Oxidized ATM-mediated Glycolysis Enhancement In Breast Cancer-associated Fibroblasts Contributes To Tumor Invasion Through Lactate As Metabolic Coupling

Background: The tumor environment(TME)is extremely important in the malignant biological process of tumors.CAFs(cancer-associated fibroblasts)play a pivotal role as the most important stromal cells.In the early stage,we analyzed the microarray data and found that compared with breast NFs(normal fibroblasts),there is abnormal activation of ATM(ataxia telangiectasia mutation)gene signaling pathway in breast cancer CAFs,and its metabolic gene profile has also alterated(upregulation expressions of genes associated with the glycolysis process,down-regulation expression of genes related with oxidative phosphorylation).Purpose: investigate(1)The changes of major metabolic pattern in CAFs.(2)Whether CAFs have ATM activation which is different from the activation of traditional DSBs(DNA double-breaks)pathway(oxidized ATM).(3)The mediation of oxidized ATM in the glycolysis process in CAFs.(4)How the oxidized ATM regulates the downstream genes to play a role in the glycolysis of CAFs.(5)Whether the metabolites(lactate)produced by oxidized ATM-mediated glycolysis in CAFs promote the migration and invasion of cancer cells.(6)The specific mechanism of glycolysis products lactate in promoting the migration and invasion of cancer cells.(7)The effect of oxidized ATM-mediated glycolysis on the growth and metastasis of tumors in CAFs.Methods:(1)Three pairs of NFs and CAFs were cultured from the specimens of clinical patients with breast cancers,and Q-PCR,WB(Western blotting)and immunofluorescence techniques were used to identify the markers.(2)Detection the OCR(oxygen consumption rate)and ECAR(extracellular acidification rate)of NFs and CAFs and measurement for mitochondrial membrane potential of NFs and CAFs using the JC-1 kit.(3)WB(Western blotting)and immunofluorescence staining confirmed the activation of oxidized ATM in CAFs under hypoxic conditions;after using KU60019 and shRNA to reduce the activity of oxidized ATM,the glucose consumption and lactate production were determined by kits respectively.(4)Mass spectrometry identified phosphorylation of downstream protein GLUT1 S490 by oxidized ATM,co-IP(immunoprecipitation)assay,in vitro kinase assay,WB assay were used for proving the phosphorylation of GLUT1 S490 by oxidized ATM;immunofluorescence staining and WB assay of membrane protein showed that the oxidized ATM phosphorylation of GLUT1 S490 site affected its translocation to the cell membrane;WB assay and immunofluorescence staining were used to detect the regulation of PKM2 protein expression by oxidized ATM.(5)The expression of ATM protein in CAFs were knocked down by shRNA and collected conditioned medium,then co-culturied with cancer cells(MDA-MB-231 and BT-549),Transwell assay were used to detect the invasion ability of cancer cells,exogenous lactate was added to the conditioned medium of CAF/sh-ATM and detected the invasive ability of the cancer cells that co-cultured;further interfered the glycolytic process of CAFs(phosphorylation inactivation of GLUT1 S490,shRNA were used to stably knockdown PKM2 expression)and detected glucose consumption and lactate generation of cells,conditioned medium were collected and co-cultured with cancer cells,the invasive ability of cancer cells was measured;blocked the process of transportation of lactate from CAFs to cancer cells(stable knockdown of MCT1 protein in CAFs,stable knockdown of MCT1 protein in cancer cells by shRNA),co-cultured the conditioned medium with cancer cells and the invasion ability was evaluated.(6)The changes of invasion-related signaling pathways in cancer cells treated above were detected by WB,whether lactate promotes invasion through activation of signaling pathway was evaluated;the shRNA is used to stably knock down the expression of ATM in CAFs,and the conditioned medium is co-cultured with cancer cells to detect mitochondrial oxidative phosphorylation activity of cancer cells;Exogenous lactate was added to the conditioned medium of CAF-shATM,the cancer cells were co-cultured and the mitochondrial oxidative phosphorylation activity was detected again.The anti-mitochondrial drug oligomycin was used to treat the cancer cells,and the mitochondrial oxidative phosphorylation activity and invasion ability were determined.(7)Divided nude mice into groups with different treatment,using engineered cells CAF/sh-Ctrl and CAF/sh-ATM,CAF/sh-MCT4 mixed with breast cancer cells MDA-MB-231,An animal model was established under subcutaneous tumor formation,and the drug 2-DG was used to interfere with the CAFs glycolysis process,or CHC was used to block cancer cells from absorbing lactate or restored the supply of lactate.The size and growth curve of nude mice were detected.HE staining was used to observe and count the lung metastasis of mice.The activity of related signaling pathways in tumor tissues was detected by WB technique and immunohistochemical staining.Results:(1)Compared with NFs,the oxygen consumption rates of in CAFs decreased,the extracellular acidification rates of glycolysis increased,and the membrane potential of CAFs were reduced when detected with JC-1 kit,indicating that the metabolic pattern of CAFs is more reliable on glycolysis.(2)Under the condition of hypoxia for 8 h,the activation of ATM protein in CAFs which reflected by the expression of p-ATM(s1981)protein was detected,and there was no DNA double-strand breaks in the cells(the relevant marker proteins ?H2AX and CHK2(T68)were not detected).After treatment of CAFs with cisplatin as a positive control for DSBs,the expression of DSBs-associated marker proteins in CAFs(?H2AX and CHK2(T68))were detected by WB.Immunofluorescence staining revealed DNA double-strand breaks in cells(nuclear fluorescence of 53BP1 and ?H2AX were formed in CAFs),but not in the CAFs treated with hypoxia for 8 h.The use of antioxidants to scavenge ROS produced by hypoxia in CAFs reduces oxidative activation of ATM.All of the above results indicated that under hypoxia for 8 h,ATM were activated with hypoxiainduced ROS(oxidized ATM)that did not participate in the DSBs repair process.After hypoxia treatment of CAFs,oxidized ATM were activated,glucose consumption and lactate generation increased,the glycolysis was enhanced,and inhibition of oxidized ATM activity by specific inhibitor KU60019 or ATM-targeted shRNA,glucose consumption and lactate generation decreased,the glycolysis was weakened,and these results suggest that oxidized ATM mediates the enhanced glycolysis of CAFs.(3)Mass spectrometry identified the oxidized ATM phosphorylation of the GLUT1 protein S490 site,and this site is highly conserved in humans and other species.Using CO-IP experiments,we confirmed that oxidized ATM can phosphorylate GLUT1 protein.In vitro kinase assay also confirmed that hypoxia-activated oxidized ATM can directly phosphorylate GLUT1 S490,and GLUT1 undergoes point mutation to make its site S490 to S490A(phosphorylation inactive form),the oxidized ATM loses phosphorylation at the site of GLUT1.The protein level of p-GLUT1(S490)was increased in CAFs under hypoxia detected by WB,and the protein level was decreased after using the inhibitor KU60019.The point-mutation technique was used to construct plasmid and transfected CAFs that endogenous GLUT1 were knocked down,and genetically engineered recombinant cells CAF-ecto WT and CAF-ecto S490 A were obtained.Compared with normoxic condition,the level of p-GLUT1(S490)protein in CAF-ecto WT increased under hypoxic condition,while the protein level of p-GLUT1(S490)in CAF-ecto S490 A both were very low under normoxia or hypoxia.The above results indicate that the oxidized ATM is capable of phosphorylating the S490 site of GLUT1.Using immunofluorescence staining,we found that GLUT1 translocated to the cell membrane in CAFs after hypoxic treatment,and GLUT1 translocated to the cell membrane after the inhibitor KU60019.Similarly,under hypoxic conditions,GLUT1 in CAF-ecto WT was translocated to the cell membrane,and the inhibitor KU60019 could inhibit this phenomenon.In CAF-ecto S490 A,GLUT1 could not translocated to cell membrane under normoxic,hypoxic conditions neither treatment with inhibitors.At the same time,glucose consumption and lactate generation were measured,the glucose consumption and lactate generation were increased with membrane translocation of GLUT1.The hypoxic-activated oxidized ATM promotes its translocation to the cell membrane by phosphorylating the S490 site of GLUT1 was also found with the detection of GLUT1 expression in membrane proteins using WB.The results of WB and immunofluorescence staining showed the activity of PI3K/AKT pathway was increased and the expression of PKM2 was up-regulated in CAFs under hypoxia.The expression of PKM2 was decreased after expression of inhibitor or knockdown of ATM,and the activation of PI3K/AKT signaling pathway was also decreased,that means oxidized ATM could regulated the expression of PKM2 through PI3K/AKT signaling pathway.(4)Using shRNA to knockdown the expression of ATM protein in CAFs,the invasive ability of cancer cells(MDA-MB-231 and BT-549)cocultured with CM was decreased,and exogenous lactate was added to the conditioned medium of CAF/sh-ATM,the invasive ability of cancer cells cocultured with it was determined.Interruption of the glycolysis process or blockage of the transportation process of lactate,the acquirements of lactate in cancer cells which co-cultured were reduced,and their invasive ability was correspondingly weakened.(5)The changes of TGF?1/p38 MAPK/MMP2/9 signaling pathway in cancer cells were detected after coculture with conditioned medium under the above conditions.The results showed that the signal transduction activity was decreased after co-culture of cancer cells with conditioned medium derived from CAF/sh-ATM.After the exogenous lactate was supplemented in the conditioned medium,the signal pathway activity was restored;interruption of the glycolysis process or blockage of the transportation process of lactate,the signal transduction activity of cancer cells under co-culture is decreased,which is consistent with the descendant invasive ability.These results suggest that lactate derived from oxidized ATM-mediated glycolysis in CAFs promotes cancer cell invasion by activating the TGF?1/p38 MAPK/MMP2/9 signaling pathway.The conditioned medium of CAF-shATM was co-cultured with cancer cells,and the mitochondrial oxidative phosphorylation activity of cancer cells was decreased;the exogenous lactate was added to the conditioned medium CAF-shATM,and the mitochondrial oxidative phosphorylation activity of the cancer cells was restored;the antimitochondrial drugs oligomycin were utilized on cancer cells,its mitochondrial oxidative phosphorylation activity is decreased,and its invasive ability is correspondingly weakened.The results show that lactate can promote its invasion by activating mitochondrial oxidative phosphorylation activity of cancer cells.(6)In vivo studies revealed knockdown of ATM expression in CAFs or inhibition of glycolysis using 2-DG,and knockdown of MCT1 expression in CAFs using shRNA or blocking of lactate transport in CAFs to cancer cells using CHC,the ability of xenograft growth and lung tissue transfer in nude mice were significantly reduced,and the ability of tumor growth and lung metastasis could be restored after the addition of exogenous lactate to restore lactate supply.Conclusions:(1)The metabolism of CAFs is more dependent on the glycolysis process.(2)Under hypoxic conditions,CAFs is oxidative activation of ATM(oxidized ATM)with hypoxic activated and no participate in the DSBs approach.(3)Oxidized ATM phosphorylates the S490 site of GLUT1 to promote its translocation to the cell membrane and regulate the expression of PKM2 through the PI3K/AKT pathway.(4)S490 site of GLUT1 phosphorylation by oxidized ATM promote its translocation to the cell membrane and regulation of PKM2 expression via PI3K/AKT pathway.(5)Oxidized ATM mediates the glycolysis of CAFs to produce lactate to promote malignant growth and metastasis of tumors.