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Rho Family GTPases and Cellular Transformation

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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.

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.

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...)

Membrane Asscociation Is Critical for Rho GTPase Function

Like Ras oncoproteins, Rho proteins also undergo COOH-terminal posttranslational modification by isoprenoid lipids, a process critical for their biological activity. Both Ras and Rho GTPases terminate in a carboxyl-terminal CAAX tetrapeptide motif (where C = cysteine, A = aliphatic amino acid, and X = terminal amino acid) that is necessary and sufficient for posttranslational modification. In the first step, a farnesyltransferase (FTase; when X = serine, methionine) or the related geranylgeranyltransferaseI (GGTaseI; when X = leucine), catalyze the covalent addition of a C15 farnesyl or a C20 geranylgeranyl isoprenoid moiety, respectively (fig. 2). Although the majority of Rho GTPases are modified by GGTaseI (e.g., RhoA, Rac1, and Cdc42), others are processed solely by FTase (e.g., RhoD and RhoE/Rnd3), or by both enzymes (such as RhoB). Prenylation is followed by a proteolytic activity that cleaves the terminal AAX residues prior to carboxymethylation of the terminal prenylated cysteine. The CAAX-signaled modifications, together with an additional membrane-targeting motif directly upstream of the CAAX motif, anchor Rho GTPases to membranes, a necessary step for GTPase function.22

In light of the critical importance of prenylation for the function of Ras and Rho GTPases, pharmacologic inhibitors of FTase (FTIs) or GGTaseI (GGTIs) have been developed as novel anti-cancer therapeutics (fig. 2). Initially developed as anti-Ras drugs, the anti-tumor activity of FTIs is speculated to be mediated, in part, by targeting other GTPases such as RhoB.23 Nevertheless, FTIs have demonstrated potent anti-oncogenic activity in cell culture and animal models, and are now under evaluation in phase III clinical trials.24 Similarly, inhibitors of GGTaseI (GGTIs) have been developed as inhibitors of Rho GTPases and have also shown anti-tumor activity in experimental models and tumor xenograft studies.25

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.

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 fashion27-29 (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.

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.

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 Regulate Actin Cytoskeletal Organization

The best-characterized cytoplasmic function of Rho family proteins is their role as regulators of actin cytoskeletal organization.3,6,8 For example, Rac1 promotes the reorganization of filamentous actin into membrane structures called lamellipodia which serve as new adhesive contacts typically found at the leading edge of migrating fibroblasts.41 On the other hand, RhoA promotes the formation of actin stress fibers and focal adhesions, promoting cell attachment, whereas RhoE causes a disruption of these structures and causes cell rounding.42-44 Other GTPases such as Cdc42 promote the protrusion of finger-like actin-rich projections that act as sensors of the extracellular environment.45

Additional processes regulated by Rho proteins and requiring cytoskeletal changes extend to membrane transport, organization of tight junctions, chemotaxis, cytokinesis, and cell polarity.46-54 Collectively, these dynamic RhoGTPase-controlled cytoskeletal rearrangements promote shape adjustment and regulate cell-cell and cell-matrix contacts, processes that may influence invasion and metastasis.

Rho GTPases Regulate Transcription Factor Function and Gene Expression

A second major function of Rho family GTPases involves their regulation of the activity of a variety of transcription factors.55 For instance, the three GTPases Rac1, RhoA, and Cdc42 activate the serum response factor (SRF).56 SRF is a transcription factor that cooperates with ternary complex factors (e.g., Elk-1) to modulate the activity of serum response element (SRE)-containing promoters of many early response genes such as c-fos.57 Rho proteins also stimulate the activation of various other transcription factors such as NF-κB, E2F-1, c-Jun, ATF-2, Elk-1, AP-1, and STAT3.4,58-67 The diverse multitude of transcription factors that are regulated by Rho GTPases emphasizes that changes in gene expression form an important outcome and may be a critical mediator of Rho GTPase-induced transformation.

Rho GTPases Regulate Cell Cycle Progression and Cell Proliferation

In light of their ability to promote uncontrolled growth, it is not surprising that Rho GTPases, like Ras, also regulate cell cycle progression.5 Dominant negative Rac1, RhoA, and Cdc42 block, whereas activated forms of these GTPases stimulate, progression through the G1 phase of the cell cycle.68,69 Rho GTPases promote cell cycle progression by modulating the activity of both positive as well as negative regulators of the Rb tumor suppressor, which functions in regulating G1 progression. For example, RhoA and Rac1 up-regulate cyclin D1 gene expression34 and the transforming activity of various Dbl family oncoproteins correlated with their ability in promote cyclin D1 expression.70 Conversely, RhoA has been found to antagonize the expression of two negative regulators of G1 progression, the cyclin-dependent kinase inhibitors p21CIP1 and p27KIP1.71 For example, RhoA inhibition of p21CIP1 has been described as one mechanism by which Rho GTPases may facilitate the growth promoting actions of Ras.72

Rho GTPases and Regulation of Apoptosis

Rho GTPases have also been shown to regulate cell survival by either promoting or antagonizing apoptosis depending on the cell type and signalling contexts. Several reports have indicated that RhoA, RhoC, Rac1, Rac2 and Cdc42 proteins were involved in apoptotic stimulation in cells such as fibroblasts, thymocytes, neurons, and epithelial cells. NIH 3T3 cells expressing Rac1 seem to be sensitized to serum withdrawal through a mechanism involving up-regulation of Fas ligand transcription.73 Overexpression of RhoA also correlates with an increased ceramide production, which is a second messenger for apoptosis.74,75 In addition, RhoG is thought to mediate the toxic effects of FTI treatment and Taxol.77,76 On the other hand, there is also considerable evidence for the anti-apoptotic activities of Rho GTPases. For example, Rac1 appears to protect Rat-1 fibroblasts from serum deprivation-induced apoptosis, or MDCK epithelial cells from matrix deprivation-induced apoptosis.78,79 Inhibition of RhoA, but not of Rac1, caused apoptosis of RET/PTC1-expressing thyroid cells.80 Similarly, Cdc42 inhibition through caspase-mediated degradation of Cdc42 was suggested to facilitate Fas ligand-induced apoptosis in T47D breast cancer cell line.81 While oncogenesis clearly involves increased cell survival, how Rho GTPase regulation of apoptosis factors into Rho GTPase-mediated oncogenesis is presently not clear.

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 growth29,82,83 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.

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...)

Rho GTPases are Involved in Tyrosine Kinase Oncoprotein Transformation

The implication of Ras in transformation caused by tyrosine kinase oncoproteins has also associated Rho GTPases with this process. For example, the BCR/Abl oncoprotein renders 32D mouse myeloid cells resistant to IL-3 deprivation-induced growth cessation and apoptosis, in part, by activation of Ras. Coexpression of dominant negative Rac1 rendered Ras-transformed 32D cells sensitive to IL-3 withdrawal growth inhibition and impaired invasion in vitro and leukemogenesis in vivo.94 Along the same line, tyrosine oncoproteins rely on multiple Rho GTPases to cause transformation via Ras. For example, Fps/Fes transformation of Rat-2 fibroblasts was impaired not only by dominant negative Ras, but also by Rac1(N17) and Cdc42(N17).93

A Ras-independent requirement of Rho GTPases in tyrosine kinase-mediated transformation has also been documented. For example, although Ras was not found to be required for the transforming activity of an avian viral mutant of EGFR, dominant negative RhoA, but not Rac1 nor Cdc42, was found to block its transforming activity.137 Thus, while tyrosine kinases can cause activation of Ras, the signalling pathways that link tyrosine kinase oncoproteins with Rho GTPases may not always involve Ras activation. This possibility is supported by earlier observations that oncogenic but not normal Ras triggers the activation of Rho GTPase cascades.

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) oncogenes106,114,139,140 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.

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 Gα13 with the NH2-terminal RGS (regulator of G protein signalling) domain up-regulates the DH catalytic activity towards RhoA124,150 (fig. 7). In agreement, constitutively activated variants of Gα13 and Lsc have also been shown to cause RhoA-dependent transformation of NIH 3T3 cells.151 Other GPCRs coupled to the Gα13 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.

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...)

Aberrant Expression of Rho GTPases in Oncogenesis

Evidence for the direct involvement of Rho GTPases in human cancer development, although less well documented, is rapidly accumulating (Table 1). For example, human breast, colon, and lung tumors exhibit high protein expression levels of Rac1, Cdc42, and in particular RhoA protein, when compared to normal tissue from the same patient.155 Increased levels of RhoA mRNA was detected in testicular germ cell tumor tissue and correlated with tumor progression.156 Similarly, a recent study also indicated that RhoA and Rac2 were largely overexpressed in head and neck cancer tissues and proposed their use as markers for squamous cell carcinoma of the head and neck.157 In addition, Rac3 was found to be hyperactive in human breast tumor cell lines and appears to be required for breast cancer cell proliferation.158

A role for another Rho family member, RhoC, in human cancer metastasis and invasion has been uncovered in melanoma cell lines.159 RhoC overexpression was found in 90% of inflammatory breast cancer and may promote tumor angiogenesis.160,161 RhoC was also found to be overexpressed in adenocarcinomas of the pancreas and its high levels of expression correlated with poor prognosis.162

A recent addition to the Rho family, a Cdc42-homolog termed Wrch-1, was found to be regulated by Wnt-1 oncogenic signalling in human mammary tumors.163 Overexpression of Wrch-1, like Wnt-1, promoted morphological transformation of mouse mammary epithelial cells. Taken together, Wrch-1 could mediate the transforming effects of Wnt-1 signalling in the regulation of cell morphology, cytoskeletal organization, and cell proliferation.

The identification of mutant Rho proteins in human cancer specimens has so far been limited to two examples. The first represents a fusion of the Rho family member RhoH/TTF to the lymphoma-associated LAZ3/BCL6 gene, a mutation identified in nonHodgkin's lymphoma (NHL) cell lines164,165 and was later found in patients with NHL and multiple myelomas.166 The second example is that of Rac1b, a novel splice variant of Rac1, found to be aberrantly expressed in human colorectal167 and breast168 carcinomas, establishing a direct link between Rho GTPase activation in general, and Rac in particular, and tumor development in humans. Rac1b contains an additional 19 amino acid insert just before the switch II region of Rac1, and in vitro analyses indicated that this variant exhibits a fast-cycling guanine nucleotide exchange activity, suggesting that it may be an activated variant of Rac1.

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.

RhoA Transformation and ROCK

Effector domain mutants have provided critical clues regarding what effector signalling pathway is important for RhoA transformation. These analyses have dissociated transformation from a variety of effector-mediated signalling pathways such as stress fiber formation and SRE activation.170,171 Interestingly, these studies have also shown strong correlations between the ability of RhoA to bind ROCK, and its ability to cooperate with Raf to cause transformation of NIH 3T3 cells.170 Indeed, treatment with the Y-27632 pharmacologic inhibitor of ROCK impaired RhoA transforming activity, as well as the transforming activity of oncoproteins (Ras, Dbl, and Net) that activated RhoA.170 In addition, a role for ROCK in facilitating RhoA-mediated invasion has been documented. Itoh and colleagues showed that dominant active ROCK promoted invasion of MM1 hepatoma cells, whereas inhibition of ROCK activity by Y-27632 reduced the ability of these cells to invade in mice.172 On the other hand, the fact that activated ROCK does not reproduce the same transforming activity seen with activated RhoA suggests that additional effectors are likely to contribute to RhoA transformation. This may involve, for example, an effector(s) that interacts with the unique insert domain of RhoA, a region required for transformation but not for ROCK binding.173

Role of Reactive Oxygen Species (ROS) NF- κB and Par-6 in Rac1 Transformation

Effector domain mutants have also been used to address the importance of specific effector signalling functions in Rac1 transformation. PAK binding, as well as induction of lamellipodia, activation of the JNK and p38 MAPKs, SRF, the MEKK1 and p70 S6 kinases, were all shown to be dispensable for Rac1-induced transformation of NIH 3T3 cells.34,174,175 PAK and JNK activation were also dissociated from the ability of Rac1 to induce cell cycle progression.176 Since a role in growth induction as well as apoptosis has been established for several of these pathways, it is likely that multiple signalling pathways may also contribute to Rac1-induced cellular transformation.

Rac1 is distinguished from RhoA and Cdc42 in its ability to activate the phagocyte NADPH oxidase system177,178 by interacting with and modulating the activity of the p67 subunit of the complex.179 Rac also activates a related oxidase system in nonphagocytic cells by an as yet to be identified effector. The reactive oxygen species (ROS) by-products of this reaction induce NF-κB activation, a process inhibited by ROS inhibitors such as N-acetylcystine (NAC).58 ROS have been shown to be important second messengers for Ras-mediated transformation180 and Rac-mediated cell cycle progression.181 Deletion of the Rho insert region of Rac1 impaired its mitogenic activity and ROS production in certain cell types. This finding, together with the observation that inhibitors of ROS production inhibit Rac1 mitogenic activity, support an important contribution of ROS in Rac transformation, possibly by its ability to activate the NF-κB-mediated cell survival pathway. NF-κB was found to be required for the transforming activity of two Dbl oncoproteins, Dbl and Dbs (GEFs for RhoA and Cdc42), providing further evidence for the importance of this signalling pathway in Rho GTPase transformation.182

A novel effector for Rac1 and Cdc42 has been identified as the human homologue of the cell polarity determinant in Caenorhabditis elegans, hPar-6, and has also been linked to cellular transformation.183,184 Rac1 and Par-6 associate with an atypical protein kinase C isoform, PKCζ, an interaction that releases the kinase activity of PKCζ leading to transformation. Additionally, Par-6 has been suggested to play an important role in the polarization of mammalian cells by functioning as an adaptor protein that links activated Rac and Cdc42 to PKCζ signalling.185,186 While it was found that PKCζ potentiated Rac1 transformation, whether PKCζ is a critical effector of Rac and Cdc42 transformation remains to be clarified.183

Cross-Talk between Vesicular Transport and Cdc42 Transformation

Cerione and colleagues identified the γ-subunit (γ-COP) of COPI, a cytoplasmic ‘coatomer’ protein complex as an effector important for Cdc42 transformation.187 The coatomer coats membrane regions that bud and form vesicles, a process controlled by the Arf small GTPase. Interactions between Arf and COPI direct the formation of vesicles that are involved in the selective transport of proteins from the Golgi complex back to the endoplasmic reticulum (ER) and in protein and lipid recycling from the plasma membrane through endosomes.3 Interestingly, Cdc42 mutants that no longer bound to γ-COP or affect coatomer interaction with the Golgi (or perhaps endosomal compartments) were also unable to cause growth transformation. However, the nontransforming insert mutant of Cdc42188 retained the ability to bind γ-COP and promote vesicle trafficking, suggesting that γ-COP may not be sufficient to mediate transformation by 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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