Research Article

Microphthalmia-Associated Transcription Factor Is a Critical Transcriptional Regulator of Inhibitor of Apoptosis in

Jasmin N. Dynek,1 Sara M. Chan,2 Jinfeng Liu,3 Jiping Zha,2 Wayne J. Fairbrother,1 and Domagoj Vucic1

Departments of 1Protein Engineering, 2Pathology, and 3Bioinformatics, Genentech, Inc., South San Francisco, California

Abstract execution of programmed cell death (6, 7). X –linked Melanoma inhibitor of apoptosis (ML-IAP) is a potent IAP (XIAP) is an IAP family member that can bind to and potently inhibit caspase-3, caspase-7, and caspase-9 (8). After cells inhibitor of apoptosis, which is highly expressed in melano- mas and likely contributes to their resistance to chemother- receive death stimuli, second mitochondria-derived activator of apeutic treatments. Herein, we show that the lineage survival caspases (SMAC) is released from mitochondria into the cytosol oncogene microphthalmia-associated transcription factor where it binds to XIAP and abrogates its caspase-inhibitory (MITF) is a critical regulator of ML-IAP transcription in activity (9, 10). ML-IAP, on the other hand, seems to primarily melanoma cells. The ML-IAP promoter contains two MITF exert its prosurvival properties by binding to SMAC via its consensus sites, and analysis of MITF and ML-IAP mRNA levels conserved baculovirus IAP repeat (BIR) domain, overcoming revealed a high correlation in melanoma tumor samples and SMAC antagonism of XIAP-mediated caspase inhibition, and thus effectively halting apoptosis (2, 4, 11). cell lines. In reporter assays, MITF promoted a strong ML-IAP stimulation of transcriptional activity from the ML-IAP At present, little is known about how tissue- and tumor- specific expression is achieved. In this study, we focused specifically promoter, and MITF bound the endogenous ML-IAP promoter on elucidation of a molecular mechanism for transcriptional in melanoma cells by chromatin immunoprecipitation and regulation of ML-IAP in melanoma. Melanoma is a neoplasm that electrophoretic mobility shift assay. Strikingly, small interfer- originates from melanocytes and is highly resistant to chemother- ing RNA (siRNA)–mediated knockdown of MITF in melanoma apeutic and radiological treatments (12). ML-IAP has been shown cells led to a dramatic decrease in ML-IAP mRNA and protein to be highly expressed in the majority of melanoma patient levels, establishing that ML-IAP expression in melanoma cells samples surveyed, as well as in a number of melanoma cell lines is MITF dependent. Additionally, cyclic AMP–mediated induc- (2, 13). However, in the same studies, ML-IAP expression was not tion of MITF expression in melanocytes resulted in increased detected in benign melanocytic proliferation or normal melano- ML-IAP expression, suggesting that melanocytes can express cytes (2, 13). Mechanisms describing how ML-IAP expression may ML-IAP when MITF levels are heightened. Disruption of MITF be up-regulated during melanocytic transformation have not yet by siRNA led to a decrease in melanoma cell viability, which been reported, although it is noteworthy that ML-IAP maps to could be rescued by ectopic expression of ML-IAP. Collectively, 20q13.3, a region frequently amplified in melanomas (14). these findings implicate MITF as a major transcriptional Understanding the regulation of ML-IAP expression in melanomas regulator of ML-IAP expression in melanomas, and suggest may contribute to the ongoing efforts to overcome resistance to that ML-IAP contributes to the prosurvival activity of MITF in apoptosis in melanoma through apoptosis-based therapeutics (15). melanoma progression. [Cancer Res 2008;68(9):3124–32] Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix-leucine zipper protein (16). Nine different Introduction isoforms of MITF with various tissue specific expression patterns Inhibitor of apoptosis (IAP) are a family of have been reported, including the melanocyte-specific isoform antiapoptotic regulators that block cell death in response to MITF-M (17). The MITF-M isoform is expressed specifically in diverse stimuli through interactions with inducers and effectors of melanocytes and melanomas where it transcriptionally regulates apoptosis (1). Melanoma IAP (ML-IAP; also known as Livin, KIAP, for melanogenesis, cell survival, and differentiation (16). A and BIRC7) is a potent antiapoptotic protein that is highly nonfunctional mutation of the Mitf in mice leads to a expressed in melanomas and other cancers, but not in most adult complete absence of melanocytes and consequent lack of coat tissues (2–5). IAP proteins inhibit apoptotic stimuli that signal color (16). In humans, MITF mutation causes Waardenburg either intrinsically, such as intracellular damage, or extrinsically, syndrome type IIA, resulting in melanocyte deficiencies in the in the case of signaling via death receptor complexes, pathways skin and inner ear, hypopigmentation, and deafness (18). The (1). These stimuli lead to activation of caspases, cysteine- MITF gene is amplified in 15% to 20% of metastatic melanomas dependent aspartyl-specific proteases that are critical for the (19), and it is considered a lineage survival oncogene due to its requirement for melanocyte proliferation and differentiation and high expression level in the majority of melanomas (16, 20). As a Note: Supplementary data for this article are available at Cancer Research Online member of the MYC family of transcription factors, MITF binds (http://cancerres.aacrjournals.org/). Requests for reprints: Domagoj Vucic, Department of Protein Engineering, to the consensus E box sequence CA(C/T)GTG (21–23) within Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080. Phone: 650-225- target genes such as cyclin-dependent kinase 2 (CDK2) and the 8839; Fax: 650-225-6127; E-mail: [email protected]. BCL-2 I2008 American Association for Cancer Research. antiapoptotic factor (24, 25). To date, both CDK2 and Bcl-2 doi:10.1158/0008-5472.CAN-07-6622 have been implicated as important mediators of the effects of

Cancer Res 2008; 68: (9). May 1, 2008 3124 www.aacrjournals.org MITF Regulates ML-IAP Transcription in Melanomas

MITF on melanoma proliferation and survival (24, 25). However, the RNeasy MiniKit (all Qiagen). For real-time PCR analysis, 100 ng of total there are likely additional MITF targets that also contribute to the RNA were used per standard 50-AL reaction volume. Fold change in gene role of MITF in melanomas. expression was calculated using the comparative CT method of relative Here we report that the ML-IAP promoter contains two quantitation. Standard curves were generated for all primer probe sets to confirm linearity of signals and relative efficiency over the experimentally functional MITF binding sites and that ML-IAP and MITF-M measured ranges. The following sequences were used: for detection of ML- mRNA levels are well correlated in melanoma patient samples IAP, 5¶-TTCCCCGACCACCGC (F), 5¶-AAACAGGTACAGTTAAGCCATCCC (R), ML-IAP and cell lines. We show that MITF binds to the promoter and 5¶-FAM-AGGAGGCCCTTGCTTGGCGTG-TAMRA (probe); for ML-IAP and transcriptionally regulates expression using luciferase MITF-M, 5¶-TCTCTTTGCCAGTCCATCTTCA (F), 5¶-CTGCACCTGATAGT- reporter assays, chromatin immunoprecipitation, electrophoretic GATTATATTCTAGCA (R), and 5¶-FAM-ACCGTCTCTCACTGGATTGG- mobility shift assay, and small interfering RNA (siRNA) knock- TGCCA-TAMRA (probe); for Axin2, 5¶-CTCCGTGTGTCGCCCTAT (F), 5¶- down of MITF in melanoma cells. Additionally, up-regulation of CATGACAAAAGTCATTGAGTACAAGA (R), and 5¶-FAM-TTGAGGGCT- MITF expression in melanocytes and melanoma cells was CAAGCTTTCCCTTGT-TAMRA (probe); for p21, 5¶-AACACCTTC- ¶ ¶ sufficient to elevate ML-IAP mRNA levels. Lastly, MITF modula- CAGCTCCTGTAACATA (F), 5 -GGGAACCAGGACACATGGG (R), and 5 - ¶ tion of ML-IAP expression was shown to play a vital role in FAM-TGGCCTGGACTGTTTTCTCTCGGC-TAMRA (probe); for MCP-1, 5 - GGGAAATTGCTTTTCCTCTTGA (F), 5¶-CTTTGTATTAATGATTCTTGCAAA- melanoma cell survival. GACC (R), and 5¶-FAM-CCACAGTTCTACCCCTGGGATGTTTTGA-TAMRA (probe); and for hRPL19, 5¶-AGCGGATTCTCATGGAACA (F), 5¶- Materials and Methods CTGGTCAGCCAGGAGCTT (R), and 5¶-FAM-TCCACAAGCTGAAGGCAGA- Cell culture and media. A375, A2058, SK-MEL-28, and UACC62 human CAAGG-TAMRA (probe). melanoma and Hek293T human embryonic kidney cells were obtained from Construction and mutagenesis of ML-IAP reporter constructs. A ¶ American Type Culture Collection and maintained in 50:50 RPMI 1640/ 2.8-kb fragment containing the 5 -untranslated region and upstream DMEM with 10% fetal bovine serum (FBS). 888 and 624 melanoma cells sequence of ML-IAP was amplified from human BAC clone 3243M2 ¶ were maintained in DMEM with 10% FBS in the absence of antibiotics (as (Invitrogen) using primers 5 -CACCCCACGGTATCATCCTGCAAAGCTGC ¶ described in ref. 2). Normal human epidermal melanocytes were purchased (F) and 5 -CTTGGCACTGTCTTTAGGTCCCATGGAG (R). The smaller from Cambrex and maintained in MBM-4 medium supplemented with the 1.6-kb fragment ML-IAP Pro 4 was subsequently amplified from the ¶ MGM-4 bullet kit (all Cambrex). 2.8-kb fragment using the primers 5 -GACTCTCGAGGAGC- ¶ In situ hybridization. PCR primers were designed to amplify a 447- CTTCTCTGCTTCCG (F) and 5 -GCTAAAGCTTGGAGGGAACACTGG- bp fragment of human ML-IAP spanning nucleotides (nt) 21 to 447 of CTCTG (R). The other ML-IAP promoter constructs were amplified from NM_139317.1 (upper, 5¶-CAAGTGCCTGCACCGTGGACC-3¶;lower,5¶- either BAC clone or genomic DNA (Roche) using the same reverse primer GGGGAACCACTTGGCATGCTC-3¶) or a 451-bp fragment of human MITF as used for ML-IAP Pro 4 and the following forward primers: ML-IAP ¶ spanning nt 1 to 450 of NM_ 000248 (upper, 5¶-ATGCTGGAAATGCTA- Pro 3, 5 -GACTCTCGAGCCTGGCTTCTGGGCTCTATC; ML-IAP Pro 2, ¶ ¶ GAATAT-3¶; lower, 5¶-GACAGGCAACGTATTTGCCAT-3¶). Primers included 5 -GCTACTCGAGCTGGCCTGAGGACAAGAG; and ML-IAP Pro 1, 5 - extensions encoding 27-nt T7 or T3 RNA polymerase initiation sites to GACTCTCGAGGAGCCTTCTCTGCTTCCG. PCR products were digested Xho Hin allow in vitro transcription of sense or antisense probes, respectively, with I and dIII and inserted into pGL4.10 (Promega). Site-directed from the amplified products. Formalin-fixed, paraffin-embedded sections mutagenesis was done using the QuickChange II (Stratagene) kit according (5 Am thick) were deparaffinized, deproteinated in 10 Ag/mL proteinase to the manufacturer’s recommendations. Mutagenesis primers were ¶ GAGGTG K for 45 min at 37jC, and further processed for in situ hybridization as designed as follows: for M1, 5 -CCCGCTGCACAGAG ACCCCA- 33 ¶ CTCCTC previously described (26). [ P]UTP-labeled sense and antisense probes AGAGGC and 5 GCCTCTGGGGTCAC TGTGCAGCGGG; for M2, ¶ CACCTC ¶ were hybridized to the sections at 55jC overnight. Unhybridized probe 5 -CAGGCAGCACAGCT TCCTTCCTTGAGCCTTC and 5 - was removed by incubation in 20 Ag/mL RNase A for 30 min at 37jC, GAAGGCTCAAGGAAGGAGAGGTGAGCTGTGCTGCCTG. Â 5 followed by a high stringency wash at 55jCin0.1Â SSC for 2 h and Luciferase reporter assay. Hek293T cells, seeded at 8 10 per well in dehydration through a graded ethanol series. The slides were dipped in six-well dishes, were transfected the following day using Lipofectamine 2000 NTB nuclear track emulsion (Eastman Kodak), exposed in sealed plastic with ML-IAP reporter constructs, pTopglow, or pGL4.1 together with either slide boxes containing dessicant for 4 wk at 4jC, developed, and pEGFP-MITF or pEGFP alone, or in combination with lymphoid enhancer counterstained with H&E. Hu-ML-IAP (P13P14) sequences align with ref factor-1 (LEF-1) or h-catenin S45Y constructs and pRL-Renilla (Promega). A sequence NM_139317.1 at nt 194 to 600, and Hu-MITF (p5P6) sequences total of 4 Ag of DNA were used for each transfection. Cell lysates were align with ref sequence NM_000248.2, nt 122 to 571, according to the prepared 48 h posttransfection; the activities of firefly and Renilla luciferase BLAST search. were assayed using a Dual Luciferase kit (Promega) according to the Bioinformatics. Transcription factor binding site analysis of the ML-IAP manufacturer’s instructions, and signals were normalized to the internal promoter was done using TRANSFAC from BIOBASE.4 mRNA expression Renilla controls for transfection efficiency. levels of MITF, ML-IAP, and CDK2 were extracted from the Gene Logic Chromatin immunoprecipitation. Chromatin immunoprecipitation expression database of Affymetrix U133 Plus 2.0 data, assays were done using the EZ ChIP kit (Upstate) according to the representing 51 normal skin and 29 melanoma samples. The following probe manufacturer’s instructions, with 5 Ag of mouse monoclonal anti-MITF sets were chosen to represent the expression of the transcripts: 226066_at for antibody (Calbiochem) or 5 Ag of mouse immunoglobulin IgG1 (BD MITF, 220451_s_at for ML-IAP, and 204252_at for CDK2. Pearson’s product- Biosciences). PCR primers used for amplification for the ML-IAP E1 site moment correlation coefficients and the associated P values were calculated were 5¶-GTGCAGTCAGCGGCATCC (F) and 5¶-AACACTGGCTCTGAC- to assess the correlation of gene expression between gene pairs. CAGGTTTG (R); PCR primers for heat shock protein 70 (Hsp70) were as Real-time quantitative PCR. The indicated cell lines were treated with described (27). 10 Amol/L forskolin (Sigma) for 24 h, 30 Ag/mL Wnt3a for 24 h, 10 Amol/ Electrophoretic mobility shift assays. Nuclear extracts from 624 Letoposide (Sigma) for 5 h, or 50 ng/AL tumor necrosis factor a (TNFa; cells were prepared using the NE-PER kit (Pierce) according to Genentech, Inc.) for 5 h. RNA was extracted with QIAshredder columns and the manufacturer’s instructions. Synthesis and biotin end-labeling of probes were carried out at Genentech, and single-stranded complemen- tary oligos were annealed to produce the double-stranded probe. The sequences of the probe containing the MITF E1 site in the ML-IAP promoter used to detect sequence-specific MITF binding 4 http://www.biobase-international.com in vitro were 5¶-CGCTGCACAGAGCATGTGACCCCAGAGGCC and www.aacrjournals.org 3125 Cancer Res 2008; 68: (9). May 1, 2008 Cancer Research

5¶-GGCCTCTGGGGTCACATGCTCTGTGCAGCG. Binding reactions were CAAdTdT and 5¶-TTGTTGTACAGGTTAGGTCdTdT; 4, 5¶-AGACGGAGCA- done using the LightShift kit (Pierce). Briefly, 4 Ag of 624 nuclear extract CACTTGTTAdTdT and 5¶-TAACAAGTGTGCTCCGTCTdTdT; and 5, 5¶- and binding buffer were incubated on ice for 20 min in a volume of 20 AL, GGTGAATCGGATCATCAAGdTdT and 5¶-CTTGATGATCCGATT- then the labeled probe (20 fmol) was added, and the reaction was allowed CACCdTdT. Scramble II (Dharmacon) siRNA was used for a negative to incubate for an additional 20 min at room temperature. For control in siRNA transfections. competition and supershift analyses, unlabeled DNA probe (in 25- to Western blot analysis, immunoprecipitation, and antibodies. 200-fold excess), anti-MITF antibody (Calbiochem; 1 Ag per reaction), or Western blot analyses and immunoprecipitation were done as previously mouse immunoglobulin IgG1 (BD Biosciences; 1 Ag per reaction) was described (2). Monoclonal antibodies against h-actin (ICN Biomedicals), added to the reaction mixture during the initial 20-min incubation. The CDK2 (Lab Vision), MITF (Calbiochem), Myc (Upstate), and FLAG (Sigma) reaction products were separated on a 4% native polyacrylamide gel run were purchased. Monoclonal antibodies against human ML-IAP were in 0.5 Â Tris-borate EDTA. Following electrophoresis, the DNA-protein generated at Genentech. complexes were transferred onto nylon membranes and detected by Viability and apoptosis assays. 888 melanoma cells were seeded at a chemiluminescence (LightShift kit, Pierce). density of 5 Â 104 per well in 24-well plates or 1 Â 104 per well in 96-well Expression vectors, siRNAs, and transfection. pEGFP-MITF was plates and cotransfected with expression vectors and siRNA as described constructed by transferring the ultimate ORF clone IOH45654 insert into above. Twenty-four hours posttransfection, apoptosis was measured by pcDNA-DEST53 using LR clonase (all Invitrogen) according to the analyzing caspase-3/caspase-7 activity using the Homogeneous ApoOne manufacturer’s protocol. pCANmycLEF1, pCANmych-catenin S45Y, and Caspase-3/7 Assay kit (Promega) according to the manufacturer’s pTopglow were kindly provided by Bonnee Rubinfeld. Constructs instructions. Cell viability was measured 48 h posttransfection by Neutral expressing Myc-ML-IAP, FLAG-ML-IAP, and FLAG-ML-IAP K138A have Red uptake as described (28). previously been described (2, 11). For siRNA and DNA transfections, Lipofectamine 2000 (Invitrogen) was used according to the manufac- Results turer’s instructions. siRNA oligonucleotides were synthesized at Genen- ML-IAP tech. The following siRNA pairs were used for MITF knockdown MITF is a candidate transcriptional regulator of . experiments: 1, 5¶-GAACGAAGAAGAAGATTTAdTdT and 5¶- To identify transcription factors that could influence the TAAATCTTCTTCTTCGTTCdTdT; 2, 5¶-GCAGATGGATGATGTAATCdTdT melanoma-specific expression of ML-IAP, the putative promoter and 5¶-GCAGATGGATGATGTAATCdTdT; 3, 5¶-GACCTAACCTGTACAA- region encompassing 1 kb upstream of the translation start site

Figure 1. MITF is a candidate transcriptional regulator of the human ML-IAP locus. A, schematic representation of the promoter region upstream of the human ML-IAP locus containing two MITF consensus E box elements, named E1 and E2. TSS, transcriptional start site. B, ML-IAP and MITF mRNA levels are well correlated in human melanomas, but not in normal skin. mRNA expression levels of MITF and ML-IAP from microarray data values were analyzed as described in Materials and Methods. Data plots of MITF versus ML-IAP expression in melanoma (open circles) and normal skin (crosses) samples are shown. The straight line represents the least squares regression line of MITF expression on ML-IAP expression in melanoma samples. Pearson’s correlation coefficient (r) = 0.68, P = 0.00006; melanoma sample, n = 29; normal skin sample, n = 51. C, Correlation of ML-IAP and MITF-M mRNA levels in human melanoma cell lines. Total RNA was extracted, ML-IAP and MITF-M transcript levels were measured by real-time PCR analysis, expression levels were normalized to that of hRPL19 (human ribosomal protein L19), and relative abundance levels were calculated with 888 melanoma cells as the reference.

Cancer Res 2008; 68: (9). May 1, 2008 3126 www.aacrjournals.org MITF Regulates ML-IAP Transcription in Melanomas

Figure 2. ML-IAP and MITF expression is concordant in human melanomas. A and B, in situ hybridization using 33P-labeled RNA probes for MITF (A) or ML-IAP (B) mRNA expression in tissue sections from the same melanoma tissue sample (ID #16148). C and D, corresponding H&E histology of melanoma tissue sections in A and B, respectively.

of the ML-IAP gene was searched against the TRANSFAC gene deemed nonconcordant. In 81% (58 of 72 total) of the melanoma regulation database using the TRANSFAC patch (pattern-based) cases surveyed, ML-IAP and MITF expression patterns were program. Two sites that matched the E box consensus-binding found to be significantly concordant (P = 0.00000003, Fisher’s site CA(C/T)GTG of MITF were identified within the ML-IAP exact test; Fig. 2; Table 1). For the 19 cases that were scored as promoter region. One site resides 46 bp and another 282 bp 2+ for MITF, 17 (89%) of those were also positive for ML-IAP upstream of the ML-IAP translation start site (Fig. 1A), herein (Supplementary Table S2). These data, together with the referred to as site E1 and E2, respectively. experimental findings presented below, strongly suggest that Because ML-IAP and MITF are both highly expressed in the MITF is a critical regulator of ML-IAP expression. However, majority of melanomas studied thus far (2, 13, 29), we sought to whereas 15% (11 of 72) of cases positive for MITF were negative explore a potential similarity between ML-IAP and MITF for ML-IAP, 4% (3 of 72) of cases negative for MITF were transcript expression in normal skin and melanoma samples. positive for ML-IAP, indicating the possibility of additional Analysis of microarray expression data using Pearson’s correlation mechanisms in the regulation of ML-IAP expression (Table 1 and coefficient revealed that there was a statistically significant linear Supplementary Table S2). Our discovery that MITF and ML-IAP relationship between ML-IAP and MITF expression in melanoma expression is significantly correlated in human melanoma cells samples (r = 0.68, P = 0.00006, n = 29), whereas no correlation and tumor samples, together with the classification of MITF as a was detected in normal skin samples (Fig. 1B; Supplementary lineage survival oncogene, particularly in melanomas, suggested Table S1). In addition, specifically in melanomas but not in that MITF is a transcription factor well poised to regulate normal skin samples, transcript levels of ML-IAP correlated ML-IAP expression in melanoma. strongly with those of CDK2, a known transcriptional target of MITF binds and regulates ML-IAP promoter. To assess MITF in melanomas (ref. 24; Supplementary Table S1). To further whether MITF-M can directly regulate ML-IAP expression, investigate the correlation between MITF and ML-IAP expression luciferase reporter constructs containing the upstream ML-IAP patterns, mRNA levels of ML-IAP and MITF-M, the melanocyte- region were transiently transfected into Hek293T cells with or specific isoform of MITF, were surveyed in a panel of six without MITF-M. Cotransfection of MITF-M with ML-IAP melanoma cell lines by real-time PCR analysis. This analysis promoter constructs containing either one (ML-IAP Pro 1) or revealed a strong correlation between ML-IAP and MITF-M two (ML-IAP Pro 2) MITF consensus sites resulted in strong expression levels (Fig. 1C). reporter activation (Fig. 3A). This MITF-dependent transcriptional We extended our correlation analysis by evaluating the status regulation required the presence of intact E box MITF binding of ML-IAP and MITF expression in archived melanoma patient sites because disrupting both E boxes simultaneously (ML-IAP samples by in situ hybridization. The concordance of MITF and Pro 2 M1 M2) or mutating the single E1 site in the shorter ML-IAP expression was defined as follows: when (a) both genes construct (ML-IAP Pro 1 M1) resulted in a complete loss of MITF- are negative; (b) both genes are positive, irrespective of levels of M–dependent regulation (Fig. 3A). It seems that both MITF sites positive signals; and (c) both genes are equivocal for expression. contribute equally to the transcriptional regulation of ML-IAP,as Samples positive for one gene but negative for the other are evidenced by the similar MITF-M responsiveness of the two www.aacrjournals.org 3127 Cancer Res 2008; 68: (9). May 1, 2008 Cancer Research constructs in which either single MITF site is mutated (compare suggesting that ML-IAP is a target of MITF-M in melanocytes in ML-IAP Pro 2 M1 and ML-IAP Pro 2 M2 in Fig. 3A). Similar response to signaling stimuli. results were also obtained when ML-IAP luciferase reporter Expression of ML-IAP is not regulated by Wnt, DNA constructs were cotransfected with MITF-M in 888 melanoma damage, or TNF-mediated signaling in melanoma cells. The cells (Supplementary Fig. S1). Thus, MITF-M can bind to and Wnt/h-catenin signaling pathway has been implicated in the regulate the ML-IAP promoter region in an E-box–dependent formation of malignant melanoma (31), in part, through its fashion. transcriptional regulation of the MITF-M promoter (32–34). To determine whether the endogenous ML-IAP promoter is MITF has also been shown to bind to and cooperate with bound by MITF, 888 cells were analyzed by a chromatin h-catenin and LEF-1 in transcriptional regulation of target genes immunoprecipitation assay with an anti-MITF antibody. The MITF (27, 35). Thus, we investigated whether Wnt signaling could E1 site–containing region of the ML-IAP promoter was amplified contribute to ML-IAP expression in melanomas. Two potential from cross-linked chromatin from 888 cells that was immunopre- LEF-1 consensus-binding sites CTTTG[A/T][A/T] were identified cipitated with anti-MITF antibody but not with isotype control in the ML-IAP promoter region upstream from the MITF E1 and antibody, or no template control (Fig. 3B). PCR amplification with E2 sites at 483 bp (CTCTGAT; note mismatch at the third primers specific for the Hsp70 promoter region served as a negative nucleotide position) and 1,303 bp (CTTTGAT) away from the control for the chromatin immunoprecipitation assay. Thus, MITF ML-IAP translation start site. ML-IAP reporter constructs specifically binds the ML-IAP promoter region. None of the containing either one (ML-IAP Pro 3) or both LEF-1 sites available antibodies recognize endogenous MITF in immunoblots, (ML-IAP Pro 4), or lacking LEF-1 sites (ML-IAP Pro 2 and ML- but we confirmed the functionality of the MITF antibody used in IAP Pro 1), did not show any responsiveness when either a chromatin immunoprecipitation experiments by immunoprecipi- stabilized h-catenin mutant or LEF-1 was ectopically expressed tating overexpressed GFP-MITF (Supplementary Fig. S2). Further- alone, together, or with MITF-M (Fig. 4A). Moreover, treatment more, MITF was shown to specifically bind to the ML-IAP of melanoma cell lines with Wnt3a led to no appreciable change promoter by an electrophoretic mobility shift assay (Fig. 3C). In in ML-IAP mRNA levels as assessed by real-time PCR analysis summary, MITF was found to regulate the transcriptional activity (Supplementary Table S3). At the same time, mRNA levels of a of ML-IAP promoter reporter constructs in an E-box–dependent bona fide target of Wnt signaling, Axin2 (36), were significantly manner, bind to the ML-IAP promoter in vitro, and occupy the elevated (Supplementary Table S3). endogenous ML-IAP promoter in melanoma cells. Both p53 and nuclear factor-nB family member transcription Up-regulation of MITF via activation of the cyclic AMP factors have been shown to regulate expression of several inhibitors pathway in melanocytes leads to increased ML-IAP expres- of apoptosis (37–39). In addition, the ML-IAP promoter region sion. In contrast to its wide expression in the majority of contains potential binding sites for these transcription factors melanomas, ML-IAP is usually not expressed in benign melano- (data not shown). Therefore, ML-IAP mRNA levels were monitored cytic lesions and normal melanocytes (2, 13). If MITF-M regulates in melanoma cells treated with either the DNA-damaging agent ML-IAP expression in melanomas, then increasing the level of etoposide or TNFa. Although the mRNA levels of p21 and MCP-1, MITF-M in melanocytes could be sufficient to allow for ML-IAP well-established p53 and nuclear factor-nB target genes, increased expression in melanocytes. To this end, normal human epidermal accordingly with treatment, ML-IAP levels were unchanged (Fig. 4B melanocytes were treated with the cyclic AMP (cAMP) agonist and C). Thus, unlike our findings for MITF, ML-IAP expression was forskolin. Forskolin mimics the action of natural signaling not affected by Wnt, p53, or TNF signaling in the melanoma cell through the melanocortin receptor that up-regulates the cAMP lines surveyed. pathway and increases MITF expression during melanocyte ML-IAP transcription in melanoma cells is dependent on growth and differentiation (30). Following a 24-hour treatment MITF. If ML-IAP is a true transcriptional target of MITF in with forskolin, ML-IAP mRNA levels increased >2-fold in normal melanoma cells, decreasing MITF expression should lead to human epidermal melanocytes, coincident with increased expres- concordant lower levels of ML-IAP mRNA and ML-IAP protein. To sion of MITF-M and CDK2 (Fig. 3D). Therefore, ML-IAP test this hypothesis, 888 melanoma cells were transiently trans- expression in melanocytes can be induced via activation of the fected with multiple siRNAs targeting MITF. Significantly lower cAMP pathway, which also increases the expression of MITF-M, protein levels of ML-IAP, as well as that of the known MITF target

Table 1. Prevalence of MITF and ML-IAP expression in melanomas

MITF ISH cases (n = 72) ML-IAP ISH cases (n = 72)

No. positive cases No. negative cases No. positive/negative cases

No. positive cases 39* 11 0 No. negative cases 3 18* 0 No. positive/negative cases 0 0 1*

Abbreviation: ISH, in situ hybridization. *Indicates concordant expression of both genes in identical tumor samples.

Cancer Res 2008; 68: (9). May 1, 2008 3128 www.aacrjournals.org MITF Regulates ML-IAP Transcription in Melanomas

Figure 3. MITF-M binds to the ML-IAP promoter and ML-IAP mRNA levels in melanocytes are affected by MITF-M up-regulation. A, MITF-M transcriptional activation of the ML-IAP promoter requires intact MITF-binding E box sequences. ML-IAP promoter constructs, with or without point mutations in the E1 and E2 MITF consensus sites, were cotransfected for 48 h with MITF-M or vector control into Hek293T cells. Firefly luciferase activities were normalized to Renilla luciferase activities, and the ratios between firefly and Renilla luciferase (Luc/Ren) are shown. B, MITF binds to the endogenous ML-IAP promoter in melanoma cells. Chromatin immunoprecipitations from 888 melanoma cells transfected for 48 h with GFP-MITF were done as described in Materials and Methods. PCR products were resolved on a 3% agarose gel, and DNA from lysates before immunoprecipitation was used as a positive control. The E1 primer set specifically amplifies a region in the ML-IAP promoter including the MITF E1 site, whereas the Hsp70 primer set amplifies a region of the Hsp70 promoter region, which is included as a negative control. C, MITF binds to the ML-IAP promoter by electrophoretic mobility shift assay. An electrophoretic mobility shift assay shows sequence-specific DNA binding of MITF to the ML-IAP promoter site in vitro. Lane 1, biotinylated probe containing the ML-IAP E1 site in the absence of 624 cell nuclear extract; lane 2, binding of MITF in 624 nuclear extract to the biotinylated probe containing the ML-IAP E1 site. Excess unlabeled probe was used at increasing concentrations (25-, 50-, 100-, and 200-fold in lanes 3–6, respectively) to compete out the bandshift. Addition of anti-MITF antibody, but not IgG1 control, was sufficient to supershift the complex (lanes 7 and 8). Asterisk, supershift, showing the specificity of the complex. D, cAMP increases expression of MITF-M and ML-IAP mRNA in melanocytes. Normal human embryonic melanocytes were stimulated for 24 h with the cAMP agonist forskolin; total RNA was extracted; ML-IAP, CDK2, and MITF-M transcript levels were measured by real-time PCR analysis; and expression levels were normalized to that of hRPL19. Columns, mean from duplicate experiments; bars, SE.

gene CDK2, were observed with four of five MITF siRNAs tested MITF and ML-IAP are critical for melanoma survival. Loss or with a pool of all five MITF siRNAs (Fig. 5A). The siRNA- of MITF function, either by siRNA knockdown or expression of a mediated knockdown of MITF was confirmed for the four dominant negative allele, leads to reduced melanoma growth successful individual and pool siRNAs by real-time PCR to and decreased melanocyte cell viability (25, 40). If regulation of measure MITF-M mRNA levels (Fig. 5A). ML-IAP mRNA levels ML-IAP expression by MITF is required for melanoma cell were also lowered significantly as assessed by real-time PCR viability, then overexpression of ML-IAP from a constitutive analysis, as were those of CDK2 (Fig. 5A). MITF siRNA promoter should be able to rescue MITF siRNA–induced cell transfection of another melanoma cell line, 624, led to a sizeable death. Indeed, transient cotransfection of ML-IAP measurably decrease in ML-IAP protein and RNA levels as well (Fig. 5B). rescued the cell death induced by MITF siRNA in 888 melanoma Additionally, transient overexpression of MITF-M in A2058 and cells (Fig. 5C). MITF knockdown–stimulated cell death was UACC62 melanoma cells, both of which have very low expression accompanied by elevated caspase activity and nuclear conden- levels of ML-IAP and MITF, was sufficient to induce increased sation, confirming its apoptotic nature (Fig. 5C and Supplemen- ML-IAP mRNA levels (Supplementary Table S4). Thus, ML-IAP tary Fig. S3A). Again, ectopic expression of ML-IAP inhibited expression is MITF dependent and regulated by MITF at the these apoptotic features of MITF siRNA–induced cell death transcriptional level in melanoma cells. (Fig. 5C and Supplementary Fig. S3A). For ML-IAP to exert its www.aacrjournals.org 3129 Cancer Res 2008; 68: (9). May 1, 2008 Cancer Research antiapoptotic function, it must retain a functional interaction regulation of ML-IAP expression in non–small-cell lung cancer with SMAC, which can be abolished by the mutation of cells reported that ML-IAP is a target of h-catenin/T-cell factor aspartate to alanine at position 138 within the ML-IAP BIR signaling (42). However, the Wnt pathway did not enhance the domain (2, 41). Notably, cotransfection of a flag-tagged ML-IAP transcriptional activation of ML-IAP or alter ML-IAP mRNA harboring the D138A mutation was unable to rescue MITF levels in reporter assays or by stimulation of the Wnt pathway in siRNA–induced cell death in 888 melanoma cells, whereas in the melanoma cells, likely reflecting differential regulation of ML-IAP same experiment, cotransfection of wild-type FLAG-ML-IAP led in melanomas versus lung cancer cells. to a significant rescue of melanoma cell viability (Supplementary Up-regulation of MITF expression, through stimulation of the Fig. S3B). These data establish ML-IAP as a functionally cAMP pathway, led to a significant increase in ML-IAP mRNA important target of MITF, and suggest that ML-IAP contributes levels in normal melanocytes. Treatment of melanocytes with to the prosurvival effects of MITF-M in melanoma cells. cAMP-elevating agents has previously been shown to result in increased MITF expression (43), as well as up-regulation of target genes with somewhat paradoxically opposing functions. On one Discussion hand, cAMP pathway stimulation of MITF activity in melano- In the present work, we identify ML-IAP as a new transcriptional cytes up-regulates the expression of hypoxia-inducible factor 1a target of MITF, a melanocyte master regulator and lineage survival (HIF1a) and the hepatocytye growth factor receptor MET, which oncogene in melanoma cells. promote cell survival (44, 45). On the flip side, MITF also up- CIP1 We report that MITF binds to and activates transcription from regulates the inhibitors of cell cycle progression, p21 and INK4A the ML-IAP promoter, and that ML-IAP expression is dependent p16 (46, 47). Likewise, in melanomas, MITF targets a on MITF-M in melanoma cells. Other candidate transcriptional number of genes with antagonistic behaviors, including genes regulators of ML-IAP including p53, nuclear factor nB, h-catenin, such as CDK2 and BCL-2, which promote cell cycle progression CIP1 INK4A and LEF-1 were also explored. This is particularly relevant for and survival, as well as p21 and p16 , which halt the cell h-catenin and LEF-1 because a recent study investigating the cycle (24, 25, 46, 47). The decision of whether MITF will exert a

Figure 4. ML-IAP expression is not influenced by Wnt pathway transcription factors, DNA damage, or TNF-mediated signaling. A, neither h-catenin nor LEF-1 activates MITF-M–dependent or MITF-M–independent transcription of ML-IAP. ML-IAP promoter constructs were cotransfected with the indicated plasmids for 48 h in Hek293T cells. Firefly luciferase activities were normalized to Renilla luciferase activities, and the ratios between firefly and Renilla luciferase normalized to vector-transfected samples are shown. The h-catenin/LEF-1 responsive Topglow reporter is included as a positive control for transcriptional activation by h-catenin and LEF-1. B, DNA damage does not stimulate ML-IAP expression in melanoma cells. Indicated cell lines were treated with 10 Amol/L etoposide for 5 h. Total RNA was extracted, ML-IAP and p21 transcript levels were measured by real-time PCR analysis, and expression levels were normalized to that of hRPL19. n.d., not detected, undetectable mRNA levels in both untreated and treated samples. C, TNF signaling does not regulate ML-IAP levels in melanoma cells. Indicated cell lines were treated with 20 ng/mL TNFa for 5 h. Real-time PCR analysis for ML-IAP and MCP-1 mRNA levels was done as in B. n.d., mRNA levels in both untreated and treated samples was not detected.

Cancer Res 2008; 68: (9). May 1, 2008 3130 www.aacrjournals.org MITF Regulates ML-IAP Transcription in Melanomas

Figure 5. MITF-M transcriptionally regulates endogenous ML-IAP in melanoma cells and MITF transcriptional regulation of ML-IAP is critical for melanoma survival. A, down-regulation of ML-IAP protein and mRNA levels by siRNA-mediated depletion of MITF in 888 melanoma cells. Melanoma cells were transfected with individual siRNA duplexes 1 to 5, or a pool of siRNAs 1 to 5 (denoted P), targeting MITF, or control siRNA. Left, 48 h posttransfection, cells were harvested and lysates were subject to Western blot analysis with anti–ML-IAP, anti-CDK2, and anti-actin antibodies. Right, 24 h posttransfection, total RNA was extracted, ML-IAP, CDK2, and MITF-M transcript levels were measured by real-time PCR analysis, and expression levels were normalized to hRPL19. B, ML-IAP protein and mRNA levels are lowered by siRNA-mediated depletion of MITF in 624 melanoma cells. Melanoma cells were transfected with a pool of MITF siRNAs (1–3 and 5 from A and B), targeting MITF, or control siRNA. Left, 48 h posttransfection, cells were harvested and lysates were subject to Western blot analysis as in A. Right, 24 h posttransfection, total RNA was extracted and real-time PCR analysis was done as in A. C, overexpression of a ML-IAP rescues loss of melanoma cell viability and increased caspase activity induced by MITF siRNA. Left, 888 melanoma cells were cotransfected with MITF or control siRNA and myc-tagged ML-IAP or vector DNA. Forty-eight hours posttransfection, lysates were prepared for Western blot analysis with anti–ML-IAP, anti-MYC, and anti-actin antibodies. Middle, cell viability was assessed by Neutral Red staining as described in Materials and Methods. Columns, mean from at least five independent experiments normalized to control siRNA– and vector DNA–trransfected cells; bars, SE. Right, caspase-3/caspase-7 activity was assayed 24 h after transfection. Columns, mean from at least five independent experiments normalized to control siRNA– and vector DNA–transfected cells expressed as relative fluorescent units (RFLU); bars, SE. prosurivival or growth inhibitory effect in melanocytes and metastatic lesions and the very infrequent expression of ML-IAP in melanoma growth is not fully understood at present, but it is benign melanocytic proliferation, using a combination of both likely that the cellular context and microenvironment are anti–ML-IAP and anti-MITF antibodies could prove useful in the important factors. Our studies suggest that MITF regulation of clinical diagnosis of melanoma. Interestingly, in primary cultures ML-IAP favors a prosurvival outcome in melanoma, and that derived from melanoma patients, a correlation between ML-IAP ML-IAP may play a role in melanocytic transformation. expression and chemotherapeutic drug resistance has been The high degree of correlation of ML-IAP and MITF mRNA levels reported (49). Whether the expression of ML-IAP in melanomas we report in this study provokes the question about whether has any prognostic or therapeutic value needs to be addressed by ML-IAP may be a useful biomarker in melanoma diagnosis. future studies examining the influence of ML-IAP expression in Currently, a MITF-specific antibody is clinically used as a highly melanoma on survival outcome in patients. sensitive immunohistochemical marker in melanoma diagnosis Melanoma is a highly aggressive cancer that is resistant to (48). However, the expression of MITF only serves as a melanocytic current anticancer treatments including chemotherapy and other lineage marker and has no prognostic value in discriminating strategies that require induction of apoptosis (12). We propose benign from malignant melanoma lesions (16). Perhaps, given the that the transcriptional regulation of ML-IAP by MITF plays an specific expression of ML-IAP in the majority of primary and important role in promoting survival during melanocytic www.aacrjournals.org 3131 Cancer Res 2008; 68: (9). May 1, 2008 Cancer Research transformation and melanoma progression. ML-IAP has been Acknowledgments proposed as a promising tumor-specific target in cancer therapy Received 12/11/2007; revised 2/11/2008; accepted 3/6/2008. due to its powerful antiapoptotic function and preferential Conflict of interest: All authors are employees and shareholders of Genentech, Inc. expression in a number of tumor types (15, 50). Understanding The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance the mechanism(s) of ML-IAP up-regulation during melanocytic with 18 U.S.C. Section 1734 solely to indicate this fact. transformation as well as the modulation of ML-IAP expression in We thank Eugene Varfolomeev, Sarah Wayson, Bonnee Rubinfeld, Venita De Almeida, Paul Polakis, Adam Eldridge, Karen O’Rourke, Laszlo Komuves, William melanoma cells may aid in designing therapies targeting ML-IAP Forrest, and the Oligo Synthesis and Sequencing facilities at Genentech that provided in malignant melanomas. help with insightful discussions, suggestions and reagents.

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