Mining Gene Expression Signature for the Detection of Pre-Malignant Melanocytes and Early Melanomas with Risk for Metastasis Camila Ferreira de Souza1, Patrı´cia Xander2, Ana Carolina Monteiro1, Amanda Gon c¸alves dos Santos Silva3, De´ bora Castanheira Pereira da Silva4, Sabine Mai5, Viviane Bernardo6, Jose´ Daniel Lopes7, Miriam Galvonas Jasiulionis1* 1 Departamento de Farmacologia, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil, 2 Departamento de Cieˆncias Biolo´ gicas, Universidade Federal de Sa˜o Paulo, Diadema, Sa˜o Paulo, Brazil, 3 CIPE, Hospital A.C. Camargo, Sa˜o Paulo, SP, Brazil, 4 Departamento de Oncologia Cutaˆnea, Hospital A.C. Camargo, Sa˜o Paulo, Sa˜o Paulo, Brazil, 5 Manitoba Institute of Cell Biology, The University of Manitoba, Cancer Care Manitoba, Winnipeg, Manitoba, Canada, 6 Departamento de Informa´tica em Sau´ de, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil, 7 Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil Abstract Background: Metastatic melanoma is a highly aggressive skin cancer and currently resistant to systemic therapy. Melanomas may involve genetic, epigenetic and metabolic abnormalities. Evidence is emerging that epigenetic changes might play a significant role in tumor cell plasticity and metastatic phenotype of melanoma cells. Principal findings: In this study, we developed a systematic approach to identify genes implicated in melanoma progression. To do this, we used the Affymetrix GeneChip Arrays to screen 34,000 mouse transcripts in melan-a melanocytes, 4C pre-malignant melanocytes, 4C112 non-metastatic and 4C11+ metastatic melanoma cell lines. The genome-wide association studies revealed pathways commonly over-represented in the transition from immortalized to pre-malignant stage, and under-represented in the transition from non-metastatic to metastatic stage. Additionally, the treatment of cells with 10 mM 5-aza-29-deoxycytidine (5AzaCdR) for 48 hours allowed us to identify genes differentially re- expressed at specific stages of melan-a malignant transformation. Treatment of human primary melanocytes with the demethylating agent 5AzaCdR in combination to the histone deacetylase inhibitor Trichostatin A (TSA) revealed changes on melanocyte morphology and gene expression which could be an indicator of epigenetic flexibility in normal melanocytes. Moreover, changes on gene expression recognized by affecting the melanocyte biology (NDRG2 and VDR), phenotype of metastatic melanoma cells (HSPB1 and SERPINE1) and response to cancer therapy (CTCF, NSD1 and SRC) were found when Mel-2 and/or Mel-3-derived patient metastases were exposed to 5AzaCdR plus TSA treatment. Hierarchical clustering and network analyses in a panel of five patient-derived metastatic melanoma cells showed gene interactions that have never been described in melanomas. Significance: Despite the heterogeneity observed in melanomas, this study demonstrates the utility of our murine melanoma progression model to identify molecular markers commonly perturbed in metastasis. Additionally, the novel gene expression signature identified here may be useful in the future into a model more closely related to translational research. Citation: Souza CFd, Xander P, Monteiro AC, Silva AGdS, Silva DCPd, et al. (2012) Mining Gene Expression Signature for the Detection of Pre-Malignant Melanocytes and Early Melanomas with Risk for Metastasis. PLoS ONE 7(9): e44800. doi:10.1371/journal.pone.0044800 Editor: Andrzej T. Slominski, University of Tennessee, United States of America Received March 13, 2012; Accepted August 14, 2012; Published September 11, 2012 Copyright: 2012 Souza et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo [09/51462-9 to C.F.S. & 06/61293-1 and 2006/61293-1 AND 11/12306-1 to M.G.J.]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mjasiulionis@gmail.com Introduction Metastatic melanoma is a highly aggressive and drug-resistant skin cancer and has a poor prognosis, with a 1-year survival rate ranging from 33% to 62% [1,2]. The current methodology to diagnose primary melanomas is based on histomorphologic features, however such classification system has remained of limited clinical relevance once metastases have developed. The current prevailing opinion is that melanomas are more variable at the molecular level than can be observed macro/microscopically [3,4]. For anti-melanoma strategies to be successful, it is important the better understanding about the molecular mechanisms that mediate phenotype-switching in melanomas, since these biomark- ers could drive metastatic disease. According to the genetic model to explain the pathways towards metastasis, the acquisition of mutations that promote invasiveness leads to cells migrating away from the primary tumor before arriving in a new site where they divide to form a new metastasis. As the genetic lesions are PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44800