| Objective: In the world,pancreatic cancer is a highly fatal malignancy with a poor prognosis and an overall 5-year survival rate that is less than 5% after diagnosis.Surgical resection is still the main treatment for pancreatic cancer.However,only10-20% of the patients can have surgery to remove the tumor,about 50% of the patients suffer from distant metastasis,and about 35% of the patients have local invasion(unresectable).Although the medical field is still dedicated to the study of the formation and evolution of pancreatic cancer and its invasion process,the clinical efficacy of pancreatic cancer is not optimistic,and the specific molecular biological mechanism of invasion and metastasis of pancreatic cancer remains still to be clarified.Integrin α6β4 is a kind of cell adhesion molecule,which combines with laminin in extracellular matrix,thus forming hemidesmosomes.In many human malignant tumors,the overexpression of integrin α6β4 is related to tumor invasion and poor prognosis.In the process of tumor development,integrin α6β4 is released from hemidesmosomes to promote tumor development,including maintenance of proliferation signals,invasion and metastasis,avoidance of apoptosis,and stimulation of angiogenesis.Integrins amplify downstream signal transduction pathways,such as PI3 K,AKT,MAPK and small GTPs that belong to Rho family by cooperating with growth factor receptors such as EGFR,ErbB-2 and c-Met.In addition,it regulates DNA demethylation that occurs on promoters of proteins related to invasion and metastasis,such as S100A4 and autotaxin,and upregulates and activates key tumor promoting transcription factors,such as NFAT and NF-κB.It is well known that the specific phosphorylation of protein sites plays an important role in regulating cell function.Therefore,it is necessary to make clear the phosphorylation function of tumor cells and draw protein phosphorylation profile.It is reported that there is no report on homologous cell lines(PC-1.0,PC-1)and their protein phosphorylation profiles in the field of pancreatic cancer.In this study,proteins with different expression levels in PC-1.0 and PC-1 cells were screened by phosphorylation protein chip technology,and 582 phosphorylation sites on 452 proteins were compared reasonably.The phosphorylation rate of tyrosine1510(Tyr1510)in integrin β4(ITGB4)of PC-1.0 cells was higher than that in PC-1cells.However,the phosphorylation sites of ITGB4 are usually concentrated on Threonine 1356,1360 and 1364,which can promote the release of integrin α6β4 from hemidesmosomes(HDs)and promote tumor progression.Moreover,the down-regulation of ITGB4 can inhibit the metastasis of pancreatic cancer cells.In addition,ITGB4 inhibits MAPK pathway by targeting p-MEK-1(Thr292)and ERK1/2in pancreatic cancer cells.Moreover,a mechanism study has shown that the bidirectional effects of MAPK and TGF-β pathway contribute to the invasion of pancreatic cancer cells induced by ITGB4.Using The Human Protein Atlas Database,we found that patients with lower ITGB4 mRNA levels had higher overall survival rates.In general,this study showed that the site-specific phosphorylation of ITG4 on Tyr1510 can promote the invasion and metastasis of pancreatic cancer,and provide a treatment strategy for ITGB4 related cancer.Methods: Hamster pancreatic cancer cell lines PC-1 and PC-1.0 and human pancreatic cancer cell lines Aspc-1 and Capan-2 were used.The pyrolysis products of PC-1 and PC-1.0 cells were determined by probe antibody chips(Full Moon Biosystems,Sunnyvale,CA,USA).The extracted cell lysates(containing different proteases and phosphatase inhibitors)were analyzed by using the antibody array tool.The operation was performed according to the requirements and instructions of the manufacturer.GenePix 4000 scanner was used to detect and analyze the chip,and GenePix Pro6.0(Molecular Devices,Sunnyvale,CA,USA)was used to process the image.The chip results were verified by Western blot.Western blot and PCR were used to detect the gene expression of ITGB4 in PC-1.0 and Aspc-1 before and after siRNA transient transfection.Transwell and Wound Healing experiments were used to detect the changes of invasion and migration ability of PC-1.0 and Aspc-1 before and after ITGB4-siRNA transient transfection.The morphological changes of PC-1.0 and Aspc-1cells before and after transient transfection with ITGB4-siRNA were observed under microscope.Western blot was used to detect the expression of MEK1,MEK2,p-MEK1(Thr292),p-MEK1(Thr386)and p-MEK2(Thr394),ERK1/2,p-ERK1/2.Results: 1.1318 targets were analyzed by using phosphorylated protein chip,and the difference of 2-fold expression was taken as the inclusion standard.A total of 57 proteins were selected,and the expression level of 32 proteins in PC-1.0 was significantly higher than that of PC-1,while the expression level of the remaining 25 proteins was relatively lower.2.In order to test the final phosphorylation result of the protein chip,we selected six kinds of protein and detected the actual expression level by Western blot.The results showed that the expression of 6 kinds of proteins in PC-1.0cells increased significantly,which was consistent with the results of the chip.ITGB4 was hyperphosphorylated at Tyr1510 in pancreatic cancer(PC-1.0 and Aspc-1)cells with high invasion and metastasis.3.After transfection with si-ITGB4,the phosphorylation level of ITGB4 Tyr1510 site in highly invasive and metastatic pancreatic cancer cells(PC-1.0 and Aspc-1)was down-regulated.4.The migration and invasion of highly invasive and metastatic pancreatic cancer cells(PC-1.0 and Aspc-1)transfected with ITGB4-siRNA were inhibited.5.PC-1.0 and Aspc-1 cells with ITGB4 knockdown grew in the way of aggregation or clustering in the culture dish,while the pseudopods of cells were weakened or disappeared.6.In PC-1.0 and Aspc-1 cells,the expression of p-MEK-1(Thr292)and ERK1/2 was significantly lower than that in the control group when ITGB4 was knocked down(P < 0.05).Conclusion: 1.The Tyr1510 site of ITGB4 is hyperphosphorylated in highly aggressive metastatic cell lines(PC-1.0 and Aspc-1).2.Inhibition the phosphorylation of ITGB4 Tyr1510 expression can down-regulate the invasion and metastasis of pancreatic cancer cells.3.ITGB4 inhibits MAPK pathway by targeting p-MEK-1(Thr292)and ERK1/2 in pancreatic cancer cells. |
