Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL

Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation.Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10% to 30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment.We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes.Recognition that breast cancer is a heterogeneous disease has helped shape advances in therapy, leading to more targeted therapeutic strategies and improved survival rates in discrete disease subgroups [1]. This is exemplified by the advent of therapeutic agents targeting estrogen-receptor positive (ER+) and HER2-positive (HER2+) breast cancers, which make up approximately 70% of all breast tumours [2,3]. Despite