Abstract
Ataxia-telegiectasia mutated (ATM), phosphatase and tensin homolog (PTEN), and p85α 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 p85α, CK2, and XIAP was accomplished using siRNAs. Stable p85α 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, p85α, and XIAP (X-linked inhibitor of apoptosis protein) was evaluated in 525 human ovarian cancers using immunohistochemistry. ATM regulated PTEN is p85α dependant. ATM also controls CK2α level which in turn phosphorylates and stabilizes PTEN. In addition, p85α physically interacts with CK2α and protects CK2α from ATM regulated degradation. ATM deficiency resulted in accumulation of XIAP/p-XIAP levels which ubiquitinated PTEN and CK2α thereby directing them to degradation. ATM depletion in the context of p85α 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, p85α, 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 throμgh XIAP mediated proteasome degradation.
Highlights
Platinating chemotherapeutic agents, such as cisplatin and carboplatin, are frequently used in ovarian cancer therapy [1,2]
Ataxia-telegiectasia mutated (ATM) depletion or inhibition and phosphatase and tensin homolog (PTEN) degradation in p85α deficient cells: As shown in Figure 1A, the LN18 cell line is p85α deficient compared to the LN229 cell line which is p85α proficient
In the current study we provide evidence that an ATM-p85α-CK2α-PTEN network operates in ovarian tumours that influence platinum sensitivity in patients
Summary
Platinating chemotherapeutic agents, such as cisplatin and carboplatin, are frequently used in ovarian cancer therapy [1,2]. Althoμgh the response rates to platinum based chemotherapy can be up to 60–80%, significant toxicities including nausea, vomiting, peripheral neuropathy, nephrotoxicity, and ototoxicity remain a significant clinical problem [1,2]. If the DNA damaging adducts are not repaired, the cells will accumulate such toxic lesions which lead to cell cycle arrest, generate DNA double strand breaks during replication and cell death [3]. DNA repair deficient tumour cells will be sensitive to platinum therapy. On the other hand, enhanced DNA repair capacity in tumours is a frequent cause of resistance to therapy which negatively impacts on clinical outcomes. The development of predictive biomarkers to personalize ovarian cancer therapy remains a high priority
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