ORIGINAL ARTICLE Notch1-induced mammary tumor development is cyclin D1-dependent and correlates with expansion of pre-malignant multipotent duct-limited progenitors H Ling 1 , J-R Sylvestre 1 and P Jolicoeur 1,2,3 1 Laboratory of Molecular Biology, Clinical Research Institute of Montreal, Montre´al, Que´bec, Canada; 2 Division of Experimental Medicine, McGill University, Montre´al,Que´bec, Canada and 3 Department of Microbiology and Immunology Universite´de Montre´al, Montre´al,Que´bec,Canada Members of the Notch family are involved in the development of breast cancer in animal models and in humans. In young transgenic mice, expressing intracel- lular activated Notch1 (N1 IC ) in mammary cells, we found that CD24 þ CD29 high progenitor cells had enhanced survival, and were expanded through a cyclin D1- dependent pathway. This expansion positively correlated with the later cyclin D1-dependent formation of basal-like ductal tumors. This expanded population exhibited abnormal differentiation skewed toward the basal cells, showed signs of pre-malignancy (low PTEN/p53 and high c-myc) and contained stem cells with impaired self- renewal in vivo, and more numerous multipotent, ductal- restricted progenitors. Our data suggest that N1 IC can favor transformation of progenitor cells early in life through a cyclin D1-dependent pathway. Oncogene (2010) 29, 4543–4554; doi:10.1038/onc.2010.186; published online 21 June 2010 Keywords: mammary tumors; stem cells; Notch1 Introduction The mammary gland is made up of differentiated epithelial cell types that can be distinguished by their cytokeratin (CK) staining: the ductal and alveolar luminal epithelial cells (CK8 þ , CK18 þ , CK19 þ ) con- stitute the inner layer, which is surrounded by basal or myoepithelial cells (CK14 þ ). The luminal and myo- epithelial cell types originate from lineage-restricted progenitors, which are themselves produced by common multipotent epithelial progenitors enriched in the line- age-negative (Ter119 À CD45 À CD31 À ) CD24 þ CD29 high cell subset (Shackleton et al., 2006; Stingl et al., 2006). Notch signaling is required for maintaining luminal cell fate and preventing uncontrolled basal cell proliferation (Buono et al., 2006; Bouras et al., 2008). Notch3 was also found to be required for the differentiation of human bipotent progenitor cells to luminal lineage (Raouf et al., 2008). The Notch pathway has been found to be deregulated in breast cancers (Callahan and Egan, 2004). Notch4 and Notch1 are frequent targets of mouse mammary tumor virus (MMTV) provirus insertion in mouse mammary tumors, resulting in overexpression of mutant truncated intracellular forms of these proteins having transforming properties in vitro (Callahan and Egan, 2004). Mouse IC activated Notch4 (N4 IC ) (reviewed in Callahan and Egan, 2004) and N1 IC (Hu et al., 2006), and human N1 IC (Kiaris et al., 2004) also cause mammary tumors in transgenic (Tg) mice. Moreover, overexpression of Notch1 and Notch3 was documented in human breast tumors, and was associated with poor prognosis (Reedijk et al., 2005; Stylianou et al., 2006). Conflicting results have been obtained on the effects of high Notch signaling in vitro. Enhanced Notch signaling achieved through exogenous ligands promoted human breast stem cell proliferation in mammospheres and affected their differentiation, in particular by increasing the number of myoepithelial progenitors (Dontu et al., 2004). In contrast, short-term culture of mouse mam- mary stem/progenitor CD24 þ CD29 high cells in vitro, followed by their transduction with activated Notch1 led to their commitment to luminal cell fate after re- transplantation in vivo (Bouras et al., 2008). It is not known whether breast stem/progenitor cells are themselves the target of oncogenic N1 IC in vivo. In the present study, we used the MMTV/N1 IC Tg mice expressing N1 IC and developing mammary tumors at high incidence (Hu et al., 2006) to gain insights into the mechanisms of Notch1-induced mammary tumors and to investigate the impact of N1 IC overexpression on mammary stem/progenitor cells in vivo. Results Expansion of the mammary CD24 þ CD29 high cells in MMTV/N1 IC Tg mice Similar to published data (Shackleton et al., 2006; Stingl et al., 2006), four distinct lineage-negative À (CD31 À CD45 À Ter119 À ) cell sub-populations could be detected Received 19 October 2009; revised 11 March 2010; accepted 20 April 2010; published online 21 June 2010 Correspondence: Dr P Jolicoeur, Laboratory of Molecular Biology, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montre´al, Que´bec, Canada H2W 1R7. E-mail: paul.jolicoeur@ircm.qc.ca Oncogene (2010) 29, 4543–4554 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc