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Carcinogenesis |
Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709 [W. P. B., J. F. C., K. S. K.]; and Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892 [W. P. H., H. E. V.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) knockout (ERKO) mice, which lack the gene encoding ER, and mouse mammary virus tumor (MMTV)-Wnt-1 transgenic mice (Wnt-1 TG), which develop mammary hyperplasia and neoplasia due to ectopic production of the Wnt-1 secretory glycoprotein. We have crossed these lines to ascertain the effects of ER deficiency on mammary gland development and carcinogenesis in mice expressing the Wnt-1 transgene. Introduction of the Wnt-1 transgene into the ERKO background stimulates proliferation of alveolar-like epithelium, indicating that Wnt-1 protein can promote mitogenesis in the absence of an ER-mediated response. The hyperplastic glandular tissue remains confined to the nipple region, implying that the requirement for ER in ductal expansion is not overcome by ectopic Wnt-1. Tumors were detected in virgin ERKO females expressing the Wnt-1 transgene at an average age (48 weeks) that is twice that seen in virgin Wnt-1 TG mice (24 weeks) competent to produce ER. Prepubertal ovariectomy of Wnt-1 TG mice also extended tumor latency to 42 weeks. However, pregnancy did not appear to accelerate the appearance of tumors in Wnt-1 TG mice, and tumor growth rates were not measurably affected by late ovariectomy. Small hyperplastic mammary glands were observed in Wnt-1 TG males, regardless of ER gene status; the glands were similar in appearance to those found in ERKO/Wnt-1 TG females. Mammary tumors also occurred in Wnt-1 TG males; latency tended to be longer in the heterozygous ER and ERKO males (86 to 100 weeks) than in wild-type ER mice (ca. 75 weeks). We conclude that ectopic expression of the Wnt-1 proto-oncogene can induce mammary hyperplasia and tumorigenesis in the absence of ER in female and male mice. The delayed time of tumor appearance may depend on the number of cells at risk of secondary events in the hyperplastic glands, on the carcinogenesis-promoting effects of ER signaling, or on both. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,2
a member of the superfamily of nuclear receptors that can function as ligand-inducible transcription factors (4)
. A second gene encoding another ER, termed ERß, has been identified and may mediate estrogen signaling in some tissues (5, 6, 7, 8)
.
During puberty, mammary epithelial cells divide and migrate into the stromal fat pad as TEBs in response to estradiol and growth hormone in a process called ductal morphogenesis (9, 10, 11, 12)
. Mitosis occurs in the cap cell layer of the TEBs that appear to be devoid of ER; ER is present, however, in the ductal epithelium and mammary stromal cells (9)
. ERß mRNA is detectable in human and rat mammary tissue (8
, 13, 14, 15)
; however, its role in mammary development remains inconclusive.
The estrogen/ER signaling pathway may stimulate proliferation of both ER-positive and -negative mammary epithelium (16
, 17)
. Estrogens can act directly on epithelial cells and/or stimulate stromal cells to secrete growth factors that induce mitogenesis in the epithelium (18
, 19
). Furthermore, estrogens can stimulate the secretion of pituitary prolactin that induces mitogenesis in mammary epithelium (20
, 21)
. Therefore, estrogens may affect the growth of both ER-positive and -negative mammary tumors (11
, 22)
.
To assess the role of ER in mammary gland carcinogenesis, we have introduced into ERKO mice an MMTV-Wnt-1 transgene known to induce mammary hyperplasia and carcinomas (23)
. Wnt genes encode secretory glycoproteins that normally act in autocrine and paracrine fashions to stimulate cell proliferation and differentiation (24)
. Several members of the Wnt gene family are expressed in the mouse mammary gland during various stages of development (24, 25, 26)
. Although Wnt-1 is not normally produced in the mammary gland, insertional activation of the Wnt-1 gene by MMTV proviruses initiate mammary carcinogenesis (27
, 28)
, and a transgene that simulates an insertionally activated locus produces mammary hyperplasia in both male and female animals (23)
. Virtually all female mice with the MMTV-Wnt-1 transgene develop mammary tumors within the first year of life, with half the animals displaying tumors at 
5–6 months of age. In addition, 15–30% of the male Wnt-1 TGs have mammary tumors by the age of 1 year (23)
.
In previous crosses of the Wnt-1 TG mice to genetically altered mice, we found that other oncogenes (e.g., FGF-3) and tumor suppressor genes (e.g., p53) affect Wnt-1-induced tumorigenesis (29
, 30)
. Here, we report that the Wnt-1 transgene can overcome a genetic deficiency of ER to produce mammary hyperplasia and tumors, but the hyperplasia is limited by impaired ductal morphogenesis and the onset of tumors is significantly delayed. In addition, the effect on tumor latency can be mimicked by prepubertal ovariectomy. Thus, estrogen-/ER-mediated signaling is not required for Wnt-1-induced proliferation and oncogenic conversion of mammary epithelium. Estrogenic stimulation of the mammary gland can affect mammary carcinogenesis in this model system by determining the number of cells at risk of neoplastic conversion, directly promoting the growth of tumor cells, or both.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene (ER+/-). First-generation HET/Wnt-1 TG males were then crossed with HET females. The offspring consisted of mice with the six possible genotypes. The mice were monitored biweekly for tumors of 0.5 cm in diameter, and their ages were recorded at the time of tumor discovery.
Genotyping.
Genomic DNA was isolated from a tail biopsy using a procedure described previously (31)
, and mouse genotypes were identified by PCR. Three pairs of PCR primers were synthesized to detect the intact ER gene, the disrupted ER gene and the Wnt-1 transgene. Primers used to detect the intact ER gene (forward, 5'-CGGTCTACGGCCAGTCGGGCATC-3'; and reverse, 5'-GTAGAAGGCGGGAGGGCCGGTGTC-3') anneal to sites that flank the neo gene insertion site used to disrupt the ER gene in exon 2 (32)
. These primers generate a 239-bp PCR product from the intact ER gene. The neo gene primers (forward, 5'-GTGTTCCGGCTGTCAGCGCA-3'; reverse, 5'-GTCCTGATAGCGGTCCGCCA-3') anneal to the neo gene used to disrupt the ER gene and generate a PCR product of 555 bp. To detect the Wnt-1 transgene, the forward Wnt-1 primer (5'-GGACTTGCTTCTCTTCTCATAGCC-3') anneals to the 3' end of the Wnt-1 gene and the reverse SV40 primer (5'-CCACACAGGCATAGAGTGTCTGC-3') anneals to the SV40 polyadenylation sequence inserted immediately downstream of the Wnt-1 gene in the TG construct (23)
. This primer pair generates a 407-bp PCR product. All three primer sets (5 pmol of ER primers, 10 pmol of Wnt-1 primers, and 50 pmol of neo primers) were used in a single-tube reaction. The PCR conditions were 95°C denaturing, 65°C annealing., and 72°C extension for 30 cycles using Taq polymerase and a DNA Thermal Cycler from Perkin-Elmer. The PCR products were resolved by electrophoresis in 2% Nusieve low melting agarose-0.7% agarose-1x T Tris-borate EDTA gels (FMC Bioproducts).
Ovariectomy and Pregnancy Studies.
To determine whether tumors could continue to grow after estradiol deprivation, mice were ovariectomized under anesthesia by Veterinary Medicine (National Institute of Environmental Health Sciences, Research Triangle Park, NC) after a tumor of 0.5–1.0-cm diameter was observed. The mice were monitored weekly for tumor enlargement and then sacrificed when the tumors grew a further 3-fold in diameter. To determine whether estradiol at puberty is required for tumor development, mice were ovariectomized before puberty (15 days old) and then monitored for the development of tumors 0.5 cm in diameter. To determine whether pregnancy could accelerate tumor onset, Wnt-1 TG females were continuously mated beginning at 8 weeks of age and monitored for tumor development.
Kaplan-Meier Analysis.
The compiled data of mouse age at the time of tumor appearance were subjected to Kaplan-Meier analysis and plotted as a function of the probability of a mouse being tumor free versus its age in weeks. This analysis also takes into account the time a mouse on study remains tumor free and then is removed from study before tumor appearance due to death by old age, sickness, and so on. The various plots were then compared to each other using a life table test (33)
to determine whether the rate of tumor development was statistically different among the Wnt-1 TG mice with different ER genotypes.
Mammary Whole-Mount and Histological Analyses.
Mammary glands were stained and whole mounted according to a modified procedure from Russo et al. (34)
. Inguinal mammary fat pads were excised from euthanized mice and placed in a tissue histocassette containing a biopsy foam pad. The fat pads were placed in 10% formalin for at least 24 h and then defatted in acetone over 5–7 days. The fat pads were rehydrated in a graded series of alcohols to distilled water and then stained in a 0.035% toluidine blue dye solution for 30–60 min. The fat pads were destained in methanol for 30–60 min, 70% ethanol for 30 min, and postfixed in 4% ammonium molybdate for 30 min. After remaining in distilled water overnight, the fat pads were dehydrated in a graded series of alcohols to xylene and then mounted using Permount (Fisher). Mammary glands and tumors excised for histological analyses were fixed in 10% formalin, embedded in paraffin, sectioned at 5-µm thickness, and stained with H&E.
RPA.
Radiolabeled antisense RNA probes for mouse Wnt-1, keratin 18, and cyclophilin were generated from linearized cDNA templates in pBluescript (Wnt-1 and keratin 18) and pTRI-cyclophilin (Ambion) using T7 RNA polymerase and [32P]CTP (Amersham) according to the Maxiscript kit protocol (Ambion). The length of protected probe fragments were: Wnt-1, 404 nt; keratin 18, 547 nt; cyclophilin, 103 nt.
RPA reactions consisting of 5 x 104 cpm of each riboprobe, mammary gland RNA (1 µg), and yeast tRNA (24 µg) were mixed and precipitated in ethanol overnight at -70°C. The pellets were further processed according to the Hybspeed RPA kit (Ambion) protocol. Protected probe fragments were resolved by electrophoresis using a 1.5-mm 6% bis-acrylamide, 8.3 M urea, and 1x Tris-borate EDTA gel (National Diagnostics). The gels were fixed, dried, and exposed to a Phosphorimager screen followed by exposure to X-ray film. RPA results were analyzed using the Phosphorimager Storm 860 and ImageQuant software (Molecular Dynamics).
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RESULTS |
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gene on Wnt-1-induced mammary neoplasia, the ERKO mouse line was crossed with the Wnt-1 TG line. Three Wnt-1 TG males were mated with six females heterozygous for the ER gene (ER+/-, HET) to generate HET/Wnt-1 TG males, which were then crossed with HET females to generate wild-type, ERKO (ER-/-), Wnt-1 TG, and ERKO/Wnt-1 TG mice. Mammary gland morphology, mammary tumor incidence, and the effect of ovarian hormones on tumor development were examined in these mice.
Inguinal mammary glands from mice bearing the Wnt-1 transgene in the presence or absence of the ER gene were analyzed at 10 weeks of age. Virgin wild-type female mice progressed through mammary ductal morphogenesis as indicated by the presence of bulbous TEBs and an ordered ductal network (Fig. 1A)
. As reported previously (23)
, mammary glands from Wnt-1 TG females showed excessive hyperplasia, consisting of extensive ductal side-branching and the development of aberrant lobular-like structures (Fig. 1C)
. The ERKO female mammary gland was undeveloped, as illustrated by the rudimentary ductal network that remained confined to the nipple region and lacked both TEBs and alveolar structures (Fig. 1B)
. In contrast, the ERKO/Wnt-1 TG mammary gland was comprised of a hyperplastic rudimentary ductal structure without TEBs (Fig. 1D)
.
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affected progression of Wnt-1-induced hyperplasia, mammary glands from 6-month-old virgin Wnt-1 TG and ERKO/Wnt-1 TG female mice were examined. At 6 months, the Wnt-1 TG female mammary gland was comprised of hyperplastic epithelium that occupied the entire fat pad (Fig. 1E)
. In contrast, mammary glands from 6 month ERKO/Wnt-1 TG mice exhibited hyperplasia (Fig. 1F)
that was similar to that seen in glands from the 10 week old mice (Fig. 1D)
. Hyperplasia remained confined to the nipple region of ERKO/Wnt-1 TG glands, with no directed growth into the inguinal fat pad beyond the lymph node (Fig. 1F)
. This restricted mammary hyperplasia was maintained with age in ERKO/Wnt-1 females.
Mammary gland tissue sections were analyzed by H&E staining to determine whether cellular morphology was altered due to Wnt-1 expression. Wnt-1 TG female mammary glands consisted of closely spaced ducts and more periductal connective tissue, indicative of ductal and lobuloalveolar hyperplasia, compared to the normal ductal spacing and adipose stroma of wild-type mammary glands (compare Fig. 2, A and B
). The ERKO/Wnt-1 TG mammary gland contained regions of lobuloalveolar hyperplasia at the periphery of the gland. Ductal hyperplasia and a dense connective tissue stroma were evident in the interior of the gland (Fig. 2C)
.
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Gene Status.
. In contrast, mammary glands from both Wnt-1 TG and ERKO/Wnt-1 TG male mice exhibited epithelial hyperplasia (Fig. 3, C and D
, respectively), which appeared similar to the ERKO/Wnt-1 TG female mammary glands (Fig. 1F)
. Cellular morphology of Wnt-1 TG and ERKO/Wnt-1 TG male mammary glands (data not shown) was similar to that seen in female ERKO/Wnt-1 TG mammary glands (Fig. 2C)
.
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Delays Wnt-1-induced Mammary Tumor Development. gene levels on Wnt-1-induced mammary tumor incidence and latency, we recorded the ages at which mammary tumors (
0.5-cm diameter) were discovered and subjected the data to Kaplan-Meier analysis. Wnt-1 TG and HET/Wnt-1 TG females developed mammary tumors at the same rate; tumors appeared in one-half of the mice in each group by 24 weeks of age (Fig. 4A)
. In contrast, the onset of tumors in ERKO/Wnt-1 TG females was delayed, with one-half of the mice developing tumors by 48 weeks (Fig. 4A)
. The onset of tumors in Wnt-1 TG males of all three ER genotypes was more delayed than in the ERKO/Wnt-1 TG females, despite similar levels of hyperplasia. One-half of the Wnt-1 males in each ER gene group developed tumors by the following ages: 75 weeks (wild-type), 86 weeks (HET), and 100 weeks (ERKO; Fig. 4B
). The absence of ER did not appear to affect the type of mammary tumors that developed. The tumors that arose in mice of both sexes were lobuloalveolar adenocarcinomas as reported previously (23)
, regardless of ER gene copy number (data not shown).
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Ovariectomy was performed on Wnt-1 TG females at 15 days of age to determine whether estradiol production at puberty is required for mammary tumorigenesis. This experiment also permitted a comparison of systemic estradiol deprivation versus an ER-negative environment on mammary hyperplasia and tumor development. Prepubertal ovariectomy of Wnt-1 TG females did not prevent Wnt-1-induced mammary hyperplasia, which was morphologically similar to that seen in surgically intact ERKO/Wnt-1 TG females (see Fig. 1, D and F
). Ovariectomized Wnt-1 TG females developed tumors at an average age of 42 weeks, which is delayed compared to the intact Wnt-1 TG females (24 weeks) but similar to the intact ERKO/Wnt-1 TG group (48 weeks; Fig. 5
). In addition, eight ERKO/Wnt-1 TG females were ovariectomized before puberty and exhibited mammary hyperplasia similar in morphology to their surgically intact counterparts (Fig. 1, D and F)
. Four of these mice developed mammary tumors at an age (mean ± SD) of 60 ± 11 weeks, whereas four mice died at the ages of 80, 81, 87, and 112 weeks without developing tumors.
Estrogen Action Is Not Mediated by ERß, and Wnt-1 Gene Expression Is Not Altered in the Absence of ER.
As described above, ERKO/Wnt-1 TG females developed tumors, and tumor latency was increased in these mice by prepubertal ovariectomy. Possible tumor-promoting actions of estradiol and progesterone may be mediated by ERß and PR, respectively, in the ERKO/Wnt-1 TG mammary gland. RPAs demonstrated that ERß mRNA was not detected in wild-type or ERKO mammary glands, confirming a previous report (35)
. Furthermore, ERß gene expression was apparently not induced by Wnt-1 because ERß mRNA was not detected in hyperplastic mammary or tumor tissue from Wnt-1 TG and ERKO/Wnt-1 TG mice (data not shown). In contrast, PR mRNA was readily observable by RPA in hyperplastic mammary and tumor tissue from Wnt-1 TG and ERKO/Wnt-1 TG mice (data not shown).
The possibility remained that Wnt-1 transgene expression may be reduced in mammary epithelium lacking ER, accounting for the increased latency of mammary tumors in ERKO/Wnt-1 TG mice. Normalizing the level of Wnt-1 mRNA in bulk mammary tissue to keratin 18 mRNA, an epithelial cell marker, can distinguish differences in Wnt-1 gene expression that are due either to variations in epithelial cell abundance or to altered expression within the epithelial compartment (29
, 36)
. As expected, keratin 18 mRNA was detected in the mammary glands of wild-type females but not in ERKO females, and Wnt-1 mRNA was not detected in wild-type or ERKO mammary glands from either sex (Fig. 6)
. When normalized to keratin 18 mRNA, Wnt-1 mRNA levels were essentially the same in Wnt-1 TG and ERKO/Wnt-1 TG mammary tissues from either sex (Fig. 6)
. Wnt-1 mRNA levels were increased in mammary tumors compared to hyperplastic glands; however, keratin 18 mRNA was also increased, indicating epithelial cell abundance (Fig. 6)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-deficient background, we have begun to dissect the complex relationship that exists between the hormonal factors that normally govern organ development and the oncogenic lesions that produce tumors in that organ. Here, it is apparent that the proliferative and tumor-inducing effects of ectopic Wnt-1 expression in the mammary gland can occur in the absence of ER. Mammary epithelial cells responsive to Wnt-1 are produced without estrogenic stimulation. Furthermore, the Wnt-1 signaling pathway, which has been shown to use several transcription factors (37, 38, 39, 40, 41)
, does not require any contribution from ER. However, tumor latency is prolonged in ERKO/Wnt-1 TG mice and in mice ovariectomized before sexual maturity. This effect may be ascribed to the reduced size of the mammary glands, direct effects of estrogen on tumor promotion, or both.
Estrogen/ER signaling is essential for mammary ductal morphogenesis and lobuloalveolar development by inducing estrogen-responsive gene products that contribute to these processes (42)
. The rudimentary ductal network emanating from the nipple in the ERKO female clearly demonstrates the importance of estrogen/ER action in mammary gland development. The ERKO/Wnt-1 TG female mammary gland was comprised of hyperplastic epithelium in the nipple region of the gland. Therefore, the Wnt-1 signal did not require a functional ER to stimulate proliferation or hyperplasia. However, the hyperplasia did not progress into the inguinal mammary fat pad beyond the lymph node, indicating that ductal elongation did not occur as a result of Wnt-1 action in the ERKO female. Therefore, Wnt-1 cannot substitute for ER-mediated ductal morphogenesis involving cap cell division and TEB formation. A previous report demonstrated that reconstituted mammary epithelium expressing Wnt-1 from a retrovirus vector did not develop TEBs, and the rate of hyperplastic growth was reduced after ovariectomy (43)
. However, another study showed that mammary epithelium transplanted from 3-month-old Wnt-1 TG females into ovariectomized, athymic nude mice developed hyperplasia that occupied the entire mammary fat pad (44)
. Because ovariectomy was performed during adulthood in those studies, the host mice were exposed to estradiol before transplant which may account for the expanded mammary hyperplasia. Our results demonstrate that prepubertal ovariectomy and/or the lack of ER in Wnt-1 TG females prevents hyperplastic progression beyond the inguinal lymph node.
Wild-type male mammary ductal rudiments generally undergo regression in response to fetal testicular androgens on days 14–16 of gestation (45)
. The lack of ER in male mammary cells did not appear to have an impact on this apoptotic process because ERKO male mice did not develop the mammary ductal rudiments seen in ERKO females. However, Wnt-1 TG males possessing or lacking the ER gene retained a hyperplastic mammary rudiment indicating that male mammary regression was impaired and/or the rate of cellular proliferation was greater than apoptosis. Indeed, Wnt-1-induced hyperplasia was shown to be evident in mammary rudiments at day 18 of gestation (46)
.
ERKO/Wnt-1 TG females exhibit extensive mammary hyperplasia and likely possess the elevated concentration of serum estradiol associated with the female ERKO phenotype (47)
. Yet, the onset of tumors was delayed in ERKO/Wnt-1 TG mice compared to Wnt-1 TG females. Furthermore, ovariectomized Wnt-1 TG females possessed hyperplastic mammary glands and a tumor latency that was similar to intact ERKO/Wnt-1 TG mice. These data suggest that ER may promote mammary tumor growth in a ligand (estradiol)-dependent manner, which is absent in ERKO/Wnt-1 TG females. The ERKO/Wnt-1 TG and ovariectomized Wnt-1 TG females possess a reduced number of mammary epithelial cells at risk of neoplastic transformation, which may also contribute to increased tumor latency compared to intact Wnt-1 TG females. In addition, ductal morphogenesis is absent in ERKO/Wnt-1 TG and ovariectomized Wnt-1 TG females. Dividing cap cells, which may be very susceptible to neoplastic conversion (11)
, are probably lacking in these mammary glands, as noted by the absence of TEBs. Therefore, defective ductal morphogenesis may also contribute to tumor latency.
Ovariectomized Wnt-1 TG mice developed tumors earlier than did ovariectomized ERKO/Wnt-1 TG mice. These results indicate that the presence of ER in Wnt-1 TG mice may also promote tumorigenesis in the absence of estradiol, suggesting a role for ligand-independent activation of ER. Growth factors acting through their tyrosine kinase receptors have been shown to indirectly activate ER (48, 49, 50, 51, 52)
, and the mitogen-activated protein kinase pathway has been implicated in this process (53
, 54)
. A Wnt-1-induced neoplastic environment may possess aberrant growth factor signaling that indirectly influences ER signaling.
Prepubertal ovariectomy appeared to increase tumor latency in ERKO/Wnt-1 TG females, suggesting that ER-negative mammary glands could still respond to estradiol. A recently identified second ER, termed ERß (5)
, may mediate estrogen signaling in certain adult tissues. ERß mRNA has been detected in rat mammary glands and human breast tissue, tumors, and immortalized mammary cancer cell lines using RT-PCR and RPA (8
, 13, 14, 15)
. ERß mRNA has also been detected in mouse mammary tissue by RT-PCR (55)
, but an RPA did not detect ERß mRNA in mammary glands from wild-type or ERKO female mice (35)
. In our study, ERß mRNA expression was not detected in Wnt-1-expressing mammary or tumor tissue of either sex. Therefore, ERß is unlikely to mediate the apparent tumor-promoting effects of estradiol in ERKO/Wnt-1 TG mammary glands. However, progesterone circulates at near normal levels in ERKO female mice (47)
and could provide a growth stimulus to ERKO/Wnt-1 TG mammary glands that express the PR gene.
Unlike the female cohort, male mice expressing the Wnt-1 transgene possessed a similar degree of mammary hyperplasia regardless of ER genotype, and there was little difference in the circulating sex steroid levels of wild-type and ERKO males (56
, 57)
. Therefore, differences in mammary tumor latency among the Wnt-1 TG males could be attributed more directly to the promotional effects of ER. An apparent ER gene dosage effect on mammary tumor development was noted in TG Wnt-1 males, i.e., a decrease in ER gene levels resulted in increased tumor latency. Wnt-1 TG males of each ER genotype developed mammary tumors later than ERKO/Wnt-1 TG females despite similar levels of mammary hyperplasia. This difference in tumor onset may be due to the tumor-promoting effect of ovarian hormones and to other sex-specific factors that contribute to the susceptibility of female mammary epithelium to carcinogenesis.
Differences in tumor latency between ER-positive and negative mammary tissue were not due to altered levels of Wnt-1 transgene expression. Wnt-1 mRNA levels were dependent on mammary epithelial cell abundance, and neither ER gene levels nor the sex of the mouse influenced Wnt-1 expression. Expression of the Wnt-1 transgene is dependent on the Wnt-1 promoter and the MMTV LTR enhancer, which provides epithelial cell-specific expression (23
, 58)
. Our results are consistent with the fact that the MMTV LTR is not inducible by estrogen (59)
. Although the MMTV LTR is progesterone inducible (59
, 60)
, pregnancy did not appear to accelerate tumor onset in Wnt-1 TG females in this study.
The action of ovarian hormones, particularly estradiol, is strongly associated with the development of breast cancer (2
, 61)
. Approximately 70% of primary breast tumors in women are ER positive and exhibit estrogen-dependent growth (62)
. The most malignant mammary tumors, which are ER negative and exhibit estrogen-independent aggressive growth, are thought to progress from an ER-positive state (11
, 22)
. However, only 10–25% of human and mouse mammary epithelial cells possess ER, raising the possibility that tumors could initiate directly from either ER-positive or -negative epithelium (16
, 17)
. ER is also present in mammary stromal cells, which are thought to secrete growth factors that stimulate mitogenesis in the epithelial compartment (18
, 19)
. Therefore, the ERKO/Wnt-1 TG mouse model has demonstrated that hyperplasia and tumorigenesis can be induced in mammary glands deficient in estrogen signaling. Furthermore, ER-negative mammary tumors do not necessarily have to progress from an ER-positive state but can be initiated directly in ER-negative epithelium.
Finally, many other TG mice expressing different oncogenes develop mammary tumors (36)
. On the basis of our mouse model, it may be useful to determine whether the absence of ER has any impact on the development and growth of mammary tumors in these TG lines.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, NIH, MD B302, P.O. Box 12233, Research Triangle Park, NC 27709. Phone: (919) 541-3512; Fax: (919) 541-0696. 
2 The abbreviations used are: ER, estrogen receptor; TEB, terminal end bud; ERKO, ER knockout; MMTV, mouse mammary tumor virus; TG, transgenic; HET, heterozygous; nt, nucleotide(s); RPA, RNase protection assay; PR, progesterone receptor; LTR, long terminal repeat.
Received 9/25/98. Accepted 2/17/99.
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REFERENCES |
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(ER) and estrogen receptor-ß (ERß) messenger ribonucleic acid in the wild-type and ER-knockout mouse. Endocrinology, 138: 4613-4621, 1997. and ß. Endocrinology, 139: 111-118, 1998.This article has been cited by other articles:
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 |
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  X.-Y. Wang, Y. Yin, H. Yuan, T. Sakamaki, H. Okano, and R. I. Glazer Musashi1 Modulates Mammary Progenitor Cell Expansion through Proliferin-Mediated Activation of the Wnt and Notch Pathways Mol. Cell. Biol., June 1, 2008; 28(11): 3589 - 3599. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Huang, S. V. Fernandez, S. Goodwin, P. A. Russo, I. H. Russo, T. R. Sutter, and J. Russo Epithelial to Mesenchymal Transition in Human Breast Epithelial Cells Transformed by 17 -Estradiol Cancer Res., December 1, 2007; 67(23): 11147 - 11157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kuorelahti, S. Rulli, I. Huhtaniemi, and M. Poutanen Human Chorionic Gonadotropin (hCG) Up-Regulates wnt5b and wnt7b in the Mammary Gland, and hCG{beta} Transgenic Female Mice Present with Mammary Gland Tumors Exhibiting Characteristics of the Wnt/{beta}-Catenin Pathway Activation Endocrinology, August 1, 2007; 148(8): 3694 - 3703. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Silva and M. A. Shupnik Integration of Steroid and Growth Factor Pathways in Breast Cancer: Focus on Signal Transducers and Activators of Transcription and Their Potential Role in Resistance Mol. Endocrinol., July 1, 2007; 21(7): 1499 - 1512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Labbe, L. Lock, A. Letamendia, A. E. Gorska, R. Gryfe, S. Gallinger, H. L. Moses, and L. Attisano Transcriptional Cooperation between the Transforming Growth Factor-{beta} and Wnt Pathways in Mammary and Intestinal Tumorigenesis Cancer Res., January 1, 2007; 67(1): 75 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Russo, S. V. Fernandez, P. A. Russo, R. Fernbaugh, F. S. Sheriff, H. M. Lareef, J. Garber, and I. H. Russo 17-Beta-estradiol induces transformation and tumorigenesis in human breast epithelial cells FASEB J, August 1, 2006; 20(10): 1622 - 1634. [Abstract] [Full Text] [PDF]
|
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L. Blavier, A. Lazaryev, F. Dorey, G. M. Shackleford, and Y. A. DeClerck Matrix metalloproteinases play an active role in wnt1-induced mammary tumorigenesis. Cancer Res., March 1, 2006; 66(5): 2691 - 2699. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Han, F. J. DeMayo, J. Xu, S. Y. Tsai, M.-J. Tsai, and B. W. O'Malley Steroid Receptor Coactivator (SRC)-1 and SRC-3 Differentially Modulate Tissue-Specific Activation Functions of the Progesterone Receptor Mol. Endocrinol., January 1, 2006; 20(1): 45 - 55. [Abstract] [Full Text] [PDF]
|
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|
 |
|
 D. J. Mulholland, S. Dedhar, G. A. Coetzee, and C. C. Nelson Interaction of Nuclear Receptors with the Wnt/{beta}-Catenin/Tcf Signaling Axis: Wnt You Like to Know? Endocr. Rev., December 1, 2005; 26(7): 898 - 915. [Abstract] [Full Text] [PDF]
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S.-Q. Kuang, L. Liao, S. Wang, D. Medina, B. W. O'Malley, and J. Xu Mice Lacking the Amplified in Breast Cancer 1/Steroid Receptor Coactivator-3 Are Resistant to Chemical Carcinogen-Induced Mammary Tumorigenesis Cancer Res., September 1, 2005; 65(17): 7993 - 8002. [Abstract] [Full Text] [PDF]
|
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M. S. Frech, E. D. Halama, M. T. Tilli, B. Singh, E. J. Gunther, L. A. Chodosh, J. A. Flaws, and P. A. Furth Deregulated Estrogen Receptor {alpha} Expression in Mammary Epithelial Cells of Transgenic Mice Results in the Development of Ductal Carcinoma In situ Cancer Res., February 1, 2005; 65(3): 681 - 685. [Abstract] |
, HET, n = 38; 
, ERKO, n = 26. Statistical comparisons were made of the following plots (significance in parentheses): wild-type versus HET (P = 0.957), wild-type versus ERKO (P = 0.001), and HET versus ERKO (P = 0.001). B, Wnt-1 TG males with the following ER
, female ERKO/Wnt-1 TG, n = 26. Statistical comparisons were made of the following plots (significance in parentheses): wild-type versus HET (P = 0.237), wild-type versus ERKO (P = 0.057), and HET versus ERKO (P = 0.352). Statistical comparisons of the female ERKO/Wnt-1 TG plot versus the following male Wnt-1 plots were made (significance in parentheses): wild-type (P = 0.154), HET (P = 0.002), and ERKO (P = 0.003).
