title: Loss of MEF2D expression inhibits differentiation and contributes to oncogenesis in rhabdomyosarcoma cells.
abstract: BACKGROUND
  Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the defects present in these cells that block myogenesis.
  
  
  METHODS
  Protein and RNA analysis identified the loss of MEF2D in RMS cells. MEF2D was expressed in RD and RH30 cells by transient transfection and selection of stable cell lines, respectively, to demonstrate the rescue of muscle differentiation observed. A combination of techniques such as proliferation assays, scratch assays and soft agar assays were used with RH30 cells expressing MEF2D to demonstrate the loss of oncogenic growth in vitro and xenograft assays were used to confirm the loss of tumor growth in vivo.
  
  
  RESULTS
  Here, we show that one member of the MEF2 family of proteins required for normal myogenesis, MEF2D, is largely absent in RMS cell lines representing both major subtypes of RMS as well as primary cells derived from an embryonal RMS model. We show that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find that MyoD and myogenin are bound with their dimerization partner, the E proteins, to the promoters of muscle specific genes in RMS cells. However, we cannot detect MEF2D binding at any promoter tested. We find that exogenous MEF2D expression can activate muscle specific luciferase constructs, up regulate p21 expression and increase muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility and anchorage independent growth in vitro. We have confirmed the inhibition of tumorigenicity by MEF2D in a tumor xenograft model, with a complete regression of tumor growth.
  
  
  CONCLUSIONS
  Our data indicate that the oncogenic properties of RMS cells can be partially attributed to the loss of MEF2D expression and that restoration of MEF2D may represent a useful therapeutic strategy to decrease tumorigenicity.
authors.forename: Meiling
authors.initials: M
authors.lastname: Zhang
authors.forename: Jamie
authors.initials: J
authors.lastname: Truscott
authors.affiliation: Department of Biochemistry and Molecular Biology and Simmons Cancer Institute, Southern Illinois University School of Medicine, 229 Neckers Building, 1245 Lincoln Dr, Carbondale, IL 62901, USA. jdavie@siumed.edu.
authors.forename: Judith
authors.initials: J
authors.lastname: Davie
mesh_terms: D000818:Animals; D063646:Carcinogenesis / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement; D002471:Cell Transformation, Neoplastic; D015536:Down-Regulation; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D064326:MEF2 Transcription Factors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D012208:Rhabdomyosarcoma / Q000378:metabolism / Q000473:pathology*; D047368:Tumor Burden
chemical_list: D064326:MEF2 Transcription Factors; C578043:MEF2D protein, human