Negative correlation of single-cell <i>PAX3:FOXO1</i> expression with tumorigenicity in rhabdomyosarcoma

Abstract

Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage <i>Myf6Cre,Pax3:Foxo1,p53</i> mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor <i>Tp53</i> (<i>Myf6Cre,Pax3:Foxo1,p53</i>), exhibit marked heterogeneity in <i>PAX3:FOXO1</i> (<i>P3F</i>) expression at the single cell level. In mouse RMS cells, <i>P3F</i> expression is directed by the <i>Pax3</i> promoter and coupled to <i>eYFP</i> YFP^low/P3F^low mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFP^low/P3F^low compared with YFP^high/P3F^high cells. Both YFP^low/P3F^low and YFP^high/P3F^high cells gave rise to mixed clones in vitro, consistent with fluctuations in <i>P3F</i> expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFP^low/P3F^low RMS cells. Heterogeneous expression of <i>PAX3:FOXO1</i> at the single cell level may provide a critical advantage during tumor progression.