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

Article metrics

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Wellbrock, C., Karasarides, M. & Marais, R. The RAF proteins take centre stage. Nat. Rev. Mol. Cell Biol. 5, 875–885 (2004).

  2. 2

    Dhillon, A.S., Hagan, S., Rath, O. & Kolch, W. MAP kinase signalling pathways in cancer. Oncogene 26, 3279–3290 (2007).

  3. 3

    Bollag, G. et al. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 467, 596–599 (2010).

  4. 4

    Flaherty, K.T. et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N. Engl. J. Med. 363, 809–819 (2010).

  5. 5

    Joseph, E.W. et al. The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner. Proc. Natl. Acad. Sci. USA 107, 14903–14908 (2010).

  6. 6

    King, A.J. et al. Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885. Cancer Res. 66, 11100–11105 (2006).

  7. 7

    Tsai, J. et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl. Acad. Sci. USA 105, 3041–3046 (2008).

  8. 8

    Yang, H. et al. RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models. Cancer Res. 70, 5518–5527 (2010).

  9. 9

    Hatzivassiliou, G. et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464, 431–435 (2010).

  10. 10

    Heidorn, S.J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140, 209–221 (2010).

  11. 11

    Poulikakos, P.I., Zhang, C., Bollag, G., Shokat, K.M. & Rosen, N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464, 427–430 (2010).

  12. 12

    Murphy, E.A. et al. Disruption of angiogenesis and tumor growth with an orally active drug that stabilizes the inactive state of PDGFRβ/B-RAF. Proc. Natl. Acad. Sci. USA 107, 4299–4304 (2010).

  13. 13

    Alavi, A., Hood, J.D., Frausto, R., Stupack, D.G. & Cheresh, D.A. Role of Raf in vascular protection from distinct apoptotic stimuli. Science 301, 94–96 (2003).

  14. 14

    Alavi, A.S., Acevedo, L., Min, W. & Cheresh, D.A. Chemoresistance of endothelial cells induced by basic fibroblast growth factor depends on Raf-1-mediated inhibition of the proapoptotic kinase, ASK1. Cancer Res. 67, 2766–2772 (2007).

  15. 15

    McGlynn, L.M. et al. Ras/Raf-1/MAPK pathway mediates response to tamoxifen but not chemotherapy in breast cancer patients. Clin. Cancer Res. 15, 1487–1495 (2009).

  16. 16

    Mikula, M. et al. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J. 20, 1952–1962 (2001).

  17. 17

    Weber, C.K., Slupsky, J.R., Kalmes, H.A. & Rapp, U.R. Active Ras induces heterodimerization of cRaf and BRaf. Cancer Res. 61, 3595–3598 (2001).

  18. 18

    Borysov, S.I., Cheng, A.W. & Guadagno, T.M. B-Raf is critical for MAPK activation during mitosis and is regulated in an M phase–dependent manner in Xenopus egg extracts. J. Biol. Chem. 281, 22586–22596 (2006).

  19. 19

    Borysov, S.I. & Guadagno, T.M. A novel role for Cdk1/cyclin B in regulating B-raf activation at mitosis. Mol. Biol. Cell 19, 2907–2915 (2008).

  20. 20

    Borysova, M.K., Cui, Y., Snyder, M. & Guadagno, T.M. Knockdown of B-Raf impairs spindle formation and the mitotic checkpoint in human somatic cells. Cell Cycle 7, 2894–2901 (2008).

  21. 21

    Cui, Y., Borysova, M.K., Johnson, J.O. & Guadagno, T.M. Oncogenic B-Raf(V600E) induces spindle abnormalities, supernumerary centrosomes, and aneuploidy in human melanocytic cells. Cancer Res. 70, 675–684 (2010).

  22. 22

    Cui, Y. & Guadagno, T.M. B-Raf(V600E) signaling deregulates the mitotic spindle checkpoint through stabilizing Mps1 levels in melanoma cells. Oncogene 27, 3122–3133 (2008).

  23. 23

    Malumbres, M. & Barbacid, M. Cell cycle kinases in cancer. Curr. Opin. Genet. Dev. 17, 60–65 (2007).

  24. 24

    Nigg, E.A. Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell Biol. 2, 21–32 (2001).

  25. 25

    Taylor, S. & Peters, J.M. Polo and Aurora kinases: lessons derived from chemical biology. Curr. Opin. Cell Biol. 20, 77–84 (2008).

  26. 26

    Macůrek, L. et al. Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Nature 455, 119–123 (2008).

  27. 27

    Petronczki, M., Lenart, P. & Peters, J.M. Polo on the rise-from mitotic entry to cytokinesis with Plk1. Dev. Cell 14, 646–659 (2008).

  28. 28

    Seki, A., Coppinger, J.A., Jang, C.Y., Yates, J.R. & Fang, G. Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry. Science 320, 1655–1658 (2008).

  29. 29

    Knauf, J.A. et al. Oncogenic RAS induces accelerated transition through G2/M and promotes defects in the G2 DNA damage and mitotic spindle checkpoints. J. Biol. Chem. 281, 3800–3809 (2006).

  30. 30

    Luo, J. et al. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 137, 835–848 (2009).

  31. 31

    McInnes, C. et al. Inhibitors of Polo-like kinase reveal roles in spindle-pole maintenance. Nat. Chem. Biol. 2, 608–617 (2006).

  32. 32

    Santamaria, A. et al. Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis. Mol. Biol. Cell 18, 4024–4036 (2007).

  33. 33

    Steegmaier, M. et al. BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo. Curr. Biol. 17, 316–322 (2007).

  34. 34

    Strebhardt, K. Multifaceted polo-like kinases: drug targets and antitargets for cancer therapy. Nat. Rev. Drug Discov. 9, 643–660 (2010).

  35. 35

    Hüser, M. et al. MEK kinase activity is not necessary for Raf-1 function. EMBO J. 20, 1940–1951 (2001).

  36. 36

    Hindley, A. & Kolch, W. Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases. J. Cell Sci. 115, 1575–1581 (2002).

  37. 37

    Kamata, T. et al. BRAF inactivation drives aneuploidy by deregulating CRAF. Cancer Res. 70, 8475–8486 (2010).

Download references

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.

Author information

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.

Correspondence to David A Cheresh.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

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

Rights and permissions

Reprints and Permissions

About this article

Further reading