The Expression And Roles Of Integrin ?6 In Cholangiocarcinoma

BackgroundCholangiocarcinoma(CCA)was first reported by Durand-Fardel in 1840.The tumor arises from the ductular epithelium of the biliary tree and is usually of highly malignancy.CCAs could be divided into intrahepatic cholangiocarcinoma(ICC)and extrahepatic cholangiocarcinoma due to different position according to the 7th edition of AJCC.ICC derives from the epitheliums of intrahepatic bile duct and its branches.ECC could be further divided into perihilar cholangiocarcinoma(PHCC,also called Klatskin tumor)and distal cholangiocarcinoma(DCC)bordered by the meet of ductus cysticus and common hepatic duct.CCA accounts for 3%of all gastrointestinal cancers worldwide,but it is the second commonest primary hepatic tumor.And the morbidity and mortality of this has been increasing these recent years,especially the ICC.Radical resection of the tumor is the only way for the radical treatment of this tumor.However,the disease is notoriously difficult to diagnose and is usually fatal because of its late clinical presentation and the metastatic property of the tumor.In addition,CCA is usually not sensitive to chemotherapy and radiotherapy,and no evidence has been shown that assistant chemotherapy or radiotherapy may reduce the recurrent risk.All these make CCA a devastating malignancy,with the 5-year survival around 5%.Therefore,exploring the key factors that essential for the tumorigenesis and progress,especially for the tumor invasiveness and metastasis,and a detailed understanding of the related mechanism behind the invasion and metastasis is doom to be helpful in the treatment of CCA and thereby reduce mortality rate.Integrins belong to a family of cell surface adhesion molecule,which is composed of a and ? subunits associated non-covalently.Integrin ?v?6 is the only heterodimer that ?6 subunit can form,it 'is highly expressed during the process of embryogenesis,tissue repair and tumorigenesis,and is absent or lowly expressed in normal epithelium,benign epithelial tumor and other types of malignant tumors.It has been reported that integrin av(36 is highly expressed in a number of malignant digestive tumors such as gastric cancer,colon cancer and PD AC.It's expression is closely correlated with tumor invasiveness and metastasis,and it promotes the development and progress of tumors via activating various of classical oncogenic signaling pathways.Recently we demonstrated that ?v?6 shuttles between the plasma membrane and the cytoplasma through the endocytosis cycling,rather than fixed expressed on the plasma membrane.This shuttling is of great importance to the interaction between the tumor cell and ECM and tumor cell migration.Moreover,we also found a direct link between the ?6 subunit and extracellular signal-regulated kinase 2(ERK2),which could further promote the invasiveness of tumor cells via activating the related downstream signaling and mediating the expression of matrix metalloproteinases(MMPs).In addition,by cooperating with chemotactic factors such as SDF-1 and IL-8,integrin ?6 participates in the target metastasis of tumor cells.The property of tumor strict expression and its role in promoting tumor progression makes integrin ?6 a potential target for the diagnosis and treatment of tumors.However,as a key molecule that contributes to the malignant digestive tumors,the role and related mechanisms of integrin ?6 in CCA is still largely unknown.Racl is a member of the Rho family of small GTPases which includes Racl,Rac2,Rac3 and Rac1b,which share 90%of the homology and they all contain a structure of RhoGTPase.Among all these four members,most research are focus on Racl so far.Racl acts as a molecular switch and is important to maintaining normal cell functions through regulating various of signaling pathways.It has been reported that Rac1 is up-regulated or activated in a number of tumors such as gastric cancer,colon cancer and pancreatic duct adenocarcinoma(PDAC).Racl has been implicated in a wide variety of malignant behaviors of tumor cells,including tumor proliferation,tumor cell apoptosis,tumor migration,tumor invasion and metastasis.The role of Racl in promoting tumor migration and invasion has drawn a lot of attentions.The process of migration and invasion incorporates the formation of the pseudopodia,cell motility,the adhesion between cell to cell and cell to extracellular matrix(ECM),the degradation of ECM etc.Research have found that Racl is highly-expressed in a number of CCA cells,and the expression of Racl is positively correlated with the metastatic potential of CCA cells.Rac1 has been reported to be involved in the formation of tumor microenvironment and the process of chemoresistance.However,the role and related mechanisms of Racl in promoting the invasiveness and metastasis is rarely known.In order to explicit the expression condition and role of integrin P6 in CCA and provide theoretical support for designing ?6-targeted treatment strategy,here we conducted the research through the following four aspects.(1)We collected 95 CCA tissue sample and corresponding clinical data from Qilu Hospital of Shandong University,then detected the expression condition of integrin ?6 in CCA tissues and adjacent normal tissues by immunohistochemistry(IHC).Followed by the analysis between ?6 expression condition and clinicopathological parameters.Moreover,we also demonstrated that integrin ?6 was identified as a biomarker for the diagnosis of CCA and an indicator of lymph node metastasis.(2)A series of in vitro experiments were conducted and demonstrated that integrin ?6 could markedly promote the proliferation,migration and invasion capacities of CCA cells.(3)Further study revealed that integrin ?6 promotes the migration and invasion of CCA cells through activating Racl-GTPase,which resulting in the polymerization of F-actin and upregulation of MMP9.(4)Finally,we verified the effect of integrin ?6 on promoting the progression of CCA by establishing subcutaneous tumor model.Here we report Rac1 as a key target of the p6-mediated tumor development and progression.In view of the vital role of Racl in tumorigenesis,we proposed that ?6-targeted therapy is expected to suppress the invasiveness and metastasis of CCA and improve the prognosis of CCA patients,which is of great importance and perspectives to the comprehensive treatment of CCA.Part ?The expression condition and clinicopathological significance of integrin ?6 in CCA tissuesObjectiveTo explicit the expression condition of integrin ?6 in CCA tissues,analyze the relationship between ?6 expression and clinicopathological parameters,and explore the clinicopathological significance of ?6 expression condition.Methods1.CCA tumor tissues,corresponding adjacent normal tissues and clinical data of the patients at Qilu Hospital of Shandong University range from 2006 to 2013 were collected.Clinical data includes gender,age,serum CA19-9 level,serum CEA level,postsurgical pathological data(including depth of invasion,lymph node metastasis,tumor differentiation etc.)and distant metastasis.2.The expression of integrin ?6 in CCA tumor tissues and adjacent normal tissues was detected by IHC after informed consent.IHC staining was independently evaluated by two experienced pathologists in a blinded manner.Chi-square test was conducted to evaluate the associations between ?6 expression and clinicopathological parameters.3.Further ROC analysis was conducted to assess the diagnostic value of ?6 in CCA as well as its sensitivity and specificity in predicting lymph node metastasis.Results1.95 CCA pathological tissues and related adjacent normal tissues were collected.IHC results revealed that moderate-high expression of ?6 staining was observed in tumor tissues,with the major expression on the plasma membrane,and a little in the cytoplasm.While ?6 expression was almost negative in the non-tumorous tissues.2.Integrin ?6 expression was significantly associated with lymph node metastasis(P=0.0003)and distant metastasis(P=0.0126),while no significant association was observed between ?6 expression and age,gender,CA19-9 level,CEA level,depth of invasion and tumor differentiation(P>0.05),indicating that patient with high ?6 expression were easier to have lymph node metastasis and distant metastasis.3.ROC analysis was conducted to evaluate the capacity of ?6 expression to diagnose CCA,and results showed that high integrin ?6 expression could serve as an IHC marker in the diagnosis of CCA(AUC=0.876,P<0.001),with the estimated sensitivity and specificity of 78.95%and 93.68%,respectively.While after analyzing of ?6 expression between CCA tissues with or without lymph node metastasis,results showed that ?6 expression could effectively predict lymph node metastasis when IHC score>5(AUC=0.701,p<0.001),with an estimated sensitivity and specificity of 77.55%and 63.04%respectively.Conclusions and significanceIntegrin ?6 expression was significantly up-regulated in CCA tumor tissues compare to that in adjacent normal tissues,and ?6 expression was significantly associated with lymph node metastasis and distant metastasis.Integrin ?6 may serves as an IHC marker for the diagnosis of CCA.and when the IHC score of ?6 expression>5,there is a high possibility for the CCA patients to have lymph node metastasis.Therefore,we suggest IHC ?6 staining should be routinely done for the assistant diagnoses of CCA and prediction of lymph node metastasis,as well as deducing tumor malignancy.All these results indicated that integrin ?6 may play an important role in the invasiveness and metastasis of CCA,which provide theoretical support to further investigate the effect of ?6 on the malignant biological behaviors of CCA cells and explore the related mechanisms.Part ?The effect of integrin ?6 on the malignant biological behaviors of CCA cellsObjectiveTo explicit the effect of integrin ?6 on the malignant biological behaviors of CCA cells after confirming that integrin ?6 was markedly up-regulated in CCA tumor tissues and closely correlated to lymph node metastasis and distant metastasis.Methods1.Quantitative Real-time PCR and western blot were used to detect the expression of integrin ?6 in two CCA cell lines RBE and QBC939.2.The CCA lines RBE and QBC939 were divided into four groups respectively,NC group,si?6 group,Mock group and ?6 transfectant group.Quantitative Real-time PCR and western blot were used to detected the silencing and overexpression efficiency,which made preparations for the next steps of experiments.3.CCK8 assay was used to detect cell viability after silencing or overexpressing ?6.4.MTT assay was used to detect the changes of cell proliferation after silencing or overexpressing(36.5.Exploring the difference of migration capacity between the aforementioned groups by wound healing assay and Transwell migration assay.6.Matrigel pre-coated Transwell invasion assay was used to detect the changes of invasion capacity of CCA cells after after silencing or overexpressing P6.Results1.Moderate ?6 expression was shown at both mRNA level and protein level in RBE and QBC939 cells.?6 expression was significantly decreased at both the mRNA level and protein level after transfection with ?6-specific siRNA,while ?6 expression was significantly increased after transfection of ?6 overexpressing plasmid.2.CCK8 assay revealed that ?6 silencing in RBE and QBC939 cells markedly suppressed cell viability 48 hours after transfection in a time-dependent manner,while ?6 overexpressing resulted in the opposite effect.3.MTT assay showed that P6 downregulation in RBE and QBC939 cells was associated with decreased cell proliferation,while ?6 overexpression was associated with increased cell proliferation.4.The results of wound healing assay and Transwell migration assay showed that ?6 silencing significantly suppressed the migration ability of CCA cells,and ?6 overexpression markedly promoted cell migration capacity.5.By the using of Matrigel pre-coated Transwell cells,we found that ?6 silencing suppressed the invasion of tumor cells and ?6 overexpression promoted tumor cell invasion.Conclusions and significanceWe explored the effect of integrin ?6 on the malignant biological behaviors of CCA cells in this section.Firstly,we found that ?6 was moderate expressed at both mRNA and protein levels in RBE and QBC939 cells.So we treated the two cell lines with both ?6 siRNA and ?6 overexpressing plasmid.Then CCK8 assay,MTT assay,wound healing assay and Transwell assay were conducted to investigate the role of integrin ?6 in the cell viability,cell proliferation,migration and invasion of the two CCA cells.The results showed that integrin ?6 could significantly promote the proliferation,migration and invasion of CCA cells,indicating that(36 might be of great importance in the development and progression of CCA.All these results updates our understanding of the mechanisms of CCA progression,replenished the role of P6 in CCA,which lays a foundation for the further research on the mechanisms of how integrin ?6 promotes CCA invasiveness and metastasis.Part IIIIntegrin ?6 promoted the invasiveness and metastasis of CCA cells via activating RaclObjectiveIt has been reported that Rac1 plays an important role in tumor progression,especially in tumor invasiveness and metastasis.We have also confirmed that integrin?6 is closed correlated to the invasiveness and metastasis of CCA.Here we aim at exploring whether integrin(36 promotes the invasiveness and metastasis of CCA cells via Rac1 pathway.Methods1.Detecting Racl activity using PAK-PBD pull down assay after ?6 silencing or overexpression.2.Transwell assay was used to detect the migration and invasion capacity of CCA cells after the treatment with different concentrations of NSC23766,a specific Rac1 inhibitor.3.After the treatment of 50 ?M NSC23766,transfect CCA cells with the Mock or ?6 overexpressing plasmid,then Transwell assay was used to detect tumor cell migration and invasion capacity.4.The CCA cells RBE and QBC939 were divided into four groups and transfected with NC/siRac1 and Mock/?6 overexpressing plasmid,then detect tumor cell migration and invasion capacity by Transwell assay.5.Real-time PCR and ELISA assay were used to detect the expression of uPA,MMP2,MMP3,MMP9 in the two CCA cells and their supernatant after(36 silencing or overexpression.6.Real-time PCR and ELISA assay were used to detect the expression of uPA,MMP2,MMP3,MMP9 in the two CCA cells and their supernatant after Racl silencing.7.Transfect CCA cells with si?6 or ?6 plasmid after the treatment of NSC23766,then detect the expression of MMP9 in the supernatant of tumor cells.8.After ?6 silencing,fluorescently-labeled phalloidin was used to stain the cytoskeleton F-actin of CCA cells,then observe the polymerization of F-actin using confocal microscopy.9.Transfect CCA cells with si?6 or ?6 plasmid after the treatment of NSC23766,then fluorescently-labeled phalloidin was used to stain the cytoskeleton F-actin of CCA cells and confocal microscopy was used to observe the polymerization of F-actin.Results1.?6 silencing significantly decreased Racl-GTPase activity,while ?6 overexpression increased Racl activity in CCA cells.The expression of total Racl was not affected by integrin ?62.NSC23766,a specific Racl inhibitor,significantly suppressed the migration and invasion of CCA cells in a dose-dependent manner.3.Overexpression of integrin ?6 could effectively promote the migration and invasion capacity of CCA cells,whereas NSC23766 significantly inhibited this effect.Racl silencing also had the similar effect.4.The results of real-time PCR showed that ?6-specific siRNA significantly downregulated MMP9 mRNA expression in CCA cells,and ?6 overexpression markedly increased MMP9 mRNA expression.No effect was observed on uPA,MMP2 and MMP3.5.The results of ELISA showed that the secreted MMP9 in the supernatant of CCA cells was significantly decreased after the suppression of ?6 expression,while ?6 overexpression increased MMP9 secretion in the supernatant.6.The results of real-time PCR indicated that Rac1 silencing significantly decreased the expression of MMP9 at mRNA level.while Racl silencing had no effect on the expression of uPA,MMP2 and MMP3.7.ELISA results showed that Racl silencing significantly decreased the expression of MMP9 at protein level.8.The results of ELISA showed that ?6 silencing decreased MMP9 expression,while ?6 overexpression increased the expression of MMP9.However,after pretreatment with a Rac1 activity inhibitor,the effects of both ?6 silencing and overexpression were abolished.9.The results of Phalloidin fluorescent staining showed that F-actin was organized into the cytoskeleton in the NC group,while in the ?6-silenced group,F-actin was depolymerized in most cells.Statistical analysis revealed that ?6 silencing could significantly promote F-actin depolymerization.10.The results of Phalloidin fluorescent staining showed that F-actin was significantly depolymerized after treatment with ?6 siRNA and ?6 overexpression increased F-actin polymerization.However,after treatment with NSC23766,the F-actin depolymerization increased in different groups and the aforementioned effect was abolished.Conclusions and significanceWe aimed at exploring the detailed mechanisms of how integrin ?6 promoted the invasiveness and metastasis of CCA in this section.First of all,we demonstrated that integrin ?6 significantly promoted the activation of Racl in CCA cells,while had no effect on total Racl expression.Further experiments proved that Racl is essential for the migration and invasion of CCA cells.Then we confirmed that Rac1 is involved in the ?6-mediated CCA cell migration and invasion by the using of Racl inhibitor and Rac1 siRNA.Moreover,we found that ?6 induced the expression of MMP9 via Rac1 signaling pathway.Phalloidin fluorescent staining revealed that integrin could promote the polymerization of F-actin by activating Rac1.Here we found that Rac1 acts as a key target that(36 promoted the migration and invasion of CCA cells,and demonstrated that integrin ?6 induced the migration and invasion of CCA cells by activating Racl,which subsequently promoted F-actin polymerization and MMP9 secretion.Due to the pivotal role of Racl in the tumor invasion and metastasis,there is no doubt that all these results enriched the related theory of integrin ?6 and targeting ?6 may be a novel strategy for suppressing the invasiveness and metastasis of CCA.Part IVIntegrin ?6 promoted tumor growth and MMP9 expression of tumor cells in nude miceObjectivePrevious experiments have already demonstrated the driving effect of integrin ?6 on the migration and invasion of CCA cells and revealed the corresponding mechanisms in vitro,here we aimed at testify the role of integrin ?6 in promoting the progression of CCA in vivo.Methods1.The integrin ?6 silencing and overexpression lentiviral vectors were constructed.2.RBE cells were cultured and transfected with ?6 silencing and overexpression lentiviral vectors respectively,then subcutaneous tumor model was conducted.The volume of the tumors were measured during the formation of the tumor and tumor growth curve was drawn.3.The volume and weight of the tumors from different groups were measured.Then the tumor tissues were embedded with paraffin and cut into slides,IHC was conducted to detect the expression of integrin ?6 and MMP9 within the tumor tissues.Results1.Compared with that of the NC group,silencing of ?6 markedly suppressed the growth of RBE xenograft tumors,the overall mean tumor volume and tumor weight of the LV-si?6 group were significantly smaller than those of the LV-NC group.While ?6 overexpression promoted the growth of tumors and tumors in the LV-?6 group had a relatively larger volume and weight.2.The results of IHC showed that compared with the LV-NC group,?6 silencing markedly decreased the ?6 expression and MMP9 expression in the xenograft tumors,while ?6 overexpression significantly increased the ?6 expression and MMP9 expression.Conclusions and significanceIn this section,we further testified the results of in vitro experiments using the subcutaneous tumor model.Here we demonstrated that integrin ?6 not only promoted the migration and invasion of CCA cells in vitro,but also markedly promoted the tumor growth and MMP9 expression inside the tumor in vivo,indicating that tumor tissues that with high ?6 expression had stronger invasive capacity.All these results further confirmed the driving effect of ?6 on the invasiveness and metastasis of CCA.