BRD7 Inhibits the Warburg Effect and Tumor Progression Through

Niu et al. Cell Death and Disease (2018) 9:519 DOI 10.1038/s41419-018-0536-7 Cell Death & Disease

ARTICLE Open Access

Corrected: Retraction BRD7 inhibits the Warburg effect and tumor progression through inactivation of HIF1α/LDHA axis in breast cancer Weihong Niu1,2,3,YanweiLuo2,3,XinyeWang2,3, Yao Zhou2,3,HuiLi4,HeranWang1,3, Yaojie Fu5, Shanshan Liu5, Shanghelin Yin5, Jianglei Li5,RanZhao2,3, Yukun Liu2,3,SongqingFan4,ZhengLi2,3,6, Wei Xiong2,3, Xiaoling Li2,3, Guiyuan Li1,2,3, Caiping Ren3,MingTan 7 andMingZhou1,2,3

Abstract The bromodomain-containing protein 7 (BRD7) was first identified as a tumor suppressor in nasopharyngeal carcinoma and has critical roles in cancer development and progression. However, the regulatory roles and mechanisms of BRD7 in cancer metabolism are still unknown. In this study, we demonstrated that BRD7 was lowly expressed in breast cancer tissues and was identified as a poor prognostic factor in breast cancer. Meanwhile, BRD7 could suppress cell proliferation, initiate cell apoptosis and reduce aerobic glycolysis, suggesting that BRD7 plays a tumor suppressive roles in breast cancer. Mechanistically, BRD7 could negatively regulate a critical glycolytic enzyme LDHA through directly interaction with its upstream transcription factor, HIF1α, facilitating degradation of HIF1α mediated by ubiquitin–proteasome pathway. Moreover, restoring the expression of LDHA in breast cancer cells could reverse the effect of BRD7 on aerobic glycolysis, cell proliferation, and tumor formation, as well as the expression of cell cycle and apopotosis related molecules such as cyclin D1, CDK4, P21, and c-PARP both in vitro and in vivo. Taken

1234567890():,; 1234567890():,; together, these results indicate that BRD7 acts as a tumor suppressor in breast cancer and represses the glycolysis and tumor progression through inactivation of HIF1α/LDHA transcription axis.

Introduction that BRD7 is involved in growth inhibition of NPC by Bromodomain-containing proteins are a class of inhibiting cell cycle progression from G1 to S, and thus evolutionarily conserved proteins that includes was identified as a tumor suppressor in NPC4,5. BRD7 is bromodomain-containing protein 7 (BRD7). Since our also involved in multiple physiological processes, includ- group first cloned this gene from nasopharyngeal carci- ing the regulation of spermatogenesis and cognitive – noma (NPC) cells in 2000, numerous studies have shown behavior6 8. BRD7 is downregulated in multiple types of that BRD7 plays critical roles in cancer development and tumor specimen and cancer cells, and might be involved – progression1 3. Our previous studies have demonstrated in the development and progression of multiple types of cancers, including breast cancer and prostate cancer1. BRD7 can bind to BRCA1 in breast cancer cells, which is Correspondence: Ming Tan ([email protected]) or Ming Zhou required for the BRCA1-mediated transcriptional regula- ([email protected]) tion of the estrogen receptor (ER), suggesting a close 1The Affiliated Tumor Hospital of Xiangya Medical School, Central South ’ relationship between BRD7 and breast cancer develop- University, Changsha, Hunan 410013, People s Republic of China 9 2The Key Laboratory of Carcinogenesis of the Chinese Ministry of Health, The ment . However, so far there is still no direct evidence to Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry show that BRD7 plays a role in breast cancer. of Education, Xiangya Hospital, Central South University, Changsha, Hunan Interaction of BRD7 and p53 is essential for the tran- 410078, People’s Republic of China Full list of author information is available at the end of the article scriptional activation of some target genes of p53, Edited by: A. Finazzi-Agrò

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including p21 and MDM2, which is required for p53- progression through inactivation of HIF1α/LDHA tran- dependent oncogene-induced senescence10,11. Notably, scription axis. one study shows that BRD7 is capable of regulating X-box binding protein 1 (XBP1) nuclear translocation and Materials and Methods interacts with the regulatory subunits of phosphatidyl- Tissue samples and clinical data inositol3-kinase (PI3K) to increase the nuclear translo- All the human tissues were collected from the Second cation of both p85α/β and XBP1s. Reinstating BRD7 Xiangya Hospital of Central South University from levels in the liver restores XBP1s nuclear translocation, November 2001 to September 2012. This study was improves glucose homeostasis12, and ultimately reduces approved by the Committee on the Ethics of Central South the blood glucose levels in obese and diabetic mouse University. All of the individuals participating in this study models13, which is also consistent with the result of the have provided written informed consent. The samples recent paper, the role of BRD7 in embryo development were compiled into a tissue microarray as previously and glucose metabolism, suggesting that BRD7 is impli- described, which included 225 cases of breast cancer tis- – cated in cellular energy metabolism mechanisms such as sues and 61 cases of non-cancerous breast tissues18 20. glycolysis. However, the role of BRD7 in regulating cancer Clinicopathologic characteristics of breast cancer patients cell metabolism has not been systematically investigated. were recorded, including gender, age, clinical tumor node Hypoxia-inducible factor 1α (HIF1α) takes part in the metastasis (TNM) stage, pathology diagnosis, and mole- reprogramming of cancer metabolism by regulating key cular subtype. The profile of clinicopathologic character- molecules, including LDHA, SLC2A1, SLC2A3, HK1, istics of the breast cancer patients is shown in Table 1. HK2, and MCT4 in glucose metabolism14,15. The Patients’ ages ranged from 23 to 71 years old. All of the expression of HIF1α is high in multiple types of cancers, 225 breast cancer patients had valid follow-up data, and such as lung cancer, prostate cancer, breast cancer, and the longest survival time was 120 months. The overall colonic adenocarcinoma. In tumor progression, insulin, survival (OS) was defined as the time from diagnosis to the insulin-like growth factor (IGF)-1 or IGF-2, v-Src, lactate, date of death or the date last known alive. pyruvate, and genetic alterations such as oncogene activation or tumor suppressor gene inactivation lead to Cell culture HIF1α overexpression14,16. Human breast cancer cell lines MCF-7, MDA-MB-231, Mounting evidence accumulated in cancer research has MDA-MB-453, and the human embryonic kidney cell line demonstrated that dysregulated cancer cell bioenergy plays an important role in the development and pro- 17 Table 1 Clinicopathologic characteristics of breast cancer gression of breast cancer . While currently there is no patients direct evidence to support a role of BRD7 in breast cancer and it is not clear whether BRD7 regulates cancer cell Age metabolism. Therefore, in this study we investigated Mean ± S.D. 46 ± 0.66 whether BRD7 indeed plays a role in breast cancer progression and explored whether BRD7 regulates breast Sex (%) cancer cell metabolism. We revealed that BRD7 showed Male 0.4 (1/225) low expression in breast cancer tissues compared to Female 99.6 (224/225) normal tissue, and loss of BRD7 expression in breast Tumor size (%) cancer was identified as a poor prognostic factor. More- – over, ectopic expression of BRD7 in breast cancer cells T1 2 62.7 (141/225) suppressed cell proliferation, initiated cell apoptosis, and T3–4 37.3 (84/225) decreased glycolysis. Furthermore, we found that lactate Nodal metastasis (%) dehydrogenase A (LDHA) was negatively regulated by Present 74.2 (167/225) BRD7 through promoting proteasomal degradation of HIF1α, and restoring the expression of LDHA in BRD7- Absent 25.8 (58/225) overexpressed or breast cancer cells could reverse the Distant metastasis (%) effect of BRD7 on aerobic glycolysis, cell proliferation, and Present 9.3 (21/225) apoptosis, as well as the expression of cell cycle and Absent 81.7 (204/225) apopotosis related molecules such as cyclin D1, CDK4, P21, and c-poly-ADP-ribose polymerase (c-PARP) both TNM stage (%) in vitro and in vivo. Taken together, these results indicate I–II 65.8 (148/225) that BRD7 functions as a tumor suppressor gene in breast III–IV 34.2 (77/225) cancer and BRD7 inhibits aerobic glycolysis and tumor

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HEK-293T were obtained from the American Type Cul- citric acid buffer (pH 6.0), heated in a microwave oven for ture Collection (Manassas, VA, USA). All cells were cul- 10 min, and then treated with 3% H2O2 for 15 min and tured in high glucose DMEM medium (Invitrogen Life washed with Tris-buffered saline containing 1% Tween 20 Technologies, Carlsbad, CA, USA) containing 10% fetal (TBST) three times each for 5 min. After a 60 min incu- bovine serum (FBS) (Gibco, Thermo Fisher Scientific, bation in normal goat serum (Boster, Wuhan, China), the Waltham, MA, USA), 100 units/ml penicillin and 100 mg/ slides were incubated with primary antibodies overnight ml streptomycin. The cells were incubated at 37 °C in 5% at 4°C. The antibodies included polyclonal rabbit anti- CO2. For hypoxic condition, cells were cultured in a BRD7 (1:500 dilution), polyclonal rabbit anti-LDHA Hypoxic Workstation (Don Whitley, West Yorkshire, UK) (1:2000 dilution) from Proteintech (Wuhan, China), that was infused with a mixture of 1% O2,5%CO2 and polyclonal rabbit polyclonal rabbit anti-Cyclin D1 (1:200 at 37 °C temperature. Cells were lysed for extraction of dilution) from Santa Cruz (Dallas, TX, USA), polyclonal protein and RNA in the workstation to avoid rabbit anti-c-PARP (1:200 dilution) and polyclonal rabbit reoxygenation. anti-p21 (1:500 dilution) from Cell Signaling Technology (Danvers, MA), and polyclonal rabbit anti-Ki67 (1:200 Transfection dilution) from Bioworld Technology, Inc. (St. Louis, For ectopic expression of BRD7 in MCF-7, MDA-MB- Minnesota, USA). The slides were washed three times 231, and MDA-MB-453, the cells were transfected with with TBST (each for 15 min) and then incubated with a pIRES2-EGFP-3Flag/BRD7 using Lipofectamine 3000 secondary goat anti-rabbit antibody (Maixin, Fujian, transfection reagent (Invitrogen, Carlsbad, CA) according China) for 60 min at 37 °C. Then, the slides were visua- to the manufacturer’s instructions; the cells transfected lized with 3,3′-diaminobenzidine (DAB) (Zhongshan Gold with pIRES2-EGFP were used as a negative control. The Bridge, Beijing, China) for 5 min and counterstained with positive cells with ectopic expression of BRD7 or blank hematoxylin for 45 s. The slides were mounted and pho- control were further sorted by GFP-mediated flow cyto- tographed with an Olympus BX51 microscope (Olympus, metry technology and extended culture. As for the Japan). recovery experiment for LDHA, the MCF-7, MDA-MB- 231, and MDA-MB-453 cells with BRD7 overexpression Evaluation of staining were further transiently transfected with pENTER/LDHA To evaluate the expression of BRD7 in the tissue or pENTER, and the cells transfected with pIRES2-EGFP microarrays, two independent pathologists who blinded to and pENTER vectors were used as a control. To knock clinicopathologic features and clinical scored the staining down BRD7 or LDHA expression in MCF-7 and MDA- under a light microscope (BX51; Olympus, Japan). MB-231 cells, the cells were transfected with BRD7 or BRD7 staining intensity was scored as 0 (negative, −), 1 LDHA small interfering RNA (siRNA) oligonucleotides (weak, +), 2 (moderate, ++), and 3 (strong, +++). The (Ribobio, Guang zhou; 50 nmol/l). The cells transfected extent of staining was scored as 0–1.0 (0–100%). The final with control siRNA oligonucleotides were used as a staining score (0–3) was calculated as the multiplication negative control. of the intensity score and extent score. The value of final score ≥1 was defined as high expression, otherwise was Inhibition of glycolysis with oxamate and 2-DG defined as low expression, which was referred to the To inhibit aerobic glycolysis in breast cancer cells, the previous publication18,19. glycolytic inhibitors oxamate sodium (Oxamate; Sigma- To semi-quantify the expression of BRD7, LDHA, Aldrich, St. Louis, MO, USA) and 2-deoxy-D-glucose (2- Cyclin D1, p21, c-PARP, and Ki67 in the tumor tissues DG) (Sigma-Aldrich, St. Louis, MO, USA) were used to from nude mice, Image-J (NIH) was used to access the treat the breast cancer cells (MCF-7 and MDA-MD-231) integral optical density (IOD) of immunoreactivity. Data with ectopic expression of BRD7 sorted by GFP-mediated were acquired from eight sections/animal and averaged to flow cytometry technology at different concentrations. For produce a single value per subject. oxamate, the cells were treated with a concentration of 50 or 80 mM for different lengths of time (48 or 72 h). For 2- Immunoprecipitation DG, the cells were treated with various concentrations (2, After transfection, MCF-7 cells were cultured in a 4, 6 mM) of 2-DG for 48 h. Then, the cell viability was hypoxic workstation with 1% O2 for 24 h. Then the media measured using an MTT assay, and the data were pre- covering the cells were washed once with phosphate- sented as the percentage of viability inhibition. buffered saline (PBS). Cells were then scraped from the plates in 500 μl of ice-cold RIPA lysis buffer containing Immunohistochemistry protease and phosphatase inhibitors (Roche). The lysed The paraffin-embedded slides were routinely depar- cells were pipetted into a 1.5 ml Eppendorf tube, incu- affinized and hydrated. The slides were then retrieved in bated on ice for 30 min, and microcentrifuged at 13,000

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rpm for 15 min at 4 °C. Clarified lysates were incubated anti-UB (1:3000 dilution) from Santa Cruz (Dallas, Texas, with 2 μg indicated antibody overnight at 4 °C, and then USA); mouse monoclonal anti-HIF1α (1:1000 dilution) incubated for 2 h with 30 μl Protein A/G Agarose. Beads from Abcam (Cambridge, USA). After washing with 1× were extensively washed in lysis buffer, and bound pro- TBST three times for 8 min each, the membranes were teins were eluted in moderate 2× SDS sample buffer for incubated with the corresponding secondary antibodies 100 °C 10 min and subjected to western blot analysis. for 1 h at 37 °C, and then washed with 1× TBST for three times again, and finally the bands were visualized using an Ubiquitination assay ECL kit (Millipore). Signals were quantified by Image-J MCF-7 cells were transiently transfected with the indi- software and normalized to β-tubulin. cated plasmids and treated with MG132 (10 µM) for 8 h prior to cell lysis and in a hypoxic workstation with 1% O2 Lactate production assay and LDH activity assay for 24 h. Subsequently, cells were washed with ice-cold Cells were seeded on six-well plates at a density of 1 × PBS and lysed in ice-cold RIPA lysis buffer containing 105 cells per well, and the culture medium was changed to protease and phosphatase inhibitors. Next, cell lysates fresh DMEM medium after incubation overnight. Lactate were cleared by a spin (13,000 rpm, 15 min, 4 °C) and a concentrations in the culture medium were collected and fixed amount of whole-cell lysates was incubated with the measured after 24 or 48 h using a Lactate Assay Kit HIF1α antibodies (1:1000 dilution) from Abcam (Cam- (Jiancheng Bioengineering, Nanjing, China). Meanwhile, bridge, MA) overnight. Then bound proteins were eluted the cells were lysed in isolation buffer provided in the in moderate 2× SDS sample buffer at 100 °C for 10 min LDH activity assay kit (Jiancheng Bioengineering, Nanj- and subjected to western blot analysis. ing, China), and the LDH activity was determined according to the manufacturer’s instructions. Total Protein stability and half-life assay enzyme activity was normalized to the protein content of MCF-7 cells were transiently transfected with the indi- the cell lysate. cated plasmids. Twenty-four hours later, the cells were treated with 10 µM MG132 (Selleckchem, Houston, USA) Measurement of ATP content and pyruvate and 150 μM CoCl2 (Sigma-Aldrich, St. Louis, MO, USA) dehydrogenase activity prior to cell lysis for indicated time (0, 4, 8, 12, 16 h). To The ATP concentration was measured by a firefly test the half-life of HIF1α, the transfected MCF-7 cells luciferase-based Bioluminescence ATP assay kit (Beyo- were firstly treated with 150 μM CoCl2 for 24 h, then the time Biotechnology) following the manufacturer’s proto- cells were incubated with 50 μg/ml cycloheximide (CHX; col21. For immune-captured pyruvate dehydrogenase MedChemExpress, USA), a kind of protein synthesis (PDH) activity, the pyruvate dehydrogenase Elisa assay inhibitor, for the indicated periods of time (0, 1, 3, 6 h). (Jiancheng Bioengineering, Nanjing, China) was used Finally, the above whole-cell lysate was subjected to according to the manufacturer’s instructions22. The PDH western blot analysis. activity and ATP concentration detected by using a PARADIGM Detection Platform (Beckman, Brea, CA). Western blot Values were normalized to the protein content of the cell Protein was extracted from indicated cells by using lysate and expressed as a fold increase over vector control. RIPA lysis buffer. Protein concentrations were determined using a BCA Protein Assay kit (Thermo Fisher Cell viability measurement Scientific, Rockford, IL, USA). A total of 60 μg of protein One thousand cells were seeded into each well of a was separated on a 10% SDS-PAGE gel and transferred 96-well plate and cultured for 0, 24, 48, 72, and 96 h onto polyvinylidene difluoride membranes (Millipore, continuously. At each time point, 200 μl of MTT (Sigma- Billerica, MA, USA), which were soaked in 5% nonfat milk Aldrich, St. Louis, MO, USA) at a 0.5 mg/ml final con- for 1 h and then incubated with corresponding primary centration was added into each well and incubated for 4 h antibodies overnight at 4 °C. Antibodies used in this at 37 °C. Then 150 μl of DMSO (Sigma-Aldrich) was study: rabbit polyclonal anti-BRD7 (1:500 dilution), rabbit added to each well and incubated for 10 min at room polyclonal anti-LDHA (1:20,000 dilution), and rabbit temperature. Finally, the optical density (OD) at 490 nm polyclonal anti-β-Tubulin (1:3000 dilution) from Pro- was detected by using a PARADIGM Detection Platform teintech (Wuhan, China); rabbit polyclonal anti-t-PARP (Beckman, Brea, CA). The experiments were repeated (1:1000 dilution), rabbit polyclonal anti-c-PARP (1:500 three independent times. dilution), and rabbit polyclonal anti-p21 (1:1000 dilution) from Cell Signaling Technology (Danvers, MA); rabbit Colony formation assay polyclonal anti-Cyclin D1 (1:500 dilution), rabbit poly- A total of three hundred cells in each group were added clonal anti-CDK4 (1:500 dilution), and rabbit polyclonal to each well of a six-well plate in triplicate for 10–12 days.

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Then, the colonies were fixed and stained with crystal approximately 200–240 bp length. The primers of each violet. The colony number was counted in each group. fragments are as follows: P1: 5′-TGGCCCACCCCTAA- Each experiment had three duplicates and was conducted ACTTTC-3′ (Forward), 5′-ACTCATGCCTGTAATCC- independently. CAGCA-3′ (Reverse); P2: 5′-GCTGGGATTCAGGC ATGAGT-3′ (Forward), 5′-AGATAGCCTCAGGAAGGA- Cell cycle analysis GC-3′ (Reverse); P3: 5′- GCTCCTTCCTGAGGCTATCTA- Cells from each group were washed with cold PBS and 3′ (Forward), 5′-ACCTCTGGGCCTGTATTCT T-3′ collected. The cells were fixed in 70% alcohol at 4 °C (Reverse); P4: 5′-CAAGAATACAGGCCCAGAGG-3′ (For- overnight. Then, the cells were treated with RNase A ward), 5′-CCTGCC TCAGCCTCCTGAG-3′ (Reverse); P5: (TaKaRa, Japan) for 30 min at 37 °C and stained with 5′-TTCTCAGGAG GCTGAGGCAG-3′ (Forward), 5′- propidium iodide (PI) (KeyGEN Biotech) for 15 min, ATGGCTTCCTGCAGCCTCG-3′ (Reverse); P6: 5′- protected from light. The cell cycle was analyzed by using GCTTGTAGT CCCAGTCACTTCG-3′ (Forward), BD LSRFortessa (Becton, Dickinson and Company) and 5′-ACCCCACTGGCATCTCATCTC-3′ (Reverse); P7: 5′- Mod fit LT software (Verity Software House, Topsham, CTGACAGTTCTTGGGTATGGTTG-3′ (Forward), 5′- ME). This assay was repeated three independent times. GAAG AAAATGCCTAGCAT CAGTC-3′ (Reverse); P8: 5′-CTGACTGCTAGGCATTTTC TTC-3′ (Forward), Cell apoptosis assay 5′-CAACCACA GAAGCCGGGGT-3′ (Reverse); P9: 5′- An Annexin V-PE/7-AAD Apoptosis Detection Kit (BD, ACT CAGGCTCATGGCTCCG-3′ (Forward), 5′-AGC- Biosciences) was used to detect cell apoptosis. After the TATGCTGACGTCAGCGG-3′ (Reverse). The primer indicated treatment, the cells were collected and washed pair from the binding region of BIRC2, 5′-CACAGTGG twice with cold PBS and then resuspended in 100 μlof1× TTGCTGTCAAGG-3′ (Forward) and 5′-CTGGCCC- binding buffer. A volume of 5 μl of Annexin V-PE and 5 μl TTTAACCCTGTCT-3′ (Reverse) was used as a positive of 7-AAD was added, mixed well, and incubated for 10 control5. The PCR products were resolved electro- min, protected from light. Cells were analyzed using BD phoretically on a 1.5% agarose gel and visualized using LSRFortessa (Becton, Dickinson and Company, Franklin ethidium bromide staining. Lakes, New Jersey, USA). This assay was repeated three independent times. Immunofluorescence staining For detection of co-localization of BRD7 and HIF1α, Luciferase reporter gene assays after fixation with 4% paraformaldehyde and permeabili- A 1.8 kb LDHA promoter sequence from −1683 to zation in 0.3% TritonX-100, cells were incubated with the +138 bp relative to the transcription initiation site was indicated BRD7 and HIF1α antibodies (1:200 dilution) for inserted into the pGL3-echancer vector (Promega) to 16 h at 4 °C. The samples were immunostained with Alexa generate a recombinant luciferase reporter gene (pluc- Fluor 488-conjugated anti-rabbit IgG polyclonal (1:1000 LDHA). HEK293, MCF-7, and MDA-MB-231 cells were dilution) and Alexa Fluor 594-conjugated anti-mouse IgG transfected with the pLuc-LDHA alone or co-transfected (1:1000 dilution) from Life Technology (Carlsbad, CA, with a BRD7 expressed plasmid or vector control. The USA), for 1 h at 37 °C. Then cells were incubated with cells transfected with empty pGL3-echancer vector were 4′,6-diamidino-2-phenylindole (DAPI; 1:1000 dilution) for used as a control. At 36 h after transfection, cells were 10 min at room temperature. Images were collected using collected and lysed. A volume of 100 μl of the super- a confocal fluorescence microscope from UltraView Vox, natants was used to detect luciferase activities using a PerkinElmer, USA. luciferase reporter gene assay kit (Promega, Madison, WI, USA) on the PARADIGM Detection Platform (Beckman, In vivo tumorigenicity assays Brea, CA). Xenograft tumorigenicity assays were approved by the Institutional Animal Care and Use Committee of Central Chromatin immunoprecipitation assay South University (Changsha, China). Five-week-old MCF-7 cells were crosslinked in 1% formaldehyde for female athymic nude mice were divided into three 10 min at 37 °C. DNA from fixed chromatin cells was then groups, each group containing five female athymic nude subjected to immunoprecipitation using a ChIP assay kit mice. Mice were injected intramuscularly with 0.2 mg of (Millipore) and antibodies against Flag (Sigma-Aldrich, St. 17-β-estradiol in 10 μl of ethyl alcohol (Tokyo Chemical Louis, MO, USA) or anti-mouse IgG (Santa Cruz, Dallas) Industry, Tokyo, Japan) every 2 days for 6 days before according to the manufacturer’s protocol. On the basis of injection of MCF-7 cells. The three groups of MCF-7 the requirement of the ChIP assay, the 1.8-kb promoter cells were prepared as described in the above in intro region of LDHA was divided into nine fragments based on experiments, including MCF-7 cells transfected with the DNA walking method. Each of the fragments was pIRES2-EGFP-3Flag/BRD7 alone, co-transfected with

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Fig. 1 BRD7 expression was negatively correlated with clinical stage and poor prognosis in breast cancer. a Representative images of immunohistochemistry staining for BRD7 in non-cancerous breast tissues and different clinical TNM stage I–IV breast cancer tissues. Upper bar = 200 μm; lower bar = 50 μm. b The score of BRD7 expression in normal adjacent tissues and breast cancer tissues. c The relative BRD7 expression in normal adjacent tissues and different clinical TNM stages of breast cancer tissues. d Kaplan–Meier curves showed worse overall survival rates for breast cancer patients with low expression of BRD7 compared to patients with high expression of BRD7 (P = 0.0004). Data are expressed as the mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001 pIRES2-EGFP-3Flag/BRD7 and pENTER/LDHA, and the statistical analysis. The data were presented as the mean cells transfected with pIRES2-EGFP and pENTER were ± standard deviation. The contingency data were analyzed used as a control. After the indicated treatment, 5 × 106 using the chi-squared test. The OS estimates over time MCF-7 cells in 150 μl of normal saline were injected were calculated using the Kaplan–Meier method with log- subcutaneously into the subaxillary of the right forelimb. rank test. Student’s t-test and ANOVA were used Mice were examined, and tumor volumes were measured depending on experimental conditions. A value of P < 0.05 every 2 days. Twenty days after injection, all mice were was considered to be statistically significant. sacrificed to examine the final tumor weight and the expression of key molecules related to cell cycle and Results apoptosis. The tumor volume was equivalent to 1/2 Low expression of BRD7 predicts a poor prognosis in (length × width2). Tumor weight was also recorded when patients with breast cancer mice executed. All animals were allowed access to stan- We detected the expression of BRD7 in breast cancer dard chow diet and water ad libitum and were housed in a and non-cancerous breast tissues by immunohistochem- pathogen-free barrier facility with a 12 L:12D cycle. The istry and the clinicopathologic characteristics of breast mice were sacrificed by CO2 asphyxiation. cancer patients are shown in Table 1. As a result, BRD7 was diffusely expressed in the cytoplasm and nucleus of Statistical analysis breast cancer cells but mainly expressed in the nucleus GraphPad Prism 6 software (Graphpad Software, Inc., (Fig. 1a). The average expression of BRD7 in breast cancer La Jolla, CA, USA) and SPSS 18.0 were used to perform tissues was lower than that in the non-cancerous breast

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Table 2 Association between BRD7 expression and BRD7 expression was significantly associated with the clinicopathologic characteristics in breast cancer tumor size (P = 0.025), clinical TNM stage (P = 0.002), ER expression (P = 0.02), and HER2 expression Variables BRD7 expression χ2 test P (P = 0.039). We also analyzed the association between High (score≥1), Low (score<1), BRD7 expression and survival of the patients. All 225 n=132 n=93 breast cancer patients were included in the survival curves. The survival time ranged from 2 to 120 months. Age 46.13 ± 0.92 (58.7) 46.73 ± 0.94 (41.3) 0.65 The OS rate of patients with high BRD7 expression is Sex (%) 1.00 significantly higher than the survival of patients with low P = = Male 1 (0.7) 0 (0) BRD7 expression ( 0.0004, Hazard ratio 2.83, 95% CI of ratio: 1.59 to 5.05, Fig. 1d). These results indicate Female 131 (99.3) 93 (100) that BRD7 has an essential role in the development of Tumor size (%) 0.025* breast cancer. T1–2 91 (68.9) 50 (53.7) T3–4 41 (31.1) 43 (46.2) Ectopic expression of BRD7 inhibits cell proliferation and induces apoptosis Nodal metastasis (%) 0.87 To further investigate the tumor suppressive role of Present 97 (73.5) 70 (75.3) BRD7 in breast cancer, we firstly detected the expression Absent 35 (26.5) 23 (24.7) of exogenous BRD7 and the effect of ectopic expression of Distant metastasis (%) 0.061 BRD7 on cell proliferation and apoptosis in breast cancer MCF-7 and MDA-MB-231 cells. The result showed a Present 8 (6.1) 13 (14.0) significant increase in BRD7 expression compared with Absent 124 (93.9) 80 (86.0) the control group (Fig. 2a). As we expected, over- TNM stage (%) 0.002** expression of BRD7 significantly inhibited cell prolifera- I–II 98 (74.2) 50 (53.8) tion and colony formation (Fig. 2b, c), arrested cell cycle progression at the G1/S phase (Fig. 2d), and initiated III–IV 34 (25.8) 43 (46.2) apoptosis (Fig. 2e) in both MCF-7 and MDA-MB-231 cell ER expression (%) 0.021* lines. As BRD7 is correlated with HER2 (Table 2), while Positive 78 (59.1) 40 (43.0) both of MCF-7 and MDA-MB-231 are HER2-negative Negative 54 (40.9) 53 (57.0) cells, we chose another HER2-positive MDA-MB-453 cell line to repeat our results. As a result, ectopic expression of PR expression (%) 0.227 BRD7 significantly inhibited cell proliferation, arrested Positive 72 (54.5) 43 (46.2) cell cycle progression at the G1/S phase, and initiated Negative 60 (45.5) 50 (53.8) apoptosis (Supplementary Fig. 2a–c) in MDA-MB-453 HER2 expression (%) 0.039* cells, which is consistent with the results shown in HER2- negative breast cancer cells. Altogether, these results Positive 71 (53.8) 63 (67.7) indicate that BRD7 functions as a tumor suppressor Negative 61 (46.2) 30 (32.3) whether in HER2-negative or positive breast cancer TNM Tumor Node Metastasis, ER estrogen receptors, PR progesterone receptors, cells. HER human epidermal growth factor receptor-2, P pathological. *P < 0.05, **P < 0.01 Effects of altered BRD7 expression on aerobic glycolysis We detected the mRNA and protein expressions of control tissues (Fig. 1b), and the percentage of high BRD7 BRD7 in MCF-7 and MDA-MB-231 cells transfected with expression in the breast cancer and the non-cancerous BRD7 siRNAs (siRNA #1 or siRNA#2). The results show breast tissue was 58.7% (132/225) and 83.6% (51/61), that the expression of BRD7 was significantly reduced in respectively. In addition, the expression of BRD7 MCF-7 and MDA-MB-231 cells compared with the con- decreased with an increase in the clinical TNM stage of trol group (Fig. 3a, b). To determine the effect of altered breast cancer patients (Fig. 1a, c and Table 2). BRD7 expression on aerobic glycolysis in breast cancer We further investigated the association between the cells, overexpression experiment of BRD7 were performed expression of the BRD7 protein and clinicopathological in HER2-negative MCF-7 and MDA-MB-231 cells and features of breast cancer. As shown in Table 2, no sig- HER2-positive MDA-MB-453 cells. As a result, ectopic nificant differences were observed between BRD7 protein expression of BRD7 decreased lactate production, LDH expression and age, gender, lymph node metastasis, or activity and increased ATP production, PDH activity in PR expression of breast cancer (P > 0.05). However, breast cancer cells (Fig. 3c, e, g, supplementary 3a–c).

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Fig. 2 BRD7 inhibits cell proliferation and induces cell apoptosis in breast cancer cells. a Western blot analysis of the expression of BRD7 in MCF-7 (left) and MDA-MB-231 (right) cells transfected with pIRES2-EGFP-3Flag/BRD7. b MTT assay in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 or pIRES2-EGFP. c Colony formation assay was used to determine the colony forming ability in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 or pIRES2-EGFP and quantification. d Cell cycle analysis in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 or pIRES2-EGFP and quantification. e Flow cytometric analysis for cell apoptosis in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 or pIRES2-EGFP and quantification. Data are expressed as the mean ± S.D. **P < 0.01, ***P < 0.001 vs vector

Conversely, loss of BRD7 in MCF-7 and MDA-MB-231 BRD7 functions as a negative regulator of LDHA cells did the opposite (Fig. 3d, f, h). Several studies have LDHA is a key checkpoint of glycolysis that catalyzes the reported glycolytic inhibitors, 2-DG and oxamate, are conversion of pyruvate to lactate24. We previously showed useful in the treatment of solid tumors by attacking that inhibition of LDHA by inhibitors such as oxamate or anaerobic cells23. We wondered whether BRD7 expres- siRNA can enhance the sensitivity of drug-resistant breast sion is correlated with the sensitivity of cells to glycolytic cancers to trastuzumab or taxol treatment25,26. To inves- inhibitors. As expected, overexpression of BRD7 tigate whether dynamic expression of BRD7 could impact enhanced the inhibitory effects of these two glycolytic the expression of the key glycolysis gene LDHA, we ana- inhibitors on cell proliferation (Fig. 3i, j), and knockdown lyzed the mRNA and protein expression of LDHA using BRD7 presented the the opposite results in MDA-MB-231 qPCR and western blotting analysis in MCF-7, MDA-MB- cells (Supplementary Fig. 3d, e). Therefore, the above 231, and MDA-MB-453 cells after ectopic expression of results demonstrate that BRD7 could reduce aerobic BRD7. The results demonstrate that the mRNA and pro- glycolysis and increase the sensitivity of breast cancer cells tein levels of LDHA in BRD7-overexpressed breast cancer to glycolytic inhibitors. cells were signiﬁcantly lower than those in control cells

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Fig. 3 BRD7 inhibits lactate production and LDH activity in breast cancer cells. a qPCR analysis of the expression of BRD7 in MCF-7 and MDA- MB-231 cells transfected with BRD7 siRNAs (siRNA #1 or siRNA#2). b Western blot analysis of the expression of BRD7 in MCF-7 (left) and MDA-MB-231 (right) cells transfected with BRD7 siRNAs (siRNA #1 or siRNA#2). c The lactate concentrations were determined in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 or pIRES2-EGFP. d The lactate concentrations were determined in MCF-7 and MDA-MB-231 cells transfected with BRD7 siRNA pools (siRNA#1 and siRNA#2) or scramble siRNA (NC). e LDH activity was determined in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 or pIRES2-EGFP. f The LDH activity was determined in MCF-7 and MDA-MB-231 cells transfected with BRD7 siRNA pools (siRNA#1 and siRNA#2) or scramble siRNA (NC). g ATP concentrations and PDH activity was determined in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 or pIRES2-EGFP. h ATP concentrations and PDH activity were determined in MCF-7 and MDA-MB-231 cells transfected with BRD7 siRNA pools (siRNA#1 and siRNA#2) or scramble siRNA (NC). i MCF-7 and MDA-MB-231 cells with ectopic expression of BRD7 were treated with oxamate sodium (Oxamate) for 48 or 72 h. The cell viability was measured using an MTT assay, and the cells transfected with pIRES2-EGFP were used as a negative control. j MCF-7 and MDA-MB-231 cells with ectopic expression of BRD7 were treated with various concentrations of 2-DG for 48 h. The cell viability was measured using an MTT assay, and the cells transfected with pIRES2-EGFP were used as a negative control. Data are presented as the percentage of viability inhibition. *P < 0.05, **P < 0.01, ***P < 0.001

(Fig. 4a, b, Supplementary Fig. 1a–c). Conversely, regulates LDHA expression at both the mRNA and pro- BRD7 silencing upregulated the expression of LDHA in tein levels in breast cancer cells. MCF-7 and MDA-MB-231 cells (Supplementary Fig. 1d, To further explore the mechanism of BRD7 regulating e). Thus, our data clearly reveal that BRD7 negatively LDHA expression, we ﬁrst analyzed the promoter

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Fig. 4 BRD7 inhibits the expression of LDHA at the mRNA and protein level in breast cancer cells. a qPCR analysis of the expression of BRD7 (left) and LDHA (right) in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 or pIRES2-EGFP. b Western blot analysis of the expression of BRD7 and LDHA in MCF-7 (left) and MDA-MB-231 (right) cells transfected with pIRES2-EGFP-3Flag/BRD7. c The dual-luciferase reporter system assay evaluated the activity of the potential LDHA promoter in MCF-7, MDA-MB-231, and HEK293 cells. d The dual-luciferase reporter system assay evaluated the effect of overexpression of BRD7 on the LDHA promoter activity in MCF-7, MDA-MB-231, and HEK293 cells. e A schematic diagram showing the location of the LDHA potential promoter region on chromosome 11 and the locations of the PCR primers for the ChIP assay. f ChIP-PCR analysis of BRD7 binding of fragments of the LDHA potential promoter sequence. BIRC2 was used as a positive control. P1–P9 represent the nine fragments from the LDHA potential promoter, respectively. The error bars are presented as the mean ± S.E.M. **P < 0.01, ***P < 0.001

sequence of LDHA according to the literatures reported, any region of the nine fragments of this LDHA promoter and found that the sequence from −1683 to 138 bp might (Fig. 4f), suggesting that BRD7 negatively regulates LDHA be the potential promoter region, and thus constructed a expression in an indirect manner. luciferase reporter plasmid integrated with this sequence (pLuc-LDHA). We then transfected pLuc-LDHA alone or BRD7 destabilizes HIF1α through the co-transfected the BRD7 expression plasmid and pLuc- ubiquitin–proteasome pathway LDHA into HEK293, MCF-7, and MDA-MB-231 cells. As To find out the candidate transcription factors that expected, the region from −1683 to 138 bp has significant could directly regulate LDHA expression and be simul- luciferase activity compared to the negative control taneously regulated by BRD7, we consulted the related (Fig. 4c), and upregulation of BRD7 greatly decreased the literature and designed series of experiments. It is inter- luciferase activity of the LDHA promoter (Fig. 4d). These esting to find that overexpression of BRD7 can decreased results indicate that BRD7 negatively regulates LDHA HIF1α protein levels with no effect on HIF1α mRNA expression through inhibiting the activity of the LDHA levels under hypoxia condition (Fig. 5a, b). Meanwhile, promoter at the transcriptional level. Then we further HIF1α has been reported to be a critical oncogenetic explored the exact region that BRD7 binds to in the transcription factor that directly regulates LDHA tran- LDHA promoter. The promoter sequence of LDHA was scription14. Consistent with previous results, over- divided into nine fragments based on the DNA walking expression of HIF1α could upregulate the mRNA and method, and each of the fragments was approximately protein expression of LDHA in MCF-7 cells (Supple- 200–240 bp in length (Fig. 4e). The ChIP-PCR analysis mentary Fig. 4a, b). HIF1α is extremely unstable in protein showed that BRD7 could directly bind to the transcrip- level for bearing the ubiquitin–proteosome degradation tional regulation region of BIRC2, which was used as a in normoxic conditions. Hypoxia and CoCl2 were able positive control in this experiment5, but could not bind to to stabilize HIF1α protein levels and lead to its rapid

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Fig. 5 BRD7 destabilizes HIF1α through the ubiquitin-mediated degradation. a The mRNA expression of BRD7, LDHA, HIF1α, and β-tubulin were analysised by reverse transcription-PCR (RT-PCR) under hypoxia in BRD7-overexpressing cells. b Western blot analysis of BRD7/Flag, HIF1α, and

β-tubulin expression in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 under hypoxia (1%O2) for 24 h. c Western blot analysis of HIF1α and β-tubulin expression in MCF-7 cells transfected with pIRES2-EGFP-3Flag/BRD7 and treated with CoCl2 (150 μM) and MG132 (10 μM) for the indicated periods of time (0, 4, 8, 12, 16 h) (left). Quantitative results also were shown (right). d MCF-7 cells transfected with pIRES2-EGFP-

3Flag/BRD7 or pIRES2-EGFP for 24 h and treated with CoCl2 (150 μM) for 24 h. Then the cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated periods of time (0, 1, 3, 6 h) (left). Lysates were harvested from the cells and analyzed by western blotting (left panel). Quantitation of HIF1α protein levels were shown in the right pane. e The MCF-7 cells transfected with pIRES2-EGFP-3Flag/BRD7 and pCDNA3.1-HIF1α were under

hypoxia (1% O2) for 24 h. Cellular co-localization of BRD7 and HIF1α in MCF-7 cells was analyzed by immunofluorescence staining with anti-BRD7 and anti-HIF1α under hypoxia (1% O2) for 24 h. f MCF-7 cells transfected with the indicated pIRES2-EGFP-3Flag/BRD7 and pCDNA3.1-HIF1α were subject to immunoprecipitation (IP) respectively with anti-Flag antibodies (upper) and anti-HIF1α (down) under hypoxia (1% O2) for 24 h. g The MCF-7 cells transfected with pIRES2-EGFP-3Flag/BRD7 and pCDNA3.1-HIF1α were under hypoxia (1%O2) for 24 h before collection to immunoprecipitation (IP) with anti-HIF1α antibodies accumulation27. In addition, BRD7 decreased LDHA compared to the control (Fig. 5c). To prove that BRD7 expression in protein level, and restoring the expression of affects HIF1α protein stability, we treated the cells with HIF1α in the BRD7-overexpressing cells could recover the protein synthesis inhibitor CHX, and found that the LDHA protein level (Suplementary Fig. 4c). These results half-life of HIF1α protein was obviously shortened in suggest that BRD7 decreases LDHA expression through BRD7-overexpressing cells (Fig. 5d). These results inactivation of the HIF1α/LDHA transcription axis. demonstrate that BRD7 destabilizes HIF1α protein We then explored the mechanism by which BRD7 through proteasome system. To investigate the mechan- downregulates the protein levels of HIF1α. As a result, in ism of BRD7 destabilizing HIF1α protein, we assessed the the presence of proteasome inhibitor MG132, HIF1α co-localization of BRD7 and HIF1α by confocal micro- protein levels were strongly blocked over time and BRD7 scopy. Interestingly, the co-localization was observed promote the degradation of HIF1α protein under the between green fluorescent protein BRD7 and red fluor- condition of hypoxia treated with CoCl2 in MCF-7 cells escent protein HIF1α in MCF-7 cells during hypoxia

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Fig. 6 (See legend on next page.)

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(see figure on previous page) Fig. 6 Overexpression of LDHA rescues the suppressive effects of BRD7 on MCF-7 and MDA-MB-231 cells. a MTT assay was used to determine the cell viability in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7, or co-transfected with pIRES2-EGFP-3Flag/BRD7 plus pENTER/LDHA. b Colony formation assay was used to determine the colony-forming ability in MCF-7 and MDA-MB-231 cells transfected with pIRES2- EGFP-3Flag/BRD7, or co-transfected with pIRES2-EGFP-3Flag/BRD7 plus pENTER/LDHA and quantification. c Cell cycle analysis by flow cytometry in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7, or co-transfected with pIRES2-EGFP-3Flag/BRD7 plus pENTER/LDHA and quantification. d Cell apoptosis analysis by flow cytometry in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7, or co- transfected with pIRES2-EGFP-3Flag/BRD7 plus pENTER/LDHA and quantification. The cells transfected with pIRES2-EGFP + pENTER were used as a control. The data are presented as the mean ± S.E.M. **P < 0.01, ***P < 0.001. All experiments were performed in triplicate

(Fig. 5e). Subsequently, co-IP applied to verify protein cycle and apoptosis. As expected. ectopic expression of interaction between BRD7 and HIF1α. The result showed BRD7 increased the expression of c-PARP and p21, and that BRD7 and HIF1α were co-immunoprecipitated with decreased the expression of cyclin D1 and CDK4 com- each other (Fig. 5f). Furthermore, poly-ubiquitinated pared with the control group. Meanwhile, restoring the HIF1α was upregulated in the BRD7-overexpressing expression of LDHA at least partially recovered the MCF-7 breast cancer cells (Fig. 5g), indicating that expression of the above molecules related to the cell cycle BRD7 promoted the degradation of HIF1α in a and apoptosis (Fig. 7a and Supplementary Fig. 2d). In ubiquitination-dependent manner. Collectively, these contrast, the silencing of BRD7 had the opposite effect, results suggest that BRD7 downregulates LDHA expres- and restoring the expression of LDHA in BRD7 knock- sion through increasing HIF1α ubiquitination, which down cells also recovered the expression of cell cycle promotes the degradation of HIF1α by the proteasome. and apoptosis related molecules (Fig. 7b). These data demonstrate that BRD7 functions as a tumor suppressor LDHA antagonizes the effects of BRD7 on glycolysis and through an LDHA-dependent mechanism, suggesting that cell growth the tumor suppressive role of BRD7 in breast cancer cells As LDHA is a key enzyme that catalyzes the conversion might be associated with aerobic glycolysis. of pyruvate to lactate, we further examined whether LDHA plays a critical role in BRD7-mediated inhibition of LDHA can rescue the tumor suppressive effect of BRD7 on aerobic glycolysis. As a result, BRD7-mediated inhibitory tumor growth in vivo effects on aerobic glycolysis, lactate production, and LDH Since BRD7 inhibits cell proliferation through regula- activity were clearly attenuated by ecoptic expression of tion of LDHA expression in vitro, MCF-7-xenografted LDHA in BRD7-overexpressing cells. Furthermore, tumor models were performed to corroborate the above restoring the expression of LDHA in BRD7 knockdown results. As a result, upregulation of BRD7 significantly cells completely recovered the effects of BRD7 silencing suppressed the tumor growth compared with the control on glycolysis (Supplementary Fig. 5). These results suggest group, while LDHA restoration dramatically increased the that LDHA is critical for BRD7-mediated inhibition of tumor growth rate and weight of the xenografted tumors aerobic glycolysis. compared with those of the MCF-7/BRD7 group (Fig. 7c, Since BRD7 could significantly inhibit cell proliferation d). We also examined the expression of BRD7, LDHA, and initiate apoptosis in breast cancer cells, we further Cyclin D1, and p21 by immunohistochemistry analysis. investigate the effect of LDHA on BRD7-mediated tumor Consistent with the results of experiments in vitro, BRD7 suppressive roles. Intriguingly, restoring the expression of overexpression increased the protein level of p21 and LDHA in BRD7-overexpressed breast cancer cells appar- c-PARP and decreased the expression of Cyclin D1 ently recovered the inhibitory roles of BRD7 in regulating and Ki67, while restoring the expression of LDHA in cell proliferation, colony formation (Fig. 6a, b), and cell BRD7-overexpressing MCF-7 cells reversed the expres- cycle progression from the G1 to S phase (Fig. 6c) sion of the above molecules related to cell proliferation compared with the BRD7-overexpressed MCF-7 and and apoptosis (Fig. 7e). MDA-MB-231 cells. Moreover, restoration of LDHA in BRD7-overexpressed breast cancer cells also reversed Discussion BRD7-induced cell apoptosis (Fig. 6d). The same result BRD7 was initially identified as a tumor suppressor in was obtained in MDA-MB-453 cells (Supplementary NPC by involved in multiple pathways such as ras/MEK/ Fig. 2a–c). Accordingly, depletion of LDHA suppressed ERK, Rb/E2F, and Wnt/beta-catenin, and might be a the effects of BRD7 silencing on cancer cell growth, cell prognostic biomarker28,29. Here, we demonstrated that cycle, and apoptosis (Supplementary Fig. 6). Furthermore, BRD7 is downregulated in breast cancer tissues compared we also examined several key molecules related to the cell with normal tissues and that low expression of BRD7 is a

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Fig. 7 BRD7 regulates the expression of key molecules related to the cell cycle and apoptosis and inhibits tumor growth in vivo through regulation of LDHA. a Western blot analysis of BRD7, LDHA, c-PARP, t-PARP, Cyclin D1, CDK4, and P21 in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 or co-transfected with pIRES2-EGFP-3Flag/BRD7 plus pENTER/LDHA. The cells transfected with pIRES2-EGFP + pENTER were used as a control. b Western blot analysis for BRD7, cleaved-PARP, P21, and Cyclin D1 in MCF-7 and MDA-MB-231 cells transfected with BRD7 siRNA pools (siRNA #1 and siRNA#2), or co-transfected with BRD7 siRNA pools (siRNA #1 and siRNA#2) plus LDHA siRNA. The cells transfected with scramble control were used as a negative control (NC). c The cells with ectopic expression of BRD7 used for in vitro experiments were sorted by GFP-mediated flow cytometry technology to ensure the transfection efficiency. As for the recovery experiment for LDHA, the sorted cells with ectopic expression of BRD7 were further transfected with pENTER/LDHA or pENTER. Then the indicated cells were injected into nude mice. Measurement of the tumor weight across groups. d The tumor volume was monitored every 2 days after injection.The tumors were obtained from the mice. e Representative images of immunohistochemical staining for BRD7, LDHA, p21, and Cyclin D1, and the markers of proliferation and apoptosis Ki67 and c-PARP in tumor tissues, and quantification of integral optical density (IOD).The error bars are presented as the S.E.M. **P < 0.01, ***P < 0.001. All experiments were performed in triplicate

poor prognostic factor in breast cancer. Moreover, over- exhibits crucial roles in tumor progression31. As yet, there expression of BRD7 in breast cancer cells exhibits a dra- have been no reports demonstrating the association matic tumor suppressive effect assessed by a series of between BRD7 and glycolysis in cancer cells. In our study, in vitro and in vivo experiments. Thus, these ﬁndings we demonstrated that ectopic expression of BRD7 suggest that BRD7 is a novel tumor suppressor in breast reduced LDH activity and lactate production, and cancer. increased ATP production and PDH activity, while One of the key characteristics of tumor cells is dereg- reduced expression of BRD7 did the opposite, suggesting ulating cellular energetics30. It has been reported that that BRD7 is a critical negative regulator of glycolysis in aberrant expression and activity of metabolic enzymes breast cancer. Dysregulated metabolism, which is con- contribute to the development and progression of cancers sidered a hallmark of cancer, is frequently observed in and that they are hallmarks of cancer. An increasing breast cancer and is associated with tumor progression amount of evidence has conﬁrmed that aerobic glycolysis and drug resistance in cancer therapy32. Genetic

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regulators such as c-Myc and PI3k/Akt regulate meta- polyubiquitination and subsequent proteasomal degrada- – bolism as well as the ER pathway33 35. tion48,49. Therefore, whether BRD7 facilitates HIF1α In our study, we found that BRD7 overexpression could degradation by promoting the function of VHL or pro- decrease LDHA expression through indirect effect on the moting the hydroxylation of HIF1α remains to be further activity of LDHA promoter at the transcriptional level studied. validated by chromatin immunoprecipitation. Therefore, Furthermore, our study shows that overexpression of the problem to be solved is how BRD7 decreases LDHA in BRD7 in breast cancer cells exhibits the tumor suppres- transcription level. LDHA is the enzyme that catalyzes the sive effects that are assessed by cell viability, cell cycle, conversion of pyruvate to lactate, which is considered to apoptosis, and xenograft tumor model, and restoring the be a key checkpoint of glycolysis24. Several transcription expression of LDHA could reverse the inhibitory effect of factors, including HIF1α, KLF4, and FOXM1, have been BRD7 on cell proliferation, apoptosis, and tumor formation reported to regulate LDHA expression, which results in a in breast cancer cells both in vitro and in vivo. It has been – shift towards the Warburg effect36 38. And HIF1α plays a reported that LDHA was upregulated in clinical samples of vital role in coordinating the activities of many tran- esophageal squamous cell carcinoma and that LDHA scription factors and signaling molecules, which impacts knockdown could inhibit cell growth and migration in vitro tumor growth and angiogenesis39,40. The protein levels of and tumorigenesis in vivo by increasing cleavage of PARP HIF1α are degraded through the ubiquitination- and caspase 8 and downregulating cyclin D1 and activation dependent proteasomal pathway by von Hippel-Lindau of AKT50,51. Therefore, BRD7 acts its tumor suppressive (VHL)-mediated polyubiquitination and subsequent pro- function through inhibition of LDHA expression and its teasomal degradation in normoxia41,42. In recent years, downstream critical molecules or pathway. lots of reports illuminate various transcription factors take The mechanisms that alter the bioenergetic metabolism – participate in polyubiquitination degradation of HIF1α43 in cancer cells are still not fully understood. However, it is 46. The ER is a ligand-activated transcription factor, and generally accepted that increased glycolysis offers cancer its could induce cell proliferation in breast cancer cells. cells an advantage to allow them to better proliferate, The ER is a ligand-activated transcription factor, and its survive, and invade. We show that BRD7 inhibits cancer activation could induce cell proliferation in breast cancer cell glycolysis and growth by targeting glycolysis-related cells. Our results show that the expression of BRD7 is critical molecule LDHA. The upregulation of LDHA in associated with that of ER in breast cancer tissues shown BRD7-overexpressing cells can promote glycolysis and in Table 2, which fits with the finding that depletion of cell proliferation and inhibit cell apoptosis, but the BRD7 results in loss of ERα expression in breast cancer opposite result was obtained with the loss of LDHA in cells47, while ERα signaling pathway has also been BRD7 knockdown cells. 2-DG, a non-metabolizable glu- reported to directly regulate HIF1α expression9, appearing cose analog, competitively inhibits the production of BRD7 could positively regulate HIF1α through activation glucose 6-phosphate from glucose at the phosphoglucoi- of the ERα signaling pathway. However, BRD7 was found somerase level, which disrupts glycolysis and induces a to function as a tumor suppressor in breast cancer whe- state of glucoprivation. And oxamate is a structural analog ther in ER-positive breast cancer MCF-7 cells or ER- of pyruvate that directly inhibits the converting process of negative MDA-MB-231 and MDA-MB-453 cells, sup- pyruvate to lactate catalyzed by LDH, therefore, both of porting that BRD7 function as tumor suppressor inde- them inhibits cell glycolysis52. Thus, we further confirmed pendent of ER but other mechanism, including the the effect of BRD7 on the glycolysis-mediated growth regulation of BRD7 on HIF1α. As expected, BRD7 could inhibition, and found that ectopic expression of BRD7 decrease the protein stability of HIF1α via ubiquitin- increased the sensitivity of breast cancer cells to 2-DG and mediated proteasome pathway, and recovering the oxamate. Therefore, our data are consistent with the expression of HIF1α could reverse the expression of notion that increased glycolysis contributes to the main- LDHA, and thus restoring the expression of LDHA tenance of cancer cell malignant phenotypes, suggesting could reverse the inhibition effect of BRD7 on glucose that BRD7 plays suppressive roles in breast cancer mainly glycolysis and the tumor malignant phenotype, supporting or partially through the inhibition of glycolysis. that BRD7 inhibits the Warburg effect and tumor pro- In conclusion, we demonstrated that BRD7 inhibits gression through facilitating degradation of HIF1α medi- both aerobic glycolysis and tumor growth by blocking ated by ubiquitin–proteasome system. Under normoxia, HIF1α/LDHA axis in breast cancer. To our knowledge, HIF1α is hydroxylated on prolines 402 and 564 in the this is the first report that BRD7 downregulates oxygen-dependent degradation domain by dioxyOgenase LDHA through promoting HIFα degradation under prolyl hydroxylases, and this hydroxylated form can then hypoxic condition in human breast cancer cells, interact with the VHL protein, which is part of an E3 thereby suppressing the Warburg effect and preventing ubiquitin ligase complex, thus resulting in HIF1α tumorigenesis.

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Acknowledgements 10. Burrows, A. E., Smogorzewska, A. & Elledge, S. J. Polybromo-associated BRG1- This work was supported by the grants from the National Natural Science associated factor components BRD7 and BAF180 are critical regulators of p53 Foundation of China (grant Nos. 81772990 and 81572748), the Natural Science required for induction of replicative senescence. Proc. Natl. Acad. Sci. USA 107, Foundation of Hunan Province (grant no. 2015JJ2148), the National Key R&D 14280–14285 (2010). Program of China (grant no. 2016YFC1201803), the “111” project (grant no.111- 11. Drost, J. et al. BRD7 is a candidate tumour suppressor gene required for p53 2-12), the Fundamental Research Funds for the Central Universities of Central function. Nat. Cell Biol. 12,380–389 (2010). South University (grant no. 2016zzts114), and the Open-End Fund for the 12. Kim,Y.,Andres,S.H.M.,Herrema,H.,Delibasi,T.&Park,S.W.TheroleofBRD7 Valuable and Precision Instruments of Central South University (grant no. in embryo development and glucose metabolism. J. Cell Mol. Med. 20, CSUZC201743). 1561–1570 (2016). 13. Park, S. W. et al. BRD7 regulates XBP1s’ activity and glucose homeostasis Author details through its interaction with the regulatory subunits of PI3K. Cell Metab. 20, 1The Affiliated Tumor Hospital of Xiangya Medical School, Central South 73–84 (2014). University, Changsha, Hunan 410013, People’s Republic of China. 2The Key 14. Semenza, G. L. HIF-1: upstream and downstream of cancer metabolism. Curr. Laboratory of Carcinogenesis of the Chinese Ministry of Health, The Key Opin. Genet. Dev. 20,51–56 (2010). Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of 15. Iyer, N. V. et al. Cellular and developmental control of O2 homeostasis by Education, Xiangya Hospital, Central South University, Changsha, Hunan hypoxia-inducible factor 1α. Genes Dev. 12, 149 (1998). 410078, People’s Republic of China. 3Cancer Research Institute, Central South 16. MR, P., Wang, L. & CJ, H. STAT3 and HIF1α cooperatively activate HIF1 target University, Changsha, Hunan 410078, People’s Republic of China. 4The Second genes in MDA-MB-231 and RCC4 cells. Oncogene 33, 1670 (2014). Xiang-Ya Hospital, Central South University, Changsha, Hunan 410011, People’s 17. Zhao, Y. et al. Abstract 3208: overcoming cancer therapeutic resistance by Republic of China. 5Medical school of Xiangya, Central South University, targeting dysregulated glucose metabolism. Cancer Res. 72,3208–3208 (2012). Changsha, Hunan 410013, People’s Republic of China. 6High Resolution Mass 18. Luo, Y. et al. High Bak expression is associated with a favorable prognosis in Spectrometry Laboratory of Advanced Research Center, Central South breast cancer and sensitizes breast cancer cells to paclitaxel. PLoS ONE 10, University, Changsha, Hunan 410013, People’s Republic of China. 7Mitchell e0138955 (2015). Cancer Institute, University of South Alabama, Mobile, AL 36604, USA 19. Fan, S. Q. et al. Differential expression of Epstein-Barr virus-encoded RNA and several tumor-related genes in various types of nasopharyngeal epithelial Conflict of interest lesions and nasopharyngeal carcinoma using tissue microarray analysis. Hum. – The authors declare that they have no conflict of interest. Pathol. 37,593 605 (2006). 20. Tang, X. et al. MicroRNA-16 sensitizes breast cancer cells to paclitaxel through suppression of IKBKB expression. Oncotarget 7,23668–23683 (2016). 21. Jiang, X. R. et al. RFT2 is overexpressed in esophageal squamous cell carci- Publisher's note noma and promotes tumorigenesis by sustaining cell proliferation and pro- Springer Nature remains neutral with regard to jurisdictional claims in tecting against cell death. Cancer Lett. 353,78(2014). fi published maps and institutional af liations. 22. Li, M. et al. The effects of fulvic acid on microbial denitrification: promotion of NADH generation, electron transfer, and consumption. Appl. Microbiol. Bio- Supplementary Information accompanies this paper at https://doi.org/ technol. 100, 5607 (2016). 10.1038/s41419-018-0536-7. 23. Wang, Z., Wang, N., Chen, J. & Shen, J. Emerging glycolysis targeting and drug discovery from Chinese medicine in cancer therapy. Evid. Based Com- Received: 22 November 2017 Revised: 13 March 2018 Accepted: 15 March plementary Altern. Med. 2012, 873175 (2012). 2018 24. Miao,P.,Sheng,S.,Sun,X.,Liu,J.&Huang,G.LactatedehydrogenaseAin cancer: a promising target for diagnosis and therapy. IUBMB Life 65,904–910 (2013). 25. Billiard, J. et al. Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells. Cancer Metab. 1,19 (2013). References 26. Zhao, Y. et al. Overcoming trastuzumab resistance in breast cancer by tar- 1. Yu,X.,Li,Z.&Shen,J.BRD7:anoveltumorsuppressorgeneindifferent geting dysregulated glucose metabolism. Cancer Res. 71, 4585–4597 (2011). cancers. Am.J.Transl.Res.8,742–748 (2016). 27. Toffoli, S. Hypoxia and CoCl2 protect HepG2 cells against serum deprivation- 2. Yu,Y.etal.Analysisandmolecularcloning of differentially expressing genes in and t-BHP-induced apoptosis: a possible anti-apoptotic role for HIF-1. Exp. Cell nasopharyngeal carcinoma. ShengWuHuaXueYuShengWuWuLiXueBao Res. 295, 340 (2004). (Shanghai) 32, 327–332 (2000). 28. Zhou, J. et al. BRD7, a novel bromodomain gene, inhibits G1-S progression by 3. Mantovani,F.,Drost,J.,Voorhoeve,P.M.,Del,S.G.&Agami,R.Generegulation transcriptionally regulating some important molecules involved in ras/MEK/ and tumor suppression by the bromodomain-containing protein BRD7. Cell ERK and Rb/E2F pathways. J. Cell Physiol. 200,89–98 (2004). Cycle 9,2777–2781 (2010). 29. Peng, C. et al. BRD7 suppresses the growth of nasopharyngeal carcinoma cells 4. Kaeser,M.D.,Aslanian,A.,Dong,M.Q.,Yates,J.R.&Emerson,B.M.BRD7,a (HNE1) through negatively regulating β-catenin and ERK pathways. Mol. Cell novel PBAF-specific SWI/SNF subunit, is required for target gene activation Biochem. 303,141–149 (2007). and repression in embryonic stem cells. J. Biol. Chem. 283, 32254–32263 30. Hanahan,D.&Weinberg,R.A.Hallmarksofcancer:thenextgeneration.Cell (2008). 144,646–674 (2011). 5. Xu, K. et al. Integrating ChIP-sequencing and digital gene expression profiling 31. Gatenby, R. A. & Gillies, R. J. Why do cancers have high aerobic glycolysis? Nat. to identify BRD7 downstream genes and construct their regulating network. Rev. Cancer 4, 891–899 (2004). Mol. Cell Biochem. 411,57–71 (2016). 32. Xiao, X. et al. The miR-34a-LDHA axis regulates glucose metabolism and tumor 6. Xu, Y. et al. Inactivation of BRD7 results in impaired cognitive behavior and growth in breast cancer. Sci. Rep. 6, 21735 (2016). reduced synaptic plasticity of the medial prefrontal cortex. Behav. Brain Res. 33. Wilde, B. R. & Ayer, D. E. Interactions between Myc and MondoA transcription 286,1–10 (2015). factors in metabolism and tumourigenesis. Br. J. Cancer 113,1529–1533 7. Wang, H. et al. Knockout of BRD7 results in impaired spermatogenesis and (2015). male infertility. Sci. Rep. 6, 21776 (2016). 34. Barthel, A. et al. Regulation of GLUT1 gene transcription by the serine/ 8. Zhao R. et al. BRD7 plays an anti-inflammatory role during early acute threonine kinase Akt1. J. Biol. Chem. 274, 20281–20286 (1999). inflammation by inhibiting activation of the NF-кB signaling pathway. Cell. 35. Matoba, S. et al. p53 regulates mitochondrial respiration. Science 312, Mol. Immunol. 14,830–841 (2017) 1650–1653 (2006). 9. Harte, M. T. et al. BRD7, a subunit of SWI/SNF complexes, binds directly to 36.Cui,J.etal.FOXM1promotesthewarburgeffectandpancreaticcancer BRCA1 and regulates BRCA1-dependent transcription. Cancer Res. 70, progression via transactivation of LDHA expression. Clin. Cancer Res. 20, 2538–2547 (2010). 2595–2606 (2014).

Ofﬁcial journal of the Cell Death Differentiation Association Niu et al. Cell Death and Disease (2018) 9:519 Page 17 of 17

37. Shi, M. et al. A novel KLF4/LDHA signaling pathway regulates aerobic glyco- 45. Yu, B. et al. c-Jun protects hypoxia-inducible factor-1alpha from degradation lysis in and progression of pancreatic cancer. Clin. Cancer Res. 20,4370–4380 via its oxygen-dependent degradation domain in a nontranscriptional man- (2014). ner. Cancer Res. 69,7704–7712 (2009). 38. Semenza, G. L. et al. Hypoxia response elements in the aldolase A, enolase 1, 46. Cho, S. et al. Binding and regulation of HIF-1α by a subunit of the proteasome and lactate dehydrogenase A gene promoters contain essential binding sites complex, PSMA7. FEBS Lett. 498,62(2001). for hypoxia-inducible factor 1. J. Biol. Chem. 271, 32529–32537 (1996). 47. Jun,Y.etal.Estrogenreceptor-α directly regulates the hypoxia-inducible factor 39. Balamurugan, K. HIF-1 at the crossroads of hypoxia, inﬂammation, and cancer. 1 pathway associated with antiestrogen response in breast cancer. Proc. Natl. Int. J. Cancer 138, 1058 (2016). Acad. Sci. USA 112,15172–15177 (2015). 40. Semenza, G. L. HIF-1 and tumor progression: pathophysiology and ther- 48. Epstein,A.C.etal.C.elegans EGL-9 and mammalian homologs deﬁne a family apeutics. Trends Mol. Med. 8, S62 (2002). of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107,43–54 41. Salceda,S.&Caro,J.Hypoxia-induciblefactor1α (HIF-1α) protein is rapidly (2001). degraded by the ubiquitin-proteasome system under normoxic conditions. Its 49. Huang, L. E., Gu, J., Schau, M. & Bunn, H. F. Regulation of hypoxia-inducible stabilization by hypoxia depends on redox-induced changes. J. Biol. Chem. factor 1alpha is mediated by an O2-dependent degradation domain via the 272, 22642–22647 (1997). ubiquitin-proteasome pathway. Proc.Natl.Acad.Sci.USA95,7987(2015). 42. Tanimoto, K., Makino, Y., Pereira, T. & Poellinger, L. Mechanism of regulation of 50. Rong, Y. et al. Lactate dehydrogenase A is overexpressed in pancreatic cancer the hypoxia-inducible factor-1α by the von Hippel-Lindau tumor suppressor and promotes the growth of pancreatic cancer cells. Tumour Biol. 34, protein. EMBO J. 19, 4298–4309 (2000). 1523–1530 (2013). 43. Lee, S. H. et al. Runx2 protein stabilizes hypoxia-inducible factor-1α through 51. Sheng, S. L. et al. Knockdown of lactate dehydrogenase A suppresses tumor competition with von Hippel-Lindau protein (pVHL) and stimulates angio- growth and metastasis of human hepatocellular carcinoma. FEBS J. 279, genesis in growth plate hypertrophic chondrocytes. J. Biol. Chem. 287, 14760 3898–3910 (2012). (2012). 52. Chang,G.G.,Huang,S.M.&Chiou,S.H.Kineticmechanismoftheendo- 44. Zhang, L. et al. The bidirectional regulation between MYL5 and HIF-1α pro- genous lactate dehydrogenase activity of duck ϵ-crystallin☆. Arch. Biochem. motes cervical carcinoma metastasis. Theranostics 7,3768–3780 (2017). Biophys. 284, 285 (1991).

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