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ARF1 controls proliferation of breast cancer cells by regulating the retinoblastoma protein

Oncogene volume 30, pages 3846–3861 (2011)Cite this article

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Abstract

The ADP-ribosylation factors (ARFs) 1 and 6 are small GTP-binding proteins, highly expressed and activated in several breast cancer cell lines and are associated with enhanced migration and invasiveness. In this study, we report that ARF1 has a critical role in cell proliferation. Depletion of this GTPase or expression of a dominant negative form, which both resulted in diminished ARF1 activity, led to sustained cell-growth arrest. This cellular response was associated with the induction of senescent markers in highly invasive breast cancer cells as well as in control mammary epithelial cells by a mechanism regulating retinoblastoma protein (pRB) function. When examining the role of ARF1, we found that this GTPase was highly activated in normal proliferative conditions, and that a limited amount could be found in the nucleus, associated with the chromatin of MDA-MB-231 cells. However, when cells were arrested in the G0/G1 phase or transfected with a dominant negative form of ARF1, the total level of activated ARF1 was markedly reduced and the GTPase significantly enriched in the chromatin. Using biochemical approaches, we demonstrated that the GDP-bound form of ARF1 directly interacted with pRB, but not other members of this family of proteins. In addition, depletion of ARF1 or expression of ARF1T31N resulted in the constitutive association of pRB and E2F1, thereby stabilizing the interaction of E2F1 as well as pRB at endogenous sites of target gene promoters, preventing expression of E2F target genes, such as cyclin D1, Mcm6 and E2F1, important for cell-cycle progression. These novel findings provide direct physiological and molecular evidence for the role of ARF1 in controlling cell proliferation, dependent on its ability to regulate pRB/E2F1 activity and gene expression for enhanced proliferation and breast cancer progression.

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Acknowledgements

We would like to thank Dr Tony Kouzarides (The Gurdon Institute, University of Cambridge, UK) for GST-pRB. We thank Raphaëlle Lambert from genomic platform of IRIC (Montreal, Canada). We are grateful to Dr Christian Beauséjour (University of Montreal, Montreal, Canada) for reagents and helpful discussion. We thank Dr Stéphane A Laporte (McGill University, Montreal, Canada) for the use of his confocal microscope and Dr Denis de Blois and Julie-Émilie Huot-Marchand (University of Montreal, Montreal, Canada) for the use of their photo-microscopy plateform. We thank Dr Véronique Bourdeau (University of Montreal, Montreal, Canada) for helpful discussion and critical reading of the paper. This work was supported by the Canadian Institutes of Health Research (CIHR) grant MOP-79470 to AC. PLB is the recipient of a Frederick Banting and Charles Best PhD Research Award from the CIHR. AC is the recipient of a New Investigator Award from the CIHR.

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  1. Department of Pharmacology, Faculty of Medicine, University of Montreal, Montréal, Québec, Canada,

    P-L Boulay, S Schlienger, S Lewis-Saravalli & A Claing

  2. Institut des Neurosciences Cellulaires et Intégratives Unité Propre de Recherche-3212 Centre National de la Recherche Scientifique/Université de Strasbourg, Strasbourg, France

    N Vitale

  3. Department of Biochemistry, Faculty of Medicine, University of Montreal, Montréal, Québec, Canada,

    G Ferbeyre & A Claing

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  1. P-L Boulay
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Boulay, PL., Schlienger, S., Lewis-Saravalli, S. et al. ARF1 controls proliferation of breast cancer cells by regulating the retinoblastoma protein. Oncogene 30, 3846–3861 (2011). https://doi.org/10.1038/onc.2011.100

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