Review

Acetylation: A Novel Link between Double-Strand BreakRepair and Autophagy

Ghadeer Shubassi1, Thomas Robert1, Fabio Vanoli1, Saverio Minucci2,3, and Marco Foiani1,3

AbstractHistone deacetylase (HDAC) inhibitors are clinically relevant because they are used as anticancer drugs. Recent

evidence sheds light on an intriguing connection among the DNA damage response (DDR), protein acetylation,and autophagy. HDAC inhibitors have been shown to counteract key steps in the cellular response to double-strand break formation by affecting checkpoint activation, homologous recombination–mediated repair of DNAlesions, and stability of crucial enzymes involved in resection of DNA ends. The degradation of the resectionfactors depends on autophagy, which plays a detrimental role when cells are in a hyperacetylated state andexperience treatment with radiomimetic anticancer drugs. Future work will be required to further investigate themechanisms underlying the link between acetylation, autophagy, and the DDR, as well as the significance ofmTORC1 inhibitors, which are potent inducers of autophagy that are now used in cancer treatment. Cancer Res;72(6); 1332–5. �2012 AACR.

IntroductionOne hallmark of cancer and aging cells is an unstable

genome, which results from DNA damage. Eukaryotic cellsdeveloped intricate surveillance mechanisms to maintaintheir genome integrity and to repair DNA lesions. Followinggenotoxic stress, the evolutionary conserved DNA damageresponse (DDR) is activated and, in turn, coordinates DNArepair with cell-cycle events. In Saccharomyces cerevisiae, theDDR is mediated by two phosphoinositide 3-kinases: Mec1(ATR in humans) and Tel1 (ATM in humans). ATR sensessingle-stranded DNA (ssDNA) coated with replication proteinA (RPA), whereas ATM monitors the presence of DNA breaks.This mediation of the DDR results in activation of signaltransduction pathways, which involves the Rad53 (Chk2 inhumans) and Chk1 (Chk1 in humans) kinases (1). Thesecascades end in phosphorylation of several substrates involvedin cell-cycle arrest and DNA repair (2). Defects in the DDRpathway components can lead to genome instability andcancer. In fact, several studies showed that replication stressand double-strand breaks (DSB), which lead to activation ofDDR, occur in early tumor lesions (3, 4).

DSBs can arise spontaneously as a consequence of certainmetabolic reactions, can be induced by extrinsic genotoxicevents, such as ionizing radiation, or can occur in a pro-grammedmanner as inmeiosis. These lesionsmust be prompt-ly repaired to prevent loss of genetic material and maintaingenome integrity. Induction of a single DSB is sufficient toactivate the DDR in mitotically growing cells (5). In G2, DSBsare typically repaired by the homologous recombination (HR)pathway, which is initiated by 50-end resection followed by thecoating of 30-ssDNA overhangs with RPA. The RPA–ssDNAnucleofilaments are thought to be the signal for ATR activation(6). S. cerevisiae has been instrumental in elucidating theconserved pathways involved in DSB repair. Several proteinsmediate the resection process, including the MRX complexthat consists of Mre11-Rad50-Xrs2, Sae2, Exo1, Sgs1, as well asDna2 (7). RPA is then exchanged with Rad51, the RecA strandexchange protein, which in turn promotes DNA recombinationprocesses. Inefficient and/or faulty DSB repair inevitably leadto genome rearrangements and cancer. A new phenomenon,called chromothripsis, has recently been described in cancercells (8). It involves the rearrangement of broken chromosomesand can be acquired from one cellular catastrophic event.Remarkably, cells can survive such a crisis by selectivelyrearranging their genomes, although the consequence iscancer.

Regulation of expression and activity of key DNA repair andcheckpoint proteins is essential and is mediated partly byposttranslational modifications (PTM), such as acetylation,phosphorylation, or ubiquitination. Cross-talk between differ-ent PTMs is key for regulation of DNA repair pathways.Acetylation, which is mediated by acetyltransferases (HAT)and histone deacetylases (HDAC), not only affects gene expres-sion through its modulation of histone tails but also hasrecently been implicated in regulating nonhistone proteins.Intriguingly, a large class of acetylated proteins is involved in

Authors' Affiliations: 1Fondazione Istituto FIRC di Oncologia Molecolare(IFOM); 2European Institute of Oncology; 3DSBB-Universit�a degli Studi diMilano, Milan, Italy

Note:Current address for T. Robert, Institut deG�en�etiqueHumaine CNRS-UPR1142, �Equipe M�eiose et Recombinaison, Montpellier, France; andcurrent address for F. Vanoli, Memorial Sloan-Kettering Cancer Center,New York, New York.

Corresponding Author:Marco Foiani, FIRC Institute of Molecular Oncol-ogy, Via Celoria 26, Milan 20133, Italy. Phone: 39-02-574-303221; Fax: 39-02-574-303231; E-mail: marco.foiani@ifom-ieo-campus.it

doi: 10.1158/0008-5472.CAN-11-3172

�2012 American Association for Cancer Research.

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DNA damage and repair; examples include p53, ATM, Ku70,and Exo1 (9). The biologic significance of protein acetylation islargely not understood.

ValproicAcidReveals a LinkbetweenAcetylationand Double-Strand Break RepairHDAC inhibitors are currently used in cancer treatment (9).

To investigate the importance of protein acetylation in theyeast DSB repair pathway, Robert and colleagues (10) usedvalproic acid (VPA), a U.S. Food and Drug Administration–approved antiepileptic drug shown to be anHDAC class I and IIinhibitor and, therefore, now used in cancer clinical trials (11).Cells experiencing a single irreparable DSB were treatedwith VPA to induce a hyperacetylated state. Intriguingly, VPAtreatment counteracted DDR activation. The authors thendissected the steps of DSB repair that are defective in VPA-treated cells and found that DSB resection rates were slower inVPA. They found that, whereas loading ofMre11 at theDSB sitewas unaffected, its unloading seemed to be defective in VPA.This phenotype resembled that of sae2 cells (12), raising thepossibility that Sae2 endonuclease might be nonfunctional inVPA. Indeed, the levels of Sae2 protein in VPA-treated cellssignificantly declined. This finding not only applies to Sae2 butalso to Exo1, suggesting that the DSB repair defect in VPA isdue to the absence of at least these 2 critical nucleasesimplicated in resection. Consistently, Sae2 protein is hyper-acetylated in VPA-treated cells.A previous study by Kaidi and colleagues showed that CtIP,

the mammalian counterpart of Sae2, is also acetylated inmammalian cells (13), indicating that acetylation as a PTMon Sae2/CtIP is highly conserved. Interestingly, also in mam-mals, a hyperacetylated state induced by treatments withHDAC inhibitors led to DSB repair defects, although CtIPdegradation was not reported. Although acetylation of Exo1was described in mammals (9), it was not found in yeast (10).

A Role for Autophagy in DNA Damage ResponseAn interesting study by Jeong and colleagues showed that

mutantHuntingtin (Htt) protein, which undergoes acetylation,is degraded by macroautophagy (referred to as autophagyhereon; ref. 14). Autophagy is a highly conserved catabolicprocess involved in degradation of cytoplasmic components inthe vacuole (lysosomes in humans). During autophagy, adouble-membrane vesicle, named the autophagosome, formsand nonspecifically engulfs the cytoplasmic material thattypically includes organelles, pathogens, or protein aggregates.Upon fusion of the autophagosome with the vacuole, thecontent is released and consequently gets degraded by lyso-somal enzymes. This degradation allows recycling of aminoacids andnutrients that are necessary for survival of cells understress conditions.Much of our knowledge of the molecular mechanisms

underlying autophagy comes from S. cerevisiae (15). Althoughautophagy largely occurs on a nonspecific basis, several selec-tive types of autophagy also exist; examples include pexophagy,mitophagy, and the cytoplasm to vacuole (CvT) pathway. CvTtakes place in nutrient-rich conditions and mediates the

transport of the precursor vacuolar hydrolases aminopepti-dase 1 and a-mannosidase from the cytoplasm into the vac-uole. In S. cerevisiae, 34 autophagy (ATG) genes have beenidentified to date, some of which are shared among all autop-hagy subpathways and constitute the core machinery neededfor membrane formation. Autophagy is initiated by activationof the essential Ser/Thr kinase Atg1 and is induced mostcommonly by nutrient starvation and in response to severalstimuli, such as treatment with the mTORC1 inhibitor rapa-mycin, which is currently used in the clinic as an anticanceragent, suggesting that inhibition of mTOR might have impor-tant therapeutic values in cancer prevention (16).

The link between mutant Htt protein acetylation and itsautophagy-dependent degradation suggested that autophagycould also account for Sae2 degradation. Indeed, genetic andchemical inactivation of autophagy, more specifically the CvTpathway, rescued Sae2 degradation in VPA. Moreover, induc-tion of autophagy by rapamycin resulted in Sae2 degradation.These lines of evidence support the notion that autophagynegatively influences DSB repair in a hyperacetylated state.

Sae2 Is Regulated by Gcn5, Hda1, and Rpd3To uncover the identity of HDACs and HATs that mediate

regulation of Sae2 acetylation status, the authors monitoredSae2 stability in strains deleted for both HDA1 (class 1) andRPD3 (class 2). Indeed, Sae2 is destabilized in hda1 rpd3 cells.This finding recapitulates the effect of VPA, validating themechanism of action of VPA through HDAC inhibition andindicating that Hda1 and Rpd3 are at least 2 HDAC targets ofVPA involved in the DDR. Gcn5 HAT, a member of the SAGAHAT family in mammals, opposes the effect of Hda1 and Rpd3(17). Consistently, deletion ofGCN5 resulted in the suppressionof Sae2 degradation in VPA and rapamycin. These findingssuggest that interplay among Gcn5, Hda1, and Rpd3 regulatesthe acetylation status of Sae2 and, thus, its stability during DSBrepair. Interestingly, in mammals, the SIRT6 HDAC has beenimplicated in CtIP acetylation and in DSB repair (13). SIRT6belongs to class III HDACs, suggesting a role of other HDACs inthe DDR pathways.

A Working ModelRobert and colleagues proposed the following model on

the basis of their study (Fig. 1A): upon DSB formation, theMRX complex is recruited to DSB sites, followed by Sae2 andExo1, which remain in a deacetylated state in an Hda1/Rpd3-dependent manner. Once resection takes place, Gcn5 (andlikely other HATs) mediate hyperacetylation of Sae2 (Ac-Sae2), triggering its degradation by autophagy. Becauseautophagy takes place in the cytoplasm, it is likely thatAc-Sae2, along with Exo1 and additional unknown targets, isexported into the cytoplasm through the nuclear pore.Piecemeal microautophagy of nucleus (PMN) has beenreported in S. cerevisiae and has been shown to take placeat nucleus–vacuole junctions (18). PMN depends on severalautophagy genes, including ATG1. Another possibility, whichremains to be addressed, is that Ac-Sae2 degradation takesplace in a PMN-dependent manner.

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PerspectivesA future challenge is to further elucidate the contribution of

the different autophagy pathways in regulating the DDR.Autophagy is involved in degradation of long-lived proteinsand in recycling of macromolecular components. Perhapsacetylated Sae2 and other DSB repair proteins (including Exo1)form large complexes and, thus, difficult substrates for ubi-quitylation. Hence, after their use, autophagy might representthe only degradation pathway mediating their recycling. Infact, the acetylome study conducted in human cells showedthat large protein complexes undergo acetylation (9), whichmay signal their degradation by autophagy.

Increasing evidence in the literature shows that autop-hagy is induced in response to DNA damage (19). In additionto the recent link between autophagy and DNA damage inS. cerevisiae (10), Dyavaiah and colleagues (20) showed thata subunit of ribonucleotide reductase Rnr1 is also regulatedin an autophagy-dependent manner (20). It is important tonote that DNA-damaging agents also cause protein damage(21), which in turn may induce stress pathways such asautophagy as a survival mechanism to degrade damagedproteins or aggregates.

The physiologic role of autophagy has recently been a topicof debate (22). Autophagy has a cytoprotective role and isneeded for survival during development, aging, and pathogeninvasion. Defects in autophagy genes lead to predisposition tocancer and neurodegenerative diseases (22). In fact, a strongcorrelation exists between autophagy induction and tumorsuppression. On the other hand, autophagy can have a cyto-toxic effect because tumor cells can rely on it for survival orhealthy cells can be killed by excessive autophagy. The yeast

studies suggest that autophagy has a negative impact on cellsurvival when cells in a hyperacetylated state are exposed toradiomimetic drugs (10).

The study by Robert and colleagues has raised many ques-tions. Pathways that regulate autophagy are well characterizedand include TOR1, Ras-PKA, Snf1 AMP kinase (AMPK inhumans), as well as Sch9 kinase (S6K and/or PKB in humans;ref. 23). It will be interesting to investigate the mechanism(s)underlying autophagy induction by VPA or other HDAC inhi-bitors that are currently in clinical development. In mamma-lian cells, VPA activates autophagy in an mTOR-independentmanner (24, 25); however, in yeast, the target pathway of VPAis still unknown. Moreover, the precise mechanism underly-ing the CvT-mediated degradation of Sae2 and Exo1 (andlikely other targets) should be explored further. Cross-talkbetween autophagy and the proteasome has been reported(26), and whether it takes place during DSB repair should beinvestigated.

One study conducted in breast cancer cells showed thatrapamycin affects DSB repair pathways, HR, and nonhomol-ogous end joining (27). Consistently, the report by Robert andcolleagues further pinpoints that the DDR can be counteractedby rapamycin and HDAC inhibitors. Rapamycin and its ana-logs, rapalogues, have effective antitumor activity and arecurrently used either alone or in combination with anticancerdrugs (16). Ongoing clinical trials that rely on combiningrapamycin with genotoxic drugs should be evaluated in lightof these recent findings.

Although much attention has been granted to DNA damagerepair pathways, protein damage likely plays a significant rolein response to genotoxic stress. Thus, the theme of nutritional

A B

Figure 1. A, workingmodel based on findings reported by Robert and colleagues (10). Upon DSB induction, theMRX complex forms at DSB ends, followed bythe consecutive recruitment of Sae2 and Exo1 nucleases, which remain hypoacetylated in an Hda1- and Rpd3-mediatedmanner. Resection takes place, andultimately theMec1-Ddc2complex is activated, followedby autophosphorylation ofRad53 kinase.Sae2 (and likely other targets) undergoesGcn5-dependentacetylation, which may trigger the formation of a large protein complex that is exported from the nucleus and becomes destined for vacuolar degradation bythe CvT autophagy subpathway. B, both rapamycin and HDAC inhibitors, including VPA, trigger autophagy, which then counteracts DSB repair. Themechanism(s) underlying VPA-induced autophagy remain(s) to be elucidated butwill be important to unravel, given the therapeutic value of HDAC inhibitors incancer treatment.

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pathways such as autophagy will becomemore common in thegenome integrity field. From the therapeutic perspective,mechanistic studies aimed at understanding the interplayamong autophagy, DSB resection and acetylation, and drugintervention designed for autophagy and/or DDR cross-talkmight aid in the development of more effective cancertreatments.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsWe thank Chiara Lucca, Elisa Ferrari, Mong Sing Lai, and all members of our

laboratories for comments and discussions.

Grant SupportWork in our laboratories is supported by FIRC, Associazione Italiana per la

Ricerca sul Cancro (AIRC), American Institute for Cancer Research (AICR),Telethon-Italy, Ministry of Health, Italy, and the European Union.

Received September 22, 2011; revised November 11, 2011; accepted November16, 2011; published online March 15, 2012.

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