USP49-FKBP5-AKT Signaling Pathway And The Mechanism Of Pancreatic Carcinoma Drug Resistance

Objective: Serine/threonine protein kinase AKT(also known as PKB),the key regulation factor of cell growth,takes part in a variety of cell signaling pathway and has extensive biological effect.Hyperphosphorylation of AKT not only causes the cell malignant transformation,but plays an important role in tumor cell migration,adhesion,angiogenesis and the degradation of extracellular matrix.In addition,it's superactivation will lead to tumorigenesis and chemotherapy tolerance.Previous research has shown that PHLPP negatively regulates AKT through direct dephosphorylation of Ser473.FKBP5,as a scaffolding protein,could enhance PHLPP-AKT interaction and facilitate PHLPP-mediated dephosphorylation of Akt Ser473,negatively regulating Akt activation.Therefore,FKBP5 plays an important role in inhibiting tumorigenesis through negatively regulating Akt activation.However,the regulation of FKBP5-PHLPP-AKT pathway remains unclear.Here,we mainly look for FKBP5 regulation protein and study the regulation mechanism of FKBP5,besides,The question how the protein regulates tumor cells growth and chemotherapytolerance through the FKBP5-PHLPP-AKT pathway will be also explored.Methods :(1)We look for FKBP5 specific binding proteins by using the tandem affinity purification and mass spectrometry(TAP-MS)assay.(2)Gene transduction and sh RNA technology to overexpress or knockdown FKBP5 specific binding protein in pancreatic cancer cells,the following to detect the FKBP5 protein level by western blot.(3)Using ub assay to explore how FKBP5 binding protein effects FKBP5 ubiquitin level in vivo or vitro.(4)Using western blot to detect the expression of phosphorylation of AKT and phosphorylation of downsream protein of AKT in pancreatic cancer cells stably expressing indicated sh RNA.(5)Using MTT assay and soft agar assay to study the mechanism how the binding protein regulats cell proliferation.(6)Constructing animal model to validate the effect of the binding protein on tumor growth.(7)Using Immunohistochemistry assay to detect the expression of FKBP5,P473-AKT and the binding protein in tissue microarray.(8)To test how USP49 regulates pancreatic cancer chemoresponse through AKT pathway.Results:(1)Using tandem affinity purification and mass spectrometry(TAP-MS)assay,We identified deubiquitination enzyme USP49 as a interaction protein of FKBP5.(2)Knockdown of USP49 downregulated FKBP5 level and decreased FKBP5 stability.(3)Downregulation of USP49 increased FKBP5 ubiquitination and overexpression of USP49 increased FKBP5 ubiquitination.(4)Downregulation of USP49 reduced the phosphorylation of AKT on Serine 473 and the phosphorylation of downstream substrates of Akt.but their phosphorylation level has no further decline followed by together with downregulation of FKBP5.(5)Downregulation of USP49 markedly increased pancreatic cancer cell proliferation,while USP49 knockdown had no apparent effect on proliferation in cells depleted of FKBP51.(6)In vivo,Downregulation of USP49 induced the growth of tumor,but restoring FKBP51 expression reversed the tumor-promoting effect of USP49 sh RNA.(7)Immunohistochemical result showd that USP49 is downregulated in pancreatic cancers,which is positively correlated with FKBP51 expression and negatively correlated with AKT phosphorylation on Ser473.(8)USP49 enhanced pancreatic cancer cell responsiveness to gemcitabine in a AKT dependent manner.Conclusion: Here we showed a deubiquitinase,USP49 is a new regulator of the AKT pathway.Mechanically,USP49 deubiquitinates and stabilizes FKBP51,which in turn enhancing PHLPP'scapability to dephosphorylate of Akt.Furthermore,USP49 inhibited pancreatic cancerproliferation and enhanced pancreatic cancer cell responsiveness to gemcitabine in a FKBP51-AKT dependent manner.Clinically,we found that USP49 downregulation in pancreatic cancer patients was associated with decreased FKBP51 expression and increased AKT phosphorylation.Overall,our findings establish USP49 as a novel regulator of AKT pathway with a critical role in tumorigenesis and chemo response in pancreatic cancer.