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Vanderbilt University Medical Center August 14, 1993 The Council for Tobacco Research U.S.A. Inc. 900 Third Avenue New York, N.Y. 10022 Dear Sir or Madam, Please find enclosed a copy of my preliminary application proposal entitled "The Role of Prostaglandin EP3 Receptor in Tumor Metastasis". This proposal is for a three year study with a first year direct costs of $55,000. If you have any questions, I can be contacted by phone at (615) 343-0257, and by fax at (615) 343-7156. Thank you very much for your consideration. Sincerely, • . reyer, Ph.'D~'~ Assistant Professor of Medicine and Pharmacology 40008918

THE ROLE OF PROSTAGLANDIN EP3 RECEPTOR IN TUMOR METASTASIS Beck~'ound and Si~nif/c~nce. Prostaglandin E2 (POE2) is a potent modulator of a v,~de variety of physiological responses including inflammation, vascular tone, water m-~d ion transport as well as cell growth and division. Increased PGE2 synthesis has been associated with proliferation of tumor cells in human lung cancer, breast cancer and colon cancer as well as in rodent model systems (1, 2, 3). In pat/ents with lung cancer, there is an increased PGE2 synthesis in cancerous versus normal t/ssue (4). This increase was more pronounced in lung cancers of patients who smoked cigarettes as compare~I with nonsmokers. In other studies an inverse correlation b~tween elevations in PGE2 synthesis and mean survival time has been observed for patients with breast cancer, and to a lesser e×tent in lung and colonic cancers (5). In patients with familial polyposis, the adm/nist.rat~on of nons.teroidal anti-inflammatory drugs (NSAIDs) which are potent inhibitors of PGE2 synthesis have been shown to cause polyp regression, and withdra~val of the NSAIDs reverse this effect (6, 7). These findings support the notion that PGE2 mediated effects may play an important role in cell transformation, tumor groxvth and/or metastasis (8). Unlike classical circulating hormones, such as insulin and corticosteroids, prostaglandins are autacoids, acting locally on the tissues in which they are synthesized or on adjacent tissues. Accumulating evidence suggests that PGE2 exerts its cellular action through specific receptors coupled to their intracellular effectors via guanine nucleotide regulatory proteins (G-proteins) (9, 10, 11). Based upon their pharmacological ligand binding properties and physiologic effects in smooth muscle (12) at least three classes of PGE2 receptor have been proposed. These three E-Prostanoid receptors are designated EPI, EP2 and EP3. It'is of interest that PGE2 receptors have bee~ ~'hown to couple to three separate signalling pathways, via G-protein mediated PI (EP1) turnover and cAMP generation via Gs (EP2) and Gi (EP3). In one model of mouse mammary tumor metastasis cell lines 66 and 4526 have been demonstrated to express a high affinity PGE2 receptor (1). Stimulation of this receptor leads to an increase in cAMP levels (i.e. it is EP2-1ike). Blockade of this receptor with PGE2 selective antagonists leads to a decrease in cAMP generation and an increase in the lung metastasis of this tumor in vivo. (1). Our laboratory has recently clones a family of alternatively spliced variants of the EP3 receptor. These receptors are presumably coupled in vivo to the Gi pathway. As described below, we believe that each of these receptors will hav~ identical ligand binding properties but different signal transduction characteristics. We will test the ability of each of these receptors to modulate cyclic AMP levels in vitro and metastasis in vivo. Specific Aims. The focus of this proposal is to evaluate the role of EP3 receptor subtypes in a mouse model of metastasis. In principle, stimulation of the Gi coupled EP3 receptor should lower cAMP levels in the same manner as EP2 receptor blockade. Thus stimulation of EP3 receptor might be expected to increase metastatic potential of these tumor cells. The aims of this proposal are to : Evaluate the effect of expression of EP3 receptor subtypes 72A, 74A 77A~ and 80A in murine mammary tumor lines 66 and 4526 on cAMP generation and lung metastasis. Preliminary_ Studies In settling on a target tissue in which to characterize the PGE2 receptor, we have initially focused on the kidney. Not only is PGE2 the major renal cyelooxygenase metabolite of arachidonic acid but the kidney is one of the richest sources of PGE2 receptors. We amplified rabbit eDNA sequences by reverse transcription PCR (RT-PCR) using poly (A)+ RNA isolated from rabbit renal cortex and oligonucleotide primers derived from the cloned mouse EP3o~ receptor (13). Overlapping PCR products representing putative tmnsmembrane regions III through VII were generated, cloned and sequenced. Overall the deduced amino acid sequence from these fragments is 84% homologous with the predicted amino acid sequence for the mouse receptor. Using these PCR fragments as probes, northem hybridization analysis revealed multiple transcripts of 1.8, 2.6 and 7 kb expressed in both renal cortex and medulla. Moreover the levels of expression of these transcripts ~vere modulated by in vivo pretreatment of animals with indomethacin, suggesting feedback modulation of receptor message levels by depletion of the endogenous ligand. 40008919

COOH Figure 1. The deduced amino acid sequence of the rabbit EP receptor 72A, showing the typical seven transmembrane structure of a G-protein coupled receptor. The transmembrane spanning regions of G-protein coupled receptors largely determine the ligand binding specificity. The intracellular third loop and the C-termlnal tall have been implicated in coupling specificity and regulation. PJ. : I~.~rd M. Breyer~ Ph.D. We probed a rabbit renal cortc× cDNA library with PCR fragments of the rabbit EP receptor ~nd isolated five independent clones. The deduced amino acid sequence of one clone, 72A, is shown in Fig. 1. This clone contains a cDNA of 2061 nt and has an open reading frame of 361 amino acids. Analysis of the deduced amino acid sequence indicates that the polypeptide encoded by this eDNA has seven transmembrane regions as represented in Figures 1 and is 82% homologous to the mouse EP3 receptor. When transfected into COS1 cells, clone 72A confers [3HI PGE2 to cell membranes, with a KD of 300 pM, and an order of agonist affinity typical of an EP3 receptor: M&B 28767 > sulprostone > PGE2 = PGE1 > PGF2a > PGD2 > 8-epi PGF2 Sequencing of the .remaining clones indicates that they fall into four classes (Fig. 2). Each class appears to be nearly identical up to nucleotide 1065 (using the clone 72A numbering system) where the four classes of receptors clearly diverge in sequence. At the amino acid level, the divergent reg!on corresponds to amino acid 355, ten amtno acids C-terminal to transmembrane VII on the cytoplasmic surface . We currently have clones containing the complete coding regions for only two of the four classes of sequence, but preliminary data indicates that • the receptors will be identical, or very nearly throughout the seven transmembrane domain region, and diverge at the C-terminal tail. This region is implicated in regulation of G-protein coupled receptors by a number of protein kinases. It may be that the functional effect of these different C-terminal sequences is to cause differential regulation by the regulatory kinases. Functionally this would imply that the ligand binding portion of each receptor is similar and only the signalling differs. EXPERIMENTAL DESIGN AND PROCEDURES Based upon Scatchard analysis cell lines 66 and 4526 appear to have a single EP receptor and functional studies indicate that this is an EP2 subtype. We will confirm the absence of EP3 receptor message in cell lines 66 and 4526 by Northem Blot using a rabbit EP3 common region probe. This probe has previously been sho~vn to cross hybridize to the mouse EP3 receptor message present in normal tissues. Cells will be evaluated for the effects of EP3 selective agonists. Initially.we will utilize M&B 28767 (EP3 selective ) and sulprostone (EP1/3 selective). Both of these ligands have a high affinity for the EP3 receptor (see accompanying manuscript). Cells will be pretreated with these compounds at saturating concentrations (-~0.1 I.tM) and assayed for their ability to stimulate cAMP formation, or inhibit forskolin stimulated cAMP generation. Increased metastasis will be evaluated as described: after pretreatment, cells will be injected into Balb/c mice in PBS i. v. in the tail vein. Three weeks later, mice will be sacrificed and lung tissue will be examined for the presence of surface colonies(l). Cell lines will be transfected with each of the full length receptor constructs, 72A, 77A, 80A and 74A individually. Expression plasmid constructs encoding clones 77A and 72A constructs already exist in the laboratory and we are currently engaged in constructing full length 80A and 74A. Cell lines will be assayed for their ability to bind EP3 selective ligands, inhibit cAMP generation and the presence of the transfected receptor message. Clones expressing each receptor subtype isolated by standard cloning methodology. Each subtype will be tested for its ability to inhibit cAMP generation and to increase metastasis in response to EP3 selective ligands. 2 40008920

The R~ ~f Pt'c~L~gI~d~ EP3 Receptor in Tumor Met~t~i~ Start ~ Stop -18~ 290 400 600 800 ~0[ 1200 1400 1600 1800 2000 77A 74A 80A Fig 2. upperpanel The alignment of four EP receptor clones representing the four classes of sequence. 72A appears to be a full ienglh cDNA~ 77A h~s lhe entire coding region~ and clones 74A and 80A are fragments encoding only a portion of the corresponding receptor, lo~erpand The deduced amino acid sequence of the divergent lmrtion of the four EP3 receptor subtypes. Pl. : R|chard/%L Breyer~ Ph.D. Differential regulation of these receptor subtypes may ~ of potential functional significance in determining thc rcsponsv of a given cell type of tissue to PGE2. One receptor done, 72A, has the C-terminal sequence RKILLRKFCQEEF%VEK • This C-terminal "'tail" is notable in that it is exceptionally short and is different from each of the other receptor clones in that it contains no scrine or threonine residues. Both serine and threonine are targets for G-protein coupled receptor regulation via phosphorylation by a number of protein kinases including receptor-specific ldnases (14, 15). It might be predicted that changes in the relative fraction of the 72A receptor subtype will change the susceptibility of PGE2 receptors to regulation by protein kinases. Thus far, studies on the effects of prostanoids and cancer have focused on the generation of cyclooxygenase metabolites of arachidonic acid, and their inhibition by NSAIDs. With the cloning of individual PGE2 receptors it is now possible to determine the effects of PGE2 at specific receptor subtypes, and to characterize the relative abundance of the different receptor isoforms under various conditions Understanding the effects of stimulation or blockade of particular EP receptor subtypes on cell growth in normal and pathophysiologie states may ultimately lead our understanding of the role PGE2 plays in tumor promotion, growth and metastasis. Literature Cited 1. Fulton, A. M.. Zhang, S.-z.. and Chong. Y. C. (1991) CancerRes. 51, 2047-2050 2. Thun, M. J., Namboodiri, 13. S., and I-Iealh Jr., C. W. (1991) N. Engl. J. Meal. 325, 1593-1596 3. Noguchi, M., Taniya, T., Koyasaki, N., Kumaki, T., Miyazaki, I., and Mizu -kami, Y. (1991) CancerRes. 51, 2683-2689 4. McLemore, T. L., Hubbard, W. C., Littcrst, C. L., Liu, M. C., Miller, S., McMahon, N. A., Eggleston, J. C., and Boyd, M. R. (1988) CancerRes. 48, 3140-3147 5. 13ennett, A., Stamford, I. F., and Cooper, D. (1990) Adv. Prostaglandin, Thromboxane and Leukotriene Res. 21, 875-878 6. Klein, W. A., Miller, H. H., Anderson, M. A., and DeCosse, J. J. (1987) Cancer 60, 2863-2868 7. Rigau, L, Piqut, J. M., Rubio, E., Pl.qnas, R.. Tarrech, J. M., and Bordas. J. M. (1991) Ann. Intern. Meal. 115, 952-954 8. Honn, K. V., Boclanan, R. S., and Marnett, L. J. (1981) Prostaglandins 21, 833-865 9. Haga, T., Ross, E. M., Anderson, I-I. J., and Gilman, A. G. (1977) Proc. Natl. Acad. Sei. 74, 2016-2020 10. Lefkowitz, R. J., Mulliken, D., Wood, C. L., Gore, T. B., and Mukherjee, C. (1977) J. Biol. Chem. 252, 5295-5303 11. Watanabe, T., Umegaki, K., and Smith, W. L. (1986) Y. BioL Chem. 261, 13430-13439 12. Gardiner, P. J. (1990) Adv. in Prostaglandin, Thromboxane, and Leukotriene Res. 20, 110-118 13. Sugimoto, Y., Namba, T., Negishi, kL Ichikawa, A., and Narumiya, S. (1992) J. Biol. Chem. 267, 6463-6466 14. Bouvier, M., Hausdorf, W. P.o Deblasi, A., O'Dowd, B. F., Kobilka, B. K., Caron, M. G., and Lefkowitz, R. J. (1988) Nature 333, 370-373 15. Hausdorf, W. P., Caron, M. G., and Lelkowitz, R. J. (1990) FASEB Y. 4, 2881-2889 40008921

BIOGRAPHICAL SKETCH Give the f~'J::~-;,~g inf:rma~L.~,~ f:r the ~:~y psrs~l ~ ~n~:tan~ and ~b:rato:s. ~g~n ~ ~e Frln:~ I~'~gat~r/Prc~ram D;re:t~r. Fhat:a~p] th~s p~5~ fzr ea:h person. FOSffIDN TITLE R[ch~rd M. Breyer Assistant Professor of M~c~ne and Pharmacology EDUCATION (~n w;th b~ure~te ~r other ~nlt~.t profe~s~:n31 education ~uch ~s ~ur~q, a~d include p~stff~ctor~l ~a~nlnq ) INSTITUTION AND LOCATION DEGREE Y~R RELD OF STUDY University of Michigan, Ann Arboh M{chigan B.S. 1978 Microbiology Massachuse~s Instit~e of Technology, S.M. 1982 Biology Cambridge, Mas~chuse~s Massachuse~s Institute of Technology Ph.D. 1988 Biochemist~ RESEARCH AND PROFESSIONAL EXPERIENCE. Concluding with present position, list, in chronological order, previous employment, experience and honors. Key personnel include the principal investigator and any other individuals who participate in the scientific development or execution of the projecl. Key personnel typically will include all individuals with doctoral or other professional degree~, but in some projects will include individual~ at the masters or baccalaureate level provided they contribute in a substantive way to the scientific development or execution of the project, Include present membership on any Federal Government public advisory committee. List, in chronological order, the titles, all authors, and complete references to all publications during the past three years and to representative earlier publications pertinent to this application. DO NOT EXCEED TWO PAGES. Professional Experience: 1982-1984 1988-1990 1990-1991 1991 -Present 1991 -Present Research Assistant, Repligen Corporation, Cambridge. Massachusetts Postdoctoral Fellow, Biologie Mol~culaire des R6cepteurs, Institut Pastuer, Paris, France Postdoctoral Fellow, Laboratoire d'lmmuno-Pharmacologie Mol~culaire, Institute Cochin de G~netique Mol~culaire, Institute Cochin de G6netique Mol~culaire, Pads, France Assistant Professor of Medicine, Division of Nephrology, Vanderbilt University, Nashville, Tennessee Assistant Professor of Pharmacology, Vanderbilt University, Nashville, Tennessee Honors and Awards: 1973 1978 1978 1987-1988 1988-1989 1989-1990 1990-1991 1992 William Branstrom Freshman Prize, University of Michigan James B. Angell Scholar, University of Michigan Phi Beta Kappa, University of Michigan Centocor Research Fellow, Predoctoral Fellowshlp Foundation de la Recherche Medicale, Postdoctoral Fellowship Association pour la Recherche sur le Cancer, Postdoctoral Fellowship Agence Nationale sur le SIDA, Postdoctoral Fellowship National Kidney Foundation Young Investigator Award Publications: Reilly, E.B., Reilly, R.M., Breyer, R.M., Sauer, R.T., and Eisen, H.N. (1984) Amino Acid and Nucleotide Sequences of Variable Regions of Mouse Immunoglobulin Light Chains of the Z :3 Subtype. J. Immunol., 133:471-475. Colbert, D., Anilionis, A., Gelep, P., Farley, J., and Breyer, R. (1984) Molecular Organization of the Protein AGene and its Expression in Recombinant Host Organisms. J. BioL Resp. Modif., 3:255-259. Breyer, R.M. and Sauer, R.T. (1989) Production and Characterization of Monoclonal Antibodies to the N-terminal Domain of ~. Represser. J. BioL Chem., 26~/:13348-13354. Breyer, R.M. and Sauer, R.T. (1989) Mutational Analysis of the Fine Specificity of Binding of Monoclonal Antibody 51F to Z Represser. J. BioL Chem., 264:13355-13369. 5. Brever. R.M., Strosberg, A.D., and Guillet, J.-G. (1990) Mutational Analysis of Ligand Binding Activity of 82 Adrenergic 40008922

6o Reidhaar-OIson, J.F., Bowie, J.U., Breyer, R.M., Hu, J.C., Kn:'ght, K.L, Lim, W.A., Mossing, M.C., Pars~ll, D.A., Sho2rnaker, K.R., and Seuer, R.T. (1991) Random Mutagenes~s of Protein Sequ_=nces Using O][gonucteotide Cass~.ttes. Methods Enz}~-nol 20~:565-585. 7o Bert[n, B., Fre[ssmuth, M., Brevet. R.M.. Strosberg, A.D., and Maru]Io, S. Functional expression of the 5HTla receptor In Escherichfa coil: Ligand binding properties and interaction with recombinant G-proteins (1992) J. Biol. Chem. 267:8200-8206. Breyer, R.M., Emeson, R.B., Breyer, M.D., Davis, LS., Abromson, R.A., and Ferrenbach, S.M. (1993) Alternative Splicing Generates Multiple Isoforms of a Rabbit Prostaglandin E? Receptor (Submi[ted) Breyer, M.D., Jacobson, H.R., Davis, LS., and Breyer, R.M. (1993) In situ Hybridization and Localization of mRNA for the Rabbit Prostaglandin-E2, EP3 Receptor (Submitted) Abstracls: Breyer, R.M., Guillet, J.-G., and Strosberg, A.S. (1991) Identification of B2 Adrenergic Receptor Mutants with Altered Agonist Binding. JASN 2:449. Breyer, R.M., Ferrenbach, S., Breyer, M.D., and Emeson, R,B. (1992) PCR amplification of a Putative Rabbit Prostaglandin Receptor. JASN 3(3):486. Breyer, M.D., Jacobson, H.R., Noland, T.D., Moffatt, L.S., Fredin, D., Redha, R., and.Breyer, R.M. (1992) Evidence that Separate Prostaglandin E2 (PGE2) Receptors Couple to Cyclic AMP (cAMP) Generation and Intracellular Calcium ([Ca+ +]) in the Rabbit Cortical Collection Duct (CCD). JASN 3(3):452. Breyer, M.D., Superdock, K.R., Emeson, R., Prie D., Ronco P., and Breyer, R.M. (1993) Amplification of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA from the rabblt kidney and its presence in cortical collecting duct (CCD). FASEB (New Orleans, MD). 40008923

CURRENTLY ACTIVE GP.A~NTS, CONTRACTS and OTHER SOURCES of FUNDS List financi~_~ support (direct costs, only) from al._~l sources, in:luding own institution. Title of Project Characterization of Renal Prostaglandin E2 Receptors Renal Disease Research Researeh-Nephrology Sources (give grant numbers) IROI DK 46205- OIAI Gifts N/A Total Value of Grant (direct , costs) $506,905 1,000 Pays balanc iby NIH gran Current Arm~al Amount Available to You $121,136 1,000 e of salary n( t. Dateof Termination of Grant 7131197 613o/94 t covered Identi~ and describe any overlap of this applicat'ion wi~ the above grants: The projects listed above have no scientific overlap with this proposal. Indicate the total' annual funds available to you this year for all research projects under your supervision. $ PENDING OR PLANNED Title of Project Sources f.give grant numbers) Total Value of Grant (direct costs) Identify and describe'any'overlap of this application with the abovi'project. Avg. Annual Amount Available to You Total Duration (give inclusive dates) CONFIDENTIAL 40008924

july 20, 1993 Alternative SpIicing Generates Multiple Isoforms of a Rabbit Prostaglandin E2 Receptor (Aitemati4e splicing/Dinoprostone/Kidney/Receptor) Running Title: Multiple EP3 Receptor Isoforms Richard M. Breyer*~r§, Ronald B. Emeson+, Matthew D. Breyer *:~, Linda S. Davis*, Richard M. Abromson*, & Suzanne M. Ferrenbach* *Division of Nephrology, +Department of Pharmacology ~tVeterans Administration Medical Center, and Vanderbilt University School of Medicine Nashville, Tennessee USA 37232-2372 Telephone: (615) 343-8496 FAX: (615) 343-7156 The nucleotide seqfitences reported in this paper have been submitted to the Genbank/EMBL Data Bank with accession numbers 1111, 2222, 3333 and 4444 § Author to whom correspondence should be addressed 40008925

R.I~[. Breyer ~ a[. ~ SUMI~£ARY Four cDNA clones homologous wihh a murine prostaglandin P-2 receptor have been isolated from a rabbit kidney cortex eDNA library. These cDNAs encode related proteins which d.~ffer only in the,_'r carboxyl- terminal sequences. Southern blot analysis of rabbit genomic DNA has indicated that these receptor cDNAs represent alternatively spliced variants derived from a single gene. Transient expression of a novel full length eDNA in COS1 ceils confirmed the ligand binding profile typical of an EP3 receptor sub['ype. Ribonuclease protection assays have indicated that the gene encoding these receptors is most highly expressed in kidney, adrenal, and stomach, with lower bu.t significant expression in uterus, lung, heart, ileum, spleen and brain. Moreover, each of the cloned isoforms is expressed in the kidney. In situ hybridization analyses of rabbit kidney demonstrated hhat the level of expression is highest in the outer medulla ~vith lesser expression in the cortex and no detectable expression in the inner medulla. The ligand binding profile and tissue distribution of these receptors is consistent with a functional role for this ~amily of EP3 receptbrs in mediadng the renal acdons of prostaglandins as well as the effects of prostaglandins on gastric acid secretion and adrenal function. 40008926

In situ Hybridization and Localization of mRNA for the Rabbit Prostaglandin EP3 Receptor. Matthew D. Breyer, Harry R. Jacobson, Linda S. Davis, and Richard M. Breyer Department of Veterans Affairs Medical Center, Nashville Division of Nephrology and Department of Medicine Vanderbilt University F427-ACRE Bldg. Nashville, TN. 37212 SHORT TITLE: In situ hybridization of EP3 receptor Address correspondence to: Dr. Matthew D. Breyer Division of Nephrology and Department of Medicine Vanderbilt University F427-ACRE Bldg. Department of Veterans Affairs Medical Center Nashville, TN. 37212 pH (615) 343-9867 FAX: (615) 343-7156 40008927