A truncating mutation of HDAC2 in human cancersconfers resistance to histone deacetylase inhibitionSantiago Ropero1, Mario F Fraga1, Esteban Ballestar1, Richard Hamelin2, Hiroyuki Yamamoto3,Manuel Boix-Chornet1, Rosalia Caballero1, Miguel Alaminos1, Fernando Setien1, Maria F Paz1,Michel Herranz1, Jose Palacios4, Diego Arango5, Torben F Orntoft6, Lauri A Aaltonen7,Simo Schwartz Jr5 & Manel Esteller1

Disruption of histone acetylation patterns is a common featureof cancer cells, but very little is known about its genetic basis.We have identified truncating mutations in one of the primaryhuman histone deacetylases, HDAC2, in sporadic carcinomaswith microsatellite instability and in tumors arising inindividuals with hereditary nonpolyposis colorectal cancersyndrome. The presence of the HDAC2 frameshift mutationcauses a loss of HDAC2 protein expression and enzymaticactivity and renders these cells more resistant to the usualantiproliferative and proapoptotic effects of histone deacetylaseinhibitors. As such drugs may serve as therapeutic agents forcancer, our findings support the use of HDAC2 mutationalstatus in future pharmacogenetic treatment of these individuals.

Widespread changes in DNA methylation1,2 and post-translationalmodifications of histones occur in cancer cells3,4, and both of theseprocesses are critical for chromatin packaging and gene expres-sion1,2,5,6. We are largely ignorant of the mechanisms underlying thedisruption of the epigenetic landscape in transformed cells. Onepossibility is that the enzymes that epigenetically modify DNA andhistones, and the transcription factors that ‘read’ these marks, maythemselves be targets of genetic disruption, as occurs in the case of thep300 histone acetyltransferase7,8.

To explore the presence of inactivating mutations in the so-called‘epigenetic modifier genes’, it is useful to consider tumors showingmicrosatellite instability, both in the context of hereditary nonpoly-posis colon cancer (HNPCC) associated with germline mutations inthe mismatch repair genes9 and in sporadic cancers associated withhMLH1 inactivation by promoter CpG island methylation9,10. Tumorswith microsatellite instability progress along a genetic pathway with ahigh rate of insertion and deletion mutations in mononucleotiderepeats, which often result in the generation of premature stop

codons. Illustrative target genes include the growth-control geneTGFBRII (ref. 11) and the proapoptotic gene BAX12.

We first screened six colorectal (RKO, SW48, LoVo, HCT-15, Co115and HCT-116) and four endometrial (AN3CA, SKUT-1, SKUT-1Band HEC1B) cancer cell lines with microsatellite instability for thepresence of mutations in all the exonic mononucleotide repeatspresent in the coding sequences of histone deacetylases (HDAC1and HDAC2), histone acetyltransferases (pCAF), histone methyltrans-ferases (G9a), DNA methyltransferases (DNMT1 and DNMT3b) andmethyl-CpG binding proteins (MBD1, MBD2 and MeCP2). Thelocation of the corresponding repeats and the PCR primers used areshown in Supplementary Table 1 online. We detected only wild-typesequences for all the genes described, with the single notable exceptionof HDAC2 (Fig. 1a,b). We found a frameshift mutation in HDAC2in the A9 coding microsatellite repeat of exon 1, consisting of thedeletion of an A in two colorectal cell lines (RKO and Co115) and twoendometrial cell lines (AN3CA and SKUT-1). We analyzed the RKOand Co115 cell lines and found no evidence of the HDAC2 protein innuclear extracts (Fig. 1c) or by immunofluorescence staining(Fig. 1c). Most notably, we found that histone deacetylase enzymaticactivity was lost in HDAC2-immunoprecipitated cell extracts of RKOand Co115 cells (compared with HCT-116, SW48 and LoVo cells,which have the wild-type HDAC2 coding repeat; Fig. 1c), thusdemonstrating functional abrogation of HDAC2 in RKO and Co115.As the two alleles of HDAC2 in RKO and Co115 cells are retained inFISH analysis (Supplementary Fig. 1 online), and we found onlymutant alleles when we sequenced multiple clones, these observationsimply the biallelic inactivation of HDAC2 by the described mutation.In contrast, the two endometrial cancer cell lines were heterozygousfor the HDAC2 mutation (Supplementary Fig. 2 online). For all celllines, we did not observe any significant differences in the levels ofHDAC2 mRNA (Fig. 1c) or any evidence of HDAC2 mutations in

Received 18 January; accepted 8 March; published online 16 April 2006; doi:10.1038/ng1773

1Cancer Epigenetics Laboratory, Spanish National Cancer Centre (CNIO), 28029 Madrid, Spain. 2INSERM U434, Centre d’Etude du Polymorphisme Humain, 75010Paris, France. 3First Department of Internal Medicine, Sapporo Medical University, Sapporo 060-8543, Japan. 4Laboratory of Breast and Gynaecological Cancer,Spanish National Cancer Centre (CNIO), 28029 Madrid, Spain. 5Molecular Oncology and Aging Research, Centre d’Investigacions en Bioquimica i Biologia Molecular,Hospital Universitari Vall d’Hebron, Barcelona 08035, Catalonia, Spain. 6Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus UniversityHospital/Skejby, Brendstrupgaardsvej 100, DK-8200 Aarhus N, Denmark. 7Department of Medical Genetics, Haartmaninkatu 8, Biomedicum Helsinki, University ofHelsinki, Helsinki, Finland. Correspondence should be addressed to M.E. (mesteller@cnio.es).

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colorectal (SW480, SW620, CaCo2 and COLO250) and endometrial(KLE) cancer cell lines that lacked microsatellite instability.

Once the presence of an inactivating mutation of HDAC2 in cancercells had been confirmed, it became very important to establishwhether the abolition of HDAC2 function altered the biochemicaland cellular effects mediated by the histonedeacetylase (HDAC) inhibitors. This could bea potentially relevant clinical issue, as HDACinhibitors are drugs that have significantanticancer activities at doses that are welltolerated by patients in clinical trials13. Inthe case of colorectal cancer, HDAC inhibi-tors induce tumor growth inhibition incolon cancer cell lines13,14 and in the polyp-prone Apc (min) mice15. Could the detec-tion of an HDAC2 inactivation mutation ina given tumor predict the response toHDAC inhibitors?

To address these issues, we assessed theeffects of three classical HDAC inhibitors,the hydroxamic acid trichostatin A and thecarboxylic acids butyrate and valproate, onfive colorectal cancer cell lines: RKO andCo115, which have mutant HDAC2, andHCT-116, SW48 and LoVo, which havewild-type HDAC2. We analyzed the acetyla-tion levels at histones H3 and H4 usingprotein blotting with antibodies raised againsttetraacetylated peptides of histones H3 andH4 (ref. 3) and by high-performance capillaryelectrophoresis (HPCE)3. We found thatalthough butyrate and valproate were ableto induce hyperacetylation of histones H3and H4 in all colorectal cell lines irrespective

of their HDAC2 status, trichostatin A was unable to induce thehyperacetylation of either histone in the HDAC2-deficient RKO andCo115 cell lines, but it was effective in the cells with wild-type HDAC2(Fig. 2a and Supplementary Fig. 3 online). The HDAC2-deficient cellswere resistant to trichostatin A action from both a biochemical and a

E1

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Figure 1 Biochemical and biological effects of HDAC2 mutations in human cancer. (a) Schematic representation of the HDAC2 gene, with the location

of the A9 repeat and the amino acid sequence of wild-type and mutant HDAC2 proteins. Black and green arrows represent transcriptional and translation

start sites, respectively. (b) Electropherograms of HDAC2 wild-type (normal colon and HCT-116) and mutant (RKO and CO115) cells. (c) HDAC2 protein

expression, as analyzed by protein blot (left) and immunofluorescence (right), is lost in the mutant RKO and CO115 cells, but not in the other colon cancer

cell lines, which have a wild-type sequence. HDAC1 protein and HDAC2 mRNA levels cells are not significantly different. Below, HDAC2 enzymatic activity

analyzed in the HDAC2-immunoprecipitated extracts is depleted in RKO and Co115 cells.

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Figure 2 The effect of HDAC inhibitors varies according to HDCA2 mutational status. (a) Quantification

of histone H3 and H4 acetylation levels by protein blot after treatment with HDAC inhibitors. The

hydroxamic acid trichostatin A (TSA) does not induce histone hyperacetylation in the HDAC2-deficient

RKO and CO115 cells, whereas in HDAC2-proficient cells (HCT-116, SW48 and LoVo), TSA does

induce hyperacetylation. The carboxylic acids valproate (Val) and butyrate (But) induce hyperacetylation

in all cells. (b) Top: HDAC2 activity of immunoprecipitated extracts of RKO transfected with empty

vector or HDAC2. Center: HDAC2 and HDAC1 expression levels. Bottom: quantification of H4

acetylation by HPCE. RKO cells transfected with HDAC2 show enhanced sensitivity to histone

acetylation mediated by TSA. (c) Top: protein blot of HCT116 cells in which HDAC2 has been knocked

down by siRNA. Bottom: quantification of histone H4 acetylation by HPCE. HCT116 cells with

knocked-down HDAC2 are resistant to histone acetylation mediated by TSA.

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cellular point of view. We found that valproate and butyrate inducedblockage of the cell cycle at G2/M, significantly inhibited proliferationand induced apoptosis in all cancer cell lines independently of theirHDAC2 status, whereas trichostatin A produced these effects only inthe HDAC2-proficient cell lines, and RKO and Co115 remained highlyresistant (Supplementary Fig. 3 online). We reproduced these resultsin xenografted nude mouse models: butyrate induced tumor shrinkagein all cancer cell lines independent of their HDAC2 status, whereastrichostatin A produced these effects in only the HDAC2-proficient cellline (HCT-116), and RKO and Co115 remained highly resistant(Supplementary Fig. 4 online). The potential mechanisms underlyingthe different effects of distinct HDAC inhibitors upon HDAC2inactivation are largely unknown. It is possible that the loss ofHDAC2 renders these cells more dependent upon the activity ofother HDACs, as it occurs with HDAC1 (ref. 16). In this scenario,small differences in the opening of the HDAC catalytic site17 couldaccount for the observed differences, particularly for inhibitors, such astrichostatin A, that have a higher number of interacting sites thanbutyrate and valproate. The targeted individual and combined dis-ruption of all four class I HDACs (HDAC1, HDAC2, HDAC3 andHDAC8) could be used to test this hypothesis.

To further establish a link between HDAC2 mutations and thephenotypes observed, we reconstituted HDAC2 function in HDAC2-deficient cancer cells (RKO) or knocked down HDAC2 in HDAC2-proficient cells (HCT-116). Transfection of wild-type HDAC2 in RKOcells restored HDAC2 activity (Fig. 2b) and rendered these cells moresensitive to the actions of trichostatin A (Fig. 2b). Most notably, theectopic expression of HDAC2 in these HDAC2-deficient cells induced

tumor suppressor–like features such as growth inhibition in xeno-grafted nude mice and reduced colony formation (SupplementaryFig. 5 online). In sharp contrast, the downregulation of HDAC2 byRNA interference in cells with wild-type HDAC2 (HCT-116) wasassociated with a resistance to the effects of trichostatin A (Fig. 2c).

Finally, once we had demonstrated the functional molecular andcellular consequences of harboring an inactivating mutation ofHDAC2 in cancer cells, we sought to measure the frequency of thedescribed HDAC2 disruption in human primary tumors. We assessedthe HDAC2 mutational status of 228 human primary malignancieswith microsatellite instability, including colorectal tumors fromHNPCC patients (n ¼ 47) and sporadic colorectal (n ¼ 127), gastric(n ¼ 38) and endometrial (n ¼ 16) neoplasms (Table 1). We foundthat the frameshift mutation in the HDAC2 gene in the A9 codingmicrosatellite repeat of exon 1 was present in 21% (48 of 228) of theprimary tumors analyzed. No significant differences in HDAC2mutation frequency were found between inherited and sporadictumors or between tumor types (Table 1). For 17 cases ofsporadic colon tumors with microsatellite instability, we conducteda double-blind immunohistochemical analysis of HDAC2 mutationalstatus (Fig. 3). In all cases with wild-type HDAC2 (n ¼ 11), HDAC2protein was strongly expressed. In contrast, of the six HDAC2mutant tumors, five (83%) completely lacked HDAC2 expression,whereas a heterogeneous pattern of loss of expression was observedin the remaining case. Laser microdissection analysis of theHDAC2-stained sections confirmed that the presence of the HDAC2mutation was always associated with the loss of HDAC2 signal. Thedescribed HDAC2 mutation was not present in primary colorectaltumors without microsatellite instability (0/40), normal colorectalmucosa (0/50) or in normal lymphocytes from healthy donors(0/50; Table 1).

In summary, we have demonstrated the presence of an inactivatingmutation in HDAC2 in human cancer cell lines and primary tumorswith microsatellite instability that impairs these transformed cells’biochemical and cellular responses to trichostatin A, the archetypicalhydroxamic acid with histone deacetylase inhibitory activity. Thisfinding supports the role of epigenetics, and especially of histonemodifications, in tumorigenesis, and it may have potential relevancefor the pharmacogenetic selection of cancer patients treated withhistone deacetylase inhibitors.

METHODSCell lines and primary tumor samples. Human colorectal and endometrial

cancer cell lines were obtained from the American Type Culture Collection.

HDAC inhibition treatment involved the addition

of 0.25 mM trichostatin A, 10 mM sodium valpro-

ate or 10 mM sodium butyrate to the culture

medium for 24 h. We obtained DNA samples from

primary tumors at the time of clinically indicated

surgical procedures.

Mutation analysis. Genomic DNA from cell lines

and primary tumors and cDNA from the cell lines

was amplified by PCR. PCR products and recom-

binant plasmids from ten clones of every sample

were sequenced in an automated ABI Prism 3700

sequencer. The genes studied, their locations and

the primers used are described in Supplementary

Table 1 online.

FISH analysis. FISH was performed by standard

methods, including denaturation steps, overnight

hybridization at 37 1C and two washes, one in

Figure 3 Immunohistochemistry of HDAC2 in sporadic colon tumors with microsatellite instability.

Left: strong HDAC2 expression in two tumors with wild-type sequences. Right: loss of HDAC2 staining

in two tumors harboring the HDAC2 mutation.

Table 1 Frequency of HDAC2 mutations in cancer cell lines, primary

tumors and normal tissues

Sample type Cell lines Tissue samples

Colon tumors from HPNCC - 8/47 (17%)

Sporadic colon tumors (MSI+) 2/6 (33.3%)a 26/127 (20.4%)b

Sporadic gastric tumors (MSI+) - 11/38 (28.9%)

Sporadic endometrial tumors (MSI+) 2/4 (50%)c 3/16 (18.7%)

Sporadic colon tumors (MSI–) 0/4 0/40

Normal colon - 0/50

Normal lymphocytes - 0/50

MSI+, tumors with microsatellite instability; MSI–, tumors without microsatellite instability.aHomozygous mutations. bFor six cases analyzed, five homozygous mutations and oneheterozygous mutation were observed. cHeterozygous mutations.

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0.4� SSC at 75 1C and another in 2� SSC at 24 1C. The BAC clone containing

the HDAC2 gene (RP11 456N11) was a gift from M. Rocci (University of

Bari, Italy).

Protein blotting, immunolocalization and immunohistochemistry assays.

For protein blotting of HDAC2 expression, nuclear extracts were immuno-

probed with antibodies to HDAC2 (1:1,000) and HDAC1 (1:1,000; both from

Abcam). The acetylated forms of histones 3 and 4 were analyzed as previously

described3. We extracted the histones directly from the cell pellets with 0.25 M

HCl. We fractionated the acid-extracted proteins on a 15% SDS-PAGE gel.

Transferred membranes were immunoprobed with antibodies to acetylated H4

(1:2,000; Upstate) and acetylated H3 (1:50,000; Upstate). We used antibody to

H4 (1:3,000; Upstate) as a loading control. For immunolocalization experi-

ments, we grew cells on coverslips and stained them with antibodies against

HDAC2 and HDAC1 (Abcam). Immunohistochemical staining of HDAC2 was

performed using a polyclonal antibody (Abcam) at a 1:1,500 dilution.

High-performance capillary electrophoresis (HPCE) quantification of global

histone acetylation. We prepared histones in accordance with established

protocols18, and we quantified global histone H4 acetylation as previously

described19. We prepared individual histone fractions from cell nuclei and

purified them by reverse-phase HPLC on a Jupiter C18 column (Phenomenex)

with an acetonitrile gradient (20�60%) in 0.3% trifluoroacetic acid, using

an HPLC gradient system (Beckman-Coulter). We resolved non-, mono, di-,

tri- and tetraacetylated histone H4 derivatives by HPCE.

HDAC2 activity. For HDAC2 activity determinations, HDAC2 was immuno-

precipitated as described elsewhere20 from nuclear extracts using the same

antibody used for protein blotting and immunolocalization experiments. We

then determined the HDAC2 activity by measuring released 3H-labeled acetate

in a scintillation counter after 1-h incubations of HDAC2 immunoprecipitates

at 37 1C with 3H-labeled histones.

Apoptosis, cell cycle analysis and viability assay. The percentage of apoptotic

cells was determined by flow cytometry using the Vybrant Apoptosis Assay Kit

#4 (YO-PRO-1 and propidium iodide; Molecular Probes/Invitrogen). To

analyze the cell cycle profiles, we stained with propidium iodide and deter-

mined the percentage of cells in G2/M by flow cytometry. Cell viability was

determined by the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium

bromide (MTT) assay.

Analysis of HDAC2 CpG island promoter methylation. DNA samples were

treated with sodium bisulfite and we used primers spanning the CpG island of

the HDAC2 promoter for bisulfite genomic sequencing. Primer sequences and

PCR conditions are available upon request. We analyzed 18 clones per sample.

HDAC2 transfection and colony formation assay. The HDAC2 expression

vector pcDNA3-HDAC2 was constructed by cloning the cDNA corresponding

to the gene HDAC2 into a pcDNA3 vector (Invitrogen). Transfection of RKO

cells was performed by electroporating 107 cells in 0.8 ml PBS with 40 mg of the

vector at 250 V and 975 mF. After electroporation, cells were washed with PBS

and seeded in fresh medium containing 20% FBS. Clones expressing HDAC2

were selected in complete medium supplemented with 1 mg ml–1 G418. For

colony formation experiments, stable G418–resistant colonies were fixed and

stained with 2% methylene blue in 60% methanol.

HDAC2 siRNA. The HDAC2-specific small interfering RNA (siRNA) was

designed and synthesized by Qiagen. Two siRNA duplexes, recognizing

two different sequences, were used against the HDAC2 gene (Supplementary

Table 1). We used Scramble siRNA (Qiagen) as a control. Transfection was

carried out using oligofectamine (Invitrogen). HDCA2 content was analyzed by

protein blotting, and histone acetylation was quantified by HPCE.

Mouse xenograft model. Six-week-old female athymic nude mice were used

for tumor xenografts. Animals were randomly separated in three groups of

seven specimens each (those treated with PBS as a control, those treated with

trichostatin A and those treated with butyric acid). Both flanks of each animal

were injected subcutaneously with 106 (HCT116) or 2 � 106 (RKO and

CO115) cells. For transfection assays, both flanks of each animal were injected

subcutaneously with 2 � 106 RKO (left) or RKO-HDAC2-transfected (right)

cells in a total volume of 200 ml of PBS. Tumor development at the site of

injection was evaluated daily.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSThis work was supported, in part, by the Health and Science Departmentsof the Spanish Government and the Spanish Association AgainstCancer (AECC).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturegenetics

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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