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Cyclooxygenase-2-derived Prostaglandin E2 Stimulates Id-1 Transcription*

Cyclooxygenase-2 (COX-2) and Id-1 are overexpressed in a variety of human malignancies. Recently, each of these genes was found to play a role in mediating breast cancer metastasis to the lungs, but their potential interdependence was not evaluated. Hence, the main objective of the current study was to determine whether COX-2-derived prostaglandin (PGE2) activated Id-1 transcription, leading in turn to increased invasiveness of mammary epithelial cells. In MDA-MB-231 cells, treatment with PGE2 induced Id-1, an effect that was mimicked by an EP4 agonist. PGE2 via EP4 activated the epidermal growth factor receptor (EGFR) → ERK1/2 pathway, which led to increased expression of Egr-1. PGE2 stimulated EGFR signaling by inducing the release of amphiregulin, an EGFR ligand. The ability of PGE2 to activate Id-1 transcription was mediated by enhanced binding of Egr-1 to the Id-1 promoter. Silencing of COX-2 or pharmacological inhibition of COX-2 led to reduced PGE2 production, decreased Id-1 expression, and reduced migration of cells through extracellular matrix. A similar decrease in cell migration was found when Id-1 was silenced. The interrelationship between COX-2, PGE2, Id-1, and cell invasiveness was also compared in nontumorigenic SCp2 and tumorigenic SCg6 mammary epithelial cells. Consistent with the findings in MDA-MB-231 cells, COX-2-derived PGE2 induced Id-1, leading in turn to increased cell invasiveness. Taken together, these results suggest that PGE2 via EP4 activated the EGFR → ERK1/2 → Egr-1 pathway, leading to increased Id-1 transcription and cell invasion. These findings provide new insights into the relationship between COX-2 and Id-1 and their potential role in metastasis.

This article has been withdrawn by the authors. In Fig. 1A, the first two lanes of the 18S rRNA panel were reused in the last two lanes. In Fig. 1D, the last two lanes of the Id-1 panel were reused as 18S rRNA. Also, the first two lanes of the Id-1 panel in Fig. 1D were reused in cancer cells. Notably, PGE 2 via EP 4 activated a signal transduction pathway comprised of EGFR 3 ERK1/2 3 Egr-1 resulting in enhanced Id-1 gene expression. The increase in Id-1 expression mediated by PGE 2 led in turn to increased invasiveness of breast cancer cells.
Cell Lines-MDA-MB-231 human breast cancer cells (American Type Culture Collection; Manassas, VA) were maintained in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% sodium pyruvate. SCp2 and SCg6 murine mammary epithelial cell lines, obtained from Dr. Mina Bissell (Lawrence Berkeley National Laboratory, Berkeley, CA), were grown in Dulbecco's modified Eagle's medium F-12 medium supplemented with 10% Leibovitz medium. All cells were grown to 60% confluence in a 5% CO 2 , water-saturated incubator at 37°C before being placed in serum-free medium for 24 h. Subsequently, treatments were carried out in serum-free medium. PGE 2 Production-Cells were plated in 6-well dishes and grown to 60% confluence. The amount of PGE 2 released by cells was measured by enzyme immunoassay. Production of PGE 2 was normalized to protein concentrations.
Western Blotting-Cell lysates were prepared by treating cells with lysis buffer (150 mM NaCl, 100 mM Tris (pH 8.0), 1% Tween 20, 50 mM diethyldithiocarbamate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml trypsin inhibitor, and 10 g/ml leupeptin). Lysates were prepared by sonicating cells for 20 s on ice and centrifuged at 10,000 ϫ g for 10 min to sediment the particulate material. The protein concentration of the supernatant was measured by the method of Lowry et al. (44). SDS-PAGE was performed under reducing conditions on 10% polyacrylamide gels as described by Laemmli (45). The resolved proteins were transferred onto nitrocellulose sheets as detailed by Towbin et al. (46). The nitrocellulose membrane was then incubated with primary antibodies. Secondary antibody to IgG conjugated to horseradish peroxidase was used. The blots were probed with the ECL Western blot detection system according to the manufacturer's instructions.
Northern Blotting-Total cellular RNA was isolated from cell monolayers using an RNA isolation kit from Qiagen Inc. 10 g of total cellular RNA per lane were electrophoresed in a formaldehyde-containing 1.2% agarose gel and transferred to nylon-supported membranes. After baking, membranes were prehybridized overnight in a solution containing 50% formamide, 5ϫ sodium chloride/sodium phosphate/EDTA buffer (SSPE), 5ϫ Denhardt's solution, 0.1% SDS, and 100 g/ml single-stranded salmon sperm DNA and then hybridized for 12 h at 42°C with radiolabeled cDNA probes. Probes were labeled with [ 32 P]CTP by random priming. After hybridization, membranes were washed twice for 20 min at room temperature in 2ϫ SSPE, 0.1% SDS, twice for 20 min in the same solution at 55°C, and twice for 20 min in 0.1ϫ SSPE, 0.1% SDS at 55°C. Washed membranes were then subjected to autoradiography.
Transient Transfections-Cells were seeded at a density of 5 ϫ 10 4 cells/well in 6-well dishes and grown to 50 -60% confluence. For each well, 2 g of plasmid DNA were introduced into cells using 8 g of Lipofectamine as per the manufacturer's instructions. After 7 h of incubation, the medium was replaced with basal medium. The activities of luciferase and ␤-galactosidase were measured in cellular extract. Transfection of siRNAs was carried out using a similar methodology.
Electrophoretic Mobility Shift Assay-Cells were harvested, and nuclear extracts were prepared. For binding studies, oligonucleotides containing different response elements in the Id-1 promoter were used. The complementary oligonucleotides were annealed in 20 mM Tris (pH 7.6), 50 mM NaCl, 10 mM MgCl 2 , and 1 mM dithiothreitol. The annealed oligonucleotide was phosphorylated at the 5Ј-end with [␥-32 P]ATP and T4 polynucleotide kinase. The binding reaction was performed by incubating 5 g of nuclear protein in 20 mM HEPES (pH 7.9), 10% glycerol, 300 g of bovine serum albumin, and 1 g of poly (dI⅐dC) in a final volume of 10 l for 10 min at 25°C. The labeled oligonucleotide was added to the reaction mixture and allowed to incubate for an additional 20 min at 25°C. The samples were electrophoresed on a 4% nondenaturing polyacrylamide gel. The gel was then dried and subjected to autoradiography at Ϫ80°C. Top, cellular protein (100 g/lane) was subjected to immunoblot analysis. The blot was probed with antibodies to Id-1 and ␤-actin, respectively. Bottom, total cellular RNA was isolated from cells. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized Id-1 mRNA and 18 S rRNA. B, cells were transfected with 1.8 g of Id-1 promoter-luciferase construct (1.5 BV) and 0.2 g of pSV␤gal. 24 h after transfection the cells were treated with vehicle or the indicated concentrations of PGE 2 for 24 h. Cells were then lysed, and reporter activities were measured. Luciferase activity represents data that have been normalized to ␤-galactosidase activity. Columns, means; bars, S.D.; n ϭ 6. p Ͻ 0.01 (*) and p Ͻ 0.001 (**) compared with vehicle-treated cells. C, MDA-MB-231 cells were treated with vehicle, 1 M celecoxib, or 1 M indomethacin for 24 h. After treatment, the medium was collected, and the amount of PGE 2 was quantified. Production of PGE 2 was determined by enzyme immunoassay. Columns, means; bars, S.D.; n ϭ 6. **, p Ͻ 0.001 compared with vehicle-treated cells. Inset, immunoblot demonstrating that COX-2 is expressed in this cell line. D and E, MDA-MB-231 cells were treated with vehicle or the indicated concentrations of celecoxib (D) or indomethacin (E) for 24 h. Top panels, cellular protein (100 g/lane) was subjected to immunoblot analysis. The blots were probed with antibodies to Id-1 and ␤-actin, respectively. Bottom panels, total cellular RNA was isolated from cells. 10 g of RNA was added to each lane. The blots were hybridized with probes that recognized Id-1 mRNA and 18 S rRNA. F, MDA-MB-231 cells were transfected with 1.8 g of Id-1 promoter-luciferase and 0.2 g of pSV␤gal. 24 h after transfection, cells were treated with indicated concentrations of celecoxib, indomethacin (Indo), celecoxib (Cel) plus 0.5 M PGE 2 , or indomethacin plus 0.5 M PGE 2 . 24 h after treatment the cells were lysed, and reporter activities were measured. Luciferase activity represents data that have been normalized to ␤-galactosidase activity. Columns, means; bars, S.D.; n ϭ 6. **, p Ͻ 0.001 compared with celecoxib (1 M) or indomethacin-treated cells. G, MDA-MB-231 cells were transfected with 0.9 g of Id-1 promoter-luciferase construct and 0.2 g of pSV␤gal. The column labeled Control represents cells that received 0.9 g of empty vector; the column labeled GFP siRNA represents cells that also received 0.9 g of siRNA to GFP; the column labeled COX-2 siRNA represents cells that received 0.9 g of siRNA to COX-2; the column labeled COX-2 siRNA plus PGE 2 represents cells that received 0.9 g of siRNA to COX-2 plus 0.5 M PGE 2 . 24 h after transfection the cells were treated with vehicle or 0.5 M PGE 2 as indicated. 24 h later the cells were lysed, and reporter activities were measured. Luciferase activity represents data that have been normalized to ␤-galactosidase activity. Columns, means; bars, S.D.; n ϭ 6. **, p Ͻ 0.001 compared with cells transfected with COX-2 siRNA. Inset, Western blot analysis shows suppression of COX-2 in cells treated with siRNA to COX-2.
Chromatin Immunoprecipitation (ChIP) Assay-ChiP assay was performed with a kit (Upstate Biotechnology) according to the manufacturer's instructions. 1 ϫ 10 6 cells were cross-linked in a 1% formaldehyde solution for 10 min at 37°C. Cells were then lysed in 200 l of SDS buffer and sonicated to generate 200 -1000-bp DNA fragments. After centrifugation, the cleared supernatant was diluted 10-fold with ChIP buffer and incubated with 1.5 g of the indicated antibody at 4°C. Immune complexes were precipitated, washed, and eluted as recommended. DNA-protein cross-links were reversed by heating at 65°C for 4 h, and the DNA fragments were purified and dissolved in 50 l of water. 10 l of each sample were used as a template for PCR amplification. Id-1 oligonucleotide sequences for PCR primers were 5Ј-AGCGGAGAATGCTCC-AGCCCAGTTTT-3Ј (forward) and 5Ј-AGGCCTCCGAGCA-AGCTCTCCCT-3Ј (reverse). This primer set encompasses the Id-1 promoter segment from nucleotide Ϫ932 to Ϫ1156 bp, which includes the Egr-1 and cAMP-response element (CRE) binding sites. PCR was performed at 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s for 30 cycles. The PCR products generated from the ChIP template were sequenced, and the identity of the Id-1 promoter was confirmed.
were harvested and resuspended to 5 ϫ 10 5 /ml in basal medium and added to the matrix-coated insert, and the insert was placed in a 24-well plate containing conditioned medium. After incubation at 37°C and 5% CO 2 for 20 h, the membrane was fixed and stained per the manufacturer's instructions. The number of cells that migrated through the matrix-coated insert was counted.
Statistics-Comparisons between groups were made by Student's t test. A difference between groups of p Ͻ 0.05 was considered significant. 2 Induces the Expression of Id-1-Initially, we investigated the effects of exogenous PGE 2 on Id-1 levels in a human breast cancer cell line, MDA-MB-231. Treatment with PGE 2 led to a concentration-dependent increase in levels of Id-1 protein, mRNA (Fig.  1A), and promoter activity (Fig. 1B). Next, a pharmacological approach was used to determine whether endogenous PGE 2 regulated the expression of Id-1. Treatment with celecoxib, a selective COX-2 inhibitor, or indomethacin, a dual inhibitor of COX-1 and COX-2, suppressed the production of PGE 2 (Fig. 1C), inhibited the expression of Id-1 protein (Figs. 1, D and E), Id-1 mRNA (Figs. 1, D and E), and downregulated Id-1 promoter activity (Fig. 1F). The suppressive effects of celecoxib and indomethacin on Id-1 promoter activity were reversed by the addition of exogenous PGE 2 (Fig. 1F). Consistent with these pharmacological findings, treatment with siRNA to COX-2 suppressed Id-1 promoter activity, an effect that was reversed by the addition of PGE 2 (Fig. 1G).

COX-2-derived PGE
The Egr-1 Site Is Necessary for the Induction of Id-1 by PGE 2 -We next were interested in identifying the region of the Id-1 promoter that was important for mediating the inductive effects of PGE 2 . Transient transfections were performed with a series of human Id-1 promoter-deletion constructs ( Fig. 2A). Treat- Cold-chase experiments were performed by incubating nuclear protein (5 g) from cells treated with 0.5 M PGE 2 with a 32 P-labeled oligonucleotide containing the Egr-1 site of the Id-1 promoter and a 100-, 75-, or 50-fold excess of unlabeled Egr-1 oligonucleotide, 100-fold excess of CREB oligonucleotide, or 100-fold excess of E box oligonucleotide. The protein-DNA complexes that formed were separated on a 4% polyacrylamide gel. B, ChIP assays were performed. MDA-MB-231 cells were treated with vehicle or 0.5 M PGE 2 for 3 h. Chromatin fragments were immunoprecipitated with Egr-1 antibody, and the Id-1 promoter region was amplified by PCR. DNA sequencing was carried out, and the PCR product was confirmed to be the Id-1 promoter. The Id-1 promoter (Ϫ931 to 1156 bp) was not detected when normal IgG was used or antibody was omitted from the immunoprecipitation step. C, MDA-MB-231 cells were transfected with 0.9 g of Id-1 promoter construct and 0.2 g of pSV␤gal. The column labeled GFP siRNA represents cells that also received 0.9 g of siRNA to GFP, and the column labeled Egr-1 siRNA represents cells that received 0.9 g of siRNA to Egr-1. The total amount of transfected DNA for each condition was kept constant at 2 g by using corresponding empty expression vector. 24 h after transfection the cells were treated with vehicle (Control) or 0.5 M PGE 2 . 24 h post-treatment cellular lysates were isolated and subjected to either Western blotting (top) or measurements of reporter activities (bottom). In the top panel the blot was probed with antibodies to Egr-1 and ␤-actin, respectively. In the bottom panel luciferase activity represents data that have been normalized to ␤-galactosidase activity. Columns, means; bars, S.D.; n ϭ 6. **, p Ͻ 0.001 compared with cells transfected with GFP siRNA. DECEMBER 5, 2008 • VOLUME 283 • NUMBER 49 ment of MDA-MB-231 cells with PGE 2 led to a severalfold increase in Id-1 promoter activity when the Ϫ1575 bp deletion construct (1.5 BV) was used (Fig. 2B). The magnitude of PGE 2mediated induction of Id-1 promoter activity remained essentially constant until the Ϫ927 bp deletion construct (5Јdel3) was used. The Ϫ927 bp Id-1 promoter construct was not stim-ulated by PGE 2 . This result implies that one or more promoter elements located between Ϫ927 bp and Ϫ1147 bp is necessary for PGE 2 -mediated induction of Id-1 promoter activity. CREB, E-box, and Egr-1 sites are found within this region of the Id-1 promoter (Fig. 2C). To determine which promoter element(s) was important for mediating the induc-

COX-2-derived Prostaglandin E 2 Stimulates Id-1 Transcription
tive effects of PGE 2 , transient transfections were performed utilizing Id-1 promoter constructs in which these enhancer elements were mutagenized. As shown in Fig. 2D, the induction of Id-1 promoter activity by PGE 2 was abrogated by mutagenizing the Egr-1 site. By contrast, mutagenizing the CREB and E-box sites had no effect on PGE 2 -mediated stimulation of Id-1 promoter activity.
Electrophoretic mobility shift assays were performed to identify the transcription factor that contributed to the induction of Id-1 by PGE 2 (Fig. 3A). Increased binding of nuclear proteins to the Egr-1 site of the Id-1 promoter was detected after treatment with PGE 2 . Binding was prevented when nuclear extract was incubated with an excess of Egr-1 cold probe. By contrast, binding was unaffected when nuclear extract was incubated with an excess of CREB or E-box containing cold probes. ChIP assays were also done. Protein-DNA complexes were immunoprecipitated with an antibody to Egr-1, and bound DNA fragments were recovered and subjected to semiquantitative PCR with oligonucleotides specific for the Id-1 promoter. The binding of Egr-1 to the Id-1 promoter was enhanced by incubation with PGE 2 (Fig. 3B). Transient transfections were carried out to further evaluate the functional importance of Egr-1 in mediating the activation of the Id-1 promoter in response to treatment with PGE 2 . siRNA to Egr-1 suppressed PGE 2 -mediated induction of Egr-1 protein and blocked PGE 2 -mediated induction of Id-1 promoter activity (Fig. 3C).
Defining the Signaling Mechanism by which PGE 2 Induces Id-1-We next attempted to define the signal transduction pathway by which PGE 2 stimulated Id-1 transcription. PGE 2 exerts its effects by binding to G protein-coupled receptors known as EP 1 , EP 2 , EP 3 , and EP 4 . Small inhibitory RNA was used successfully to knockdown each of the four EP receptors (Fig. 4A). Importantly, the inductive effects of PGE 2 on the Id-1 promoter were abrogated by siRNA to EP 4 (Fig. 4B). By contrast, siRNAs to EP 1 , EP 2 , and EP 3 did not suppress the stimulatory effects of PGE 2 (Fig. 4B). Additional experiments were carried out to evaluate the role of EP 4 in PGE 2 -mediated induction of Id-1. Notably, treatment with PGE1 alcohol, an EP 4 agonist, reversed the inhibition of Id-1 promoter activity that resulted from silencing of COX-2 or treatment with indomethacin (Fig. 4C). Treatment with PGE1 alcohol induced Id-1 mRNA (Fig. 4D) and protein (Fig. 4E), thereby mimicking the effects of PGE 2 . Moreover, GW627368X, an EP 4 antagonist, suppressed PGE 2 -mediated induction of Id-1 mRNA (Fig. 4F) and protein (Fig. 4G). Cross-talk between EP receptors and EGFR signaling occurs (47). Previously, stimulation of ERK1/2 MAPK, a downstream component of the EGFR signaling cascade, was found to induce Egr-1 (48). Hence, it was logical to determine whether PGE 2 -mediated induction of Id-1 was a consequence of activation of the EGFR 3 ERK1/2 MAPK 3 Egr-1 pathway. As shown in Fig. 5A, treatment with PGE 2 led to increased phosphorylation of EGFR. Experiments were next done to determine whether EGFR activation was causally linked to the induction of Id-1. AG1478, a small molecule inhibitor EGFR tyrosine kinase, was used. Treatment with AG1478 blocked PGE 2 -mediated induction of both Id-1 and Egr-1 (Figs.  5, B and C). Because EGFR can be activated by either extracellular or intracellular mechanisms, we next investigated whether an antibody to the EGFR ligand binding site suppressed PGE 2mediated induction of Id-1 and Egr-1. As shown in Fig. 5D, the induction of Id-1 and Egr-1 by PGE 2 was suppressed by this neutralizing antibody. In contrast, control IgG did not suppress the induction of Id-1 or Egr-1. Importantly, EGFR can be activated by several different ligands including amphiregulin. Treatment with 0.5 M PGE 2 led to a 2.5-fold increase in the production of amphiregulin (p Ͻ 0.001). To investigate whether this increase in amphiregulin production was causally linked to the induction of Egr-1 and Id-1, a neutralizing antibody to amphiregulin was used. As shown in Fig. 5E, an antibody to amphiregulin abrogated the induction of Egr-1 and Id-1 by PGE 2 . Pharmacological and genetic approaches were next employed to evaluate the link between ERK1/2 MAPK activity and the induction of Id-1 by PGE 2 . In the first experiment, we found that treatment with PGE 2 stimulated ERK1/2 MAPK activity (Fig. 6A). Next we utilized PD98059, a specific inhibitor of MAPK kinase, which prevents activation of ERK1/2. Treatment with PD98059 suppressed PGE 2 -mediated induction of Id-1 mRNA (Fig. 6B) and protein (Fig. 6C). To further investigate the importance of MAPK in mediating the induction of Id-1, transient transfections were performed. Consistent with the pharmacological findings, overexpressing a dominant negative for ERK1 suppressed PGE 2 -mediated activation of the Id-1 promoter (Fig. 6D). PD98059 also suppressed PGE 2 -mediated induction of Egr-1 (Fig. 6E). To evaluate whether PGE 2mediated activation of EGFR led to increased ERK1/2 activity,

FIGURE 4. EP 4 is important for PGE 2 -mediated induction of Id-1.
A, MDA-MB-231 cells were transfected with 2 g of siRNAs to GFP or EP 1-4 and allowed to grow for 36 h before analysis. Total RNA was prepared from cells and subjected to northern blotting (10 g/lane). The blots were hybridized sequentially with the indicated probes. B, cells were transfected with 0.9 g of Id-1 promoter-luciferase and 0.2 g of pSV␤gal. The column labeled GFP siRNA represents cells that also received 0.9 g of siRNA to GFP, and the columns labeled EP 1-4 siRNA represent cells that also received 0.9 g of siRNA to EP 1-4 . The total amount of transfected DNA for each condition was kept constant at 2 g by using corresponding empty expression vector. 36 h after transfection the cells were treated with vehicle (Control) or 0.5 M PGE 2 . Treatments were for 24 h. Subsequently, luciferase activity was measured in cell lysates, and the activities represent data that have been normalized to ␤-galactosidase activity. Columns, mean (n ϭ 6); bars, S.D. **, p Ͻ 0.001 compared with cells transfected with GFP siRNA. C, cells were transfected with 0.9 g of Id-1 promoter-luciferase and 0.2 g of pSV␤gal. The column labeled GFP siRNA represents cells that also received 0.9 g of siRNA to GFP; the columns labeled COX-2 siRNA represents cells that received 0.9 g of siRNA to COX-2. In all other columns the cells also received 0.9 g of empty vector. 36 h after transfection, cells received fresh medium containing vehicle, 1 M indomethacin (Indo), 0.2 M PGE1 alcohol, or combinations of these treatments as indicated. Treatments were for 24 h. Subsequently, luciferase activity was measured in cell lysates, and the activities represent data that have been normalized to ␤-galactosidase activity. Columns, mean (n ϭ 6); bars, S.D. **, p Ͻ 0.001 versus COX-2 siRNA or Indo-treated cells, respectively. D and E, MDA-MB-231 cells were treated with vehicle or the indicated concentration of PGE1 alcohol for 24 h. In D, total cellular RNA was isolated from cells. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized Id-1 mRNA and 18S rRNA. In E, cellular protein (100 g/lane) was subjected to immunoblot analysis. The blot was probed with antibodies to Id-1 and ␤-actin, respectively. F and G, MDA-MB-231 cells were treated with vehicle, 0.5 M PGE 2 , or 0.5 M PGE 2 plus the indicated concentrations of an EP 4 receptor antagonist (GW 627368X) for 24 h. In F, total cellular RNA was isolated from cells. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized Id-1 mRNA and 18 S rRNA. In G, cellular protein (100 g/lane) was subjected to immunoblot analysis. The blot was probed with antibodies to Id-1 and ␤-actin.
AG1478 was utilized (Fig. 6F). The induction of ERK1/2 activity by PGE 2 was attenuated in cells treated with the inhibitor of EGFR-tyrosine kinase. Treatment with AG1478 also inhibited PGE1 alcohol-mediated induction of ERK1/2 activity.
Evidence That COX-2-derived PGE 2 Stimulates Cell Invasiveness by an Id-1-dependent Mechanism-SCp2 cells are a phenotypically normal murine mammary epithelial cell line that becomes invasive when Id-1 is overexpressed (49,50). SCg6 cells share a common lineage with SCp2 cells but are transformed and exhibit invasive behavior (51). Cell transformation stimulates COX-2 transcription and PGE 2 production (8). Hence, we next evaluated the role of the COX-2 and PGE 2 as determinants of Id-1 expression and cell invasiveness in SCg6 and SCp2 cells. Interestingly, tumorigenic SCg6 cells expressed higher levels of COX-2 (Fig. 7A) and produced more PGE 2 (Fig. 7B) than nontumorigenic SCp2 cells. A corresponding increase in Id-1 levels was found in SCg6 cells (Fig. 7A). To determine whether COX-derived PGE 2 contributed to the higher levels of Id-1 in SCg6 cells, additional experiments were performed. Similar to what was found in MDA-MB-231 cells (Fig. 1), treatment of SCg6 cells with celecoxib or indomethacin led to more than an 80% decrease in PGE 2 production (data not shown) and suppressed expression of Id-1 (Fig. 7C). Moreover, treatment of nontumorigenic SCp2 cells with exogenous PGE 2 led to a concentration-dependent increase in amounts of Id-1 mRNA and protein (Fig. 7D). Consistent with the findings in MDA-MB-231 cells, treatment with PGE1 alcohol caused dosedependent induction of Id-1 mRNA (Fig. 7E). Moreover, GW627368X, an EP 4 antagonist, suppressed PGE 2 -mediated induction of Id-1 (data not shown).
The results described above indicate that COX-derived PGE 2 induces Id-1 in both human (MDA-MB-231) and murine (SCg6 and SCp2) cells. Hence, we next investigated whether PGE 2mediated induction of Id-1 was important for cell invasiveness. In transformed SCg6 cells, silencing of COX-2 with siRNA or treatment with celecoxib led to more than a 50% reduction in PGE 2 production (p Ͻ 0.01) and a corresponding decrease in the migration of cells through ECM (Fig. 8A). Notably, silencing of Id-1 led to a similar reduction in the ability of these cells to traverse the ECM-coated inserts (Fig. 8A). By contrast, treatment with 0.5 M PGE 2 enhanced the migration of cells through ECM (Fig. 8A). In nontumorigenic SCp2 cells (Fig. 8B), COX-2 overexpression led to more than a 2-fold increase in PGE 2 production (p Ͻ 0.01) and a severalfold increase in migration of cells through ECM. Treatment with PGE 2 also enhanced the migration of cells through ECM. Importantly, silencing of Id-1 attenuated the increased in cell invasion mediated by overexpressing COX-2 or treatment with exogenous PGE 2 . Because COX-2-derived PGE 2 induced Id-1 transcription in MDA-MB-231 cells, we also evaluated cell invasion. Silencing of COX-2 or treatment with celecoxib caused more than a 50% decrease in PGE 2 production (p Ͻ 0.01) and a marked reduction in the ability of cells to migrate through ECM (Fig. 8C). Silencing of Id-1 mimicked this inhibitory effect, whereas treatment with PGE 2 enhanced cell invasion. Taken together, these results suggest that COX-2-derived PGE 2 induces Id-1, which contributes in turn to enhanced cell invasiveness.

FIGURE 5. PGE 2 -mediated induction of Id-1 is dependent on EGFR activation.
A, MDA-MB-231 cells were treated with indicated concentrations of PGE 2 for 1 h. Cell lysate protein (100 g) was subjected to Western blotting. The blots were probed with antibodies to phospho-EGFR (pEGFR) or EGFR. B, cells were treated with vehicle, 0.5 M PGE 2 , or PGE 2 plus the indicated concentration of AG1478 for 24 h. Total cellular RNA was isolated from cells. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized Id-1 mRNA and 18 S rRNA. C, cells were treated with vehicle, 0.5 M PGE 2 , or PGE 2 plus the indicated concentration of AG1478 for 24 h. Cell lysate protein (100 g) was subjected to Western blotting. The blot was probed with antibodies to Egr-1 and ␤-actin. D, cells were treated with vehicle, 0.5 M PGE 2 , 0.5 M PGE 2 plus IgG, or 0.5 M PGE 2 plus neutralizing antibody (Ab) to EGFR for 24 h. E, cells were treated with vehicle, 0.5 M PGE 2 , 0.5 M PGE 2 plus neutralizing antibody to amphiregulin, or 0.5 M PGE 2 plus IgG for 24 h. In D and E, total cellular RNA was isolated from cells. 10 g of RNA was added to each lane. The blots were hybridized with probes that recognized Egr-1, Id-1, and 18 S rRNA. Data shown are representative of three independent experiments.
Because the EP 4 receptor is responsible for the induction of Id-1 by PGE 2 , we next evaluated the role of this receptor in mediating cell invasiveness. Small inhibitory RNA was used to knockdown each of the four EP receptors in SCp2 cells (Fig.  9A). Next we investigated the effect of silencing these receptors on PGE 2 -mediated induction of cell invasion. Treatment with PGE 2 enhanced the migration of SCp2 cells through ECM (Fig.  9B), an effect that was suppressed by silencing EP 4 or treatment with GW627368X, an EP 4 antagonist. In contrast, silencing EP 1-3 did not affect the increase in cell invasiveness mediated by PGE 2 (Fig. 9B). Consistent with these findings, treatment with PGE1 alcohol, an EP 4 agonist, also stimulated the migration of SCp2 cells through ECM. Similar experiments were carried out in MDA-MB-231 cells (Fig. 9C). Once again, silencing of EP 4 or treatment with GW627368X suppressed the increase in cell invasion mediated by PGE 2 . Additionally, PGE1 alcohol stimulated the migration of cells through ECM (Fig. 9C). To complement the above studies, we investigated whether PGE1 alcohol, the EP 4 receptor agonist, could reverse the suppression of cell invasion mediated by genetic or pharmacological inhibi- Cell lysate protein (100 g) was subjected to Western blotting. The blot was probed with antibodies to phospho-ERK1/2 (pERK1/2) and ERK1/2. B and C, cells were treated with vehicle, 0.5 M PGE 2 , or 0.5 M PGE 2 plus the indicated concentrations of PD98059, a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor for 24 h. In B, total cellular RNA was isolated from cells. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized Id-1 and 18S rRNA. In C, cell lysate protein (100 g) was subjected to Western blotting. The blot was probed with antibodies to Id-1 and ␤-actin. D, MDA-MB-231 cells were transfected with 0.9 g of Id-1 promoter construct and 0.2 g of pSV␤gal. The column labeled ERK1 DN represents cells that were transfected with 0.9 g of a dominant negative form of ERK1. The total amount of transfected DNA was kept constant at 2 g by using a corresponding empty expression vector. Treatment with vehicle (control) or 0.5 M PGE 2 was for 24 h. Luciferase activity represents data that have been normalized to ␤-galactosidase activity. Columns, means; bars, S.D.; n ϭ 6. **, p Ͻ 0.001 versus PGE 2 treated cells. E, cells were treated with vehicle, 0.5 M PGE 2 or 0.5 M PGE 2 plus the indicated concentrations of PD98059 for 24 h. Cell lysate protein (100 g) was subjected to Western blotting. The blot was probed with antibodies to Egr-1 and ␤-actin, respectively. F, cells were treated with vehicle, 0.5 M PGE 2 , 0.2 M PGE1 alcohol, or these compounds plus 0.5 M AG1478 for 1 h. Cell lysate protein (100 g) was subjected to Western blotting. The blot was probed with antibodies to pERK1/2 and ERK1/2. tion of COX-2 in both SCg6 (Fig. 9D) and MDA-MB-231 cells (Fig. 9E). In both cell lines the EP 4 receptor agonist reversed the suppressive effects related to COX-2 inhibition. Collectively, these results suggest that COX-2-derived PGE 2 binds to EP 4 , resulting in enhanced expression of Id-1, which contributes in turn to increased cell invasiveness.

DISCUSSION
In the current study we show that COX-2-derived PGE 2 induced Id-1 gene expression and cell invasion. PGE 2 via EP 4 activated the EGFR 3 ERK1/2 3 Egr-1 pathway leading to enhanced Id-1 transcription. Several observations support a critical role for EP 4 in PGE 2 -mediated induction of Id-1. First, an agonist of EP 4 mimicked PGE 2 by inducing Id-1. Moreover, an EP 4 receptor antagonist or silencing of EP 4 suppressed the inductive effects of PGE 2 . The EGFR also played a central role in mediating the inductive effects of PGE 2 . Treatment with PGE 2 stimulated the phosphorylation of EGFR. Additionally, we found that AG1478, an EGFR-tyrosine kinase inhibitor, or antibody blockade of the ligand binding site of EGFR abrogated PGE 2 -mediated induction of Id-1. EGFR can be activated via either intracellular or extracellular mechanisms (52)(53)(54). The fact that a neutralizing antibody to EGFR blocked the induction of Id-1 by PGE 2 suggests an extracellular process. Consistent with this notion, treatment with PGE 2 stimulated the release of amphiregulin, a ligand of the EGFR. Most importantly, an antibody to amphiregulin blocked the induction of Id-1 by PGE 2 . Notably, these findings are consistent with prior evidence that PGE 2 can induce amphiregulin and thereby activate EGFR signaling (53,55). Our results are also consistent with growing evidence that cross-talk between EP receptors and EGFR is likely to be important for regulating a number of cellular functions that are relevant in carcinogenesis (47,(53)(54)(55). Additional experiments were carried out to define the signal transduction pathway downstream of EGFR that mediated the induction of Id-1. The data suggest an important role for ERK1/2 in mediating the induction of Id-1 by PGE 2 . First, the activity of ERK1/2 was increased by treatment with PGE 2 . Second, an inhibitor of MAPK kinase blocked the induction of Id-1 in PGE 2 -treated cells. Third, overexpression of dominant negative ERK1 suppressed the activation of Id-1 promoter activity by PGE 2 .
Id-1 gene expression can potentially be regulated by either Egr-1 or CREB (43,56). Previous studies have shown that PGE 2 can regulate gene expression by either Egr-1 or CREB-dependent mechanisms (48,57,58). We report that induction of Id-1 promoter activity by PGE 2 was mediated through an Egr-1 binding site. Several findings support a role for Egr-1 in mediating the induction of Id-1 in cells treated with PGE 2 . Electrophoretic mobility shift assays showed increased binding of Egr-1 to the Id-1 promoter in response to exposure to PGE 2 . This result was confirmed by ChIP analysis. The functional importance of Egr-1 was established because mutagenizing the Egr-1 site or silencing of Egr-1 abrogated the activation of the Id-1 promoter by PGE 2 . Previously, PGE 2 was found to activate ERK1/2 leading to induction of Egr-1 (57). In the current study we showed that inhibitors of the EGFR 3 ERK1/2 pathway suppressed the induction of Egr-1 by PGE 2 . Collectively, these FIGURE 7. COX-2-derived PGE 2 contributes to the increased levels of Id-1 found in SCg6 cells. A, total RNA was isolated from SCp2 and SCg6 cells. 10 g of RNA was added to each lane. The blots were hybridized with probes that recognized COX-2, Id-1, and ␤-actin. B, medium was collected from SCp2 and SCg6 cells, and amounts of PGE 2 were measured by enzyme immunoassay. Columns, means; bars, S.D.; n ϭ 6. C, SCg6 cells were treated with vehicle or the indicated concentrations of celecoxib or indomethacin for 24 h. Total cellular RNA was isolated. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized Id-1 mRNA and 18 S rRNA. D, SCp2 cells were treated with vehicle or the indicated concentrations of PGE 2 for 24 h. In the top panel cell lysate protein (100 g) was subjected to Western blotting. The blot was probed with antibodies to Id-1 and ␤-actin. In the bottom panel total RNA was isolated from cells. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized Id-1 mRNA and 18 S rRNA, respectively. E, SCp2 cells were treated with vehicle or the indicated concentrations of PGE1 alcohol for 24 h. Total cellular RNA was isolated from cells. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized Id-1 mRNA and 18 S rRNA.
Both COX-2/PGE 2 and Id-1 have been suggested to play a role in tumor metastases (9,31,32,41,42,50). Our results suggest that COX-2/PGE 2 -mediated induction of Id-1 contrib- Control, cells treated with vehicle; GFP siRNA, cells that received 2 g of GFP siRNA; COX-2 siRNA, cells that received 2 g COX-2 siRNA; Id-1 siRNA, cells that received 2 g Id-1 siRNA; PGE 2 , cells that were treated with 0.5 M PGE 2 ; celecoxib, cells that were treated with 1 M celecoxib. Panel B, top, Western blot shows SCp2 cells stably overexpressing COX-2 (left panel) and suppression of Id-1 in COX-2 overexpressing cells treated with siRNA to Id-1 (right panel). Panel B, bottom, labeling of bars is as follows: Control, cells that were stably transfected with vector alone; COX-2, cells that were stably transfected with murine COX-2 expression vector; COX-2 ϩ GFP siRNA, COX-2 overexpressing cells that received 2 g of GFP siRNA; COX-2 ϩ Id-1 siRNA, COX-2 overexpressing cells that received 2 g of Id-1 siRNA; PGE 2 , cells that were treated with 0.5 M PGE 2 ; PGE 2 ϩ GFP siRNA and PGE 2 ϩ Id-1 siRNA, cells transfected with 2 g of GFP siRNA or Id-1 siRNA, respectively, and then treated with 0.5 M PGE 2 . Panel C (top), Western blot shows suppression of Id-1 in MDA-MB231 cells treated with siRNA to Id-1. Panel C, bottom, labeling of bars is as follows: Control, cells that were treated with vehicle; GFP siRNA, cells that received 2 g of GFP siRNA; COX-2 siRNA, cells that received 2 g COX-2 siRNA; Id-1 siRNA, cells that received 2 g Id-1 siRNA; PGE 2 , cells that were treated with 0.5 M PGE 2 ; celecoxib, cells that were treated with 1 M celecoxib. Columns, means; bars, S.D.; n ϭ 6. **, p Ͻ 0.001 versus respective controls. The data shown are representative of three independent experiments. FIGURE 9. EP 4 is important for PGE 2 -mediated induction of cell invasion. A, SCp2 cells were transfected with 2 g of siRNAs to GFP or murine EP 1-4 and allowed to grow for 36 h before analysis. Total RNA was prepared from cells and subjected to northern blotting (10 g/lane). The blots were hybridized sequentially with the indicated probes. In panels B-E (SCp2; B), (MDA-MB-231; C and E), (SCg6; D), cells were aliquoted into inserts containing ECM-coated filters. Cells were transfected as indicated 36 h before being aliquoted into inserts. The inserts including the indicated treatments were then placed into wells containing conditioned medium and incubated at 37°C for 20 h. The cells that migrated into the lower well were counted after being fixed and stained. In panels B and C, the labeling of bars is as follows: Control, cells treated with vehicle; PGE 2 , cells treated with 0.5 M PGE 2 ; PGE1 alc., cells treated with 0.2 M PGE1 alcohol; PGE 2 ϩ GFP siRNA, cells transfected with 2 g of GFP siRNA were treated with 0.5 M PGE 2 ; PGE 2 ϩ EP 4 siRNA, cells transfected with 2 g of EP 1 -EP 4 siRNAs were treated with 0.5 M PGE 2 . In C, PGE 2 ϩ EP 1-4 siRNA2 represents cells that were transfected with a second siRNA to EP 4 ; PGE 2 ϩ 100 nM or 250 nM GW627368X represents cells that were co-treated with 0.5 M PGE 2 and 100 nM GW627368X or 0.5 M PGE 2 and 250 nM GW627368X, respectively. In panels D and E, the labeling of bars is as follows: Control, cells treated with vehicle; GFP siRNA, cells received 2 g of GFP siRNA; COX-2 siRNA, cells received 2 g of COX-2 siRNA; Cel, cells treated with 1 M celecoxib; Indo, cells treated with 1 M indomethacin; COX-2 siRNA ϩ PGE1 alc., cells transfected with COX-2 siRNA were treated with 0.2 M PGE1 alcohol; Cel ϩ PGE1 alc. represents cells co-treated with 1 M celecoxib and 0.2 M PGE1 alcohol; Indo ϩ PGE1 alc., cells were co-treated with 1 M indomethacin and 0.2 M of PGE 1 alcohol. The data shown are representative of three independent experiments. Columns, means; bars, S.D.; n ϭ 6.

COX-2-derived Prostaglandin E 2 Stimulates Id-1 Transcription
utes to cell invasion through ECM. Several findings support this conclusion. In the non-transformed mammary epithelial cell line SCp2, overexpression of COX-2 or treatment with PGE 2 , an inducer of Id-1, stimulated cell invasiveness through ECM. These effects were attenuated by silencing Id-1. In transformed SCg6 cells, inhibition of COX-2 activity led to a reduction in both amounts of Id-1 and cell invasiveness; silencing of Id-1 led to a comparable decrease in cell invasion. A similar cascade was observed in MDA-MB-231 cells in which high levels of endogenously expressed COX-2-mediated an Id-1-dependent increase in cell invasiveness. Both genetic and pharmacological studies suggested a critical role for EP 4 in PGE 2 -mediated stimulation of cell invasion. Recently, high levels of Id-1 and the related family member Id-3 were found to be required for metastatic progression in a subline of MDA-MB-231 called LM2 (59). Knockdown of Id-1 and Id-3 in LM2 resulted in a block of tumor re-initiation in the lung in vivo with little effect on invasiveness through an endothelial cell monolayer in vitro or extravasation in vivo. The Id-1-dependent invasiveness through ECM reported here and in previous studies (50) may reflect an invasive process in the local microenvironment of the metastatic niche that contributes to the observed tumor-reinitiation phenotype in the Id knockdown experiment. Further experiments will be required to explore this possibility.