Rho GTPases: Regulators of Cell Migration, Metastasis and More

Cancer cells spread from the initial site of tumour growth to distant sites by first invading the surrounding tissues, then entering the blood stream and finally exiting it by binding to endothelial cells and crossing the blood vessel wall. Leukocytes in the blood also have to attach to endothelial cells in order to cross the blood vessel wall either during immune surveillance or in response to inflammation. Leukocytes may contribute to cancer metastasis in several ways, for example by secreting pro-invasive molecules at the site of primary tumour growth, or by binding to cancer cells in the bloodstream and enhancing their transmigration across endothelial cells.

Many different signalling proteins contribute to cell migration, from transmembrane receptors to transcription factors, but the Rho GTPases act as a focal point for migration signalling. They act in multiple ways to regulate cell migration, affecting actin and microtubule dynamics, cell-cell and cell-extracellular matrix adhesion, and intracellular trafficking of proteins required for cells to move (reviewed in 1). There are 22 Rho family members in humans, but the best-studied members are RhoA, Rac1 and Cdc42. Each Rho GTPase induces its own unique response in cells by activating several downstream targets, including protein kinases, lipid-modifying enzymes and protein scaffolds. For example, RhoA interacts with the serine/threonine kinases ROCK I and ROCK II, which then stimulate the phosphorylation of proteins such as myosin phosphatase, leading to increased interaction of myosin with actin filaments and cell contraction. We are investigate how Rho GTPases and their interacting partners affect the various steps of cell migration leading to cancer metastasis, using cell biological approaches.

By studying epithelial cells, leukocytes and endothelial cells 2-4, we have shown that RhoA and Rac1 act together to coordinate cell migration. Rac1 acts at the front of migrating cells to stimulate actin-mediated membrane protrusion and attachment of protrusions to the extracellular matrix, whereas RhoA acts primarily at the rear of cells to induce forward movement of the nucleus and cell body, and to mediate detachment of the back of the cell from the extracellular matrix (Figure 1). Cdc42 is important for cell polarization and directionality of movement , and depending on the cell type can also contribute to cell speed by enhancing Rac-mediated membrane protrusion at the front of cells 4. Cdc42 stimulates actin polymerization by binding to WASP; however we have shown that WASP activity is also regulated by phosphorylation 5,6, and thus WASP acts as a node, receiving signals from multiple inputs (Figure 2). This may be a pardigm for other Rho GTPase targets.

Fig. 1 Rho proteins in cell migration

Fig. 2 Multiple ways of regulating WASP

Recently we have been investigating the function of a less well-characterized member of the Rho family, RhoE. RhoE is unusual in that it does not hydrolyse GTP 7, and therefore is not a classic ‘GTPase switch’. Instead, RhoE function may be regulated by altering its expression: it is upregulated in a number of cancers, and we have found it is increased in response to growth factors and by cisplatin, which induces DNA damage 8,9. RhoE acts in opposition to RhoA, inducing a decrease in actin stress fibres (Figure 3) and enhancing epithelial cell migration 7. We have found that RhoE specifically interacts with the RhoA target ROCK I and prevents it from stimulating assembly of actin and myosin filaments to form stress fibres (Figure 3) 8. Interestingly, sustained upregulation of RhoE expression inhibits cell cycle progression and cell proliferation, in part by preventing accumulation of the cell cycle regulator Cyclin D1 9. RhoE therefore has two functions – one in cell migration and one in cell cycle control. Previous work has suggested that RhoA and Rac1 also have a dual role in regulating these two cellular responses.

Fig. 3 Micrograph of RhoE-injected cell. [Figure courtesy of Dr. Kirsi Riento]

In the future we aim to characterize how RhoE contributes to cell cycle arrest and how its expression is induced by DNA damage. In addition, we are investigating how Rho GTPases and their targets contribute to cancer cell invasion and migration across endothelial cells. In this way, we will be able to pinpoint which Rho GTPases and which of their targets are good candidates for therapeutic intervention in cancer metastasis and/or growth.

Back row: Aleks Ivetic, Jaime Millan, Michael Bright; Middle row: Parag Bhavsar, Ana Maria Pajari, Jenny MacKenzie, Ann Wheeler, Steve Smith; Front row: Maria Christodoulou, Eva Cernuda Morollon, Anne Ridley, Priam Villalonga Smith. (Absent: Kirsi Riento, Ritu Garg, Nicholas Reymond, Jana Grunewald, Sarah Heasman)

References

  1. Ridley, A.J., Schwartz, M.A., Burridge, K., Firtel, R.A., Ginsberg, M.H., Borisy, G., Parsons, J.T., Horwitz, A.R. (2003) Cell Migration: Integrating Signals from Front to Back. Science 302, 1704-1709.
  2. Potempa, S. and Ridley, A.J. (1998) Activation of both MAP kinase and phosphatidylinositide 3-kinase by Ras is required for HGF/SF-induced adherens junction disassembly. Mol. Biol. Cell 9, 2185-2200.
  3. Allen, W.E., Zicha, D., Ridley, A.J., Jones, G.E. (1998) A Role for Cdc42 in Macrophage Chemotaxis. J. Cell Biol 141, 1147-1157.
  4. Wojciak-Stothard, B., Ridley, A.J. (2003) Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases. J. Cell Biol. 161, 429-439.
  5. Cory, G.O.C., Garg, R., Cramer, R., Ridley, A.J. (2002) Phosphorylation of Tyrosine 291 Enhances the Ability of WASp to Stimulate Actin Polymerization and filopodium formation. J. Biol. Chem 277, 45115-45121.
  6. Cory, G.O.C., Cramer, R., Blanchoin L., Ridley, A.J. (2003) Phosphorylation of the WASp-VCA Domain Increases its Affinity for the Arp2/3 Complex and Enhances Actin Polymerization by WASp. Mol. Cell 11, 1229-1339.
  7. Guash, R.M., Scambler, P., Jones, G.E., Ridley, A.J. (1998) RhoE regulates actin cytoskeleton organization and cell migration. Mol. Cell. Biol. 18, 4761-71.
  8. Riento, K., Guasch, R.M., Garg, R., Bouquin, J., Ridley, A.J. (2003) RhoE binds to ROCKI and inhibits downstream signalling. Mol. Cell Biol. 23, 4219-4229.
  9. Villalonga, P., Guasch, M.G., Riento, K., Ridley, A.J. (2004) RhoE inhibits cell cycle progression and Ras-induced transformation. Mol. Cell. Biol. 24, 7829-7840.