The Role And Its Potential Mechanisms Of Oxidized ATM In Regulating The Proliferation And Energy Metabolism Reprogramming Of Breast CAFs

Chapter One The role and its Potential Mechanisms of Oxidized ATM in Regulating the Abnormal Proliferation of Breast CAFsObjective:This chapter was to investigate the function and its inner mechanisms of oxidized ATM in regulating the proliferation of CAFs derived from breast cancer.Methods:(1) Based on the immortalized breast NFs and CAFs cell lines, the abnormal proliferation of breast CAFs was tested using the MTT method, Cell density assay, Flow cytometry, and Western blot. (2) The protein expression levels of p-ATM (s1981), ATM, and yH2AX (s139) in NFs and CAFs were determined with Western blot, Immunocytofluorescent test, and Immunohistochemistry. After treating CAFs with ROS inhibitors, the changes of ROS levels in CAFs were assessed using the DCFH probe and the oxidation and activation of ATM in CAFs were tested using Western blot. (3) The function of oxidized ATM was inhibited by the ATM specific inhibitor KU60019 or siRNA, the impact of ATM deficiency on the proliferation of breast CAFs was observed. (4) After treating breast CAFs with KU60019 and NAC together, We detected the effect of NAC on the CAFs proliferation defect caused by KU60019. (5) Western blot tested the effect of KU60019 on the activity of MEK-MAPK, PI3K-AKT, and WNT signaling pathways. After breast CAFs were treated with U0126, LY294002, and XAV939, the impact of these inhibitors on CAFs proliferation was assessed. (6) Western blot tested the role of KU60019,U0126, LY294002, and XAV939 in the protein expression of GSK3β, β-catenin, and c-Myc in breast CAFs. ChIP assay detected the impact of these inhibitors on the transcription activity of β-catenin. The effect of siRNA-mediated knockdown of c-Myc on CAFs proliferation was tested.Results:The results of the MTT method, Cell density assay, Flow cytometry, and Western blot showed that breast CAFs had a more rapid proliferation potential, a higher cell density, a more proportion of cells in S phase, and a stronger expression of positive cell cycle regulation proteins than NFs. (2) The expression levels of p-ATM (s1981), ATM, and p-AKT (s473) in CAFs were higher than that in NFs, but yH2AX (139) with no change. Only NAC, DPI, and rotenone reduced the ROS levels and oxidization-dependent activation of ATM in CAFs. (3) Loss of oxidized ATM function by KU60019 or siRNA suppressed the proliferation of breast CAFs, suggesting oxidized ATM promotes CAFs proliferation. (4) NAC reversed the increased ROS levels in CAFs with KU60019, but only partially alleviated the CAFs proliferation defect resulted from KU60019 treatment. (5) The results of Western blot revealed that KU60019 severely suppressed the activities of ERK-MAPK, PI3K-AKT, and WNT proliferation signaling pathways in CAFs. Individual or a combination of U0126, LY294002, and XAV939 inhibited CAFs proliferation. (6) The results of Western blot showed KU60019, U0126, LY294002, and XAV939 all increased the activity of GSK3β kinase and decreased the protein expression of β-catenin and c-Myc. The results of ChIP uncovered KU60019, U0126, LY294002, and XAV939 all blocked the transcription of β-catenin. SiRNA-mediated knockdown of c-Myc suppressed CAFs proliferation.Conclusions:(1) Breast CAFs have an abnormal proliferation phenotype compared with NFs. (2) The oxidization and activation of ATM happens in CAFs independent of DSBs. (3) Oxidized ATM promotes breast CAFs proliferation. (4) Redox homeostasis maintenance function of oxidized ATM plays a minor role in regulating breast CAFs proliferation. (5) Oxidized ATM promotes the proliferation of breast CAFs through enhancing the activities of ERK-MAPK, PI3K-AKT, and WNT signaling pathways. (6) The axis of β-Catenin/c-Myc mediates the role of oxidized ATM in promoting CAFs proliferation.Chapter Two The Role and its Mechanisms of Oxidized ATM in Regulating the Hypoxia-Induced EMR of Breast CAFsObjective:This section aimed to explore the role and its mechanisms of oxidized ATM in regulating the hypoxia-induced EMR of CAFs.Methods:(1) The differentially expressed genes involved in energy metabolism in breast CAFs were screened through analyzing the mRNA microarray data of breast CAFs/NFs and next reproved by qRT-PCR. Compared with normoxic NFs, the impact of hypoxia on the EMR of CAFs were assessed by the Glucose consumption, Lactate generation, Western blot, Mitochondrial activity, and electron microscope assays. (2) The effect of NAC and rotenone on hypoxic CAFs EMR was detected compared with the solvent control. (3) Western blot tested the influence of NAC and rotenone on the oxidation and activation of hypoxic breast CAFs. The impact of loss of oxidized ATM function on hypoxic CAFs EMR was detected. (4) The potential substrates of oxidized ATM in hypoxic breast CAFs were screened using phospho-proteomics and bioinformatics technologies.Results:(1) The results of breast CAFs/NFs mRNA microarray data screening and qRT-PCR showed that many glycolysis related genes were upregulated in CAFs relative to NFs, with OP related genes downregulated and energy metabolism regulation signaling pathways activated. Compared with normoxic NFs, the glucose consumption rate, the lactate generation rate and the expression of energy metabolism related proteins in CAFs were significantly increased, with mitochondrial activity significantly inhibited and mitochondrial number/cristae obviously decreased. These results showed hypoxia maked CAFs have a EMR behaviour. (2) The results of DCFH probe, Western blot, and metabolic assays showed suppressed ROS levels increase and EMR of hypoxic CAFs.(3) The results of Western blot uncovered that NAC and rotenone inhibited oxidation and activation of ATM in hypoxic CAFs. Loss of oxidized ATM impaired hypoxic CAFs EMR. (4) The results of phosphoproteomic assay and bioinformatics analysis showed that oxidized ATM has many novel energy metabolism related substrates in hypoxic CAFs.Conclusions:(1) Hypoxia maked breast CAFs have a typical EMR phenotype. (2) Mitochondria-derived ROS mediated the hypoxia-induced breast CAFs EMR. (3) Mitochondria-derived ROS oxidized and activated ATM in breast CAFs. Oxidized ATM promoted the EMR of breast CAFs induced by hypoxia. (4) Oxidized ATM may promote the hypoxia-induced EMR of breast CAFs through phosphorylating new energy metabolism related substrates.