Journal Article

Atovaquone-induced activation of the PERK/eIF2α signaling axis mitigates metabolic radiosensitisation

Chambers, Sarah, Islam, Rayhanul, Feng, Jie, Pathak, Varun, Coulter, Jonathan A., Wang, Tongchuan, Byrne, Niall M., Medina, Reinhold J.

2025

Overview

Background Hypoxia, a key feature of most solid tumours, including head and neck cancer, reduces radiotherapy efficacy by promoting radiation resistance through micro-environmental and genomic alterations. Addressing these resistance mechanisms is crucial, as radiotherapy remains central to managing locally advanced disease. Atovaquone, a mitochondrial electron transport chain complex III inhibitor, is reported to reduce tumour hypoxia in preclinical models, however, this response does not consistently enhance radiation sensitivity. This work examines the potential of atovaquone to modify the hypoxic response in models of head and neck squamous cell carcinoma (HNSCC), uncovering an adaptive resistance mechanism driven by integrated stress response (ISR) signaling that limits the radiosensitising potential of this approach. Methods The bioenergetic response of HNSCC cells to atovaquone was assessed using the Seahorse XFe96 Analyzer with the XF Cell Mito Stress Test. Radiation dose modifying effects of atovaquone were tested by clonogenic survival assays, while ROS yields were analysed by flow cytometry. Western blotting and quantitative reverse transcription-PCR were employed to study activation of ISR signaling and the overall influence of atovaquone on the hypoxic response. Finally, the role of the ISR activation in modulating radiosensitivity was investigated using both siRNA and pharmacological inhibition of eIF2α, a central regulator of the ISR. Results Herein we report that atovaquone significantly disrupts mitochondrial respiration, triggering phosphorylation of eIF2α, a pivotal regulator of the ISR, and a master regulator of protein synthesis. Notably, atovaquone also increased the autophagic load under hypoxia, while autophagy inhibition significantly enhanced apoptosis, improving radiation sensitivity. Combined eIF2α inhibition and atovaquone promotes cell cycle redistribution and significantly enhances mitochondrial ROS production and compared to atovaquone alone, restoring atovaquone mediated radiosensitisation. Conclusions Our data highlight dual counter opposing impacts of atovaquone, serving as a hypoxic radiosensitiser though oxidative phosphorylation (OXPHOS) inhibition, but also in promoting stress induced ISR signaling, conferring resistance to radiation treatment. Importantly, if ISR activation is impeded, the metabolic radiosensitising properties of atovaquone is restored. These data provide a new insight to a molecular response that could help counteract hypoxia-induced radioresistance. Plain English summary Solid tumours, including head and neck cancer, have poorly oxygenated regions, termed hypoxic, making them less responsive to radiotherapy. Hypoxia not only changes the local tumour environment but also alters the genetic behaviour of the tumour, contributing to treatment resistance. Atovaquone, a drug that inhibits oxygen dependent respiration with the mitochondria, has been shown to sensitise tumour models to radiation by reducing oxygen consumption, a strategy referred to as metabolic radiosensitisation. However, this response is inconsistent between tumour models. In this study, we explored how atovaquone affects the response of head and neck cancer cells to radiation under both normoxic (21% O 2 ) and hypoxic (0.5% O 2 ) conditions. We found that while atovaquone disrupts mitochondrial energy production, it also activates a stress response pathway called the integrated stress response (ISR). This highly conserved pathway helps protect tumour cells against the metabolism altering properties of atovaquone, ultimately reducing the effectiveness of radiation. Using inhibitors (both drug and genetic) of key proteins in the integrated stress response signaling pathway, tumour cells were rendered more sensitive to radiation, enhancing cell death. These results reveal that while atovaquone has the potential to enhance radiation therapy by reducing hypoxia, it simultaneously triggers protective stress response mechanisms within the tumour cells. By targeting the ISR, we could improve the effectiveness of atovaquone and radiation therapy together. This research offers new strategies to tackle hypoxia-related treatment resistance in cancer.

• Publisher: BioMed Central,Springer Nature B.V,BMC
• Subject: Apoptosis / Atovaquone / Atovaquone - pharmacology / Autophagy / Biomedical and Life Sciences / Cancer therapies / Cell Biology / Cell cycle / Cell Line, Tumor / Cells / Cellular stress response / Cytokines and Growth Factors / Drug dosages / eIF-2 Kinase - metabolism / Electron transport chain / ER stress / Eukaryotic Initiation Factor-2 - metabolism / Flow cytometry / Head & neck cancer / Head and neck carcinoma / Humans / Hypoxia / Kinases / Life Sciences / Metabolic rate / Metabolism / Mitochondria / Mitochondria - drug effects / Mitochondria - metabolism / Oxidative phosphorylation / Phosphorylation / Polyclonal antibodies / Protein biosynthesis / Protein-Ligand Interactions / Radiation therapy / Radiation Tolerance - drug effects / Radiation-Sensitizing Agents - pharmacology / Radioresistance / Radiosensitisation / Radiosensitivity / Radiosensitization / Radiosensitizers / Reactive oxygen species / Reactive Oxygen Species - metabolism / Receptors / Respiration / Reverse transcription / Signal Transduction - drug effects / siRNA / Solid tumors / Squamous cell carcinoma / Squamous Cell Carcinoma of Head and Neck - metabolism / Squamous Cell Carcinoma of Head and Neck - pathology / Treatment resistance / Western blotting