PAX-FKHR function as pangenes by simultaneously inducing and inhibiting myogenesis

Abstract

Alveolar rhabdomyosarcomas (ARMS) escape terminal differentiation despite exhibiting a skeletal muscle phenotype. To understand the role of the ARMS-specific PAX-FKHR proteins in myogenesis, we characterized their regulation of MyoD expression and function. Reporter assays show that PAX-FKHR transactivate MyoD expression through its 258 bp core enhancer. Gel-shift assays confirm that PAX-FKHR bind to core enhancer sequences showing similarity to consensus PAX3/PAX-FKHRbinding sites. We show that while PAX3-FKHR activates the expression of endogenous MyoD and myogenin proteins in transduced NIH3T3 fibroblasts, it inhibits them from terminally differentiating as shown by low myogenin and myosin heavy chain expression, and lack of myotube formation. Attenuation of MyoD transcriptional activity via phosphorylation coupled to the lack of cell cycle arrest is the underlying mechanism for the differentiation block. Lastly, we show that fibroblast growth factor receptor signaling likely mediates the inhibition of differentiation by PAX3-FKHR. In a single experimental system we demonstrate that PAX3-FKHR can simultaneously induce myogenesis while preventing its completion. We propose a model whereby PAX-FKHR commit a yet undefined precursor cell to the myogenic lineage while at the same time inhibit terminal differentiation, thereby contributing to ARMS formation.

A Chimeric RNA Characteristic of Rhabdomyosarcoma in Normal Myogenesis Process

“…On the other hand, the transience of PAX3-FOXO1 RNA is consistent with the normal process of muscle differentiation, as prolonged production of PAX3-FOXO1 RNA leads to constant expression of MYOD and MYOG , which then blocks terminal differentiation ( 13,14 ). We reasoned that a posttranscriptional process, such as trans -splicing of precursor mRNAs, can give rise to transient, temporal appearance of the fusion product ( 16,17 ), whereas gene fusions caused by chromosomal translocation result in constitutive synthesis of chimeric PAX3-FOXO1 RNA, and in turn MYOD and MYOG expression that permanently arrests myocytes at immature forms, which can potentially lead to ARMS if combined with secondary mutations.…”

“…Studies in fi broblast and rhabdomyosarcoma cell lines have demonstrated that the PAX3-FOXO1 protein simultaneously induces myogenesis while blocking differentiation to mature muscle, thereby contributing to the formation of ARMS ( 13,14 ). To test the effect of constant expression of PAX3-FOXO1 in the muscle differentiation setting, MSC cells were infected with retroviral vectors that overexpress PAX3-FOXO1 ( Fig.…”

AKT and PAX3-FKHR cooperation enforces myogenic differentiation blockade in alveolar rhabdomyosarcoma cell

“…A typical feature of ARMS also includes the myogenic phenotype with expression of MyoD, 14 a bona fide target of PAX3-FKHR, 13,15 which acts as a key regulator of myogenic gene expression, leading to terminal differentiation. 16 However, they fail to complete the terminal myogenic differentiation program.…”

Uncovering metabolism in rhabdomyosarcoma

“…Pax3-Foxo1 orchestrates the expression of numerous Pax3 downstream genes with increased amplitude and without feedback control, impairing the apoptosis and differentiation processes [49][50][51][52][53] and conferring a G2 checkpoint-dependent resistance to irradiation in vitro and in vivo. 54 For example, the Pax3-Foxo1-dependent transcription of genes like hepatocyte growth factor receptor (HGFR or c-MET), FGFR4, IGF-2 and C-X-C chemokine receptor type 4 (CXCR4) contributes to increase tumor aggressiveness and metastasis recurrence.…”