Summary Screens for agents that specifically kill epithelial cancer stem cells (CSCs) have not been possible due to the rarity of these cells within tumor cell populations and their relative instability in culture. We describe here a novel approach to screening for agents with epithelial CSC-specific toxicity. We have implemented this method in the context of a chemical screen and have discovered compounds showing selective toxicity for breast CSCs. In functional assays, one compound (salinomycin) reduced the proportion of CSCs by >100-fold relative to paclitaxel, a commonly used breast cancer chemotherapeutic drug. Treatment of mice with salinomycin inhibits mammary tumor growth in vivo and induces increased epithelial differentiation of tumor cells. In addition, global gene expression analyses show that salinomycin but not paclitaxel treatment results in the loss of expression of breast CSC genes previously identified by analyses of breast tissues isolated directly from patients. This study demonstrates that it is possible to identify agents with specific toxicity for epithelial CSCs as well as providing a practical approach for doing so.

Cancer cells within individual tumors often exist in distinct phenotypic states that differ in functional attributes. While cancer cell populations typically display distinctive equilibria in the proportion of cells in various states, the mechanisms by which this occurs are poorly understood. Here, we study the dynamics of phenotypic proportions in human breast cancer cell lines. We show that subpopulations of cells purified for a given phenotypic state return towards equilibrium proportions over time. These observations can be explained by a Markov model in which cells transition stochastically between states. A prediction of this model is that, given certain conditions, any subpopulation of cells will return to equilibrium phenotypic proportions over time. A second prediction is that breast cancer stem-like cells arise de novo from non-stem-like cells. These findings contribute to our understanding of cancer heterogeneity and reveal how stochasticity in single-cell behaviors promotes phenotypic equilibrium in populations of cancer cells.

Current models of stem cell biology assume that normal and neoplastic stem cells reside at the apices of hierarchies and differentiate into nonstem progeny in a unidirectional manner. Here we identify a subpopulation of basal-like human mammary epithelial cells that departs from that assumption, spontaneously dedifferentiating into stem-like cells. Moreover, oncogenic transformation enhances the spontaneous conversion, so that nonstem cancer cells give rise to cancer stem cell (CSC)-like cells in vitro and in vivo. We further show that the differentiation state of normal cells-of-origin is a strong determinant of posttransformation behavior. These findings demonstrate that normal and CSC-like cells can arise de novo from more differentiated cell types and that hierarchical models of mammary stem cell biology should encompass bidirectional interconversions between stem and nonstem compartments. The observed plasticity may allow derivation of patient-specific adult stem cells without genetic manipulation and holds important implications for therapeutic strategies to eradicate cancer.breast cancer | dedifferentiation

Introduction The phenotypic and functional differences between cells that initiate human breast tumors (cancer stem cells) and those that comprise the tumor bulk are difficult to study using only primary tumor tissue. We embarked on this study hypothesizing that breast cancer cell lines would contain analogous hierarchical differentiation programs to those found in primary breast tumors.

The study of normal breast epithelial morphogenesis and carcinogenesis in vivo has largely used rodent models. Efforts at studying mammary morphogenesis and cancer with xenotransplanted human epithelial cells have failed to recapitulate the full extent of development seen in the human breast. We have developed an orthotopic xenograft model in which both the stromal and epithelial components of the reconstructed mammary gland are of human origin. Genetic modification of human stromal cells before the implantation of ostensibly normal human mammary epithelial cells resulted in the outgrowth of benign and malignant lesions. This experimental model allows for studies of human epithelial morphogenesis and differentiation in vivo and underscores the critical role of heterotypic interactions in human breast development and carcinogenesis.

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Women with inherited mutations in the BRCA1 gene have increased risk of developing breast cancer, but also exhibit a predisposition for the development of aggressive basal-like breast tumors. We report here that breast epithelial cells derived from patients harboring deleterious mutations in BRCA1 (BRCA1mut/+) give rise to tumors with increased basal differentiation relative to cells from BRCA1+/+ patients. Molecular analysis of disease-free breast tissues from BRCA1mut/+ patients revealed defects in progenitor cell lineage commitment even before cancer incidence. Moreover, we discovered that the transcriptional repressor Slug is an important functional regulator of human breast progenitor cell lineage commitment and differentiation and that it is aberrantly expressed in BRCA1mut/+ tissues. Slug expression is necessary for increased basal-like phenotypes prior to and following neoplastic transformation. These findings demonstrate that the genetic background of patient populations, in addition to affecting incidence rates, significantly impacts progenitor cell fate commitment and, therefore, tumor phenotype.

The aggressive clinical behavior of melanoma has led to the hypothesis that the developmental origins of melanocytes in the neural crest might be relevant for their metastatic propensity. We demonstrate that primary human melanocytes, transformed using a specific set of introduced genes, form melanomas that frequently metastasize to multiple secondary sites, while human fibroblasts and epithelial cells transformed using an identical set of genes generate primary tumors that rarely do so. Importantly, these melanomas exhibit a metastasis spectrum similar to that observed in human patients. These observations indicate that part of the metastatic proclivity of melanoma is attributable to lineage-specific factors expressed in melanocytes and not in other cell types analyzed. Analysis of microarray data from human nevi reveals that Slug, a master regulator of neural crest cell specification and migration, correlates in its expression pattern with other genes that are important for neural crest cell migrations during development. Moreover, Slug is required for the metastasis of the transformed melanoma cells. These findings indicate that melanocyte-specific factors present prior to neoplastic transformation can play a pivotal role in governing melanoma's progression.