Lei Lang1, Yixuan Hou1,2, Yanlin Chen1, Gang Tu3, Jing Tao4, Dan Yang1, Lei Xi1, Lixin Fu1, Kexin Sun1, Jiali Yin1, Rui Chen3, Meixi Peng1, Shuiqing Liu1, Manran Liu5. 1. Key Laboratory of Laboratory Medical Diagnostics, Chinese Ministry of Education, Chongqing Medical University, Chongqing, China. 2. Experimental Teaching Center of Basic Medicine Science, Chongqing Medical University, Chongqing, China. 3. Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. 4. Department of Rheumatology and Immunology, Children's Hospital of Chongqing Medical University, Chongqing, China. 5. Key Laboratory of Laboratory Medical Diagnostics, Chinese Ministry of Education, Chongqing Medical University, Chongqing, Chinamliu-hncq@hotmail.com.
Abstract
BACKGROUND/AIMS: The ataxia-telangiectasia mutated (ATM) protein kinase is critical for the maintenance of genomic stability and acts as tumor suppressor. Although evidence shows that a DNA damage-independent ATM (oxidized ATM) may be involved in cancer progression, the underlying mechanism is still unclear. METHODS: Immunohistochemistry, immunofluorescence and western blotting were applied to detect the levels of oxidized ATM. Transwell assay was used to detect the cell migration and invasion abilities in different treatments. Quantitative phosphoproteome analysis was performed using hypoxic BT549 cells, in the presence or absence of Ku60019, a specific inhibitor of ATM kinase. The phosphorylated cortactin, the target protein of oxidized ATM, was confirmed by immunoprecipitation-western blots and in vitro kinase assay. The functions of phosphorylated cortactin were studied by specific short hairpin RNA, site-directed mutation, transwell assay, and actin polymerization assay. RESULTS: Enhanced oxidized ATM proteins were present not only in the advanced and invasive breast tumor tissues but also malignant hypoxic breast cancer cells, in the absence of DNA damage. Loss of ATM expression or inhibiting oxidized ATM kinase activity reduced breast cancer cell migration and invasion. Using quantitative phosphoproteomics approach, 333 oxidized ATM target proteins were identified, some of these proteins govern key signaling associated with gap junction, focal adhesion, actin cytoskeleton rearrangement. Cortactin, one of the biggest changed phospho-protein, is a novel oxidized ATM-dependent target in response to hypoxia. Mechanically, we reveal that hypoxia-activated ATM can enhance the binding affinity of cortactin with Arp2/3 complex by phosphorylating cortactin at serine 113, and as a result, in favor of breast cancer cell migration and invasion. CONCLUSION: Oxidized ATM can phosphorylate cortactin at serine 113, playing a critical role in promoting breast tumor cell mobility and invasion via actin polymerization.
BACKGROUND/AIMS: The ataxia-telangiectasia mutated (ATM) protein kinase is critical for the maintenance of genomic stability and acts as tumor suppressor. Although evidence shows that a DNA damage-independent ATM (oxidized ATM) may be involved in cancer progression, the underlying mechanism is still unclear. METHODS: Immunohistochemistry, immunofluorescence and western blotting were applied to detect the levels of oxidized ATM. Transwell assay was used to detect the cell migration and invasion abilities in different treatments. Quantitative phosphoproteome analysis was performed using hypoxic BT549 cells, in the presence or absence of Ku60019, a specific inhibitor of ATM kinase. The phosphorylated cortactin, the target protein of oxidized ATM, was confirmed by immunoprecipitation-western blots and in vitro kinase assay. The functions of phosphorylated cortactin were studied by specific short hairpin RNA, site-directed mutation, transwell assay, and actin polymerization assay. RESULTS: Enhanced oxidized ATM proteins were present not only in the advanced and invasive breast tumor tissues but also malignant hypoxic breast cancer cells, in the absence of DNA damage. Loss of ATM expression or inhibiting oxidized ATM kinase activity reduced breast cancer cell migration and invasion. Using quantitative phosphoproteomics approach, 333 oxidized ATM target proteins were identified, some of these proteins govern key signaling associated with gap junction, focal adhesion, actin cytoskeleton rearrangement. Cortactin, one of the biggest changed phospho-protein, is a novel oxidized ATM-dependent target in response to hypoxia. Mechanically, we reveal that hypoxia-activated ATM can enhance the binding affinity of cortactin with Arp2/3 complex by phosphorylating cortactin at serine 113, and as a result, in favor of breast cancer cell migration and invasion. CONCLUSION: Oxidized ATM can phosphorylate cortactin at serine 113, playing a critical role in promoting breast tumor cell mobility and invasion via actin polymerization.
Authors: Kelly L Harris; Meagan B Myers; Karen L McKim; Rosalie K Elespuru; Barbara L Parsons Journal: Environ Mol Mutagen Date: 2019-10-06 Impact factor: 3.216