Self-renewal pathways in normal and malignant human mammary stem cells.

Proc Amer Assoc Cancer Res, Volume 47, 2006 SY17-02 The mammary gland epithelial components are thought to arise from stem cells which have a unique capacity for self-renewal as well as for differentiation into lineages which comprise the lobular alveolar structure of the adult gland. Stem cells may undergo either symmetric or asymmetric division during tissue regeneration and repair respectively. There is increasing evidence that these stem and early progenitor cells may be targets for transformation during breast carcinogenesis. Normal stem cells and cancer cells share many characteristics including the ability to self-renew and to differentiate, albeit abberently in the case of cancer cells. In addition, both cells are long lived, express telomerase, are resistant to apoptosis, and are able to migrate and metastasize. Since normal stem cells and cancer cells share these characteristics, we and others have postulated that a key event in transformation may be the disregulation of pathways that regulate normal stem cell self-renewal. We have recently described an in vitro system for the propagation of human mammary stem and progenitor cells in suspension culture. We demonstrated that human mammary cells isolated from reduction mammoplasties generate spherical colonies in suspension that we have termed “mammospheres” which are highly enriched in stem and progenitor cells capable of self-renewal and multi-lineage differentiation. We postulate that the sphere forming cells have stem cell characteristics while the majority of cells within a mammosphere are differentiating progenitor cells which retain multi-lineage differentiation capacity. We have utilized this culture system as well as an in vivo NOD-SCID mouse model to investigate the molecular pathways that regulate stem cell self-renewal including Hedgehog, Notch, and Bmi-1. Components of both the Hedgehog and Notch pathway are over-expressed in mammospheres compared to differentiated cells cultured on a collagen substratum. The activation of either pathway is able to increase mammosphere formation as well as mammosphere size. We demonstrate cross-talk between Notch and Hedgehog signaling pathways with subsequent activation of the polycomb gene Bmi-1. In order to demonstrate a functional role of Bmi-1 in stem cell self-renewal, we have utilized siRNAs that down-regulate Bmi-1 expression and have demonstrated that this construct abrogates the effects of Notch and Hedgehog signaling. In order to examine the effect of disregulation of Hedgehog signaling on mammary development, we have utilized an in vivo system in which mammospheres are implanted into the cleared fat pads of NOD-SCID mice which have been humanized with irradiated normal human mammary fibroblasts. Over-expression of the Hedgehog target gene Gli2 leads to the production of ductal hyperplasia’s in this model. We have previously demonstrated that human mammary cancers may be driven by a subcomponent displaying stem cell properties and displaying the phenotype CD44+ CD24 low lin-. As few as 200 cells with this phenotype are able to produce tumors in NOD-SCID mice, whereas 20,000 cells isolated from the same tumor that do not display this phenotype, are not tumorigenic. Furthermore, tumors derived from the tumorigenic component recapitulate the phenotypic heterogeneity of the initial tumor. We have utilized this system to investigate the expression of self-renewal pathway genes in tumor stem cells. We find that CD44+ CD24 low lin- cells show Hedgehog activation and Bmi-1 over-expression compared to nontumorigenic cells. Together these studies support a tumor stem cell model in which transformation involves the disregulation of self-renewal of mammary stem or progenitor cells. This disregulation may involve activation of Hedgehog or Notch signaling with subsequent over-expression of the polycomb gene Bmi-1. Interventions aimed at disrupting these pathways represent a rationale therapeutic strategy for the prevention and therapy of breast cancer.