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Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis

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Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis Research Article Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis Chen Chen Jiang, Li Hua Chen, Susan Gillespie, Yu Fang Wang, Kelly A. Kiejda, Xu Dong Zhang, and Peter Hersey Immunology and Oncology Unit, Newcastle Misericordiae Hospital, Newcastle, New South Wales, Australia Abstract lumen and cause so-called ER stress (1–3). The ER responds to the Past studies have shown that activation of mitogen-activated stress conditions by activation of a range of stress response protein kinase/extracellular signal-regulated kinase (ERK) signaling pathways to alter transcriptional and translational kinase (MEK)/ERK is a common cause for resistance of mela- programs, which couples the ER protein folding load with the ER noma cells to death receptor–mediated or mitochondria- protein folding capacity and is termed the unfolded protein mediated apoptosis.We report in this study that inhibition of response (UPR; refs. 1–3). the MEK/ERK pathway also sensitizes melanoma cells to The UPR of mammalian cells is initiated by three ER endoplasmic reticulum (ER) stress-induced apoptosis, and this transmembrane proteins, activating transcription factor 6 (ATF6), is mediated, at least in part, by caspase-4 activation and is inositol-requiring enzyme 1 (IRE1), and double-stranded RNA- associated with inhibition of the ER chaperon glucose- activated protein kinase-like ER kinase (PERK), which act as regulated protein 78 (GRP78) expression.Treatment with proximal sensors of ER stress. Under unstressed conditions, the the ER stress inducer tunicamycin or thapsigargin did not luminal domains of these sensors are occupied by the ER chaperon induce significant apoptosis in the majority of melanoma glucose-regulated protein 78 (GRP78; refs. 1–3). On ER stress, cell lines, but resistance to these agents was reversed by sequestration of GRP78 by unfolded proteins activates these the MEK inhibitor U0126 or MEK1 small interfering RNA sensors by inducing phosphorylation and homodimerization of (siRNA).Induction of apoptosis by ER stress when MEK was IRE1 and PERK and relocalization of ATF6 to the Golgi where it inhibited was caspase dependent with caspase-4, caspase-9, is cleaved by site 1 and site 2proteases (S1P and S2P)leading to and caspase-3 being involved.Caspase-4 seemed to be the its activation as a transcriptional factor (1–3). apical caspase in that caspase-4 activation occurred before The UPR is fundamentally a cytoprotective response. However, activation of caspase-9 and caspase-3 and that inhibition of excessive or prolonged UPR results in apoptotic cell death. Pro- caspase-4 by a specific inhibitor or siRNA blocked activation of cessing of caspase-2, caspase-3, caspase-4, caspase-7, caspase-8, caspase-9 and caspase-3, whereas inhibition of caspase-9 caspase-9, and caspase-12has been observed in ER stress-induced or caspase-3 did not inhibit caspase-4 activation.Moreover, apoptosis (4–7). Among them, caspase-12is thought to be a overexpression of Bcl-2 inhibited activation of caspase-9 and key mediator as caspase-12–deficient mouse cells are partially caspase-3 but had minimal effect on caspase-4 activation. resistant to ER stress-induced apoptosis (8). However, caspase-12 Inhibition of MEK/ERK also resulted in down-regulation of is expressed only in rodents. Its human homologue is silenced by GRP78, which was physically associated with caspase-4, before several mutations during evolution (9). Human caspase-4 has been and after treatment with tunicamycin or thapsigargin.In shown to fulfill the function of caspase-12and plays an important addition, siRNA knockdown of GRP78 increased ER stress- role in ER stress-induced apoptosis of human neuroblastoma and induced caspase-4 activation and apoptosis.Taken together, HeLa cells (7). Moreover, the c-Jun NH2-terminal kinase (JNK), the these results seem to have important implications for new transcription factor CAAT/enhancer binding protein homologous treatment strategies in melanoma by combinations of agents protein (CHOP), and deregulation of Bcl-2and inhibitor of that induce ER stress and inhibitors of the MEK/ERK pathway. apoptosis protein family members have all been suggested to play [Cancer Res 2007;67(20):9750–61] roles in ER stress-induced apoptosis (5, 6, 10–13). There is increasing evidence to show that the UPR is activated Introduction in various solid tumors, perhaps due to nutrient deprivation and Several cellular stress conditions, such as nutrient deprivation, hypoxia [e.g., elevated expression of GRP78 has been reported hypoxia, alterations in glycosylation status, and disturbances of in several cancers, such as breast cancer and prostate cancer calcium flux, lead to accumulation and aggregation of unfolded (14–17)]. Moreover, GRP78 expression has been shown in some and/or misfolded proteins in the endoplasmic reticulum (ER) cases to be associated with tumor development and growth and correlated with resistance to certain forms of chemotherapy. It seems that some cancer cells may have adapted to ER stress by activation of the UPR without resulting in apoptosis (15, 16, 18). Note: Supplementary data for this article are available at Cancer Research Online The central feature of this adaptive response has been suggested (http://cancerres.aacrjournals.org/). to be maintenance of expression of proteins that facilitate Requests for reprints: Peter Hersey or Xu Dong Zhang, Immunology and survival, such as GRP78, without persistence of proapoptotic Oncology Unit, Newcastle Misericordiae Hospital, Room 443, David Maddison Clinical Sciences Building, Corner King and Watt Streets, Newcastle, New South Wales 2300, proteins, such as CHOP (19). In addition, other survival signaling Australia. Phone: 61-2-49-236828; Fax: 61-2-49236184; E-mail: Peter.Hersey@newcastle. pathways, such as the phosphatidylinositol 3-kinase/Akt and edu.au or [email protected]. I2007 American Association for Cancer Research. mitogen-activated protein kinase (MAPK)/extracellular signal- doi:10.1158/0008-5472.CAN-07-2047 regulated kinase (ERK) kinase (MEK)/ERK pathways, may also Cancer Res 2007;67: (20). October 15, 2007 9750 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. MEK Inhibition Sensitizes Melanoma to Apoptosis play roles in counteracting the apoptosis-inducing potential of ER electrophoresis revealed the inactive form as two cleaved fragments and the stress (14, 20). active form as a noncleaved fragment. Previous studies have shown that constitutive activation of the Caspase activity assay. Measurement of caspase activities by fluoro- MEK/ERK pathway is a common cause for resistance of melanoma metric assays was done as described previously (28). The specific substrates z-DEVD-AFC, Ac-LEVD-AFC, and z-LEHD-AFC were used to measure cells to apoptosis mediated by the death receptor and mitochon- caspase-3, caspase-4, and caspase-9 activities, respectively (Calbiochem). drial apoptotic pathways (21–24). In view of this, we examined the The generation of free AFC was determined using FLUOstar OPTIMA potential interaction between the UPR and the MEK/ERK pathway (Labtech) set at an excitation wavelength of 400 nm and an emission in regulation sensitivity of melanoma cells to ER stress-induced wavelength of 505 nm. apoptosis. We show in this report that inhibition of the MEK/ERK Immunoprecipitation. Methods used were as described previously with signaling pathway also sensitizes cultured human melanoma minor modification (29). Briefly, 100 AL of lysates were precleared by cells to ER stress-induced apoptotic cell death. This is mediated, incubation with 20 AL of a mixture of protein A-Sepharose and protein at least in part, by activation of caspase-4 that in turn leads to G-Sepharose packed beads (Santa Cruz Biotechnology) in a rotator at 4jC A j mitochondria-dependent apoptosis. We demonstrate, for the first for 2h and then with 20 L of fresh packed beads in a rotator at 4 C time, that inhibition of MEK/ERK down-regulates GRP78 expres- overnight. Twenty micrograms of anti-GRP78 antibody or control immunoglobulin were then added to the lysate and rotated at 4jCfor sion and blocks its induction by the UPR. This may play a role in 2h. The beads were then pelleted by centrifugation and washed five times activation of caspase-4 and enhancement of sensitivity of the cells with ice-cold lysate buffer before elution of the proteins from the beads in to ER stress-induced apoptosis. lysate buffer at room temperature for 1 h. The resulted immunoprecipitates were then subjected to SDS-PAGE and Western blot analysis. Plasmid vector and transfection. Stable Mel-RM transfectants of Bcl-2 Materials and Methods were established by electroporation of the PEF-puro vector carrying human Cell lines. Human melanoma cell lines Mel-RM, MM200, IgR3, Mel-CV, Bcl-2 provided by Dr. David Vaux (Walter and Eliza Hall Institute, Me4405, Sk-Mel-28, Mel-FH, Sk-Mel-110, and Me1007 have been described Melbourne, Victoria, Australia) and described elsewhere (21, 29). previously (21, 25). They were cultured in DMEM containing 5% FCS Real-time PCR. Total RNA was isolated with SV total RNA isolation (Commonwealth Serum Laboratories). Melanocytes were kindly provided system (Promega). RT-PCR was carried out using Moloney murine leukemia by Dr. P. Parsons (Queensland Institute of Medical Research, Brisbane, virus transcriptase and oligo(dT), and the resulting cDNA products were Queensland, Australia) and cultured in medium supplied by Clonetics. The used as templates for real-time PCR assays. Real-time RT-PCR was done cell line of human embryonic fibroblasts, FLOW 2000, was cultured in using the ABI Prism 7700 sequence detection system (PE Applied DMEM containing 5% FCS as described previously (26). Biosystems). Mixture (25 AL) was used for reaction, which contains 5 AL A A Antibodies, recombinant proteins, and other reagents. Tunicamycin cDNA sample (0.5–1 g/ L) and 9 mmol/L MgCl2. Primers and probes for and thapsigargin were purchased from Sigma Chemical Co.
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