Afshin Raouf

Transcriptome Analysis of the Normal Human Mammary Cell Commitment and Differentiation Process

Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process, but the molecular mechanisms involved, particularly in the human mammary gland, are poorly understood. To address this issue, we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified, and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together, these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation.

A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels, which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2–8 weeks, the gels contain bilayered mammary epithelial structures, including luminal and myoepithelial cells, their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations.

Epithelial progenitors in the normal human mammary gland.

The human mammary gland is organized developmentally as a hierarchy of progenitor cells that become progressively restricted in their proliferative abilities and lineage options. Three types of human mammary epithelial cell progenitors are now identified. The first is thought to be a luminal-restricted progenitor; in vitro under conditions that support both luminal and myoepithelial cell differentiation, this cell produces clones of differentiating daughter cells that are exclusively positive for markers characteristic of luminal cells produced in vivo (i.e., keratins 8/18 and 19, epithelial cell adhesion molecule [EpCAM] and MUC1). The second type is a bipotent progenitor. It is identified by its ability to produce “mixed” colonies in single cell assays. These colonies contain a central core of cells expressing luminal markers surrounded by cells with a morphology and markers (e.g., keratin 14+) characteristic of myoepithelial cells. Serial passage in vitro of an enriched population of bipotent progenitors promotes the expansion of a third type of progenitor that is thought to be myoepithelial-restricted because it only produces cells with myoepithelial features. Luminal-restricted and bipotent progenitors can prospectively be isolated as distinct subpopulations from freshly dissociated suspensions of normal human mammary cells. Both are distinguished from many other cell types in mammary tissue by their expression of EpCAM and CD49f (α6 integrin). They are distinguished from each other by their differential expression of MUC1, which is expressed at much higher levels on the luminal progenitors. To relate the role of these progenitors to the generation of the three-dimensional tubuloalveolar structure of the mammary tree produced in vivo, we propose a model in which the commitment to the luminal versus the myoepithelial lineage may play a determining role in the generation of alveoli and ducts.

Y-box binding protein-1 induces the expression of CD44 and CD49f leading to enhanced self-renewal, mammosphere growth, and drug resistance.

Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor expressed in >40% of breast cancers, where it is associated with poor prognosis, disease recurrence, and drug resistance. We questioned whether this may be linked to the ability of YB-1 to induce the expression of genes linked to cancer stem cells such as CD44 and CD49f. Herein, we report that YB-1 binds the CD44 and CD49f promoters to transcriptionally upregulate their expressions. The introduction of wild-type (WT) YB-1 or activated P-YB-1(S102) stimulated the production of CD44 and CD49f in MDA-MB-231 and SUM 149 breast cancer cell lines. YB-1-transfected cells also bound to the CD44 ligand hyaluronan more than the control cells. Similarly, YB-1 was induced in immortalized breast epithelial cells and upregulated CD44. Conversely, silencing YB-1 decreased CD44 expression as well as reporter activity in SUM 149 cells. In mice, expression of YB-1 in the mammary gland induces CD44 and CD49f with associated hyperplasia. Further, activated mutant YB-1(S102D) enhances self-renewal, primary and secondary mammosphere growth, and soft-agar colony growth, which were reversible via loss of CD44 or CD49f. We next addressed the consequence of this system on therapeutic responsiveness. Here, we show that paclitaxel induces P-YB-1(S102) expression, nuclear localization of activated YB-1, and CD44 expression. The overexpression of WT YB-1 promotes mammosphere growth in the presence of paclitaxel. Importantly, targeting YB-1 sensitized the CD44(High)/CD24(Low) cells to paclitaxel. In conclusion, YB-1 promotes cancer cell growth and drug resistance through its induction of CD44 and CD49f.

Ets transcription factors and targets in osteogenesis.

Bone formation in vivo is a complex phenomenon whereby recruitment and replication of mesenchymal precursors of osteoblasts, differentiation into preosteoblasts, osteoblasts, and mature osteoblasts ultimately result in the accumulation and mineralization of the extracellular matrix. MC3T3-E1, a clonal osteoblastic cell line, was derived from mouse calvaria and undergoes an ordered and time dependent developmental sequence leading to formation of multilayered bone nodules over a 30–35 day period. This developmental pattern is characterized by the replication of preosteoblasts followed by growth arrest and expression of mature osteoblastic characteristics such as matrix maturation and eventual formation of multilayered nodules with a mineralized extracellular matrix. We have found that Ets1 is expressed in proliferating preosteoblastic cells whereas Ets2 is expressed by differentiating and mature osteoblasts. In addition, the expression of Ets1 can be induced in MC3T3-E1 and fetal rat calvaria cells by retinoic acid (RA) which is known to exert profound effects on skeletal growth and development, bone turnover, and induce specific cellular responses in bone cells. Thus the multiple functions of RA in bone cells are likely to be mediated in part by Ets1. Also, Ets2 transgenic mice develop multiple neurocranial, viserocranial, and cervical skeletal abnormalities. Significantly, these abnormalities are similar to the skeletal anomalies found in trisomy-16 mice and in humans with Downs syndrome, wherein the dosage of Ets2 is known to be increased. These results indicate that Ets2 has an important role in skeletal development and that Ets2 overexpression in transgenics is responsible for the genesis of the same type of skeletal abnormalities that are seen in Downs syndrome. Thus the genetic programs regulated by Ets1 and Ets2 may significantly affect the development and differentiation of osteoblasts, and in fact, Ets1 has been shown to interact with the ‘quintessential’ osteoblast transcription factor CbfA1. This review will examine in detail the role and possible targets of Ets1 and Ets2 in osteoblast differentiation and bone formation.

Deciphering the mammary epithelial cell hierarchy.

Clonal assays offer a powerful approach to dissecting the many events involved in the generation and maintenance of complex tissues from an undifferentiated stem cell pool. The application of such quantitative functional methodologies to studies of the hematopoietic system have been key to defining the hierarchy of progenitor subsets that reflect an irreversible stepwise process of lineage restriction. Recent studies now suggest that a similar paradigm applies to the normal mammary gland. The adult mouse mammary gland maintains a population of stem cells that generate biologically distinct and physically separable subpopulations of mammary epithelial progenitors which, in turn, generate terminally differentiated cells. Suggestive parallels between mouse and human mammary cells point to the likelihood that a similarly structured multi-step differentiation program characterizes the mammary gland from both species.

Developmental changes in the in vitro activated regenerative activity of primitive mammary epithelial cells.

Mouse fetal mammary cells display greater regenerative activity than do adult mammary cells when stimulated to proliferate in a new system that supports the production of transplantable mammary stem cells ex vivo.

The Ets1 proto-oncogene is upregulated by retinoic acid: characterization of a functional retinoic acid response element in the Ets1 promoter.

The v-ets oncoprotein and its progenitor Ets1 belong to a family of transcription factors that are related by an 85 amino acid conserved DNA binding domain, the ets domain. Ets1 plays important role(s) in control of cell proliferation, differentiation and apoptosis. Abnormal expression of Ets1 could lead to disruption of these processes and contribute to development of malignancy. Retinoic acid (RA) inhibits proliferation, induces differentiation and regulates apoptosis in many different cell types. Here, we demonstrate that RA treatment increases the expression of Ets1 mRNA, but not that of Ets2, Elk1 or Fli1 in MC3T3-E1 cells. Ets1 induction is detectable after 4 h, can be maintained for at least 14 days, and is inhibited by Actinomycin D, which suggests that RA regulation of Ets1 occurs at the transcriptional level. The promoter region of Ets1 contains four retinoic acid response element (RARE) half sites located at −94, −152, −1765 and −2252 from the translation start site. We show that RARβ is expressed by MC3T3-E1 cells in the presence of RA and demonstrate that it binds to the −94 RARE half site. Furthermore, RA induces transcription of Ets1 promoter-reporter constructs containing this RARE half site.

Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets

Significance Our study reveals lineage-specific mechanisms of ROS control and associated sensitivity to oxidative DNA damage in the basal and luminal progenitor-enriched subsets of normal human mammary cells. We show that the primitive luminal cells contain more mitochondria, show greater uptake of O2, sustain and withstand higher levels of ROS, and have mechanisms that allow them to accrue mutagenic levels of oxidative DNA damage. These findings support a growing body of data suggesting the involvement of primitive luminal cells in the generation of human breast cancers. Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated “steady-state” normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.

The biology of human breast epithelial progenitors

Current evidence suggests that similar to other tissues in the human body mammary epithelia cells are being maintained by the unique properties of stem cells, undifferentiated as well as lineage-restricted progenitors. Because of their longevity, proliferation and differentiation potentials these primitive breast epithelial cells are likely targets of transforming mutations that can cause them to act as cancer initiating cells. In this context, understanding the molecular mechanisms that regulate the normal functions of the human breast epithelial stem cells and progenitors and how alterations to these same mechanisms can confer a cancer stem cell phenotype on these rare cell populations is crucial to the development of new and more effective therapies again breast cancer. This review article will examine the current state of knowledge about the isolation and characterization of human breast epithelial progenitors and their relevance to breast cancer research.