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 1

Time-dependent radioresistance is associated with increased DNA damage and delayed γ-H2AX and DNA repair after irradiation in glioma CSCs.

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Time-dependent radioresistance is associated with increased DNA damage a...
(A) Human patient-derived IN528 CSCs and the matched non–cancer stem cells (non-CSCs) were cultured in serum-free stem cell medium and either irradiated by 5-Gy x-ray or were not irradiated. The number of viable cells was determined using cell viability assays. The data are presented as a percentage based on the number of viable nonirradiated cells of the same type (mean ± SEM, n = 3). (B) IN528 CSCs and matched non-CSCs were irradiated with 5-Gy x-ray. Cells were harvested at different times after irradiation. DNA damage was assessed using the single-cell gel electrophoresis comet assay with a neutral condition. Representative images and quantification of the percentage of DNA in the comet tails (mean ± SD). Scale bar: 50 μm. (C) IN528 CSCs and matched non-CSCs and glioma U87 cells were irradiated with 5-Gy x-ray. Cell lysates were immunoblotted with anti–P-H2AX-Ser139 (γ-H2AX) and anti-GAPDH antibodies. Representative blots. γ-H2AX band intensity was quantified and expressed as a percentage of its expression level before radiation. (D) CD133+ CSCs and CD133– non-CSCs were isolated from IN528 GBM cells and irradiated with 5-Gy x-ray. Cell lysates were immunoblotted with anti–P-H2AX-Ser139 (γ-H2AX) and anti-GAPDH antibodies. (E) IN528 CSCs and matched non-CSCs were irradiated with 5-Gy x-ray. Nuclear extracts were incubated with linearized DNA in NHEJ reaction buffer, followed by electrophoresis and gel imaging. The reaction mixtures were immunoblotted using an anti-PCNA antibody. Band intensity of multimers was analyzed.