Unraveling the role of polycomb group protein EZH2 in PAX3-FOXO1 alveolar rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. It is characterized by the expression of skeletal muscle markers but without any sign of differentiation. There are two RMS main histotypes: embryonal and alveolar. The latter, characterized in 80% of cases by the expression of the oncogenic fusion protein PAX3-FOXO1 (P3F), has a greater predisposition to metastasize and shows a dismal prognosis. Restoring the differentiation of RMS cells towards a skeletal muscle phenotype is an accepted strategy to facilitate the reversion of transformed phenotype and has a strong therapeutic impact. Enhancer of Zeste Homolog 2 (EZH2) is a histone methyltransferase working as the catalytic subunit of Polycomb Repressor Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3). This type of histone methylation is a hallmark of PRC2-mediated gene repression and plays a major role during embryo development and tissue differentiation. Accordingly, EZH2 is capable to inhibit myogenesis by repressing the expression of differentiation genes. EZH2 expression is deregulated in many types of human cancers and is correlated to prognosis. Recent studies show that transcripts of EZH2 are abnormally expressed in cell lines and primary tumors of RMS, irrespective of the fusion status. Moreover, in agreement with its role in muscle development, silencing of EZH2 in an embryonal RMS cell line grown in a pro-differentiating medium promotes cell cycle arrest and formation of muscle-like structures. However, the role of EZH2 in P3F-bearing alveolar RMS remained unknown. The aim of this study is to understand the molecular mechanisms involved in EZH2 deregulation in alveolar RMS expressing P3F. Surprisingly, EZH2 depletion by gene silencing or by chemical inhibition in two alveolar P3F RMS cell lines, RH30 and RH4, induced programmed cell death in vitro and prevented tumor growth in vivo. In this context, we demonstrate the derepression of FBXO32 (Atrogin1/ MAFbx), a tumor suppressor gene coding for an ubiquitin ligase involved in muscle homeostasis, as the key factor following the depletion of EZH2. Simultaneous knockdown of FBXO32 and EZH2 in P3F RMS cells impaired the pro-apoptotic response, whereas overexpression of FBXO32 facilitated apoptosis in EZH2-depleted cells. These results were fenocopied using DZnep, which promotes EZH2 protein degradation, or a new class of catalytic inhibitors of EZH2, MC1945. Collectively, these results suggest that EZH2 plays a key role in RMS cell proliferation and survival and its depletion could be considered an adjuvant strategy in the treatment of this tumor.