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.

Aldehyde dehydrogenase and estrogen receptor define a hierarchy of cellular differentiation in the normal human mammary epithelium.

INTRODUCTION: Although estrogen and progesterone play a key role in normal mammary development and in breast cancer, the potential for proliferation and lineage differentiation as well as origin of cells that express the estrogen receptor (ER) in normal breast epithelium are not known. Some evidence suggests that normal human mammary stem/progenitor cells are ER-, but the identity of these cells and the cellular hierarchy of breast epithelium are still subjects of controversy. It is likely that elucidation of these aspects will bring insight into the cellular origin of breast cancer subtypes. METHODS: We used fluorescence-activated cell sorting of primary human mammary epithelial cells along with in vitro and in vivo functional assays to examine the hierarchic relation between cells with aldehyde dehydrogenase enzymatic activity (ALDH+ cells) and ER+ cells in the normal human breast epithelium. We assessed the proliferation and lineage differentiation potential of these cells in vitro and in vivo. A gene reporter assay was used to separate live ER+ and ER- mammary epithelial cells. With shRNA-mediated knockdown, we investigated the role of ALDH isoforms in the functionality of mammary epithelial progenitor cells. RESULTS: We describe a cellular hierarchy in the normal human mammary gland in which ER-/ALDH+ cells with functional properties of stem/progenitor cells generate ER+ progenitor cells, which in turn give rise to cells of luminal lineage. We show that the ALDH1A1 isoform, through its function in the retinoic acid metabolism, affects the proliferation and/or early differentiation of stem/progenitor cells and is important for branching morphogenesis. CONCLUSIONS: This study presents direct evidence that ER+ cells are generated by ER-/ALDH+ stem/progenitor cells. We also show that ER+ cells are able to generate cell progeny of luminal lineage in vitro and in vivo. Loss of ALDH1A1 function impairs this process, as well as branching morphogenesis and clonogenicity in suspension culture. This latter effect is reversed by treatment with retinoic acid.

BRCA1 regulates human mammary stem/progenitor cell fate.

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.

MicroRNAs: shortcuts in dealing with molecular complexity?

Recent studies from Clarke's group published in the journal Cell indicate that miRNAs may be the elusive universal stem cell markers that the field of cancer stem cell biology has been seeking. Distinct profiles of miRNAs appear to reflect the state of cell differentiation not only in breast cancer cells, but also in normal mammary epithelial cells. Moreover, they are conserved across tissues and species. The authors of this work also show evidence that downregulation of miRNA-200c in normal and malignant breast stem cells and in embryonal carcinoma cells has functional relevance, being responsible for the proliferative potential of these cells in vitro and in vivo.

Targeting breast stem cells with the cancer preventive compounds curcumin and piperine.

The cancer stem cell hypothesis asserts that malignancies arise in tissue stem and/or progenitor cells through the dysregulation or acquisition of self-renewal. In order to determine whether the dietary polyphenols, curcumin, and piperine are able to modulate the self-renewal of normal and malignant breast stem cells, we examined the effects of these compounds on mammosphere formation, expression of the breast stem cell marker aldehyde dehydrogenase (ALDH), and Wnt signaling. Mammosphere formation assays were performed after curcumin, piperine, and control treatment in unsorted normal breast epithelial cells and normal stem and early progenitor cells, selected by ALDH positivity. Wnt signaling was examined using a Topflash assay. Both curcumin and piperine inhibited mammosphere formation, serial passaging, and percent of ALDH+ cells by 50% at 5 microM and completely at 10 microM concentration in normal and malignant breast cells. There was no effect on cellular differentiation. Wnt signaling was inhibited by both curcumin and piperine by 50% at 5 microM and completely at 10 microM. Curcumin and piperine separately, and in combination, inhibit breast stem cell self-renewal but do not cause toxicity to differentiated cells. These compounds could be potential cancer preventive agents. Mammosphere formation assays may be a quantifiable biomarker to assess cancer preventive agent efficacy and Wnt signaling assessment can be a mechanistic biomarker for use in human clinical trials.

Breast cancer stem cell markers - the rocky road to clinical applications.

Lately, understanding the role of cancer stem cells in tumor initiation and progression became a major focus in stem cell biology and in cancer research. Considerable efforts, such as the recent studies by Honeth and colleagues, published in the June issue of Breast Cancer Research, are directed towards developing clinical applications of the cancer stem cell concepts. This work shows that the previously described CD44+CD24- stem cell phenotype is associated with basal-type breast cancers in human patients, in particular BRCA1 inherited cancers, but does not correlate with clinical outcome. These very interesting findings caution that the success of our efforts in translating cancer stem cell research into clinical practice depends on how thorough and rigorous we are at characterizing these cells.

Cancer stem cells in breast: current opinion and future challenges.

There is increasing evidence for the cancer stem cell hypothesis, which holds that cancers are driven by a cellular subcomponent that has stem cell properties, that is, self-renewal, tumorigenicity and multilineage differentiation capacity. The cancer stem cell hypothesis modifies our conceptual approach of oncogenesis and shall have implications in breast cancer prevention, detection and treatment, especially in metastatic breast cancer for which no curative treatment exists. Given the specific stem cell features, novel therapeutic pathways can be targeted. Following this approach, new molecules are currently in development. Focusing on the cross-talk between stem cells and their microenvironment is also a promising way to explore how to better target cancer stem cells and be curative.

Cancer stem cells: an old idea--a paradigm shift.

Although the concept that cancers arise from "stem cells" or "germ cells" was first proposed about 150 years ago, it is only recently that advances in stem cell biology have given new impetus to the "cancer stem cell hypothesis." Two important related concepts of this hypothesis are that (a) tumors originate in either tissue stem cells or their immediate progeny through dysregulation of the normally tightly regulated process of self-renewal. As a result of this, (b) tumors contain a cellular subcomponent that retains key stem cell properties. These properties include self-renewal, which drives tumorigenesis, and differentiation albeit aberrant that contributes to cellular heterogeneity. Recent experimental evidence in a variety of tumors has lent strong support to the cancer stem cell hypothesis that represents a paradigm shift in our understanding of carcinogenesis and tumor cell biology. This hypothesis has fundamental implications for cancer risk assessment, early detection, prognostication, and prevention. Furthermore, the current development of cancer therapeutics based on tumor regression may have produced agents that kill differentiated tumor cells while sparing the rare cancer stem cell population. The development of more effective cancer therapies may thus require targeting this important cell population.

Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells.

The epithelial components of the mammary gland are thought to arise from stem cells with a capacity for self-renewal and multilineage differentiation. Furthermore, these cells and/or their immediate progeny may be targets for transformation. We have used both in vitro cultivation and a xenograft mouse model to examine the role of hedgehog signaling and Bmi-1 in regulating self-renewal of normal and malignant human mammary stem cells. We show that hedgehog signaling components PTCH1, Gli1, and Gli2 are highly expressed in normal human mammary stem/progenitor cells cultured as mammospheres and that these genes are down-regulated when cells are induced to differentiate. Activation of hedgehog signaling increases mammosphere-initiating cell number and mammosphere size, whereas inhibition of the pathway results in a reduction of these effects. These effects are mediated by the polycomb gene Bmi-1. Overexpression of Gli2 in mammosphere-initiating cells results in the production of ductal hyperplasia, and modulation of Bmi-1 expression in mammosphere-initiating cells alters mammary development in a humanized nonobese diabetic-severe combined immunodeficient mouse model. Furthermore, we show that the hedgehog signaling pathway is activated in human breast "cancer stem cells" characterized as CD44+CD24-/lowLin-. These studies support a cancer stem cell model in which the hedgehog pathway and Bmi-1 play important roles in regulating self-renewal of normal and tumorigenic human mammary stem cells.

Breast cancer stem cells-research opportunities utilizing mathematical modeling.

There is increasing evidence for the "cancer stem cell hypothesis" which holds that cancers originate in tissue stem cells or progenitor cells. As a result of this, cancers are driven by a cellular subcomponent that retains stem cell properties. Among these properties are self-renewal and multi-lineage differentiation. The biological processes which account for stem cell properties are currently being elucidated. Cancer stem cells maintain many of the same characteristics of their normal counterparts. The combination of biological research with mathematical modeling may provide for a greater understanding of the complex picture of breast cancer stem cells and assist cancer biologists and clinical oncologists in designing and testing novel therapeutic strategies.

[The cancer stem cell: the breast cancer driver]. / La cellule souche cancéreuse: un pilote aux commandes du cancer du sein.

Recent research in a large variety of tumors, including breast cancer, has given support to the "cancer stem cell hypothesis". Based on this, tumors contain and are driven by a cellular subcomponent that retains key stem cell properties. These include self-renewal, which drives tumorigenesis, and the capacity to generate cellular heterogeneity. Recently, different techniques have been used to isolate potential breast cancer stem cells with the cell surface phenotype CD44+CD24-/low lin- or expressing Aldehyde dehydrogenase. This model has fundamental implications for breast cancer treatment. The development of specific therapeutics that target this population is an important focus for the future.

Mammary stem cells, self-renewal pathways, and carcinogenesis.

The mammary gland epithelial components are thought to arise from stem cells that undergo both self-renewal and differentiation. Self-renewal has been shown to be regulated by the Hedgehog, Notch, and Wnt pathways and the transcription factor B lymphoma Mo-MLV insertion region 1 (Bmi-1). We review data about the existence of stem cells in the mammary gland and the pathways regulating the self-renewal of these cells. We present evidence that deregulation of the self-renewal in stem cells/progenitors might be a key event in mammary carcinogenesis. If 'tumor stem cells' are inherently resistant to current therapies, targeting stem cell self-renewal pathways might provide a novel approach for breast cancer treatment.

Breast cancer, stem/progenitor cells and the estrogen receptor.

Recently, substantial progress has been made in the identification and characterization of stem and progenitor cells in the mouse and human mammary gland. Furthermore, there is accumulating evidence that these cells might be targets for transformation during mammary carcinogenesis. On the basis of this stem cell concept, we propose a model in which the transformation of different subsets of stem and progenitor cells results in the diversity of breast cancer phenotypes, including expression of the estrogen receptor in breast cancers subtypes. This model has important implications for understanding mammary carcinogenesis. Furthermore, the concept of breast cancer as a disease of mammary stem and progenitor cells has profound implications for the development of new strategies for breast cancer prevention and therapy.

Stem cells in mammary development and carcinogenesis: implications for prevention and treatment.

Recently, substantial progress has been made in the identification and characterization of stem and progenitor cells in the mouse and human mammary gland. Furthermore, there is increasing evidence that a variety of neoplasms, including breast cancer, may result from transformation of normal stem and progenitor cells. Consistent with this model of carcinogenesis, a breast cancer stem cell population, with the phenotype CD24-CD44+ lineage, was recently identified utilizing flow-cytometry based cell sorting and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografts. As few as 200 cells of this cancer stem cell population were capable of generating tumors in animals, whereas the bulk of the tumor population was tumorigenic only when implanted in high numbers. Like their normal counterparts, the cancer stem cells have the ability to self-renew, driving tumorigenicity and possibly recurrence and metastasis, and have the ability to differentiate, generating the heterogeneity of the tumors. This stem cell model of carcinogenesis has important implications for understanding the basic biology of breast cancer, as well as other cancers. Furthermore, the concept of cancer as a disease of stem and progenitor cells has profound implications for the development of new strategies for cancer prevention and therapy.

Modeling the breast cancer bone metastatic niche in complex three-dimensional cocultures.

Despite advances in early detection, prevention and treatment of breast cancer, the mortality of breast cancer patients did not decrease considerably in the last years. Metastatic breast cancer remains incurable. There is compelling evidence that dissemination of breast cancer cells at distant sites is an early event. At the time of detection and diagnosis, patients have disseminated breast cancer cells in the bone marrow. Only in half of these patients the disseminated cells proliferate and generate metastases, typically in 3-5 years for ER negative breast tumors and 10-15 years for ER positive breast tumors. In other patients metastases never develop. The ability to predict which patients will develop metastases and to devise strategies to interfere with this process hinges on understanding the mechanisms underlying growth at the metastatic site. In turn, this requires novel experimental systems that model in vitro the survival, dormancy and proliferation of disseminated cancer cells.We have established such experimental systems that model the bone microenvironment of the breast cancer metastatic niche. These systems are based on 3D complex cultures of human bone marrow stromal cells and breast cancer cell lines in collagen biomatrices. We identified conditions in which cancer cells are dormant, and conditions in which they proliferate and we validated the results in vivo. Dormant cancer cells were able to proliferate upon transfer into supportive microenvironment or upon manipulation of signaling pathways that control dormancy. These experimental systems will be instrumental in screening new compounds for metastasis studies and particularly in studying the pathways that control cellular dormancy. We provide in this chapter detailed protocols for these complex 3D coculture systems.

Models of breast morphogenesis based on localization of stem cells in the developing mammary lobule.

Characterization of normal breast stem cells is important for understanding their role in breast development and in breast cancer. However, the identity of these cells is a subject of controversy and their localization in the breast epithelium is not known. In this study, we utilized a novel approach to analyze the morphogenesis of mammary lobules, by combining one-dimensional theoretical models and computer-generated 3D fractals. Comparing predictions of these models with immunohistochemical analysis of tissue sections for candidate stem cell markers, we defined distinct areas where stem cells reside in the mammary lobule. An increased representation of stem cells was found in smaller, less developed lobules compared to larger, more mature lobules, with marked differences in the gland of nulliparous versus parous women and that of BRCA1/2 mutation carriers versus non-carriers.

Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells.

INTRODUCTION: Notch signaling has been implicated in the regulation of cell-fate decisions such as self-renewal of adult stem cells and differentiation of progenitor cells along a particular lineage. Moreover, depending on the cellular and developmental context, the Notch pathway acts as a regulator of cell survival and cell proliferation. Abnormal expression of Notch receptors has been found in different types of epithelial metaplastic lesions and neoplastic lesions, suggesting that Notch may act as a proto-oncogene. The vertebrate Notch1 and Notch4 homologs are involved in normal development of the mammary gland, and mutated forms of these genes are associated with development of mouse mammary tumors. METHODS: In order to determine the role of Notch signaling in mammary cell-fate determination, we have utilized a newly described in vitro system in which mammary stem/progenitor cells can be cultured in suspension as nonadherent 'mammospheres'. Notch signaling was activated using exogenous ligands, or was inhibited using previously characterized Notch signaling antagonists. RESULTS: Utilizing this system, we demonstrate that Notch signaling can act on mammary stem cells to promote self-renewal and on early progenitor cells to promote their proliferation, as demonstrated by a 10-fold increase in secondary mammosphere formation upon addition of a Notch-activating DSL peptide. In addition to acting on stem cells, Notch signaling is also able to act on multipotent progenitor cells, facilitating myoepithelial lineage-specific commitment and proliferation. Stimulation of this pathway also promotes branching morphogenesis in three-dimensional Matrigel cultures. These effects are completely inhibited by a Notch4 blocking antibody or a gamma secretase inhibitor that blocks Notch processing. In contrast to the effects of Notch signaling on mammary stem/progenitor cells, modulation of this pathway has no discernable effect on fully committed, differentiated, mammary epithelial cells. CONCLUSION: These studies suggest that Notch signaling plays a critical role in normal human mammary development by acting on both stem cells and progenitor cells, affecting self-renewal and lineage-specific differentiation. Based on these findings we propose that abnormal Notch signaling may contribute to mammary carcinogenesis by deregulating the self-renewal of normal mammary stem cells.

Stem cells in normal breast development and breast cancer.

The main focus of this review is the role of mammary stem cells in normal breast development and carcinogenesis. We have developed a new in vitro culture system that permits, for the first time, the propagation of mammary stem and progenitor cells in an undifferentiated state, which should facilitate the elucidation of pathways that regulate normal mammary stem-cell self-renewal and differentiation. Furthermore, we propose a model in which transformation of stem cells, or early progenitor cells, results in carcinogenesis. A key event in this process is the deregulation of normal self-renewal in these cells. Transformed mammary stem or progenitor cells undergo aberrant differentiation processes that result in generation of the phenotypic heterogeneity found in human and rodent breast cancers. This phenotypic diversity is driven by a small subset of mammary tumour stem cells. We will discuss the important implications of this mammary tumour stem-cell model.

Growth hormone is secreted by normal breast epithelium upon progesterone stimulation and increases proliferation of stem/progenitor cells.

Using in vitro and in vivo experimental systems and in situ analysis, we show that growth hormone (GH) is secreted locally by normal human mammary epithelial cells upon progesterone stimulation. GH increases proliferation of a subset of cells that express growth hormone receptor (GHR) and have functional properties of stem and early progenitor cells. In 72% of ductal carcinoma in situ lesions, an expansion of the cell population that expresses GHR was observed, suggesting that GH signaling may contribute to breast cancer development.