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. 2008 Feb 5;105(5):1680-5.
doi: 10.1073/pnas.0711613105. Epub 2008 Jan 29.

BRCA1 regulates human mammary stem/progenitor cell fate

Suling Liu  1 , Christophe Ginestier, Emmanuelle Charafe-Jauffret, Hailey Foco, Celina G Kleer, Sofia D Merajver, Gabriela Dontu, Max S Wicha
Affiliations

BRCA1 regulates human mammary stem/progenitor cell fate

Suling Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Although it is well established that women with germ-line mutations in the BRCA1 gene have a greatly increased lifetime incidence of breast and ovarian cancer, the molecular mechanisms responsible for this tissue-specific carcinogenesis remain undefined. The majority of these breast cancers are of the basal-like phenotype characterized by lack of expression of ER, PR, and ERBB2. Because this phenotype has been proposed to resemble that of normal breast stem cells, we examined the role of BRCA1 in human mammary stem cell fate. Using both in vitro systems and a humanized NOD/SCID mouse model, we demonstrate that BRCA1 expression is required for the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Knockdown of BRCA1 in primary breast epithelial cells leads to an increase in cells displaying the stem/progenitor cell marker ALDH1 and a decrease in cells expressing luminal epithelial markers and estrogen receptor. In breast tissues from women with germ-line BRCA1 mutations, but not normal controls, we detect entire lobules that, although histologically normal, are positive for ALDH1 expression but are negative for the expression of ER. Loss of heterozygosity for BRCA1 was documented in these ALDH1-positive lobules but not in adjacent ALDH1-negative lobules. Taken together, these studies demonstrate that BRCA1 plays a critical role in the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Because BRCA1 also plays a role in DNA repair, our work suggests that loss of BRCA1 may result in the accumulation of genetically unstable breast stem cells, providing prime targets for further carcinogenic events.

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Conflict of interest statement

Conflict of interest statement: M.S.W. has financial holdings in and is a scientific advisor for OncoMed Pharmaceuticals.

Figures

Fig. 1.
Fig. 1.
BRCA1 expression increases during mammary differentiation. Knockdown of BRCA1 increases mammary stem/progenitor cells and decreases ER expression. (A) Level of BRCA1 mRNA and protein in mammospheres compared with differentiated cells (cultured for 7 days on collagen) measured by real-time RT-PCR and Western blot analysis, respectively. (B) Level of BRCA1 mRNA and protein measured by real-time RT-PCR and Western blot analysis in BRCA1 knockdown mammary epithelial cells compared with GFP-control-infected mammary epithelial cells. We used two independent BRCA1 siRNA lentiviruses and a GFP-control lentivirus to infect the cells. (C) BRCA1 knockdown increases mammosphere number upon serial passage. (D) BRCA1 knockdown has no effect on mammospheres' size. (E) ALDH1 enzymatic activity as assessed by the ALDEFLUOR assay and flow cytometry demonstrates that BRCA1 knockdown increases the ALDEFLUOR-positive population in vitro. (F) As assessed by flow cytometry, BRCA1 knockdown decreases ER expression in BRCA1 knockdown cells compared with DsRed-control primary mammary epithelial cells. SUM-44 ER-positive and SUM-159 ER-negative breast cancer cell lines serve as positive and negative controls.
Fig. 2.
Fig. 2.
BRCA1 knockdown blocks epithelial differentiation in vitro. (A) Level of BRCA1 mRNA measured by real-time RT-PCR in the four populations defined by the expression of the luminal marker (ESA) and the myoepithelial marker (CD10). (B) Knockdown of BRCA1 blocks epithelial differentiation in vitro. BRCA1 siRNA or GFP-control-infected cells were induced to differentiate by culturing cells on collagen plates. Expression of lineage-specific markers was determined by flow cytometry at different time points (0, 7, 12, 26, and 35 days). CD10 is a marker of myoepithelial cells, and ESA is a marker of luminal epithelial cells. FACS analysis scatter plots according to ESA and CD10 expression are presented for the two groups, days 0 and 35. Evolution of the four populations for the two groups is plotted as a function of the number of culture days.
Fig. 3.
Fig. 3.
BRCA1 knockdown blocks epithelial differentiation in NOD/SCID mice xenografts. Mammosphere-initiating cells transduced with GFP-control lentivirus or BRCA1 siRNA lentivirus were introduced into the humanized cleared fat pads of NOD/SCID mice. Mammary structures formed were stained by H&E or examined by immunohistochemistry for expression of GFP, ALDH1, CK18, SMA, and ER. (i, iii, vii, and ix) Fatpads injected with GFP-control-infected cells display mammary epithelial duct structures (i) from human origin (iii, GFP-positive, red staining) that comprised two cell layers with the inner layer expressing the luminal marker CK18 (vii, brown staining), and the outsider layer expressing the myoepithelial marker SMA (ix, red staining). (v and xi) No ALDH1 expression was detected (v), and some cells display ER expression (xi, brown staining). (ii, iv, vi, viii, x) BRCA1 knockdown cells identified by GFP positivity (iv, red staining) produced abnormal structures composed of a single cell layer (ii) that was ALDH1-positive (vi, red staining) and negative for the expression of luminal markers CK18 (viii), and Estrogen Receptor, and with variable expression of the myoepithelial marker SMA (x, red staining). (Scale bars: 100 μm.)
Fig. 4.
Fig. 4.
Model depicting the proposed role of BRCA1 in mammary, stem, and progenitor cell fate. BRCA1 is required for the differentiation of ALDH1-positive/ER-negative stem/progenitor cells into ER-positive luminal epithelial cells. Loss of BRCA1 function results in aberrant luminal differentiation and also may have an effect on the survival of the luminal cells. Moreover, the loss of BRCA1 function results in the accumulation of ALDH1-positive/ER-negative stem/progenitor cells.
Fig. 5.
Fig. 5.
Phenotypic characterization of ALDH1-positive lobules in BRCA1 mutation carrier samples. (i) Immunostaining for ALDH1 (red staining) was performed in samples obtained from prophylactic mastectomy specimens of women with confirmed BRCA1 mutations. Foci of ALDH1-positive cells comprising entire acini were detected (red circle) in samples obtained from 5 of 13 patients. Double staining with ALDH1 (i, red staining) and CK18 (ii, brown staining) or CK14 (iii, brown staining) and ER (iv, brown staining) of morphologically normal breast epithelium from BRCA1 carrier patients showed ALDH1-expressing lobules that displayed absence of expression of CK18 and ER (red circles) and a reduced expression of CK14, whereas ALDH1-negative acini (blue circles) were composed of a continuous outer layer of CK14-positive myoepithelial cells surrounding an inner layer of CK18-positive and ER-positive epithelial cells.
Fig. 6.
Fig. 6.
Stem/progenitor cell expansion in BRCA1 mutation carriers is associated with LOH at the BRCA1 locus. (A) LOH analysis at the BRCA1 locus in ALDH1-positive acini versus ALDH1-negative acini from BRCA1 mutation carriers containing ALDH1-positive acini. ALDH1-positive and adjacent ALDH1-negative lobules were isolated by using laser capture microdissection. Extracted DNA from each microdissected sample was analyzed for four microsatellites in and telemeric to BRCA1 loci (D17S855, D17S1323, D17S1325, and D17S806). In each of the four BRCA1 mutation carrier patients analyzed, LOH at the BRCA1 locus was demonstrated in at least one of the BRCA1 microsatellite markers in ALDH1-positive acini but not in ALDH1-negative acini. (B) LOH analysis at the BRCA1 locus in ALDH1-negative acini versus stromal cells from BRCA1 mutation carriers with no detectable ALDH1-positive lobules. LOH analysis performed as previously comparing ALDH1-negative acini and adjacent stromal cells. In each of the six BRCA1 mutation carrier patients analyzed, no LOH at the BRCA1 locus was observed (LOH, loss of heterozygocity; ROH, retention of heterozygocity; NI, noninformative).
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