The c-MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1

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The c-MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1, and the SIRT1 deacetylase form a positive feedback loop Antje Menssen a,1 , Per Hydbring b,2,3 , Karsten Kapelle c,2 , Jörg Vervoorts c , Joachim Diebold d , Bernhard Lüscher c , Lars-Gunnar Larsson b , and Heiko Hermeking a,1 a Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, D-80337 Munich, Germany; b Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, 171 77 Stockholm, Sweden; c Institute of Biochemistry and Molecular Biology, Medical School, Rheinisch-Westfälische Technische Hochschule, Aachen University, D-52074 Aachen, Germany; and d Institute of Pathology, Kantons Spital Luzern, 6000 Luzern 16, Switzerland Edited by Edward V. Prochownik, Childrens Hospital of Pittsburgh of University of Pittsburg Medical Center, Pittsburgh, PA, and accepted by the Editorial Board November 21, 2011 (received for review April 4, 2011) Silent information regulator 1 (SIRT1) represents an NAD + -depen- dent deacetylase that inhibits proapoptotic factors including p53. Here we determined whether SIRT1 is downstream of the proto- typic c-MYC oncogene, which is activated in the majority of tumors. Elevated expression of c-MYC in human colorectal cancer correlated with increased SIRT1 protein levels. Activation of a conditional c- MYC allele induced increased levels of SIRT1 protein, NAD + , and nicotinamide-phosphoribosyltransferase (NAMPT) mRNA in several cell types. This increase in SIRT1 required the induction of the NAMPT gene by c-MYC. NAMPT is the rate-limiting enzyme of the NAD + salvage pathway and enhances SIRT1 activity by increasing the amount of NAD + . c-MYC also contributed to SIRT1 activation by sequestering the SIRT1 inhibitor deleted in breast cancer 1 (DBC1) from the SIRT1 protein. In primary human broblasts previously immortalized by introduction of c-MYC, down-regulation of SIRT1 induced senescence and apoptosis. In various cell lines inactivation of SIRT1 by RNA interference, chemical inhibitors, or ectopic DBC1 enhanced c-MYC-induced apoptosis. Furthermore, SIRT1 directly bound to and deacetylated c-MYC. Enforced SIRT1 expression in- creased and depletion/inhibition of SIRT1 reduced c-MYC stability. Depletion/inhibition of SIRT1 correlated with reduced lysine 63- linked polyubiquitination of c-Myc, which presumably destabilizes c-MYC by supporting degradative lysine 48-linked polyubiquitina- tion. Moreover, SIRT1 enhanced the transcriptional activity of c-MYC. Taken together, these results show that c-MYC activates SIRT1, which in turn promotes c-MYC function. Furthermore, SIRT1 suppressed cellular senescence in cells with deregulated c-MYC ex- pression and also inhibited c-MYCinduced apoptosis. Constitutive activation of this positive feedback loop may contribute to the development and maintenance of tumors in the context of de- regulated c-MYC. tumor metabolism | immortalization | p53 | tumor suppression | acetylation T he protein product of the proto-oncogene c-MYC is at the center of a transcription factor network that regulates cellular proliferation, replicative potential, cellcell competition, cell size, differentiation, metabolism, and apoptosis (13). Expression of c-MYC is induced rapidly by diverse mitogens and is down-regulated during differentiation. Deregulation of c-MYC activity has been implicated in the genesis of the majority of human cancers, and its inhibition represents a possible alternative to current cancer treat- ments (4, 5). The oncogenic activation of c-MYC often is caused by constitutive expression of c-MYC resulting from mutations in up- stream regulators, such as the components of the adenomatous polyposis coli (APC)β-cateninTCF4 pathway, or genomic alter- ations, such as amplications and translocations. In addition, the turnover rate of the c-MYC protein often is affected in tumors. Although several E3 ubiquitin ligases and signaling pathways have been reported to regulate ubiquitination and degradation of c-MYC, additional mechanisms likely contribute to this phenome- non. Activation of the c-MYC proto-oncogene antagonizes repli- cative and Ras-induced senescence and is sufcient for cellular immortalization (69). Furthermore, elevated levels of c-MYC may induce replication stress and reactive metabolites that elicit apo- ptosis or premature senescence through p53-dependent or -in- dependent pathways (1013). c-MYC directly induces the human telomerase reverse tran- scriptase (htert) gene, which encodes the catalytic subunit of telomerase (7). However, htert expression may prolong the rep- licative lifespan of cells to only a limited extent (8). Therefore, we hypothesized that c-MYC may regulate other factors that antag- onize cellular senescence and mediate cellular immortalization. The human silent information regulator 1 (SIRT1) gene enc- odes an NAD + -dependent protein deacetylase, which is involved in epigenetic silencing, heterochromatin formation, regulation of metabolism, DNA repair, and cellular stress responses. These functions are mediated by deacetylation of histones, transcription factors, chromatin-modifying enzymes, and other nuclear proteins (14, 15). Recently, the NAD + salvage pathway and its rate-limiting enzyme, nicotinamide phosphoribosyltransferase (NAMPT), have been implicated in the activation of SIRT1 (16). In contrast, the deleted in breast cancer 1 (DBC1) gene product negatively regu- lates SIRT1 activity through binding to its active site and thereby inhibiting SIRT1substrate interaction (17, 18). Moreover, DBC1 was shown to be involved in the induction of apoptosis in response to TNF-α (19). In yeast, Drosophila, and Caenorhabditis elegans ectopic expression of SIR2, the orthologue of SIRT1, extends life- span (20). However, these effects recently were shown to depend on the genetic background in Drosophila and C. elegans (21). In ad- dition, SIRT1 extends the replicative lifespan of human cells (22), an effect that can be attributed, at least in part, to the SIRT1-me- diated deacetylation and inhibition of p53 (2325). Furthermore, other proapoptotic factors such as Foxo transcription factors, Smad7, Ku70, p73, and poly(ADP-ribose) polymerase 1 (PARP1) Author contributions: A.M., B.L., L.-G.L., and H.H. designed research; A.M., P.H., K.K., J.V., and H.H. performed research; J.D. contributed new reagents/analytic tools; A.M., P.H., K.K., J.V., J.D., B.L., L.-G.L., and H.H. analyzed data; and A.M., B.L., L.-G.L., and H.H. wrote the paper. The authors declare no conict of interest. This article is a PNAS Direct Submission. E.V.P. is a guest editor invited by the Editorial Board. 1 To whom correspondence may be addressed. E-mail: [email protected]. de or [email protected]. 2 P.H. and K.K. contributed equally to this work. 3 Present address: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215. See Author Summary on page 1007. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1105304109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1105304109 PNAS | January 24, 2012 | vol. 109 | no. 4 | E187E196 MEDICAL SCIENCES PNAS PLUS

Transcript of The c-MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1

Page 1: The c-MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1

The c-MYC oncoprotein, the NAMPT enzyme, theSIRT1-inhibitor DBC1, and the SIRT1 deacetylaseform a positive feedback loopAntje Menssena,1, Per Hydbringb,2,3, Karsten Kapellec,2, Jörg Vervoortsc, Joachim Dieboldd, Bernhard Lüscherc,Lars-Gunnar Larssonb, and Heiko Hermekinga,1

aExperimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, D-80337 Munich, Germany; bDepartment ofMicrobiology, Tumor and Cell Biology, Karolinska Institute, 171 77 Stockholm, Sweden; cInstitute of Biochemistry and Molecular Biology, Medical School,Rheinisch-Westfälische Technische Hochschule, Aachen University, D-52074 Aachen, Germany; and dInstitute of Pathology, Kantons Spital Luzern, 6000 Luzern16, Switzerland

Edited by Edward V. Prochownik, Children’s Hospital of Pittsburgh of University of Pittsburg Medical Center, Pittsburgh, PA, and accepted by the EditorialBoard November 21, 2011 (received for review April 4, 2011)

Silent information regulator 1 (SIRT1) represents an NAD+-depen-dent deacetylase that inhibits proapoptotic factors including p53.Here we determined whether SIRT1 is downstream of the proto-typic c-MYC oncogene, which is activated in themajority of tumors.Elevated expression of c-MYC in human colorectal cancer correlatedwith increased SIRT1 protein levels. Activation of a conditional c-MYC allele induced increased levels of SIRT1 protein, NAD+, andnicotinamide-phosphoribosyltransferase (NAMPT) mRNA in severalcell types. This increase in SIRT1 required the induction of theNAMPT gene by c-MYC. NAMPT is the rate-limiting enzyme of theNAD+ salvage pathway and enhances SIRT1 activity by increasingthe amount of NAD+. c-MYC also contributed to SIRT1 activation bysequestering the SIRT1 inhibitor deleted in breast cancer 1 (DBC1)from the SIRT1 protein. In primary human fibroblasts previouslyimmortalized by introduction of c-MYC, down-regulation of SIRT1induced senescence and apoptosis. In various cell lines inactivationof SIRT1 by RNA interference, chemical inhibitors, or ectopic DBC1enhanced c-MYC-induced apoptosis. Furthermore, SIRT1 directlybound to and deacetylated c-MYC. Enforced SIRT1 expression in-creased and depletion/inhibition of SIRT1 reduced c-MYC stability.Depletion/inhibition of SIRT1 correlated with reduced lysine 63-linked polyubiquitination of c-Myc, which presumably destabilizesc-MYC by supporting degradative lysine 48-linked polyubiquitina-tion. Moreover, SIRT1 enhanced the transcriptional activity ofc-MYC. Taken together, these results show that c-MYC activatesSIRT1, which in turn promotes c-MYC function. Furthermore, SIRT1suppressed cellular senescence in cells with deregulated c-MYC ex-pression and also inhibited c-MYC–induced apoptosis. Constitutiveactivation of this positive feedback loop may contribute to thedevelopment and maintenance of tumors in the context of de-regulated c-MYC.

tumor metabolism | immortalization | p53 | tumor suppression |acetylation

The protein product of the proto-oncogene c-MYC is at thecenter of a transcription factor network that regulates cellular

proliferation, replicative potential, cell–cell competition, cell size,differentiation, metabolism, and apoptosis (1–3). Expression ofc-MYC is induced rapidly by diversemitogens and is down-regulatedduring differentiation. Deregulation of c-MYC activity has beenimplicated in the genesis of the majority of human cancers, and itsinhibition represents a possible alternative to current cancer treat-ments (4, 5). The oncogenic activation of c-MYC often is caused byconstitutive expression of c-MYC resulting from mutations in up-stream regulators, such as the components of the adenomatouspolyposis coli (APC)–β-catenin–TCF4 pathway, or genomic alter-ations, such as amplifications and translocations. In addition, theturnover rate of the c-MYC protein often is affected in tumors.Although several E3 ubiquitin ligases and signaling pathways havebeen reported to regulate ubiquitination and degradation of

c-MYC, additional mechanisms likely contribute to this phenome-non. Activation of the c-MYC proto-oncogene antagonizes repli-cative and Ras-induced senescence and is sufficient for cellularimmortalization (6–9). Furthermore, elevated levels of c-MYCmayinduce replication stress and reactive metabolites that elicit apo-ptosis or premature senescence through p53-dependent or -in-dependent pathways (10–13).c-MYC directly induces the human telomerase reverse tran-

scriptase (htert) gene, which encodes the catalytic subunit oftelomerase (7). However, htert expression may prolong the rep-licative lifespan of cells to only a limited extent (8). Therefore, wehypothesized that c-MYC may regulate other factors that antag-onize cellular senescence and mediate cellular immortalization.The human silent information regulator 1 (SIRT1) gene enc-

odes an NAD+-dependent protein deacetylase, which is involvedin epigenetic silencing, heterochromatin formation, regulation ofmetabolism, DNA repair, and cellular stress responses. Thesefunctions are mediated by deacetylation of histones, transcriptionfactors, chromatin-modifying enzymes, and other nuclear proteins(14, 15). Recently, the NAD+ salvage pathway and its rate-limitingenzyme, nicotinamide phosphoribosyltransferase (NAMPT), havebeen implicated in the activation of SIRT1 (16). In contrast, thedeleted in breast cancer 1 (DBC1) gene product negatively regu-lates SIRT1 activity through binding to its active site and therebyinhibiting SIRT1–substrate interaction (17, 18). Moreover, DBC1was shown to be involved in the induction of apoptosis in responseto TNF-α (19). In yeast, Drosophila, and Caenorhabditis elegansectopic expression of SIR2, the orthologue of SIRT1, extends life-span (20).However, these effects recently were shown to dependonthe genetic background in Drosophila and C. elegans (21). In ad-dition, SIRT1 extends the replicative lifespan of human cells (22),an effect that can be attributed, at least in part, to the SIRT1-me-diated deacetylation and inhibition of p53 (23–25). Furthermore,other proapoptotic factors such as Foxo transcription factors,Smad7, Ku70, p73, and poly(ADP-ribose) polymerase 1 (PARP1)

Author contributions: A.M., B.L., L.-G.L., and H.H. designed research; A.M., P.H., K.K., J.V.,and H.H. performed research; J.D. contributed new reagents/analytic tools; A.M., P.H.,K.K., J.V., J.D., B.L., L.-G.L., and H.H. analyzed data; and A.M., B.L., L.-G.L., and H.H. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. E.V.P. is a guest editor invited by the EditorialBoard.1To whom correspondencemay be addressed. E-mail: [email protected] or [email protected].

2P.H. and K.K. contributed equally to this work.3Present address: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston,MA 02215.

See Author Summary on page 1007.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1105304109/-/DCSupplemental.

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are negatively regulated by SIRT1 (15, 26). In summary, theseproperties of SIRT1 led us to hypothesize that SIRT1 may playa role downstream of c-MYC.Here we report that c-MYC activates the SIRT1 enzyme, which

critically contributes to suppression of senescence in cells withderegulated c-MYC and suppression of c-MYC–induced apo-ptosis. Furthermore, we could delineate two mechanisms in-volving NAMPT and DBC1 by which c-MYC enhances SIRT1activity. In addition, we identified a positive feedback loop be-tween c-MYC and SIRT1, supporting an important role forSIRT1 in c-MYC–driven tumorigenesis.

ResultsPosttranscriptional Activation of SIRT1 by c-MYC.Here we sought todetermine whether SIRT1 represents an effector of the c-MYConcoprotein. In line with this hypothesis, SIRT1 protein ex-pression increased as early as 9 h after activation of a conditionalc-Myc-estrogen receptor (ER) fusion protein in c-myc–deficientRAT1 fibroblasts or of a conditional c-MYC allele in the P493-6B-cell line (Fig. 1A and SI Appendix, Fig. S1 A and B). SIRT1mRNA was not affected by c-MYC activation in these cells,whereas known c-MYC target genes were clearly induced (Fig. 1B and C) (2). SIRT1 mRNA levels were not affected by acuteactivation of c-MYC in two other cellular systems tested (SIAppendix, Fig. S1C). Furthermore, down-regulation of c-MYCexpression by an inducible shRNA directed against c-MYC wasfollowed by a decrease in SIRT1 protein in the colorectal cancercell line LS-174T (Fig. 1D). When c-MYC expression was in-duced by restimulation with serum, SIRT1 levels increased inRAT1 fibroblasts but not in c-myc–deficient RAT1 cells (Fig.1E). Again, the SIRT1 mRNA was unaffected by experimentalmodulation of c-MYC activity in LS-174T and RAT1 fibroblasts

(SI Appendix, Fig. S1 C and D). Taken together, these resultsdemonstrate that c-MYC activation is sufficient for posttran-scriptional induction of SIRT1 protein and is necessary for theincrease in SIRT1 protein after mitogenic stimulation.To validate whether the abundance of SIRT1 protein is reg-

ulated at the posttranscriptional level, MCF-7– and humandiploid fibroblast (HDF)-derived cell lines constitutively expres-sing a SIRT1-GFP fusion protein or GFP under control of anLTR promoter were analyzed. SIRT1-GFP protein expressionwas down-regulated after serum starvation in both cell types(Fig. 1F and SI Appendix, Fig. S1E). After readdition of serum,which generally is accompanied by an increase in c-MYC ex-pression, SIRT1-GFP but not GFP expression was induced.Moreover, inhibition of proteasomal degradation induced SIRT1expression in c-myc–deficient RAT1 fibroblasts, whereas in wild-type c-myc RAT1 cells the SIRT1 level was affected only mar-ginally by MG132 treatment (Fig. 1G). Taken together, thesefindings suggest that the c-MYC–mediated increase in the SIRT1protein may be caused by inhibition of the proteasomal degra-dation of SIRT1.To determine whether the regulation of SIRT1 by c-MYC also

occurs in vivo, we analyzed colorectal cancer specimens, whichgenerally show high c-MYC expression because of mutationalactivation of the APC–β-catenin pathway and/or K-Ras or B-Rafmutations (27–29). In primary colorectal cancer biopsies derivedfrom 15 patients, carcinoma cells with high c-MYC expressionconsistently showed elevated SIRT1 expression, whereas adjacentnormal colonic crypts displayed barely detectable expression ofSIRT1 and c-MYC (Fig. 1H and SI Appendix, Fig. S2). Therefore,the expression of SIRT1 may be regulated by c-MYC in normaland malignant tissues.

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Fig. 1. c-MYC induces SIRT1 protein expression. Ly-sates were prepared at the indicated time points andsubjected to Western blot analysis (A and D–G). De-tection of α-tubulin or β-actin served as loading con-trols. (A) RAT1 c-myc−/− (HO15) fibroblasts (82) stablyexpressing c-Myc-ER were starved for 48 h at 0.1% FBSand then treated with 300 nM 4-hydroxytamoxifen (4-OHT) for activation of Myc-ER. As a control, serumstarvation was continued for another 48 h (right lane).(B) RAT1 c-myc−/− (HO15) fibroblasts stably expressingc-Myc-ER were starved for 48 h at 0.1% FBS and thentreated with 300 nM 4-OHT for activation of c-Myc-ER.At the indicated time points mRNA expression ofc-MYC target genes and SIRT1 was analyzed by quan-titative PCR (qPCR) [SIRT1 primer pair 1 (p1) and 2 (p2)].Western blot analysis of these cells is shown in A. Barsrepresent mean values of biological triplicates (SIRT1)or duplicates with SE. LDHa, lactate dehydrogenase A;NPM, nucleophosmin; ODC, ornithine decarboxylase.(C) qPCR analysis of SIRT1 expression and direct c-MYCtarget genes 11 h after c-MYC activation in P493-6 cells,a pre–B-cell line that harbors a conditional c-MYC allele(83). Bars represent mean values of biological tripli-cates with SE. CDK4, cyclin-dependent kinase 4; DKC,dyskerin. Five additional cell lines were analyzed withsimilar results after induction of a conditional c-MYCallele or after activation of endogenous c-MYCwith upto two different SIRT1-specific primer pairs (SI Appen-dix, Fig. S1 C and D). (D) LS-174T colorectal cancer cellswere treated with 1 μg/mL DOX for the indicatedperiods to activate the expression of c-MYC–specificshRNA (84). (E) c-myc+/+ (TGR) and c-myc−/− RAT1fibroblasts (HO15) were kept for 48 h at 0.1% FBS andthen restimulated with 8% (vol/vol) FBS for the indicated periods. (F) HDF expressing either SIRT1-GFP or GFP were serum starved at 0.1% FBS for 48 h and thenrestimulatedwith 10% (vol/vol) FBS for the indicated periods. See also SI Appendix, Fig. S1E. (G) HO15 (myc−/−) and TGR-1 (myc+/+) cells were treatedwithMG132(10 μM) for the indicated periods. (H) Expression of c-MYC and SIRT1 protein in colorectal cancer and adjacent normal colonic cells. Shown are representativeresults obtained by immunohistochemical analysis of consecutive sections derived from one colorectal biopsy (of 15) with antibodies directed against c-MYC andSIRT1. (Magnification: 200×.) N, normal tissue; T, tumor. See also SI Appendix, Fig. S2.

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c-MYC Induces SIRT1 Deacetylase Activity. SIRT1 is known todeacetylate p53 at lysine residue 382 (K382), thereby attenuatingp53 activity (23–25). We also observed deacetylation of DNA-damage–induced p53 by ectopic SIRT1 expression (Fig. 2A).Similarly, ectopic expression of c-MYC resulted in deacetylationof p53K382 in MCF-7 cells, whereas a dominant-negative mutantc-MYC protein (MADMYC) did not influence p53 acetylationafter induction of DNA damage by γ-irradiation (examples areshown in Fig. 2A): Of 50 MCF-7 cells that stained positive forectopic c-MYC protein, 38 cells (76%) showed decrease or loss ofacetylation of p53K382, whereas expression of a MADMYC fu-sion protein affected the acetylation of only 10 of 50 cells (20%).Induction of SIRT1 activity was observed after c-Myc activationin P493-6 cells (SI Appendix, Fig. S3). When SIRT1 activity wasinhibited by treatment with nicotinamide (NAM), which is anendogenous SIRT1 inhibitor, p53K382 acetylation was enhancedin the presence of an activated c-MYC allele (Fig. 2B). Takentogether, these results show that the c-MYC–induced accumula-tion of SIRT1 protein is accompanied by an increase in SIRT1activity and affects critical downstream targets such as p53.

Induction of NAMPT by c-MYC Mediates SIRT1 Activation. Becausethe deacetylase activity of SIRT1 depends on NAD+, we hy-

pothesized that the increased SIRT1 activity induced by c-MYCmay be caused by c-MYC–mediated changes in metabolism af-fecting the NAD+/NADH ratio. Indeed, c-MYC activationprovoked an increase in NAD+ and a decrease in the NADHlevels, resulting in a more than fourfold increase in the NAD+/NADH ratio (Fig. 2 C–E and SI Appendix, Table S1). Activationof either glycolysis or the NAD+ salvage pathway, which is theprimary means of cellular NAD+ regeneration (30), may inducesuch changes. Interestingly, the addition of FK866 (31), a specificinhibitor of NAMPT, which is the rate-limiting enzyme of thesalvage pathway, resulted in a pronounced decrease in NAD+

before and after c-MYC activation, whereas chemical inhibitionof glycolysis did not affect the NAD+ levels significantly (SIAppendix, Fig. S4 A and B) but reduced NADH by ∼50% when itwas applied for 18 h. As shown previously (32), inhibition ofglycolysis by 2-deoxyglucose resulted in an increase of theNAD+/NADH ratio, arguing against a major contribution ofglycolysis to the overall NAD+ levels and to SIRT1 activation.Because the inhibition of NAMPT activity had such pronouncedeffects on NAD+, our data suggest that the contribution of thesalvage pathways to the NAD+ pool in the cell exceeds theamount of NAD+ generated by lactate dehydrogenase-A (whichis induced by c-MYC) during glycolysis. We therefore hypothe-

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Fig. 2. c-MYC activates SIRT1: effects on p53 and mediationby NAMPT. (A) Cells were transfected with plasmids encodingthe indicated proteins and treated with etoposide (20 μM) for7 h (HCT-116 cells; Left) or 10 Gy γ-irradiation (MCF-7 cells;Center and Right) and fixed after 4 h. Expression of ectopicMYC-HA and a c-MYC mutant with the transactivation do-main replaced by the repressive Sin3 domain of MAD1(MADMYC-HA) was detected with an anti-HA antibody (Cen-ter and Right), and SIRT1-GFP expression was detected by GFPfluorescence (Left). Acetylation (ac) of p53K382 was detectedby indirect immunofluorescence. Arrows indicate the posi-tions of cells positive for ectopic proteins (SIRT1-GFP, c-MYC-,or MADMYC-HA). (B) P493-6 c-MYC tetracycline-off cells werekept in the absence or presence of tetracycline. After 72 h celllysates were subjected to immunoblot analysis of the in-dicated proteins. As documented by β-actin detection, differ-ent amounts of protein were loaded to adjust for comparablep53 levels in all samples. (C) NADH and (D) NAD+ concen-trations were determined, as described previously (85), inRAT1 myc−/− fibroblasts stably expressing c-Myc-ER. After se-rum starvation for 48 h in 0.1% FBS, c-Myc-ER was activatedfor 24 h by addition of 300 nM 4-OHT. Cell lysates were sub-jected to the cycling reaction (for details see SI Appendix, SIMethods). The graphs show mean values ± SD of three in-dependent experiments. Results of the individual measure-ments are given in SI Appendix, Table S1. (E) Mean values ofNAD+/NADH ratios of MYC OFF (−4-OHT) and MYC ON (+4-OHT) are given with SDs. Data were taken from the analysisshown in C and D and SI Appendix, Table S1. (F) Schematicrepresentation of the NAD+ salvage pathway. NAD+ is gen-erated by NAMPT-mediated conversion of NAM to NMN,which is converted to NAD+ by the enzymes nicotinamide/nicotinic acid mononucleotide adenyltransferase (NMAT) 1–3.(G) Schematic representation of human, mouse, and ratNAMPT promoter regions. The transcription start site is in-dicated by “+1”, E-box positions (gray squares) and E-boxmotifs conserved between species are indicated. qPCR ampli-con positions are indicated by pairs of arrows. (H) ChIP analysisof c-MYC binding to the NAMPT promoter in serum (ser)-stimulated MCF-7 cells. After starvation with 0.1% FBS for 48 h, half of the cells were restimulatedwith 10% (vol/vol) FBS for 12 h. The assay was performed in triplicate using a polyclonal anti-MYC antibody and rabbit IgGs as control. c-MYC enrichment at E-boxes was determined by qPCR with primer pairs flanking E-boxes (see G). The DNA input was normalized with a genomic amplicon devoid of E-boxes. (I)qPCR analysis of NAMPT mRNA expression upon c-MYC activation in P493-6 cells. RNA was isolated 12 and 24 h after removal of tetracycline. (J) qPCR analysisof NAMPT mRNA expression upon DOX (1 μg/mL)-mediated induction of a conditional c-MYC allele for the indicated periods in the MCF-7 cell line PJMMR1(86). RNA samples for the 0 (noninduced) and 12-h time points were harvested simultaneously. Analyses were performed in triplicates. (K) Induction ofNAMPT protein upon activation of c-MYC-ER in HDF. Cells were serum starved for 48 h and then stimulated with 4-OHT for the indicated times, lysed si-multaneously, and subjected to Western blot analysis. β-actin served as loading control. (L) Induction of SIRT1 by c-Myc is dependent on NAMPT. HDF-MYC-ERcells were transfected with the indicated siRNAs and then starved as in K and treated with 4-OHT for 48 h. Cells were lysed simultaneously and analyzed byWestern blotting for the expression of the indicated proteins. β-actin served as loading control.

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sized that these changes could result from a c-MYC–inducedactivation of NAMPT that promotes the conversion of NAM toNAD+ via nicotinamide mononucleotide (NMN) (Fig. 2F).Analysis of the NAMPT promoter sequence revealed threeconserved, noncanonical E-boxes in the vicinity of the tran-scriptional start site (Fig. 2G), two of which are CACGCGmotifs. Binding of c-MYC to CACGCG motifs in promoterregions of other genes has been reported previously (33). ChIPanalysis revealed binding of endogenous c-MYC to a regionencompassing the noncanonical E-boxes in the NAMPT pro-moter. Occupancy by c-MYC was enhanced after mitogenicstimulation by serum, which is known to activate expression of c-MYC. The E-box located ∼2.5 kbp upstream of the transcriptionstart site did not display occupation by c-MYC (Fig. 2H). Inaddition, inspection of publicly available ChIP-Seq analyses ofgenome-wide c-MYC binding confirmed the presence of c-MYCat the NAMPT promoter coinciding with histone modificationsindicating transcriptional activity (SI Appendix, Fig. S5 A and B).Upon c-MYC activation, increased expression of NAMPTmRNA was observed in the B-cell line P493-6 and in MCF-7cells (Fig. 2 I and J). The NAMPT protein level also increasedafter activation of c-MYC-ER in HDF (Fig. 2K). Down-regula-tion of NAMPT expression by siRNAs largely prevented an in-crease in SIRT1 after c-MYC activation (Fig. 2L). Takentogether, these results establish the NAMPT gene as a mediatorof SIRT1 activation by c-MYC.

c-MYC–DBC1 Association Facilitates Activation of SIRT1. We pre-viously identified the DBC1 protein as a c-MYC–interactingprotein (34). Interestingly, the functionally important MYC Box-II domain is essential for the c-MYC–DBC1 interaction. Fur-thermore, DBC1 recently was shown to inhibit the SIRT1 enzymeby binding to its active site (17, 18). This observation raised thequestion whether DBC1 may play a role in the regulation ofSIRT1 by c-MYC. Indeed, SIRT1- and DBC1-GST fusion pro-teins were found to associate directly with recombinant c-MYC invitro, suggesting that these interactions may affect each other invivo (Fig. 3A). When increasing amounts of ectopic c-MYC wereexpressed in HEK-293 cells, the amount of endogenous DBC1bound to ectopic SIRT1 gradually decreased (Fig. 3B). Becausethe amount of c-MYC associated with SIRT1 did not increaseproportionally to the amount of expressed c-MYC, c-MYC pre-sumably sequesters DBC1 away from SIRT1 rather than com-peting withDBC1 for binding to SIRT1 (Fig. 3B). It is conceivablethat a competition betweenDBC1 andMYC for binding to SIRT1may occur preferentially when c-MYC is expressed at lower orintermediate levels. In summary, these observations show that, inaddition to affecting the NAD+/NADH ratio, c-MYC may con-tribute to the activation of SIRT1 by binding directly to the SIRT1inhibitor DBC1 and preventing its interaction with SIRT1.

SIRT1 Suppresses c-MYC–Induced Apoptosis. Next we investigatedwhether SIRT1 inactivation affects apoptosis induced by acuteactivation of conditional c-MYC alleles. Indeed, apoptosis in-duced by c-MYC-ER was augmented substantially by the additionof NAM in RAT1A cells (Fig. 4A). Also, when ectopic c-MYCwas expressed in U2OS cells, a pronounced increase in apoptosiswas observed when SIRT1 was down-regulated simultaneously byinduction of a microRNA (miRNA) directed against its 3′-UTR(Fig. 4B). In these cells, ectopic c-MYC combined with down-regulation of SIRT1 enhanced acetylation of p53K382 (Fig. 4C)accompanied by an up-regulation of p53 and its targets PUMAand p21. Therefore, as also documented in P493-6 cells (Fig. 2B),c-MYC–induced SIRT1 deacetylates p53 and thereby attenuatesthe transcriptional activity of p53 after c-MYC activation, result-ing in decreased expression of proapoptotic genes and therebypresumably attenuating c-MYC–induced apoptosis.In Fig. 3B we show that enforced expression of c-MYC

sequesters the SIRT1 inhibitor DBC1 from SIRT1, possibly sup-

porting SIRT1 activity. Therefore, we investigated whether mod-ulation of DBC1 expression has an effect on c-MYC–inducedapoptosis. Indeed, ectopic expression of DBC1 increased c-MYC–induced apoptosis and decreased proliferation, whereas suppres-sion of DBC1 by a DBC1-specific miRNA resulted in decreasedapoptosis and increased proliferation in U2OS cells (Fig. 4 D andE). Similarly, c-MYC-ER activation resulted in less apoptosis inhuman fibroblasts when DBC1 was down-regulated by specificsiRNAs (SI Appendix, Fig. S6). Interestingly, in the presence ofDBC1-specific siRNAs, c-MYC-ER activation resulted in in-creased expression of the c-MYC target gene product CDK4 (Fig.4F), a result that is in line with increased proliferation. Therefore,the degree of DBC1-mediated inhibition of SIRT1 may be a criti-cal determinant of the outcome of c-MYC activation.The cells used in the apoptosis assays described above (RAT1-

Myc-ER, U2OS, HDF-c-MYC-ER) express wild-type p53, asdocumented by sequence analysis and/or by increased p53 accu-mulation upon etoposide treatment or c-MYC activation (SI Ap-pendix, Fig. S7 A and B). To determine whether the antiapoptoticand protective effects of SIRT1 also may be mediated by deace-tylation of SIRT1 substrates other than p53, human U937 mon-oblast cells, which express mutant p53, were analyzed. The SIRT1-specific inhibitor EX527 (35) or sirtinol, which is a synthetic small-molecule inhibitor of SIRT1 and SIRT2 (36), induced apoptosis inU937 cells stably expressing a v-myc gene but not in parental U937cells (Fig. 4G). As shown for EX527, this effect resulted in re-duced proliferation (Fig. 4 H and I). Therefore, at least some ofthe protective effects of SIRT1 observed after c-MYC activationmay be mediated via SIRT1 substrates other than p53.

Role of SIRT1 in c-MYC–Immortalized HDF. HDFs immortalized byretroviral introduction of constitutive expression of c-MYC (6)showed increased expression of SIRT1 protein (Fig. 5A). Wenext asked whether c-MYC–induced SIRT1 activity is requiredto maintain these cells. Only in c-MYC–immortalized but not inprimary HDFs down-regulation of SIRT1 by siRNAs induceda pronounced increase in cell size and senescence-associatedβ-galactosidase activity at pH 6, two markers of cellular senes-cence (Fig. 5B). Furthermore, inactivation of SIRT1 resulted inan increased fraction of apoptotic cells in c-MYC–immortalizedbut not of hTERT-immortalized or primary HDFs (Fig. 5C).

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Fig. 3. Interplay between c-MYC, DBC1, and SIRT1. (A) GST-tagged fusionproteins of DBC1 or SIRT1 and GST protein as control were used to detectinteractions with in vitro transcribed and translated c-MYC. An aliquot of thec-MYC protein was analyzed as a control (input). (B) HEK-293 cells werecotransfected with a vector encoding a SIRT1-vesicular stomatitis virus (VSV)fusion protein in combination with a plasmid encoding c-MYC–HA at theindicated molar ratios. Binding of endogenous DBC1 to SIRT1-VSV was an-alyzed by coimmunoprecipitation and immunoblot analysis 24 h aftertransfection. Ectopic SIRT1 was immunoprecipitated with an antibody di-rected against the VSV tag.

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Similarly, the addition of sirtinol resulted in an increase of ap-optosis only in c-MYC–immortalized cells (Fig. 5D). Taken to-gether, these results suggest that SIRT1 plays a role in thesuppression of apoptosis and cellular senescence in cells pre-viously immortalized by c-MYC. Therefore, increased SIRT1activity may be relevant for the long-term survival and expansionof cancer cells exhibiting deregulation of c-MYC.

c-MYC Is an SIRT1 Substrate. As mentioned above, we detecteda direct interaction of recombinant or ectopic c-MYC and SIRT1

proteins (Fig. 3 A and B). Coimmunoprecipitation analysis ofendogenous proteins confirmed that this interaction also occursbetween endogenous c-MYC and SIRT1 (Fig. 6A). When c-MYCwas acetylated by cAMP-response element binding protein (CBP)in vitro and then was exposed to recombinant SIRT1, efficientdeacetylation of c-MYC was observed that was dependent on thepresence of NAD+ and was blocked by NAM (Fig. 6B). Treat-ment of cells with NAM resulted in hyperacetylation of c-MYC,whereas treatment with trichostatin A (TSA), an inhibitor ofhistone deacetylases, had no effect on c-MYC acetylation

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Fig. 4. SIRT1 and DBC1 modulate c-MYC–induced apoptosis. (A) RAT1 HO15.19 myc−/− cells expressing Myc-ER were treated with 4-OHT in the presence of8% FBS in DMEM. NAM (5 mM) was added for 72 h before cells were harvested. Apoptotic sub-G1 cells were quantified by flow cytometric analysis of DNAcontent. The average of three independent experiments is depicted; bars indicate SD. (B) U2OS cells harboring pEMI and RTS vectors expressing the indicatedmiRNAs or proteins were treated with DOX (0.1 μg/mL). As a control, a nonsilencing miRNA or an empty RTS vector was used. After 48 h the percentage ofcells in sub-G1 was determined by propidium iodide staining and FACS analysis. The mean values and SEs obtained in two independent experiments areshown. (C) As in B, pools of U2OS cells harboring DOX-inducible pEMI and RTS vectors expressing the indicated miRNAs or proteins were generated. Total celllysates were prepared 48 h after addition of DOX (0.1 μg/mL) and subjected to Western blot analysis of the indicated proteins. As control a pEMI vectorencoding a nonsilencing miRNA (miR-ctrl.) or an empty RTS vector (vector) was used. (D) As described in B, the percentage of cells in sub-G1 was determined bypropidium iodide staining and FACS analysis after 72 h of DOX treatment. The mean values and SD of three samples are shown. (E) U2OS cells cotransfectedwith RTS or pEMI vectors encoding the indicated proteins or miRNAs (see B) were subjected to real-time impedance measurements using an x-CELLigencedevice (Roche). DOX (0.1 μg/mL) was added 14 or 17 h after cell seeding. This time point was used for cell index normalization. The cell index representsrelative cellular impedance, which indicates relative cell numbers. Parallel end-point analysis by conventional cell counting confirmed the results shown here.(F) HDF were transfected with DBC1-specific siRNA, serum starved for 48 h, and then stimulated with 4-OHT for activation of c-MYC-ER. After 24 h cells werelysed and subjected to Western blot analysis. (Cellular impedance measurements of these cells are shown in SI Appendix, Fig. S4B.) (G) v-Myc–expressing andparental human U937 monoblasts were treated with sirtinol (30 μM) or EX527 (1 μM) for 6 d. Apoptosis was determined as enrichment of cytoplasmicnucleosomes using a quantitative cell death detection ELISA kit (Roche). (H) Effect of EX527 (1 μM) on the proliferation of MYC-transformed or (I) parentalhuman U937 monoblasts. Cell numbers were determined at the indicated time-points.

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(Fig. 6C). In line with these results, CBP-mediated acetylation ofc-MYC was reversed by SIRT1 but not by an inactive SIRT1mutant or SIRT2 in HEK-293 cells (Fig. 6D). In summary, theseresults establish c-MYC as substrate of the SIRT1 deacetylase.

SIRT1 Stabilizes c-MYC. Acetylation of the c-MYC protein alsoregulates its ubiquitination and thereby affects the rate of pro-teasomal degradation (37, 38). In line with a role of SIRT1-mediated deacetylation in the regulation of c-MYC degradation,the c-MYC protein levels in subconfluent sirt1−/− mouse embryofibroblasts (MEFs) were reduced compared with subconfluentsirt1+/+ MEFs (Fig. 7A), whereas c-myc mRNA expression re-mained unchanged (Fig. 7B). Accordingly, c-MYC target genesalso were expressed at higher levels in the sirt1+/+ MEFs than inSIRT1-deficient MEFs (Fig. 7B). As shown by [35S]methioninepulse-chase analysis and densitometric quantification, ectopicexpression of SIRT1 increased the half-life of endogenous c-MYC from 25 to 40 min in MCF-7 cells (Fig. 7C). Unexpectedly,ectopic expression of SIRT1 in combination with cycloheximide(CHX) treatment resulted in increased c-MYC turnover (SIAppendix, Fig. S8A). Therefore, CHX treatment combined withectopic SIRT1 expression presumably causes a nonphysiologicaldegradation of c-MYC, whereas SIRT1 expression alone in-creases the half-life of c-MYC. These conclusions also weresupported by experiments in which miRNA- or shRNA-medi-ated down-regulation of SIRT1 reduced the half-life of endog-enous c-MYC from 32 to 23 min in U2OS cells (Fig. 7D and SIAppendix, Fig. S8B). Moreover, administration of tenovin-6,a recently described SIRT1 inhibitor (39), reduced the c-MYChalf-life from 34 to 25 min in HCT-116 cells (Fig. 7E). Asexpected, c-MYC had slightly varying half-lives in the differentcell lines. Taken together, these results suggest that the half-lifeof c-MYC is increased by SIRT1-mediated deacetylation.

SIRT1 Promotes Lysine-63–Linked Polyubiquitination of c-MYC. Be-cause acetylation is known to affect the ubiquitination of ly-sine residues, we tested whether SIRT1-mediated deacetylationmodulates ubiquitination of c-MYC. The net amount of poly-ubiquitinated c-MYC protein was reduced in HCT-116 cellsectopically expressing wild-type c-MYC and HA-tagged ubiq-uitin after treatment with tenovin 6 (Fig. 8A), despite increased

turnover of c-MYC (Fig. 7E). c-MYC is known to be conju-gated with both lysine-48 (K48)- and lysine-63 (K63)-linkedpolyubiquitin chains (40) that mediate proteolytic and non-proteolytic functions, respectively (41). To investigate whattypes of ubiquitin ligation are affected by SIRT1 inhibition,cells were transfected with HA-tagged K48R- or K63R-ubiq-uitin mutants. A reduction of polyubiquitinated c-MYC in re-sponse to tenovin-6 was observed with the K48R ubiquitinmutant, as with wild-type ubiquitin, but not with the K63Rmutant (Fig. 8A). This result suggested that the decrease inc-MYC half-life observed after SIRT1 inhibition may be causedby reduced K63 polyubiquitination, which normally couldstabilize c-MYC by competing with K48-linked degradativeubiquitination of lysine residues. Also, after miRNA-mediatedreduction of SIRT1 expression in U2OS cells, the total amountof c-MYC–conjugated polyubiquitin as well as non–K48-linkedpolyubiquitin chains decreased when equal amounts of immu-noprecipitated c-MYC were analyzed (SI Appendix, Fig. S8C).Furthermore, mono, bi-, and polyvalences of K63-linked ubiq-uitin molecules conjugated to c-MYC were diminished asa result of the SIRT1 knockdown (SI Appendix, Fig. S8C). Toidentify the involved critical lysine residues of c-MYC, a mutantwith six lysines (K298, K317, K323, K326, K341, and K365)changed to arginine (Myc-K6R), previously shown to be defectivein homologous to E6-AP carboxy-terminus H9 (HectH9)-medi-ated K63-linked ubiquitination (40), was analyzed. Interestingly,polyubiquitination of the Myc-K6R mutant was reduced afterSIRT1 inhibition by Tenovin-6 in a manner similar to wild-typec-MYC (Fig. 8B and SI Appendix, Fig. S8D). The same pattern ofpolyubiquitination of Myc-K6R and of wild-type c-MYC also wasobtained using K48R and K63R ubiquitin mutants in response toTenovin-6. Therefore, the six lysines mutated in Myc-K6R pre-sumably are not targeted by SIRT1 for deacetylation. In summary,our results suggest that SIRT1-mediated deacetylation increasesconjugation of K63-linked ubiquitin chains to c-MYC; thesechains do not support degradation but may lead to stabilization ofc-MYC by preventing K48-linked degradative ubiquitination.

SIRT1 Increases Transcriptional Activity of c-MYC. To evaluate thefunctional consequences of SIRT1-mediated deacetylation andstabilization of c-MYC, wemeasured the transcriptional activity of

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Fig. 5. SIRT1 suppresses apoptosis and senescence in c-MYC–immortalized HDF. (A) Expression of SIRT1 in primary (passage 16) HDF and in c-MYC (passage77) and hTERT-immortalized HDF (passage 92) as determined by Western blot analysis. (B) Four days after transfection of primary and c-MYC–immortalizedHDFs with the indicated siRNAs, cells were stained for senescence-associated β-galactosidase at pH 6 as described (87). (Right) Representative phase-contrastimages of the cells before fixation. (Magnification: 200×.) (C) Effect of siRNA-mediated down-regulation of SIRT1 in HDF immortalized by c-MYC. Four daysafter transfection with the indicated siRNAs, the percentage of cells in sub-G1 was determined by flow cytometric analysis of DNA content. (D) Effect ofinhibition of SIRT1 activity on c-MYC–immortalized HDFs. Sirtinol (50 μM), a small molecule inhibitor of SIRT1, was added to primary c-MYC–immortalized andhTERT-immortalized HDFs for 24 h. The percentage of cells in sub-G1 was determined by flow cytometric analysis of DNA content. Mean values ± SD for threeindependent experiments are shown.

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c-MYC in reporter assays (Fig. 8C and SI Appendix, Fig. S8 E andF). Coexpression of SIRT1 resulted in a pronounced increase inthe transactivation of an E-box-containing reporter construct byc-MYC, whereas a catalytically inactive SIRT1 mutant did notenhance c-MYC activity. Analysis of the cell lysates used for thereporter assay confirmed stabilization of c-MYC in the presence ofcatalytically active SIRT1 but not by SIRT1-H363Y. Furthermore,mutation of c-MYC-K323, a lysine residue previously implicatedin the regulation of c-MYC activity (42), did not abolish the sta-bilizing, stimulatory effect of SIRT1 on the c-MYC protein (Fig.8C). In addition, the expression of endogenous c-MYC targetgenes was elevated by ectopic expression of SIRT1 or reducedby shRNA-mediated down-regulation of SIRT1 (Fig. 8D andSI Appendix, Fig. S8G). In summary, SIRT1 positively regulatestransactivation by c-MYC, presumably by increasing the abun-dance of c-MYC via interfering with its degradation. In combi-nation with the effects of c-MYC on SIRT1 described above,

c-MYC and SIRT1 therefore seem to be connected by a positivefeedback loop involving several factors and mechanisms.

DiscussionOur results show that the activation of SIRT1 by c-MYC and thesubsequent deacetylation of p53 or other SIRT1 substrates pro-vide a mechanism for promoting c-MYC–induced cellular pro-liferation by suppressing apoptosis and senescence. These findingssuggest that endogenous SIRT1 exerts protumorigenic activities inthe context of c-MYC–driven tumor development, at least in partbecause of the ability to prevent or attenuate cellular senescenceand/or c-MYC–induced apoptosis. A number of studies linkSIRT1 to cancer-relevant substrates, including p53, E2F1, BAX,hypermethylated in cancer (HIC1), and FOXO3 (15). The de-acetylation of these proteins by SIRT1 results in inhibition ofapoptosis and senescence, and therefore may favor the growth of

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Fig. 6. c-MYC is a SIRT1 substrate. (A) Coimmunoprecipitation of endoge-nous c-MYC and SIRT1. SIRT1 was immunoprecipitated from lysates ofU2OS cells using a polyclonal SIRT1-specific antibody. Rabbit IgG served ascontrol. Coprecipitated proteins were detected by Western blot analysisusing the indicated antibodies. (B) Recombinant c-MYC fused to maltose-binding protein (MBP-c-MYC) was incubated with recombinant, baculovirus-expressed His-CBP and [14C]-acetyl-CoA. MBP-c-MYC was deacetylated withtandem-affinity purification-tagged SIRT1 purified from HEK-293 cells. Theamount of the released acetyl ADP-ribose in the supernatant was measuredby scintillation counting. As a control NAD+ was omitted or 10 mM NAM wasadded to the deacetylation assay as indicated. (C) HEK-293 cells were tran-siently transfected with a vector encoding FLAG-c-MYC and were incubatedwith TSA or NAM or were left untreated. Acetylation of immunoprecipitatedFLAG-c-MYC was detected by Western blot analysis using a pan–acetyl-ly-sine–specific antibody. (D) FLAG-tagged c-MYC was coexpressed in HEK-293cells with the indicated proteins or an empty vector. c-MYC was immuno-precipitated using a FLAG-specific antibody and was detected by immuno-blot analysis using a c-MYC– or a pan–acetyl-lysine–specific antibody.

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Fig. 7. Regulation of c-MYC expression and half-life by SIRT1. (A) Expo-nentially proliferating, subconfluent MEFs were lysed and subjected to im-munoblot analysis of the indicated proteins. (B) Exponentially proliferating,subconfluent MEFs (as in A) were analyzed by qPCR for mRNA expression ofc-MYC and target genes. The mean values ± SD of biological triplicates areshown. (C) MCF-7 cells stably expressing a SIRT-VSV fusion protein (right fourlanes) or the vector backbone (left four lanes) were analyzed. Cells werepulse-labeled with [35S]methionine followed by chase in medium containingcold methionine for the indicated time periods. Cell lysates were immuno-precipitated with anti-MYC antibody (N262) and subjected to SDS/PAGEfollowed by quantification with a phospho-imager. (D) U2OS cells harboringDOX-inducible pEMI vectors encoding a SIRT1-specific miRNA were analyzedafter addition of DOX (0.1 μg/mL) for 72 h. The corresponding controlsample expressed a nonsilencing miRNA pEMI vector. Pulse chase andanalysis were done as in C. (E) HCT-116 cells were treated with DMSO or anSIRT1 inhibitor, tenovin-6 (10 μM) for 8 h followed by a CHX chase for theindicated time points (minutes). Cell lysates were subjected to immuno-precipitation with a polyclonal c-MYC antibody (N262). Precipitated c-MYCwas determined by immunoblot analysis with the monoclonal c-MYC anti-body (C33). In C–E, the densitometric quantification of the remaining pro-tein expressed as percentage of the starting amount is shown in thediagrams (Right).

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tumors (23, 26, 43–45). Furthermore, the elevated expression ofSIRT1 protein in primary human tumors and its association withpoor prognosis in diffuse large B-cell lymphoma, as well as inprostate, gastric, and breast cancer support a role for SIRT1 in themaintenance of cancer cells (46–49).We provide evidence that c-MYC regulates SIRT1 activity by at

least two mechanisms: (i) by induction of the NAMPT gene,leading to an increase in the SIRT1 cofactor NAD+, and (ii) viasequestration of the SIRT1 inhibitor DBC1. This dual regulation

results in an increased amount and activity of the SIRT1 protein.The molecular details of SIRT1 stabilization are still unknown,although it has been shown that augmented activity of SIRT1 isaccompanied by an increase in the amount of SIRT1 protein (22,50). In line with this observation, many enzymes accumulate uponactivation because they are engaged in stabilizing complexes whenbinding to substrates (51). In the case of SIRT1, a plausible sce-nario is a conformational tightening of the ternary complex of theSir2 apoenzyme with NAD+ and an acetylated substrate whichappears to be more compact than the binary complex and there-fore less accessible to proteolytic degradation (52).NAMPT may stimulate SIRT1 activity via two routes (53):

Besides enhancing NAD+ production, NAMPT also reducesthe concentration of cellular NAM, the main endogenous SIRT1inhibitor. The NAMPT-mediated activation of SIRT1 has beenimplicated in the regulation of differentiation, stress response,and metabolism and in the promoting the extension of thecellular life span (22, 30). SIRT1 accomplishes these roles bydeacetylation of substrates, such as the p53 and PARP proteins,and by protection from PARP-induced and/or p53-dependentcell death (22, 26, 30). Therefore, c-MYC–induced NAMPT ex-pression may contribute to cell proliferation in part by activatingSIRT1 and inhibiting various proapoptotic factors. Intriguingly,activation of lymphocytes, which is associated with elevatedc-MYC levels, leads to enhanced NAMPT expression (54).Moreover, the subcellular localization of NAMPT was shown tobe regulated in a cell cycle-dependent manner (55), indicative ofan additional mode of regulation by c-MYC. Furthermore, in-creased NAMPT expression in tumors correlates with cancerprogression and bad prognosis (56, 57). These oncogenic prop-erties of NAMPT are consistent with its induction by c-MYC.NAMPT inhibitors are being tested currently in clinical cancertrials (58). In mouse tumors inhibition of NAMPT affects notonly NAD+ levels but also glycolysis (59). Therefore, in additionto inducing glycolytic enzymes, c-MYC also may enhance gly-colysis via the activation of NAMPT and the salvage pathway.Recently, DBC1 was identified as a negative regulator of

SIRT1 (17, 18, 60), but how and in which physiological contextthe DBC1–SIRT1 interaction is regulated remained elusive. Theresults shown here suggest that the elevated or constitutivec-MYC expression found in tumor cells may interfere with theDBC1-mediated inhibition of SIRT1 via sequestration of DBC1by c-MYC, resulting in increased SIRT1 activity and ultimatelyin elevated c-MYC levels and activity. However, our experimentsdo not rule out the possibility that the observed effect also maybe caused by c-MYC interfering with the inhibitory effect ofDBC1 on other enzymes, such as histone deacetylase 3 andSuvH39 (61, 62). Intriguingly, a decrease in the interaction be-tween DBC1 and SIRT1 has been found recently in mammarycarcinoma cell lines (63), which are known to display elevatedc-MYC expression and c-MYC amplifications.We showed that c-MYC–activated SIRT1 feeds back to c-MYC

by deacetylating c-MYC, in turn increasing the stability of en-dogenous c-MYC. Deacetylation through SIRT1 presumablyrenders target lysines available for K63-mediated polyubiquiti-nation. It was shown previously that K63-linked ubiquitination ofc-MYC is mediated by HectH9 and recruits cofactors such as p300and thereby enhances c-MYC’s transcriptional activity (40). Inaccordance with our results, K63-linked ubiquitination mediatedby HectH9 has been shown to lead to increased c-MYC function(40). K63-linked ubiquitin chains usually do not promote protea-somal degradation but may result in protein stabilization byreplacing degradative K48-linked chains, as recently shown for thec-JUN coactivator RACO-1 (64). The typical half-life of c-MYC isless than 30 min (65, 66), but in cancer c-MYC exhibits an ex-tended half-life (67–69). In some cancers, these high c-MYC levelshave been associated with impaired c-MYC turnover throughFbw7/Cdc4mutation (70). However, so far Fbw7/CDC4 is the onlyE3 ligase of c-MYC that has been shown to be a target for muta-

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Fig. 8. SIRT1 regulates ubiquitination and activity of c-MYC. (A) HCT-116cells were transiently transfected with wild-type c-MYC together with theindicated HA-tagged ubiquitin constructs. Forty hours after transfection,cells were treated with DMSO or the SIRT1 inhibitor tenovin-6 (10 μM) for 8 hfollowed by immunoprecipitation of c-MYC and immunoblot detection ofHA-ubiquitin using a monoclonal HA-specific antibody (12CA5). Immuno-precipitated c-MYC was detected using a monoclonal c-MYC–specific anti-body (C33). (B) Densitometric analysis of c-MYC ubiquitination. As in A, HCT-116 cells coexpressing wild-type c-MYC or the Myc-K6R were cotransfectedwith wild-type ubiquitin or mutant ubiquitin (K48R-Ub, K63R-Ub) constructs.The signal intensities of the blots were quantitated with a CCD camera. (C)HEK-293 cells were cotransfected with the M4–min-tk–luc reporter construct(38) with four MYC/MAX binding sites and expression plasmids encoding c-MYC, c-MYC-K323R, HA-SIRT1, and HA-SIRT1-H363Y. Mean values of threeindependent experiments performed in duplicate are shown. Western blotanalysis of the indicated proteins detected in reporter assay lysates is shownin the lower panel. (D) qPCR analysis of the c-MYC target gene DKC 11 hafter activation of a tetracycline-regulatable c-MYC allele in P493-6 B cellsstably expressing pINCO-SIRT1 or the vector backbone pINCO as a control.Error bars represent SD of biological triplicates.

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tional inactivation, albeit with low frequency. Therefore, the in-creased half-life of c-MYC resulting from SIRT1-mediateddeacetylation may explain the elevated c-MYC protein levelsfound in certain tumor types and may contribute to tumor for-mation. Our results are in contradiction to recently published workby Yuan et al. (42), because we did not observe a direct inductionof SIRT1 mRNA expression by c-MYC or a negative feedbackinhibition of c-MYC by SIRT1. In our study, we used multipleexperimental systems and analyses that generally are accepted asrequired for the confirmation of direct regulation by c-MYC in thefield (71, 72). However, none of the different systems used hererevealed an induction of SIRT1 mRNA following c-MYC activa-tion. Furthermore, the published microarray or serial analysis ofgene expression studies investigating transcriptional regulation byc-MYC did not reveal a significant mRNA induction of SIRT1 byc-MYC (73, 74). However, this result does not exclude the possi-bility that c-MYC could affect SIRT1mRNA expression indirectlyunder certain conditions. Another result conflicting with theresults reported by Yuan et al. (42) is the influence of SIRT1 onc-MYC stability. Although Yuan et al. found that enforced SIRT1expression increases the rate of c-MYC turnover, our resultssuggest that SIRT1 stabilizes c-MYC. This discrepancy likely arisesfrom the different experimental approaches. Furthermore, thehalf-lives of c-MYC reported by Yuan et al. in the absence ofSIRT1 deviate substantially from those reported in the literature[ca. 3 h vs. 20–40 min (65, 66)], whereas our results are well withinthe range of previously published data (67–69). When we com-pared half-life measurements of c-MYC in the presence of ectopicSIRT1 expression, we found that results obtained after CHXtreatment oppose those obtained by 35S pulse-chase experiments.Therefore, we suspect that CHX treatment in this context causesan artificial degradation of c-MYC. Although Yuan et al. (42)consistently used CHX treatment and found a negative effect ofSIRT1 on c-MYC half-life, our S35 pulse-chase analyses show thatSIRT1 increases the half-life of c-MYC. The latter results areconsistent with the increased steady-state levels and transcrip-tional activity of c-MYC we observed in response to increasedSIRT1 expression and with the reduced c-MYC stability caused byknockdown, enzymatic inhibition, or deletion of SIRT1. Further-more, in our hands mutation of K323 in c-MYC did not affectSIRT1-mediated changes in c-MYC stability and c-MYC–driventranscription. Yuan et al. (42) found that mutation of K323influences c-MYC stability and transactivation. The reason forthese discrepancies will have to be resolved in the future.Our results support a tumor-promoting function of SIRT1 in

the context of c-MYC activation. Other studies also have providedevidence for a protumorigenic function of SIRT1 (39, 45, 75–77).For example, the tumor suppressor HIC1, which is epigeneticallysilenced in cancer, functions as an inhibitor of SIRT1 expression(75). Furthermore, SIRT1 inhibitors have antitumor activity invivo (39, 45). Nonetheless, recent mouse tumor models arguefor a tumor-suppressive role of SIRT1 (78). Since these mousemodels did not represent c-MYC–induced tumors, it will be im-portant to analyze the role of SIRT1 in MYC-driven tumormodels and to clarify this point in the future. It should be men-tioned, however, that at present there is no evidence for genetic or

epigenetic alterations affecting SIRT1 in cancer. Therefore, theSIRT1 gene itself does not seem to represent a proto-oncogene ora tumor-suppressor gene. Only recently it has been shown, thatSIRT1 has species-specific functions that also are dependent onthe genetic background (21, 79). Therefore, it seems that SIRT1 isinvolved in oncogenic or tumor-suppressive signaling in a species-,tumor type-, and context-dependent manner. In the future com-parative analysis of mouse and human SIRT1 functions mayreveal explanations for the ambivalent role of SIRT1 in thepathogenesis of human and mouse tumors.Taken together, the positive feedback loop linking c-MYC,

NAMPT, DBC1, and SIRT1 described here suggests that ther-apeutic inhibition of NAMPT or SIRT1 enzymatic activities maybe a suitable approach to sensitize human cancer cells withderegulated expression of c-MYC, regardless of their p53 status.

MethodsCell Culture and Conditional Systems. P493-6 cells, RAT1 (TGR-1), and c-Myc−/−

RAT1 (HO15.19) fibroblasts were maintained as described previously (74).U2OS cells, MCF-7 cells, MCF-7 cells with an inducible c-MYC allele (PJMMR1),MEFs (sirt+/+,−/−), HDF cells, and c-MYC–immortalized HDF cells were kept inDMEM containing 10% (vol/vol) FBS. c-Myc−/− RAT1 (subclone HO15.19) stablyexpressing Myc-ER were kept in phenol-red free DMEM supplemented with8% (vol/vol) FBS. HDF-Myc-ER (immortalized by hTERT) cells were grown inphenol-red free DMEM and 10% (vol/vol) FBS. To down-regulate endogenousc-MYC in the PJMMR1 (MCF-7) cell line, the antiestrogen ICI 182,780 (1 μM)was added to the cells for 60 h before activation of DOX-inducible c-MYC.

RNA Interference. To generate pEMI vectors targeting DBC1 or SIRT1, thespecific hairpins from the pSM2c library (80) were subcloned into the pEMIvector as described previously (81). siRNAs were transfected at a final con-centration of 5–40 nM using the fast-forward protocol (Qiagen). Sequencesof shRNAs, miRNAs, and siRNA oligonucleotides are available on request.

Western Blot Analysis. Cells were lysed in RIPA buffer [50mMTris·HCL (pH 7.4),1% Nonidet P-40, 0.1% SDS, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mMEDTA, 1 mM PMSF, 1 mM Na3VO4, and protease inhibitor mixture (CompleteMini; Roche)]. To detect p53 acetylation, NAM (5 mM) and TSA (1 μM) wereadded to the lysis buffer. The signals were obtained with enhanced chem-iluminescence reagent (Perkin-Elmer) and recorded with a 440CF imagingsystem (Kodak). Antibodies used are given in SI Appendix, SI Methods.

All other methods, further details, and associated references are providedonline in SI Appendix, SI Methods.

ACKNOWLEDGMENTS. We thank Carla Grandori, Roy Frye, Tony Kouzarides,Dirk Eick, John Sedivy, Bert Vogelstein, Yoichi Taya, Hans Clevers, AxelUllrich, Martin Eilers, Fuyuki Ishikawa, and Raul Mostoslavsky for providingantibodies, plasmids, and cell lines; Sonia Lain and Jo Campbell for Tenovin-6; Heike Koch for generating DBC1-related reagents; Robert Huber, StefanMüller, and Sybille Mazurek for helpful discussions; Ruwin Pandtihage andRichard Lilischkis for help with the initial MYC-SIRT1 deacetylase assays; FuadBahram for help with the initial MYC degradation assays; and Andrea Sen-delhofert and Anja Heier for immunohistochemistry. A.M. thanks Axel Ull-rich for support. This work was supported by the Max-Planck-Society and theDeutsche Krebshilfe (H.H.), by the Deutsche Forschungsgemeinschaft andthe START program of the Medical School of the RWTH Aachen University(B.L.), and by the Olle Enkvist’s Foundation, the Swedish Cancer Society,the Swedish Childhood Cancer Foundation, and the Swedish ResearchCouncil (L.-G.L.).

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