The Effect And Mechanism Of FKBP4 In Regulating Lung Adenocarcinoma Progression

Background Lung cancer is the most common cause of cancer-related mortality worldwide.Approximately 85%of diagnosed cases are classified as non-small cell lung cancer(NSCLC),with lung adenocarcinoma(LUAD)being the predominant histological subtype.Despite recent advances in treatments such as platinum therapy and radiotherapy,the clinical outcomes of LUAD patients remain unsatisfactory due to late diagnosis and high rates of metastasis.Therefore,deciphering gene alterations and mechanisms underlying the initiation and progression of LUAD are beneficial to discovering novel diagnostic biomarkers and potential therapeutic targets.Activation of nuclear factor-?B(NF-?B)signaling has been shown to contribute to tumorigenesis and development in various cancers.In resting cells,NF-?B heterodimers reside in the cytoplasm due to the interaction with the inhibitory factor I?B.The canonical pathway is normally triggered tumor necrosis factor-?(TNF-?)and interleukin-1(IL-1)that activate the tripartite I?B kinase complex(IKK complex;IKK?/IKK?/IKK?).Under certain stimuli,activated IKK complex directly phosphorylates I?B? at Ser32/36 and induces its degradation via the proteasome.Subsequently,the liberated NF-?B heterodimers,consisting predominantly of RelA(p65)and NF-?B1(p50)subunits,translocate into the nucleus and mediate the transcription of target genes.FK506-binding proteins(FKBPs)belong to the family of immunophilins,which are structurally characterized by the existence of peptidyl prolyl isomerase(PPIase)domains.In addition to their well-established role in immunosuppression,FKBPs are involved in numerous cellular processes,such as protein trafficking,transcriptional regulation,protein folding,and signal transduction.The PPIase domains are located at the N-terminal end of FKBPs and consist of FKBP 12-like domains 1 and 2(FK1 and FK2).Only the FK1 domain confers PPIase enzymatic activity that can be inhibited by the immunosuppressants FK506 and rapamycin,while the FK2 domain seems to play a structural role.High-molecular weight immunophilins,such as FKBP4 and FKBP5,possess C-terminal tetratricopeptide repeat(TPR)domains,which are able to form complexes with the molecular chaperone Hsp90 and participate in protein-protein interactions.As a co-chaperone,FKBP4 modulates the activity of steroid receptors(SRs),including glucocorticoid(GR),mineralocorticoid(MR),androgen(AR),progesterone receptors(PR),as well as estrogen receptor(ER).This is particularly important for the development of hormone-dependent breast cancer and prostate cancer.Notably,FKBP4 interacts with RelA and is recruited to the promoter region of NF-?B target genes in 293T cells.ObjectivesIn this study,we characterized the biological role and regulatory mechanism of FKBP4 overexpression in LUAD progression through bioinformatics analysis,clinical LUAD specimens,LUAD in vitro and in vivo cellular models,which may provided a novel therapeutic target for LUAD managing LUAD patients.CHAPTER 1.Expression and prognostic value of FKBP4 in LUADMethods1.Bioinformatics analysis The differential mRNA expression of FKBP4 in LUAD tissues and normal tissues were performed using TCGA and Oncomine database.The relationship between FKBP4 mRNA expression and clinical characteristics was determined according to TCGA database.The association between the level of FKBP4 mRNA expression and prognosis of LUAD patients were detected by TCGA database and Kaplan-Meier plotter online software.2.Immunohistochemistry(IHC)The immunohistochemistry staining was employed to detect the FKBP4 protein expression in 94 specimens of LUAD tissues and 86 specimens of normal tissues.The relationship between FKBP4 protein expression and clinical characteristics/overall survival time were also detected.Results1.The results of Bioinformatics analysis The results showed that FKBP4 mRNA expression was significantly increased in LUAD tissues compared with normal tissues form TCGA and Oncomine database.Moreover,we found that the FKBP4 mRNA expression levels were notably associated with gender,tumor size and clinical TNM stage according to TCGA database.KaplanMeier curves showed that patients with high FKBP4 mRNA expression had a worse overall survival(OS)than those with low FKBP4 expression according to TCGA database and Kaplan-Meier plotter online software.2.The results of immunohistochemistry staining The results showed that FKBP4 protein expression was significantly increased in LUAD tissues compared with normal tissues.FKBP4 expression was positively correlated with the pathological grade and TNM stage.Kaplan-Meier analysis further confirmed that high expression of FKBP4 shortened the overall survival time of LUAD patients.Conclusions FKBP4 expression was upregulation in LUAD tissues,and the upregulation was correlated with poor prognosis.Therefore,FKBP4 might serve as a biomarker of poor prognosis for LUAD patients.CHAPTER 2.The effect of FKBP4 on malignant behaviors in LUADMethods1.Cell transfection We used specific siRNAs to knock down the expression of FKBP4 in PC9 and H1975 cell lines transiently.FKBP4 overexpression plasmid was transiently into BEAS-2B cells.PC9 and H1975 cells stably transfected with FKBP4targeting shRNA.2.Western blot assays Western blot were employed to detect the expression of FKBP4.Furthermore,we determined the expression of cell cycle related proteins(CyclinD1,CDK6),invasion related proteins(MMP9)and proliferation related proteins(c-Myc)using western blot.3.CCK-8 and colony formation assays The effect of FKBP4 on cell proliferation was detected by CCK-8 and colony formation assays.4.Cell cycle assays The effect of FKBP4 on cell cycle was detected by cell cycle assays.5.Transwell migration and invasion assays The effect of FKBP4 on cell migration and invasion were detected by transwell migration and invasion assays.6.Nude mice subcutaneous tumor formation assays The effect of FKBP4 on tumor growth in vivo was detected by subcutaneous tumor formation assays.7.Nude mice tail vein injection assays The effect of FKBP4 on tumor metastasis in vivo was detected by tail vein injection assays.Results1.FKBP4 downregulation inhibits the proliferation,migration and invasion of LUAD cells in vitro CCK-8 and colony formation assays showed that the proliferation and colonyformation capabilities were significantly decreased in FKBP4-inhibited cells compared with their control counterparts.Cell cycle assays showed that the percentage of FKBP4depleted cells was significantly increased in G0/G1 phases coupled with reduction in G2/M phases.These results demonstrated that FKBP4 inhibition impaired cell proliferation by regulating the cell cycle in LUAD.Moreover,FKBP4 depletion abrogated the migratory and invasive phenotypes of LUAD cells.2.FKBP4 upregulation promotes the proliferation,migration and invasion of LUAD cells in vitro CCK-8 and colony formation assays showed that the proliferation and colonyformation capabilities were significantly increased in FKBP4-overexpression cells compared with their control counterparts.The percentage of FKBP4-overexpression cells was significantly decreased in G0/G1 phases coupled with increased in G2/M phases.These results demonstrated that FKBP4 overexpression promotes cell proliferation by regulating the cell cycle in LUAD.Moreover,FKBP4 upregulation contributed to the migratory and invasive cells phenotypes.3.FKBP4 knockdown inhibits the proliferation and metastasis of LUAD cells in vivo Nude mice subcutaneous tumor formation assays showed that the volumes and weights of tumors excised from the FKBP4-knockdown group were strikingly reduced compared with those excised from the control group.Nude mice tail vein injection assays showed that the mice injected with FKBP4-inhibited cells developed fewer lung metastatic nodules compared with mice injected with control cells.Conclusions High FKBP4 expression contributes to the proliferation,migration and invasion of LUAD cells in vitro and in vivo.CHAPTER 3.FKBP4 promotes LUAD progression via IKK/NF-?B signaling Methods1.Transcriptome sequencing(RNA-seq)Transcriptome sequencing was employed to obtain the differentially expressed genes and pathway related to FKBP4 downregulation.2.BioID and mass spectrometry experiments We investigated FKBP4 interactome through the BioID technique and mass spectrometry experiments.3.Dual-luciferase reporter assay Dual-luciferase reporter assays were used to assess the effects of differential FKBP4 expressions on NF-?B transcriptional activity.Dual-luciferase reporter assays were used to assess whether FKBP4 regulates NF-?B signaling through IKK?.Dual-luciferase reporter assays were used to assess whether the interaction between FKBP4 and Hsp90/IKK could promote NF-?B transcriptional activity.Dual-luciferase reporter assays were used to assess whether FKBP4 regulates NF-?B signaling through RelA.4.Co-immunoprecipitation(Co-IP)We investigated the endogenous/exogenous interaction between FKBP4 and Hsp90,IKK complex by Co-IP assays.Co-IP assays were used to detect the effect of FKBP4 on regulation of Hsp90/IKK complex interaction.Co-IP assays were used to detect the effect of FKBP4 on regulation of IKK complex assembly.Co-IP assays were used to define the interacting domains between FKBP4 and IKK?/?.5.GST pull-down assays GST pull-down assays were used to detect the interaction between FKBP4 and Hsp90,IKK complex in vitro.GST pull-down assays were used to detect the interaction between FKBP4 and Hsp70,RelA in vitro.6.Immunofluorescence assays We used immunofluorescence assays to detect the colocalization of FKBP4 with RelA.7.Western blot We used western blot assays to detect the effect of FKBP4 on regulating NF-?B pathway related proteins.Results1.FKBP4 potentiates NF-?B pathway and transcriptional activity through FKBP4/Hsp90/IKK complex 1.1 FKBP4 potentiates NF-?B pathway and transcriptional activity through IKK?The RNA sequencing data revealed that FKBP4 downregulation inhibited the IKK/NF?B pathway and transcriptional activity.Western blot assays showed that FKBP4 obviously promoted the phosphorylation of IKK?/?,I?B?,as well as RelA.Dualluciferase reporter assays showed that FKBP4 potently enhanced NF-?B activity.Furthermore,FKBP4 significantly increased IKK?-activated NF-?B.These results suggest that FKBP4 regulates NF-?B signaling through IKK?.1.2 The association between FKBP4 and Hsp90,IKK complex We investigated FKBP4 interactome through the BioID technique.Among these proximal proteins,we focused on Hsp90 as an additional component of the IKK complex,which is required for IKK phosphorylation.Co-IP assays revealed that FKBP4 bound to all three subunits of IKK complex,IKK?,IKK? and IKK?,as well as Hsp90 under transfection/endogenous conditions.GST pull-down assays revealed that FKBP4 directly interact with IKK?,IKK? as well as Hsp90,while the interaction with IKK? wasn't be detected.It is feasible that the interaction of FKBP4 with IKK? is IKK?/IKK?/Hsp90 mediated.1.3 FKBP4 promotes Hsp90/IKK association and IKK complex assembly We found that the association between IKK and Hsp90 was impaired in 293T cells transfected with shRNA FKBP4,and vice versa.Consistent with these results,endogenous Co-IP assays in H1975 cells further ascertained that FKBP4 knockdown blocked the interaction between IKK and Hsp90.Moreover,FKBP4 knockdown dramatically reduced the binding of IKK? to IKK?,suggesting the role of this protein in facilitating IKK complex assembly.1.4 TPR domain of FKBP4 is required for the interaction with IKK complex and the interaction is dependent on Hsp90 existing Co-IP assays revealed that the full-length FKBP4 and the PPIase domain-truncated mutants were capable of binding both Hsp90 and IKK,while the TPR domain-truncated mutant of FKBP4 failed to do so.Notably,the interaction between FKBP4 PPIase domain truncated form and IKKy was significantly reduced compared with FKBP4 fulllength and IKK?,suggesting the involvement of the PPIase domain in FKBP4/IKKy interaction.We generated a TPR domain point mutant,Flag-FKBP4 K354A,which was unable to interact with Hsp90.Interestingly,no interaction was detected between the mutant FKBP4 and IKK.It is feasible that the association between FKBP4 and IKK is dependent on Hsp90 existing.1.5 The interaction between FKBP4 and Hsp90/IKK promotes NF-?B transcriptional activity Luciferase reporter assays showed that the stimulatory action of 293T cells transfected with FKBP4 point mutant(K354A)was weakened compared with that of 293T cells transfected with wild-type FKBP4.In addition,the FKBP4 point mutant(K354A)still generated stimulatory action on NF-?B activity compared with the control cells.Based on these results,we wondered other molecular mechanisms were existing of FKBP4 to regulate NF-?B signaling.2.FKBP4 potentiates NF-?B pathway and transcriptional activity through FKBP4/Hsp70/RelA complex 2.1 FKBP4 potentiates NF-?B transcriptional activity through RelA Dual-luciferase reporter assays showed that depletion of FKBP4 decreased RelAinduced NF-?B activity,whereas overexpression of FKBP4 increased RelA-induced NF-?B activity.2.2 FKBP4 promotes NF-?B nuclear translocation and the levels of NF-?B downstream targets Western blot assays showed that FKBP4 promoted the level of RelA in nucleus and the levels of downstream targets of NF-?B,including MMP9,c-Myc,CyclinD1 and CDK6.2.3 The association between FKBP4 and Hsp70,RelA Immunofluorescence assays revealed that the colocalization of FKBP4 with RelA were increased upon TNF-? stimulation,suggesting the potential existence of nuclear interactions.GST pull-down assays indicated the direct association between FKBP4 and Hsp70/RelA.Co-IP assays results revealed the endogenous interaction between FKBP4 and Hsp70/RelA.3.FKBP4 potentiates LUAD cell invasion via upregulation of NF-?B signaling Transwell invasion assays showed that FKBP4 promoted LUAD cell invasion via positive regulation of NF-?B signaling.Moreover,the interaction between FKBP4 and Hsp90 promoted LUAD cell invasion.Conclusions FKBP4 integrates FKBP4/Hsp90/IKK with FKBP4/Hsp70/RelA complex to promote lung adenocarcinoma progression via IKK/NF-?B signaling.