The Role of Glycogen Synthase Kinase 3 beta In Regulating the Function of PAX3-FOXO1 by Phosphorylation

Abstract

Alveolar rhabdomyosarcoma (ARMS) is a type of rhabdomyosarcoma, which is the most common soft tissue sarcoma in pediatric patients. Nearly 70% of ARMSs express the fusion protein PAX3-FOXO1, which is linked to poor prognosis and increased tumor aggressiveness. Previous studies have shown that the glycogen synthase kinase 3 beta (GSK3β) inhibitor TWS119 can inhibit cell proliferation in alveolar rhabdomyosarcoma cells, and that GSK3β can phosphorylate PAX3-FOXO1 in vitro. However, the specific nature of these phosphorylation events and physiological relevance of these events for TWS119 activity are not known. In this study, site-directed mutagenesis is used to evaluate a putative phosphorylation site located at the junction of the PAX3 and FOXO1 domains in the fusion protein for the site’s importance in PAX3-FOXO1 functional activity. Our results show that this site can regulate PAX3-FOXO1 functional activity and strongly suggests it may be a phosphorylation site for GSK3β. These studies provide insight to the role of PAX3-FOXO1 function in ARMS cells, important since this fusion protein is indicative of a more aggressive cancer phenotype that is resistant to conventional chemotherapy and radiotherapy. Novel strategies in treating these aggressive ARMS types might include modulating the activity of the fusion protein’s target genes as well as the fusion protein’s transcriptional activity, particularly through inhibiting GSK3β.