Abstract 1600: Simvastatin targets breast cancer stem-like cells by inhibiting intracellular signaling pathways leading to radiosensitization of inflammatory breast cancer cells.
Authors: Wolfe Adam R. , Lacerda Lara , Larson Richard , Debeb Bisrat G. , Xu Wei , Ueno Naoto T. , Woodward Wendy A. 
Published: August 2014

Abstract
Background: Inflammatory breast cancer (IBC) is responsible for 10% of breast cancer deaths although contributing to a small percentage (∼1%) of total breast cancer cases each year. The hallmarks of IBC are skin involvement and a high propensity to metastasize. It is thought that a small population of stem-like cancer cells is responsible for the metastatic potential and treatment resistance to therapies such as radiation therapy in IBC. In this study we explored the radiosensitization of several breast cancer cell lines in vitro with simvastatin and the mechanism for this.
Methods: In order to determine cell proliferation, breast cancer cell lines SUM149, SUM159 and MCF-7 were grown in 96 well plates with different concentrations of simvastatin for one week. In order to determine the effect of simvastatin in stem-like cancer cells, cells were seeded in serum free, growth factor enriched, anchorage independent, treated with simvastatin for 24 hours and mammosphere formation was quantified a week later. In addition, SUM149, SUM159 and MCF-7 cells were cultured in standard monolayer cultures and in stem cell enriching anchorage independent cultures with simvastatin and treated with increasing concentrations of radiation. Survival curves were generated using Sigmaplot 8.0 and t-test was used to compare surviving fraction (SF) of groups. Finally, cell lysates were obtained from cultures untreated and treated with simvastatin and radiation and analyzed through western blot.
Results: Simvastatin significantly decreased cancer cell proliferation in vitro in all cell lines (p&lt;0.002). Cell lines SUM 159 and MCF 7 grown in mammosphere enriched media showed significant decreased mammosphere formation when grown in the presence of simvastatin (p&lt;0.05). Moreover, simvastatin radiosensitized all cell lines in both monolayer and anchorage independent clonogenic assays. The triple-negative IBC cell line SUM149 had the greatest response to combined treatment regardless of the radiation dose used in monolayer cultures (SF2: 0.417 vs 0.319, SF4: 0.136 vs 0.075, SF6: 0.026 vs 0.018, in control vs treated respectively, all p&lt;0.0001) and in stem cell enriching cultures (SF2: 0.880 vs 0.762, SF4: 0.863 vs 0.492, SF6: 0.920 vs 0.492, in control vs treated respectively, all p&lt;0.0001). The triple-negative non-IBC cell line SUM159 was more sensitive to combined treatment in monolayer cultures (p&lt;0.001) than in stem cell enriching cultures. Combination of simvastatin and radiation therapy decreased protein expression of phospho-rho A, VEGF-A, and phospho-ras.
Conclusions: IBC remains a difficult disease to treat with a high rate of local recurrence after radiation therapy. Statins have been shown to increase tumor free survival in breast cancer. Herein we show simvastatin targets breast cancer stem-like cells resulting in radiosensitizing of IBC in vitro.
Citation Format: Adam R. Wolfe, Lara Lacerda, Richard Larson, Bisrat G. Debeb, Wei Xu, Naoto T. Ueno, Wendy A. Woodward. Simvastatin targets breast cancer stem-like cells by inhibiting intracellular signaling pathways leading to radiosensitization of inflammatory breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1600. doi:10.1158/1538-7445.AM2013-1600