ATM Regulated PTEN Degradation Is XIAP E3 Ubiquitin Ligase Mediated in p85? Deficient Cancer Cells and Influence Platinum Sensitivity.
Ataxia-telegiectasia mutated (ATM), phosphatase and tensin homolog (PTEN), and p85? are key tumour suppressors. Whether ATM regulates PTEN expression and influence platinum sensitivity is unknown. We generated ATM knockdowns (KD) and CRISPR knock outs (KO) in glioblastoma (LN18, LN229) and ovarian cancer cells (OVCAR3, OVCAR4). Doxycycline inducible PTEN expression was ... generated in LN18 and LN229 cells. Transient KD of p85?, CK2, and XIAP was accomplished using siRNAs. Stable p85? knock-in was isolated in LN18 cells. Molecular biology assays included proteasome activity assays, PCR, flow cytometry analysis (cell cycle, double strand break accumulation, apoptosis), immunofluorescence, co-immunoprecipitation, clonogenic, invasion, migration, and 3D neurosphere assays. The clinicopathological significance of ATM, PTEN, p85?, and XIAP (X-linked inhibitor of apoptosis protein) was evaluated in 525 human ovarian cancers using immunohistochemistry. ATM regulated PTEN is p85? dependant. ATM also controls CK2? level which in turn phosphorylates and stabilizes PTEN. In addition, p85? physically interacts with CK2? and protects CK2? from ATM regulated degradation. ATM deficiency resulted in accumulation of XIAP/p-XIAP levels which ubiquitinated PTEN and CK2? thereby directing them to degradation. ATM depletion in the context of p85? deficiency impaired cancer cell migration and invasion reduced 3D-neurosphere formation and increased toxicity to cisplatin chemotherapy. Increased sensitivity to platinum was associated with DNA double strand breaks accumulation, cell cycle arrest, and induction of autophagy. In ovarian cancer patients, ATM, PTEN, p85?, and XIAP protein levels predicted better progression free survival after platinum therapy. We unravel a previously unknown function of ATM in the regulation of PTEN thro?gh XIAP mediated proteasome degradation.
Mesh Terms:
Ataxia Telangiectasia Mutated Proteins, Blotting, Western, Caspase 3, Caspase 7, Cell Line, Tumor, Cell Proliferation, Cisplatin, Doxycycline, Female, Flow Cytometry, HeLa Cells, Humans, Ovarian Neoplasms, PTEN Phosphohydrolase, Plasmids, Platinum, Protein Stability, Real-Time Polymerase Chain Reaction, Tissue Array Analysis, Ubiquitin-Protein Ligases, X-Linked Inhibitor of Apoptosis Protein
Ataxia Telangiectasia Mutated Proteins, Blotting, Western, Caspase 3, Caspase 7, Cell Line, Tumor, Cell Proliferation, Cisplatin, Doxycycline, Female, Flow Cytometry, HeLa Cells, Humans, Ovarian Neoplasms, PTEN Phosphohydrolase, Plasmids, Platinum, Protein Stability, Real-Time Polymerase Chain Reaction, Tissue Array Analysis, Ubiquitin-Protein Ligases, X-Linked Inhibitor of Apoptosis Protein
Cells
Date: Dec. 18, 2018
PubMed ID: 31635307
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