cbx4 suppresses metastasis via recruitment of hdac3 to the ... · cbx4 suppresses metastasis via...

Tumor and Stem Cell Biology

CBX4 Suppresses Metastasis via Recruitment ofHDAC3 to the Runx2 Promoter in ColorectalCarcinomaXin Wang, Liping Li, Yuanzhong Wu, Ruhua Zhang, Meifang Zhang, Dan Liao,Gang Wang, Ge Qin, Rui-hua Xu, and Tiebang Kang

Abstract

Polycomb chromobox (CBX) proteins participate in the poly-comb repressive complex (PRC1) that mediates epigenetic genesilencing and endows PRC1 with distinct oncogenic or tumorsuppressor functions in a cell-type–dependent manner. In thisstudy, we report that inhibition of cell migration, invasion, andmetastasis in colorectal carcinoma requires CBX4-mediatedrepression of Runx2, a key transcription factor that promotescolorectal carcinoma metastasis. CBX4 inversely correlatedwith Runx2 expression in colorectal carcinoma tissues, andthe combination of high CBX4 expression and low Runx2expression significantly correlated with overall survival, moreso than either CBX4 or Runx2 expression alone. Mechanisti-

cally, CBX4 maintained recruited histone deacetylase 3(HDAC3) to the Runx2 promoter, which maintained a deace-tylated histone H3K27 state to suppress Runx2 expression. Thisfunction of CBX4 was dependent on its interaction withHDAC3, but not on its SUMO E3 ligase, its chromodomain,or the PRC1 complex. Disrupting the CBX4–HDAC3 interac-tion abolished Runx2 inhibition as well as the inhibition of cellmigration and invasion. Collectively, our data show that CBX4may act as a tumor suppressor in colorectal carcinoma, andstrategies that stabilize the interaction of CBX4 with HDAC3may benefit the colorectal carcinoma patients with metastases.Cancer Res; 76(24); 7277–89. �2016 AACR.

IntroductionPolycomb group (PcG) proteins have been documented to be

major transcriptional repressors that mediate epigenetic genesilencing (1, 2). In general, PcG proteins organize to form twodominant polycomb -repressive complexes (PRC), PRC1 andPRC2 (3, 4), which are mainly involved in regulating develop-ment (5), senescence (6), stemness (7), and cancer progression(1, 4). PcG proteins are well known to be frequently dysregulatedin various cancer types (8). However, because PcG complexescontain multiple subunits and PcG proteins carry out variousfunctions, the exact regulatory elements of PcG proteins thatregulate certain types of cancer have not yet been identified(4). Notably, several polycomb chromobox (CBX) proteins,including CBX2, CBX4, CBX6, CBX7, and CBX8, have also beenshown to participate in the PRC1 complex and endow PRC1 withdistinguish functions (9), suggesting that CBX proteinsmay act asan oncogene or tumor suppressor in a cell-type–dependent man-ner. For instance, CBX8 has been recently reported to exert

paradoxical effects, promoting proliferation while suppressingmetastasis, in colorectal carcinoma progression (10); CBX7 func-tions as a tumor suppressor in lung carcinoma by recruitingHDAC2 to the CCNE1 promoter to suppress CCNE1 expression(11), whereas it is an oncogene in gastric cancer and lymphoma(12, 13). Therefore, the functionof eachCBXprotein in any cancermust be assessed separately.

CBX4 (also known as polycomb 2, Pc2) is a special chromoboxprotein because it is not only a transcriptional repressor but also aSUMO E3 ligase (14, 15). The N-terminal chromodomain andtwoSUMO-interactingmotifs (SIM) conferCBX4withpolycomb-and SUMO E3 ligase–dependent functions, respectively (6, 16).For instance, CBX4 protects slow-cycling human epidermal stemcells (epSC) from senescence via its chromodomain, whereas itprevents active proliferation and terminal differentiation in epSCsvia its SUMOylation activity (6). Recently, CBX4 has been iden-tified as a new therapeutic target for hepatocellular carcinoma(HCC; refs. 9, 17), which is one of the most prevalent malignan-cies resistant to current chemotherapies or radiotherapies (18).CBX4 increases the transcriptional activity of HIF-1a, hypoxia-induced VEGF expression and angiogenesis by promoting HIF-1aSUMOylation at K391 and K477, and this function depends onthe two SUMO-interacting motifs of CBX4 (9). In addition, highCBX4 expression predicts poor overall survival in patients withHCC (9, 17). Colorectal carcinoma is one of the most commoncauses of cancer-related death worldwide (19, 20). Thus, betterunderstanding the molecular mechanisms of colorectal carcino-ma tumorigenesis and identifying new therapeutic targets forcolorectal carcinoma are urgent research objectives. Here, weprovide evidence to show that CBX4 may serve as a tumorsuppressor in colorectal carcinoma by recruiting HDAC3 to the

State Key Laboratory of Oncology in South China, Collaborative InnovationCenter for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou,China.

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

Corresponding Author: Tiebang Kang, Sun Yat-sen University Cancer Center,651 Dongfeng East Road, Guangzhou 510060, China. Phone: 8620-8734-3183;Fax: 8620-8734-3170; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-2100

�2016 American Association for Cancer Research.

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on October 25, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst November 18, 2016; DOI: 10.1158/0008-5472.CAN-16-2100

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Runx2 promoter to impede Runx2 expression and that this novelfunction of CBX4 is independent of its SUMO E3 ligase, itschromodomain, and the PRC1 complex.

Materials and MethodsMicroarray data analysis

Briefly, samples (HCT116 cells transfected with control orCBX4 shRNA) were used to synthesize cRNA, and cRNA waslabeled and hybridized to Gene Expression Hybridization Kit(cat# 5188-5242, Agilent Technologies) in Hybridization Oven(cat# G2545A, Agilent Technologies), according to the manufac-turer's instructions, and samples were scanned by Agilent Micro-array Scanner (cat# G2565CA, Agilent Technologies) with defaultsettings (dye channel: green, scan resolution¼ 5 mm, PMT 100%,10%, 16 bit). Datawere extractedwith Feature Extraction software10.7 (Agilent Technologies). Raw data were normalized byQuan-tile algorithm, Gene Spring Software 11.0 (Agilent Technologies).Data are available via Gene Expression Omnibus (GEO)GSE87778, and in Supplementary Data S1.

Cell cultureThe HCT116, DLD1, LOVO, SW620, SW480, and 293T embry-

onic kidney cells were obtained from the ATCC in 2013. TheTHC8307 cell line was a gift from the Institute of Hematology(CAMS & PUMC, Tianjin, China) in 2013. All cell lines werecultured in DMEM (Life Technologies) supplemented with 10%FBS (Life Technologies)with 5%CO2at 37�C.All cell lines used inthis study were authenticated using short-tandem repeat profilingless than 6 months ago when this project was initiated in 2013,and the cells have not been in culture for more than 2 months.

Plasmid constructionThe flag-HDAC constructs were gifts from Prof. Binhua P. Zhou

(University of Kentucky, Lexington, KY). pBABE-puro-CDM,pBABE-puro-DSIM1/2, and pBABE-puro-CBX4 were generouslyprovided by Prof. Guo-Qiang Chen (Shanghai Jiao TongUniversity School of Medicine, China). Myc-tagged CDM,DSIM1/2, and CBX4 were cloned into the pCDNA3.1 vector.HDAC1, HDAC2, and HDAC3 were cloned into the pSIN-EF2-puro vector. The promoter regions of Runx1, Runx2, TGFB2,Slug, VIM, FN1, SOX2, VCAN, and ITGB8 were cloned into thepGL3-basic vector. The PLKO.1-puro vector was used to clonethe shRNAs targeting CBX4, Runx2, or Slug. The sequences usedfor cloning the indicated shRNAs are shown in SupplementaryMaterials and Methods.

RNAi treatmentThe sequence targeting HDAC3, 50-GCATTGATGACCA-

GAGTTA-30, has been used previously (21). Transfection wasperformed according to the manufacturer's instructions usingLipofectamine RNAiMAX transfection reagent (Invitrogen) and50 nmol/L siRNA.

RNA extraction and qRT-PCRBriefly, total RNA was isolated using TRIzol reagent (Invitro-

gen) according to the manufacturer's instructions. First-strandcDNA was synthesized using the Revert AidTM First StrandcDNA Synthesis Kit (MBI Fermentas). The primers used toamplify the indicated genes are shown in SupplementaryMaterials and Methods.

The luciferase reporter assayBriefly, the cells were plated in 12-well plates at a density of

1.4� 105 cells per well then transfected with 0.8 mg of promoter-luciferase plasmid. To normalize the transfection efficiency, thecells were also cotransfected with 8 ng of pRL-CMV (Renillaluciferase). After transfection for 48 hours, the luciferase activitywas measured using a Dual-Luciferase Assay kit (Promega). Threeindependent experiments were performed, and the calculatedmeans and SDs are presented. The primers used for cloning theindicated promoters are shown in Supplementary Materials andMethods.

Western blotting and coimmunoprecipitationBriefly, cells were collected and lysed by RIPA buffer (150

mmol/L NaCl, 0.5% EDTA, 50 mmol/L Tris, 0.5% NP40) andcentrifuged for 20 minutes at 12,000 rpm and 4�C. Fifty micro-grams of harvested total protein was loaded and separated on the8% SDS–polyacrylamide gradient gel. The proteins were thentransferred onto polyvinylidene difluoride membranes andblocked with 5% non-fat milk for 2 hours at room temperature.The membranes were incubated with primary antibody andhorseradish peroxidase–conjugated secondary antibody, and pro-teinswere thendetected using the ECL chemiluminescence system(Pierce).

For coimmunoprecipitation, the clarified supernatants werefirst incubated with anti-FLAG-agarose (Sigma Aldrich) gels for2 hours or overnight at 4�C, and the precipitates were washed fivetimes with RIPA. To investigate the interaction between endog-enous CBX4 and HDAC3, the clarified supernatants were firstincubated with an anti-CBX4 antibody for 2 hours at 4�C. ProteinA/G-agarose was then added for 2 hours or overnight, and theprecipitates were washed five times with RIPA and analyzed byWestern blotting. The antibodies used in this work are shown inSupplementary Materials and Methods.

Chromatin immunoprecipitation assayThis procedure was performed as described by the ChIP kit

(Millipore, 17-10085 & 17-10086). Briefly, 15-cm plates wereseeded with cells of each of the tested cell lines and allowed togrow to 70%–80% confluence. To fix cells, complete cell fixativesolution (1/10th the volume of growth medium volume) wasadded to the existing culture medium. The fixation reaction wasstopped by adding stop solution (1/20th the volume of growthmedium volume) to the existing culture medium. The cells werecollected by centrifugation, and the nuclear pellet was resus-pended in chromatin immunoprecipitation (ChIP) buffer. Thecell lysate was subjected to sonication and then incubated with 5mg of antibodies overnight, followed by incubation with theprotein A/G agarose beads overnight at 4�C. BoundDNA–proteincomplexes were eluted, and cross-links were reversed after a seriesof washes. The purified DNA was resuspended in TE buffer forPCR. The primers for the indicated promoters are shown inSupplementary Materials and Methods.

MTT assayA 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bro-

mide (MTT) assay was used to measure cell viability. Briefly,HCT116, THC8307, or DLD1 cells were seeded at a density of2,000 cells per well in a 96-well microplate. The cells wereincubated with MTT for 4 hours, and the optical density (OD)was detected at 490 nm with the microplate reader once per day

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for 5 days. The results are presented as the mean � SD of threeindependent experiments.

Soft agar colony formation assayHCT116, THC8307, or DLD1 cells in a single-cell suspension

were plated in 0.3%agarose over the 0.6%agarose bottom layer in6-well plates at the density of 1 � 104 cells per well, and wereincubated for 10 days. The colonies containingmore than 50 cellswere counted.

Migration and invasion assaysTranswell assays using Boyden chambers containing 24-well

Transwell plates (BD Biosciences) with 8-mmpore size were usedto evaluate the migration and invasiveness of cells. All experi-ments were performed in duplicate and repeated three times.For the migration assay, the cell culture inserts were seeded with1.5� 105 (HCT116 cells), 3� 104 (THC8307 cells), and 1� 105

(DLD1 cells) in 200 mL of serum-free DMEM without an extra-cellular matrix coating. DMEMmedium containing 20% FBS wasadded to the bottom chamber. After approximately 24 hours ofincubation, the cells on the bottom surface of the filter were fixed,stained, and examined using amicroscope. For the invasion assay,the membrane was coated with 50 mL of 1:8 diluted Matrigel (BDBiosciences). After theMatrigel had solidified at 37�C for 2 hours,3 � 105 (HCT116 cells), 5 � 104 (THC8307 cells), of 2 � 105

(DLD1 cells) in 200 mL of serum-free DMEM were added to thecell culture inserts, whereas the bottom chamber was filled withDMEM containing 20% FBS. The Boyden chamber was thenincubated at 37�C in 5% CO2 for approximately 24 hours. Thecells were then stained and observed as described for the migra-tion assays.

Animal experimentsAnimal care and experiments were performed in strict accor-

dance with the "Guide for the Care and Use of LaboratoryAnimals" and the "Principles for the Utilization and Care ofVertebrate Animals" and were approved by the Animal ResearchCommittee of Sun Yat-sen University Cancer Center.

All animals were obtained from the Shanghai Institutes forBiological Sciences (Shanghai, China). Lung and liver metastasismodels were used. For the lung metastatic model, the indicatedcells were harvested andwashed twice with 1� PBS. The cells werethen suspended in PBS. Approximately 2 � 106 cells in 150-mLPBS were injected into the tail veins of 5-week-old male athymicmice. For the liver metastatic model, the spleen was accessed via a1-cm incision in the upper left lateral abdomen, and 2� 106 cellssuspended in 50 mL of PBS were injected into the distal tip of thespleen using an insulin syringe. The spleen was then placed backinto the abdomen, and the abdominal cavity was closed withsutures. All mice were sacrificed 10 weeks after the injection, andthe lungs or livers were harvested. The metastatic nodules in eachlung or liver were counted.

Human tissue specimensA total of 212 paraffin-embedded primary specimens were

obtained from the recruited colorectal carcinoma patients. Thepatients were diagnosed according to their clinicopathologiccharacteristics at the Sun Yat-sen University Cancer Center(Guangzhou, China), from 2001 to 2009 (115 colon carcinomapatients with ages from 19 to 83 years, 97 rectum carcinomapatients with ages from 25 to 75 years). No patients had received

radiotherapy and/or chemotherapy prior to surgery. Tumors werestaged according to the Union for International Cancer ControlTNM staging system. Resected specimens were macroscopicallyexamined to determine the location and size of a tumor, andspecimens for histology were fixed in 10% (v/v) formalin andprocessed for paraffin embedding. Informed consent wasobtained from all patients and approved by the research medicalethics committee of Sun Yat-sen University Cancer Center.

Immunohistochemical stainingImmunohistochemical staining was performed on 3-mm sec-

tions. The primary antibodies against CBX4or Runx2were diluted1:200 or 1:1,000, respectively, and then incubated at 4�C over-night in a humidified container. After three washes with PBS, thetissue slides were treated with a non-biotin horseradish peroxi-dase detection system according to manufacturer's instructions(Dako). The immunohistochemical staining was evaluated bytwo independent pathologists. The protein expression levels ofCBX4 and Runx2 were evaluated on the basis of thirteen scores.Generally, the CBX4 and Runx2 signals were detected in thenucleus. The staining intensity of CBX4 or Runx2 was stratifiedinto four classes in colorectal carcinoma tissues, namely 0, 1, 2,and 3, which were designated as absent, weak, moderate, andstrong signals, respectively. The percentage of stained cells wascategorized as 0, 1, 2, 3, and 4 to indicate no staining, 1%–10%,11%–50%, 51%–80% and 81%–100% stained cells, respectively.The score for each tissue was calculated by multiplying thestaining value by the percentage category value, and the averagescore from the two pathologists (M.F. Zhang and M. Li) was usedas the final score. The immunohistochemical cut-off for high orlowexpressionof indicatedmoleculewas determined through theROC curve analysis. The sensitivity and specificity for discrimi-nating dead or alive was plotted as IHC score, thus generating aROC curve. The cut-off value was established to be the point onthe ROC curve where the sum of sensitivity and specificity wasmaximized. Cancers with scores above the obtained cut-off valuewere considered to have high expression of indicated moleculeand vice versa.

Statistical analysisThe SPSS software (version 16.0, SPSS Inc.) was used for the

statistical analysis. The significance of differences was assessedusing two-tailed Student t test or a c2 test, as appropriate. Therelationship betweenCBX4expression, Runx2 expression, and theclinicopathologic parameters was examined using the Pearson c2

tests. The correlations between CBX4 expression, Runx2 expres-sion, and overall survival curves were assessed using Kaplan–Meier plots and compared with the log-rank test. Univariate andmultivariate Cox regression analyses were used to evaluate sur-vival data. Differences were considered significant when the Pvalues were <0.05.

Study approvalThe animal experimentswere approved by the Animal Research

Committee of Sun Yat-sen University Cancer Center and wereperformed in accordance with the established guidelines. The useof human colorectal carcinoma tissues was reviewed andapproved by the ethical committee of Sun Yat-sen UniversityCancer Center, and informed consent was obtained. The sampleswere retrospectively acquired from the surgical pathology archivesof Sun Yat-sen University Cancer Center.

Role of CBX4 in the Colorectal Carcinoma Metastasis

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ResultsCBX4 negatively regulates cancer metastasis in colorectalcarcinoma

CBX proteins may act as an oncogene or tumor suppressor in acell-type–dependent manner (4), and we have recently reportedthat CBX8 exerts paradoxical effects in colorectal carcinoma,promoting proliferation while suppressing metastasis (10).Therefore, we sought to determine the role of each CBX proteinin colorectal carcinoma metastasis because the molecularmechanisms of colorectal carcinoma progression urgently needto be elucidated (19, 20). Initially, following the strategy used inHCC (9), we ectopically expressed Flag-tagged CBX2, CBX4,CBX6, CBX7, or CBX8 in DLD1 cells and found that only CBX4suppressed cell migration (Supplementary Fig. S1A–S1C). In fact,the CBX4 protein levels differed among the tested colorectalcarcinoma cell lines (Fig. 1A). Next, we used two pairs of shorthairpin RNAs (shRNA) to dramatically reduce endogenous CBX4expression (Supplementary Fig. S2A and S2B). This knockdownsignificantly enhanced the migration and invasion of bothHCT116 and THC8307 cells, which endogenously express highlevels of CBX4 (Fig. 1B and C; Supplementary Fig. S2C and S2D).Conversely, the knockdown of CBX4marginally affected both cellviability and soft agar colony formation ability in these cell lines(Supplementary Fig. S2E–S2H). In contrast, the stably ectopicexpression of CBX4 dramatically inhibited the migration andinvasion in DLD1 cells, which endogenously express low levelsof CBX4 (Fig. 1D; Supplementary Fig. S3A and S3B). Moreover,the stable ectopic expression of CBX4 did affect neither theviability nor soft agar colony formation ability of these cells(Supplementary Fig. S3C and S3D). Knocking down CBX4 notonly consistently increased the number of metastatic nodules butalso enhanced the tumor size (Fig. 1E and F) when HCT116 cellswere injected into the tail veins of mice, and this increase wasrecapitulated in the hepatic metastasis in vivo animal model usingthese cells (Supplementary Fig. S4A and S4B). Taken together,CBX4 negatively regulates cell migration, invasion, and cancermetastasis, but not the cell proliferation, in colorectal carcinoma.

CBX4occupies theRunx2 promoter and represses its expressionAs a transcription repressor, CBX4 frequently targets the pro-

moters of downstream genes and mediates their epigenetic mod-ification (6). To investigate the molecular mechanism of CBX4 incolorectal carcinoma, the global transcriptomes were analyzed inHCT116 cells inwhichCBX4was knocked downwith shRNA, andthese transcriptomes were compared with those of HCT116 cellstransfected with scrambled shRNA (GEO accession number:GSE87778). Intriguingly, gene ontology (GO) or Kyoto Encyclo-pedia ofGenes andGenomes (KEGG)pathway analyses indicatedthat the genes upregulated by knocking down CBX4 were mainlyassociated with cell adhesion, migration, and the extracellularmatrix (Fig. 2A; Supplementary Fig. S5A and S5B; SupplementaryData S1). Indeed, the expression of factors that are well knownto be related to metastasis, such as Slug, vimentin, and fibronectin(FN), increased by knocking down CBX4 in colorectal carcinomacells, as shown in both the qRT-PCR assay (Fig. 2B) and theWestern blots (Fig. 2C). Accordingly, the ectopic expression ofCBX4 decreased the levels of Slug, vimentin, and FN in DLD1 cells(Fig. 2D).

A luciferase reporter assay was then used to further identify thedirect downstream target of CBX4. Among the upregulated genes

shown in Fig. 2B, only the promoter activity of Runt-relatedtranscription factor 2 (Runx2) was significantly increased uponCBX4 knockdown (Supplementary Fig. S5C). Furthermore, asshown in Fig. 2E, CBX4 was associated with the Runx2 promoteraccording to a chromatin immunoprecipitation (ChIP) assayusing the p16 (INK4a/ARF) promoter and the GAPDH promoteras positive and negative controls (6, 10). As shown in Fig. 2F,changes in CBX4 expression (knockdown and ectopic expres-sion), respectively, increased and decreased the Runx2 levels incolorectal carcinoma cells. Taken together, these data indicate thatCBX4 can downregulate Runx2 by occupying its promoter incolorectal carcinoma cells.

The effect of CBX4 on colorectal carcinomametastasis dependson Runx2

Next, we tested the dependence of the observed increase inmetastasis due to CBX4 knockdown on Runx2. Knocking downRunx2 using two pairs of shRNAs reduced the expression of Slug(Supplementary Fig. S6A and S6B), a downstream gene of Runx2(22), and, as expected, silencing Runx2 abolished the increase inSlug at both the protein and mRNA levels induced by knockingdown CBX4 in colorectal carcinoma cells (Fig. 3A and B). Moreimportantly, silencing Runx2 abrogated the increase in cell migra-tion, invasion, andmetastasis inducedby knockingdownCBX4 inHCT116 cells (Fig. 3C–E; Supplementary Fig. S7). Consistently,silencing Slug also reversed the increase of cell migration andinvasion induced by knocking down CBX4 (Supplementary Fig.S8). These results demonstrate that knocking down CBX4 pro-motes metastasis by abrogating its inhibition of Runx2.

CBX4 and Runx2 expression inversely correlates in colorectalcarcinoma tissues, andCBX4may be a newmarker of colorectalcarcinoma

The clinical significanceof the regulationofRunx2byCBX4wasthen evaluated in 212 colorectal carcinoma tissues by immuno-histochemical (IHC) staining. Using both anti-CBX4 and anti-Runx2 antibodies, which were highly specific (SupplementaryFig. S9A and S9B), 120 of 212 tissues were shown to express highlevels of CBX4, whereas 104 of 212 tissues expressed high levels ofRunx2. Moreover, the levels of these two proteins inverselycorrelated (Fig. 4A; Supplementary Table S1). In addition, higherlevels of CBX4 and Runx2 were associated with better and pooreroverall survival in colorectal carcinoma patients, respectively(Fig. 4B and C). More interestingly, the combination of a highlevel of CBX4 and a low level of Runx2 more accuratelypredicted the overall survival of colorectal carcinoma patients(Fig. 4D). In addition, the c2 test revealed that the CBX4 levelwas related to differentiation (P ¼ 0.010) and vital status (P ¼0.019; Supplementary Table S2), and the univariate and mul-tivariate analyses revealed that the CBX4 expression level (P ¼0.047), with a HR of 0.592 and a 95% CI of 0.353–0.992, wasan independent prognostic factor in patients with colorectalcarcinoma (Supplementary Table S3). These results indicatethat CBX4 may be a new marker to predict clinical outcomes incolorectal carcinoma patients.

CBX4 may act in colorectal carcinoma by decreasing H3K27acetylation (H3K27-Ac) at the Runx2 promoter

Because the functions of CBX proteins primarily depend ontheir chromodomain (23) and because SUMOE3 ligase activity iscritical for the key roles of CBX4 in HCC (9, 24), we sought to

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investigatewhether the functions of CBX4 in colorectal carcinomadepend on its SUMO E3 ligase or its polycomb role. As illustratedin Fig. 5A, the mutant of DSIM1/2-CBX4, in which the SUMO-interacting motifs (DSIM1/2) was depleted, lacked SUMO E3

ligase activity (9, 25), whereas the chromodomain mutant ofCDM-CBX4, which harbors a CBX4F11A and CBX4W35L doublemutation, no longer bound trimethylated H3K27 (H3K27-Me3)but maintained the proper structure of the protein (9, 16). Using

Figure 1.

CBX4 represses the migration, invasion, and metastasis of colorectal carcinoma cells. A, The CBX4 protein level was visualized in the indicated celllines by a Western blot analysis. B–D, The migration and invasion of HCT116 (B), THC8307 (C), and DLD1 (D) cells were determined as described in Materialsand Methods. Bars, SD. The results are expressed as the mean � SD of three independent experiments. � , P < 0.05; �� , P < 0.01 using Student t test. E and F,The lung metastasis of the indicated stable cell lines was measured in vivo in nude mice as described in Materials and Methods. E, Representative results ofgross (left) and hematoxylin and eosin (H&E) staining (middle, scale, �40; right, scale, �200) of the metastatic lung nodules. F, The statistical results (n ¼ 8).Bars, SD. � , P < 0.05 using Student t test.






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DLD1 cells that stably overexpressedwild-type CBX4 (WT-CBX4),DSIM1/2-CBX4, or CDM-CBX4, we unexpectedly found that bothmutants of CBX4 similarly inhibited Runx2 and its downstreameffector Slug, and cell migration and invasion among these cell

lines were comparable with the migration and invasion ofWT-CBX4 cells (Fig. 5B–D; Supplementary Fig. S10). Consistentwith these results, knocking down CBX4 had little effect on theH3K27-Me3 of the Runx2 promoter in HCT116 cells. However,

Figure 2.CBX4 directly downregulates Runx2 by occupying its promoter. A, Representative heatmaps from a global comparative transcriptome analysis indicating genesthat are upregulated upon CBX4 depletion. B, The relative mRNA levels of the indicated genes were normalized to the GAPDH level in the HCT116 cellsstably transfected with control or shRNA-CBX4 as determined by qRT-PCR. Bars, SD. The results are expressed as the mean � SD of three independentexperiments. � , P < 0.05; �� , P < 0.01 using Student t test. C, The indicated proteins were analyzed by Western blotting in HCT116 and THC8307 cells stablyexpressing control or shRNA-CBX4, as indicated. D, The indicated proteins were analyzed by Western blotting in DLD1 cells stably expressing pBABE-Vector orpBABE-CBX4. E, The ChIP assay was performed with HCT116 cells using an anti-CBX4 antibody or IgG antibody, as indicated. The p16 and GAPDH promoterswere used as the positive and negative controls, respectively. F, The indicated proteins were analyzed by Western blotting in HCT116 and THC8307 cells stablyexpressing control or shRNA-CBX4 and in DLD1 cells stably expressing pBABE-Vector or pBABE-CBX4.

Figure 3.

The promotion of cell migration and invasion after CBX4 knockdown primarily depends on Runx2. A and B, HCT116 cells stably expressing control,shRNA-CBX4, shRNA-Runx2, or both, as indicated, were analyzed by Western blotting (A) and qRT-PCR (B). C, A cell migration and invasion assay wasused in HCT116 cells stably expressing control, shRNA-CBX4, shRNA-Runx2, or both, as indicated. Bars, SD. The results are expressed as the mean � SD of threeindependent experiments. � , P < 0.05; ��, P < 0.01 using Student t test. D and E, An in vivo lung metastasis model was established in nude mice using HCT116cells stably expressing control, shRNA-CBX4, shRNA-Runx2, or both, as indicated, as described inMaterials andMethods.D,Representative results of gross (left) andH&E staining (middle, scale,�40; right, scale,�200) ofmetastatic lung nodules. E, The statistical results (n¼ 8). Bars, SD. � , P <0.05; �� , P <0.01 using Student t test.






the binding of H3K27-Ac, a super-enhancer marker, in the Runx2promoter was significantly enhanced by knocking down CBX4 inthese cells, which was confirmed by the dramatic increase of RNApolymerase II (Pol II) in the Runx2 promoter (Fig. 5E). Takentogether, CBX4may repress Runx2 expression, cell migration, andinvasion in colorectal carcinoma by decreasing H3K27-Ac at theRunx2 promoter locus, and this effect is independent of both theSUMO E3 ligase activity and the chromodomain of CBX4.

HDAC3 associates with CBX4 and specifically represses theRunx2 promoter via histone deacetylation

Histone deacetylase (HDAC) is mainly responsible for thedeacetylation of histone H3K27 (26), and HDAC2 has beenreported to be recruited to the CCNE1 promoter by CBX7 tosuppress CCNE1 expression in lung carcinoma (11). Weattempted to identify HDACs that may be recruited to the Runx2promoter by CBX4 to repress Runx2 expression. Interestingly,

among all 11 HDACs (fromHDAC1 to HDAC11), CBX4 stronglyboundHDAC1, HDAC2,HDAC3, andHDAC9when eachHDACwas tagged with Flag and cotransfected with Myc-tagged CBX4into 293T cells (Supplementary Fig. S11). Furthermore, HDAC1,HDAC2, and HDAC3, but not HDAC9, were detectable in thetested colorectal carcinoma cell lines (Supplementary Fig. S12).Moreover, HDAC3, but not HDAC1 or HDAC2, dramaticallyreduced the Runx2 promoter activity (Fig. 6A). Indeed, as shownin Fig. 6B and C, Runx2 expression was significantly reduced atboth the mRNA and protein levels in HCT116 cells stably over-expressingHDAC3, but not inHCT116 cells stably overexpressingHDAC1. Accordingly, the interaction between CBX4 and HDAC3was already evident at endogenous expression levels (Fig. 6D),and both mutants of CBX4-DSIM1/2 and CBX4-CDM did notaffect its binding with HDAC3 (Supplementary Fig. S13). Col-lectively, these data show that CBX4may interact with HDAC3 toinhibit the Runx2 promoter activity and repress Runx2 expression.

Figure 4.

The combination of CBX4 with Runx2 correlates with clinical prognosis in colorectal carcinoma. A, Representative immunohistochemical staining ofCBX4 and Runx2 from 212 paraffin-embedded colorectal carcinoma tissues. Scale bar, 100 mm. B and C, Overall survival curves were generated on the basisof the protein level of CBX4 (B) or Runx2 (C) in 212 paraffin-embedded colorectal carcinoma tissues. D, Comparison of the overall survival between 57 CBX4high/Runx2low and 73 CBX4low/Runx2high tissues. Actuarial probabilities were calculated using the Kaplan–Meier method and compared using the log-rank test.

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We also examined whether the HDAC3-mediated suppres-sion of the Runx2 promoter activity depends on CBX4. As shownin Fig. 6E, the knockdown of CBX4 abolished the inhibitionof Runx2 promoter activity by HDAC3. Moreover, using theChIP-qPCR assay, both HDAC1 and HDAC3, but not HDAC2,bound the Runx2 promoter, and the binding affinity of HDAC3,but not HDAC1, with the Runx2 promoter was dramaticallydecreased and increased by knocking down and ectopic expres-sion of CBX4, respectively (Fig. 6F; Supplementary Fig. S14Aand S14B). Indeed, silencing HDAC3 could reverse the sup-pression of Runx2 and H3K27-Ac mediated by overexpressionof CBX4 (Fig. 6G). However, cell migration and invasion werenot further enhanced by the overexpression of CBX4 in DLD1cells silenced of HDAC3, as the knockdown of HDAC3 alreadyinhibited the cell migration and invasion in DLD1 cells (Fig.6H; Supplementary Fig. S15). Therefore, HDAC3 is essential forthe functions of CBX4 in colorectal carcinoma. These resultsdemonstrate that CBX4 represses Runx2 expression by recruitingHDAC3 to sustain the deacetylation status of H3K27 at theRunx2 promoter.

The suppression of Runx2, cell migration, and invasion byCBX4 depends on its binding with HDAC3 in colorectalcarcinoma

To further assess the importance of the physical interactionwithHDAC3 for the functions of CBX4, we mapped the interactionregions of CBX4 for their ability to bind with HDAC3. A series offragments of CBX4 were generated, as illustrated in Fig. 7A, andtheir interactionwithHDAC3was tested.We found three separateregions of CBX4, residues 161–179 (I), 260–269 (II), and 551–560 (III), that are important for its binding with HDAC3 (Fig. 7A;Supplementary Fig. S16A–S16D). To determine the key regions ofCBX4 that are essential for this interaction, we further generatedseveral mutants of CBX4 by differently depleting regions I, II, andIII, as illustrated in Fig. 7B. The binding ofHDAC3with CBX4wascompletely abolished by DTM-CBX4, which lacked these threeregions, and this interaction was only partially diminished byDDM1-CBX4 and DDM2-CBX4, which each only lacked two ofthese three regions, whereas DDM3-CBX4 could not abolish thisinteraction, as shown in Fig. 7C.Notably, region III ofCBX4 is alsoinvolved in transcriptional silencing and binding to other PcG

Figure 5.

A decrease in the H3K27-Ac marker on the Runx2 promoter is responsible for the functions of CBX4 in colorectal carcinoma. A, The schematic illustrationfor CBX4 and its mutants used in this study. B and C, The indicated proteins or genes were analyzed by Western blotting (B) or qRT-PCR (C) in DLD1 cells stablyoverexpressing vector, WT-CBX4, DSIM1/2-CBX4, or CDM-CBX4, as indicated. Bars, SD. The results are expressed as the mean � SD of three independentexperiments. � , P < 0.05; �� , P < 0.01; �� , P < 0.001 using Student t test. D, DLD1 cells stably overexpressing vector, WT-CBX4, DSIM1/2-CBX4, or CDM-CBX4, asindicated, were subjected to migration and invasion assays, as described in Materials and Methods. Bars, SD. The results are expressed as the mean � SDof three independent experiments. �� , P < 0.01; �� , P < 0.001 using Student t test. E, The ChIP-qPCR analysis of the occupancies of H3K27-Ac, H3K27-Me3,and Pol II on the Runx2 promoter in stably transfected HCT116 cells, as indicated. Bars, SD. The results are expressed as the mean � SD of three independentexperiments. � , P < 0.05 using Student t test.






Figure 6.

CBX4 recruits HDAC3 to suppress the expression of Runx2. A, HCT116 cells were cotransfected with the Runx2-Luc reporter and Flag-HDAC1, Flag-HDAC2, orFlag-HDAC3 for 48 hours and then subjected to a luciferase activity assay as described in Materials and Methods. B and C, HCT116 cells stably expressing pSIN-Vector,pSIN-HDAC1, or pSIN-HDAC3 were analyzed by qRT-PCR (B) and Western blotting (C). Bars, SD. The results are expressed as the mean � SD of threeindependent experiments. �� , P < 0.001 using Student t test. D, The co-IP assay was performed in HCT116 cells expressing endogenous levels of CBX4 usinganti-CBX4 antibody or anti-IgG antibody as indicated. E, HCT116 cells stably expressing control or shRNA-CBX4 were cotransfected with the Runx2-Luc reporterandFlag-HDAC3 for 48hours and then subjected toa luciferase activity assay asdescribed inMaterials andMethods.Bars, SD. The results are expressedas themean� SDof three independent experiments. �� , P < 0.01 using Student t test. F, The ChIP-qPCR analysis of the occupancies of HDAC1, HDAC2, or HDAC3 on theRunx2 promoter inHCT116cells stably expressingcontrolor shRNA-CBX4.Bars, SD. The resultsare expressedas themean�SDof three independent experiments. �� ,P<0.01usingStudent ttest.G andH,DLD1 cells stably overexpressing Vector or CBX4were transfectedwith control orHDAC3 siRNA for 48 hours, then the indicated proteinswere analyzed byWestern blotting (G) and the cell migration and invasion were determined as described in Materials and Methods (H).


Wang et al.




Figure 7.

The key regions that facilitate binding between CBX4 and HDAC3 are required for the functions of CBX4 in colorectal carcinoma. A, Schematic illustration ofCBX4 mutants with the indicated HDAC3-binding capability. þ, binding; �, no binding based on the results shown in Supplementary Fig. S16A–S16D. Thethree HDAC3 binding regions of CBX4 are indicated by a blue box. I, residues 161–170; II, residues 260–269; and III, residues 551–560. B, The schematic illustration ofCBX4 mutants in which the indicated regions were depleted; DC represents the depletion of III, DDM1 represents the depletion of I and II, DM2 represents thedepletion of II and III, DDM3 indicates the depletion of I and III, and DTM represents the depletion of I, II, and III. C,HEK293T cells were cotransfectedwith Myc-HDAC3with vector, Flag-CBX4-WT, Flag-CBX4-DDM1, Flag-CBX4-DTM, Flag-CBX4-DDM2, or Flag-CBX4-DDM3 for 48 hours and then lysed and analyzed byimmunoprecipitation (IP) using Flag-agarose and Western blotting. D, The indicated proteins were analyzed by Western blotting in DLD1 cells stablyexpressing vector, CBX4-WT, CBX4-DDM1, CBX4-DTM, or CBX4–DC, as indicated. E, Cell migration and invasion were assessed in DLD1 cells stably expressingvector, CBX4-WT, CBX4-DDM1, CBX4-DTM, or CBX4–DC, as indicated and as described in Materials and Methods. Bars, SD. The results are expressed as themean � SD of three independent experiments. � , P < 0.05; �� , P < 0.01; �� , P < 0.001, using Student t test. F, A proposed model illustrating the regulation ofcolorectal carcinoma metastasis by CBX4. The repressive complex containing CBX4 and HDAC3 sustains the deacetylation status of H3K27 in the Runx2promoter to repress Runx2 expression in colorectal carcinoma. However, the downregulation of CBX4 in colorectal carcinoma reduces HDAC3 in thisrepressive complex to maintain a high level of H3K27-Ac, which consequently activates Runx2 transcription to promote metastasis. CRC, colorectal carcinoma.






proteins (27). Indeed, DC-CBX4 lacked region III and was able toabolish the binding of CBX4 with Bmi1 (SupplementaryFig. S17A), which is the indicator for CBX4 within the PRC1complex. Conversely, DDM1-CBX4, which lacked both regions Iand II, did not affect the binding of CBX4 with Bmi1 (Supple-mentary Fig. S17B). Because region III of CBX4 is required forCBX4 toparticipate in the PRC1 complex (14, 28),we investigatedwhether the participation of CBX4 in the PRC1 complex is crucialfor the function of CBX4 in colorectal carcinoma. Using DLD1cells stably overexpressing WT-CBX4, DDM1-CBX4, DC-CBX4, orDTM-CBX4, we found that bothWT-CBX4 andDC-CBX4 similarlyinhibited Runx2, cell migration, and invasion. In contrast, theseeffects were abrogated in both DDM1-CBX4 and DTM-CBX4 cells(Fig. 7D and E; Supplementary Fig. S18). Taken together, theseresults strongly indicate that the suppression of Runx2, cellmigration, and invasion in colorectal carcinoma byCBX4 stronglydepend on its binding with HDAC3, but not on its binding withthe PRC1 complex.

DiscussionIn this report, we demonstrated that CBX4 may function as a

tumor suppressor to impair Runx2 transcription by recruitingHDAC3 to deacetylate histone H3K27-Ac at the Runx2 pro-moter in colorectal carcinoma. This evidence is the first toshow that CBX4 exerts a noncanonical function by recruitingHDAC3 to mediate histone deacetylation, which inhibits genetranscription. Furthermore, this novel function of CBX4 isindependent on its SUMO E3 ligase, its chromodomain, andthe PRC1 complex.

Traditionally, the PRC2 complex promotes H3K27-Me3, andthe PRC1 complex then binds to H3K27-Me3 via its chromobox(CBX) proteins (1, 4). Therefore, CBX4 participates in the PRC1complex, and its functions largely depend on the chromodomain(4, 6). For instance, CBX4 has been shown to repress transcriptionby binding the promoters of several target genes, such as p16(INK4a/ARF; ref. 6) andGATA4/GATA6 (29).However, it has beenreported that certain CBX proteins, such as CBX4 (6, 9) and CBX8(30), can associate with protein complex other than PRC1,thereby playing a PRC1-independent role in transcriptional reg-ulation (23, 30). Intriguingly, we have shown here that CBX4represses Runx2 transcription in colorectal carcinoma by inhibit-ing H3K27-Ac markers but not promoting H3K27-Me3 markersand by recruiting HDAC3 at the Runx2 promoter, which conse-quently impairs cellmigration and invasion. Both the recruitmentof HDAC3 and the repression of cell migration and invasion byCBX4 are independent of its SIMs, its chromodomain, and thePRC1 complex. These new findings strongly suggest that theinteraction with HDAC3 is pivotal for the function of CBX4 incolorectal carcinoma metastasis, as proposed in Fig. 7F. Notably,DC-CBX4 had a much higher protein level than WT-CBX4, indi-cating that the C-terminal 10 amino acidsmay be critical for CBX4protein stability. Because these 10 amino acids are essential forbinding with the PRC1 core subunits, such as Ring1a, Ring1b andBmi1, we speculate that the E3 ligase activity of Ring1a and/orRing1b or an unknown E3 ligase may degrade CBX4 in the intactPRC1 complex. This phenomenon is currently under investiga-tion in our laboratory.

Because of its exclusive SUMO E3 ligase activity, CBX4 alsopromotes the SUMOylation of HIF-1a, CtBP1, and ZEB1 (9, 15,31). Recently, CBX4 has been proposed to play an oncogenic role

in HCC by positively regulating proliferation (17), angiogenesis(9) and cancer metastasis (24), and this function appears to beinvolved in two SIMs of CBX4 (9). However, our results showedthat CBX4 may be a tumor suppressor in colorectal carcinoma bytargeting Runx2 because knocking down and ectopically over-expressing CBX4 in colorectal carcinoma cell lines, respectively,increased and decreased cell migration, invasion, and metastasis.The reverse correlation between CBX4 and Runx2was observed incolorectal carcinoma tissues, and the combination of CBX4high

and Runx2low is a good marker to better predict the overallsurvival of patients with colorectal carcinoma. In fact, CBX4 exertsan antioncogenic effect by repressing the expression of c-myc andcellular transformation (32). Furthermore, CBX4 associates withE2F and Rb to block entry into mitosis by inhibiting cyclin A andcdc2 transcription, which in turn negatively regulate cell prolif-eration (33). Therefore, CBX4 can likely exert both oncogenic andantioncogenic functions depending on the cancer type and itsinteracting partners. This pleiotropic nature is not surprisingbecause CBX7 also plays diverse roles in different cancer types(11–13, 34). CBX7 recruitsHDAC2 to the promoter of theCCNE1gene and inhibits histone acetylation to consequently suppressCCNE1 expression and lung carcinoma progression (11). Con-versely, CBX7 is oncogenic in lymphoma and gastric cancerbecause it represses the p16 (INK4a/ARF) tumor suppressor locus(12, 13).

Cancer metastasis is a common cause of cancer-related death,and agents targeting the epithelial-to-mesenchymal transition(EMT) or angiogenesis, which are crucial for metastasis, arepotential drugs for cancer therapy (22, 35). Runx family pro-teins play an important role in cancer progression (35). MostRunx family genes, such as Runx1 and Runx3, have been shownto have tumor suppressor activities (35–37), whereas Runx2 isunique due to its oncogenic features. Runx2 has been welldocumented to induce invasion and metastasis in differentcancers, such as breast cancer (35), prostate cancer (38), thyroidcancer (39, 40), and colorectal carcinoma (19, 41), and thiseffect is likely mediated by the regulation of EMT-relatedmolecules (Slug and Twist), MMPs, and angiogenic factors(VEGFA and VEGFC; ref. 22). Our results showed that CBX4recruits HDAC3, but not HDAC1, to the Runx2 promoter toimpede cell migration and invasion in colorectal carcinoma.Furthermore, our data demonstrate that the interactionbetween CBX4 and HDAC3 is required for aforementionedfunctions in colorectal carcinoma, indicating that stabilizingthe binding of CBX4 with HDAC3 with a specific agent, such asa small molecule, may be a good strategy to treat colorectalcarcinoma patients harboring metastases.

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

Authors' ContributionsConception and design: X. Wang, T. KangDevelopment of methodology: T. KangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Wang, L. Li, Y. Wu, G. Wang, G. Qin, T. KangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. WangWriting, review, and/or revision of themanuscript: X.Wang, R.-H. Xu, T. KangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Zhang, M. Zhang, D. Liao, T. KangStudy supervision: T. Kang

Wang et al.





AcknowledgmentsWe thank both Prof. Binhua P. Zhou (University of Kentucky) and Prof. Guo-

Qiang Chen (Shanghai Jiao Tong University School of Medicine, China) for thekind gifts of plasmids.

Grant SupportThis work received support from the Yangtze River Scholarship (85000-

52121100 to T. Kang), theNational Key Research andDevelopment Program of

China (no: 2016YFA0500304 to T. Kang), and the National Nature ScienceFoundation in China (NSFC; 81530081, 31571395 to T. Kang).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 1, 2016; revised October 14, 2016; accepted October 31,2016; published OnlineFirst November 18, 2016.

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2016;76:7277-7289. Published OnlineFirst November 18, 2016.Cancer Res Xin Wang, Liping Li, Yuanzhong Wu, et al. Runx2 Promoter in Colorectal CarcinomaCBX4 Suppresses Metastasis via Recruitment of HDAC3 to the

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