Pharmacological Inhibition of cFLIP Targets Breast Cancer Stem Cells

Therapeutic targeting of tumour initiating, cancer stem cells (CSCs), offers the potential to improve long-term responses to cancer treatments. The apoptosis related protein Cellular FLICE-Like Inhibitory Protein (cFLIP) has previously been reported to protect breast cancer cells and breast CSCs from the cytotoxic effects of chemotherapy and apoptosis-inducing agents. We recently described the development of a small-molecule protein-protein inhibitor of cFLIP (OH14) that sensitizes refractory breast cancer cells to the death-receptor agonist Tumour Necrosis Factor Alpha Receptor Apoptosis Inducing Ligand (TRAIL). Here we investigated whether the pharmacological inhibition of cFLIP also targeted breast CSCs. Human breast cancer cell lines and primary-derived breast cancer samples were subjected to OH14 with or without TRAIL and assessed for bCSC viability by colony-formation and tumoursphere assay in vitro and xenograft tumour initiation in vivo. OH14 potentiated a reduction in the number of bCSCs after TRAIL treatment, mimicking the sensitizing effects previously observed with epigenetic silencing of cFLIP. Moreover, prolonged inhibition of cFLIP alone, either by shRNA knockdown or treatment with OH14, reduced the bCSC pool, an outcome that was independent of caspases. These data provide proof-of-principle for the use of pharmacological inhibitors of cFLIP to target bCSCs and highlights for the first time both apoptosis-dependent and independent mechanisms for cFLIP-mediated regulation of the breast cancer stem cell pool.