Pigment Cell Melanoma Res. 21; 665–676 ORIGINAL ARTICLE Novel MITF targets identified using a two-step DNA microarray strategy Keith S. Hoek1, Natalie C. Schlegel1, Ossia M. Eichhoff1, Daniel S. Widmer1, Christian Praetorius2, Steingrimur O. Einarsson2,3, Sigridur Valgeirsdottir3, Kristin Bergsteinsdottir2, Alexander Schepsky2,4, Reinhard Dummer1 and Eirikur Steingrimsson2 1 Department of Dermatology, University Hospital of Zrich, Zrich, Switzerland 2 Department of Biochemistry KEYWORDS microphthalmia-associated transcrip- and Molecular Biology, Faculty of Medicine, University of Iceland, Reykjavik, Iceland 3 NimbleGen Systems of tion factor ⁄ microarray ⁄ melanoma ⁄ melanocyte ⁄ Iceland LLc, Reykjavik, Iceland 4 Signalling and Development Lab, Marie Curie Research Institute, Oxted, Surrey, transcription ⁄ correlation UK PUBLICATION DATA Received 20 June 2008, CORRESPONDENCE Keith S. Hoek, e-mail: [email protected] revised and accepted for publication 18 August 2008 doi: 10.1111/j.1755-148X.2008.00505.x Summary Malignant melanoma is a chemotherapy-resistant cancer with high mortality. Recent advances in our under- standing of the disease at the molecular level have indicated that it shares many characteristics with develop- mental precursors to melanocytes, the mature pigment-producing cells of the skin and hair follicles. The development of melanocytes absolutely depends on the action of the microphthalmia-associated transcription factor (MITF). MITF has been shown to regulate a broad variety of genes, whose functions range from pigment production to cell-cycle regulation, migration and survival. However, the existing list of targets is not sufficient to explain the role of MITF in melanocyte development and melanoma progression. DNA microarray analysis of gene expression offers a straightforward approach to identify new target genes, but standard analytical procedures are susceptible to the generation of false positives and require additional experimental steps for validation. Here, we introduce a new strategy where two DNA microarray-based approaches for identifying transcription factor targets are combined in a cross-validation protocol designed to help control false-positive generation. We use this two-step approach to successfully re-identify thirteen previously recorded targets of MITF-mediated upregulation, as well as 71 novel targets. Many of these new targets have known relevance to pigmentation and melanoma biology, and further emphasize the critical role of MITF in these processes. Introduction highly resistant to treatment and patients rarely survive longer than 10 months. The microphthalmia-associated Malignant melanoma is an aggressive form of cancer transcription factor (MITF) is a member of the basic with a high mortality rate. Although early stage disease helix-loop-helix leucine-zipper transcription factor fam- is easily treatable, advanced stages of the disease are ily and has been shown to be a critical regulator of Significance While the microphthalmia-associated transcription factor (MITF) is recognized as a master regulator for both melanocyte development and melanoma progression the number of known target genes is relatively small. This high throughput study of gene expression in melanoma cell lines serves to identify with high probability novel candidates for MITF-mediated activation in a melanocytic context. ª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard 665 Hoek et al. melanocyte development and survival (Steingrimsson involved in pigmentation. Despite this variety of known et al., 2004). Various isoforms of MITF are also impor- targets MITF must regulate multiple other genes to tant for the development and homeostasis of other cell account for all aspects of MITF function during melano- types including retinal pigment epithelia, osteoclasts and cyte development and melanoma progression. Thus, in mast cells (Kawaguchi and Noda, 2000; Nechushtan order to further characterize the role of MITF in melano- et al., 1997; Planque et al., 2004). In melanoma, MITF is cyte and melanoma development, it is important to reported to be a critical factor in regulating proliferation identify additional MITF target genes. (Carreira et al., 2006; Hoek et al., 2008), and amplifica- Endogenous expression of MITF is driven by signals tion of the MITF gene is associated with poor patient which likely also activate other genes. Experiments survival (Garraway et al., 2005; Ugurel et al., 2007). where a constitutively active expression vector is used Table 1 shows that MITF is reported to regulate the to drive MITF expression short-circuit this background expression of a broad variety of genes, many of them of normally co-regulated factors. This changes the Table 1. Verification of reported Mitf target genes Symbol Title References Cell type Folda Cob ACP5 Acid phosphatase 5, tartrate resistant Luchin et al., (2000) Osteoclast 209 7 BCL2 B-cell CLL ⁄ lymphoma 2 McGill et al., (2002) Melanoma <2 7 BEST1 Bestrophin 1 Esumi et al., (2007) RPE <2 7 BIRC7 Baculoviral IAP repeat-containing 7 Dynek et al., (2008) Melanoma 214 7 CDK2 Cyclin-dependent kinase 2 Du et al., (2004) Melanocyte 3 7 CLCN7 Chloride channel 7 Meadows et al., (2007) Osteoclast 4 7 DCT Dopachrome tautomerase Yasumoto et al., (2002) Melanocyte 218 7 EDNRB Endothelin receptor type B Sato-Jin et al., (2007) Melanocyte 15 7 GPNMB Glycoprotein (transmembrane) nmb Loftus et al., (2008) Melanoblast 251 7 GPR143 G protein-coupled receptor 143 (Oa1) Vetrini et al., (2004) Melanocyte 19 6 MC1R Melanocortin 1 receptor Aoki and Moro, (2002) Melanocyte 16 4 MLANA Melan-A Du et al., (2003) Melanocyte 173 7 OSTM1 Osteopetrosis-associated transmembrane protein 1 Meadows et al., (2007) Osteoclast 5 7 RAB27A RAB27A, member RAS oncogene family Chiaverini et al., (2008) Melanocyte 13 7 SILV Silver homolog (mouse) Du et al., 2003) Melanocyte 217 7 SLC45A2 Solute carrier family 45, member 2 Du and Fisher, (2002) Melanocyte 222 7 TBX2 T-box 2 Carreira et al., (2000) Melanocyte 4 5 TRPM1 Transient receptor potential cation channel, M1 Miller et al., (2004) Melanocyte 137 6 TYR Tyrosinase Hou et al., (2000) Melanocyte 267 7 TYRP1 Tyrosinase-related protein 1 Fang et al., (2002) Melanocyte <2 7 CADM1 Cell adhesion molecule 1 Ito et al., (2003) Mast cell <2 1 CDKN1A Cyclin-dependent kinase inhibitor 1A (p21) Carreira et al., (2005) Melanocyte <2 0 CDKN2A Cyclin-dependent kinase inhibitor 2A (p16) Loercher et al., (2005) Melanocyte <2 1 Cma1 Chymase 1, mast cell Morii et al., (1997) Mast cell 7 0 CTSK Cathepsin K Motyckova et al., (2001) Osteoclast <2 1 DIAPH1 Diaphanous homolog 1 Carreira et al., (2006) Melanoma <2 1 GZMB Granzyme B Ito et al., (1998) Mast cell 10 0 HIF1A Hypoxia-inducible factor 1, alpha subunit Busca et al., (2005) Melanoma 2 0 ITGA4 Integrin, alpha 4 Kim et al., (1998) Melanocyte 3 0 KIT v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene Tsujimura et al., 1996) Mast cell <2 2 Mcpt2 Mast cell protease 2 Ge et al., (2001) Mast cell <2 0 Mcpt4 Mast cell protease 4 Jippo et al., (1999) Mast cell <2 0 Mcpt9 Mast cell protease 9 Ge et al., (2001) Mast cell <2 0 MET Met proto-oncogene McGill et al., (2006) Melanocyte <2 3 NDST2 N-deacetylase ⁄ N-sulfotransferase 2 Morii et al., (2001) Melanocyte <2 0 NGFR Nerve growth factor receptor (p75) Jippo et al., (1997) Mast cell 3 0 OSCAR Osteoclast associated, immunoglobulin-like receptor So et al., (2003) Osteoclast 4 0 PRKCB1 Protein kinase C, beta 1 Park et al., (2006) Melanocyte 2 1 SERPINE1 Serpin peptidase inhibitor E1 (PAI-1) Murakami et al., (2006) Mast cell 6 0 SLC11A1 Solute carrier family 11, member 1 Gelineau-van Waes et al., (2008) RPE 4 1 TPH1 Tryptophan hydroxylase 1 Ito et al., (1998) Mast cell <2 0 Tpsb2 Tryptase beta 2 Morii et al., (1996) Mast cell <2 1 aFold change up on transformation of SK-MEL-28 with a Mitf-expressing vector (P < 0.05). bNumber of data sets in which correlation exceeds 0.5 with 207233_s_at (MITF). RPE, retinal pigment epithelium. 666 ª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard Novel MITF targets context in which MITF operates and, in the absence of co-regulated activators and suppressors, potentially activates biologically irrelevant target genes. Alterna- tively, identification of novel MITF targets via correla- tion of mRNA expression patterns is complicated by factors which are co-regulated with MITF. This poten- tial confusion of causality and correlation also risks generating false positives. However, each of these approaches serve to complement the other. Exogenous upregulation of MITF is unlikely to activate endoge- nously co-regulated factors, and analysis of endo- genous expression patterns serves to filter out biologically irrelevant transcription activated by exoge- nous MITF expression. We show here how such a Figure 1. MAPK ⁄ JNK signalling and MITF expression. Cell two-step strategy may detect additional targets for extracts from a library of melanoma cultures were subject to MITF-driven transcription. western blotting against phosphorylated Erk1 ⁄ Erk2, phosphorylated JNK, phosphorylated p38, and b-tubulin. MITF levels, as well as BRAF and NRAS mutation status were reported previously (Hoek Results et al., 2006). These data show that neither MAPK ⁄ JNK activation nor BRAF ⁄ NRAS mutation status correlate with Mitf expression in Endogenous MAPK signalling does not affect Mitf melanoma. protein levels If a transcription factor’s function is primarily regulated by post-transcriptional or post-translational mechanisms sensitive. Nevertheless,