Rac1 and Rac3 Are Targets for Geranylgeranyltransferase I Inhibitor-Mediated Inhibition of Signaling, Transformation, and Membrane Ruffling

[CANCER RESEARCH 63, 7959–7967, November 15, 2003] Rac1 and Rac3 Are Targets for Geranylgeranyltransferase I Inhibitor-Mediated Inhibition of Signaling, Transformation, and Membrane Ruffling

Patricia L. Joyce and Adrienne D. Cox Departments of Pharmacology and Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

ABSTRACT mapped to chromosome band 17q25.3 near a region that is commonly deleted in breast and ovarian cancers, suggesting possible transcrip- Rac1, a Rho family GTPase, is a mediator of diverse cellular functions tional dysregulation in these diseases (15, 16). Additionally, Rac3 but including membrane ruffling, cell cycle progression, and transformation. not Rac1 was shown to be hyperactivated in breast cancer cells, and Rac3, a close relative of Rac1, is less well characterized. Posttranslational addition of geranylgeranyl isoprenoid lipids to Rac proteins is required inhibition of Rac3 was shown to impair breast cancer cell proliferation for biological activity. Inhibitors of geranylgeranyl transferase I (GGTIs) (17). These results suggest that Rac proteins may be attractive targets are currently under investigation as a possible anticancer therapy, al- for anticancer drugs. though the targets of GGTIs have not been determined. We created Rac/Rho proteins and Ras proteins are both modified posttransla- COOH-terminal mutants of Rac1 and Rac3 that are farnesylated and used tionally by isoprenoid lipids. Addition of isoprenoid groups is re- them to characterize Rac1 and Rac3 as physiological targets of GGTIs. quired for proper localization and function of Rho/Ras family proteins We show that, like Rac1, activated Rac3 causes transformation and leads (18). Rac and Rho are modified by GGTase I, whereas Ras proteins to membrane ruffling. Farnesylated versions of Rac1 and Rac3 retain the are modified by FTase (18). FTIs are in clinical trials as potential ability to signal to the transcription factor c-Jun and cause membrane anticancer drugs, whereas GGTIs are still in preclinical development ruffling and transformation, indicating that switching isoprenoid modifi- (19, 20). Although GGTIs have been shown to arrest human tumor cation does not alter function. Finally, treatment with GGTIs led to the inhibition of membrane-ruffling and transforming activities of both acti- cell growth in vitro (21, 22) and to reduce tumor growth in animal vated and wild-type Rac1 and Rac3. However, the farnesylated versions of models (23), the physiologically relevant downstream targets of both activated and wild-type Rac1 and Rac3 were resistant to the inhib- GGTIs have not been determined. Most Rho family proteins, includ- itory effects of GGTIs. These results illustrate that Rac1 and Rac3 are ing the Rac GTPases, are substrates for GGTase I and are logical potential physiological targets for these novel drugs. targets for GGTIs (18). GG isoprenoids are added to cysteine residues at the COOH terminus of proteins whose CAAX motifs terminate in leucine (X ϭ L). The Rac1 CAAX motif is CLLL, and Rac1 is a known INTRODUCTION substrate for GGTase I (24, 25). Interestingly, the CAAX motif of Rac3 is CTVF, which suggests that Rac3 may be a substrate for both The small GTPases Rac1 and Rac3 are members of the Rho family. GGTase I and FTase (26). Like Ras proteins, Rho family proteins cycle between active GTP- Indications that Rac3 is specifically hyperactivated and required for bound and inactive GDP-bound states (1–3). They are positively proliferation in breast cancer cells, as well as its sequence similarity regulated by Dbl family guanine nucleotide exchange factors, which to Rac1, suggest that Rac3 has oncogenic potential. To address the promote the GTP-bound state. Negative regulation is achieved by issue of whether Rac1 and Rac3 are physiologically important targets GTPase-activating proteins, which enhance the hydrolysis of GTP, of GGTIs as anticancer agents, we created COOH-terminal mutants of and guanine nucleotide dissociation inhibitors, which sequester Rac WT and activated forms of Rac1 and Rac3 that render them exclu- proteins away from their active site at the membrane. Oncogenic sively geranylgeranylated, farnesylated, or UN (no isoprenoid group forms of Rac proteins are constitutively GTP bound and active. is added). These mutants were used to characterize the sensitivity of Rac1 function is important in G cell cycle progression (4), in actin 1 Rac1 and Rac3 to GGTIs and FTIs in signaling, membrane ruffling, cytoskeleton organization through the formation of lamellipodia and and transformation. Our results suggest that, in contrast to what has membrane ruffles (5, 6) and has been shown to regulate downstream been reported for RhoA (27), Rac1 and Rac3 are sufficient to mediate gene expression through a variety of pathways including those involv- the inhibitory effects of GGTIs on transformation and membrane ing cyclin D1, E2F-1, nuclear factor ␬B, c-Jun, and SRF1 (7–12). ruffling and appear to be potential physiological targets for this class Rac1 has also been shown to be transforming in rodent fibroblast of drugs. models and is required for transformation induced by Ras (13, 14). Rac3 is less well characterized. Rac3 has 92% overall amino acid identity with Rac1, with the majority of the differences occurring in MATERIALS AND METHODS the COOH-terminal membrane targeting region and in regions sur- Prenylation Mutants of rac1 and rac3. Human WT rac1 and rac3 in the rounding and within the Rho insert domain (15). Rac3 has been vector pcDNA3.1ϩ were obtained from the Guthrie cDNA Resource (Sayre, PA). A Q61L mutation was introduced into Rac3 by the QuickChange Site- Received 6/6/03; revised 8/20/03; accepted 9/12/03. Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manu- Grant support: NIH Grants T32-CA71341 (to P. L. J.) and U19-CA67771 (to facturer’s instructions. All primers were generated by the Nucleic Acids Core A. D. C.). Facility at the University of North Carolina at Chapel Hill. The Rac1 Q61L The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with construct has been described previously (11). The CAAX motifs of human Rac1 18 U.S.C. Section 1734 solely to indicate this fact. and Rac3 in either WT or oncogenically activated versions (12V or 61L) were Requests for reprints: Adrienne D. Cox, Department of Radiation Oncology, CB# mutated by PCR by methods previously published for Ras family member 7512, University of North Carolina, Chapel Hill, North Carolina 27599-7512. Phone: proteins (28). Additionally, a G12V mutation was created within the 5Ј primer (919) 966-7713; Fax: (919) 966-7681; E-mail: [email protected]. Ј 1 The abbreviations used are: SRF, serum response factor; GGTase I, geranylgeranyl- for rac3. Primers at the 3 ends were designed to alter the CAAX motif of Rac1 transferase I; FTase, farnesyltransferase; FTI, farnesyltransferase inhibitor; GGTI, gera- from the parental CLLL (X ϭ L; GG) to CVLS (X ϭ S; F), and SLLL (C to nylgeranyltransferase inhibitor; WT, wild-type; GG, geranylgeranyl; F, farnesyl; UN, S; not prenylated, UN) and to alter the CAAX motif of Rac3 from the parental unprocessed; P, parental; HA, hemagglutinin; EGFP, enhanced green fluorescent protein; CTVF (X ϭ F; possibly GG or F) to CTVL (X ϭ L; GG), CTVS (X ϭ S; F), P/S, penicillin-streptomycin; PDGF, platelet-derived growth factor; JNK, c-Jun NH2- terminal kinase; %AUC, percentage of the area under the curve; PAK, p21-activated and STVF (C to S; UN). PCR products of rac1 and rac3 were digested with kinase. BamHI (all restriction enzymes; Invitrogen, Carlsbad, CA) at the sites intro- 7959

duced at the 5Ј and 3Ј ends and ligated in-frame with the HA epitope tag into Santa Cruz, CA) for 30 min at 4¡C. Beads were collected and washed, and the vector pCGN-hyg for expression in mammalian cells (28). PCR products protein samples were prepared as described previously (30). SDS-PAGE were also digested with BamHI EcoRI and ligated in-frame with an EGFP tag analysis and immunoblot for HA were performed as described above. The P:S into the mammalian vector pEGFP-C1 (Clontech, Palo Alto, CA), which was ratio was derived by dividing the %AUC generated for the P100 sample by the cut previously with BglII and EcoRI. Expression plasmids were thus generated %AUC generated for the S100 sample for each condition. The %AUC was in pCGN and in pEGFP-C1 for each of the following: -rac1(WT or 61L)-P determined with the program Molecular Analyst 2.1.2 (Bio-Rad). Dividing the (CAAX ϭ CLLL); -rac1(WT or 61L)-F (CVLS); -rac1(WT or 61L)-UN P:S ratio of the treated samples by the P:S ratio of the vehicle-treated cells and (SLLL); -rac3(WT, 12V or 61L)-P (CTVF); -rac3(WT, 12V or 61L)-GG subtracting from one [1 Ϫ (P:S treated/P:S untreated)] yields the percentage of (CTVL); -rac3(WT, 12V or 61L)-F (CTVS); and -rac3(WT, 12V or 61L)-UN protein that has been moved into the cytosol by drug treatment. (STVF). All mutations and ligation junctions were verified by forward and Reporter Gene Assays. For transient luciferase assays, NIH 3T3 cells in reverse sequencing. 35-mm, 6-well plates were cotransfected with 1 ␮g of pCGN-hyg vector, Cell Culture and Transfections. NIH 3T3 mouse fibroblasts were grown pCGN-rac1(61L) prenylation mutants [Rac1(61L)-P, -F, -UN], or 100 ng of in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with pCGN-rac3(61L) prenylation mutants [Rac3(61L)-P, -GG, -F, -UN] and 125 10% calf serum (Life Technologies, Inc.) and 1% P/S (complete medium) and ng of pJ-luc, a c-Jun luciferase reporter construct (a gift of Silvio Gutkind;

maintained in 10% CO2 at 37°C. Cells were plated the day before transfection NIH, Bethesda, MD). All transfections were performed in duplicate. Cells at 5 ϫ 105 cells/100-mm dish, 2.5 ϫ 105 cells/60-mm dish, or 1 ϫ 105 were placed in DMEM containing 0.5% calf serum containing either DMSO cells/35-mm 6-well plate. NIH 3T3 cells were transfected by calcium phos- vehicle or 1 ␮M GGTI-2166 immediately after glycerol shock and were grown phate coprecipitation for 3–5 h followed by glycerol shock for 3 min as for 20Ð24 h. The cells were then rinsed with 1ϫ PBS and lysed in 1ϫ lysis described previously (28) or with LipofectAMINE and Plus reagents (Invitro- buffer (Amersham Biosciences), and luciferase activity was measured with gen) following the manufacturer’s instructions. Stable cell lines were created enhanced chemiluminescence reagents (Amersham Biosciences) in a Mono- in NIH 3T3 cells after transfection with 200 ng of pCGN-hyg constructs light 2010 luminometer (Analytical Luminescence, San Diego, CA). expressing the Rac1 and Rac3 prenylation mutants. Two days after transfec- Transformation Assays. For focus formation assays, NIH 3T3 cells were tion, one-third of the cells were split into complete medium containing 200 plated in 60-mm dishes and cotransfected with 200 ng of either pZIP vector or ␮g/ml hygromycin B (Roche, Indianapolis, IN) in 100-mm dishes. Cells were pZIP-raf22W and 500 ng of pCGN-hyg vector, pCGN-rac1(61L), or pCGN- maintained in hygromycin B for 10–12 days, after which colonies were pooled rac3(12V) prenylation mutants [Rac1(61L)-P, -F, -UN; Rac3(12V)-P, -GG, -F, for use. To prevent loss of protein expression, stable cell lines were maintained -UN]. All transfections were performed in duplicate. Cells were grown in continuously in hygromycin B until they were split for experiments. complete medium containing no drugs or in complete medium containing Swiss 3T3 mouse fibroblasts, generously provided by Krister Wennerberg either DMSO vehicle or 5 ␮M GGTI-2166. Medium was replaced every other and Keith Burridge (University of North Carolina at Chapel Hill), were grown day. After 14Ð21 days, cells were photographed under the ϫ10 objective, in DMEM supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) washed with 1ϫ PBS, fixed with 3:1 (v/v) methanol:acetic acid, and stained

and 1% P/S and maintained in 10% CO2 at 37°C. Cells were plated as with 0.4% crystal violet in 20% ethanol. Stained foci were then counted for described above for NIH 3T3 cells. Transfections were carried out with quantitation of transforming activity. FuGENE 6 (Roche) according to the manufacturer’s instructions. For soft agar assays, NIH 3T3 cells stably expressing pCGN-hyg vector, Western Immunoblotting. Stable cell lines were plated onto 60-mm pCGN-rac1(61L), and pCGN-rac3(12V) prenylation mutants [Rac1(61L)-P, dishes and allowed to grow for 2 days. Cells were lysed in 300 ␮l of TX-100 -F, -UN; Rac3(12V)-P, -GG, -F, -UN] were prepared as described above. 5 lysis buffer [50 mM Tris (pH 7.5), 100 mM NaCl, 1% (v/v) Triton X-100, 5 Single cell suspensions (1 ϫ 10 cells/60-mm dish) of each stable cell line ␮g/ml aprotinin, 10 ␮M leupeptin, 20 nM ␤-glycerophosphate, 12 mM p- were plated in complete medium containing 0.4% agar and DMSO vehicle, 10 nitrophenylphosphate, and 0.1 mM sodium vanadate]. Lysates were cleared by ␮M FTI-2153, or 10 ␮M GGTI-2166 on top of a bottom layer of complete centrifugation at 12,000 rpm for 10 min at 4°C, and protein concentration was medium containing 0.6% agar. Colonies were allowed to form for 14Ð21 days, determined with a colorimetric assay (Bio-Rad, Hercules, CA). Samples were after which they were photographed under the ϫ4 objective. prepared in 5ϫ Laemmli sample buffer, and 20 ␮g of protein from each sample Localization Assays and Fluorescent Microscopy. NIH 3T3 cells were were run on 15% SDS-PAGE gels. Proteins were transferred at 100 V for 1 h plated on glass coverslips in 35-mm, 6-well plates. For visualization of to polyvinylidene difluoride (Immobilon-P; Millipore, Bedford, MA). Mem- subcellular localization and formation of lamellipodia and membrane ruffles, branes were blocked in 5% nonfat dry milk for1hatroom temperature and cells were transiently transfected with 1 ␮g of pEGFP-C1 vector, pEGFP- then incubated for1hineither 1:1,000 anti-HA antibody (Covance, Philadel- rac1(61L), or pEGFP-rac3(61L) prenylation mutants [Rac1(61L)-P, -F, -UN; phia, PA) or 1:5,000 anti-␤-actin (Sigma, St. Louis, MO) and then washed. Rac3(61L)-P, -GG, -F, -UN]. After glycerol shock, cells were placed in Membranes were incubated for1hin1:30,000 antimouse IgG-horseradish complete medium containing DMSO vehicle, 10 ␮M FTI-2153, or 10 ␮M peroxidase antibody (Amersham Biosciences, Arlington Heights, IL), washed GGTI-2166. After 48 h, live cells were visualized with a fluorescent micro- extensively, and developed with SuperSignal West Dura Extended Duration scope (Axioskop; Zeiss, Thornwood, NY), and images were captured under the substrate (Pierce, Rockford, IL). ϫ20 objective with the MetaMorph digital imaging software (Universal Im- Cell Fractionation and Immunoprecipitation. NIH 3T3 cells in 100-mm aging Corp., Downington, PA). dishes were transfected with 3 ␮g of pCGN-rac1(61L), -rac3(61L), or Swiss 3T3 cells were plated on glass coverslips in 35-mm, 6-well plates. -rac3(61L)-GG with LipofectAMINE and Plus reagent (Invitrogen). Three h Before transfection with FuGENE 6, cells were placed in medium (DMEM after transfection, the medium was replaced with medium containing DMSO, ϩ10% fetal bovine serum, 1% P/S) with DMSO vehicle, 10 ␮M FTI-2153, or ␮ ␮ GGTI-2166 (1, 5, or 10 ␮M), or FTI-2153 (10 ␮M) and grown for 48 h. GGTI- 10 M GGTI-2166. Cells were transfected with 1 g of pEGFP-C1 vector, 2166 and FTI-2153 are both gifts from Saõ¬d M. Sebti (University of South Florida, pEGFP-rac1(WT), or pEGFP-rac3(WT) prenylation mutants [Rac1(WT)-P, Tampa, FL) and Andrew D. Hamilton (Yale University, New Haven, CT; Ref. 29). -F, -UN, Rac3(WT)-P, -GG, -F, -UN]. After 24 h, the cells were placed in serum-free medium containing DMSO vehicle, FTI, or GGTI at the above The in vitro IC50s for GGTI-2166 for GGTase I and FTase are 21 and 5600 nM, ␮ concentrations. After an additional 24 h, cells were treated with either vehicle respectively. The IC50 for GGTI-2166 for Rap1 processing in cells is 0.3 M, and Ͼ ␮ (4 mM HCl and 0.1% BSA) or 20 ng/ml PDGF (BB homodimer; Sigma). After for Ras, it is 30 M. The in vitro IC50s for FTI-2153 for GGTase I and FTase are 1700 and 1.4 nM, respectively. The IC for FTI-2153 for Rap1 processing in cells 30 min of treatment, live cells were visualized with a fluorescent microscope, 50 ϫ is Ͼ30 ␮M, and for Ras, it is 0.01 ␮M (29). and images were captured under the 20 objective. Cells were harvested in PBS, resuspended in hypotonic buffer, and disrupted with a homogenizer as described previously (30). Disrupted cells were fractionated into S100 and P100 fractions by centrifugation at 100,000 ϫ g.A RESULTS 450-␮l sample was removed to represent the total protein before fractionation. The total protein, S100, and P100 samples were all immunoprecipitated with Both Geranylgeranylated and Farnesylated Forms of Rac1 and anti-HA antibody (Covance) at 1:150 for 1 h at 4¡C, followed by the addition Rac3 Are Expressed Equivalently in NIH 3T3 Cells. To validate of 20 ␮l of protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, the use of the prenylation mutants in the experiments shown, it was 7960

first necessary to determine their expression levels. Constructs were (P100) and increase within the cytosolic fraction (S100), as shown by made to express Rac1 with a parental CAAX motif, which should be the decrease in the P:S ratio from DMSO-treated samples to those geranylgeranylated, or a mutated CAAX motif that made it either F or treated with increasing doses of GGTI. Loss of protein from the UN. Due to the possibility that the parental CAAX motif of Rac3, membrane fraction indicates loss of prenylation because prenylation is CTVF, could be a target for either GGTase I or FTase (26), constructs necessary to anchor Rho family proteins in the membrane (18). were made to express Rac3 with the parental CAAX motif or a mutated Comparison of the P:S ratio of GGTI-treated samples with the P:S CAAX that made it exclusively GG, exclusively F, or UN. As shown ratio of the DMSO-treated samples shows that for Rac1, 67%, 87%, in Fig. 1A, all prenylated mutants were expressed in stable cell lines and 97% of the protein was moved to the cytosolic fraction after at levels approximately equivalent to that of the Rac proteins with the treatment with 1, 5, and 10 ␮M GGTI, respectively. For Rac3, 69%, parental CAAX motifs. Interestingly, Rac3-UN was expressed at sig- 75%, and 83% was moved at 1, 5, and 10 ␮M GGTI, respectively, and nificantly higher levels than any other Rac3 protein (Fig. 1A). This for Rac3-GG, 87%, 78%, and 89% was moved at 1, 5, and 10 ␮M held true in at least six independently isolated cell lines. Although the GGTI, respectively. Conversely, with 10 ␮M FTI treatment, the basis for the differential expression is not clear, expression of amount of Rac1, Rac3, or Rac3-GG protein in the membrane (P100) Rac3-UN appeared to enhance cell viability, as reflected in a much fraction appears similar to the amount with DMSO treatment, indi- larger number of colonies obtained from antibiotic selection for cating that prenylation of geranylgeranylated forms of Rac proteins is Rac3-UN compared with any other Rac3 protein (data not shown). not sensitive to the inhibition of FTase by FTIs. When the percentages Geranylgeranylated Forms of Rac1 and Rac3 Show Dose-De- are compared, more protein was moved from the membrane to the pendent Sensitivity to GGTIs. To determine whether geranylgera- cytosol for Rac1 than either Rac3 or Rac3-GG at the highest dose of nylated forms of Rac1 and Rac3 are sensitive to GGTIs, cells tran- GGTI, but there was also more protein in the P100 fraction compared siently expressing activated Rac1, Rac3, or Rac3-GG were treated with the S100 fraction for Rac1 than either Rac3 or Rac3-GG. This with increasing doses of GGTI or with FTI. Cells were crudely suggests there may be some differences between Rac1 and Rac3 in fractionated into cytosolic S100 and membrane-containing P100 frac- partitioning between the membrane and cytosol and/or in general tions. Fig. 1B shows that with increasing doses of GGTI, Rac1, Rac3, sensitivity to GGTI treatment. and Rac3-GG proteins all decrease within the membrane fraction Both Geranylgeranylated and Farnesylated Forms of Rac1 and Rac3 Signal to c-Jun, but Only Signaling from the Geranylgera- nylated Rac Proteins Is Sensitive to GGTIs. Activated Rac3 has been reported to activate JNK kinase activity (15, 17), suggesting that it should also be able to activate the downstream target of JNK, c-Jun. We evaluated the ability of the prenylation mutants of activated Rac1 and Rac3 to signal to the c-Jun pathway in the presence or absence of GGTIs by using a c-Jun luciferase reporter assay. Fig. 2 shows that, as expected, both activated Rac1 and Rac3 can signal robustly to c-Jun, and both prenyl groups (GG or F) support signaling activity. To determine whether the ability of Rac1 and Rac3 to transcrip- tionally transactivate c-Jun is sensitive to GGTI-mediated inhibition, we performed the reporter assays in the presence of 1 ␮M GGTI. Decreases in activation by Rac1, Rac3, and Rac3-GG (Fig. 2, A and B) demonstrate that all three are sensitive to GGTI. From Fig. 1B,we determined that 67% of Rac1, 69% of Rac3, and 87% of Rac3-GG has been moved from the membrane fraction at the dose of GGTI used, which may account for incomplete inhibition of c-Jun transactivation. Rac3 with the parental CAAX motif was just as sensitive to GGTI as Rac3 with a CAAX motif that should only be geranylgeranylated (Rac3-GG). This argues against the notion that the CTVF CAAX motif of Rac3 is normally a target for both GGTase I and FTase and suggests that the majority of Rac3 is indeed geranylgeranylated. The inhibition of the signaling activity of geranylgeranylated Rac1 and Rac3 suggests that they could be targets of GGTI activity. In contrast, the signaling activity of farnesylated Rac1 and Rac3 is not sensitive to GGTI. Farnesylated and Geranylgeranylated Forms of Rac1 and Rac3 Fig. 1. Expression of geranylgeranylated and farnesylated forms of activated Rac1 and Rac3 and dose-dependent sensitivity of geranylgeranylated forms Rac1 and Rac3 to Are Both Morphologically Transforming. The similarity of Rac3 to GGTIs. A, NIH 3T3 cells were stably transfected with pCGN-hyg constructs containing Rac1 suggests that it has oncogenic potential, yet the transforming activated mutants of rac1 or rac3 genes in-frame with an NH2-terminal HA tag. The expression levels of Rac1 and Rac3 with parental CAAX motifs (CLLL and CTVF, activity of Rac3 has not been demonstrated. It has been shown that respectively) or with CAAX motifs that had been mutated to give the indicated prenylation activated Rac1 cooperates with activated Raf in focus formation mutants were compared by Western immunoblot with an antibody to the HA tag. The assays (13, 14). To determine whether Rac3 could also cooperate with same blot was also probed for ␤-actin to show equal protein loading. The results shown are representative of at least six independently created sets of stable lines. B, NIH 3T3 Raf to form foci, we cotransfected plasmids encoding activated Rac1 cells were transiently transfected with pCGN-rac1(61L), -rac3(61L), or -rac3(61L)-GG or Rac3 with a truncated and activated version of Raf into NIH 3T3 ␮ and allowed to grow for 48 h in the presence of DMSO; 1, 5, or 10 M GGTI-2166; or cells. We also cotransfected prenylation mutants of activated Rac1 or 10 ␮M FTI-2153. Cells were crudely fractionated into cytosolic S100 (S) or membrane- containing P100 (P) fractions by centrifugation at 100,000 ϫ g. The total (T) lane Rac3 with active Raf to determine whether the prenylation mutants represents the total sample before fractionation. Fractionated samples were concentrated could cooperate with Raf to support focus formation. by immunoprecipitation with an anti-HA antibody, and samples were compared by Western immunoblot with the same anti-HA antibody. P:S ratios were determined from Consistent with its ability to activate c-Jun transcriptional trans- the intensity of the bands for each condition. activation like Rac1, Rac3 was also able to cooperate with Raf to 7961

Farnesylated Rac Rescues Cells from GGTI-Mediated Inhibi- tion of Anchorage-Independent Growth. If Rac proteins are physiologically relevant targets of GGTI, then a GGTI-resistant form of Rac should rescue GGTI-mediated inhibition of transformation. Due to the unexpected finding that GGTI inhibited the focus formation of Raf (Fig. 3C), we were unable to use the cooperative focus formation assay to answer this question. Instead, we turned to colony formation in soft agar, a transformed phenotype for which oncogenic Rac1 protein does not require coexpression of Raf (13). Therefore, we determined whether activated Rac3 alone could promote anchorage- independent growth in a soft agar colony-forming assay. As shown in Fig. 4, NIH 3T3 cells stably transfected with pCGN-hyg vector were not able to form colonies when suspended in soft agar. However, cells stably expressing activated Rac3 formed many colonies in soft agar, demonstrating that activated Rac3 alone could promote anchorage- independent growth. To confirm that GGTI could disrupt soft agar colony formation by Rac3-expressing cells, we seeded NIH 3T3 cells expressing activated Rac3 and Rac3-GG onto soft agar in the presence of DMSO vehicle or 10 ␮M GGTI, a dose in which 97% of Rac1 and 83% and 89% of Rac3 and Rac3-GG, respectively, have been moved out of the membrane fraction as shown in Fig. 1B. As expected, GGTI totally ablated the ability of Rac3 and Rac3-GG to form colonies, whereas FTI did not (Fig. 4). This confirmed that Rac3-transforming activity is sensitive to GGTI but resistant to FTI and that Rac3 could be targeted by GGTI. If Rac3 is a physiologically relevant target of GGTI, then a GGTI- resistant form of Rac3 should rescue GGTI-mediated inhibition of transformation. Therefore, we seeded NIH 3T3 cells stably expressing activated Rac3-F onto soft agar in the presence of DMSO, GGTI, or FTI. Overall, colony formation by Rac3-F was lower than that of Rac3-WT or Rac3-GG, but Rac3-F was able to overcome growth inhibition by GGTI to form colonies, whereas it formed no colonies in the presence of FTI (Fig. 4). Similar results were seen with activated Rac1 (data not shown). Farnesylated Rac Rescues Cells from GGTI-Mediated Inhibi- tion of Cell Spreading and Ruffling. To determine whether the Fig. 2. GGTI inhibits signaling to c-Jun from geranylgeranylated Rac proteins. NIH membrane localization and membrane ruffling activity of Rac1 and 3T3 fibroblasts were transiently cotransfected with a c-Jun luciferase reporter construct and pCGN-hyg constructs containing either activated mutants rac1 with the parental Rac3 could be targeted by GGTIs, we transiently transfected activated CAAX motif or the indicated prenylation mutations (A) or activated mutants of rac3 with versions of parental Rac1 and Rac3 and their prenylation mutants the parental CAAX motif or the indicated prenylation mutations (B). Immediately after transfection, cells were placed in low-serum medium containing either DMSO vehicle or expressed from the vector pEGFP-C1 into NIH 3T3 cells. As can be GGTI-2166 for 24 h and then lysed for analysis of luciferase activity. Luciferase activity seen in Fig. 5, in the presence of vehicle control, activated parental from the c-Jun reporter is expressed as fold activation over the pCGN-hyg control and is Rac1 and Rac3 and their prenylation mutants showed significant shown as mean Ϯ SE. Results are representative of four independent experiments. membrane ruffling and localization to the areas of ruffling with clear nuclear exclusion. However, in the presence of GGTI, parental Rac1 form foci (Fig. 3A). Rac1 and Rac3 foci were easily distinguished and Rac3 and the Rac3-GG mutant showed dramatically decreased from Raf foci due to the presence of enlarged, rounded refractile ruffling activity and accumulation of Rac protein in the nucleus. cells that were not found in the tightly arrayed refractile cells in Farnesylated versions of Rac1 and Rac3 were still able to ruffle in the Raf foci (Fig. 3B). The prenylation mutants of activated Rac1 and presence of GGTI and had nuclear exclusion similar to vehicle con- Rac3 were able to form numbers of foci relatively similar to their trol. However, farnesylated Rac1 and Rac3 were now sensitive to FTI, parental counterparts in cooperation with Raf, with morphology with decreased ruffling and increased accumulation in the nucleus. that was indistinguishable from that of the parental Rac1 and Rac3 Activated Rac1- and Rac3-UN showed no ruffling activity and had (Fig. 3A; data not shown). Activated Rac1- and Rac3-UN were diffuse localization throughout the cytoplasm and nucleus, much like unable to form foci to any significant degree, with numbers equiv- pEGFP-C1 vector (Fig. 5; data not shown). alent only to that of the Raf-only control (Fig. 3A). We were unable WT Rac3-F Rescues PDGF-Mediated Ruffling from GGTI In- to test the ability of Rac1 and Rac3 to form foci in cooperation with hibition. Because mutationally activated versions of Rac proteins Raf in the presence of GGTIs due to the unexpected result that the have not been identified in human tumors, it is thought that overex- GGTIs inhibited the formation of Raf foci, leading to nonspecific pression of WT forms or their hyperactivation may account for the decreases in foci formed for Raf with or without coexpression of contribution of Rac proteins to oncogenesis (31, 32). To determine activated Rac1 or Rac1-F, thus confounding interpretation of the whether Rac proteins are physiologically relevant targets for GGTIs, results (Fig. 3C). However, this may suggest that another target of we used WT versions of Rac3 with parental and mutated prenylation GGTI, perhaps another geranylgeranylated protein, plays an im- sites in pEGFP-C1 to transfect Swiss 3T3 cells. Cells treated with portant role in Raf transformation. DMSO, FTI, or GGTI were also treated with or without PDGF, a 7962

Fig. 3. Rac3 and prenylation mutants of both Rac1 and Rac3 are able to cooperate with Raf to transform NIH 3T3 cells in a focus formation assay. NIH 3T3 cells were transiently cotransfected with either pZIP vector only or pZIP containing an activated mutant of raf, and either pCGN-hyg vector or pCGN-hyg constructs containing activated mutants of rac1 and rac3 with either the parental CAAX motif or motifs that were mutated to give the indicated prenylation mutants. Cells were fed with medium either containing no drugs (A) or with DMSO vehicle and 5 ␮M GGTI-2153 (C). A, after approximately 21 days of growth, plates were stained with crystal violet and foci were quantitated. Results are expressed as mean Ϯ SD from duplicate plates. B, before staining, representative foci were photographed under a ϫ10 objective. C, crystal violet-stained plates demonstrate the negative effect of GGTI on Raf focus formation. Quantitation and photographs are representative of two to three independent experiments. known activator of Rac1, to determine whether subsequent mem- to flattened cells and an increase in nuclear localization. Ruffling still brane-ruffling activity was still subject to inhibition by GGTIs. As occurred in the presence of FTI in these cells. Cells transfected with seen in Fig. 6, PDGF induces membrane ruffling and nuclear exclu- farnesylated Rac3 were resistant to the inhibitory action of GGTI and sion for WT Rac3 with either a parental (Rac3-P) or mutated preny- had nuclear accumulation and the prevention of membrane ruffling lation site (Rac3-GG and Rac3-F). The ability to induce ruffling is only in the presence of FTI. Similar results were seen with WT Rac1 inhibited for Rac3-P and Rac3-GG in the presence of GGTI, leading constructs (data not shown). These results indicate that both Rac1 and 7963

Fig. 4. Farnesylated Rac3 rescues cells from GGTI-mediated inhibition of anchorage-independent growth. NIH 3T3 cells were seeded into soft agar stably expressing pCGN-hyg vector or pCGN-hyg constructs containing activated mutants of rac3 with either the parental CAAX motif or motifs mutated to express exclusively GG- or F-modified proteins. Colonies were allowed to form for 18Ð21 days in the presence of DMSO vehicle, FTI, or GGTI, after which they were photographed under a ϫ4 objective. Results are representative of three independent experiments with independently isolated stable cell lines.

Rac3 can mediate PDGF responsiveness and that GGTI-resistant Rac gence of Rac1 and Rac3 in evolution (36) suggests that functional proteins can overcome GGTI inhibition of a physiologically important distinctions must exist. Amino acids in and around the Rho insert function of WT Rac proteins. region of Rac1 and other Rho family members are known to contribute to effector binding (8, 37Ð39). Possible distinctions between Rac1 DISCUSSION and Rac3 may also lie in the COOH-terminal hypervariable domain, a region that dictates isoprenoid modification and is important for Rac1 proteins are involved in membrane ruffling, morphological membrane localization of small GTPases and for biological activity and growth transformation, and signaling to a variety of downstream (18, 40, 41). ␬ transcription factors, including c-Jun, SRF, and nuclear factor B Rac1 is known to be modified by a GG isoprenoid lipid (25). The (1, 33, 34). Considerably less is known about the consequences of CAAX motif of Rac3, CTVF, with F in the X position, suggests that it Rac3 activation. Rac3 has been reported to activate JNK (15) and could be a potential target for either GGTase I or FTase (26). Our PAK (17) and to up-regulate DNA synthesis in a PAK-dependent, results demonstrate that Rac3 is likely to be mostly geranylgerany- JNK-independent manner (17). Rac3 GTP levels have been reported lated in cells. Individual small GTPases may differ in their require- to be elevated in breast cancer cell lines and primary tumor tissues ment for modification by a specific isoprenoid moiety for function. (17). These results, along with the sequence similarity of Rac3 and For example, WT H-Ras is growth inhibitory only when modified by Rac1, suggest that Rac3 could also have transforming activity. In a GG group instead of its native F group (42), but the biological support of this idea, we show here that constitutively activated mutant forms of Rac3 cause both focus formation and growth in soft agar in activity of activated farnesylated RhoA is indistinguishable from that NIH 3T3 cells, in a manner very similar to that of Rac1. Additionally, of the authentically geranylgeranylated RhoA (27). We have shown we show here that both WT Rac1 and Rac3 induce membrane ruffling here that both oncogenic and WT Rac1 and Rac3 appear to be tolerant in response to PDGF. Thus, Rac3 is functionally similar to Rac1 in of modification by either a GG group or F group for transformation terms of responsiveness to growth factor stimulation and transforming and membrane-ruffling activities. Thus, the consequences of alternate ability. lipid modification of Rac1 and Rac3 are more similar to what has been Our data from the experiments outlined above suggest that Rac1 shown for activated RhoA than for WT H-Ras, suggesting that far- and Rac3 function similarly. Although there is substantial sequence nesylated Rac proteins are useful tools to investigate whether Rac1 identity in the classical effector domain region of these highly related and Rac3 are physiological targets for inhibition of GGTase I by proteins, other important elements of sequence divergence exist be- GGTIs, the basis for a novel anticancer therapy. tween Rac1 and Rac3 (15). For example, differences exist in and FTase, the enzyme that attaches the F group to Ras, RhoB, and a around the Rho insert domain, a sequence that is unique to Rho family subset of other small GTPases, has long been a target for rational drug proteins (35), and in the hypervariable region at the COOH terminus design (19, 20, 43Ð45). FTIs are in Phase IÐIII clinical trials for that could dictate functional distinctions. Furthermore, the early emer- anticancer treatment, although the identity of the most critical targets 7964

Fig. 5. Farnesylated Rac rescues cells from GGTI-mediated inhibition of cell spreading and ruffling. NIH 3T3 cells plated on coverslips were transiently transfected with pEGFP-C1 constructs containing either activated mutants of rac1 with a parental CAAX motif or with motifs mutated to express F-modified or UN Rac1 (A) or activated mutants of rac3 with a parental CAAX motif or with motifs mutated to express GG- or F-modified or UN Rac3 (B). Immediately after transfection, cells were placed in medium containing DMSO vehicle, FTI, or GGTI and allowed to express protein for 48 h. Live cells expressing green fluorescent protein-tagged Rac1 and Rac3 proteins were viewed with a fluorescent microscope and photographed under a ϫ20 objective. Photographs shown are representative of four experiments. that can explain FTI antitumor activity are still under investigation. with the development of new inhibitors to block geranylgeranylation GGTase I modifies many proteins in the Rho family of small (19, 20). GGTIs have been shown to arrest human tumor cell growth GTPases, including Rac1 and Rac3, by attaching a GG group. in vitro (21, 22) and to reduce tumor growth in animal models (23), GGTase I has also recently become a target for rational drug design, yet the physiologically relevant downstream targets of GGTIs have 7965

Fig. 6. WT Rac3-F rescues PDGF-mediated ruffling from GGTI inhibition. Swiss 3T3 cells plated on coverslips were transiently transfected with pEGFP-C1 constructs containing WT rac3 with a parental CAAX motif (A), WT rac3 with a CAAX motif mutated to express GG-modified protein (B), or WT rac3 with a CAAX motif mutated to express F-modified protein (C). After transfection, cells were grown continually in the presence of DMSO vehicle, FTI, or GGTI for 24 h in complete medium and for an additional 24 h in serum-free medium. Cells were then treated for 20 min with vehicle or with 20 ng/ml PDGF, viewed with a fluorescent microscope, and photographed under a ϫ20 objective. Results are representative of two independent experiments.

not been determined. Candidate downstream targets include the gera- native geranylgeranylated RhoA, is unable to restore RhoA activity in nylgeranylated members of Rho family GTPases. the presence of GGTI (27). These results suggest that although RhoA Inhibition of RhoA by GGTI led to an increase in p21waf1/cip1 may be an important and necessary target of GGTIs, it may not be expression, which is normally repressed by RhoA (46). This may help sufficient to mediate the inhibitory action of GGTIs. to mediate the G1 arrest that is seen with GGTI treatment (21, 22). We used the GGTI-insensitive, farnesylated versions of Rac1 and However, farnesylated RhoA, although functionally equivalent to the Rac3 to determine whether Rac proteins are biologically important 7966

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. Rac1 AND Rac3 ARE TARGETS OF GGTIS downstream targets for GGTIs. If a GGTI-insensitive form of Rac can 18. Cox, A. D., and Der, C. J. Protein prenylation: more than just glue? Curr. Opin. Cell rescue cells from GGTI-mediated growth inhibition, then Rac is likely Biol., 4: 1008Ð1016, 1992. 19. Sebti, S. M., and Hamilton, A. D. Farnesyltransferase and geranylgeranyltransferase to be either an important mediator of that inhibition or downstream of I inhibitors in cancer therapy: important mechanistic and bench to bedside issues. a critical GGTase I target. Therefore, we investigated the ability of Expert Opin. Investig. Drugs, 9: 2767Ð2782, 2000. farnesylated forms of oncogenic Rac1 and Rac3, which we demon- 20. Sebti, S. M., and Hamilton, A. D. Farnesyltransferase and geranylgeranyltransferase I inhibitors and cancer therapy: lessons from mechanism and bench-to-bedside strated to be GGTI insensitive, to rescue cells from GGTI-mediated translational studies. Oncogene, 19: 6584Ð6593, 2000. inhibition of anchorage-independent growth and from inhibition of 21. Vogt, A., Qian, Y., McGuire, T. F., Hamilton, A. D., and Sebti, S. M. Protein membrane-ruffling activity. These results suggested that Rac proteins geranylgeranylation, not farnesylation, is required for the G1 to S phase transition in mouse fibroblasts. Oncogene, 13: 1991Ð1999, 1996. may play an important role in the cellular response to GGTIs. How- 22. Vogt, A., Sun, J., Qian, Y., Hamilton, A. D., and Sebti, S. M. The geranylgeranyl- ever, oncogenically mutated forms of Rac and Rho proteins have not transferase-I inhibitor GGTI-298 arrests human tumor cells in G0/G1 and induces been found in human cancer cells; instead, it is thought that amplifi- p21(WAF1/CIP1/SDI1) in a p53-independent manner. J. Biol. Chem., 272: 27224Ð 27229, 1997. cation of Rho family proteins or activation of their upstream regula- 23. Sun, J., Qian, Y., Hamilton, A. D., and Sebti, S. M. Both farnesyltransferase and tors such as exchange factors contribute to the ability of these geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-Ras GTPases to influence the transformed phenotype (31, 32). Therefore, prenylation but each alone is sufficient to suppress human tumor growth in nude mouse xenografts. Oncogene, 16: 1467Ð1473, 1998. we also investigated whether a WT version of farnesylated Rac was 24. Didsbury, J., Weber, R. F., Bokoch, G. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. Rac1 and Rac3 Are Targets for Geranylgeranyltransferase I Inhibitor-Mediated Inhibition of Signaling, Transformation, and Membrane Ruffling

Patricia L. Joyce and Adrienne D. Cox

Cancer Res 2003;63:7959-7967.

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