Rho GTPases and Breast Cancer

Cancer is the leading cause of death in most countries and its consequences result in huge economic, social and psychological burden. Breast cancer is the most frequently diagnosed cancer type and the leading cause of cancer death among females. In this book, we discussed characteristics of breast cancer cell, role of microenvironment, stem cells and metastasis for this deadly cancer. We hope will contribute to the development of novel diagnostic as well as therapeutic approaches.


Introduction
The Rho GTPases is a subfamily of molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state within the Ras superfamily. In the past, members of the Rho subfamily were mainly thought to be involved in the regulation of cytoskeletal organization in response to extracellular growth factors. However, a number of studies over the past few years have revealed that the Rho GTPases play crucial roles in a wide spectrum of cellular functions related to cell adhesion to the extracellular matrix, cell morphology, cell cycle progression, malignant transformation, invasion and metastasis. Alterations of the expression levels to Rho GTPases have been detected in many types of human tumors and, in some cases, up-regulation and/or overexpression of Rho protein correlates with poor prognosis. This article reviews the evidence of aberrant Rho signaling and the cellular effects elicited by Rho GTPases signaling in human breast tumors.
formation and tumorigesis when expressed in NIH-3T3 cells (Eva and Aaronson 1985). It has 29% sequence identity with the Saccharomyces cerevisiae cell division protein Cdc24, which is found upstream of the yeast small GTP-binding protein Cdc42 in the bud assembly pathway (Ron, Zannini et al. 1991). This was the first clue that DB1 functions as a GEF. Biochemical study has confirmed that Db1 is able to release GDP from the human homolog of Cdc42 in vitro. Further study suggested that the DH domain is essential and sufficient for the catalytic activity and that this domain was also necessary to induce oncogenicity (Zheng, Zangrilli et al. 1996). After the discovery of Dbl, a number of mammalian proteins containing DH and PH domain have been studied (Cerione and Zheng 1996). Many of these have been identified as oncogenes in transfection assays. Tiam, however, was first identified as an invasioninducing gene using proviral tagging and in vitro selection for invasiveness (Habets, Scholtes et al. 1994). Two other members of the DH/PH-containing protein family, Fgd1 and Vav, have been shown to be essential for normal embryonic development (Pasteris, Cadle et al. 1994;Tarakhovsky, Turner et al. 1995). Moreover, some members of the DH protein family (such as Dbl) have been shown to exhibit exchange activity in vitro for a broad range of Rho-like GTPases, whereas others appear to be more specific. For example, Lbc and oncoproteins Lfc and Lsc, are specific for Rho, whereas Fgd1 is specific for Cdc42 (Glaven, Whitehead et al. 1996). Although Vav was originally identified as an activator of Ras (Gulbins, Coggeshall et al. 1993), it has been demonstrated more recently to function as a GEF for members of the Rho family (Crespo, Schuebel et al. 1997;Han, Das et al. 1997).

GAPs
The first GAP protein specific for the Rho family GTPases was purified from cell extracts using recombinant Rho. This protein, designated p50Rho-GAP, was shown to have GAP activity toward Rho, Cdc42 and Rac in vitro (Hall 1990;Lancaster, Taylor-Harris et al. 1994). Since then, a growing number of proteins that present GAP activity for Rho GTPases have been identified in mammalian cells, all of which share a related GAP domain that spans 140 amino acids without significant resemblance to Ras GAP. In addition to accelerating the hydrolysis of GTP, Rho GAPs also mediate other downstream functions of Rho proteins in mammalian systems. For example, it has been reported that the p190GAP plays a role in cytoskeletal rearrangement (Chang, Gill et al. 1995).

GDIs
The ubiquitously expressed protein Rho GDI was the first GDI identified for the members of the Rho family. It was isolated as a cytosolic protein that preferentially associated with the GDP-bound form of RhoA and RhoB and thereby inhibited the dissociation of GDP (Fukumoto, Kaibuchi et al. 1990;Ueda, Kikuchi et al. 1990). Rho GDI was found to be active on Cdc42 and Rac as well (Abo, Pick et al. 1991;Leonard, Hart et al. 1992). Further studies demonstrated that Rho GDI also associated weakly with the GTP-bound form of Rho, Rac, and Cdc42 (Hart, Maru et al. 1992;Chuang, Xu et al. 1993), leading to an inhibition of the intrinsic and GAP-stimulated GTPase activity of the Rho GTPases. Therefore, Rho GDI appears to be a molecule capable of blocking both the GDP/GTP exchange step and the GTP hydrolytic step. It was also reported that the Rho GDIs play a crucial role in the translocation of the Rho GTPases between membranes and the cytoplasm. In resting cells, the Rho proteins are found in the cytosol as a complex with Rho GDIs, which inhibit their www.intechopen.com Breast Cancer -Focusing Tumor Microenvironment, Stem Cells and Metastasis 562 GTP/GDP exchange ratio, but are released from the GDI and translocated to the membranes during the course of cell activation (Takai, Sasaki et al. 1995).

Effectors of the Rho GTPases
The Rho GTPases have been implicated in a wide varity of cellular processes, including cytoskeletal organization, cell adhesion to the substratum, cell polarity, and transcriptional activation. Several lines of evidence indicate that Rho GTPases link plasma membrane receptors to the assembly and organization of the actin cytoskeleton. Rho GTPases control individual aspects of the actin cytoskeleton through distinct effector proteins. In fact, over 60 targets of the three common Rho GTPases (Rho, Rac, Cdc42) have been found (Fig. 2). Transmembrane receptors activate Rho GTPases through GEFs such as Tiam-1 or adaptor proteins. Activated Rho GTPases bind to and activate protein kinases, including these of the MRCK, PAK and ROCK families. The effector proteins then interact with several proteins with distinct effects on the actin cytoskeleton and cellular morphology. See text for details. www.intechopen.com

Rho signaling
Rho was originally studied for its role in regulate the formation of stress fibers and focal adhesion (FA) complexes  which precursors actomyosin assembly and contractile potential, both of which are required for the cellular movement. Rho is also involved in cell-cell adhesion. In particular, inactivation of RhoA by C3 transferase disrupts the organization of actin filaments at cell-cell contact, leading to the inhibition of the proper formation of both adherens junctions (AJs) and tight junctions (TJs) (Braga, Machesky et al. 1997;Takaishi, Sasaki et al. 1997). For example, in normal mammary epithelial cells, MCF10 cells, E-cadherin cytoskeletal links in AJs was disrupted by C3 transferase. In addition, inhibition of Rho blocks the formation of new AJs in MCF10 cells (Zhong, Kinch et al. 1997). It has been suggested that the function of Rho can be either promoted or antagonized by Rac and Cdc42, depending on different variables, such as cellular context, stimulus, and extracellular matrix (ECM) Narumiya and Morii 1993;. In Swiss 3T3 fibroblasts, the Rho GTPases have been placed in a hierarchical order where Cdc42 activates Rac, and Rac activates Rho ; however, in N1E-115 neuroblastoma and Madine-Darby canine kidney (MDCK) cells, constitutively activated Rac down-regulates Rho (Leeuwen, Kain et al. 1997;Michiels and Collard 1999). Rho is widely studied for its involvement in the acquisition of migratory, invasive, and metastatic phenotypes. Expression of a dominant negative form of RhoA led to the attenuation of membrane ruffling, lamellipodia formation and migration (O'Connor, Nguyen et al. 2000). In addition, RhoA localization to lamellipodia was blocked by inhibiting phosphodiesterase activity while enhanced by inhibiting cAMP-dependent protein kinase activity (O'Connor, Nguyen et al. 2000). Furthermore, activation of Rho either by LPA treatment or by stimulating the actomyosin system has been associated with the migratory ability of tumor cells. For example, in an experimental metastasis model, NIH3T3 fibroblasts expressing a constitutively active form of RhoA were injected into the tail vein of nude mice and formed increased number metastasis nodules in the lung (del Peso, Hernandez-Alcoceba et al. 1997). Moreover, in the absence of serum, activated RhoA is capable of promoting invasion of cultured rat MM1 hepatoma cells through a mesothelial cell monolayer (Yoshioka, Matsumura et al. 1998). Although these are not oncogenes by themselves, RhoA and RhoC are frequently found to be overexpressed in clinical cancers (Sahai and Marshall 2002), and RhoC has been repeatedly associated with metastasis. For example, the expression of RhoA, RhoB and RhoC in 33 pancreatic ductal adnocarcinoma cases were examined in a study (Suwa, Ohshio et al. 1998), it was found that the expression level of RhoC was higher in tumors than in non-malignant tissues, higher in metastatic lesions than in primary tumors, and correlated with perineural invasion and lymph node metastasis as well as poorer prognosis. Although early studies showed that RhoB has a positive role in cell growth, more recent studies suggested that RhoB is down-regulated in human tumors, and its expression inversely correlates with tumor aggressiveness. For example, RhoB protein is found expressed in normal lung tissue and is lost progressively throughout lung cancer progression (Mazieres, Antonia et al. 2004). In line with this, higher expression of RhoB is associated with favorable prognosis in bladder cancer (Kamai, Tsujii et al. 2003). It has been suggested that RhoB can act as a tumor suppressor, since it is activated in response to several stress stimuli, such as DNA damage and hypoxia, inhibits tumor growth, cell migration, and invasion, and has proapoptotic functions in cells (Huang and Prendergast 2006).

Effectors of Rho
There are two major effectors that are downstream of Rho: Rho associated coiled-coil forming kinase (ROCK/Rho kinase/ROK) (Leung, Manser et al. 1995;Ishizaki, Maekawa et al. 1996) and mammalian homolog of Drosophila diaphanous (mDia) (Watanabe, Madaule et al. 1997;Wasserman 1998). While mDia is a formin molecule that can catalyze actin nucleation, polymerization,and produce long, straight actin filaments (Goode and Eck 2007), ROCK is a serine/threonine kinase that phosphorylates a number of substrates (Riento and Ridley 2003). The actions of ROCK and mDia on actin and myosin are believed to work together to induce actomyosin bundles in cells. Expression of an active form of mDia induces stress fibers in cultured cells, and treatment of these cells with a specific ROCK inhibitor, Y-27632 (Narumiya, Ishizaki et al. 2000), causes dissolution of the bundles, leaving the cells with diffusely distributed actin filaments (Watanabe, Kato et al. 1999). It has also been reported that ROCK and mDia are required in contractile ring formations (Kosako, Yoshida et al. 2000;Watanabe, Ando et al. 2008). At least six substrates of ROCK are known to play roles in actin cytoskeletal reorganization, including myosin light chain (MLC), myosin-binding subunit of MLC phosphatase, LIMkinase, adducin, ezrin/radixin/moesin (ERM) family of proteins, and Na + /H + exchange protein (NHE1). Among the six substrates, MLC-phosphatase, MLC, and LIM-kinase, are the three best studied ROCK effectors and have been found to play important roles in driving ROCK's physiological function on the actin cytoskeleton. ROCK inactivates myosinbinding subunit of MLC-phosphatase by phosphorylation (Kimura, Ito et al. 1996;Uehata, Ishizaki et al. 1997). ROCK is also able to phosphorylate myosin light chain directly (Maekawa, Ishizaki et al. 1999). These two actions of ROCK increase the myosin light chain phosphorylation, stimulate cross-linking of actin by myosin and enhance actomyosin contractility. ROCK also phosphorylates and activates LIM-kinase, which in turn phosphorylates and inactivates actin-depolymerizing and severing factor, cofilin (Amano, Ito et al. 1996). The later action of ROCK results in stabilization of existing actin filaments and increase in their content. The ROCK effectors adducin and the ERM family of proteins regulate actin cytoskeleton in a more direct way. ROCK has been shown to phosphorylate adducin (Kimura, Fukata et al. 1998;Fukata, Oshiro et al. 1999), which, together with spectrin, is an important component of the cortical actin network underlying the plasma membrane (Gardner and Bennett 1987). ROCK-phosphorylated adducin interacts with filamentous-actin (F-actin), and its localization suggests a role in regulating cellular migration. In HGF/SF-stimulated MDCK cells, phosphoadducin localizes to membrane ruffles, and ROCK-phosphorylated adducin localizes to the leading edge of migrating NRK49F fibroblasts in wound healing assays (Fukata, Oshiro et al. 1999); while the introduction of nonphosphorylatable adducin into MDCK and NRK49F cells inhibited membrane ruffling and migration, as did a dominant negative ROCK mutant (Fukata, Oshiro et al. 1999). ROCK can also phosphorylate the ERM proteins that are important for linking actin filaments to the plasma membrane (Matsui, Maeda et al. 1998). Interestingly, it has been demonstrated that the TSC1 tumor suppressor hamartin regulates cell adhesion to cell substrates through the ERM family of actin-binding proteins and RhoA (Lamb, Roy et al. 2000). Finally, NHE1 is well known as a ubiquitous Na + /H + exchange protein that enables stress fiber formation (Tominaga, Ishizaki et al. 1998).

Rac and Cdc42 signaling
In classical Swiss 3T3 fibroblast model, activation of Cdc42 leads to filopodia formation, Rac results in lamellipodia formation and membrane ruffling, and Rho results in stress fibers formation . The cytoskeletal rearrangements caused by Rho GTPases activation play a key role in cell motility. In addition to their effects on the actin cytoskeleton and motility, Rac and Cdc42 also play a role in cell-cell adhesion in epithelial cells. Expression of a constitutively active form of Rac in MDCK cells or keratinocytes leads to an increase in E-cadherin complex members and F-actin at cell-cell contacts, while a dominant negative mutant was found to disrupt cell-cell adhesions (Braga, Machesky et al. 1997;Takaishi, Sasaki et al. 1997;Jou and Nelson 1998). A number of studies have suggested that Cdc42 plays an important role in establishing the initial polarization of epithelial cells, which is required for the proper formation of cell-cell adhesions. For example, transfection of a dominant negative form of Cdc42 in MDCK cells results in the selective depolarization of basolateral membrane proteins due to inhibition of membrane transport (Kroschewski, Hall et al. 1999). Expression of a constitutively active form of Cdc42 in MDCK cells increased AJs and blocked cellular migration induced by HGF/SF (Kodama, Takaishi et al. 1999). Given the importance of Rac and Cdc42 in the regulation of cell cytoskeletal, adhesion and motility, it has been widely considered that they play important roles in cellular processes related to invasion and metastasis. The first evidence of Rac's role in invasion was obtained when Rac-specific GEF T-lymphoma invasion and metastasis (Tiam-1) was identified in a retroviral insertional mutagenesis screen. Virus-infected T-lymphoma cells were repeatedly selected for in vitro invasion through a layer of fibroblasts and the proviral insertions in i n v a s i v e c l o n e s w e r e u s e d t o i d e n t i f y t h e Tiam-1 gene (Habets, Scholtes et al. 1994). Subsequently, Rac, and later Cdc42, were shown to also confer an invasive potential to these T-lymphoma cells (Michiels, Habets et al. 1995;Stam, Michiels et al. 1998). More evidence for Rac and Cdc42's involvement in invasion and metastasis has been provided since then. Expression of the laminin-receptor 6 4 integrin in the melanoma cell line MDA-MB-435 promotes invasiveness in a Rac and PI3-kinase-dependent manner (Shaw, Rabinovitz et al. 1997). In addition, constitutively active forms of Rac and Cdc42 in breast carcinoma cell line T47D promote invasion through a collagen matrix. However, this invasion can be blocked by PI3-Kinase inhibitors, indicating that PI3-kinase acts downstream of Rac and Cdc42 (Keely, Westwick et al. 1997).

Effectors of Rac and Cdc42
A number of Rac and Cdc42 effectors have been identified. Some of these have been found to specifically mediate cell motility, whereas others play a more prominent role in mediating cell adhesion. It is well established that WASP and MRCKs are Cdc42 specific effectors that regulate actin organization and filopodia formation which promotes a more motile phenotype (Aspenstrom, Lindberg et al. 1996;Miki, Miura et al. 1996). In addition, members of the p21-activated kinase family (PAK), downstream of Rac and Cdc42, play important roles in cytoskeletal-mediated changes that affect motility (Manser, Leung et al. 1994). The scalffold proteins IQGAP and Par-6, both of which can be activated by Cdc42 and rac, promote cell polarization and contribute to cell-cell adhesion. The scaffold protein N-WASP binds to Arp2/3 complexes that are crucial for the assembly of within filopodia (Kolluri, Tolias et al. 1996). It has been shown that both N-WASP and Arp2/3 complexes are required for Cdc42 to trigger actin filament assembly (Welch, DePace et al. 1997;Miki, Sasaki et al. 1998). Therefore, N-WASP may promote cellular motility through proper filopodia formation. MRCKs and are Cdc42 specific effectors that can phosphorylate MLC via a ROCK-like kinase domain (Leung, Chen et al. 1998). It is well accepted that phosphorylation of MLC is required for actomyosin complex assembly and contraction. Overexpression of MRCK and Cdc42 synergizes to promote filopodia formation, while a MRCK kinase-deficient mutant inhibits the formation of Cdc42-induced filopodia (Leung, Chen et al. 1998). Therefore, MRCDs are believed to play important roles in cytoskeletal organization and contraction, and contribute to migration. PAK, a protein kinase downstream of Rac and Cdc42, plays a crucial role in actin dynamics and adhesion (Manser, Leung et al. 1994). PAK has been demonstrated to phosphorylate and inactivate MLCK, subsequently causing a decrease in MLC phosphorylation (Sanders, Matsumura et al. 1999). Thus, inactivation of MLCK leads to stress fiber and focal adhesion disassembly. Moreover, PAK controls the actin cytoskeletion through the phosphorylation and subsequent activation of LIM-kinase. Phosphor-LIM-kinsae can further phosphorylate and inactivate the actin-depolymerizing protein cofilin, thus inhibiting actin depolymerization when Rac is activated and causing extreme membrane ruffling (Arber, Barbayannis et al. 1998;Yang, Higuchi et al. 1998). The IQGAP1 and IQGAP2 scaffolding effectors of Cdc42 and Rac regulate cell-cell adhesion through actin polymerization and sequestration ofcatenin (Kuroda, Fukata et al. 1996;Erickson, Cerione et al. 1997). In vitro, IQGAP oligomerizes and cross-links F-catin it has also been found to complex with Cdc42 and Factin in vivo (Fukata, Kuroda et al. 1997). In addition, one study has shown that the IQGAP protein also competes with -catenin for binding to -catenin, thus preventing Ecadherin/ -catenin/ -catenin complex from attaching to the actin cytoskeleton, and thereby disrupting cell-cell contacts (Erickson, Cerione et al. 1997). Another scaffolding protein, Par-6, was identified using activated Cdc42 and TC10 mutants as baits in yeast two-hybrid screens (Joberty, Petersen et al. 2000;Qiu, Abo et al. 2000). It is known that Par-6 binds to a second scaffolding protein, Par-3, and both Par-6 and Par-3 bind independently to atypical protein kinase C (aPKC) isioforms (Lin, Edwards et al. 2000). In addition, endogenous Par-3 localizes to TJs in MDCK cells, overexpression of Par-6 or the N-terminal portion of Par-3 (the Par-6-interaction responsible region) disrupts TJ formation (Joberty, Petersen et al. 2000).

Expression of Rho GTPases in breast tumors
Aberrant Rho signalling resulting from alterations in Rho GTPase protein level, changes in activation status, and abnormal quantity of effector proteins are found in a large variety of human tumors. of GTPases: the Rho family (RhoA, RhoB and RhoC), the Rac family (Rac1, Rac2 and Rac3) and the Cdc42 family, in order to avoid repetitions.

Rho GTPases in breast tumors
Overexpression of RhoC has been found in inflammatory breast cancer (IBC), an aggressive form of breast cancer that is highly infiltrative and metastatic with poor prognosis for the patients, using in situ hybridization (van Golen, Davies et al. 1999). Compared to normal untransformed parental cells, RhoC-transformed cells produce and secrete high levels of proangiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-6 (IL-6), and interleukin-8 (IL-8). when compared to normal untransformed parental cells (van Golen, Wu et al. 2000). In addition, microarray analysis has shown that MCF10A breast cells stably transfected with wild type RhoC or a constitutively active mutant of RhoC overexpress genes associated with invasion and metastasis (Wu, Wu et al. 2004). Other RhoGTPases are also involved in breast tumors. RhoA is found overexpressed in breast tumor tissues but not in the normal tissue (Fritz, Brachetti et al. 2002). The expression of dominant negative RhoA in rat mammary adenocarcinoma cells affects tumor cell growth in vivo and reduces intravasation into the peripheral blood, resulting in decrease in lung colonization ability (Fritz, Just et al. 1999). Other studies have indirectly shown an important role of RhoA in breast carcinogenesis. For example, highly metastatic MDA-MB-231 cells that were treated with HMG-CoA reductase inhibitor, namely cerivastain, showed reduced proliferation and invasion through Matrigel, in a RhoA-but not Ras-dependent manner (Denoyelle, Vasse et al. 2001). However, poorly metastatic breast cancer cells such as MCF-7 are less sensitive to cerivastain treatment, indicating that RhoA might be more significantly overexpressed in late stages of breast cancer as with other tumors. Rho proteins are also important players in breast tumor progression and metastasis exerted by the CD44 hyaluronan receptor (Bourguignon 2001). CD44 is expressed in human breast tumors and promotes cell growth and metastasis in tumor cells. Studies have found that RhoA and CD44 directly interact with each other in vivo in highly metastatic human breast cancer cell lines. Accordingly, inhibition of Rho signaling results in the abrogation of the metastatic phenotype elicited by CD44 (Bourguignon, Zhu et al. 1999). RhoA has also been found to be involved in insulin signaling via Shc in human breast cancer (Finlayson, Chappell et al. 2003). Overexpression of insulin receptors correlates with development, progression and outcome of breast cancer, and insulin signaling involves hyperphosphorylation of Shc. Hence, Shc leads to the activation of geranyl transferases, which results in an increased amount of prenylated RhoA in the tumor tissue compared with normal mammary tissue (Finlayson, Chappell et al. 2003). Furthermore, RhoA has been reported to increase the metastatic potential of tumor cells via its ability to promote tumor angiogenesis through the downregulation of thrombospodin-1 (Tsp-1) (Watnick, Cheng et al. 2003). Rho pathway is part of the downstream signaling cascade that is activated by PI3K and leads to ROCK stimulation, Myc phosphorylation and Tsp-1 repression.

Rac GTPases in breast tumors
The involvement of Rac GTPases in breast cancer was first reported in rodents (Bouzahzah, Albanese et al. 2001). Expression of a dominant negative Rac1 mutant indicated that Rac1 affects tumor cell growth and metastasis in vivo. Deregulation of Rac3, closely related to Rac1, has also been detected in breast cancer (Mira, Benard et al. 2000). Rac3 maps to chromosome band 17q25.3, a region known to contain candidate tumor suppressor genes both in breast and ovarian cancers (Morris, Haataja et al. 2000). Highly proliferative breast cancer cells, T47D and MCF-7, but not normal breast cell lines, contain constitutively active Rac3 in a Ras-independent manner (Morris, Haataja et al. 2000). It has also been shown that expression of a dominant negative mutant Rac3 (N17) leads to inhibition of S-phase entry and cellular proliferation in breast tumor cells, which indicate that Rac3 may promote cell growth (Leung, Nagy et al. 2003). Further, the Rac-PAK signaling pathway is essential for receptor tyrosine kinase ErbB2-mediated transformation of human breast epithelial cancer cells (Mazieres, Antonia et al. 2004). Activation of Rac-PAK1 pathway by ErbB2 homodimers can induce growth factor-independent proliferation and promote disruptions to the three-dimensional (3D) mammary acinar-like structures, via activation of the Erk and Akt pathways (Mazieres, Antonia et al. 2004). Moreover, Rac1 enhances estrogen receptor (ER ) transcriptional activity, resulting in increased proliferation in breast cancer cells (Rosenblatt, Garcia et al. ;Folkman 1972).

Cdc42 family in breast tumors
Cdc42 is overexpressed in some breast cancers and there is accumulating evidence that activated Cdc42 contributes to the accumulation of ErbB1 in cells through the regulation of c-Cbl function (Abraham, Kuriakose et al. 2001;Marionnet, Lalou et al. 2003). The view that Cdc42 is involved in human breast carcinogenesis is supported by a rodents model of breast carcinoma where the expression of a dominant negative mutant of Cdc42 reduced the number of focal contacts, inhibited colony formation in soft agar and affected cell growth in vivo (Fritz, Just et al. 1999). The dominant negative Cdc42 also reduced intravasation of tumor cells into peripheral blood and ability to form lung metastasis. In addition, through the activation of Cdc42, transforming growth factor (TGF-) mediates the invasion of MDA-MB-231 cells into 3-D collagen matrices by initiating the formation of protrusions into collagen. (Kamai, Tsujii et al. 2003;Fisher, Sacharidou et al. 2009). Further, another study has shown that membrane-type-1 matrix metalloproteinase (MT1-MMP) and Cdc42 are fundamental components of a co-associated invasion-signaling complex that controls directed single-cell invasion of 3D collagen matrices (Fisher, Sacharidou et al. 2009).

Multiple functions mediated by Rho GTPases in breast cancer
Rho GTPases mediate housekeeping aspects of cell biology including cell growth, cell polarity, cell adhesion, membrane trafficking and motility. They function as signaling switches that regulate lipid metabolism, microtubules-and actin-based structures, epithelial cell-junctions, cell cycle and apoptosis regulatory proteins, and transcription factors.

Rho GTPases and cytoskeleton organization
Eukaryotic cellular morphology and attachment to the substratum in response to extracellular signals are largely dependent on rearrangements of the actin cytoskeleton. Cell motility, cytokinesis and phagocytosis all rely on coordinated regulation of the actin cytoskeleton (Small 1994;Zigmond 1996). Filamentous actin can be organized into several discrete structures: (a) filopodia, finger-like protrusions that contain a tight bundle of long actin filaments in the direction of the protrusion. These are found primarily in motile cells and neuronal growth cones. (b) lamellipodia, thin protrusive actin sheets that dominate the edges of cultured fibroblasts and many other motile cells. Membrane ruffles observed at the leading edge of the cell result from lamellipodia that lift up off the substrate and fold backward. (c) actin stress fibers, bundles of actin filaments that traverse the cell and are linked to the ECM through focal adhesion (Van Aelst and D'Souza-Schorey 1997). The actin polymerization is tightly regulated by Rho GTPases. Rho activation in fibroblasts is known to stimulate the assembly of contractile actin/myosin filaments, the formation of stress fibers, and the clustering of integrins involved in the formation of focal adhesion complexes. Activation of Rac facilitates actin polymerization at the cell periphery to generate protrusive actin-rich lamellipodia and membrane ruffling. And activation of Cdc42 results in actin polymerization to form peripheral actin microspikes and filopidia. As described previously, a number of proteins have been identified as targets www.intechopen.com of Rho, Rac and Cdc42 (Fig. 3). Most of them are involved in Rho GTPases mediated cytoskeletal rearrangements (Tang, Olufemi et al. 2008). Fig. 3. A model of the cellular migratory process. See text for detailed explanation of motility phases.

Rho GTPases in cell migration
Cell migration is a multistep process involving polarization, sequential cell protrusion and adhesion formation in the direction of migration, cells body contraction, and tail detachment (Pinner and Sahai 2008). During the migration process, cells move with extending protrusions at the front and a retracting tail at the rear, both regulated by members of the Rho GTPases family (Ridley, Schwartz et al. 2003). The idea that Rho family GTPases could regulate cell migration derives from observations that they mediate the formation of specific actin containing structures. In addition, Rho proteins regulate several other processes that are relevant to cell migration, including cell-substrate adhesion, cell-cell adhesion, protein secretion, vesicle trafficking, and transcription.

Cell polarization and lamellipodium extension at the leading edge
An asymmetrical organization of intracellular activities is required for a cell to move, that means the molecular processes at the leading and trailing edges of a moving cell must be different. Establishing and maintaining cell polarity in response to extracellular stimuli appear to be mediated by Rho family GTPases. Cdc42 is well accepted as a master regulator of cell polarity in eukaryotic organisms ranging from yeast to human.Cdc42 was first studied in a budding yeast model for its involvement in cell polarity. During the cell cycle, yeast cells adopt alternative states of growth to nonfocused isotropic growth. In the absence of Cdc42, Saccharomyces cerevisiae fail to establish focused apical growth and, cells expand isotropically (Pruyne and Bretscher 2000). Cdc42 regulates cell polarity by deciding the location of lamellipodia formation (Srinivasan, Wang et al. 2003). In addition, Cdc42 directs the localization of the microtubule-organizing center (MTOC) and Golgi apparatus to the front of the nucleus, oriented toward the direction of movement. MTOC orientation at the leading edge then facilitates the delivery of Golgi derived vesicles to the leading edge and microtubule growth into the lamellipodium (Rodriguez, Schaefer et al. 2003). It has been further studied that Cdc42 exerts its effect on MTOC through its downstream effector, PAK1 (Li, Hannigan et al. 2003).

Protrusion formation
Inherent polarity drives the formation of membrane protrusions, and the organization of filaments depends on the type of protrusion. Actin filaments form a branching dendritic network in lamellipodia, but form long parallel bundles in filopodia (Pollard, Blanchoin et al. 2000). The dendritic organization of lamelipodia that provides a tight brush-like structure, formed via the actin-nucleating activity of the actin-related proteins 2/3 (Arp2/3) protein complex (Urban, Jacob et al.). Rac stimulates new actin polymerization by acting on Arp2/3 complexes, which binds to pre-existing filaments (Campellone and Welch). Activation of Arp2/3 complexes by Rac is carried out through its target IRSp53. Upon activation, IRSp53 interacts with WAVE through its SH3 domain, it then binds to and activates Arp2/3 complexes (Chesarone and Goode 2009). It has also been reported that IRSp53 binds to Cdc42 through a separate domain (Miki, Yamaguchi et al. 2000). So, IRSp53 can serve as a direct link between Cdc42 and Rac, which explains how Cdc42 induces Rac involvement in lamellipodium formation. Furthermore, IRSp53 can bind to a Rho target, Dial, which might underlie the capability of Rho to facilitate lamellipodium extension (Cox and Huttenlocher 1998;Fujiwara, Mammoto et al. 2000).

Cell-substrate adhesions
Newly formed focal adhesion complexes are localized in the lamellipodia of most migrating cells. Once the lamellipodium attach to the ECM, integrins come into contact with ECM ligands and cluster in the cell membrane where they interact with FAK, -actin, and talin (Cox and Huttenlocher 1998). All these proteins can bind to adaptor proteins through Srchomologous domain 2 and 3 (SH2, SH3) as well as proline rich domains to more actin binding proteins (vinculin, paxillin and -actin) and regulatory molecules PI3K to focal complexes (Zamir and Geiger 2001). Rac is required for focal complex assembly, and Rac itself can be activated by cell-substrate ECM adhesion (Rottner, Hall et al. 1999). It is suggested that the adhesion assemblies in migrating cells begin with small-scale clustering and the speed of the cell migration is dependent on ECM composition, which determines the relative activated levels of Rho, Rac and Cdc42 (Price, Leng et al. 1998). Therefore, interactions between ECM and integrins at the leading edge of cells play an important role in maintaining the level of active Rac. This indicates the existence of a positive feedback loop that allows continuous crosstalk between integrins and Rac, and allows cells to respond to changing ECM composition.

Cell body contraction by actomyosin complexes
Cell body contraction is driven by actomyosin contractility and the force transmitted to sites of adhesion derives from myosin II. Myosin II, which is predominantly induced by Rho and its downstream effector ROCK, controls stress fiber assembly and contraction. Rho acts via ROCKs to affect MLC phosphorylation by inhibiting MLC phosphatase or the MLC phosphorylation. MLC phosphorylation is also regulated by MLCK, which is controlled by both intracellular calcium concentration and ERK MAPKs (Fukata, Amano et al. 2001).
ROCKs and MLCK have been suggested to act in concert to regulate different aspects of cell contractility, since ROCK appears to be required for MLC phosphorylation which are associated with actin filaments in the cell body, and MLCK is required at the cell periphery (Totsukawa, Yamakita et al. 2000).

Adhesion disassembly and tail detachment
Tail detachment occurs when cell-substrate linkages are preferentially disrupted at the rear of a migrating cell, while the leading edge remains attached to the ECM and continues to elongate (Palecek, Huttenlocher et al. 1998). Mechanisms underlying the focal complex disassembly and tail detachment depend on the type of cell and strength of adhesion to the extracellular matrix at the trailing edge (Wear, Schafer et al. 2000). In slow moving cells, tail detachment depend on the action of a calcium-dependent, non-lysosomal cysteine protease calpain that cleaves focal complex components like talin and cytoplasmic tail of 1 and 3 integrins along the trailing edge (Potter, Tirnauer et al. 1998). Strong tension forces exerted across the cells at the rear adhesions is required to break the physical link between integrin and the actin cytoskeleton. Rho and myosin II are involved in this event. Furthermore, Rho plays important roles in reducing adhesion and promoting tail detachment in fibroblasts, which have relatively large focal adhesion complexes (Cox and Huttenlocher 1998).

Rho GTPases and transcriptional activation
A number of studies have suggested that Rho family GTPases are involved in the regulation of nuclear signaling. Rac and Cdc42, but not Rho, have been demonstrated to regulate the activation of JNK and reactivate kinase p38RK in certain cell types (Seger and Krebs 1995). Expression of constitutively active forms of Rac and Cdc42 in HeLa, NIH-3T3, and Cos cells stimulates JNK and p38 activity (Coso, Chiariello et al. 1995). Furthermore, these same effects were observed with oncogenic GEFs for these Rho proteins (Minden, Lin et al. 1995). However in human kidney 293 T cells, Cdc42 and the Rho protein, but not Rac, induces the activation of JNK (Teramoto, Crespo et al. 1996). Upon activation, JNKs and p38 translocate to the nucleus where they phosphorylate transcription factors, including c-Jun, ATF2, and Elk (Derijard, Hibi et al. 1994;Gille, Strahl et al. 1995). Further, Rac has been shown to activate PEA3, a member of the Ets family, in a JNK-dependent manner (O'Hagan, Tozer et al. 1996). Activated p38 phosphorylates ATF2, Elk, Max, and the cAMP response element binding protein.
PAKs are the farthest known upstream kinases that connect Rho GTPases to JNK and p38 through GTP-dependent bindings to Rac and Cdc42 in vitro and are activated after binding to activated Rac and Cdc42. (Manser, Chong et al. 1995). In addition, certain constitutively active forms of PAK can activate JNK and p38 (Zhang, Han et al. 1995). Further, a mutant effector of Rac that cannot bind to PAK remains a potent JNK activator (Westwick, Lambert et al. 1997). These observations suggest that other kinases, in addition to PAK, participate in the signalling from Rho GTPases to JNK. Supporting this, MLK3 and MEKK4 are found to be regulated by Cdc42 and Rac, and selectively activate the JNK pathway (Gerwins, Blank et al. 1997). It has also been reported that Cdc42/Rac can bind to MLK3 both in vitro and in vivo and that the coexpression of activated Cdc42/Rac mutants elevates the enzymatic activity of MLK3 in Cos-7 cells (Teramoto, Coso et al. 1996;Gerwins, Blank et al. 1997). In addition, Rho, Rac and Cdc42 stimulate the activation of the serum responsive factor (SRF) (Hill, Wynne et al. 1995). SRF forms a complex with TCF/Elk proteins to stimulate transcription with serum response elements (SREs) at their promoter enhancer regions, for example the Fos promoter (Treisman 1990).

Rho GTPases and cell growth control
Several lines of evidence have suggested that Rho family members play important roles in several aspects of cell growth. The Rho proteins have been shown to increase expression of cyclin D1, a cell cycle regulator that controls the transition from G 1 phase to S phase, in Swiss 3T3 fibroblasts (Yamamoto, Marui et al. 1993;Olson, Ashworth et al. 1995) and in mammary epithelial cells (Liberto, Cobrinik et al. 2002). Overexpression of RhoE inhibits cell cycle progression by inhibiting translation of cyclin D1 mRNA (Villalonga, Guasch et al. 2004). In fibroblasts, RhoA is involved in ERK activation and subsequent cyclin D1 expression (Roovers and Assoian 2003). RhoA also downregulates cdk inhibitors p21 and p27 during the G1 phase of the cell cycle (Weber, Hu et al. 1997). Rac 1 is capable of regulating the cell cyle through the activation of a number of distinct intra-cellular pathways, including the NFκB pathway. In contrast to other Rho proteins, Rac1 can directly activate cyclin D1 expression (Page, Li et al. 1999). Furthermore, Rho, Rac, and Cdc42 have been demonstrated to possess transforming and oncogenic potential in some cell lines. For example, cells with constitutively active forms of Rac and Rho display enhanced anchorage independent growth ability, and initiate tumor formation when inoculated into nude mice (Khosravi-Far, Solski et al. 1995). The observation that Tiam, a Rac GEF, can transform NIH-3T3 cells suggests a role for Rac in transformation ( van Leeuwen, van der Kammen et al. 1995). While expression of constitutively activated Rac is sufficient to cause malignant transformation of rodent fibroblasts , this is not the case with Rho , suggesting that the growth-promoting effects of the Rho GTPases are specific to cell type. Evidence of Cdc42's role in cell growth has been provided in fibroblasts. The constitutively active mutant of Cdc42 stimulates anchorage independent growth and proliferation in nude mice (Qiu, Abo et al. 1997). Using a Cdc42 mutant, Cdc42(F28L), that can undergo GTP-GDP exchange in the absence of GEF, one study demonstrated that cells stably tranfected with Cdc42(F28L) exhibited not only anchorage-independent growth but also lower dependence on serum for growth (Lin, Bagrodia et al. 1997). A role for Cdc42 in Ras transformation has also been established in Rat 1 fibroblasts. Coexpression of a dominant negative form of Cdc42, Cdc42N17, with oncogenic Ras results in inhibition of RasV12-induced focus formation and anchorage-independent growth, and reversed the change in morphology in RasV12-transformed cells (Qiu, Abo et al. 1997).

Rho GTPases and angiogenesis
Beside their roles in multiple processes of cellular control, tumor growth, progression and metastasis, the Rho proteins have also been shown to be involved in angiogenesis, a process Where new blood vessels arise from existing mature vessels. This process is controlled by a number of pro-angiogenic and anti-angiogenic factors at different stages (Folkman 1972). The major pro-angiogenic factors are comprised of vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF), platelet derived growth factor-(PDGF ), angiopoietins 1 and 2 (Ang-1 and 2), tumor necrosis factor (TNF), interleukin 6 and 8 (IL-6 and 8), and epidermal growth factor (EGF). The main anti-angiogenic foctors include the thrombospondins (TSPs), angiostatin, and endostain (Merajver and Usmani 2005). The Rho proteins are believed to be capable of altering the expression and activity of pro-angiogenic and anti-angiogenic factors during angiogenesis.

Regulation of VEGF and hypoxia inducible factor-1 (HIF1)
It has been reported that hypoxia increases the expression and activity of Cdc42, Rac1 and RhoA in renal cell carcinoma cell lines and a human microvascular endothelial cell line (Turcotte, Desrosiers et al. 2003). This study demonstrated that reactive oxygen species (ROS) are responsible for the upregulation of Rho proteins and that RhoA is required for the accumulation of HIF-1 (Turcotte, Desrosiers et al. 2003), a transcription factor induced by hypoxia that plays important roles in the process of angiogenesis by inducing the transcription of crucial mediators, including VEGF, PDGF and Ang-2 (Gleadle and R a t c l i f f e 1 9 9 8 ) . I n c o n t r a s t , R a c 1 i s s h o w n t o b e i n v o l v e d i n h y p o x i a -i n d u c e d P I 3 K activation of HIF-1 through a different mechanism (Hirota and Semenza 2001). Hypoxiainduced expression of Rac1 also contributes to the upregulation of HIF-1 and, subsequently, VEGF in gastric and hepatocellular cancer cells (Xue, Bi et al. 2004). VEGF has been reported to increase RhoA activity and localization to the cell membrane, and the RhoA /ROCK pathway has been implicated in the VEGF-mediated angiogenesis (van Nieuw Amerongen, Koolwijk et al. 2003). In addition, RhoA activation also increases tyrosine phosphorylation of the primary VEGF receptor, VEGFR-2 (Gingras, Lamy et al. 2000). Overexpression of RhoC in human mammary epithelial cells (HME) and a highly aggressive breast cancer cell line, SUM-149, increases VEGF expression (van Golen, Wu et al. 2000). Similar finding were found in the MCF10A cells (Wu, Wu et al. 2004), further suggesting that RhoC plays a role in, further suggesting that RhoC plays a role in increasing VEGF in mammary neoplasis.

IL-6 and IL-8 expression
IL-6 is a multifunctional cytokine that is involved in many different biological process, including immunological and inflammatory processes, tumor growth and angiogenesis (Hirano, Akira et al. 1990;Mateo, Reichner et al. 1994). IL-8 is another important cytokine that acts as a pro-angiogenic factor. Both of these cytokines can be induced by hypoxia (Yan, Tritto et al. 1995;Mizukami, Jo et al. 2005) and have been shown to upregulate VEGF mRNA expression (Cohen, Nahari et al. 1996). Studies indicate that active Rho proteins upregulate the expression of NFκB components in NIH-3T3 cells (Perona, Montaner et al. 1997;Montaner, Perona et al. 1998). Consistent with Rho-mediated activation of NFκB, HKG-CoA reductase inhibitors had been reported to reduce IL-6 expression by inhibiting Rho proteins (Ito, Ikeda et al. 2002). Rac1 has been shown to mediate the activation of a potential oncogen, STAT3, through NFκB regulated IL-6 signaling (Faruqi, Gomez et al. 2001). IL-8 expression has also been found to be regulated by Rho proteins. In human endothelial cells, it has been shown that inhibition of RhoA, Rac1 and Cdc42 decreases NFκB activation and, therefore, decreases IL-8 mRNA and IL-8 protein expression (Hippenstiel, Soeth et al. 2000;Warny, Keates et al. 2000). In addition, RhoC has been shown to increase IL-6 and IL-8 expression in aggressive breast cancer cell lines (Xue, Bi et al. 2004). These evidences suggest that different Rho proteins modulate IL-6 and IL-8 through distinct signaling pathways.

FGF activation
FGF1 and FGF2 are the two earliest characterized members of the FGF family of growth factors. FGF is an angiogenic factor that is frequently overexpressed in breast and prostate cancers. Rac1 and Cdc42 have been reported to increase FGF1 expression by stimulating the FGF1 gene promoter region (Chotani, Touhalisky et al. 2000). One study demonstrated that Rac1 activity is required for FGF2-induced activation of Ras/MAPK signaling in human breast cell line MCF7 (Liu, Chevet et al. 1999). In addition, medium collected from RhoC stably transfected HME and SUM149 cells present higher level of FGF2, in comparison to those collected from control transfected HME cells (van Golen, Wu et al. 2000). However, how Rho proteins regulate FGF expression remains unclear.

Repression of Tsp-1
The anti-angiogenic molecule Tsp-1 is capable of inhibiting metalloproteinase-9 (MMP9) from releasing the VEGF sequestered in ECM. The oncoprotein Ras has been reported to increase VEGF expression and inhibit Tsp-1 expression. One study showed that the inhibitory function of Ras on Tsp-1 via PI3K pathway also involve RhoA and RhoC in human embryonic kidney cell lines, human mammary cell lines, and breast cancer cell lines (Watnick, Cheng et al. 2003). And the suppression of Tsp-1 always correlates with promotion of angiogenesis.

Conclusion
It is apparent that individual members of Rho GTPases play specific roles in different aspects in breast cancer development (Fig. 4). Aberrant expression and activity of Rho proteins contribute to the transformation from normal epithelial phenotype, increases in proliferation, the promotion of angiogenesis, elevated motility, and metastasis to distant organs. RhoA, RhoC and Rac1 are frequently overexpressed in metastatic breast cancers. Manipulating the Rho GTPases' regulatory proteins and their effectors can induce activation of Rho proteins, , leading to aberrant transcription factor activation, including that of NFκB, that contribute to invasive phenotypes. All this evidence suggests that Rho GTPases could be targets in cancer therapy. Therefore, better knowledge of the the regulation mechanisms of Rho GTPases in breast cancer may be critical for a more in-depth understanding of tumor biology, facilitating development of novel approaches for cancer treatment.