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

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 1 alpha (IRE1 alpha) and activation of transcription factor 6 (ATF6), respectively, seemed to be involved. In one melanoma line, there was clear evidence of activation of the IRE1 alpha pathway, and small interfering RNA (siRNA) knockdown of IRE1 alpha substantially reduced the up-regulation 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-112 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-112 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.