**4. 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.

#### **4.1 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

et al. 1997; Miki, Sasaki et al. 1998). Therefore, N-WASP may promote cellular motility through proper filopodia formation. MRCKs α and β 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 MRCKα and Cdc42 synergizes to promote filopodia formation, while a MRCKα 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 of βcatenin (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 α-catenin for binding to β-catenin, thus preventing Ecadherin/α-catenin/β-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).

**4. Expression of Rho GTPases in breast tumors** 

**4.1 Rho GTPases in breast tumors** 

Rac2 and Rac3) and the Cdc42 family, in order to avoid repetitions.

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,

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.

#### **4.2 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

Rho GTPases and Breast Cancer 569

of Rho, Rac and Cdc42 (Fig. 3). Most of them are involved in Rho GTPases mediated

Fig. 3. A model of the cellular migratory process. See text for detailed explanation of motility

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

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

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

**5.2.1 Cell polarization and lamellipodium extension at the leading edge** 

cytoskeletal rearrangements (Tang, Olufemi et al. 2008).

phases.

**5.2 Rho GTPases in cell migration** 

secretion, vesicle trafficking, and transcription.

appear to be mediated by Rho family GTPases.

Akt pathways (Mazieres, Antonia et al. 2004). Moreover, Rac1 enhances estrogen receptor α (ERα) transcriptional activity, resulting in increased proliferation in breast cancer cells (Rosenblatt, Garcia et al. ; Folkman 1972).
