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Cyclin-dependent kinase 4 phosphorylates and positively regulates PAX3-FOXO1 in human alveolar rhabdomyosarcoma cells.

Liu L, Wu J, Ong SS, Chen T - PLoS ONE (2013)

Bottom Line: Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1.In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430).Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.

ABSTRACT
Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo. This prompted us to examine the signaling pathways required to activate the function of PAX3-FOXO1 and to explore the functional interaction between the Ink4a/ARF and PAX3-FOXO1 signaling pathways. Here we report that inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin (a small molecule selective inhibitor of Cdk4/cyclin D1 that we identified in a screen for compounds that inhibit PAX3-FOXO1) led to inhibition of the transcriptional activity of PAX3-FOXO1 in ARMS cell line Rh30. Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1. In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430). Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells. Our findings indicate that Cdk4 phosphorylates and positively regulates PAX3-FOXO1 and suggest that inhibition of Cdk4 activity should be explored as a promising avenue for developing therapy for ARMS.

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Identification of kinase inhibitors that inhibit PAX3-FOXO1 transcriptional activity.Rh30_PRS9 cells were treated with increasing concentrations (2.8 nM to 56 µM) of compounds for 2 h before determination of A) PRS9 reporter activity and B) viable cells (percentage relative to DMSO-treated cells). C) Fascaplysin decreased the expression of CPT1A and PTN in a PAX3-FOXO1-dependent manner. Rh30, Rh41, NIH3T3, and RD cells were treated with 1 µM fascaplysin or DMSO for 2 h. CPT1A or PTN mRNA levels in samples treated with fascaplysin were normalized to that of cells treated with DMSO (samples treated with DMSO were arbitrarily set as 1). * p<0.05; *** p<0.001; ns p>0.05.
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pone-0058193-g001: Identification of kinase inhibitors that inhibit PAX3-FOXO1 transcriptional activity.Rh30_PRS9 cells were treated with increasing concentrations (2.8 nM to 56 µM) of compounds for 2 h before determination of A) PRS9 reporter activity and B) viable cells (percentage relative to DMSO-treated cells). C) Fascaplysin decreased the expression of CPT1A and PTN in a PAX3-FOXO1-dependent manner. Rh30, Rh41, NIH3T3, and RD cells were treated with 1 µM fascaplysin or DMSO for 2 h. CPT1A or PTN mRNA levels in samples treated with fascaplysin were normalized to that of cells treated with DMSO (samples treated with DMSO were arbitrarily set as 1). * p<0.05; *** p<0.001; ns p>0.05.

Mentions: Rh30_PRS9 were seeded at 5,000 cells per well in 25 µl of medium into solid white 384-well tissue culture-treated plates 24 h before compound treatment. Then, 140 nL of compound at either 1 mM or 10 mM was added to each well (final compound concentration was 5.6 µM or 56 µM; final DMSO concentration was 0.56%), and the plates were incubated for 2 h before a luciferase reporter assay using the SteadyLiteHTS Luciferase Assay System (PerkinElmer, Waltham, MA) and cell viability assay using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) as described previously [8], [12]. The compounds used for the screen contained 160 protein kinase inhibitors from the InhibitorSelect 384-Well Protein Kinase Inhibitor Library I (EMD Millipore, Billerica, MA). Data are expressed as percentage of reporter activity of firefly luciferase or percentage of viable cells in the presence of compound compared with DMSO vehicle control (set as 100%) as previously described [8]. At 5.6 µM, 2 compounds [AGL 2043 (CAS 226717-28-8, a PDGFR inhibitor) and VEGFR2 inhibitor IV (CAS 216661-57-3)], and at 56 µM, 5 additional compounds [Syk inhibitor III (CAS 1485-00-3), DMBI (CAS 5812-07-7, a PDGFR inhibitor), fascaplysin (CAS 114719-57-2, a selective Cdk4/cyclin D1 inhibitor), Chelerythrine Chloride (CAS 3895-92-9, a PKC inhibitor), and GSK-3β inhibitor I] showed >50% of inhibition in luciferase reporter activity and the difference in inhibition of luciferase reporter activity and inhibition of viability was greater than 30%. Inhibitors of PKC and GSK-3β have previously been reported to inhibit the transcriptional activity of PAX3- FOXO1 [1], [8]. For dose response analysis, compounds were first diluted in DMSO (1∶3 serial dilutions from 10 mM; final compound concentrations ranged from 2.8 nM to 56 µM) and added to cells as described above. Four compounds significantly inhibited the luciferase activity in a dose-dependent manner but only slightly decreased cell viability (the difference in maximal inhibition of luciferase reporter activity and the maximal inhibition of viability was greater than 30%) are shown in Figure 1.


Cyclin-dependent kinase 4 phosphorylates and positively regulates PAX3-FOXO1 in human alveolar rhabdomyosarcoma cells.

Liu L, Wu J, Ong SS, Chen T - PLoS ONE (2013)

Identification of kinase inhibitors that inhibit PAX3-FOXO1 transcriptional activity.Rh30_PRS9 cells were treated with increasing concentrations (2.8 nM to 56 µM) of compounds for 2 h before determination of A) PRS9 reporter activity and B) viable cells (percentage relative to DMSO-treated cells). C) Fascaplysin decreased the expression of CPT1A and PTN in a PAX3-FOXO1-dependent manner. Rh30, Rh41, NIH3T3, and RD cells were treated with 1 µM fascaplysin or DMSO for 2 h. CPT1A or PTN mRNA levels in samples treated with fascaplysin were normalized to that of cells treated with DMSO (samples treated with DMSO were arbitrarily set as 1). * p<0.05; *** p<0.001; ns p>0.05.
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pone-0058193-g001: Identification of kinase inhibitors that inhibit PAX3-FOXO1 transcriptional activity.Rh30_PRS9 cells were treated with increasing concentrations (2.8 nM to 56 µM) of compounds for 2 h before determination of A) PRS9 reporter activity and B) viable cells (percentage relative to DMSO-treated cells). C) Fascaplysin decreased the expression of CPT1A and PTN in a PAX3-FOXO1-dependent manner. Rh30, Rh41, NIH3T3, and RD cells were treated with 1 µM fascaplysin or DMSO for 2 h. CPT1A or PTN mRNA levels in samples treated with fascaplysin were normalized to that of cells treated with DMSO (samples treated with DMSO were arbitrarily set as 1). * p<0.05; *** p<0.001; ns p>0.05.
Mentions: Rh30_PRS9 were seeded at 5,000 cells per well in 25 µl of medium into solid white 384-well tissue culture-treated plates 24 h before compound treatment. Then, 140 nL of compound at either 1 mM or 10 mM was added to each well (final compound concentration was 5.6 µM or 56 µM; final DMSO concentration was 0.56%), and the plates were incubated for 2 h before a luciferase reporter assay using the SteadyLiteHTS Luciferase Assay System (PerkinElmer, Waltham, MA) and cell viability assay using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) as described previously [8], [12]. The compounds used for the screen contained 160 protein kinase inhibitors from the InhibitorSelect 384-Well Protein Kinase Inhibitor Library I (EMD Millipore, Billerica, MA). Data are expressed as percentage of reporter activity of firefly luciferase or percentage of viable cells in the presence of compound compared with DMSO vehicle control (set as 100%) as previously described [8]. At 5.6 µM, 2 compounds [AGL 2043 (CAS 226717-28-8, a PDGFR inhibitor) and VEGFR2 inhibitor IV (CAS 216661-57-3)], and at 56 µM, 5 additional compounds [Syk inhibitor III (CAS 1485-00-3), DMBI (CAS 5812-07-7, a PDGFR inhibitor), fascaplysin (CAS 114719-57-2, a selective Cdk4/cyclin D1 inhibitor), Chelerythrine Chloride (CAS 3895-92-9, a PKC inhibitor), and GSK-3β inhibitor I] showed >50% of inhibition in luciferase reporter activity and the difference in inhibition of luciferase reporter activity and inhibition of viability was greater than 30%. Inhibitors of PKC and GSK-3β have previously been reported to inhibit the transcriptional activity of PAX3- FOXO1 [1], [8]. For dose response analysis, compounds were first diluted in DMSO (1∶3 serial dilutions from 10 mM; final compound concentrations ranged from 2.8 nM to 56 µM) and added to cells as described above. Four compounds significantly inhibited the luciferase activity in a dose-dependent manner but only slightly decreased cell viability (the difference in maximal inhibition of luciferase reporter activity and the maximal inhibition of viability was greater than 30%) are shown in Figure 1.

Bottom Line: Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1.In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430).Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.

ABSTRACT
Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo. This prompted us to examine the signaling pathways required to activate the function of PAX3-FOXO1 and to explore the functional interaction between the Ink4a/ARF and PAX3-FOXO1 signaling pathways. Here we report that inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin (a small molecule selective inhibitor of Cdk4/cyclin D1 that we identified in a screen for compounds that inhibit PAX3-FOXO1) led to inhibition of the transcriptional activity of PAX3-FOXO1 in ARMS cell line Rh30. Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1. In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430). Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells. Our findings indicate that Cdk4 phosphorylates and positively regulates PAX3-FOXO1 and suggest that inhibition of Cdk4 activity should be explored as a promising avenue for developing therapy for ARMS.

Show MeSH
Related in: MedlinePlus