title: Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS.
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.
authors.affiliation: Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S, Onofrio 4, 00165 Rome, Italy. roberta.ciarapica@yahoo.com.
authors.forename: Roberta
authors.initials: R
authors.lastname: Ciarapica
authors.forename: Elena
authors.initials: E
authors.lastname: Carcarino
authors.forename: Laura
authors.initials: L
authors.lastname: Adesso
authors.forename: Maria
authors.initials: M
authors.lastname: De Salvo
authors.forename: Giorgia
authors.initials: G
authors.lastname: Bracaglia
authors.forename: Pier Paolo
authors.initials: PP
authors.lastname: Leoncini
authors.forename: Alessandra
authors.initials: A
authors.lastname: Dall'agnese
authors.forename: Federica
authors.initials: F
authors.lastname: Verginelli
authors.forename: Giuseppe M
authors.initials: GM
authors.lastname: Milano
authors.forename: Renata
authors.initials: R
authors.lastname: Boldrini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Inserra
authors.forename: Stefano
authors.initials: S
authors.lastname: Stifani
authors.forename: Isabella
authors.initials: I
authors.lastname: Screpanti
authors.forename: Victor E
authors.initials: VE
authors.lastname: Marquez
authors.forename: Sergio
authors.initials: S
authors.lastname: Valente
authors.forename: Antonello
authors.initials: A
authors.lastname: Mai
authors.forename: Pier Lorenzo
authors.initials: PL
authors.lastname: Puri
authors.forename: Franco
authors.initials: F
authors.lastname: Locatelli
authors.forename: Daniela
authors.initials: D
authors.lastname: Palacios
authors.forename: Rossella
authors.initials: R
authors.lastname: Rota
mesh_terms: D000293:Adolescent; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002648:Child; D002675:Child, Preschool; D004195:Disease Models, Animal; D000071221:Enhancer of Zeste Homolog 2 Protein; D005260:Female; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D009362:Neoplasm Metastasis; D009367:Neoplasm Staging; D063151:Polycomb Repressive Complex 2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018233:Rhabdomyosarcoma, Embryonal / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D047368:Tumor Burden; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2