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
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