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Cancer Research 67, 5285, June 1, 2007. doi: 10.1158/0008-5472.CAN-07-0828
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Extracellular Signal-Regulated Kinase Is a Target of Cyclooxygenase-1-Peroxisome Proliferator-Activated Receptor-{delta} Signaling in Epithelial Ovarian Cancer

Takiko Daikoku1, Susanne Tranguch1,2, Anindita Chakrabarty1, Dingzhi Wang3, Dineo Khabele5, Sandra Orsulic6, Jason D. Morrow4, Raymond N. DuBois2,3 and Sudhansu K. Dey1,2,4

Departments of 1 Pediatrics, 2 Cell and Developmental Biology, 3 Medicine, and 4 Pharmacology, Division of Reproductive and Development Biology, Vanderbilt University Medical Center; 5 Department of Obstetrics and Gynecology, Meharry Medical College, Nashville, Tennessee; and 6 Massachusetts General Hospital Center for Cancer Research, Harvard Medical School, Charlestown, Massachusetts

Requests for reprints: Sudhansu K. Dey, Division of Reproductive and Developmental Biology, Vanderbilt University, Nashville, TN 37232-2678. Phone: 615-322-8642; Email: sk.dey{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The underlying causes of epithelial ovarian cancer (EOC) are unclear, and treatment options for patients with advanced disease are limited. There is evidence that the use of nonsteroidal anti-inflammatory drugs is associated with decreased risk of developing EOC. Nonsteroidal anti-inflammatory drugs inhibit cyclooxygenase (COX)-1 and COX-2, which catalyze prostaglandin biosynthesis. We previously showed that mouse and human EOCs have increased levels of COX-1, but not COX-2, and a COX-1–selective inhibitor, SC-560, attenuates prostaglandin production and tumor growth. However, the downstream targets of COX-1 signaling in EOC are not yet known. To address this question, we evaluated peroxisome proliferator-activated receptor {delta} (PPAR{delta}) expression and function in EOC. We found that EOC cells express high levels of PPAR{delta}, and neutralizing PPAR{delta} function reduces tumor growth in vivo. More interestingly, aspirin, a nonsteroidal anti-inflammatory drug that preferentially inhibits COX-1, compromises PPAR{delta} function and cell growth by inhibiting extracellular signal-regulated kinases 1/2, members of the mitogen-activated protein kinase family. Our study, for the first time, shows that whereas PPAR{delta} can be a target of COX-1, extracellular signal-regulated kinase is a potential target of PPAR{delta}. The ability of aspirin to inhibit EOC growth in vivo is an exciting finding because of its low cost, lack of cardiovascular side effects, and availability. [Cancer Res 2007;67(11):5285–92]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The hallmark of epithelial ovarian cancers (EOC), arising from the ovarian surface epithelium (OSE), is the rapid growth and spread of solid i.p. tumors frequently associated with ascites and other complications. EOC is the major cause of death in patients with gynecologic malignancies and is the fourth leading cause of cancer death in the United States (1–3). Patient mortality is high because diagnosis of the disease at early stages is difficult and its underlying causes are poorly understood.

Cyclooxygenase (COX)-1 and COX-2 catalyze the conversion of arachidonic acid (AA) to prostaglandins. Although overwhelming evidence suggests a role for COX-2 in a variety of cancers, the contribution of COX-1 to cancers remains undefined or controversial (4). We recently showed that human EOC overexpresses COX-1, but not COX-2, and that prostacyclin (PGI2) and prostaglandin E2 (PGE2) are the major prostanoids generated by COX-1 in human ovarian cancer cells, OVCAR3 (1). We have also shown that genetically engineered mouse OSE cells lacking p53 but expressing c-myc and Akt, or c-myc and K-ras, produce tumors that overexpress COX-1 with little or no COX-2 expression (4). These OSE cells primarily produce PGI2 via COX-1, and treatment with SC-560, a selective COX-1 inhibitor, attenuates PGI2 production and OSE cell growth in vitro and tumor growth in vivo (4). More importantly, various mouse models of EOC also show high COX-1 overexpression when compared with COX-2, suggesting that the participation of COX-1 in EOC is not unique to specific genetic and oncogenic alterations, but rather is a general characteristic of EOC (5). Aspirin is a nonsteroidal anti-inflammatory drug and a nonspecific COX inhibitor that preferentially inhibits COX-1 over COX-2 (6). Aspirin has an added advantage of being readily available and inexpensive. Some epidemiologic studies have found an association between the consumption of aspirin and a reduced risk of many cancers, including EOC (7–9).

The downstream targets of COX-1–derived prostaglandins in EOC are not yet known. PGI2 and PGE2, in addition to their interactions with cell surface G-protein–coupled receptors IP and EP, respectively, can also activate peroxisome proliferator-activated receptor {delta} (PPAR{delta}). Although PGI2 can directly bind and activate PPAR{delta}, PGE2 transactivates PPAR{delta} via the Wnt/ß-catenin pathway in colorectal cancer (10, 11). Each PPAR isoform (PPAR{delta}, PPAR{alpha}, and PPAR{gamma}) binds to sequence-specific DNA response elements as a heterodimer with one of the retinoic acid X receptors (RXR{alpha}, RXRß, or RXR{gamma}), a promiscuous partner (12). PPAR{delta} was also shown to play important roles in embryo implantation, placentation, epidermal maturation, wound healing, fatty acid metabolism, repression of atherogenic inflammatory responses, and regulation of apoptosis (13–18).

Although PPAR{delta} has been implicated in tumorigenesis, its exact role remains unclear (19–21). In mammary and hepatocellular cancers, PPAR{delta} activation stimulates cell proliferation and tumor growth (22, 23). In addition, PPAR{delta} is overexpressed in human colorectal tumors (24), head and neck squamous carcinomas (25), endometrial adenocarcinomas (26), and human breast cancer cell lines (27). Although we have shown that activation of PPAR{delta} promotes intestinal tumor growth in mice, there is also evidence that its activation attenuates tumor growth (19–21, 28). These disparate results suggest that the contribution of PPAR{delta} to tumor growth depends on the cellular context and perhaps the genetic makeup of the species being studied. To our knowledge, the expression profile of PPAR{delta} and its role in EOC have not yet been examined.

In this study, we found that PPAR{delta} is highly expressed in mouse and human EOC tumors and that neutralization of PPAR{delta} activity results in reduced tumor growth in mouse models of EOC. We also provide new evidence that aspirin compromises PPAR{delta} function by lowering prostaglandin levels and attenuates EOC cell growth by inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. These results, and our previous observations of heightened expression of COX-1 in EOC, suggest that COX-1-PPAR{delta}-ERK signaling plays a critical role in EOC, and inhibition of this signaling cascade by aspirin provides a potential treatment option for EOC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. Immunocompromised Rag2/{gamma}C null female mice were used for all experiments. Homozygous Rag2/{gamma}C null females, hemizygous {gamma}C null, and homozygous Rag2 null males derived on a B6 background were purchased from Taconic. We also generated Rag2/{gamma}C null mice on a CD1 background (F7) for experiments. All mice were used between 6 and 12 weeks of age and housed in the Vanderbilt Institutional Animal Care Facility according to NIH and Institutional guidelines on the care and use of laboratory animals.

Cell lines and culture. To generate C1 (genotype: p53–/–, c-myc, K-ras) and C2 (genotype: p53–/–, c-myc, Akt) cell lines, ovarian explants from K5-TVA/p53–/– mice were infected with different combinations of RCAS viruses carrying human c-myc, mouse K-rasG12D, and mouse myr-Akt1 oncogenes as previously described (2, 4). Cell cultures were passaged in the presence of virus for 3 weeks, after which they consisted of a pure population of transformed OSE cells. T1 and T2 cell lines were derived by isolating cells from tumors that were generated by orthotopic placement of C1 and C2 cells into nude mice, respectively. C1, C2, T1, and T2 cell lines develop tumors when transplanted into immunocompromised mice. OSE cells were propagated in DMEM containing 10% fetal bovine serum, 100 units/mL of penicillin, and 100 µg/mL of streptomycin. OVCAR3 human ovarian cancer cells (American Type Culture Collection) were cultured in RPMI 1640 (American Type Culture Collection) supplemented with 20% fetal bovine serum and antibiotics. For ERK phosphorylation assays, T2 (5 x 104 cells/60 mm dish) or OVCAR3 (105 cells/60 mm dish) cells were seeded and incubated for 24 or 72 h, respectively, followed by 24 h of serum starvation. Cells were then treated with AA (Cayman Chemical), carbaprostacyclin (cPGI, Cayman Chemical) or GW501516 (Synthelab AB) at indicated doses for different time points. Treatment with COX inhibitors (SC-560, aspirin, or celecoxib) was started 2 h prior to AA treatment.

Hybridization probes. cDNA clones for COX-1, PPAR{delta}, and RXR{alpha} have previously been described (5, 13). For in situ hybridization, sense and antisense 35S-labeled cRNA probes were generated using Sp6 and T7 RNA polymerases, respectively. For Northern hybridization, antisense 32P-labeled cRNA probes were generated. rPL7 was used as a housekeeping gene. Probes had specific activities of 2 x 109 dpm/µg.

RNA isolation and Northern blot analysis. Total RNA was extracted from cultured cells or tissue specimens using Trizol reagent (Invitrogen). Total RNA (6 µg) was denatured, separated by formaldehyde/agarose gel electrophoresis, transferred to nylon membranes, and UV cross-linked. Blots were prehybridized, hybridized, and washed as we have previously described (13).

In situ hybridization. In situ hybridization was carried out as previously described (13). Sections were prehybridized and hybridized at 45°C for 4 h in 50% formamide hybridization buffer containing 35S-labeled antisense or sense cRNA probes. RNase A–resistant hybrids were detected by autoradiography. Sections were poststained with H&E. Sections hybridized with sense probes did not show any positive signals and served as negative controls.

Western blot analysis. Western blot analysis was done as previously described (4). Membranes were blocked with 10% milk in TBST and probed with antibodies against mouse PPAR{delta} (1:1,000; ref. 13), human PPAR{delta} (1:250; Santa Cruz Biotechnology), COX-1 (1:1,000; kindly provided by David Dewitt, Michigan State University; ref. 5), actin (1:100; Santa Cruz Biotechnology), ERK (1:1,000; Cell Signaling), or phospho-ERK (1:500, Cell Signaling) overnight at 4°C. After thorough washing, blots were incubated in peroxidase-conjugated donkey/anti-goat IgG or donkey/anti-rabbit IgG (Jackson ImmunoResearch Lab, Inc.), followed by washing. Protein signals were detected using chemiluminescent reagents (Amersham).

Transfection and luciferase assay. T2 cells (3 x 104 cells/well) were seeded in 24-well plates and inoculated in growth media for 16 h. Cells were transfected with 0.3 µg UAS-tk-luc/0.3 µg PPAR{delta}-GAL4/2.5 ng pRL-SV40 (Promega) or 0.3 µg PPAR-responsive element (PPRE)-tk-luc/2.5 ng pRL-SV40 using LipofectAMINE and Plus reagent according to the instructions of the manufacturer (Invitrogen). OVCAR3 cells (1.5 x 105 cells/well) were seeded in 12-well plates and inoculated in growth media for 16 h. Cells were transfected with 0.4 µg UAS-tk-luc/0.4 µg PPAR{delta}-GAL4/5 ng pRL-SV40 using LipofectAMINE and Plus reagent. After 3 h of incubation, serum-free fresh medium was added, and cells were incubated for an additional 3 h, harvested and lysed. Relative light units from firefly luciferase activity were determined by using a luminometer (MGM Instruments) and normalized to relative light units from renilla luciferase using a Dual Luciferase kit (Promega). All constructs were kindly provided by Barry Forman (Gonda Diabetes Center).

Prostaglandin assays. OVCAR3 (2 x 105 cells/well) or T2 (105 cells/well) cells were incubated in 24-well plates in growth media for 16 h. Cells were then incubated in serum-free media for 24 h. After changing to serum-free media containing AA and/or aspirin, cells were incubated for an additional 4 h. Media were collected and analyzed for prostaglandin levels by gas chromatography negative ion chemical ionization mass spectrometric assay (13). AA and aspirin were dissolved in ethanol and DMSO, respectively. Controls contained appropriate concentrations of vehicles.

Cell growth assay. MTT colorimetric assays per manufacturer's instructions (Promega) were monitored at an absorbance of 490 nm as an index of cell viability/proliferation.

Generation of polyclonal cell lines expressing dominant-negative human PPAR{delta}. T2 cells were transfected with pcDNA3.1(zeo) vector or pcDNA3.1(zeo)–dominant-negative human PPAR{delta} (DNhPPAR{delta}; ref. 21) using LipofectAMINE (Invitrogen). Transfected cells were selected with 600 µg/mL of zeocin (Invitrogen), and cells were pooled to generate polyclonal cell lines.

Allografting and xenografting OSE cells for tumor growth studies. For aspirin studies, a suspension of T2 (107 cells) or OVCAR3 (6 x 106 cells) cells was s.c. injected under the dorsal skin of 6- to 12-week-old female Rag2–/–/{gamma}C–/– mice. Tumor growth was recorded every 4 to 5 days by direct measurement of tumor dimensions. Aspirin at 500 ppm or 3,000 ppm mixed with pulverized food was kindly provided by Clinton Grubbs, University of Alabama, Birmingham, AL. Mice were fed with aspirin-containing or control diet from the day of tumor grafting until sacrifice. To assess the effect of PPAR{delta} on tumor growth, suspensions of polyclonal T2 cells (107) expressing DNhPPAR{delta} (pDN) or empty vector (pVEC) were placed under the dorsal skin of 6- to 12-week-old female Rag2–/–/{gamma}C–/– mice. Tumor growth was measured weekly. Tumor volume was calculated according to the equation V = 0.5 x (LW2), where V, volume; L, length; and W, width (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PPAR{delta} is highly expressed in EOC. To determine whether PPAR{delta} has a role in EOC, we first examined its expression using mouse models of EOC as we have previously described (2). The derivation of C1, C2, T1, and T2 mouse ovarian cancer cell lines is described in Materials and Methods. Northern hybridization (Fig. 1A, top ) and Western blotting (Fig. 1A, bottom) show that both PPAR{delta} mRNA and protein are expressed in all four cell lines. We next examined the expression of PPAR{delta} in tumors generated in vivo from these cell lines. Northern (Fig. 1B, top) and Western blot analyses (Fig. 1B, bottom) show that PPAR{delta} is expressed in tumors generated from each cell line. In contrast, whereas PPAR{gamma} mRNA was detected at low levels in only C1 and T1 cell lines and tumors, PPAR{alpha} mRNA was low to undetectable in all four cell lines and tumors (data not shown).


Figure 1
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  		Figure 1. PPAR{delta} is expressed in EOC. A, Northern blot analysis of PPAR{delta} in C1, C2, T1 and T2 OSE cell lines (top). rPL7 is a housekeeping gene. Western blot analysis of PPAR{delta} in OSE cell lines (bottom). Actin serves as a loading control. B, Northern blot analysis of PPAR{delta} in EOC tumors derived from C1, C2, T1, and T2 OSE cells (top). Western blot analysis of PPAR{delta} in these tumor samples (bottom). C, in situ hybridization of PPAR{delta} and RXR{alpha} in mouse tumor sections and normal ovary. D, in situ hybridization of COX-1 and PPAR{delta} in human ovarian tumors and nontumorous ovaries. Pink grains, sites of hybridization. Bar, 400 µm.

 
To determine cell-specific localization of PPAR{delta}, we did in situ hybridization in tumors generated from each cell line (Fig. 1C). Again, PPAR{delta} was expressed in all tumors, whereas its expression in normal ovaries was low to undetectable. Microscopic examination of the hybridized sections poststained with H&E under bright-field showed that hybridization signals were primarily restricted to epithelial-like cells. In contrast, expression of PPAR{alpha} and PPAR{gamma} was low to undetectable, although some focal expression was noted in some tumors (data not shown). Furthermore, RXR{alpha}, a heterodimeric partner of PPARs for transcriptional activity, is also expressed in these tumors (Fig. 1C). We also evaluated a limited number (n = 9) of human ovarian tumors or nontumorous ovaries by in situ hybridization. Although all EOC samples expressed PPAR{delta}, nontumorous ovarian samples showed little to no PPAR{delta} expression (Fig. 1D). More importantly, PPAR{delta} expression overlapped with that of COX-1 in human EOC (Fig. 1D, top). PPAR{delta} mRNA was also detected in OVCAR3 cells and tumors generated from this cell line (data not shown). Collectively, our results show that PPAR{delta} expression is associated with mouse and human EOC.

Aspirin attenuates COX-1-PGI2-PPAR{delta} signaling in EOC. cPGI, a stable analogue of PGI2, and GW501516, a selective synthetic agonist of PPAR{delta}, activate PPAR{delta} in a variety of cell lines (24, 30, 31). Therefore, we examined the ability of cPGI or GW501516 to activate PPAR{delta} in T2 and OVCAR3 cell lines using a PPAR{delta}-LBD-GAL4 ligand–binding reporter assay (24, 32). As shown in Fig. 2A , reporter activity is enhanced by both cPGI and GW501516 in a dose-dependent manner in both cell lines. These data suggest that GW501516 or cPGI can bind to PPAR{delta} to enhance its activity. To determine whether these agonists can enhance PPAR{delta} function, we used the PPRE-tk-luc system (21, 24, 32). This reporter contains three tandem repeats of the PPRE from the promoter of the acyl-CoA oxidase gene. GW501516 and cPGI each increases the reporter activity compared with vehicle-treated cells (Fig. 2B). As previously shown (13), the reporter activity was significantly enhanced when cells were treated with a combination of GW501516 or cPGI with 9-cis-retinoic acid, a natural ligand for RXR (Fig. 2B). These results suggest that GW501516 and cPGI are each capable of binding PPAR{delta} and enhancing its activity in EOC.


Figure 2
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  		Figure 2. Ligand activation of PPAR{delta} in EOC cells. A, PPAR{delta} binding activity assay. T2 or OVCAR3 cells were transiently transfected with UAS-tk-luciferase, PPAR{delta}-GAL4, and pRL-SV40. Cells were treated with DMSO (vehicle), cPGI or GW501516 in serum-free media for 20 h. Experiments were run in triplicate for T2 cells and in duplicate for OVCAR3 cells. Columns, mean fold activation; bars, SE (*, P < 0.05, unpaired t test). B, PPAR{delta} functional activity assay. T2 cells were transiently transfected with PPRE3-tk-luciferase and pRL-SV40. Cells were treated with DMSO (vehicle), GW501516, cPGI, or 9-cis retinoic acid (9-cis-RA) in serum-free media for 16 h. Experiments were run in five replicates. Columns, mean fold activation; bars, SE (*, P < 0.05, unpaired t test). C, aspirin reduces PGI2 formation in T2 and OVCAR3 cells. Cells were treated with DMSO plus ethanol (vehicle), 20 µmol/L of AA with or without aspirin for 4 h. Columns, mean from experiments run in duplicate; bars, SE. D, OVCAR3 cells were transiently transfected with UAS-tk-luciferase, PPAR{delta}-GAL4, and pRL-SV40. Cells were treated with vehicle, AA, or aspirin in serum-free media for 20 h. Experiments were run in triplicate. Columns, mean fold activation; bars, SE. Whereas, AA significantly increased activity, aspirin (5 µmol/L) inhibited this activation (P < 0.05, unpaired t test).

 
The ability of cPGI to enhance PPAR{delta} activity is especially interesting because PGI2 is the main prostaglandin produced in T2 cells and its production is attenuated by a COX-1 inhibitor, SC-560 (4). We also observed that OVCAR3 cells produce PGI2, and SC-560 reduces its production, whereas celecoxib (a COX-2–selective inhibitor) had little effect (Supplemental Fig. S1). Although SC-560 attenuates prostaglandin production in EOC cells and their growth in vitro and in vivo (4), this compound is not being developed for clinical purposes. On the other hand, aspirin, known to preferentially inhibit COX-1, is widely available at a low cost (6). This provoked us to examine whether aspirin would have an effect on EOC. This hypothesis is consistent with recent epidemiologic evidence indicating that regular consumption of aspirin is associated with reduced risk of several cancers, including EOC (7–9).

As shown in Fig. 2C, aspirin lowers PGI2 levels in both T2 and OVCAR3 cells in a dose-dependent manner. It also attenuates PPAR{delta}-LBD-GAL4 transactivation with AA in OVCAR3 cells (Fig. 2D). These data suggest that aspirin, similar to SC-560, inhibits prostaglandin production, and implicates PPAR{delta} as a downstream target of COX-1–derived prostaglandins.

Inhibition of COX-1-PPAR{delta} signaling attenuates tumor growth in vitro and in vivo. Because cPGI or GW501516 enhances PPAR{delta} activity and aspirin attenuates prostaglandin production and PPAR{delta} activity, we next asked whether aspirin affects EOC cell growth. As shown in Fig. 3A , aspirin decreases the growth of T2 and OVCAR3 cells in a dose-dependent manner as determined by MTT assays. To examine the role of PPAR{delta}, we used a DNhPPAR{delta} construct because PPAR{delta}-selective inhibitors are not currently available. T2 cells were stably transfected with DNhPPAR{delta} construct or empty vector and selected with antibiotics (zeocin) to establish a polyclonal cell line (pDN or pVEC). Western blotting results confirmed that the transfection was successful because DNhPPAR{delta} is expressed in pDN cells, but not in pVEC cells (Fig. 3B, inset). As expected, endogenous mouse PPAR{delta} and COX-1 proteins are expressed in pDN and pVEC cell lines (Fig. 3B, inset). We used these transfected cell lines to examine whether DNhPPAR{delta} attenuates EOC cell growth. As shown in Fig. 3B, pDN cells grow at a slower rate when compared with pVEC.


Figure 3
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  		Figure 3. Aspirin inhibits PPAR{delta}-induced EOC cell proliferation in vitro. A, aspirin reduces cell growth of mouse and human EOC cells. T2 cells (5 x 103 cells/well) or OVCAR3 cells (15 x 103 cells/well) were seeded in 96-well plates and grown in growth media for 16 h. Cells were treated with DMSO (vehicle) or various doses of aspirin in serum-free media for 72 h. Experiments were run in quadruplicate. Data are shown as mean ± SE, (*, P < 0.05, unpaired t test). B, silencing of PPAR{delta} function attenuates OSE cell proliferation in vitro. Western blot analysis of DNhPPAR{delta}, endogenous mouse PPAR{delta} and COX-1 expression in T2 cells (inset). Actin serves as a loading control. Growth of pVEC or pDN cells was assessed by the MTT assay. Columns, mean; bars, SE (*, P < 0.05, unpaired t test).

 
We next examined whether aspirin reduces EOC growth in vivo in our mouse models. T2 cells were s.c. allografted to immunocompromised Rag2–/–/{gamma}C–/– mice. These mice were allowed to consume diets with or without aspirin (3,000 ppm) from the day of the allograft up to 6 to 8 weeks. As shown in Fig. 4A , mice consuming the aspirin-containing diet had significantly slower rates of tumor growth. We did similar experiments using OVCAR3 cells that were s.c. xenografted to immunocompromised Rag2–/–/{gamma}C–/– mice and tumor growth was monitored. Again, tumor growth was significantly compromised in mice consuming aspirin (Fig. 4B). No apparent toxic side effects were noted in the treatment group. The inhibitory effects of aspirin on tumor growth were significant even when a lower dose (500 ppm) of aspirin was used (Fig. 4C). Because PPAR{delta}-specific antagonists are not available, we used pDN (dominant negative) cells to define the role of this transcription factor in EOC growth. Indeed, immunocompromised Rag2–/–/{gamma}C–/– mice allografted s.c. with pDN cells showed dramatically reduced rates of tumor growth when compared with mice bearing the same numbers of pVEC cells (Fig. 4D). Collectively, these results suggest that whereas the enhancement of COX-1-PPAR{delta} signaling pathway promotes cell and tumor growth in EOC, inhibition of this signaling by aspirin or DNPPAR{delta} reduces such growth.


Figure 4
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  		Figure 4. Mouse and human EOC growth in vivo is inhibited by aspirin or DNPPAR{delta}. Aspirin consumption attenuates mouse EOC growth arising from T2 cells (A) or growth of human EOC arising from OVCAR3 cells (B and C) in vivo. T2 (107) or OVCAR3 (6 x 106) cells were s.c. engrafted under the dorsal skin of female Rag2–/–/{gamma}C–/– mice. Tumor growth was measured every 4 to 5 d by direct measurement of tumor volume (n = 4–12 mice/group). Arrowheads, the day when aspirin consumption was initiated. Points, mean tumor volume; bars, SE (*, P < 0.05, unpaired t test). D, silencing of PPAR{delta} attenuates EOC growth in vivo. Rag2–/–/{gamma}C–/– female mice were allografted with pVEC or pDN cells (107) under the dorsal skin. Tumor growth was recorded every 7 d for a period of 6 or 8 wks (n = 9–10 mice/group). Points, mean tumor volume; bars, SE (*, P < 0.05, unpaired t test).

 
COX-1-PPAR{delta} signaling regulates cell growth through ERK1/2 activation. The ERK signaling pathway is constitutively activated in many types of cancers, most often promoting cell cycle progression (reviewed in ref. 33). Although it is not known whether the COX-PPAR{delta} signaling pathway can activate ERK signaling, there is evidence that PGE2 enhances cell proliferation via ERK1/2 phosphorylation in colorectal cancer cells (34), and GW501516 induces ERK phosphorylation in lung cancer cells (35). These findings raise the possibility that the COX-1-PPAR{delta} signaling pathway affects ERK phosphorylation. Indeed, we observed that an ERK-phosphorylation inhibitor (PD98059) decreases cell viability in both T2 and OVCAR3 cells in a dose-dependent manner (Supplemental Fig. S2). We also asked whether altering the COX-1-PPAR{delta} signaling pathway would affect ERK activation in EOC. As shown in Fig. 5A , AA induces ERK phosphorylation in T2 and OVCAR3 cells in a dose-dependent manner. This AA-induced ERK phosphorylation in these cells is attenuated after their exposure to SC-560 or aspirin, but not celecoxib (Fig. 5B). We also observed that cPGI or GW501516 induces ERK phosphorylation in these cells in a dose-dependent manner (Fig. 5C). More importantly, ERK activation with cPGI was not observed in pDN cells (Fig. 5D). Overall, our results suggest that prostaglandins produced by COX-1 activate PPAR{delta} and promote ERK1/2 phosphorylation which increases EOC growth. The observed ERK activation was quite rapid. The earliest response we observed was between 5 and 10 min, albeit with some experimental variation (data not shown). All results shown are for the 30 min time point.


Figure 5
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  		Figure 5. COX-1-PPAR{delta} signaling induces ERK1/2 activation in EOC cells. A, AA induces ERK1/2 phosphorylation in T2 and OVCAR3 cells. B, SC-560 or aspirin, but not celecoxib, attenuates AA-induced ERK1/2 phosphorylation. C, cPGI or GW501516 induces ERK1/2 phosphorylation. D, cPGI fails to induce ERK1/2 phosphorylation in T2 cells expressing a PPAR{delta} dominant negative construct (pDN), whereas cPGI activates ERK1/2 in cells expressing the control (pVEC).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present investigation highlights that PPAR{delta} is a downstream target of COX-1–derived prostaglandins, and this signaling activates ERK to stimulate EOC growth. Although COX-2–derived prostaglandins were shown to induce PPAR{delta} signaling (21, 24), our results showing that COX-1–derived prostaglandins target ERK phosphorylation via PPAR{delta} activation and influence tumor growth in mouse and human models of EOC are novel. We found that either reducing the levels of PGI2 serving as a PPAR{delta} ligand or reducing PPAR{delta} function intervenes with EOC growth. These observations provide strong evidence that growth-promoting effects are mediated by COX-1–derived prostaglandins affecting PPAR{delta} function. This is supported by our observation of enhanced PPAR{delta} activity in EOC cells by cPGI or GW501516.

ERK signaling is known to be activated in numerous cancers and is considered a prime target for cancer therapy (33, 36–38). Indeed, several inhibitors of this pathway have been tested in clinical trials (37). Ras signaling is heightened in a variety of cancers, and one of the downstream effectors in the Ras cascade is ERK activation (reviewed in refs. 33, 39, 40). It is known that many extracellular stimuli bind to their membrane receptors to recruit Ras to initiate its signal transduction cascade with eventual ERK phosphorylation (33, 34). However, studies addressing whether and how PPAR{delta} induces ERK phosphorylation are very limited. There is evidence that PGE2 can transactivate PPAR{delta} and activate Ras-ERK signaling cascades in colorectal cancer cells (34). A recent study also shows that GW501516 phosphorylates ERK in lung cancer cells (35). Our studies showing induction of ERK phosphorylation in EOC cells by cPGI or GW501516, and its attenuation by inhibition of COX-1 or PPAR{delta} function provide strong evidence that COX-1-PPAR{delta}-ERK signaling plays an important role in EOC. Further studies are required to determine how PPAR{delta} ligands stimulate ERK phosphorylation. Although COX-2–derived PGE2 is known to influence the growth of many solid tumors by activating PPAR{delta} and canonical Wnt signaling (10, 21), our studies showing the involvement of COX-1, PPAR{delta}, and ERK activation in mouse and human models of EOC suggest that signaling cascades for promoting tumor growth is complex and tissue context–dependent.

Although rapid ERK phosphorylation by cPGI or GW501516 is interesting, the mechanism by which this is achieved is not presently clear. Because of the rapidity of the response, it is assumed that this activation does not involve protein synthesis. There is evidence that a consensus mitogen-activated protein kinase site located within the A/B domain of hPPAR{gamma} regulates its transcriptional activity through phosphorylation (41). If such a kinase domain is present in PPAR{delta}, it may be possible that phosphorylation of PPAR{delta} following its activation in turn transphosphorylates other substrates within the cell. It has also been shown that PPAR{gamma} ligands can rapidly activate the MEK/ERK pathway via phosphoinositide-3-kinase or epidermal growth factor receptor, a phenomenon similar to that observed in rapid signaling by steroid hormones for their "nongenomic" effects (42–44). Whether this is true for PPAR{delta} remains to be determined. The mechanism by which PPAR{delta} activation rapidly turns on ERK phosphorylation is currently under investigation in our laboratory.

Although aspirin as a potential anticancer drug has been considered, its efficacy as a therapeutic agent remains controversial because of inconclusive recommendations regarding dose, duration of treatment, and/or age of patients (45). A recent case study found that the use of many nonsteroidal anti-inflammatory drugs, including aspirin, within 5 years of diagnosis or interview, was associated with a reduced risk of ovarian cancer (7). This study, however, was limited in its ability to detect beyond the 5-year time frame. Aspirin was also effective in reducing colorectal cancer risk by 35% if taken for >10 years (46–48). These preliminary clinical studies and our current findings showing that aspirin inhibits the COX-1-PPAR{delta}-ERK signaling cascade reinforces the importance of further investigating the role of aspirin as a potential treatment for EOC.


   Acknowledgments
 
Grant support: USPHS grants, P01-CA-77839 (R.N. DuBois), R37 HD12304 (S.K. Dey), Mary Kay Ash Charitable Foundation (S.K. Dey), and Gynecologic Cancer Foundation Awards/Ann Schreiber Ovarian Cancer Research Grant (T. Daikoku), and VICC-Meharry U54CA091405-06 grant. S. Tranguch is supported by an NIH NRSA individual fellowship from NIDA (F31 DA021062).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Fuhua Xu for his help with statistical analysis and Jessica Potts for her assistance in tumor measurement.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 3/ 1/07. Revised 4/ 9/07. Accepted 4/24/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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