Cell culture and reagents
Human BC MDA-MB-231, MDA-MB-468, CAL-148, MDA-MB-453, MDA-MB-157, MDA-MB-436, and HCC1937 cell lines were obtained from Nanjing CoBioer Biosciences (CoBioer, China) in 2018. The SUM149 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The human embryonic kidney HEK293T cell line was kindly provided by Prof. Guo-Hong Hu (Shanghai Institutes for Biological Sciences) in 2014. All of the above cell lines were authenticated by DNA profiling (short tandem repeat, STR), morphology, cell viability, isoenzymes, and mycoplasma assays. MDA-MB-436 cells were maintained in Leibovitz’s L15 medium (BasalMedia, L620) with 10 µg/ml insulin and 16 µg/ml glutathione, HCC1937 cells were maintained in RPMI 1640 medium (BasalMedia, L210), and SUM149 cells were maintained in DMEM/F12 (BasalMedia, L310) with 1 µg/ml hydrocortisone and 5 µg/ml insulin. The other cells were maintained in DMEM (BasalMedia, China, L110). All media were supplemented with 10% fetal bovine serum (Gibco, USA, 10270-106) and 1% penicillin-streptomycin (BasalMedia, S110B), and the cells were not passaged more than six times from collection to use. Olaparib (AZD2281, S1060), veliparib (ABT-888, S1004), palbociclib (PD-0332991, S1579), and ribociclib (LEE011, S7440) were purchased from Selleck (USA). For the administration of various inhibitors, cells were treated with 10 μM H-89 from Med Chem Express (MCE, USA, HY-15979) and 10 μM MG-132 (MCE, HY-13259) as indicated.
Acquired olaparib-resistant cell lines were generated by culturing cells in media supplemented with increasing concentrations of olaparib for 8 months starting at 0.5 µM and reaching a final dose of 20 µM. Resistant cells were maintained in media supplemented with 20 µM olaparib.
Cell viability assay and determination of drug synergy
The cells of interest (1×103~3×103 cells per well) were seeded into 96-well plates in triplicate overnight in 100 ml of complete growth medium and then treated with the indicated drugs for the designed time. Cell viability was tested using the cell counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Japan, CK04) as the manufacturer's instructions. The combination index (CI) was used to evaluate the synergistic effect of the two drugs and was calculated by the Chou-Talalay method with CompuSyn software[21].
Flow cytometry analysis
Flow cytometry analysis was used to assess cell apoptosis and the cell cycle. The cells of interest were treated with the designed drugs for 3 days and digested with EDTA-free trypsin. For apoptosis evaluation, the cells were collected and stained using an Annexin V-FITC/PI apoptosis kit (MultiSciences, Hangzhou, China, AP101). For cell cycle analysis, the cells were fixed using 70% precooled ethanol at 4°C overnight, washed with PBS and stained with PI solution (MultiSciences, AP101-60-PI). The above cells were all identified and quantified by a flow cytometer (Beckman Cytomics FC 500 BD FACSCanto II) according to the manufacturer’s instructions, and the data were analyzed by FlowJo v10 software.
Migration assay
The cells used for the Transwell (Corning, USA) migration assay were pretreated with specific drugs for 3 days. Then, a total of 5~10×104 cells were resuspended in the upper compartment of each chamber with 100 µl serum-free medium, and the lower chamber was filled with 600 µl medium containing 20% fetal bovine serum. The Transwell plates were incubated in a humidified environment with 95% air and 5% CO2 at 37°C for 18 h (HCC1937) or 24 h (MDA-MB-436). The chambers were washed with PBS, fixed with formaldehyde and stained with 0.5% crystal violet. The migrated cells were imaged and counted using ImageJ (National Institutes of Health, Bethesda, MD, USA).
Senescence assay
Following the manufacturer’s protocol, a β-Galactosidase Staining Kit (Solarbio, Beijing, China, G1580) was used for the detection of cell senescence according to Dimri et al[22]. Images were taken in transmitted light by a computerized imaging system consisting of a Leica charge-coupled device (CCD) DFC420 camera and a Leica DM IRE2 microscope (Leica Microsystems Imaging Solutions Ltd).
Colony formation assay
For the colony formation assay, 500 cells were plated into 6-well or 12-well plates. After overnight incubation, the cells were treated with the designed drugs for 12–17 days, and the medium was replaced every 3 days. The colonies were fixed in formaldehyde and stained with 0.5% crystal violet. Colonies consisting of 50 or more cells were included in the count.
Immunofluorescent staining
Immunofluorescent staining was carried out as described previously[23,24]. Briefly, after drug treatment, the adherent cells were washed in PBS, fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and blocked in 1% bovine serum albumin (BSA) in PBS. The cells were incubated with an anti-ɣH2AX (Abcam, England,ab26350, 1:250) and anti-RAD51 (Abcam, ab133534, 1:500) or anti-β-Catenin (Cell Signaling Technology, USA, D10A8 #8480, 1:100) antibody in 1% BSA at 4°C overnight, washed three times in PBS for five minutes each, and then incubated with the appropriate secondary antibody (Jackson ImmunoResearch, USA, 115-095-003/111-585-003). DNA staining was performed using Gold Antifade Mountant with DAPI (Invitrogen, USA, P36931). Leica SP5 confocal laser scanning microscopy (Leica Microsystems, Buffalo Grove, USA) was used for immunofluorescence imaging.
HR assay
The HR assay has been described in previous research[25,26]. In brief, a clone stably expressing pDR-GFP was generated and validated by analyzing GFP-positive cells. For HR assays, cells stably expressing DR-GFP were transfected with ISceI-GR plasmids. Twenty-four hours post transfection, the cells were treated with 10 μmol/L triamcinolone acetonide (TA) and cultured for another 48 h[27]. To study the effect of drugs on HR, we added a specific concentration of drugs together with TA. The proportion of GFP-positive cells was evaluated using flow cytometry, and the efficiency of HR was calculated.
RNA-Seq analysis
Total RNA from MDA-MB-436 and HCC1937 cells treated with DMSO, 5 µM olaparib, 5 µM palbociclib, or their combination (Ola/Palb) for 24 h was extracted using TRIzol Reagent (Invitrogen). RNA quantity and quality were assessed by an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agarose gel. One microgram of total RNA with an RNA integrity number (RIN) value above 6.5 was used for cDNA library construction. The cDNA libraries created from the cell lines (sixteen samples in total, with duplicate libraries for each sample) were run on a HiSeq 2500 instrument according to the manufacturer’s instructions (Illumina, San Diego, CA, USA).
Gene set enrichment analysis (GSEA) was performed by the JAVA program using MSigDB Hallmark gene set collection. One thousand random sample permutations were carried out, and the significance threshold was set at NES absolute value>1, NOM p-value<0.05 and FDR q-value<0.05.
Real-time quantitative reverse transcription PCR (qRT-PCR)
Total RNA was isolated from specimens or cells using the RNeasy Plus Mini Kit (QIAGEN, Germany) following the manufacturer’s protocol. cDNA was synthesized using a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Japen, RR047A). Real-time qRT-PCR was carried out using SYBR Premix Ex Taq (TaKaRa, RR420A) in triplicate on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). All primers were synthesized by Sangon Biotech, and the sequences of the primers are available in Supplementary Table S1. The results were analyzed with SDS v2.1 software and the 2−ΔΔCT method by normalization to GAPDH levels.
Western blot (WB) analysis
The WB protocol has been described in detail previously[28]. In short, cells were lysed in Pierce T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific Inc.) containing protease and phosphatase inhibitors (Bimake, USA, B14001, B15001A+B). The lysates were centrifuged at 12000 rpm for 15 min, the supernatants were collected, and protein concentrations were determined with a bicinchoninic (BCA) protein assay kit (Solarbio, PC0020). A total of 20–30 μg of protein was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA, IPVH00010, ISEQ00010). The primary and secondary antibodies are described in Supplementary Table S2. For the immunoprecipitation (IP) analysis, the cells were lysed in NP-40 lysis buffer (Beyotime, China, P0013F) containing protease and phosphatase inhibitors. The supernatants were incubated with 1–3 μg primary antibodies overnight at 4°C on a rotating platform, followed by immunoblotting analysis. ImageJ was used to quantify the immunoblotting results by measuring the protein band densities.
Expression vectors, plasmid transfection and lentiviral infection
The CTNNB1 (NM_001904) and MYC (NM_002467) cDNAs were obtained from GeneChem (Shanghai, China) and subcloned into the Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin vector (GV492) to generate the Flag-gcGFP-CTNNB1 and Flag-gcGFP-MYC expression vectors, respectively. Site-directed mutations CTNNB1S675A and CTNNB1S675D were generated by PCR-based mutagenesis and verified by DNA sequencing. Human CTNNB1 short hairpin RNAs (shRNAs) in the U6-MCS-Ubiquitin-Cherry-IRES-puromycin vector (GV298) and MYC shRNAs in the U6-MCS-Ubiquitin-EGFP-IRES-puromycin vector (GV248) were purchased from GeneChem. Detailed information concerning DNA constructs and the primers used for molecular cloning is provided in Supplementary Table S1. To generate stable cell lines expressing cDNAs or shRNAs, each lentiviral expression vector was transfected into HEK293T cells with polyethyleneimine (PEI). The supernatant containing viruses was collected 48 h after transfection, filtered, and used to infect target cells in the presence of 10 μg/ml polybrene (Sigma-Aldrich, USA, H9268) prior to drug selection with 1-2 μg/ml puromycin for one week. Overexpression (OE) and knockdown (KD) efficiencies were validated by immunoblotting after transfection.
The lentiCas9-Blast (Addgene, USA, #52962) and lentiGuide-Puro (Addgene, #52963) vectors were provided by the Feng Zhang laboratory. The MYC knockout (KO) cell line was generated using the CRISPR/Cas9 system[29] and was validated by Sanger sequencing and immunoblotting analysis. The individual sgRNA sequences are provided in Supplementary Table S3.
Xenograft in vivo model
All animal experiments were performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals (http://oacu.od.nih.gov/regs/index.htm) and were approved by the Fudan Animal Ethics Committee (approval number, 201911004S). MDA-MB-436 and HCC1937 cells (8 × 106 per mouse) were harvested and resuspended in 50 μL of PBS and 50 μL of Corning® Matrigel® (BD Biocoat, USA, 354248). Then the cells were injected directly into the mammary fat pads of six-week-old female NOD-SCID mice weighing 15 to 16 g. Tumor volumes were monitored via caliper measurements every 3–4 days and were calculated as follows: length × width 2/2. When tumors reached approximately 75 mm3, the mice were randomly allocated into four groups administered control solvent, olaparib, palbociclib, or their combination (Ola/Palb) once a day. Olaparib was dissolved in 4% DMSO+30% PEG300+ddH2O for intraperitoneal administration and dosed at 50 mg/kg/day; palbociclib was dissolved in 0.9% warm saline and administered via oral gavage at 100 mg/kg/day. After 21 days, the tumors were collected.
Immunohistochemistry (IHC) analysis
Details on the IHC protocol have been described previously [30-32]. For hematoxylin and eosin (H&E) staining, slides were stained with Mayer’s hematoxylin (Sigma-Aldrich) and 0.1% sodium bicarbonate and were counterstained with Eosin Y solution (Sigma-Aldrich). For IHC, the primary and peroxidase (HRP)-conjugated secondary antibodies were listed in Supplementary Table S2. The positive-staining density was measured by the computerized imaging system mentioned above.
Statistical analysis
Quantification and statistical analysis were performed with GraphPad Prism 7.0 (GraphPad Software, Inc.), SPSS version 22.0 (SPSS, Chicago, IL) or R software version 3.5.3. utilizing the statistical tests described in the text and figure legends. The unpaired two tailed Student’s t test was used to compare data between two groups, and correlation coefficients were calculated using the Pearson test. All p-values were two-sided, and p-values less than 0.05 were considered statistically significant.