Tunicamycin Sensitizes Human Melanoma Cells to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis by Up-regulation of TRAIL-R2 via the Unfolded Protein Response

Abstract

We have reported previously low expression of death receptors for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in fresh isolates and tissue sections of melanoma. This seemed to correlate with relative resistance of freshly isolated melanoma cells to TRAIL-induced apoptosis. We show in this study that the endoplasmic reticulum (ER) stress inducer, tunicamycin, selectively up-regulated the cell surface expression of TRAIL-R2, but not other members of the TNF receptor family, and enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. Tunicamycin-mediated sensitization of melanoma cells to TRAIL-induced apoptosis was associated with increased activation of the caspase cascade and reduction in mitochondrial membrane potential and was inhibited by a recombinant TRAIL-R2/Fc chimeric protein. Up-regulation of TRAIL-R2 on the melanoma cell surface was associated with increased transcription of TRAIL-R2 and its total protein levels. Two signaling pathways of the ER stress-induced unfolded protein response mediated by inositol-requiring transmembrane kinase and endonuclease 1A (IRE1A) and activation of transcription factor 6 (ATF6), respectively, seemed to be involved. In one melanoma line, there was clear evidence of activation of the IRE1A pathway, and small interfering RNA (siRNA) knockdown of IRE1A substantially reduced the upregulation of TRAIL-R2. Similarly, there was evidence for the activation of the ATF6 pathway, and siRNA knockdown of ATF6 had a delayed effect on TRAIL-R2 expression in one but not another melanoma cell line. Moreover, the transcription factor CCAAT/enhancer-binding protein homologous protein seemed to be involved in the up-regulation of TRAIL-R2 by tunicamycin, but its role varied between different melanoma lines. Taken together, our results suggest that agents that induce ER stress may enhance TRAIL-R2 expression and increase the therapeutic response to TRAIL in melanoma.

RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy

“…Upon activation, ERN1 cleaves XBP1 mRNA, producing a splicing form that encodes a transcription factor. 81 Activated ATF6, as a transcription factor, activates downstream target genes of the UPR such as XBP1. 82 The conjunction of the ERN1 and ATF6 arms of the UPR on XBP1 suggests that XBP1 may be responsible for the increase in HSF1 triggered by the UPR.…”

Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein

“…Methods used were as described previously with minor modification (20). Melanoma cells were seeded onto sterile glass coverslips in 24-well plates (Falcon 3047; Becton Dickinson) for 16 to 24 h. Mitotraker Red CMXRos (50 nmol/L; Molecular Probes) was added to the culture medium for 30 min before washing cells with PBS, followed by fixation with 2% paraformaldehyde for 10 min.…”

Novel SMAC-mimetics synergistically stimulate melanoma cell death in combination with TRAIL and Bortezomib

“…Two inducers of endoplasmic reticulum (ER) stress, Thapsigargin and Tunicamycin, have been suggested to sensitise Me1007 to TRAIL by upregulation of TRAIL-R2 Jiang et al, 2007) and caspase-8 (Chen et al, 2008). As Bortezomib can also cause ER stress (Boccadoro et al, 2005), we checked whether it upregulated caspase-8 in Me1007, but no evidence for this was observed, even in response to elevated doses of Bortezomib ( Figure 3B).…”