Interaction of hedgehog and notch pathways, and Bmi-1 in the regulation of human breast stem cell self-renewal

2043

The mammary gland epithelial components are thought to arise from stem cells capable of self-renewal and multilineage differentiation. There is increasing evidence that stem/early progenitor cells are the targets for transformation during breast carcinogenesis. Since normal stem cells and cancer cells share many characteristics, it is postulated that a key transformational event may be the deregulation of normal stem cell self-renewal. We have demonstrated that human mammary epithelial cells (HMECs) isolated from reduction mammoplasties generate spherical colonies in suspension which are termed “mammospheres”, which are highly enriched in mammary stem/progenitor cells and capable of self-renewal and differentiation. We utilized this culture system to investigate pathways regulating stem cell self-renewal, including Hedgehog (Hh) and Notch, and Bmi-1. We utilized real-time PCR to compare the expression of Hh pathway components in mammospheres to these in differentiated cells on collagen derived from the same patient. The expression of Indian Hh, PTCH1, Gli1, and Gli2 was increased in undifferentiated HMECs compared to their differentiated counterparts. In suspension culture, exposure to recombinant Hh ligands (3μg/ml) increased mammosphere formation and their size, and induced the expression of receptors PTCH1 and SMO, and Hh signaling targets Gli1 and Gli2. These effects were blocked by 300 nM of cyclopamine, a specific Hh inhibitor. The overexpression of Gli1 or Gli2 in mammosphere initiating cells by retroviral infection increased mammosphere formation and their size, and induced PTCH1. These results suggest that Hh signaling regulates mammary stem cell self-renewal as well as progenitor cell proliferation. In order to investigate the interaction between Hh and Notch signaling, we examined the effect of recombinant Hh ligands on the induction of the Notch effector HES1. We found that activation of Hh signaling induced HES1. In addition, activation of the Notch pathway by Notch ligand DSL induced Hh pathway components PTCH1, Gli1 and Gli2. These results suggest an intricate interaction involving a positive feedback loop between Notch and Hh pathways. Interestingly, we found that activation of either Notch or Hh signaling resulted in induction of Bmi-1. Since Bmi-1 was shown to regulate stem cell self-renewal in hematopoietic and neuronal stem cells, this suggests both Notch and Hh may regulate stem cell renewal via regulating Bmi-1. Transgenic models of mammary carcinogenesis have demonstrated that deregulation of Hh, Notch, or Bmi-1 results in mammary tumor formation. Together, these results support the hypothesis that transformation of mammary stem/progenitor cells involves deregulation of key pathways, including Hedgehog, Notch and Bmi-1. Tumor stem cells resulting from this deregulation may be susceptible to strategies aimed at targeting these self-renewal pathways.