AUTHOR'S VIEW Molecular & Cellular Oncology 3:1, e1030538; January 2016; © 2016 Taylor & Francis Group, LLC
Autophagy is critically required for DNA repair by homologous recombination David A Gillespie1 and Kevin M Ryan2,* 1
Centre for Biomedical Research of the Canary Islands; Department of Anatomy; Anatomical Pathology and Physiology; Faculty of Medicine; Universidad de La Laguna; Tenerife, Spain; 2Cancer Research UK Beatson Institute; Garscube Estate, Switchback Rd, Glasgow, UK
Keywords: autophagy, DNA repair, Chk1, homologous recombination
Autophagy delivers damaged cytoplasmic constituents to lysosomes for degradation, thus preserving cellular integrity and protecting against disease. Remarkably, autophagy-deficient cells also exhibit aberrant DNA damage responses with therapeutic implications, such as suppression of checkpoint kinase-1 function, impaired DNA double-strand break repair by homologous recombination, and increased reliance on error-prone non-homologous end-joining for survival.
Autophagy is a group of cellular processes that serve to deliver cytoplasmic cargoes to lysosomes for degradation. Macroautophagy is the best-studied form and is commonly and hereafter simply referred to as autophagy. During the initiation of autophagy, bulk cytoplasm, proteins, and organelles are recruited to autophagosomes, the characteristic organelles of autophagy.1 These ball-like structures can then fuse with endosomes or multivesicular bodies, but it is the ultimate fusion with lysosomes that leads to degradation of the contents of the autophagosome by lysosomal acidic hydrolases.1 The degraded products are then recycled back into the cytoplasm where they can be used in biosynthetic pathways, or under some circumstances further catabolized to generate energy. Virtually all, if not all, cells have a basal rate of autophagy. In this context autophagy acts to degrade damaged and misfolded proteins as well as damaged organelles. In fact, autophagy is the only process responsible for the turnover of organelles.1 As a result of this fundamental role in protecting cellular fidelity, autophagy is very important for cellular health and its absence can lead to various forms of disease including diabetes, neurodegenerative diseases, and cancer.2 Interestingly, although autophagy is a cytoplasmic process, reports have shown that loss of autophagy leads to genomic
damage.3 To some level this will undoubtedly be caused by decreased protein integrity, which will result in the accumulation of genotoxic levels of reactive oxygen species. All of our cells would, however, accumulate high levels of spontaneous genomic damage without the actions of multiple DNA repair mechanisms, therefore we considered the possibility that loss of autophagy might impair the activity of one or more of these mechanisms. We directly assessed the impact of autophagy loss on DNA repair by isolating fibroblasts from mouse embryos that were homozygous for a loxP-modified (“floxed”) allele of autophagy-related 7 (Atg7), an essential autophagy gene.1 The Atg7 allele was then recombined in vitro by infection with a retrovirus expressing Cre recombinase, leading to rapid and efficient deletion of the floxed Atg7 alleles and loss of Atg7 protein expression. Immediately following ablation of Atg7, cells were exposed to g-irradiation and assayed for DNA repair proficiency. We adopted this approach to limit the time period during which spontaneous DNA damage could accumulate in the autophagy-deficient cellular environment so that the extent of exogenously induced DNA damage would be equivalent in wild type and Atg7-null cells following irradiation. We used these autophagy-deficient and control cell cultures, in combination with previously described plasmid-based
reporter assays, to measure the proficiency of DNA repair via homologous recombination (HR) and non-homologous end joining (NHEJ), the 2 principal mechanisms used by cells to repair DNA double-strand breaks,4 and found that loss of autophagy has no impact on NHEJ whereas HR is greatly diminished in cells lacking Atg7.5 In line with the decreased HR,4 we also found that autophagy-deficient cells exhibit increased levels of cell death by apoptosis (as assessed by sub-G1 DNA content) and a higher incidence of micronuclei,5 suggesting that the shift to reliance on NHEJ leads to spontaneous genomic instability even in the absence of exogenous DNA damage. Cell cycle stage and progression can profoundly affect HR proficiency, but since these parameters were not perturbed by loss of Atg7, we questioned whether a specific functional component critical for HR was somehow affected. We therefore examined several factors known to be required for HR and discovered that autophagy-deficient cells exhibit aberrant regulation and expression of checkpoint kinase-1 (Chk1).5 Immediately (days) after Atg7 deletion we observed that the total level of Chk1 protein was minimally changed in the absence of autophagy, yet its activation in response to radiation- or etoposide-induced DNA damage (as determined by phosphorylation of the critical serine 345 [S345] regulatory site)
*Correspondence to: Kevin M. Ryan; Email: [email protected] Submitted: 03/10/2015; Revised: 03/11/2015; Accepted: 03/11/2015 http://dx.doi.org/10.1080/23723556.2015.1030538
www.tandfonline.com
Molecular & Cellular Oncology
e1030538-1
was markedly attenuated. Furthermore, at later times after recombination (weeks) we observed that autophagy-deficient cells expressed substantially lower levels of total Chk1 as a result of increased proteasomal degradation in addition to attenuated activation. Thus, loss of autophagy results in a rapid, acute, defect in Chk1 activation in response to DNA damage that is followed by a more profound state of Chk1 hypomorphism in autophagy-deficient cells in the long term (Fig. 1). Chk1 is a key effector of multiple aspects of the DNA damage responses and in particular its activation and function is intimately linked to DNA repair by HR.6 The primary signal for Chk1 activation by the upstream kinase ATR (ataxia telangiectasia and Rad3 related) is singlestranded DNA (ssDNA), which in the context of double-strand breaks (DSBs) is generated by strand resection.7 Tracts of ssDNA generated by strand resection also serve to initiate DSB repair by HR through strand invasion of the undamaged sister chromatid in association with Rad51. Activated Chk1 has been shown to phosphorylate Rad51 and promote its recruitment to sites of damage to initiate recombination,8 and crucially, we observed that autophagy-deficient cells failed to form Rad51 foci after irradiation. We believe it is highly probable that the failure to activate Chk1 efficiently in
response to DNA damage in autophagydeficient cells is linked to the observed defect in DNA repair by HR. This linkage could be direct, through the observed decrease in recruitment of Rad51 to sites of damage, although we cannot rule out the possibility that the strand resection process itself or other DNA repair factors in addition to Chk1 are also affected by loss of autophagy. Finally, we hypothesized that, as a result of diminished HR, autophagy-deficient cells should be hyperdependent on NHEJ for repair of DNA double-strand breaks. Indeed, we found that inhibition of NHEJ in Atg7-null cells (by inhibition of DNA-PK, see Fig. 1) resulted in the persistence of genomic damage and increased cell death upon exposure to g-irradiation or treatment with etoposide,5 a chemotherapeutic drug that causes DNA double-strand breaks.9 Moreover, we also found that autophagy-deficient cells are hypersensitive to camptothecin,5 a chemotherapeutic drug that causes “single-ended” DNA double-strand breaks during replication that can only be repaired by HR.10 In summary, our recent findings identified another important impact of the loss of autophagy—diminished HR. Since autophagy-deficient cells are more dependent on the error-prone process of NHEJ this may be one reason why
these cells accumulate spontaneous DNA damage. In addition, our study also identified a synthetic lethal strategy to kill autophagy-deficient cells through inhibition of NHEJ. Moreover, although our study focused on DSB repair by HR, it is likely that Chk1 functional hypomorphism in the absence of autophagy will result in additional vulnerabilities. For example, under conditions of replication stress, thought to be inherent in many tumor cells and exacerbated by genotoxic therapies such as gemcitabine and hydroxyurea, activation of Chk1 is required to prevent replication fork collapse and premature entry into mitosis with unreplicated DNA, outcomes that lead rapidly to cell death. Autophagy deficiency and Chk1 hypomorphism may therefore create additional synthetic lethal interactions that could be exploited therapeutically in addition to the inhibition of NHEJ that we have described. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Funding
Work in the Ryan lab is funded by Cancer Research UK. DAG is an IMBRAIN investigator at the Universidad de La Laguna (FP7-REGPOT-2012CT2012–31637-IMBRAIN) supported by the EU FP7 program and the Gobierno de Canarias. References
Figure 1. Loss of autophagy impairs DNA repair by homologous recombination and uncovers a synthetic lethal strategy to kill autophagy-deficient cells. Cells with deletion of the essential autophagy gene Atg7 exhibit degradation and attenuated activation of checkpoint kinase 1 (Chk1) and diminished repair of DNA double-strand breaks by homologous recombination. Consequently, cells lacking autophagy become hyperdependent on non-homologous end joining for repair of DNA double-strand breaks. As a result, inhibition of non-homologous end joining prior to induction of DNA-damage causes persistence of genetic lesions and synthetic lethal killing of autophagy-deficient cells. DNA-PK, DNA-dependent protein kinase.
e1030538-2
Molecular & Cellular Oncology
1. Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nat Cell Biol 2007; 9:1102-9; PMID:17909521; http://dx.doi.org/ 10.1038/ncb1007-1102 2. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008; 132:27-42; PMID:18191218; http://dx.doi.org/10.1016/j.cell.2007.12.018 3. Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G, Mathew R, Jin S, White E. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes and Dev 2007; 21:1621-35; PMID:17606641; http://dx.doi.org/10.1101/ gad.1565707 4. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 2004; 73:39-85; PMID:15189136; http:// dx.doi.org/10.1146/annurev. biochem.73.011303.073723 5. Liu EY, Xu N, O’Prey J, Lao LY, Joshi S, Long JS, O’Prey M, Croft DR, Beaumatin F, Baudot AD, et al.
Volume 3 Issue 1
Loss of autophagy causes a synthetic lethal deficiency in DNA repair. Proc Natl Acad Sci U S A 2015; 112:7738; PMID:25568088; http://dx.doi.org/10.1073/ pnas.1409563112 6. Smith J, Tho LM, Xu N, Gillespie DA. The ATMChk2 and ATR-Chk1 pathways in DNA damage signaling and cancer. Adv Cancer Res 2010; 108:73-112; PMID:21034966; http://dx.doi.org/10.1016/B978-012-380888-2.00003-0 7. Symington LS, Gautier J. Double-strand break end resection and repair pathway choice. Annu
www.tandfonline.com
Rev Genet 2011; 45:247-71; PMID:21910633; http://dx.doi.org/10.1146/annurev-genet-110410132435 8. Sorensen CS, Hansen LT, Dziegielewski J, Syljuasen RG, Lundin C, Bartek J, Helleday T. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nat Cell Biol 2005; 7:195-201; PMID:15665856; http://dx.doi.org/ 10.1038/ncb1212 9. Baldwin EL, Osheroff N. Etoposide, topoisomerase II and cancer. Curr Med Chem Anticancer Agents 2005;
Molecular & Cellular Oncology
5:363-72; PMID:16101488; http://dx.doi.org/ 10.2174/1568011054222364 10. Arnaudeau C, Lundin C, Helleday T. DNA doublestrand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells. J Mol Biol 2001; 307: 1235-45; PMID:11292338; http://dx.doi.org/ 10.1006/jmbi.2001.4564
e1030538-3
Autophagy is critically required for DNA repair by homologous recombination David A Gillespie1 and Kevin M Ryan2,* 1
Centre for Biomedical Research of the Canary Islands; Department of Anatomy; Anatomical Pathology and Physiology; Faculty of Medicine; Universidad de La Laguna; Tenerife, Spain; 2Cancer Research UK Beatson Institute; Garscube Estate, Switchback Rd, Glasgow, UK
Keywords: autophagy, DNA repair, Chk1, homologous recombination
Autophagy delivers damaged cytoplasmic constituents to lysosomes for degradation, thus preserving cellular integrity and protecting against disease. Remarkably, autophagy-deficient cells also exhibit aberrant DNA damage responses with therapeutic implications, such as suppression of checkpoint kinase-1 function, impaired DNA double-strand break repair by homologous recombination, and increased reliance on error-prone non-homologous end-joining for survival.
Autophagy is a group of cellular processes that serve to deliver cytoplasmic cargoes to lysosomes for degradation. Macroautophagy is the best-studied form and is commonly and hereafter simply referred to as autophagy. During the initiation of autophagy, bulk cytoplasm, proteins, and organelles are recruited to autophagosomes, the characteristic organelles of autophagy.1 These ball-like structures can then fuse with endosomes or multivesicular bodies, but it is the ultimate fusion with lysosomes that leads to degradation of the contents of the autophagosome by lysosomal acidic hydrolases.1 The degraded products are then recycled back into the cytoplasm where they can be used in biosynthetic pathways, or under some circumstances further catabolized to generate energy. Virtually all, if not all, cells have a basal rate of autophagy. In this context autophagy acts to degrade damaged and misfolded proteins as well as damaged organelles. In fact, autophagy is the only process responsible for the turnover of organelles.1 As a result of this fundamental role in protecting cellular fidelity, autophagy is very important for cellular health and its absence can lead to various forms of disease including diabetes, neurodegenerative diseases, and cancer.2 Interestingly, although autophagy is a cytoplasmic process, reports have shown that loss of autophagy leads to genomic
damage.3 To some level this will undoubtedly be caused by decreased protein integrity, which will result in the accumulation of genotoxic levels of reactive oxygen species. All of our cells would, however, accumulate high levels of spontaneous genomic damage without the actions of multiple DNA repair mechanisms, therefore we considered the possibility that loss of autophagy might impair the activity of one or more of these mechanisms. We directly assessed the impact of autophagy loss on DNA repair by isolating fibroblasts from mouse embryos that were homozygous for a loxP-modified (“floxed”) allele of autophagy-related 7 (Atg7), an essential autophagy gene.1 The Atg7 allele was then recombined in vitro by infection with a retrovirus expressing Cre recombinase, leading to rapid and efficient deletion of the floxed Atg7 alleles and loss of Atg7 protein expression. Immediately following ablation of Atg7, cells were exposed to g-irradiation and assayed for DNA repair proficiency. We adopted this approach to limit the time period during which spontaneous DNA damage could accumulate in the autophagy-deficient cellular environment so that the extent of exogenously induced DNA damage would be equivalent in wild type and Atg7-null cells following irradiation. We used these autophagy-deficient and control cell cultures, in combination with previously described plasmid-based
reporter assays, to measure the proficiency of DNA repair via homologous recombination (HR) and non-homologous end joining (NHEJ), the 2 principal mechanisms used by cells to repair DNA double-strand breaks,4 and found that loss of autophagy has no impact on NHEJ whereas HR is greatly diminished in cells lacking Atg7.5 In line with the decreased HR,4 we also found that autophagy-deficient cells exhibit increased levels of cell death by apoptosis (as assessed by sub-G1 DNA content) and a higher incidence of micronuclei,5 suggesting that the shift to reliance on NHEJ leads to spontaneous genomic instability even in the absence of exogenous DNA damage. Cell cycle stage and progression can profoundly affect HR proficiency, but since these parameters were not perturbed by loss of Atg7, we questioned whether a specific functional component critical for HR was somehow affected. We therefore examined several factors known to be required for HR and discovered that autophagy-deficient cells exhibit aberrant regulation and expression of checkpoint kinase-1 (Chk1).5 Immediately (days) after Atg7 deletion we observed that the total level of Chk1 protein was minimally changed in the absence of autophagy, yet its activation in response to radiation- or etoposide-induced DNA damage (as determined by phosphorylation of the critical serine 345 [S345] regulatory site)
*Correspondence to: Kevin M. Ryan; Email: [email protected] Submitted: 03/10/2015; Revised: 03/11/2015; Accepted: 03/11/2015 http://dx.doi.org/10.1080/23723556.2015.1030538
www.tandfonline.com
Molecular & Cellular Oncology
e1030538-1
was markedly attenuated. Furthermore, at later times after recombination (weeks) we observed that autophagy-deficient cells expressed substantially lower levels of total Chk1 as a result of increased proteasomal degradation in addition to attenuated activation. Thus, loss of autophagy results in a rapid, acute, defect in Chk1 activation in response to DNA damage that is followed by a more profound state of Chk1 hypomorphism in autophagy-deficient cells in the long term (Fig. 1). Chk1 is a key effector of multiple aspects of the DNA damage responses and in particular its activation and function is intimately linked to DNA repair by HR.6 The primary signal for Chk1 activation by the upstream kinase ATR (ataxia telangiectasia and Rad3 related) is singlestranded DNA (ssDNA), which in the context of double-strand breaks (DSBs) is generated by strand resection.7 Tracts of ssDNA generated by strand resection also serve to initiate DSB repair by HR through strand invasion of the undamaged sister chromatid in association with Rad51. Activated Chk1 has been shown to phosphorylate Rad51 and promote its recruitment to sites of damage to initiate recombination,8 and crucially, we observed that autophagy-deficient cells failed to form Rad51 foci after irradiation. We believe it is highly probable that the failure to activate Chk1 efficiently in
response to DNA damage in autophagydeficient cells is linked to the observed defect in DNA repair by HR. This linkage could be direct, through the observed decrease in recruitment of Rad51 to sites of damage, although we cannot rule out the possibility that the strand resection process itself or other DNA repair factors in addition to Chk1 are also affected by loss of autophagy. Finally, we hypothesized that, as a result of diminished HR, autophagy-deficient cells should be hyperdependent on NHEJ for repair of DNA double-strand breaks. Indeed, we found that inhibition of NHEJ in Atg7-null cells (by inhibition of DNA-PK, see Fig. 1) resulted in the persistence of genomic damage and increased cell death upon exposure to g-irradiation or treatment with etoposide,5 a chemotherapeutic drug that causes DNA double-strand breaks.9 Moreover, we also found that autophagy-deficient cells are hypersensitive to camptothecin,5 a chemotherapeutic drug that causes “single-ended” DNA double-strand breaks during replication that can only be repaired by HR.10 In summary, our recent findings identified another important impact of the loss of autophagy—diminished HR. Since autophagy-deficient cells are more dependent on the error-prone process of NHEJ this may be one reason why
these cells accumulate spontaneous DNA damage. In addition, our study also identified a synthetic lethal strategy to kill autophagy-deficient cells through inhibition of NHEJ. Moreover, although our study focused on DSB repair by HR, it is likely that Chk1 functional hypomorphism in the absence of autophagy will result in additional vulnerabilities. For example, under conditions of replication stress, thought to be inherent in many tumor cells and exacerbated by genotoxic therapies such as gemcitabine and hydroxyurea, activation of Chk1 is required to prevent replication fork collapse and premature entry into mitosis with unreplicated DNA, outcomes that lead rapidly to cell death. Autophagy deficiency and Chk1 hypomorphism may therefore create additional synthetic lethal interactions that could be exploited therapeutically in addition to the inhibition of NHEJ that we have described. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Funding
Work in the Ryan lab is funded by Cancer Research UK. DAG is an IMBRAIN investigator at the Universidad de La Laguna (FP7-REGPOT-2012CT2012–31637-IMBRAIN) supported by the EU FP7 program and the Gobierno de Canarias. References
Figure 1. Loss of autophagy impairs DNA repair by homologous recombination and uncovers a synthetic lethal strategy to kill autophagy-deficient cells. Cells with deletion of the essential autophagy gene Atg7 exhibit degradation and attenuated activation of checkpoint kinase 1 (Chk1) and diminished repair of DNA double-strand breaks by homologous recombination. Consequently, cells lacking autophagy become hyperdependent on non-homologous end joining for repair of DNA double-strand breaks. As a result, inhibition of non-homologous end joining prior to induction of DNA-damage causes persistence of genetic lesions and synthetic lethal killing of autophagy-deficient cells. DNA-PK, DNA-dependent protein kinase.
e1030538-2
Molecular & Cellular Oncology
1. Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nat Cell Biol 2007; 9:1102-9; PMID:17909521; http://dx.doi.org/ 10.1038/ncb1007-1102 2. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008; 132:27-42; PMID:18191218; http://dx.doi.org/10.1016/j.cell.2007.12.018 3. Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G, Mathew R, Jin S, White E. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes and Dev 2007; 21:1621-35; PMID:17606641; http://dx.doi.org/10.1101/ gad.1565707 4. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 2004; 73:39-85; PMID:15189136; http:// dx.doi.org/10.1146/annurev. biochem.73.011303.073723 5. Liu EY, Xu N, O’Prey J, Lao LY, Joshi S, Long JS, O’Prey M, Croft DR, Beaumatin F, Baudot AD, et al.
Volume 3 Issue 1
Loss of autophagy causes a synthetic lethal deficiency in DNA repair. Proc Natl Acad Sci U S A 2015; 112:7738; PMID:25568088; http://dx.doi.org/10.1073/ pnas.1409563112 6. Smith J, Tho LM, Xu N, Gillespie DA. The ATMChk2 and ATR-Chk1 pathways in DNA damage signaling and cancer. Adv Cancer Res 2010; 108:73-112; PMID:21034966; http://dx.doi.org/10.1016/B978-012-380888-2.00003-0 7. Symington LS, Gautier J. Double-strand break end resection and repair pathway choice. Annu
www.tandfonline.com
Rev Genet 2011; 45:247-71; PMID:21910633; http://dx.doi.org/10.1146/annurev-genet-110410132435 8. Sorensen CS, Hansen LT, Dziegielewski J, Syljuasen RG, Lundin C, Bartek J, Helleday T. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nat Cell Biol 2005; 7:195-201; PMID:15665856; http://dx.doi.org/ 10.1038/ncb1212 9. Baldwin EL, Osheroff N. Etoposide, topoisomerase II and cancer. Curr Med Chem Anticancer Agents 2005;
Molecular & Cellular Oncology
5:363-72; PMID:16101488; http://dx.doi.org/ 10.2174/1568011054222364 10. Arnaudeau C, Lundin C, Helleday T. DNA doublestrand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells. J Mol Biol 2001; 307: 1235-45; PMID:11292338; http://dx.doi.org/ 10.1006/jmbi.2001.4564
e1030538-3
