clinical human cancer biology cancer...

Human Cancer Biology

miR-34a Induces the Downregulation of Both E2F1 and B-MybOncogenes in Leukemic Cells

Giorgio Zauli1, Rebecca Voltan2, Maria Grazia di Iasio2, Raffaella Bosco2, Elisabetta Melloni2,Maria Elena Sana2, and Paola Secchiero2

AbstractPurpose: To elucidate new molecular mechanisms able to downregulate the mRNA levels of key

oncogenes, such as B-Myb and E2F1, in a therapeutic perspective.

Experimental Design: B-Myb and E2F1mRNA levels were evaluated in primary B chronic lymphocytic

leukemia (B-CLL, n¼ 10) and acute myeloid leukemia (AML, n¼ 5) patient cells, in a variety of p53wild-type

and p53mutated/deleted leukemic cell lines, as well as in primary endothelial cells and fibroblasts. Knockdown

experiments with siRNA for p53 and E2F1 and overexpression experiments withmiR34a were conducted to

elucidate the role of these pathways in promoting B-Myb downregulation.

Results: In vitro exposure to Nutlin-3, a nongenotoxic activator of p53, variably downregulated the

expression of B-Myb in primary leukemic cells and in p53wild-type myeloid (OCI, MOLM) and lympho-

blastoid (SKW6.4, EHEB) but not in p53mutated (NB4, BJAB, MAVER) or p53deleted (HL-60) leukemic cell

lines. The transcriptional repression of B-Myb was also observed in primary normal endothelial cells and

fibroblasts. B-Myb downregulation played a critical role in the cell-cycle block in G1 phase induced by

Nutlin-3, as shown by transfection experiments with specific siRNA. Moreover, we have provided

experimental evidence suggesting that miR-34a is a central mediator in the repression of B-Myb both

directly and through E2F1.

Conclusions: Owing to the role of B-Myb and E2F1 transcription factors in controlling cell-cycle

progression of leukemic cells, the downregulation of these oncogenes bymiR-34a suggests the usefulness of

therapeutic approaches aimed to modulate the levels of miR-34a. Clin Cancer Res; 17(9); 2712–24. �2011

AACR.

Introduction

Nutlin-3a represents a potent and selective small mole-cule inhibitor of the p53–MDM2 interaction (1), whichraises the levels of p53 protein and induces subsequentcytostatic or cytotoxic effects in a variety of tumor cells (1).Taking into consideration that p53 is mutated in less than15% of hematological malignancies at diagnosis (2), it isnoteworthy that Nutlin-3 has shown in vitro promisingtherapeutic potential in both myeloid and lymphoid leu-

kemia characterized by a p53wild-type (p53wt) status (3–9).However, because p53 mediates different cellular func-tions, such as in particular cell-cycle arrest and apoptosis(3) the critical molecular determinants mediating/modu-lating the therapeutic activity of Nutlin-3 in leukemic cellsare not completely understood.

Previous studies have shown that several solid tumorsare characterized by overexpression of B-Myb, also knownas MYBL2 (10–13), a member of the vertebrate MYBfamily of nuclear transcription factors, which recognizeand bind to the DNA consensus sequence [PyAAC(G/T)G] to promote gene transcription (14). B-Myb, unlike C-Myb and A-Myb, is expressed in virtually all proliferatingcells as a regulator of cell-cycle progression and cellsurvival (15) and it plays an essential role in vertebratedevelopment (16). Indeed, the knock-down of murineB-Myb causes early embryonic lethality (E4.5-6.5) result-ing from unsuccessful inner cell mass formation (16).Interestingly, it has also been shown that B-Myb chromo-somal locus (20q13) is amplified and/or highly expressedin a variety of tumor types including neuroblastomasand breast, prostate, liver and ovarian carcinomas, andin most cases this high expression is associated with apoor prognosis (10–13).

Authors' Affiliations: 1Institute for Maternal and Child Health, IRCCS"Burlo Garofolo," Trieste and 2Department of Morphology and Embryologyand LTTA Centre, University of Ferrara, Ferrara, Italy

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

G. Zauli and R. Voltan contributed equally to the work.

Corresponding Author: Rebecca Voltan, Department of Morphology andEmbryology and LTTACentre, University of Ferrara, Via Fossato di Mortara66, 44121 Ferrara, Italy. Phone: 39-0532-455846; Fax: 39-0532-455950.E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-3244

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(9) May 1, 20112712

Cancer Research. on February 20, 2019. © 2011 American Association forclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 2, 2011; DOI: 10.1158/1078-0432.CCR-10-3244

http://clincancerres.aacrjournals.org/

On these bases, the aim of the present study was toinvestigate the baseline expression levels and the potentialregulation and/or involvement of B-Myb in the therapeuticresponse to Nutlin-3 in myeloid and lymphoid leukemias,as well as in primary stromal cells.

Materials and Methods

Cell culturesPeripheral blood samples were collected in heparin-

coated tubes from 5 patients affected by acute myeloidleukemia (AML: 2 M4, 1 M5, and 2 M4/M5), 10 patientsaffected by B-chronic lymphocytic leukemia (B-CLL), and10 healthy blood donors following informed consent, inaccordance with the Declaration of Helsinki and inagreement with institutional guidelines. Peripheral bloodmononuclear cells (PBMC) from AML and B-CLLpatients and from healthy donors were isolated by gra-dient centrifugation with lymphocyte cell separationmedium (Cedarlane Laboratories). Specific CD19þ andCD34þ cell populations were selected from normalPBMC by using immunomagnetic microbeads and theAutoMACS system (Miltenyi Biotech) in accordancewith the manufacturer’s instructions, obtaining a purityexceeding 90% for each subpopulation. For analysisof CD19þ cells, RNA preparations were obtained from5 independent donors. For analysis of CD34þ cells, eachRNA preparation (n ¼ 4 in total) was obtained bypooling CD34þ cells purified from at least 3 differentdonors.The p53wt (OCI, MOLM, SKW6.4, EHEB), p53mutated

(NB4, BJAB, MAVER), and p53deleted (HL-60) leukemiccell lines were purchased from the ATCC (American TypeCulture Collection) or obtained from DSMZ (DeutscheSammlung von Mikroorganismen und ZellkulturenGmbH). Control PBMC and patient cells were culturedin RPMI-1640 (Gibco BRL) containing 10% FBS, L-gluta-mine, and penicillin/streptomycin (Gibco BRL). The cell

lines were cultured in RPMI-1640, or in Alpha MEM(OCI) (LONZA) supplemented with 10% FBS andL-glutamine and penicillin/streptomycin (Gibco BRL).Cells were seeded at a density of 1 � 106 cells/mL beforetreatment with Nutlin-3 (used in the range of 0.1–10mmol/L; Cayman Chemical) or Chlorambucil (used in therange of 0.10–10 mmol/L; Sigma-Aldrich). The HCT116human colon cancer cells, with either p53wt or p53knockout (p53�/�), were cultured in Dulbecco’s modifiedEagle’s medium (DMEM) media supplement with 10%FBS (17).

Human umbilical vein endothelial cells (HUVEC) werepurchased from BioWhittaker and grown as previouslydescribed (18). For the evaluation of cytostatic and/or toxiceffects, HUVEC and fibroblasts were seeded andgrown to subconfluence before treatment with Nutlin-3(10 mmol/L). Neonatal human dermal fibroblasts werepurchased from LONZA and grown in DMEM (LONZA)containing 20% FBS, L-glutamine and penicillin/strepto-mycin (Gibco BRL).

Assessment of cell viability, apoptosis, and cell cycleCell viability was examined by Trypan blue dye exclu-

sion, and induction of apoptosis was quantified byAnnexin V-FITC/propidium iodide (PI) staining (Immu-notech) followed by flow cytometry analysis (19). Toavoid nonspecific fluorescence from dead cells, live cellswere gated tightly by using forward and side scatter, asdescribed (20). To analyze the degree of apoptosis inthe entire cell population of HUVEC and fibroblastcultures, substrate-attached cells were harvested by tryp-sin treatment and pooled with floating cells for thestaining.

Cell-cycle profile was analyzed by flow cytometric ana-lysis, as previously described (21).

Real time PCRTotal RNA was extracted from cells by using the Qia-

gen RNeasy Plus mini kit (Qiagen) according to thesupplier’s instructions. Total RNA was transcribed intocDNA, using the QuantiTect Reverse Transcription kit(Qiagen). Levels of miR-34a expression were evaluatedby using the miScript PCR System (Qiagen), composedof the miScript Reverse Transcription kit and themiScript SYBR Green PCR kit for cDNA amplifica-tion with specific miScript Primer Assays for miR34a(Qiagen). The expression of miR-34a values was normal-ized by using specific primers to RNU6B (Qiagen) asendogenous reference RNA.

Western blot analysesCells were lysed in ice-cold radioimmunoprecipitation

assay buffer [50 mmol/L Tris (pH 7.5), 150 mmol/LNaCl, 0.1% SDS, 1% Nonidet P-40, 0.25% sodium des-oxycholate) supplemented with protease inhibitors(Complete, Roche) on ice for 1 hour. Protein determina-tion was carried out by BCA Protein Assay (Thermo Scien-tific) and then added with loading buffer [250 mmol/L

Translational Relevance

B-Myb oncogene was found overexpressed in bothprimary acute myeloid leukemia blasts. The small mole-cule inhibitor of MDM2–p53 interactions, Nutlin-3,efficiently repressed both B-Myb and E2F1 in a varietyof p53wild-type leukemic cell types as well as in primaryendothelial cells and fibroblasts. Gene knockdown andoverexpression experiments showed that Nutlin-3 upre-gulated miR34a, which in turn induced the down-regulation of B-Myb both directly and through thedownregulation of E2F1. These data strengthen thenotion that promoting the upregulation of miR34amight have important clinical applications for the treat-ment of human leukemias.

miR34a Downregulates B-Myb Expression

www.aacrjournals.org Clin Cancer Res; 17(9) May 1, 2011 2713




Tris (pH 6.8), 2% SDS, 40% Glycerin, 20% b-mercap-toethanol] and boiled for 2 minutes (22). Equal amountsof protein for each sample were migrated in acrylamidegels and blotted onto nitrocellulose filters, as previouslydescribed. The following monoclonal antibodies wereused in our experiments: anti-p53 (DO-1; Santa CruzBiotechnology), anti-E2F1 (KH95; Santa Cruz Biotech-nology), anti-pRb (G3-245; BD Biosciences Pharmingen),

and anti-tubulin (Sigma-Aldrich). After incubation withperoxidase-conjugated anti-mouse immunoglobulin G,specific reactions were revealed with the ECL Lightningdetection kit (Perkin Elmer). Densitometric values wereestimated by the ImageQuant TL software (GE Health-care). Multiple film exposures were used to verify thelinearity of the samples analyzed and to avoid saturationof the film.

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Figure 1. Transcriptionalrepression of B-Myb and E2F1 byNutlin-3 in p53wt leukemic celllines. Primary B-CLL and AMLleukemic cells and in the indicatedp53wt or p53mutated/deleted

(p53mut/del) lymphoid and myeloidleukemic cell lines were either leftuntreated or exposed to Nutlin-3(10 mmol/L). After 24 hours oftreatment, levels of B-Myb mRNA(A and B) and E2F1 (C) wereassessed by quantitative RT-PCR,and results were expressed as foldof B-Myb and E2F1 modulation inNutlin-3–treated cultures withrespect to the control untreatedcultures, which were set to 1(hatched line). D, B-Myb and E2F1RNA modulation by Nutlin-3 wasanalyzed in OCI cells transientlytransfected with p53 siRNA or withcontrol scramble (scr.) siRNA andin the isogenic-paired cell linesHCT116 p53wt and HCT116p53�/�. A, Horizontal bars aremedian, top and bottom edges ofbox are 75th and 25th percentiles,lines extending from box are 10thand 90th percentiles. B–D, dataare reported as mean � SD ofresults from at least 3experiments, each conducted intriplicate. *, P < 0.05 with respectto the untreated cultures or, forOCI siRNA-transfectionexperiments (D), with respect tocells transfected with thescramble siRNA.

Zauli et al.

Clin Cancer Res; 17(9) May 1, 2011 Clinical Cancer Research2714




Transfection experimentsCells (1� 106) were resuspended into 0.1 mL of Nucleo-

fector solution of human nucleofector kit NHDF or V(Amaxa). Two micrograms of plasmid DNA (green fluor-escent protein construct) or 1 mg of siRNA (see Supple-mentary Material and Methods) were mixed with the0.1 mL of cell suspension, transferred into a 2.0 mmelectroporation cuvette, and nucleofected by an AmaxaNucleofector II apparatus, following the manufacturer’sguidelines. After transfection, cells were immediately trans-ferred into complete medium and cultured in T175 flasks at37�C. Transfection efficiency was estimated in each experi-ment by scoring the number of GFP-positive cells byfluorescence microscopy examination and by flow cytome-try analysis.InmiR-34a functional studies, cells were transfected with

the hsa-miR-34a precursor oligo, for mimicking the over-expression of this miRNA, or with the hsa-miR-34a anti-miR oligo, for the inhibition of the activity of endogenousmiR-34a. In parallel, cells were transfected with the FAM-

labeled miR negative control oligo as a nontargeting nega-tive control and for monitoring transfection efficiency. Alloligos were from Ambion.

Statistical analysisData are calculated and shown as mean � SD or as

median and interquartile range, according to the distribu-tion. The results were evaluated by using ANOVA withsubsequent comparisons by Student’s t test and with theMann–Whitney rank-sum test. Correlations were tested bySpearman’s correlation coefficient. Statistical significancewas defined as P < 0.05. To investigate the effect of Chlor-ambucil plus Nutlin-3 combination, cells were then treatedwith serial dilutions (from 10 to 0.1 mmol/L) of Chlor-ambucil or Nutlin-3, individually or in combination, byusing a constant ratio (Chlorambucil/Nutlin) 1:1. Resultswere analyzed by the CalcuSyn software program (Biosoft),which uses the method of Chou and Talalay (23). Acombination index (CI) of 1 indicates an additive effect,whereas a CI less than 1 indicates synergism.

Figure 2. Transcriptionaldownregulation of B-Myb andE2F1 by Nutlin-3 in leukemic cellsis related to cell-cycle block ratherthan to apoptosis induction. Thep53wt EHEB and SKW6.4 cellslines, and the p53mutated BJAB cellline were exposed to Nutlin-3 andChlorambucil. A, cells wereexposed to 10 mmol/L of Nutlin-3or Chlorambucil before analysis ofp53 protein levels byWestern blot.Tubulin staining is shown asloading control. B, cells wereexposed to Nutlin-3 andChlorambucil, both used in therange of 0.1 to 10 mmol/L, beforeassessing the percentage of cellviability and the induction ofapoptosis, quantitativelyevaluated after Annexin V/PIstaining.

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Results

Transcriptional repression of B-Myb and E2F1 byNutlin-3 in p53wild-type leukemic cells

In the first group of experiments, B-Myb mRNA levelswere analyzed by quantitative Real Time (RT)-PCR insamples obtained from AML and B-CLL patients in com-parison with primary normal CD34þ progenitor cells andCD19þ B cells, respectively (Supplementary Fig. S1). AMLsamples showed significantly (P < 0.01) greater levels ofB-Myb mRNA as compared with normal CD34þ cells(Supplementary Fig. S1). Also B-CLL showed an increaseof B-Myb mRNA endogenous levels with respect to pri-mary CD19þ B cells but the difference did not reach

statistically significance. Because a constitutive expressionof B-Myb can bypass p53-mediated G1 arrest and isrequired for recovery from the DNA damage-inducedG2 checkpoint in p53 mutant cells (24), we investigatedthe potential modulation of B-Myb in response to Nutlin-3, a nongenotoxic activator of the p53 pathway. The invitro exposure to Nutlin-3 (10 mmol/L) for 24 hoursvariably downregulated the mRNA levels of B-Myb inprimary leukemic cells (Fig. 1A). To explore the molecularmechanisms underlining the Nutlin-3–induced downre-gulation of B-Myb, we have used a series of leukemic celllines, characterized by a different p53 status. After 24hours of Nutlin-3 treatment, B-Myb levels were clearly(P < 0.01) decreased in all p53wt leukemic cells of both

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Figure 2. (Continued ) C, distribution of cells in the different phases of the cell cycle was calculated from the flow cytometry dot plots after BrdU/PIstaining, and expressed as the percentage of the total population. D, representative cell-cycle profiles of cells, either left untreated or treated asindicated, analyzed by flow cytometry are shown. In each panel, the rectangle represents the cells in S phase of the cell cycle, which have incorporatedBrdU. E, E2F1 protein levels were analyzed by Western blot. Tubulin staining is shown as loading control. F, levels of B-Myb and E2F1 mRNA wereanalyzed by quantitative RT-PCR. Results are expressed as fold of B-Myb and E2F1modulation by Chlorambucil, after 24 hours of treatment, with respect tothe control untreated cultures set to 1 (hatched line). A and E, representative examples of Western blot results out of 3 to 5 independent experiments areshown. B and F, data are reported as mean � SD of results from at least 4 experiments, each conducted in triplicate. *, P < 0.05 with respect to theuntreated cultures. Unt., untreated; Nutl., Nutlin-3; Chl., Chlorambucil.

Zauli et al.





myeloid (OCI, MOLM) and lymphoid (EHEB, SKW6.4)origin (Fig. 1B). On the contrary, in p53mutated myeloid(NB4) and lymphoid (BJAB, MAVER) cell lines, Nutlin-3

treatment did not affect B-Myb mRNA levels (Fig. 1B).Similarly, lack of B-Myb downregulation was observed inthe myeloid p53deleted HL-60 cell line (Fig. 1B).

Figure 3. Transcriptionaldownregulation of B-Myb andE2F1 by Nutlin-3 also infibroblasts (Fibrobl.) andendothelial (Endoth.) cells. Primaryfibroblasts and endothelial cellswere exposed to Nutlin-3 (10mmol/L). A, levels of B-Myb andE2F1 mRNA were analyzed byquantitative RT-PCR. Results areexpressed as fold of B-Myb andE2F1 modulation by Nutlin-3(Nutl.), after 24 hours of treatment,with respect to the controluntreated (Unt.) cultures set to 1(hatched line). Data are reportedas mean � SD of results from atleast 3 experiments, eachconducted in triplicate. In parallel,E2F1 protein levels were analyzedby Western blot. Tubulin stainingis shown as loading control.Representative examples ofWestern blot results, out of 3independent experiments, areshown. B, representative cell-cycle profiles of cells, either leftuntreated or treated with Nutlin-3,analyzed by flow cytometry areshown. In each panel, therectangle represents the cells in Sphase of the cell cycle, which haveincorporated BrdU. C, fibroblastswere transfected with eithercontrol scrambled (scr.) siRNA orB-Myb siRNA. Efficiency oftransfection was monitored byimmunofluorescence microscopyand flow cytometry, by using GFP-constructs, whereas efficiency ofB-Myb knockdown wasdocumented by analyzing levels ofB-Myb mRNA assessed byquantitative RT-PCR. Levels of B-Myb mRNA were expressed asfold of modulation with respect tothe scrambled (scrmbl.) controltransfected cultures. At 48 hoursposttransfection, distribution ofcells in the different phases of thecell cycle was calculated from flowcytometry dot plots after BrdU/PIstaining, and expressed aspercentage of the total population.Data are reported as mean � SDof results from 3 independentexperiments. *, P < 0.05 withrespect to the untreated culturesor to the scrambled controltransfected cultures.

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Figure 4. Inverse correlation between Nutlin-induced upregulation of miR-34a and downregulation of B-Myb and E2F1. A, prediction of hsa-mir-34a bindingsite in the 30-UTR of B-Myb gene. B, modulation of miR-34a mRNA by Nutlin-3 was analyzed by quantitative RT-PCR in the indicated cell lines as well as infibroblasts (Fibrobl.) and endothelial (Endoth.) cells. C, the p53wt EHEB cell line was either left untreated or exposed to Nutlin-3 (10 mmol/L). At the indicatedtime points, levels of E2F1 and B-Myb and miR-34a were simultaneously assessed by RT-PCR. D, distribution of cells (EHEB) in the different phases of the cellcycle was calculated from the flow cytometry dot plots after BrdU/PI staining, and expressed as the percentage of the total population. C, levels of E2F1, B-Myb, and miR-34a were expressed as fold of modulation with respect to the control untreated cultures (time ¼ 0). Data are reported as mean � SD of resultsfrom 3 independent experiments. *, P < 0.05 with respect to the untreated cultures.

Zauli et al.





As it has been shown that B-Myb is a transcriptionaltarget of E2F1 (25, 26) and previous studies have shownthat E2F1 is downmodulated by Nutlin-3 in solid tumormodels (27) in parallel, we have analyzed the effectof Nutlin-3 on E2F1 expression. As shown in Figure1C, treatment with Nutlin-3 strikingly downregulatedalso E2F1 RNA levels in the p53wt, but not inp53mutated/deleted, leukemic cell lines.To clarify the p53-dependence of the observed gene

modulations, we have then used isogenically matched setof cells differing only for p53 status. For this purpose, wehave analyzed the effect of Nutlin-3 in OCI cells tran-siently transfected with p53 siRNA (Fig. 1D). Becausetransfection efficiency is approximately 40% in OCI cells,p53 is not silenced but significantly knocked down, asevaluated by quantitative RT-PCR assay (data notshown). The ability of Nutlin-3 to decrease both B-Myb and E2F1 RNA levels was significantly (P < 0.05)attenuated with respect to OCI cells transfected withcontrol scramble siRNA (Fig. 1D). Moreover, the depen-dence from p53 of Nutlin-3–mediated downregulationof B-Myb was independently confirmed by examiningB-Myb and E2F1 RNA modulation by Nutlin-3 by usingthe p53 isogenic-paired cell lines HCT116 p53wt andHCT116 p53�/� (Fig. 1D).

Transcriptional downregulation of B-Myb and E2F1by Nutlin-3 is related to cell-cycle block ratherthan to apoptosis inductionGiven that both B-Myb and E2F1 have been involved in

modulating either the survival or the proliferation oftumor cells (28–30), we have next investigated whetherthe downregulation of B-Myb and E2F1 by Nutlin-3 wasrelated to the induction of apoptosis, cell-cycle arrest, orboth events. For this purpose, we have followed 2 inde-pendent approaches: (i) we have compared the effects ofNutlin-3 (in terms of transcriptional modulation of E2F1,B-Myb, cell-cycle arrest, and apoptosis induction) withthose of Chlorambucil, a chemotherapeutic drug com-monly used for the treatment of hematological malignan-cies, and (ii) we have analyzed the effects of Nutlin-3(again, in terms of transcriptional modulation of E2F1,B-Myb, cell-cycle arrest, and apoptosis induction) also inprimary normal endothelial cells and fibroblasts.Treatment with Nutlin-3 and Chlorambucil (both used

at 10 mmol/L) induced a comparable accumulation of p53protein in the p53wt EHEB and SKW6.4 cells lines, whereasp53mutated BJAB cells were characterized by constitutivehigh levels of p53 that were unaffected by either Nutlin-3 or Chlorambucil treatment (Fig. 2A). Interestingly,Nutlin-3 and Chlorambucil (both used in the range of0.1–10 mmol/L) induced comparable levels of cytotoxicity,as evaluated by assessing the percentage of cell viability andapoptosis induction, in the p53wt SKW6.4 and EHEB cells,but not in p53mutated BJAB cells (Fig. 2B). However, inp53wt cells, Nutlin-3 and Chlorambucil exhibited strikinglydifferent effects on cell-cycle progression of these cell lines(Fig. 2C and D). Indeed, although both drugs induced cell-

cycle arrest, as indicated by reduction in cell counts, Nutlin-3 exposure induced the accumulation of cells in G1 andalmost abrogated S phase in p53wt leukemic cells, Chlor-ambucil increased the percentage of cells in S and G2/Mphases, as clearly assessed by bromodeoxyuridine (BrdU)incorporation (Fig. 2C and D). These effects of Nutlin-3were accompanied by drastic reduction of E2F1 protein inboth p53wt EHEB and SKW6.4 cell lines, but not inp53mutated BJAB (Fig. 2E). On the contrary, exposure toChlorambucil did not reduce (and rather, it tended toincrease) E2F1mRNA and protein levels in the p53wt EHEBand SKW6.4 or p53mutated leukemic cells (Fig. 2E and F).Similarly, Chlorambucil treatment did not affect thesteady-state mRNA levels of B-Myb in the 3 B-cell linesinvestigated (Fig. 2F).

When cultures were treated with serial concentrations ofNutlin-3 plus Chlorambucil (in the range of 0.1–10 mmol/L) at a constant Nutlin-3/Chlorambucil ratio 1:1 (for dataanalysis by the method of Chou and Talalay; ref. 23), asynergistic cytotoxicity (average CI values less than 1) ofcombination treatment was observed in p53wt, but not inp53mutated, cell lines (Supplementary Fig. S2A). Even in thecombined treatment Nutlin-3 þ Chlorambucil, as in thetreatment with Nutlin-3 alone, both E2F1 and B-Myb weredecreased (Supplementary Fig. S2B), and cell cycle wasblocked in G1 phase, as documented by BrdU incorpora-tion (data not shown) and analysis of pRb in Western blot(Supplementary Fig. S2A).

In parallel experiments, we have investigated whetherNutlin-3 affected the levels of B-Myb and E2F1 also inprimary fibroblasts and endothelial cells. Treatmentwith Nutlin-3 of fibroblasts and endothelial cellsinduced a striking transcription repression of B-Myb,as well as of E2F1 (Fig. 3A), a finding confirmed forE2F1 also at the protein level (Fig. 3A). Similar to thedata obtained on leukemic cell lines, Nutlin-3 induced acell-cycle block in G1 also in fibroblasts and endothelialcells (Fig. 3B), but with negligible effects on the back-ground levels of apoptosis (data not shown). Takentogether, these findings suggested that the transcriptiondownregulation of B-Myb by Nutlin-3 was related to itsability to block cell cycle, rather than to its ability toinduce apoptosis. To further elucidate the potentialcause–effect relationship between downmodulation ofB-Myb and cell-cycle arrest, we have conducted knock-down experiments of endogenous B-Myb by using fibro-blast cultures, which were used for their high efficiencyof transfection. As shown in Figure 3C, at 24 to 48hours after B-Myb siRNA transfection we observed amarked, and specific, decrease of B-Myb mRNA, butnot of p53 (analyzed for control; data not shown),accompanied by a significant (P < 0.01) decrease ofthe percentage of cell in S-phase (Fig. 3C).

Key role of miR-34a in mediating the downregulationof B-Myb by Nutlin-3

To characterize the mechanism of B-Myb transcrip-tion repression by Nutlin-3, we have conducted a target






prediction analysis by NBmiRTar, an in silico approachbased on machine learning by a Naive Bayes classifier(31). The program identified a potential binding site formiR-34a in the 30 untranslated region (UTR) of B-Myb(Fig. 4A; chr 20:41778338–41778361). Because differentreports have shown that one pathway through which p53regulates cell growth is through regulation of a set ofmiRNA, including miR-34a (32, 33), we have investigatedthe hypothesis of a role for miR-34a in the downregula-tion of B-Myb by Nutlin-3. For this purpose, we firstmeasured the modulation of miR-34a in the cell modelsin which we had previously documented transcriptionalrepression of B-Myb by Nutlin-3 (Fig. 1B–D). As shown inFigure 4B, we have observed a significant induction ofmiR-34a in response to Nutlin-3 treatment in all p53wt,

but not in the p53mutated/deleted, cells. Moreover, in time-course experiments we found that exposure to Nutlin-3induced a rapid (within 10 hours) induction of miR-34aconcomitant to the transcriptional decrease of B-Myb andE2F1 (Fig. 4C), and to the induction of cell-cycle arrest(Fig. 4D). These data showed an inverse correlationbetween induction of miR-34a and downmodulation ofB-Myb and E2F1, but did not prove yet that the Nutlin-3–mediated downregulation of B-Myb and/or E2F1 ismediated by miR-34a. Thus, to functionally test thehypothesis that miR-34a decreases B-Myb expression,we transfected primary fibroblasts with the precursor tomiR-34a (designated miR-34a mimic). As a control, somecells were transfected with scrambled miRNA. As shownin Figure 5A, the precursor to miR-34a significantly

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Figure 5. Role of miR-34a in thedownregulation of B-Myb byNutlin-3. Fibroblasts weretransfected with the either controlscrambled (scr.) or the hsa-miR-34a precursor (mimic) or hsa-miR-34a inhibitor (anti-miR) oligos.After transfections, upmodulation(A) and downmodulation (C) ofmiR-34a were documented byquantitative RT-PCR and areexpressed as folds of modulationwith respect to the scrambledcontrol transfected cultures (set to1). A and B, cells transfected asindicated were analyzed for levelsof B-Myb, E2F1, p53, and p21mRNA that were assessed in thesame samples by quantitative RT-PCR. C, cells transfected asindicated were left untreated orexposed to Nutlin-3 (10 mmol/L)and analyzed for levels of B-Myband E2F1 mRNA. Results wereexpressed as fold of B-Myb andE2F1 modulation in Nutlin-3–treated cultures with respect tothe control-untreated cultures,which were set to 1 (hatched line).Data are reported as mean � SDof results from 3 independentexperiments. *, P < 0.05 withrespect to scrambled controltransfected cultures.

Zauli et al.





(P < 0.01) decreased B-Myb levels. Consistently withprevious studies (34, 35) overexpression of miR-34a alsodownmodulated E2F1 expression (Fig. 5B). On the con-trary, it did not affect p53 and p21 expression (Fig. 5B). Inadditional experiments, the downregulation of miR34awith a specific antago-miR significantly (P < 0.05) coun-teracted the ability of Nutlin-3 of downregulating bothE2F1 and B-Myb (Fig. 5C).By computational analysis (as predicted by SABios-

ciences’ Text Mining Application and the UCSC GenomeBrowser) both E2F1 and B-Myb promoters exhibit several

p53 consensus DNA binding sites, which have previouslysuggested a gene regulation/repression occurring throughp53 binding directly to the consensus DNA bindingsequences (36, 37). In addition, E2F1 DNA binding siteshave been found in the B-Myb regulatory region, indicat-ing that a potential regulation of B-Myb is also mediatedby E2F1 (36–38). This was confirmed in our cell model byE2F1 knockdown experiments. Indeed, as shown inFigure 6A, siRNA specific for E2F1 induced a significant(P < 0.05) downregulation of B-Myb, confirming theimportant role of E2F1 upstream of B-Myb. Of interest,

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the analysis by in silico prediction by miRecords, whichintegrates the predicted targets of different miRNA targetprediction tools (39), identify a binding site for miR-34ain 30-UTR region of E2F3 and E2F5, but not in E2F1. Thisobservation supports the proposed regulation of E2F1 bymiR-34a through an indirect mechanism (34, 35). In nextanalysis, all leukemic samples were divided in 2 groupson the basis of the endogenous levels of miR-34a (belowor above the median value; Fig. 6B). The comparison ofthe endogenous levels of B-Myb and E2F1 between the 2groups showed that the samples characterized by lowermiR-34a levels exhibited significantly (P < 0.05) higherB-Myb and E2F1 levels (Fig. 6B). In addition, analysisof Nutlin-3 modulation (expressed as folds) in all sam-ples assayed showed a significant inverse correlationbetween the modulation of miR-34a and B-Myb (R ¼�0.55, P < 0.01), as well as between miR-34a and E2F1(R ¼ �0.65, P < 0.01), whereas a significant positivecorrelation was observed between the modulation ofB-Myb and E2F1 (R ¼ 0.81, P < 0.01). It should beemphasized that our present data suggest a previouslyuncharacterized mechanism of negative regulation ofB-Myb, through miR-34a, that together with E2F1, med-iates p53-suppression of cell proliferation in response toNutlin-3 treatment (Fig. 6C).

Discussion

Starting from the observation that B-Myb was over-expressed in primary AML patients cells in comparisonwith normal CD34þ progenitor cells, we have shown for

the first time that the nongenotoxic activator of p53pathway, Nutlin-3, significantly downregulated transcrip-tion of both B-Myb and E2F1 in p53wt leukemic cells, butnot in p53mutated/deleted leukemic cell lines. The ability ofNutlin-3 to downregulate B-Myb and E2F1 in AML cells isparticularly noteworthy because it has been previouslyshown that high levels of expression of B-Myb (40, 41)and E2F1 (42, 43) contribute to the block of maturationcharacterizing AML. On the contrary, the levels of B-MybmRNA in primary B-CLL were not significantly differentfrom that of primary CD19þ B cells and Nutlin-3 induceda modest downregulation of B-Myb in B-CLL cells. Ascirculating B-CLL cells are primarily in G1 phase of the cellcycle, these findings are consistent with a predominantinvolvement of Nutlin-3–mediated downregulation of B-Myb in the cell-cycle block, rather than in the induction ofapoptosis.

The primary role of B-Myb downregulation by Nutlin-3in cell-cycle block was strongly supported by the differ-ential effect of Nutlin-3 and Chlorambucil with respect toinduction of apoptosis and cell-cycle arrest. In fact,Nutlin-3 and Chlorambucil induced a comparable levelof apoptosis in p53wt leukemic cell lines, but the effect ofChlorambucil was strikingly different from that of Nutlin-3 with respect to cell cycle, because Nutlin-3 promotedthe accumulation of leukemic cells in G1, whereas Chlor-ambucil induced the accumulation of leukemic cells inG2/M. Consistently with these different effects, treatmentwith Chlorambucil did not decrease E2F1 and B-Mybexpression in p53wt leukemic cells. The observationsobtained in leukemic cells were extended also to normal

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Zauli et al.





endothelial cells and fibroblasts. In this respect, it isparticularly noteworthy that E2F1 has been recentlyinvolved in promoting proangiogenic pathways (44).Therefore, the ability to downregulate E2F1 in endothe-lial cells can contribute to antiangiogenic activity ofNutlin-3 (45) independently of its effects on primarytumor cells.In agreement with previous data obtained in other cell

models (36–38), our present findings also suggested thatE2F1 is involved in B-Myb downregulation at transcrip-tional level. First of all, we could show that the modula-tion of mRNA levels of E2F1 and B-Myb transcriptionfactors, assessed in response to Nutlin-3 in all cell typesinvestigated, significantly and positively correlated. More-over, we have observed that transcriptional E2F1 down-regulation anticipated B-Myb decline. Finally, the knock-down of endogenous E2F1 determined downregulationof B-Myb. Nevertheless, the most striking and novel find-ing of our study in terms of molecular mechanismsinvolved in the control of B-Myb regulation is the invol-vement of miR-34a upregulation in mediating the effectof Nutlin-3. Consistently with recent reports indicatingthat some of the p53- downregulated genes might beassociated with miRNA expression (31, 32), we haveshown for the first time that miR-34a is a key determinantin mediating B-Myb downregulation by Nutlin-3. The useof an antagomiR specific for miR-34a significantly coun-teracted the Nutlin-3–mediated downregulation of bothE2F1 and B-Myb. However, target prediction analysis by insilico approach identified a potential binding site for miR-34a in the 30-UTR of B-Myb and in 30-UTR region of E2F3and E2F5, but not in E2F1. This observation suggeststhat the downregulation of E2F1 by miR-34a might beindirectly mediated by other transcription factors (34,35). In this respect, it has been recently reported thatmiR-34a, regulated by C/EBPa during granulopoiesis,

modulates directly E2F3 and indirectly E2F1,a majortranscriptional target of E2F3, showing an inversecorrelation between miR-34a and E2F (both E2F3 andE2F1) levels in AML with CEBPA mutations (46). Inaddition, we found an inverse correlation between theendogenous miR34a levels and those of B-Myb and ofE2F1, both before and after Nutlin-3 treatment.

In conclusion, we have shown for the first time thatmiR34a induces the transcriptional repression of B-Myb inp53wt leukemic cells and we propose that such down-regulation is a key determinant in the biological activityof Nutlin-3. In a therapeutic perspective, it is noteworthythat Nutlin-3, but not Chlorambucil, was able to promoteE2F1 and B-Myb downregulation in p53wt leukemic cells,suggesting that it might display superior therapeutic effi-cacy as compared with commonly used chemotherapeuticdrugs. The ability of miR34a to downregulate B-Myb wasclearly related to cell-cycle arrest rather than apoptosisinduction in both malignant cells and primary normalfibroblasts and endothelial cells, suggesting that therapeu-tic approaches able to raise the levels of miR34a will likelyresults in antiangiogenic activity.

Disclosure of Potential Conflicts of Interest

The authors declare no potential conflicts of interest.

Grant Support

This study was supported by grants from the Italian Association forCancer Research (AIRC; to G. Zauli), Beneficientia Foundation (to G. Zauli),and CariFe Foundation (to P. Secchiero).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 6, 2010; revised February 16, 2011; accepted Feb-ruary 20, 2011; published OnlineFirst March 2, 2011.

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Zauli et al.





2011;17:2712-2724. Published OnlineFirst March 2, 2011.Clin Cancer Res Giorgio Zauli, Rebecca Voltan, Maria Grazia di Iasio, et al.

Oncogenes in Leukemic CellsB-Myb and E2F1miR-34a Induces the Downregulation of Both

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