Research Article Fabrice Sircoulomb et al. Figure 1. ZNF703 is a target gene of the 8p12 amplification in luminal B breast tumours. A. Supervised analyses of aCGH data comparing amplifications in 41 luminal B tumours and 59 luminal A tumours. Genomic segments are ordered on the X-axis from chromosome 1 to 22 and on the Y-axis according to their association with luminal A or B molecular subtype ( log( p- value)). The grey zone contains genomic seg- ments that are not significantly associated with any of the two molecular subtypes ( p > 0.01). Red dots represent genomic segments significantly amplified in luminal B breast tumours ( p < 0.01). These segments are contained in 8p12, 8q22, 11q13, 17q24, and 20q13 chromosomal regions. We did not identify genomic segments signifi- cantly amplified in luminal A tumours. B. Combined genomic and transcriptional analysis for each gene from the 8p12 amplicon identified in luminal B breast tumours. Genes are ordered from telomere (left) to centromere (right) and the frequency of luminal B tumours presenting an increase in gene copy number and mRNA level is represented. Blue bars label genes that present a statistically significant association between gene amplification and overexpression (Fisher’s exact test, p-value 0.05). C. Box-plot representing ZNF703 expression distri- bution according to molecular subtypes of 1172 breast tumours. ZNF703 expression varies across the five molecular subtypes (ANOVA, p ¼ 1.2 10 53) and is higher in luminal B tumours compared to luminal A tumours (t-test, p ¼ 4 10 6). D. Kaplan–Meier survival curves according to ZNF703 gene expression status. ZNF703 gene expression is associated with DFS ( p ¼ 0.0136). To determine whether ZNF703 overexpression had clinical impact, we investigated the association with clinical and pathologic features in 561 luminal tumours (A and B). ZNF703 overexpression was not correlated with any histocli- nical factor (Supplementary Table 6). However, a high level of ZNF703 mRNA was associated with a decrease in disease-free survival (DFS) with a 5-years DFS of 68% versus 78% for a low level of ZNF703 ( p ¼ 0.0136) (Fig 1D). Taken together, our results identiﬁed ZNF703 as a plausible oncogene candidate, prompting us to study its functional contribution to luminal B oncogenesis. Oestrogen receptor regulates ZNF703 transcription The oestrogen receptor (ER) is the master transcriptional regulator of luminal tumour phenotype (Hurtado et al, 2008). Interestingly, several studies have described ZNF703 as a target gene of ER transcriptional activity and have identiﬁed oestrogen response elements (ERE) in its promoter region (Lin et al, 2004, 2007). To ﬁrmly establish that ZNF703 expression is under the control of ER transcriptional activity, we cultured MDA-MB-134 and HCC1500 breast cancer cell lines in medium depleted or supplemented in 17b-estradiol (E2). In these cells, ZNF703 transcription and protein production were induced by the addition of E2 in a time-dependent manner (Supplementary Fig 3). Thus, ZNF703 is a potential oncogene under the control of ER activity in luminal B tumours. Identification and characterization of ZNF703 in the nucleus To identify signalling pathways regulated by ZNF703, we overexpressed ZNF703 in MCF7, a luminal breast cancer cell line that does not present 8p12 ampliﬁcation, utilizing a GFP- ZNF703 fusion protein expression vector. As a control, we www.embomolmed.org EMBO Mol Med 3, 153–166 2011 EMBO Molecular Medicine 155