Background
Pediatric rhabdomyosarcoma (RMS) is a locally invasive soft-tissue sarcoma with a predisposition to metastasize that accounts for ~ 30% of all soft-tissue sarcomas (STS) and for 7-8% of all solid tumors in childhood [
1]. Embryonal RMS is the major histopathologic subtype, accounting for 60% of all RMS cases and, when nonmetastatic, shows a 5-year overall survival of 70% [
2]. Childhood cancer statistics show that the outcome for young patients with RMS has tremendously improved from 53% in 1975–1978 to 68% in 1979–1982 [
3], but unfortunately current treatments for embryonal RMS in the metastatic form often do not respond to therapy. Indeed, metastatic or relapsed forms, even if they can undergo complete remission with secondary therapy, are often characterized by poor long-term prognosis and dismal outcome [
4‐
6]. Moreover, children who relapse need to be closely monitored for a long time as anti-cancer therapy side effects may persist or develop months or years after treatment. Therefore, novel more specific and less toxic treatment approaches, such as molecular targeted therapies, are under study. Since RMS cells share characteristics of skeletal muscle precursors, the most reliable theory about the origin of RMS suggests that perturbations of the normal mesenchymal development of the skeletal muscle lineage might have a causative role [
7]. Consistently, results from some groups and ours recently suggest that a differentiation therapy seems to represent an alternative way to reduce the aggressiveness of cancer cells, not by exerting cytotoxicity but by restoring the differentiation fate of tumor cells [
8‐
12]. Indeed, under specific treatments, RMS cells progress toward less proliferating myoblast-like cells that are capable to develop myotube-like structure. The methyltransferase Polycomb Group (PcG) protein Enhancer of zeste homolog 2 (EZH2), the catalytic factor of the Polycomb Repressor Complex 2 (PRC2), represses gene transcription by silencing target genes through methylation of histone H3 on lysine 27 (H3K27me3) and it has been shown to prevent cell differentiation and promote cell proliferation in several tissues [
13]. Increasing evidence demonstrates that EZH2 is not only aberrantly expressed in several types of human cancers, but often behaves as a molecular biomarker of poor prognosis [
14‐
21]. EZH2 was clearly shown to act as a negative regulator of skeletal muscle differentiation favoring the proliferation of myogenic precursors [
22‐
24]. This function results from an EZH2-dependent direct repression of genes related to myogenic differentiation [
22]. We previously reported that EZH2 is markedly expressed in the RMS context, both in cell lines and primary tumors compared to their normal counterparts [
25]. The first evidence of the role of EZH2 as a main player in the inability of RMS cells to undergo differentiation has been recently reported
in vitro for the embryonal RMS cell line RD, established from a tumor recurrence, through EZH2 genetic silencing upon serum withdrawal [
26].
Here, after having shown that EZH2 was de-regulated in a cohort of primary embryonal RMS, we evaluated whether it was possible to boost the differentiation capability of embryonal RMS RD cells after EZH2 inhibition even in serum-enriched culture conditions. As an additional promising approach, we investigated whether pharmacological inhibition of EZH2 in RD cells by either reducing its expression or catalytically inhibiting its activity might be detrimental for cancer cell proliferation both in vitro and in vivo. Our data demonstrate that EZH2 down-regulation restores the myogenic differentiation of RD cells with no need to reduce serum (cultured in growth medium), and that pharmacological inhibition of EZH2 is a feasible way to restrain the tumor-promoting potential in embryonal RMS.
Methods
Additional file
1: Supplementary Methods.
Cell lines
RD embryonal RMS cell line was obtained from American Type Culture Collection (Rockville, MD). A204 and RH18 embryonal RMS cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). Normal Human Skeletal Muscle cells (SkMC; myoblasts) were obtained from PromoCell (Heidelberg Germany).
Nuclear fraction-enrichment
Cells were lysed and assayed as previously reported [
10]. Briefly, cells were lysed in cytoplasm lysis buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.2 mM EDTA, 1 mM DTT), containing protease inhibitors, 0.5 mM phenylmethylsulfonylfluoride (PMSF) and 0.6% Nonidet P-40 (Sigma Chemical Co., St Louis, MO, USA). Lysates were centrifuged at 10.000 rpm 10 min at 4°C and the supernatants (cytoplasmic fractions) were split into aliquots and rapidly frozen. The nuclear pellet was washed in buffer A without Nonidet P-40 and finally resuspended in nuclear lysis buffer B (20 mM HEPES pH 7.9, 0.4 M NaCl, 2 mM EDTA, 1 mM DTT), containing protease inhibitors and 1 mM PMSF (Sigma Chemical Co., St Louis, MO, USA). Samples were incubated on ice 30 min and centrifuged at 13.000 rpm 10 min at 4°C; the supernatants (nuclear fractions) were split into aliquots and rapidly frozen or used for western blot analysis.
Western blotting
Western blotting was performed on whole-cell lysates and histone extracts as previously described [
27,
28]. Briefly, cells were lysed in RIPA buffer (50 mM Tris–HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1% D.O.C. (Na), 0,1% SDS, 1% Triton X-100) containing protease inhibitors (Sigma Chemical Co., St Louis, MO, USA). Lysates were sonicated, incubated on ice 30 min and centrifugated at 10,000 g 20 min at 4°C. Supernatants were used as total lysates. Protein concentrations were estimated with the BCA protein assay (Pierce, Rockford, IL). EZH2 was detected using the EZH2 antibody (612666; Transduction LaboratoriesTM, BD, Franklin Lakes, NJ). Antibodies against Myogenin (F5D) and Myosin Heavy Chain (Meromyosin, MF20) were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa (DSHB, Iowa City, IA). Antibodies against p21Cip1 (sc-397), β-actin (sc-1616) and all secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibodies against Troponin I (4002) were obtained from Cell Signaling (Beverly, MA). The antibody against the Topoisomerase IIβ was obtained from Sigma Aldrich (Sigma Chemical Co., St Louis, MO, USA). Antibody against against Histone 3 (H3), H3K27me3 (Lys27) and H3K4me3 (Lys4) were obtained from Millipore (EMD Millipore Corporation, Billerica, MA, USA). Antibody against α-tubulin (ab4074) was from Abcam (Cambridge, UK). All the antibodies were used in accordance with the manufacturer’s instructions.
Cells were harvested and washed twice with ice-cold Phosphate Buffered saline (PBS) 1X supplemented with 5 mM Sodium Butyrate and resuspended in Triton Extraction Buffer (TEB: PBS, 0.5% Triton X 100 (v/v)) containing 2 mM PMSF and 0.02% (w/v) NaN3 (107 cells/ml) and lysated on ice for 10 min. Lysates were centrifuged at 2000 rpm for 10 min at 4°C and the pellets were washed in half volume of TEB and centrifuged.Histones were extracted O/N at 4°C from pellets resuspended in 0.2 N HCl (4×107 cells/ml). Samples were then centrifuged and supernatants were used for western blot analysis.
Transient RNA interference
Cells were sequentially transfected by 2 subsequent rounds (24 h), to secure efficient cell silencing, with ON-TARGETplus SMART pool siRNA targeting different regions of the EZH2 transcript (L-004218-00) or non-targeting siRNA (control; D-001206-13), previously validated in other publications [
14,
29,
30] (both from Dharmacon, Thermo Fisher Scientific, Lafayette, CO).
Real time qRT-PCR
Total RNA was extracted using TRizol (Invitrogen, Carlsbad, CA) and analyzed by real-time RT-qPCR for relative quantification of gene expression [
27] using Taqman gene assays (Applied Biosystems, Life Technologies, Carlsbad, CA) for GAPDH (Hs99999905_m1), EZH2 (Hs01016789_m1), Myogenin (Hs01072232_m1), MCK (Hs00176490_m1) and p21 (Hs00355782_m1). For the relative quantification of Murine Ezh2 and MHC mRNA the SYBR-green method was used (Applied Biosystems, Life Technologies, Carlsbad, CA) with primers previously reported [
31] or available on request. The values were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. An Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA) was used for measurements.
Murine Ezh2 over-expression
Flag-tagged murine Ezh2, cloned into the pMSCV retroviral vector (Addgene, Cambridge, MA) or control empty vector, both co-expressing the Green Fluorescent Protein (GFP) as reporter gene, were kindly obtained from G. Caretti. Phoenix ampho cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS (growth medium, GM).Transient transfection of Phoenix ampho cells were performed using lipofectamine reagent (Invitrogen, Carlsbad, MA) and viral particles were collected after 48 h. Supernatant containing viral particles were used to infect RD cells O/N in the presence of 8 ug/ml of polybrene.
Immunofluorescence for MHC detection
Immunofluorescence to visualize MHC was performed as previously described using the MF-20 antibody (Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA) [
10]. Briefly, cells were washed 3 times in PBS, fixed 10 min in 4% PFA and permealized 5 min with 0.2% Triton X-100 in PBS. After 30 min in PBS containing 3% bovine serum albumin, slides were incubated 1 h at room temperature with the MF-20 antibody against myosin heavy chain (MHC; Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA). After 2 washing in PBS, cells were treated with a rhodamine-conjugated secondary antibody (Millipore, Temecula, CA). After being counterstained with DAPI, chamber slides were mounted in GelMount (Biomeda, Foster City, CA, USA). Images were acquired with an Eclipse E600 fluorescence microscope, through LUCIA software version 4.81 (Nikon, Sesto Fiorentino, Firenze, Italy).
Cell cycle and apoptosis assays
Cells were transfected 24 h after seeding (Day 0) with siRNAs and after 24 h transfected again. Then, they were harvested and counted at the reported time points. For pharmacological treatments RD cells were treated with the S-adenosyl-L-homocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) and MC1945 for 24 h, 48 h, 72 h and 96 h. For cell cycle assay, cells were harvested by trypsinization at the indicated time points, washed in ice-cold PBS, fixed in 50% PBS and 50% acetone/methanol (1:4 v/v) for at least 1 h and, after removing alcoholic fixative, stained in the dark with a solution containing 50 μg/ml Propidium Iodide (PI) and 100 μg/ml RNase (Sigma) for 30 min at room temperature. For quantification of apoptosis, cells were harvested, washed twice with ice-cold PBS and stained in calcium-binding buffer with APC-conjugated Annexin V and 7-Aminoactinomycin D (7-AAD) using Annexin V apoptosis detection kit (BD Pharmingen, San Diego, CA), according to manufacturer’s recommendations. Samples were analyzed within 1 h. The stained cells were analyzed for both cell cycle and apoptosis by fluorescence-activated cell sorting using a FACSCantoII equipped with a FACSDiva 6.1 CellQuest software (Becton Dickinson Instrument, San Josè, CA).
Chromatin immunoprecipitation (ChIP)
ChIP assay was performed as previously described (70) with minor modifications. Briefly, chromatin was cross-linked in 1% formaldehyde for 15 min at room temperature and quenched by addition of glycine at 125 mM final concentration for 5 min at room temperature before being placed on ice. Cells were washed twice with ice-cold PBS containing 1 mM PMSF and 1X protease inhibitors, resuspended in ice-cold cell lysis buffer (10 mM Tris–HCl pH 8, 10 mM NaCl, 0.2% NP-40, 1 mM PMSF and 1X protease inhibitors) and incubated on ice for 20 minutes. After centrifugation at 4000 rpm for 5 min, nuclei were resuspended in ice-cold nuclear lysis buffer (50 mM TrisHCl pH 8.1; 10 mM EDTA; 1% SDS, 1 mM PMSF and 1X protease inhibitors) and left on ice for 10 min. Chromatin was then sonicated to an average fragment size of 200–300 bp using a Bioruptor and diluted ten times with IP dilution buffer (16.7 mM Tris–HCl pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01%SDS, 1.1% Triton X-100, 1 mM PMSF and 1X protease inhibitors). Diluted chromatin was pre-cleared using protein G-agarose magnetic beads (Invitrogen) for 1 hour at 4°C and incubated with the corresponding antibodies O/N at 4°C. The following antibodies were used: anti-acetylated histone H3, anti-trimethyl Lysine 27 histone H3 and anti-trimethyl Lysine 4 histone H3 (EMD Millipore Corporation, Billerica, MA, USA) and anti-Ezh2 (Diagenode s.a. Liège, Belgium). Immunoprecipitated chromatin was recovered by incubation with protein G-agarose magnetic beads (Invitrogen, Carlsbad, CA) for 2 hours at 4°C. Beads were washed twice with low salt washing buffer (20 mM Tris–HCl pH8, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 150 mM NaCl), twice with high salt washing buffer (20 mM Tris–HCl pH8, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 500 mM NaCl) and twice with TE before incubating them with elution buffer (10 mM Tris–HCl pH8 1 mM EDTA, 1% SDS) for 30 minutes at 65°C. Cross-linking was then reverted O/N at 65°C and samples were treated with proteinase K for 2 hours at 42°C. The DNA was finally purified by phenol: chloroform extraction in the presence of 0.4 M LiCl and ethanol precipitated. Purified DNA was resuspended in 50 μl of water. Real-time PCR was performed on input samples and equivalent amounts of immunoprecipitated material with the SYBR Green Master Mix (Applied Biosystems, Life Technologies, Carlsbad, CA). Primer sequences are available on request.
Xenograft experiments and immunohistochemistry
Athymic 6-week-old female BALB/c nude mice (nu + \nu+) were purchased from Charles River. Procedures involving animals and their care were conformed to institutional guidelines that comply with national and international laws and policies (EEC Council Directive 86\609, OJ L 358, 12 December 1987). RD cell suspensions in PBS (10×10
6 cells in 100 μl) were injected subcutaneously into the posterior flanks of nude mice. When the tumors became palpable, i.e., about approximately 70–80 mm
3, mice were intraperitoneally injected with MC1945 (2.5 mg/Kg) or control vehicle (DMSO) twice daily, 3 days per week for 3 weeks when mice were sacrificed. No visible signs of toxicity such as weight loss or behavioral change were seen with the compound dose and treatment timing used, as already reported [
32,
33]. Tumor volume was measured by caliper with the following formula: tumor volume (mm3) = L × S2 × π/6 wherein L is the longest and S the shorter diameter and π/6 is a constant to calculate the volume of an ellipsoid, as described [
10]. Representative tumor growth data were obtained from 3 mice per treatment/group. In a parallel experiment, 3 mice per treatment/group were sacrificed 12 days after the first treatment, i.e. the exponential tumor growth phase, and xenografts removed after tumor volume measurement. Portions of the excised tumors embedded in paraffin were used for immunohistochemical analysis. Sections of 10 μm cut from xenograft blocks were stained with hematoxylin/eosin. Five μm serial sections were subjected to immunohistochemistry for the expression of EZH2 and Ki67 with methods and antibodies reported below for primary human RMS samples. The MF-20 antibody (DSHB, USA) was used to detect the expression of MHC. Counterstaining was carried out with Gill’s hematoxyline (Bio-Optica, MI, Italy). Sections were dehydrated and mounted in non-aqueous mounting medium. Images were acquired under an Eclipse E600 microscope (Nikon) through the LUCIA software, version 4.81 (Nikon) with a Nikon Digital Camera DXM1200F.
Immunohistochemistry on RMS primary tissues
Archival, de-identified formalin-fixed, paraffin-embedded RMS and control tissues were obtained from the Department of Pathology of Ospedale Pediatrico Bambino Gesù in Roma, (Italy) after approval of the Institutional Review Boards. Clinicopathological characteristics of the cohort are reported in Table
1. Histopathological features of the tumors were reviewed for the present study by a Pathologist (R. B) blinded to the results of immunohistochemical analysis. Sections from RMS samples and 3 control muscle tissues were cut at 3–5 μM, deparaffinized in xylene and rehydrated through graded ethanol. Antigen retrieval was performed for 25 min at 98°C. After endogenous peroxidase blocking with 3% H2O2 in Tris-buffered saline (TBS) for 30 min at room temperature (RT), 3% to 5% BSA in TBS was applied for 1 hour at room temperature for non-specific background blocking. Sections were treated with Biotin Blocking System (DAKO, Carpinteria, CA) for additional blocking, according to the manufacturer’s instructions. Sections were incubated with primary antibodies for EZH2 (Transduction LaboratoriesTM, BD, Franklin Lakes, NJ), as reported [
34] and Ki67 (Novocastra; Newcastle upon Tyne, UK), and then with secondary antibodies EnVision System-HRP (Power vision Plus method, Zymed, San Francisco, CA, USA) and Biotinilated link (DAKO, Carpintera, CA), respectively. Positive reactions were visualized by staining with 3-amino-9-ethylcarbazolo (AEC) and 3,3′-diamminobenzidine (DAB) (DAKO Carpintera, CA), respectively, and then sections were slightly counter-stained with Gill’s hematoxylin (Bio-Optica, Milan, Italy). Negative controls were stained in parallel by treating serial cross-sections simultaneously either with isotype non-specific IgG or omitting the primary antibody. Positive staining was defined as well-localized nuclear pattern. Levels of expression were semi-quantitatively quantified by scoring the percentage of positive nuclei stained for each specific molecule per microscopic field in at least 5 fields per section by 2 blinded observers and, in rare cases of discrepancy, by an additional third independent observer. Differences in intensity of immunoreactivity were not taken into account. Each section was scored using an Eclipse E600 microscope (Nikon, Sesto Fiorentino, Firenze, Italy) at 400× magnification. Images were acquired through LUCIA software, version 4.81 (Nikon, Sesto Fiorentino, Firenze, Italy) with a Nikon Digital Camera DXM1200F.
Table 1
Clinical and histopathological features of pediatric patients with embryonal rhabdomyosarcoma (RMS) (n=19)
Sex | |
Male | 11 (58) |
Female | 8 (42) |
Age (years) | |
< 10 | 14 (74) |
≥ 10 | 5 (26) |
Localisation | |
Orbit-genitourinary tract-head and neck$
| 9 (47) |
Cranial paramenigeal-extremity-other$$
| 10 (53) |
Tumor volume | |
< 5 cm | 7 (37) |
≥ 5 cm | 12 (63) |
IRS stage | |
I | 2 (10) |
II | 3 (16) |
III | 11 (58) |
IV | 3 (16) |
Metastasis | |
No | 16 (84) |
Yes | 3 (16) |
Recurrence | |
No | 12 (63) |
Yes | 7 (37) |
Outcome | |
Alive | 13 (68) |
DOD | 6 (32) |
Expression of markers | |
EZH2 (positive cells/microscopic field) | 40 (range 29-44) |
Ki67 (positive cells/microscopic field) | 20 (range 17-29) |
Statistical analysis
The Student’s t-test was done to assess the difference between various treatments. Statistical significance was set at a two-tailed P value less than 0.05. All analyses were performed with SPSS 11.5.1 for Windows Package (© SPSS, Inc., 1989–2002 and © LEADTOOLS 1991–2000, LEAD Technologies, Inc., Chicago, IL).
Discussion
In the last decade, to trace the way for developing innovative anti-cancer therapies, several groups focused their pre-clinical research on the modulation of epigenetic regulators often aberrantly expressed in cancer. Due to the fact that epigenetic processes are key players in cell tissue specification during the embryonal life, this approach seems to be particularly captivating for those cancers, such as pediatric embryonal RMS, in which the pathogenic mechanisms involve the deregulation of genes controlling the lineage commitment [
41]. Among these, the histone methyltransferase EZH2 is a fundamental negative regulator of myogenic precursor differentiation by repressing the expression of myogenic genes through the H3K27me3 mark deposition on the promoters of myogenic genes [
22,
28]. We recently reported that EZH2 transcripts were aberrantly expressed in both embryonal RMS primary tumors and in the RD cell line [
25,
35]. In this study, we report that, as for transcripts, EZH2 protein is aberrantly over-expressed in 19 out of 19 embryonal RMS primary tumors compared to normal muscle tissues, thus indicating that the high level of expression of EZH2 is a common molecular lesion of embryonal RMS neoplasia.
Moreover, a recent report indicates that the RD cell line, derived from an embryonal RMS local recurrence and thus representative of an aggressive tumor [
38], may reactivate muscle-specific genes and develop a partial recovery of myocyte phenotype following EZH2 knockdown when depleted of serum [
26]. We show here that it is possible to revert the tumor phenotype of the RD cell line by silencing EZH2 even under proliferative stimuli such as in a serum-enriched molecular context. The final result is the acquisition of a myogenic phenotype, by the de-repression of myogenic genes
Myogenin and
MCK, which can be rescued by the over-expression of a murine Ezh2 not targeted by the used siRNA oligos. More importantly, as a proof-of-concept we report that in these pro-proliferative conditions, pharmacological inhibition of EZH2 by two different approaches,
i.e. by decreasing its availability or hampering its activity, is capable to prevent the proliferation and allow the recovery of myogenic differentiation of these cells
in vitro and
in vivo. In line with the inability of RD cells to undergo terminal differentiation in conditions that induce myotube formation in normal, non-tumorigenic, myoblasts (REF), low-serum differentiation medium did not potentiate the effect of EZH2 depletion/inactivation on the myogenic-like characteristics
vs growth medium. Consistently, EZH2 expression is not modulated by serum deprivation in RD cells (data not shown). Small molecule inhibitors of histone methyltransferases are emerging [
42] and a number of novel EZH2 inhibitors are under preclinical evaluation in other types of cancer [
43‐
45].
Here we treated RD RMS cells with the prototype inhibitor of PRC2, deazaneplanocin A (DZNep), which acts through an indirect mechanism by reducing the level of EZH2 protein [
17,
46]. Recently, DZNep has been reported to be effective in several preclinical studies favoring apoptosis and/or differentiation of tumor cells [
39,
47‐
49]. We found that DZNep arrested RD proliferation in a dose-dependent manner with a concomitant down-regulation of EZH2 protein levels and a decrease in global levels of H3K27me3, while the levels of the other repressive mark H3K9me3 remained unchanged, suggesting an EZH2-specific effect at the doses utilized. Strikingly, in the same growth condition DZNep induced the appearance of MHC-positive multinucleated myotube-like structure in RD cells, accompanied by the activation of myogenic genes such as
Myogenin and
MCK, and with no signs of apoptosis. The observation that in RMS DZNep induces myogenic differentiation instead of apoptosis, the general effect that DZNep has in other human cancer, suggests that its inhibition toward EZH2 is quite specific being pro-differentiative. However, since DZNep may affect other methyltransferases, we enrolled in our study also two molecules belonging to a new class of catalytic inhibitors, validated against a panel of histone methyltransferases [
32,
40], MC1948, which has been already validate as EZH2 inhibitor in myoblasts [
28] and a new, more effective, derivative, MC1945. Both MC inhibitors phenocopied the effects of DZNep and EZH2 genetic depletion
in vitro, indicating a common mechanism of action. More importantly we observed that MC1945 was able to restrain tumor growth of RD xenografts in nude mice inducing tumor cells differentiation
in vivo. Pharmacological inhibition of EZH2 by using a new EZH2 inhibitor has been recently shown to induce anti-tumoral effects in malignant rhabdoid tumor (MRT) cells deleted for
SMARCB1[
50]. Importantly, this result highlights the dependency of
SMARCB1-mutant/deleted MRT tumorigenicity on EZH2. However, the Authors showed no effects of the inhibitor on
SMARCB1-wild-type RD cells that were cultured in medium replenished with the drug on day 4 [
50]. Differently, we treated RD cells with new doses of inhibitors every day since this approach was defined as effective during preliminary experiments. As a consequence, in our experimental protocol tumor cells were in contact with fresh drug each 24 h. These diverse approaches could be responsible for the difference in the response to pharmacological inhibitors.
In summary, here we present a preclinical study in which the experimental evidence indicates that the pharmacological targeting of EZH2 might represent a way to reduce the aggressiveness of RMS, promoting a more differentiated phenotype and thus enlarging the scenery of the future clinical intervention to treat this type of tumors.
Acknowledgments
We thank E. Giorda for FACS analysis. Myogenin (Wright WE), and MF20 (Fishman DA) antibodies were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. We wish to thank G. Caretti for the Ezh2 murine plasmid and control. SS is a Chercheur National of the Fonds de la Recherche en Santé du Quebec.
Grant support
Associazione Italiana per la Ricerca sul Cancro (AIRC, 10338) and Italian Ministry of Health Ricerca Corrente (RR); Association for International Cancer Research (AICR-UK, 12–0168) (DP); AIRC 5 per mille (FL); NIH Intramural Research Program, National Cancer Institute, CCR (VEM); PRIN 2009PX2T2E, FIRB RBFR10ZJQT, and FP7 Project BLUEPRINT/282510 (AM).
Competing interests
The authors indicate no competing financial interests.
Authors’ contributions
RC participated in the design of the study, participated in statistical analysis and in manuscript writing. RC, EC, LA, GB, PPL, AD and FV participated in the in vitro experiments. MDS participated in the in vitro and in vivo studies. GMM, RB and AI carried out primary samples and clinical data collection of RMS patients. SS and IS participated with reagents and discussion. VEM, SV and AM produced DZNep and MC inhibitors and participated with discussion and data analysis. PLP was involved in the design of the study, wrote and reviewed the manuscript. FL participated in the design of the study, discussion of clinical and research data and reviewed the manuscript. DP participated in the study design, experimental procedures, data analysis and manuscript writing. RR was the responsible of the conception and design of the study, coordinated the study, was involved in manuscript writing and reviewed the final version. LA, MDS and GB contributed equally as second co-authors. All authors read and approved the manuscript.