Slugging their way to immortality: Driving mammary epithelial cells into a stem cell-like state

Delineating the molecular factors that define and maintain the mammary stem cell state is vital for understanding normal development and tumourigenesis. A recent study by Guo and colleagues identifies two master transcriptional regulators of mammary stem cells, Slug and Sox9, ectopic expression of which confers stem cell attributes on differentiated mammary epithelial cells. Slug and Sox9 expression was also shown to determine in vivo metastatic potential of human breast cancer cell lines. Understanding these factors in the context of normal lineage differentiation is an important step toward elucidating the mammary epithelial cell hierarchy and the origins of cancer stem cells

The receptor protein tyrosine phosphatase PTPRB negatively regulates FGF2-dependent branching morphogenesis

PTPRB is a transmembrane protein tyrosine phosphatase known to regulate blood vessel remodelling and angiogenesis. Here we demonstrate that PTPRB negatively regulates branching morphogenesis in the mammary epithelium. We show that Ptprb is highly expressed in adult mammary stem cells and also, although at lower levels, in estrogen receptor positive luminal cells. During mammary development Ptprb expression is down-regulated during puberty, a period of extensive of ductal outgrowth and branching. In vivo shRNA knockdown of Ptprb in the cleared mammary fat pad transplant assay resulted in smaller epithelial outgrowths with an increased branching density and also increased branching in an in vitro organoid assay. Organoid branching was dependent on stimulation by FGF2, and Ptprb knockdown in mammary epithelial cells resulted in a higher level of FGFR activation and ERK1/2 phosphorylation, both at baseline and following FGF2 stimulation. Therefore, PTPRB regulates branching morphogenesis in the mammary epithelium by modulating the response of the FGFR signalling pathway to FGF stimulation. Considering the importance of branching morphogenesis in multiple taxa, our findings have general importance outside mammary developmental biology

Annexin A8 identifies a subpopulation of transiently quiescent c-kit positive luminal progenitor cells of the ductal mammary epithelium

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ~15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G0/G1 arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin

Slugging their way to immortality: driving mammary epithelial cells into a stem cell-like state

Delineating the molecular factors that define and maintain the mammary stem cell state is vital for understanding normal development and tumourigenesis. A recent study by Guo and colleagues identifies two master transcriptional regulators of mammary stem cells, Slug and Sox9, ectopic expression of which confers stem cell attributes on differentiated mammary epithelial cells. Slug and Sox9 expression was also shown to determine in vivo metastatic potential of human breast cancer cell lines. Understanding these factors in the context of normal lineage differentiation is an important step toward elucidating the mammary epithelial cell hierarchy and the origins of cancer stem cells

Mouse mammary stem cells express prognostic markers for triple-negative breast cancer

Introduction Triple negative breast cancer (TNBC) is a heterogeneous group of tumours in which chemotherapy, the current mainstay of systemic treatment, is often initially beneficial but with a high risk of relapse and metastasis. There is currently no means of predicting which TNBC will relapse. We tested the hypothesis that the biological properties of normal stem cells are re-activated in tumour metastasis and that, therefore, the activation of normal mammary stem cell-associated gene sets in primary TNBC would be highly prognostic for relapse and metastasis. Methods Mammary basal stem and myoepithelial cells were isolated by flow cytometry and tested in low dose transplant assays. Gene expression microarrays were used to establish expression profiles of the stem and myoepithelial populations; these were compared to each other and to our previously established mammary epithelial gene expression profiles. Stem cell genes were classified by Gene Ontology (GO) analysis and the expression of a subset analysed in the stem cell population at single cell resolution. Activation of stem cell genes was interrogated across different breast cancer cohorts and within specific subtypes and tested for clinical prognostic power. Results A set of 323 genes was identified that was expressed significantly more highly in the purified basal stem cells compared to all other cells of the mammary epithelium. 109 out of 323 genes had been associated with stem cell features in at least one other study in addition to our own, providing further support for their involvement in the biology of this cell type. GO analysis demonstrated an enrichment of these genes for an association with cell migration, cytoskeletal regulation and tissue morphogenesis, consistent with a role in invasion and metastasis. Single cell resolution analysis showed that individual cells co-expressed both epithelial- and mesenchymal-associated genes/proteins. Most strikingly, we demonstrated that strong activity of this stem cell gene set in TNBCs identified those tumours most likely to rapidly progress to metastasis. Conclusions Our findings support the hypothesis that the biological properties of normal stem cells are drivers of metastasis and that these properties can be used to stratify patients with a highly heterogeneous disease such as TNBC

Aurora A Kinase Regulates Mammary Epithelial Cell Fate by Determining Mitotic Spindle Orientation in a Notch-Dependent Manner

Cell fate determination in the progeny of mammary epithelial stem/progenitor cells remains poorly understood. Here, we have examined the role of the mitotic kinase Aurora A (AURKA) in regulating the balance between basal and luminal mammary lineages. We find that AURKA is highly expressed in basal stem cells and, to a lesser extent, in luminal progenitors. Wild-type AURKA expression promoted luminal cell fate, but expression of an S155R mutant reduced proliferation, promoted basal fate, and inhibited serial transplantation. The mechanism involved regulation of mitotic spindle orientation by AURKA and the positioning of daughter cells after division. Remarkably, this was NOTCH dependent, as NOTCH inhibitor blocked the effect of wild-type AURKA expression on spindle orientation and instead mimicked the effect of the S155R mutant. These findings directly link AURKA, NOTCH signaling, and mitotic spindle orientation and suggest a mechanism for regulating the balance between luminal and basal lineages in the mammary gland

Mouse mammary stem cells express prognostic markers for triple-negative breast cancer

Introduction Triple negative breast cancer (TNBC) is a heterogeneous group of tumours in which chemotherapy, the current mainstay of systemic treatment, is often initially beneficial but with a high risk of relapse and metastasis. There is currently no means of predicting which TNBC will relapse. We tested the hypothesis that the biological properties of normal stem cells are re-activated in tumour metastasis and that, therefore, the activation of normal mammary stem cell-associated gene sets in primary TNBC would be highly prognostic for relapse and metastasis. Methods Mammary basal stem and myoepithelial cells were isolated by flow cytometry and tested in low dose transplant assays. Gene expression microarrays were used to establish expression profiles of the stem and myoepithelial populations; these were compared to each other and to our previously established mammary epithelial gene expression profiles. Stem cell genes were classified by Gene Ontology (GO) analysis and the expression of a subset analysed in the stem cell population at single cell resolution. Activation of stem cell genes was interrogated across different breast cancer cohorts and within specific subtypes and tested for clinical prognostic power. Results A set of 323 genes was identified that was expressed significantly more highly in the purified basal stem cells compared to all other cells of the mammary epithelium. 109 out of 323 genes had been associated with stem cell features in at least one other study in addition to our own, providing further support for their involvement in the biology of this cell type. GO analysis demonstrated an enrichment of these genes for an association with cell migration, cytoskeletal regulation and tissue morphogenesis, consistent with a role in invasion and metastasis. Single cell resolution analysis showed that individual cells co-expressed both epithelial- and mesenchymal-associated genes/proteins. Most strikingly, we demonstrated that strong activity of this stem cell gene set in TNBCs identified those tumours most likely to rapidly progress to metastasis. Conclusions Our findings support the hypothesis that the biological properties of normal stem cells are drivers of metastasis and that these properties can be used to stratify patients with a highly heterogeneous disease such as TNBC

ANXA8 expression arrests Kim2A8 cells in G₀.

<p>(<b>A</b>) Kim2A8 and Kim2RTS cells were grown in 6-well plates with or without 100ng/ml of dox for 48 hours. The graph shows the average percentage numbers of cells in G₀/G₁, S and G₂/M as quantified by FACS from three independent experiments. (<b>B</b>) Kim2A8 and Kim2RTS cells grown in chamber slides with or without 100ng/ml dox for six days were fixed and stained for Ki67 antigen. EGFP was used as a reporter of ANXA8 expression. (<b>C</b>) Graph showing the percentage of Ki67-positivity in the EGFP positive and negative populations of Kim2A8 cells grown with or without 100ng/ml dox. At least 1000 cells were analyzed in each population. (<b>D</b>) Western blot showing Ki67 and ANXA8 protein expression in cells after six days in culture. Actin was used as a loading control. Numbers show the relative intensities of ANXA8 and Ki67 bands respectively (normalised to actin) determined by measuring area pixel intensities using AIDA Image Analyzer software. The reduction of Ki67 levels (∼50%) is consistent with the reduced number of Ki67+ve Dox-treated Kim2A8 cells seen in (<b>B</b>).</p