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.
Highlights
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood and adolescence, accounting for about 5% of malignant paediatric tumours [1]
In accordance with previously published data [31], DNMT1 mRNA levels were significantly higher in alveolar RMS (ARMS) and embryonal RMS (ERMS) tumours compared to normal skeletal muscle (NSM) (30.5±9.0, data not shown)
Our results indicate that the inhibition of DNMT3B expression is a crucial step for reverting RMS cancer phenotype towards skeletal muscle differentiation, by restricting the expression of proliferative markers and up-regulating myogenic genes
Summary
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood and adolescence, accounting for about 5% of malignant paediatric tumours [1]. There is a growing evidence that epigenetic mechanisms play an important role in cancer development, with changes in DNA methylation being a crucial event in the control of gene expression [5]. Recent evidences suggest that a deregulation of the de novo DNMT enzymes contributes to the development of paediatric brain tumours [20]. DNMTs inhibit gene expression by recruiting transcriptionally repressive complexes, which include methyl-CpG binding domain (MBD) factors, polycomb group (PcG) proteins and repressor factors, to specific DNA sites [23,24,25,26,27]. Increased DNMT1 activity in RMS was previously reported [31] and DNA methylation signatures have been recently described in ARMS and ERMS cases [32], suggesting that aberrant DNA methylation may contribute to the development of RMS
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