Cell culture and chemicals
T47D, BT474 and MCF-7 breast cancer cell lines were obtained from Dr. Charles V. Clevenger (Department of Pathology, Virginia Commonwealth University, Richmond, VA, USA) and MCF10A immortalized normal mammary epithelial cells were purchased from The American Type Culture Collection (ATCC, Manassas, VA, USA). T47D, BT474 and MCF-7 are ER+/PR+ cell lines; T47D has the highest PR expression of the three cell lines . T47D, BT474 and MCF-7 cells were maintained in phenol free MEM supplemented with 10 % FBS (Atlanta Biologicals, Norcross, GA, USA), 2 mM L-glutamine, 1 % MEM-NEAA, 0.075 % Sodium bicarbonate and 100 units/mL of penicillin, 100 μg/mL of streptomycin and 25 μg/mL of Fungizone® in a humidified incubator at 37 °C and 5 % CO2. MCF10A cells were grown in DMEM/F12 containing 5 % horse serum, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 100 ng/mL cholera toxin (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/mL insulin, and 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of Fungizone®. Cell growth media and all of the cell culture supplements were purchased from Gibco® (Carlsbad, CA, USA) unless indicated. Estradiol (E2), progesterone (P4), 17α-hydroxy-6α-methylprogesterone acetate (MPA) and Mifepristone (RU486) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Promegestone (R5020) was obtained from PerkinElmer (Santa Clara, CA, USA). 17α-acetoxy-21 methoxy-11β[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione (telapristone acetate, TPA; CDB4124) was provided by Repros Therapeutics (The Woodlands, TX, USA). E2, and progestogens (P4, MPA and R5020) were reconstituted in ethanol and TPA in DMSO. All solvents were cell culture grade and the working solutions were stored at −20 °C.
Cell viability assay
The viability of T47D cells was evaluated by MTT assay according to the manufacturer’s instructions (Roche Life Science, Indianapolis, IN, USA). 5,000–10,000 cells were plated per well of a 96-well plate in 200 μL of growth media supplemented with 5 % charcoal-stripped FBS (CHS/FBS, Atlanta Biologicals, Norcross, GA, USA) and incubated for 24 h. These hormone-starved cells were then treated with 10 nM P4, 10 nM MPA, 10 nM R5020 ± TPA (0.1 μM, 1 μM) alone or in combination with 1 nM E2. Control cells received ethanol. Cell viability at 24, 48 and 72 h was determined by measuring metabolic activity of living cells as relative colorimetric changes. All experiments were repeated at least three times. Two-way analysis of variance (ANOVA) was used to determine the significant differences between treatments. The Bonferroni test was used to analyze multiple comparisons. All statistical tests were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA).
Proliferation and apoptosis
Apoptosis and Cell proliferation were examined using Annexin V (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA, Cat# A23204) and Ki-67 (BD Biosciences, San Jose, CA, USA, cat# 561126) labeling respectively. T-47D cells were cultured in regular media as described above. At 80–85 % cell confluence, the cell cycle was synchronized by serum starvation. Following that, treatment with vehicle, R5020 (10nM), and R5020 with TPA (1 μM) for 0 h, 24 h, 48 h, and 72 h in 5 % charcoal stripped FBS, phenol red free MEM (Atlanta Biologicals, Norcross, GA, USA) was performed. The treated cells were then disassociated, counted, aliquoted in two sets and incubated with Annexin V or Ki-67 as per manufacturer’s recommendations. Cell cycle was analyzed using BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and data analysis was performed using Graphpad Prism Ver 6.0 (San Diego, CA, USA). Two-way ANOVA was utilized to determine the significance of the differences over the time course of the experiments and Tukey’s test to determine significance between treatments at individual time points.
3 × 105 cells of T47D and BT474 were hormone-starved for 24 h. T47D cells were then treated with 10 nM R5020 for 24 h. BT474 cells were incubated with 1 nM E2 for 72 h, washed twice with growth media, and treated with 10 nM R5020 for 24 h. Cells were harvested and whole proteins extracted in RIPA buffer (Pierce, Rockford, IL, USA) including protease inhibitor cocktail and EDTA. Protein concentration was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA) and identical amounts of protein were separated in 10 % NuPAGE Bis-Tris SDS/PAGE Protein Gels (Invitrogen, Carlsbad, CA, USA) followed by transfer onto a polyvinylidene difluoride membrane (Invitrogen, Carlsbad, CA, USA). The membrane was probed with anti-PR antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA) followed by incubation with a secondary goat anti-mouse antibody (Pierce, Rockford, IL, USA). The blots were developed using the ECL Prime Western Blotting Detection Reagent (Amersham, Piscataway, NJ, USA). Anti-GAPDH antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA) were used for loading controls of proteins.
Cell cycle analysis
Cell cycle distribution was examined by measuring the cellular DNA content using propidium iodide (PI) and flow cytometry. T47D cells, growing in the exponential phase were hormone-starved for 24 h in growth media containing 5 % CHS/FBS; and BT474 cells, after 72 h exposure to E2, were treated with 10 nM P4, 10 nM MPA, 10 nM R5020 ± TPA (0.1 μM, 1 μM) alone or in combination with 1 nM E2 for 24 h. After incubation, cell pellets were collected by centrifugation, washed twice with PBS, fixed in 70 % (v/v) ice-cold ethanol for 24 h at −20 °C and then stained with PI (50 μg/mL) containing RNase (100 μg/mL) and 0.1 % Triton X-100 for 30 min in the dark at 37 °C. Cell cycle was analyzed using BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo vX (FlowJo, LLC, Ashland, OR, USA).
Measurement of PRE promoter activity
The PRE-luciferase reporter plasmid was a generous gift from Dr. Dean P. Edwards (Baylor College of Medicine, TX). T47D, BT474 and MCF-7 cells (1.2 × 105 cells) were plated in a 24-well plate and hormone-starved for 24 h. Cells were then transfected with 0.8 μg of PRE-luc reporter plasmid along with phRl-TK (0.01 μg) Renilla control plasmid using Lipofectamine 2000 (Life technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The transfected T47D cells were treated with 10 nM P4, 10 nM MPA, 10 nM R5020 ± TPA (10 nM, 100 nM, 1 μM) alone or in combination with 1nM E2. Control cells received ethanol and DMSO as vehicle. Cells were processed and the luminescence from firefly and Renilla luciferase was measured using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA) and the Synergy HT microplate reader (BioTek, Winooski, VT, USA). The relative PRE- luciferase activity was expressed as the ratio of the firefly luciferase/Renilla luciferase unit (RLU).
Microarray analysis and statistical analysis
Three separate T47D cell cultures were used for microarray analysis. The experimental treatments were vehicle, 10 nM R5020, 1 μM TPA, and 10 nM R5020 with 1 μM TPA. All RNA samples were processed at the Genomics Core Facility in the Center for Genetic Medicine at Northwestern University (Chicago, IL). The quality of total RNA was evaluated using the Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). 150 ng of each RNA sample, with 260/280 and 28S/18S ratio of greater than 1.8, was used to make double-stranded cDNA. Gene expression analysis was performed using the Illumina Human HT-12v4 Expression BeadChip. Quality checks and probe level processing of the Illumina microarray data were further made with the R Bioconductor package lumi (http://www.bioconductor.org/packages/release/bioc/html/lumi.html). Data was quantile normalized, and hierarchical clustering and Principal Component Analysis were performed on the normalized signal data to assess the sample relationship and variability. Probes absent in all samples were filtered out according to Illumina’s detection p-values in the downstream analysis. Differential gene expression between the different conditions was assessed by a statistical linear model analysis using the bioconductor package limma (http://www.bioconductor.org/packages/release/bioc/html/limma.html). The moderated t-statistic p-values derived from the limma analysis above were further adjusted for multiple testing by Benjamini and Hochberg’s method to control false discovery rate (FDR) . The lists of differentially expressed genes were obtained by the FDR criteria of <5 % and fold change cutoff of > ± 1.5. Data obtained from the microarray was further analyzed by MetaCore (Thompson Reuters; https://portal.genego.com) and Ingenuity Pathway Analysis (IPA; Qiagen, http://www.ingenuity.com).
Validation of gene expression for selected 16 genes
Cell cycle regulating genes responding to both R5020 and TPA (microarray data) were compared with cell cycle genes upregulated by progesterone in luteal phase of normal breast tissue (RNA-Seq data)  and 16 genes that were significantly differentially expressed were identified. The expression of these 16 genes was validated with reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Briefly, RNA from the gene arrays was reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Life technologies, Carlsbad, CA, USA). Real-time qPCR was performed using an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Life technologies, Carlsbad, CA, USA). The geometric mean of housekeeping gene (GAPDH and β-Actin) was used as an internal control to normalize the variability in expression levels. PCR primers used for real-time PCR were purchased from integrated DNA technologies (Coralville, IA, USA) and the list of the primers is provided in Additional file 1: Table S4. Expression data of the 16 genes was normalized to housekeeping genes GAPDH and β-Actin to control the variability in expression levels and were analyzed using the 2-ΔΔCT method described by Livak and Schmittgen . The expression of the 16 genes was validated by real-time PCR using T47D and MCF10A cells. 6.0 × 105 cells of T47D and MCF10A were hormone-starved for 24 h. Cells were then treated with 10 nM P4, 10 nM MPA, 10 nM R5020 ± TPA for 24 h. Vehicle treated cells were used as a control. Total RNA from samples was extracted using Trizol reagent (Life technologies, Carlsbad, CA, USA). 2 μg of total RNA was converted to cDNA using SuperScriptVILO master mix (Life technologies, Carlsbad, CA, USA) according to the manufacturer’s instruction. Real-time PCR and data analysis were as above. Two-way analysis of variance (ANOVA) was used to determine the significant differences between treatments. The Sidak correction was applied to analyze multiple comparisons. All statistical tests were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA).
Regulation of expression of the selected 16 genes
Motif analysis was performed using HOMER (v4.8) to identify common sequences in the promoters among the 16 genes of interest (Salk Institute, La Jolla, CA, USA; http://homer.salk.edu/homer/). The ENCODE transcription factor (TF) binding site tracks were enabled for the MCF-7 cell line to determine if promoters of the selected 16 genes are bound by the same TFs (https://www.genome.ucsc.edu/ENCODE/).