Research Article ZNF703 in luminal B breast cancer Figure 4. ZNF703 overexpression regulates CSC biology in MCF7 cell line. A. Gene expression supervised analysis identified ZNF703 co-regulated genes. Gene expression profiles of two independent MCF7 GFP and two MCF7 GFP-ZNF703 stably expressing clones were compared. The 100 top genes regulated by ZNF703 overexpression are represented on hierarch- ical clustering. This gene list is highly enriched in stem cell-related genes. B. To determine whether genes induced by ZNF703 overexpression were related to stem cell biology, we utilized IPA using Ingenuity1 software. Bar plot represents enrichment for each of the network components identified, where the strength of the association is represented by the log10 ( p-value). IPA identified five significant networks associated with ZNF703 overexpression, including three directly related to stem cell biology and development process (in bold). C. Using FACS analysis, we measured CD49f expression in MCF7 GFP com- pared to MCF7 GFP-ZNF703 cells. We found seven times more CD49f- positive cells in MCF7 GFP-ZNF703 cells compared to MCF7 GFP cells. D. MCF7 GFP-ZNF703 and GFP cells were cultivated in suspension and tumoursphere formation was evaluated in the presence or absence of 17-b estradiol (E2). ZNF703 overexpression increases primary (t-test, p ¼ 2.8 10 6) [left part] and secondary tumourosphere (t-test, p ¼ 1.4 10 6) [right part] formation under E2 treatment. modulate the activity of master transcriptional regulators, such as ER or E2F1, and thus regulate key cell processes (Battaglia et al, 2010; Perissi et al, 2010). Both ER and E2F1 have been implicated in the control of differentiation and self-renewal programmes in stem cells (Dontu et al, 2004; Galderisi et al, 2006; Orford and Scadden, 2008). Therefore, we speculated that ZNF703 overexpression may regulate breast CSC biology through the regulation of ER and E2F1 transcriptional activities. To test this hypothesis, we ﬁrst studied ER protein expression by immunostaining in MCF7 GFP-ZNF703 cells. We observed a decrease of ER expression in cells with ZNF703 dot-like structures: 80% of cells with ZNF703 dot-like structures displayed low ER expression, whereas 60% of cells with diffuse cytoplasmic ZNF703 expression displayed high ER expression (Fig 5A). Second, we measured ER transcriptional activity using an ER reporter system that consisted in a vector (p17M-ERE- Luc) coding for the luciferase protein under the control of 17 ERE. Once again, we observed a decrease of ER transcriptional activity in MCF7 GFP-ZNF703 compared to MCF7 GFP cells, independently of E2 induction (Fig 5B). Third, we found that MCF7 GFP-ZNF703 cells presented a decrease in expression for the two main ER-associated proteins, FOXA1 and GATA3 (Fig 5C), and we conﬁrmed a lower ER expression compara- tively to MCF7 GFP cells. These results indicate that ZNF703 modulates ER transcriptional activity and may control breast CSC differentiation process through this regulatory mechanism. We next examined the effect of ZNF703 overexpression on the other classical nuclear target of co-repressors, E2F1. We used two different E2F1 reporter systems (p3XE2F1-Luc and pCCNE- Luc). The vectors code, respectively, for the luciferase protein under the control of three E2F1 responsive elements or under the control of Cyclin E (CCNE1) promoter, a direct E2F1 target. We found similar results for both reporter assays with an increase of E2F1 transcriptional activity induced by ZNF703 overexpression (Fig 5D). We further tested the functional activation of E2F1 by western blot analysis of RB1 and P27Kip1 protein status. Both proteins are implicated in E2F1 regulation. RB1 binds E2F1 and represses cell cycle progression; phosphor- ylations inactivate RB1 that releases E2F1 leading to proteolysis of P27Kip1 and to transcription of several target genes implicated in cell cycle progression from G1 to S (Orford and Scadden, 2008). We found an increase of RB1 phosphorylation and a decrease of P27Kip1 protein expression in MCF7 GFP-ZNF703 cells, attesting of E2F1 activation induced by ZNF703 over- expression (Fig 5E). It is interesting to note that the PHB1/ NCOR1 complex is known to repress E2F1 transcriptional activity (Wang et al, 2002), whereas the ZNF703/PHB2/NCOR2 complex seems to positively regulate E2F1 activity. Altogether, these results suggest that ZNF703 may be implicated in the regulation of ER and E2F1 transcriptional regulators and thus may modulate gene expression programmes implicated in breast CSC biology through these two major pathways. Luminal B tumours present an activation of E2F1 transcriptional programme and a decrease in the activity of ER-related transcription factors We sought to extend these results to clinical samples of luminal B primary tumours. We used the GSEA algorithm to screen the activated transcription factors from the Broad Institute (MSigDB C3: motif gene sets TFT). We evaluated the different gene sets that share a transcription factor binding site deﬁned in the TRANSFAC database enriched in luminal B tumours compared 158 2011 EMBO Molecular Medicine EMBO Mol Med 3, 153–166 www.embomolmed.org