Abstract 3352: Therapeutic targeting of the Notch pathway in breast cancer stem cells

Recent identification of breast cancer stem cells (CSCs) suggest that a subpopulation of tumor cells with a stem cell phenotype may initiate and maintain tumors. Breast CSCs are characterized by their ability to self-renew and generate tumors in serial xenotransplantation assays in mice. The Notch pathway is an evolutionally conserved signaling pathway and Notch activity has been implicated in normal and malignant mammary stem cell maintenance. Therefore, we investigated the role of Notch in breast CSCs by utilizing a number of CSC specific assays and a mouse xenograft model. Upon activation, the Notch intracellular domain (NICD) is cleaved by the gamma secretase complex and translocates to the nucleus. Subsequently, it binds to the transcriptional complex (RBP-jk), to activate target genes such as the HES and HEY family of proteins. The purpose of this study is to test the Notch activity in CSCs from breast cancer cell lines including basal, luminal, and HER2-amplified subtypes. We hypothesize that cells with higher Notch activity may be enriched for cells with stem cell properties. To study the Notch pathway in breast cancer cell lines we used a Notch reporter that contains multiple copies of the Notch response elements upstream of the mCMV promoter that turns on GFP and luciferase expression upon Notch activation. We measured Notch activity by flow cytometry of cells or by bioluminescent imaging of tumors in mice utilizing Notch responsive GFP and luciferase expression, respectively. In addition, we utilized a gamma secretase inhibitor (GSI) which inhibits the cleavage of NICD and nuclear translocation. We treated mice with GSI, docetaxel, or GSI and docetaxel in combination. We have found that basal cell lines such as Sum 159 and MDA-MB-436 have a higher expression of Notch activity compared to the ER+ luminal cell lines MCF7, T47D, and ZR-75-1. We demonstrated that Notch activated cells show a stem cell phenotype with co-localization of expression of the Notch target gene HES1 and the stem cell marker ALDH1. In addition, Notch active cells were able to generate tumors in serial xenotransplantations in mice and to generate both Notch GFP-positive and Notch-GFP negative cells in vitro and in vivo. In contrast, Notch-GFP negative cells neither gave rise to Notch-GFP positive cells nor did they generate tumors in serial xenotransplantation assays. GSI treatment of mice with breast tumors decreased the percentage of Notch-GFP positive cells in the tumor, reducing the cancer stem cell population and decreasing tumorigenesis in serial passages. Furthermore, the combination of docetaxel with GSI further reduced tumor size by targeting the bulk tumor cell populations. These studies strongly suggest that the Notch pathway plays an essential role in breast CSC maintenance and may be an attractive target for more effective treatment. The combination therapy of GSI with cytotoxic chemotherapy is currently being evaluated in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3352. doi:10.1158/1538-7445.AM2011-3352