Past studies have shown that activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK is a common cause for resistance of melanoma cells to death receptor-mediated or mitochondriamediated apoptosis. We report in this study that inhibition of the MEK/ERK pathway also sensitizes melanoma cells to endoplasmic reticulum (ER) stress-induced apoptosis, and this is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperon glucoseregulated protein 78 (GRP78) expression. Treatment with the ER stress inducer tunicamycin or thapsigargin did not induce significant apoptosis in the majority of melanoma cell lines, but resistance to these agents was reversed by the MEK inhibitor U0126 or MEK1 small interfering RNA (siRNA). Induction of apoptosis by ER stress when MEK was inhibited was caspase dependent with caspase-4, caspase-9, and caspase-3 being involved. Caspase-4 seemed to be the apical caspase in that caspase-4 activation occurred before activation of caspase-9 and caspase-3 and that inhibition of caspase-4 by a specific inhibitor or siRNA blocked activation of caspase-9 and caspase-3, whereas inhibition of caspase-9 or caspase-3 did not inhibit caspase-4 activation. Moreover, overexpression of Bcl-2 inhibited activation of caspase-9 and caspase-3 but had minimal effect on caspase-4 activation. Inhibition of MEK/ERK also resulted in down-regulation of GRP78, which was physically associated with caspase-4, before and after treatment with tunicamycin or thapsigargin. In addition, siRNA knockdown of GRP78 increased ER stressinduced caspase-4 activation and apoptosis. Taken together, these results seem to have important implications for new treatment strategies in melanoma by combinations of agents that induce ER stress and inhibitors of the MEK/ERK pathway.

Purpose: Given that inhibitors of mitogen-activated protein (MAP)/extracellular signalregulated kinase (ERK) kinase (MEK) are being introduced into treatment for melanoma, the present study was carried out to better understand the mechanism by which they may induce apoptosis of melanoma cells. Experimental Design: A panel of human melanoma cell lines and fresh melanoma isolates was assessed for their sensitivity to apoptosis induced by the MEK inhibitor U0126. The apoptotic pathways and regulatory mechanisms involved were examined by use of the inhibitor and small interfering RNA (siRNA) techniques. Results: Inhibition of MEK induced apoptosis in the majority of melanoma cell lines through a mitochondrial pathway that was associated with the activation of Bax and Bak, release of mitochondrial apoptogenic proteins, and activation of caspase-3. However, apoptosis was independent of caspases and instead was associated with mitochondrial release of AIF as shown by the inhibition of apoptosis when AIF was knocked down by siRNA. Inhibition of MEK resulted in the up-regulation of the BH3-only proteins PUMA and Bim and down-regulation of the antiapoptotic protein Mcl-1. These changes were critical for the induction of apoptosis by U0126 as siRNA knockdown of PUMA or Bim inhibited apoptosis, whereas siRNA knockdown of Mcl-1 increased apoptosis particularly in the apoptosis-resistant cell lines. Conclusions: Apoptosis of melanoma cells induced by the inhibition of the MEK/ERK pathway is mediated by the up-regulation/activation of PUMA and Bim and down-regulation of Mcl-1. Release of AIF rather than the activation of caspases seems to be the mediator of apoptosis. Our results suggest that cotargeting Mcl-1 and the MEK/ERK pathway may further improve treatment results in melanoma.

Taxanes have a broad spectrum of activity against various human cancers, including melanoma. In this study, we have examined the molecular mechanism of docetaxelinduced apoptosis of human melanoma. We report that docetaxel induced varying degrees of apoptosis in a panel of melanoma cell lines but not in normal fibroblasts.

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.

We have previously reported that sensitivity of melanoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is largely correlated with the levels of expression of TRAIL death receptors, in particular, TRAIL-R2 on the cell surface. However, fresh melanoma isolates and melanoma tissue sections express, in general, low levels of death receptors for TRAIL. We show in this study that the endoplasmic reticulum stress inducer, thapsigargin (TG), selectively up-regulated TRAIL-R2 and enhanced TRAIL-induced apoptosis in melanoma cells. However, the TRAIL-R2 pathway did not appear to be involved in induction of apoptosis by TG alone. Up-regulation of TRAIL-R2 appeared to be cooperatively mediated by the inositol-requiring transmembrane kinase and endonuclease 1alpha (IRE1alpha)- and activation of transcription factor (ATF)-6-signaling pathways of the unfolded protein response (UPR) and the transcription factor CCAAT/enhancer-binding protein-homologous protein (CHOP). The latter played a critical role in the initial phase of the increase in TRAIL-R2 as small interfering RNA (siRNA) knockdown of CHOP blocked up-regulation of TRAIL-R2 only at a relatively early stage (16 h) after exposure to TG. In contrast, IRE1alpha and ATF6 appeared to be crucial in maintaining the increased levels of TRAIL-R2 in that siRNA knockdown of IRE1alpha or ATF6 had no effect on the increase in TRAIL-R2 at the initial phase, but blocked TRAIL-R2 up-regulation at a relatively late stage (36 h). Our results indicate that modulation of the UPR may be useful in sensitizing melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-R2.