The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx)

• Published in: Oncogene Vol. 33; no. 32; pp. 4173 - 4184
• Main Authors: Ciarapica, R, De Salvo, M, Carcarino, E, Bracaglia, G, Adesso, L, Leoncini, P P, Dall'Agnese, A, Walters, Z S, Verginelli, F, De Sio, L, Boldrini, R, Inserra, A, Bisogno, G, Rosolen, A, Alaggio, R, Ferrari, A, Collini, P, Locatelli, M, Stifani, S, Screpanti, I, Rutella, S, Yu, Q, Marquez, V E, Shipley, J, Valente, S, Mai, A, Miele, L, Puri, P L, Locatelli, F, Palacios, D, Rota, R
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 07-08-2014; Nature Publishing Group
• Subjects: 631/67/1798; 631/67/2332; 631/80/86; Adolescent; Apoptosis; Cancer; Cell Biology; Cell Differentiation; Cell Line, Tumor; Cell Nucleus - metabolism; Cell Proliferation; Cell Survival; Child; Enhancer of Zeste Homolog 2 Protein; Female; Forkhead Box Protein O1; Forkhead Transcription Factors - metabolism; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Methyltransferases; Histone-Lysine N-Methyltransferase - metabolism; Homeostasis; Human Genetics; Humans; Internal Medicine; Male; Medicine; Medicine & Public Health; Muscle Proteins - antagonists & inhibitors; Muscle Proteins - physiology; Oncology; original-article; Paired Box Transcription Factors - metabolism; PAX3 Transcription Factor; Polycomb Repressive Complex 2 - physiology; Proteins; Rhabdomyosarcoma, Alveolar - metabolism; SKP Cullin F-Box Protein Ligases - antagonists & inhibitors; SKP Cullin F-Box Protein Ligases - physiology; Stem cells; PAX3-FOXO1; histone methyltransferases; FBXO32; rhabdomyosarcoma; Polycomb proteins; EZH2
• ISSN: 0950-9232; 1476-5594; 1476-5594
• DOI: 10.1038/onc.2013.471

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.