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A MEK-independent role for CRAF in mitosis and tumor progression

Nature Medicine volume 17, pages 1641–1645 (2011)Cite this article

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Abstract

RAF kinases regulate cell proliferation and survival and can be dysregulated in tumors1,2. The role of RAF in cell proliferation has been linked to its ability to activate mitogen-activated protein kinase kinase 1 (MEK) and mitogen-activated protein kinase 1 (ERK). Here we identify a MEK-independent role for RAF in tumor growth. Specifically, in mitotic cells, CRAF becomes phosphorylated on Ser338 and localizes to the mitotic spindle of proliferating tumor cells in vitro as well as in murine tumor models and in biopsies from individuals with cancer. Treatment of tumors with allosteric inhibitors, but not ATP-competitive RAF inhibitors, prevents CRAF phosphorylation on Ser338 and localization to the mitotic spindle and causes cell-cycle arrest at prometaphase. Furthermore, we identify phospho-Ser338 CRAF as a potential biomarker for tumor progression and a surrogate marker for allosteric RAF blockade. Mechanistically, CRAF, but not BRAF, associates with Aurora kinase A (Aurora-A) and Polo-like kinase 1 (Plk1) at the centrosomes and spindle poles during G2/M. Indeed, allosteric or genetic inhibition of phospho-Ser338 CRAF impairs Plk1 activation and accumulation at the kinetochores, causing prometaphase arrest, whereas a phospho-mimetic Ser338D CRAF mutant potentiates Plk1 activation, mitosis and tumor progression in mice. These findings show a previously undefined role for RAF in tumor progression beyond the RAF-MEK-ERK paradigm, opening new avenues for targeting RAF in cancer.

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Figure 1: CRAF is required for mitotic progression.
Figure 2: Phospho-Ser338 CRAF is upregulated in mitosis and localizes to the mitotic spindles in human cell lines and tumor biopsies.
Figure 3: CRAF interacts with Plk1 and promotes its activation and accumulation to the kinetochores at prometaphase.
Figure 4: The phospho-mimetic CRAF S338D alteration drives tumor growth and activates Plk1 in vivo.

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Acknowledgements

We thank K. Elliott and J. Lesperance for their assistance with the mouse experiments and immunohistochemistry, and M. Schmid, M. Kaulich and J. Desgrosellier for discussions. We thank K. Lee (US National Institutes of Health National Cancer Institute) and R. Erikson (Harvard University) for providing Plk1 constructs. D.A.C. was supported by grants CA78045, CA119335, CA95262 and CA104898 from the US National Institutes of Health.

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Authors and Affiliations

  1. Department of Pathology, Moores Cancer Center, University of California San Diego, La Jolla, California, USA

    Ainhoa Mielgo, Laetitia Seguin, Miller Huang, Maria Fernanda Camargo, Sudarshan Anand, Aleksandra Franovic, Sara M Weis, Eric A Murphy & David A Cheresh

  2. Department of Radiation Oncology, University of California San Diego, La Jolla, California, USA

    Sunil J Advani

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Contributions

A.M. and D.A.C. designed the studies. E.A.M. designed and provided KG5. A.M., L.S., M.H., M.F.C., S.A., A.F. and S.J.A. performed experiments. A.M., L.S., M.H., M.F.C., S.A., A.F., S.J.A. and S.M.W. analyzed data. A.M. and D.A.C. wrote the manuscript. D.A.C. supervised the project.

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Correspondence to David A Cheresh.

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Supplementary Figures 1–14 and Supplementary Table 1 (PDF 2655 kb)

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Mielgo, A., Seguin, L., Huang, M. et al. A MEK-independent role for CRAF in mitosis and tumor progression. Nat Med 17, 1641–1645 (2011). https://doi.org/10.1038/nm.2464

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