Chemicals and reagents
Primary antibodies against PD-L1 (#13684), p-JNK (#9251), JNK (#9252), p-c-Jun (#9261), c-Jun (#9165), HDAC3 (#3949), ubiquitin (#3933) were obtained from Cell Signaling Technology (USA). PE conjugated primary antibody PD-L1 (#393608) and CD3 (#300308) were purchased from Biolegend (USA). Purified human PD-L1 antibody (#329747) used for PD-L1 blocking was obtained from BioLegend (USA). CD3+ T cells positive selection kits (#130–050-101) were purchased from Miltenyi Biotech (Germany), Purified anti-CD3 (#566685, Clone: OKT3) and anti-CD28 antibodies (#555728, Clone: CD28.2) were purchased from BD Biosciences (USA). Carboxyfluorescein succinimidyl ester (CFSE, #C34554), DAPI (#D21490) and lipofectamine 3000 (#L3000015) were purchased from Invitrogen (USA). JNK inhibitor SP600125 (#S1460) and proteasome inhibitor MG132 (#S2619) were purchased from Selleck Chemicals (USA); JNKs agonist anisomycin (#HY-18982) was purchased from Medchem Express (USA). PrimeScript® RT reagent Kit (#DRR037A) and SYBR® Premix Ex TaqTM (#DRR420A) were products of TaKaRa. E.Z.N.A® HP (Japan). Total RNA Kit (#R1034) was purchased from Omega Bio-Tek (USA). Small interfering RNA (siRNA) against human c-Jun, c-Fos, S6K (ribosomal protein S6 kinase), Stat1 (signal transducers and activators of transcription 1), Stat3 (signal transducers and activators of transcription 3), IRF1 (interferon regulatory factor 1) and negative control were purchased from RiboBio (China). The plasmid vector (pEnter), Flag- and His- tagged c-Jun overexpression plasmid (pEnter-c-Jun, #CH836318) and COP1 overexpression plasmid (pEnter-COP1, #CH884210) were purchased from Vigene Biosciences (China). The lentiviral null vector (pReceiver-Lv233) and HDAC3 overexpression vector (pReceiver-Lv233-HDAC3) were purchased from GeneCopoeia Inc. (USA).
Cell lines and cell culture
Parental cancer cells (A549, MCF-7, HepG2) and their drug-resistant counterparts (A549/CDDP, MCF-7/ADR, and HepG2/ADR) were kindly provided by the Cancer Institute & Hospital, Chinese Academy of Medical Sciences (Beijing, China). All cells were cultured in RPMI-1640 or DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, USA) in a humidified atmosphere of 5% CO2 at 37 °C.
Patients and tumor tissues
To examine PD-L1 protein expression in NSCLC tissues sensitive and resistant to cisplatin treatment, we retrospectively obtained 90 cases of NSCLC tissues from the First Affiliated Hospital of Sun Yat-sen University during 2014 ~ 2017. NSCLC tissues were paraffin embedded and sectioned for immunohistochemistry, and all tumor tissues were pathologically diagnosed as NSCLC according to the WHO classification criteria. All patients received 1 to 3 courses of cisplatin treatment before surgery. The clinical characteristics of the patients are shown in Additional file 1: Table S1. According to the Response Evaluation Criteria In Solid Tumors (RECIST), 45 tumor samples from patients with a 30% or more decrease in the entire tumor burden after cisplatin therapy were considered to be sensitive to cisplatin, while another 45 tumor samples from patients with a 20% increase in the entire tumor burden or appearance of new lesions after cisplatin therapy were considered to be resistant to cisplatin [10]. This study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University, and all methods were carried out in accordance with the approved guidelines. Written informed consent was signed and documented by all the patients who participated in the study.
Cell transfection
For transfection, cells were seeded on a 6-well plate (2 × 105 cells/well) and cultured for 12 h. Then cells were transfected with 2 μg plasmid or 100 pmol siRNA mixed with lipofectamine 3000 reagent in the complete medium with 10% FBS according to the manufacturer’s instructions, and then incubated for indicated time before harvest.
Quantitative real-time PCR
Quantitative real-time PCR was performed as previously described [17]. The primer sequences used in each reaction were listed as Additional file 1: Table S2.
Western blot analysis
Western blotting was performed as previously described [18]. Notably, c-Jun and COP1 were detected in the western blotting assays using anti-c-Jun and anti-COP1 antibodies, respectively, but not anti-His or anti-Flag antibodies when the overexpression plasmids were used.
For nuclear and cytoplasmic protein detection, nuclear and cytoplasmic proteins were extracted using a nuclear/cytosol fractionation kit (#P0028, Beyotime, China) according to the manufacturer’s instructions, and the samples were then examined by western blotting.
To detect c-Jun ubiquitination, cells were treated with or without MG132 (10 μM) for 8 h. Subsequently, the cells were lysed and immunoprecipitated with a primary antibody against c-Jun or rabbit control IgG, and equal amounts of immunoprecipitates were then subjected to immunoblot analysis using an anti-ubiquitin mAb to detect ubiquitin.
Flow cytometry
To detect antigens on the cell membrane by flow cytometry, cell suspensions were washed with PBS and then directly incubated with indicated antibodies (such as anti-PD-L1 antibodies) or isotype controls for 1 h at 4 °C. Subsequently, the cells were washed and resuspended with PBS, and then fluorescence data were collected on a flow cytometry machine (Millipore, USA). The data were analyzed using FlowJo 7.6.1 software.
Immunofluorescence microscopy
To detect the localization and expression of p-c-Jun in cancer cells, cells were seeded on 96-well plate (3 × 103 cells/well) overnight, and then were fixed with 4% paraformaldehyde for 20 min and permeated by 1% Triton-X100 for 15 min. Subsequently, cells were blocked with 10% normal goat serum for 30 min at 37 °C and incubated with antibodies against p-c-Jun (1:100 dilution) at 4 °C overnight. After washing with PBS, slides were incubated with FITC conjugated secondary antibodies (1:1000 dilution) and counter stained with DAPI (10 mg/ml). The expression of p-c-Jun was detected by High Throughput Screening equipment (ArrayScan VTI 600 plus, Thermo).
Chromatin immunoprecipitation
Chromatin immunoprecipitation assays were performed according to the manufacturer’s instructions of the Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit (#17–245, Millipore, USA). Briefly, cells were crosslinked by 1% formaldehyde incubation and then sonicated on ice to shear the DNA to lengths between 200 and 1000 base pairs. Soluble chromatin fragments of 200 to 1000 bp in length were incubated with 5 μg of anti-acetyl-histone H3 antibodies at 4 °C overnight. Normal rabbit IgG was used as a negative control for validating the ChIP assay. Isolated DNA fragments were purified, and quantitative PCR was performed using 2 μl of DNA in triplicate. ChIP primers covering 1800 bp upstream of the human PD-L1 gene start codon were designed by NCBI-Blast software. Amplicons were between 60 and 150 base pairs, and the primers were as follows: primer 1 (− 1178 bp to − 1117 bp), forward 5′- GCT GGG CCC AAA CCC TAT T − 3′ and reverse 5′-TTT GGC AGG AGC ATG GAG TT-3′; primer 2 (− 455 bp to − 356 bp), forward 5′-ATG GGT CTG CTG CTG ACT TT-3′ and reverse 5′-GGC GTC CCC CTT TCT GAT AA-3′; primer 3 (− 105 bp to − 32 bp), forward 5′-ACT GAA AGC TTC CGC CGA TT-3′ and reverse 5′-CCC AAG GCA GCA AAT CCA GT-3′. ChIP-qPCR result was calculated using the ΔΔct method. Briefly, each ChIP fractions’ Ct value was normalized to the input DNA fraction Ct value to account for chromatin sample preparation differences (Δct normalized ChIP). Fold changes in H3 acetylation in the PD-L1 promoter of drug-resistant cancer cells were calculated by 2−ΔΔCt, where ΔΔCt = ΔCt [drug-resistant cancer cells: normalized ChIP] − ΔCt [drug-sensitive cancer cells: normalized ChIP].
Immunohistochemical examination
Immunohistochemical examination was performed as previously described [ 17]. Immunohistochemical sections were observed and images were captured for 5 random fields by two pathologists without knowing the patients’ clinical information under a light microscope (Nikon, Japan) at a magnification × 20. The staining intensity was assessed using a modified quickscore method on a scale of 0–3 as negative (0), weak (1), medium (2) or strong (3). The extent of the staining, defined as the percentage of positive stained areas of cancer cells per the whole tumor area, was scored on a scale of 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). An overall protein expression score (overall score range, 0–12) was calculated by multiplying the intensity and positivity scores according to our previous study [19].
Establishment of A549/CDDP cells with stable HDAC3 overexpression
A549/CDDP cells were transfected with lentiviruses with HDAC3 expression vectors (pReceiver-HDAC3) or their control null vectors (pReceiver) at a multiplicity of infection of 100 transfecting units per cell in the presence of 5 mg/ml polybrene. The transfected A549/CDDP cells were selected with puromycin (1 μg/ml) for 10~14 days. The surviving cells were then picked out and reseeded into a 96-well plate for cell clone formation and expansion. The expanded monoclonal cell populations (named A549/CDDPHDAC3 and A549/CDDPpReceiver) were collected and stored for further study.
Animal studies
Six-week-old female BALB/C nude mice were obtained from the Animal Experimental Center of Southern Medical University (Guangzhou, China). The procedures for the handling and care of the mice were approved by the Animal Experimentation Ethics Committee of Southern Medical University. In total, 1 × 107 A549, A549/CDDP, A549/CDDPpReceiver or A549/CDDPHDAC3 cells in Matrigel (BD Biosciences, USA) were injected into the right flanks of nude mice to form xenograft tumors. When the tumor volumes reached ~ 100 mm3, mice bearing A549/CDDP tumors were treated with SP600125 (15 mg/kg) in PPCES vehicle (30% PEG-400, 20% polypropylene glycol, 15% Cremophor EL, 5% ethanol and 30% saline) or PPCES vehicle alone every 4 days by intragastrical gavage for 2 weeks. At the end, the tumors were collected and then digested to prepare a single cell suspension for cell surface PD-L1 detection and CD3+ T cell proliferation assays.
CD3+ T cell proliferation assay
A CD3+ T cell proliferation assay was performed as previously described [20]. Briefly, CD3+ T cells were isolated from healthy donor PBMCs using positive selection kits and labeled with CFSE. Then, 3 × 105 CFSE-labeled CD3+ T cells were cocultured with 1 × 103 cultured or tumor-derived cancer cells in 96-well plates. Next, the cocultured T cells were stimulated by the addition of anti-CD3 (3 μg/ml) and anti-CD28 antibodies (3 μg/ml). After 3 days, the cells were harvested and stained with a PE-conjugated anti-CD3 antibody, and T cell proliferation was determined by measuring the CFSE dilution using flow cytometry after gating on the CD3+ cell populations.
Statistical analysis
Results were expressed as mean ± standard deviation (SD) of three independent experiments unless otherwise specified. Student’s t test and one-way ANOVA were performed to compare the differences between groups. The correlations of PD-L1, c-Jun and HDAC3 expression in tumor tissues were analyzed by Pearson’s correlation coefficient. Statistical analyses were performed using GraphPad Prism Software Version 5.0 (GraphPad Software Inc., CA, USA). All experiments were performed independently in triplicate. P < 0.05 was considered as statistically significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.