Characterization of rat mammary epithelial cells (RMECs) based on cell surface and intracellular markers.

<p>(<b>A</b>) Representative flow cytometric histograms and dot plots showing gating for propidium iodide (PI)-negative (live) cells (left panel); exclusion of endothelial cells and leukocytes based on CD31 and CD45 expression, respectively (middle left panel); CD61 expression in CD45–CD31– RMECs (middle right panel); CD24 and CD29 expression in CD45–CD31– RMECs identifies two major populations indicated with a red or blue circle (right panel). (<b>B</b>) Dot plots of intracellular cytokeratin (CK) 14 and CK19 expression in CD45–CD31– RMECs (upper left panel); intracellular smooth muscle actin (SMA) staining with phalloidin and CD29 expression in CD45–CD31– RMECs (upper right panel); overlay of dot plots showing CD24 and CD29 expression in CK14+CK19- cells and CK19+CK14- cells (lower left panel); overlay of dot plots of phalloidin bright cells on CD24 and CD29 expression in CD45–CD31– RMECs (lower right panel). Based on CK14, CK19, and SMA expression, the luminal (red) and basal (blue) populations in CD45–CD31– RMECs are identified. (<b>C</b>) Contour plot showing binding of Peanut Lectin (PNL) or anti-Thy-1 in CD45–CD31– RMECs (left panel), overlaid histograms showing CD29 expression on PNL+Thy1-, PNL-Thy-1+ cells (middle left panel), contour plots showing anti-Thy-1 (middle right panel) or PNL binding in CD29med or CD29hi cells (right panel). For all panels, rats of 12 weeks of age were used.</p

Differences between the RMECs from mammary glands of untreated control rats and mammary carcinomas from rats exposed to 7,12-dimethylbenz(a)anthracene (DMBA) or <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU).

<p>(<b>A</b>) Representative pseudo-color dot plots showing CD24 and CD29 expression in the RMECs from the mammary gland of an age-matched (22 weeks of age) untreated control rat (upper left panel) and a DMBA- (upper middle panel) or MNU-induced (upper right panel) carcinoma; bar graphs (lower panel) quantifying mean ± sem percentage cells in the CD24hiD29hi gate within the total (CD45–CD31–) RMECs. A significantly different percentage comparing carcinomas to mammary glands is indicated with an asterisk (p<0.05). (<b>B</b>) Bar graphs showing the mean ± sem percentage of RMECs containing >2n cellular DNA (actively dividing cells in S/G2+M phase of cell cycle). Significantly different percentage comparing RMECs from carcinomas to control mammary glands is indicated with an asterisk (p<0.05). (<b>C</b>) Representative overlaid histograms showing upregulation of CD29 expression (upper left panel), upregulation of CD49f expression (upper middle panel) and downregulation of CD61 expression (upper right panel) in RMECs of a DMBA-induced or MNU-induced carcinoma as compared to a control mammary gland; bar graphs quantifying the mean fluorescence intensity (MFI) in artificial units (a.u.) ± sem of CD29 (lower left panel), CD49f (lower middle panel) and CD61 (lower right panel) on RMECs from control mammary glands and carcinomas. Significantly different MFI is indicated with an asterisk (p<0.05). (<b>D</b>) Representative pseudo-color dot plot showing gating for CD29 and focal adhesion kinase (FAK) in RMECs from a control mammary gland (upper left panel), a DMBA-induced (upper middle panel) and MNU-induced (upper right panel) carcinoma; bar graph (lower panel) quantifying mean ± sem percentage of CD29hiFAK+ cells. A significantly different percentage comparing carcinomas to control mammary glands is indicated with an asterisk (p<0.05). (<b>E</b>) Representative pseudo-color dot plot showing gating for CD29 and Y397-phosphorylated focal adhesion kinase (pFAK) in RMECs from a control mammary gland (upper left panel), a DMBA-induced (upper middle panel) and MNU-induced (upper right panel) carcinoma; bar graph (lower panel) quantifying mean ± sem percentage of CD29hi pFAK+ cells. A significantly different percentage comparing carcinomas to control mammary glands is indicated with an asterisk (p<0.05). In the entire <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g004" target="_blank">figure 4</a>, age-matched untreated control mammary glands: n = 16, DMBA-induced mammary carcinomas: n = 10 and MNU-induced mammary carcinomas: n = 10.</p

Features of the actively dividing cells in CD45–CD31– RMECs.

<p>(<b>A</b>) Flow cytometric histogram showing gating for actively dividing cells (cells in S/G2+M phase of the cell cycle) by having >2n cellular DNA content (left panel); representative dot plot showing the actively dividing cells overlaid on CD24 and CD29 expression in the RMECs (right panel). (<b>B</b>) Representative dot plot showing gating for RMECs expressing high levels of both CD24 and CD29 (CD24hiCD29hi gate; left panel); bar graph (right panel) showing mean ± sem percentage of cells in S/G2+M phase of the cell cycle in total RMECs or CD24hiCD29hi-gated cells (n = 24 each). A significant enrichment of actively dividing cells was detected in the CD24hiCD29hi-gated cells (p<0.05; indicated with an asterisk). (<b>C</b>) Overlaid histogram showing CD49f expression in the total RMECs and actively dividing cells (left panel); bar graph (right panel) showing mean fluorescence intensity (MFI) in artificial units (a.u.) ± sem of CD49f in the total RMECs and actively dividing cells (n = 14 each). Significantly different MFI is indicated with an asterisk (p<0.05). (<b>D</b>) Representative dot plot showing CD49f expressing cells (CD49f+) overlaid on CD24 and CD29 expression of the RMECs (left panel); bar graph showing mean ± sem percentage of CD24hiCD29hi-gated cells in total RMECs or the CD49f expressing population (right panel). A significant enrichment of CD24hiCD29hi-gated cells was detected in the CD49f expressing population (p<0.05; indicated with an asterisk). For all panels, rats of 12 weeks of age were used.</p

Epithelial cell differentiation in mammary glands and carcinomas from 7,12-dimethylbenz(a)anthracene (DMBA)-or <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU)-exposed rats.

<p>(<b>A</b>) Schematic representation of the modulation of RMEC differentiation 1 week after exposure of rats to the mammary carcinogens DMBA or MNU. DMBA exposure increases CD49f expression and proliferation (not shown here). MNU exposure disrupts the luminal and basal homeostasis. (<b>B</b>) Schematic representation of the changes of RMEC differentiation in carcinomas as compared to mammary gland from untreated age-matched (22 weeks of age) control rats. Note that the RMECs from animals of 22 weeks of age have a higher percentage of luminal cells as compared to younger animals (comparing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g001" target="_blank">Fig. 1A</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026145#pone-0026145-g004" target="_blank">Fig. 4A</a>). In both panels cell surface expression of CD24, CD29, CD49f and CD61 and intracellular expression of focal adhesion kinase (FAK) and Y397-phosphorylated FAK (pFAK) in basal, luminal and CD24hiCD29hi-gated cells are shown.</p

The rat mammary carcinoma susceptibility locus <i>1a</i> (<i>Mcs1a</i>) is located in a gene desert and confers resistance to three distinctly acting carcinogenic treatments.

<p>A) Genetic map of the congenic lines contributing to the positional identification of the <i>Mcs1a</i> locus on rat chromosome <i>2</i>. Each congenic line, as defined by genotyping the genetic markers indicated along the vertical scale bar (also listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549.s005" target="_blank">Table S1</a>), represents a segment from the resistant Copenhagen (Cop) inbred strain introgressed into the susceptible Wistar-Furth (WF) genetic background. The critical interval for the <i>Mcs1a</i> resistance allele is defined by resistant congenic lines (filled bars) showing a <i>7,12</i>-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma multiplicity that is lower than that of the susceptible congenic control line (WF.Cop), and susceptible congenic lines (open bars) showing a DMBA-induced mammary carcinoma multiplicity not different than that of the susceptible congenic control line. The grey boxes illustrate the areas of recombination. The coordinates (in bp) along the vertical axis are from the 2004 version of the rat genome (UCSC Genome Browser, rn4). B) DMBA-induced mammary carcinoma multiplicity phenotype for <i>Mcs1a</i> resistant congenic lines Q (n = 83), R3 (n = 24), V4 (n = 24), W4 (n = 28), Y4 (n = 45), and W5 (n = 41) and susceptible congenic lines P5 (n = 16), V5 (n = 56), R5 (n = 30), A4 (n = 24), Y3 (n = 38) and WF.Cop (n = 44). Congenic line Q originally defined the <i>Mcs1a</i> interval in our previous publication and is used as a reference for resistance <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549-Haag1" target="_blank">[33]</a>. C) <i>N</i>-methyl-<i>N</i>-nitrosourea (MNU)-induced mammary carcinoma multiplicity phenotype of resistant congenic lines W4 (n = 20) and W5 (n = 23), susceptible congenic line R5 (n = 14) and the susceptible congenic control line WF.Cop (n = 28). D) Mammary carcinoma multiplicity phenotype induced by mammary ductal infusion of retrovirus expressing the activated <i>HER2/neu</i> oncogene (<i>HER2/neu</i>) for resistant congenic line R3 (n = 15) and susceptible congenic line A4 (n = 14). In all graphs, resistant congenic lines are displayed as filled bars, susceptible congenic lines are displayed as open bars. Significant difference (P<0.05) from the susceptible congenic control line (panels B and C) or from susceptible congenic line A4 (panel D) is indicated by an asterisk.</p

Reduced <i>NR2F1</i> transcript levels in human breast cancer correlate with histological grade 3 tumors.

<p>A) Average (+/− sem) of the normalized median <i>NR2F1</i> probe intensities, obtained from a total of 12 breast cancer expression studies (with 120+ samples each study) available through Oncomine. G = Histological grade, ER = Estrogen receptor, PR = Progestrone receptor, HER2 = Human epidermal growth factor receptor 2, TN = Triple-negative. B) Average (+/− sem) of the normalized <i>NR2F1</i> probe intensity in sample groups organized by grade and ER status (from GSE3494; left panel) or by grade and HER2 status (from GSE5460; right panel). Left panel: ER+G1 n = 62, ER+G2 n = 116, ER+G3 n = 33, ER-G2 n = 11, ER-G3 n = 21. Right panel: HER2+G2 n = 7, HER2+G3 n = 23, HER2-G1 n = 27, HER2-G2 n = 24, HER2-G3 n = 46. Significantly different <i>NR2F1</i> transcript level (P<0.05) is indicated by an asterisk.</p

The non-protein coding <i>Mcs1a</i> resistance locus regulates transcript levels of <i>Nr2f1</i> in the mammary gland, mammary epithelium and mammary carcinomas.

<p>A–C) Q-PCR analysis of <i>Nr2f1</i> transcript levels in mammary gland (MG; panel A), rat mammary epithelial cell (RMEC, panel B) and DMBA- or MNU-induced mammary carcinoma (carc.; panel C) samples from resistant congenic (res.; n = 54 panel A, n = 18 panel B, n = 12 each panel C) and WF.Cop susceptible congenic control (susc.; n = 19 panel A, n = 11 panel B, n = 6 each panel C) rat lines. Data derived from both the W4 and W5 congenic lines are used in the <i>Mcs1a</i> resistant congenic data points. <i>Nr2f1</i> transcript levels are shown relative to the transcript level of the <i>ActB</i> endogenous control gene. D) Chromosome conformation capture (3C) assay for the <i>Nr2f1</i> promoter and the <i>Mcs1a</i> critical interval. The region is shown as a UCSC Genome Browser view (version rn4 of rat genome) and the location of the <i>Mcs1a</i> critical interval in indicated as a horizontal black line. The evolutionary sequence conservation track is also shown. The locations of the 3C assay primers are shown as vertical purple lines. The fixed primer in the <i>Nr2f1</i> promoter is shown with respect to <i>Bgl</i>II restriction sites in the <i>Nr2f1</i> gene span. Graphed is the average relative interaction frequency (+/− sem) of the <i>Bgl</i>II fragment in the <i>Nr2f1</i> promoter containing the fixed primer with each of the <i>Bgl</i>II fragments in <i>Mcs1a</i> containing the 3C assay primers (n = 4 or more templates). Significantly increased relative interaction frequency is indicated with 1 asterisk for a background cut-off interaction frequency of 0.05 and 2 asterisks for a background cut-off interaction frequency of 0.1. The horizontal axis indicates the genomic distance (in Kb) from the 3C assay primers in <i>Mcs1a</i> to the fixed primer in the <i>Nr2f1</i> promoter. The main peak in the interaction profile coincides with blocks of strong evolutionary sequence conservation (to zebrafish and frog, <i>X. tropicalis</i>). Sequence variation within the interacting <i>Mcs1a</i> region is outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549.s002" target="_blank">Figure S2</a>. E) Schematic drawing of the higher-order chromatin interaction of <i>Mcs1a</i> with the <i>Nr2f1</i> promoter. The <i>Mcs1a</i> critical interval is indicated as a thick area in the black line that represents the DNA. The green, orange and red shapes represent the putative DNA-binding proteins involved in the structure.</p

<i>Mcs1a</i> affects rat mammary epithelial cell (RMEC) growth and differentiation.

<p>A) Results of the limiting-dilution RMEC transplantation assay. Graphed is the percentage of transplant sites with an outgrowth versus the number of RMECs transplanted. Vertical bars represent lower and upper limits of a 95%-confidence interval for a proportion. Outgrowth potential of RMECs from the susceptible congenic control (susc.; light grey) and <i>Mcs1a</i> resistant congenic (res.; dark grey) line (lines W4 and W5 combined) is not significantly different. B) Pseudocolor dot plots from a susceptible congenic control sample, representing the gating strategy used to enrich for clonogenic luminal RMECs using cell sorting on the BD FACS Aria. CD31–CD45− RMECs were divided into luminal and basal cells based on CD24 and CD29 expression (left panel). Based on staining with anti-CD61 and peanut lectin (PNL), the clonogenic luminal cell population (CD45–CD31-CD24hiCD29medCD61+PNLhi; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003549#pgen.1003549.s003" target="_blank">Figure S3</a>) was selected for Matrigel assays (right panel). C) Graphed is the average (+/− sem) number of spherical mammary colonies formed in Matrigel assays plating 10,000 clonogenic RMECs sorted from the susceptible congenic control (n = 12) and <i>Mcs1a</i> resistant congenic line (n = 10; line W4 only). In the lower panel, a representative picture of a spherical mammary colony in a methylene blue-stained Matrigel is shown. Significantly different colony-forming ability (P<0.05) is indicated by an asterisk.</p

Proposed model for the <i>Mcs1a</i> breast cancer risk-affecting mechanism.

<p>The <i>Mcs1a</i> resistance allele displays increased mammary <i>Nr2f1</i> transcript levels as compared with the susceptible allele. Lower <i>Nr2f1</i> transcript levels in the mammary gland are associated with susceptibility, a lower percentage of luminal rat mammary epithelial cells (RMEC), a higher percentage of basal RMECs and increased colony-forming ability of the clonogenic luminal RMEC population, indicative of increased proliferation.</p

FACS analysis of rat and mouse MEC populations.

<p>A) Quantification of luminal and basal RMEC populations derived from susceptible congenic control (susc.; open bars; n = 6; WF.Cop) and <i>Mcs1a</i> resistant congenic (res.; filled bars; n = 7; Line W4) rats. B) Quantification of the CD61hi and PNLhi gates in RMEC populations derived from susc. (open bars; n = 11; WF.Cop) and res. (filled bars; n = 24; Lines W4 and W5). These gates were quantified in all CD31–CD45− RMECs as well as in the luminal RMECs. PNL = peanut lectin. C) Quantification of luminal and basal MMEC populations derived from wild type (WT; filled bars; n = 30) and megadeletion (MD; open bars; n = 27) mice (FVB and B6 pooled). D) Quantification of mature luminal (ML), luminal progenitor (LP) and mammary stem cell (MaSc) populations derived from WT (filled bars; n = 30) and MD (open bars; n = 27) mice (FVB and B6 pooled). FACS pseudocolor dot plot or histograms in each panel's upper figure illustrate the gating strategies used to quantify specific MEC populations. These dot plots were taken from a susc. sample (RMEC), or from a WT (FVB) sample (MMEC). Graphed in panels A–C are the average (+/−sem) percentages of populations among CD45–CD31− MECs. Graphed in panel D is the average (+/− sem) percentage of population among CD45–CD31− MECs, expressed relative to the WT run on the same day. In all graphs, significantly different (P<0.05) percentages of cells between susc. and <i>Mcs1a</i> or between WT and MD are indicated with an asterisk.</p