Role Of YAP In The Migration,Invasion And Angiogenesis In Human Pancreatic Ductal Adenocarcinoma

Pancreatic cancer,also known as pancreatic ductal adenocarcinoma(PDAC),is one of the most aggressive tumors with a five-year survival rate lower than 5%.The incidence rate of PD AC is annually increasing because of the obesity,high-fat diets,diabetes,smoking,alcoholic dependence,and chronic pancreatitis.Surgical resection for PD AC is the only therapy with curative potential,but only 15%to 20%of patients have the opportunity for surgery.The mortality of PD AC is mainly caused by delayed diagnosis,early metastasis,and resistance to systemic chemotherapy and radiotherapy.Therefore,elucidating the underlying mechanisms of PDAC metastasis and developing novel therapeutic strategies are critical.The Hippo pathway regulates organ development and stem cells via inhibiting cell proliferation and promoting apoptosis.Recent studies have shown that the Hippo pathway is strongly involved in the process of tumor initiation,progression and metastasis.The Yes-associated protein 1(YAP),a transcriptional co-activator,is the most critical component of the Hippo pathway.The Hippo pathway involves a cascade of kinases which mediates YAP phosphorylation leading to YAP degradation in cytoplasm.The unphosphorylated YAP translocates to the nucleus and transactivates the downstream genes,such as TEADs,SMAD,RUNX and CTGF.YAP is involved in malignant behaviors of tumors,such as cancer stem cell properties,epithelial-mesenchymal transition(EMT),increased potential for migration and metastasis and resistance to chemotherapy.YAP is highly expressed in various malignant tumors including pancreatic cancer.The high expression of YAP is shown to participate in the development,progression and recurrence of PDAC.However,few studies have investigated the role of the Hippo pathway in remodeling of tumor microenvironments and angiogenesis.Hypoxic microenvironments exist in solid tumors and are associated with tumor invasion and distant metastasis,extracellular matrix(ECM)degradation,and EMT.Hypoxia-inducible factor-la(HIF-la)is the most important transcription factor caused by hypoxia.Hypoxic microenvironments and HIF-1α are critical drivers of PDAC metastasis and progression.In pancreatic cancer,the expression of HIF-la is upregulated and associated with tumor progression,metastasis,EMT and extracellular matrix degradation.YAP activity is also linked to oxygen availability.However,the mechanism by which YAP regulates tumor invasion and metastasis under hypoxia needs to be further illustrated.Verteporfin is a photosensitizer clinically used for photodynamic therapy to treat neovascularization caused by age-related macular degeneration.Recently,studies have shown that verteporfin could inhibit YAP activation by disrupting YAP-TEAD interactions and preventing YAP induced oncogenic growth.However,the role of verteporfin in pancreatic cancer is unclear.Thus,further studies of verteporfin might provide insights into potential approaches in PDAC therapy.In this study,we investigated the expression of YAP in pancreatic cancer and the correlation between YAP expression and clinicopathlogical feature.We also explored the role of YAP in migration,invasion and angiogenesis.In addition,we focused on the mechanism that the Hippo pathway deactivated in hypoxia.At last,we investigated the antitumor effect of verteporfin in vitro and in vivo.Therefore,YAP may represent a novel therapeutic target in pancreatic cancer treatment.Part 1.The expression of YAP in pancreatic canerObjective:We aimed to examine the expressions of YAP mRNA and protein in pancreatic cancer and adjacent tumor tissues.We further investigated the YAP expression and subcellular location and the correlation between YAP expression and clinicopathologic features.In addition,we also investigated the relation of YAP expression and prognosis.Material and methods:Nineteen PDAC tumor specimens and matched adjacent non-tumor specimens were saved in liquid nitrogen and obtained from the Qianfoshan Hospital of Shandong Province between October 2014 and December 2015.Human pancreatic cancer tissue microarrays(TMAs)were used to analysis the correlation between YAP expression and clinicopathologic features and prognosis.1.The expressions of YAP mRNA and protein in tumor tissues and adjacent tumor tissues were examined by qRT-PCR,Western Blot and immunofluorescence.2.The YAP expression in the tissues was evaluated by immunohistochemistry(IHC).The χ2 or Fisher exact test was used to analyze the correlation between the YAP expression and the clinicopathologic features.The Kaplan-Meier method and log-rank test were used to evaluate the survival curves.Results:1.The expressions of YAP mRNA and protein were upregulated in PDAC.2.The nuclear YAP was positively expressed in 58.7%of tumor tissues and 35.1%of adjacent non-tumor tissues.A significant difference in the positive rate of the nuclear YAP was found between tumor tissues and non-tumor tissues(P<0.05).Nuclear YAP expression was correlated with tumor differentiation.3.The overall survival time of patients with high nuclear YAP expression was significantly shorter than those with negative and low nuclear YAP expression.Conclusions:1.The expression of YAP was elevated in PDAC and nuclear YAP expression was correlated with tumor differentiation.2.Nuclear YAP was associated with poor prognosis and might be a novel target for PDAC treatment.Part 2.The role of YAP in the migration,invasion and angiogenesis in PD ACObjective:We aimed to explore the role of YAP in the migration,invasion and angiogenesis in PD AC in vitro.Material and methods:1.The expressions of YAP in pancreatic cancer cells(PANC-1,BxPC-3,SW1990,Mia PaCa-2 and Panc 02.03)and HPDE6 were examined by qRT-PCR and Western Blot to select high YAP expression cell lines for further research.2.The PANC-1 and BxPC-3 were transfected with shYAP and selected stable cells.The wound healing and transwell assays was used to evaluate PANC-1 and BxPC-3 migration and invasion.3.The expressions of E-cadherin,Vimentin,Snail,MMP2 and MMP13 were examined by Western Blot.4.The HUVECs were transfected with shYAP and full-length human YAP ectopic vector.The capillary tube formation ability of HUVECs was evaluated by transwell and tube formation assay.The expression of angiopoietin-2(Ang2)was detected by Western Blot.Results:1.YAP mRNA and protein levels were higher in PD AC cells than those in HPDE6.2.The results of qRT-PCR and Western blot analysis revealed that shYAP significantly inhibited YAP expression.The wound healing and transwell assays demonstrated that depletion of YAP decreased the cells migration and invasion.3.Western blot analysis showed that depletion of YAP downregulated Vimentin,Snail,MMP2,and MMP13 and upregulated E-cadherin.4.Depletion of YAP inhibited,while overexpression of YAP promoted,the migration of HUVECs.Depletion of YAP impaired,while overexpression of YAP,promote the capillary tube formation ability of HUVECs.Conclusions:1.YAP was upregulated in PD AC cells and enhanced PDAC progression.2.YAP promoted the invasion of PDAC cells via EMT-and MMP-mediated ECM degradation.3.YAP transcriptionally regulated Ang2 expression to enhance angiogenesis.Part 3.Hypoxia deactivates Hippo pathway and induces YAP nuclear translocation to promote the invasion of PDAC cellsObjective:We aimed to investigate the relationship between HIF-1α and YAP expressions and explore the mechanism how hypoxia promoted PDAC cells invasion via Hippo pathway.Material and methods:1.The expressions of HIF-la and YAP in 19 PDAC specimens were detected by qRT-PCR and the correlation between HIF-la and YAP mRNA expressions were analyzed by Pearson test.2.The wound healing and transwell assays were used to evaluate the effect of hypoxia on migration and invasion.The mRNA and protein levels of EMT-related markers,MMP2,and MMP13 were examined.3.We investigated whether hypoxia affected the Hippo pathway by Western Blot.Nuclear and cytosolic YAP were detected to illustrate the distribution of YAP in hypoxia by Western Blot and confocal microscopy.4.The migration and invasion were examined by wound healing and transwell assays after silencing HIF-la or YAP.The expressions of E-cadherin,Vimentin,Snail,MMP2,MMP13 and Hippo pathway molecular were examined by Western Blot.Results;1.HIF-la mRNA levels were upregulated in PDAC tissues and HIF-la mRNA expression was positively correlated with that of YAP in tumor tissues.2.Hypoxia promoted the invasion of PDAC cells.The mRNA and protein levels of EMT-related markers,Vimentin,Snail,MMP2,and MMP13 were increased,while E-cadherin was decreased,by hypoxia.3.Western blot analysis suggested that hypoxia induced HIF-la upregulation.The expression of pYAP was decreased and the expression of TEAD was increased under hypoxia.The ratio of nuclear YAP to cytosolic YAP was also increased under hypoxia.4.Depletion of HIF-1α or YAP inhibited the invasion of PDAC cells under hypoxia.Knockdown of HIF-la or YAP decreased the expressions of Vimentin,Snail,MMP2,and MMP13,and increased the expression of E-cadherin as shown by qRT-PCR and Western blot.Depletion of HIF-la had little effect on,but depletion of YAP significantly inhibited,the expression of total YAP,pYAP and TEAD.Conclusions:1.YAP and HIF-1α expressions were upregulated in tumor tissues.HIF-1α mRNA expression was positively correlated with that of YAP in tumor tissues.2.Hypoxia promoted the invasion of PDAC cells via EMT.3.Hypoxia deactivated Hippo pathway and induced YAP nuclear translocation.4.Nuclear YAP interacted with HIF-la and activated Snail transcription to participate in EMT-and MMP-mediated remodeling of tumor microenvironments.Part 4.Hippo pathway inhibitor verteporfin induces PDAC apoptosis and suppresses angiogenesisObjective:We aimed to investigate the antitumor effect and mechanism of verteporfin on PDAC in vitro and in vivo.Material and methods:1.Cell viability was determined with CCK-8;cell cycle was analyzed cytometrically;a plate colony formation assay was performed to examine the effect of verteporfin on clonogenicity.2.Cell apoptosis after verteporfin treated was examined by Transmission electron microscopy,TUNEL assay and flow cytometry.3.The expressions of cyclinDl,cyclinE1,PARP,cleave PARP,Bax,Bcl-2,YAP,pYAP and TEAD was examined by Western Blot after verteporfin treated.4.The ability of angiogenesis was evaluated by transwell assay and in vitro tube formation assay.The expression of Ang2 was examined by Western Blot.5.The transwell asssy and tube formation assay were performed to evaluate vasculogenic mimicry.Three markers for VM(VE-cadherin,a-SMA and MMP2)were detected by Western Blot.6.The effect of verteporfin on cell proliferation,apoptosis,angiogenesis and vasculogenic mimicry were examined in PDAC cells xenograft models.The expressions of cyclinD1,cyclinEl,Ki-67,Ang2,VE-cadherin,α-SMA,MMP2 and CD31 were detected by immunohistochemistry.TUNEL assay and PAS/CD31 double staining were also performed.Results:1.Verteporfin reduced the viability of PDAC cells in dose-and time-dependent manners.Verteporfin also increased G1 phase cells and decreased the percentage of cells in S phase.2.Verteporfin induced cell apoptosis.3.Verteporfin inhibited the expression of cyclinDl and cyclinEl in a dose-dependent manner.Verteporfin also increased the expression of Bax,decreased the expression of Bcl-2,and activated PARP.Verteporfin inhibited the expression of total YAP,phosphoYAP and TEAD in a dose-dependent manner.4.Verteporfin inhibited PDAC angiogenesis and vasculogenic mimicry.Verteporfin also suppresses the migration of PDAC cells and formation of VM via downregulating MMP2,VE-cadherin and a-SMA expression.5.The weight and volume of tumors from verteporfin-treated mice were significantly lighter and smaller than those from vehicle-treated controls.The expressions of cyclinDl,cyclinEl,Ki-67,Ang2,VE-cadherin,a-SMA,MMP2 and CD31 were decreased.Fewer Ki-67 positive-cells and vasculogenic mimicry were observed in tumors treated with verteporfin compared with control tumors.TUNEL assay showed increased apoptosis in verteporfin-treated tumors.Conclusions:1.Verteporfin suppressed the proliferation of human PD AC cells by arresting cells at G1 phase,and inducing apoptosis in dose-and time-dependent manners.2.Treatment with verteporfin led to downregulation of cyclinDl and cyclinEl,modulation of Bcl-2 family proteins and activation of PARP.Verteporfin impaired YAP and TEAD interaction to suppress the expression of targeted genes.3.Verteporfin exhibited an inhibitory effect on angiogenesis and vasculogenic mimicry via suppressing Ang2,MMP2,VE-cadherin,and a-SMA expression in vitro and in vivo.