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.