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Abstract
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS can be parsed based on clinical outcome into two subtypes, fusion-positive RMS (FP-RMS) or fusion-negative RMS (FN-RMS) based on the presence or absence of either PAX3-FOXO1 or PAX7-FOXO1 gene fusions. In both RMS subtypes, tumor cells show histology and a gene expression pattern resembling that of developmentally arrested skeletal muscle. Differentiation therapy is an attractive approach to embryonal tumors of childhood including RMS; however, agents to drive RMS differentiation have not entered the clinic and their mechanisms remain unclear. MicroRNA-206 (miR-206) expression increases through normal muscle development and has decreased levels in RMS compared with normal skeletal muscle. Increasing miR-206 expression drives differentiation of RMS, but the target genes responsible for the relief of the development arrest are largely unknown. Using a combinatorial approach with gene and proteomic profiling coupled with genetic rescue, we identified key miR-206 targets responsible for the FN-RMS differentiation blockade, PAX7, PAX3, NOTCH3, and CCND2. Specifically, we determined that PAX7 downregulation is necessary for miR-206-induced cell cycle exit and myogenic differentiation in FN-RMS but not in FP-RMS. Gene knockdown of targets necessary for miR-206-induced differentiation alone or in combination was not sufficient to phenocopy the differentiation phenotype from miR-206, thus illustrating that miR-206 replacement offers the ability to modulate a complex network of genes responsible for the developmental arrest in FN-RMS. Genetic deletion of miR-206 in a mouse model of FN-RMS accelerated and exacerbated tumor development, indicating that both in vitro and in vivo miR-206 acts as a tumor suppressor in FN-RMS at least partially through downregulation of PAX7. Collectively, our results illustrate that miR-206 relieves the differentiation arrest in FN-RMS and suggests that miR-206 replacement could be a potential therapeutic differentiation strategy.
Highlights
Because of the resemblance to developing skeletal muscle, RMS is often viewed through the prism of normal muscle
These changes were accomplished with a physiologic increase in miR-206 levels in cells maintained under high serum growth conditions still below the expression level of miR-206 in differentiated myotubes (Figure 1c and Supplementary Figure S1a)
G6PD24 did have a subtle but significant decrease in CKM expression but no detectable difference in myosin heavy chain (MHC) expression by ICC (Supplementary Figures S5a–d). These results suggest that downregulation of PAX7, PAX3, CCND2 and NOTCH3 is necessary for the miR-206induced differentiation of fusionnegative RMS (FN-RMS) cells
Summary
Because of the resemblance to developing skeletal muscle, RMS is often viewed through the prism of normal muscle. While miR-1 is expressed more abundantly in cardiac muscle, miR-206 is expressed nearly exclusively in mature skeletal muscle with increasing expression during myogenesis driven by MyoD1 and Myogenin.[14,15,16] Genetic deletion of miR-206 in mice has revealed a role of miR-206 in the regeneration of the neuromuscular synapsis and skeletal muscle regeneration following injury.[17,18,19] In both FN- and FP-RMS, decreased miR-206 expression has been demonstrated in patient tumors compared with normal skeletal muscle.[20,21] Higher miR-206 expression correlated to increased patient survival in FN-RMS but not in FP-RMS.[21] To gain insight into the biological relevance of miR-206 in RMS, several groups overexpressed miR-206 in RMS cell lines and illustrated decreased proliferation and migration as well as an induction of differentiation.[21,22,23,24] viral expression of miR-206 in RMS cell line xenografts in mice decreased tumor growth.[22,25] This recent work has highlighted a few exciting targets of miR-206 in RMS;[26] the necessity and/or sufficiency of these putative miR-206 target genes in mediating RMS differentiation remained unexplored. Using a combinatorial approach with microarrays, large-scale proteomics, and prediction algorithms, we identified the crucial miR-206 targets responsible for the developmental arrest in RMS
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