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

A Morlé,O Micheau,C Garrido

doi:10.1038/cddis.2015.12

A Morlé, O Micheau + Show 1 more

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https://doi.org/10.1038/cddis.2015.12

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Abstract

TRAIL is involved in immune tumor surveillance and is considered a promising anti-cancer agent owing to its limited side effects on healthy cells. However, some cancer cells display resistance, or become resistant to TRAIL-induced cell death. Hyperthermia can enhance sensitivity to TRAIL-induced cell death in various resistant cancer cell lines, including lung, breast, colon or prostate carcinomas. Mild heat shock treatment has been proposed to restore Fas ligand or TRAIL-induced apoptosis through c-FLIP degradation or the mitochondrial pathway. We demonstrate here that neither the mitochondria nor c-FLIP degradation are required for TRAIL-induced cell death restoration during hyperthermia. Our data provide evidence that insolubilization of c-FLIP, alone, is sufficient 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 cleavage and apoptosis. Hyperthermia-induced c-FLIP depletion was independent of c-FLIP DED2 FL chain assembly motif or ubiquitination-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 the TRAIL DISC, thus inhibiting TRAIL-induced apoptosis during hyperthermia. Altogether our results demonstrate that c-FLIP is a thermosensitive protein whose targeting by hyperthermia allows restoration of apoptosis induced by TNF ligands, including TRAIL. Our findings suggest that combining TRAIL agonists with whole-body or localized hyperthermia may be an interesting approach in cancer therapy.

Highlights

Targeting c-FLIP has clearly emerged as an important issue for cancer therapies
Regardless of the isoform, c-FLIP proteins are co-recruited within the DISC of death domain (DD)-containing receptors of the TNF superfamily and prevent the release of active caspase-8 to the cytosol, inhibiting apoptosis induced by these death receptors.[8,9]
Hyperthermia restores TRAIL-induced apoptosis in tumor cells[17,23,24] but not in normal cells.[25]. In line with these findings, incubating resistant cancer cell lines of various origin for 1 h at 42 °C (HS) in the presence of TRAIL followed by subsequent incubation at 37 °C for 5 h (Figure 1a), significantly increased apoptosis triggered by TRAIL as compared with a 6 h incubation time at 37 °C (Figure 1b)

Summary

Introduction

Targeting c-FLIP has clearly emerged as an important issue for cancer therapies. So far, three isoforms have been described. C-FLIP isoforms are repressed by transcription factors including E2F1 or c-Myc,[10,11] or induced by NF-kB.[12,13]. Regulation of c-FLIP expression by NF-kB plays a central role in protecting cells from TNF-induced cell death.[14] This pro-inflammatory signaling pathway contributes to sustained expression of c-FLIP in primary tumors and confers resistance to apoptosis induced by death receptors.[15,16] At the posttranslational level, c-FLIP proteins are regulated through the ubiquitin-proteasomal pathway. 166, leading to c-FLIP ubiquitination on lysine 167 and degradation by the proteasome.[18] Several ubiquitin ligases contribute to c-FLIP ubiquitination including itch, c-Cbl and AIP4.20–22 Consistent with the increasing body of evidence

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