Keywords: Ras, Rho, Rac, cdc42, Ral, epithelial cells, fibroblasts, tumorigenesis, cell migration, basement membrane

Abstract

With over 100 members in humans, the Ras superfamily is a diverse group of monomeric G proteins participating in many cellular processes. Members of the Ras, Rho, and Arf families have been shown to regulate cell motility in fibroblasts and epithelial cells. Ras and Rho family members are also widely involved in human tumorigenesis, either through activating mutations or by overexpression. In this review, tools for studying carcinoma cell migration are discussed and evidence for regulation of carcinoma cell motility by specific Ras superfamily members is summarized. Novel emerging mechanisms of migration in carcinoma cells involving RhoC and Ral are also discussed.

INTRODUCTION

The complexity of human cancers has resisted easy classification, with hundreds of different tumor types arising from every tissue and in every organ. This is additionally complicated by cancer cells' ability to invade surrounding tissues and metastasize to distant organs. As a primary classification scheme, cancer biologists and oncologists now generally group together cancers by tissue of origin, regardless of organ, emphasizing the similarities in cellular structure and function among these tumors. In this scheme, carcinomas are the group of tumors originating from epithelial cells and typically represent >80% of diagnosed human cancer each year [1]. Recent advances in oligonucleotide array technology have also led to the ability to classify cancer in terms of global gene expression patterns [2], with hopes of linking such molecular tumor “fingerprints” to more targeted therapeutic strategies.

Upon a diagnosis of carcinoma, a crucial distinction arises for both physician and patient. Superficial tumors, or carcinoma in situ, remain localized to the tissue of origin, often constrained by an intact basement membrane. Such tumors often respond well to treatment, resulting in a good prognosis for the patient. In contrast, invasive carcinomas, by disrupting basement membranes and growing into surrounding tissues, are more difficult to treat successfully. Additionally, since invasion is usually a prerequisite for metastasis, which is the ultimate cause of most cancer-related deaths [3], even when the local lesion is treated, the prognosis is often poor. Elucidating the processes underlying carcinoma invasion and developing specific strategies to block them could therefore lead to new, more efficacious treatments for cancer patients.

Although each invasive carcinoma is the unique evolutionary outcome of a complex set of interactions between tumor genotype and environment [4], there do seem to be a set of processes commonly associated with the invasive phenotype in many carcinomas. These processes are summarized in Table 1. Invasive carcinoma cells often lack adherens junctions and desmosomes, resulting in a loss of the polarized structure characteristic of epithelial cells. This phenotypic shift appears to be a form of the epithelial-mesenchymal transition (EMT) first described as a component of morphogenesis during animal development. The role of EMTs in tumor progression has recently been reviewed [5] as have the associated changes in C-Met tyrosine kinase signaling and E-cadherin expression [6, 7]. Also recently reviewed have been the functions of matrix metalloproteinases (MMPs) [8] and tissue inhibitors of metalloproteinases (TIMPs) [9] in regulating extracellular proteolysis in tumor invasion and metastasis. Anoikis, the programmed death of epithelial cells detached from the extracellular matrix, and its evasion by carcinoma cells has also been the subject of recent reviews [10, 11]. The focus of this review is on mechanisms of carcinoma cell motility and in particular their regulation by members of the Ras superfamily of small G proteins.

Carcinoma cell motility

In discussing carcinoma cell motility, it is important to distinguish between different modes of cell movement and how they are measured, since assuming these different modes are equivalent may lead to apparent contradictions in the literature. Chemokinesis and chemotaxis are both types of cell motility generated in response to soluble molecules such as growth factors or cytokines, with chemokinesis referring to non-directional movement triggered by uniformly distributed signal molecules and chemotaxis indicating directional movement along a concentration gradient of signal molecules. Following the same pattern with reference to directionality, haptokinesis and haptotaxis are forms of non-directional and directional cell movement, respectively, in response to extracellular matrix (ECM) molecules such as fibronectin or hyaluronan. The distinction between chemokinesis/taxis and haptokinesis/taxis can be determined experimentally in vitro, but may not be so clear in vivo, given interactions between growth factors and ECM molecules, such as with heparin-binding EGF-like growth factor [12], as well as interactions between growth factor receptors and integrins [13]. While cancer cell motility is often described as a rate-limiting step in invasion, the relative contributions of each of the four type of induced cell movement to carcinoma invasion in vivo remains to be determined.

The molecular dissection of carcinoma cell motility has depended upon several in vitro assays of cell movement. Direct microscopic observation of cultured cell motility, while philosophically straightforward, is the most technically challenging of the methods, assuming multiple cells are to be assayed in an experiment. This can involve sophisticated stage manipulation and image acquisition/analysis capabilities, as well as the need to maintain culture conditions such as temperature, humidity, and CO2 during longer term observations, the expense of which have no doubt limited the use of this approach. However, by altering the concentrations of stimulating molecules, all four forms of cell motility can be determined this way, including measurements of velocity and persistence of direction [14]. By expressing fluorescently tagged proteins such as fusions to Green Fluorescent Protein (GFP), the cellular locations of specific proteins during locomotion can be determined [15, 16]. Whether a specific protein is involved in motility can be assayed both genetically and pharmacologically. Also, this approach has been modified to detect cell movement in artificial 3-dimensional matrices, which may reflect better than 2-dimensional measurements the in vivo motility of carcinoma cells [17].

Cell motility can also be assayed in vitro using transwell, or Boyden, chambers. In this case, motile cells placed on the upper surface of a porous filter crawl through pores in the filter to reach the lower surface. After a period of time, counting the number of cells (either directly through microscopic observation or indirectly through biochemical markers [18]) on the lower surface of the filter and dividing it by the total number of cells placed in the chamber creates a measure of cell motility, or motility index. By altering the concentration of growth factor or cytokine in the upper and lower chambers, chemokinesis and chemotaxis can be discriminated, as can haptokinesis and haptokinesis by coating the upper and lower surfaces of the filter with different concentrations of ECM components. Coating the filter with ECM components is also often used to create an in vitro model of invasion [19]. Cells can be genetically modified prior to transwell chamber assays and these assays can be performed in the presence of drug inhibitors, allowing the involvement of specific proteins in movement to be determined [18]. However, since this is an endpoint assay, location of GFP-fusion proteins in moving cells cannot be determined.

Another commonly used in vitro assay of cell motility is the artificial wound assay. Confluent cell monolayers are scraped to create an acellular area, which is then monitored to see cells move into the wound. While convenient, this assay suffers from limitations. Since gradients are difficult to establish in this system, neither chemotaxis not haptotaxis is easily studied in a wound assay, nor is haptokinesis, since creating the wound would also remove a coating of ECM components. The usual endpoint of a wound assay is complete wound closure by a control group of cells; therefore it is easier to study inhibition of motility than stimulation. Even though normally only used to study chemokinesis, wound assays are still convenient and powerful ways to study cell motility, allowing for both genetic and pharmacologic manipulation [20, 21] as well as visualization of GFP-fusion protein localization in motile cells [22].

Using these in vitro assays in combination with genetic and pharmacologic manipulation, a molecular model of mammalian adherent cell motility has begun to emerge, involving four fundamental processes: lamellipod extension, contraction, leading edge attachment, and uropod detachment [23, 24]. At the leading edge of a moving cell, localized remodeling of the actin cytoskeleton results in the extension of a single lamellipodium, or in some cases, multiple smaller filopodia. Dozens of proteins that interact with actin monomers and/or microfilaments and modulate their structures and activities have been shown to participate in this process [25]. Other cytoskeletal changes behind the leading edge result in contraction, which pulls the cell body forward. Contraction is driven by changes in actin/myosin interactions, regulated at least in part by myosin light chain modifying proteins such as protein kinases and phosphatases [26]. In order for contraction to result in forward cell movement, new contacts between the cell and its substratum must be established at the leading edge. These contacts involve integrins and other transmembrane receptors for extracellular matrix (ECM) components and must be dynamically altered in composition and/or activity for a cell to continue moving forward [27]. Finally, at the uropod, or trailing edge, a moving cell detaches by breaking contact with ECM, either by proteolytic cleaving of adhesion proteins (via calpain or other proteases) or by membrane recycling of contact components [28]. While this model of cell motility is based primarily upon experiments on fibroblasts, similar processes appear to be important for epithelial cell movement as well, with perhaps some differences in the molecules involved [29, 30]. As discussed earlier, carcinoma cells have characteristics of both epithelial cells and fibroblasts, so mechanisms of carcinoma cell motility may include aspects of both fibroblast and epithelial models of cell movement. Perhaps more importantly, carcinoma cell motility may involve novel mechanisms and/or molecules that would represent promising new targets for therapeutic intervention in cancer.

The Ras superfamily

Whether a moving cell is a fibroblast, an epithelial cell, or a carcinoma cell, the processes of cell motility just described together involve both simultaneous and ordered changes in the activities and/or locations of hundreds of proteins. Such complexity indicates an extensive level of coordinate regulation, at least some of which comes from the participation of members of the Ras superfamily of small G proteins in most, if not all, of these processes. The Ras superfamily in humans comprises over a hundred related small (20-30 kDa), monomeric guanine nucleotide binding proteins. Although there is a lack of consensus as to their classification, one common approach places them into six families: Ras [31, 32], Rho [33, 34], Arf [35], Rab [36], Ran [37], and Rad. As seen in Table 2, Ras superfamily members participate in many cellular processes, primarily as signal transducers and/or regulators of membrane traffic.

Underlying the functional diversity in the Ras superfamily is a common mechanism called the GTP/GDP cycle. As shown in Figure 1, these proteins cycle between a GTP-bound state and a GDP-bound state due to their GTP hydrolysis activity and a higher affinity for GTP than GDP. In vivo, this cycle is regulated by GTPase activator proteins (GAPs), which increase the rate of GTP hydrolysis [38], and guanine nucleotide exchange factors (GEFs), which stimulate the exchange of GDP for GTP [39]. Members of the Rho, Rab, and Ran families are also regulated by GDP dissociation inhibitors (GDIs) [40-42], which, by binding to the GDP-bound form, inhibit nucleotide exchange. In general, there are two consequences of the GTP/GDP cycle. The GTP-bound form adopts a different conformation than the GDP-bound form, allowing interaction with effector molecules. Also, the GTP-bound form is usually found more tightly associated with membranes or at different membrane locations than the GDP-bound form. As a result of these changes, the GTP-bound form of Ras superfamily monomeric G proteins is often referred to as the active form, whereas the GDP-bound form is called the inactive form. This can be misleading, since in some cases, the GDP-bound form does bind to separate effectors and thereby exert effects on cellular activity [43, 44]. Nevertheless, another name for the ratio of the amount of GTP-bound G protein to total G protein is the activation state, which represents the complex and dynamic output of both the intrinsic properties of each G protein and the signal transduction events converging on the associated GAPs, GEFs, and GDIs.

Tools for studying the Ras superfamily

Summarized in Table 3 are some genetic and pharmacologic tools available to study the involvement of Ras superfamily members in cell motility. The G12V and Q61L versions of H-Ras were initially discovered as the consequences of oncogenic mutations in H-Ras genes and later shown to result in a constitutively active form of H-Ras [45]. Identical amino acid changes in homologous positions in other Ras superfamily members, i.e. RhoA G14V or RalA G23V, have the same effect. This constitutive activation is due to a severe reduction in GTPase activity, slowing the GTP/GDP cycle in vivo, and resulting in the accumulation of the active, GTP-bound form. Alternative activated forms of Ras and cdc42 have been developed, F28L, which have an increased rate of nucleotide exchange without affecting GTP hydrolysis [46, 47]. They also accumulate in the GTP-bound in vivo, but without attenuating the GTP/GDP cycle. Since F28 is a widely conserved residue in the Ras superfamily, activated and cycling forms of other G proteins may be possible. While not commonly used, these activated and cycling Ras superfamily members may help to clarify some paradoxical results involving constitutively active G proteins and cell motility that will be discussed below.

Dominant negative versions of many of the Ras superfamily, such as Ras S17N or RhoA T19N members are also available. These versions bind to GEFs with a higher affinity than wild type proteins without having their nucleotide exchange stimulated. Therefore, they accumulate in the GDP-bound form and also inhibit nucleotide exchange of their endogenous counterparts through inhibition of GEFs. Since GEFs are somewhat promiscuous in their substrate specificity within each family, expression of a particular dominant negative G protein in cells may actually result in the inhibition of activation of more than one family member [48]. Ideally, using both constitutively active and dominant negative forms should help identify which specific members of a family are involved in cell motility. Alternatively, the development of RNA interference (RNAi) technology should result in the specific inhibition of small G protein function in mammalian cells [49, 50].

Ras superfamily member function can also be perturbed pharmacologically. For example, farnesyltransferase inhibitors (FTIs), which block the addition of farnesyl, a 15 carbon isoprene derivative, to the C-terminus of Ras proteins, were developed as specific inhibitors of Ras function, since this modification is necessary for Ras function and other superfamily members contain the 20 carbon geranylgeranyl C-terminal modification. While it is clear that FTIs do inhibit Ras function in cells, they also alter the function of RhoB, which normally exists in separate farnesylated and geranylgeranylated pools [51]. Rho family member functions are inhibited by a variety of toxins produced by bacteria in the genus Clostridium, but the effects of these toxins is also not totally specific for individual Rho family members [52]. Brefeldin A, a fungal toxin, inhibits activity of Arf family members by inactivating ArfGEFs [53]. At best, pharmacologic approaches can therefore only implicate a group of family members as being involved in cell motility, without identifying a particular culprit.

The Ras superfamily and carcinoma cell motility
Rho family

The evidence for Rho family member involvement in human cancer has been recently reviewed. While Rho family gene mutations in tumors are rare, overexpression is more common [34]. For example RhoA is overexpressed in head and neck squamous carcinomas [54] as well as in lung, colon, and breast tumors [55]. In the case of breast tumors, increasing RhoA expression correlated with increasing tumor grade, suggesting a role for RhoA in tumor progression. RhoC overexpression is found in breast carcinoma [56] and pancreatic adenocarcinoma [57]. Breast cancer is also associated with overexpression of Rac1 and cdc42 [55], as well as alternative splicing of Rac1 [58], which is also found in colon cancer [59]. Dysfunctional regulation of Rho family member activation may also contribute to cancer, in that many Rho GEFs have transforming ability [60, 61] and p190 RhoGAP is a tumor suppressor [62]. Reduced expression of RhoGDI2 has recently been shown to correlate with increasing invasive and metastatic ability in human bladder carcinoma lines [63]. Which Rho family members are the targets of dysregulation in human cancer remains to be determined.

Among its many functions, RhoA has been shown to play a key role in epithelial cell motility, at least in part through its effector Rho-kinase and its regulation of contraction via myosin light chain phosphorylation status [64]. Rho-kinase can also affect cell motility by regulating formation of stress fibers and focal adhesions in many cell types [65]. Another RhoA effector, mDia, has been shown to cooperate with Rho-kinase in promoting stress fiber formation in HeLa cells [66] and in MDCK epithelial cells [67], while it antagonizes the ability of Rho-kinase to inhibit Rac1 activation in Swiss 3T3 fibroblasts [68]. Such results indicate that RhoA, like many other Ras superfamily members, may utilize multiple effectors in regulating cell motility, and that some of these effector interactions may be cell type specific.

Recent evidence has indicated a probable role for RhoA in carcinoma cell motility. In clone A colon carcinoma cells, laminin stimulated both haptotaxis and RhoA activation through a6b4 integrin [69]. This haptotaxis was inhibited by expression of dominant negative RhoA (DN-RhoA), but not dominant negative Rac1. Expression of DN-RhoA also inhibited epidermal growth factor (EGF) stimulated chemokinesis in T24 bladder carcinoma cells [20] and urokinase-type plasminogen activator (uPA) stimulated chemotaxis in MCF-7 breast carcinonoma cells [70]. EGF stimulated invasion/chemotaxis of PANC-1 pancreatic carcinoma cells was inhibited by C3 exoenzyme [71]. In MM1 rat hepatoma cells, expression of constitutively active RhoA stimulated in vitro invasion, as did overexpression of wild type RhoA [72]. Taken together, these data point toward RhoA as a necessary motility component in a variety of carcinoma cell lines as well as a contributing factor to the invasive phenotype, although the effects of DN-RhoA and C3 exoenzyme may be mediated by other Rho family members. Since RhoD has been shown to participate in fibroblast motility [73] and RhoE is involved in the motility of MDCK epithelial cells [74], the involvement of other Rho proteins in carcinoma cell motility warrants further investigation.

Most studies on downstream effectors of RhoA in carcinoma cell motility have focused on Rho-kinase. The Rho-kinase inhibitor, Y-27632, has been shown to inhibit chemotaxis of PC3 prostate carcinoma cells [75] and MCF-7 breast carcinoma cells [70]. Reduction of Rho-kinase expression through use of antisense oilgonucleotides inhibited chemotaxis of PANC-1 pancreatic carcinoma cells [76]. In vitro migration of Met-1 breast cancer cells was inhibited by expression of a dominant negative form of Rho-kinase [77]. While Rho-kinase does appear to mediate at least some of the downstream effects of RhoA on carcinoma cell motility, what remains unclear is whether these effects are targeting such processes as contraction, stress fiber formation, focal adhesion dynamics, or Rac activation. Whether mDia or other RhoA effectors are also participating in carcinoma cell motility is also unclear.

RhoC was recently identified as an essential regulator of melanoma metastasis and cell motility [78]. In this study, increased RhoC expression correlated with increasing metastatic behavior generated in an in vivo selection scheme. This correlation was shown to have functional consequences in that overexpression of RhoC in weakly metastatic melanoma cells increased their metastatic ability and expression of DN-RhoA, presumed to also inhibit RhoC activation, in highly metastatic cells reduced their ability to form metastases. In terms of cell motility, overexpression of RhoC stimulated melanoma cell motility to a greater degree than RhoA overexpression. Overexpression of wild type RhoC has also been shown to stimulate motility and in vitro invasion in breast carcinoma cells [79] and this stimulation was reduced by inhibitors of MAP kinases [80]. The role of RhoC in "normal" cell motility has not been studied, so this leaves open the possibility that RhoC may be playing a unique role in carcinoma motility, given its overexpression not only in metastatic melanoma and breast cancer but also in pancreatic cancer progression, especially since in each of these cases, high levels of RhoC expression correlate with invasive and/or metastatic phenotypes.

Closely related, Rac1 and cdc42 are thought to be indispensable to both fibroblast and epithelial cell motility, due to their ability to regulate actin cytoskeletal dynamics and lamellipod extension [81], in part by signaling through effectors p21 activated kinases (PAKs), IRSp53, and WASPs to actin remodeling proteins such as Arp2/3 and cofilin [82]. Rac1 also influences cell motility through effects on focal adhesion dynamics [83] and both Rac1 and cdc42 modulate cadherin/catenin containing adherens junctions via their effector IQGAP, which is also an actin binding protein [84]. While extensive crosstalk between Rac1, cdc42, and RhoA through shared effectors and regulation of each others’ activation states makes it challenging to dissect out specific pathway positions for each protein in cell motility or any other cellular process [85], almost all models of mammalian adherent cell motility have Rac1 and cdc42 as necessary participants.

Results on the role of Rac1 and cdc42 in carcinoma cell motility are less conclusive. Chemotaxis of MDA-MB-435 breast carcinoma toward lysophosphatidic acid (LPA) was inhibited by expression of dominant negative (T17N) Rac1, as was EGF driven chemotaxis of A431 squamous carcinoma cells [86]. However, expression of constitutively active Rac1 (G12V) reduced chemokinesis of HeLa cervical carcinoma cells toward serum [87] and had a similar inhibitory effect on migration and chemotaxis/invasion of clearCa-28 renal carcinoma cells [88]. This inhibitory effect of constitutively active Rac1 may be due to a need for Rac1 cycling between the GTP-bound and GDP-bound forms to stimulate motility. Alternatively, this inhibitory effect may be due to an overabundance of GTP-bound form inhibiting motility. Use of an active and cycling form might help to distinguish between these possibilities and determine whether stimulation of carcinoma cell motility requires Rac1 cycling or proper levels of Rac1-GTP. As mentioned earlier, expression of DN-Rac1 had no effect on laminin-stimulated chemotaxis in clone A carcinoma cells [69]. EGF-stimulated chemokinesis in T24 bladder carcinoma cells was unaffected by expression of DN-Rac1 or DN-cdc42 [20] and there was also no detectable activated Rac1 or cdc42 in these cells. Although these results may reflect differences in cell type and/or stimulus, they raise the intriguing possibility that, in contrast to fibroblasts and epithelial cells, at least some carcinomas have developed mechanisms of cell motility independent of Rac1 and cdc42. Further dissection of these mechanisms and identification of the proteins involved could therefore result in new specific targets for stopping invasive cancer in its tracks.

Ras family
The involvement of Ras family members in human cancer is without question. Activating Ras gene mutations are estimated to occur in 30% of human tumors, with K-Ras mutations almost ubiquitous in pancreatic tumors and common in lung and colorectal cancer, H-Ras mutations found in bladder and kidney cancer and N-Ras mutations common in leukemias [89]. In the absence of Ras gene mutations, Ras proteins are often overexpressed in tumors [90, 91]. Dysregulation of Ras activation state also contributes to human cancer, with many growth factor receptors and Ras GEFs having transforming properties and both NF1, a Ras GAP, and RASSF1A, a putative Ras inhibitor, acting as tumor suppressors [92, 93].

Most models of cell motility involving Ras family monomeric G proteins place them as upstream regulators of Rho family proteins, regulating the activation states of RhoA, Rac1, and cdc42 in response to growth factor or cytokine stimulation [94]. For example, constitutively active K-Ras was recently shown to be approximately twice as efficient as activated H-Ras in stimulating both cell motility and Rac1 activation in fibroblasts [95]. However, Ras mediated downstream activation of a MAP kinase cascade in COS cells was shown to be necessary for cell motility, at least in part through effects on myosin light chain phosphorylation status [96]. Activation of a Ras-Raf-MAP kinase cascade was also shown to stimulate motility in fibroblasts through calpain activation and subsequent rear detachment [97]. In breast epithelial cells, activated R-Ras and TC21 were shown to stimulate migration and this stimulation was dependent upon phosphatidylinositol-3-kinase and protein kinase C but not MAP kinase [98]. Given the growing number of Ras effectors [99], it is not surprising that regulation of cell motility by Ras may involve many downstream pathways.

Expression of dominant negative H-Ras (S17N) has been shown to inhibit motility in many carcinoma cells, including serum-stimulated chemotaxis of T24 bladder carcinoma cells [18], hepatocyte growth factor (HGF) stimulated chemotaxis of various ovarian carcinoma cells [100], and chemotaxis of MCF-7 breast cancer cells toward uPA [101]. In the latter two cases, the effects of H-Ras on cell motility appeared to be through activation of a MAP kinase cascade, presumably via the Ras effector Raf. In pancreatic carcinoma cells, the FTI manumycin inhibited haptotaxis toward fibronectin, particularly in cells that had an activating K-Ras mutation [102]. While both H-Ras and K-Ras appear to be involved in carcinoma cell motility, what is not clear is whether their participation indicates carcinoma specific mechanisms of cell motility, which will require further delineation of the downstream effectors of H-Ras and K-Ras stimulation of cell motility in carcinomas.

RalA and RalB are closely related and relatively understudied members of the Ras family. While no direct role for RalA or RalB in fibroblast or epithelial cell motility has been demonstrated, among their effectors are RalBP1, a GAP for Rac1 and cdc42 [103], and filamin, an actin binding and regulatory protein [104], both of which potentially connect Ral to cell motility regulation. A Ras-RalGEF-Ral signaling cascade has also been recently shown to be important in stimulating an invasive phenotype in both fibroblasts and epithelial cells [105]. In T24 bladder carcinoma cells, expression of dominant negative RalA (S28N) inhibited EGF-stimulated chemokinesis [20]. Since DN-RalA would presumably work by inhibiting one or more RalGEFs, which would also therefore inhibit RalB, it is not clear whether RalA or RalB or both are necessary for motility in these cells. As previously mentioned, this chemokinesis was also inhibited by DN-RhoA, but not by DN-Rac1 and DN-cdc42. Brefeldin A, an inhibitor of ArfGEFs, also inhibited this cell motility, indicating the involvement of one or more Arf family members in bladder carcinoma cell motility. In these cells, chemokinesis was also reduced by n-butanol, an inhibitor of phospholipase D (PLD), consistent with RhoA, RalA and Arf all using PLD1 as an effector [106]. Given the involvement of RhoA, RalA, Arf, and PLD, all known to be regulators of endocytosis and/or exocytosis [107, 108], in bladder carcinoma chemokinesis, it is interesting to speculate on the involvement of vesicular transport in carcinoma cell motility.

Other families
Recent results have shown a role for Arf6 in epithelial cell migration [109, 110], potentially linking vesicle transport and cell motility in these cells. As mentioned above, Arf involvement in bladder carcinoma cell motility has been indicated through inhibition by Brefeldin A [20]. Along with the involvement of Rab proteins in regulating both exocytosis [111] and epithelial actin cytoskeletal dynamics [112], this may indicate that many more Ras superfamily members are participating in cell motility than previously thought, which may also point toward their involvement in carcinoma cell motility.

Conclusion

It is somewhat surprising that, with all the research done on the roles of various members of the Ras superfamily of monomeric G proteins in human cancer and in cell motility, that so much remains unknown about the functions of the Ras superfamily in carcinoma cell motility. Still unresolved are issues of specificity; such as how different are the roles of H-Ras vs. K-Ras, RhoA vs. RhoC, or RalA vs. RalB? Also awaiting clarification are the relationships between cellular localization of these G proteins, effector specificity, and directionality of cell movement. Application of a new generation of molecular tools for cell biology, such as the previously mentioned RNAi and fluorescence resonance energy transfer (FRET), which can detect protein-protein interaction in living cells [113], will no doubt contribute to a deeper understanding of carcinoma cell motility. With such knowledge, there may be new hope to reduce the deadly toll of invasive carcinoma.

Acknowledgements

The authors wish to thank Drs. Alan Horwitz and Thomas Parsons and members of the Theodorescu laboratory for helpful suggestions and comments.

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Table 1. Processes associated with carcinoma cell invasion

Table 2. The Ras Superfamily

Table 3. Tools to study Ras superfamily member involvement in cellular function

Figure 1. The GTP/GDP cycle

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