Expression and Role of the ErbB3-Binding Protein 1 in ... · Cancer Therapy: Preclinical Expression and Role of the ErbB3-Binding Protein 1 in Acute Myelogenous Leukemic Cells Le

Cancer Therapy: Preclinical

Expression and Role of the ErbB3-BindingProtein 1 in Acute Myelogenous Leukemic CellsLe Xuan Truong Nguyen, Li Zhu, Yunqin Lee, Lynn Ta, and Beverly S. Mitchell

Abstract

Purpose: The ErbB3-binding protein 1 (Ebp1) has been impli-cated in diverse cancers as having either oncogenic or tumorsuppressor activities. The present study was undertaken to deter-mine the effects of Ebp1 expression in AML cells and to determinethe mechanisms by which Ebp1 promotes cell proliferation inthese cells.

Experimental Design: The expression of Ebp1 was studied inmononuclear cells obtained from the peripheral blood of54 patients with AML by Western blot analysis. The effects ofEbp1 expression on proliferating cell nuclear antigen (PCNA)expression and cell proliferation was measured using Westernblot analysis, immunoprecipitation, in vitro ubiquitination,and colony-forming assays. The role of Ebp1 in promoting

rRNA synthesis and cell proliferation was evaluated by mea-suring the level of pre-rRNA and the recruitment of Pol I torDNA.

Results: Ebp1 is highly expressed in acute myelogenous leu-kemia (AML) cells and regulates the level of ribosomal RNA(rRNA) synthesis by binding to RNA Polymerase I (Pol I) andenhancing the formation of the Pol I initiation complex. Ebp1also increases the stability of PCNA protein by preventing itsinteraction with Mdm2, for which it is a substrate.

Conclusions: These results demonstrate an important role ofEbp1 in promoting cell proliferation in AML cells through theregulation of both rRNA synthesis and PCNA expression. ClinCancer Res; 22(13); 3320–7. �2016 AACR.

IntroductionErbB3-binding protein 1 (Ebp1), the human homologue of

the mouse protein p38-2G4, is ubiquitously expressed in avariety of normal and malignant human tissues and cell lines(1,2) and has been shown to play important roles in theregulation of cell proliferation and differentiation (2,3). Ebp1encodes two alternatively spliced isoforms, p48 and p42, thathave different functions (4). The p48 isoform is located pre-dominantly in the nucleolus and cytoplasm and promotes cellproliferation and cell survival (4–6), whereas the p42 isoformis predominantly cytoplasmic and inhibits cell growth (4,7).Ebp1 also interacts with a variety of other important regulatoryproteins that include Akt and Nucleophosmin 1 (NPM1; 8,9).

Ribosomal RNA synthesis is a prerequisite for cellular pro-liferation and is tightly regulated in response to diverse growthand inhibitory stimuli (reviewed in 10, 11). It requires therecruitment of RNA polymerase I (Pol I) and several transcrip-tion initiation factors including Transcription Initiation Factor I(TIF-IA), upstream binding factor (UBF), and the TATA-bindingprotein (TBP)-containing factor TIF-IB/SL1 to the ribosomalDNA (rDNA) promoter to generate a productive transcription

initiation complex. Although the p48 isoform of Ebp1 has beenlocalized to the nucleolus and shown to bind to RNA, it had notpreviously been known to participate in the increasingly com-plex process of regulating rRNA synthesis. We have recentlyshown that Ebp1 p48 binds to TIF-IA to regulate its intracellularlocalization and activity (12). Our present studies demonstratethat Ebp1 is also involved in the formation of the rRNAtranscription initiation complex, contributing to its ability toregulate rRNA synthesis.

Proliferating cell nuclear antigen (PCNA) is a ring-shapedheterotrimeric clamp that provides a platform for a variety ofproteins that are involved in DNA replication and repair and isfrequently increased in expression in malignant and cycling cells.PCNA binds to the E3 ligase HDM2 that regulates its turnoverduring the normal cell cycle (13, 14). Higher levels of PCNAexpression have been found to correlate with more proliferativedisease (15) and with a poorer response to treatment in acutemyeloid leukemias (16) and in myelodysplastic syndromes (17).

In investigating the role of Ebp1 in acute myelogenousleukemia (AML), we found that it is expressed at high levelsin the great majority of patient samples and that its expressioncorrelates with and is directly responsible for increased expres-sion of PCNA. In addition, Ebp1 expression is an importantdeterminant of rRNA synthesis in these cells, demonstratingthat it contributes to cell proliferation by two independent, butpotentially complementary pathways.

Materials and MethodsHuman patient samples

Mononuclear cells were isolated from the bone marrow orthe peripheral blood of patients obtained under IRB-approvedprotocols using density-gradient centrifugation with Ficoll–Paque Plus (GE Healthcare). Cells at the interface wereremoved and washed with PBS. Cell pellets were viably frozen

Departments of Medicine and Chemical and Systems Biology,Stanford Cancer Institute, Stanford University School of Medicine,Stanford, California.

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

Corresponding Author: Beverly S. Mitchell, Stanford University School ofMedicine, Lorry Lokey Building, 265 Campus Drive, Suite G2167, Stanford, CA94305-5456. Phone: 650-736-7716; Fax: 650-736-0607; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-15-2282

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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at �80 �C until use. All cultures were carried out in definedmedium as previously referenced (18). The patient character-istics of 13 samples (SU37 to SU49) are shown on Supple-mentary Table S3.

MLL-AF9 and MLL-AF10 mouse leukemia cellsC57BL/6 mice were obtained from Jackson Laboratory. All

studies were performed in accordance with protocols approvedby the Stanford University (Stanford, CA) Institutional AnimalCare and Use Committee. Mouse bone marrow cells were har-vested from leg bones and c-kitþ cells were purified by auto-MACS using anti-CD117 microbeads (Miltenyi Biotec). Purifiedcells were transduced with recombinant retroviruses made frompMSCV-neo constructs encoding MLL-AF9 and MLL-AF10, andplated inmethylcellulose media (M3231, Stemcell Technologies)containing SCF, IL3, IL6, andGM-CSF. Transformed cells (1�106)from methylcellulose cultures were transplanted intravenouslyinto lethally irradiated C57BL/6 mice (900 rads) with 2�105

syngeneic bone marrow cells (19). Mice were subsequently sacri-ficed and bonemarrow cells from control animals and fromMLL-AF9 and MLL-AF10 mice were collected for analysis by Westernblot analysis.

Synthetic siRNA oligonucleotidesThe siGENOME SMARTpool for si-RNA was purchased

from Thermo Scientific. Scrambled control RNA (SCR) was

Translational Relevance

Previous studies have indicated that Ebp1 plays a variety ofroles in human cancer cells that are dependent in part on thetype of cell in which it is expressed. Its role in acute leukemiahas not been investigated. The results of the present studyconfirm that Ebp1 is expressed at high levels in themajority ofAML clinical samples and further demonstrate that p48 Ebp1regulates two parameters pivotal to cell proliferation, rRNAsynthesis throughdirect interactionwith the Pol I transcriptioninitiation complex and PCNA expression through enhancingprotein stability. These previously unrecognized effects ofEbp1 raise the possibility that it could constitute a newtherapeutic target in the treatment of a subset of AML.

Figure 1.Relative expression of Ebp1 and PCNA in primary AML cells as compared with normal marrow mononuclear cells. Western blot analyses of cell lysate (30 mg)from 12 normal bone marrows and 54 AML samples were probed with anti-Ebp1, anti-PCNA, and anti-tubulin antibodies as indicated by the arrowhead.The bottom panels were performed with same loading of cell lysate (30 mg) on different blots.

Role of Ebp1 in AML Cells

www.aacrjournals.org Clin Cancer Res; 22(13) July 1, 2016 3321




used as a control. The target sequences for Ebp1 p48 siRNA areshown in the Supplementary Table S1.

ChIP assayChromatin immunoprecipitation (ChIP) was performed as

described by the manufacturer (Pierce). Precleared chromatinwas incubated overnight by rotation with 4 mg of Pol I antibodyor IgG antibody as a negative control. Inmunoprecipitates wereresuspended in 50 mL TE buffer. Inputs and immunoprecipitatedDNA samples were quantified by qPCR on a 7900T Fast real-time

PCR system (Applied Biosystems). Primers are listed in Supple-mentary Table S2.

RNA labeling and analysisThe cells were washed and incubated in phosphate-free DMEM

(Gibco) supplemented with 10%FBS for 2 hours followed by onehour label with 0.5 mCi [32P] orthophosphate (PerkinElmer).Total RNA was extracted with TRIzol (Life technology) followingthe manufacture's protocol. Equal amounts of RNA (10 mg) wereseparated on a 1.2% MOPS formaldehyde gel. The gel was driedand visualized by autoradiography.

Figure 2.Regulation of Mdm2-induced PCNA ubiquitination and degradation by Ebp1. A, effect of Ebp1 depletion on ubiquitination and expression of PCNAprotein in K562 leukemic cells. Cells were cotransfected with HA-Ub and SCR or siEbp1 (20 nmol/L) for 36 hours. Top, after treatment with MG132 (10mmol/L) for 8 hours, cell lysate was immunoprecipitated with anti-PCNA and immunoblotted with anti-HA antibodies; (bottom) cell lysate wasimmunoblotted with the antibodies shown. B, effects of Ebp1 overexpression on PCNA ubiquitination and protein expression in MEF cells (Mdm2þ/þ,p53�/�), (Mdm2�/�, p53�/�), and (Mdm2 C464, p53�/�). Cells were transfected with vector control or GFP-Ebp1 for 24 hours. The cells weretreated continuously with MG132 for 8 hours (top) or untreated (bottom) and the cell lysate was immunoprecipitated and immunoblotted as shown.C and D, effect of the level of Ebp1 expression on the interaction of PCNA with Mdm2 in leukemic cells. C, K562 cells were transfected with SCR orsiEbp1 (20 nmol/L) for 36 hours and the lysate was immunoprecipitated and immunoblotted as indicated. D, ten AML patient samples were divided into twogroups with low (n ¼ 5) and high (n ¼ 5) expression of Ebp1 based on the immunoblot analysis shown in Fig. 1. Left, the combined cell lysates wereimmunoprecipitated and immunoblotted as indicated; (right) the cells were treated with MG132 (10 mmol/L) for 8 hours and the combined lysateswere immunoblotted with anti-PCNA antibody. E, effect of Ebp1 overexpression on the interaction of Mdm2 with PCNA and on PCNA ubiquitinationin MEF Ebp1�/� cells. The MEF Ebp1�/� cells were transfected with vector control or GFP-Ebp1 (left) or cotransfected with HA-Ub and vector controlor GFP-Ebp1 (right) for 24 hours. The lysate was immunoprecipitated and immunoblotted with the antibodies shown.

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Statistical analysisWhere indicated, results were compared using the unpaired

Student t testwith values obtained fromat least three independentexperiments. P < 0.05 was considered significant.

ResultsEbp1 expression correlates with the PCNA expression in AMLcells

We compared the level of expression of Ebp1 protein infrozen normal mononuclear cells obtained from normal bonemarrow by Ficoll separation with that in mononuclear cellsobtained from the bone marrow or peripheral blood of 54patients with AML. The expression of Ebp1 protein was signif-icantly higher in the majority of AML samples (Fig. 1, secondlanes; Supplementary Fig. S1A) and was associated with anincrease in the expression of PCNA (Fig. 1, second and thirdlanes). Of note, however, a few SU samples expressed very littleEbp1 for unknown reasons and these samples had a corre-spondingly low level of PCNA expression (Fig. 1, second lanes;Supplementary Fig. S1A). There was also a relative increasein Ebp1 and PCNA expression in leukemic cells obtainedfrom two murine AML models in which either of twoMLL fusion proteins (MLL-AF9 or MLL-AF10) is expressed

(Supplementary Fig. S1B; ref. 26). These results further suggesta relationship between the expression of these two proteins.

Regulation of PCNA ubiquitination and degradation by Ebp1Previous studies have shown that p48 Ebp1 binds to and

stabilizes the HDM2 E3 ligase through Akt signaling (20).PCNA is known to be regulated by HDM2 and previous studieshave indicated that the interaction of a signaling protein, ERK8,with HDM2 prevents PCNA ubiquitination and degradation,thereby stabilizing PCNA levels (14). We therefore askedwhether the interaction of p48 with HDM2 provided an expla-nation for the association of Ebp1 expression with that ofPCNA. Depletion of Ebp1 in K562 leukemia cells using siRNAincreased the ubiquitination of PCNA and decreased PCNAexpression (Fig. 2A), whereas overexpression of Ebp1 in murineembryonic fibroblasts deficient in p53 (Mdm2þ/þ, p53�/�)inhibited PCNA ubiquitination, as well as its interaction withMdm2 (Fig. 2B, left). In contrast, there was no change in theubiquitination or expression of PCNA in the presence orabsence of exogenously expressed Ebp1 in MEFs that weredeficient in Mdm2 (Mdm2�/�, p53�/�) or expressed a cat-alytically inactive form of Mdm2 (Mdm2 C464, p53�/�;Fig. 2B, middle and right). K562 leukemic cells that were

Figure 3.Regulation of rRNA synthesis and cell proliferation by Ebp1. A, quantitative expression of (1) Ebp1 mRNA, (2) pre-rRNA synthesis, (3) Pol I occupancy of therDNA promoter, (4) OD absorbance by MTS assay, and (5) immunoblot analysis of Ebp1 and PCNA protein in bone marrow mononuclear cells from normalindividuals and from patients with AML. The bar indicates the average value and the ends of the whiskers represent minimum and maximum values.Significance was determined using the Student t test. B, effects of Ebp1 depletion on rRNA synthesis and cell proliferation in K562 leukemic cells. Panel 1,cells were transfected with SCR or siEbp1 (20 nmol/L) for 36 hours. RNA was extracted for measurement of 50ETS pre-rRNA synthesis and RNAlabeling with [32P]. Panel 2, recruitment of Pol I to the rDNA promoter; Panel 3, MTS and colony forming assays; Panel 4, Western blot analysis of K562cells depleted of Ebp1.






depleted of Ebp1 by siRNA (Fig. 2C) or primary AML cells withlow Ebp1 expression (Fig. 2D) demonstrated increased bindingof Mdm2 and PCNA (Fig. 2C and D, left lanes), an increase inPCNA ubiquitination (Fig. 2A, top and 2D, right), and adecrease in PCNA expression (Fig. 2C and D). The binding ofMdm2 with PCNA was also greater in MEF Ebp1�/� cells ascompared with MEF Ebp1þ/þ cells, whereas overexpression ofEbp1 reversed this interaction (Fig. 2E, left) and inhibitedPCNA ubiquitination (Fig. 2E, right). Collectively, these datashow that Ebp1 stabilizes PCNA by inhibiting its interactionwith and ubiquitination by Mdm2.

Regulation of rRNA transcription and cell proliferation byEbp1 in AML cells

The high level of Ebp1 expression in primary AML cells wasreflected in highly significant increases in the expression ofEbp1 mRNA, as well as in increased levels of rRNA synthesisand cell proliferation as determined by MTS assays (Fig. 3A,1–2, 4; Supplementary Fig. S2A). Depletion of endogenousEbp1 expression in K562 leukemic cells strongly inhibited bothPCNA expression and cell proliferation (Fig. 3B, 3 and 4;Supplementary Fig. S2B), suggesting that Ebp1 expression isintegral to both these processes. In addition, those leukemic

cells that expressed higher levels of Ebp1 demonstrated signif-icantly higher levels of rRNA synthesis as determined by thelevel of 50ETS pre-rRNA and Pol I recruitment to rDNA whencompared with normal mononuclear cells (Fig. 3A, 1–3),whereas reducing Ebp1 expression in K562 leukemic cellssignificantly reduced rRNA synthesis (Fig. 3B, left) and cellproliferation, as determined by colony-forming assays (Fig. 3B,third). Similar results demonstrating increased levels of Ebp1mRNA correlating with increased rRNA synthesis were obtainedfrom murine MLL-AF9 and MLL-AF10 leukemic cells (Supple-mentary Fig. S3). Depletion of Ebp1 in K562 leukemic cells alsoslightly increased cell death, as measured by Annexin V staining(Supplementary Fig. S2C). Further analysis of the cell-cycleresponse to Ebp1 depletion revealed a decrease in the numberof cells entering S phase and a corresponding increase in cells inG1 (Supplementary Fig. S2D).

To obtain additional data in primary AML cells, 24 AMLsamples were divided into low (n ¼ 8) and high Ebp1 (n ¼16) expression groups based on the Western blot analyses shownin Fig. 1. Those samples with higher expression of Ebp1 alsodisplayed higher levels of rRNA synthesis, PCNA expression, andcell proliferation (Fig. 4). Similarly, MEF Ebp1þ/þ cells hadhigher levels of rRNA synthesis and higher rates of proliferationthan did MEF Ebp1�/� cells, whereas overexpression of Ebp1 inMEF Ebp1 �/� cells significantly enhanced both rRNA synthesisand cell proliferation (Supplementary Fig. S4). Taken together,these data strongly suggest that the expression of Ebp1 plays animportant role in regulating both rRNA synthesis and cell prolif-eration in AML cells.

Interaction of Ebp1with the Pol I complex and enhancement ofthe formation of the Pol I transcription initiation complex

Previous studies have indicated that the p48 but not the p42isoform of Ebp1 can be found in the nucleolus as well as in thecytoplasm (4). Immunofluorescent and FISH staining of 293Tcells demonstrated colocalization of p48 Ebp1 with Pol I, UBF,and rDNA, as shown in densitometry scans (Fig. 5A and Supple-mentary Fig. S5). To determine more specifically whether Ebp1associates with the Pol I transcription initiation complex, Ebp1was immunoprecipitated from AML cell lysate and the samplesprobed for Pol I, UBF, and PAF53. Each of these proteins copre-cipitated with Ebp1 (Fig. 5B), strongly suggesting that Ebp1 isinvolved in the regulation of rRNA transcription. Decreasing theexpression of Ebp1 in K562 leukemic cells (Fig. 5C, left) or in theprimary AML cells with lesser expression of Ebp1 (Fig. 5C, right)decreased the binding of both UBF and TIF-IA to Pol I. MEFEbp1�/� cells also demonstrated less binding of TIF-IA and UBFto Pol I than did wild-type MEF cells, while reexpression of Ebp1increased the interaction of both proteins with Pol I (Fig. 5D).These results are consistent with the extent both of Pol I recruit-ment to rDNA and of rRNA synthesis in these cells (Supplemen-tary Fig. S4). These data demonstrate that p48 Ebp1 is a compo-nent of the Pol I initiation complex and suggest that Ebp1 plays anintegral and direct role in the regulation of rRNA synthesis. Aschema that demonstrates the effects of Ebp1 on both PCNAstability and rRNA synthesis is shown in Fig. 6.

DiscussionOur data show that Ebp1 is expressed at higher levels in AML

cells than in normal bone marrow mononuclear cells and that

Figure 4.Relationship between the level of Ebp1 expression, rRNA synthesis, andcell proliferation in primary AML cells. AML patient samples were dividedby Ebp1 expression levels relative to the average value for all samples intolow and high expression groups. Numbers of contributing samples areshown. A and B, RNA was extracted for measurement of 50ETS pre-rRNAand ChIP assay was performed to measure the level of Pol I recruited torDNA promoter; C and D, MTS assay and Western blot analysis.

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Figure 5.Interaction of Ebp1 with the Pol I transcription initiation complex. A, colocalization of p48 Ebp1 with Pol I, UBF, and rDNA. 293T cells were transfected with GFP- p48Ebp1 for 24 hours. Left, cells were immunostained with anti-Pol I (top) and anti-UBF (middle) antibodies. rDNA was labeled with an rDNA probe (bottom).Fluorescence intensitywasmeasured along the line shown. Scale bar, 5mm. B, interaction of Ebp1with Pol I, UBF, and PAF53 in AML cells. Lysate from combinedAMLcells (n ¼ 10) was immunoprecipitated with anti-IgG or anti-Ebp1 antibody and immunoblotted with anti-Pol I (top), anti-UBF (middle), or anti-PAF53 (bottom)antibodies, as shown. C, effects of Ebp1 expression onUBFandTIF-IAbinding to Pol I inAML cells. Left, K562 cellswere transfectedwith SCRor siEbp1 (20nmol/L) for36 hours; Right, 24 AML patient samples were divided into groups with low (n ¼ 8) and high (n ¼ 16) expression of Ebp1. Combined cell lysate wasimmunoprecipitatedwith anti-UBF (first lanes) or anti-TIF-IA (second lanes) antibody and immunoblottedwith anti-Pol I antibody. D, effects of Ebp1 overexpressionon UBF and TIF-IA binding with Pol I in MEF Ebp1�/� cells. Lysates from MEF Ebp1 þ/þ cells, GFP-control and GFP-Ebp1-transfected MEF Ebp1�/� cells wereimmunoprecipitated with anti-UBF (left) or anti-TIF-IA (right) antibody and immunoblotted with anti-Pol I antibody.






cell proliferation depends on Ebp1 expression. In addition, andnot previously recognized, Ebp1 appears to be a component ofthe Pol I transcription initiation complex and to facilitate rRNAsynthesis.

The regulation of rRNA gene transcription is central toribosome production and therefore to cell growth and prolif-eration and the growth-dependent regulation of rRNA synthesisis evolutionarily conserved (11). The synthesis of pre-ribosom-al RNA that reflects the overall level of rRNA transcription isincreased in various human cancers and indeed has beenconsidered as one of the hallmarks of cancer (reviewed inref. 21). The transcription of pre-ribosomal rRNA by Pol Irequires the formation of a preinitiation complex containingthe promoter selectivity factor (SL1) complex, UBF, and TIF-IA.There are many oncogenic events including protein overexpres-sion, mutations, and translocation caused by the alterations ofcellular signaling pathways such as PI3K/Akt/mTOR, Myc, andRb that directly act on the proteins essential for rRNA tran-scription. For example, activated Akt enhances rRNA synthesisand consequently promotes cell proliferation in AML cellsthrough the stabilization and activation of TIF-IA (22–25). Thedata reported here support the concept that Ebp1 is one of thekey players in the regulation of rDNA transcription through itsinteraction with the Pol I initiation complex. This finding issupported by the presence of p48 Ebp1 in the nucleolus, whereEbp1 has been shown to bind to a number of RNAs includingmRNA, rRNA, and ribosomal subunits (2, 25). Ebp1 is also acomponent of ribonucleoprotein complexes and associateswith double-stranded RNA (2). In addition, Ebp1 interactswith the nucleolar protein nucleophosmin 1 (NPM1) andcontributes to NPM1-regulated ribosome biogenesis (9). Thesedata indicate that Ebp1 plays an important and multifactorialrole in the nucleolus.

p48 Ebp1 is the predominant form of Ebp1 expressed in manycell types (4–6,8). Western blot analyses of AML cells using anantibody that also detects the p42 isoform show only a single

band of 48 kDa. The use of an Ebp1 siRNA construct that targetstheN-terminal sequence that is specific for the p48 isoform largelydepletes Ebp1 protein, further suggesting that p48 accounts forthe great majority of Ebp1 expression in AML cells. A number ofstudies have indicated that p48 may play an important role inoncogenesis. For example, p48 Ebp1 interacts with nuclear PKB/Akt to prevent apoptotic cell death in neuronal cells (8). Highexpression of Ebp1 in breast cancers predicts a poor clinicaloutcome, whereas tamoxifen treatment of MCF-7 breast cancercells markedly decreases both the transcription and proteinexpression of p48 Ebp1 (26). It has also been shown recentlythat the phosphorylation of p48 Ebp1 by CDK2 is required for itstumorigenic function (6). Although further studies are needed toprove that p48 Ebp1 is an oncogenic protein, the findings to datestrongly suggest it has tumor-promoting activities. The correlationof Ebp1 with PCNA expression and proliferation support apotential role for Ebp1 in the progression of AML. However, wehave not found an association between the level of Ebp1 geneexpression and overall survival in published databases from AMLpatients.

Previous studies have shown that p48 Ebp1 binds to andstabilizes Hdm2 (20) and enhances Hdm2-induced p53 deg-radation (5). In contrast, our results indicate that Ebp1 expres-sion reduces the ubiquitination and degradation of PCNA byMdm2 (Fig. 2). This study also suggests that the increase inPCNA protein induced by Ebp1 is independent of the mech-anism by which Ebp1 downregulates p53 (5). By using MEFcells deficient in p53 or in both Mdm2 and p53, we havedemonstrated that Ebp1 enhances the interaction of Mdm2with PCNA in the absence of p53, indicating that p53 is notrelevant to this interaction. Previous studies have similarlydemonstrated that the chromatin-bound kinase ERK8 actsindependently from p53 in preventing Hdm2-induced PCNAdegradation (14). Although it remains possible that additionalfactors are involved in these interactions, the data obtained todate indicate that Ebp1 differentially regulates the expression of

Figure 6.Schematic model showing therelationship of p48 Ebp1 to rRNAsynthesis and PCNA expression basedon the data shown.


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p53 and PCNA through Mdm2. Although technically difficult,the direct relationship between Ebp1 expression, PCNA expres-sion, and cell proliferation could be further confirmed bydepleting Ebp1 in cells overexpressing PCNA. Nonetheless, thepresent data strongly support a role of p48 Ebp1 as a promoterof cell proliferation and, potentially, as an enhancer of suscep-tibility to cellular transformation.

In summary, Ebp1 plays an important and previously unrec-ognized role in regulating rRNA synthesis and cell proliferationin AML cells. These results provide preliminary evidence thatEbp1 might constitute an additional therapeutic target forthe treatment of AML.

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

Authors' ContributionsConception and design: L.X. Truong Nguyen, B.S. MitchellDevelopment of methodology: L.X. Truong Nguyen, L. Ta

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Zhu, Y. LeeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.X. Truong Nguyen,Writing, review, and/or revision of the manuscript: L.X. Truong Nguyen,L. Zhu, B.S. MitchellAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. ZhuStudy supervision: B.S. Mitchell

Grant SupportThis work was supported by a translational research grant and by a SCOR

award from the Leukemia and Lymphoma Society.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 18, 2015; revisedDecember 7, 2015; acceptedDecember21, 2015; published OnlineFirst January 26, 2016.

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2016;22:3320-3327. Published OnlineFirst January 26, 2016.Clin Cancer Res Le Xuan Truong Nguyen, Li Zhu, Yunqin Lee, et al. Myelogenous Leukemic CellsExpression and Role of the ErbB3-Binding Protein 1 in Acute

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