Download Tatsuya Ishibe

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
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
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.
FGF
Function
cell growth, angiogenesis, differentiation, etc.
FGFR
Association with tumor
FGF3, 4, 5: originally reported by transforming activity
FGF8: prostate and breast cancer
Autophosphorylation
Signal transduction
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
Expression of FGF genes in cell lines (semi-quantitative RT-PCR)
SS
FGF 1
FGF 2
FGF 3
FGF 4
FGF 5
FGF 6
FGF 7
FGF 8
FGF 9
FGF 10
FGF 11
FGF 12
FGF 13
FGF 14
FGF 16
FGF 17
FGF 18
FGF 19
FGF 20
FGF 21
FGF 22
FGF 23
b actin
FGF 2, 8, 9, 11 and 18
genes were expressed
in all five SS cell lines.
Expression of FGFR genes in cell lines
SS
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.
Expression of FGF and FGFR genes in primary tumors
Other soft tissue tumors
Synovial sarcoma
Biphasic
SYT-SSX1(+)
FGF 18
FGF 8
FGF 2
FGF 9
FGF 11
FGFR 1
FGFR 2b
FGFR 2c
FGFR 3
FGFR 4
β actin
Monophasic
SYT-SSX1(+)
Monophasic
SYT-SSX2(+)
Mitogenic effect of rhFGF 8 &18 in SS cells
BrdU incorporation assay
400
rhFGF8
0
100ng/ml
%BrdU uptake
300
200
100
0
YaFuSS
HS-SY-II
SYO-1
Fuji
1273/99
400
rhFGF18
%BrdU uptake
300
Mitogenic effect of
200
rhFGF8 in all SS cells,
100
0
and rhFGF18 in one
YaFuSS
HS-SY-II
SYO-1
Fuji
1273/99
was confirmed.
Signal transduction through MAP kinases by rhFGF 8 &18
YaFuSS
ERK1/2
FGF8
HS-SY-II
SYO-1
1273/99
-
10
100
(ng/ml)
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
FGFR specific inhibitors
SU5402
PD166866
FGFs
(FGF8,18)
FGFR
Ras
MAPK
IC50 10-20 M
IC50 50nM
Both compounds inhibit
FGF receptor tyrosine kinase.
Mohammadi, et al. Science (276) 1997
Cell growth
Panek, et al. JPET, (286) 1998
Growth of SS cells were inhibited by FGFR inhibitor
SU5402
0
40mM
100
80
60
40
20
0
synovial sarcoma
Similar results were obtained in low serum condition
100
80
60
40
20
0
SS
Growth inhibitory effect of FGFR inhibitor was through the inhibition of
auto- or paracrine growth signals in SS.
Effect of FGFR inhibitor in the phosphorylation of ERK1/2 and p38MAPK
SS cells
YaFuSS
HS-SY-II
SYO-1
HT1080
COLO205
1273/99
Fuji
SU5402
p-ERK1/2
ERK1
p-p38
p38
other cells
p-ERK1/2
ERK1
p-p38
p38
NMS-2
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
Vehicle (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
SU5402 (20uM, 48hrs)
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
intensity
intensity
reduced S phase,
SYO-1
subG1 : 1.1±0.3
G1
: 56.3±5.1
S
: 22.3±2.8
G2/M : 12.8±2.1
intensity
subG1 : 9.7±1.9
G1
: 74.6±4.1
S
: 9.7±1.6
G2/M : 5.0±1.9
intensity
**
**
**
**
inducing G1 arrest
in SS cells.
Effect of FGFR inhibitor in vivo
(xenograft model, nude mice)
FGFR inhibitor (PD166866) i.p.
Vehicle
SS xenograft (SYO-1 cell)
8,000
Tumor volume (mm3)
0.5mg/day
Fibrosarcoma xenograft (HT1080)
5,000
4,500
7,000
4,000
6,000
pERK1/2
3,500
ERK1
3,000
5,000
4,000
2,500
3,000
2,000
2,000
1,000
0
0.1mg/day
1
3
*
5
*
*** **
* **
8
10
12
15
**
*
*
1,500
1,000
500
Day
0
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.
Proposed mechanism in the growth of synovial sarcoma cells
FGFR
FGF8,18
Ras
FGFR inhibitor
(SU5402 or PD166866)
FGF8,18
P
MEK1/2
P
ERK1/2
Transcriptional
activation
Cell cycle
progression
Cell Cell
cyclegrowth
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.