Members of the Ras-related Rho family of GTPases act as key signalling nodes that regulate a variety of crucial cellular activities including morphology, motility, and proliferation. Their aberrant expression causes uncontrolled growth and transformation, and they are required for the transforming actions of Ras and other oncoproteins. However, whereas the identification of mutated Ras proteins in human cancers prompted immediate and intense interest in investigating their role in tumor formation, the evidence for the involvement of Rho GTPases in oncogenesis has come mainly from experimental cell culture and animal studies. Nevertheless, novel evidence for a direct link between Rho proteins and cancer development is beginning to accumulate, rendering them potential targets for drug discovery and cancer intervention.

Introduction

Malignant transformation is a complex multifaceted process involving systematic alterations in a variety of cellular properties. Cancer cells are characterized by deregulated cell cycle control, reduced contact inhibition, loss of matrix-dependent growth regulation, increased cell survival, morphologic alteration, increased motility, and acquisition of invasive and metastatic properties. Rho family GTPases act as key regulators of all of these cellular processes and mounting evidence implicates aberrant Rho GTPase function in human oncogenesis. Like the Ras oncoproteins, Rho GTPases function as regulated GDP/GTP switches that relay extracellular cues to cytoplasmic signalling cascades. In contrast to Ras proteins however, where direct mutational activation of Ras is prevalent in human cancers, much of the evidence linking Rho GTPases to oncogenesis is indirect, and implicates Rho proteins as critical downstream signalling components of a diverse array of oncoproteins. Recent reviews have provided excellent overviews on various facets of normal Rho GTPase function.^1-5 Therefore, in this review we have especially focused on a summary of the evidence linking Rho GTPases to oncogenesis.

The Rho Family of GTPases Is a Major Branch of the Ras Superfamily

The Rho family constitutes one of the major branches of the Ras superfamily of small GTPases. Presently, 18 mammalian members have been described that share significant sequence identity ranging from 50 to 90%, with an overall 25% identity with Ras. This family is further subdivided into several groups and include the Rho (isoforms A, B, and C), Rac (isoforms 1, 2, 3), Cdc42 (and Wrch-1), TC10 (and TCL), Rnd (isoforms 1, 2, and 3), RhoD, RhoG, and TTF subfamilies. The most heavily studied and best characterized members are Rac1, RhoA, and Cdc42.^6-8 Rho GTPase function is conserved in evolution, with members identified in yeast, plants, slime mold, worms, and flies.

Rho GTPases Function As Membrane-Associated GDP/GTP-Regulated Molecular Switches

In addition to sharing strong primary amino acid sequence identity, Ras and Rho GTPases share substantial structural, biochemical, and functional similarities. In the sections below, we have summarized these similarities (as well as differences) with regards to their function as regulated GDP/GTP signal transducers.

The Rho GDP/GTP Cycle Is Regulated by GEFs and GAPs

Like Ras, Rho GTPases act as binary switches that cycle between active GTP-bound and inactive GDP-bound states (fig. 1). The GTP-bound forms complex with and activate multiple downstream effector target molecules to stimulate cytoplasmic signalling cascades. Subsequently, an intrinsic GTP hydrolysis activity regenerates the GDP-bound form terminating the active state.

Figure 1

Rho GTPases are regulated molecular switches. Similar to Ras, Rho family proteins shuttle between the inactive GDP-bound and the active GTP-bound states. Activation is supported by guanine nucleotide exchange factors (GEFs) which promote exchange of GDP (more...)

The intrinsic GTP hydrolysis and the GDP/GTP nucleotide exchange reactions of Rho and other Ras superfamily GTPases are both very slow, and require the enzymatic assistance of two families of regulators: GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) (fig. 1). GAPs enhance the intrinsic GTP hydrolysis rate and therefore attenuate Rho protein activity by promoting the GDP-bound state.⁹ More than 20 Rho GAPs have been identified to date.¹⁰ Among these is BCR, which was identified originally as the translocation partner for the Abl tyrosine kinase in the Philadelphia Chromosome translocation found in a majority of human chronic myelogenous and acute lymphocytic leukemias.¹¹

GEFs act as positive modulators by catalyzing the exchange of GDP for GTP, thereby increasing the GTP-bound protein levels in the cell (fig. 1). Rho GEFs, like Ras GEFs, eject the bound nucleotide (GDP or GTP) to promote the formation of the nucleotide-free form of the GTPase. However, since there is about 15-fold excess of GTP versus GDP in the cell, the net result of GEF-mediated exchange is to enhance formation of the GTP-bound protein.¹² Rho GEFs are also referred to as Dbl family proteins and are named after the founding member characterized originally as a transforming protein from a human diffuse B-cell lymphoma (Dbl) cell line.^13,14 To date, over 60 Dbl family proteins have been identified, and like Dbl, many were identified initially in screens for novel transforming oncoproteins. All Dbl family proteins contain a Dbl homology (DH) domain that catalyses GDP/GTP exchange, as well as a pleckstrin homology (PH) domain that regulates DH domain function.^15,16 The tandem DH/PH domains are typically flanked by different modules (SH2, SH3, calponin homology (CH) domains, kinase domains, etc.) that contribute to the DH domain activation through diverse signalling activities.

Unlike Ras, Rho GTPases are under the control of a third functional class of regulators called guanine nucleotide dissociation inhibitors (GDIs) comprising three members: GDI1 (RhoGDI or GDIα), GDI2 (D4/Ly-GDI or GDIβ), and GDI3 (GDIγ).¹⁷ GDIs inhibit Rho GTPase signalling by exerting three concerted functions (fig.1 and fig.2). First, they inhibit the nucleotide dissociation and block the GDP/GTP exchange, therefore antagonizing GEF activity.^18-20 Second, GDIs interfere with GTP hydrolysis and hinder GAP activity.²¹ Finally, perhaps the most important function of GDIs involves their stimulation of the release of Rho GTPases from cellular membranes (fig. 2). The net effect is the transfer of the Rho protein from membrane compartments, where it is active, to the cytosol, shutting down the GTPase activation altogether.

Figure 2

Regulation of Rho proteins by membrane association. Rho proteins undergo posttranslational modification by farnesyltransferases (FTase) (e.g., RhoE/Rnd3), geranylgeranylases (e.g., Rac1), and carboxymethyltransferase. FTase and GGTaseI recognize carboxyl (more...)

Rho GTPase Mutants Are Molecular Tools for Dissection of Function

Alterations of specific residues in Rho GTPases can genetically modify their ability to interact with their regulators, effectors, or with membranes.²⁶ Depending on the residue, these mutations can either generate dominant activated, inactive, or dominant negative GTPase. In particular, two classes of mutants have provided powerful reagents to evaluate the signalling and biological functions of Rho GTPases (fig. 3).

Figure 3

Activated versus dominant negative Rho GTPases. The GAP-insensitive (G12V and Q61L) mutants are constitutively GTP-bound, and have therefore been used to characterize the different cellular functions of a variety of GTPases. By inhibiting Rho GEF function, (more...)

Mutations analogous to those found in tumor-associated mutants of Ras (e.g., G12V and Q61L) interfere with the intrinsic GTP hydrolysis reaction, and de-sensitize the G-protein to GAP regulation, resulting in constitutively active GTP-bound proteins that are activated in a ligand-independent fashion^27-29 (fig. 4). A second type of activated mutants is based on the Ras(F28L) variant, which shows decreased affinity for guanine nucleotides and results in a “fast-cycling” protein.³⁰ Their increased intrinsic nucleotide exchange rate, coupled with excess in cellular GTP, favor the formation of GTP-bound proteins. Like the GAP-deficient mutants, F28 versions of Ras, Cdc42, RhoA, and Rac1 are constitutively activated and transforming.³¹

Figure 4

Deregulated Rho GTPase mutants are tools for functional studies. Dominant, gain-of-function (G12V, F28L, and Q61L), or dominant-negative (S17N) mutants of Rho GTPases have been developed based on Ras mutants. The majority of studies have evaluated the (more...)

Another important class of GTPase mutants is those that display a dominant-negative phenotype.³² These mutants have been essential reagents that implicated Rho GTPases as mediators of oncogenesis.³³ In particular, the S17N mutant of Ras has been the prototype for analogous versions in Rho proteins. Introducing an aspargine at position 17 (17N) favors formation of nucleotide-free GTPases that bind GEFs with higher affinity than their wild type counterparts. Hence, they prevent GTPase activation via inhibition of GEF function. When introduced in cells, Rac1(17N) for instance, would compete with the endogenous Rac for GEF binding, forming tight complexes unable to relay effector-mediated downstream signalling. As described in the following sections, both dominant activating and dominant negative mutants of Rho GTPases have been instrumental in linking Rho GTPases to oncogenesis.

A third functional class of mutants are those that are impaired in downstream effector interaction²⁶ (fig. 4). As described below, Rho GTPase function is mediated through multiple downstream effector targets. Although all effector interactions are dependent on a core effector domain (corresponding to Ras residues 32-40), select missense mutations in the Rho protein core effector domain can result in differential impairment in effector binding (e.g., see refs. 34 and 35). Such mutants have provided powerful reagents to assess the contribution of a particular effector to a specific GTPase signalling and/or function.

In contrast to the Ras superfamily of proteins, Rho GTPases possess an additional ∼13 amino acid “insert region” positioned between amino acids corresponding to Ras residues 122 and 123.³⁶ Although the insert region is distant some 18 _om the switch regions and the effector domain, it still participates in effector-mediated functions, such as cytoskeletal remodeling.³⁷ The sequence and structural divergence of the insert domain between different Rho GTPases suggests it may serve very distinct roles, particularly in transformation.^37-39

Rho Family GTPases Are Critical Components of Diverse Signalling Pathways

A large and diverse variety of cell surface agonists utilize Rho GTPases to modify cellular behavior.⁴⁰ An expanding body of evidence has implicated Rho proteins in growth factor, cytokine, cell-cell as well as cell-extracellular matrix signalling. The ligand-activated receptors are thought to regulate Rho GTPase activation mainly via activation of Rho GEFs, although in some situations regulation of Rho GAP activity has been described. Once activated, the GTP-bound Rho GTPase interacts with a wide spectrum of downstream effectors, initiating signalling cascades that regulate events in the cytoplasm and the nucleus (fig. 5). In the following sections, we have described the cellular responses regulated by signalling cascades that may contribute to the aberrant growth properties of cancer cells.

Figure 5

Extracellular signal activation of Rho family GTPases. Rho GTPases function downstream of a variety of extracellular stimuli. For example, Rho proteins are activated by ligand-stimulated G protein-coupled serpentine receptors (GPCR), receptor tyrosine (more...)

Rho GTPases Promote Oncogenic Transformation

Several lines of evidence have implicated Rho GTPases in cellular transformation. First, Rho GTPases are required for Ras-mediated oncogenesis.^29,82-88 Rho proteins are also required for the functions of other oncoproteins such as polyoma virus middle T antigen, tyrosine kinases such as Abl, Met, Fps, BCR-Abl, RET, epidermal growth factor receptor, insulin-like growth factor receptor, G protein-coupled receptors (Mas, G2A, M1-muscarinic receptor, and Par-1), and Dbl family oncoproteins (see below).^80,89-101 Finally, Rho GTPases are associated with tumor suppressor function. For example, the NF2 tumor suppressor merlin may mediate its actions, in part, by inhibiting the functions of Rac.¹⁰² Moreover, the hematopoietic-specific Rho GTPase Rac2 was shown to cooperate with Ras to promote the proliferation of mast cells heterozygous for the tumor suppressor neurofibromin (NF1).¹⁰³

Second, ectopic expression of dominant active forms of Rho GTPases, analogous to the oncogenic mutants of Ras (e.g., G12V and Q61L), cause growth and morphologic transformation, and tumorigenic and metastatic growth of rodent fibroblasts.^{29,82,83,85,104,105} Similarly, activated Rac1 was shown to induce anchorage-independent growth^29,82,83 and to promote motility, invasion, and metastasis in a variety of cell types, including fibroblasts, T lymphoma, epithelial, and melanoma cells.^106-109 In the following sections below, we provide a more detailed discussion of the evidence that implicates Rho GTPases in the transforming functions of Ras, Dbl family oncoproteins, and GPCRs, as well as the emerging evidence that Rho GTPases themselves are aberrantly expressed in human cancers (see below).

Rho GTPases Are Critical for Ras-Mediated Oncogenesis

The evidence implicating Rho GTPases as critical mediators of oncogenic Ras function has come mainly from studies of NIH 3T3 and Rat-1 rodent fibroblast cell lines. First, it was found that dominant negative versions of Rac1, RhoA, RhoB, RhoG, Cdc42, and TC10 blocked Ras-induced transformation of rodent fibroblasts.^29,82-87,110 Second, constitutively activated mutants of Rho GTPases were shown to cooperate with activated Raf to cause synergistic transformation of rodent fibroblasts. Similarly, coordinate expression of activated Rac1 and MEK1 caused synergistic growth transformation of FRTL-5 rat thyroid epithelial cells.¹¹¹ Finally, like Ras, Rac and Raf cooperated with E1A to cause transformation of primary BRK rat epithelial cells.¹¹² These observations suggested a model where Ras activation of Rho GTPases via a Raf-independent effector(s) pathway can promote various aspects of Ras transformation of both fibroblasts and epithelial cells.

Studies with activated or dominant negative Rho GTPase mutants have delineated the contribution of specific Rho family GTPases to distinct aspects of the transformed phenotype caused by oncogenic Ras. Although activated Rho GTPases are not sufficient to reproduce the drastic morphologic transformation caused by activated Ras or Raf, NIH 3T3 cells stably-expressing activated RhoA or Rac1, or Rat-1 cells expressing activated Cdc42, grow in anchorage-independent conditions and form tumors in nude mice, indicating that each GTPase alone can contribute to the growth-promoting actions of oncogenic Ras.^29,31,84 Furthermore, analyses of Ras transformation of Rat-1 fibroblasts indicated that while RhoA, Rac1 and Cdc42 all contribute to Ras induction of anchorage-independent growth, Rac1 contributed to the reduced growth factor requirements, whereas Cdc42 and RhoA were required for morphologic transformation.^82-84

Interestingly, there is evidence that Rac1 may also contribute to Ras-mediated transformation by blocking apoptosis.¹¹³ Similarly, Symons and colleagues determined that Rac1 protected MDCK epithelial cells⁷⁹ and Rat1 cells⁷⁸ against apoptosis induced by matrix deprivation. Finally, activated Rac1 can promote the invasion and metastasis of a variety of cell types, suggesting that the invasive properties of Ras may be mediated, in part, by activation of Rac1.^107,114

Whether Rho GTPases are found to be constitutively activated in Ras-transformed rodent fibroblasts, and whether Rho GTPases are important for Ras transformation of epithelial cells, are issues that have been addressed with conflicting results, and are yet to be fully resolved. Earlier microinjection analyses showed that transient expression of oncogenic Ras caused activation of Rac, which in turn caused activation of RhoA.^41,42 However, Ras-transformed rodent fibroblasts lack well-developed actin stress fibers, an observation that is inconsistent with an up-regulated RhoA activity. In fact, activated RhoA restored stress fibers to Ras-transformed cells, arguing for a down-regulated Rho function.¹¹⁵ Similarly, inhibition of RhoA/B/C function in mouse T cells by the C3 toxin promoted the development of malignant thymic lymphoblastic lymphomas, consistent with the model that the loss of Rho function supports oncogenesis.¹¹⁶ On the other hand, Rho activation in Ras-transformed MDCK cells was found to contribute to their transformation from an epithelial to a mesenchymal morphology, an observation consistent with RhoA up-regulation in Ras-transformed cells.¹¹⁷ Finally, as described below, observations from RhoA-specific GEF studies as well as others using pharmacologic inhibition of RhoA signalling, provide further evidence that up-regulation, rather than down-regulation, of RhoA activity is consistent with promoting oncogenesis.

Similarly, Ras-transformed cells show increased membrane ruffling, which corresponds to an up-regulated Rac1 activity by oncogenic Ras.¹¹⁸ Furthermore, transient expression of oncogenic Ras was shown to increase Rac1-GTP levels in COS-1 cells.¹¹⁹ In contrast, Collard and colleagues determined that sustained activation of Ras down-regulated Rac activity in MDCK epithelial cells.¹¹⁷ Interestingly, activated Rac inhibited cell migration in these cells, suggesting an invasion-suppressing, rather than promoting, function for Rac.¹²⁰ It was argued that while transient Ras activation can cause Rac activation, sustained Ras stimulation leads instead to down-regulation of Rac activity. Finally, Nur-E-Kamal and colleagues showed that Cdc42-GTP levels were indeed up-regulated in Ras-transformed NIH 3T3 cells and that stimulated receptor tyrosine kinases that activate Ras also activate Cdc42.¹²¹ In accordance, inhibition of Cdc42 by the Cdc42-specific GTPase binding domain of ACK-1 blocked Ras transforming activity. Thus, similar to Ras, Rho GTPases may have positive or negative roles in growth regulation that will be influenced by cell type differences and/or genetic context.

The signalling pathways that link Ras with Rho family GTPases are currently incompletely understood.^122-124 Several possible links between Ras and Rac have been suggested (fig. 6). First, both Ras and Rac1 associate with the two subunits of phosphatidylinositol 3-kinase (PI3K) p110 and p85, respectively. Ras binding to p110 catalytic subunit may regulate the association of p85 with Rac1, which may determine Rac1 function.^125,126 Additionally, Ras activation of PI3K leads to the production of lipid products such as phosphatidylinositol 3,4,5-trisphosphate (PIP₃), which in turn may interact with the PH domain of Dbl family proteins to promote GEF activity. For example, PI3K activation has been described to cause activation of Vav and Tiam1.^127,128 Second, the fact that the Ras exchange factor Son of sevenless (Sos), which in addition to the Cdc25 Ras GEF domain also contains a functional DH/PH unit that exhibits some activity towards Rac1 suggests that Sos-dependent Ras activation may potentially lead to Sos-dependent Rac1 activation.¹²⁹ Another mechanism has been proposed where the activation of Grb2/Sos1 leads to the formation of a complex (Eps8-E3b1-Sos1) able to bind and possibly modulate Rac1 activity.¹³⁰ Third, Ras activation of its effector RalGDS (for Ral guanine nucleotide dissociation stimulator) leads to the activation of the Ral small GTPase, which in turn binds a Rac/Cdc42 GAP (RalBP1/RIP1/RLIP76), and consequently, may modulate its activity towards Rac1.^131,134 Several reports have emphasized the importance of the RalGDS pathway in Ras transformation of mouse and human cells.^135,136 Depending on the cell type and the mechanisms used, these observations place Rho GTPases at the point of convergence of a multitude of membrane-triggered events ultimately determining cellular fate.

Figure 6

Linking Ras to Rho. Three main pathways relate Ras signalling events to Rho. Growth factor mediated activation of Sos leads to Ras activation which can (1) activate RalGEF/Ral pathway, leading to activation of a RhoGAP and inhibition of Rho activation. (more...)

Dbl Family Oncoproteins Are Activators of Rho GTPases

Further evidence that up-regulated Rho GTPase activity promotes oncogenesis comes from the observations that many Dbl family proteins were identified initially as oncoproteins before they were characterized as Rho GEFs.^15,16,138 Many Dbl family proteins were identified initially in biological screens for novel transforming (e.g., Dbl, Vav, Ect2, Tim, Net1, Lfc, Lsc) or invasion-inducing (Tiam1) oncogenes^{106,114,139,140} and in most cases, structural alterations were identified as the mechanism of activation of their transforming activities. For example, amino terminal truncation of negative regulatory sequences accounts for the formation of constitutively activated and transforming mutants of Dbl, Vav, Ect2, and Tiam1. However, despite their identification in screens of tumor cell-derived DNA or RNA, these genetic alterations occurred during the transfection procedure and did not originate from the patient tumor sample.

Despite the tantalizing links between Dbl family proteins and oncogenesis, evidence that Dbl family proteins are aberrantly activated in human cancers has been limited to three examples (Table 1). Recently, mutations in Tiam1 have been reported in human renal carcinomas leading to constitutive activation of the protein.¹⁴¹ The two other Dbl family members were identified initially as proteins whose genes are rearranged in human leukemias. First, the product of the breakpoint cluster region (BCR) gene is truncated and forms a fusion protein with the Abl tyrosine kinase caused by the Philadelphia chromosome translocation, an aberration associated with about 95% of chronic myelogenous leukemias.¹⁴² Although some versions of the BCR-Abl fusion protein do contain the Rho GEF catalytic domain, these versions are less transforming than those that lack this domain. While BCR-Abl has been found to activate Rac, this has been attributed to its association with and activation of the Vav Dbl family protein.¹⁴³ Second, the leukemia associated RhoGEF (LARG) was also identified as a gene rearranged in mixed lineage leukemia (MLL) and encoded a chimeric fusion protein with MLL.¹⁴⁴ LARG has been subsequently shown to cause growth transformation of rodent fibroblasts via activation of its target GTPase RhoA.^145,146 While these rearrangements mimic those that render other Dbl family proteins constitutively activated, there is no current evidence that these BCR or LARG fusion proteins promote oncogenesis by causing persistent activation of Rho GTPases.

Table 1

Aberrant Rho GTPase activity in human cancers.

Finally, there are preliminary observations that altered expression of RhoGDI may be involved in oncogenesis (Table 1). Decreased mRNA expression of RhoGDI2 was associated with a more invasive variant of the T24 bladder cancer cell line¹⁴⁷ suggesting a role for Rho activation in metastasis and invasion. In contrast, proteomic analyses identified RhoGDI protein overexpression in invasive ovarian cancers, suggesting that loss of Rho activity may promote tumor progression.¹⁴⁸ However, whether Rho GTPase activity was altered by changes in RhoGDI activity were not determined in these studies.

Transforming G Protein-Coupled Receptors Are Linked to Rho GTPases

Investigations into the mechanisms of receptor signalling have uncovered a pivotal role for Rho GTPases in heterotrimeric G protein-coupled receptor (GPCR) oncogenesis. The orphan GPCR Mas, initially identified in a genetic screen for transforming proteins, provided the first evidence for the transforming potential of GPCRs.¹⁴⁹ Mas was later shown to cause lamellipodia formation as well as Rac-dependent transformation of NIH 3T3 cells.⁹⁹ However, the fact that Mas is normally expressed in nonmitotic neural cells raises the possibility that it may not play an important role in oncogenesis.

The link between GPCRs and Rho GTPases has been best delineated for the p115 RhoGEF/ Lsc, where association of activated G_α13 with the NH₂-terminal RGS (regulator of G protein signalling) domain up-regulates the DH catalytic activity towards RhoA^124,150 (fig. 7). In agreement, constitutively activated variants of G_α13 and Lsc have also been shown to cause RhoA-dependent transformation of NIH 3T3 cells.¹⁵¹ Other GPCRs coupled to the G_α13 heterotrimeric subunit are G2A and Par-1. Like Mas, G2A and Par-1 were also identified in NIH 3T3 expression library screens for novel transforming proteins. Both G2A and Par-1 activate RhoA and their transforming activities were shown to require Rho GTPase activation.^100,152 Interestingly, G2A expression is up-regulated by BCR-Abl, and Par-1 overexpression has been associated with tumor cell invasion.^153,154

Figure 7

Heterotrimeric G protein activation of Rho. The lysophosphatidylcholine-stimulated G2A GPCR causes activation of RhoA by activation of the p115RhoGEF/Lsc Dbl family protein. The G2A-activated Gα₁₃ subunit binds the regulator of G protein signalling (more...)

Rho GTPases Mediate Transformation through Multiple Effectors

The conformations adopted by switch 1 and switch 2 in the active GTP-bound state enable high affinity interactions with multiple downstream molecules termed effectors which transmit their signals downstream.^1,6,7,169 For example, over 25 known or candidate effectors of Rac1 have been identified to date. In addition to the complexity provided by multiple effectors, the fact that effector utilization can be shared by different Rho GTPases adds another level of regulatory complication to Rho GTPase function. For instance, despite their distinct functions, Rac1 and Cdc42 share a wide spectrum of effectors.¹ The majority of these effector-mediated signalling pathways remain to be fully delineated, but enough evidence points to the involvement of few key effectors in Rho GTPase transformation. In the following sections, we summarize what is known regarding the effectors and signalling activities important for the transforming actions of RhoA, Rac1, and Cdc42.

Conclusions and Future Directions

While establishing the importance of Rho GTPases in human carcinogenesis will require further experimental support and validation, the vital role these GTPases play in diverse cellular functions establish them as key components of malignant tumor progression. As mediators of a variety of oncoproteins, Rho proteins represent attractive targets for intervention and inhibition of oncogene function. GGTIs represent one possible approach for blocking the action of Rho GTPases. However, in light of the many other cellular proteins that are also substrates for GGTaseI, it remains to be determined whether more precise approaches will be needed to block Rho GTPase function. Information regarding the effectors and downstream signalling pathways that promote transformation by Rho GTPases remains limited. This task is certain to be complicated by the expanding ensemble of effectors and the plethora of cellular functions attributed to Rho GTPases. Regulation of gene expression is an important consequence of Rho GTPase function and the critical gene targets that mediate Rho GTPase activities will need to be identified. Further delineation of these pathways may characterize more precise points of therapeutic intervention. Finally, while most of the attention has been focused on Rac1, RhoA, and Cdc42, the emergence of other GTPases such as RhoC and Rac3 as important GTPases in cancer suggest that the involvement of other “nonclassical” members of the family deserves more attention.

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