ATG5 is induced by DNA-damaging agents and promotes mitotic catastrophe independent of autophagy.
Anticancer drug therapy activates both molecular cell death and autophagy pathways.Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death.Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.
Affiliation: Institute of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland.
Anticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.
- Antineoplastic Agents/pharmacology*
- Autophagy/drug effects*/genetics
- DNA Damage/drug effects*/genetics
- Microtubule-Associated Proteins/biosynthesis/genetics/metabolism*
- Mitosis/drug effects*/genetics
- Active Transport, Cell Nucleus
- Aurora Kinase B/metabolism
- Cell Line, Tumor
- Cell Nucleus/metabolism
- HSP90 Heat-Shock Proteins/metabolism
- HeLa Cells
- Inhibitor of Apoptosis Proteins/metabolism
- Jurkat Cells
- M Phase Cell Cycle Checkpoints/drug effects/genetics
- RNA Interference
- RNA, Small Interfering
- Signal Transduction/drug effects
© Copyright Policy
f8: Nuclear ATG5 physically interacts with survivin.(a) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide (48-h cultures) were immunoprecipitated with anti-ATG5 or antisurvivin antibodies. A physical interaction between ATG5 and survivin, absent in untreated or GFP-expressing control cells, was detected reciprocally. Full-length immunoblots are provided in Supplementary Fig. S12. (b) Jurkat T cells overexpressing ATG5-ΔNES exhibited no ATG5/survivin molecular interaction. (c) The ATG5-K130R mutant did show coprecipitation of survivin with ATG5. (d) Calpain-mediated, truncated ATG5 (tATG5), that is, the N-terminal part of ATG5, failed to bind survivin. The ATG5-K130R mutant was the control. (e) Confocal microscopy. MDA-MA-231 cells were etoposide treated or transduced as indicated (48-h cultures) and stained. In untreated cells, survivin and ATG5 were in the cytosol with no colocalization. ATG5-transduced cells exhibited ATG5/survivin colocalization in the nucleus. ATG5-ΔNES-transduced cells showed survivin, but not ATG5, in the nuclei of cycling cells. Numerical analysis was performed and Pearson’s correlation coefficients are indicated in the images. Scale bar, 10 μm. Right: Statistical analysis (analysis of variance) is presented in which the results of ten representative cells within each group were integrated. Values are means±s.d. (f) The physical interaction between Aurora B and survivin was analysed reciprocally and reduced in cells expressing high levels of nuclear ATG5. Neither ATG5 nor HSP90 were detectable in the Aurora B-containing immune precipitate, but ATG5 molecularly interacted with survivin. Results representative of three independent experiments. (g) Confocal microscopy. HeLa cells treated with etoposide or transduced as indicated (48-h cultures) were stained with centromere-specific antiserum and anti-Aurora B (top panels) or antisurvivin antibodies (lower panels). In untreated or ATG5-ΔNES transduced cells, both Aurora B and survivin were recruited to prometaphase centromeres and located at the central spindle during anaphase where they no longer colocalized with centromeres. In contrast, etoposide-treated or ATG5-transduced cells recruited much less Aurora B to prometaphase centromeres. Such recruitment, however, was observed during anaphase. Compared with cells in normal mitosis, survivin, although reduced, was recruited to centromeres in prometaphase, but, similar to Aurora B, remained attached to centromeres during anaphase. Such mislocation of Aurora B and survivin was associated with severe chromosome alignment and segregation defects. Colocalization images were prepared using Imaris software. Numerical analysis was performed and correlation coefficients are indicated. Scale bar, 2 μm. (h) Confocal microscopy. Sections of lung (non-small-cell lung cancer) and oesophageal carcinoma stained for ATG5 and survivin expression. In untreated patients, ATG5 expression in cancer cells was slight and mainly cytosolic. Following radio- and/or chemotherapy, ATG5 was preferentially expressed in the nucleus and colocalized with survivin. Overlay images present the average Pearson’s correlation coefficient as calculated with Imaris. Results are representative of three independent experiments. Scale bar, 10 μm.
To test the possibility that survivin interacts physically with ATG5 following etoposide treatment or ectopic overexpression, we performed coimmunoprecipitation assays using antibodies for both ATG5 and survivin. The presence of survivin in the ATG5 immunoprecipitate and, reciprocally, ATG5 in the survivin immunoprecipitate, was unambiguous both with ATG5 overexpression and etoposide treatment of Jurkat T cells (Fig. 8a). In a further coimmunoprecipitation experiment, we demonstrated that ATG5-ΔNES failed to interact with survivin (Fig. 8b), suggesting that the interaction takes place in the nucleus, where ATG5-ΔNES is absent, or, alternatively, that an interaction with survivin depends on the mutated leucine-rich sequence modified in ATG5-ΔNES. In contrast to ATG5-ΔNES, the ATG5-K130R mutant, which is able to mediate mitotic catastrophe (Fig. 3c), interacted with survivin (Fig. 8c). Moreover, calpain-mediated 24-kDa truncated ATG5 (ref. 23) was unable to interact with survivin (Fig. 8d), suggesting that the amino-terminal part of ATG5 is most likely not responsible for the interaction with survivin.