Transcriptome analysis of mammary epithelial subpopulations identifies novel determinants of lineage commitment and cell fate

Kendrick, Howard; Regan, Joseph L.; Magnay, Fiona-Ann; Grigoriadis, Anita; Mitsopoulos, Costas; Zvelebil, Marketa; Smalley, Matthew J.

doi:10.1186/1471-2164-9-591

Cited by 157 publications

(231 citation statements)

References 103 publications

(130 reference statements)

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“…Looking at individual genes in more detail, analysis of the expression patterns of Krt14, Krt18 and Esr1 largely supported the K14, K18 and ER staining on slides, although in populations which showed no staining for these markers, low levels of gene expression could always be detected, consistent with previous observations (Sleeman et al, 2007;Kendrick et al, 2008). As expected, the basal populations expressed the highest levels of Krt14, while the luminal populations expressed very low levels.…”

Section: Resultssupporting

confidence: 87%

“…Consistent with the transcriptomic data (Kendrick et al, 2008), the majority (up to 90%) of luminal Sca-1 À progenitors were c-Kit þ (equal to 50% of all mammary epithelial cells). A small number of these (designated luminal Sca-1 À c-Kit þ /High ) had very high levels of c-Kit expression.…”

Section: Resultssupporting

confidence: 85%

“…Grey boxes indicate samples in which the gene was undetectable. (c) Fold expression ± 95% confidence intervals in mammary epithelial subpopulations (n ¼ 3 independent cell preparations) over the comparator population (as above) of genes associated with the basal (Krt14, Itga6 and Trp63/DNp63) and luminal (Krt18, Esr1) lineages (Kendrick et al, 2008). *Gene expression was undetectable in these populations in all three independent isolates.…”

Section: Resultsmentioning

confidence: 99%

“…To investigate further the relationships between the populations, a qPCR-based gene expression analysis was carried out on a panel of 33 genes. The panel included genes previously shown to be specifically expressed in basal and luminal cells, as well as genes thought to be expressed uniformly throughout the mammary epithelium (Kendrick et al, 2008). Unsupervised hierarchical clustering of the data was carried out (Figure 2b; the full data set including relative expression levels and 95% confidence intervals for all genes in all populations is given in Supplementary Table 1; the expected luminal or basal expression pattern of each gene based on our previous transcriptomic and qPCR analysis is also shown) and patterns of expression of individual genes were analysed (Figure 2c).…”

Section: Resultsmentioning

confidence: 99%

“…Gene expression analysis showed that Kit was highly expressed by luminal ER À progenitors in both mouse (Kendrick et al, 2008) and human (Lim et al, 2009). Furthermore, Brca1 loss in mouse mammary epithelial cells resulted in defective differentiation and increased c-Kit expression (Smart et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

et al. 2011

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BRCA1 mutation-associated breast cancer originates in oestrogen receptor-alpha-negative (ER À ) progenitors in the mammary luminal epithelium. These cells also express high levels of the Kit gene and a recent study demonstrated a correlation between Brca1 loss and Kit over-expression in the mammary epithelium. However, the functional significance of c-Kit expression in the mammary gland is unknown. To address this, c-Kit À and c-Kit þ mammary epithelial subsets were isolated by flow cytometry, characterised for expression of lineage-specific cell markers and functionally analysed by in vitro colony forming and in vivo transplantation assays. The results confirm that the majority of luminal ER À progenitors are c-Kit þ , but also that most stem cells and the differentiated cell populations are c-Kit À . A subset of c-Kit þ cells with high proliferative potential was found in the luminal ER þ population, however, suggesting the existence of a distinct luminal ER þ progenitor cell type. Analysis of mouse Brca1 mammary tumours demonstrated that they expressed Kit and its downstream effector Lyn at levels comparable to the most strongly c-Kit þ luminal ER À progenitors. Consistent with c-Kit being a progenitor cell marker, in vitro threedimensional differentiation of c-Kit þ cells resulted in a loss of c-Kit expression, whereas c-Kit over-expression prevented normal differentiation in vivo. Furthermore, c-Kit was a functional marker of proliferative potential, as c-Kit inhibition by short hairpin knockdown prevented normal epithelial growth and caused cells to undergo apoptosis. Therefore, c-Kit defines distinct progenitor populations in the mammary epithelium and is critical for mammary progenitor survival and proliferation. Importantly, c-Kit is only the second mammary epithelial stem/progenitor marker to be shown to have a functional role in the mammary epithelium and the first marker to be shown to be required for progenitor cell function. The c-Kit signalling network has potential as a target for therapy and/or prevention in BRCA1-associated breast cancer.

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Section: Resultssupporting

confidence: 87%

Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

et al. 2011

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Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines

et al. 2012

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The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells. ' 2012 International Society for Advancement of Cytometry Key terms CD90; Thy-1; breast cancer; cancer stem cell; CD14; breast cancer stem cell

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Does FACS perturb gene expression?

2015

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Fluorescence activated cell sorting is the technique most commonly used to separate primary mammary epithelial sub-populations. Many studies incorporate this technique before analyzing gene expression within specific cellular lineages. However, to our knowledge, no one has examined the effects of fluorescence activated cell sorting (FACS) separation on short-term transcriptional profiles. In this study, we isolated a heterogeneous mixture of cells from the mouse mammary gland. To determine the effects of the isolation and separation process on gene expression, we harvested RNA from the cells before enzymatic digestion, following enzymatic digestion, and following a mock FACS sort where the entire cohort of cells was retained. A strict protocol was followed to minimize disruption to the cells, and to ensure that no subpopulations were enriched or lost. Microarray analysis demonstrated that FACS causes minimal disruptions to gene expression patterns, but prior steps in the mammary cell isolation process are followed by upregulation of 18 miRNA's and rapid decreases in their predicted target transcripts. V C 2015 International Society for Advancement of Cytometry Key terms FACS; mammary epithelial cells; miRNA MURINE mammary glands are highly branched tubular organs that develop mostly postnatally, within subcutaneous fat pads. The ducts that comprise the mammary gland arise from multipotent stem cells that generate all of the lineages found in the mature ductal tree (1,2). Differentiated mammary epithelial cells (MECs) can be broadly classified into two populations: luminal epithelial cells that line the ducts, and myoepithelial cells that form a layer surrounding the luminal cells. However, there are multiple cell types within each of these two populations; for example some luminal cells express the estrogen receptor and some do not.To identify the mechanisms that regulate lineage commitment, researchers have conducted transcriptional analysis of discrete mammary cell populations isolated by fluorescence activated cell sorting (FACS) (3,4). These studies all rely on the assumption that robust differences in gene expression between separate lineages are due to endogenous differences in gene expression. However, to our knowledge, this assumption has never been tested. Our study now demonstrates that FACS produces minimal short-term transcriptional artifacts, but also emphasizes the need for care in the methods used to isolate primary cells. MATERIALS AND METHODS MiceA 8-9 week old C3H mice were ordered from Harlan laboratories. Our handling and use of these mice strictly adhered to ACUC-approved protocol (M/12/080). Cell Preparation and RNA ExtractionMammary tissue from the 3rd, 4th, and 5th mammary fat pads was collected (minus the lymph nodes). Each replicate consisted of pooled tissue from three mice. Each mock sorting experiment contained three biological replicates from nine mice.

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Transcriptome analysis of mammary epithelial subpopulations identifies novel determinants of lineage commitment and cell fate

Cited by 157 publications

References 103 publications

c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines

Does FACS perturb gene expression?

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