ZBTB7A governs estrogen receptor alpha expression in breast cancer

ZBTB7A governs estrogen receptor alpha expression in breast cancer Abstract ZBTB7A, a member of the POZ/BTB and Krüppel (POK) family of transcription factors, has been shown to have a context-dependent role in cancer development and progression. The role of ZBTB7A in estrogen receptor alpha (ERα)-positive breast cancer is largely unknown. Approximately 70% of breast cancers are classified as ERα-positive. ERα carries out the biological effects of estrogen and its expression level dictates response to endocrine therapies and prognosis for breast cancer patients. In this study, we find that ZBTB7A transcriptionally regulates ERα expression in ERα-positive breast cancer cell lines by binding to the ESR1 promoter leading to increased transcription of ERα. Inhibition of ZBTB7A in ERα-positive cells results in decreased estrogen responsiveness as demonstrated by diminished estrogen-response element-driven luciferase reporter activity, induction of estrogen target genes, and estrogen-stimulated growth. We also report that ERα potentiates ZBTB7A expression via a post-translational mechanism, suggesting the presence of a positive feedback loop between ZBTB7A and ERα, conferring sensitivity to estrogen in breast cancer. Clinically, we find that ZBTB7A and ERα are often co-expressed in breast cancers and that high ZBTB7A expression correlates with improved overall and relapse-free survival for breast cancer patients. Importantly, high ZBTB7A expression predicts a more favorable outcome for patients treated with endocrine therapies. Together, these findings demonstrate that ZBTB7A contributes to the transcriptional program maintaining ERα expression and potentially an endocrine therapy-responsive phenotype in breast cancer. ZBTB7A, ERα, breast cancer, endocrine therapies Introduction ZBTB7A, also known as FBI-1 (Pessler et al., 1997) and LRF (Liu et al., 2004), belongs to the POZ/BTB and Krüppel (POK) family of transcription factors. There are 40 members of the POK family encoded in the human genome, several of which play various roles in both cancer progression and inhibition (Perez-Torrado et al., 2006). ZBTB7A plays diverse roles in many cellular processes including differentiation and development (Laudes et al., 2008; Maeda et al., 2007, 2009). Although ZBTB7A was initially described as a proto-oncogene due to its ability to transcriptionally repress the tumor suppressor ARF (Maeda et al., 2005), recent studies suggest that its role in cancer may be context dependent. Evidence has demonstrated an oncogenic role of ZBTB7A in non-small cell lung cancer and hepatocellular carcinoma (Fang et al., 2012; Potenza et al., 2017); however, it is frequently mutated in myeloid leukemia resulting in an anti-proliferative effect (Faber et al., 2016; Hartmann et al., 2016). Furthermore, loss of Zbtb7a in the PTEN-deficient model of prostate cancer led to accelerated tumorigenesis (Wang et al., 2013). Our lab has shown that ZBTB7A acts as a tumor suppressor by transcriptionally inhibiting glycolysis in human cancers thereby impeding tumor metabolism (Liu et al., 2014, 2016). In human breast cancers, ZBTB7A is often overexpressed (Aggarwal et al., 2010), although its involvement in the development and progression of the disease as well as its role in mammary gland development remain unclear. Breast cancer is one of the most common malignancies in women. In the clinic, pathological markers are used to determine treatment decisions, of which estrogen receptor alpha (ERα) is the most common, making up two-thirds of breast cancer diagnoses. Patients whose tumors are ERα-positive have improved disease-free and overall survival (OS) compared to those that lack ERα. The molecular mechanisms maintaining ERα expression in breast cancer, however, are not fully understood (Thomas and Gustafsson, 2011). Transcriptional regulation of ERα (deGraffenried et al., 2002, 2004) as well as protein modification and turnover (Reid et al., 2002; Miyoshi et al., 2010) have been described as mechanisms of ERα expression and maintenance in breast cancer. Uncovering the molecular determinants of ERα expression in breast cancer could result in improved therapeutic approaches, resulting in improved outcomes for breast cancer patients. Here we describe a novel role for ZBTB7A in breast cancer in which it controls ERα expression. Mechanistically, we provide evidence that ZBTB7A transcriptionally regulates ERα by directly binding to GC-rich regions in its promoter. Our data also suggest a positive feedback loop in which ERα leads to enhanced ZBTB7A protein stability, thus further maintaining ERα expression in breast cancer. Similar to human breast tumors, ZBTB7A is expressed at higher levels in luminal than basal cell populations within the normal mouse mammary gland. However, loss of ZBTB7A does not significantly affect ERα expression in the normal mammary gland nor does it interfere with normal mammary gland development, suggesting that the regulatory network dictating the relationship between ZBTB7A and ERα are predominant only in breast cancers. Of clinical significance, we find that ZBTB7A is highly expressed in patient samples of ERα-positive breast cancers and its expression correlates with improved relapse-free survival (RFS) and OS. Overall these findings may have important implications for future prognostic and treatment options for ERα-positive breast cancers. Results Clinical relevance of ZBTB7A expression in breast cancer To investigate the impact of ZBTB7A expression in breast cancer patients, we utilized the publically available RNASeq expression data in The Cancer Genome Atlas (TCGA) for 1093 primary breast tumors and 112 morphologically healthy surrounding breast tissues. ZBTB7A was shown to be overexpressed in tumor tissue compared to surrounding tissue (Figure 1A), which was in line with the previous findings by Aggarwal et al. (2010). Surprisingly, this high expression of ZBTB7A was associated with increased OS in the TCGA cohort (1091 patients stratified by median expression of ZBTB7A) (Figure 1B). Because ZBTB7A was proposed as a proto-oncogene in breast cancer (Qu et al., 2010), we further utilized KM plotter online tool to investigate the clinical relevance of ZBTB7A in large, microarray-based studies stratifying patients based on ‘best cut-off’ value (Gyorffy et al., 2010; Mihaly et al., 2013). Contrary to a study published by Qu et al. (2010), we found that high expression of ZBTB7A was predictive (n = 3951, P = 1.4e−15) of favorable RFS across all patients (Figure 1C) as well as patients who received endocrine therapy (n = 1064, P = 0.032) (Figure 1D). This contradicting finding led us to investigate ZBTB7A expression in different molecular subtypes of breast cancer. Breast cancers have been classified into at least five different intrinsic molecular subtypes (Luminal A, Luminal B, HER2, basal, and normal-like), which contribute to the heterogeneity of the disease and distinct clinical outcomes (Perou et al., 2000; Sorlie et al., 2001). While Luminal A and Luminal B subtypes are associated with good outcomes, HER2 and basal type breast cancers typically have a poor prognosis (Dai et al., 2015). The results indicated that ZBTB7A expression was significantly higher (P < 0.001) in Luminal A and Luminal B compared to basal-like breast tumors (Figure 1E) and higher expression is predictive of favorable RFS (Supplementary Figure S2A). Additionally, we found that ZBTB7A expression was significantly higher in ERα-positive tumors compared to ERα-negative tumors and normal tissue (Figure 1F). Figure 1 View largeDownload slide Clinical relevance of ZBTB7A in breast cancer. (A) mRNA expression level in primary tumor and surrounding breast tissue in TCGA cohort. Tumor samples are stratified into high (red) and low (green) expression of ZBTB7A based on median expression. (B) OS in TCGA patients stratified by median ZBTB7A expression (median, log-rank test). (C) RFS in KM Plotter cohort 213303_x_at probe (n = 3951) and ‘best cut-off’. (D) RFS in patients who received endocrine therapy (n = 1064). (E) ZBTB7A expression level in breast cancer molecular subtypes. (F) ZBTB7A expression in surrounding tissue, ER-positive and ER-negative tumors in TCGA cohort. Figure 1 View largeDownload slide Clinical relevance of ZBTB7A in breast cancer. (A) mRNA expression level in primary tumor and surrounding breast tissue in TCGA cohort. Tumor samples are stratified into high (red) and low (green) expression of ZBTB7A based on median expression. (B) OS in TCGA patients stratified by median ZBTB7A expression (median, log-rank test). (C) RFS in KM Plotter cohort 213303_x_at probe (n = 3951) and ‘best cut-off’. (D) RFS in patients who received endocrine therapy (n = 1064). (E) ZBTB7A expression level in breast cancer molecular subtypes. (F) ZBTB7A expression in surrounding tissue, ER-positive and ER-negative tumors in TCGA cohort. To further investigate the molecular role of ZBTB7A in breast cancer, we performed differential expression analysis between tumors with high and low expression of ZBTB7A. We utilized GiTools 2.2 TCGA BRCA collection with expression profiles for 889 patients. To perform differential expression analysis, we stratified patients into ‘low ZBTB7A’ (<−0.5) and ‘high ZBTB7A’ (>0.5) expression (121 and 83 patients, respectively). Surprisingly, one of the top differentially expressed genes was ESR1 (Supplementary Table S1), and its expression was positively correlated with ZBTB7A (Figure 2A and Supplementary Figure S1A), again contradicting previous findings by Qu et al. (2010) in a smaller (125 patient) cohort. We also performed gene-set enrichment analysis (GSEA) across all the primary tumors using ZBTB7A expression level as continuous phenotype. This analysis revealed that both estrogen receptor early and late response genes were significantly enriched in the TCGA cohort (Figure 2B and Supplementary Figure S1B). Figure 2 View largeDownload slide ZBTB7A and ERα expressions are correlated in breast cancer samples. (A) Heatmap showing top genes differentially expressed in breast cancer samples with high and low expression of ZBTB7A. (B) Gene sets that are correlated with increased ZBTB7A expression in TCGA cohort. (C) ZBTB7A expression in ER-positive and ER-negative breast cancer cell lines. (D) ZBTB7A protein expression in human breast cancer cell lines (MCF-7, T47D, MDA-MB-231, MDA-MB-468). Figure 2 View largeDownload slide ZBTB7A and ERα expressions are correlated in breast cancer samples. (A) Heatmap showing top genes differentially expressed in breast cancer samples with high and low expression of ZBTB7A. (B) Gene sets that are correlated with increased ZBTB7A expression in TCGA cohort. (C) ZBTB7A expression in ER-positive and ER-negative breast cancer cell lines. (D) ZBTB7A protein expression in human breast cancer cell lines (MCF-7, T47D, MDA-MB-231, MDA-MB-468). Next, we tested whether ZBTB7A–ESR1 correlation observed in human tumor samples was also present in breast cancer cell lines. We utilized expression levels deposited in Cancer Cell Line Encyclopedia (CCLE) and confirmed higher expression of ZBTB7A in ER-positive (ER+) cell lines (Figure 2C) and positive correlation between ESR1 and ZBTB7A expression (Supplementary Figure S1C). We next analyzed the protein levels of ZBTB7A in ER-positive (MCF-7, T47D) and ER-negative (MDA-MB-231, MDA-MB-468) breast cancer cell lines and found that ZBTB7A protein levels were higher in ERα-positive breast cancer cell lines (Figure 2D). Hence, within human breast cancer tumor specimens and breast cancer cell lines, expression of ZBTB7A correlated with ERα levels, suggesting they may be regulating each other’s expression. ZBTB7A drives expression of ERα in luminal breast cancers To explore the functional role of ZBTB7A in hormone-receptor positive breast cancer, we used MCF-7 and T47D cells as model systems. Upon silencing of ZBTB7A using two different oligonucleotide constructs, we observed a significant reduction in ESR1 mRNA levels (Figure 3A). ERα protein levels were also reduced following ZBTB7A silencing in MCF-7 cells, under both basal and 17β-estradiol-stimulated conditions (Figure 3B). To further explore the mechanism through which ZBTB7A regulates ERα expression, we used a luciferase reporter driven by the promoter of ERα, containing 3500 base pairs upstream from the promoter start site (deGraffenried et al., 2004). ZBTB7A binds in GC-rich DNA sequences (Maeda et al., 2005). Promoter motif analyses were used to recognize four potential ZBTB7A-binding sites in the ERα promoter sequence, located at −1381, −2079, −2729, and −3253 base pairs from the transcription start site. Mutations of these putative ZBTB7A-binding sites were made to the ERα promoter luciferase construct and transfected into HEK293T cells with Renilla luciferase as a control. Cells were lysed and luciferase activity assessed 24 h later. Mutations of these putative ZBTB7A-binding sites resulted in significant reduction of basal luciferase activity (Figure 3C), suggesting that the binding of ZBTB7A to these sites is important for ERα transcriptional activity. Figure 3 View largeDownload slide ZBTB7A controls ERα expression in luminal breast cancers. (A) ESR1 mRNA level decreased in MCF-7 and T47D cell lines 48 h after transfection with ZBTB7A siRNA. The data represent three independent experiments and are presented as mean ± SEM. (B) ERα protein level in MCF-7 cells decreased 48 h following ZBTB7A siRNA treatment. The western blot is representative of three independent experiments. The quantification was done using Image J and represents mean ± SEM of three independent experiments. (C) Diminished ERα-promoter luciferase activity following mutation in putative ZBTB7A-binding sites. The putative ZBTB7A-binding sites within the ERα promoter are shown. HEK293T cells were transfected with the wild-type ER-promoter luciferase or mutant constructs and Renilla luciferase, and then collected 24 h later. Data from three independent experiments are represented as mean ± SEM. (D) ZBTB7A binds to the ERα promoter in MCF-7 cells. ChIP assays were performed in MCF-7 cells with anti-ZBTB7A antibody or control IgG. The abundance of DNA within the ERα promoter was assessed using qPCR. Data from three independent experiments are shown as mean ± SEM. Figure 3 View largeDownload slide ZBTB7A controls ERα expression in luminal breast cancers. (A) ESR1 mRNA level decreased in MCF-7 and T47D cell lines 48 h after transfection with ZBTB7A siRNA. The data represent three independent experiments and are presented as mean ± SEM. (B) ERα protein level in MCF-7 cells decreased 48 h following ZBTB7A siRNA treatment. The western blot is representative of three independent experiments. The quantification was done using Image J and represents mean ± SEM of three independent experiments. (C) Diminished ERα-promoter luciferase activity following mutation in putative ZBTB7A-binding sites. The putative ZBTB7A-binding sites within the ERα promoter are shown. HEK293T cells were transfected with the wild-type ER-promoter luciferase or mutant constructs and Renilla luciferase, and then collected 24 h later. Data from three independent experiments are represented as mean ± SEM. (D) ZBTB7A binds to the ERα promoter in MCF-7 cells. ChIP assays were performed in MCF-7 cells with anti-ZBTB7A antibody or control IgG. The abundance of DNA within the ERα promoter was assessed using qPCR. Data from three independent experiments are shown as mean ± SEM. Our data suggest that ZBTB7A modulates ERα expression at the transcriptional level. To further elucidate how ZBTB7A regulates ERα, we used chromatin-immunoprecipitation (ChIP) to analyze the recruitment of ZBTB7A to the ESR1 promoter in ER-positive breast cancer cells. The interaction between ZBTB7A and the ESR1 promoter was confirmed by ChIP assay. The results indicated that ERα promoter fragments were precipitated with ZBTB7A antibody at the −1381 base pair site but not with control IgG, supporting a direct binding of ZBTB7A to the promoter of ERα (Figure 3D). Together, these data suggest that ZBTB7A upregulates ERα expression through direct binding to GC boxes in its promoter. Next, we explored whether ZBTB7A regulation of ERα affects downstream activity of ERα target genes. Inhibition of ZBTB7A using siRNA abrogated E2-induced estrogen-response element (ERE) luciferase reporter activity in MCF-7 cells (Figure 4A). As a member of the POK family of transcription factors, ZBTB7A mediates its effects on transcription by binding to DNA through its zinc finger domain. To assess what region of the ZBTB7A protein was necessary for ERE-luciferase activity, ZBTB7A mutants were generated with point mutations in either the BTB or zinc finger domain (Liu et al., 2016). The K424N and K424T mutations in the second zinc finger domain resulted in significant reduction in ERE-luciferase activity, consistent with the requirement of the zinc finger domain-mediated ZBTB7A DNA binding necessary for induction of ERα expression and its transcriptional activity (Figure 4B). To assess the effects of ZBTB7A inhibition on the expression of ERα target genes, we treated MCF-7 cells with ZBTB7A siRNA in the presence and absence of 17β-estradiol. Indeed, 17β-estradiol-mediated induction of ERα target genes was reduced by silencing ZBTB7A including progesterone receptor (PGR), trefoil factor 1(TFF1), GATA binding protein 3 (GATA3), and C-MYC mRNA (Figure 4C). These data indicate that ZBTB7A is required for ERα expression and subsequent activation of ERα target genes. Figure 4 View largeDownload slide Abrogation of ERα function following ZBTB7A inhibition. (A) Decreased ERα function as evidenced by decreased ERE-luciferase activity following ZBTB7A siRNA treatment. MCF-7 cells were stripped of hormone for 3 days, transfected with siRNA, treated with E2 (10 nM) at 24 h post-transfection, and collected at 36–48 h. Data from three independent experiments are shown as mean ± SEM. (B) ERE-luciferase activity was diminished following transfection with 424 ZBTB7A mutant plasmids. Cells were transfected and collected 24 h later. Data from three independent experiments are shown as mean ± SEM. (C) Diminished E2-induced induction of ER target genes following inhibition of ZBTB7A with siRNA. Cells were stripped of hormone for 3 days, transfected with siRNA, treated with E2 (10 nM) at 24 h post-transfection, and collected at 36–48 h. Data from three independent experiments are shown as mean ± SEM. (D) E2-induced growth is abrogated in MCF-7 cells following ZBTB7A siRNA treatment. Cells were stripped of hormone for 3 days, transfected with siRNA, and plated for growth assay 24 h later. Data are representative of three independent experiments done in triplicate. Figure 4 View largeDownload slide Abrogation of ERα function following ZBTB7A inhibition. (A) Decreased ERα function as evidenced by decreased ERE-luciferase activity following ZBTB7A siRNA treatment. MCF-7 cells were stripped of hormone for 3 days, transfected with siRNA, treated with E2 (10 nM) at 24 h post-transfection, and collected at 36–48 h. Data from three independent experiments are shown as mean ± SEM. (B) ERE-luciferase activity was diminished following transfection with 424 ZBTB7A mutant plasmids. Cells were transfected and collected 24 h later. Data from three independent experiments are shown as mean ± SEM. (C) Diminished E2-induced induction of ER target genes following inhibition of ZBTB7A with siRNA. Cells were stripped of hormone for 3 days, transfected with siRNA, treated with E2 (10 nM) at 24 h post-transfection, and collected at 36–48 h. Data from three independent experiments are shown as mean ± SEM. (D) E2-induced growth is abrogated in MCF-7 cells following ZBTB7A siRNA treatment. Cells were stripped of hormone for 3 days, transfected with siRNA, and plated for growth assay 24 h later. Data are representative of three independent experiments done in triplicate. ERα mediates growth and proliferation through activation of its downstream target genes; as such, we next sought to determine whether ZBTB7A inhibition would affect MCF-7 cell proliferation. As shown in Figure 4D, siRNA-mediated downregulation of ZBTB7A led to a reduction in 17β-estradiol-stimulated proliferation in MCF-7 cells. Together, these data suggest that the pro-proliferative signal of 17β-estradiol is blunted following the inhibition of ZBTB7A through its effects on ERα expression. ERα regulates ZBTB7A post-translational stability In human breast cancer, ZBTB7A and ERα are highly correlated (Figure 2A). ERα has been shown to participate in positive cross-regulatory loops with other transcription factors such as Gata3, in which ERα and Gata3 reciprocally regulate each other’s transcription (Eeckhoute et al., 2007). To investigate the hypothesis that ERα may be regulating the expression of ZBTB7A in breast cancer, we inhibited ERα in MCF-7 cells using siRNA. The results showed that reduction of ERα led to a decrease of ZBTB7A protein levels (Figure 5A and Supplementary Figure S3), but had little effect on ZBTB7A mRNA levels 48 h post siRNA treatment (Figure 5B and Supplementary Figure S3). We then asked whether ZBTB7A downregulation due to ERα siRNA was occurring via protein degradation by the proteasome. Treatment with the proteasome inhibitor MG132 prevented the downregulation of ZBTB7A protein levels in the presence of ERα siRNA, indicating that protein degradation, at least partially, contributed to the observed alterations in ZBTB7A levels (Figure 5C). Next, to assess whether ZBTB7A half-life is altered following ERα siRNA treatment, the protein level of ZBTB7A was analyzed in the presence of cycloheximide. The addition of ERα siRNA significantly decreases ZBTB7A half-life at 6 h and 8 h following cycloheximide treatment (Figure 5D and E). Together, these data suggest that ERα plays a role in ZBTB7A protein stability. Figure 5 View largeDownload slide ERα regulates ZBTB7A protein level and stability in breast cancer. (A and B) ERα siRNA causes decreased ZBTB7A protein level. MCF-7 cells were transfected with ERα siRNA and 48 h later were subjected to western blot (A) and qPCR (B) analyses. ERα siRNA treatment does not lead to reduced ZBTB7A mRNA levels. Graphs show mean ± SEM of three independent experiments. (C) Western blot demonstrating transfection of ERα siRNA leads to downregulation of ZBTB7A protein that is rescued by MG132 treatment. MCF-7 cells were transfected with siRNA, 48 h later treated with MG132 for 6 h, and then collected for western blot analysis. The blot is representative of three independent experiments. Quantification was done using Image J and includes three independent experiments, mean ± SEM. (D) Western blot of MCF-7 cells treated with control siRNA or ERα siRNA for 48 h followed by cycloheximide (100 μg/ml) treatment at the indicated time points. (E) Quantification of D including two independent experiments, mean ± SEM using Image J. Figure 5 View largeDownload slide ERα regulates ZBTB7A protein level and stability in breast cancer. (A and B) ERα siRNA causes decreased ZBTB7A protein level. MCF-7 cells were transfected with ERα siRNA and 48 h later were subjected to western blot (A) and qPCR (B) analyses. ERα siRNA treatment does not lead to reduced ZBTB7A mRNA levels. Graphs show mean ± SEM of three independent experiments. (C) Western blot demonstrating transfection of ERα siRNA leads to downregulation of ZBTB7A protein that is rescued by MG132 treatment. MCF-7 cells were transfected with siRNA, 48 h later treated with MG132 for 6 h, and then collected for western blot analysis. The blot is representative of three independent experiments. Quantification was done using Image J and includes three independent experiments, mean ± SEM. (D) Western blot of MCF-7 cells treated with control siRNA or ERα siRNA for 48 h followed by cycloheximide (100 μg/ml) treatment at the indicated time points. (E) Quantification of D including two independent experiments, mean ± SEM using Image J. The role of ZBTB7A in the normal mammary gland ERα is essential for normal mammary gland development. Mice lacking ERα exhibit deficiencies in ductal morphogenesis of the mammary gland, including an inability to form terminal end buds (TEBs) and defects in ductal invasion (Mallepell et al., 2006; Feng et al., 2007). Additionally, ZBTB7A has been shown to play an important role in the differentiation processes of various cell types (Laudes et al., 2008; Maeda et al., 2009). Since we have observed that ZBTB7A controls ERα expression in luminal breast cancer cells, we next wanted to investigate its function in the normal mammary gland. First, we set out to determine whether there is a difference in ZBTB7A expression levels in luminal vs. basal cells in the mouse mammary gland. In accordance with human tumor data (Figure 2B), we found that the luminal cell population displayed increased ZBTB7A mRNA expression compared to basal cells isolated from the mouse mammary gland (Figure 6A). Next, we used a loss of function approach to study the role of ZBTB7A in the mammary gland. Zbtb7a-null mice display embryonic lethality due to anemia caused by apoptosis of late stage erythroblasts (Maeda et al., 2009). We therefore used a CRE/LOXP recombination system to conditionally knockout Zbtb7a in the mammary gland. Mice with a floxed Zbtb7a gene (Maeda et al., 2007) were crossed with constitutively active MMTV-CRE mice, which express CRE in the mammary gland, skin, and salivary glands (Wagner et al., 1997). This conditional deletion approach rescued embryonic lethality observed in Zbtb7a-null mice, as homozygous Zbtb7a-floxed mice carrying the MMTV-CRE transgene were born in normal Mendelian ratios (data not shown). The genotypes of mice harboring the Zbtb7a-floxed alleles were determined by PCR using genomic DNA isolated from ear clips of the mice (Figure 6B). Mammary glands were isolated from 6-week-old female mice to determine Zbtb7a expression level in wild-type vs. Zbtb7aflox/flox mammary glands. ZBTB7A expression was significantly lower in mice harboring the Zbtb7a-floxed allele and the MMTV-CRE allele compared to wild-type littermates (Figure 6C). However, in contrast to the breast cancer cell lines, Esr1 mRNA was not impacted by decreased expression of Zbtb7a in the normal mammary gland. To examine ductal morphogenesis, mammary glands from wild-type and Zbtb7aflox/flox mice were whole-mounted at 4 and 6 weeks of age. Zbtb7aflox/flox mammary glands displayed normal pubertal and post-pubertal ductal morphogenesis (Figure 6D). The histoarchitecture of the TEBs and ducts from wild-type and Zbtb7aflox/flox mice were indistinguishable by hematoxylin and eosin staining. In addition, pups from Zbtb7aflox/flox female mice were born in numbers and of weights comparable to wild-type mice. Together these data suggest that ZBTB7A is not responsible for normal mammary gland growth and development, but is implicated in the growth of cancerous breast epithelial cells. Figure 6 View largeDownload slide ZBTB7A expression in the normal mammary gland. (A) ZBTB7A expression in luminal and basal mammary cell populations. Mammary epithelial cells were extracted from 8- to 10-week-old female mice and subjected to FACS analysis. (B) Representative PCR genotyping resulting from wild-type, ZBTB7Aflox/flox, and ZBTB7AWT/flox mice. (C) ZBTB7A and ESR1 mRNA levels from 8- to 10-week-old wild-type and ZBTB7Aflox/flox mice (n = 4–6). (D) Representative whole-mount staining from 4- and 6-week-old wild-type and ZBTB7Aflox/flox mice (n = 8–10). Scale bar, 10 mm. (E) Ductal length from 4- and 6-week-old wild-type and ZBTB7Aflox/flox mice (n = 4). Graph shows mean ± SEM. Figure 6 View largeDownload slide ZBTB7A expression in the normal mammary gland. (A) ZBTB7A expression in luminal and basal mammary cell populations. Mammary epithelial cells were extracted from 8- to 10-week-old female mice and subjected to FACS analysis. (B) Representative PCR genotyping resulting from wild-type, ZBTB7Aflox/flox, and ZBTB7AWT/flox mice. (C) ZBTB7A and ESR1 mRNA levels from 8- to 10-week-old wild-type and ZBTB7Aflox/flox mice (n = 4–6). (D) Representative whole-mount staining from 4- and 6-week-old wild-type and ZBTB7Aflox/flox mice (n = 8–10). Scale bar, 10 mm. (E) Ductal length from 4- and 6-week-old wild-type and ZBTB7Aflox/flox mice (n = 4). Graph shows mean ± SEM. Discussion ERα is one of the most critical biomarkers dictating treatment decisions and prognosis for breast cancer patients. Extensive research on the downstream functions of ERα has led to targeted treatment strategies such as endocrine therapies. However, the mechanism through which ERα itself is regulated and maintained at high levels in breast cancers remains incompletely understood. Here we uncover a link between ZBTB7A and ERα that may in part explain how ERα levels are maintained in breast tumors (Supplementary Figure S4). Recent studies from our lab and others have suggested a context specific role for ZBTB7A in cancer. Few studies, however, have looked at the role of ZBTB7A in breast cancer. Zu et al. (2011) reported that ZBTB7A was expressed at elevated levels in breast tumors compared to the normal breast tissue. Due to the heterogeneity of breast cancer, we sought to assess ZBTB7A expression within the various breast cancer subtypes. In our current study, we find that ZBTB7A is more highly expressed in the luminal vs. basal breast cancer subtypes (Figure 1E). In agreement with this observation, we find that ZBTB7A is more highly expressed in ER-positive compared to ER-negative human breast cancers (Figure 1F). Differential gene expression analysis of human tumor samples expressing high vs. low levels of ZBTB7A also suggested that there is a strong relationship between ZBTB7A and genes including DEK, ESR1 and KDM4B. DEK expression has been associated with β-catenin activity and a more invasive phenotype in breast cancer (Privette Vinnedge et al., 2011). Although the relationship between DEK and ZBTB7A is unknown, our analysis found a negative correlation between DEK and ZBTB7A supporting ZBTB7A’s association with less aggressive breast cancers. KDM4B has been associated with ERα signaling cascade in breast cancer (Gaughan et al., 2013) and was positively correlated with ZBTB7A. Because ERα signaling is important in breast cancer, we focused on the strong positive correlation observed between ESR1 and ZBTB7A. ZBTB7A’s association with less aggressive, more differentiated breast cancer subtypes led us to further examine its overall functional role in breast cancer, specifically in ERα-positive disease. Our results in human breast cancer cell lines argue a role for ZBTB7A in maintaining ERα expression in breast cancer. We find that ZBTB7A expression is higher in ERα-positive breast cancer cell lines and that knockdown of ZBTB7A leads to diminished ERα expression at the transcript and protein level. This decreased ERα expression imparted by ZBTB7A knockdown resulted in reduced ERα target gene expression following estrogen treatment. Functionally, depletion of ZBTB7A reduced E2-induced growth of ER-positive breast cancer cells. Mechanistically, we find that ZBTB7A controls ERα transcriptional activity by directly binding to the ERα promoter. Transcription of ERα can be initiated from multiple promoters upstream of the ESR1 gene locus (Grandien et al., 1997). Normal mammary epithelial cells utilize the more distal promoters while breast cancer cells typically activate the more proximal promoters (Grandien et al., 1995). ChIP analyses suggest that ZBTB7A is recruited to the proximal promoter of ESR1 (Figure 3D). Further, mutations in the predicted ZBTB7A-binding sites in the ERα proximal promoter led to decreased activity of ESR1 reporter constructs (Figure 3C). Together these data provide evidence that ZBTB7A initiates ESR1 transcription via direct binding to the ERα promoter. Our data also provide evidence of a positive feedback loop between ERα and ZBTB7A in breast cancer. Knockdown of ESR1 lowered ZBTB7A protein expression with no significant effect on ZBTB7A mRNA levels. Inhibition of the ubiquitin–proteasome pathway by MG132 rescued ZBTB7A downregulation following ESR1 knockdown, suggesting that ERα can block the degradation of ZBTB7A by the proteasome. Overall, this feedback loop provides further evidence indicating that ZBTB7A and ERα are tightly connected in breast cancer and may be responsible for the maintenance of ER expression levels resulting in tumor growth. Due to the observed relationship between ZBTB7A and ERα in breast cancer, we next sought to assess the role of ZBTB7A in the normal mammary gland. Both genomic and mammary gland-specific ERα knockout mouse models give rise to developmental defects of the mammary gland, specifically arrested ductal growth in the pre-pubertal stage of development (Mallepell et al., 2006; Feng et al., 2007). To assess whether ZBTB7A governs ERα expression in the normal mammary gland, we utilized a mouse model in which ZBTB7A was depleted specifically in the mammary gland. Homozygote Zbtb7aflox/flox mice did not display any discernable phenotype and loss of ZBTB7A did not affect the expression of ERα in the mammary gland. Although it has been reported that different mouse strains exhibit variations in ductal morphology (Montero Girard et al., 2007; Aupperlee et al., 2009), it is unlikely that the lack of phenotype observed in ZBTB7A-null mammary glands is due to the background of the mice. ZBTB7A has been shown to play a role in various aspects of development including regulating the determination of B vs. T lymphoid fate (Maeda et al., 2007), as well as in the early stages of preadipocyte differentiation (Laudes et al., 2004). The lack of phenotype observed in the Zbtb7a-null mammary gland may be due to functional redundancy of other ZBTB family members. It has been suggested that some ZBTB proteins may play functionally redundant roles following depletion of other ZBTB family members (Kelly and Daniel, 2006). It is possible that an unidentified ZBTB member was able to compensate for the loss of ZBTB7A in the mammary epithelial cells thus preventing any disruption in ERα expression and signaling. Other ZBTB members such as ZBTB4 have been reported to play roles in breast cancer (Kim et al., 2012), however none have yet been reported to alter ERα expression in breast cancer or the normal mammary gland. Alternatively, the observed results may suggest that the link between ERα and ZBTB7A may not play a role in the transcriptional program that governs mammary epithelial cell differentiation and is only relevant in breast cancer cells. Importantly, our study reveals the potential clinical significance of ZBTB7A for breast cancer patients. We demonstrate that ZBTB7A levels in human breast tumors correlated with improved overall and RFS (Figure 1B and C) for breast cancer patients. Although these results are contrary to previously published studies (Qu et al., 2010; Zu et al., 2011), our analysis utilized a larger sample size, which likely accounts for these discrepancies. In addition, the methods used to assess the level of ZBTB7A were different. Our study used RNA expression from microarray data to determine ZBTB7A level, while the previous studies used immunohistochemical analysis to score ZBTB7A protein intensity, potentially accounting for the differences in observed outcomes. We also demonstrate that ZBTB7A levels correlated with improved outcomes for patients harboring luminal A and B type tumors (Supplementary Figure S2). Clinically, we propose that high ZBTB7A expression may result in more favorable outcomes for patients treated with endocrine therapies as the major determinant of response to these therapies is ERα expression (Pinzone et al., 2004; Thomas and Gustafsson, 2011). In support of this notion, we also present evidence that patients with high ZBTB7A have improved response to endocrine therapies. Overall, the current study demonstrates a novel relationship between ZBTB7A and ERα, suggesting that ZBTB7A may be used to predict breast cancer patient outcomes and have important clinical implications for the treatment of ERα-positive breast tumors in the future. Methods Cell culture MCF-7, MDA-MB-231, MDA-MB-468, and Hek293T cells were cultured in DMEM (Corning, Cellgro) plus 10% fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin G, and 100 μg/ml streptomycin (Corning, Cellgro). T47D cells were cultured in RPMI (Corning, Cellgro) plus 10% FBS, 100 U/ml penicillin G, and 100 μg/ml streptomycin. All cells were cultured in a 37°C, 5% CO2 incubator. Antibodies and reagents Anti-ZBTB7A antibody (Santa Cruz Biotechnology, 13E9), anti-ERα antibody (Santa Cruz Biotechnology, HC-20), anti-alpha tubulin antibody (Cell Signaling Technologies, 2144), and anti-GAPDH antibody (Cell Signaling Technologies, 2118) were used for western blot; anti-ZBTB7A antibody (Bethyl Laboratories, A300-549A) and normal Rabbit IgG (Cell Signaling Technologies, 2729) were used for ChIP. Immunoblot Cells were lysed in buffer (50 mM Tris, at pH 8.0, 150 mM NaCl, 0.5% NP-40). Protein concentrations of the lysates were measured by Bradford assay (Bio-Rad). The lysates were then resolved by SDS-PAGE and immunoblotted with the indicated antibodies. qPCR to quantify gene expression Total RNA was extracted using TRIZOL according to the manufacturer’s instructions (Ambion). RNA was reversed transcribed using iScript cDNA synthesis kit (Bio-Rad). qPCR was performed using diluted cDNA, appropriate primers, and PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) in StepOnePlus real-time PCR system (Applied Biosystems). The following primers were used: ESR1_F-TCCTCATCCTCTCCCACATC, ESR1_R-TCCAGCAGCAGGTCATAGAG, ZBTB7A_F-GCTTGGGCCGGTTGAATGTA, ZBTB7A _R-GGCTGTGAAGTTACCGTCGG, 18S_F-GTAACCCGTTGAACCCCATT, 18S_R-CCATCCAATCGGTAGTAGCG, GATA3_F-TACAGCTCCGGACTCTTCCC, GATA3_R-CCCACAGTTCACACACTCCC, TFF1_F-GCCACCATGGAGAACAAGGT, TFF1_R-CAATTCTGTCTTTCACGGGG, MYC_F-GCCACGTCTCCACACATCAG, MYC_R-TCTTGGCAGCAGGATAGTCCT, PGR_F-TGGTGTCCTTACCTGTGGGA, PGR_R-ACGATGCAGTCATTTCTTCCA. siRNA-based knockdown For siRNA transfections, 3 × 105 cells were reverse transfected with siRNA and INTERFERin (Polyplus Transfection) and collected 48 h following transfection. siRNAs for ZBTB7A were manufactured by Sigma (siRNA ID for ZBTB7A#1 SASI_Hs01_00244764, ZBTB7A#2 SASI_Hs01_00244765). ESR1 siRNA1 was manufactured by Sigma (SASI_Hs01_00078592) and ESR1 siRNA2 was manufactured by Thermo Fisher Scientific (Silencer Select siRNA s4824). Mutagenesis Mutagenesis was performed using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer’s instructions. The following primers were used for site-directed mutagenesis: MUT1_F-GTCCAGCAGGTTTTTGTAGGGAGGGAAGC, MUT1_R-ACACACGTTCCAGCCGTG, MUT2_F-CTGCAATACTATATATCTTTCAAAGCACAAAAACATATATTTG, MUT2_R-TGTTTGAGCACAGGAGAG, MUT3_F-TTCAGAGACTATATACTAGGGCCAGTAAGG, MUT3_R-CAGAGGCATTGCCATGCC, MUT4_F-TGTTCTCCATATATATATGTGTGTGTCTCCTTTTTC, MUT4_R-GCAATCCTCATCTCCCTG. The resulting mutations were then confirmed by sequencing. Chromatin-immunoprecipitation assays ChIP was performed according to the manufacturer’s instructions (Cell Signaling Technology, 9003S). Briefly, the chromatin/DNA protein complexes were prepared from MCF-7 cells plated 48 h before collection. Crosslinking of DNA to proteins was carried out using 1% formaldehyde for 10 min at room temperature. The crosslinking was quenched by the addition of glycine (0.125 M) for 5 min at room temperature and followed by two washes with ice-cold PBS. Cells were scraped into PBS containing protease inhibitor cocktail (PIC). Cells were pelleted and mixed by inverting the tube every 3 min in buffer A, DTT, and PIC, followed by incubation on ice for 10 min. The nuclei were dissolved in buffer B, DTT, and micrococcal nuclease and incubated for 20 min at 37°C with frequent mixing to digest DNA. The lysate was then sonicated using the SONIFIER 450. The lysate was incubated with ZBTB7A/FBI-1 antibody (Bethyl Laboratories, A300-549A) or normal rabbit IgG (Cell Signaling Technologies, 2729), followed by incubation with ChIP-grade protein G magnetic beads. DNA was purified and subjected to qPCR analysis. Mouse strains Generation of mice containing the floxed Zbtb7a allele was previously described (Maeda et al., 2007). Cre-recombinase was expressed under the control of the mouse mammary tumor virus (MMTV) promoter (JAX stock #003553) (Wagner et al., 1997) in the FVB background. Genotyping was carried out by PCR. All animal experiments were conducted in accordance with the IACUC approved protocols following Harvard guidelines. Evaluation of cell populations in mouse mammary gland Mammary glands were collected from female mice and prepared into single-cell suspensions as previously described (Shackleton et al., 2006). FACS analysis and cell sorting of mammary stem cell-enriched/basal (CD29hiCD24+) and luminal (CD29loCD24+) cell populations were performed as previously described (Fu et al., 2015). Mammary gland whole-mount preparation Tissues were fixed and stained as previously described (Plante et al., 2011). Briefly, glands were fixed to slides using Carnoy’s fixative (100% EtOH, chloroform, glacial acetic acid; 6:3:1) overnight at 4°C, washed in 70% EtOH, rehydrated, stained in carmine alum overnight and cleared in xylene. Survival analysis For OS analysis in TCGA cohort, samples were stratified by median expression by RNA-Seq log-rank analysis was performed. For survival analysis based on microarray derived gene expression data, we utilized online KM plotter (Gyorffy et al., 2010). To test the RFS, we used 213303_x_at probe that allowed analysis of the highest number of patients and we stratified patients based on ‘best cut-off’ (Mihaly et al., 2013). Statistical analysis If not indicated, statistical analyses were completed using GraphPad Prism 7 software. The differential gene expression on RNA-seq data was performed on expression data obtained from GiTools 2.2. Samples were stratified into ‘low ZBTB7A’ (<−0.5) and ‘high ZBTB7A’ (>0.5) expression. Differential expression analysis was performed using t-test, followed by false discovery rate (FDR) correction. Transcripts with q (FDR-corrected P-value) < 0.001 and fold change log2(high/low) >2 were considered differentially expressed genes. Heatmap was generated with GiTools 2.2. The list of differentially expressed transcripts is included in Supplementary Table S1. GSEA (Broad Institute) (Mootha et al., 2003; Subramanian et al., 2005) was performed across all tumors downloaded from Board Institute for TCGA BRCA Collection. The ZBTB7A expression level was used as a continuous phenotype and Pearson correlation was used as metrics for ranking the genes (Hallmark gene sets h.all.v6.0.symbols.gmt). The expression levels for surrounding tissue and primary breast tumors were retrieved together with clinical data from Board Institute for TCGA BRCA Collection (gdac.broadinstitute.org_BRCA.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800). Expression levels are reported as RNA-Seq by Expectation-Maximization (Li and Dewey, 2011). Expression levels for ZBTB7A and ESR1 in cell lines were retrieved via cBioPortal (Cerami et al., 2012) from Cancer Cell Line Encyclopedia (Barretina et al., 2012) and estrogen receptor status from the study by Jiang et al. (2016). Only cell lines with established estrogen receptor status were used for analysis (Supplementary Table S2). Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. 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Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

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Abstract

Abstract ZBTB7A, a member of the POZ/BTB and Krüppel (POK) family of transcription factors, has been shown to have a context-dependent role in cancer development and progression. The role of ZBTB7A in estrogen receptor alpha (ERα)-positive breast cancer is largely unknown. Approximately 70% of breast cancers are classified as ERα-positive. ERα carries out the biological effects of estrogen and its expression level dictates response to endocrine therapies and prognosis for breast cancer patients. In this study, we find that ZBTB7A transcriptionally regulates ERα expression in ERα-positive breast cancer cell lines by binding to the ESR1 promoter leading to increased transcription of ERα. Inhibition of ZBTB7A in ERα-positive cells results in decreased estrogen responsiveness as demonstrated by diminished estrogen-response element-driven luciferase reporter activity, induction of estrogen target genes, and estrogen-stimulated growth. We also report that ERα potentiates ZBTB7A expression via a post-translational mechanism, suggesting the presence of a positive feedback loop between ZBTB7A and ERα, conferring sensitivity to estrogen in breast cancer. Clinically, we find that ZBTB7A and ERα are often co-expressed in breast cancers and that high ZBTB7A expression correlates with improved overall and relapse-free survival for breast cancer patients. Importantly, high ZBTB7A expression predicts a more favorable outcome for patients treated with endocrine therapies. Together, these findings demonstrate that ZBTB7A contributes to the transcriptional program maintaining ERα expression and potentially an endocrine therapy-responsive phenotype in breast cancer. ZBTB7A, ERα, breast cancer, endocrine therapies Introduction ZBTB7A, also known as FBI-1 (Pessler et al., 1997) and LRF (Liu et al., 2004), belongs to the POZ/BTB and Krüppel (POK) family of transcription factors. There are 40 members of the POK family encoded in the human genome, several of which play various roles in both cancer progression and inhibition (Perez-Torrado et al., 2006). ZBTB7A plays diverse roles in many cellular processes including differentiation and development (Laudes et al., 2008; Maeda et al., 2007, 2009). Although ZBTB7A was initially described as a proto-oncogene due to its ability to transcriptionally repress the tumor suppressor ARF (Maeda et al., 2005), recent studies suggest that its role in cancer may be context dependent. Evidence has demonstrated an oncogenic role of ZBTB7A in non-small cell lung cancer and hepatocellular carcinoma (Fang et al., 2012; Potenza et al., 2017); however, it is frequently mutated in myeloid leukemia resulting in an anti-proliferative effect (Faber et al., 2016; Hartmann et al., 2016). Furthermore, loss of Zbtb7a in the PTEN-deficient model of prostate cancer led to accelerated tumorigenesis (Wang et al., 2013). Our lab has shown that ZBTB7A acts as a tumor suppressor by transcriptionally inhibiting glycolysis in human cancers thereby impeding tumor metabolism (Liu et al., 2014, 2016). In human breast cancers, ZBTB7A is often overexpressed (Aggarwal et al., 2010), although its involvement in the development and progression of the disease as well as its role in mammary gland development remain unclear. Breast cancer is one of the most common malignancies in women. In the clinic, pathological markers are used to determine treatment decisions, of which estrogen receptor alpha (ERα) is the most common, making up two-thirds of breast cancer diagnoses. Patients whose tumors are ERα-positive have improved disease-free and overall survival (OS) compared to those that lack ERα. The molecular mechanisms maintaining ERα expression in breast cancer, however, are not fully understood (Thomas and Gustafsson, 2011). Transcriptional regulation of ERα (deGraffenried et al., 2002, 2004) as well as protein modification and turnover (Reid et al., 2002; Miyoshi et al., 2010) have been described as mechanisms of ERα expression and maintenance in breast cancer. Uncovering the molecular determinants of ERα expression in breast cancer could result in improved therapeutic approaches, resulting in improved outcomes for breast cancer patients. Here we describe a novel role for ZBTB7A in breast cancer in which it controls ERα expression. Mechanistically, we provide evidence that ZBTB7A transcriptionally regulates ERα by directly binding to GC-rich regions in its promoter. Our data also suggest a positive feedback loop in which ERα leads to enhanced ZBTB7A protein stability, thus further maintaining ERα expression in breast cancer. Similar to human breast tumors, ZBTB7A is expressed at higher levels in luminal than basal cell populations within the normal mouse mammary gland. However, loss of ZBTB7A does not significantly affect ERα expression in the normal mammary gland nor does it interfere with normal mammary gland development, suggesting that the regulatory network dictating the relationship between ZBTB7A and ERα are predominant only in breast cancers. Of clinical significance, we find that ZBTB7A is highly expressed in patient samples of ERα-positive breast cancers and its expression correlates with improved relapse-free survival (RFS) and OS. Overall these findings may have important implications for future prognostic and treatment options for ERα-positive breast cancers. Results Clinical relevance of ZBTB7A expression in breast cancer To investigate the impact of ZBTB7A expression in breast cancer patients, we utilized the publically available RNASeq expression data in The Cancer Genome Atlas (TCGA) for 1093 primary breast tumors and 112 morphologically healthy surrounding breast tissues. ZBTB7A was shown to be overexpressed in tumor tissue compared to surrounding tissue (Figure 1A), which was in line with the previous findings by Aggarwal et al. (2010). Surprisingly, this high expression of ZBTB7A was associated with increased OS in the TCGA cohort (1091 patients stratified by median expression of ZBTB7A) (Figure 1B). Because ZBTB7A was proposed as a proto-oncogene in breast cancer (Qu et al., 2010), we further utilized KM plotter online tool to investigate the clinical relevance of ZBTB7A in large, microarray-based studies stratifying patients based on ‘best cut-off’ value (Gyorffy et al., 2010; Mihaly et al., 2013). Contrary to a study published by Qu et al. (2010), we found that high expression of ZBTB7A was predictive (n = 3951, P = 1.4e−15) of favorable RFS across all patients (Figure 1C) as well as patients who received endocrine therapy (n = 1064, P = 0.032) (Figure 1D). This contradicting finding led us to investigate ZBTB7A expression in different molecular subtypes of breast cancer. Breast cancers have been classified into at least five different intrinsic molecular subtypes (Luminal A, Luminal B, HER2, basal, and normal-like), which contribute to the heterogeneity of the disease and distinct clinical outcomes (Perou et al., 2000; Sorlie et al., 2001). While Luminal A and Luminal B subtypes are associated with good outcomes, HER2 and basal type breast cancers typically have a poor prognosis (Dai et al., 2015). The results indicated that ZBTB7A expression was significantly higher (P < 0.001) in Luminal A and Luminal B compared to basal-like breast tumors (Figure 1E) and higher expression is predictive of favorable RFS (Supplementary Figure S2A). Additionally, we found that ZBTB7A expression was significantly higher in ERα-positive tumors compared to ERα-negative tumors and normal tissue (Figure 1F). Figure 1 View largeDownload slide Clinical relevance of ZBTB7A in breast cancer. (A) mRNA expression level in primary tumor and surrounding breast tissue in TCGA cohort. Tumor samples are stratified into high (red) and low (green) expression of ZBTB7A based on median expression. (B) OS in TCGA patients stratified by median ZBTB7A expression (median, log-rank test). (C) RFS in KM Plotter cohort 213303_x_at probe (n = 3951) and ‘best cut-off’. (D) RFS in patients who received endocrine therapy (n = 1064). (E) ZBTB7A expression level in breast cancer molecular subtypes. (F) ZBTB7A expression in surrounding tissue, ER-positive and ER-negative tumors in TCGA cohort. Figure 1 View largeDownload slide Clinical relevance of ZBTB7A in breast cancer. (A) mRNA expression level in primary tumor and surrounding breast tissue in TCGA cohort. Tumor samples are stratified into high (red) and low (green) expression of ZBTB7A based on median expression. (B) OS in TCGA patients stratified by median ZBTB7A expression (median, log-rank test). (C) RFS in KM Plotter cohort 213303_x_at probe (n = 3951) and ‘best cut-off’. (D) RFS in patients who received endocrine therapy (n = 1064). (E) ZBTB7A expression level in breast cancer molecular subtypes. (F) ZBTB7A expression in surrounding tissue, ER-positive and ER-negative tumors in TCGA cohort. To further investigate the molecular role of ZBTB7A in breast cancer, we performed differential expression analysis between tumors with high and low expression of ZBTB7A. We utilized GiTools 2.2 TCGA BRCA collection with expression profiles for 889 patients. To perform differential expression analysis, we stratified patients into ‘low ZBTB7A’ (<−0.5) and ‘high ZBTB7A’ (>0.5) expression (121 and 83 patients, respectively). Surprisingly, one of the top differentially expressed genes was ESR1 (Supplementary Table S1), and its expression was positively correlated with ZBTB7A (Figure 2A and Supplementary Figure S1A), again contradicting previous findings by Qu et al. (2010) in a smaller (125 patient) cohort. We also performed gene-set enrichment analysis (GSEA) across all the primary tumors using ZBTB7A expression level as continuous phenotype. This analysis revealed that both estrogen receptor early and late response genes were significantly enriched in the TCGA cohort (Figure 2B and Supplementary Figure S1B). Figure 2 View largeDownload slide ZBTB7A and ERα expressions are correlated in breast cancer samples. (A) Heatmap showing top genes differentially expressed in breast cancer samples with high and low expression of ZBTB7A. (B) Gene sets that are correlated with increased ZBTB7A expression in TCGA cohort. (C) ZBTB7A expression in ER-positive and ER-negative breast cancer cell lines. (D) ZBTB7A protein expression in human breast cancer cell lines (MCF-7, T47D, MDA-MB-231, MDA-MB-468). Figure 2 View largeDownload slide ZBTB7A and ERα expressions are correlated in breast cancer samples. (A) Heatmap showing top genes differentially expressed in breast cancer samples with high and low expression of ZBTB7A. (B) Gene sets that are correlated with increased ZBTB7A expression in TCGA cohort. (C) ZBTB7A expression in ER-positive and ER-negative breast cancer cell lines. (D) ZBTB7A protein expression in human breast cancer cell lines (MCF-7, T47D, MDA-MB-231, MDA-MB-468). Next, we tested whether ZBTB7A–ESR1 correlation observed in human tumor samples was also present in breast cancer cell lines. We utilized expression levels deposited in Cancer Cell Line Encyclopedia (CCLE) and confirmed higher expression of ZBTB7A in ER-positive (ER+) cell lines (Figure 2C) and positive correlation between ESR1 and ZBTB7A expression (Supplementary Figure S1C). We next analyzed the protein levels of ZBTB7A in ER-positive (MCF-7, T47D) and ER-negative (MDA-MB-231, MDA-MB-468) breast cancer cell lines and found that ZBTB7A protein levels were higher in ERα-positive breast cancer cell lines (Figure 2D). Hence, within human breast cancer tumor specimens and breast cancer cell lines, expression of ZBTB7A correlated with ERα levels, suggesting they may be regulating each other’s expression. ZBTB7A drives expression of ERα in luminal breast cancers To explore the functional role of ZBTB7A in hormone-receptor positive breast cancer, we used MCF-7 and T47D cells as model systems. Upon silencing of ZBTB7A using two different oligonucleotide constructs, we observed a significant reduction in ESR1 mRNA levels (Figure 3A). ERα protein levels were also reduced following ZBTB7A silencing in MCF-7 cells, under both basal and 17β-estradiol-stimulated conditions (Figure 3B). To further explore the mechanism through which ZBTB7A regulates ERα expression, we used a luciferase reporter driven by the promoter of ERα, containing 3500 base pairs upstream from the promoter start site (deGraffenried et al., 2004). ZBTB7A binds in GC-rich DNA sequences (Maeda et al., 2005). Promoter motif analyses were used to recognize four potential ZBTB7A-binding sites in the ERα promoter sequence, located at −1381, −2079, −2729, and −3253 base pairs from the transcription start site. Mutations of these putative ZBTB7A-binding sites were made to the ERα promoter luciferase construct and transfected into HEK293T cells with Renilla luciferase as a control. Cells were lysed and luciferase activity assessed 24 h later. Mutations of these putative ZBTB7A-binding sites resulted in significant reduction of basal luciferase activity (Figure 3C), suggesting that the binding of ZBTB7A to these sites is important for ERα transcriptional activity. Figure 3 View largeDownload slide ZBTB7A controls ERα expression in luminal breast cancers. (A) ESR1 mRNA level decreased in MCF-7 and T47D cell lines 48 h after transfection with ZBTB7A siRNA. The data represent three independent experiments and are presented as mean ± SEM. (B) ERα protein level in MCF-7 cells decreased 48 h following ZBTB7A siRNA treatment. The western blot is representative of three independent experiments. The quantification was done using Image J and represents mean ± SEM of three independent experiments. (C) Diminished ERα-promoter luciferase activity following mutation in putative ZBTB7A-binding sites. The putative ZBTB7A-binding sites within the ERα promoter are shown. HEK293T cells were transfected with the wild-type ER-promoter luciferase or mutant constructs and Renilla luciferase, and then collected 24 h later. Data from three independent experiments are represented as mean ± SEM. (D) ZBTB7A binds to the ERα promoter in MCF-7 cells. ChIP assays were performed in MCF-7 cells with anti-ZBTB7A antibody or control IgG. The abundance of DNA within the ERα promoter was assessed using qPCR. Data from three independent experiments are shown as mean ± SEM. Figure 3 View largeDownload slide ZBTB7A controls ERα expression in luminal breast cancers. (A) ESR1 mRNA level decreased in MCF-7 and T47D cell lines 48 h after transfection with ZBTB7A siRNA. The data represent three independent experiments and are presented as mean ± SEM. (B) ERα protein level in MCF-7 cells decreased 48 h following ZBTB7A siRNA treatment. The western blot is representative of three independent experiments. The quantification was done using Image J and represents mean ± SEM of three independent experiments. (C) Diminished ERα-promoter luciferase activity following mutation in putative ZBTB7A-binding sites. The putative ZBTB7A-binding sites within the ERα promoter are shown. HEK293T cells were transfected with the wild-type ER-promoter luciferase or mutant constructs and Renilla luciferase, and then collected 24 h later. Data from three independent experiments are represented as mean ± SEM. (D) ZBTB7A binds to the ERα promoter in MCF-7 cells. ChIP assays were performed in MCF-7 cells with anti-ZBTB7A antibody or control IgG. The abundance of DNA within the ERα promoter was assessed using qPCR. Data from three independent experiments are shown as mean ± SEM. Our data suggest that ZBTB7A modulates ERα expression at the transcriptional level. To further elucidate how ZBTB7A regulates ERα, we used chromatin-immunoprecipitation (ChIP) to analyze the recruitment of ZBTB7A to the ESR1 promoter in ER-positive breast cancer cells. The interaction between ZBTB7A and the ESR1 promoter was confirmed by ChIP assay. The results indicated that ERα promoter fragments were precipitated with ZBTB7A antibody at the −1381 base pair site but not with control IgG, supporting a direct binding of ZBTB7A to the promoter of ERα (Figure 3D). Together, these data suggest that ZBTB7A upregulates ERα expression through direct binding to GC boxes in its promoter. Next, we explored whether ZBTB7A regulation of ERα affects downstream activity of ERα target genes. Inhibition of ZBTB7A using siRNA abrogated E2-induced estrogen-response element (ERE) luciferase reporter activity in MCF-7 cells (Figure 4A). As a member of the POK family of transcription factors, ZBTB7A mediates its effects on transcription by binding to DNA through its zinc finger domain. To assess what region of the ZBTB7A protein was necessary for ERE-luciferase activity, ZBTB7A mutants were generated with point mutations in either the BTB or zinc finger domain (Liu et al., 2016). The K424N and K424T mutations in the second zinc finger domain resulted in significant reduction in ERE-luciferase activity, consistent with the requirement of the zinc finger domain-mediated ZBTB7A DNA binding necessary for induction of ERα expression and its transcriptional activity (Figure 4B). To assess the effects of ZBTB7A inhibition on the expression of ERα target genes, we treated MCF-7 cells with ZBTB7A siRNA in the presence and absence of 17β-estradiol. Indeed, 17β-estradiol-mediated induction of ERα target genes was reduced by silencing ZBTB7A including progesterone receptor (PGR), trefoil factor 1(TFF1), GATA binding protein 3 (GATA3), and C-MYC mRNA (Figure 4C). These data indicate that ZBTB7A is required for ERα expression and subsequent activation of ERα target genes. Figure 4 View largeDownload slide Abrogation of ERα function following ZBTB7A inhibition. (A) Decreased ERα function as evidenced by decreased ERE-luciferase activity following ZBTB7A siRNA treatment. MCF-7 cells were stripped of hormone for 3 days, transfected with siRNA, treated with E2 (10 nM) at 24 h post-transfection, and collected at 36–48 h. Data from three independent experiments are shown as mean ± SEM. (B) ERE-luciferase activity was diminished following transfection with 424 ZBTB7A mutant plasmids. Cells were transfected and collected 24 h later. Data from three independent experiments are shown as mean ± SEM. (C) Diminished E2-induced induction of ER target genes following inhibition of ZBTB7A with siRNA. Cells were stripped of hormone for 3 days, transfected with siRNA, treated with E2 (10 nM) at 24 h post-transfection, and collected at 36–48 h. Data from three independent experiments are shown as mean ± SEM. (D) E2-induced growth is abrogated in MCF-7 cells following ZBTB7A siRNA treatment. Cells were stripped of hormone for 3 days, transfected with siRNA, and plated for growth assay 24 h later. Data are representative of three independent experiments done in triplicate. Figure 4 View largeDownload slide Abrogation of ERα function following ZBTB7A inhibition. (A) Decreased ERα function as evidenced by decreased ERE-luciferase activity following ZBTB7A siRNA treatment. MCF-7 cells were stripped of hormone for 3 days, transfected with siRNA, treated with E2 (10 nM) at 24 h post-transfection, and collected at 36–48 h. Data from three independent experiments are shown as mean ± SEM. (B) ERE-luciferase activity was diminished following transfection with 424 ZBTB7A mutant plasmids. Cells were transfected and collected 24 h later. Data from three independent experiments are shown as mean ± SEM. (C) Diminished E2-induced induction of ER target genes following inhibition of ZBTB7A with siRNA. Cells were stripped of hormone for 3 days, transfected with siRNA, treated with E2 (10 nM) at 24 h post-transfection, and collected at 36–48 h. Data from three independent experiments are shown as mean ± SEM. (D) E2-induced growth is abrogated in MCF-7 cells following ZBTB7A siRNA treatment. Cells were stripped of hormone for 3 days, transfected with siRNA, and plated for growth assay 24 h later. Data are representative of three independent experiments done in triplicate. ERα mediates growth and proliferation through activation of its downstream target genes; as such, we next sought to determine whether ZBTB7A inhibition would affect MCF-7 cell proliferation. As shown in Figure 4D, siRNA-mediated downregulation of ZBTB7A led to a reduction in 17β-estradiol-stimulated proliferation in MCF-7 cells. Together, these data suggest that the pro-proliferative signal of 17β-estradiol is blunted following the inhibition of ZBTB7A through its effects on ERα expression. ERα regulates ZBTB7A post-translational stability In human breast cancer, ZBTB7A and ERα are highly correlated (Figure 2A). ERα has been shown to participate in positive cross-regulatory loops with other transcription factors such as Gata3, in which ERα and Gata3 reciprocally regulate each other’s transcription (Eeckhoute et al., 2007). To investigate the hypothesis that ERα may be regulating the expression of ZBTB7A in breast cancer, we inhibited ERα in MCF-7 cells using siRNA. The results showed that reduction of ERα led to a decrease of ZBTB7A protein levels (Figure 5A and Supplementary Figure S3), but had little effect on ZBTB7A mRNA levels 48 h post siRNA treatment (Figure 5B and Supplementary Figure S3). We then asked whether ZBTB7A downregulation due to ERα siRNA was occurring via protein degradation by the proteasome. Treatment with the proteasome inhibitor MG132 prevented the downregulation of ZBTB7A protein levels in the presence of ERα siRNA, indicating that protein degradation, at least partially, contributed to the observed alterations in ZBTB7A levels (Figure 5C). Next, to assess whether ZBTB7A half-life is altered following ERα siRNA treatment, the protein level of ZBTB7A was analyzed in the presence of cycloheximide. The addition of ERα siRNA significantly decreases ZBTB7A half-life at 6 h and 8 h following cycloheximide treatment (Figure 5D and E). Together, these data suggest that ERα plays a role in ZBTB7A protein stability. Figure 5 View largeDownload slide ERα regulates ZBTB7A protein level and stability in breast cancer. (A and B) ERα siRNA causes decreased ZBTB7A protein level. MCF-7 cells were transfected with ERα siRNA and 48 h later were subjected to western blot (A) and qPCR (B) analyses. ERα siRNA treatment does not lead to reduced ZBTB7A mRNA levels. Graphs show mean ± SEM of three independent experiments. (C) Western blot demonstrating transfection of ERα siRNA leads to downregulation of ZBTB7A protein that is rescued by MG132 treatment. MCF-7 cells were transfected with siRNA, 48 h later treated with MG132 for 6 h, and then collected for western blot analysis. The blot is representative of three independent experiments. Quantification was done using Image J and includes three independent experiments, mean ± SEM. (D) Western blot of MCF-7 cells treated with control siRNA or ERα siRNA for 48 h followed by cycloheximide (100 μg/ml) treatment at the indicated time points. (E) Quantification of D including two independent experiments, mean ± SEM using Image J. Figure 5 View largeDownload slide ERα regulates ZBTB7A protein level and stability in breast cancer. (A and B) ERα siRNA causes decreased ZBTB7A protein level. MCF-7 cells were transfected with ERα siRNA and 48 h later were subjected to western blot (A) and qPCR (B) analyses. ERα siRNA treatment does not lead to reduced ZBTB7A mRNA levels. Graphs show mean ± SEM of three independent experiments. (C) Western blot demonstrating transfection of ERα siRNA leads to downregulation of ZBTB7A protein that is rescued by MG132 treatment. MCF-7 cells were transfected with siRNA, 48 h later treated with MG132 for 6 h, and then collected for western blot analysis. The blot is representative of three independent experiments. Quantification was done using Image J and includes three independent experiments, mean ± SEM. (D) Western blot of MCF-7 cells treated with control siRNA or ERα siRNA for 48 h followed by cycloheximide (100 μg/ml) treatment at the indicated time points. (E) Quantification of D including two independent experiments, mean ± SEM using Image J. The role of ZBTB7A in the normal mammary gland ERα is essential for normal mammary gland development. Mice lacking ERα exhibit deficiencies in ductal morphogenesis of the mammary gland, including an inability to form terminal end buds (TEBs) and defects in ductal invasion (Mallepell et al., 2006; Feng et al., 2007). Additionally, ZBTB7A has been shown to play an important role in the differentiation processes of various cell types (Laudes et al., 2008; Maeda et al., 2009). Since we have observed that ZBTB7A controls ERα expression in luminal breast cancer cells, we next wanted to investigate its function in the normal mammary gland. First, we set out to determine whether there is a difference in ZBTB7A expression levels in luminal vs. basal cells in the mouse mammary gland. In accordance with human tumor data (Figure 2B), we found that the luminal cell population displayed increased ZBTB7A mRNA expression compared to basal cells isolated from the mouse mammary gland (Figure 6A). Next, we used a loss of function approach to study the role of ZBTB7A in the mammary gland. Zbtb7a-null mice display embryonic lethality due to anemia caused by apoptosis of late stage erythroblasts (Maeda et al., 2009). We therefore used a CRE/LOXP recombination system to conditionally knockout Zbtb7a in the mammary gland. Mice with a floxed Zbtb7a gene (Maeda et al., 2007) were crossed with constitutively active MMTV-CRE mice, which express CRE in the mammary gland, skin, and salivary glands (Wagner et al., 1997). This conditional deletion approach rescued embryonic lethality observed in Zbtb7a-null mice, as homozygous Zbtb7a-floxed mice carrying the MMTV-CRE transgene were born in normal Mendelian ratios (data not shown). The genotypes of mice harboring the Zbtb7a-floxed alleles were determined by PCR using genomic DNA isolated from ear clips of the mice (Figure 6B). Mammary glands were isolated from 6-week-old female mice to determine Zbtb7a expression level in wild-type vs. Zbtb7aflox/flox mammary glands. ZBTB7A expression was significantly lower in mice harboring the Zbtb7a-floxed allele and the MMTV-CRE allele compared to wild-type littermates (Figure 6C). However, in contrast to the breast cancer cell lines, Esr1 mRNA was not impacted by decreased expression of Zbtb7a in the normal mammary gland. To examine ductal morphogenesis, mammary glands from wild-type and Zbtb7aflox/flox mice were whole-mounted at 4 and 6 weeks of age. Zbtb7aflox/flox mammary glands displayed normal pubertal and post-pubertal ductal morphogenesis (Figure 6D). The histoarchitecture of the TEBs and ducts from wild-type and Zbtb7aflox/flox mice were indistinguishable by hematoxylin and eosin staining. In addition, pups from Zbtb7aflox/flox female mice were born in numbers and of weights comparable to wild-type mice. Together these data suggest that ZBTB7A is not responsible for normal mammary gland growth and development, but is implicated in the growth of cancerous breast epithelial cells. Figure 6 View largeDownload slide ZBTB7A expression in the normal mammary gland. (A) ZBTB7A expression in luminal and basal mammary cell populations. Mammary epithelial cells were extracted from 8- to 10-week-old female mice and subjected to FACS analysis. (B) Representative PCR genotyping resulting from wild-type, ZBTB7Aflox/flox, and ZBTB7AWT/flox mice. (C) ZBTB7A and ESR1 mRNA levels from 8- to 10-week-old wild-type and ZBTB7Aflox/flox mice (n = 4–6). (D) Representative whole-mount staining from 4- and 6-week-old wild-type and ZBTB7Aflox/flox mice (n = 8–10). Scale bar, 10 mm. (E) Ductal length from 4- and 6-week-old wild-type and ZBTB7Aflox/flox mice (n = 4). Graph shows mean ± SEM. Figure 6 View largeDownload slide ZBTB7A expression in the normal mammary gland. (A) ZBTB7A expression in luminal and basal mammary cell populations. Mammary epithelial cells were extracted from 8- to 10-week-old female mice and subjected to FACS analysis. (B) Representative PCR genotyping resulting from wild-type, ZBTB7Aflox/flox, and ZBTB7AWT/flox mice. (C) ZBTB7A and ESR1 mRNA levels from 8- to 10-week-old wild-type and ZBTB7Aflox/flox mice (n = 4–6). (D) Representative whole-mount staining from 4- and 6-week-old wild-type and ZBTB7Aflox/flox mice (n = 8–10). Scale bar, 10 mm. (E) Ductal length from 4- and 6-week-old wild-type and ZBTB7Aflox/flox mice (n = 4). Graph shows mean ± SEM. Discussion ERα is one of the most critical biomarkers dictating treatment decisions and prognosis for breast cancer patients. Extensive research on the downstream functions of ERα has led to targeted treatment strategies such as endocrine therapies. However, the mechanism through which ERα itself is regulated and maintained at high levels in breast cancers remains incompletely understood. Here we uncover a link between ZBTB7A and ERα that may in part explain how ERα levels are maintained in breast tumors (Supplementary Figure S4). Recent studies from our lab and others have suggested a context specific role for ZBTB7A in cancer. Few studies, however, have looked at the role of ZBTB7A in breast cancer. Zu et al. (2011) reported that ZBTB7A was expressed at elevated levels in breast tumors compared to the normal breast tissue. Due to the heterogeneity of breast cancer, we sought to assess ZBTB7A expression within the various breast cancer subtypes. In our current study, we find that ZBTB7A is more highly expressed in the luminal vs. basal breast cancer subtypes (Figure 1E). In agreement with this observation, we find that ZBTB7A is more highly expressed in ER-positive compared to ER-negative human breast cancers (Figure 1F). Differential gene expression analysis of human tumor samples expressing high vs. low levels of ZBTB7A also suggested that there is a strong relationship between ZBTB7A and genes including DEK, ESR1 and KDM4B. DEK expression has been associated with β-catenin activity and a more invasive phenotype in breast cancer (Privette Vinnedge et al., 2011). Although the relationship between DEK and ZBTB7A is unknown, our analysis found a negative correlation between DEK and ZBTB7A supporting ZBTB7A’s association with less aggressive breast cancers. KDM4B has been associated with ERα signaling cascade in breast cancer (Gaughan et al., 2013) and was positively correlated with ZBTB7A. Because ERα signaling is important in breast cancer, we focused on the strong positive correlation observed between ESR1 and ZBTB7A. ZBTB7A’s association with less aggressive, more differentiated breast cancer subtypes led us to further examine its overall functional role in breast cancer, specifically in ERα-positive disease. Our results in human breast cancer cell lines argue a role for ZBTB7A in maintaining ERα expression in breast cancer. We find that ZBTB7A expression is higher in ERα-positive breast cancer cell lines and that knockdown of ZBTB7A leads to diminished ERα expression at the transcript and protein level. This decreased ERα expression imparted by ZBTB7A knockdown resulted in reduced ERα target gene expression following estrogen treatment. Functionally, depletion of ZBTB7A reduced E2-induced growth of ER-positive breast cancer cells. Mechanistically, we find that ZBTB7A controls ERα transcriptional activity by directly binding to the ERα promoter. Transcription of ERα can be initiated from multiple promoters upstream of the ESR1 gene locus (Grandien et al., 1997). Normal mammary epithelial cells utilize the more distal promoters while breast cancer cells typically activate the more proximal promoters (Grandien et al., 1995). ChIP analyses suggest that ZBTB7A is recruited to the proximal promoter of ESR1 (Figure 3D). Further, mutations in the predicted ZBTB7A-binding sites in the ERα proximal promoter led to decreased activity of ESR1 reporter constructs (Figure 3C). Together these data provide evidence that ZBTB7A initiates ESR1 transcription via direct binding to the ERα promoter. Our data also provide evidence of a positive feedback loop between ERα and ZBTB7A in breast cancer. Knockdown of ESR1 lowered ZBTB7A protein expression with no significant effect on ZBTB7A mRNA levels. Inhibition of the ubiquitin–proteasome pathway by MG132 rescued ZBTB7A downregulation following ESR1 knockdown, suggesting that ERα can block the degradation of ZBTB7A by the proteasome. Overall, this feedback loop provides further evidence indicating that ZBTB7A and ERα are tightly connected in breast cancer and may be responsible for the maintenance of ER expression levels resulting in tumor growth. Due to the observed relationship between ZBTB7A and ERα in breast cancer, we next sought to assess the role of ZBTB7A in the normal mammary gland. Both genomic and mammary gland-specific ERα knockout mouse models give rise to developmental defects of the mammary gland, specifically arrested ductal growth in the pre-pubertal stage of development (Mallepell et al., 2006; Feng et al., 2007). To assess whether ZBTB7A governs ERα expression in the normal mammary gland, we utilized a mouse model in which ZBTB7A was depleted specifically in the mammary gland. Homozygote Zbtb7aflox/flox mice did not display any discernable phenotype and loss of ZBTB7A did not affect the expression of ERα in the mammary gland. Although it has been reported that different mouse strains exhibit variations in ductal morphology (Montero Girard et al., 2007; Aupperlee et al., 2009), it is unlikely that the lack of phenotype observed in ZBTB7A-null mammary glands is due to the background of the mice. ZBTB7A has been shown to play a role in various aspects of development including regulating the determination of B vs. T lymphoid fate (Maeda et al., 2007), as well as in the early stages of preadipocyte differentiation (Laudes et al., 2004). The lack of phenotype observed in the Zbtb7a-null mammary gland may be due to functional redundancy of other ZBTB family members. It has been suggested that some ZBTB proteins may play functionally redundant roles following depletion of other ZBTB family members (Kelly and Daniel, 2006). It is possible that an unidentified ZBTB member was able to compensate for the loss of ZBTB7A in the mammary epithelial cells thus preventing any disruption in ERα expression and signaling. Other ZBTB members such as ZBTB4 have been reported to play roles in breast cancer (Kim et al., 2012), however none have yet been reported to alter ERα expression in breast cancer or the normal mammary gland. Alternatively, the observed results may suggest that the link between ERα and ZBTB7A may not play a role in the transcriptional program that governs mammary epithelial cell differentiation and is only relevant in breast cancer cells. Importantly, our study reveals the potential clinical significance of ZBTB7A for breast cancer patients. We demonstrate that ZBTB7A levels in human breast tumors correlated with improved overall and RFS (Figure 1B and C) for breast cancer patients. Although these results are contrary to previously published studies (Qu et al., 2010; Zu et al., 2011), our analysis utilized a larger sample size, which likely accounts for these discrepancies. In addition, the methods used to assess the level of ZBTB7A were different. Our study used RNA expression from microarray data to determine ZBTB7A level, while the previous studies used immunohistochemical analysis to score ZBTB7A protein intensity, potentially accounting for the differences in observed outcomes. We also demonstrate that ZBTB7A levels correlated with improved outcomes for patients harboring luminal A and B type tumors (Supplementary Figure S2). Clinically, we propose that high ZBTB7A expression may result in more favorable outcomes for patients treated with endocrine therapies as the major determinant of response to these therapies is ERα expression (Pinzone et al., 2004; Thomas and Gustafsson, 2011). In support of this notion, we also present evidence that patients with high ZBTB7A have improved response to endocrine therapies. Overall, the current study demonstrates a novel relationship between ZBTB7A and ERα, suggesting that ZBTB7A may be used to predict breast cancer patient outcomes and have important clinical implications for the treatment of ERα-positive breast tumors in the future. Methods Cell culture MCF-7, MDA-MB-231, MDA-MB-468, and Hek293T cells were cultured in DMEM (Corning, Cellgro) plus 10% fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin G, and 100 μg/ml streptomycin (Corning, Cellgro). T47D cells were cultured in RPMI (Corning, Cellgro) plus 10% FBS, 100 U/ml penicillin G, and 100 μg/ml streptomycin. All cells were cultured in a 37°C, 5% CO2 incubator. Antibodies and reagents Anti-ZBTB7A antibody (Santa Cruz Biotechnology, 13E9), anti-ERα antibody (Santa Cruz Biotechnology, HC-20), anti-alpha tubulin antibody (Cell Signaling Technologies, 2144), and anti-GAPDH antibody (Cell Signaling Technologies, 2118) were used for western blot; anti-ZBTB7A antibody (Bethyl Laboratories, A300-549A) and normal Rabbit IgG (Cell Signaling Technologies, 2729) were used for ChIP. Immunoblot Cells were lysed in buffer (50 mM Tris, at pH 8.0, 150 mM NaCl, 0.5% NP-40). Protein concentrations of the lysates were measured by Bradford assay (Bio-Rad). The lysates were then resolved by SDS-PAGE and immunoblotted with the indicated antibodies. qPCR to quantify gene expression Total RNA was extracted using TRIZOL according to the manufacturer’s instructions (Ambion). RNA was reversed transcribed using iScript cDNA synthesis kit (Bio-Rad). qPCR was performed using diluted cDNA, appropriate primers, and PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) in StepOnePlus real-time PCR system (Applied Biosystems). The following primers were used: ESR1_F-TCCTCATCCTCTCCCACATC, ESR1_R-TCCAGCAGCAGGTCATAGAG, ZBTB7A_F-GCTTGGGCCGGTTGAATGTA, ZBTB7A _R-GGCTGTGAAGTTACCGTCGG, 18S_F-GTAACCCGTTGAACCCCATT, 18S_R-CCATCCAATCGGTAGTAGCG, GATA3_F-TACAGCTCCGGACTCTTCCC, GATA3_R-CCCACAGTTCACACACTCCC, TFF1_F-GCCACCATGGAGAACAAGGT, TFF1_R-CAATTCTGTCTTTCACGGGG, MYC_F-GCCACGTCTCCACACATCAG, MYC_R-TCTTGGCAGCAGGATAGTCCT, PGR_F-TGGTGTCCTTACCTGTGGGA, PGR_R-ACGATGCAGTCATTTCTTCCA. siRNA-based knockdown For siRNA transfections, 3 × 105 cells were reverse transfected with siRNA and INTERFERin (Polyplus Transfection) and collected 48 h following transfection. siRNAs for ZBTB7A were manufactured by Sigma (siRNA ID for ZBTB7A#1 SASI_Hs01_00244764, ZBTB7A#2 SASI_Hs01_00244765). ESR1 siRNA1 was manufactured by Sigma (SASI_Hs01_00078592) and ESR1 siRNA2 was manufactured by Thermo Fisher Scientific (Silencer Select siRNA s4824). Mutagenesis Mutagenesis was performed using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer’s instructions. The following primers were used for site-directed mutagenesis: MUT1_F-GTCCAGCAGGTTTTTGTAGGGAGGGAAGC, MUT1_R-ACACACGTTCCAGCCGTG, MUT2_F-CTGCAATACTATATATCTTTCAAAGCACAAAAACATATATTTG, MUT2_R-TGTTTGAGCACAGGAGAG, MUT3_F-TTCAGAGACTATATACTAGGGCCAGTAAGG, MUT3_R-CAGAGGCATTGCCATGCC, MUT4_F-TGTTCTCCATATATATATGTGTGTGTCTCCTTTTTC, MUT4_R-GCAATCCTCATCTCCCTG. The resulting mutations were then confirmed by sequencing. Chromatin-immunoprecipitation assays ChIP was performed according to the manufacturer’s instructions (Cell Signaling Technology, 9003S). Briefly, the chromatin/DNA protein complexes were prepared from MCF-7 cells plated 48 h before collection. Crosslinking of DNA to proteins was carried out using 1% formaldehyde for 10 min at room temperature. The crosslinking was quenched by the addition of glycine (0.125 M) for 5 min at room temperature and followed by two washes with ice-cold PBS. Cells were scraped into PBS containing protease inhibitor cocktail (PIC). Cells were pelleted and mixed by inverting the tube every 3 min in buffer A, DTT, and PIC, followed by incubation on ice for 10 min. The nuclei were dissolved in buffer B, DTT, and micrococcal nuclease and incubated for 20 min at 37°C with frequent mixing to digest DNA. The lysate was then sonicated using the SONIFIER 450. The lysate was incubated with ZBTB7A/FBI-1 antibody (Bethyl Laboratories, A300-549A) or normal rabbit IgG (Cell Signaling Technologies, 2729), followed by incubation with ChIP-grade protein G magnetic beads. DNA was purified and subjected to qPCR analysis. Mouse strains Generation of mice containing the floxed Zbtb7a allele was previously described (Maeda et al., 2007). Cre-recombinase was expressed under the control of the mouse mammary tumor virus (MMTV) promoter (JAX stock #003553) (Wagner et al., 1997) in the FVB background. Genotyping was carried out by PCR. All animal experiments were conducted in accordance with the IACUC approved protocols following Harvard guidelines. Evaluation of cell populations in mouse mammary gland Mammary glands were collected from female mice and prepared into single-cell suspensions as previously described (Shackleton et al., 2006). FACS analysis and cell sorting of mammary stem cell-enriched/basal (CD29hiCD24+) and luminal (CD29loCD24+) cell populations were performed as previously described (Fu et al., 2015). Mammary gland whole-mount preparation Tissues were fixed and stained as previously described (Plante et al., 2011). Briefly, glands were fixed to slides using Carnoy’s fixative (100% EtOH, chloroform, glacial acetic acid; 6:3:1) overnight at 4°C, washed in 70% EtOH, rehydrated, stained in carmine alum overnight and cleared in xylene. Survival analysis For OS analysis in TCGA cohort, samples were stratified by median expression by RNA-Seq log-rank analysis was performed. For survival analysis based on microarray derived gene expression data, we utilized online KM plotter (Gyorffy et al., 2010). To test the RFS, we used 213303_x_at probe that allowed analysis of the highest number of patients and we stratified patients based on ‘best cut-off’ (Mihaly et al., 2013). Statistical analysis If not indicated, statistical analyses were completed using GraphPad Prism 7 software. The differential gene expression on RNA-seq data was performed on expression data obtained from GiTools 2.2. Samples were stratified into ‘low ZBTB7A’ (<−0.5) and ‘high ZBTB7A’ (>0.5) expression. Differential expression analysis was performed using t-test, followed by false discovery rate (FDR) correction. Transcripts with q (FDR-corrected P-value) < 0.001 and fold change log2(high/low) >2 were considered differentially expressed genes. Heatmap was generated with GiTools 2.2. The list of differentially expressed transcripts is included in Supplementary Table S1. GSEA (Broad Institute) (Mootha et al., 2003; Subramanian et al., 2005) was performed across all tumors downloaded from Board Institute for TCGA BRCA Collection. The ZBTB7A expression level was used as a continuous phenotype and Pearson correlation was used as metrics for ranking the genes (Hallmark gene sets h.all.v6.0.symbols.gmt). The expression levels for surrounding tissue and primary breast tumors were retrieved together with clinical data from Board Institute for TCGA BRCA Collection (gdac.broadinstitute.org_BRCA.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800). Expression levels are reported as RNA-Seq by Expectation-Maximization (Li and Dewey, 2011). Expression levels for ZBTB7A and ESR1 in cell lines were retrieved via cBioPortal (Cerami et al., 2012) from Cancer Cell Line Encyclopedia (Barretina et al., 2012) and estrogen receptor status from the study by Jiang et al. (2016). Only cell lines with established estrogen receptor status were used for analysis (Supplementary Table S2). Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. 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Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Published: Mar 21, 2018

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