SS18-SSX IS A CELL-CONTEXT-DEPEDENT EPIGENETIC REGULATOR: IMPLICATION FOR CELL-OF-ORIGIN OF SYNOVIAL...
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SS18-SSX IS A CELL-CONTEXT-DEPEDENT EPIGENETIC REGULATOR: IMPLICATION FOR CELL-OF-ORIGIN OF SYNOVIAL SARCOMAS. Sakura Tamaki 1,2 , Makoto Fukuta 1,2,3 , Kazuo Hayakawa 1,2,3 , Tomohisa Kato Jr. 1 , and Junya Toguchida 1,2,4.

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Synovial Sarcoma (SS)

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Synovial sarcoma ss

SS18-SSX IS A CELL-CONTEXT-DEPEDENT EPIGENETIC REGULATOR: IMPLICATION FOR CELL-OF-ORIGIN OF SYNOVIAL SARCOMAS

Sakura Tamaki1,2, Makoto Fukuta1,2,3, Kazuo Hayakawa1,2,3, Tomohisa Kato Jr.1, and Junya Toguchida1,2,4

1Dept. Tissue Regeneration, Inst. Frontier Med. Sci., Kyoto Univ. 2Dept. Growth and Differentiation, CiRA, Kyoto Univ.

3Dept. Orthop. Surg., Grad. School Med. Sci, Nagoya City Univ. 4Dept. Orthop, Surg., Grad. School Med., Kyoto Univ.


Synovial sarcoma ss

Synovial Sarcoma (SS)

SSX

SS18

(SSX1, SSX2, SSX4)

Xp11

18q11

18q11-qter

Xp11-qter

SS18-SSX


Synovial sarcoma ss

Function of SS18-SSX protein

Transcriptional activator

Transcriptional repressor

BRM

PcG

p300

SS18-SSX

DD

◆No DNA binding motif

NH2

SNH

QPGY

RD

COOH

Chromatin modulator

Up- or down-regulate target genes


Synovial sarcoma ss

Search for genes up-regulated by SS18-SSX

◆FZD10 is expressed only in placenta among adult normal tissues.

(Sited from LSBM)

◆FZD10 is specifically up-regulated in SS, but not in other types

of soft tissue sarcomas(STS).

MSC

LS

SS

SS cell lines

MFH

LMS

MPNST

6

1

2

3

4

5

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

FZD10

b2MG

The FZD10 gene is a candidate as gene

up-regulated by SS18-SSX

[Nagayama et.al., Cancer Res., 2002]


Synovial sarcoma ss

Objective

Understand how SS18-SSX regulates the downstream genes using the

FZD10 gene as a representative


Synovial sarcoma ss

SS18-SSX binds core promoter region of FZD10

Luc

Luc

Luc

Luc

Luc

Luc

Luc

Luc

FZD10 (-3029)

FZD10 (-2715)

FZD10 (-1332)

FZD10 (-780)

FZD10 (-360)

FZD10 (-118)

SYO-1

FZD10 (-65)

ChIP on FZD10 core promoter

Relative Luc activity

U2OS

Relative to Input (%)

FZD10

SS18-SSX2

**

Relative expression

to SYO-1

HA

rIgG

IP:

U2OS/EV

SS18-

SSX2

SS18-

SSX2

U2OS/HA-SS18-SSX2

( ** p<0.01)

EV

EV


Synovial sarcoma ss

SS18-SSX directly regulates the FZD10 gene

SS cell lines

1273/99

SYO-1

SS18-SSX2

FZD10

( * P<0.05, ** p<0.01)


Synovial sarcoma ss

SS18-SSX failed to induce FZD10 in hDF

hDF49

SS-18SSX1

YaFu-SS

SS18-SSX2

SYO-1

EV

SS18-SSX

SS18-SSX1

SS18-SSX2

FZD10

Human dermalfibroblast

ACTB

(FZD10-negative)

→ Are hDFs not appropriate cells?→ If so, what kind of cells are appropriate?


Synovial sarcoma ss

SS shares the gene expression profile with MPNST

Create “Cluster Trees”

1204 genes

577

558

391

606

712

446

298

623

357

697

406

679

734

508

330

407

551

181

327

543

341

416

580

602

325

456

370

267

473

403

350

188

646

487

582

334

190

737

259

248

213

438

397

316

53

97

44

SS

SS+MPNST

MPNST

MFH

LMS

PLS or DLS

Expressed in >90% of 47 STS cases

Those expression ratio varied by SD>1

1,204 Genes

(Nagayama et al., Cancer Res., 2002)


Synovial sarcoma ss

SS shares the protein expression profile with CCS

(Suehara et al., Proteomics 6: 4402-9, 2006)

Clear cell sarcoma

(CCS)

Synovial sarcoma

MPNST


Synovial sarcoma ss

SS is also a neural crest-derive tumor?

?

?

Neural crest

SSprecursor

Schwann cell

Melanocyte

SS

MPNST

CCS


Synovial sarcoma ss

Application of iPS cells for sarcoma research

: investigation for the cell-of-origin of

synovial sarcoma

Makoto Fukuta1,2,3, Kazuo Hayakawa1,2,3, Sakura Tamaki1,2,

Kazuya Sekiguchi1,2, Shingo Sato1,Makoto Ikeya2,

Takanobu Otsuka3, Knut Woltjen4,Junya Toguchida1,2

SS18-SSX2

SS18-SSX2

SS18-SSX2

NCC

Skin

fibroblast

+ Dox

+ Dox

+ Dox

Bone

Fat

Muscle

NC-derived

Mesenchymalstem cell

Pluripotent stem cell (PSC)

Neural crest cell

Cartilage

NC-MSC


Synovial sarcoma ss

Property of NCC

CD271(p75)

Relative expression

PAX6/Ap2α/DAPI

Relative expression


Synovial sarcoma ss

Property of NC-MSC

Osteogenic differentiation

Chondrogenic differentiation

Alucian Blue staining

Alizarin Red staining


Synovial sarcoma ss

Induction of the FZD10 in ESC and NCC

Stuffer

SS18-SSX2

(** p<0.01)

KhES1

SS18-SSX2

FZD10

**

**

**

Relative expression

to SYO-1

DOX (ug/ml)

0

0.03

0.1

1.0

0

0.03

0.1

1.0

0.3

0.3

NCC-KhES1

SS18-SSX2

FZD10

**

**

Relative expression

to SYO-1

0

0.1

0.3

1.0

0

0.1

0.3

1.0


Synovial sarcoma ss

Induction of the FZD10 in NC-MSC

NC-MSC-KhES1

Stuffer

SS18-SSX2

SS18-SSX2

FZD10

Relative expression

to SYO-1

DOX (ug/ml)

0.1

0.3

1.0

5.0

0

0.1

0.3

1.0

5.0

0

SYO-1

Relative expression

to SYO-1

DOX (ug/ml)

0

0

1.0

1.0

0

0

0

1.0

1.0

1.0

0

1.0

SYO-1

(hr)

48

72

96

48

72

96


Synovial sarcoma ss

Change in H3K27me3 at the FZD10 locus by SS18-SSX2

ATG

-2kb

-1kb

+457

ORF

FZD10

5’UTR

1

2

3

4

H3K27me3

Stuffer

SS18-SSX2

NCC-KhES1

KhES1

NC-MSC-KhES1

FZD10;

Induced

Induced

Not induced

Relative to Input (%)

1

2

3

4

1

2

3

4

Region

1

2

3

4


Synovial sarcoma ss

Change in H3K4me3 at the FZD10 locus by SS18-SSX2

ATG

-2kb

-1kb

+457

ORF

FZD10

5’UTR

1

2

3

4

Stuffer

SS18-SSX2

H3K4me3

NCC-KhES1

KhES1

NC-MSC-KhES1

FZD10;

Induced

Induced

Not induced

Relative to Input (%)

1

2

3

4

1

2

3

4

Region

1

2

3

4


Synovial sarcoma ss

Change in H3Ac at the FZD10 locus by SS18-SSX2

ATG

-2kb

-1kb

+457

ORF

FZD10

5’UTR

1

2

3

4

H3Ac

Stuffer

SS18-SSX2

NCC-KhES1

KhES1

NC-MSC-KhES1

FZD10;

Induced

Induced

Not induced

Relative to Input (%)

1

2

3

4

1

2

3

4

Region

1

2

3

4


Synovial sarcoma ss

NCC

Importance of H3Ac for the FZD10 induction

TSA (HDAC inhibitor)

Occupancy of H3Ac at FZD10 locus

Relative expression

(to SYO-1)

Relative to Input (%)

TSA (uM);

0

0.01

0.03

0.1

0.3

1.0

Region

1

2

3

4

SS18-SSX2

FZD10

DMSO

TSA


Synovial sarcoma ss

Conclusion

SS18-SSX2

SS18-SSX2

SS18-SSX2

NC-MSC

ESC

NCC

(-)

(-)

(-)

FZD10

(+)

(+)

(-)

FZD10

H3K4me3

H3K27me

H3Ac


Synovial sarcoma ss

Toguchida’s Lab. in IFMS & CiRA, Kyoto Univ.


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