In the last decades, several signaling pathways have been identified as key molecular regulators of cell behavior, among them the Rho GTPases. The Rho family of GTPases is a family of small (~21 kDa) monomeric G proteins that belong to the Ras superfamily of GTPases. The Ras superfamily of GTPases comprises more than 50 members that share several common features: their molecular weight (18–28 kDa), their C-terminal polyisoprenylation region and the property to bind to and hydrolyze guanine nucleotides (1).

Rho GTPases are key regulators of cytoskeletal structure and dynamics influencing cell adhesion, morphology and progression through the cell cycle. They act as a molecular switch cycling between an inactive GDP-bound and an active GTP-bound state. The Rho GTPases form 8 subfamilies; one subfamily comprises Rac1, Rac2, Rac3 and RhoG; the second subfamily contains Cdc42, TC10 (also known as RhoQ) and TC10-like protein (TCL; also known as RhoJ); a third subfamily includes CHP (also known as RhoV) and WNT1-responsive Cdc42 homologue-1 (WRCH1; also known as RhoU); a fourth subfamily involves RhoH; a fifth subfamily embraces RhoBTB1 and RhoBTB2; a sixth subfamily embodies RhoA, RhoB and RhoC; a seventh subfamily consists of RND1, RND2 and RND3 (also known as RhoE); and the last subfamily is composed by RAP1-interacting factor-1 (RIF; also known as RhoF) and RhoD (2).

Up to date three members of the Rho GTPase family have been studied in detail: Cdc42, Rac1 and RhoA. These are all the classical Rho GTPases that cycle between active GTP-bound forms and inactive GDP-bound forms.

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in a process dependent of Mg²⁺. In most cases, members of the Ras superfamily when they are in their active state, they transmit signals by binding to a specific set of proteins termed effectors, leading to a signaling cascade. The Rho GTPases have functional domains in common, similar to those of Ras. Such domains consist of four regions that take part in the binding and hydrolysis of guanine nucleotides (G1, G3, G4 and G5), a G2 region involved in the interaction with effectors (3) and a terminal CAAX box (being C a cysteine residue, A an aliphatic residue and X any other amino acid) (4). The CAAX box acts as a signal for protein prenylation which involves the addition of C₁₅ (farnesyl) or C₂₀ (geranyl-geranyl), a phenomenon which is catalyzed by protein prenyl-transferases. Following prenylation, additional enzymatic processing including proteolysis and methylation occurs (5). While not as common, some Rho family members need the covalent addition of a palmitate acyl chain to their C-terminal hypervariable domain found immediately adjacent to the CAAX box. Similar to prenylation, palmitoylation plays a pivotal role in directing these Rho family members to membrane-associated states (6). Post-translational modifications do not affect GTPase activity but are essential for their biological function since they control their anchorage to cell membrane, therefore, their localization inside the cell (7).

Rho proteins are highly regulated. There are three types of regulatory proteins: i) GEFs: which stands for guanine-nucleotide exchange factors that catalyze the release of GDP and the subsequent uptake of GTP. ii) GAPs: (also known as GTPase-activating proteins or GTPase-accelerating proteins) are a family of regulatory proteins whose members can bind to activated G proteins and stimulate their GTPase activity, with the result of terminating the signaling event; and iii): GDIs or GDP-dissociation inhibitors which regulate the GDP/GTP exchange reaction of the Rho proteins by inhibiting the dissociation of GDP from them (8).

The small GTPases of the Rho family are master regulators of actin cytoskeleton rearrangements and cell morphology. RhoA can be activated by extracellular ligands (such as lysophosphatidic acid) and its activation results in the assembly of stress fibers and focal adhesion complexes. Rac1 is activated in response to various stimuli including growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or insulin, leading to lamellipodia formation and the constitution of a motile cell surface that contains a meshwork of newly polymerized actin filaments known as membrane ruffling or cell ruffling. Rac1 also plays a pivotal role in promoting the formation of nascent focal complexes (9).

Integrin-mediated cell-extracellular matrix adhesion activates Rac1, which directly binds and activates p21-activated kinases (PAKs) and other effectors including phosphatidylinositol-4-phosphate 5-kinase, Nap125, PIR121 and IRSp53, resulting in increased membrane protrusions and actin polymerization. On the other hand, activated Cdc42 phosphorylates PAK1 and PAK2, which in turn lead to filopodia formation (10–13). These changes either by themselves or integrated can affect gene expression, cell cycle progression and apoptosis. Although, different cell processes are modulated by Rho GTPases, it is important to highlight that the cellular events are regulated by integrated signaling networks and not by a single signaling cascade. Then, the same stimuli in different cell contexts could produce different responses.

An example of cross talk between Rho GTPases and other regulating pathways is the regulation of Rac1-GAP β2-chimaerin by the protein kinase C (PKC). Rac1-GAPs β2-chimaerins are activated by the lipid second messenger diacylglycerol upon activation of tyrosine kinase receptors. It has been described that epidermal growth factor (EGF) causes rapid phosphorylation of β2-chimaerin via PKCδ retaining it in the cytosol and preventing its translocation to membranes (14). Also, there is evidence of intercommunication between the different Rho GTPases, whereas RhoA and Rac1 regulate each other, then modulating different cell processes (15,16).

Approximately 1% of the human genome encodes proteins that either regulate or are regulated by interactions with members of the Rho family of small GTPases. The wide variety of biological functions of the different members of the Rho GTPases is due to the binding to different effectors. More than 50 effectors of RhoA, Rac1 and Cdc42 are known including serine-threonine kinases, tyrosine-kinases, lipid-kinases, lipases, oxidases and structural proteins (scaffold proteins) (17).

At least two Rho effectors have been studied in detail: the Rho-associated protein kinases (ROCKs) and the formin mDia1 (mammalian homolog of the Drosophila Diaphanous protein) (18). ROCK1 and ROCK2 share 65% overall homology and 92% homology in the kinase domain (19). Both are serine/threonine kinases that are downstream targets of the small GTPases RhoA, RhoB and RhoC. Structurally they contain a catalytic kinase domain at the N-terminus, a central coiled-coil domain, which includes the Rho-binding domain (RBD) and a C-terminal pleckstrin-homology (PH) domain. ROCKs are involved in diverse cellular activities including actin cytoskeleton organization, cell adhesion and motility, proliferation, apoptosis, remodeling of the extracellular matrix (ECM) and smooth muscle cell contraction. Formins also regulate the actin and microtubule cytoskeleton and are involved in various cellular functions such as cell polarity, cytokinesis, cell migration and serum response factor (SRF) transcriptional activity. Some formins can contain a GTPase-binding domain (GBD) required to bind to Rho GTPases and a C-terminal conserved DRF auto-regulatory domain (Dia-autoregulatory domain or DAD). The GBD domain is a bifunctional auto-inhibitory domain that interacts with and is regulated by activated Rho family members. DAD induces actin filament formation, stabilizes microtubules and activates serum-response mediated transcription (20).

On the other hand, effector proteins of Cdc42 as WASP (Wiskott-Aldrich syndrome protein) and N-WASP are involved in the formation of filopodia. WASP is only expressed in hematopoietic cells. N-WASP, which is ubiquitously expressed, shares around 50% homology with WASP. Both proteins possess several domains: a PH domain that binds phosphatidylinositol (4,5) bisphosphate, a Cdc42-binding (GBD) domain, a proline-rich region, a G-acting binding verprolin homology (V) domain, a domain (C) with homology to the actin-depolymerizing protein cofilin and finally a C-terminal acidic segment. Both WASP and N-WASP induce actin polymerization when overexpressed in fibroblasts. WASP proteins bind directly to acting monomers and activate the Arp2/3 complex; they comprise a core mechanism that directly connects signal transduction pathways to the stimulation of acting polymerization (21).

Furthermore, Rac1 is also able to activate the Arp2/3 complex and this is one of the core regulatory pathways driving membrane protrusion. Activation of WAVE (WASP-like verprolin-homologous protein) complex requires simultaneous interactions with prenylated Rac1-GTP and acidic phospholipids, as well as a specific state of phosphorylation. Together, these signals promote full activation in a highly cooperative process leading to the formation of lamellipodia. Also another major effector of both Rac1 and Cdc42 is PAK1. PAK1 phosphorylates LIM-kinase at threonine 508 within LIM-kinase's activation loop, increasing LIM-kinase-mediated phosphorylation of the actin-regulatory protein cofilin. Activated GTPases can thus regulate actin depolymerization through PAK1 and LIM-kinase modulating microfilaments length (22).

Another process regulated by Rho GTPases is the intracellular organization of microtubules, playing a central role in cell polarity and mitotic spindle assembly (23). The microtubule tip protein CLIP-170 interacts with the Cdc42/Rac1 effector IQGAP and mediates transient capture of microtubules. IQGAP1 is a scaffold protein essential for cellular signaling in response to external cues, linking dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein SKAP (24).

It is important also to point out the role played by stathmin which is crucial for the regulation of the microtubule cytoskeleton. Stathmin interacts with two molecules of dimeric α- and β-tubulin to form a tight ternary complex called the T2S complex. When stathmin sequesters tubulin into the T2S complex, tubulin becomes non-polymerizable. Without tubulin polymerization, there is no microtubule assembly. Stathmin also promotes microtubule disassembly by acting directly on the microtubule ends. Regulation of stathmin is cell cycle-dependent and controlled by the cell's protein kinases in response to specific cell signals. Stathmin phosphorylation increases the concentration of tubulin available in the cytoplasm for microtubule assembly. For cells to assemble the mitotic spindle, stathmin phosphorylation must occur. At cytokinesis, the last phase of the cell cycle, rapid dephosphorization of stathmin occurs to block the cell from entering back into the cell cycle until it is ready (25).

In addition to their cytoskeletal effects, Rho GTPases regulate several signal transduction pathways that lead to alterations in gene expression affecting several transcription factors such as SRF, NF-κB, JNK (c-jun N-terminal kinase) and p38 MAP kinase (17).

Also, Rho GTPases are able to modulate different enzymatic activities. For instance, Rac1 plays a crucial role in activation of Nox family NADPH oxidases in enzymes dedicated to production of reactive oxygen species such as superoxide. The phagocyte oxidase Nox2, crucial for microbicidal activity during phagocytosis, is activated in a Rac1-dependent manner. Rac1 in the GTP-bound form directly binds to the oxidase activator p67 (PHOX), which in turn interacts with NOX2, leading to superoxide production (26,27).

Furthermore, Rho GTPases have also been shown to regulate cell cycle entry and cell cycle progression, in particular by regulating expression of a number of genes involved in G1/S transition, such as cyclin D1 or p21waf1. Rho GTPases are also critically involved in mitosis. Indeed, at mitosis onset, RhoA activity increases and the resulting activation of its effector ROCK, mediates cortical retraction during mitotic cell rounding. During early mitosis, depending on the cell type, either the GEF-H1/RhoA/mDia1 or the Ect2/Cdc42/mDia3 pathways are needed for spindle assembly and attachment of microtubules to kinetochores. Later in mitosis, Rho GTPases are directly involved in cytokinesis by regulating the actin and myosin contractile ring, which eventually forms the cleavage furrow to separate daughter cells (28).

It is important to highlight that cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as ECM stiffness or confined adhesiveness. By mechano-transduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behavior, but how rigidity sensing is ultimately linked to activity of nuclear transcription factors just started to be understood. Downstream events involving Rho GTPases in response to cytoskeleton tensioning include activation of YAP (yes-associated protein) and TAZ (transcriptional coactivator with PDZ binding motif) transcriptional regulators as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape (29).

In every physiological process each signaling element has an exquisite temporal and spatial regulation; at the same time deregulated expression leads to the development of many diseases among them cancer, the focal point of the present study (30). As mentioned, Rho GTPases through a series of complex biochemical networks control some of the most fundamental processes of the cell. Mutations or alterations of this complex signaling system could lead to cancer development. Traditionally, overactivation of the Rho GTPase pathways has been associated with malignant transformation. Recently, it has been described in a gain-of-function mutation in the rac1 gene in sun-exposed melanomas, although rac1 has been rarely found mutated in other human cancers (30–32). Rac1 upregulation is mostly due to alterations of its regulatory proteins. Notably, GEF activation is the most common mechanism for signal-mediated GTPase activation. This activation is commonly driven by aberrant signaling from growth factor receptors (RTKs) and GPCRs (G protein coupled receptors) and upregulation or mutation of GEFs (33). In this regard, many GEFs present a relevant role in cancer such as Ect2, Tiam1, Vav family, P-Rex1, DOCK family among others (34–37).

First evidence of malignant transformation by Rho GTPases was provided by experiments where constitutively activated Rac1, and in a lesser extent RhoA, induced malignant transformation in fibroblasts and tumorigenicity in athymic mice (38,39). In line with this idea, Rac1 is implicated in epithelial-mesenchymal transition and in mesenchymal-epithelial transition (40–43). Currently, alterations in the Rho GTPase pathways can be found in many cancer types. Among them we can mention malignant tumors of pancreas, breast, skin, prostate, testicle, colon, liver, lung, stomach as well as in leukemia, osteosarcoma, neuroblastoma, glioblastoma and head and neck cancer (44–47). Although Rho GTPase overexpression is commonly observed in cancer, downregulation of some Rho family members has also been linked to cancer (48).

Loss of cell polarity is a hallmark of cancer, with the loss of epithelial cell polarity being common among carcinoma cells (49,50). For cells to keep their polarity, cell-to-cell contact must be maintained, being Rac1, RhoA and Cdc42 indispensable for keeping a normal state (51). Regarding cell motility the Rho/ROCK pathway plays a central role in the acquisition of migratory and invasive properties in tumor cells (52), since they are implicated in the remodeling of the actin cytoskeleton, in intracellular adhesion mediated by cadherins and in the remodeling of the extracellular matrix (ECM) primarily by the release of matrix metalloproteinases (MMP), mainly MMP-9 (53).

It has been found that Rac1 is a key protein in the invasive process of pediatric medulloblastoma (54). In models of glioblastoma multiforme Rac1, Rac3 and some GEFs mediate the migratory and invasive capacity of these tumors (36,55,56).

Once a tumor is established, neovascularization of the pathological tissue is essential (57). In vitro and in vivo studies have identified Rho proteins as essential in signaling for vascular endothelial growth factor (VEGF)-dependent capillary formation (58). Regarding the regulatory proteins of the GTPases involved in cancer, one of the most studied examples is the Rac1-GEF Tiam1 (T-cell lymphoma invasion and metastasis 1). Although GTPases are not considered oncogenes, Tiam1 was confirmed as one, since it is implicated in the oncogenic transformation of fibroblasts (59,60). The Tiam1 gene was identified by insertional mutagenesis in a model of T cell lymphoma, carrying out an in vitro selection of highly invasive clones. In this way it was established that Tiam1 increases the invasive capacity and the metastatic potential of tumor cells (59). Additionally, Tiam1 levels are increased and could be correlated with the prognosis of human prostate carcinomas, nasopharyngeal carcinomas and with the degree of progression of breast tumors (61).

In line with this idea, Vav1, another Rac1 GEF, is over-expressed in pancreatic adenocarcinoma and metastatic melanoma (62).

Also, the expression levels of Rac1-GAP β2-chimaerin are significantly diminished in mammary tumors (63). Furthermore, it has been shown that overexpression of β2-chimaerin in a murine mammary carcinoma model produces the reduction of tumor growth rate and its invasive and metastatic capacity (64).

Another hallmark exhibited by cancer cells is the limitless replicative potential that is mainly due to the activity of telomerase. This holoenzyme maintains telomere length, adding TTAGGG repetitions at the end of chromosomes in each cell division (65). In addition to this function, there are extratelomeric roles of telomerase that are involved in cancer promoting events (66). It has been shown that telomerase reconstitution increased fibroblast migration through activation of CXCL12/CXCR4 axis and Rho family proteins (67).

Yeh et al (68) demonstrated that Cdc42/Rac1 participates in the control of telomerase activity in human nasopharyngeal cancer cells (NPC-076). They investigated the effects of inhibiting Cdc42 and Rac1 on the telomerase activity on cells. Treatment of NPC-076 cells with antisense oligonucleotides against Cdc42 or Rac1 produced an inhibition of telomerase activity. Similarly, transient expression of dominant-negative mutants of Cdc42 or Rac1, also produced an inhibition of telomerase activity in NPC-076 cells. This inhibition of telomerase activity was not associated with a transcriptional downregulation of hTERT, the key regulator of telomerase (69). This suggests that Cdc42/Rac1 participate in the post-transcriptional control of telomerase activity in NPC-076 cells. It also was founded that RAC3 overexpression is required to maintain telomerase activity (70). Further analysis suggested that exogenous expression of hTERT may promote invasiveness and metastasis through upregulation of MMP9 and RhoC (71).

Since telomerase is a key target against cancer, many molecules have been developed against it. Lately, it has been postulated that telomerase regulation could be better than direct inhibition itself (72). It remains to know if Rho GTPases could exert this action in a more effective way that the ones that are known so far.

Stathmin is also a protein of clinical relevance in cancer. Stathmin's role in regulation of the cell cycle causes it to be oncoprotein 18 (op18). Op18/stathmin can cause uncontrolled cell proliferation when mutated and not functioning properly. If stathmin is unable to bind to tubulin, it allows for constant microtubule assembly and therefore constant mitotic spindle assembly. With no regulation of the mitotic spindle, the cell cycle is capable of cycling uncontrollably resulting in the unregulated cell growth characteristic of cancer cells (73).

Among the many different effectors of Rho GTPases, four of them are directly related with malignant transformation mediated by Rho proteins. First, we can mention the WASP family, effectors of Rac1 and Cdc42, related with actin cytoskeleton rearrangements, therefore associated to cell migration, filopodia, podosomes as well as membrane ruffles regulation. Another described effector is IQGAP, which by its interaction with Rac1 and Cdc42 is implicated in membrane ruffles and in the cell-cell unions mediated by E-cadherin (74,75).

Thirdly, we should mention that the role of PAK in cancer has been widely described. They have more than 40 possible different substrates and mediate biological processes such as cell proliferation, cell motility and survival, angiogenesis and substrate-independent growth, among others (76). It has been described that by Rac1 activation, PAK1 is able to activate PKCγ, a necessary event for the interaction with the actin-bundling protein fascin, mediating cell migration in a model of human colon carcinoma (77).

Finally, ROCK1 directly interacts with and stabilizes the oncogene c-Myc protein leading to the increased oncomir miR-17-92 cluster expression in breast cancer and prostate cancer (78); ROCK family members are direct effectors also of RhoA (79).

It is important to highlight that Rac1 may also drive anti-tumorigenic effects (80). Recently Zandvakili et al (81) reviewed the role of Rho GTPases with potential tumor suppressing roles, however, most of that data emerged from cancer genomic studies. In those studies the concept of mutation penetrance and expressivity have been not sufficiently analyzed to be conclusive about the subject (82). In any case, the redundant function among Rho GTPases, their many regulators and effectors added to the interplays of feedback signaling loops may explain such contradictions.

Although we just gave an overview on how different Rho-GTPases and their associated proteins may play a defined role in cancer, it should be taken into account that given the overlap between its physiological and pathological functions as well as its often contrasting effects in cellular processes, these different molecules could be, at times, playing a similar role.

The substantial body of evidence that relates Rho GTPases with cancer has made them an attractive therapeutic target at the molecular level. With an incredible level of complexity and as more knowledge was being incorporated to the true nature of Rho GTPases, many groups started the search of inhibitors that could influence negatively the characteristics of tumor cell growth. Some strategies used to develop inhibitors are shown in Fig. 1 and described below.

The redundancy and plasticity of the different pathways allow tumor cells to adapt and to overcome different difficult conditions either of therapeutic or environmental origin, implying that therapies with a unique highly specific target not always reach the levels of efficacy that are desirable. That suggests that different concomitant therapeutic strategies may be needed (126). An option is to combine drugs that modulate multiple signaling pathways. The most effective combinations may include agents that inhibit survival signals in multiple signal transduction pathways.

Targeting Rho GTPases represent a very interesting therapeutic opportunity. As shown, several inhibitors have been developed. While some of these compounds show promising results, it is evident that more potent inhibitors are yet to be identified. However, advancements in rational drug design together with our expanding knowledge of the mechanisms involved in regulating Rho GTPases signaling pave the way toward the development of highly specific and context dependent Rho-GTPase inhibitors.