Role of Decreased Levels of Lipid Phosphate Phosphatase-1 in Accumulation of Lysophosphatidic Acid in Ovarian Cancer1

3534 Vol. 9, 3534–3545, September 1, 2003 Clinical Cancer Research

Featured Article Role of Decreased Levels of Lipid Phosphate Phosphatase-1 in Accumulation of Lysophosphatidic Acid in Ovarian Cancer1

Janos L. Tanyi, Yutaka Hasegawa, leukin 8. Although ovarian cancer cells can produce LPA, Ruth Lapushin, Andrew J. Morris, this may not be the major reason for altered LPA levels in Judith K. Wolf, Andrew Berchuck, Karen Lu, ascites. We have demonstrated that the major mechanism of degradation of LPA by ovarian cancer cells is through a David I. Smith, Kimberly Kalli, lipid phosphate phosphatase (LPP)-like activity. We demon- Lynn C. Hartmann, Karen McCune, strate herein that LPP-1 mRNA is decreased in the majority David Fishman, Russell Broaddus, of ovarian cancers. This is recapitulated in ovarian cancer Kwai W. Cheng, Edward N. Atkinson, cell lines, where LPP-1 RNA levels are lower than those in Jose M. Yamal, Robert C. Bast, Edward A. Felix, normal ovarian epithelium and immortalized ovarian epi- 2 thelial cells. Introduction of LPP-1 into ovarian cancer cell Robert A. Newman, and Gordon B. Mills lines results in increased LPA hydrolysis, which is associated Departments of Molecular Therapeutics [J. L. T., Y. H., R. L., with a marked inhibition of cell proliferation and colony- K. W. C., G. B. M.], Gynecological Oncology [J. K. W., K. L.], Experimental Therapeutics [R. C. B., E. A. F., R. A. N.], Pathology forming activity and a marked increase in apoptosis. Thus, [R. B.], and Biomathematics [E. N. A., J. M. Y.], University of Texas the LPA-rich environment of the ovarian cancer cell in vivo M. D. Anderson Cancer Center, Houston, Texas 77030; Department and the subsequent effects of cellular pathophysiology may of Cell and Developmental Biology [A. J. M.], University of North be a consequence of both increased LPA production and Carolina, Chapel Hill, North Carolina; Department of Obstetrics and decreased LPA metabolism by ovarian cancer cells. Gynecology, [A. B.], Duke University Medical Center, Durham, North Carolina; Department of Laboratory Medicine and Pathology [D. I. S.], Endocrine Research Unit [K. K.], Mayo Foundation, Introduction Rochester, Minnesota; Department of Obstetrics, Gynecology and LPA3 (1-acyl-2-lyso-sn-glycero-3-phosphate) is present in Reproductive Sciences and Cancer Research Institute [K. S-M.], University of California at San Francisco, San Francisco, California; elevated levels in the ascites of ovarian cancer patients. LPA, at and Department of Obstetrics and Gynecology, Northwestern the concentrations present in ovarian cancer ascites (up to 80 University Hospital, Chicago, Illinois [D. F.] ␮M), is sufficient to induce growth promotion, anchorage-independent growth, increase production of growth and neovascu- larization factors, including interleukin 8, vascular endothelial Abstract growth factor, and LPA itself, and prevent apoptosis and anoikis The levels of lysophosphatidic acid (LPA) are consis- (1–6). LPA also increases the production and action of proteases tently elevated in the ascites of ovarian cancer patients, and increases invasiveness of ovarian cancer cells, suggesting suggesting that ovarian cancer cells are exposed to an LPA that it may contribute to spread of ovarian cancer (6, 7). Taken replete environment. LPA stimulates cell proliferation, cell together, these data suggest that an LPA-rich environment in the survival, resistance to cisplatin, production and activation of patient may contribute to pathophysiology of the disease and proteases, invasiveness and production of the neovascular- may have an adverse effect on outcome. izing factors, vascular endothelial growth factor, and inter- The source of LPA in ascites fluid from ovarian cancer patients remains to be fully elucidated. Activated platelets, adipocytes, leukocytes, fibroblasts, and endothelial cells can produce LPA (2, 4, 6, 8–10). We and others (11, 12) have Received 8/2/02; revised 3/6/03; accepted 3/14/03. demonstrated that ovarian cancer cells, but not breast or normal The costs of publication of this article were defrayed in part by the ovarian epithelial cells, can produce LPA in culture. Phorbol payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to esters and LPA itself have been demonstrated to increase LPA indicate this fact. production by ovarian cancer cells but not by breast cancer cells 1 The work has been supported by the National Research and Scientific (11, 12). The accumulation of LPA in the supernatants of cells Base Foundation of Hungary Grant OTKA F 29426 (to J. L. T.), the in culture could be attributable to increased production or de- W. M. Keck Center for Cancer Gene Therapy Development Award, University of Texas M. D. Anderson Cancer Center (to J. W., G. B. M., creased metabolism or a combination of both. J. L. T.), the SPORE in Ovarian Cancer Grant, University of Texas LPA is removed by being acylated to produce phosphatidic M. D. Anderson Cancer Center Grant CA 83639 (to J. W., G. B. M., acid, deacylated by lysophospholipases to produce glycerol J. L. T.), and the Basic Biology of Ovarian Cancer PPG Grant CA phosphate, or dephosphorylated by LPPs also known as phos- 64602. K. W. C. was supported by Houston Endowment Scientific Achievement Award by M. D. Anderson Cancer Center, and G.B.M. was supported by the M. D. Anderson Cancer Center Core Grant P30 CA16671. 2 To whom requests for reprints should be addressed, at University of 3 The abbreviations used are: LSA, lysophosphatidic acid; LPP, lipid Texas, M. D. Anderson Cancer Center, Department of Molecular Ther- phosphate phosphatase; LPAAT, lysophosphatidic acid acyltransferase; apeutics, Box 317, 1515 Holcombe Boulevard, Houston, TX 77030. LPL1, lysophospholipase-1; PLA, phospholipase A; CMV, cytomega- Phone: (713) 745-1134; Fax: (713) 745-1184; E-mail: gmills@mail. lovirus; GFP, green fluorescent protein; PI, propidium iodide; NOE, mdanderson.org. normal epithelial cell line. Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2003 American Association for Cancer Research. Clinical Cancer Research 3535

phatidic acid phosphohydrolases to produce monoacylglycerol, To allow comparison across multiple arrays and the two which is inactive on LPA receptors (2–4, 6, 10). The most likely databases, the mRNA expression data of each gene was normal- candidates for hydrolysis of extracellular LPA, including LPP-1, ized to the expression detected in normal epithelial cells. The LPP-2, and LPP-3, are six transmembrane domain containing overall expression levels in the two datasets were comparable, integral membrane proteins with their catalytic site and func- allowing combination the data. tions on the extracellular surface of the cell (13–15). We have Tumor Samples and RNA Preparation. Twenty-six demonstrated that the major pathway for degradation of LPA by fresh frozen primary ovarian cancers were obtained from Duke ovarian cancer cells is through a lysophosphatidic acid phos- University (9 endometrioid), the Mayo Clinic (9 stage I serous), phohydrolase-like activity that is regulated by GnRH (16). Fur- and M. D. Anderson Cancer Center (8 stage III serous). Four thermore, LPPs can attenuate LPA-induced cell responses (14, pools of normal ovarian surface scrapings from 13 patients were 15, 17, 18). LPAAT-␣ and LPAAT-␤ catalyze the conversion of obtained from Northwestern University. The normal scrapings LPA to PA, however, these are intracellular proteins (19). The were collected using a cytobrush, and then the cells were resus- human lysophospholipase homologue, HU-K5, human LPL1, pended and frozen in RNeasy Lysis Buffer (Qiagen, Inc., Va- and lecithin cholesterol acyltransferase-like lysophospholipase lencia, CA). RNA was extracted using the RNeasy Kit (Qiagen, can remove the acyl chain from LPA (20, 21), however, their Inc.). contribution to the metabolism of extracellular LPA is unknown. Gene Expression and Data Analysis. The Affymetrix We and others (12, 22) have implicated phospholipase D GeneChip Human Genome U95 set was used to obtain gene and cellular and secreted PLA2 in the production of LPA by expression data. This series tests the expression of Ͼ60,000 ovarian cancer cells. The recently identified phosphatidylserine- human genes and expressed sequence tags. The cRNA prepara- and phosphatidylcholine-specific PLA1s (23, 24) and lysophos- tion, hybridization, and scanning of the microarrays were per- pholipase D (25) may also contribute to the production of LPA formed according to manufacturer’s protocols at the Mayo Clin- by ovarian cancer cells. In particular, PLA1 may lead to the ic’s microarray facility. Microarray data were analyzed using production of polyunsaturated sn-2 LPAs that are preferential the dChip software (30).5 ligands for LPA3 receptor (also known as Edg 7, endothelial Tissue Samples, Cell Cultures, and G418 Resistance. differentiation gene 7 receptor; Ref. 26). OVCAR-3, SKOV3, SKOV3 IP1, HEY, and A 2780 (human In this article, we demonstrate that LPP-1 mRNA levels are epithelial ovarian carcinoma cell lines) were cultured in RPMI decreased in ovarian cancer cells as compared with normal 1640 supplemented with 10% fetal bovine serum (Atlanta Bio- epithelium and cancer cells from other lineages. This translates logicals, Norcross, GA). The IOSE 80 and IOSE 29 (SV40 into a decreased level of LPP-1 mRNA in ovarian cancer cell T-antigen-semi-immortalized ovarian surface epithelial cell lines as compared with normal ovarian epithelial cells. Intro- lines) were kindly provided by Nelly Auersperg (Department of duction of LPP-1 into ovarian cancer cell lines results in in- Obstetrics and Gynecology, University of British Columbia, creased ectoLPA hydrolysis, which is associated with decreased Vancouver, British Columbia, Canada; Ref. 31). The IOSE cells colony-forming cell activity and increased apoptosis. Thus, de- were grown in medium 199/MCDB 105 supplemented with creased LPP-1 levels in ovarian cancer cells may contribute to 10% fetal bovine serum. All of the cell lines were tested to elevated LPA levels in ascites and play a role in the pathophys- evaluate G418 (Invitrogen Corp., Carlsbad, CA) resistance. iology of ovarian cancer. G418 was applied in concentration from 200 to 1000 ␮g/ml. Medium contained the appropriate amount of G418 and was Materials and Methods changed every third day. The lowest concentration of G418 that Analysis of Gene Expression Profiles in Ovarian Tissue killed all of the cells was applied later in the experiments. Samples from Published Microarray Hybridization Data. Twenty stage III, primary human epithelial ovarian tumor sam- RNA extraction and hybridization on oligonucleotide microar- ples and 6 human normal ovarian surface epithelial samples rays were performed and published on 27 primary human ovar- were provided by the Basic Biology of Ovarian Cancer Program ian cancer and other normal and tumors samples by previous Project Grant (CA 64602) and the SPORE in Ovarian Cancer studies (27–29). Affymetrix U95 array hybridization data are (CA 83639) tumor banks. available online (27–29).4 The control samples consisted of 36 First-Strand cDNA Synthesis and Semiquantitative Re- normal adult epithelial tissues (27). We combined these results verse Transcriptase-PCR of LPP-1. The mRNA expression with our own analysis of expression arrays from an additional of LPP-1 was determined using semiquantitative reverse tran- 26 ovarian cancers (17 from serous and 9 from endometrioid scriptase-PCR. Oligonucleotide primers were used for: hLPP-1, histological subtypes). All samples were assessed and demon- 5Ј-CGCGGATCCATGTTTGACAAGACGCG and 5Ј-TGGC- strated to contain at least 80% tumor with limited stromal TATGTAGTTATTCCTG (304 bp); and control glyceraldehyde- contamination. Control samples consisted of four separate pools 3-phosphate dehydrogenase primers, 5Ј-CCCATGGCAAATTC- of normal ovarian epithelial cells scraped directly from the CATGGCACCG and 5Ј-GTCATGGATGACCTTGGCCAGGGG surface of the ovary from 13 different patients into preservative, (344 bp). The RNA samples were treated with DNase before providing an indication of mRNA levels in normal ovarian first-strand cDNA synthesis or reverse transcriptase-PCR reaction epithelial cells in vivo. following the manufacturer’s instructions (DNA free, DNase Treat-

4 Internet address: http://www.gnf.org/cancer/epican. 5 Internet address: http://www.biostat.harvard.edu/complab/dchip/.

ment and Removal Kit; Ambion, Austin, TX). In some cases, A total of 1.5 ␮g of expression vector was mixed and incubated reverse transcriptase-PCR was performed as a single step with for an additional 30 min at room temperature. Cotransfection the reverse transcriptase-PCR reaction mixture consists 1 ␮gof with 0.5 ␮g of GFP-expressing vector was performed as a total RNA, 0.4 ␮M sense and antisense primers, 0.2 mM de- control for transfection efficiency. This mixture was applied into oxynucleotide triphosphates, 0.5 units of reverse transcriptase the 60-mm plate containing 3 ml of medium. and TaqDNA polymerase enzymes (in enzyme mix), 5 mM DTT Quantitation of transfection efficiency was done by flow

solution, 5 units of RNase inhibitor, 1.5 mM MgCl2 in final cytometry using a FACScan with Cellquest 3.3 software pack- volume of 50 ␮l (Titan One Tube RT-PCR System; Roche age (Becton Dickinson, San Jose, CA). GFP was identified Molecular Biochemicals, Mannheim, Germany). The reverse using a single parameter histogram display of log green fluo- transcriptase reaction was performed at 60°C for 30 min fol- rescent. Cells transfected with an empty vector were used as a lowed by 35 cycles of PCR in a Thermal Cycler (Perkin-Elmer negative control to develop a gate to determine the percentage of 480, Atlanta, GA). Each cycle of PCR included 30 s of dena- GFP-positive transfected cells. The transfection efficiency var- turation at 94°C, 2 min of annealing at 56°C, and 1 min of ied from 4–21%. To create stable lines, 48 h after transient extension at 72°C. transfection, medium containing the appropriate amount of The reverse transcriptase PCR was performed as a two step G418 (selection medium) was changed every third day for 6–8 approach in the case of the primary tumor samples and the weeks. ovarian surface epithelial samples. As the first step, first-strand GFP-targeted Cell Cycle Progression Assay with Tran- cDNA synthesis was done with SuperScript First-Strand Syn- sient Transfection. Cells were transiently transfected using thesis System (Invitrogen Corp.) according to the manufactur- FuGene 6 when they were 60–80% confluent. The parental er’s instruction using 5 ␮g of total RNA from each primary lines were transfected with either pcDNA3.1-LacZ-HisA, ovarian tumor. PCR was then performed as described above pc-hTERT-DNA3.1-HisA, pcDNA3.1-LPP-1-His A, or pchTERT- using one-fifth of the reverse transcriptase product. Reverse DNA3.1-LPP-1-HisA constructs separately and cotransfected transcriptase-PCR with glyceraldehyde-3-phosphate dehydro- with pGFP. Forty-eight h later, the medium was removed, and genase (29 cycles) was used to demonstrate equal amounts of the cells were washed twice in PBS and trypsinized. Both RNA in each sample and used to normalize results. PCR prod- floating and adherent cells were harvested and subjected to flow ucts were visualized by electrophoresis in a 2% agarose gel with cytometry. Cells were fixed with 0.25% paraformaldehyde ethidium bromide. (Poly-Scientific, Bay Shore, NY) in PBS solution followed by Analysis of Total LPP and LPP-1 Activity in Ovarian DNA staining with PI staining buffer (10 ␮g/ml PI, 0.1% Tween Cancer Cell Lines. Cells were grown in 35-mm diameter 20, and 100 ␮g/ml RNase A in PBS). To assess cell cycle dishes to ϳ80% confluence, washed once in PBS, and lysed by progression, a two-color cytometric analysis was performed on scraping in buffer containing 20 mM Tris (pH 7.4), 2 mM EDTA, a FACScan flow cytometer (Becton Dickinson) using Cellquest and protease inhibitors (benzamidine and phenylmethylsulfonyl 3.3 software for acquisition and analysis. GFP-positive cells fluoride). The cells were disrupted by brief probe sonication. were gated and analyzed for cellular DNA content. LPP activity in the lysates was determined using [32P]LPA Colony Formation Assay. The colony formation assay presented in mixed micelles of Triton X-100 as described pre- was performed as previously described with slight modifications viously (32, 33). For the data shown in Fig. 3 assays contained with both transiently and stably transfected cell lines (36, 37). 1–5 ␮g of protein. Immunoprecipitations were performed as Two days after transient transfection, cells were trypsinized, described previously (32–34). LPP activity in washed immune washed in PBS twice, and counted and 3 ϫ 104 cells were complexes was determined as above using [32P]LPA. reseeded. For stable-transfected cells, 2 ϫ 103 stably transfected The LPP-1 cDNA Containing Vectors Driven by CMV cells were plated into 30-mm 6-well plates. Two weeks later, and hTERT Promoters. The complete cDNA of LPP-1 (33) colonies were stained with 0.1% Coomassie blue (Bio-Rad, was amplified using primers with the sequences 5-CGCGGATC- Hercules, CA) in 30% methanol and 10% acetic acid. The CATGTTTGACAAGACGCG-3 (forward) and 5-GCTCTA- number of the colonies (Ͼ300 cells) was counted by two inde- GAAGGCTGGTGATTGCTCG-3 (reverse) using PCR, cut with pendent investigators, and the variation was Ͻ10%. BamHI and XbaI, and inserted into BamHI and XbaI restriction Analysis of the Bystander Effect of hLPP-1 Using Tran- sites of the pcDNA3.1-HisA epitope-tagged eukaryotic expression sient and Stable Transfection. Two days after transient vector (Invitrogen Corp.). The CMV promoter was replaced in the transfection, cells were trypsinized and counted. Cells trans- pcDNA3.1-HisA vector with the hTERT promoter (35) using BglII fected with hLPP-1 protein were mixed with equal numbers and KpnI. The validity of all PCR products was confirmed by (3 ϫ 104) of neo-transfected control cells and cultured in 30-mm sequencing and of the vectors by restriction endonuclease analyses. dishes the presence of G418. Stably transfected SKOV3 cells Transient and Stable Expression of LPP-1. SKOV3 or (2 ϫ 103) were mixed with an equal number of nontransfected OVCAR-3 cells were plated in 60-mm plastic dishes at a density SKOV3 cells and plated into 30-mm dishes in the absence of of 0.5 ϫ 106 cells/dish. After 48 h, when they were ϳ60–80% G418. Two weeks later, colonies were stained and counted as confluent, cells were washed twice in PBS (pH 7.4; Sigma, St. described above. Louis, MO). Cell transfection was carried out using FuGene 6 Migration Assays of LPP-1 Overexpressing SKOV3 carrier (Roche Molecular Biochemicals, Indianapolis, IN) ac- Cells. Migration assays were performed using a transwell cording to the manufacturer’s instructions with minor modifi- chamber membrane (8-␮m pore size; Biocoat, Becton Dickin- cations. Briefly, 10 ␮l of FuGene 6 were added into 100 ␮lof son Labware, Franklin Lakes, NJ). LPA (10 ␮M) was added to serum-free medium and incubated 10 min at room temperature. the lower chambers. Cells were initially starved of serum. Cells

(5 ϫ 104) were added to the upper chamber and allowed to tions were performed as described previously (34, 40). The only migrate for 24 h at 37°C. Cells that had not migrated were modification was that we used 12.5% SDS-polyacrylamid gel removed from the upper chamber with a cotton swab. The instead of the previously published 7.5% (34, 40). remaining cells were fixed and stained with crystal violet. LPA Determination in Cell Supernatants. LPA 18:1 (1 Cell Preparation for Analysis of LPP-1 Activity. For ␮M) was added to SKOV3 cells, and LPP-1-transfected SKOV3 studies using SKOV3 cell membranes or detergent-extracted and cell supernatants were collected at the indicated times. An membrane proteins, the monolayer of infected cells was washed unnatural LPA, 17:0, was added to the supernatants after col- gently with PBS and lysed by the addition of 4 ml of ice-cold lection to monitor efficiency of isolation and detection of LPA. lysis buffer [20 mM Tris (pH 7.5), 5 mM EGTA, 0.1 mM LPA was extracted from 1 ml of cell supernatant using Waters benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride] and Oasis HLB (1 ml) and 30 mg of solid phase extraction cartridges scraping (34). The cell suspension was transferred to a 15-ml (Milliford, CT) preconditioned with 1 ml of methanol and 1 ml conical tube, and the cells were disrupted by sonication (Vertis of water. Cartridges were washed twice with 1 ml of water and Systems Sonifier), 10-s pulses on ice. The disrupted cells were dried under vacuum for 5 min. LPA was eluted from the ϫ centrifuged at 20,000 g at 4 °C for 20 min. The cytosolic cartridges using 1 ml of 95:5:5 methanol:chloroform:1 M fraction was removed, and the membrane fraction was resus- ␮ NH4OH. Twenty-five l of eluant were injected into the liquid pended in ice-cold lysis buffer. Detergent extracts were prepared chromatograph/mass spectrometer/mass spectrometer using a from the membranes by the addition of Triton X-100 to final Waters XTerra 3.5-␮m C18 2 ϫ 100-mm micropore column in concentration of 1% followed by incubation at 4°C with con- an Agilent 1100 binary HPLC. The column was run in the stant rocking for 1 h. The solubilized material was centrifuged isocratic mode using a mobile phase of 90:5:5 methanol:chlo- ϫ at 26,000 g at 4°C for 30 min, and the supernatant was roform:1 M ammonium hydroxide. LPA isoforms were detected removed. using a MicroMass QuattroUltima triple quadrapole mass spec- 32 Preparation of LPA as Substrate. [ P]LPA was pre- trometer (Beverly, MA) using electrospray-negative ionization pared by phosphorylation of oleoyl monoacylglycerol (mono- with the instrument operating in a multiple reaction-monitoring lein) (Avanti Polar Lipids, Alabaster, AL) using Escherichia mode. Specific transitions for each LPA are as follows: LPA coli diacylglycerol kinase (Calbiochem, San Diego, CA) and 18:1 is 435.24 Ͼ 152.8 and LPA 17:0 423 Ͼ 152.8. Instrument 32 [␥ P]ATP (ICN Pharmaceuticals, Costa Mesa, CA). The reac- settings are as follows: cone voltage, 50 V; capillary voltage, tion was terminated by extraction with acidified CHCl3 and 3.00 kV; and collision energy, 22. methanol, and the dried organic phases obtained were resus- Statistical Analyses. Unpaired continuous outcomes pended in 0.4 ml of 20:9:1 CHCl3/methanol/H2O (solvent A) were compared using Wilcoxon rank-sum tests and exact per- and neutralized by the addition of a small volume of 20% mutation tests. Paired continuous outcomes were compared us- NH4OH in methanol. This material was applied to an Econosil ing Wilcoxon sign-rank tests. Proportions were compared using 2 NH2 5 units of high-pressure liquid chromatography column ␹ analyses. Significance was set at P Ͻ 0.05. (250 ϫ 4.2 mm; Alltech Associates, Baulkham Hills, NSW, Australia). The column was washed with 20 ml of solvent A and then eluted with a 40-ml linear gradient of 0–1 M ammonium Results acetate in solvent A. A total of 0.5-ml fractions of the eluant was LPP-1 and Human Lysophospholipase Homologue collected, and associated radioactivity was determined by liquid mRNA Expression Is Decreased in Ovarian Carcinomas. scintillation counting. 32P-labeled products were pooled and Using Affymetrix array data, we were able to analyze levels of extracted from the eluant by the addition of 3 M HCl and CHCl3 expression of a number of enzymes involved in hydrolysis of to give two phases. LPA in multiple tumor types (Table 1). LPP-1 mRNA levels in LPP-1 Assays and Immunoprecipitation. The assay ovarian cancer samples were decreased ϳ5-fold as compared procedures used were adapted from previous descriptions (38, with normal epithelium. Furthermore, LPP-1 RNA levels were 39). In brief, assays were performed in medium containing 20 lower in ovarian cancers as compared with other tumor lineages. mM Tris (pH 7.5), 1 mM EGTA, and 2 mM EDTA, and in some For example, the levels of LPP-1 RNA in prostate and kidney cases, the MgCl2 concentration of the assay medium was varied samples were at least 10-fold higher than the levels in ovarian 32 by the addition of MgCl2 as indicated. P-labeled LPA was cancer cells. In contrast, LPP-2 and LPP-3 RNA levels were dried under vacuum and resuspended in 0.1% BSA. The assay similar between ovarian cancer samples and normal epithelium. volume was 100 ␮l of serum-free medium, with final concen- The overall total of LPP-1, LPP-2, and LPP-3 RNA levels was 32 tration of 100 ␮M P-labeled lipid substrate. Detergent- lower in ovarian cancer than in any other tumor type analyzed, extracted membrane proteins (generally 0.1–5 ␮g of protein) compatible with decreased LPP and particularly LPP-1 levels were added directly to these incubations. Assays were per- contributing to the elevated LPA levels in ovarian cancer formed at 37°C and were terminated by addition of ice-cold 10 patients. mg/ml BSA and 10% trichloroacetic acid. The samples were The human lysophospholipase homologue (HU-K5) 32 2- ϳ centrifuged for 5 min in a microcentrifuge, and [ P]PO4 mRNA expression was decreased in ovarian cancers 2.5-fold released into the supernatant was quantitated by liquid scintil- when compared with normal epithelium, however, it was not lation counting. This assay was validated by demonstrating that decreased as compared with several other tumor lineages. the water-soluble radioactivity released from the substrate was LPAAT-␤ mRNA expression was decreased ϳ30% in ovarian 32 2- [ P]PO4 by quantitative extraction with ammonium molyb- cancer cells, whereas no significant changes were detectable in date. Immunoprecipitation, SDS-PAGE, and protein determina- LPAAT-␣ or LPL1.

Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2003 American Association for Cancer Research. 3538 Decreased LPP-1 Expression in Ovarian Cancer Lung/ normal Breast/ normal Ovary/ normal a 8) ϭ ( n Bladder a 6) ϭ ( n Pancreas a 11) ϭ Kidney ( n Fig. 1 Human LPP-1, LPP-2, LPP-3, and HU-K5 gene expression in

a ovarian cancer. The normalized values of hybridization signal of LPP-1,

26) LPP-2, LPP-3, and HU-K5 transcripts from 53 ovarian carcinoma sam-

ϭ ples (see “Materials and Methods”). The average of the expression of the Prostate ( n transcripts in the normal epithelial tissues normalized to 1 is shown for comparison as a horizontal dashed line. a 4 23) ϭ Colon ( n Averages Ratios (%) We evaluated changes in LPP and HU-K5 mRNA levels in individual patients by combining data from the published data- a 27)

Analysis of the DNA array data base with our own data using the same U95 Affymetrix arrays ϭ

Lung (Fig. 1). This combined analysis of 53 ovarian cancer samples ( n compared with four separate pools of normal epithelial cells

Table 1 scraped directly from the ovaries of 13 patients demonstrated a 25) that LPP-1 mRNA levels were markedly decreased in all sam- ϭ Ͼ

Breast ples, and in many cases, LPP-1 levels were decreased 10-fold. ( n LPP-1 expression was decreased at least 2-fold in 89% of the

a tumors as compared with normal epithelium. When LPP-2 and

27) LPP-3 mRNA levels were considered, 2 of 3 of patients showed ϭ some decrease in levels (Ͼ30%). HU-K5 mRNA levels were Ovarian ( n decreased at least 2-fold in 77% of ovarian cancers. Semiquantitative Reverse Transcriptase-PCR Analysis of Human Ovarian Carcinoma Cell Lines and Primary Ovarian Tumor Samples. On the basis of the microarray Normal epithelium data, we decided to further evaluate LPP-1 mRNA expression levels in ovarian cancer cells as compared with normal epithelium. We characterized the expression of LPP-1 in 20 primary ovarian tumor samples, all with Ͼ80% tumor cell content, 6

Genbank normal surface epithelium samples, 1 normal surface epithelial accession no. U 56417 490 812 773 632 757 595 725 653 623 1.7 1.6 1.3 U 56418 544 359short-term 481 411 culture (NOE), 953 and 399 5 ovarian 640 carcinoma 473 cell 347 lines, 0.7 0.9 0.8

including OVCAR-3, SKOV3, SKOV3 IP1, HEY, and A 2780. Expression levels were normalized to that of a SKOV3 LPP-1 transfectant, which was run on all gels and designated as 1. As

(hLPP-2) AFO 35959 230 264 642indicated 509 in Fig. 718 2, LPP-1 230 levels in 103 normal 856 ovarian 457 epithelium 1.1 2.8 2.2 Enzyme

Values at the medians (range) of expressed DNA array results in each tumor type in Ref. 27 – 29. from multiple individuals were tightly clustered. All ovarian a tumors samples and ovarian cancer cell lines were lower than PAP-2a1 (hLPP-1)PAP-2b (hLPP-3) AFO 14402PAP-2 ␥ LPAAT ␣ AFO 17786 388 634 91 663 163 404 199 686 119 311 2310 1767 980 3009 284 612 614 782 0.2 1.0 0.4 0.6 0.5 1.1 LPAAT ␤ HU-K5LPL1 U 67963 AFO 81281 339the 512 average 151 458 of the 109 665 NOE. 296 417 Using 292the 561 lowest 106 576 normal 815 248 ovarian 494 355 88 941 0.4 0.9 0.3 1.3 0.9 0.8

levels on the ability of ovarian cancer cells to proliferate as indicated by colony-forming cell activity, a sensitive method to determine effects of mediators on cellular function (36, 37). We attempted to establish stable cell lines expressing LPP-1 in IOSE 29, IOSE 80, OVCAR-3, HEY, and A2780. Despite multiple attempts and a ready ability to develop neomycin-resistant lines, we were unable to develop stable LPP-1-expressing cell lines based on these lines. This suggests that LPP-1 decreases the growth or survival of ovarian epithelial and ovarian cancer cell lines. We have demonstrated that the SKOV3 cell line releases high levels of LPA into cell supernatants (12). Consistent with this observation, we were able to develop stable LPP-1-expressing cell lines in

SKOV3 and in SKOV3 IP1, a variant of SKOV3 selected for the ability to grow i.p. These cells demonstrated a ϳ4.9-fold increase in LPP-1 RNA levels as assessed by reverse transcriptase-PCR analysis (data not presented). However, even on the SKOV3 background, LPP-1 mRNA levels and activity deceased during cell passage. Thus, for analysis of LPP-1 activity, early passage-transfected cells were used. We examined the ability of stably transfected and parental SKOV3 cell lines to hydrolyze LPA presented in the medium. Fig. 2 Decreased LPP-1 mRNA expression in ovarian carcinoma cell This assay reflects the activity of all of the LPP isoforms on the lines and tissue samples. The mRNA expression data of normal epithelium and malignant ovarian tissue samples and cell lines after normal- cell surface. The LPA hydrolysis activity of LPP-1 stably trans- ization (NOE). The average of the expression of LPP-1 in normal fected SKOV3 was 7.7 times higher (32 Ϯ 2.3 versus 246 Ϯ 4.3 epithelial tissues normalized to 1 is shown for comparison as a hori- pmol LPA hydrolyzed/30 min/mg protein) than in the nontrans- zontal double line. Reverse transcriptase-PCR reaction and normaliza- fected parental line. As indicated by immunoprecipitation of cell tion are described in “Materials and Methods.” lysates with LPP-1-specific antibodies (34), SKOV3 cell lysates demonstrated decreased LPP-1 enzyme activity (10 Ϯ 1.1 versus 23.1 Ϯ 1.9 pmol/30 min/mg protein immunoprecipitated) as epithelial sample as a cutoff, 16 of 20 ovarian cancer samples compared with NOE. Stably transfected SKOV3 (late passage) and 4 of 5 ovarian cancer cell lines demonstrated decreased demonstrated 27.0 Ϯ 0.9 pmol/30 min/mg protein immunopre- LPP-1 RNA levels, with some of the levels markedly decreased. cipitated LPP-1. This activity data were compatible with the These data suggested the possibility that decreased LPP-1 levels 4.9-fold increase in LPP-1 mRNA expression in the stable lines. contribute to the increased LPA levels in ascites of ovarian Thus, ectopic expression of LPP-1 in ovarian cancer cells results cancer patients and to the increased levels of LPA in ovarian in a marked increase in the ability of cells to hydrolyze extra- cancer cell supernatants. cellular LPA. LPP-1 Activity Is Decreased in Ovarian Cancer Cell The increased ability of LPP-1-transfected SKOV3 to hy- Lines. As indicated in Fig. 3, HEY, OVCAR-3, and SKOV3 drolyze radiolabeled LPA suggested that LPP-1 transfection demonstrated lower total LPP activity, representing the activity would decrease the concentration of LPA in the culture media. of all of the LPP isoforms than the nontumorigenic ovarian When 1 ␮M 18:1 LPA was added to media alone, there was no surface epithelial cell line (IOSE). When LPP-1 was immuno- detectable change in LPA concentration over time (time 0, 780 precipitated with LPP-1-specific antibodies (the antibodies ef- nM; 10 min, 740 nM; 1 h, 845 nM; and 8 h, 819 nM). SKOV3 fectively and specifically immunoprecipitate LPP-1 but do not induced hydrolysis of extracellular LPA cells [(time 0, 727 nM; function as efficiently in Western blotting), ϳ10% of the LPP-1 10 min, 580 nM;1h,555nM;and8h,Ͻ100 nM) levels above activity present in IOSE was accounted for by LPP-1. Strik- 100 nM were readily detectable in calibration curves]. The ingly, HEY, OVCAR-3, and SKOV3 demonstrated markedly expression of LPP-1 in SKOV3 cells resulted in a marked lower levels of LPP-1 activity than did IOSE. Indeed, the increase in the hydrolysis of LPA (time 0, 701 nM; 10 min, 633 difference in LPP-1 activity between IOSE and the tumor cells nM; 1 h, 364 nM;and8h,Ͻ100 nM), compatible with increased was much greater than that for total LPP activity, compatible LPP-1 activity. Thus, the extracellular concentration of LPA is with the contention that LPP-1 activity is selectively decreased markedly decreased by transfected of LPP-1. in ovarian cancer cells. The LPP-1 activity correlated with that As indicated by the ability to form colonies, stable of the mRNA levels with SKOV3 having the lowest levels in expression of LPP-1 decreased the growth of the SKOV3 both assays. ovarian cancer cells ϳ1.9-fold (49 Ϯ 8.4 versus 26 Ϯ 4.3, LPP-1 Decreases Colony-forming Ability of Ovarian P ϭ 0.0002; Fig. 4B). Because of the difficulty in obtaining Carcinoma Cells. LPA is a potent regulator of multiple high-level LPP-1 expression in stable cells lines, we assessed activities in ovarian cancer cells. To evaluate the potential the effect of transient transfection of LPP-1 on colony-form- role of decreased LPP-1 levels in the pathophysiology of ing cell activity of SKOV3 and OVCAR-3 ovarian cancer cell ovarian cancer, we assessed the effect of increasing LPP-1 lines. As indicated in Fig. 4A, transient expression of LPP-1

Fig. 3 Determination of LPP activity in ovarian cancer cell lysates and LPP-1 immune precipi- tates. Data normalized to 1 ␮g of protein in lysates. The data shown are means Ϯ SD of tripli- cate determinations from a single representative experiment.

Fig. 4 Growth inhibitory effect of LPP-1 overexpression assessed by colony-forming cell activity. A, 2 days after transient transfection, cells were trypsinized, washed in PBS twice, and counted. A total of 3 ϫ 104 cells was seeded into 30-mm 6-well plates. The selection medium supplemented with G418 was changed every third day. Two weeks later, colonies were stained by 0.1% Coomassie blue in 30% methanol and 10% acetic acid. Average number of colonies/dish is presented. B, growth inhibition effect of LPP-1 in stably transfected SKOV3 cell line. A total of 2 ϫ 103 of cells from the stable SKOV3 line was seeded into 30-mm 6-well plates. Complete culture medium supplemented with G418 was changed every third day. Two weeks later, colonies were stained by 0.1% Coomassie blue in 30% methanol and 10% acetic acid. The data are the mean of six separate experiments Ϯ SD.

caused a marked growth inhibition in both OVCAR-3 and 0.0022, P ϭ 0.0416, P ϭ 0.0022), respectively. Thus, ectopic SKOV3. The inhibitory effect ranged from 2.3- to 5.8-fold expression of LPP-1 strongly inhibits the ability of ovarian when LPP-1 was driven by the CMV and hTERT promoters cancer cell lines to proliferate as indicated by colony-forming in the OVCAR-3 and SKOV3 cell lines (P ϭ 0.0022, P ϭ cell activity in vitro.

Fig. 5 LPP-1 increases apoptosis in ovarian cancer cell lines. Forty-eight h after transient transfection, cells were harvested and fixed by 0.25% paraformaldehyde in PBS solution followed by PI (10 ␮g/ml) for DNA staining. Two-color cytometric analysis was performed, and the percentages of hypodiploid cells as an indication of apoptosis determined using CellQuest software. Primary flow cytometric graphs from SKOV3 cell line transfected with hTERT-driven constructs are presented. M1 represents hypodiploid cells. Percent- age of cells in G1, S, and G2-M is presented with (right) and without (left) correction for hypodiploid cells. The data shown in the bar graph are the mean of three separate experiments Ϯ SD.

LPP-1 Increases the Apoptosis Rate in Ovarian Carci- be the sum of the number of colonies produced by parental cells noma Cell Lines. The decreased ability to form colonies could and the number of colonies produced by the transfected cells reflect decreased cell cycle progression or increased rates of apop- cultured separately. As indicated in Fig. 6, A and B, with either tosis. There were no obvious differences in cell cycle progression transient transfection or stable cell lines, there was a marked as indicated by number of cells in G1,S,orG2-M in control or decrease in the number of colonies observed compared with the LPP-1-expressing SKOV3 or OVCAR-3 cells (Fig. 5). However, expected number of colonies (P ϭ 0.01, P ϭ 0.01). Therefore, as indicated by an accumulation of a hypodiploid peak on cell cycle LPP-1-transfected cells were able to decrease the proliferation analysis, LPP-1 expression increased the rate of apoptosis in both of parental cells compatible with the effect of LPP-1 related to SKOV3 and OVCAR-3 (Fig. 5). Thus, the decreased ability of the effects of an extracellular mediator, likely LPA. LPP-1-expressing ovarian cancer cells to form colonies is associ- LPP-1 Overexpression Decreases the Ability of the Tu- ated with an increased rate of cellular apoptosis. mor Cells to Migrate. LPA has been demonstrated to mark- LPP-1 Decreases the Growth of Nontransfected By- edly increase cellular migration. We thus assessed whether stander Cells. The ability of hLPP-1 expression to increase overexpression of LPP-1 in ovarian cancer cells would also the hydrolysis of extracellular LPA suggests that LPP-1 may decrease LPA-induced cellular migration. As indicated by the function to decrease extracellular LPA concentrations. If this ability of cells to migrate toward LPA in a transwell assay (Fig. were the case, expression of LPP-1 in one population of ovarian 7), LPA (10 ␮M) stimulated migration of empty vector cancer cells could decrease the colony-forming cell activity of pcDNA3.1-transfected SKOV3 cells. In contrast, LPA failed to bystander-nontransfected cells. To assess this possibility, we induce migration in LPP-1-overexpressing SKOV3 cells. Thus, performed a series of cell mixing assays with LPP-1-transfected the increased hydrolysis of LPA of LPP-1 is translated into cells and control parental cells. alterations in cellular motility as well as in proliferation, colony Our preliminary data indicated that the number of colonies formation, and survival, compatible with the decreases in LPP-1 formed demonstrated essentially a linear relationship related to in ovarian cancer cells contribution to these important compo- the number of cells plated, i.e., there were no effects related nents of the transformation cascade. to cell crowding at higher concentrations (data not presented). To assess the effects of LPP-1-transfected cells on the growth of nontransfected cells, we combined equal numbers of stably or Discussion transiently LPP-1-transfected cells and parental cells. The pre- LPA levels are consistently elevated in the ascites of ovar- dicted number of colonies in the combination experiment would ian cancer patients (41, 42) and likely contributing to the patho-

Fig. 6 LPP-1 decreases the colony-forming ability in bystander cells. PcDNA3.1-transfected control cells (I), or twice as many cells (II) or LPP- 1-expressing cells (3 ϫ 104 cells with transient or 2 ϫ 103 cells with stable transfection) were plated separately. To determine the bystander effect, 1 unit of control cells (3 ϫ 104 cells with transient or 2 ϫ 103 cells with stable transfection) was mixed with equal amounts of LPP-1-expressing cells, and 2 weeks later, the number of colonies was counted. The predicted value presents the number of the colonies, which is the sum of the colonies when the control and LPP-1-expressing line were seeded separately. The actual value presents the end number of the colonies in the experiment when the control cells and LPP-1-expressing cells were plated together. A, the bystander effect of LPP-1 using transient transfection. (see “Materials and Methods”). B, bystander effect of the LPP-1 enzyme on stably transfected SKOV3 cells. The data are the mean Ϯ SD of six independent experiments.

physiology of ovarian cancer (6). The mechanisms leading to integral membrane protein that likely degrades extracellular or the elevated levels of LPA in ovarian cancer ascites are not membrane associated LPA, playing a major role in limiting currently clear. Although ovarian cancer cells can release LPA responses to LPA (11, 17, 44–46). LPA levels are low intra- into cell supernatants (11, 12), it is not known whether ovarian cellularly, in the cell membrane, and in plasma from normal cancer cells or other cellular components in ascites are the individuals, suggesting that LPA-degrading enzymes are highly primary source of LPA. Furthermore, it is not known whether active (2, 3, 47). the increased levels are because of increased rates of production LPP-1 mRNA levels are decreased in the majority of or decreased rates of degradation. We have demonstrated that ovarian cancers. In contrast, LPP-1 and total LPP levels are the major mechanism of LPA degradation by ovarian cancer markedly elevated in kidney and prostate cancers. The RNA cells is through an LPP-like activity (16). expression data would support the contention that the LPPs are The LPP family includes at least three members, LPP-1, not acting redundantly, and LPP-1 has a function distinct from LPP-2, and LPP-3 isoforms (13, 38, 39). LPP-like properties LPP-2 and LPP-3, although the genes are expressed in overlap- serve to terminate the receptor-directed signaling functions of ping patterns, and the proteins appear to be able to catalyze the LPA and hydrolyze LPA, PA, ceramide 1-phosphate, and sphin- same reactions. Thus, the effects of decreased LPP-1 levels may gosine 1-phosphate (14, 15, 38, 43). Little is known about the be relatively selective to ovarian cancers and may also argue biological function or reason for existence of multiple LPP that the initiation or progression of ovarian cancer may be isoforms (13). particularly dependent on the action of LPA. The decrease in Based upon the decrease in LPP-1 mRNA expression in LPP-1 levels may contribute to the ability of ovarian cancer cell ovarian cancers as compared with normal epithelium and other lines to produce high amounts of LPA as compared with normal tumor lineages indicated by transcriptional profiling and semi- ovarian epithelium or breast tumor samples (11, 12). We and quantitative reverse transcriptase-PCR analysis combined with others (47) have demonstrated that ovarian cancer cells exhibit the decreased LPP-1 enzyme activity in ovarian cancer cell increased expression of LPA2 (edg4) and LPA3 (edg7) LPA lines, we focused on the LPP-1 isoform as a potential contrib- receptors. The increased LPA levels in ascites, decreased ability utor to the increased levels of LPA in ascites and supernatants of to hydrolyze membrane-associated LPA because of decreased ovarian cancer cells. LPP-1 demonstrates modest selectivity for LPP-1 levels, and altered LPA receptor expression may act LPA with a rank order preference of LPAϾPAϾsphingosine together to contribute to the transformation of ovarian cancer 1-phosphateϾceramide 1-phosphate (17, 33, 34). LPP-1 is an cells. In particular, the altered LPA degradation and receptor

Fig. 7 LPA-induced migration of ovarian cancer cell lines. A represents the migration after LPA stimulation on the empty vector (pCDNA3.1)-transfected cell and B the LPP-1-overexpressing cells. Left pictures show migrated cells without, whereas right pictures show with 10 ␮M of LPA, respectively.

expression could contribute to the ability of ovarian cancer cells striking in terms of long-term cell growth because we were to survive in suspension in ascites (anoikis conditions) to pro- unable to develop stable overexpressing LPP-1 ovarian epithe- liferate and to implant and invade in the peritoneal cavity. Taken lial cell lines in any but the SKOV3 background despite multiple together, this suggests that LPA production, degradation, or attempts. SKOV3, as compared with other ovarian cancer cells, action are potential targets for therapy for this devastating produces high levels of LPA (12), potentially contributing to the disease. ability to establish stable cell lines expressing LPP-1. Even with Intriguingly, differentiation of preadipocytes into mature the SKOV3 cell line, we were able to only modestly overexpress adipocytes is associated with a decrease in the ability to hydro- LPP-1 (4.9-fold increase in RNA levels, 7.7-fold increase in lyze extracellular LPA and a decrease in LPP-1, LPP-2, and ectoLPP activity). Furthermore, the overexpression proved un- LPP-3 mRNA levels (44). This appears to be similar to the case stable, with LPP levels declining with cell passage. We estab- in ovarian cancer wherein LPP and in particular LPP-1 levels lished multiple SKOV3-overexpressing cell lines on several are markedly decreased. However, in contrast to ovarian cancer occasions in all cases, overexpression of LPP-1 was associated cells where LPA2 and LPA3 receptors (2) are increased on the with a decrease in cell proliferation and colony-forming cell cell surface rendering the cells more sensitive to LPA (7), activity. Transient transfection studies demonstrated, however, mature adipocytes exhibit a marked down-regulation of LPA that the effects observed with stably overexpressing SKOV3 cell receptor expression (48). Thus, the net outcome during differ- lines could be generalized to other ovarian cancer cell lines. entiation of adipocytes as compared with ovarian cancer cells Thus, the decrease in LPP-1 in ovarian cancer may contribute may be decreased LPA signaling. to increased survival, proliferation, and colony-forming cell Compatible with the decreased LPP-1 levels in ovarian activity. cancer cells playing a part in the pathophysiology of ovarian In addition to changes in cellular proliferation, LPA can cancer, ectopic expression of LPP-1 decreased the ability of also induce increases in cellular motility (25). This became ovarian cancer cells to form colonies, a sensitive assay for readily apparent with the cloning of the lysophospholipase D cellular proliferation (36, 37). This effect was associated with an responsible for the production and LPA and the demonstration increased apoptotic rate rather than a decrease in cell cycle that lysophospholipase D is identical to the previously cloned progression, suggesting that a major role for LPA in this system autocrine motility factor, autotaxin (25). Compatible with the is maintenance of cell viability. The effects of LPP-1 were effects of LPP-1 on cellular proliferation and extracellular LPA

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Janos L. Tanyi, Yutaka Hasegawa, Ruth Lapushin, et al.

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