Candidate Luminal B Breast Cancer Genes Identified by Genome, Gene Expression and DNA Methylation Profiling

Gene Expression and DNA Methylation Profiling. PLoS ONE 9(1): e81843. doi:10.1371/journal.pone.0081843 Candidate Luminal B Breast Cancer Genes Identified by Genome, Gene Expression and DNA Methylation Profiling Ste phanie Cornen 0 Arnaud Guille 0 Jose Ade lade 0 Lynda Addou-Klouche 0 Pascal Finetti 0 Marie-Rose Saade 0 Marwa Manai 0 Nadine Carbuccia 0 Ismahane Bekhouche 0 Anne Letessier 0 Ste phane Raynaud 0 Emmanuelle Charafe-Jauffret 0 Jocelyne Jacquemier 0 Salvatore Spicuglia 0 Hugues de The 0 Patrice Viens 0 Fran cois Bertucci 0 Daniel Birnbaum 0 Max Chaffanet 0 Chad Creighton, Baylor College of Medicine, United States of America 0 1 Marseille Cancer Research Center, UMR1068 Inserm, Institut Paoli-Calmettes (IPC), Department of Molecular Oncology, Equipe labellise e Ligue Contre le Cancer , Marseille , France , 2 Biotoxicology Laboratory, Djillali Liabes University, Sidi-Bel-Abbe`s, Algeria, 3 Biochemistry and Molecular Biology Unit, Biology Dpt, Faculty of Sciences El Manar, Tunis, Tunisia, 4 Institut Paoli-Calmettes (IPC), Department of Biopathology , Marseille , France , 5 Aix-Marseille Universite , Marseille, France, 6 Inserm U1090, TAGC, Marseille , France , 7 UMR 944/7212 INSERM/CNRS/Universite Paris 7, Ho pital St Louis, Paris, France, 8 Institut Paoli-Calmettes (IPC), Department of Medical Oncology , Marseille , France Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal Bspecific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a metaanalysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype. - Funding: Financial support for this study came from Inserm, Institut Paoli-Calmettes, and grants from Ligue Nationale Contre le Cancer (Label DB), GEFLUC (MC), Institut National du Cancer (ACI2007, AO2008, AO/R-TRANS09 identification, caracterisation et validation clinique de nouvelles cibles cellulaires et moleculaires dans les cancers du sein de mauvais pronostic IVOIRES (INCA)) and DGOS (SIRIC label: grant INCa-DGOS-Inserm 6038). LAK and IB were supported by a fellowship from the Franco-Algerian program of high level formation in France (PROFAS) and the Algerian government, respectively. MM was supported by a fellowship from the AVEROES European program. 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. . These authors contributed equally to this work. Breast cancer (BC) is a complex and heterogeneous disease whose therapeutic approach must be refined in view of recent studies allowing better classification and/or prognosis assessment [1]. DNA microarray-based expression profiling has identified clinically and biologically relevant intrinsic molecular subtypes (luminal A, luminal B, ERBB2, basal, and normal-like) [24] and prognostic and/or predictive gene expression signatures [5]. Genomic studies have also suggested a prognostic impact of genomic data [68]. Combining expression and genomic data allowed the identification of candidate BC genes [6,919]. The status of DNA methylation may also contribute to improve BC molecular classification [2024]. The identification of new fusion genes by RNA-seq approaches [25,26] and of driver mutations in cancer genes in various molecular and clinical BC entities [2731] will also help design targeted treatments. Luminal B BCs have a poor prognosis [32]. Although they express hormone receptors, their metastatic risk and resistance to hormone therapy and to conventional chemotherapy demand to develop appropriate therapies. Some proteins (e.g. CITED2, NCOR2) or molecular networks associated with BCAR (breast cancer anti-estrogen resistance) genes are involved in the resistance to anti-estrogen therapy and in the progression of these cancers [3235]. Luminal B cancers exhibit various mutated genes, TP53 and PIK3CA being the most frequent (29% each) [28]. To further define molecular alterations associated with the luminal B subtype we studied DNA copy number aberrations (CNAs), DNA promoter methylation alterations (DPMAs), gene expression deregulation (EXP), and selected gene mutations in 188 primary BC samples. These analyses identified luminal B-specific candidate genes. Materials and Methods Ethics statement The study was approved by our institutional review board: the Comite dOrientation Strategique of the Institut Paoli Calmettes (IPC) (Marseille, France). Each patient gave a written informed consent for research use. Breast cancer samples Pre-treatment tumor tissues were collected from 188 patients with invasive adenocarcinomas. Patients underwent surgical biopsies or initial surgery at the Institut Paoli-Calmettes between 1987 and 2007. The main histoclinical, biological and subtype characteristics were established for the 188 BCs as described [12,1719]. They are listed in Table S1A and illustrated in Figure S1. Gene profiling and data analysis DNA and RNA were extracted as previously described [12,17 19] and controled on Agilent Bioanalyzer (Agilent Technologies, Massy, France). Genomic profiles of the 188 BCs were established by using array-comparative genomic hybridization (aCGH) onto high-resolution 244K CGH microarrays (Hu-244A, Agilent Technologies, Massy, France). A pool of 13 normal male DNA was used as reference. Gene expression data from the same 188 BCs and 4 normal breast (NB) samples, whic (...truncated)