Effects of PAX3-FKHR on malignant phenotypes in alveolar rhabdomyosarcoma

doi:10.1016/j.bbrc.2007.11.017

Biochemical and Biophysical Research Communications

Volume 365, Issue 3, 18 January 2008, Pages 568-574

https://doi.org/10.1016/j.bbrc.2007.11.017 Get rights and content

Abstract

The malignancy of alveolar rhabdomyosarcoma (ARMS) has been linked to expression of the PAX3-FKHR chimeric gene. To understand the effect of this gene, we used RNAi to knock down its expression (without affecting the expressions of either PAX3 or FKHR) in human ARMS cell lines. Down-regulating PAX3-FKHR caused (a) tumor cells to accumulate in the G1 phase, inhibiting the rate of cellular proliferation, (b) a reduction in the levels of the MET, reducing cell motility stimulated by HGF, and (c) induction of the myogenic differentiation gene, myogenin, and muscle differentiation (morphologic change and the expression of muscle specific proteins, desmin, and myosin heavy chain). These results suggest that PAX3-FKHR in ARMS cells promotes malignant phenotypes such as proliferation, motility, and to suppress differentiation.

Section snippets

Materials and methods

Cell culture and reagents. Human ARMS cell lines, SJ-Rh4 (Rh4), SJ-Rh30 (Rh30), and SJ-Rh41 (Rh41) were used. Cells were maintained in RPMI 1640 containing 10% fetal bovine serum at 37 °C in a 5% CO₂ incubator. We used the panel of 10 antibodies in Supplement Table 1. Human hepatocyte growth factor (HGF) was purchased from PeproTech.

Sequencing of the PAX3-FKHR mRNA. RNA extraction was performed with the RNeasy Mini Kit (Qiagen). Total RNA was transcribed into cDNA using SuperScript First-Strand

Efficiency and specificity of PAX3-FKHR siRNA

The sequences on both sides of the fusion point were found to be identical in each of the three ARMS cell lines. We selected the chimeric PAX3-FKHR mRNA as a target and designed three siRNAs that spanned the fusion point. We conducted BLAST searches of the human genome with this sequence. Aside from the 19-base matches with the fusion transcript, no human sequence matched more than 16 bases (Fig. 1A).

To identify highly efficient PAX3-FKHR siRNAs we performed an siRNA scan of the PAX3-FKHR

Discussion

Clinically, ARMS tumors have a more aggressive behavior than embryonal RMS, probably because of the presence of PAX3-FKHR fusion protein in the alveolar subtype. Ectopic expression of PAX3-FKHR in embryonal RMS cells was shown to increase cell proliferation [8], while ectopic expression of PAX3-FKHR, but not PAX3, in fibroblasts was found to accelerate the G0/G1 to S cell cycle transition [13]. However, these results were obtained from experiments using the method of ectopic expression, and

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^☆: This study was supported in part by the fund of Children’s Cancer Association of Japan in 2006: #37, and a Grant-in-Aid (17-13) in Cancer Research, Ministry of Health, Labor and Welfare of Japan.

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Effects of PAX3-FKHR on malignant phenotypes in alveolar rhabdomyosarcoma☆

Abstract

Section snippets

Materials and methods

Efficiency and specificity of PAX3-FKHR siRNA

Discussion

J. Biol. Chem.

Cell

Am. J. Pathol.

Methods

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Am. J. Pathol.

Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma

Nat. Genet.

PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children’s oncology group

J. Clin. Oncol.

The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3

Mol. Cell. Biol.

The hybrid PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma transforms fibroblasts in culture

Proc. Natl. Acad. Sci. USA