Lysophosphatidic Acid Induces Urokinase Secretion by OvarianCancer Cells1

Terri B. Pustilnik, Veronica Estrella,Jon R. Wiener, Muling Mao, Astrid Eder,Mary-Anne V. Watt, Robert C. Bast, Jr., andGordon B. Mills 2

The University of Texas M. D. Anderson Cancer Center, Houston,Texas 77030

ABSTRACTLysophosphatidic acid (LPA) is present at high concen-

trations in ascites from ovarian cancer patients and haspotent mitogenic propertiesin vitro. Urokinase plasminogenactivator (uPA), a critical component of the metastatic cas-cade, is also found at high concentrations in ovarian ascitesand ovarian cancers, and the levels of uPA correlate in-versely with prognosis. Because LPA stimulates the invasionof both hepatoma and lung cell lines, we investigatedwhether LPA could induce uPA secretion by ovarian epi-thelial cells and whether this process was associated withmalignant transformation of ovarian epithelial cells. As in-dicated by zymography and Western blotting, physiologi-cally relevant concentrations of LPA equivalent to thosepresent in ovarian cancer ascites stimulated uPA secretionin the ovarian cancer cell lines OVCAR-3, SKOV-3, OVCA429, OVCA 432, and OVCA 433, but not from establishednormal ovarian epithelial (NOE) cells as indicated by nor-mal epithelial cell lines NOE 033 and NOE 035 or from SV40large T antigen-immortalized normal epithelial cell linesIOSE 29 and IOSE 80. 18:1 LPA, but not 18:0 LPA, 16:0LPA, or lysophosphatidylcholine, induced uPA secretion,concordant with previous studies of LPA receptor selectiv-ity. Expression of the edg-2 LPA receptor was not consis-tently different between normal epithelial cell lines andovarian cancer cell lines. In contrast, expression of the edg-4LPA receptor was markedly increased in ovarian cancer celllines as compared with NOE cell lines, raising the possibilitythat the edg-4 LPA receptor contributes to the ability ofovarian cancer cells but not NOE cells to produce uPA inresponse to LPA. LPA induced a consistent increase in uPApromoter activity and mRNA levels, suggesting that in-creased uPA production is, at least in part, transcriptional.

Malignant transformation may alter LPA-induced cell acti-vation by altering the pattern of LPA receptors present andmay possibly lead to more aggressive behavior by up-regu-lating LPA-mediated uPA secretion and stimulating extra-cellular stromal breakdown and invasion.

INTRODUCTIONOvarian cancer is predominantly detected at a late stage

after extensive intra-abdominal spread and is often accompaniedby the formation of ascites (1). Many growth-promoting factorsare known to be present in ovarian cancer ascites, frequently atlow levels. LPA,3 in contrast, is found at significant levels(2–80mM) in ovarian ascites (2, 3) and may play an importantrole in the development or progression of ovarian cancer. LPAfunctions as an extracellular signaling molecule, and its specificfunction is likely to depend on the local environment, the celllineage, and the spectrum of LPA receptors expressed by the cell(4). LPA induces a plethora of cellular responses includingsmooth muscle contraction, neurite retraction, and focal adhe-sion assembly (4, 5). LPA also has potent mitogenic propertiesleading to increases in proliferation in ovarian and breast cancercell lines (2, 6). LPA may also be involved in the metastaticprocess because it has recently been shown to stimulate invasionin a rat ascites hepatoma and a small cell lung cancer cell line(7).

uPA contributes to metastasis and migration because itcatalyzes the conversion of plasminogen to plasmin, thus lead-ing to degradation of the basement membrane (8). It also stim-ulates cellular migration and proliferation (9) potentially bybinding to its specific cell surface receptor (uPA receptor). uPAhas been linked to malignant transformation in ovarian (10),breast (11), and colon (12) cell lineages. In ovarian cancer,cellular uPA levels correlate inversely with prognosis (13, 14).In striking contrast to other cell lineages, uPA production can belocalized to ovarian cancer cellsin vivo (15). uPA is also presentin significant levels in ascites (15) and is secreted by humanovarian carcinoma cell lines (10, 16, 17). Furthermore, levels ofthe uPA receptor are increased in ovarian cancers (18).

Because LPA and uPA are both present at elevated levelsin ovarian cancer, and LPA can induce invasiveness in other celllineages, we investigated whether LPA could induce uPA se-cretion in normal and ovarian cancer cell lines. We demonstratethat LPA induces an increase in uPA promoter activity, mRNAlevels, protein levels, and enzyme activity in ovarian cancer cell

Received 5/15/99; revised 8/2/99; accepted 8/3/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported by NIH Grant CA64602.2 To whom requests for reprints should be addressed, at The Universityof Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard,Box 317, Houston, TX 77030. Phone: (713) 792-7770; Fax: (713)794-1807; E-mail: gmills@notes.mdacc.tmc.edu.

3 The abbreviations used are: LPA, lysophosphatidic acid; uPA, uroki-nase plasminogen activator; LPC, lysophosphatidylcholine; NOE, nor-mal ovarian epithelial; ATCC, American Type Culture Collection; SFM,serum-free medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide; GAPDH, glyceraldehyde-3-phosphate dehy-drogenase.

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lines but not in NOE cell lines. This increase is associated witha marked increase in expression of the edg-4 LPA receptor inovarian cancer cells.

MATERIALS AND METHODSReagents. Synthetic lysophospholipids (18:1, 18:0, and

16:0 LPA and LPC) were purchased from Avanti Polar LipidInc. (Alabaster, AL). A polyclonal rabbit antibody against hu-man urokinase (uPA) was obtained from American Diagnostica,Inc. (Greenwich, CT). Plasminogen was obtained from Boeh-ringer Mannheim (Indianapolis, IN).

Cell Culture. OVCAR-3 and SKOV-3 ovarian cancercells and SKBR 3 breast cancer cells were obtained from ATCC(Manassas, VA). They were cultured in RPMI 1640 (Life Tech-nologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated FCS (Sigma), 1% penicillin, and 1% streptomycin(Life Technologies, Inc.). NOE cells were cultured from freshsurgical specimens according to an institutional review board-approved protocol at The University of Texas M. D. AndersonCancer Center. Cells were scraped from the surface and cultured

in a 1:1 mixture of MCDB 105 medium (Sigma) and Medium199 (Life Technologies, Inc.) supplemented with 10 ng/ml epi-dermal growth factor (Sigma), 10% FCS, 10%L-glutamine, 1%penicillin, and 1% streptomycin (NOE media; Ref. 19). Immor-talized NOE cells (IOSE 029 and IOSE 80; Ref. 19) wereobtained from Dr. N. Auersperg (University of British Colum-bia, Vancouver, Canada) and were also maintained in the mediaused to culture NOE cells, but without the addition of epidermalgrowth factor and the reduction of FCS from 10% to 5%.NIH3T3 cells (ATCC) were cultured in DMEM (Life Technol-ogies, Inc.) containing 10% FCS, 10%L-glutamine, 1% peni-cillin, and 1% streptomycin. OVCA 429, OVCA 432, andOVCA 433 (a generous gift from Dr. R. Bast, University ofTexas M. D. Anderson Cancer Center) were maintained inRPMI 1640 supplemented with 10% heat-inactivated FCS, 1%penicillin, and 1% streptomycin.

LPA Stimulation. A 20 mM LPA stock solution wasprepared in chloroform (Sigma) and stored at220°C. Immedi-ately before use, the required aliquot was dried down understerile conditions, and the LPA pellet was gently resuspended in1% delipidated BSA (Sigma) for 30 min on ice. The cell lineswere cultured to near confluence in complete medium, washedwith SFM, and then starved in SFM for 16–24 h. Fresh SFMwas replaced before LPA addition.

Zymography. Zymography was performed by themethod outlined by Heussen and Dowdle (20). Serum-free su-pernatants from each cell culture, after normalization for viablecell number by the MTT assay (21), were analyzed undernonreducing conditions on 7.5% SDS-PAGE (Sigma) contain-ing 0.1% casein and 4 mg of plasminogen. The gel was thenincubated at room temperature in a 2.5% Triton X-100 solutioncontaining 0.05% sodium azide and 0.05M Tris (pH 7.5; Sigma)followed by incubation at 37°C in 0.1M Tris-HCl (pH 8.3)/0.5M sodium chloride solution for 16 h. The gel was then stainedwith a 0.25% Coomassie Blue/1.1% acetic acid/45.5% methanolsolution for 40 min, followed by destaining in a 25% methanol/10% acetic acid mixture. Enzyme activity was visualized asclear zones on a blue background after conversion of plasmin-ogen to plasmin by uPA. UMSCC (a squamous carcinoma cellline kindly provided by Dr. T. Carey, University of Michigan,Ann Arbor, MI) was used as a positive control.

Western Blot Analysis. Serum-free cell culture superna-tant was concentrated 10–30-fold by Centricon 10 columns(Amicon, Beverly, MA). Sample volumes were normalized forcell number by the MTT dye conversion assay (19) and/or BCAprotein quantification of cell supernatants (Pierce, Rockford, IL)as indicated. Supernatant proteins were separated by 12.5%SDS-PAGE, transferred to Immobilon-P membrane (Millipore,Bedford, MA), blocked with 3% BSA or 4% skim milk powder,and probed using a 1:1000 dilution of rabbit polyclonal antibodyto uPA. The membrane was incubated with horseradish perox-idase-conjugated antirabbit IgG (Bio-Rad, Hercules, CA), andimmunoreactive bands were visualized with enhanced chemilu-minescence (Amersham, Arlington, IL). As a positive control,25 ng of recombinant-uPA provided by Dr. J. Henkin (AbbottLaboratories, Chicago, IL) were included.

Northern Blot Analysis. Northern blot analysis for thequantitation of uPA-specific mRNA was performed as describedpreviously (22). Briefly, total RNA isolated from OVCAR-3

Fig. 1 LPA induces uPA production by ovarian cancer cells but not bynormal ovarian epithelium. Cells were starved overnight in the absenceof serum. Conditioned SFM was collected from the NOE cells NOE033, NOE 035, IOSE 29, and IOSE 80 and the ovarian cancer cell linesOVCAR-3 and SKOV-3 after a 24-h incubation with 20mM LPA. Thesupernatants were normalized based on the viable cell number using aMTT assay and then concentrated 30-fold. Samples were electrophore-sed on a 12.5% SDS-PAGE gel and electroblotted onto an Immobilon-Pmembrane. The blot was then probed with a polyclonal rabbit antibodyto uPA after blocking with 3% BSA and probed using a 1:1000 dilutionof rabbit polyclonal antibody to uPA. The uPAarrow on the rightrepresents the migration of recombinant uPA in a parallel lane.

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cells by the method of Chomczynski and Sacchi (23) waselectrophoresed (10mg/lane) through 1% agarose gels contain-ing 0.22M formaldehyde (Sigma). uPA (ATCC), edg-2, edg-4,and GAPDH probes were radiolabeled by random prime label-ing using the Redi-Prime labeling kit (Amersham). Membraneswere incubated with radiolabeled probes in 50% formamide, 53Denhardt’s solution, 0.5% SDS, 50 mM HEPES (pH 7.0), 4.83SSC, and 100mg/ml salmon sperm single-strand DNA (Sigma)at 42°C for 18 h. Blots were washed three times in 0.13 SSCcontaining 0.1% SDS at room temperature and then washedthree times (for 20 min each) at 50°C in 0.13 SSC/0.1% SDSbefore autoradiography at280°C for 3 days with one screen orwith a phosphorimager. Stripped blots were probed for GAPDHexpression to standardize RNA loading.

Transient Transfection and the Luciferase ReporterAssay. Trypsinized and washed ovarian cancer cells(OVCAR-3 and SKOV-3 cells) were resuspended in completemedium at 53 106 cells/ml, and 700ml were reserved for eachelectroporation. The cells were gently mixed with 10mg of theluciferase construct (pRSV-luc or puPA-luc; provided by Dr. T.Silverman, NIH, Bethesda, MD) and pulsed at 250 V/940mFusing a Gene Pulser II system (Bio-Rad). The cells were thenrecovered in 10 ml of complete medium and cultured in dishesfor 24 h. The cells were starved in SFM for 12 h before LPA

addition in fresh SFM. LPA stimulation was performed over anadditional 12-h incubation. After a 48-h total incubation, the cellmonolayer was washed times three with ice-cold PBS and lysedusing Promega (Madison, WI) reporter lysis buffer for 10 minon ice. The cells were scraped and collected in a microfuge tubeand vortexed vigorously, and the lysate was recovered afterfull-speed centrifugation at 4°C for 10 min. For the luciferaseassay, 15mg of total lysate protein, as determined by the BCAassay, were resuspended in 50ml of lysis buffer and au-tosampled using the Analytical Luminescence Monolight 2010(San Diego, CA).

RESULTSLPA induces a striking increase in the level of uPA in the

supernatants of OVCAR-3 and SKOV-3 cells, as shown byWestern blot analysis (Fig. 1) and by zymography. Low butsignificant levels of uPA protein are present in resting superna-tants of SKOV-3 cells, but not OVCAR-3 cells. This may reflectthe constitutive production of LPA by SKOV-3 cells but notOVCAR-3 cells (data not shown). LPA-induced uPA secretioncould be detected by 12 h, increased until 24 h (Fig. 2), andpersisted for at least 48 h. LPA also induced an increase in uPAproduction by other ovarian cancer cell lines including OVCA

Fig. 2 Time dependence of LPA-induced uPA production. Cells were starved overnight in SFM. Supernatants were collected from OVCAR-3 andSKOV-3 cells incubated in 20mM LPA in SFM for the indicated times. Supernatants were normalized for viable cell number as assessed by MTTand concentrated 30-fold. The samples were then electrophoresed on a 12.5% SDS-PAGE gel and electroblotted onto an Immobilon-P membrane. Theblot was then probed with a polyclonal rabbit antibody to uPA after blocking with 3% BSA and probed using a 1:1000 dilution of rabbit polyclonalantibody to uPA. The location of the uPA band was determined by running recombinant uPA in a parallel lane.

Fig. 3 Concentration depend-ence of LPA-induced uPA pro-duction. Varying concentrationsof 18:1 LPA were added to se-rum-starved OVCAR-3 cellscultured in SFM as described in“Materials and Methods,” andthe cells were incubated for 24h. The supernatants were col-lected, concentrated 10-fold, andrun on a 12.5% SDS-PAGE gel.After transfer to an Immobilon-Pmembrane, the blot was probedwith polyclonal rabbit antibody touPA at a 1:1000 dilution. The lo-cation of the uPA band was deter-mined by running recombinantuPA in a parallel lane.

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429, OVCA 432, and OVCA 433. LPA-induced increases inuPA secretion by OVCAR-3 cells were detectable upon expo-sure to 10mM LPA, with optimal concentrations ranging from20–80mM (Fig. 3). Similar concentrations of LPA have previ-ously been shown to induce significant proliferative effects onovarian cancer cells and to parallel the LPA concentrationsfound in the serum and ascites of ovarian cancer patients (2, 3, 6).

We have demonstrated previously that 18:1 LPA effi-ciently induces cellular changes in cytosolic calcium and cellu-lar proliferation in ovarian cancer cells, whereas 16:0 LPA and18:0 LPA are relatively inefficient in mediating such effects (2,6). In the ovarian cancer cell line OVCAR-3, LPA containing anunsaturated fatty acyl chain (18:1 LPA) effectively stimulateduPA secretion, whereas LPA with a saturated fatty acyl chain

Fig. 4 LPA structure is critical for uPA production. Cells were serum-starved overnight. Conditioned medium was collected after a 24-h treatmentwith 18:1 LPA, 18:0 LPA, 16:0 LPA, or LPC and concentrated 10-fold. Samples were normalized for viable cell number by the MTT assay and runon a 12.5% SDS-PAGE gel. After transfer to an Immobilon-P membrane, the blot was probed with uPA antibody. In contrast to 18:1 LPA, whichinduced an increase in uPA secretion, other LPA forms and LPC had no effect. The location of the uPA band was determined by running recombinantuPA in a parallel lane.

Fig. 5 LPA increases uPAmRNA levels. OVCAR-3 cellswere serum-starved overnightand incubated with 20mM LPAas indicated. Total RNA wasisolated as described in “Mate-rials and Methods.” RNA (10mg/lane) was electrophoresedthrough a 1% agarose gel con-taining 0.22 M formaldehydeand transferred to an Immo-bilon-P membrane. The mem-brane was then probed with uPAfollowed by GAPDH aftermembrane stripping. LPA stim-ulates uPA transcription maxi-mally at 48 h.

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(18:0 LPA and 16:0 LPA) had no effect (Fig. 4). Alteration ofthe phosphate group (LPC) rendered the lysophospholipid inef-fective in increasing uPA secretion (Fig. 4). This suggests thatthe structure of LPA is important in regulating function andpotential interaction with the LPA receptors, mediating uPAproduction in ovarian cancer cells.

In striking contrast to the consistent response of ovariancancer cell lines to LPA, LPA did not induce detectable increasesin uPA secretion by NOE cell lines NOE 033 and NOE 035 or viralSV40 large T antigen-immortalized NOE cell lines IOSE 29 andIOSE 80 (Ref. 19; Fig. 1). LPA also did not induce uPA secretionby the SKBR 3 breast cancer cell line or by NIH3T3 murinefibroblasts, despite the ability of LPA to induce the proliferation ofthese cells (data not shown). Thus, LPA specifically up-regulatesuPA secretion in ovarian cancer cell lines, but not in NOE celllines. Because LPA increases the level of secreted and active uPAprotein, we investigated whether LPA altered the expression ofuPA transcripts. LPA induced a 5.5-fold increase in the level ofuPA mRNA in OVCAR-3 cells within 24 h of treatment, as shownby densitometry (Fig. 5). After 48 h of LPA stimulation, a marked35-fold increase in uPA mRNA levels was seen (Fig. 5). This issimilar to the time course of LPA-induced uPA secretion (Fig. 1).Taken together, these findings strongly suggest that LPA stimula-tion induces increased steady-state uPA-specific mRNA levels andpromotes the concomitant elevation of uPA protein synthesis andsecretion.

The effect of LPA on the nuclear transcription of uPA wasthen evaluated using a promoter-linked luciferase transfectionassay. A 2–3-fold increase in luciferase activity was consistentlyseen in the presence of LPA (Fig. 6). The modest but consistentresponse of the uPA promoter to LPA treatment indicates thatLPA-induced increases in uPA transcription likely contribute tothe ability of LPA to increase uPA levels, mRNA levels, andprotein levels in cellular supernatants.

The ability of LPA to consistently induce uPA secretion byovarian cancer cells but not by normal ovarian epithelium led usto explore the expression of the edg-1, edg-2, and edg-4 LPAreceptors (4) by ovarian epithelial cell lines. mRNA levels ofedg-1, which mediates responses to LPA and sphingosine-1-phosphate (4), are low in the ovarian cancer cell lines studied(data not shown). As indicated in Fig. 7, edg-2 mRNA levelsdiffered markedly between normal and malignant ovarian epi-thelial cell lines. Although edg-2 mRNA levels were low inNOE 033 and NOE 035, similar edg-2 mRNA levels werepresent in SKOV-3 and OVCAR-3, which secreted uPA inresponse to LPA, and IOSE80, which did not produce uPA inresponse to LPA. Thus edg-2 expression was not sufficient toexplain the ability of LPA to induce uPA secretion. In contrast,edg-4 mRNA levels were consistently increased in ovariancancer cell lines as compared with NOE cell lines. Thus, theability of LPA to induce the secretion of uPA from ovarian celllines correlates with edg-4 mRNA levels but not with edg-1 oredg-2 mRNA levels.

DISCUSSIONAs in other cancers, the presence of the uPA correlates with

the aggressiveness of ovarian cancer (10, 13–15, 24, 25). Forexample, a correlation was noted between higher uPA levels and

increased metastatic lymph node involvement, increased ascites,and higher-grade ovarian tumors (10, 13–15, 24, 25). Addition-ally, higher tissue and ascites levels of uPA were associated withmalignant transformation of ovarian epithelium (10). uPA isimportant in the metastatic process because it disrupts the base-ment membrane and facilitates spread (26). Once secreted, uPAbinds to its specific receptors, and the ligand receptor complexinduces the conversion of plasminogen to plasmin (27). Plasminthen degrades the surrounding stroma, which contains fibrin,fibronectin, proteoglycans, laminin, and collagen type IV (28).However, the mechanisms regulating uPA production by ovar-ian cancer cells are not well characterized.

LPA is markedly elevated in ascites and plasma of patientswith ovarian cancer when compared with normal healthywomen (2, 29). It has recently been suggested that LPA mayprovide an early diagnostic marker for ovarian cancer (29). LPAhas multiple biological roles but has only recently been shownto stimulate invasion in certain cancer cell lines. Our datasuggest that LPA is involved in invasion and metastases, be-cause LPA consistently induces the secretion of uPA by ovarian

Fig. 6 Induction of uPA promoter activity by LPA. Cells were mixedwith a luciferase construct (pRSV-luc or puPA) and electroporated at250 V. After allowing 24 h for recovery, cells were starved in SFM.LPA was then added for 12 and 24 h. Cells were washed and lysed withthe Promega reporter lysis buffer, and the lysate was recovered aftercentrifugation. Total lysate protein (15mg) was analyzed for luciferaseactivity. The data are presented as relative luciferase activity, normal-izing the RLU (relative light units) to that of puPA in the absence ofLPA. A 100% increase indicates a doubling from control levels. LPAdid not alter the activity of a pRSV-luc construct, indicating specificityfor uPA-luc.

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cancer cell lines including OVCAR-3, SKOV-3, OVCA 429,OVCA 432, and OVCA 433. As shown by Western blotting inFig. 1, SKOV-3, but not OVCAR-3, secretes significant levelsof uPA protein in the absence of exogenous LPA. Strikingly,SKOV-3, but not OVCAR-3 or normal ovarian epithelium,constitutively produces low levels of LPA (data not shown).NOE cells have been reported to secrete uPA, but at levels17–34-fold lower than those of ovarian cancer cell lines (30).LPA does not, however, increase the secretion of uPA by NOEcells. Malignant transformation may render ovarian cancer cellssusceptible to LPA-induced increases in uPA secretion, contrib-uting to invasion and metastases. mRNA expression levels ofthe edg-1 and edg-2 LPA receptors did not correlate with theability of LPA to induce uPA expression. In contrast, higherlevels of edg-4 were present in ovarian cancer cells than innormal ovarian epithelium, correlating with the ability of LPAto induce uPA secretion. Thus, signaling through edg-4 may

account for the ability of LPA to induce uPA secretion byovarian cancer cells. Alternatively, LPA receptors may link todifferent signaling and physiological responses in ovarian can-cer cells than in normal ovarian epithelium.

LPA also demonstrates structural specificity because only18:1 LPA induced uPA secretion, whereas forms of LPA withsaturated fatty acyl chains did not. This implies that lipid struc-ture is tightly linked to function and is likely regulated througha highly specific cellular receptor. LPA induced a significantincrease in uPA mRNA steady-state levels and a modest in-crease in activity of the uPA promoter, suggesting that sustainedelevations in uPA protein levels in cell supernatants are a resultof increased uPA promoter activation. However, the modestincrease in promoter activity is much less than the increase inuPA mRNA levels induced by LPA, suggesting that LPA mayalso alter mRNA stability.

In conclusion, LPA likely plays a role in the invasiveness and

Fig. 7 RNA levels of edg-2 and edg-4. RNA from normal ovarian epithelium and ovarian cancer cell lines was probed for the presence of edg-2 andedg-4.A, representative Northern blots.B andC, densitometry from several Northern blots normalized for 18S RNA. To compare across Northernblots, OVCAR-3 was run on each blot, and samples were normalized to the intensity of the signal for OVCAR-3.

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outcome of ovarian cancer through the production of uPA. LPAmay thus be involved in the metastatic process because it inducesproliferation and stimulates secretion of uPA in ovarian cancercells. Mechanisms targeting LPA production or action might thusbe useful in treating this aggressive cancer. Indeed, we and others(2) have identified potential LPA receptor antagonists.

Note Added in ProofA novel LPA receptor, edg-7, with selectivity for LPA with unsat-

urated fatty acyl chains has been recently identified (31). The require-ment of LPA-induced uPA secretion for LPA with an unsaturated fattyacyl chain (Fig. 4), combined wth a markedly increased edg-7 mRNAexpression in ovarian cancer cells as compared with normal ovarianepithelium, suggests that edg-7 may contribute to LPA-induced uPAsecretion.

ACKNOWLEDGMENTSWe thank Dr. Douglas Boyd for helpful discussion.

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3710LPA-mediated uPA Secretion

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1999;5:3704-3710. Clin Cancer Res   Terri B. Pustilnik, Veronica Estrella, Jon R. Wiener, et al.   Ovarian Cancer CellsLysophosphatidic Acid Induces Urokinase Secretion by

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