Author contributions: A.C., M.M., and V.P.: collection and assembly of data; J.J.: provision of study material; D.M. and M.A.D.: conception and design; M.M.F.: conception and design and manuscript writing; M.A.G.: conception and design, financial support, and manuscript writing. All authors contributed to data analysis and interpretation and final approval of the manuscript. M.M.F. and M.A.G contributed equally to this article.
Institut Curie, Centre de Recherche, Paris, France
Mammary epithelium comprises a layer of luminal cells and a basal myoepithelial cell layer. Both mammary epithelial compartments, basal and luminal, contain stem and progenitor cells, but only basal cells are capable of gland regeneration upon transplantation. Aberrant expansion of stem/progenitor cell populations is considered to contribute to breast tumorigenesis. Germline deletions of p53 in humans and mice confer a predisposition to tumors, and stem cell frequency is abnormally high in the mammary epithelium of p53-deficient mice. However, it is unknown whether stem/progenitor cell amplification occurs in both, basal and luminal cell populations in p53-deficient mammary tissue. We used a conditional gene deletion approach to study the role of p53 in stem/progenitor cells residing in the mammary luminal and basal layers. Using two- and three-dimensional cell culture assays, we showed that p53 loss led to the expansion of clonogenic stem/progenitor cells in both mammary epithelial cell layers. Moreover, following p53 deletion, luminal and basal stem/progenitor cells acquired a capacity for unlimited propagation in mammosphere culture. Furthermore, limiting dilution and serial transplantation assays revealed amplification and enhanced self-renewal in the basal regenerating cell population of p53-deficient mammary epithelium. Our data suggest that the increase in stem/progenitor cell activity may be, at least, partially mediated by the Notch pathway. Taken together, these results strongly indicate that p53 restricts the propagation and self-renewal of stem/progenitor cells in both layers of the mammary epithelium providing further insight into the impact of p53 loss in breast cancerogenesis. Stem Cells2013;31:1857-1867
The mammary epithelium is composed of two cell layers: a luminal layer lining the lumen of ducts and alveoli, and an external basal myoepithelial layer. The remarkable proliferative potential of the mammary epithelium, allowing lobulo-alveolar development in subsequent pregnancies and repopulation of the cleared mammary fat pad upon transplantation of tissue fragments or dispersed cells, strongly suggests that stem and progenitor cells are present in this tissue . Transplantation experiments with sorted epithelial cells from adult mouse mammary gland revealed that only basal cells (BCs) could regenerate bilayered mammary ducts and alveoli [2-4]. On the basis of these experiments, it was assumed that the mammary BC layer harbored multipotent stem cells responsible for adult gland homeostasis. However, lineage-tracing studies have shown that, although early mammary progenitors display BC characteristics (i.e., they express the BC cytokeratins, K5 and K14) during the postnatal period, the BC compartment makes little or no contribution to the luminal cell layer [5-7], and that both the luminal and basal mammary epithelial compartments are maintained by their specific unipotent stem cells . Furthermore, the possible existence of a bipotent stem/progenitor cell giving rise to alveoli in subsequent pregnancies remains a matter of debate .
Precise molecular markers of mammary stem cells remain to be defined, and the mechanisms controlling the maintenance and self-renewal of these cells are only partly understood. Wnt/β-catenin, RANKL/RANK, and Notch pathways and transcription factors, such as cEBPβ and Myc, have been implicated in the regulation of stem and progenitor cell function in the mammary gland [8-13].
The transcription factor p53 controls multiple tumor suppressor pathways and is frequently mutated in breast cancer . Inactivating mutations of the Tp53 gene have been found in most basal-like mammary carcinomas, a type of tumor thought to originate from mammary stem or progenitor cells [14-16]. Germline mutations of the gene encoding p53 in humans and mice confer a predisposition to cancer [17-20]. Consistently, somatic deletion of p53 from the mouse mammary epithelium leads to frequent estrogen receptor (ER)-positive or -negative cancers, the hormonal status of the tumor depending on the cell population targeted for gene deletion [21, 22].
p53 has been implicated in the control of stem cell function in various tissues, including hematopoietic and neural systems and the mammary gland (reviewed in [23, 24]). The regenerative potential of normal mammary epithelium declines markedly after a few serial transplantations, whereas fragments of p53-deficient mouse glands can be retransplanted in an almost unlimited manner . The frequency of regenerating cells has been shown to be higher in the mammary glands of p53-null mice than in normal mammary tissue [26, 27]. These studies led to the conclusion that p53 restricts the proliferation and self-renewal of mammary gland-resident stem cells. However, the functions of p53 in the maintenance of the distinct stem and progenitor cell populations residing in the luminal and basal mammary epithelial compartments were not studied specifically.
In this study, we used a conditional gene deletion approach to study p53 functions in basal and luminal mammary cell populations. We found that K5 promoter-driven Cre expression resulted in the deletion of the Trp53 gene from both mammary epithelial layers, leading to (a) the expansion of clonogenic stem/progenitor cell populations in luminal and basal compartments and the acquisition of a capacity for unlimited propagation in cell culture; (b) amplification and enhanced self-renewal of the regenerating BC population. Additionally, our data suggest that Notch pathway can contribute to the enhanced stem/progenitor cell activity observed in luminal and basal compartments of the p53-deficient mammary epithelium.
Materials and Methods
Transgenic mice expressing the Cre recombinase under the control of the K5 promoter (K5-Cre) were kindly provided by Dr. J. Jorcano , Rosa26LacZ reporter strain, carrying a loxP-stop-loxP-lacZ cassette, by Dr. P. Soriano . The generation of Trp53F/F mice has been described previously . All mice were bred in a 129SV/C57BL6 genetic background. Trp53F/+ or Trp53F/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).
Whole-Mount Mammary Gland Analyses
For whole-mount Carmine-Alum staining, dissected mammary fat pads were spread onto glass slides, fixed in a 1/3/6 mixture of acetic acid/chloroform/methanol and stained as described elsewhere . 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). For histological analyses, glands were embedded in paraffin, and 7 μm-thick sections were cut, de-waxed, and counterstained with Fast Red. In competition experiments, following X-gal staining, glands were dehydrated, postfixed in acetone for 30 minutes, rehydrated, and stained with Carmine-Alum.
Primary Mammary Epithelial Cells Preparation
The inguinal mammary glands of three to five 16- to 20-week-old virgin females were pooled for the preparation of a single-cell suspension, and cells were processed for flow cytometry, as described elsewhere [2, 4, 5]. The following conjugated antibodies were used: anti-CD24-phycoerythrin (PE) (clone M1/69; BD Biosciences, San Diego, CA, http://www.bdbiosciences.com), anti-CD49f-fluorescein isothiocyanate (clone GoH3; BD Biosciences), anti-CD45-Allophycocyanin (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; e-Biosciences, San Diego, CA, http://www.ebiosciences.com). Labeled cells were analyzed and sorted on a FACSVantage flow cytometer (BD Biosciences). 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 83% and 92%.
Sorted BCs were resuspended in 10 μL of 50% growth factor-reduced Matrigel (BD Biosciences) and injected into the inguinal fat pads of 3-week-old nude BALB/C females cleared of endogenous epithelium as described elsewhere [5, 32]. Repopulating unit frequency was calculated with Extreme Limiting Dilution Analysis software . In competition assays, before transplantation, BCs isolated from K5Cre;Rosa26 mouse glands were mixed in a 1:1 ratio with either control (Trp53F/F) or mutant (K5Cre;Trp53F/F) BCs.
For serial transplantation assays, 450 BCs per fat pad were injected to obtain primary outgrowths; 10 weeks after transplantation, half the transplanted fat pads were stained with Carmine-Alum to visualize the outgrowths, whereas another half was pooled to isolate BCs and retransplant them sequentially at a density of 100 cells per fat pad to obtain second- and third-generation outgrowths.
For mammosphere transplantation assays, BC mammospheres were dissociated and injected into the cleared mammary fat pads (1,000 cells per fat pad). The outgrowths were analyzed 10–12 weeks after transplantation. In all experiments, control and mutant cells were grafted into two contralateral fat pads of the same recipient mouse.
Cell Culture Assays
Sorted luminal cells were cultured at a density of 250 cells per well as described elsewhere . Sorted BCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing 1% fetal calf serum, B27 (Gibco, Invitrogen, Saint Aubin, France, http://www.invitrogen.com) at a density of 1,000 cells per well . After 1 week of culture, colonies were incubated with 5 μM BrdU (5-bromo-2-deoxyuridine, Sigma-Aldrich, Saint-Quentin Fallavier, France, http://www.sigmaaldrich.com) for 1 hour and fixed with 70% ethanol at 4°C. Prior to immunolabeling with anti-BrdU antibodies, cells were treated with 2N HCl for 20 minutes at room temperature and next, with 0.1 M borax buffer.
For mammosphere cultures, freshly isolated cells were seeded on ultralow-adherence 24-well plates (Corning, NY, http://www.corning.com/lifesciences) at the density of 5,000 cells per well, in mammosphere media: DMEM/F12 medium supplemented with B27 (Gibco), 20 ng/mL epidermal growth factor, (Invitrogen), 20 ng/mL basic fibroblast growth factor (Gibco), 4 μg/mL heparin (Sigma-Aldrich), 10 μg/mL insulin (Sigma-Aldrich), and 2% Matrigel (BD Pharmingen, San Diego, http://www.bdbiosciences.com) as described elsewhere . Every 2 weeks, mammospheres were dissociated with 0.05% trypsin (Gibco) and reseeded as described above. When mentioned, to inhibit the Notch pathway, 5 μM N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) solution in dimethyl sulfoxide (DMSO) (or, as control, equal amount of DMSO) was added to culture medium. To evaluate mammosphere size, ImageJ software was used. Mammospheres of 200–400 pixels were defined as small; 400–600 pixels, medium; > 600 pixels, large.
For the mammosphere BrdU incorporation analysis, first-generation mammospheres were dissociated and seeded at a density of 30,000 cells/mL overnight in mammosphere culture conditions. Next, cells were incubated with BrdU for 5 hours, spun down at 1,500 rpm for 5 minutes, fixed, and proceeded for immunolabeling.
For immunofluorescence analysis, mammospheres were resuspended in 50 μL Matrigel and incubated for 3 hours at 37°C. They were then fixed in a 1/3/6 mixture of acetic acid/chloroform/methanol and embedded in paraffin. Sections (7 μm) were cut and dewaxed for immunolabeling, as described elsewhere . Fixed colonies and cytospinned cells were incubated for 1 hour at 37°C with primary antibodies, for 1 hour at room temperature with secondary antibodies and for 3 minutes with 4′,6-diamidino-2-phenylindole (DAPI).
The following primary antibodies were used: rabbit polyclonal anti-K5, and anti-K8 (Covance, Princeton, NJ, http://www.covance.com), anti pan-keratin (Dako, Trappes, France, http://www.dako.fr), anti-BrdU (BD Pharmingen). Alexafluor-conjugated secondary antibodies (1/1,000; Invitrogen) were used for immunofluorescence labeling.
Reverse Transcription-Polymerase Chain Reaction
RNA was reverse-transcribed with MMLV H(-) Point reverse transcriptase (Promega, Madison, WI, http://www.promega.com), and quantitative PCR (qPCR) was performed by monitoring, in real time, the increase in fluorescence of the SYBR Green dye on an LightCycler 480 Real-Time PCR System (Roche Applied Science, Basel, Switzerland, http://www.roche.com). The values obtained were normalized to Gapdh levels. The primers used for qPCR analysis are listed in the Supporting Information section.
Statistical Analysis of the Data
All values are shown as mean ± SEM. p values were determined using Student's t test with two-tailed distribution and unequal variance.
K5 Promoter-Driven Cre Expression Leads to Trp53 Gene Deletion and an Increase in Cell Number in the Mammary Basal and Luminal Epithelial Cell Layers
To study the effects of p53 deletion on mammary stem and progenitor cells, K5Cre mice were crossed with mice carrying conditional alleles of the Trp53 gene (Trp53F/F). Overall, the mammary glands of K5Cre;Trp53F/F mice developed normally as estimated by morphological analyses (Fig. 1A; Supporting Information Fig. S1A, S1B). However, mature virgin K5Cre;Trp53F/F mice had a higher duct density, with more side branches, than control littermates (Fig. 1A; Supporting Information Fig. S1A). Whole-mount and histological analyses did not reveal any difference between mammary glands of control and mutant females at day 15 of pregnancy (Supporting Information Fig. S1B and data not shown). Moreover, expression levels of milk protein genes and myoepithelial cell markers were similar in control and mutant epithelium of 15-day-pregnant mice, indicating that mammary differentiation was not affected by the deletion of p53 (Supporting Information Fig. S1C). The mutant mice did not develop mammary tumors within the 12-month observation period, and their mammary epithelium displayed normal bilayered organization (Supporting Information Fig. S1D).
The K5 promoter is active in early mammary progenitors suggesting that K5-driven Cre expression should lead to conditional (floxed) allele excision from the entire mammary epithelium . We analyzed the distribution of Trp53-deficient cells in mutant mammary epithelium, by crossing control and K5Cre;Trp53F/F mice with the Rosa26-LacZ-reporter mouse strain (R26). As expected, all BCs and most of the luminal cells were LacZ-positive in control K5Cre;R26 mice (Fig. 1B). Similarly, in mutant mouse mammary glands, all the BCs were LacZ-positive, and the number of LacZ-positive cells in the luminal layer was even greater than that in the control (Fig. 1B; Supporting Information Fig. S1E). These data strongly suggest that p53 was deleted from both cell layers of the K5Cre;Trp53F/F mammary epithelium.
Flow cytometry cell sorting and the isolation of BCs and luminal cells from the mammary tissue of control and mutant mice revealed that both compartments of the mutant mammary epithelium contained larger absolute numbers of cells than those of control animals (Fig. 1C, 1D; Supporting Information Fig. S1F). However, the increase in cell number was greater for the luminal compartment, resulting in a slight change in the proportions of BCs and luminal cells in the mutant epithelium (Fig. 1E). Consistent with the results of X-gal assay (Fig. 1B), a qPCR analysis of Trp53 expression suggested that the gene was deleted from both mammary epithelial layers in mutant mice (Fig. 1F).
p53 Deletion Leads to the Expansion of the Luminal Progenitor Population, with Increases in Proliferative Potential and Self-Renewal Capacity
To analyze the effects of p53 deletion on the luminal progenitor population, we examined in freshly sorted luminal cells using qPCR, the expression of several genes known to be differentially regulated in progenitors and mature luminal cells. Mutant luminal cells had higher transcript levels for the Csn2, Hey1, Elf5, and Kit genes (encoding β-casein, Hey1, ELF5, and c-Kit, respectively), characteristic of progenitor-enriched population, and lower levels of transcripts for the Pgr, Prlr, Esr1, and Prom1 genes (encoding progesterone receptor, prolactin receptor, ERα, and prominin 1, respectively), which are known to be expressed by mature ductal luminal cells (Fig. 2A). Notably, levels of Mki67 (Ki67) expression were higher in mutant luminal cells than in control cells, strongly suggesting the presence of a larger number of proliferating cells (Fig. 2A).
The luminal cell fraction negative for Sca-1 has been reported to be enriched in progenitors, whereas hormone receptor-expressing cells have been found essentially in the Sca-1-positive fraction . Consistently with the qPCR data, flow cytometry analysis of Sca-1 expression in mammary luminal cells revealed that the progenitor-enriched Sca-1-negative population was larger in mutant epithelium (Fig. 2B).
Functional evaluation of progenitor cell content in control and mutant luminal cells was carried out using colony formation and mammosphere assays. Mutant luminal cells, when seeded on a fibroblast feeder layer formed more and larger colonies than control cells (Fig. 2C). The percentage of colony-forming cells was 16.8 ± 1.4 for control cultures and 22.2 ± 1.8 for mutant cultures. Consistently, the number of BrdU-incorporating cells in the colonies formed by mutant p53-deficient luminal cells was found to be about twice that in the control colonies (Fig. 2D). The Sca-1-positive (enriched in mature luminal cells) and Sca-1-negative fractions (enriched in progenitors) from the mutant mice displayed increased colony formation capacities, however, for the Sca-1-negative fraction, the difference in the percentage of colony-forming cells between control and mutant was not statistically significant (Supporting Information Fig. S2A).
Wahl's team recently described a method for the evaluation of stem/progenitor cell content in sorted mammary cell populations grown in the presence of 2% Matrigel . Using this technique, we found that freshly isolated luminal cells deficient for p53 formed larger mammospheres, with greater numbers of proliferating cells than those formed by control luminal cells (Fig. 2E; Supporting Information S2B), and the percentage of mammosphere-forming cells was 2.8 ± 1.1 for control and 6.6 ± 1.6 for mutant luminal cell preparations. When control mammospheres were dissociated and the cells obtained were reseeded in the Matrigel-containing medium, they formed only a few second-generation mammospheres of similar size, and further passages resulted in smaller cell aggregates only (Fig. 2E–2G). By contrast, the mammospheres formed by p53-deficient luminal cells could be maintained in culture for at least 25 generations (Fig. 2F, and data not shown). The primary mammospheres formed by control or mutant luminal cells consisted essentially of cells expressing the luminal cytokeratin K8 and contained only a few K5+ cells (Fig. 2G). A small number of K5+ cells were detected in the mammospheres formed by mutant luminal cells, in all passages (Fig. 2G and data not shown).
p53 Deletion Leads to Expansion and Enhanced Self-Renewal in the Basal Progenitor Population
Like mutant luminal cells, the p53-deficient BC population contained a higher proportion of colony-forming cells than the control BC population (2.7 ± 0.5% and 4.9 ± 0.8% for control and mutant BC preparations, respectively, Fig. 3A). The colonies that developed from mutant BCs were significantly larger than control colonies and contained a higher proportion of proliferating cells, as evaluated by BrdU incorporation assays (Fig. 3A, 3B). Similarly, the mammospheres formed by mutant BCs were more numerous and larger than those formed by control cells and contained larger numbers of BrdU-incorporating cells (Fig. 3C–3E; Supporting Information Fig. S3A). Control BC mammospheres could be passaged once, any further passaging being accompanied by substantial decreases in the number and size of mammospheres. In the fourth generation, control BCs formed only small-cell aggregates (Fig. 3C–3E). By contrast, mutant BC mammospheres could be maintained in culture for at least 25 generations. The percentage of mammosphere-forming cells in sorted BC preparations (first generation of mammospheres) was 1.7 ± 0.6 for control cells and 5.8 ± 1.4 for mutant cells. Of note, K5-positive cells were detected in most mutant BC mammospheres, in all generations, whereas K5 expression decreased markedly during the passaging of control BC mammosphere cultures (Fig. 3E, 3F).
Recent studies have suggested that activation of the epithelium-to-mesenchyme transition (EMT) program in mammary epithelial cells is associated with stem cell properties [35, 36]. Mammary BCs are capable of gland regeneration and express EMT-driving transcription factors such as Snai1, Snai2, Zeb1 [35-37]. Consistently with the higher amount of K5+ (basal) cells, the expression levels of several EMT-associated genes (Vim, Snai1, Snai2, Zeb1) were higher in mutant BC mammospheres than in control mammospheres, whereas the level of the Cdh1 (E-cadherin) transcript was lower (Supporting Information Fig. S3B).
Analysis of gene expression in freshly isolated mutant BCs by qPCR revealed decreased transcript levels of p53 targets Cdkn1a and Bbc3 (encoding p21 and Puma, respectively), and, consistently with the increased proliferation detected in the colony and mammosphere assays, increased Mki67 levels (Fig. 3G). Interestingly, levels of transcript of Hey1, a Notch target gene, were found to be elevated in mutant basal cells suggesting activation of the Notch pathway (Fig. 3G).
Wnt/β-catenin signaling pathway has been implicated in the control of mammary stem cell population [8, 38]. However, expression levels of β-catenin target genes Axin2 and Lgr5 were similar in control and mutant basal cells (Fig. 3G). The expression of the transcription factors Slug and CEBPβ (Snai2 and Cebpb, respectively) known regulators of mammary stem cells [13, 35] was not affected in mutant BCs. Moreover, myoepithelium-specific genes were expressed at similar levels in control and mutant cells suggesting that basal cell differentiation was not impaired in virgin mutant mouse epithelium (Supporting Information Fig. S3C). Altogether these results indicate that basal cells depleted of p53 display increased progenitor activity accompanied with the activation of the Notch pathway and apparently normal differentiation.
Notch Activity Contributes to Mammosphere Formation by Luminal Cell and BC
It has recently been reported that p53 represses mammosphere formation by unsorted, nonadherent mammary cells, by inhibiting the Notch pathway . We therefore examined effects of Notch inhibitor DAPT on the mammosphere formation by sorted luminal and BCs. First, qPCR analysis of the Notch targets Hey1, Hey2, and Hes1 confirmed that the pathway was efficiently inhibited after DAPT treatment (Supporting Information Fig. S4A). In the presence of DAPT, control luminal cell and BCs formed fewer mammospheres (Fig. 4A, 4C). The DAPT-induced inhibition of sphere formation was dose-dependent (Supporting Information Fig. S4B, S4C). In mutant cell cultures, mammosphere formation also was affected by DAPT treatment, with fewer mammospheres formed in the presence of DAPT by sorted luminal and BCs (Fig. 4A, 4C; Supporting Information Fig. S4B, S4C). The size of the mammospheres was also altered, particularly in control cell cultures, with significantly smaller mammospheres in the cultures treated with DAPT (Fig. 4B, 4D). Interestingly, we found that after DAPT treatment, independently of p53 status, the mammospheres generated by BCs and luminal cells, consisted mainly of K5-positive (basal) cells (Fig. 4E,F) indicating that the inhibition of Notch perturbed essentially the luminal cell amplification within the mammospheres. These data suggest that under the conditions of the experiment, the Notch pathway contributed to mammosphere formation from luminal and BCs, regardless of p53 status.
Mammary BCs Depleted of p53 Display Enhanced Stem Cell Activity and Self-Renewal
We evaluated the effects of p53 deletion on the regenerative capacity of BCs, by carrying out four series of transplantation experiments: (a) cotransplantation of freshly isolated control and mutant BCs into the cleared mammary fat pads; (b) transplantation of BCs at limiting dilutions; (c) serial transplantation of sorted BCs; (d) transplantation of cells obtained after the dissociation of control or mutant BC mammospheres.
In the first series of experiments, we mixed equal numbers of freshly isolated unlabeled control or mutant BCs with LacZ-labeled control BCs isolated from K5Cre;R26 mouse mammary glands and transplanted the resulting cell mixtures into cleared fat pads. Ten weeks after transplantation, to analyze the origin of the developed outgrowths, the transplanted fat pads were consecutively stained with X-gal and Carmine-Alum in whole-mount. As expected, in the fat pads transplanted with a mixture of labeled and unlabeled control BCs, LacZ-positive and LacZ-negative outgrowths, each, occupied approximately half of the total transplanted fat pad surface (Fig. 5A, left panels and data not shown). By contrast, the outgrowths developed in the fat pads transplanted with a mixture of p53-deficient unlabeled and control LacZ-labeled BCs were almost entirely LacZ-negative, with only a few small blue branches observed in some fat pads (Fig. 5A, right panels and data not shown). These data suggest that p53-deficient BCs have higher levels of repopulating activity and an advantage over p53-positive BCs.
To compare the frequency of the repopulating cells, we transplanted control and p53-deficient BCs at limiting dilutions (Fig. 5B). The repopulating unit frequency in BC populations was found to be 1/20 (1/31-1/13) for control and 1/7 (1/5-1/11) for mutant (CI 95%, p value .000194). Thus, the deletion of p53 led to an expansion of the regenerating BC population.
We then analyzed the role of p53 in stem cell self-renewal, by performing serial transplantations of sorted BCs. In the first transplant generation, the rate of engraftment was 100% for both control and mutant BCs. In the second transplant generation, when 100 BCs per fat pad were transplanted, the engraftment rate appeared to be below 50% for the control, whereas mutant BCs developed elaborate outgrowths in all transplanted fat pads (Fig. 5C). Furthermore, in the third transplant generation, engraftment was observed in 1 and 8 of the 23 transplanted fat pads, for control and mutant BCs, respectively (Fig. 5C). These data suggest that p53-deficient mammary stem cells have an enhanced self-renewal capacity.
Finally, we investigated whether a cell population with repopulating capacity was maintained in the mammospheres, by dissociating second-generation mammospheres developed from control and p53-deficient BCs and transplanting them into cleared mammary fat pads. Cells from p53-deficient BC mammospheres had markedly higher levels of stem cell activity than cells from control mammospheres (Fig. 5D). The regenerating cell population was maintained in the mammospheres formed from p53-deficient BCs after numerous passages (Supporting Information Fig. S5).
In this study, we used a Cre-Lox approach for conditional deletion of the Trp53 gene from mouse mammary epithelium and analyzed the effects of p53 loss on stem cell function in luminal and BC populations, separately. Several previous studies have shown that the mammary epithelium of p53-deficient mice displays enhanced stem cell activity [25-27]. However, they did not determine whether the loss of p53 affected stem cells in both the luminal and basal compartments of the mammary epithelium. In addition, in the case of germline p53 deletion, the possibility of stromal and systemic effects on stem cell function cannot be excluded, making it difficult to define epithelium-specific alterations in these mouse mutants.
We found that p53 was deleted from both mammary epithelial layers of K5Cre;Trp53F/F mice, the basal and the luminal. These data are consistent with the results of lineage-tracing experiments suggesting that the K5 promoter is active in early mammary progenitors (, and this study). We previously used a similar approach to delete the myc gene from the mammary epithelium (K5Cre;MycF/F mice, ). Unlike p53, Myc was deleted from the basal layer but not from luminal cells .
Thus, in the context of the developing mammary gland, the efficiency of Cre-Lox-mediated gene deletion does not depend exclusively on the specificity of the promoter delivering Cre. It also depends on the properties of the protein encoded by the targeted gene. The deletion of p53 conferred an advantage to mutant cells, in that they proliferated more rapidly. By contrast, the absence of Myc limited cell propagation. Both p53 and Myc have been implicated in cell competition, a biological phenomenon involving the selection of cells with a growth or survival advantage within a particular cellular compartment [39, 40]. Lower levels of p53 and higher levels of Myc make cells highly competitive, probably accounting for the complete p53 and partial Myc deletion from the mammary epithelial bilayer of mouse mutants with Cre expression driven by the basal K5 promoter.
Two-dimensional colony-formation and nonadherent mammosphere assays are generally used to analyze the function of mammary stem/progenitor cells in vitro [2, 4, 26, 41]. Classical nonadherent mammosphere assays are performed with total unsorted mammary cells, and the identity of the cells generating such mammospheres is unknown. In this study, we used colony-formation assays and a recently described mammosphere-formation assay adapted for sorted luminal and basal cells . Like the colony-formation assay, this method can be used to evaluate clonogenic cell number and proliferative potential within tested populations, with mammosphere passaging making it possible to evaluate the self-renewal capacity of stem/progenitor cells.
We found that the deletion of p53 led to an increase in clonogenic cell number and proliferative capacity and conferred an unlimited capacity for self-renewal to luminal and basal stem/progenitor cells in mammosphere culture, suggesting that p53 restricts the amplification of stem/progenitor cells residing in the luminal and basal compartments of the mammary epithelium.
Mammospheres formed by cells retaining the membrane dye PKH26 isolated from p53-deficient mouse glands have been reported to contain several stem cells, whereas control mammospheres contain only one stem cell each . We observed a rapid loss of expression of the basal marker K5 from control BC mammospheres upon passaging, whereas in p53-deficient BC mammospheres, basal K5-positive cells were detected at all passages. Recent studies have suggested that activation of the EMT program is associated with the acquisition of stem cell properties, whereas mammary BCs, which have regenerative potential, express numerous genes relating to EMT, including the Slug transcription factor [35, 37]. Consistent with this observation and the presence of BCs in the mammospheres, we were able to detect the upregulation of EMT-associated genes in mutant BC mammosphere cultures. It would have been interesting to determine whether the potential for self-renewal and the regenerative capacity were associated with this basal K5+ cell population within the mammospheres. However, expression of the surface markers used to isolate BC by flow cytometry, the α6 and β1 integrins, was upregulated in all cells within the mammospheres, making it impossible to isolate a specific BC population. We are currently trying to identify other surface markers that could be used for the isolation of BC from the mammospheres, to determine whether clonogenic activity and regenerative potential are associated with this cell population.
An evaluation of the regenerative potential of isolated BCs revealed that p53 loss increased the capacity of the cells to repopulate the mammary fat pad and led to expansion of the regenerating BC population. Furthermore, serial transplantations with limited numbers of cells suggested that the self-renewal capacity of the regenerating BCs was enhanced by p53 deletion. Interestingly, the regenerative capacity of p53-deficient BCs declined in serial transplantations, although it remained markedly higher than that of control cells. By contrast, the mammosphere-forming potential of basal p53-deficient cells did not decrease with numerous passages. Thus, to maintain the stem cell properties, sorted p53-deficient BCs, when transplanted into the cleared mammary fat pad, may require some additional factors such as the presence of luminal cells and growth factors provided in the mammosphere assays, or, when tissue fragments are transplanted.
It has been suggested that mutations leading to abnormal function of stem and progenitor cells can lead to cancer . Two studies have provided evidence that mammary luminal progenitors are at the origin of basal-like Brca1-associated breast cancer [15, 16]. Most basal-like breast tumors are characterized by inactivating mutations of p53 . Thus, our data, connecting p53 loss to luminal progenitor amplification and the acquisition of unlimited self-renewal potential, provide new insight into the cellular and molecular events leading to breast tumorigenesis.
Studies of p53 functions in embryonic stem cells and various adult tissues have suggested that p53 may control the amplification and self-renewal of stem/progenitor populations via several mechanisms, including the modulation of Wnt signaling, the inhibition of rapid cell cycle progression, the promotion of asymmetric stem cell divisions and prevention of the EMT program activation [23, 27, 43]. In addition, it has been shown that p53 inhibits Notch signaling in mammary epithelial cells .
Notch factors are essential mammary stem cell regulators [9, 45]. Notch activation has been reported to induce the expansion of human cell derived mammospheres in nonadherent culture [46, 47], and a recent study by Tao et al.  suggested that p53 restricts mammary stem/progenitor cell activity by affecting the Notch pathway. In transplantation assays, interference with Notch signaling inhibits normal ductal growth . In agreement with these data, we found increased expression levels of the Notch target gene Hey1 in the p53-deficient mammary basal and luminal cells, indicating that an enhanced Notch pathway activation may contribute to the increased stem/progenitor cell activity in luminal and basal compartments of mutant epithelium.
It remains to be defined how the Notch pathway enhances mammary stem cell activity. Surprisingly, we found that Notch inhibition affected mammosphere formation from BCs and luminal cells, essentially, by restricting expansion of K8-positive (luminal) cells. These data are in agreement with the results reported by Bouras et al.  suggesting that Notch activation induced expansion of luminal rather than basal mammary epithelial compartment. Thus, one of the options could be that Notch enhances stem cell activity, as assessed in mammosphere or transplantation assays, mostly by mediating luminal cell propagation necessary for formation of spheres or outgrowths.
We found that treatment with DAPT diminished mammosphere formation from control and mutant cells, whereas, Tao et al.  reported that only p53-deficient, but not control cells were affected by Notch inhibition. One plausible explanation for this contradiction is a significant difference in the experimental conditions. In contrast to the study by Tao et al., where the mammosphere assay was performed in nonadherent cell culture with unsorted mammary cells, that is, mixed population consisting of luminal, BCs, and stromal cells, we assessed the clonogenic capacity of sorted BCs or luminal cells in 2% Matrigel (according to Spike et al. ). Spike et al.  have observed that in nonadherent conditions, sorted mammary epithelial cells displayed very low sphere-forming capacity.
Although our results suggest that the changes in stem/progenitor cell properties observed in p53-deficient cells are, at least, partially mediated by the Notch pathway, contribution of other pathways cannot be excluded. Further investigations are required to establish the precise molecular mechanisms underlying the connection between p53 loss and stemness.
In summary, in this study, we show that loss of p53 led to an expansion of the clonogenic BC and luminal cell populations in vivo, and the acquisition of unlimited self-renewal capacity in vitro, in mammosphere culture, by basal and luminal stem/progenitor cells. In addition, p53 depletion from mammary epithelium resulted in an increased number of regenerating BCs and their enhanced self-renewal capacity in vivo. Altogether, these observations revealing the functions of p53 in the maintenance of specific mammary stem and progenitor pools provide further insights into the impact of p53 loss in breast cancer.
We are particularly grateful to A. Di Cicco and C. Lambert 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 Drs. J.L. Jorcano and P. Soriano for providing mouse strains, and Dr. G. Wahl for sharing the mammosphere protocol with us before publication. This 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 MAG. A.C. and M.M. received funding from Association pour la Recherche sur le Cancer; A.C., from Institut Curie and Servier Laboratories; M.A.G. is Directeur de Recherche, M.M.F. and M.A.D., Chargé de Recherche at the Institut National de la Santé et de la Recherche Médicale (INSERM).
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.