Lysophosphatidic Acid Down-Regulates Stress Fibers and Up-Regulates Pro–Matrix Metalloproteinase-2 Activation in Ovarian Cancer Cells

Thuy-Vy Do,1 Jay C. Symowicz,1 David M. Berman,3 Lance A. Liotta,3 Emanuel F. Petricoin III,4 M. Sharon Stack,2 and David A. Fishman5

Departments of 1Obstetrics and Gynecology and 2Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois; 3Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland; 4Office of the Director, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland; and 5Department of Obstetrics and Gynecology, Gynecologic Oncology, NewYork, NewYork

Abstract Introduction Epithelial ovarian cancer (EOC) is asymptomatic at Epithelial ovarian cancer (EOC) is the most lethal early stages and is often diagnosed late when tumor cells gynecologic malignancy among women in the Western world. are highly metastatic. Lysophosphatidic acid (LPA) has More than two-thirds of women are diagnosed during later been implicated in ovarian oncogenesis as levels of this stages when 5-year survival rates are only 20% to 30%. lipid are elevated in patient ascites and plasma. Because However, when ovarian cancer is diagnosed early, the long- the underlying mechanism governing LPA regulation term survival rate is almost 90% (1-3). of matrix metalloproteinase-2 (MMP-2) activation remains Lysophosphatidic acid (LPA) is a bioactive lipid that has undefined, we investigated the relationship between been shown in preliminary studies to be a promising early LPA-induced changes in actin microfilament diagnostic and prognostic biomarker for EOC (4, 5). LPA organization and MMP-2 enzymatic activity. We report exerts its effects upon binding to cognate G protein–coupled that when cells were cultured at a high density, LPA receptors encoded by the endothelial cell differentiation gene mediated stress fiber and focal adhesion disassembly (Edg) family members. Several high-affinity LPA receptors and significantly repressed RhoA activity in EOC have been identified, including Edg-2/LPA1, Edg-4/LPA2, and cells. Inhibition of Rho-kinase/ROCK enhanced both Edg-7/LPA3. Following receptor binding, LPA elicits a diverse LPA-stimulated loss of stress fibers and pro–MMP-2 range of biological responses, including platelet aggregation, activation. In contrast, expression of the constitutively smooth muscle contraction, mitogenesis, and cell shape active RhoA(G14V) mutant diminished LPA-induced changes (6). pro–MMP-2 activation. LPA had no effects on membrane LPA plays a unique role in the pathobiology of ovarian type 1–MMP or tissue inhibitor of metalloproteinase-2 cancer. Ascitic fluid from the peritoneal cavity of patients with expression, but up-regulated MMP-2 levels, contributing ovarian cancer induces a robust mitogenic response in EOC to the induction of MMP-2 activation. Interestingly, when cells, and LPA has a significant role in this response (6, 7). cells were cultured at a low density, stress fibers were Interestingly, LPA levels are substantially higher (2-80 Amol/L) present after LPA stimulation, and ROCK activity was in ascites (7, 8) and plasma (3, 4, 9) from patients with early- required for EOC cell migration. Collectively, these and late-stage ovarian cancer when compared with levels in results were consistent with a model in which LPA healthy subjects or in patients with breast cancer and leukemia. stimulates the metastatic dissemination of EOC cells by By comparison, activated platelets produce and release initiating loss of adhesion and metalloproteinase lysophospholipids (10), resulting in circulating serum LPA activation. (Mol Cancer Res 2007;5(2):121–31) levels of 1 to 5 Amol/L (11). Although LPA is not secreted at significant levels by normal ovarian epithelial cells, EOC cells produce substantially higher levels of LPA. LPA can also stimulate some EOC cells to produce LPA, generating autocrine loops that perpetuate and amplify oncogenic pathways (12, 13).

Received 9/26/06; revised 11/29/06; accepted 12/4/06. Finally, the LPA receptors, LPA2 and LPA3, were overex- Grant support: NIH grants (RO1 CA89503, RO1 CA82562, and RO1 pressed in EOC cells when compared with normal cells CA01015) and National Cancer Institute grants (UO1 CA85133, P50 CA83639). (12, 14-16), and the activity of autotaxin/lysophospholipase 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 D, an enzyme involved in LPA production, was elevated in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ovarian cancer ascites (17). Note: Current address for L.A. Liotta and E.F. Petricoin III: Center for Applied Another well-recognized role of LPA is the ability to activate Proteomics and Molecular Medicine, George Mason University, Manassas, VA. Requests for reprints: David A. Fishman, Gynecologic Oncology, Department RhoA, a small GTPase that mediates changes in actin of Obstetrics and Gynecology, 550 First Avenue, New York, NY 10016. E-mail: microfilament organization necessary for adhesion, motility, [email protected] Copyright D 2007 American Association for Cancer Research. and cell shape changes. RhoA activates mDia and ROCKI/Rho- doi:10.1158/1541-7786.MCR-06-0319 kinase to regulate the formation of stress fibers and focal

adhesions. ROCK has been shown to directly phosphorylate the regulatory light chain of myosin II as well as myosin phosphatase, promoting RhoA-regulated actomyosin contractility. Another important player in mediating actomyosin contractility is myosin light chain kinase (MLCK), a Ca2+/ calmodulin-regulated enzyme that directly phosphorylates regulatory light chains. The balance between MLCK activity (of ROCK and MLCK) and myosin phosphatase activity coordinates regulatory light chain phosphorylation (18). Matrix metalloproteinase-2 (MMP-2) is present in ovarian cancer ascites, cells, and tissue in which its proteolytic activity is thought to play an important role in i.p. invasion. Ovarian cancer cells respond to LPA by inducing the processing of pro– metalloproteinase-2 (pro–MMP-2) to its active form, thereby potentiating MMP-dependent cellular migration and invasion (19). MMPs are zinc-dependent endopeptidases that are activated when the zymogen (pro–MMP) is processed at the cell surface (20) via the formation of a ternary complex consisting of the zymogen, tissue inhibitor of metalloproteinase-2 (TIMP-2), and membrane type 1 (MT1)-MMP (21). An adjacent MT1-MMP molecule not bound to TIMP-2 then cleaves the pro–MMP-2 molecule within this complex, yielding an intermediate form. The intermediate MMP-2 species is finally cleaved via a concentration-dependent autolytic event, requiring another MMP-2 molecule, that generates active MMP-2 (22). Because LPA can regulate both the actin cytoskeleton and pro–MMP-2 activation, we tested the hypothesis that LPA modulates MMP-2 activity via changes in actin microfilament organization. We report that LPA exposure resulted in sustained disruption of stress fibers and increased peripheral filament bundling. Correspondingly, RhoA activity was significantly repressed after LPA treatment. Furthermore, LPA treatment increased MMP-2 levels, promoting pro–MMP-2 activation. Restoration of RhoA activity by expression of the constitutively active mutant, RhoA(G14V), resulted in the down-regulation of LPA-induced pro–MMP-2 activation. Our findings allowed us to construct a model in which LPA plays an active role in EOC FIGURE 1. LPA triggers loss of stress fibers and an increase in peripheral actin filament bundles. Texas red – phalloidin immunofluores- dissemination by concurrently promoting loss of adhesion and cence (n = 3) images depict actin microfilament organization in three EOC MMP activation, thereby potentiating metastasis. cell lines: DOV13, OVCAR3, and OVCA429. Cells were treated with either vehicle (0.1% BSA/PBS) or 80 Amol/L of LPA for 5 min (arrowheads, peripheral actin filaments; arrows, stress fibers; bar, 100 Am). Results LPA Promotes Stress Fiber Disassembly and Enhances disassembly of stress fibers increases with prolonged exposure Peripheral Filament Formation to LPA, and was very prominent by 24 h (see Fig. 4B). Because LPA mediates changes in actin microfilament organization by LPA-induced changes in the actin cytoskeleton of EOC cells activating the small GTPase, RhoA (23, 24). To analyze the were distinct from other epithelia, in which stress fibers form in effects of LPA on actin cytoskeleton morphology, EOC cells response to LPA, phalloidin immunofluorescence was evaluated were treated with pathophysiologic levels of LPA (80 Amol/L) in three EOC cell lines: DOV13, OVCAR3, and OVCA429. for 5 min, and then stained with Texas red–phalloidin to Loss of stress fibers was observed in all cell lines, and this visualize actin filaments. Cells were cultured at high density phenotype was sustained for up to 24 h (see Fig. 4B). We used (confluent) and low density (no cell-cell contacts). When EOC the DOV13 cell line for all subsequent experiments in this study. cells were plated at a low density, stress fibers were present in Because these data suggest a loss of focal adhesions in vehicle- and LPA-treated cells (Fig. 1). However, when EOC response to LPA, changes in the association of the focal cells were plated at a high density, LPA exposure began to adhesion components, vinculin and talin, with the actin trigger the loss of stress fibers, as well as an increase in cytoskeleton were evaluated. Sequential detergent extraction peripheral filaments when compared with vehicle-treated cells was done on cell monolayers using buffers containing saponin, (Fig. 1). Some loss of stress fibers was first visible after 5 min of Triton X-100, and SDS. Cells were first solubilized in saponin exposure to LPA, but it was not a complete loss (Fig. 1). This to extract soluble cytoplasmic proteins (S), and Triton X-100

was then used to solubilize membrane-associated proteins (TX). talin associated with the cytoskeleton (Fig. 2A, top, lane 12) The remaining insoluble pellet consisting of proteins associated was severely diminished after 120min of exposure to LPA with the cytoskeleton (P) was solubilized in buffer containing compared with time 0(Fig. 2A, lane 3). Densitometric analysis SDS. Consistent with the loss of stress fibers seen using was done to quantitate the distribution of vinculin and talin in phalloidin immunofluorescence, the amount of vinculin and the various detergent pools in response to LPA treatment over

FIGURE 2. LPA induces focal adhesion disassembly and represses RhoA activity. A. Focal adhesion components shift from the cytoskeletal to the cytoplasmic pool after LPA treatment. Top, anti-talin and anti-vinculin immunoblots using lysates from cells extracted sequentially with buffers containing the detergents saponin (S), Triton X-100 (TX), and SDS (P). Cells were exposed to 80 Amol/L of LPA for the times indicated before sequential detergent extraction was done. Bottom, corresponding densitometric analysis of talin and vinculin expression in the saponin-, Triton X-100 – , and SDS-soluble pools. For each time point, talin and vinculin expression was represented as a percentage of total protein expression (sum of density from all detergent pools). Columns, means relative to time 0 min; bars, SE (n = 6). Columns labeled with different letters are statistically significant as analyzed by repeated measures ANOVA, followed by Tukey’s multiple comparison test (P < 0.05). B. Cellular RhoA-GTP bound to RBD-agarose was assayed in cells treated with 80 Amol/L of LPA for 0, 30, 120, and 300 min. RhoA activity was quantified as the amount of RhoA bound to RBD normalized to the amount of RhoA in input cell lysates. Points, mean RhoA activity values relative to the activity at 0 min; bars, SE (n = 3). Student’s t test was done relative to time 0 (a, P < 0.01; b, P < 0.05). C. Representative anti-RhoA immunoblot (n = 3) showing endogenous levels of RhoA-GTP acquired from RBD-agarose affinity-capture assays using lysates from cells treated with 80 Amol/L of LPA for 0, 30, 120, and 300 min (left). The corresponding input lysates were also immunoblotted with anti-RhoA as a loading control (right).

the time course. Talin levels increased 1.4-fold (P < 0.05) and expression from cells treated with LPA was evaluated by 1.7-fold (P < 0.001) in the saponin-soluble pool after 30 and reverse transcription-PCR. No change in MMP-2 RNA levels 120min of LPA treatments, respectively (Fig. 2A, top). Also, was observed with LPA treatment (Fig. 3A). In addition, MMP- talin levels decreased by 36% (P < 0.05) and 45% (P < 0.01) in 2 expression in whole cell lysates and conditioned medium was the insoluble pool (SDS) after 30and 120min of LPA assayed by ELISA. LPA stimulation resulted in 1.8-fold higher treatments, respectively (Fig. 2A, top). Correspondingly, after levels of MMP-2 in cell lysates (Fig. 3B, top) and 1.7-fold 120min of exposure to LPA, vinculin levels increased 2-fold higher levels of MMP-2 in conditioned medium (Fig. 3B, (P < 0.01) in the saponin-soluble pool, and decreased 66% bottom) compared with vehicle treatment. In addition, the time (P < 0.001) in the insoluble pool (Fig. 2A, bottom). Talin and course for LPA induction of pro–MMP-2 activation was vinculin levels in the Triton-soluble pool did not change in a determined over a 24-h period. Active MMP-2 was detected statistically significant manner over the time course of LPA after 4 h of LPA treatment and continued to increase up to treatment (Fig. 2A). These results were consistent with the 24 h (Fig. 3C, top). Accumulation of the active MMP-2 species phalloidin immunofluorescence data and suggest that LPA in response to LPA over the time course was quantified by initiates the stress fiber and focal adhesion disassembly. densitometry. Because active MMP-2 was first detected after Furthermore, the loss of stress fibers suggests that the RhoA/ 4 h of LPA treatment, secretion of active MMP-2 was ROCK pathway is down-regulated upon LPA stimulation. quantified relative to this time point. Active MMP-2 increased 6.7-fold (P < 0.05) and 19.8-fold (P < 0.001) after 12 and LPA Attenuates RhoA Activity 24 h of treatment, respectively (Fig. 3C, bottom). MT1-MMP Because the loss of stress fibers upon LPA stimulation was cell surface expression was also assayed over a time course indicative of possible repression of the RhoA pathway, of LPA treatment for up to 24 h. A representative anti– experiments were done to evaluate the effects of LPA on RhoA MT1-MMP immunoblot is shown in Fig. 3D (top). Densito- activity. RhoA activation assays were done using the Rhotekin metric analysis revealed that MT1-MMP expression increased Rho-binding domain (RBD) conjugated to agarose beads. 1.8-fold after 6 and 8 h of LPA treatment (P < 0.05), relative to Rhotekin, a Rho effector protein, binds only to Rho-GTP 0h (Fig. 3D, middle). The 44 kDa autocatalytic product of (active Rho) and not Rho-GDP (inactive Rho; ref. 25). RBD- MT1-MMP, often detected when enhanced MT1-MMP activity agarose was used to affinity-capture Rho-GTP from cells stimulates pro–MMP-2 processing (28-30), was never detected treated with 80 Amol/L of LPA for 0, 30, 120, and 300 min. As with LPA treatment. Furthermore, secreted TIMP-2 expression shown in Fig. 2B, RhoA activity decreased in a statistically did not change dramatically in LPA-treated cells (Fig. 3D). This significant manner to 51% after 120min of exposure to LPA suggests that LPA stimulates pro–MMP-2 processing by up- (Fig. 2B and C, compare lanes 1 and 3) and then to 30% after regulating MMP-2 and MT1-MMP protein levels, but not by 300 min (Fig. 2B and C, compare lanes 1 and 4) relative to changes in MT1-MMP processing. time 0(P < 0.01 and P < 0.05, respectively). Because LPA stimulation significantly decreased RhoA GTPase The levels of inactive RhoA may also be examined by activity, we wanted to determine if increasing RhoA activity by assaying the levels of RhoA/Rho GDP dissociation inhibitor ectopic expression of a constitutively active RhoA mutant, complexes (26). The GDP dissociation inhibitors are thought RhoA(G14V), would repress LPA-stimulated pro–MMP-2 to inhibit Rho GTPase activity by antagonizing the actions of activation. DOV13 cells were transiently transfected with guanine nucleotide exchange factors and GTPase-activating either a RhoA(G14V) construct or the vector, pcDNA3.1(+), proteins, and by controlling the translocation of the GTPase and then treated with either vehicle or LPA. Interestingly, from the cytosol to the membrane (27). The Rho GDP cells transfected with RhoA(G14V) exhibited a decrease in dissociation inhibitor was immunoprecipitated from lysates LPA-induced pro–MMP-2 activation when compared with prepared after DOV13 cells were treated with 80 Amol/L of cells transfected with empty vector (Fig. 3E, top, compare LPA for 0, 30, 120, and 300 min. However, no dramatic change lanes 2 and 4). Densitometric analysis revealed that in the amount of RhoA associated with Rho GDP dissociation RhoA(G14V) transfection reduced LPA-induced MMP-2 acti- inhibitor was observed for up to 300 min after LPA treatment vation by 90% (P < 0.05) relative to vector-transfected cells when compared with time 0(data not shown). (Fig. 3E, bottom). Collectively, the results indicate that LPA exposure significantly represses RhoA activity through a pathway that was not ROCK Activity Inhibits LPA-Stimulated Pro–MMP-2 mediated by Rho GDP dissociation inhibitor. The down- Activation and Stress Fiber Disassembly regulation of RhoA activity was consistent with the loss of Because expression of RhoA(G14V) negatively regulates stress fibers and the dissociation of focal adhesion components pro–MMP-2 processing, the effects of inhibiting ROCK and from the cytoskeleton observed. MLCK (two enzymes that regulate actin microfilament organization) on MMP processing were evaluated. LPA Up-Regulates MMP-2,and RhoA Activity Down- Regulatory light chain phosphorylation has been shown to be Regulates LPA-Induced Pro–MMP-2 Activation both necessary and sufficient for the formation of stress fibers Pro–MMP-2 activation may occur by several mechanisms and focal adhesions in 3T3 fibroblasts. Both MLCK and ROCK that affect the trimolecular complex formation required for are known to directly phosphorylate regulatory light chains (31). zymogen processing at the cell surface. These mechanisms Given that loss of central stress fibers and increased peripheral include the up-regulation of MMP-2 or MT1-MMP levels, and actin filament bundling was observed upon LPA treatment, the down-regulation of TIMP-2 levels (21). MMP-2 RNA the effect of inhibiting MLCK and ROCK using ML-7 and

FIGURE 3. LPA up-regulates MMP-2 levels, and RhoA can modulate pro – MMP-2 activation. A. MMP-2 RNA expression was assayed by reverse transcription-PCR in cells treated with either vehicle (0.1% BSA/PBS) or LPA (80 Amol/L) for 24 h (n = 3). As a negative control, PCR was done using reverse transcription reactions in which no reverse transcriptase was added (ÀRT); RPL-19 was used as a loading control. B. MMP-2 protein levels in lysates and medium from cells treated with either vehicle (0.1% BSA/PBS) or 80 Amol/L of LPA for 24 h, as assayed by ELISA. Columns, means relative to vehicle treatment; bars, SE (n = 3); Student’s t test (a and b, P < 0.0001). C. Gelatin zymography of conditioned medium from cells treated with LPA (80 Amol/L) for 0, 4, 8, 12, and 24 h (top). Corresponding densitometric analysis of the active MMP-2 bands from gelatin zymograms over the time course of LPA treatment (bottom). Columns, means relative to the 4-h time point when active MMP-2 was first detected; bars, SE (n = 3); columns labeled with different letters are statistically significant as analyzed by repeated measures ANOVA, followed by Tukey’s multiple comparison test (P < 0.05). D. Anti-MT1-MMP immunoblot of cell surface – biotinylated proteins from cells treated with LPA (80 Amol/L) for 0, 2, 4, 6, 8, 12, and 24 h (top). Corresponding densitometric analysis of MT1- MMP expression over the time course of LPA treatment (middle). Data are represented as means F SE relative to 0 h (n = 4); columns labeled with different letters are statistically significant, as analyzed by repeated measures ANOVA, followed by Tukey’s multiple comparison test (P < 0.05). Anti – TIMP-2 immunoblot of conditioned medium from cells treated with LPA (80 Amol/L) for 24 h (n =3;bottom). E. Gelatin zymography of conditioned medium from cells transfected with either the empty vector, pcDNA3.1(+), or RhoA(G14V) construct, and then treated with vehicle (0.1% BSA/PBS) or 80 Amol/L of LPA for 24 h (top). Corresponding densitometric analysis of active MMP-2 bands from gelatin zymograms of vector-transfected and RhoA(G14V)-transfected conditioned medium (bottom). Columns, mean relative to vector-transfected values; bars, SE (n = 3). Student’s t test (*, P < 0.05).

Y-27632, respectively, on LPA-stimulated pro–MMP-2 activa- In addition, inhibition of ROCK with Y-27632 decreased central tion was determined. Quiescent DOV13 cells were treated with stress fibers and enhanced LPA-induced loss of stress fibers and either Y-27632 or ML-7, in the presence or absence of LPA, and focal adhesions (Fig. 4B). In contrast, inhibition of MLCK conditioned medium was subjected to gelatin zymography to activity did not significantly affect LPA-induced pro–MMP-2 evaluate pro–MMP-2 processing. When cells were treated with activation (Fig. 4A, bottom). Densitometric analysis showed increasing concentrations of Y-27632 in conjunction with that LPA-stimulated MMP-2 activation (white column) did not 80 Amol/L of LPA (Fig. 4A, top left, lanes 4-6), we observed change in a statistically significant manner with any dose of a dose-dependent increase in pro–MMP-2 activation when ML-7 (gray columns) used (Fig. 4A, bottom). Furthermore, compared with LPA treatment alone (Fig. 4A, top left, lane 2). MLCK inhibition (with ML-7) decreased peripheral actin Treatment with 10 Amol/L of Y-27632 and LPA increased filament bundles compared with control cells and attenuated MMP-2 activation by 1.8-fold (P < 0.05) when compared with LPA-induced peripheral actin filament bundling when compared LPA treatment alone (Fig. 4A, top, compare columns 1 and 4). with cells treated with LPA alone (Fig. 4B).

FIGURE 4. ROCK and MLCK play different roles in pro – MMP-2 processing and actin microfilament organization. A. Gelatin zymography of conditioned medium from cells treated with Y-27632 (0.2, 1, and 10 Amol/L) and ML-7 (0.2, 5, and 10 Amol/L) in the presence or absence of 80 Amol/L LPA for24h(left). Graphs depicting corresponding densitometric analysis of the active MMP-2 bands from LPA- treated cells (1) and cells treated with increasing concentrations of inhibitor (Y-27632 or ML-7) and LPA (2-4)on gelatin zymograms (right). Columns, means relative to LPA treatment alone; bars, SE (n = 3 for each inhibitor). Columns labeled with different letters are statistically significant as analyzed by repeated measures ANOVA, followed by Tukey’s multiple comparison test (P < 0.05). B. Texas red – phalloidin immunofluorescence of cells treated with the following for 24 h: vehicle (0.1% BSA/PBS/DMSO); 80 Amol/L of LPA; 10 Amol/L of ML7; 10 Amol/L of ML-7/80 Amol/L of LPA; 10 Amol/L of Y- 27632; and 10 Amol/L of Y-27632/80 Amol/L of LPA (n = 3). Arrows, central stress fibers; arrowheads, peripheral actin filaments; bar, 100 Am. Bottom, bright-field images of cells treated with either vehicle (0.1% BSA/PBS) or 80 Amol/L LPA for 24 h (n = 3). Note that LPA-treated cells round upand control cells are more flattened (arrowheads, single cell within each bright-field image; bar, 50 Am.).

Collectively, these results suggest that, in DOV13 cells, Quiescent DOV13 cells were treated with cytochalasin D (cyD), MLCK plays a role in peripheral actin filament formation, which disrupts actin filaments, for 24 h and then conditioned whereas ROCK plays a role in central stress fiber formation. medium was subjected to gelatin zymography. Treatment of Also, disruption of ROCK function and not MLCK function cells with cyD alone was sufficient to stimulate processing of enhances LPA-stimulated pro–MMP-2 activation. pro–MMP-2 to its intermediate form (Fig. 5A, top left, lane 3). CyD also enhanced LPA-regulated pro–MMP-2 activation in a Disruption of Actin Filaments Augments LPA-Induced concentration-dependent manner (Fig. 5A, top left, lanes 4-6) Pro–MMP-2 Activation,but Stabilization of Actin Filaments when compared with LPA treatment alone (Fig. 5A, top left, Represses Pro–MMP-2 Activation lane 2). When compared with LPA treatment alone, the addition Because LPA induces pro–MMP-2 activation and changes in of 1 and 3 Amol/L of cyD increased LPA-induced active MMP-2 the actin cytoskeleton, the effects of disrupting versus stabilizing secretion by 1.8-fold and 2.0-fold (P < 0.01), respectively actin filaments on pro–MMP-2 activation were evaluated. (Fig. 5A, top right, compare column 1 with columns 3 and 4).

Furthermore, treatment with cyD disrupted central stress fibers The effects of restoring stress fiber formation on LPA- and peripheral filaments, with some cortical filaments still triggered pro–MMP-2 activation were also assessed by visible at the cell periphery (Fig. 5B). Peripheral filament treating cells with phalloidin. Phalloidin treatment diminished staining was more intense in cells treated with both cyD and LPA-stimulated pro–MMP-2 activation in a dose-dependent LPA than in cells treated with cyD alone (Fig. 5B). CyD manner (Fig. 5A, middle left, lanes 4-6) when compared with was much more efficient at disrupting central stress fibers than LPA treatment alone (Fig. 5A, middle left, lane 2). Treatment Y-27632, which may explain why cyD treatment alone was with 20 Ag/mL of phalloidin decreased LPA-dependent pro– sufficient to promote more robust processing of pro–MMP-2 MMP-2 activation by 40% (P < 0.05) when compared with to its intermediate form. LPA treatment alone (Fig. 5A, middle right, compare columns

FIGURE 5. Effects of cytoskeletal modulators on pro – MMP-2 processing and actin microfilament organization. A. Gelatin zymography of conditioned medium from cells treated with cyD (cyD, 0.1, 1, and 3 Amol/L), phalloidin (Phall, 2, 5, and 20 Amol/L), and nocodazole (Noc, 10 Amol/L) in the presence or absence of 80 Amol/L LPA for 24 h (left). Conditioned medium from cells treated with vehicle (BSA/PBS/DMSO) was used as a control. Right, graphs depicting corresponding densitometric analysis of the active MMP-2 bands from cells treated with LPA alone (1) and cells treated with both inhibitor (cyD, Phall, or Noc) and LPA (2-4). Columns, means relative to LPA treatment alone; bars, SE (n = 3). For cyD and phalloidin treatment, columns labeled with different letters are statistically significant as analyzed by repeated measures ANOVA, followed by Tukey’s multiple comparison test (P < 0.05). For nocodazole treatment, paired Student’s t test was done (*, P <0.05).B. Texas red – phalloidin immunofluorescence showing actin microfilament organization of quiescent cells treated with the following for 24 h: vehicle (0.1% BSA/PBS/DMSO), 80 Amol/L of LPA, 1 Amol/L of cyD, 1 Amol/L of cyD/80 Amol/L of LPA, 20 Amol/L of phalloidin, 20 Amol/L of phalloidin/80 Amol/ L of LPA, 10 Amol/L of nocodazole, and 10 Amol/L of nocodazole/80 Amol/L of LPA (n = 3). Arrows, central stress fibers; arrowheads, peripheral actin filaments; and arrowheads in Noc panels, focal contacts (bar, 100 Am).

1 and 4). Phalloidin treatment also restored stress fiber formation in LPA-treated cells, although the stress fibers seemed thinner when compared with those in vehicle-treated cells (Fig. 5B). To ensure that the induction of pro–MMP-2 processing was not simply due to the disruption of the cytoarchitecture of the cell, we also treated cells with nocodazole, a compound that destabilizes microtubules. Unlike cyD, nocodazole treatment alone had no effect on pro–MMP-2 processing (Fig. 5A, bottom left, lane 3), but significantly inhibited LPA-stimulated pro–MMP-2 activation (Fig. 5A, bottom left, lane 4) when compared with LPA treatment alone (Fig. 5A, bottom left, lane 2). Nocodazole treatment repressed LPA-induced active MMP- 2 secretion by 80% (P < 0.05) relative to LPA treatment alone (Fig. 5A, bottom, compare columns 1 and 2). Interestingly, nocodazole treatment induced the formation of peripheral focal contacts and restored some stress fiber formation in LPA-treated cells (Fig. 5B). Collectively, these data suggest that loss of stress fibers and focal adhesions enhances LPA-induced pro– MMP-2 processing, whereas stabilization of actin filaments represses LPA-induced pro–MMP-2 processing.

ROCK Activity Is Required for LPA-Induced Haptotactic Migration in Cells Cultured at Low Density Our results show that when cells were cultured at a high density, LPA down-regulates RhoA/ROCK activity, which contributes to pro–MMP-2 activation. However, when cells were cultured at a low density, stress fibers were present even after LPA stimulation (Fig. 1). We previously reported that LPA induces EOC haptotactic cell migration (19). Because stress FIGURE 6. ROCK activity is required for LPA-stimulated haptotactic fibers are present in cells treated with LPA at a low density, we cell migration in EOC cells cultured at low density. Cells were cultured at low density on coverslips coated with colloidal gold and type I collagen, next wanted to determine if the RhoA/ROCK pathway plays a and then allowed to migrate for 24 h before quantifying phagokinetic role in LPA-induced EOC cell migration. DOV13 cells were tracks. A. Bright-field images of phagokinetic tracks made by individual migrating cells on colloidal gold – coated coverslips treated with vehicle, 80 cultured at a low density on glass coverslips coated with Amol/L of LPA, 20 Amol/L of Y-27632, and 20 Amol/L of Y-27632/80 Amol/L colloidal gold and type I collagen, and the area of phagokinetic of LPA. B. Average areas of phagokinetic tracks for individual cells treated tracks of individual migrating cells was quantitated. LPA with Y-27632 (1, 10, and 20 Amol/L) in the presence or absence of 80 Amol/L of LPA, relative to control (vehicle) treatment. Columns, means stimulated EOC migration 1.9-fold (P < 0.001) relative to relative to vehicle; bars, SE (n = 3). Columns labeled with different letters vehicle treatment, and inhibition of ROCK activity with Y- are statistically significant as analyzed by repeated measures ANOVA, 27632 repressed LPA-induced cell migration (in a dose- followed by Tukey’s multiple comparisons test (P < 0.05). dependent manner) down to basal levels (Fig. 6B). The stimulation of cell migration by LPA, in addition to the autocrine loops that promote tumorigenic pathways such as repression of LPA-stimulated cell migration by Y-27632, could survival, proliferation, migration, invasion, and angiogenesis be visualized by the images of the phagokinetic tracks (black (6). The studies presented here investigated the relationship area on the colloidal gold background) made by the cells from between LPA-mediated changes in actin microfilament each treatment (Fig. 6A). organization and MMP activation. We found that, in EOC Taken together, these results show that LPA exerts cells cultured to a high density, LPA induced the loss of stress differential effects on actin microfilament organization depend- fibers and focal adhesions, as well as the formation of ing on EOC cell density. These differential effects may promote peripheral actin filaments. Consistent with the observed metastatic dissemination by promoting the loss of focal disassembly of stress fibers, RhoA activity decreased signif- adhesion and pro–MMP-2 activation in cells at a high density, icantly in response to LPA. These findings show that LPA and migration in cells at a low density. stimulation attenuated RhoA activity in DOV13 cells, in contrast with results reported for other quiescent normal and malignant epithelium, which form stress fibers as a result of Discussion RhoA activation by LPA (32-34). LPA is thought to play a unique role in the pathobiology of LPA has different effects on the actin cytoskeleton of ovarian cancer because levels of this bioactive lipid are DOV13 cells cultured at a low density (stress fibers remain) elevated (2-80 Amol/L) in the plasma and ascites of patients versus high density (loss of stress fibers). The unique changes with early- and late-stage ovarian cancer (4, 7, 8). In ovarian in actin microfilament organization induced by LPA may be due cancer cells, LPA can induce LPA production, generating to the fact that EOC cells possess both epithelial and

mesenchymal characteristics (35). Ovarian surface epithelia when LPA treatment relaxes mechanical tension in EOC cells express smooth muscle actin, a mesenchymal marker, and can by inducing the disassembly of stress fibers and focal contract a collagen gel lattice in vitro (36). EOC cells also adhesions. In addition, LPA treatment up-regulated MMP-2 produce LPA and overexpress LPA receptors, which may affect expression, which can facilitate trimolecular complex formation microfilament organization (6, 7). It will be critical to elucidate and autolytic cleavage of the intermediate MMP-2 form the factors, such as cadherins, that dictate how LPA required for zymogen activation at the cell surface. differentially modulates RhoA activity in high-density versus Collectively, our data are consistent with a model in which low-density conditions. Cadherin family members have been LPA promotes EOC metastasis by triggering loss of adhesion implicated in the regulation of MMP expression and invasive and activation of MMPs, or haptotactic migration. Further potential of tumor cells (37-39). elucidation of mechanisms by which LPA induces modifica- High-density cultures can be thought of as a simplified two- tions of the microfilament system and processing of proteinases dimensional model of cells within a solid tumor, whereas low- may lead to new insights in ovarian oncogenesis and the design density cultures can be thought of as metastatic populations that of novel therapeutic targets. may exist as single cells or as small colonies. We propose a model in which LPA is elevated in the ovarian cancer cell microenvi- ronment and has different effects on EOC cells depending on the Materials and Methods pathobiological status of malignant epithelia. LPA may promote Cell Culture,Treatments,and Transfections loss of stress fibers and focal adhesions as well as pro–MMP-2 The DOV13 cell line, a generous gift from Dr. Robert Bast, activation in cells within a solid tumor. Loss of adhesion would Jr. (M.D. Anderson Cancer Center, Houston, TX), was cultured facilitate the dissociation of single cells or small groups of cells as previously described (51). OVCAR3 and OVCA429 cells from the primary tumor, allowing EOC cells to attach to the i.p. were grown in RPMI 40supplemented with 20%fetal bovine cavity and, hence, metastasize. In fact, we have reported that LPA serum and MEM supplemented with 10% fetal bovine serum stimulated MMP-dependent EOC haptotactic migration and (Invitrogen, Carlsbad, CA), respectively. For all experiments, invasion (19). In the current study, stress fibers and focal cells were cultured to confluency and serum-starved for adhesions were present after LPA treatment of EOC cells 24 h before treatment, except when low-density conditions Â 3 cultured at a low density, indicating that RhoA was active. were specified, cells were plated at 1.5 10 cells/mL. Cells Furthermore, Rho-kinase/ROCK function is required for LPA- were pretreated with phalloidin (Molecular Probes, Eugene, induced haptotactic migration on thin layer type I collagen. OR) during the 24-h serum starvation period. For all other Several studies have shown that disruption of the actin inhibitors, cells were pretreated with inhibitor for 1 h before A cytoskeleton by cyD promotes the activation of pro–MMP-2 culturing cells in the presence of inhibitor and 80 mol/L of (40-42). LPA may work through a similar mechanism to 18:1 LPA (Avanti Polar Lipids, Alabaster, AL) for 24 h. ML-7, activate pro–MMP-2 by triggering the disassembly of stress nocodazole, and cyD were from Sigma-Aldrich (St. Louis, fibers. In DOV13 cells, ROCK regulates central stress fiber MO), whereas Y-27632 was from EMD Biosciences (San formation, whereas MLCK regulates peripheral filament Diego, CA). For transient transfections, cells were maintained formation. Inhibition of MLCK activity did not significantly in Opti-MEM after reaching confluency, and then transfected A affect LPA-mediated pro–MMP-2 activation, but if ROCK using Lipofectamine 2000 Reagent (Invitrogen) and 1.6 gof activity is inhibited, further augmenting LPA-induced loss of DNA [pcDNA3.1(+) or pcDNA3.1(+)_RhoA(G14V)] for each stress fibers, then an increase in LPA-triggered pro–MMP-2 well of a 12-well plate, according to the instructions of the activation is observed. Also, counteracting LPA repression of manufacturer. A second transfection was done 24 h later, and A RhoA by expression of the constitutively active mutant, cells were treated on the same day with 80 mol/L of LPA for RhoA(G14V), down-regulated pro–MMP-2 activation. 24 h. Transfection efficiencies were determined by cotransfec- A Mechanical stress relayed by the extracellular matrix and the tion of the pEGFP construct (0.1 g). organization of the actin microfilament system has been shown to play a role in MMP expression and activation. When Immunofluorescence and Imaging Techniques fibroblasts were grown on substrates that promote cell Cells were cultured on glass coverslips and fixed in 4% spreading, such as attached collagen substrates, stress fibers paraformaldehyde in 20mmol/L of HEPES (pH 7.4) and 150 and focal adhesions form and most of the MMP produced is mmol/L of NaCl for 20min, permeabilized in 0.1%Triton X- inactive (43-45). In contrast, relaxation of mechanical tension 100 in PBS for 10 min, blocked with 1% bovine serum albumin by either cyD treatment or culture on a relaxed collagen gel (BSA)/PBS for 1 h, and then incubated at room temperature for changes the cell shape (cell retraction or rounding) and up- 1 h with Texas red–phalloidin (Molecular Probes) at 1:100 in regulates MMP expression and activation in multiple cell types blocking solution. Images were acquired on a Nikon Eclipse (42, 46, 47). Similarly, giant cell tumor cells that are exposed to TE2000-U microscope using the SPOT digital camera and soluble RGD peptides, which disrupt integrin binding and cause Metamorph 5.0software. cell rounding, process pro–MMP-2 to its active form (48). Fibroblasts grown in mechanically relaxed collagen lattices Gelatin Zymography exhibit microfilament morphology similar to that of LPA- To analyze pro–MMP-2 processing, cells were cultured to treated EOC cells: cortical filaments are present, but central confluency in 12-well plates (200,000 cells/mL), grown over- stress fibers are severely diminished (43, 45, 49, 50). This is night, and then treated as described above. Conditioned medium analogous to the induction of pro–MMP-2 activation observed was subjected to SDS-PAGE (52) under nonreducing conditions,

using 9% polyacrylamide gels containing 0.1% gelatin. Gels mmol/L NaCl, 2 mmol/L EGTA, 5 mmol/L EDTA, 1 mmol/L were washed twice in 2.5% Triton X-100 for 30 min, Na3VO4, 25 mmol/L NaF] for 30min, scraped, and incubated in 20mmol/L of glycine, 10mmol/L of CaCl 2, and centrifuged at 15,000 rpm for 30 min. The supernatant was 1 Amol/L of ZnCl2 (pH 8.3) at 37jC for 24 h, and then stained in designated the saponin-soluble pool (S), and the pellet was Coomassie blue to detect areas of gelatinolytic activity. resuspended in Triton X-100 buffer (same composition as saponin buffer, but with 1% Triton X-100 instead of saponin). RhoA Activation Assay The Triton X-100 lysate was centrifuged for 30 min, and the To assess RhoA activation, the amount of RhoA-GTP bound to resulting supernatant was designated the Triton-soluble pool the Rhotekin RBD was determined using the Rho Activation (TX). The remaining pellet (P) was resuspended in Laemmli Assay Kit (Upstate Biotechnology, Lake Placid, NY) according to sample buffer. Whole cell lysates were prepared by incubating the instructions of the manufacturer. Briefly, DOV13 cells were cells in Laemmli sample buffer for 20min at 4 jC and then cultured to confluency, made quiescent, and then treated with boiling for 5 min. Biotinylation of cell surface proteins was 80 Amol/L of LPA before solubilizing with Mg2+ lysis buffer done by incubating cells with 0.5 mg/mL of Sulfo-NHS-LC- [125 mmol/L HEPES (pH 7.5), 750mmol/L NaCl, 5% Igepal Biotin (Pierce, Rockford, IL) for 30min at 4 jC. Free biotin was then quenched with 100 mmol/L of glycine and cells CA-630, 50 mmol/L MgCl2, 5 mmol/L EDTA, 25 mmol/L NaF, were lysed [50mmol/L Tris (pH 7.4), 150mmol/L NaCl, 1 mmol/L Na3VO4, and 10% glycerol]. Protein concentration was determined using the DC Protein Assay (Bio-Rad, Hercules, CA) 5 mmol/L EDTA, 1% Triton X-100, and 0.1% SDS] and then and equal amounts of protein were incubated with RBD-agarose clarified by centrifugation. Biotinylated proteins were isolated j for 45 min at 4jC. Agarose beads were washed thrice with lysis by incubation with streptavidin beads (Pierce) at 4 C for buffer, resuspended in Laemmli sample buffer, and boiled for 16 h and then boiled in Laemmli sample buffer. 5 min. Lysates were immunoblotted with anti-RhoA. Cell extracts were subjected to SDS-PAGE on a 7% or 15% polyacrylamide gel. Proteins were transferred to nitrocellulose, Densitometry blocked for 1 h in either TPBS (0.005% Tween 20 in PBS) or 2% Densitometric analysis was done on (a) the active MMP-2 milk/TPBS, and blotted with anti-vinculin (Sigma), anti-talin bands from gelatin zymograms; (b) anti-MT1-MMP, anti-talin, (Sigma), or anti-RhoA (Santa Cruz Biotechnology, Santa Cruz, and anti-vinculin immunoblots, (c) and RhoA activity results CA) at 1:1,000 dilutions. Blots were washed thrice for 5 min in from at least three independent experiments using Molecular TPBS, incubated with anti-mouse antibody conjugated to horse- Analyst 1.5 and Image J 1.34s software. For gelatin zymo- radish peroxidase (1:5,000) for 1 h, washed twice for 5 min in grams, the densities of the active MMP-2 bands from different TPBS and twice for 5 min in PBS, and then developed with treatment conditions were compared, and these analyses SuperSignal West Pico Chemiluminescent Substrate (Pierce). assumed that all bands were within the linear range. All statistical analyses were done using Prism 4 software. Haptotactic Migration Assay For cell migration assays, cells were plated at 1.5 Â 103 MMP-2 ELISA cells/mL (low density conditions) onto 25 mm square glass Analysis of pro- and active MMP-2 protein concentration in coverslips coated with type I collagen (10 Ag/mL) and gold whole cell lysates and conditioned media were done using the chloride trihydrate (Sigma) as described by Albrecht-Buehler Quantikine MMP-2 (total) ELISA kit (R&D Systems, Minne- (53). Cells were allowed to adhere for 2 h before treatment and apolis, MN) according to the instructions of the manufacturer. then allowed to migrate for 24 h before fixation with 0.1% Transfection-conditioned media were concentrated 3-fold. formaldehyde. The area of a phagokinetic track from a single Samples from three independent experiments were assayed in cell was quantified using a Wesco Verta VU 7000 series duplicate and absorbance values were acquired on a Spectramax inverted microscope and NIH Scion Image software (version Plus 384 plate reader (Molecular Devices, Sunnyvale, CA). 4.0). The areas of 50 phagokinetic tracks per coverslip, from duplicate sets of coverslips for each treatment, were averaged Reverse Transcription-PCR for each experiment; three independent experiments were done. Total RNA was isolated from DOV13 cells using Qiagen’s RNeasy kit according to the instructions of the manufacturer (Qiagen, Valencia, CA). Reverse transcription reactions were Acknowledgments We thank Shankar Srinivasan, Monica Antenos, and Niti Jetly for critical reading carried out using 2 Ag of RNA (DNaseI-treated) and the of this manuscript. RETROScript kit according to the protocols of the manufacturer. The primers used to amplify MMP-2 were: 5¶-TTGAG AAGGATGGCAAGTACGGCT-3¶ and 5¶-AGAGCTCCT- References 1. Bast RC, Jr. Status of tumor markers in ovarian cancer screening. J Clin Oncol GAATGCCCTTGATGT-3¶. The primers used to amplify 2003;21:200 – 5. RPL19 were: 5¶-ATGAAATCGCCAATGCCAACTCCC 3¶ 2. Society AC. Cancer facts and figures 2003. and 5¶-CTGCCTTCAGCTTGTGGATGTGTT-3¶. 3. Cvetkovic D. Early events in ovarian oncogenesis. Reprod Biol Endocrinol 2003;1:68. Cellular Extracts,Cell Surface Biotinylation,and Immu- 4. Xu Y, Shen Z, Wiper DW, et al. Lysophosphatidic acid as a potential noblotting biomarker for ovarian and other gynecologic cancers. JAMA 1998;280:719 – 23. 5. Sutphen R, Xu Y, Wilbanks GD, et al. 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Mol Cancer Res 2007;5(2). February 2007 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Lysophosphatidic Acid Down-Regulates Stress Fibers and Up-Regulates Pro−Matrix Metalloproteinase-2 Activation in Ovarian Cancer Cells

Thuy-Vy Do, Jay C. Symowicz, David M. Berman, et al.

Mol Cancer Res 2007;5:121-131.

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