title: DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation.
abstract: Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway.Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS.
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University of Rome, Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Megiorni
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University of Rome, Rome, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Camero
authors.affiliation: Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Ceccarelli
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University of Rome, Rome, Italy.|Department of Oncology, Alder Hey Children's NHS Foundation Trust, Liverpool, United Kingdom.
authors.forename: Heather P
authors.initials: HP
authors.lastname: McDowell
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University of Rome, Rome, Italy.
authors.forename: Olga
authors.initials: O
authors.lastname: Mannarino
authors.affiliation: Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, L'Aquila, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Marampon
authors.affiliation: Department of Oncology, Alder Hey Children's NHS Foundation Trust, Liverpool, United Kingdom.
authors.forename: Barry
authors.initials: B
authors.lastname: Pizer
authors.affiliation: Department of Perinatal and Paediatric Pathology, Alder Hey Children's NHS Foundation Trust, Liverpool, United Kingdom.
authors.forename: Rajeev
authors.initials: R
authors.lastname: Shukla
authors.affiliation: Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Pizzuti
authors.affiliation: Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Marchese
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University of Rome, Rome, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Clerico
authors.affiliation: Department of Paediatrics and Infantile Neuropsychiatry, Sapienza University of Rome, Rome, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Dominici
mesh_terms: D002070:Butadienes; D002453:Cell Cycle; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D066298:In Vitro Techniques; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D009570:Nitriles; D010641:Phenotype; D018232:Rhabdomyosarcoma, Alveolar / Q000235:genetics*; D018233:Rhabdomyosarcoma, Embryonal / Q000235:genetics*; D000097584:DNA Methyltransferase 3B
chemical_list: D002070:Butadienes; D004248:DNA (Cytosine-5-)-Methyltransferases; D009570:Nitriles; D000097584:DNA Methyltransferase 3B; C113580:U 0126
keywords: DNMT3B; MEK/ERK signalling; RNA interference; myogenic differentiation; rhabdomyosarcoma