TY - JOUR
AU - Thiele, Carol J.
AB - Abstract Background: Ewing’s sarcoma cells express c-kit, a receptor tyrosine kinase, and its ligand, stem cell factor (SCF), creating a potential autocrine loop that may promote tumor survival. We thus examined whether the specific tyrosine kinase inhibitor imatinib mesylate (hereafter imatinib; formerly STI571) could inhibit the proliferation of Ewing’s sarcoma cells in vitro and in vivo. Methods: The effect of imatinib on c-kit expression and phosphorylation in Ewing’s sarcoma cells was examined by immunoblotting. The effect of imatinib on cell growth and apoptosis was examined with an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and with a morphologic test and Annexin V staining, respectively. The effect of imatinib oral therapy (every 12 hours for 5–7 days) on primary tumor growth was assessed in Ewing’s sarcoma xenografts in SCID/bg mice (5 or 10 mice per group). Results: All Ewing’s sarcoma cell lines tested were sensitive to imatinib-mediated apoptosis with a concentration inhibiting growth by 50% (IC50) of 10–12 μM. Imatinib inhibited SCF-mediated c-kit phosphorylation (IC50 = 0.1–0.5 μM). In the xenograft model, imatinib treatment resulted in the regression or control of primary Ewing’s sarcomas. After 6 days of treatment, the mean lower extremity volume including xenograft tumor was 3744 mm3 (95% confidence interval [CI] = 3050 to 4437 mm3), 1442 mm3 (95% CI = 931 to 1758 mm3), and 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. Conclusions: Imatinib interferes with growth of all Ewing’s sarcoma cell lines tested in vitro and in vivo. Targeted inhibition of tyrosine kinase-dependent autocrine loops, therefore, may be a viable therapeutic strategy for Ewing’s sarcoma. Tyrosine kinases are important intracellular mediators of proliferation, survival, and differentiation signals and have been shown to play a role in the growth of several malignancies (1,2). In some cases, constitutive activation of a tyrosine kinase is a sentinel event in neoplastic transformation. The bcr-abl gene fusion (i.e., BCR-ABL) on the Philadelphia chromosome in patients with chronic myelogenous leukemia results in the constitutive expression of an abnormal protein tyrosine kinase (3), and a mutation in the c-kit gene (i.e., KIT; encoding the stem cell factor [SCF] receptor, a receptor tyrosine kinase) expressed in gastrointestinal stromal tumors leads to constitutive activity of this receptor tyrosine kinase (4). Even in the absence of proximal transforming events involving tyrosine kinases, tyrosine kinase signaling may contribute to the survival advantage of transformed cells. For example, overexpression of epithelial growth factor receptors have been implicated in the growth of breast and ovarian cancers (1,5), and growth loops involving the insulin-like growth I receptor have been shown to play a role in several malignancies (2). An autocrine growth loop resulting from the coexpression of the c-kit receptor tyrosine kinase and its ligand, SCF, is found in Ewing’s sarcoma and in small-cell lung cancer cells and likely contributes to the growth of these transformed cells (6,7). Ewing’s 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 Ewing’s sarcoma succumb to recurrent disease, which highlights the need for effective alternative therapies directed at minimal residual Ewing’s sarcoma. Interaction of c-kit with its ligand, SCF, has been implicated in the growth, survival, and metastatic potential of Ewing’s sarcoma (6,8). Cell lines and fresh isolates from the Ewing’s 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 Ewing’s sarcoma cells. Blockade of c-kit signaling by monoclonal antibodies or antisense oligonucleotides inhibits cell growth and leads to apoptosis in cultures of Ewing’s 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 (11–13). 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 Ewing’s sarcoma cell lines, the induction of apoptosis in these lines, and the in vivo growth of Ewing’s sarcoma xenografts. Materials and Methods Cell Lines and Cell Growth The following Ewing’s 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 Ewing’s 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 mMl-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) 24–72 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 EDTA•4Na (Life Technologies) and washed in flow cytometry buffer (phosphate-buffered saline [PBS] containing 2% bovine serum albumin and 0.1% NaN3). Approximately 1–5 × 105 cells were stained with phycoerythrin-conjugated anti-CD117 (c-kit), anti-CD140a (PDGFRα), or anti-CD140b (PDGFRβ) (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 Tris–HCl [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 2× Tris glycine/sodium dodecyl sulfate (SDS) buffer (Invitrogen, Carlsbad, CA), and proteins were separated by SDS–polyacrylamide 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 Tris–HCl [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 Ewing’s 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 21–28 days after inoculation. At a tumor volume of 100–500 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 5–7 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 × d2/6) × π, 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 Institute’s 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. Results Surface Expression of c-kit and PDGFRs by Ewing’s Sarcoma Cell Lines Flow cytometry was used to confirm the presence of receptor tyrosine kinases on our panel of Ewing’s 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 PDGFRβ also varied among these Ewing’s sarcoma cell lines, with seven of 10 lines expressing PDGFRβ. Like the majority of established Ewing’s sarcoma lines tested, the early-passage cell line EWS-011 consistently expressed c-kit and PDGFRβ. Conversely, we were not able to detect cell surface expression of PDGFRα in this panel of Ewing’s sarcoma cell lines (Fig. 1). Growth Inhibition of Ewing’s Sarcoma Cell Lines Because of previous data showing that the c-kit-mediated SCF autocrine loop was important for cell survival and growth in Ewing’s sarcoma (8), we tested the effects of imatinib on the growth of Ewing’s sarcoma cell lines. When Ewing’s sarcoma cell lines were treated with imatinib from 0 to 20 μM during the linear growth phase, the proliferation of each Ewing’s 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 Ewing’s 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 10–12 μM for the 10 Ewing’s sarcoma cell lines tested (five experiments). To determine whether growth inhibition reflected cytostasis or cell death, cultures were microscopically inspected after treatment with imatinib. We observed morphologic changes consistent with the induction of cell death, such as loss of adherence properties, rounded cells, and nuclear condensation (Fig. 2, B). Hoechst dye staining further revealed nuclear condensation (data not shown). To verify apoptosis, cells were harvested at regular intervals and assessed for apoptosis by Annexin V staining. Treatment with imatinib at concentrations greater than 7.5 μM produced many Annexin V-positive, PI-negative apoptotic cells (Fig. 3). Apoptosis was observed as early as 12 hours after treatment with imatinib at concentrations greater than its IC50 value, and the peak level of apoptosis occurred between 36 and 72 hours after imatinib treatment (Fig. 3, B). The degree of Annexin V staining at peak apoptosis was dependent on the concentration of imatinib (Fig. 3, C and D). By 4 days after treatment, all cells cultured with greater than 10 μM imatinib were nonviable (data not shown). Thus, imatinib induced apoptosis in Ewing’s sarcoma with an IC50 value of 10–12 μM. Inhibition of c-kit Phosphorylation by Imatinib in Ewing’s Sarcoma Cell Lines Because all Ewing’s 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 Ewing’s sarcoma cell lines cultured in 0.1% fetal calf serum. Treatment with SCF (100 μg/mL) substantially increased c-kit phosphorylation in Ewing’s 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 0.1–0.5 μM (Fig. 4, B). These results confirmed c-kit expression and signaling activity in Ewing’s sarcoma. Inhibition of Tumor Growth In Vivo The in vivo activity of imatinib was tested by administering imatinib orally to mice with Ewing’s sarcoma xenografts. SCID/bg mice with a palpable Ewing’s 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 Ewing’s sarcoma in vivo. Discussion Imatinib has had remarkable success in early clinical trials targeting tumors in which sentinel, transforming events involve constitutive activation of susceptible tyrosine kinases. It remains unknown whether targeting pathways that contribute to growth and/or survival advantages of neoplastic cells with imatinib will result in a clinical benefit. Our studies provide evidence that imatinib can impact tumor survival even in tumors lacking sentinel, transforming c-kit mutations. These results suggest that targeted inhibition of tyrosine kinase-dependent autocrine growth loops may be a viable therapeutic strategy for Ewing’s sarcoma. All Ewing’s 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 Ewing’s 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 Ewing’s sarcoma. Fas resistance in Ewing’s 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 Ewing’s 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 Ewing’s 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 Ewing’s 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 Ewing’s sarcoma cells at these low doses. Another possibility is that the action of imatinib in Ewing’s sarcoma may involve inhibition of other tyrosine kinases. Immunohistochemical studies found that only approximately 30% of Ewing’s sarcoma clinical specimens were strongly positive for c-kit, although up to 71% of Ewing’s 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 Ewing’s 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 PDGFRβ is expressed on the surface of some but not all Ewing’s sarcoma cell lines (25). However, in these studies, we observed no evidence for cell surface PDGFRα expression, a component that is apparently necessary for PDGF-C signaling (26,27). Of note, cell lines that did not express surface PDGFRα or PDGFRβ 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 Ewing’s 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 Ewing’s sarcoma is currently under study. The fact that higher concentrations of imatinib were required to kill Ewing’s 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 Ewing’s sarcoma may allow the design of related compounds with increased specificity to induce death of Ewing’s sarcoma cells. Table 1. Surface expression of receptor tyrosine kinases on Ewing’s sarcoma cell lines* Cell line  CD117 (c-kit)  CD140a (PDGFRα)  CD140b (PDGFRβ)  *Cells were directly stained with phycoerythrin-labeled antibodies against CD117, CD140a, and CD140b and analyzed by flow cytometry. PDGFR = platelet-derived growth factor receptor; + = positive staining above background; ++ = positive staining with a mean fluorescence intensity greater than two orders of magnitude above background staining; − = coincident with background staining.  Ewing’s sarcoma            5838  ++  −  −      A4573  +  −  +      EWS-021  +  −  +/−      EWS-011  +  −  −      EWS-94  +  −  +      EWS-925  ++  −  −      EWS-95  +  −  +      RD-ES  +  −  +      TC32  ++  −  +      TC71  +  −  +  Neuroblastoma            SY5Y  +  +  +  Rhabdomyosarcoma            RD4A  −  −  +  Cell line  CD117 (c-kit)  CD140a (PDGFRα)  CD140b (PDGFRβ)  *Cells were directly stained with phycoerythrin-labeled antibodies against CD117, CD140a, and CD140b and analyzed by flow cytometry. PDGFR = platelet-derived growth factor receptor; + = positive staining above background; ++ = positive staining with a mean fluorescence intensity greater than two orders of magnitude above background staining; − = coincident with background staining.  Ewing’s sarcoma            5838  ++  −  −      A4573  +  −  +      EWS-021  +  −  +/−      EWS-011  +  −  −      EWS-94  +  −  +      EWS-925  ++  −  −      EWS-95  +  −  +      RD-ES  +  −  +      TC32  ++  −  +      TC71  +  −  +  Neuroblastoma            SY5Y  +  +  +  Rhabdomyosarcoma            RD4A  −  −  +  View Large Fig. 1. View largeDownload slide Surface expression of CD117 (c-kit), CD140a (platelet-derived growth factor receptor a [PDGFRα]), and CD140b (PDGFRβ) on the Ewing’s sarcoma cell lines indicated to the left. Ewing’s sarcoma cells lines were cultured in RPMI-1640 containing 10% fetal calf serum, harvested, and stained. Direct immunofluorescence was studied with phycoerythrin (PE)-labeled anti-CD117, antiCD140a, or anti-CD140b (solid histograms). Background staining with a PE-conjugated isotype control is also shown (open histograms). The positive control for CD140a was the neuroblastoma cell line SY5Y (data not shown). Counts indicate number of events. Fig. 1. View largeDownload slide Surface expression of CD117 (c-kit), CD140a (platelet-derived growth factor receptor a [PDGFRα]), and CD140b (PDGFRβ) on the Ewing’s sarcoma cell lines indicated to the left. Ewing’s sarcoma cells lines were cultured in RPMI-1640 containing 10% fetal calf serum, harvested, and stained. Direct immunofluorescence was studied with phycoerythrin (PE)-labeled anti-CD117, antiCD140a, or anti-CD140b (solid histograms). Background staining with a PE-conjugated isotype control is also shown (open histograms). The positive control for CD140a was the neuroblastoma cell line SY5Y (data not shown). Counts indicate number of events. Fig. 2. View largeDownload slide Concentration-dependent growth inhibition of Ewing’s sarcoma cell lines after treatment with imatinib. A) Multiple Ewing’s sarcoma cell lines (solid lines) or a control neuroblastoma cell line (CHP-212, solid squares and dashed line) were incubated with imatinib as indicated. An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was performed 60 hours later, and the results were plotted as the percentage of control untreated cells. Data are the mean (±95% confidence intervals) of results from four wells from one of five similar experiments. Representative Ewing’s sarcoma cell lines A4573 (open squares), EWS-95 (triangles), EWS-011 (inverted triangles), TC32 (open circles), and TC71 (diamonds) are shown. B) Altered growth and morphology of Ewing’s sarcoma cell lines were evident after treatment with imatinib. Ewing’s sarcoma cell lines were incubated in 96-well plates with imatinib as indicated, and 76 hours later, photomicrographs were taken with a phase-contrast Nikon microscope (Nikon, Inc., Melville, NY). Scale bar = 50 μm. Fig. 2. View largeDownload slide Concentration-dependent growth inhibition of Ewing’s sarcoma cell lines after treatment with imatinib. A) Multiple Ewing’s sarcoma cell lines (solid lines) or a control neuroblastoma cell line (CHP-212, solid squares and dashed line) were incubated with imatinib as indicated. An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was performed 60 hours later, and the results were plotted as the percentage of control untreated cells. Data are the mean (±95% confidence intervals) of results from four wells from one of five similar experiments. Representative Ewing’s sarcoma cell lines A4573 (open squares), EWS-95 (triangles), EWS-011 (inverted triangles), TC32 (open circles), and TC71 (diamonds) are shown. B) Altered growth and morphology of Ewing’s sarcoma cell lines were evident after treatment with imatinib. Ewing’s sarcoma cell lines were incubated in 96-well plates with imatinib as indicated, and 76 hours later, photomicrographs were taken with a phase-contrast Nikon microscope (Nikon, Inc., Melville, NY). Scale bar = 50 μm. Fig. 3. View largeDownload slide Imatinib induces apoptosis in Ewing’s sarcoma cell lines. Ewing’s sarcoma cells were stained with Annexin V coupled to fluorescein isothiocyanate (FITC) and propidium iodide (PI) after 60 hours of culture with imatinib and examined by flow cytometry. A) TC32 Ewing’s sarcoma cells cultured in medium alone show a majority of double-negative live cells (lower left quadrant). B) TC32 Ewing’s sarcoma cells treated with 20 μM imatinib have increased numbers of Annexin V-positive PI-negative apoptotic cells (lower right quadrant) and increased double-positive dead cells (upper right quadrant). C and D) Histograms represent increased apoptotic Ewing’s sarcoma cells from cultures treated with imatinib. PI-negative gated cells show increased Annexin V staining after a 60-hour treatment with 20 μM imatinib (solid histograms) or 10 μM imatinib (dashed histograms) compared with medium alone (shaded histograms). Representative imatinib-sensitive Ewing’s sarcoma cell lines TC71 (C) and A4573 (D) and the imatinib-resistant neuroblastoma cell line CHP-212 (E) are shown. Counts = number of events. Fig. 3. View largeDownload slide Imatinib induces apoptosis in Ewing’s sarcoma cell lines. Ewing’s sarcoma cells were stained with Annexin V coupled to fluorescein isothiocyanate (FITC) and propidium iodide (PI) after 60 hours of culture with imatinib and examined by flow cytometry. A) TC32 Ewing’s sarcoma cells cultured in medium alone show a majority of double-negative live cells (lower left quadrant). B) TC32 Ewing’s sarcoma cells treated with 20 μM imatinib have increased numbers of Annexin V-positive PI-negative apoptotic cells (lower right quadrant) and increased double-positive dead cells (upper right quadrant). C and D) Histograms represent increased apoptotic Ewing’s sarcoma cells from cultures treated with imatinib. PI-negative gated cells show increased Annexin V staining after a 60-hour treatment with 20 μM imatinib (solid histograms) or 10 μM imatinib (dashed histograms) compared with medium alone (shaded histograms). Representative imatinib-sensitive Ewing’s sarcoma cell lines TC71 (C) and A4573 (D) and the imatinib-resistant neuroblastoma cell line CHP-212 (E) are shown. Counts = number of events. Fig. 4. View largeDownload slide Phosphorylation of c-kit is inhibited by imatinib. A) Ewing’s sarcoma cell lines A4573, EWS-011, TC71, RDES, 5838, and TC32 or the neuroblastoma (NB) cell line KCNR were incubated for 30 minutes with 10–15 μM imatinib. Lysates were prepared, c-kit was immunoprecipitated, and immunoblots of these immunoprecipitates were probed with anti-phosphotyrosine (α-pY99) antibodies to reveal status of c-kit signaling after treatment with imatinib and/or stem cell factor (SCF). The immunoblots were also probed with an anti-CD117 (α-ckit) antibody to show that comparable amounts of c-kit were loaded in each lane. B) EWS-95 cells were incubated for 30 minutes with 0–2 μM imatinib as indicated and subjected to immunoprecipitation and immunoblot analysis as in A. IP = immunoprecipitation; IB = immunoblot. Fig. 4. View largeDownload slide Phosphorylation of c-kit is inhibited by imatinib. A) Ewing’s sarcoma cell lines A4573, EWS-011, TC71, RDES, 5838, and TC32 or the neuroblastoma (NB) cell line KCNR were incubated for 30 minutes with 10–15 μM imatinib. Lysates were prepared, c-kit was immunoprecipitated, and immunoblots of these immunoprecipitates were probed with anti-phosphotyrosine (α-pY99) antibodies to reveal status of c-kit signaling after treatment with imatinib and/or stem cell factor (SCF). The immunoblots were also probed with an anti-CD117 (α-ckit) antibody to show that comparable amounts of c-kit were loaded in each lane. B) EWS-95 cells were incubated for 30 minutes with 0–2 μM imatinib as indicated and subjected to immunoprecipitation and immunoblot analysis as in A. IP = immunoprecipitation; IB = immunoblot. Fig. 5. View largeDownload slide Oral therapy with imatinib inhibits Ewing’s sarcoma tumor growth in vivo. Xenografts were established with TC71 Ewing’s sarcoma cells in female SCID/bg mice 17–21 days before imatinib treatment. Therapy was begun when tumor was palpable, with a lower extremity volume of 200–1000 mm3. Twice-daily oral gavages of imatinib at 50 mg/kg (solid triangles, n = 5 mice), imatinib at 100 mg/kg (solid circles, n = 10 mice), or vehicle alone (open squares, n = 10 mice) were given for 7 days. Mice were withdrawn from the study when major toxicity was detected or when individual tumors reached a volume greater than 5000 mm3. At necropsy, four of 10 mice treated with imatinib doses of 100 mg/kg had no evidence of gross tumor. Data are the mean ± 95% confidence intervals and represent two experiments. *, P = .038; **, P = .007, both versus vehicle by one-way analysis of variance using the Bonferroni comparison of groups. All statistical tests were two-sided. Fig. 5. View largeDownload slide Oral therapy with imatinib inhibits Ewing’s sarcoma tumor growth in vivo. Xenografts were established with TC71 Ewing’s sarcoma cells in female SCID/bg mice 17–21 days before imatinib treatment. Therapy was begun when tumor was palpable, with a lower extremity volume of 200–1000 mm3. Twice-daily oral gavages of imatinib at 50 mg/kg (solid triangles, n = 5 mice), imatinib at 100 mg/kg (solid circles, n = 10 mice), or vehicle alone (open squares, n = 10 mice) were given for 7 days. Mice were withdrawn from the study when major toxicity was detected or when individual tumors reached a volume greater than 5000 mm3. At necropsy, four of 10 mice treated with imatinib doses of 100 mg/kg had no evidence of gross tumor. Data are the mean ± 95% confidence intervals and represent two experiments. *, P = .038; **, P = .007, both versus vehicle by one-way analysis of variance using the Bonferroni comparison of groups. All statistical tests were two-sided. We thank Andrew Reff for his technical assistance and Dr. Chand Khanna from the Pediatric Oncology Branch, National Cancer Institute, for contributing the patient-derived cell line EWS-011. We also thank Drs. Pamela Cohen and Elisabeth Buchdunger from Novartis Pharmaceuticals for their helpful discussions. References 1 Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science  1989; 244: 707–12. Google Scholar 2 Rubin R, Baserga R. Insulin-like growth factor-I receptor. Its role in cell proliferation, apoptosis, and tumorigenicity. Lab Invest  1995; 73: 311–31. Google Scholar 3 Kozbor D. Expression of a translocated c-abl gene in hybrids of mouse fibroblasts and chronic myelogenous leukaemia cells. Nature  1986; 319: 331–3. Google Scholar 4 Nakahara M. A novel gain-of-function mutation of c-kit gene in gastrointestinal stromal tumors. Gastroenterology  1998; 115: 1090–5. Google Scholar 5 Hines SJ, Organ C, Kornstein MJ, Krystal GW. Coexpression of the c-kit and stem cell factor genes in breast carcinomas. Cell Growth Differ  1995; 6: 769–79. Google Scholar 6 Landuzzi L, De Giovanni C, Nicoletti G, Rossi I, Ricci C, Astolfi A, et al. The metastatic ability of Ewing’s sarcoma cells is modulated by stem cell factor and by its receptor c-kit. Am J Pathol  2000; 157: 2123–31. Google Scholar 7 Krystal GW, Honsawek S, Litz J, Buchdunger E. The selective tyrosine kinase inhibitor STI571 inhibits small cell lung cancer growth. Clin Cancer Res  2000; 6: 3319–26. Google Scholar 8 Ricotti E, Fagioli F, Garelli E, Linari C, Crescenzio N, Horenstein AL, et al. c-kit is expressed in soft tissue sarcoma of neuroectodermic origin and its ligand prevents apoptosis of neoplastic cells. Blood  1998; 91: 2397–405. Google Scholar 9 Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med  1996; 2: 561–6. Google Scholar 10 Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ, et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther  2000; 295: 139–45. Google Scholar 11 Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med  2001; 344: 1031–7. Google Scholar 12 Joensuu H, Roberts PJ, Sarlomo-Rikala M, Andersson LC, Tervahartiala P, Tuveson D, et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med  2001; 344: 1052–6. Google Scholar 13 Joensuu H. Treatment of inoperable gastrointestinal stromal tumor (GIST) with Imatinib (Glivec, Gleevec). Med Klin  2002; 97 Suppl 1: 28–30. Google Scholar 14 Kontny HU, Lehrnbecher TM, Chanock SJ, Mackall CL. Simultaneous expression of Fas and nonfunctional Fas ligand in Ewings’s sarcoma. Cancer Res  1998; 58: 5842–9. Google Scholar 15 Kalebic T, Judde JG, Velez-Yanguas M, Knutsen T, Helman LJ. Metastatic human rhabdomyosarcoma: molecular, cellular and cytogenetic analysis of a novel cellular model. Invasion Metastasis  1996; 16: 83–96. Google Scholar 16 Thiele C. Neuroblastoma. In: Masters J, Palsson B, editors. Human cell culture. Vol 1. Boston (MA): Kluwer Academic Publishers; 1999. p. 21–53. Google Scholar 17 Sjoblom T, Shimizu A, O’Brien KP, Pietras K, Dal Cin P, Buchdunger E, et al. Growth inhibition of dermatofibrosarcoma protuberans tumors by the platelet-derived growth factor receptor antagonist STI571 through induction of apoptosis. Cancer Res  2001; 61: 5778–83. Google Scholar 18 Miller RG. Simultaneous statistical inference. Springer Series in Statistics. 2nd ed. New York (NY): Springer-Verlag; 1981. p. 15–6. Google Scholar 19 Kontny HU, Hammerle K, Klein R, Shayan P, Mackall CL, Niemeyer CM. Sensitivity of Ewings’s sarcoma to TRAIL-induced apoptosis. Cell Death Differ  2001; 8: 506–14. Google Scholar 20 Buchdunger E, Zimmermann J, Mett H, Meyer T, Muller M, Druker BJ, et al. Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative. Cancer Res  1996; 56: 100–4. Google Scholar 21 Hornick JL, Fletcher CD. Immunohistochemical staining for KIT (CD117) in soft tissue sarcomas is very limited in distribution. Am J Clin Pathol  2002; 117: 188–93. Google Scholar 22 Smithey BE, Pappo AS, Hill DA. C-kit expression in pediatric solid tumors: a comparative immunohistochemical study. Am J Surg Pathol  2002; 26: 486–92. Google Scholar 23 Zwerner JP, May WA. PDGF-C is an EWS/FLI induced transforming growth factor in Ewing family tumors. Oncogene  2001; 20: 626–33. Google Scholar 24 Zwerner JP, May WA. Dominant negative PDGF-C inhibits growth of Ewing family tumor cell lines. Oncogene  2002; 21: 3847–54. Google Scholar 25 Uren A, Sun CJ, Vitolo M, Sun YF, Tsokos M, Passaniti A, et al. Expression of functional beta-PDGF receptors by Ewings sarcoma. Proc Annu Meet Am Assoc Cancer Res  2002; 43: 830. Google Scholar 26 Gilbertson DG, Duff ME, West JW, Kelly JD, Sheppard PO, Hofstrand PD, et al. Platelet-derived growth factor C (PDGF-C), a novel growth factor that binds to PDGF alpha and beta receptor. J Biol Chem  2001; 276: 27406–14. Google Scholar 27 Li X, Ponten A, Aase K, Karlsson L, Abramsson A, Uutela M, et al. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor. Nat Cell Biol  2000; 2: 302–9. Google Scholar 28 Krystal GW. Mechanisms of resistance to imatinib (STI571) and prospects for combination with conventional chemotherapeutic agents. Drug Resist Updat  2001; 4: 16–21. Google Scholar 29 Deininger MW, Goldman JM, Lydon N, Melo JV. The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells. Blood  1997; 90: 3691–8. Google Scholar © Oxford University Press
TI - Potential Use of Imatinib in Ewing’s Sarcoma: Evidence for In Vitro and In Vivo Activity
JF - JNCI: Journal of the National Cancer Institute
DO - 10.1093/jnci/94.22.1673
DA - 2002-11-20
UR - https://www.deepdyve.com/lp/oxford-university-press/potential-use-of-imatinib-in-ewing-s-sarcoma-evidence-for-in-vitro-and-Pzee0EA0CP
SP - 1673
EP - 1679
VL - 94
IS - 22
DP - DeepDyve
ER -