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Strong evidences support the inhibitory activity of cellular FLICE-inhibitory protein (FLIP) in the apoptotic signalling by death receptors in tumor cells. However, little is known about the role of FLIP in the regulation of apoptosis in non-transformed cells. In this report, we demonstrate that FLIPL plays an important role as a survival protein in non-transformed breast epithelial cells. Silencing of FLIPL by siRNA methodology enhances TRAIL-R2 expression and activates a caspase-dependent cell death process in breast epithelial cells. This cell death requires the expression of TRAIL, TRAIL-R2, FADD and procaspase-8 proteins. A mitochondria-operated apoptotic pathway is partially required for FLIPL siRNA-induced apoptosis. Interestingly, FLIPL silencing markedly abrogates formation of acinus-like structures in a three-dimensional basement membrane culture model (3D) of the human mammary MCF-10A cell line through a caspase-8 dependent process. Furthermore, over-expression of FLIPL in MCF-10A cells delayed lumen formation in 3D cultures. Our results highlight the central role of FLIP in maintaining breast epithelial cell viability and suggest that the mechanisms regulating FLIP levels should be finely controlled to prevent unwanted cell demise., This work was supported by grants from Ministerio de Educación y Ciencia (SAF2006-00633 and SAF2009-07163), Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD06/0020/0068) and Junta de Andalucía (CTS-211 and CVI-4497) to AL-R. RY and CP were supported by contracts from Instituto de Salud Carlos III and Junta de Andalucía, respectively.