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.
Page 1: 40008903
V .derbilt 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.
d M. Breyer, Pit. DI,/
Assistant Professor of Medicine and Pharmacology
Page 2: 40008904
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
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
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
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
~d metastasis in vivo.
Specific Aims. ~e focus of ~is proposM is to evMuate the role of EP3 receptor subtypes in a
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
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.
Page 3: 40008905
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
the receptors will be identical, or very ne .arl.y throughout the seven transmembrane domain region,
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
to cause differential regulation by the regulatory kinases. Functionally this would imply that the
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
confirm the absence of EP3 receptor message in cell lines 66 and 4526 by Northern Blot using a
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).
of these ligands have a high affinity for the EP3 receptor (see accompanying manuscript). Cells will
pretreated with these compounds at saturating concentrations (--0.1 ~tM) and assayed for their
stimulate cAMP formation,~ or inhibit forskolin stimulated cAMP generation. Increased metastasis
be evaluated as described: after pretreatment, cells will be injected into Balb/c mice in PBS i. v.
tail vein. Three weeks later, mice will be sacrificed and lung tissue will be examined for the
Cell lines will be transfected with each of the full length receptor constructs, 72A, 77A, 80A
individually. Expression plasmid constructs encoding clones 77A and 72A constructs already exist in
laboratory and we are currently engaged in constructing full length 80A and 74A. Cell lines ,.viii
assayed for their ability to bind EP3 selective ligands, inhibit cAMP generation and the presence of
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.
Page 4: 40008906
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.
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
. 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
phosphorylation by a
number of protein kinases including receptor-specific ldnases (14, 15). It might be predicted that
in the relative fraction of the 72A receptor subtype will change the susceptibility of PGE2
regulation by protein kinases. Thus far, studies on the effects of prostanoids and cancer have
the generation of cyclooxygenase metabolites of arachidonic acid, and their inhibition by NSAIDs.
the cloning of individual PGE2 receptors it is now possible to determine the effects of PGE2 at
receptor subtypes, and to characterize the relative abundance of the different receptor isoforms
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
the role PGE2 plays in tumor promotion, growth and metastasis.
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,
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,
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
Page 5: 40008907
INSTITUTION A.t',[D LOCAT[ON
DEGREE YEAR FIELD OF STUDY
University of Michigan, Ann Arbor, Michigan
Massachusetts Institute of Technology,
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.
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:
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
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
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~ __
Page 6: 40008908
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.~
Shaemaker, K.R., and Sauer, R.T. (1991) Random Mutagenesls of Protein Sequences Using
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.
Breyer, R.M., Emeson, R.B., Breyer, M.D., Davis, L.S., Abromson, R.A., and Ferrenbach, S.M. (1993)
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)
,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
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 Prostagtandin E2 (PGE2) Receptors Couple to Cyclic AMP (cAMP) Generation and Intracellular
([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
(CCD). FASEB (New Orleans, MD).
Page 7: 40008909
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
Characterization of Renal
Prostaglandln E2 Receptors
Renal D~.sease Research
. costs) .....
Iby NIH gran
e of salary nc
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
Id;ntify and des~;ibe any overlapof this application with the above project.