histone deacetylase inhibitors repress tumoral expression of the … · obtained from dr. adriana...

Therapeutics, Targets, and Chemical Biology

Histone Deacetylase Inhibitors Repress TumoralExpression of the Proinvasive Factor RUNX2Valentina Sancisi1, Greta Gandolfi1, Davide Carlo Ambrosetti2, and Alessia Ciarrocchi1

Abstract

Aberrant reactivation of embryonic pathways occurs common-ly in cancer. The transcription factor RUNX2 plays a fundamentalrole during embryogenesis and is aberrantly reactivated duringprogression and metastasization of different types of humantumors. In this study, we attempted to dissect the molecularmechanisms governing RUNX2 expression and its aberrant reac-tivation. We identified a new regulatory enhancer element, locat-ed within the RUNX2 gene, which is responsible for the activationof the RUNX2 promoter and for the regulation of its expression incancer cells. Furthermore, we have shown that treatment with the

anticancer compounds histone deacetylase inhibitor (HDACi)results in a profound inhibition of RUNX2 expression, which isdetermined by the disruption of the transcription-activating com-plex on the identified enhancer. These data envisage a possibletargeting strategy to counteract the oncongenic function ofRUNX2 in cancer cells and provide evidence that the cytotoxicactivity of HDACi in cancer is not only dependent on the reac-tivation of silenced oncosuppressors but also on the repression ofoncogenic factors that are necessary for survival and progression.Cancer Res; 75(9); 1868–82. �2015 AACR.

IntroductionRUNX2, a transcription factor belonging to the Runt-related

family, is necessary for osteoblast differentiation and skeletalmorphogenesis (1, 2). Factors that are crucial during embryogen-esis are often hijacked during cancer progression, and RUNX2 isnot an exception. RUNX2 is increasingly recognized in cancerbiology for its oncogenic properties and a large number of articleslink the deregulation of RUNX2 expression with progression andmetastasization of different types of epithelial tumors (1, 3–18).Regulation of RUNX2 may occur at multiple levels and throughmultiple signaling pathways. However, the mechanisms thatregulate the reactivation of this factor in cancer are still unknown.It has been reported that RUNX2 expression is higher in tumorthan in normal tissue and that the level of expression of thisprotein has a negative prognostic value in a number of cancertypes (16, 19–22). The RUNX2 gene encodes for two isoformsstarting from two separate promoters: the proximal P2 promoterexpresses RUNX2 isoform I and the distal P1 promoter expressesisoform II (23, 24). The two isoforms differ for only a fewaminoacids at the N-terminal regions, even though differencesin their activity were reported. The existence of two alternativepromoters suggests that the expression of the two isoforms is

regulated through different mechanisms (24–27). Indeed, evi-dence exists that the use of the two promoters is context depen-dent. In particular, our group and others have shown that RUNX2isoform I is the major RUNX2 isoform in tumor cells, whileisoform II seems to be restricted to skeletal cells (16, 26). Notice-ably, the majority of molecular signals known to control RUNX2act through the P1 promoter, while the regulation of the P2promoter remains largely unknown (28–30). Dissecting themechanisms controlling RUNX2 P2 promoter is a necessary stepto elucidate the complex network of molecular determinants thatgovern RUNX2 expression in tumor and it may help designing anappropriate therapeutic approach to counteract the prometastaticfunction of RUNX2.

Histone deacetylase inhibitors (HDACi) are a class of chemicalcompounds that block the activity of Zn-dependent HDAC,inducing the hyperacetylation of a number of proteins (31).Histone acetylation is associated with open chromatin structureand active transcription. Thus, it is believed that the majoranticancer effect of HDACi is due to the reactivation of silentoncosuppressor genes. However, increasing evidence indicatesthat, besides histones, HDACi treatment affects acetylation andfunction of a number of nonhistonic proteins, opening newexplanations to the mechanisms of action of these cancer drugs(32). Recent studies have shown that HDACi suppress the expres-sion of a number of oncogenes that are highly expressed in cancer,supporting the hypothesis that besides reinducing suppressorgenes, these drugs may impair cancer cell growth by blockingoncogenic signals that are necessary for tumor survival andprogression (33–35).

In this study, we demonstrate for the first time that HDACiprofoundly impairs the expression of RUNX2 isoform I in tumorcells. In the attempt to dissect the mechanism responsible for theHDACi-dependent RUNX2 inhibition, we identified a newenhancer within the RUNX2 gene, which is responsible for theactivation of the P2 promoter in tumor cells and contains theHDACi-responsive elements. We have shown that c-JUN binds tothe ENH3 and is a positive regulator of RUNX2 expression.

1Laboratory of Translational Research, Research and Statistic Infra-structure, Arcispedale S. Maria Nuova-IRCCS, Reggio Emilia, Italy.2Laboratory of Molecular Biology, Department of Pharmacology andBiotechnology (FaBiT), University of Bologna, Bologna, Italy.

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

Corresponding Authors: Alessia Ciarrocchi, Laboratory of TranslationalResearch, Arcispedale S. Maria Nuova-IRCCS, Viale Risorgimento 80, 42123Reggio Emilia, Italy. Phone: 39-0522-295668; Fax: 39-0522-295454; E-mail:[email protected]; and Valentina Sancisi,[email protected]

doi: 10.1158/0008-5472.CAN-14-2087

�2015 American Association for Cancer Research.

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Published OnlineFirst March 13, 2015; DOI: 10.1158/0008-5472.CAN-14-2087

http://cancerres.aacrjournals.org/

Finally, we have shown that inhibition of HDAC6 is implicated inthe repressive effects of HDACi.

Materials and MethodsCell cultures and treatments

BCPAP and TPC1 cell lines were obtained from Prof. Mas-simo Santoro (University of Naples, Naples, Italy). MCF7,A549, H1299, HCT116, PC3, DU145, and SAOS2 cell lineswere obtained from Dr. Massimo Broggini (IRCCS-Istituto diRicerche Farmacologiche Mario Negri, Milan, Italy). A375,MDA-MB-231 cell lines and colon H2998 cell line wereobtained from Dr. Adriana Albini and Dr. Bruno Casali, respec-tively (Arcispedale Santa Maria Nuova-IRCCS, Reggio Emilia,Italy). All cell lines were authenticated by SNP profiling atMultiplexion GmbH; dates of last authentication reports areDecember 9, 2014, and November 12, 2014. All cell lines weregrown at 37�C/5% CO2. A549, H1299, PC3, DU145, andH2998 were grown in RPMI with 10% fetal bovine serum.HCT116 were grown in Iscove medium with 10% fetal bovineserum. The remaining lines were grown in DMEM with 10%fetal bovine serum.

Unless otherwise specified, cells were treated for 24 hourswith trichostatin A (TSA) 1 mmol/L or suberoylanilide hydro-xamic acid (SAHA; Vorinostat) 10 mmol/L (Sigma-Aldrich).Tubacin, 5-Aza-20-deoxycytidine (5AZA), actinomycin D, cyclo-heximide, and DMSO were purchased from Sigma-Aldrich.

Quantitative real time-PCRTotal RNA was purified with RNAeasy Mini Kit (Qiagen) and

retrotranscribed using the iScript cDNA kit (Bio-Rad). Quantita-tive real time-PCR (qRT-PCR) was conducted using Sso FastEvaGreen Super Mix (Bio-Rad) in the CFX96 Real Time PCRDetection System (Bio-Rad).

Western blot analysisWestern blot analysis was performed as previously described

(16). Antibodies used were goat anti-RUNX2 (AF2006; R&D Sys-tems), rabbit anti-c-JUN (H-79, sc-1694; Santa Cruz Biotechno-logy, DBA Italia), mouse anti-actin (AC-15; Sigma-Aldrich),horseradish peroxidase–conjugated anti-goat (sc-2350; Santa CruzBiotechnology), anti-rabbit and anti-mouse (GE Healthcare).

Transfection and luciferase assayCellswere cotrasfectedwith apGL3 reporter vector and thepRL-

TK vector (Promega) as a control, using Lipofectamine LTX (LifeTechnologies). Forty-eight hours after transfection, cells wereharvested and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) in a GloMax 20/20Luminometer (Promega), according to the manufacturer'sinstructions. For each sample, firefly luciferase activity was nor-malized on Renilla luciferase activity and transactivation of thevarious reporter constructs was expressed as fold induction onpGL3-basic activity.

Bioinformatic analysisFor the identification of putative enhancers, we analyzed a 300-

kb genomic region spanning the RUNX2 gene using ENCODEproject annotations (36), integrated in Genome Browser (http://genome.ucsc.edu/; refs. 37, 38). Putative enhancer regionswere identified on the basis of conservation in mammals, the

presence of DNAse I hypersensitivity clusters, and the presence ofspecific histone modifications associated with enhancer regions(H3K27Ac and H3K4Me1). Transcription factors–binding sitesanalysis on the ENH3 core was performed by merging the datafrom the open-source JASPAR database (http://jaspar.genereg.net/) and by the data retrieved from the TRANSFAC Professionaldatabase (http://www.biobase-international.com/) using theMatch algorithm.

DNase sensitivity assayChromatin accessibility of the P2 promoter and ENH1, ENH2,

and ENH3 was assessed using the EpiQ Chromatin Analysis Kit(Bio-Rad) according to the manufacturer's protocol.

Chromatin immunoprecipitationAfter cross-linking with 1% formaldehyde, chromatin was

sonicated using a Sonopuls HD2070 sonicator (Bandelin) andprecipitated with anti-RNA-PolII antibodies (ab817; Abcam),rabbit anti-c-JUN antibodies (H-79, sc-1694X; Santa Cruz Bio-technology), or with mouse and rabbit IgG (Santa Cruz Biotech-nology) as control, and the immunoprecipitated DNA fragmentswere quantified by qPCR.

Electromobility shift assayS3A wild-type (WT) and mutant probes were obtained by

Integrated DNA Technologies. Probe was radiolabeled with[g-32P]-ATP (PerkinElmer) using T4 polynucleotide kinase (NewEngland Biolabs) and purified on a Chromaspin-30 column(Clontech Laboratories). Radiolabeled probe was incubated withor without nuclear extract in the presence of 1 mg of dI-dC (Sigma-Aldrich). Binding reactions were resolved on a nondenaturantpolyacrylamide gel.

DNA pulldownFifty microliters of streptavidin-conjugated Dynabeads M-280

(Life Technologies) was incubated with 20 pmol of biotinylatedWTorMut1þ2S3Aprobe (IntegratedDNATechnologies). Boundproteins were eluted in SDS sample buffer and resolved onSDS-PAGE.

siRNA transfectionsc-JUN Stealth RNAi (20 nmol/L) and control oligos (Life

Technologies) or 30 nmol/L of TriFECTa RNAi against HDAC6and control oligos (Integrated DNA Technologies) were trans-fected using RNAiMax Lipofectamine (Life Technologies). Forcotransfection of plasmids and siRNA, Lipofectamine 2000 (LifeTechnologies)wasused and the cellswereharvested48hours aftertransfection for luciferase assay.

Statistical analysisStatistical analysis was performed using GraphPad Prism Soft-

ware (GraphPad). Statistical significance was determined usingthe Student t test. Each experiment was replicated from a minu-mum of two times to a maximum of five times. CooperationbetweenMut1 andMut2 was assessed by F statistics in an ANOVAsetting.

Additional materials and methods are provided in the Supple-mentary Data.

HDACi Repress RUNX2 Expression in Tumor Cells

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Sancisi et al.

Cancer Res; 75(9) May 1, 2015 Cancer Research1870




ResultsHDACi inhibit RUNX2 expression in thyroid cancer cell lines

Reactivation of crucial factors during cancer progressionmaybedetermined by changes in chromatin organization. We evaluatedthe effect of chromatin remodeling agents on RUNX2 expressionin two different papillary thyroid cancer (PTC) cell lines. TPC1and BCPAP cells were treated with 5AZA, inhibitor of DNAmethyltransferases, andwith TSA and SAHA, inhibitors ofHDACs(Fig. 1A–D). In both cell lines, treatment with TSA and SAHAresulted in a strong inhibition of RUNX2 expression at themRNAand protein level, whereas 5AZA displayed only marginal effectsas compared with control.

We analyzed the kinetic of HDACi effect on RUNX2 expression(Fig. 1E and F). In both cell lines, RUNX2mRNA levels decreasedrapidly after TSA exposure and the effect was already detectableafter 3 hours. To assess the reversibility of the HDACi effect onRUNX2 expression, TPC1 and BCPAP cells were treated with TSAfor 6 hours and thenmoved to TSA-free medium (Fig. 1G andH).In both cell lines, inhibition of RUNX2 was completely rescuedafter 6 hours from TSA withdrawal. The rapidity of the TSAmediated inhibition and the ready reversibility of the effectsuggested thatHDACi control RUNX2 expression through a directmechanism. To test this hypothesis, BCPAP and TPC1 cells weretreated with TSA in the presence or absence of actinomycin D thatinhibits transcription, and cycloheximide that blocks proteinsynthesis (Fig. 1I and J). Blockade of transcription by actinomycinD completely abolishes the effect of TSA on RUNX2 expression inboth cell lines, demonstrating that repression of RUNX2 by TSArequires active transcription. In contrast, the inhibition of proteinsynthesis by cycloheximide did not block the TSA-mediatedRUNX2 repression, demonstrating that the HDACi effect wasmostly direct and did not require synthesis of new factors.However, in BCPAP cells, cycloheximide treatment slightlyreduced the repressive effect of TSA, suggesting the possibleinvolvement of additional undirect events in this mechanism.Next, we investigated the effect of HDACi on RUNX2 mRNAstability. TPC1 and BCPAP cells were pretreated with TSA for 30minutes and then actinomycin D was added to halt transcription(Fig. 1K and L). In both cell lines, degradation rate was similar,independently from the TSA pretreatment, indicating that HDACido not have significant effect on RUNX2 mRNA stability. Inaddition, luciferase assay has shown that TSA did not target theRUNX2 30-UTR, thus ruling out the possibility that HDACi

induced the expression of noncoding RNAs that may affectRUNX2 stability (Supplementary Fig. S1A and S1B).

Next, we tested the effect of TSA on RUNX2 promoter activity.We previously have shown that isoform I is the major RUNX2isoform expressed in thyroid cancer patients (16). RT-PCR anal-ysis confirmed that isoform II was not detectable in either TPC1orBCPAPuntreated cells and that TSAdidnot affect its expression. Incontrast, isoform I was strongly repressed by TSA in both cell lines(Fig. 2A andB). Thus,we focusedour analysis on the P2promoter.The full-lengthRUNX2P2promoter, and a shorter regionof about400 bp encompassing the transcriptional starting site (TSS; sP2)were cloned upstream of a luciferase reporter gene and theirtranscriptional activity was assessed in the presence or absenceof TSA (Fig. 2C). Surprisingly, TSA treatment induced a strongactivation of both the full-length and sP2 promoter in both celllines. This observationwas in conflictwith the expressiondata andsuggested that the HDACi-responsive elements were not locatedwithin the P2 promoter.We also noticed that the P2promoterwasnot able to induce a significant activation of the reporter gene inuntreated cells, and that there was no significant difference in theactivity of the full-length and of the short version of the P2promoter.

A new enhancer controls RUNX2 expression in thyroid cancercells

On the basis of this evidence, we hypothesized the existence ofunknown DNA regulatory regions outside the P2 promoter. Weanalyzed the annotation data of the ENCODE project to identifyDNA regions within the RUNX2 locus with features of regulatoryelements (36–38). Three potential enhancers (ENH) were iden-tified and named ENH1, ENH2, and ENH3 (Fig. 2D). Figure 2Eshows the chromatin accessibility of these regions assessed byDNase sensitivity assay. In both cell lines, ENH1 and ENH2showed partial accessibility, whereas ENH3 displayed a fullyaccessible chromatin structure, comparablewith the P2 promoter.Next, the DNA fragments corresponding to ENH1, 2, and 3 werecloned into the pGL3-P2 vector downstream of the luciferasereporter gene and their ability to activate the P2 promoter wasassessed by luciferase assay (Fig. 2F). Only ENH3 determined astrong induction of the reporter gene, suggesting a potent activityof this element on the P2 promoter. Noticeably, the activatingeffect of ENH3 on the P2 promoter was stronger in TPC1 than inBCPAP cells, in line with the higher level of RUNX2 expressiondetected in TPC1 as compared with BCPAP cells (Fig. 2B; ref. 16).

Figure 1.HDACi repress RUNX2 expression. A and B, qRT-PCR analysis of RUNX2 mRNA levels in TPC1 and BCPAP cells treated with two concentrations of 5AZA for theindicated time. The bars represent the average fold change of RUNX2 in treated cells as compared with untreated cells (DMSO, Mock). C and D, qRT-PCRanalysis of RUNX2mRNA levels in TPC1 andBCPAP cells treatedwith three concentrations of TSA andSAHA for 24 hours. The bars represent the average fold changeof RUNX2 in treated cells as compared with untreated cells (Mock). Insets, Western blot analysis of RUNX2 and actin levels in TPC1 and BCPAP cellsuntreated (Mock) or treatedwith TSAor SAHA. E and F, qRT-PCR analysis of RUNX2mRNA levels in TPC1 andBCPAP cells treatedwith 1mmol/L TSA for the indicatedtime. The bars represent the average fold change of RUNX2 in TSA-treated cells as compared with untreated cells (Mock). G and H, qRT-PCR analysis ofRUNX2mRNA levels in TPC1 and BCPAP cells treated (gray bars) or untreated (black bars) with TSA. Cells were exposed to TSA or Mockmedium. After 6 hours, thetreatment was removed and RUNX2 expression was analyzed at the indicated times. For each time point, the gray bars represent the RUNX2 average foldchange in TSA-treated cells as compared with Mock (black bars). I and J, qRT-PCR analysis of RUNX2 mRNA levels in TPC1 and BCPAP cells untreated (black bars)or treated with TSA (gray bars) for 24 hours, in the presence or absence of actinomycin D (ActD; 5 mg/mL) or cycloheximide (CHX; 50 mg/mL). NT,cells that were not treated either with actinomycin D or cycloheximide. Bars represent the average fold change of RUNX2 in treated cells as compared with cellsthat did not receive any treatment (Mock NT). RUNX2 levels in TSA-treated BCPAP cells subjected to actinomycin D and cycloheximide are significantlydifferent as comparedwith NT cells (P

Figure 2.A new ENH element is required to induce activation of the P2 promoter in thyroid cancer cells. A, schematic representation of RUNX2 isoform I and isoform II.B, RT-PCR analysis of RUNX2 isoform I and isoform II expression in BCPAP and TPC1 cells treated or untreated with TSA. GAPDH expression was analyzed as acontrol. M, molecular weight marker. C, luciferase analysis of RUNX2 P2 promoter activity in TPC1 and BCPAP cells. (Continued on the following page.)

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Furthermore, ENH3 maintained the ability of activating thereporter gene when cloned in the inverted orientation andwhen cloned in the pGL3 vector containing the sP2 promoter.Finally, we have shown by chromatin immunoprecipitation(ChIP) experiments that the binding of RNA Polymerase II(RNA PolII) on the ENH3 was significantly enriched as com-pared with two downstream RUNX2 exons (exons 5 and 7; Fig.3A and B). This observation is in agreement with the hypothesisthat enhancer elements may function as RNA PolII reservoir,facilitating the access of the enzyme on the target promoter(39, 40).

A minimal 30-bp element represents the ENH3 coreWith the intent of identifying the minimal DNA regulatory

elements (ENH3 core) of ENH3, we performed a functionaldissection of this region (Fig. 3C). We generated sequentialdeletion mutants of the ENH3 and their activity on the sP2promoter was assessed by luciferase assay (Fig. 3D–F). Thisanalysis restricted the ENH3 activity to the ENH3.C3 fragmentin the central region of the ENH3 (Fig. 3D and E). This region wasfurther divided into four fragments (Fig. 3F). Two overlappingfragments ENH3.C3A and ENH3.C3B retained most of the activ-ity. We reasoned that the 30-bp overlapping region contained thefunctional sites of ENH3 and represented the ENH3 core. Notice-ably, this region has shown high-grade conservation across spe-cies, suggesting a selective pressure to preserve this element duringevolution (Supplementary Fig. S1C). In order to understandwhich nucleotides had a relevant function for the activity ofENH3, we introduced three independent point mutations withinthe ENH3 core in the full-length ENH3.C3 fragment (Fig. 4A andB). G>T substitution in position 1149 (Mut1) and A>T substitu-tion in position 1156 (Mut2) strongly repressed the activity ofENH3.C3. In contrast, C>G mutation in position 1157 (Mut3)showed a trend (even if not statistically significant) towardinduction, suggesting the possibility that this site negativelycontrol ENH3 activity. Doublemutation G>T and A>T (Mut1þ2)further decreased the activity of the ENH3.C3 with a significantinteraction effect, suggesting a positive cooperation between thesetwo sites.

Next, we performed electromobility shift assay (EMSA) usinga probe of 47 bp containing the ENH3 core (S3A probe). Figure4C shows that the radiolabeled S3A probe was bound by aprotein complex in both TPC1 and BCPAP cells and that theinteraction was specific because the addition of increasingamounts of a cold WT competitor abolished the binding. Wealso performed competition assays using increasing amounts ofMut1, Mut2, and Mut1þ2 cold competitors. Mut1 displayedsimilar competition efficiency as the WT competitor in both cell

lines. In contrast, Mut2 displayed partial efficiency in compet-ing for the binding as compared with the WT competitor andthe competition efficiency was even further decreased whenboth positions were mutated in the Mut1þ2 double-mutantcompetitor. Also, Mut3 had reduced competition efficiency ascompared with the WT probe, suggesting the possibility thatthis site participate to the binding of the protein complex toENH3 (Fig. 4D and Supplementary Fig. S1D).

We used two independent databases (TRANSFAC and JASPAR)to predict which transcription factors were likely to bind thisregion. Thirty-five and 39 binding sites were predicted respec-tively. From these lists, we sorted transcription factors whosebinding was abolished by Mut1 and Mut2 and that was notaffected by Mut3. Noticeably, Mut1 and Mut2 affected the bind-ing of two completely distinct sets of transcription factors andnone of the predicted sites was commonly abolished by bothmutations. Therefore, we concluded that the ENH3 core is com-posed of two adjacent functional sites (sites 1 and 2), whichcooperate in controlling ENH3 activity and RUNX2 expression(Fig. 4E and F). This model was strongly supported by luciferaseand EMSA data, in which simultaneous mutation of both sites(Mut1þ2) had a stronger effect on the ENH3 activity than thesingle Mut1 and Mut2 substitutions (Fig. 4B and C). Next, wecrossed the results obtained by TRANSFAC and JASPAR databasesin order to find binding sites that were predicted by bothapproaches. From this search, we obtained a list of five transcrip-tion factors (ETS1, ELK1, ERG, FLI1 belonging to the ETS family,and NFAT1 belonging to the Rel family) that were predicted tobind to site 1, and AP1 that was predicted to bind to site 2. Weperformed supershift assay using anti-c-JUN antibodies to con-firm that AP1 binds to site 2. In both cell lines, the addition ofc-JUN antibodies determined a reduction of the binding of theprotein complex to the S3A probe (Fig. 4G). Furthermore, ChIPexperiments using anti-c-JUN antibodies showed that c-JUNactively binds to ENH3 in a context of structured chromatin(Fig. 4H). Finally, we used the S3A probe or the Mut1þ2 dou-ble-mutant probe to precipitate binding proteins in a DNApulldown assay. Western blot analysis using anti-c-JUN antibo-dies showed that c-JUN is actively precipitated by the S3A probewhile, double mutation strongly inhibited the binding (Fig. 4I).All together, these experiments indicate that the ENH3 transcrip-tional activity is controlled by a multiprotein complex that bindsto a bipartite site within the ENH3 core and showed that AP1 isone of the components of this functional complex.

The ENH3 contains the HDACi-responsive elementsWe tested whether ENH3 was able to mediate the HDACi-

repressive effect on RUNX2 expression (Fig. 5A). Noticeably, in

(Continued.) A fragmentofabout4,500bp, corresponding to the full-lengthRUNX2P2promoter, andashorter regionofabout400bpencompassing theTSS(sP2)werecloned into the pGL3 basic vector upstream of a luciferase reporter gene. Cells were transfected with the indicated pGL3 constructs and left untreated(black bars) or treated with TSA (gray bars). For each treatment, the bars represent the average fold change of luciferase activity in pGL3-P2 and pGL3-sP2 vector-transfected cells relative to pGL3-empty vector-transfected cells. TSA or DMSO (Mock) were added to cell culture medium 24 hours after transfection and luciferaseactivity was measured after 24 hours from TSA treatment. Each sample was run in triplicate. D, schematic representation of RUNX2 genomic locus showing theposition of the putative ENHs. Tracks corresponding to the chromatin features thatwere used for the identification of the ENHs are displayed. The diagramwas obtainedbymodification of the genomebrowser view (http://genome.ucsc.edu). E, DNase sensitivity assay of the P2 promoter and of ENH1, ENH2, and ENH3genomic regions inTPC1 and BCPAP cells. Chromatin accessibility is expressed as a percentage on a scale ranging from a constitutively expressed gene (GAPDH) to a constitutivelyrepressed gene (rhodopsin). F, luciferase analysis of ENH1, ENH2, and ENH3 activity in TPC1 and BCPAP cells. Cells were transfectedwith the indicated pGL3 constructs.The bars represent the average fold change of luciferase activity in cells transfected with the pGL3-P2/ENHs vectors as compared with cells transfected with thepGL3-P2 vector. G, luciferase analysis of ENH3 properties in TPC1 and BCPAP cells. Cells were transfected with the indicated pGL3 constructs. The bars represent theaverage fold change of luciferase activity normalized to cells transfected with the pGL3-empty vector. All the luciferase data (C, F, and G) were normalized to Renillaluciferase activity as transfection efficiency control and are expressed as mean values � SEM (n ¼ 3). �� , P � 0.001; �� , P � 0.01; � , P � 0.05.






Figure 3.Mapping of the ENH3 core. A and B, ChIP analysis of RUNX2 P2, ENH3, exon 5, and exon 7 regions with anti-RNA PolII antibodies in TPC1 and BCPAP cells.ChIP with mouse IgG was performed as a control. An unrelated DNA region upstream of the P2 promoter was used as a negative control. The bars represent theaverage enrichment of the indicated genomic regions in the immunoprecipitated DNA expressed as percentage of the input C, schematic representationof ENH3 deletion mutants. The ENH3.C3 fragment is highlighted in black. D–F, luciferase analysis of ENH3 deletion mutants in TPC1 and BCPAP cells. Cells weretransfected with the indicated pGL3 constructs. The bars represent the average fold change of luciferase activity in cells transfected with the pGL3-sP2/ENH3mutants vectors as compared with cells transfected with the pGL3-sP2 vector. All the luciferase data (D–F) were normalized to Renilla luciferase activity astransfection efficiency control and are expressed as mean values � SEM (n ¼ 3). �� , P � 0.001; �� , P � 0.01; � , P � 0.05.

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both cell lines, TSA determined a strong inhibition of thereporter gene in the presence of the ENH3. The ENH3.C3region that contains the ENH3 core was sufficient to mediatethe HDACi repression, while single mutation in site 1 or site 2or double mutation in both sites abolished the response toHDACi, most likely as a consequence of their deleterious effecton the ENH3 activity (Fig. 5A and Supplementary Fig. S1E). Incontrast, Mut3 did not affect the repressive effect of HDACi onENH3, suggesting that this site is not directly involved in theHDACi-mediated RUNX2 regulation. These results indicatedthat the ENH3 and, in particular, the ENH3 core contains theelements necessary to mediate the HDACi-repressive effect onRUNX2 expression.

To test the possibility that HDACi alter the formation of thetranscriptional complex on the ENH3, we performed EMSA withthe S3A probe in both TPC1 and BCPAP cells in the presence orabsence of TSA (Fig. 5B). Noticeably, the addition of TSA abol-ished the binding of the protein complex on the ENH3 core,pointing to a possible mechanism to explain the HDACi-medi-ated RUNX2 repression. On the basis of these observations, wepropose the following model. In unperturbed cancer cells,RUNX2 expression is sustained by ENH3 that stimulates thetranscriptional activity of the otherwise inert P2 promoter. Whencells are exposed to HDACi, these drugs alter the formation of thetranscriptional complex on ENH3, likely by changing the acety-lation status of one or more of its components, thus shortcuttingthe ENH3 effect on the P2 promoter and abolishing RUNX2expression (Fig. 5C). Indeed, when we silenced c-JUN usingspecific siRNAs, we observed a significant reduction of RUNX2levels in both TPC and BCPAP cells, demonstrating that c-JUN is apositive regulator of RUNX2 expression (Fig. 5D). This observa-tion strongly supports the proposed model. However, we cannotexclude that additionalmechanismsmay impinge on theHDACi-mediated effect on ENH3.

HDAC6 contributed to ENH3 activity and to RUNX2 expressionregulation

Recently, a functional link between HDAC6 and RUNX2 incancer cells has been proposed (41). To assess the possibilitythat HDAC6 controls RUNX2 expression in cancer cells, wetreated TPC1 and BCPAP cells with increasing amounts oftubacin, which is a selective inhibitor of HDAC6. Tubacinstrongly repressed RUNX2 expression in TPC1 cells in adose-dependent manner. In contrast, this drug had only min-imal effect on RUNX2 levels in BCPAP cells (Fig. 6A and B). Weconfirmed this observation by siRNA-mediated inhibition ofHDAC6 expression. As expected, anti-HDAC6 siRNA deter-mined a profound reduction of RUNX2 expression in TPC1cells, but not in BCPAP cells, confirming the results obtainedwith the chemical inhibitor (Fig. 6C and D). In order tomeasure the effect of HDAC6 knockdown on the activity ofthe ENH3, we performed luciferase assay in TPC1 and BCPAPcells transfected with anti-HDAC6 siRNA or control siRNA.Indeed, while in BCPAP cells no effect was detected, in TPC1cells, the siRNA-mediated HDAC6 inhibition resulted in asignificant reduction of the transcriptional activity of both thefull-length ENH3 and the ENH3.C3 fragment, indicating thatHDAC6 participates to the regulation of the transcriptionalcomplex controlling ENH3 (Fig. 6E and F). However, ChIPexperiments using anti-HDAC6 antibodies failed to detectHDAC6 on either the RUNX2 P2 promoter or the ENH3,

suggesting that this deacetylase controls RUNX2 expressionwithout physically interacting with its regulatory regions (datanot shown). These observations demonstrate for the first timethat HDCA6 has a function in controlling RUNX2 expression inthyroid cancer cells and that its inhibition accounts for most ofthe repressive effect of the HDACi in TPC1 cells. However, thesedata also point toward the existence of cell-specific mechan-isms, involving other HDACs, in controlling RUNX2 expres-sion. The different sensitivity of TPC1 and BCPAP to HDAC6inhibition was confirmed by proliferation assay (Fig. 6G andH). Tubacin induced a complete growth arrest in TPC1 cells,which was already detectable after 24 hours. In contrast, thisdrug determined only a partial reduction of the proliferationrate in BCPAP cells. Meanwhile, TSA determined a strong celldeath effect in both cell lines.

Inhibition of ENH3 activity is a general mechanism of action ofHDACi in different types of cancer

Besides thyroid cancer, RUNX2 has also been proposed tosustain progression in other types of cancer. Thus, we investigatedthe HDACi effect on RUNX2 expression in 10 cancer cell linesfrom different tumors. In six cell lines (A549, H1299, MCF7,MDA-MB-231, PC3, and SAOS2), the TSA treatment determined asignificant repression of RUNX2 expression (>1.5-fold; Fig. 7A).Comparable results were obtained with SAHA (data not shown).Recent evidence indicates that the cytotoxic effect of HDACi oncancer cells is also mediated by the repression of highly expressedoncogenes. Thus, we analyzed whether the degree of HDACi-mediated RUNX2 repressionwas associatedwith the basal level ofRUNX2 expression in cancer cells (Fig. 7B). Regression analysisshowed a striking correlation between the expression levels ofRUNX2and the strength of TSA-repressing effect. This observationsuggests that the HDACi-mediated RUNX2 repression takes placeonly in those tumors in which this transcription factor is highlyexpressed. Finally, we tested whether the TSA-repressive effect wasmediated by repression of ENH3 as we have shown in thyroidcancer cells. In all cell lines tested, the presence of ENH3 down-stream of the sP2 promoter determined the activation of thereporter gene as already observed in TPC1 and BCPAP cells (Fig.7C). Similarly, upon TSA treatment, the ENH3 activity wasrepressed in all cell lines, suggesting the possibility to extend toother cancer types the model of ENH3 functioning that weproposed in thyroid cancer.

DiscussionTumor development and progression are the consequence of

the deregulation of many pathways, which are usually orches-trated by the aberrant function of transcription factors. RUNX2has been described as driver of aggressiveness and majordeterminant of metastatic spreading in cancer (1, 3–7, 9, 12, 13, 16). However, the regulatory mechanisms thatlead to RUNX2 reactivation during cancer development andprogression are still to be unraveled. In this respect, this studyprovides a completely new model of RUNX2 regulation andopens new perspectives. We have shown that the P2 promoter,which is responsible for the expression of the major RUNX2isoform in cancer, is not sufficient to drive RUNX2 expression.This observation is in agreement with other articles, whichinvestigated the function of RUNX2 promoters and described ahighly regulated P1 promoter and a rather constitutive P2







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promoter with a constant basal activity, which is not altered bythe presence of regulatory stimuli (24, 28–30). Our resultsdemonstrate that the major transcriptional activity resides in anewly identified enhancer element, located in an intronicregion of the RUNX2 gene, which is able to enhance theactivity of the P2 promoter and to respond to external regu-latory stimuli. Our data indicate that this element is active inseveral types of tumors in which RUNX2 is overexpressed. It isknown that the expression of genes involved in critical pro-cesses is extremely refined and it may be regulated by theprecise spatiotemporal activation of multiple regulatory ele-ments. Thus, it is likely that, besides ENH3, other elementsmay contribute to the complicate regulation of RUNX2expression.

Being the point of convergence of multiple oncogenic process-es, RUNX2 appears to be a promising target for developingtherapeutic strategies to counteract tumor progression. However,targeting transcription factors in cancer has been proven to beextremely challenging (42). In this study, we demonstrated for thefirst time that the HDACi strongly repress the expression ofRUNX2 in cancer cells, suggesting a possible therapeutic strategyto counteract the prooncogenic effect of this factor in cancerpatients.

To our knowledge, this is one of the first works describing theeffect of HDACi on RUNX2 expression in cancer. Nevertheless,our observations appear to be in contrast with previous works inwhich a positive effect of these drugs on RUNX2 expression wasreported in skeletal cells (43, 44). Currently, there are no exper-imental data to explain such discrepancy, but the different cellularcontext is likely to play a relevant role. In bone and cartilage cells,both RUNX2 isoforms are expressed, whereas in cancer, onlyisoform I is observed. It is possible that the increased expressionof RUNX2 detected upon HDACi treatment in skeletal cells is theresult of a combinatory effect on the expression of both isoforms.It is also reasonable to suppose that in a nontumoral context, suchas the skeletal tissue, the ENH3 is not active and RUNX2 expres-sion is under the control of different elements on which HDACiexert a different regulation.

One of the major effects of HDACi on cancer cells is theremodeling of chromatin, which usually leads to the reexpressionof epigenetically silenced oncosuppressor. For many years, thishas been considered as the main mechanism of action of thesedrugs as anticancer therapies (31). Reversible acetylation is awidespread phenomenon that affects many signaling pathways

and thereby many cellular processes. The growing number ofidentified acetylable targets beyond chromatin provides a wholenew world of potential regulatory effects of these drugs, eventhough most of these are likely to converge on controlling geneexpression (32). Indeed, it has been demonstrated that HDACitreatment induces expression changes in 2% to 10% of all humangenes with almost an equal amount of induced and repressedgenes (45, 46). This effect cannot be explainedwithout taking intoconsideration the role of HDACi as fine modulators of transcrip-tion through modification of nonhistonic proteins. We observedthat treatment with HDACi abolishes the formation of a proteincomplex on the ENH3 core, thus abrogating its activating effect onthe P2promoter.Wemapped abipartite binding site, in the ENH3core, in which three families of transcription factors are predictedto bind. The ETS family and the Rel family, would bind to site 1,making complex with AP1, which is predicted to bind site 2. Allthese factors have been implicated in cancer progression, in linewith our data that suggest their possible role in supporting theexpression of the prooncogenic factor RUNX2. Noteworthy, theability of AP1 to form transcriptional complexes withmembers ofboth ETS and NFAT families is well established, and all thesefactors have been shown to be directly or indirectly controlled byHDACs (47, 48).

We also demonstrated that c-JUN, component of the AP1complex physically interactswith ENH3and is a positive regulatorof RUNX2 expression. To our knowledge, this is the first reportdescribing a direct link between c-JUN activity and RUNX2expression. Acetylation changes the electrostatic state of lysinefrom positive to neutral, thus reducing the affinity for negativelycharged molecules like DNA. Similarly, acetylation of lysineresidues can create new dock sites for protein–protein interaction.Therefore, it is likely that changes in the acetylation profile of oneor more of the factors of the transcriptional complex binding theENH3 affect the affinity of the interactions disassembling thecomplex. Previous studies have shown that c-JUN is acetylatedand that its acetylation status affects transcriptional activity. Vriesand colleagues (49, 50) observed that the hyperacetylation ofLys271 of c-JUN is necessary for the repression of the collagenasepromoter in the presence of the E1A protein.

Many HDACs have been shown to bind to RUNX2 and tocontrol its transcriptional activity. Of these, HDAC6 is the onlyHDAC that has been described to cooperate with RUNX2 andmediate its prooncogenic role in cancer (41). In this work, wedemonstrated for the first time that HDAC6 is required for the

Figure 4.Functional analysis of the ENH3 core elements. A, schematic representation of the point mutations in the ENH3 core. Gray bar, the position of the S3A probe.B, luciferase analysis of the effect of targeted point mutations in the ENH3 core. Cells were transfected with the indicated pGL3 constructs. The barsrepresent the average fold change of luciferase activity in cells transfected with the pGL3-sP2/ENH3 mutants vectors as compared with cells transfectedwith the pGL3-sP2 vector. Luciferase data were normalized to Renilla luciferase activity as transfection efficiency control and are expressed as mean values� SEM(n ¼ 3). �� , P � 0.001; �� , P � 0.01; � , P � 0.05. Interaction effect between Mut1 and Mut2 on ENH3.C3 activity, assessed by ANOVA, was significant in bothTPC1 (P < 0.001) and BCPAP (P ¼ 0.003) cells. C and D, EMSA analysis of the transcriptional complex bound to ENH3 core region. Nuclear extracts from TPC1 andBCPAP cells were incubated with 32P-labeled S3A probe in the absence or presence of increasing amount of the indicated cold competitors. Black arrow, thespecific protein complex bound to the probe. � , unspecific bands. E, diagram of the workflow used for the identification of transcription factors bindingsites in the ENH3 core region. F, sequences of the WT and mutated ENH3 core in which the mutated nucleotides are displayed. Boxes highlight the site 1 and site 2sequences. Consensus diagram of the predicted transcription factor binding sites is reported above the alignment and matrix scores (TRANSFAC) are indicated inbrackets. G, supershift analysis of c-JUN binding to S3A probe. Nuclear extract from TPC1 and BCPAP cells was incubated with 32P-labeled S3A probe in thepresence or absence of 4 mg of c-JUN antibodies or control IgG. Arrowhead, the specific complex bound to the probe. � , unspecific bands. H, ChIP analysis of RUNX2P2 promoter and ENH3 with anti-c-JUN antibodies in TPC1 and BCPAP cells. ChIP with rabbit IgG was performed as a control. An unrelated DNAregion upstream of the P2 promoter was used as a negative control. The bars represent the average enrichment of the indicated genomic regions in theimmunoprecipitated DNA expressed as percentage of the input. I, DNA pulldown assay. Western blot analysis with anti-c-JUN antibodies of nuclear extractsprecipitated using a biotin-labeled S3A probe or Mut1þ2 double-mutant probe, immobilized on streptavidin-conjugated magnetic beads. Input represents2.5% of the nuclear extract used for precipitation.






RUNX2 transcription in thyroid cancer, because its inhibitionresulted in the repression of the ENH3 activity and of RUNX2levels. This observation implies the existence of an additionallevel of positive regulation of HDAC6 on RUNX2 in cancercells, besides the functional cooperation between these twoproteins.

We have shown that the inhibitory effect of HDACi on RUNX2expression is not restricted to thyroid cancer cells but is common

to other cancer types in which RUNX2 has been implicated. Wehave also shown that the mechanism through which this inhibi-tion takes place is similar in all cell lines tested and involves theinhibition of ENH3 transcription activity. In contrast, the obser-vation that HDAC6 inhibition does not affect expression ofRUNX2 in BCPAP seems to suggest that different HDACs maycontribute to this regulation depending on the cellular context oron the genetic background of the tumors.

Figure 5.HDACi repress RUNX2 expression by inhibiting the ENH3 function. A, luciferase analysis of the effect of TSA on ENH3 activity in TPC1 and BCPAP cells. Cells weretransfected with the indicated pGL3 constructs and left untreated (black bars) or treated with TSA (gray bars). For each set of samples (TSA treated orMock), the bars represent the average fold change of luciferase activity in cells transfected with indicated pGL3-ENH3.C3 vectors relative to pGL3-sP2 vectortransfected cells. Data were normalized to Renilla luciferase activity as transfection efficiency control and are expressed as mean values � SEM (n ¼ 3). P valueindicates the statistical significance of the difference between TSA treated and untreated samples. �� , P � 0.001; �� , P � 0.01; � , P � 0.05. B, EMSA analysisof the effect of TSA on the binding of the transcriptional complex to the ENH3 core. Nuclear extracts from TPC1 and BCPAP cells treated or untreated with TSAwereincubated with 32P-labeled S3A probe. Black arrow, the specific protein complex bound to the probe. � , unspecific bands. C, putative model of HDACimechanism of action on RUNX2 gene expression. D, qRT-PCR analysis of c-JUN and RUNX2 mRNA levels in TPC1 and BCPAP cells transfected with siRNAagainst c-JUN (gray bar) or scramble oligos (black bars). The bars represent the average fold change of c-JUN and RUNX2 expression in c-JUN siRNA-transfectedcells as compared with control. Expression data were normalized to cyclophilin A.

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Figure 6.HDAC6 controls ENH3-mediated RUNX2 expression in TPC1 cells. A and B, qRT-PCR analysis of RUNX2 mRNA levels in TPC1 and BCPAP cells treated with tubacin(TUB) for 24 hours. The bars represent the average fold change of RUNX2 in tubacin-treated cells as compared with untreated cells (Mock). C and D,qRT-PCR analysis of HDAC6 and RUNX2mRNA levels in TPC1 and BCPAP cells transfectedwith siRNA against HDAC6 (gray bar) or scramble oligos (black bars). Thebars represent the average fold change of HDAC6 and RUNX2 expression in HDAC6 siRNA-transfected cells as compared with control. Expression data werenormalized to cyclophilin A (A and B) or GUSB (C and D) levels and are expressed as mean values� SEM (n¼ 3). E and F, luciferase analysis of the effect of HDAC6siRNA on ENH3 activity in TPC1 and BCPAP cells. Cells were transfected with the indicated pGL3-sP2 constructs together with HDAC6 siRNA (gray bars) orscramble oligos (balck bars). The bars represent the average fold change of luciferase activity normalized to cells transfectedwith the pGL3-sP2 vector togetherwithscramble oligo. Luciferase datawere normalized toRenilla luciferase activity as transfection efficiency control and are expressed asmeanvalues�SEM (n¼ 3). G andH, proliferation curve of TPC1 and BCPAP cells untreated or treated with TSA or tubacin. Mean values � SEM (n ¼ 3). �� , P � 0.001; �� , P � 0.01; � , P � 0.05.






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

Authors' ContributionsConception and design: V. Sancisi, A. CiarrocchiDevelopment of methodology: V. Sancisi, D.C. Ambrosetti

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. GandolfiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V. Sancisi, G. Gandolfi, A. CiarrocchiWriting, review, and/or revision of themanuscript: V. Sancisi, D.C. Ambrosetti,A. CiarrocchiStudy supervision: A. Ciarrocchi

Figure 7.ENH3mediates HDACi-repressive effect in different types of cancer. A, qRT-PCR analysis of RUNX2mRNA levels in the indicated cancer cell lines. Cell lines from lung(A549 and H1299), breast (MCF7 and MDA-MB-231), colon (HTC116 and HCC2998), prostate (PC3 and DU145) carcinoma, osteosarcoma (SAOS2), andmelanoma (A375) were treatedwith TSA for 24 hours. The bars represent the average fold change of RUNX2 in TSA-treated (gray bars) as comparedwith untreatedcells (Mock, black bars). Expression data were normalized to cyclophilin A levels and are expressed as mean values� SEM (n¼ 3). �� , P� 0.001; �� , P� 0.01; � , P�0.05. The induction observed in HCT116 upon TSA treatment is most likely a normalization artefact due to the basal low level of RUNX2 expression in thesecells. B, correlation analysis between RUNX2 expression levels and TSA-induced repression expressed as fold change in a panel of cancer cell lines. C, luciferaseanalysis of the effect of TSA on ENH3 activity in the indicated cell lines. Cells were transfected with pGL3-sP2 or with pGL3-sP2/ENH3 construct in thepresence (gray bars) or absence (Mock, black bars) of TSA. For each set of samples (TSA treated or Mock), the bars represent the average fold change of luciferaseactivity in cells transfected with indicated pGL3-sP2/ENH3 vector relative to pGL3-sP2 vector transfected cells. Luciferase data were normalized to Renillaluciferase activity as transfection efficiency control and are expressed as mean values � SEM (n ¼ 3). �� , P � 0.001; �� , P � 0.01; � , P � 0.05.

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AcknowledgmentsThe authors thank Drs. Giovanna Damia, Massimo Broggini, and Alfredo

Budillon for constructive discussion and helpful suggestions about the experi-ments. The authors also thankDrs.Marco Tigano, Valentina Fragliasso, andMilaGugnoni for critical revision of the article.

Grant SupportThis work was supported by the Italian Association for Cancer Research

(AIRC, MFAG10745 and IG15862).

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 indicatethis fact.

Received July 21, 2014; revised January 4, 2015; accepted January 20, 2015;published OnlineFirst March 13, 2015.

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2015;75:1868-1882. Published OnlineFirst March 13, 2015.Cancer Res Valentina Sancisi, Greta Gandolfi, Davide Carlo Ambrosetti, et al. Proinvasive Factor RUNX2Histone Deacetylase Inhibitors Repress Tumoral Expression of the

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histone deacetylase inhibitors repress tumoral expression of the … · obtained from dr. adriana...

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