The human ovarian cancer cell lines ES-2 (cat. no. CRL-1978) and SK-OV-3 (cat. no. HTB-77), were purchased from the American Type Culture Collection (ATCC). All cell lines were tested every 6 months, or new ATCC cell lines were obtained every 6 months. SK-OV-3 and ES-2 were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum and 1% antibiotic mixture (10,000 U/ml penicillin and 10 mg/ml streptomycin) and cultured with 5% CO₂ at 37°C.

Negative control (siControl) and YAP-1-specific (siYAP-1, siYAP-2) siRNA were purchased from Invitrogen; Thermo Fisher Scientific, Inc. (sequences: siYAP-1: 5′-CAGCAGAAUAUGAUGAACUCGGCUU-3′; siYAP-2: 5′-GGAAGGAGAUGGAAUGAACAUAGAA-3′). Peptide 17 was purchased from Selleck Chemicals. YAP, phospho-Akt (Ser473), phospho-PI3K p85 (Tyr458)/p55 (Tyr199), mTOR, and Akt2 antibody were obtained from Cell Signaling Technology. The GSK3-β and GAPDH antibodies were obtained from Proteintech Group, Inc. The Ki-67, PI3K p85 beta, phospho-GSK3-β (Ser9), and phospho-mTOR (Ser2448) antibodies were obtained from Abcam. Matrigel was purchased from BD Biosciences. The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG (1:5,000; Antgene). The complete information on all the antibodies used in the present study is provided in supplementary Table SI.

Transwell plates (8-µm pore size; Corning, Inc.) were used for the migration assay as described previously (12). Cells transfected with siControl or siYAP or treated with peptide 17 (250 nM) were harvested and re-suspended with serum-free McCoy's 5A and seeded at a cell density of 2×10⁴ into the upper chamber. Complete media (500 µl) were added to the bottom chambers. The cells were incubated at 37°C for 24 h. After 1 day of incubation, the cells that migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde for 15 min, then washed with phosphate-buffered saline (PBS) for 3 times and stained with 0.05% crystal violet solution for 15 min. Non-migrating cells on the top surface of the filter were carefully wiped off with a cotton swab, and the migrating cells were quantified by counting five random fields using a phase contrast microscope.

In vitro cell invasion was investigated using a Transwell assay (8.0 µm, Corning, Inc.) coated with basement membrane Matrigel (BD Biosciences). Cells transfected with siControl or siYAP or treated with peptide 17 (250 nM) were harvested and seeded at a cell density of 2×10⁴ into the upper chamber. Complete media (500 µl) were added to the bottom chambers. After 48 h of incubation, non-invading cells on the top surface of the filter were carefully wiped off with a cotton swab, and the invading cells were quantified by counting five random fields using a phase contrast microscope.

Cells transfected with siControl or siYAP or treated with peptide 17 (250 nM) were seeded in 6-well plates in triplicate. After the cells had reached ~100% confluence, the monolayers were manually scratched using a sterile 10-µl pipette tip and cultured for 24 h. The area of the scratch was photographed under a microscope (Olympus Corporation) and measured using ImageJ software (National Institutes of Health). The results were expressed as a percentage of the original area.

Cell viability was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) at defined intervals. Cells transfected with siControl or siYAP (4×10³ cells per well) were placed in 96-well plates in triplicate and incubated for 72 h. After treatment, cell viability was detected using the CCK-8 assay according to the manufacturer's instructions. After incubating with CCK-8 dye at 37°C for 2 h in the dark, the absorbance value was measured by SpectraMax190 (Molecular Devices, LLC) at a wavelength of 450 nm.

The EdU assay was carried out using the Cell-Light™ EdU imaging detecting kit according to the manufacturer's instructions (RiboBio).

Normal ovaries, normal fallopian tubes and OC tissues were collected from patients undergoing surgery at the Department of Gynecological Oncology of Tongji Hospital (Wuhan, China). IHC staining was conducted as previously described (12,13).

Total RNA was isolated from cells or tissues using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. NanoDrop (Thermo Fisher Scientific, Inc.) was used to assess the quality and quantity of the total RNA. Total RNA (1 µg) was used to synthesize cDNA using M-MLV reverse transcriptase (Takara Bio, Inc.). RT-qPCR detection was performed using the Bio-Rad CFX96 system with SYBR-Green to determine the expression level of mRNA of interest (Bio-Rad Laboratories, Inc.). The primers used were as follows: GAPDH forward, 5′-ATGGAAATCCCATCACCATCTT-3′ and reverse, 5′-CGCCCCACTTGATTTTGG-3′; YAP forward, 5′-TAGCCCTGCGTAGCCAGTTA-3′ and reverse, 5′-TCATGCTTAGTCCACTGTCTGT-3′. The comparative Cq method was used to calculate the relative mRNA expression levels. The expression level of GAPDH was used as a loading control.

After washing twice with cold PBS, the cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology) containing 1X protease inhibitor cocktail (Roche Diagnostics). Proteins were separated and electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, transferred to nitrocellulose membranes by a wet transfer apparatus, and then blocked with TBST buffer (25 mmol/l Tri-HCl, pH 7.5, 137 mmol/l NaCl, 2.7 mmol/l KCl and 0.05% Tween-20) with 5% bovine serum albumin for 1 h at 37°C. The blots were probed with the indicated antibodies in blocking buffer overnight at 4°C. GAPDH was used as the internal control. After the membranes were washed with TBST 3 times for 20 min each time, HRP-linked secondary antibody (Antgene) was applied to the membrane for 60 min. The protein was finally detected using enhanced chemiluminescence (ECL; Bio-Rad Laboratories, Inc.). Band density was measured by ImageJ software (National Institutes of Health) and normalized to GAPDH.

Six-week-old female BALB/c-nu mice (n=10 weight: 17–19 g) were obtained from the Shanghai Animal Experimental Center. All mice were housed under pathogen-free conditions in the Animal Research Center of Tongji Hospital at 22°C with 12-h light/dark cycles and free access to water and food. SK-OV3-ip3-luc (1×10⁶) cells were injected orthotopically under the ovarian bursa. One week after surgery, the mice were randomized into the control (saline) and treatment (peptide 17) experimental groups (n=5 per group). Peptide 17 was administered i.p. (0.2 mg/kg) daily and saline was used as control. The mice were anesthetized with pentobarbital sodium (75 mg/kg) after 28 days of treatment, and imaged with the IVIS SPECTRUM system (Caliper; Xenogen) 10 min after i.p. injection of 100 mg/kg D-luciferin substrate. Living Image software version 4.3.1 (Caliper; Xenogen) was used for the analysis. The tumor-bearing mice were sacrificed by cervical dislocation, and the tumors were removed for assessment.

Cells transfected with siControl or siYAP were seeded in 6-well plates and incubated at 37°C for 10 days. For the peptide 17 experiment, cells were plated in 6-well plates and treated with peptide 17 (250 nM) for 10 days; the medium was replaced every 48 h to ensure constant levels of the drug. The cells were stained with 0.1% (w/v) crystal violet solution.

The Kaplan-Meier plotter tool (http://kmplot.com/analysis/) was used to generate survival curves combining YAP1 (Affymetrix probe 224895_at) mRNA data from all public ovarian cancer datasets (14).

All data analyses in the present study were performed using GraphPad Prism 6 (GraphPad Software, Inc.). Data are expressed as the means ± standard deviation from at least 3 independent experiments. Two experimental groups were compared using a paired Student's t-test for paired data or a Student's t-test for unpaired data. Analysis of variance was used to compare differences among multiple groups (post hoc test: Turkey's multiple comparisons test). Kruskal-Wallis test (post hoc test: Dunn's multiple comparisons test) was used to compare differences in YAP expression among normal ovaries, normal fallopian tubes, primary tumors and metastases. P-values <0.05 were considered to indicate statistically significant differences.

To explore the role of YAP in OC, YAP expression was first determined in normal ovaries, normal fallopian tubes and paired primary tumors and metastases of epithelial ovarian cancer (EOC) by IHC. The information on different tissues is provided in the supplementary Table SII. Compared with normal ovaries and normal fallopian tubes, YAP expression was significantly higher in OC primary tumors and metastases (Fig. 1A and B) (primary vs. normal ovary, P=0.0051; primary vs. normal fallopian tube, P=0.0371; metastases vs. normal ovary: P<0.0001; metastases vs. normal fallopian tube, P<0.0001). Notably, higher nuclear YAP levels were observed in metastases compared with primary tumors (Fig. 1C). Furthermore, Kaplan-Meier plot analysis of YAP expression in OC was performed to determine whether YAP is correlated with prognosis in OC patients. The result indicated an association of high YAP expression with poor progression-free survival (PFS) in OC patients (Fig. 1D). Thus, these results confirmed that YAP is upregulated in OC tissues and is correlated with patient survival, suggesting that YAP may affect OC progression.

To further explore the function of YAP in OC, two siRNAs targeting YAP and siControl were employed to transfect SK-OV-3 and ES-2 cells. qPCR and western blot analyses demonstrated that transfection of siYAP-1 and siYAP-2 efficiently inhibited YAP expression at both the mRNA and protein levels, particularly siYAP-1 (Fig. 2A and B). Transwell assays indicated that the migratory ability of both SK-OV-3 and ES-2 cells transfected with siYAP was markedly inhibited compared with cells transfected with siControl (Fig. 2C). Consistently, wound healing assays also confirmed that silencing of YAP diminished the migration of OC cells (Fig. 2D and E). We also demonstrated that YAP silencing inhibited the invasion of OC cells, as assessed by Transwell assays (Fig. 2F). Cell viability assay demonstrated that silencing of YAP inhibited the proliferation of OC cells (Fig. 2G). Similarly, colony formation assays confirmed that the colony-forming ability of OC cells was reduced upon transfection with siRNA of YAP (Fig. 2H). The EdU assay also supported these conclusions (Fig. 2I). Thus, the findings demonstrated that silencing of YAP diminished the migration, invasion and proliferation of OC cells, indicating that YAP may be a potential target for the treatment of OC.

To further explore the mechanism through which silencing of YAP inhibits the malignant behavior of OC cells, we evaluated the activation of the PI3K/Akt/mTOR pathway, a key signaling pathway involved in tumor progression. The phosphorylation status of PI3K, Akt and mTOR, the key signaling proteins of the PI3K/Akt/mTOR pathway, was assessed by western blotting. Compared with siControl-transfected cells, the phosphorylation of PI3K was significantly lower in siYAP-transfected SK-OV-3 and ES-2 cells (Fig. 3A and B). Similarly, downregulation of YAP expression decreased the phosphorylation of Akt and mTOR in both types of OC cells (Fig. 3A, C and D). Moreover, the activation of GSK-3β was assessed in both cell lines, since glycogen synthase kinase (GSK)-3β is another downstream regulator of the PI3K/Akt pathway. However, no significant changes in the phosphorylation status of GSK-3β were observed between siControl-transfected and siYAP-transfected cells (Fig. 3A and E). These results indicate that silencing of YAP may inhibit the activation of the PI3K/Akt/mTOR pathway, suggesting that this be the mechanism underlying YAP-regulated OC progression.

As YAP silencing significantly inhibited the malignant behavior of OC cells, we next investigated whether peptide 17, a YAP inhibitor, exerted the same effect. Wound healing assays indicated that peptide 17 markedly impaired the migration of SK-OV-3 and ES-2 cells compared with the control group (Fig. 4A and B). Peptide 17 also inhibited the invasive ability of both SK-OV-3 and ES-2 cells, as evidenced by Transwell assays (Fig. 4C). The colony formation assay demonstrated that peptide 17 significantly inhibited colony formation by both SK-OV-3 and ES-2 cells (Fig. 4D). Similar results were also obtained by the EdU assay (Fig. 4E). These results demonstrated that peptide 17 inhibited the malignant behavior of OC cells. Considering that silencing YAP inhibited activation of the PI3K/Akt/mTOR pathway, we further investigated whether peptide 17 exerted a similar effect. Western blot analysis demonstrated that phosphorylation of PI3K, Akt and mTOR was lower in cells treated with peptide 17 compared with the control group (Fig. 4F). These results demonstrated that peptide 17 markedly attenuated the malignant behavior of OC cells and modulated the PI3K/Akt/mTOR pathway, suggesting that peptide 17 may be a potential therapeutic strategy for OC.

Next, to verify whether inhibition of YAP with peptide 17 restrains OC progression in vivo, an orthotopic ovarian cancer model utilizing SK-OV-3-ip3-luc cells was employed. Consistent with the in vitro results, mice treated with peptide 17 developed significantly smaller tumors compared with mice treated with saline (Fig. 5A). To better understand these results, the tumors were removed at the end of the experiment, and further experiments were conducted. Subsequently, tumors from peptide 17-treated mice exhibited increased Ki-67 staining, as evidenced by IHC, compared with those in the saline group (Fig. 5B), indicating that peptide 17 inhibited the growth of OC in vivo. Since it was determined that peptide 17 could regulate the activation of the PI3K/Akt/mTOR signaling pathway in OC cells, phospho-Akt (Ser473) and phospho-mTOR (S2448) expression were detected in the obtained tumor sections. Mechanistically, IHC analysis revealed significantly diminished staining of phospho-Akt (Ser473) (Fig. 5C) and phospho-mTOR (S2448) in peptide 17-treated mice, compared with the saline group (Fig. 5D). As shown by western blot analysis, the expression of phospho-PI3K (Tyr458), phospho-Akt (Ser473) and phospho-mTOR (S2448) was found to be markedly impaired in tumors from peptide 17-treated mice compared with those from saline-treated mice (Fig. 5E-F). Thus, these data demonstrated that peptide 17 significantly attenuated OC progression through inhibition of the PI3K/Akt/mTOR signaling pathway.