**5.2.4 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).

(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

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

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, α-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 α-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

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).

MTOC through its downstream effector, PAK1 (Li, Hannigan et al. 2003).

and Huttenlocher 1998; Fujiwara, Mammoto et al. 2000).

**5.2.4 Cell body contraction by actomyosin complexes** 

**5.2.2 Protrusion formation** 

**5.2.3 Cell-substrate adhesions** 

to changing ECM composition.

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).

### **5.2.5 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 β1 and β3 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).

#### **5.3 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

Rho GTPases and Breast Cancer 573

proteins are believed to be capable of altering the expression and activity of pro-angiogenic

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-1α (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, PDGFβ and Ang-2 (Gleadle and Ratcliffe 1998). In contrast, Rac1 is shown to be involved in hypoxia-induced PI3K activation of HIF-1α through a different mechanism (Hirota and Semenza 2001). Hypoxiainduced expression of Rac1 also contributes to the upregulation of HIF-1α 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.

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

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

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

and anti-angiogenic factors during angiogenesis.

2000).

mammary neoplasis.

**5.5.2 IL-6 and IL-8 expression** 

signaling (Faruqi, Gomez et al. 2001).

through distinct signaling pathways.

**5.5.1 Regulation of VEGF and hypoxia inducible factor-1 (HIF1)** 

with serum response elements (SREs) at their promoter enhancer regions, for example the Fos promoter (Treisman 1990).

### **5.4 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 G1 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 (Qiu, Chen et al. 1995), this is not the case with Rho (Qiu, Chen et al. 1995), 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).

### **5.5 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-β (PDGFβ), 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

with serum response elements (SREs) at their promoter enhancer regions, for example the

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 G1 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

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 (Qiu, Chen et al. 1995), this is not the case with Rho (Qiu, Chen et al. 1995), 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

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-β (PDGFβ), 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

Fos promoter (Treisman 1990).

**5.4 Rho GTPases and cell growth control** 

activate cyclin D1 expression (Page, Li et al. 1999).

RasV12-transformed cells (Qiu, Abo et al. 1997).

**5.5 Rho GTPases and angiogenesis** 

proteins are believed to be capable of altering the expression and activity of pro-angiogenic and anti-angiogenic factors during angiogenesis.
