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Expression during Tumor Progression
Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, NIH, Institutes of Health, Bethesda, Maryland 20892 [K. Y., M-A. S., C. C., J. E. G.], and Receptor Biology Section, Laboratory of Reproduction and Developmental Toxicology, National Institute of Environmental Health Science, National Institute of Health Sciences, NIH, Research Triangle Park, North Carolina 27709 [K. S. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) expression do not appear to modify the
level of transgene expression. The C3(1)/TAG transgene
becomes transcriptionally active in mammary ductal target cells at 3
weeks of age after the estrogen-induced differentiation of the mammary
epithelial anlage to the ductal outgrowth stage. Complete
maturation of the mammary ductal tree, however, is not required for
cancer development because tumors arise in animals where ductal
branching and terminal end bud formation have been prematurely arrested
by ovariectomy. Mammary tumorigenesis in this model is promoted by
increased estrogen exposure with the development of significantly more
mammary intraepithelial neoplastic lesions and carcinomas associated
with accelerated malignant conversion. The promotion of mammary tumors
in this model appears to occur through an estrogen-induced
proliferation and increase in the number of available target cells for
transformation at the terminal ductal lobular units, as has been
postulated to occur in women who receive hormone replacement therapy
and/or by additional molecular mechanisms. We show, for the first time
in a transgenic mouse model, that mammary tumor progression is
associated with the loss of ER expression, as has been often
observed in human breast cancers with important clinical significance.
Estrogen signaling may, therefore, serve different functions, depending
upon the stage of tumorigenesis. ERß expression is up-regulated
during tumor progression, although the functional significance of this
remains to be determined. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ER4
status is an important
factor in assessing prognosis and in determining therapeutic strategies
for treating breast cancer. Approximately 70% of human breast cancers
stain positively for ER. ER expression is associated with more
differentiated and less aggressive tumors. On the other hand, tumors
lacking ER are generally associated with a more aggressive disease
course and poorer clinical outcome (4
, 5)
. Tumors from the
same patient often progress from an ER-positive to an ER-negative
state. Therefore, breast cancers likely evolve into a
hormone-unresponsive state and become poorly responsive to endocrine
therapies.
To gain further insights into the role of estrogen signaling in mammary carcinogenesis, we have studied how alterations in estrogen exposure might influence the development of mammary cancer in the C3(1) /TAG transgenic mouse model in which 100% of female mice develop mammary carcinomas, as reported previously (6) . In this model, both large T- and small t-antigens of the SV40 early region are expressed under the transcriptional control of the 5' flanking region of the C3(1) component of the rat prostate steroid binding protein gene. Although male mice carrying this transgene develop prostate cancer, the C3(1) /TAG transgene is also expressed in both mammary epithelial ductal cells and the TLDUs without the need for pregnancy or hormone stimulation to drive transgene expression, as is often required for other transgenic mammary models (reviewed in Ref. 7 ). The mammary lesions arising in these transgenic mice develop over a predictable time course with histological similarities to human breast cancer (8 , 9) .
Unlike other transgenic models that use promoters that are highly
responsive to hormone stimulation, we demonstrate that the C3(1)
transgene is not estrogen responsive in the mammary gland. However,
estrogen appears to promote mammary tumorigenesis in this model. The
generation of C3(1)
/TAG mice lacking the ER receptor
demonstrates that mammary oncogenesis in this model requires the
differentiation of the mammary ductal epithelium through the ductal
outgrowth stage. Complete development and differentiation of the
mammary ductal tree, however, are not required for tumorigenesis. In
this study, we are the first to demonstrate, in a transgenic mouse
model, that ER expression appears to be lost during mammary tumor
progression, as often occurs in human breast cancer. This transgenic
model should provide further insights into the mechanisms of how ER
expression is lost during mammary cancer development and how estrogen
may stimulate mammary oncogenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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plasmid (pSG5-MOR) using the FuGENE6
transfection reagent according to the manufacturer’s protocol
(Boehringer Mannheim, Indianapolis, IN). The cells were treated with 0,
1, 2, 5, 10, or 20 nM of E2 for 48 h.
Western Blotting.
Cells were lysed with radioimmunoprecipitation assay buffer (1x PBS,
1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and 10 mg/ml
phenylmethylsulfonyl fluoride). Twenty µg of total protein were
loaded onto 8% Tris-glycine gels (Novex, San Diego, CA) and
transferred to nitrocellulose membranes (Novex, San Diego, CA).
Membranes were incubated with 3% bovine serum albumin (Sigma),
followed by the addition of primary antibody against SV40
TAG (Ab-2, at a concentration of 1.0 µg/ml; Calbiochem,
La Jolla, CA) or rabbit polyclonal anti-ER antibody (MC20, at a
dilution of 1:250; Santa Cruz Biotechnology, Santa Cruz, CA).
Monoclonal anti-actin antibody (N350, at a dilution of 1:500; Amersham
Life Science, Arlington Heights, IL) served as an internal control.
Blots were then washed and incubated for 1 h at room temperature
with horseradish peroxidase-conjugated goat antimouse or antirabbit IgG
(CalTAG; San Francisco, CA). Autoradiography was
performed using the enhanced chemiluminescence method (NEN Life Science
Products, Inc., Boston, MA).
Hormone Manipulations of C3(1)
/TAG Transgenic Mice.
Transgenic mice carrying the C3(1)
/TAG transgene
have been described previously by our laboratory and have been
maintained in the FVB/NCr background (6)
. All studies were
performed with female mice heterozygous for the transgene. The effects
of altered estrogen signaling on tumor development were determined in
response to various manipulations of the mice. For studies to determine
the short-term effect of estrogen on transgene expression in
vivo, mice were ovariectomized at 10 weeks of age. One week later,
the mice received injections intraperitoneally with 3 mg/kg body weight
of E2 dissolved in 10% ethanol and 90% peanut oil (Sigma)
or with peanut oil alone. Mice were sacrificed at 8, 16, or 24 h
after injection, and mammary tissues were excised and frozen at
-80°C for further analyses. Ten-week-old normal FVB/N female mice
were implanted with pellets that release 0.23 mg of E2
daily and sacrificed after 1 or 8 days for mammary gland analyses.
For studies to determine the long-term effects of hormone manipulations on tumorigenesis, groups of mice were ovariectomized prior to maturation but during a stage of ductal outgrowth at either 3 weeks of age (OVX3W), after puberty at 8 weeks of age (OVX8W), or left intact and implanted with placebo pellets (Innovative Research of America, Sarasota, FL). Additional groups of animals ovariectomized after sexual maturation at 8 weeks of age were either implanted with pellets (Innovative Research of America) containing 7.5 mg of E2 released over 60 days (OVX E2) or implanted with pellets containing 25 mg of TAM released over 60 days (Innovative Research of America). Pellets were replaced every 60 days. All manipulations of mice were performed in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, 1985, under animal protocol LC-059). Individual body weights were recorded weekly. Mammary tumor size was measured twice weekly using a caliper, and tumor volume was calculated using the following formula: the largest diameter x (the smallest diameter)2 x 0.4 (10) . Mice were euthanized by CO2 asphyxiation.
Generation of C3(1)
/TAG Transgenic Mice Carrying
Mutant ER Alleles.
To study the effect of ER on mammary tumorigenesis in
C3(1)
/TAG transgenic mice,
C3(1)
/TAG+/- transgenic male mice in the FVB/N
background were crossed with wild-type C57BL/6 female mice, and male
offspring carrying the C3(1)
/TAG+/- transgene
were identified by slot blot. These hybrid C3(1)
/TAG
(FVB/N x C57BL/6) male mice were crossed with
ER+/-;C57BL/6 female mice. Males from the
second cross with the
C3(1)
/TAG+/-;ER+/-
genotype were back crossed with ER+/-;C57BL/6
female mice. C3(1)
/TAG+/- female progeny with
similar genetic backgrounds carrying either ER+/+,
ER+/-, or ER-/-
genotypes were used for analyses. Genotypes were identified from tail
DNA by slot blot analysis using a probe for TAG
(6)
and PCR to determine the ER genotypes as described
previously (11)
.
Whole-Mount Preparation, Histopathology, and
Immunohistochemistry.
Mammary gland whole-mount preparations were spread on a glass
slide, fixed in 70% ethanol, rehydrated in distilled water, stained
with 0.2% carmine and 0.5% aluminium potassium sulfate, redehydrated
in 100% ethanol, cleared in xylene, and mounted with coverslips using
Permount (Fisher Scientific, Fair Lawn, NJ). Mammary tissue and tumor
samples were dissected from C3(1)
/TAG transgenic female or
age-matched nontransgenic FVB/N females. The sizes of the lesions were
recorded. A portion of each mammary tissue sample was also immediately
frozen on dry ice for subsequent molecular biological analyses. The
remaining mammary tissue was fixed in 4% paraformaldehyde or 10%
phosphate-buffered formalin, embedded in paraffin, cut at a thickness
of 4 µm, and stained with H&E for histopathological examination.
Histopathological lesions were quantitated by summing the number of lesions in sections from two axillary and one inguinal mammary gland from each mouse. Glands from the same locations in all mice were used for these studies.
For selected immunohistochemical analysis, the sections were
heated by microwave in distilled water for antigen retrieval,
hybridized with primary antibody, and processed using the avidin-biotin
complex method (Vectastain ABC Elite kit; Vector Laboratories,
Burlingame, CA). Anti-SV40 TAG mouse monoclonal antibody
(PAB 101; PharMingen, San Diego, CA) was used at a dilution of 1:50.
Anti-ER rabbit polyclonal antibody (MC20; Santa Cruz Biotechnology)
was used at a dilution of 1:500; and anti-proliferating cell nuclear
antigen mouse monoclonal antibody (PC-10; Dako Corp., Carpinteria, CA)
was used at a dilution of 1:500.
RNA Extraction, Northern Blotting, and Reverse RT-PCR Assay.
RNA was extracted twice with RNA STAT-60 reagent (Tel-Test "B",
Inc., Friendswood, TX) according to the manufacturer’s protocol.
RNA from mammary glands of intact FVB/NCr female mice were analyzed and
used as normal controls. A 1.8-kb EcoRI fragment of ER
fragment from pSG5-MOR was labeled with [-32P]dCTP by
random oligonucleotide-primed synthesis. Poly(A) RNA was isolated using
the MicroPoly(A) Pure kit (Ambion, Austin, TX) according to the
manufacturer’s protocol, and 5 µg of mRNA were fractionated on a
1.0% formaldehyde agarose gel, transferred onto a nylon membrane, and
fixed by UV cross-linking. Membranes were hybridized with a
32P-labeled probe and washed using standard protocols. The
membranes were then exposed to X-ray film at -70°C for varying
periods of time.
For RT-PCR, 2 µg of total RNA were incubated with DNAse I, then
incubated with reverse transcriptase using oligo (dT)12–18
primer and Super Script II, and subsequently incubated with RNaseH
(Life Technologies, Inc.) according to the manufacturer’s protocol.
cDNA was amplified using a thermal cycler (PTC-100; M. J. Research,
Inc., Watertown, MA). The conditions and the sequences for each primer
set were as follows: TAG: 32 cycles at 94°C for 1 min,
60°C for 2 min, and 72°C for 3 min; the 5' primer corresponds to
SV40 TAG nucleotides 4198–4219
(5'-gcagacactctatgcctgtgtg-3') and the 3' primer corresponds to
nucleotides 5040–5018 (5'-catcctgataaaggaggagatg-3'); ERß: 35 cycles
at 94°C for 1 min, 60°C for 2 min, and 72°C for 3 min; the 5'
primer corresponds to mouse ERß (U81451) nucleotides 39-60
(5'-aactacagtgttcccagcagca-3') and the 3' primer corresponds to
nucleotides 311-290 (5'-atccctcttggcgcttggacta-3'); TGF-: 32 cycles
at 94°C for 1 min, 57°C for 2 min, and 72°C for 3 min; the 5'
primer corresponds to mouse TGF- (U65016) nucleotides 117-137
(5'-cctgctagcgctgggtatcct-3') and the 3' primer corresponds to
nucleotides 343-323 (5'-ctgcatgctcacagcgaacac-3'); EGFR: 32 cycles at
94°C for 1 min, 57°C for 2 min, and 72°C for 3 min; the 5' primer
corresponds to mouse EGFR (X59698) nucleotides 256-276
(5'-ggcacaagtaacaggctcacc-3') and the 3' primer corresponds to
nucleotides 532-512 (5'-agttggacaggatggctaagg-3'); her-2/neu: 32 cycles
at 94°C for 1 min, 57°C for 2 min, and 72°C for 3 min; the 5'
primer corresponds to rat her-2/neu (X03362) nucleotides 1487-1504
(5'-cggaacccacatcaggcc-3') and the 3' primer corresponds to nucleotides
2179-2159 (5'-tttcctgcagcagcctacgc-3'); c-myc: 32 cycles at
94°C for 1 min, 57°C for 2 min, and 72°C for 3 min; the 5' primer
corresponds to mouse c-myc (X01023) nucleotides 1301-1324
(5'-gggccagccctgagcccctagtgc-3') and the 3' primer corresponds to mouse
nucleotides 1456-1433 (5'-atggagatgagcccgactccgacc-3'); and G3PDH: 30
cycles at 94°C for 45 s, 60°C for 45 sec, and 72°C for 2
min; the 5' primer used corresponds to mouse G3PDH (M32599) nucleotides
51-76 (5'-tgaaagtcggtgtgaacggatttggc-3') and the 3' primer to
nucleotides 1033-1010 (5'-catgtaggccatgaggtccaccac-3').
Statistical Analysis.
The two-sided Student’s t test was used to determine
whether significant differences existed between the mean values of the
groups analyzed. The log-rank test was used to evaluate the survival
curves.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, Lanes
1–5). However, M6 cells express low
levels of endogenous ER (Fig. 1
, Lane 1), which is
down-regulated by the addition of E2 in a dose-dependent
manner (Fig. 1
A, Lanes 1–5), demonstrating that the ER
signaling pathway is still operative in these cells.
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, which
could limit the response of the transgene to stimulation by
E2, we performed experiments where high levels of murine
ER were expressed after transfection of the plasmid pCMVmER.
Despite the overexpression of ER in M6 cells under these conditions,
the addition of up to 20 nM E2 did not lead to
a change in TAG levels (Fig. 1
A, Lanes 6–10).
These results demonstrate that although estrogen signaling is active in
the M6 cells as evidenced by reduced ER expression in response to
E2 (Fig. 1
, Lanes 1–5), transcriptional
activity of the C3(1)
5' flanking sequence is not influenced by ER
signaling in these cells (Fig. 1
, Lanes 1–10).
The effect of E2 stimulation of the transgene was also
studied in vivo. Because E2 stimulation of cell
proliferation, either by direct stimulation of transgene expression or
through other physiological mechanisms, would lead to an expansion of
mammary gland lesions expressing TAG protein, absolute
levels of TAG would not necessarily reflect an increase in
transcriptional activity of the C3(1)
5' flanking sequence. Therefore,
to examine how transcriptionally responsive the C3(1)
/TAG
transgene was to E2 in vivo, it was necessary to
perform trans-activation experiments over a relatively short
time frame before significant proliferation could occur. Ten-week-old
intact transgenic females were ovariectomized 1 week prior to being
given a single intraperitoneal injection of 3 mg/kg body weight of
E2. TAG expression in the mammary gland did not
change after ovariectomy or within 24 h after injection of
E2 (Fig. 1
B, Lanes 1–8). ER expression
increased with ovariectomy alone but significantly decreased in
ovariectomized mice given E2 supplementation (Fig. 1
B, Lanes 2, 6–8). These results demonstrate that although
the addition of estrogen reduces ER expression in the mammary tissue
as expected, C3(1)
is not transactivated by E2 in
vivo. Thus, phenotypic effects observed in C3(1)
/TAG
mice given estradiol supplementation do not result from increased
transgene expression.
Alterations in the Natural History and Histopathology of Mammary
Tumor Formation in C3(1)
/TAG Mice by Estrogen
Signaling.
TAG was not detectable by immunohistochemical analysis at
1, 8, or 14 days after birth (data not shown) but was detectable at 3
weeks of age in normal-looking epithelium of intact
C3(1)
/TAG transgenic female mice (Fig. 2A)
. The number of
TAG-positive mammary epithelial cells increases with age
and is associated with multifocal low-grade MIN lesions
(12)
in the ducts and TDLUs of the mammary gland beginning
at about 8 weeks of age (Fig. 2B)
. These lesions progress to
advanced MIN by 14 weeks of age (Fig. 2C)
. There is a
progressive increase in the number of MIN lesions that begin to develop
into invasive mammary adenocarcinomas at about 17 weeks of age. These
tumors continue to express high levels of TAG (Fig. 2D)
. Gross tumors were generally observed after 16 weeks of
age, with mice dying by 7 months of age because of tumor burden. The
mammary carcinomas are morphologically designated as mixed solid and
glandular carcinomas with necrosis and fibrosis (12)
and
appear to resemble what has been called poorly to moderately
differentiated human invasive ductal adenocarcinomas.
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The administration of supplemental E2 to normal intact
FVB/N females at 10 weeks of age resulted in proliferative effects in
the TDLUs, leading to increased proliferation and cellularity (Fig. 3)
without expansive lobuloalveaolar
formation as is seen in pregnancy. Proliferating cell nuclear antigen
positivity by immunohistochemical staining rose from 3.7% of mammary
epithelial cells in untreated mice, to 9.0 and 21.4% of cells in mice
treated with E2 for 1 or 8 days, respectively. This result
demonstrates that estrogen can provide a proliferative signal to the
mammary epithelium and increase the number of target cells, where the
C3(1)
/TAG transgene is transcribed.
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.
Ovariectomy before puberty (3 weeks of age) resulted in a dramatic
decrease in mammary duct side-branching and alveolar bud formation
(Fig. 4, B and G)
with a reduction in the number
of MIN lesions compared with intact animals (Fig. 5A)
. Ovariectomy after puberty
(8 weeks of age) resulted in a more modest reduction in duct and TDLU
formation and MIN formation (Fig. 4, C and H)
. At
14 weeks of age, the number of MIN lesions in the OVX3W and OVX8W
groups were significantly lower than that of the intact group
(P = 0.00031 and P = 0.0077, respectively; Fig. 5D
). There was also a
trend toward a lower number of MIN lesions in OVX3W animals compared
with that of OVX8W, although this was not statistically significant
(P = 0.054).
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, which was also associated
with a higher number of MIN lesions in the E2-supplemented
group (Fig. 4I)
compared with the other groups (Fig. 5D
; P = 0.0026 versus
intact; P = 0.00003 versus OVX 3W;
P = 0.00013 versus OVX8W; and
P = 0.0037 versus TAM). Tamoxifen
treatment given to intact females at 8 weeks of age moderately
inhibited TDLU formation (Fig. 4E)
and tended to reduce MIN
formation (Fig. 4J)
compared with intact animals, although
this was not statistically significant (P = 0.075; Fig. 5A
).
Ovariectomized female mice given E2 supplementation died
significantly earlier than intact mice (P = 0.0157; Fig. 5B
) because of accelerated tumor formation
associated with increased tumor multiplicity (P = 0.016; Fig. 5C
) and tumor volumes (Fig. 5D)
. The OVX E2 group had significantly larger
tumors than did the intact group (P = 0.0266)
at 20 weeks of age, but there was no significant difference among the
other groups (Fig. 5D)
. Although both the 3-week-old and
8-week old ovariectomized groups that did not receive E2
had a reduced number of MIN lesions at 14 weeks of age compared with
controls, the actual number of palpable tumors was not significantly
different from that observed in the intact group (Fig. 5C)
.
By 20 weeks of age, 15 of 16 OVX3W mice had developed palpable mammary
tumors. This suggests that by 3 weeks of age, the number of target
cells for transgene expression leading to the development of gross
tumor formation at 20 weeks of age was not influenced by ovariectomy.
However, estrogen supplementation promoted a large increase in the
number of MIN lesions as well as an acceleration of tumor development.
E2 supplementation to ovariectomized C3(1) /TAG mice also resulted in more aggressive lesion formation as assessed by histopathology. E2 given to mice ovariectomized at 8 weeks of age induced significantly more high-grade MIN lesions and invasive carcinomas than untreated ovariectomized mice (data not shown). Small, early MIN lesions showed a higher grade of cellular atypia with a loose pattern of cell-cell interaction compared with early lesions arising in intact mice. In a subgroup (17%) of the mice supplemented with E2, ectopic chondrocyte and bone formation was noted. This incidence is much higher than the occasional ectopic chondrocyte and bone formation observed in mammary glands from control C3(1) /TAG female mice (<1%; Ref. 6 ).
Effect of the Loss of ER Alleles on Mammary Tumor Progression.
Because the prepubertal removal of estrogen by ovariectomy at 3 weeks
of age did not significantly reduce palpable mammary tumor formation
(Fig. 5C)
, experiments were performed to determine whether
mammary tumor development induced by the C3(1)
/TAG
transgene could occur in rudimentary mammary ductal anlage
lacking functional ER. Mice genetically engineered to lack
functional ER (ERKO) exhibit mammary gland hypoplasia with
development arrested prior to ductal branching (13
, 14)
.
To determine whether the C3(1)
/TAG transgene would be
expressed and lead to tumor formation in this mammary epithelium where
development was arrested at an early stage prior to ductal branching.
C3(1)
/TAG mice were generated in the ERKO background.
Previous studies using MMTV-int-1-ERKO mice have demonstrated that
mammary tumors develop in the absence of ER (15)
.
C3(1)
/TAG-ER+/+ females in the FVB/N-C57BL6
mixed background lived significantly longer than
C3(1)
/TAG-ER+/+ females in the FVB/N
background, suggesting that there are important strain differences that
modulate tumor development. Whole-mount preparations of the mammary
glands of C3(1)
/TAG-ER-/- (ERKO) female
mice demonstrated that a primitive mammary ductal system developed with
the formation of only secondary branches near the nipples (Fig. 6A)
. TAG
expression was not detected by immunohistochemistry or by RT-PCR in the
rudimentary mammary ductal epithelium of these mice up to 9 months of
age (data not shown). No proliferative lesions were observed in these
rudimentary mammary structures by histopathological analysis at any
time during the 10-month period of study. These results indicate that
the C3(1)
/TAG transgene is not expressed in the very
rudimentary mammary ductal structures prior to the onset of ductal
branching. The transgene is expressed when the mammary epithelial cells
progress to a more differentiated state at 3 weeks of age. ER is
required for this early differentiation and outgrowth of the ductal
cells. C3(1)
/TAG mice that were heterozygous or wild-type
for ER developed normal mammary glands, and mice from both genotypes
exhibited a similar incidence of mammary gland tumor formation (56.7%
tumor incidence for C3(1)
/TAG-ER+/- and
67.8% tumor incidence for C3(1)
/TAG-ER+/+
mice).
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ERKO females, tumors developed in other organs
of C3(1)
/TAG-ERKO mice similar to those observed in
C3(1)
/TAG ER+/+ in the FVB/N background.
C3(1)
/TAG-ERKO female mice developed foot pad tumors
(23.5%) and salivary gland tumors (5.9%), which expressed high levels
of TAG (Fig. 6B)
. This observation further
suggests that the ER signaling pathway is not required for expression
of the C3(1)
/TAG transgene. However, unlike
C3(1)
/TAG mice in the FVB/N background, carcinomas of the
nasal structures were more frequently observed in the hybrid
FVB/N x C57BL6 background (<1% versus
64.7%, respectively). Animals were sacrificed because of the morbidity
associated with these nonmammary tumors.
There was no significant difference in survival rates among
C3(1)
/TAG transgenic mice with either the
ER+/+, ER+/-, or
ER-/- genotypes. All C3(1)
/TAG
mice died before 10 months of age, regardless of the ER genotype.
Death was either because of the development of mammary tumors in mice
carrying the ER+/+ or
ER+/- genotypes or because of salivary and
nasal tumors in C3(1)
/TAG mice carrying the
ER-/-.
ER Expression Is Reduced during Mammary Tumor Progression.
ER expression was observed only in mammary epithelial cells with
normal morphology (Fig. 2E)
and low-grade MIN lesions in
C3(1)
/TAG transgenic mice (Fig. 2F)
. There was a
dramatic decrease in ER expression in high-grade MIN lesions, with
some cells demonstrating weak nuclear staining, although normal
neighboring luminal cells stained strongly positively for ER (Fig. 2G)
. ER expression in invasive carcinomas was low to
undetectable (Fig. 2H)
. A similar reduction in ER
expression was observed during mammary tumor progression, as assessed
by Northern and Western blot analyses (Fig. 7)
. Expression of TAG protein
increased during mammary tumor progression, as determined by Western
blot analysis (Fig. 7A)
, whereas ER expression was
dramatically decreased in tumors compared with normal FVB/N mammary
glands or preneoplastic lesions. Northern blotting also demonstrated
that steady-state levels of a 6.6-kb ER transcript were
significantly lower in tumors compared with normal tissue and
preneoplastic lesions, suggesting that the down-regulation of ER
occurs at the RNA level (Fig. 7B)
. Because normal mammary
tissue and tissues with MIN lesions contain a much smaller percentage
of epithelial cells compared with the carcinomas, the relative
reduction in ER expression in the tumor cells is presumably even
greater than that depicted by these results.
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was inversely related to estrogen exposure.
ER was elevated in tumors that developed with reduced exposure to
estrogen (OVX) compared with tumors from unmanipulated transgenic
animals (Figure 1
B, Lane 1 compared with Lane 2).
E2 supplementation to OVX mice tended to suppress
expression of ER (Fig. 1
B, Lane 5 compared with
Lane 2). ER expression tended to be increased in the TAM
group compared with that of intact animals (data not shown).
TAG expression in mammary tumors from intact and OVX3W mice
was similar, further demonstrating that transgene expression was not
dependent upon estrogen signaling (Fig. 1C)
.
ERß expression as determined by RT-PCR was barely detectable in
normal mammary tissue but appeared to increase during mammary tumor
progression (Fig. 8)
. ERß expression
was not significantly altered in tumors arising from the
hormone-manipulated groups.
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, and
EGFR. All the reactions were semiquantitatively confirmed by serial
dilution of template cDNA samples using the same number of cycles.
Expression of TGF- and EGFR appear to increase in early lesions,
whereas expression of c-myc and neu appears to increase at the invasive
carcinoma stage (Fig. 8)
. There was no difference in the expression of
these genes in tumors derived from mice in which estrogen levels were
increased or decreased compared with tumors derived from intact mice
(data not shown). However, we do not know whether expression of these
genes might have been altered by changes in estrogen levels at earlier
stages of tumor progression. |
DISCUSSION |
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The C3(1)
/TAG transgene, however, is active in ductal
epithelial cells, terminal end buds, and the TLDUs in virgin mammary
glands. In this study, we have demonstrated both in vitro
and in vivo that the C3(1)
5' flanking region is not
trans-activated by estrogen. This is consistent with a
previous study where the endogenous expression of C3(1)
in the rat was
shown to be stimulated by androgen but not by estrogen
(17)
. Earlier studies of the C3(1) gene
demonstrated that the major hormone response element is contained
within the first intron (18
, 19)
, which is not contained
in the C3(1)
/TAG transgene used in this model. Our results
demonstrate that the C3(1)
5' flanking region is not responsive to
estrogen stimulation as evidenced by the following: (a)
TAG expression is maintained in tumors and tumor cell lines
despite the loss of ER expression; (b) estrogen does not
stimulate transgene expression in transgenic mammary glands or
transgenic mammary cells lines, even with the cotransfection of ER.
ER signaling is maintained, however, as evidenced by the
down-regulation of endogenous ER expression in response to
E2 and the up-regulation ER in the presence of
anti-estrogen therapy; (c) C3(1)
/TAG expression
and tumor formation occur in several different organs in genetically
modified mice completely lacking functional ER; and (d)
prepubertal ovariectomy resulted in similar rates of gross tumor
formation as that observed in intact animals.
Expression of the C3(1)
/TAG transgene occurs only in
mammary target cells that have reached the ductal outgrowth stage. Mice
containing the transgene but lacking ER develop only vestigial ducts
present at the nipples without ductal outgrowth (20)
. The
C3(1)
/TAG transgene is not transcribed in these rudimentary
mammary anlagen, and tumors do not develop in contrast to
MMTV-int-1/ERKO mice where tumors arise (15)
. However, in
mice containing ER where ductal outgrowth occurs, the
C3(1)
/TAG transgene is expressed, and tumors form when mice
are ovariectomized as early as 3 weeks of age. Thus, full maturation of
the mammary epithelium is not necessary for tumor formation.
Although E2 did not increase expression of the transgene, E2 induced significantly more MIN lesions, invasive carcinomas, larger tumors, and reduced survival. These effects of estrogen may be attributable to an increase in the number of target cells that express the transgene and/or by promotion of tumorigenesis through other stimulatory mechanisms. It is also possible that the stimulation of tumorigenesis by estrogen is mediated through paracrine mechanisms involving the stroma. We have demonstrated that E2 causes proliferative changes in the mammary epithelium, in particular by increasing cell numbers in the TDLU. Because these cells are targets for C3(1) /TAG expression, it seems likely that increased numbers of lesions induced by E2 results at least in part from the expansion of the number of target cells. There is strong evidence that a similar phenomenon occurs in human breast cancer, where higher density of breast epithelium is associated with an increased risk of breast cancer (21) . Hormone replacement therapy is associated with increased breast density (22 , 23) , which may be part of the mechanism of hormone replacement therapy in elevating the cumulative risk of developing breast cancer (2 , 21) .
The histological features of mammary lesions in the C3(1) /TAG transgenic females share resemblance to human ductal carcinoma in situ and infiltrating ductal adenocarcinomas (8 , 9) . In addition to accelerated growth of mammary lesions, estrogen stimulation induced histological changes in the tumors. More severe nuclear atypism within MIN lesions was noted with E2 treatment. From these results, it appears that E2 accelerates the process of "malignant conversion" in this model. Although there was a trend toward a reduction in the number of MIN lesions and tumors arising in animals given TAM, this was not statistically significant. It is likely that the major population of target cells for transgene expression was already developed by 8 weeks of age when TAM treatment was begun, and that TAM, therefore, had little effect on lesion development compared with intact mice.
ER is expressed in normal mammary ductal cells and in low-grade MIN
lesions in which TAG is expressed. However, in high-grade
MIN and invasive carcinomas, the expression of ER mRNA and protein
is significantly reduced, as determined by Northern blotting,
immunohistochemistry, and Western blotting. It, therefore, appears that
reduced ER expression occurs as part of the process of tumor
progression and is not the result of a clonal outgrowth of epithelial
cells that are initially ER negative. To our knowledge, this is the
first report demonstrating the loss of ER expression during tumor
progression in a transgenic model for mammary cancer. Human breast
cancers often progress to an ER-negative state that is generally
associated with a more aggressive disease course and poorer clinical
outcome because they rarely respond to hormone therapies
(24)
.
The expression of the C3(1)
/TAG transgene does not increase
in response to E2 in vitro and in
vivo, but E2 does reduce endogenous ER expression
as expected. These data suggest that despite an intact estrogen
signaling pathway, estrogen does not affect transcription of the
C3(1)
/TAG transgene. The expression of TAG did
not appear to interfere with estrogen signaling because ER
expression diminished in response to exogenous E2, despite
the presence of TAG.
Although it appears paradoxical that estrogen promotes tumorigenesis in
this model whereas expression of ER is reduced during tumor
progression, these findings suggest that estrogen signaling may play
different roles in oncogenesis, depending upon tumor stage. In this
model, it appears that estrogen can stimulate proliferation of the
target tissue for transgene expression and may also result in the
up-regulation of cellular factors that promote tumorigenesis following
the initiation of oncogenesis by TAG. Once this interaction
occurs during the MIN stage, further progression to invasive carcinoma
may no longer require the influence of estrogen and expression of
ER. Given the fact that ER expression is consistently reduced
during advanced stages of tumorigenesis, it is possible that there is
selective pressure to down-regulate ER expression to provide a
growth advantage for invasive tumor development.
A second ER, ERß, has been cloned from the rat prostate (25) . ERß mRNA is expressed in both normal and neoplastic human breast tissues (26 , 27) . In this study, ERß expression was not detected in normal mammary glands but was detectable in transgenic mammary tumors. The role of ERß in mammary tumorigenesis in this model will require further study.
We have demonstrated previously that mammary tumor progression in this
model involves several genetic alterations including amplification and
overexpression of Ki-ras
(28)
,6
loss of
p53, Rb, and p21 function (9
, 29)
7
and loss of
protective apoptotic mechanisms (30). In this study, we
have explored whether the expression of other genes relevant to human
breast cancer are altered during mammary tumor progression in this
model. The expression of her2/neu mRNA appeared to be slightly elevated
in carcinomas, but expression in tumors was not affected by hormone
manipulations. Both TGF- and EGFR were up-regulated at the MIN
stage, suggesting that an autocrine/paracrine mechanism involving this
pathway might be an early event in this model of mammary cancer
development. Expression of c-myc is up-regulated in the carcinomas but
not in MIN lesions in this transgenic model, suggesting that c-myc
overexpression may be a late event in mammary oncogenesis in this
model. Hormone manipulations did not induce a change in the expression
of c-myc in the mammary tumors, suggesting that the ER pathway is
not a major regulator of c-myc in this model.
In summary, mammary oncogenesis in the C3(1)
/TAG transgenic
model appears to be promoted by estrogen through a mechanism that does
not involve up-regulation of transgene expression by estrogen. The
mechanism for this stimulatory effect of estrogen on tumorigenesis will
be the focus of future studies. Further insights into how ER
expression is lost during tumor progression in this model may be highly
relevant to understanding the transition in human breast cancer from an
ER-positive to an ER-negative state.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 K. Y. is a Japan Society for the Promotion
of Science Research Fellow in Biomedical and Behavioral Research at
NIH. 
2 To whom requests for reprints should be
addressed, at Laboratory of Cell Regulation and Carcinogenesis,
National Cancer Institute, NIH, Building 41, Room C629, 41 Library
Drive, Bethesda, MD 20892. Phone: (301) 435-5193; Fax: (301) 496-8395
E-mail: jegreen{at}nih.gov
3 The abbreviations used are: ER, estrogen
receptor; E2, 17ß-estradiol; MIN, mammary intraepithelial
neoplastic; TAM, tamoxifen; OVX3W and OVX8W, ovariectomy at 3 and 8
weeks of age, respectively; TDLU, terminal ductal lobular unit; RT-PCR,
reverse transcription-PCR; TGF, transforming growth factor; EGFR,
epidermal growth factor receptor; MMTV, mouse mammary tumor virus;
G3PDH, glyceraldehyde-3-phosphate dehydrogenase.
4 C. L. Jorcyk, M. Anver, and J. E. Green,
manuscript in preparation.
5 M-L. Liu, M-A. Shibata, F. C. Von Lintig, W.
Wang, S. Cassenaer, G. Boss, and J. E. Green, manuscript in
preparation.
6 M. A. Shibata, C. L. Jorcyk, and J. E. Green.
Suppression of mammary carcinoma growth in vitro and
in vivo by inducible expression of the Cdk inhibitor p21.
Cancer Gene Therapy, in press, 2000.
7 Present address: Osaka Medical College, Osaka
569-8686, Japan.
Received 5/30/00. Accepted 10/18/00.
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