Human ovarian carcinoma cell lines ES-2, SKOV3, 293T, HEY, COV318 and OVCAR3 were provided by the Shanghai Cancer Institute (Shanghai, China). ES-2 and SKOV3 cells were used in assessing the function of YWHAZ on cellular behaviors and the activation of glycolysis. Other cell lines were only used for the detection of YWHAZ expression levels. All cells were cultured according to the instructions of the American Type Culture Collection (ATCC; Manassas, VA, USA). Antibodies used in this study included YWHAZ (cat. no. ab51129; Abcam, Cambridge, MA, USA), AKT (cat. no. ab18785; Abcam), p-AKT1 (cat. no. ab133458; Abcam), PI3K-p85α (cat. no. 13666), vimentin (cat. no. 5741), LDHA (cat. no. 2012), PFKP (cat. no. 5412), tubulin (cat. no. 2146; all were from Cell Signaling Technology, Danvers, MA, USA).

RNA isolation and real-time qPCR were performed as previously described (6). Total RNA of ES-2 and SKOV3 cells was extracted using TRIzol reagent (Takara Bio Inc., Shiga, Japan). cDNA was synthesized by PrimeScript RT-PCR kit (Takara Bio Inc.) based on the manufacturer's protocols. Quantitative real-time PCR was run with SYBR Premix Ex Taq (Takara Bio Inc.) on the 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cDNA was diluted at 1:20 before use. Primers used for YWHAZ were: 5′-AGGAGCCCGTAGGTCATCTT-3′ (forward) and 5′-TGCTTGTGAAGCATTGGGGA-3′ (reverse). Primers used for 18S were: 5′-TGCGAGTACTCAACACCAACA-3′ (forward) and 5′-GCATATCTTCGGCCCACA-3′ (reverse). The cycling settings were as follows: 95°C for 10 min; 95°C for 10 sec, 60°C for 30 sec for 45 cycles; 40°C for 30 sec. Expression level and fold change were calculated using the ΔΔCq method (20).

Two stable plasmids for opposite functions were constructed. For stable YWHAZ interference, short hairpin RNA (shRNA) targeting YWHAZ or control shRNA was constructed into the PLKO.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.). The sequences of the shRNAs were designed based on the sequence of siRNA-1 of YWHAZ (si1). For stable overexpression of YWHAZ, the cDNA encoding YWHAZ was subcloned into the pCDH-CMV-MCS-EF1-Puro (CD510B-1) vector to generate the YWHAZ-overexpressing plasmid. Then C510B-1-YWHAZ-Linker-HA plasmids which had been constructed were packaged as required. Virus packaging was performed in 293T cells after cotransfection of constructed plasmids with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 11668-019). Viruses were harvested at 48 and 72 h after transfection, and virus titers were determined. ES-2 and SKOV3 cells, two target cell lines, were infected with 1×10⁸ recombinant lentivirus-transducing units plus 8 µg/ml polybrene (Sigma-Aldrich, Shanghai, China). Cells were selected and maintained in the presence of 2 µg/ml puromycin. The transfection efficiency was assessed by RT-PCR and western blotting at both the mRNA and protein levels. Subsequent experiments were performed after 2 weeks of puromycin selection.

Lipofectamine RNAiMAX transfection reagent (Invitrogen Thermo Fisher Scientific, Inc.) was used for transfecting the designed small interfering RNAs (siRNAs) into ES-2 and SKOV3 cells. The transfection steps were carried out following the manufacturer's instructions. Diluted siRNA was added into diluted Lipofectamine RNAiMAX reagent for a 5-min incubation at room temperature. Then, the siRNA-lipid complex was added to the targeted cells. The final siRNA applied per well was 25 pmol in one 6-well plate. The sequences were: GGAGAUUACUACCGUUACUdTdT (hs-YWHAZ-si-1), GAGCUGAAUUAUCCAAUGAdTdT (hs-YWHAZ-si-2) and CGUCUCAAGUAUUGAACAAdTdT (hs-YWHAZ-si-3). Non-targeting scrambled siRNA was used as a control in the transfection. The silencing effects were verified by qRT-PCR and western blot analysis.

Cell proliferation assay was performed as previously described (21). ES-2 and SKOV3 cells were seeded in 96-well plates at the concentration of 5,000 cells in 100 µl complete medium/well before transfection. After the cells were attached, transient transfection was performed according to the protocols. At 24, 48 and 72 h after siRNA transfection, 10 µl of Cell Counting Kit-8 (CCK-8) reagents (Share-bio, Shanghai, China) was added to each well for 1 h of incubation. The absorbance was measured at 450 nm using a microplate reader. All experiments were performed in triplicate.

ES-2 and SKOV3 cell migration was determined using 8-mm pore size cell culture inserts within a 24-well plate (Corning Inc., Corning, NY, USA) based on the protocols. Cells (2×10⁴) in 100 µl serum-free medium were added into the upper chamber with 700 µl serum medium in the lower chamber and incubated for 24 h. The migrated cells were immobilized with 4% paraformaldehyde and stained with 0.1% crystal violet. The quantity of migrated cells was counted under a light microscope in six random fields. Each experiment was performed in triplicate.

ES-2 and SKOV3 cells were seeded on 6-well plates. Cell death was assessed by double-labelling with Annexin V-FITC and propidium iodide (PI) according to the protocols. After induction of apoptosis by serum-free media, the required cells were harvested and washed in cold phosphate-buffered saline (PBS) followed by resuspending the cells in Annexin-binding buffer. After addition of 5 µl of Annexin V to each 100 µl of cell suspension, the cells were incubated for 15 min at room temperature. Then propidium iodide (PI) was added 5 min before detection on a flow cytometer (Becton-Dickinson; BD Biosciences, San Jose, CA, USA).

Total protein was extracted from ES-2 and SKOV3 cells using a total protein extraction buffer (Beyotime Institute of Biotechnology, Haimen, China). BCA Protein Assay kit was used to measure the protein concentration. Total protein (50 µg) was added into each sample well. Then the same amounts of proteins were separated by 12% SDS-PAGE and transferred to NC membranes. The membranes were blocked with 5% non-fat dry milk for 1 h and washed twice with TBS wash buffer. Then the membranes were incubated at 4°C overnight with the following primary antibodies: YWHAZ (dilution 1:1,000), p-Akt1 (dilution 1:1,000), tubulin (dilution 1:2,000), PI3K (dilution 1:2,000), vimentin (dilution 1:1,000), LDHA (dilution 1:2,000) and PFKP (dilution 1:1,000). After washing with TBS wash buffer, HRP-conjugated anti-rabbit (cat. no. ab205718; Abcam) and anti-mouse (cat. no. ab205719; Abcam) secondary antibodies were diluted (dilution 1:10,000) and added for incubation for 1 h at room temperature. The protein bands were detected by an ECL kit (Share-bio).

For H&E staining, the tissue samples were stained with hematoxylin and eosin. The staining procedures were performed routinely. Firstly, the sections which were pretreated were rinsed in to distilled water. Then the nuclei were stained with hematoxylin for 10 min. After washing in running tap water for 5 min, the sections were differentiated with 0.3% acid alcohol for 1 min. For bluing, the sections were inserted in 0.2% ammonia water later following by a 5-min wash with water. Then the sections were counterstained in eosin for 1 min. Finally, dehydration, clearing and mounting of the sections were performed.

Mice were supplied by and cultivated in the SPF Animal Laboratory of East China Normal University according to the protocols approved by the Animal Care Commission and received humane care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Sciences and published by the National Institutes of Health. A total of 20 female BALB/C nude mice (6-weeks old) were divided into four groups for the metastasis experiments. They lived in standard housing conditions with 10 h of light and 14 h of dark cycle. The indoor temperature was maintained at 26°C and the relative humidity was maintained at 40–60%. The particles in the air did not exceed >0.3 µm. The food and water were sterilized by special treatment. We treated ES2 and SKOV3 cell lines with stable transfection in advance. After a 2-week puromycin selection, 2×10⁶ SKOV3 or ES2 cells expressing Lenti-shcontrol or Lenti-shYWHAZ were injected into the tail vein of nude mice (n=5 each group). The mice were sacrificed by neck dislocation 4 weeks later according to common protocols of the metastasis assay used in numerous literatures and the number of metastatic nodules in the lung was counted under an inverted microscope (Olympus Corporation, Tokyo, Japan). The harvested lungs were fixed and stained by H&E for histologic assessment.

Lactate production was detected as previously described using the Lactate Assay kit (cat. no. ABIN411683; BioVision Inc., Mountain View, CA, USA) (6). Lactate production was measured at the absorbance (570 nm) with a microplate reader.

The extracellular acidification rate (ECAR) of control or YWHAZ-knockdown ovarian cancer cells was assayed on the Seahorse Extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA) to evaluate glycolysis according to the manufacturer's instructions (6). SKOV3 and ES2 cells were seeded on a XF96-well plate at a density of 4×10⁴/well and allowed to attach overnight, followed by transfection with the negative-control siRNA or siRNAs targeting YWHAZ. After 24 h, cells were incubated in a non-CO₂ 37°C incubator with unbuffered medium for 1 h followed by a sequential injection of 10 mM glucose, 1 µM oligomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 50 mM 2-deoxyglucose (2-DG; Sigma-Aldrich; Merck KGaA; D8375). Measurements were normalized to total protein content and reported as mpH/min for ECAR. Each datum was determined in triplicate.

For immunohistochemical staining of YWHAZ and analysis of its correlation with clinicopathological features, an ovarian cancer tissue microarray containing 83 cases of ovarian cancer samples and 18 cases of normal ovarian samples was used. These tissues were obtained from the Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University from May 2006 to January 2009. All human ovarian tissues were obtained with informed consent and all protocols were approved by the Ethics Review Committee of the Research Ethics Committee of the Fifth People's Hospital of Shanghai, Fudan University Hospital. Expression score was determined by staining intensity and immunoreactive cell percentage. Scoring was conducted according to the ratio and intensity of positive staining cells: 0–5% scored 0; 6–35% scored 1; 36–70% scored 2; and >70% scored 3. The final score of YWHAZ expression was designated as low or high expression group as follows: Low expression, score 0–1; and high expression, score 2–3. All the scores of YWHAZ expression were performed in a blinded manner and determined independently by two senior pathologists.

All data were analyzed as the mean ± standard deviation (SD). Statistical analyses were conducted using SPSS 14.0 software (SPSS Inc., Chicago, IL, USA). We performed the Pearson's χ² test in cross tables to assess the relationships between the expression levels of YWHAZ and the clinicopathological factors. Overall survival (OS) was calculated using the Kaplan-Meier method. Comparison between groups was analyzed by one-way analysis of variance (ANOVA) followed by a post hoc test (Student-Newman-Keuls method). Values of P<0.05 were considered to indicate a statistically significant result.

To investigate the relationship between YWHAZ and the biological characteristics of ovarian cancer, we analyzed YWHAZ expression using the cBioPortal database (www.cbiopotal.org). The number of copies of the YWHAZ gene was increased in most tumors, such as breast, prostate and ovarian cancer (Fig. 1A). Using the TCGA database (http://cancergenome.nih.gov/), we analyzed YWHAZ copy number and mRNA levels in ovarian cancers compared with normal ovarian tissue. We found that YWHAZ mRNA expression and copy number in cancer tissues was similarly increased (Fig. 1B and C). Then, we performed an immunohistochemical (IHC) analysis of a tissue microarray that contained 83 epithelial ovarian cancer (EOC) tissue samples and 20 normal ovarian tissues. As shown in Fig. 1D, strong YWHAZ staining was observed in most EOC samples (61.4 and 51/83), whereas staining was minimal in normal ovarian tissues (16.7 and 3/18). Using Kaplan-Meier analysis, patients with high YWHAZ expression were found to be significantly associated with reduced overall survival (Fig. 1E).

Then, we analyzed the relevance of YWHAZ expression based on patient clinicopathological parameters. We found that patients with high YWHAZ expression exhibited higher tumor stage and worse prognosis (Table I).

To select suitable cell lines for further research, we assessed YWHAZ expression in five ovarian cell lines (HEY, CAOV3, ES2, COV318 and SKOV3). The results showed that ES2 and SKOV3 cells that exhibit high metastatic potential expressed the highest levels of YWHAZ (Fig. 2A). Therefore, ES2 and SKOV3 cells were transfected with siRNAs targeting YWHAZ or a scrambled siRNA (labeled as control). RT-PCR and western blotting were used to validate the silencing effects of the siRNAs in the cell lines (Fig. 2B and C). The results verified that YWHAZ expression levels were significantly decreased by the three siRNAs against YWHAZ.

Next, we investigated the effect of YWHAZ on cell viability. After transfection with siRNAs, the Cell Counting Kit-8 (CCK-8) assay was used to detect ES2 and SKOV3 proliferation. The results showed that ES2 and SKOV3 cell proliferation was suppressed by silencing of YWHAZ (Fig. 3A and B).

To explore whether YWHAZ affects ovarian cancer cell apoptosis, ES2 and SKOV3 cells transfected with siRNAs against YWHAZ were used to detect cell apoptosis by flow cytometry. The results showed that YWHAZ silencing promoted ES2 and SKOV3 cell apoptosis (Fig. 3C and D).

To further study the functional role of YWHAZ in ovarian cancer, we investigated the effects of YWHAZ on ovarian cancer cell migration in vitro. Silencing of YWHAZ expression significantly inhibited ES2 and SKOV3 cell migration in vitro as assessed by Transwell migration assay (Fig. 4A and B), whereas YWHAZ overexpression promoted ES2 and SKOV3 cell migration in vitro (Fig. 4C and D) compared with the control group. Then, we constructed stable ES-2 and SKOV3 cell lines expressing shYWHAZ. The knockdown efficiency was determined by qRT-PCR and western blotting (Fig. 4E and F). Then, we injected these cells into the tail vein of BALB/C nude mice. Thirty-five days after injection, we examined tumor metastases in the mice by histological H&E staining analyses. Compared with the control group, the number of metastatic lung nodules was significantly reduced in the interference group (Fig. 4G and H). Consistent with our findings in vitro, genetic silencing of YWHAZ significantly reduced the tumor burden of EOC in the xenograft metastasis model in nude mice (Fig. 4G and H).

Considering that YWHAZ modulates cellular glycolysis in other cancers, we next investigated whether YWHAZ is involved in ovarian cancer glycolysis. First, we measured glycolysis-related enzymes upon YWHAZ silencing in ovarian cancer cells. In YWHAZ-silenced ES-2 and SKOV3 cells, the mRNA and protein expression levels of glycolysis-related enzymes, including lactate dehydrogenase A (LDHA), human platelet phosphofructokinase (PFKP) and glyceraldehyde-3-dehydrogenase (PGD), were significantly reduced (Fig. 5A and B). Then, we examined lactate production, extracellular acidification rates (ECAR) and NADH production to probe glycolysis. YWHAZ silencing significantly reduced lactate, ECAR and NADH production in ES-2 and SKOV3 cells (Fig. 5C and D). These data suggest that YWHAZ can promote ovarian cancer cell glycolysis by regulating the expression of related metabolic enzymes. YWHAZ binds numerous effectors with a phosphoserine or phosphothreonine motif and participates in cancer development and progression by regulating a large spectrum of both general and specialized signaling pathways. Importantly, YWHAZ interacts with Akt1 (22), a crucial factor involved in the regulation of Warburg effect by targeting glycolytic enzymes (6). This information prompted us to explore the possibility that YWHAZ may stimulate the Warburg effect through its regulation of Akt1 in EOC cells. As shown in Fig. 5E, levels of phosphorylated Akt1 were reduced after genetic inhibition of YWHAZ. In contrast, YWHAZ overexpression upregulated Akt1 phosphorylation (Fig. 5F). Notably, YWHAZ also regulated the phosphorylation of PI3K (Fig. 5F), suggesting that YWHAZ also affected Akt1 through PI3K signaling. Furthermore, the expression level of vimentin, a downstream substrate of Akt1, was downregulated by YWHAZ silencing and upregulated by YWHAZ overexpression. These data suggest that YWHAZ promotes the Warburg effect by modulating PI3K/Akt1 signaling.

Collectively, these results demonstrated that YWHAZ promotes ovarian cancer growth and metastasis, which is probably mediated by regulating PI3K/Akt1/vimentin signaling and glycolysis.