Blockade of Notch Signaling with Gamma-Secretase Inhibitors Induces Apoptosis in the Stem-Like Population of Breast Cancer Cells.

Abstract

Abstract BACKGROUND: Numerous studies have identified stem-like cells, termed cancer stem cells (CSCs), in breast tumors and established cell lines. These self-renewing CSCs are believed to be responsible for breast cancer formation, progression and recurrence; therefore, a deeper understanding of the signaling pathways regulating their survival will benefit development of novel therapeutic strategies. Notch signaling, which is known to be dysregulated in breast cancer and has been implicated in mammary stem cell self-renewal, can be effectively blocked by gamma-secretase inhibitors (GSIs). While GSIs are currently in clinical trials for breast cancer, it is unclear how these compounds will affect CSCs or if CSCs from different breast cancer phenotypes (estrogen receptor-alpha (ER)-positive, ER negative, Her2/neu overexpressing) will be differentially affected. We previously reported that stem-like cells derived from the T47D breast cancer cell line showed elevated levels of Notch activity and Notch inhibition using GSIs or Notch decoy proteins abolished secondary mammosphere formation.METHODS: We have expanded our studies to breast cancer cell lines of different phenotypes (T47D-A18, T47D-C42, MCF7, SKBR3, BT474, MBA-MD231) and five primary clinical specimens. We studied expression of a panel of Notch pathway genes including Notch receptors, ligands and immediate downstream targets of Notch activation in CSCs and non-CSCs. We also determined the effect of Notch inhibition on secondary mammosphere formation (a measure of self-renewal) and clonogenicity. We evaluated cell death using TUNEL assays.RESULTS: We report that CSCs possess elevated levels of Notch activation compared to non-CSCs regardless of breast cancer phenotype or sample origin (cell culture or primary tumor). However, the specific Notch target genes most abundantly expressed vary depending on the cell line, and may suggest that CSC from different subtypes of breast cancer have different “Notch signatures”. Blockade of Notch signaling with a GSI abolished secondary mammosphere formation, eventually resulting in apoptosis, a finding that was consistent for ER-positive, ER-negative, and Her2/neu overexpressing cancer cells and primary tumor samples from heavily pre-treated, late-stage patients. Furthermore, GSIs blocked colony formation in soft agar.CONCLUSIONS: These novel findings support a role for Notch signaling in CSC self-renewal and proliferation, and they suggest Notch inhibition with agents currently in clinical development may prove beneficial in targeting CSCs in a wide variety of breast cancers. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1154.