The expression of Yes-associated protein (YAP) has been reported to be dysregulated in pancreatic cancer. However, its contributions to tumor formation and progression remain to be elucidated. The present study demonstrated that YAP overexpression promoted the epithelial-mesenchymal transition (EMT) in a manner associated with pancreatic cancer invasion
Pancreatic cancer is the fourth leading cause of cancer-associated mortality worldwide (
Yes-associated protein (YAP) overexpression has been reported for several human tumor entities, including prostate, ovarian, colon, liver, lung and pancreatic cancer (
The present study revealed that YAP overexpression promoted the epithelial-mesenchymal transition (EMT) of pancreatic cancer cells and increased drug resistance. The role of YAP on the sensitivity of pancreatic cancer cells to gemcitabine was investigated and the present study explored the mechanisms, which may mediate such an effect. The findings of the present study suggested that YAP induces the EMT and regulates the sensitivity of pancreatic cancer cells to gemcitabine by activating AKT and raises the possibility that YAP may be a promising target to improve the efficacy of therapy for pancreatic cancer.
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
The cells were treated with the AKT inhibitor ipatasertib (0.5
Cell migration and invasion were investigated using a Transwell migration assay and a matrigel invasion assay (8
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
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 (
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 (
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 (
Based on the association between the expression of YAP and the invasion of pancreatic cancer
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 (
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
YAP is a multifunctional molecule that regulates cell survival, proliferation, migration and differentiation in several human cancer types (
Increasing evidence from experimental and clinical studies suggest that the EMT is important in tumor invasion, migration and metastasis (
In models of chemotherapy resistant cancer types, EMT gene signatures have been hypothesized to be involved in the presence of chemotherapy resistance, and regulation of EMT transcriptional regulators modulates resistance to chemotherapeutic agents (
In conclusion, the results of the present study revealed that YAP is expressed in pancreatic cancer tissues and is positively correlated with tumor progression. The overexpression of YAP may contribute to the invasiveness of pancreatic cancer cells. Additionally, the present study provided evidence of a molecular and phenotypic association between the YAP-induced EMT phenotype and gemcitabine-resistance of pancreatic cancer cells. YAP expression reduces the sensitivity to gemcitabine in pancreatic cancer cells. Taken together, YAP is important for the pathogenesis pancreatic cancer and may be a biomarker for predicting response to gemcitabine treatment.
epithelial-mesenchymal transition
Yes-associated protein
YAP is upregulated in human pancreatic cancer. The relative protein expression levels of YAP was determined in (A) different pancreatic cancer cell lines, (B) tumor and peritumoral tissues from patients with pancreatic cancer and (C) in fresh-frozen specimens from pancreatic cancer and matched peritumoral tissues by western blot analysis. (D) Densitometric quantitation of the western blots is also shown for the fresh-frozen specimens. β-actin was used as a loading control. T, tumor; P, peritumoral; YAP, YES-associated protein.
Overexpression of YAP promotes pancreatic cancer invasion
YAP overexpression mediates the EMT of the pancreatic cancer cells. (A and B) EMT markers were observed between high and low expression YAP cells (Capan-1-YAP, vs. Capan-1-Mock and PANC-1-YAP shRNA, vs. PANC-1-NC shRNA). YAP, YES-associated protein; EMT, epithelial-mesenchymal transition.
YAP-mediated pancreatic cancer cell EMT by the hyperactivation of AKT. (A) The expression of p-AKT is downregulated by the knockdown of YAP in pancreatic cancer cells. (B) The forced expression of YAP upregulated the expression of p-AKT in pancreatic cancer cells. (C) The AKT specific inhibitor, ipatasertib, reversed the EMT conferred by YAP overexpression in Capan-1 cells. YAP; YES-associated protein; EMT, epithelial-mesenchymal transition; p-, phosphorylated; sh, short hairpin.
YAP induces the resistance to gemcitabine of pancreatic cancer cells. (A) Overexpression of YAP in Capan-1 cells reduced the sensitivity of the cells to gemcitabine. (B) Knockdown of the expression of YAP in PANC-1 cells increased the sensitivity of the cells to gemcitabine. The data are expressed as the mean ± standard deviation (n=3; *P<0.05 and **P<0.01). YAP, YES-associated protein.