The human laryngeal cancer Hep2 cell line (Hep2 cells) was obtained from Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences. Hep2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) (both from Gibco, Bedford, MA, USA) and 1% penicillin/streptomycin at 37°C under 5% CO₂.

Cell proliferation was measured with the CCK-8 assay kit (Gibco). Briefly, a total density of 3×10³ Hep2 cells/well, growing in logarithmic phase, in 100 µl of DMEM were plated into the 96-well plates. Then, 10 µl of CCK-8 was added into every well at the specific time points. After culture for 2 h at 37°C under 5% CO₂, OD₄₅₀ absorbance values were monitored. In the present study, the proliferation of T-Hep2 cells following treatment with the selective inhibitor of AURKA (VX680) (Selleck Chemicals, Houston, TX, USA), which was dissolved in dimethyl sulfoxide (DMSO), was measured at 0, 24 and 48 h for 2 days.

Hep2 cells treated with VX680 for 48 h were the experimental group and the cells treated with DMSO or without any treatment were considered the controls. Cells at a density of 2×10³ were layered into 6-well plates. After culture for 2 weeks with DMEM containing 10% FBS, the cells were washed with phosphate-buffered saline (PBS) 3 times and fixed with crystal violet for 30 min at room temperature. The formed colonies were counted in every well.

Hep2 cells treated with VX680 for 48 h or without any treatment, at the density of 5×10⁴, were seeded into Millicell^® EZ Slides (Millipore, Bedford, MA, USA). The cells were fixed with 4% paraformaldehyde (PFA) for 30 min when they adhered to the wall of the slides. Then, the slides were rinsed 3 times with 1X PBS for 5 min each time, followed by blocking with primary cocktail (Cell Signaling Technology, Danvers, MA, USA) (diluted 1:100 as indicated on the datasheet in antibody dilution buffer) to examine the expression of EMT markers, vimentin and E-cadherin. Following overnight incubation at 4°C, the blocking solution was aspirated and the slides were washed 3 times with 1X PBS for 5 min each time and were then incubated with Alexa Fluor^® 555 and Alexa Flour^® 488 (Cell Signaling Technology) (diluted 1:100 as indicated on the datasheet in antibody dilution buffer) for 1 h at room temperature in the dark. Then, the slides were rinsed 3 times with 1X PBS for 5 min each. For immunofluorescence staining of p-AURKA (anti-p-AURKA; 1:100; Cell Signaling Technology), blocking with 5% BSA in PBS containing 0.05% Triton for 1 h at room temperature was the only difference in the protocol. Vimentin and E-cadherin were analyzed using fluorescence microscopy magnification, ×10 (U-ULS100HG; Olympus Optical Co., Ltd., Tokyo, Japan) (magnification, ×10).

Hep2 cells, treated with VX680, the inhibitor of FAK (TAE226), and the inhibitor of PI3K (omipalisib) (both from Selleck Chemicals), respectively, were lysed with RIPA buffer containing 1% protease inhibitor cocktail (100:1) and quantified by the BCA protein assay kit (all from Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Harvested proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) for 2 h. Then, the membranes were blocked with 5% non-fat milk dissolved in 1X TBST (150 mM NaCl, 0.05% Tween-20, 10 mM Tris-HCl, pH 8.0) for 2 h, and incubated with the primary antibodies at 4°C overnight including anti-E-cadherin (1:2,000), anti-N-cadherin (1:2,000), anti-vimentin (1:2,000), anti-slug (1:2,000) (all from Proteintech Group, Inc., Rosemont, IL, USA), anti-p-AURKA (1:2,000), anti-AURKA (1:2,000), anti-FAK (1:2,000), anti-p-FAK (Tyr397, 1:2,000) (all from Cell Signaling Technology), anti-PI3K (1:2,000), anti-p-PI3K (1:2,000) and GAPDH (1:5,000) (all from Abcam). After overnight incubation, the PVDF membranes were washed with 1X TBST 3 times and incubated with a secondary antibody (1:5,000; Cell Signaling Technology) for 2 h at room temperature. Lastly, the membranes were detected with an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA).

Hep2 cells treated with TAE226 and omipalisib were considered the experimental groups, and groups treated with DMSO or without any treatment were the control groups. Hep2 cells at a density of 1×10⁶ cell/well were seeded into 6-well plates. After overnight culture, the Hep2 cells were scratched with 20-µl pipette tips to form straight lines. The floating cells were removed using 1X PBS 3 times. Finally, the wound lines were photographed at 0, 24 and 48 h at a magnification of ×2.

A density of 2×10⁵ cell/well treated with TAE226, omipalisib, DMSO or without any treatment suspended in 200 µl of serum-free DMEM were placed into the top of Transwell chambers (Corning, Tewksbury, MA, USA) and the lower chambers were filled with 600 µl of DMEM with 10% FBS. After overnight culture, non-migratory Hep2 cells were removed with a cotton swab from the top surface of the chamber and the lower filter of the chamber was stained with crystal violet solution for 30 min at 37°C. Then, the migratory Hep2 cells were photographed using microscopy at magnification, ×20. Briefly, the experimental produces of the cell invasion and the cell migration assays were identical except that BD Matrigel™ (Becton-Dickinson Labware, Bedford, MA, USA) covered the membrane of the top chamber.

Data of all experiments were analyzed by GraphPad Prism 6 software (GraphPad, San Diego, CA, USA) and are shown as mean ± SD. The Student's t-test was performed to assess the differences between experimental groups and controls. The level of significance was set at P<0.05, and a high level of significance was set at P<0.01.

Our previous study showed that AURKA was overexpressed in Hep2 cells and promoted the metastasis of LSCC (13,14). We downregulated AURKA with VX680 (80 nm/ml) (28), a selective inhibitor of AURKA, in the Hep2 cells for 24 and 48 h (Hep2/VX680). Hep2 cells treated with DMSO (Hep2/ctrl) and without any treatment (Hep2/parental) were the control groups. The results of the CCK-8 assay showed less proliferation in the Hep2/VX680 cells compared with the Hep2/ctrl and Hep2/parental cells (P<0.05; Fig. 1A). The plate colony-formation assay showed parallel results in the Hep2/VX680 (404±18.95), Hep2/ctrl (601±16.74) and Hep2/parental cells (607±24.11) (P<0.01; Fig. 1B and C). These results indicated that AURKA significantly promoted cell proliferation and that VX680 effectively reduced the function of AURKA.

To investigate whether AURKA is involved in EMT to promote the metastasis of LSCC, the EMT-related proteins E-cadherin, N-cadherin, vimentin and slug were assessed in the Hep2 cells treated with VX680 at specific times. The results of immunofluorescence staining showed that the expression of p-AURKA and vimentin in the Hep2 cells was lower, while the expression of E-cadherin was higher when compared with these levels in the control group (Fig. 2A). Subsequently, the results of western blotting also revealed that the expression of p-AURKA was decreased almost 3-fold (P<0.01; Fig. 2B and C) and the EMT-related protein levels were altered in a time-dependent manner along with the alteration of AURKA. Expression of E-cadherin was increased and expression of N-cadherin, vimentin and slug was decreased (Fig. 2B and C). All of the aforementioned observations indicated that AURKA may induce EMT to promote the metastasis of LSCC.

Given that the FAK pathway is regarded as a regulatory factor in tumor metastasis (29–31) and FAK is involved in PI3K-promoted EMT in lung cancer (32), we aimed to demonstrate that AURKA regulates those factors to induce EMT. As expected, the expression of p-FAK (Tyr397) and p-PI3K was reduced following the diminution of p-AURKA in the Hep2 cells (P<0.01; Fig. 3A and B), which demonstrated that FAK and PI3K were regulated by AURKA and played a key role in contributing to the tumorigenesis of LSCC.

To explore whether the FAK/PI3K pathway plays a key role in the induction of EMT in LSCC, we utilized the FAK inhibitor TAE226 (2.1 µM/ml) (33), and PI3K inhibitor omipalisib (500 nm/ml) (34) dissolved in DMSO. Treatment with TAE226 altered the EMT-related proteins in a time-dependent manner. The level of E-cadherin was increased, while N-cadherin, vimentin and slug were decreased, indicating that FAK caused more mesenchymal-like characteristics (Fig. 4A and B). p-AURKA, AURKA, p-FAK (Tyr397), FAK, p-PI3K and PI3K in the Hep2/TAE226 cells were also examined. The expression of p-FAK (Tyr397) was nearly decreased 2-fold after treatment with TAE226 for 24 h (P<0.05; Fig. 4C and D). In addition, the expression of p-PI3K was decreased (P<0.01; Fig. 4C and D), whereas p-AURKA, AURKA, FAK and PI3K were not changed, indicating that FAK is a downstream factor of AURKA.

EMT-related proteins were also inhibited in the Hep2/omipalisib cells. These observations showed that E-cadherin was increased, while N-cadherin, vimentin and slug were decreased, which also demonstrated that inhibition of PI3K caused less mesenchymal-like characteristics (Fig. 4E and F). Moreover, p-AURKA, AURKA, p-FAK (Tyr397), FAK, p-PI3K and PI3K were assessed after using omipalisib for 0, 6 and 12 h. From the results of the western blotting, p-PI3K was markedly decreased (P<0.01; Fig. 4G and H), whereas p-AURKA, AURKA, p-FAK (Tyr397), FAK and PI3K were not changed, which illustratied that PI3K is a downstream factor of AURKA/FAK (Fig. 4G and H).

To further demonstrate that activation of the FAK/PI3K pathway may be crucial for AURKA-induced EMT in LSCC metastasis, wound healing, migration and invasion assays were performed. We down-regulated FAK and PI3K with TAE226 (Hep2/TAE226) and omipalisib (Hep2/omipalisib), respectively. Similarly, Hep2 cells treated with DMSO (Hep2/ctrl) and without any treatment (Hep2/parental) were considered the control groups. First the wound was scratched and observed for 0, 24 and 48 h. The results showed that Hep2/TAE226 and Hep2/omipalisib cells moved slightly, while Hep2/ctrl and Hep2/parental cells almost reached the middle of the scratch, indicating that FAK and PI3K caused more mesenchymal-like characteristics and markedly promoted Hep2 cell mobility (P<0.05; Fig. 5A and B).

Similarly, the results of the migration assay indicated that the numbers of Hep2/TAE226 (101±7.23) and Hep2/omipalisib cells (101±12.7) migrating through the Transwell chamber were significantly less than these numbers in the Hep2/parental (203±8.76) and Hep2/ctrl cells (203±13.33). The invasion assay also showed parallel results with Hep2/TAE226 (88±4.10), Hep2/omipalisib (105±3.93), Hep2/parental (130±3.46), Hep2/ctrl cells (135±2.89) (Fig. 5C and D). These observations indirectly illustrated that FAK and PI3K acquired more mesenchymal-like characteristics and increased mobility, migration and invasion.