LSD1 Destabilizes FBXW7 Independent Of Its Demethylase Activity And Negatively Regulates FBXW7 Functions

BackgroundFBXW7(F-box and WD-40 domain protein 7)is a substrate recognition subunit of SCF(SKP1-Cullinl-F-box protein)E3 ubiquitin ligase.It is one of the most well-characterized family member of F-box proteins.FBXW7 negatively regulates few significant biological processes,such as cell proliferation,survival and migration by promoting ubiquitylation and degradation of oncogenic substrates,including c-MYC,Notch 1,Cyclin E,c-JUN and MCL-1.The FBXW7 gene is located in chromosome 4q32 where genomic deletion frequently occurs.FBXW7 inactivation by point mutation and promoter hypermethylation have also been frequently observed in many types of human cancers,leading to accumulation of oncogenic substrates to promote proliferation and survival of cancer cells.Not surprisingly,a number of studies have demonstrated that the FBXW7 leve]is negatively correlated with the tumor progression and poor patient survival.LSD1(Lysine-specific demethylase 1)is the first identified histone demethylase,as one of the member of flavin adenine dinucleotide dependent amine oxidase superfamily.LSD1 specifically catalyzes the demethylation of Histone 3 lysine 4(H3K4)and lysine 9(H3K9)by redox reaction.Through regulating the methylation status of H3K4/H3K9,LSD1 serves as a master regulator of gene transcription,and is actively involved in vast range of biological processes,such as cell proliferation,apoptosis,chromosome segregation,stem cell pluripotent regulation and embryonic development.ObjectiveLysine-specific demethylase 1(LSD1)is a well-characterized histone demethylase,so far,most of LSD1 related studies were focused on its demethylase activity.Whether LSD1 has demethylase-independent activity remains elusive.Similarly,FBXW7 related researches were mainly focused on discovery of its downstream substrates.However,the upstream regulation network of FBXW7 is largely unknown.In this study,we mainly demonstrate that LSD1 negatively regulates the stability of FBXW7 in a demethylase independent manner.Thus,targeting LSD1 would stabilize FBXW7 for its tumor suppressive functions.Our study,therefore,has translational implication by providing a novel strategy in the anti-cancer drug development via disrupting FBXW7-LSD1 interaction.MethodsIn order to determine that LSD1 regulates FBXW7 protein expression at post-translational level,Western Blotting(WB)and qRT-PCR(Quantitative Real-time PCR)were used to detect the effects by LSD1 manipulation on FBXW7 protein and mRNA expression levels,respectively.The interaction between FBXW7 and LSD1 was confirmed by Co-immunoprecipitation(Co-IP)and in vitro protein pull-down assay.Protein half-life and in vivo ubiquitylation assays showed that FBXW7 did not promote ubiquitylation and affect the protein stability of LSD1.In contrast,LSD1 promotes the self-ubiquitylation of FBXW7,thus negatively regulating its stability.The in vivo and in vitro protein dimerization assay were used to show that LSD1 negatively regulates FBXW7 via disrupting FBXW7 dimerization,and promotes its self-ubiquitylated degradation.The constructs,expressing FBXW7 mutant with dimerization domain deletion and LSD1 mutant with CPD degron site deleted were made to determine whether LSD1 regulates FBXW7 in a manner dependent on their interaction,but independent of LSD1 demethylase activity.Co-IP,proteasome inhibitors(MG132)and lysosome inhibitors(CQ,Choroloquine)were used to show that the self-ubiquitylated FBXW7 was subjected to degradation by proteasome as well as lysosome in a manner dependent of autophagy protein p62/SQSTM1.For the biological related experiments,ATPlite,colony formation assay,micro-irradiation laser assay,NHEJ linearized plasmid assay and clonogenic survival assay were used to detect the effects of LSD1 on FBXW7's biological functions,including cell growth suppression,NHEJ repair and radiation protection.ResultsIn this study,we demonstrate that LSD1 directly binds FBXW7 to destabilize FBXW7 independent of its demethylase activity.Specifically,LSD1 is a pseudo-substrate of FBXW7 and LSD1-FBXW7 binding does not trigger LSD1 ubiquitylation,instead promotes FBXW7 self-ubiquitylation by preventing FBXW7 dimerization.The self-ubiquitylated FBXW7 is subjected to degradation by proteasome and lysosome in a manner dependent of autophagy protein p62/SQSTM1.Biologically,LSD1 destabilizes FBXW7 to abrogate its functions in growth suppression,NHEJ repair and radioprotection.ConclusionsLSDI functions as a pseudo substrate of FBXW7,which directly binds FBXW7 to destabilize FBXW7 independent of its demethylase activity.Our study revealed a previously unknown activity of LSD1,which likely contributes to its oncogenic function.Targeting LSD1 protein,rather than its demethylase activity,might be a unique approach for LSD1-based drug discovery for anti-cancer application.