histone deacetylase inhibitors induce growth arrest and ...histone deacetylase inhibitors induce...

Cancer Therapy: PreclinicalSee commentary by Woodman, p. 323

Histone Deacetylase Inhibitors Induce Growth Arrest andDifferentiation in Uveal Melanoma

Solange Landreville1, Olga A. Agapova1, Katie A. Matatall1, Zachary T. Kneass2, Michael D. Onken1,Ryan S. Lee1, Anne M. Bowcock3, and J. William Harbour1

AbstractPurpose: Metastasis is responsible for the death of most cancer patients, yet few therapeutic agents are

available which specifically target the molecular events that lead to metastasis. We recently showed that

inactivating mutations in the tumor suppressor gene BAP1 are closely associated with loss of melanocytic

differentiation in uveal melanoma (UM) and metastasis. The purpose of this study was to identify

therapeutic agents that reverse the phenotypic effects of BAP1 loss in UM.

Experimental Design: In silico screens were done to identify therapeutic compounds predicted to

differentiate UM cells using Gene Set Enrichment Analysis and Connectivity Map databases. Valproic acid

(VPA), trichostatin A, LBH-589, and suberoylanilide hydroxamic acidwere evaluated for their effects onUM

cells using morphologic evaluation, MTS viability assays, bromodeoxyuridine incorporation, flow cyto-

metry, clonogenic assays, gene expression profiling, histone acetylation and ubiquitination assays, and a

murine xenograft tumorigenicity model.

Results:Histonedeacetylase (HDAC) inhibitors inducedmorphologic differentiation, cell-cycle exit, and

a shift to a differentiated,melanocytic gene expressionprofile in culturedUMcells. VPA inhibited the growth

of UM tumors in vivo.

Conclusions: These findings suggest that HDAC inhibitors may have therapeutic potential for

inducing differentiation and prolonged dormancy of micrometastatic disease in UM. Clin Cancer Res;

18(2); 408–16. �2011 AACR.

Introduction

Metastasis is responsible for the death of most cancerpatients, yet there are few therapies available that effectivelytarget the metastatic process. This is particularly true formetastatic melanoma, which is notoriously resistant totreatment. Because metastatic melanoma may remainasymptomatic in a state of dormancy for months to yearsbefore becoming clinically detectable, one therapeutic strat-egy is to prevent or delay micrometastatic disease fromescaping dormancy by inducing senescence or differentia-tion (1). However, such a strategy has been hampered by aninadequate understanding of the genetic events drivingmetastasis and, consequently, a lack of rational drug targets.

Uveal melanoma (UM) is a highly aggressive form ofmelanoma that exhibits a strong tendency for lethal hema-togenous metastasis to the liver and other sites (2). At thetime the primary eye tumor is diagnosed, less than 4% ofpatients have detectable metastatic disease (3). Yet up tohalf of these individuals will eventually succumb to metas-tasis, despite successful treatment of the primary tumor,indicating that they harbored subclinical micrometastasesat initial presentation. The most accurate method for iden-tifying UM patients who are at high risk of metastasis is bygene expression profiling of the primary tumor, which canbe done on a fine-needle biopsy using a validated 15-geneassay (4). This method separates UMs into 2 classes. Class 1tumors,whichhave a very lowmetastatic risk, are composedofmoredifferentiated tumor cells, and their gene expressionprofile is highly similar to that of normal differentiatedmelanocytes (5). In contrast, class 2 tumors, which have ahigh risk of metastasis, contain cells that have lost mor-phologic features of melanocytic differentiation, and theirgene expression signature is enriched for genes expressed inprimitive neuroectodermal cells (5).

We recently showed that inactivating somatic mutationsin the tumor suppressor geneBAP1, located at chromosome3p21, and concomitant loss of the other copy of chromo-some 3 are present in the vast majority of class 2 tumors butnot class 1 tumors (6). Depletion of BAP1 in cultured class 1

Authors' Affiliations: Departments of 1Ophthalmology & Visual Sciences,2Otolaryngology, and 3Genetics, Washington University School of Medi-cine, St. Louis, Missouri

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: J. William Harbour, Department of Ophthalmol-ogy, Washington University School of Medicine, Campus Box 8096, 660South Euclid Avenue, St. Louis, MO 63110. Phone: 314-362-3315; Fax:314-747-5073; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-0946

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(2) January 15, 2012408

on June 18, 2020. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 28, 2011; DOI: 10.1158/1078-0432.CCR-11-0946

http://clincancerres.aacrjournals.org/

UM cells induced a loss of melanocytic differentiation andacquisition of a class 2 gene expression profile, suggestingthat the loss of BAP1 may be mechanistically linked tometastasis. Functional studies have confirmed the tumorsuppressor activity of BAP1 (7, 8), and several recent reportshave linked germline BAP1 mutations to a spectrum offamilial cancers, including UM and cutaneous melanomaand mesothelioma (9–11). The link between BAP1 anddifferentiation in UM suggests that therapeutic compoundswhich trigger differentiation in UM cells may have clinicalvalue, particularly in the adjuvant setting in which the goalwould be to induce a prolonged latency of micrometastaticdisease. Thus, we carried out 2 complementary in silicoscreens to identify therapeutic compounds predicted toshift UM cells from the class 2 to the class 1 signature.Histone deacetylase (HDAC) inhibitors were ranked at ornear the top of candidate compound lists in both screens.We analyzed the effects of 4 different HDAC inhibi-

tors, including valproic acid (VPA), trichostatin A (TSA),LBH-589, and suberoylanilide hydroxamic acid (SAHA) inestablished UM cell lines and in primary UM cells in short-term culture. These compounds induced a proliferationblock through G1 cell-cycle arrest, as well as morphologicand transcriptomic changes consistent with melanocyticdifferentiation. VPA inhibited the growth of UM tumorsin vivo. We conclude that HDAC inhibitors may play a rolein the adjuvant therapy of patients with UM by inducingdifferentiation and prolonged dormancy of micrometa-static disease.

Materials and Methods

Tumor samplesThis study was approved by the Institutional Review

Board of Washington University in St. Louis and adheredto the tenets of the Declaration of Helsinki. Primary UMsamples were collected at the time of enucleation as previ-ously described (12). Informed consent was obtained foreach patient. All samples were confirmed to be UMs by

pathologic evaluation. Tumor samples were snap-frozenfor DNA and RNA isolation or collected in HAM’S F-12medium for tissue culture. Primary UM cells were grown inserum-free MDMF medium on collagen-covered tissueculture plates in 5% CO2 and 4% O2 (13). Elimination ofcontaminating cells was confirmed by microscopic inspec-tion and Melan-A staining. The multigene prognostic assayfor assignment of tumors to class 1 or class 2 groups wasdone as described elsewhere (4). To determine BAP1 statusof primary tumors, genomic DNA was subjected to Sangersequencing of all BAP1 exons as described elsewhere (14).The UM cell lines 92.1, OCM1A, and Mel202 (kindlyprovided by Drs. M. Jager, J. Kan-Mitchell and B. Ksander,respectively) were grown in RPMI-1640 supplemented with10% FBS, L-glutamine, and antibiotics at 5% CO2. TheseUM cell lines are all BAP1 wildtype with a gene expressionprofile similar to class 1 tumors.

Gene set enrichment analysis and connectivitymapping

Gene expression profiles from 14 class 1 and 10 class2 UMswere obtained on the Affymetrix U133A platform, aspreviously described (15). Curated gene sets enriched inclass 1 UM tumors versus class 2 UM tumors were identifiedwith gene set enrichment analysis (GSEA; version 2.0.4;Broad Institute), using a significance threshold set at anominal P value of less than 0.01 (16), as we previouslydescribed (5). To identify compounds that could potential-ly induce differentiation of class 2UM cells, we interrogatedthe connectivity map database (Broad Institute; ref. 17) forcompounds that caused gene expression changes that close-ly resembled the gene expression differences between nor-mal uveal melanocytes versus class 2 UM cells, as well asclass 1 UM cells versus class 2 UM cells. The 100 mostdifferentially expressed genes were chosen using Studentt test. This gene set was compared with those from theconnectivity map database, and potential compounds werechosen using the following parameters (e.g., positiveenrichment score with a P < 0.05).

Chemosensitivity assaysThe effects of 4 HDAC inhibitors, VPA (Sigma), TSA

(Sigma), LBH-589 (Selleck), and SAHA (Selleck) were stud-ied in UM cell lines and primary UM cells. Drug concent-rations ranged from 0.5 to 2 mmol/L for VPA, 50 to250 nmol/L for TSA, 5 to 60 nmol/L for LBH-589, and0.5 to 2.5 mmol/L for SAHA. Dimethyl sulfoxide was addedas vehicle in TSA, LBH-589, and SAHA untreated controls.

MTS assays were done according to manufacturer’sinstructions (CellTiter 96 AQueous Assay reagent; Pro-mega). Bromodeoxyuridine (BrdU; 1:1,000; AmershamBiosciences) incorporation assays were done using ananti-BrdU-peroxidase–conjugated antibody according tothe manufacturer’s instructions (Roche; dilution 1:75).Tetramethylbenzidine (Sigma) was added as substrate forsignal detection, and colorimetric changesweremeasured at370 nm using a Microplate spectrophotometer (Spectra-MAX 190; Molecular Devices). Flow cytometry was done

Translational Relevance

Uveal melanoma (UM) is highly metastatic and, onceit has disseminated, it is highly recalcitrant to availabletherapies. Recently, we discovered that inactivatingmutations in the histone H2A ubiquitin hydrolase BAP1are strongly associated with metastasis in UM. In thisstudy, we show that histone deacetylase (HDAC) inhi-bitors reverse the biochemical effects of BAP1 loss,induce melanocytic differentiation and cell-cycle arrest,and inhibit the growth of UM tumors in vivo in axenograft model. These findings suggest that HDACinhibitors may be effective in an adjuvant setting forinducing differentiation and prolonging the dormancyof micrometastatic disease in UM.

HDAC Inhibition in Uveal Melanoma

www.aacrjournals.org Clin Cancer Res; 18(2) January 15, 2012 409




after treatment of cells for 48 hours using a standardpropidium iodide staining protocol as previously described(18) using a FACScan analyzer (BD Biosciences). The per-centage of cells in each phase was determined with theVenturiOne software (Applied Cytometry). For clonogenicassays, flow cytometry (MoFlo; Cytomation) was used toseed one viable cell per well in ultralow attachment 96-wellplates containing MDMF medium only or MDMF withdrug.Cellsweremonitoredwithphase contrastmicroscope,and cell number was assessed for each well at 7 days. Cellmorphology was assessed by phase contrast microscopyand morphologic changes were quantified by counting thenumber of dendritic arborizations per cell in primary UMcells or by determining the maximal cell length/width ratio(spindle morphology index) in UM cell lines using ImageJ.Higher values are consistent with increasing melanocyticdifferentiation.

Multigene prognostic assay and qPCRTotal RNA extracted with TRIzol (Invitrogen) was DNase

treated and reverse transcription was done using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosys-tems). cDNA was preamplified for 14 cycles with pooledprimers according to the manufacturer’s protocol (AppliedBiosystems). Themultigene prognostic assay was done withRNA from primary tumor cells treated with VPA, LBH-589,and SAHA, and molecular class was determined usingsupport vector machine (SVM), as described elsewhere(4). BAP1 mRNA levels were measured by qPCR as pre-viously described (6).

BAP1 knockdownBAP1 was depleted in the 92.1 UM cell line by using a

lentiviral-based short hairpin RNA (shRNA). LentiviralpLKO.1 shRNA vectors for GFP (clonetechGfp-438s1c1)and BAP1 [NM_004656.2-321s1c1; developed by theRNAi Consortium (TRC)] were purchased from the Chil-dren’s Discovery Institute/Genome Sequencing Center atWashington University in St. Louis. Viral production andinfections were carried out according to The RNAi Consor-tium recommendations (Broad Institute). Lentivirusesencoded by the pLKO.1 shRNA vectors were packaged in293FT cells (Invitrogen) after cotransfection of the shRNAplasmids with pCMV-dR8.2 dvpr and pCMV-VSV-G lenti-viral plasmids (Addgene plasmids #8454 and #8455) usingTransIT-LT1 (Mirus Bio; ref. 19). 92.1 UM cell line wasinfected for 24 hours with lentiviral supernatants in thepresence of 8 mg/mL polybrene. Puromycin (3 mg/mL) wasadded to the cells at 24 hours postinfection for selection.

Western blotting and immunofluorescenceWestern blotting was done as previously described (18)

using anti-BAP1 (1:250; Santa Cruz), anti–a-tubulin (load-ing control, 1:1000; Sigma), anti-acetyl-histone H3Lys9(1:200; Cell signaling), anti-histone H3 (1:200; Cell signal-ing), anti–ubiquityl-histone H2A (1:100; Millipore), andanti-histone H2A (1:200; Millipore) antibodies. Histoneproteins for Western blotting were extracted from cells

according to previously published protocol (20). Westernblotting densitometrywas carried outwith theAlphaEaseFCsoftware using the Spot Denso Analysis Tool. Intensity ofeach Ub-H2A and total H2A bands was measured using arectangular spot and Ub-H2A was normalized to total H2Aafter background subtraction. Immunofluorescence wasdone by plating 5 � 103 92.1 cells on Lab-Tek 8-wellPermanox chamber slides (Nunc) and using anti-BAP1,anti–acetyl-histone H3Lys9 and anti–ubiquityl-histoneH2A antibodies.

Animal studiesAnimal experiments were approved by the Washington

University in St. Louis Animal Studies Committee. A totalof 1 � 106 92.1 UM cells were resuspended in Cultrex(Trevigen) and injected subcutaneously into the flanks ofNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ JAX [nonobese diabeticsevere-combined immunodeficient (NOD SCID) gamma]mice (Jackson Laboratory). Mice were then given intraper-itoneal injection of VPA (0.1 mg/g of body weight) everyother day, beginning at day 7. Tumor volume was moni-tored once a week and the mice were euthanized after42 days. Tumorswere collected and volumemeasured usingthe ellipsoid volume formula (p xyz/6 mm3).

Statistical analysisExcept where otherwise noted, data were analyzed for

statistical significance using MedCalc software, version9.5.1.0.

Figure 1. Gene set enrichment analysis. Enrichment plot of gene setPEART_HISTONE_UP. The gene set that was most similar to the genesupregulated in class 1 UMs (relative to class 2) using GSEA wasPEART_HISTONE_UP, which consisted of genes upregulated by theHDAC inhibitors SAHA and depsipeptide. Genes that areoverrepresented in class 1 tumors show a peak enrichment score (ES)that is positive and to the left of the plot, and those that areoverrepresented in class 2 tumors show a peak ES that is negative and tothe right of the plot.

Landreville et al.

Clin Cancer Res; 18(2) January 15, 2012 Clinical Cancer Research410




Results

In silico screening for compounds that reverse theeffects of BAP1 lossBAP1 loss in UM cells results in morphologic and tran-

scriptomic changes consistent with a loss of melanocytic

differentiation and a shift from class 1 to class 2 transcrip-tomic profile (6). Thus, we sought to identify therapeuticcompounds that may reverse the effects of BAP1 loss byrestoring a more differentiated, class 1-like transcriptomicprofile. We used 2 complementary in silico approaches–GSEA andConnectivityMapping–to compare genes that are

Figure 2. Effects of HDAC inhibitorson UM cell lines. Cells were eitheruntreated (UT) or treated with VPA,TSA, and LBH-589. A, MTS cellviability assays after 72 hourstreatment. The absorbance ofcontrol cells at 490 nm was taken as100%. B, BrdU incorporation assaysafter 72 hours treatment. Theabsorbance at 370 nm of controlcells was taken as 100%. C, cell-cycle analysis by flow cytometryusing propidium iodide staining.Cells were either UT or HDACinhibitor–treated for 48 hours; x-axisrepresents DNA content and y-axisrepresents cell number. D, single-cell clonogenic assays. OCM1Awere either UT or HDAC inhibitor–treated and cell number for each wellcontaining a colony was assessedafter 7 days. �, P < 0.05.






differentially expressed between class 1 and class 2 UMs tocurated gene sets associated with perturbation of cancercells with therapeutic compounds. UsingGSEA, the gene setthat was most similar to the genes upregulated in class 1UMs (relative to class 2) was PEART_HISTONE_UP (Fig. 1),which consisted of genes upregulated by the HDAC inhi-bitors SAHA and depsipeptide (21). We obtained similarresults with the Connectivity Mapping, which identified 3HDAC inhibitors (VPA, TSA, and SAHA) among its topmatches (Supplementary Table S1).

HDAC inhibition blocks proliferation of UM cellsInitially, we chose VPA to test the effects of HDAC

inhibition in UM cells. As expected, VPA caused adose-dependent increase in histone H3 acetylation (Sup-plementary Fig. S1). In all 3 UM cell lines (92.1, OCM1A,and Mel202), VPA inhibited proliferation but did notsignificantly reduce the fraction of viable cells, induced aG1 cell-cycle arrest, and markedly reduced the clonogeni-city of UM cells (Fig. 2). The spindle morphology indexincreased after treatment with the HDAC inhibitors(Supplementary Fig. S2). Similar changes were seen withTSA and LBH-589, except that these agents significantlyreduced the fraction of viable cells and increased theproportion of cells undergoing apoptosis (Fig. 2), con-sistent with increased cytotoxicity.

VPA inhibits UM tumor growth in vivoBecause VPA induced cell-cycle arrest rather than cell

death, we predicted that it may have therapeutic potentialin an adjuvant setting to block the growth of micrometa-static disease. To model this situation, we established smallsubcutaneous flank tumors in NODSCID gammamice andtested whether VPA could prevent the growth of thesetumors. Treatment with VPA or control was initiated whenthe tumors became barely palpable (usually about 7 daysafter injection). VPA markedly reduced the growth andfinal volumeof these tumordeposits comparedwith control(Fig. 3).

BAP1 loss sensitizes UM cells to HDAC inhibitionBAP1 is the catalytic subunit of the PR-DUB complex

that deubiquitinates histone H2A (22). As expected, RNAi-mediated knockdownofBAP1 inUMcells (Fig. 4A) induceda marked increase in H2A ubiquitination (Fig. 4B, toppanel). Because a recent report showed that HDAC inhibi-tors decrease histone H2A ubiquitination through tran-scriptional repression of the PRC1 component BMI1(23), we wished to determine whether HDAC inhibitionmay reverse this H2A hyperubiquitination in BAP1-defi-cient UM cells. Indeed, VPA markedly reduced the levelsof histone H2A ubiquitination in BAP1-depleted cells(Fig. 4B, bottom panel and Fig. 4C top panel), whereastotal histone H2A level remained unchanged (Fig. 4C,bottom panel). We showed in Fig. 2A that VPA inhibitedproliferation of BAP1 wildtype UM cell lines, but it did notreduce the fraction of viable cells. However, when we stablydepleted BAP1 in UM cells using lentiviral shRNA, VPA

significantly reduced the fraction of viable cells comparedwith control cells (Fig. 4D), indicating that BAP1 losssensitized the UM cells to HDAC inhibition.

We then investigated whether HDAC inhibition couldreverse the phenotypic consequences of BAP1 loss: lossof melanocytic differentiation and acquisition of the class2 gene expression profile (6). Because UM cell linesundergo genetic and epigenetic artifacts in long-termculture, for these experiments we used primary UM cellsfrom 5 class 2 tumors (MM137, MM138, MM151,MM161, and MM162) and 1 class 1 tumor (MM131)which were obtained immediately after surgical resectionand placed in short-term culture. Although only 4 of theclass 2 samples contained detectable BAP1 mutations,all 5 showed low BAP1 RNA levels compared with theclass 1 tumors (Supplementary Table S2 and Supplemen-tary Fig. S3). In cells treated with VPA for 3 and 7 days, cellbodies became enlarged and flattened, and there was amarked increased in the number of dendritic arboriza-tions, consistent with melanocytic differentiation (Fig. 5A

Figure 3. VPA inhibits the growth ofUM tumors in vivo. A, growth curves of92.1 cell xenografts in NOD SCID gamma mice, either UT or treated withVPA (0.1 mg/g of body weight, injected intraperitoneally every other day,from day 7 to day 41; n¼ 10 animals for each condition). B, volume of UTandVPA-treated flank tumors at the time of necropsy (day 42).Middle linerepresents median; box, 25th to 75th percentiles; outer bars, minimumand maximum values. �, P < 0.05.

Landreville et al.





and B). Untreated or VPA-treated cells were subjected toamultigene expression profile that classifies UMs as class 1or class 2 (4). The results of this assay were reported as aSVM discriminant score; the more negative the score, themore class 1 the expression profile, the more positivethe more class 2. VPA shifted all of the class 2 UM cellstoward a class 1 profile, and it even shifted the class 1 UMcells toward a more class 1 profile (Fig. 5C and Supple-mentary Fig. S4). This effect was dose dependent, andsimilar results were observed with LBH-589 (Supplemen-tary Figs. S4 and S5C). The genes most affected byVPA were HTR2B (downregulated by VPA) and LMCD1(upregulated by VPA; Supplementary Fig. S5), both strongindicators of metastasizing UMs (4). A fourth HDACinhibitor, SAHA, induced effects similar to VPA in UMcell lines and primary UM cells (Supplementary Fig. S6).

Discussion

Here, we show that clinically achievable concentrationsof several HDAC inhibitors can reprogramhighly aggressiveUM cells to a low-grade, differentiated state. This is consis-tent with similar findings in other cancers (24). These

findings suggest a potential role for HDAC inhibitors inpatients with high-risk class 2 UMs. Most of these patientsharbor undetectablemicrometastases at the time of primaryeye tumor diagnosis, and the goal of HDAC inhibitortherapy would be to induce prolonged clinical dormancy(and longer patient survival) by driving these micrometa-static cells into a differentiated, quiescent state.

We have not yet found a UM cell line with naturallyoccurring BAP1mutations (data not shown), despite manyof these cell lines being derived from metastasizing UMsthat likely contained cells with mutant BAP1. This suggeststhat BAP1 mutant UM cells may not grow well in culture.This has hindered our ability to study the effects of HDACinhibitors on BAP1 mutant UM cells in vivo. However,in short-term cultures, HDAC inhibitors did indeedreverse the biochemical, transcriptomic, and morphologicconsequences of BAP1 loss in primary UM cells. Knock-down of BAP1 caused an increase in histone H2A ubiqui-tination, consistent with its H2A ubiquitin carboxy-termi-nal hydrolase activity (7, 22), which is critical for its tumorsuppressor function (7, 8). HDAC inhibitors reversedthe H2A hyperubiquitination caused by BAP1 loss, andthey shifted the gene expression profile of class 2 cells

Figure 4. VPA counteracts the abnormal ubiquitination of histone H2A in BAP1-depleted UM cells. A, BAP1 Western blot (left) and immunofluorescence(right, magnification 200�) of 92.1 control (shGFP) and BAP1-depleted (shBAP1) cells. B, ubiquityl-histone H2A (Ub-H2A) immunofluorescence of UT orVPA-treated 92.1 control (shGFP) and BAP1-depleted (shBAP1) cells. Magnification 400�. C, ubiquityl-histone (Ub-H2A) and total histone H2AWesternblots (left) of UT or VPA-treated 92.1 BAP1-depleted cells (shBAP1); corresponding densitometry (right). D, MTS cell viability assays after 72 hourstreatment with VPA 2 mmol/L. The absorbance of control cells at 490 nm was taken as 100%. �, P < 0.05.






toward a class 1 profile. Furthermore, HDAC inhibitorsinduced morphologic changes consistent with melanocyticdifferentiation. These findings suggest that BAP1 normallyfunctions, at least in part, to maintain melanocyte differ-entiation in UM cells and that this function can be at leastpartially restored in the absence of BAP1 by increasinghistone H3 acetylation. HDAC inhibitors reverse the bio-chemical defect caused by BAP1 loss (i.e., H2A hyperubi-quitination), which may explain why BAP1-deficientUM cells were more sensitive to HDAC inhibition thanBAP1-competent cells. However, because HDAC inhibitorsact through multiple mechanisms of action, it is not sur-prising that they also had a differentiating effect on BAP1wildtype tumor cells.

Histones undergo extensive posttranslational modifica-tions, including acetylation,methylation, phosphorylation,

ubiquitination, and ribosylation, that comprise a complexcombinatorial code that regulates gene expression (25).This system has been of particular interest in understandingthe coordinated expression of transcriptional programsduring development and differentiation. Among thesemodifications, acetylation has been one of the most thor-oughly studied. The balance between acetylation and dea-cetylation is determined by the relative activities of histoneacetyltransferases and HDACs, with increased acetylationpromoting greater chromatin accessibility for gene expres-sion. Histone ubiquitination is less well understood.Monoubiquitination of histone H2A on Lys119 plays animportant role in X-chromosome inactivation, Polycombgroup-dependent gene silencing, and other developmentalprocesses (26, 27). Distinct histone modifications also canfunction together in a coordinated fashion to regulate

Figure 5. VPA induces differentiation and a shift to class 1 signature.A, representative phase contrast images of primary UM cells from aclass 2 tumor (MM151) and a class 1 tumor (MM131) UT or VPA-treated for 7 days. Scale bar equal 50 mm. B,mean number of dendriticarborizations in primary cultured UM cells that were untreated (UT) orVPA-treated. C, SVM discriminant scores showing the effect of VPAtreatment (2mmol/L) ongeneexpression signature. Themorenegativethe number, the more class 1-like; the more positive the number,the more class 2-like. MM137, MM138, and MM151 cells were fromclass 2 tumors; MM131 was from a class 1 tumor. Red spheresrepresent the UT cells and blue spheres the VPA-treated cells.�, P < 0.05.

Landreville et al.





chromatin structure and gene expression. For example, thehistone deubiquitinase 2A-DUB regulates transcription bycoordinatinghistone acetylation anddeubiquitination, anddestabilizing the association of linker histone H1 withnucleosomes (28). Furthermore, inhibition of HDAC activ-ity has been shown to decrease histone ubiquitinationthrough transcriptional repression of the PRC1 componentBMI1 (23). This may explain the efficacy of HDAC inhibi-tors in UM cells.VPA and LBH-589 have both shown therapeutic poten-

tial as monotherapy or in combination with other anti-tumor drugs in solid and hematologic malignancies (29).As expected, on the basis of their mechanism of action,HDAC inhibitors have not been shown to be effective as acytotoxic therapy in patients with advanced metastaticmelanoma (30), but the findings herein suggest that theymay have a role in preventing the progression of micro-metastatic disease. We focused here on VPA because it hada more prominent effect on cell-cycle arrest and differ-entiation, compared with TSA and LBH-589, which weremore cytotoxic. The effect of SAHA was similar to VPA.VPA is a well-characterized compound that has been usedfor almost 30 years in the treatment of epilepsy and, morerecently, as an anticancer agent (29). The toxicity profileof VPA would allow its use as an adjuvant agent in high-risk cancer patients. Taken together, these factors suggestthat a clinical trial of adjuvant VPA or a similar HDACinhibitor in high-risk class 2 UM patients may be war-ranted. Gene expression profiling can allow enrollmentof high-risk class 2 patients, about half of whom will

develop overt metastatic disease within 3 years of eyetumor diagnosis (4).

Disclosure of Potential Conflicts of Interest

J.W. Harbour andWashington University may receive income based on alicense of related technology by theUniversity to Castle Biosciences, Inc. Thiswork was not supported by Castle Biosciences, Inc.

Acknowledgments

The authors thankWilliamEades, JonChristopherHolley, and JacquelineHughes in the Siteman Cancer Center High Speed Sorter Core Facility (NCICancer Center Support Grant #P30 CA91842).

Grant Support

This work was supported by Fonds de la Recherche en Sant�e du Qu�ebecPostdoctoral Training Award (S. Landreville), NIH/NIDCDT32 ResearchTraining Program for Otolaryngology (Z.T. Kneass), Alvin J. Siteman CancerCenter Summer Undergraduate Research Fellowship program (R.S. Lee),NIH R01 AR050266 (A.M. Bowcock), NIH R01 CA125970 (J.W. Harbour),NIHR01EY13169 (J.W.Harbour), Barnes-JewishHospital Foundation (J.W.Harbour), Kling Family Foundation (J.W. Harbour), Horncrest Foundation(J.W.Harbour) and Research to Prevent Blindness (J.W.Harbour), and by anunrestricted grant to the Department of Ophthalmology and Visual Sciencesfrom a Research to Prevent Blindness, Inc. and the NIH Vision Core GrantP30 EY02687.

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 April 12, 2011; revised October 15, 2011; accepted October 20,2011; published OnlineFirst October 28, 2011.

References1. Wikman H, Vessella R, Pantel K. Cancer micrometastasis and tumour

dormancy. APMIS 2008;116:754–70.2. Landreville S, Agapova OA, Harbour JW. Emerging insights into the

molecular pathogenesis of uveal melanoma. Future Oncol 2008;4:629–36.

3. Finger PT, Kurli M, Reddy S, Tena LB, Pavlick AC.Whole body PET/CTfor initial staging of choroidal melanoma. Br J Ophthalmol 2005;89:1270–4.

4. OnkenMD,Worley LA, TuscanMD,Harbour JW.An accurate, clinicallyfeasiblemulti-gene expression assay for predictingmetastasis in uvealmelanoma. J Mol Diagn 2010;12:461–8.

5. ChangSH,Worley LA,OnkenMD,Harbour JW.Prognostic biomarkersin uvealmelanoma: evidence for a stemcell-like phenotype associatedwith metastasis. Melanoma Res 2008;18:191–200.

6. Harbour JW,OnkenMD,RobersonED,DuanS,Cao L,Worley LA, et al.Frequent mutation of BAP1 in metastasizing uveal melanomas. Sci-ence 2010;330:1410–3.

7. Jensen DE, Proctor M, Marquis ST, Gardner HP, Ha SI, Chodosh LA,et al. BAP1: a novel ubiquitin hydrolase which binds to the BRCA1RING finger and enhances BRCA1-mediated cell growth suppression.Oncogene 1998;16:1097–112.

8. Ventii KH, Devi NS, Friedrich KL, Chernova TA, Tighiouart M, Van MeirEG, et al. BRCA1-associated protein-1 is a tumor suppressor thatrequires deubiquitinating activity and nuclear localization. Cancer Res2008;68:6953–62.

9. Abdel-Rahman MH, Pilarski R, Cebulla CM, Massengill JB, Christo-pher BN, Boru G, et al. Germline BAP1 mutation predisposes to uvealmelanoma, lung adenocarcinoma, meningioma, and other cancers.J Med Genet 2011 Sep 22. [Epub ahead of print].

10. Testa JR, Cheung M, Pei J, Below JE, Tan Y, Sementino E, et al.Germline BAP1mutations predispose tomalignantmesothelioma. NatGenet 2011;43:1022–5.

11. Wiesner T, Obenauf AC, Murali R, Fried I, Griewank KG, Ulz P, et al.Germline mutations in BAP1 predispose to melanocytic tumors. NatGenet 2011;43:1018–21.

12. OnkenMD,Worley LA, Person E, Char DH, Bowcock AM, Harbour JW.Loss of heterozygosity of chromosome 3 detected with single nucle-otide polymorphisms is superior to monosomy 3 for predicting metas-tasis in uveal melanoma. Clin Cancer Res 2007;13:2923–7.

13. Landreville S, Agapova OA, Kneass ZT, Salesse C, Harbour JW.ABCB1 identifies a subpopulation of uveal melanoma cells with highmetastatic propensity. Pigment Cell Melanoma Res 2011;24:430–7.

14. Wood LD, Parsons DW, Jones S, Lin J, Sjoblom T, Leary RJ, et al. Thegenomic landscapes of human breast and colorectal cancers. Science2007;318:1108–13.

15. Onken MD, Worley LA, Ehlers JP, Harbour JW. Gene expressionprofiling inuvealmelanoma reveals twomolecular classesandpredictsmetastatic death. Cancer Res 2004;64:7205–9.

16. SubramanianA, TamayoP,Mootha VK,Mukherjee S, Ebert BL,GilletteMA, et al. Gene set enrichment analysis: a knowledge-based approachfor interpreting genome-wide expression profiles. Proc Natl Acad SciU S A 2005;102:15545–50.

17. Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, et al.The Connectivity Map: using gene-expression signatures toconnect small molecules, genes, and disease. Science 2006;313:1929–35.

18. Delston RB, Matatall KA, Sun Y, Onken MD, Harbour JW. p38 phos-phorylates Rb on Ser567 by a novel, cell cycle-independent






mechanism that triggers Rb-Hdm2 interaction and apoptosis. Onco-gene 2011;30:588–99.

19. Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, et al.Lentivirus-delivered stable gene silencing by RNAi in primary cells.RNA 2003;9:493–501.

20. Shechter D, Dormann HL, Allis CD, Hake SB. Extraction, purificationand analysis of histones. Nat Protoc 2007;2:1445–57.

21. Peart MJ, Smyth GK, van Laar RK, Bowtell DD, Richon VM, Marks PA,et al. Identification and functional significance of genes regulated bystructurally different histone deacetylase inhibitors. ProcNatl Acad SciU S A 2005;102:3697–702.

22. ScheuermannJC,deAyalaAlonsoAG,OktabaK, Ly-HartigN,McGintyRK, Fraterman S, et al. Histone H2A deubiquitinase activity of thePolycomb repressive complex PR-DUB. Nature 2010;465:243–7.

23. Bommi PV, Dimri M, SahasrabuddheAA, Khandekar JD, Dimri GP. Thepolycomb group protein BMI1 is a transcriptional target of HDACinhibitors. Cell Cycle 2010;9:2663–73.

24. GottlicherM,Minucci S, ZhuP, KramerOH,Schimpf A,Giavara S, et al.Valproic acid defines a novel class of HDAC inhibitors inducingdifferentiation of transformed cells. EMBO J 2001;20:6969–78.

25. van Leeuwen F, van Steensel B. Histone modifications: from genome-wide maps to functional insights. Genome Biol 2005;6:113.

26. deNapolesM,Mermoud JE,WakaoR, TangYA, EndohM, AppanahR,et al. Polycomb group proteins Ring1A/B link ubiquitylation of histoneH2A to heritable gene silencing and X inactivation. Dev Cell 2004;7:663–76.

27. Wang H, Wang L, Erdjument-Bromage H, Vidal M, Tempst P, JonesRS, et al. Role of histone H2A ubiquitination in Polycomb silencing.Nature 2004;431:873–8.

28. ZhuP, ZhouW,Wang J,Puc J,Ohgi KA, Erdjument-BromageH, et al. Ahistone H2A deubiquitinase complex coordinating histone acetylationand H1 dissociation in transcriptional regulation. Mol Cell 2007;27:609–21.

29. Tan J, Cang S, Ma Y, Petrillo RL, Liu D. Novel histone deacetylaseinhibitors in clinical trials as anti-cancer agents. J Hematol Oncol2010;3:5.

30. Rocca A, Minucci S, Tosti G, Croci D, Contegno F, Ballarini M, et al. Aphase I-II study of the histone deacetylase inhibitor valproic acid pluschemoimmunotherapy in patients with advanced melanoma. Br JCancer 2009;100:28–36.

Landreville et al.





2012;18:408-416. Published OnlineFirst October 28, 2011.Clin Cancer Res Solange Landreville, Olga A. Agapova, Katie A. Matatall, et al. Differentiation in Uveal MelanomaHistone Deacetylase Inhibitors Induce Growth Arrest and

Updated version

10.1158/1078-0432.CCR-11-0946doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2011/10/27/1078-0432.CCR-11-0946.DC1

http://clincancerres.aacrjournals.org/content/suppl/2012/01/10/1078-0432.CCR-11-0946.DC2Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/18/2/408.full#ref-list-1

This article cites 29 articles, 11 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/18/2/408.full#related-urls

This article has been cited by 18 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/18/2/408To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-11-0946

http://clincancerres.aacrjournals.org/content/suppl/2012/01/10/1078-0432.CCR-11-0946.DC2 http://clincancerres.aacrjournals.org/content/suppl/2011/10/27/1078-0432.CCR-11-0946.DC1

http://clincancerres.aacrjournals.org/content/suppl/2012/01/10/1078-0432.CCR-11-0946.DC2 http://clincancerres.aacrjournals.org/content/suppl/2011/10/27/1078-0432.CCR-11-0946.DC1

http://clincancerres.aacrjournals.org/content/18/2/408.full#ref-list-1

http://clincancerres.aacrjournals.org/content/18/2/408.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/18/2/408


histone deacetylase inhibitors induce growth arrest and ...histone deacetylase inhibitors induce...

Documents