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[Cancer Research 63, 7959-7967, November 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rac1, a Rho family GTPase, is a mediator of diverse cellular functions including membrane ruffling, cell cycle progression, and transformation. Rac3, a close relative of Rac1, is less well characterized. Posttranslational addition of geranylgeranyl isoprenoid lipids to Rac proteins is required for biological activity. Inhibitors of geranylgeranyl transferase I (GGTIs) are currently under investigation as a possible anticancer therapy, although the targets of GGTIs have not been determined. We created COOH-terminal mutants of Rac1 and Rac3 that are farnesylated and used them to characterize Rac1 and Rac3 as physiological targets of GGTIs. We show that, like Rac1, activated Rac3 causes transformation and leads to membrane ruffling. Farnesylated versions of Rac1 and Rac3 retain the ability to signal to the transcription factor c-Jun and cause membrane ruffling and transformation, indicating that switching isoprenoid modification does not alter function. Finally, treatment with GGTIs led to the inhibition of membrane-ruffling and transforming activities of both activated and wild-type Rac1 and Rac3. However, the farnesylated versions of both activated and wild-type Rac1 and Rac3 were resistant to the inhibitory effects of GGTIs. These results illustrate that Rac1 and Rac3 are potential physiological targets for these novel drugs.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The small GTPases Rac1 and Rac3 are members of the Rho family. Like Ras proteins, Rho family proteins cycle between active GTP-bound and inactive GDP-bound states (1, 2, 3) . They are positively regulated by Dbl family guanine nucleotide exchange factors, which promote the GTP-bound state. Negative regulation is achieved by GTPase-activating proteins, which enhance the hydrolysis of GTP, and guanine nucleotide dissociation inhibitors, which sequester Rac proteins away from their active site at the membrane. Oncogenic forms of Rac proteins are constitutively GTP bound and active.

Rac1 function is important in G1 cell cycle progression (4) , in actin cytoskeleton organization through the formation of lamellipodia and membrane ruffles (5 , 6) and has been shown to regulate downstream gene expression through a variety of pathways including those involving cyclin D1, E2F-1, nuclear factor {kappa}B, c-Jun, and SRF1 (7, 8, 9, 10, 11, 12) . Rac1 has also been shown to be transforming in rodent fibroblast 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 the COOH-terminal membrane targeting region and in regions surrounding and within the Rho insert domain (15) . Rac3 has been mapped to chromosome band 17q25.3 near a region that is commonly deleted in breast and ovarian cancers, suggesting possible transcriptional dysregulation in these diseases (15 , 16) . Additionally, Rac3 but not Rac1 was shown to be hyperactivated in breast cancer cells, and inhibition of Rac3 was shown to impair breast cancer cell proliferation (17) . These results suggest that Rac proteins may be attractive targets for anticancer drugs.

Rac/Rho proteins and Ras proteins are both modified posttranslationally by isoprenoid lipids. Addition of isoprenoid groups is required for proper localization and function of Rho/Ras family proteins (18) . Rac and Rho are modified by GGTase I, whereas Ras proteins are modified by FTase (18) . FTIs are in clinical trials as potential anticancer drugs, whereas GGTIs are still in preclinical development (19 , 20) . Although GGTIs have been shown to arrest human tumor cell growth in vitro (21 , 22) and to reduce tumor growth in animal models (23) , the physiologically relevant downstream targets of GGTIs have not been determined. Most Rho family proteins, including the Rac GTPases, are substrates for GGTase I and are logical targets for GGTIs (18) . GG isoprenoids are added to cysteine residues at the COOH terminus of proteins whose CAAX motifs terminate in leucine (X = L). The Rac1 CAAX motif is CLLL, and Rac1 is a known substrate for GGTase I (24 , 25) . Interestingly, the CAAX motif of Rac3 is CTVF, which suggests that Rac3 may be a substrate for both GGTase I and FTase (26) .

Indications that Rac3 is specifically hyperactivated and required for proliferation in breast cancer cells, as well as its sequence similarity to Rac1, suggest that Rac3 has oncogenic potential. To address the issue of whether Rac1 and Rac3 are physiologically important targets of GGTIs as anticancer agents, we created COOH-terminal mutants of WT and activated forms of Rac1 and Rac3 that render them exclusively geranylgeranylated, farnesylated, or UN (no isoprenoid group is added). These mutants were used to characterize the sensitivity of Rac1 and Rac3 to GGTIs and FTIs in signaling, membrane ruffling, and transformation. Our results suggest that, in contrast to what has been reported for RhoA (27) , Rac1 and Rac3 are sufficient to mediate the inhibitory effects of GGTIs on transformation and membrane ruffling and appear to be potential physiological targets for this class of drugs.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Prenylation Mutants of rac1 and rac3.
Human WT rac1 and rac3 in the vector pcDNA3.1+ were obtained from the Guthrie cDNA Resource (Sayre, PA). A Q61L mutation was introduced into Rac3 by the QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. All primers were generated by the Nucleic Acids Core Facility at the University of North Carolina at Chapel Hill. The Rac1 Q61L construct has been described previously (11) . The CAAX motifs of human Rac1 and Rac3 in either WT or oncogenically activated versions (12V or 61L) were mutated by PCR by methods previously published for Ras family member proteins (28) . Additionally, a G12V mutation was created within the 5' primer for rac3. Primers at the 3' ends were designed to alter the CAAX motif of Rac1 from the parental CLLL (X = L; GG) to CVLS (X = S; F), and SLLL (C to S; not prenylated, UN) and to alter the CAAX motif of Rac3 from the parental CTVF (X = F; possibly GG or F) to CTVL (X = L; GG), CTVS (X = S; F), and STVF (C to S; UN). PCR products of rac1 and rac3 were digested with BamHI (all restriction enzymes; Invitrogen, Carlsbad, CA) at the sites introduced at the 5' and 3' ends and ligated in-frame with the HA epitope tag into the vector pCGN-hyg for expression in mammalian cells (28) . PCR products were also digested with BamHI EcoRI and ligated in-frame with an EGFP tag into the mammalian vector pEGFP-C1 (Clontech, Palo Alto, CA), which was cut previously with BglII and EcoRI. Expression plasmids were thus generated in pCGN and in pEGFP-C1 for each of the following: -rac1(WT or 61L)-P (CAAX = CLLL); -rac1(WT or 61L)-F (CVLS); -rac1(WT or 61L)-UN (SLLL); -rac3(WT, 12V or 61L)-P (CTVF); -rac3(WT, 12V or 61L)-GG (CTVL); -rac3(WT, 12V or 61L)-F (CTVS); and -rac3(WT, 12V or 61L)-UN (STVF). All mutations and ligation junctions were verified by forward and reverse sequencing.

Cell Culture and Transfections.
NIH 3T3 mouse fibroblasts were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% calf serum (Life Technologies, Inc.) and 1% P/S (complete medium) and maintained in 10% CO2 at 37°C. Cells were plated the day before transfection at 5 x 105 cells/100-mm dish, 2.5 x 105 cells/60-mm dish, or 1 x 105 cells/35-mm 6-well plate. NIH 3T3 cells were transfected by calcium phosphate coprecipitation for 3–5 h followed by glycerol shock for 3 min as described previously (28) or with LipofectAMINE and Plus reagents (Invitrogen) following the manufacturer’s instructions. Stable cell lines were created in NIH 3T3 cells after transfection with 200 ng of pCGN-hyg constructs expressing the Rac1 and Rac3 prenylation mutants. Two days after transfection, one-third of the cells were split into complete medium containing 200 µg/ml hygromycin B (Roche, Indianapolis, IN) in 100-mm dishes. Cells were maintained in hygromycin B for 10–12 days, after which colonies were pooled for use. To prevent loss of protein expression, stable cell lines were maintained continuously in hygromycin B until they were split for experiments.

Swiss 3T3 mouse fibroblasts, generously provided by Krister Wennerberg and Keith Burridge (University of North Carolina at Chapel Hill), were grown in DMEM supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and 1% P/S and maintained in 10% CO2 at 37°C. Cells were plated as described above for NIH 3T3 cells. Transfections were carried out with FuGENE 6 (Roche) according to the manufacturer’s instructions.

Western Immunoblotting.
Stable cell lines were plated onto 60-mm dishes and allowed to grow for 2 days. Cells were lysed in 300 µl of TX-100 lysis buffer [50 mM Tris (pH 7.5), 100 mM NaCl, 1% (v/v) Triton X-100, 5 µg/ml aprotinin, 10 µM leupeptin, 20 nM ß-glycerophosphate, 12 mM p-nitrophenylphosphate, and 0.1 mM sodium vanadate]. Lysates were cleared by centrifugation at 12,000 rpm for 10 min at 4°C, and protein concentration was determined with a colorimetric assay (Bio-Rad, Hercules, CA). Samples were prepared in 5x Laemmli sample buffer, and 20 µg of protein from each sample were run on 15% SDS-PAGE gels. Proteins were transferred at 100 V for 1 h to polyvinylidene difluoride (Immobilon-P; Millipore, Bedford, MA). Membranes were blocked in 5% nonfat dry milk for 1 h at room temperature and then incubated for 1 h in either 1:1,000 anti-HA antibody (Covance, Philadelphia, PA) or 1:5,000 anti-ß-actin (Sigma, St. Louis, MO) and then washed. Membranes were incubated for 1 h in 1:30,000 antimouse IgG-horseradish peroxidase antibody (Amersham Biosciences, Arlington Heights, IL), washed extensively, and developed with SuperSignal West Dura Extended Duration substrate (Pierce, Rockford, IL).

Cell Fractionation and Immunoprecipitation.
NIH 3T3 cells in 100-mm dishes were transfected with 3 µg of pCGN-rac1(61L), -rac3(61L), or -rac3(61L)-GG with LipofectAMINE and Plus reagent (Invitrogen). Three h after transfection, the medium was replaced with medium containing DMSO, GGTI-2166 (1, 5, or 10 µM), or FTI-2153 (10 µM) and grown for 48 h. GGTI-2166 and FTI-2153 are both gifts from Saïd M. Sebti (University of South Florida, Tampa, FL) and Andrew D. Hamilton (Yale University, New Haven, CT; Ref. 29 ). The in vitro IC50s for GGTI-2166 for GGTase I and FTase are 21 and 5600 nM, respectively. The IC50 for GGTI-2166 for Rap1 processing in cells is 0.3 µM, and for Ras, it is >30 µM. The in vitro IC50s for FTI-2153 for GGTase I and FTase are 1700 and 1.4 nM, respectively. The IC50 for FTI-2153 for Rap1 processing in cells is >30 µM, and for Ras, it is 0.01 µM (29) .

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 x g. A 450-µl sample was removed to represent the total protein before fractionation. The total protein, S100, and P100 samples were all immunoprecipitated with anti-HA antibody (Covance) at 1:150 for 1 h at 4°C, followed by the addition of 20 µl of protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at 4°C. Beads were collected and washed, and protein samples were prepared as described previously (30) . SDS-PAGE analysis and immunoblot for HA were performed as described above. The P:S ratio was derived by dividing the %AUC generated for the P100 sample by the %AUC generated for the S100 sample for each condition. The %AUC was determined with the program Molecular Analyst 2.1.2 (Bio-Rad). Dividing the P:S ratio of the treated samples by the P:S ratio of the vehicle-treated cells and subtracting from one [1 - (P:S treated/P:S untreated)] yields the percentage of protein that has been moved into the cytosol by drug treatment.

Reporter Gene Assays.
For transient luciferase assays, NIH 3T3 cells in 35-mm, 6-well plates were cotransfected with 1 µg of pCGN-hyg vector, pCGN-rac1(61L) prenylation mutants [Rac1(61L)-P, -F, -UN], or 100 ng of pCGN-rac3(61L) prenylation mutants [Rac3(61L)-P, -GG, -F, -UN] and 125 ng of pJ-luc, a c-Jun luciferase reporter construct (a gift of Silvio Gutkind; NIH, Bethesda, MD). All transfections were performed in duplicate. Cells were placed in DMEM containing 0.5% calf serum containing either DMSO vehicle or 1 µM GGTI-2166 immediately after glycerol shock and were grown for 20–24 h. The cells were then rinsed with 1x PBS and lysed in 1x lysis buffer (Amersham Biosciences), and luciferase activity was measured with enhanced chemiluminescence reagents (Amersham Biosciences) in a Monolight 2010 luminometer (Analytical Luminescence, San Diego, CA).

Transformation Assays.
For focus formation assays, NIH 3T3 cells were plated in 60-mm dishes and cotransfected with 200 ng of either pZIP vector or pZIP-raf22W and 500 ng of pCGN-hyg vector, pCGN-rac1(61L), or pCGN-rac3(12V) prenylation mutants [Rac1(61L)-P, -F, -UN; Rac3(12V)-P, -GG, -F, -UN]. All transfections were performed in duplicate. Cells were grown in complete medium containing no drugs or in complete medium containing either DMSO vehicle or 5 µM GGTI-2166. Medium was replaced every other day. After 14–21 days, cells were photographed under the x10 objective, washed with 1x PBS, fixed with 3:1 (v/v) methanol:acetic acid, and stained with 0.4% crystal violet in 20% ethanol. Stained foci were then counted for quantitation of transforming activity.

For soft agar assays, NIH 3T3 cells stably expressing pCGN-hyg vector, pCGN-rac1(61L), and pCGN-rac3(12V) prenylation mutants [Rac1(61L)-P, -F, -UN; Rac3(12V)-P, -GG, -F, -UN] were prepared as described above. Single cell suspensions (1 x 105 cells/60-mm dish) of each stable cell line were plated in complete medium containing 0.4% agar and DMSO vehicle, 10 µM FTI-2153, or 10 µM GGTI-2166 on top of a bottom layer of complete medium containing 0.6% agar. Colonies were allowed to form for 14–21 days, after which they were photographed under the x4 objective.

Localization Assays and Fluorescent Microscopy.
NIH 3T3 cells were plated on glass coverslips in 35-mm, 6-well plates. For visualization of subcellular localization and formation of lamellipodia and membrane ruffles, cells were transiently transfected with 1 µg of pEGFP-C1 vector, pEGFP-rac1(61L), or pEGFP-rac3(61L) prenylation mutants [Rac1(61L)-P, -F, -UN; Rac3(61L)-P, -GG, -F, -UN]. After glycerol shock, cells were placed in complete medium containing DMSO vehicle, 10 µM FTI-2153, or 10 µM GGTI-2166. After 48 h, live cells were visualized with a fluorescent microscope (Axioskop; Zeiss, Thornwood, NY), and images were captured under the x20 objective with the MetaMorph digital imaging software (Universal Imaging Corp., Downington, PA).

Swiss 3T3 cells were plated on glass coverslips in 35-mm, 6-well plates. Before transfection with FuGENE 6, cells were placed in medium (DMEM +10% fetal bovine serum, 1% P/S) with DMSO vehicle, 10 µM FTI-2153, or 10 µM GGTI-2166. Cells were transfected with 1 µg of pEGFP-C1 vector, pEGFP-rac1(WT), or pEGFP-rac3(WT) prenylation mutants [Rac1(WT)-P, -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 concentrations. After an additional 24 h, cells were treated with either vehicle (4 mM HCl and 0.1% BSA) or 20 ng/ml PDGF (BB homodimer; Sigma). After 30 min of treatment, live cells were visualized with a fluorescent microscope, and images were captured under the x20 objective.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Both Geranylgeranylated and Farnesylated Forms of Rac1 and Rac3 Are Expressed Equivalently in NIH 3T3 Cells.
To validate the use of the prenylation mutants in the experiments shown, it was first necessary to determine their expression levels. Constructs were made to express Rac1 with a parental CAAX motif, which should be geranylgeranylated, or a mutated CAAX motif that made it either F or UN. Due to the possibility that the parental CAAX motif of Rac3, CTVF, could be a target for either GGTase I or FTase (26) , constructs were made to express Rac3 with the parental CAAX motif or a mutated CAAX that made it exclusively GG, exclusively F, or UN. As shown in Fig. 1ACitation , all prenylated mutants were expressed in stable cell lines at levels approximately equivalent to that of the Rac proteins with the parental CAAX motifs. Interestingly, Rac3-UN was expressed at significantly higher levels than any other Rac3 protein (Fig. 1A)Citation . This held true in at least six independently isolated cell lines. Although the basis for the differential expression is not clear, expression of Rac3-UN appeared to enhance cell viability, as reflected in a much larger number of colonies obtained from antibiotic selection for Rac3-UN compared with any other Rac3 protein (data not shown).



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  		Fig. 1. Expression of geranylgeranylated and farnesylated forms of activated Rac1 and Rac3 and dose-dependent sensitivity of geranylgeranylated forms Rac1 and Rac3 to GGTIs. A, NIH 3T3 cells were stably transfected with pCGN-hyg constructs containing 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, respectively) or with CAAX motifs that had been mutated to give the indicated prenylation mutants were compared by Western immunoblot with an antibody to the HA tag. The same blot was also probed for ß-actin to show equal protein loading. The results shown are representative of at least six independently created sets of stable lines. B, NIH 3T3 cells were transiently transfected with pCGN-rac1(61L), -rac3(61L), or -rac3(61L)-GG and allowed to grow for 48 h in the presence of DMSO; 1, 5, or 10 µM GGTI-2166; or 10 µM FTI-2153. Cells were crudely fractionated into cytosolic S100 (S) or membrane-containing P100 (P) fractions by centrifugation at 100,000 x g. The total (T) lane represents the total sample before fractionation. Fractionated samples were concentrated 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 the intensity of the bands for each condition.

 
Geranylgeranylated Forms of Rac1 and Rac3 Show Dose-Dependent Sensitivity to GGTIs.
To determine whether geranylgeranylated forms of Rac1 and Rac3 are sensitive to GGTIs, cells transiently expressing activated Rac1, Rac3, or Rac3-GG were treated with increasing doses of GGTI or with FTI. Cells were crudely fractionated into cytosolic S100 and membrane-containing P100 fractions. Fig. 1BCitation shows that with increasing doses of GGTI, Rac1, Rac3, and Rac3-GG proteins all decrease within the membrane fraction (P100) and increase within the cytosolic fraction (S100), as shown by the decrease in the P:S ratio from DMSO-treated samples to those treated with increasing doses of GGTI. Loss of protein from the membrane fraction indicates loss of prenylation because prenylation is necessary to anchor Rho family proteins in the membrane (18) . Comparison of the P:S ratio of GGTI-treated samples with the P:S ratio of the DMSO-treated samples shows that for Rac1, 67%, 87%, and 97% of the protein was moved to the cytosolic fraction after treatment with 1, 5, and 10 µM GGTI, respectively. For Rac3, 69%, 75%, and 83% was moved at 1, 5, and 10 µM GGTI, respectively, and for Rac3-GG, 87%, 78%, and 89% was moved at 1, 5, and 10 µM GGTI, respectively. Conversely, with 10 µM FTI treatment, the amount of Rac1, Rac3, or Rac3-GG protein in the membrane (P100) fraction appears similar to the amount with DMSO treatment, indicating that prenylation of geranylgeranylated forms of Rac proteins is not sensitive to the inhibition of FTase by FTIs. When the percentages are compared, more protein was moved from the membrane to the cytosol for Rac1 than either Rac3 or Rac3-GG at the highest dose of GGTI, but there was also more protein in the P100 fraction compared with the S100 fraction for Rac1 than either Rac3 or Rac3-GG. This suggests there may be some differences between Rac1 and Rac3 in partitioning between the membrane and cytosol and/or in general sensitivity to GGTI treatment.

Both Geranylgeranylated and Farnesylated Forms of Rac1 and Rac3 Signal to c-Jun, but Only Signaling from the Geranylgeranylated 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. 2Citation 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.



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  		Fig. 2. GGTI inhibits signaling to c-Jun from geranylgeranylated Rac proteins. NIH 3T3 fibroblasts were transiently cotransfected with a c-Jun luciferase reporter construct and pCGN-hyg constructs containing either activated mutants rac1 with the parental CAAX motif or the indicated prenylation mutations (A) or activated mutants of rac3 with 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 GGTI-2166 for 24 h and then lysed for analysis of luciferase activity. Luciferase activity from the c-Jun reporter is expressed as fold activation over the pCGN-hyg control and is shown as mean ± SE. Results are representative of four independent experiments.

 
To determine whether the ability of Rac1 and Rac3 to transcriptionally transactivate c-Jun is sensitive to GGTI-mediated inhibition, we performed the reporter assays in the presence of 1 µM GGTI. Decreases in activation by Rac1, Rac3, and Rac3-GG (Fig. 2, A and B)Citation demonstrate that all three are sensitive to GGTI. From Fig. 1BCitation , 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 Are Both Morphologically Transforming.
The similarity of Rac3 to Rac1 suggests that it has oncogenic potential, yet the transforming activity of Rac3 has not been demonstrated. It has been shown that activated Rac1 cooperates with activated Raf in focus formation assays (13 , 14) . To determine whether Rac3 could also cooperate with Raf to form foci, we cotransfected plasmids encoding activated Rac1 or Rac3 with a truncated and activated version of Raf into NIH 3T3 cells. We also cotransfected prenylation mutants of activated Rac1 or Rac3 with active Raf to determine whether the prenylation mutants could cooperate with Raf to support focus formation.

Consistent with its ability to activate c-Jun transcriptional transactivation like Rac1, Rac3 was also able to cooperate with Raf to form foci (Fig. 3A)Citation . Rac1 and Rac3 foci were easily distinguished from Raf foci due to the presence of enlarged, rounded refractile cells that were not found in the tightly arrayed refractile cells in Raf foci (Fig. 3B)Citation . The prenylation mutants of activated Rac1 and Rac3 were able to form numbers of foci relatively similar to their parental counterparts in cooperation with Raf, with morphology that was indistinguishable from that of the parental Rac1 and Rac3 (Fig. 3ACitation ; data not shown). Activated Rac1- and Rac3-UN were unable to form foci to any significant degree, with numbers equivalent only to that of the Raf-only control (Fig. 3A)Citation . We were unable to test the ability of Rac1 and Rac3 to form foci in cooperation with Raf in the presence of GGTIs due to the unexpected result that the GGTIs inhibited the formation of Raf foci, leading to nonspecific decreases in foci formed for Raf with or without coexpression of activated Rac1 or Rac1-F, thus confounding interpretation of the results (Fig. 3C)Citation . However, this may suggest that another target of GGTI, perhaps another geranylgeranylated protein, plays an important role in Raf transformation.



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  		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 µM 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 ± SD from duplicate plates. B, before staining, representative foci were photographed under a x10 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.

 
Farnesylated Rac Rescues Cells from GGTI-Mediated Inhibition 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)Citation , 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. 4Citation , 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.



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  		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 18–21 days in the presence of DMSO vehicle, FTI, or GGTI, after which they were photographed under a x4 objective. Results are representative of three independent experiments with independently isolated stable cell lines.

 
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 µM 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. 1BCitation . As expected, GGTI totally ablated the ability of Rac3 and Rac3-GG to form colonies, whereas FTI did not (Fig. 4)Citation . 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)Citation . Similar results were seen with activated Rac1 (data not shown).

Farnesylated Rac Rescues Cells from GGTI-Mediated Inhibition of Cell Spreading and Ruffling.
To determine whether the membrane localization and membrane ruffling activity of Rac1 and Rac3 could be targeted by GGTIs, we transiently transfected activated versions of parental Rac1 and Rac3 and their prenylation mutants expressed from the vector pEGFP-C1 into NIH 3T3 cells. As can be seen in Fig. 5Citation , in the presence of vehicle control, activated parental Rac1 and Rac3 and their prenylation mutants showed significant membrane ruffling and localization to the areas of ruffling with clear nuclear exclusion. However, in the presence of GGTI, parental Rac1 and Rac3 and the Rac3-GG mutant showed dramatically decreased ruffling activity and accumulation of Rac protein in the nucleus. Farnesylated versions of Rac1 and Rac3 were still able to ruffle in the presence of GGTI and had nuclear exclusion similar to vehicle control. However, farnesylated Rac1 and Rac3 were now sensitive to FTI, with decreased ruffling and increased accumulation in the nucleus. Activated Rac1- and Rac3-UN showed no ruffling activity and had diffuse localization throughout the cytoplasm and nucleus, much like pEGFP-C1 vector (Fig. 5Citation ; data not shown).



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  		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 x20 objective. Photographs shown are representative of four experiments.

 
WT Rac3-F Rescues PDGF-Mediated Ruffling from GGTI Inhibition.
Because mutationally activated versions of Rac proteins have not been identified in human tumors, it is thought that overexpression of WT forms or their hyperactivation may account for the contribution of Rac proteins to oncogenesis (31 , 32) . To determine whether Rac proteins are physiologically relevant targets for GGTIs, we used WT versions of Rac3 with parental and mutated prenylation sites in pEGFP-C1 to transfect Swiss 3T3 cells. Cells treated with DMSO, FTI, or GGTI were also treated with or without PDGF, a known activator of Rac1, to determine whether subsequent membrane-ruffling activity was still subject to inhibition by GGTIs. As seen in Fig. 6Citation , PDGF induces membrane ruffling and nuclear exclusion for WT Rac3 with either a parental (Rac3-P) or mutated prenylation site (Rac3-GG and Rac3-F). The ability to induce ruffling is inhibited for Rac3-P and Rac3-GG in the presence of GGTI, leading to flattened cells and an increase in nuclear localization. Ruffling still occurred in the presence of FTI in these cells. Cells transfected with farnesylated Rac3 were resistant to the inhibitory action of GGTI and had nuclear accumulation and the prevention of membrane ruffling only in the presence of FTI. Similar results were seen with WT Rac1 constructs (data not shown). These results indicate that both Rac1 and Rac3 can mediate PDGF responsiveness and that GGTI-resistant Rac proteins can overcome GGTI inhibition of a physiologically important function of WT Rac proteins.



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  		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 x20 objective. Results are representative of two independent experiments.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Rac1 proteins are involved in membrane ruffling, morphological and growth transformation, and signaling to a variety of downstream transcription factors, including c-Jun, SRF, and nuclear factor {kappa}B (1 , 33 , 34) . Considerably less is known about the consequences of Rac3 activation. Rac3 has been reported to activate JNK (15) and PAK (17) and to up-regulate DNA synthesis in a PAK-dependent, JNK-independent manner (17) . Rac3 GTP levels have been reported to be elevated in breast cancer cell lines and primary tumor tissues (17) . These results, along with the sequence similarity of Rac3 and Rac1, suggest that Rac3 could also have transforming activity. In support of this idea, we show here that constitutively activated mutant forms of Rac3 cause both focus formation and growth in soft agar in NIH 3T3 cells, in a manner very similar to that of Rac1. Additionally, we show here that both WT Rac1 and Rac3 induce membrane ruffling in response to PDGF. Thus, Rac3 is functionally similar to Rac1 in terms of responsiveness to growth factor stimulation and transforming ability.

Our data from the experiments outlined above suggest that Rac1 and Rac3 function similarly. Although there is substantial sequence identity in the classical effector domain region of these highly related proteins, other important elements of sequence divergence exist between Rac1 and Rac3 (15) . For example, differences exist in and around the Rho insert domain, a sequence that is unique to Rho family proteins (35) , and in the hypervariable region at the COOH terminus that could dictate functional distinctions. Furthermore, the early emergence of Rac1 and Rac3 in evolution (36) suggests that functional distinctions must exist. Amino acids in and around the Rho insert region of Rac1 and other Rho family members are known to contribute to effector binding (8 , 37, 38, 39) . Possible distinctions between Rac1 and Rac3 may also lie in the COOH-terminal hypervariable domain, a region that dictates isoprenoid modification and is important for membrane localization of small GTPases and for biological activity (18 , 40 , 41) .

Rac1 is known to be modified by a GG isoprenoid lipid (25) . The CAAX motif of Rac3, CTVF, with F in the X position, suggests that it could be a potential target for either GGTase I or FTase (26) . Our results demonstrate that Rac3 is likely to be mostly geranylgeranylated in cells. Individual small GTPases may differ in their requirement for modification by a specific isoprenoid moiety for function. For example, WT H-Ras is growth inhibitory only when modified by a GG group instead of its native F group (42) , but the biological activity of activated farnesylated RhoA is indistinguishable from that of the authentically geranylgeranylated RhoA (27) . We have shown here that both oncogenic and WT Rac1 and Rac3 appear to be tolerant of modification by either a GG group or F group for transformation and membrane-ruffling activities. Thus, the consequences of alternate lipid modification of Rac1 and Rac3 are more similar to what has been shown for activated RhoA than for WT H-Ras, suggesting that farnesylated Rac proteins are useful tools to investigate whether Rac1 and Rac3 are physiological targets for inhibition of GGTase I by GGTIs, the basis for a novel anticancer therapy.

FTase, the enzyme that attaches the F group to Ras, RhoB, and a subset of other small GTPases, has long been a target for rational drug design (19 , 20 , 43, 44, 45) . FTIs are in Phase I–III clinical trials for anticancer treatment, although the identity of the most critical targets that can explain FTI antitumor activity are still under investigation. GGTase I modifies many proteins in the Rho family of small GTPases, including Rac1 and Rac3, by attaching a GG group. GGTase I has also recently become a target for rational drug design, with the development of new inhibitors to block geranylgeranylation (19 , 20) . GGTIs have been shown to arrest human tumor cell growth in vitro (21 , 22) and to reduce tumor growth in animal models (23) , yet the physiologically relevant downstream targets of GGTIs have not been determined. Candidate downstream targets include the geranylgeranylated members of Rho family GTPases.

Inhibition of RhoA by GGTI led to an increase in p21waf1/cip1 expression, which is normally repressed by RhoA (46) . This may help to mediate the G1 arrest that is seen with GGTI treatment (21 , 22) . However, farnesylated RhoA, although functionally equivalent to the native geranylgeranylated RhoA, is unable to restore RhoA activity in the presence of GGTI (27) . These results suggest that although RhoA may be an important and necessary target of GGTIs, it may not be sufficient to mediate the inhibitory action of GGTIs.

We used the GGTI-insensitive, farnesylated versions of Rac1 and Rac3 to determine whether Rac proteins are biologically important downstream targets for GGTIs. If a GGTI-insensitive form of Rac can rescue cells from GGTI-mediated growth inhibition, then Rac is likely to be either an important mediator of that inhibition or downstream of a critical GGTase I target. Therefore, we investigated the ability of farnesylated forms of oncogenic Rac1 and Rac3, which we demonstrated to be GGTI insensitive, to rescue cells from GGTI-mediated inhibition of anchorage-independent growth and from inhibition of membrane-ruffling activity. These results suggested that Rac proteins may play an important role in the cellular response to GGTIs. However, oncogenically mutated forms of Rac and Rho proteins have not been found in human cancer cells; instead, it is thought that amplification of Rho family proteins or activation of their upstream regulators such as exchange factors contribute to the ability of these GTPases to influence the transformed phenotype (31 , 32) . Therefore, we also investigated whether a WT version of farnesylated Rac was resistant to the effects of GGTI. Farnesylated versions WT of Rac1 and Rac3 were both resistant to the effects of GGTI and continued to form ruffles after PDGF stimulation. These results suggest that in a physiological setting such as a human tumor, in which Rac proteins are unlikely to contain activating mutations, Rac proteins can still be functionally targeted by GGTIs.


   ACKNOWLEDGMENTS
 
We thank Christyn M. Gable for technical assistance, Drs. Saïd M. Sebti and Andrew D. Hamilton for provision of FTIs and GGTIs, and Drs. Krister Wennerberg and Keith Burridge for provision of Swiss 3T3 cells.


   FOOTNOTES
 
Grant support: NIH Grants T32-CA71341 (to P. L. J.) and U19-CA67771 (to A. D. C.).

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 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Adrienne D. Cox, Department of Radiation Oncology, CB# 7512, University of North Carolina, Chapel Hill, North Carolina 27599-7512. Phone: (919) 966-7713; Fax: (919) 966-7681; E-mail: adrienne_cox{at}med.unc.edu

1 The abbreviations used are: SRF, serum response factor; GGTase I, geranylgeranyltransferase I; FTase, farnesyltransferase; FTI, farnesyltransferase inhibitor; GGTI, geranylgeranyltransferase inhibitor; WT, wild-type; GG, geranylgeranyl; F, farnesyl; UN, unprocessed; P, parental; HA, hemagglutinin; EGFP, enhanced green fluorescent protein; 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 kinase. Back

Received 6/ 6/03. Revised 8/20/03. Accepted 9/12/03.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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