Marco B E Schaaf1, Barry Jutten1, Tom G Keulers1, Kim G M Savelkouls1, Hanneke J M Peeters1, Twan van den Beucken2, Frederik-Jan van Schooten3, Roger W Godschalk3, Marc Vooijs1, Kasper M A Rouschop4. 1. Department of Radiation Oncology (Maastro Lab), GROW School for Oncology & Developmental Biology, Maastricht University Medical Center+, The Netherlands. 2. Department of Toxicogenomics, The Netherlands. 3. Department of Toxicology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University, The Netherlands. 4. Department of Radiation Oncology (Maastro Lab), GROW School for Oncology & Developmental Biology, Maastricht University Medical Center+, The Netherlands. Electronic address: Kasper.Rouschop@maastrichtuniversity.nl.
Abstract
BACKGROUND: (Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in human cancer cell lines. MATERIALS AND METHODS: Changes in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown. RESULTS: IR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated γ-H2AX formation and DNA damage repair. CONCLUSION: Our data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation.
BACKGROUND: (Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in humancancer cell lines. MATERIALS AND METHODS: Changes in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown. RESULTS: IR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated γ-H2AX formation and DNA damage repair. CONCLUSION: Our data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation.
Authors: Barry Jutten; Tom G Keulers; Hanneke J M Peeters; Marco B E Schaaf; Kim G M Savelkouls; Inge Compter; Ruud Clarijs; Olaf E M G Schijns; Linda Ackermans; Onno P M Teernstra; Marijke I Zonneveld; Resi M E Colaris; Ludwig Dubois; Marc A Vooijs; Johan Bussink; Julio Sotelo; Jan Theys; Guido Lammering; Kasper M A Rouschop Journal: Autophagy Date: 2018-01-29 Impact factor: 16.016