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Cancer Therapy: Preclinical

Disruption of Fibroblast Growth Factor Signal Pathway Inhibits the Growth of Synovial Sarcomas: Potential Application of Signal Inhibitors to Molecular Target Therapy

Tatsuya Ishibe, Tomitaka Nakayama, Takeshi Okamoto, Tomoki Aoyama, Koichi Nishijo, Kotaro Roberts Shibata, Yasuko Shima, Satoshi Nagayama, Toyomasa Katagiri, Yusuke Nakamura, Takashi Nakamura and Junya Toguchida
DOI: 10.1158/1078-0432.CCR-04-2057 Published April 2005

Abstract

Purpose: Synovial sarcoma is a soft tissue sarcoma, the growth regulatory mechanisms of which are unknown. We investigated the involvement of fibroblast growth factor (FGF) signals in synovial sarcoma and evaluated the therapeutic effect of inhibiting the FGF signal.

Experimental Design: The expression of 22 FGF and 4 FGF receptor (FGFR) genes in 18 primary tumors and five cell lines of synovial sarcoma were analyzed by reverse transcription-PCR. Effects of recombinant FGF2, FGF8, and FGF18 for the activation of mitogen-activated protein kinase (MAPK) and the growth of synovial sarcoma cell lines were analyzed. Growth inhibitory effects of FGFR inhibitors on synovial sarcoma cell lines were investigated in vitro and in vivo.

Results: Synovial sarcoma cell lines expressed multiple FGF genes especially those expressed in neural tissues, among which FGF8 showed growth stimulatory effects in all synovial sarcoma cell lines. FGF signals in synovial sarcoma induced the phosphorylation of extracellular signal–regulated kinase (ERK1/2) and p38MAPK but not c-Jun NH2-terminal kinase. Disruption of the FGF signaling pathway in synovial sarcoma by specific inhibitors of FGFR caused cell cycle arrest leading to significant growth inhibition both in vitro and in vivo. Growth inhibition by the FGFR inhibitor was associated with a down-regulation of phosphorylated ERK1/2 but not p38MAPK, and an ERK kinase inhibitor also showed growth inhibitory effects for synovial sarcoma, indicating that the growth stimulatory effect of FGF was transmitted through the ERK1/2.

Conclusions: FGF signals have an important role in the growth of synovial sarcoma, and inhibitory molecules will be of potential use for molecular target therapy in synovial sarcoma.

  • Synovial sarcoma
  • Fibroblast Growth Factor
  • Extracellular signal regulated kinase
  • Tyrosine Kinase Inhibitor

Synovial sarcoma is the most frequent soft-tissue sarcoma (STS) among patients in the third to fourth decade of life (1) and accounts for about 7% to 10% of all human STSs (2). It predominantly affects the lower extremities but can occur in any part of the body. Surgical resection with an adequate surgical margin is the definitive choice of treatment for primary tumors and has been shown to control local recurrence (3, 4). Disseminated distant metastasis is the major cause of poor outcome, and several reports describing the results of current therapy showed a 5-year survival rate of around 50% to 60% (3, 4). The efficacy of adjuvant chemotherapy is still a matter of debate, and the development of a new therapeutic approach is required to improve the prognosis.

Despite little progress in clinical treatment during the last 20 years, cytogenetic and molecular genetic analyses have greatly improved the understanding of this type of tumor, especially with the discovery of the reciprocal translocation t(18;X)(q11;p11) creating an SYT-SSX fusion gene as a synovial sarcoma–specific genetic alteration (5, 6). Thus far, three SSX genes (SSX1, SSX2, and SSX4) have been identified as a partner of the SYT gene, and >95% of synovial sarcoma tumors carried one of these fusion genes (7). Although the precise function and the mechanism of oncogenesis are not yet clearly shown, the high sensitivity and specificity of the SYT-SSX fusion gene in synovial sarcoma have proven to be useful for molecular diagnosis (8). In particular types of tumors with a specific reciprocal translocation such as PML-RARα in acute promyelocytic leukemia (9) and BCR-ABL in chronic myelogenous leukemia (10), fusion gene products themselves serve as targets for the therapy. Although immunotherapy using a peptide derived from SYT-SSX protein as a specific vaccine has been investigated (11), no therapeutic approach has been discovered for directly targeting the fusion protein or its function.

Gene expression profiling of tumors has been shown a powerful tool with which to isolate a molecular target for therapy (12). We have done a gene expression analysis of synovial sarcoma using a genome-wide cDNA microarray and found that synovial sarcoma shared its molecular signature with malignant peripheral nerve sheath tumor, of which the precursors were neural crest–derived cells, and also identified a set of genes commonly up-regulated in synovial sarcoma including the fibroblast growth factor 18 (FGF18) gene (13). The FGF signaling pathway seems to play significant roles in tumor development and progression (14–16), and recently FGF18 was identified as an autocrine growth factor involved in colon cancers (17). Thus far, 22 genes have been identified as members of the FGF family, and our cDNA microarray contained 10 FGF genes (FGF2, FGF3, FGF4, FGF7, FGF9, FGF11, FGF12, FGF13, FGF18, and FGF19). We found that some FGF genes other than FGF18 were also expressed in synovial sarcoma (data not shown). Based on these results, we have done an intensive analysis of FGF and its receptor (FGFR) genes in synovial sarcoma and also investigated whether inhibition of the FGF signal is a new therapeutic modality for synovial sarcoma.

Materials and Methods

Tissue samples and cell lines. Tumor tissues were obtained at either biopsy or resection surgery and kept at −80°C. Informed consent was obtained from each patient, and tumor samples were approved for analysis by the Ethics Committee of the Faculty of Medicine, Kyoto University. Five human synovial sarcoma cell lines (YaFuSS, HS-SY-II, SYO-1, Fuji, and 1273/99) were used in this study. YaFuSS and HS-SY-II cells have the SYT-SSX1 fusion gene and the others have the SYT-SSX2 fusion gene (data not shown). YaFuSS was established in our laboratory from a monophasic synovial sarcoma in a 28-year-old male. HS-SY-II was a gift from H. Sonobe (Kochi University, Japan; ref. 18), SYO-1 from A. Kawai (Okayama University, Japan; ref. 19), Fuji from S. Tanaka (Hokkaido University, Japan; ref. 20), and 1273/99 from O. Larsson (Karolinska Institute, Sweden). Among control cell lines, NMS-2 (malignant peripheral nerve sheath tumor; ref. 21) was provided by A. Ogose (Niigata University, Japan), and Saos2 (osteosarcoma), HT1080 (fibrosarcoma), COLO205 (colon cancer), and SW480 (colon cancer) cells were purchased from American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, MI) for Fuji, SW480, and COLO205 with 10% fetal bovine serum (FBS, HyClone, Road Logan, UT) or DMEM (Sigma, St. Louis, MO) for other cells with 10% FBS. OPTI-MEM I, which contains insulin and transferrin as protein supplements (Invitrogen, Carlsberg, CA), was used in serum-free cultures.

Reagents and antibodies. The FGFR-specific tyrosine kinase inhibitor SU5402 was purchased from Calbiochem (La Jolla, CA; ref. 22). PD166866 was provided by Pfizer Global Research and Development (Groton, CT; ref. 23). The MAPK/extracellular signal–regulated kinase (ERK) kinase (MEK) inhibitor U0126 was purchased from Promega (Madison, WI; ref. 24). Recombinant human FGF18 (rhFGF18) was purchased from Wako Pure Chemical Industry (Osaka, Japan), rhFGF8 from PeproTech (London, United Kingdom), and rhFGF2 from Oncogene Research Products (San Diego, CA). Anti-ERK1 (M12320), anti-phosphorylated-p38 (P39520), anti-pan-p38 (P19820), anti-phosphorylated-JNK1/SAPK1 (S37220), and anti-pan-JNK1/SAPK1 (M54920) antibodies were purchased from BD Biosciences PharMingen (San Diego, CA); anti-phosphorylated-ERK1/2 (sc-7383), anti-FGFR3 (sc-123), and peroxidase-conjugated anti-mouse IgG (sc-2005) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phosphotyrosine (PY20) from Zymed Laboratories (South San Francisco, CA); anti-FGF18 (MAB667) from R&D Systems (Minneapolis, MN); peroxidase-conjugated anti-rabbit immunoglobulin (P0399) from DakoCytomation (Glostrup, Denmark).

Reverse transcription-PCR. Total RNA was extracted using TRIzol reagent (Invitrogen) following the manufacturer's instructions. After treatment with DNase I (Nippon Gene, Osaka, Japan), 1 μg of total RNA was reverse transcribed for single-stranded cDNAs using oligo(dT) primer and Superscript II reverse transcriptase (Invitrogen). PCR was done using 1 μL of RT product in a final volume of 25 μL containing 20 pmol each of the sense and antisense primers, 2.5 mmol/L MgCl2, 0.2 mmol/L of each deoxynucleotide triphosphate, and 1 unit of rTaq polymerase (TOYOBO, Osaka, Japan). All PCRs were done using GeneAmp 9700 (PE Applied Biosystems, Foster City, CA). Information of the primers are available upon request.

Quantitative reverse transcription-PCR (RT-PCR) analyses were done in selected FGF and FGFR genes with ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). One microliter of RT product in a final volume of 25 μL containing 12.5 μL of 2× SYBR Green Mastermix (PE Applied Biosystems). Information for primers are available upon requests. The mean of triplicated data was used to calculate the ratio of target gene/β-actin expression. Statistical analysis was done by t test after logarithmic transformation.

Western blot analyses. To detect FGF18 protein in the supernatant, cells were cultured up to 80% confluency in 100-mm dish. After washing the dish, cells were further incubated in 5 mL OPTI-MEM I for 4 days. The supernatant was harvested, mixed with 8 mL ice-cold acetone, and kept at −80°C for 1 hour. The mixture was centrifuged at 10,000 × g for 15 minutes, and the precipitate was suspended by lysis buffer (100 μL) containing aprotinin (1 μg/mL), leupeptin (1 μg/mL), pepstatin A (1 μg/mL), and phenylmethylsulfonyl fluoride (1 mmol/L) followed by sonication and centrifugation. Twenty microliters of the supernatant were electrophoresed on a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking with 3% skim milk, membranes were probed with anti-FGF18 antibody at 1:1,000 dilutions for 1 hour and with peroxidase-conjugated anti-mouse IgG 1:2,000 for 1 hour. Immunoreactive bands were detected with Enhanced Chemiluminescence Plus (Amersham Biosciences, Little Chalfont Buckinghamshire, United Kingdom).

To detect FGFR3 protein in cell lysate, cells were harvested by the same lysis buffer mentioned above. The lysate (40 μg) was electrophoresed using 8% SDS-polyacrylamide gel, blotted, and blocked by 5% skim milk. The membrane was probed with anti-FGFR3 antibody at 1:500 dilutions and with peroxidase-conjugated anti-rabbit immunoglobulin at 1:2,000.

Phosphorylation analyses. To evaluate the phosphorylation status of FGFR3, serum-starved cells were treated by 20 μmol/L SU5402 for 30 minutes followed by the treatment with rhFGF18 or vehicle. The cells were harvested by 1 mL modified radioimmunoprecipitation assay buffer containing protease inhibitors and sodium orthovanadate, sonicated, and centrifuged at 15,000 × g for 10 minutes. After preclearing by 100 μL protein A agarose (Upstate, Lake Placid, NY), 1 mg of the protein was added with 4 μg anti-FGFR3 antibody and rotated for 2 hours at 4°C, and the immunocomplex was mixed with 100 μL protein A agarose and rotated for another 2 hours. The mixture was washed twice by PBS, and the precipitate was boiled for 5 minutes with 60 μL of 2× sample buffer, centrifuged, and the supernatant was used for Western blotting using anti-FGFR3 antibody (1:500) or anti-phosphotyrosine (1:500).

To evaluate the phosphorylation of MAPKs, cells (4 × 105) were seeded on 60-mm dishes and incubated overnight with medium containing 10% FBS followed by serum starvation for 24 hours. After treatment with each reagent for 15 minutes in the serum-free medium, cells were lysed in a buffer (100 μL) containing sodium orthovanadate (5 mmol/L) and protease inhibitors, sonicated, and centrifuged. Proteins (15 μg) were used for Western blotting.

Bromodeoxyuridine incorporation assay. Cells (4 × 103) were seeded on 96-well plates and incubated overnight with the medium containing 10% FBS. Then the medium was replaced with the serum-free medium, in which cells were further incubated with each reagent for 48 hours. Bromodeoxyuridine (BrdUrd, 10 μmol/L) was added to the culture medium during the last 4 hours, and the incorporated BrdUrd was detected with BrdUrd Detection Kit (Roche Molecular Biochemicals, Manheim, Germany), according to the manufacturer's instructions. Experiments were done in triplicate at least.

Cell cycle analysis. Cells (1 × 105) were incubated overnight in 60-mm dishes in DMEM with 10% FBS, followed by serum starvation for 24 hours. After treatment with SU5402 (20 μmol/L) for 30 minutes, FBS (1%) was added to the medium and cells were further incubated for 48 hours. Cells were then harvested, washed once with PBS, and fixed in 70% ethanol overnight. After an incubation in PBS containing propidium iodide (10 μg/mL) and RNaseA (10 μg/mL) for 30 minutes at 37°C, the DNA content of these cells was analyzed using FACScan and CELL Quest Software (Becton Dickinson, San Jose, CA). The data from 10,000 cells were collected and analyzed.

In vivo growth assay. All animal studies were approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University and done according to the Guideline for Animal Experiments of Kyoto University. First, the effect of PD166866 on phosphorylated ERK1/2 in vivo was investigated. SYO-1 (5 × 106) cells suspended in 100 μL of PBS were s.c. injected into the hind flank region of male BALB/c nu/nu athymic mice at 5 weeks of age (Japan SLC, Hamamatsu, Japan). PD166866 (0.1 or 0.5 mg) suspended in 50 μL of DMSO was given i.p. when the tumor volume was ∼1,500 mm3. Tumors were dissected 30 minutes after the injection, and tissue blocks (5 × 5 × 5 mm) were prepared from the central and peripheral portion of tumors. Whole cell lysates were prepared from each tissue block and used for Western blot. Growth inhibition experiments were done using SYO-1 and HT1080. Cells (5 × 106) were inoculated as described above. When the tumor volume reached about 50 mm3 (usually 11-12 days after inoculation), PD166866 was given i.p., a treatment which was repeated thereafter once a day, 6 days a week, for 2 weeks. Tumor size was measured with vernier calipers, and the volume was calculated as π/6 × length × width × height.

Proliferating cell nuclear antigen staining. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) was done using EPOS anti-PCNA/horseradish peroxidase system (DakoCytomation). On day 13 (the day of the last administration), tumor tissues were dissected, fixed in 10% formalin, and embedded in paraffin. Sections (5-μm-thick) were probed with anti-PCNA antibody for 1 hour and counterstained with hematoxylin. To quantify the PCNA-positive area, microscopic images were digitally acquired, the color blue (hematoxylin) was subtracted, and the brown area (PCNA-positive nucleus) was calculated as a digital value using Scion Image software. Five different sites were randomly acquired and the average value for the PCNA-positive area was calculated.

Statistical analysis. Significant differences between experimental values were determined using t test.

Results

Synovial sarcoma cell lines expressed multiple fibroblast growth factor and fibroblast growth factor receptor genes. The mRNA expression of 22 subtypes of FGF genes was analyzed by RT-PCR in five synovial sarcoma and five other cell lines (Fig. 1A). None of the five synovial sarcoma cell lines expressed the FGF5, FGF6, FGF12, FGF14, FGF16, FGF17, FGF22, or FGF23 gene, whereas the FGF2, FGF8, FGF9, FGF11, and FGF18 genes were expressed in all five synovial sarcoma cell lines. Expression of the remaining nine FGF genes varied among cell lines. The expression profile in NMS-2 (malignant peripheral nerve sheath tumor) was similar to that in synovial sarcoma, which may relate to the overall similarity of the gene expression profile between synovial sarcoma and malignant peripheral nerve sheath tumor (13). HT1080 (fibrosarcoma) expressed a similar set of FGF genes to malignant peripheral nerve sheath tumor but at much lower levels, and the expression profile of Saos2 (osteosarcoma) was quite different being positive only for FGF1 and FGF13. Interestingly, synovial sarcoma shared with colon carcinoma cell lines the expression of some FGF genes such as FGF3 and FGF18 genes, which were not expressed in mesenchymal cell lines. Secretion of these FGF proteins by synovial sarcoma cell lines was confirmed by the Western blotting of culture supernatant. FGF18 proteins were clearly detected in culture supernatant of synovial sarcoma cell lines, although the level of protein was not completely agreed with the level of RNA expression (Fig. 1C).

Fig. 1.
Fig. 1.

Expression of FGF and FGFR in cell lines. mRNA expression of 22 FGF genes (A) and four FGFR genes including splicing variants (B) was analyzed by RT-PCR in five synovial sarcoma cell lines as well as cell lines of malignant peripheral nerve sheath tumor (NMS-2), fibrosarcoma (HT1080), osteosarcoma (Saos2), and colon carcinoma (COLO205 and SW480). C, Western blotting for FGF18 using concentrated supernatant of synovial sarcoma cells. D, Western blotting for FGFR3 using cell lysates of synovial sarcoma cells and NMS-2. Two isoforms (125 and 135 kDa) corresponding to FGFR3b and FGFR3c were detected in synovial sarcoma cells but not in NMS-2.

In contrast with the heterogeneous expression pattern of ligands, the expression profile of receptors (FGFR) was relatively homogeneous among synovial sarcoma cell lines, being positive for all subtypes analyzed except FGFR2b, which is an epithelia-specific FGFR expressed in colon carcinomas (Fig. 1B). In addition to FGFR2b, NMS-2 was negative for FGFR3, and HT1080 was negative for FGFR2c. Expression of FGFR3 in synovial sarcoma cell lines was confirmed at the protein level by the Western blotting using cell lysates of each cell line (Fig. 1D).

Expression of fibroblast growth factor and fibroblast growth factor receptor genes in tumor tissues. The FGF2, FGF8, FGF9, FGF11, and FGF18 genes, which were commonly expressed in the five synovial sarcoma cell lines, were investigated in tumor tissues of 18 synovial sarcomas and 11 STSs of other types (Fig. 2A). In accordance with the results in the cell lines, almost all 18 synovial sarcoma tumors expressed all of these FGF genes (Fig. 2A). Among five FGF genes, the FGF2, FGF9, and FGF11 genes were also expressed in some tumors other than synovial sarcoma. In contrast, expression of FGF8 and FGF18 genes was only weakly detected in other tumors. Expression profiles of these five FGF genes were further analyzed by the quantitative RT-PCR (Fig. 2B). The level of expression of FGF8 and FGF18 genes in synovial sarcoma tumors were significantly higher than in tumor tissues of other type of tumors, whereas those of FGF2, FGF9, and FGF11 genes showed no difference.

Fig. 2.
Fig. 2.

Expression of FGF and FGFR genes in tumor tissues. A, RT-PCR analysis of the expression of five FGF genes (FGF2, FGF8, FGF9, FGF11, and FGF18) in tumor tissues. Among 18 synovial sarcoma (SS) tumors, seven were SYT-SSX1-positive biphasic, six were SYT-SSX1-positive monophasic, and five were SYT-SSX2-positive monophasic tumors. Abbreviations: PLS, pleomorphic liposarcoma; LMS, leiomyosarcoma; MFH, malignant fibrous histiocytoma; MPNST, malignant peripheral nerve sheath tumor. B, quantitative RT-PCR analyses of the expression of five FGF genes. •, tumors other than synovial sarcoma; ○, synovial sarcoma tumors. C, RT-PCR analysis of the expression of FGFR genes. D, quantitative RT-PCR analyses of the expression of FGFR2b, FGFR2c, and FGFR3 genes in tumor tissues. •, tumors other than synovial sarcoma; ○, monophasic synovial sarcoma tumors; Embedded Image, biphasic synovial sarcoma tumors. **, P < 0.01.

As for receptor genes, synovial sarcoma tumors expressed all types of FGFR genes analyzed including the FGFR2b gene (Fig. 2C), which was not detected in cell lines (Fig. 1B). In contrast with the uniform expression pattern observed in synovial sarcoma, expression of the FGFR genes in other types of tumors seemed to vary among tumors. For example, FGFR3 was expressed in one of three leiomyosarcomas and one of two malignant fibrous histiocytoma. The quantitative RT-PCR analyses showed that the expression level of FGFR2b and FGFR3 but not FGFR2c genes were significantly higher in synovial sarcoma than those in other types of STS (Fig. 2D). As expected, the expression level of FGFR2b gene was higher in biphasic than in monophasic tumors, although some monophasic tumors expressed this gene at the same level observed in biphasic tumors.

Growth stimulatory effects of fibroblast growth factors in synovial sarcoma cell lines. Expression of FGF and FGFR genes and proteins suggested the involvement of FGFs as autocrine/paracrine growth factors in synovial sarcoma. Among FGF genes expressed in synovial sarcoma both in vitro and in vivo, we focused on the FGF2, FGF8, and FGF18 genes in subsequent experiments. The FGF2 gene was selected as a gene commonly up-regulated in STS (Fig. 2A). FGF8 and FGF18 genes were selected as synovial sarcoma–specific up-regulated FGF genes (Fig. 2A).

Growth stimulatory effects of each rhFGF protein were analyzed under serum-starved conditions. Recombinant hFGF8 showed a dose-dependent growth-promoting effect in all synovial sarcoma cell lines (Fig. 3A). On the treatment with rhFGF18, only HS-SY-II showed a dose-dependent increase (Fig. 3B). A heterogeneous response to FGF was more clearly observed in the case of rhFGF2, in which the growth of HS-SY-II showed a dose-dependent increase, whereas a growth inhibitory effect was observed in the case of YaFuSS (Fig. 3C).

Fig. 3.
Fig. 3.

Growth stimulatory effect of FGFs in synovial sarcoma cell lines. Synovial sarcoma cells were treated with FGF8 (A), FGF18 (B), or FGF2 (C) at the indicated concentration, and the amount of incorporated BrdUrd in each sample was presented as a relative BrdUrd uptake value using the uptake of a control sample treated with DMSO as a standard value (100%). *, P < 0.05 and **, P < 0.01 compared with each control sample.

Activation of mitogen-activated protein kinases by fibroblast growth factor signals. One of major signal pathways located downstream of FGFR is the MAPKs, for which three subtypes are known: ERK, p38 MAPKs, and c-Jun NH2-terminal kinases (JNK; ref. 25). To investigate the events downstream of activated FGFRs, the phosphorylation of these MAPKs was investigated. In all synovial sarcoma cell lines analyzed, ERK1/2 was phosphorylated under serum-free conditions, suggesting the presence of endogenous stimulatory signals (Fig. 4A). The amount of phosphorylated ERK1/2 (pERK1/2) increased on treatment with FGF8 in a dose-dependent manner in all four cell lines analyzed, with the response of HS-SY-II being the highest (Fig. 4A). FGF18 also induced phosphorylation in all cell lines (Fig. 4A). An induction by FGF2 was also observed in HS-SY-II and 1273/99, but not remarkably in YaFuSS and SYO-1 (Fig. 4A).

Fig. 4.
Fig. 4.

Activation of ERK1/2 and p38 by FGFs in synovial sarcoma cell lines. A, Western blot analysis of total ERK1 and phosphorylated ERK1/2 (pERK1/2) in synovial sarcoma cell lines after treatment with FGF8, FGF18, or FGF2. B, Western blot analysis of total p38 and phosphorylated p38 (pp38) in synovial sarcoma cell lines after treatment with FGF8, FGF18, or FGF2.

As in the case of ERK1/2, phosphorylated p38MAPK (pp38) was observed in all synovial sarcoma cell lines under serum-free conditions (Fig. 4B). The level of pp38 increased on treatment with FGF8 in all synovial sarcoma cell lines, among which HS-SY-II showed the clearest induction (Fig. 4B). FGF18 also induced pp38 in HS-SY-II and other cell lines but at much lower levels. A dose-dependent increase in pp38 upon treatment with FGF2 was observed in SYO-1 (Fig. 4B). Phosphorylation of JNK was not observed under serum-free conditions or after the treatment with FGFs in any synovial sarcoma cell lines (data not shown). Therefore, exogenous FGF signals were transmitted through both ERK1/2 and p38MAPK in synovial sarcoma cell lines.

Fibroblast growth factor receptor–specific inhibitors reduce the growth of synovial sarcoma cell lines in accordance with inactivation of fibroblast growth factor receptor. MAPKs in synovial sarcoma are activated by intrinsic pathways, and exogenous FGFs can stimulate the growth of synovial sarcoma, suggesting that inhibition of the intrinsic FGF signal pathway may inhibit the growth of synovial sarcoma. SU5402 is known to inhibit FGFR autophosphorylation at an IC50 of 10 to 20 μmol/L (22). To confirm the effect of SU5402 on synovial sarcoma cell lines, the phosphorylation status of FGFR3 was analyzed by the immunoprecipitation and Western blotting in three synovial sarcoma cell lines (YaFuSS, SYO-1, and 1273/99; Fig. 5A). Phosphorylated FGFR3 was detected in these cell lines with serum-starved condition, and the treatment with rhFGF18 protein increased the amount of phosphorylated FGFR3 (Fig. 5A), which was compatible with the autocrine/paracrine model of FGFs in synovial sarcoma cells. Treatment with SU5402 effectively decreased the phosphorylation of FGFR3 into almost undetectable level (Fig. 5A), indicating that SU5402 can be used as an inhibitor of FGFR in synovial sarcoma cells. By the treatment with SU5402, the growth of all five synovial sarcoma cells was inhibited in a dose-dependent manner (Fig. 5B), and the IC50 value ranged from 8.5 to 17.2 μmol/L (Table 1). The growth inhibitory effect of SU5402 for cell lines other than synovial sarcoma was not remarkable. The same experiments were done under low-serum conditions (1% FBS, Fig. 5C), resulting in similar growth inhibition in all synovial sarcoma cell lines. The IC50 was almost identical in the three cell lines (YaFuSS, HS-SY-II, and SYO-1), whereas the other two cell lines (Fuji and 1273/99) required a higher concentration of SU5402 (Table 1). Growth inhibitory effects of another FGFR-specific inhibitor, PD166866 (23), was also investigated under the low-serum conditions. All five synovial sarcoma cell lines had an IC50 value (0.08-4.7 μmol/L) much lower than those of other cell lines (>10 μmol/L, Table 1). Similar to the data obtained using SU5402, IC50 values in YaFuSS, HS-SY-II, and SYO-1 were much lower than those in Fuji and 1273/99.

Fig. 5.
Fig. 5.

Antiproliferative effect of FGFR inhibitor on synovial sarcoma cell lines. A, inhibition of phosphorylation of FGFR3 by SU5402. Immunoprecipitated FGFR3 was hybridized with anti-phosphotyrosine antibody (top) and anti-FGFR3 antibody (bottom) in three synovial sarcoma cell lines. Amount of incorporated BrdUrd was analyzed after the treatment with FGFR inhibitor SU5402 in synovial sarcoma cell lines as well as other cell lines, and the relative uptake value of each sample was calculated using the value of a control as a standard (100%). Experiments were done in the culture medium with 10% FBS (B) or 1% FBS (C). *, P < 0.05 and **, P < 0.01.

View this table:
Table 1.

IC50 (μmol/L) of FGFR inhibitors in STS cell lines

Growth inhibition of fibroblast growth factor receptor inhibitor associated with the reduction of phosphorylation of extracellular signal–regulated kinase 1/2 but not of p38. To investigate which signals may contribute to the growth of synovial sarcoma, the phosphorylation of ERK1/2 and p38 before and after the treatment with SU5402 was investigated. In three synovial sarcoma cell lines (YaFuSS, HS-SY-II, and SYO-1), which were relatively sensitive to the growth inhibitory effect of SU5402, phosphorylation of ERK1/2 under low-serum conditions was completely inhibited by SU5402, whereas the amount of phosphorylated p38 showed no change (Fig. 6A). A small amount of pERK1/2 was still observed after SU5402 treatment in 1273/99, whereas no significant decrease was observed in Fuji, both of which showed no significant change in the pp38 (Fig. 6A). In cell lines other than synovial sarcoma, SU5402 showed no significant changes in the amount of pERK1/2 or pp38 (data not shown). These results suggested that the signal to induce the phosphorylation of ERK1/2 in synovial sarcoma was sent mainly through the FGF and FGFR pathway and that although the FGF signal induced the phosphorylation of both ERK1/2 and p38, the growth stimulatory effects were transmitted through ERK1/2. To further confirm that the growth inhibition of SU5402 was due to the reduction in pERK1/2, the function of MAPK/ERK kinase 1/2 in synovial sarcoma, which is a kinase of ERK1/2, was inhibited by U0216. Phosphorylation of ERK1/2 was effectively inhibited in all five cell lines, in which 1273/99 still had a small amount of pERK1/2 at the lower concentration (Fig. 6B). BrdUrd uptake was inhibited in a dose-dependent manner in all five cell lines, in which 1273/99 was relatively resistant at the lower concentration, consistent with the status of pERK1/2 (Fig. 6C). These results further indicated that the growth inhibition of SU5402 in synovial sarcoma was achieved through the inhibition of signals through ERK1/2.

Fig. 6.
Fig. 6.

Effect of FGFR inhibitor on down stream molecules and cell cycle profiles in synovial sarcoma cells. A, amount of ppERK1/2 and pp38MAPK after the treatment with SU5402 was analyzed by Western blotting in synovial sarcoma cell lines. Total ERK1 and p38 were also analyzed. B, Western blot analysis of pERK1/2 and ERK1 after treatment with the MAPK/ERK kinase inhibitor U0126 in synovial sarcoma cell lines. C, BrdUrd uptake of synovial sarcoma cell lines after treatment with U0126. The relative uptake value of each sample was calculated using the value of a control as a standard (100%). **, P < 0.01. D, effect of FGFR inhibitor on cell cycle profiles of synovial sarcoma cell line. DNA contents of YaFuSS after treatment with vehicle (a) or 20 μmol/L SU5402 (b) were analyzed by fluorescence-activated cell sorting. *, P < 0.05 and **, P < 0.01.

Growth inhibition by fibroblast growth factor receptor inhibitor in synovial sarcoma is associated with cell cycle arrest. The fraction of each cell cycle phase was analyzed before and after the treatment with SU5402 in YaFuSS cells. SU5402 significantly increased the G1 and subG1 fractions, and decreased the S fraction (Fig. 6D). These data suggested that the growth inhibition of synovial sarcoma by SU5402 was due to its induction of G1 arrest as previously reported for other tyrosine kinase inhibitors (26).

Fibroblast growth factor receptor inhibitors reduced the growth of synovial sarcoma cells in vivo. For in vivo study, we used SYO-1 because of its consistency in developing tumor, and PD166866 was used because of its lower IC50 value. To confirm its efficacy in vivo, tumor tissue was taken 30 minutes after the injection of PD166866. We found that 0.1 mg of PD166866 was enough to reduce the phosphorylated ERK1/2 in the tumor (Fig. 7A). Based on this result, the growth inhibitory effect of PD166866 (0.1 and 0.5 mg/body) was analyzed in vivo. Although PD166866 was unable to stop the growth of the tumor completely, a significant inhibition of growth was observed during the period of administration (P < 0.01), and the effect was retained even at day 21 at the higher dose (P = 0.02, Fig. 7B). No definite reduction of body weight or pathologic changes in vital organs such as liver and kidney was observed (data not shown). No growth inhibitory effect was observed when HT1080 was used instead of SYO-1 even at the higher dose (data not shown).

Fig. 7.
Fig. 7.

Effect of FGFR inhibitor on the growth of synovial sarcoma cells in vivo. A, Western blot of phosphorylated ERK1/2 obtained using tissue blocks dissected from the peripheral (P) and central (C) portion of SYO-1 tumors in vivo. B, growth curve of SYO-1 tumors in vivo. PD166866 (0.1 or 0.5 mg) was injected intraperitoneally on the days indicated by an arrow. C and D, immunohistochemistry for PCNA in SYO-1 tumors. Tumor tissues treated with vehicle (C) or 0.5 mg PD166866 (D) were dissected at day 13, and processed for the staining of PCNA. Original magnification, ×400.

Histologic examination of tumor tissues showed no significant morphologic changes or focal necrosis. The number of PCNA-positive cells, however, was clearly smaller in PD166866-treated tumors (19.3 ± 6.6%, Fig. 7D) than control tumors (53.0 ± 2.0%, Fig. 7C), indicating that PD166866 reduced the number of cells in S phase. No such difference was observed when HT1080 was used instead of SYO-1 (data not shown).

Discussion

FGFs may activate genetic programs which promote cell growth by at least one of three general mechanisms: first, as mitogens for the tumor cells themselves; second, by promoting angiogenesis to supply a growing tumor; and third, by inhibiting apoptosis and allowing tumor cells to continue to grow beyond normal constraints. We found that synovial sarcoma cells expressed a number of FGF genes, of which FGF2, FGF8, FGF9, FGF11, and FGF18 were commonly expressed. The involvement of FGF2 in malignant tumors as an autocrine growth factor and/or a paracrine angiogenic factor was first shown in brain tumors (27), and subsequently in non–small cell carcinoma (14). FGF8 was originally identified as an androgen-induced growth factor found in conditioned medium of androgen-dependent mouse mammary carcinoma SC-3 cells and reported to have a growth stimulatory effect (28). The human FGF8 gene was expressed in breast and prostate cancers (15, 16, 29) and associated with anchorage-independent proliferation and invasion (30, 31). FGF9 was originally purified from the conditioned medium of the glial cell line NMC-G1 and designated as glia-activating factor due to its mitogenic activity toward glial cells (32). FGF11 was one of four FGF homology factors, which shared structural homology with classic FGFs but failed to activate FGFRs (33). Recently, FGF11 was identified as one of the gene products enriched in neuronal precursors during the development of the nervous system (34). FGF18 is cloned by its homology to FGF8 and FGF9 (35, 36). FGF18, in association with FGF8, is expressed at various times and places during embryogenesis, especially neurogenesis (33, 37). Mitotic activity was shown in colon carcinomas, in which the expression of FGF18 was up-regulated by WNT signals (17). Overall, we found that synovial sarcoma expressed several FGF genes, particularly those expressed in neural tissues, which may further suggest the neural origin of precursor cells of synovial sarcoma as we previously proposed (13). Mitotic activity of exogeneous FGFs was not remarkable in some cell lines (Fig. 3). The serum-free medium used in this study contained insulin, which is known to potentiate the effect of FGF, and the further addition of insulin showed no increase of the BrdUrd uptake (data not shown). It is possible that the extent of autocrine stimulation may already be saturating so that further FGF stimulation does not lead to additional growth.

The expression of FGFR1, FGFR2c, FGFR3, and FGFR4, to which FGF2 (1c, 3c and 4), FGF8 (2c, 3c, 4), and FGF18 (2c, 3c, 4) preferentially bind (38, 39), was confirmed in both tumor tissues and cell lines. The expression of FGFR2b gene, which was detected in tumor tissues but not in cell lines, is intriguing. FGFR2b is a receptor for keratinocyte growth factor (FGF7), and expressed in epithelial cells (40). As expected, all biphasic synovial sarcomas expressed the FGFR2b gene, whereas the expression was low in most of the monophasic synovial sarcomas and other STSs. It is, however, intriguing that some monophasic synovial sarcomas with no apparent epithelial structures expressed the FGFR2b gene at a level comparable with that in biphasic synovial sarcoma (Fig. 2D). All five synovial sarcoma cell lines used in this study including SYO-1, which was established from a biphasic synovial sarcoma, were negative for the FGFR2b gene. These results suggested that synovial sarcoma tumor cells may have an intrinsic mechanism to express FGFR2b in vivo, which was somehow inhibited or disappeared in vitro. Alternatively, synovial sarcoma cells positive for FGFR2b may have growth disadvantages, and therefore only negative cells were established as cell lines. Recently, single nucleotide polymorphism in FGFR4 is reported to associate with poor prognosis in high-grade soft tissue sarcomas including synovial sarcomas (41) and both FGF8 and FGF18 bind to FGFR4. Although the status of polymorphism was not investigated in our samples, FGF signals through FGFR4 may contribute to the aggressiveness of synovial sarcoma.

The binding of FGF to FGFR leads to receptor dimerization and tyrosine autophosphorylation, followed by downstream activation of PLCγ, Crk, and SNT-1/FRS2 signaling pathways among which the SNT-1/FRS2 is the major pathway for mitogenic signals (25). Phosphorylated SNT-1/FRS2 directly binds to the GRB2-SOS complex, and then membrane-associated RAS recruits RAF-1, which in turn activates MAPKs (42). MAPKs are composed of three well-characterized subfamilies, ERK, p38 MAPK, and JNK (25), and we found that the growth-promoting signal of FGF was transmitted mainly through ERK1/2. Although ERK1/2 are activated by signals through other receptor tyrosine kinases (43), phosphorylation of ERK1/2 was completely inhibited by FGFR inhibitors, which indicated that the major signal to activate ERK1/2 in synovial sarcoma is FGF signaling, suggesting the important role of FGF in synovial sarcoma.

We have no clear explanation for the heterogeneous response in synovial sarcoma cell lines to FGF2, which stimulated the growth of HS-SY-II, but inhibited the growth of YaFuSS. In contrast with many studies describing the role of FGF2 as a mitogen, several reports showed that FGF2 acted as a growth inhibitor of tumors of Ewing's sarcoma family, which are supposed to be of neuroectodermal origin (44, 45). Multiple FGF signals are involved in the biological process in synovial sarcoma, and the response may depend on the cell specificity. Nevertheless, the disruption of FGF signals was found to cause growth inhibition in all synovial sarcoma cell lines, suggesting that an inhibitory molecule for FGFR will be a promising tool for molecular target therapy in synovial sarcoma.

Molecular therapy targeted to FGFR has been investigated with hematologic malignancies carrying a translocation involving the FGFR gene such as chronic myeloid leukemia with the BCR-FGFR1 gene (46) and multiple myeloma with the MMSET-FGFR3 fusion gene (47). In both instances, SU5402 was found to effectively inhibit the growth of cell lines with an IC50 equivalent to that in synovial sarcoma. For experiments in vivo, we used PD166866 instead of SU5402 because of its lower IC50 value and found that i.p. given PD166866 efficiently inhibited the phosphorylation of ERK1/2 in s.c. tumor causing an inhibition of growth without significant side effects. As in the case of other tyrosine kinase inhibitors, PD166866 did not strongly induce apoptosis or necrosis, and therefore no complete tumor remission was observed. However, cell cycle arrest caused by the treatment with PD166866 may enhance the cytotoxic effects of anticancer drugs as previously shown in some instances (48, 49), and therefore further in vivo studies using the treatment combined with suitable anticancer drugs may provide a promising approach for clinical application.

Acknowledgments

We thank Drs. H. Sonobe, A. Kawai, S. Tanaka, O. Larsson, and A. Ogose for providing cell lines and Y. Furukawa for useful comments.

Footnotes

  • Grant support: Ministry of Education, Culture, Sports, Science, and Technology scientific research grant 11177101 (J. Toguchida).

  • 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.

    • Accepted January 13, 2005.
    • Received October 7, 2004.
    • Revision received January 4, 2005.

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Clinical Cancer Research: 11 (7)
April 2005
Volume 11, Issue 7

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Disruption of Fibroblast Growth Factor Signal Pathway Inhibits the Growth of Synovial Sarcomas: Potential Application of Signal Inhibitors to Molecular Target Therapy
Tatsuya Ishibe, Tomitaka Nakayama, Takeshi Okamoto, Tomoki Aoyama, Koichi Nishijo, Kotaro Roberts Shibata, Yasuko Shima, Satoshi Nagayama, Toyomasa Katagiri, Yusuke Nakamura, Takashi Nakamura and Junya Toguchida
Clin Cancer Res April 1 2005 (11) (7) 2702-2712; DOI: 10.1158/1078-0432.CCR-04-2057
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