Functional characterization of mammary stem cells in development and breast cancer

Breast cancer is a genetically and clinically heterogeneous disease. Whether different target cells contribute to this heterogeneity, and which cell types are most susceptible to oncogenesis is still not well understood. Identifying mammary cell lineage markers is a prerequisite for elucidating the function of stem cells in mammary development and tumorigenesis, and especially for understanding preneoplastic progression. Our laboratory has used genetically engineered mice coupled with fluoresence-activated cell sorting analysis and transplantation into the cleared mammary fat pad, as a model system in which to isolate and characterize functional mammary progenitors and stem cells. Taking advantage of approaches similar to those employed to isolate and characterize hematopoietic and epidermal stem cells, we identified a population of self-renewing, label retention cells, which excluded Hoechst dye and were present on fluoresence-activated cell sorting analysis as side-population (SP) cells. The SP cells and label retention cells represented approximately 0.5% of mammary epithelial cells in the immature mouse. DNA microarrays have been employed to determine the gene expression profiles of these SP cells in comparison with the non-SP population. Approximately 75% of the SP cells expressed stem cell antigen-1 (Sca-1). Sca-1 cells have been localized to the terminal end buds of growing ducts and as few as 1000 Sca-1⁺ cells were able to generate mammary gland outgrowths containing luminal, alveolar and myoepithelial cells. In addition, no outgrowths were observed in Sca-1–mammary epithelial cell transplants. In different genetically engineered mice mammary tumor models Sca-1 expression levels, as well as several other putative markers of progenitors, including keratin-6, have dramatically altered expression profiles. For example, Wnt-1 tumors express these markers and contain at least two populations of tumor cells, epithelial cells and myoepithelial cells that share secondary mutations such as loss of p53 and Pten, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing β-catenin and c-myc, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells as well as cells expressing keratin 6; however, progenitor cell markers and myoepithelial cells are lacking in mammary tumors from transgenic mice expressing Neu, H-Ras or polyoma middle T antigen. Interestingly, K6 expression was also detected in the ductal epithelium of C/EBPβ null mice, suggesting that germline deletion of this bZIP transcription factor alters mammary epithelial cell fate. These data suggest that the genetic heterogeneity observed in breast cancer results from the activation of specific oncogene and/or tumor suppressor-regulated signaling pathways in specific mammary progenitors. Finally, recent studies in several other laboratories have identified a comparable population of multipotent stem cells in the normal human breast as well as a population of tumorigenic stem cells in some breast cancers. Understanding the differences between normal and cancer stem cells may provide new therapeutic targets for the treatment of breast cancer.