METHODS FOR SCREENING FOR SUBSTANCES WHICH INHIBIT FP PROSTANOID RECEPTOR INTERACTION WITH A COMPOUND HAVING PGF2.ALPHA.ALPHAACTIVITY AND METHODS OF TREATING CANCER

PROCEDES DE CRIBLAGE POUR DES SUBSTANCES QUI INHIBENT L'INTERACTION D'UN RECEPTEUR DE PROSTANOIDE FP AVEC UN COMPOSE A ALPHA-ACTIVITE PGF2.ALPHA. ET METHODES DE TRAITEMENT DU CANCER

English Abstract

The present invention provides mehtods for screening for substances which
inhibit the interaction between a FP prostanoid receptor and a compoudn having
PGF2.alpha.alphaactivity, methods for inhibiting the interaction, methods of
inhibiting signaling mediated by .beta.beta-catenin, and methods of treating
cancer.

French Abstract

La présente invention concerne des procédés de criblage pour des substances qui inhibent l'interaction entre un récepteur de prostanoïde FP et un composé présentant une activité PGF¿2.alpha.?, des procédés d'inhibition de cette interaction, des procédés d'inhibition de la signalisation à médiation de .beta.-caténine, ainsi que des méthodes de traitement du cancer.

Claims

Claims:
1. A method of screening for substances which inhibit the interaction between
a FP prostanoid
receptors and a compound having PGF2.alpha. activity comprising
contacting a cell expressing the FP prostanoid receptors with the substance to
be screened;
contacting said cell with said PGF2.alpha. compound; and
assaying the presence or absence of interaction between FP prostanoid
receptors and PGF2.alpha.
compound, wherein the absence of an interaction between FP prostanoid
receptors and PGF2.alpha.
compound indicates the substance inhibits the interaction.
2. The method of Claim 1, wherein said assaying comprises analyzing the
morphology of the
cell; wherein said morphology is selected from the group consisting of cell
rounding, loss of
filopodia, and formation of cell aggregates; wherein an absence of a change in
cell
morphology compared to a cell not contacted with PGF2.alpha. compound
indicates inhibition of
the interaction.
3. The method of Claim 1, wherein the assaying comprises measuring apoptosis
in said cell.
4. The method of Claim 1, wherein said cell endogenously expresses the FP
prostanoid receptor.
5. The method of Claim 1, wherein said assaying comprises measuring the
transcription activity
of a Tcf/Lef responsive promoter.
6. The method of Claim 5, wherein said assaying comprises a detection method
selected from
the group consisting of RT-PCR, Northern blot, luciferase reporter gene,
.beta.-gal reporter gene,
and other reporters.
7. The method of Claim 1, wherein said assaying comprises measuring the level
of
phosphorylation of .beta.-catenin in the cell, wherein an increased level of
phosphorylation
compared to the .beta.-catenin in a cell not contacted with said substance
indicates the inhibition
of the interaction.
19

8. The method of Claim 1, wherein said substance is an antibody.
9. The method of Claim 8, wherein said antibody binds to the FP prostanoid
receptor.
10. The method of Claim 8, wherein said antibody binds to PGF2.alpha.
compound.
11. The method of Claim 1, wherein said PGF2.alpha. compound is PGF2.alpha..
12. The method of Claim 1, wherein said assaying comprises measuring changes
in at least one
member selected from the group consisting of inositol phosphate stimulation,
activation of
Rho, stress fiber formation and phosphorylation of P125.
13. The method of Claim 1, wherein said FP prostanoid receptor is FP B.
14. A method of screening for substances which inhibit the interaction between
a FP prostanoid
receptor and a compound having PGF2° activity comprising
introducing and expressing a polynucleotide which encodes the FP prostanoid
receptor;
contacting said cell expressing the FP prostanoid receptor with the substance
to be screened;
contacting said cell with said PGF2.alpha. compound; and
assaying the presence or absence of interaction between FP prostanoid receptor
and PGF2.alpha.
compound, wherein the absence of an interaction between FP prostanoid receptor
and PGF2.alpha.
indicates the substance inhibits the interaction.
15. A method of inhibiting the interaction between FP prostanoid receptor and
a compound
having PGF2.alpha. activity comprising contacting said FP A with a substance
which is capable of
inhibiting said interaction.
16. A method of inhibiting .beta.-catenin signaling comprising contacting a
cell expressing FP
prostanoid receptor with a substance which is capable of inhibiting the
interaction between FP
prostanoid receptor and a compound having PGF2.alpha. activity.

17. A method of inhibiting G12 and G13 mediated signaling comprising
contacting a cell
expressing FP prostanoid receptor with a substance which is capable of
inhibiting the
interaction between FP prostanoid receptor and a compound having PGF2.alpha.
activity.
18. A method of treating cancer comprising administering to a patient in need
thereof a substance
which inhibits the interaction between FP prostanoid receptor and a compound
having PGF2.alpha.
activity in an amount sufficient to inhibit said interaction.
19. The method of Claim 18, wherein said cancer is colorectal cancer.
20. A method of screening for substances which inhibit .beta.-catenin
signaling comprising
contacting a cell expressing FP prostanoid receptor with the substance to be
screened;
contacting said cell with a compound having PGF2.alpha. activity ; and
assaying the signaling activity, phosphorylation and/or the subcellular
localization of the .beta.-
catenin; wherein a change in one or more of the signaling activity,
phosphorylation and/or the
subcellular localization is lower than the signaling activity, phosphorylation
and/or the
subcellular localization compared to a cell not contacted with the substance
indicates the
substance inhibits the interaction .beta.-catenin signaling.
21. A method of screening for a substance for their ability to inhibit cancer
cell growth
comprising
contacting a cell expressing FP prostanoid receptor with the substance to be
screened;
contacting said cell with a compound having PGF2.alpha. activity ; and
assaying the change in cell growth, wherein a decrease in cell growth is
indicates an
inhibition of cancer cell growth.
22. The method of Claim 21, wherein said assaying comprises analyzing the
morphology of the
cell; wherein said morphology is selected from the group consisting of cell
rounding, loss of
filopodia, and formation of cell aggregates; wherein an absence of a change in
cell morphology
21

compared to a cell not contacted with said PGF2.alpha. compound indicates
inhibition of the
interaction.
22. The method of Claim 21, wherein the assaying comprises measuring apoptosis
in said cell.
23. The method of Claim 21, wherein said cell endogenously expresses the FP
prostanoid
receptor.
24. The method of Claim 21, wherein said assaying comprises measuring the
transcription
activity of a Tcf/Lef responsive promoter.
25. The method of Claim 24, wherein said assaying comprises a detection method
selected from
the group consisting of RT-PCR, Northern blot, luciferase reporter gene,
.beta.-gal reporter gene,
and other reporters.
26. The method of Claim 21, wherein said assaying comprises measuring the
level of
phosphorylation of .beta.-catenin in the cell, wherein an increased level of
phosphorylation
compared to the .beta.-catenin in a cell not contacted with said substance
indicates the inhibition
of the interaction.
27. The method of Claim 21, wherein said substance is an antibody.
28. The method of Claim 27, wherein said antibody binds to the FP prostanoid
receptor.
29. The method of Claim 27, wherein said antibody binds to PGF2.alpha.
compound.
30. The method of Claim 21, wherein said PGF2.alpha. compound is PGF2.alpha..
31. The method of Claim 21, wherein said assaying comprises measuring changes
in at least one
member selected from the group consisting of inositol phosphate stimulation,
activation of
Rho, stress fiber formation and phosphorylation of P125.
32. The method of Claim 21, wherein said FP prostanoid receptor is FP B.
33. The method of Claim 21, wherein said inhibiting cancer cell growth
comprises treating
cancer.
22

34. The method of Claim 21, wherein said method is performed in vitro or in
vivo.
23

References
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26

Description

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Title of the Invention
METHODS FOR SCREENING FOR SUBSTANCES WHICH INHIBIT FP PROSTANOID
RECEPTOR INTERACTION WITH A COMPOUND HAVING PGFZa ACTIVITY AND
METHODS OF TREATING CANCER
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention provides methods for screening for substances which
inhibit the
interaction between FP prostanoid receptors and a compound having PGFza
activity, methods for
inhibiting the interaction, methods of inhibiting signaling mediated by (3-
catenin, and methods of
treating cancer.
Discussion of the Background
The primary amino acid sequences of the ovine FPA and FPB prostanoid receptor
isoforms
are the same throughout their amino termini and seven membrane spanning
domains, but the FPB
isoform is truncated and lacks the last 46 carboxyl terminal amino acids
present in the FPA
isoform (1). This is very similar to the EP3 (2) and thromboxane A2 (3)
prostanoid receptors in
which alternative mRNA splicing gives rise to a variety of isoforms in humans
and in other
species (4). The physiological significance of these receptor isoforms is not
clear, although
differences have been shovv~m to exist with respect to second messenger
coupling and receptor
desensitization. The inventor has discovered that the FPA and FPB receptor
isoforms have similar
pharmacological properties and that prostaglandin FZa (PGFZa)' stimulates
phosphoinositide
turnover to a similar extent in cells expressing these isoforms (1). In
addition, stimulation of FPA
or FPB expressing cells with PGFZa activates Rho leading to the formation of
actin stress fibers,
phosphorylation of p125 focal adhesion kinase and cell rounding (5). Cell
rounding involves the
retraction of filopodia and a change from an isolated dendritic appearance to
one in which the
cells are rounded and form small cobblestone-like aggregates (see Figure 1A).
Following the

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removal of PGFza, however, FPA expressing cells return to their original
dendritic morphology,
but the FPB expressing cells do not and remain rounded (6). Here we show that
Tcf/(3-catenin
mediated transcriptional activation is elevated 16 hours after an initial 1
hour treatment of FPB
expressing cells with PGFZa. This is not observed in FPA expressing cells and
suggests that FPB
expressing cells remain rounded because of persistent activation of a Tcf/(3-
catenin signaling
pathway.
Previous studies by others have established that stimulation of this pathway
is strongly
associated with the development of colorectal and other cancers. It is also
well known that the
inhibition of cyclooxygenase enzymes by nonsteroidal anti-inflammatory drugs
(NSAIDs) is
beneficial for the treatment and prevention of colorectal cancer. This
beneficial effect is assumed
to result from the decreased biosynthesis of prostanoid metabolites, however,
since dozens of
metabolites are affected, the specific mechanism whereby this decrease
produces a benefit is
unknown. Our data are the first to provide an unambiguous receptor-based
mechanism whereby a
decrease in a specific prostanoid metabolite, PGFZa, could account for the
beneficial effects of
NSAIDs in the prevention and treatment of colorectal cancer. In terms of this
receptor-based
mechanism, it can be predicted with virtual certainty that a FP prostanoid
receptor antagonist
would have the same functional consequence as selectively decreasing PGFZa
Thus, there is a
reasonable expectation that FP prostanoid receptor antagonists would be
effective as drugs in the
treatment and prevention of colorectal cancer and possibly other cancers as
well. It is also clearly
evident that the use of recombinant FP prostanoid receptors in functional
screens would be an
effective means of discovering existing and novel substances that could be
used as such drugs.
The present technology based on the use of NSAIDs is nonspecific because
NSAIDs block the
key enzymes (cyclooxygenases) required for the biosynthesis of all prostanoid
metabolites.
Because so many metabolites are affected, it is actually very uncertain as to
how the NSAIDs are
producing their beneficial actions. In addition, the use of NSAIDs is
associated with a number of
adverse effects related to their widespread effects on prostanoid metabolite
biosynthesis.
Presently, as it concerns the treatment and prevention of cancer, there is no
existing technology
based on the pharmacological blockade of a specific prostanoid receptor
subtype or isoform.
Furthermore, there are no existing data that we are aware of that would even
support such an
approach if it were contemplated. Our disclosure is novel because it clearly
establishes the

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feasibility of using prostanoid receptor antagonists for the treatment and
prevention of cancer. It
would be expected that such prostanoid receptor antagonists would have the
potential to be more
efficacious with fewer adverse side effects. In addition, the use of
recombinant FP receptors for
the discovery of potential anti-cancer drugs is unprecedented because until
now there was no
obvious reason to expect that FP receptors might be involved with the
pathophysiology of cancer.
The use of recombinant FP receptors for such a purpose would have the
significant advantages
because the present technology for the discovery of potential anti-colorectal
cancer drugs are
highly nonspecific and do not take into account this receptor=based mechanism
for the treatment
of this disease.
SUMMARY OF THE INVENTION
One object of the present invention is a method of screening for substances
which inhibit
the interaction between a FP prostanoid receptors and a compound having PGFza
activity
including contacting a cell expressing the FP prostanoid receptors with the
substance to be
screened, contacting the cell with said PGFZa compound, and assaying the
presence or absence of
interaction between FP prostanoid receptors and said PGFZa compound, wherein
the absence of
an interaction between FP prostanoid receptors and said PGFZa compound
indicates the substance
inhibits the interaction.
In a preferred embodiment the FP prostanoid receptor is FPB. and said PGF2a
compound is
PGFza.
Another object of the present invention is assaying with a detection method
selected from RT-
PCR, Northern blot, luciferase reporter gene, [3-gal reporter gene, and other
reporters.
Another object of the present invention is where the inhibiting substance is
an antibody. Such
an antibody can bind to FP prostanoid receptors or said PGFZa compound.
Another object of the present invention is a method of screening for
substances which inhibit
the interaction between a FP prostanoid receptors and a compound having PGFZa
activity by
introducing and expressing a polynucleotide which encodes the FP prostanoid
receptors;

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contacting the cell expressing the FP prostanoid receptors with the substance
to be screened;
contacting said cell with said PGF2« compound; and assaying the presence or
absence of
interaction between FP prostanoid receptors and said PGFZ« compound, wherein
the absence of
an interaction between FP prostanoid receptors and said PGF2« compound
indicates the substance
inhibits the interaction.
Another object of the present invention is a method of inhibiting the
interaction between FP
prostanoid receptors and a compound having PGFZ« activity ound by contacting
said FP
prostanoid receptors with a substance which is capable of inhibiting the
interaction.
Another object of the present invention is a method of inhibiting (3-catenin
signaling by
contacting a cell expressing FP prostanoid receptors with a substance which is
capable of
inhibiting the interaction between FP prostanoid receptors and a compound
having PGFZ«
activity.
Another object of the present invention is a method of inhibiting G12 and G13
mediated
signaling by contacting a cell expressing FP prostanoid receptors with a
substance which is
capable of inhibiting the interaction between FP prostanoid receptors and a
compound having
PGFZ« activity.
Another object of the present invention is a method of treating cancer by
administering to a
patient a substance which inhibits the interaction between FP prostanoid
receptors and a
compound having PGFz« activity in an amount sufficient to inhibit the
interaction.
Another object of the present invention is a method of screening for
substances which inhibit
[3-catenin signaling by contacting a cell expressing FP prostanoid receptors
with the substance to
be screened; contacting said cell with a compound having PGFz« activity;
assaying the signaling
activity, phosphorylation and/or the subcellular localization of the (3-
catenin; wherein a change in
one or more of these properties indicates the substance inhibits the
interaction ~i-catenin
signaling.
Another object of the present invention is method of screening for a substance
for their ability
to inhibit cancer cell growth by contacting a cell expressing FP prostanoid
receptors with the

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substance to be screened; contacting the cell with PGFza; assaying the change
in cell growth,
wherein a decrease in cell growth is indicative of the substance usefulness
for the treatment of
cancer. In one embodiment the inhibition of cancer cell growth includes
screening for substances
which are useful for treating cancer.
A more complete appreciation of the invention and many of the attendant
advantages thereof
will be readily obtained as the same becomes better understood by reference to
the following
detailed description when considered in connection with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. A, phase contrast microscopy (x225) of FPA and FPB expressing cells
after treatment
with either vehicle (panels a and c) or 1 ~,M PGFZa (panels b and d) for 1
hour at 37°C. B, (3-
catenin FITC immunofluorescence (green) and nuclear DAPI fluorescence (blue)
microscopy
(x225) of FPA and FPB cells after the same treatment. Cells were labeled and
prepared for
microscopy as described in Experimental Procedures. The results are
representative of more than
three experiments.
Figure 2. A, Immunoblot of [3-catenin in particulate and cytosolic fractions
prepared from FPA
and FPB expressing cells after treatment with either vehicle (lanes a and c)
or 1 ~,M PGFZa (lanes
b and d) for 1 hour at 37°C. B, RT-PCR of (3-catenin and control
glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA from FPA and FPB expressing cells after the same
treatment.
Immunoblotting (10 ~,g of protein per sample) and RT-PCR were done as
described in
Experimental Procedures. Results for both the immunoblotting and RT-PCR are
representative of
three independent experiments.
Figure 3. Immunoblot (IB) of j3-catenin ((3-cat) in cytosolic fractions and
nuclear extracts; and
immunoblot of serine/threonine phosphorylated (PS/PT) cytosolic (3-catenin,
from FPA and FPB
expressing cells after treatment with either vehicle (lanes a and c) or 1 ~,M
PGFZa (lanes b and d)
for 1 hour at 37°C. Cytosolic fractions were prepared as described in
Experimental Procedures
and samples (100 ~.g protein) were immunoprecipitated (IP) with antibodies to
(3-catenin and

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were first probed with antibodies to phosphoserine and phosphothreonine (upper
panel); and then
were stripped and reprobed with antibodies to (3-catenin (middle panel).
Immunoblotting of
nuclear extracts (lower panel) was done with 10 ~,g protein per sample without
prior
immunoprecipitation. Results are representative of three independent
experiments.
Figure 4. A, phase contrast microscopy (x225) and B, stimulation of Tcf/Lef
responsive
luciferase reporter gene activity after FPA and FPB expressing cells were
treated with either
vehicle or 1 ~,M PGFZa for 1 hour and were washed extensively drug-free media
and incubated
for an additional 16 hours at 37 °C in drug-free media. The
transfection conditions, drug washout,
and luciferase assay are provided in Experimental Procedures. Luciferase data
are normalized to
the vehicle treated FP,, cells and are the means +/- the standard errors of
three independent
experiments each performed in duplicate.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art of
molecular biology.
Although methods and materials similar or equivalent to those described herein
can be used in the
practice or testing of the present invention, suitable methods and materials
axe described herein.
All publications, patent applications, patents, and other references mentioned
herein are
incorporated by reference in their entirety. In case of conflict, the present
specification, including
definitions, will control. In addition, the materials, methods, and examples
are illustrative only
and are not intended to be limiting.
Reference is made to standard textbooks of molecular biology that contain
definitions and
methods and means for carrying out basic techniques, encompassed by the
present invention. See,
for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor
Laboratory, New York (1982) and Sambrook et al., Molecular Cloning: A
Laboratory Manual,
Cold Spring Harbor Laboratory, New York (1989) and the various references
cited therein.
As used herein PGFza is understood to mean prostaglandin FZa, analogues of
PGFza, and
substances which mimic the action of PGFZa at the FP prostanoid receptor; GPCR
is understood
to mean G-protein coupled receptor; Tcf is understood to mean T-cell factor;
Lef is understood to

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mean lymphoid enhancer factor; FITC is understood to mean fluorescein
isothiocyanate; DAPI is
understood to mean 4',6-diamidino-2-phenylindole; RT is understood to mean
reverse
transcription; PCR is understood to mean polymerase chain reaction; GSK-3(3 is
understood to
mean glycogen synthase kinase-3~i; and APC is understood to mean adenomatous
polyposis coli.
This invention provides a method for identifying substances potentially useful
for the
treatment and prevention of pre-cancerous and cancerous lesions in mammals.
In performing the present method use of cells either endogenously or
exogenously
expressing the FP prostanoid receptors can be used. In the case where the cell
does not
endogenously express the FP prostanoid receptors a suitable vector carrying
the gene which
encodes the FPA receptor can be introduced into the cell by procedure known in
the art. The
vector should be suitably constructed so as to facilitate expression of the FP
prostanoid receptor
gene upon introduction. The gene may be maintained episomally or may be
integrated into the
cellular chromosomes using methods known in the art. The FP prostanoid
receptor gene which
can be used in accordance with the present methods are those which are
isolated from mammalian
species, particularly, mouse, rat, human, sheep, cow and the like.
The methods of screening substances can be performed i~ vitro or in vivo.
Types of assays which are embodied within the present invention include
analyzing the
morphology of the cell; wherein said morphology is selected from the group
consisting of cell
rounding, loss of filopodia, and formation of cell aggregates; wherein an
absence of a change in
cell morphology compared to a cell not contacted with PGFZa indicates
inhibition of the
interaction; measuring apoptosis in said cell; assaying comprises measuring
the transcription
activity of a Tcf/Lef responsive promoter; or measuring the level of
phosphorylation of (3-catenin
in the cell, wherein a increased level of phosphorylation compared to the (3-
catenin in a cell not
contacted with said substance indicates the inhibition of the interaction.
Additional detection
methods for determining whether a substance successfully inhibits FP
prostanoid receptors and
PGFZa embodied within the present invention include inositol phosphate
stimulation, activation of
Rho, stress fiber formation, and phosphorylation of P125 (see reference 6).
In one embodiment of the present method the cells are transfected with a
reporter
construct as are known in the art. Such reporter constructs are preferably
sensitive to changes in
-catenin signaling efficacy;~typically by including a responsive promoter,
e.g., a Tcf/Lef

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promoter. Levels of activity from the reporter construct can be determined by
measuring changes
in transcript levels, e.g, using Northern blots, dot-blots, primer extensions,
RNase protections,
RT-PCR and the like. Alternatively, the responsive promoter is functionally
linked to a reporter
gene whereby the levels of activity are measured by assaying changes in
enzymatic, fluorescence
or colorimetric activity of the reporter gene. Such reporter genes are known
in the art and some
examples include (3-galactosidase, luciferase, green fluorescence protein and
the like. These and
other methods, genes, and vectors are described in, for example, Maniatis et
al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982)
and
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New
York (1989) and the various references cited therein.
In one embodiment of the present invention the inhibition of the interaction
between FP
prostanoid receptors and PGFza also inhibits signaling mediated by the G-
proteins G12 and G13.
This invention relates to a novel method for screening test substances for
their ability to
treat and prevent neoplasia, especially pre-cancerous lesions, safely. In
particular, the present
invention provides a method for identifying test substances that can be used
to treat and prevent
neoplasia, including precancerous lesions, with minimal side effects
associated with inhibition
and other non-specific interactions.
The method of this invention is useful to identify substances that can be used
to treat or
prevent neoplasms, and which are not characterized by the serious side effects
of conventional
NSAIDs.
Cancer and precancer may be thought of as diseases that involve unregulated
cell growth.
Cell growth involves a number of different factors. One factor is how rapidly
cells proliferate,
and another involves how rapidly cells die. Cells can die either by necrosis
or apoptosis
depending on the type of environmental stimuli. Cell differentiation is yet
another factor that
influences tumor growth kinetics. Resolving which of the many aspects of cell
growth is affected
by a test substance is important to the discovery of a relevant target for
pharmaceutical therapy.
Screening assays based on this selectivity can be combined with tests to
determine which
substances having growth inhibiting activity.
"Precancerous lesion" includes syndromes represented by abnomnal neoplastic,
including
dysplastic, changes of tissue. Examples include dysplastic growths in colonic,
breast, prostate or

CA 02435510 2003-07-21
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lung tissues, or conditions such as dysplastic nevus syndrome, a precursor to
malignant
melanoma of the skin. Examples also include, in addition to dysplastic nevus
syndromes,
polyposis syndromes, colonic polyps, precancerous lesions of the cervix (i.e.,
cervical dysplasia),
esophagus, lung, prostatic dysplasia, prostatic intraneoplasia, breast and/or
skin and related
conditions (e.g., actinic keraosis), whether the lesions are clinically
identifiable or not.
"Carcinoma" or "cancer" refers to lesions which are cancerous. Examples
include
malignant melanomas, breast cancer, prostate cancer and colon cancer. As used
herein, the terms
"neoplasia" and "neoplasms" refer to both cancerous and pre-cancerous lesions.
In an alternate embodiment, the screening method of the present invention
involves
further determining whether the substance reduces the growth of tumor cells.
Various cell lines
can be used in the sample depending on the tissue to be tested. For example,
these cell lines
include: colonic adenocaxcinoma; lung adenocarcinoma carcinoma; breast
adenocaxcinoma;
melanoma line; keratinocytes; prostrate carcinoma and other cancer model cell
lines commonly
used in the axt. Cytotoxicity data obtained using these cell lines are
indicative of an inhibitory
effect on neoplastic lesions. These and other cell lines are well
characterized, and are used
commonly used in the art for screening for new anti-cancer drugs.
One embodiment of the present method of screening for substances which is
useful for
selecting substances for the treatment of cancer include the tumor progression
model. This model
includes the induction of cells into a cancerous state by applying TPA. The
subsequent or
concurrent administration of the tested substance and reduction in tumor
progression would be
indicative of the successful inhibition of the interaction between the FP
prostanoid receptor and
PGFZ«.
Significant tumor cell growth inhibition greater than about 50% at a dose of
100 ~,M or
below is further indicative that the substance is useful for treating
neoplastic lesions. Preferably,
an ICS° value is determined and used for comparative purposes. This
value is equivalent to the
concentration of drug needed to inhibit tumor cell growth by 50% relative to
the control.
Preferably, the ICS° value should be less than 100 ~.M for the
substance to be considered further
for potential use for treating neoplastic lesions.

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One measure of successful inhibition is to assay the presence or absence of
apoptosis in
the cell carrying the FP prostanoid receptor. Methods of detecting apoptosis
include the TUNEL
assay and ELISA assay. These and other methods are disclosed in Tomei, L. D.
and Cope, F. O.
Apoptosis: The Molecular Basis of Cell Death (1991) Cold Spring Harbor Press,
N.Y.; Tomei, L.
D. and Cope, F. O. Apoptosis II: The Molecular Basis of Apoptosis in Disease
(1994) Cold
Spring Harbor Press, N.Y.; Duvall and Wyllie (1986) Immun. Today 7(4):115-119
and Sambrook
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,
New York
(1989).
Examples of useful substances capable of inhibiting the interaction between FP
prostanoid
receptors and PGFZa are antibodies that bind to either FP prostanoid receptors
or PGFZa. In a
preferred embodiment antibodies binding to the FP prostanoid receptors bind to
a extracellular
potion of the receptor. Such antibodies are readily obtainable by one of skill
in the art using
conventional antibody isolation and production methods. Such methods are
described in Harlow
and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, 1988, which
is hereby incorporated by reference.
Upon successful isolation of a substance which can inhibit the interaction
between FP
prostanoid receptors and PGFza it will be useful to formulate this substance
into a pharmaceutical
composition suitable for administration to a animal, preferably a human. Such
pharmaceutical
compositions typically include pharmaceutically acceptable carriers. The
pharmaceutically
acceptable carrier which can be used in the present invention is not limited
particularly and
includes an excipient, a binder, a lubricant, a colorant, a disintegrant, a
buffer, an isotonic agent, a
preservative, an anesthetic, and the like which can be used in a medical
field.
The pharmaceutical composition can be applied by any suitable administration
method
depending on the purpose of treatment and selected from injection
(subcutaneous, intracutaneous,
intravenous, intraperitoneal, etc.), eye dropping, instillation, percutaneous
administration, oral
administration, inhalation, and the like.
The dosage form such as injectable preparations (solutions, suspensions,
emulsions, solids
to be dissolved when used, etc.), tablets, capsules, granules, powders,
liquids, liposome
inclusions, ointments, gels, external powders, sprays, inhalating powders, eye
drops, eye
ointments, suppositories, pessaries, and the like can be selected
appropriately depending on the
to

CA 02435510 2003-07-21
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administration method, and the inhibiting substance of the present invention
can be accordingly
formulated. Formulation in general is described in Chapter 25.2 of
Comprehensive Medicinal
Chemistry, Volume 5, Editor Hansch et al, Pergamon Press 1990.
The dose of the medicine of the present invention should be set up
individually depending
on the purpose of administration (prevention, maintenance (prevention of
aggravation),
alleviation (improvement of symptom) or cure); the kind of disease; the
symptom, sexuality and
age of patient; the administration method and the like and is not limited
particularly.
Having generally described this invention, a further understanding can be
obtained by
reference to certain specific examples which are provided herein for purposes
of illustration only,
and are not intended to be limiting unless otherwise specified.
EXAMPLES
Experimental Procedures
ImmuuofZuorescefZCe Microscopy. HEK-293 cells stably expressing the ovine FPA
and FPB
prostanoid receptor isoforms (5) were split and grown in six-well plates
containing 22-mm round
glass cover slips for 3-4 days. Cells were treated with either vehicle (sodium
carbonate, 0.002%
final) or 1 ~M PGFZa and were rapidly washed, fixed, and incubated with a
1:1000 dilution of a
mouse monoclonal antibody to (3-catenin (Transduction Laboratories). They were
then washed
and incubated with a 1:4000 dilution of an FITC-conjugated goat anti-mouse
secondary antibody
(Sigma). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma).
Cells were
visualized by phase-contrast and epifluorescence microscopy as previously
described (6).
ImmuhoprecipitatiosZ and Blotting. Cells were scraped and sonicated in a lysis
buffer consisting
of 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 2 mM EGTA, 2 mM
phenylmethylsulfonylfluoride,
0.1 mg/ml leupeptin and 2 mM sodium vanadate. Samples were centrifuged (16,000
x g) for 15
minutes at 4°C and the supernatant (cytosolic fi°action) was
removed and the pellet (particulate
f~actioh) was solubilized with lysis buffer containing 0.2% Triton X-100 and
then centrifuged
again to remove insoluble debris. For immunoprecipitation, samples were
rotated for 2 hours at
11

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4°C with antibodies to (3-catenin, followed by addition of protein-G
sepharose (Amersham) and
rotation for another hour. The sepharose was washed with lysis buffer and then
resuspended with
SDS-PAGE sample buffer and boiled. Samples were electrophoresed on 7.5% SDS-
polyacrylamide gels, transferred to nitrocellulose membranes, and incubated
with either
antibodies to [3-catenin or with a mixture of mouse monoclonal antibodies to
phosphoserine
(Sigma) and phosphothreonine (Sigma). The membranes were washed and incubated
with
horseradish peroxidase-conjugated goat anti-mouse secondary antibodies and
were visualized by
enhanced chemiluminescence (SuperSignal, Pierce). Nuclear extracts were
prepared according to
the method of Dignam as modified by Westin et al. (7).
RT PCR. RT was done using the Superscript Preamplification System (Life
Technologies) and 1
~g of RNA/sample that had been pretreated with DNase I. This was followed by
PCR using an
initial incubation at 94°C for 5 minutes, followed by 20 cycles of
94°C, 60°C, and 68°C each for 2
minutes, and a final incubation at 68°C for 10 minutes. The human [3-
catenin and GAPDH primer
pairs were exactly according to Rezvani ~c Liew (8). Product sizes were 521 by
for [3-catenin and
737 by for GAPDH and were resolved by electrophoresis on 1.5% agarose gels.
Preliminary
experiments were done to find the optimal conditions for quantitative
amplification of (3-catenin
and GAPDH mRNA.
TcflLef Reporter Cehe Assay. Cells were split into 10-cm dishes and the next
day were
transiently transfected using FuGENE-6 (Roche) and either 10 ~,g/dish of the
wildtype Tcf/Lef
reporter plasmid, TOPflash, or the mutant plasmid, FOPflash. FOPflash differs
from TOPflash by
mutation of its Tcf binding sites and serves to differentiate Tcfl(3-catenin
mediated signaling from
background (Upstate Biotechnology). Cells were incubated overnight and were
treated for 1 hour
at 37°C with either vehicle or 1 ~.M PGFZa. They were then rapidly
washed three times each with
2 ml of Opti-MEM (Life Technologies) as previously described (6) and incubated
for 16 hours at
37°C in 10 ml of Opti-MEM containing 250 ~.g/ml geneticin, 200 ~.g/ml
hygromycin B, and 100
~g/ml gentamicin. Cells were placed on ice, rinsed twice with ice cold PBS,
and extracts were
prepared using the Luciferase Assay System (Promega). Luciferase activity in
the extracts 0500
ng protein/sample) was measured using a Turner TD-20/20 luminometer and was
corrected for
background by subtraction of FOP-FLASH values from corresponding TOP-FLASH
values.
12

CA 02435510 2003-07-21
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Results
Figure 1A shows phase contrast microscopy of HEK cells stably expressing
either the
ovine FPA prostanoid receptor (panels a and b) or the ovine FPB prostanoid
receptor (panels c and
d) following 1 hour treatment with either vehicle (panels a and c) or 1 ~M
PGFz« (panels b and
d). It can be appreciated that in both FPA and FPB expressing cells treatment
with PGFz« resulted
in morphological changes consisting of a loss of filopodia and formation of
cell aggregates. We
have previously shown that these morphological changes involve the activation
of Rho and
phosphorylation of p125 focal adhesion kinase (5). However, following the
removal of PGFz« the
FPA expressing cells show a rapid (within 1 hour) reversal of these
morphological changes,
whereas the FPB expressing cells remain rounded even after 48 hours (6). To
investigate the
possible role of other adhesion proteins in this process we used
immunofluorescence microscopy
to examine the localization of E-cadherin and (3-catenin in HEK cells stably
expressing either the
FPA or FPB isoforms following treatment with 1 ~.M PGFZ«. Although effects on
E-cadherin
localization were not apparent (data not shown), Figure 1 B shows that PGFZ«
treatment resulted
in a marked accumulation of (3-catenin in regions of cell-to-cell contact in
FPB expressing cells
(panels c and d), but not in FPA expressing cells (panels a and b). Both cells
lines, however,
showed agonist dependent cell rounding following treatment with PGFz« (Figure
1A) indicating
that the process of cell rounding itself was not responsible for the increased
contiguous
accumulation of (3-catenin in the FPB expressing cells.
Besides its role in cell adhesion (3-catenin is well recognized as a signaling
molecule that
undergoes stimulus dependent translocation from the cytosol to the nucleus
where it is involved
in the regulation of Tcf/Lef mediated gene transcription (9-11). We,
therefore, used
immunoblotting to examine both particulate and cytosolic fractions for changes
in ~3-catenin
expression following treatment of FPA and FPB expressing cells with PGFZ«.
Figure 2A shows
that the expression of J3-catenin is higher in both the particulate and
cytosolic fractions from FPB
expressing cells as compared with FPA expressing cells. Furthermore, treatment
with PGFz«
increased the levels of cytosolic (3-catenin in both the FPA and FPB
expressing cells, but had little
effect on the levels of [3-catenin in the particulate fraction. Reverse
transcription (RT) followed
by the polymerase chain reaction (PCR) was used to determine if there were any
differences in (3-
13

CA 02435510 2003-07-21
WO 02/058546 PCT/US02/00522
catenin mRNA levels under these same experimental conditions. Figure 2B shows
that (3-catenin
and GAPDH mRNA levels were the same for both cell lines and were not affected
by PGFZa,
indicating that the observed differences in (3-catenin expression appear to be
the result of changes
in translation and/or protein turnover.
Serine/threonine phosphorylation of [3-catenin by glycogen synthase kinase-3(3
(GSK-3[3)
marks (3-catenin for degradation and is a critical factor in the regulation of
its signaling activity
(12,13). Thus, under most conditions cytosolic ~i-catenin is phosphorylated
leading to an
association with the tumor suppressor protein, adenomatous polyposis coli
(APC), and the
scaffolding protein, axin, which is then followed by ubiquitination and
proteasomal degradation
(14). Using immunoprecipitation and immunoblotting we examined
serine/threonine
phosphorylation of [3-catenin following treatment of either FPA or FPB
expressing cells with
PGFZa. Figure 3 shows that in FPA expressing cells the vehicle control levels
of cytosolic (3-
catenin are very low and there is no detectable phosphorylation (lane a).
Following treatment with
PGFZa the levels of cytosolic [3-catenin increase and there is a marked
increase in phosphorylation
(lane b). In FPB expressing cells the vehicle control levels of cytosolic (3-
catenin are already
elevated and so is phosphorylation (lane c). This probably reflects endogenous
GSK-3 (3 activity
and tight coupling to the elevated levels of cytoplasmic (3-catenin. After
treatment of FPB
expressing cells with PGFZa, however, there is a further increase in cytosolic
(3-catenin, but a
dramatic fall in phosphorylation (lane d), suggestive of an uncoupling or
decrease in GSK-3 (3
activity. It would, therefore, be expected that degradation of cytosolic [3-
catenin would be favored
at the expense of nucleax translocation in FPA expressing cells, whereas, the
opposite would be
true in FPB expressing cells. This appears to be confirmed in Figure 3 where
immunoblotting of
nuclear extracts shows significantly higher levels of (3-catenin in FPB
expressing cells following
treatment with PGFZa (lane d) as compaxed with FPA expressing cells (lane b).
Following nucleax translocation, [3-catenin is known to interact with members
of the
Tcf/Lef family of transcription factors (15), which in many instances serves
as a switch for
cellular differentiation and transformation. Because of this signaling
potential, we were interested
in the possibility that the failure of FPB expressing cells to return to their
original dendritic
morphology following removal of PGFZa might represent a transformation event
induced by a (3-
14

CA 02435510 2003-07-21
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catenin mediated switch in gene expression. To examine this we transiently
transfected either FPA
or FPB expressing cells with a Tcf/Lef responsive reporter plasmid (16) and
measured luciferase
reporter gene activity following treatment with 1 p,M PGFZa. Initially we
found that basal levels
of luciferase activity were elevated (~3 fold) in FPB expressing cells as
compared with FPA
expressing cells and that measurement of reporter gene activity immediately
following a 1 hour
treatment with PGFZa did not stimulate luciferase activity in either cell line
(data not shown).
However, as shown in Figure 4A, the morphological effects of PGFZa on FPB
expressing cells
persist long after its removal. Thus, when cells are examined 16 hours after
an initial 1 hour
treatment with PGFza (followed by washout and replacement with fresh media),
the FPA
expressing cells show a return to their original dendritic morphology (panel
b), whereas, the FPB
expressing cells remain rounded and aggregated (panel d). We, therefore,
examined Tcf/Lef
reporter gene activity at the same time point and the results are shown in
Figure 4B. In a
remarkable parallel to the morphological findings, FPB expressing cells show a
persistent
activation of luciferase activity (column d) that is roughly 6.5 fold higher
than either the vehicle
control (column c) or PGFZa treated FPA cells (column b). We have previously
reported that the
failure of FPB expressing cells to show reversal of cell rounding is not
because of changes in the
kinetics of PGFza binding or in its removal during the washout procedure (6).
Discussion
The present inventor has sho~m that FPB expressing cells differ in several
important
regards from FPA expressing cells in terms of their potential for activation
of Tcf/~3-catenin
mediated signaling. First FPB expressing cells show PGFZa stimulated
accumulation of (3-catenin
at their contiguous cell boundaries that is not evident in FPA expressing
cells. Second, while both
FPA and FPB expressing cells show PGFZa stimulated increases in cytosolic (3-
catenin, in FPA
expressing cells this is accompanied by increased (3-catenin phosphorylation
and in FPB
expressing cells by decreased (3-catenin phosphorylation. Third, FPB
expressing cells show a
profound stimulation of Tcf/Lef reporter gene activity 16 hours after agonist
removal that is
essentially absent in FPA expressing cells. Obviously a key control point
could be in the
differential phosphorylation of ~i-catenin. Thus, it is possible that the
agonist stimulated
accumulation of (3-catenin at the contiguous cell boundaries of FPB cells
results in enhanced
is

CA 02435510 2003-07-21
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adhesive interactions with E-cadherin. In turn, this could initiate E-cadherin
outside-in signaling
leading to the sequential activation of phosphatidylinositol 3-kinase and Akt
kinase (17). This is
potentially meaningful because phosphorylation of GSI~-3(3 by Akt kinase is
inhibitory (18) and
could lead to the decreased phosphorylation of (3-catenin found in agonist
treated FPB cells.
Recently Meigs et al. reported that constitutively active mutants of Gale and
G«l3 interact
with the cytoplasmic domain of E-cadherin resulting a release of (3-catenin
and stimulation of
Tcf/Lef reporter gene activity in a mutant cell line lacking APC (19). This is
an intriguing finding
since it is the first report of a link between heterotrimeric G-proteins and
the Tcf/[3-catenin
signaling pathway. However, because of the altered nature of their model, its
physiological
relevance might be questioned. In light of the present findings, though, it
appears likely that both
GPCRs and heterotrimeric G-proteins will be involved with activation of this
important signaling
pathway. We previously showed that FP receptors activate Rho and hypothesized
that this
occurred through activation of Gl2 and/or Gl3 (5). Both receptor isoforms were
equally effective
in this regard and, therefore, it would appear unlikely that activation of Gl2
and/or Gl3 could be
solely responsible for the present findings since persistent activation of
Tcf/(3-catenin signaling
was only observed for cells expressing the FPB isoform.
One possible mechanism for that activation of Tcf/(3-catenin signaling by
PGFZa in cells
expressing the FPB receptor is responsible for a phenotypic transformation
that is morphologically
similar, but fundamentally different from the cell rounding observed in
agonist treated FPA cells.
Thus, maintenance of shape change in FPA expressing cells depends upon
continuous stimulation
by PGFZa and following its removal the cells revert back to their original
morphology. In contrast,
while shape change in FPB expressing cells is initiated by PGFZa, its
maintenance is independent
of fiuther PGFZa stimulation and may not even require the continued presence
of the receptor
following the initial agonist stimulation. In this manner the FPB prostanoid
receptor is functioning
as one would expect of a trigger in a developmental or malignant
transformation pathway.
This has considerable significance for the signaling potential of FP
prostanoid receptors
and possibly for other GPCRs as well. For example, in sheep and cattle it is
known that PGFZa is
the physiological signal for regression of the corpus luteum, but only during
a short window of
the luteal cycle. Thus, if pregnancy occurs the corpus luteum is maintained
and loses sensitivity
16

CA 02435510 2003-07-21
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to the luteolytic actions of PGFZa (20). Interestingly the expression of FP
receptors does not
appear to change during this transition, however, these receptors are
represented almost entirely
by the FPA isoform (21). Brief expression of a small population of FPB
receptors during the
sensitive phase luteal cycle could explain the luteolytic actions of PGFZa.
Indeed the FPB isoform
was cloned from a midphase ovine corpus luteum cDNA library where the
predominant isoform
was the FPA ( 1 ).
Another condition that might involve the FPB isoform or a homologue is in
colorectal
cancer. It is well established that aberrant activation of Tcf/(3-catenin
signaling is strongly
associated with the development of this disease (22-24) and that inhibition of
cyclooxygenase by
nonsteroidal antiinflammatory drugs (NSAIDs) can slow tumor progression (25).
However, the
specific mechanism of this beneficial effect is vague because of the large
number of prostanoid
metabolites that are affected by the inhibition of cyclooxygenase. The
disclosure provided in the
present application support a mechanism in which NSAID mediated decreases in
PGFza, would
result in decreased Tcf/(3-catenin signaling by FPB prostanoid receptors. This
conclusion is
supported by animal models of skin carcinogenesis in which PGFZa reversed the
anti-tumor
promoting activity of indomethacin and was the only prostanoid tested that
increased the tumor
promoting activity of phorbol esters (26). Although a human homologue of the
ovine FPB
receptor has not yet been identified, it is easy to imagine genetic or even
posttranslational
mechanisms that could give rise to functional FPB isoforms. Thus, much like
the known
mutations of APC, truncation of the human FPA receptor by allelic variation,
somatic mutations,
or proteolytic cleavage could give rise to receptors capable of producing
persistent activation of
Tcf/[3-catenin signaling. The possible role of FPB receptors in these and
other physiological
processes is intriguing and awaits future studies.
Another condition that might involve the FPB isoform or a homologue is in the
control of
hair growth. Thus, it has been well documented that in some patients receiving
latanoprost (an
analogue of PGFza), for the treatment of glaucoma, there is hypertrichosis of
the eyelashes and
adjacent hair in the treated eye, but not in the untreated eye (27-28). The
mechanism of this
curious side effect is unknown; however, recent studies have indicated that
Wnt signaling and
activation of Tcf/Lef transcription complexes is critical to hair follicle
development and
m

CA 02435510 2003-07-21
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differentiation (29-30). The disclosure provided in the present application
supports a mechanism
in which activation of the FPB receptor or a homologue would stimulate a
Tcf/(3-catenin signaling
pathway in the hair follicle leading to increased hair growth.
Obviously, numerous modifications and variations on the present invention are
possible in
light of the above teachings. It is therefore to be understood that within the
scope of the
appended claims, the invention may be practiced otherwise than as specifically
described herein.
i8