Fibroblast growth factor signals as potential molecular targets in synovial sarcoma.

1. Fibroblast growth factor signals as potential molecular targets in synovial sarcoma. Tatsuya Ishibe, Tomitaka Nakayama, Takeshi Okamoto, Tomoki Aoyama, Koichi Nishijo, Satoshi Nagayama, Takashi Nakamura, and Junya Toguchida Department of Orthopaedic Surgery and Surgery, Institute for Frontier Medical Sciences, Kyoto University, Japan

2. Backgrounds Gene expression profiling of 47 soft tissue sarcomas by cDNA microarray (23,040 genes) Identified 26 genes as up-regulated genes commonly in synovial sarcomas, including Fibroblast Growth Factor 18 gene Fibroblast growth factor 18 gene Other soft tissue sarcomas synovial sarcoma (11/13) Nagayama et al. Cancer Res. 2002

3. Fibroblast Growth Factor (FGF) FGF polypeptide growth factors, a large family with 22 members which share 120 amino acids. FGF receptor (FGFR) FGF binds one of five subtypes of FGFR, inducing the tyrosine kinase activity, and transmit the signal by sequential phosphorylation of down-stream kinases. Function cell growth, angiogenesis, differentiation, etc. Association with tumor FGF3, 4, 5: originally reported by transforming activity FGF8: prostate and breast cancer FGF FGFR Auto- phosphorylation Signal transduction

4. Objectives To investigate the role of FGF signal in synovial sarcoma (SS), and to evaluate the therapeutic effect of FGFR inhibitors. Methods 1) FGF&FGFR gene expression semi-quantitative RT-PCR 2) Mitogenic effect of rhFGF proteins BrdU incorporation assay 3) FGF signal transduction phosphorylation specific Western blotting 4) Growth inhibition by FGFR inhibitors BrdU incorporation assay 5) Cell cycle analysis FACS 6) in vivo study nude mouse xenograft

5. Expression of FGF genes in cell lines (semi-quantitative RT-PCR) SS COLO205 HT1080 HS-SY-II SW480 NMS-2 YaFuSS 1273/99 SYO-1 Saos2 Fuji FGF 1 FGF 2 FGF 3 FGF 4 muscle FGF 5 FGF 6 FGF 7 FGF 8 FGF 9 FGF 10 FGF 11 FGF 12 FGF 13 FGF 14 FGF 16 FGF 2, 8, 9, 11 and 18 genes were expressed in all five SS cell lines. FGF 17 FGF 18 FGF 19 FGF 20 FGF 21 FGF 22 FGF 23 b actin

6. Expression of FGFR genes in cell lines SS COLO205 HS-SY-II 1273/99 HT1080 YaFuSS NMS-2 SaOS2 SW480 SYO-1 hMSC Fuji FGFR1 FGFR2b FGFR2c FGFR3 FGFR4 β actin All SS cell lines expressed all subtypes of FGFR genes except FGFR2b gene, which is an epithelia-specific FGFR.

7. Expression of FGF and FGFR genes in primary tumors Synovial sarcoma Other soft tissue tumors Biphasic SYT-SSX1(+) Monophasic SYT-SSX1(+) Monophasic SYT-SSX2(+) PLS LMS MFH MPNST FGF 18 FGF 8 FGF 2 FGF 9 FGF 11 FGFR 1 FGFR 2b FGFR 2c FGFR 3 FGFR 4 β actin

8. Fuji Fuji YaFuSS YaFuSS HS-SY-II HS-SY-II SYO-1 SYO-1 1273/99 1273/99 Mitogenic effect of rhFGF 8 &18 in SS cells BrdU incorporation assay rhFGF8 400 0 100ng/ml 300 200 %BrdU uptake 100 0 rhFGF18 400 300 Mitogenic effect of rhFGF8 in all SS cells, and rhFGF18 in one was confirmed. 200 %BrdU uptake 100 0

9. Signal transduction through MAP kinases by rhFGF 8 &18 YaFuSS HS-SY-II SYO-1 1273/99 ERK1/2 (ng/ml) - 10 100 FGF8 Phospho-ERK1/2 ERK1 FGF18 Phospho-ERK1/2 ERK1 p38MAPK FGF8 Phospho-p38MAPK p38MAPK FGF18 Phospho-p38MAPK p38MAPK Signals from FGFR are transduced through both ERK1/2 and p38MAPK

10. FGFR specific inhibitors SU5402 PD166866 FGFs (FGF8,18) FGFR Ras IC50 10-20μM IC50 50nM Both compounds inhibit FGF receptor tyrosine kinase. MAPK Mohammadi, et al. Science (276) 1997 Cell growth Panek, et al. JPET, (286) 1998

11. Growth of SS cells were inhibited by FGFR inhibitor SU5402 0 40mM 100 80 60 40 20 0 Fuji SYO-1 HT1080 Saos2 YaFuSS NMS-2 HS-SY-II COLO205 1273/99 SW480 synovial sarcoma

12. Similar results were obtained in low serum condition 100 80 60 40 20 0 Fuji Saos2 SYO-1 NMS-2 HT1080 1273/99 YaFuSS HS-SY-II SW480 COLO205 SS Growth inhibitory effect of FGFR inhibitor was through the inhibition of auto- or paracrine growth signals in SS.

13. Effect of FGFR inhibitor in the phosphorylation of ERK1/2 and p38MAPK SS cells YaFuSS HS-SY-II SYO-1 1273/99 Fuji SU5402 p-ERK1/2 ERK1 p-p38 p38 other cells NMS-2 HT1080 COLO205 Activation of ERK1/2, not of p38, is important in the growth of SS, which largely depends on the signal from FGFR. p-ERK1/2 ERK1 p-p38 p38

14. Cell cycle profile before & after the treatment of FGFR inhibitor Vehicle (48hrs) SU5402 (20uM, 48hrs) HS-SY-II * subG1 : 3.2±0.6 G1 : 61.4±6.0 S : 21.8±4.8 G2/M : 9.6±2.4 subG1 : 7.3±2.2 G1 : 71.4±1.8 S : 8.1±1.8 G2/M : 9.9±0.8 * * * FGFR inhibitor increased G1 and reduced S phase, inducing G1 arrest in SS cells. intensity intensity SYO-1 * * subG1 : 9.7±1.9 G1 : 74.6±4.1 S : 9.7±1.6 G2/M : 5.0±1.9 subG1 : 1.1±0.3 G1 : 56.3±5.1 S : 22.3±2.8 G2/M : 12.8±2.1 * * * * * * intensity intensity

15. Effect of FGFR inhibitor in vivo (xenograft model, nude mice) FGFR inhibitor (PD166866) i.p. Vehicle 0.1mg/day 0.5mg/day SS xenograft (SYO-1 cell) Fibrosarcoma xenograft (HT1080) 8,000 5,000 4,500 7,000 vehicle 0.1 mg 0.5 mg 4,000 6,000 3,500 pERK1/2 5,000 3,000 ERK1 Tumor volume (mm3) 4,000 2,500 2,000 3,000 * 1,500 * 2,000 * * 1,000 * * * 1,000 * 500 * * * * * Day * 0 0 1 3 5 8 10 12 15 17 19 21 1 3 5 8 10 12 15 17 19 21 Growth inhibitory effect of FGFR inhibitor is not through inhibition of angiogenesis, and may relate to the growth mechanism specific in SS.

16. FGFR inhibitor (SU5402 or PD166866) Cell cycle progression Transcriptional activation Cell cycle arrest Proposed mechanism in the growth of synovial sarcoma cells FGFR FGF8,18 Ras FGF8,18 P MEK1/2 P Cell cycle progression ERK1/2 Transcriptional activation Cell growth

17. Conclusion FGF/FGFR/ERK signal has important roles in the growth of synovial sarcoma, and is a good candidate for molecular target therapy. FGFR inhibitors were not cytocidal drugs, but induced cell cycle arrest in G1 phase, suggesting the promising role for combination therapy for synovial sarcoma.