Hyperthermia restores apoptosis induced by death receptors through aggregation-induced c-FLIP cytosolic depletion

TRAIL is involved in immune tumor surveillance and is considered a promising anti-cancer agent owing to its limited side effectson healthy cells. However, some cancer cells display resistance, or become resistant to TRAIL-induced cell death. Hyperthermiacan enhance sensitivity to TRAIL-induced cell death in various resistant cancer cell lines, including lung, breast, colon or prostatecarcinomas. Mild heat shock treatment has been proposed to restore Fas ligand or TRAIL-induced apoptosis through c-FLIPdegradation or the mitochondrial pathway. We demonstrate here that neither the mitochondria nor c-FLIP degradation are requiredfor TRAIL-induced cell death restoration during hyperthermia. Our data provide evidence that insolubilization of c-FLIP, alone, issufficient to enhance apoptosis induced by death receptors. Hyperthermia induced c-FLIP depletion from the cytosolic fraction,without apparent degradation, thereby preventing c-FLIP recruitment to the TRAIL DISC and allowing efficient caspase-8 cleavageand apoptosis. Hyperthermia-induced c-FLIP depletion was independent of c-FLIP DED2 FL chain assembly motif orubiquitination-mediated c-FLIP degradation, as assessed using c-FLIP point mutants on lysine 167 and 195 or threonine 166,a phosphorylation site known to regulate ubiquitination of c-FLIP. Rather, c-FLIP depletion was associated with aggregation,because addition of glycerol not only prevented the loss of c-FLIP from the cytosol but also enabled c-FLIP recruitment within theTRAIL DISC, thus inhibiting TRAIL-induced apoptosis during hyperthermia. Altogether our results demonstrate that c-FLIP is athermosensitive protein whose targeting by hyperthermia allows restoration of apoptosis induced by TNF ligands, includingTRAIL. Our findings suggest that combining TRAIL agonists with whole-body or localized hyperthermia may be an interestingapproach in cancer therapy.