Small GTPases of the Rho family are intracellular signaling molecules, best known for their role in regulating the actin cytoskeleton, in vesicle trafficking, cell cycle regulation and transcriptional reprogramming (Cain & Ridley 2009).
Fig. 1.6: Regulation of small GTPases of the Rho family. Inactive Rho-GTPases are activated by the exchange of GDP for GTP mediated by GEFs. GAPs inactivate Rho-GTPases by catalyzing the hydrolysis of GTP to GDP. GDIs bind inactive Rho-GTPases in the cytosol and prevent the nucleotide exchange. Modified from Lawson & Burridge 2014
Rho GTPases exist in either an inactive GDP-bound form or in an active GTP-bound form (Fife et al. 2014). Three different classes of regulatory molecules modulate the activity of the Rho-family small GTPases: guanine nucleotide exchange factors (GEFs), GTPase-activating Proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs) (Lawson & Burridge 2014). While GEFs catalyze the exchange of bound GDP for GTP, GAPs stimulate the intrinsic GTPase activity, turning the Rho-family proteins into an inactive state. GDIs maintain
26 a cytosolic pool of inactive Rho-family small GTPases. If required, they can be transported to cell membranes, where the nucleotide exchange takes place (Leung et al. 1995; Zheng 2001; Rossman et al. 2005; Bos et al. 2007) (Fig. 1.6).
Among Rho GTPases, the best-studied members are RhoA, Rac1 and Cdc42 and their role during cell motility (Burridge & Wennerberg 2004). For a long time, RhoA was thought to be inhibitory for cell migration, since it promotes stress fiber formation and strong adherence mediated by focal adhesions (Lawson & Burridge 2014). But RhoA was also found to be active at the leading edge of migrating cells, where it is responsible for membrane ruffling and the formation of lamellipodia (O’Connor et al. 2000; Machacek et al. 2009; El-sibai et al.
2009). The switch between stress fiber formation and lamellipodia formation is not well understood but might be mediated by two different GEFs, whereby one potentially activates Rho at the leading edge and the other one at the rear of the cell (Sadok & Marshall 2014).
The two homologs RhoA and RhoC were described to have different roles in cell migration since they act through different targets (Vega et al. 2011). While RhoC inhibits the development of lamellipodia through the formin FMNL3, RhoA promotes tail retraction via its effectors mDia (mammalian homolog of Drosophila diaphanous) and ROCK (Rho-associated kinase) (Narumiya et al. 2009; Vega et al. 2011). Both are well described regarding their roles in stress fiber formation. The activation of mDia and ROCK constitutes the trigger for the assembly of actomyosin filaments (Hall 2012). Here, ROCK phosphorylates and inactivates the myosin phosphatase and activates myosin light chain, resulting in an enhancement of actomyosin contractility (Kimura et al. 1996). Subsequently, contraction leads to bundling of actin filaments and clustering of integrins into focal adhesions (Narumiya et al. 2009). Actin polymerization itself is then stimulated by mDia.
During cell motility, RhoA acts in concert with Rac1 and Cdc42 (El-sibai et al. 2009;
O’Connor et al. 2012). In contrast to RhoA, Rac1 and Cdc42 promote Arp2/3-based actin polymerization and branching in the lamellipodium by activating the WAVE or WASP protein complexes (Bid et al. 2013; Blanchoin et al. 2014). Effectors of Rac1 and Cdc42 are, among others, p21-activated kinase (PAK), WAVE/WASP, IQGAP1 (IQ motif containing GTPase activating protein 1) and IQGAP2 (IQ motif containing GTPase activating protein 2) (Kuroda et al. 1999). Rac1 activates the p21-activated kinases PAK1, PAK2 and PAK3, which themselves activate the actin-binding LIM kinases LIMK1 and LIMK2. These in turn phosphorylate the actin binding protein cofilin, thereby inactivating its activity of converting F-actin into G-F-actin and allowing F-actin growth (Ridley 2006; Bid et al. 2013). Rac1 and Cdc42 were also described to recruit mDia to RhoA, thus facilitating its activity during lamellae formation (Kurokawa & Matsuda 2005).
Introduction
27
Abb 1.7: Rho-family of small GTPases regulating actin remodeling. RhoA promotes actomyosin contractility via ROCK. ROCK itself phosphorylates LIMK, thereby leading to inhibition of cofilin. RhoA also affects mDIA, which in turn promotes actin polymerization. Rac1 antagonizes RhoA´s function. It activates PAK and WAVE. WAVE-dependent activation of Arp2/3 leads to actin polymerization, whereas PAK-mediated activation of LIMK results in actin turnover. Cdc42 activates Arp2/3 via WASP resulting in actin polymerization. Adapted from Sadok & Marshall 2014.
Altered expression or dysregulated activity of the Rho-family of small GTPases is frequently associated with tumorigenesis and the development of different cancer types including colorectal cancer (Mack et al. 2011). While no mutations for Rho GTPases have been described so far, the Rac1-specific GEF TIAM 1 (T-lymphoma invasion and metastasis-inducing protein-1) and the RhoA-specific GEF RGNEF (p190RhoGEF) were shown to be up-regulated in colorectal cancer (Leve & Morgado-Díaz 2012). TIAM 1 was identified as an invasion and metastasis gene and shown to be required for the initiation of colon cancer growth (Cook et al. 2013). In addition to invasion and metastasis, a mitotic role was also suggested for TIAM 1, since it was found to localize to mitotic centrosomes antagonizing the function of Eg5 in centrosome separation during prophase (Whalley et al. 2015). In axons, TIAM 1 was shown to localize to microtubules via MAP1B (Montenegro-Venegas et al.
2010). TRIO constitutes another Rac1-GEF with possible functions in mitosis. TRIO was identified as a microtubule plus-end binding protein in neurons (Van Haren et al. 2014). In these cells, binding is achieved via EB1/Nav1 complexes and requires dynamic microtubules. High expression of TRIO is found in different tumor types, including breast and lung cancer and glioblastoma and is associated with poor patient prognosis (Schmidt &
Debant 2014).
A variety of tumor cells exhibit a deregulated expression or activity of Rho GTPases (Boettner & Van Aelst 2002). Especially Rac1 hyperactivation is associated with aggressive tumor growth (Bid et al. 2013). Aggressive tumor growth is accompanied by a high migration and invasion potential. These processes require the formation of certain cell surface extensions like lamellipodia or invadopodia, which emerge from Rac1-mediated
28 reorganization of the actin cytoskeleton (Parri & Chiarugi 2010). Therefore, Rac1 or other members of the Rac1 pathway would represent interesting therapeutic targets for anti-cancer therapy (Bid et al. 2013).