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Keywords:

  • Mammary gland;
  • Stem cells;
  • Self-renewal;
  • Progenitors;
  • Myc

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The mammary epithelium comprises two major cell lineages: basal and luminal. Basal cells (BCs) isolated from the mammary epithelium and transplanted into the mouse mammary fat pad cleared from the endogenous epithelium regenerate the mammary gland, strongly suggesting that the basal epithelial compartment harbors a long-lived cell population with multipotent stem cell potential. The luminal cell layer is devoid of the regenerative potential, but it contains cells with clonogenic capacity, the luminal progenitors. Mammary BCs and luminal progenitors express high levels of the transcription factor Myc. Here, we show that deletion of Myc from mammary basal epithelial cells led to impaired stem cell self-renewal as evaluated by limiting dilution and serial transplantation assays. Luminal progenitor population was significantly diminished in mutant epithelium suggesting control by the BC layer. Colony formation assay performed with isolated BCs showed that clonogenic capacity was abolished by Myc deletion. Moreover, transplanted BCs depleted of Myc failed to produce epithelial outgrowths. Stimulation with ovarian hormones estrogen (E) and progesterone (P) partially rescued the repopulation capacity of Myc-depleted BCs; however, the Myc-deficient mammary epithelium developed in response to E/P treatment lacked stem and progenitor cells. This study provides the first evidence that in the mammary gland, Myc has an essential nonredundant function in the maintenance of the self-renewing multipotent stem cell population responsible for the regenerative capacity of the mammary epithelium and is required downstream from ovarian hormones, for the control of mammary stem and progenitor cell functions. STEM CELLS2012;30:1246–1254


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The transcription factor proto-oncogene Myc regulates the expression of numerous genes involved in the control of cellular metabolism, growth, and proliferation [1]. Myc is required for the maintenance of embryonic stem cells [2]. Several studies implicate Myc in the regulation of self-renewal and differentiation of hematopoietic stem cells [3] and the maintenance of stem/progenitor cell function in the epidermis and intestine [4, 5]. The related factor N-Myc plays an important role in the regulation of neural stem/progenitor cells [6].

The over-expression of Myc is one of the common anomalies associated with human cancer. Myc expression is often deregulated in breast cancer, in particular, in the triple-negative subtype [7, 8]. Moreover, Myc is an estrogen receptor-α (ERα) target, and there is a significant overlap between ERα- and Myc-regulated genes associated with the control of proliferation [9].

The mammary epithelium is organized as a bilayer, with a layer of basal myoepithelial cells and a luminal cell layer. In mice and humans, the basal and luminal lineages segregate during perinatal period (reviewed in [10]). Both mammary lineages originate from embryonic precursor cells that express the basal-type cytokeratins 5 and 14 (K5 and K14, respectively). In adult tissue, the mammary basal cells (BCs), rather than the luminal cells, are able to regenerate mammary gland upon transplantation into mouse mammary fat pad cleared of endogenous epithelium. This strongly suggests that the basal epithelial compartment harbors a long-lived cell population with multipotent stem cell properties [11, 12]. The luminal cell layer contains clonogenic cells, the luminal progenitors, which are thought to give rise to secretory alveoli during pregnancy [11–13]. Lineage tracing experiments have suggested that the basal and luminal compartments are independently maintained by their respective unipotent stem/progenitor cells in adult animals [14].

In the mammary gland, high levels of Myc expression are characteristic of BCs and of luminal progenitors [15]. Myc over-expression in the luminal layer of the mouse mammary gland leads to unscheduled lobulo-alveolar development and tumorigenesis [16, 17]. Myc deletion from the differentiated luminal cells affects milk secretion and moderately reduces the repopulating capacity of the mammary epithelium as evaluated by transplantation of tissue fragments [18]. However, neither the functions of Myc in the mammary basal epithelial compartment nor the effects of Myc deletion on distinct mammary stem/progenitor populations have been studied so far.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Mice

Transgenic mice expressing the Cre recombinase under the control of the K5 promoter (K5-cre) were kindly provided by Dr. J. Jorcano [19], Rosa26LacZ reporter strain, carrying a loxP-stop-loxP-lacZ cassette, by P. Soriano [20], mycF/F strain, by I.M. de Alboran [21]. All mice were bred in a 129SV/C57BL6 genetic background. mycF/+ or mycF/F mice were used as controls unless indicated otherwise. Experiments were conducted in accordance with French Veterinary Guidelines and those formulated by the Council of Europe for experimental animal use (L358-86/609EEC).

Estrogen/Progesterone Stimulation

For hormone stimulation, silicon tubes containing estrogen/progesterone (E/P) were implanted subcutaneously 5 weeks after transplantation as previously described [22]. Mammary glands were analyzed 4 weeks after tube implantation.

Preparation of Mammary Epithelial Cells and Cell Cycle Analysis

The inguinal mammary glands of four to six 20-week-old virgin females were pooled to prepare single-cell suspensions, and cells were processed for flow cytometry as described [12, 13, 23]. The following conjugated antibodies were used: anti-CD24-PE (clone M1/69; BD Pharmingen, San Diego, CA, http://www.bdbiosciences.com), anti-CD49f-FITC (clone GoH3; BD Pharmingen), anti-CD45-APC (clone 30-F11; Biolegend, San Diego, CA, http://www.biolegend.com), anti-CD31-APC (clone MEC13.3; Biolegend), and anti-Ly6A/E-PE-Cy5 (clone D7; eBiosciences, San Diego, CA, http://www.ebiosciences.com). Labeled cells were analyzed and sorted on a FACSVantage flow cytometer (Becton Dickinson, San Jose, CA). Sorted cell populations were routinely reanalyzed and found to be 94%–98% pure. As estimated by trypan blue exclusion, cell viability after sorting was between 80% and 88%. For cell cycle studies, cells isolated from six pooled mammary fat pads were incubated with Hoechst 33342 (Sigma Aldrich, Saint-Quentin Fallavier, http://www.sigmaaldrich.com/france; 6 μg/ml) for 1 hour at 37°C before cell surface labeling and flow cytometry. Cell cycle analysis was performed using FLowJo software. Data from one of three independent experiments are shown.

Transplantation Assays

Fragments of mammary epithelium or 1,000–5,000 sorted BCs in 10 μl of 50% growth-factor-reduced matrigel (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com) were implanted into the inguinal fat pads of 3-week-old nude BALB/c females cleared of endogenous epithelium as described elsewhere [23]. In limiting dilution assays, 500–100,000 of total unsorted mammary cells per fat pad were transplanted in matrigel. Mutant and control epithelial fragments and cells were grafted into two contralateral fat pads of the same recipient mouse, and the outgrowths were analyzed 10 weeks after transplantation. Repopulating unit frequency was calculated using the Extreme Limiting Dilution Analysis software [24].

Cell Culture Assays

Sorted luminal cells were cultured at a density of 250 cells per well as described elsewhere [13]. Sorted BCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing 1% fetal calf serum (FCS), B27 at a density of 500–1,000 cells per well [11]. For cultures on matrigel, 15,000 control or 45,000 mutant BCs were seeded on LabtekII slides (Nalge/Nunc International, Rochester, NY, http://www.nalgenenunc.com) coated with matrigel (BD Pharmingen) and grown in 2% FCS DMEM/F12 medium.

X-Gal Test

For whole-mount X-gal staining, mammary glands were fixed in 2.5% paraformaldehyde in phosphate buffered saline (PBS), pH 7.5, for 1 hour at 4°C, and stained overnight at 30°C (Biology of the Mammary Gland, http://mammary.nih.gov).

Alternatively, cryosections (10–15-μm thick) were cut from mammary gland pieces embedded in Tissue-Tek (Miles Diagnostic Division, Elkhart, IN) and frozen in isopentane cooled by liquid nitrogen. Cryosections were thawed at room temperature for 30 minutes, fixed for 15 minutes in 2% formaldehyde, 0.2% glutaraldehyde, 0.02% Nonidet P40 in PBS, washed with PBS, and incubated with the X-gal staining solution (0.025% X-gal, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, 1.5 mM MgCl2, 15 mM NaCl, 40 mM Hepes, and pH 8) overnight at 30°C. The sections were then postfixed with 4% formaldehyde and counterstained with nuclear Fast Red. X-gal staining of colonies was performed as for cryosections after fixation in 2% formaldehyde and 0.2% glutaraldehyde at 4°C for 4 minutes.

Whole-Mount Analysis, Histology, and Immunostaining

Dissected mammary fat pads were spread onto glass slides, fixed in a 1:3:6 mixture of acetic acid/chloroform/methanol, and stained with Carmine in whole mount. For histological analysis, glands were embedded in paraffin. Seven micrometer-thick sections were cut and dewaxed prior to hematoxylin and eosin staining or immunolabeling performed as described elsewhere [25]. Before incubation with primary antibodies, sections were treated with 5% fetal calf serum in PBS for 1 hour. Sections were incubated overnight at 4°C with primary antibodies, 1 hour at room temperature with secondary antibodies, and 3 minutes with 4',6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: mouse monoclonal anti-α-smooth muscle actin (αSMA; Sigma) and anti-ERα (Dako, Trappes, France, http://www.dako.fr); rabbit polyclonal anti-K5, anti-K8 (Covance, Princeton, NJ, http://www.covance.com), anti-Ki67 (Novocastra Labs., Newcastle upon Tyne, UK, http://www.novocastra.co.uk), and anti-signal transducer and activator of transcription 5A (STAT5A, Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com). Alexafluor-conjugated secondary antibodies (1/1,000; Invitrogen Saint Aubin, France, http://www.invitrogen.com) were used for immunofluorescence labeling. The Envision+ System HRP kit (Dako) was used for immunohistochemistry.

Reverse Transcription Polymerase Chain Reaction

RNA was retro-transcribed with Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Madison, WI, http://www.promega.com), and quantitative RT-PCR (q-PCR) was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). The primers used for q-PCR analysis are listed in Supporting Information.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Deletion of Myc from BCs Leads to Diminished Proliferation Rates in both Mammary Epithelial Cell Layers and Gland Hypoplasia

To investigate the role of Myc in the control of mammary stem and progenitor cell function, we deleted Myc from mammary basal epithelial cells. K5-Cre transgenic mice were bred with mice carrying conditional myc alleles (mycF/F). As reported previously, K5Cre;mycF/F mice are viable and fertile [4]. Mammary glands were found to be slightly hypoplastic in adult virgin K5Cre;mycF/F females, if compared with those of age-matched control animals (Fig. 1A). Most K5Cre;mycF/F mothers lost part of their litters, and the weight of surviving pups was lower than that of pups fed by control females (Supporting Information Fig. S1A). In lactating mice, the amount of secretory alveoli was found to be lower in mutants than in controls especially in the second and third pregnancies (Fig. 1A and data not shown). Consistently, the levels of milk protein gene transcripts were decreased in mutant glands as compared to control (Fig. 1B).

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Figure 1. Mammary development in K5Cre;mycF/F mice. (A): Mammary glands from control and K5Cre;mycF/F mice stained with Carmine in whole mount. Upper panels, 4-month-old virgin; lower panels, 1-day-lactating mice, third pregnancy. Bars = 1.5 mm in upper panels and 0.5 mm in lower panels. (B):csn2 (β-casein) and wap (WAP) gene expression in control and mutant mammary glands on the second day of lactation, third pregnancy. Q-PCR. *, p < .05 and .02 for csn2 and wap, respectively. Three control and three mutant mice were analyzed. The values reported are normalized to gapdh expression. (C): Sections through control, K5Cre;Rosa26, and mutant, K5Cre;Rosa26;mycF/F, mouse mammary glands X-gal-stained in whole mount. Upper panels, 4-month-old virgin; lower panels, 1-day-lactating mice. Bar = 100 μm. Abbreviation: L, lumen.

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To analyze the distribution of Myc-deficient cells in mutant mammary epithelium, control and K5Cre;mycF/F mice were bred with the Rosa26LacZ-reporter (R26) mouse strain. Thus, LacZ served as BC genetic marker permitting lineage tracing and, in addition, monitoring of the Myc deletion in mutant tissue. All BCs and approximately 80% of luminal cells in control virgin K5Cre;R26 mice were LacZ-positive indicating that K5-driven Cre expression led to recombination in mammary stem/early progenitor cells (Fig. 1C). As could be expected, in control lactating mouse glands, most alveolar cells were LacZ-positive (Fig. 1C). Strikingly, in mutant virgin mice, LacZ expression was limited to the BC layer with most luminal cells being LacZ-negative, whereas during lactation, most mammary alveoli comprised LacZ-negative luminal cells (Fig. 1C). Thus, in mutant mammary epithelium, myc was deleted from BCs only, and in pregnancy, secretory alveoli were developed from Myc-positive progenitors. We suggest that the luminal cell population in mutant epithelium originated during early stages of the gland development, from cells committed to luminal lineage that switched off the K5 promoter and escaped Myc deletion. In agreement with this hypothesis, in mutant E18-embryos, in contrast to control, cells that escaped recombination and remained LacZ-negative were found in the central (future luminal) part of the developing mammary ducts (Supporting Information Fig. S1B). Many of these cells displayed luminal features, that is, expressed K8 and were negative for K5 (Supporting Information Fig. S1B).

Next, we separated mammary basal and luminal cells using flow cytometry cell sorting (Fig. 2A). The full gating strategy used to sort mammary basal and luminal cells is shown in Supporting Information Figure S2. In agreement with the results of whole-mount and histological analyses, the number of basal and luminal cells was found to be decreased in mutant mouse glands (Fig. 2B). Moreover, the ratio between basal and luminal cell populations was altered in K5Cre;mycF/F mammary epithelium. In control, BCs (CD31−/CD45−/CD24+/CD49f-high) represented 42.4% ± 6.6% of the total mammary epithelial population (CD31−/CD45−/CD24+), whereas in the mutant, 20.4% ± 5.9% only (Fig. 2C).

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Figure 2. Mammary epithelial cell populations in K5Cre;mycF/F mice. (A): Dot plot showing separation of luminal (CD31−/CD45−/CD24+/CD49f-low) and basal (CD31−/CD45−/CD24+/CD49f-high) epithelial cells from 4-month-old virgin mouse mammary glands by flow cytometry. Red ovals indicate separated luminal and basal cell (BC) populations, arrows point to BC population. (B): Diagram showing the absolute number of basal and luminal cells per gland isolated from mammary glands of control and mutant 4-month-old virgin mice. The values obtained in four independent cell-sorting experiments are shown as mean ± SEM. *, p < 10−5 for BCs and .023 for luminal cells. (C): Diagram showing percentages of CD49f-high cells in CD45−/CD31−/CD24+ cell populations. The values obtained in seven independent cell-sorting experiments are shown as mean ± SEM. *, p < .001. (D): q-PCR analysis of gene expression in freshly isolated basal and luminal mammary epithelial cells from 4-month-old virgin mice. The values normalized to gapdh and shown as mean ± SEM were obtained in five and three independent experiments for myc and mki67, respectively. * indicates p < .008 for myc and p < .05 for mki67. (E): Flow cytometry cell cycle analysis of freshly isolated mammary epithelial cells from 4-month-old virgin mice (a representative experiment). Percentages of cells in G0/G1, S, and G2/M are indicated.

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Cre expression was found only in BCs from mutant glands (Supporting Information Fig. S2B). Myc was efficiently deleted from the BC population, as its transcript levels were 15- to 20-fold lower in mutant BCs (Fig. 2D). In luminal cells from mutant mammary epithelium, myc expression levels were only 20%–25% lower than the control value, confirming that myc was not deleted from most luminal cells (Fig. 2D). mki67 expression was markedly decreased not only in basal but also in luminal cells from the mutant mammary glands suggesting that proliferation was inhibited in both compartments of K5Cre;mycF/F mammary epithelium (Fig. 2D). This was confirmed by flow cytometry cell cycle analyses of mammary epithelial cells isolated from virgin mouse glands. In basal and luminal populations, the proportions of cells in S-G2-M cell cycle phases in the mutants were approximately half those in controls (Fig. 2E). Thus, deletion of Myc from mammary BCs led to a moderate gland hypoplasia, a decrease of the BC population size, and diminished proliferation rates in both basal and luminal compartments.

Deletion of Myc from Mammary Epithelial BCs Affects Colony-Forming Stem/Progenitor Populations in Basal and Luminal Compartments

Sorted Myc-deficient BCs were able to proliferate, when seeded on matrigel-coated surface at high density; however, the percentage of Ki67-positive cells was 2.4-fold lower in mutant than control BC cultures (Fig. 3A). Mammary BCs have been reported to form clonal colonies on a fibroblast feeder layer, a property attributed to stem and progenitor cells [11]. Colony-forming cells constituted 2.5% ± 0.2% and 2.7% ± 0.5% of total BC population in 4- and 18-month-old control mice, respectively, whereas Myc-deficient BCs did not form colonies (Fig. 3B).

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Figure 3. Deletion of Myc from mammary epithelial basal cells (BCs) affects colony-forming stem/progenitor populations in basal and luminal compartments. (A): Double immunofluorescence labeling of mammary BCs seeded on matrigel with anti-Ki-67 and anti-αSMA antibodies. Arrows indicate Ki67-positive nuclei. The values shown in the diagram represent means ± SEM from three independent experiments; *, p < .04. Bar = 70 μm. (B): Colonies formed by 1,000 mammary BCs isolated from 4- (upper panels) or 18- (lower panels) month-old virgin mice. (C): Colonies formed by 250 mammary luminal cells isolated from 4-month-old virgin mice. The values shown in the diagram represent means ± SEM from four independent experiments; *, p < .002. (D): Colonies formed by 250 luminal cells isolated from 18-month-old virgin mice. The values shown in the diagram represent means ± SEM from three independent experiments; *, p < .025. (E): Separation of luminal progenitor-enriched cell populations from 4-month-old control and K5Cre;mycF/F mouse mammary epithelia, flow cytometry. CD45−/CD31−/CD24-high (luminal) cell fraction is shown. (F): q-PCR analysis of gene expression in luminal cells isolated from 4-month-old control and K5Cre;mycF/F mouse mammary glands. Three luminal cell preparations isolated in independent cell-sorting experiments were analyzed. (G): Immunolabeling of sections through 4-month-old control or mutant virgin mouse mammary gland with antiestrogen receptor antibodies. The values shown in the diagram represent means ± SEM for four control and four mutant mice; *, p < .01. Bar = 70 μm.

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Interestingly, luminal cell population was affected by Myc deletion from BCs. Amount of luminal colony-forming cells (luminal progenitors) was found to be decreased approximately threefold in mutant epithelium from 4-month-old females (Fig. 3C). In K5Cre;R26;mycF/F epithelium, all colony-forming cells were LacZ-negative, that is, expressed Myc (Supporting Information Fig. S3). Strikingly, luminal cells isolated from 18-month-old mutant mice did not exhibit any clonogenic capacity (Fig. 3D).

Luminal progenitors have been reported to be enriched in Sca1-negative luminal cell fraction, whereas Sca1-positive population has been found to contain essentially mature luminal cells that express hormone receptors [13]. Consistently with the low clonogenic capacity, in luminal cells isolated from mutant glands, Sca1-positive cell population was significantly increased when compared with control (Fig. 3E). In agreement with this result, analysis of gene expression by q-PCR revealed higher levels of ERα (esr1) and PrlR (prlr) transcripts and diminished expression of kit1 (kit) and Elf5 (elf5), characteristic of luminal progenitors, in luminal cells from mutant mice when compared with those from control animals. The Notch pathway has been implicated in the control of the luminal progenitor amplification [26]. Luminal cells from K5Cre;mycF/F mice displayed low expression levels of Hey1 (hey1), a Notch pathway target, indicating impaired Notch signaling. Expression levels of the transcription factor Gata3 (gata3) known to regulate luminal cell differentiation [27, 28] were not altered, whereas BRCA1 (brca1) transcript levels were diminished in luminal cells from K5Cre;mycF/F mice (Fig. 3F). In addition, q-PCR analysis revealed elevated expression levels of p16 (cdnk2a), marker of senescence, in luminal cells isolated from mutant mouse mammary epithelium (Fig. 3F). In agreement with the PCR data, immunohistochemical analysis demonstrated that luminal cell layer from mutant mammary epithelium contained significantly more ERα-positive cells than control (Fig. 3G). Taken together, these data clearly indicate that deletion of Myc from BCs affected clonogenic stem/progenitor cell function in both mammary epithelial layers, basal, and luminal.

Deletion of Myc from the Mammary BC Layer Severely Impairs Stem Cell Function

To analyze the effects of Myc deletion on stem cell function in the mammary epithelium, we performed three series of experiments: (a) serial transplantation of mammary epithelial fragments; (b) limiting dilution transplantation of mammary cells obtained by tissue dissociation; and (c) transplantation of sorted BCs.

All grafted epithelial fragments produced outgrowths in the first transplant; however, the density of the ductal network developed in virgin or pregnant hosts was lower for mutant than control tissue (Fig. 4A; Supporting Information Fig. S4). The regenerative potential of the mutant epithelium was exhausted after two transplantations suggesting that deletion of Myc from the BC layer affected the self-renewal potential of the stem cell population(s) present in the adult mammary epithelium (Fig. 4B).

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Figure 4. Deletion of Myc from the basal cell (BC) layer affects the stem cell self-renewal. (A): Outgrowths developed from control and K5Cre;mycF/F mammary epithelial fragments transplanted into the cleared fat pads. The diagram presents quantitative evaluation of the ductal branching complexity in control and mutant outgrowths (11 transplanted mice, mean ± SEM levels are presented, *, p < .001). Bar = 3 mm. (B): Take rate and fat pad filling in serial transplants of control and K5Cre;mycF/F mammary epithelial fragments. (C): Mammary epithelial outgrowths developed from isolated mammary BCs transplanted into the cleared mammary fat pads. Arrowheads indicate the host mouse mammary fat pad area containing the aggregates formed by Myc-deficient mammary BCs. Bar = 3 mm. (D): Sections through virgin mouse mammary fat pad transplanted with Myc-deficient BCs. Left and central panels, H&E; right panel, double immunofluorescence labeling of sections through Myc-KO cell aggregates with anti-K5 and anti-K8 antibodies. Arrows indicate bilayered mammary acinus-like structure. Bar = 0.22 mm, left panel, and 38 μm, other panels.

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To evaluate the functional stem cell content, mammary cells from control and mutant glands were transplanted at limiting dilutions. K5Cre;mycF/F mouse mammary cells produced only few outgrowths (Table 1). The repopulating unit frequency in the total mammary cell population was 1/361 (1/1227-1/107) for controls and 1/76,146 (1/145582-1/39828) for the mutant (CI 95%, p = 4.34e-276). Thus, the number of functional multipotent stem cells in the adult virgin mutant mouse mammary gland was 200-fold lower than that in controls. Consistently with these data and the results of the colony formation assays, transplanted Myc-deficient BCs (1,000–5,000 of sorted BCs per fat pad, 20 transplanted mice, and three independent experiments) failed to develop outgrowths suggesting that multipotent stem cell population was affected by Myc deletion (Fig. 4C). Small dense cell aggregates were visible at transplantation sites, and immunolabeling of the sections through these areas revealed small-cell clusters, with more or less visible lumen, comprising luminal (K8+/K5) and basal (K8−/K5+) cells (Fig. 4D). Altogether, these data reveal that Myc is essential for the self-renewal and function of the longed-lived mammary stem cell population residing in the basal epithelial compartment.

Table 1. Limiting dilution transplantation of mammary cells from control and K5Cre/mycF/F epithelium
  1. Repopulating unit frequency in total mammary epithelial cell population, as determined in limiting dilution transplantations was of 1/361 (1/1,227-1/107) for control and of 1/76,146 (1/145,582-1/39,828) for mutant; CI 95%, p value 4.34e-276.

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Ovarian Hormones Confer a Limited Repopulating Potential to Myc-Deficient BCs, but Fail to Restore Functional Stem and Progenitor Cell Populations

Two recent studies have shown that the key regulators of mammary development, the ovarian hormones E and P, control mammary stem cell function via paracrine mechanisms [29, 30]. The induction of receptor activator of NF-κB-ligand (RANKL, a member of the tumor necrosis factor family) in luminal cells, in response to E/P-stimulation, leads to the amplification of multipotent stem cells in the basal mammary compartment. We, therefore, examined that the regenerative potential of Myc-deficient mammary BCs could be rescued by E/P stimulation of the transplant hosts. In eight out of the 14 animals stimulated with E/P, isolated Myc-deficient BCs produced mammary ductal outgrowths occupying 50%–100% of the fat pad (Fig. 5A, 5B). However, when retransplanted into secondary hosts, the epithelial fragments dissected from the Myc-deficient outgrowths, in most cases, did not give rise to new epithelial ducts (Fig. 5B). These data indicate that E/P stimulation did not rescue the self-renewing multipotent stem cell population affected by Myc deletion.

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Figure 5. Ovarian hormones confer a limited repopulating potential to Myc-deficient basal cells (BCs) but fail to rescue functional stem/progenitor cell populations. (A): Mammary outgrowths produced by control and Myc-deficient (Myc-KO) mammary BCs in hosts stimulated with E/P. Left panels, fragments of the outgrowths Carmine stained in whole mount; right panels, section through whole-mount X-gal-stained outgrowths. Bars = 0.8 mm, left panels and 0.2 mm, right panels. (B): Serial transplantation of control and Myc-deficient BCs in E/P-stimulated hosts. First transplant generation, outgrowths developed from 1,000 transplanted BCs; second transplant generation, outgrowths developed from primary outgrowth fragments retransplanted into new hosts. Primary and secondary hosts were E/P-treated as described in Materials and Methods. (C): q-PCR analysis of Myc expression in basal and luminal cell isolated from the outgrowths developed in E/P-stimulated hosts (a representative experiment). The values reported are normalized to gapdh expression. (D): Colonies formed by 1,000 BCs isolated from the outgrowths developed in E/P-stimulated hosts. (E): q-PCR analysis of cdkn2a and mycn expression in BCs isolated from the outgrowths developed in E/P-stimulated hosts (a representative experiment). The values were normalized to gapdh expression. (F): Colonies formed by 250 luminal cells isolated from the outgrowths developed in E/P-stimulated hosts. (G): q-PCR analysis of gene expression in luminal cells isolated from the outgrowths developed in E/P-stimulated hosts (representative experiments). The values reported are normalized to gapdh expression. (H): Fragments of mammary outgrowths developed from transplanted control or mutant BCs in 19-day-pregnant host. The entire outgrowths are shown in Supporting Information Figure S5. Arrows point to lateral branches and aberrant bud-like structures in mutant tissue. Bar = 0.8 mm. (I): Double immunofluorescence labeling of sections through the outgrowths developed from control or Myc-deficient BCs in pregnant host with anti-STAT5A and anti-αSMA antibodies. Arrows point to alveoli with bright nuclear STAT5 staining in control and weak diffuse staining in mutant tissue. Bar = 175 μm.

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Of note, unlike the K5Cre;mycF/F mouse mammary epithelium (Figs. 1C, 2D), in the outgrowths developed from Myc-deficient BCs, both, basal and luminal cell layers were depleted of Myc. X-gal-staining and q-PCR analyses confirmed that myc expression was abolished not only in basal cells but also in luminal cells (Fig. 5A, 5C). Therefore, the mammary epithelium developed from Myc-deficient BCs is referred to as Myc-KO.

BCs isolated from Myc-KO outgrowths developed in hormone-stimulated hosts were able to produce colonies, albeit to a significantly lesser extent than control BCs, and displayed elevated p16 (cdkn2a) expression suggesting senescence (Fig. 5D, 5E). Three Myc proteins, Myc, L-Myc (mycl), and N-Myc (mycn), have partially redundant functions [1]. We found that N-Myc was expressed at much lower levels than Myc in mammary BCs, whereas L-Myc levels were below the detection threshold. In BCs isolated from Myc-KO outgrowths, N-Myc transcript levels were higher than those in control BCs (Fig. 5E), possibly accounting for the partial rescue of repopulating capacity, whereas L-Myc was undetectable.

Isolated luminal cells depleted of Myc were almost completely devoid of clonogenic capacity indicating a lack of luminal progenitors (Fig. 5F). In agreement with these data, expression of the genes coding for Elf5 (elf5), β-casein (csn2), and α-lactalbumin (lalba) previously shown to be upregulated in luminal progenitors [31] was very low in mutant luminal cells (Fig. 5G). Myc-deficient luminal cells displayed low expression levels of Notch pathway target, Hey1 (hey1), and a Notch ligand, Jag2 (jag2), indicating impaired Notch signaling (Fig. 5G). These results suggest that Myc contributes to the control of intercellular signaling interactions in mammary epithelium.

Pregnancy also partially rescued the repopulating capacity of Myc-deficient BCs (Fig. 5H, 5I; Supporting Information Fig. S5A--S5C), but at parturition, only few if any alveoli were found in Myc-KO outgrowths, whereas expression of genes characteristic of luminal progenitors was much lower than in control outgrowths (Fig. 5H; Supporting Information Fig. S5A). These data implicate Myc in the control of the luminal progenitor function during pregnancy. Thus, even though ovarian hormone stimulation conferred a limited repopulating potential to Myc-deficient BCs, mammary epithelial outgrowths depleted of Myc lacked self-renewing multipotent stem cells and luminal progenitors of high proliferative potential giving rise to alveoli in pregnancy.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Consistent with other studies, our lineage tracing experiments indicated that early progenitor/stem cells giving rise to both layers of the mammary epithelium in the developing mouse gland express K5 [14]. Therefore, it could be expected that K5-promoter-driven Cre expression should lead to deletion of Myc from basal and luminal mammary epithelial layers. However, we observed that in K5cre;mycF/F mice, Myc was deleted from the BC layer only. In contrast to luminal cells, BCs continuously express K5 throughout development and in adult permitting complete deletion of Myc in the basal compartment. Luminal cells emerging early in development lose K5 expression. Myc has been implicated in cell competition phenomenon [32], and, therefore cells that lost K5 expression to acquire luminal phenotype and escaped Myc deletion could out-compete Myc-negative cells within the luminal epithelial compartment.

Deletion of Myc led to a significant reduction of the BC population and moderate but obvious gland hypoplasia. Even though Myc was deleted from the BCs only, luminal cells displayed decreased proliferation rates, and luminal progenitor population was diminished in mutant epithelium. A recent study provided evidence that BCs contribute to the control of mammary branching morphogenesis probably due to production of soluble growth factors stimulating proliferation of luminal cells [33]. We, therefore, suggest that a lack of paracrine or direct intercellular signaling between basal and luminal layers due to low BC number or/and impaired gene expression in Myc-deficient BCs could account for the diminished proliferation and low progenitor cell content in the luminal compartment and decreased branching complexity observed in K5cre;mycF/F mammary epithelium. The nature of the signals from basal to luminal cells serving for the maintenance of the luminal progenitor population remains to be defined. Candidate growth factors and pathways include FGFs, Notch, and Eph/ephrin signaling.

Myc regulates the expression of numerous genes essential for various cell functions, including metabolism, replication, protein synthesis, and cell cycle, all converging on the capacity of the cell to proliferate [1, 34]. One of the most striking results of this study was the impaired capacity of isolated Myc-deficient BCs to repopulate the mammary fat pad. Notably, microarray analysis of gene expression in Myc-deficient mammary BCs revealed that glycolysis and oxidative phosphorylation pathways were affected in mutant cells (data not shown). Therefore, inefficient energy metabolism may, at least, in part, account for the impaired proliferative potential of the mutant cells and their progeny, making impossible intensive cell amplification required for the outgrowth formation from transplanted cells and, in particular, for alveologenesis. Furthermore, transplanted Myc-deficient BCs may be unable to reconstitute the microenvironment required for the initiation of ductal morphogenesis, as Myc-regulated genes include those responsible for interactions between the epithelium and stroma [34].

Stimulation of the transplant host by ovarian hormones provided a limited repopulating capacity to Myc-deficient BCs; however, the mammary epithelial outgrowths completely depleted of Myc failed to repopulate the fat pad in serial transplants, lacked clonogenic progenitor cells in both basal and luminal compartments, and could not undergo lobulo-alveolar development. These observations strongly indicate that Myc is required downstream from ovarian hormones for the control of mammary stem and progenitor cell functions.

In addition to ovarian hormones, Wnt signaling has been shown to regulate the mammary stem cell pool by promoting self-renewal [35]. Myc is a transcriptional target of canonical Wnt signaling (http://www.stanford.edu/group/nusselab/cgi-bin/wnt), therefore, Myc may mediate the effects of Wnts on mammary stem cells.

We found that Myc deletion targeted to the mammary epithelial BC layer had a relatively moderate effect on mammary gland morphogenesis, in most cases, permitting the development of secretory tissue, albeit hypoplastic, in, at least, three successive pregnancies. On the contrary, lobulo-alveolar development was severely inhibited in Myc-KO epithelium comprising both epithelial layers, basal and luminal, depleted of Myc. These data suggest that, despite the clear importance of Myc for normal homeostasis, the cell type-restricted deletion of Myc does not necessarily lead to severe tissue damage, indicating that Myc could be targeted in cancer treatment.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The transcription factor proto-oncogene Myc is a master regulator of cell metabolism and proliferation, and its over-expression is often associated with breast cancer. Myc is a target of ERα and it is thought to mediate, at least, in part, pro-proliferative ERα function. This study provides the first evidence that in the mammary gland, the proto-oncogene Myc is essential for maintenance of the self-renewing multipotent stem cell population responsible for the regenerative capacity of the mammary epithelium and acts downstream from ovarian hormones to induce progenitor cell amplification required for alveologenesis in pregnancy. It has been suggested that deregulated expansion of stem cells or committed progenitors could be at the origin of mammary tumorigenesis [36]. Further studies are required to define whether Myc activity contributes to the maintenance and amplification of tumor-initiating cell populations in breast cancer.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We are particularly grateful to A. Di Cicco for expert technical assistance, to Dr. I. Grandjean and the personnel of the animal facilities at Institut Curie for taking care of the mice, and to Z. Maciorowski and A. Viguier for excellent assistance with fluorescence-activated cell-sorting analyses. We also thank Dr. J.L. Jorcano, P. Soriano, I.M. de Alboran for providing mouse strains and Dr. D. Medina for valuable discussions. The work was supported by La Ligue Nationale Contre le Cancer (Equipe Labelisée 2009) and a grant from Agence Nationale de la Recherche ANR-08-BLAN-0078-01 to M.A.G. M.M., A.C., and A.G. received funding from Association pour la Recherche sur le Cancer; A.C., from Institut Curie and Servier Laboratories; A.G. from the Instituto de Salud Carlos III (Spain); M.A.G. is Directeur de Recherche, M.M.F., M.A.D., and A.G. are Chargé de Recherche at the Institut National de la Santé et de la Recherche Médicale (INSERM).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
SC_12-0109_sm_SupplFigure1.pdf143KSupplementary Figure 1
SC_12-0109_sm_SupplFigure2.pdf165KSupplementary Figure 2
SC_12-0109_sm_SupplFigure3.pdf87KSupplementary Figure 3
SC_12-0109_sm_SupplFigure4.tif2948KSupplementary Figure 4
SC_12-0109_sm_SupplFigure5.pdf132KSupplementary Figure 5
SC_12-0109_sm_SupplMethods.pdf8KSupplementary Data

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