Expression Profiling Reveals Novel Pathways in the Transformation of Melanocytes to Melanomas

[CANCER RESEARCH 64, 5270–5282, August 1, 2004] Expression Profiling Reveals Novel Pathways in the Transformation of Melanocytes to Melanomas

Keith Hoek,1 David L. Rimm,2 Kenneth R. Williams,1 Hongyu Zhao,3 Stephan Ariyan,4 Aiping Lin,1 Harriet M. Kluger,5 Aaron J. Berger,2 Elaine Cheng,6 E. Sergio Trombetta,7 Terence Wu,1 Michio Niinobe,8 Kazuaki Yoshikawa,8 Gregory E. Hannigan,9 and Ruth Halaban6 Departments of 1Molecular Biophysics and Biochemistry, 2Pathology, 3Epidemiology, 4Surgery, 5Internal Medicine, 6Dermatology, and 7Cell Biology, Yale University School of Medicine, New Haven, Connecticut; 8Division of Regulation of Macromolecular Functions, Institute for Protein Research, Osaka University, Osaka, Japan; and 9Laboratory Medicine and Pathobiology, Cancer Research Program, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

ABSTRACT silencing through promoter hypermethylation (6). The loss of p16INK4a expression correlates with the invasive stage of melanoma Affymetrix and spotted oligonucleotide microarrays were used to assess tumor progression (7–9), suggesting that its absence confers growth global differential gene expression comparing normal human melanocytes advantage. Retinoblastoma protein is also likely to be inactivated by with six independent melanoma cell strains from advanced lesions. The data, validated at the protein level for selected genes, confirmed the signaling pathways that stimulate mitogen-activated protein kinase, overexpression in melanoma cells relative to normal melanocytes of sev- such as activating mutation in B-Raf and N-Ras (10, 11), and consti- eral genes in the growth factor/receptor family that confer growth advan- tutive stimulation of cell membrane receptors, such as the fibroblast tage and metastasis. In addition, novel pathways and patterns of associ- growth factor (FGF) receptor 1 (12) and integrin pathway (13). One of ated expression in melanoma cells not reported before emerged, including the consequences of retinoblastoma protein inactivation is the release the following: (a) activation of the NOTCH pathway; (b) increased Twist of E2F transcription factor, leading to aberrant expression of E2F- expression and altered expression of additional transcriptional regulators regulated genes that promote cell cycle progression (reviewed in Refs. implicated in embryonic development and epidermal/mesenchymal tran- 14 and 15). sition; (c) coordinated activation of cancer/testis antigens; (d) coordinated Several high-throughput approaches have been applied to melano- down-regulation of several immune modulation genes, in particular in the mas to assess chromosomal aberrations and gene expression patterns IFN pathways; (e) down-regulation of several genes implicated in membrane trafficking events; and (f) down-regulation of growth suppressors, in an unbiased fashion. For example, comparative genome hybridiza- such as the Prader-Willi gene NECDIN, whose function was confirmed by tion identified several chromosomal and genetic changes. These in- overexpression of ectopic Flag-necdin. Validation of differential expres- clude losses of chromosomes 6q, 8p, and 10 and gains in copy number sion using melanoma tissue microarrays showed that reduced ubiquitin of chromosomes 1q, 6p, 7, and 8 in primary melanomas (16–22); COOH-terminal esterase L1 in primary melanoma is associated with amplification of the cyclin D1 locus in acral melanoma (17, 19); worse outcome and that increased expression of the basic helix-loop-helix frequent deletion of chromosome 13 and 17p in melanomas arising in protein Twist is associated with worse outcome. Some differentially ex- chronically sun-damaged skin; and frequent amplification of chromo- pressed genes reside on chromosomal regions displaying common loss or some 12q in mucosal melanomas that can affect metastatic potential gain in melanomas or are known to be regulated by CpG promoter (23, 24). Global gene expression profiling of several melanoma sub- methylation. These results provide a comprehensive view of changes in types has implicated dysregulation of the Wnt pathway in contributing advanced melanoma relative to normal melanocytes and reveal new targets that can be used in assessing prognosis, staging, and therapy of to melanoma invasion and motility (25, 26), which was validated by melanoma patients. ectopic expression of Wnt5a (26). More recently, serial analysis of gene expression of three melanoma tumors indicated up-regulation of intracellular calcium and G-protein signaling and increased expres- INTRODUCTION sion of CD74 (27). Most gene expression analysis studies did not include normal Wide-spread aberrations in gene expression due to losses and gains human melanocytes. Therefore, we set out to monitor global gene in genetic material, suppressive or activating mutations, and dysregu- expression in normal melanocytes compared with melanoma cells. As lation of transcription factors are rampant in cancer cells, including reported below, gene expression profiling, validated at the protein INK4a melanomas. Inactivation of p16 , the cyclin-dependent kinase level for selected gene products, provides a comprehensive view of inhibitor (cyclin-dependent kinase 4/6) that leads to suppression of molecular changes in malignant melanocytes and reveals possible retinoblastoma protein, is an early event commonly observed in mel- underlying molecular pathogenesis not identified before. anomas and other cancer cells as well (reviewed in Refs. 1 and 2). P16INK4a is eliminated in most melanomas not only by point muta- MATERIALS AND METHODS tions, but also by deletion (Refs. 3 and 4; reviewed in Ref. 5) or Cells. Normal human melanocytes from newborn foreskins were grown in Received 3/1/04; revised 5/10/04; accepted 5/21/04. Ham’s F-12 medium (GibcoBRL, Invitrogen Corp., Grand Island, NY) sup- Grant support: USPHS Grant CA44542 (R. Halaban), AR41942 Yale Skin Diseases plemented with serum [7% fetal bovine serum from Gemini Bio-Products Research Center; R. E. Tigelaar, Program Investigator, the Patrick and Catherine Weldon (Woodland, CA)], termed basal medium, which was further enriched with Donaghue Investigator Award (D. L. Rimm); the C. J. Swebilius Award (H. M. Kluger); and NIH/National Institute of General Medical Sciences Medical Scientist Training several ingredients required for optimal proliferation. They included 85 nM Program Grant GM07205 (A. J. Berger). 12-O-tetradecanoyl phorbol-13-acetate (TPA), 0.1 mM 3-isobutyl-1-methyl- The costs of publication of this article were defrayed in part by the payment of page ␮ 6 Ј xanthine, 2.5 nM cholera toxin, 1 M Na3VO4, and 0.1 mM N ,2 -O-dibutyry- charges. This article must therefore be hereby marked advertisement in accordance with ladenosine 3:5-cyclic monophosphate (all from Sigma-Aldrich, St. Louis, 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Cancer Research Online MO), termed TICVA (28). Early-passage melanocyte cultures (passage 1–2) (http://cancerres.aacrjournals.org)andathttp://info.med.yale.edu/microarray/pub࿝serv. pooled from three to six Caucasian donors were used to reduce individual htm. K. Hoek is currently in the Department of Dermatology, University Hospital of donor variations. The normal melanocytes proliferated at a population dou- Zurich, Zurich, Switzerland. bling time of of 3–4 days, were highly differentiated, and expressed all known Requests for reprints: Ruth Halaban, Department of Dermatology, Yale University School of Medicine, 15 York Street, P. O. Box 208059, New Haven, CT 06520-8059. melanocyte-specific genes (12). Phone: (203) 785-4352; Fax: (203) 785-7637; E-mail: [email protected]. The human primary melanoma WW165 (F, 2.25-mm lesion from the back) 5270

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. NOVEL PATHWAYS IN MELANOMAS and metastatic YUHEIK melanoma cells (F, maxillary gingiva, lymph node number, title, and chromosomal location using NetAffx, a web interface metastasis) were grown in basal medium supplemented with 3-isobutyl-1- program from Affymetrix Inc. For analysis of the Affymetrix data, values in methylxanthine required for optimal proliferation. The metastatic melanomas the range of 2.5–5-fold change were considered moderate, values in the MNT1 (M, lymph node metastasis; Ref. 29), YUMAC (M, recurrent metastasis 5–10-fold range were considered high, and values above the 10-fold range at the site of excision), YUCAL (F, soft tissue metastasis), YUSAC2 (M, soft were termed extremely high. tissue metastasis), YUSIT1 (site unknown), 501 mel (site unknown; Ref. 30), Initial processing of the fluorescent hybridization signal from OHU16K and YUGEN8 (F, brain) were maintained in basal medium as described microarray slides was performed with QuantArray 2.0 (Perkin-Elmer, Boston, previously (28). The YUHEIK, YUMAC, and YUCAL melanoma cells were MA) and Qamerge12 (Hongyu Zhao; Laboratory of Statistical Genomics and used during early passage in culture (passage 3–8). Proteomics, Yale Medical School, New Haven, CT). The OHU16K data were The normal and malignant melanocytes were harvested during the logarith- normalized using the Intensity Dependent (Lowess) method. The values of the mic growth phase to avoid differences due to cell cycle distribution because raw data were set to be 5-fold higher than the background level of 200. Genes normal melanocytes become arrested in the absence of specific growth factors that were differentially expressed between normal human melanocytes and under conditions that allow metastatic melanoma cells to proliferate; likewise, melanoma cells were generated using t tests with a significance level of 0.05. metastatic melanoma cells are arrested in the presence of TPA, N6,2Ј-O- The two types of microarrays were compared to find the common genes dibutyryladenosine 3:5-cyclic monophosphate, and cholera toxin (31, 32). using UniGene as a common identifier and the KARMA program13 (Keck The immortalized mouse melanocytes derived from a B10BR-black mouse Microarray Resource, Yale Center for Medical Informatics, Yale University). were grown in Ham’s F-12 medium supplemented with antibiotics, 10% horse Only the overlapped UniGene clusters were considered for the comparisons. serum, and TPA (33). When a UniGene cluster ID has more than one gene, the ratio for that cluster RNA Isolation and Application to DNA Microarrays. Approximately was calculated using the average value of the corresponding genes. 20–30 million cells (normal human melanocytes and melanoma cells/each) Hierarchical cluster analysis was performed by using the agglomerative were used for mRNA extraction per hybridization. High-quality total RNA was clustering algorithm QT_Clust (37) to determine associations between coex- prepared with the TRIzol reagent (Invitrogen Life Technologies, Inc., Invitro- pressed genes allowing for a correlation coefficient of 0.97 as described gen Corp., Carlsbad, CA). The quality of RNA products was determined online.14 visually by 1% denaturing agarose gel, and RNA concentration was measured Western Blot Analysis. Normal melanocytes and melanoma cells were using a spectrophotometer. Poly(A) mRNA was further isolated from ϳ300 ␮g lysed in radioimmunoprecipitation assay buffer (PBS, 1% NP40, 0.5% sodium total RNA/sample using the PolyATtract mRNA isolation system IV (Pro- deoxycholate, and 0.1% SDS) supplemented with a mixture of protease (Com- mega, Madison, WI) following the manufacturer’s instructions. This additional plete Boehringer Mannheim Corp., Roche Molecular Biochemicals, Indianap- step is required due to the presence of melanin in the total RNA preparation, olis, IN) and phosphatase (100 mM NaF, 10 mM Na4P2O7,and1mM Na3VO4) especially in normal human melanocytes, that suppresses PCR hybridization inhibitors. Total cell extracts (35 ␮g proteins/lane), measured by the Bio-Rad reactions (our experience as well as that of others; see, for example, Ref. 34). protein assay (Bio-Rad Laboratories, Hercules, CA), were fractionated in The quality and quantity of the poly(A) RNA products were determined by precast gels composed of 8% polyacrylamide Tris-glycine or 10% polyacryl- Bioanalyzer (Agilent Technologies) and spectrophotometric analysis. amide NuPAGE Bis-Tris (Novex, San Diego, CA) and Western blotted ac- The human genome Affymetrix GeneChip system HG-U133A microarray cording to standard protocols (38). (Affymetrix Inc., Santa Clara, CA) containing 11 pairs of match/mismatch Antibodies raised against the following proteins were used for Western 25-mer oligonucleotide probes for each of ϳ14,500 transcripts of known genes blotting: UCHL1 (ubiquitin COOH-terminal esterase L1, also known as pro- and the OHU16K human library oligonucleotide array comprising a single tein gene product 9.5; polyclonal antibody AB1761), Chemicon International, 70-mer oligonucleotide probe in duplicate spots for each of 16,659 different Inc.; fibronectin (ab299) and connective tissue growth factor [CTGF gene transcripts (printed by the Keck Microarray Resource at Yale) were used. (ab6992)], abcam (Cambridge, United Kingdom); tenascin C (H-300), IGBP7 A description of these slides can be found online.10 (C-16), ErbB-3 (C-17), anti-Twist (sc-15393), and anti-ZEB/TCF8 (E-020), For the HG-U133A chip, cDNA templates were generated from purified Santa Cruz Biotechnology (Santa Cruz, CA); ATPase Naϩ/Kϩ transporting ␤1 mRNAs for in vitro transcription of biotin-labeled amplified RNAs. Protocols polypeptide, Upstate Biotechnology (Lake Placid, NY); integrin-linked kinase- for hybridizing fragmented biotin-labeled cRNAs, washing unhybridized ma- associated serine/threonine phosphatase 2C (ILKAP) and Rab27A, BD Trans- terial from the chips, and scanning them with a GeneArray Scanner (Agilent duction Laboratories (San Diego, CA); IFN-inducible MX1/MxA (from Dr. Technologies) were followed as recommended by Affymetrix Inc. Mark A. McNiven; Center for Basic Research in Digestive Diseases and For the OHU16K microarray we used the two-color hybridization protocols Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, of Drs. Patrick O. Brown and Geoff Childs (35) optimized by the Keck MN); and ISG15 (from Dr. Ernest C. Borden; Departments of Microbiology Resource. They are available online.11 The slides were scanned by the Keck and Cancer Center, Medical College of Wisconsin, Milwaukee, WI). Antibod- Microarray Resource using a GenePix 4000A scanner (Axon Instruments). The ies against capping protein (gelsolin-like, CapG, anti-peptide WKGRKANEK- results appear as whole array images containing signal intensity information ERQAALQVAE) were raised in rabbits by Proteintech Group, Inc. (Chicago, for both fluorescent Cy dyes. The primary data tables and the image files are IL) and against necdin (NC243) by Michio Niinobe (39); anti-RAB32 anti- available from the Yale Microarray Database. bodies were from Dr. R. J. Haslam (Department of Pathology and Molecular Data Extraction and Statistical Analysis. The total HG-U133A signal Medicine, McMaster University, Ontario, Canada). Anti-tyrosinase (mono- was normalized to an arbitrary signal intensity value of 500 using MAS 5.0 clonal antibody T311) was used as a marker for differentiation, and anti-␤- software (Affymetrix Inc.). Subsequent analyses for both HG-U133A and actin (A2066; polyclonal antibody; Sigma-Aldrich) was used as a measure for OHU16K experiment data sets were performed using GeneSpring 6.0 (Silicon protein loading in each well. In several cases, the same membrane was Genetics, Inc., Redwood City, CA). Comparisons were made between normal successively blotted with two or three antibodies, thus sharing the ␤-actin human melanocytes and melanoma cells. We first applied a fold change filter control. of 2.5 between average control and average melanoma cells, and only those Melanoma Tissue Microarrays (TMAs). The TMA comprised 570 tissue probesets with a minimal fold change of 2.5 combined with an average signal cores (553 melanomas, 17 normal skin samples) measuring 0.6 mm in diam- intensity of 500 or higher in either melanocyte or melanoma cells were eter, spaced 0.8 mm apart on a single glass slide. The cohort was constructed included in the analysis. The data were then further restricted using the from paraffin-embedded formalin-fixed tissue blocks obtained from the ar- GeneSpring confidence filter, which used the cross-gene error model to obtain chives of Yale University Department of Pathology as described previously a t test P statistic for each probeset and the Benjamini and Hochberg False (40, 41). The specimens were resected between 1959 and 1994, with a Discovery Rate for multiple testing correction (36). Probesets exceeding a t test follow-up period ranging between 2 months and 38 years. The TMA slides P of 0.05 were not included. Genes were annotated with GenBank Accession were treated, probed, and scored as described previously (42). The primary

10 http://keck.med.yale.edu/affymetrix/ and http://keck.med.yale.edu/dna_arrays.htm. 12 http://bioinformatics.med.yale.edu/. 11 http://cmgm.Stanford.edu/pbrown/ and http://info.med.yale.edu/wmkeck/dnaarrays/ 13 http://ymd.med.yale.edu/karma/cgi-bin/karma.pl. protocols.htm. 14 http://www.genome.org/cgi/content/full/9/11/1106. 5271

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. NOVEL PATHWAYS IN MELANOMAS antibodies anti-UCHL1 and anti-Twist, validated by Western blotting, were and published information. Of these, 315 and 274 genes were up- and applied at 1:500 and 1:100 dilution, respectively. The prognostic significance down-regulated, respectively, in the Affymetrix U133A data set, and of the parameters was assessed for predictive value using the Cox proportional 99 and 87 genes were up- and down-regulated, respectively, in the hazards model with overall survival as an end point. Survival curves were OHU16K data set. calculated using the Kaplan-Meier method, with significance evaluated using A comparison between the gene expression data from the Af- the Mantel-Cox log-rank test. fymetrix U133A and OHU16K microarrays using a P of Ͻ0.05 Plasmid and Transfection. The mouse HEY1/HESR1-pIRES2-EGFP plasmid (43) was from Drs. C. C. Hughes and M. Henderson (Department of without fold change cutoff restriction showed that the expression Molecular Biology and Biochemistry, University of California Irvine, Irvine, levels of only five genes were altered in the opposite direction in the CA), and pcDNA3.Flag-necdin (44) was from Drs. Timothy M. Wright and Bo two types of microarrays. Among these genes, ISG15 (IFN-␣-induc- Hu (Department of Medicine, University of Pittsburgh, Pittsburgh, PA). The ible protein clone IFI-15K, also known as G1P2) showed significantly HEY1/HESR1-pIRES2-EGFP plasmid was transfected into immortalized reduced expression in five of six melanoma cell strains compared with TPA-dependent normal mouse melanocytes (33), and the pcDNA3.Flag- normal melanocytes in the Affymetrix data set (Ϫ21-fold) but was necdin was transfected into human melanoma cells (501 mel) using Lipo- slightly up-regulated in the OHU16K microarray (1.44-fold). As fectAMINE 2000 reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA) shown in Fig. 5B, Western blot analysis revealed up-regulation of following the manufacturer’s instructions. Parallel cultures were transfected ISG15 in the melanoma cells, consistent with previously published with pcDNA.GFP as a control. Transfection efficiencies were in the order of 30% as evaluated by sorting for green fluorescing cells. The level of the data (46). The change in expression of the other four genes was in the ectopic protein was also assessed 1 day or 2 days after transfection by Western range of 1.3–2, below that used in our analysis. blotting of cell lysates (35 ␮g protein/assay) with anti HEY1/HESR1 antibod- Hierarchical clustering of the Affymetrix U133A data set indicated ies (AB5714; Chemicon International, Inc.) or anti-Flag M2 monoclonal that the two independent cultures of normal human melanocytes antibody (Sigma-Aldrich). The HEY1/HESR1-transfected mouse melanocytes grown from different donors closely resembled each other (Fig. 1A, were grown in medium deprived of growth factor (TPA) 4 days after trans- NM). Therefore, the average expression values for specific genes of fection for over 3 months to evaluate changes in growth properties and the two data sets were used for further comparisons. In addition, the differentiation. The growth properties of pcDNA3.Flag-necdin as compared hierarchical clustering showed that among the six melanoma cell with green fluorescent protein transfectants were evaluated by DNA synthesis strains, the MNT1 gene expression pattern was the least divergent assay (28) after 2 days of incubation in G418-supplemented medium (400 from that of normal human melanocytes, followed by the primary ␮g/ml; 30,000 cells/well; 24-well plate in triplicates) applied 2 days after transfection and by cell proliferation after 8 days of selection in G418 starting melanoma WW165 and the metastatic melanomas YUMAC, with an equal cell number (200,000 cells each, seeded in 55-cm2 Petri dishes). YUSIT1, YUHEIK, and YUCAL (Fig. 1A). The similarity between Each transfection was repeated three times with similar results. the metastatic MNT1 melanoma cells and normal melanocytes could Immunofluorescence Microscopy. Melanoma cells and normal human not be attributed solely to differentiated state, being highly pigmented, melanocytes were grown on coverslips and processed for immunofluorescence and expressing normal levels of melanocyte-specific proteins such as analysis with anti-necdin and anti-Flag antibodies as described previously (45). tyrosinase (Fig. 1B) because YUHEIK and YUMAC melanoma cells Reverse Transcription-PCR. Poly(A) tracked mRNA was isolated from have similar characteristics (Fig. 1B). total RNA using the PolyATtract mRNA isolation system IV (Promega) The bioinformatic and QT_Clust analysis of the combined data set following the manufacturer’s instructions. Complementary DNA template was revealed differential expression, in some cases in a coordinated man- generated from the poly(A)-selected RNA using Invitrogen SuperScript II Reverse Transcriptase according to the manufacturer’s instructions, and the ner, of genes representing several functional groups. We report here levels of Rab33A and FGF13 gene transcripts compared with ␤-actin control the analysis derived mostly from the Affymetrix data set, as corrob- were assessed by PCR using the following primer pairs synthesized by the orated or not conflicted by the OHU16K data set when applicable. The oligonucleotide synthesis facility at the Department of Pathology, Yale Uni- Affymetrix chip incorporates mismatch controls for cross-hybridiza- versity School of Medicine (Table 1). tion and was thus deemed more reliable. DNA Sequencing. Sequencing of selected cDNA amplified from normal Below is a description of changes in selected functional groups melanocytes and melanoma cells with gene-specific primers by PCR was following, in most cases, the guidance set by Gene Ontology. The full performed by the Keck DNA Sequencing Resource at Yale. scope of differential gene expression can be accessed in the supplementary data. RESULTS Global Changes in Gene Expression and Receptor Activity Hierarchical Clustering The oligonucleotide microarray results showed changes in the Global probing for differences in gene expression between normal expression of receptors, ligands, cell surface and adhesion mole- human melanocytes and melanoma cells showed statistically signifi- cules that affect proliferation, angiogenesis, tissue remodeling, cant altered expression of 589 genes in the Affymetrix U133A data set cell-cell communication, ion channels, attachment, motility, and using a 2.5-fold change cutoff and of 186 genes in the OHU16K invasion (Fig. 2A; Supplementary Table 1, A and B). Some of these arrays using a 2-fold change cutoff. These cutoffs were determined as changes, as observed also at the protein level (Fig. 2B), confirmed the least stringent when compared with expression of known genes published observations. These include integrin signaling (47), insulin-like growth factor (IGF1) receptor (48), FGF receptor 1 (reviewed in Ref. 31), transforming growth factor ␤ (49), ErbB-3 Table 1 Primer sequences (50), EphA3 (reviewed in Ref. 51), melanoma-inhibitory activity Gene Primer Product size (bp) (52), N-cadherin and protocadherin, down-regulation of E and Rab33A Fa:5Ј-GGAGATGGCGCAGCCCATCCT-3Ј 835 P-cadherins (53, 54), and the transmembrane protease dipeptidyl- B: 5Ј-TTTTATTTTGATATCAATTTGTATGGAAAC-3Ј peptidase IV (55). However, there were also changes in the ex- FGF13 F: 5Ј-CACCAAAGATGAGGACAGCACTTAC-3Ј 429 pression of additional genes, in some cases in a coordinated B: 5Ј-TTCAGCACGCCAGAGACACTTC-3Ј ␤-Actin F: 5Ј-GTGGGGCGCCCCAGGCACCA-3Ј 540 manner, that had not been observed previously. B: 5Ј-CTCCTTAATGTCACGGCACGATTTC-3Ј We found increased expression of NOTCH2 and HEY1/HERS1, the a F, forward; B, backward. transcription factor activated by NOTCH signaling (Ref. 56; Fig. 2; 5272

scored by the overexpression of several integrins, in particular ITGA7, ␣ ␣ ␣ ␤ ITGA4, ITGA6, and ITGB3 (integrins 7, 4, 6, and 3; Fig. 2C); the four integrin ligands TNC1, FN1, CTGF, and Cyr61 (cysteine-rich); and two tetraspanins, TM4SF13 and TM4SF1, known to complex with integrins (Fig. 2D) and the underexpression of ILKAP (Fig. 2E; Supplementary Table 1, A and B). Here again we confirmed the overexpression of CTGF and the underexpression of ILKAP at the protein level, the latter correlated nicely with the Affymetrix gene transcript hybridization intensity (compare Fig. 2F, ILKAP with Fig. 2E). There was up-regulation of IGF1 (ϳ4-fold, in four of six melanoma cells) and several insulin-like growth factor-binding proteins (IG- FBPs), with IGFBP3 being the most prominent (Fig. 3). The expression of IGFBPs appeared to be nonrandom, with higher levels in metastatic cells, such as MNT1, YUHEIK, YUSIT1 and YUCAL, and to a lesser degree, YUMAC, compared with normal melanocytes and the primary melanoma WW165 (Fig. 3). Interestingly, the cytokines Cyr61 and CTGF discussed above also belong to the family of IGFBPs (IGFBP10 and IGFBP8, respectively), and their expression is similarly coordinated with the other IGBPs (Fig. 3). We show for the first time the up-regulation of FGF13/FHF2 (Fig. 4A), confirmed by semiquantitative reverse transcription-PCR (Fig. 4C). FGF13 maps to chromosomal region Xq26.3, and QT_Clust analysis revealed that it is coordinately expressed in the different melanoma cell strains in a pattern similar to that of MAGE gene family members located on Xq28 (Fig. 4A), suggesting a similar mechanism of activation. FGF2, another member of this family of growth factors up-regulated in melanoma cells (57, 58) and known to confer growth advantage (59, 60), was not identified here in the microarray analysis probably due to low mRNA levels (57).

Cancer-Testis Antigens (CTAs) The results not only confirmed the up-regulation of several genes encoding CTAs (reviewed in Ref. 61) but also unraveled different patterns of coexpression for MAGE and GAGE family members (Fig. 4, A and B). The reexpression of at least some of these antigens in Fig. 1. Hierarchical clustering and differentiation marker. A, cluster map and phylo- tumors is driven by demethylation of selected CpG in the promoter genetic tree resulting from a hierarchical cluster analysis of normalized intensity data regions (reviewed in Refs. 61–63), and the unique pattern of expres- across two control (melanocytes; NM) and six sample (melanoma, as indicated) data sets. Genes were selected for clustering according to a fold change and signal intensity cutoff sion for each group suggests a coordinated chromatin modification. protocol described in the text. Clustering was performed using GeneSpring 6.0 (Silicon Although the function of these antigens is not known, they are Genetics, Inc.). Because the data distribution is parametric, we used a standard correlation algorithm for both gene and condition tree analyses. B, Western blot of cell extracts with recognized by autologous CTLs and are targets for CTL-directed anti-tyrosinase monoclonal antibody T311 and anti-␤-actin as a control. The normal immunotherapy (reviewed in Ref. 61). The coordinated expression of melanocyte (NM) and melanoma cells (YUHEIK, YUMAC, and MNT1) with high specific CTA members revealed by our data may help in assessing tyrosinase levels were also highly pigmented. The other melanoma cells (WW165, YUCAL, YUSIT1, 501 mel, YUSAC, and YUGEN8) were amelanotic or very slightly antigen expression and selection of patients for immunotherapy. pigmented. Immune Modulation

Supplementary Table 1). NOTCH2 levels were coordinately up- In this category, we included the IFN-regulated genes, immune regulated with genes belonging to the family of receptor activity, complements, and histocompatibility antigens, as well as genes en- such as Tenascin C (TNC1), Fibronectin 1 (FN1), Tissue inhibitor of coding proteins in the vesicular and ubiquitin pathways (Supplemen- metalloproteinase 3 (TIMP3), and other adhesion/receptor molecules tary Tables 2–4). The latter two categories may provide melanoma (Fig. 2A). However, overexpression of HEY1/HESR1-pIRES2-EGFP cells with an immune escape mechanism by reducing the level of in immortalized mouse melanocytes did not facilitate release from antigenic peptides (see, for example, Ref. 64). TPA-dependent mode of growth, suggesting that, by itself, HEY1/ IFN-Regulated Genes. Genes that belong to the IFN pathway HERS1 does not confer growth advantage. were predominantly down-regulated (21 of 22 genes), composing The immunoblotting confirmed the general trend of up-regulation ϳ8% of all down-regulated genes (Supplementary Table 2A). Fur- of this class of genes (Fig. 2B). However, there was no consistent thermore, 14 of these genes were coordinately down-regulated, as correlation between transcript levels, as reflected by hybridization revealed by QT_Clust analysis (Fig. 5A). The down-regulation of intensity and cellular proteins. TNC1, for example, was up-regulated MX1/IFI78 was confirmed by Western blotting, showing nice corre- in all six melanoma cell strains examined with the Affymetrix mi- lation with the Affymetrix data set. MX1/IFI78 was expressed in croarray (Fig. 2A), but the protein was not detected in YUHEIK and normal melanocytes (NM) and YUHEIK (Fig. 5B), the only mela- MNT1 melanoma cells (Fig. 2B). noma cell strain with intensity levels similar to that in normal mela- The role of integrin signaling in melanoma progression was under- nocytes (Fig. 5A, gray diamonds). On the other hand, the expression 5273

Fig. 2. Differential expression of genes in the category of receptor activity. The figure shows differences in the expression of a wide spectrum of genes meeting fold change and intensity minimum requirements and identified as belonging to receptor activity. To accommodate the large span in hybridization intensity values, they are plotted here and in most subsequent figures in log scale. A, coordinated regulation of receptor activity genes. Data were subjected to QT_Clust analysis in Gene- Spring 6.0, as described in the Fig. 1 legend. Min- imum cluster size used was 2, minimum correlation was 0.98, and a standard correlation similarity measure was used. FN1, fibronectin 1; TNC, tenascin C1 (hexabrachion); TNFRSF12A, tumor necrosis factor receptor superfamily member 12A; TIMP3, tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory); BPAG1, bullous pemphigoid antigen 1, 230/240 kDa; PCOLCE2, procollagen C-endopeptidase en- hancer 2; SDC2, syndecan 2 (heparan sulfate pro- teoglycan 1, cell surface-associated, fibroglycan); ␣ ITGA6, integrin, 6; COL9A3, collagen, type IX, ␣ 3. B, Western blots of cell extracts. ATP1B1, ATPase, Naϩ/Kϩ transporting ␤1 polypeptide; ErbB3, v-erb-B2 erythroblastic leukemia viral oncogene homolog 3; CapG, capping protein (actin filament), gelsolin-like. Others terms are as defined in A. ␤-Actin was used as a control. Normal human melanocytes (NM) were grown continuously in the presence of growth factor (ϩ) or overnight without growth factors. The same membrane was used to blot successively for ATP1B1 and FN1 and for integrin-linked kinase-associated serine/threonine phosphatase 2C and CapG. CϪE, expression of genes belonging to the integrin pathway. ITGA7, ␣ ␣ ␣ ITGA4, ITGA6, and ITGB3, integrin 7, 4, 6, and ␤ 3, respectively; CTGF, connective tissue growth factor; Cyr61, cysteine-rich; TM4SF1 and TM4SF13, tetraspanin 1 and 13, respectively; ILKAP, integrin-linked kinase-associated serine/ threonine phosphatase 2C. F, Western blots of cell extracts using antibodies against CTGF, integrin- linked kinase-associated serine/threonine phosphatase 2C, and ␤-actin as a control.

of ISG15, which was conflicted between the Affymetrix HG-U133A on the hybridization intensity data, Rab38, Rab27A, and Rab32 were and the Operon 70-mer 16K data sets (see above), was shown to be the most abundant, and Rab31 and Rab33A were the least abundant up-regulated (Fig. 5B), in agreement with previously published re- family members in normal human melanocytes (Fig. 5D). As con- ports (46). firmed by Western blotting, Rab38 and Rab27A were down-regulated Leukemia Inhibitory Factor (LIF). The immune modulator LIF in several melanomas but remained at high levels in the melanotic (also known as melanoma-derived LPL inhibitor) was up-regulated in MNT1 melanoma cells, the line least divergent from normal human four of six melanoma cell strains (Fig. 5C; Supplementary Table 2B). melanocytes (Fig. 5B). In contrast, reverse transcription-PCR analysis LIF is a cytokine that activates monocytes and macrophages and revealed that Rab33A was easily detected in normal melanocytes induces chemotaxis in immune cells (reviewed in Ref. 65). Its pro- but not in the five melanoma cell strains tested, including MNT1 duction in melanomas was suggested to induce cancer cachexia (66, (Fig. 5E). 67). LIF also stimulated the human HLA-G promoter in JEG3 chori- RAGA (Ras-related GTP-binding protein, also known as FIP-1) was ocarcinoma cells (68). In our QT_Clust analysis, LIF expression also about 3-fold down-regulated in the six melanoma cell strains strongly correlated with the expression of genes encoding several tested (Supplementary Table 3). RAGA is involved in transport of class II major histocompatibility complexes, HLA-DQB1, HLA-DRA, viruses and cytoplasmic organelles and in chromosome segregation HLA-DMA, and HLA-DRB4 (Fig. 5C), suggesting a common mech- through its binding to the light chain of cytoplasmic dynein (72), and anism by which this group of genes is regulated. therefore its down-regulation may abrogate these processes. Because Vesicular Pathway. Several genes in the membrane transport RAGA localizes to 9p21.3, a region commonly lost in melanomas, we pathway were down-regulated. These include VAMP8, TRS20, HPS1, sequenced the cDNA from normal melanocytes and several melanoma and several Ras-related GTP-binding proteins (Fig. 5D; Supplemen- cell strains (501 mel, YUMAC, and YUSIT1), but we ruled out the tary Table 3). The products of some of these genes participate in the presence of any mutation. transport of proteins into melanosomes and/or the transport of mela- Ubiquitin Pathway/Proteolysis. The ubiquitin pathway encom- nosomes to the plasma membrane (reviewed in Refs. 69–71). Based passes an additional group of genes that was predominately down- 5274

Fig. 3. Up-regulation of insulin-like growth factor-binding protein (IGFBs). The histogram shows that six members of the IGFBP group (Molecular Function ontology identifier 5520) were significantly up-regulated. Inset, higher magnification to better demonstrate the differences in the expression of IGFBs.

regulated in melanoma cells compared with normal melanocytes (12 genes are sequentially activated during embryogenesis, setting in of 14 genes; Supplementary Table 4). motion correct embryonic patterning (see, for example, Ref. 74), GRAIL (also known as RNF128), an E3 ubiquitin ligase, was suggesting that the down-regulation in melanoma may be the reversal up-regulated in five of the seven melanoma cell strains tested (Fig. of this process. 6A). Western blot analysis also showed up-regulation at the protein The Affymetrix data showed that Twist was up-regulated in five of level in five of nine melanoma cell strains (Fig. 6B). The two data sets six melanoma cell strains and displayed a pattern of expression similar were in general agreement, with the exception of YUSIT1 (Fig. 6, to that of TBX3 (Fig. 7B). Western blot analysis revealed that TWIST compare hybridization intensity levels in A and protein level in B). Down-regulation of UCHL1 at the gene transcription level, as reflected by the Affymetrix hybridization intensity values, correlated nicely with the protein level (compare NM, YUHEIK, and YUCAL expression in Fig. 6, A and B, panel marked UCHL1). Assessment of UCHL1 expression in the 553 case melanoma TMA showed a trend toward decreased survival with decreased levels that was not statistically significant (Fig. 6C; P ϭ 0.0671). However, in primary melanomas, there was a significant association between poor survival and decreased UCHL1 expression (Fig. 6D; P ϭ 0.0361). High UCHL1 expression was more abundant in primary lesions compared with metastases (P ϭ 0.01) and in lesions thinner than 2 mm (P ϭ 0.04), suggesting that it is lost as malignancy progresses (see Fig. 6E for histospots representing high and low UCHL1 expression). There was no significant association between UCHL1 expression and Clark level, presence of ulceration, patient age, and gender. On multivariate analysis, UCHL1 was not an independent predictor of survival among primary melanomas (P ϭ 0.1), and the only two independent predic- tors were Breslow depth and Clark level.

Transcription Factors and Growth Suppressors There were changes in the expression of genes encoding highly conserved families of transcriptional regulators critical for develop- mental programs, mesoderm/ectoderm transition, and differentiation (Supplementary Table 5). In addition to HEY1/HERS1 mentioned above, ASXL1, SHOX2, and FOXD1 were persistently up-regulated in melanoma cells (Fig. 7A; Supplementary Table 5). FOXD1 transcription factor regulates the expression of growth factors secreted by stromal cells that modulate the differentiation of neighboring epithelial cells, such as vascular endothelial growth factor and placental Fig. 4. Differential expression of cancer-testis antigen group. Signal intensity data growth factor (73). In our QT_Clust analysis, FOXD1 expression corresponding to the cancer-testis antigens were subject to QT_Clust analysis in Gene- clearly correlated with vascular endothelial growth factor and heparin- Spring 6.0. The results show different patterns of expression for MAGE (A) and GAGE binding epidermal growth factor-like growth factor (Fig. 7A, VEGF (B) family members. Notice that FGF13 genes expression pattern is clustered within the MAGE group. C, expression of FGF13 gene transcript compared with ␤-actin using 30 and HBEGF), implying a cause and effect. On the other hand, four cycles of reverse transcription-PCR. Numbers on the left indicate size markers of a 1-kb HOX genes were down-regulated (Supplementary Table 4). The HOX DNA ladder. 5275

Fig. 5. Differential expression of genes in the immune modulation pathway. A, QT_Clust analysis of the insulin-responsive genes. B, Western blots of cell extracts with antibodies against MX1/ IFI78, ISG15, and Rab32, using ␤-actin as a control. NT, not tested. C, coordinated expression of leukemia inhibitory factor (LIF) and HLA (MHC class II types as indicated) genes as determined by QT_Clust analysis. D, coordinated down-regulation of genes in the vesicular pathway. VAMP8, vesicle-associated membrane protein 8 (endobre- vin). TRS20 is also known as LOC51693; RAB38, RAB27A, RAB32, RAB33A, and RAB31, Ras- related proteins. E, expression of Rab33A gene transcript compared with ␤-actin using 35 cycles of reverse transcription-PCR. Numbers on the left indicate size markers of a 1-kb DNA ladder.

was up-regulated in five of nine melanoma cell strains (Fig. 7C). Here ing of the anti-Necdin antibodies to the tissues on the microarray (data again, there was no direct correlation between the Affymetrix inten- not shown). Overexpression of Flag-tagged Necdin in melanoma cells sity values and the levels of the protein in two melanoma cell strains (501 mel cells, Fig. 8C, inset) suppressed growth, as observed by a (YUHEIK and YUSIT1). Probing of the melanoma TMA with anti- 30% reduction in [3H]thymidine incorporation 4 days after transfec- Twist antibodies indicated that Twist overexpression was associated tion (data not shown) and by a cell count determined 10 days after with worse patient survival in the entire cohort (P ϭ 0.0037) and transfection (Fig. 8C). Immunofluorescence analysis revealed that within the subset of primary melanomas (P ϭ 0.0028; Fig. 7, D and endogenous necdin was cytoplasmic in normal and malignant cells but E), highlighting the significance of its involvement in tumorigenesis displayed a patchy and aggregated pattern in melanoma compared in vivo. Intense Twist staining was more prominent in metastatic with even distribution in normal pigment cells (Fig. 8E). However, lesions (including nodal and distant metastases) than in primary sequencing NECDIN in 501 mel cells did not indicate any mutation, lesions (P ϭ 0.0053; Fig. 7E). Among the primary lesions, there was suggesting that the different pattern was due to other factors. no significant association between high Twist expression and Clark level, Breslow depth, presence of ulceration in the lesion, patient age, DISCUSSION or gender. On multivariate analysis using the Cox model, high Twist expression was an independent predictor of poor outcome (hazard We report here a comprehensive analysis of changes in gene ratio, 3.2; 95% confidence interval, 1.16–8.99; P ϭ 0.024), as were expression in human melanoma cells compared with normal melano- Breslow depth of Ͼ1 mm and a Clark level of IV or V. Other variables cytes at a scope not demonstrated previously. These changes were included in this analysis that were not of significant predictive value observed by analyzing proliferating melanoma cells from six ad- were presence of ulceration on primary tumors, patient age, and vanced lesions and normal human melanocytes grown in culture. The gender. melanoma cells were selected to represent strains with high and low Three genes encoding growth suppressors, NECDIN (neurally dif- levels of pigmentation expressing a wide range of tyrosinase, the ferentiated embryonal carcinoma cell-derived factor), NBL1 (neuro- melanocyte-specific gene product, as a measure of differentiation. blastoma suppressor of tumorigenicity 1 precursor), and CIRBP (cold Several of the differences between normal and malignant melanocytes inducible RNA-binding protein) were also down-regulated in mela- detected by our analysis have already been reported in cells and noma cells (Supplementary Table 5), but in an uncoordinated manner tumors, supporting the notion that most of them were not generated (Fig. 8A). Reduced Necdin expression was confirmed by Western during growth in culture and do not reflect a response to ingredients blotting in seven of nine melanoma cell strains (Fig. 8, B and D), but in the culture medium. The use of proliferating normal and malignant attempts to stain melanoma TMA failed due to the nonspecific bind- melanocytes proved that several critical changes in gene expression 5276

Fig. 6. Differential expression of genes in the proteasomal pathway. A, Affymetrix hybridization intensity pattern for grail (E) and UCHL1 (F). Intensity scales on the left and right are for grail and UCHL1, respectively. B, Western blots of cell extracts with anti-grail and anti-UCHL1 compared with ␤-actin control. NT, not tested. C and D, UCHL1 Kaplan-Meier survival curves over a 30- year period. Results are based on immunostaining of melanoma tissue microarray with anti-UCHL1 polyclonal antibodies. The entire cohort of melanoma patients (C) and primary melanoma lesions only (D) are displayed separately. E, histospots showing cytoplasmic staining of UCHL1 with high (score of 3) and low (score of 0) expression scores. Pictures were taken at ϫ10 magnification (insets, ϫ60).

characteristic to the malignant state are independent of proliferation integrins (ITGA7, ITGA4, ITGA6, ITGB3), integrin ligands (Cyr61, status. TNC1, FN1, CTGF), and two tetraspanins (TM4SF13 and TM4SF1), Hierarchical clustering showed that the primary melanoma cell and demonstrated down-regulation of the serine/threonine phospha- strain WW165 isolated from an advanced lesion was more similar to tase ILKAP. ILKAP selectively suppresses the activity of integrin- the metastatic melanoma cells than to normal melanocytes, confirm- linked kinase 1 (78), a mediator of integrin signal transduction that ing an observation made almost two decades ago (75). Indeed, the promotes angiogenesis (see, for example, Ref. 79). More recently, it WW165 cells, unlike primary melanoma cells from superficial spread- was demonstrated that integrin-linked kinase 1 protein expression ing or thin lesions, proliferate in culture independently of most of the increases as melanoma progresses, with strong positive correlation growth factors required for normal human melanocytes (76). The between expression and lymph node invasion and with decreased MNT1 melanoma cells, on the other hand, isolated from lymph node 5-year survival (80). Suppression of ILKAP by small interfering RNA metastasis, were the least divergent from normal human melanocytes. increased entry of cells into S phase, and overexpression of ILKAP The similarity included not only high pigmentation, which was ob- inhibited anchorage-independent growth of LNCaP prostate carci- served in at least two other melanoma cell strains (YUHEIK and noma cells (81). Reduced ILKAP expression, therefore, in combina- YUMAC), but also genes in the receptor activity group, such as tion with high integrin-linked kinase expression, is likely to enhance TNC1, TNFRSF12A, TIMP3, ITGA6, SDC2, and in the Rab family the aggressive state of melanomas. (Rab38 and Rab27A). MNT1 is a melanoma cell strain that was Other changes in the integrin pathway shown here have been established in culture more than a decade ago (29), and information observed previously in melanoma tumors, such as Tenascin C (82– regarding the patient’s clinical outcome is not available. 84). Tenascin C is also present in the serum of patients with stage IV Although extensive activation of cell surface receptors in mela- melanoma, but not in healthy donors (85), suggesting that it can be noma cells has been reported previously, our analysis revealed several used as a marker for early transformation and metastatic load. Cyr61, novel details. We show, for example, the increased expression of on the other hand, is expressed in uveal melanomas (86). It is an FGF13 as a possible mediator of FGF receptor 1 stimulation. FGF13 angiogenic factor that induces migration and stimulates mitogenesis was originally cloned from an ovarian cancer cell library, and its by interacting with integrins (87). Cyr61 confers resistance to apo- expression is particularly high in the brain. The growth factor is likely ptosis by increasing nuclear factor-␬B activity (88), and the expres- to be mitogenic for the melanoma cells because it stimulates the sion of Cyr61 and CTGF, in addition to vascular endothelial growth mitogen-activated protein kinase pathway (Ref. 77 and the references factor, is likely to contribute to the angiogenic activity of melanoma therein). cells. These three factors are produced by other cancer cells as well We also demonstrated that several components of the integrin and are targets for tumor therapy [see, for example, Ref. 89; Lester F. pathway are overexpressed in a synergistic manner, which include Lau (University of Illinois, Chicago, IL and FibroGen, South San 5277

Fig. 7. Expression pattern of transcriptional regulators and their affected genes. A and B, QT_Clust analysis. FOXD1, forkhead box D1; HBEGF, heparin-binding epidermal growth factor-like growth factor (also known as diphtheria toxin receptor); VEGF, vascular endothelial growth factor; TBX3, T-box 3 (ulnar mammary syndrome); TWIST, twist homolog (acrocephalosyndactyly 3; Saethre-Chotzen syndrome). C, anti-Twist Western blot of cell extracts, using ␤-actin as a control. D and E, Twist Kaplan-Meier survival curves based on immunostaining of melanoma tissue microarray with anti-Twist polyclonal antibodies. The entire cohort of melanoma patients (D) and primary melanoma lesions only (E) are displayed separately. F, histospots showing cytoplasmic staining of Twist with high (score of 3) and low (score of 0) expression scores. Pictures were taken at ϫ10 magnification (insets, ϫ60).

Francisco, CA)]. TM4SF1 is also being evaluated as a diagnostic products affect immune response. In particular, we observed ex- marker for colorectal and gastric cancers and as a target for therapy tensive suppression of IFN-regulated genes (21 of 22 genes; 14 with monoclonal antibodies (90, 91). were suppressed in a coordinated manner). Among the down- We demonstrated overexpression at the gene transcript level of regulated genes was OAS-1. OAS-1 activity is required for RNase NOTCH2 and HEY1, the latter of which is a downstream effector of endoribonuclease activation and the antiviral and apoptotic activity the NOTCH receptor. The increased HEY1/HERS1 expression in the of IFNs (Refs. 95 and 96; reviewed in Refs. 97 and 98). Several least divergent melanocytic lesions, i.e., MNT1 and the primary mutations in RNase endoribonuclease are associated with prostate melanoma WW165, suggests a role in early transformation. Based on cancer (98), and IFN-responsive genes are down-regulated in colon a limited microarray analysis, Seykora et al. (92) also found increased polyps and tumors as compared with normal colon tissue (99, 100). NOTCH2 expression in two advanced primary melanoma tumors The repression of IFN signaling genes might reflect immortaliza- compared with benign nevi. However, ectopic expression of HEY1/ tion because the predominant genes silenced by DNA methylation HERS1 in immortalized benign mouse melanocytes did not release in spontaneously immortal Li-Fraumeni fibroblasts belonged to the cells from their dependence on the external growth factor TPA, this pathway (46% of 85 genes; Ref. 101). The coordinated down- suggesting that HEY1/HERS1, by itself, is not sufficient to confer regulation of IFN-responsive genes is of particular importance ␣ ␤ malignancy. This observation is in agreement with previously pub- because IFN 2 is frequently administered as an adjuvant in lished results showing that collaborating genetic events are required immunotherapy for melanoma (102, 103). The effectiveness of this for tumorigenic transformation by NOTCH, in particular Ras-medi- treatment is likely to be conditional on the ability to induce the ated activation of Erk/mitogen-activated protein kinase and phosphati- expression of at least some of these genes. dylinositol 3Ј-kinase (Ref. 93; reviewed in Ref. 94). We also demonstrated that genes in the ubiquitin pathway were A surprising finding was the extent of changes in genes whose mostly down-regulated (12 of 14 genes). One of them, UCHL1, was 5278

expression correlated with poor survival, and we can only speculate that it contributes to melanoma resistance to this type of drug. Necdin was one of the growth suppressors down-regulated in six of eight melanoma cell strains tested. Low expression in melanomas can still confer advantage for tumor growth, as shown for other tumor suppressors. For example, tumors developed in animals lacking one copy of p27Kip1 expressing half the levels of functional wild type p27Kip1 (111). Similar observations were gleaned from p53, and the phenomenon of gene dosage sensitivity is termed haploinsufficiency (Ref. 112 and the references therein). Furthermore, Necdin in melanoma cells is localized to a different cytoplasmic compartment compared with normal melanocytes, suggesting that it is inactivated by sequestration. Its growth-suppressive function was validated by ectopic overexpression that led to rapid and efficient arrest of melanoma cell proliferation. Necdin is expressed predominantly in postmitotic cells, such as neurons and muscle cells (Ref. 113 and the references therein), and in melanocytes, as demonstrated here for the first time. NECDIN is on chromosomal region 15q11.2-q12, and is one of four genes encoding the Prader-Willi syndrome. This is an autosomal dominant neurodevelopmental disorder caused by mutations or dele- tions because the respective genes on the maternal chromosome in this region are inactive through imprinting. In the case of melanoma, the expressed necdin was not mutated in the coding region. The exact function of necdin has not yet been determined. It binds to the transcription factors E2F1 and E2F4 and to p75 neurotrophin receptor (113). Ectopic coexpression of necdin with E2F1 in U2OS osteosar- coma cells suppressed E2F1-stimulated transcription. On the other hand, coexpression of necdin with E2F1 or p75 neurotrophin receptor Fig. 8. Expression patterns of transcriptional repressors and functional validation of necdin. A, Affymetrix intensity data. NDN, necdin; NBL1, neuroblastoma, suppression of in N1E-115 neuroblastoma cells sequestered the protein in the nucleus tumorigenicity 1; CIRBP, cold inducible RNA-binding protein. B, anti-necdin Western or the cell membrane, respectively (113). Therefore, identifying the blot of cell extracts, using ␤-actin as a control. C, a histogram showing the number of G418-resistant pcDNAFlag.Necdin- and pcDNA.GFP-transfected cells assessed 10 days necdin-binding proteins in melanoma cells and normal melanocytes is after transfection. Inset, anti-Flag Western blot of cell lysates derived from pcDNA.GFP- likely to shed light on the molecular basis for the growth-arresting and Flag.Necdin-expressing cells 1 day after transfection. D, anti-necdin Western blot of function of necdin in this cellular system. normal melanocytes and 501 mel melanoma cell extracts. E, necdin immunofluorescent analysis of normal human melanocytes and 501 mel melanoma cells using anti-necdin Some of the down-regulated genes are localized at chromosomal antibodies. regions known to be altered in melanomas. For example, NBL1 is on 1p36.3-p36.2, and GBP1 (guanylate-binding protein 1, IFN-inducible, 67 kDa) is on 1p22.2, regions harboring melanoma suppressor genes suppressed in all of the melanoma strains tested, and its expression (114). Another example is RRAGA (Ras-related GTP-binding pro- was also reduced in metastatic melanoma lesions, as seen by probing tein) on 9p21.3, a region frequently deleted in melanomas that may the melanoma TMA. Reduction in UCHL1 in primary melanomas was significantly associated with poor survival. UCHL1 has a dual func- possess more then one tumor suppressor gene (115). However, se- tion, a hydrolase activity that removes small COOH-terminal ubiq- quencing of RRAGA in normal melanocytes and several melanoma uitin to generate the ubiquitin monomer and a dimerization-dependent cell strains failed to identify any mutation, suggesting that if down- ubiquityl ligase activity. Several UCHL1 variants associated with regulation is due to loss of chromosomal material, then the remaining Parkinson disease may play roles in proteasomal protein degradation gene is normal. (reviewed in Ref. 104). The UCHL1 enzymatic activity most conse- Expression or reexpression of some genes might be driven by quential for melanocyte tumorigenesis is not yet clear. demethylation/methylation of selected CpG in the promoter regions, Twist was up-regulated in ϳ55% of the melanoma cell strains processes common in cancer cells (reviewed in Ref. 116). Although tested, and its overexpression in melanoma tumors correlated with the majority of the activated genes on the X chromosome encoded poor survival. Twist is a basic helix-loop-helix transcription factor CTAs known to be reexpressed due to demethylation (reviewed in that is required for mesodermal cell fates, and its pattern of expression Refs. 61–63), other X-linked genes were also activated, such as in melanomas was similar to that of TBX3, a transcriptional repressor TRAG3 (Taxol resistance-associated gene 3; Xq28) and FGF13 required for normal breast development, with mutations in the TBX3 (Xq26.3), and might be affected by the same process. DNA methyl- gene linked to ulnar-mammary syndrome (Ref. 105 and the references ation/demethylation processes of promoter CpG-rich regions might therein). TBX3 and Twist suppress senescence by down-regulating cause the change in expression of HOXB genes (HOXB2, HOXB7, ARF, a protein that inhibits MDM2-mediated proteolysis of p53 and HOXB13; 17q21.2; Ref. 117), MX1/IFI78 (12q24.2; Ref. 101), (106–108). Twist also blocks myogenic differentiation and is inap- and dipeptidylpeptidase IV (2q24.3; Ref. 99; reviewed in Refs. 100 propriately expressed in 50% of rhabdomyosarcomas (109). In our and 118–120). Indeed, preliminary sequence analysis of the promoter samples, there was no correlation between Twist expression and region and exon 1 using the MethPrime program of Li and Dahiya differentiation, as determined by the levels of tyrosinase and pigmen- (121) available online15 revealed that 64% (of 35 genes) and 70% (of tation. In another report, increased Twist protein levels were associ- 48 genes) of the top down- and up-regulated genes, respectively, ated with resistance to Taxol and vincristine in nasopharyngeal, blad- der, ovarian, and prostate cancers (110). We showed that Twist 15 http://itsa.ucsf.edu/ϳurolab/methprimer/index1.html. 5279

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. NOVEL PATHWAYS IN MELANOMAS contain at least one CpG-rich island (data not shown). Monitoring 17. Bastian BC, Kashani-Sabet M, Hamm H, et al. Gene amplifications characterize CpG methylation patterns and the genes they affect can be used for acral melanoma and permit the detection of occult tumor cells in the surrounding skin. Cancer Res 2000;60:1968–73. assessing tumor progression, drug resistance, and immunological re- 18. Harvell JD, Bastian BC, LeBoit PE. Persistent (recurrent) spitz nevi: a histopatho- sponses. This information is particularly needed to assess drug effi- logic, immunohistochemical, and molecular pathologic study of 22 cases. Am J Surg Pathol 2002;26:654–61. cacy of novel DNA demethylating agents, such as 5-aza-2-deoxycy- 19. Sauter ER, Yeo UC, von Stemm A, et al. Cyclin D1 is a candidate oncogene in tidine (commercially known as decitabine), antisense to DNA cutaneous melanoma. Cancer Res 2002;62:3200–6. methyltransferases such as DNMT1 (MG98), and histone deacetylase 20. Bastian BC, Xiong J, Frieden IJ, et al. Genetic changes in neoplasms arising in congenital melanocytic nevi: differences between nodular proliferations and mela- inhibitor depsipeptide FR901228, which are in Phase II trials (see, for nomas. Am J Pathol 2002;161:1163–9. example, Refs. 122 and 123; reviewed in Ref. 124). 21. Balazs M, Adam Z, Treszl A, et al. Chromosomal imbalances in primary and In summary, we have identified a number of novel genes and metastatic melanomas revealed by comparative genomic hybridization. Cytometry 2001;46:222–32. pathways that are up- or down-regulated in metastatic melanoma cell 22. Bastian BC, Olshen AB, LeBoit PE, Pinkel D. Classifying melanocytic tumors based strains compared with normal melanocytes. The data provide new on DNA copy number changes. Am J Pathol 2003;163:1765–70. insights into immune modulation and possible causes for failed IFN or 23. Bastian BC. Understanding the progression of melanocytic neoplasia using genomic analysis: from fields to cancer. Oncogene 2003;22:3081–6. chemotherapy treatments common in melanomas. We identified 24. Takata M, Maruo K, Kageshita T, et al. Two cases of unusual acral melanocytic FGF13 as a new autocrine factor, Twist as clinically valuable prog- tumors: illustration of molecular cytogenetics as a diagnostic tool. Hum Pathol nostic markers for patients with melanomas, and Necdin as a potent 2003;34:89–92. 25. Bittner M, Meltzer P, Chen Y, et al. Molecular classification of cutaneous malignant melanoma growth suppressor. We demonstrated coordinated expres- melanoma by gene expression profiling. Nature (Lond) 2000;406:536–40. sion of genes representing several functional groups, including recep- 26. Weeraratna AT, Jiang Y, Hostetter G, et al. Wnt5a signaling directly affects cell tor activity, IFN-responsive pathway, CTAs, Rab family members, motility and invasion of metastatic melanoma. Cancer Cell 2002;1:279–88. 27. Weeraratna AT, Becker D, Carr KM, et al. Generation and analysis of melanoma and genes involved in embryonic tissue remodeling. These data can SAGE libraries: SAGE advice on the melanoma transcriptome. Oncogene 2004;23: serve as a basis for further evaluation of pathways leading to malig- 2264–74. nant transformation, prognostic markers, and/or drug targets. 28. von Willebrand M, Zacksenhaus E, Cheng E, Glazer P, Halaban R. The tyrphostin AG1024 accelerates the degradation of phosphorylated forms of retinoblastoma protein (pRb) and restores pRb tumor suppressive function in melanoma cells. ACKNOWLEDGMENTS Cancer Res 2003;63:1420–9. 29. Cuomo M, Nicotra MR, Apollonj C, et al. Production and characterization of the We thank Dr. Mark A. McNiven for the anti-MX1/MxA antibodies; Dr. murine monoclonal antibody 2G10 to a human T4-tyrosinase epitope. J Investig Dermatol 1991;96:446–51. Ernest C. Borden for the anti-ISG15 antibodies; Dr. R. J. Haslam for the 30. Zakut R, Perlis R, Eliyahu S, et al. KIT ligand (mast cell growth factor) inhibits the anti-RAB32 antibodies; Drs. Timothy M. Wright and Bo Hu for the HEY1/ growth of KIT-expressing melanoma cells. Oncogene 1993;8:2221–9. HESR1-pIRES2-EGFP plasmid; Dr. Shrikant Mane (Affymetrix Resource) for 31. Halaban R, Miglarese MR, Smicun Y, Puig S. Melanomas, from the cell cycle point help in conducting the microarray experiments; Dr. Janet Hager (Microarray of view. Int J Mol Med 1998;1:419–25. 32. Halaban R. The regulation of normal melanocyte proliferation. Pigment Cell Res Resource) for critical review of the raw OHU16K data; Donna LaCivita, who 2000;13:4–14. is in charge of the Cell Culture Core facility at the Yale Skin Disease Research 33. Tamura A, Halaban R, Moellmann G, et al. Normal murine melanocytes in culture. Center, for the normal human melanocytes and melanoma cells; and the Yale In Vitro Cell Dev Biol 1987;23:519–22. Melanoma Unit for discussions and support. 34. Giambernardi TA, Rodeck U, Klebe RJ. Bovine serum albumin reverses inhibition of RT-PCR by melanin. Biotechniques 1998;25:564–6. 35. Eisen MB, Brown PO. DNA arrays for analysis of gene expression. Methods REFERENCES Enzymol 1999;303:179–205. 36. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and 1. Cannon-Albright LA, Kamb A, Skolnick M. A review of inherited predisposition to powerful approach to multiple testing. J R Stat Soc B 1995;57:289–300. melanoma. Semin Oncol 1996;23:667–72. 37. Heyer LJ, Kruglyak S, Yooseph S. Exploring expression data: identification and 2. Dracopoli NC, Fountain JW. CDKN2 mutations in melanoma. Cancer Surv 1996; analysis of coexpressed genes. Genome Res 1999;9:1106–15. 26:115–32. 38. Ausubel FM, Brent R, Kingston R, Moore RE, Moore DD, Seidman JG, Smith JA, 3. Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially Struhl K. Current protocols in molecular biology. New York: John Wiley & Sons; involved in genesis of many tumor types. Science (Wash DC) 1994;264:436–40. 1995. 4. Kamb A, Shattuck-Eidens D, Eeles R, et al. Analysis of the p16 gene (CDKN2) as 39. Niinobe M, Koyama K, Yoshikawa K. Cellular and subcellular localization of a candidate for the chromosome 9p melanoma susceptibility locus. Nat Genet necdin in fetal and adult mouse brain. Dev Neurosci 2000;22:310–9. 1994;8:23–6. 40. Camp RL, Chung GG, Rimm DL. Automated subcellular localization and quanti- 5. Tucker MA, Goldstein AM. Melanoma etiology: where are we? Oncogene 2003; 22:3042–52. fication of protein expression in tissue microarrays. Nat Med 2002;8:1323–8. 6. van der Velden PA, Metzelaar-Blok JA, Bergman W, et al. Promoter hypermethy- 41. Kielhorn E, Provost E, Olsen D, et al. Tissue microarray-based analysis shows lation: a common cause of reduced p16INK4a expression in uveal melanoma. Cancer phospho-beta-catenin expression in malignant melanoma is associated with poor Res 2001;61:5303–6. outcome. Int J Cancer 2003;103:652–6. 7. Reed JA, Loganzo F, Shea CR, et al. Loss of expression of the p16/cyclin-dependent 42. Berger AJ, Kluger HM, Li N, et al. Subcellular localization of activating transcrip- kinase inhibitor 2 tumor suppressor gene in melanocytic lesions correlates with tion factor 2 (ATF2) in melanoma specimens predicts patient survival. Cancer Res invasive stage of tumor progression. Cancer Res 1995;55:2713–8. 2003;63:8103–7. 8. Piccinin S, Doglioni C, Maestro R, et al. p16/CDKN2 and CDK4 gene mutations in 43. Henderson AM, Wang SJ, Taylor AC, Aitkenhead M, Hughes CC. The basic sporadic melanoma development and progression. Int J Cancer 1997;74:26–30. helix-loop-helix transcription factor HESR1 regulates endothelial cell tube forma- 9. Alonso SR, Ortiz P, Pollan M, et al. Progression in cutaneous malignant melanoma tion. J Biol Chem 2001;276:6169–76. is associated with distinct expression profiles: a tissue microarray-based study. Am J 44. Hu B, Wang S, Zhang Y, et al. A nuclear target for interleukin-1alpha: interaction Pathol 2004;164:193–203. with the growth suppressor necdin modulates proliferation and collagen expression. 10. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Proc Natl Acad Sci USA 2003;100:10008–13. Nature (Lond) 2002;417:949–54. 45. Halaban R, Patton RS, Cheng E, et al. Abnormal acidification of melanoma cells 11. Alsina J, Gorsk DH, Germino FJ, et al. Detection of mutations in the mitogen- induces tyrosinase retention in the early secretory pathway. J Biol Chem 2002;277: activated protein kinase pathway in human melanoma. Clin Cancer Res 2003;9: 14821–8. 6419–25. 46. Padovan E, Terracciano L, Certa U, et al. Interferon stimulated gene 15 constitu- 12. Halaban R, Cheng E, Smicun Y, Germino J. Deregulated E2F transcriptional activity tively produced by melanoma cells induces e-cadherin expression on human den- in autonomously growing melanoma cells. J Exp Med 2000;191:1005–15. dritic cells. Cancer Res 2002;62:3453–8. 13. Seftor RE, Seftor EA, Hendrix MJ. Molecular role(s) for integrins in human 47. Van Belle PA, Elenitsas R, Satyamoorthy K, et al. Progression-related expression of melanoma invasion. Cancer Metastasis Rev 1999;18:359–75. beta3 integrin in melanomas and nevi. Hum Pathol 1999;30:562–7. 14. Kaelin WG Jr. E2F1 as a target: promoter-driven suicide and small molecule 48. Easty DJ, Herlyn M, Bennett DC. Abnormal protein tyrosine kinase gene expression modulators. Cancer Biol Ther 2003;2:S48–54. during melanoma progression and metastasis. Int J Cancer 1995;60:129–36. 15. Ma D, Zhou P, Harbour JW. Distinct mechanisms for regulating the tumor suppres- 49. Reed JA, McNutt NS, Prieto VG, Albino AP. Expression of transforming growth sor and antiapoptotic functions of Rb. J Biol Chem 2003;278:19358–66. factor-beta 2 in malignant melanoma correlates with the depth of tumor invasion. 16. Bastian BC, LeBoit PE, Hamm H, Brocker EB, Pinkel D. Chromosomal gains and Implications for tumor progression. Am J Pathol 1994;145:97–104. losses in primary cutaneous melanomas detected by comparative genomic hybrid- 50. Bodey B, Kaiser HE, Goldfarb RH. Immunophenotypically varied cell subpopula- ization. Cancer Res 1998;58:2170–5. tions in primary and metastatic human melanomas. Monoclonal antibodies for 5280

diagnosis, detection of neoplastic progression and receptor directed immunotherapy. 80. Dai DL, Makretsov N, Campos EI, et al. Increased expression of integrin-linked Anticancer Res 1996;16:517–31. kinase is correlated with melanoma progression and poor patient survival. Clin 51. Easty DJ, Bennett DC. Protein tyrosine kinases in malignant melanoma. Melanoma Cancer Res 2003;9:4409–14. Res 2000;10:401–11. 81. Kumar AS, Naruszewicz I, Wang P, Leung-Hagesteijn C, Hannigan GE. ILKAP 52. Garbe C, Leiter U, Ellwanger U, et al. Diagnostic value and prognostic significance regulates ILK signaling and inhibits anchorage independent growth. Oncogene of protein S-100beta, melanoma-inhibitory activity, and tyrosinase/MART-1 reverse 2004;23:3454–61. transcription-polymerase chain reaction in the follow-up of high-risk melanoma 82. Tuominen H, Kallioinen M. Increased tenascin expression in melanocytic tumors. J patients. Cancer (Phila) 2003;97:1737–45. Cutan Pathol 1994;21:424–9. 53. Li G, Herlyn M. Dynamics of intercellular communication during melanoma devel- 83. Tuominen H, Pollanen R, Kallioinen M. Multicellular origin of tenascin in skin opment. Mol Med Today 2000;6:163–9. tumors–an in situ hybridization study. J Cutan Pathol 1997;24:590–6. 54. Herlyn M, Berking C, Li G, Satyamoorthy K. Lessons from melanocyte develop- 84. Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasis reveals ment for understanding the biological events in naevus and melanoma formation. an essential role for RhoC. Nature (Lond) 2000;406:532–5. Melanoma Res 2000;10:303–12. 85. Burchardt ER, Hein R, Bosserhoff AK. Laminin, hyaluronan, tenascin-C and type 55. Wesley UV, Albino AP, Tiwari S, Houghton AN. A role for dipeptidyl peptidase IV VI collagen levels in sera from patients with malignant melanoma. Clin Exp in suppressing the malignant phenotype of melanocytic cells. J Exp Med 1999;190: Dermatol 2003;28:515–20. 311–22. 86. Walker TM, Van Ginkel PR, Gee RL, et al. Expression of angiogenic factors Cyr61 56. Maier MM, Gessler M. Comparative analysis of the human and mouse Hey1 and tissue factor in uveal melanoma. Arch Ophthalmol 2002;120:1719–25. promoter: Hey genes are new Notch target genes. Biochem Biophys Res Commun 87. Kunz M, Moeller S, Koczan D, et al. Mechanisms of hypoxic gene regulation of angiogenesis factor Cyr61 in melanoma cells. J Biol Chem 2003;25:45651–60. 2000;275:652–60. 88. Lin MT, Chang CC, Chen ST, et al. Cyr61 expression confers resistance to apoptosis 57. Halaban R, Kwon BS, Ghosh S, Delli Bovi P, Baird A. bFGF as an autocrine growth in breast cancer MCF-7 cells by a mechanism of NF-kappaB-dependent XIAP factor for human melanomas. Oncogene Res 1988;3:177–86. up-regulation. J Biol Chem 2004;279:24015–23. 58. Albino AP, Davis BM, Nanus D. Induction of growth factor RNA expression in 89. Huang J, Frischer JS, Serur A, et al. Regression of established tumors and metastases human malignant melanoma: markers of transformation. Cancer Res 1991;51:4815– by potent vascular endothelial growth factor blockade. Proc Natl Acad Sci USA 20. 2003;100:7785–90. 59. Yayon A, Ma YS, Safran M, Klagsbrun M, Halaban R. Suppression of autocrine cell 90. Schiedeck TH, Wellm C, Roblick UJ, Broll R, Bruch HP. Diagnosis and monitoring proliferation and tumorigenesis of human melanoma cells and fibroblast growth of colorectal cancer by L6 blood serum polymerase chain reaction is superior to factor transformed fibroblasts by a kinase-deficient FGF receptor 1: evidence for the carcinoembryonic antigen-enzyme-linked immunosorbent assay. Dis Colon Rectum involvement of Src- family kinases. Oncogene 1997;14:2999–3009. 2003;46:818–25. 60. Becker D, Lee PL, Rodeck U, Herlyn M. Inhibition of the fibroblast growth factor 91. Kaneko R, Tsuji N, Kamagata C, et al. Amount of expression of the tumor- receptor 1 (FGFR-1) gene in human melanocytes and malignant melanomas leads to associated antigen L6 gene and transmembrane 4 superfamily member 5 gene in inhibition of proliferation and signs indicative of differentiation. Oncogene 1992;7: gastric cancers and gastric mucosa. Am J Gastroenterol 2001;96:3457–8. 2303–13. 92. Seykora JT, Jih D, Elenitsas R, Horng WH, Elder DE. Gene expression profiling of 61. Scanlan MJ, Gure AO, Jungbluth AA, Old LJ, Chen YT. Cancer/testis antigens: an melanocytic lesions. Am J Dermatopathol 2003;25:6–11. expanding family of targets for cancer immunotherapy. Immunol Rev 2002;188:22– 93. Fitzgerald K, Harrington A, Leder P. Ras pathway signals are required for notch- 32. mediated oncogenesis. Oncogene 2000;19:4191–8. 62. Sigalotti L, Coral S, Nardi G, et al. Promoter methylation controls the expression of 94. Nam Y, Aster JC, Blacklow SC. Notch signaling as a therapeutic target. Curr Opin MAGE2, 3 and 4 genes in human cutaneous melanoma. J Immunother 2002;25:16– Chem Biol 2002;6:501–9. 26. 95. Carr DJ, Al-khatib K, James CM, Silverman R. Interferon-beta suppresses herpes 63. Coral S, Sigalotti L, Altomonte M, et al. 5-Aza-2Ј-deoxycytidine-induced expression simplex virus type 1 replication in trigeminal ganglion cells through an RNase of functional cancer testis antigens in human renal cell carcinoma: immunothera- L-dependent pathway. J Neuroimmunol 2003;141:40–6. peutic implications. Clin Cancer Res 2002;8:2690–5. 96. Al-khatib K, Williams BR, Silverman RH, Halford W, Carr DJ. The murine 64. Marincola FM, Wang E, Herlyn M, Seliger B, Ferrone S. Tumors as elusive targets double-stranded RNA-dependent protein kinase PKR and the murine 2Ј,5Ј-oligoad- of T-cell-based active immunotherapy. Trends Immunol 2003;24:335–42. enylate synthetase-dependent RNase L are required for IFN-beta-mediated resist- 65. Metcalf D. The unsolved enigmas of leukemia inhibitory factor. Stem Cells 2003; ance against herpes simplex virus type 1 in primary trigeminal ganglion culture. 21:5–14. Virology 2003;313:126–35. 66. Mori M, Yamaguchi K, Honda S, et al. Cancer cachexia syndrome developed in 97. Samuel CE. Antiviral actions of interferons. Clin Microbiol Rev 2001;14:778–809. nude mice bearing melanoma cells producing leukemia-inhibitory factor. Cancer 98. Silverman RH. Implications for RNase L in prostate cancer biology. Biochemistry Res 1991;51:6656–9. 2003;42:1805–12. 67. Paglia D, Oran A, Lu C, et al. Expression of leukemia inhibitory factor and 99. Karpf AR, Jones DA. Reactivating the expression of methylation silenced genes in interleukin-11 by human melanoma cell lines: LIF, IL-6, and IL-11 are not coregu- human cancer. Oncogene 2002;21:5496–503. lated. J Interferon Cytokine Res 1995;15:455–60. 100. Chawla-Sarkar M, Lindner DJ, Liu YF, et al. Apoptosis and interferons: role of 68. Bamberger AM, Jenatschke S, Schulte HM, Loning T, Bamberger MC. Leukemia interferon-stimulated genes as mediators of apoptosis. Apoptosis 2003;8:237– inhibitory factor (LIF) stimulates the human HLA-G promoter in JEG3 choriocar- 49. cinoma cells. J Clin Endocrinol Metab 2000;85:3932–6. 101. Kulaeva OI, Draghici S, Tang L, et al. Epigenetic silencing of multiple interferon 69. Dell’Angelica EC. Melanosome biogenesis: shedding light on the origin of an pathway genes after cellular immortalization. Oncogene 2003;22:4118–27. obscure organelle. Trends Cell Biol 2003;13:503–6. 102. Gray RJ, Pockaj BA, Kirkwood JM. An update on adjuvant interferon for melanoma. 70. Seabra MC, Mules EH, Hume AN. Rab GTPases, intracellular traffic and disease. Cancer Control 2002;9:16–21. Trends Mol Med 2002;8:23–30. 103. Kirkwood JM, Richards T, Zarour HM, et al. Immunomodulatory effects of high- dose and low-dose interferon alpha2b in patients with high-risk resected melanoma: 71. Hammer JA III, Wu XS. Rabs grab motors: defining the connections between Rab the E2690 laboratory corollary of intergroup adjuvant trial E1690. Cancer (Phila) GTPases and motor proteins Curr Opin Cell Biol 2002;14:69–75. 2002;95:1101–12. 72. Lukashok SA, Tarassishin L, Li Y, Horwitz MS. An adenovirus inhibitor of tumor 104. Chung KK, Dawson VL, Dawson TM. New insights into Parkinson’s disease. necrosis factor alpha-induced apoptosis complexes with dynein and a small GTPase. J Neurol 2003;250:III15–24. J Virol 2000;74:4705–9. 105. Davenport TG, Jerome-Majewska LA, Papaioannou VE. Mammary gland, limb and 73. Zhang H, Palmer R, Gao X, et al. Transcriptional activation of placental growth yolk sac defects in mice lacking Tbx3, the gene mutated in human ulnar mammary factor by the forkhead/winged helix transcription factor FoxD1. Curr Biol 2003;13: syndrome. Development (Camb) 2003;130:2263–73. 1625–9. 106. Lingbeek ME, Jacobs JJ, van Lohuizen M. The T-box repressors TBX2 and TBX3 74. Forlani S, Lawson KA, Deschamps J. Acquisition of Hox codes during gastrulation specifically regulate the tumor suppressor gene p14ARF via a variant T-site in the and axial elongation in the mouse embryo. Development (Cambr) 2003;130:3807– initiator. J Biol Chem 2002;277:26120–7. 19. 107. Maestro R, Dei Tos AP, Hamamori Y, et al. Twist is a potential oncogene that 75. Herlyn M, Balaban G, Bennicelli J, et al. Primary melanoma cells of the vertical inhibits apoptosis. Genes Dev 1999;13:2207–17. growth phase: similarities to metastatic cells. J Natl Cancer Inst (Bethesda) 1985; 108. Brummelkamp TR, Kortlever RM, Lingbeek M, et al. TBX-3, the gene mutated in 74:283–9. ulnar-mammary syndrome, is a negative regulator of p19ARF and inhibits senes- 76. Halaban R, Ghosh S, Duray P, Kirkwood JM, Lerner AB. Human melanocytes cence. J Biol Chem 2002;277:6567–72. cultured from nevi and melanomas. J Investig Dermatol 1986;87:95–101. 109. Carlson H, Ota S, Song Y, Chen Y, Hurlin PJ. Tbx3 impinges on the p53 pathway 77. Greene JM, Li YL, Yourey PA, et al. Identification and characterization of a to suppress apoptosis, facilitate cell transformation and block myogenic differenti- novel member of the fibroblast growth factor family. Eur J Neurosci 1998;10: ation. Oncogene 2002;21:3827–35. 1911–25. 110. Wang X, Ling MT, Guan XY, et al. Identification of a novel function of TWIST, a 78. Leung-Hagesteijn C, Mahendra A, Naruszewicz I, Hannigan GE. Modulation of bHLH protein, in the development of acquired Taxol resistance in human cancer integrin signal transduction by ILKAP, a protein phosphatase 2C associating with cells. Oncogene 2004;23:474–82. the integrin-linked kinase, ILK1. EMBO J 2001;20:2160–70. 111. Fero ML, Randel E, Gurley KE, Roberts JM, Kemp CJ. The murine gene p27Kip1 79. Tan C, Cruet-Hennequart S, Troussard A, et al. Regulation of tumor angiogenesis by is haplo-insufficient for tumour suppression. Nature (Lond) 1998;396:177–80. integrin-linked kinase (ILK). Cancer Cell 2004;5:79–90. 112. Sherr CJ. Principles of tumor suppression. Cell 2004;116:235–46. 5281

113. Kuwako KI, Taniura H, Yoshikawa K. Necdin-related MAGE proteins differentially 119. Ballestar E, Paz MF, Valle L, et al. Methyl-CpG binding proteins identify novel sites interact with the E2F1 transcription factor and the p75 neurotrophin receptor. J Biol of epigenetic inactivation in human cancer. EMBO J 2003;22:6335–45. Chem 2004;279:1703–12. 120. Lu R, Au WC, Yeow WS, Hageman N, Pitha PM. Regulation of the promoter 114. Gillanders E, Hank Juo SH, Holland EA, et al. Localization of a novel melanoma activity of interferon regulatory factor-7 gene. Activation by interferon and silencing susceptibility locus to 1p22. Am J Hum Genet 2003;73:301–13. by hypermethylation. J Biol Chem 2000;275:31805–12. 115. Pollock PM, Welch J, Hayward NK. Evidence for three tumor suppressor loci on 121. Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinfor- chromosome 9p involved in melanoma development. Cancer Res 2001;61:1154–61. matics 2002;18:1427–31. 116. Esteller M, Herman JG. Cancer as an epigenetic disease: DNA methylation and 122. Robert MF, Morin S, Beaulieu N, et al. DNMT1 is required to maintain CpG chromatin alterations in human tumours. J Pathol 2002;196:1–7. methylation and aberrant gene silencing in human cancer cells. Nat Genet 2003;33: 117. Flagiello D, Poupon MF, Cillo C, Dutrillaux B, Malfoy B. Relationship between 61–5. DNA methylation and gene expression of the HOXB gene cluster in small cell lung 123. Reid GK, Besterman JM, MacLeod AR. Selective inhibition of DNA methyltrans- cancers. FEBS Lett 1996;380:103–7. ferase enzymes as a novel strategy for cancer treatment. Curr Opin Mol Ther 118. Karpf AR, Lasek AW, Ririe TO, et al. Limited gene activation in tumor and normal 2002;4:130–7. epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2Ј-deoxycy- 124. Goffin J, Eisenhauer E. DNA methyltransferase inhibitors-state of the art. Ann tidine. Mol Pharmacol 2004;65:18–27. Oncol 2002;13:1699–716.

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