(Gelsi-Boyer et al, 2005) and others (Kwek et al, 2009; Ray et al, 2004) have previously dissected the 8p12 ampliﬁcation and identiﬁed up to four subamplicons, A1–A4. We have shown here that ZNF703, which is included in A1, is signiﬁcantly ampliﬁed in luminal B tumours. Moreover, ZNF703 mRNA expression is associated with luminal B tumours and deﬁnes patients with poor clinical outcome. A different study has been performed on a large series of breast tumours and has similarly identiﬁed ZNF703 as the most prominent gene in the 8p12 amplication occurring frequently in luminal B samples (Holland et al, 2011). ZNF703 satisﬁes the main criterion for being a driver of the A1 amplicon, i.e. it is overexpressed when ampliﬁed. We found it was the most regularly ampliﬁed and overexpressed 8p11-12 gene in both our cell lines and tumour panels. This was also observed by previous studies (Letessier et al, 2006; Ray et al, 2004). Other potential oncogenes of 8p11-12 have been identiﬁed, including FGFR1 (Turner et al, 2010), TC-1/C8orf4 (Yang et al, 2007; Zhang et al, 2009), PPAPDC1P (Bernard- Pierrot et al, 2008), RCP/RAB11FIP1 (Zhang et al, 2009) and WHS1L1 (Yang et al, 2010). Various assays, mostly proliferation assays, have shown that these genes may function as oncogenes. They could act in collaboration with ZNF703. ZNF703 has four important features that may explain its involvement in mammary oncogenesis. First, we observed an induction of proliferation when we overexpressed ZNF703 gene in MCF7 cells independently of E2 stimulation, whereas non- modiﬁed MCF7 cells need E2 stimulation to boost their proliferation. Reciprocally, when we used siRNA to deplete ZNF703 in MDA-MB-134, and tested the capacity of the ZNF703- depleted cells to proliferate, we observed a reduction of the capacity to proliferate of ZNF703-depleted cells. Second, ZNF703 transcription is induced by oestrogen stimulation. In agreement, ZNF703 has been identiﬁed as an ER-regulated gene (Lin et al, 2004, 2007). In contrast, the other 8p12 potential oncogenes have not been shown to be regulated by ER. This could explain the association of A1 (ZNF703) ampliﬁcation with the luminal B subtype. The 8p11-12 region is often co-ampliﬁed with the 11q13 region (Kwek et al, 2009; Ollendorff et al, 1992; Szepetowski et al, 1992), which comprises the Cyclin D- encoding locus CCND1. CCND1 is also under the control of ER (Cicatiello et al, 2004). ZNF703 and CCND1 may thus function in the same pathway and potentiate each other. Indeed, CCND1 overexpression increases ZNF703 expression (Kwek et al, 2009). Third, the ZNF703 protein may be a cofactor in a nuclear co- repressor complex composed of DCAF7, PHB2 and NCOR2. Co- repressors control the action of many transcription factors including ER (Battaglia et al, 2010; Perissi et al, 2010). Interestingly, ZNF703 overexpression represses the expression of luminal markers and ER-associated proteins such as FOXA1 and GATA3. ZNF703 would uncouple ER-related differentiation and proliferation programmes. Moreover, ZNF703-overexpres- sing cells display RB1 phosphorylation, P27Kip1 downregulation, E2F1 activation and CCNE1 activation. ZNF703 interaction with DCAF7 may explain its role on E2F1 signalling. DCAF7 is a CUL4/DDB1 interacting protein (Jin et al, 2006) that activates SKP2 to induce P27Kip1 proteolysis (Bondar et al, 2006). Research Article Fabrice Sircoulomb et al. Interestingly, the DCAF7 gene is located in chromosomal region 17q23, which we found often gained or ampliﬁed in luminal B tumours (Supplementary Table 2). GSEA showed that the E2F1 activation/ER repression balance is prominent in luminal B tumours. Fourth, ZNF703 overexpression increases both the CD49f-positive cell population and the formation of primary and secondary tumourspheres suggesting a role in the control of CSC self-renewal. Moreover, the gene expression programme activated by ZNF703 overexpression is enriched in genes related to stem cell biology. It has been proposed that luminal tumours might arise through transformation of an ER-positive luminal progenitor (Dontu et al, 2004). The oncogenic capacity of ZNF703 could thus be exerted on ER-positive mammary progenitors and contribute to re-activation of a self-renewal programme. All these speciﬁc features may explain both the association of ZNF703 ampliﬁcation with the luminal B subtype and its frequent association with CCND1. We propose that ZNF703 and Cyclin D1 are two ER-regulated components of a pathway leading to activation of CCNE and cell cycle progression from G1 to S past the R restriction point (Fig 6B). This latter has been deﬁned as an important cell cycle stage controlling stem cell fate allowing equilibrium between self-renewal and committed cell- fate decision (Orford and Scadden, 2008). In self-renewing cells, CCNE is constitutively active throughout the cell cycle, which allows the transition of stem cells from M phase directly to late G1. The resulting absence of the Cyclin D-dependent early G1 phase shortens the G1 phase and the entire cell cycle. Thus, tempering with the R point would modify the balance between self-renewal and differentiation (Orford and Scadden, 2008). In luminal tumours, the ‘tempering pathway’ would include ZNF703 and CCND1. It could be set into action and kept overfunctioning by the ampliﬁcation of 8p12 ZNF703 or 11q13 CCND1, or by 8p12/11q13 co-ampliﬁcation. Alterations in other components of the pathway, such as loss of function of RB1 (Herschkowitz et al, 2008) or overexpression of CCNE1 (Keyomarsi et al, 2002) can also activate this pathway but can occur in any subtype since these proteins are not regulated by ER. We therefore propose that ZNF703 ampliﬁcation represses luminal differentiation and increases self-renewal, thus sustaining a luminal B phenotype. This does not prevent other genes of the ampliﬁed regions to exert an effect. The number of 8p11-12 subamplicons and validated candidates (e.g. DDHD2, ERLIN2, FGFR1, PPAPDC1P, RCP/RAB11FIP1, TC-1/ C8orf4 and WHS1L1) indicates that the 8p11-12 region contains several closely-linked oncogenic targets that function in cooperation after ampliﬁcation. This cooperative effect could be to sustain both self-renewal and proliferation of the luminal progenitor. Thus, our results explain all the three enigmatic features of the 8p11-12 ampliﬁcation that are the association with luminal B tumours, the plethora of candidate oncogenes and the frequent co-ampliﬁcation with CCND1 at 11q13. From a more general point of view, our study describes another type of cancer gene. If ampliﬁed genes may be considered as potential oncogenes, not all of these oncogenes should be viewed as solely proliferation boosters but some may be considered as transcriptional re-programmers controlling www.embomolmed.org EMBO Mol Med 3, 153–166 2011 EMBO Molecular Medicine 161