MDA-MB-231, Hs578T and BT20 cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Canoga Park, CA) + 10% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA) + 1% Antibiotic-Antimycotic mix (Thermo Fisher Scientific). CAL51 was obtained from The Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany) and cultured in DMEM + 20% FBS. Cell cultures were maintained and propagated in cell culture flasks (Corning Inc., Corning, NY) in a humidified incubator at 37 °C and 5% CO2. To mimic in vivo conditions more closely, cells were also cultured in low glucose (1000 mg/L) DMEM (Thermo Fisher Scientific) and under low oxygen conditions (2%). For tumorsphere suspension culture, 20,000 cells per well were cultured in MammoCult culture medium (STEMCELL Technologies, Vancouver, Canada) in ultra-low adherence 6-well culture plates (Corning Inc.) for up to 14 days.
Stable and transient transfection
Lipofectamine 2000 reagent (Thermo Fisher Scientific) was used for stable and transient gene knockdown (KD) according to the manufacturer's protocol. Before transfection, cells were seeded at 3 × 105 cells per well in 6-well plates or 1 × 105 cells per well in 24-well plates and incubated with transfection medium for up to 48 h. For stable knockdown EP300 and scramble shRNA vectors were used (Thermo Fisher Scientific). For selection of cell clones with successful DNA integration, KD cells were further cultured as single cells in 96-well plates under the addition of G418 (geneticin) at 50 µg/ml. siRNA transient gene KD for EP300 and negative control were performed in duplicate using Silencer Select siRNAs (Thermo Fisher Scientific). As a control for transfection efficiency, the cells were simultaneously transfected with BLOCK-iT Alexa Fluor Red Fluorescent Oligo (Thermo Fisher Scientific) and red fluorescence expression was validated after 24 h transfection using a Zeiss Axiovert 200 inverted microscope. Gene knockdown was validated via qRT-PCR.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA extraction was performed using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. First strand synthesis was performed using the qScript cDNA Supermix (Quantabio, Beverly, MA) and 1 µg of total RNA per reaction. The following reaction conditions were used on a T100 thermal cycler (Bio-Rad, Irvine, CA): 25 °C for 5 min, 42 °C for 30 min, 85 °C for 5 min. Samples were prepared as duplicates unless stated otherwise.
For gene expression quantification, SYBR Green RT-PCR master mix was used (Quantabio). Reaction were performed in 96-well plates (Bio-Rad), using 2 µl cDNA, 12.5 µl SYBR Green master mix, 1µL each forward and reverse primer probes and 8.5 µl nuclease free deionized water. The following reaction conditions were used on a MyiQ or CFX96 Real-Time system (Bio-Rad): 95 °C for 3 min, 40 cycles of 95° C for 20 s, 60 °C for 20 s, 72 °C for 30 s. Primer sequences were obtained via the Harvard primer bank and synthesized by ValueGene (San Diego, CA). To ensure gene-specific priming, the primer sequences were validated using the NCBI BLAST tool and only exon-junction spanning primers used. Primer sequences and corresponding genes are listed in supplementary table S2.
Fluorescence Activated Cell Sorting (FACS)
Cells were harvested using non-enzymatic cell dissociation buffer (Sigma Aldrich, St. Louis, MO). FACS was performed on a BD LSRFortessa (BD Biosciences, San Jose, CA) using a 405 nm excitation wavelength. Gating was performed based on negative controls as described below. Cell cycle analysis: Cells were washed twice in ice-cold FACS buffer (PBS + 2% FBS) and centrifuged at 1000 rpm for 5 min between washes. After the second wash, the cell pellets were re-suspended in ice cold 80% ethanol. The ethanol was added dropwise under constant vortex agitation, to a final volume of 500 µl ethanol per 1 × 106 cells. Samples were stored at 4 °C until further use. For analysis, 10 µl of a 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) nuclear dye solution was added to a 100 µl ethanol cell suspension.
Side population assay: The cell number was adjusted to 1 × 106 cells per mL and re-suspended in complete culture medium (DMEM + 10% FBS + 1% pen strep). Five µg of Hoechst 33342 dye (Sigma Aldrich) was added to each sample. For negative controls, samples were prepared in the same way, plus the addition of 100 µM verapamil (Sigma-Aldrich) to block dye efflux. The samples were incubated for 2 h at 37 °C in a cell culture incubator and gently agitated every 30 min. Following incubation, samples were immediately placed on ice and centrifuged at 1000 rpm at 4 °C for 5 min. Cell pellets were re-suspended in 300 µl cold PBS containing 5 µg/ml propidium iodide (PI) (Thermo Fisher Scientific) for dead cell exclusion.
Protein quantification
Cells were washed twice with ice-cold PBS and subsequently scraped into ice-cold PBS plus protease inhibitor (Merck Millipore, Billerica, MA) and 1 µM Dithiothreitol (DTT) (Sigma-Aldrich), pelleted at 1000 rpm for 5 min and stored and − 80 °C until further use.
For protein extraction, the cell pellets were thawed on ice and processed using the Pierce M-PER Extraction Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. To ensure loading of equal amounts of protein for each sample, colorimetric protein quantification was performed using a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) and a SpectraMax M3 spectrophotometer (Molecular Devices, Sunnyvale, CA). For standard curve values, an albumin standard (Thermo Fisher Scientific) dilution serious was used. Protein extracts were mixed 1:1 with 2x Laemmli Sample Buffer (Bio-Rad) containing 5% of 2-mercaptoethanol (Sigma-Aldrich) and boiled for 5 min. The samples were loaded onto pre-cast 4–20% gradient gels (PAGEr Gold Precast gels, Lonza, Basel, Switzerland) including a Precision Plus Protein Dual Color Standard (Bio-Rad) for size determination. After gel electrophoresis (running buffer: 25 mM Tris HCl, 193 mM glycine, 0.1% sodium dodecyl sulfate (SDS), pH 8.3) the separated protein fractions were transferred onto nitrocellulose membranes (Bio-Rad) (transfer buffer: 80% Tris-Borate buffer [0.089 M Tris, 0.089 M Borate, pH 8.3], 20% methanol, 25 mM Tris HCl, 193 mM glycine) at 4 °C overnight. The membranes were then blocked in 5% skim milk for 60 min. Primary antibody incubation was performed in blocking solution over night at 4 °C. Primary antibodies: polyclonal rabbit anti-human p300 (N-15), monoclonal mouse anti-human ACTB (H-102) (both from Santa Cruz Biotechnologies). Secondary antibody incubation was performed for 60 min at room temperature. Secondary antibodies: goat anti-mouse IgG HRP conjugated; and goat anti-rabbit IgG HRP conjugated (both from Santa Cruz Biotechnologies). Subsequently the membranes were incubated with HRP substrate (GE Healthcare Life Sciences, Pittsburgh, PA) for 5 min. and chemiluminescence was recorded using a ChemiDoc MP gel imaging system (Bio-Rad). ImageJ software was used for quantification. Samples were prepared in technical duplicates.
Animal experiments
Eight to ten weeks old female NOD scid gamma (NSG) mice (NOD-scid IL2Rgammanull, The Jackson Laboratory, Bar Harbor, Maine) were used for all xenograft experiments. The animals were housed at the USC Zilkha Neurogenetic Institute vivarium, according to IACUC requirements (protocol number 11204). For tumor cell injection, the mice were anesthetized with 2.5-4% isoflurane (Santa Cruz Biotechnology, San Diego, CA) under continuous infusion via a nose cone. At the experimental endpoint (tumor size ~ 1500mm3 or mice showing significant signs of sickness due to cumulative tumor burden) mice were sacrificed using carbon dioxide in an enclosed chamber, followed by cervical dislocation. All animal procedures adhered to the ARRIVE guidelines [20].
Tumor cell line subcutaneous (s.c.) and tail vein injection: For subcutaneous xenograft experiments, cells were prepared at a concentration of 1 × 106 cells per 100 µl in a 1:1 mix of 75 µL cell culture medium (DMEM/ F12) and 75 µl Matrigel basement membrane (BD Biosciences, San Jose, CA), for a final volume of 150 µl slurry. A 100 µl bolus of cell slurry was injected subcutaneously into the left hind flank region of each mouse and the puncture site sealed using degradable tissue adhesive (3M Health Care, St. Paul, MN). Tumor growth was monitored weekly using a digital caliper (VWR, Radnor, PA). Tumor volume was calculated using the formula Volume = (Width × 2 × Length)/2. To investigate whether micro-environmental or paracrine factors affect tumor growth of MDA-MB-231EP300KD cells, wild type cells expression mCherry and MDA-MB-231EP300KD clone 1 (GFP positive) were injected as a 1:1 mix for a total of 1 × 106 cells per mouse.
For tail vein injections, the mice were placed under a heating lamp for 2 min to dilate blood vessels. After subsequent immobilization in a rodent holder (Kent Scientific, Torrington, CT) 1 × 106 cells in 100 µl culture medium were injected per mouse using hypodermic syringes.
In vivo imaging: Tumor growth and spread after tail vein injection was monitored intra-vitally once per week for a total of 5 weeks. Luciferin (Promega, Madison, WI) was prepared at a final concentration of 300 mg/ml. After induction of anesthesia as described above, 300 µl of luciferin was injected intraperitoneally per mouse and allowed to distribute through the body of the animal for 15 min before imaging. Imaging was performed using an IVIS Spectrum pre-clinical in vivo imaging system (PerkinElmer, Waltham, MA) under continued anesthesia. Images were taken after 10 s, 1 min and 3 min each for supine and prone position. Bioluminescence was quantified using the Living Image 4.2 software (PerkinElmer).
Gene association and survival analysis
For gene association studies publicly available data sets from the The Cancer Genome Atlas (TCGA) breast cancer cohort were used and analyzed via Ingenuity pathway (IPA) tool and cBioPortal [21–23]. The Kaplan-Meier (KM) plotter web interface was used to assesses the effect of EP300 gene expression on survival in breast cancer patients [24]. KM plotter is a meta-analysis-based biomarker assessment tool using a manually curated gene expression database downloaded from Gene Expression Omnibus (GEO), European Genome-phenome Archive (EGA) and The Cancer Genome Atlas (TCGA). The auto selected cut-off was used to define high vs. low expression, which computes the best threshold between the upper and lower quartiles based on sample selection. Recurrence free survival (RFS) was reported at 60-month survival.
Statistical analysis
GraphPad PRISM (GraphPad Software Inc., La Jolla, CA) was used for statistical analysis of all experiments. Non-normal distribution was assumed, and appropriate non-parametric statistical tests were used; for experiments with single variables and groups of two Mann-Whitney test was used; for three or more groups, non-parametric one-way ANOVA (Kruskal-Wallis and Dunn’s multiple comparison) was used. For two variables and groups of two or more two-way ANOVA was used. Statistical hypothesis testing (Sidak test) was used to correct for multiple comparison. Data for repeats were presented as means and standard deviation. A two-tailed statistical significance level (alpha) of 0.05 for all statistical tests was considered meaningful to reject the null hypothesis. For survival analysis after tail vain injection, Kaplan-Meier curves were created and log-rank (Mantel-Cox) tests were used.