Cytoplasmic levels of cFLIP determine a broad susceptibility of breast cancer stem/progenitor-like cells to TRAIL

Abstract

BackgroundThe clinical application of TRAIL receptor agonists as a novel cancer therapy has been tempered by heterogeneity in tumour responses. This is illustrated in breast cancer, where TRAIL is cytotoxic in cell lines of mesenchymal origin but refractory in lines with an epithelial-like phenotype. However, it is now evident that intra-tumour heterogeneity includes a minority subpopulation of tumour-initiating stem/progenitor-like cells (CSCs) that possess mesenchymal characteristics. We hypothesised therefore that TRAIL may target these phenotypically distinct CSC-like cells that are common to most - if not all - breast cancers, thus impacting on the source of malignancy in a much broader range of breast tumour subtypes than previously envisaged.MethodsWe used colony formation, tumoursphere, flow cytometry and xenograft tumour initiation assays to observe the TRAIL sensitivity of CSC-like cells in a panel of two mesenchymal-like (TRAIL-sensitive) and four epithelial-like (TRAIL-resistant) breast cancer cell lines. Subcellular levels of the endogenous TRAIL inhibitor, cFLIP, were determined by western blot and immunofluorescence microscopy. The effect of the subcellular redistribution of cFLIP on TRAIL sensitivity and Wnt signalling was determined using cFLIP localisation mutants and the TOPFlash reporter assay respectively.ResultsTRAIL universally suppressed the clonal expansion of stem/progenitors in all six of the breast cancer cell lines tested, irrespective of their phenotype or overall sensitivity to TRAIL. A concomitant reduction in tumour initiation was confirmed in the TRAIL-resistant epithelial cell line, MCF-7, following serial dilution xenotransplantation. Furthermore TRAIL sensitivity of breast CSCs was inversely proportional to the relative cytoplasmic levels of cFLIP while overexpression of cFLIP in the cytosol using subcellular localization mutants of cFLIP protected these cells from cytotoxicity. The accumulation of nuclear cFLIP on the other hand did not influence TRAIL cytotoxicity but instead promoted Wnt-dependent signalling.ConclusionThese data propose a novel role for TRAIL as a selective CSC agent with a broad specificity for both epithelial and mesenchymal breast tumour subtypes. Furthermore we identify a dual role for cFLIP in the maintenance of breast CSC viability, dependent upon its subcellular distribution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0478-y) contains supplementary material, which is available to authorized users.

The regulatory protein GADD34 inhibits TRAIL-induced apoptosis via TRAF6/ERK-dependent stabilization of myeloid cell leukemia 1 in liver cancer cells

“…In addition, c-FLIP (cellular FLICE (FADD-like interleukin-1␤converting enzyme)inhibitory protein) is involved in the resistance of cancer cells to TRAIL-induced cell death (28,29). Levels of c-FLIP may affect the sensitivity of cancer cells to TRAIL (30). In the current study, we show that GADD34 prevents TRAIL-induced apoptosis through TRAF6-and ERK-mediated stabilization of the BCL-2 family member MCL-1.…”

TRAIL-induced variation of cell signaling states provides nonheritable resistance to apoptosis

“…In addition to survival pathway activation, fractional killing by TRAIL has also been explained by variation in pro and anti-apoptotic protein abundance in tumor cells because of genetic aberrations or nongenetic mechanisms (Zhang & Fang, 2005). Studies characterized nongenetic mechanisms of variation in levels of anti-apoptotic protein cellular FLICE (c-FLIP), which binds to FADD and prevents formation of DISC, in spatial organization of DR4/5 and in rate and duration of initiator caspase activation by DISC (French et al, 2015; Marconi et al, 2013; Roux et al, 2015; Twomey et al, 2015). Recent work indicates that multiple mechanisms might be at play simultaneously to result in intercellular variation and, therefore, cell states of resistance (Ramirez et al, 2016; Salgia & Kulkarni, 2018).…”

Engineered human mesenchymal stem cells for neuroblastoma therapeutics

“…Resistance to TRAIL killing may have different causes, including high expression of decoy receptors and downregulation or upregulation of apoptotic proteins. Expression of the intracellular apoptotic inhibitor c-FLIP can confer TRAIL resistance in different types of cancer cell lines and it may be involved in the protection of neuroblastoma cells from the cytotoxic effect of TRAIL (52). Deficient expression of caspases, in particular caspase-8, essential with FADD to form the death receptor complex DISC, may contribute to TRAIL resistance (53).…”