Oxidized ATM-EMR-Acetyl-CoA/SIRT1 Axis Facilitates TNBC-CSCs Enrichment And Stemness Maintenace

Objective:To uncover the potential functions and mechanisms of oxidized ATM to the TNBC-CSCs enrichment and stemness maintenance.Methods:(1)FACS was used to analyze the ratio of CD44+/CD24-cells in both of Hs578T and BT549 cell population.Mammosphere formation efficiency was determined by mammosphere assay in both Hs578T and BT549 cells cultured under 1%O₂ condition.CSCs markers were measured by qRT-PCR and Western Blot.(2)Hs578T and BT549cells were suspension culture in CSCs special medium under normaxia(21%O₂)or hypoxia(1%O₂).The protein expression levels of ATM?p-ATM(s1981)and?H2AX(s139)in mammosphere cells were determined with Western Blot.Mammosphere formation efficiency was measured using mammosphere assay and the indicated CSC-associated gene expressions were assessed using qRT-PCR and Western Blot in mammosphere cells treated with or without ATM inhibitor KU60019.(3)The impact of oxidized ATM on the EMR of TNBC-CSCs were measured by the glucose consumption,pyruvate and lactate production,acetyl-CoA and citrate concentration,?-KG and succinate levels,transmission electron microscope and mitochondrial membrane potential analysis.(4)qRT-PCR and Western blotting tested the glycolysis-related gene expressions in mammospheres after treatment with KU60019.The roles of knockdown of ATM or Glut1,2-Deoxy-D-glucose,(2-DG,5 mM),UK5099(15?M),BMS203141(50?M),acetate(10 mM)alone or in combine with KU60019(10?M)on TNBC-CSCs enrichment and relative Ac-CoA productions were determined by mammosphere assay and Acetyl-CoA Concentration Assay Kit.(5)qRT-PCR and Western Blot tested the influence of oxidized ATM on the expressions of SIRT1 and USP13.COIP assay analyzed the interactive relationship between SIRT1 and USP13.(6)Western blot and immunofluorescence assay checked acetylated-H4(H4ac)levels in Hs578T and BT549 mammospheres treated with or without KU60019.Western blot verified the roles of treatments,including 2DG,UK5099 and BMS303141,in H4ac and CSC-associated gene levels.The status of H4 acetylation and stemness markers were determined using Western blot in Hs578T and/or BT549 mammospheres suffered to different treatments,including treated with acetate(10 mM),SIRT1 activator SRT1720(5?M),KU60019(10?M),SIRT1 inhibitor EX527(20?M),and acetate(10mM)alone or in combine.(7)The role of oxidized ATM on tumorigenesis in vivo was investigated.We established TNBC-CSC xenograft models and then treated tumor-bearing mice with different administrations.The concentration of acetyl-CoA of the relevant xenograft tumors was tested by Acetyl-CoA Concentration Assay Kit.IHC analyses were performed to test GLUT1,HK2,SIRT1,H4ac and SOX2 expression levels in residual tumors administrated with or without drug treatment.Results:(1)The result of FACS showed that the percentage of CD44+/CD24-cells were markedly increased when Hs578T and BT549cells were exposed to 1%O2 for 24h.Similarly,the mammosphere formation and mammosphere sizes were also significantly increased by hypoxia in both Hs578T and BT549 cells.(2)We observed an oxidized ATM(p-ATM,s1981,a hallmark of ATM activation by oxidative stress)existed in TNBC-CSCs in normoxic CSCs,and more significantly enhanced p-ATM in the hypoxic CSCs.And almost no?H2AX(a biomarker of DSBs)was detected under hypoxic stimulation.Functional inhibition of oxidized ATM using ATM inhibitor KU60019 significantly reduced mammosphere formation of TNBC cells under both normoxic and hypoxic condition,such as the sphere numbers and sizesin a dose-dependent manner.Inhibition of oxidized AMT using KU60019decreased the expressions of indicated stemness markers in mammospheres of Hs578T and BT549 in mRNA and protein levels.Knockdown of ATM caused a dramatical drop in mammospheres formation formed from both Hs578T and BT549 cells as well.(3)The levels of cellular ATP in hypoxic TNBC-CSCs were robustly reduced under 2-DG(a glucose analog that inhibits glycolysis)treatment,meanwhile,cellular ATP were mildly reduced under Metformin(an inhibitor of electron transfer chain(ETC))treatment,and the ATP productions were further decreased under combine using 2-DG and Metformin.The decreased glucose consumption and pyruvate production were detected in the hypoxic spheres treated with KU60019 compared with the spheres untreated with KU60019 under hypoxic condition.Interestingly,there was no significant change in lactate production.The?-KG and succinate had slightly decreased as oxidized ATM activation be lost.The large and damaged mitochondria were in the mammospheres treated with ATM inhibitor.KU60019 treatments led to decreased mitochondrial activation of hypoxic mammospheres.(4)It was found that glycolysis-genes,Glut1,Hk2 and Pkm2 were down-regulated and Ldha was up-regulated in oxidized ATM inactivated mammosphere cells;the TCA-related genes,only Pdha encoding an enzyme catalyzing pyruvate into acetyl-CoA was decreased,no other genes(including Pdk,Pdhb,Cs,Acly,Idh and Scs)in mammospheres had significant changes after inhibition of oxidized ATM.Knockdown of Glut1,application of 2DG,UK-5099(an inhibitor of the mitochondrial pyruvate transporter)or BMS203141(inhibiting the activation of ACLY,a key enzyme catalyzing citrate into acetyl-CoA in cytoplasm)to the hypoxic mammospheres,the reduced spheroid formations were observed in both BT549 and Hs578T cells.Addition of acetate(a precursor of acetyl-CoA)to medium promoted the TNBC-CSCs enrichment in a dose-dependent manner and partly rescued the TNBC-CSCs enrichment blunted by KU60019.Accordingly,the acetyl-CoA levels in the hypoxic sphere cells had the same changes under the treatments.(5)SIRT1 was confirmed to be up-regulated in mammospheres of Hs578T and BT549 treated with ATM inhibitor,but the levels of SIRT1 mRNA had no change.The increased USP13 expression was detected in hypoxic spheres treated with KU60019 compared with that untreated.Immunoprecipitation was employed to unravel the interaction between USP13 and SIRT1.Knockdown or inhibition of endogenous USP13 significantly elevated the polyubiquitilation of SIRT1 in mammospheres formed from Hs578T and BT549 cells.(6)The mammosphere cells treated with the inhibitor of oxidized ATM,the acetylation of total histone H4 not H3 was decreased.Each treatment with these three inhibitors(2DG,UK5509 and BMS301341)caused a significant loss in the acetylation of H4K8,K12 and K16 in Hs578T mammospheres.In line with these findings,the protein levels of CSC stemness markers were markedly suppressed in the sphere cells treated with 2DG,UK5099and BMS301341,respectively.The addition of acetate elevated the acetylation of histone H4 and the CSC-associated gene expressions.We treated cells with SIRT1 specific activator SRT1720 and found that it down-regulated the acetylation levels of histone H4 and CSC-associated gene expressions simultaneously.Suppression of H4 histone acetylation by KU60019 was rescued by EX527,an inhibitor of SIRT1,and co-administration of EX527 and acetate further rescued the H4 histone acetylation.Accordingly,the levels of CSC-associated genes in the hypoxic sphere cells had the same changes under the treatments.(7)Oxidized ATM promoted the growth of TNBC-CSC xenograft tumors,and KU60019suppressed the growth of tumors and as well as inhibition of MPC by UK5099.In contrast to blockage of oxidized ATM by KU60019 alone,combined administrating mice with KU60019 and EX527(SIRT1 inhibitor)could diminish the suppressive efficiencies to tumor growth,addition of acetate further restored tumor growth.In line with this,the Ac-CoA levels of the relevant xenograft tumors had the same changes.IHC assay showed that KU60019-mediated CSCs abolishment correlated with decreased GLUT1,HK2,H4ac and SOX2 expression and increased SIRT1 expression in xenograft tumors.Conclusions:(1)Hypoxia facilitates enrichment of CSCs derived from triple negative cancer cells(TNBC-CSCs).(2)Oxidized ATM is required for TNBC-CSCs enrichment.(3)Oxidized ATM induces EMR.(4)Oxidized ATM-mediated EMR fuels acetyl-CoA production.(4)Oxidized ATM decreases SIRT1 activation through USP13 in TNBC-CSCs.(5)acetyl-CoA and SIRT1 regulate acetylation of histone H4 and CSC-associated gene expressions.(6)acetyl-CoA and SIRT1 regulate acetylation of histone H4 and CSC-associated gene expressions.(7)Oxidized ATM promotes tumor formation of CSCs in mice Xenografts.