β-Arrestin/Ral Signaling Regulates Lysophosphatidic Acid–Mediated Migration and Invasion of Human Breast Tumor Cells

The lipid mediator lysophosphatidic acid (LPA) plays a role in cancer progression and signals via specific G protein–coupled receptors, LPA1-3. LPA has been shown to enhance the metastasis of breast carcinoma cells to bone. However, the mechanisms by which LPA receptors regulate breast cancer cell migration and invasion remain unclear. Breast cancer cell proliferation has been shown to be stimulated by Ral GTPases, amember of the Ras superfamily. Ral activity can be regulated by the multifunctional protein β-arrestin. We now show that HS578T and MDA-MB-231 breast cancer cells and MDA-MB-435 melanoma cells have higher expression of β-arrestin 1 mRNA compared with the nontumorigenic mammary MCF-10A cells. Moreover, we found that the mRNA levels of LPA1, LPA2, β-arrestin 2, and Ral GTPases are elevated in the advanced stages of breast cancer. LPA stimulates the migration and invasion of MDA-MB-231 cells, but not of MCF-10A cells, and this is mediated by pertussis toxin–sensitive G proteins and LPA1. However, ectopic expression of LPA1 in MCF-10A cells caused these cells to acquire an invasive phenotype. Gene knockdown of either β-arrestin or Ral proteins significantly impaired LPA-stimulated migration and invasion. Thus, our data show a novel role for β-arrestin/Ral signaling in mediating LPA-induced breast cancer cell migration and invasion, two important processes in metastasis. (Mol Cancer Res 2009;7 (7):1064–77) Introduction Lysophosphatidic acid (LPA), in addition to being a key intermediate in de novo lipid synthesis, is a known regulator of diverse cellular processes such as migration, cytoskeletal reorganization, survival, and proliferation (1). In humans, LPA concentration in serum is relatively high (1-5 μmol/L; ref. 2). It has been shown that cell surface G protein–coupled receptors (GPCR) mediate the cellular effects of LPA. At least three types of GPCRs, Edg-2/LPA1/vzg-1, Edg-4/LPA2, and Edg-7/LPA3, which belong to the Edg (endothelial cell differentiation gene) family, have been identified as specific receptors for LPA and share 50% to 57% amino acid identities (3). More recently, four other LPA receptors, LPA4-7, have been identified. These receptors share 35% amino acid homology with each other but are structurally distinct from the Edg family of LPA receptors and are more closely related to the P2Y family of nucleotide receptors (4). The P2Y subfamily of LPA receptors also have a more restrictive distribution pattern compared with LPA1-3 (4-7), and although reports have suggested that they may be responsive to LPA, further study is required to validate these observations. LPA and its receptors, LPA1 and LPA2, have been implicated in breast cancer (8). Autotaxin, a key enzyme in LPA production in blood (1, 8), is overexpressed in various human malignancies, including breast cancer, implying the involvement of LPA production and signaling in a variety of human tumors. LPA1 expression has been linked to the motility and invasiveness of several metastatic cell lines (9-11). In an in vivo model, LPA1 activity promoted breast carcinoma cell metastasis to the bone, and inhibition by a pharmacologic antagonist or silencing of LPA1 expression significantly reduced the progression of osteolytic bone metastases (12). LPA2 receptors are elevated in invasive ductal carcinomas (13). Although these studies have indicated an important role for LPA in breast cancer, the specific mechanisms by which LPA and its receptors mediate breast cancer cell migration and invasion, two important processes in cancer metastasis, remain unclear. The Ras-like GTPases, RalA and RalB, are highly similar proteins (85% amino acid identity) which participate in diverse cellular functions and processes such as endocytosis, exocytosis, actin cytoskeletal dynamics, cell migration, as well as transcription (14). Signaling downstream of Ral can be mediated through effectors such as RalBP1, Sec5, filamin, and phospholipase D1. Additionally, the roles of Ral proteins in tumorigenesis and cancer progression have been identified (14, 15). Ral proteins have been shown to stimulate breast cancer cell proliferation, which can be inhibited by the suppression of Ral activity (16). Furthermore, Received 1/6/09; revised 3/27/09; accepted 4/8/09; published 7/16/09. Grant support: This study was funded by the Canadian Institutes of Health Research. The following are recipients of a salary award and studentships: M. Bhattacharya, NSERC University Faculty Award; A.V. Babwah, CIHR New Investigator Award; and T.T. Li, CIHR Strategic Training Program (London Regional Cancer Program) Studentship. 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. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Requests for reprints:MoshmiBhattacharya,Department of Physiology andPharmacology, The University of Western Ontario, London, ON, Canada N6A 5C1. Phone: 519-661-2111, ext. 82970; Fax: 519-661-3827. E-mail: Moshmi.Bhattacharya@ schulich.uwo.ca Copyright © 2009 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-08-0578 Mol Cancer Res 2009;7(7). July 2009 1064 on April 13, 2017. © 2009 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from breast cancer cells that are estrogen-independent do not proliferate when a dominant-negative Ral mutant is expressed (17). Ral has also been implicated in the metastatic process (18); elevated levels of Ral have been found in human metastatic bladder cancer cells compared with nonmetastatic variants of the same cells. However, the role of Ral in breast cancer cell invasion or metastasis is unknown. In addition to Ras-dependent Ral activation, Ral can also be activated independently of Ras via Ca influx orβ-arrestins (14, 19). As key regulators of GPCR signaling, β-arrestins carry out many important roles in G protein–regulated biological functions (20, 21). The β-arrestins (β-arrestin 1 and β-arrestin 2) are ubiquitously expressed proteins that are instrumental in attenuating GPCR signaling (21) and have been shown to regulate the internalization of LPA1 (22). β-Arrestins also function as scaffolds for the organization of signaling complexes, including Src family members, mitogen-activated protein kinases and RalGDS (19, 21). Additionally, novel roles for β-arrestins in cell migration have emerged from genetic studies demonstrating impaired CXCR-mediated motility in lymphocytes from β-arrestin 2 knockout mice (23). Here, we report that the expression of LPA receptors (LPA1 and LPA2), β-arrestins, and Rals is elevated in breast cancer tissue in advanced stages of the disease. Moreover, we show for the first time thatβ-arrestins are highly expressed in the highly metastatic MDA-MB-231 breast cancer cells, and LPA1 associates with Ral and can stimulate Ral activity to mediate migration and invasion via a β-arrestin/Ral pathway. Results LPA Receptors, β-Arrestins, and Rals Are Aberrantly Expressed in Human Breast Tumors The expression of β-arrestins (β-arrestin 1 and β-arrestin 2), RalA and RalB, and their guanine-nucleotide exchange factor, RalGDS, has not been examined in breast cancer tumors. To date, one study has shown that LPA2 levels are elevated in invasive ductal carcinoma (13). Therefore, we examined the expression of these genes by quantitative real-time PCR (qPCR) in a cDNA array comprised of 48 tissue samples taken from patients with stages 0 to IV breast cancer. LPA1, and in particular LPA2 transcripts, were significantly more abundant in primary tumor samples from the more advanced stages of breast cancer, compared with noninvasive stage 0 breast tumors (Fig. 1A). We found that β-arrestin 2 and both Ral genes were highly expressed in stages I to IV, relative to expression in stage 0 (Fig. 1B and C). On the other hand, mRNA expression of FIGURE 1. Transcripts for LPA receptors, β-arrestins, and Rals are aberrantly expressed in tissue from advanced stages of breast cancer. Breast cancer tissue expresses elevated levels of transcripts for LPA receptors (A), β-arrestin 1 and β-arrestin 2 (B), RalA and RalB (C), and RalGDS (D) compared with stage 0 tissue samples as determined by quantitative real-time PCR. Points, log-transformed relative mRNA expression compared with the mean expression of stage 0 samples. Samples were prenormalized to β-actin expression by the manufacturer. a. P < 0.05 compared with stage 0 expression; b. P < 0.01 compared with stage 0 expression. LPA Mediates Breast Carcinoma Invasion via β-Arrestin Mol Cancer Res 2009;7(7). July 2009 1065 on April 13, 2017. © 2009 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from RalGDS was not significantly different between stages (Fig. 1D). Taken together, our results suggest that in addition to LPA receptors,β-arrestins, RalA, and RalB are also aberrantly expressed in human breast cancer tissue as the disease progresses. Elevated Expression of LPA1 Receptors and β-Arrestins in Human Breast Cancer Cells We next determined whether or not the mRNA expression of the LPA receptors, β-arrestins, and Ral genes was altered in breast cancer cells by qPCR analysis. Using three well established cancer cell lines, the MDA-MB-231 and HS578T breast cancer cells, and MDA-MB-435 melanoma cells, we compared the expression of these genes to the levels detected in the nontumorigenic mammary epithelial cell line, MCF-10A. We found that MDA-MB-231, HS578T, and MDA-MB-435 cells express significantly higher levels of LPA1 receptor transcript (∼86-fold, ∼6-fold, and ∼16-fold increases, respectively) compared with the MCF-10A cells (Fig. 2A). HS578T have been reported to only express LPA1 receptors (24). The levels of LPA2 and LPA7 receptor mRNA were found to be very low in mammary cells MCF-10A, MDA-MB-231, and HS578T (Supplementary Fig. S1) whereas transcripts of LPA3-6 were undetectable (data not shown). Western blot analysis of receptor expression in these cell lines could not be conducted due to a lack of effective antibodies against endogenous receptors. We observed that MDA-MB-231, HS578T, and MDA-MB-435 cells expressed significantly higher levels of β-arrestin 1 mRNA (∼24-fold, ∼13-fold, and ∼46-fold, respectively) than MCF-10A cells (Fig. 2B). In contrast, β-arrestin 2 mRNA was significantly elevated only in MDA-MB-435 cells relative to MCF-10A cells (Fig. 2C). However, elevated expression of both β-arrestin 1 and β-arrestin 2 was observed at the protein level in both MDA-MB-231 and MDA-MB-435 cells compared with MCF-10A cells. Expression of β-arrestin 2, but not β-arrestin 1 protein, was detected in HS578T. The relative protein expression of RalA and RalB were each found to be equivalent between the cell lines (Supplementary Fig. S2). LPA1 Activation Stimulates Breast Cancer Cell Migration and Invasion in Three-Dimensional Cultures Both normal and malignant breast cells can be cultured in reconstituted extracellular matrix as a three-dimensional model, mimicking the in vivo microenvironment (25). When MCF10A cells were suspended in three-dimensional Matrigel cultures, the nonmalignant cells formed spheroid-shaped colonies, a characteristic of normal mammary epithelial cells (Fig. 3A). In contrast, MDA-MB-231 cells formed complex, stellate structures that invaded into the extracellular matrix. When the overlaying medium was supplemented with Ki16425, a LPA1/3 antagonist, the number of MDA-MB-231 colonies that formed invasive structures was greatly reduced with ∼55% of the colonies retaining a spheroidal-shape resembling the ones formed by MCF-10A cells. Growing the cells in the presence of the vehicle (0.1% DMSO) did not affect the formation of stellate structures. Because our qPCR results indicated that MDAMB-231 cells primarily express LPA1 receptors, our studies suggest a critical role for LPA1 activity for MDA-MB-231 cells migration and invasion in a three-dimensional context. HS578T breast cancer cells, which we determined express lower mRNA levels of LPA1 compared with MDA-MB-231 cells, also invaded through Matrigel, although to a lesser extent than MDA-MB231 cells, and hence, all subsequent studies were conducted with the more aggressive MDA-MB-231 cells. We next examined the effect of ectopic expression of LPA1 in the normal mammary epithelial cells, MCF-10A, which have low levels of endogenous receptors. We found that MCF-10A cells stably expressing LPA1 also formed stellate structures when cultured in reconstituted extracellular matrix (Fig. 3B). Immunostaining of the three-dimensional cultures verified the expression of Flag-LPA1 in cells (Fig. 3B). FIGURE 2. Cancer cell lines express elevated levels of LPA1, β-arrestin 1, and β-arrestin 2 compared with nonmalignant mammary epithelial cells. Relative mRNA expression of LPA1 (A), β-arrestin 1 (B), and β-arrestin 2 (C) was determined by quantitative real-time PCR as described in Materials and Methods. **, P < 0.005; ***, P < 0.0001 compared with expression in MCF-10A cells from three to four independent experiments. Endogenous expression of β-arrestin 1 or β-arrestin 2 protein in each cell line was determined by Western blot. Li et al. Mol Cancer Res 2009;7(7). July 2009 1066 on April 13, 2017. © 2009 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from To determine whether LPA stimulates cell migration and invasion, we did Transwell chamber cell motility and invasion assays. Medium containing 10% serum was used as a positive control for cell migration; additionally, LPA is a major constituent of serum. We observed that serum-starved MDA-MB-231 cells only showed significant migration when LPA was placed in the bottom chamber compared with that observed when serum-free medium was used (Fig. 4A). In contrast, the nontumorigenic MCF-10A cells did not migrate towards LPA nor in medium containing serum (Fig. 4A). MCF-10A cells stably expressing Flag-LPA1 also did not significantly migrate towards LPA or serum either (data not shown). Interestingly, although MDA-MB-231 cells endogenously express LPA1 predominantly, stable expression of Flag-tagged LPA1 or LPA2 in these cells further stimulated LPA-mediated migration of cells compared with nontransfected cells (Fig. 4A). Doseresponse studies indicated that LPA-mediated migration of MDA-MB-231 began to plateau at the 10 μmol/L LPA concentration, with no considerable differences at higher concentrations of LPA (Fig. 4B), and thus, all further experiments were conducted using this concentration of LPA, as reported in other studies (9, 26). A role for Gi/o signaling pathways in LPA-stimulated cell migration has been reported for ovarian, prostate, and pancreatic cancer cells (9-11). To determine the possible mechanisms by which LPA stimulates breast cancer cell migration, MDA-MB231 cells were pretreated with Ki16425 or with pertussis toxin (PTX) to antagonize LPA1/3 signaling or inhibit Gi/o activity, respectively. Migration ofMDA-MB-231 cells towards 10 μmol/L of LPA was significantly inhibited by either pretreatment with FIGURE 3. LPA1 mediates the invasion of normal mammary epithelial and breast cancer cells. A. Normal mammary epithelial MCF-10A cells maintain spheroidal morphology when suspended in three-dimensional Matrigel culture assays. MDA-MB-231 cells form stellate structures that invade into the surrounding matrix and invasion can be inhibited by treatment with 10 μmol/L of Ki16425. Phase-contrast images are of representative three-dimensional colonies from independent experiments. Bar, 40 μm. Colony shape of MDA-MB-231 cells was scored as being either stellate or spheroidal after growth in Matrigel. ***, P < 0.0001 compared with cells treated with vehicle (0.1% DMSO; n = 4). B. Normal mammary epithelial MCF-10A cells stably expressing Flag-LPA1 form stellate structures in three-dimensional Matrigel cultures. Bar, 40 μm. MCF-10A colonies express Flag-LPA1 receptors (red). Bar, 20 μm. LPA Mediates Breast Carcinoma Invasion via β-Arrestin Mol Cancer Res 2009;7(7). July 2009 1067 on April 13, 2017. © 2009 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Ki16425 (∼6% of total cells migrated) or with PTX (∼5% of total cells migrated; Fig. 4C). Pretreatment of cells with these agents did not affect cell viability as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Supplementary Fig. S3). Furthermore, LPA was observed to stimulate MDA-MB-231 cells (∼20%) to invade through Matrigel-coated Transwell chambers, and invasion was significantly blocked by pretreating the cells with either Ki16425 or PTX (∼0.9% and ∼0.8%, respectively; Fig. 4D). LPA also significantly stimulated HS578Tcells to migrate (Supplementary Fig. S4A) and invade (Supplementary Fig. S4B). LPA Receptor Activation Stimulates Breast Cancer Cell Cytoskeletal Rearrangement Cytoskeletal rearrangement is critical for the processes of cell migration and invasion. However, a role for LPA in regulating the cytoskeleton of breast cancer cells has not been described. MDA-MB-231 cells grown under serum-free conditions were stimulated with LPA. Within a few minutes, extensive cytoskeletal reorganization was observed with the majority of the cells retracting and forming retraction fibers, followed by membrane ruffling (Fig. 5). Pretreatment of the cells with the antagonist Ki16425 blocked this cytoskeletal rearrangement, indicating a role for the endogenous LPA1 receptors in mediating these dynamic processes. In order to determine the localization of LPA1 receptors in MDA-MB-231 cells, cell lines stably expressing Flag-tagged LPA1 were generated and receptors were detected by immunostaining. In serum-starved cells in the absence of agonist, LPA1 was primarily localized at the cell surface (Fig. 6A, i). Within 5 minutes of LPA stimulation, the cells quickly retracted and formed retraction fibers that were positively stained for LPA1. Three-dimensional image projections by confocal microscopy revealed that these retraction fibers were found to be parallel to the surface of the culture dish (Fig. 6A, ii). Similarly, retraction fibers were positive for filamenFIGURE 4. LPA induces the migration of breast cancer cells but not normal mammary epithelial cells. Stable expression of Flag-LPA1 or Flag-LPA2 in MDA-MB-231 cells increases LPA-mediated cell migration. A. Migration was determined using Transwell chamber assays. In all cases, medium containing 10% serum, serum-free medium, or serum-free medium containing 10 μmol/L of LPA were placed in the lower chamber. a, P < 0.0001 compared with MCF10A cells migrating towards LPA; b, P < 0.0001 compared with MDA-MB-231 cells under serum-free conditions; c, P < 0.05 compared with LPA-treated nontransfected MDA-MB-231 cells. Data from three independent experiments each in duplicate. B. Increasing LPA concentrations increases MDA-MB-231 cell migration. a, P < 0.001 compared with either 10 or 100 nmol/L of LPA; b, P < 0.01 compared with 1 μmol/L of LPA; c, P < 0.001 compared with 1 μmol/L of LPA. Data from four independent experiments. Migration towards concentrations of 10, 30, and 50 μmol/L of LPA were not significantly different from each other. C. LPA-mediated breast cancer cell migration is inhibited by LPA receptor antagonist (Ki16425) or PTX treatment. MDA-MB-231 cells pretreated with 10 μmol/L of Ki16425 or 100 ng/mL of PTX were plated into Transwell chambers and cells allowed to migrate towards serum-free medium containing 10 μmol/L of LPA. **, P < 0.005 compared with untreated MDA-MB-231 cells and represent three to five independent experiments. D. LPA-stimulated breast cancer cell invasion can be inhibited by Ki16425 or PTX treatment. Cell invasion was measured by Matrigel-coated Transwell chamber assays. MDA-MB-231 cells pretreated with 10 μmol/L of Ki16425 or 100 ng/mL of PTX were plated into Matrigel-coated Transwell chambers and with serum-free medium containing 10 μmol/L of LPA in the lower chamber. ***, P < 0.0001 compared with serum-free conditions from three independent experiments. Li et al. Mol Cancer Res 2009;7(7). July 2009 1068 on April 13, 2017. © 2009 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from tous actin as confirmed using fluorescent-tagged phalloidin (Fig. 6B). Comparable results were observed in MDA-MB231 cells stably expressing Flag-LPA2 receptors (Supplementary Fig. S5). We also examined whether or not Ral GTPases were localized in the LPA-stimulated membrane retraction fibers. MDAMB-231 cells transfected with green fluorescent protein (GFP)–tagged RalA were serum-starved and cytoskeletal changes observed by live-cell confocal microscopy in response to stimulation with 10 μmol/L of LPA for specified intervals. In the absence of agonist, RalA was localized at the plasma membrane and in membrane ruffles (Fig. 6C, i). Shortly upon LPA stimulation, extensive retraction fibers were formed and were positive for RalA. Ral proteins are activated by guanine-nucleotide exchange factors such as RalGDS (27). Cytosolic RalGDS is inactive and must translocate to the plasma membrane to function as a Ral-specific guanine-nucleotide exchange factor (16, 28). Therefore, we tested whether GFP-RalGDS translocates to the plasma membrane in serum-starved MDA-MB231 cells. In response to LPA receptor activation, GFP-RalGDS FIGURE 6. Flag-LPA1 receptors are localized in retraction fibers in response to LPA. A.MDA-MB-231 cells stably expressing Flag-tagged LPA1 receptors were grown on glass coverslips and incubated in the absence (0 min) or presence (10 μmol/L) of LPA for 5 and 15 min. Cells were then subjected to immunofluorescent staining for Flag (red) and nuclei stained with 0.1% Hoechst 33258 dye (blue). Bar, 20 μm. ii, a three-dimensional projection of FlagLPA1 MDA-MB-231 cells stimulated for 5 min with 10 μmol/L of LPA. B. MDA-MB-231 cells form filamentous actin–containing retraction fibers in response to LPA. Serum-starved MDA-MB-231 cells were incubated in the absence (0 min) or presence (10 μmol/L) of LPA for the indicated times. Filamentous actin was stained with phalloidin conjugated to AlexaFluor-546 (red) and the nuclei stained with 0.1% Hoechst 33258 dye (blue). Bar, 20 μm.C. LPA stimulates Ral and translocation of RalGDS and β-arrestin 1. Flag-LPA1 stably-transfected MDA-MB-231 cells were transfected with GFP-RalA (i), GFP-RalGDS (ii), or β-arrestin 1–GFP (iii). Serum-starved cells were then stimulated and live cell images taken. Representative micrographs from independent experiments. Bar, 10 μm. FIGURE 5. LPA stimulates cytoskeletal rearrangement in breast cancer cel ls . i , MDA-MB-231 ce l ls contract and form retraction fibers in response to LPA. Cells pretreated with Ki16425 do not respond to LPA stimulation. Serum-starved MDA-MB-231 cells were grown on glass-bottomed culture dishes and stimulated with 10 μmol/L of LPA in the absence or presence of 10 μmol/L of Ki16425. DIC images were taken at specified times after stimulation. Bar, 20 μm. ii, magnified image of retraction fibers formed after LPA stimulation. LPA Mediates Breast Carcinoma Invasion via β-Arrestin Mol Cancer Res 2009;7(7). July 2009 1069 on April 13, 2017. © 2009 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from was redistributed from the cytosol to the plasma membrane within a minute of stimulation by LPA and persisted at the membrane even after 10 minutes, as visualized by live cell confocal microscopy (Fig. 6C, ii). Agonist-induced activation of GPCRs leads to the recruitment of β-arrestins to the plasma membrane that mediates, among other things, GPCR internalization (20). As observed with RalGDS, stimulation with LPA induced the redistribution of a β-arrestin 1–GFP fusion protein to the plasma membrane with noticeable clearing of the cytosol within 10 minutes (Fig. 6C, iii). Similar results were observed when breast cancer cells were transfected with β-arrestin 2–GFP (data not shown). Together, these results indicate for the first time that LPA stimulation can activate RalGDS and β-arrestins in breast cancer cells. Ral GTPases Associate with Flag-LPA1 in MDA-MB-231 Cells In order to determine whether or not LPA receptors associate with Ral GTPases, we conducted coimmunoprecipitation experiments. Both endogenous RalA and GFP-RalA associate constitutively with Flag-LPA1 (Fig. 7i) and Flag-LPA2 (Fig. 7ii) in these breast cancer cells, and stimulation of cells with LPA did not seem to further alter the interaction between the receptors and Ral. LPA-Induced Breast Cancer Cell Migration and Invasion Is Mediated by β-Arrestin/Ral Signaling As Ral and β-arrestin have been implicated in the reorganization of the actin cytoskeleton and the regulation of chemotaxis (29, 30), we examined whether or not these proteins regulated LPA-mediated breast cancer cell migration and invasion. Transwell chamber assays showed that MDA-MB-231 cells stably expressing the constitutively active Ral mutant, RalA (27), display significantly reduced cell migration towards LPA (Fig. 8A, i). A role for β-arrestins in regulating cell migration has been reported but the molecular mechanism by which this occurs is poorly understood (23). A possible mechanism by which this can occur is through activation of Ral proteins because β-arrestins can modulate the activity of Ral GTPases through RalGDS (19). Stable MDA-MB-231 cells were generated expressing HA-RalGDS, an HA-tagged, dominantnegative RalGDS mutant, consisting of the minimum region of RalGDS required to bind β-arrestin and shown previously to block cytoskeletal rearrangement (ref. 19; Fig. 8A, ii). Expression of the RalGDS dominant-negative mutant significantly reduced the migration of MDA-MB-231 cells to LPA (Fig. 8A, i). Furthermore, MDA-MB-231 cells stably expressing HA-RalGDS did not invade into the surrounding matrix in three-dimensional morphogenesis assays, but rather formed spherical colonies similar to those formed by the nonmalignant MCF-10A cells (Fig. 8B, i). Quantification of these three-dimensional cultures showed that significantly fewer colonies expressing HA-RalGDS formed stellate structures (∼33%) compared with controls. To verify that the spheroidal colonies were expressing the RalGDS mutant, the three-dimensional cultures were fixed and the cells immunostained using an anti-HA antibody. We observed that the cells forming the spheroidal colonies expressed HA-RalGDS in the cytosol (Fig. 8B, ii). Expression of HA-RalGDS was also verified by Western blot analysis using an anti-HA antibody (data not shown). Knockdown of Endogenous Ral and β-Arrestin Reduces Stellate Structure Formation in Three-Dimensional Assays In order to validate a role for β-arrestin and Ral signaling in breast cancer cell migration and invasion, MDA-MB-231 cells stably expressing short hairpin RNA against β-arrestin 1, β-arrestin 2, RalA, or RalB were generated, as well as double knockdowns for β-arrestin 1/2 or RalA/B. Each gene was targeted using two individual and independent shRNA constructs and stable cell lines expressing each construct were used for subsequent assays. Stable expression of each construct significantly reduced the expression of their respective targets and their specific isoform at mRNA and protein levels, as determined by qPCR and Western blot analysis, respectively (Figs. 9A and 10A). Single knockdown did not affect the expression of their corresponding isoform (Supplementary Fig. S6), nor did the expression of a scrambled sequence affect the expression of individual targets compared with nontransfected cells (data not shown). FIGURE 7. LPA receptors associate with RalGTPases. LPA1 and LPA2 receptors interact with RalA. MDA-MB-231 cells stably expressing Flag-LPA1 (i) or Flag-LPA2 (ii) receptors were transfected with GFP-RalA and serum-starved for 4 h before being stimulated with 10 μmol/L of LPA for specified times. MDAMB-231 cells transfected with Flag vector were used as a control. Lysates were used for coimmunoprecipitation with anti-Flag monoclonal antibodies and expression of Ral immunoblotted with a monoclonal anti-Ral antibody by Western blot. Lysates from Flag-LPA1 stably transfected cells were used to examine receptor interaction with endogenous RalA. β-Actin was used to control for equal loading of individual samples. Representative blots (n = 3). Li et al. Mol Cancer Res 2009;7(7). July 2009 1070 on April 13, 2017. © 2009 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Knockdown of either β-arrestin 1 or β-arrestin 2 in MDAMB-231 cells significantly reduced LPA-mediated breast cancer cell migration compared with the scrambled shRNA control (Fig. 9B).We did not observe any further reduction in cell migration when bothβ-arrestin 1 andβ-arrestin 2 (β-arrestin 1/2) were simultaneously knocked down. We also examined the effects of β-arrestin and Ral knockdown on breast cancer cell invasion using three-dimensional morphogenesis assays. Amarked reduction in the number of stellate colonies was observed in MDAMB-231 stable cell lines expressing β-arrestin 1, β-arrestin 2, or β-arrestin 1/2 shRNA (Fig. 9C). Approximately 70% of total cell colonies retained a spheroidal morphology when either β-arrestin 1 orβ-arrestin 2 was individually knocked down compared with the scrambled control (Fig. 9D). We did not observe a further reduction in the number of stellate colonies with MDA-MB231 cells expressing β-arrestin 1/2 shRNA as ∼60% of colonies remained spheroidal. As β-arrestins can modulate the activity of Ral GTPases, we next sought to determine whether knockdown of β-arrestin had an effect on Ral activation in MDA-MB-231 cells. The content of active GTP-bound Ral was measured in serum-starved MDAMB-231 cells through its ability to bind to its effector protein RalBP1. The amount of GTP-bound RalA was significantly increased in MDA-MB-231 cells by LPA within 30 seconds of stimulation and was detected, but not significant, following 5 minutes of stimulation (Fig. 9E, i and ii). Similarly, MDAMB-231 cells stably expressing β-arrestin 1 or β-arrestin 2 shRNA significantly increased GTP-bound Ral in response to LPA (data not shown). However, LPA-stimulated Ral activation was significantly blocked in cells expressing both βarrestin 1/2 shRNA (Fig. 9E, iii and iv). Taken together, these data indicate that LPA stimulates Ral activity via a β-arrestin–dependent mechanism in breast cancer cells. Furthermore, we found that the formation of invasive stellate structures was significantly reduced in MDA-MB-231 cells expressing RalA, RalB, or RalA/B shRNA in three-dimensional morphogenesis assays (Fig. 10B). Single knockdown of RalA or RalB resulted in ∼50% of the total colonies remaining spheroidal (Fig. 10C). Knockdown of both RalA/B did not significantly reduce the number of stellate colonies compared with results from individual knockdowns. Discussion To date, one study has shown that LPA can activate Ral in Rat-2 fibroblasts in a Ras-independent manner (31). In the present study, we have identified, for the first time, that LPA stimulates Ral activity in breast cancer cells to regulate cell migration and invasion and that LPA1 interacts with Ral GTPases. Additionally, our data also indicate a novel role for FIGURE 8. LPA-stimulated MDA-MB-231 cell migration is significantly reduced by the expression of Ral or RalGDS mutants. A. MDA-MB-231 cells stably expressing either a constitutively active Ral mutant (RalA) or a dominant-negative RalGDS mutant (HARalGDS) were subjected to Transwell chamber assays with serum-free medium containing 10 μmol/L of LPA in the lower chamber. ii, RalGDS mutant consists of the minimum binding domain required to bind β-arrestin. ***, P < 0.0001 from three to four independent experiments. B. Stable expression of RalGDS mutant (HA-RalGDS) in MDA-MB-231 cells reduces their ability to invade as seen in three-dimensional Matrigel assays (i). Bar, 40 μm. Colony shape was scored as being either stel late or spheroidal after growth in Matrigel (ii). *, P < 0.05, compared with cells transfected with a vector (n = 3). MDA-MB-231 spheroids express HA-RalGDS 768 (iii). Bar, 20 μm. LPA Mediates Breast Carcinoma Invasion via β-Arrestin Mol Cancer Res 2009;7(7). July 2009 1071 on April 13, 2017. © 2009 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from