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 NOR 3 NH 2 COOH EWS protein (EWing Sarcoma) 1656 NH 2 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 NOR1 protein (Neuron-derived Orphan Receptor) NH 2 COOH DNA BD AF1LZAF2 - 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 1949 NH 2 COOH AF1LZAF2 EWS-NTD DNA BD 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 gene promoters NOR1/NGFI-B/NURR1 bind to and constitutively activate transcription from the NBRE, what about EWS/NOR1 ? 175 EWS/NOR1NOR1 83 kDa NBRE 5 AAAGGTCA 3 mNBRE 5 AGAGGTCA 3 EWS/NOR1NOR1 competitor 5X20X NBRE 5X20X mNBRE 5X20X NBRE 5X20X mNBRE 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 RLU COSC20 NOR COSC20 EWS/NOR1 265 aa626 aa EWS65/NOR1 EWS132/NOR1 EWS204/NOR % RLU 100 EN65132204 EWS/NOR1 265 aa626 aa EWS/NOR1AF2 NOR1 NOR1AF2 AF % RLU 100 ENAF % RLU 100 NAF2 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 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/NOR1AF2 4) GST/NOR1LZ 5) GST/NOR1E5 6) GST/NOR1DBD 1239 DBDLZAF kDa SL input SLSLSLSLSLSL AF1 1) GST 2) GST/EWS/NOR1 3) GST/EWS/NOR1LZ 4) GST/EWS/NOR1DBD 5) GST/EWS/NOR1AF1 6) GST/EWS/NOR1NOR DBDLZAF EWSAF1 kDa SL input SLSLSLSLSLSL 1332 1) SIX3 2) SIX3C 3) SIX3HD HDSD kDa L input 231L gst/nor1 231L gst/ ews/nor1 23 Protein-protein interactions in vivo : mammalian two-hybrid system GAL4 DRE TATA LUCIFERASE EXPRESSION + GAL4 DRE TATA LUCIFERASE DBD ACT + GAL4 DRE TATA LUCIFERASE +++ DBD ACTXY 1) DBD 2) DBD/NOR1 3) ACT 4) ACT/SIX3 5) ACT/SIX3HD 1147 DBDLZAF AF HDSD ,25 0,5 RLU X 1.5X X 3.0X 1) DBD 2) DBD/EWS/NOR1 3) ACT 4) ACT/SIX3 5) ACT/SIX3HD 1147 DBDLZAF AF HDSD EWS 2,5 5 RLU X 50X X 75X Transient transfections of human chondrocyte cells with a NOR1 expression vector, a NBRE/luciferase reporter vector, and increasing amounts of a SIX3 or SIX3HD expression vector 0,05 0,1 RLU 0,5 pcDNASIX3NOR 1 SIX3 1:1 NOR1 SIX3HD 1:1 NOR1 SIX3 1:2 NOR1 SIX3HD 1:2 NOR1 SIX3 1:3 NOR1 SIX3HD 1:3 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 SIX3HD expression vector RLU 45 pcDNASIX3EN SIX3 1:1 EN SIX3HD 1:1 EN SIX3 1:2 EN SIX3HD 1:2 EN SIX3 1:3 EN SIX3HD 1:3 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/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 RT-PCR EN1EN20pcDNA WESTERN EN1EN20pcCFK2 0,5 1,0 RLU 1,5 EN1EN20pcCFK2 0,7X 194X 532X RT EWS/NOR1 Cell line G 0 /G 1 (%) S (%) G 2 /M (%) CFK pcDNA EN EN pcDNA EN1 pcDNA EN1 CFK2pcEN1EN20 CDCDCDCD collagen type II CFK2 EN1 pcDNA EN20 proteoglycan staining Tetracycline-regulated expression of EWS/NOR1 in CFK2 cells tetVP16 CMV pJMF2 vector EWS/NOR1VRE VP16 EWS/NOR1VRE Tetracycline EWS/NOR1 expression + _ _ +++ EWS/NOR1 18S ENinpJ _ + _ + EWS/NOR1 FMRP ENinpJ _ + _ + NORTHERN WESTERN TET SERUM- AND GLUCOCORTICOID-REGULATED KINASE (SGK) SGK 18S ENinpJ _ + _ + M CPC 12 HCC 12 EMC 12C SGK GAPDH NORTHERN RT-PCR TET Cycling Confluence SGK 18S EWS/NOR1 GAPDH SGK 18S CFK2 pc1pc2EN1EN20 SIX3 expression in EN1 and EN20 cell lines SIX3 EN1EN20EN1EN20C cyclingconfluence 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 Courjal Maxime Tremblay Cynthia Laflamme Hugo Poulin Research Assistants Johanne Bussires Christine Filion 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 sant du Qubec