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.Oncogene ( Keywords: EZH2; FBXO32; histone methyltransferases; PAX3-FOXO1; rhabdomyosarcoma; Polycomb proteins INTRODUCTION Rhabdomyosarcomas (RMS) are heterogeneous highly malignant tumors, which account for 7-8% of all pediatric malignancies and over half of the soft-tissue sarcomas in children. RMS are classically subdivided in two major histotypes: embryonal (around 70-80%) and alveolar (around 20-30%), the latter often metastatic at diagnosis and showing a high risk of recurrence. 1 In 70% of cases, alveolar RMS is characterized by the chromosomal translocation t(2;13) or t(1;13), resulting in

Loss of H3K27 trimethylation distinguishes malignant peripheral nerve sheath tumors from histologic mimics

“…15 EZH2 has been shown to be with PAX3-FOXO1 fusion, EZH2 was also found to be overexpressed, leading to increased proliferation and survival and offering a potential target for EZH2 inhibitor therapies. 17 Both oncogenic and tumor suppressor functions have been attributed to EZH2; it is likely that a delicately balanced gene dosage of the polycomb group proteins together with biologic context are crucial for stem cell homeostasis and determine oncogenic or tumor suppressor functions. 9,11 In contrast, EED and SUZ12 thus far appear to act predominantly as tumor suppressors; [7][8][9] however, additional studies are required to clarify the role of each polycomb group protein in different cellular contexts.…”

Roles of enhancer of zeste homolog 2: From skeletal muscle differentiation to rhabdomyosarcoma carcinogenesis

“…156 Furthermore, recent studies investigated the role of EZH2 in PAX3-FOXO1 aRMS. 157 EZH2 depletion in aRMS cell lines highly affects their cell survival leading to apoptosis. The apoptotic effect of EZH2 knockdown is partially due to transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis.…”

Helicase CHD4 is an epigenetic coregulator of PAX3-FOXO1 in alveolar rhabdomyosarcoma

“…We analyzed 4 independent FLAG-affinity purifications of PAX3-FOXO1 together with controls ( Figure 1B) by MS, which confirmed the presence of PAX3-FOXO1 with a minimum coverage of 54% in each replicate experiment including PAX3-FOXO1 breakpoint-specific peptides (Supplemental Figure 1B). Considering only proteins found with a or indirect target genes downstream of PAX3-FOXO1 (29)(30)(31)(32). In contrast, epigenetic mechanisms that would orchestrate the network of aberrant transcriptional activity at the level of PAX3-FOXO1 itself are largely unknown.…”