Regulation of FANCD2 by the mTOR Pathway Contributes to ... · PI3K–mTOR–AKT pathway in Rh30...

Therapeutics, Targets, and Chemical Biology

Regulation of FANCD2 by the mTOR Pathway Contributes totheResistanceofCancerCells toDNADouble-StrandBreaks

Changxian Shen1, Duane Oswald1, Doris Phelps1, Hakan Cam1, Christopher E. Pelloski2, Qishen Pang3, andPeter J. Houghton1

AbstractDeregulation of the mTOR pathway is closely associated with tumorigenesis. Accordingly, mTOR inhibitors

such as rapamycin and mTOR-selective kinase inhibitors have been tested as cancer therapeutic agents.Inhibition of mTOR results in sensitization to DNA-damaging agents; however, the molecular mechanism isnot well understood. We found that an mTOR-selective kinase inhibitor, AZD8055, significantly enhancedsensitivity of a pediatric rhabdomyosarcoma xenograft to radiotherapy and sensitized rhabdomyosarcoma cellsto the DNA interstrand cross-linker (ICL)melphalan. Sensitization correlated with drug-induced downregulationof a key component of the Fanconi anemia pathway, FANCD2 through mTOR regulation of FANCD2 genetranscripts via mTORC1-S6K1. Importantly, we show that FANCD2 is required for the proper activation ofATM-Chk2 checkpoint in response to ICL and that mTOR signaling promotes ICL-induced ATM-Chk2checkpoint activation by sustaining FANCD2. In FANCD2-deficient lymphoblasts, FANCD2 is essential tosuppress endogenous and induced DNA damage, and FANCD2-deficient cells showed impaired ATM-Chk2 andATR-Chk1 activation, which was rescued by reintroduction of wild-type FANCD2. Pharmacologic inhibition ofPI3K–mTOR–AKT pathway in Rh30 rhabdomyosarcoma cells attenuated ICL-induced activation of ATM,accompanied with the decrease of FANCD2. These data suggest that the mTOR pathway may promote therepair of DNA double-strand breaks by sustaining FANCD2 and provide a novel mechanism of how the Fanconianemia pathway modulates DNA damage response and repair. Cancer Res; 73(11); 3393–401. �2013 AACR.

IntroductionRecent data have shown that cyclin-dependent kinases

(CDK) are required for the processing of damaged DNA endsand checkpoint activation (1–3), but themolecularmechanismbywhichCDKs act is unknown. PI3K–AKT andRAS–MAPKarethe predominant growth-promoting signaling pathways,which enhance CDK activity and increase cell-cycle progres-sion. Cancer genome projects showed that PI3K–AKT andRAS–MAPK are among the most frequently mutated signalingpathways in cancers (4), and both pathways converge onmTOR signaling (5). mTOR lies at the hub of intracellular andextracellular signal transduction networks via integrating andprocessing multiple signals, and dictates the rates of macro-molecule synthesis and hence cell growth, proliferation, and

survival (6–8). Aberrant activity of mTOR signaling has beenfound in most cancers, making mTOR an important target forcancer drug development (7, 9, 10). We previously found thatthe TOR pathway promotes cell survival at the cost of elevatedmutation rate in response to DNA replication stress and DNAdamage in yeast (11). Data also show that inhibiting the mTORpathway sensitizes many cancer cells to chemotherapy andradiotherapy; however, the molecular mechanism by whichthis occurs is largely unclear (12–14).

These observations suggest that mTOR signaling maintainssome components of the DNA damage response (DDR) andrepair response. Besides homologous recombination, most ifnot all DNA repair systems are error prone and the majorfunction of DNA damage checkpoint and repair systems is tomaximize cell survival. It was intriguing, therefore, to testwhether mTOR signaling modulates DDR and enhances DNAdamage repair in cancer cells.

In this study, using pediatric rhabdomyosarcoma models invitro and in vivo, we have investigated the functional linkagebetween the mTOR pathway and DDR, and found that mTORsignaling sustains FANCD2-mediated signaling pathways topromote DDR via potentiating the ATM checkpoint activationin response DNA double-strand breaks (DSB).

Materials and MethodsDrugs

AZD8055, MK2206, and PD0332991 were provided by Astra-Zeneca, Merck, and Pfizer, respectively. Rapamycin was from

Authors' Affiliations: 1Center for Childhood Cancer & Blood Diseases,Nationwide Children's Hospital; 2Department of Radiation Oncology,Arthur G. James Comprehensive Cancer Center and Richard L. SoloveResearch Institute, Ohio State University, Columbus; and 3Division ofExperimental Hematology and Cancer Biology, the Cincinnati Children'sHospital Medical Center, Cincinnati, Ohio

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Peter J. Houghton, Center for Childhood Cancer &Blood Diseases, The Research Institute, Nationwide Children's Hospital, 700Children'sDrive,ResearchBuilding II,Columbus,OH43205.Phone:614-355-2670; Fax: 614-355-2927; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-4282

�2013 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst April 30, 2013; DOI: 10.1158/0008-5472.CAN-12-4282

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the National Cancer Institute drug repository (Frederick, MD).Melphalan, cisplatin, mitomycin C, cycloheximide, andhydroxyurea were purchased from Sigma.

Solid tumor xenografts studiesCB17SC scid�/� female mice (Taconic Farms) were used to

propagate subcutaneously implanted rhabdomyosarcoma. Allmice were maintained under barrier conditions and experi-ments were carried out using protocols and conditionsapproved by the Institutional Animal Care and Use Committee.AZD8055 was administered per os daily at 20 mg/kg/day.Rapamycin was dissolved in dimethyl sulfoxide (5% final con-centration) and diluted in 5% Tween-80 in water and admin-istered intraperitoneally daily at a doseof 5mg/kg.Tumorswereharvested after treatment on day 1 or 4 at different timepoints.

Cells, siRNA, and plasmidsRhabdomyosarcoma Rh30 cells were cultured in RPMI-1640

(GIBCO) supplemented with 10% heat-inactivated FBS(GIBCO). Control and ON-TARGETplusSMARTpool siRNAs ofCDK4, CDK6, mTOR, and FANCD2 were purchased fromDharmacon. Plasmid pcDNA3 Myr-AKT1 was a gift of WilliamSellers (Novartis, Cambridge MA; Addgene plasmid 9008).pcDNA-DEST40 and pcDNA-DEST40-FANCD2 plasmids wereprovided by Niall G. Howlett (Moffitt Cancer Center). S6K1knockout mouse embryonic fibroblast (MEF) was provided byGeorge Thomas (University of Cincinnati, Cincinnati, OH).MEF cells were cultured in Dulbecco's Modified Eagle Medium(GIBCO) supplementedwith 10%heat-inactivated FBS (GIBCO).Lipofectamine 2000 was from Invitrogen and transfection ofsiRNA or plasmids in Rh30 cells was conducted according to themanufacturer's instructions.

Immunoblotting and immunofluorescence stainingCells were lysed on ice in radioimmunoprecipitation assay

lysis buffer (Cell Signaling Technology) supplemented withprotease inhibitors and phosphatase inhibitor (Roche), and 1mmol/L phenylmethylsulfonylfluoride (Sigma). Immunoblotswere probed with the following antibodies: S6, pS6 (S235/236),AKT, pAKT (S473), ATM, pATM (S1981), ATR, DNA-PKcs,CHK1, pCHK1 (S345), CHK2, pCHK2 (T68), p53, p53-S15,mTOR, S6K1, pS6K1 (T389), GSK3b, pGSK3b (S9), p4E-BP1(T37/46), b-actin, Myc, GAPDH, Rb, pRb (S780); Cell SignalingTechnology); pH2AX (S139; Upstate); PARP1, cleaved PARP1(Abnova); FANCD2 (Epitomics); FANCC, FANCA (Abcam);RAD51, CDK4, CDK6 (Santa Cruz). For immunofluorescencestaining, cells grown on sterile cover slips were fixed in 100%cold methanol, washed with TBS, and blocked with 1% bovineserum albumin (BSA) in PBS. Cells were incubated with thegH2AX antibody (Clone JBW301, UPSTATE) diluted in 1% BSAin TBS for 60 minutes, and washed in 1� TBS. The slides wereadditionally incubated with the FANCD2 antibody diluted in1% BSA in TBS for 60 minutes and washed in 1� TBS.Secondary antibodies Alexa Fluor 488 goat anti-mouse immu-noglobulin G (IgG) and rhodamine (TRIT)-conjugated goatanti-rat IgG were from Invitrogen. Sample was simultaneouslystained with 40, 6-diamidino-2-phenylindole (DAPI) to detectnucleus. The fluorescence was viewed with a Zeiss axioskop 2microscope equipped with differential interference contrast,

epi-fluorescence, and UV-blocking filter sets. Images wereacquired with a Micromax CCD camera (Princeton Instru-ments Inc.,) and IP lab software (Scanalytics).

RNA isolation, cDNA synthesis, and reversetranscriptase-PCR

Total RNA from cultured cells was extracted with mirVanamiRNA Isolation Kit (Ambion) according to the total RNAisolation protocol. Reverse transcriptionwere conducted usingthe High Capacity RNA-to-cDNA Kit (Applied Biosystems)according to the manufacturer's instructions. Real-time PCRwas conducted on the 7900HT Fast Real-Time PCR Systemusing the TaqMan Universal Mastermix II. Human and mouseFANCD2 expression was quantified in real time with FANCD2-specific FAM dye-labeled MGB probes and normalized toGAPDH, MYCN, or b-actin (Applied Biosystems).

Cell-cycle analysis and colony formation assayFollowing treatment, cells were trypsinized, fixed with

70% ethanol, and stored at 4�C for subsequent fluorescence-activated cell sorting analysis of DNA content. For colonyformation assay, 500 cells were plated in a 10 cm Petri dish.Two weeks after treatment, cells were washed with PBS,fixed with cold methanol for 15 minutes under �20�C, andstained with 1% Crystal Violet (Fisher) in 25% methanol for15 minutes. The dishes were washed with water extensivelyand the blue colonies were counted.

ResultsAn mTOR kinase inhibitor sensitizes cancer cells toradiotherapy and chemotherapy

Several mTOR-selective kinase inhibitors have recently beenreported that in comparison with rapalogs, abrogate mTORcomplex 1 (mTORC1)-mediated phosphorylation of 4E-BP1(15, 16). We previously showed that AZD8055 is a potent andspecificmTOR kinase inhibitor (17). To test the involvement ofmTOR in DDR, we examined the effect of AZD8055 on ionizingradiation using theRh30 xenograftmodel.Micewere irradiated(2 Gy/tumor day) usingwhole body shielding to a range of totaldoses (from 10–40 Gy) with or without AZD8055 at variousstarting volumes. In the radiation alone group, radiationtreatment induced tumor volume regressions, but tumorsregrew and progressed in 14 of the 18 mice (78% failure rate)with a radiation dose per volume of tumor of 60 Gy/cm3. Incontrast, only in 4 of the 15 mice did tumors regrow in thecombination XRT-AZD8055 treatment arm (Table 1) with dose

Table 1. Sensitization to ionizing radiation byAZD8055

GroupMeandose

Failures/total

Failurerate

XRT 60 Gy/CC 14/18 78%XRT þ AZD 27 Gy/CC 4/15 27%

Abbreviations: XRT, X-ray treatment; AZD, AZD8055.

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per volume of 27 Gy/cm3; a more than 50% reduction inradiation dose with a similar reduction in failure rate. In Rh30xenografts, AZD8055 has limited single-agent activity, slowingtumor progression, but does not induce either stable disease ortumor regression (17). Thus, this mTOR kinase inhibitor maybe a promising radiosensitizer.We previously reported that rapamycin enhanced the activ-

ity of the bifunctional alkylating agent, cyclophosphamide,against several pediatric tumor models (18). To extend thisobservation in vitro, we checked whether AZD8055 sensitizescultured Rh30 cells to DNA interstrand cross-linker (ICL)melphalan, which produces DNA DSB. We pretreated Rh30cells with AZD8055 for 16 hours, and transiently exposed thecells to melphalan for 2 hours. Drugs were washed away, andsurvival was assayed by colony formation. AZD8055 or tran-sient insult withmelphalan resulted in 16% and 18% loss of cellviability, respectively. Pretreatment with AZD8055 followed bytransient melphalan exposure led to a significant decrease incolony formation (66%; Fig. 1A and B). Thus, targeting mTORkinase sensitizes cancer cells to ICL-based chemotherapies.

The mTOR pathway controls FANCD2The above data suggest that mTOR signaling plays an

important role for cancer cells to survive DSB-producingagents. This may be not due to the decrease of ATM, ATRand DNA-PKcs, as AZD8055 did not alter the protein levels of

ATR, RAD51, and DNA-PKcs but slightly increased ATM (Fig.1C). Of note, exposure to AZD8055 resulted inmarked decreasein FANCD2, but not other components of the Fanconi anemiapathway (FANCC, FANCA). Fanconi anemia pathway-deficientcells display phosphorylation of H2AX, amarker of DNA strandbreaks, and defect of DNA damage checkpoint in response toDNA damage (19, 20). Fanconi anemia cells, such as FANCD2-deficient PD20 lymphoblast cells, display high levels of gH2AX,and reexpression of FANCD2 in PD20 cells reduced gH2AXlevels (Fig. 1D; refs. 19, 20). Similarly, knockdown of FANCD2bysiRNA led to increased gH2AX in Rh30 cells (Fig. 1E). Inter-estingly, AZD8055 downregulated FANCD2 and inducedgH2AX, whereas ectopic overexpression of FANCD2 partiallyattenuated AZD8055-induced gH2AX (Fig. 1F). These resultsled us to hypothesize that mTOR signaling may regulate DDRand hence DSB repair by controlling FANCD2 signaling.AZD8055 treatment reduced the levels of FANCD2 in atime-dependent manner (Supplementary Fig. S1A). Similarly,knockdown ofmTORby siRNA decreased FANCD2 (Fig. 1G). Incontrast to melphalan that causes ICL, inhibition of mTORsignaling did not result in the monoubiquitination of FANCD2in Rh30 cells (Fig. 1H). In addition, overexpression of wild-typeAKT1 (Supplementary Fig. S1B) or myristoylated AKT1 (myr-AKT1; Supplementary Fig. S1C) in Rh30 cells increasedFANCD2 and was associated with enhanced activity of theAKT pathway as shown by the elevation of the pGSK3b-S9

Figure 1. ThemTORpathwaypromotes cell survival in response tomephalan by sustaining FANCD2. A, five hundred cellswere plated in 10 cmdish for 6 hours,then AZD8055 (2 mmol/L) was applied for 16 hours. Melphalan (0.2 mg/mL) was added to untreated or AZD8055-treated cells for 2 hours. The drugs werewashed away. Two weeks later, the colonies were counted. Shown is a representative of 3 independent experiments. B, quantification of the colonies inFig. 1A. Error bars, mean� SD (n¼ 3). C, Rh30 cells were treated with AZD8055 (2 mmol/L) for 16 hours. Total proteins were extracted for immunoblotting. D,lymphoblast PD20 cells derived from a patient with FANCD2 were stably transfected with empty vector (pMMP-Puro) or wild-type FANCD2 plasmid (pMMP-wt-FANCD2). FANCD2 and gH2AX were detected by immunoblotting.�, pMMP-Puro;þ, pMMP-wt-FANCD2. E, Rh30 cells were transfected with control orFANCD2 siRNA. Forty-eight hours later, total proteins were extracted for immunoblotting. F, Rh30 cells were transfected with vector or FANCD2 plasmid.Twenty-four hours later, AZD8055 (2 mmol/L) was added for additional 24 hours. Total proteins were extracted for immunoblotting. G, Rh30 cells weretransfected with control or mTOR siRNA. Forty-eight hours later, total proteins were extracted for immunoblotting. H, Rh30 cells were treated with AZD8055(AZD, 2 mmol/L) or melphalan (MP, 2 mg/mL) for the time indicated. Total proteins were extracted for immunoblotting. UT, untreated. Upper band of FANCD2staining showed the monoubiquitination of FANCD2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and b-actin served as loading controls.

Regulation of FANCD2 by mTOR

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signal. These data showed that AKT–mTOR signaling controlsthe levels of FANCD2. To test this further, we treated culturedrhabdomyosarcoma Rh30 cells with mTORC1-specific inhib-itor rapamycin, AZD8055 and AKT kinase inhibitor MK2206(21). Rapamycin inhibited the phosphorylation of pS6K1(T389), a marker of mTORC1 activity, but increased the phos-phorylation of AKT at Ser473 due to the mTORC1-S6K1-IRS–negative feedback loop (22). Treatmentwith either AZD8055 orMK2206 resulted in absence of the phosphorylation of bothpS6K1 (T389) and pAKT (S473), indicating inhibition of bothmTORC1 and mTORC2. Similar to AZD8055, MK2006 reducedFANCD2 although the effect of rapamycinwas less pronounced(Supplementary Fig. S1D), showing that AKT–mTOR pathwaycontrols FANCD2.

Our data suggest that mTOR signaling may be involved inthe DDR and repair of DSB by regulating the Fanconi anemiasignaling pathway. To test this in vivo, we next checkedFANCD2 protein levels of the mouse xenografts of Rh18, Rh30,and Rh10 following treatment with AZD8055. Mice receivedAZD8055 daily for 4 days, and tumorswere harvested 1, 4, 8, and24 hours after the final dose. AZD8055 efficiently and rapidlyinhibited both the mTORC1 and mTORC2, as shown by thedecrease of p4E-BP1-T37/46, pS6-S235/6, and pAKT-S473 sig-nals 1 hour after treatment (Fig. 2A and B). At 24 hours afterAZD8055 dosing, FANCD2wasmarkedly reduced in all of the 3tumor models (Fig. 2C). As AZD8055 inhibits both mTORC1and mTORC2, we additionally checked the FANCD2 of themouse tumor xenografts Rh30 and Rh18 treated with rapa-mycin. Similar to AZD8055, inhibition of mTORC1 by rapa-mycin resulted in apparent downregulation of FANCD2 in vivo(Fig. 2D). These in vivo and in vitro data suggest that FANCD2 isregulated by the mTORC1 pathway.

mTORC1-S6K1 signaling controls transcription ofFANCD2

The above results showed that inhibition ofmTORC1 led todecreased FANCD2 both in tumor xenografts and culturedcells. As TOR signaling has been implicated in both transla-tion and transcription, we probed the mechanism by whichthis pathway regulates FANCD2 levels. We determined themRNA levels of FANCD2 following inhibition of mTOR sig-naling by real-time reverse transcriptase PCR (RT-PCR).Treatment of Rh30 cells with AZD8055 resulted in a progres-sive decrease in FANCD2 mRNA (Fig. 3A). Similarly, rapamy-cin and MK2206 also decreased FANCD2 mRNA (Fig. 3B).Conversely, overexpression of AKT1 increased the mRNA ofFANCD2 (Fig. 3C). Thus mTOR signaling seems to regulateeither transcription or transcript stability of FANCD2 gene.

From yeast tomammalian cells, the TOR pathway promotescell-cycle progression by increasing the activity of CDKs (8).Indeed, AZD8055 robustly reduced both total and pRb-S780(Fig. 3D). Recently, an essential role for CDKs in processing ofdamaged DNA ends and checkpoint activation has beendescribed (1). Our data show that mTOR coordinately controlsthe proteins and mRNAs of FANCD2. These observationssuggest that mTOR controls the gene expression of FANCD2by regulating CDKs. To further test this observation, we treatedRh30 cells with rapamycin, AZD8055 or MK2206, in parallel

with a CDK4/6-specific inhibitor PD0332991 (23, 24), anddetermined FANCD2 by immunoblotting. Similar to AZD8055and MK2206, PD0332991 significantly reduced FANCD2 (Fig.3E). We further analyzed the FANCD2 mRNAs of Rh30 cellstreatedwith PD0332991. Consistentwith the decreased proteinlevels, PD0332991 reduced FANCD2mRNAs (Fig. 3F), showingthat the gene transcription/transcript stability of FANCD2 isindirectly controlled by CDKs. We next reduced CDK4 andCDK6 by siRNAs in Rh30 cells. Knockdown of CDK4 led toreduction of both FANCD2 protein (Supplementary Fig. S2A)and mRNA (Supplementary Fig. S2B). Interestingly, downre-gulation of CDK6 by siRNA increased both the protein andmRNA levels of FANCD2, accompanied by an enhanced pRb-S780 signal (Supplementary Fig. S2A). These data showed thatFANCD2 is positively controlled by CDK4, which is consistentwith the finding that FANCD2 is regulated by Rb-E2F1 (25).

We further tested whether FANCD2 mRNA is controlled bymTORC1 signaling, by examining whether S6K1 knock outMEF cells (S6K1�/� MEF). S6K1�/� MEFs showed significantdownregulation of FANCD2 protein (Fig. 3G) and mRNA (Fig.3H), and rapamcyin did not further downregulate FANCD2 in

Figure 2. FANCD2 is controlled by mTOR signaling in pediatricrhabdomyosarcoma in vivo. A andB, pediatric rhabdomyosarcomaRh10and Rh30 tumor xenograft models were propagated subcutaneously insevere combined immunodeficient mice and were treated with mTORkinase inhibitor AZD8055 at 20 mg/kg/d. Tumors were harvested 1, 4, 8,and24hours after treatment onday4andwerepulverized under liquidN2.

Total proteins were extracted for immunoblotting. C, mice bearingsubcutaneous rhabdomyosarcoma xenografts, Rh18, Rh10, and Rh30tumor xenografts, were treated with mTOR kinase inhibitor AZD8055 (20mg/kg daily). Tumors were harvested 24 hours after the fourth doseadministered. Total proteins were extracted for immunoblotting to detectFANCD2. D, pediatric rhabdomyosarcoma Rh18 and Rh30 tumorxenograft models were propagated subcutaneously in severe combinedimmunodeficient mice and were treated with rapamycin at 5 mg/kg/d.Tumors were harvested 24 hours after treatment on day 1. Total proteinswere extracted for immunoblotting. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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S6K1�/�MEFs. Thus, mTOR kinase seems to control FANCD2principally via the mTORC1–S6K1 pathway.

FANCD2 is required for the activation of both ATM-Chk2and ATR-Chk1 checkpointsIt was recently reported that FANCD2 mediates the nucleo-

lytic incisions near the ICL and leads to DNA DSB (26, 27),which may result in ATM-Chk2 activation and phosphoryla-tion of H2AX. This model suggests that FANCD2 may beessential for ATM-Chk2 activation in the early steps of Fanconianemia signaling-mediated repair of ICL-induced DNA lesions.To test this hypothesis, we first compared the status of ATR-Chk1 and ATM-Chk2 activation of the lymphoblast PD20 cellsderived from a patient with mutant FANCD2 and the isogenicPD20 cells reintroduced with wild-type FANCD2 by immuno-blotting. Similar to previous findings (28, 29), FANCD2-defi-cient PD20 cells showed high levels of gH2AX, an indication ofDNA strand breaks, whereas reintroduction of wild-type

FANCD2 into PD20 cells suppressed gH2AX signal (see Fig.1D). Reintroduction ofwild-type FANCD2 into PD20 cells led toan increase of pChk1-S345, and a slight increase in pChk2-T68but not pATM-S1981 signals, indicating the activation of ATR-Chk1 checkpoint (Fig. 4A). As the central function of ATR-Chk1is to promote DNA damage repair (30), these data suggest thatFANCD2 functions to enhance ATR-Chk1 checkpoint andsuppress spontaneous DNA damage under normal growthconditions.

FANCD2 is required for repair of ICL-induced DNA damageand intra-S phase checkpoint activation. To test whetherFANCD2 is required for intra-S-phase checkpoint activation,we examined ATR-Chk1 and ATM-Chk2 activation in responseto hydroxyurea in PD20 cells and the PD20 cells reintroducedwith wild-type FANCD2 by immunoblotting. Hydroxyurea-treated PD20 cells displayed decreased pChk1-S345, pATM-S1981, and pChk2-T68 signals compared with those of PD20with FANCD2 reintroduced (Fig. 4B). We additionally tested

Figure 3. The mTOR pathway regulates FANCD2 at transcription level. A, Rh30 cells were treated with AZD8055 (2 mmol/L) for the times indicated.The total RNA was extracted to detect FANCD2 mRNA by real-time RT-PCR with GAPDH as internal control. Relative quantity (Ln) of FANCD2 mRNA wasplotted. B, Rh30 cells were treated with rapamycin or MK2206 for 12 hours. Total RNA was extracted to detect FANCD2 mRNA by real-time RT-PCRwith GAPDH as internal control. Relative quantity (Ln) of FANCD2 mRNA was plotted. C, Rh30 cells were transfected with vector or AKT1-wt plasmid.Forty-eight hours later, the total RNA was extracted to detect FANCD2mRNA by real-time RT-PCR withGAPDH as internal control. Relative quantity (Ln) ofFANCD2 mRNA was plotted. D, Rh30 cells were treated with AZD8055 for 16 hours. Total proteins were extracted for immunoblotting. E, Rh30 cells weretreated with rapamycin, AZD8055, MK2206, or PD03332991 (1 mmol/L) for 16 hours. Total proteins were extracted for immunoblotting. F, Rh30 cellswere treated with PD03332991 for 12 hours. The total RNA was extracted to detect FANCD2 mRNA by real-time RT-PCR with GAPDH as internal control.Relative quantity (Ln) of FANCD2mRNA was plotted. G, wild-type (MEF WT) and S6K1 KOMEF cells were treated with rapamycin (100 ng/mL) for 24 hours.Total proteins were extracted to detect FANCD2 by immunoblotting with b-actin as loading controls. H, cells were treated as in G; total mRNA wasextracted to detect mouse FANCD2 mRNA by real-time RT-PCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Relativequantity (Ln) of FANCD2 mRNA was plotted. Error bars, mean � SD (n ¼ 3).






the status of ATR-Chk1 and ATM-Chk2 checkpoint in PD20cells in response tomelphalan by immunoblotting. In responsetomelphalan, there was apparent impairment of the activationof both ATR-Chk1 and ATM-Chk2 checkpoints in PD20 cells,shown by the significant low levels of pChk1-S345, pATM-S1981, and pChk2-T68 signals compared with those of PD20-expressing wild-type FANCD2 (Fig. 4C), these data show thatFANCD2 is required for the full activation of both DNAreplication and damage checkpoints under normal growthconditions and in response to genotoxins.

The mTOR pathway is essential for proper ATM-Chk2checkpoint activation in response to ICL

The model that FANCD2-mediated nucleolytic incisionnear the ICL results in DNA DSB (26, 27) also predicts thatactivation of ATR-Chk1 precedes the activation of ATM-Chk2.To explore thismodel, we treatedRh30 cells withmelphalan fora different timepoint and monitored the dynamics of gH2AXand pChk1-S345 signals. Chk1 was activated in 1 hour andreached its full activation 2 hours following melphalan treat-ment. In contrast, phosphorylation of H2AX displayed a sig-nificant delay (Fig. 5A), which correlated with PARP1 cleavage.Similar to the delayed phosphorylation of H2AX, ATM alsoshowed delayed phosphorylation (Fig. 5B). We next knockeddown FANCD2 in Rh30 cells by siRNA, transiently insultedwithdifferent concentrations of melphalan, and detected thegH2AX and pChk1-S345 signals by immunoblotting. Down-regulation of FANCD2 by siRNA did not effect the activation ofChk1, but apparently attenuated the phosphorylation of H2AXby melphalan (Fig. 5C). We further examined gH2AX byimmunocytochemistry of Rh30 treated with FANCD2 siRNAplus melphalan or AZD8055 plus melphalan. Both AZD8055and FANCD2 siRNA reduced the phosphorylation of H2AXinduced by melphalan (Fig. 5D). These results showed thatduring the early response to ICL, FANCD2 is required for the

proper phosphorylation of H2AX and hence activation of ATM,but not essential for ATR-Chk1 activation, providing evidencefor the proposedmodel of the function of FANCD2 in responseto ICL (26, 27).

Our observations, that FANCD2 is required for the timelyATM-Chk2 checkpoint activation in response to ICL andFANCD2 is controlled by mTOR signaling, suggest that themTOR pathway is essential for the timely activation of ATM-Chk2 checkpoint in response to ICL. To test this, we treatedPD20 cells with AZD8055 for 16 hours, followed by addition ofmelphalan for 5 hours. Although FANCD2-deficient PD20 cellsdisplayed impaired Chk2 activation in response to melphalan(Fig. 4C), PD20 cells rescued by wild-type FANCD2 showedrobust Chk2 activation, which was abolished by AZD8055accompanied with reduction of FANCD2 protein (Fig. 6A).Similarly, in Rh30 cells, melphalan-induced pATM-S1981 sig-nalswere reduced by either AZD8055 orMK2206, accompaniedwith downregulation of FANCD2 (Fig. 6B). In addition tomelphalan, AZD8055 also abolished the activation of Chk2 bymitomycin C (Fig. 6C). This reduction seems independent ofthe proteasome pathway because MG132 did not affectAZD8055-induced reduction of FANCD2 andmelphalan-medi-ated Chk2 activation (Fig. 6C). Thus, these data support thehypothesis that mTOR pathway is required for an early step(s)in ATM-Chk2 checkpoint activation in response to ICL.

DiscussionIn this study, we observed that an mTOR-selective kinase

inhibitor sensitized pediatric rhabdomyosarcoma xenograftsto radiotherapy and rhabdomyosarcoma cells to ICL. Controlof FANCD2 transcripts and protein levels by mTOR signaling(Supplementary Fig. S3A) may, at least partially, contribute tothe sensitization of cancer cells. Furthermore, FANCD2 isrequired for timely ATM-Chk2 checkpoint activation inresponse to ICL in Rh30 cells, suggesting that one of themechanisms that Fanconi anemia signaling promotes cellsurvival of ICL is through potentiation of ATM-Chk2 activation(Supplementary Fig. S3B).

Importantly, therapy-using agents that damage DNA andradiotherapy are essential components of many clinical pro-tocols, and form the backbone for curative treatment of severalchildhood malignancies. ICL-based chemotherapies, such ascisplatin or cyclophosphamide, are first-line drugs for treat-ment of many cancers. Our observation that cancer cells aresensitized to melphalan and ionizing radiation by an mTORkinase inhibitor provides a strategy for cancer therapy bycombination of targeting mTOR signaling with these DNA-damaging modalities.

Our findings may provide the explanation for recent reportsabout the involvement of mTOR signaling in DDR. Severalgroups found that inhibition of mTOR signaling resulted ingH2AX and impairment of DDR following exposure to DNA-damaging agents in cultured cells (31–33). The key function ofDDR is tomaximize cell survival by promotingDNA repair (30).Induction of gH2AX following inhibition of mTOR signalingsuggests impairment of DNA strand break repair. Moreover,inhibition of mTOR signaling in MCF-7 cells was shown toresult in impaired recruitment of BRCA1 and RAD51 to DNA

Figure 4. FANCD2 is required for ATR-Chk1 and ATM-Chk2 activation inresponse to DNA replications stress and damage in lymphoblast PD20cells. A, total protein extracts from lymphoblast PD20 cells from patientwith FANCD2 stably transfectedwith empty vector (pMMP-Puro) or wild-type FANCD2 plasmid (pMMP-wt-FANCD2) were used forimmunoblotting. UT, untreated. B and C, lymphoblast PD20 cells as in Awere treated with hydroxyurea (HU, 2 mmol/L) or melphalan (MP,2 mg/mL) for 6 hours; total protein extracts were for immunoblotting.�, pMMP-Puro; þ, pMMP-wt-FANCD2. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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repair foci (34). BRCA1 and RAD51 promote repair of DNADSBby homologous recombination (HR). Fanconi anemia signalingis essential for cells to survive DNA ICL by coordinating DNAdamage repair through translesion DNA synthesis (TLS),nucleotide excision repair (NER), and HR (35). Therefore,downregulation of FANCD2 by inhibition of mTOR signalingmay, at least partially, explain the impaired DDR in response toDNA-damaging agents.Recently, it was shown that ATM is synthetically lethal with

Fanconi anemia (28). Fifteen FANC genes have been identifiedin Fanconi anemia or Fanconi anemia-like patients (35, 36).FANCD2 is the key component of Fanconi anemia signaling.Activation of Fanconi anemia pathway by ICL leads to mono-ubiquitination of FANCI-FANCD2 by Fanconi anemia core E3ubiquitin ligase complex. Activated FANCI-FANCD2 isrecruited to DNA damage sites, and helps endonucleases tocut both sides of ICL to generate DNA strand breaks, andpromotes TLS, NER, and Rad51-mediated HR together with

BRCA1 and BRCA2 (FANCD1; ref. 37). FANCD2-mediatednucleolytic incisions near the ICL result in a DNA DSB (26,27), predicted to activate ATM-Chk2. In support of this model,our data show that FANCD2 is required for ATM-Chk2 acti-vation in the early steps of Fanconi anemia signaling-mediatedrepair of ICL-induced DNA lesions.

Our observation that FANCD2 is controlled by mTOR sig-naling and mTOR inhibition led to attenuation of ATM-Chk2checkpoint activation by ICL provides the molecular mecha-nism by which inhibition of mTOR signaling sensitizes cancercells to radiotherapy and chemotherapy. FANCD2 is requiredfor repair of DSB and intra-S-phase checkpoint activation(20, 35). Although the Fanconi anemia pathway is essentialfor cells to survive ICL, it can be activated by most DNAdamage including DSB, single-strand breaks, base damage,intra- and interstrand DNA crosslinks, and DNA replicationblock (37). We found attenuated activation of ATR-Chk1 andATM-Chk2 in PD20 cells under normal growth conditions,

Figure 5. ATR-Chk1 precedes the activation of ATM-Chk2 in response to ICL. A, Rh30 cells were treated with 2 mg/mL melphalan for the time asindicated. Total proteins were extracted for immunoblotting to detect Chk1, pChk1-S345, gH2AX, and PARP1. B, Rh30 cells were treated with 2 mg/mLmelphalan for the time as indicated. Total proteins were extracted for immunoblotting to detect pChk1-S345, ATM, and pATM-S1981. C, Rh30 cells weretransfected with control or FANCD2 siRNA. Forty-eight hours later, melphalan with the indicated concentrations was added for 8 hours. Total proteinswere extracted for immunoblotting of gH2AX, pChk1-S345, and FANCD2. D, Rh30 cells were transfected with FANCD2 siRNA for 48 hours or AZD8055(2 mmol/L) for 16 hours. A total of 2 mg/mL melphalan was added alone or in the cells with AZD8055 or FANCD2 siRNA for 8 hours. FANCD2 and gH2AX wasdetected by immunocytochemistry. DAPI-stained nuclei. Scare bar, 20 mmol/L.






hydroxyurea-induced DNA replication stress, and melphalan-mediated DNA damage. ATR-Chk1 and ATM-Chk2 check-points are the central genome surveillance systems tomaintaincell survival in response to endogenous and exogenous DNAdamage. DNA damage or replication stress leads to ATR-Chk1and ATM-Chk2 checkpoints activation to maintain cell sur-vival by stabilizing stalled DNA replication forks, preventingthefiring of late origins and promotingDNAdamage repair (38,

39). Thus FANCD2-dependent activation of ATM checkpointmay be one of the mechanisms by which Fanconi anemiasignaling promotes cell survival in response to ICL.

In addition, our data showed that FANCD2 is positivelyregulated by CDK4 while being apparently negatively con-trolled by CDK6 in Rh30 cells (Supplementary Fig. S2). Theseresults could suggest a distinct role of CDK4 and CDK6 inregulating FANCD2, although this may be context specific.Finally, AZD8055 also decreased the protein levels of FANCD2in PD20 cells, which was rescued by wild-type FANCD2 via aretroviral vector (Fig. 6A). These results indicate that mTORsignaling also regulates FANCD2 at posttranscriptionallevels.

In summary, we show that FANCD2 is under tight controlof the mTOR pathway and FANCD2 is required for the timelyactivation of the ATM checkpoint in response to ICL inpediatric rhabdomyosarcoma cells. Our data provide the basisfor the sensitization of cancer cells toDNA-damaging agents bytargeting the mTOR pathway. The observation that mTORsignaling protects cells from therapeutic modalities used intreatment of human malignancies, gives insight into potentialstrategies that may enhance therapeutic activity or reducesequellae that result from high-dose therapies, particularly inchildren.

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

Authors' ContributionsConception and design: C. Shen, P.J. HoughtonDevelopment of methodology: C. Shen, D.A. PhelpsAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Shen, D. Oswald, H. Cam, C.E. PelloskiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Shen, D. Oswald, D.A. Phelps, C.E. Pelloski,P.J. HoughtonWriting, review, and/or revision of the manuscript: C. Shen, D. Oswald,C.E. Pelloski, Q. Pang, P.J. HoughtonAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C. Shen, Q. PangStudy supervision: P.J. Houghton

AcknowledgmentsThe authors thank Niall G. Howlett for plasmid pcDNA-DEST40-FANCD2,

George Thomas for p70S6K1 knockout MEFs, William Sellers for plasmid-pcDNA3Myr-AKT1, and Alan D. D'Andrea for PD20 cells.

Grant SupportThis work was supported by NIH grants CA77776 (to P.J. Houghton) and a

Hope on Wheels grant from the Hyundai Car Company of America (to C.E.Pelloski).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 28, 2012; revised February 20, 2013; accepted March 17,2013; published OnlineFirst April 30, 2013.

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2013;73:3393-3401. Published OnlineFirst April 30, 2013.Cancer Res Changxian Shen, Duane Oswald, Doris Phelps, et al. Resistance of Cancer Cells to DNA Double-Strand BreaksRegulation of FANCD2 by the mTOR Pathway Contributes to the

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