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Inhibition of cyclin-dependent kinase 6 suppresses cell proliferation and enhances radiation sensitivity in medulloblastoma cells

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Abstract

Medulloblastoma accounts for 20 % of all primary pediatric intracranial tumors. Current treatment cures 50–80 % of patients but is associated with significant long-term morbidity and thus new therapeutic targets are needed. One such target is cyclin-dependent kinase 6 (CDK6), a serine/threonine kinase that plays a vital role in cell cycle progression and differentiation. CDK6 is overexpressed in medulloblastoma patients and is associated with an adverse prognosis. To investigate the role of CDK6 in medulloblastoma, we assayed the effect of CDK6 inhibition on proliferation by depleting expression with RNA interference (RNAi) or by inhibiting kinase function with a small molecule inhibitor, PD0332991. Cell proliferation was assessed by colony focus assay or by the xCELLigence system. We then investigated the impact of CDK6 inhibition on differentiation of murine neural stem cells by immunofluorescence of relevant markers. Finally we evaluated the effects of PD0332991 treatment on medulloblastoma cell cycle and radiosensitivity using colony focus assays. Gene expression analysis revealed that CDK6 mRNA expression is higher than normal cerebellum in fifteen out of sixteen medulloblastoma patient samples. Inhibition of CDK6 by RNAi significantly decreased medulloblastoma cell proliferation and colony forming potential. Interestingly, CDK6 inhibition by RNAi increased differentiation in murine neural stem cells. PD0332991 treatment significantly decreased medulloblastoma cell proliferation and led to a G0/G1 cell cycle arrest. Furthermore, PD0332991 pretreatment sensitized medulloblastoma cells to ionizing radiation. Our findings suggest that targeting CDK6 with small molecule inhibitors may prove beneficial in the treatment of medulloblastoma, especially when combined with radiation.

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Acknowledgments

This work was supported by NINDS K08 NS059790 (RV) and the Morgan Adams Foundation (RV, NKF). Imaging experiments were performed in the University of Colorado Anschutz Medical Campus Advance Light Microscopy Core supported in part by NIH/NCRR Colorado CTSI Grant Number UL1 RR025780.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Pediatrics, Children’s Hospital Colorado and University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA

    Susan L. Whiteway, Peter S. Harris, Sujatha Venkataraman, Irina Alimova, Andrew M. Donson, Nicholas K. Foreman & Rajeev Vibhakar

  2. Department of Pediatric Neurosurgery, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA

    Diane K. Birks

Authors
  1. Susan L. Whiteway
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  2. Peter S. Harris
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  3. Sujatha Venkataraman
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  4. Irina Alimova
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  5. Diane K. Birks
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  6. Andrew M. Donson
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  7. Nicholas K. Foreman
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  8. Rajeev Vibhakar
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Corresponding author

Correspondence to Susan L. Whiteway.

Electronic supplementary material

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Supplementary Fig. 1

Quantifying bar graphs show significant decrease in CDK6 mRNA expression in Daoy and ONS-76 medulloblastoma cell lines after transfection with shRNA targeting CDK6. Error bars represent SEM. (***P<0.001) (TIFF 19241 kb)

Supplementary Fig. 2

Subgroup analysis of microarray data in 120 medulloblastoma patients show CDK6 mRNA expression is statistically higher in the Wnt subgroup than Group 3 and Group 4. (**P<0.01, ***P<0.001) (TIFF 15944 kb)

Supplementary Fig. 3

Nonlinear regression lines for Daoy and ONS-76 cells treated with PD0332991 followed by radiation. The R2 values for each line are listed below their respective graphs (TIFF 19707 kb)

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Whiteway, S.L., Harris, P.S., Venkataraman, S. et al. Inhibition of cyclin-dependent kinase 6 suppresses cell proliferation and enhances radiation sensitivity in medulloblastoma cells. J Neurooncol 111, 113–121 (2013). https://doi.org/10.1007/s11060-012-1000-7

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  • DOI: https://doi.org/10.1007/s11060-012-1000-7

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