Cell culture
T47D were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 2% penicillin-streptomycin (Life Technologies), and insulin (10 μg/ml). Cos7 cells were maintained in DMEM, supplemented with 10% FBS and 2% penicillin-streptomycin (Life Technologies). All cell lines were grown in a humidified atmosphere with 5% CO2 at 37 °C, authenticated by Eurofins and tested for Mycoplasma infection (Lonza, Rockland, ME, USA).
Prior to experiments, when it was indicated, cells were grown in phenol red-free medium supplemented with 10% charcoal-stripped serum (Biowest). Cells were then treated with 10 nM of R5020 (Perkin Elmer) or E2 (Sigma) for the indicated times.
Generation of CRISPR ERα-36 KO cell lines
Electroporation of T47D cells
Cells were grown at subconfluence and electroporated with CRISPR reagents after cell dissociation using the Neon electroporator Invitrogen 1750 V-20 ms-1pulse. Electroporated cells were cultured as single cells to obtain pure clonal populations.
Strategy
Guide RNAs were designed using an in-house genOway’s tool, and those with the highest score were selected.
Targeted sequences were as follows: #1: 5′ TTAATAAGTACACACCGCAG AGG 3′; #2: 5′ CTGTGAGGCCTTATGACCAG AGG 3′.
These guide RNAs were designed to induce the deletion of an ERα-36-specific sequence by cutting into intron 8 and downstream of exon 9 (intron 31 and exon 32 with genOway’s numbering). ERα-36 isoform-specific knock-out clones were amplified, and isolated DNA was characterized by PCR amplification, as the 393-bp deleted sequence includes the ERα-36-specific exon 9 splice acceptor site, coding sequence, and STOP codon.
Antibodies
Information of primary antibodies
Antibodies
|
Supplier
|
Origin
|
Dilution for WB
|
Dilution for PLA
|
Dilution for IHC
|
---|
ERα-36
|
Homemade
|
Rabbit
|
1/1000
|
1/100
|
1/50
|
PR (AB8)
|
Thermo Scientific
|
Mouse
| |
1/500
| |
PR (H190)
|
Santa Cruz Biotechnology
|
Rabbit
|
1/2000
| | |
p-PRS294
|
Cell Signaling Technology
|
Rabbit
|
1/1000
|
1/500
| |
p-PR S345
|
Cell Signaling Technology
|
Rabbit
|
1/1000
|
1/500
| |
Tubulin
|
Sigma
|
Mouse
|
1/10000
| | |
V5-tag (D3H8Q)
|
Cell Signaling Technology
|
Rabbit
|
1/1000
| | |
Flag M2
|
Sigma
|
Mouse
|
1/1000
| | |
p44/42 MAPK (Erk1/2)
|
Cell Signaling Technology
|
Rabbit
|
1/1000
| | |
Phospho p44/42 MAPK (Erk1/2)
|
Cell Signaling Technology
|
Rabbit
|
1/1000
| | |
Luciferase reporter assay
HeLa cells (7.5 × 104) were plated in 24-well plates 24 h prior to transfection. The transfected DNA included 100 ng of reporter plasmid and 25 ng of pRL-TK Renilla luciferase vector (Promega) used as an internal control, together with various amounts of expression vectors, as indicated. Total transfected DNA was kept constant by adding empty pSG5-Flag vectors. The cells were induced with 10 nM R5020 24 h following transfection, then harvested after an additional 24 h and assayed for luciferase activity following the manufacturer’s instructions. Luciferase activities were normalized against the activity of the internal control Renilla luciferase.
Immunofluorescence
T47D cells (2 × 105) were grown on coverslips in 12-well plates. After treatment, cells were fixed in methanol for 2 min and washed twice in PBS. Non-specific binding was blocked using a 1% gelatin solution for 30 min at room temperature. Cells were incubated with PR antibody for 1 h at 37 °C, subsequently with the secondary antibodies Alexa Fluor 488 anti-mouse (Jackson ImmunoResearch, Cambridge, UK) (1:2000e) and Alexa Fluor 568 anti-rabbit (Invitrogen, Carlsbad, USA) (1:1000e) in Dako diluent for 1 h. Finally, coverslips were mounted on glass slides in mounting solution (Dako, Carpinteria, CA, USA). The fluorescent slides were viewed under the Nikon NIE microscope.
Immunoprecipitation and Western blot analysis
Cells were lyzed using RIPA buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.25% deoxycholate) supplemented with protease inhibitor tablets (Roche Molecular Biochemicals) and phosphatase inhibitors (1 mM NaF, 1 mM Na3VO4, and 1 mM β-glycerophosphate). Protein extracts were incubated with primary antibodies overnight at 4 °C on a shaker. Protein G-Agarose beads were added, and the mixture was incubated for 2 h at 4 °C. The immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot, then visualized by electrochemiluminescence (ECL, Roche Molecular Biochemicals).
Chromatin immunoprecipitation
ChIP experiments were performed according to the manufacturer’s protocol (SingleChIP enzymatic chromatin IP Kit - Cell signaling) with antibodies against PR, ERα, and IgG. Results are expressed relative to the signal obtained with chromatin input. Primer sequences are indicated in the Additional file 1.
RNA extraction and real-time RT-qPCR analysis
Total RNA (1 μg) was extracted and purified using TRI Reagent (Sigma-Aldrich, USA), prior to being reverse-transcribed using 100 ng of random primers following the Superscript II (Thermo Fisher, USA) protocol. Real-time PCR was performed with SYBR Green qPCR master mix (BioRad) in a Step One plus real-time PCR detection system (Applied Biosystems). All amplifications were performed in triplicate. Mean values of triplicate measurements were calculated according to the −ΔΔCt quantification method and were normalized against the expression of 28S ribosomal mRNA as reference. Data were presented as mean ± SEM. Sequences of the oligonucleotides used are listed in Additional file 2.
Proximity ligation assay, image acquisition, and analysis
This technology exposes protein/protein interactions in situ [15]. Briefly, cells were seeded and fixed with cold methanol. After saturation, the different couples of primary antibodies were incubated for 1 h at 37 °C. The proximity ligation assay (PLA) probes consisting of secondary antibodies conjugated with complementary oligonucleotides were incubated for 1 h at 37 °C. The amplification step followed the ligation of nucleotides for 100 min at 37 °C. Samples were subsequently analyzed under fluorescence microscopy.
The hybridized fluorescent slides were viewed under a Nikon Eclipse Ni microscope. Images were acquired under identical conditions at × 60 magnification. Image acquisition was performed by imaging DAPI staining at a fixed Z Position while a Z stack of ± 5 μm at 1 μm intervals was carried out. The final image was stacked to a single level before further quantification. On each sample, at least one hundred cells were counted. Analysis and quantification of these samples were performed using the ImageJ software (free access). PLA dots were quantified on 8-bit images using the “Analyse Particles” command, while cells were counted using the cell counter plugin.
IHC images were also acquired using a Nikon Eclipse Ni microscope at × 40 magnification, and PLA dots were quantified as described above.
Glutathione transferase pull-down assay
ERα-expressing plasmids were transcribed and translated in vitro using T7-coupled reticulocyte lysate in the presence of [35S] methionine. Glutathione transferase (GST) fusion proteins were incubated with labeled proteins in 200 μl of binding buffer (Tris 20 mM pH 7.4, NaCl 0.1 M, EDTA 1 mM, glycerol 10%, Igepal 0.25% with 1 mM DTT and 1% milk) for 2 h at room temperature. After washing, bound proteins were separated by SDS-PAGE and visualized by autoradiography.
Proliferation studies
4 × 103 cells seeded onto a 96-well plate were plated 5 h before incubation with the different hormones (E2, R5020, or ethanol). Images were acquired using an IncuCyte ZOOM over 7 days, and cell proliferation was measured as the percentage of cell density observed over this period. Results are represented as graphs indicating the rate of proliferation over time, extrapolated from at least three independent experiments, each performed in triplicate.
Wound healing assay
Cells were plated in duplicate in 6-well plates and grown to confluence. Wounds were then performed with a p200 pipette tip. After washes to remove cellular debris, three images of each well were taken. The width of the wound was measured at 3 places and recorded as t = 0. The cells were then allowed to migrate back into the wounded area. After 16 h, the width of the open area was measured. Cell migration was expressed as the percentage of the gap (t = 16) relative to the primary width of the open area (t = 0). Images were acquired on a phase contrast microscope (Zeiss, Axiovert 25). All experiments were performed in triplicate.
Patient population
We screened 200 consecutive female patients with operable breast cancers who had undergone radical surgery and received adjuvant/neoadjuvant therapy in the Centre Léon Bérard between January 1999 and December 2001. Paraffin blocks of tumor tissue were available for 182 patients. Among these, we failed to assess ERα-36 in 22 tumor specimens as a result of insufficient tumor or tissue loss during TMA preparation. Therefore, a total of 160 specimens were analyzed in this study.
Patients underwent radical surgery (either modified radical mastectomy (MRM) or breast-conserving surgery (BCS) with axillary lymph node (LN) staging). ERα-66 and PR were detected by immunohistochemistry, and tumors were considered positive if they display nuclear staining in 10% or more of the tumor cells. HER2 expression was determined using immunohistochemistry, and tumors were considered positive if they reached 3+ staining by immunohistochemistry or 2+ staining with HER2 amplification detected by FISH.
The data exported from patient files for analysis included age, histological subtype, maximum tumor size, number of LNs involved, SBR grade, ER, PR, HER2 status, date of diagnosis, date of relapse, and date of death or last clinical visit. Tumor samples and clinical data were obtained with the approval of the Institutional Review Board. This study is reported according to the REMARK criteria [16].
Immunohistochemical analysis
The breast tumor samples were inserted as triplicates using a 600-μm needle into 4 tissue microarray (TMA) blocks. The blocks containing invasive carcinoma were sectioned at a thickness of 4 μm. After deparaffinization and rehydration, endogenous peroxidases were blocked by incubating the slides in 5% hydrogen peroxide in sterile water. For heat-induced antigen retrieval, tissue sections were boiled in 10 mM citrate buffer pH 6.0 (Dako, Trappes, France) using a water bath at 98 °C for 50 min.
The slides were then incubated at room temperature for 1 h with the antibodies against ERα-36 (rabbit polyclonal antibody). These antibodies were diluted using an antibody diluent solution (Chemmate, Dako, Trappes, France) at 1/50. After rinsing in PBS, the slides were incubated with a biotinylated secondary antibody bound to a streptavidin peroxidase conjugate (LSAB+ Kit, Dako, Trappes, France). Bound antibodies were detected by adding the substrate 3,3-diamino-benzidine. Sections were counterstained with hematoxylin.
Blinded to the clinical data, biomarker expression was evaluated by 2 observers who assessed both the percentage and the intensity of the membranous staining for ERα-36 in the infiltrative carcinomatous cells only (faint cytoplasmic staining which was found in almost all malignant cells was not considered).
For scoring purposes, the highest intensity of staining in malignant cells was divided into 3 levels (0, no staining; 1, weak staining; 2, moderate to strong staining), and the percentage of stained cells was also classified into 3 levels (0, no stained cells; 1, staining in less than half of the malignant cells; 2, staining in half or more of the malignant cells). Then, both intensity and percentage scores were added to obtain a single score (from 0 to 4) in a manner similar to the Allred score for ER and PR staining [17]. For the purpose of correlation and survival analyses, tumors were considered to have a low expression for ERα-36 if they scored between 0 and 2 and were considered to have high expression above 2.
Statistical analysis
The correlation between ERα-36 expression and clinico-pathologic characteristics was determined using Pearson’s chi-square test (or Fisher’s exact test). Distant metastasis-free survival (DMFS) was defined as the time from the date of histological diagnosis of breast cancer to the date of distant metastasis or death. Disease-free survival (DFS) was defined as the time from the date of histological diagnosis of breast cancer to the date of any cancer recurrence (local, distant, or contralateral) or death. Overall survival (OS) was defined as the time from the date of histological diagnosis of breast cancer to the date of death. The database was locked at 12 years of follow-up, and patients who were event-free at the last follow-up visit (or at 12 years) were censored.
Survival curves, median DMFS, DFS, and OS (if reached) in addition to 8-year DMFS, DFS, and OS (with 95%CIs) were derived from Kaplan-Meier estimates, and the curves were compared using log-rank test. Hazard ratios and 95%CIs were calculated using Cox regression model. Cox multivariate analysis was performed to determine whether a factor is an independent predictor of DMFS, DFS, or OS after adjusting for other significant factors at the univariate level. All statistical tests were two-sided, and the p value was considered statistically significant if inferior to 5%. Statistical analyses were performed using SPSS 20.0 statistics package.