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Yves Labelle

Functional analysis of the EWS/NOR1 fusion protein expressed in extraskeletal myxoid chondrosarcoma tumors. Yves Labelle. Department of medical biology Laval University Quebec City. t(9;22). Chr 22 EWS. Chr 9 NOR1. 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 2. 3. 4. 5. 6. 7. 8. 1. 2.

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Yves Labelle

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  1. Functional analysis of the EWS/NOR1 fusion protein expressed in extraskeletal myxoid chondrosarcoma tumors Yves Labelle Department of medical biology Laval University Quebec City

  2. t(9;22) Chr 22 EWS Chr 9 NOR1 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 3 4 5 6 7 8 5’ 3’ NH2 COOH

  3. EWS protein (EWing Sarcoma) 1 656 NH2 COOH RNA BD homology with the CTD of the LS 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

  4. NOR1 protein (Neuron-derived Orphan Receptor) 1 626 NH2 COOH DNA 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 ?

  5. EWS/NOR1 protein 1 949 NH2 DNA BD COOH EWS-NTD AF1 LZ AF2 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

  6. 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 gene promoters NOR1/NGFI-B/NURR1 bind to and constitutively activate transcription from the NBRE, what about EWS/NOR1 ?

  7. NOR1 EWS/NOR1 kDa 75 175 NBRE 5’ AAAGGTCA 3’ mNBRE 5’ AGAGGTCA 3’ 48 83 NOR1 EWS/NOR1 NBRE mNBRE NBRE mNBRE 5X 20X 5X 20X 5X 20X 5X 20X competitor

  8. Transient transfections of COS and human chondrocyte cells with a NOR1 or EWS/NOR1 expression vector, a NBRE/luciferase reporter vector, and a beta-galactosidase normalizing vector NOR1 EWS/NOR1 15 5000 RLU 10 2500 5 250 COS C20 COS C20

  9. EWS/NOR1 265 aa 626 aa 100 EWS∆65/NOR1 75 EWS∆132/NOR1 % RLU 50 25 EWS∆204/NOR1 EN ∆65 ∆132 ∆204

  10. 100 EWS/NOR1 75 265 aa 626 aa % RLU 50 AF2 25 EWS/NOR1∆AF2 EN ∆AF2 100 NOR1 75 % RLU 50 NOR1∆AF2 25 N ∆AF2

  11. CONCLUSION EWS/NOR1 MAY PARTICIPATE IN THE DEVELOPMENT OF EMC TUMORS BY OVER-ACTIVATING NOR1-TARGET GENES INVOLVED IN CELL PROLIFERATION AND/OR CELL DEATH

  12. 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 ?

  13. RT-PCR

  14. 1 239 1) GST 2) GST/NOR1 3) GST/NOR1∆AF2 4) GST/NOR1∆LZ 5) GST/NOR1∆E5 6) GST/NOR1∆DBD AF1 DBD LZ AF2 1 626 611 443 359 288 input 1 2 3 4 5 6 kDa S L S L S L S L S L S L S L 66 45 31

  15. 1 239 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 EWS AF1 DBD LZ AF2 1 949 766 611 444 264 input 1 2 3 4 5 6 kDa S L S L S L S L S L S L S L 66 45 31

  16. SD HD 1 332 1) SIX3 2) SIX3∆C 3) SIX3∆HD gst/ ews/nor1 input gst/nor1 kDa L 1 2 3 L 1 2 3 L 1 2 3 66 45 31

  17. Protein-protein interactions in vivo : mammalian two-hybrid system LUCIFERASE EXPRESSION TATA GAL4 DRE LUCIFERASE + ACT DBD TATA GAL4 DRE LUCIFERASE + X Y ACT DBD +++ TATA GAL4 DRE LUCIFERASE

  18. 1 147 1) DBD 2) DBD/NOR1 3) ACT 4) ACT/SIX3 5) ACT/SIX3∆HD AF1 DBD LZ AF2 1 626 1 46 SD HD 1 332 181 14.3X 0,5 RLU 0,25 3.1X 3.0X 1.5X 1+3 1+4 2+3 2+4 2+5

  19. 1 147 1) DBD 2) DBD/EWS/NOR1 3) ACT 4) ACT/SIX3 5) ACT/SIX3∆HD EWS AF1 DBD LZ AF2 1 949 1 46 SD HD 1 332 181 160X 5 RLU 75X 2,5 50X 2X 1+3 1+4 2+3 2+4 2+5

  20. Transient transfections of human chondrocyte cells with a NOR1 expression vector, a NBRE/luciferase reporter vector, and increasing amounts of a SIX3 or SIX3∆HD expression vector 0,5 0,1 RLU 0,05 pcDNA SIX3 NOR1 NOR1 SIX3 1:1 NOR1 SIX3∆HD 1:1 NOR1 SIX3 1:2 NOR1 SIX3∆HD 1:2 NOR1 SIX3 1:3 NOR1 SIX3∆HD 1:3

  21. Transient transfections of human chondrocyte cells with an EWS/NOR1 (EN) expression vector, a NBRE/luciferase reporter vector, and increasing amounts of a SIX3 or SIX3∆HD expression vector 45 30 RLU 15 pcDNA SIX3 EN EN SIX3 1:1 EN SIX3∆HD 1:1 EN SIX3 1:2 EN SIX3∆HD 1:2 EN SIX3 1:3 EN SIX3∆HD 1:3

  22. 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

  23. 3) Identification of a putative EWS/NOR1-regulated gene • Cellular model in which the oncogenic properties of EWS/NOR1 • are expressed • EMC : most probably consist of primitive mesenchymal • cells 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

  24. EWS/NOR1 RT-PCR 532X 1,5 EN1 EN20 pcDNA RT + - + - + - 1,0 RLU 194X 0,5 WESTERN EN1 EN20 pc CFK2 0,7X CFK2 pc EN1 EN20

  25. Cell line G0/G1 (%) S (%) G2/M (%) CFK2 60 10 30 pcDNA 59 9 32 EN1 61 11 28 EN20 59 12 29

  26. pcDNA EN1

  27. pcDNA EN1

  28. CFK2 pc EN1 EN20 C D C D C D C D collagen type II CFK2 pcDNA proteoglycan staining EN20 EN1

  29. Tetracycline-regulated expression of EWS/NOR1 in CFK2 cells pJMF2 vector CMV VP16 EWS/NOR1 expression tet VP16 Tetracycline _ + VRE EWS/NOR1 VP16 _ VRE EWS/NOR1 +++

  30. ENin pJ _ _ + + TET EWS/NOR1 18S NORTHERN ENin pJ _ _ + + TET EWS/NOR1 FMRP WESTERN

  31. SERUM- AND GLUCOCORTICOID-REGULATED KINASE (SGK) ENin pJ _ _ + + TET SGK NORTHERN 18S CPC HCC EMC M 1 2 1 2 1 2 C SGK RT-PCR GAPDH

  32. CFK2 pc1 pc2 EN1 EN20 Cycling SGK 18S Confluence SGK 18S EWS/NOR1 GAPDH

  33. SIX3 expression in EN1 and EN20 cell lines cycling confluence EN1 EN20 EN1 EN20 C SIX3 RT-PCR GAPDH

  34. 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

  35. 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 ?

  36. 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 ?

  37. CREDITS Students Frank Courjal Maxime Tremblay Cynthia Laflamme Hugo Poulin • Collaborators • Mary Goldring, Harvard Institutes of Medicine • Marc Ladanyi, Sloan-Kettering Cancer Center • Julia Bridge, Nebraska Medical Center • David 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 Research Assistants Johanne Bussières Christine Filion

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