miR-34a induces the down-regulation of both E2F1 and B-Myb ... fileGiorgio Zauli1§, Rebecca...

CCR-10-3244.R1

miR-34a induces the down-regulation of both E2F1 and B-Myb oncogenes in leukemic cells

Giorgio Zauli1§, Rebecca Voltan2§, Maria Grazia di Iasio2, Raffaella Bosco2, Elisabetta Melloni2,

Maria Elena Sana2, Paola Secchiero2

1Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste, Italy; 2Department of

Morphology and Embryology and LTTA Centre, University of Ferrara, Ferrara, Italy.

§These author equally contributed to the work

Corresponding author: Dr. Rebecca Voltan, Department of Morphology and Embryology and

LTTA Centre, University of Ferrara, Via Fossato di Mortara 66, 44121 Ferrara, Italy. Tel: 39-0532-

455846; Fax: 39-0532-455950; E-mail: [email protected]

Running title: miR34a down-regulates B-Myb expression

Keywords: B-Myb, E2F1, p53, miR34, Nutlin-3.

Research. on May 28, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 2, 2011; DOI: 10.1158/1078-0432.CCR-10-3244

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Statement of Translational Relevance: B-Myb oncogene was found overexpressed in both

primary acute myeloid leukemia blasts. The small molecule inhibitor of MDM2/p53 interactions

Nutlin-3 efficiently repressed both B-Myb and E2F1 in a variety of p53 wild-type leukemic cell

types as well as in primary endothelial cells and fibroblasts. Gene knock-down and overexpression

experiments demonstrated that Nutlin-3 up-regulated miR34a, which in turn induced the down-

regulated B-Myb both directly and through the down-regulation of E2F1. These data strengthen the

notion that promoting the up-regulation of miR34a might have important clinical applications for

the treatment of human leukemias.




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Abstract

Purpose: To elucidate new molecular mechanisms able to down-regulated the mRNA levels of key

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

Experimental Design: B-Myb and E2F1 mRNA levels were evaluated in primary B chronic

lymphocytic leukemia (B-CLL, n=10), acute myeloid leukemia (AML, n=5) patient cells, in a

variety of p53wild-type and p53mutated/deleted leukemic cell lines, as well as in primary endothelial cells

and fibroblasts. Knock-down experiments with siRNA for p53 and E2F1 and overexpression

experiments with miR34a were performed to elucidate the role of these pathways in promoting B-

Myb down-regulation.

Results: In vitro exposure to Nutlin-3, a non-genotoxic activator of p53, variably down-regulated

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

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

leukemic cell lines. The transcriptional repression of B-Myb was observed also in primary normal

endothelial cells and fibroblasts. B-Myb down-regulation played a critical role in the cell cycle

block in G1 phase induced by Nutlin-3, as demonstrated 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: Due to the role of B-Myb and E2F1 transcription factors in controlling cell cycle

progression of leukemic cells, the down-regulation of these oncogenes by miR-34a suggests the

usefulness of therapeutic approaches aimed to modulate the levels of miR-34a.




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Introduction

Nutlin-3a represents a potent and selective small molecule inhibitor of the p53-MDM2

interaction (1), which raises the levels of p53 protein and induces subsequent cytostatic or cytotoxic

effects in a variety of tumor cells (1). Taking into consideration that p53 is mutated in less than 15%

of hematological malignancies at diagnosis (2), it is noteworthy that Nutlin-3 has shown in vitro

promising therapeutic potential in both myeloid and lymphoid leukemia characterized by a p53wild-

type status (3-9). However, since p53 mediates different cellular functions, such as in particular cell

cycle arrest and apoptosis (3) the critical molecular determinants mediating/modulating the

therapeutic activity of Nutlin-3 in leukemic cells are incompletely understood.

Previous studies have shown that several solid tumors are characterized by overexpression of

B-Myb, also known as MYBL2 (10-13), a member of the vertebrate MYB family of nuclear

transcription factors, which recognize and 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

proliferating cells as a regulator of cell cycle progression and cell survival (15) and it plays an

essential role in vertebrate development (16). Indeed, knocking-down murine B-Myb causes early

embryonic lethality (E4.5-6.5) resulting from unsuccessful inner cell mass formation (16).

Interestingly, it has also been shown that B-Myb chromosomal locus (20q13) is amplified and/or

highly expressed in a variety of tumor types including neuroblastomas, breast, prostate, liver and

ovarian carcinomas, and in most cases this high expression is associated with a poor prognosis (10-

13).

On these bases, the aim of the present study was to investigate the baseline expression levels

and the potential regulation and/or involvement of B-Myb in the therapeutic response to Nutlin-3 in

myeloid and lymphoid leukemias, as well as in primary stromal cells.




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Materials and Methods

Cell cultures

Peripheral blood samples were collected in heparin-coated tubes from 5 patients affected by

acute myeloid leukemia (AML: 2 M4, 1 M5 and 2 M4/M5), 10 patients affected by B-chronic

lymphocytic leukemia (B-CLL) and from 10 healthy blood donors following informed consent, in

accordance with the Declaration of Helsinki and in agreement with institutional guidelines.

Peripheral blood mononuclear cells (PBMC) from AML and B-CLL patients and from healthy

donors were isolated by gradient centrifugation with lymphocyte cell separation medium (Cedarlane

Laboratories, Hornby, ON). Specific CD19+ and CD34+ cell populations were selected from normal

PBMC using immunomagnetic micro-beads and the AutoMACS system (Myltenyi Biotech,

Auburn, CA) in accordance with the manufacturer’s instructions, obtaining a purity exceeding 90%

for each subpopulation. For analysis of CD19+ cells, RNA preparations were obtained from 5

independent donors. For analysis of CD34+ cells, each RNA preparation (n=4 in total) was obtained

by pooling CD34+cells purified from at least 3 different donors.

The p53wild-type (OCI, MOLM, SKW6.4, EHEB), p53mutated (NB4, BJAB, MAVER) or

p53deleted (HL-60) leukemic cell lines were purchased from the ATCC (American Type Culture

Collection, Manassas, VA) or obtained from DSMZ (Deutsche Sammlung von Mikroorganismen

und Zellkulturen GmbH, Braunschweig, Germany). Control PBMC and patient cells were cultured

in RPMI-1640 (Gibco BRL, Grand Island, NY) containing 10% FBS, L-glutamine and

penicillin/streptomycin (Gibco BRL). The cell lines were cultured in RPMI-1640, or in Alpha

MEM (OCI) (LONZA, Verviers, Belgium) supplemented with 10% FBS and L-glutamine and

penicillin/streptomycin (Gibco BRL). Cells were seeded at a density of 1x106 cells/ml before

treatment with Nutlin-3 (used in the range of 0.1-10 μM; Cayman Chemical, Ann Arbor, MI) or

Chlorambucil (used in the range of 0.10-10 μM; Sigma-Aldrich, St Louis, MO). The HCT116

human colon cancer cells, with either p53 wild-type (p53wt) or p53 knockout (p53-/-), were cultured




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in DMEM media supplement with 10% FBS (17).

Human umbilical vein endothelial cells (HUVEC) were purchased from BioWhittaker

(Walkersville, MD) and grown as previously described (18). For the evaluation of cytostatic and/or

toxic effects, HUVEC and fibroblasts were seeded and grown to subconfluence before treatment

with Nutlin-3 (10 μM). Neonatal human dermal fibroblasts were purchased from LONZA and

grown in DMEM (LONZA) containing 20% FBS, L-glutamine and penicillin/streptomycin (Gibco

BRL).

Assessment of cell viability, apoptosis and cell cycle

Cell viability was examined by Trypan blue dye exclusion, and induction of apoptosis was

quantified by Annexin V-FITC/propidium iodide (PI) staining (Immunotech, Marseille, France)

followed by flow cytometry analysis (19). To avoid non-specific fluorescence from dead cells, live

cells were gated tightly using forward and side scatter, as described (20). In order to analyze the

degree of apoptosis in the entire cell population of HUVEC and fibroblast cultures, substrate-

attached cells were harvested by trypsin treatment and pooled with floating cells for the staining.

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

Real time-PCR

Total RNA was extracted from cells by using the Qiagen RNeasy Plus mini kit (Qiagen,

Hilden, Germany) according to the supplier’s instructions. Total RNA was transcribed into cDNA,

using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). Levels of miR-34a

expression were evaluated by using the miScript PCR System (Qiagen), composed of a miScript

Reverse Transcription kit, an amplification miScript SYBR Green PCR kit for cDNA with miScript

Primer Assays including a specific validated primer sets for miR-34a (QuantiTech primer assay,

Qiagen). The expression of miR-34a values was normalized by using specific primers to RNU6B

(Qiagen) as endogenous reference RNA.




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Western blot analyses

Cells were lysed in ice-cold Ripa buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 1%

Nonidet P-40, 0.25% sodium desoxycholate) supplemented with protease inhibitors (Complete,

Roche; Mannheim, Germany) on ice for 1 hour. Protein determination was performed by BCA

Protein Assay (Thermo Scientific, Rockford, IL) and then additioned with loading buffer (250 mM

Tris pH 6.8, 2% SDS, 40% Glycerin, 20% beta-mercaptoethanol) and boiled for 2 min (22). Equal

amounts of protein for each sample were migrated in acrylamide gels and blotted onto nitrocellulose

filters, as previously described. The following mAb were used in our experiments: anti p53 (DO-1;

Santa Cruz Biotechnology, Santa Cruz, CA), anti E2F1 (KH95; Santa Cruz Biotechnology), anti

pRb (G3-245; BD Biosciences Pharmingen, Franklin Lakes, NJ) and anti tubulin (Sigma-Aldrich, St

Louis, MO). After incubation with peroxidase-conjugated anti mouse IgG, specific reactions were

revealed with the ECL Lightning detection kit (Perkin Elmer, Waltham, MA). Densitometric values

were estimated by the ImageQuant TL software (GE Healthcare, Buckinghamshire, UK). Multiple

film exposures were used to verify the linearity of the samples analyzed and to avoid saturation of

the film.

Transfection experiments

Cells (1x106) were resuspended into 0.1 ml of NucleofectorTM solution of human nucleofector

kit NHDF or V (Amaxa, Cologne, Germany). Two μg of plasmid DNA (GFP-construct) or 1 μg of

siRNA (see Supplementary Material and Methods) were mixed with the 0.1 ml of cell suspension,

transferred into a 2.0 mm electroporation cuvette, and nucleofected using an Amaxa Nucleofector II

apparatus, following the manufacturer guidelines. After transfection, cells were immediately

transferred into complete medium and cultured in T175 flasks at 37°C. Transfection efficiency was

estimated in each experiment by scoring the number of GFP-positive cells by fluorescence

microscopy examination and by flow cytometry analysis.




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In miR-34a functional studies, cells were transfected with the hsa-miR-34a precursor oligo,

for mimicking the overexpression of this miRNA, or with the hsa-miR-34a anti-miR oligo, for the

inhibition of the activity of endogenous miR-34a. In parallel, cells were transfected with the FAM-

labeled miR negative control oligo as a nontargeting negative control and for monitoring

transfection efficiency. All oligos were from Ambion.

Statistical analysis

Data are calculated and shown as mean+SD or as median and Interquartile Range (IQR),

according to the distribution. The results were evaluated by using analysis of variance with

subsequent comparisons by Student’s t-test and with the Mann-Whitney rank-sum test. Correlations

were tested by Spearman’s correlation coefficient. Statistical significance was defined as p<0.05. In

order to investigate the effect of Chlorambucil plus Nutlin-3 combination, cells were then treated

with serial dilutions (from 10 to 0.1 μM) of Chlorambucil or Nutlin-3, individually or in

combination using a constant ratio (Chlorambucil:Nutlin) 1:1. Results were analyzed using the

CalcuSyn software program (Biosoft, Cambridge, United Kingdom), which uses the method of

Chou and Talalay (23). A combination index (CI) of 1 indicates an additive effect, while a CI below

1 indicates synergism.




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Results

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

In the first group of experiments, B-Myb mRNA levels were analyzed by quantitative RT-

PCR in samples obtained from AML and B-CLL patients in comparison with primary normal

CD34+ progenitor cells and CD19+ B cells, respectively (Supplementary Figure 1). AML samples

showed significantly (p<0.01) greater levels of B-Myb mRNA as compared to normal CD34+ cells

(Supplementary Figure 1). Also B-CLL showed an increase of B-Myb mRNA endogenous levels

with respect to primary CD19+ B cells but the difference did not reach statistically significance.

Since a constitutive expression of B-Myb can bypass p53-mediated G1 arrest and is required for

recovery from the DNA damage-induced G2 checkpoint in p53 mutant cells (24), we investigated

the potential modulation of B-Myb in response to Nutlin-3, a non-genotoxic activator of the p53

pathway. The in vitro exposure to Nutlin-3 (10 μM) for 24 hours variably downregulated the

mRNA levels of B-Myb in primary leukemic cells (Figure 1A). In order to explore the molecular

mechanisms underlining the Nutlin-3-induced down-regulation of B-Myb, we have used a series of

leukemic cell lines, characterized by a different p53 status. After 24 hours of Nutlin-3 treatment, B-

Myb levels were clearly (p<0.01) decreased in all p53wild-type leukemic cells of both myeloid (OCI,

MOLM) and lymphoid (EHEB, SKW6.4) origin (Figure 1B). On the other hand, in p53mutated

myeloid (NB4) and lymphoid (BJAB, MAVER) cell lines, Nutlin-3 treatment did not affect B-Myb

mRNA levels (Figure 1B). Similarly, lack of B-Myb down-regulation was observed in the myeloid

p53deleted HL-60 cell line (Figure 1B).

Since it has been demonstrated that B-Myb is a transcriptional target of E2F1 (25, 26) and

previous studies have shown that E2F1 is down-modulated by Nutlin-3 in solid tumor models (27)

in parallel, we have analyzed the effect of Nutlin-3 on E2F1 expression. As shown in Figure 1C,

treatment with Nutlin-3 strikingly down-regulated also E2F1 RNA levels in the p53wild-type, but not

in p53mutated/deleted leukemic cell lines.




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In order to clarify the p53-dependence of the observed gene modulations, we have then used

isogenically matched set of cells differing only for p53 status. For this purpose, we have analyzed

the effect of Nutlin-3 in OCI cells transiently transfected with p53 siRNA (Figure 1D). Since in

OCI cells transfection efficiency is approximately 40%, p53 is not silenced but significantly

knocked-down, as evaluated by quantitative RT-PCR assay (data not shown). 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 with control scramble siRNA (Figure 1D). Moreover, the dependence from

p53 of Nutlin-3-mediated down-regulation of B-Myb was independently confirmed by examining

B-Myb and E2F1 RNA modulation by Nutlin-3 using the p53 isogenic-paired cell lines HCT116

p53wt and HCT116 p53-/- (Figure 1D).

Transcriptional down-regulation of B-Myb and E2F1 by Nutlin-3 is related to cell cycle block

rather than to apoptosis induction

Given that both B-Myb and E2F1 have been involved in modulating either the survival or the

proliferation of tumor cells (28-30), we have next investigated whether the down-regulation of B-

Myb and E2F1 by Nutlin-3 was related to the induction of apoptosis, cell cycle arrest or to both

events. For this purpose, we have followed two independent approaches: i) we have compared the

effects of Nutlin-3 (in terms of transcriptional modulation of E2F1, B-Myb, cell cycle arrest and

apoptosis induction) with those of Chlorambucil, a chemotherapeutic drug commonly used for the

treatment of hematological malignancies, 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

in primary normal endothelial cells and fibroblasts.

Treatment with Nutlin-3 and Chlorambucil (both used at 10 μM) induced a comparable

accumulation of p53 protein in the p53wild-type EHEB and SKW6.4 cells lines, while p53mutated BJAB

cells were characterized by constitutive high levels of p53 that were unaffected by either Nutlin-3 or

Chlorambucil treatement (Figure 2A). Interestingly, Nutlin-3 and Chlorambucil (both used in the




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range of 0.1-10 μM) induced comparable levels of citotoxicity, as evaluated by assessing the

percentage of cell viability and apoptosis induction, in the p53wild-type SKW6.4 and EHEB cells but

not in p53mutated BJAB cells (Figure 2B). However, in p53wild-type cells, Nutlin-3 and Chlorambucil

exhibited strikingly different effects on cell cycle progression of these cell lines (Figure 2C-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 and almost abrogated S phase in p53wild-

type leukemic cells, Chlorambucil increased the percentage of cells in S and G2/M phases, as clearly

assessed by BrdU incorporation (Figure 2C-D). These effects of Nutlin-3 were accompanied by

drastic reduction of E2F1 protein in both p53wild-type EHEB and SKW6.4 cell lines but not in

p53mutated BJAB (Figure 2E). On the other hand, exposure to Chlorambucil did not reduce (and

rather, it tended to increase) E2F1 mRNA and protein levels in the p53wild-type EHEB and SKW6.4

or p53mutated leukemic cells (Figure 2E-F). Similarly, Chlorambucil treatment did not affect the

steady-state mRNA levels of B-Myb in the three B-cell lines investigated (Figure 2F).

When cultures were treated with serial concentrations of Nutlin-3 plus Chlorambucil (in the

range of 0.1-10 μM) at a constant Nutlin-3:Chlorambucil ratio 1:1 (for data analysis by the method

of Chou and Talalay) (23), a synergistic citotoxicity (average CI values less than 1) of combination

treatment was observed in p53wild-type but not in p53mutated cell lines (Supplementary Figure 2A).

Even in the combined treatment Nutlin-3+Chlorambucil, as in the treatment with Nutlin-3 alone,

both E2F1 and B-Myb were decreased (Supplementary Figure 2B), and cell cycle was blocked in

G1 phase, as documented by BrdU incorporation (data not shown) and analysis of pRb in Western

blot (Supplementary Figure 2A).

In parallel experiments, we have investigated whether Nutlin-3 affected the levels of B-Myb

and E2F1 also in primary fibroblasts and endothelial cells. Treatment with Nutlin-3 of fibroblasts

and endothelial cells induced a striking transcription repression of B-Myb, as well as of E2F1

(Figure 3A), a finding confirmed for E2F1 also at the protein level (Figure 3A). Similarly to the

data obtained on leukemic cell lines, Nutlin-3 induced a cell cycle block in G1 also in fibroblasts




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and endothelial cells (Figure 3B), but with negligible effects on the background levels of apoptosis

(data not shown). Taken together, these findings suggested that the transcription down-regulation of

B-Myb by Nutlin-3 was related to its ability to block cell cycle, rather than to its ability to induce

apoptosis. To further elucidate the potential cause-effect relationship between down modulation of

B-Myb and cell cycle arrest, we have performed knockdown experiments of endogenous B-Myb by

using fibroblast cultures, which were used for their high efficiency of transfection. As shown in

Figure 3C, at 24-48 hours after B-Myb siRNA transfection we observed a marked, and specific,

decrease of B-Myb mRNA, but not of p53 (analyzed for control; data not shown), accompanied by

a significant (p<0.01) decrease of the percentage of cell in S-phase (Figure 3C).

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

To characterize the mechanism of B-Myb transcription repression by Nutlin-3, we have

performed a target prediction analysis by NBmiRTar, an in silico approach based on machine

learning by a Naïve Bayes classifier (31). The program identified a potential binding site for miR-

34a in the 3’UTR of B-Myb (Figure 4A; chr 20:41778338-41778361). Since different reports have

shown that one pathway through which p53 regulates cell growth is through regulation of a set of

miRNA, including miR-34a (32, 33), we have investigated the hypothesis of a role for miR-34a in

the down-regulation of B-Myb by Nutlin-3. For this purpose, we first measured the modulation of

miR-34a in the cell models in which we had previously documented transcriptional repression of B-

Myb by Nutlin-3 (Figure 1B-D). As shown in Figure 4B, we have observed a significant induction

of miR-34a in response to Nutlin-3 treatment in all p53wild-type, but not in the p53mutated/deleted cells.

Moreover, in time-course experiments we found that exposure to Nutlin-3 induced a rapid (within

10 hours) induction of miR-34a concomitant to the transcriptional decrease of B-Myb and E2F1

(Figure 4C), and to the induction of cell cycle arrest (Figure 4D). These data demonstrated an

inverse correlation between induction of miR-34a and down-modulation of B-Myb and E2F1, but

did not prove yet that the Nutlin-3-mediated down-regulation of B-Myb and/or E2F1 is mediated by




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miR-34a. Thus, to functionally test the hypothesis that miR-34a decreases B-Myb expression, we

transfected primary fibroblasts with the precursor to miR-34a (designated miR-34a mimic). As a

control, some cells were transfected with scrambled miRNA. As shown in Figure 5A, the precursor

to miR-34a significantly (p<0.01) decreased B-Myb levels. Consistently with previous studies (34,

35) overexpression of miR-34a also down-modulated E2F1 expression (Figure 5B). On the other

hand, it did not affect p53 and p21 expression (Figure 5B). In additional experiments, the down-

regulation of miR34a with a specific antago-miR significantly (p<0.05) counteracted the ability of

Nutlin-3 of down-regulating both E2F1 and B-Myb (Figure 5C).

By computational analysis (as predicted by SABiosciences' Text Mining Application and the

UCSC Genome Browser) both E2F1 and B-Myb promoters exhibit several p53 consensus DNA

binding sites, which have previously suggested a gene regulation/repression occurring through p53

binding directly to the consensus DNA-binding sequences (36, 37). In addition, E2F1 DNA-binding

sites have been found in the B-Myb regulatory region, indicating a potential regulation of B-Myb

mediated also by E2F1 (36-38). This was confirmed in our cell model by E2F1 knockdown

experiments. Indeed, as shown in Figure 6A, siRNA specific for E2F1 induced a significant

(p<0.05) down-regulation of B-Myb, confirming the important role of E2F1 upstream of B-Myb. Of

interest, the analysis by in silico prediction using miRecords, which integrates the predicted targets

of different miRNA target prediction tools (39), identify a binding site for miR-34a in 3’ UTR

region of E2F3 and E2F5, but not in E2F1. This observation supports the proposed regulation of

E2F1 by miR-34a through an indirect mechanism (34, 35). In next analysis all leukemic samples

were divided in two groups based on the endogenous levels of miR-34a (below or above the median

value) (Figure 6B). The comparison of the endogenous levels of B-Myb and E2F1 between the two

groups showed that the samples characterized by lower miR-34a levels exhibited significantly

(p<0.05) higher B-Myb and E2F1 levels (Figure 6B). In addition, analysis of Nutlin-3 modulation

(expressed as folds) in all samples assayed showed a significant inverse correlation between the

modulation of miR-34a and B-Myb (R= -0.55, p<0.01) as well as between miR-34a and E2F1 (R= -




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0.65, p<0.01), while a significant positive correlation was observed between the modulation of B-

Myb and E2F1 (R= 0.81, p<0.01). It should be emphasized that our present data suggest a

previously uncharacterized mechanism of negative regulation of B-Myb, through miR-34a, that

together with E2F1, mediates p53-suppression of cell proliferation in response to Nutlin-3 treatment

(Figure 6C).




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Discussion

Starting from the observation that B-Myb was over-expressed in primary AML patients cells,

in comparison with normal CD34+ progenitor cells, we have demonstrated for the first time that the

non-genotoxic activator of p53 pathway, Nutlin-3, significantly down-regulated transcription of

both B-Myb and E2F1 in p53wild-type leukemic cells, but not in p53mutated/deleted leukemic cell lines.

The ability of Nutlin-3 to down-regulate B-Myb and E2F1 in AML cells is particularly noteworthy

since it has been previously shown that high levels of expression of B-Myb (40, 41) and E2F1 (42,

43) contribute to the block of maturation characterizing AML. On the other hand, the levels of B-

Myb mRNA in primary B-CLL were not significantly different from that of primary CD19+ B cells

and Nutlin-3 induced a modest down-regulation of B-Myb in B-CLL cells. Since circulating B-CLL

cells are primarily in G1 phase of the cell cycle, these findings are consistent with a predominant

involvement of Nutlin-3-mediated down-regulation of B-Myb in the cell cycle block, rather than in

the induction of apoptosis.

The primary role of B-Myb down-regulation by Nutlin-3 in cell cycle block was strongly

supported by the differential effect of Nutlin-3 and Chlorambucil with respect to induction of

apoptosis and cell cycle arrest. In fact, Nutlin-3 and Chlorambucil induced a comparable level of

apoptosis in p53wild-type leukemic cell lines, but the effect of Chlorambucil was strikingly different

from that of Nutlin-3 with respect to cell cycle, since Nutlin-3 promoted the accumulation of

leukemic cells in G1, while Clorambucil induced the accumulation of leukemic cells in G2/M.

Consistently with these different effects, treatment with Chlorambucil did not decrease E2F1 and B-

Myb expression in p53wild-type leukemic cells. The observations obtained in leukemic cells were

extended also to normal endothelial cells and fibroblasts. In this respect, it is particularly

noteworthy that E2F1 has been recently involved in promoting pro-angiogenic pathways (44).

Therefore, the ability to down-regulate E2F1 in endothelial cells can contribute to anti-angiogenic

activity of Nutlin-3 (45) independently of its effects on primary tumor cells.




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In agreement with previous data obtained in other cell models (36-38), also our present

findings suggested that E2F1 is involved in B-Myb down-regulation at transcriptional level. First of

all, we could demonstrate that the modulation of mRNA levels of E2F1 and B-Myb transcription

factors, assessed in response to Nutlin-3 in all cell types investigated, significantly and positively

correlated. Moreover, we have observed that transcriptional E2F1 down-regulation anticipated B-

Myb decline. Finally, knocking-down endogenous E2F1 determined down-regulation of B-Myb.

Nevertheless, the most striking and novel finding of our study in terms of molecular mechanisms

involved in the control of B-Myb regulation is the involvement of miR-34a up-regulation in

mediating the effect of Nutlin-3. Consistently with recent reports indicating that some of the p53-

down-regulated genes might be associated with microRNA expression (31, 32), we have

demonstrated for the first time that miR-34a is a key determinant in mediating B-Myb down-

regulation by Nutlin-3. The use of an antagomiR specific for miR-34a significantly counteracted the

Nutlin-3-mediated down-regulation of both E2F1 and B-Myb. However, target prediction analysis

by in silico approach identified a potential binding site for miR-34a in the 3’UTR of B-Myb and in

3’ UTR region of E2F3 and E2F5, but not in E2F1. This observation suggests that the down-

regulation of E2F1 by miR-34a might be indirectly mediated by other transcription factors (34, 35).

In this respect, it has been recently reported that miRNA-34a, regulated by C/EBPa during

granulopoiesis, modulates directly E2F3 and indirectly E2F1, a major transcriptional target of

E2F3, showing an inverse correlation between miR-34a and E2F (both E2F3 and E2F1) levels in

AML with CEBPA mutations (46). In addition, we found an inverse correlation between the

endogenous miR34a levels and those of B-Myb and of E2F1, both before and after Nutlin-3

treatment.

In conclusion, we have demonstrated for the first time that miR34a induces the transcriptional

repression of B-Myb in p53wild-type leukemic cells and we propose that such down-regulation is a

key determinant in the biological activity of Nutlin-3. In a therapeutic perspective, it is noteworthy

that Nutlin-3 but not Chlorambucil was able to promote E2F1 and B-Myb down-regulation in




CCR-10-3244.R1

17

p53wild-type leukemic cells, suggesting that it might display superior therapeutic efficacy as compared

to commonly used chemotherapeutic drugs. The ability of miR34a to down-regulate B-Myb was

clearly related to cell cycle arrest rather than apoptosis induction in both malignant cells and

primary normal fibroblasts and endothelial cells, suggesting that therapeutic approaches able to

raise the levels of miR34a will likely results in anti-angiogenic activity.




CCR-10-3244.R1

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Acknowledgments

This study was supported by grants from: the Italian Association for Cancer Research (AIRC) and

Beneficientia Foundation (both to G.Z.) and by CariFe Foundation (to P.S.).




CCR-10-3244.R1

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Figure Legends

Figure 1. Transcriptional repression of B-Myb and E2F1 by Nutlin-3 in p53wild-type leukemic

cell lines. Primary B-CLL and AML leukemic cells and in the indicated p53wild-type (p53wt) or

p53mutated/deleted (p53mut/del) lymphoid and myeloid leukemic cell lines were either left untreated or

exposed to Nutlin-3 (10 μM). After 24 hours of treatment, levels of B-Myb mRNA (A-B) and E2F1

(C) were assessed by quantitative RT-PCR, and results were expressed as fold of B-Myb and E2F1

modulation in Nutlin-3 treated-cultures with respect to the control untreated-cultures, which were

set to 1 (hatched line). In D, B-Myb and E2F1 RNA modulation by Nutlin-3 was analyzed in OCI

cells transiently transfected with p53 siRNA or with control scramble (scr.) siRNA and in the

isogenic-paired cell lines HCT116 p53wt and HCT116 p53-/-. In A, Horizontal bars are median,

upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and

90th percentiles. In B-D, data are reported as means+SD of results from at least three experiments,

each performed in triplicate. Asterisks, p<0.05 with respect to the untreated cultures or, for OCI

siRNA-transfection experiments (D), with respect to cells transfected with the scramble siRNA.

Figure 2. Transcriptional down-regulation of B-Myb and E2F1 by Nutlin-3 in leukemic cells is

related to cell cycle block rather than to apoptosis induction. The p53wild-type EHEB and SKW6.4

cells lines, and the p53mutated BJAB cell line were exposed to Nutlin-3 and Chlorambucil. In A, cells

were exposed to 10 μM of Nutlin-3 or Chlorambucil before analysis of p53 protein levels by

Western blot. Tubulin staining is shown as loading control. In B, cells were exposed to Nutlin-3 and

Chlorambucil, both used in the range of 0.1-10 μM, before assessing the percentage of cell viability

and the induction of apoptosis, quantitatively evaluated after Annexin V/PI staining. In C,

distribution of cells in the different phases of the cell cycle was calculated from the flow cytometry

dot plots after BrdU/PI staining, and expressed as the percentage of the total population. In D,




CCR-10-3244.R1

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representative cell-cycle profiles of cells, either left untreated or treated as indicated, analyzed by

flow cytometry are shown. In each panel, the rectangle represents the cells in S phase of the cell

cycle, which have incorporated BrdU. In E, E2F1 protein levels were analyzed by Western blot.

Tubulin staining is shown as loading control. In F, levels of B-Myb and E2F1 mRNA were

analyzed by quantitative RT-PCR. Results are expressed as fold of B-Myb and E2F1 modulation by

Chlorambucil, after 24 hours of treatment, with respect to the control untreated cultures set to 1

(hatched line). In A and E, representative examples of Western blot results out of three-five

independent experiments are shown. In B and F, data are reported as means+SD of results from at

least four experiments, each performed in triplicate. Asterisks, p<0.05 with respect to the untreated

cultures.

Figure 3. Transcriptional down-regulation of B-Myb and E2F1 by Nutlin-3 also in fibroblasts

and endothelial cells. Primary fibroblasts and endothelial cells were exposed to Nutlin-3 (10 μM).

In A, levels of B-Myb and E2F1 mRNA were analyzed by quantitative RT-PCR. Results are

expressed as fold of B-Myb and E2F1 modulation by Nutlin-3, after 24 hours of treatment, with

respect to the control untreated cultures set to 1 (hatched line). Data are reported as means+SD of

results from at least three experiments, each performed in triplicate. In parallel, E2F1 protein levels

were analyzed by Western blot. Tubulin staining is shown as loading control. Representative

examples of Western blot results, out of three independent experiments, are shown. In B,

representative cell-cycle profiles of cells, either left untreated or treated with Nutlin-3, analyzed by

flow cytometry are shown. In each panel, the rectangle represents the cells in S phase of the cell

cycle, which have incorporated BrdU. In C, fibroblasts were transfected with either control

scrambled (scr.) siRNA or B-Myb siRNA. Efficiency of transfection was monitored by

immunofluorescence microscopy and flow cytometry, by using GFP-constructs, while efficiency of

B-Myb knockdown was documented by analyzing levels of B-Myb mRNA assessed by quantitative

RT-PCR. Levels of B-Myb mRNA were expressed as fold of modulation with respect to the




CCR-10-3244.R1

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scrambled control transfected cultures. At 48 hours post transfection, distribution of cells in the

different phases of the cell cycle was calculated from flow cytometry dot plots after BrdU/PI

staining, and expressed as percentage of the total population. Data are reported as means+SD of

results from three independent experiments. Asterisks, p<0.05 with respect to the untreated cultures

or to the scrambled control transfected cultures.

Figure 4. Inverse correlation between Nutlin-induced up-regulation of miR-34a and down-

regulation of B-Myb and E2F1. In A, prediction of hsa-mir-34a binding site in the 3’ UTR of B-

Myb gene. In B, modulation of miR-34a mRNA by Nutlin-3 was analyzed by quantitative RT-PCR

in the indicated cell lines as well as in fibroblasts (Fibrobl.) and endothelial (Endoth.) cells. In C,

the p53wt EHEB cell line was either left untreated or exposed to Nutlin-3 (10 μM). At the indicated

time points, levels of E2F1 and B-Myb and miR-34a were simultaneously assessed by RT-PCR,

and (D) distribution of cells in the different phases of the cell cycle was calculated from the flow

cytometry dot plots after BrdU/PI staining, and expressed as the percentage of the total population.

In C, levels of E2F1, B-Myb and miR-34a were expressed as fold of modulation with respect to the

control untreated cultures (time=0). Data are reported as means+SD of results from three

independent experiments. Asterisks, p<0.05 with respect to the untreated cultures.

Figure 5. Role of miR-34a in the down-regulation of B-Myb by Nutlin-3. Fibroblasts were

transfected with the either control scrambled (scr.) or the hsa-miR-34a precursor (mimic) or hsa-

miR-34a inhibitor (anti-miR) oligos. After transfections, up-modulation (A) and down-modulation

(C) of miR-34a were documented by quantitative RT-PCR and are expressed as folds of modulation

with respect to the scrambled control transfected cultures (set to 1). In A and B, cells transfected as

indicated were analyzed for levels of B-Myb, E2F1, p53 and p21 mRNA that were assessed in the

same samples by quantitative RT-PCR. In C, cells transfected as indicated were left untreated or

exposed to Nutlin-3 (10 µM) and analyzed for levels of B-Myb and E2F1 mRNA. Results were




CCR-10-3244.R1

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expressed as fold of B-Myb and E2F1 modulation in Nutlin-3 treated-cultures with respect to the

control untreated-cultures, which were set to 1 (hatched line). Data are reported as means+SD of

results from three independent experiments. Asterisks, p<0.05 with respect to scrambled control

transfected cultures.

Figure 6. Role of miR34a and E2F1 in B-Myb down-regulation. In A, Fibroblasts were

transfected with either control scrambled (scr.) siRNA or E2F1 siRNA. Efficiency of E2F1

knockdown was documented by analyzing levels of E2F1 mRNA and proteins, assessed by

quantitative RT-PCR and Western blot analysis respectively. Levels of E2F1 and B-Myb mRNA

were assessed in the same samples and are expressed as fold of modulation with respect to the

scrambled control transfected cultures. Data are reported as means+SD of results from three

independent experiments. Asterisks, p<0.05 with respect to scrambled control transfected cultures.

In B, endogenous levels of B-Myb and E2F1 mRNA in leukemic cells (primary cell samples plus

cell lines; n=18) were analyzed in relationship to the endogenous levels of miR-34a (lower and

higher that the median value). Horizontal bars are median, upper and lower edges of box are 75th

and 25th percentiles, lines extending from box are 10th and 90th percentiles. In C, scheme of

proposed mechanism for p53-regulation of E2F1 and B-Myb by miR-34a, upon Nutlin-3 treatment.




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Figure 6

C

Nutlin-3

C

p53 MDM2

miR-34a

+

B Myb

E2F1

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B-Myb promoter: 4 binding

E2F1 promoter: 5binding sites for p53

B-Myb sites for p53 and 2 for E2F1

Cell cycle progressionand apoptosis

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Published OnlineFirst March 2, 2011.Clin Cancer Res Giorgio Zauli, Rebecca Voltan, Maria Grazia di Iasio, et al. oncogenes in leukemic cellsmiR-34a induces the down-regulation of both E2F1 and B-Myb

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