Affiliations of authors: Y.-L. Hu, M.-K. Tee, R. B. Jaffe (Center for Reproductive Sciences), E. J. Goetzl (Departments of Medicine and MicrobiologyImmunology), University of California, San Francisco; N. Auersperg, Departments of Anatomy and Obstetrics and Gynecology, University of British Colombia, Vancouver, Canada; G. B. Mills, The University of Texas M. D. Anderson Cancer Center, Houston; N. Ferrara, Department of Cardiovascular Research, Genentech, Inc., South San Francisco, CA.
Correspondence to: Robert B. Jaffe, M.D., Center for Reproductive Sciences, Box 0556, University of California, San Francisco, 505 Parnassus Ave., San Francisco, CA 94143 (e-mail: jaffer{at}obgyn.ucsf.edu).
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ABSTRACT |
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INTRODUCTION |
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Angiogenesis is necessary for tumor growth, and a transition from limited to rapid growth accompanies neovascularization (12,13). The development of new blood vessels depends on the production of angiogenic factors released from tumor cells and/or cells in the tumor microenvironment. Among these angiogenic factors, VEGF, also known as vascular permeability factor, binds to vascular endothelial cells and is a potent inducer of angiogenesis. Cancer patients have increased serum VEGF, and elevated VEGF messenger RNA (mRNA) levels occur in the majority of human cancers (14). Ovarian carcinoma is the most lethal gynecologic cancer in U.S. women. Ovarian carcinomas are predominantly derived from the ovarian surface epithelium and are characterized by widespread intraperitoneal carcinomatosis and formation of large volumes of ascitic fluid. We and others have reported that VEGF mRNA and protein are extensively expressed in ovarian tumors and ovarian cancer cell lines (1519), and VEGF also plays a pivotal role in ascites formation associated with ovarian cancer by increasing vascular permeability (19).
Human OVCAR-3 cancer cells express VEGF mRNA and protein (16), and after receiving an intraperitoneal injection of OVCAR-3 cells, female athymic mice develop intraperitoneal carcinomatosis and massive ascites similar to clinical ovarian cancer (19,20). We hypothesized that LPA promotes ovarian tumor growth by increasing angiogenesis via VEGF. Therefore, we used OVCAR-3 cells as a model to investigate the stimulation of VEGF gene expression by LPA and mechanism(s) involved in LPA induction of VEGF expression. Because LPA stimulates ovarian tumor cell growth, we also explored Edg2/Edg4 expression in ovarian cancer cell lines and in normal ovarian surface epithelial cells.
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MATERIALS AND METHODS |
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The following items were purchased: 1-oleoyl-LPA and fatty acidfree bovine serum albumin (Sigma Chemical Co., St. Louis, MO) and pGL2-basic vector and all restriction enzymes (Promega Corp., Madison, WI). All cell culture reagents were obtained from the Cell Culture Facility, University of California, San Francisco. The ovarian cancer cell lines OVCAR-3, SKOV-3, and CAOV-3 were obtained from American Type Culture Collection (Manassas, VA). The ovarian cancer cell line DOV-13 was provided by Dr. Robert Bast (The University of Texas M. D. Anderson Cancer Center, Houston). IOSE-29 was a simian virus 40 T-antigen-immortalized ovarian surface epithelial cell line (provided by Dr. Nelly Auersperg), and normal ovarian surface epithelial cells were obtained by scraping the ovarian surface during surgery for nonmalignant disorders.
Cell culture and LPA stimulation.
OVCAR-3 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 Dulbecco's modified Eagle medium with 10% FCS. IOSE-29 and normal ovarian surface epithelial cells were cultured in medium 199/MCDB 105 with 10% FCS as described previously (21,22). For LPA stimulation, cells were seeded on six-well plates and cultured in complete growth medium. Upon reaching confluence, the cells were washed twice with prewarmed phosphate-buffered saline and cultured in serum-free medium overnight. LPA was added to the culture at a concentration of 20 µM, and incubation was carried out at 37 °C for various times up to 24 hours. In a different experiment, various concentrations of LPA (0.0220 µM) were added to the culture, and incubation was carried out at 37 °C for 24 hours. After incubation, the conditioned medium was aspirated and saved for VEGF enzyme-linked immunosorbent assay (ELISA). The cells were harvested and used to isolate cellular total RNA for northern blotting analysis.
RNA extraction and northern blotting analysis.
Total cellular RNA was isolated from the ovarian epithelial cancer cell lines and from the normal ovarian surface epithelial cells with the use of a High Pure RNA Isolation Kit (Boehringer Mannheim Biochemicals, Indianapolis, IN) according to the manufacturer's 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. The membranes were UV cross-linked and hybridized in the Expresshyb hybridization buffer (Clontech Laboratories, Inc., Palo Alto, CA) at 68 °C for 2 hours with 32P-labeled VEGF, Edg2, Edg4, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNA (cDNA) probes synthesized with a random primer rediPrime DNA labeling kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) according to the manufacturer's instructions. The blots were washed at room temperature in 2x SSC (i.e., 300 mM sodium chloride and 30 mM sodium citrate)/0.05% sodium dodecyl sulfate (SDS) for 20 minutes three times and then at 50 °C in 0.1x SSC/0.1% SDS for 30 minutes twice. The washed membranes were then exposed to Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY) with two intensifying screens for 12 days at -70 °C.
VEGF ELISA.
The ovarian cancer cell lines were each incubated in serum-free medium overnight and then in the presence or absence of LPA. Conditioned media were centrifuged at 1100g for 10 minutes at 4 °C to remove debris and stored at -70 °C until analysis. The VEGF protein level in the conditioned media was determined with the use of ELISA (23).
Stability of VEGF mRNA.
To determine the stability of VEGF mRNA, we incubated OVCAR-3 cells in serum-free medium overnight and then in the presence or absence of LPA (20 µM) for 24 hours. Thereafter, cells were incubated with 5 µg/mL dactinomycin (i.e., actinomycin D). Total RNA was extracted 0, 0.25, 0.5, 1, 2, and 4 hours later. Northern blotting analysis was performed for VEGF mRNA expression. Relative VEGF mRNA levels represent arbitrary units normalized to GAPDH mRNA levels.
Preparation of VEGF promoterluciferase fusion constructs.
The 5.1-kilobase (kb) VEGF promoter fragment, containing the first two exons of the human VEGF gene and 3.4-kb sequence upstream to the translation initiation site, was provided by Dr. D. Leitman (University of California, San Francisco) (24). The 5.1-kb fragment was digested with KpnI and NheI restriction enzymes, and the resulting 2324-base-pair (bp) VEGF promoter fragment (-2274 to +50 bp relative to the transcription start site) was cloned into KpnI and NheI sites of the promoter-less luciferase reporter pGL2-basic vector to generate pGL2-2274VEGF. A series of other VEGFluciferase constructs containing different lengths of the VEGF promoter fragment were prepared by digestion with the appropriate restriction enzymes and insertion into the polylinker of pGL2-basic vector in the 5'- to 3'-orientation with respect to the coding region of the luciferase reporter gene. (The short construct [pGL2-25VEGF] contains a 75-bp [from -25 to +50 bp] VEGF promoter fragment.) The sequences of the VEGFluciferase constructs were confirmed by multiple digestions with convenient restriction enzymes. All of the fusion constructs have the same 3'-end in the NheI site of the pGL2-basic vector.
Cell transfection and luciferase activity measurements.
For cell transfections, OVCAR-3 cells were seeded in 12-well cluster plates in triplicate. When reaching about 70% confluence, the cells were transfected for 2 hours with 1 µg of VEGFluciferase fusion constructs, 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). After transfection, the medium was replaced by fresh normal growth medium, and the cells were incubated for 24 hours. After starvation in serum-free medium for 8 hours, the cells were incubated in the presence or absence of LPA (20 µM) for another 24 hours. The cells were harvested with passive lysis buffer, and the luciferase activities were determined with the use of a Dual-Luciferase Reporter Assay Kit (Promega Corp.) according to protocols provided by the manufacturer. In cotransfection experiments, 0.5 µg of pCMV-Fos and 0.5 µg of pCMV-Jun expression plasmids were cotransfected with VEGFluciferase fusion constructs and pRL-CMV plasmid at the same time; 48 hours after transfection, the cells were harvested and luciferase activities were determined.
Statistical analysis.
Each experiment was performed in duplicate or triplicate. All experiments were repeated at least three times on different occasions. The results are presented as means ± 95% confidence intervals of all the values. A paired Student's t test was used to evaluate statistically significant differences in VEGF protein levels between the LPA treatment groups and the vehicle control group. P<.05 was selected as the statistically significant value. All statistical tests and corresponding P values were two-sided.
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RESULTS |
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We first performed northern blotting to determine whether LPA affects the levels of VEGF mRNA in vitro. OVCAR-3 cells were incubated with or without LPA from 2 to 24 hours. LPA (20 µM) stimulation of VEGF mRNA in OVCAR-3 cells occurred after a 4-hour incubation, reaching a maximum level after 16 hours, then declining after 24 hours, but VEGF mRNA levels were still substantially higher than control levels (0 time) (Fig. 1, A). LPA also increased VEGF mRNA levels in OVCAR-3 cells in a dose-dependent fashion (Fig. 1
, B). VEGF mRNA levels were markedly enhanced by 2 µM, with a further increase induced by 20 µM, concentrations that are present physiologically.
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VEGF Protein Induction by LPA in Ovarian Cancer Cell Lines
OVCAR-3 cells were incubated in the absence or presence of various concentrations of LPA and for various times (Fig. 2, A and B). Conditioned media were assayed for secreted VEGF by ELISA. VEGF production induced by LPA paralleled that of LPA stimulation of VEGF mRNA in a time- and dose-dependent manner. After LPA stimulation, secreted VEGF levels increased progressively up to 3.2-fold above basal levels from 4 to 16 hours of incubation, then diminished at 24 hours, but still were 2.9-fold above the control level. VEGF secretion also rose progressively when OVCAR-3 cells were treated with various concentrations of LPA.
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Mechanism of LPA Stimulation of VEGF Expression
We first investigated VEGF mRNA stability by examining its half-life. OVCAR-3 cells were incubated with or without LPA for 24 hours. Dactinomycin was then added at various times. Total RNA was isolated for northern blotting. The half-life of VEGF mRNA in both untreated and treated OVCAR-3 cells was similar (Fig. 3, A), indicating that LPA-evoked increases were not attributable to increased mRNA stability by prolonging its half-life. We speculated that LPA might increase the VEGF transcription rate by stimulating VEGF promoter activity. Therefore, two VEGF promoterreporter constructs were transfected transiently into OVCAR-3 cells. The luciferase activity of the pGL22274VEGF construct, which contains the entire VEGF promoter, was considerably increased by twofold after LPA treatment. However, LPA had no effect on the luciferase activity of the shorter construct (pGL225VEGF, which contains a minimal promoter fragment [25 to +50 bp relative to the transcription start site]) or pGL2-basic vector (Fig. 3
, B).
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LPA Receptors in Normal Ovarian Surface Epithelial Cells, IOSE-29 Cells, and Ovarian Cancer Cell Lines
We further analyzed the expression of LPA receptors in normal ovarian surface epithelial cells (passages 3 and 4), IOSE-29 cells, and ovarian cancer cell lines. Edg4 (1.8-kb transcript) was readily detectable by northern blotting in all cancer cell lines studied but not in normal ovarian surface epithelial or in IOSE-29 cells. In contrast, Edg2 (4.6-kb transcript) was expressed at similar levels by all cell lines (although at a low level in OVCAR-3) (Fig. 4). Similar results were obtained by reverse transcriptionpolymerase chain reaction and by western blotting with antibodies to Edg2 and Edg4 (data not shown).
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DISCUSSION |
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We demonstrated that LPA enhances VEGF mRNA and protein levels in ovarian carcinoma cell lines. LPA increases the VEGF transcription rate rather than the mRNA stability. However, VEGF levels in untreated and LPA-treated DOV-13 cells were undetectable. This result is not unexpected, since this is the only cell line that does not have gene amplification of phosphoinositol-3 kinase (PI3-K) among about 80% of ovarian cancer cell lines and primary ovarian tumors, and both LPA and VEGF signal via a PI3-K pathway (26). Although the increase in VEGF protein is modest (1.5-fold to threefold), a twofold to fourfold increase in VEGF leads to considerable tumorigenic competence in colorectal cancer (27). LPA stimulation of ovarian cancer but not of IOSE-29 cell proliferation in vitro can be associated with VEGF induction by LPA. Because we examined only one IOSE cell line, it would be of interest to confirm these observations with the use of other IOSE cell lines. Thus, our present data suggest that LPA stimulation of VEGF expression is a common response of ovarian cancer cell lines and that LPA stimulates tumor growth indirectly through increasing expression of VEGF and perhaps other angiogenic factors (e.g., interleukin 8 and basic fibroblast growth factor [bFGF]) to provide the blood supply essential both for progressive growth of primary malignancies and for development of metastatic disease.
We demonstrated that LPA specifically stimulates the VEGF promoter activity, and we are continuing to localize LPA response element(s) in the promoter region. Analysis of the VEGF promoter region reveals potential binding sites for transcription factors AP1, AP2, and SP1, which are involved in the activation of VEGF expression by platelet-derived growth factor, tumor necrosis factor-, bFGF, and transforming growth factor-
(24,2830). Our data also demonstrate that forced expression of c-jun and c-fos cDNAs can stimulate VEGF promoter activity fourfold in OVCAR-3 cells. Interaction of c-Jun with SP1 transcription factor stimulates expression of cardiac-specific atrial natriuretic factor (31). Such an interaction might also occur in the VEGF promoter and accounts, in part, for the fourfold increase in promoter activity. To examine this possibility, we created two 5' deletional promoterluciferase constructs at 268 and 131 that have potential AP2 and SP1 sites but lack three AP1 sites. After either LPA treatment or forced expression with c-jun and c-fos cDNAs, the luciferase activities of these constructs were intermediate between constructs pGL22274VEGF and pGL225VEGF (data not shown). This observation suggests that LPA stimulation of ovarian tumor growth via VEGF can occur through activation of both AP1 and SP1. AP1 promotes tumor progression and metastasis (3234). However, the specific mechanisms by which LPA induces VEGF via Fos-Jun and Jun-Sp1 interaction with the VEGF promoter are still unclear and await further exploration with the use of site-directed mutagenesis and gel mobility shift assays. LPA may also activate other transcription factors (e.g., HIF-1) to further induce VEGF expression, since LPA can activate a PI3-K (2), which is involved in hypoxia induction of VEGF expression through HIF-1 (35).
The four ovarian cancer cell lines that we studied, as well as OV202 primary ovarian cancer cells (36), express Edg2 and Edg4, while normal and immortalized ovarian surface epithelial cells express only Edg2. LPA stimulates DNA synthesis and activates serum response elementluciferase reporter activity in ovarian cancer cells but not in IOSE-29 cells. Furthermore, pertussis toxin and mitogen-activated protein kinase kinase inhibitor (PD98059), which block LPA receptor-signaling pathways, suppress ovarian cancer cell responses to LPA (36). Thus, our data suggest that Edg4, but not Edg2, may be a key receptor involved in LPA stimulation of ovarian tumor growth, together with the activation of VEGF expression. Other Edg receptors may also be involved, and the factor(s) that induce Edg4 expression in ovarian cancer cells remain to be explored.
Results of treatment of ovarian epithelial cancer have changed minimally (37), likely because of the failure to detect the disease at an early stage. Our data show that Edg4 was detected at relatively high levels in ovarian cancer cell lines but not in normal ovarian surface epithelial cells. Thus, Edg4 may be a novel biomarker for ovarian cancer, as LPA may be. However, the levels of LPA receptors, especially of Edg4, do not appear to correlate precisely with the VEGF response of ovarian cancer cells to LPA. We speculate that Edg4 is necessary but not sufficient (e.g., a PI3-K-signaling pathway also is needed) for LPA stimulation of VEGF in ovarian cancer cells. Since LPA can stimulate ovarian tumor growth by inducing angiogenesis through increasing VEGF expression, inhibition of cell growth could be achieved by creating soluble dominant negative Edg4 mutant(s) that can block the effects of LPA. Thus, an Edg4 dominant negative mutant(s) and/or antisense Edg4 sequence may be useful in inhibiting ovarian tumor growth in vivo and in vitro.
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NOTES |
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We thank Kyu Hong and Gloria Meng for technical assistance in performing the VEGF ELISA.
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Manuscript received August 14, 2000; revised February 21, 2001; accepted March 6, 2001.
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