The Effect And Mechanism Of FKBP51 On Cell Biological Behavior And Chemosensitivity Of AML-M5

BackgroundAcute myeloid leukemia(AML)is a highly heterogeneous malignant clonal hematological disorder that accounts for about 80%adult acute leukemia and represents the most frequent malignant myeloid disease in the adult population.It’s characterized by uncontrolled proliferation of immature,abnormal blast cells and impaired production of normal blood cells,the destruction of blood tissue and,therefore,to profound pancytopenia,severe bleeding,and infection.Among the AML subtypes,acute monocytic leukaemia(AML-M5)is a common subtype in adults.AML-M5 is characterized by a large number of myeloid derived monoblasts,promonocytes,and monocytes in the bone marrow and peripheral blood.Based on the relative proportion of monoblasts and promonocytes,AML-M5 is classified as M5a or M5b.Clinically,AML-M5 is manifested by hyperleukocytosis,extramedullary infiltration,and coagulation abnormalities and is considered to have a worse prognosis than other subtypes of AML.The etiology of AML is complex and not fully elucidated,among these are genetic factors,environmental factors and lifestyle,drugs,and antecedent blood disorders.However,in majority of cases,it appears as a de novo malignancy in previously healthy individuals.Regardless of its etiology,the pathogenesis of AML involves the abnormal proliferation and differentiation of a clonal population of myeloid stem cells.Some cell signaling pathways play an important role in AML survival,proliferation and self-renewal properties and are abnormally activated or suppressed.This includes the NF-κB,PI3K/AKT pathways and inhibitors of these pathways were regarded as a potential new anti-AML drugs.The standard treatment for AML is the "7+3" regimen of the DNA-targeting drugs cytarabine(Ara-C)and daunorubicin,which has remained essentially unchanged for almost 50 years.However,the progression and patient response to chemotherapy in AML-M5 vary greatly.One-third of AML patients do not achieve complete remission after standard chemotherapy,and even when complete remission is achieved,～70%of AML patients relapse within 5 years.The resistance of leukemic cells to chemotherapeutic drugs is one of the main reasons for treatment failure.Hence,there is an urgent demand to elucidate the mechanisms underlying drug resistance in AML and identify new biomarkers and therapeutic targets to enhance the chemotherapy efficacy.FK506 binding protein 51(FKBP51)is a 51-kDa protein that belongs to a family of immunophilins.Human FKBP5 is highly expressed in multiple tissues,including kidney,skeletal muscle,liver,placenta,heart,and peripheral blood.Structurally,FKBP51 contains two N-terminal FKBP like domains 1 and 2(FK1 and FK2)and C-terminal tetratricopeptide repeat(TPR)domains.The FK1 domain has peptidylprolyl cis-trans isomerization(PPIase)activity and immunosuppressant agent binding ability and is thus involved in immunoregulation,protein folding and trafficking.The FK2 domain seems to have only retained protein interaction ability.The C-terminal TPR domain binds to heat shock protein 90(Hsp90)and assembles with all steroid receptors.It regulates steroid hormone receptor signaling,including receptor maturation,hormone binding,and nuclear translocation.FKBP51 protein expression can be induced by steroid hormone(glucocorticoid,progesterone and androgen).Hormone response elements in the introns of FKBP5 regulate the induction of expression by glucocorticoids,progesterone,and androgens.In addition to participating in the regulation of steroid hormones responses and hormone receptor activities,increasing evidence indicates that FKBP51 plays a role in the abnormal cell growth of cancers,and it could be considered a promising new marker of tumor progression and response to radio/chemotherapy.Both overexpression and downregulation of FKBP5 have been observed in human cancers.According to the Oncomine,FKBP5 is overexpressed in prostate cancer,lymphoma,head and neck cancer,and endometrial adenocarcinoma.On the other hand,FKBP5 has also been shown to be downregulated in pancreatic cancer,melanoma,colon cancer,and testicular cancer.Similar to its expression pattern,FKBP5 has been shown to either promote or suppress tumor growth through its regulation of different signaling pathways in a specific tissue environment.In addition,FKBP51 has been shown to be involved in regulating the sensitivity of pancreatic,breast cancer cells and leukomonocyte to chemotherapeutic drugs.FK506 binding protein 5 has been shown to be involved in several different signaling pathways,including steroid receptor signaling pathways,NF-kB,and AKT-PHLPP pathways,all of which have important roles in tumorigenesis and response to antineoplastic chemotherapy.However,the role of FKBP51 in human haematological malignancies has not been understood until now.ObjectivesIn this study,we aimed to investigate the role of FKBP51 in the development of AML-M5 and cytarabine chemosensitivity,as well as the underlying molecular mechanisms.At the same time,the regulation of dexamethasone on the expression of FKBP51 in AML-M5 cells and its potential value in the treatment of AML-M5 were explored.It may provide a novel direction for diagnosis and clinical targeted treatment of AML-M5.Part Ⅰ.The effect and mechanism of FKBP51 on cell biological behavior in AMLM5Methods1.Bioinformatics analysis The association between the level of FKBP51 mRNA expression and overall survival time of AML-M5 patients was detected by TARGET database and KaplanMeier plotter method.2.Detection of FKBP51 protein expression in peripheral blood monocyte of AML-M5 patients The westernblot was employed to detect the FKBP51 protein expression in monocytes of 4 specimens of AML-M5 patients without any radiotherapy and chemotherapy and 4 specimens of normal monocytes.3.Establishment of FKBP51 overexpression and knockdown cell lines Two AML-M5 cell lines,THP-1,U937,were selected for their relatively high and low FKBP51 expression.The constructed FKBP51 overexpression lentivirus was used to infect THP-1 cells and to establish FKBP51 up-regulated cell models.The constructed shRNA-FKBP51 plasmids were transfected into U937 cells to establish cell lines that stably knock-down FKBP51 expression.4.Detection of biological behavior of AML-M5 cells The effects of FKBP51 on cells proliferation,cycle,apoptosis,migration and invasion of THP-1 and U937 were detected by CCK-8 proliferation assay,cell cycle analysis,apoptosis assay,transwell migration and invasion assay.5.NOD/SCID mice subcutaneous tumor formation assays The model of AML-M5 xenograft tumor was established by subcutaneously injecting OE-FKBP51 cells or NC-OE cells in NOD/SCID mice to detect the effect of FKBP51 on the proliferation of AML-M5 cells in vivo.6.Detection of AKT pathway molecules in AML-M5 cells The total AKT,p-AKT(Ser473)and AKT downstream target proteins FOXO1,GSK3β,P21,P27,BCL-2 and BAX were detected by Western blot after up-regulation or down-regulation of FKBP51 in AML-M5 cells.At the same time,the effect of FKBP51 inhibitor SAFit2 on AKT and p-AKT(Ser473)levels was detected to explore the effect of FKBP51 on AKT pathway and activity of AML-M5 cells.7.Detection of the role of AKT in the proliferation of AML-M5 cells Different concentrations of AKT1/2 kinase inhibitor A6730 were added to NC-sh and sh-FKBP51 cells,then cells proliferation,cell cycle and AKT pathway molecules were detected.8.The effect of AKT on the expression of FKBP51 in AML-M5 cells Western blot was used to detect the changes of FKBP51 protein expression in U937 cells treated with different concentrations of AKT inhibitor A6730 for 48 hours.9.Detection of NF-κB pathway activity Western blot was used to detect the protein levels of IκB,p-IKK,IKK and P65 in the cytoplasm and nucleus of cell lines,meanwhile,dual luciferase assay was used to detect the effect of FKBP51 on the transcriptional activity of NFκB.10.Detection of invasion-related protein Western blot was used to detect the changes of migration-related proteins MMP2,MMP9 and Timp1 after up-regulation or down-regulation of FKBP51.In addition,the protein level of MMP9 in sh-FKBP51 cells and control cells was detected after cultured with NF-κB inhibitor BAY 11-7082.Results1.The results of Bioinformatics analysis Kaplan-Meier curves showed that patients with low FKBP51 mRNA expression had a worse overall survival(OS)than those with high FKBP51 expression according to TARGET database.2.Decreased expression of FKBP51 protein in peripheral blood monocytes in patients with AML-M5 The expression of FKBP51 protein in peripheral blood monocytes of AMLM5 patients was significantly decreased compared with health donors.3.Establishment of cell lines with FKBP51 over expression or knockdown The baseline expression levels were determined by western blot,FKBP51 protein levels were markedly higher in the U937 cells and lower in THP-1 cells.After infection with lentivirus,the FKBP51 up-regulated cell model of THP-1 cells was successfully constructed;the FKB51 downregulated U937 cell model was successfully constructed by shRNA plasmid transfection.2.FKBP51 inhibits malignant behavior of AML-M5 cell line In vitro,when THP-1 cell line overexpressed FKBP51,the cell cycle process was blocked,the proliferation,invasion and migration abilities of cells were decreased,and the apoptosis ability was enhanced.On the contrary,When FKBP51 knocked down in U937 cells,the cell cycle progression was accelerated,the proliferation,invasion and migration abilities of the cells were significantly enhanced,and the apoptosis ability was reduced.At the same time,the results of in vivo xenograft tumor experiments showed that compared with mice inoculated with control cells,the tumor volume and tumor weight of mice inoculated with FKBP51 overexpressing cells were significantly reduced.It is suggested that the up-regulation of FKBP51 expression can inhibit the proliferation ability of AML-M5 cells in vivo.3.FKBP51 inhibits the activity of AKT pathway in AML-M5 cells Western blot results showed that the phosphorylation of AKT on Ser473 was decreased in FKBP51 overexpressed THP-1 cells compared with that in negative control cells,and there was impaired phosphorylation of GSK3β on serine 9 and FOXO1A on serine 256,decreased BCL-2 expression and elevated P21 and P27 expression,which are related to cell cycle progression,cell proliferation and apoptosis.The opposite results were observed in FKBP51-knockdown U937 cells.In addition,p-AKT(Ser473)and total AKT protein expression could be downregulated by the FKBP51 inhibitor SAFit2.These results implied that FKBP51 negatively regulates p-AKT(Ser473)and its downstream target proteins.4.The proliferation of AML-M5 cells is regulated by AKT Different concentrations of AKT inhibitor A6730 were added to U937 sh-FKBP51 and control cells NC-sh,and it was found that A6730 caused cell cycle arrest and inhibited cell proliferation.Furthermore,we found that the pro-proliferative ability caused by down-regulation of FKBP51 was abolished by A6730 at low concentrations.This indicates that AKT pathway plays an important role in the proliferation of AML-M5 cells,and FKBP51 regulates cell proliferation through AKT pathway.5.AKT pathway positively regulates the expression of FKBP51 Western blot results showed that the protein level of FKBP51 decreased in a dose-dependent manner after U937 cells were treated with AKT inhibitor A6730,indicating that AKT pathway can positively regulate the expression of FKBP51.6.FKBP51 inhibits the activation of NF-κB pathway Western blot results showed that after up-regulation of FKBP51,IκB increased,the ratio of p-IKK/IKK and nuclear P65 decreased.In contrast,down-regulation of FKBP51 decreased IκB levels,increased p-IKK/IKK ratio and nuclear P65 levels.The results of the dual luciferase assay showed that compared with the control group,the up-regulation of FKBP51 inhibited the transcriptional activity of NF-κB,and the down-regulation of FKBP51 enhanced the transcriptional activity of NF-κB,whether at the basal level or stimulated by TNF-α.It indicated that FKBP51 inhibited the activation of NF-κB pathway in AML-M5 cells.7.FKBP51 inhibits the invasion-related genes by inhibiting the NF-κB pathway Compared with THP-NC cells,the levels of invasion-related proteins MMP2 and MMP9 in OE-FKBP51 cells decreased,while the level of tissue inhibitor of metalloproteinase 1(Timpl)increased.In contrast,down-regulation of FKBP51 increased the expression of MMP2,MMP9,and decreased Timp1 levels.After administration of NF-κB inhibitor BAY 11-7082,the MMP9 levels of sh-FKBP51 and control cells were significantly decreased.The above shows that FKBP51 can inhibit the level of invasion-related proteins by inhibiting the NF-κB pathway.Summary1.FKBP51 expression decreased in monocytes of AML M5 patients,and the survival time of patients with low expression of FKBP51 was shortened.2.FKBP51 inhibits the proliferation,invasion and migration of AML-M5 cell lines(THP-1 and U937)and promotes the apoptosis of tumor cells.3.FKBP51 inhibits the activity of AKT pathway by inhibiting AKT(Ser473)phosphorylation,induces cell cycle arrest and inhibits AML-M5 cell lines(THP-1 and U937)proliferation.4.FKBP51 inhibits AML-M5 cells lines(THP-1 and U937)invasion and migration by inhibiting the NF-κB pathway.Part Ⅱ.The regulation of dexamethasone on FKBP51 in AML-M5 cells and its effect on the sensitivity of cytarabineMethods1.The effect of dexamethasone on the expression of FKBP51 in AML-M5 cells THP-1 and U937 cells were incubated with different concentrations of dexamethasone(Dex),the protein expression level of FKBP51 was detected by Western blot,and the mRNA level of FKBP51 was detected by qPCR.2.The effect of FKBP51 on the chemosensitivity of AML-M5 cells with cytarabine Two groups of AML-M5 cell lines with FKBP51 stable overexpression or knockdown were cultured with different concentrations of cytarabine(Ara-C).The cell viability was detected by CCK-8 assay and the apoptosis ratio was detected by flow cytometry.3.The effect of dexamethasone on the proliferation of AML-M5 cells AML-M5 cells cultured with different concentrations of Dex,and CCK-8 assay was used to detect the effect of Dex on cells proliferation.4.Effects of dexamethasone on Ara-C sensitivity of AML-M5 cells in vitro Dex and different concentrations of Ara-C were added to two groups of AML-M5 cell lines with FKBP51 stably overexpressed or knocked down,and the cell viability was detected by CCK-8.5.Mechanism of Dex enhancing AML-M5 cell sensitivity to Ara-C The changes of FKBP51 levels in OE-FKBP51/THP-NC and sh-FKBP51/NC-sh cell lines after adding dexamethasone were detected by Western blot and qPCR.6.The effect of dexamethasone on the cytotoxicity of Ara-C in vivo The first step was the establishment of AML mice xenograft models,then all mice with successful tumor-forming were divided into four groups including the vehicle group,Ara-C group,Dex group and AraC/Dex combination group.When tumors had a tactile sensation,each group began to receive medication using intraperitoneal injection.After 10 days of drug treatment,the study was terminated.7.The effect of dexamethasone on the migration and invasion of AML-M5 cells After incubation of AML-M5 cells with Dex,the migration and invasion abilities of the cells were detected by transwell migration and invasion assays.Results1.Dexamethasone up-regulated the expression of FKBP51 in AML-M5 cells The discovery and research motivation of FKBP51 are closely related to the research on steroid hormones.Previous studies have shown that FKBP51 upregulation can inhibit the proliferation,invasion and migration of monocytic leukemia cells.Here,we explored the regulation of FKBP51 in monocytic leukemia cells by dexamethasone to explore its clinical application value.Western blot and qPCR results showed that Dex upregulated the expression of FKBP51 in a dose-dependent manner,and reached a peak at 1μM and this concentration was selected for subsequent functional experiments.2.Dexamethasone inhibits the proliferation of AML-M5 cells The results of CCK-8 showed that the AML-M5 cells was not sensitive to Dex,and Dex itself only slightly inhibited cell proliferation.3.FKBP51 enhances the sensitivity of AML-M5 cells to Ara-C The results showed that the survival rate and the half inhibitory concentration(IC50)of OE-FKBP51 cells decreased significantly compared with NC-OE cells,and the proportion of apoptosis increased.On the contrary,sh-FKBP51 cell viability and IC50 significantly increased while apoptosis decreased.This indicates that FKBP51 enhances the sensitivity of Ara-C,and the down-regulation of FKBP51 makes AML-M5 resistant to Ara-C.4.Dexamethasone sensitizes AML-M5 cells to Ara-C by upregulating FKBP51 The CCK-8 results showed that compared with the solvent control group,the cell survival rate and IC50 of THP-NC and OE-FKBP51 groups were significantly reduced.It indicates that Dex enhances the efficacy of Ara-C in vitro.This phenomenon also appeared in the NC-sh group,but the sensitization effect of DEX on Ara-C disappeared in the sh-FKBP51 cell line.Western blot results showed that the protein and mRNA levels of FKBP51 in THP-1 cells were upregulated by Dex in both the control group and the overexpression group,which was consistent with the up-regulation of Ara-C chemotherapy sensitivity by Dex.In U937 cells,the level of FKBP51 in the control group was up-regulated by Dex,but the up-regulation of FKBP51 on protein and mRNA disappeared after knockout of FKBP51.This proves from both positive and negative aspects that Dex sensitizes AML-M5 cells to Ara-C by upregulating FKBP51.5.Dexamethasone enhances AML-M5 cells sensitivity to Ara-C in vivo The NOD/SCID mice xenograft models showed that Ara-C alone could inhibit the growth of tumors,while the combined application of Dex significantly enhanced the effect of Ara-C,and the tumor volume and weight were further reduced.At the same time,Dex alone also showed antitumor effect in vivo.6.Dexamethasone inhibits invasion and migration of AML-M5 cellsThe results of transwell migration and invasion assay showed that Dex inhibited the migration and invasion of THP-1 and U937 cells.Summary1.Dexamethasone sensitizes AML-M5 cells to Ara-C by upregulating FKBP51.2.Dexamethasone inhibits the migration and invasion of AML-M5 cells.ConclusionsIn this study,bioinformatics analysis,clinical sample study,cell model and NOD/SCID mouse xenograft model were used to explore the effects of FKBP51 on the biological behavior and cytarabine sensitivity of AML-M5 and its molecular mechanism.The summary is as follows:1.FKBP51 is low-expression in monocyte cells in AML-M5 patients and is positively correlated with the overall survival of patients.It may become an indicator for judging prognosis of AMLM5 patients.2.FKBP51 inhibits the proliferation,migration and invasion of monocytic leukemia cells in vivo and in vitro,and plays a significant anti-tumor effect in AML-M5.3.Dexamethasone sensitizes acute monocytic leukemia cells to Ara-C by upregulating FKBP51.