**3.1.1 GEFs**

GEFs for Rho GTPases belong to a rapidly growing family of proteins that share common minimal functional units, including a Db1-homolog (DH) domain followed by a pleckstrin homology (PH) domain (Cerione and Zheng 1996). The DH domain is the catalytic site required for GDP-GTP exchange, whereas the PH domain contributes to protein-protein, protein-cytoskeleton, and protein-lipid interactions that help regulate the intracellular localization of GEFs as well as their catalytic activity. Db1 oncogene product is the prototype for the DH domain, and was originally discovered because of its ability to induce focus

Like all members of the Ras superfamily, the activity of the Rho GTPases is tightly controlled by the ratio of their GTP/GDP-bound forms in the cell (Fig. 1)(Scheffzek and

The cycle of activation/inactivation of Rho family GTPases is under the regulation of three distinct families of proteins: GEFs, guanine nucleotide exchange factors catalyze nucleotide exchange when activated by upstream signals; GAPs, GTPase-activating proteins promote the GTP hydrolisis; GDIs, guanine nucleotide dissociation inhibitors block both nucleotide hydrolisis and exchange and participate in Rho GTPase movement between cytosol and

Rho-specific guanine nucleotide exchange factors (RhoGEFs) activate Rho proteins by facilitating the exchange of GDP for GTP. Rho GTPase activating proteins (RhoGAPs) stimulate the intrinsic rate of hydrolysis of Rho proteins, thus converting them into their inactive state. While Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) compete with RhoGEFs for binding to GDP-bound Rho proteins, and sequester Rho in the

GEFs for Rho GTPases belong to a rapidly growing family of proteins that share common minimal functional units, including a Db1-homolog (DH) domain followed by a pleckstrin homology (PH) domain (Cerione and Zheng 1996). The DH domain is the catalytic site required for GDP-GTP exchange, whereas the PH domain contributes to protein-protein, protein-cytoskeleton, and protein-lipid interactions that help regulate the intracellular localization of GEFs as well as their catalytic activity. Db1 oncogene product is the prototype for the DH domain, and was originally discovered because of its ability to induce focus

**3. Regulators and effectors in Rho GTPases signaling** 

**3.1 Regulators of the Rho GTPases** 

Fig. 1. Regulation of Rho family proteins.

Ahmadian 2005).

membranes.

**3.1.1 GEFs** 

inactive state (Olofsson 1999).

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).

#### **3.1.2 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).

#### **3.1.3 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

Rho GTPases and Breast Cancer 563

Rho was originally studied for its role in regulate the formation of stress fibers and focal adhesion (FA) complexes (Nobes and Hall 1995) 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) (Zhang, Nie et al. ; Narumiya and Morii 1993; Nobes and Hall 1995). In Swiss 3T3 fibroblasts, the Rho GTPases have been placed in a hierarchical order where Cdc42 activates Rac, and Rac activates Rho (Nobes and Hall 1995); 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

**3.2.1 Rho signaling** 

and Prendergast 2006).

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).
