Recent lineage-tracing studies demonstrated that the mammary luminal lineage (including ductal and alveolar luminal cells) is maintained by lineage-restricted luminal stem cells (LuSCs), rather than by multipotent basal mammary stem cells. However, the identity of LuSCs and their role in breast cancer remain elusive. By linage-analysis using Wap-Cre mice coupled with a conditional YFP reporter (R26Y), we found that in virgin females, Wap-Cre marks a luminal mammary epithelial cell (MEC) subpopulation that expresses luminal progenitor (LP) markers (e.g., CD61) and is independent of ovarian hormones for their emergence and maintenance. By microarray analysis, we found these Wap-Cre-marked MECs cluster together with LPs at the molecular level. Importantly, by using a novel pulse-chase lineage-tracing approach based on intraductal injection of Wap-Cre adenovirus (Ad-Wap-Cre), we found that Ad-Wap-Cre-marked nulliparous MECs (CD61+) clonally gave rise to CD61- differentiating alveolar luminal cells during pregnancy/lactation and could maintain themselves long-term as CD61+ MECs after pregnancy in parous females. These data suggest that Wap-Cre marks a unipotent LuSC population committed to the alveolar fate [i.e., alveolar stem cells (ASCs)]. To test whether Wap-Cre-marked ASCs can serve as cells of origin of breast cancer, we performed lineage-tracing in breast cancer mouse models (coupled with R26Y reporter) by intraductal injection of Ad-Wap-Cre. In one model based on Cre-mediated activation of an ETV6-NTRK3 (EN) fusion oncogene, Ad-Wap-Cre injection led to development of mammary tumors (with basal differentiation) with full penetrance. By analyzing YFP-marked premalignant MECs in this model, we found that although normally Wap-Cre-marked ASCs only differentiate to alveolar luminal cells (upon pregnancy), when transformed by EN, they give rise to both luminal and basal cells (independent from pregnancy). In another model (MMTV-PyMT) that only develops luminal tumors, upon Ad-Wap-Cre injection at a young age (to mark its cells of origin), we observed clonally derived YFP+ premalignant cells and tumor cells 1-3 weeks later and these YFP+ cells were exclusively luminal cells. It was shown recently that mammary tumors developed in Wap-Cre;EN and MMTV-PyMT mice resemble human HER2-Enriched and Luminal B breast cancers at the molecular level, respectively (Genome Biol. 14:R125); our data thus directly support that a common unipotent ASC population can give rise to distinct subtypes of breast cancer, when transformed by different oncogenic events. Overall, our study provides definitive evidence for the existence of an alveolar-restricted LuSC population in the virgin mammary gland that can serve as cells of origin of heterogeneous mammary tumors, and should have important implications for understanding cells of origin and heterogeneity of breast cancer.

Citation Format: Maaike van Bragt, Luwei Tao, Zhe Li. An alveolar-restricted stem cell population in the mammary luminal lineage revealed by lineage tracing serves as cells of origin of heterogeneous mammary tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3033. doi:10.1158/1538-7445.AM2014-3033