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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,

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fibroblast growth factor signals as potential molecular targets in synovial sarcoma
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

slide2

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

slide3

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

slide4

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

slide5

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

slide6

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.

slide7

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

slide8

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

slide9

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

slide10

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

slide11

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

slide12

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.

slide13

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

slide14

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

slide15

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.

slide16

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

slide17

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.