Abstract
Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage Myf6Cre,Pax3:Foxo1,p53 mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor Tp53 (Myf6Cre,Pax3:Foxo1,p53), exhibit marked heterogeneity in PAX3:FOXO1 (P3F) expression at the single cell level. In mouse RMS cells, P3F expression is directed by the Pax3 promoter and coupled to eYFP YFPlow/P3Flow 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 YFPlow/P3Flow compared with YFPhigh/P3Fhigh cells. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Heterogeneous expression of PAX3:FOXO1 at the single cell level may provide a critical advantage during tumor progression.
Highlights
Rhabdomyosarcoma, the most common soft tissue sarcoma in children and adolescents, comprises two main genotypes defined by the presence or absence of PAX gene rearrangements [1, 2]
In mouse U23674 and U21459 cells, expression of PAX3: FOXO1 (P3F) is directed by the Pax3 promoter and coupled to an eYFP fluorescent marker, which is activated as a second cistron downstream from an encephalomyocarditis virus–derived internal ribosome entry site (IRES) [17]
To explore whether similar cell-tocell variability of P3F transcript levels may occur in human RMS, single-cell digital droplet PCR was performed to quantify the absolute number of P3F and GAPDH mRNA molecules per single cell in three human RMS patient–derived primary cell cultures (IC-pPDX35, RMSZH003, and SJRHB013759_X1) and in two human cell lines (Rh41 and Rh30) (Figs 1A and B and S1B and Table S1)
Summary
Rhabdomyosarcoma, the most common soft tissue sarcoma in children and adolescents, comprises two main genotypes defined by the presence or absence of PAX gene rearrangements [1, 2]. Canonical PAX translocations juxtapose the N-terminus of the paired-box genes PAX3 or PAX7 with the C terminus of the transcription factor FOXO1 [3]. PAX3: FOXO1 (P3F) has been detected in 55% and PAX7:FOXO1 (P7F) in 22% of alveolar histology RMS tumors [4]. Both PAX gene fusions act as major oncogenic drivers. P3F knockdown in human and mouse RMS cell lines was linked to a decrease in proliferation rates [6, 7]. Poor survival rates call for a deeper understanding of the biology of P3F+ RMS [2]
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