CCAAT/enhancer binding protein beta regulates stem cell activity and specifies luminal cell fate in the mammary gland - PubMed (original) (raw)

CCAAT/enhancer binding protein beta regulates stem cell activity and specifies luminal cell fate in the mammary gland

Heather L LaMarca et al. Stem Cells. 2010.

Abstract

The bZIP transcription factor C/EBP beta is important for mammary gland development and its expression is deregulated in human breast cancer. To determine whether C/EBP beta regulates mammary stem cells (MaSCs), we employed two different knockout strategies. Using both a germline and a conditional knockout strategy, we demonstrate that mammosphere formation was significantly decreased in C/EBP beta-deficient mammary epithelial cells (MECs). Functional limiting dilution transplantation assays indicated that the repopulating ability of C/EBP beta-deleted MECs was severely impaired. Serial transplantation experiments demonstrated that C/EBP beta deletion resulted in decreased outgrowth potential and premature MaSC senescence. In accord, fluorescence-activated cell sorting analysis demonstrated that C/EBP beta-null MECs contained fewer MaSCs, the loss of luminal progenitors and an increase in differentiated luminal cells as compared with wild-type. Gene profiling of C/EBP beta-null stem cells revealed an alteration in cell fate specification, exemplified by the expression of basal markers in the luminal compartment. Thus, C/EBP beta is a critical regulator of both MaSC repopulation activity and luminal cell lineage commitment. These findings have critical implications for understanding both stem cell biology and the etiology of different breast cancer subtypes.

Keywords: C/EBP beta; alveolar; differentiation; lineage commitment; luminal; mammary stem cell; progenitor cell.

PubMed Disclaimer

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Figure 1

Figure 1

Conditional deletion of C/EBPβ results in altered ductal morphogenesis and decreased lobuloalveolar development. MECs were isolated from 10-week old wildtype (C/EBPβ+/+;R26R) and C/EBPβfl/fl;R26R mice, transduced with Ad.Cre1 in vitro and Cre-mediated recombination was examined (n=3). (A) qPCR for C/EBPβ showed that the expression of C/EBPβ was decreased >300-fold in transduced C/EBPβfl/fl;R26R MECs as compared to wildtype. (B–G) Transduced cells were transplanted into syngeneic hosts and stained with X-gal after 10 weeks of outgrowth. Large, dilated ducts were evident in virgin C/EBPβ-deleted glands as compared to wildtype by whole mount analysis (B,C) and H&E staining of paraffin-embedded sections (D,E). H&E staining of pregnant glands (P16–17) illustrated decreased alveolar development in C/EBPβ-null glands (G) as compared to wildtype (F), the latter of which contained lipid droplets. Uniform lacZ expression was observed throughout the mammary gland, indicative of a high extent of Cre-mediated recombination. Scale bars, 5 mm (B,C) or 20μm (D–G).

Figure 2

Figure 2

Decreased mammosphere formation in C/EBPβ-null MECs. Graphs illustrate the number of secondary mammospheres formed per 5000 cells expressed as mammosphere efficiency using either (A) conditional wildtype (C/EBPβ+/+;R26R) and C/EBPβfl/fl;R26R cells transduced with Ad.Cre1 or (B) germline wildtype (C/EBPβ+/+) and C/EBPβ−/− MECs. The ability of C/EBPβ-null cells to form secondary mammospheres was significantly inhibited using both these models (*p<0.0001). Data represent mean ± SEM of 1 representative experiment and the experiment was performed 3 times.

Figure 3

Figure 3

Decreased MRUs in C/EBPβ-null outgrowths when transplanted at limiting dilution. MECs were isolated from wildtype (C/EBPβ+/+;R26R) and C/EBPβfl/fl;R26R glands, transduced with Ad.Cre1 and transplanted into syngeneic hosts at limiting dilutions. (A) Outgrowth potential was significantly decreased in C/EBPβ-deleted glands as compared to wildtype (p=0.017). The ability of C/EBPβ-null MECs to fill the fat pad was also significantly impaired as compared to wildtype (p=0.0005). Photomicrographs depict whole mount analysis of glands injected with 250 of wildtype (B) or C/EBPβ-null (C) cells. Scale bars, 5 mm.

Figure 4

Figure 4

Decreased outgrowth of C/EBPβ−/− serial transplants. CFP+ epithelia were dissected from wildtype (C/EBPβ+/+;CFP+) or germline C/EBPβ−/−; CFP+ glands and transplanted sequentially for 3 consecutive generations, harvesting tissue at each generation after 8 weeks of outgrowth. (A) While take rate remained similar between wildtype and C/EBPβ−/−; CFP+ tissue, the ability to reconstitute the fat pad significantly decreased with subsequent generations (p=0.002) in C/EBPβ-null glands as compared to wildtype (Gen 2 p=0.003, Gen 3 p=0.004). (B) Fluorescent micrographs (inverted images) depict representative images of generation 3 outgrowths of wildtype and C/EBPβ−/−; CFP+ glands in mature virgin mice (top) or late pregnant (P19) glands (bottom). (C) Images depict generation 4 outgrowths from wildtype and stunted C/EBPβ−/− CFP+ donor glands. The number of outgrowths represented by each micrograph is depicted below each image. Scale bars, 5 mm.

Figure 5

Figure 5

Altered stem/progenitor cell populations in C/EBPβ−/− mammary glands. (A) Dot plots depict that the percentage of LIN−CD24+CD29hi cells, which contain MRUs, is decreased in C/EBPβ−/− MECs, while the LIN−CD24hiCD29lo subpopulation is increased. (B) The LIN−CD24hiCD29lo subpopulation was gated and CD61 expression was examined within this subpopulation. C/EBPβ−/− MECs express a lower percentage of LIN−CD24hiCD29loCD61+ cells as compared to wildtype (C/EBPβ+/+). (C) The LIN−CD24hiSca1hi subpopulation is increased in C/EBPβ−/− glands as compared to wildtype, which is accompanied by the loss of LIN−CD24loSca1− cells. For all FACS plots, lineage-positive cells were excluded using a mouse lineage panel kit plus biotin-conjugated CD31 and CD140a antibodies. Each dot/contour plot represents 1 experiment, and bar graphs depict the mean ± SEM of 4 independent experiments (*p<0.0001).

Figure 6

Figure 6

Decreased colony formation in C/EBPβ-null MECs. Colony forming ability on cultured feeder layers was significantly decreased in C/EBPβ−/−;CFP+ MECs as compared to wildtype (C/EBPβ+/+;CFP+). Data represent mean ± SEM of 3 independent experiments (*p<0.0001). Images depict crystal violet-stained colonies in wildtype and C/EBPβ−/−;CFP+ cells, and insets demonstrate a single CFP+ colony. Scale bars, 5 mm (large panels) and 250 μm (insets).

References

    1. Asselin-Labat ML, Sutherland KD, Barker H, et al. Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol. 2007;9:201–209. - PubMed
    1. Shackleton M, Vaillant F, Simpson KJ, et al. Generation of a functional mammary gland from a single stem cell. Nature. 2006;439:84–88. - PubMed
    1. Sleeman KE, Kendrick H, Robertson D, et al. Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland. J Cell Biol. 2007;176:19–26. - PMC - PubMed
    1. Stingl J, Eaves CJ, Zandieh I, et al. Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue. Breast Cancer Res Treat. 2001;67:93–109. - PubMed
    1. Stingl J, Eirew P, Ricketson I, et al. Purification and unique properties of mammary epithelial stem cells. Nature. 2006;439:993–997. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources