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Published OnlineFirst August 31, 2010; DOI: 10.1158/0008-5472.CAN-10-1604

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RB-E2F1 Pathway Regulates Autophagy

Jiang1, Vanesa Martin1, Candelaria Gomez-Manzano1, David G. Johnson2, Marta Alonso1,
hite1, Jing Xu1, Timothy J. McDonnell3, Naoki Shinojima1, and Juan Fueyo1
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ophagy is a protective mechanism that renders cells viable in stressful conditions. Emerging evidencets that this cellular process is also a tumor suppressor pathway. Previous studies showed that cyclin-dent kinase inhibitors (CDKI) induce autophagy. Whether retinoblastoma protein (RB), a key tumorssor and downstream target of CDKIs, induces autophagy is not clear. Here, we show that RB triggersagy and that the RB activators p16INK4a and p27/kip1 induce autophagy in an RB-dependent man-B binding to E2 transcription factor (E2F) is required for autophagy induction and E2F1 antagonizesuced autophagy, leading to apoptosis. Downregulation of E2F1 in cells results in high levels ofagy. Our findings indicate that RB induces autophagy by repressing E2F1 activity. We speculate that
autoph
this newly discovered aspect of RB function is relevant to cancer development and therapy. Cancer Res; 70(20);7882–93. ©2010 AACR.

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ophagy is a cellular process that engulfs organelles andasmic contents to digest and recycle these materials ton cellular metabolism (1). In addition to providing a ba-tabolic function, autophagy is also used by the cell toith stressful conditions to improve survival (2). Emerg-idence suggests that autophagy is a tumor suppressoray (2, 3), although the detailed mechanisms governingathway are largely unknown. Indeed, several tumoressor proteins, including phosphatase and tensin ho-ue (PTEN) and p53 (2), induce autophagy. Conversely,le oncogenes, including Bcl-2 and the AKT/target ofycin (TOR) pathway, inhibit autophagy (2). Of particu-portance, the inactivation of several key autophagy reg-s, such as Beclin 1 and ATG4, results in spontaneouss (4) or increases the formation of tumors by carcino-n mice (5).noblastoma protein (RB) is considered the prototype ofsuppressor proteins, and as such, inactivation of RB isin an ample array of cancers (6). In tumors with wild-B gene, RB is frequently inactivated by the abnormal

tivators, such as the p16INK4a (p16) protein,, including cyclin D1 and cyclin-dependent

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Cell cHum

2), heMG) cin DM(FBS;100 μggene,FBS an

t ions: 1Brain Tumor Center, Departments ofnd 3Hematopathology, The University of Texas M.D.enter, Houston, Texas

ry data for this article are available at Cancer Researchrres.aacrjournals.org/).

uthor: Hong Jiang, Department of Neuro-Oncology,iversity of Texas M.D. Anderson Cancer Center, 1515rd, Houston, TX 77030. Phone: 713-834-6203; Fax:ail: [email protected].

5472.CAN-10-1604

ssociation for Cancer Research.

20) October 15, 2010

Research. on August 30, 20cancerres.aacrjournals.org ded from

-4 (6). Interestingly, loss of RB in cancer cells leadsreased sensitivity to therapy (7–9) and activation ofrough transfer of p16 increases chemoresistancehe mechanisms underlying this RB-mediated resis-are not completely understood. Recently, autophagyeen connected to resistance to cancer therapy (2).e basis of previous studies in which cyclin-dependentinhibitors (CDKI), activators of RB, were shown toautophagy (11, 12), we hypothesized that RB, a keysuppressor and downstream target of CDKIs, induceshagy.reover, the physical interaction between RB and E2ription factor 1 (E2F1) is perhaps the best-understoodnism of how RB functions. These two proteins play op-roles in the regulation of the cell cycle and apoptosis.omotes growth arrest, whereas E2F1 positively regu-he transition from the G1 to the S phase (13); RB pro-from apoptosis (7), whereas E2F1 induces apoptosishus, we asked whether RB and E2F1 also play antago-roles in the regulation of autophagy.his study, we examined the role of RB/E2F1 in the reg-n of autophagy. Our data indicate that RB induceshagy by repressing E2F1 activity, and E2F1 antagonizestivity to prevent autophagy.

rials and Methods

ulturean osteosarcoma (U-2 OS), sarcoma osteogenic (Saos-

patoma (Hep3B), and glioblastoma-astrocytoma (U-87ells (American Type Culture Collection) were culturedEM/F-12 supplemented with 10% fetal bovine serumHyclone Laboratories, Inc.), 100 μg/mL penicillin, and/mL streptomycin (Invitrogen). The 293 cell line (Qbio-
Inc.) was cultured in DMEM supplemented with 10%d antibiotics. Brain tumor (MDNSC23) stem cells were
18. © 2010 American Association for Cancer

http://cancerres.aacrjournals.org/

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ished from acute cell dissociation of surgical specimensman glioblastoma multiforme and maintained in/F-12 supplemented with B27 (Invitrogen), epidermalfactor, and basic fibroblast growth factor (20 ng/mL

Sigma-Aldrich) according to procedures describedere (15).

nts and antibodiesidine orange was obtained from Sigma-Aldrich. Rabbitonal p16 (N-20), rabbit polyclonal E2F1 (C-20), and goatonal actin (I-19) antibodies were obtained from Santaiotechnology. Rabbit polyclonal Bcl-2, light chain 3B), and Beclin 1 antibodies were obtained from Cell Sig-Technology. Mouse monoclonal RB was obtained from, and monoclonal anti–α-tubulin (B-5-1-2) was

ed from Sigma-Aldrich.

virusesconstruct recombinant adenoviruses expressing RBts, we made the Δ22 deletion (amino acids 738–775;) and R661W mutation (17) required for E2F binding.te-directed mutagenesis for the deletion and mutationonducted with the use of the QuikChange II XL site-ed mutagenesis kit (Stratagene) in pXCJL-RB, a shuttlethat includes the expression cassette for wild-type RBby human cytomegalovirus (CMV) promoter (18). Wemed the mutations in RB cDNA and the fidelity of theenesis reaction by sequencing the whole RB cDNA re-n the resultant pXCJL-RBΔ22 and pXCJL-RB R661Wids. We then cotransfected the pXCJL-RBΔ22 or-RB R661W plasmid with pBHG10 (Microbix Biosys-Inc.) into 293 cells to generate an AdCMV-RBΔ22 orV-RB R661W adenovirus. The recombinant adeno-s were prepared as previously described (19). Briefly,uses were amplified in 293 cells, purified by three suc-e bandings on cesium, and stored at −80°C. The viralas assayed by the tissue culture infection dose 50

50) and determined as plaque-forming units (pfu) perter, according to the validated method developed byum Biotechnology. Other recombinant adenovirusesin the study included AdCMV-RB (18), AdRSV-p16AdCMV, AdCMV-green fluorescent protein (GFP),V-E2F1 (20), AdMH4p27 (11), and AdCMV-β-gal (allred by the Keck Institutional Vector Core at M.D.son Cancer Center).

noprecipitation and immunoblottingcells were suspended and lysed in ice-cold PBS plus40 and a protease inhibitor cocktail (Sigma-Aldrich).ch sample, 2 μg of anti-E2F1 antibody and 40 μL ofn A-agarose beads (Upstate Biotechnology) wereto 500 μg of cell lysate proteins. After 2 hours of im-precipitation at 4°C, the beads were pelleted andd. The precipitated proteins or cell lysate proteinsdissolved in 1× SDS loading buffer, separated byAGE, and probed with antibodies. Finally, the protein
were visualized with the ECL Western blot detection(Amersham Pharmacia Biotech).
Kit (QTURB

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Research. on August 30, 20cancerres.aacrjournals.org Downloaded from

llular localization of light chain-3 fusion proteinss-2 and U-87 MG cells were transfected with enhancedfluorescent protein-light chain 3 (EGFP-LC3; a kind giftDr. S. Kondo, The University of Texas M.D. Andersonr Center) or mRFP-EGFP-LC3 (Addgene; ref. 21) fusionn–expressing plasmid by FuGENE 6 (Roche Molecularemicals). The cells were then screened with G418g/mL for Saos-2, 700 μg/mL for U-87 MG). Positivewere selected and maintained in culture medium plusThe cells expressing EGFP-LC3 were first fixed in 4%rmaldehyde in PBS for 30 minutes at 4°C. Saos-P-EGFP-LC3 cells were observed alive. Images wereand processed on a Zeiss Axiovert Zoom fluorescencescope (Carl Zeiss, Inc.) equipped with an AxioCamcamera and a 40×/1.3 oil EC Plan-NEOFLUAR objec-sing Immersol (n = 1.518) at room temperature. Thesition software was AxioVision Release 4.7.1 (Carl. Images were processed by Photoshop (Adobe) withe of proportional adjustments. We counted 150 tolls from three independent experiments for quantita-alysis.

on microscopyples were fixed with a solution containing 3% glutaral-e plus 2% paraformaldehyde in 0.1 mol/L cacodylate(pH 7.3) for 1 hour. After fixation, the samples wered and treated with 0.1% Millipore-filtered cacodylate-ed tannic acid (Sigma-Aldrich), postfixed for 1 hour% buffered osmium tetroxide (Sigma-Aldrich), and thend with 1% Millipore-filtered uranyl acetate (Sigma-h). The samples were dehydrated in increasing concen-s of ethanol, infiltrated, and then embedded in Spurr'sscosity medium. The samples were polymerized in anor 2 days at 70°C. Ultrathin sections were cut in a Leicaut microtome (Leica), stained with uranyl acetate anditrate in a Leica EM stainer, and examined by using a010 transmission electron microscope (JEOL USA,t an accelerating voltage of 80 kV. Digital images wereed with the use of an imaging system from Advancedscopy Techniques.

nterferencesiRNA for Beclin 1 depletion was obtained from SantaBiotechnology. We purchased custom and controls from Dharmacon, Inc. The E2F1 siRNA (siE2F1)nce was as follows: 5′-GGCCCGAUCGAUGUUUUC-′ (22). The cells were mock or siRNA transfected withFERin (Polyplus-transfection, Inc.) according to thefacturer's protocol. To knock down RB expression inMG cells, we transfected the cells with shGFP- orexpressing plasmids (23) by FuGENE 6. The cells wereed, and stable cell clones were maintained in culturem plus 0.5 μg/mL puromycin.

hagy PCR arrayal RNA was extracted from cells with the RNeasy Mini
iagen, Inc.). The RNA samples were then treated withO DNA-free kit to remove any DNA contamination
Cancer Res; 70(20) October 15, 2010 7883



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on) and sent to SABiosciences for analysis with thehagy PCR Array.

rase assays were seeded in 24-well plates (3 × 104 cells per well)fected with adenovirus vectors expressing GFP, RB, ortants (100 pfu/cell). Twenty-four hours later, wesfected the cells with 250 ng of the E2F1 reporter plas-4) and 1 ng of pRL-CMV (Promega Life Science) as al for transfection efficiency with FuGENE 6 (Rocheular Biochemicals). Twenty-four hours after transfec-

ads). Bar, 500 nm. D, immunoblot of LC3, Beclin 1, and RB. The cells werefected with AdCMV-RB (100 pfu/cell) for 72 h. Actin was used as loading

he cells were lysed, and luciferase activity was assayedluminometer.

considmean

r Res; 70(20) October 15, 2010


tification of apoptotic cells with AnnexinC stainingptotic cells were stained with Annexin V-FITC usingnexin V-FITCApoptosis Detection Kit I (BD Biosciences)ing to the manufacturer's instructions. Stained cellshen analyzed by flow cytometry with a FACScan cyt-r and CellQuest software (both from Becton Dickinson).

tical analysesperformed a two-tailed Student's t test to determineatistical significance of the experiments. P < 0.05 was

nsfected with the indicated siRNA (10 nmol/L). After 24 h, the cellsl. CsiRNA, control siRNA; siBeclin 1, siRNA against Beclin 1.

1. RB induces autophagy responses in RB-deficient cells. A, immunoblot analysis of LC3 and RB. Cells were infected with AdCMV-RB (100 pfu/cell).h, cell lysates were collected. Note the accumulation of LC3-II in RB-expressing cells but not in mock-treated or GFP-expressing cells. Actin

ed as a loading control. AdCMV-GFP was used as a control for viral infection and nonspecific protein expression. B, green fluorescence punctation incells constitutively expressing EGFP-LC3. The cells were infected with AdCMV-RB (100 pfu/cell). Seventy-two hours later, the cells were fixedn examined by fluorescence microscopy. Left, representative images of the cells with the indicated treatments. Right, quantification of the cells withC3 dots (columns, mean; bars, SD). *, P = 0.003; **, P > 0.05. AdCMV (Vector) was used as a control for viral infection. Bar, 20 μm. C, representativeission electron microscopy images of autophagosome/autolysosome accumulation in Saos-2 cells. The cells were infected with AdCMV-RBu/cell) for 72 h. AdCMV-GFP was used as a control for viral infection and nonspecific protein expression. Membrane-bordered vacuoles containsmic components (arrows) in the cytoplasm of RB-expressing cells (bottom left) but not in mock-treated or GFP-expressing cells (top). A close-up ofuoles revealed double membrane in the autophagosome with darker content or single membrane in autolysosome with lighter content (bottom,

ered statistically significant. Data were given as± SD.

Cancer Research



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duces autophagic responses in RB-deficient cellsest our hypothesis that RB induces autophagy, we firstuced RB into the following RB-defective human cancersarcoma osteogenic cells (Saos-2), hepatoma cells
B), and brain tumor stem cells (MDNSC23; ref. 15).ogenous RB protein induced autop
RB, b

for viral infection.

acrjournals.org


Fig. 1; Supplementary Fig. S1). Specifically, we exam-hether the transfer of RB induced the conversion oficrotubule-associated protein LC3-I to LC3-II, aemical marker of autophagy that is correlated withrmation of autophagosomes. Immunoblotting analysesed an accumulation of LC3-II in the cells treated with
ut not in the mock-treated or GFP-treated cells
hagy in all three cell (Fig. 1A). Consistently, RB also triggered the formation of

2. RB mediatesagosome maturation incells. Saos-2 cellstively expressing mRFP-C3 fusion protein werewith AdCMV-RB at/cell in the absence ore of bafilomycin A1 (BA1).ays later, live cells wered by fluorescenceopy. A, representativeof the cells with thed treatments. Bar, 20 μm.tification of the cells withrescent dots (columns,bars, SD). *, P = 0.008.(Vector) was used as a




autopEGFP-sion pcells, Ecent dplayedwere ca tranlationautopHep3BWe

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Figure(300 pfB, greeexaminEGFP-LshRB-e(300 pfD, immunoblot analysis of LC3. Cells were infected with AdMH4p27 at the indicated doses (pfu/cell). Seventy-two hours later, cell lysates were collected forimmuno specifi

Jiang et al.

Cance7886


hagosomes/autolysosomes visualized by punctateLC3 in Saos-2 cells that constitutively express the fu-rotein (Fig. 1B). In more than 80% of the RB-treatedGFP-LC3 was not soluble and presented green fluores-ots, whereas less than 20% of the control cells dis-the same pattern (Fig. 1B). Ultimately, these dataonfirmed by an ultrastructural study of the cells withsmission electron microscope, which revealed accumu-of membrane-rimmed vacuoles with characteristics ofhagosomes/autolysosomes in Saos-2 (Fig. 1C) and(Supplementary Fig. S1) cells after RB expression.then challenged the autophagy mechanism by down-lating Beclin 1, a key activator of the pathway. Asted, silencing Beclin 1 in Saos-2 cells with siRNAsed the RB-mediated processing of the formation of

blot analysis. AdCMV-β-gal (100 pfu/cell) was added as a control for non

hagosomes/autolysosomes and membrane-bound(Fig. 1D; Supplementary Fig. S2). In agreement with

mRFPdetect

r Res; 70(20) October 15, 2010


results was our observation that RB expression incells, in a PCR-based array, stimulated the expressioneral autophagy-related genes (Supplementary Fig. S3),as ATG4A and ATG7. However, Bcl-2, which inhibitstophagy process and whose gene is a well-studied tran-ional target of E2F1 (25, 26), was down-modulatedlementary Fig. S3). Overall, our data indicate that RBation in RB-deficient cells triggers autophagy.

omotes autophagosome formationaturationaccumulation of autophagosomes and LC3-II in theould be due to either the increased formation or thesed turnover of vesicles. To determine how RB affectstophagy flux, we took advantage of the double-tagged

c protein expression. Actin was used as a loading control.

3. CDKIs trigger autophagy in an RB-dependent manner. A, immunoblot analysis of LC3 and p16. The cells were infected with AdRSV-p16u/cell) for 72 h. AdCMV-GFP was used as a control for viral infection and nonspecific protein expression. Actin was used as a loading control.n fluorescence punctation in U-87-EGFP-LC3. The cells were infected with AdRSV-p16 (300 pfu/cell); 72 h later, the cells were fixed and thened by fluorescence microscopy. Top, representative images of the cells with the indicated treatments. Bottom, quantification of the cells withC3 dots (columns, mean; bars, SD). *, P = 0.004. Bar, 20 μm. C, top, immunoblot analysis of RB in U-87 MG cells transfected with shGFP orxpressing plasmid. α-Tubulin was used as a loading control. Bottom, immunoblot analysis of LC3 and p16. The cells were infected with AdRSV-p16u/cell) for 72 h. AdCMV-β-gal was used as a control for viral infection and nonspecific protein expression. Actin was used as a loading control.

-EGFP-LC3 construct (ptfLC3; ref. 21), which ised as yellow fluorescence (green merged with red) in

Cancer Research



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idic structures (autophagosomes and amphisomes)s red only in autolysosomes due to the quenching ofin these acidic structures. As shown in Fig. 2, more0% of the RB-transduced cells displayed tremendousorescence punctation, whereas only less than 10% ofock- or AdCMV-treated cells showed this phenotype.er, when the cells were treated with bafilomycin A1h inhibits the fusion of autophagosomes with lyso-) to stop the turnover of autophagosomes, differenthe cells transduced with RB, we found an extraordi-mount of punctate yellow fluorescence in the cyto-(Fig. 2A). These data indicate that RB increases the
ort of mRFP-EGFP-LC3 to acidic lysosomes (i.e., for- Bec
lls were infected with the indicated vectors for 72 h. The numbers on the topn (pfu/cell). Actin was used as a loading control.

acrjournals.org


nation of the cells revealed that bafilomycin A1 causedcumulation of vacuoles in RB-expressing cells thatmost of the space in the cytoplasm, whereas theles in mock or GFP-expressing cells occupied less thanf the space in the cytoplasm (Supplementary Fig. S4),ting that RB stimulated a higher rate of autophago-formation. Taken together with the previous observa-we conclude that RB mediates the initiation andation of autophagosomes instead of inhibiting theof autophagosomes and lysosomes.

s cause autophagy in an RB-dependent manner
ause RB activity is positively regulated by CDKIs, such as
n of autolysosomes). Moreover, electron microscopic p16 and p27/kip1 (p27), we asked whether activation of the RB

4. RB mutants deficient for E2F binding are unable to induce autophagy in Saos-2 Cells. A, immunoprecipitation analysis of RB and its mutantsing with E2F1. The cells were infected with AdCMV-RB (100 pfu/cell) and AdCMV-E2F1 (5 pfu/cell), and cell lysates were collected forprecipitation analysis after 48 h. B, effect of RB and RB mutants on E2F1 activity. The cells were infected with the indicated vectors (100 pfu/cell).h, the cells were transfected with a luciferase-reporting plasmid with an E2F1 promoter. Twenty-four hours later, the luciferase activity ofples was determined (columns, mean; bars, SD). *, P = 0.0007; #, P = 0.0004; $, P = 0.003; **, P = 0.4. C, green fluorescence punctation inC3–expressing Saos-2 cells. The cells were infected with adenovirus vectors expressing RB and its mutants (100 pfu/cell) for 72 h. Left,ntative images of the cells with the indicated treatments. Right, quantification of the cells with EGFP-LC3 dots (columns, mean; bars, SD)..0002; **, P > 0.05. Bar, 20 μm. D, immunoblot analysis of RB and LC3 in Saos-2 cells infected with adenoviruses expressing RB or its mutants.

of the protein bands represent the doses of the viruses for




pathwthis e(U-87expreslipidatlines (LC3 cecencecontrowhenexpres(Fig. 3the coU-87 MIn a

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Jiang et al.

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ay by CDKIs would result in autophagy in these cells. Tond, we transferred p16 to glioblastoma-astrocytomaMG) and human osteosarcoma (U-2 OS) cell lines thats wild-type RB. The ectopic p16 protein triggered theion of LC3, resulting in an increase of LC3-II in both cellFig. 3A). Accordingly, more than 60% of the U-87-EGFP-lls showed a marked punctate pattern of green fluores-after transduction of p16, whereas less than 20% of thel cells showed a similar pattern (Fig. 3B). Moreover,we transferred p16 to U-87-shRB cells, in which RBsion had been knocked down by shRNA against RBC), we observed that down-modulation of RB preventednversion of LC3-I to LC3-II, indicating that it renderedG cells resistant to p16-mediated autophagy (Fig. 3C).ddition to p16, the best-documented CDKI that is in-in the positive regulation of autophagy is p27 (11,

ion. Columns, mean from at least three independent experiments; bars, S

nlike p16, p27 has other targets in addition to RB). When we transferred p27 to U-2 OS and Saos-2

to indally fo

r Res; 70(20) October 15, 2010


U-2 OS cells were much more sensitive to p27 in aependent manner, resulting in a conversion of LC3-I3-II that was not observed in Saos-2 cells (Fig. 3D),sting that RB might be one of the factors involved-mediated autophagy.lectively, these data indicate that RB expression ised for p16-mediated autophagy and that RB could bef the downstream targets involved in p27-inducedhagy. These data therefore reinforce the role of thethway in autophagy regulation.

ng to E2F is required for RB to trigger autophagybest-known mechanistic model of RB indicates theal binding and repression of E2F transcription factors,ing E2F1 (30). Thus, we speculated whether RB mutantsre deficient for E2F binding would lose the capability

= 0.007; #, P = 0.001.

5. RB/E2F1 interaction regulates autophagy and apoptosis. A, green fluorescence punctation in EGFP-LC3–expressing Saos-2 cells. The cells werected with AdCMV-RB (100 pfu/cell). After 24 h, the cells were infected with AdCMV-E2F1 (50 pfu/cell) for another 48 h. Top, representativeof the cells with the indicated treatments. Bar, 20 μm. Bottom, quantification of the cells with EGFP-LC3 punctation (columns, mean; bars, SD)..03. B, immunoblot analysis of LC3, E2F1, and RB in Saos-2 cells. The cells were first infected with AdCMV-RB (100 pfu/cell). After 24 h, theere infected with AdCMV-E2F1 (50 pfu/cell). Proteins from the cell lysates were collected for analysis after 48 h. C, cells were treated as describedd were stained with Annexin V and analyzed by flow cytometry. AdCMV-β-gal was used as a control for viral infection and nonspecific protein

uce autophagy. We selected two RB mutants natur-und in cancer cells that are deficient in binding E2F.

Cancer Research



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unoprecipitation assays showed that both RB mu-Δ22 (16) and R661W (17) displayed impaired abilityding E2F1 (Fig. 4A). Accordingly, the two RB mutantseficient in blocking the transactivation of the E2F1nsible element, as quantified by luciferase assaysB). Then, we examined autophagy occurrence incells transduced with the two mutants. We found

he expression of Δ22 or R661W resulted in EGFP-unctation in less than 20% of the cells, whereasype RB induced green fluorescence dots in more thanf the cells (Fig. 4C). Consistently, neither Δ22 norinduced significant LC3-I to LC3-II conversion,

ing to immunoblot analysis (Fig. 4D). Therefore, RBts with impaired binding to E2F are deficient forhagy induction, indicating that RB binding to E2F ised for RB-induced autophagy.

antagonizes RB-mediated autophagyand E2F1 exert contrary functions in the regulation ofll cycle and apoptosis (7, 13). We previously reportedpression of ectopic E2F1 overrides the cell cycle arrestd by RB (20). In this study, we were interested in as-ning whether the expression of E2F1 would be suffi-to antagonize RB-mediated autophagy. Thus, weerred RB and then E2F1 into Saos-2 cells 24 hours later.nation of the cells 48 hours after the transfer of E2F1ed that E2F1 was able to partially override RB functionrevent the formation of the green-dot phenotype and
nversion of LC3-I to LC3-II (Fig. 5A and B). These stud-wed a progressive decrease in the percentage of cells
BecE2F1

control.

acrjournals.org


unctate green fluorescence: from almost 100% in cellsd with RB, to 40% in cells treated with both RB andto less than 20% in cells treated with E2F1 aloneA). Consistently, Bcl-2 protein, an autophagy inhibitor,ownregulated by RB but was upregulated by E2F1B).ause expression of E2F1 induces apoptosis (20), wewhether the cells that were transduced by E2F1 weregoing cell death through apoptosis. Optical microscopyd that although RB-transduced cells displayed a flatology and remained attached to the culture dish, somecells transduced with both RB and E2F1 became round-efringent, and detached from the dish (data not shown),ly suggesting decreased viability of the culture. To de-e whether the cause of the cell death was apoptosis, welow cytometry to examine the ability of the cells to bindnexin V, a reagent to detect the translocation of therane phospholipid phosphatidylserine from the innerouter leaflet of the plasma membrane—one of thet indications of apoptosis. Although the percentage ofhagic cells was less than 10% in E2F1-treated cellsA), the treatment caused about 40% of the cells togo apoptosis (Fig. 5C). Thus, we conclude that E2F1nizes RB-mediated autophagy and leads to apoptosis.results suggest that RB and E2F1 proteins play oppositen the control of both autophagy and apoptosis.

regulation of E2F1 results in autophagy
ause RB needs to bind to E2F to induce autophagy, andantagonizes RB-mediated autophagy, we hypothesized
6. Downregulation of E2F1in autophagy. A, greenence punctation in U-87ls constitutively expressingC3. The cells wereted with siRNA (20 nmol/L). Representative imagesells with the indicatednts. B, quantification ofs with dotted EGFP-LC3ns, mean; bars, SD)..002; **, P = 0.3 (Student'sBar, 20 μm. C, immunoblotof LC3 and E2F1 in U-87

ls treated as describedTubulin was used as a




that ceto spothesecontrdown-the E2as docEGFP-and Ba dottysis reand caresultstriggersing audown-E2F1 a

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lls lacking E2F1 expression would be more susceptiblentaneous autophagy and that the levels of autophagy incells would be significantly higher than the levels inol cells. To test this hypothesis, we used siRNA tomodulate E2F1 expression in U-87 MG cells. SilencingF1 gene resulted in the formation of autophagic vacuoles,umented by a dramatic lipidation of LC3 that causedLC3 punctation in more than 60% of the cells (Fig. 6A). In contrast, less than 10% of the control cells showeded pattern (Fig. 6B). Consistently, immunoblotting anal-vealed that E2F1 siRNAdownregulated E2F1 protein levelused the conversion of LC3-I to LC3-II (Fig. 6C). Thesesuggest that down-modulation of E2F1 is sufficient toautophagy and that E2F1 plays an active role in suppres-tophagy. Thus, the increased level of autophagy in E2F1–
modulated cells indirectly points to the inhibition ofs the mechanism for RB-mediated autophagy.
quiredactivit

r Res; 70(20) October 15, 2010


ssion

RB/E2F1 pathway, which is crucial in regulating cellh and apoptosis, is disrupted in virtually all humanrs (31). Here, we report that RB positively regulateshagy by repressing E2F1 activity. Our results indicatenstead of preventing autophagosome turnover in theRB activity, or lack of E2F1 activity, promotes autop-lux, from the formation of autophagosomes to their fu-ith lysosomes. Our data reveal a previously unknownon of the RB/E2F1 pathway that might contribute to itscancer suppression and resistance to cancer therapy.

alanced RB/E2F1 pathway is required to maintain cel-omeostasis. We found that excessive RB activity orf E2F1 causes autophagy, and RB/E2F interaction is re-
for RB-mediated autophagy. Moreover, excessive E2F1
y antagonizes RB-induced autophagy, leading to apoptosis.

ureandptouresy reuceause apoptosis.

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7. Rheostat model for the interplay ofE2F1 to regulate autophagy and

sis. Balanced RB and E2F1 activityhomeostasis in the cell. Strong stimuli

sult in either excessive RB activity toautophagy or immoderate E2F1 function

Cancer Research

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ThereregulamodelulatesE2F1 atomaicell. Gsis (2)whichvolvemmutatsuggepathwpressoing morganit hastophatheir ntransfhad deformedicatethe crand ininhibiautopsupprAsid

act asstressfstituteavoiding ceapoptoptosiscentrain thecross-top of(Fig. 7well. Mvascuautophbility isporadthan bylationtion paggresing RBsurvivstancephagyexpresof RB,showin a R

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fore, in addition to the opposite roles of RB and E2F1 inting the cell cycle and apoptosis (30), our data suggest ain which the interplay between RB and E2F1 also reg-autophagy (Fig. 7). We speculate that adequate RB/ctivity ensures a basal level of autophagy that is criticalntaining normal cellular andmolecular processes in theiven the negative effects of autophagy on tumorigene-, autophagy could be part of the mechanism throughRB acts as a tumor suppressor. In this regard, the in-ent of PTEN, p53, and RB, the three most frequentlyed genes in human cancers, in regulating autophagysts a strong mechanistic link between stress-sensingays, autophagy, and tumor suppression. Tumor sup-rs, such as RB, may induce autophagy as a housekeep-echanism to recycle damaged macromolecules andelles to ensure the stability of the genome (32). In fact,been reported that the basal levels of proteolysis or au-gic degradation in cancer cells were lower than those inormal counterparts (33). In this regard, cells that wereormed by SV40, which can inactivate RB and p53,creased levels of proteolysis compared with nontrans-d or nontreated cells (34). Lately, autophagy was in-d as an effector mechanism of senescence (35). Givenitical role that RB plays during cellular senescence (36)the response to cancer therapeutics such as CDK

tors (37), there might be a link between RB-mediatedhagy and senescence and its consequential tumoression.e from its housekeeping function, autophagy can alsoa protective mechanism to prolong cell survival underul conditions (38). In certain scenarios, autophagy con-s a stress adaptation that suppresses apoptosis tocell death (39). In addition to its central role in inhibit-ll proliferation, RB also plays a crucial role in inhibitingsis (7). Studies in RB-mutant mice show massive apo-in neurons (40). Interestingly, Atg7 deficiency in thel nervous system also causes massive neuronal losscerebral and cerebellar cortices (41), suggesting a

talk between autophagy and apoptosis (39). Thus, onautophagy/apoptosis regulation by RB/E2F1 interplay), RB-mediated autophagy might inhibit apoptosis asoreover, during its growth, the tumor is usually poorly

larized in the center area, and in this environment,agy has a more prominent role in sustaining cell via-n cancer cells (42). Previous work has shown that mostic cancers inactivate RB by phosphorylation, rathery mutations in the RB gene itself (7). Because phosphor-is reversible, altering this posttranslational modifica-athway of RB not only allows cancer cells to growsively but also gives these cells the option of reactivat-for protection against apoptotic stimuli to exploit the

al advantage conferred by RB under stress (7). For in-, glucose-regulated stress, a potent inducer of auto-(12), causes stabilization of p27 (12) and decreasedsion of cyclin D1 (43), resulting in hypophosphorylationwhich is the active form of the protein (6). Our data
that CDKIs, such as p27 and p16, induce autophagyB-dependent manner (Fig. 3), indicating the involve-
Howenormo

acrjournals.org


of RB-mediated autophagy in the stress response.it is of clinical relevance to determine whether RBty in cancer cells expressing wild-type RB is partiallyed under these conditions to induce autophagy tont apoptotic cell death. Furthermore, results from ex-ental anticancer therapies suggest that autophagy activa-presents a cellular attempt to survive the stress inducedotoxic agents (2). In fact, it has been reported thatation of the RB protein in RB-deficient cells results in re-ce to DNA-damaging agents including cisplatin, etopo--fluorouracil, and doxorubicin (44, 45). In addition,sion of p16 is associated with chemoresistance in gliomaladder cancer cells (10, 46). Overall, our study suggestsB-induced autophagy provides a possible new mechan-nk between RB, cancer cell survival, and resistance tor therapy.ough the role of RB in modulating autophagy seems toll defined here, the role of E2F1 could be more complex.t, E2F1 has a dual role as a tumor suppressor and anene and, as such, can positively regulate both cell pro-ion and cell death (47). Thus, E2F1 may exert a contex-le in autophagy as well. A weak E2F1 transactivation ofhagy-related genes was observed in a drug-inducedexpressing system (48). However, our data show thattivated E2F1 blocked RB-induced autophagy and ledptosis (Fig. 5). One of the possible molecular targets1 in the suppression of autophagy is Bcl-2, whichautophagy by binding Beclin 1 and prevents activationPI3Kc3 complex (25). Our previously published resultsed that Bcl-2 is a transcriptional target of E2F1 (26).we show that repression of E2F1 by RB leads tomodulation of Bcl-2 (Supplementary Fig. S3; Fig. 5),buting to the triggering of autophagy. The mammalianmTOR) pathway is a key negative regulator of autop-(49). We examined the phosphorylation status ofK, a substrate of mTOR (50). We did not observe anyes in the total p70S6K level and its phosphorylation lev-r introducing RB into Saos-2 and MDNSC23 cells orto U-87 MG and U-2 OS cells (data not shown). Wee that mTOR does not play a role under this circum-. Thus, RB-induced autophagy is more relevant tomediated Bcl-2 expression than affecting the mTORay. However, it is now clear that E2F1 transactivatesthan 2,000 genes and that the role of E2F1 in regulatingription is highly dependent on context (47). Therefore,verly simplistic to identify one or two players to com-explain the delicate balance between RB and E2F1 in

gulation of autophagy. Besides, further study will be re-to determine whether RB/E2F1 modulation of autop-s independent of its role in cell cycle regulation.the other hand, RB interacts with more than 200ns (6), one of which is the hypoxia-inducible factor,is a potent inducer of autophagy under hypoxicions (51). In these extreme circumstances, RB pro-ems to negatively modulate hypoxia-inducible factor–ed autophagy by attenuating BNIP3 induction (52).
ver, in our system, transfer of RB to cancer cells underxia resulted in the upregulation of BNIP3 (Supplementary



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3), suggesting a marked difference between thetion of autophagy under normoxic and hypoxicions.ummary, our data unequivocally show that the RB/pathway plays a role in the regulation of autophagy.ver, the existence of such a rheostat-like module tote the delicate balance between autophagy and apo-(Fig. 7) provides a new interpretation for the role oftumor suppression during development, cell survival
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phosphorylation, viral oncoprotein association, and nuclear teth-ng. Proc Natl Acad Sci U S A 1991;88:3033–7.hmann DR, Brandt B, Hopping W, Passarge E, Horsthemke B.

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r Res; 70(20) October 15, 2010


owledgments

hank Dr. Jinsong Liu (Department of Pathology, M.D. Anderson) for thelasmid, Dr. Seiji Kondo for EGFP-LC3 plasmid (Brain Tumor Center,nderson), Tamara K. Locke and Joseph Munch (Department ofic Publications, M.D. Anderson) for editorial assistance, and Kenneth, Jr. for electron microscopy analysis.

Support

onal Cancer Institute (NIH/P50CA127001 to J. Fueyo), The Marcustion (to J. Fueyo), The University of Texas M.D. Anderson Cancer Cen-itutional Research Grant to J. Fueyo), and the High Resolution Electronopy Facility (Core Grant CA-16672 to M.D. Anderson Cancer Center).costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 05/04/2010; revised 07/06/2010; accepted 07/20/2010; publishedirst 08/31/2010.

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2010;70:7882-7893. Published OnlineFirst August 31, 2010.Cancer Res Hong Jiang, Vanesa Martin, Candelaria Gomez-Manzano, et al. The RB-E2F1 Pathway Regulates Autophagy

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