YPEL3 suppresses epithelial–mesenchymal transition and metastasis of nasopharyngeal carcinoma cells through the Wnt/β-catenin signaling pathway

Human NPC cell lines (CNE-1, CNE-2, SUNE-1, HNE-1, HONE-1, 5-8 F, 6-10B) were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10 % (v/v) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The human immortalized nasopharyngeal epithelial NP69 cell line was grown in keratinocyte serum–free medium (Invitrogen, Carlsbad, CA, USA) supplemented with bovine pituitary extract (BD Biosciences, San Jose, CA, USA). We maintained 293FT cells in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10 % FBS. In addition, 22 freshly frozen NPC biopsy samples and 12 normal nasopharyngeal epithelium samples were collected from Sun Yat-sen University Cancer Center. This study was approved by the Institutional Ethical Review Boards of our center, and written informed consent was obtained from each patient.

Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and quantified at 260 nm by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Total RNA (2 μg) was reverse-transcribed to complementary DNA (cDNA) using an RT kit (Promega, Madison, WI, USA). Quantitative PCR was performed in triplicate using Platinum SYBR Green qPCR Super Mix-UDG reagents (Invitrogen, Carlsbad, CA, USA) on a CFX96 Touch™ sequence detection system (Bio-Rad, Hercules, CA, USA). The following primer sequences were used for amplification: YPEL3 forward, 5′-CCACGACGACCTCATCTC-3′; reverse, 5′-CATATTTCCAGCCCAAAGT-3′; E-cadherin forward, 5′-GAAGAGGACCAGG ACTTTGAC-3′; reverse, 5′- GTAGTCATAGTCCTGGTCTTTGTC-3′; Vimentin forward, 5′- TCAGACAGGATGTTGACAATGC-3′; reverse, 5′- TCATATTGCTGACGTACGTCAC-3′. GAPDH was used as the endogenous control, and the comparative threshold cycle (2^-ΔΔCT) equation was used to calculate the relative expression levels.

Cultured cells were washed twice with ice-cold phosphate-buffered saline (PBS), solubilized in a lysis buffer containing 1 mmol/L protease inhibitor cocktail (FDbio Science, Hangzhou, China) on ice, and quantified using the bicinchoninic acid method. Cell lysate protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5 % skim milk in Tris-buffered saline–Tween (TBST) buffer (10 mmol/L Tris–HCl [pH 7.4], 150 mmol/L NaCl, 0.1 % Tween 20) for 2 h. Protein expression was detected following overnight incubation at 4 °C using primary antibodies against HA (1:2000, Sigma-Aldrich, USA); YPEL3 (1:100, Abcam, Cambridge, MA, USA), β-catenin (1:500, Proteintech, Wuhan, China), c-MYC (1:2000, Proteintech, Wuhan, China), cyclin D1 (1:500, Proteintech, Wuhan, China), α-catenin (1:500, BD Biosciences, San Jose, CA, USA), E-cadherin (1:500, BD Biosciences, San Jose, CA, USA), vimentin (1:500, BD Biosciences, San Jose, CA, USA), GSK3β(1:1000, Proteintech, Wuhan, China), TBP (1:800, Proteintech, Wuhan, China), and GAPDH (1:500, Proteintech, Wuhan, China). Thereafter, the membranes were washed and incubated for 1 h at room temperature with the appropriate horseradish peroxidase–conjugated secondary antibody. After the membranes were washed with TBST buffer three times, the proteins were visualized with an enhanced chemiluminescence reagent (Beyotime, Shanghai, China). The bands were analyzed using Image J software.

CNE-2 and SUNE-1 cells were seeded onto a 6-well culture plate and cultured to a subconfluent state in complete medium. After 24-h starvation in serum-free medium, cell monolayers were linearly scraped with a P-200 pipette tip. Cells that had detached from the bottom of the wells were gently aspirated and incubated in serum-free medium for 24 h. The width of the scratch was monitored under a microscope and quantified in terms of the difference between the original width of the wound and the width after cell migration.

Transwell migration and invasion assays were carried out using Transwell chambers (Corning, Tewksbury, MA, USA) with 8-μm pore polyethylene membranes. For the migration assay, cells were placed in the upper chamber of each insert that had not been coated with Matrigel (BD Biosciences, San Jose, CA, USA). For the invasion assay, cells were placed in the upper chamber of inserts that had been pre-coated with Matrigel. Plasmid- or siRNA-transfected CNE-2 and SUNE-1 cells (5 × 10⁴ for the migration assay;1 × 10⁵ for the invasion assay) were added to the upper chamber in serum-free medium, and the lower chamber contained culture medium with 20 % FBS to act as a chemoattractant. The cells were incubated for 12 h or 24 h at 37 °C in 5 % CO₂, and then they were fixed and stained. Cells on the undersides of the filters were observed and counted under × 200 magnification.

CNE-2 and SUNE-1 cells were cultured on coverslips in 6-well plates, washed with PBS, fixed with 4 % paraformaldehyde for 30 min, and permeabilized with 0.5 % Triton X-100 for 10 min. After blocking with 1 % bovine serum albumin for 1 h, the cells were immunostained with antibody against E-cadherin or vimentin at a 1:500 dilution. The cells were washed with PBS and incubated with Alexa Fluor–conjugated goat anti-mouse secondary antibody (1:200, A11011; Invitrogen). After washing with PBS, the nuclei were stained with DAPI (Invitrogen,) for 15 min, and fluorescence images were obtained using a confocal scanning microscope (OLYMPUS FV1000; Olympus, Tokyo, Japan) and analyzed using Image-Pro Plus 6.0 software.

Female, pathogen-free, athymic nude mice were purchased from Charles River Laboratories (Beijing, China) (n = 9mice per group). SUNE-1 cells (1 × 10⁶) stably overexpressing vector or YPEL3 that had been resuspended in 200 μL serum-free medium were injected intravenously into the tail veins of the mice. After 8 weeks, the mice were sacrificed and the lung tissues were fixed for calculating the numbers of macrometastatic nodes formed on the surface of lungs. Then, the tissues were paraffin-embedded, serial 5-μm tissue sections were cut, and one of every ten sections was stained with hematoxylin–eosin (HE) for examination of micrometastatic nodes formed in the lungs as previously described [18, 19]. All animal research was conducted in accordance with the detailed rules approved by the Animal Care and Use Ethnic Committee of Sun Yat-sen University Cancer Center and all efforts were made to minimize animal suffering.

All statistical analyses were carried out with SPSS software (standard version 17.0, Chicago, IL, USA). All experiments were performed in three independent experiments and all data are presented as the mean ± SD. Significant differences between two groups were analyzed using two-tailed unpaired Student’s t-test and a P-value <0.05 was considered statistically significant.

The mRNA and protein expression levels of YPEL3 in the normal nasopharyngeal epithelial NP69 cell line and seven NPC cell lines were assessed by quantitative RT-PCR and western blotting. Compared to NP69 cells, YPEL3 was significantly downregulated in the NPC cell lines at both mRNA and protein level (Fig. 1a, b). We also investigated the YPEL3 mRNA levels in 12 frozen NPC tissues and 12 normal nasopharyngeal epithelial tissues, and observed that YPEL3 mRNA was expressed at considerably lower levels in the NPC tissues (Fig. 1c). Western blotting validated the decreased YPEL3 protein levels in the NPC tissues (Fig. 1d). These results indicate that decreased YPEL3 expression may promote NPC development and progression.

To study whether YPEL3 affects the migratory and invasive abilities of NPC cells, we performed wound healing, Transwell migration, and invasion assays using CNE-2 and SUNE-1 cells stably overexpressing YPEL3 or vector. As shown in Fig. 2a, Western blotting validated that YPEL3 protein level was obviously elevated after stably overexpressing YPEL3 in NPC cells. The wound healing assay demonstrated that the migratory ability of CNE-2 and SUNE-1 cells stably overexpressing YPEL3 was much lower than that of cells expressing vector plasmid (Fig. 2b). Overexpressing YPEL3 suppressed the migratory (Fig. 2c) and invasive (Fig. 2d) abilities of the NPC cells as determined by the Transwell migration and invasion assays. These findings indicate that YPEL3 inhibits the migratory and invasive abilities of NPC cells in vitro.

To further investigate whether silencing of YPEL3 affects the migratory and invasive abilities of NPC cells, we transiently transfected CNE-2 and SUNE-1 cells with siYPEL3 or control siRNA, and performed wound healing, Transwell migration, and invasion assays. As shown in Fig. 3a, Western blotting confirmed that the YPEL3 protein level was remarkably decreased after silencing of YPEL3 in NPC cells. The wound healing and Transwell migration assays showed that cells transfected with siYPEL3 migrated more slowly than cells transfected with control siRNA (Fig. 3b, c). Knocking down YPEL3 promoted NPC cell invasive capability as determined by the Transwell invasion assay (Fig. 3d). These findings indicate that silencing YPEL3 promotes the migratory and invasive abilities of NPC cells in vitro.

As EMT is important for the acquisition of metastatic potential in tumors, we determined the expression levels of the epithelial markers E-cadherin and α-catenin, and that of the mesenchymal marker vimentin using immunofluorescence staining and western blotting. Immunofluorescence staining revealed that YPEL3 overexpression significantly rescued E-cadherin expression but inhibited vimentin expression (Fig. 4a); western blotting revealed similar changes in epithelial and mesenchymal marker expression in the CNE-2 and SUNE-1 cells overexpressing of YPEL3 (Fig. 4b). Conversely, silencing YPEL3 suppressed E-cadherin and α-catenin expression but increased vimentin expression (Fig. 4c). In addition, Spearman’s correlation analysis showed thatYPEL3 mRNA expression was positively correlated with E-cadherin expression, and inversely correlated with vimentin expression in 10 NPC tissue samples (r = 0.803 and r = -0.754, respectively; both p < 0.001, Fig. 4d). These observations demonstrate that YPEL3 suppresses NPC cell EMT.

To determine the role of YPEL3 in NPC cell metastasis in vivo, we constructed a lung metastasis model by injecting SUNE-1 cells stably overexpressing YPEL3 or vector into the tail veins of nude mice. After 8 weeks, fewer macroscopic metastatic nodes were seen on the lung surfaces of the YPEL3-overexpressing group as compared with the vector group (Fig. 5a, b). In addition, there were smaller and fewer microscopic metastatic nodules in the YPEL3-overexpressing group than in the vector group (Fig. 5c, d). These results indicate that YPEL3 suppresses NPC cell metastasis, suggesting YPEL3 functions as a negative regulator of metastasis.

As Wnt/β-catenin plays a critical in EMT induction and maintenance, we examined the protein expression of the Wnt/β-catenin signaling genes for GSK3β, β-catenin, c-MYC, and cyclin D1 in CNE-2 and SUNE-1 cells overexpressing YPEL3 or in which YPEL3 had been silenced to explore the YPEL3 mechanism underlying NPC metastasis. Overexpressing YPEL3 elevated the protein expression of GSK3β and suppressed the protein expression of β-catenin, c-MYC, and cyclin D1(Fig. 6a). Conversely, silencing YPEL3 inhibited the protein expression of GSK3β and increased the protein expression of β-catenin, c-MYC, and cyclin D1 the genes (Fig. 6b). Moreover, subcellular protein fraction study confirmed that YPEL3 overexpression decreased nuclear β-catenin expression and increased cytoplasmic β-catenin expression (Fig. 6c), which indicates that the suppressive effect of YPEL3 may take place through the inhibition of β-catenin nuclear translocation. These data demonstrate that YPEL3 plays a critical role in mediating the Wnt/β-catenin signaling pathway.