Human Breast Stem Cells Research Articles

Evaluation of chemotherapeutic and cancer-protective properties of sphingosine and C2-ceramide in a human breast stem cell derived carcinogenesis model.

The overall goal of the present study was to evaluate the chemotherapeutic and cancer-protective properties of D-erythro-sphingosine (sphingosine) and C2-ceramide using a human breast epithelial cell (HBEC) culture system, which represents multiple-stages of breast carcinogenesis. The HBEC model includes Type I HBECs (normal stem), Type II HBECs (normal differentiated) and transformed cells (immortal/non-tumorigenic cells and tumorigenic cells, which are transformed from the same parental normal stem cells). The results of the present study indicate that sphingosine preferentially inhibits proliferation and causes death of normal stem cells (Type I), tumorigenic cells, and MCF7 breast cancer cells, but not normal differentiated cells (Type II). In contrast to the selective anti-proliferative effects of sphingosine, C2-ceramide inhibits proliferation of normal differentiated cells as well as normal stem cells, tumorigenic cells, and MCF7 cancer cells with similar potency. Both sphingosine and C2-ceramide induce apoptosis in tumorigenic cells. Among the sphingosine stereoisomers (D-erythro, D-threo, L-erythro, and L-threo) and sphinganine that were tested, L-erythro-sphingosine most potently inhibits proliferation of tumorigenic cells. The inhibition of breast tumorigenic/cancer cell proliferation by sphingosine was accompanied by inhibition of telomerase activity. Sphingosine at non-cytotoxic concentrations, but not C2-ceramide, induces differentiation of normal stem cells (Type I), thereby reducing the number of stem cells that are more susceptible to neoplastic transformation. To the best of our knowledge, the present study demonstrates one of the first results that sphingosine can be a potential chemotherapeutic and cancer-protective agent, whereas C2-ceramide is not an ideal chemotherapeutic and cancer-protective agent due to its anti-proliferative effects on Type II HBECs and its inability to induce the differentiation of Type I to Type II HBECs.

Cadmium Exposure Inhibits Branching Morphogenesis and Causes Alterations Consistent With HIF-1α Inhibition in Human Primary Breast Organoids.

Developmental cadmium exposure in vivo disrupts mammary gland differentiation, while exposure of breast cell lines to cadmium causes invasion consistent with the epithelial-mesenchymal transition (EMT). The effects of cadmium on normal human breast stem cells have not been measured. Here, we quantified the effects of cadmium exposure on reduction mammoplasty patient-derived breast stem cell proliferation and differentiation. Using the mammosphere assay and organoid formation in 3D hydrogels, we tested 2 physiologically relevant doses of cadmium, 0.25 and 2.5 µM, and tested for molecular alterations using RNA-seq. We functionally validated our RNA-seq findings with a hypoxia-inducible factor (HIF)-1α activity reporter line and pharmaceutical inhibition of HIF-1α in organoid formation assays. 2.5 µM cadmium reduced primary mammosphere formation and branching structure organoid formation rates by 33% and 87%, respectively. Despite no changes in mammosphere formation, 0.25 µM cadmium inhibited branching organoid formation in hydrogels by 73%. RNA-seq revealed cadmium downregulated genes associated with extracellular matrix formation and EMT, while upregulating genes associated with metal response including metallothioneins and zinc transporters. In the RNA-seq data, cadmium downregulated HIF-1α target genes including LOXL2, ZEB1, and VIM. Cadmium significantly inhibited HIF-1α activity in a luciferase assay, and the HIF-1α inhibitor acriflavine ablated mammosphere and organoid formation. These findings show that cadmium, at doses relevant to human exposure, inhibited human mammary stem cell proliferation and differentiation, potentially through disruption of HIF-1α activity.

Heterogeneity of Human Breast Stem and Progenitor Cells as Revealed byTranscriptional Profiling.

SummaryDuring development, the mammary gland undergoes extensive remodeling driven by stem cells. Breast cancers are also hierarchically organized and driven by cancer stem cells characterized by CD44+CD24low/− or aldehyde dehydrogenase (ALDH) expression. These markers identify mesenchymal and epithelial populations both capable of tumor initiation. Less is known about these populations in non-cancerous mammary glands. From RNA sequencing, ALDH+ and ALDH−CD44+CD24− human mammary cells have epithelial-like and mesenchymal-like characteristics, respectively, with some co-expressing ALDH+ and CD44+CD24− by flow cytometry. At the single-cell level, these cells have the greatest mammosphere-forming capacity and express high levels of stemness and epithelial-to-mesenchymal transition-associated genes including ID1, SOX2, TWIST1, and ZEB2. We further identify single ALDH+ cells with a hybrid epithelial/mesenchymal phenotype that express genes associated with aggressive triple-negative breast cancers. These results highlight single-cell analyses to characterize tissue heterogeneity, even in marker-enriched populations, and identify genes and pathways that define this heterogeneity.

Fentanyl Inhibits Tumorigenesis from Human Breast Stem Cells by Inducing Apoptosis

Fentanyl is an opioid analgesic that it is widely used in cancer patients. Since there have been reports of effects of analgesic medications on the recurrence and development of resistance to treatment, influences of of fentanyl on MCF-7 and HEK293 cells were evaluated. Cell viability and apoptosis were assessed by MTT assay and flow cytometry, respectively. Gene expression analysis was performed by quantitative real-time PCR assay for the Oct4, Sox2 and Nanog genes as stem cell markers and Bax, Bcl2, and p53 genes as apoptosis markers. MTT assay results showed that fentanyl significantly inhibited the growth of MCF-7 cells in a dose-and time-dependent manner while significantly increasing apoptosis. In contrast, decrease was noted in HEK-293 cells. In MCF-7 derived cancer stem cells, fentanyl treatment decreased the expression of Bax, Bcl2, Oct4, Sox2, Nanog genes when compared to untreated cells. In HEK-293 stem cells, decrease was noted for Sox2, Nanog and Bax, but increase for Oct4. Our study supports an antitumor role of fentanyl by inducing apoptosis and reducing numbers of cancer stem cells in the MCF-7 human breast adenocarcinoma line.

Decreased Mitochondrial Mutagenesis during Transformation of Human Breast Stem Cells into Tumorigenic Cells.

Rare stochastic mutations may accumulate during dormancy of stem-like cells, but technical limitations in DNA sequencing have limited exploring this possibility. In this study, we employed a recently established deep-sequencing method termed Duplex Sequencing to conduct a genome-wide analysis of mitochondrial (mt) DNA mutations in a human breast stem cell model that recapitulates the sequential stages of breast carcinogenesis. Using this method, we found significant differences in mtDNA among normal stem cells, immortal/preneoplastic cells, and tumorigenic cells. Putative cancer stem-like cell (CSC) populations and mtDNA copy numbers increased as normal stem cells become tumorigenic cells. Transformed cells exhibited lower rare mutation frequencies of whole mtDNA than did normal stem cells. The predicted mtDNA rare mutation pathogenicity was significantly lower in tumorigenic cells than normal stem cells. Major rare mutation types in normal stem cells are C>T/G>A and T>C/A>G transitions, while only C>T/G>A are major types in transformed cells. We detected a total of 1,220 rare point mutations, 678 of which were unreported previously. With only one possible exception (m10342T>C), we did not find specific mutations characterizing mtDNA in human breast CSCs; rather, the mitochondrial genome of CSCs displayed an overall decrease in rare mutations. On the basis of our work, we suggest that this decrease (in particular T>C/A>G transitions), rather than the presence of specific mitochondrial mutations, may constitute an early biomarker for breast cancer detection. Our findings support the hypothesis that the mitochondrial genome is altered greatly as a result of the transformation of normal stem cells to CSCs, and that mtDNA mutation signatures may aid in delineating normal stem cells from CSCs. Cancer Res; 76(15); 4569-78. ©2016 AACR.

Transcriptomic profiling of curcumin-treated human breast stem cells identifies a role for stearoyl-coa desaturase in breast cancer prevention.

Curcumin is a potential agent for both the prevention and treatment of cancers. Curcumin treatment alone, or in combination with piperine, limits breast stem cell self-renewal, while remaining non-toxic to normal differentiated cells. We paired fluorescence-activated cell sorting with RNA sequencing to characterize the genome-wide changes induced specifically in normal breast stem cells following treatment with these compounds. We generated genome-wide maps of the transcriptional changes that occur in epithelial-like (ALDH+) and mesenchymal-like (ALDH-/CD44+/CD24-) normal breast stem/progenitor cells following treatment with curcumin and piperine. We show that curcumin targets both stem cell populations by down-regulating expression of breast stem cell genes including ALDH1A3, CD49f, PROM1, and TP63. We also identified novel genes and pathways targeted by curcumin, including downregulation of SCD. Transient siRNA knockdown of SCD in MCF10A cells significantly inhibited mammosphere formation and the mean proportion of CD44+/CD24- cells, suggesting that SCD is a regulator of breast stemness and a target of curcumin in breast stem cells. These findings extend previous reports of curcumin targeting stem cells, here in two phenotypically distinct stem/progenitor populations isolated from normal human breast tissue. We identified novel mechanisms by which curcumin and piperine target breast stem cell self-renewal, such as by targeting lipid metabolism, providing a mechanistic link between curcumin treatment and stem cell self-renewal. These results elucidate the mechanisms by which curcumin may act as a cancer-preventive compound and provide novel targets for cancer prevention and treatment.

Abstract A29: Detection of ultra-rare mutations during breast stem cell tumor progression by Duplex Sequencing

Abstract Most studies have mainly investigated clonal mutations due to the high background error frequencies of conventional next generation sequencing (NGS) methods. We have established a new method termed Duplex Sequencing (DS) that detects mutations with unprecedented accuracy and enables us to detect sub-clonal and rare mutations as well as clonal mutations. Using DS, we present our first genome-wide analysis of mitochondrial (mt) DNA mutations during the transformation of human normal breast stem cells to tumorigenic cells. We used a model system to dissect sequential steps in the progression of normal stem cells to tumorigenic cells. Significant differences have been found among normal stem cells, immortal cells, and tumorigenic cells. These cells differ in putative breast cancer stem cell (CSC) populations, mtDNA copy numbers, and the frequencies and types of mutations. We found that putative CSCs and mtDNA copy numbers increase as normal stem cells become tumorigenic cells. The vast majority of mutations identified in our study are stochastic (rare mutations) and the frequency of rare mutations is lower in transformed cells than in normal stem cells. This lower rare mutation load in transformed cells induces lower scores of predicted pathogenicity in transformed cells than in normal stem cells. C>T/G>A and A>G/T>C transitions are the major mutation types in normal stem cells, while only C>T/G>A are the major mutation types in transformed cells. Using DS, we detected a total of 1220 rare mutations and 678 of these variants have not been reported previously. The mtDNA mutation spectra of breast tumorigenic cells correlate with published nuclear DNA mutation signatures, which are found in the majority of tumors. We have identified mutations that can serve as potential biomarkers that indicate the different stages of breast carcinogenesis. These significant differences in mutation profiles could aid in characterizing different phenotypes between normal stem cells and transformed cells. Citation Format: Eun Hyun Ahn, Kensen Hirohata, Seung Hyuk Lee, Joon Yup Kim, Brendan F. Kohrn, Edward J. Fox, Chia-Cheng Chang, Lawrence A. Loeb. Detection of ultra-rare mutations during breast stem cell tumor progression by Duplex Sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A29.

Of mice and women: a comparative tissue biology perspective of breast stem cells and differentiation.

Tissue based research requires a background in human and veterinary pathology, developmental biology, anatomy, as well as molecular and cellular biology. This type of comparative tissue biology (CTB) expertise is necessary to tackle some of the conceptual challenges in human breast stem cell research. It is our opinion that the scarcity of CTB expertise contributed to some erroneous interpretations in tissue based research, some of which are reviewed here in the context of breast stem cells. In this article we examine the dissimilarities between mouse and human mammary tissue and suggest how these may impact stem cell studies. In addition, we consider the differences between breast ducts vs. lobules and clarify how these affect the interpretation of results in stem cell research. Lastly, we introduce a new elaboration of normal epithelial cell types in human breast and discuss how this provides a clinically useful basis for breast cancer classification.

Steroid hormones, steroid receptors, and breast cancer stem cells.

The ovarian hormones progesterone and estrogen play important roles in breast cancer etiology, proliferation, and treatment. Androgens may also contribute to breast cancer risk and progression. In recent years, significant advances have been made in defining the roles of these steroid hormones in stem cell homeostasis in the breast. Stem cells are potential origins of breast cancer and may dictate tumor phenotype. At least a portion of breast cancers are proposed to be driven by cancer stem cells (CSCs), cells that mimic the self-renewing and repopulating properties of normal stem cells, and can confer drug resistance. Progesterone has been identified as the critical hormone regulating normal murine mammary stem cell (MaSC) populations and normal human breast stem cells. Synthetic progestins increase human breast cancer risk; one theory speculates that this occurs through increased stem cells. Progesterone treatment also increases breast CSCs in established breast cancer cell lines. This is mediated in part through progesterone regulation of transcription factors, signal transduction pathways, and microRNAs. There is also emerging evidence that estrogens and androgens can regulate breast CSC numbers. The evolving concept that a breast CSC phenotype is dynamic and can be influenced by cell signaling and external cues emphasizes that steroid hormones could be crucial players in controlling CSC number and function. Here we review recent studies on steroid hormone regulation of breast CSCs, and discuss mechanisms by which this occurs.

Reduced tumorigenicity and drug resistance through the downregulation of octamer-binding protein 4 and Nanog transcriptional factor expression in human breast stem cells.

Breast cancer is the most common type of malignancy among females. Previous studies examining breast cancer tissue have demonstrated the presence of stem cells, and have detected octamer-binding protein 4 (Oct4) and Nanog transcription factor expression. In the present study, breast cancer stem cells (CSCs) were isolated and enriched from MDA-MB-231 breast cancer cell lines, and were defined as MDA-MB-231 stem cells using flow cytometry. The expression of Oct4 and Nanog in breast CSCs were detected by quantitative polymerase chain reaction and western blotting. RNA interference (RNAi) was used in order to downregulate the expression of Oct4 and Nanog. Drug resistance and tumor-initiating capability following in vivo injection of MDA-MB-231 stem cells transduced with negative RNAi, Oct4 RNAi and Nanog RNAi were compared with that of MDA-MB-231 stem cells without siRNA transfection as a control group. In addition the capability of MDA-MB-231 breast cancer cells to initiate tumor formation in mice was compared with that of MDA-MB-231 stem cells. A paclitaxel inhibition test was also conducted in order to detect resistance of MDA-MB-231 breast cancer stem cells to this treatment. The MDA-MB-231 stem cells were revealed to exhibit elevated percentages of the cluster of differentiation (CD)44+CD24−/low subset, high tumorigenicity and resistance to chemotherapy, all of which are characteristic stem cell properties. In addition, the MDA-MB-231 stem cells were more tumorigenic in vivo. Furthermore, the breast CSCs also expressed high levels of the Oct4 and Nanog transcription factors. Therefore, downregulation of Oct4 or Nanog expression may reduce chemotherapeutic drug resistance and tumorigenicity in breast CSCs. In conclusion, Oct4 and Nanog expression may be a key factor in the development of resistance to chemotherapy and tumor growth of breast CSCs. This finding indicates that Oct4 or Nanog-targeted therapy may be a promising means of overcoming resistance to chemotherapy and inhibiting tumor growth in breast cancer treatment.

Evolution of energy metabolism, stem cells and cancer stem cells: how the Warburg and Barker hypothesis might be linked.

Evolution of energy metabolism, stem cells and cancer stem cells: how the Warburg and Barker hypothesis might be linked.

Epigenetic Regulation of Cancer Stem Cell Genes in Triple-Negative Breast Cancer

Expression of specific breast cancer stem cells (BCSCs) is seen in aggressive tumors, but their regulation is unclear. Epigenetic changes influence gene expression and are implicated in breast cancer progression. We hypothesized that promoter methylation regulates specific BCSC-related genes [CD44, CD133, CD24, MSH1 (alias, Musashi-1), and ALDH1] and that this epigenetic profile can identify aggressive subtypes, such as triple-negative breast cancer (TNBC). Methylation analysis was performed using MassARRAY EpiTYPER sequencing; CpG-rich sites were identified in the promoter regions of BCSC genes, except ALDH1. These sites were screened by treatment with 5-aza-2'-deoxycytidine in four TN and five non-TNBC cell lines. The specific regulatory CpG site demonstrating the most significant inverse correlation between CpG site methylation and mRNA expression was identified for CD44, CD133, and Musashi-1, but not for CD24. Methylation of CD44, CD133, and Musashi-1 was evaluated in 91 American Joint Committee on Cancer stage I to III primary breast cancer tumors, and these sites were significantly hypomethylated in TNBC versus non-TNBC. The IHC staining of primary tumors with the highest and lowest methylation levels revealed the strongest staining in hypomethylated specimens, suggesting that hypomethylation leads to gene activation. We demonstrate that methylation is a significant mechanism regulating CD44, CD133, and Musashi-1, and that gene hypomethylation correlates with TNBC. Assessment of epigenetic changes in BCSC genes may provide a more accurate classification of TNBC and could be developed as potential therapeutic targets.

Human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones

Human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones

Cancer Stem Cells in the Human Mammary Gland and Regulation of Their Differentiation by Estrogen

The identification and characterization of normal and breast cancer stem cells have provided a new vision of breast tumorigenesis. Cancer stem cells may be responsible for breast tumor initiation, progression and development of resistance to therapy. Most breast cancers express the estrogen receptor, and several studies have linked long-term estrogen exposure to enhanced breast cancer risk; however, estrogen receptor-positive tumors usually present a better prognosis than estrogen receptor-negative ones. The finding that estrogen reduces the pool of human breast stem cells may explain the more differentiated phenotype observed in estrogen receptor-positive tumors. In this article, our current understanding of the complex role of estrogen in human breast stem cells is discussed in the context of breast malignancy.

Abstract 5559: Long-term exposure of breast cell lines to ethanol affects the transcriptional signature for some oncogenic gene families, but has little effect on this phenotype in mammospheres or on the expression of stem cell markers

Abstract Objectives. Chronic alcoholism is a risk factor for breast cancer but the mechanism of its potential oncogenic effects is unknown. Stem cells have been found in the epithelium of normal breast tissue and are postulated to convert into cancer stem cells driving tumor generation and metastasis. Breast stem cells are assumed to be enriched within multiple-cell spheroids named mammospheres. Here we aimed to determine whether ethanol, or its metabolite, acetaldehyde, stimulate the in vitro proliferation and malignant transformation of human breast stem cells within epithelial cell cultures and mammospheres, and whether this process is defined by specific gene expression signatures denoting oncogenesis, stemness and/or alcohol-regulation. Methods. Cultures of monolayer attached cells and mammospheres from MCF-12 (“normal”) and MCF-7 (“malignant”) cell lines were incubated +/- 25 mM ethanol and subjected to DNA microarray analysis, or in triplicate with increasing concentrations of ethanol (0-25 mM) or acetaldehyde (0-12.5 mM) for 6 days, followed by immunocytofluorescence, quantitative western blot for stem cell markers Oct4 and nanog, and by RT-PCR, the formazan assay and growth in soft agar. Results. Low levels of mRNAs for stem cell markers were seen by DNA microarrays in all cultures, but except for Sox4 and Jag1 were not responsive to ethanol. Oct4 and nanog proteins were detected in the nuclei of MCF-7 attached cells, and expressed respectively as 45 (Oct4a) and 38 kDa bands for the active isoforms, whereas in MCF-12 attached cells they were detected as the variant 33 (Oct4b) and 55 kDa proteins. Ethanol reduced Oct4a moderately in MCF-7 attached cells and acetaldehyde increased it, but no changes occurred in mammospheres. Ethanol increased Oct4b and reduced nanog in MCF-12 attached cells, with no change (Oct4) or an increase (nanog) in mammospheres. In both MCF-7 and MCF-12 attached cells, but not in mammospheres, ethanol upregulated by >2 several alcohol-responsive metallothioneins (mainly 1F, 1X, and 2A), and related trans-membrane proteases serine (TMPRSS), as well as centromer-associated protein and PAI 1, while reducing other serpines and caspase 4, all malignancy-associated genes. Conclusions. Although stem cell markers were expressed in MCF-7 and MCF-12 attached cells, they were not increased in mammospheres, raising doubts about the putative stem cell enrichment in these spheroids. The inconsistent modulation of stem cell number by ethanol and acetaldehyde requires confirmation by cell sorting. The identification of several ethanol-responsive, malignancy-associated genes in both MCF-7 and MCF-12 attached cells may support an oncogenic risk for high alcohol levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5559. doi:10.1158/1538-7445.AM2011-5559

Effects of estrogen on the proportion of stem cells in the breast

There is increasing evidence that breast cancers contain tumor-initiating cells with stem cell properties. The importance of estrogen in the development of the mammary gland and in breast cancer is well known, but the influence of estrogen on the stem cell population has not been assessed. We show that estrogen reduces the proportion of stem cells in the normal human mammary gland and in breast cancer cells. The embryonic stem cell genes NANOG, OCT4, and SOX2 are expressed in normal breast stem cells and at higher levels in breast tumor cells and their expression decreases upon differentiation. Overexpression of each stem cell gene reduces estrogen receptor (ER) expression, and increases the number of stem cells and their capacity for invasion, properties associated with tumorigenesis and poor prognosis. These results indicate that estrogen reduces the size of the human breast stem cell pool and may provide an explanation for the better prognosis of ER-positive tumors.

536 Modeling the human breast stem cell and the breast cancer niche

536 Modeling the human breast stem cell and the breast cancer niche

Stem Cells in the Human Breast

The origins of the epithelial cells participating in the development, tissue homeostasis, and cancer of the human breast are poorly understood. However, emerging evidence suggests a role for adult tissue-specific stem cells in these processes. In a hierarchical manner, these generate the two main mammary cell lineages, producing an increasing number of cells with distinct properties. Understanding the biological characteristics of human breast stem cells and their progeny is crucial in attempts to compare the features of normal stem cells and cancer precursor cells and distinguish these from nonprecursor cells and cells from the bulk of a tumor. A historical overview of research on human breast stem cells in primary tissue and in culture reveals the progress that has been made in this area, whereas a focus on the cell-of-origin and reprogramming that occurs during neoplastic conversion provides insight into the enigmatic way in which human breast cancers are skewed toward the luminal epithelial lineage.

Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.

BackgroundCancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages.MethodologySingle cell suspensions derived from human breast ‘organoids’ were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres.Principal FindingsWe show that primary mammospheres contain a distinct side-population (SP) that displays a CD24low/CD44low phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44high/CD24low cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated β-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture.ConclusionsThus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.

Musashi1 Modulates Mammary Progenitor Cell Expansion through Proliferin-Mediated Activation of the Wnt and Notch Pathways

The RNA-binding protein Musashi1 (Msi1) is a positive regulator of Notch-mediated transcription in Drosophila melanogaster and neural progenitor cells and has been identified as a putative human breast stem cell marker. Here we describe a novel functional role for Msi1: its ability to drive progenitor cell expansion along the luminal and myoepithelial lineages. Expression of Msi1 in mammary epithelial cells increases the abundance of CD24(hi) Sca-1(+), CD24(hi) CD29(+), CK19, CK6, and double-positive CK14/CK18 progenitor cells. Proliferation is associated with increased proliferin-1 (PLF1) and reduced Dickkopf-3 (DKK3) secretion into the conditioned medium from Msi-expressing cells, which is associated with increased colony formation and extracellular signal-regulated kinase (ERK) phosphorylation. Treatment with the MEK inhibitor U0126 inhibits ERK activation and decreases Notch and beta-catenin/T-cell factor (TCF) reporter activity resulting from Msi1 expression. Reduction of DKK3 in control cells with a short hairpin RNA (shRNA) increases Notch and beta-catenin/TCF activation, whereas reduction of PLF1 with a shRNA in Msi1-expressing cells inhibits these pathways. These results identify Msi1 as a key determinant of the mammary lineage through its ability to coordinate cell cycle entry and activate the Notch and Wnt pathways by a novel autocrine process involving PLF1 and DKK3.