Temporal DNA-PK activation drives genomic instability and therapy resistance in glioma stem cells
Yanling Wang, Haineng Xu, Tianrun Liu, Menggui Huang, Param-Puneet Butter, Chunsheng Li, Lin Zhang, Gary D. Kao, Yanqing Gong, Amit Maity, Constantinos Koumenis, Yi Fan
Yanling Wang, Haineng Xu, Tianrun Liu, Menggui Huang, Param-Puneet Butter, Chunsheng Li, Lin Zhang, Gary D. Kao, Yanqing Gong, Amit Maity, Constantinos Koumenis, Yi Fan
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Research Article Oncology Stem cells

Temporal DNA-PK activation drives genomic instability and therapy resistance in glioma stem cells

Abstract

Cancer stem cells (CSCs) — known to be resistant to genotoxic radiation and chemotherapy — are fundamental to therapy failure and cancer relapse. Here, we reveal that glioma CSCs are hypersensitive to radiation, but a temporal DNA repair mechanism converts the intrinsic sensitivity to genomic instability and treatment resistance. Transcriptome analysis identifies DNA-dependent protein kinase (DNA-PK) as a predominant DNA repair enzyme in CSCs. Notably, DNA-PK activity is suppressed after irradiation when ROS induce the dissociation of DNA-PKcs with Ku70/80, resulting in delayed DNA repair and radiosensitivity; subsequently, after ROS clearance, the accumulated DNA damage and robust activation of DNA-PK induce genomic instability, facilitated by Rad50-mediated cell-cycle arrest, leading to enhanced malignancy, CSC overgrowth, and radioresistance. Finally, we show a requisite in vivo role for DNA-PK in CSC-mediated radioresistance and glioma progression. These findings identify a time-sensitive mechanism controlling CSC resistance to DNA-damaging treatments and suggest DNA-PK/Rad50 as promising targets for CSC eradication.

Authors

Yanling Wang, Haineng Xu, Tianrun Liu, Menggui Huang, Param-Puneet Butter, Chunsheng Li, Lin Zhang, Gary D. Kao, Yanqing Gong, Amit Maity, Constantinos Koumenis, Yi Fan

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Figure 5

Delayed DNA-PK activation induces genomic instability and enhances cell malignancy in CSCs.

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Delayed DNA-PK activation induces genomic instability and enhances cell ...
(A–C) IN528 CSCs and matched non-CSCs were either irradiated with 5-Gy x-ray or were not irradiated (sham control). (A) At 2 days after irradiation, the cells were subjected to micronucleus analyses. Representative images and quantification of data (mean ± SD, n = around 1,000 cells). Scale bar: 50 μm. (B and C) Cells were analyzed by flow cytometry for aneuploidy. (B) Representative sorting. (C) Quantified data (mean ± SEM, n = 3). (D) IN528 CSCs were pretreated with 1 μM KU60019 (inhibitor of ATM [iATM]), NU7441 (inhibitor of DNA-PK [iDNA-PK]), or control 0.1% DMSO, and irradiated with 5-Gy x-ray. At different times after irradiation, cells were subjected to aneuploidy analysis by flow cytometry (mean ± SEM, n = 3). (E–I) IN528 CSCs were transduced with lentivirus that expresses shRNAs targeting control GFP, DNA-PK, Rad50, or ATM, followed by puromycin selection for stable expression cell lines. Cells were irradiated by 5-Gy x-ray. (E) Cells were analyzed by immunoblot. (F) 24 hours after irradiation, cells were stained with propidium iodide and analyzed by flow cytometry for cell cycle. Representative sorting and quantitative results (mean ± SEM, n = 3). (G) 72 hours after irradiation, cells were stained with propidium iodide and analyzed by flow cytometry for cell aneuploidy (mean ± SEM, n = 3). (H) Cells were subjected to viability analysis at different time points after radiation (mean ± SEM, n = 3-6). (I) At 2 weeks after irradiation, cells were subjected to neurosphere formation/survival fraction analysis (mean ± SEM, n = 3–5).