MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line.

Fiorella Faggi,Federica Bono,Chiara Fiorentini,Silvia Codenotti,Alessandra Zanola,Nicola Chiarelli,Charles Keller,Manuela Cominelli,Harriet P Lo,Marika Vezzoli,Alessandro Fanzani,Pietro Luigi Poliani,Robert G Parton,Marina Colombi,Giovanni Tulipano,Eugenio Monti

doi:10.1371/journal.pone.0130287

Abstract

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS.

Highlights

Rhabdomyosarcoma (RMS) is a myogenic tumor classified as the most common soft-tissue malignancy of childhood [1,2,3]
In this work we have investigated whether Muscle-Restricted Coiled-coil (MURC)/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Cav-3 in heart and muscle tissues [51, 52], may be expressed and play a role in RMS
By evaluating the variability in the expression levels of MURC/CAVIN-4, CAV-3 and myosin heavy chain (MHC), as detected in aRMS (n = 11) and eRMS (n = 7) tumors and skeletal muscle samples (n = 3) (Fig 1A), we found that the medians of CAV-3 and MHC in three groups were statistically different with a p-value of 0.0398 and 0.0222, respectively, while the levels of MURC/CAVIN-4 in tumor samples did not significantly differ from those observed in skeletal muscle (p-value = 0.1013) (Fig 1B)

Summary

Introduction

Rhabdomyosarcoma (RMS) is a myogenic tumor classified as the most common soft-tissue malignancy of childhood [1,2,3]. ARMS is dominated by a t(2;13)(q35; q14) chromosomal translocation that juxtaposes the DNA binding domains of the PAX3 (paired box 3) gene in frame with the partial DNA binding domain and full transactivation domain of the FOXO1 (forkhead box O1) gene, resulting in the expression of the fused Pax3-Foxo transcription factor [30]. This factor drives transcription of numerous Pax-3 downstream genes in a deliberate manner, contributing to suppress apoptosis and differentiation processes [31, 32] and conferring resistance to stress conditions such as irradiation in vitro and in vivo [33]. The presence of a PAX3–FOXO1 gene fusion is a strong indicator of poor prognosis as fusion-negative aRMS have better resolution mimicking the clinical course of eRMS in the majority of patients [34, 35]

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Conclusion