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

Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of

Melanoma Cells by the ARC Protein

Li Hua Chen, Chen Chen Jiang, Ralph Watts, Rick F. Thorne, Kelly A. Kiejda,Xu Dong Zhang, and Peter Hersey

Immunology and Oncology Unit, Calvary Mater Newcastle Hospital, Newcastle, New South Wales, Australia

Abstract

We have shown previously that most melanoma cell linesare insensitive to endoplasmic reticulum (ER) stress–induced apoptosis, but resistance can be reversed throughactivation of caspase-4 by inhibition of the MEK/ERKpathway. We report in this study that apoptosis was inducedby the ER stress inducer thapsigargin or tunicamycin via acaspase-8–mediated pathway in the melanoma cell lineMe1007, although the MEK/ERK pathway was activated inthis cell line. The high sensitivity of Me1007 to ER stress–induced apoptosis was associated with low expression levelsof the apoptosis repressor with caspase recruitmentdomain (ARC) protein that was expressed at relatively highlevels in the resistant melanoma cell lines. Transfection ofcDNA encoding ARC into Me1007 cells inhibited bothcaspase-8 activation and apoptosis induced by thapsigarginor tunicamycin. In contrast, inhibition of ARC by smallinterfering RNA knockdown sensitized the resistant melano-ma cell lines to ER stress–induced apoptosis, which wasinhibitable by blockage of caspase-8 activation. Bothexogenous and endogenous ARC seemed to predominantlylocate to the cytoplasm and mitochondria and could becoimmunoprecipitated with caspase-8. Taken together, ERstress can potentially activate multiple apoptosis signalingpathways in melanoma cells in a context-dependent manner.Whereas the MEK/ERK signaling pathway plays an impor-tant role in inhibiting ER stress–induced caspase-4 activa-tion, ARC seems to be critical in blocking activation ofcasapse-8 in melanoma cells subjected to ER stress. [CancerRes 2008;68(3):834–42]

Introduction

A number of cellular stress conditions, such as nutrientdeprivation, hypoxia, alterations in glycosylation status, anddisturbances of calcium flux, lead to accumulation and aggregationof unfolded and/or misfolded proteins in the endoplasmicreticulum (ER) lumen and cause so-called ER stress (1–3). TheER responds to the stress conditions by activation of a range ofstress-response signaling pathways to alter transcriptional andtranslational programs, which couples the ER protein folding load

with the ER protein folding capacity and is termed the unfoldedprotein response (1–3).The unfolded protein response is fundamentally a cytopro-

tective response. However, excessive or prolonged unfoldedprotein response results in apoptotic cell death. Althoughcaspase-12 in rodents and its human homologue caspase-4 arethought to be key mediators (4–6), activation of other caspases,including caspase-2, caspase-3, caspase-7, caspase-8, and caspase-9, has been reported to be involved in ER stress–inducedapoptosis (4, 7–9). Several recent studies have challenged therole of caspase-12 and caspase-4 as ER stress was found toinduce similar degrees of apoptosis regardless of the presence orabsence of these caspases (10, 11). Nevertheless, we have shownthat ER stress induces apoptosis in human melanoma cell linesby activation of caspase-4 when the MEK/ERK signaling pathwayis inhibited (12). The majority of melanoma cell lines areotherwise relatively resistant to ER stress–induced apoptosis,except that one line, Me1007, seems to be exceptionally sensitiveto apoptosis induced by ER stress inducers although the MEK/ERK pathway is also constitutively activated in the cells (12).This suggests that, besides activation of caspase-4, ER stress canpotentially initiate other apoptotic mechanism(s), which is lesssensitive to the inhibitory effect of the MEK/ERK pathway, butnevertheless blocked in most melanoma cell lines.Apoptosis repressor with caspase recruitment domain (ARC) is

an endogenous inhibitor of apoptosis that was initially thought tobe primarily expressed in terminally differentiated cells, such ascardiac and skeletal myocytes and neurons (13). ARC seems to bedistinct from most endogenous apoptosis inhibitors in that itcannot only inhibit death receptor–mediated apoptotic signalingby binding to and inhibiting caspase-8 but also block themitochondrial apoptotic pathway by physically interacting withBax and inhibiting its activation (13–15). Moreover, ARC can alsobind to FADD, Fas, and caspase-2, which protects cells fromapoptosis induced by varying stimuli (13, 14). Recently, ARC wasreported to be expressed by various human cancer cells and to belargely located in nuclei (16, 17). However, the functionalsignificance of endogenous ARC in cancer cells remains to be fullyelucidated.In the present report, we examined the mechanisms by which

ER stress induces apoptosis in the melanoma cell line Me1007and explored the potential role of ARC in protection of themajority of melanoma lines from ER stress–induced apoptosis.We show that the ER stress inducer thapsigargin or tunicamycininduced apoptosis of Me1007 cells by activation of caspase-8,which was inhibitable by overexpression of ARC. In addition,relatively high levels of endogenous ARC expression seemed toplay a role in inhibition of caspase-8 activation and apoptosisinduced by ER stress in most melanoma cell lines. In contrast toprevious reports, both endogenous and exogenous ARC in

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).Requests for reprints: Peter Hersey or Xu Dong Zhang, Room 443, David

Maddison Clinical Sciences Building, corner King and Watt Streets, Newcastle, NewSouth Wales 2300, Australia. Phone: 61-2-49-236828; Fax: 61-2-49236184; E-mail:[email protected] or [email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-07-5056

Cancer Res 2008; 68: (3). February 1, 2008 834 www.aacrjournals.org

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melanoma was predominantly located in the cytoplasm andmitochondria.

Materials and Methods

Cell lines and fresh melanoma isolates. Human melanoma cell linesMel-RM, MM200, IgR3, Mel-CV, Me4405, Sk-Mel-28, Mel-FH, and Me1007

have been described previously (12, 18). They were cultured in DMEM

containing 5% FCS (Commonwealth Serum Laboratories). Melanocytes

were kindly provided by Dr. P. Parsons (Queensland Institute of MedicalResearch) and cultured in medium supplied by Clonetics (Edward Kellar).

Antibodies, recombinant proteins, and other reagents. Tunicamycinand thapsigargin were purchased from Sigma Chemical Co. They weredissolved in DMSO and made up in stock solutions of 1 mmol/L,

respectively. The cell-permeable general caspase inhibitor Z-Val-Ala-

Asp(OMe)-CH2F (z-VAD-fmk), the caspase-3 specific inhibitor Z-Asp(OMe)-

Glu(OMe)-Val-Asp(OMe)-CH2F (z-DEVD-fmk), the caspase-9 specific inhi-bitor Z-Leu-Glu(Ome)-His-Asp(Ome)-CH2F (z-LEHD-fmk), the caspase-8

specific inhibitor Z-lle-Glu(Ome)-Thr-Asp(Ome)-CH2F (z-IETD-fmk), and

the caspase-2 specific inhibitor Z-Val-Ala-Asp(OMe)-Val-Ala-Asp(OMe)-

CH2F (z-VDVAD-fmk) were purchased from Calbiochem. The caspase-4specific inhibitor Z-Leu-Glu-Val-Asp-FMK (z-LEVD-fmk) was from BioVi-

sion. The rabbit polyclonal antibodies against caspase-3, caspase-8, caspase-

2, and caspase-9 were from Stressgen. The mouse monoclonal antibody

(mAb) against caspase-4 was from Abcam. The rabbit mAb against GRP78was purchased from Santa Cruz Biotechnology. The rabbit polyclonal

antibody against ARC was from Cayman. The mouse mAb against Flag M2

was purchased from Sigma. Isotype control antibodies used were the ID4.5(mouse IgG2a) mAb against Salmonella typhi supplied by

Dr. L. Ashman (Institute for Medical and Veterinary Science), the 107.3

Figure 1. ER stress–induced apoptosis of Me1007 cells is caspase-dependent. A, left, thapsigargin (TG ) or tunicamycin (TM) induces the ER stress response.Whole-cell lysates from Me1007 and Mel-RM cells treated with thapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for indicated periods were subjected to Westernblot analysis of GRP78 expression. The data shown are representative of three individual experiments. Right, kinetics of thapsigargin-induced or tunicamycin-inducedapoptosis of Me1007 cells. Me1007 and Mel-RM cells treated as above were subjected to measurement of apoptosis by the propidium iodide method using flowcytometry. Points, mean of three individual experiments; bars, SE. B, Me1007 cells were treated with the general caspase inhibitor z-VAD-fmk (20 Amol/L), thecaspase-2 inhibitior z-VDVAD-fmk (50 Amol/L), the caspase-3 inhibitor z-DEVD-fmk (20 Amol/L), the caspase-4 inhibitor z-LEVD-fmk (30 Amol/L), the caspase-8 inhibitorz-IETD-fmk (20 Amol/L), or the caspase-9 inhibitor z-LEHD-fmk (20 Amol/L) for 1 h before adding thapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for a further 36 h.Apoptosis was measured by the propidium iodide method using flow cytometry. Columns, mean of three individual experiments; bars, SE. C, whole-cell lysates fromMe1007 cells treated with thapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for indicated periods were subjected to Western blot analysis of caspase-3, caspase-4,caspase-8, and caspase-9. The lower parts of the caspase-8 and caspase-3 graphs were obtained from the same membranes with longer exposure, respectively, tobetter visualize the cleaved forms of caspase-8 and caspase-3. The arrow-pointed bands in figures for caspase-4 and caspase-3 are presumably either nonspecificbands or intermediately cleaved capsase-4 and caspase-3, respectively. The data shown are representative of three individual experiments.

ARC Protects Melanoma from ER Stress–Induced Apoptosis

www.aacrjournals.org 835 Cancer Res 2008; 68: (3). February 1, 2008



mouse IgG1 mAb purchased from PharMingen, and rabbit IgG from SigmaChemical Co.

Apoptosis. Quantitation of apoptotic cells by measurement of sub-G1DNA content using the propidium iodide method was carried out as

described elsewhere (12, 18). 4¶,6-Diamidino-2-phenylindole (DAPI) stainingwas performed according to the manufacture’s instructions (Molecular

Probes) as described elsewhere (18, 19).Confocal microscopy. 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 theculture medium for 30 min before washing cells with PBS, followed by

fixation with 2% paraformaldehyde for 10 min. Cells were then

permeabilized in 0.05% saponin diluted in PBS containing 10% human

antibody serum, followed by incubation with primary antibodies, whichwere then detected with Alexa Fluor 488–conjugated antimouse or

antirabbit immunoglobulin. Coverslips were mounted with SlowFade Gold

medium (Invitrogen), and confocal images were acquired using a Zeiss LSM510 scanner fitted to an Axiovert 100 M microscope. Dual-color analysis was

performed with appropriate single-color controls with individual channels

recorded sequentially. Instrument settings were adjusted to obtain minimal

saturated pixels, and the settings were kept constant on wheresemiquantitative comparisons were performed.

Caspase activity assay. Measurement of caspase activities by fluoro-

metric assays was performed as described previously (12). The specific

substrates z-DEVD-AFC, Ac-LEVD-AFC, and z-LEHD-AFC were used tomeasure caspase-3, caspase-4, and caspase-8 activities, respectively

(Calbiochem). The generation of free AFC was determined using Fluostar

OPTIMA (LABTECH) set at an excitation wavelength of 400 nm and anemission wavelength of 505 nm.

Western blot analysis. Western blot analysis was carried out as

described previously (12, 18). Labeled bands were detected by Immun-Star

horseradish peroxidase chemiluminescent kit, and images were capturedand the intensity of the bands was quantitated with the Bio-Rad VersaDoc

image system (Bio-Rad).

Immunoprecipitation. Methods used were as described previously withminor modification (12). Briefly, 100 AL of lysates were precleared byincubation with 20 AL of a mixture of protein A and protein G Sepharose

packed beads (Santa Cruz) in a rotator at 4jC for 2 h and then with 20 AL offresh packed beads overnight. Twenty micrograms of anti-Flag M2 antibody,anticaspase-8 antibody, or control immunoglobulin was then added to the

lysates and rotated at 4jC for 2 h. The beads were then pelleted by

centrifugation and washed five times with ice-cold lysate buffer before

elution of the proteins from the beads in lysate buffer at room temperaturefor 1 h. The resulted immunoprecipitates were then subjected to SDS-PAGE

and Western blot analysis.

Reverse transcription–PCR. Total RNA was extracted from cells by

using SV total RNA isolation system (Promega). One microgram of

RNA was subjected to reverse transcription. PCR was carried out in a

two-step protocol using Moloney murine leukemia virus transcriptase

(Invitrogen) and Taq DNA polymerase (Promega) according to the

manufacturers’ instruction. The primer sequences for caspase-8 are

forward, 5-TGCCCTCAAGTTCCTGTGCTTGGA-3; reverse, 5-GGATG-

CTAAGAATG-TCATCTCC-3. Twenty-five microliters of mixture were used

for reaction, which contains 100 ng cDNA sample, 3 Amol/L MgCl2,

400 nmol/L deoxynucleotide triphosphate, 400 nmol/L primer mix, and

5 units/AL Taq DNA polymerase for 35 cycles with the annealing

temperature of 60jC.Plasmid vector and transfection. The pcDNA3-ARC-Flag was kindly

provided by Dr G. Nunez (University of Michigan Medical School) and was

described previously (13). Melanoma cells were seeded at 1 � 104 cells per

well in 24-well plates 24 h before being transfected with 0.8 Ag ARC plasmidin Opti-MEM medium (Invitrogen) with 5% FCS using Lipofectamine

reagent (Invitrogen) according to the manufacturer’s transfection protocol.

After transfection for 6 h, cells were switched to fresh medium for another

24 h. Cells were then passaged at 1:10 into fresh medium for further24 h followed by selection G418 (Invitrogen).

Small RNA interference. Melanoma cells were seeded at 3.5 � 104 cellsper well in 24-well plates and allowed to reachf50% confluence on the day of

transfection. The small interfering RNA (siRNA) constructs used were

obtained as the siGENOME SMARTpool reagents (Dharmacon), the

siGENOME SMARTpool ARC (D-015682-00), and the siGENOME SMARTpoolcaspase-8 (M-003466-01-04). The nontargeting siRNA control, SiConTRol-

Nontargeting siRNA pool (D-001206-13-20), was also obtained from

Dharmacon. Cells were transfected with 50 to 100 nmol/L siRNA in Opti-

MEM medium (Invitrogen) with 5% FCS using Oligofectamine reagent(Invitrogen) according to themanufacturer’s transfection protocol. Efficiency

of siRNA was measured by Western blot analysis 24 h after transfection.

Results

ER stress–induced apoptosis in the melanoma cell lineMe1007 is caspase dependent. Our previous studies have shownthat, in contrast to most melanoma cell lines that are resistant to ERstress–induced apoptosis, Me1007 is highly sensitive to apoptosisinduced by the ER stress inducers thapsigargin and tunicamycin(12). To further study the susceptibility of this melanoma line toapoptosis induced by ER stress, we treated the cells withthapsigargin at 1 Amol/L or tunicamycin at 3 Amol/L for varyingperiods. The resistant melanoma line Mel-RMwas used as a control.Figure 1A shows that whereas thapsigargin and tunicamycininduced rapid up-regulation of the ER chaperon GRP78 in both celllines, indicative of activation of the ER stress response, they onlyinduced significant apoptosis in Me1007 cells, which could beobserved by 6 h and peaked at 36 h after treatment. This difference insensitivities between the two cell lines was further evidenced inassays with DAPI staining (Supplementary Fig. S1).We examined involvement of caspases in ER stress–induced

apoptosis in Me1007 cells by treating the cells with the generalcaspase inhibitor z-VAD-fmk and specific inhibitors z-VDVAD-fmk against caspase-2, z-DEVD-fmk against caspase-3, z-LEVD-fmk against caspase-4, z-IETD-fmk against caspase-8, andz-LEHD-fmk against caspase-9, 1 h before the addition thapsi-gargin or tunicamycin. As shown in Fig. 1B , z-VAD-fmk and theinhibitors against caspase-8, caspase-4, caspase-9, or caspase-3inhibited thapsigargin-induced or tunicamycin-induced apoptosisto varying degrees. In contrast, the inhibitor against caspase-2exhibited only minimal inhibitory effects on thapsigargin-inducedor tunicamycin-induced apoptosis in Me1007 cells but blockedapoptosis of MM200 cells induced by TRAIL, which was used asa positive control for the caspase-2 inhibitor (Fig. 1B and datanot shown).Consistent with results in assays with the caspase inhibitors,

Western blot analysis showed that caspase-8, caspase-4, caspase-9,and caspase-3 were activated by thapsigargin or tunicamycin inMe1007, but not Mel-RM, cells (Fig. 1C and data not shown).Whereas activation of caspase-8, caspase-4, and caspase-3 couldbe detected as early as at 6 h, caspase-9 activation could only beobserved at or after 24 h. Of note, the proenzyme of caspase-8 wasdetected at very low levels in Me1007 cells before treatmentbut was markedly induced by thapsigargin or tunicamycin.Up-regulation of procasapse-8 by the ER stress inducers was notobserved in Mel-RM cells (data not shown). Activation of caspase-8 and caspase-4 by thapsigargin or tunicamycin in Me1007cells was also shown in fluorometric assays detecting activitiesof the caspases by specific substrates in whole-cell lysates(Supplementary Fig. S2).Caspase-8 plays an important role in ER stress–induced

apoptosis of Me1007 cells. We tested if activation of capasse-8 or

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caspase-4 is an upstream factor in ER stress–induced apoptosis inMe1007 cells by monitoring caspase activation in the cells treatedwith the caspase-8 inhibitor z-IETD-fmk or the caspase-4 inhibitorz-LEVD-fmk, followed by the addition of thapsigargin or tunica-mycin. Figure 2A shows that the caspase-8 inhibitor significantlyblocked activation of both caspase-8 and caspase-4, but thecaspase-4 inhibitor inhibited only caspase-4 activity with minimaleffects on caspase-8 activation.To further confirm the role of caspase-8 in ER stress–induced

apoptosis of Me1007 cells, we silenced caspase-8 by a specific

siRNA pool in the cells. As shown in Fig. 2B , the caspase-8 siRNAsignificantly inhibited up-regulation of caspase-8 by thapsigarginbut had no effect on the levels of capsase-4. Figure 2Cshows that siRNA knockdown of caspase-8 not only inhibitedthapsigargin-induced apoptosis but also blocked activation ofcaspase-4 and caspase-3. Thus, activation of caspase-8 seemedto be an initiating factor in ER stress–induced apoptosis inMe1007 cells.We studied the mechanism(s) by which procaspase-8 is up-

regulated by examining caspase-8 mRNA expression in Me1007

Figure 2. Caspase-8 activation is critical in ER stress–induced apoptosis of Me1007 cells. A, the caspase-8 inhibitor z-IETD-fmk inhibited activation of caspase-4induced by tunicamycin or thapsigargin. Me1007 cells were treated with z-IETD-fmk (30 Amol/L) or the caspase-4 inhibitor z-LEVD-fmk (30 Amol/L) for 1 h beforeadding thapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for a further 16 h. Whole-cell lysates were harvested and subjected to fluorometric assays for caspase-4 andcaspase-8 activities. The values of activity in the cells without treatment were arbitrarily designated as 1. The values of activity in cells treated with tunicamycin orthapsigargin were compared with those in cells without treatment and are expressed as the fold increases. Columns, mean of three individual experiments; bars, SE.B, siRNA knockdown of caspase-8 inhibits its expression. Me1007 cells were transfected with the control or caspase-8 siRNA. Twenty-four hours later, cells weretreated with thapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for 6 h. Whole-cell lysates were subjected to Western blot analysis of the expression of caspase-8 andcaspase-4. The arrow-pointed bands in figures for caspase-4 are presumably either nonspecific bands or intermediately cleaved capsase-4. The data shown arerepresentative of three individual experiments. C, siRNA knockdown of caspase-8 inhibits apoptosis (left ) and activation of caspase-8, caspase-4, and caspase-3 (right )induced by thapsigargin or tunicamycin. Me1007 cells were transfected with the control or caspase-8 siRNA. Twenty-four hours later, cells were treated withthapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for a further 24 (left ) or 16 h (right ) before measurement of apoptosis by the propidium iodide method using flowcytometry or Western analysis of caspase-8, caspase-4, and caspase-3. Columns, mean of three individual experiments (bars, SE; left ) or representative of threeindividual experiments (right ).





before and after exposure to thapsigargin or tunicamycin. Theresults show that caspase-8 mRNA seemed to be low in Me1007before treatment, but after exposure to thapsigargin or tunicamy-cin, there was an increase in the levels of caspase-8 mRNA, whichcould be detected as soon as 3 h and reached a peak by 6 h(Supplementary Fig. S3). This suggests that caspase-8 in Me1007cells could be up-regulated by ER stress at the transcriptional level.

Endogenously expressed ARC contributes to resistance ofmelanoma to ER stress–induced apoptosis. To understand themechanism(s) by which ER stress–induced caspase-8 activation isinhibited in most melanoma cell lines but not in Me1007, westudied the expression of ARC in a panel of melanoma cell lines. Asshown in Fig. 3A , ARC was detected at varying but generally higherlevels in melanoma cell lines in comparison with those in

Figure 3. Endogenously expressed ARC contributes to resistance of melanoma to ER stress–induced apoptosis. A, top, ARC is expressed in melanoma cell lines.Whole-cell lysates from melanocytes and a panel of melanoma cell lines were subjected to Western blot analysis of ARC expression. The data shown arerepresentative of three individual experiments. Bottom, ER stress induces a decrease in ARC expression in Me1007 cells. Whole-cell lysates from Me1007 andMel-RM cells treated with thapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for indicated periods were subjected to Western blot analysis of ARC expression.The data shown are representative of three individual experiments. B, top, Mel-RM and MM200 cells were transfected with the control or ARC siRNA. Twenty-fourhours later, whole-cell lysates were subjected to Western blot analysis of ARC expression. The data shown are representative of three individual experiments.Bottom, Mel-RM and MM200 cells were transfected with the control or ARC siRNA. Twenty-four hours later, the cells were treated with thapsigargin (1 Amol/L)or tunicamycin (3 Amol/L) for a further 36 h. Apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are the mean F SEof three individual experiments. C, siRNA knockdown of ARC enhances thapsigargin-induced or tunicamycin-induced caspase-8 activation. Twenty-four hourslater after transfection with the control or ARC siRNA, the cells were treated with thapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for a further 16 h. Whole-celllysates were subjected to Western blot analysis of the expression of caspase-8. The data shown are representative of three individual experiments. D, thecaspase-8 inhibitor z-IETD-fmk partially inhibits thapsigargin-induced or tunicamycin-induced apoptosis in Mel-RM and MM200 cells with ARC being knockeddown. Twenty-four hours later, after transfection with the control or ARC siRNA, the cells were treated with z-IETD-fmk (30 Amol/L) for 1 h before the addition ofthapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for a further 36 h. Apoptosis was measured by the propidium iodide method. Columns, mean of three individualexperiments; bars, SE.

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melanocytes. Of interest, the levels of ARC in Me1007 seemed to bethe lowest among the melanoma cell lines. Figure 3A also showsthat exposure to thapsigargin or tunicamycin did not cause anynotable change in the levels of ARC in Mel-RM cells but resulted ina moderate decrease in ARC expression in Me1007 cells that couldbe observed as soon as 6 h after exposure to thapsigargin ortunicamycin.To study if endogenous ARC plays a role in protection of

melanoma cells from ER stress–induced apoptosis, we inhibitedARC with a specific siRNA pool in Mel-RM and MM200 cells. Asshown in Fig. 3B , siRNA knockdown of ARC markedly inhibited itsexpression in both cell lines and resulted in an increase in

apoptosis induced by thapsigargin or tunicamycin. This wasassociated with induction of caspase-8 activation as shown inboth Western blot analysis and fluorometric assays (Fig. 3C andSupplementary Fig. S4). Figure 3D shows that pretreatment withthe caspase-8 inhibitor z-IETD-fmk partially inhibited induction ofapoptosis by thapsigargin or tunicamycin in Mel-RM and MM200cells with ARC being knocked down by siRNA.Exogenous ARC inhibits ER stress–induced apoptosis in

Me1007 cells. To study if the high sensitivity of Me1007 cells to ERstress–induced apoptosis is associated with the low levels of ARCexpression, we transfected cDNA encoding Flag-tagged ARC intoMe1007 cells (Fig. 4A). The levels of thapsigargin-induced or

Figure 4. Overexpression of ARC inhibits ER stress–induced apoptosis in Me1007 cells. A, left, whole-cell lysates from Me1007 cells stably transfected with c-DNA forFlag-tagged ARC or vector alone were subjected to Western blot analysis of the expression of ARC. The data shown are representative of three individual experiments.Right, Me1007 cells stably transfected with c-DNA for Flag-tagged ARC or vector alone were treated with thapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for36 h before measurement of apoptosis by the propidium iodide method. Columns, mean of duplicate assays in two individual experiments; bars, SE. B, overexpressionof ARC inhibits ER stress–induced activation of caspase-8. Me1007 cells stably transfected with c-DNA for Flag-tagged ARC or vector alone were treated withthapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for 16 h before whole-cell lysates were harvested. Left, caspase-8 activities were measured by fluorometric assays.The values of activity in the cells without treatment were arbitrarily designated as 1. The values of activity in cells treated with tunicamycin or thapsigargin werecompared with those in cells without treatment and are expressed as the fold increase. Columns, mean of three individual experiments; bars, SE. Right, whole-celllysates were subjected to Western blot analysis of the expression of caspase-8. The data shown are representative of three individual experiments. C, exogenous ARCis physically associated with caspase-8. Whole-cell lysates from Me1007 cells transfected with cDNA encoding Flag-tagged ARC with or without treatment withthapsigargin (1 Amol/L) or tunicamycin (3 Amol/L) for 16 h were subjected to immunoprecipitation with the antibody against Flag M2. The precipitates were subjected toSDS-PAGE and probed with antibodies against ARC and caspase-8. The data shown are representative of three individual experiments. D, endogenous ARC isphysically associated with caspase-8. Whole-cell lysates from Mel-RM and MM200 cells were subjected to immunoprecipitation with an antibody against caspase-8.The precipitates were subjected to SDS-PAGE and probed with antibodies against ARC and caspase-8. The data shown are representative of three individualexperiments.





tunicamycin-induced apoptosis in ARC-transfected cells weresignificantly decreased in comparison with those in vector alone–transfected cells (P < 0.05, two-tailed Student’s t test; Fig. 4A). Asshown in Fig. 4B , thapsigargin-induced or tunicamycin-inducedcaspase-8 activation was also reduced in ARC-transfected cells.ARC is physically associated with caspase-8 in melanoma

cells. To understand the mechanism(s) by which exogenous ARCprotects Me1007 cells from ER stress–induced apoptosis, wecarried out immunoprecipitation using the anti-Flag M2 antibodyin whole-cell lysates from Me1007 cells transfected with Flag-tagged ARC before and after exposure to thapsigargin ortunicamycin. As shown in Fig. 4C , ARC, but not caspase-8, wasdetected in protein complexes precipitated with the anti-Flagantibody before treatment. In contrast, caspase-8 was readily seenalong with ARC in the precipitates obtained from cells treated withthapsigargin or tunicamycin. The antibody M2-matched isotypecontrol mouse IgG did not produce precipitates containing eitherARC or caspase-8 (data not shown).We next studied if the endogenous ARC was associated with

caspase-8 in Mel-RM and MM200 cells. As shown in Fig. 4D , ARCwas detected in caspase-8 complexes obtained with an antibody

against caspase-8 in the whole lysates from Mel-RM and MM200cells before and after exposure to thapsigargin or tunicamycin. Incontrast, the ARC protein was not precipitated from whole-celllysates using the isotype-matched control mouse IgG (data notshown). These results indicate that ARC can bind to caspase-8 inmelanoma cells.ARC in melanoma is located to the cytoplasm and

mitochondria. We studied the subcellular localization of ARCusing confocal microscopy as shown in Fig. 5A . The endogenousARC in Mel-RM and MM200 cells stained with an anti-ARCantibody seemed to locate to the cytoplasm, with a proportion ofthe protein showing colocalization with mitochondria labeled withMitotracker. A similar distribution was shown for exogenous ARCin Me1007 cells transfected with ARC-Flag and detected by theanti-Flag M2 antibody. No nuclear staining pattern of ARC wasobserved in the cell lines. Exposure to thapsigargin or tunicamycindid not cause any notable change in the subcellular localization ofARC (data not shown).We confirmed absence of ARC in the nucleus and its mitochon-

drial localization in melanoma cells by Western blot analysis ofisolated nuclear and mitochondrial fractions in Mel-RM cells and

Figure 5. ARC in melanoma is located to the cytoplasmand mitochondria. A, Mel-RM and MM200 cells andMe1007 cells transfected with ARC-Flag growing oncoverslips were labeled for mitochondria by addingMitotracker red CMXRos for 30 min. Cells werepermeabilized before staining with an antibody againstARC and the anti-Flag M2 antibody, respectively, followedby labeling with Alex 488 secondary antibody (green ), andwere then subjected to analysis in confocal microscopy.Yellow and/or orange color in the merged images indicatescolocalization. Magnification, 594�. The data shown arerepresentative of two individual experiments. B, the nuclear(N ) and mitochondrial (M ) fractions isolated from Mel-RMcells and Me1007 cells overexpressing ARC-Flag weresubjected to Western blot analysis of ARC expression.Western blot analysis of histone H1 and cytochrome c wasincluded as controls for relative purity of the nuclear andmitochondrial fractions, respectively. The data shown arerepresentative of two individual experiments.

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Me1007 cells expressing ARC-Flag. As shown in Fig. 5B , ARC wasobserved in the mitochondrial fractions, but not in the nuclearfractions from both cell lines. This further confirms the presence ofARC in mitochondria but not in nuclei in melanoma cells.

Discussion

As reported elsewhere, most melanoma lines are resistant to ERstress–induced apoptosis, although cultured melanocytes andfibroblasts undergo apoptosis under the same conditions (12). Itwas therefore of much interest that the melanoma cell line Me1007was highly sensitive to apoptosis induced by the ER stress inducerthapsigargin or tunicamycin. This led us to a closer look at themechanisms involved. In the present study, apoptosis of Me1007cells induced by thapsigargin or tunicamycin seemed to depend onactivation of caspase-8 that in turn gave rise to activation ofcaspase-4, caspase-3, and caspase-9 as shown by kinetic studies,experiments with specific caspase inhibitors, and knockdown ofcaspase-8 by siRNA.Investigation of factors known to inhibit caspase-8 led to studies

on ARC, which was found to be expressed at relatively high levels inmost melanoma lines but was at low levels in Me1007 cells andcultured melanocytes. Moreover, when ARC was transfected intoMe1007 cells, they became resistant to ER stress–induced apoptosisand activation of caspase-8 was also inhibited. The more generalimportance of the role of ARC in protection of melanoma cellsfrom ER stress–induced apoptosis was shown by siRNA knock-down of ARC in two typical resistant melanoma lines, whichshowed that these cell lines became sensitive to ER stress–inducedapoptosis that was dependent on activation of caspase-8.Immunoprecipitation studies clearly showed that both exogenousand endogenous ARC was physically associated with caspase-8.These results indicate that ARC is involved in protection ofmelanoma cells against ER stress–induced apoptosis.

ARC was identified as an endogenous CARD containing proteinthat inhibits apoptosis by selectively binding to and inhibitingcaspase-8 and caspase-2, but not caspase-1, caspase-9, andcaspase-3 (13). It can also interact with Bax and inhibits itsactivation and subsequently mitochondrial apoptotic events(14, 15). In addition, binding of ARC with Fas and FADD has beenreported to play a role in inhibiting death receptor-mediatedapoptosis (14). However, most of these observations were made interminally differentiated cells, such as cardiac and skeletalmyocytes, and neurons, which were initially believed to be theonly cell types that express ARC (13–15, 21–26). It was not untilrecently that ARC has been found to be expressed in varioushuman cancer cells (16, 17). The present study seemed to be thefirst to show that endogenous ARC has a functional role in mostmelanoma cell lines.The levels of ARC expression in the apoptosis sensitive Me1007,

but not those in the resistant melanoma lines, were rapidlydecreased by treatment with thapsigargin or tunicamycin. Thissuggests that removal of the inhibitory effect of ARC may berequired to ensure optimal induction of apoptosis by ER stress(14, 24, 27, 28). In support of this, degradation of ARC by theubiquitin-proteasome pathway has been reported to be aninitiating event in apoptosis induced by varying stimuli (27, 28).Alternatively, the decrease in ARC may be a consequence ofinduction of apoptosis. ARC was found to be phosphorylated atThr149 by protein kinase CK2 and that this was required to targetARC to mitochondria (29). We found in this study that ARC inmelanoma cells was predominantly located to the cytoplasm andmitochondria, but it is not yet known whether ARC is phosphor-ylated by protein kinase CK2 in melanoma cells. The cytoplasmicand mitochondrial localization of ARC in melanoma cells seemedto be different from previous reports that showed ARC to be mainlylocalized to nuclei in cancer cells (16). This difference may be dueto the different types of cells used in the studies.Involvement of caspase-8 in the ER stress–induced apoptosis of

Me1007 was unexpected, as we have previously found that this cellline lacks caspase-8 expression (30). The latter is a frequent findingin neuroblastoma due to hypermethylation of its promoter (31).Inhibition of caspase-8 by gene hypermethylation has also beenreported in some melanoma cell lines (32). However, whether thesame applies in the Me-1007 line is not clear. The rapid kinetics ofcaspase-8 up-regulation may argue against demethylation of itspromoter as a cause (31, 32). This is supported by the finding thatthere was no notable change in methylation status of caspase-8gene in Me1007 cells before and after exposure to tunicamycin orthapsigargin using methylation-specific PCR (data not shown).Caspase-8 has been proposed to undergo autoactivation byproximity-driven dimerization (33, 34). Overexpression of cas-pase-8 is also known to induce apoptosis (13, 33). It seems in thisstudy that, although caspase-8 was expressed in most melanomacell lines, it was bound to by ARC, which prevented it fromautoactivation. In contrast, the relatively low concentration of ARCin Me1007 would presumably allow caspase-8, when induced, toundergo autoactivation. However, there was no general correlationof the relative concentrations of ARC and capsase-8 with sensitivityto ER stress–induced apoptosis in the panel of melanoma cell lines(data not shown). This suggests that factors other than ARC mayalso operate to control caspase-8 activation in melanoma cellssubjected to ER stress.Many other mechanisms have been implicated in the induction

of apoptosis by ER stress, such as CHOP-mediated up-regulation

Figure 6. A schematic illustration of mechanisms responsible for resistance ofmelanoma cells to ER stress–induced apoptosis. ER stress can potentiallyinduce apoptosis by activation of caspase-4 and caspase-8 in melanoma cells,which are, however, inhibited by GRP78 and ARC, respectively. Anothermechanism that may involve in induction of apoptosis by ER stress in melanomacells is activation of BH3-only proteins, which can be inhibited by antiapoptoticBcl-2 family members (36, 38). In addition, ER stress can cause calcium flux.The latter is conceivably bound by ARC and GRP78 and prevented from inducingapoptosis (9, 25). Question marks at the arrow points indicate that moremechanisms may be found to involve in ER stress–induced apoptosis inmelanoma cells, and accordingly more resistance mechanisms are yet to bediscovered.





of TRAIL-R2 and/or down-regulation of Bcl-2 (8, 35), up-regulationof the BH3-only protein PUMA, Noxa, and Bim (36–38), andactivation of caspase-12 in murine systems and its counterpartcaspase-4 in human cells (4–6). The present finding that ER stressinduces apoptosis of melanoma cells by activation of caspase-8 when ARC is deficient, along with our previous studies showingthat ER stress can induce apoptosis of melanoma cells byactivation of caspase-4 when the MEK/ERK pathway is inhibited(12), indicates that ER stress can potentially activate multipleapoptosis signaling pathways. Other mechanisms involved mayinclude calcium flux and activation of BH3-only proteins, such asPUMA, Noxa, and Bim (9, 25, 36, 38). It is conceivable thatmelanoma cells may have adapted to ER stress by inhibitingmultiple apoptotic pathways as illustrated schematically in Fig. 6.The present study reveals that expression of the ARC protein is

another adaptive mechanism by which melanoma cells areprotected from ER stress–induced apoptosis and suggests thattargeting ARC may improve treatment results of clinicallyavailable chemotherapeutic drugs and those in development forclinical use that can induce ER stress in melanoma cells, such ascisplatin and sorafenib (39, 40).

Acknowledgments

Received 8/28/2007; revised 11/19/2007; accepted 11/29/2007.Grant support: New South Wales State Cancer Council, Melanoma and Skin

Cancer Research Institute Sydney, Hunter Melanoma Foundation (New South Wales),and National Health and Medical Research Council (Australia). X.D. Zhang is a CancerInstitute New South Wales Fellow.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

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2008;68:834-842. Cancer Res Li Hua Chen, Chen Chen Jiang, Ralph Watts, et al. Apoptosis of Melanoma Cells by the ARC Protein

Induced−Inhibition of Endoplasmic Reticulum Stress

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

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