The Function And Regulation Of FAK In Pancreatic Cancer Malignant Phenotypes And Associated Molecular Mechanisms

BackgroundPancreatic cancer(PC)is a refractory disease that has difficulty with early diagnosis,and is not sensitive to existing treatments.It is one of the solid tumors with the highest mortality-to-incidence ratio,and is known for its poor prognosis,with an overall 5-year survival rate of only 9%.In recent years,the incidence and mortality of PC have increased drastically in China,which has become a serious health problem in our country.It is generally known that PC is prone to invasion and metastasis,and local vascular invasion and distant metastasis indicate a poor prognosis.Therefore,lucubrating the molecular principles underlying the invasion and metastasis of PC might help optimize treatment targets and further improve patient survival.Focal adhesion kinase(FAK)is a non-receptor tyrosine kinase with sophisticated functions.Extensive studies found FAK as a key regulator in cancer progression,especially in the invasion and metastasis of cancer cells.However,the relevant research on the molecular mechanisms of FAK regulating the invasion and metastasis of PC is still insufficient.Literature research showed that FAK promoted malignant phenotypes,including tumor invasion and metastasis,via regulating the mammalian target of rapamycin(mTOR)signaling pathway and metabolic reprogramming in several types of cancers,which has not been investigated in PC.Therefore,this study aims to conduct in-depth research on the above issues in order to optimize PC treatment targets and provide a potentially effective way to improve the prognosis of patients with PC.ObjectiveThe purpose of this study is to explore the role of FAK in regulating the mTOR signaling pathway and its impact on the proliferation,invasion and metastasis of PC,to investigate the influence of FAK on the metabolic reprogramming and its effect on invasion and metastasis of PC,and to evaluate the prognostic value of FAK in patients with PC.MethodsThe expression of FAK in PC cell lines and normal pancreatic ductal epithelial cell line was investigated using quantitative real-time PCR(qRT-PCR)and western blot.The effect of FAK on the proliferation,migration and invasion of PC cells in vitro and its impact on the mTOR signaling pathway were assessed after FAK transiently knockdown.Using lentiviral vectors transfection,FAK knockdown,overexpression and corresponding control stable transfected PC cell lines were screened and established.The effect of FAK on the proliferation of PC cells was evaluated using CCK-8 assays,colony formation assays and subcutaneous xenograft models in nude mice.The effect of FAK on the migration and invasion of PC cells was assessed using transwell assays and wound healing assays.The regulation of FAK on the mTOR signaling pathway was verified using stable transfected PC cell lines,and rescue experiment was performed to evaluate the effect of FAK on the malignant phenotypes of PC cells via the mTOR signaling pathway.The impact of FAK on the sensitivity of mTOR inhibitor in PC was investigated using cytotoxicity assays.The possible effect of FAK on the metabolic reprogramming of PC was investigated using transcriptome sequencing and non-targeted metabolomics.The expression of the key enzymes in the related metabolic pathways were assessed,which was verified using rescue experiment.Using immunohistochemical methods,the expression patterns of FAK in PC tissues and adjacent normal tissues were evaluated,which were verified via ONCOMINE and Gene Expression Profiling Interactive Analysis(GEPIA),including its relationships with focal adhesion regulating proteins.The relationships between FAK expression and the clinicopathological parameters,as well as the prognosis of patients with PC were analyzed.The statistical methods applied in this study included Student's t test,Mann-Whitney U test,Pearson chi-square test,Fisher exact test,Kaplan-Meier survival analysis,and Cox regression analysis.P<0.05 is considered a significant difference.Results1.The effect of FAK on the malignant phenotypes of PC and the regulation of mTOR signaling pathwayThe expression level of FAK in the majority of the PC cell lines was higher than that in the normal pancreatic ductal epithelial cell line.Transiently knock down FAK significantly inhibited the proliferation,migration and invasion of AsPC-1 and PANC-1 cell lines.The above results were verified in FAK knockdown and overexpression stable transfected PC cell lines,that is,FAK knockdown inhibited proliferation,colony formation,migration,invasion and wound healing of AsPC-1 and PANC-1 cell lines,whereas overexpression of FAK had the opposite effects.2.The molecular mechanism of FAK-mTOR pathway regulating the invasion and migration of PCTransiently knock down FAK downregulated the phosphorylation levels of AKT and mTOR compared with the control group.These results were verified in FAK knockdown and overexpression stable transfected PC cell lines,that is,FAK knockdown downregulated the phosphorylation of AKT,mTOR and its downstream molecules,whereas overexpression of FAK had the opposite effects.GSK2256098,a FAK inhibitor,also inhibited the phosphorylation of AKT and mTOR signaling pathway,and the malignant phenotypes of PC cells.The rescue experiments showed that rapamycin,a mTOR complex inhibitor,inhibited the phosphorylation of p70S6K,S6 and eIF 4EBP1,and partially overcame the effects of FAK promoting proliferation,migration and invasion of PC cells.In addition,FAK inhibition increased the sensitivity of mTOR inhibitor in PC.Transcriptome sequencing indicated that FAK might regulate the metabolic reprogramming of PC,and metabolomics analyses showed that FAK might have potential impact on the choline metabolism of PC.Further studies revealed that FAK promoted the migration and invasion of PC cells by upregulating CHKa,a key enzyme in the choline metabolic pathway.In addition,FAK was found to be significantly associated with TIAM2,a focal adhesion regulator,in PC.3.The prognostic value of FAK in patients with PCThe results of immunohistochemical staining of clinical tissue samples found that the expression of FAK in PC tissues was significantly higher than that in adjacent normal tissues,and high expression of FAK was significantly associated with the pathological N stage.In addition,low FAK expression was significantly associated with longer cancer-specific survival,and FAK expression was found to be prognostic in 7 subgroups of patients with PC.Furthermore,the independent prognostic value of FAK was established in multivariate analyses.ConclusionFAK promoted the proliferation,migration and invasion of PC cells via activating the mTOR signaling pathway.FAK regulated the choline metabolism of PC,and promoted the invasion and metastasis of PC through upregulating the expression of CHKa.FAK might be applied as a prognostic indicator,and combined targeting FAK and mTOR signaling pathway might serve as a potential therapeutic strategy for patients with PC.