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. 2010 Jan 1;391(1):1049-55.
doi: 10.1016/j.bbrc.2009.12.017. Epub 2009 Dec 6.

Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells

Fu-Yue Zeng  1 , Hanqing Dong, Jimmy Cui, Lingling Liu, Taosheng Chen
Affiliations

Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells

Fu-Yue Zeng et al. Biochem Biophys Res Commun. .

Abstract

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies.

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Figures

Fig 1
Fig 1
Identification of compounds that preferentially block the growth of Rh30. Cells were exposed to (A), 10 μM or (B), 2 μM of drugs as indicated for 72 h before determining viable cells (%). (C) Soft agar colony formation assay. Colony formed in vehicle-treated control was set as 100%. Data represent 2 independent experiments, each performed in triplicate.
Fig. 2
Fig. 2
(A) and (B) TWS119 induces differential activation of caspases 3 and 7 in Rh30 and RD cells. The activity of caspases 3 and 7 (A) was determined as described in Materials and methods. Data represent 3 independent experiments, each performed in triplicate. In the PARP cleavage assay (B), Rh30 and RD cells were exposed to TWS119 for 72 h before Western blot analysis, using anti-PARP antibody and anti β-actin antibody (as loading control). (C) TWS119 inhibits activation of GSK3. Rh30 and RD cells were exposed to DMSO or TWS119 for 24 h and analyzed by Western blot, using indicated specific antibodies. (D) and (E) PAX3-FKHR sensitizes TWS119-mediated inhibition of cell proliferation in RMS cells. Cells were exposed to indicated concentrations (D) or 5 μM (E) of TWS119 for 72 h and viable cells (%) was determined. Data represent 3 independent experiments, each performed in triplicate.
Fig. 3
Fig. 3
Down-regulation of GSK3 attenuates transcriptional activity of PAX3-FKHR. (A) Inhibition of transcriptional activity of PAX3-FKHR by TWS119. Rh30 cells stably transfected with pGL4.14-6 × PRS9-tk were treated with indicated concentrations of TWS119 for 16 h. Cell viability was determined by the CellTiter-Glo® assay. (B) Verification of GSK3α and GSK3β expression by Western blot analysis with anti-GSK3 antibody. Rh30 cells were treated with NT siRNA (NT) or gene-specific siRNA as indicated. (C) Gene reporter assay. Twenty four hours after transfection with NT siRNA (control) or gene-specific siRNA, luciferase activity was determined. Reporter activity from cells transfected with siRNA against PAX3-FKHR is set as 0% activation and NT siRNA as 100% activation. Data represent 3 independent experiments, each performed in triplicate.
Fig. 4
Fig. 4
GSK3 phosphorylates PAX3-FKHR in vitro. HEK293T cells were transiently transfected with EGFP-vector or EGFP-PAX3-FKHR plasmids. (A) The amount of EGFP and EGFP-PAX3-FKHR (EGFP-PF) proteins immunoprecipitated by anti-GFP agarose beads were revealed by staining with SimplyBlue. Purified GFP (Millipore) was included as a control. (B) In vitro kinase assays. (C) EGFP and EGFP-PF were revealed by Western blot, using anti-GFP antibody.
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