The melanoma-associated antigen MAGE-D2 suppresses TRAIL receptor 2 and protects against TRAIL-induced apoptosis in human melanoma cells

Carcinogenesis, Oct 2012

Emerging evidence has pointed to biological roles of melanoma-associated antigens (MAGEs) in cancer development, progression and resistance to treatment. However, the mechanisms involved remain to be fully elucidated. In this report, we show that one of the MAGE proteins, MAGE-D2, suppresses the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 2 (TRAIL-R2) and plays an important role in protecting melanoma cells from apoptosis induced by TRAIL. MAGE-D2 was commonly expressed at increased levels in melanoma cells compared with melanocytes. Although its inhibition by small interfering RNA (siRNA) did not cause cell death, it rendered melanoma cells more sensitive to TRAIL-induced apoptosis. This was associated with enhanced formation of TRAIL death-inducing signaling complex and up-regulation of TRAIL-R2, and was blocked by a recombinant TRAIL-R2/Fc chimeric protein or siRNA knockdown of TRAIL-R2. Regulation of TRAIL-R2 by MAGE-D2 appeared to be mediated by p53, in that knockdown MAGE-D2 did not up-regulate TRAIL-R2 in p53-null or mutant p53 melanoma cells. In addition, inhibition of MAGE-D2 did not result in up-regulation of TRAIL-R2 in wild-type p53 cell lines with p53 inhibited by short hairpin RNA. Indeed, knockdown of MAGE-D2 led to up-regulation of p53 due to a transcriptional increase. The regulatory effect of MAGE-D2 on TRAIL-R2 expression and TRAIL-induced apoptosis was recapitulated in studies on fresh melanoma isolates. Taken together, these results identify the expression of MAGE-D2 as an important mechanism that inhibit TRAIL-induced apoptosis and suggest that targeting MAGE-D2 may be a useful strategy in improving the therapeutic efficacy of TRAIL in melanoma.

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The melanoma-associated antigen MAGE-D2 suppresses TRAIL receptor 2 and protects against TRAIL-induced apoptosis in human melanoma cells

Hsin-Yi Tseng 1 2 Li Hua Chen 1 2 Yan Ye 1 2 Kwang Hong Tay 1 2 Chen Chen Jiang 1 2 Su Tang Guo 0 Lei Jin 3 Peter Hersey 3 Xu Dong Zhang 1 2 0 Department of Molecular Biology, Shanxi Cancer Hospital and Institution , Taiyuan, Shanxi 030013, China 1 Cancer Research Program, Hunter Medical Research Institution , New Lambton Heights, NSW 2310, Australia 2 School of Medicine and Public Health, Priority Research Center for Cancer Research, University of Newcastle , Newcastle, NSW 2300, Australia 3 Immunology and Oncology, Kolling Institute for Medical Research , St Leonard, NSW 2065, Australia *To whom correspondence should be addressed. Tel:+61 2 49138174; Fax: +61 2 49138184; Email: The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: - Emerging evidence has pointed to biological roles of melanoma-associated antigens (MAGEs) in cancer development, progression and resistance to treatment. However, the mechanisms involved remain to be fully elucidated. In this report, we show that one of the MAGE proteins, MAGE-D2, suppresses the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 2 (TRAIL-R2) and plays an important role in protecting melanoma cells from apoptosis induced by TRAIL. MAGE-D2 was commonly expressed at increased levels in melanoma cells compared with melanocytes. Although its inhibition by small interfering RNA (siRNA) did not cause cell death, it rendered melanoma cells more sensitive to TRAIL-induced apoptosis. This was associated with enhanced formation of TRAIL death-inducing signaling complex and up-regulation of TRAIL-R2, and was blocked by a recombinant TRAIL-R2/Fc chimeric protein or siRNA knockdown of TRAIL-R2. Regulation of TRAIL-R2 by MAGE-D2 appeared to be mediated by p53, in that knockdown MAGE-D2 did not up-regulate TRAIL-R2 in p53-null or mutant p53 melanoma cells. In addition, inhibition of MAGE-D2 did not result in up-regulation of TRAIL-R2 in wild-type p53 cell lines with p53 inhibited by short hairpin RNA. Indeed, knockdown of MAGE-D2 led to up-regulation of p53 due to a transcriptional increase. The regulatory effect of MAGE-D2 on TRAIL-R2 expression and TRAIL-induced apoptosis was recapitulated in studies on fresh melanoma isolates. Taken together, these results identify the expression of MAGE-D2 as an important mechanism that inhibit TRAIL-induced apoptosis and suggest that targeting MAGE-D2 may be a useful strategy in improving the therapeutic efficacy of TRAIL in melanoma. Introduction Melanoma-associated antigens (MAGEs) are a class of the cancer/testis antigen family that consists of more than 50 closely related proteins and can be further divided into two types, MAGE-I and MAGE-II, according to tissue-specific expression patterns and gene structures (13). Members of MAGE-I are products of a number of chromosome X-clustered genes including MAGE-A, -B and -C that are commonly expressed in cancer cells of different origins but not in adult tissues Abbreviations: DISC, death-inducing signaling complex; mAbs, mouse monoclonal antibodies; MAGEs, melanoma-associated antigens; mRNA, messenger RNA; PARP, poly (ADP ribose) polymerase; PI, propidium iodide; qPCR, quantitative PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; z-VAD-FMK, Z-Val-Ala-Asp(OMe)-CH2F; Z-DEVD-FMK, Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-FMK; z-IETD-FMK, Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F. These authors contributed equally to thiswork. except for germ-line cells in testes, ovaries and placenta. As such, they are potentially involved in cancer immune responses and appear to be attractive targets for vaccine-based cancer immunotherapy (47). In contrast, members of MAGE-II including MAGE-D variants have no defined chromosome clustering and are not specific to cancers as they are expressed almost universally in normal adult tissues in addition to germ-line cells (2,4). There is increasing evidence showing that MAGE family members play essential roles in physiological processes such as embryonic development and germ cell growth (1,2,8). Some MAGE proteins have also been demonstrated to impinge on cell survival, proliferation and apoptosis in cancers (915). For example, interaction of MAGE-A2, -A3, -A6 or-C2 with the E3 ligase TRIM28 resulted in ubiquitylation and degradation of the tumor suppressor p53, which has been suggested to contribute to tumorigenesis (12). MAGE-A2 could also inhibit p53 transcriptional activity by recruiting histone deacetylases to p53 transcription sites (13). Similarly, the type-II MAGE protein necdin interacted directly with p53, thus conferring resistance of cells to apoptosis (14). Consistent with their functional roles, elevated expression levels of some MAGE proteins in cancer cells have been reported to associate with disease progression and poor prognosis of patients (1618). Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) appears to be a promising candidate for cancer therapeutics because of its ability to preferentially induce apoptosis in malignant cells (1922). Induction of apoptosis by TRAIL is mediated by its interaction with two death domain containing receptors, TRAIL-R1 and -R2 (20,23). This in turn orchestrates activation of the caspase cascade (20,23). Recombinant TRAIL and agonistic antibodies against its death receptors are currently in clinical evaluation for the treatment of various cancers (2426). We have shown previously that sensitivity of cultured melanoma cells to TRAIL-induced apoptosis is in general correlated with the levels of the cell surface expression of TRAIL death receptors, in particular, TRAIL-R2 (26). However, fresh melanoma isolates are relatively resistant to TRAIL-induced apoptosis due to low levels of TRAIL-death receptor expression (2729). These findings indicate that melanoma may not respond to treatment with TRAIL unless given with agents that increase the cell surface expression of TRAIL death receptors (3032). In this report, we show that the expression of the type-II MAGE protein, MAGE-D2, is an important mechanism that suppresses TRAIL-R2, and that targeting MAGE-D2 may be a useful strategy in improving the therapeutic efficacy of TRAIL in melanoma. Materials and methods Human melanoma cell lines Mel-JD, MM200, Mel-FH, ME4405, Mel-RM, Mel-CV, Mel-RMu, ME1007 and IgR3 were obtained as described previously (26,30). All cell lines were cultured in Dulbeccos modified Eagles medium supplemented with 5% fetal calf serum (Commonwealth Serum Laboratories, Melbourne, Australia). Individual melanoma cell line authentication was confirmed using the AmpFlSTR Identifiler PCR Amplification Kit obtained from Applied Biosystems and GeneMarker V1.91 software. Apanel of 16 markers was tested, and each cell line had a distinct individual set of markers present. The human melanocyte cell line HEMn-MP was purchased from Banksia Scientific (Bulimba, Queensland, Australia) and cultured in melanocyte medium (Gibco, Invitrogen, Australia). Fresh melanomaisolates Isolation of melanoma cells from fresh surgical specimens was carried out as described previously (27). Studies using human tissues were approved by the human research ethics committees of University of Newcastle, Australia. Antibodies, recombinant proteins and otherreagents The mouse monoclonal antibodies (mAbs) against TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and Fas were supplied by Immunex (Seattle, WA). The cell permeable general caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F (z-VAD-FMK), the caspase-3-specific inhibitor Z-Asp(OCH3)-Glu(OCH3)Val-Asp(OCH3)-FMK (Z-DEVD-FMK) and the caspase-8-specific inhibitor Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (z-IETD-FMK) were purchased from Calbiochem (La Jolla, CA); the rabbit polyclonal antibodies (pAbs) against caspase-3 and -9 and the mouse mAb against caspase-8 were from Stressgen (Victoria, British Columbia, Canada); the mouse mAb against cleaved form of poly (ADP ribose) polymerase (PARP) were from BD Pharmingen (NSW, Australia); the rabbit pAbs against Bcl-2 interacting protein (Bid) and p53-upregulated modulator of apoptosis (PUMA) were from Cell Signaling Technology (Beverly, MA); the mouse mAbs against p53 and p21 were from Upstate (Billerica, MA); the rabbit pAbs against Fas-associated death domain (FADD) was from Santa Cruz Biotechnology (Santa Cruz, CA); the rabbit antibody against cytochrome c and the mouse antibody against Cyclooxygenase (COX) IV were from Clontech (Mountain View, CA), the rabbit pAb against MAGE-D2 and -actin were from Abcam (Cambridge, MA). Apoptotic cells were quantified by measurement of sub-G1 DNA content using propidium iodide (PI) in a flow cytometer (Becton Dickinson, Sunnyvale, CA) as described elsewhere (30,31). Briefly, cells were seeded at 1 105 cells/well onto 24-well culture plates and allowed to grow for 24 h followed by the desired treatment. Cells were harvested with PI buffer (PI, 50g/ml, in 0.1% sodium tri-citrate and 0.1% Triton X-100), transferred to fluorescence-activated cell sorting tubes and incubated overnight at 4C in the dark before analysis. Gates were set to record only the events representing intact cells as determined by forward and side scatter parameters and cell debris was excluded. Cell viabilityanalysis Cells were seeded at 5000 cells/well onto flat-bottomed 96-well culture plates and allowed to grow for 24 h followed by the desired treatment. Cells were then labeled with the VisionBlue reagent (BioVision, Mountain View, CA) and detected by Synergy 2 multi-detection microplate reader (BioTek, VT). Colony formationassays Cells were seeded at 2000 cells/well onto 6-well culture plates and allowed to grow for 24 h followed by the desired treatment. Cells were then allowed to grow for a further 12days before fixation with methanol and staining with 0.5% crystal violet. The images were captured with Bio-Rad VersaDoc image system (Bio-Rad, NSW, Australia). Mitochondrial membrane potential (m) was quantitated with MitoProbe JC-1 assay kit (Invitrogen, Carlsbad, CA) according to manufacturers instructions as described previously (31). Analysis for m was performed as reported previously (33). Preparation of mitochondrial and cytosolic fractions Subcellular fractionation was done by using Qproteome Mitochondrial isolation kit (Qiagen, Doncaster, VIC, Australia) following the manufacturers protocol as described previously (31). Immunoprecipitation and western blotanalysis Immunoprecipitation and western blot analysis were carried out as described previously (31,34). Briefly, cells were harvested and disrupted on ice in NP-40 lysis buffer. Equal amounts of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred to membranes and immunoblotted with specific primary and secondary antibodies. Labeled bands were detected by Luminata Crescendo Western HRP substrate (Millipore, Billerica, MA), and images were captured with ImageReader LAS-4000 (Fujifilm Corporation, Tokyo, Japan) . Cell surfacestaining Immunostaining on intact cells was carried out as described previously (31). In brief, adherent cells were harvested and fixed with 4% paraformaldehyde before blocking with 10% fetal calf serum. Cells were then stained with respective primary antibodies followed by R-phycoerythrin-conjugated secondary antibody and read by FACScanto (Becton Dickinson). Quantitative reverse transcription and real-time PCR(qPCR) Quantitative PCR (qPCR) was carried out as described previously (31,34). In brief, total RNA was isolated using RNeasy mini kit (Qiagen, Doncaster, VIC, Australia) and reverse transcribed into complementary DNA using Superscript III (Invitrogen, Carlsbad, CA) following the manufacturers instructions. qPCR was performed using the ABI Prism 7900 sequence detection system (Applied Biosystem, Mulgrave, VIC, Australia) with specific gene primers (TRAIL-R2 forward, 5-CGC TGC ACC AGG TGT GAT T-3; TRAIL-R2 reverse, 5-GTG CCT TCT TCG CAC TGA CA-3; p53 forward, 5-TGG AAA CTA CTT CCT GAA AAC AAC-3; p53 reverse, 5-GGG TCT TCA GTG AAC CAT TG-3; -actin forward, 5-GGC ACC CAG CAC AAT GAA G-3; -actin reverse, 5-GCC GAT CCA CAC GGA GTA CT-3). The following PCR conditions were used: fast cycle 95C for 20 s, 40 cycles of 95C for 1 s and 60C for 20 s using Fast SYBR Green mastermix (Applied Biosystem, Mulgrave, VIC, Australia). Small interfering RNA (siRNA) knockdown The small interfering RNA (siRNA) constructs used were obtained as the siGENOME SMARTpool reagents (Dharmacon, Lafayette, CO). The following siRNA constructs were used: TRAIL-R2 siGENOME SMARTpool (M-004448-00-0010), MAGE-D2 siGENOME SMARTpool (M-017284-00-0010) and non-targeting siRNA pool (D-001206-13-20) as control. Transfection of siRNA pools was carried out as described previously (31). Short hairpin RNA (shRNA) stable knockdown Sigma MISSION Lentiviral Transduction Particles (Sigma-Aldrich, Castle Hill, NSW, Australia) for short hairpin RNA (shRNA)-mediated knockdown of p53 were used for knockdown of p53 according to the manufacturers instruction as described previously (35). Plasmid vector transfection Stable melanoma transfectants of Bcl-2 were established by electroporation of the pEF-puro vector carrying human Bcl-2 as described elsewhere (36). Luciferase reporterassays To measure TRAIL-R2 promoter activity, the core fragment of the TRAIL-R2 gene (TNFRSF10B) promoter was cloned by genomic PCR using human genomic DNA as template. The fragment was cloned into promoter-less luciferase reporter plasmid pGL3-Basic Luciferase Vector (Promega, Hawthorn, VIC, Australia). Cells were transiently transfected with pGL3-vector or pGL3-TRAIL-R2 with or without co-transfection of control or MAGE-D2 siRNA. The luciferase activity was measured using the Dual-Luciferase Reported Assay System (Promega, Hawthorn, VIC, Australia) by Synergy 2 multi-detection microplate reader (Biotek, Winooski, VT). To measure p53 promoter assay, we used a p53 reporter assay kit according to the manufacturers instructions (SABioscience, Frederick, MD). Briefly, cells were transiently transfected with negative control or p53 reporter construct with or without co-transfection of control or MAGE-D2 siRNA. The luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega by Synergy 2 multi-detection microplate reader (Biotek, Winooski, VT). Statistical analysis was carried out using GraphPad Prism 4.0. Two-tailed Student's t-test was used to assess differences in values between experimental groups. P 0.05 was considered to be statistical significant. Results siRNA knockdown of MAGE-D2 sensitizes melanoma cells to TRAILinduced apoptosis Our initial studies in a panel of melanoma cell lines and a melanocyte line by western blot analysis demonstrated that the MAGE-D2 protein was expressed at varying levels in melanoma cell lines, which were in general higher than those in melanocytes (Figure1A). To investigate if MAGE-D2 has a role in regulating melanoma cell survival and proliferation, we inhibited its expression in Mel-RM and MM200 cells by siRNA (Figure 1B). Knockdown of MAGE-D2 moderately inhibited melanoma cell survival and proliferation and did not result in significant apoptosis (<10% apoptotic cells) (Figure 1C and 1D). Consistently, there is no significant activation of caspase-3 and cleavage of its substrate PARP in cells with MAGE-D2 knocked down (Figure1D). Nevertheless, siRNA inhibition of MAGE-D2 markedly sensitized Mel-RM and MM200 cells to apoptosis induced by TRAIL, which was associated with enhanced activation of caspase-3 and cleavage of PARP (Figure1C and 1D). This appeared to be specific, in that inhibition of MAGE-D2 did not alter sensitivity of melanoma cells to apoptosis induced by the DNA-damaging agent cisplatin, the microtubule-targeting drug, docetaxol, or the Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 in Mel-RM and MM200 cells, nor did it result in increased induction of apoptosis by the mutant B-Rapidly accelerated fibrosarcoma (BRAF) inhibitor PLX4720 in BRAFV600E melanoma cells (MM200 and IgR3) (Supplementary Figure 1 and data not shown) (37,38). The sensitization to TRAILinduced apoptosis was confirmed in another wild-type p53 melanoma cell line (data not shown), but notably, knockdown of MAGE-D2 did not enhance TRAIL-induced apoptosis in the p53-null ME4405 and the mutant p53 Mel-FH melanoma lines, nor did it cause inhibition of survival and proliferation in these cells (Figure1E and Supplementary Figure2) (39). Sensitization of melanoma cells to TRAIL-induced apoptosis is associated with enhanced activation of the TRAIL death-inducing signaling complex(DISC) The mitochondrial apoptotic pathway is known to play an important role in regulating sensitivity of melanoma cells to TRAIL-induced apoptosis (36,40), we, therefore, examined if knockdown of MAGE-D2 impinges on activation of mitochondrial apoptotic signaling by TRAIL in wild-type p53 melanoma cells. As shown in Figure2A, sensitization to TRAIL-induced apoptosis by inhibition of MAGE-D2 was associated with enhanced reduction in the m and mitochondrial release of cytochrome c and Smac/DIABLO. Similarly, knockdown of MAGE-D2 did not significantly alter the cleavage of caspase-8, caspase-9 and Bid, but it enhanced TRAIL-induced caspase-8 and caspase-9 activation and Bid cleavage (Figure2B). The importance of the mitochondrial pathway was confirmed by inhibition of TRAIL-induced apoptosis even when MAGE-D2 was knocked down in Mel-RM and MM200 sublines stably over-expressing Bcl-2 (Figure2C) (41). We also treated Mel-RM and MM200 cells with the general caspase inhibitor z-VAD-FMK, the caspase-3 inhibitor, z-DEVD-FMK, and the caspase-8 inhibitor, z-IETD-FMK, before the addition of TRAIL. Similar to z-VAD-FMK and z-DEVD-FMK, z-IETD-FMK efficiently blocks the sensitization (Figure2D). Together, these results suggested that MAGE-D2 interfered with TRAIL death-inducing signaling complex (DISC) formation in melanoma cells, which were confirmed by increased recruitment of caspase-8 and FADD into the TRAIL DISC precipitated by an antibody against TRAIL-R2 (Figure2E). siRNA knockdown of MAGE-D2 triggers transcriptional up-regulation ofTRAIL-R2 As shown in Figure 3A, inhibition of MAGE-D2 up-regulated TRAIL-R2 expression on the surface of Mel-RM and MM200 cells. In contrast, there was no increase in the expression of the other TRAIL death receptor, TRAIL-R1, and the TRAIL decoy receptors, TRAIL-R3 and -R4, nor was there any change in the expression of the other TNF receptor family members TNF-R1, TNF-R2 and Fas on the cell surface (Figure3A and data not shown). The increase in TRAIL-R2 on the cell surface was associated with an increase in its total protein levels as shown by western blot analysis of TRAIL-R2 in whole-cell lysates (Figure3B). Inhibition of MAGE-D2 also resulted in an increase in TRAIL-R2 messenger RNA (mRNA) levels (Figure 3C). This was due to transcriptional up-regulation rather than a change in TRAIL-R2 mRNA stability, as the levels of TRAIL-R2 mRNA were reduced by the general transcription inhibitor actinomycin D with similar kinetics and to comparable degrees in cells transfected with the control siRNA and those with the MAGE-D2 siRNA (Figure3D). In support, knockdown of MAGE-D2 increased the TRAIL-R2 promoter activity as demonstrated in Mel-RM and MM200 cells transfected with luciferase reporter plasmids of the TRAIL-R2 core promoter (Figure3E). Notably, unlike in Mel-RM and MM200 cells that expressed wild-type p53, knockdown of MAGE-D2 did not up-regulate TRAIL-R2 at either the mRNA or protein level in ME4405 cells that are p53-null and Mel-FH cells that harbored mutant p53 (Figure3F and Supplementary Figure3) (39). Sensitization to TRAIL-induced apoptosis by inhibition of MAGE-D2 is associated with an increase in interaction between TRAIL andTRAIL-R2 We examined the role of the interaction between TRAIL-R2 and TRAIL in sensitization of melanoma cells to TRAIL-induced apoptosis by knockdown of MAGE-D2. As shown in Figure 4A, 4a TRAIL-R2/Fc chimeric protein efficiently blocked TRAIL-induced apoptosis in Mel-RM and MM200 cells with or without MAGE-D2 knocked down (*P < 0.05, two-tailed Students t-test). Similarly, it markedly inhibited TRAIL-induced cleavage of PARP and reduction in m even when MAGE-D2 was inhibited (Figure 4B and 4C). However, there was no significant change when cells were pretreated with TRAIL-R1/Fc chimeric protein (data not shown). The importance of up-regulation of TRAIL-R2 in knockdown of MAGED2-mediated sensitization of melanoma cells to TRAIL-induced apoptosis was confirmed by co-introducing TRAIL-R2 siRNA into Mel-RM and MM200 cells (Figure4DF). Transcriptional up-regulation of TRAIL-R2 by knockdown of MAGE-D2 is mediated byp53 Given the results showing that knockdown of MAGE-D2 did not cause transcriptional up-regulation of TRAIL-R2 in p53-null and mutant p53 melanoma cells (Figure 3E), it is possible that regulation of TRAIL-R2 by MAGE-D2 is mediated by p53. As shown in Figure 5A, when MAGE-D2 was inhibited by siRNA, there were increases in the levels of p53 and its transcriptional target p21 in Mel-RM and MM200 cells. However, some other p53 downstream targets including PUMA and Noxa were not up-regulated, suggesting that p53-mediated transcriptional activation of its targets in melanoma cells when MAGE-D2 is inhibited is highly selective. Up-regulation of p53 by knockdown of MAGE-D2 is associated with an increase in its transcript, which could be inhibited by actinomycin D to the similar extent in cells transfected with the control siRNA and those transfected with the MAGE-D2 siRNA, indicating that up-regulation of p53 is due to a transcriptional increase (Figure5B). To confirm this, we co-introduced p53 reporter plasmid into Mel-RM and MM200 cells with the control or MAGE-D2 siRNA. This showed that knockdown of MAGE-D2 increased p53 activity (Figure5C). We next took advantage of p53-deficient Mel-RM and MM200 sublines that had been established by lentiviral transduction of p53 shRNA (Figure 5D) (35). Introduction of MAGE-D2 siRNA into these p53-deficient melanoma cells did not cause transcriptional up-regulation of TRAIL-R2 (Figure5D and 5E). Consistent with this, knockdown of MAGE-D2 in melanoma cells deficient in p53 did not significantly enhance TRAIL-induced apoptosis (Figure5E). Inhibition of MAGE-D2 up-regulates TRAIL-R2 and enhances TRAIL-induced apoptosis in wild-type p53 fresh melanomaisolates We examined the expression of MAGE-D2 in a panel of wild-type p53 fresh melanoma isolates, which may reflect more closely the MAGE-D2 expression status in melanoma cells in vivo. Figure 6A shows that six of six fresh melanoma isolates all expressed moderate to high levels of MAGE-D2 compared with melanocytes, consolidating the observation that MAGE-D2 is commonly expressed in melanoma cells. As shown in Figure 6B and 6C, siRNA knockdown of MAGE-D2 similarly up-regulated TRAIL-R2 and enhanced TRAILinduced apoptosis in fresh melanoma isolates (*P<0.05, two-tailed Students t-test). Moreover, the TRAIL-R2/Fc chimeric protein efficiently blocked TRAIL-induced apoptosis in fresh isolates with MAGE-D2 knocked down (Figure6D), further confirming the importance of MAGE-D2-mediated regulation of TRAIL-R2 in regulating sensitivity of melanoma cells to apoptosis induced byTRAIL. As tumor-associated antigens, MAGE proteins have attracted considerable interest in developing novel approaches of vaccine-based cancer immunotherapy (4,6,7). On the other hand, accumulating evidence has pointed to biological functions of these proteins in development and progression of various cancers (1,2,911). Indeed, increasing numbers of MAGE proteins have been reported to play roles in regulating cell survival, proliferation and sensitivity to apoptosis induced by chemotherapeutic drugs (911). Our finding in this study that MAGE-D2 protects melanoma cells from TRAIL-induced apoptosis is of particular interest in that TRAIL is expressed by many types of immune cells such as activated CD4 and CD8 T lymphocytes, NK cells and dendritic cells, and plays an important role in killing of melanoma cells by the immune system (40,42,43). It is conceivable that while MAGE-D2 may serve as a tumor-associated antigen that elicits immune responses, its protection against TRAIL-induced apoptosis may blunt the responses by enabling melanoma cells to evade immune cell-induced apoptosis. Sensitization of melanoma cells to TRAIL-induced apoptosis by inhibition of MAGE-D2 was associated with increased recruitment of caspase-8 and FADD to the TRAIL DISC and up-regulation of TRAIL-R2 on the cell surface and could be efficiently blocked by a TRAIL-R2/Fc chimera or siRNA knockdown of TRAIL-R2. These results identified MAGE-D2 as a therapeutic target in sensitizing melanoma cells to TRAIL-induced apoptosis. Importantly, up-regulation of TRAIL-R2 by inhibition of MAGE-D2 seemed to be highly selective because MAGE-D2 knockdown did not up-regulate the other TNF receptor family members, TRAIL-R1, TRAIL-R3, and TRAIL-R4, TNF-R1 and TNF-R2, and Fas. This selectivity of MAGE-D2 in regulation of TRAIL-R2 would be an advantage for potential clinical use of targeting MAGE-D2 as a means to enhance TRAIL-induced apoptosis. Up-regulation of TRAIL-R2 on the cell surface was associated with elevated TRAIL-R2 total protein levels and increased TRAIL-R2 gene transcription, which appeared to be mediated by p53 and was specific to inhibition of MAGE-D2 in melanoma cells, in that our previous studies have shown that, unlike in many other cell types, activation of p53 by agents such as DNA-damaging chemotherapeutic drugs did not increase the TRAIL-R2 expression in melanoma cells (30,32,44). Similarly, p53-mediated up-regulation of its transcriptional targets when MAGE-D2 was inhibited also appeared to be selective, as unlike TRAIL-R2 and p21, some other downstream targets of p53 including PUMA and Noxa were not altered in expression when MAGE-D2 was inhibited. Many mechanisms are known to contribute to promoter selectivity of p53 target genes, such as post-translational modifications of p53, single-nucleotide polymorphisms in putative p53 response elements and interactions of p53 with cofactors (4547). Indeed, MAGE-D2 has been reported to directly interact with p53 (48). Whether this and any of other mechanisms are involved in the specific regulation of TRAIL-R2 in melanoma cells when MAGE-D2 is inhibited requires furtherstudies. Our results also demonstrated that MAGE-D2 plays a role in repressing p53 expression in melanoma cells, as inhibition of MAGE-D2 resulted in up-regulation of p53 expression levels. This was associated with an increase in the p53 transcript that could be efficiently inhibited by actinomycin D, suggesting that up-regulation of the p53 protein is at least partially due to a transcriptional increase. On the other hand, although various MAGE-I proteins including MAGE-A2, -A3, -A6 or-C2 can interact with the E3 ubiquitin ligase TRIM28 causing ubiquitination and degradation of p53 (12), type-II MAGE proteins can also bind to and activate RING E3 ubiquitin ligases (12). It is likely that reduced ubiquitination and degradation of the p53 protein may also contribute to the increased p53 expression when MAGE-D2 is inhibited (Supplementary Figure4). MAGE-D2 has been reported to locate to cytoplasm as well as the nucleus (48). MAGE-D2 expression levels appeared to be increased in both melanoma cell lines and fresh melanoma isolates. Although whether the expression of MAGE-D2 in melanoma cells is up-regulated with melanoma development and progression needs to be established in studies with large cohorts of melanocytic tumor tissues, our results strongly suggest that the increased MAGE-D2 expression is a mechanism to inactivate p53 that is advantageous to melanoma cell proliferation and survival. Interestingly, it has been recently reported that MAGE-D2 is expressed at higher levels in melanoma cells with the active BRAFV600E mutation compared with those harboring wild-type BRAF (4951), even though we did not find a significant difference in MAGE-D2 expression between BRAFV600E and wild-type BRAF melanoma cell lines (data not shown), conceivably due to the relatively small number of cell lines and fresh isolates included in this study. Whether the increased MAGE-D2 is a direct consequence of activation of the RAF/MEK/ERK pathway requires further studies. Similarly, whether epigenetic mechanisms such as DNA demethylation that is involved in up-regulation of many MAGE proteins in cancer cells also contributes to the increase in MAGE-D2 in melanoma cells needs further investigations. The finding that inhibition of MAGE-D2 could sensitize fresh melanoma isolates to TRAIL-induced apoptosis by up-regulation of TRAIL-R2 is of particular importance because this may reflect more closely the in vivo status of TRAIL death receptor and MAGE-D2 expression in melanoma cells and their susceptibility to TRAILinduced apoptosis. Although evaluation in more normal cells is needed to assess the potential toxicity of the combination of targeting MAGE-D2 and TRAIL before in vivo investigations are carried out, results from this study point to a novel approach that may increase the therapeutic potential of TRAIL in melanoma. Supplementarymaterial Supplementary Figures14 can be found at http://carcin.oxfordjournals.org/ Funding References New South Wales State Cancer Council (RG 11-10 to X.D.Z.); National Health and Medical Research Council (APP1026458 toX.D.Z). Conflict of Interest Statement: None declared.


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Hsin-Yi Tseng, Li Hua Chen, Yan Ye, Kwang Hong Tay, Chen Chen Jiang, Su Tang Guo, Lei Jin, Peter Hersey, Xu Dong Zhang. The melanoma-associated antigen MAGE-D2 suppresses TRAIL receptor 2 and protects against TRAIL-induced apoptosis in human melanoma cells, Carcinogenesis, 2012, 1871-1881, DOI: 10.1093/carcin/bgs236