ERα promotes transcription of tumor suppressor gene ApoA-I by establishing H3K27ac-enriched chromatin microenvironment in breast cancer cells

ERα promotes transcription of tumor suppressor gene ApoA-I by establishing H3K27ac-enriched chromatin microenvironment in breast cancer cells ERα通过在乳腺癌细胞中建立富含H3K27ac修饰的染色质微环境促进抑癌基因ApoA-I的转录 1 first-author 1 2 1 corresp Li TAN, litan@fudan.edu.cn Li TAN, litan@fudan.edu.cn litan@fudan.edu.cn https://orcid.org/0000-0001-5961-0450 Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, and Shanghai Key Laboratory of Medical Epigenetics, Institutes of Biomedical Sciences, Fudan University, Shanghai200032, China Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, and Shanghai Key Laboratory of Medical Epigenetics, Institutes of Biomedical Sciences, Fudan University, Shanghai200032, China Colorectal Cancer Center, Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai200032, China Colorectal Cancer Center, Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai200032, China Apolipoprotein A-I (ApoA-I), the main protein component of high-density lipoprotein (HDL), plays a pivotal role in reverse cholesterol transport (RCT). Previous studies indicated a reduction of serum ApoA-I levels in various types of cancer, suggesting ApoA-I as a potential cancer biomarker. Herein, ectopically overexpressed ApoA-I in MDA-MB-231 breast cancer cells was observed to have antitumor effects, inhibiting cell proliferation and migration. Subsequent studies on the mechanism of expression regulation revealed that estradiol (E2)/estrogen receptor α (ERα) signaling activates ApoA-I gene transcription in breast cancer cells. Mechanistically, our ChIP-seq data showed that ERα directly binds to the estrogen response element (ERE) site within the ApoA-I gene and establishes an acetylation of histone 3 lysine 27 (H3K27ac)‍-enriched chromatin microenvironment. Conversely, Fulvestrant (ICI 182780) treatment blocked ERα binding to ERE within the ApoA-I gene and downregulated the H3K27ac level on the ApoA-I gene. Treatment with p300 inhibitor also significantly decreased the ApoA-I messenger RNA (mRNA) level in MCF7 cells. Furthermore, the analysis of data from The Cancer Genome Atlas (TCGA) revealed a positive correlation between ERα and ApoA-I expression in breast cancer tissues. Taken together, our study not only revealed the antitumor potential of ApoA-I at the cellular level, but also found that ERα promotes the transcription of ApoA-I gene through direct genomic effects, and p300 may act as a co-activator of ERα in this process. 目的 2 载脂蛋白A-I（apolipoproteinA-I，ApoA-I）是高密度脂蛋白的主要蛋白成分。已有研究表明ApoA-I除了参与胆固醇逆转运，还能够抑制炎症反应以及抑制肿瘤的生长、侵袭和代谢活动。另外，多种癌症病人的血清中ApoA-I的水平相较健康人是降低的。ApoA-I作为潜在的抗肿瘤因子具有重要的研究价值。本研究旨在探究ApoA-I对乳腺癌细胞生物学行为的影响以及对乳腺癌发生发展起重要作用的雌激素/雌激素受体（E2/ERα）信号通路是否参与了ApoA-I的表达调控。 创新点 2 本研究在细胞层面上证明了ApoA-I具有抑癌功能，另外结合体外实验以及TCGA数据分析，首次发现乳腺癌细胞中 ApoA-I 是ERα的直接靶基因，这为理解ERα在乳腺癌中的双刃剑作用提供了新的切入点。同时，我们猜测雌激素替代疗法联合ApoA-I模拟肽给药可能会增强luminal（ERα表达阳性）型乳腺癌的治疗效果。 方法 2 通过干预E2/ERα信号通路（用雌激素受体拮抗剂ICI182780降解ERα；激素剥夺后加入E2激活E2/ERα信号通路；在三阴性乳腺癌细胞MDA-MB-231中过表达ERα），观察 ApoA-I 转录水平的变化；通过用ERα抗体进行ChIP-seq结合序列分析探究ERα是否直接调控 ApoA-I 转录；在激素剥夺或ICI182780处理的条件下用组蛋白H3赖氨酸27位点乙酰化（H3K27ac）抗体进行ChIP-seq或ChIP-qPCR，结合乙酰转移酶p300抑制剂处理，探究ERα调控 ApoA-I 转录的具体机制。 结论 2 ERα能够直接结合在 ApoA-I 基因的最后一个外显子上促进 ApoA-I 的转录，且该结合位点上有一个雌激素响应元件（ERE）；p300可能作为ERα的一个共激活因子，通过建立富含H3K27ac的染色质微环境来激活 ApoA-I 的转录。 Apolipoprotein A-I (ApoA-I) Estrogen receptor α (ERα) Acetylation of histone 3 lysine 27 (H3K27ac) p300 Breast cancer 载脂蛋白A-I（ApoA-I） ERα 组蛋白H3赖氨酸27位点乙酰化（H3K27ac） 乙酰转移酶p300 乳腺癌 1 Introduction 1 According to the 2020 World Health Organization (WHO) report, the number of new breast cancer cases surpassed that of lung cancer for the first time, which means that breast cancer had become the world's most common cancer. Based on molecular characteristics, breast cancers are divided into hormone receptor (estrogen receptor (ER)/progesterone receptor (PR))‍-positive/Erb-B2 receptor tyrosine kinase 2 (ERBB2)‍-negative (70% of patients), ERBB2-positive (15%–20%), and triple-negative subtypes (15%) ( Waks and Winer, 2019 Waks and Winer, 2019 ). Estrogen signaling modulates a variety of physiological processes through the direct or indirect regulation of target gene transcription ( Vrtačnik et al., 2014 Vrtačnik et al., 2014 ). The abnormal overactivation of ERs may lead to the development of breast cancer, and the specific underlying mechanisms include: (1) amplification of ER coactivators ( Anzick et al., 1997 Anzick et al., 1997 ); (2) inhibition of ER corepressors ( Mussi et al., 2006 Mussi et al., 2006 ); (3) overexpressed bridge proteins promoting the recruitment of coactivators by ERs ( Zwijsen et al., 1998 Zwijsen et al., 1998 ; McMahon et al., 1999 McMahon et al., 1999 ); (4) the mutations of ER enabling it to be activated at a lower concentration of estrogen ( Herynk and Fuqua, 2004 Herynk and Fuqua, 2004 ); and (5) ER localization on the plasma membrane and the subsequent signaling cascade activation ( Levin and Pietras, 2008 Levin and Pietras, 2008 ). Breast cancer patients with ERα-positive tumors are treated with endocrine therapy; however, most of them will develop endocrine resistance ( Hanker et al., 2020 Hanker et al., 2020 ). This prompts us to conduct a deeper investigation about the complex role of ERα in breast cancer. It has been reported that ERα can inhibit programmed death-ligand 1 ( PD-L1 ) expression in breast cancer cells ( Liu et al., 2018 Liu et al., 2018 ), which suggests the double-edged sword role of ERα. Investigations of ERα regulation of cancer-related genes will contribute to the clinical treatment of breast cancer. Apolipoprotein A-I (ApoA-I) is the main protein component of high-density lipoprotein (HDL) ( Shao and Heinecke, 2018 Shao and Heinecke, 2018 ). In addition to its well-known function in reverse cholesterol transport (RCT) and protective effect against cardiovascular diseases, ApoA-I is involved in inflammatory and immune responses ( Gordon et al., 2011 Gordon et al., 2011 ). Many recent studies have discovered a reduction of serum ApoA-I in various types of cancer ( Georgila et al., 2019 Georgila et al., 2019 ). The decrease of serum ApoA-I level has been related to the progression and metastasis of breast cancer ( Gonçalves et al., 2006 Gonçalves et al., 2006 ). Besides, a positive correlation between the level of serum ApoA-I secreted in tumor tissue fluid and chemotherapy sensitivity was observed in breast cancer ( Cortesi et al., 2009 Cortesi et al., 2009 ; Zhang et al., 2016 Zhang et al., 2016 ). ApoA-I contains ten consecutive helical domains, which are essential for the biophysical properties of proteins that spontaneously dissolve lipids in an aqueous environment ( Li et al., 1988 Li et al., 1988 ). Based on the characteristics of these amphiphilic helical sequences, researchers have synthesized various peptides that do not share any sequence homology, but all mimic the function of ApoA-I ( Reddy et al., 2014 Reddy et al., 2014 ). The ApoA-I mimetic peptide D-4F has been proved effective in reducing proliferative response induced by oxidized low-density lipoprotein (oxLDL) in human breast adenocarcinoma cells ( Cedó et al., 2016 Cedó et al., 2016 ). Although numerous studies have shown that ApoA-I is a potential biomarker with antitumor effects, the regulatory mechanism of its expression in breast cancer remains unclear. Exploring the regulation of ApoA-I expression will help us to better manipulate its level and function, which may contribute to prevention and treatment strategies for breast cancer. Herein, we explored the biological effect of ApoA-I on breast cancer cells and uncovered the effect of estradiol (E2)/ERα signaling on ApoA-I transcription in breast cancer. 2 Materials and methods 1 2.1 Vector construction and cell transfection 2 The human breast cancer cell lines MCF7 and MDA-MB-231 were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The human breast cancer cell line BT549 was obtained from Dr. Sulin LIU (Fudan University Cancer Hospital, Shanghai, China). The 293T cell line was kindly provided by Dr. Degui CHEN (Shanghai Institution of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China). All cell lines used in this study were cultured in Dulbecco's modified Eagle's medium (DMEM) high-glucose medium (HyClone, Marlborough, MA, USA) supplemented with 10% (volume fraction) fetal bovine serum (FBS; BI, Israel) and 1% (volume fraction) penicillin/streptomycin (Gibco, Carlsbad, CA, USA). The cells were placed in a humidified incubator at 37 °C under 5% CO 2 atmosphere. The following compounds were used in the corresponding experiments: 500 nmol/L ICI 182780 treatment for 7 d (Fulvestrant, Sigma-Aldrich, Darmstadt, Germany); 1 nmol/L E2 treatment for 2 d (Sigma-Aldrich) after 5 d of hormone deprivation; 20 μmol/L histone acetyltransferase inhibitor II (Selleck, Houston, Texas, USA). 2.2 Cell culture and treatment procedure 2 The coding sequences (CDSs) of ApoA-I and ERα amplified from the complementary DNAs (cDNAs) of MCF7 cells were separately inserted into a pLenti6.2/v5/TEV vector (Clonetech, Waltham, MA, USA). The recombinant plasmids and packaging plasmids were transfected into 293T cells using Lipofectamine TM 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. After 48 h of transfection, the cell supernatant was collected and filtered. Virus-containing supernatant containing 0.1% (volume fraction) polybrene (Sigma-Aldrich) was added to 1.5×10 6 MDA-MB-231 cells seeded in a six-well plate. After 48 h of infection, cells were screened using 10 μg/mL Blasticidin S HCl (Gibco). 2.3 qRT-PCR analysis 2 Total RNA was extracted from breast cancer cells using the TRIzol ® reagent (Ambion, Life Technologies, Carlsbad, CA, USA) and quantified by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Total RNA was reverse-transcribed into cDNA using a Primescript TM RT Reagent Kit with genomic DNA (gDNA) eraser (TaKaRa, Kusatsu, Japan) according to the manufacturer's protocol. The cDNA was subjected to quantitative real-time polymerase chain reaction (qRT-PCR) amplification by using the SYBR Green Master Mix (Roche, Basel, Switzerland) and performing in triplicates on a Roche LightCycler 480 II system. Table 1 Table 1 shows the primer sequences used in this study. GAPDH : glyceraldehyde-3-phosphate dehydrogenase; ApoA-I : apolipoprotein A-I; GREB1 : growth regulation by estrogen in breast cancer 1; ERα : estrogen receptor α; F: forward; R: reverse. Target gene Primer sequence (5'→3') 2 GAPDH F: CTGACTTCAACAGCGACACC R: GTGGTCCAGGGGTCTTACTC 2 ApoA-I F: TGAGGCTCTCAAGGAGAACG R: CCTCACTGGGTGTTGAGCTT 2 GREB1 F: GACCAGCTTCTGATCACCCC R: TACCTAAAGCCGATGGTCGC 2 ERα F: CCTCCTCATCCTCTCCCACA R: ATGCGATGAAGTAGAGCCCG 2 ApoA-I _ChIP-qPCR F: GCCTTCAAACTGGGACACAT R: CATTTCTGGCAGCAAGATGA 2.4 Western blot 2 Cells were harvested and lysed using sodium dodecyl sulfate (SDS) lysis buffer containing protease inhibitors. Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membrane using a wet-transfer system (Bio-Rad, Hercules, CA, USA). After 1 h of blocking, the membranes were placed in bovine serum albumin (BSA) solution at 4 ℃ overnight with the following primary antibodies: anti-‍β‍-actin (Proteintech, Rosemont, IL, USA; 66009-1-Ig), anti-vinculin (Cell Signaling Technology, Boston, MA, USA; 13901), anti-ApoA-I (Abcam, Cambridge, UK; ab52945), anti-ERα (Cell Signaling Technology; 8644), and anti-H3K27ac (Active Motif, Shanghai, China; 39133). After washing with Tris-buffered saline (TBS)/Tween buffer, the appropriate secondary antibodies (L3012: horseradish peroxidase (HRP)‍-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody; L3032: HRP-conjugated goat anti-mouse IgG secondary antibody) were used for blocking at room temperature for 1 h. The expression levels of proteins were detected by the ChemiDoc Touch Imaging System (Bio-Rad). 2.5 CCK-8 and transwell assay 2 The cell counting kit-8 (CCK-8) experiments were carried out with 2000 cells in accordance with the kit instructions (Enhanced Cell Counting Kit-8; Beyotime, Shanghai, China). Specifically, 2000 cells were planted in each well of the 96-well plate. After 0, 1, 2, 3, and 4 d, the medium was removed, and 100 μL of medium containing 10 µL CCK-8 solution was added to react in the cell incubator for 2 h. The absorbance was subsequently measured at 450 nm. For the transwell assay, 5×10 4 cells were placed on the upper layer of a cell culture insert with a cell permeable membrane (Millicell Hanging Cell Culture Inserts, 8.0 μm polyethylene terephthalate (PET); Millipore, Massachusetts, USA), and DMEM high-glucose medium containing 20% (volume fraction) FBS (BI) and 1% (volume fraction) penicillin/streptomycin (Gibco) was placed below the membrane. Following an 8-h incubation period, the cells that had migrated through the membrane were stained and counted. 2.6 ChIP-seq and ChIP-qPCR 2 This experiment was performed as previously described ( Kong et al., 2016 Kong et al., 2016 ). In particular, the monolayer of cultured cells was fixed with 1% (volume fraction) formaldehyde for 8 min, which process was terminated by 2.5 mol/L glycine solution for 5 min. After washing with phosphate-buffered saline (PBS), cells were harvested and lysed using a cocktail containing high-salt and lysis buffer (50 mmol/L 4-(2-hydroxyerhyl)piperazine-1-erhanesulfonic acid (HEPES; pH 7.5), 500 mmol/L NaCl, 1 mmol/L ethylenediamine tetraacetic acid (EDTA), 0.001 g/mL Na-deoxycholate, 1% (volume fraction) Triton X-100, 0.001 g/mL SDS) (Roche). Next, the cell lysate was sonicated for 30 min (30 s on, 40 s off, 95% amplify) using a Bioruptor ® sonicator (Diagenode, Belgium). The 10% (volume fraction) supernatant was taken out as the control group. The 2 μL of ERα or H3K27ac antibody (Active Motif) was added to the remaining sonicated chromatin for rotating overnight at 4 ℃. Then, 15 μL protein A/G magnetic beads (Invitrogen) were added to bind to the antibody at 4 °C for 2 h. The beads were washed according to the following method: thrice with high-salt and lysis buffer, twice with low-salt buffer (10 mmol/L Tris-Cl (pH 8.0), 250 mmol/L LiCl, 1 mmol/L EDTA, 0.001g/mL Na-deoxycholate, and 0.5% (volume fraction) NP-40), and once with TE buffer (2 mmol/L EDTA, 10 mmol/L Tris-Cl (pH 8.0)). The "beads" and the control group were resuspended in 100 μL elution and de-crosslink buffer (50 mmol/L Tris-Cl (pH 8.0), 10 mmol/L EDTA, and 0.01g/mL SDS) and incubated at 65 ℃ on the oscillator for 6 h. Chromatin was eluted by 60 μL of DNA elution buffer (10 mmol/L Tris-Cl, pH 8.0) containing 20 μg of proteinase A for 2 h at 37 ℃ and 20 μg of protease K for 1 h at 55 ℃. The DNA was purified using a MinElute ® PCR purification kit (Qiagen, Valencia, CA, USA). A ChIP-seq library was constructed using KAPA HyperPrep Kits (KAPA, Boston, MA, USA) according to the manufacture's protocol. Sequencing was completed by Basepair Biotechnology Co., Ltd. (Suzhou, China). The ChIP-qPCR experiment was performed as described above. Our own raw and processed nucleic acid sequencing data (ERα and H3K27ac ChIP-seq) were deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) ( https://www.ncbi.nlm.nih.gov/geo https://www.ncbi.nlm.nih.gov/geo ) under the accession number GSE136673. 2.7 TCGA data analysis 2 The data analysis was carried out by the method described in the previous article published by our group ( Liu et al., 2018 Liu et al., 2018 ). In short, The Cancer Genome Atlas (TCGA) data were downloaded from http://www.cbioportal.org. http://www.cbioportal.org. The expression of ERα in tumor samples was determined based on clinical data. 2.8 Statistical analysis 2 The results derived from three independent experiments were presented as mean±standard deviation (SD). The significance of the data was analyzed with GraphPad Prism 8.2.1 by a two-tailed unpaired t -test. The difference was considered significant when P < 0.05. 3 Results 1 3.1 Proliferation and migration of MDA-MB-231 cells inhibited by ectopic overexpression of ApoA-I 2 In order to investigate the effect of ApoA-I on the biological behavior of breast cancer cells, we first determined the messenger RNA (mRNA) levels of ApoA-I in three breast cancer cell lines ( Fig. 1 Fig. 1 a). Since the mRNA level of ApoA-I in MDA-MB-231 cells is the lowest, we constructed an ectopic ApoA-I-overexpression system in MDA-MB-231 cells ( Fig. 1 Fig. 1 b). Then, we performed CCK-8 and transwell experiments to detect the effect of ApoA-I overexpression on MDA-MB-231 cells. The CCK-8 test result showed that the proliferation rate of ApoA-I-overexpressed MDA-MB-231 cells was significantly lower than that of the control group ( Fig. 1 Fig. 1 c). Furthermore, ApoA-I inhibited the migration of MDA-MB-231 cells (Figs. 1d 1d and 1e 1e ). This result indicated that ApoA-I may be a tumor suppressor that can inhibit the proliferation and migration of breast cancer cells. 3.2 ApoA-I transcription induced by E2/ERα signaling in breast cancer cells 2 In order to explore whether E2/ERα signaling modulates ApoA-I transcription, we exposed ERα‍-positive MCF7 cells to estrogen antagonist ICI 182780 to abolish the ERα functions. Accompanied by a decrease of ERα ( Fig. 2 Fig. 2 a), the mRNA levels of growth regulation by estrogen in breast cancer 1 ( GREB1 ; the classic ERα target gene) and ApoA-I in MCF7 cells were significantly downregulated by ICI 182780 ( Fig. 2 Fig. 2 b). 1;2; Given that the ER inhibitor downregulated the ApoA-I mRNA level in MCF7 cells, we speculated that the activation of ERα signaling would promote the transcription of ApoA-I . We performed hormone deprivation on ER-positive MCF7 cells, and then added an equal volume of ethanol (EtOH, vehicle control) or E2. Our results showed that the transcription of both GREB1 and ApoA-I was dramatically upregulated by E2 ( Fig. 2 Fig. 2 c). Compared with ER-positive MCF7 cells, triple-negative BT549 and MDA-MB-231 cells had lower ApoA-I mRNA levels ( Fig. 1 Fig. 1 a). We hypothesized that ectopic expression of ERα would also activate ApoA-I transcription in ER-negative breast cancer cells. As expected, the western blotting and qRT-qPCR data showed that the restoration of ERα expression in MDA-MB-231 cells upregulated the mRNA level of ApoA-I (Figs. 2d 2d and 2e 2e ). 3.3 ERα directly binds to last exon of ApoA-I gene 2 In order to examine whether ERα has a direct role in regulating ApoA-I transcription, ChIP-seq was conducted using anti-ERα antibodies. We observed that ERα was enriched in the last exon of the ApoA-I gene in both MCF7 and ERα-overexpressed MDA-MB-231 cells ( Fig. 3 Fig. 3 a). Notably, when challenging hormone-depleted MCF7 cells with E2, the ChIP signal intensity of ERα on the ApoA-I gene was more significant than that of hormone-depleted MCF7 cells treated with EtOH ( Fig. 3 Fig. 3 a). In addition, by analyzing the ERα‍-enriched region, we found that an estrogen response element (ERE) was located right in the center of the ERα peak ( Fig. 3 Fig. 3 b). 3.4 p300-mediated H3K27ac is involved in the transcriptional regulation of ApoA-I through ERα 2 Aimed at exploring the mechanism by which ERα regulates ApoA-I transcription, we analyzed the epigenetic characteristics of the ApoA-I gene. The increased H3K27ac signal intensity was observed in hormone-depleted MCF7 cells when treated with E2 ( Fig. 4 Fig. 4 a). To further investigate the relationship between ERα and H3K27ac on the ApoA-I gene, we performed ChIP-qPCR using anti-H3K27ac antibodies. Our data showed that ICI 182780 significantly reduced the abundance of H3K27ac on the ApoA-I gene in MCF7 cells ( Fig. 4 Fig. 4 b). These data suggested that the binding of ERα on the ApoA-I gene is positively correlated with the abundance of H3K27ac modification. Subsequently, we tested the role of acetyltransferase p300 in this process. With the intervention of p300 inhibitor (p300i), the mRNA level of ApoA-I decreased significantly along with the decline of H3K27ac level (Figs. 4c 4c and 4d 4d ). Specifically, challenging MCF7 cells with p300i for 24 and 72 h made the ApoA-I mRNA level drop by more than 90% and 98%, respectively ( Fig. 4 Fig. 4 d). 3.5 Positive correlation between mRNA expression levels of ERα and ApoA-I in breast cancer 2 In order to support our findings with clinical data, we analyzed the TCGA data for breast cancer (TCGA-BRCA) uploaded by Perou ( Ciriello et al., 2015 Ciriello et al., 2015 ). According to the examined clinical data, we separated samples into ERα‍-negative and ERα‍-positive groups ( Fig. 5 Fig. 5 a). The average mRNA level of ApoA-I in the ERα-positive group was significantly higher than that in the ERα‍-negative group ( Fig. 5 Fig. 5 b). Moreover, the regression analysis revealed a positive correlation between the mRNA expression levels of ERα and ApoA-I ( Fig. 5 Fig. 5 c). 4 Discussion 1 Numerous studies have suggested the downregulation of serum and tissue ApoA-I in various cancer types ( Zamanian-Daryoush and Didonato, 2015 Zamanian-Daryoush and Didonato, 2015 ). Therefore, it is of high importance to understand the role of this phenomenon and the associated regulatory mechanism. In this study, we demonstrated that ApoA-I can inhibit the proliferation and migration of breast cancer cells. Moreover, ERα could directly bind to the last exon of ApoA-I gene to active its transcription, and p300 might be a co-activator of ERα by establishing an H3K27ac-enriched chromatin microenvironment in this process. The ApoA-I mimetic peptide D-4F has been proven to inhibit the proliferation of human breast cancer cells ( Peng et al., 2017 Peng et al., 2017 ). We found that the direct overexpression of human ApoA-I protein arrived at the same result. The antitumor effect of ApoA-I may be achieved through a cellular autonomic mechanism, or its impact on immune responses ( Gordon et al., 2011 Gordon et al., 2011 ), or both, which remains an elusive topic. In the cellular autonomic mechanism, ApoA-I can induce changes in the expression of cancer-related genes, thus producing anti-cancer effects. For example, ApoA-I induces the downregulation of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α expression, thereby inhibiting the proliferation and migration of ovarian cancer cells ( Gao et al., 2011 Gao et al., 2011 , 2012 2012 ). In colorectal adenocarcinoma cells, ApoA-I inhibits the activity of cancer cells by downregulating cyclooxygenase-2 (COX-2) expression ( Aguirre-Portolés et al., 2018 Aguirre-Portolés et al., 2018 ). In our in vitro model, the anti-cancer effect is more likely to be achieved through the cellular autonomic mechanism due to the absence of an immune system. Nonetheless, the relevant specific mechanism remains to be further studied. Although most researches have indicated that ApoA-I levels are negatively correlated with the occurrence and progression of various types of cancer, there are also reports showing a positive correlation ( Chen et al., 2013 Chen et al., 2013 ; Martin et al., 2015 Martin et al., 2015 ; Shi et al., 2018 Shi et al., 2018 ; Zografos et al., 2019 Zografos et al., 2019 ). It is not clear whether the positive correlation between ApoA-I levels and cancer parameters as reported by a number of studies reflects the cancer-promoting effects of ApoA-I in these special cases. Many transcription factors implicated in the regulation of ApoA-I promotor have been identified, including peroxisome proliferator-activated receptor γ (PPARγ), hepatocyte nuclear factor 4 (HNF4), liver receptor homologue-1 (LRH1), and ApoA-I regulatory protein 1 (ARP1)/nuclear receptor subfamily 2 group F member 2 (NR2F2) ( Kardassis et al., 2014 Kardassis et al., 2014 ). Besides, an endogenously expressed long noncoding antisense transcript, ApoA1-AS , can also modulate ApoA-I transcription through recruiting histone methylation enzymes ( Halley et al., 2014 Halley et al., 2014 ). Herein, for the first time, ERα was demonstrated to be an activator for ApoA-I gene transcription in breast cancer cells: we observed a positive correlation between the expression of ERα and ApoA-I mRNAs, in both breast cancer cells cultured in vitro and breast tumor samples. Estrogen can exert its function through both genomic and non-genomic signaling. Our ChIP-seq data displayed an ERα enrichment signal within the ApoA-I gene, suggesting that ERα promoted the transcription of ApoA-I gene through genomic signaling. Genomic signaling can be divided into direct and indirect types. In direct genomic signaling, dimerized ERα binds directly to specific DNA sequences known as EREs ( Klinge, 2001 Klinge, 2001 ). By analyzing the sequence of ERα enrichment locus on the ApoA-I gene, we found the sequence AGGTCACGCTGTCCC that has proved to be an ERE ( Bourdeau et al., 2004 Bourdeau et al., 2004 ). ERα seems to modulate ApoA-I transcription through direct genomic signaling, while this is suggested by the experiment through the process focusing on the influence of ERE sequence mutation. Recruiting various co-regulators to form an ER complex is the intrinsic mechanism of estrogen signaling ( Heldring et al., 2007 Heldring et al., 2007 ). The activity of ERs depends on co-regulators within this complex. We found that H3K27ac abundance on the ApoA-I gene is tightly dependent on ERα. Moreover, the inhibitor of acetyltransferase p300 could suppress ApoA-I mRNA expression. We speculate that histone acetyltransferase p300 may be a co-activator of ERα by making the chromatin region containing ApoA-I more accessible to transcriptional factors. A recent study revealed that Ajuba, the LIM protein in MCF7 and T47D breast cancer cells, could recruit deleted in breast cancer 1 (DBC1) and cAMP-regulated enhancer-binding protein (CREB)‍-binding protein (CBP)/p300 to enhance the regulatory activity of ERα on target genes by acetylating ERα ( Xu et al., 2019 Xu et al., 2019 ). We hypothesize that p300 may combine with ERα to enhance the transcriptional activity of ERα on ApoA-I , which process requires further verification. 5 Conclusions 1 This study revealed the tumor suppressive role of ApoA-I in breast cancer cells and identified ERα as a new activator of ApoA-I gene transcription through directly binding to the last exon of the ApoA-I gene. This is the site where an ERE sequence is found, and p300 may serve as a co-activator of ERα in activating ApoA-I expression by establishing an H3K27ac-enriched chromatin microenvironment. Our study demonstrated the mechanism of ERα promoting ApoA-I gene transcription in breast cancer cells, providing further supporting evidence of the double-edged nature of the E2/ERα signaling in tumorigenesis. Moreover, given the positive regulatory effect of ERα on ApoA-I expression and the tumor suppressive role of ApoA-I in various types of cancer, such as melanoma ( Zamanian-Daryoush et al., 2013 Zamanian-Daryoush et al., 2013 ), breast cancer ( Cedó et al., 2016 Cedó et al., 2016 ), colon cancer ( Gkouskou et al., 2016 Gkouskou et al., 2016 ), and pancreatic cancer ( Peng et al., 2017 Peng et al., 2017 ), it is considered that the administration of ApoA-I mimetic peptides may enhance the efficacy of systemic therapy for luminal breast cancer. References Aguirre-Portolés C , Feliu J , Reglero G , et al. , 2018 . ABCA1 overexpression worsens colorectal cancer prognosis by facilitating tumour growth and caveolin-1-dependent invasiveness, and these effects can be ameliorated using the BET inhibitor apabetalone . Mol Oncol , 12 ( 10 ): 1735 - 1752 . https://doi.org/10.1002/1878-0261.12367 https://doi.org/10.1002/1878-0261.12367 ABCA1 overexpression worsens colorectal cancer prognosis by facilitating tumour growth and caveolin-1-dependent invasiveness, and these effects can be ameliorated using the BET inhibitor apabetalone Anzick SL , Kononen J , Walker RL , et al. , 1997 . A1B1, a steroid receptor coactivator amplified in breast and ovarian cancer . Science , 277 ( 5328 ): 965 - 968 . https://doi.org/10.1126/science.277.5328.965 https://doi.org/10.1126/science.277.5328.965 A1B1, a steroid receptor coactivator amplified in breast and ovarian cancer Bourdeau V , Deschênes J , Métivier R , et al. , 2004 . Genome-wide identification of high-affinity estrogen response elements in human and mouse . Mol Endocrinol , 18 ( 6 ): 1411 - 1427 . https://doi.org/10.1210/me.2003-0441 https://doi.org/10.1210/me.2003-0441 Genome-wide identification of high-affinity estrogen response elements in human and mouse Cedó L , García-León A , Baila-Rueda L , et al. , 2016 . ApoA-I mimetic administration, but not increased apoA-I-containing HDL, inhibits tumour growth in a mouse model of inherited breast cancer . Sci Rep , 6 : 36387 . https://doi.org/10.1038/srep36387 https://doi.org/10.1038/srep36387 ApoA-I mimetic administration, but not increased apoA-I-containing HDL, inhibits tumour growth in a mouse model of inherited breast cancer Chen CL , Lin TS , Tsai CH , et al. , 2013 . Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics . J Proteomics , 85 : 28 - 43 . https://doi.org/10.1016/j.jprot. 2013.04.024 https://doi.org/10.1016/j.jprot.2013.04.024 Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics Ciriello G , Gatza ML , Beck AH , et al. , 2015 . Comprehensive molecular portraits of invasive lobular breast cancer . Cell , 163 ( 2 ): 506 - 519 . https://doi.org/10.1016/j.cell. 2015.09.033 https://doi.org/10.1016/j.cell.2015.09.033 Comprehensive molecular portraits of invasive lobular breast cancer Cortesi L , Barchetti A , de Matteis E , et al. , 2009 . Identification of protein clusters predictive of response to chemotherapy in breast cancer patients . J Proteome Res , 8 ( 11 ): 4916 - 4933 . https://doi.org/10.1021/pr900239h https://doi.org/10.1021/pr900239h Identification of protein clusters predictive of response to chemotherapy in breast cancer patients Gao F , Vasquez SX , Su F , et al. , 2011 . L-5F, an apolipoprotein A-I mimetic, inhibits tumor angiogenesis by suppressing VEGF/basic FGF signaling pathways . Integr Biol , 3 ( 4 ): 479 - 489 . https://doi.org/10.1039/c0ib00147c https://doi.org/10.1039/c0ib00147c L-5F, an apolipoprotein A-I mimetic, inhibits tumor angiogenesis by suppressing VEGF/basic FGF signaling pathways Gao F , Chattopadhyay A , Navab M , et al. , 2012 . Apolipoprotein A-I mimetic peptides inhibit expression and activity of hypoxia-inducible factor-1α in human ovarian cancer cell lines and a mouse ovarian cancer model . J Pharmacol Exp Ther , 342 ( 2 ): 255 - 262 . https://doi.org/10.1124/jpet. 112.191544 https://doi.org/10.1124/jpet.112.191544 Apolipoprotein A-I mimetic peptides inhibit expression and activity of hypoxia-inducible factor-1α in human ovarian cancer cell lines and a mouse ovarian cancer model Georgila K , Vyrla D , Drakos E , 2019 . Apolipoprotein A-I (ApoA-I), immunity, inflammation and cancer . Cancers , 11 ( 8 ): 1097 . https://doi.org/10.3390/cancers11081097 https://doi.org/10.3390/cancers11081097 Apolipoprotein A-I (ApoA-I), immunity, inflammation and cancer Gkouskou KK , Ioannou M , Pavlopoulos GA , et al. , 2016 . Apolipoprotein A-I inhibits experimental colitis and colitis-propelled carcinogenesis . Oncogene , 35 ( 19 ): 2496 - 2505 . https://doi.org/10.1038/onc.2015.307 https://doi.org/10.1038/onc.2015.307 Apolipoprotein A-I inhibits experimental colitis and colitis-propelled carcinogenesis Gonçalves A , Esterni B , Bertucci F , et al. , 2006 . Postoperative serum proteomic profiles may predict metastatic relapse in high-risk primary breast cancer patients receiving adjuvant chemotherapy . Oncogene , 25 ( 7 ): 981 - 989 . https://doi.org/10.1038/sj.onc.1209131 https://doi.org/10.1038/sj.onc.1209131 Postoperative serum proteomic profiles may predict metastatic relapse in high-risk primary breast cancer patients receiving adjuvant chemotherapy Gordon SM , Hofmann S , Askew DS , et al. , 2011 . High density lipoprotein: it's not just about lipid transport anymore . Trends Endocrinol Metab , 22 ( 1 ): 9 - 15 . https://doi.org/10.1016/j.tem.2010.10.001 https://doi.org/10.1016/j.tem.2010.10.001 High density lipoprotein: it's not just about lipid transport anymore Halley P , Kadakkuzha BM , Faghihi MA , et al. , 2014 . Regulation of the apolipoprotein gene cluster by a long noncoding RNA . Cell Rep , 6 ( 1 ): 222 - 230 . https://doi.org/10.1016/j.celrep.2013.12.015 https://doi.org/10.1016/j.celrep.2013.12.015 Regulation of the apolipoprotein gene cluster by a long noncoding RNA Hanker AB , Sudhan DR , Arteaga CL , 2020 . Overcoming endocrine resistance in breast cancer . Cancer Cell , 37 ( 4 ): 496 - 513 . https://doi.org/10.1016/j.ccell.2020.03.009 https://doi.org/10.1016/j.ccell.2020.03.009 Overcoming endocrine resistance in breast cancer Heldring N , Pike A , Andersson S , et al. , 2007 . Estrogen receptors: how do they signal and what are their targets . Physiol Rev , 87 ( 3 ): 905 - 931 . https://doi.org/10.1152/physrev.00026.2006 https://doi.org/10.1152/physrev.00026.2006 Estrogen receptors: how do they signal and what are their targets Herynk MH , Fuqua SAW , 2004 . Estrogen receptor mutations in human disease . Endocr Rev , 25 ( 6 ): 869 - 898 . https://doi.org/10.1210/ER.2003-0010 https://doi.org/10.1210/ER.2003-0010 Estrogen receptor mutations in human disease Kardassis D , Mosialou I , Kanaki M , et al. , 2014 . Metabolism of HDL and its regulation . Curr Med Chem , 21 ( 25 ): 2864 - 2880 . https://doi.org/10.2174/0929867321666140303153430 https://doi.org/10.2174/0929867321666140303153430 Metabolism of HDL and its regulation Klinge CM , 2001 . Estrogen receptor interaction with estrogen response elements . Nucleic Acids Res , 29 ( 14 ): 2905 - 2919 . https://doi.org/10.1093/nar/29.14.2905 https://doi.org/10.1093/nar/29.14.2905 Estrogen receptor interaction with estrogen response elements Kong LC , Tan L , Lv RT , et al. , 2016 . A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands . Nucleic Acids Res , 44 ( 18 ): 8682 - 8692 . https://doi.org/10.1093/nar/gkw529 https://doi.org/10.1093/nar/gkw529 A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands Levin ER , Pietras RJ , 2008 . Estrogen receptors outside the nucleus in breast cancer . Breast Cancer Res Treat , 108 ( 3 ): 351 - 361 . https://doi.org/10.1007/s10549-007-9618-4 https://doi.org/10.1007/s10549-007-9618-4 Estrogen receptors outside the nucleus in breast cancer Li WH , Tanimura M , Luo CC , et al. , 1988 . The apolipoprotein multigene family: biosynthesis, structure, structure‒function relationships, and evolution . J Lipid Res , 29 ( 3 ): 245 - 271 . https://doi.org/10.1016/S0022-2275(20)38532-1 https://doi.org/10.1016/S0022-2275(20)38532-1 The apolipoprotein multigene family: biosynthesis, structure, structure‒function relationships, and evolution Liu L , Shen YH , Zhu XG , et al. , 2018 . ERα is a negative regulator of PD-L1 gene transcription in breast cancer . Biochem Biophys Res Commun , 505 ( 1 ): 157 - 161 . https://doi.org/10.1016/j.bbrc.2018.09.005 https://doi.org/10.1016/j.bbrc.2018.09.005 ERα is a negative regulator of PD-L1 gene transcription in breast cancer Martin LJ , Melnichouk O , Huszti E , et al. , 2015 . Serum lipids, lipoproteins, and risk of breast cancer: a nested case-control study using multiple time points . J Nat Cancer Inst , 107 ( 5 ): djv032 . https://doi.org/10.1093/jnci/djv032 https://doi.org/10.1093/jnci/djv032 Serum lipids, lipoproteins, and risk of breast cancer: a nested case-control study using multiple time points McMahon C , Suthiphongchai T , DiRenzo J , et al. , 1999 . P/CAF associates with cyclin D1 and potentiates its activation of the estrogen receptor . Proc Natl Acad Sci USA , 96 ( 10 ): 5382 - 5387 . https://doi.org/10.1073/pnas.96.10.5382 https://doi.org/10.1073/pnas.96.10.5382 P/CAF associates with cyclin D1 and potentiates its activation of the estrogen receptor Mussi P , Liao L , Park SE , et al. , 2006 . Haploinsufficiency of the corepressor of estrogen receptor activity (REA) enhances estrogen receptor function in the mammary gland . Proc Natl Acad Sci USA , 103 ( 45 ): 16716 - 16721 . https://doi.org/10.1073/pnas.0607768103 https://doi.org/10.1073/pnas.0607768103 Haploinsufficiency of the corepressor of estrogen receptor activity (REA) enhances estrogen receptor function in the mammary gland Peng MY , Zhang Q , Cheng YN , et al. , 2017 . Apolipoprotein A-I mimetic peptide 4F suppresses tumor-associated macrophages and pancreatic cancer progression . Oncotarget , 8 ( 59 ): 99693 - 99706 . https://doi.org/10.18632/oncotarget.21157 https://doi.org/10.18632/oncotarget.21157 Apolipoprotein A-I mimetic peptide 4F suppresses tumor-associated macrophages and pancreatic cancer progression Reddy ST , Navab M , Anantharamaiah GM , et al. , 2014 . Apolipoprotein A-I mimetics . Curr Opin Lipidol , 25 ( 4 ): 304 - 308 . https://doi.org/10.1097/MOL.0000000000000092 https://doi.org/10.1097/MOL.0000000000000092 Apolipoprotein A-I mimetics Shao BH , Heinecke JW , 2018 . Quantifying HDL proteins by mass spectrometry: how many proteins are there and what are their functions? Expert Rev Proteomics , 15 ( 1 ): 31 - 40 . https://doi.org/10.1080/14789450.2018.1402680 https://doi.org/10.1080/14789450.2018.1402680 Quantifying HDL proteins by mass spectrometry: how many proteins are there and what are their functions? Shi FY , Wu H , Qu K , et al. , 2018 . Identification of serum proteins AHSG, FGA and APOA-I as diagnostic biomarkers for gastric cancer . Clin Proteomics , 15 : 18 . https://doi.org/10.1186/s12014-018-9194-0 https://doi.org/10.1186/s12014-018-9194-0 Identification of serum proteins AHSG, FGA and APOA-I as diagnostic biomarkers for gastric cancer Vrtačnik P , Ostanek B , Mencej-Bedrač S , et al. , 2014 . The many faces of estrogen signaling . Biochem Med , 24 ( 3 ): 329 - 342 . https://doi.org/10.11613/BM.2014.035 https://doi.org/10.11613/BM.2014.035 The many faces of estrogen signaling Waks AG , Winer EP , 2019 . Breast cancer treatment: a review . JAMA , 321 ( 3 ): 288 - 300 . https://doi.org/10.1001/jama.2018.19323 https://doi.org/10.1001/jama.2018.19323 Breast cancer treatment: a review Xu BH , Li Q , Chen N , et al. , 2019 . The LIM protein Ajuba recruits DBC1 and CBP/p300 to acetylate ERα and enhances ERα target gene expression in breast cancer cells . Nucleic Acids Res , 47 ( 5 ): 2322 - 2335 . https://doi.org/10.1093/nar/gky1306 https://doi.org/10.1093/nar/gky1306 The LIM protein Ajuba recruits DBC1 and CBP/p300 to acetylate ERα and enhances ERα target gene expression in breast cancer cells Zamanian-Daryoush M , Didonato JA , 2015 . Apolipoprotein A-I and cancer . Front Pharmacol , 6 : 265 . https://doi.org/10.3389/fphar.2015.00265 https://doi.org/10.3389/fphar.2015.00265 Apolipoprotein A-I and cancer Zamanian-Daryoush M , Lindner D , Tallant TC , et al. , 2013 . The cardioprotective protein apolipoprotein A1 promotes potent anti-tumorigenic effects . J Biol Chem , 288 ( 29 ): 21237 - 21252 . https://doi.org/10.1074/jbc.M113.468967 https://doi.org/10.1074/jbc.M113.468967 The cardioprotective protein apolipoprotein A1 promotes potent anti-tumorigenic effects Zhang JW , Cai Y , Hu HB , et al. , 2016 . Nomogram basing pre-treatment parameters predicting early response for locally advanced rectal cancer with neoadjuvant chemotherapy alone: a subgroup efficacy analysis of FOWARC study . Oncotarget , 7 ( 4 ): 5053 - 5062 . https://doi.org/10.18632/oncotarget.6469 https://doi.org/10.18632/oncotarget.6469 Nomogram basing pre-treatment parameters predicting early response for locally advanced rectal cancer with neoadjuvant chemotherapy alone: a subgroup efficacy analysis of FOWARC study Zografos E , Anagnostopoulos AK , Papadopoulou A , et al. , 2019 . Serum proteomic signatures of male breast cancer . Cancer Genomics Proteomics , 16 ( 2 ): 129 - 137 . https://doi.org/10.21873/cgp.20118 https://doi.org/10.21873/cgp.20118 Serum proteomic signatures of male breast cancer Zwijsen RML , Buckle RS , Hijmans EM , et al. , 1998 . Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1 . Genes Dev , 12 ( 22 ): 3488 - 3498 . https://doi.org/10.1101/gad.12.22.3488 https://doi.org/10.1101/gad.12.22.3488 Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1 Bingjie WANG performed the experimental research and TCGA data analysis, wrote and edited the manuscript. Yinghui SHEN performed the ChIP-seq experiment. Tianyu LIU contributed to the data analysis, writing and editing of the manuscript. Li TAN contributed to the study design and editing of the manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Natural Science Foundation of China (Nos. 81672785, 31871291, and 82073113 to Li TAN) and the National Key R&D Project of China (No. 2016YFA0101800 to Li TAN). Li TAN was also supported by the Innovative Research Team of High-level Local University in Shanghai. We thank Dr. Xuguo ZHU (Institutes of Biomedical Sciences, Fudan University, Shanghai, China) for constructing the ERα‍-overexpressed MDA-MB-231 cells, Dr. Ruitu LV (Department of Chemistry, University of Chicago, Chicago, IL, USA) for analyzing the ChIP-seq data, and Prof. Yujiang Geno SHI (Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital, Harvard Medical School, Boston, USA) for his valuable comments. 10.1631/jzus.B2100393 1673-1581（2021）12-1034-11 This work was supported by the National Natural Science Foundation of China (Nos. 81672785, 31871291, and 82073113 to Li TAN) and the National Key R&D Project of China (No. 2016YFA0101800 to Li TAN). 2021-04-27 2021-07-14 2021-12-15 2022-05-09 2021-11-15 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2021 2021 浙江大学出版社 Bingjie WANG, Yinghui SHEN, Tianyu LIU, and Li TAN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 12 22