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2014;74:3043-3053.Published OnlineFirst March 31,2014.Cancer Res Phyllis S.Y.Chong,Jianbiao Zhou,Lip-Lee Cheong,et al.in Acute Myelogenous LeukemiaLEO1 Is Regulated by PRL-3 and Mediates Its Oncogenic Properties Updated version 10.1158/0008-5472.CAN-13-2321doi:Access the most recent version of this article at:MaterialSupplementary http:/cancerres.aacrjournals.org/content/suppl/2014/04/01/0008-5472.CAN-13-2321.DC1.htmlAccess the most recent supplemental material at:Cited Articles http:/cancerres.aacrjournals.org/content/74/11/3043.full.html#ref-list-1This article cites by 32 articles,14 of which you can access for free at:E-mail alerts related to this article or journal.Sign up to receive free email-alerts SubscriptionsReprints and .pubsaacr.orgTo order reprints of this article or to subscribe to the journal,contact the AACR Publications Department at Permissions .permissionsaacr.orgTo request permission to re-use all or part of this article,contact the AACR Publications Department aton September 23,2014.2014 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 31,2014;DOI:10.1158/0008-5472.CAN-13-2321 on September 23,2014.2014 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 31,2014;DOI:10.1158/0008-5472.CAN-13-2321 Molecular and Cellular PathobiologyLEO1 Is Regulated by PRL-3 and Mediates Its OncogenicProperties in Acute Myelogenous LeukemiaPhyllis S.Y.Chong1,Jianbiao Zhou1,Lip-Lee Cheong2,Shaw-Cheng Liu1,Jingru Qian3,Tiannan Guo3,Siu Kwan Sze3,Qi Zeng4,and Wee Joo Chng1,2,5AbstractPRL-3,an oncogenic dual-specificity phosphatase,is overexpressed in 50%of acute myelogenous leukemia(AML)and associated with poor survival.We found that stable expression of PRL-3 confers cytokineindependence and growth advantage of AML cells.However,how PRL-3 mediates these functions in AML isnot known.To comprehensively screen for PRL3-regulated proteins in AML,we performed SILAC-basedquantitative proteomics analysis and discovered 398 significantly perturbed proteins after PRL-3 over-expression.We show that Leo1,a component of RNA polymerase IIassociated factor(PAF)complex,is anovel and important mediator of PRL-3 oncogenic activities in AML.We described a novel mechanism whereelevated PRL-3 protein increases JMJD2C histone demethylase occupancy on Leo1 promoter,therebyreducing the H3K9me3 repressive signals and promoting Leo1 gene expression.Furthermore,PRL-3 andLeo1 levels were positively associated in AML patient samples(N 24;P 0.01).On the other hand,inhibitionof Leo1 reverses PRL-3 oncogenic phenotypes in AML.Loss of Leo1 leads to destabilization of the PAFcomplex and downregulation of SOX2 and SOX4,potent oncogenes in myeloid transformation.In conclusion,we identify an important and novel mechanism by which PRL-3 mediates its oncogenic function in AML.Cancer Res;74(11);304353.?2014 AACR.IntroductionPRL-3,encoded by the PTP4A3 gene,is a small dual-specificity phosphatase characterized by the conservedC(X5)R catalytic domain,and a unique C-terminal prenyla-tion domain essential for its proper subcellular localization(1,2).PRL-3 has been shown to promote cellular processes,such as cell motility,invasion,cell growth,and survival,through various mechanisms(27).PRL-3 was first linked tocancer when it was consistently found at elevated levels incolorectal cancer metastases,but at much lower levels inmatched early-staged tumor and normal colorectal epithe-lium(8).Since then,elevated expression of PRL-3 has beenimplicated in the progression and metastasis of an array ofcancer types,including gastric,ovarian,cervical,lung,liver,and breast,and PRL-3 protein was overexpressed in anaverage of 22.3%of 1,008 human carcinoma samples exam-ined using immunohistochemistry(9,10).Together with thefact that it has a highly restricted basal pattern of expressionin adult tissues(11),PRL-3 is deemed as an attractivetherapeutic target that spares normal tissues.The potentialof this target has been demonstrated using PRL-3specificantibodies in an in vivo model(12).In recent years,accumulating evidence suggests that PRL-3is also a novel therapeutic target and biomarker in leukemia(13,14).WewerethefirsttoreportthatelevatedPRL-3proteinexpression occurs in about 47%of human acute myelogenousleukemia(AML)cases while absent from normal myeloid cellsinbone marrow(13).Inaddition,alarge-scalegene expressionprofiling study of 454 primary AML samples demonstratesthat high PRL-3 levels is an independent negative prognosticfactor in AML,both for overall survival and event-free survival(14).These reports collectively suggest that PRL-3 may be ofbiologic and clinical relevance in AML and warrants furtherinvestigation.In the present study,we created a TF-1 AML cell line stablyoverexpressing PRL-3(TF1-hPRL3)as a model to study thebiologic relevance of PRL-3 in AML,and we use a quantitativeproteomic strategy to profile on a global scale changes inprotein expression induced by PRL-3 in AML.We found thatPRL-3 has pro-oncogenic properties in AML,and Leo1,acomponentofthehumanRNApolymeraseIIassociatedfactor(PAF)complex,is one of the most differentially expressedproteins induced by PRL-3.Further,we showed that PRL-3Authors Affiliations:1Cancer Science Institute of Singapore;2Depart-ment of Medicine,Yong Loo Lin School of Medicine,National University ofSingapore;3Department of Biological Sciences,Nanyang TechnologicalUniversity;4Institute of Molecular and Cell Biology(A?STAR);and5Depart-ment of Haematology-Oncology,National University Cancer Institute ofSingapore,National University Health System,SingaporeNote:Supplementary data for this article are available at Cancer ResearchOnline(http:/cancerres.aacrjournals.org/).P.S.Y.Chong and J.Zhou contributed equally to this work.Corresponding Author:Wee-Joo Chng,National University Health Sys-tem,1EKentRidgeRoad,NUHSTowerBlock,Level10,Singapore119228.Phone:65-6772-4612;Fax:65-6777-5545;Email:mdccwjnus.edu.sgdoi:10.1158/0008-5472.CAN-13-2321?2014 American Association for Cancer Research.CancerResearchwww.aacrjournals.org3043on September 23,2014.2014 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 31,2014;DOI:10.1158/0008-5472.CAN-13-2321 upregulates Leo1 through a novel epigenetic mechanism.Onthe other hand,knockdown of Leo1 significantly diminishesthe oncogenic effects of PRL-3.Our current work implicates apro-oncogenic role of PRL-3 in AML,and reveals Leo1 as anovel downstream molecule required for PRL-3 oncogenicfunction in leukemia.Materials and MethodsCell cultureHEK293T cells were cultured in Dulbeccos Modified EagleMediumwith10%fetalcalfserum(FCS).TF1-derivedcelllineswereculturedinRPMI1640mediumcontaining10%FCS(R10)supplemented with 5 ng/mL human interleukin(IL)-3(Milte-nyi Biotec).Molm-14,HEL,and HL-60 were cultured in R10.HumanCD34cellsweregrowninStemSpanSFEMIImediumsupplemented with StemSpan CC100 cytokine cocktail(Stem-Cell Technologies).Primary AML cells were grown in sameconditions,with the addition of granulocyte macrophagecolony-stimulating factor(20 ng/mL).Cell lines were obtainedfrom American Type Culture Collection and authenticated.Plasmid details are available in Supplementary Methods.SILAC-based mass spectrometryTF-1 wascultured inlight stable isotope labeling by aminoacids in cell culture(SILAC)medium containing normal lysineand arginine amino acids,whereas the TF1-hPRL3 was grownin heavy SILAC medium with stable isotope-labeled13C6lysine(6-Da shift)and13C615N4arginine(10-Da shift;Thermo Scientific).The cellular lysates were combined andproteolytically digested by trypsin followed by tandem massspectrometry(MS)identification.Peptides were subjected totarget-decoy database search strategy with a false discoveryrate(FDR)of less than 1%(1.5-fold;Supple-mentary Fig.S1B).To gain a functional understanding of the dataset,wesubjected the differentially expressed proteins(up-or down-regulated)to gene ontology analysis.Upregulated proteins areenriched for processes such as nucleic acid metabolism(19.7%),protein metabolism(15.1%),and pre-mRNA proces-sing(5.7%),whichare novel biologicprocesses associated withBright fieldGFPTF1-pEGFPTF1-hPRL314121086420*TF1-EGFPTF1-EGFPTF1-EGFPTF1-hPRL3TF1-hPRL3TF1-pEGFPTF1-hPRL3TF1-hPRL3Relative expressionTF1-EGFPTF1-hPRL3PRL-3b-Actin120100806040200Viable cells(%)Day 0Day 2Day 4Day 66050403020100Cell number(105)Without additional cytokine250200150100500Number of colonies(/well)1,4001,2001,0008006004002000Tumor volume(mm3)*ADBECFigure 1.Establishment of stable PRL-3 cell line.A,images of TF1-pEGFP and TF1-hPRL3 cells in bright field and GFP channel under an invertmicroscopy.B,qRT-PCR and Western blot analysis of PRL-3 mRNA and protein expression in TF1-pEGFP and TF1-hPRL3 cells.b-Actin was used as aloading control in the protein analysis.C,after IL-3 starvation,TF1-pEGFP and TF1-hPRL3 cells were stained and assessed with Trypan BlueExclusion methods on a hemacytometer.The percentages of viable cells at 72 hours were calculated relative to before IL-3 withdrawal(0 hour).Toconstruct the cell growth curve,20?105TF1-hPRL3 cells were initially seeded in standard medium without additional human cytokines.Cells werecounted and reseeded in fresh medium every 2 days for up to 6 days.In both figures,three independent replicates were conducted(mean?SD).D,colony-forming assay of TF1-pEGFP and TF1-hPRL3.The experiments were duplicated and representative pictures are presented.E,mouse xenograftmodels of TF1-pEGFP and TF1-hPRL3.Five million TF1-pEGFP and TF1-hPRL3 cells were subcutaneously injected into loose skin between theshoulder blades and the left and right front leg of NOD/SCID recipient mice,respectively.At 4 weeks after injection,tumors were harvested andmeasurement of tumor volume was taken.The tumor volume is shown as the mean?SD(mm3).Arrows,the TF1-hPRL3 tumor mass at right andno tumor at left(TF1-pEGFP cell injected site)in one mouse.?,P 0.005.LEO1 Mediates PRL-3 Oncogenic Function in AMLwww.aacrjournals.orgCancer Res;74(11)June 1,20143045on September 23,2014.2014 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 31,2014;DOI:10.1158/0008-5472.CAN-13-2321 PRL-3,as well as known involvement in cell-cycle regulation(7.1%)andcell motility(6%)asdepicted in Supplementary Fig.S1C.Amongst the differentially regulated proteins,45.5%ofthem are nuclear proteins,whereas 54.5%are cytoplasmicproteins(Supplementary Fig.S1D).Validation of MS resultsExpression of several representative proteins,includingLeo1,stathmin,HSP-90,hnRNPE1,and HDAC2,was com-pared between TF1-pEGFP and TF1-hPRL3 cells(Fig.2A),and concordance was observed between the Western blotand MS results.Unexpectedly,we did not detect PRL-3 inour liquid chromatography/tandem mass spectrometry(LC/MS-MS)result.This is likely due to the trypsin cleavagesites distribution in the PRL-3 that generates peptideseither too long or too short for detection by LC-MS/MS.Nonetheless,PRL-3 overexpression was confirmed at boththe mRNA(data not shown)and protein levels by Westernblot(Fig.2A).We further performed lentiviral-based delivery of PRL-3shRNA into two additional human AML cells,Molm-14 andHEL,which expresses high endogenous level of PRL-3.The knockdown of PRL-3 reduced the levels of Leo1,stathmin,HSP90,and hnRNPE1(Fig.2B).An exception wasHDAC2,where although MS identifies it as one of theupregulated proteins by PRL-3,Western blot was not sen-sitive enough to detect the differences(Fig.2B).Theseexperiments confirmed the general validity of our proteo-mic analysis.Leo1 is upregulated by PRL-3Leo1,a component of the human PAF complex,was thetop candidate from the SILAC LC-MS/MS analysis with themost significant increase in protein abundance.We there-fore decided to focus on this protein in our subsequentexperiments.To confirm that Leo1 is a downstream target ofPRL-3,we introduced a Tet-On myc-tagged PRL-3 constructinto 293T cells.Forty-eight hours after transfection,293Tcells were exposed to increasing concentrations of thetetracycline derivative,doxycycline,to induce PRL-3 expres-sion.PRL-3 protein accumulated in a dose-dependent man-ner,and a corresponding increase in Leo1 protein levels wasobserved(Fig.2C)and quantified(Supplementary Fig.S2).To complement the overexpression studies,we also per-formed knockdown by transfecting PRL-3 siRNA into 293Tcells.The degree of PRL-3 inhibition correlated to the dose ofsiRNA added,and Leo1 was downregulated(Fig.2D).Col-lectively,these observations corroborated the MS resultsshowing that Leo1 expression is positively correlated withPRL-3 levels.TF1TF1-PRL3Molm-14Molm14-PRL3 KDHEL-PRL3 KDHELLeo1Leo1Leo1Leo1StathminStathminHSP-90HSP-90hnRNPE1hnRNPE1HDAC2HDAC2PRL-3PRL-3PRL-3b-Actinb-Actinb-ActinActinEmp0220(mg/mL Dox)Myc(PRL3)Mock1.4 mg siRNA2.8 mg siRNA0.7 mg siRNA0.3 mg siRNAABCDFigure 2.Western blot validation ofcandidate proteins identified byMS.A,expression of severalrepresentative upregulatedproteins is compared betweenTF-1 and TF1-hPRL3.B,knockdown of endogenous PRL-3inAMLcelllinesMolm-14andHEL.The same panel of proteins isexamined by Western blot.C,293Tcells were stably transfected withan inducible Tet-on myc-taggedPRL-3 construct,and increasingconcentration of doxycyclin wasused to induce PRL-3 expression.D,293T cells were transfected withspecifieddosesof PRL-3siRNA for48 hours before analysis withWestern blot.Chong et al.Cancer Res;74(11)June 1,2014Cancer Research3046on September 23,2014.2014 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 31,2014;DOI:10.1158/0008-5472.CAN-13-2321 Abrogation of Leo1 reduces PRL3-mediatedproliferation and cytokine independence of AMLTo determine whether Leo1 contributes to PRL3-inducedleukemic growth,we used lentiviral-based delivery of shRNAsto deplete Leo1 expression and looked at growth properties.qRT-PCR and immunoblotting analysis demonstrated a 5-foldreduction in Leo1 mRNA levels and a marked reduction in theLeo1protein levels upon treatmentwith Leo1shRNA(Fig.3A).Notably,reductioninLeo1levelssignificantlyimpairedgrowthof TF1-hPRL3 cells to growth rates comparable with TF-1parental cells(Fig.3B).To determine whether loss of Leo1 affects the survival ofTF1-hPRL3,we plated the cells in the absence of human IL-3 for 72 hours,followed by flow cytometric analysis forapoptotic markers Annexin V-phycoerythrin and 7-ami-noactinomycin D(7-AAD).Although approximately 90%of TF1-hPRL3 population was able to survive withoutsupplementation of cytokine,Leo1 knockdown notablyincreased the apoptosis of TF1-hPRL3 induced by cytokinewithdrawal(Fig.3C),indicating that Leo1 abrogationremoves the protective effect of PRL-3 toward cytokinedeprivation.Leo1 knockdown in PRL-3 cells resulted insignificantly fewer colonies,indicating a loss of clonogenicsurvival of these cells(Fig.3D).These