The pancreatic cancer cell lines, PANC-1, MIA PaCa-2, BxPC-3, Capan-1, T3M4 and colo357, were purchased from American Type Culture Collection (Rockville, MD, USA). The BxPC-3 cells were grown in RPMI-1640 medium, containing 10% fetal bovine serum (FBS) and penicillin/streptomycin (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The PANC-1, MIA PaCa-2, T3M4 and colo357 cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.), containing 10% FBS and penicillin/streptomycin.

Fresh-frozen specimens of human normal pancreatic tissues and primary pancreatic cancer tissues were obtained along with written informed consent and pathology reports from the Henan Provincial People's Hospital (Henan, China), and were used for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Sample collection was performed following approval from the institutional Ethics Review Committee of the Henan Provincial People's Hospital. No patient had undergone chemotherapy prior to surgery.

The cells were lysed in cell lysis buffer for western and IP (Beyotime Institute of Biotechnology, Haimen, China) to obtain the total cellular protein. The protein concentrations were determined using an Enhanced BCA protein assay kit (Beyotime Institute of Biotechnology) and were subsequently boiled for 10 min at 100°C. The protein samples (2 µg/µl; 30 µg) were separated by 12% SDS-PAGE (Beyotime Institute of Biotechnology) and were subsequently transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were rinsed in Tris-buffered saline, containing 0.1% Tween-20 and blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology) for 2 h at room temperature. Following blocking, the membranes were incubated with the following primary antibodies at 4°C overnight: Mouse monoclonal anti-YAP (1:500; cat. no. sc-376830; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit monoclonal anti-E-cadherin (1:1,000; cat. no. 3195; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-N-cadherin (1:1,000; cat. no. 13116; Cell Signaling Technology, Inc.), rabbit monoclonal anti-snail (1:1,000; cat. no. 3879; Cell Signaling Technology, Inc.), rabbit monoclonal anti-phosphorylated (p)-AKT (1:1,000; cat. no. 4060; Cell Signaling Technology, Inc.) and mouse monoclonal anti-β-actin (1:1,000; cat. no. sc-130065; Santa Cruz Biotechnology, Inc.). The membranes were rinsed in phosphate-buffered saline containing 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated goat anti-mouse (cat. no. A0216) and goat anti-rabbit (cat. no. A0208) secondary antibodies (1:1,000; Beyotime Institute of Biotechnology) for 2 h at room temperature. Following washing, the proteins were detected using enhanced chemiluminescence (Beyotime Institute of Biotechnology).

The cells were treated with the AKT inhibitor ipatasertib (0.5 µM; Anpei, Nanjing, China) for 24 h. Subsequently, cell lysates were prepared and western blotting was performed.

Cell migration and invasion were investigated using a Transwell migration assay and a matrigel invasion assay (8 µm pore size; BD Falcon, San Jose, CA, USA), as described previously (23). Briefly, for the Transwell migration assay, 5×10⁴ cells were suspended in 200 µl serum-free DMEM and placed in the cell culture insert of a Transwell plate, and warmed culture medium supplemented with 10% FBS was placed in the well. Cells in serum-free DMEM were seeded in the upper chamber and medium containing FBS was seeded in the lower chamber. For the matrigel invasion assay, 2×10⁵ cells were suspended in 200 µl DMEM without FBS and then placed in the cell culture insert precoated with 1 µg/µl Matrigel (BD Biosciences). Warmed culture medium containing 10% FBS was added to the well. The cells were cultured for 12 h at 37°C in an atmosphere containing 5% CO₂ and were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma-Aldrich). The number of migrated cells in five randomly selected fields was counted under a light microscope (magnification, ×100; Olympus, Tokyo, Japan).

To determine drug sensitivity, the cells were seeded into 96-well plates at a density of 2×10³ cells/well. Following incubation for 24 h, the cells were placed in complete medium, containing different concentrations of gemcitabine (0.2, 1, 5, 25, 125 µM; Jiangsu Hansoh Pharmaceutical Co., Ltd., Lianyugang, China). Following incubation for a further 72 h, the sensitivity of the cells to gemcitabine was measured using a cell counting kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan).

The YAP and YAP short hairpin (sh)RNA expression lentivirus were purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China) and the target shRNA sequences were as follows: 5′-CTC AGG ATG GAG AAA TTTA-3′ and 5′-CGT GCC CCA GAC CGT GCCC-3′. The lentiviral vector was transfected into cells, as described previously (24), cancer cells were infected with lentivirus plus 6 µg/ml polybrene (Sigma-Aldrich) for 24 h, and transfection was confirmed by immunoblotting.

Statistical analysis was performed using SPSS 12.0 software (SPSS, Inc., Chicago, IL, USA). The data are expressed as the mean ± standard deviation. The data were examined using analysis of variance and the least significant differences method for multisample comparisons, or Student's t-test for two-sample comparisons. Kaplan-Meier curves were plotted to assess the effects of YAP expression on the progression-free survival. Survival curves were compared using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

To explore the role of YAP in pancreatic cancer progression, the expression of YAP was assessed in various human pancreatic cancer cell lines, pancreatic cancer and matched peritumoral tissues. The expression of YAP in 30 pancreatic cancer and matched peritumoral tissues was analyzed by western blotting. Compared with the peritumoral samples, semi-quantitative analysis revealed that the protein expression levels of YAP were markedly higher in the cancer tissues (Fig. 1A and B). By contrast, in normal pancreatic tissues, little YAP expression was observed. YAP expression in early-stage (I-II) and advanced-stage (III-IV) pancreatic cancer tissues was significantly higher compared with that in normal pancreatic tissues (P<0.01). In addition, YAP expression in advanced-stage (III-IV) was significantly higher compared with in early-stage (I-II) pancreatic cancer tissues (P<0.01; Fig. 1C).

To elucidate the role of YAP in pancreatic cancer progression, YAP shRNAs were used to reduce the expression of YAP in the human PANC-1 pancreatic cancer cells, which exhibit a high level of YAP protein expression (Fig. 1A). YAP shRNAs significantly reduced the expression of YAP, as well as the invasion of PANC-1 cells (P<0.01; Fig. 2A and C). To further evaluate whether YAP upregulation promoted tumor invasion, lentivirus-mediated delivery of YAP cDNA was used to increase the expression of YAP in human Capan-1 pancreatic cancer cells, which exhibit low protein expression of YAP (Fig. 1A). Upregulation of the regulation of YAP was observed in YAP infectants (Fig. 2B). YAP upregulation significantly increased the invasion ability of Capan-1 cells compared with the mock control (Fig. 2D). Collectively, the data from the in vitro assays revealed that YAP significantly contributed to tumor invasion of pancreatic cancer.

Based on the association between the expression of YAP and the invasion of pancreatic cancer in vitro, and since that the EMT is considered a striking feature of most cancer types and has a vital role in cancer migration and invasion, the present study compared the expression of epithelial and mesenchymal markers, as well as other molecules thought to induce EMT in cancer cells. As shown in Fig. 3A, Capan-1-YAP cells expressed a lower level of the epithelial gene, E-cadherin, compared with Capan-1-Mock cells. The mesenchymal genes, snail and N-cadherin, were significantly upregulated in Capan-1-YAP cells compared with Capan-1-Mock cells. Notably, the level of E-cadherin was higher in PANC-1-YAP shRNA compared with PANC-1-NC shRNA cells, while mesenchymal-associated genes, snail and N-cadherin, were downregulated in PANC-1-YAP shRNA cells (Fig. 3B).

It has been previous confirmed that the induction of the EMT may be an important mechanism of constitutive AKT signaling activation in various cancer types. To further understand whether the YAP-mediated EMT process in pancreatic cancer cells was dependent on the activation of the AKT pathway, western blotting analysis was performed to assess the activation of the components of the AKT pathway in YAP-knockdown or -overexpressing pancreatic cancer cells. The results indicated that shRNA-mediated YAP downregulation in PANC-1 cells markedly reduced the expression of p-AKT (Fig. 4A), whereas YAP overexpression in Capan-1 cells increased the expression of p-AKT (Fig. 4B). Finally, the present study analyzed the effect of ipatasertib-mediated p-AKT inhibition on the expression levels of E-cadherin, N-cadherin and snail in pancreatic cancer cells. Notably, the expression levels of N-cadherin and snail were markedly downregulated, while the expression of E-cadherin was markedly upregulated in cancer cells treated with ipatasertib (Fig. 4C). These results indicated that YAP induced the EMT by way of hyperactivation of AKT signaling in pancreatic cancer cells.

The present study further investigated whether increasing or inhibiting the expression of YAP modulated the sensitivity of pancreatic cancer cells to gemcitabine, which is currently used as the first line treatment for pancreatic cancer. Following exogenous expression of YAP in Capan-1 cells, the cells were treated with a series of concentrations of gemcitabine (0.2, 5, 25 and 125 µM). The effect of YAP on the chemoresistance of Capan-1 cells is shown in Fig. 5A. The half-maximal inhibitory concentrations (IC₅₀) of gemcitabine on Capan-1-Mock and Capan-1-YAP cells were 8.52±1.88 and 21.56±3.03 µM, respectively (P<0.05). These results indicated that the introduction of YAP notably reduced the chemosensi-tivity of Capan-1 cells to gemcitabine. In addition, the inhibition of PANC-1 cell growth with gemcitabine was significantly increased by transfection with YAP shRNA. The IC₅₀ values of gemcitabine on PANC-1-NC shRNA-2 and PANC-1-YAP shRNA cells were 14.22±1.45 and 4.88±0.61 µM, respectively (P<0.05; Fig. 5B).