Research Article Fabrice Sircoulomb et al. Figure 3. ZNF703 modulation regulates cancer cell proliferation. A. MCF7 GFP and MCF7 GFP-ZNF703 cells were cultured in the presence or absence of 17-b estra- diol (E2) and cell number was measured during 7 days. GFP-ZNF703 overexpression increases cell proliferation independently of E2 stimulation compared to MCF7 GFP cells cultured in the absence of E2, whereas E2 treatment boosts cell proliferation in MCF7 GFP only. B. To confirm the role of ZNF703 in the proliferation process, we inhibited ZNF703 expression via a ZNF703-siRNA in the MDA-MB-134 cell line, which presents amplification and overexpression of the ZNF703 gene. An empty siRNA was used as control (si-control). We started to measure cell number 30 h after siRNA lipofection and over 7 days of culture. MDA-MB-134 cells proliferate only in the presence of E2 and ZNF703 knock-down reduces MDA-MB-134 proliferation. gene expression similarities across 432 published gene expres- sion datasets analysed with Affymetrix GeneChip1 from cell lines and primary tumours studies, we found that NCOR2 was the most co-expressed gene with ZNF703 (Supplementary Table 8). Altogether, our results suggest that nuclear ZNF703 is a co- factor of a nuclear co-repressor complex and may contribute to transcriptional regulation. ZNF703 expression regulates cell proliferation To evaluate the role of ZNF703 overexpression as a potential oncogene, we measure the kinetic of cell proliferation in MCF7 GFP compared to MCF7 GFP-ZNF703 as assessed by an MTT assay. This test was done in the presence or absence of E2. As expected, MCF7 GFP cell proliferation was highly stimulated by the presence of E2. ZNF703 overexpression induced a signiﬁcant cell proliferation independently of E2 stimulation (Fig 3A). Using a ZNF703-siRNA, we inhibited ZNF703 expression in the MDA-MB-134 cell line, which presents an ampliﬁcation and overexpression of ZNF703 (Supplementary Fig 7) and measured the kinetic of cell proliferation. Depletion of ZNF703 expression in MDA-MB-134 cells reduced cell proliferation (Fig 3B). These observations indicate that ZNF703 is implicated in the regulation of cell proliferation. ZNF703 overexpression increases cancer stem cell population To further characterize the effect of ZNF703 overexpression, we analysed the modiﬁcation of gene expression programmes in MCF7 GFP-ZNF703 cells compared to MCF7 GFP cells using DNA microarrays. A total of 189 genes were differentially expressed between the two types of cells, with 160 genes induced and 29 genes downregulated by ZNF703 overexpres- sion (Fig 4A and Supplementary Table 9). Among the 160 genes induced by ZNF703, several genes were related to WNT (such as LEF1, TCF12, WNT4) or NOTCH (such as ASCL1, HEY2, TLE4) signalling pathways. Activation of these pathways is known to regulate the self-renewal programme in breast CSC (Harrison et al, 2010; Korkaya and Wicha, 2009; Ponti et al, 2005). To conﬁrm that genes induced by ZNF703 overexpression were related to stem cell biology, we utilized an Ingenuity pathway analysis (IPA) using Ingenuity1 software (Fig 4B). IPA identiﬁed ﬁve signiﬁcant networks associated with ZNF703 overexpression, including three directly related to stem cell biology and development process. Moreover, to better char- acterize the association between genes induced by ZNF703 and stem cell-related genes, we measured the enrichment of 26 published stem cell-related gene sets in genes differentially expressed in MCF7 GFP-ZNF703. Ten out of twenty-six gene sets were enriched in the ZNF703-related genes (Supplementary Table 10). These results suggest that ZNF703 overexpression may be implicated in the regulation of breast CSCs. To test this hypothesis, we ﬁrst measured the proportion of CD49f-positive cells in MCF7 GFP and MCF7 GFP-ZNF703 cell lines. The CD49f- positive population has been described to contain the CSC population in the MCF7 cell line (Cariati et al, 2008). Overexpression of ZNF703 induces an expansion of the CD49f-positive population with seven times more CD49f- positive cells in the MCF7 GFP-ZNF703 cell line than in the MCF7 GFP cell line (Fig 4C). Second, we used an in vitro tumoursphere assay, a powerful surrogate method to evaluate cancer stem/progenitor cells (Ponti et al, 2005). We cultivated MCF7 GFP and MCF7 GFP-ZNF703 cells in adherent conditions in the presence or absence of E2, then in suspension culture at clonogenic density. When stimulated by E2, MCF7 GFP-ZNF703 cells showed an increase in primary tumoursphere formation. Similar results were observed for secondary tumoursphere formation (Fig 4D). These results suggest that ZNF703 over- expression increases the CSC population and may act as a regulator of the self-renewal programme in ER-positive cells. ZNF703 overexpression regulates ER and E2F1 transcriptional activity The above observations suggested that ZNF703 could regulate breast CSC self-renewal activity by interacting with the PHB2/ NCOR2 nuclear co-repressor complex. Nuclear co-repressors www.embomolmed.org EMBO Mol Med 3, 153–166 2011 EMBO Molecular Medicine 157