- Email: [email protected]
Role of Rho-family proteins in cell adhesion and cancer
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Role of Rho-family proteins in cell adhesion and cancer Angeliki Malliri and John G Collardy Rho-family proteins control signalling pathways that regulate a wide range of biological processes. In vitro studies implicating Rho proteins in cell adhesion, migration, transcriptional activation, cell-cycle progression and transformation suggested roles for these proteins in the formation and progression of tumours in vivo. Studies using different recombinant mouse models have recently confirmed this idea. Rho signalling pathways crosstalk with different oncogenic signalling cascades, including those downstream of Ras and Wnt, and contribute to various aspects of tumourigenesis, including survival, growth and progression of tumour cells. Addresses The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands e-mail: [email protected] y e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:583–589 This review comes from a themed issue on Cell-to-cell contact and extracellular matrix Edited by Eric Brown and Elisabetta Dejana 0955-0674/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00098-X
Abbreviations AJ adherens junction APC adenomatous polyposis coli Asef APC-stimulated GEF Dvl Dishevelled Fz Frizzled GAP GTPase-activating protein GDI guanine nucleotide dissociation inhibitor GEF guanine nucleotide exchange factor GSK glycogen synthase kinase TCF T-cell factor
Introduction Rho GTPases comprise a large subfamily of the Ras superfamily and include Cdc42, Rac and Rho proteins. Similarly to Ras proteins, Rho GTPases are guanine nucleotide binding proteins that cycle between an active, GTP-bound and an inactive, GDP-bound state (Figure 1). The activity of Rho proteins is controlled by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs activate small GTPases by promoting the exchange of GDP for GTP, whereas GAPs enhance the intrinsic rate of hydrolysis of bound GTP to GDP, leading to inactivation. In cells, Rho GTPases exist predominantly in their inactive, GDP-bound form in complex with GDP dissociation inhibitors (GDIs). Actiwww.current-opinion.com
vation of Rho GTPases results in binding to various effector molecules that elicit downstream responses. Because Rho proteins control a wide range of signalling pathways that regulate various biological processes, it is not surprising that dysregulation of their activities can result in diverse aberrant cellular phenotypes found in different diseases (reviewed in [1]). Cell surface receptors use different GEFs to activate specific GTPases, and more than 50 potential GEFs have been identified within mammalian genomes (reviewed in [2]). GEFs not only activate Rho GTPases but also participate in the selection of downstream effectors by either binding to effectors directly or to scaffold proteins that complex with components of effector pathways (Figure 1). For example, the Rac activator a-PIX/Cool-2 also binds to Pak [3], a downstream effector of Rac and Cdc42, while the Rac activator Tiam1 binds IB2/JIP2, a scaffold for the p38 mitogenactivated protein kinase (MAPK) cascade, as well as the scaffold spinophilin. Spinophilin binding enhances Tiam1’s ability to activate p70 S6 kinase, while simultaneously suppressing its ability to activate Pak [4,5]. Evidence implicating aberrant Rho signalling in cancer has been obtained from in vitro studies that focused on specific aspects of tumour cell biology and to a lesser extent from mutations found in genes encoding Rho signalling components (both reviewed in [1,6]). More recently, in vivo studies using recombinant mice lacking or overexpressing Rho signalling proteins have provided direct evidence for the involvement of Rho proteins in cancer, which will be the focus of this review.
Rho proteins and cellular transformation The first findings to link Rho molecules with cancer came from in vitro foci formation assays that model the growthfactor- and anchorage-independent growth of tumour cells. Many Rho GEFs, such as Dbl, Ect2, Vav and Lfc, were identified as 50 -end-truncated proto-oncogenes in NIH3T3 transformation assays (reviewed in [7,8]). Moreover, it was shown that constitutively active Rac1 (V12Rac) and, to a lesser extent, RhoA were able to induce an oncogenic phenotype in fibroblasts, albeit much less efficiently than constitutively active Ras (V12Ras) [9,10]. Rho proteins appear to act downstream of Ras in oncogenic transformation of cells, since dominant-negative mutants of Rho GTPases inhibited Ras-induced focus formation, growth in soft agar and tumour formation in nude mice. In addition, V12Rac1 and V14RhoA cooperated with constitutively active Raf (RafCAAX) in focus formation [11,12], indicating that oncogenic V12Ras drives both Raf!MAPK and Rac/Rho-mediated pathways. Current Opinion in Cell Biology 2003, 15:583–589
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Figure 1
GEF
GTP
Scaffold Rho–GDP
Rho–GDP
Rho–GDI
Rho–GTP Effector
Pi GAP Cytoskeletal changes Gene expression Current Opinion in Cell Biology
The Rho GTPase cycle. Rho proteins cycle between an inactive, GDPbound, and an active, GTP-bound, state. Transitions between the two states are controlled by GEFs, GAPs and GDIs. Additionally, GEFs select downstream effectors either directly or through recruitment of scaffold proteins (see text for full details). Pi, phosphate.
Studies using dominant-active Rac and Rho effector mutants indicated that the transforming capacity of Rho GTPases was in general associated with the capacity of these mutants to induce cyclin D1 expression rather than cytoskeletal rearrangements [13,14].
Rho GTPases in cell adhesion and cell migration Further interest in Rho proteins was raised by their ability to induce reorganisation of the actin cytoskeleton. Since actin cytoskeletal changes are required for migratory behaviour of cells in response to growth factor stimulation or matrix interactions, subsequent studies addressed the contribution of Rho molecules to motility and invasion of tumour cells. Experiments performed primarily using fibroblasts showed that Cdc42, Rac and Rho cooperate to regulate the cytoskeletal changes required for migratory behaviour of cells (reviewed in [15,16]). To summarise the findings of numerous studies here, Cdc42 regulates the polarity of cell migration, whereas Rac regulates the formation of membrane protrusions at the leading edge of migrating cells, required for forward movement. RhoA is required for the generation of contractile force leading to rounding of the cell body. Rho proteins also regulate microtubule polymerisation involved in cell migration. In epithelial cells, Rho GTPases regulate the formation and maintenance of specialised junctional adhesion complexes — tight junctions and adherens junctions (AJs) — required for the barrier function of epithelial layers and to establish apical-basolateral polarity (reviewed in [17,18]). Loss of AJs, in particular, has been correlated with the malignant progression of epithelial tumours (see [19,20]). In addition to promoting intercellular adhesion, Rho proteins can also stimulate epithelial cell migration downCurrent Opinion in Cell Biology 2003, 15:583–589
stream of certain integrin–extracellular-matrix (ECM) interactions [21]. It is now considered that whether an epithelial cell migrates or not represents an integration of migration-inhibitory cell–cell interactions and migration-promoting cell–substrate interactions. These interactions are influenced by numerous external and intrinsic factors, including the nature of the ECM, the relative levels of activity of Rac and Rho, and possibly the specific exchange factors used to activate the Rho proteins (see later for APC-stimulated GEF [Asef]). For example, activation of Rac by Tiam1 promoted migration of Madin–Darby canine kidney (MDCK) cells on a collagen substrate but inhibited migration on a fibronectin or laminin substrate [22]. Epithelial–mesenchymal transition induced by expression of the Ras oncogene in MDCK cells was caused by a shift in balance between Rac and Rho activity [23,24]. Since Tiam1/Rac signalling is required in MDCK cells for E-cadherin-mediated adhesion [25], downregulating the activity of Rac and consequently activating Rho, results in loss of an epithelial phenotype and acquisition of a mesenchymal one (Figure 2). The mechanism by which Rac antagonises Rho involves Rac-mediated production of reactive oxygen species (ROS) that inhibit the low-molecular-weight protein tyrosine phosphatase, resulting in phosphorylation and activation of p190Rho GAP, a negative regulator of Rho activity [26]. Figure 2
Ras
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Cell–cell adhesion Cell–substrate adhesion Cell spreading
Acto-myosin contraction Stress fibers Cell rounding
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Balance between Rac and Rho activity determines cell phenotype. Activated Rac (pink) stimulates cell–cell adhesions, promoting an epithelial phenotype. By contrast, activated Rho (green) stimulates cell contraction and a mesenchymal phenotype. Rac downregulates Rho activity by stimulating Rho GAP activity through reactive oxygen species (ROS) production. ROS production inhibits the low molecular weight protein tyrosine phosphatase (LMW-PTP), resulting in phosphorylation and activation of Rho GAP, which in turn inhibits Rho activity. Rac and Rho activity is also influenced by upstream signalling. Low levels of Ras activate Rac, whereas high levels of Ras inhibit Rac activity (see text for full details). www.current-opinion.com
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The cell matrix might also influence growth factor signalling and cell proliferation. Loss of matrix attachment leads to inhibition of growth-factor-induced activation of the Ras!Raf!extracellular-signal-regulated kinase (ERK) cascade [27], explaining why normal cell proliferation is dependent on integrin-mediated anchorage to cell matrices. Adhesion to fibronectin leads to activation of both Rac and Rho, and as a consequence to increased cyclin D1 levels and to downregulation of the cyclindependent kinase inhibitor p21WAF1 [28,29]. Both events promote cell proliferation. Thus, cooperation exists between growth factor receptors and integrins in the activation of Ras- and Rho-mediated signalling pathways to regulate proliferation and migration of cells. How Rho signalling could affect the development and progression of tumours in vivo is difficult to predict from in vitro studies alone. Recent studies employing recombinant mice directly implicate Rho-family proteins in all stages of tumourigenesis, and reveal both tumour-promoting and -suppressor functions (see below).
Rho signalling and Ras-induced skin tumours in mutant mice Studies using knockout mice have demonstrated that deregulated Rho signalling influences various aspects of tumourigenicity. A two-stage chemical skin carcinogenesis protocol has been applied to two knockout mouse lines, namely RhoB- and Tiam1-deficient mice. The protocol entails tumour initiation in epidermal keratinocytes by treatment with the carcinogen 7,12-dimethylbenzanthracene, which induces oncogenic activation of the c-Ha-Ras gene. Subsequent repeated treatments with the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) result in the outgrowth and progression of initiated cells. Applying this protocol to RhoBdeficient mice resulted in the development of increased numbers of skin tumours compared with wild-type mice [30]. Moreover, E1A- and Ras-transformed RhoBdeficient primary mouse embryonic fibroblasts were more resistant to apoptosis induced by DNA-damaging agents. These findings imply that RhoB functions normally to suppress tumourigenesis by promoting apoptosis following cellular stress. By contrast, application of the same protocol to Tiam1deficient mice resulted in much fewer and smaller tumours compared with wild-type mice [31]. Tiam1 deficiency was associated with increased Ras-induced apoptosis during tumour initiation and with impeded TPA-induced proliferation during tumour promotion. These results show that Tiam1/Rac signalling cooperates with Ras during tumour initiation and promotion by positively influencing cell survival and growth (Figure 3). The effect of Tiam1/Rac signalling on cell growth could be mediated by D-type cyclins, since Rac has been shown to induce expression of cyclin D1 and D2 (see [14]), and cyclin D1-deficient mice display a www.current-opinion.com
Figure 3
Ras
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Tiam1/Rac
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?
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?
Migration Current Opinion in Cell Biology
Interactions between Ras and Rho pathways in tumourigenesis based on analysis of RhoB and Tiam1 mutant mice. RhoB-deficient mice develop more Ras-induced skin tumours than wild-type mice. Also, RhoB-deficient cells are more resistant to apoptosis. These findings imply that RhoB functions normally to suppress cell survival following cellular stress and thus to suppress tumourigenesis. In contrast, Tiam1deficient mice develop fewer and smaller Ras-induced skin tumours than wild-type mice. This is related to the positive effects of Tiam1/Rac signalling on cell survival and proliferation. However, Tiam1-deficient tumours progressed more frequently to malignancy. This can be explained by in vitro and in vivo results showing that high levels of Ras signalling (Ras""), as seen in Ras-transformed cells and advanced skin malignancies, downregulate Tiam1 expression and thereby Rac activity, which is required for cell–cell adhesion. Dashed lines indicate hypothetical links. (See text for full details.)
similar resistance to the development of skin and mammary tumours [32,33]. Other studies also support a role for Rac in cell survival. Rac was found to protect cells from Ras- induced apoptosis, through activation of NF-kB [34]. Thus, the studies with Tiam1 mutant mice are consistent with conclusions drawn from in vitro models and strongly suggest that Rac signalling is required for Ras-induced tumourigenesis through the stimulation of cell growth and enhancing cell survival in the presence of cellular stress. The fact that RhoB exerts an opposite effect on Ras-induced skin tumours and apoptosis might represent another example of Rac and Rho signalling being mutually antagonistic. This has been shown in several biological processes such as epithelial–mesenchymal transition [24] and neurite formation [35,36]. Interestingly, the few tumours in Tiam1-deficient mice progressed more frequently to malignancy than those in wild-type mice, suggesting that Tiam1 deficiency promotes malignant conversion. Indeed, analysis of Tiam1 expression in tumours of wild-type mice revealed that benign papillomas maintained high levels of Tiam1 expression, whereas expression was reduced in squamous Current Opinion in Cell Biology 2003, 15:583–589
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cell carcinomas and was completely lost in highly invasive spindle cell carcinomas [31]. Thus, while Tiam1 function appears to be essential for the initiation and promotion of Ras-induced skin tumours, these data suggest that subsequent loss of Tiam1 expression promotes the malignant conversion of benign tumours. Ras can activate Tiam1 in two ways: through a phosphatidylinositol 3-kinase-dependent mechanism [22,37]; or through direct binding of active GTP-bound Ras to a Rasbinding domain located within the Tiam1 protein [38]. Interestingly, the increased Ras signalling associated with advanced skin malignancies that results from amplification of the mutated Ras allele [39] seems to be responsible for the reduction or loss of Tiam1 expression in the later stages of tumour progression, as demonstrated in vitro for Ras-transformed MDCK cells [24]. As discussed above, loss of Tiam1 expression and the resultant decrease in Rac activity in advanced tumours could lead to decreased E-cadherin-mediated adhesion, promoting invasiveness of epithelial tumour cells (Figure 3).
Rho proteins and lymphoid tumours In addition to a role for Rho molecules in the development of Ras-induced epithelial skin tumours, Rho proteins and their regulators have also been implicated in the development of lymphoid tumours, although the oncogenic pathways targeted have not yet been identified. Transgenic mice, in which the expression of C3 toxin is driven by the thymocyte-specific Lck promoter, develop aggressive malignant thymic lymphomas [40]. Because C3 toxin inactivates RhoA, -B and -C, it is not clear which of the inhibited Rho proteins play a role in promoting tumourigenesis. Tiam1 was found to be activated by retroviral insertions in T lymphomas induced by Moloney murine leukaemia virus infection of transgenic Em-Pim1 mice, indicating that Tiam1 cooperates with Pim1 in lymphomagenesis [41]. Interestingly, a few examples of mutations in Rho GTPases have been found in human lymphoid tumours. In non-Hodgkin’s lymphoma, a t(3;4)(q27;p11-13) translocation was found that generates a fusion protein between the RhoH/TTF GTPase and LAZ3/BCL3 [42]. Mutations in RhoH were also found in B cell diffuse large cell lymphomas [43]; however, the potential function of RhoH in the formation of these lymphoid tumours remains to be established.
Rho proteins and Wnt signalling Recently, Rho proteins have also been implicated in Wnt-signalling pathways (Figure 4). Aberrant Wnt!bcatenin!T-cell factor (TCF) signalling frequently leads to the development of various types of tumours, including intestinal and mammary epithelial tumours. The regulatory step in the Wnt!b-catenin pathway is degradation of b-catenin, controlled by the destruction complex comCurrent Opinion in Cell Biology 2003, 15:583–589
prising axin, adenomatous polyposis coli (APC) and the serine/threonine kinase glycogen synthase kinase-3b (GSK3b; reviewed in [44]). b-catenin is phosphorylated by GSK3b, which earmarks it for ubiquitination and subsequent proteolytic degradation. Binding of Wnt factors to their transmembrane Frizzled (Fz) receptors inactivates GSK3b through the adaptor protein Dishevelled (Dvl). Through inactivation of GSK3b or mutations in proteins of the Wnt signalling pathway, b-catenin becomes stabilised and shuttles to the nucleus, where it binds to DNA-binding proteins of the TCF family, to serve as a coactivator of transcription of Wnt-responsive genes. In tumours, components of the Wnt pathway (most frequently APC) are often mutated, leading to constitutive activation of the b-catenin!TCF pathway, overexpression of Wnt target genes, and malignant transformation. Preliminary data from our laboratory indicate that Tiam1 is a Wnt-responsive gene and that its deficiency impairs the development of intestinal tumours in APC mutant mice, similarly as shown before (A Malliri et al., unpublished data) for Ras-induced skin tumours. These data suggest that Tiam1 and Rac play a role in the oncogenic Wnt!bcatenin signalling pathway. Another recently identified Wnt transcriptional target is Wrch-1, a novel Cdc42-like GTPase whose overexpression phenocopies Wnt-1 in morphological transformation of mouse mammary epithelial cells [45]. Whether Wrch-1 plays a role in Wnt/b-catenin/ TCF-induced tumours in vivo remains to be established. In addition to a potential role in b-catenin/TCF-induced tumour formation, Rho proteins and their regulators influence cell motility and polarity by interacting directly with components of Wnt signalling pathways. The Racspecific GEF Asef was cloned because of its ability to interact with the armadillo repeat domain of APC [46]. This interaction leads to the activation of the GEF function of Asef, through relief of its auto-inhibition. Overexpression of Asef decreases E-cadherin-mediated cell–cell adhesion and promotes the migration of MDCK cells [47]. Truncated APC proteins expressed in colorectal tumour cells stimulate both these activities of Asef. This suggests that truncated APC, apart from affecting tumour cell proliferation by activating b-catenin/TCF signalling, also contributes to the aberrant migratory properties of tumour cells through stimulation of the activity of Asef. The effect of Asef on cell migration is intriguing, especially since another Rac-specific GEF, Tiam1, inhibits migration of most epithelial cells, including MDCK cells [25]. These data, showing two different Rac GEFs having opposite effects on cell migration, support the concept discussed above that GEFs might determine the signalling downstream of Rho GTPases by binding to specific effector or adaptor molecules. APC also interacts with the plus ends of microtubules, and this interaction is essential for cell polarisation. Cdc42 www.current-opinion.com
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Figure 4
Wnt Frizzled
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Dvl Dvl
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Rho proteins and Wnt signalling. Binding of Wnt ligands to their receptors Frizzled activates several signalling cascades. The Wnt!b-catenin (b-cat)!TCF pathway results in altered gene expression that can lead to cellular transformation. Two such Wnt-responsive genes are Tiam1 and Wrch-1. In another cascade, activation of Dvl leads independently to activation of Rho and Rac. In the case of Rho, this requires the formin-homology protein Daam1 and results in ROCK activation. Dvl activation of Rac is independent of Daam1 and triggers activation of c-Jun amino-terminal kinase (JNK). Both pathways cooperate to control cell motility during vertebrate gastrulation. APC, a key component of the Wnt!b-catenin!TCF pathway, interacts with the Rac GEF Asef and promotes cell motility by weakening AJs, whereas Tiam1–Rac complexes exert the opposite effect. APC interaction with microtubules is also regulated through inhibition of GSK3b by the Cdc42–Par6–PKCz complex, contributing to cell polarisation. (See text for full details.) p, phosphate; PKC, protein kinase C.
is required to establish cellular asymmetry during directed migration, and it acts through a Par6-aPKC (atypical protein kinase C) complex to inhibit the activity of GSK3b. Inhibition of GSK3b, in turn, allows association of APC with the plus ends of microtubules in cellular protrusions, resulting potentially in their stabilisation [48]. Additionally, it was shown recently that Wnt/Fz signalling activates Rac and Rho independently of each other and of b-catenin/TCF signalling. The adaptor protein Dvl plays an important role in this Wnt-induced activation of Rac and Rho, which controls morphogenetic movements during vertebrate gastrulation [49]. The GEFs involved in Dvl-mediated activation of Rac and Rho are still unknown, and whether or how these pathways regulate motility of tumour cells or other aspects of tumourigenesis still remains to be established.
Conclusions Data have emerged that directly implicate Rho signalling in the aetiology of cancer. Recent studies using mutant mouse models directly demonstrate the involvement of www.current-opinion.com
Rho proteins in multiple stages of tumour initiation and progression, and underscore in vitro studies that suggested roles for these GTPases in the regulation of cell growth and survival, as well as in the invasive behaviour of tumour cells. Both in vivo and in vitro studies have connected Rho proteins with the Ras and Wnt oncogenic signal transduction pathways. This even involves direct interaction of Rho molecules or their regulators with key mediators of Ras and Wnt signalling cascades. The involvement of Rho proteins in lymphomagenesis and the collaboration of Tiam1 and Pim1 in inducing lymphomas in vivo point to a broader involvement of Rhofamily proteins in various oncogenic signalling pathways. This is supported by studies of human malignancies that have identified mutations in Rho proteins and their regulators or shown that expression of these molecules is altered (reviewed in [1,6]). However, further studies are required to establish which of these mutational events play a causal role in tumourigenesis and to detail the full contribution of deregulated Rho signalling to human cancer. This would require identifying the critical effector Current Opinion in Cell Biology 2003, 15:583–589
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molecules and downstream signalling events that mediate the tumourigenic effects of Rho proteins. These effectors, together with Rho molecules and their regulators, could prove to be targets for the development of diagnostic tools or novel anti-cancer therapies.
Acknowledgements We thank Rob Roovers and Adam Hurlstone for critical reading of the manuscript. The Dutch Cancer Society, the European Community, and the Association for International Cancer Research support the research of JG Collard.
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35. van Leeuwen FN, Kain HE, Kammen RA, Michiels F, Kranenburg OW, Collard JG: The guanine nucleotide exchange factor Tiam1 affects neuronal morphology; opposing roles for the small GTPases Rac and Rho. J Cell Biol 1997, 139:797-807.
46. Kawasaki Y, Senda T, Ishidate T, Koyama R, Morishita T, Iwayama Y, Higuchi O, Akiyama T: Asef, a link between the tumor suppressor APC and G-protein signaling. Science 2000, 289:1194-1197.
36. Kozma R, Sarner S, Ahmed S, Lim L: Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol Cell Biol 1997, 17:1201-1211. 37. Fleming IN, Gray A, Downes CP: Regulation of the Rac1-specific exchange factor Tiam1 involves both phosphoinositide 3-kinase-dependent and -independent components. Biochem J 2000, 351:173-182. 38. Lambert JM, Lambert QT, Reuther GW, Malliri A, Siderovski DP, Sondek J, Collard JG, Der CJ: Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism. Nat Cell Biol 2002, 4:621-625. This study shows that the Rac activator Tiam1 is able to bind to activated Ras, providing a direct link between Ras- and Rac signalling. 39. Frame S, Balmain A: Integration of positive and negative growth signals during Ras pathway activation in vivo. Curr Opin Genet Dev 2000, 10:106-113. 40. Cleverley SC, Costello PS, Henning SW, Cantrell DA: Loss of Rho function in the thymus is accompanied by the development of thymic lymphoma. Oncogene 2000, 19:13-20.
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47. Kawasaki Y, Sato R, Akiyama T: Mutated APC and Asef are involved in the migration of colorectal tumour cells. Nat Cell Biol 2003, 5:211-215. This paper shows that the binding of truncated adenomatous polyposis coli (APC) to Asef decreases E-cadherin-mediated cell–cell adhesion and promotes the migration of epithelial MDCK cells and colorectal cancer cells. These results suggest that the APC–Asef complex might contribute to aberrant migratory behaviour of colorectal tumour cells. 48. Etienne-Manneville S, Hall A: Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature 2003, 421:753-756. Cdc42-dependent phosphorylation of glycogen synthase kinase-3b occurs specifically at the leading edge of migrating cells, and induces the interaction of adenomatous polyposis coli protein with the plus ends of microtubules, which is required for cell polarisation and directed cell migration. 49. Habas R, Dawid IB, He X: Coactivation of Rac and Rho by Wnt/ Frizzled signaling is required for vertebrate gastrulation. Genes Dev 2003, 17:295-309. This paper shows that Wnt/Fz signalling activates Rac and Rho through the cytoplasmic Dishevelled protein. Wnt/Fz activation of Rac and Rho is proposed to be required for cell polarity and movements during vertebrate gastrulation.
Current Opinion in Cell Biology 2003, 15:583–589
Role of Rho-family proteins in cell adhesion and cancer Angeliki Malliri and John G Collardy Rho-family proteins control signalling pathways that regulate a wide range of biological processes. In vitro studies implicating Rho proteins in cell adhesion, migration, transcriptional activation, cell-cycle progression and transformation suggested roles for these proteins in the formation and progression of tumours in vivo. Studies using different recombinant mouse models have recently confirmed this idea. Rho signalling pathways crosstalk with different oncogenic signalling cascades, including those downstream of Ras and Wnt, and contribute to various aspects of tumourigenesis, including survival, growth and progression of tumour cells. Addresses The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands e-mail: [email protected] y e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:583–589 This review comes from a themed issue on Cell-to-cell contact and extracellular matrix Edited by Eric Brown and Elisabetta Dejana 0955-0674/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00098-X
Abbreviations AJ adherens junction APC adenomatous polyposis coli Asef APC-stimulated GEF Dvl Dishevelled Fz Frizzled GAP GTPase-activating protein GDI guanine nucleotide dissociation inhibitor GEF guanine nucleotide exchange factor GSK glycogen synthase kinase TCF T-cell factor
Introduction Rho GTPases comprise a large subfamily of the Ras superfamily and include Cdc42, Rac and Rho proteins. Similarly to Ras proteins, Rho GTPases are guanine nucleotide binding proteins that cycle between an active, GTP-bound and an inactive, GDP-bound state (Figure 1). The activity of Rho proteins is controlled by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs activate small GTPases by promoting the exchange of GDP for GTP, whereas GAPs enhance the intrinsic rate of hydrolysis of bound GTP to GDP, leading to inactivation. In cells, Rho GTPases exist predominantly in their inactive, GDP-bound form in complex with GDP dissociation inhibitors (GDIs). Actiwww.current-opinion.com
vation of Rho GTPases results in binding to various effector molecules that elicit downstream responses. Because Rho proteins control a wide range of signalling pathways that regulate various biological processes, it is not surprising that dysregulation of their activities can result in diverse aberrant cellular phenotypes found in different diseases (reviewed in [1]). Cell surface receptors use different GEFs to activate specific GTPases, and more than 50 potential GEFs have been identified within mammalian genomes (reviewed in [2]). GEFs not only activate Rho GTPases but also participate in the selection of downstream effectors by either binding to effectors directly or to scaffold proteins that complex with components of effector pathways (Figure 1). For example, the Rac activator a-PIX/Cool-2 also binds to Pak [3], a downstream effector of Rac and Cdc42, while the Rac activator Tiam1 binds IB2/JIP2, a scaffold for the p38 mitogenactivated protein kinase (MAPK) cascade, as well as the scaffold spinophilin. Spinophilin binding enhances Tiam1’s ability to activate p70 S6 kinase, while simultaneously suppressing its ability to activate Pak [4,5]. Evidence implicating aberrant Rho signalling in cancer has been obtained from in vitro studies that focused on specific aspects of tumour cell biology and to a lesser extent from mutations found in genes encoding Rho signalling components (both reviewed in [1,6]). More recently, in vivo studies using recombinant mice lacking or overexpressing Rho signalling proteins have provided direct evidence for the involvement of Rho proteins in cancer, which will be the focus of this review.
Rho proteins and cellular transformation The first findings to link Rho molecules with cancer came from in vitro foci formation assays that model the growthfactor- and anchorage-independent growth of tumour cells. Many Rho GEFs, such as Dbl, Ect2, Vav and Lfc, were identified as 50 -end-truncated proto-oncogenes in NIH3T3 transformation assays (reviewed in [7,8]). Moreover, it was shown that constitutively active Rac1 (V12Rac) and, to a lesser extent, RhoA were able to induce an oncogenic phenotype in fibroblasts, albeit much less efficiently than constitutively active Ras (V12Ras) [9,10]. Rho proteins appear to act downstream of Ras in oncogenic transformation of cells, since dominant-negative mutants of Rho GTPases inhibited Ras-induced focus formation, growth in soft agar and tumour formation in nude mice. In addition, V12Rac1 and V14RhoA cooperated with constitutively active Raf (RafCAAX) in focus formation [11,12], indicating that oncogenic V12Ras drives both Raf!MAPK and Rac/Rho-mediated pathways. Current Opinion in Cell Biology 2003, 15:583–589
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Figure 1
GEF
GTP
Scaffold Rho–GDP
Rho–GDP
Rho–GDI
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Pi GAP Cytoskeletal changes Gene expression Current Opinion in Cell Biology
The Rho GTPase cycle. Rho proteins cycle between an inactive, GDPbound, and an active, GTP-bound, state. Transitions between the two states are controlled by GEFs, GAPs and GDIs. Additionally, GEFs select downstream effectors either directly or through recruitment of scaffold proteins (see text for full details). Pi, phosphate.
Studies using dominant-active Rac and Rho effector mutants indicated that the transforming capacity of Rho GTPases was in general associated with the capacity of these mutants to induce cyclin D1 expression rather than cytoskeletal rearrangements [13,14].
Rho GTPases in cell adhesion and cell migration Further interest in Rho proteins was raised by their ability to induce reorganisation of the actin cytoskeleton. Since actin cytoskeletal changes are required for migratory behaviour of cells in response to growth factor stimulation or matrix interactions, subsequent studies addressed the contribution of Rho molecules to motility and invasion of tumour cells. Experiments performed primarily using fibroblasts showed that Cdc42, Rac and Rho cooperate to regulate the cytoskeletal changes required for migratory behaviour of cells (reviewed in [15,16]). To summarise the findings of numerous studies here, Cdc42 regulates the polarity of cell migration, whereas Rac regulates the formation of membrane protrusions at the leading edge of migrating cells, required for forward movement. RhoA is required for the generation of contractile force leading to rounding of the cell body. Rho proteins also regulate microtubule polymerisation involved in cell migration. In epithelial cells, Rho GTPases regulate the formation and maintenance of specialised junctional adhesion complexes — tight junctions and adherens junctions (AJs) — required for the barrier function of epithelial layers and to establish apical-basolateral polarity (reviewed in [17,18]). Loss of AJs, in particular, has been correlated with the malignant progression of epithelial tumours (see [19,20]). In addition to promoting intercellular adhesion, Rho proteins can also stimulate epithelial cell migration downCurrent Opinion in Cell Biology 2003, 15:583–589
stream of certain integrin–extracellular-matrix (ECM) interactions [21]. It is now considered that whether an epithelial cell migrates or not represents an integration of migration-inhibitory cell–cell interactions and migration-promoting cell–substrate interactions. These interactions are influenced by numerous external and intrinsic factors, including the nature of the ECM, the relative levels of activity of Rac and Rho, and possibly the specific exchange factors used to activate the Rho proteins (see later for APC-stimulated GEF [Asef]). For example, activation of Rac by Tiam1 promoted migration of Madin–Darby canine kidney (MDCK) cells on a collagen substrate but inhibited migration on a fibronectin or laminin substrate [22]. Epithelial–mesenchymal transition induced by expression of the Ras oncogene in MDCK cells was caused by a shift in balance between Rac and Rho activity [23,24]. Since Tiam1/Rac signalling is required in MDCK cells for E-cadherin-mediated adhesion [25], downregulating the activity of Rac and consequently activating Rho, results in loss of an epithelial phenotype and acquisition of a mesenchymal one (Figure 2). The mechanism by which Rac antagonises Rho involves Rac-mediated production of reactive oxygen species (ROS) that inhibit the low-molecular-weight protein tyrosine phosphatase, resulting in phosphorylation and activation of p190Rho GAP, a negative regulator of Rho activity [26]. Figure 2
Ras
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Acto-myosin contraction Stress fibers Cell rounding
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Balance between Rac and Rho activity determines cell phenotype. Activated Rac (pink) stimulates cell–cell adhesions, promoting an epithelial phenotype. By contrast, activated Rho (green) stimulates cell contraction and a mesenchymal phenotype. Rac downregulates Rho activity by stimulating Rho GAP activity through reactive oxygen species (ROS) production. ROS production inhibits the low molecular weight protein tyrosine phosphatase (LMW-PTP), resulting in phosphorylation and activation of Rho GAP, which in turn inhibits Rho activity. Rac and Rho activity is also influenced by upstream signalling. Low levels of Ras activate Rac, whereas high levels of Ras inhibit Rac activity (see text for full details). www.current-opinion.com
Role of Rho-family proteins in cell adhesion and cancer Malliri and Collard 585
The cell matrix might also influence growth factor signalling and cell proliferation. Loss of matrix attachment leads to inhibition of growth-factor-induced activation of the Ras!Raf!extracellular-signal-regulated kinase (ERK) cascade [27], explaining why normal cell proliferation is dependent on integrin-mediated anchorage to cell matrices. Adhesion to fibronectin leads to activation of both Rac and Rho, and as a consequence to increased cyclin D1 levels and to downregulation of the cyclindependent kinase inhibitor p21WAF1 [28,29]. Both events promote cell proliferation. Thus, cooperation exists between growth factor receptors and integrins in the activation of Ras- and Rho-mediated signalling pathways to regulate proliferation and migration of cells. How Rho signalling could affect the development and progression of tumours in vivo is difficult to predict from in vitro studies alone. Recent studies employing recombinant mice directly implicate Rho-family proteins in all stages of tumourigenesis, and reveal both tumour-promoting and -suppressor functions (see below).
Rho signalling and Ras-induced skin tumours in mutant mice Studies using knockout mice have demonstrated that deregulated Rho signalling influences various aspects of tumourigenicity. A two-stage chemical skin carcinogenesis protocol has been applied to two knockout mouse lines, namely RhoB- and Tiam1-deficient mice. The protocol entails tumour initiation in epidermal keratinocytes by treatment with the carcinogen 7,12-dimethylbenzanthracene, which induces oncogenic activation of the c-Ha-Ras gene. Subsequent repeated treatments with the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) result in the outgrowth and progression of initiated cells. Applying this protocol to RhoBdeficient mice resulted in the development of increased numbers of skin tumours compared with wild-type mice [30]. Moreover, E1A- and Ras-transformed RhoBdeficient primary mouse embryonic fibroblasts were more resistant to apoptosis induced by DNA-damaging agents. These findings imply that RhoB functions normally to suppress tumourigenesis by promoting apoptosis following cellular stress. By contrast, application of the same protocol to Tiam1deficient mice resulted in much fewer and smaller tumours compared with wild-type mice [31]. Tiam1 deficiency was associated with increased Ras-induced apoptosis during tumour initiation and with impeded TPA-induced proliferation during tumour promotion. These results show that Tiam1/Rac signalling cooperates with Ras during tumour initiation and promotion by positively influencing cell survival and growth (Figure 3). The effect of Tiam1/Rac signalling on cell growth could be mediated by D-type cyclins, since Rac has been shown to induce expression of cyclin D1 and D2 (see [14]), and cyclin D1-deficient mice display a www.current-opinion.com
Figure 3
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?
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Interactions between Ras and Rho pathways in tumourigenesis based on analysis of RhoB and Tiam1 mutant mice. RhoB-deficient mice develop more Ras-induced skin tumours than wild-type mice. Also, RhoB-deficient cells are more resistant to apoptosis. These findings imply that RhoB functions normally to suppress cell survival following cellular stress and thus to suppress tumourigenesis. In contrast, Tiam1deficient mice develop fewer and smaller Ras-induced skin tumours than wild-type mice. This is related to the positive effects of Tiam1/Rac signalling on cell survival and proliferation. However, Tiam1-deficient tumours progressed more frequently to malignancy. This can be explained by in vitro and in vivo results showing that high levels of Ras signalling (Ras""), as seen in Ras-transformed cells and advanced skin malignancies, downregulate Tiam1 expression and thereby Rac activity, which is required for cell–cell adhesion. Dashed lines indicate hypothetical links. (See text for full details.)
similar resistance to the development of skin and mammary tumours [32,33]. Other studies also support a role for Rac in cell survival. Rac was found to protect cells from Ras- induced apoptosis, through activation of NF-kB [34]. Thus, the studies with Tiam1 mutant mice are consistent with conclusions drawn from in vitro models and strongly suggest that Rac signalling is required for Ras-induced tumourigenesis through the stimulation of cell growth and enhancing cell survival in the presence of cellular stress. The fact that RhoB exerts an opposite effect on Ras-induced skin tumours and apoptosis might represent another example of Rac and Rho signalling being mutually antagonistic. This has been shown in several biological processes such as epithelial–mesenchymal transition [24] and neurite formation [35,36]. Interestingly, the few tumours in Tiam1-deficient mice progressed more frequently to malignancy than those in wild-type mice, suggesting that Tiam1 deficiency promotes malignant conversion. Indeed, analysis of Tiam1 expression in tumours of wild-type mice revealed that benign papillomas maintained high levels of Tiam1 expression, whereas expression was reduced in squamous Current Opinion in Cell Biology 2003, 15:583–589
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cell carcinomas and was completely lost in highly invasive spindle cell carcinomas [31]. Thus, while Tiam1 function appears to be essential for the initiation and promotion of Ras-induced skin tumours, these data suggest that subsequent loss of Tiam1 expression promotes the malignant conversion of benign tumours. Ras can activate Tiam1 in two ways: through a phosphatidylinositol 3-kinase-dependent mechanism [22,37]; or through direct binding of active GTP-bound Ras to a Rasbinding domain located within the Tiam1 protein [38]. Interestingly, the increased Ras signalling associated with advanced skin malignancies that results from amplification of the mutated Ras allele [39] seems to be responsible for the reduction or loss of Tiam1 expression in the later stages of tumour progression, as demonstrated in vitro for Ras-transformed MDCK cells [24]. As discussed above, loss of Tiam1 expression and the resultant decrease in Rac activity in advanced tumours could lead to decreased E-cadherin-mediated adhesion, promoting invasiveness of epithelial tumour cells (Figure 3).
Rho proteins and lymphoid tumours In addition to a role for Rho molecules in the development of Ras-induced epithelial skin tumours, Rho proteins and their regulators have also been implicated in the development of lymphoid tumours, although the oncogenic pathways targeted have not yet been identified. Transgenic mice, in which the expression of C3 toxin is driven by the thymocyte-specific Lck promoter, develop aggressive malignant thymic lymphomas [40]. Because C3 toxin inactivates RhoA, -B and -C, it is not clear which of the inhibited Rho proteins play a role in promoting tumourigenesis. Tiam1 was found to be activated by retroviral insertions in T lymphomas induced by Moloney murine leukaemia virus infection of transgenic Em-Pim1 mice, indicating that Tiam1 cooperates with Pim1 in lymphomagenesis [41]. Interestingly, a few examples of mutations in Rho GTPases have been found in human lymphoid tumours. In non-Hodgkin’s lymphoma, a t(3;4)(q27;p11-13) translocation was found that generates a fusion protein between the RhoH/TTF GTPase and LAZ3/BCL3 [42]. Mutations in RhoH were also found in B cell diffuse large cell lymphomas [43]; however, the potential function of RhoH in the formation of these lymphoid tumours remains to be established.
Rho proteins and Wnt signalling Recently, Rho proteins have also been implicated in Wnt-signalling pathways (Figure 4). Aberrant Wnt!bcatenin!T-cell factor (TCF) signalling frequently leads to the development of various types of tumours, including intestinal and mammary epithelial tumours. The regulatory step in the Wnt!b-catenin pathway is degradation of b-catenin, controlled by the destruction complex comCurrent Opinion in Cell Biology 2003, 15:583–589
prising axin, adenomatous polyposis coli (APC) and the serine/threonine kinase glycogen synthase kinase-3b (GSK3b; reviewed in [44]). b-catenin is phosphorylated by GSK3b, which earmarks it for ubiquitination and subsequent proteolytic degradation. Binding of Wnt factors to their transmembrane Frizzled (Fz) receptors inactivates GSK3b through the adaptor protein Dishevelled (Dvl). Through inactivation of GSK3b or mutations in proteins of the Wnt signalling pathway, b-catenin becomes stabilised and shuttles to the nucleus, where it binds to DNA-binding proteins of the TCF family, to serve as a coactivator of transcription of Wnt-responsive genes. In tumours, components of the Wnt pathway (most frequently APC) are often mutated, leading to constitutive activation of the b-catenin!TCF pathway, overexpression of Wnt target genes, and malignant transformation. Preliminary data from our laboratory indicate that Tiam1 is a Wnt-responsive gene and that its deficiency impairs the development of intestinal tumours in APC mutant mice, similarly as shown before (A Malliri et al., unpublished data) for Ras-induced skin tumours. These data suggest that Tiam1 and Rac play a role in the oncogenic Wnt!bcatenin signalling pathway. Another recently identified Wnt transcriptional target is Wrch-1, a novel Cdc42-like GTPase whose overexpression phenocopies Wnt-1 in morphological transformation of mouse mammary epithelial cells [45]. Whether Wrch-1 plays a role in Wnt/b-catenin/ TCF-induced tumours in vivo remains to be established. In addition to a potential role in b-catenin/TCF-induced tumour formation, Rho proteins and their regulators influence cell motility and polarity by interacting directly with components of Wnt signalling pathways. The Racspecific GEF Asef was cloned because of its ability to interact with the armadillo repeat domain of APC [46]. This interaction leads to the activation of the GEF function of Asef, through relief of its auto-inhibition. Overexpression of Asef decreases E-cadherin-mediated cell–cell adhesion and promotes the migration of MDCK cells [47]. Truncated APC proteins expressed in colorectal tumour cells stimulate both these activities of Asef. This suggests that truncated APC, apart from affecting tumour cell proliferation by activating b-catenin/TCF signalling, also contributes to the aberrant migratory properties of tumour cells through stimulation of the activity of Asef. The effect of Asef on cell migration is intriguing, especially since another Rac-specific GEF, Tiam1, inhibits migration of most epithelial cells, including MDCK cells [25]. These data, showing two different Rac GEFs having opposite effects on cell migration, support the concept discussed above that GEFs might determine the signalling downstream of Rho GTPases by binding to specific effector or adaptor molecules. APC also interacts with the plus ends of microtubules, and this interaction is essential for cell polarisation. Cdc42 www.current-opinion.com
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Figure 4
Wnt Frizzled
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Dvl Dvl
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Rho proteins and Wnt signalling. Binding of Wnt ligands to their receptors Frizzled activates several signalling cascades. The Wnt!b-catenin (b-cat)!TCF pathway results in altered gene expression that can lead to cellular transformation. Two such Wnt-responsive genes are Tiam1 and Wrch-1. In another cascade, activation of Dvl leads independently to activation of Rho and Rac. In the case of Rho, this requires the formin-homology protein Daam1 and results in ROCK activation. Dvl activation of Rac is independent of Daam1 and triggers activation of c-Jun amino-terminal kinase (JNK). Both pathways cooperate to control cell motility during vertebrate gastrulation. APC, a key component of the Wnt!b-catenin!TCF pathway, interacts with the Rac GEF Asef and promotes cell motility by weakening AJs, whereas Tiam1–Rac complexes exert the opposite effect. APC interaction with microtubules is also regulated through inhibition of GSK3b by the Cdc42–Par6–PKCz complex, contributing to cell polarisation. (See text for full details.) p, phosphate; PKC, protein kinase C.
is required to establish cellular asymmetry during directed migration, and it acts through a Par6-aPKC (atypical protein kinase C) complex to inhibit the activity of GSK3b. Inhibition of GSK3b, in turn, allows association of APC with the plus ends of microtubules in cellular protrusions, resulting potentially in their stabilisation [48]. Additionally, it was shown recently that Wnt/Fz signalling activates Rac and Rho independently of each other and of b-catenin/TCF signalling. The adaptor protein Dvl plays an important role in this Wnt-induced activation of Rac and Rho, which controls morphogenetic movements during vertebrate gastrulation [49]. The GEFs involved in Dvl-mediated activation of Rac and Rho are still unknown, and whether or how these pathways regulate motility of tumour cells or other aspects of tumourigenesis still remains to be established.
Conclusions Data have emerged that directly implicate Rho signalling in the aetiology of cancer. Recent studies using mutant mouse models directly demonstrate the involvement of www.current-opinion.com
Rho proteins in multiple stages of tumour initiation and progression, and underscore in vitro studies that suggested roles for these GTPases in the regulation of cell growth and survival, as well as in the invasive behaviour of tumour cells. Both in vivo and in vitro studies have connected Rho proteins with the Ras and Wnt oncogenic signal transduction pathways. This even involves direct interaction of Rho molecules or their regulators with key mediators of Ras and Wnt signalling cascades. The involvement of Rho proteins in lymphomagenesis and the collaboration of Tiam1 and Pim1 in inducing lymphomas in vivo point to a broader involvement of Rhofamily proteins in various oncogenic signalling pathways. This is supported by studies of human malignancies that have identified mutations in Rho proteins and their regulators or shown that expression of these molecules is altered (reviewed in [1,6]). However, further studies are required to establish which of these mutational events play a causal role in tumourigenesis and to detail the full contribution of deregulated Rho signalling to human cancer. This would require identifying the critical effector Current Opinion in Cell Biology 2003, 15:583–589
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molecules and downstream signalling events that mediate the tumourigenic effects of Rho proteins. These effectors, together with Rho molecules and their regulators, could prove to be targets for the development of diagnostic tools or novel anti-cancer therapies.
Acknowledgements We thank Rob Roovers and Adam Hurlstone for critical reading of the manuscript. The Dutch Cancer Society, the European Community, and the Association for International Cancer Research support the research of JG Collard.
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Current Opinion in Cell Biology 2003, 15:583–589