JARID2 is a direct target of the PAX3-FOXO1 fusion protein and inhibits myogenic differentiation of rhabdomyosarcoma cells

Abstract

Rhabdomyosarcomas (RMS) are the most frequent soft-tissue sarcoma in children and characteristically show features of developing skeletal muscle. The alveolar subtype is frequently associated with a PAX3-FOXO1 fusion protein that is known to contribute to the undifferentiated myogenic phenotype of RMS cells. Histone methylation of lysine residues controls developmental processes in both normal and malignant cell contexts. Here we show that JARID2, that encodes a protein known to recruit various complexes with histone methylating activity to their target genes, is significantly overexpressed in RMS with PAX3-FOXO1 compared to fusion gene negative RMS (t test p<0.0001). Multivariate analyses showed higher JARID2 levels are also associated with metastases at diagnosis, independent of fusion gene status and RMS subtype (n= 120; p=0.039). JARID2 levels were altered by silencing or over-expressing PAX3-FOXO1 in RMS cell lines with and without the fusion gene, respectively. Consistent with this, we demonstrated that JARID2 is a direct transcriptional target of the PAX3-FOXO1 fusion protein. Silencing JARID2 resulted in reduced cell proliferation coupled with myogenic differentiation including increased expression of MYOGENIN (MYOG) and MYOSIN LIGHT CHAIN (MYL1) in RMS cell lines representative of both the alveolar and embryonal subtypes. Induced myogenic differentiation was associated with a decrease in JARID2 levels and this phenotype could be rescued by overexpressing JARID2. Furthermore, we that showed JARID2 binds to and alters the methylation status of histone H3 lysine 27 in the promoter regions of MYOG and MYL1 and that the interaction of JARID2 at these promoters is dependent upon EED, a core component of the Polycomb Repressive Complex 2 (PRC2). Therefore JARID2 is a downstream effector of PAX3-FOXO1 that maintains an undifferentiated myogenic phenotype that is characteristic of RMS. JARID2 and other components of PRC2 may represent novel therapeutic targets for treating RMS patients.

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

“…37,38 Moreover, gene expression profiling data for 101 RMS previously described patient samples in the ITCC/CIT data set (Innovative Therapies for Children with Cancer/Carte d'Identité des Tumeurs) 1 within the R2 online platform 39 relative to publicly available Affymetrix expression profiling data for skeletal muscle samples (GSE9103), indicated that FBXO32 expression is much lower in RMS patient samples relative to skeletal muscle (Figure 5b), which is in direct contrast to EZH2 levels in RMS patient samples where EZH2 is overexpressed, as previously reported by our group. 30 Finally, the analysis of the RMS patient sample set also showed a significant negative correlation between FBXO32 and EZH2 expression in RMS patient samples (R ¼ À 0.5, P ¼ 1.0e À 07), once again indicating an inverse relationship between the levels of these two proteins that supports their functional interactions ( Figure 5c). …”

“…EZH2 protein levels are elevated in human PAX3-FOXO1 RMS primary samples and cell lines We first evaluated whether, as we recently reported for EZH2 transcripts, 30,31 EZH2 protein levels were higher in PAX3-FOXO1 RMS tumor samples than in normal muscle tissue. EZH2 was studied using immunohistochemical analysis on 10 primary samples from PAX3-FOXO1-bearing alveolar RMS patients (Supplementary Table 1).…”

“…[24][25][26][27][28][29] Collectively, these findings demonstrate a role of EZH2 as a potential prognostic marker and therapeutic target in cancer. We and others have recently reported that (i) as compared with the normal myoblasts and muscle tissues EZH2 is markedly overexpressed in RMS 30,31 and (ii) EZH2 downregulation in vitro leads to myogenic-like differentiation of an embryonal RMS cell line. 32 However, the role of EZH2 in the aggressive PAX3-FOXO1 alveolar RMS cells has not been evaluated, and the effect of EZH2 inhibition on the tumorigenic phenotype remains to be tested.…”

“…30,31 Moreover, EZH2 knockdown has been shown to induce in vitro differentiation of cells of the embryonal RMS subtype. 32 However, the effects of EZH2 downregulation in PAX3-FOXO1 alveolar RMS, clinically and molecularly distinct from the embryonal subtype 1,46-48 and considered as the highest-risk form of RMS, 2 remained unknown.…”

Rhabdomyosarcoma: Current Challenges and Their Implications for Developing Therapies

“…Hope for future anti-RMS therapies may rest in large part on interventions that recruit antitumor immune mechanisms, induce myogenic differentiation, or interfere with specific cellular and molecular mechanisms that drive the malignant behavior of RMS cells (Ciarapica et al 2013;Highfill et al 2014;Walters et al 2014). Unfortunately, the preclinical anti-RMS activity of targeted agents such as mTOR inhibitors (Hosoi et al 1999) and neutralizing IGF1 receptor antibodies (Mayeenuddin et al 2010) have not translated thus far into durable objective response rates in early-phase clinical trials (Geoerger et al 2012;Pappo et al 2014;Weigel et al 2014).…”