Glycogen synthase kinase 3β represses MYOGENIN function in alveolar rhabdomyosarcoma |
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Authors: | M G Dionyssiou S Ehyai E Avrutin M K Connor J C McDermott |
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Institution: | 1.Department of Biology, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada;2.Department of Kinesiology, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada;3.Centre for Research in Mass Spectrometry, York University, Toronto, Ontario M3J 1P3, Canada;4.Muscle Health Research Centre, York University, Toronto, Ontario M3J 1P3, Canada;5.Centre for Research in Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada |
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Abstract: | MYOGENIN is a member of the muscle regulatory factor family that orchestrates an obligatory step in myogenesis, the terminal differentiation of skeletal muscle cells. A paradoxical feature of alveolar rhabdomyosarcoma (ARMS), a prevalent soft tissue sarcoma in children arising from cells with a myogenic phenotype, is the inability of these cells to undergo terminal differentiation despite the expression of MYOGENIN. The chimeric PAX3-FOXO1 fusion protein which results from a chromosomal translocation in ARMS has been implicated in blocking cell cycle arrest, preventing myogenesis from occurring. We report here that PAX3-FOXO1 enhances glycogen synthase kinase 3β (GSK3β) activity which in turn represses MYOGENIN activity. MYOGENIN is a GSK3β substrate in vitro on the basis of in vitro kinase assays and MYOGENIN is phosphorylated in ARMS-derived RH30 cells. Constitutively active GSK3β(S9A) increased the level of a phosphorylated form of MYOGENIN on the basis of western blot analysis and this effect was reversed by neutralization of the single consensus GSK3β phosphoacceptor site by mutation (S160/164A). Congruently, GSK3β inhibited the trans-activation of an E-box reporter gene by wild-type MYOGENIN, but not MYOGENIN with the S160/164A mutations. Functionally, GSK3β repressed muscle creatine kinase (MCK) promoter activity, an effect which was reversed by the S160/164A mutated MYOGENIN. Importantly, GSK3β inhibition or exogenous expression of the S160/164A mutated MYOGENIN in ARMS reduced the anchorage independent growth of RH30 cells in colony-formation assays. Thus, sustained GSK3β activity represses a critical regulatory step in the myogenic cascade, contributing to the undifferentiated, proliferative phenotype in alveolar rhabdomyosarcoma (ARMS). |
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Keywords: | alveolar rhabdomyosarcoma PAX3-FOXO1 MYOGENIN GSK3β cell proliferation tumorigenicity |
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