RESUMEN

DNA damage has been implicated in the stimulation of the type 1 interferon (T1IFN) response. Here, we show that downregulation of the DNA repair protein, polynucleotide kinase/phosphatase (PNKP), in a variety of cell lines causes robust phosphorylation of STAT1, upregulation of interferon-stimulated genes and persistent accumulation of cytosolic DNA, all of which are indicators for the activation of the T1IFN response. Furthermore, this did not require damage induction by ionizing radiation. Instead, our data revealed that production of reactive oxygen species (ROS) synergises with PNKP loss to potentiate the T1IFN response, and that loss of PNKP significantly compromises mitochondrial DNA (mtDNA) integrity. Depletion of mtDNA or treatment of PNKP-depleted cells with ROS scavengers abrogated the T1IFN response, implicating mtDNA as a significant source of the cytosolic DNA required to potentiate the T1IFN response. The STING signalling pathway is responsible for the observed increase in the pro-inflammatory gene signature in PNKP-depleted cells. While the response was dependent on ZBP1, cGAS only contributed to the response in some cell lines. Our data have implications for cancer therapy, since PNKP inhibitors would have the potential to stimulate the immune response, and also to the neurological disorders associated with PNKP mutation.

Asunto(s)

Enzimas Reparadoras del ADN , ADN Mitocondrial , Interferón Tipo I , Fosfotransferasas (Aceptor de Grupo Alcohol) , Radiación Ionizante , Especies Reactivas de Oxígeno , Humanos , Interferón Tipo I/metabolismo , Interferón Tipo I/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Reparación del ADN , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Daño del ADN , Línea Celular , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Transducción de Señal , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Fosforilación , Citosol/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética

RESUMEN

Cellular adaptations to hypoxia promote resistance to ionizing radiation (IR). This presents a challenge for treatment of head and neck cancer (HNC) that relies heavily on radiotherapy. Standard radiosensitizers often fail to reach diffusion-restricted hypoxic cells, whereas nitroimidazoles (NIs) [such as iodoazomycin arabinofuranoside (IAZA) and fluoroazomycin arabinofuranoside (FAZA)] can preferentially accumulate in hypoxic tumours. Here, we explored if the hypoxia-selective uptake of IAZA and FAZA could be harnessed to make HNC cells (FaDu) susceptible to radiation therapy. Cellular response to treatment was assessed through clonogenic survival assays and by monitoring DNA damage (immunofluorescence staining of DNA damage markers, γ-H2AX and p-53BP1, and by alkaline comet assay). The effects of reoxygenation were studied using the following assays: estimation of nucleoside incorporation to assess DNA synthesis rates, immunofluorescent imaging of chromatin-associated replication protein A as a marker of replication stress, and quantification of reactive oxygen species (ROS). Both IAZA and FAZA sensitized hypoxic HNC cells to IR, albeit the former is a better radiosensitizer. Radiosensitization by these compounds was restricted only to hypoxic cells, with no visible effects under normoxia. IAZA and FAZA impaired cellular adaptation to reoxygenation; high levels of ROS, reduced DNA synthesis capacity, and signs of replication stress were observed in reoxygenated cells. Overall, our data highlight the therapeutic potentials of IAZA and FAZA for targeting hypoxic HNC cells and provide rationale for future preclinical studies.