ANTICANCER RESEARCH 26: 2857-2862 (2006)

Prostaglandin E2 Receptor Subtype 2 (EP2) Null Mice are Protected Against Murine Lung Tumorigenesis

ROBERT L. KEITH1,2, MARK W. GERACI2, S. PATRICK NANA-SINKAM2, RICHARD M. BREYER3, TYLER M. HUDISH1, AMY M. MEYER4, ALVIN M. MALKINSON4 and LORI D. DWYER-NIELD4

1Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, Denver VA Medical Center, 1055 Clermont St., Box 111A, Denver, CO 80220; 2Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine and 4Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262; 3Department of Medicine, Division of Nephrology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, U.S.A.

Abstract. Background: Manipulating prostaglandin (PG) acid by the action of cyclooxygenase (COX) enzymes and are production modulates tumor development. Elevated PGI2 important in cancer biology. The need for better lung cancer production prevents murine lung cancer, while decreasing PGE2 chemopreventive strategies beyond smoking cessation, content protects against colon cancer. PGE2 receptor subtype 2 coupled with the impressive reduction in colon cancer (EP2)-deficient mice were hypothesized to be resistant to lung observed with cyclooxygenase (COX) inhibition, has tumorigenesis. Materials and Methods: EP2 null BALB/c mice stimulated considerable interest in manipulating the and their wild-type littermates were exposed to an initiation- pulmonary prostaglandin content to modify lung cancer risk. promotion carcinogenesis protocol and lung tumorigenesis was Prostaglandin E2 (PGE2) mediates several hallmarks of examined. Chronic lung inflammation was induced to determine cancer. During early neoplastic transformation, PGE2 whether EP2 ablation influenced inflammatory cell infiltration. activates the epidermal growth factor receptor (EGFR) to Results: Tumor multiplicity in EP2 null mice was 34% lower enhance cell proliferation (2) and up-regulates survivin than in their wild-type littermates (21.9±3.0 vs. 14.5±2.9 concentrations in tumors to decrease apoptosis (3). At later tumors/mouse, p<0.001). The lung tumor burden, an indicator stages of progression, PGE2 up-regulates the biosynthesis of of growth rate, also declined (57%, p<0.05). All the mice vascular endothelial growth factor (VEGF) expression, which exhibited similar inflammatory cell infiltration. Conclusion: stimulates angiogenesis (4), increases IL-10 biosynthesis to PGE2, acting through EP2, enhanced lung tumorigenesis inhibit adaptive antitumor immune responses (5, 6) and through a mechanism that may be distinct from its pro- augments protease expression, thereby promoting invasion inflammatory activity. Thus, EP2 is a potential target for novel (7). Many cancers exhibit increased expression of the chemoprevention strategies. cyclooxygenase enzymes, COX1 and COX2, that provide the PGH2 substrate for PGE2 synthase (PGES) (8). Non-specific Lung cancer is the leading cause of cancer death in the COX inhibitors, including conventional non-steroidal anti- United States, and the majority of lung cancers are diagnosed inflammatory drugs (NSAIDs) and COX2-specific inhibitors, in former smokers (1). The large number of current and have been applied to pre-clinical models of various cancers former smokers, coupled with poor 5-year survival rates, and are used therapeutically against a hereditary form of emphasizes the need for effective chemopreventive agents. colon cancer (9, 10). Clinical lung cancer trials in which Prostaglandins are lipid mediators derived from arachidonic celecoxib is applied along with conventional platinum and taxane cytotoxic agents are on-going (11-14). However, recent studies using a mouse model of adenocarcinoma (AC), the most common form of human lung cancer, suggest that Correspondence to: Lori D. Dwyer-Nield, Department of downstream targeting of the PGE pathways may be more Pharmaceutical Sciences, University of Colorado Health Sciences 2 Center, 4200 E. Ninth Ave., Denver, CO 80262, U.S.A. effective. Celecoxib administration did not reduce the multiplicity of chemically-induced mouse lung tumors and

Key Words: EP2, two-stage carcinogenesis, chronic pulmonary actually enhanced their growth rate (15). This may be, at least inflammation, downstream prostaglandin receptors. in part, because PGH2 is also the substrate for prostaglandin

0250-7005/2006 $2.00+.40 2857 ANTICANCER RESEARCH 26: 2857-2862 (2006)

I2 synthase (PGI2 synthase, PGIS). Mice that overexpress We report here, fewer lung tumors in EP2 null mice PGIS are protected from lung tumor induction by both compared to their wild-type littermates, as well as a chemical carcinogens and cigarette smoke (16, 17). Cell lines significant reduction in the rate of tumor growth. The EP2 derived from mouse lung tumors produce more PGE2 and less mutation did not affect leukocyte infiltration in response to PGI2 than non-tumorigenic cell lines (18). The PGIS content chronic BHT administration, suggesting that PGE2- in human AC is usually down-regulated while the PGES mediated inflammation is not integral to the pro- content rises but, importantly, a small subset of AC patients tumorigenic mechanism mediated by EP2. whose lung tumors retained PGIS expression exhibited significantly improved survival (19). These results suggest that Materials and Methods specifically interfering with PGE2 function downstream from COX, while maintaining PGI2 production, would be an Tumorigenesis experiments. EP2 null mice and their wild-type littermates effective chemoprevention strategy for human lung cancer. were bred in the animal care facility at Vanderbilt University, USA, PGE exhibits potent immunosuppressive effects (5, 6) and genotyped by Southern analysis, as previously published (38). The 2 mice were transferred to the Denver VAMC animal care facility at 8 through distinct G protein coupled E-prostanoid receptors, weeks of age for the tumorigenesis experiments. The mice were EP1-4. EP receptors can increase cellular cAMP (EP2 and maintained in a 12-h light/dark cycle on hardwood bedding, and fed EP4), decrease intracellular cAMP (EP3), or mobilize standard chow and water ad libitum. The mice used in the intracellular calcium (EP1) (20). EP receptors are inflammation studies were bred at the Denver VAMC animal facility differentially expressed based on the cell type and regulate and genotyped and maintained as described above. B and T cell development (21, 22), macrophage cytokine Seventeen wild-type and 16 EP2-null mice (10-12 weeks old) were release (23) and modify the post-surgical immune response injected intraperitoneally (i.p.) with 15 mg 3-methylcholanthrene (MCA)/kg body weight dissolved in Mazola® corn oil. In nine wild- (24). Genetically modified mice that lack specific EP type and eight EP2 null mice, this was followed by six weekly i.p. receptors have led to an improved understanding of their injections of butylated hydroxytoluene (BHT, 150 mg BHT/kg body specific roles (reviewed in (25)). In a murine colon cancer weight administered week 1; 200 mg/kg thereafter) dissolved in corn model, EP1 and EP4 receptor knockout mice developed oil. The remaining wild-type and EP2 null mice received six weekly fewer and smaller tumors, while EP2 and EP3 ablation did corn oil injections. Twenty weeks after MCA exposure, the mice were sacrificed by lethal pentobarbital injection and their lungs were not affect the tumor number or size (26, 27). EP3 is also believed to play a role in lung carcinogenesis by reinforcing excised. Lung tumors were counted under a dissecting microscope (5X magnification) and their diameters measured using digital the activation of the ras signaling pathway (28) and Src calipers. The volume of each individual tumor was determined using signaling in cultured A549 (lung adenocarcinoma) cells (29). the formula "Volume = 4/3r3." The tumor burden per animal was However, the effects of genetic ablation of individual calculated as the sum of these volumes. To confirm the pulmonary receptors are organ-specific. For example, EP2 null mice adenoma pathology, tumors and surrounding uninvolved lung were develop fewer skin tumors in response to chemical paraffin-embedded and sectioned, followed by H&E staining. All carcinogens than do their wild-type littermates (30). In slides were reviewed with a pathologist to confirm findings. pancreatic cancer cell lines, PGE induced VEGF 2 Chronic inflammation studies. Homozygous EP (–/–, n=6), production via EP signaling (4). Down-regulation of EP in 2 2 2 heterozygous (+/–, n=15), and wild-type (+/+, n=13) male and immortalized human keratinocytes reduced paxillin female littermates (8-10 weeks of age) were exposed to four weekly expression, thereby decreasing progression to an invasive i.p. BHT injections (150 mg BHT/kg body weight administered week 1; phenotype (31). In the lung, EP2 is important in 200 mg/kg thereafter) to induce a pulmonary inflammatory response, bronchodilation (32) and fibroproliferative lung disease (33, as described previously in BALB mice (39). The control mice received four weekly vehicle injections. Six days after the last treatment, the 34). While the role of PGE2 receptors in human lung cancer is unknown, growth inhibition of human non-small lung mice were euthanized and bronchoalveolar lavage (BAL) performed. The trachea was cannulated with an 18g angiocatheter and three serial cancer (NSCLC) cells by peroxisome proliferator-activated 1 ml aliquots of 10 mM K2HPO4 (pH 7.4), 150 mM NaCl, 0.6 mM receptor gamma (PPAR-Á) ligands is mediated through EDTA instilled and aspirated back into the syringe. Cells from all inhibiting EP2 expression (35). We therefore hypothesized three aliquots were pooled, quantified, cytospun onto glass slides and that mice null for EP2 would exhibit impaired lung tumor sent for routine cell counts with differentials (performed by the development when subjected to a chemical carcinogenesis Clinical Laboratory at the University of Colorado Hospital, USA). model. A defective EP2 receptor mutation was bred into BALB/c mice (36), an inbred strain highly susceptible to two- Statistical analysis. All values are expressed as means±SEM. For tumor multiplicity and cell counts, the data were normally stage carcinogenesis initiated by the tobacco carcinogen, 3- distributed and statistical analyses were performed using GraphPad methylcholanthrene (MCA) and promoted by the food Prism 4.0 for Windows (GraphPad Software for Science Inc., San additive, butylated hydroxytoluene (BHT) (37). Diego, CA, USA). Data was considered significant at the p<0.05 Phenotypically, these EP2 null mice exhibit salt-sensitive level. Groups were compared by ANOVA followed by Student hypertension and decreased fertility (38). Newman Keuls post-hoc analysis.

2858 Keith et al: Reduced Lung Tumor Formation in EP2-null Mice

Figure 1. Average lung tumor multiplicity and burden in wild-type (white bars) and EP2 null (black bars) mice treated with MCA or MCA + BHT. EP2 null mice displayed significantly reduced tumor multiplicity (A) and tumor burden (B) when subjected to an initiation (MCA)-promotion (BHT) tumorigenesis model. *p<0.05 vs. wild-type (MCA + BHT)-treated mice.

Results

Tumor growth. Tumor multiplicities and burdens from wild- type and EP2 null mice induced with MCA or MCA + BHT were determined (Figure 1A, 1B). EP2 null mice developed 34% fewer tumors in response to the two-stage MCA + BHT protocol (21.9±3.0 vs. 14.5±2.9 tumors/mouse, p<0.001). Both groups developed a small number of tumors in response to MCA alone (1.1±0.4 (EP2 null) vs. 1.8±0.4 (wild-type) tumors/mouse, p=NS). No significant differences in tumor incidence between MCA-treated EP2 null and wild-type mice (6/8 vs. 7/8) were observed and all mice exposed to MCA + BHT developed tumors (100% incidence). The tumors in EP null mice were significantly smaller 2 Figure 2. BAL macrophages in vehicle vs. BHT-treated wild-type (white than in their wild-type littermates (0.69±0.03 vs. 0.81±0.03 bars), EP2 heterozygote (gray bars) and EP2 null (black bars) mice. mm tumor diameter, p<0.05). EP2 null animals exhibited a Chronic BHT increases BAL macrophage content, compared to vehicle nearly three-fold reduction in tumor burden compared to alone.*p<0.03 vs. vehicle. Genetic modulation of EP2 does not affect their wild-type littermates (5.1±2.1 vs. 14.3±4.2 mm3/mouse, BAL macrophage content after chronic exposure to BHT. p<0.05) (Figure 1B). The tumor burden reflects growth rate.

Chronic inflammation. To determine if loss of functional EP2 in human trials. Orthologous gene expression studies of altered the pulmonary inflammatory response and, thereby, human and chemically-induced murine adenocarcinomas accounted for the diminished tumor multiplicity and growth, revealed significant similarities (19), validating the use of homozygous and heterozygous EP2 knockouts and wild-type murine models. Based on the many pro-tumorigenic littermates were exposed to four weekly injections of BHT. properties of PGE2, we hypothesized that EP2 null mice This insult typically results in a substantial influx of would be less susceptible to chemical lung carcinogenesis. macrophages (39). Differential cell counts were performed on When EP2 null mice and their wild-type littermates were BAL fluid to determine the number and types of subjected to a widely used initiation-promotion lung tumor inflammatory cells present. Macrophage cell counts were model, the genetically modified mice exhibited significant similar in EP2 null, EP2 heterozygote and wild-type mice reductions in tumor multiplicity and burden (Figures 1A (Figure 2). No differences in lymphocyte, neutrophil, or and 1B). eosinophil counts were observed (data not shown). In this lung carcinogenesis model, chronic pulmonary inflammation mediates tumor formation (40). Inbred strains Discussion such as BALB/c that are sensitive to BHT-tumor promotion, are also characterized by a macrophage predominant influx Preclinical modeling of lung cancer is critical for evaluating following BHT treatment (39). When pulmonary macrophage novel prevention strategies and to better predict outcomes infiltration during MCA/BHT carcinogenesis in BALB mice

2859 ANTICANCER RESEARCH 26: 2857-2862 (2006)

was reduced, tumor multiplicity decreased (40). PGE2 is a 4 Eibl G, Bruemmer D, Okada Y, Duffy JP, Law RE, Reber HA and Hines OJ: PGE(2) is generated by specific COX-2 activity and potent mediator of inflammation, and EP3 receptor null mice exhibit attenuated inflammation when exposed to topical increases VEGF production in COX-2-expressing human pancreatic cancer cells. Biochem Biophys Res Commun 306: 887- arachidonic acid (41). We hypothesized that one explanation 897, 2003. for differences in tumor multiplicity may have been altered 5 Huang M, Stolina M, Sharma S, Mao JT, Zhu L, Miller PW, responses to a lung inflammatory insult. EP2 null, heterozygous Wollman J, Herschman H and Dubinett SM: Non-small cell lung (to assess for the presence of a critical level of EP2 receptor cancer cyclooxygenase-2-dependent regulation of cytokine balance expression), and their wild-type littermates did not differ in in lymphocytes and macrophages: up-regulation of interleukin 10 BAL macrophage cell counts (Figure 2), however. Therefore, and down-regulation of interleukin 12 production. Cancer Res 58: 1208-1216, 1998. the effects of EP2 ablation on tumorigenesis may be explained by alternative mechanisms. EP may suppress the immune 6 Stolina M, Sharma S, Lin Y, Dohadwala M, Gardner B, Luo J, 2 Zhu L, Kronenberg M, Miller PW, Portanova J, Lee JC and response to lung cancer by altering the activity of T regulatory Dubinett SM: Specific inhibition of cyclooxygenase 2 restores cells (Treg). Tumor-derived PGE2 induces expression of antitumor reactivity by altering the balance of IL-10 and IL-12 Foxp3, a Treg-specific transcription factor, and Foxp3 synthesis. J Immunol 164: 361-370, 2000. expression is ablated in the absence of EP2 (42). A decreased 7 Dohadwala M, Batra RK, Luo J, Lin Y, Krysan K, Pold M, production of these immune suppressor cells upon ablating Sharma S and Dubinett SM: Autocrine/paracrine prostaglandin E2 production by non-small cell lung cancer cells regulates matrix EP2 may explain observed differences in tumor number and growth rate. The EP -independent activities of PGE include metalloproteinase-2 and CD44 in cyclooxygenase-2-dependent 2 2 invasion. J Biol Chem 277: 50828-50833, 2002. PGE stimulating colon cancer growth by inactivating and 2 8 Buchanan FG, Wang D, Bargiacchi F and Dubois RN: releasing glycogen synthase 3‚ from its complex with axin, Prostaglandin E2 regulates cell migration via the intracellular which leads to the activation of ‚-catenin (43). activation of the epidermal growth factor receptor. J Biol Chem Efforts are currently underway to systematically examine 278: 35451-35457, 2003. lung tumor formation in BALB/c mice null for EP1, EP3 and 9 Thun MJ, Namboodiri MM and Heath CW Jr: Aspirin use and reduced risk of fatal colon cancer. N Engl J Med 325: 1593-1596, EP4, since specific antagonists and agonists that act at each 1991. of these receptors are available. Modulating PGE2 pro- tumorigenic activities by inhibiting individual EP receptors 10 Giardiello FM, Yang VW, Hylind LM, Krush AJ, Petersen GM, Trimbath JD, Piantadosi S, Garrett E, Geiman DE, Hubbard W, may complement other lung chemopreventive strategies, Offerhaus GJ and Hamilton SR: Primary chemoprevention of especially those that up-regulate the PGI2 pathway. Since familial adenomatous polyposis with sulindac. N Engl J Med 346: EP2 is downsteam from COX, the pleiotopic effects of COX 1054-1059, 2002. inhibition can thereby be avoided. EP2 is thus an attractive 11 Altorki NK, Keresztes RS, Port JL, Libby DM, Korst RJ, Flieder target, singly or in combination with other preventive DB, Ferrara CA, Yankelevitz DF, Subbaramaiah K, Pasmantier strategies, for future pre-clinical and human trials. MW and Dannenberg AJ: Celecoxib, a selective cyclo-oxygenase- 2 inhibitor, enhances the response to preoperative paclitaxel and carboplatin in early-stage non-small-cell lung cancer. J Clin Oncol Acknowledgements 21: 2645-2650, 2003. 12 Csiki I, Morrow JD, Sandler A, Shyr Y, Oates J, Williams We thank Amanda Osterberg and Jake Smith for technical MK, Dang T, Carbone DP and Johnson DH: Targeting assistance. This work was supported by NCI grant CA 33497; Dr. cyclooxygenase-2 in recurrent non-small cell lung cancer: a Keith was supported by Advanced Career Development and Merit phase II trial of celecoxib and docetaxel. Clin Cancer Res 11: Review awards from the Department of Veterans' Affairs. 6634-6640, 2005. 13 Gasparini G, Meo S, Comella G, Stani SC, Mariani L, References Gamucci T, Avallone A, Lo VS, Mansueto G, Bonginelli P, Gattuso D and Gion M: The combination of the selective 1 Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, cyclooxygenase-2 inhibitor celecoxib with weekly paclitaxel is a Feuer EJ and Thun MJ: Cancer statistics, 2005. CA Cancer J safe and active second-line therapy for non-small cell lung Clin 55: 10-30, 2005. cancer: a phase II study with biological correlates. Cancer J 11: 2 Johnson FM, Yang P, Newman RA and Donato NJ: 209-216, 2005. Cyclooxygenase-2 induction and prostaglandin E2 accumulation 14 Nugent FW, Mertens WC, Graziano S, Levitan N, Collea R, Gajra in squamous cell carcinoma as a consequence of epidermal A, Marshall J and McCann J: Docetaxel and cyclooxygenase-2 growth factor receptor activation by imatinib mesylate. J Exp inhibition with celecoxib for advanced non-small cell lung cancer Ther Oncol 4: 317-325, 2004. progressing after platinum-based chemotherapy: a multicenter 3 Baratelli F, Krysan K, Heuze-Vourc'h N, Zhu L, Escuadro B, phase II trial. Lung Cancer 48: 267-273, 2005. Sharma S, Reckamp K, Dohadwala M and Dubinett SM: PGE2 15 Kisley LR, Barrett BS, Dwyer-Nield LD, Bauer AK, Thompson confers survivin-dependent apoptosis resistance in human DC and Malkinson AM: Celecoxib reduces pulmonary monocyte-derived dendritic cells. J Leukoc Biol 78: 555-564, inflammation but not lung tumorigenesis in mice. Carcinogenesis 2005. 23: 1653-1660, 2002.

2860 Keith et al: Reduced Lung Tumor Formation in EP2-null Mice

16 Keith RL, Miller YE, Hoshikawa Y, Moore MD, Gesell TL, 30 Sung YM, He G and Fischer SM: Lack of expression of the EP2 Gao B, Malkinson AM, Golpon HA, Nemenoff RA and Geraci but not EP3 receptor for prostaglandin E2 results in suppression MW: Manipulation of pulmonary prostacyclin synthase of skin tumor development. Cancer Res 65: 9304-9311, 2005. expression prevents murine lung cancer. Cancer Res 62: 734- 31 Konger RL, Scott GA, Landt Y, Ladenson JH and Pentland AP: 740, 2002. Loss of the EP2 prostaglandin E2 receptor in immortalized 17 Keith RL, Miller YE, Hudish TM, Girod CE, Sotto-Santiago S, human keratinocytes results in increased invasiveness and Franklin WA, Nemenoff RA, March TH, Nana-Sinkam SP and decreased paxillin expression. Am J Pathol 161: 2065-2078, 2002. Geraci MW: Pulmonary prostacyclin synthase overexpression 32 Sheller JR, Mitchell D, Meyrick B, Oates J and Breyer R: EP(2) chemoprevents tobacco smoke lung carcinogenesis in mice. receptor mediates bronchodilation by PGE(2) in mice. J Appl Cancer Res 64: 5897-5904, 2004. Physiol 88: 2214-2218, 2000. 18 Dwyer-Nield L, Srebernak MC, Barrett BS, Ahn J, Cosper P, 33 White ES, Atrasz RG, Dickie EG, Aronoff DM, Stambolic V, Mak Meyer A, Kisley L, Bauer A, Thompson DC and Malkinson A: TW, Moore BB and Peters-Golden M: Prostaglandin E(2) inhibits Cytokines differentially regulate the synthesis of prostanoid and fibroblast migration by E-prostanoid 2 receptor-mediated increase nitric oxide mediators in tumorigenic vs. non-tumorigenic mouse in PTEN activity. Am J Respir Cell Mol Biol 32: 135-141, 2005. lung epithelial cell lines. Carcinogenesis 26: 1196-1206, 2005. 34 Moore BB, Ballinger MN, White ES, Green ME, Herrygers AB, 19 Stearman RS, Dwyer-Nield L, Zerbe L, Blaine SA, Chan Z, Wilke CA, Toews GB and Peters-Golden M: Bleomycin-induced Bunn PA Jr, Johnson GL, Hirsch FR, Merrick DT, Franklin WA, E prostanoid receptor changes alter fibroblast responses to Baron AE, Keith RL, Nemenoff RA, Malkinson AM and Geraci prostaglandin E2. J Immunol 174: 5644-5649, 2005. MW: Analysis of orthologous gene expression between human 35 Han S and Roman J: Suppression of prostaglandin E2 receptor pulmonary adenocarcinoma and a carcinogen-induced murine subtype EP2 by PPARgamma ligands inhibits human lung model. Am J Pathol 167: 1763-1775, 2005. carcinoma cell growth. Biochem Biophys Res Commun 314: 20 Breyer MD, Jacobson HR and Breyer RM: Functional and 1093-1099, 2004. molecular aspects of renal prostaglandin receptors. J Am Soc 36 Yang L, Yamagata N, Yadav R, Brandon S, Courtney RL, Nephrol 7: 8-17, 1996. Morrow JD, Shyr Y, Boothby M, Joyce S, Carbone DP and 21 Fedyk ER, Harris SG, Padilla J and Phipps RP: Prostaglandin Breyer RM: Cancer-associated immunodeficiency and dendritic receptors of the EP2 and EP4 subtypes regulate B lymphocyte cell abnormalities mediated by the prostaglandin EP2 receptor. J activation and differentiation to IgE-secreting cells. Adv Exp Clin Invest 111: 727-735, 2003. Med Biol 433: 153-157, 1997. 37 Malkinson AM, Koski KM, Evans WA and Festing MF: 22 Rocca B, Spain LM, Pure E, Langenbach R, Patrono C and Butylated hydroxytoluene exposure is necessary to induce lung FitzGerald GA: Distinct roles of prostaglandin H synthases 1 and tumors in BALB mice treated with 3-methylcholanthrene. Cancer 2 in T-cell development. J Clin Invest 103: 1469-1477, 1999. Res 57: 2832-2834, 1997. 23 Nataraj C, Thomas DW, Tilley SL, Nguyen MT, Mannon R, 38 Kennedy CR, Zhang Y, Brandon S, Guan Y, Coffee K, Funk Koller BH and Coffman TM: Receptors for prostaglandin E(2) CD, Magnuson MA, Oates JA, Breyer MD and Breyer RM: Salt- that regulate cellular immune responses in the mouse. J Clin sensitive hypertension and reduced fertility in mice lacking the Invest 108: 1229-1235, 2001. prostaglandin EP2 receptor. Nat Med 5: 217-220, 1999. 24 Strong VE, Mackrell PJ, Concannon EM, Naama HA, Schaefer 39 Bauer AK, Dwyer-Nield LD, Hankin JA, Murphy RC and PA, Shaftan GW, Stapleton PP and Daly JM: Blocking Malkinson AM: The lung tumor promoter, butylated prostaglandin E2 after trauma attenuates pro-inflammatory hydroxytoluene (BHT), causes chronic inflammation in promotion- cytokines and improves survival. Shock 14: 374-379, 2000. sensitive BALB/cByJ mice but not in promotion- resistant CXB4 25 Hata AN and Breyer RM: Pharmacology and signaling of mice. Toxicology 169: 1-15, 2001. prostaglandin receptors: multiple roles in inflammation and 40 Bauer AK, Dwyer-Nield LD, Keil K, Koski K and Malkinson immune modulation. Pharmacol Ther 103: 147-166, 2004. AM: Butylated hydroxytoluene (BHT) induction of pulmonary 26 Watanabe K, Kawamori T, Nakatsugi S, Ohta T, Ohuchida S, inflammation: a role in tumor promotion. Exp Lung Res 27: 197- Yamamoto H, Maruyama T, Kondo K, Ushikubi F, Narumiya S, 216, 2001. Sugimura T and Wakabayashi K: Role of the prostaglandin E 41 Goulet JL, Pace AJ, Key ML, Byrum RS, Nguyen M, Tilley SL, receptor subtype EP1 in colon carcinogenesis. Cancer Res 59: Morham SG, Langenbach R, Stock JL, McNeish JD, Smithies O, 5093-5096, 1999. Coffman TM and Koller BH: E-prostanoid-3 receptors mediate 27 Mutoh M, Watanabe K, Kitamura T, Shoji Y, Takahashi M, the proinflammatory actions of prostaglandin E2 in acute Kawamori T, Tani K, Kobayashi M, Maruyama T, Kobayashi K, cutaneous inflammation. J Immunol 173: 1321-1326, 2004. Ohuchida S, Sugimoto Y, Narumiya S, Sugimura T and 42 Sharma S, Yang SC, Zhu L, Reckamp K, Gardner B, Baratelli F, Wakabayashi K: Involvement of prostaglandin E receptor subtype Huang M, Batra RK and Dubinett SM: Tumor cyclooxygenase- EP(4) in colon carcinogenesis. Cancer Res 62: 28-32, 2002. 2/prostaglandin E2-dependent promotion of FOXP3 expression 28 Yano T, Zissel G, Muller-Qernheim J, Jae SS, Satoh H and and CD4+ CD25+ T regulatory cell activities in lung cancer. Ichikawa T: Prostaglandin E2 reinforces the activation of Ras Cancer Res 65: 5211-5220, 2005. signal pathway in lung adenocarcinoma cells via EP3. FEBS Lett 43 Castellone MD, Teramoto H, Williams BO, Druey KM and 518: 154-158, 2002. Gutkind JS: Prostaglandin E2 promotes colon cancer cell growth 29 Yamaki T, Endoh K, Miyahara M, Nagamine I, Thi Thu HN, through a Gs-axin-beta-catenin signaling axis. Science 310: 1504- Sakurai H, Pokorny J and Yano T: Prostaglandin E2 activates Src 1510, 2005. signaling in lung adenocarcinoma cell via EP3. Cancer Lett 214: Received April 4, 2006 115-120, 2004. Accepted May 22, 2006

2861