Aberrant Proliferation of Differentiating Alveolar Cells Induces Hyperplasia in Resting Mammary Glands of SV40-TAg Transgenic Mice

WAP-T1 transgenic mice express SV40-TAg under control of the whey acidic protein (WAP) promoter, which directs activity of this strong viral oncogene to luminal cells of the mammary gland. Resting uniparous WAP-T1 glands develop hyperplasia composed of TAg positive cells prior to appearance of advanced tumor stages. We show that cells in hyperplasia display markers of alveolar differentiation, suggesting that TAg targets differentiating cells of the alveolar compartment. The glands show significant expression of Elf5 and milk genes (Lalba, Csn2, and Wap). TAg expressing cells largely co-stain with antibodies to Elf5, lack the epithelial marker Sca1, and are hormone receptor negative. High expression levels of Elf5 but not of milk genes are also seen in resting glands of normal BALB/c mice. This indicates that expression of Elf5 in resting WAP-T1 glands is not specifically induced by TAg. CK6a positive luminal cells lack TAg. These cells co-express the markers prominin-1, CK6a, and Sca1, and are positive for hormone receptors. These hormone sensitive cells localize to ducts and seem not to be targeted by TAg. Despite reaching an advanced stage in alveolar differentiation, the cells in hyperplasia do not exit the cell cycle. Thus, expression of TAg in conjunction with regular morphogenetic processes of alveologenesis seem to provide the basis for a hormone independent, unscheduled proliferation of differentiating cells in resting glands of WAP-T1 transgenic mice, leading to the formation of hyperplastic lesions.


INTRODUCTION
Breast cancer, a progressive disease of mammary gland epithelia, is marked by consecutive appearance of different alterations, namely hyperplastic lesions, carcinoma in situ, and invasive, malignant adenocarcinomas. The mutational signature of human breast cancer supports the view of a direct evolutionary relationship between pre-neoplastic and malignant lesions (1)(2)(3)(4)(5). Mutations that inactivate functions of tumor suppressor proteins and thereby decrease genomic stability seem to be basic to initiation of breast cancer. Transgenic mouse strains were developed to mimic this process at an experimental level and to decipher the mechanisms initiating breast cancer and promoting progression to malignant stages. Major questions concern the cell types targeted by oncogenes in these models as this might determine the phenotype of tumor cells appearing at advanced stages. Here, we asked whether non-malignant, hyperplastic lesions appearing in resting glands of WAP-T1 transgenic mice (6) reflect selection of a distinct epithelial cell type or whether these lesions already display a heterogeneous cell composition as is seen in advanced tumor stages.
WAP-T1 mice express SV40-T-antigen (TAg) under control of the whey acidic protein (WAP) promoter, which directs activity of this strong viral oncogene specifically to epithelial cells of the gland. The transgene encodes both, the large (LT) and small (t) T-antigen. They cooperate to inactivate the tumor suppressor proteins RB and p53, alter expression of cell cycle regulating genes, promote unscheduled transition through G1/S, and impair DNAdamage response mechanisms, events that are known to decrease genomic stability (7)(8)(9).
The WAP promoter responds to lactotrophic hormones and thus is inactive in glands of virgin mice, active during lactation in differentiated luminal epithelial cells of the lobulo-alveolar complex, and at a lower level also active in the resting gland during estrous cycle (10)(11)(12)(13)(14). In accordance, epithelial cells of glands in virgin mice lack TAg whereas differentiated epithelial cells of the lactating gland show high levels of this protein. TAg positive cells of the lactating gland disappear with regression of lobulo-alveolar structures during involution (15)(16)(17). Sections of resting glands derived from uniparous mice show re-appearance of T-antigen positive epithelial cells at about 30 days post-weaning (p.w.). They constitute small hyperplastic lesions, which gradually increase in size and completely pervade the gland at 100 days p.w. Similar to human breast cancer, these hyperplastic lesions develop prior to appearance of carcinoma in situ and advanced tumor stages WAP-T1 (2)(3)(4)(5)(6). Malignant tumors develop late at low frequency in glands of WAP-T1 mice. They reveal a gene expression profile that recapitulates the phenotype of aggressive human cancers (18).
The data suggest that carcinogenesis in resting glands of WAP-T1 is largely delayed or halted at the stage of hyperplastic lesions. The cellular composition of hyperplasia and the status of TAg expressing epithelial cells in these lesions compared to lactating glands and advanced tumor stages are not defined. It was speculated that TAg expression in WAP-T1 selects for certain epithelial cell types. A gene expression analysis showed that WAP-T1 tumor samples are enriched in transcription factors relevant for embryonic stem cell maintenance. It led one to assume that TAg expression may favor survival and proliferation of cells displaying features of epithelial stem or progenitor cells (19). But not only stem or progenitor cells but also cells at advanced stages of differentiation have been proposed to generate hyperplastic lesions in transgenic mouse models (20)(21)(22)(23)(24).
Epithelia of the mouse mammary gland reveal a complex composition, marked by stem and progenitor cells, terminally differentiated cells, and regulatory units, such as hormone sensing cells (25). They rapidly change composition and functional status of the layer in dependence of developmental stages and environmental signals. This raises the question whether oncogenic activity of TAg in WAP-T1 mice at the early stage of hyperplasia randomly targets epithelial cells or promotes selection of a distinct cell type. Gene expression analysis of advanced WAP-T1 tumors identified at least two different tumor entities, which completely differ in marker expression: (i) low grade tumors, exhibiting a basal-like and morphologically differentiated phenotype with loss of chromosomes 2 and 19 and (ii) high grade tumors marked by strong expression of the Met gene and by co-expression of keratin 8/18, keratin 6, and the mesenchymal marker vimentin (26). But, a heterogeneous cell composition of advanced tumors does not necessarily contradict the idea that TAg selects for a distinct epithelial cell type. Data obtained with a tumor cell line derived from WAP-T1 glands showed that tumor cells are equipped with phenotypic plasticity, which for instance allows these cells to acquire a mesenchymal or an epithelial phenotype depending on the tumor environment (27).
Our data show that hyperplasia in resting glands of WAP-T1 mice are uniformly composed of cells differentiating along the alveolar lineage. The results suggest that expression of the viral oncogene in luminal epithelial cells pre-disposed to alveologenesis induces unscheduled proliferation of differentiating cells and thereby causes formation of hyperplasia.

MICE
Inbred BALB/c and the transgenic WAP-SV40 early region mouse line T1 (6) were housed under SPF conditions in accordance with official regulations for care and use of laboratory animals (UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia) and approved by Hamburg's Authority for Health (Nr. 24/96).

PREPARATION OF MOUSE MAMMARY GLANDS AND ISOLATION OF LUMINAL CELL SUBPOPULATIONS
Mammary glands were collected at indicated time points from virgin mice, lactating mice, and uniparous mice of the BALB/c or WAP-T1 strains, respectively. Lymph nodes and tumors sometimes present at late stages in WAP-T1 mice were removed. Mammary glands to be used for RNA extraction were snap-frozen in liquid nitrogen. Glands intended for immunofluorescence were embedded in Shandon Cryomatrix (Thermo Scientific) and frozen at −80°C. To extract cells for subsequent FACS-sorting all mammary glands from one mouse were pooled in L15 Medium (Sigma-Aldrich), transferred to a sterile Petri dish and minced with scalpels. The organoid suspension was digested in serum-free L15 Medium with 3 mg/ml Collagenase Type I (Life Technologies) and 1.5 mg/ml trypsin (Sigma-Aldrich) for 1 h at 37°C. Cells were collected by centrifugation at 300 × g for 5 min and washed once in L15 + 10% fetal calf serum (FCS). Red blood cells were lysed by two rounds of incubation with red blood cell lysis buffer (Sigma-Aldrich) and after two washes with PBS/0.02% EDTA cells were incubated for 15 min at 37°C in SMEM Medium (Life Technologies). Cells were collected by centrifugation and incubated for 2 min at 37°C in 2 ml of 0.25% trypsin/0.2% EDTA in HANKS balanced salt solution (Sigma-Aldrich); Cells were resuspended in 5 ml L15 medium, 400 U/ml Dnase I (Sigma-Aldrich) were added, and the cells were incubated for another 5 min at 37°C before the reaction was stopped by addition of 5 ml L15 + 10% FCS. To obtain a single cell suspension, the cells were passed through a 30 µm filter (Miltenyi Biotech). Lineage depletion was performed using a mouse Lineage Cell Depletion Kit (Miltenyi Biotech), which selects for CD5, CD45R (B220), CD11b, Gr-1 (Ly-6G/C), 7-4, and Ter-119 positive cells and permits subsequent isolation of Sca1 positive cells.
Sizes of subpopulations were derived from independent FACS experiments (BALB/c n = 10; WAP-T1 n = 23). In each experiment, the percentage of total cells was determined for each subpopulation and mean values ± SEM were calculated from all experiments. Statistical significance was assessed using Student's t -test and p-values <0.05 were considered as statistically significant.

RNA EXTRACTION AND QUANTITATIVE REAL TIME PCR
Total RNA was purified using peqGOLD TriFast (Peqlab) according to the manufacturer's protocol. FACS-sorted cells were lysed directly in TRIfast, frozen mouse tissue was carefully cut into small pieces on dry ice, transferred to a lysing matrix tube (MP Biomedicals), and lysed in TRIfast by two cycles of 5 s in a FastPrep instrument (MP Biomedicals). DNA was removed by DNAse I digestion (QIAGEN) followed by another round of RNA extraction using TRIfast. Reverse transcription was performed with the High Capacity RT kit (Applied Biosystems) according to the manufacturer's protocol. PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) in a standard program (10 min 95°C; 15 s 95°C, 1 min 60°C; 40 cycles) running in an ABI 7900HT Fast Thermal Cycler (Applied Biosystems). PCR reactions for each sample were repeated in triplicates. The integrity of the amplified products was confirmed by melting-curve analysis. PCR primers (Table S1 in Supplementary Material) were designed using the Primer3 web tool (http://biotools.umassmed.edu/bioapps/primer3_www.cgi). PCR efficiency was measured for each primer pair using serial dilutions of cDNA. 18S rRNA or HSC70 were used as endogenous controls and relative quantitation of transcript levels was performed based on the 2 −ddCt method.
For the gene expression analysis in whole mammary glands (Figure 5), 10 BALB/c and 10 WAP-T1 mammary glands from five mice each were analyzed and fold changes relative to a BALB/c mammary gland were calculated. Mean values and SEM were calculated from the 10 single values for BALB/c and WAP-T1 mice. Statistical significance was assessed using Student's t -test and p-values <0.05 were considered statistically significant. : CD61 + Sca + ) were calculated and data from single experiments were summarized as mean values ± SEM. Statistical significance was assessed using Student's t -test and p-values <0.05 were considered as statistically significant.

IMMUNOHISTOCHEMISTRY
Tissue specimens were fixed overnight with 4% formaldehyde and 1% acetic acid, and stored in 4% formaldehyde at 4°C. Fixed tissue was embedded in Paraplast X-TRA (Sherwood Medical). Deparaffinated sections were stained with H&E (Sigma) according to standard laboratory protocols. For labeling with antibodies deparaffinated sections were treated with an antigen retrieval solution (Citra Plus, Biogenex). Sections were blocked with normal serum for 1 h at room temperature and stained with primary antibodies at appropriate dilutions overnight at 4°C. Bound antibodies were detected using alkaline phosphatase-, respectively peroxidaselabeled anti Ig detection systems (Envision, DakoCytomation). Alkaline phospatase activity was visualized using naphthol AS-BIphosphate and New Fuchsin (Fuchsin plus substrate-chromogene, DakoCytomation) as substrate; peroxidase activity was identified using diaminobenzidine (DAB) as substrate. Sections were counterstained with hemalum and embedded in Mowiol. Photographs were taken with a LEICA DMI6000B.
For preparation of cryosections glands embedded in cryomatrix were sectioned in a LEICA CM 3050 cryotome at −30°C. Sections were attached to glass cover slips and stored at −20°C. Sections attached to glass coverslips were incubated with −20°C acetone for 10 min, acetone was evaporated at room temperature for 5 min, and sections were rehydrated in PBS for 10 min at room temperature. Staining was done at room temperature in a dark chamber. Sections were blocked with 5% normal serum for 45 min, incubated with the primary antibody for 1 h, washed with PBS three times for 10 min each, stained with fluorochrome labeled secondary antibody for 1 h, and washed again with PBS three times 10 min each. Nuclei were visualized by counterstaining with DAPI or DRAQ5. Stained sections were embedded in Mowiol. Photographs were taken with a Zeiss LSM 501 confocal microscope or with epifluorescence microscopes (LEICA DMI6000B and LEICA DMRA).

RESTING UNIPAROUS WAP-T1 GLANDS SHOW FEATURES OF ALVEOLOGENESIS
Immunohistochemical studies performed on paraffin sections of resting uniparous WAP-T1 glands showed local clusters of TAg positive luminal epithelial cells in ducts ( Figure 1A). These clusters were often associated with processes of side bud formation ( Figure 1B, arrow) Condensing chromatin in individual TAg positive cells (see Figure 1C, arrows) suggested mitotic activity in these lesions. Reaction of individual TAg positive cells with antibodies to phospho-histone H3, a marker of condensing chromosomes in mitosis, corroborates this suggestion ( Figure 1D). The data suggest that TAg expressing cells are involved in morphogenetic processes inducing lobulo-alveolar structures.
To characterize TAg positive cells in relation to functional stages of luminal epithelia we performed an IF double labeling study with antibodies to Sca1 and SV40-TAg on cryosections of WAP-T1 glands. Sca1 is a GPI-anchored protein that was originally identified in hematopoietic stem cells. In the mouse mammary gland, it was reported to mark luminal epithelial cells in ducts and bipotent luminal progenitor cells of the lobulo-alveolar compartment, but not terminally differentiated alveolar cells (28)(29)(30). In our labeling study, we included glands from virgin mice, lactating mice, and uniparous mice (120 days p.w.). As shown in Figure 2A, luminal epithelia of virgin WAP-T1 glands contained Sca1 positive and negative cells, but were generally devoid of TAg. Luminal epithelia of lactating glands were TAg positive but Sca1 negative ( Figure 2B). In similar, cells in hyperplastic lesions of resting uniparous WAP-T1 glands which stained positively for TAg were also Sca1 negative (Figures 2C,D). Sca1 positive epithelial cells in glands were confined to luminal epithelia of ducts and did not stain with antibodies to TAg. These findings suggest that TAg expression is confined to Sca1 negative luminal epithelial cells.
To substantiate a relationship of TAg expression with alveologenesis, we assayed resting uniparous WAP-T1 glands (120 days p.w.) for expression of ELF5, a transcription factor known as master regulator of alveologenesis (31). Immunofluorescence staining on cryosections demonstrated that TAg positive cells co-expressed www.frontiersin.org ELF5 (Figure 3). But, expression of TAg and ELF5 in epithelia of hyperplasia did not totally overlap. A proportion of TAg positive cells showed only weak staining or was ELF5 negative (Figure 3).
The data suggest that TAg and Sca1 mark different functional entities of cells in luminal epithelia of WAP-T1 glands: TAg + /Sca1 − cells representing the differentiating lobulo-alveolar compartment, and TAg − /Sca1 + identifying cells of the ductal epithelium.
To determine whether TAg expressing cells in hyperplasia of WAP-T1 glands proliferate, we labeled cryosections with antibodies to Mcm2 and to Ki67, both of which are nuclear markers that are expressed in cycling cells. Mcm2 is a member of the licensing protein family, which facilitates coordinated transition from G1 into S-phase and is down-regulated, when cells exit the cell cycle (32). Human mammary gland epithelia show Mcm2 in nuclei of differentiating but not in terminally differentiated cells (33). Ki67 is expressed in G1, S, G2, and M-phase of the cell cycle but is absent from cells resting in G0. Thus, Ki67 is  Figure 4B). TAg positive cells also co-stained with antibodies to Ki67 (Figure 4C). These results indicate that TAg expressing epithelial cells that accumulate in hyperplasia of resting WAP-T1 glands do not exit the cell cycle.

RESTING UNIPAROUS WAP-T1 GLANDS SHOW LACTOGENIC ACTIVITY
Next, we asked whether formation of lobulo-alveolar structures in resting WAP-T1 glands correlated with lactogenic activity, which is marked by expression of the milk genes WAP, Csn2 (beta-casein), and Lalba (lactalbumin). For comparison, we included in our analysis one gland from each, a lactating WAP-T1 mouse, a BALB/c Frontiers in Oncology | Molecular and Cellular Oncology virgin mouse, and a WAP-T1 virgin mouse. qRT-PCR analysis performed with whole glands (summarized in Figure 5) revealed high expression levels of ELF5 and milk genes in glands of lactating WAP-T1 mouse (T1 Lak, Figures 5A-D), and low levels in glands from virgin mice (T1vir and BALB/c vir, Figures 5A-D). Resting glands of WAP-T1 (T1 120 pw) showed prominent Elf5 expression (Figure 5A), clearly exceeding the level in resting BALB/c glands (BALBc 120 pw, Figure 5A) by 1.7-fold (p < 0.01). Milk gene expression was detectable in all glands, but showed clear differences between WAP-T1 and BALB/c. While Csn2 expression was only slightly (~twofold, n.s.) upregulated in resting WAP-T1 glands compared to resting BALB/c glands (see BALB/c 120 pw and T1 120 pw in Figure 5B), Lalba and Wap expression levels were increased 40-and 10-fold, respectively (p < 0.001 and p < 1.00E-05) (Figures 5C,D). These data suggest enhanced alveologenesis in resting uniparous WAP-T1 glands.

TAg EXPRESSING CELLS REACH AN ADVANCED STAGE IN ALVEOLAR DIFFERENTIATION
Based on our in situ analysis, we asked if we could use Sca1 as marker to isolate luminal cell populations from WAP-T1 glands in order to define their status by RT-PCR. Single cell populations obtained after proteolytic digestion of glands and lineage depletion were fractionated into subpopulations applying fluorescent activated cell sorting. A first approach, trying to isolate the bulk of epithelial cells directly by use of CD24 did not reveal clear subpopulations in samples from WAP-T1 glands. Thus, we selected CD29 as marker to separate luminal (CD29 low ) from basal epithelial cell populations (CD29 high ). The CD29 low population was then fractionated into Sca1 + and Sca1 − subpopulations. In addition, we included CD61 as marker in our sorting strategy (Figure 6A). CD61 was described to mark luminal progenitor cells, endowed with the potential to differentiate into alveolar or ductal cells (35).Our sorting strategy is similar to that previously described by Shehata et al. (36), who also used Sca1 to isolate luminal cell subpopulations. But in difference to our approach they isolated epithelial cells directly by use of Epcam and substituted CD61 for CD49b.
Sorting yielded four different luminal subpopulations (Figure 6B), which were assayed for gene expression by qRT-PCR: the subpopulations CD29 low /CD61 + /Sca1 + and CD29 low /CD61 − /Sca1 + , which we expected to be enriched with TAg negative cells, and the subpopulations CD29 low /CD61 + /Sca1 − and CD29 low /CD61 − /Sca1 − , which we expected to be enriched with TAg positive cells differentiating along the alveolar lineage. qRT-PCR ( Figure 6C) verified that cells expressing the myoepithelial marker CK14 clearly separated with the CD29 hi subpopulation. Thus, basal epithelial cells were separated efficiently from luminal cell subpopulations in this approach. In accordance, immunofluorescence staining showed CK14 positive cells only in CD29 hi subpopulations (data not shown). The relative proportion of CD61 + cells in samples isolated from resting glands of BALB/c and WAP-T1 mice was rather identical (Figure 6D). But compared to BALB/c, the proportion of CD61 − /Sca1 + cells was significantly decreased (15.6 vs. 27.9%, p < 0.05) and that of CD61 − /Sca1 − cells was significantly increased (54.2 vs. 39.2%, p < 0.05) in WAP-T1 samples. This suggests a relative shift in population sizes to differentiating alveolar cells in WAP-T1 glands.
Then, we asked if ELF5 and milk gene expression observed in Sca − subpopulations from resting WAP-T1 glands (T1 in Figures 7A-C) is also detectable in luminal cell subpopulations from resting BALB/c glands. qRT-PCR analysis revealed significant expression of Elf5, Lalba, and Wap in Sca1 − subpopulations from BALB/c but only basal expression levels in Sca1 + subpopulations (BALB/c in Figures 7A-C). Remarkably, Elf5 expression ( Figure 7A) in the Sca1 − /CD61 + subpopulation reached the same high level as in WAP-T1, but was decreased 2.5-fold (p < 0.01) in the Sca1 − /CD61 − subpopulation compared to WAP-T1. This indicates that Elf5 expression in differentiating alveolar cells of BALB/c does not reach the same high level as in WAP-T1. Overall expression levels of milk genes (Figures 7B,C) in Sca1 − subpopulations from BALB/c were low compared to WAP-T1: Lalba expression was reduced by~twofold (n.s.) in CD61 + /Sca1 − and~10-fold (p < 0.01) in CD61 − /Sca1 − subpopulations; Wap expression was reduced~70-and 100-fold (p < 0.05 and p < 0.01), respectively. www.frontiersin.org The data indicate that alveolar differentiation marked by Elf5 also takes place in resting uniparous BALB/c glands and thus is not a specific feature of resting uniparous WAP-T1 glands. But, ELF5 expression in subpopulations enriched with differentiated alveolar cells (CD61 − /Sca1 − ) was significantly higher in WAP-T1 than in BALB/c mice. Furthermore, Sca1 negative subpopulations from WAP-T, but not from BALB/c showed significant expression of milk genes. This suggests that alveolar cells in resting uniparous WAP-T1 glands (120 days p.w.) reach a more advanced stage of differentiation marked by enhanced lactogenic activity.

TAg DOES NOT TARGET CK6a POSITIVE LUMINAL EPITHELIAL CELLS
Luminal epithelia of the mouse mammary gland contain CK6a positive cells, which were repeatedly discussed to represent putative progenitor cells of the ductal and alveolar lineage (20,37). Furthermore, they were described as potential targets of activated oncogenes in transgenic mice giving rise to mammary gland tumors (22,23). High grade tumors in WAP-T1 mice showed significant expression of CK6 as assayed by gene expression analysis (26). Thus, we asked whether TAg targets CK6a positive cells in resting uniparous WAP-T1 glands.
IF studies on cryosections of resting uniparous WAP-T1 glands indicated that CK6a positive cells are present in luminal epithelia of ducts, but absent from hyperplasia composed of TAg positive cells (Figure 8A).There was no evidence for CK6a positive cells co-expressing TAg. In ducts, Ck6a positive luminal cells were often seen in close proximity to TAg expressing cells ( Figure 8B). These data suggest that TAg does not target CK6a positive cells.

TAg EXPRESSING CELLS ARE ESTROGEN AND PROGESTERONE RECEPTOR NEGATIVE
It is well-known that proliferation and differentiation of luminal epithelia into lobulo-alveolar structures are under control of hormone receptors (38)(39)(40)(41). Thus, we asked whether high TAg levels in luminal cell subpopulations derived from resting uniparous WAP-T1 glands correlated with high expression of either the estrogen (Esr1) or progesterone receptor (Pgr). qRT-PCR analysis showed prominent Esr1 expression in Sca1 + subpopulations and a~sevenfold lower expression in Sca1 − subpopulations from WAP-T1 (T1 in Figures 9A,B). A similar pattern was observed in subpopulations from resting uniparous BALB/c glands (BALB/c in Figures 9A,B). But, compared to WAP-T1, Esr1 receptor expression was significantly higher (1.6-fold, p < 0.01 for CD61 + /Sca1 + ; twofold, p < 0.01 for CD61 − /Sca1 + ). Interestingly, WAP-T1 subpopulations with high Esr1 levels showed low expression of TAg, Elf5, and milk genes (compare with Figure 7), suggesting that TAg and estrogen receptor mark different cells. In accordance by immunofluorescence, we found no overlap between estrogen receptor (ER) and TAg staining in luminal epithelia of glands (see Figure 10A).
Pgr expression levels in luminal cell subpopulations from BALB/c glands generally exceeded those in subpopulations from WAP-T1 glands (BALB/c and T1 in Figure 9B). Pgr levels were most prominent in subpopulations enriched in CD61 − /Sca1 + cells with a tendency to be higher (~twofold, n.s.) in BALB/c compared to WAP-T1. Sca1 − subpopulations of BALB/c and WAP-T1 mice exhibited strongly reduced expression levels of Pgr. Thus, Pgr expression also shows a negative correlation with expression of TAg, Elf5, and milk genes as observed for Esr1. In situ, progesterone (PR) positive cells were present in luminal epithelia of ducts and generally showed no overlapping staining with antibodies to TAg (Figures 10B,B ). Taken together, these data indicate that TAg expressing luminal epithelial cells are estrogen and progesterone receptor negative.

HORMONE RECEPTOR POSITIVE CELLS LOCALIZE TO DUCTAL EPITHELIA AND EXPRESS CK6a, PROMININ-1, AND Sca1
Previous studies of others on adult virgin mice showed that estrogen and progesterone receptor expression are confined to "hormone sensing cells" located in the ductal epithelium; these cells were shown to express prominin-1 (CD133) and Sca1 and revealed a relatively differentiated phenotype (42). In line with www.frontiersin.org these data, our qRT-PCR analysis showed high expression of Prom1 specifically in the CD61 − /Sca + subpopulation both, from WAP-T1 and BALB/c, and this population also exhibited high expression levels of Esr1 and Pgr (BALB/c and T1 in Figures 9C,D).
Krt6a expression was also most prominent in this subpopulation. Therefore, we asked whether CK6a and prominin-1 localized to identical epithelial cells. IF studies performed on cryosections of resting uniparous WAP-T1 glands demonstrated that CD133 (prominin-1) and CK6a antibodies marked the same cells in luminal epithelia of ducts ( Figure 11A). CD133 positive cells were also positive for the estrogen (Figure 11B), respectively progesterone receptor (Figure 11C), but definitely negative for TAg. Our data indicate that CK6a, prominin-1, and Sca1 are common markers of hormone receptor positive cells in luminal epithelia of ducts. These cells proved to be absent from hyperplasia in resting uniparous WAP-T1 glands, but in ducts they often localized in close proximity to TAg expressing cells.

RESTING GLANDS OF AGED WAP-T1 VIRGIN MICE ARE NOT SENSITIZED TO ALVEOLOGENESIS
At this point, we asked whether WAP-T1 specific changes seen in resting glands of parous animals are already pre-determined at the virgin stage. We assumed that significant alterations should accumulate with time and thus be most prominent in aged virgin mice. Therefore, we extended our gene expression analysis to Frontiers in Oncology | Molecular and Cellular Oncology   Figure 7A).The milk genes Wap and Lalba were barely expressed (see BALB/c vir and T1 vir in Figure 7B). Esr1 and Pgr levels in luminal cell subpopulations of aged virgins were virtually identical in both mouse strains (see BALB/c vir and T1 vir in Figures 9A,B) and similar to the level in resting (120 days p.w.) glands of normal BALB/c mice (see BALB/c in Figures 9A,B). They reached the highest levels in CD61 − /Sca1 + subpopulations, and like in resting glands these populations also showed highest expression of Prom1 and Krt6a (BALB/c vir and T1 vir in Figures 9C,D). These results demonstrate that glands of aged virgin mice display a hormone receptor status similar to that of resting glands. However, pathways leading to expression of ELF5 and milk genes seem not to be active at the virgin stage in both mouse strains. Therefore, changes observed in resting uniparous WAP-T1 glands are not already pre-determined in virgin mice. The data indicate that passage through pregnancy, lactation, and involution sensitizes luminal cell populations in resting glands of parous BALB/c and WAP-T1 mice to pathways of alveologenesis.

DISCUSSION
Our data show that hyperplasia composed of TAg expressing epithelial cells in resting uniparous WAP-T1 glands display features of lobulo-alveolar cells. The cells not only express ELF5, a transcription factor known to specify secretory alveolar cell fate www.frontiersin.org of CD61 + precursors in the mature alveolar epithelium (43,44), but are secretory as indicated by expression of milk genes. This suggests they reach an advanced stage in alveolar differentiation.
As shown by immunofluorescence, TAg positive cells in lactating WAP-T1 glands and in hyperplasia of resting uniparous WAP-T1 glands did not express Sca1; Sca1 positive cells were clearly confined to ductal epithelia. It suggests that Sca1 is a marker which separates ductal cells from cells with an alveolar cell fate in the mouse mammary gland. In accordance, ELF5 and milk gene expression clearly separated with Sca1 negative subpopulations in our RT-PCR analysis.
TAg was expressed at a high level not only in CD61 − /Sca1 − , but also in CD61 + /Sca1 − cells. This led us to assume that TAg targets luminal epithelial cells early during alveolar differentiation. Possibly, TAg expression is initiated with the onset of ELF5 expression, as this master regulator of alveologenesis was shown to induce WAP expression (43). Additional markers are needed to precisely decipher alveolar differentiation of luminal cells in relation to TAg expression. Progress is being made in this area as a recent study identified hitherto unknown epithelial cell lineages that regulate spatial placement of tertiary branches as well as formation of alveolar clusters in ducts of mammary glands (45). It would be interesting to see if the onset of TAg expression coincides with early priming to the alveolar lineage.
Elf5 expression was not only seen in resting uniparous WAP-T1 glands, but also in resting uniparous BALB/c glands. We show that CD61 + /Sca1 − subpopulations from WAP-T1 and BALB/c glands exhibited nearly identical, high levels of Elf5. Thus, alveologenesis seen in resting WAP-T1 glands is not specifically induced by TAg. Alveolar differentiation and formation of lobulo-alveolar structures are initiated regularly in rodent mammary glands during estrous cycle (46). Normally, these structures regress during diestrus. Thus, we assume that development of hyperplasia in WAP-T1 mice is initiated by mechanisms normally inducing transient alveologenesis in parous mice. Considering that ELF5 induces the Wap promoter (43), it is conceivable that ELF5 sustains TAg expression in differentiating alveolar cells. Although the cells show lactogenic activity, they seem not to reach a terminal stage of alveolar differentiation, as they stay in cell cycle which is indicated by positive reaction with antibodies to Mcm2 and Ki67. Both of these markers are not expressed in differentiated cells (33,34).
It has been well-established that differentiation into alveolar, secretory active cells is regulated by a complex regulatory network comprising hormone receptors and transcription factors (43,44,47,48). Apparently, estrogen (ER) and progesterone receptor (PR) positive cells compose a hormone sensing compartment within the luminal epithelium of ducts that induces differentiation and proliferation of hormone receptor negative cells through paracrine mechanisms. In addition, proliferation of hormone sensitive cells seems to be stimulated directly by progesterone and estrogen (42,49). Recently, a relatively differentiated subpopulation of luminal cells has been identified in glands of adult virgin mice displaying the markers CD24 high /Sca1 + /prominin-1 + (CD133)/CK18 + and expressing estrogen, progesterone, and prolactin receptors. These cells represent little stem cell activity and show no proliferative activity, suggesting they constitute a hormone sensitive compartment (42). Here, we demonstrate for WAP-T1 glands that these cells localize to luminal epithelia of ducts, are TAg negative, but in close proximity to TAg positive cells. Furthermore, we show for the first time that ductal cells positive for hormone receptors and prominin-1 (CD133) are identical with those expressing cytokeratin CK6a. CK6a has been discussed to mark a population of luminal mammary progenitor cells (30,50,51), which lack repopulation activity (37). A progenitor cell function is compatible with changes in the relative proportion of cells during mammary gland development. These cells are found at significant quantity in mammary ducts of virgin mice, reduced in number during pregnancy, www.frontiersin.org apparently absent from epithelia of lactating glands, and reappear in resting glands after involution (37).
Our qRT-PCR data further corroborate a relationship between expression of CK6a, prominin-1, and hormone receptor genes. Prom1 and Krt6a expression were prominent in the same Sca1 positive subpopulation of luminal cells. This subpopulation also proved to be unique with regard to expression of the progesterone receptor, a hormone receptor known to drive mammary secretory differentiation via induction of ELF5 in luminal progenitors (47). Thus, we speculate that CK6a + /prominin + /Sca1 + /ER + /PR + luminal cells have a unique function in induction of TAg and ELF5 leading to the formation of TAg positive, estrogen, and progesterone receptor negative hyperplasia. Crossing of WAP-T1 mice with conditional progesterone receptor knock-out mice would be an interesting approach to investigate this hypothesis by testing for the requirement of this hormone receptor for tumor formation.
Features described here for WAP-T1 mice may also apply to other transgenic mouse models of mammary carcinogenesis that display hyperplasia with an alveolar phenotype. Similar to WAP-T1, C3(1)/SV40-T transgenic mice showed TAg expression in terminal duct lobular units (TDLU), an increased number of TDLU proliferative lesions and side ducts, and at later stages expansion of cells into the ductal lumen with multistage progression to carcinoma (52). Mice transgenic for the polyomavirus middle T-antigen (PyV-mT) under control of the MMTV promoter showed focal pre-malignant lesions and an enhanced number of abortive side buds with lumen that was positive for milk proteins (WAP and OPN) (53). Mice transgenic for Wnt-1, Int-2, Cyclin D, or TGF α under control of mammary specific promoters (MMTV, WAP, ß-lactoglobulin) also developed alveolar hyperplasia (54)(55)(56). In MMTV-neu transgenic mice, parity induced epithelial cells endowed with the potential to differentiate into alveolar or ductal cells were identified as targets for induction of tumorigenesis (24). Thus, hormone dependent activation of an oncogene in differentiating luminal epithelial cells of the mammary gland could generally be a crucial step to induction of aberrant proliferative activity leading to the formation of hyperplastic lesions.

AUTHOR CONTRIBUTIONS
Timo Quante, Florian Wegwitz, Wolfgang Deppert, and Wolfgang Bohn conceived and designed the experiments. Timo Quante and Florian Wegwitz designed and performed FACS and qRT-PCR analysis; Julia Abe and Alessandra Rossi were responsible for animal work and immunofluorescence analysis; Timo Quante, Wolfgang Deppert, and Wolfgang Bohn wrote the paper. The authors read and approved the final manuscript.

ACKNOWLEDGMENTS
We thank Gundula Pilnitz-Stolze, Arne Duesedau, Kerstin Reumann, and Daniela Indenbirken for excellent technical support. The Heinrich-Pette-Institute is financially supported by Freie und Hansestadt Hamburg and Bundesministerium für Gesundheit. This work was supported by grants from Deutsche Krebshilfe to Wolfgang Bohn and Wolfgang Deppert (Forschungsverbund "Tumorstammzellen"). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.