Cell cultures and reagents
HG-SOC primary cells were obtained from freshly-isolated ascitic fluid from HG-SOC patients undergoing surgery for ovarian tumor by laparotomy or paracentesis at the Gynecological Oncology of our Institute. The study protocol for tissue collection and clinical information was approved by the institutional review board (IRB) and patients provided written informed consent authorizing the collection and use of the tissue for study purposes. Patient-derived (PD)-HG-SOC cells were isolated and characterized as previously reported [14]. In this study, we employed the early passage PD-HG-SOC PMOV10 cell line, where PM stands for Preclinical Models, OV stands for ovarian serous cancer, and # is the order in which the cell line was established. In addition, we used HG-SOC cell lines, OVCAR-3 (HTB-161) and Kuramochi, which were obtained from American Type Culture Collection (ATCC) and Japanese Collection of Research Bioresources (JCRB) Cell Bank, respectively. Cells were validated by short tandem repeat (STR) profiling. PMOV10 and Kuramochi cells were cultured in RPMI 1640 (Gibco, ThermoFisher Scientific, USA) containing 1% penicillin–streptomycin and 10% fetal bovine serum, whereas OVCAR-3 cells were cultured in RPMI-1640 containing 1% penicillin–streptomycin, 20% fetal bovine serum and 0.01 mg/ml bovine insulin, under a humidified atmosphere of 5% CO2 at 37 °C. Cells were tested routinely for cell proliferation, as well as mycoplasma contamination. Before each experiment, cells were serum starved by incubation in serum-free medium for 24 h. ET-1 was used at 100 nM and was purchased from Sigma-Aldrich (Germany). Macitentan, also known as ACT-064992 or N-(5-[4-bromophenyl]-6-{2-[5-bromopyrimidin-2-yloxy]-ethoxy}-pyrimidin-4-yl)-N′-propylsulfamide, was added 30 min before ET-1 at a dose of 1 μM and was kindly provided by Actelion Pharmaceuticals, Ltd. (Switzerland). BQ123 (Bachem, Switzerland) and BQ788 (Peninsula Laboratories, USA) were added 30 min before ET-1 at a dose of 1 µM.
Small interfering RNA (siRNA) transfection
For transient knockdown, PMOV10, OVCAR-3, and Kuramochi cells were transfected for 48–72 h with Dharmacon SMARTPool ON-TARGETplus siRNA oligonucleotides specific for ZEB1 (si-ZEB1, L-006564–01-0050), YAP1 (si-YAP, L-012200–00-0050), JUN (si–c-JUN, L-003268–00-0020), or with ON-TARGETplus Non-targeting Control Pool (SCR, D-001810–10) (GE Healthcare Life Sciences, USA). In addition, PMOV10 and Kuramochi cells were transfected with a Negative Control DsiRNA (#51–01-14–04), or with a siRNA pre-designed and validated for ILK (si-ILK, hs.Ri.ILK.13.2), purchased from IDT (USA). siRNAs were used at a final concentration of 50–100 nM and Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific) was employed according to the manufacturer’s protocol.
Immunoblotting (IB) and immunoprecipitation (IP)
NE-PER nuclear and cytoplasmic extraction reagents kit (Thermo Scientific) was used to separate cytoplasmic and nuclear fractions. Whole cell lysates were prepared using a modified RIPA buffer (50 mM Tris–HCl pH 7.4, 250 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing a mixture of protease and phosphatase inhibitors. Protein content of the extracts was determined using Bio-Rad Protein Assay Kit (Bio-Rad, USA). IB for anti-PCNA and anti-tubulin antibodies (Abs) were used as loading control and to assess the purity of the nuclear and cytoplasmic fractions, respectively. IB for β-actin was used as loading control for whole cell lysates. Cell lysates were resolved by SDS/PAGE. Membranes were blocked in TTBS (TBS with 0.1% Tween 20) containing either 5% dry milk or BSA and incubated with primary antibodies (Abs) overnight at 4 °C. All Abs used in IB assays are listed in Additional file 1: Table S1. After washing, the appropriate secondary peroxidase conjugated Abs were added to membranes and incubated for 1 h. For IP, 200 μg of pre-cleared nuclear cell fractions were incubated with anti-YAP (1A12, Cell Signaling Technology), anti-ZEB1 (H3, Santa Cruz Biotechnology, USA), or anti-mouse IgG Isotype Control (ThermoFisher Scientific) Abs and protein G Sepharose 4 Fast Flow beads (Cytiva, Sweden) at 4 °C overnight. The IP and input (3% of the total nuclear extracts) samples were boiled for 5 min in SDS loading buffer, loaded onto pre-casted 10% or 4–20% SDS/PAGE (Bio-Rad), transferred by using Trans-Blot transfer pack (Bio-Rad), and IB with different Abs as before. To obtain clean and specific IB signals of TEAD4 and JUN, which run very close to heavy chain of IgG, we used HRP-conjugated protein A peroxidase (Pierce, ThermoFisher Scientific) instead of HRP-conjugated secondary Abs. Blots were developed with the enhanced chemiluminescence detection system (Clarity Western ECL Substrate Bio-Rad) or LiteAblot turbo extrasensitive chemiluminescent substrate (Euroclone, Italy). IB signals were quantified using ImageJ software (https://imagej.nih.gov/ij/).
Proximity ligation assay (PLA)
PMOV10 or Kuramochi cells (4 × 104) were seeded in 24-well plate, and after 24 h of starvation, were stimulated with ET-1 for 6 h. Cells were then washed in PBS, fixed with formaldehyde 4% in PBS for 10 min, permeabilized with Triton X-100 0.4% in PBS for 20 min, blocked with BSA 0.5% in PBS for 30 min and stained with anti-ZEB1 (D80D3, 1:20, cat. #3396, Cell Signaling Technology) together with anti-YAP (G-6, 1:20, SC-376830, Santa Cruz Biotechnology) or anti-ZEB1 (H-3, 1:20, sc-515797, Santa Cruz Biotechnology) together with anti-c-JUN (60A8, 1:20, cat. #9165S, Cell Signaling Technology) primary Abs at 4 °C overnight. PLA was performed with the Duolink in situ Detection Reagents Orange (Sigma-Aldrich, USA), according to the manufacturer’s protocol. Anti-mouse PLUS (Sigma-Aldrich) and anti-rabbit MINUS (Sigma-Aldrich) PLA probes were used for 1 h at 37 °C. Then, coverslips were washed in PBS and then incubated with a DNA ligase diluted in Ligation buffer for 30 min at 37 °C. After washing, coverslips were incubated with a DNA polymerase diluted in the amplification buffer for 100 min at 37 °C. Nuclei were stained using 4′,6′-diamidino-2-phenykindole (DAPI). Coverslips were mounted with Vectashield mounting medium for fluorescence (Vector Laboratories Ltd., UK). Fluorescence signals were captured by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by Leica FW4000 deconvolution software (Leica) using an oil 63 × objective. The number of dots per nuclei was quantified using ImageJ software.
Chromatin immunoprecipitation (ChIP)
Chromatin was extracted from 5 × 106 cells of PMOV10 cells. Briefly, cells were crosslinked with formaldehyde 1% in PBS for 8 min at room temperature. After washing in PBS, chromatin was sheared by sonication, centrifuged and diluted in 50 mM Tris pH 8.0, 0.5% NP-40, 0.2 M NaCl, 0.5 mM EDTA. One-twentieth of the precleared chromatin was used as the input for the ChIP assay. The precleared chromatin was rotated overnight with primary Ab or IgG. The primary Abs used were as follows: anti-ZEB1 (2 µg/µl, clone H3, sc-515797, Santa Cruz Biotechnology), anti-YAP (2 µg/µl, H-125, cat. no. sc-15407, Santa Cruz Biotechnology), anti-c-JUN (2 µg/µl, 60A8, cat. #9165S, Cell Signaling Technology), anti-rabbit IgG Isotype Control (ThermoFisher Scientific), and anti-mouse IgG Isotype Control (ThermoFisher Scientific). The next day, 40 μl of 50% salmon sperm-saturated protein A Sepharose (Cytiva) was added to immune complexes, and the mixtures were rotated at 4 °C for 30 min. The beads were washed with 20 mM Tris pH 8.0, 0.1% SDS, 1% NP-40, 2 mM EDTA, 500 mM NaCl and with 1 × Tris/EDTA. Immune complexes in 1 × Tris/EDTA containing 1% SDS and protein-DNA cross-links were reverted by incubation at 65 °C for 4 h. DNA–protein complexes were digested with Proteinase K at 37 °C for 1 h. DNA was purified through phenol/chloroform extraction, precipitated in ethanol, and resuspended in water. The binding between ZEB1, YAP, and JUN with AP-1 motif or a negative region in the EDN1 promoter was examined through qPCR by using AmpliTaq polimerase (Applied Biosystems, USA). The primers used are listed in Additional file 2: Table S2.
Luciferase reporter gene assay
Luciferase assays were carried out in PMOV10 and OVCAR-3 cells (6 × 104) seeded in 12-well plates and transfected with 500 ng of reporter plasmid by using Lipofectamine 2000 (ThermoFisher Scientific), according to manufacturer’s instructions. Transcriptional activity of AP-1 was studied by using a pAP1-Luc Cis-Reporter plasmid (cat. 219,074, Agilent Technologies, USA). To analyze the ZEB1 and ET-1 promoter activities we used a reporter construct containing a 900 bp sequence from ZEB1 promoter, synthesized by TEMA Ricerca (Italy), and a reporter construct containing a 1500 bp sequence from ET-1 promoter, kindly provided by Dr. Z. Zhang (University of California San Diego School of Medicine, La Jolla, Ca), respectively. All plasmids were co-transfected with 250 ng of pCMV-β-galactosidase vector (Promega) and 100 nM of siRNAs as indicated. After 24 h of transfection, cells were stimulated with ET-1 and/or macitentan for additional 24 h. Reporter activity was measured using the Luciferase assay system (Promega) and normalized to β-galactosidase activity.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was isolated using the Trizol (ThermoFisher Scientific), according to the manufacturer’s protocol. RNA integrity was confirmed through agarose gel electrophoresis, and RNA concentration and purity were determined with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). RNA was reversed transcribed using the Wonder RT cDNA Synthesis kit (Euroclone, Italy). The expression of ET-1, CTGF, CYR61, ANKRD1, E-cadherin, N-cadherin, vimentin, ZEB1, and cyclophilin-A mRNA was evaluated by using Luna Universal qPCR Master Mix (New England Biolabs, USA) on QuantStudio 6-Flex (Thermo Fisher Scientific), according to the manufacturer's instructions. The mRNA expression levels were determined by normalizing to cyclophilin-A mRNA expression and expressed as relative mRNA level (2^ΔΔct). Data are presented as means ± SD. Primer sequences are provided in Additional file 2: Table S2.
ELISA assay
PMOV10 (1 × 106) cells were seeded in 100 mm dish. After 24 h of siRNA transfection cells medium was replaced with serum-free medium containing or not macitentan. After 48 h of incubation, the conditioned media were collected, centrifuged and stored in aliquots at -80 °C. The release of ET-1 was measured with Quantikine ELISA kit (R&D Systems, USA), according to the manufacturer’s instructions. ET-1 was measured in the range of 0–25 pg/ml.
Chemoinvasion assay
Invasion assays were carried out using Boyden Chambers consisting of transwell filter inserts with 8 μm size polycarbonate membrane (Corning, USA) placed in a 24-well plate and precoated with 50 μl of cultrex (R&D Systems). After 48 h of siRNA transfection, OVCAR-3, PMOV10, or Kuramochi cells (3 × 104) were seeded with serum-free medium in the upper chamber and serum-free medium containing or not ET-1 in combination or not with macitentan was added to the lower chamber. Cells were left to invade overnight at 37 °C. Cells on the upper part of the membrane were scraped using a cotton swab and invaded cells were stained using Diff-Quick kit (Merz-Dade, Switzerland). From every transwell, several images were taken under a phase-contrast with Olympus I × 70 microscope (Olympus Corporation, Japan) at 10 × magnification for Kuramochi cells or at 20 × magnification for PMOV10 and OVCAR-3 cells.
Vasculogenic mimicry assay
After 48 h of siRNA transfection, OVCAR-3 (3.5 × 104) and Kuramochi (3 × 104) cells were seeded in a 96-well culture plate precoated with 50 μl/well of Cultrex (R&D Systems) and stimulated with serum-free medium or ET-1 in combination or not with macitentan. Cells were left overnight at 37 °C. The day after, tubule-like structure formations were visualized with an inverted microscope with a 20 × magnification. Representative images were captured with a ZOE Fluorescent Cell Imager (BioRad Laboratories). Tube formation was analyzed by using Angiogenesis Analyzer for ImageJ (NIH) measuring the number of nodes and the tube length.
Animal study
Female athymic (nu + /nu +) mice, 4–6 weeks of age (Charles River Laboratories, Italy) were injected intraperitoneally with 2.5 × 106 viable OVCAR-3 cells following the guidelines for animal experimentation of the Italian Ministry of Health. One weeks after cells injection, OVCAR-3 xenografts were randomized into two different groups of ten mice undergoing the following treatments: control (Ctr; vehicle) vs. macitentan (MAC; 30 mg/kg/oral daily) for 5 weeks. At the end of treatment, all mice were euthanized and intraperitoneal (i.p.) organs throughout the peritoneal cavity (including intestine, mesentery, liver and spleen) were analyzed. The number of visible metastases was counted and the removed i.p. nodules were carefully dissected, frozen and processed for IB analyses. Values represent the mean ± SD of ten mice in each group for OVCAR-3 xenografts from two independent experiments.
Bioinformatic analyses
The Kaplan–Meier plotter [26] was used to investigate the correlation between the expression of ETAR, ILK, ZEB1, YAP, and AP-1 mRNA levels with the prognosis of serous ovarian cancer patients. Overall survival (OS) analysis was performed in the following cohorts of patients: GSE9891, GSE18520, GSE26193, GSE30161, and GSE63885 (523 patients). In parallel, progression-free survival (PFS) analysis was performed in the following cohorts of patients: GSE9891, GSE26193, GSE30161, and GSE63885 (483 patients). The employed gene probes are as follow: 216235_s_at and 204464_s_at for ETAR; 201234_at for ILK; 217836_s_at for YAP; 201464_x_at, 201465_s_at, 201466_s_at, 203751_x_at, 214326_x_at, 203752_s_at, 202768_at and 209189_at for AP1; 210875_s_at, 212764_at, 239952_at and 212758_s_at for ZEB1. OC samples were divided into ‘low’ and ‘high’ according to gene mRNA expression using the auto select best cutoff value. Subsequently, OS and PFS for the two groups were compared with a Kaplan–Meier survival plot on the webpage (http://kmplot.com/analysis/index.php?p=service&cancer=ovar).
Statistical analysis
The significance of KM curves was evaluated by log-rank test, hazard ratio (HR) with 95% confidence intervals. Except for the animal study, each experiment was repeated at least three times. Data are presented as mean ± SD. Statistical analysis was performed using Student’s two tailed t-test to compare two groups of independent samples. Statistical tests were carried out using GraphPad Prism 8 software (San Diego). P < 0.05 was considered statistically significant.