Rho GDP dissociation inhibitors as potential targets for anticancer treatment

Drug Resistance Updates 9 (2006) 134–141

Rho GDP dissociation inhibitors as potential targets for anticancer treatment Baolin Zhang ∗ Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, 29 Lincoln Dr., Bldg. 29A, Rm. 2B-24, Bethesda, MD 20892, USA Received 22 May 2006; received in revised form 31 May 2006; accepted 1 June 2006

Abstract The Rho GDP dissociation inhibitors (RhoGDIs) are a major class of regulators of Rho GTPases and play essential roles in normal cell growth and malignant transformation. Although RhoGDIs are known to inhibit Rho activities, recent studies indicate that RhoGDIs can also act as positive regulators through their ability to target Rho GTPases to specific subcellular membranes or to protect the GTPases from degradation by caspases. RhoGDIs are aberrantly expressed in human tumors and this may contribute to Rho-induced cancer progression. This review will discuss the dual roles of RhoGDIs in the regulation of Rho GTPases, highlighting a possible role in regulating tumorigenicity. In addition, the potential for targeting RhoGDIs for anticancer therapy will be discussed. © 2006 Elsevier Ltd. All rights reserved. Keywords: RhoGDI; D4-GDI; Rho GTPases; Cancer; Metastasis

1. Introduction Rho GTPases, including isoforms of Rho, Rac1 and Cdc42, play essential roles in normal cell function. Aberrant signaling through these molecules, which is commonly found in a high percentage of human cancers, has been implicated in facilitating virtually all aspects of the malignant phenotype: increased tumor cell proliferation, promotion of angiogenesis, and acquisition of invasive and metastatic activities (Benitah et al., 2004; Burbelo et al., 2004; Gomez et al., 2005; Lin and van Golen, 2004; Ridley, 2004). Accordingly, Rho GTPases and their signaling components are viewed as attractive targets for the development of anticancer therapies (Aznar et al., 2004; Walker and Olson, 2005). As is the case for Ras protein (Blum and Kloog, 2005), the biological activities of Rho proteins are mediated through a tightly regulated GDP/GTP cycle, which is stimulated by guanine nucleotide exchange factors (RhoGEFs) and terminated by GTPase-activating proteins (RhoGAPs). Rho GTPases are subject to an additional layer of regulation by ∗

Tel.: +1 301 827 1784; fax: +1 301 480 3256. E-mail address: [email protected].

1368-7646/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.drup.2006.06.001

the Rho GDP dissociation inhibitors (RhoGDIs), whereas endogenous RasGDIs do not appear to exist. Only three human RhoGDIs have been identified: RhoGDI (also known as RhoGDI-␣ or RhoGDI-1), D4-GDI (Ly-GDI or RhoGDI␤ or RhoGDI-2), and RhoGDI-3 (RhoGDI-␥). Each acts on GTPases of the Rho family, which consists of as many as 22 members. The biochemical functions of RhoGDIs are described in several excellent reviews (DerMardirossian and Bokoch, 2005; Dovas and Couchman, 2005; Dransart et al., 2005; Olofsson, 1999). In general, RhoGDI proteins are thought to regulate Rho GTPases in three different ways: (a) by inhibiting the dissociation of the bound guanine nucleotide (usually GDP) from the partner GTPase; (b) by controlling the binding to other regulators (GEFs and GAPs) or downstream effectors; and (c) by acting as a shuttle protein to recruit Rho GTPases to their effector site and to recycle them back to cytosol. Many studies have described a role for RhoGDIs in inhibiting Rho GTPase function. For instance, when exogenously introduced into cells (by transfection or microinjection) RhoGDIs induce disruption of Rho-dependent cellular activities, including cytoskeleton organization and motility (Miura et al., 1993; Takaishi et al., 1993). In addition, cardiac-specific overexpression

B. Zhang / Drug Resistance Updates 9 (2006) 134–141

135

Fig. 1. Dual roles of RhoGDI in the regulation of Rho/Rac GTPases. (A) In an inhibitory mode, RhoGDI forms stable complexes with Rac GTPase, thus holding Rac in its inactive, cytosolic form. The dissociation of the complex is a pre-requisite for activation of Rac, a process that is driven by phosphorylation of RhoGDI or displacement factors such as members of the ERM family. Once released, Rac-GDP translocates to membrane compartments where it is anchored via its isoprenylated hydrophobic C-terminus, known as the ‘CAAX’ motif. The RhoGEFs, which are membrane-associated, stimulate GTP/GDP exchange and formation of active, GTP-bound Rac. This initiates an interaction with effector targets such as the NADPH oxidase for production of superoxide or the p21-activated kinase (PAK1) for activation of the JNK/P38 pathway. GTP-hydrolysis catalyzed by RhoGAPs terminates the signal and allows membrane dissociation promoted by RhoGDI, thereby recycling Rac back to the cytosolic pool. (B) In a stimulatory mode, RhoGDI acts as a carrier protein to direct Rac to distinct membrane compartments, which is crucial for interaction with effectors. This might be achieved by association with a component of a larger protein complex and through the assistance of specific localization signals, in particular membrane domains (Dovas and Couchman, 2005). On the other hand, the positive role of RhoGDI may also derive from its ability to protect Rac from proteolytic cleavage by apoptotic caspases.

of RhoGDI disrupts cardiac morphogenesis and inhibits cardiomyocyte proliferation, leading to embryonic lethality (Miura et al., 1993; Takaishi et al., 1993; Wei et al., 2002). The biological significance of RhoGDIs is highlighted by the striking phenotype changes observed in transgenic mice lacking D4-GDI (Yin et al., 1997) or RhoGDI (Togawa et al., 1999); in the latter, the mice developed age-dependent degeneration of kidney functions leading to renal failure and death, with additional defects in reproductive organs. Recent evidence points to a positive role for RhoGDIs in Rho GTPase function. It has been proposed that RhoGDIs can act as an escort to direct Rho GTPases to specific membraneassociated signaling complexes, which is crucial for coupling the GTPases to their downstream effector proteins. For instance, Rac GTPases are reported to bind to NADPH oxidase (Abo et al., 1991; Bromberg et al., 1994) as a complex with RhoGDI. Importantly, Cdc42-induced cellular transformation is likely to require not only the binding of activated Cdc42 to RhoGDI but also the recruitment of Cdc42 by RhoGDI to a specific cellular site (Lin et al., 2003). Our recent studies identified a novel function of RhoGDI in protecting Rac GTPase from proteolytic cleavage (Zhang et al., 2005).

This protective effect appears to be important for maintaining Rac GTPases in their intact, functional state, particularly in the context of apoptosis. A diagram illustrating the dual roles of RhoGDIs in the regulation of Rho GTPases is shown in Fig. 1. The complexity of the regulation implies a high incidence of aberrance in Rho signaling, as perturbation may occur at any of the regulatory loops. Indeed, altered Rho activity has been attributed to several different mechanisms: overexpression of Rho protein itself, mutations that render the protein resistant to normal regulatory systems (e.g. Rac1b), alterations in RhoGEFs and RhoGAPs, or downstream effectors. Evidence is accumulating that RhoGDIs are aberrantly expressed in many human cancers. For example, RhoGDI is elevated in primary ovarian cancers as well as in chemoresistant cancer cell lines (Poland et al., 2002; Sinha et al., 2000). In the latter, overexpression of RhoGDI appears to play a role in protecting cancer cells against drug-induced apoptosis. The altered RhoGDIs may make a significant contribution to Rhoinduced cancer initiation, progression, and metastasis. This review will survey recent findings on the regulatory roles of each of the three known RhoGDIs and its implications for human tumors.

136

B. Zhang / Drug Resistance Updates 9 (2006) 134–141

2. RhoGDI (RhoGDI␣/RhoGDI-1)—a regulator for most Rho GTPases 2.1. Altered RhoGDI expression in human cancers RhoGDI is ubiquitously expressed and binds to most of the Rho GTPases examined thus far, including RhoA, Cdc42, Rac1 and Rac2. However, its overexpression in human tumors was recognized only recently. Table 1 summarizes the accumulated evidence for the altered expression of RhoGDIs in carcinomas versus normal tissues. Fritz et al. (1999, 2002) provided the first evidence of the disbalance between Rho GTPases and RhoGDI expression in human breast cancers. In these studies, the protein expression of RhoGDI in 15 cases of human breast cancer was shown to be significantly enhanced when compared to normal tissue from the same patient. RhoA GTPase was also overexpressed in breast tumors. While the expression level of RhoA correlates with malignancy, the amount of RhoGDI did not seem to vary among the grade I and III tumors. There is also evidence of an opposite pattern of RhoGDI expression in breast cancers (Jiang et al., 2003). A realtime polymerase chain reaction (RT-PCR) analysis of 120 breast cancer specimens and 32 normal tissues revealed that RhoGDI as well as RhoGDI-3 is underexpressed in tumors relative to normal tissues. The authors also showed that the expression of RhoA and RhoB was similar between tumor and normal tissues; instead, the levels of RhoC and RhoG transcripts were significantly higher in tumors than in normal tissues. The reason for this discrepancy is unclear, but may be related to a difference in the sample sizes and the origin of the normal tissues (from the same patient or not). Thus, there remains a question about the expression of RhoGDIs and aberrant Rho signaling in breast cancer. Further studies with large numbers of paired samples and confirmation at both the mRNA and protein levels are warranted. There are several other types of human tumors that show alterations in RhoGDI expression. For instance, ovarian cancers display high levels of RhoGDI (Berman et al., 2004; Jones et al., 2002). A proteomics analysis revealed an approx-

imately four-fold increase of RhoGDI in invasive human ovarian cancer when compared to the low malignant potential cells from this cancer (Jones et al., 2002). In line with this, the protein amount of RhoGDI is higher in ovarian carcinoma than its ovarian epithelium counterpart, as determined through a rapid affinity-capture method (Berman et al., 2004). In ovarian cancer cells, RhoGDI was found to be complexed with Rac1, Cdc42, and five other unknown proteins. In addition, Rac1-RhoGDI was shown to be the protein complex with the highest differential of expression between malignant and normal ovary tissues. RhoGDI has been shown to be underexpressed in primary non-small cell lung cancer (NSCLC) and malignant gliomas (Blackhall et al., 2004; Wigle et al., 2002). Results from a cDNA microarray analysis showed that the level of RhoGDI decreases with tumor grade, and high expression of RhoGDI is associated with poorer prognosis. Furthermore, the expression level of RhoGDI and two other proteins (HIF1A and Citron K21) were prognostic for disease-free survival in a cohort of 130 patients with NSCLC. In addition, recent work with a proteomic analysis of 27 astrocytoma samples of different grades has shown that RhoGDI is significantly underexpressed in grade III astrocytomas compared to normal tissue and lower grade tumors (Chumbalkar et al., 2005). Since it has a broad selectivity for Rho GTPases, the misexpressed RhoGDI may have impact on pathways regulated by many different Rho proteins. When underexpressed, the insufficient RhoGDI may result in a sustained activation of Rho GTPases in cancers where their expression is excessive. This may constitute a major mechanism for the aberrant Rho activation in cancer cells, because members of the Rho family are frequently up-regulated in human cancers (Gomez et al., 2005). However, it should be noted that not only the protein level but also the phosphorylation status (DerMardirossian et al., 2004) of RhoGDI determines its interaction with Rho GTPases. In addition, the dissociation of RhoGDI–Rho complexes, which is essential for Rho activation, is regulated by proteins such as the ERM family (Takahashi et al., 1997) or the p75 neurotrophin receptor (Yamashita and Tohyama, 2003). Furthermore, although some Rho proteins (e.g. RhoA

Table 1 Evidence for altered expression of mRNA and/or protein levels of RhoGDIs in carcinomas vs. normal tissues Tumor

RhoGDIs Overexpressed

Bladder Breast

a

Underexpressed D4-GDI

Gildea et al. (2002), Seraj et al. (2000), Theodorescu et al. (2004), Yao et al. (2004)

RhoGDI RhoGDI-3

Fritz et al. (1999, 2002) Zhang and Zhang (2006) Jiang et al. (2003) Jiang et al. (2003)

RhoGDI RhoGDI

Chumbalkar et al. (2005) Blackhall et al. (2004), Wigle et al. (2002)

RhoGDI D4-GDI

Glioma Lung (NSCLC)a Ovarian

Reference

RhoGDI D4-GDI

NSCLC: non-small cell lung carcinoma.

Berman et al. (2004), Jones et al. (2002) Tapper et al. (2001)

B. Zhang / Drug Resistance Updates 9 (2006) 134–141

and RhoC) have been implicated in promotion of carcinogenesis (Gomez et al., 2005), others (e.g. RhoB and RhoBTB) are involved in tumor suppression (Prendergast, 2001), underscoring the complexity of the RhoGDI-regulated Rho signaling pathways that end up with a particular biological outcome. Thus, the contribution of the mis-expressed RhoGDI to aberrant Rho activation awaits further confirmatory results. Further studies of RhoGDI gene and protein alterations in various human tumors are necessary to elucidate the significance of this regulatory protein in tumorigenesis. 2.2. RhoGDI in drug resistance The idea that RhoGDI may be involved in the development of drug resistance came originally from the observation that RhoGDI is highly expressed in stable chemoresistant cancer cell lines such as melanoma (Poland et al., 2002; Sinha et al., 2000) and ovarian cancer (Takano et al., 2003). In line with this, treatment of non-small cell lung carcinoma or myeloid leukemia cells with apoptosis-inducing agents was associated with a down-regulation of RhoGDI levels (Kovarova et al., 2000; MacKeigan et al., 2003; Wan et al., 2001). Our recent studies have demonstrated that the overexpression of RhoGDI could be a significant determinant of the responsiveness of cancer cells to chemotherapeutic agents (Zhang et al., 2005). We found that overexpression of RhoGDI increased resistance of MDA-MB-231 human breast cancer cells and JLP-119 lymphoma cells to the induction of apoptosis by etoposide and doxorubicin. Conversely, targeted disruption of RhoGDI expression by DNA vector-mediated RNA interference (small interfering RNA, siRNA) sensitized MDA-MB-231 cells to drug-induced apoptosis. Resistance to apoptosis was restored by reintroduction of RhoGDI protein expression. These results indicate that RhoGDI overexpression gives cancer cells a survival mechanism against these antitumor chemotherapies. The mechanism by which RhoGDI inhibits apoptosis is not yet established, but there are several possibilities. • One possibility is that the overexpressed RhoGDI may interfere with actin structure or the formation of apoptotic signaling complexes. It is well established that RhoGDI controls actin cytoskeleton through Rho GTPases. A recent study demonstrated that RhoGDI as well as RhoA are involved in the formation of an apoptotic signaling complex within membrane lipid rafts in drug-treated leukemic cells (Gajate and Mollinedo, 2005). Disruption of lipid rafts and interference with actin cytoskeleton prevented Fas clustering and apoptosis (Dimanche-Boitrel et al., 2005; Mollinedo and Gajate, 2006). • On the other hand, the anti-apoptotic effect of RhoGDI may derive from its ability to protect Rac1 GTPase from apoptotic cleavage. Rac1 has been shown to be an anti-apoptotic molecule that inhibits apoptosis induced by different factors such as tumor necrosis factor and antitumor drugs (Pervaiz et al., 2001; Ruggieri et al., 2001; Zhang et al.,

137

2003, 2004). Our recent studies showed that Rac1 is a substrate for caspases and becomes cleaved during apoptosis, which allows maximal cell death following drug treatment (Zhang et al., 2003). Cleavage of native Rac1 occurs at VVGD11/G, a site that is fully covered when Rac1 is complexed to RhoGDI. Accordingly, Rac1 became completely resistant to caspase cleavage when in a 1:1 complex with RhoGDI. Knockdown of RhoGDI by siRNA rendered the cells more sensitive to drug-induced apoptosis (Zhang et al., 2005). Furthermore, expression of the caspase-3-resistant mutant Rac1D11E restored the resistance of RhoGDI-depleted cells to VP-16-induced cytotoxicity (Zhang et al., 2003). Thus, it is likely that RhoGDI inhibits drug-induced apoptosis, at least in part, by blocking the apoptotic cleavage of Rac GTPases. • RhoGDI knockdown experiments also demonstrated a role for RhoGDI in regulating the subcellular localization of Rho GTPases. Rac1 and Cdc42 reside predominantly in the cytosol of wild-type MDA-MB-231 cells; however, they are found primarily in plasma membrane fraction in cells lacking RhoGDI (Zhang and Zhang, unpublished data). These results indicate that RhoGDI is necessary for proper membrane translocation of Rho GTPases. Thus, knockdown of RhoGDI results in mis-localization of these proteins and, likely, their subsequent activation. Further experiments will determine the downstream signaling involved in the induction of apoptosis as a result of RhoGDI depletion. The availability of the RhoGDI−/− cells will help in these investigations.

3. D4-GDI (Ly-GDI/RhoGDI-2 or RhoGDI-␤)—a RhoGDI complexed to an unidentified Rho GTPase 3.1. Unique features of D4-GDI D4-GDI and RhoGDI share a 78% amino acid identity and are thought to interact in similar ways with Rho GTPases. Both are made up of two structural domains: a flexible N-terminal domain (1–69 in RhoGDI) and a C-terminal domain (70–204), which adopts an immunoglobulin-like fold. The N-terminal domain is essential for the binding of RhoGDI to Rac1 and Cdc42, and for D4-GDI binding to Rac2 (Golovanov et al., 2001; Olofsson, 1999). D4-GDI has several additional features that are distinct among RhoGDI proteins: • Unlike RhoGDI, which is ubiquitously expressed, D4GDI appeared, until recently, to be selectively expressed in hematopoietic tissues and in B- and T-cell lines (Lelias et al., 1993; Scherle et al., 1993). • D4-GDI appears to have a narrower selectivity and a lower binding affinity for Rho GTPases. Although purified recombinant D4-GDI inhibits GDP dissociation from Rac1 and RhoA, this function has not yet been confirmed in vivo. While Rho/Rac proteins are found complexed to

138

B. Zhang / Drug Resistance Updates 9 (2006) 134–141

RhoGDI when isolated from the cytosol of resting cells, none of these proteins has yet been found in complexes with D4-GDI. As a result, the Rho protein(s) regulated by D4-GDI in vivo are still unknown. It has been suggested that D4-GDI may be associated with an uncharacterized Rho protein in cells (Olofsson, 1999). • D4-GDI is well characterized as a substrate for caspases and becomes cleaved in various cells during apoptosis. This is in contrast to RhoGDI, which is completely resistant to degradation during apoptosis (Zhang et al., 2005). Caspase-3 cleaves D4-GDI at Asp19 (DELD19 S), leaving a fragment of 23 kDa that translocates into the nucleus of apoptotic cells (Essmann et al., 2000; Krieser and Eastman, 1999; Thiede et al., 2002). Cleavage by caspase-1 at Asp55 (LLGD55 G) occurs in inflammatory leukocytes (Danley et al., 1996). Although the significance of this unique feature is uncertain, a recent study showed that the stable expression of a C-terminal-depleted human D4-GDI (
LyGDI, 166–201 deletion) induces pulmonary metastasis in 1-1ras1000 recipient cells (Ota et al., 2004).

et al., 2004). Consequently, it has been proposed that D4-GDI is a metastasis suppressor gene in bladder cancers (Gildea et al., 2002; Seraj et al., 2000; Theodorescu et al., 2004). The underlying mechanism is unknown, but may involve D4-GDI-induced inhibition of expression of endothelin-1, a potent vasoconstrictor that has been implicated in facilitating metastasis to the lung (Titus et al., 2005). A cDNA microarray analysis revealed that the mRNA level of D4-GDI is significantly higher in adenocarcinoma than in benign adenoma, irrespective of the clinical tumor stage, so D4-GDI might contribute to the metastasis of this type of tumor (Tapper et al., 2001). In line with this, the induction of motility of cancer cells by the autocrine motility factor appeared to be mediated through up-regulation of D4-GDI mRNA and protein in vitro (Yanagawa et al., 2004). More recently, our laboratory demonstrated that D4-GDI is overexpressed in human breast cancer cell lines (Zhang and Zhang, 2006). Overexpression of D4-GDI correlates with the features typical of a malignant phenotype in these cells, including increased invasion and motility (Table 2). For instance, highly metastatic MDA-MB-231 cells overexpress D4-GDI, while poorly metastatic MCF-7 cells do not express D4-GDI. In contrast, RhoGDI is expressed at essentially the same level in the same cell lines. MDA-MB-231 cells with forced disruption of D4-GDI have impaired motility and invasion through Matrigel. Strikingly, forced depletion of D4-GDI reverts the cells to a normal breast epithelial phenotype characterized by the formation of cysts with a central lumen. Knockdown of RhoGDI, on the other hand, had no effect on the invasive morphology of MDA-MB-231 cells (Zhang and Zhang, unpublished data). This is somewhat surprising because RhoGDI, as described above, can be either up- or down-regulated in primary breast cancers (Fritz et al., 1999, 2002; Jiang et al., 2003). The results may suggest that D4-GDI, but not RhoGDI, is more highly overexpressed in late stages of breast, and thus may play an important role in the regulation of breast cancer metastasis. However, since no correlative prognostic information is available, proof of

3.2. D4-GDI in metastasis D4-GDI was originally found only in hematopoietic tissues and cells, but recent studies indicate that it is also expressed in cells of non-hematopoietic neoplasms including bladder, ovarian, and breast cancers. Theodorescu and coworkers (Seraj et al., 2000) showed that D4-GDI (also called RhoGDI2) mRNA was expressed in the T24 human bladder cancer cell line, but was absent in the more aggressive T24T lineage. These authors further showed that D4-GDI protein is lower or undetectable in primary bladder carcinoma when compared to normal tissues (Gildea et al., 2002; Theodorescu et al., 2004). Furthermore, the reduced expression of D4-GDI protein was shown to be associated with a poor prognosis of patients with advanced bladder cancer. In line with this, in a carcinogen-induced mouse model of bladder cancer, D4-GDI was found to be underexpressed in tumor tissues compared with normal bladder epithelium (Yao Table 2 D4-GDI protein and mRNA expression in breast cancer cell lines Breast cancer cell line

Matrigel morphologya,b

Chemoinvasionb,c

D4-GDI RNAd,e

D4-GDI proteine,f

MDA-MB-231 BT549 MCF7 BT474 MDA-MB-468 SKBR3 Hs578Bst MCF-12A

INV INV SPH SPH SPH SPH nd SPH

+++++ +++++ ++ + + + − −

+ + − + + + − +

+ + − + + + − −

a b c d e f

Morphology in Matrigel: INV, invasive branching network formation; SPH, spherical colony or non-invasive cluster formation; nd, not determined. Data taken from Coopman et al. (2000) and Hashimoto et al. (2004). Activity in Boyden chamber chemoinvasion assay. As determined by RT-PCR. Data taken from Zhang and Zhang (2006). As determined by Western blotting using anti-D4-GDI antibodies.

B. Zhang / Drug Resistance Updates 9 (2006) 134–141

the involvement of D4-GDI in breast cancer metastasis will require additional, more comprehensive studies. The seemingly conflicting results regarding the contribution of D4-GDI to metastasis make it difficult to draw a firm conclusion concerning the exact role for the protein in metastasis. The different patterns of D4-GDI expression in different cancer cells may reflect the diversity of genetic backgrounds and/or the tumor stages, as well as activation versus inhibition of Rho GTPases in the course of cancer progression. It is possible that D4-GDI acts as a metastasis suppressor in some types of cancer, but as a promoter in other types. There is little information about the molecular basis for D4-GDI regulation of tumor metastasis. Our studies showed that D4-GDI depletion is associated with a down-regulation of the cell surface protein ␤1-integrin and a loss of cellmatrix adhesion, suggesting that D4-GDI induces cell invasion by controlling ␤1-integrin expression (Zhang and Zhang, 2006).

4. RhoGDI-3 (RhoGDI␥), a Golgi-associated RhoGDI RhoGDI-3 was first found to be preferentially expressed in brain tissues and, to a lesser extent, in lung, kidney, and testis. Compared with RhoGDI and D4-GDI, which reside exclusively in the cytoplasm, RhoGDI-3 appears to be largely associated with the Golgi apparatus through its unique N-terminal ␣ helix domain (Brunet et al., 2002). The most likely partners for RhoGDI-3 are RhoB and RhoG, as they interact with RhoGDI-3 in a yeast two-hybrid system (Zalcman et al., 1996). However, RhoG seems to be the only Rho protein that binds to RhoGDI-3 in vivo (Brunet et al., 2002). Complex formation between RhoGDI-3 and RhoG inhibits RhoG activation and targets this protein to Golgi. The alteration of RhoGDI-3 expression has been linked to the progression of breast cancers. As mentioned, the levels of RhoGDI-3 transcripts are significantly lower in breast cancer tissues than in normal tissues, and are lower in cells from patients with recurrent disease or with metastasis, compared to cells from disease-free patients (Jiang et al., 2003). Interestingly, the reduced expression of RhoGDI-3 is associated with an increased expression of RhoG. As discussed above, this may result in a sustained activation of RhoG in breast cancer cells. Although the relevance of these alterations to breast carcinogenesis remains to be determined, RhoG has been implicated in the regulation of apoptotic process (Murga et al., 2002), via the phosphatidylinositol 3 -kinase and Akt pathways.

5. Perspective The available information on expression of RhoGDIs in human cancers provides sufficient justification for further

139

research on this topic. However, the involvement of other regulators (RhoGEFs and RhoGAPs) and downstream effectors must also be taken into consideration. In light of the complex roles of the Rho GTPases in the carcinogenic process, it appears that a direct measurement of Rho activities, other than merely the expression levels, may provide a deeper understanding of the role of these proteins in tumorigenesis. The challenge will be to develop a sensitive assay that measures simultaneously the activities of different Rho proteins. In principle, the active conformations of individual Rho GTPases can be detected by exploiting the selective binding to downstream effectors; e.g. the p21-activated kinase (PAK1) for Rac1 (Zhang et al., 2003). As such, a combination of protein microarray technology and utilization of the Rho-specific effector-binding domains may prove to be useful. To date, therapeutic strategies targeting the Ras and Rho GTPase signaling pathways have been largely focused on inhibiting the activities of downstream effectors and the GTPases themselves. Although small-molecule inhibitors targeting the post-translational modification enzymes (e.g. farnesyl transferase and geranylgeranyl transferase) are now in advanced-stage clinical trials, their utility and efficacy as single agents seem to be limited (Dancey and Sausville, 2003). RhoGDIs are pivotal regulators of Rho GTPase function. Growing evidence of their linkage to human cancers makes them attractive candidates as therapeutic targets for altering the elevated Rho signaling in tumors. As an alternative to blocking the post-translational modifications of Rho GTPases, it might be possible to stabilize the interaction between Rho and RhoGDI by interfacial inhibitors, such as brefeldin A (Pommier and Cherfils, 2005). In this way, Rho GTPase would be trapped in dead-end complexes that are no longer able to complete their biological function (Dransart et al., 2005). In the event that a beneficial effect of reduced RhoGDIs on tumor progression can be established, the development of specific, small-molecule inhibitors of these isoforms would follow. Depletion of RhoGDIs by siRNA technology may represent an attractive first step in this strategy. The approach has been used successfully to knock down RhoGDI and D4-GDI in cultured breast cancer cells and to alter the susceptibility of the cells to anti-cancer agents (Zhang et al., 2005; Zhang and Zhang, 2006). In light of the broad selectivity of RhoGDI towards Rho GTPases, this approach might also be advantageous for targeting multiple Rho proteins. Alternatively, the beneficial effect of reduced RhoGDI might be achieved by facilitating the release of Rho GTPases from the complexes with RhoGDI. The availability of the structural information on these complexes should allow establishing high-throughout screens to identify selective compounds or isoform-specific bioactive peptides that destabilize Rho–RhoGDI complexes. In the case of D4-GDI, however, this must await the identification of its partner GTPase(s) in cancer cells.

140

B. Zhang / Drug Resistance Updates 9 (2006) 134–141

Acknowledgements The author thanks Dr. Emily Shacter for critical reading of the manuscript and helpful suggestions. I also apologize to the authors of many interesting studies that were omitted due to limited space. References Abo, A., Pick, E., Hall, A., Totty, N., Teahan, C.G., Segal, A.W., 1991. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature 353, 668–670. Aznar, S., Fernandez-Valeron, P., Espina, C., Lacal, J.C., 2004. Rho GTPases: potential candidates for anticancer therapy. Cancer Lett. 206, 181–191. Benitah, S.A., Valeron, P.F., Van Aelst, L., Marshall, C.J., Lacal, J.C., 1705. Rho GTPases in human cancer: an unresolved link to upstream and downstream transcriptional regulation. Biochim. Biophys. Acta, 121–132. Berman, D.M., Shih, I., Burke, L.A., Veenstra, T.D., Zhao, Y., Conrads, T.P., Kwon, S.W., Hoang, V., Yu, L.R., Zhou, M., Kurman, R.J., Petricoin, E.F., Liotta, L.A., 2004. Profiling the activity of G proteins in patient-derived tissues by rapid affinity-capture of signal transduction proteins (GRASP). Proteomics 4, 812–818. Blackhall, F.H., Wigle, D.A., Jurisica, I., Pintilie, M., Liu, N., Darling, G., Johnston, M.R., et al., 2004. Validating the prognostic value of marker genes derived from a non-small cell lung cancer microarray study. Lung Cancer 46, 197–204. Blum, R., Kloog, Y., 2005. Tailoring Ras-pathway-inhibitor combinations for cancer therapy. Drug Resist. Update 8, 369–380. Bromberg, Y., Shani, E., Joseph, G., Gorzalczany, Y., Sperling, O., Pick, E., 1994. The GDP-bound form of the small G protein Rac1 p21 is a potent activator of the superoxide-forming NADPH oxidase of macrophages. J. Biol. Chem. 269, 7055–7058. Brunet, N., Morin, A., Olofsson, B., 2002. RhoGDI-3 regulates RhoG and targets this protein to the Golgi complex through its unique N-terminal domain. Traffic 3, 342–357. Burbelo, P., Wellstein, A., Pestell, R.G., 2004. Altered Rho GTPase signaling pathways in breast cancer cells. Breast Cancer Res. Treat. 84, 43–48. Chumbalkar, V.C., Subhashini, C., Dhople, V.M., Sundaram, C.S., Jagannadham, M.V., Kumar, K.N., et al., 2005. Differential protein expression in human gliomas and molecular insights. Proteomics 5, 1167–1177. Coopman, P.J., Do, M.T., Barth, M., Bowden, E.T., Hayes, A.J., Basyuk, E., Blancato, J.K., Vezza, P.R., McLeskey, S.W., Mangeat, P.H., Mueller, S.C., 2000. The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells. Nature 406, 742–747. Dancey, J., Sausville, E.A., 2003. Issues and progress with protein kinase inhibitors for cancer treatment. Nat. Rev. Drug Discov. 2, 296–313. Danley, D.E., Chuang, T.H., Bokoch, G.M., 1996. Defective Rho GTPase regulation by IL-1 beta-converting enzyme-mediated cleavage of D4 GDP dissociation inhibitor. J. Immunol. 157, 500–503. DerMardirossian, C., Bokoch, G.M., 2005. GDIs: central regulatory molecules in Rho GTPase activation. Trends Cell Biol. 15, 356–363. DerMardirossian, C., Schnelzer, A., Bokoch, G.M., 2004. Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase. Mol. Cell 15, 117–127. Dimanche-Boitrel, M.T., Meurette, O., Rebillard, A., Lacour, S., 2005. Role of plasma membrane events in chemotherapy-induced cell death. Drug Resist. Update 8, 5–14. Dovas, A., Couchman, J.R., 2005. RhoGDI: multiple functions in the regulation of Rho family GTPase activities. Biochem. J. 390, 1–9. Dransart, E., Olofsson, B., Cherfils, J., 2005. RhoGDIs revisited: novel roles in Rho regulation. Traffic 6, 957–966.

Essmann, F., Wieder, T., Otto, A., Muller, E.C., Dorken, B., Daniel, P.T., 2000. GDP dissociation inhibitor D4-GDI (Rho-GDI 2), but not the homologous rho-GDI 1, is cleaved by caspase-3 during drug-induced apoptosis. Biochem. J. 346 (Pt 3), 777–783. Fritz, G., Brachetti, C., Bahlmann, F., Schmidt, M., Kaina, B., 2002. Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters. Br. J. Cancer 87, 635–644. Fritz, G., Just, I., Kaina, B., 1999. Rho GTPases are over-expressed in human tumors. Int. J. Cancer 81, 682–687. Gajate, C., Mollinedo, F., 2005. Cytoskeleton-mediated death receptor and ligand concentration in lipid rafts forms apoptosis-promoting clusters in cancer chemotherapy. J. Biol. Chem. 280, 11641–11647. Gildea, J.J., Seraj, M.J., Oxford, G., Harding, M.A., Hampton, G.M., Moskaluk, C.A., Frierson, H.F., Conaway, M.R., Theodorescu, D., 2002. RhoGDI2 is an invasion and metastasis suppressor gene in human cancer. Cancer Res. 62, 6418–6423. Golovanov, A.P., Chuang, T.H., DerMardirossian, C., Barsukov, I., Hawkins, D., Badii, R., Bokoch, G.M., Lian, L.Y., Roberts, G.C., 2001. Structure–activity relationships in flexible protein domains: regulation of Rho GTPases by RhoGDI and D4 GDI. J. Mol. Biol. 305, 121–135. Gomez, d.P., Benitah, S.A., Valeron, P.F., Espina, C., Lacal, J.C., 2005. Rho GTPase expression in tumourigenesis: evidence for a significant link. Bioessays 27, 602–613. Hashimoto, S., Onodera, Y., Hashimoto, A., Tanaka, M., Hamaguchi, M., Yamada, A., Sabe, H., 2004. Requirement for Arf6 in breast cancer invasive activities. Proc. Natl. Acad. Sci. U.S.A. 101, 647– 6652. Jiang, W.G., Watkins, G., Lane, J., Cunnick, G.H., Douglas-Jones, A., Mokbel, K., Mansel, R.E., 2003. Prognostic value of rho GTPases and rho guanine nucleotide dissociation inhibitors in human breast cancers. Clin. Cancer Res. 9, 6432–6440. Jones, M.B., Krutzsch, H., Shu, H., Zhao, Y., Liotta, L.A., Kohn, E.C., Petricoin III, E.F., 2002. Proteomic analysis and identification of new biomarkers and therapeutic targets for invasive ovarian cancer. Proteomics 2, 76–84. Kovarova, H., Hajduch, M., Korinkova, G., Halada, P., Krupickova, S., Gouldsworthy, A., Zhelev, N., Strnad, M., 2000. Proteomics approach in classifying the biochemical basis of the anticancer activity of the new olomoucine-derived synthetic cyclin-dependent kinase inhibitor, bohemine. Electrophoresis 21, 3757–3764. Krieser, R.J., Eastman, A., 1999. Cleavage and nuclear translocation of the caspase 3 substrate Rho GDP-dissociation inhibitor, D4-GDI, during apoptosis. Cell Death. Differ. 6, 412–419. Lelias, J.M., Adra, C.N., Wulf, G.M., Guillemot, J.C., Khagad, M., Caput, D., Lim, B., 1993. cDNA cloning of a human mRNA preferentially expressed in hematopoietic cells and with homology to a GDP-dissociation inhibitor for the Rho GTP-binding proteins. Proc. Natl. Acad. Sci. U.S.A. 90, 1479–1483. Lin, M., van Golen, K.L., 2004. Rho-regulatory proteins in breast cancer cell motility and invasion. Breast Cancer Res. Treat. 84, 49–60. Lin, Q., Fuji, R.N., Yang, W., Cerione, R.A., 2003. RhoGDI is required for Cdc42-mediated cellular transformation. Curr. Biol. 13, 1469–1479. MacKeigan, J.P., Clements, C.M., Lich, J.D., Pope, R.M., Hod, Y., Ting, J.P., 2003. Proteomic profiling drug-induced apoptosis in non-small cell lung carcinoma: identification of RS/DJ-1 and RhoGDIalpha. Cancer Res. 63, 6928–6934. Miura, Y., Kikuchi, A., Musha, T., Kuroda, S., Yaku, H., Sasaki, T., Takai, Y., 1993. Regulation of morphology by Rho p21 and its inhibitory GDP/GTP exchange protein (Rho GDI) in Swiss 3T3 cells. J. Biol. Chem. 268, 510–515. Mollinedo, F., Gajate, C., 2006. Fas/CD95 death receptor and lipid rafts: new targets for apoptosis-directed cancer therapy. Drug Resist. Update 9, 53–75. Murga, C., Zohar, M., Teramoto, H., Gutkind, J.S., 2002. Rac1 and RhoG promote cell survival by the activation of PI3K and Akt, indepen-

B. Zhang / Drug Resistance Updates 9 (2006) 134–141 dently of their ability to stimulate JNK and NF-kappaB. Oncogene 21, 207–216. Olofsson, B., 1999. Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling. Cell Signal. 11, 545–554. Ota, T., Maeda, M., Suto, S., Tatsuka, M., 2004. LyGDI functions in cancer metastasis by anchoring Rho proteins to the cell membrane. Mol. Carcinog. 39, 206–220. Pervaiz, S., Cao, J., Chao, O.S., Chin, Y.Y., Clement, M.V., 2001. Activation of the RacGTPase inhibits apoptosis in human tumor cells. Oncogene 20, 6263–6268. Poland, J., Schadendorf, D., Lage, H., Schnolzer, M., Celis, J.E., Sinha, P., 2002. Study of therapy resistance in cancer cells with functional proteome analysis. Clin. Chem. Lab. Med. 40, 221–234. Pommier, Y., Cherfils, J., 2005. Interfacial inhibition of macromolecular interactions: nature’s paradigm for drug discovery. Trends Pharmacol. Sci. 26, 138–145. Prendergast, G.C., 2001. Actin’ up: RhoB in cancer and apoptosis. Nat. Rev. Cancer 1, 162–168. Ridley, A.J., 2004. Rho proteins and cancer. Breast Cancer Res. Treat. 84, 13–19. Ruggieri, R., Chuang, Y.Y., Symons, M., 2001. The small GTPase Rac suppresses apoptosis caused by serum deprivation in fibroblasts. Mol. Med. 7, 293–300. Scherle, P., Behrens, T., Staudt, L.M., 1993. Ly-GDI, a GDP-dissociation inhibitor of the RhoA GTP-binding protein, is expressed preferentially in lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 90, 7568–7572. Seraj, M.J., Harding, M.A., Gildea, J.J., Welch, D.R., Theodorescu, D., 2000. The relationship of BRMS1 and RhoGDI2 gene expression to metastatic potential in lineage related human bladder cancer cell lines. Clin. Exp. Metastasis 18, 519–525. Sinha, P., Kohl, S., Fischer, J., Hutter, G., Kern, M., Kottgen, E., Dietel, M., Lage, H., Schnolzer, M., Schadendorf, D., 2000. Identification of novel proteins associated with the development of chemoresistance in malignant melanoma using two-dimensional electrophoresis. Electrophoresis 21, 3048–3057. Takahashi, K., Sasaki, T., Mammoto, A., Takaishi, K., Kameyama, T., Tsukita, S., Takai, Y., 1997. Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J. Biol. Chem. 272, 23371– 23375. Takaishi, K., Kikuchi, A., Kuroda, S., Kotani, K., Sasaki, T., Takai, Y., 1993. Involvement of Rho p21 and its inhibitory GDP/GTP exchange protein (Rho GDI) in cell motility. Mol. Cell Biol. 13, 72–79. Takano, M., Goto, T., Sakamoto, M., et al., 2003. Identification of paclitaxel-resistance related genes by differential display using cDNA microarray in ovarian cancer cell lines. Proc. Am. Soc. Clin. Oncol. 22, 462–463. Tapper, J., Kettunen, E., El Rifai, W., Seppala, M., Andersson, L.C., Knuutila, S., 2001. Changes in gene expression during progression of ovarian carcinoma. Cancer Genet. Cytogenet. 128, 1–6. Theodorescu, D., Sapinoso, L.M., Conaway, M.R., Oxford, G., Hampton, G.M., Frierson Jr., H.F., 2004. Reduced expression of metastasis suppressor RhoGDI2 is associated with decreased survival for patients with bladder cancer. Clin. Cancer Res. 10, 3800–3806.

141

Thiede, B., Siejak, F., Dimmler, C., Rudel, T., 2002. Prediction of translocation and cleavage of heterogeneous ribonuclear proteins and Rho guanine nucleotide dissociation inhibitor 2 during apoptosis by subcellular proteome analysis. Proteomics 2, 996–1006. Titus, B., Frierson Jr., H.F., Conaway, M., Ching, K., Guise, T., Chirgwin, J., Hampton, G., Theodorescu, D., 2005. Endothelin axis is a target of the lung metastasis suppressor gene RhoGDI2. Cancer Res. 65, 7320–7327. Togawa, A., Miyoshi, J., Ishizaki, H., Tanaka, M., Takakura, A., Nishioka, H., Yoshida, H., et al., 1999. Progressive impairment of kidneys and reproductive organs in mice lacking Rho GDIalpha. Oncogene 18, 5373–5380. Walker, K., Olson, M.F., 2005. Targeting Ras and Rho GTPases as opportunities for cancer therapeutics. Curr. Opin. Genet. Dev. 15, 62–68. Wan, J., Wang, J., Cheng, H., Yu, Y., Xing, G., Oiu, Z., Qian, X., He, F., 2001. Proteomic analysis of apoptosis initiation induced by all-trans retinoic acid in human acute promyelocytic leukemia cells. Electrophoresis 22, 3026–3037. Wei, L., Imanaka-Yoshida, K., Wang, L., Zhan, S., Schneider, M.D., DeMayo, F.J., Schwartz, R.J., 2002. Inhibition of Rho family GTPases by Rho GDP dissociation inhibitor disrupts cardiac morphogenesis and inhibits cardiomyocyte proliferation. Development 129, 1705–1714. Wigle, D.A., Jurisica, I., Radulovich, N., Pintilie, M., Rossant, J., Liu, N., Lu, C., et al., 2002. Molecular profiling of non-small cell lung cancer and correlation with disease-free survival. Cancer Res. 62, 3005–3008. Yamashita, T., Tohyama, M., 2003. The p75 receptor acts as a displacement factor that releases Rho from Rho-GDI. Nat. Neurosci. 6, 461–467. Yanagawa, T., Watanabe, H., Takeuchi, T., Fujimoto, S., Kurihara, H., Takagishi, K., 2004. Overexpression of autocrine motility factor in metastatic tumor cells: possible association with augmented expression of KIF3A and GDI-beta. Lab. Invest. 84, 513–522. Yao, R., Lemon, W.J., Wang, Y., Grubbs, C.J., Lubet, R.A., You, M., 2004. Altered gene expression profile in mouse bladder cancers induced by hydroxybutyl(butyl) nitrosamine. Neoplasia 6, 569–577. Yin, L., Schwartzberg, P., Scharton-Kersten, T.M., Staudt, L., Lenardo, M., 1997. Immune responses in mice deficient in Ly-GDI, a lymphoidspecific regulator of Rho GTPases. Mol. Immunol. 34, 481–491. Zalcman, G., Closson, V., Camonis, J., Honore, N., Rousseau-Merck, M.F., Tavitian, A., Olofsson, B., 1996. RhoGDI-3 is a new GDP dissociation inhibitor (GDI). Identification of a non-cytosolic GDI protein interacting with the small GTP-binding proteins RhoB and RhoG. J. Biol. Chem. 271, 30366–30374. Zhang, B., Zhang, Y., Dagher, M.C., Shacter, E., 2005. Rho GDP dissociation inhibitor protects cancer cells against drug-induced apoptosis. Cancer Res. 65, 6054–6062. Zhang, B., Zhang, Y., Shacter, E., 2003. Caspase 3-mediated inactivation of Rac GTPases promotes drug-induced apoptosis in human lymphoma cells. Mol. Cell Biol. 23, 5716–5725. Zhang, B., Zhang, Y., Shacter, E., 2004. Rac1 inhibits apoptosis in human lymphoma cells by stimulating Bad phosphorylation on Ser-75. Mol. Cell Biol. 24, 6205–6214. Zhang, Y., Zhang, B., 2006. D4-GDI, a Rho GTPase regulator, promotes breast cancer cell invasiveness. Cancer Res. 66, 5592–5598.