Investigating the Failure of Myogenesis in the Pediatric Tumor Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is a pediatric tumor of skeletal muscle that fails to undergo terminal differentiation, even though it expresses the myogenic regulatory factor MyoD, which should be sufficient for that process. We have previously provided evidence for multiple inhibitory transcription factors in the tumors acting to oppose the activity of MyoD. Even so, we have found that it is possible to restore MyoD activity by forcing it to interact with one of its protein dimer partners, which leads to differentiation of the cells, and downregulation of the inhibitors. This work now demonstrates that differentiation of RMS can be achieved by expressing other transcription factors that play positive roles in myogenesis, RUNX1 and RP58, and that all these mechanisms of differentiation result in the increase of a single microRNA, miR-206, that is itself sufficient to differentiate RMS. One of the inhibitory factors we previously found as opposing MyoD, MSC, acts at the promoter of miR-206 to interfere with the MyoD activity necessary for the microRNA's expression. Other analyses of expression and gene regulation suggest the existence of an epistatic relationship between MyoD, RUNX1, RP58, and miR-206, with MyoD positively regulating all the other targets, RUNX1 assisting with the activation of RP58 and miR-206, and miR-206 the target of all the other factors. Genome-wide analysis of DNA binding by MyoD and MSC demonstrates that both factors bind throughout the genome of RMS, with both distinct and overlapping binding. Comparison of MyoD binding in RMS to that of MyoD in primary human cells reveals differences in the binding sites for possible cooperative factors, including RUNX1, but an overall similarity in the MyoD binding between RMS and human myotubes. Taken as a whole, the data suggests that RMS represent an arrested state of development balanced between myoblast and myotube, and that manipulation of components of the myogenic gene program can `tip the balance' and restore their ability to differentiate.