Notch Signaling Is Important in the Survival, Proliferation, and Self-Renewal of the Putative Breast Cancer Stem Cell Population

Numerous studies have identified stem-like cells, termed cancer stem cells (CSCs), in breast tumors and established cell lines. It has been hypothesized that CSCs are responsible for breast cancer formation, progression and recurrence; therefore, a deeper understanding of the signaling pathways regulating CSC survival will benefit development of novel therapeutic strategies. Notch signaling, which is dysregulated in breast cancer and has been implicated in mammary stem cell self-renewal, and can be effectively blocked by gamma-secretase inhibitors (GSIs). While GSIs are currently in clinical trials for breast cancer, it is not fully understood how these compounds will affect CSCs or if CSCs from different breast cancer phenotypes will be differentially affected. The stem cell-like population was isolated using several different methods including side population, mammosphere formation and aldehyde dehydrogenase activity. Notch pathway activity was analyzed in CSCs derived from cell lines and primary tumors of different breast cancer phenotypes using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and confirmed by Western blotting. The effect of Notch inhibition on secondary mammosphere formation and colony formation in soft agar was evaluated. Furthermore, apoptosis was confirmed after treatment with one GSI, MRK003, by TUNEL assay and Annexin V - propidum iodide staining. The mechanism of apoptosis was explored utilizing a RT-qPCR based approach as well as several kinase inhibitors including those that inhibit c-Jun N-terminal kinase and the p38 mitogen activated kinase. The CSCs possessed elevated levels of Notch activation compared to the non-CSCs regardless of breast cancer phenotype, sample origin (cell culture or primary tumor) or method of isolation / enrichment. Blockade of Notch signaling with three structurally distinct GSIs, a specific Notch decoy protein or an siRNA targeting Notch-1 abolished secondary mammosphere formation and/or blocked colony formation in soft agar and decreased the side-population. These findings support a role for Notch signaling in CSC self-renewal and proliferation, and they suggest Notch inhibition may have clinical benefits in targeting CSCs. Furthermore, our data shows that Notch may be important in the survival of mammospheres and the c-Jun N terminal kinase and/or p38 MAPK pathway may play a role in MRK003- induced apoptosis