Rac1 in the driver’s seat for melanoma (2012)

Ten years ago, the genetics of melanoma were revolutionized by the identification of frequent BRAF mutations (Davies et al., 2002). Remarkably, within this time frame, there is now a licensed treatment for patients carrying this mutation showing how quickly molecularly targeted therapies can be translated into the clinic. Since these studies, high content sequencing methods have advanced, decreasing the cost and the speed of whole genome and/or exome sequencing and permitting sequencing of larger cohorts of tumours. Thus, tumour genome sequencing has begun to provide significant insights into somatic mutations that can cooperate with the already well-known primary oncogenes and tumour suppressors that drive cancer progression as well as allowing us to understand the unique genetic forces driving specific subsets of the same type of tumour (e.g. melanoma on sun-exposed versus sun-shielded areas). Here, we highlight two studies that found a recurrent potential driver mutation in RAC1, a gene encoding a small GTPase of the Rho-family, which signals to cell motility and cell cycle progression via multiple pathways (Hodis et al., 2012; Krauthammer et al., 2012; Figure 1). These studies suggest the exciting possibility that RAC1 and its downstream targets may represent potential new drug targets that could be inhibited in combination with key oncogenes such as BRAF (Poulikakos and Rosen, 2011; Roring and Brummer, 2012). RAC1 connects with RAS and downstream pathways in control of cell cycle progression and signalling. The diagram shows an example of a generic tyrosine kinase receptor (such as a growth factor receptor) signalling to Ras and downstream to selected targets such as RAC1 and BRAF. RAS signals to BRAF leading to activation of downstream kinases MEK and ERK, which trigger changes in gene expression and cell cycle progression (Roring and Brummer, 2012). Ras also signals to phosphatidylinositol 3-kinase (PI3K) to activate RAC1. RAC1 in turn feeds back to further enhance PI3K signalling as well as activating several downstream targets (Murga et al., 2002) such as the Scar/WAVE Regulatory complex which triggers actin assembly at the plasma membrane (Insall and Machesky, 2009); RAC1 controls membrane localization of the TORC1 and TORC2 complexes (Saci et al., 2011); RAC1 activates PAK1, leading to JNK activation and gene expression changes and cell cycle progression (Brown et al., 1996); Rac1 also activates NFκB signalling to the nucleus (Frost et al., 2000; Jefferies and O’neill, 2000). Halaban and colleagues sequenced 147 human melanomas and discovered a frequently occurring new activating mutation of the gene encoding the RAC1 small GTPase (Krauthammer et al., 2012). RAC1 was found to be mutated at a highly conserved residue in the switch I loop, proline 29, with a change to serine (Krauthammer et al., 2012). The second study, by Garraway, Chin et al. (Hodis et al., 2012) also identified this same Rac1 mutation as a recurrent potential driver from exome sequences of paired tumour and normal tissues from 135 patients with melanoma. While RAC1 is known to be activated in melanoma cells in vitro (Sanz-Moreno et al., 2008) and its activity was reported to be higher in a recent study of urinary tract cancer (Kamai et al., 2010), this is the first report of a potentially tumour promoting mutation in RAC1 and as such suggests that Rac1 and its downstream effectors may be valuable targets for future therapy in melanoma and other cancers. Interestingly, both studies found that RAC1 mutations were caused by a particular cytidine to thymidine transition, which is characteristic of a UV-induced mutation. This mutation occurred in around 4% of melanomas (Hodis et al., 2012) and 9% of sun-exposed melanomas (Krauthammer et al., 2012) studied and was found by both studies to be the third most frequently mutated hot spot after NRAS and BRAF. The mutation was also highly statistically significantly more commonly found in men than in women, likely due to increased sun exposure in the male cohort (Krauthammer et al., 2012). Halaban et al. solved the crystal structure of the mutant RAC1 protein and demonstrated that the mutation confers a unique conformational change in the switch I loop that resembles the conformation of this loop in active Ras (Krauthammer et al., 2012). This was further supported by biochemical data showing increased interaction of P29S RAC1 with PAK1 and MLK3 downstream kinase targets (Krauthammer et al., 2012), increased interaction with PAK1 p21-binding domain (Hodis et al., 2012) and functional studies in cells showing enhanced proliferation and migration of melanocytes expressing Rac1 P29S (Krauthammer et al., 2012). The RAC1 mutation occurred in both NRAS and BRAF mutant melanomas (Hodis et al., 2012; Krauthammer et al., 2012), suggesting that it could be driving events independently of these oncogenes. It would be interesting to know how frequently this mutation occurs in naevi, as both NRAS and BRAF mutations occur in benign naevi (Blokx et al., 2010). Among the 20+ Rho-family GTPases in mammals (Wherlock and Mellor, 2002), Rac1 is well known as a guanine nucleotide regulated switch that triggers actin assembly underneath the plasma membrane leading to protrusion and increased migration (Heasman and Ridley, 2008). RAC1 also regulates cell cycle progression and activity of the p21-activated (PAK) kinases (Heasman and Ridley, 2008). The mechanism by which RAC1 controls migration is thought to mainly be via direct interaction with the Scar/WAVE complex (or WAVE regulatory complex), a 5-protein conserved complex that activates actin assembly (Insall and Machesky, 2009). Cell cycle progression may be controlled via activation of PAK kinases, which are direct RAC targets and in turn signal to JNK and MAPK (Ong et al., 2011). It is not yet clear whether the RAC1 P29S mutation affects metastatic spread, or is solely implicated in primary tumour formation/progression. Motile cells in melanomas display some aspects of reversion to an embryonic melanoblastic state (Yang and Weinberg, 2008), and thus, there may be a relationship between melanoblasts and melanoma metastasis. Deletion of RAC1 in melanoblasts greatly diminishes their migration in the developing embryo, resulting in large patches devoid of melanocytes in the adult (Li et al., 2011). RAC1 loss also slows down cell cycle progression of both normal and tumour cells (Li et al., 2011). Conditional deletion of RAC1 or treatment with Rac1 inhibitors in a tumour transplant model halted tumour growth and spread to lymph nodes (Li et al., 2012). Thus, inhibition of RAC1 might be therapeutically useful, in the case of NRAS driven melanomas, where Rac1 may be activated downstream of Ras and also in cases where RAC1 is mutated. The activity of RAC1 is regulated by multiple guanine nucleotide exchange proteins (GEFs), including Vav1-3, pRex1-2 and Dock proteins, all of which have been implicated in cancer progression and metastasis (Lazer and Katzav, 2011). PREX1 is important for melanoblast migration and proliferation during embryogenesis and mice deficient in PREX1 also show reduced melanoma metastasis (Lindsay et al., 2011). PREX2 has been identified in exome sequencing as mutated in 15–45% of human metastatic melanoma biopsies (Berger et al., 2012). The mutations were diverse, leaving the molecular mechanism of its importance unclear (Berger et al., 2012). Moreover, in contrast to PREX1, where expression is clearly deregulated during melanomagenesis (Lindsay et al., 2011), the expression of PREX2 during this process is unclear. PREX GEFs are large multidomain proteins that impinge on multiple signalling pathways, including PI3-kinase signalling and activation of Rac GTPases. Dock10, Vav-1 and Vav-2 have been implicated in melanoma cell invasion in vitro (Bartolome et al., 2006; Gadea et al., 2008). GEFs thus could represent another potentially useful area to target RAC1 in melanoma, but the diversity of GEFs and the large multidomain structure of these proteins presents challenges. Thus far, attempts to inhibit Ras GTPases with small molecules have not yielded any clinically useful drugs (Gysin et al., 2011), possibly due to the difficulty of targeting the GTP-binding site but as methods for drug discovery and screening evolve, these hurdles may be overcome. Two RAC1 inhibitors NSC23766 and EHT1864 are currently commercially available. It is unclear how well humans would tolerate long- or medium-term global RAC1 inhibition. RAC1 is essential during mouse gastrulation (Sugihara et al., 1998), but short-term treatment with the currently commercially available Rac1 inhibitors seems to be well tolerated and effective in mice (Li et al., 2012 and our unpublished data). Given the caveat that RAC1 may prove difficult to target pharmacologically, there are inhibitors in preclinical development or in the clinic for many of its downstream effectors. PAK1 (p21 activated kinase) is downstream of RAC1 in many systems, and there are a now a number of inhibitors in preclinical development to this kinase (Ong et al., 2011). Moreover, there are inhibitors to many of other RAC1 target pathways, such as mTOR, JNK and NFkB signalling (Figure 1), for which it will be important to re-evaluate their usefulness against melanomas harbouring activating RAC1 mutations. Taken together these studies highlight the crucial role of Rac1 in melanoma. Many parallels can be drawn to the discovery of RAF mutations and the hope would be that over the next 10 yr drugs would be in the clinic that will be able to target RAC1 mutated melanoma analogous to the way that BRAF mutation led to the recent breakthroughs in melanoma therapy.