Expression levels and role of the de novo DNA methyltransferases in rhabdomyosarcoma

Aberrant DNA methylation has been frequently observed in human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, representing approximately 5% of malignant solid tumour in the paediatric population. However, the specific mechanisms and targets are still poorly understood. We showed the up-regulation of DNA methyltransferase (DNMT) family members in 14 RMS primary tumour biopsies and 4 RMS cell lines in comparison to normal skeletal muscle (NSM). Our study focused on DNMT3B gene, which exhibited particularly high levels in RMS samples, in order to establish its individual role in this malignancy. RNA interference-mediated DNMT3B knock-down 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 p21 and p27 checkpoint regulators. DNMT3B depleted cells also showed a decreased migratory capacity and clonogenic potential in comparison to mocked controls. Interestingly, DNMT3B silencing was able to reactivate the skeletal muscle differentiation program in embryonal RMS (ERMS) cells, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, leading to cell cycle arrest and myogenic terminal differentiation, this supporting the methyltransferase as a down-stream molecule of the MEK/ERK oncogenic pathway. Moreover, we showed that DNMT3B is a target of specific miR-29 family members in RMS cells and that the restoration of miRNA expression levels, by miRNA mimic transfection, lead to decreased cell proliferation and migration and to G1 cell cycle arrest. Finally, DNMT3B silencing radiosensitizes ERMS cells by altering DNA damage response signalling. Taken together, our data shed further light on RMS development, underlying a pivotal role of DNMT3B gene in myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel and promising strategy against RMS tumour able to ameliorate traditional therapies in order to improve the survival rate for patients with this soft tissue sarcoma.