C/EBPα mediates the growth inhibitory effect of progestins on breast cancer cells
2019; Springer Nature; Volume: 38; Issue: 18 Linguagem: Inglês
10.15252/embj.2018101426
ISSN1460-2075
AutoresA. Silvina Nacht, Roberto Ferrari, Roser Zaurín, Valentina Scabia, José Carbonell‐Caballero, François Le Dily, Javier Quilez, Alexandra Leopoldi, Cathrin Brisken, Miguel Beato, Guillermo P. Vicent,
Tópico(s)RNA modifications and cancer
ResumoArticle2 August 2019free access Source DataTransparent process C/EBPα mediates the growth inhibitory effect of progestins on breast cancer cells A Silvina Nacht Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Roberto Ferrari Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Roser Zaurin Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Valentina Scabia orcid.org/0000-0002-2797-7087 Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author José Carbonell-Caballero Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Francois Le Dily Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Javier Quilez Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Alexandra Leopoldi Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Cathrin Brisken Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Miguel Beato Corresponding Author [email protected] orcid.org/0000-0002-2878-2222 Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Guillermo P Vicent Corresponding Author [email protected] orcid.org/0000-0002-0554-2226 Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author A Silvina Nacht Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Roberto Ferrari Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Roser Zaurin Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Valentina Scabia orcid.org/0000-0002-2797-7087 Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author José Carbonell-Caballero Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Francois Le Dily Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Javier Quilez Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Alexandra Leopoldi Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Cathrin Brisken Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Miguel Beato Corresponding Author [email protected] orcid.org/0000-0002-2878-2222 Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Guillermo P Vicent Corresponding Author [email protected] orcid.org/0000-0002-0554-2226 Center for Genomic Regulation (CRG), Barcelona, Spain Barcelona Institute for Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Author Information A Silvina Nacht1,2, Roberto Ferrari1,2, Roser Zaurin1,2, Valentina Scabia3, José Carbonell-Caballero1,2, Francois Le Dily1,2, Javier Quilez1,2,†, Alexandra Leopoldi1,2,†, Cathrin Brisken3, Miguel Beato *,1,2,4 and Guillermo P Vicent *,1,2,† 1Center for Genomic Regulation (CRG), Barcelona, Spain 2Barcelona Institute for Science and Technology (BIST), Barcelona, Spain 3Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 4Universitat Pompeu Fabra (UPF), Barcelona, Spain †Present address: Division of Life Sciences, Clarivate Analytics, Barcelona, Spain †Present address: Institute of Molecular Biotechnology (IMBA), Vienna, Austria †Present address: Molecular Biology Institute of Barcelona (IBMB-CSIC), Barcelona, Spain *Corresponding author. Tel: +34 933160119; E-mail: [email protected] *Corresponding author. Tel: +34 933160115; E-mail: [email protected] EMBO J (2019)38:e101426https://doi.org/10.15252/embj.2018101426 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Steroid hormones are key gene regulators in breast cancer cells. While estrogens stimulate cell proliferation, progestins activate a single cell cycle followed by proliferation arrest. Here, we use biochemical and genome-wide approaches to show that progestins achieve this effect via a functional crosstalk with C/EBPα. Using ChIP-seq, we identify around 1,000 sites where C/EBPα binding precedes and helps binding of progesterone receptor (PR) in response to hormone. These regions exhibit epigenetic marks of active enhancers, and C/EBPα maintains an open chromatin conformation that facilitates loading of ligand-activated PR. Prior to hormone exposure, C/EBPα favors promoter–enhancer contacts that assure hormonal regulation of key genes involved in cell proliferation by facilitating binding of RAD21, YY1, and the Mediator complex. Knockdown of C/EBPα disrupts enhancer–promoter contacts and decreases the presence of these architectural proteins, highlighting its key role in 3D chromatin looping. Thus, C/EBPα fulfills a previously unknown function as a potential growth modulator in hormone-dependent breast cancer. Synopsis Steroid hormones, such as progestins, control proliferation of breast cancer cells via their intracellular receptors. Here, the transcription factor C/EBPα is shown to facilitate open chromatin conformation and loading of ligand-activated progesterone receptor (PR) at key cell cycle genes, revealing functional crosstalk between PR and C/EBPα in repression of hormone-dependent cancer growth. Progestins increase C/EBPα expression in human breast cancer cells. C/EBPα enhances progesterone-induced growth inhibition in vitro and delays tumorigenesis in vivo. C/EBPα occupies around 1,000 active enhancer regions and maintains chromatin accessibility prior to PR binding. C/EBPα exerts this function by establishing chromatin loops with architectural proteins (such as YY1), mediator and the cohesin complex. Introduction The steroid hormones estrogens and progesterone acting via their intracellular receptors (ER and PR, respectively) control the proliferation of breast cancer cells in a very different way. While estrogens are inducers of cell proliferation that activate cyclin D1 (Musgrove et al, 1994; Planas-Silva & Weinberg, 1997; Planas-Silva et al, 1999) and decrease the expression of CDK inhibitors (Prall et al, 1997), progestins promote a single cell cycle followed by proliferation arrest at G1/S, that correlates with a delayed activation of CDK inhibitor p21WAF1 (Owen et al, 1998). Studies using PR-positive breast cancer cell lines have demonstrated a biphasic cellular response to progestins, with an immediate proliferative burst followed by a sustained growth arrest (Groshong et al, 1997; Musgrove et al, 1998; Skildum et al, 2005). C/EBPα is a member of the C/EBP family of transcription factors, which plays a critical role in the regulation of mitotic growth arrest and differentiation in numerous cell types, including pre-adipocytes, myeloid cells, hepatocytes, keratinocytes, and pneumocytes (Cao et al, 1991; Freytag et al, 1994; Wang et al, 1995; Flodby et al, 1996; Radomska et al, 1998). Despite the fact that C/EBPα is expressed in many tissues, its function has been best characterized only in adipocytes and in the hematopoietic system. Regulation of C/EBPα expression is fundamental for maintaining homeostasis of both embryonic and adult tissues (Schuster & Porse, 2006). Deletion of C/EBPα in mice results in pleiotropic disorders and the mice die shortly after birth due to the failure of the liver to store glycogen (Wang et al, 1995). Animals lacking C/EBPβ are viable but sterile and more susceptible to infections due to defects in the immune system (Screpanti et al, 1995). These mice exhibit also multiple defects in mammary gland development, including cystic, enlarged mammary ducts with decreased secondary branching (Seagroves et al, 1998). In addition, interactions between C/EBPα and CBP/p300 histone acetyltransferases or SWI/SNF chromatin remodeling complexes have been shown to regulate C/EBPα target genes involved in tissue specification (Erickson et al, 2001; Pedersen et al, 2001). Though it is known that C/EBPs are involved in the regulation of cell proliferation and cell differentiation of the mammary gland (Johnson, 2005), the role of C/EBPs in breast cancer cells has not been characterized. Although functional C/EBPα mutations have not been found in solid tumors, C/EBPα levels are found down-regulated in various types of cancer, indicating that inactivation of C/EBPα might be a requirement for tumor development (Lourenco & Coffer, 2017). In contrast, some studies performed in hepatocellular carcinomas and breast cancer cells have shown that increased expression of C/EBPα correlated with increased proliferation and disease progression (Tomizawa et al, 2007; Lu et al, 2010; Ming et al, 2015). However, the mechanism by which C/EBPα fulfills this function in hormone-dependent processes and in particular in breast cancer cells is unknown. Regulation of enhancer–promoter interactions is a fundamental mechanism underlying differential transcriptional control. Cell-specific 3D chromatin folding brings specific enhancers in contact with their target promoters in cis to regulate gene expression in a cell type-specific manner. Genome-wide studies have shown that the architectural proteins CTCF and its frequent associated partner cohesin are important for partition of the genome into largely conserved topologically associating domains or TADs on the megabase (Dixon et al, 2012; Nora et al, 2012; Phillips-Cremins et al, 2013). These architectural proteins along with the large mediator complex contribute directly to enhancer–promoter communication by mediating loop formation (Malik & Roeder, 2010; Ong & Corces, 2014). In embryonic stem (ES) cells and in mouse embryonic fibroblasts, RNAi studies showed that cohesin and the mediator complex are necessary for enhancer–promoter interactions of pluripotency genes (Kagey et al, 2010). Although the role of transcription factors in mediating association between specific regulatory elements in the genome is well characterized, the nature of the protein complexes required for establishing and maintaining these interactions has remained elusive and identifying the functional interactions in the context of the large number of noisy contacts remains a real challenge. Changes in the levels of architectural proteins have been associated with disease. For instance, RAD21 expression was shown to be significantly lower in invasive breast cancers compared with their in situ counterparts (Xu et al, 2011). Moreover, transcription factors can interact with architectural proteins and contribute to the cell-specific 3D genome topology (Faure et al, 2012; Yan et al, 2013). In this study, we describe a functional crosstalk between PR and C/EBPα, which acts as inhibitor of breast cancer cells proliferation. Progestins induced the expression of C/EBPα, which participates in hormone regulation in different ways. In around 1,000 DNA enhancer regions C/EBPα assists PR binding by maintaining the chromatin in an open conformation. In these sites, C/EBPα is also required to establish and to maintain promoter–enhancer contacts that assure the progestin regulation of key genes involved in cell proliferation, such as DUSP1. C/EBPα fulfills this function via interactions with RAD21, YY1, CTCF, and the Mediator complex. Moreover, we identify topoisomerase IIα (Top2α) as a previously unknown factor in C/EBPα function, required for both mechanisms of cooperation with PR. Our results demonstrate that PR and C/EBPα cooperate in a gene expression program that enables controlled cell growth in the presence of progestins. Results C/EBPα is a hormone-target gene that modulates hormonal gene regulation To address the possible role of C/EBPα in breast cancer cells, we measure its expression in the ER+ and PR+ T47D cell line exposed to hormones. In cells cultured for 4 h in medium containing 10% serum deprived of hormone by treatment with dextran-coated charcoal (CS-FBS), we found an 8-fold increase in C/EBPα expression after 6-h exposure to the progestin R5020 (Fig 1A, top left panel). Time curve experiments showed that the levels of C/EBPα mRNA increased already after 3 h, reached a peak at 6 h, and decreased gradually thereafter (Fig 1A, top right panel). ChIP-seq of PR after exposure to hormone for 60 min showed two strong peaks around the C/EBPα gene region located at −8.2 kb and −5.4 kb from the TSS, as well as two weak peaks inside the gene (Fig 1A, lower panel), indicating that the C/EBPα gene could be a direct target of PR. The C/EBPα protein levels increased after 6 h of hormone exposure and reached a peak after 19 h (Fig EV1A, left panel). The levels of the related protein C/EBPβ also increase in the presence of hormone, but this increment is moderate and delayed compared to that of C/EBPα (Fig EV1A, right panel and Fig EV1B). Figure 1. In breast cancer cells, C/EBPα is a hormone-target gene and modulates hormonal gene regulation and cell proliferation Top left panel: Hormone-induced fold change in the expression of mRNA for C/EBPα in T47D cells exposed to vehicle (T0) or to progestin (10 nM R5020) for 6 h (+R5020). The fold change relative to T0 is expressed as mean ± SD from three experiments performed in duplicate. Top right panel: Time kinetics of C/EBPα mRNA expression after hormone induction in T47D cells. The fold change relative to T0 is expressed as mean ± SD from three experiments performed in duplicate. Lower panel: Snapshot of the genome browser showing the profile of PR ChIP-seq at T0 and after 60 min of hormone exposure (+R5020) around the C/EBPα gene shown in the upper right corner. The direction of transcription indicated. T47D cells transfected with control or C/EBPα siRNAs were treated with 10 nM R5020 for the indicated time periods; cDNA was generated and used as template for real-time PCR with DUSP1 (upper panel)-specific and PGR (lower panel)-specific primers. The values are given as mean ± SD from three experiments performed in duplicate. The inset shows the level of C/EBPα depletion by Western blot, using LSD1 as loading control. P-values were obtained by Student's t-test and are relative to time zero (*P ≤ 0.05 and **P ≤ 0.01) Effect of C/EBPα knockdown on global hormonal gene regulation. T47D cells transfected with control or C/EBPα siRNAs were incubated with 10 nM R5020 for 6 h, and RNA-seq experiments were performed as described in the Materials and Methods. The number of significant (q < 0.01) differentially expressed genes upon exposure to hormone identified in each of the siControl and siC/EBPα systems is shown. Differential expression analysis between treated and control conditions was done with R (https://www.R-project.org/) package DESeq2 (Love et al, 2014), selecting as significant those genes with an adj. P < 0.01 and a fold change > 2 between conditions. Effect of C/EBPα depletion in T47D cells on hormone-induced entry in S phase. Cells were treated with Ethanol or R5020 for different time periods and subjected to flow cytometric analysis. Data are represented as mean ± SD from three experiments performed in duplicate. The P-values were obtained using ANOVA followed by Tukey test. Left panel: Levels of C/EBPα expression in cells transfected with a Dox-inducible TetO-C/EBPα vector (T47DindC/EBPα) incubated with different concentrations of doxycycline, as detected by Western blot. Middle panel: T47DindC/EBPα were induced with doxycycline (1 μg/ml Dox) for 16 h and the expression of C/EBPα was detected by immune fluorescence microscopy. Right panel: Quantitation of the percentage of T47DindC/EBPα cells entering in S phase upon exposure to Dox (1 μg/ml) for 1 or 3 h followed by hormone induction for 18 h. Data are represented as mean ± SD from three experiments performed in duplicate. The P-values were obtained using ANOVA followed by Tukey test. Source data are available online for this figure. Source Data for Figure 1 [embj2018101426-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Hormone induces the expression of C/EBPα in breast cancer cells Time kinetics of C/EBPα expression after hormone induction in T47D cells. T47D cells treated with ethanol or with 10 nM R5020 as indicated were lysed and analyzed by immunoblot with α-C/EBPα (left panel) or with α-C/EBPβ (right panel). An antibody to PARP was used as loading control. Quantitation of the bands shown in panel (A) using densitometric analysis and ImageJ software. Values were normalized to time zero (T0). Each bar represents the mean ± SD from two experiments. T47D cells transfected with control or C/EBPα siRNAs and treated or not with 10 nM R5020 as indicated were lysed and analyzed by immunoblot with specific antibodies for PR, ER, and C/EBPα. GO analysis of C/EBPα-dependent genes in T47D cells. We used g:Profiler (Reimand et al, 2016) to identify Gene Ontology Biological Process (GO:BP) in the differentially expressed genes. Source data are available online for this figure. Download figure Download PowerPoint To test whether CEBPα influences the regulation of hormone-dependent genes, T47D cells were transfected with control siRNAs and with siRNAs against C/EBPα. We found that progestin induction of the Dual Specificity Phosphatase 1 (DUSP1) gene and progestin repression of the PR gene (PGR) were significantly reduced by C/EBPα knockdown (Figs 1B and EV1C). To explore the generality of these observations, we performed RNA-seq experiments in cells transfected with siRNA against C/EBPα or with control siRNA and exposed to hormone for 6 h. In siControl cells, we found 1,187 up-regulated and 819 genes down-regulated by hormone (q < 0.01) (Fig 1C). Of the hormone-induced genes, 20% were C/EBPα dependent, while for the hormone-repressed genes the proportion was 33.5%, suggesting that C/EBPα plays a more general role during hormonal gene regulation, particularly in down-regulated genes (Fig 1C). Analysis of the Gene Ontology (GO) categories revealed that the C/EBPα-dependent up-regulated genes were primarily implicated in cellular metabolic process, RNA splicing and processing, while the down-regulated genes were involved in the regulation of cell cycle, mitotic cell cycle, cellular response to DNA damage and DNA repair (Fig EV1D). Among them, we found 49 genes associated with cell cycle and cell proliferation such as the cell cycle modulators CNTD1, CKS2, and the cyclin-dependent kinase 4 inhibitor CDKN2D; the G2 mitotic-specific cyclin B2 (CCNB2), the transcription factor involved in cell cycle E2F1, and genes associated with DNA repair such as XRCC2, PCNA, BRCA1, and its associated RING domain protein BARD1 and BRIP1 (Appendix Fig S1A). Confirming their proliferative function, the expression of these 49 genes increased proportionally with the breast cancer tumor grade (Appendix Fig S1B, left panel) and its expression is associated with a decreased overall survival of the patients (Appendix Fig S1B, right panel). We also found several up-regulated genes with anti-proliferative functions such as DUSP1, DUSP2, STAT4, IL12, GAS7, and RASAL2 that could complement the cell growth arrest program triggered by progestins. Expression of these genes tends to be anti-correlated to tumor grade, though not significantly (Appendix Fig S1C, left panel) and was associated with longer survival (Appendix Fig S1C, right panel). C/EBPα mediates the inhibitory effect of progestins on cell proliferation Previous studies using PR-positive breast cancer cell lines demonstrated a biphasic cellular response to progestins, with an immediate proliferative burst followed by a sustained growth arrest (Groshong et al, 1997; Musgrove et al, 1998; Skildum et al, 2005). We confirmed these findings in T47D cells and found that progestin-induced cell proliferation was significantly increased by C/EBPα knockdown (Fig 1D). Cells depleted of C/EBPα showed a delayed growth arrest phase, suggesting an anti-proliferative role for C/EBPα (Gery et al, 2005). To expand this finding, we generated a T47D cell line that expresses recombinant CEBPα under the control of doxycycline (Dox) (T47DindC/EBPα). The increase in C/EBPα (Fig 1E, left and middle panels) correlated with a decrease in cell number (Fig EV2A, upper panel) as well as in the percentage of cells in S phase detected after hormone (Fig 1E, right panel). The effect of overexpressed C/EBPα was also observed in MCF7 and BT474 cells (Fig EV2A, middle and lower panels), although these breast cancer cell lines express different levels of estrogen receptor (ER) and PR (Fig EV2A, inset). Moreover, in MCF7 cells, we confirmed that C/EBPα knockdown increased cell growth as observed in T47D cells (Fig EV2B). Click here to expand this figure. Figure EV2. Overexpression of C/EBPα decreases cell proliferation in BT474, MCF7, and T47D breast cancer cells T47DindC/EBPα (upper panel), BT474indC/EBPα (middle panel), and MCF7indC/EBPα cells (lower panel) and expressing recombinant C/EBPα under the control of doxycycline (DOX) were used in proliferation assays. Data are represented as mean and SD from three experiments performed in duplicate. P-values were obtained by Student's t-test. Inset: The levels of expression of PR and ER for each of the three cell lines are shown. MCF-7 cells expressing shControl or shC/EBPα were subjected to proliferation assays. Data are represented as mean ± SD from three experiments performed in duplicate. The P-values obtained using Student's t-test are indicated. T47D and MCF-7 cells were treated with 10 nM 17β-estradiol (+E2) as indicated; cDNA was generated and used as template for real-time PCR with C/EBPα and GAPDH specific primers. Each bar represents the mean ± SD from two experiments. Different italic letters (a,b) are significantly different from each other (P ≤ 0.05) using Student's t-test. Overlap between down- and up-regulated transcripts. Transcripts are scored as significant (q < 0.01) differentially expressed transcripts (DETs) upon treatment with DOX for either 8 or 16 h (see Materials and Methods). DETs are further partitioned into those showing a decrease (down-regulated) or increase (up-regulated) in expression after the treatment. Gene ontology enrichment analysis. Gene Ontology:Biological Process (GO:BP) and KEGG significantly enriched (P < 0.01) in DETs inferred in each of the two DOX treatments. Overlap between R5020- and DOX-DETs. Observed-to-expected ratios for the different transcript categories (see Materials and Methods) are shown. The dashed line denotes a ratio of 1 and, as expected, it matches the ratio seen for transcripts scored as non-regulated in R5020- and DOX-DETs sets of data. Source data are available online for this figure. Download figure Download PowerPoint In contrast to progestins, estrogens induce continuous proliferation of breast cancer cells. We found that estrogens did not increase but rather decreased the levels of CEBPα by 40–50% in T47D and MCF-7 cells (Fig EV2C). This could in part explain the strong proliferative action of estrogens in breast cancer cells. To identify the genes responsible for the anti-proliferative role of CEBPα, we performed RNA-seq in T47D cells expressing Dox-inducible CEBPα untreated or treated for 8 and 16 h with Dox. We found 407 and 262 genes up- and down-regulated, respectively, at these time points (Fig EV2D). Gene ontology analysis revealed categories associated with the regulation of cell proliferation, metabolic process, signaling, developmental processes, and cell differentiation (Fig EV2E). We compared the RNA-seq data from T47D cells exposed to hormone with T47DindC/EBPα cells treated with Dox for 8 h. We found that 13% of the down- and 17% of the up-regulated genes identified after Dox induction—around 8- and 6-fold more than expected by chance, respectively—showed the same trend in cells exposed to R5020 (Fig EV2F). C/EBPα acts as a cell growth modulator in vivo During the development of the mammary gland, progestins stimulate the growth of stem and progenitor cells (Joshi et al, 2010). In hormone-responsive breast cancers, progestins increase the stem cell-like population by converting ER+/PR+ cells to receptor negative cells that acquire expression of the tumor-initiating markers CD44 and cytokeratin 5 (CK5; Cittelly et al, 2013). Therefore, we explored whether C/EBPα is involved in the hormone-dependent dedifferentiation of luminal breast cancer cells. To address this point, we used the cell line expressing Dox-inducible C/EBPα and measured the proportion of CD44high/CD24low cells by FACS sorting. Upon overexpressing CEBPα, the proportion of cells expressing CD44high/CD24low was reduced by 5-fold (Fig 2A, right panel). Exposure of T47D cells to R5020 for 24 h increased by approximately 3-fold the cell population expressing stem cell markers. Upon overexpression of CEBPα, the dedifferentiated cells increased with hormone, but the proportion of these cells remained very low (Fig 2A, left panel). Thus, high levels of C/EBPα compromise the ability of hormones to induce dedifferentiation, suggesting that C/EBPα may function as a growth modulator in breast cancer cells. Figure 2. C/EBPα inhibits hormone-induced expression of stem cell markers and acts as a cell growth modulator in vivo T47DindC/EBPα cells induced or not with Dox were untreated or exposed to R5020 for 24 h, and the expression of CD44 and CD24 was measured using specific antibodies (CD44-APC and CD24-P, respectively) and fluorescence-activated cell sorting analysis. Data are represented as mean and SD from three experiments performed in duplicate. The P-values were obtained by ANOVA followed by Tukey test. Left panel: Scheme of the Mouse Intra-Ductal (MIND) xenograft approach: tumor cells expressing luciferase are injected intraductally via the teat (Sflomos et al, 2016). Seven days after injection, CEBPα expression is induced with 2 mg/ml doxycycline in the drinking water maintained throughout the experiment. Tumor growth is assessed by bioluminescence (IVIS). Right panel: Tumor growth of T47DindC/EBPα-MIND marked with luciferase (Sflomos et al, 2016) treated or not with Dox was assessed by bioluminescence. Results are shown as means ± SEM. Five mice were used for each condition. In total, 17 and 19 glands were analyzed from control and C/EBPα. P-values were obtained by Student's t-test and were calculated relative to control animals (*P ≤ 0.05) Upper panels: T47DindC/EBPα and T47DyindC/EBPα (PR-) cells expressing inducible recombinant C/EBPα were exposed to Doxycycline (1 μg/ml) for different times, and then, the number of cells was monitored. Data are represented as mean ± SD from two experiments performed in triplicate. P-values were obtained by Student's t-test and were calculated relative to untreated (−DOX). The numbers in parenthesis correspond to the average of the ratio +DOX over −DOX for each time point. Lower panels: T47D- and T47Dy-inducible C/EBPα cells were treated with doxycycline (1 μg/ml Dox) for 24 h, and the expression of C/EBPα was monitored by immune fluorescence microscopy (left) and Western blot (right). The Western blot confirms that T4Dy cells do not express PR. HP1γ was used as loading control. The band with a molecular weight around 55 kDa correspond to the TetO-C/EBPα ds Tomato fusion protein. Source data are available online for this figure. Source Data for Figure 2 [embj2018101426-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint An analysis of a cohort of 21 human invasive breast carcinomas and 90 i
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