EZH2/EZH1 downregulation impacts Rhabdomyosarcoma differentiation

Inglese

Rhabdomyosarcoma (RMS) is a childhood tumour that develops from muscle cell precursors. Skeletal muscle development is a complex of highly specialized and organized processes that is not yet fully understood. Embryo development, preservation of stemness, and advancement of differentiation are all regulated by epigenetic processes. To control muscle development, stemness signals are silenced by Polycomb group proteins. We focused our attention on histone hypermethylation. Enhancer of Zeste Homolog 2 (EZH2) and Enhancer of Zeste Homolog 1 (EZH1) are the catalytic subunits of Polycomb Repressive Complex 2 (PRC2). EZH2 is able to trimethylate lysine 27 of histone H3 (H3K27me3), which is highly expressed in RMS, whereas the role of EZH1 is not yet fully known. We evaluated the effects of EZH2 downregulation in RMS knock-down lines. The results showed that downregulation of EZH2 leads to an increase in myogenic regulatory factors (MRFs), resulting in partial restoration of the muscle phenotype. Furthermore, the comparison of DZNep (a molecule inhibiting EZH2 expression) and GSK126 (a molecule inhibiting EZH2 catalytic activity) in RD lines showed that the improved recovery of the muscle phenotype was associated more with the downregulation of EZH2 and not with the reduction of H3K27me3 levels alone, indicating a possible binding of EZH1 in PRC2 in favour of myoblastic differentiation. Co-IP experiments showed that there is competition between EZH2 and EZH1 as catalytic subunits of PRC2; when EZH2 is depleted, EZH1-associated PRC2 levels increase. Therefore, in order to understand the role of EZH1 in RMS, we evaluated the effects of downregulation of EZH1/EZH2 simultaneously and the results showed a reduction in the expression of myogenic markers compared to downregulation of EZH2 alone where levels were elevated. In conclusion, EZH1 plays an active and pro-differentiating role, therefore, increasing EZH1 levels together with decreasing EZH2 levels could be an important new factor for the treatment of RMS.