1. Kyoto University, Japan
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. Fibroblast growth factor 18 gene Nagayama et al. Cancer Res. 2002
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)
　 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)
YaFuSS
HS-SY-II
1273/99
NMS-2
HT1080
COLO205
SYO-1
Saos2
SW480
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
YaFuSS
HS-SY-II
1273/99
NMS-2
HT1080
COLO205
SYO-1
SaOS2
SW480
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. BrdU incorporation assay
Mitogenic effect of rhFGF 8 &18 in SS cells
BrdU incorporation assay
rhFGF8
400
100ng/ml
300
200
%BrdU uptake
100
YaFuSS
HS-SY-II
SYO-1
Fuji
1273/99
rhFGF18
400
300
Mitogenic effect of rhFGF8 in all SS cells, and rhFGF18 in one was confirmed.
200
%BrdU uptake
100
YaFuSS
HS-SY-II
SYO-1
Fuji
1273/99

9. Signal transduction through MAP kinases by rhFGF 8 &18
YaFuSS
HS-SY-II
SYO-1
1273/99
ERK1/2
(ng/ml)
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. Both compounds inhibit FGF receptor tyrosine kinase.
FGFR specific inhibitors
SU5402
PD166866
FGFs
(FGF8,18)
FGFR
Ras
IC μM
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
40mM
100 80 60 40 20
Fuji
YaFuSS
HS-SY-II
SYO-1
NMS-2
HT1080
Saos2
1273/99
COLO205
SW480
synovial sarcoma

12. Similar results were obtained in low serum condition
100 80 60 40 20
Fuji
SYO-1
NMS-2
Saos2
YaFuSS
HS-SY-II
1273/99
HT1080
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. SS xenograft (SYO-1 cell)
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)
8,000
Fibrosarcoma xenograft (HT1080)
5,000
7,000
4,500
vehicle
0.1 mg
0.5 mg
4,000
6,000
pERK1/2
3,500
5,000
ERK1
3,000
Tumor volume (mm3)
4,000
2,500
2,000
3,000 *
1,500 *
2,000
* *
1,000 *
1,000
* * *
500
* *
* * *
Day * 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. Proposed mechanism in the growth of synovial sarcoma cells
FGFR
FGF8,18
Transcriptional
activation
Cell cycle
progression
FGFR inhibitor
(SU5402 or PD166866)
Cell cycle arrest
Ras
FGF8,18 P
MEK1/2 P
ERK1/2
Transcriptional
activation
Cell cycle
progression
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.