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Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle

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Abstract

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G1 phase. Aberrant expression of CDK4 and CDK6 is a hallmark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM (the calcein acetoxymethyl-ester) is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G1 phase. The metabolic effects of calcein AM on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phosphate pathway was significantly altered. To elucidate whether these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.

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Abbreviations

Calcein AM:

Calcein acetoxymethyl-ester

CDK:

Cyclin-dependent kinase

DMEM:

Dulbecco’s modified eagle medium

FCS:

Fetal calf serum

Ct MEF:

Mouse embryonic fibroblast

PBS:

Phosphate buffer saline

PPP:

Pentose phosphate pathway

pRb:

Retinoblastoma protein

TKO MEF:

Mouse embryonic fibroblast knockout for CDK4, CDK6 and CDK2

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Acknowledgments

The authors thank Mrs Ursula Valls for her technical support in the experiments and Dr David Santamaria for his help in MEF procedures. MEF cells were a generous gift from Dr Mariano Barbacid, CNIO-Madrid (Spain). This study was supported by the projects SAF2008-00164 (to MC) and SAF2007-60491 (to NA) and by RD06/0020/0046 (to MC), RD06/0020/0010 (to OB) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III, all of them funded by the Ministerio de Ciencia e Innovación-Spanish government and European Regional Development Funds (ERDF) “Una manera de hacer Europa”. It has also received financial support from the European Union-funded project ETHERPATHS (FP7-KBBE-222639) (http://www.etherpaths.org/) and from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR)-Generalitat de Catalunya (2009SGR01308 and predoctoral fellowship of M.Z.). Mass spectrometry facility was supported by NIH grants to WNP Lee from UCLA Center of Excellence (PO1 AT003960-01) and from Harbor-UCLA GCRC (MO1 RR00425-33). MC acknowledges the support received through the prize “ICREA Academia” for excellence in research, funded by ICREA foundation-Generalitat de Catalunya.

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Author notes

  1. Antonio Ramos-Montoya

    Present address: Uro-Oncology Research Group, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK

  2. Oscar Villacañas

    Present address: Intelligent Pharma S.L, C/Baldiri Reixac 4, 08028, Barcelona, Spain

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Faculty of Biology (Edifici Nou), University of Barcelona, Av. Diagonal 645, 08028, Barcelona, Spain

    Miriam Zanuy, Antonio Ramos-Montoya, Anibal Miranda, Esther Aguilar, Silvia Marin & Marta Cascante

  2. Institute of Biomedicine of the Universitat de Barcelona (IBUB) and CSIC Associated Unit, Barcelona, Spain

    Miriam Zanuy, Antonio Ramos-Montoya, Anibal Miranda, Esther Aguilar, Silvia Marin & Marta Cascante

  3. Department of Physical Chemistry, Institut de Recerca en Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Martí i Franqués 1, 08028, Barcelona, Spain

    Oscar Villacañas & Jaime Rubio-Martinez

  4. Department of Cell Biology, Immunology and Neurosciencies, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Faculty of Medicine, Universitat de Barcelona, Casanova 143, 08036, Barcelona, Spain

    Nuria Canela, Neus Agell & Oriol Bachs

  5. Department of Pharmacology and Therapeutic Chemistry, CSIC Associated Unit, Faculty of Pharmacy, Universitat de Barcelona, Joan XXIII, s/n, 08028, Barcelona, Spain

    Maria Dolors Pujol

  6. Department of Pediatrics, Los Angeles Biomedical Research Institute at the Harbor-UCLA Medical Center, RB1, 1124 West Carson Street, Torrance, CA, 90502, USA

    Wai-Nang P. Lee

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  1. Miriam Zanuy
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Corresponding author

Correspondence to Marta Cascante.

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Miriam Zanuy and Antonio Ramos-Montoya contributed equally to this work.

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Zanuy, M., Ramos-Montoya, A., Villacañas, O. et al. Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle. Metabolomics 8, 454–464 (2012). https://doi.org/10.1007/s11306-011-0328-x

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