fibroblast growth factor signals as potential molecular targets in synovial sarcoma
DESCRIPTION
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, - PowerPoint PPT PresentationTRANSCRIPT
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
Nagayama et al. Cancer Res. 2002
Fibroblast growth factor 18 gene
synovial sarcoma (11/13)Other soft tissue sarcomas
BackgroundsBackgrounds
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
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
Fibroblast Growth Factor (FGF)
FGFR
FGF
Auto-phosphorylation
Signal transduction
ObjectivesObjectives
To investigate the role of FGF signal in synovial sarcoma (SS),
and to evaluate the therapeutic effect of FGFR inhibitors.
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
MethodsMethods
YaFu
SSH
S-SY
-IISY
O-1
NM
S-2
HT1
080
SW48
0
Fuji
1273
/99
FGF 20FGF 21FGF 22FGF 23
FGF 7FGF 8FGF 9FGF 10FGF 11
FGF 13FGF 14FGF 16FGF 17FGF 18FGF 19
FGF 12
FGF 3FGF 4FGF 5
FGF 1
FGF 6
FGF 2
actin
Saos
2
CO
LO20
5
mus
cle
Expression of FGF genes in cell lines (semi-quantitative RT-PCR)
FGF 2, 8, 9, 11 and 18
genes were expressed
in all five SS cell lines.
SS
YaFu
SSH
S-SY
-IISY
O-1
NM
S-2
HT1
080
SW48
0hM
SC
Fuji
1273
/99
SaO
S2C
OLO
205
FGFR1
FGFR2b
FGFR3
FGFR4
β actin
All SS cell lines expressed all subtypes of FGFR genes except FGFR2b gene, which is an epithelia-specific FGFR.
FGFR2c
Expression of FGFR genes in cell lines
SS
PLS LMS MFH MPNSTBiphasic
SYT-SSX1(+)MonophasicSYT-SSX2(+)
MonophasicSYT-SSX1(+)
β actin
FGF 8
FGF 18
FGFR 2b
FGFR 2c
FGF 2
FGFR 1
FGFR 3
FGFR 4
FGF 9
FGF 11
Other soft tissue tumors Synovial sarcoma
Expression of FGF and FGFR genes in primary tumors
0
100
200
300
400
0
100
200
300
400
rhFGF18
YaFuSS HS-SY-II SYO-1 Fuji 1273/99
YaFuSS HS-SY-II SYO-1 Fuji 1273/99
%B
rdU
upt
ake
%B
rdU
upt
ake
0 100ng/ml
Mitogenic effect of rhFGF 8 &18 in SS cells
Mitogenic effect of
rhFGF8 in all SS cells,
and rhFGF18 in one
was confirmed.
rhFGF8
BrdU incorporation assay
ERK1/2 HS-SY-IIYaFuSS 1273/99SYO-1
Phospho-ERK1/2
ERK1
Phospho-ERK1/2
ERK1
FGF8
FGF18
(ng/ml)
p38MAPK
Phospho-p38MAPK
p38MAPK
Phospho-p38MAPK
p38MAPK
FGF8
FGF18
Signal transduction through MAP kinases by rhFGF 8 &18
Signals from FGFR are transduced through both ERK1/2 and p38MAPK
- 10 100
FGFR specific inhibitors
Mohammadi, et al. Science (276) 1997
Panek, et al. JPET, (286) 1998
PD166866SU5402
IC50 10-20μM IC50 50nM
Both compounds inhibit FGF receptor tyrosine kinase.
FGFR
Ras
FGFs (FGF8,18)
MAPK
Cell growth
0
20
40
60
80
100
YaFuS
S
SYO-1
Fuji
1273
/99
HS-SY-II
NMS-2
HT108
0
Saos2
COLO
205
SW48
0
0 40μM
SU5402
Growth of SS cells were inhibited by FGFR inhibitor
synovial sarcoma
YaFuS
S
SYO-1
Fuji
1273
/99
HS-SY-II
NMS-2
HT108
0
Saos2
COLO
205
SW48
0
Similar results were obtained in low serum condition
0
20
40
60
80
100
SS
Growth inhibitory effect of FGFR inhibitor was through the inhibition of auto- or paracrine growth signals in SS.
HS-SY-IIYaFuSS 1273/99SYO-1 FujiSU5402
HT1080NMS-2 COLO205
p-ERK1/2
ERK1
p-p38
p38
p-ERK1/2
ERK1
p-p38
p38
Effect of FGFR inhibitor in the phosphorylation of ERK1/2 and p38MAPK
SS cells
other cells
Activation of ERK1/2,
not of p38, is important in the
growth of SS,
which largely depends on the
signal from FGFR.
Cell cycle profile before & after the treatment of FGFR inhibitor
subG1 : 3.2±0.6G1 : 61.4±6.0S : 21.8±4.8G2/M : 9.6±2.4
subG1: 7.3±2.2G1 : 71.4±1.8S : 8.1±1.8G2/M : 9.9±0.8
subG1 : 1.1±0.3G1 : 56.3±5.1S : 22.3±2.8G2/M : 12.8±2.1
subG1: 9.7±1.9G1 : 74.6±4.1S : 9.7±1.6G2/M : 5.0±1.9
*
** *
* *
* ** ** *
SU5402 (20uM, 48hrs)Vehicle (48hrs)
SYO-1
HS-SY-II
intensity intensity
intensity intensity
FGFR inhibitor
increased G1 and
reduced S phase,
inducing G1 arrest
in SS cells.
Effect of FGFR inhibitor in vivo (xenograft model, nude mice)
Vehicle 0.1mg/day 0.5mg/dayFGFR inhibitor (PD166866) i.p.
Growth inhibitory effect of FGFR inhibitor is not through inhibition of angiogenesis, and may relate to the growth mechanism specific in SS.
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
1 3 5 8 10 12 15 17 19 210
500
1,000
1,500
2,000
2,500
3,000
3,500
4,000
4,500
5,000
1 3 5 8 10 12 15 17 19 21
Day
Tu
mo
r vo
lum
e (m
m3 )
ERK1
pERK1/2
vehi
cle
0.1
mg
0.5
mg
SS xenograft (SYO-1 cell) Fibrosarcoma xenograft (HT1080)
**
* * * * * ** *
* * **
Proposed mechanism in the growth of synovial sarcoma cells
FGFR FGF8,18
Ras
ERK1/2Transcriptional
activation
Cell growth
FGF8,18
MEK1/2
Cell cycleprogression
P
P Transcriptionalactivation
Cell cycleprogression
FGFR inhibitor(SU5402 or PD166866)
Cell cycle arrest
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.