Research Article ZNF703 in luminal B breast cancer self-renewal capacity. Association of proliferation genes and self-renewal regulators constitute an ideal combination to initiate and sustain tumour progression. MATERIALS AND METHODS Ethics statement Each patient gave informed consent and the study was approved by our institutional review board. Cell culture A total of eleven breast cell lines (BCL) derived from normal or malignant epithelium were analysed: 184B5, CAMA-1, HCC1500, HME-1, MCF7, MCF10A, MDA-MB-134, T47D, ZR-75–1 obtained from ATCC (http://www.atcc.org/), SUM52 obtained from Dr S. Ethier (http:// www.asterand.com/asterand/BIOREPOSITORY/hbreastcancercellline- s.aspx), and S68 obtained from V. Catros (Universit´e de Rennes, Rennes, France). BCLs were grown using the recommended culture conditions. For experiments with oestrogen induction, cells were washed with PBS and precultured in phenol red-free RPMI 1640 medium supplemented with 10% charcoal-filtered FCS (Hyclone, Thermo Fisher Scientific) for 24 h. For rescue experiments, MDA-MB- 134 and HCC1500 cells were treated with 17b-estradiol (1 or 10 nM) (E2, Sigma–Aldrich, E8875). Tumour samples Tumour tissues were collected from 266 patients with invasive adenocarcinoma who underwent initial surgery at the Institut Paoli- Calmettes (Marseille, France) between 1987 and 2007, and from 22 samples from the Jules Bordet Institute previously described (Loi et al, 2007). Samples were macrodissected and frozen in liquid nitrogen within 30 min of removal. DNA and RNA extraction DNA and RNA were extracted from frozen tumour samples by using guanidium isothiocynanate and cesium chloride gradient as pre- viously described (Bertucci et al, 2006). RNA and DNA integrity was controlled on Agilent Bioanalyzer (Agilent Technologies) and agarose gel electrophoresis, respectively. DNA and RNA form BCLs were isolated utilizing RNeasy Mini Kit (QIAGEN) and used for QPCR and RTQPCR in a LightCycler FastStart DNA Masterplus SYBR Green I (Roche, Mannheim, Germany) (supplementary method). Gene expression analysis Expression profiles were analysed on a set of 266 tumours, 4 normal breast samples and 31 cell lines using Affymetrix U133 Plus 2.0 human oligonucleotide microarrays as previously described (Charafe- Jauffret et al, 2006; Irizarry et al, 2003). Scanning was done with Affymetrix GeneArray scanner. Data were analysed by ‘Robust Multi- chip Average’ (RMA) with the non-parametric quantile algorithm as normalization parameter in R/Bioconductor and associated packages (Benito et al, 2004). The five molecular subtypes related to the intrinsic breast cancer tumour classification were determined for the 266 samples using the single sample predictor (SSP) classifier based on list of 306 genes and 315 samples (Adler et al, 2009) associated to ‘distance weighted discrimination’ (DWD) as data set adjustment (Subramanian et al, 2005). Co-expression search for ZNF703 (probeset 222760_at) was done using MEM web tool (Chen and Sharp, 2004) as described in supplementary methods. Pathway-based analysis was done using IPA (Ingenuity1 Systems, www.ingenuity.com). Gene expression data are deposited in Gene expression Omnibus (GEO) public database (www.ncbi.nlm.nih.gov/geo) and sample accession number are reported in supplementary Table 12. Additional expression data set To determine ZNF703 gene expression in luminal breast cancer, we collected gene expression data from several public databases. Eleven public data sets were included, collected from eleven independent studies (Supplementary Table 4) representing a total of 1172 different samples. For each sample, molecular subtypes were defined as described above (Supplementary Table 5). To compare ZNF703 gene expression across data sets and to exclude bias from population heterogeneity, expression levels were standardized within data sets using the luminal A population as referential. Student’s t-test and one- way ANOVA test were used to evaluate ZNF703 expression level difference between luminal B and luminal A population and across the five molecular subtypes, respectively. Gene set enrichment analysis Gene set enrichment analysis (GSEA) {http://www.broadinstitute.org/ gsea/} (Subramanian et al, 2005) was used to identify a priori defined sets of genes that were differentially expressed between luminal A and luminal B samples. GSEA was applied on the IPC series with 138 luminal BCs (i.e. the 49 luminal B and 89 luminal A). Prior the GSEA, data were filtered to remove low and poorly measured expression probe sets as defined by an expression value inferior to 6.64 log2 units across the 138 BCs. GSEA was done using signal-to-noise metric for ranking genes, weighted enrichment statistic to computed enrichment score (ES) of each gene sets tested and 1000 phenotype permutations to evaluate significance. As gene sets database, we chose the transcription factor targets list (C3, TFT) from the Molecular Signatures database (http://www.broadinstitute.org/gsea/msigdb). The C3 TFT list contains gene sets that share a transcription factor-binding site defined in the TRANSFAC database {http://www.gene-regulation.com}. Gene sets were defined as significant at the 5% level with a false discovery rate (FDR) under 25%. Relation between each transcription factor and E2F1 or ESR1/ERa was established by mining PubMed abstracts using Chilibot (Chen and Sharp, 2004). Array-comparative genomic hybridization Genome profiling was established on a subset of 100 luminal breast tumours (59 luminal A and 41 luminal B) for which RNA and DNA samples were both available (Supplementary Table 1). Genomic imbalances of 100 breast tumours were analysed by aCGH using 244K CGH Microarrays (Hu-244A, Agilent Technologies) following a described protocol (Etienne et al, 2007). The final data set contained 225 388 unique probes covering 22 509 genes and intergenic part following the hg17 human genome mapping. aCGH data processing is described in Supplementary method section. Plasmids constructs and clone selection Human ZNF703 cDNA full length (IMAGE: 5527569) was obtained by ImaGenes (Berlin, Germany). cDNA sequence was PCR-amplified using 162 2011 EMBO Molecular Medicine EMBO Mol Med 3, 153–166 www.embomolmed.org