Cancer Biology and Signal Transduction NotchReporterActivity inBreastCancerCell Lines Identifies a Subset of Cells with Stem Cell Activity

Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor develop-ment. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notchþ) or reduced activity (Notch) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplan-tation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expres-sion of breast CSC markers. Interestingly Notchþ cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notchþ cells demonstrated tumor initiation capacity at serial dilutions inmouse xenografts, whereas Notch cells failed to generate tumors. g-Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effec-tively targets these Notchþ cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics. Mol Cancer Ther; 1–9. 2015 AACR