Roles Of IRS-1 And Context Signaling Pathways In Pancreatic Cancer

Introduction:Pancreatic carcinoma is a highly lethal malignancy worldwide and has a very poor prognosis,with an overall 5-year survival rate less than 5% after the diagnosis.Resection remain primary treatment.However,only 10–20 % of patients are candidates for resection as approximately 50 % present with metastatic and 35 % with locally advanced surgically unresectable disease.Pancreatic cancer remains a therapeutic challenge,the cellular and molecular mechanisms of invasion/metastasis have not been elucidated clearly.However,protein post-translation modifications in pancreatic cancer cell lines,which may play essential roles in the regulation of cellular responses,have not been clearly demonstrated.It is therefore important to identify any phosphorylation events and to determine whole protein phosphorylation profiles of tumor cells.However,as far as we know,studies have not analyzed the phosphoproteome profiles of the two homogenous cell lines(PC-1 with a low,and PC-1.0 with a high potential of invasion and metastasis)in pancreatic cancer research.Our aim is to compare changes on 582 phosphorylation sites of 452 proteins between PC-1 and PC-1.0 cells by utilizing Phospho Explorer Antibody Array technology.In addition,the pathways and networks that related to phosphoproteins identified in our study show important variations in their components.The PI3K/PTEN/Akt/mTORC1 and Raf/MEK/ERK are key pathways activated in pancreatic cancer.Deregulation of these pathways can result in continuous cell growth,prevention of apoptosis and senescence and chemotherapeutic drug resistance.The MAPK signaling pathway is a highly conserved pathway that transfers extracellular signals to nuclear.The MAPK pathway triggers a genetic signaling cascade to the nucleus,resulting in regulation of cell proliferation,differentiation,apoptosis,gene expression and cellular response to the external environment.Targeting molecules in these pathways may be a therapeutic approach to treat pancreatic and other cancers.A assay focused on culture filtrate proteins different expression between PC-1 and PC-1.0 was carried out by LC-MS based on silac labeling.LAR,also named Protein Tyrosine Phosphatase,Receptor Type,F(PTPRF),was a factor screened out two fold high in PC-1 cells.Protein tyrosine phosphatase(PTP)are known to be signaling molecules that regulate a variety of cellular processes including cell growth,differentiation,mitotic cycle,and oncogenic transformation.In our previous study,protein phosphorylation level differences between PC-1.0 and PC-1 cells were examined using the phosphor explorer antibody array method.The ratio of PC-1 phosphorylation at Ser636 of IRS-1 in to PC-1.0 is 0.43.The result suggest that IRS-1 may play a significant role in signal pathways of pancreatic cancer.Here,we examined the role of IRS-1 in proliferation migration and invasion of PC cells in vitro and relationships of upstream and downstream.In order to determine the function of IRS-1 in pancreatic cancer cells,the expression level of the IRS-1 gene was downregulated using IRS-1-specific small interfering RNA(siRNA).Subsequently the proliferation,apoptosis,invasion and migration were analyzed.Additionally,the present study analyzed the association between these cell functions with the MAPK signaling pathway and PI3 K signaling pathway,in order to identify molecular mechanisms in the pathogenesis of pancreatic cancer.IRs-1 may serve as a useful predictor of the outcome of PC patients and could be a new target for clinical therapy.Material and Methods MaterialCell linesHamster pancreatic cancer cells PC-1 and PC-1.0;human pancreatic cancer cells Capan-2 and Aspc-1 were achieved from Kumamoto University as a gift.Methods1.Phospho-protein profiling by Phospho Explorer Antibody Array:Cell lysates obtained from PC-1 and PC-1.0 cells were applied to a Phospho Explorer Antibody Array.cell lysates were extracted with Antibody Array Assay Kit that contained a protease inhibitor cocktail and phosphatase inhibitor,and were performed according to the manufacturer’s protocol.The slides were scanned on a GenePix 4000 scanner and the images were analyzed with GenePix Pro 6.0.Protein phosphorylation data were confirmed by western blot.2.Western blot:total and phosphoralytion protein expression level of IRS-1,LAR,AKT,MEK1 and MEK2 were analyzed by western blot.3.Gene expression level of IRS-1 and LAR were preformed by real-time PCR.4.Cell morphology were observed under microscope in siIRS-1 cells.5.Invasion of siIRS-1 cells were identified by transwell assay.6.Metastasis of siIRS-1 cells were demonstrated by wound healing assay.7.Proliferation of siIRS-1 cells were indicated by CCK-8 assay.8.Statistical analysis and graphics were undertaken using SPSS 13.0.Results are presented as mean ± standard error of mean(SEM).Comparisons of quantitative data were analyzed by Student’s t test or ANOVA between two groups(two-tailed;P-values < 0.05 were considered to be statistically significant).Results1.Of the 1318 antibodies analyzed in microarray experiments,a total of 57 proteins showed differential expression using a fold ratio>2 as the cutoff criterion.Of these 57 proteins,the expression levels of 32 proteins were markedly increased in PC-1.0 as compared with PC-1 cells.2.To validate protein phosphorylation data,the expression levels of six randomly selected proteins from comparisons of PC-1.0 and PC-1 cells were re-examined by western blot.Western blots revealed that all six proteins showed increased phosphorylation in PC-1.0 cells consistent with our array data,thus confirming the latter’s reliability.3.A strong inhibition of migration was observed for FOS,IRS-1 and RAF1 using siRNA or antibody blocking,and regarding invasion,FOS,IRS-1 and RAF1 also caused a significant reduction in the invasion ability.4.Since the result of the phosphor explorer antibody array method were from hamster pancreatic cancer cell lines.First of all,Human pancreatic cancer cells Aspc-1 and Capan-2 which morphological characteristics and functions are similar to PC-1.0 and PC-1 are used to determine if the results from hamster are coincide with human pancreatic cancer cells.There is no significant difference among the viabilities of the cells.5.The IRS-1-knockdown PC-1.0 and Aspc-1cells grew in an aggregated or clumped pattern in culture meanwhile cellular pseudopod were attenuated or inexistent.6.Blocking IRS-1 in pancreatic cancer cells inhibited invasion ability.7.Blocking IRS-1 in pancreatic cancer cells inhibited metastasis ability.8.Blocking IRS-1 in pancreatic cancer cells inhibited proliferation ability.9.LAR negatively regulated total and phosphoralytion expression level of IRS-1.10.IRS-1 positively regulated MEK1,MEK2 and AKT.11.IRS-1 regulated invasion and metastasis of pancreatic cancer cells by targeting MEK1 and MEK2;IRS-1 regulated proliferation of pancreatic cancer cells by targeting MEK1 and AKT.Conclusion1.Identified differentially expressed signaling-associated phosphorylated proteins between highly(PC-1.0)and weakly(PC-1)invasive and metastatic cancer cells.2.There were no significant difference on expression level between the hamster and human pancreatic cancer cells.3.Blocking IRS-1 down regulated proliferation,invasion and metastasis of pancreatic cancer cells.4.IRS-1 positive regulated MEK1,MEK2 and AKT,but were negative induced by LAR.5.IRS-1 regulated proliferation,invasion and metastasis of pancreatic cancer cells by targeting MEK1,MEK2 and AKT.