Functional analysis of the EWS/NOR1 fusion protein expressed in extraskeletal myxoid

chondrosarcoma tumors

Yves Labelle

Department of medical biologyLaval University

Quebec City

t(9;22)

Chr 22EWS

Chr 9NOR1

1 2 3 4 5 6 7 8 9 1 2 43 5 6 87

1 2 3 4 5 6 7 43 5 6 87

5’

3’

NH2 COOH

EWS protein (EWing Sarcoma)

1 656NH2 COOHRNA BD

homology with the CTD of theLS of the RNA pol II complex

RGG

- nuclear RNA binding protein associated with the transcriptional machinery

- expression pattern : ubiquitous

- involved in different tumors through chromosome translocations generating fusion proteins

NOR1 protein (Neuron-derived Orphan Receptor)

1 626NH2 COOHDNA

BD

AF1 LZ AF2

- member of the NGFI-B/NURR1subfamily of orphan nuclear receptors

- immediate-early gene product induced by mitogens, involved in apoptosis

- expression pattern : central nervous system, muscle

- target genes ?

EWS/NOR1 protein

1 949NH2 COOH

AF1 LZ AF2EWS-NTD

DNABD

1) EWS/NOR1 can act as a transcriptional regulator

2) Characterization of a cofactor interacting with EWS/NOR1

3) Identification of a putative EWS/NOR1-regulated gene

1) EWS/NOR1 can act as a transcriptional regulator

NBRE : NGFI-B DNA Response Element for the NOR1/NGFI-B/NURR1 receptors

5’ AAAGGTCA 3’

Found in several NURR1 and NGFI-B target genepromoters

NOR1/NGFI-B/NURR1 bind to and constitutivelyactivate transcription from the NBRE, what aboutEWS/NOR1 ?

175

EWS/NOR1NOR1

83

kDa

75

48

NBRE 5’ AAAGGTCA 3’

mNBRE 5’ AGAGGTCA 3’

EWS/NOR1NOR1

competitor 5X 20X

NBRE5X 20X

mNBRE5X 20X

NBRE5X 20X

mNBRE

Transient transfections of COS and human chondrocyte cells with aNOR1 or EWS/NOR1 expression vector, a NBRE/luciferase reporter vector, and a beta-galactosidase normalizing vector

5

10

15

RLU

COS C20

NOR1

250

2500

5000

COS C20

EWS/NOR1

EWS/NOR1

265 aa 626 aa

EWS∆65/NOR1

EWS∆132/NOR1

EWS∆204/NOR125

50

75

% R

LU

100

EN ∆65 ∆132 ∆204

EWS/NOR1

265 aa 626 aa

EWS/NOR1∆AF2

NOR1

NOR1∆AF2

AF2 25

50

75

% R

LU

100

EN ∆AF2

25

50

75

% R

LU

100

N ∆AF2

CONCLUSION

EWS/NOR1 MAY PARTICIPATE IN THE DEVELOPMENT OF EMCTUMORS BY OVER-ACTIVATING NOR1-TARGET GENES INVOLVEDIN CELL PROLIFERATION AND/OR CELL DEATH

2) Characterization of a cofactor interacting with EWS/NOR1

OHKURA ET AL : PHYSICAL INTERACTION BETWEEN NOR1 AND THE HOMEOTIC TRANSCRIPTION FACTOR SIX3

SIX3 EXPRESSED PREDOMINANTLY IN THE MAMMALIAN DEVELOPING BRAIN AND REQUIRED FOR THE FORMATION AND PATTERNING OF THE VERTEBRATE EYE

IS SIX3 EXPRESSED IN EMC ?

RT-PCR

1) GST

2) GST/NOR1

3) GST/NOR1∆AF2

4) GST/NOR1∆LZ

5) GST/NOR1∆E5

6) GST/NOR1∆DBD

1 239

DBD LZ AF2626

1

611

443

359

288

kDa

66

45

31

S L

input

S L S L S L S L S L S L

1 2 3 4 5 6

AF1

1) GST

2) GST/EWS/NOR1

3) GST/EWS/NOR1∆LZ

4) GST/EWS/NOR1∆DBD

5) GST/EWS/NOR1∆AF1

6) GST/EWS/NOR1∆NOR1

1 239

DBD LZ AF2949

1

766

611

444

264

EWS AF1

kDa

66

45

31

S L

input

S L S L S L S L S L S L

1 2 3 4 5 6

1 332

1) SIX3

2) SIX3∆C

3) SIX3∆HD

HDSD

kDa

66

45

31

1L

input

2 3 1L

gst/nor1

2 3 1L

gst/ews/nor1

2 3

Protein-protein interactions in vivo : mammalian two-hybrid system

GAL4 DRE TATA LUCIFERASE

LUCIFERASEEXPRESSION

+

GAL4 DRE TATA LUCIFERASE

DBDACT

+

GAL4 DRE TATA LUCIFERASE +++

DBDACTX Y

1) DBD

2) DBD/NOR1

3) ACT

4) ACT/SIX3

5) ACT/SIX3∆HD

1 147

DBD LZ AF2626

1 AF1

1

332

HDSD

181

1 46

0,25

0,5

RLU

1+3 1+4

3.1X 1.5X

2+3

14.3X

3.0X

2+4 2+5

1) DBD

2) DBD/EWS/NOR1

3) ACT

4) ACT/SIX3

5) ACT/SIX3∆HD

1 147

DBD LZ AF2949

1 AF1

1

332

HDSD

181

1 46

EWS

2,5

5

RLU

1+3 1+4

2X

50X

2+3

160X

75X

2+4 2+5

Transient transfections of human chondrocyte cells with aNOR1 expression vector, a NBRE/luciferase reporter vector, and increasing amounts of a SIX3 or SIX3∆HDexpression vector

0,05

0,1

RLU

0,5

pcDNA SIX3 NOR1

NOR1SIX31:1

NOR1SIX3∆HD1:1

NOR1SIX31:2

NOR1SIX3∆HD1:2

NOR1SIX31:3

NOR1SIX3∆HD1:3

Transient transfections of human chondrocyte cells with anEWS/NOR1 (EN) expression vector, a NBRE/luciferase reporter vector, and increasing amounts of a SIX3 or SIX3∆HDexpression vector

15

30

RLU

45

pcDNA SIX3 EN ENSIX31:1

ENSIX3∆HD1:1

ENSIX31:2

ENSIX3∆HD1:2

ENSIX31:3

ENSIX3∆HD1:3

CONCLUSION

SIX3 MAY DIFFERENTIALLY REGULATE THE TRANSCRIPTIONAL ACTIVITIES OF NOR1 AND EWS/NOR1 IN EMC TUMORS, THE NET RESULT BEING TO DEREGULATE THE EXPRESSION OF SPECIFIC TARGET GENES AND PUSH THE EQUILIBRIUM TOWARD UNCONTROLLED CELL PROLIFERATION

3) Identification of a putative EWS/NOR1-regulated gene

Cellular model in which the oncogenic properties of EWS/NOR1are expressed

EMC : most probably consist of primitive mesenchymalcells occasionnally expressing chondrocytic and/or neuroendocrine differentiation markers

CFK2 cell line :

- immortalized chondrogenic cell line derived from fetal rat cartilage cells

- sub-confluence : fibroblastic morphology

- at confluence : form chondrogenic-like nodules expressing cartilage-specific proteoglycan and collagen type II

RT-PCR

EN1 EN20 pcDNA+ - + - + -

WESTERN

EN1 EN20 pc CFK2

0,5

1,0

RLU

1,5

EN1 EN20pcCFK2

0,7X

194X

532X

RT

EWS/NOR1

0

200000

400000

600000

800000

1000000

CE

LL

NU

MB

ER

0 3 6 9

12

15

18

DAYS

EN20

EN1

pc4

pc2

CFK2

Cell line G0/G1 (%) S (%) G2/M (%)

CFK2 60 10 30pcDNA 59 9 32EN1 61 11 28EN20 59 12 29

pcDNA

EN1

pcDNA

EN1

CFK2 pc EN1 EN20C D C D C D C D

collagen type II

CFK2

EN1

pcDNA

EN20

proteoglycanstaining

Tetracycline-regulated expression of EWS/NOR1 in CFK2 cells

tet

VP16

VP16CMV

pJMF2vector

EWS/NOR1VRE

VP16

EWS/NOR1VRE

TetracyclineEWS/NOR1expression

+

_

_

+++

EWS/NOR1

18S

ENin pJ_ + _ +

EWS/NOR1

FMRP0

2

4

6

8

RELATIV

E L

IGH

T U

NIT

S

+ -

TETRACYCLINE

12.6XENin pJ

_ + _ +

NORTHERN

WESTERN

TET

TET

SERUM- AND GLUCOCORTICOID-REGULATED KINASE (SGK)

SGK

18S

ENin pJ_ + _ +

MCPC

1 2HCC

1 2EMC

1 2 C

SGK

GAPDH

NORTHERN

RT-PCR

TET

Cycling

Confluence

SGK

18S

EWS/NOR1

GAPDH

SGK

18S

CFK

2

pc1 pc2 EN1EN20

SIX3 expression in EN1 and EN20 cell lines

SIX3

EN1 EN20 EN1 EN20 C

cycling confluence

GAPDH

RT-PCR

CONCLUSIONS

- ONE ROLE OF EWS/NOR1 IN EMC MAY BE TO ACTIVATE THE EXPRESSION OF THE SGK GENE

- THE DEGREE OF ACTIVATION MAY BE FINELY TUNED BY THE INTERACTION WITH SIX3

Serum- and glucocorticoid-regulated kinase

- Originally cloned as a glucocorticoid-upregulated gene in a rat mammary tumor cell line

- Serine/threonine kinase showing ~50% homology to the AKT kinase

- Activated by phosphorylation (Thr 256 and Ser 422) by the phosphoinositide dependent protein kinase-1 (PDK-1), itself activated by the phophatidylinositol 3-kinase (PI 3-kinase) cascade

- Involved in cell survival and proliferative responses

- Translocation to the nucleus upon growth factor stimulation : targets ?

FUTURE STUDIES

- Protein expression of SGK in EMC tumors and CFK2 cell lines

- Link between SGK and transformation ?

- Does SIX3 repress EWS/NOR1 in cycling EN1 and EN20 cell lines ?

- Is SGK a direct target of EWS/NOR1 and NOR1 ?

CREDITS

Students

Frank CourjalMaxime TremblayCynthia LaflammeHugo Poulin

Research Assistants

Johanne BussièresChristine Filion

Collaborators

Mary Goldring, Harvard Institutes of MedicineMarc Ladanyi, Sloan-Kettering Cancer CenterJulia Bridge, Nebraska Medical CenterDavid Goltzman, McGill University

Support

- Canadian Institutes for Health Research- Natural Sciences and Engineering Research Council of Canada- Fonds de la recherche en santé du Québec