Rala and Ralb: Antagonistic Relatives in Cancer Cell Migration

Research Article

RalA and RalB: Antagonistic Relatives in Cancer Cell Migration

Gary Oxford, Charles R. Owens, Brian J. Titus, Tonia L. Foreman, Mikael C. Herlevsen, Steven C. Smith, and Dan Theodorescu

Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia

Abstract cells by three other classes of proteins: guanine nucleotide The Ral family of small G proteins has been implicated in exchange factors (GEF; ref. 7), which promote the GTP-bound, tumorigenesis, invasion, and metastasis. However, little active conformation; GTPase-activating proteins (GAP; ref. 8), emphasis has been placed on clarifying the individual roles which promote the GDP-bound, inactive conformation; and of the two Ral proteins, RalA and RalB, in these processes in guanine nucleotide dissociation inhibitors (GDI; ref. 9), which view of their high sequence homology. Here we analyze the block activation and also facilitate transfer of G proteins between separate contributions of RalA and RalB in regulating cell membrane compartments. It is estimated that there are at least as migration, a necessary component of the invasive phenotype, many, if not more, GEFs, GAPs, and GDIs as there are monomeric G in two human cancer cell lines; UMUC-3, a bladder carcinoma proteins, which contributes greatly to the complexity of under- line, and the prostate carcinoma line, DU145. Although standing G protein function and regulation. Although there is no inhibiting RalA protein expression by f80% with two different consensus, a common classification scheme splits the superfamily small interfering RNA duplexes had no effect on migration, into six families—Ras, Rho, Arf, Rab, Ran, and Rad/Gem (6). inhibiting RalB expression to the same extent with two Members of the Ras family, particularly H-Ras, K-Ras, and N-Ras, different duplexes resulted in a marked reduction in migra- are known to contribute widely to human tumorigenesis through tion. Inhibiting RalB expression did trigger a significant loss both activating mutations and overexpression (10, 11). Rho family of actin cytoskeleton fibers in UMUC-3 that was not seen with members such as RhoA, RhoC, Rac1, and cdc42, are also involved in inhibition of RalA expression. Interestingly, simultaneous many human cancers, primarily by overexpression (12). Recent inhibition of RalA and RalB expression had no effect on data also suggest that members of the Arf (13) and Rab (14) migration. However, dual inhibition of RalA and RalB families might contribute to tumor cell invasion and metastasis. expression in UMUC-3 did result in an almost total loss of Overexpression of GEFs (15) and loss of expression and/or actin fibers as well as a reduction in proliferation, particularly inactivation of GAPs (16) and GDIs (17) also occur in human in reduced serum conditions. These results suggest that RalA cancer. It is therefore not surprising that Ras and Rho family and RalB have different roles in cell migration and that they members and their regulators are now targets for new strategies of may in fact act as antagonists with regard to this phenotype. therapeutic intervention in cancer (18, 19). As further verification of this hypothesis, we found that Within the Ras family of monomeric G proteins, RalA and RalB expression of constitutively active RalA inhibited migration, are highly similar proteins (85% amino acid identity) that whereas expression of constitutively active RalB stimulated participate in diverse cellular functions (20). Among the processes migration, consistent with this model. In summary, we present regulated by Ral proteins are endocytosis, exocytosis, actin the first demonstration that despite their significant sequence cytoskeletal dynamics, and transcription (21). In some cases, Ral homology, RalA and RalB have nonoverlapping and opposing involvement in these processes has been shown to be mediated functions in cancer cell migration but overlapping functions through effectors such as RalBP1, Sec5, filamin, and phospholi- in cell growth. (Cancer Res 2005; 65(16): 7111-20) pase D1; in other cases, particular Ral-effector interactions responsible for downstream functions remain to be determined. Introduction Recent results have also indicated roles for Ral proteins in tumorigenesis and cancer progression (22, 23). For example, GEFs With >100 members in humans, the Ras superfamily of for Ral seem to be the most crucial determinants downstream of monomeric G proteins (1) contribute to the regulation of many, Ras in transforming human cells (24, 25). Also, using small if not most, cellular functions; from gene expression (2), to cell interfering RNA (siRNA) duplexes specific for RalA or RalB, it was division (3), membrane traffic (4), and cytoskeletal dynamics (5). In shown that RalA and RalB have separate, nonoverlapping roles in general, these functions are dependent upon the ability of these regulating anchorage-independent proliferation and survival, proteins to cycle between two conformations by binding and respectively, in several human cancer cell lines (26). We have hydrolyzing GTP. These two conformations usually differ in both previously shown that Ral is necessary for epidermal growth their affinity for other protein effectors and their cellular factor–stimulated migration in human bladder cancer cells (27). localization, hence allowing these G proteins to alter cellular Here, we address the critical issue of whether RalA and RalB have pathways both temporally and spatially (6). The intrinsic GTP similar or different contributions on cell migration and find that hydrolase (GTPase) activity of these proteins is further regulated in RalA and RalB play opposing roles in regulating this process, with RalB stimulating and RalA inhibiting motility. In contrast to migration, we describe overlapping roles for RalA and RalB in Requests for reprints: Dan Theodorescu, Department of Molecular Physiology and Biological Physics, Box 422, University of Virginia Health Sciences Center, regulating cancer cell proliferation. These results suggest the Charlottesville, VA 22908. Phone: 434-924-0042; Fax: 434-982-3652; E-mail: dt9d@ existence of distinct migration and proliferation signaling net- virginia.edu. I2005 American Association for Cancer Research. works downstream of Ral family proteins which have fundamen- doi:10.1158/0008-5472.CAN-04-1957 tal implications for the development of therapeutics. www.aacrjournals.org 7111 Cancer Res 2005; 65: (16). August 15, 2005

Materials and Methods after transfections, cells were fixed with 4% paraformaldehyde for 15 minutes, stained with Hoechst 33342 (1:200) and Phalloidin-AlexaFluor 594 Cells. UMUC-3 human bladder cancer cells were obtained from (1:200; both from Molecular Probes) for 20 minutes and allowed to air-dry American Type Culture Collection (Rockville, MD) and cultured in MEM overnight. Cells transfected with FLAG-Ral alleles were also visualized by with 2 mmol/L L-glutamine and Earle’s balanced salt solution adjusted to FLAG immunofluorescence using M2 anti-FLAG mouse monoclonal contain 1.5 g/L sodium bicarbonate, 1.0 mmol/L sodium pyruvate, and 10% antibody (1:7,500; Sigma) and Oregon Green conjugated anti-mouse IgG fetal bovine serum (FBS). DU145 human prostate cancer cells were also (1:200; Molecular Probes). from American Type Culture Collection and cultured in MEM with 2 mmol/L Growth curves and cell cycle analysis. Twenty four hours after siRNA L-glutamine and Earle’s balanced salt solution adjusted to contain 1.5 g/L transfection as previously described, UMUC-3 cells were harvested, counted sodium bicarbonate, 1.0 mmol/L sodium pyruvate, 0.1 mmol/L nonessential using a hemacytometer, and resuspended in media with either 2% or 10% amino acids, and 10% FBS. HeLa human cervical cancer cells were from the FBS. One thousand cells/well were added to wells of 96-well plates and University of Virginia Tissue Culture Facility and were cultured in the same plates were then incubated at 37jC, 5% CO2 for 24 to 96 hours. After media as DU145 above. incubation, cell numbers were estimated using CyQuant. For evaluation of Plasmids. pcDNA 3.1+/RalB was obtained from the Guthrie cDNA cell cycle and detection of sub-G0 populations, 120 hours after siRNA Resource Center (Sayre, PA, http://www.cdna.org/). RalA cDNA was transfection and after 4 days in reduced (2%) serum, cells were amplified from bladder cancer cell RNA by RT-PCR using the following permeabilized with 70% ethanol for 1 hour and stained with propidium primers: forward, 5V-GCTCGGATCCACCATGGCTGCAAATAAGCCCAAGG- iodide (0.1% sodium citrate, 0.1% Triton X-100, 50 Ag/mL propidium iodide G-3V; reverse, 5V-TAGACTCGAGTTATAAAATGCAGCATCTTTCTCTG-3V, and and 100 units/mL RNase) for 30 minutes. Flow cytometry was done on a cloned into pcDNA 3.1+ using BamHI/XhoI digestion and ligation. FACScalibur system using excitation light at 488 nm and Cell Quest Constitutively active mutants (G23V) of RalA and RalB were made using software (Becton Dickinson). a QuikChange mutagenesis kit (Stratagene, La Jolla, CA) with corresponding primers. Wild-type and activated mutants of RalA and RalB were NH2- terminally FLAG-tagged by cloning into pFLAG-CMV-4 (Sigma, St. Louis, Results MO) using HindIII/XbaI sites. Plasmids were transfected into cells in six- Whereas previous results have indicated the importance of Ral well plates using FuGENE (Roche, Indianapolis, IN) according to the proteins in cell motility (27, 28), these studies have assumed manufacturer’s instructions. complete functional overlap between RalA and RalB and have been Small interfering RNA. siRNA duplexes were chemically synthesized, deprotected and annealed by Dharmacon (Lafayette, CO), with the following largely based upon the use of dominant-negative alleles of RalA or target sequences: RalA I, 5V-GACAGGUUUCUGUAGAAGA-3V; RalA II, 5V- RalB. These dominant-negative Ral proteins are postulated to CAGAGCUGAGCAGUGGAUU-3V; RalB I, 5V-GGUGAUCAUGGUUGGCAGC-3V; inhibit endogenous Ral function by preventing Ral activation by RalB II, 5V-AAGCUGACAGUUAUAGAAA-3V; RalA/B, 5V-GACUAUGAACCUAC- tying up GEFs (29). Because the specificities of the Ral GEFs for CAAAG-3V; luciferase (GL2), 5V-CGTACGCGGAATACTTCGA-3V.RalA RalA versus RalB are not well understood, it must be assumed that duplexes I and II and RalB duplex I are the same as described in Chien any dominant-negative Ral allele likely inhibits both RalA and RalB and White (26); RalA/B duplex, which inhibits both RalA and RalB activation. Therefore, to better understand the individual roles of expression in UMUC-3, is the same as their RalB duplex II. Transfection with RalA and RalB in bladder cancer cell migration, we undertook a siRNA duplexes (200 nmol/L) was in six-well plates using OligofectAMINE siRNA approach. Chien and White (26) developed siRNA duplexes (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. that specifically inhibit the expression of either RalA or RalB. They Western blotting and Ral activation assays. Either 24 hours (DNA) or used these to show that inhibition of RalB expression triggers 72 hours (siRNA) posttransfection, cells were lysed in 50 mmol/L Tris-HCl apoptosis and cell death in selected cancer cell lines, but that non– (pH 7.5), 200 mmol/L NaCl, 1% Igepal CA-630 (Sigma), 10 mmol/L MgCl2. Lysates were cleared by centrifugation and protein amounts were cancer cell lines did not undergo apoptosis in response to RalB quantitated by bicinchoninic acid assay (Pierce, Rockford, IL). Proteins knockdown. Surprisingly, they showed that simultaneous inhibition were separated by SDS-PAGE, transferred to polyvinylidene difluoride of RalA and RalB expression reversed the apoptotic and cell death membrane, and probed with antibodies against RalA and RalB (PharMin- effects of RalB knockdown alone. gen, San Diego, CA) or FLAG (Sigma). Because the RalB antibody became We chose to focus initially on two highly motile human cancer unavailable, experiments involving HeLa cells used a RalB antibody cell lines which have a strong invasive and metastatic phenotype; generously provided by Dr. Raj Bhullar. Blots were developed using Super UMUC-3, a bladder cancer line isolated from a high-grade, invasive Signal Femto Chemiluminescence (Pierce) and results were visualized and transitional cell carcinoma, and DU145, a prostate cancer line quantitated using an AlphaInnotech (San Leandro, CA) imaging system. Ral isolated from a brain metastasis. Figure 1A and B shows that these activation assays were done as previously described (27) using RalBP1- agarose to pull down activated RalA and RalB (Upstate, Charlottesville, VA). two cell lines have similar levels of RalA and RalB, and that the Cell migration and plating efficiency assays. Either 24 hours (DNA) or activation levels of RalA under standard growth conditions are 72 hours (siRNA) after transfection, cells were harvested, counted in a similar between these cell lines. RalB activation is somewhat lower hemacytometer, and resuspended in serum-free media. Cells (20,000) were in UMUC-3 than in DU145. The effectiveness and specificity of added in triplicate to the upper chambers of transwell filters (8.0 Am pores, siRNA-mediated reduction in RalA and RalB expression in these Becton Dickinson, Franklin Lakes, NJ) in a 24-well tissue culture plate. The two cell lines is shown in Fig. 1C and D. Two different duplexes for lower chambers contained media with 2% FBS. Plating control assays were RalA reduced RalA expression at the protein level by f80% in both done in triplicate in adjacent wells containing the same media but with no cell lines, whereas having little effect on RalB expression. Similarly, transwell filters. After 6 hours (for UMUC-3) or 18 hours (for DU145 and two different duplexes targeting RalB reduced its expression by HeLa), cells remaining on the upper surface of the filters were removed with 80%, with little change in RalA expression. Similar results were cotton swabs and cells on the lower surface were fixed with 100% methanol, obtained in HeLa cells, in which these siRNA duplexes had been stained with crystal violet, and counted with the aid of a gridded coverslip. Cell numbers in plating control assay wells were estimated using CyQuant previously shown to be effective (26), although RalB depletion (Molecular Probes, Eugene, OR) according to manufacturer’s instructions. in HeLa was less pronounced than in UMUC-3 or DU145 (Fig. 1D). Actin cytoskeleton visualization. UMUC-3 cells were plated in six-well A nonspecific control siRNA (GL2, which targets firefly luciferase; plates containing coverslips and transfected with either siRNA or FLAG-Ral ref. 30) did not have any effect on either RalA or RalB expression. In expression constructs as described above. One day (DNA) or 3 days (siRNA) all cases, the level of protein inhibition reached a maximum

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Figure 1. Effectiveness and specificity of siRNA duplexes targeting RalA and RalB. A, results of RalA and RalB activation assays on UMUC-3 and DU145. Total lysates (20 Ag protein) and RalBP-1 pull-downs (from 1 mg protein) were Western-blotted using antibodies specific for RalA and RalB. B, quantitation of RalA and RalB activation assays from three separate experiments using AlphaInnotech software. C, 72 hours after transfection with siRNA duplexes, UMUC-3, DU145, and HeLa cell lysates were harvested and equal protein amounts were analyzed for RalA and RalB expression by Western blotting. In each case, equal loading was verified by Western blotting for a-tubulin (data not shown). D, results from three biologically separate experiments, showing mean levels of RalA and RalB expression F SD resulting from Ral siRNA transfection relative to levels from cells transfected with control siRNA (GL2).

between 48 and 72 hours after transfection and persisted until at assay. In all cases, there was no detectable motility in the absence least 120 hours posttransfection (data not shown). of FBS (data not shown). Because of data showing that RalB siRNA Using the aforementioned duplexes, we proceeded to evaluate can trigger cell death via apoptosis in cancer cells (26), we sought the effects of inhibiting RalA or RalB expression on cell migration. to determine whether the effect of RalB siRNA on motility was due Relative to the control siRNA, inhibition of RalA expression with to a specific effect on migration or a more general reduction in either RalA duplex I or II had no effect on motility toward 2% FBS plating efficiency and/or viability. Plating control assays done in (Fig. 2A). In contrast, inhibition of RalB expression with either RalB parallel to migration assays indicated that, in both the RalA and duplex I or II significantly reduced transwell migration (35-55% RalB siRNA–transfected cells, there was no reduction in plating reduction) in both UMUC-3 and DU145 (Fig. 2C). However, neither efficiency relative to control siRNA-transfected cells (Fig. 2B RalA nor RalB depletion had any effect on HeLa motility in this and D). Nor were there any changes in gross cell morphology www.aacrjournals.org 7113 Cancer Res 2005; 65: (16). August 15, 2005

Figure 2. Effects of RalA and/or RalB depletion on transwell migration. Triplicate Boyden chambers were loaded with 20,000 siRNA-transfected UMUC-3, DU145, or HeLa cells in serum-free media and cells were allowed to migrate toward 2% serum for 6 hours (UMUC-3) or 18 hours (DU145 and HeLa). After removing nonmotile cells from the upper surface of the filter, cells on the lower surface were fixed, stained, and counted. Columns, means; bars, F SD. Plating control wells did not contain transwell chambers and were quantitated using CyQUANT (Molecular Probes). Data shown are representative of three separate experiments. In each case, Western blotting of duplicate siRNA transfections indicated reduction in RalA or RalB expression of >75%. A and B, cells transfected with RalA siRNA duplexes. C and D, cells transfected with RalB siRNA duplexes (*, P < 0.01; t test). upon siRNA-mediated depletion of RalA or RalB (data not shown). RalB depletion. A priori, three results seem possible. RalA depletion Taken together, these data indicate that the effect of RalB depletion could have no effect on the ability of RalB depletion to inhibit on cell migration in UMUC-3 and DU145 is specific and not due to motility, suggesting that RalA plays no role in motility. Alterna- the production of either nonadherent or nonviable cells. Inhibition tively, RalA depletion could further inhibit motility in the context of of motility by RalB, but not RalA, siRNA points toward RalB having RalB depletion, which could be interpreted that RalB plays a a specific promigratory role in these cells. dominant role in motility that RalA can partially fill in the absence Because apoptosis triggered by RalB siRNA in cancer cells can be of RalB. Thirdly, RalA depletion could reverse the RalB siRNA blocked by simultaneous transfection of RalA and RalB siRNA (26), inhibition of motility, indicating a possible antagonistic relation- we sought to determine the effect on motility of both RalA and ship between RalA and RalB in regulating motility. In Fig. 3A,

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. RalA and RalB in Cancer Cell Migration siRNA-mediated depletion of both RalA and RalB is shown. RalA/B translation (31). Interestingly, from our results, it seems that this is is a single siRNA duplex that, in UMUC-3, is able to reduce a cell type–specific phenomenon. Thus, to address this issue, we expression of both RalA and RalB. In DU145, however, this duplex is transfected both siRNA duplexes and achieved >80% protein much less effective at reducing RalA expression. This is consistent reduction in this cell line (Fig. 3A). When we looked at the effect with it being used previously as a RalB-specific siRNA (26), of simultaneous RalA and RalB depletion on motility, we found although it has only a single base mismatch with RalA mRNA. It that, in UMUC-3, normal motility was restored using either a single has been shown that siRNA duplexes with single base mismatches siRNA duplex targeting both RalA and RalB or individual duplexes are often inefficient at targeting mRNA destruction via DICER, but for each. In DU145, only the two-duplex approach actually reduced can still inhibit protein expression, presumably by interfering with RalA and RalB expression to levels similar to that found in single

Figure 3. Effect of simultaneous RalA and RalB depletion on transwell migration. A, Western blots of UMUC-3 and DU145 transfected with siRNA duplexes targeting RalA and RalB for 72 hours. B, columns, means; bars, F SD. C, as in Fig. 2. Representative of three separate experiments. In each case, Western blotting of duplicate siRNA transfections indicated reduction in RalA and RalB expression of >70%.

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Figure 4. Effects of constitutively active RalA and RalB on transwell migration. UMUC-3 cells were transfected with expression constructs (in pFLAG vector) for either wild-type (wt) or constitutively active (act) RalA and RalB. A, Ral activation assays showing that act-RalA and act-RalB have much higher activation levels than wt-RalA and wt-RalB. B, quantitation of results in (A) using AlphaInnotech software. C, 24 hours after transfection, triplicate Boyden chambers were loaded with 20,000 cells in serum-free media and allowed to migrate toward 2% serum for 6 hours, as in Fig. 2. Plating controls as described in Fig. 2. Columns, means; bars, F SD (*, P < 0.01; **, P < 0.05; t test). Data shown are representative of three separate experiments. Inset, expression levels of wt- and act-RalA and RalB (FLAG) by Western blot.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. RalA and RalB in Cancer Cell Migration knockdowns, and in that case, normal motility was also restored. reduction of RalA and RalB expression with either one or two Because the RalB depletion in the double knockdowns is of a duplexes reduced proliferation in medium with 10% serum, and similar magnitude to that of the RalB single knockdowns, this does had an even more profound effect on growth in reduced serum not seem to be due to inefficient RalB depletion. The lack of effect conditions (Fig. 6C). on motility by simultaneous RalA and RalB protein depletion suggests that continued RalA expression is critical for the inhibition of migration triggered by RalB depletion, suggesting that these two Discussion proteins have antagonistic roles in regulating cell motility. In this report, we describe an antagonistic relationship between Having put forward this provocative model involving antago- RalA and RalB in regulating motility of bladder and prostate nistic regulation of cell motility by RalA and RalB, we went on to cancer cells. Although Ral proteins have previously been shown to further test this model using a complementary approach to siRNA. be important for the motility of skeletal myoblasts and bladder Because of our longstanding interest in bladder cancer cell cancer cells (27, 28), this is the first indication that RalA and RalB motility, we chose to focus our remaining studies on UMUC-3. have nonoverlapping functions in cell motility. That these Therefore, GTPase-deficient, constitutively active alleles of RalA nonoverlapping functions may actually be antagonistic in cell and RalB (6) were transfected into UMUC-3 cells and their effects migration is surprising and novel. Interestingly, our results showing on migration examined. In Fig. 4A and B, Ral activation assays that RalB is promigratory, whereas RalA inhibits motility, stand were carried out and show that constitutively activating somewhat in contrast to recent results showing that RalA mutations (G23V) result in markedly higher levels of Ral-GTP in activation is necessary for the formation of lamellipodia in moving these cells. Transfection of wild-type RalA or RalB (Fig. 4C) had no COS-7 and Madin-Darby canine kidney cells (34). Whether this effect on the migration of UMUC-3 cells relative to vector controls; indicates different roles for RalA and RalB in normal versus cancer however, transfection of constitutively active RalA reduced cells awaits further experimentation, although the lack of an effect motility by f50%, indicating a negative role for RalA in motility on motility upon RalB depletion in HeLa cells indicates that (Fig. 4C). In contrast, transfection of constitutively active RalB RalB is not universally required for cancer cell motility. What also stimulated motility by f50% (Fig. 4C), consistent with the model remains unclear is why migration is not stimulated in cells de- of RalB being promigratory. It is important to note that RalA and pleted of RalA. RalB alleles were expressed at similar levels and that no effect on So how might RalA and RalB antagonistically regulate cell plating controls was seen with any of the Ral alleles (Fig. 4C, motility? Of the known Ral effectors—RalBP1, Sec5, Exo84, Filamin, inset). Importantly, simultaneous expression of constitutively and phospholipase D1 (21)—none are known to bind selectively to active RalA and RalB did not affect motility, giving additional RalA or RalB. In fact, RalBP1, Sec5, and Exo84 have been shown to support to the model that RalA and RalB have opposite effects on bind to both RalA and RalB, at least in yeast two-hybrid studies cell migration (Fig. 4C). (35, 36). Recently, it has been shown that RalA binds the exocyst Actin cytoskeletal organization and dynamics have repeatedly complex more effectively than RalB, at least in Madin-Darby canine been shown to be crucial in the motility of cells both cancerous kidney cells cells, allowing RalA to target E-cadherin delivery to the and normal (32). In light of this, we looked at actin-based basolateral membrane (37). They also described nonoverlapping structures in UMUC-3 cells transfected with Ral siRNAs, as shown localizations of RalA and RalB, with RalA, but not RalB, associated in Fig. 5A. Cells transfected with control siRNA (GL2) show many with recycling endosomes. This still leaves open the possibility of prominent actin stress fibers running most of the length or width selective interaction of RalA and RalB with other known effectors of the cell. These fibers were also numerous and prominent in in vivo, as well as undiscovered Ral effectors that might specifically untransfected UMUC-3 (data not shown). In cells that had been interact with RalA or RalB. Alternatively, this antagonism may depleted of RalA by siRNA, there was little change in the number or reflect the importance of intracellular protein localization in cell prominence of these stress fibers. In contrast, there were markedly motility. Perhaps different cellular localizations of RalA and RalB, fewer and less prominent stress fibers in cells that had been particularly in a moving cell, allow for different subsets of effector depleted of RalB, suggesting that RalB has a role in the interactions that result in opposing effects on cell migration. Given organization and/or maintenance of these actin fibers. In cells that all the known Ral effectors have functions consistent with where both RalA and RalB were depleted, stress fibers were almost roles in motility (21), dissecting out the relative affinities of RalA nonexistent, and many of the cells were much more elongated than and RalB for these effectors in cells, particularly moving cells normal. Because motility was also altered in UMUC-3 cells (if possible), should be quite informative. expressing constitutively active RalA and RalB, actin-based We have also shown that RalB depletion has a much more structures were also visualized in these cells, as shown in Fig. 5B. profound effect than RalA depletion on actin cytoskeletal The number and prominence of stress fibers in cells expressing act- organization in UMUC-3 cells, causing a dramatic reduction in RalA or act-RalB seemed unchanged compared with vector- stress fibers. However, this probably doesn’t explain why RalB transfected cells or untransfected neighbors. depletion inhibits motility, because depletion of both RalA and Because Ral proteins have been shown to play important roles RalB, which restores normal motility, causes an even greater loss in regulating cancer cell proliferation (26, 33), we studied the of stress fibers, as well as a shift to a more elongated shape in effect of selective inhibition of RalA and/or RalB expression on many cells. The hypothesis that RalB depletion inhibits motility cell proliferation by doing growth curves on UMUC-3 cells independent of its effect on the actin cytoskeleton is further depleted of RalA and/or RalB by siRNA transfection. Reducing supported by the observation that expression of act-RalA, which either RalA (Fig. 6A) or RalB expression (Fig. 6B) had no effect inhibits motility, and act-RalB, which stimulates motility, both on UMUC-3 cells’ ability to grow in medium containing normal have no visible effect on the actin cytoskeleton. Given the dra- serum (10%) and only minor effects on their ability to grow matic differences in actin cytoskeleton organization and morphol- under reduced serum conditions (2%). In contrast, simultaneous ogy, and yet similar motility, between control siRNA-transfected www.aacrjournals.org 7117 Cancer Res 2005; 65: (16). August 15, 2005

Figure 5. Effects of RalA and/or RalB depletion on the actin cytoskeleton. A, UMUC-3 cells were plated on glass coverslips and transfected with Ral siRNAs. Seventy-two hours after transfection, cells were fixed and stained with phalloidin-AlexaFluor 594 (red) to visualize actin filaments and Hoechst 33342 (blue) to visualize nuclei. Magnification, 630Â. B, UMUC-3 cells were plated on glass coverslips and transfected with act-Ral expression constructs. Twenty-four hours after transfection, cells were fixed and stained with M2-anti-FLAG antibody to stain transfected cells (green), phalloidin-AlexaFluor 594 (red) to visualize actin filaments and Hoechst 33342 (blue) to visualize nuclei. Magnification, 630Â.

cells and cells depleted for both RalA and RalB, two alternatives what roles, if any, RalA and RalB play in these two modes of tumor present themselves. Either these differences in cell shape and actin cell invasion. fibers have no effect on motility, or cells with reduced levels of We have also shown that the growth of UMUC-3 cells, RalA and RalB have acquired a different mode of motility. Recent particularly in reduced serum conditions, is inhibited by results indicate that cancer cells can shift between two modes simultaneous depletion of RalA and RalB. This suggests that RalA of invasion; an elongated, lamellipodia-driven mode, dependent and RalB may have an overlapping yet essential role in regulating upon extracellular proteases, and a rounded mode dependent cell proliferation, with the presence of at least one Ral protein upon ROCK and ezrin (38). Further experiments may elucidate necessary for optimal growth. These findings may help to explain

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. RalA and RalB in Cancer Cell Migration previous observations showing involvement of Ral in the growth opposing functions, highlighting the need for careful study using, of breast cancer cells (33) and in regulating cyclin D1 expression wherever possible, specific tools to study specific protein function. (39). This effect of RalA and RalB depletion on proliferation does In this case, such an approach has uncovered novel, opposing not seem to be due to triggering apoptosis because cell cycle roles for RalA and RalB in cancer cell migration, which seem analysis of siRNA-transfected cells showed no increase in distinct from their overlapping roles in cell proliferation. apoptotic cells (

Figure 6. Effects of RalA and/or RalB depletion on cell proliferation. UMUC-3 cells were transfected with siRNA duplexes targeting RalA and/or RalB. Twenty-four hours after transfection, cells were harvested, resuspended in medium containing either 10% or 2% FBS, reseeded in 96-well plates, and allowed to grow an additional 24 to 96 hours. Cell numbers were estimated by CyQuant fluorescence. In each case, Western blotting of duplicate siRNA transfections indicated reduction in RalA and/or RalB expression of >75%. A, cells transfected with RalA siRNA duplexes. B, cells transfected with RalB siRNA duplexes. C, cells transfected with siRNA duplexes for both RalA and RalB. www.aacrjournals.org 7119 Cancer Res 2005; 65: (16). August 15, 2005

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