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. Author manuscript; available in PMC: 2011 Feb 28.
Published in final edited form as: Oncogene. 2007 Jul 23;27(6):794–802. doi: 10.1038/sj.onc.1210674

Activation of estrogen signaling pathways collaborates with loss of Brca1 to promote development of ERα-negative and ERα-positive mammary preneoplasia and cancer

LP Jones 1,1,2, MT Tilli 1,1, S Assefnia 1,1, K Torre 1, ED Halama 1,3, A Parrish 1,4, EM Rosen 1, PA Furth 1
PMCID: PMC3045705  NIHMSID: NIHMS267133  PMID: 17653086

Abstract

BRCA1 can regulate estrogen receptor-α (ERα) activity. This study tested the hypotheses that Brca1 loss in mammary epithelium alters the estrogenic growth response and that exposure to increased estrogen or ERα collaborates with Brca1 deficiency to accelerate preneoplasia and cancer development. Longer ductal extension was found in mammary glands of Brca1f/f;MMTV-Cre mice during puberty as compared to wild-type mice. Terminal end bud differentiation was impaired in Brca1 mutant mice with preservation of prolactin-induced alveolar differentiation. Exogenous estrogen stimulated an abnormal sustained increase in mammary epithelial cell proliferation and the appearance of ERα-negative preneoplasia in postpubertal Brca1 mutant mice. Carcinogenesis was investigated using Brca1f/f;MMTV-Cre mice hemizygous for p53. Exogenous estrogen increased the percentage of mice with multiple hyperplastic alveolar nodules. Targeted conditional ERα overexpression in mammary epithelial cells of mice that were Brca1 mutant and hemizygous for p53 increased the percentage of mice exhibiting multiple hyperplastic nodules, invasive mammary cancers and cancer multiplicity. Significantly more than half of the preneoplasia and cancers were ERα negative even as their initiation was promoted by ERα overexpression.

Keywords: breast cancer, mouse model, brca1, estrogen, ERα

Introduction

Women carrying germ-line mutations of the breast cancer susceptibility gene (BRCA1) are predisposed to breast and ovarian cancers (Miki et al., 1994). While most BRCA1 mutation-related breast cancers are estrogen receptor-α (ERα) and progesterone receptor (PR) negative (Foulkes et al., 2004), there is a role for estrogen in early stages of cancer progression. Ovariectomy decreases breast cancer in women (Rebbeck et al., 2002) and mice (Bachelier et al., 2005). BRCA1 represses ERα-mediated gene transcription (Fan et al., 2002), inhibits estrogen signaling to extracellular signal-regulated protein kinase and reduces cell proliferation (Razandi et al., 2004). Tamoxifen-mediated ERα agonist activity promotes cancer development in Brca1-deficient mice (Jones et al., 2005).

Mammary gland development is synchronized by hormones and growth factors (Fendrick et al., 1998). Estrogen signaling through ERα promotes cellular proliferation and differentiation and stimulates terminal end bud (TEB) formation and ductal development (Bocchinfuso and Korach, 1997). TEBs are the growing ends of mammary ducts that appear during puberty and differentiate into terminal ductal ends when pubertal mammary gland development is completed. Cell proliferation is transient in mammary epithelial cells with downregulation by negatively regulating factors including the transforming growth factor-β (TGF-β) family (Grimm and Rosen, 2006). Placental lactogen and prolactin govern alveolar differentiation during pregnancy (Dembinski and Shiu, 1987).

Like humans, Brca1 mutant mammary cancers that develop in mice are predominantly ERα negative (Xu et al., 1999; Brodie et al., 2001; Bachelier et al., 2005). Human BRCA-1 mutant breast cancers frequently exhibit p53 mutation (Gudmundsdottir and Ashworth, 2006), and p53 haploinsufficiency increases cancer incidence in mouse models. Brca1 loss in mice produces an exaggerated proliferative response to exogenous progesterone (Ma et al., 2006) and mifepristone is cancer protective (Poole et al., 2006). Finally, Brca1-deficient mammary glands demonstrate increased ANG1 (Furuta et al., 2006), Igf-1, Irs-1 and Igf-1r (Shukla et al., 2006), indicating that multiple pathways implicated in breast carcinogenesis are altered by Brca1 loss.

This study tested the hypotheses that Brca1 loss in mammary epithelium alters the estrogenic growth response and that increased estrogen signaling collaborates with Brca1 deficiency to accelerate preneoplasia and cancer development. Two experimental approaches were employed: exogenous estrogen exposure and a targeted conditional increase in ERα expression in mammary epithelial cells (Frech et al., 2005a, b). Results supported the hypothesis that estrogen signaling plays a role in early stages of Brca1 mutation-related breast cancer development.

Results

Mammary glands of Brca1f/f;MMTV-Cre mice exhibit longer ductal extension during puberty and impaired terminal end bud differentiation

To test if Brca1 loss leads to an abnormal growth response to endogenous estrogen during puberty, ductal extension was analysed on mammary gland whole mounts from 3- to 6-week-old Brca1f/f;MMTV-Cre and wild-type mice. At 3 weeks of age, ductal extension was comparable in Brca1-deficientand wild-type mice (Figures 1a and b); however at age 6 weeks ductal extension was significantly longer in Brca1f/f;MMTV-Cre as compared to wild-type mice (39.8±3.6 vs 17.3±2.9, t-test, P <0.006; Figures 1e–g). To test if Brca1 loss compromised the differentiation response of TEBs to estrogen and progesterone, 3-week-old Brca1f/f;MMTV-Cre and wild-type mice were exposed to continuous 17β-estradiol and progesterone for 2 weeks. Wild-type mice exhibited the expected TEB differentiation into terminal ductal ends (Figure 1d) while TEBs from Brca1f/f;MMTV-Cre mice did not (Figure 1c). In contrast, both Brca1f/f;MMTV-Cre and wild-type mice exhibited normal alveolar differentiation (Figures 2a–d) when exposed to prolactin in organ culture. Because increased p27 expression is found in transgenic mice expressing dominant-negative Brca1 (Deans et al., 2004), p27, p21 and estrogen signaling-associated gene products c-myc, cyclin d1 and cyclin e were examined but no significant differences were found with Brca1 loss (Figure 3). In summary, Brca1 loss impaired estrogen-and progesterone-induced differentiation but not the different developmental stage of prolactin-induced differentiation.

Figure 1.

Figure 1

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Mammary glands of Brca1f/f;MMTV-Cre mice exhibit longer ductal extension during puberty and impaired TEB differentiation. Mammary gland whole mounts of 3-week (a and b), 17β-estradiol (E) + progesterone (P)-treated 5-week (c and d) and 6-week-old (e and f) Brca1f/f;MMTV-Cre and wild-type female mice. Long thin arrows indicate extent of ductal elongation (a and b). Black arrowheads indicate retained TEBs (c and inset). Thin white arrows indicate differentiated terminal ductal ends (d and inset). Bar graph comparing ductal elongation (g). Magnification, × 0.5; scale bar, 1000 µm.

Figure 2.

Figure 2

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Normal prolactin-induced alveolar differentiation in Brca1f/f;MMTV-Cre and wild-type mice. Mammary gland whole mounts (a, b, e and f) and H&E (c, d, g and h) sections from Brca1f/f;MMTV-Cre and wild-type female mice after 7 days in organ culture medium containing insulin (I) + aldosterone (A) + hydrocortisone (H) with (a–d) or without (e–h) prolactin (P). Arrows indicate alveolar differentiation in higher magnification insets (thick arrows) (a and b), TEBs (thin arrows) (e) or terminal ductal ends (arrowhead) (f). Whole mount: magnification, × 0.5; scale bar, 1000 µm. H&E: magnification, × 10; scale bar, 100 µm.

Figure 3.

Figure 3

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Expression patterns of c-myc, p27, p21, cyclin d1 and cyclin e are similar in TEBs of wild-type, Brca1f/f;MMTV-Cre and Brca1f/f;MMTV-Cr/p53+/− mice. Immunohistochemical staining of c-myc (a–c), p21 (d–f), p27 (g–i), cyclin d1 (j–l) and cyclin e (m–o) in TEBs of 6-week-old wild-type (a, d, g, j and m), Brca1f/f;MMTV-Cre (b, e, h, k and n) and Brca1f/f;MMTV-Cre P53+/− (c, f, i, l and o) mice. Arrows indicate nuclear-localized c-myc, p21, p27, cyclin d1 or cyclin e expression. Large images, × 20; small insets, × 40. Medium insets are cross sections, × 20; scale bars, 20 µm.

Exogenous estrogen stimulated a sustained increase in mammary epithelial cell proliferation and abnormal growth in postpubertal Brca1f/f;MMTV-Cre mice

To test if Brca1 loss altered estrogen response, postpubertal Brca1f/f;MMTV-Cre and wild-type mice were implanted with a 17β-estradiol or placebo pellet and mammary glands were analysed 8 weeks later. A sustained increase in cellular proliferation was found in Brca1f/f;MMTV-Cre mice (1.2±0.4 vs 11.2±1.8, placebo vs 17β-estradiol-treated, Mann–Whitney, P <0.0001; Figures 4a and b) unlike wild-type mice (2.9±1.7 vs 1.6±0.3, placebo vs 17β-estradiol-treated, Mann–Whitney, P = 0.96; Figures 4c and d). Brca1f/f;MMTV-Cre/p53+/− mice showed the same response as Brca1f/f;MMTV-Cre mice (data not shown). Whole mount analyses demonstrated dense mammary epithelial cell growth in estrogen- treated intact and ovariectomized Brca1f/f;MMTV-Cre (Figures 5a, b, e and f) but not in wild-type mice (Figures 5c, d, g and h). Progesterone may contribute to the more uniform growth pattern in intact (Figure 5b) as compared to ovariectomized mice (Figure 5f). Percentages of nuclear-localized ERα were not different between placebo-treated Brca1f/f;MMTV-Cre and wild-type mice (5.7±0.9 vs 3.5±0.6, Mann–Whitney, P <0.17; Figures 4e and g). ERα was downregulated in Brca1f/f;MMTV-Cre and wild-type mice following estrogen treatment (Figures 4f and h). No significant differences in the cell percentages demonstrating nuclear-localized ERβ or PR expression were found (Figures 4e–h). The proliferative response did not alter epithelial cell organization (Figure 6). Steady-state RNA expression levels of Tgfβ1, Tgfβ2, Tgfβr-1, Tgfβr-2 (analysis of variance (ANOVA), P <0.0001), Tgfβ3 (ANOVA, P <0.02) and Igfbp-4 (ANOVA, P <0.04) were lower in mammary glands from Brca1f/f;MMTV-Cre compared to wild-type mice (Supplementary data). Brca1 loss did not alter expression levels of Vegfa, Flt4, Kdr, Figf, Kit, Tgfα, C-met, Hgf, Fgf7 and Fgfr2.

Figure 4.

Figure 4

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Exogenous estrogen stimulated an abnormal sustained increase in mammary epithelial cell proliferation in postpubertal Brca1f/f;MMTV-Cre mice. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA), estrogen receptor-α (ERα), progesterone receptor (PR, insets) and estrogen receptor-β (ERβ, insets) in mammary gland sections from 6-month-old placebo (a, c, e and g) and 17β-estradiol-treated (b, d, f and h) Brca1f/f;MMTV-Cre and wild-type female mice. Arrows indicate cells demonstrating nuclear-localized PCNA, ERα, PR or ERβ expression. Magnification, × 40; scale bars, 20 µm.

Figure 5.

Figure 5

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Exogenous estrogen stimulated abnormal growth in postpubertal Brca1f/f;MMTV-Cre mice. Mammary gland whole mounts of 6-month-old placebo (a, c, e and g) and 17β-estradiol-treated (b, d, f and h) intact (a–d) and ovariectomized (ovex) (e–h) Brca1f/f;MMTV-Cre and wild-type female mice. Thick arrowheads indicate areas of abnormal ductal growth found in the 17βestradiol-treated Brca1f/f;MMTV-Cre mice. Thin arrows indicate normal-appearing ductal structures in the placebo-treated Brca1f/f;MMTV-Cre and placebo and 17β-estradiol-treated wild-type mice. Magnification, × 4; scale bar, 200 µm.

Figure 6.

Figure 6

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Organization of the mammary epithelium was not altered by the abnormal proliferative response. Hematoxylin and eosin (H&E)-stained sections (a and b) and immunohistochemical detection of retinoid X receptor-α (RXR) (thick arrows) (c, d and g) and smooth-muscle actin (SMA, thin arrows) (e, f and h) in mammary gland sections from 6-month-old placebo (a, c, e, g and h) and 17β-estradiol-treated (b, d and f) Brca1f/f;MMTV-Cre and wild-type female mice. Magnification, × 40; scale bars, 20 µm.

p53 deficiency cooperates with estrogen stimulation in the development of hyperplastic alveolar nodules (HANs)

p53 haploinsufficiency increases the percentage of Brca1f/f;MMTV-Cre mice that develop invasive mammary adenocarcinomas (Xu et al., 1999). HANs are noninvasive preneoplastic lesions identified in mammary gland whole mounts that reflect early events in cancer development(Heppner et al., 2000). Estrogen exposure increased the mean number of HANs/gland and the percentage of mice with HANs similar to the effects of p53 haploinsufficiency. p53 haploinsufficiency increased the mean number of HANs from 0.3±0.1 in Brca1f/f;MMTV-Cre mice to 2.0±0.5 in Brca1f/f;MMTV-Cre/p53+/− mice (t-test, P <0.013) and percentage of mice with HANs from 19 to 45% (χ2, P <0.011). Estrogen exposure increased the mean number of HANs in Brca1f/f;MMTV-Cre mice to 2.0±0.8 (t-test, P <0.004) and percentage of mice with HANs to 86% (χ2, P <0.003). The combination of p53 haploinsufficiency and estrogen exposure increased mean number of HANs to 13.0±2.6 (t-test, P <0.0001) and the prevalence to 92% (χ2, P <0.002) as compared to untreated Brca1f/f;MMTV-Cre/p53+/− mice. Study duration was limited by development of ureteral obstruction (Arbeit et al., 1996) 1–5 months after pellet placement in 71% of the Brca1f/f;MMTV-Cre and 1–3 months after pellet placement in 25% of the Brca1f/f;MMTV-Cre/p53+/− mice. Fourteen percent of estrogen-exposed Brca1f/f;MMTV-Cre mice and 25% of the Brca1f/f;MMTV-Cre/p53+/− mice developed invasive mammary cancer similar to percentages found in untreated mice (Xu et al., 1999; Jones et al., 2005).

Targeted ERα overexpression in mammary epithelial cells of Brca1f/f;MMTV-Cre/p53+/−CERM mice increased the percentage of mice exhibiting multiple hyperplastic nodules and invasive multiple mammary cancers

To increase estrogen signaling pathway activity specifically in mammary epithelial cells and avoid toxicity from systemic estrogen exposure, the conditional estrogen receptor α in mammory tissue (CERM) system (Frech et al., 2005a, b) was introduced into Brca1f/f;MMTV-Cre mice to generate Brca1f/f;MMTV-Cre/CERM and Brca1f/f;MMTV-Cre/p53+/−/CERM mice. Brca1f/f;MMTV-Cre/CERM mice demonstrated a modest nonsignificant increase in the mean number of HANs/gland (0.6±0.4) and HAN prevalence (40%) as compared to Brca1f/f;MMTV-Cre mice. ERα overexpression in Brca1f/f;MMTV-Cre/p53+/−/CERM mice increased mean number of HANs/gland (14.3±3.7, t-test, P <0.0001) and HAN prevalence (100%, χ2, P <0.023) compared to Brca1f/f;MMTV-Cre/p53+/− mice. Levels were comparable to estrogen-treated Brca1f/f;MMTV-Cre/p53+/− mice. The percentage of Brca1f/f;MMTV-Cre/p53+/−/CERM mice developing invasive cancer was increased (100%, χ2, P <0.023) as compared to control Brca1f/f;MMTV-Cre/p53+/− mice (53%) and mean number of palpable invasive cancers per mouse was increased to 2.6±0.4 in mice from 0.6±0.6 in (t-test, P <0.0001). The percentage of mice with multiple invasive cancers was higher in Brca1f/f;MMTV-Cre/p53+/−/CERM mice (86%) as compared to Brca1f/f;MMTV-Cre/p53+/− mice (10%). In summary increasing the activity of the estrogen signaling pathway through upregulation of ERα expression promoted the development of both HANs and invasive mammary cancer in the setting of p53 haploinsufficiency.

ERα-negative preneoplasia and cancers developed in both Brca1f/f;MMTV-Cre/p53+/− and Brca1f/f;MMTV-Cre p53+/−/CERM mice

ERα-negative preneoplasia and cancers developed in Brca1f/f;MMTV-Cre/p53+/− (Figures 7q and r) and Brca1f/f;MMTV-Cre/p53+/−/CERM mice (Figures 7f and n). The fact that increased ERα expression facilitated the appearance of HANs (Figure 7a, inset), preneoplasia (Figures 7a and e) and invasive cancers (Figures 7i and m) raised the possibility that preneoplasia and cancers would be ERα positive. ERα immunohistochemistry was performed on both preneoplasia (Figures 7b and f) and invasive cancers (Figures 7j, n and r) fromBrca1f/f;MMTV-Cre/p53+/− and Brca1f/f;MMTV-Cre/p53+/−/CERM mice. Preneoplasia and cancers from Brca1f/f;MMTV-Cre/p53+/− mice were ERα negative (Figures 7r). In contrast, some (Figures 7b and j), but not all (Figures 7f and n), preneoplasia and invasive cancers from Brca1f/f;MMTV-Cre/p53+/−/CERM mice demonstrated nuclear-localized ERα. Expression of the ERα target gene PR followed the ERα staining pattern (Figures 7d, h, l, p and t). Cre expression was found in Brca1f/f;MMTV-Cre/p53+/−/CERM (Figures 7c, g, k and o) and Brca1f/f;MMTV-Cre/p53+/− (Figure 7s) mice with parallel expression patterns of ERα and Cre in ERα-positive Brca1f/f;MMTV-Cre/p53+/−/CERM mice (Figures 7c and k), suggesting that some cells simultaneously lost full-length Brca1 and expressed ERα. However, even when ERα overexpression promoted development of mammary cancer, ERα-negative preneoplasia and cancer still emerged.

Figure 7.

Figure 7

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ERα-negative preneoplasia and cancer in Brca1f/f;MMTV-Cre/p53+/− and ERα-negative and -positive preneoplasia and cancer in Brca1f/f;MMTV-Cre/p53+/−/CERM mice. Hematoxylin and eosin (H&E)-stained sections (a, e, i, m and q), representative HAN from a mammary gland whole mount(a, inset) and immunohistochemical detection of nuclear-localized estrogen receptor-α (ERα; b and j), Cre (c, g, k, o, s and w), nuclear-localized progesterone receptor (PR) (d and l) in preneoplasia (a–h, w) and invasive cancer (i–t and v) from Brca1f/f;MMTV-Cre/p53+/−/CERM and Brca1f/f;MMTV-Cre/p53+/− mice. Representative ERα-and PR-negative preneoplasia (f and h) and invasive cancer (n and p) from Brca1f/f;MMTV-Cre/p53+/−/CERM mice and invasive cancer (r and t) from Brca1f/f;MMTV-Cre/p53+/− mice. Negative controls: no Cre antibody (u and v). Magnification, × 40.

Discussion

Loss of full-length Brca1 in mammary epithelial cells in mice was associated with an abnormal response to estrogen stimulation that correlated with enhanced development of preneoplasia and invasive mammary cancer. Impaired differentiation responses during puberty and after exogenous estrogen and progesterone exposure were identified. Reduction of BRCA1 by RNA interference in a 3D culture system also results in failure of differentiation into mammary acini and increased cell proliferation (Furuta et al., 2005). This impaired differentiation response could be one reason why pregnancy does not appear to be as protective against the development of breast cancer in women who carry a BRCA1 mutation as other populations (Cullinane et al., 2005). Anomalous but not absent grow th and differentiation responses are consistent with the fact that either loss of full-length Brca1 or BRCA1 mutation does not abort mammary gland development; instead it produces a gland that functions normally in many ways but is predisposed toward cancer development.

Estrogen stimulated sustained proliferation of ERα-negative mammary epithelial cells when Brca1 function was impaired. Estrogen-induced cell proliferation is normally mediated through paracrine signaling pathways including epidermal growth factor (EGF)- and insulin growth factor (IGF)-associated pathways (Fendrick et al., 1998; Silberstein, 2001). BRCA1 can inhibit EGF and IGF signaling (Razandi et al., 2004) and loss of Brca1 in mouse mammary glands is associated with increased expression levels of Igf-1, Irs-1 and Igf-1r (Shukla et al., 2006). This study showed that loss of Brca1 was associated with increased expression levels of Igfbp4 and decreased expression levels of both receptors and ligands from the inhibitory TGF-β signaling pathway. This suggests a model in which loss of Brca1 leads to increased activity of IGF signaling pathways and decreased activity of the TGF-β pathways resulting in an abnormal and sustained proliferative response to estrogen. The target cell for this proliferative response was not identified; however, it is known that over half of the adenocarcinomas that develop in this model demonstrate gene expression patterns reminiscent of human basal cell cancers, a type of human cancer that is overrepresented in BRCA1 mutation carriers (Herschkowitz et al., 2007).

Retention of retinoid X receptor-α (RXRα) expression in the proliferating cells suggests that RXR ligands, perhaps in combination with PPARγ, could be a chemopreventive approach for Brca1 mutation-related cancer through differentiation and/or reduced IGF levels (Decensi et al., 2003).

Expression of PR is regulated by the estrogen signaling pathway in mammary tissue and mediates some estrogen-induced actions (Fendrick et al., 1998). The PR inhibitor mifepristone is cancer protective in a different but related p53 null mouse model of Brca1 mutation-related breast cancer (Poole et al., 2006). Mifepristone increases TGF-β1 expression, decreases cell proliferation and promotes apoptosis in ERα- and PR-positive and -negative breast cancer cell lines (El Etreby et al., 1998; Liang et al., 2003). In tumor explants, progesterone increases IGF availability (Krzysiek et al., 2003) and mifepristone reduces this by stimulating secretion of IGFBP-3 (Milewicz et al., 2005). It is possible that mifepristone may reduce cancer development by upregulation of inhibitory factors such as TGF-β1 and/or downregulation of stimulatory pathways including IGF. Progesterone and IGF increase ductal elongation in the mammary gland (Ruan et al., 2005) and progesterone and the PR-B isoform has been shown to increase insulin receptor substrate-2 expression (Vassen et al., 1999; Cui et al., 2003). One can speculate that the enhanced ductal elongation observed in the Brca1-deficient mice was secondary to increased PR and IGF signaling in the pubertal mice.

Estrogen pathway stimulation was as effective as p53 haploinsufficiency in promoting the development of preneoplasia in the Brca1f/f;MMTV-Cre mice butt he combination of estrogen pathway activation with p53 haploinsufficiency in Brca1f/f;MMTV-Cre/p53+/− and Brca1f/f;MMTV-Cre/p53+/−/CERM mice was more potent than either intervention alone. Increasing ERα expression levels in the BRCA1f/f;MMTV-Cre/p53+/−/CERM mice promoted development of both ERα-positive and -negative invasive mammary cancer. This stands in contrast to another CERM-related model in which TAg and ERα overexpression are combined to produce uniformly ERα-positive mammary cancers (Tilli et al., 2003). The results in the Brca1 mouse model are compatible with data from human BRCA1 mutation carriers where approximately two-thirds of the breast cancers are ERα negative (Foulkes et al., 2004).

The study suggests a model in which loss of Brca1 function results in an aberrant proliferative response to estrogen signaling leading to development of preneoplasia and cancer. In this model, p53 insufficiency is a collaborating factor. It is possible that decreased expression levels of inhibitory factors such as TGF-β family members and increased expression of stimulatory factors including the IGF signaling pathway contribute to the abnormal estrogen-induced growth response.

Materials and methods

Mice

Female C57Bl/6 Brca1f/f;MMTV-Cre, Brca1f/f;MMTV-Cre/p53+/−, Brca1f/f;MMTV-Cre/CERM, Brca1f/f;MMTV-Cre/p53+/−/CERM and wild-type mice were identified using PCR. Mice were maintained under guidelines approved by the Georgetown University Animal Care and Use Committee. MMTV-Cre transgene expression initiates during embryogenesis (Wagner et al., 2001). Subcutaneous pellet placement and ovariectomy (Raafat et al., 1999) was performed under isofluorane anesthesia. Mammary gland organ culture was carried out as described (Ginsburg and Vonderhaar, 2000). Mice were scored as having multiple invasive cancers when more than one palpable cancer was present. Supplementary data provides experimental groups and numbers of mice.

Mammary gland whole mounts, histology and immunohistochemistry

Mounted #4 mammary glands of 3- to 6-week-old mice were measured for ductal extension. In older mice, HANs were counted and prevalence calculated as the percentage of mice/group exhibiting one or more HANs/#4 mammary gland. The other #4 mammary gland from each mouse was fixed in 10% buffered formalin, embedded in paraffin using standard techniques and 5 µm sections stained using hematoxylin and eosin (H&E) or immunohistochemical detection performed: proliferating cell nuclear antigen (PCNA) (1:100; U7032; DAKO, Carpinteria, CA, USA), ERα (1:50; IM2133; Beckman Coulter Immunotech, Miami, FL, USA), ERβ (1:100; GTX70174; GeneTex Inc., San Antonio, TX, USA), PR (1:100; NCL-L-PG-AB; Novacastra, UK), RXRα (1:100; sc-553; Santa Cruz, Santa Cruz, CA, USA), smooth-muscle actin (SMA, 1:100; 1A4, MS-113-P; NeoMarkers, Fremont, CA, USA), c-Myc (1:50; sc-764; Santa Cruz), P21 (1:50; ms-387; NeoMarkers), P27 (1:50; sc-1641; Santa Cruz), cyclin D1 (1:50; sc-8396; Santa Cruz) and cyclin E (1:300; sc-481; Santa Cruz) Cre (1:400; 69050; Novagen, San Diego, CA, USA). Proliferative index and percent ERα-, ERβ- and PR-positive cells were calculated. Digital photographs were taken using a Nikon Eclipse E800M microscope with DMX1200 camera (Nikon Instruments Inc., Melville, NY, USA).

Statistical analyses

Means and s.e. were analysed using Student’s t-test and two-way ANOVA tests (Graphpad Software, San Diego, CA, USA). Nonparametric data were analysed using Mann–Whitney two-tailed and χ2-tests (SPSS Inc., Chicago, IL, USA). P <0.05 was considered statistically significant.

Supplementary Material

Supplementary Data

Acknowledgements

This study was supported by NIH NCI MAO/RFP NO1-CN-05024 (PAF and LPJ), RO1CA08000 (EMR, PAF and KT), 2T32CA09686-08 (LPJ) and the Susan G Komen Breast Cancer Foundation PDF040244 (LPJ and SA) and PDF0503642 (MTT).

Footnotes

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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