Review Article Rho GTPases and cancer
Hui Li Karine Peyrollier € lcan Kilic Gu Cord Brakebusch*
University of Copenhagen, BRIC, BMI, 2200 Copenhagen, Denmark
Abstract Rho GTPases are a family of small GTPases, which play an important role in the regulation of the actin cytoskeleton. Not surprisingly, Rho GTPases are crucial for cell migration and therefore highly important for cancer cell invasion and the formation of metastases. In addition, Rho GTPases are involved in growth and survival of tumor cells, in the interaction of tumor cells with their environment, and they are vital for the
cancer supporting functions of the tumor stroma. Recent research has significantly improved our understanding of the regulation of Rho GTPase activity, the specificity of Rho GTPases, and their function in tumor stem cells and tumor stroma. This review summarizes these novel findings and tries C 2013 to define challenging questions for future research. V BioFactors, 40(2):226–235, 2014
Keywords: cancer; Rho GTPases
1. Rho GTPases Rho GTPases are a subfamily of the small G proteins with 20 members [1]. In their active GTP-bound form, Rho GTPases can interact with a large and diverse number of more than 70 effector molecules including scaffold molecules, actin binding proteins, and kinases that mediate the biological effects, which range from actin cytoskeletal re-organization to differentiation and regulation of stemness [2]. Activation of Rho GTPases requires GTP exchange factors (GEFs), which downstream of many cell surface receptors replace GDP by GTP. More than 70 GEFs have been identified and for many of them it was shown that they can activate more than Rho GTPase [3]. Different cell surface receptor families can activate GEFs including receptor tyrosine kinases and integrins. Inactivation of classical Rho GTPases occurs by hydrolysis of GTP to GDP, catalyzed by the Rho GTPase itself with the help of GTPase activating proteins (GAPs). This group of Rho GTPase regulators is also large with more than 50 members [4]. Nonclassical Rho GTPases, however, have lost the GTPase function and are constantly in the active conformation [5].
All Rho GTPases are regulated at the level of gene expression, protein turnover, protein modification, and interaction with inhibitory proteins [6]. Guanosine nucleotide dissociation inhibitors (GDIs), for example, bind to several Rho GTPases and prevent the dissociation of GDP thus keeping the Rho GTPases in the inactive state. Rho GTPases can therefore integrate information from many different signaling pathways. Furthermore, Rho GTPases crosstalk with each other at the level of Rho GTPase activation, Rho GTPase stability, and regulation of downstream signaling pathways, resulting in a complex network of interactions and coordinated regulation of Rho GTPases in different biological processes [7]. Regulation of Rho GTPases is therefore rather complex and cell type specific. Also downstream signaling of Rho GTPases is complex, as activation of a specific Rho GTPase can induce many different Rho GTPase effector pathways. However, it is not clear whether all these pathways are equally important in a given cell or whether dominant regulation by other signaling mechanisms or preformed complexes of Rho GTPases with specific regulators and effectors reduce the complexity of Rho GTPase signaling. Recent data discussed below seem to support such ideas.
C 2013 International Union of Biochemistry and Molecular Biology V
Volume 40, Number 2, March/April 2014, Pages 226–235 *Address for correspondence: Cord Brakebusch, Ph. D., BRIC, Ole Maalïes Vej 5, 2200 Copenhagen, Denmark. Tel.: 145 353 25619; Fax: 145 353 25669; E-mail: [email protected]. Received 16 August 2013; accepted 14 November 2013 DOI 10.1002/biof.1155 Published online 21 December 2013 in Wiley Online Library (wileyonlinelibrary.com)
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2. Rho GTPase Function in Cancer Studies with tumor cell lines showed that Rho GTPases regulate cell polarity and promote proliferation, survival, production of angiogenic factors, and migration of cancer cells [8]. Such pro-tumorigenic function of many Rho GTPases is supported by the increased expression of Rho GTPases in many tumors and by a limited number of studies with genetically
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modified mice (Table 1). However, reduced expression of RhoB in lung and head and neck squamous cell carcinoma [9,10] or spontaneous development of hepatocellular cancers in mice lacking Cdc42 in hepatocytes [11] indicates that the Rho GTPase signaling network is complex and that the relative in vivo importance of specific molecular mechanisms of Rho GTPase action described in vitro is mostly unknown and probably highly cell type specific. Rho GTPases are important regulators of cancer cell proliferation, survival, invasion, and metastasis. More recently, crucial functions of Rho GTPases in the regulation of tumor stroma, including endothelial cells, immune cells, and cancer associated fibroblasts, as well as in the formation of microvesicles have been reported (Fig. 1). The aim of this review is to describe how research of the last years has improved our understanding of Rho GTPase regulation and function in cancer and to define open questions for future studies.
TABLE 1
Rho GTPase expression in tumors.
Rho GTPase
Expression in tumors
RhoA
"
[12–21]
RhoB
"#
[9,10,12,14]
RhoC
"
[12,14–16,21–33]
Rnd1
"
[24]
"#
[34–37]
Rac1
"
[12,17,18,24,38–42]
3. Regulation of Rho GTPase Activity by Oncogenes
Rac2
"
[13,25,42]
Rac3
"
[43]
Aberrant regulation of Rho GTPase activity and corresponding effector pathways is quite likely a hallmark of human tumors. First, the increased expression of, for example, RhoA, RhoC, Rac1, and Cdc42 in different types of cancer suggests that increased activity of these Rho GTPases promotes tumor progression, although the increased Rho GTPase expression may not necessarily correlate with an increase in activity, which is under complex regulation as described above. Second, oncogenic activation of tyrosine kinases such as ERBB2 or MET should result in increased activation of Rho GTPases in cancer even in the absence of any changes in expression of Rho GTPase genes, as tyrosine kinases are major activators of Rho GTPases [52]. Finally, several cancer promoting effects of miRNAs have been associated with altered expression of Rho GTPases [6]. However, more in vivo data are required to prove a direct relationship. Interestingly, Rho GTPase activation has hardly been checked in human tumors, probably since the most robust methods available, pull-down of activated Rho GTPases and ELISA, require fresh tumor material. Antibodies specifically recognizing the activated forms of RhoA, Rac1 and Cdc42 have been generated, but these antibodies have up to now not been used for larger screening studies of human material. A recent addition to the Rho GTPase activating oncogenes is the transcription factor human pituitary tumor-transforming gene 1 (hPTTG1), which is overexpressed in many metastatic tumors [53]. One of the target genes of hPTTG1 is the GEF-H1, which activates RhoA and increases invasion of MDA-MB-231 breast cancer cells in vitro and in vivo. As inhibitors of RhoA and ROCK prevented hPTTG1 induced cytoskeletal changes in vitro, it was suggested that RhoA/ROCK signaling is crucial for the pro-invasive effect of hPTTG1. ROCK is highly important for the regulation of cell contraction, suggesting a function in
RhoG
"
[24]
Cdc42
"
[12,17]
RhoH
#
[44–47]
RhoBTB1
#
[48]
RhoBTB2
#
[49–51]
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References
Rnd2 Rnd3 Rif RhoD
TC10 TCL Chp Wrch1
This table lists the Rho GTPase gene family and reported changes in gene expression observed in human tumors.
cell migration and metastasis. Indeed, several cell culture and mouse studies support this hypothesis, but the role of ROCK in human cancer is less clear and ROCK inhibitors clinically used for treatment of different diseases are not applied in cancer therapy or trials [54,55].
4. Regulators of Rho GTPase Activity The oncoprotein gankyrin is upregulated by Ras induced transformation and increased in lung cancer with Ras mutations. In vitro data suggest that gankyrin promotes tumor cell survival by decreasing RhoA activation [56]. Gankyrin promoted the binding of RhoA to Rho GDIa, which reduced the amount of active GTP-bound RhoA and decreased ROCK activity. As ROCK is required for PTEN activity, reduced RhoA/
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FIG 1
Overview of tumor relevant functions of Rho GTPases. Simplified scheme showing major regulation and effector pathways of Rho GTPases in cancer with a focus on Rho, Rac, and Cdc42. Important effector pathways are indicated beside the corresponding arrows. Rho GTPases regulate invasion and metastasis particularly by controlling cytoskeletal organization. Rho GTPasedependent gene expression controls stemness, angiogenesis, and immune response, while proliferation and survival are affected by cytoskeleton and gene expression. Rho-dependent cell contraction is important for microvesicle and CAF formation (RTKs: receptor tyrosine kinases; ROS: reactive oxygen species; CAF: cancer associated fibroblasts).
ROCK activity resulted in prolonged Akt activation. This study therefore implies that reduction of RhoA and ROCK activity promotes tumor progression. Reduced ROCK activity in cancer has been suggested already earlier, since it was found that Ras signaling reduces ROCK activity. In that case, however, ROCK activity was decreased independent of RhoA via sustained MekErk signaling [57,58]. Mouse models should be applied to test the relative importance of these pathways in vivo. GAPs and GDIs are known to inhibit Rho GTPase activation by increasing GTPase activity or maintaining inactive Rho GTPases in the cytosol, respectively. Yet two recent articles indicate they could also contribute to increased Rho GTPase activity, at least in cancer. The first study analyzes p190B, which is a GAP for RhoA, Rac1, and Cdc42. Overexpression of p190B in mammary epithelium of MMTV-neu induced tumors, however, resulted in increased activation of Rac1, while amounts of GTP-bound Cdc42 and RhoA were not changed [59]. This correlated with increased tumor multiplicity and metastasis formation. Neither tumor cell proliferation nor apoptosis were altered, although activation of Pak1/2, AKT, and ERK were even decreased, which normally correlates with
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decreased proliferation and survival. The second paper demonstrates an unexpected role for GDIa in cancer cells. Knockout of GDIa decreases protein amounts of RhoA, Rac1, and Cdc42 in vivo [60]. However, in the prostate carcinoma cell line PC-3, GDIa was shown to increase the half life time of the associated RhoA and apparently also RhoA signaling, as anchorage independent growth was decreased similar to overexpression of RhoA [61]. No change was observed for protein levels of Rac1 and Cdc42. This complex behavior of GDIs is also reflected in cancer patients, where GDI expression was found to be increased or decreased depending on the type of malignancy [62]. Some Rho GTPases such as Rnd1, 2, 3 have lost the GTPase function and are constitutively active. They do not bind to GDIs, but recently it was shown that they can be inhibited by binding to 14-3-3, which sequestrates the Rnd’s in the cytosol. This interaction is regulated by ROCK mediated phosphorylation of the C-terminus of Rnd [63]. Interestingly, increased 14-3-3 expression correlates with poor survival of cancer patients [64]. As Rnd’s are inhibiting Rho/ROCK signaling, increased 14-3-3 levels could cause increased Rho/ROCK
Rho GTPases and Cancer
activity, which might promote cancer progression by Rho/ ROCK dependent pathways.
5. Rho GTPase Mutations in Cancer Although Rho GTPases are frequently overexpressed in malignant tumors, suggesting although not proving that increased Rho GTPase activation might promote tumor progression, no constitutively active mutants of Rho GTPases have been found in cancer until recently. Two recent papers, however, described an activated Rac1 mutant with a P29S exchange in sun-exposed melanoma [65,66]. In fact, this mutation was the most frequent one after mutations in BRAF or NRAS. Activating mutations were also found for Rac2 and Cdc42. In head and neck squamous cell carcinoma, and esophageal and pancreatic cancers, Rac1 P29S mutations were found as a rare event. As Rac1 was shown to regulate proliferation and migration of melanoblasts in vivo [67], the presence of a Rac1 P29S mutant is conceivably highly relevant for cancer progression. The recently shown activation of p110b PI3K by Rac1 and Cdc42 might also contribute to the oncogenic potential of the Rac1 P29S mutation [68].
6. Channeled Activation of Rho GTPase Signaling In vitro studies showed that Rho GTPases can affect a large number of biological processes via multiple mechanisms, yet display at the same time also a high extent of cell type specificity. A simple explanation for this observation could be the differential expression of Rho GTPase effectors. Yet recent data suggest that also channeled activation of specific effectors, and crosstalk to other Rho GTPases and other signaling pathways might contribute to the specificity of Rho GTPases. Expression of constitutively active Rho GTPase mutants in cells often leads to the parallel activation of different downstream pathways initiated by the interaction of the Rho GTPases with corresponding effector proteins. However, physiological activation of Rho GTPases might be much more restricted with respect to downstream signaling (Fig. 2). This was beautifully illustrated in a recent study on integrin signaling, where av containing integrins activate RhoA through GEF-H1 resulting in activation of mDia, but not of ROCK and myosin II [69]. Activation of a5b1 integrin, conversely, leads to activation of the RhoA/ ROCK pathway with little mDia mediated stress fiber formation. One explanation for this specificity could be the formation of complexes containing GEFs, Rho GTPases, and effector proteins, which channel the activated Rho GTPases into restricted effector pathways. Alternatively, Rho GTPases or their effectors proteins are post-translationally modified by Rho GTPase independent pathways to favor certain Rho GTPase–effector interactions. In any case, these data suggest that of the many theoretically possible Rho GTPase–effector interactions in a cancer cell maybe only very few are actually used, depending on the path of Rho GTPase activation.
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7. Cell Type Specificity of the Atypical Rho GTPase Rnd3 The non-classical constitutively active Rho GTPase Rnd3, enhances inactivation of Rho by p190RhoGAP and directly inhibits the Rho effector ROCK1 [70]. Depending on the type of cancer, Rnd3 expression is increased or decreased. In liver and gastric cancer, specificRnd3 is downregulated [34,64,65]. Earlier it was shown that Rnd3 inhibits cell cycle progression and transformation in NIH 3T3 cell independent of RhoA and ROCK [71]. More recently, knockdown of Rnd3 in two liver cancer cell lines increased invasiveness in vitro and in vivo and promoted plasma membrane blebbing, characteristic for ameboid migrating cells [72]. This correlated with increased activation of ROCK and the fact that the ROCK inhibitor Y27632 decreased cell migration. Pulmonary metastasis, however, was not significantly changed. Another report also found that decrease of Rnd3 in HCC cells promotes invasiveness, but correlated it to decreased E-cadherin expression [73]. Despite the different mechanistic explanations, both studies indicate that loss of Rnd3 increases invasiveness of HCC and suggest that it might have a similar function in other tumors where expression of Rnd3 is decreased. However, Rnd3 is not always downregulated in cancer. In pancreatic cancer and NSLC, for example, it is overexpressed, indicating that depending on the cellular context Rnd3 might function as an oncogene or as a tumor suppressor [35,36]. In fact, it was suggested that even differences in the genetic background might explain contrasting results of Rnd3 function in HCC [72].
8. Specific function of Rho GTPase subfamily members As many effectors are shared between different Rho GTPases, Rho GTPases might have overlapping functions. Nevertheless, specific biological effects can be mediated. The Rho subfamily of Rho GTPases has three members, RhoA, RhoB, and RhoC, which bind to a highly overlapping set of effectors, although maybe with slight different affinities resulting in a certain degree of specificity [74]. Different intracellular location of RhoA, B and C further contributes to specific functions. RhoB is found mainly at endosomal membranes, whereas the active forms of RhoA and RhoC are present at the cell membrane. This correlated with the reported tumor suppressor role of RhoB and the mostly tumor promoting effects of RhoA and C. Probably the different intracellular localization is contributing to the specific actions of RhoB compared to RhoA and C. With respect to human cancer, RhoC seems to be more important than RhoA, as it is overexpressed in a wide range of malignant tumors correlating with malignant progression, while RhoA is less frequently overexpressed. RhoC-null mice displayed normal development and develop mammary tumors induced by polyoma middle T antigen
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FIG 2
“Channeled” versus “effector determined” Rho GTPase signaling. A. In the “channeled” model GEFs and effectors are coupled, either by complex formation, localization, or preferred interaction of post-translationally modified Rho GTPases with certain GEFs and effectors. Furthermore, specific GEFs might be coupled to specific receptors. In such a model, the biological effect of Rho GTPase activation is dependent on the pathway of Rho GTPase activation. B. In the “effector determined” model, different pathways lead to activation of a certain Rho GTPase, which then interacts with all corresponding effectors present in the cell. The biological effect of Rho GTPase activation is therefore dependent on the type and relative amounts of Rho GTPase effector molecules present.
similar to wild-type mice. However, growth and formation of metastases were significantly reduced [75]. Knockdown experiments in PC-3 cells suggested that RhoC promotes invasion by specific binding to Fmnl3, an actin polymerization inducing formin [76]. RhoA cannot bind to Fmnl3, but instead interacts with ROCK2 resulting in inhibition of Rac1 and decreased
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invasion. This could explain a RhoC specific contribution to invasion and would predict a more invasive phenotype of RhoA-deficient tumor cells. An earlier study using breast carcinoma cells suggested a pro-invasive function of RhoC and an anti-invasive role of RhoA due to inhibition of Rac1 [77]. Other data, however, indicate pro-invasive functions for both RhoA
Rho GTPases and Cancer
and RhoC in different cancer cell lines [78–80]. Interestingly, deletion of the RhoA gene in mouse resulted in reduced activation of Rac1 and Cdc42 in vitro, while both Rho GTPases showed unchanged activation in RhoA-null epidermis in vivo [81]. These data highlight the cell type specificity of Rho GTPases and their high dependency on the environmental conditions. Clearly, data obtained with tumor cells growing on rigid cell culture dishes in 2D in special growth media do not always model Rho GTPase signaling in vivo.
9. Cross-talk Between Rho GTPases It has been observed that knockdown and knockout models of one Rho subfamily member will lead to increased protein amounts of the remaining Rho subfamily members. GDIa seems to play an important role in this crosstalk. In PC-3 cells, knockdown of RhoC prevents the formation of RhoC–GDIa complexes. Instead, RhoA binds to GDIa, leading to increased levels of RhoA protein and increased RhoA signaling, including activation of ROCK and p38 and expression of the tumor suppressive secreted factor NAG-1 [61]. Surprisingly, the authors demonstrated that the biological effects of the RhoC knockdown are largely caused by the increased RhoA signaling. Knockdown of RhoA, conversely, increased RhoC protein, but had no effect on anchorage independent growth and NAG-1 dependent gene expression. These data indicate quite specific functions for RhoA and RhoC at least in PC-3 cells. Neither expression nor activity of Rac1 or Cdc42 were altered when RhoA or RhoC were knocked down, which is in partial contrast to the study discussed earlier that also used PC-3 cells [76]. Probably, the relative amounts of Rho GTPases, GDIs, and of other Rho GTPase binding molecules within a cell determine how Rho GTPases cross-talk, which has to be considered when overexpressing or knocking downRho GTPases [82].
10. Signaling Downstream of Actin Polymerization Actin polymerization releases the transcriptional co-activator MAL, which promotes SRF dependent gene expression [83]. Although many Rho GTPases regulate actin polymerization, it appears that there is a cell type specific preference for certain Rho GTPases as major regulators of MAL/SRF signaling. A recent example for this preference concerns the extravasation of cancer cells. Extravasation of tumor cells from the blood vessels can be modeled in vitro by assessing binding of cancer cells to an endothelial monolayer, opening of endothelial cell–cell contacts, and intercalation of cancer cells into the endothelial cell layer. Using a siRNA screen it was shown now that several Rho GTPases including RhoA, Rac1, and Cdc42 are important for adhesion of PC3 prostate cancer cells to HUVEC endothelial cells [84]. Of these, knockdown of Cdc42 impaired most efficiently cancer cell intercalation. Also in vivo, Cd42 was required for extravasation of PC-3 cells and consequently for
Li et al.
metastasis. Depletion of Cdc42 protein decreased SRF/MAL dependent expression of b1 integrin expression, probably via decreasing F-actin polymerization. This decrease in b1 integrin was the reason for the impaired intercalation of the Cdc42 knockdown cells. Cdc42 could therefore be an interesting drug target to reduce metastasis.
11. Rho GTPases and Tumor Stem Cells Evidence is accumulating that often a small population of cancer stem cells is required and sufficient for tumor formation [85,86]. Furthermore, cancer stem cells seem to be important for tumor metastasis [87]. In genetic mouse models, Cdc42 and Rac1 have been shown to be important for the maintenance of stem cells in skin and brain [88–90]. It seems therefore possible that these Rho GTPases play a functional role in cancer stem cells. Yet even other Rho GTPases might be important. For example, in human breast cancer, the breast cancer stem cell marker ALDH1 is tightly correlated with RhoC expression [91]. In vitro analysis of breast cancer cell lines with knockdown and overexpression of RhoC further supported an association of RhoC expression with ALDH1 and metastatic properties. Interestingly, in this case no change in the level of the RhoA protein was observed in response to the decreased RhoC expression.
12. Rac1 Function in Tumor Stem Cells In the glioma cell lines U87 and U373, Rac1 activation was increased in sphere cultures compared to 2D monolayers [92]. As sphere growth is considered to depend on glioma stem cells, this finding suggested a crucial function for Rac1 in maintenance of stemness. Indeed, knockdown of Rac1 reduced sphere formation and expression of the stem cell markers CD133, nestin, and musashi-1. Also invasiveness was decreased. Growth in monolayer culture, conversely, was not affected by knockdown of Rac1. Knockdown of Rac1 in human non small cell lung cancer cell lines decreased proliferation and invasion [93]. Stem cell like cells identified by weakly staining with Hoechst 33342 (side population) showed higher levels of Rac1 activation and increased metastatic potential. Knockdown of Rac1 decreased metastasis formation of both side population and non-side population cells. These data indicate that cancer stem cells might express higher levels of Rac1 than the other cells of the tumor and suggest that Rac1 might be important for the maintenance and function of cancer stem cells. However, functional experiments with cancer stem cells and control cell populations directly isolated from tumors are essential to confirm this notion.
13. Rho GTPases and Tumor Stroma Tumor growth and progression are not only dependent on the properties of the tumor cells but also on the tumor stroma, which consists of extracellular matrix, endothelial cells, fibroblasts, and immune cells. Cancer cells can manipulate the
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stroma and induce angiogenesis or the formation of cancer associated fibroblasts (CAFs), thus creating a tumor-supporting environment [94]. Novel stroma-directed cancer therapies aim to prevent the formation of new blood vessels or CAFs or to stop the production of tumor promoting growth factors by CAFs and immune cells. Rho GTPases seem to be important for the development as well as the function of the tumor stroma.
tumor load at later stages. Elegant tumor transplantation experiments revealed that the impaired tumor growth in RhoB-null mice was stroma dependent, as loss of RhoB delayed the formation of tumor vasculature. This was most probably caused by decreased VEGF-A expression by RhoBnull tumor cells and by the endothelial cell specific role of RhoB in the initial phase of angiogenesis [99].
14. Rho GTPase Function in Cancer Associated Fibroblasts
17. Stroma-Dependent Rho GTPase Activation
The origin and development of CAFs is not well understood and probably complex, but similarities of CAFs with myofibroblasts in wounds suggest that TGFb and cell contraction are important for their establishment [94]. Rho/ROCK dependent regulation of cell contraction could therefore be important for their generation. Moreover, Rho/ROCK-dependent contraction of CAF cells was found in a organotypic 3D cell culture model to be crucial for the generation of tracks within the extracellular matrix [95]. Cancer cells follow the fibroblasts along these tracks in a contraction independent manner, suggesting that during cancer cell invasion CAFs lead the way. Since Rac1 function in fibroblasts is required for the induction of myofibroblasts by bleomycin in vivo, Rac1 might also be essential for the formation of CAFs [96].
Stroma cells can be important for activation of Rho GTPases in cancer cells as, for example, during perineural invasion, when tumor cells invade surrounding nerve bundles causing pain and late onset of metastases. Semaphorin 4D secreted by neurons binds to plexin-B1 receptors on tumor cells, which will activate RhoA and ROCK and promote cell migration [100]. Plexin-B1 is a R-RAS specific GAP, but binds in addition also the Rho GEFs PDZ Rho GEF and leukemia-associated Rho GEF, which stimulate RhoA. Indeed, several neurotropic tumors show increased expression of semaphorin 4D, supporting in vivo importance of this mechanism. Also in leukemia stroma cells can be crucial for Rho GTPase signaling in malignant cells. Close contact of chronic lymphocytic leukemia (CLL) cells with stromal cells is considered to support their survival and proliferation in vitro and in vivo. Murine CLL cells lacking the constitutively active Rho GTPase RhoH, which is expressed only in hematopoietic cells, showed decreased homing to the bone marrow, and reduced cell–cell interaction with stromal cells [101]. This correlated with aberrant regulation of RhoA and Rac1 activation in RhoH-null CLL cells. Treatment of human CLL cells with the therapeutic drug lenalidomide decreased RhoH expression and resulted in a phenotype comparable to murine RhoH-null CLL cells, suggesting that RhoH might be an interesting drug target in CLL therapy.
15. Rho GTPases and Microvesicles Cancer cells can influence their microenvironment by secretion of soluble mediators and extracellular matrix molecules. In addition, they can form microvesicles, which contain proteins, mRNAs, and miRNAs. These microvesicles can fuse with stroma cells and thus transfer tumor specific molecules. Microvesicles promote the formation of a stroma, which supports tumor growth, invasion, and angiogenesis. In the breast cancer cell line MDA-MB-231 RhoA activity was found to be crucial for microvesicle formation, while Rac1, Cdc42, and interestingly also RhoC did not play a role [97]. Unexpectedly, microvesicle formation did not require MLC dependent cell contraction, but inhibition of the F-actin severing cofilin activity, which is controlled through RhoA/ROCK/LIMK.
16. Rho GTPase Function in Tumor Angiogenesis Recently RhoB expression was reported to be increased in endothelial cells of the tumor stroma compared to adjacent normal tissue [98]. Knockout experiments support a role for RhoB in tumor angiogenesis. RhoB-deficient cancer cells isolated from mammary tumors in PymT RhoB2/2 mice showed anchorage independent growth and increased proliferation in vitro corresponding to increased tumor incidence and activation of Akt. However, although more tumors formed initially in RhoB-null mice, they showed only poor growth resulting in a decreased
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18. Stroma Activation by Rho GTPase Signaling in Cancer Cells Rho GTPase signaling in cancer cells might also contribute to the formation of the tumor stroma. Recently, a Rho GTPase dependent cross-talk between epithelial and immune cells was shown in keratinocytes, where loss of Rac1 reduced F-actin polymerization leading to increased expression of the IFNc signal transducer Stat1 and the IFNc target gene CXCL10, thus promoting an inflammatory response [102]. Such pathways could also be involved in the interaction of cancer cells with immune cells, affecting cancer immune surveillance and cancer promoting effects of innate immune cells.
19. Outlook Chemical inhibitors have been found that block specific GEFs [103], or interfere with the interaction of subfamilies of Rho
Rho GTPases and Cancer
GTPases with GEFs [104] or Rho GTPase effectors, thus enabling different possibilities to interfere with Rho GTPase signaling in cancer. As tamoxifen-induced deletion of Cdc42 inhibited tumor growth in immunocompromised mice [105], blocking of Rho GTPases could indeed be useful in cancer therapy. Yet a major stumbling block on the way to Rho GTPase directed tumor therapy is the complexity of Rho GTPase signaling and the high cell type specificity. As cancer is heterogenous, the chance of therapy resistant cells within a malignant tumor might be high. In addition, most biological effects of Rho GTPases have only been described in tumor cells in vitro. Only animal models and patient studies will confirm the importance of these pathways in vivo. Murine cancer models closely modeling the human disease in combination with tissue specific and inducible mutations of GEFs, Rho GTPases, and Rho GTPase effectors will be crucial tools in answering these questions. Another important topic is the role of Rho GTPases in the formation and function of the tumor stroma, which is important for tumor growth and malignant progression. Murine cancer models with stroma specific mutations could address these issues. Conceivably, these in vivo studies will modify our perception of Rho GTPase function in cancer.
Acknowledgement This work was supported by the Novo Nordisk Foundation, the Lundbeck Foundation and the Danish Research Council for Health.
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235
Hui Li Karine Peyrollier € lcan Kilic Gu Cord Brakebusch*
University of Copenhagen, BRIC, BMI, 2200 Copenhagen, Denmark
Abstract Rho GTPases are a family of small GTPases, which play an important role in the regulation of the actin cytoskeleton. Not surprisingly, Rho GTPases are crucial for cell migration and therefore highly important for cancer cell invasion and the formation of metastases. In addition, Rho GTPases are involved in growth and survival of tumor cells, in the interaction of tumor cells with their environment, and they are vital for the
cancer supporting functions of the tumor stroma. Recent research has significantly improved our understanding of the regulation of Rho GTPase activity, the specificity of Rho GTPases, and their function in tumor stem cells and tumor stroma. This review summarizes these novel findings and tries C 2013 to define challenging questions for future research. V BioFactors, 40(2):226–235, 2014
Keywords: cancer; Rho GTPases
1. Rho GTPases Rho GTPases are a subfamily of the small G proteins with 20 members [1]. In their active GTP-bound form, Rho GTPases can interact with a large and diverse number of more than 70 effector molecules including scaffold molecules, actin binding proteins, and kinases that mediate the biological effects, which range from actin cytoskeletal re-organization to differentiation and regulation of stemness [2]. Activation of Rho GTPases requires GTP exchange factors (GEFs), which downstream of many cell surface receptors replace GDP by GTP. More than 70 GEFs have been identified and for many of them it was shown that they can activate more than Rho GTPase [3]. Different cell surface receptor families can activate GEFs including receptor tyrosine kinases and integrins. Inactivation of classical Rho GTPases occurs by hydrolysis of GTP to GDP, catalyzed by the Rho GTPase itself with the help of GTPase activating proteins (GAPs). This group of Rho GTPase regulators is also large with more than 50 members [4]. Nonclassical Rho GTPases, however, have lost the GTPase function and are constantly in the active conformation [5].
All Rho GTPases are regulated at the level of gene expression, protein turnover, protein modification, and interaction with inhibitory proteins [6]. Guanosine nucleotide dissociation inhibitors (GDIs), for example, bind to several Rho GTPases and prevent the dissociation of GDP thus keeping the Rho GTPases in the inactive state. Rho GTPases can therefore integrate information from many different signaling pathways. Furthermore, Rho GTPases crosstalk with each other at the level of Rho GTPase activation, Rho GTPase stability, and regulation of downstream signaling pathways, resulting in a complex network of interactions and coordinated regulation of Rho GTPases in different biological processes [7]. Regulation of Rho GTPases is therefore rather complex and cell type specific. Also downstream signaling of Rho GTPases is complex, as activation of a specific Rho GTPase can induce many different Rho GTPase effector pathways. However, it is not clear whether all these pathways are equally important in a given cell or whether dominant regulation by other signaling mechanisms or preformed complexes of Rho GTPases with specific regulators and effectors reduce the complexity of Rho GTPase signaling. Recent data discussed below seem to support such ideas.
C 2013 International Union of Biochemistry and Molecular Biology V
Volume 40, Number 2, March/April 2014, Pages 226–235 *Address for correspondence: Cord Brakebusch, Ph. D., BRIC, Ole Maalïes Vej 5, 2200 Copenhagen, Denmark. Tel.: 145 353 25619; Fax: 145 353 25669; E-mail: [email protected]. Received 16 August 2013; accepted 14 November 2013 DOI 10.1002/biof.1155 Published online 21 December 2013 in Wiley Online Library (wileyonlinelibrary.com)
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2. Rho GTPase Function in Cancer Studies with tumor cell lines showed that Rho GTPases regulate cell polarity and promote proliferation, survival, production of angiogenic factors, and migration of cancer cells [8]. Such pro-tumorigenic function of many Rho GTPases is supported by the increased expression of Rho GTPases in many tumors and by a limited number of studies with genetically
BioFactors
modified mice (Table 1). However, reduced expression of RhoB in lung and head and neck squamous cell carcinoma [9,10] or spontaneous development of hepatocellular cancers in mice lacking Cdc42 in hepatocytes [11] indicates that the Rho GTPase signaling network is complex and that the relative in vivo importance of specific molecular mechanisms of Rho GTPase action described in vitro is mostly unknown and probably highly cell type specific. Rho GTPases are important regulators of cancer cell proliferation, survival, invasion, and metastasis. More recently, crucial functions of Rho GTPases in the regulation of tumor stroma, including endothelial cells, immune cells, and cancer associated fibroblasts, as well as in the formation of microvesicles have been reported (Fig. 1). The aim of this review is to describe how research of the last years has improved our understanding of Rho GTPase regulation and function in cancer and to define open questions for future studies.
TABLE 1
Rho GTPase expression in tumors.
Rho GTPase
Expression in tumors
RhoA
"
[12–21]
RhoB
"#
[9,10,12,14]
RhoC
"
[12,14–16,21–33]
Rnd1
"
[24]
"#
[34–37]
Rac1
"
[12,17,18,24,38–42]
3. Regulation of Rho GTPase Activity by Oncogenes
Rac2
"
[13,25,42]
Rac3
"
[43]
Aberrant regulation of Rho GTPase activity and corresponding effector pathways is quite likely a hallmark of human tumors. First, the increased expression of, for example, RhoA, RhoC, Rac1, and Cdc42 in different types of cancer suggests that increased activity of these Rho GTPases promotes tumor progression, although the increased Rho GTPase expression may not necessarily correlate with an increase in activity, which is under complex regulation as described above. Second, oncogenic activation of tyrosine kinases such as ERBB2 or MET should result in increased activation of Rho GTPases in cancer even in the absence of any changes in expression of Rho GTPase genes, as tyrosine kinases are major activators of Rho GTPases [52]. Finally, several cancer promoting effects of miRNAs have been associated with altered expression of Rho GTPases [6]. However, more in vivo data are required to prove a direct relationship. Interestingly, Rho GTPase activation has hardly been checked in human tumors, probably since the most robust methods available, pull-down of activated Rho GTPases and ELISA, require fresh tumor material. Antibodies specifically recognizing the activated forms of RhoA, Rac1 and Cdc42 have been generated, but these antibodies have up to now not been used for larger screening studies of human material. A recent addition to the Rho GTPase activating oncogenes is the transcription factor human pituitary tumor-transforming gene 1 (hPTTG1), which is overexpressed in many metastatic tumors [53]. One of the target genes of hPTTG1 is the GEF-H1, which activates RhoA and increases invasion of MDA-MB-231 breast cancer cells in vitro and in vivo. As inhibitors of RhoA and ROCK prevented hPTTG1 induced cytoskeletal changes in vitro, it was suggested that RhoA/ROCK signaling is crucial for the pro-invasive effect of hPTTG1. ROCK is highly important for the regulation of cell contraction, suggesting a function in
RhoG
"
[24]
Cdc42
"
[12,17]
RhoH
#
[44–47]
RhoBTB1
#
[48]
RhoBTB2
#
[49–51]
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References
Rnd2 Rnd3 Rif RhoD
TC10 TCL Chp Wrch1
This table lists the Rho GTPase gene family and reported changes in gene expression observed in human tumors.
cell migration and metastasis. Indeed, several cell culture and mouse studies support this hypothesis, but the role of ROCK in human cancer is less clear and ROCK inhibitors clinically used for treatment of different diseases are not applied in cancer therapy or trials [54,55].
4. Regulators of Rho GTPase Activity The oncoprotein gankyrin is upregulated by Ras induced transformation and increased in lung cancer with Ras mutations. In vitro data suggest that gankyrin promotes tumor cell survival by decreasing RhoA activation [56]. Gankyrin promoted the binding of RhoA to Rho GDIa, which reduced the amount of active GTP-bound RhoA and decreased ROCK activity. As ROCK is required for PTEN activity, reduced RhoA/
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FIG 1
Overview of tumor relevant functions of Rho GTPases. Simplified scheme showing major regulation and effector pathways of Rho GTPases in cancer with a focus on Rho, Rac, and Cdc42. Important effector pathways are indicated beside the corresponding arrows. Rho GTPases regulate invasion and metastasis particularly by controlling cytoskeletal organization. Rho GTPasedependent gene expression controls stemness, angiogenesis, and immune response, while proliferation and survival are affected by cytoskeleton and gene expression. Rho-dependent cell contraction is important for microvesicle and CAF formation (RTKs: receptor tyrosine kinases; ROS: reactive oxygen species; CAF: cancer associated fibroblasts).
ROCK activity resulted in prolonged Akt activation. This study therefore implies that reduction of RhoA and ROCK activity promotes tumor progression. Reduced ROCK activity in cancer has been suggested already earlier, since it was found that Ras signaling reduces ROCK activity. In that case, however, ROCK activity was decreased independent of RhoA via sustained MekErk signaling [57,58]. Mouse models should be applied to test the relative importance of these pathways in vivo. GAPs and GDIs are known to inhibit Rho GTPase activation by increasing GTPase activity or maintaining inactive Rho GTPases in the cytosol, respectively. Yet two recent articles indicate they could also contribute to increased Rho GTPase activity, at least in cancer. The first study analyzes p190B, which is a GAP for RhoA, Rac1, and Cdc42. Overexpression of p190B in mammary epithelium of MMTV-neu induced tumors, however, resulted in increased activation of Rac1, while amounts of GTP-bound Cdc42 and RhoA were not changed [59]. This correlated with increased tumor multiplicity and metastasis formation. Neither tumor cell proliferation nor apoptosis were altered, although activation of Pak1/2, AKT, and ERK were even decreased, which normally correlates with
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decreased proliferation and survival. The second paper demonstrates an unexpected role for GDIa in cancer cells. Knockout of GDIa decreases protein amounts of RhoA, Rac1, and Cdc42 in vivo [60]. However, in the prostate carcinoma cell line PC-3, GDIa was shown to increase the half life time of the associated RhoA and apparently also RhoA signaling, as anchorage independent growth was decreased similar to overexpression of RhoA [61]. No change was observed for protein levels of Rac1 and Cdc42. This complex behavior of GDIs is also reflected in cancer patients, where GDI expression was found to be increased or decreased depending on the type of malignancy [62]. Some Rho GTPases such as Rnd1, 2, 3 have lost the GTPase function and are constitutively active. They do not bind to GDIs, but recently it was shown that they can be inhibited by binding to 14-3-3, which sequestrates the Rnd’s in the cytosol. This interaction is regulated by ROCK mediated phosphorylation of the C-terminus of Rnd [63]. Interestingly, increased 14-3-3 expression correlates with poor survival of cancer patients [64]. As Rnd’s are inhibiting Rho/ROCK signaling, increased 14-3-3 levels could cause increased Rho/ROCK
Rho GTPases and Cancer
activity, which might promote cancer progression by Rho/ ROCK dependent pathways.
5. Rho GTPase Mutations in Cancer Although Rho GTPases are frequently overexpressed in malignant tumors, suggesting although not proving that increased Rho GTPase activation might promote tumor progression, no constitutively active mutants of Rho GTPases have been found in cancer until recently. Two recent papers, however, described an activated Rac1 mutant with a P29S exchange in sun-exposed melanoma [65,66]. In fact, this mutation was the most frequent one after mutations in BRAF or NRAS. Activating mutations were also found for Rac2 and Cdc42. In head and neck squamous cell carcinoma, and esophageal and pancreatic cancers, Rac1 P29S mutations were found as a rare event. As Rac1 was shown to regulate proliferation and migration of melanoblasts in vivo [67], the presence of a Rac1 P29S mutant is conceivably highly relevant for cancer progression. The recently shown activation of p110b PI3K by Rac1 and Cdc42 might also contribute to the oncogenic potential of the Rac1 P29S mutation [68].
6. Channeled Activation of Rho GTPase Signaling In vitro studies showed that Rho GTPases can affect a large number of biological processes via multiple mechanisms, yet display at the same time also a high extent of cell type specificity. A simple explanation for this observation could be the differential expression of Rho GTPase effectors. Yet recent data suggest that also channeled activation of specific effectors, and crosstalk to other Rho GTPases and other signaling pathways might contribute to the specificity of Rho GTPases. Expression of constitutively active Rho GTPase mutants in cells often leads to the parallel activation of different downstream pathways initiated by the interaction of the Rho GTPases with corresponding effector proteins. However, physiological activation of Rho GTPases might be much more restricted with respect to downstream signaling (Fig. 2). This was beautifully illustrated in a recent study on integrin signaling, where av containing integrins activate RhoA through GEF-H1 resulting in activation of mDia, but not of ROCK and myosin II [69]. Activation of a5b1 integrin, conversely, leads to activation of the RhoA/ ROCK pathway with little mDia mediated stress fiber formation. One explanation for this specificity could be the formation of complexes containing GEFs, Rho GTPases, and effector proteins, which channel the activated Rho GTPases into restricted effector pathways. Alternatively, Rho GTPases or their effectors proteins are post-translationally modified by Rho GTPase independent pathways to favor certain Rho GTPase–effector interactions. In any case, these data suggest that of the many theoretically possible Rho GTPase–effector interactions in a cancer cell maybe only very few are actually used, depending on the path of Rho GTPase activation.
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7. Cell Type Specificity of the Atypical Rho GTPase Rnd3 The non-classical constitutively active Rho GTPase Rnd3, enhances inactivation of Rho by p190RhoGAP and directly inhibits the Rho effector ROCK1 [70]. Depending on the type of cancer, Rnd3 expression is increased or decreased. In liver and gastric cancer, specificRnd3 is downregulated [34,64,65]. Earlier it was shown that Rnd3 inhibits cell cycle progression and transformation in NIH 3T3 cell independent of RhoA and ROCK [71]. More recently, knockdown of Rnd3 in two liver cancer cell lines increased invasiveness in vitro and in vivo and promoted plasma membrane blebbing, characteristic for ameboid migrating cells [72]. This correlated with increased activation of ROCK and the fact that the ROCK inhibitor Y27632 decreased cell migration. Pulmonary metastasis, however, was not significantly changed. Another report also found that decrease of Rnd3 in HCC cells promotes invasiveness, but correlated it to decreased E-cadherin expression [73]. Despite the different mechanistic explanations, both studies indicate that loss of Rnd3 increases invasiveness of HCC and suggest that it might have a similar function in other tumors where expression of Rnd3 is decreased. However, Rnd3 is not always downregulated in cancer. In pancreatic cancer and NSLC, for example, it is overexpressed, indicating that depending on the cellular context Rnd3 might function as an oncogene or as a tumor suppressor [35,36]. In fact, it was suggested that even differences in the genetic background might explain contrasting results of Rnd3 function in HCC [72].
8. Specific function of Rho GTPase subfamily members As many effectors are shared between different Rho GTPases, Rho GTPases might have overlapping functions. Nevertheless, specific biological effects can be mediated. The Rho subfamily of Rho GTPases has three members, RhoA, RhoB, and RhoC, which bind to a highly overlapping set of effectors, although maybe with slight different affinities resulting in a certain degree of specificity [74]. Different intracellular location of RhoA, B and C further contributes to specific functions. RhoB is found mainly at endosomal membranes, whereas the active forms of RhoA and RhoC are present at the cell membrane. This correlated with the reported tumor suppressor role of RhoB and the mostly tumor promoting effects of RhoA and C. Probably the different intracellular localization is contributing to the specific actions of RhoB compared to RhoA and C. With respect to human cancer, RhoC seems to be more important than RhoA, as it is overexpressed in a wide range of malignant tumors correlating with malignant progression, while RhoA is less frequently overexpressed. RhoC-null mice displayed normal development and develop mammary tumors induced by polyoma middle T antigen
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FIG 2
“Channeled” versus “effector determined” Rho GTPase signaling. A. In the “channeled” model GEFs and effectors are coupled, either by complex formation, localization, or preferred interaction of post-translationally modified Rho GTPases with certain GEFs and effectors. Furthermore, specific GEFs might be coupled to specific receptors. In such a model, the biological effect of Rho GTPase activation is dependent on the pathway of Rho GTPase activation. B. In the “effector determined” model, different pathways lead to activation of a certain Rho GTPase, which then interacts with all corresponding effectors present in the cell. The biological effect of Rho GTPase activation is therefore dependent on the type and relative amounts of Rho GTPase effector molecules present.
similar to wild-type mice. However, growth and formation of metastases were significantly reduced [75]. Knockdown experiments in PC-3 cells suggested that RhoC promotes invasion by specific binding to Fmnl3, an actin polymerization inducing formin [76]. RhoA cannot bind to Fmnl3, but instead interacts with ROCK2 resulting in inhibition of Rac1 and decreased
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invasion. This could explain a RhoC specific contribution to invasion and would predict a more invasive phenotype of RhoA-deficient tumor cells. An earlier study using breast carcinoma cells suggested a pro-invasive function of RhoC and an anti-invasive role of RhoA due to inhibition of Rac1 [77]. Other data, however, indicate pro-invasive functions for both RhoA
Rho GTPases and Cancer
and RhoC in different cancer cell lines [78–80]. Interestingly, deletion of the RhoA gene in mouse resulted in reduced activation of Rac1 and Cdc42 in vitro, while both Rho GTPases showed unchanged activation in RhoA-null epidermis in vivo [81]. These data highlight the cell type specificity of Rho GTPases and their high dependency on the environmental conditions. Clearly, data obtained with tumor cells growing on rigid cell culture dishes in 2D in special growth media do not always model Rho GTPase signaling in vivo.
9. Cross-talk Between Rho GTPases It has been observed that knockdown and knockout models of one Rho subfamily member will lead to increased protein amounts of the remaining Rho subfamily members. GDIa seems to play an important role in this crosstalk. In PC-3 cells, knockdown of RhoC prevents the formation of RhoC–GDIa complexes. Instead, RhoA binds to GDIa, leading to increased levels of RhoA protein and increased RhoA signaling, including activation of ROCK and p38 and expression of the tumor suppressive secreted factor NAG-1 [61]. Surprisingly, the authors demonstrated that the biological effects of the RhoC knockdown are largely caused by the increased RhoA signaling. Knockdown of RhoA, conversely, increased RhoC protein, but had no effect on anchorage independent growth and NAG-1 dependent gene expression. These data indicate quite specific functions for RhoA and RhoC at least in PC-3 cells. Neither expression nor activity of Rac1 or Cdc42 were altered when RhoA or RhoC were knocked down, which is in partial contrast to the study discussed earlier that also used PC-3 cells [76]. Probably, the relative amounts of Rho GTPases, GDIs, and of other Rho GTPase binding molecules within a cell determine how Rho GTPases cross-talk, which has to be considered when overexpressing or knocking downRho GTPases [82].
10. Signaling Downstream of Actin Polymerization Actin polymerization releases the transcriptional co-activator MAL, which promotes SRF dependent gene expression [83]. Although many Rho GTPases regulate actin polymerization, it appears that there is a cell type specific preference for certain Rho GTPases as major regulators of MAL/SRF signaling. A recent example for this preference concerns the extravasation of cancer cells. Extravasation of tumor cells from the blood vessels can be modeled in vitro by assessing binding of cancer cells to an endothelial monolayer, opening of endothelial cell–cell contacts, and intercalation of cancer cells into the endothelial cell layer. Using a siRNA screen it was shown now that several Rho GTPases including RhoA, Rac1, and Cdc42 are important for adhesion of PC3 prostate cancer cells to HUVEC endothelial cells [84]. Of these, knockdown of Cdc42 impaired most efficiently cancer cell intercalation. Also in vivo, Cd42 was required for extravasation of PC-3 cells and consequently for
Li et al.
metastasis. Depletion of Cdc42 protein decreased SRF/MAL dependent expression of b1 integrin expression, probably via decreasing F-actin polymerization. This decrease in b1 integrin was the reason for the impaired intercalation of the Cdc42 knockdown cells. Cdc42 could therefore be an interesting drug target to reduce metastasis.
11. Rho GTPases and Tumor Stem Cells Evidence is accumulating that often a small population of cancer stem cells is required and sufficient for tumor formation [85,86]. Furthermore, cancer stem cells seem to be important for tumor metastasis [87]. In genetic mouse models, Cdc42 and Rac1 have been shown to be important for the maintenance of stem cells in skin and brain [88–90]. It seems therefore possible that these Rho GTPases play a functional role in cancer stem cells. Yet even other Rho GTPases might be important. For example, in human breast cancer, the breast cancer stem cell marker ALDH1 is tightly correlated with RhoC expression [91]. In vitro analysis of breast cancer cell lines with knockdown and overexpression of RhoC further supported an association of RhoC expression with ALDH1 and metastatic properties. Interestingly, in this case no change in the level of the RhoA protein was observed in response to the decreased RhoC expression.
12. Rac1 Function in Tumor Stem Cells In the glioma cell lines U87 and U373, Rac1 activation was increased in sphere cultures compared to 2D monolayers [92]. As sphere growth is considered to depend on glioma stem cells, this finding suggested a crucial function for Rac1 in maintenance of stemness. Indeed, knockdown of Rac1 reduced sphere formation and expression of the stem cell markers CD133, nestin, and musashi-1. Also invasiveness was decreased. Growth in monolayer culture, conversely, was not affected by knockdown of Rac1. Knockdown of Rac1 in human non small cell lung cancer cell lines decreased proliferation and invasion [93]. Stem cell like cells identified by weakly staining with Hoechst 33342 (side population) showed higher levels of Rac1 activation and increased metastatic potential. Knockdown of Rac1 decreased metastasis formation of both side population and non-side population cells. These data indicate that cancer stem cells might express higher levels of Rac1 than the other cells of the tumor and suggest that Rac1 might be important for the maintenance and function of cancer stem cells. However, functional experiments with cancer stem cells and control cell populations directly isolated from tumors are essential to confirm this notion.
13. Rho GTPases and Tumor Stroma Tumor growth and progression are not only dependent on the properties of the tumor cells but also on the tumor stroma, which consists of extracellular matrix, endothelial cells, fibroblasts, and immune cells. Cancer cells can manipulate the
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BioFactors
stroma and induce angiogenesis or the formation of cancer associated fibroblasts (CAFs), thus creating a tumor-supporting environment [94]. Novel stroma-directed cancer therapies aim to prevent the formation of new blood vessels or CAFs or to stop the production of tumor promoting growth factors by CAFs and immune cells. Rho GTPases seem to be important for the development as well as the function of the tumor stroma.
tumor load at later stages. Elegant tumor transplantation experiments revealed that the impaired tumor growth in RhoB-null mice was stroma dependent, as loss of RhoB delayed the formation of tumor vasculature. This was most probably caused by decreased VEGF-A expression by RhoBnull tumor cells and by the endothelial cell specific role of RhoB in the initial phase of angiogenesis [99].
14. Rho GTPase Function in Cancer Associated Fibroblasts
17. Stroma-Dependent Rho GTPase Activation
The origin and development of CAFs is not well understood and probably complex, but similarities of CAFs with myofibroblasts in wounds suggest that TGFb and cell contraction are important for their establishment [94]. Rho/ROCK dependent regulation of cell contraction could therefore be important for their generation. Moreover, Rho/ROCK-dependent contraction of CAF cells was found in a organotypic 3D cell culture model to be crucial for the generation of tracks within the extracellular matrix [95]. Cancer cells follow the fibroblasts along these tracks in a contraction independent manner, suggesting that during cancer cell invasion CAFs lead the way. Since Rac1 function in fibroblasts is required for the induction of myofibroblasts by bleomycin in vivo, Rac1 might also be essential for the formation of CAFs [96].
Stroma cells can be important for activation of Rho GTPases in cancer cells as, for example, during perineural invasion, when tumor cells invade surrounding nerve bundles causing pain and late onset of metastases. Semaphorin 4D secreted by neurons binds to plexin-B1 receptors on tumor cells, which will activate RhoA and ROCK and promote cell migration [100]. Plexin-B1 is a R-RAS specific GAP, but binds in addition also the Rho GEFs PDZ Rho GEF and leukemia-associated Rho GEF, which stimulate RhoA. Indeed, several neurotropic tumors show increased expression of semaphorin 4D, supporting in vivo importance of this mechanism. Also in leukemia stroma cells can be crucial for Rho GTPase signaling in malignant cells. Close contact of chronic lymphocytic leukemia (CLL) cells with stromal cells is considered to support their survival and proliferation in vitro and in vivo. Murine CLL cells lacking the constitutively active Rho GTPase RhoH, which is expressed only in hematopoietic cells, showed decreased homing to the bone marrow, and reduced cell–cell interaction with stromal cells [101]. This correlated with aberrant regulation of RhoA and Rac1 activation in RhoH-null CLL cells. Treatment of human CLL cells with the therapeutic drug lenalidomide decreased RhoH expression and resulted in a phenotype comparable to murine RhoH-null CLL cells, suggesting that RhoH might be an interesting drug target in CLL therapy.
15. Rho GTPases and Microvesicles Cancer cells can influence their microenvironment by secretion of soluble mediators and extracellular matrix molecules. In addition, they can form microvesicles, which contain proteins, mRNAs, and miRNAs. These microvesicles can fuse with stroma cells and thus transfer tumor specific molecules. Microvesicles promote the formation of a stroma, which supports tumor growth, invasion, and angiogenesis. In the breast cancer cell line MDA-MB-231 RhoA activity was found to be crucial for microvesicle formation, while Rac1, Cdc42, and interestingly also RhoC did not play a role [97]. Unexpectedly, microvesicle formation did not require MLC dependent cell contraction, but inhibition of the F-actin severing cofilin activity, which is controlled through RhoA/ROCK/LIMK.
16. Rho GTPase Function in Tumor Angiogenesis Recently RhoB expression was reported to be increased in endothelial cells of the tumor stroma compared to adjacent normal tissue [98]. Knockout experiments support a role for RhoB in tumor angiogenesis. RhoB-deficient cancer cells isolated from mammary tumors in PymT RhoB2/2 mice showed anchorage independent growth and increased proliferation in vitro corresponding to increased tumor incidence and activation of Akt. However, although more tumors formed initially in RhoB-null mice, they showed only poor growth resulting in a decreased
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18. Stroma Activation by Rho GTPase Signaling in Cancer Cells Rho GTPase signaling in cancer cells might also contribute to the formation of the tumor stroma. Recently, a Rho GTPase dependent cross-talk between epithelial and immune cells was shown in keratinocytes, where loss of Rac1 reduced F-actin polymerization leading to increased expression of the IFNc signal transducer Stat1 and the IFNc target gene CXCL10, thus promoting an inflammatory response [102]. Such pathways could also be involved in the interaction of cancer cells with immune cells, affecting cancer immune surveillance and cancer promoting effects of innate immune cells.
19. Outlook Chemical inhibitors have been found that block specific GEFs [103], or interfere with the interaction of subfamilies of Rho
Rho GTPases and Cancer
GTPases with GEFs [104] or Rho GTPase effectors, thus enabling different possibilities to interfere with Rho GTPase signaling in cancer. As tamoxifen-induced deletion of Cdc42 inhibited tumor growth in immunocompromised mice [105], blocking of Rho GTPases could indeed be useful in cancer therapy. Yet a major stumbling block on the way to Rho GTPase directed tumor therapy is the complexity of Rho GTPase signaling and the high cell type specificity. As cancer is heterogenous, the chance of therapy resistant cells within a malignant tumor might be high. In addition, most biological effects of Rho GTPases have only been described in tumor cells in vitro. Only animal models and patient studies will confirm the importance of these pathways in vivo. Murine cancer models closely modeling the human disease in combination with tissue specific and inducible mutations of GEFs, Rho GTPases, and Rho GTPase effectors will be crucial tools in answering these questions. Another important topic is the role of Rho GTPases in the formation and function of the tumor stroma, which is important for tumor growth and malignant progression. Murine cancer models with stroma specific mutations could address these issues. Conceivably, these in vivo studies will modify our perception of Rho GTPase function in cancer.
Acknowledgement This work was supported by the Novo Nordisk Foundation, the Lundbeck Foundation and the Danish Research Council for Health.
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