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V .derbilt University Medical Center De~artn~nt cfMedJClne 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, d M. Breyer, Pit. DI,/ Assistant Professor of Medicine and Pharmacology 40008903

PJ.: Richard M, Bre~r, Phi. TI-1E ROLE OF PROSTAGLANDIBI EP3 RECEPTOR IN TUMOR METASTASIS Back~ound...aud ..~ignificance. ~osmglan~n E2 ~GE2) is a potent modulator of a wide v~ety of physiological responses inclu~ng infla~adon, v~cul~ ton~, water and ion ~anspo~ as well as cell ~owth ~d division. Increased PGE2 synthesis has been associated ~th proliferation of tumor cells in human lung c~cer, breast c~cer and colon cancer as well as in rodent model systems (1, 2, 3). In patients with lung cancer, ~ere is an increased PGE2 syn~esis in cancerous versus nodal tissue (4). This increase was more pronounced in lung c~cers of patients who smok~ cig~ettes as comp~ wi~ nonsmokers. In other studies ~ inverse co,elation ~tween elevations in PGE2 synthesis and mean su~ival time has ~en obse~ed for patients wi~ breast c~eer, and to a lesser extent in lung ~d colonic cancers (5). In patien~ with familial pol)9osis, ~e ad~nis~afion of nonsteroidal anti-infl~mato~ d~gs (NSAIDs) which ~e potent inhibitors of PGE2 synthesis have ~en shown t~ cause polyp regression, ~d with~awal of ~e NSA~s reverse this effect (6, 7). ~ese findings suppo~ the notion that PGE2 mediated effects may play an impo~ant role in cell ~ansfo~ation, tumor ~owth an~or metastasis (8). Unlike classical circulating hormones, such as insulin and coaicosteroids, prostaglandins are autacoids, acting locally on the tissues in which they are synthesized or on adjacent tissues. AcCumulating evidence suggests ~at PGE2 execs its cellul~ action ~ough specific receptors coupled to their in~acellul~ effectors via gu~ine nueleot[de regulato~ proteins (G-proteins) (9, 10, 11). Based upon their ph~acologieM lig~d bin~ng prope~es ~d physiologic effects ia smooth muscle (12) at least three classes of PGE2 receptor have been proposed. These three E~rostanoid receptors are designated EP1, EP2 ~d EP3. Itis of interest ~at PGE2 receptors have ~en shown to couple to ~ree separate signMling pa~ways, via G-protein me~ated PI ~P1) turnover ~d cAMP generation via Gs (EP2) and Gi ~P3). In one model of mouse m~a~ tumor metastasis cell lines 66 ~d 4526 have ~en demonsffated to express a high affinity PGE2 receptor (1). Stimularon of ~is receptor leads to an increase in cAMP levels (i.e. it is EP2-1ike). Blockade of ~is receptor wi~ PGE2 selective ~tagonists leads to a decrease in cAMP gene~tion ~d ~ increase in ~e lung mems~sis of ~is tumor in vivo. (1). Our laborato~ has recently clones a family of altema~vely splic~ va6ants of ~e EP3 receptor. These receptors ~e presumably cgupl~ [n vivo to Se Gi pathway. As descfi~ ~low, we ~lieve that each of these receptors will have idemicM lig~d binding prope~ies but different signal ~ansduction eharaeteristles. We will test the ability of each of ~ese r~eptors to modulate cyclic AMP levels in vitro ~d metastasis in vivo. Specific Aims. ~e focus of ~is proposM is to evMuate the role of EP3 receptor subtypes in a mouse model of metastasis. In p~neiple, stimularon of ~e Gi coupl~ EP3 r~eptor should lower cAMP levels in the same manner as EP2 receptor blockade. ~us stimulation of EP3 receptor might ~ expected to increase me,static potential of these tumor cells. ~e Mms of this proposM ~e to : Evaluate the effect of expre~ion of EP3 receptor subtypes 72A, 74A 77A, and 80A in murine mammary tumor lin~ 66 and 4526 on cAMP generation and lung metast~is .... Welimina~ Studies In setOing on a ~get tissue in which to ch~actefize the PGE2 receptor, we have initially focused on the kidney. Not only is PGE2 the major renal cyclooxygenase metabolite of arachidonic acid but ~e ~dney is one of the richest sources of PGE2 receptors. We amplified rabbit eDNA sequences ~y reverse ~anscHpfion PCR (RT-PCR) using poly (A)+ RNA isolated from rabbit renal coaex and oligonucleotide primers derived from the cloned mouse EP3~ receptor (13). Overlapping PCR products representing pu~tive ~nsmembmne regions III through VII were generate, cloned and sequenced. Overall" the deduced amino acid sequence from these fragments is 84% homologous with ~e predicted ~no acid s~uence for the mouse receptor. Using these PC~ ~agments as probes, nonhero hybridization ~alysis revealed multiple ~anscfipts of 1.8, 2.6 ~d 7 kb expressed in both renal codex ~d medulla. Moreover ~e levels of expression of ~ese ~anscfpts were modulat~ by in vivo pre~ea~ent of animals with indome~acin, suggesting feedback modulation of receptor message levels by depletion of the endogenous lig~d. 40008904

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-terminal tail have been implicated in coupling specificity and regulation. P.L: Riehard M. tlreber, PhJ). VCe prob~:l a rabbit renal cortex eDNA library with PCt~ fragments of the rabbit EP receptor and isolated five independent clones. The deduced amino acid sequence of one clone, 72A, is shown in Fig. I. This clone contains a cDNA of 2051 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 transfeeted 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 region corresponds to amino acid 355, ten amino 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 ne .arl.y throughout the seven transmembrane domain region, and diverge at the C-terminal tail. This region is maplicated 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 an.d 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 Northern Blot using a rabbit EP3 common region probe. This probe has previously been shown 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 ~vill 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 ~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 ,.viii 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 L 40008905

1600 1800 80A Fig I..pperpanel The alignment of four EP receptor clones representing the four classes of sequence. 72A appears to be a full length eDNA, 77A has the entire coding region, nod clones 74A and g0A are fragments encoding only a portion of Ihe corresponding receptor, lo~erpanel The deduced amino acid sequence of the divergent portion ot the four EP3 receptor subtype~ PJ. : Richard I~L Bre}'er, Ph.D. Differential regulation of "these receptor subtypes may of potential functional 2000 significance in determining the response of a given cell type of tissue to PGE2. One receptor clone, 72A, has the C-terminal scquence RKILLRKFCQEEFWEK . 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 serine or thrconine residues. Both serine and threonine arc 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 pathophysiologic 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) Cancerges. 51, 2047-2050 2. Thun, NI. J., Namboodiri, B. S., and Heath Jr., C. W. (1991) N. Engl. J. Med. 325, 1593-1596 3. Noguchi, M,, Taniya, T., Koyasaki, N., Kumaki, T., Miyazaki, I., and Mizukami, Y. (1991) CancerRes. 5L 2683-2689 4. McImmore, T. L., Hubbard, W. C., Litterst, C. L., Liu. M. C., Miller, S., McMahon, N. A., Eggleston, J. C., and Boyd. M. R. (1988) CancerRes. 48, 3140-3147 5. Bennett, 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, J., Piqu6, J. M., Rubio, E., Planas, R., Tarrech, J. M., and Bordas, J. M. (1991) Ann. Intern. Med. I15, 952-954 8. Honn, K. V., Bockman, R. S., and Mamett, L. L (1981) Prostaglandins 21~ 833-865 9. Haga, T., Ross, E. M., Anderson, H. J., and Gilman, A. (3. (1977) Proc. Natl. Acad. ScL 74, 2016-2020 10. Le[kowitz, R. J., Ululliken, D., Wood, C. L,, (3ore, T. B., and Mukherjee. C. (1977) J. Biol, Chem. 252, 5295-5303 11. Walanabe, T., Umegaki, K., and Smith, W. L. (1986) J'. Biol. Chem. 261, 13430-13439 12. Gardiner, P. J, (1990) Adv. in Prostaglandin, Thromboxane, and LeukoMene Res. 20, 110-118 13. Sugimoto, Y., Namba, T.,Negishi, M.. Ichikawa, A., and Nammiya, S. (1992) J. Biol. Chem. 267, 6463-6466 14. Bouvier, M.. Hausdorf, W. P., 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 Lefkowitz, R. J. (1990) FASEB J. 4, 2881-2889 3 40008908

SKETCH INSTITUTION A.t',[D LOCAT[ON DEGREE YEAR FIELD OF STUDY University of Michigan, Ann Arbor, Michigan Massachusetts Institute of Technology, Cambridge, Massachusetts Massachusetts Institute of Technology B.S. 1978 Microbiology S.M. 1982 Biology Ph.D. 1988 Biochemistry RESEARCH AND PROFESSIONAL EXPERIENCE. ConcIuding with present position, list, in chronological order, previous emptoymant, experience and honors. Key personnel include the principal investigator and any other ~ndividuals who participate in the ~clenfific development or execution of the project. Key personnel typically wlil include all individuals with doctoral or other professional degrees, but in some projects will include indivldu,~fs 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. Ust, in chronological order, the titles, all authors, and complete references to all publications during the past three years and ~o representative earlier publlcations pertinent to ~his ~pp|ication. DO NOT EXCEED I~VO PAGES. Professional Experience: 1982-1984 1988-1990 1990-1991 1991-Present 1991-Present Research Assistant, Repligen Corporation, Cambridge, Massachusetts Postdoctoral Fellow, Biologie Mol~culalre des RBcepteurs, Institut Pastuer, Pads, France Postdoctoral Fellow, Laboratoire d'lmmuno-Pharmacologie Mol~culaire, Institute Cochin de Gdnetique Mol~culaire, Institute Cochin de G~netique Mol~culaire, Pads, France Assistant Professor of Medicine, Division of Nephrology, Vanderbilt University, Nashville, Tennessee Assistant Professor of Pharmacology, Vanderbilt University, Nashville, Tennessee H.__onors 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 Fellowship Foundation de la Recherche Medicale, Postdoctoral Fellowship Association pour la Recherche sur le Cancer, Postdoctoral Fellowship Agence Nationale sur le SIDA, Postdoctoral Fellowship National Kidnoy Foundation Young Investigator Award Publications: Reilly, E.B., Reiliy, R.M., Brever, R.M., Sauer, R.T., and Eisen, H.N. (1g~4) Amino Acid and Nucleotide Sequences of Variable Regions o~ Mouse Immunoglobulin Light Chains of the ~. 3 Subtype. J. Immunol., 133:471-475. Colbert, D., Anilionis, A., Gelep, P., Farley, J., and Breyer, R. (1984) Molecular Organization of the Protein A Gene and its Expression in Recombinant Host Organisms. J. Biol. Resp. ModE., 3:255-259. Breyeq R.M. and Sauer, R.T. (1989) Production and Characterization of Monoclonal Antibodies to the N-terminal Domain of ~ Repressor. J. Biol. Chem., 264:13348-13354. Breyer, R.M. and Sauer, R.T. (1989) Mutational Analysis of ~he Fine Speci(icity of Binding of Monoclonal Antibody 51F to ;k Repressor. J. Biol. Chem., 264:13355-13360. 5. Brever. R.M., 8trosberg, A.D., and Guillet, J.-G. (1990) Mutational Analysis of Ugand Binding Activity of I}2 Adrenergic FHS 3~3 (R-=~,. 9/~) F~g~ __ 40008907

FF 6o Rec.eptur Express~g in Escher~ch~a coE. E,'/,BO J., 9:~.679-26B4. Re;dhaar-OIson, J.F., Bowie, J.U., Br.ey~r, R.M., HU, J.C., Kn.~ght, K.L, Lira, W.A., Mossing, M.C.~ ParseIl, D.A.. Shaemaker, K.R., and Sauer, R.T. (1991) Random Mutagenesls of Protein Sequences Using Oligonu¢leotide Cassettes Methods Enz3,mo1208:565-585. Bertin, B., Freissmuth, M., B~rever. R.M., Strosi~erg, A.D., and MaruIlo, S. Functional expression of the 5HTla receptor in Escherichia coil: Lig~,nd binding properties and interaction with recombinant G-proteins (1992) J. Biol. Chem. 267:8200-8206. 8o 9o Breyer, R.M., Emeson, R.B., Breyer, M.D., Davis, L.S., Abromson, R.A., and Ferrenbach, S.M. (1993) Alternative Splicing Generates Multiple Isoforms of a Rabbit Prostaglandin E~ Receptor (Submitted) Breyer, M.D., J.acobson, H.R., Davis, L£., and Breyer, R.M. (1993) In situ Hybridization and Localization of mRNA for the Rabbit Prostaglandin-E2, EP3 Receptor (Submitted) Abstracts: ,BreYer, .R.M., Guillet, J.oG., and Strosberg, A.$. (lg91) 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. 3o Breyer, M.D., Jacobson, H.R., Noland, T.D., Moffatt, L.S., Fredin, D., Redha, R., and Breyer, R.M. (1992) Evidence that Separate Prostagtandin 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 Brever. R.M. (1993) Amplification of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA from the rabbit kidney and its presence in cortical collecting duct (CCD). FASEB (New Orleans, MD). 40008908

CUP.R, ENTLY ACTIVE GRA~,'rS, CONq-RACTS and OTHER SOURCES of FUNDS L~ f~r~r,:%~ support (direct costs, only) from all sources, including own institution. Title of Project (give grant numbers) Characterization of Renal Prostaglandln E2 Receptors Renal D~.sease Research Research-Nephrology IR01DK 46205- OIAI Gifts Total Value of Grant (direct . costs) ..... $506,905 1,000 IPays balanc Iby NIH gran Current Annual Amount Available to You $121,136 1,000 e of salary nc t. Date of Termination of Grant 7/31/97 6/30194 t covered Ide~tif9 and describe an~ overlap of this application with the above grants:" " The projects listed above have no scientific overlap with this proposal. Indicate'the lotal annual run'Is available to you this year f~r all research projects under your supervision. $ PENDING OR PLANNED Ti'tle of Project .... Sources T0'tal V'alue (give grant numbers) of Grant (direct costs) Avg. Annual Amount Available to You Total Duration (give inclusive dates) Id;ntify and des~;ibe any overlapof this application with the above project. CONFIDENTIAL 40008909