Open Access Highly Accessed Research article

Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS

Roberta Ciarapica1*, Elena Carcarino2, Laura Adesso1, Maria De Salvo1, Giorgia Bracaglia1, Pier Paolo Leoncini1, Alessandra Dall’Agnese2, Federica Verginelli1, Giuseppe M Milano1, Renata Boldrini3, Alessandro Inserra4, Stefano Stifani5, Isabella Screpanti6, Victor E Marquez7, Sergio Valente8, Antonello Mai8, Pier Lorenzo Puri29, Franco Locatelli110, Daniela Palacios2 and Rossella Rota1*

Author Affiliations

1 Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy

2 IRCCS Fondazione Santa Lucia, Rome, Italy

3 Departments of Pathology, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy

4 Departments of Surgery, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy

5 Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada

6 Department of Molecular Medicine, Sapienza University, Rome, Italy

7 Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, CCR, National Cancer Institute, NIH, Frederick, Maryland, USA

8 Istituto Pasteur, Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University, Rome, Italy

9 Muscle Development and Regeneration Program, Sanford-Burnham Medical Research Institute, La Jolla, California, USA

10 Dipartimento di Scienze Pediatriche, Università di Pavia, Pavia, Italy

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BMC Cancer 2014, 14:139  doi:10.1186/1471-2407-14-139

Published: 27 February 2014

Abstract

Background

Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts.

Methods

Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo.

Results

Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo.

Conclusions

These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.

Keywords:
EZH2; Histone methyltransferase; rhabdomyosarcoma; Polycomb proteins; Differentiation; DZnep; EZH2 catalytic inhibitors