Research Article ZNF703 in luminal B breast cancer Little is known about speciﬁc signalling pathways deregu- lated in luminal B tumours. This subtype is characterized by the expression of hormone receptors combined with high prolif- erative index. The speciﬁc gene expression signature associated with luminal B tumours is enriched in genes that drive the proliferation of cancer cells, such as CCNE1, MKI67 or MYBL2 (Sorlie et al, 2001). Moreover, the functional loss of the retinoblastoma tumour suppressor gene (RB1) seems to be associated with the luminal B phenotype (Herschkowitz et al, 2008). To identify master genes and molecular mechanisms implicated in luminal B biology, we used a strategy based on an integrated proﬁling study that combined high-resolution analysis of genome copy number aberrations (CNA) by array- comparative genomic hybridization (aCGH) and gene expres- sion in luminal A and luminal B primary tumours. According to the number and features of CNAs, several types of genomic proﬁles have been identiﬁed in breast cancers, including simplex, complex and ampliﬁer (also called ﬁrestorm). We and others have found that luminal B tumours are frequently associated with the ‘ampliﬁer’ type, which shows several focal ampliﬁcations (Chin et al, 2006; Fridlyand et al, 2006; Geyer et al, 2009; Hicks et al, 2006). We show here that ﬁve genomic regions are frequently ampliﬁed in luminal B tumours compared to luminal A tumours. The ampliﬁcation of chromosome region 8p12 is highly speciﬁc and among the most frequent alterations of the luminal B subtype. We identiﬁed ZNF703 as the most signiﬁcant candidate oncogene of this ampliﬁcation. The function of ZNF703 is not known. In this study, we identiﬁed and functionally validated ZNF703 protein interactors and gene expression programme activation. Our results suggest that ZNF703 plays a role in the regulation of the luminal B cancer stem cell (CSC) population via transcriptional control of key cellular processes. RESULTS 8p12 amplification is a recurrent copy number abnormality in luminal B tumours We had previously studied (Finetti et al, 2008) a series of 266 breast tumours by gene expression analysis and determined their molecular subtypes by SSP analysis (Hu et al, 2006). To identify molecular mechanisms and signalling pathways speciﬁcally deregulated in luminal B tumours, we compared the genomic proﬁles of 41 luminal B and 59 luminal A cases from this panel (Supplementary Table 1) by using high density aCGH. Because luminal B tumours have been associated with the ampliﬁer genomic proﬁle, we focused on recurrent ampliﬁca- tions. As shown in Fig 1A, ﬁve genomic regions of recurrent high-level ampliﬁcation (8p12, 8q22, 11q13, 17q24, 20q13) were associated with luminal B tumours (Fisher’s exact test, p < 0.01). The different genomic segments ampliﬁed and recurrently associated with luminal B tumours are reported in Supplementary Table 2. In contrast, with the same statistical cut-off we did not identify any genomic region speciﬁcally ampliﬁed in luminal A tumours; this was not surprising since luminal A tumours have been associated with the simplex phenotype. Several studies have already reported ampliﬁcation of these genomic regions in luminal tumours (irrespective of A and B) as compared to basal tumours (Adelaide et al, 2007; Letessier et al, 2006). Luminal B tumours exhibit ZNF703 gene amplification and overexpression We had previously characterized the ampliﬁed 8p12 region. To identify potential targeted genes that may specify luminal B biology, we focused on this 8p12 ampliﬁcation. In the past, several genes, including RCP/RAB11FIP1, PPAPDC1B or FGFR1, have been proposed as candidate oncogenes targeted by the 8p12 ampliﬁcation (Bernard-Pierrot et al, 2008; Turner et al, 2010; Zhang et al, 2009). We found a strong association between 8p12 ampliﬁcation and luminal B tumour subtype, with 32% of luminal B tumours displaying ampliﬁcation (Fig 1A, Supplementary Table 3). We searched to identify the 8p11-12 genes targeted by ampliﬁcation by comparing for each gene the association between copy number and messenger RNA (mRNA) expression level for the 38 genes comprised in the amplicon deﬁned by the aCGH analysis. These genes are localized in a 4.6 Mb region delimited by ZNF703 towards the telomere and ANK1 towards the centro- mere. We found a hot spot of ampliﬁcation frequency for the genomic segment that contains only the ZNF703 gene (Fisher’s exact test, p < 0.01) (Supplementary Table 2). Nineteen genes showed ampliﬁcation correlated with upregulation (Fisher’s exact test, p < 0.01; Fig 1B). Among them, ZNF703 was the most frequently ampliﬁed and overexpressed gene. In 11 breast cancer cell lines, we did a similar integrated analysis of genomic ampliﬁcation (using this time quantitative PCR) and gene overexpression by both DNA microarrays and reverse- transcribed (RT)-PCR for each of the 38 genes. ZNF703 expression levels measured by DNA microarrays and by quantitative RT-PCR were correlated (Pearson test, r ¼ 0.96), and correlated with an increase in protein expression level (Supplementary Fig 1). The integrated analysis on cell lines conﬁrmed the results obtained on luminal B primary tumours, with ZNF703 gene presenting the most signiﬁcant correlation between DNA copy number and mRNA expression (Pearson test, r ¼ 0.9) (Supplementary Fig 2). Ampliﬁcation and overexpression of ZNF703 were especially high in MDA-MB-134 and HCC1500 cell lines, ZNF703 was over- expressed up to 15-fold in these cell lines as compared to cell lines derived from normal breast. Probably because of a constitutively high level of proliferation due to cell culture conditions, the distinction between luminal A and B is not possible in cell lines (Charafe-Jauffret et al, 2006; Neve et al, 2006). It was therefore not possible to show whether these two cell lines may have originated from a luminal B tumour. We next examined ZNF703 expression measured by DNA microarrays according to the distribution of molecular subtypes in 1172 breast cancers from 11 different published datasets (Supplementary Tables 4 and 5). ZNF703 overexpression was associated with luminal B tumours when compared with luminal A tumours (t-test, p ¼ 4 10 6) or with other subtypes (ANOVA, p ¼ 1.2 10 53) (Fig 1C). 154 2011 EMBO Molecular Medicine EMBO Mol Med 3, 153–166 www.embomolmed.org