BETi enhance ATGL expression and its lipase activity to exert their antitumoral effects in triple-negative breast cancer (TNBC) cells

Cell lines, transfections, and treatments

Human breast cancer cell lines MDA-MB231 (referred to as MB231, human triple-negative metastatic) and Hs578T (human triple-negative primary) were purchased from ATCC. MCF7 (human breast cancer luminal) was purchased from Sigma-Aldrich. BT549 (human triple-negative primary) cell line was a kind gift from Dott.ssa Paola Bonetti at IFOM-IEO campus, Milan, Italy. Cell lines were authenticated by SNP profiling at Multiplexion GmbH, Germany. MDA-MB231 and BT549 cell lines were grown at 37 °C/5% CO2 in DMEM with 10% FBS (ThermoFisher scientific). Hs578T was grown at 37 °C/5% CO2 in DMEM (Invitrogen) medium with 10% FBS and 0.01 mg/ml human insulin (Sigma-Aldrich). MCF7 were grown at 37 °C/5% CO2 in MEM + GlutaMAX (plus Earle’s Salts, Invitrogen) medium with 10% FBS and 1% of NEAA (Invitrogen). All media were supplemented with 1% penicillin-streptomycin (Life Technologies). All cell lines were tested monthly for Mycoplasma contamination. Select small interfering RNA (siRNA) against PNPLA2 (ID: s32682, siATGL), FoxO1 (ID: s25258, siFoxO1), and negative control oligos (siCNT) (Ambion) were transfected using 25 nmol/L of siRNA with RNAiMax Lipofectamine (Thermo Fisher Scientific) reagent according with the reverse transfection protocol. Cells were harvested and analyzed 48 and 72 hours after transfection. BET inhibitors were added to siATGL, siFoxO1, and siCNT 24 h after the transfection at the time and doses indicated. Flag-FOXO1A plasmid was a gift from Stefan Koch (Addgene plasmid # 153141; http://n2t.net/addgene:153141; RRID:Addgene_153141) and was transfected with Lipofectamine2000 (Thermo Fisher Scientific) reagent according with the transfection/manufacturer’s protocol. For drug treatment, cells were plated at sub confluence density 24 hours before treatment. DMSO (referred to as CNT, Sigma-Aldrich) stocks of JQ1, OTX015, and ATGListatin (all purchased from Sigma-Aldrich) were diluted in the cell culture medium at the time and indicated concentrations. Propionic acid (PA, 402907) was purchased from Sigma-Aldrich. Oleic acid (OA) was a kind gift from Dott. Egidio Iorio, Istituto Superiore di Sanità, Rome, Italy. Fatty acids (FAs) were added to the culture medium at the indicated concentrations.

BrdU incorporation assay

In order to avoid cell–cell-contact-induced inhibition of cell proliferation due to cell confluency, we seeded 3-5 × 105 cells per well in 6 wells plate. This procedure ensured that cells were in a sub confluent state after 1 day and 3 days of incubation. 10 μM of 5-bromo-2′-deoxyruridine (BrdU, Sigma-Aldrich) was added to cell cultures for 1 hour. After 1 hour pulse, the cells were fixed in ice-cold 70% ethanol dropwise on a vortex and left at 4 °C for 30 minutes, washed twice with PBS and suspended in 2 M HCl for 30 minutes at room temperature (RT) with occasional mixing. The cells were subsequently washed twice in PBS and 2.5ul of anti-BrdU mAb (B44, BD biosciences) in BSA-PBS-Tween (PBS + 0.1% BSA + 0.2% Tween20, pH 7.4) were added to the cell pellet at RT for 30 minutes in the dark. After washing the cells twice, 2.5ul of goat anti-mouse FITC (BD555988, goat anti mouse) in PBS-Tween was added to the cells at RT for 30 minutes. The pellet was washed in PBS, 10μg/ml of RNAse (Ribonuclease A, R4642, Sigma-Aldrich) was added for 15 minutes at 37 °C and 50μg/ml of PI (Propidium Iodide, Sigma-Aldrich, #P4170) was added prior flow cytometry analysis. We collected at least 30,000 events at the BD FACS Canto II (BD Biosciences).

Cell death and CFSE detection

Following the protocol’s instruction, cell death was quantified using FITC Annexin V Apoptosis detection Kit (BD Biosciences, #559763). Briefly, 5 × 105 cells/well were plated in a 6-well plate, and both supernatant and attached cells were used for the analysis. Cells were washed with PBS, resuspended in Annexin V-binding buffer (1X in water). Cells were stained with Annexin V-FITC (Fluorescein isothiocyanate) and 7-amino-actinomycin D (7-AAD) for 15 min at + 4 °C in the dark. Analysis was performed by flow cytometry using a FACSCanto II (BD Biosciences) on FSC/SSC viable gated cells, excluding cell debris and doublets. Samples were analyzed using FACSDiva Software.

For cell duplication rate analysis, we used a CFSE (5(6)-Carboxyfluorescein N-hydroxysuccinimidylester) staining by CFSE - Cell Labeling Kit (Abcam, #ab113853) following manufacture’s protocol. Briefly 5 × 104 cells/condition were washed with PBS and resuspended in PBS/CFSE (4 μM) for 15 minutes at 37 °C in the dark. The reaction was quenched by adding an equal volume of medium for 5 minutes. The cells were washed to remove unincorporated CFSE and immediately after that After that, plated and analysis was every 24 hours by flow cytometry using a FACSCanto (BD Biosciences). Samples were analyzed using FACSDiva Software.

Cell proliferation and Colony-forming assays

For BETi efficacy, a proliferation assay was performed, plating 5 × 103 cells per well in 96-wells, and viable cells were counted 72 hours after BETi treatment using trypan blue (Sigma-Aldrich) staining and the Bürker chamber for the count. Proliferation analysis with FAs and ATGListatin were performed, seeding 2 × 103 cells per well in 96-wells. The day after, cells were treated and placed in IncuCyte S3 Live-Cell Analysis System (Essen BioScience). Cell proliferation was monitored for 96 hours, and images were collected every 12 hours using the phase-contrast confluence metric. For the colony formation assay, cells were seeded in 6-wells plates at a density of 500–600 MDA-MB231 cells/well and 1000–1200 Hs578T cells/well and cultured for 10–12 days. Then, colonies were fixed with methanol, stained with 0.5% Crystal Violet (Sigma-Aldrich) and counted with ImageJ software [22].

Lipid staining

Oil Red O (ORO) working solution (Sigma-Aldrich O-06525, ORO stock solution 0.3% in isopropanol) was prepared by diluting 6 parts of ORO stock with 4 parts of water. 3 × 105 cells were seeded in 12-well plates. After 24 hours of BETi treatment, cells were fixed in paraformaldehyde (PFA-4% solution, Sigma-Aldrich) for 10 minutes and rinsed twice with PBS. Each well was washed with 60% isopropanol (Sigma-Aldrich) and let dry completely. Next, cells were stained with 0.5 ml/well of ORO for 10 minutes at room temperature. Subsequently, wells were rinsed under running tap water until no excess stain was seen. 1 ml of hematoxylin was added into each well to counterstain the nuclei for 1 minute. Finally, wells were washed until no excess stain was seen and viewed on a phase-contrast microscope (Nikon). Lipid appeared red and nuclei blue.

BODIPY™ 500/510 C1, C12 (4,4-Difluoro-5-Methyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid, ThermoFisher scientific) stock solution, 1 mg/mL, was prepared in ethanol (Sigma-Aldrich) and kept at − 20 °C until used. Cells were incubated overnight with 1 μg/mL BODIPY™ in co-administration with the specific treatment.

Immunofluorescence (IF) and flow cytometry assays

For IF, 6 × 104 cells were seeded onto chambers slide (4 chambers, Eppendorf), washed with PBS, and fixed with 4% paraformaldehyde for 10 minutes. For BODIPY staining only, the cells were permeabilized with 0.1% Triton (Sigma-Aldrich) in PBS for 5 minutes, washed with PBS, and stained with DAPI (Invitrogen). For ATGL staining, the cells were permeabilized 10 minutes with 0.1% Triton, washed with PBS, blocked 1 h in 2% BSA/PBS and stained with 1:100 ATGL antibody (Invitrogen, PA5–17436) in 0.1% BSA (Sigma-Aldrich)/PBS overnight at 4 °C. The day after, cells were stained with secondary antibody anti-rabbit Alexa 594 (Thermo Fisher Scientific) and DAPI. Coverslips were mounted with Slowfade long antifade Mountant (Invitrogen). Immunofluorescences were detected using confocal microscopy (LEICA).

For flow cytometry, cells were seeded and treated with specific drugs for 24 hours, and BODIPY was added overnight. Afterward, cells were washed with PBS, detached, and resuspended in PBS with 3 mM EDTA, and analyzed for at least 10,000 events at the BD FACS Canto II (BD Biosciences). Median Fluorescence Intensity (MFI) was calculated by setting the gate at 50% of the CNT and analyzing the curve’s shift of the treated cells.

RNA isolation and reverse-transcription quantitative PCR assays

Total RNA for gene expression was extracted with Maxwell RSC simplyRNA Cells (Promega). Up to 1 μg of RNA was synthesized to cDNA using the iScript cDNA Kit (Bio-Rad). Reverse-transcription quantitative PCR (RT-qPCR) was conducted using Sso Fast EvaGreen SuperMix (Bio-Rad) and primers mixed at a final concentration of 250 nmol/L in the CFX96 Real-Time PCR Detection System (Bio-Rad). Fold change was calculated by the 2(−ΔΔCt) method. βACTIN (b-ACT) was used as a housekeeping gene. RT-qPCR primers are listed in Table 1.

Table 1 Primers used in this studyRNA-sequencing (RNA-seq)

RNA was extracted using TRIzol (Invitrogen) from MDA-MB231 treated with 1 μM of JQ1 for 6 hours. RNA-seq libraries were prepared using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina), starting from 1 μg of RNA. RNA-seq was performed on NextSeq 500 platform (Illumina) using a dual strand 2 × 75 approach. A minimum of 20 million reads for each sample replicate were considered. In bioinformatics data analysis, Fastq quality check was carried on using FastQC, and adapters removal and sequence alignment was conducted by Trimmomatic and STAR, respectively. Cufflink RNA-Seq workflow was applied to proceed with reads count and normalization. Differential gene expression was calculated by the Cuffdiff pipeline as fold-change. Genes with a P-value< 0.05 were considered significantly deregulated. Data analysis was supported by using R software (version 3.4.3). We plot the genes in Fig. 1C based on log2 data with an arbitrary cutoff for the validation of log2 fold change ≥1.

Fig. 1figure 1

BET inhibitor JQ1 differentially affects the expression of genes involved in lipid metabolism. A Proliferation rate of MDA-MB231, Hs578t, BT549, and MCF7 cells with two doses of JQ1 (0,5 μM and 1 μM). B Proliferation rate at day 3 represented as a percentage of the cell number rate in A. C Immunoblotting for p21 in MDA-MB231, Hs578t, BT549 and MCF7 cells after treatment with CNT or JQ1 for 24 hours. bACT served as a loading control. D Cell cycle analysis of MDA-MB231, Hs578t, BT549 and MCF7 cells treated with 1 μM JQ1 for 24 hours. BrdU and PI incorporation indicated the % of population. Here, we plotted the % of cell population in all the three different phases of the cell cycle. The significance is calculated versus the CNT treated cells. E Percentage of cell death in MDA-MB231 and Hs578t cells treated with 1 μM JQ1 for 1 day (1d) or 3 days (3d) with Annexin V staining. The significance is calculated versus the CNT treated cells. F RNA-seq analysis of MDA-MB231 treated for 6 hours with 1 μM of JQ1. The volcano plot represents the differentially regulated genes (red = significant, black = not significant), on the right are the upregulated, while on the left are the downregulated genes. G Top 5 GO (gene ontology) categories of upregulated genes analyzed with STRING. The Metabolic pathway category denotes the 25% of genes directly related to lipid metabolism. On the STRING network, different colors discriminate the diverse lipid metabolic process category: in purple the Inositol phosphate metabolism; in blue the Cholesterol biosynthesis; in yellow the Fatty acid metabolism Biosynthesis; in red the Glycerol lipid metabolism and green the Sphingolipid metabolism. H Genes in the “lipid metabolic process” identified by RNA-seq plotted for the log2 fold change value. In orange, the genes were selected for further analysis with a log2 ≥ 1. I Validation of the selected genes in panel C by RT-qPCR analysis

Lipidomic analysis

Deuterated reagents (methanol (CD3OD), chloroform (CDCl3)) and deuterium oxide (D2O) were purchased from Cambridge Isotope Laboratories, Inc.; 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TSP) was obtained from Merck & Co, Montreal, Canada. For the extraction of aqueous and organic metabolites, cell pellets were extracted according to the protocol previously described [23]. The polar phase containing water-soluble cellular metabolites was evaporated using a rotary evaporator and lyophilized while the organic fraction (lipid phase) was evaporated under nitrogen gas flow. Both phases of cell extracts were stored at − 20 °C. Lipid fraction from cells was resuspended in a CD3OD / CDCl3 solution (2:1 v/v) with 0.05%of tetramethylsilane (TMS) as internal reference. High-resolution 1H-NMR analyses were performed at 25 °C at 600 MHz (14 T Bruker AVANCE Neo spectrometer; Karlsruhe, Germany, Europe) on organic cell extracts using acquisition pulses, water pre-saturation, data processing, and peak area deconvolution as previously described [23]. Relative quantification of lipid signals in organic fractions was referred to the signal at 1.6 ppm (as a measure of total acyl chain and referred to 100). The integrals of characteristic lipid signals were compared to this value.

Mitochondrial metabolic activity by MTT assay

Cells were seeded in a 96-wells plate at 3 × 103 cells/well density. After siRNA transfection or drug treatment, 40 μl of a 5 mg/ml of MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan, Thiazolyl blue formazan- Sigma-Aldrich) solution was added to each well. After 3 hours at 37 °C in the dark, the supernatant was discarded, and 100 μl of DMSO was added to dissolve the precipitate. The optical density of formazan was detected at 560 nm to estimate the metabolic activity with GloMax Discover Microplate Reader (Promega).

ROS evaluation

Thirty minutes before the end of the experimental time (48 h of siATGL and siCNT transfection), cells were incubated with DHE (dihydroethidium) assay kit (Ab236206, Abcam) at 37 °C according to the manufacturer’s directions. Fluorescence was measured using the microplate reader (GloMax Discover Microplate Reader) at 520 nm (excitation) and 580–640 nm (emission). The fluorescence was normalized to the total number of cells.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed as previously described [24]. Briefly, chromatin was precipitated with FoxO1 antibody (rabbit mAb C29H4, Cell Signaling Technology) and the relative IgG control (rabbit IgG 2729S, Cell Signaling Technology). 1% of chromatin used for immunoprecipitation was kept as input control. Purified DNA was analyzed by RT-qPCR. Each RT-qPCR value was normalized over the appropriate input control and reported in graphs as a ratio over the IgG. The analyzed amplicon of the PNPLA2 genome mapped on FoxO1 binding site as highlighted by Jaspar TF tool in UCSC genome browser (Fig. 6D). Primers for PNPLA2 region for 5′- TTCATGGGTGAGGGTGCTTC − 3′ and rev: 5′- ACATCACTCCCTCATGGCAG − 3′.

Western blot

Cells were lysed in Passive Lysis Buffer (PLB, Promega), resuspended in Laemmli 4x buffer (Bio-Rad), and boiled at 95 °C for 10 minutes. Total protein extracts were separated using an SDS-PAGE gel (Bio-Rad), transferred to nitrocellulose membrane (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad), and blocked 5% milk/PBS-0.1% Tween 20 (Sigma-Aldrich). The membranes were incubated with primary antibodies diluted in 2% BSA/PBS tween 0.1% overnight at 4 °C on a shaker. Primary antibodies used for the study were as follows: β-actin as a loading control (Sigma-Aldrich, A1978), p21 (Abcam, ab227443), ATGL (Invitrogen, PA5–17436), DGAT1 (SCBT, sc271934), SOD1 (Cell Signaling, 71G8), CPT1a (Abcam, ab128568) and FoxO1 (Cell Signaling, C29H4). Secondary antibodies were HRP-conjugated donkey anti-rabbit and sheep anti-mouse (GE Healthcare). Membranes were incubated with secondary HRP-conjugated antibodies for 1 hour at room temperature. Clarity ECL (Bio-Rad) or ECL prime (Amersham) were used for detection using ChemiDoc Imager (Bio-Rad) following the manufacturer’s instructions. ImageJ software was used to quantify the band intensity.

Statistical analysis

Comparisons between two data groups were analyzed using an unpaired two-tailed Student t-test (GraphPad Prism 7). P-values < 0.05 were considered significant and indicated in the graphs by *. P values < 0.01 or < 0.001 were indicated by ** and ***, respectively. All data were shown as mean and SEM. Each experiment was replicated two to five times.

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