Affiliations of authors: Y.-L. Hu, R. B. Jaffe, Center for Reproductive Sciences, University of California, San Francisco; C. Albanese, R. G. Pestell, Division of Hormone Dependent Tumor Biology, Albert Einstein College of Medicine, Bronx, NY.
Correspondence to: Robert B. Jaffe, M.D., Center for Reproductive Sciences, HSW 1695, University of California, San Francisco, 505 Parnassus Ave., San Francisco, CA 941430556 (e-mail: jaffer{at}obgyn.ucsf.edu).
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ABSTRACT |
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INTRODUCTION |
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Angiogenesis is essential for tumor growth (13) and is induced by the binding of vascular endothelial growth factor (VEGF) to one of two VEGF receptors, Flt1 and KDR. Cancer patients have increased levels of serum VEGF, and elevated levels of VEGF mRNA have been observed in the majority of human cancer cells, including ovarian cancer cells (14). VEGF directly stimulates the growth of some malignancies (e.g., leukemia, lymphoma, and myeloma) that express KDR and/or Flt1 through an autocrine mechanism (15,16). We have previously demonstrated (17) that LPA stimulates VEGF expression in the ovarian cancer cell lines OVCAR-3, SKOV-3, and CAOV-3 through transcriptional activation but that LPA does not stimulate the expression of VEGF in nontumorigenic IOSE cells. Thus, LPA indirectly stimulates ovarian tumor growth, at least in part, by increasing angiogenesis via VEGF.
Some ovarian cancer cell lines (e.g., DOV-13, Hey-A8, and OCC-1), however, do not express VEGF (17,18) but are still sensitive to LPA (3,17), indicating that there is another mechanism involved in regulating their growth. LPA stimulates cell proliferation mediated by serum response element (SRE)-driven recruitment of immediate-early response genes associated with growth (6) and stimulates SRE-driven luciferase activity in ovarian cancer cells but not in IOSE cells (19). Overexpression of c-Fos, which has an SRE-binding site in its promoter region (20), increases the expression of cyclin D1 mRNA in fibroblasts (21). Cyclin D1, a member of a protein family that regulates cyclin-dependent protein kinase activity, can act as an oncogene and has been implicated in the development of several human neoplasms (22,23). It is a key regulator of the G1-phase checkpoint and promotes cell cycle progression from G1 phase to S phase. Cyclin D1 is overexpressed in various human cancers, including ovarian cancer (24,25). Antisense cyclin D1 cDNA expression abolishes growth of pancreatic, hepatocellular, and breast carcinoma cells in nude mice (2628), indicating a critical role for cyclin D1 in tumorigenesis. In our study, we investigated whether LPA directly promotes ovarian tumor growth by increasing the level of cyclin D1, which increases cell proliferation.
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METHODS |
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We purchased 1-oleoyl-lysophosphatidic acid (i.e., LPA) and fatty acid-free bovine serum albumin from Sigma Chemical Corp. (St. Louis, MO). All restriction enzymes were from Promega (Madison, WI). All cell culture reagents were from the Cell Culture Facility, University of California, San Francisco. Ovarian cancer cell lines OVCAR-3, SKOV-3, and CAOV-3 were from the American Type Culture Collection (Manassas, VA). Ovarian cancer cell lines Hey-A8, OCC-1, and DOV-13 were provided by Gordon Mills and Robert Bast (University of Texas M. D. Anderson Cancer Center, Houston). IOSE-29 (simian virus 40 T antigen-immortalized normal ovarian surface epithelial cells) and normal OSE cells were provided by Nelly Auersperg (University of British Columbia, Vancouver, Canada). Human umbilical vein endothelial cells (HUVECs) were from Clonetics (San Diego, CA). Cyclin D1, c-Jun, c-Fos, and actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). EDG4 and EDG7 antibodies were from Calbiochem (La Jolla, CA). pLuc-MCS (multiple cloning sites; control), pAP1-Luc, pNFB-Luc (where NF
B is nuclear factor
B), and pSRE-Luc cis-reporting plasmids were from Stratagene (La Jolla, CA).
Cell Culture and LPA Stimulation
OVCAR-3, Hey-A8, OCC-1, and DOV-13 cells were cultured in RPMI-1640 medium with 10% fetal calf serum (FCS). CAOV-3 and SKOV-3 cells were cultured in Dulbeccos modified Eagle medium with 10% FCS. IOSE-29 and normal OSE cells were cultured in medium 199/MCDB 105 medium with 10% FCS. For LPA stimulation, cells were plated on six-well plates and cultured in complete growth medium. When cells reached confluence, they were washed twice with prewarmed phosphate-buffered saline (PBS) and cultured in serum-free medium overnight. LPA (0.220 µM) was added to the cultures, and cultures were incubated at 37 °C, as indicated. After incubation, cells were harvested and used to isolate total cellular RNA for northern blot analysis.
RNA Extraction and Northern Blot Analysis
Total cellular RNA was isolated from ovarian epithelial cancer cell lines and normal OSE cells by use of a High Pure RNA isolation kit (Roche, Indianapolis, IN) according to the manufacturers instructions. Equal amounts of total RNA (10 µg per lane) were separated by electrophoresis on denaturing 1.2% agarose gels containing 2.2 M formaldehyde and transferred to nylon membranes. RNAs were cross-linked to the membranes with UV irradiation and hybridized in Expresshyb hybridization buffer (BD Biosciences Clontech, Franklin Lakes, NJ) at 68 °C for 2 hours with 32P-labeled cDNA probes for Flt1, KDR, cyclin D1, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) synthesized with a random primer rediPrime DNA labeling kit (Amersham Pharmacia Biotech, Piscataway, NJ), according to the manufacturers instructions. The blots were washed for three 20-minute periods at room temperature in 2x standard saline citrate (SSC) (1x SSC = 150 mM sodium chloride and 15 mM sodium citrate) containing 0.05% sodium dodecyl sulfate (SDS) and then washed for two 30-minute periods at 50 °C in 0.1x SSC containing 0.1% SDS. Washed membranes were then exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY) with two intensifying screens for 12 days at 70 °C.
Western Blot Analysis
After LPA exposure, ovarian cancer cells and IOSE-29 cells were washed with ice-cold PBS and lysed in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, aprotinin at 10 µg/mL, leupeptin at 10 µg/mL, and pepstatin at 10 µg/mL. Lysates were clarified by centrifugation at 20 800g for 20 minutes at 4 °C. Supernatants were collected, and 50 µg of total protein was subjected to SDSpolyacrylamide gel electrophoresis in 10% gels. Proteins were transferred to a polyvinylidene difluoride membrane and probed with antibodies directed against cyclin D1, EDG4, EDG7, c-Jun, c-Fos, and actin. Blots were then washed, and bands were visualized by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and enhanced chemiluminescence reagents (Amersham Pharmacia Biotech).
Preparation of Cyclin D1 Promoter-Luciferase (CD1-Luc) Fusion Constructs
A 1882-base-pair (bp) PvuII fragment of human cyclin D1 (CD1) genomic clone, which contains the entire promoter region, was subcloned into the vector pA3-Luc to form the construct 1745 CD1-Luc (29). The constructs 66 CD1-Luc, 66 CD1/ATFm-Luc, 66 CD1/NFBm-Luc, and 22 CD1-Luc were created by polymerase chain reaction with specific primers, as described previously (2931). The ATF/CRE site in the 66 CD1/ATFm-Luc construct was mutated from 5'-TAACGTCAC ACGGAC-3' to 5'-TcgCGTCcCcCGGAC-3' (where lowercase letters are mutated bases), and the NF
B site in the 66 CD1/NF
Bm-Luc construct was mutated from 5'-AGGGGAGTTTT-3' to 5'-AccccAGTTTT-3'. The 3' end of the cyclin D1 promoter in all CD1-luciferase reporter constructs is +138 bp relative to the transcription start site. Various CD1-Luc constructs (e.g., 1745 CD1-Luc, 66 CD1-Luc, and 22 CD1-Luc) that contain different lengths of the cyclin D1 promoter region were used to identify possible LPA response elements in the cyclin D1 promoter. Constructs 66 CD1/NF
Bm-Luc and 66 CD1/ATFm-Luc were used to test whether NF
B and ATF transcription factors are involved in LPA stimulation of cyclin D1 transcription.
Cell Transfection and Luciferase Activity Measurements
For cell transfection, OVCAR-3 cells were plated in 12-well cluster plates (1.5 mL of medium per well) in triplicate. When OVCAR-3 cells were approximately 70% confluent, they were transfected for 2 hours with 1 µg of a CD1-Luc fusion construct, 7.5 µL of Superfect transfection reagent (Qiagen, Valencia, CA), and 0.02 µg of pRL-CMV, an internal control plasmid containing the cytomegalovirus (CMV) promoter linked to a constitutively active Renilla luciferase reporter gene. OVCAR-3 and IOSE-29 cells were 70% confluent for cotransfection, and incubations were for 2 hours. For cotransfection experiments with OVCAR-3 cells, 0.2 µg of CMV-IB
mutant (I
B
M) plasmid or pCMX control expression plasmid, 1 µg of a CD1-Luc fusion construct, and 0.02 µg of pRL-CMV plasmid were cotransfected. For cotransfection experiments with IOSE-29 cells, 0.2 µg of a cis-reporting plasmid or 1 µg of a CD1-Luc fusion construct; 1 µg of pCDEF3 control expression vector containing the human polypeptide elongation factor 1
(EF1
) promoter (another control for EDG4 and EDG7), EDG4/EF3, or EDG7/EF3 expression constructs; and 0.02 µg of pRL-CMV plasmid were cotransfected. After transfection, the medium was replaced with fresh growth medium, cells were incubated for 24 hours, and the medium was replaced with serum-free medium. After starvation in serum-free medium for 8 hours, cells were incubated in the presence or absence of 20 µM LPA for another 24 hours. Cells were harvested with passive lysis buffer (Promega), and luciferase activity was determined with a Dual-Luciferase Reporter Assay kit (Promega), according to the manufacturers protocol. Thus, 56 hours after transfection, cells were harvested, and luciferase activity was determined.
Data Analysis
Each experiment was performed in duplicate or triplicate. All experiments were repeated at least three times on different occasions. The results are expressed as the mean and 95% confidence interval (CI).
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RESULTS |
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Because VEGF plays an important role in the autocrine growth stimulation of some neoplasms that express VEGF receptors KDR and/or Flt1 (15,16), we first determined whether the increased VEGF level induced by LPA directly stimulated the proliferation of ovarian cancer cells. Treatment with VEGF had a minimal effect on ovarian cancer proliferation in vitro (data not shown). Total RNAs from four ovarian cancer cell lines, IOSE-29 cells, primary OSE cells, and HUVECs were analyzed by northern blotting for Flt1 and KDR mRNAs. HUVECs expressed both Flt1 and KDR mRNAs; OSE cells expressed KDR mRNA at a size and level similar to that in HUVECs. Flt1 and KDR mRNAs were not detected in IOSE-29 cells and the four human ovarian cancer cell lines tested, whereas GAPDH mRNA was detected in all cell lines (Fig. 1).
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We used western blot analysis with cyclin D1 antibody to investigate whether cyclin D1 protein was overexpressed in six ovarian cancer cell lines relative to the expression in IOSE-29 cells, which have a cyclin D1 level similar to that of normal OSE cells (data not shown). Cyclin D1 protein (36 kd) was overexpressed in Hey-A8, OCC-1, DOV-13, and OVCAR-3 cells but not in SKOV-3 or CAOV-3 cells (Fig. 2). Hey-A8, OCC-1, and DOV-13 cells expressed cyclin D1 at a high level and grew more rapidly than the other cancer cell lines tested in vitro (data not shown).
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Because cyclin D1 was overexpressed in ovarian cancer cells, it might be involved in LPA-stimulated ovarian cancer growth. To investigate this possibility, we examined whether LPA affected the expression of cyclin D1 protein in vitro by western blot analysis. We used human OVCAR-3 cells in this study because after intraperitoneal injection, female athymic mice develop intraperitoneal carcinomatosis and massive ascites, similar to those seen in stage III ovarian cancer (17). When OVCAR-3 cells were treated overnight (i.e., 18 hours) with LPA at 0.2 µM, 2 µM, and 20 µM (all physiologic concentrations), cyclin D1 levels increased in an LPA dose-dependent manner compared with untreated controls (Fig. 3, A). The level of cyclin D1 was slightly increased by 0.2 µM LPA, increased substantially with 2 µM LPA, and increased further by 20 µM LPA, all compared with untreated control cells. To determine the time course of this effect, OVCAR-3 cells were incubated with or without 20 µM LPA for 224 hours. The level of cyclin D1 increased after a 2-hour incubation, reached its maximum level after 6 hours, and continued to be elevated after 18 and 24 hours, all compared with untreated control cultures (Fig. 3, B
).
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To determine whether treatment with LPA increased the level of cyclin D1 in other ovarian cancer cells, we incubated three more ovarian cancer cell lines, OCC-1, DOV-13, and CAOV-3, with 20 µM LPA for 24 hours and determined the level of cyclin D1 protein by western blot analysis. Although basal levels of cyclin D1 did vary among the three cancer cell lines, all cell lines treated with LPA contained increased levels of cyclin D1 protein compared with corresponding untreated controls (Fig. 4).
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We used northern blot analysis to determine whether LPA induced higher levels of cyclin D1 protein and mRNA in OVCAR-3 cells. Cells were treated with various concentrations of LPA for various times. The increased levels of LPA-induced cyclin D1 mRNA corresponded with the increased levels of LPA-induced cyclin D1 protein in a time- and dose-dependent fashion (Fig. 5). In the doseresponse experiment, the level of cyclin D1 mRNA (4.5 kilobases [kb]) increased progressively from 0.2 to 20 µM LPA. In a time course experiment in which OVCAR-3 cells were incubated with 20 µM LPA, the level of cyclin D1 mRNA increased progressively for 26 hours and then declined after 18 and 24 hours; levels at 18 and 24 hours, however, were still higher than the basal level.
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The cyclin D1 promoter region contains binding sites for transcription factors SP1, AP1, NFB, and ATF/CREB, all of which regulate cyclin D1 transcription (2931). Because treatment with LPA increased the level of cyclin D1 mRNA (4.5 kb) in OVCAR-3 cells in a time- and dose-dependent manner, we investigated whether LPA stimulated cyclin D1 promoter activity by using transient transfection of cyclin D1 promoter-luciferase reporter constructs into OVCAR-3 cells (Fig. 6
). Luciferase activity of construct 1745 CD1-Luc, which contains the entire cyclin D1 promoter, was increased 3.0-fold (95% CI = 2.7-fold to 3.3-fold) after LPA treatment compared with untreated controls. Luciferase activity of a promoter construct with a deletion between positions 1745 and 66 was increased 2.5-fold (95% CI = 2.1-fold to 2.9-fold) after LPA treatment compared with untreated controls. However, the luciferase activity of construct 22 CD1-Luc, a promoter construct with a minimal promoter fragment (from positions 22 to +138), was not altered by LPA treatment. These results indicate that the LPA response element is located principally between positions 66 and 22 in the cyclin D1 promoter. Luciferase activity of the construct with an ATF/CRE site mutation (at position 57) was stimulated less by LPA (2.0-fold, 95% CI = 1.6-fold to 2.4-fold), and the luciferase activity of the construct with an NF
B site mutation (at position 33) was stimulated even less (1.4-fold, 95% CI = 1.1-fold to 1.7-fold), both compared with the untreated control for each plasmid. Luciferase activity of the construct with a promoter deletion between positions 1745 and 66 and a mutation of the ATF/CRE or NF
B site was low with or without LPA treatment, indicating that these transcription factors may have important roles in the basal transcription of cyclin D1 (Fig. 6, A
).
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c-Jun and c-Fos are major components of transcription factor AP1. Both JunJun and JunFos dimers recognize AP1 site located at position 954 in the cyclin D1 promoter, whereas JunATF and FosATF complexes recognize ATF/CRE sites (33) at position 57 or the AP1 site at position 954 in the cyclin D1 promoter (29,30). Because mutation of the ATF/CRE site in the cyclin D1 promoter region and a promoter deletion (from positions 1745 to 66) containing the AP1 site partially diminished LPA activation of the cyclin D1 promoter (Fig. 6, A and B), we used western blot analysis to determine whether LPA induced the expression of c-Fos and c-Jun, which bind to these sites, in OVCAR-3 cells. When OVCAR-3 cells were incubated with 20 µM LPA, the level of c-Fos protein (62 kd) increased markedly and consistently for 26 hours and then declined by 18 and 24 hours but not to unstimulated control levels. LPA stimulation of c-Jun protein (39 kd) occurred after 2 hours, reached a maximum level after 4 hours, then declined gradually for 618 hours, and returned to unstimulated control values after 24 hours. LPA had no effect on the level of actin (internal control) (Fig. 6, C
).
EDG4 and EDG7 Expression in IOSE-29 Cells and Ovarian Cancer Cell Lines
We next analyzed the expression of the LPA receptor proteins EDG4 and EDG7 in IOSE-29 cells and six ovarian cancer cell lines by western blot analysis. Although EDG7 protein (40 kd) was detected in IOSE-29 cells and the six ovarian cancer cell lines, the highest levels of EDG7 were detected in SKOV-3 and CAOV-3 cells (Fig. 7). Levels of EDG7 were similar in normal OSE cells and IOSE-29 cells (data not shown). EDG4 (50 kd) was detected by western blot analysis in all six cancer cell lines but not in IOSE-29 cells (Fig. 7
). The levels of EDG4 expression detected are consistent with those in our previous study (17), in which we detected EDG4 mRNA in all ovarian cancer cell lines studied but not in normal OSE or IOSE-29 cells.
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We next determined whether IOSE-29 cells acquired LPA responsiveness after the forced expression of EDG4 or EDG7 cDNAs, by assessing the LPA stimulation of cyclin D1 promoter-, AP1-, NFB-, and SRE-driven luciferase transcription activity (Fig. 8
). When EDG4 or EDG7 cDNAs were cotransfected with the AP1-, NF
B-, or SRE-luciferase plasmid, both cDNAs mediated increased AP1-, NF
B-, or SRE-driven luciferase transcription activity induced by 20 µM LPA. The increase mediated by EDG4 was larger than that mediated by EDG7. Luciferase activity in IOSE-29 cells cotransfected with pCDEF3 control expression vector (containing the EF1
promoter) and an AP1-, NF
B-, or SRE-luciferase plasmid was minimally responsive to LPA; however, the luciferase activity in IOSE-29 cells cotransfected with EDG4/EF3 or EDG7/EF3 expression plasmids and a control pLuc-MCS (containing no transcription factor-binding sites) was not affected by LPA (Fig. 8, A
). Forced expression of EDG4 or EDG7 cDNAs in IOSE-29 cells increased the LPA stimulatory effect on cyclin D1 promoter activity by 2.6-fold (95% CI = 2.4-fold to 2.8-fold) or 1.8-fold (95% CI = 1.6-fold to 2.0-fold), respectively, from construct 1745 CD1-Luc but not from the shorter construct 22 CD1-Luc construct, both compared with control vector pCDEF3 (Fig. 8, B
). Thus, primarily EDG4, and to a lesser extent EDG7, are required for LPA stimulation of cyclin D1 expression in ovarian cancer cells.
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DISCUSSION |
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Other pathways can be involved in the development of various human cancers, such as the retinoblastoma (Rb)/cyclin D1/p16 pathway (35). A previous study (36) showed that cyclin D1 was overexpressed in four of six ovarian cancer cell lines and that ovarian cancer cells that coexpress endogenous Rb and p16 were insensitive to overexpression of functional p16 protein. Thus, LPA-induced cyclin D1 in ovarian cancer cell lines with endogenous p16 protein, such as CAOV-3 and OVCAR-3, might circumvent the need to disrupt p16 expression. However, other mechanisms may also be involved, because some cancer cell lines (e.g., Hey-A8, DOV-13, OCC-1, and SKOV-3) express Rb but not p16 protein (37). The lower level of cyclin D1 that we observed in CAOV-3 cells may be caused by their lack of Rb, which would ultimately lead to disassembly of the complex containing cyclin D1 and cyclin-dependent kinase 4/6 and the increased turnover of cyclin D1 (38).
We demonstrated that LPA specifically stimulates cyclin D1 promoter activity approximately threefold and that the LPA response element is located in the promoter region of the cyclin D1 gene, principally between positions 66 and 22, and contains ATF/CRE and NFB sites. LPA stimulation of cyclin D1 promoter activity was diminished more by mutation of the NF
B site than by mutation of the ATF/CRE site. I
B
M binds to NF
B and blocks translocation of NF
B into the nucleus, which prevents the induction of specific NF
B target genes (39). Because I
B
M abolished LPA-enhanced cyclin D1 promoter activity, we speculate that I
B
M inhibited both basal and LPA-inducible cyclin D1 promoter transcription, just as mutation of the NF
B site in the 66 CD1-Luc plasmid also markedly reduced both basal and LPA-inducible luciferase activity (Fig. 6, A
). LPA activates NF
B activity by inducing degradation of the I
B
inhibitor (40). When NF
B signaling is blocked by the forced expression of I
B
M, angiogenesis is inhibited and the tumorigenicity of ovarian cancer cells is reduced (41). In another study (31), cyclin D1 protein and mRNA were reduced in myoblast cells expressing I
B
M.
Our data demonstrate that mutation of the ATF/CRE site or deletion of the AP1 site (positions 1745 to 66) in the cyclin D1 promoter reduced the ability of LPA to activate the cyclin D1 promoter. Forced expression of c-Jun and c-Fos increased cyclin D1 expression through both AP1 and ATF/CRE sites (29,30,42,43). In fibroblasts derived from c-Fos-/- fosB-/- mouse embryos that have a defect in proliferation associated with selective reduction of the cyclin D1 level, induction of c-Fos expression restored both cyclin D1 expression and DNA synthesis, indicating pivotal roles for c-Fos and cyclin D1 in cell proliferation (43). c-Jun-/- fibroblasts have reduced levels of cyclins D1 and D3 (44). In this article, we demonstrated that LPA dramatically induces c-Fos and c-Jun proteins in ovarian cancer cells. Maximal stimulatory effects of LPA on the expression of c-Fos observed after 24 hours of incubation and on that of c-Jun observed after 4 hours of incubation occurred much earlier than those on cyclin D1 protein observed after 618 hours of incubation. Therefore, LPA probably stimulates the expression of cyclin D1, in part, by activating c-Jun and c-Fos, which bind to the AP1 and ATF/CRE sites and then increase the transcription of cyclin D1. Because c-Jun is also required for cyclin D3 expression (44) and because the cyclin D3 promoter region contains AP1 sites (45), LPA likely also induces cyclin D3 expression in ovarian cancer cells to further increase cell proliferation. Because the region of the cyclin D1 promoter from positions 1745 to 66 contains one AP1 site, four SP1 sites, and two STAT (signal transducer and activator of transcription) sites (29,42,46), we do not exclude the possibility that SP1 and STATs are also involved in LPA-stimulated cyclin D1 expression in ovarian cancer cells. Demonstrating this involvement will require further experiments.
Some ovarian cancer cell lines, particularly SKOV3 and CAOV3, expressed EDG7 protein at higher levels than IOSE-29 cells. However, EDG4 protein (in this article) and mRNA (17) were detected at high levels in all ovarian cancer cell lines studied but not in normal OSE or IOSE-29 cells. Forced expression of EDG4 or EDG7 cDNAs caused IOSE-29 cells to become sensitive to LPA stimulation of cyclin D1 promoter-, SRE-, AP1-, and NFB-driven gene transcription, with the effect of EDG4 being larger than that of EDG7. Ovarian cancer cells (such as OVCAR-3) are insensitive to apoptosis induced by serum starvation, in contrast to normal and immortalized OSE cells, which are sensitive to such apoptosis. We found that when IOSE-29 cells were transiently transfected with EDG4 or EDG7 cDNAs, the survival rate of the transfected cells in serum-free medium containing 20 µM LPA was substantially greater than that of nontransfected IOSE cells under similar conditions (data not shown). Thus, the expression of EDG4 or EDG7 may suppress apoptosis in ovarian epithelial cells. Recently, cyclin D1 expression was shown to be increased by platelet-derived growth factor, epidermal growth factor, and basic fibroblast growth factor through a phosphatidylinositol 3-kinase (PI3K) pathway (47,48). This PI3K pathway can be activated by LPA, resulting in increased cell proliferation (6). PI3K gene amplification occurs in more than 40% of ovarian cancer cell lines and primary ovarian tumors (49). Thus, LPA also may induce cyclin D1 expression in ovarian cancer cells through the PI3K signaling pathway.
We previously showed (17) that LPA stimulates VEGF expression through transcriptional activation in ovarian cancer cells but not in IOSE-29 cells. In this article, we did not detect the VEGF receptors Flt1 and KDR in the four ovarian cancer cell lines studied, although we detected KDR in normal OSE cells. Because VEGF has little effect on ovarian cancer cell growth in vitro and LPA increases cyclin D1 expression in ovarian cancer cells but not in IOSE-29 cells, dual mechanisms are probably involved in LPA-stimulated ovarian tumor growth in vivoan indirect endothelial cell-dependent mechanism that involves increasing angiogenesis via VEGF and a direct endothelial cell-independent mechanism that involves increasing cell proliferation via cyclin D1. Thus, blockade of the two distinct LPA mechanisms by LPA antagonist(s) and/or antisense LPA receptor RNA (particularly that for EDG4) may be a useful approach for inhibiting ovarian tumor growth.
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NOTES |
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We thank Gordon B. Mills and Robert C. Bast for Hey-A8, OCC1, and DOV-13 ovarian cancer cell lines; Nelly Auersperg for OSE and IOSE-29 cells; and Meng Kian Tee for helpful advice and discussion.
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Manuscript received July 19, 2002; revised March 7, 2003; accepted March 19, 2003.
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