ZNF703 gene amplification at 8p12 specifies luminal B breast cancer - PubMed (original) (raw)

doi: 10.1002/emmm.201100121. Epub 2011 Feb 15.

Nathalie Nicolas, Anthony Ferrari, Pascal Finetti, Ismahane Bekhouche, Estelle Rousselet, Aurélie Lonigro, José Adélaïde, Emilie Baudelet, Séverine Esteyriès, Julien Wicinski, Stéphane Audebert, Emmanuelle Charafe-Jauffret, Jocelyne Jacquemier, Marc Lopez, Jean-Paul Borg, Christos Sotiriou, Cornel Popovici, François Bertucci, Daniel Birnbaum, Max Chaffanet, Christophe Ginestier

Affiliations

ZNF703 gene amplification at 8p12 specifies luminal B breast cancer

Fabrice Sircoulomb et al. EMBO Mol Med. 2011 Mar.

Erratum in

Abstract

Luminal B breast cancers represent a fraction of oestrogen receptor (ER)-positive tumours associated with poor recurrence-free and disease-specific survival in all adjuvant systemic treatment categories including hormone therapy alone. Identification of specific signalling pathways driving luminal B biology is paramount to improve treatment. We have studied 100 luminal breast tumours by combined analysis of genome copy number aberrations and gene expression. We show that amplification of the ZNF703 gene, located in chromosomal region 8p12, preferentially occurs in luminal B tumours. We explored the functional role of ZNF703 in luminal B tumours by overexpressing ZNF703 in the MCF7 luminal cell line. Using mass spectrometry, we identified ZNF703 as a co-factor of a nuclear complex comprising DCAF7, PHB2 and NCOR2. ZNF703 expression results in the activation of stem cell-related gene expression leading to an increase in cancer stem cells. Moreover, we show that ZNF703 is implicated in the regulation of ER and E2F1 transcription factor. These findings point out the prominent role of ZNF703 in transcription modulation, stem cell regulation and luminal B oncogenesis.

Copyright © 2011 EMBO Molecular Medicine.

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Figures

Figure 1

Figure 1. ZNF703 is a target gene of the 8p12 amplification in luminal B breast tumours

  1. Supervised analyses of aCGH data comparing amplifications in 41 luminal B tumours and 59 luminal A tumours. Genomic segments are ordered on the _X_-axis from chromosome 1 to 22 and on the _Y_-axis according to their association with luminal A or B molecular subtype (−log(_p_-value)). The grey zone contains genomic segments that are not significantly associated with any of the two molecular subtypes (p > 0.01). Red dots represent genomic segments significantly amplified in luminal B breast tumours (p < 0.01). These segments are contained in 8p12, 8q22, 11q13, 17q24, and 20q13 chromosomal regions. We did not identify genomic segments significantly amplified in luminal A tumours.
  2. Combined genomic and transcriptional analysis for each gene from the 8p12 amplicon identified in luminal B breast tumours. Genes are ordered from telomere (left) to centromere (right) and the frequency of luminal B tumours presenting an increase in gene copy number and mRNA level is represented. Blue bars label genes that present a statistically significant association between gene amplification and overexpression (Fisher's exact test, _p_-value ≤ 0.05).
  3. Box-plot representing ZNF703 expression distribution according to molecular subtypes of 1172 breast tumours. ZNF703 expression varies across the five molecular subtypes (ANOVA, p = 1.2 × 10−53) and is higher in luminal B tumours compared to luminal A tumours (_t_-test, p = 4 × 10−6).
  4. Kaplan–Meier survival curves according to ZNF703 gene expression status. ZNF703 gene expression is associated with DFS (p = 0.0136).

Figure 2

Figure 2. Identification and characterization of ZNF703 nuclear domain

  1. Immunoblotting and immunofluorescence analyses of MCF7 GFP-ZNF703 and MCF7 GFP breast cancer cell lines. Immunoblotting with anti-ZNF703 and anti-GFP antibodies identifies GFP-ZNF703 fusion protein with a molecular weight of 83 kDa. α-tubulin protein expression is shown as a control. Immunofluorescence reveals nuclear dot-like structures in MCF7 GFP-ZNF703 cells, whereas GFP is detected in all subcellular structures in MCF7 GFP cells.
  2. GFP-ZNF703 interacts with DNA in the nucleus. MCF7 GFP-ZNF703 cells non-treated (control) or treated with RNAse A or DNAse I were processed for immunofluorescence with anti-GFP antibody. The GFP-ZNF703 signal in the nucleus is perturbed only when cells are treated with DNAse.
  3. MCF7 cells expressing GFP and GFP-ZNF703 proteins were subjected to immunoprecipitation with anti-GFP antibody (IP GFP) or beads only (No Ab), separated by SDS-PAGE, and stained with colloidal Coomassie blue. Three co-precipitated proteins were identified by mass spectrometry: HSP60 (accession number, NP 955472); DCAF7 (NP 005819) and PHB2 (NP 001138303), respectively. The lane on the left contains molecular weight standards (MW). The interaction between ZNF703 and HSP60 or DCAF7 was confirmed by ZNF703-immunoprecipitation, followed by Western blot analysis using anti-HSP60 or anti-DCAF7 antibody. Input represents whole-cell extract from HCC1500 and MDA-MB-134.
  4. MCF7 cells overexpressing GFP-ZNF703 were fixed and immunostained with anti-DCAF7, anti-PHB2 or anti-SMRT/NCOR2 antibody. GFP-ZNF703 (green) co-localizes in the nucleus with DCAF7, PHB2 and SMRT/NCOR2 (red). Nuclei are counterstained with Hoechst (blue).

Figure 3

Figure 3. ZNF703 modulation regulates cancer cell proliferation

  1. MCF7 GFP and MCF7 GFP-ZNF703 cells were cultured in the presence or absence of 17-β estradiol (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.
  2. 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.

Figure 4

Figure 4. ZNF703 overexpression regulates CSC biology in MCF7 cell line

  1. 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 hierarchical clustering. This gene list is highly enriched in stem cell-related genes.
  2. To determine whether genes induced by ZNF703 overexpression were related to stem cell biology, we utilized IPA using Ingenuity® 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).
  3. Using FACS analysis, we measured CD49f expression in MCF7 GFP compared to MCF7 GFP-ZNF703 cells. We found seven times more CD49f-positive cells in MCF7 GFP-ZNF703 cells compared to MCF7 GFP cells.
  4. MCF7 GFP-ZNF703 and GFP cells were cultivated in suspension and tumoursphere formation was evaluated in the presence or absence of 17-β estradiol (E2). ZNF703 overexpression increases primary (_t_-test, p = 2.8 × 10−6) [left part] and secondary tumourosphere (_t_-test, p = 1.4 × 10−6) [right part] formation under E2 treatment.

Figure 5

Figure 5. ZNF703 overexpression regulates ER and E2F1 transcriptional activity

  1. MCF7 GFP-ZNF703 cells were fixed and immunostained with anti-ER. ZNF703 (green) and ER (red) do not co-localize and are expressed in different cells (white arrowheads). Nuclei were counterstained with Hoechst (blue).
  2. Oestrogen receptor-dependent transcriptional activity was evaluated using a luciferase reporter system (p17M-ERE-Luc) in the presence and absence of 17-β estradiol (E2) in MCF7 GFP and GFP-ZNF703 breast cancer cell lines. ER-dependent transcriptional activity decreases in MCF7 GFP-ZNF703 cells as compared to GFP expressing cells, independently of E2 induction.
  3. Analysis of ER, FOXA1, and GATA3 protein expression by immunoblotting in MCF7 GFP-ZNF703 and MCF7 GFP breast cancer cell lines. MCF7 GFP-ZNF703 cells show a decrease in ER-associated proteins.
  4. E2F1-dependent transcriptional activity was evaluated using two different luciferase reporter systems. Both vectors code for the luciferase protein under the control of either three E2F1 responsive elements (pGL2-3XE2F1-Luc) or CCNE1 promoter (pGL3-CCNE1-Luc), which is a direct E2F1 target gene. E2F1-dependent transcriptional activity decreases with ZNF703 overexpression.
  5. Analysis of RB1 and P27Kip1 protein expression by immunoblotting in MCF7 GFP-ZNF703 and MCF7 GFP cells. ZNF703 overexpression induces a hyperphosphorylation of RB1 protein and a decrease in P27Kip1 protein expression. α-Tubulin protein expression was used as a control.
  6. To test E2F1 or ER transcriptional activity in luminal B primary tumours compared to luminal A, we performed a GSEA to screen the activated transcription factors from the Broad Institute (MSigDB C3: motif gene sets TFT). We represented the different gene sets that share a transcription factor binding site defined in the TRANSFAC database enriched in luminal B tumours, compared to luminal A tumours. Each gene sets is plotted according to its association to either luminal A or B tumours (−log(diff GSEA _p_-value)); E2F1-related gene sets are in red, ER-related gene sets are in blue and other gene sets are in grey (F). A total of 117 gene sets were differentially regulated between luminal B and luminal A primary tumours (FDR _q_-value < 0.25; Nominal _p_-value < 0.05). E2F1-related gene sets are significantly enriched in luminal B tumours, whereas luminal A tumours, gene expression programme is enriched in ER-related gene sets (G).

Figure 6

Figure 6. Role of ZNF703 in the regulation of cell cycle progress

  1. 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).
  2. 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).

Comment in

References

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