Research Article ZNF703 in luminal B breast cancer Figure 6. Role of ZNF703 in the regulation of cell cycle progress. A. To determine the role of ZNF703 in cell cycle regulation, we analysed the cell cycle status of synchronized cells from MCF7 GFP and MCF7 GFP-ZNF703 cell lines in the presence or absence of E2. This analysis was done every 2 h over 24 h after FBS stimulation. Results are summarized in Supplementary Fig 8. Pie charts represent cell cycle progress of synchronized cells during 24 h after FBS stimulation. MCF7 GFP cells need E2 stimulation to enter the cell cycle, whereas ZNF703 overexpression is sufficient to induce cell cycle entry independently of E2 stimulation. Interestingly, in the presence of E2, G0/G1 phase is significantly shorter (4 h) in MC7 GFP-ZNF703 cells compared to cells cultured in the absence of E2 (6 h). B. Hypothetical representation of the putative ZNF703-associated network in luminal B tumours presenting an 8p12 amplification. ZNF703 is part of a nuclear complex comprising DCAF7, PHB2 and NCOR2. This complex may act as a nuclear co-repressor modulating CSC self-renewal and differentiation. ZNF703 expression is under the control of ER/GATA3, and potentially CCND1, and activates E2F1 transcriptional activity through RB1 and P27Kip1 inactivation. Interaction with DCAF7 should induce P27Kip1 degradation. E2F1 activation induces CCNE1 transcription that controls cell cycle entry in late G1 phase. When ZNF703 gene is amplified and overexpressed, it induces a shortened G1 phase supporting CSC self-renewal; CCNE1 is constitutively active and ER-dependent differentiation and proliferation is repressed (red lines). (35.5%) were related to genes containing EREs or to ER- associated transcription factors whereas none of these gene sets was related to E2F1 transcription factor (Fig 5G). These results are consistent with our results suggesting that ZNF703 is a speciﬁc luminal B oncogene regulating E2F1 and ER transcrip- tional activity. It seems that in luminal B cancer cells the balance between E2F1 and ER activation is tilted towards an E2F1 transcriptional programme, in which ZNF703 may act as a key modulator. ZNF703 expression regulates cell cycle progression Taken together, our observations suggest a strong implication of ZNF703 in the transcriptional control of cell cycle progression. The E2F1/RB1/CCNE1 protein network controls cell cycle entry and cell cycle progression from G1 to S. This cell cycle stage has been proposed to control stem cell fate by allowing an 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, ZNF703 overexpression may modify the balance between self- renewal and differentiation by shortening the G1 phase. To evaluate the role of ZNF703 overexpression in the control of cell cycle progression, we analysed the cell cycle status of synchronized MCF7 GFP and MCF7 GFP-ZNF703 cells in the presence or absence of E2. This analysis was done every 2 h over 24 h. Results are summarized in Supplementary Fig 8. MCF7 GFP cells needed E2 stimulation to enter the cell cycle, whereas ZNF703 overexpression was sufﬁcient to induce cell cycle entry independently of E2 (Fig 6A). Interestingly, in the presence of E2, the G0/G1 phase was shorter (4 h) in MC7 GFP-ZNF703 cells compared to cells cultured in the absence of E2 (6 h). These results suggest that ZNF703 may regulate CSC biology by activating the E2F1/RB/CCNE1 network and inducing a short G1 phase that may favour self-renewal over differentiation. Consistent with our results of tumoursphere assay, combined E2 stimulation and ZNF703 overexpression seem to increase both self-renewal and proliferation in MCF7 cells, whereas cell proliferation seems only to need ZNF703 overexpression. DISCUSSION We describe here the identiﬁcation of a potential breast cancer oncogene, ZNF703, implicated in the deﬁnition of the luminal B subtype. Recent molecular portraits of breast cancer have consistently revealed biologically distinct subtypes associated with different prognosis (Sorlie et al, 2001). Luminal B tumours are associated with poor breast cancer recurrence-free and disease-speciﬁc survival in all adjuvant systemic treatment categories including hormone therapy alone (Cheang et al, 2009). The identiﬁcation of key mechanisms that regulate luminal B biology will thus lead to a better diagnostic and treatment of luminal tumours. We have shown that luminal B tumours display speciﬁc ampliﬁcations, including ampliﬁcation of the 8p12 region. Interestingly, breast cancer cell lines with 8p12 ampliﬁcation show resistance to 4-hydroxytamoxifen (Turner et al, 2010). We 160 2011 EMBO Molecular Medicine EMBO Mol Med 3, 153–166 www.embomolmed.org