title: The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).
abstract: The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Ciarapica
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: M
authors.initials: M
authors.lastname: De Salvo
authors.affiliation: IRCCS Fondazione Santa Lucia, Roma, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Carcarino
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Bracaglia
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Adesso
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: P P
authors.initials: PP
authors.lastname: Leoncini
authors.affiliation: IRCCS Fondazione Santa Lucia, Roma, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Dall'Agnese
authors.affiliation: Sarcoma Molecular Pathology, Divisions of Molecular Pathology and Cancer Therapeutics, The Institute of Cancer Research, Sutton, UK.
authors.forename: Z S
authors.initials: ZS
authors.lastname: Walters
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Verginelli
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: L
authors.initials: L
authors.lastname: De Sio
authors.affiliation: Department of Pathology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Boldrini
authors.affiliation: Department of Surgery, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Inserra
authors.affiliation: Department of Pediatrics, Oncohematology Unit, University of Padova, Padova, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Bisogno
authors.affiliation: Department of Pediatrics, Oncohematology Unit, University of Padova, Padova, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Rosolen
authors.affiliation: Medicine DIMED, Pathology Unit, University of Padova, Padova, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Alaggio
authors.affiliation: Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Ferrari
authors.affiliation: Anatomic Pathology Unit 2, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Collini
authors.affiliation: Scientific Directorate, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Locatelli
authors.affiliation: Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Stifani
authors.affiliation: Department of Molecular Medicine, Sapienza University, Roma, Italy.
authors.forename: I
authors.initials: I
authors.lastname: Screpanti
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Rutella
authors.affiliation: Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore.
authors.forename: Q
authors.initials: Q
authors.lastname: Yu
authors.affiliation: Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, CCR, National Cancer Institute, NIH, Frederick, MD, USA.
authors.forename: V E
authors.initials: VE
authors.lastname: Marquez
authors.affiliation: Sarcoma Molecular Pathology, Divisions of Molecular Pathology and Cancer Therapeutics, The Institute of Cancer Research, Sutton, UK.
authors.forename: J
authors.initials: J
authors.lastname: Shipley
authors.affiliation: Istituto Pasteur, Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University, Roma, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Valente
authors.affiliation: Istituto Pasteur, Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University, Roma, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Mai
authors.affiliation: Cancer Institute, University of Mississippi Medical Center, Jackson, MS, USA.
authors.forename: L
authors.initials: L
authors.lastname: Miele
authors.affiliation: 1] IRCCS Fondazione Santa Lucia, Roma, Italy [2] Muscle Development and Regeneration Program, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: P L
authors.initials: PL
authors.lastname: Puri
authors.affiliation: 1] Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy [2] Dipartimento di Scienze Pediatriche, Università di Pavia, Pavia, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Locatelli
authors.affiliation: IRCCS Fondazione Santa Lucia, Roma, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Palacios
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Rota
mesh_terms: D000293:Adolescent; D017209:Apoptosis; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D002648:Child; D000071221:Enhancer of Zeste Homolog 2 Protein; D005260:Female; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D008297:Male; D009124:Muscle Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism*; D063151:Polycomb Repressive Complex 2 / Q000502:physiology*; D018232:Rhabdomyosarcoma, Alveolar / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases / Q000037:antagonists & inhibitors* / Q000502:physiology
chemical_list: C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D009124:Muscle Proteins; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051761:Paired Box Transcription Factors; D000076983:Histone Methyltransferases; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D011495:Histone-Lysine N-Methyltransferase; D063151:Polycomb Repressive Complex 2; C440907:FBXO32 protein, human; D044843:SKP Cullin F-Box Protein Ligases