Ezrin Promotes Pancreatic Cancer Progression Via Regulating YAP Expression Mediated By PI3K/AKT/mTOR Signaling Pathway

BackgroundPancreatic cancer is a gastrointestinal cancer with a high degree of malignancy.As reported,the 5-year survival rate of pancreatic cancer was less than 2%.Though,great advances have been made in surgical techniques,chemotherapy,radiotherapy and biotherapy,the prognosis of pancreatic cancer still remains poor.As well known,invasion and metastasis are the hallmarks of pancreatic cancer,and the leading cause of poor prognosis.Therefore,much attention has been payed on identifying the mechanism of invasion and metastasis.Tumor metastasis is a multi-stage process.It involves the rearrangement of tumor cytoskeleton,falls off from the primary stove,surrounding extracellular matrix invasion,bloodstream invasion and enters the circulatory system.Thus,to identify the molecular mechanism underlying pancreatic cancer metastasis will provide more effective target genes for pancreatic cancer therapy,and improve survival of pancreatic cancer patients.Ezrin,also named cytovilliezrin,is a connexin for cell membrane and cytoskeleton.Ezrin is a member of the ERM family which includes Radixin and Moesin.As a multi-function protein,Ezrin participated in cell mitosis,apoptosis,adhesion,morphology and motility.Recently,it is reported that the high expression of Ezrin is closely related to the occurrence and development of variou tumors.Liang et al.found that high expression of ezrin correlated with clinical stage,TNM stage,lymph node metastasis and poor prognosis of esophagus cancer,gastric cancer and colorectal cancer,which indicated that Ezrin could be a potent prognostic marker for gastrointestinal cancers[1].In previous study,we found that Ezrin played important roles in EMT progression of cervical cancer and breast cancer,and activated AKT signaling pathway.However,it is unclear whether Ezrin involves in pancreatic cancer development and progression.YAP,also called Yes associated protein 1,is an intracellular connexin and transcriptional co-activator.As a downstream nuclear effector of the Hippo signaling pathway,YAP is involved in cell differentiation,growth,repair,and homeostasis.Liu et al.reported that high expression of YAP correlated with histological grade,lymph node metastasis and tumor recurrence of cervical cancer[2].In addition,Shao et al.also reported that YAP and K-ras co-activate transcription factor FOX to inhibit EMT progression of lung cancer.In previous study,we found that Ezrin regulates the size of skin fibroblasts and promote cell proliferation by regulating YAP expression[3]However,it is not clear whether ezrin affect tumor biological behavior,especially,EMT progression and matastasis of pancreatic cancer through regulating YAP.ObjectivesThis study is aimed to identify the expression status of Ezrin and YAP in pancreatic cancer tissues,analyze the correlation between Ezrin/YAP expression and clinicopathological parameters,and explore the effect and mechanism of Ezrin/YAP on pancreatic cancer proliferation,metastasis and angiogenesis.Materials and Methods1.Pancreatic cancer tissue and database analysis.1)The expression status of Ezrin and YAP protein in pancreatic cancer tissues and normal tissues were obtained from TCGA and Oncomine database.Then,the relations between Ezrin,YAP and survival of pancreatic cancer patients were also analyzed by TCGA and Oncomine database.2)The expression levels of Ezrin and YAP protein in pancreatic cancer and paracancerous tissues were detected by immunohistochemistry,and analyzed the correlations between Ezrin/YAP and the clinicopathological features.3)The Kaplan-Meier survival analysis was performed to identify the correlations between Ezrin,YAP expression and the prognosis of pancreatic cancer.4)ROC analysis was performed to identify the sensitivity and specificity of Ezrin and YAP in pancreatic cancer diagnosis.2.In vitro experiments.1)The expression of Ezrin protein in MiAPaca-2,BxPC-3,ASPC-1,SW1990,Capan-1 and Panc-1 pancreatic cancer cell lines were detected by Western blot.Then,the proper cell lines were selected for further study.2)Capan-1 and BxPC-3 cells were selected for Ezrin silencing or overexpression.Then,Ezrin expression level was detected in the transfected cells by using western blot.3)The effects of Ezrin silencing or overexpression on the proliferation of pancreatic cancer cells were detected by colony formation assay,EdU staining and MTT assays.4)Invasion and migration abilities were analyzed by tumor formation assay,scratch assay and transwell chamber detection in pancreatic cancer cells with Ezrin silencing or overexpression.5)Western blot and immunofluorescence were used to detect EMT-related markers(including epithelial markers E-cadherin,ZO-1 and interstitial markers Vimentin,Snail,Slug,β-Catenin),matrix metalloproteinases(MMP-2 and MMP-9)and vascular marker(VEGF)in pancreatic cancer cells with Ezrin silencing or overexpression.6)The expression of p-AKT,AKT,p-mTOR,p-S6,S6,p-4EBP1 and 4EBP-1 was detected by western blot in pancreatic cancer cells with Ezrin silencing or overexpression.7)Microtubule formation assay and vasculogenic mimicry assay were used to detect the effect of Ezrin silencing or overexpression on the blood vessels formation ability of HUVEC(human umbilical vein endothelial cells,HUVEC)and the tubule formation ability of tumor cells.8)The expression of YAP and p-YAP in nucleus and cytoplasm were detected by nuclear-plasma separation after Ezrin silencing or overexpression.9)The interaction between Ezrin and YAP protein was detected by Co-IP assay.10)The effect of PI3K inhibitor LY294003 and mTOR inhibitor rapamycin on the expression of EMT markers,and nuclear and cytoplasmic expressions of YAP protein were detected by western blot after Ezrin silencing or overexpression.11)Colony formation assay and transwell assay were performed to detect the proliferation and migration ability of BxPC-3 cells induced by PI3K inhibitor LY294003 and mTOR inhibitor Rapamycin.3.In vivo experiments.Pancreatic cancer-chick embryo chorioallantoic membrane model was established by pseudocyst method to detect the effect on the blood vessel formation abilities of blood vessels of pancreatic cancer cells after Ezrin silencing or overexpression.Results1.Ezrin and YAP were co-overexpressed in pancreatic cancers,and both correlated with poor prognosis.TCGA,Oncomine database analysis showed that Ezrin and YAP protein were highly expressed in pancreatic cancer tissues,which were closely related to the poor prognosis of patients with pancreatic cancer.The positive rate of Ezrin protein in pancreatic cancer was 86.1%(93/108),significantly higher than adjacent tissues(35.4%,P<0.001).The expression of Ezrin was related to the pathological grading,clinical stage,lymph node metastasis and invasion of nerve/vessel in pancreatic cancer(P<0.05),but not related to the gender,age,tumor location and size(P>0.05).The positive rate of YAP protein was 85.2%(92/108)in pancreatic cancer tissue,which was significantly higher than normal tissue(31.6%(25/79),P<0.001).The expression of YAP was closely related to pathological grading and lymph node metastasis(P<0.01),but,not related to the gender,age,tumor location,tumor size and clinical stage(P>0.05).Statistically,the expression level of Ezrin was positively correlated with the expression level of YAP in pancreatic cancer tissues(r=0.821.p<0.001).The Survival anaylysis showed the high expression of Ezrin and YAP was significantly associated with poor prognosis in pancreatic cancer patients.Univariate analysis showed that tumor grade,lymph node metastasis,Ezrin,YAP and clinical stage could affected the overall survival rate of patients,and multivariate analysis showed that only the expression of Ezrin protein(Wald=6.297,P=0.012)and pathological grade(Wald=5.465,P=0.019)were independent risk factors of poor prognosis;The ROC analysis showed that the area value of the Ezrin and YAP protein in the pancreatic cancer tissue was 0.782 and 0.754,respectively,indicating the significance and sensitivity in the clinical diagnosis.2.Ezrin promotes the proliferation of pancreatic cancer cells.Capan-1 was transfected with si-Ezrin,and BxPC-3 was transfected with Ezrin-overexpression plasmid.Silencing of Ezrin inhibited the proliferation and colony formation ability of pancreatic cancer cells,and overexpression of Ezrin promoted the proliferation and colony formation ability.3.Ezrin could promote the invasion and migration of pancreatic cancer cells via regulation EMT progression.Overexpression of Ezrin promoted the migration and invasion capacity of pancreatic cancer cells,and silencing of Ezrin inhibited the migration and invasion capacity of pancreatic cancer cells.We detected the expression of EMT markers after overexpression or silencing of Ezrin.The results showed that overexpression of Ezrin down-regulated the expression of epithelial marker E-cadherin,ZO-1 and up-regulated mesenchymal markers Vimentin,β-catenin,Snail,Slug and matrix metalloproteinases MMP-2,MMP-9.These results indicated that Ezrin affects pancreatic cancer migration and invasion by regulating EMT progression.Consistently,the same results were also obtained by IF staining.4.Ezrin promotes the angiogenesis of pancreatic cancer.Silencing of Ezrin protein could effectively reduce HUVEC angiogenesis and vasculogenic mimicry of tumor cells,while Ezrin overexpression enhanced HUVEC angiogenesis and promote vasculogenic mimicry of tumor cells.5.Ezrin regulates YAP phosphorylation and nuclear translocation.The expression of YAP was not significantly changed in pancreatic cancer cells after Ezrin silencing,whereas p-YAP was up-regulated.Similarly,the expression of YAP was not changed in pancreatic cancer cells after Ezrin overexpression,whereas p-YAP was down-regulated.The results of nuclear-slurry separation experiment showed that the expression of YAP protein was down-regulated in the nucleus and up-regulated in cytoplasm after Ezrin silencing.Then,the results were contrary when Ezrin was overexpressed.The expression of p-YAP showed no obvious change in cytoplasm with Ezrin silencing or overexpression,but p-YAP expression in the nuclei was up-regulated when Ezrin was silenced.Additionally,immunoprecipitation showed that Ezrin interact with YAP.6.Ezrin modulates EMT progression and YAP nuclear translocation via regulating PI3K/AKT/mTOR signaling pathway in pancreatic cancer.The expression of p-AKT,AKT,p-mTOR,mTOR,p-4EBP1,4EBP1 was down-regulated after silencing of Ezrin,whereas the results were contrary when Ezrin was overexpressed.Additionally,the treatment of PI3K inhibitor LY294002 and mTOR inhibitor Rapamycin reversed the effect of Ezrin on PI3K/AKT/mTOR signaling and EMT progression,suggesting that Ezrin participated in EMT progression through regulating PI3K/AKT/mTOR signaling.Addtionally,the expression of YAP protein in the nucleus was reduced and in cytoplasm was increased with LY294002 or Rapamycin treatment.Moreover,colony formation assay and migration assay were performed to prove the suppressive effect of this process on pancreatic cancer cell proliferation and migration.7.Ezrin overexpression promotes the angiogenesis of pancreatic cancer-chick embryo chorioallantoic membrane model.The effect of pancreatic cancer cells on the ability of chicken blastocyst angiogenesis was reduced after silencing Ezrin,meanwhile Ezrin overexpression,its effect was obvoiusly elevated.Conclusions1.Ezrin and YAP were co-overexpressed in pancreatic cancers,and both correlated with poor prognosis.2.The overexpression of Ezrin promotes the proliferation,metastasis and angiogenesis in pancreatic cancer.3.Ezrin modulates EMT progression and YAP nuclear translocation via regulating PI3K/AKT/mTOR signaling pathway in pancreatic cancer.