From the Center for Vascular Biology, Department of
Physiology, University of Connecticut Health Center, Farmington,
Connecticut 06030, the ¶ Experimental Pathology
Department, Holland Laboratory, American Red Cross, Rockville, Maryland
20855-2743, and the
Department of Biological Chemistry, UCLA,
Los Angeles, California 90095-8362
Received for publication, November 29, 2000, and in revised form, March 6, 2001
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ABSTRACT |
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The cyclooxygenase
(COX)-2 gene encodes an inducible prostaglandin
synthase enzyme that is overexpressed in adenocarcinomas and other
tumors. Deletion of the murine Cox-2 gene in
Min mice reduced the incidence of intestinal tumors,
suggesting that it is required for tumorigenesis. However, it is not
known if overexpression of Cox-2 is sufficient to induce
tumorigenic transformation. We have derived transgenic mice that
overexpress the human COX-2 gene in the mammary glands
using the murine mammary tumor virus promoter. The human Cox-2 mRNA
and protein are expressed in mammary glands of female transgenic mice
and were strongly induced during pregnancy and lactation. Female virgin
Cox-2 transgenic mice showed precocious lobuloalveolar differentiation
and enhanced expression of the The cyclooxygenase
(Cox)1 enzymes, Cox-1 and -2, catalyze the rate-limiting steps in the biosynthesis of prostaglandins
and thromboxanes, collectively known as prostanoids. Regulation of expression of Cox-1 and -2 is distinct; Cox-1 is expressed
ubiquitously, whereas Cox-2 is induced as an immediate-early gene in
most cells (1-3). Various extracellular stimuli, including growth
factors, cytokines, tumor promoters, peroxisomal proliferators, and
carcinogens, induce Cox-2 expression (1-6). Although transcriptional
regulation of Cox-2 has been studied extensively, post-transcriptional
mechanisms are also important for Cox-2 expression (1-3).
A strong correlation has been established between the use of
non-steroidal anti-inflammatory drugs (NSAIDs) and the decreased incidence of colorectal, breast, and lung cancers (4-8). In addition, NSAIDs and Cox-2 inhibitors suppress carcinogen-induced tumorigenesis in animal models (9-11). Such data have prompted the examination of
expression of Cox-1 and -2 in human cancer tissues. Cox-1 is expressed
in both normal and malignant cells; however, Cox-2 is up-regulated in a
high percentage of tumors (4-10). This finding was established
originally in gastrointestinal tumors but has been extended to
carcinomas of diverse origins (4-8, 12-14). Indeed, recent studies
have indicated that high levels of Cox-2 are expressed in human mammary
tumor tissues compared with the adjacent normal tissue (13, 14). Gene
deletion studies support the concept that Cox-2 plays a critical role
in the development of intestinal tumors (15). This work has been
confirmed not only in intestinal tumors but also in carcinogen-induced
model of skin cancer (16, 17). Surprisingly however, deletion of the
murine Cox-1 gene also reduced the incidence of both
intestinal and skin tumors (16, 17). The Cox-2 gene was
expressed in the mesenchymal compartment in the neoplastic tissue in
the mice, which is in contrast with both mesenchymal and epithelial
expression in human tumors (15). These genetic loss-of-function studies
suggest that expression of Cox-1 and -2 at the site of transformation or at some distal site is necessary for tumorigenesis.
In contrast, other studies show that inhibition of Cox-2 by NSAIDs
cannot account for their anti-tumor effects. For example, sulindac
sulfone, a derivative of sulindac which lacks Cox-1 or -2 enzymatic
inhibitory activity, induced epithelial cell apoptosis and inhibited
carcinogenesis in animal models (18, 19). Furthermore, various NSAIDs,
albeit at high concentrations, inhibited growth and tumorigenicity of
transformed mouse embryonic fibroblasts lacking both Cox-1 and Cox-2
(20). Several other targets of NSAID action have also been proposed.
First, the nuclear receptor peroxisomal proliferator-activated receptor
(PPAR) The question of whether Cox-2 overexpression is sufficient to induce
tumorigenesis has not been addressed. In this report, we describe a
genetic gain-of-function approach to examine the direct role of Cox-2
in tumorigenesis.
Generation of Transgenic Mice--
The human COX-2
gene (23) was cloned behind the murine mammary tumor virus (MMTV)
promoter (24). The linearized construct was used to derive transgenic
CD1 mice (Charles River Laboratories) in the UCHC transgenic core
facility. Transgenic mice were identified by Southern blot analysis of
tail DNA using a human Cox-2 3'-untranslated region specific probe.
Three founder animals (two males and one female) were used to derive F1
and F2 hemizygotic transgenic mice. Gene expression and phenotypic
analysis were done on female F1 and F2 mice derived from all three
founders. For the analysis of mammary glands during involution,
lactating mice at 7 days postpartum were used, and the pups were
removed to induce mammary gland involution.
Northern and Western Analysis of Mammary Glands--
Mammary
glands were dissected, total RNA was purified, and Northern analysis
for human Cox-2 (25) and mouse Arachidonic Acid Metabolism in Mammary Glands--
Mammary
glands were dissected, cut into 1-mm3 sections, washed
extensively, and incubated with 12 µM
[1-14C]arachidonic acid (PerkinElmer Life Sciences) for
30 min at 37 °C. Medium was acidified, extracted, and prostanoids
were separated and identified by TLC/autoradiography procedures as
described previously (25). In some experiments, cold arachidonic acid was used, and medium was analyzed for PGE2 synthesis by
radioimmunoassay as described (25, 27).
Whole Mount, Histological Methods, and
Immunohistochemistry--
Mammary glands were dissected and processed
for whole mount analysis as described (24, 26). Some glands were fixed
and analyzed by histological methods (28). Immunohistochemistry for
human Cox-2 was conducted as described (12, 28) using the Cox-2
monoclonal (27) and polyclonal antisera (Cayman Chemical). Specificity
of immunostaining was confirmed by competition with excess peptide
antigen as described (12, 28).
Apoptosis Assay--
Terminal nucleotidyl transferase staining
was performed as described (29) on formallin-fixed, paraffin-embedded
sections (4 µm) with a in situ cell death detection kit
(Roche, Indianapolis) according to the manufacturer's instructions.
Sections were counterstained with methyl green (Vector Laboratories,
Burlingame, CA). The percentage of apoptotic cells was calculated from
randomly selected fields (total of 4,000 cells/section) at a
magnification of ×60). Statistical analysis was done using Student's
t test.
The MMTV promoter (24) was used to direct expression of the human
COX-2 gene in transgenic mice (Fig.
1A). Three independent founder
lines, obtained after pronuclear microinjection of the linearized
construct, were bred with CD1 mice, and the hemizygous F1 and F2 mice
were characterized for the studies described below. By Northern
analysis, human Cox-2 transgenic mRNA was detected only in mammary
glands and not in liver, lung, heart, brain, spleen, or muscle tissues
(data not shown). RNA samples were isolated from the mammary gland
during progressive stages of maturation. Northern blot analysis was
conducted to determine the expression of endogenous murine Cox-1 and
Cox-2 as well as transgenic human Cox-2 mRNA. High stringency
hybridization conditions (20% formamide, 65 °C) were used to
discriminate between mouse and human Cox-2 mRNAs. Only endogenous
murine Cox-1 mRNA was expressed in the mammary glands of virgin
mice. During lactation, Cox-1 expression was induced and returned to
base line after the involution of the mammary gland in weaned mothers
(Fig. 1B). Murine Cox-2 mRNA was not detected by
Northern analysis of total RNA preparations, suggesting that endogenous
Cox-2 is expressed at extremely low levels during the development and
involution of the mammary gland. Reverse transcription-polymerase chain
reaction analysis for murine Cox-2 confirmed the Northern data and
suggests that it is expressed at extremely low levels in normal mammary
tissue (data not shown). In the human Cox-2 transgenic mice, a low
level of Cox-2 mRNA was detected in mature virgin mice, but
significantly higher levels were induced during pregnancy and
lactation. Concomitant with the regression of the mammary gland in
weaning females, the transgenic Cox-2 expression was attenuated after
removal of nursing young (Fig. 1B). The highest level of
Cox-2 expression was achieved during lactation, which is consistent
with the activity of the MMTV promoter (24). The Cox-2 transcript was
not detected by Northern analysis in non-transgenic normal mice.
Expression of the human Cox-2 mRNA did not alter the expression of
endogenous murine Cox-1 and -2.
-casein gene, which was inhibited by
the Cox inhibitor indomethacin. Mammary gland involution was delayed in
Cox-2 transgenic mice with a decrease in apoptotic index of mammary
epithelial cells. Multiparous but not virgin females exhibited a
greatly exaggerated incidence of focal mammary gland hyperplasia,
dysplasia, and transformation into metastatic tumors. Cox-2-induced
tumor tissue expressed reduced levels of the proapoptotic proteins Bax and Bcl-xL and an increase in the anti-apoptotic protein
Bcl-2, suggesting that decreased apoptosis of mammary epithelial cells contributes to tumorigenesis. These data indicate that enhanced Cox-2
expression is sufficient to induce mammary gland tumorigenesis. Therefore, inhibition of Cox-2 may represent a mechanism-based chemopreventive approach for carcinogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was induced by the APC mutation, which resulted in the
transcriptional enhancement of PPAR
via the
-catenin pathway.
High doses of NSAIDs inhibited the action of PPAR
on transcriptional
regulation of downstream genes, suggesting that it might be one of the
targets for NSAID inhibition of carcinogenesis (21). Second, NSAIDs at
high doses inhibited the activity of the nuclear factor
B pathway
and thus may promote cell death in a prostanoid independent manner
(22). These studies have raised questions about the causal role of
Cox-2 in tumorigenesis and have identified potential non-Cox-2 targets of NSAID action.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-casein (26) was done as described
previously (25). For analysis of Cox-2 polypeptide expression, mammary
glands were homogenized in the extraction buffer containing 1% Tween
20, and Western analysis was conducted as described previously (25).
The Cox-2 monoclonal antibody was described previously (27). Polyclonal
antibodies for Bax, Bcl-XL, and Bcl-2 were purchased from
Santa Cruz.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Generation of transgenic mice that
overexpress Cox-2 in mammary glands. Panel A, scheme of
the construct. The MMTV promoter was cloned upstream of the human Cox-2
gene (23, 24), and the resulting construct was used to derive
transgenic mice as described. Panel B, Northern analysis of
mammary tissues from Cox-2 transgenic mice (Cox-2) and
normal mice (NM). Mammary tissues from virgin (V,
16 weeks), pregnant (P, 18 days pregnant), lactating
(L, 7 days postpartum), and weaning (W, 14 days
postweaning) mice were used to purify RNA, and Northern analysis for
human Cox-2 and mouse Cox-1 was done as described. Panel C,
immunoblot analysis of mammary tissues. Extracts were prepared from
mammary tissues of Cox-2 transgenic and normal mice, and immunoblot
analysis was conducted using the Cox-2 or the -actin antibody as
described. Panel D, immunohistochemistry of mammary tissues
from Cox-2 transgenic mice. Mammary glands from 18-days-pregnant Cox-2
transgenic mice were fixed and analyzed by immunohistochemical
procedures for expression of the Cox-2 antigen. Note the specific
expression in mammary epithelia of alveolar and ductal structures
(asterisk, magnification ×20). The inset
shows the ×40 magnification of a developing alveolar structure
indicating perinuclear staining for Cox-2. Staining of similar sections
with an irrelevant mouse IgG or staining of non-transgenic animals with
the Cox-2 antibody was negative. Panel E, TLC analysis of
radioactive eicosanoids secreted by mammary glands. Mammary glands (200 mg) from lactating Cox-2 and normal mice animals were superfused with
12 µM [14C]arachidonic acid for 30 min, and
eicosanoids secreted into the medium were extracted, separated by TLC,
and exposed to an autoradiographic film. The bands were identified by
comigration with authentic cold standards. Panel F, mammary
tissues from Cox-2 and normal mice were superfused with 12 µM arachidonic acid for 30 min, medium was extracted, and
PGE2 was quantified by a radioimmunoassay as described.
Results represent the mean ± S.D. from mammary glands derived
from four animals.
Immunoblot analysis of mammary tissues indicates that the human Cox-2 polypeptide accumulates concomitantly with the transcript (Fig. 1C). Immunohistochemistry of mammary tissue sections was conducted using the human Cox-2-specific monoclonal antibody (Fig. 1D). The expression of the Cox-2 transgene was observed in the mammary epithelial cells of developing alveoli. In contrast, stromal cells in the mammary gland do not express the transgenic Cox-2. Prominent perinuclear immunostaining was observed, consistent with previous studies that demonstrated endoplasmic reticulum localization of the Cox-2 polypeptide (30).
The functionality of the transgenic Cox-2 polypeptide was demonstrated
by thin layer chromatorgraphic analysis of radioactive arachidonic acid
metabolites secreted from mammary gland explants. As shown in Fig.
1E, enhanced synthesis of PGE2,
6-keto-PGF1, PGD2, and PGF2
was observed in Cox-2 transgenic mammary glands compared with the
age-matched normal counterparts. Quantitative analysis of
PGE2 synthesis by mammary gland explants (Fig.
1F) was conducted next in mammary glands from normal as well
as Cox-2 transgenic mice. Mammary glands from non-transgenic mice also exhibited an increase in Cox activity during lactation, which is
consistent with the induction of endogenous Cox-1 mRNA expression. The human Cox-2 transgenic mice exhibited a higher level of
PGE2 synthesis, which correlated with the expression of the
Cox-2 mRNA and polypeptide. These data suggest that overexpression
of Cox-2 was achieved in the mammary glands of transgenic mice,
particularly during pregnancy and lactation.
The mammary glands of Cox-2 transgenic mice were analyzed by
morphological methods. Analysis of whole mount preparations of virgin
mammary glands demonstrated abnormal and hyperplastic alveolar development (Fig. 2A).
Histological analysis of mammary sections confirmed precocious
development of alveolar glands in Cox-2 transgenic mice (Fig.
2B). Northern blot analysis indicated that the expression of
the -casein, a gene normally expressed during late pregnancy and
lactation, was markedly up-regulated in virgin mammary glands of Cox-2
transgenic mice (Fig. 2C). To determine if secreted
prostanoids are involved in this phenomenon, virgin mice were treated
with indomethacin, a potent inhibitor of prostanoid synthesis (2). As
shown in Fig. 2, A, C, and D,
indomethacin treatment suppressed PGE2 synthesis,
-casein gene expression, and precocious alveolar differentiation.
These data strongly suggest that enhanced Cox-2 expression and the
concomitant secretion of prostanoids mediate the precocious mammary
gland differentiation in Cox-2 transgenic mice.
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The Cox-2 transgenic mice lactated normally and were able to nurse
their young. Histological and whole mount analysis of mammary glands
from pregnant and lactating animals showed no gross abnormalities (Fig.
3A). These data suggest that
exaggerated Cox-2 expression did not interfere with the physiological
functions of the mammary gland. We next studied the effect of Cox-2 on
mammary gland involution after weaning. As shown in Fig. 3A,
the expression of Cox-2 polypeptide decayed rapidly after weaning in
Cox-2 transgenic mice. Histological analysis of mammary tissue (Fig.
3B) indicates that a delay in mammary gland involution
occurred in Cox-2 transgenic mice. Mammary glands from control mice
showed expected rates of epithelial cell death and collapse of the
alveolar glands at 1-2 days postweaning, resulting in the regression
of the mammary gland. Although mammary glands from Cox-2 transgenic
mice regressed at 7 days, the rate was delayed at early stages (days
1-2), no signs of involution were apparent (Fig. 3B). 2 weeks after weaning, the mammary glands appeared similar in both Cox-2
transgenic and normal mice. Thus enhanced Cox-2 expression at 0-2 days
postweaning may have maintained the viability and function of the
mammary epithelial cells.
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Because cellular apoptosis contributes to the involution of the mammary gland, we measured the rates of apoptosis in mammary glands of normal and Cox-2 transgenic mice after weaning using the terminal nucleotidyl transferase method (29). As shown in Fig. 3C, significant decreases in apoptotic cells were observed in Cox-2 transgenic mice compared with non-transgenic normal mice at 2 days after weaning. These observations are consistent with the previous findings that enhanced Cox-2 expression inhibits cellular apoptosis in transfected epithelial cells (31).
Because pregnancy and lactation induced the Cox-2 transgene to very
high levels, we compared the mammary glands from multiparous females.
Focal areas of hyperplasia, also known as hyperplastic alveolar
nodules, are present with high frequency in human Cox-2 transgenic mice
that have undergone three or four cycles of pregnancy and lactation
(Fig. 4A). Histologic analyses
of such sections indicate that alveolar hyperplasia, squamous
metaplasia, and adenomatous carcinomas of the mammary epithelial cells
contributed to these lesions. In contrast, age-matched, multiparous
non-transgenic female mice did not show such tumors (Fig.
4A).
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The incidence of mammary tumors in Cox-2 transgenic mice and non-transgenic mice were analyzed by a combination of whole mount analysis and histological sections. As shown in Fig. 4B, > 85% of Cox-2 transgenic mice that have undergone multiple cycles of pregnancy and lactation show the presence of tumors in mammary glands. In contrast, a very low incidence of tumors was seen in non-transgenic mice with similar age and multiparosity. Transgenic female mice derived from all three founders showed this phenomenon, suggesting that the effects are caused by the expression of the human COX-2 gene rather than integration-dependent events. Cox-2 transgenic mice that did not undergo pregnancy and lactation did not show the increased mammary tumorigenesis. Three female Cox-2 transgenic mice (the founder female and two F1 females from a different founder) developed large mammary tumors with metastatic spread. One such animal at necropsy is shown in Fig. 4C, as an example of the presence of large metastatic tumors in and outside of the mammary glands.
Histological analyses of mammary tissue from non-transgenic mice and
from Cox-2-induced tumors are shown in Fig.
5, A and B,
respectively. In Fig. 5B, one can observe numerous
hyperplastic glandular structures, keratinizing squamous cells
(indicating metaplasia), stromal proliferation, and numerous blood
vessels. In Fig. 5C, adenomatous carcinoma was observed in
the peripheral lymph node. Well differentiated tumors appeared to have
metastasized into the lymph nodes of the Cox-2 transgenic mice. In Fig.
5D, an example of a mammary tumor with squamous metaplasia
is shown. In Fig. 5E, an example of a hyperplastic and
invasive ductal structure with hemosiderin (indicating bleeding into
the ducts) is shown. These data suggest that Cox-2 overexpression
induced various types of invasive mammary tumors, including ductal and
lobuloalveolar carcinomas.
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Mammary tumors in the Cox-2 transgenic mice were stained with the Cox-2 antibody using immunohistochemical procedures (Fig. 5F). Numerous epithelial cells in glandular structures exhibited strong immunoreactivity in the perinuclear locale. Immunoreactivity was competed by coincubation of the antibody with the Cox-2 antigenic peptide (data not shown). These data suggest that Cox-2 is expressed in the mammary tumors of Cox-2 transgenic mice.
To determine if decreased apoptosis of mammary epithelial cells
contributed to the Cox-2-induced tumorigenesis, we analyzed the
expression of apoptotic regulatory proteins Bcl-2, Bax, and Bcl-xL. Expression of Bcl-2 was induced by Cox-2
overexpression in intestinal epithelial cells (31), and Bax is known to
regulate NSAID-induced apoptosis (32). As shown in Fig.
6, high levels of Cox-2 polypeptide were
observed in the mammary tumors and lower levels were seen in the
adjacent normal tissue. In contrast, Cox-2 polypeptide was not
expressed in mammary glands of multiparous, non-transgenic mice.
Interestingly, expression of the proapoptotic proteins Bax and
Bcl-xL was reduced in Cox-2-expressing tumors. In contrast,
Bcl-2 expression was up-regulated in the tumor tissue. These data
suggest that regulation of expression of apoptosis regulatory factors
Bcl-2, Bax, and Bcl-XL may be a mechanism via which Cox-2
overexpression contributes to tumorigenesis.
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Together, these observations support the concept that overexpression of
Cox-2 alone is sufficient to transform the mammary epithelium into a
tumorigenic state.
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DISCUSSION |
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In this report, we describe a gain-of-function approach to evaluate the direct role for Cox-2 in tumorigenesis. Although studies utilizing various approaches, namely, epidemiologic, expression studies and genetic loss-of-function approaches, have implicated the functional role of Cox-2 in tumorigenesis, questions remain regarding the requirement and sufficiency of Cox-2 (4-8, 18, 19). Furthermore, NSAIDs were shown to inhibit various animal models of tumorigenesis; however, issues of nonspecificity have prevented the unequivocal interpretation of these studies (18-22). In this study we provide evidence that overexpression of Cox-2 alone is sufficient to induce tumorigenic transformation in a tissue-specific manner.
Overexpression of Cox-2 expression was achieved using the entire human COX-2 gene (23) under the control of the MMTV promoter (24). Previously, we attempted to derive transgenic mice using the keratin K14- driven human Cox-2 cDNA (33) and cytomegalovirus promoter-driven human Cox-2 cDNA in a tetracycline-regulated system (34). Both approaches were unsuccessful. In the former case, embryonic lethality appears to be induced by the developmental expression of the transgene. In the second case, the Cox-2 cDNA was not expressed after induction in two independent lines of transgenic mice. Therefore, we used the MMTV promoter, which is known to be induced postpartum in a mammary gland-enriched manner (24). In addition, we utilized the entire human COX-2 gene to include all of the post-transcriptional signals required for efficient expression of the gene (23, 25).
As shown in Fig. 1, MMTV-hCox-2 transgenic mice expressed high levels of Cox-2 transcript, protein, and enzymatic activity in the mammary glands. Specifically, expression of the transgenic Cox-2 polypeptide was induced during mammary gland development, achieved very high levels during pregnancy, and was maximal during lactation. Although the expression of Cox-2 is low in normal mammary glands, it can be induced by a high fat diet and carcinogen exposure (35, 36). However, significant expression of Cox-1 was observed in murine mammary tissue during all phases of mammary gland development. In addition, Cox-1 activity was induced during lactation.
Cox-2 enzymatic activity followed a kinetics of induction similar to
that of the mRNA and the protein, resulting in the production of
PGE2, 6-keto-PGF1, PGD2, and
PGF2
in the human Cox-2 transgenic mammary glands. After
weaning, Cox-2 expression decayed rapidly, consistent with the short
half-life of the mRNA and the protein (1-3). Thus the MMTV-hCox-2
transgenic mouse model provides an animal model in which persistent low
level expression of Cox-2 is achieved in mammary epithelial cells, and
exaggerated expression of Cox-2 can be induced during pregnancy and lactation.
Interestingly, MMTV-hCox-2 mice exhibited precocious development of the mammary glands as virgins. This is most likely the result of the secretion of prostanoids because indomethacin treatment reversed the changes. Mammary development is controlled by hormonal factors as well as by local signals (37). It is known that PGE2 induces mammary epithelial cell proliferation (38). In addition, PGE2 is known to induce aromatase, an enzyme involved in the local biosynthesis of estrogen, a potent inducer of mammary gland development (39). Thus, low level persistent expression of Cox-2 may induce precocious mammary gland development via the secretion of PGE2.
MMTV-hCox-2 transgenic mice exhibited a delayed mammary gland involution after weaning. Mammary gland involution is accompanied by rapid decay of Cox-2 expression, consistent with the knowledge that Cox-2 mRNA has a rapid turnover rate (23, 27). Cox-2 expression in epithelial cells of the gastrointestinal tract is associated with inhibition of apoptosis (31). Mammary gland involution is brought about in part by apoptosis of epithelial cells (40). Indeed, analysis of apoptotic rates by terminal nucleotidyl transferase assay indicates that Cox-2 overexpression resulted in decreased apoptotic cells. However, the effect on inhibition of mammary gland involution was transient because the Cox-2 expression decayed rapidly. These data suggest that Cox-2 overexpression may dysregulate the normal homeostatic mechanisms in the mammary gland by inhibition of apoptosis.
Mammary tumorigenesis occurred with high frequency in MMTV-hCox-2 transgenic mice after multiple rounds of pregnancy. The tumor incidence was high in all three lines of mice, suggesting that Cox-2 overexpression, rather than integration-dependent effects, were involved. The tumors were invasive and malignant, and secondary metastases were observed. The lag period of tumorigenesis may suggest the requirement for exaggerated induction of Cox-2, which occurred only during pregnancy and lactation. Indeed, low level expression of Cox-2 in virgin mammary gland did not result in spontaneous development of tumors. Alternatively, it is possible that a high level of Cox-2 induction and a second mutation are required to transform the mammary epithelium completely. Indeed, proliferating epithelial cells in the pregnant mammary gland may be more susceptible to mutagenic events of critical tumor suppressor genes. This observation is similar to the phenotype of other transgenes such as MMTV-cyclin D1 and MMTV-stromelysin (41, 42). Nevertheless, these findings point out that overexpression of Cox-2 alone is sufficient to induce tumorigenic transformation.
The molecular mechanisms responsible for Cox-2 induction of
tumorigenesis are unclear at present. Secreted prostanoids may interact
with plasma membrane-localized G protein-coupled receptors to induce
mitogenic or anti-apoptotic signals (1-3). Alternatively, nuclear
receptors such as PPAR and PPAR
, which are activated by
prostanoids, may be involved (1-3, 21). The possibility that the
peroxidase action of Cox-2 is involved in tumorigenesis also cannot be
ruled out at present (1-3). The animal model described in this study
may provide a useful system to begin to address these issues. However,
Cox-2-induced tumors expressed high levels of Cox-2, the anti-apoptotic
protein Bcl-2, and reduced levels of the pro-apoptotic proteins
Bcl-XL and Bax. This suggests that Cox-2 may regulate
expression of these death-regulatory proteins and thus inhibit
apoptosis and contribute to tumorigenesis. In agreement, the Cox-2
transgenic mice exhibit reduced apoptotic rates after mammary gland involution.
Many risk factors for human breast cancer induce Cox-2 expression. For
example, dietary n-6 fatty acids, activation of HER/Neu signaling, Wnt signaling, as well as carcinogen exposure induce Cox-2 gene expression (5, 8, 36, 43-45). In addition, data from carcinogen-induced rodent models of mammary cancer suggest that
Cox-2 inhibition reduces the incidence of mammary tumors (10, 11). Our
data are consistent with the concept that overexpression of Cox-2 in
the mammary epithelium is sufficient to induce mammary carcinogenesis.
Coupled with the strong epidemiological evidence of NSAID usage and
cancer incidence in humans (7), these findings strongly support the
notion that dysregulated and exaggerated expression of Cox-2 are strong
carcinogenic risk factors. Whether inhibition of Cox-2 in high risk
populations would confer an anti-cancer benefit in human breast cancer
warrants further study. However, efforts to define chemopreventive
agents that work via Cox-2 inhibition will be greatly facilitated by
this human Cox-2 transgenic model. Furthermore, mechanistic studies and
identification of downstream genetic targets of Cox-2 action may be
readily approached in this model.
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ACKNOWLEDGEMENTS |
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We thank M. Keough for help with mouse procedures, Dr. D. DeWitt for the gift of mouse Cox probes, Dr. Steven Clark for transgenesis protocols, Dr. Cristophe Creminon for the Cox-2 antibody, and Dr. Gilbert Jay for helpful comments.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL-49094 and HL-54710 and an established investigator grant from the American Heart Association (to T. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship from the Academy of Finland.
** To whom correspondence should be addressed: Center for Vascular Biology, Dept. of Physiology, MC-3501, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030. E-mail: hla@sun.uchc.edu.
Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M010787200
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ABBREVIATIONS |
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The abbreviations used are: Cox, cyclooxygenase; NSAID(s), non-steroidal anti-inflammatory drug(s); PPAR, peroxisomal proliferator-activated receptor; MMTV, murine mammary tumor virus; PG, prostaglandin.
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