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CANCER |
Eppley Institute for Research in Cancer (J.D.S., K.L.P., T.M.R., M.C.S., T.E.S., T.J.S., M.T.) and Departments of Biochemistry and Molecular Biology (J.D.S., T.E.S., T.J.S., M.T.) and Pathology and Microbiology (J.D.S., R.D.M.), University of Nebraska Medical Center, Omaha, Nebraska 68198-6805
Address all correspondence and requests for reprints to: Dr. James Shull, Eppley Cancer Institute, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805. E-mail: jshull{at}unmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have demonstrated that the female of the inbred ACI rat strain exhibits a unique propensity to develop mammary cancers when treated with E2; continuous treatment with E2 induced mammary cancers at an incidence of 100% and a median latency of 143 d (4, 5). In contrast, the female of the Copenhagen (COP) strain, an inbred strain closely related genetically to the ACI strain, is relatively resistant to E2-induced mammary cancers (5, 6). Interestingly, both ACI and COP rats are relatively resistant to mammary cancers induced by dimethylbenz[a]anthracene (DMBA) (7, 8, 9, 10) or N-methyl-N-nitrosourea (MNU) (7, 10, 11, 12). These data suggest that the molecular mechanisms underlying development of E2-induced mammary cancers differ from those for DMBA- or MNU-induced mammary cancers. We have recently demonstrated that the mammary epithelium of the female ACI rat proliferates in response to administered E2 to a greater extent than does the mammary epithelium of the female COP rat, as evidenced by morphometric analysis of the epithelium to adipose tissue ratio and quantification of S phase indexes (13). Together these data suggest that the differing susceptibilities of ACI and COP rats to E2-induced mammary cancers are genetically conferred and result at least in part from differences in the extent to which the mammary epithelia of these two rat strains proliferate in response to E2. A goal of this laboratory is to define the molecular bases for these strain differences in the hope that this information may provide novel insights into the mechanisms through which estrogen contributes to breast cancer etiology in humans.
As the first step in defining the genetic bases of the unique susceptibility of the ACI rat to 17ß-estradiol (E2)-induced mammary cancers, we examined how susceptibility to these cancers segregates in reciprocal crosses between the genetically related ACI and COP rat strains. The data presented herein indicate that susceptibility to E2-induced mammary cancer segregates as an incompletely dominant trait in these reciprocal intercrosses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reciprocal ACI female x COP male (cross A) and COP female x ACI male (cross B) intercrosses were performed to generate (ACI x COP)F1 (F1a) and (COP x ACI)F1 (F1b) progeny, respectively. F1 siblings from each cross were mated to generate F2a and F2b progeny. F1a and F1b males were mated to ACI females to generate backcross (BC) a and BCb progeny, respectively. All pups were weaned at 21 ± 2 d of age. Treatment with E2 was initiated when the rats were 63 ± 4 d of age. SILASTIC brand implants (Dow Corning Corp., Midland, MI) containing 27.5 mg crystalline E2 (Sigma, St. Louis, MO) were prepared and inserted sc in the interscapular region as described previously (4). Beginning approximately 5 wk after initiation of E2 treatment, each animal was examined twice weekly for the presence of palpable mammary tumors. An animal was killed by decapitation when its largest mammary tumor reached 1.5–2.0 cm in diameter or when it exhibited other treatment-related pathology. The location and size of each macroscopic mammary tumor were noted at necropsy. Mammary tissues, both grossly normal and tumors, were collected and evaluated histologically as described previously (4, 13). The pituitary gland was removed and weighed as an indicator of E2-induced lactotroph hyperplasia; pituitary weight correlates with lactotroph number and circulating PRL level (14).
Statistical analysis of data
Latency was scored to the appearance of the first palpable
mammary tumor. Phenotypic differences between different genetic
populations were assessed by the log-rank test and Wilcoxon
rank-sum/Mann-Whitney U test. Where appropriate, differences between
population means were evaluated using two-tailed t test.
P 
0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The first
mammary tumor in the treated ACI population was observed after 88
d of E2 treatment, and 100% of the population at risk exhibited at
least 1 palpable mammary tumor within 197 d of initiation of
treatment. Three of 34 E2-treated ACI rats were killed before the
appearance of palpable mammary tumors due to treatment-associated
morbidities after 144, 165, and 175 d of treatment, resulting in
an overall incidence of 91% in this population. Within the 31
tumor-positive ACI rats, the median and mean latencies to the
appearance of the first palpable tumor were 140 and 141 d,
respectively (Table 1). In contrast, the
first mammary tumor to appear in the population of E2-treated COP rats
was observed after 188 d of E2 treatment, and 44% of the
population at risk, 35% (7 of 20) of the total COP population,
ultimately developed mammary cancers (Fig. 1B). The median and mean
latencies within the tumor-positive COP population were 195 and
208 d, respectively. Log-rank analyses indicated that the ACI and
COP rat strains differed significantly with respect to latency to
appearance of the first palpable mammary cancer (P <
10-6).
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). Median and
mean latencies in the tumor-positive F1a
population were 194 and 207 d, respectively (Table 1). In the
(COP x ACI)F1 (F1b)
population, the first mammary tumor was observed after 87 d of E2
treatment, and 85% of the F1b population at
risk, 81% (35 of 43) of the total F1b
population, were tumor positive within 259 d of initiation of E2
treatment (Fig. 1C). Median and mean latencies to the appearance of the
first palpable mammary tumor in the F1b
population were 157 and 163 d, respectively (Table 1). Comparison
of the E2-treated F1a and
F1b populations by the log-rank test indicated
that they differed significantly (P = 0.02). Although
the incidence of E2-induced mammary cancers in the
F1a and F1b populations
approached that in the ACI parental strain, the latencies in both
F1 populations were significantly prolonged
relative to that of the ACI population (P
10-6, ACI vs.
F1a; P = 1.33 x
10-4, ACI vs.
F1b). Both F1 populations
also differed significantly from the COP population (P
= 1.6 x 10-3, COP vs.
F1a; P = 2.9 x
10-5, COP vs.
F1b). Because the two different
F1 populations exhibited a phenotype intermediate
compared to those of the ACI and COP rat strains, we conclude that
susceptibility to E2-induced mammary cancers is inherited as an
incompletely dominant trait in these reciprocal intercrosses between
the ACI and COP rat strains.
The ability of E2 to induce mammary carcinomas was evaluated in 134
(F1a x
F1a)F2
(F2a) and 172 (F1b x
F1b)F2 (F2b) progeny (Fig. 1D). Mammary cancers were first observed in the E2-treated
F2a and F2b populations
following 102 and 70 d of treatment, respectively, and the final
incidence in the F2a and
F2b populations at risk exceeded 99% (Fig. 1D).
The incidences of E2-induced mammary cancers in the total
F2a and F2b populations
were 86% (115 of 134) and 61% (104 of 172), respectively (Table 1).
Median and mean latencies in the tumor-positive
F2a populations were 184 and 193 d,
respectively. In the tumor-positive F2b
populations, median and mean latencies were 151 and 157 d,
respectively. Log-rank analysis indicated that the two
F2 populations did not differ significantly from
one another with respect to latency to the appearance of the first
palpable mammary cancer (P = 0.80). The combined
F2 population differed significantly from the ACI
(P < 10-6) as well as the COP
(P = 1.87 x 10-4)
populations and exhibited a prolonged latency relative to the
F1b (P = 0.041), but not the
F1a or combined F1,
populations.
Mammary cancers in the (ACI x F1a)BC (BCa)
and (ACI x F1b)BC (BCb) populations were
first observed 117 and 98 d, respectively, after initiation of E2
treatment (Fig. 1E). One hundred percent of the BCa population at risk
and the total BCa population developed at least one palpable mammary
cancer within 231 d of initiation of E2 treatment (Table 1).
Similarly, 100% of the BCb population at risk, 86% of the total BCb
population, was tumor positive by 225 d after initiation of
treatment. Log-rank analyses indicated that these two BC populations
did not differ significantly from one another (P =
0.252). Mammary tumor development in the combined BC population was
significantly delayed compared with that in the ACI population
(P = 6.03 x 10-3), whereas
tumors appeared more rapidly in the combined BC population than in the
COP (P < 10-6), combined
F1 (P = 2.36 x
10-4), or combined F2
(P < 10-6) populations.
Presented in Table 2 are the
percentages of each population exhibiting one or more mammary cancers
after 175 d of E2 treatment, the time point that most clearly
demarcates the phenotypic differences between the treated ACI and COP
populations. At this time point, 84% of the E2-treated ACI rats
exhibited mammary cancer in contrast to 0% for the COP population. The
percentages of rats in the E2-treated F1,
F2, and BC populations were 45%, 41%, and 65%,
respectively. These data closely approximate the fractions of these
populations predicted to be tumor positive by a genetic model in which
one gene, with ACI and COP alleles acting codominantly, determines
susceptibility to E2-induced mammary cancers. However, these data are
also consistent with a model in which two independently segregating
genes determine susceptibility.
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). Twenty-seven of the 31 tumor-positive ACI rats (87%)
exhibited multiple mammary tumors at necropsy. By contrast, tumor
number in the E2-treated COP population ranged from 0–2, and averaged
0.4 tumors/animal at the time the animals were killed. Only 1 of 7
(14%) tumor-positive COP rats exhibited more than 1 tumor at necropsy.
Tumor number at necropsy in the E2-treated ACI population differed
significantly (P = 0.006) from that in the treated COP
population. The average lengths of E2 treatment in the ACI and COP
populations were 171 and 271 d, respectively.
The E2-treated F1a population exhibited, on the
average, 2.6 mammary tumors/rat at the time of death, whereas the
F2b population exhibited, on the average, 1.8
tumors/rat, a difference that was statistically significant
(P = 0.028; Table 1). Tumor number in the
F1a population did not differ significantly from
that in the ACI population (P = 0.077). However, tumor
number in the F1b population was significantly
lower (P = 0.0003) than that in the E2-treated ACI
population. Both the F1a and
F1b populations exhibited significantly more
mammary tumors at the time of death than did the E2-treated COP rats
(P 0.0001 for both comparisons). Tumor number in the
F2a population averaged 2.3 tumors/rat, which was
significantly greater (P 0.01) than the average of
1.2 tumors/rat observed in the F2b population.
The average numbers of tumors observed at the time of death in the
F2a and F2b populations
were significantly less than that observed in the ACI population and
significantly greater than that observed in the COP population. The BCa
and BCb populations exhibited an average of 3.3 and 2.7 mammary
cancers/rat, respectively, at the time of death (Table 1), a difference
that was not statistically significant. The average number of mammary
tumors observed in the E2-treated BCa and BCb populations did not
differ significantly from that observed in the treated ACI population,
but was significantly greater (P 0.01 for both
comparisons) than that observed in the treated COP population. It is
noteworthy that the F1a,
F2a, and BCa progeny generated from the ACI
x COP cross survived longer, on the average, than the
F1b, F2b, and BCb progeny
from the COP x ACI cross. It is possible that the longer duration
of E2 treatment in the progeny from the ACI x COP cross
contributed to the greater mammary tumor burden observed in these
populations relative to that observed in the progeny from the COP
x ACI cross.
Histological features of estrogen-induced mammary cancers
The mammary cancers induced by E2 in ACI and COP rats and their
derived F1, F2, and BC
progeny were adenocarcinomas of the comedo, papillary, or cribriform
types. Invasive features were observed in a subset of the cancers. The
histological appearance of the tumors was similar among the different
genetic groups.
Effects of estrogen treatment on pituitary weight
Pituitary weight was measured to evaluate the possible association
between E2-induced lactotroph hyperplasia and susceptibility to
E2-induced mammary cancers. Pituitary weight in untreated,
ovary-intact, female rats averaged 11.4 mg, and continuous treatment
with E2 increased pituitary weight 5.3- to 17.2-fold in the different
genetic groups (Fig. 2). Although
pituitary weight at necropsy was increased to a similar extent in the
E2-treated ACI and COP populations, the COP population was treated an
average of 100 d longer than the ACI population. These data are
consistent with our previous report that the ACI rat strain is more
sensitive to the pituitary growth-inducing actions of estrogen than the
COP rat strain (14). Interestingly, the stimulatory effect
of E2 on pituitary growth was substantially less in the
F1a, F2a, and BCa females
derived from the ACI x COP cross than in the
F1b, F2b, and BCb progeny
derived from the COP x ACI cross (Fig. 2). No association was
apparent between pituitary weight and susceptibility to E2-induced
mammary cancers in the F2 and BC progeny from the
reciprocal crosses between the ACI and COP rat strains. In the
E2-treated F2 population, pituitary weights in
those animals exhibiting a palpable mammary cancer before 175 d of
treatment were, on the average, 16% less than those in animals
exhibiting their first palpable mammary cancers after 175 d and
46% less than those in animals that did not develop mammary cancers
(Table 3). In the E2-treated BC
population, pituitary weights were, on the average, similar in each of
the three phenotypic classes of animals. Pituitary weights in the
F2 and BC animals are plotted as a function of
duration of E2 treatment and mammary cancer phenotype in Figs. 3 and 4,
respectively. These data clearly illustrate the high degree of
variability in pituitary weights observed in the E2-treated
F2 and BC populations and the lack of correlation
between pituitary weight and susceptibility to E2-induced mammary
cancers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observation that susceptibility to E2-induced mammary cancers in
reciprocal ACI x COP intercrosses is inherited as an incompletely
dominant phenotype is consistent with multiple genetic models. In the
first model the ACI allele of a single gene, inherited through the germ
line, would confer susceptibility in F1 progeny.
Somatic loss of the COP allele of this putative tumor suppressor gene
would be required for mammary tumors to develop in
F1 animals. Consequently, latency to tumor
appearance would be prolonged, and tumor burden would be reduced in
F1 females relative to that observed in the
parental ACI strain. In this model genomic instability could contribute
to COP allele loss and mammary cancer development. We demonstrated that
the mammary cancers induced in ACI rats by E2 commonly exhibit
aneuploidy (13). Moreover, unpublished data from our
laboratory indicate widespread allelic imbalances consistent with LOH
in mammary cancers induced in (ACI x COP)F1
progeny by E2 (17). Thus, genetic instability is a common
feature of E2-induced mammary cancers, and this could contribute to
mammary carcinogenesis in this model. The phenotypic data presented
herein are also consistent with a model in which two independently
segregating genes confer susceptibility to E2-induced mammary cancers.
Using the formula: n = (µF1 -
µACI)2/4
G2
(18), where n represents the number of loci modifying a
specific trait, µF1 and
µACI represent the phenotypic means of the
F1 and ACI populations, respectively, and
G2 represents the genetic
variance of the backcross population, it is estimated that no more than
two independently segregating genes confer susceptibility to E2-induced
mammary cancers in the ACI x COP intercrosses. Using the
phenotypically defined F2 and BC populations
described herein, we have mapped to rat chromosome 5 a locus,
Emca1, that modifies susceptibility to E2-induced
mammary cancers in the reciprocal ACI x COP intercrosses
(19) (Tochacek, M., T. M. Reindl, C. R.
Murrin, E. A. VanderWoude, and J. D. Shull, manuscript in
preparation).
For the most part, the mammary cancer profiles generated in the two reciprocal crosses between the ACI and COP rat strains were very similar. The F1a population from the ACI x COP cross exhibited a significantly prolonged latency relative to the F1b population from the COP x ACI cross. However, the F2 and BC populations from the two crosses did not differ significantly from one another. The numbers of mammary tumors observed at necropsy were also somewhat higher in the F1, F2, and BC populations from the ACI x COP cross relative to the numbers observed in the corresponding populations from the COP x ACI cross. Therefore, we cannot exclude at this time the possibility that latency and/or tumor burden might be modified by either an X-linked or imprinted autosomal locus.
It has often been suggested that mammary cancers that develop in rats that are treated chronically with estrogen are secondary to the development of PRL-producing pituitary tumors and associated hyperprolactinemia (20, 21, 22). Pituitary weight in estrogen-treated rats correlates with the absolute lactotroph number (23) as well as with the circulating PRL level (14, 23). Although the ACI and COP rat strains differ markedly in susceptibility to E2-induced mammary cancers, both strains exhibit significant pituitary growth and hyperprolactinemia in response to administered estrogen (4, 6, 14). In the present study each of the E2-treated ACI, COP, F1, F2, and BC animals exhibited significantly increased pituitary weight relative to untreated female rats of the same genetic background. Moreover, in the genetically diverse F2 and BC populations, latency to appearance of mammary carcinoma did not correlate with pituitary weight at the time of death. Finally, we have demonstrated that ovariectomy markedly inhibits E2-induced mammary carcinogenesis in the ACI rat without inhibiting pituitary growth and associated hyperprolactinemia (4). Together, these data indicate that E2- induced pituitary growth and associated hyperprolactinemia are insufficient for mammary cancer development. This, in turn, implies that the mammary cancers that develop in response to continuous E2 treatment are not simply the consequence of pituitary tumor-associated hyperprolactinemia. We have recently mapped four quantitative trait loci in ACI x COP crosses that modify the pituitary growth response to administered estrogen (Strecker, T. E., T. J. Spady, A. Kaufman, F. Chen, and J. D. Shull, manuscript in preparation). These quantitative trait loci are distinct from the Emca1 modifier of susceptibility to E2-induced mammary cancers identified by us in similar crosses, indicating that the tumor-inducing actions of estrogen in the mammary gland are genetically dissociable from those in the pituitary gland.
The ACI rat appears by numerous criteria to represent a unique and physiologically relevant animal model for study of the molecular and hormonal etiology of breast cancer. Mammary tumors develop rapidly and at high incidence in response to E2. The cancers are estrogen dependent and, like most breast cancers in humans, exhibit genomic instability (13). The data presented herein indicate that this unique susceptibility of the ACI rat to E2-induced mammary cancers segregates as an incompletely dominant phenotype in crosses to the COP rat strain, which is considered to represent the paradigm of genetically conferred resistance to experimental mammary cancer. Further elucidation of the genetic bases of susceptibility to E2-induced mammary cancers in this rat model should significantly enhance our understanding of the mechanisms through which estrogens contribute to breast cancer etiology and provide insights into the genetic epidemiology of breast cancer.
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Acknowledgments |
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Footnotes |
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Abbreviations: BC, Backcross; BCa, (ACI x F1a)BC; BCb, (ACI x F1b)BC; BN, Brown Norway; COP, Copenhagen; DMBA, dimethylbenz[a]anthracene; F1, first filial; F2, second filial; MNU, N-methyl- N-nitrosourea.
Received August 1, 2001.
Accepted for publication August 28, 2001.
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,
F1a; 
, F1b. D, 
