The paper explained PROBLEM: Despite improvements in the treatment of breast cancer, resistance to conventional therapy is a major clinical problem. The heterogeneity of breast cancer renders prognosis and treatment difficult. Heterogeneity may result from various cell- of-origins and genetic alterations. The latter are numerous in breast cancer but few have been characterized and even less can be used as markers or therapeutic targets. Although both luminal A and B tumours express hormone receptors (oestrogen and progesterone), the risk of relapse in women treated by hormone therapy is greater in women with a luminal B tumour than in women with a luminal A tumour. Therefore, the elucidation of the molecular mechanisms that govern luminal B tumour biology should improve both our understanding of tumourigenesis and treatment of patients. RESULTS: We have studied 100 luminal breast tumours by combined analysis of genome CNAs and gene expression. We show that amplification of the ZNF703 gene, located in chromosomal region 8p12, preferentially occurs in luminal B tumours. We identified ZNF703 as a nuclear protein whose expression is stimulated by oestrogen and which represses ER-associated proteins. ZNF703 is part of a protein complex including DCAF7 attB1-TCACCATGAGCGATTCGCCCGCTGG-30 and attB2-CCTGGTATCC- CAGCGCCGAGG-50 oligonucleotides. The resulting PCR fragment was inserted into the vector pDONRTM/Zeo using BP-reaction gateway technology (Invitrogen). pDONRTM/Zeo-ZNF703 was then subcloned in pEGFP-C1-DEST using LR-reaction (Invitrogen). MCF7 cells were transfected with pEGFP-ZNF703 or pEGFP by electroporation as described by the manufacturer (Amaxa, Lonza Cologne, Germany). Individual clones stably expressing GFP-ZNF703 fusion protein or GFP were obtained by constant geneticin selection at 0.5 mg/ml, the resulting clones were confirmed by Western blotting. siRNA lipofection Three stealth siRNA targeted to the ZNF703 mRNA (Invitrogen) were designed along with the corresponding stealth siRNA control. MDA- MB-134 cells were lipofected with these four oligonucleotides utilizing lipofectamine RNAiMAX (Invitogen). Twenty-five thousand cells were plated in triplicate in 24 well plates with 10 pmol of siRNA in 600 ml of Opti-MEM I Medium. Cell proliferation was evaluated in triplicate at 30-h after lipofection. Cell proliferation assay For MTS assays, cells were plated in adherent conditions in 96-well plates at 5000 cells per well in the presence or absence of E2. The cell growth kinetic was estimated at different time points by addition of 20 ml MTS solution (5 mg/ml in PBS) in each well. Cells were then incubated for 1 h at 378C, followed by addition of 50 ml DMSO to each well. Absorbance was measured at 560 nm in a fluorescence plate Research Article Fabrice Sircoulomb et al. and PHB2 and its expression both stimulates cell proliferation and increases the stem cell population. Moreover, ZNF703 expression in the MCF7 cell line is associated with a shift from an ER-associated gene expression programme to an E2F1- associated programme. This transcriptional shift may lead to the activation of stem cell-related gene expression, which induces an increase in CSCs. IMPACT: We identified a new type of oncogene. ZNF703 should be not be viewed solely as a proliferation booster but may be considered as a transcriptional re-programmer controlling self-renewal capacity. Defining the role of ZNF703 in the regulation of luminal B oncogenesis may help to understand luminal B tumour biology and hormone-resistance: the asso- ciation of ZNF703 amplification with the luminal B type may serve as a marker for this subtype of poor prognosis; the cross-talk between ER and ZNF703 explains features of luminal B tumours; the role of ZNF703 on self-renewal may be used as a handle to characterize the luminal B cell-of-origin; finally, the ZNF703-associated protein complex may be used as a therapeutic target to treat luminal B tumours with 8p12 amplification. reader (Spectrafluor; Tecan). For MDA-MB-134 cells were first lipofected with siRNA and plated for MTS assays after 30 h. Nuclease treatments MCF7 GFP-ZNF703 culture cells were grown on labtek chamber, washed briefly in PBS and fixed in cold methanol/acetone for 7 min at 208C. Fixed cells were then treated, at room temperature, with RNAse A (80 mg/ml) or DNAse I (100 mg/ml) for 30 and 15 min, respectively. Cells were then washed with PBS and immunostained with an anti-GFP antibody for 1 h (Roche 11814460001, 1:500). The nuclei were counterstained with DAPI/antifade (Invitrogen) and cover- slipped. Sections were examined with a fluorescent microscope (Leica). Co-immunoprecipitation Cells were lysed in extraction buffer (1% v/v Triton X-100, 50 mM Hepes, pH 7, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 100 mM sodium fluoride, 1 mM Na3VO4 and one tablet of CompleteTM inhibitor mix (Roche Applied Science) per 25 ml of buffer and loaded onto SDS- polyacrylamide gels. For co-immunoprecipitation experiments 12 mg of protein lysates were produced, with 2 mg for SDS-PAGE analysis and 10 mg for mass spectroscopy analysis. First, lysates were precleared 1 h with 70 ml of sepharose beads at 48C. After centrifugation, 5 mg of antibody were added to the precleared lysate. Immunoprecipitation was performed overnight at 48C under constant rotation. 70 ml of protein G-sepharose or A-sepharose beads were added and incubated 1 h at 48C. Beads were collected by centrifugation (1500 rpm, 5 min) and washed once in lysis buffer and three times in HNTG buffer (Hepes www.embomolmed.org EMBO Mol Med 3, 153–166 2011 EMBO Molecular Medicine 163