RAC1 is active in melanoma
Aberrant expression of RAC or RAC activators in human cancer, and the effect of conditional ablation of rac1 or Rac activators in mouse tissues on tumour formation and progression have implicated RAC1 in tumourigenesis. While RAC mutations were not readily detected in human cancer using conventional sequencing, subsequent exome sequencing has revealed oncogenic RAC alleles. In particular, approximately 9% of cases of melanoma developing in sun-exposed sites possess RAC1 mutated at a common amino acid, Proline 29. Conversion of this residue to Serine alters the conformation of the switch I loop of RAC1 and activates the protein. RAC1 mutation can coincide with gain-of-function of BRAF or NRAS mutation in melanoma, suggesting that RAC1 co-operates with MAPK signalling to induce melanoma.

To investigate possible co-operation between RAC and RAS in melanoma formation, we combined expression of an active RAC1 allele (RAC1G12V) with oncogenic RAS (HRAS G12V) in zebrafish melanocytes. The combination was significantly more potent at inducing tumour nodules than either mutant gene alone. Surprisingly, expression of RAC1G12V alone did not perturb zebrafish melanocyte development, morphology, proliferation or migration, but in combination with HRASG12V did induce precocious proliferation and migration of transformed melanocytes, promoting tumour formation. Immunohistochemical staining of human melanoma samples demonstrated widespread overexpression and hyperactivation of RAC1.  We also revealed overexpression of the RAC activator TIAM1 (T-lymphoma invasion and metastasis protein) in nodular forms of melanoma (Dalton et al. J Invest Dermatol 2013). Thus, we conclude that RAC1 deregulation is a common event in the genesis of melanoma, which co-operates with aberrant RAS signalling to drive malignant progression.

Figure 1. RAC GTPase cycles between inactive GDP-bound and active GTP-bound states. RAC activation is facilitated by the action of GEFs, which promotes GDP dissociation from RAC and allows GTP to bind instead. In turn, GEFs are stimulated by receptor tyrosine kinases (RTK), integrins (ab), and G-protein coupled receptors (GPCR). Through the association with GAPs, the intrinsic GTPase activity of RAC is accelerated, thereby inactivating RAC. Through association with RhoGDIs (GDI) RAC can be sequestered in its inactive state. Activated RAC can also be removed through ubiquitylation-induced degradation (mediated by HACE1 following a migration stimulus) or it can be maintained following its modification by SUMO (mediated by PIAS3).

Post-translational modifications of Rac1 during cell migration
Recently, regulation by post-translational modification has emerged as a significant means of regulating RAC activity. To gain further insight into the regulation of RAC during cell migration, we performed a screen for proteins that interact with RAC following treatment of cells with a motility-inducing factor, Hepatocyte Growth Factor (HGF). This revealed the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3, as a novel RAC interacting protein. Subsequently, we demonstrated that RAC1 can be conjugated to SUMO-1 by PIAS3 in response to HGF. PIAS3 interacts better with GTP-bound RAC and the GTP-bound form of RAC is a better substrate for SUMOylation. Furthermore, we demonstrated that PIAS3-mediated SUMOylation of RAC1 controls RAC1-GTP levels and the ability of Rac1 to stimulate lamellipodia, cell migration and invasion (Castillo-Lluva et al. Nat Cell Biol. 2010).

RAC activity is also regulated through ubiquitylation and subsequent degradation. Recently, we identified the tumour suppressor HACE1 to be the E3 ubiquitin ligase responsible for RAC degradation following activation by a migration stimulus. We showed that HACE1 and RAC1 interaction is enhanced by HGF signalling and that HACE1 catalyses the poly-ubiquitylation of RAC1 at lysine 147 following its activation by HGF, resulting in its proteasomal degradation. HACE1-depletion is accompanied by increased total RAC1 levels and accumulation of RAC1 in membrane ruffles. Moreover, HACE1-depletion enhances cell migration independently of growth factor stimulation, which may have significance for malignant conversion (Castillo-Lluva et al. Oncogene 2013). Jointly, the above two studies suggest that SUMOylation and ubiquitylation of RAC1 act co-ordinately to fine-tune RAC1 activity in migrating cells, promoting RAC activity at sites where the cell membrane is advancing, while antagonising RAC at sites where membrane protrusion needs to cease.

TIAM1-RAC signalling regulates bipolar spindle assembly, chromosome congression and mitotic progression dependent on phosphorylation of TIAM1 by Cyclin B/CDK1
TIAM1 is a guanine nucleotide exchange factor that selectively activates RAC. Mice deficient for Tiam1 are resistant to the formation of skin tumours induced by chemical carcinogens and the few resulting tumours grow very slowly (Malliri et al. Nature 2002). To better understand the role of TIAM1 in promoting tumour growth we have examined its role in the cell cycle. We revealed that TIAM1 and RAC localise to centrosomes during prophase and prometaphase, and TIAM1, acting through RAC, ordinarily retards centrosome separation. TIAM1-depleted cells transit more slowly through mitosis and display increased chromosome congression errors. Significantly, suppression of the microtubule motor Kinesin-5/Eg5 in TIAM1-depleted cells rectifies not only their increased centrosome separation but also their chromosome congression errors and mitotic delay. These findings identified TIAM1-RAC signalling as an antagonist of centrosome separation during mitosis and demonstrated its requirement in balancing Eg5-induced forces during bipolar spindle assembly (Woodcock et al. Curr Biol. 2010). Subsequent to this study, we have found that TIAM1 is phosphorylated by Cyclin B/CDK1 in mitosis. This phosphorylation, while not required for TIAM1 localisation to centrosomes, is essential for its role in regulating centrosome separation. Currently, we are investigating the mechanism by which phosphorylation of TIAM1 influences its role at centrosomes.

Figure 2. RAC contributes to several cancer hallmarks that promote tumour formation and progression.

TIAM1 antagonises malignant progression
Despite their slower growth, tumours arising in Tiam1-deficient mice progressed more frequently to malignancy (Malliri et al. Nature 2002). One mechanism by which TIAM1 and RAC suppress malignant progression is through promoting cell-cell adhesion. We further investigated the function of TIAM1 and RAC at cell-cell adhesions. We identified β2-syntrophin, a component of the dystroglycan adhesion complex, as a TIAM1 binding partner. Our study (Mack et al. Nat Cell Biol. 2012) unearthed a novel role for this complex in regulating tight junctions and the generation of apicobasal polarity through the generation of a RAC activity gradient in the membrane region encompassing these junctions.

Malignant progression can entail the loss of cell-cell adhesion. The oncoprotein Src, a non-receptor tyrosine kinase, targets adherens junctions (AJ) for disassembly. Previously, we showed that Src phosphorylates TIAM1 inducing its cleavage by Calpain and its depletion from AJs. Abrogating TIAM1 phosphorylation by Src suppressed AJ disassembly (Woodcock et al. Mol Cell 2009). We have now found that TIAM1 like RAC1 is ubiquitylated and degraded upon treatment of cells with HGF. We have mapped ubiquitylation sites and identified the responsible E3 ligase. Moreover, we show that interfering with TIAM1 ubiquitylation retards the scattering and invasion of cells through delaying AJ disassembly.