utx and mll4 coordinately regulate transcriptional ......reversed by histone lysine demethylases...

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Molecular and Cellular Pathobiology

UTX and MLL4 Coordinately Regulate TranscriptionalPrograms for Cell Proliferation and Invasiveness in BreastCancer Cells

Jae-Hwan Kim1, Amrish Sharma1, Shilpa S. Dhar1, Sung-Hun Lee1, Bingnan Gu1, Chia-Hsin Chan1,Hui-Kuan Lin1,2,3,4, and Min Gyu Lee1,2

AbstractHistone methyltransferases and demethylases reversibly modulate histone lysine methylation, which is

considered a key epigenetic mark associated with gene regulation. Recently, aberrant regulation of geneexpression by histone methylation modifiers has emerged as an important mechanism for tumorigenesis.However, it remains largely unknown how histone methyltransferases and demethylases coregulate transcrip-tional profiles for cancer cell characteristics. Here, we show that in breast cancer cells, the histone H3 lysine 27(H3K27) demethylase UTX (also known as KDM6A) positively regulates gene expression programs associatedwith cell proliferation and invasion. The majority of UTX-controlled genes, including a cohort of oncogenes andprometastatic genes, are coregulated by the H3K4methyltransferase mixed lineage leukemia 4 (MLL4, also calledALR, KMT2D, and MLL2). UTX interacted with a C-terminal region of MLL4. UTX knockdown resulted insignificant decreases in the proliferation and invasiveness of breast cancer cells in vitro and in a mouse xenograftmodel. Such defective cellular characteristics of UTX-depleted cells were phenocopied byMLL4 knockdown cells.UTX-catalyzed demethylation of trimethylated H3K27 and MLL4-mediated trimethylation at H3K4 occurredinterdependently at cotarget genes of UTX andMLL4. Clinically, high levels of UTX orMLL4 were associated withpoor prognosis in patientswith breast cancer. Taken together, these findings uncover that coordinated regulationof gene expression programs by a histone methyltransferase and a histone demethylase is coupled to theproliferation and invasion of breast cancer cells. Cancer Res; 74(6); 1705–17. �2014 AACR.

IntroductionHistone lysine methylation is central to epigenetic regula-

tion of gene expression at the genome-wide level (1, 2).Methylated histone H3 lysine 27 (H3K27) and H3K4 are amongthe well-characterized histone methylation marks. MethylatedH3K27 is associated with gene repression, whereas methylatedH3K4 is linked to gene activation or poised states (1, 3–6).Histone lysine methylation, which is present in mono-, di-, ortrimethylated states at specific lysine residues, is catalyzed byhistone methyltransferases (7, 8). For example, H3K4 methyl-

ation is generated by several specific H3K4methyltransferases,such as mixed lineage leukemia (MLL) 1 to 5 and SET1A/B (3),whereas H3K27 methylation is catalyzed by EZH1- or EZH2-containing complexes. Histone lysine methylation can bereversed by histone lysine demethylases (KDM; refs. 9, 10).For instance, H3K4 methylation is removed by several H3K4demethylases (e.g., LSD1/2 and JARID1a-d), whereas H3K27methylation is demethylated by UTX (also known as KDM6A)and JMJD3 (also called KDM6B; ref. 10).

Recent studies have shown that aberrations in histone lysinemethylation may be clinically relevant to breast cancer, whichis themost commonmalignancy in women. For example, it hasbeen shown that global levels of trimethylated H3K27(H3K27me3) are decreased in many breast tumors and thatthese altered levels correlate with poor prognosis (11). Inaddition, low H3K4me2 levels may be associated with poorpatient survival (12).

It also has become evident that dysregulation of histonemethylation modifiers may be important for breast cancerphenotypes. The H3K27 methyltransferase EZH2 is signifi-cantly overexpressed in breast tumors, and high levels ofEZH2 have been shown to be associated with poor progno-ses of breast cancer, including inflammatory breast cancer(13, 14). In addition, EZH2 expression in benign breastsamples may be linked to high risk for breast cancer (15,16). Mechanistically, EZH2 may promote cancer progression

Authors' Affiliations: 1Department of Molecular & Cellular Oncology, TheUniversity of Texas MD Anderson Cancer Center; 2Cancer Biology Pro-gram, Graduate School of Biomedical Sciences, The University of TexasHealth Science Center at Houston, Houston, Texas; 3Graduate Institute ofBasic Medical Science, China Medical University, Taichung; and 4Depart-ment of Biotechnology, Asia University, Taichung, Taiwan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

J.-H. Kim and A. Sharma contributed equally to this work.

Corresponding Author:Min Gyu Lee, Department of Molecular & CellularOncology, The University of Texas MD Anderson Cancer Center, 1515HolcombeBlvd., Houston, TX 77030.Phone: 713-792-3678; Fax: 713-794-3270; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-1896

�2014 American Association for Cancer Research.

CancerResearch

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by repressing 2 key cellular senescence genes, p14ARF andp16INK4A (17), and may increase cancer cell invasion bytranscriptionally repressing the metastasis suppressor RKIP(18). Recent studies have indicated that the oncogenicfunction of EZH2 may be antagonized by the H3K27demethylase JMJD3, which de-represses p14ARF and p16INK4A

genes (19). Interestingly, it has been shown that the histoneH3K9 and H3K36 demethylase JMJD2C (alias GASC1) is apredictive marker in invasive breast cancer and is geneamplified in breast cancer samples (20, 21).

The gene encoding the H3K27 demethylase UTX oftenundergoes somatic loss-of-function mutations in multiplecancer types, such as medulloblastoma, renal carcinoma,bladder cancer, leukemia, and prostate tumors (reviewed inref. 22). In addition, it has been shown that in normalfibroblast cells, UTX transcriptionally activates Rb pathwaygenes to suppress cell growth (23). Therefore, UTX has beenthought to be a tumor suppressor in these types of tumors.In contrast, it has been reported that in breast tumors, UTXmay be overexpressed (24) and is rarely mutated (25). Tobetter understand the role of UTX in breast cancer, wesought to determine (i) the effect of UTX on breast cancercell behaviors, such as cell proliferation and invasion; (ii)which transcriptional programs are modulated by UTX; and(iii) how UTX regulates transcriptional programs in breastcancer cells.

In this study, we found that UTX contributes to cellularproliferation and invasiveness by activating oncogenic geneexpression programs in breast cancer cells. Expression of mostUTX-modulated genes was also regulated by the H3K4methyl-transferase MLL4 (also called ALR, KMT2D, and MLL2), ofwhich C-terminal region interacts with UTX. UTX-depletedcells and MLL4-depleted cells had similar defects in theproliferation and invasion of breast cancer cells. Moreover,our results indicate that UTX and MLL4 upregulate geneexpression by decreasing H3K27me3 levels and in parallelincreasing H3K4me3 levels at their cotarget genes in a coor-dinated manner. These findings suggest that UTX and MLL4cooperatively regulate gene expression programs for cell pro-liferation and invasiveness in breast cancer cells.

Materials and MethodsCell culture

The HMLE, MCF-10A, MCF-7, MDA-MB-231, MDA-MB-468,BT-474, BT-549, SUM-159, Hs578T, T-47D, 293T, andHeLa cellswere fromAmerican Type Culture Collection (ATCC) andweremaintained in low passage (<15) for this study. ATCC validatescell lines by short tandem repeat profiles that are generated bysimultaneous amplification of multiple short tandem repeatloci and amelogenin (for gender identification). The cell lines,with the exception of HMLE and MCF-10A, were cultured inDulbecco's Modified Eagle Medium (DMEM; Thermo Scien-tific) containing 10% FBS and penicillin and streptomycin(Mediatech) at 37�C in a humidified atmosphere of 5% CO2.HMLE and MCF-10A cell lines were cultured in defined mam-mary epithelial growth media (CC-2571, MEGM Bullet Kit;Lonza, Inc.) supplemented with bovine pituitary extracts andepidermal growth factor.

Plasmids, reagents, and antibodiesThe cDNA construct encoding a C-terminus (Flag-MLL4-C)

of MLL4 (GenBank protein accession no. NP_003473.3) and itsdeletion constructs [i.e., Flag-MLL4-C (N) and Flag-MLL4-C(C)] were cloned into the expression vector pFLAG-CMV2. TheshUTX6-resistant UTX and shUTX6-resistant UTX catalyticmutant (mUTX) were generated using pFLAG-CMV2-UTX(ref. 26 and Supplementary Table S1). The anti-UTX antibodywas previously described (26). Anti-MLL4/MLL2-specific anti-body (AP6183a) was from Abgent. Anti-Flag mouse monoclo-nal M2 (F3165) was from Sigma. Anti-RbBP5 (A300-109A) andanti-ASH2L (A300-107A) polyclonal antibodies were fromBethyl Laboratories. Anti-WDR5 polyclonal antibody (07-706) was from Millipore.

Cell invasion and proliferation assaysFor the cell invasion assay, the Boyden chamber assay with a

modification was performed. Cells (1� 105) in 200 mL of serumfree media containing 0.1% bovine serum albumin (BSA) wereseeded on the Matrigel-precoated membrane in the inserts.After 48 hours, cells that had invaded the Matrigel and migrat-ed to the other side of the membrane were fixed and stained.Cell numbers were counted or the relative intensities of stainedcells were quantified using ImageJ 1.46r. For cell proliferationassay, cells were seeded in triplicate at a density of 2� 104 cellsperwell in a 12-well plate andwere counted for 6 days at a 2-dayinterval. For all the assays, cells were infected with lentivirusescontaining small hairpin (sh) Luciferase (Luc), shUTX-6,shUTX-8, shMLL4-15, or shMLL4-18 (Supplementary Table S1).

Soft agar assaysFor soft agar assays, MDA-MB-231 cells (1 � 103) were

infected with lentiviruses containing shUTX-6, shUTX-8, orshLuc, resuspended in 1 mL of 0.35% (w/v) agar with DMEMcontaining 10% FBS, and overlaid onto a 0.5% (w/v) agarsolution in a well of a 6-well plate. Colonies were visible afteran incubation of 10 to 14 days and were stained with crystalviolet.

Mouse xenograft experimentsFemale athymic nu/nu mice were purchased from The

University of Texas MD Anderson Cancer Center. The careand use of all mice were approved by the Animal Care and UseCommittee at The University of Texas MD Anderson CancerCenter. Luciferase-expressing MDA-MB-231 cells (2 � 106)were treated with scrambled shRNA (shScrambled) orshUTX-6 for 72 hours. Each group of cells were injected intomice (n¼ 7) via the tail veins. The transplanted animalswere invivomonitored using an IVIS100 imaging system every 2weeks.After bioluminescence images were captured, their intensitieswere quantitated using Living Image 3.2 acquisition andanalysis software (Caliper Life Sciences). At the end of 8 weeks,lungs were collected from mouse and processed for hematox-ylin and eosin staining.

ImmunoprecipitationpFLAG-CMV2plasmids harboring cDNAs ofMLL4-C,MLL4-

C (N), or MLL4-C (C) were transiently transfected into human

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embryonic kidney 293T cells. Immunoprecipitation using anti-Flag resin was performed as previously described (26).

Gene expression analysis using microarraysRNA was extracted from virus-infected MDA-MB-231 cells

using RNeasy Mini Kit (Qiagen), and labeled cRNAs werehybridized to an oligonucleotide microarray consisting of54,675 gene probes (Affymetrix U133PPlus 2.0 GeneChip; SantaClara). RMAexpress was used to compute gene expressionsummary values. The Web-based Database for Annotation,Visualization, and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov; ref. 27)was used to analyze gene sets thatweredown- or upregulated (�2 fold change) in the microarray.

RNA isolation, cDNA synthesis, and quantitative reversetranscription PCRTotal RNAs were isolated using RNeasy Kits (Qiagen)

according to the manufacturer's instructions. cDNA was syn-thesized using the iScript cDNA Synthesis Kit (Bio-Rad)according to the manufacturer's instructions and amplifiedwith iQ SYBRGreen Supermix (Bio-Rad) using the CFX384 real-time PCR detection system (Bio-Rad). Expression levels werequantified using CFX Manager software (Bio-Rad) and nor-malized to 18s rRNA. The relative mRNA levels represent thefold changes over the control.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assays were per-

formed using the Millipore ChIP protocol with minormodifications as described previously (28). Chromatin immu-noprecipitates for proteins and methyl marks were amplifiedby quantitative PCR, normalized to input, and calculated aspercentages of inputs. Fold enrichment levels indicate the foldchanges over the negative control immunoglobulin G (IgG).The PCR primer sequences are described in SupplementaryTable S1.

Analysis of expression levels, survival, and correlationThe overall survival and correlation analysis were performed

using the dataset from The Cancer Genome Atlas (TCGA)database. For expression analysis, the Oncomine database,which represents a unifying microarray database resourcewith an integrated data-searching tool (https://www.onco-mine.org), was used in addition to TCGA.

Statistical analysis of dataThe statistical significance between 2 groups was analyzed

by Student's t-test. Survival rate and correlation analysis werecarried out by the log-rank test and Pearson correlation test inPrism 5 software (GraphPad Software Inc.), respectively. Dataare presented as themean� SEM (error bars). �, P< 0.05; ��, P<0.01; and ��, P < 0.001 denote statistically significant changes.

ResultsUTX is critical for the proliferation and invasiveness ofbreast cancer cellsIn an effort to determine the role of UTX in breast cancer, we

first assessed UTX levels in 2 nontransformed mammaryepithelial cell lines (MCF-10A and HMLE) and several breast

cancer cell lines. UTX protein levels were generally higher inbreast cancer cell lines than in the normal cell lines (Fig. 1A).Next, to examine whether UTX is critical for breast cancer cellproliferation, we depleted UTX in the estrogen receptor (ER)-positive cell line MCF-7 and 2 triple-negative (ER/PR/HER2�)breast cancer cell lines MDA-MB-231 and MDA-MB-468 usingshRNAs. UTX knockdown substantially reduced the prolifer-ation of MCF-7, MDA-MB-231, and MDA-MB-468 cells regard-less of ER status of breast cancer cell lines (Fig. 1B–D). Incontrast, the cell proliferation rate of the non-breast cancer cellline U2OS (a human osteosarcoma cell line) was not affected byUTX depletion (Fig. 1E). Previously, it has been shown thatknockdown of UTX in normal fibroblasts increased cell pro-liferation (23). This previous report and our results suggest thatUTX may regulate cell proliferation in a cell type–specificmanner. Consistent with the inhibitory effect of UTX knock-down on the proliferation of MDA-MB-231 cells, UTX knock-down also decreased colony formation abilities of MDA-MB-231 cells in a soft agar assay (Fig. 1F). Furthermore, wedetermined the effect of UTX knockdown on the invasiveabilities of 2 metastatic cell lines MDA-MB-231 and MDA-MB-468. UTX depletion remarkably inhibited the in vitroinvasive abilities of both cell lines (Fig. 2A and B), althoughit did not have any significant effect on themigration capacitiesof MDA-MB-231 cells (Supplementary Fig. S1A). These resultsindicate that UTX is important for the proliferation, anchor-age-independent growth, and invasion of breast cancer cells,such as MDA-MB-231 cells.

Cancer cell metastasis involves intravasation (entrance intoblood systems), extravasation (exit from the blood vessels), andcolonization in a distant organ (29). For these processes, theinvasive and proliferative abilities of cancer cells are essential.To further assess the role of UTX in the invasion and tumorgrowth of breast cancer cells, we treated luciferase-expressingMDA-MB-231 (MDA-MB-231-Luc) cells with shScrambled (con-trol)- or shUTX-6-containing viruses and subsequently injectedthese 2 groups of cells into mice via tail veins. In vivo imaginganalysis demonstrated that mice (n ¼ 7) injected withshScrambled-treatedMDA-MB-231-Luc cells had stronger lucif-erase signals than those (n ¼ 7) injected with shUTX-treatedMDA-MB-231-Luc cells (Fig. 2C and D). In particular, luciferasesignals between the shScrambled group and the shUTX-6 groupwere remarkably different at the 8th week (Fig. 2E). Tumorswere confirmed by hematoxylin and eosin staining (Fig. 2F).Because themouse xenograftmodels basedon tail vein injectiongenerally involve both cancer cell invasion during extravasationand tumor growth in a distant organ (primarily, lung), theseresults suggest that UTX is required for the in vivo invasivenessand tumor formation capacities of MDA-MB-231 cells.

UTX's demethylation activity contributes to theproliferative and invasive abilities of breast cancer cells

It has been reported that UTX's demethylase activity is notrequired for T-box family member-dependent gene expression(30). To examine whether the demethylation activity of UTX isnecessary for the phenotypic defects of breast cancer cells andwhether defective phenotypes of UTX knockdown cells resultfrom specific knockdown of UTX, we ectopically expressed

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Figure 1. UTX knockdown attenuates the proliferation and colony formation abilities of breast cancer cells. A, UTX levels in normal and breast cancer cell lines.UTX protein levels were examined by Western blot analysis. For comparison, the breast-unrelated cell lines HEK293T and HeLa were included. B–E,the effect of UTX knockdownon the proliferation ofMCF-7 (B),MDA-MB-231 (C),MDA-MB-468 (D), andU2OS (E) cells. Cells were treatedwith control shRNA(shLuc) and two shRNAs against UTX. Knockdown efficiencies by shRNAs were examined by quantitative RT-PCR (left). Cell numbers were countedat the indicated time points (right). Data,mean�SD (error bars) of at least three independent experiments (B, n¼ 4; C, n¼ 3; D, n¼ 3; E, n¼ 3). F, the inhibitoryeffect of UTX knockdown on colony formation of MDA-MB-231 cells in soft agar. Data, mean� SEM (error bars) of five independent experiments. �, P < 0.05;��, P < 0.01; ��, P < 0.001.

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shUTX6-resistant UTX and shUTX6-resistant UTX catalyticmutant (mUTX) in UTX-depleted MDA-MB-231 cells (Fig.3A, left). As shown in Fig. 3A and B, wild-type UTX significantlyrescued defective proliferation and invasiveness of UTX-depleted MDA-MB-231 cells, whereas the catalytic mutantmUTX weakly or insignificantly restored such cellular pheno-types. These results indicate that UTX and UTX's catalyticactivity are needed for the proliferation and invasion of breastcancer cells, such as MDA-MB-231 cells.

UTX interacts with a C-terminal region of MLL4, andMLL4 is required for the proliferation and invasivenessof breast cancer cellsThe H3K27 demethylase UTX is present in multiprotein

complexes with the H3K4 methyltransferase MLL4 (26, 31,32). We previously showed that a C-terminus of MLL4

(MLL4-C; 4507-5537aa) is responsible for MLL4's interactionwith the core subunits (e.g., ASH2L, RBBP5, and WDR5) in theMLL4 complex (28). To examinewhetherMLL4-C alsomediatesthe association between UTX and MLL4, we transfected anexpression plasmid encoding Flag-tagged MLL4-C into humanembryonic kidney 293T cells and purified Flag-tagged MLL4-Cusing anti-Flag immunoprecipitation. Western blot analysis ofimmunoprecipitation eluates showed that MLL4-C physicallyinteracted with UTX (Fig. 3C and D). To further map whatregionofMLL4-C is responsible for the associationwithUTX,wegenerated the N-terminal region of MLL4-C [MLL-C (N), 4507-4996aa] and the C-terminal region ofMLL4-C [MLL-C (C), 4997-5537aa]. Immunoprecipitation experiments demonstratedthat UTX associated with the N-terminal region of MLL4-C and that ASH2L, RBBP5, and WDR5 interacted with theC-terminal region of MLL4-C (Fig. 3C and D). These results not

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Figure 2. UTX depletionsuppresses in vitro and in vivoinvasive abilities of breast cancercells. A and B, the effect of UTXknockdown on the invasiveness ofMDA-MB-231 (A) and MDA-MB-468 (B) cells. For invasion assay,six different microscopic fields(magnification, �4 ) from at leastthree independent experimentswere examined; cells in the fieldswere counted (A, n � 3) or relativeintensities of the fields weremeasured (B, n � 3). C–F, theeffect of UTX knockdown oncellular invasiveness and tumorgrowth in a mouse xenograftmodel based on tail vein injection.Luciferase-expressing MDA-MB-231 (MDA-MB-231-Luc) cellswere treated with virusescontaining shScrambled (controlshRNA) or shUTX-6. Then,shScrambled-treated or UTX-depleted cells were injected intomice via tail veins, and tumorcolonization in the lung wasmonitored using an IVIS100imaging system. Representativebioluminescent images of tumor-harboring mice are shown inC. The luciferase signals werecompared between theshScrambled group (n¼ 7) and theshUTX-6 group (n ¼ 7; D). Inaddition, the luciferase signals forboth groups at the 8th week aftertail vein injection were individuallyplotted (E). Metastatic lesions inlungs from mice at the 8th weekwere analyzed by hematoxylin andeosin staining (F). Data, mean �SEM (error bars). �, P < 0.05;��, P < 0.001.






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only confirm the association ofUTX andMLL4, but also indicatethat UTX interacts with MLL4 via a C-terminal region of MLL4(4507–4996aa).

Because MLL4 interacts with UTX, we sought to determinewhether MLL4 has an effect on cell growth and invasion likeUTX. We depleted MLL4 in MDA-MB-231 cells using shRNAs

(Fig. 3E, left) and then performed cell proliferation and inva-sion assays. MLL4 depletion reduced the proliferation ofMDA-MB-231 cells (Fig. 3E, right) and also impeded in vitro invasiveabilities of MDA-MB-231 cells (Fig. 3F). Of interest, MLL4knockdown did have a moderate effect on the migration ofMDA-MB-231 cells (Supplementary Fig. S1B). These results

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Figure 3. UTX's demethylase activity is important for cell proliferation and invasion, and MLL4 knockdown impedes these cellular properties. A and B, therescue experiments of defective proliferation and invasiveness of UTX-depleted cells. Wild-type UTX and its catalytic mutant mUTX were ectopicallyexpressed in UTX-depleted MDA-MB-231 cells. The mRNA and protein levels of UTX and mUTX were assessed by quantitative RT-PCR and Western blotanalysis, respectively (A, left). For cell proliferation assays, cell numbers were counted at the indicated time points (A, right). For invasion assays,cells in the fields (magnification, �10) from three independent experiments were counted (B). The shLuc virus–treated cells that were transfected with theempty vector were used as controls. C, schematic representation of a C-terminal region of MLL4. PHD, plant homeodomain; SET, the catalytic domain.D, mapping of an MLL4's C-terminal region responsible for the interaction with UTX. Flag-tagged MLL4-C, MLL4-C (N) and MLL4-C (C) were expressed in293T cells. Whole cell extracts were immunoprecipitated with anti-Flag antibody, followed by Western blot analysis with anti-UTX, anti-ASH2L,anti-RBBP5, and anti-WDR5 antibodies. E, the negative effect of MLL4 knockdown on MDA-MB-231 cell proliferation. Knockdown efficiency was assessedby quantitative RT-PCR (left). Cells were counted at the indicated time points (right). F, inhibitory effect of MLL4 knockdown on the invasiveness ofMDA-MB-231 cells (magnification, �4). Data, mean � SEM (error bars) of three independent experiments. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

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indicate that similar to UTX, MLL4 is necessary for theproliferation and invasion of breast cancer cells.

Most UTX-regulated genes are comodulated by MLL4BecauseUTX-depleted cells andMLL4-depleted cells shared

similar defects in cell proliferation and invasiveness andbecause UTX physically interacts with MLL4, we reasonedthat the transcriptional coactivators, UTX and MLL4, mayregulate a similar set of genes. For this, we assessed the effectof UTX or MLL4 knockdown on gene expression profiles inMDA-MB-231 cells. Specifically, we compared the mRNAexpression levels between UTX (or MLL4)-depleted cells andcontrol (shLuc-treated) cells using whole genome mRNAexpression analysis.In particular, we analyzed genes that were at least 2-fold

down- or upregulated by UTX or MLL4 knockdown in themicroarray assays. Remarkably, 54% of shUTX-downregulated

genes (656/1,215) overlapped with 73% of shMLL4-downreg-lated genes (656/899; Fig. 4A), and 68% of shUTX-upregulatedgenes (1,065/1,576) were identical with 77% of shMLL4-upre-gulated genes (1,065/1,389; Fig. 4B). These results indicate thatmost UTX-controlled genes are coregulated by MLL4.

UTX andMLL4 coregulate transcriptional programs thatare associated with cell proliferation and invasiveness

As described above, knockdown of UTX or MLL4 inhibitedcell proliferation and invasiveness. Therefore, we carried outgene ontology analysis of our whole genomemRNA expressiondata to assess whether UTX and MLL4 coregulate gene setsthat are associated with such knockdown phenotypes. Ofinterest, a cohort of cell proliferation–associated genes andpro-invasiveness genes were significantly downregulated byUTX andMLL4 depletion (Fig. 4C; see Supplementary Table S2for each category of genes). In contrast, UTX and MLL4

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Figure 4. UTX and MLL4 positivelycoregulate gene expressionprograms related to cellproliferation and invasion. A and B,Venn diagrams for genes that aredownregulated (A) or upregulated(B) by UTX and MLL4 knockdown.MDA-MB-231 cells were treatedwith viruses containingshLuciferase, shUTX-6, orshMLL4-18. The mRNA levels inUTX- or MLL4-depleted cells weremeasured by the expressionmicroarray Affymetrix U133P andcompared with those in the controlcells. C and D, gene ontologyanalysis of genes that are co-downregulated (C) or co-upregulated (D) by UTX and MLL4knockdown. Gene groups that arerelated only to cell proliferation andinvasiveness are shown. Geneontology analysis was performedusing the program DAVID. n.s., notsignificant. E and F, quantitativeRT-PCR analysis of several genesthat are co-downregulated by UTX(E) and MLL4 (F) knockdown inMDA-MB-231 cells. Data, mean �SEM (error bars) of at least threeindependent experiments.�, P < 0.05; ��, P < 0.01;��, P < 0.001.






CAN-13-1896; 24/2/2017; 2:44:18

knockdown did not have any significant effect on the upregu-lation of these categories of genes (Fig. 4D). These resultsindicate that UTX and MLL4 contribute to breast cancer cellproliferation and invasion by activating cell proliferation–associated genes and pro-invasiveness genes.

To confirm our whole genome mRNA expression analysis,we individually analyzed expression levels of several cell pro-liferation–associated genes and pro-invasiveness genes,including MMP-9, MMP-11, ERBB3, Six1, Cyclin A1, Pim-1, andCSPG4, using quantitative reverse transcription (RT)-PCR.MMP-9 and MMP-11 have been known to promote cell inva-sion (33). ERBB3, Six1, Cyclin A1, Pim-1, and CSPG4 areinvolved in tumorigenesis by promoting cell proliferation andinvasion. Consistent with whole genome expression results,quantitative RT-PCR data demonstrated that in MDA-MB-231cells, these genes were downregulated by UTX orMLL4 knock-down (Fig. 4E and F). Similarly, UTX or MLL4 depletion alsodecreased expression levels ofMMP-9,MMP-11, Six1, Cyclin A1,and CSPG4 in MDA-MB-468 cells (Supplementary Fig. S2A andS2B). Interestingly, although UTX was shown to regulate Rbpathway genes in mouse embryonic fibroblast cells (23), ourquantitative RT-PCR and microarray results showed that Rbpathway genes (e.g., Rb and Rbbp4-6) in MDA-MB-231 cellswere mostly unaffected by UTX knockdown (SupplementaryFig. S3; data not shown).

UTX-mediated demethylation of trimethylated H3K27 islinked to MLL4-catalyzed H3K4 trimethylation atcotarget genes of UTX and MLL4

Cotarget genes of UTX and MLL4 are likely downregulatedbyUTXorMLL4 knockdown, becauseUTX andMLL4 generallyact as transcriptional coactivators. Therefore, we examinedwhether UTX andMLL4 are co-recruited to genes (e.g.,MMP-9,MMP-11, and Six1) that were downregulated by UTX andMLL4knockdown. Quantitative ChIP results showed that UTX andMLL4 co-occupied the promoters of theMMP-9,MMP-11, andSix1 genes (Fig. 5A–F, left). In particular, MLL4 occupancy atthese genes was confirmed using 2 different antibodies (Sup-plementary Fig. S4).

UTX has been shown to remove methyl groups from therepressivemarkH3K27me3 at the proximal promoters near thetranscription start sites (26). To determine whether UTXdemethylates H3K27me3 at the proximal promoters of theMMP-9, MMP-11, and Six1 genes, we examined the effect ofUTX knockdownonH3K27me3 levels at the promoters of thesegenes. Quantitative ChIP results showed that UTX depletionincreased H3K27me3 levels at the proximal promoters of theMMP-9, MMP-11, and Six1 genes (Fig. 5A–C, right). Theseresults indicate that expression of the MMP-9, MMP-11, andSix1 genes is positively regulated by UTX-catalyzed demeth-ylation of H3K27me3 at their proximal promoters.

We also assessed the effect ofMLL4 knockdown onH3K4me3levels at the proximal promoters of the MMP-9, MMP-11, andSix1 genes. Our ChIP assays showed that MLL4 depletionreduced H3K4me3 levels at the promoters of these genes. Theseresults indicate thatMLL4may upregulate theMMP-9,MMP-11,and Six1 genes by enzymatically generating H3K4me3 at theirproximal promoter regions (Fig. 5D–F, right).

To determine whether UTX affects MLL4-catalyzed H3K4methylation at theMMP-9,MMP-11, and Six1 genes, we exam-ined the effect of UTX knockdown on H3K4me3 and MLL4levels at the proximal promoters of the MMP-9, MMP-11, andSix1 genes. Our ChIP experiments demonstrated that UTXknockdown decreased MLL4 and H3K4me3 levels on thesepromoters (Fig. 6A–C). We also assessed whether MLL4 hasany effect on UTX-catalyzed demethylation of H3K27me3 atthe MMP-9, MMP-11, and Six1 genes. As shown in Fig. 6D–F,MLL4 knockdown decreased UTX levels while increasingH3K27me3 levels at these genes. Furthermore, we determinedwhether UTX (orMLL4) knockdown affects total cellular levelsof H3K4me3, H3K27me3, MLL4 (UTX in case of MLL4 knock-down). UTX or MLL4 knockdown did not result in obviouschanges in total cellular levels of H3K4me3 and H3K27me3(Supplementary Fig. S5). Interestingly, MLL4 knockdownseemed to slightly downregulate cellular UTX mRNA andprotein levels (Supplementary Fig. S6A), although UTX knock-down did not change MLL4 mRNA levels (Supplementary Fig.S6B). Therefore, decreased levels of UTX at MMP-9, MMP-11,and Six1 genes by MLL4 knockdown may be at least in partbecause of transcriptional regulation of UTX expression byMLL4. In contrast, decreased chromatin levels ofMLL4 byUTXdepletion might result from reduced formation of the UTX-MLL4 complex because of diminished UTX levels, although wecannot exclude the possibility that UTX knockdownmay affectMLL4's protein stability. Nevertheless, these results indicatethat UTX and MLL4 occupancy at their cotarget genes may beinterconnected and that UTX-catalyzed H3K27me3 demeth-ylation parallels MLL4-mediated H3K4 trimethylation at theircotarget genes.

High mRNA levels of UTX and MLL4 are associated withpoor prognosis in patients with breast cancer

As mentioned earlier, UTX mRNA levels have been reportedto be overexpressed in breast cancer samples (24). Thus, wedetermined whether both UTX and MLL4 mRNA levels areupregulated in breast tumors as compared with normal breasttissues using the following 2 publicly available databases:oncomine and TCGA. Analysis of oncomine database showedthat although their average levels were not increased in breastcancer samples in most datasets (data not shown), averageUTX andMLL4 levels were higher in the invasive subtypes thanin normal breast tissues in one study set (Fig. 7A and B; ref. 34).In the TCGA database, there were no significant differences inaverage UTX and MLL4 levels between normal breast tissuesand all breast tumor types combined, although their levels insomebreast tumor sampleswere highly increased as comparedwith their normal average levels (Fig. 7C and D).

Next, we performed the Kaplan–Meier survival analysis toassess whether high levels of UTX and MLL4 in breast tumorsare linked to the clinical outcomes of patients with breastcancer. The survival analysis using the TCGA data with patienthistory demonstrated that highmRNA levels of UTX andMLL4were linked to shorter survival in patients with breast cancer(Fig. 7E and F). In addition, we found that UTX mRNA levelshad a significant positive correlation with MLL4 mRNA levelsin patients with breast cancer (Fig. 7G). These results indicate

Kim et al.





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that high UTX and MLL4 levels may contribute to poorprognosis in patients with breast cancer.

DiscussionUTX plays an important role in regulating multiple cellular

functions, including somatic cell reprogramming (35), germcell development (35), embryo development in a sex-specificmanner (36, 37), cardiac development (38), aging (39), andmyogenesis (40). Results reported here showed that UTXknockdown suppressed cellular invasive and proliferative abil-ities of breast cancer cells, implicating a role for UTX in cellinvasion and tumor growth. In line with this, intravenousmouse xenograft experiments in this study indicated that UTXmaybe required for in vivo invasion and tumor growth of breastcancer cells. Mechanistically, UTX activates gene expressionprograms associated with cell proliferation and invasiveness.Therefore, our findings reveal a novel UTX function in which

UTX positively regulates breast cancer cell proliferation andinvasiveness by transcriptionally activating the relevant geneexpression programs.

Our results in this study also showed that similar to UTXknockdown, MLL4 depletion inhibited the proliferative andinvasive capacities of breast cancer cells. UTX and MLL4coregulated several gene expression programs, including thoseassociated with cell proliferation and invasiveness. Our addi-tional data showed a correlation of high UTX andMLL4mRNAlevels with poor survival of patients with breast cancer,strengthening the clinical importance of UTX and MLL4 inpatients with breast cancer. Furthermore, our findings thatUTX mRNA levels correlate with MLL4 mRNA levels in breasttumors support the physical and cell biological link betweenthese 2 histone methylation modifiers. Together, our workuncovers that UTX and MLL4 regulate the proliferation andinvasiveness of breast cancer cells in a cooperative manner.

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Figure 5. UTX and MLL4 are co-recruited toMMP-9,MMP-11, andSix1 genes. A–C, the effect of UTXknockdown on chromatin levels ofUTX and H3K27me3 at theproximal promoters ofMMP-9 (A),MMP-11 (B), and Six1 (C) in MDA-MB-231 cells. Fold enrichments ofUTX over the negative control IgGwere measured by quantitativeChIP (left). In addition, chromatinlevels of UTX andH3K27me3werecompared between shLuc-treated(control) cells and UTX-depletedcells using ChIP (right). D–F, theeffect of MLL4 knockdown onchromatin levels of MLL4 andH3K4me3 at the proximalpromoters ofMMP-9 (D),MMP-11(E), and Six1 (F) in MDA-MB-231cells. Fold enrichments of MLL4over IgG were measured byquantitative ChIP (left). In addition,chromatin levels of MLL4 andH3K4me3 were comparedbetween shLuc-treated (control)cells and MLL4-depleted cellsusing ChIP (right). Data, mean �SEM (error bars) of threeindependent experiments (n ¼ 3).�, P < 0.05; ��, P < 0.01;��, P < 0.001.






CAN-13-1896; 24/2/2017; 2:44:19

Histone-modifying enzymes often cooperate for generepression. The H3K4 demethylase LSD1 and the histonedeacetylases HDAC 1 and 2 co-repress gene expression (41),and the H3K4 demethylase JARID1a and the H3K9 methyl-transferase G9a cooperate for gene repression (42). Conversely,certain modifiers cooperatively activate their target genes. TheH3K9 demethylase JMJD2B and the H3K4 methyltransferaseMLL4 coordinately regulate ERa-activated transcription (43).In addition, our previous report showed that UTX and MLL4were co-recruited to regulate the same target genes, such as thedifferentiation-specific HOX cluster genes, during stem celldifferentiation (26). The notion that UTX-catalyzed H3K27demethylation is linked to MLL4-mediated H3K4 methylationis supported by our following results: (i) UTX knockdown notonly increased the UTX substrate H3K27me3 but alsodecreased MLL4 and H3K4me3 levels; (ii) MLL4 knockdownnot only decreased UTX and the MLL4's enzymatic productH3K4me3 but also increasedH3K27me3 levels.Moreover,mostof UTX-controlled genes were affected by MLL4 knockdownand vice versa. Therefore, it is likely that UTX and MLL4regulate gene expression programs in a coordinated manner.

Similar to theUTX gene, theMLL4 gene also harbors somaticmutations and deletions in several types of cancers, includingprostate cancer, medulloblastoma, and lymphoma (reviewedin ref. 22). In contrast to these types of cancer, genomicalterations of UTX and MLL4 rarely occur in human breasttumors (25). Our results indicate that UTX and MLL4 may bepositive contributors to breast tumor, because UTX or MLL4knockdown impeded the invasiveness and proliferation of

breast cancer cells. In support with our results, a recent reportindicated that UTX is upregulated in breast cancer and corre-lates with poor prognosis in patients with breast cancer (24). Inaddition, UTX is overexpressed in renal cell carcinoma (44) andleukemia (45), and its knockdown in leukemia cell lines exhi-bits an antiproliferation effect, implicating its role in thetumor-promoting function (45). Similarly, MLL4 has also beenshown to be critical for the growth of HeLa cells (31).

These conflicting functions of UTX and MLL4 in differenttypes of cancer might be explained by the following potentialscenarios. First, tumor suppressive effect and oncogenic func-tion of a histonemodifiermay not bemutually exclusive. EZH2is overexpressed and plays an oncogenic role in several types oftumors (e.g., breast and prostate cancer), whereas it undergoessomatic loss-of-function mutations in malignant myeloid dis-eases (reviewed in ref. 46). Similarly, it is possible that UTX andMLL4 act as either oncogenic factors or tumor suppressors in atissue-dependentmanner. In this regard, UTX andMLL4mightplay oncogenic roles in breast cancer, but function as tumorsuppressors in medulloblastoma, bladder carcinoma, leuke-mia, and prostate tumors in which their loss-of-functionmutation rates are significantly high. The second scenario isthat UTX and MLL4 may function in a tumor stage-specificmanner. It has been reported that UTX activates Rb-relatedtumor suppression pathways innormal humanfibroblasts (23),whereas our results showed that UTX positively regulatesoncogenic gene expression programs for cell growth andinvasion in breast cancer cells. Therefore, it is tempting tospeculate that UTX and MLL4 may act as tumor suppressors

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Figure 6. UTX-catalyzedH3K27me3 demethylation andMLL4-catalyzed H3K4methylation occur in a coordinatemanner. A–C, the effect of UTXknockdown on chromatin levels ofMLL4 and H3K4me3 at theproximal promoters ofMMP-9 (A),MMP-11 (B), and Six1 (C) in MDA-MB-231 cells. ChIP assays werecarried out with anti-MLL4 andanti-H3K4me3 after UTXknockdown. D–F, the effect ofMLL4 knockdown on chromatinlevels of UTX and H3K27me3 atthe proximal promoters ofMMP-9(D), MMP-11 (E), and Six1 (F) inMDA-MB-231 cells. ChIP assayswere performed with anti-UTX andanti-H3K27me3 after MLL4knockdown. Data, mean � SEM(error bars) of three independentexperiments. �, P < 0.05;��, P < 0.01; ��, P < 0.001.

Kim et al.





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during the normal cellular stage or initial stages of breastcancer development and become a tumor-promoting proteinin later stages and metastatic tumors. Intriguingly, this type ofscenario has also been exemplified by TGF-b, which behaves asan antiproliferative protein in normal cells and at early onco-genesis but promotes tumor invasiveness in later stages (47). Inaddition, LSD1 is frequently overexpressed in ER� breastcancer (48) but inhibits breast cancer metastasis (49).H3K27 methylation is regulated by the histone methyltrans-

ferases EZH1/2 and demethylases UTX and JMJD3. Our presentstudy and others' work support the notion that UTX likelycontributes to the oncogenic pathogenesis in the breast(24, 44, 45). Interestingly, its biochemically opposing enzymeEZH2 seems to be an oncogenic protein in the same tissue (13,14). These seeminglypuzzlingobservationsmaybea result of theregulation of different sets of genes by UTX and EZH2. UTXmayhelp cancer cells grow and invade by activating gene programsthat may promote tumor growth and metastasis, as describedherein. EZH2-containing polycomb repressive complex 2 tran-scriptionally suppresses p14ARF and p16INK4A, allowing cancercells to resist various cellular senescence signals (17). Of note, itis also possible that EZH2 would promote breast tumorigenesisvia an oncogene activation mechanism independent of its

methylation activity, as EZH2 phosphorylation by AKT1 maycontribute to castration-resistant prostate cancer by inhibitingEZH2'smethyltransferase activity and enabling EZH2 to act as atranscriptional co-activator of androgen receptor (50). Equallyinteresting, it seems as thoughUTX and JMJD3, albeit having thesame in vitro H3K27 demethylase activities, produce oppositebiological outcomes by regulating different gene expressionprograms, because JMJD3 may have a tumor-suppressive func-tion by activating p16INK4A and p14ARF (19).

In summary, our results provide evidence that a histonedemethylase and a histone methyltransferase coregulate geneexpression profiles in a coordinated manner to contribute tothe proliferation and invasiveness of breast cancer cells. Ourfindings might be relevant to the development of therapeuticagents, such as small molecule modulators of UTX or MLL4.

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

Authors' ContributionsConception and design: J.-H. Kim, M.G. LeeDevelopment of methodology: J.-H. Kim, A. Sharma, M.G. LeeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.-H. Kim, A. Sharma, S.-H. Lee, C.-H. Chan

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Figure 7. High mRNA levels of UTXand MLL4 correlate with poorsurvival in patients with breastcancer. A–D, comparisonofUTX (Aand C) and MLL4 (B and D) mRNAlevels between normal breasttissues and breast carcinoma.Oncomine (A and B) and TCGA (CandD) databaseswere used. E andF, survival analysis of patients withbreast cancer on the basis of UTX(E) and MLL4 (F) mRNA levels. Thepublicly available dataset fromTCGA (774 human breast tumors)was analyzed. High UTX and highMLL4denotes patientswithmRNAlevels that are greater than or equalto average plus 1 SD (UTX-low,n ¼ 683; UTX-high, n ¼ 91;MLL4-low, n ¼ 649; MLL4-high,n ¼ 125). G, correlation analysisbetween UTX and MLL4 mRNAlevels in the TCGA dataset.��, P < 0.001.






CAN-13-1896; 24/2/2017; 2:44:20

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.-H. Kim, A. Sharma, S.-H. Lee, B. Gu, M.G. LeeWriting, review, and/or revision of the manuscript: J.-H. Kim, A. Sharma,S.S. Dhar, M.G. LeeAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases): A. Sharma, C.-H. Chan, H.-K. Lin, M.G. LeeStudy supervision: M.G. Lee

AcknowledgmentsThe authors thank Dr. M.-C. Hung, Dr. Z. Han, and S. Zhang for their help for

bioluminescence imaging and L. Jorewicz for manuscript editing.

Grant SupportThis work was supported by grants to M.G. Lee from the NIH (R01 GM095659

and R01 CA157919) and Cancer Prevention and Research Institute of Texas(RP110183).

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 indicate thisfact.

Received July 16, 2013; revisedDecember 18, 2013; acceptedDecember 26, 2013;published OnlineFirst February 3, 2014.

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Correction

Correction: UTX and MLL4 CoordinatelyRegulate Transcriptional Programs for CellProliferation and Invasiveness in BreastCancer CellsIn this article (Cancer Res 2014;74:1705–17), which appeared in theMarch 15, 2014issue of Cancer Research (1), the two labels "Flag-tag MLL4-C (N)" and "Flag-tagMLL4-C (C)" in Fig. 3Dweremistakenly interchanged. As a result, the text for Fig. 3Dwas also incorrect.

In addition, "(4997–5537aa)" at the end of the fourth paragraph of the Resultssection should read "(4507–4996aa)."

Finally, the citation "(33–37)" in the ninth paragraph of the Results section wasinadvertently added.

The online version of the article has been corrected and no longer matches the print.The authors regret these errors.

Reference1. Kim J-H, Sharma A, Dhar SS, Lee S-H, Gu B, Chan C-H, et al. UTX and MLL4 coordinately regulate

transcriptional programs for cell proliferation and invasiveness in breast cancer cells. Cancer Res2014;74:1705–17.

Published online May 1, 2017.doi: 10.1158/0008-5472.CAN-17-0639�2017 American Association for Cancer Research.

CancerResearch

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http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-17-0639&domain=pdf&date_stamp=2017-4-19

2014;74:1705-1717. Published OnlineFirst February 3, 2014.Cancer Res Jae-Hwan Kim, Amrish Sharma, Shilpa S. Dhar, et al. Cancer CellsPrograms for Cell Proliferation and Invasiveness in Breast UTX and MLL4 Coordinately Regulate Transcriptional

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utx and mll4 coordinately regulate transcriptional ......reversed by histone lysine demethylases...

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