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.

Keywords: DNMT3B; MEK/ERK signalling; RNA interference; myogenic differentiation; rhabdomyosarcoma.

Figure 1. DNMT3B expression in RMS tumours and cell lines

A. Quantitative real time PCR (Q-PCR) analysis of DNMT3B mRNA levels in 14 RMS primary tumours (7 ARMSs and 7 ERMSs), expressed as fold increase over NSM, arbitrarily set at 1. Transcript levels were normalized to GAPDH mRNA and error bars represent SD of two independent Q-PCR reactions, each performed in triplicate. B. Q-PCR of DNMT3B mRNA levels in ARMS (RH4 and RH30) and ERMS (RD and TE671) cell lines, expressed as fold increase over NSM, arbitrarily set at 1. GAPDH was used as control. Bars represent mean values of three independent experiments, each performed in triplicate. HFM, human foetal myoblats. C. Western blot showing the expression of DNMT3B protein in RH4, RH30, RD and TE671 cell lines. Tubulin was used as loading control. Representative of three different experiments.

Figure 2. DNMT3B knock-down by RNA interfering and effects on RD cell viability and morphology

A. DNMT3B transcript levels measured by Q-PCR at 72 h in RD cells after transfection with DNMT3B siRNA (si-DNMT3B) in comparison to samples transfected with non-targeting control siRNA (si-NC), arbitrarily set at 1. Specific transcript levels were normalized to GAPDH control mRNA. Shown are the means of four independent experiments. Error bars represent standard deviations of the means. Asterisks represent the statistical significance (***, p<0.001; ns, not significant). DNMT1 and DNMT3A mRNA levels were also analysed to check the specificity of siRNA-mediated silencing. B. Western blots showing the expression of DNMT3B protein at 72 h after si-DNMT3B transfection compared to si-NC cells. Tubulin was used as loading control. DNMT1 and DNMT3A protein levels were not perturbed by si-DNMT3B delivery. Representative of three independent experiments. C. Representative immunofluorescence showing the down-regulation of DNMT3B protein levels in nuclear compartment at 72 h after si-DNMT3B transfection. High levels of DNMT3B were evident in the nuclei of si-NC cells. D. Viability of RD cells 72 h post-transfection with DNMT3B siRNA calculated with respect to control si-NC cells, assessed by trypan blue exclusion staining. Results represents the mean value of four independent experiments ± SD. Statistical significance: **, p<0.01. E. MTT assay performed to assess relative RD cell numbers at different point after si-DNMT3B or si-NC transfection (0-24-48-72 h). Each point is the mean of three replicate wells ± SD and is representative of three independent experiments. F. Cellular morphology of si-NC and si-DNMT3B RD cells was analysed under light microscope at 20x magnification at 72 and 144 h after siRNA transfection. In si-DNMT3B cultures, more elongated cellular bodies were evident, many of which formed multinucleated myotube-like structures.

Figure 3. Depletion of DNMT3B levels induces G1 arrest and alters the expression of specific cell cycle regulators in RD cells

A. Flow cytometry data showing percentages of cells in G1, S and G2 phases in si-DNMT3B and si-NC RD cells. Data are average values of three independent experiments. B. Western blot analyses of a panel of cell cycle regulatory proteins in RD cells at 72 h after si-NC or si-DNMT3B transfection. Tubulin expression was used as the internal control. Representative blots of three independent experiments. C. Immunofluorescence experiments showing the expression and localization of Cyclin B1, Cyclin D1, p21, p-RB and E2F1 proteins in RD cells at 72 h after DNMT3B or NC siRNA transfection. DAPI was used for nuclear staining. Images captured under ApoTome microscope at 40x magnification.

Figure 4. DNMT3B reduction correlates with altered migration and clonogenic ability of RD cells

A. Representative images of RD si-DNMT3B and si-NC migrated cells using the transwell migration assay (magnification of 10x). Data in the histograms are expressed as the means ± SD from three separate experiments, each performed in triplicate (**, p<0.01 compared with the respective si-NC control, arbitrarily set at 1). B. RD cells transfected with si-DNMT3B or si-NC were seeded at low concentration and allowed to grow for 8 days to examine their colony formation capacity. Representative pictures of colonies stained with crystal violet. Colony forming efficiency was calculated by crystal violet absorbance from three independent experiments, each performed in triplicate (*, p<0.05 compared with the respective si-NC control, arbitrarily set at 1).

Figure 5. DNMT3B knock-down triggers myogenic differentiation in RD cells

A. Expressionof myogenic genes, MYOD1, Myogenin and MyHC, by Q-PCR assays in RD cells transfected with DNMT3B or NC siRNAs for 72 h. Expression of each mRNA was normalized to GADPH levels and plotted as fold change relative to si-NC samples. Histograms are means ± SD. Asterisks represent the statistical significance (**, p<0.01; ***, p<0.001). B. Western blot showing the expression of MYOD1, Myogenin and MyHC proteins in si-DNMT3B and si-NC RD cells at 72-144 h post-transfection. Tubulin was used as loading control. Representative of three different experiments. C. Immunofluorescence experiments showing the expression and localization of MYOD1 and MyHC at 72 h after DNMT3B or NC siRNA transfection. DAPI was used for nuclear staining. Images captured under ApoTome microscope at 40x magnification. D. Expression of myogenic miRNAs, miR-133a and miR-206, by Q-PCR experiments in RD cells transfected with DNMT3B or NC siRNAs for 72 h. Expression of each miRNA was normalized to U6 levels and plotted as fold change relative to si-NC samples. Histograms are means ± SD. Asterisks represent the statistical significance (**, p<0.01; ***, p<0.001).

Figure 6. Inhibition of MEK/ERK pathway by U0126 down-regulates DNMT3B and induces myogenic program in RD cells

A. Western blot showing the down-regulation of DNMT3B and the up-regulation of MYOD1, Myogenin and MyHC in RD cells treated with 10 μM U0126 for 96 h. Mocked control cells were treated with DMSO. Tubulin was used as loading control. Representative of three different assays. B. Time-course experiments showing the early decreased phosphorylation status of ERK (p-ERK) and the down-stream reduced expression of DNMT3B protein in RD cells upon U0126 treatment for 0-6-12-24-48-96 h. Tubulin was used as loading control. Representative of two different assays. C. Western blot showing phosphorylated and total p38 levels in si-DNMT3B and si-NC RD cells at 72 and 144 h post-transfection.