Breast Cancer Res Treat (2014) 143:423–433 DOI 10.1007/s10549-013-2817-2

PRECLINICAL STUDY

Progesterone stimulates progenitor cells in normal human breast and breast cancer cells Heidi N. Hilton • N. Santucci • A. Silvestri • S. Kantimm • L. I. Huschtscha • J. D. Graham C. L. Clarke



Received: 14 November 2013 / Accepted: 18 December 2013 / Published online: 7 January 2014 Ó Springer Science+Business Media New York 2014

Abstract The epithelium of the human breast is made up of a branching ductal–lobular system, which is lined by a single layer of luminal cells surrounded by a contractile basal cell layer. The co-ordinated development of stem/progenitor cells into these luminal and basal cells is fundamentally important for breast morphogenesis. The ovarian steroid hormones, progesterone (P) and 17b-estradiol, are critical in driving this normal breast development, yet ovarian activity has also been shown to be a major driver of breast cancer risk. We previously demonstrated that P treatment increases proliferation and augments the number of progenitor-like cells, and that the progesterone receptor (PR) is also expressed in the bipotent progenitor-enriched subfraction. Here we demonstrate that PR is expressed in a subset of CD10? basal cells and that P stimulates this CD10? cell compartment, which is enriched for bipotent progenitor activity. In addition, we have shown that P stimulates progenitor cells in human breast cancer cell lines and expands the cancer stem cell population via increasing the stem-like CD44? population. As changes in cell type composition are one of the hallmark features of breast cancer progression, the demonstration that progenitor cells are stimulated by P in

Electronic supplementary material The online version of this article (doi:10.1007/s10549-013-2817-2) contains supplementary material, which is available to authorized users. H. N. Hilton (&)  N. Santucci  A. Silvestri  S. Kantimm  J. D. Graham  C. L. Clarke Westmead Institute for Cancer Research, Sydney Medical School, University of Sydney at Westmead Millennium Institute, Westmead, NSW 2145, Australia e-mail: [email protected] L. I. Huschtscha Children’s Medical Research Institute, Westmead, NSW 2145, Australia

both normal breast and in breast cancer cells has critical implications in discerning the mechanisms of how P increases breast cancer risk. Keywords Progesterone  Progenitor  Breast cancer  Cell lineage Abbreviations ALDH Aldehyde dehydrogenase CFC Colony forming cell CK14 Cytokeratin-14 CK18 Cytokeratin-18 CSC Cancer stem cell E 17b-estradiol ER Estrogen receptor FACS Fluorescence-activated cell sorter FITC Fluorescein isothiocyanate HRT Hormone replacement therapy IF Immunofluorescence IPX Immunoperoxidase MEC Mammary epithelial cell miRNA MicroRNA qPCR Quantitative PCR P Progesterone PE Phycoerythrin PR Progesterone receptor SD Standard deviation SE Standard error

Introduction The ovarian steroid hormones, progesterone (P) and 17bestradiol (E), are critical in the growth and proliferation of the breast during normal development, yet ovarian activity

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has also been shown to be a major driver of breast cancer risk. Through the menstrual cycle, healthy women are exposed to frequent changes in the hormonal milieu through monthly cycling levels of E and P. The follicular phase of the cycle is associated with a peak of circulating E, and is followed by the luteal phase, during which a prominent peak of serum P and a second peak of serum E occurs. This luteal phase of the cycle is characterised by increasing complexity and size of breast lobules, which reflects proliferative activity within the epithelial compartment when mitosis is highest [1–3]. The persistent hormonal influence throughout a woman’s reproductive life, and the fact that exposure to exogenous hormones, such as the use of progestins in hormone replacement therapy (HRT) and oral contraception are associated with increased breast cancer risk [4–6], highlight the importance of understanding the molecular mechanisms of ovarian hormone signalling, both in the normal breast and in the development and progression of breast cancer. The normal human breast is comprised of two major tissue compartments—the stroma and the epithelium, which is lined by a single layer of luminal epithelial cells associated with secretory activity, surrounded by a basal cell layer, consisting mostly of myoepithelial cells with contractile properties [7]. The epithelium derives from stem cells, which self-renew and give rise to uncommitted bipotent progenitors, which differentiate to lineage-committed progenitors and eventually to mature luminal or basal/myoepithelial cells. Although P has been shown to regulate the stem cell compartment in the mouse mammary gland [8, 9], the role of cycling levels of P on stem and progenitor cell expansion in the human remains unknown. A growing body of evidence suggests stem and progenitor cells are targets for carcinogenic transformation [10], and the majority of breast cancers contain only luminal epithelial cells [11, 12]. Normal stem cells may also yield cancer stem cells (CSCs), which are characterised as cells expressing CD44?/CD24- [13] or high levels of aldehyde dehydrogenase enzyme (ALDH; [14]), and are a rare population of cells with indefinite potential for self-renewal that can expand to form a tumor mass [15]. Together, these observations underline the fact that dysregulation of cell fate determination is a critical aspect of breast tumorigenesis. Because of this, much interest in recent years has been focussed on studying the individual cell subtypes within the breast. Markers for mature luminal cells (e.g. MUC1, cytokeratin-18) and myoepithelial cells (e.g. p63, cytokeratin-14) are well-established, but although a large array of markers have been reported to be associated with progenitor activity (e.g. CD49f, SSEA-4, CD133, Thy1, CD44, cytokeratin-5) [16–18], an antigen which convincingly marks stem/progenitor cells in the human breast remains to be identified. CD10 is emerging as an interesting

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candidate in that regard: it has recently been shown to be enriched in the bipotent progenitor cell compartment [19–21], CD10 is co-expressed with putative stem cells markers, and CD10? cells express both luminal and myoepithelial markers [19]. Moreover, CD10? cells display enhanced colony forming ability with a high proportion of colonies displaying a bipotent progenitor phenotype [19, 21, 22]. So despite historical data suggesting that CD10 marks myoepithelial cells in the human breast [23], more recent evidence based on contemporary analytical methods points to CD10 as a marker of stem-like or bipotent progenitorenriched populations. The ovarian steroid hormones influence the biology of the normal breast via their nuclear receptors (estrogen receptor, ER; progesterone receptor, PR), which have long been known to be expressed in mature luminal cells [24], with myoepithelial cells generally believed to be hormone receptor negative. However, we have recently demonstrated that high levels of PR transcripts are found in the CD49fhi (MUC1/CD133)-(CD10/THY1)? bipotent progenitor-enriched compartment, and that in a subset of cells, PR is co-expressed with the basal markers, cytokeratin-14 (CK14) and p63 [25]. Moreover, we showed that P augments the progenitor-like compartment in the normal breast [26]. Given that P regulates progenitor cells in the breast, and the emerging association of CD10 with bipotent progenitor activity in the normal human breast, it is crucial to determine whether P can regulate the CD10 compartment. Although it has been demonstrated that P regulates progenitor-like cells in breast cancer [27, 28], whether P directly targets CSCs remains to be shown. Here we present data demonstrating for the first time that P stimulates both CD10? bipotent progenitor-enriched cells in the normal human breast, and CD44?/CD24- CSCs in breast cancer cell lines. These findings are important for understanding the role P signalling plays in cell fate determination in the human breast, which is critical in order to determine if and how these effects may contribute to breast tumorigenesis.

Results PR expression in normal human mammary epithelial cell (MEC) subsets To determine whether PR could be detected in CD10? cells, we dissociated and fractionated primary human MECs grown in 3D culture into two major subpopulations of CD10? basal cells and MUC1? luminal cells (Fig. 1a and Supp Fig. 1a). We confirmed that these cell fractions were enriched for luminal or basal markers, by performing quantitative PCR (qPCR) to demonstrate that transcripts of

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Fig. 1 PR expression in sorted normal breast epithelial cells a Gating strategy for simultaneous detection and fractionation into two major subpopulations on the basis of MUC1 and CD10 expression. b Relative mRNA levels of PR were normalised to TBP in CD10? and MUC1? subpopulations. Graph represents mean ? SE in three independent experiments. Representative images showing c IPX staining for PR in sorted CD10? and MUC1? fractions, d dual IF staining for (i) PR (red) and CD10 (green) in sorted CD10? cells, and (ii) PR (red) and MUC1 (green) in sorted MUC1? cells. e dual IF staining for PR (red) and CK14 (green) in sorted (i) basal CD10? and (ii) luminal MUC1? MECs in 3D culture. Scale bars represent 25 lm

the basal marker, CK14, were indeed enriched in the CD10? fraction, while transcripts of the luminal marker, cytokeratin-18 (CK18), were enriched in the MUC1? fraction (Supp Fig. 1b). Importantly, the conservative fractionation strategy, while it resulted in some CD10? and MUC1? cells being stratified into the double negative CD10-/MUC1- fraction (Supp Fig. 1b), ensured that the CD10? fraction was purely basal and did not contain the luminal marker (CK18), and vice versa. Quantitation of total PR transcripts (both A and B isoforms) by qPCR in the basal and luminal fractions isolated from three separate individuals revealed PR expression in the MUC1? luminal fraction, as would be expected [24], but also showed a relatively high number of PR transcripts in the basal CD10? subfraction, although there was heterogeneous expression of PR in each subfraction between individuals (Fig. 1b). PR protein expression in the CD10? fraction was confirmed by IPX (Fig. 1c) and IF (Fig. 1d) staining. The majority of cells within the sorted CD10? fraction also expressed CK14, with a large number of PR? cells coexpressing CK14, as we have previously observed [25] (Fig. 1e). To exclude the possibility that this finding was a consequence of the cell fractionation process, we also identified PR and CD10 co-expressing cells in MECs growing in 3D culture (Fig. 2a), in freshly isolated breast epithelial cells (Fig. 2b) and in a rare subset of cells

(estimated to be \1 %) in normal human breast tissue (Fig. 2c). Taken together, these findings demonstrate that PR transcripts and protein are expressed in CD10? cells in the normal human breast. P-mediated stimulation of progenitor cells in normal human MECs We have previously shown that P expands the human breast progenitor cell compartment [26]. By scoring the number of colonies with luminal, myoepithelial or bipotent progenitor morphology [29], CD10? cells displayed enhanced colony forming ability with a high proportion of colonies displaying a bipotent progenitor phenotype (Fig. 3a; Supp Figs. 2a, b), in agreement with others [19– 22]. As Figs. 1 and 2 revealed that PR is expressed in a subset of CD10? cells, we were interested in determining whether P could stimulate the CD10? cell subset. To quantitate absolute numbers of luminal and basal cells, primary human breast cultures grown in 3D culture after 48 h of treatment with P, E, or combined E ? P treatment were flow sorted using MUC1 and CD10 cell surface markers. There was no consistent change in the number of MUC1? cells with any hormone treatment (Supp Fig. 2c), but there was a significant and reproducible increase in the number of basal CD10? cells with P treatment (p = 0.03),

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Fig. 2 Co-expression of PR and CD10 in normal breast. Representative images showing dual IF staining for PR (red) and CD10 (green) in a (i and ii) MECs in 3D culture, b uncultured normal MECs and c (and inset) normal breast tissue. White arrows indicate cells which have stained positive for both PR and CD10. All scale bars represent 20 lm

but not with E (p = 0.11) or E ? P (p = 0.78) treatment (Fig. 3b). This P-mediated increase in CD10? cells is therefore concordant with P stimulating bipotent progenitor cells. To determine whether this effect also occurred in the physiologically relevant context of the normal menstrual cycle, we exposed normal human breast cultures grown in 3D culture to sequential treatments of E and P in a regimen that mimicked the menstrual cycle. Changes in lineage composition of the acini were determined by dual IF staining of luminal (CK18) and basal (p63) markers (Fig. 3c). Scoring of the number of acini with only luminal cells, only basal cells, or both cell types, revealed that each hormone treatment regimen increased the proportion of dual lineage acini (Fig. 3c). Importantly, this trend was significant when only P treatment was included in the regimen (p = 0.02), which induced a twofold increase in the proportion of dual lineage acini compared with that of vehicle-treated cultures. Furthermore, treatment with E or P individually had a more profound effect than that of combined E ? P treatment (Fig. 3d). There was no significant change in the relative proportions of acini containing only luminal cells, or only basal cells (Supp

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Fig. 2d). As bipotent progenitors in 3D culture are able to give rise to both luminal and myoepithelial cells [26], this P-mediated increase in numbers of acini composed of both lineage types is consistent with there being an increase in the number of bipotent progenitor cells. P-mediated stimulation of breast cancer stem cell-like population These data, together with our previous demonstration that P increased the proportion of cells that formed mammospheres in suspension, and the proportion of ALDH? cells [26], prompted us to test whether the stimulation of progenitor cells by P in the normal human breast could be recapitulated in breast cancer cells. To determine the effect of P on breast cancer stem cells, we treated the PR? breast cancer cell line, T47D, which contains a very rare subset of tumorigenic CD44?/CD24- CSCs (B0.1 %; [30–32] ), with P and the synthetic progestins, ORG2058 and MPA [33]. We quantitated the proportion of cells which expressed CD44, but not CD24, by flow cytometry (using the gating strategy depicted in Fig. 4a), and revealed a statistically significant increase in the proportion of

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Fig. 3 P-mediated stimulation of CD10? progenitor-enriched cell compartment. a Proportion of different colony types yielded from CFC assays using CD10? and MUC1? sorted cells. b Fold increase in proportion of CD10? cells with indicated hormone treatments (n = 3). c Representative immunofluorescent image of normal human

mammospheres in 3D culture stained for CK18 (green) and p63 (red). Scale bar represents 100 lm. d Fold increase in proportion of dual lineage acini with indicated hormone treatments. Data are representative of the mean ? SE for three independent experiments using tissue from two individuals

CD44?/CD24- cells following all treatments (Fig. 4b). P induced an average fold increase of 4.2 (95 % CI 1.8–9.7, p = 0.045), ORG2058 induced an average fold increase of 6.9 (95 % CI 4.8–10.0, p = 0.000) and MPA induced an average fold increase of 4.2 (95 % CI 2.0–8.8, p = 0.012). This increase in CSCs was attenuated when the cells were co-treated with the PR antagonists RU486 (Fig. 4b) and onapristone (Supp Fig. 3a), confirming this effect was mediated directly by PR. Stimulation of CSCs by progestins was also recapitulated in a second PR? cell line, HCC1428 (Fig. 4c). To determine whether the P-mediated increase in CSCs was due to increased expression of the progenitor marker, CD44, we documented a significant increase in CD44 transcripts, which was attenuated by onapristone co-treatment, in cells treated with P and progestins (Supp Fig. 3b). This was confirmed by the observation that P and progestins increased the number of total CD44? cells (Fig. 4d). There was no consistent change in the proportion of CD24? cells with any treatment (data not shown). Finally, to determine whether PR may be expressed in CD44? cells, we sorted HCC1428 breast cancer cells, which display heterogeneous expression of PR, on the basis of CD44 expression (Supp Fig. 4a). We collected CD44hi and CD44- subfractions, and observed that PR was indeed present in the CD44hi fraction (Supp Fig. 4b), suggesting

that PR can be expressed in CD44? progenitor cells in breast cancer cells. In summary, the findings of this study demonstrate that in the normal human breast, PR is expressed in a subset of CD10? cells and that P also expands this CD10? population, which we postulate to be bipotent progenitor cells. In addition, we have shown that P stimulates progenitor cells in human breast cancer cell lines and expands the CSC population via increasing the stemlike CD44? population.

Discussion Although critical in normal breast development, the evidence that P is also a major driver of breast cancer risk has grown immensely over the last decade (reviewed in [34]). P has been shown to regulate the stem cell compartment in the murine mammary gland via RANK, the cognate receptor of RANKL [8, 9], a pathway important in the initiation of mouse mammary tumorigenesis [35]. Mouse mammary stem cells do not express PR [36, 37], suggesting that P regulates stem cells via paracrine mechanisms, and indeed, RANK mRNA expression is enriched in the basal cell population of the mouse mammary gland [9]. Augmentation of the murine mammary stem cell compartment by P may provide an explanation as to the

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Breast Cancer Res Treat (2014) 143:423–433 b Fig. 4 P-mediated stimulation of CSCs via CD44? cell compart-

ment. a Gating strategy for detection of CD44-positive and CD24negative (CD44? CD24-) CSCs (Q4). Quadrants denote positivity thresholds based on negative staining controls. b Average fold change in proportion of CD44? CD24- cells in T47D cells following 4 days of treatment with P (100 nM; n = 4) or progestins (10 nM; n = 6), with or without RU486 co-treatment (100 nM; n = 4). Chart represents mean fold change with upper and lower confidence limits of log transformed data. c Fold change in proportion of CD44? CD24- cells in HCC1428 cells (n = 4) following treatment with progestins (10 nM, 4 days). d Average fold change in proportion of total CD44? cells in T47Ds following 4 days of treatment with P (100 nM; n = 4) or progestins (10 nM; n = 6). Chart represents mean ? SE

transient increases in breast cancer risk that occur during pregnancy and the menstrual cycle, thus it is critical to now establish what impact P has on the stem/progenitor cell population in the human breast. We have previously demonstrated that in the normal human breast, P treatment increases proliferation and augments the size of the progenitor-like compartment [26], that PR transcripts are enriched within this compartment, and that within a subset of cells, PR is co-expressed with basal markers [25]. Importantly, we have also recently shown that changes in lineage composition correlate with increased proliferation, and are one of the earliest events in breast carcinogenesis [38], which again highlights the fact that tight regulation of cell fate determination is critical for normal development. Here, we demonstrate for the first time that PR transcripts and protein are expressed in CD10? cells, a cell compartment enriched in the bipotent progenitor subfraction. Importantly, we have also shown for the first time that P stimulates progenitor cells via expansion of this CD10? compartment in the normal human breast, and the CD44? compartment in breast cancer cell lines. CD10 has recently gained prominence as a marker of stem and progenitor cells not only in the breast, but also in organs such as the bone marrow and lung [39]. Combined with the evidence that physiologically relevant levels of cycling P treatment (in the absence of E) increases the proportion of dual lineage acini, the demonstration of a significant increase in CD10? cells by P suggests that this action is specifically targeting the bipotent progenitor cell population. Because we have shown by qPCR and immunostaining that PR can also be expressed in this CD10? population, it is tempting to speculate that this subset of basal cells expressing PR in fact comprise progenitor cells which are able to directly respond to the proliferative effects of P action via cell-intrinsic, rather than paracrine mechanisms. The level of detection of PR in the CD10? population varied considerably between individuals. This heterogeneity may reflect age differences between patients, menstrual cycle phase, exogenous hormone use, parity or

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natural inter-individual variation. However, our data clearly demonstrate the presence of PR in both MUC1? and CD10? cells in every individual tested. It is interesting to note that the P-mediated induction of both CD10? cells (Fig. 3b), and dual lineage acini, appears to be tempered when the cells are exposed to combined E ? P treatment (Fig. 3d). Precisely how this occurs is beyond the scope of this study, however E has previously been shown to have anti-tumorigenic activity when used in combination with P, such as in the classic rat mammary tumor model where P greatly accelerated tumor formation, whereas combined E ? P decreased tumor incidence [40]. This underlines the importance of balanced levels of combined E and P signalling during the menstrual cycle and pregnancy, in order to avoid inappropriate expansion of particular cell compartments due to unopposed signalling from either ovarian hormone. Also intriguing is the lower levels of PR that are often expressed in CD10? cells, relative to adjacent more intensely staining PR? cells (Fig. 2). This may be indicative of a transient progenitor cell pool which is continuously and rapidly proliferating and differentiating into PR? and PR- cells, responding to various stimuli such as microenvironment factors and hormone action. Alternatively, perhaps translation of PR transcripts is suppressed in progenitor cells due to epigenetic mechanisms, or expression of specific microRNAs (miRNAs). For example, miR-26a, miR-181a, miR-126-3p and miR-513a-5p, have all been shown to down-regulate PR expression [41–43], and because miRNAs are often expressed in a tightly cell-type specific manner, it would be interesting to determine whether these miRNAs are expressed in progenitor cells of the normal human breast. This study also provides evidence that P induces CSC properties of PR-positive breast cancer cells via stimulating the tumor-initiating CD44? population, a cell subset that has also been observed by [44, 45] to be targeted by P. That P can stimulate the expansion of CSCs is also supported by a recent study showing that P increased the proportion of ALDH? cells, a cell subset which displays increased radioresistance and tumorsphere-forming abilities [46]. The demonstration that stimulation of RANK-overexpressing human MECs by the P target, RANKL, increased the proportion of CD44? CD24- CSCs, as well as CD10? cells [47], further supports our findings. Finally, we have shown that PR can be expressed in CD44? cells, again providing the possibility that these cells could directly respond to P action via cell-intrinsic, rather than paracrine mechanisms. This supports the demonstration of low levels of PR transcripts in CD44? cells in a sample of normal breast and an invasive breast tumor [48]. It has recently been shown that the presence of CD44?/CD24- CSCs was significantly associated with the rare ER–PR? cancer subtype and tumors expressing basal markers [49], thus this

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signalling pathway may be of great clinical importance in breast cancer. Although whether CSCs arise from normal breast stem/progenitor cells, or from a differentiated progenitor cell, remains to be determined, a recent report has suggested that the CD44?/CD24- CSC markers alone could also be used to isolate cells progenitor activity in normal human MECs [50]. Therefore, perhaps P-mediated expansion of distinct cell types within the epithelial hierarchy, and particularly progenitor cells which are highly susceptible to transformation, gives rise to different subtypes of breast cancer. Treatment with P in the mouse mammary gland selectively up-regulates specific targets in either basal or luminal cells [8, 9]. We have shown that PR is expressed in both progenitor and mature luminal cells, that PR? bipotent progenitor cells are E-insensitive [25], and here we demonstrate that P stimulates progenitor cells in the human breast, via up-regulation of progenitor markers, including CD10 and CD44. We therefore hypothesise that P action is mediated via different mechanisms in progenitor and mature cells in the normal human breast, and that exogenous hormones disrupt these mechanisms and contribute to progenitor expansion in cancer. This effect of P on progenitor cells may underlie the capacity of P analogues in HRT to expand pre-existing lesions and increase breast cancer risk, highlighting the importance of now characterising the mechanisms which drive P-mediated progenitor cell expansion in order to identify new potential indicators of breast cancer risk, prognosis and/or treatment options.

Materials and methods Matrix-embedded culture of primary human mammary epithelial cells (MECs) Normal human breast tissue samples (n = 5) obtained from reduction mammoplasty specimens were collagenase digested and filtered. The resulting epithelial organoids were grown in matrix-embedded culture, as described previously [26, 51]. All samples were obtained with informed consent from donors, and approval from Human Research Ethics Committees of the Sydney West Area Health Service, the University of Sydney, and the Sydney Adventist Hospital, New South Wales, Australia. Following 9 days of culture, the acini were harvested and dissociated, as described previously [25]. Cultures were treated for 48 h with 100 nM progesterone (Sigma-Aldrich, Castle Hill, Australia), 10 nM E (Sigma-Aldrich) or vehicle. Where indicated, cultures were exposed to sequential treatments of E and P (7 days of 0.5 nM E, followed by 7 days of combined 1 nM E and 40 nM P) in a regimen

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that mimicked the menstrual cycle. In parallel, cultures were also only exposed to E or P treatment in this menstrual cycle regimen. Cell culture T47D and HCC1428 cells were purchased from the American Type Culture Collection (atcc.org, Manassas, VA, USA) and maintained in RPMI1640 medium containing 10 % fetal calf serum and 0.25 units/ml insulin. Both cell lines did not require E pre-treatment for efficient P-mediated responses. Progestin stimulation experiments involved treating cells for 4 days with progesterone (Sigma-Aldrich), ORG2058 (Amersham Biosciences; GE Healthcare, Rydalmere, Australia), MPA and/or vehicle control. Where indicated, cells were co-treated with onapristone; (13a)-11b-[4-(dimethylamino)phenyl]-17ahydroxy-17b-(3-hydroxypropyl)estra-4,9-dien-3-one (Arno Therapeutics, Inc., New Jersey, USA) or RU38486 (Roussel-Uclaf, Romainville, France). Fluorescence-activated cell sorter (FACS) analysis For MUC1/CD10 cell sorting experiments, single primary MECs from 3D cultures were stained with anti-MUC1 conjugated to fluorescein isothiocyanate (FITC) and antiCD10 conjugated to phycoerythrin (PE). Sorted fractions were then used in downstream analysis assays, including colony forming cells (CFC) assays (as described in [25]), quantitative PCR analysis and immunohistochemistry analysis (see below). For CSC experiments, cells were labelled with anti-CD44 conjugated to FITC and antiCD24 conjugated to PE under optimised conditions. All antibodies were purchased from BD Biosciences. For all flow cytometry experiments, forward scatter and side scatter plots were used to account for debris and aggregated cells, single stained samples were used as compensation controls, and control samples consisted of unstained cells and/or IgG antibodies directly conjugated to FITC and PE (BD Biosciences). Cells were analysed on a FACSCanto II or FACS Calibur, or sorted on a FACSAria III cell sorter (BD Biosciences) and performed in the Flow Cytometry Centre at the Westmead Millennium Institute. Analysis was performed using FACS Diva software (v6.1.2, BD Biosciences). RNA preparation and qPCR Sorted primary MECs were maintained at 4 °C and collected by centrifugation. RNA was harvested using the Absolutely RNA Microprep Kit (Stratagene) according to the manufacturer’s instructions, and reverse transcribed using the high-capacity cDNA Reverse Transcription kit

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(Applied Biosystems). For cell lines, RNA was harvested using the RNAqueous total RNA isolation kit (Ambion, Inc., Texas, USA) following the manufacturers’ instructions, and reverse transcribed using SuperScriptÒ III Reverse Transcriptase (Life Technologies, Inc.). For qPCR, cDNA was amplified using the Platinum SYBR Green qPCR Supermix (Life Technologies, Inc.) on a Rotor-gene 6000 real-time cycler (Corbett Research, Australia). The TATA-binding protein (TBP) was used as an internal control gene, and primers used were as described in [25]. Additional primers used were CD44 Forward: GCAGCA CTTCAGGAGGTTACAT and CD44 Reverse: CAAGAG GGATGCCAAGATGAT. Sample processing and immunohistochemistry Matrigel cultures or thrombin clots were fixed, paraffinembedded, cut into 2-lm sections, and antigens retrieved [52]. Immunoperoxidase (IPX) staining was performed as previously published [53]. For dual immunofluorescence (IF) staining, antigens were revealed sequentially by incubation of the following primary antibodies: total PR (both A and B isoforms; Novocastra), cytokeratin-18 (CK18; Sigma-Aldrich), cytokeratin-14 (CK14; Novocastra), p63 (AbCam), MUC1 (AbCam) and CD10 (Novocastra). This was followed by detection by an appropriate biotinylated secondary antibody (goat anti-mouse or antirabbit; Dakocytomation, Glostrup, Denmark) and a streptavidin-conjugated fluorescent label (Alexa 594 or 488; Life Technologies, Inc.). Sections were mounted in Prolong Gold antifade reagent containing the nuclear counterstain DAPI (Life Technologies, Inc.). To ensure antibody specificity, adjacent sections were stained without the primary antibody, using the biotinylated secondary antibody and streptavidin-conjugated fluorescent label only. For experiments where acini were stained with lineage markers (CK18 and p63), acini containing C10 cells/ nuclei were scored, and counted as dual lineage if at least 3 cells of each lineage was present in each acinus. A range of 201-664 acini were counted in 3 independent experiments. Statistical analysis Significance in population comparisons was determined using the T test (Excel, Microsoft). Where indicated, log transformations were performed to stabilise the variance of data. Error bars are presented as standard deviation (SD) or standard error of the mean (SE), as indicated. Acknowledgments We wish to thank Arno Therapeutics, Inc. for generously donating the onapristone. We wish to thank Dr Xin Maggie Wang for assistance with flow cytometry, performed in the Flow Cytometry Centre at Westmead Millennium Institute which is supported by the National Health and Medical Research Council of

431 Australia (NHMRC) and Cancer Institute New South Wales. This study was supported by an NHMRC project grant (1011496), Cure Cancer Australia Foundation and the National Breast Cancer Foundation. We gratefully acknowledge the advice and assistance of Dr. Karen Byth, Westmead Millennium Institute, with statistical analysis. Conflict of interest peting interests.

The authors declare that they have no com-

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Progesterone stimulates progenitor cells in normal human breast and breast cancer cells.

The epithelium of the human breast is made up of a branching ductal-lobular system, which is lined by a single layer of luminal cells surrounded by a ...
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