Cancer Therapy: Preclinical Disruption of Fibroblast Growth Factor Signal Pathway Inhibits the Growth of Synovial Sarcomas: Potential Application of Signal Inhibitors to MolecularTarget Therapy Ta t s u y a I s hi b e , 1, 2 Tomitaka Nakayama,2 Ta k e s h i O k a m o t o, 1, 2 Tomoki Aoyama,1Koichi Nishijo,1, 2 Kotaro Roberts Shibata,1, 2 Ya s u ko Shim a ,1, 2 Satoshi Nagayama,3 Toyomasa Katagiri,4 Yusuke Nakamura, 4 Takashi Nakamura,2 andJunya Toguchida 1 Abstract Purpose: Synovial sarcoma is a soft tissue sarcoma, the growth regulatory mechanisms of which are unknown.We investigatedthe involvement of fibroblast growth factor (FGF) signals in synovial sarcoma andevaluatedthe therapeutic effect of inhibiting the FGF signal. Experimental Design:The expression of 22 FGF and4 FGF receptor (FGFR) genes in18prima- ry tumors andfive cell lines of synovial sarcoma were analyzedby reverse transcription-PCR. Effects of recombinant FGF2, FGF8, andFGF18 for the activation of mitogen-activatedprotein kinase (MAPK) andthe 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 expressedmultiple FGF genes especially those expressed in neural tissues, among which FGF8 showedgrowth stimulatory effects in all synovial sarcoma cell lines. FGF signals in synovial sarcoma induced the phosphorylation of extracellular signal ^ regulatedkinase (ERK1/2) andp38MAPK but not c-Jun NH 2-terminal kinase. Disruption of the FGF signaling pathway in synovial sarcoma by specific inhibitors of FGFR causedcell cycle ar- rest leading to significant growth inhibition both in vitro and in vivo.Growthinhibitionbythe FGFR inhibitor was associatedwith a down-regulation of phosphorylatedERK1/2 but not p38MAPK, andan ERK kinase inhibitor also showedgrowth inhibitory effects for synovial sar- coma, 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, andinhibi- tory molecules will be of potential use for molecular target therapy in synovial sarcoma. Synovial sarcoma is the most frequent soft-tissue sarcoma chemotherapy is still a matter of debate, and the development (STS) among patients in the third to fourth decade of life (1) of a new therapeutic approach is required to improve the and accounts for about 7% to 10% of all human STSs (2). It prognosis. predominantly affects the lower extremities but can occur in Despite little progress in clinical treatment during the last any part of the body. Surgical resection with an adequate 20 years, cytogenetic and molecular genetic analyses have greatly surgical margin is the definitive choice of treatment for improved the understanding of this type of tumor, especially primary tumors and has been shown to control local with the discovery of the reciprocal translocation recurrence (3, 4). Disseminated distant metastasis is the major t(18;X)(q11;p11) creating an SYT-SSX fusion gene as a synovial cause of poor outcome, and several reports describing the sarcoma–specific genetic alteration (5, 6). Thus far, three SSX results of current therapy showed a 5-year survival rate of genes (SSX1, SSX2, and SSX4) have been identified as a partner around 50% to 60% (3, 4). The efficacy of adjuvant 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 Authors’ Affiliations: 1Institute for Frontier Medical Sciences, Departments of high sensitivity and specificity of the SYT-SSX fusion gene in 2 3 Orthopaedic Surgery, Surgery Surgical Basic Science, Graduate School of Medi- synovial sarcoma have proven to be useful for molecular cine, Kyoto University, Kyoto, Japan; and 4Institute of Medical Science, University o f To k y o , To k y o , Ja p a n diagnosis (8). In particular types of tumors with a specific Received10/7/04; revised1/4/05; accepted1/13/05. reciprocal translocation such as PML-RARa in acute promyelo- Grant support: Ministry of Education, Culture, Sports, Science, and Technology cytic leukemia (9) and BCR-ABL in chronic myelogenous scientific research grant 11177101 (J.Toguchida). leukemia (10), fusion gene products themselves serve as targets 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 for the therapy. Although immunotherapy using a peptide with 18 U.S.C. Section 1734 solely to indicate this fact. derived from SYT-SSX protein as a specific vaccine has been Requests for reprints: Junya Toguchida, Department of Tissue Regeneration, investigated (11), no therapeutic approach has been discovered Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. Phone: 81-75-751-4134; Fax: 81-75- for directly targeting the fusion protein or its function. 751-4646; E-mail: [email protected]. Gene expression profiling of tumors has been shown a F 2005 American Association for Cancer Research. powerful tool with which to isolate a molecular target for Clin Cancer Res 2005;11(7) April 1, 2005 2702 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Fibroblast Growth Factors in Synovial Sarcoma therapy (12). We have done a gene expression analysis of Reverse transcription-PCR. Total RNA was extracted using TRIzol synovial sarcoma using a genome-wide cDNA microarray and reagent (Invitrogen) following the manufacturer’s instructions. After found that synovial sarcoma shared its molecular signature with treatment with DNase I (Nippon Gene, Osaka, Japan), 1 Ag of total malignant peripheral nerve sheath tumor, of which the RNA was reverse transcribed for single-stranded cDNAs using oligo(dT) primer and Superscript II reverse transcriptase (Invitrogen). PCR was precursors were neural crest–derived cells, and also identified done using 1 AL of RT product in a final volume of 25 AL containing a set of genes commonly up-regulated in synovial sarcoma 20 pmol each of the sense and antisense primers, 2.5 mmol/L MgCl2, including the fibroblast growth factor 18 (FGF18) gene (13). The 0.2 mmol/L of each deoxynucleotide triphosphate, and 1 unit of rTaq FGF signaling pathway seems to play significant roles in tumor polymerase (TOYOBO, Osaka, Japan). All PCRs were done using development and progression (14–16), and recently FGF18 was GeneAmp 9700 (PE Applied Biosystems, Foster City, CA). Information identified as an autocrine growth factor involved in colon of the primers are available upon request. cancers (17). Thus far, 22 genes have been identified as members Quantitative reverse transcription-PCR (RT-PCR) analyses were done of the FGF family, and our cDNA microarray contained 10 FGF in selected FGF and FGFR genes with ABI PRISM 7700 Sequence genes (FGF2, FGF3, FGF4, FGF7, FGF9, FGF11, FGF12, FGF13, Detection System (PE Applied Biosystems). One microliter of RT FGF18, and FGF19). We found that some FGF genes other product in a final volume of 25 AL containing 12.5 ALof2Â SYBR than FGF18 were also expressed in synovial sarcoma (data Green Mastermix (PE Applied Biosystems). Information for primers are available upon requests. The mean of triplicated data was used to not shown). Based on these results, we have done an calculate the ratio of target gene/b-actin expression. Statistical analysis intensive analysis of FGF and its receptor (FGFR) genes in was done by t test after logarithmic transformation. synovial sarcoma and also investigated whether inhibition of Western blot analyses. To detect FGF18 protein in the supernatant, the FGF signal is a new therapeutic modality for synovial cells were cultured up to 80% confluency in 100-mm dish. After sarcoma. 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 Materials and Methods acetone, and kept at À80jC for 1 hour. The mixture was centrifuged at 10,000 Â g for 15 minutes, and the precipitate was suspended by lysis Tissue samples and cell lines. Tumor tissues were obtained at either buffer (100 AL) containing aprotinin (1 Ag/mL), leupeptin (1 Ag/mL), biopsy or resection surgery and kept at À80jC. Informed consent was pepstatin A (1 Ag/mL), and phenylmethylsulfonyl fluoride (1 mmol/L) obtained from each patient, and tumor samples were approved for followed by sonication and centrifugation. Twenty microliters of the analysis by the Ethics Committee of the Faculty of Medicine, Kyoto supernatant were electrophoresed on a 10% SDS-polyacrylamide gel University. Five human synovial sarcoma cell lines (YaFuSS, HS-SY-II, and transferred onto a polyvinylidene difluoride membrane (Millipore, SYO-1, Fuji, and 1273/99) were used in this study. YaFuSS and HS-SY-II Bedford, MA). After blocking with 3% skim milk, membranes were cells have the SYT-SSX1 fusion gene and the others have the SYT-SSX2 probed with anti-FGF18 antibody at 1:1,000 dilutions for 1 hour and fusion gene (data not shown). YaFuSS was established in our laboratory with peroxidase-conjugated anti-mouse IgG 1:2,000 for 1 hour. from a monophasic synovial sarcoma in a 28-year-old male. HS-SY-II Immunoreactive bands were detected with Enhanced Chemilumines- was a gift from H. Sonobe (Kochi University, Japan; ref. 18), SYO-1 cence Plus (Amersham Biosciences, Little Chalfont Buckinghamshire, from A. Kawai (Okayama University, Japan; ref. 19), Fuji from S. United Kingdom). Tanaka (Hokkaido University, Japan; ref. 20), and 1273/99 from To detect FGFR3 protein in cell lysate, cells were harvested by the O. Larsson (Karolinska Institute, Sweden). Among control cell lines, same lysis buffer mentioned above.