocking in 20 µL of 10% (vol/vol) protein A-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C for 2 hours and washed twice with lysis buffer followed by a single wash with H2O. Precipitates were resuspended in 20 µL of 2x Tris glycine/sodium dodecyl sulfate (SDS) buffer (Invitrogen, Carlsbad, CA), and proteins were separated by SDSpolyacrylamide gel electrophoresis and transferred to 0.45-µm (pore size) Protran membranes (Schleicher & Schuell, Keene, NH). Protein blots were incubated with anti-phosphotyrosine antibody (pY99; Santa Cruz Biotechnology) at a 1 : 1000 dilution for 1 hour at room temperature, washed with Tris-buffered saline containing Tween-20 (20 mM TrisHCl [pH 7.4], 150 mM NaCl, and 0.5% Tween-20), and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G (Santa Cruz Biotechnology) at a 1 : 2000 dilution for 1 hour. Blots were analyzed with an enhanced chemiluminescence (ECL) detection system (Amersham Corp., Piscataway, NJ). The blots were stripped of antibody in 62.5 mM Tris (pH 6), 2% SDS, and 100 mM 2-mercaptoethanol at 50 °C for 30 minutes and then reprobed with polyclonal anti-c-kit antibody (DAKO, Carpinteria, CA).
In Vivo Tumor Growth
Tumor cells were cultured to a confluence of 75%, harvested with trypsin/EDTA, and then washed twice with PBS. Two million Ewings sarcoma cells were injected in 100 µL of PBS into the gastrocnemius of 4- to 8-week-old female SCID/bg mice (Taconic, Germantown, NY). Each mouse had a single palpable tumor evident at 2128 days after inoculation. At a tumor volume of 100500 mm3, mice were randomly assigned to receive oral gavage with imatinib (100 mg/kg per dose or 50 mg/kg per dose) or vehicle alone (5 or 10 mice per group). Doses of imatinib were administered by gavage in 100 µL of distilled H2O at 12-hour intervals for 57 days, and dosages were obtained from previously reported studies (17) in which imatinib was administered to SCID mice. Tumor dimensions were measured every 1 or 2 days with digital calipers to obtain two diameters of the tumor sphere. The lower extremity volume at the site of the tumor was determined by the formula (D x d2/6) x , where D is the longer diameter and d is the shorter diameter. Lower extremity volumes without tumor were approximately 50 mm3. Xenograft studies were approved by the National Cancer Institutes Animal Care and Use Committee, and all animal care was in accordance with institutional guidelines.
Statistical Analysis
One-way analysis of variance was performed with the use of Prism 3.0 software (GraphPad Software, Inc., San Diego, CA). Tumor growth curves were compared with a post-test Bonferroni comparison of groups to reduce the overall chance of a type I error (18). Data were considered statistically significant at P<.05. All statistical tests were two-sided.
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RESULTS |
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Flow cytometry was used to confirm the presence of receptor tyrosine kinases on our panel of Ewings sarcoma cell lines. Surface c-kit expression was detected on all cell lines in this panel (Fig. 1 and Table 1
). Although c-kit expression was generally homogeneous within a given cell line, considerable variability in c-kit expression was evident among cell lines. Surface expression of PDGFR
also varied among these Ewings sarcoma cell lines, with seven of 10 lines expressing PDGFR
. Like the majority of established Ewings sarcoma lines tested, the early-passage cell line EWS-011 consistently expressed c-kit and PDGFR
. Conversely, we were not able to detect cell surface expression of PDGFR
in this panel of Ewings sarcoma cell lines (Fig. 1
).
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Because of previous data showing that the c-kit-mediated SCF autocrine loop was important for cell survival and growth in Ewings sarcoma (8), we tested the effects of imatinib on the growth of Ewings sarcoma cell lines. When Ewings sarcoma cell lines were treated with imatinib from 0 to 20 µM during the linear growth phase, the proliferation of each Ewings sarcoma cell line was inhibited in a concentration-dependent manner (Fig. 2, A). The inhibition of cell growth by imatinib was similar for all 10 Ewings sarcoma cell lines tested, including early passages of EWS-021. However, not all tumor cell lines were sensitive to imatinib at these drug concentrations, as shown by imatinib-resistant CHP-212 neuroblastoma cells (Fig. 2, A
). The concentration of imatinib inhibiting cell growth by 50% (IC50) was 1012 µM for the 10 Ewings sarcoma cell lines tested (five experiments).
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Because all Ewings sarcoma cell lines examined were sensitive to imatinib and expressed cell surface c-kit, the ability of imatinib to inhibit c-kit phosphorylation was examined. Baseline c-kit phosphorylation was detected minimally or was absent in Ewings sarcoma cell lines cultured in 0.1% fetal calf serum. Treatment with SCF (100 µg/mL) substantially increased c-kit phosphorylation in Ewings sarcoma cell lines (Fig. 4), but pretreatment with imatinib blocked SCF-mediated c-kit phosphorylation in all cell lines tested (Fig. 4, A
). The imatinib inhibition of c-kit phosphorylation was concentration-dependent, with an IC50 value of 0.10.5 µM (Fig. 4, B
). These results confirmed c-kit expression and signaling activity in Ewings sarcoma.
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The in vivo activity of imatinib was tested by administering imatinib orally to mice with Ewings sarcoma xenografts. SCID/bg mice with a palpable Ewings sarcoma xenograft tumor were given imatinib at 50 mg/kg or 100 mg/kg or water, as a control, by oral gavage every 12 hours for 7 days. Tumor dimensions were measured every 1 or 2 days and are presented as lower extremity volumes. Although tumors in sham-treated mice grew exponentially, tumors in the group receiving imatinib doses of 50 mg/kg were stable or grew only minimally throughout the course of treatment (Fig. 5), and tumors of mice receiving imatinib doses of 100 mg/kg regressed. In fact, four of 10 mice receiving imatinib doses of 100 mg/kg had no palpable tumor at the termination of treatment (lower extremity volume <50 mm3). A comparison of groups at day 6 of therapy showed a lower extremity volume, including xenograft tumor, of 3744 mm3 (95% confidence interval [CI] = 3050 to 4437 mm3), 1442 mm3 (95% CI = 931 to 1758 mm3), or 346 mm3 (95% CI = 131 to 622 mm3) in mice treated with carrier alone or with imatinib at 50 mg/kg or at 100 mg/kg, respectively. The high-dose arm of this study was prematurely stopped because of high mortality. Six of 10 mice died on therapy between days 3 and 6, and the remaining mice appeared wasted and sluggish. No gross abnormalities or metastatic tumors were found on autopsy of these animals. Similar results were obtained with imatinib doses of 100 mg/kg in the tumor-free mice and in mice xenografted with EWS-95 cells (data not shown). In contrast, no wasting and only one death were observed in the TC71 xenograft mice treated with imatinib doses of 50 mg/kg, and no death was observed in control mice treated with carrier only. Of note, in human clinical trials of imatinib, minimal toxicities were detected (11), but studies in imatinib-treated mice have shown substantially different pharmacokinetics. In spite of these difficulties, these studies provide proof of principle that imatinib has antitumor activity against Ewings sarcoma in vivo.
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DISCUSSION |
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All Ewings sarcoma cell lines tested were sensitive to the cytotoxic effects of imatinib, which causes apoptosis in a concentration-dependent manner, even though some of these Ewings sarcoma tumor lines have been shown to be chemoresistant and/or Fas resistant [(19) and data not shown]. This observation is especially important because chemoresistance is a major contributor to the rate of treatment failure in recurrent Ewings sarcoma. Fas resistance in Ewings cell lines has been attributed to decreased ligand expression or initiator caspase expression (14). The fact that Fas resistance did not alter the ability of cells to respond to imatinib implies that the apoptosis initiated by the inhibition of this growth factor is not dependent on an intact Fas pathway but effectively activates downstream caspases despite proximal deficits in the extrinsic death pathway.
The results presented in this article are from cell lines and, thus, may not be representative of parent tumors; however, several lines of evidence suggest that nascent Ewings sarcoma tumors may be targeted by imatinib. First, we have studied an early-passage cell line, EWS-021 (studied at passages 3 and 10), derived from fresh tumor and found that these cells were as sensitive as more established cell lines to imatinib-induced apoptosis. In addition, imatinib inhibition of growth in our studies was not limited to in vitro assays, as shown by the in vivo sensitivity of imatinib-treated xenografts.
Although the inhibition of c-kit phosphorylation in our studies occurred at concentrations similar to the IC50 values previously reported for tyrosine kinase inhibition (9,20), the inhibition of Ewings sarcoma cell growth required a higher concentration of imatinib (IC50 = 10 µM) than that required in studies of chronic myelogenous leukemia or gastrointestinal stromal tumors (IC50 = 1 µM). At least two possibilities may explain these results. One possibility is that a pharmacologic limitation may restrict the accessibility of imatinib to c-kit in Ewings sarcoma cells by limiting uptake or inhibiting its action once inside the cell. However, as shown in Fig. 4, inhibition of SCF-mediated c-kit phosphorylation occurred at the relatively low IC50 of 0.5 µM, providing evidence that phosphorylation of c-kit could be inhibited in Ewings sarcoma cells at these low doses. Another possibility is that the action of imatinib in Ewings sarcoma may involve inhibition of other tyrosine kinases. Immunohistochemical studies found that only approximately 30% of Ewings sarcoma clinical specimens were strongly positive for c-kit, although up to 71% of Ewings sarcomas showed at least some c-kit staining (21,22). In fact, the heterogeneity of c-kit expression, the low or absent level of baseline c-kit phosphorylation, and the uniformity of imatinib sensitivity in Ewings sarcoma cell lines would support an alternative target.
Because imatinib is also a specific inhibitor of PDGFR, a particularly attractive possibility is the inhibition of signaling by PDGF-C, a growth factor with inherent transforming properties that is induced by EWS/FLI1 transfection (23,24). Currently, it is not clear whether autocrine or paracrine growth is responsible for PDGF-C-mediated transformation (24). Our results are consistent with recent reports that PDGFR is expressed on the surface of some but not all Ewings sarcoma cell lines (25). However, in these studies, we observed no evidence for cell surface PDGFR
expression, a component that is apparently necessary for PDGF-C signaling (26,27). Of note, cell lines that did not express surface PDGFR
or PDGFR
in these studies were nonetheless sensitive to imatinib-induced apoptosis. In addition, imatinib-mediated cytotoxicity occurred at higher concentrations of imatinib than concentrations reported for PDGFR inhibition (20), making it unlikely to fully account for the induction of cell death in this setting. PDGF-C may bind to another, perhaps uncharacterized, receptor with tyrosine kinase activity that is inhibited by imatinib, or a separate growth pathway that is critical for proliferation may be inhibited by imatinib. Indeed, although imatinib is commonly cited as specifically inhibiting v-abl, bcr-abl, PDGFR, and c-kit because of their exquisite sensitivity at doses less than 1 µM, other targets have been reported (28). Epidermal growth factor-dependent growth of BALB/MK cells is inhibited by imatinib at concentrations similar to those required in our Ewings sarcoma experiments (IC50 = 12.7 µM) (20), and the SCF-mediated or insulin-like growth factor I-mediated growth of small-cell lung cancer cells is inhibited by similar concentrations of imatinib (7). Whether these or other, as yet undescribed, tyrosine kinases contribute to the effect of imatinib in Ewings sarcoma is currently under study.
The fact that higher concentrations of imatinib were required to kill Ewings sarcoma cells than other tumor cells does not preclude future clinical evaluation of imatinib in these tumors. The toxicity of imatinib at this level was reported to be minimal to nonexistent for normal cells (9,29), providing evidence that the effects observed are likely to be relatively tumor-specific. Although the IC50 values determined in these studies are twofold higher than the concentrations achieved in early clinical trials (i.e., 4.6 µM) (11), a clear, maximally tolerated dose of this drug has not been determined, and trials are underway to assess efficacy and toxicity of increased doses to treat gastrointestinal stromal tumors. Finally, even if the toxicity of higher doses of imatinib proves intolerable for clinical translation of these results, the identification of the target or targets of imatinib that lead to cytotoxicity in Ewings sarcoma may allow the design of related compounds with increased specificity to induce death of Ewings sarcoma cells.
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NOTES |
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Manuscript received April 3, 2002; revised September 3, 2002; accepted September 11, 2002.
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