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

Roberta Ciarapica, Elena Carcarino, Laura Adesso, Maria De Salvo, Giorgia Bracaglia, Pier P. Leoncini, Alessandra Dall'Agnese, Federica Verginelli, Giuseppe M. Milano, Renata Boldrini, Alessandro Inserra, Stefano Stifani, Isabella Screpanti, Victor E. Marquez, Sergio Valente, Antonello Mai, Pier L. Puri, Franco Locatelli, Daniela Palacios, Rossella Rota

Research output: Contribution to journalArticle

28 Citations (Scopus)

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.

Original languageEnglish
Article number139
JournalBMC Cancer
Volume14
Issue number1
DOIs
Publication statusPublished - Feb 27 2014

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Embryonal Rhabdomyosarcoma
Pharmacology
Cell Proliferation
Tumor Cell Line
Therapeutics
Adenosylhomocysteinase
Myogenin
Recurrence
Neoplasms
Rhabdomyosarcoma
Heterografts
Sarcoma
Cultured Cells
Intercellular Signaling Peptides and Proteins
Skeletal Muscle
Up-Regulation
Down-Regulation
Pediatrics
Cell Line
Genes

Keywords

  • Differentiation
  • DZnep
  • EZH2
  • EZH2 catalytic inhibitors
  • Histone methyltransferase
  • Polycomb proteins
  • rhabdomyosarcoma

ASJC Scopus subject areas

  • Oncology
  • Cancer Research
  • Genetics
  • Medicine(all)

Cite this

Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS. / Ciarapica, Roberta; Carcarino, Elena; Adesso, Laura; De Salvo, Maria; Bracaglia, Giorgia; Leoncini, Pier P.; Dall'Agnese, Alessandra; Verginelli, Federica; Milano, Giuseppe M.; Boldrini, Renata; Inserra, Alessandro; Stifani, Stefano; Screpanti, Isabella; Marquez, Victor E.; Valente, Sergio; Mai, Antonello; Puri, Pier L.; Locatelli, Franco; Palacios, Daniela; Rota, Rossella.

In: BMC Cancer, Vol. 14, No. 1, 139, 27.02.2014.

Research output: Contribution to journalArticle

Ciarapica, R, Carcarino, E, Adesso, L, De Salvo, M, Bracaglia, G, Leoncini, PP, Dall'Agnese, A, Verginelli, F, Milano, GM, Boldrini, R, Inserra, A, Stifani, S, Screpanti, I, Marquez, VE, Valente, S, Mai, A, Puri, PL, Locatelli, F, Palacios, D & Rota, R 2014, 'Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS', BMC Cancer, vol. 14, no. 1, 139. https://doi.org/10.1186/1471-2407-14-139
Ciarapica, Roberta ; Carcarino, Elena ; Adesso, Laura ; De Salvo, Maria ; Bracaglia, Giorgia ; Leoncini, Pier P. ; Dall'Agnese, Alessandra ; Verginelli, Federica ; Milano, Giuseppe M. ; Boldrini, Renata ; Inserra, Alessandro ; Stifani, Stefano ; Screpanti, Isabella ; Marquez, Victor E. ; Valente, Sergio ; Mai, Antonello ; Puri, Pier L. ; Locatelli, Franco ; Palacios, Daniela ; Rota, Rossella. / Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS. In: BMC Cancer. 2014 ; Vol. 14, No. 1.
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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.",
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author = "Roberta Ciarapica and Elena Carcarino and Laura Adesso and {De Salvo}, Maria and Giorgia Bracaglia and Leoncini, {Pier P.} and Alessandra Dall'Agnese and Federica Verginelli and Milano, {Giuseppe M.} and Renata Boldrini and Alessandro Inserra and Stefano Stifani and Isabella Screpanti and Marquez, {Victor E.} and Sergio Valente and Antonello Mai and Puri, {Pier L.} and Franco Locatelli and Daniela Palacios and Rossella Rota",
year = "2014",
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T1 - Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS

AU - Ciarapica, Roberta

AU - Carcarino, Elena

AU - Adesso, Laura

AU - De Salvo, Maria

AU - Bracaglia, Giorgia

AU - Leoncini, Pier P.

AU - Dall'Agnese, Alessandra

AU - Verginelli, Federica

AU - Milano, Giuseppe M.

AU - Boldrini, Renata

AU - Inserra, Alessandro

AU - Stifani, Stefano

AU - Screpanti, Isabella

AU - Marquez, Victor E.

AU - Valente, Sergio

AU - Mai, Antonello

AU - Puri, Pier L.

AU - Locatelli, Franco

AU - Palacios, Daniela

AU - Rota, Rossella

PY - 2014/2/27

Y1 - 2014/2/27

N2 - 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.

AB - 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.

KW - Differentiation

KW - DZnep

KW - EZH2

KW - EZH2 catalytic inhibitors

KW - Histone methyltransferase

KW - Polycomb proteins

KW - rhabdomyosarcoma

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