BCKDK Of BCAA Catabolism Cross-talking With The MAPK Pathway Promotes Tumorigenesis Of Colorectal Cancer

Background&Aims:Colorectal cancer(CRC)is the third most commonly diagnosed cancer in the world.Branched-chain amino acids(BCAA)catabolism plays an important role in human cancers.The branched-chain ?-keto acid dehydrogenase kinase(BCKDK)is a key negative regulation enzyme in BCAA catabolism,which plays an important role in many serious human diseases.Here we investigated the role of BCKDK in CRC tumorigenesis and the crosstalk of BCKDK with MAPK pathway.Methods:1.The anchorage-independent cell transformation assay was used to analyze whether BCAA can promote the colony formation and proliferation of JB6 C141 cells.2.BCAA kit was used to detect the BCAA concentration in colorectal cancer patients'serums,normal people serums,or paired tumors and corresponding tumor adjacent tissues.3.The levels of branched-chain a-keto acid dehydrogenase El(BCKDHA),p-BCKDHA and BCKDK were observed by immuohistochemistry staining(IHC)in paired tumors and corresponding tumor adjacent tissue collected from 117,118,or 113 patients respectively,and the correlation between the levels of these proteins and survival times of patients was assessed with Kaplan-Meier method.4.The pCMV-BCKDK-Flag and plko.l-shBCKDK plasmids were constructed,and BCKDK stable cell lines in the JB6 C141 or WiDr cells,BCKDK-knockdown cell lines in the HCT116 or DLD1 cells were built.5.The above cell lines were used to analyze for colony formation,proliferation and the levels of key molecules in MAPK signaling pathways by western blot.6.HCT116 cells with BCKDK-knockdown were grown as xenograft tumors in nude mice.Tumor growth subsequently measured and the levels of p-MEK and p-ERK were tested by IHC.7.Immunoprecipitation(IP)was used to analyze the interaction between BCKDK and MEK:the pCMV-BCKDK-Flag and pCMV-Myc-MEK1 plasmids were constructed,and transfected to HEK293T cells.The total protein(48 h)and antibodies ofanti-Flag and anti-Myc were used for in vitro IP.The total protein of HCT116 and BCKDK antibody were used for ex vivo IP.8.IP kinase assay was used to test BCKDK phosphorylating MEK1:the pet23a-MEK1(62-393 aa)-his plasmid was constructed and expressed in BL21 bacteria,the MEK1(62-393 aa)-his protein was used for substrate.The IP method was used to acquire the BCKDK kinase.The p-MEK1/2(Ser221)monoclonal antibody was used to detect the level ofp-MEK1(62-393 aa)(Ser221),9.Whether BCKDK phosphorylates the MEK in concentration dependence:different concentrations of pCMV-BCKDK-Flag plasmid and one concentration of pCMV-Myc-MEK1 plasmid were co-transfected to HEK293T cells;the level of p-MEK 1/2(Ser221)was detected.10.Phenylbutyrate(PB).a reported BCKDK inhibitor,was used to further confirm above experiments.Results:1.BCAA could not promote cell transformation in anchorage-independent cell transformation assays in JB6 C141 cells.2.The BCAA concentration between colorectal cancer patients,serums and normal people serums had no significant change,neither between paired tumors and corresponding tumor adjacent tissues.3.The levels of BCKDHA,BCKDK,or p-BCKDHA were higher in human colorectal tumor tissue than corresponding in tumor adjacent tissue(P<0.001),but only BCKDK protein level was associated with shorter survival times of patients(P=0.011).4.Overexpression of BCKDK in JB6 C141 or WiDr cells increased their proliferation ex vivo.Knockdown of BCKDK in HCT116 or DLD1 cells reduced their proliferation ex vivo.5.The level of BCKDK was positively correlated with the levels of p-MEK and p-ERK.6.Knockdown of BCKDK in HCT116 cells reduced tumor formation in mice,compared to HCT116-shMock cells.7.Immunoprecipitation and IP Kinase assays indicated that BCKDK interacted with MEK and phorsphorylated it in vitro and ex vivo.8.Phenylbutyrate(PB),a BCKDK inhibitor,inhibited the phosphorylation of MEK by BCKDK in the IP kinase assays,the proliferative responses in HCT116 cells,and the levels of p-MEK and p-ERK in HCT116 cells.Conclusions:BCKDK promotes CRC by enhancing the MAPK signaling pathway through direct MEK phosphorylation.rather than by increasing BCAA concentration.