Cancer Therapy: Preclinical

Synergistic Interaction between Histone Deacetylase and Topoisomerase II Inhibitors Is Mediated through To p o i s o m e r a s e II B Douglas C. Marchion, Elona Bicaku, Joel G. Turner, Adil I. Daud, Daniel M. Sullivan, and Pamela N. Munster

Abstract Background: DNA topoisomerase IIinhibitors and poisons are among the most efficacious drugs for the treatment of cancer. Sensitivity of cancer cells to the cytotoxic effects of topoisomerase II targeting agents is thought to depend on the expression of the topoisomerase IIa isoform, and drug resistance is often associated with loss or mutation of topoisomerase IIa. Histone deacetylase inhibitors (HDACi) are a novel class of compounds that potentiate the antitumor effects of topo- isomerase II ^ targeting agents. Methods: The interaction between HDACi and topoisomerase II ^ targeting agents in cancer cells was evaluated as a function of topoisomerase IIa and topoisomerase IIh expression. Topoi- somerase II isoforms were selectively depleted using small interfering RNA and antisense. Drug- induced formation of cleavable complexes involving topoisomerase IIa and topoisomerase IIh was evaluated by trapped-in-agarose DNA immunostaining and band depletion assays in the presence and absence of HDACi. Results: Preexposure to HDACi increased the cytotoxicity of topoisomerase II poisons.This was associated witha down-regulationof topoisomerase IIa expressionbuthadno effectsontopoiso- merase IIh. In the setting of HDACi-induced chromatin decondensation and topoisomerase IIa depletion, topoisomeraseIIpoisoncytotoxicity wasmediatedthroughtopoisomeraseIIhcleavable complex formation. The HDACi-induced sensitization was also observed in cells with target- specific resistance to topoisomerase II poisons. Conclusions: The recruitment of topoisomerase IIh as a target may overcome primary or emergent drug resistance to topoisomerase II ^ targeting agents and hence may broaden the applicability of this important class of anticancer agents.

Type II topoisomerases are essential enzymes that regulate out the cell cycle (7) and may have an important role in the topological state of DNA to facilitate several biological transcription (8). Although it was reported that topoisomerase processes. Topoisomerase II exists in two isoforms, topoiso- IIh may not be essential in mitosis, topoisomerase IIa merase IIa and topoisomerase IIh. Although these enzymes knockdown experiments have shown that topoisomerase IIh have high sequence homology (1) and a similar mechanism of may only partially substitute topoisomerase IIa during action (2), they are regulated independently and have been chromatin decondensation and cell segregation (9). associated with different cellular functions. Topoisomerase IIa Due to their essential roles, topoisomerase II enzymes have is necessary for many biological processes involving double- been successfully targeted for the treatment of cancer. To stranded DNA, including replication, mitosis, and chromatin maintain genetic integrity during the cleavage and religation of condensation (3, 4). Topoisomerase IIa is mainly located in DNA, both topoisomerase isoenzymes form transient bonds the nucleus and peaks in expression during G2-M (5, 6). In with the cleaved DNA called the cleavable complex (10). The contrast, topoisomerase IIh expression remains stable through- stabilization and persistence of these cleavable complexes by topoisomerase II poisons have been associated with DNA damage and cell death (11, 12). Multiple in vitro and in vivo

Authors’Affiliation: Experimental Therapeutics Program, Department of studies have suggested that sensitivity of tumor cells to Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, topoisomerase II poisons depends on access to the targets, as Tampa, Florida well as target location and expression levels. For clinical use, Received 5/13/05; revised 8/15/05; accepted 8/31/05. topoisomerase IIa has been considered the more relevant target Grant support: Susan G. Koman BreastCancer Foundation and Don Shula Career (13–17). DevelopmentAward. The costs of publication of this article were defrayed in part by the payment of page A class of drugs that may enhance access to DNA and thereby charges. This article must therefore be hereby marked advertisement in accordance increase the antitumor activity of topoisomerase II–targeting with 18 U.S.C. Section 1734 solely to indicate this fact. agents are the histone deacetylase inhibitors (HDACi; refs. 14, Requests for reprints: Pamela N. Munster, H. Lee Moffitt Cancer Center, 12902 18, 19). We previously reported a sequence-specific and time- Magnolia Drive, MRC 4E,Tampa, FL 33612. Phone: 813-745-8948; Fax: 813-745- 1984; E-mail: [email protected]. dependent potentiation of topoisomerase II–targeting agents F 2005 American Association for Cancer Research. by the HDACi, suberoylanilide hydroxamic acid (SAHA), and doi:10.1158/1078-0432.CCR-05-1073 valproic acid (14, 20). Our data indicated that prolonged

www.aacrjournals.org 8467 Clin Cancer Res 2005;11(23) December 1, 2005 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Therapy: Preclinical treatment of cancer cells with an HDACi resulted in the 4 hours. Epirubicin was then removed, and colonies were allowed to depletion of proteins involved in the maintenance of hetero- grow for 14 to 21 days to a maximal size of 2 to 3 mm in diameter, chromatin and led to subsequent chromatin decondensation. stained with 2% crystal violet in methanol, and counted. Colonies Chromatin decondensation increased the access of topoiso- were included in the assessment if measuring at least 0.2 mm. For single-drug samples and untreated controls, saline was used in lieu of merase II–targeting agents to the DNA substrate, which valproic acid or epirubicin or both. All experiments were done in potentiated cell death induced by topoisomerase II–targeting duplicates and repeated at least thrice. The concentration of epirubicin agents. Topoisomerase II drug cytotoxicity is largely dependent required for IC50 was determined using the CalcuSyn software as upon topoisomerase IIa expression. Here, we investigated the described previously (14). Synergistic effects versus additive effects relevance of topoisomerase IIa and topoisomerase IIh as targets were also determined by the CalcuSyn program. The fractional of topoisomerase II poisons in the presence of HDACi. We inhibition of valproic acid was depicted as the number of observed show that in the setting of HDACi pretreatment, the formation cells (treatment group) divided by the number of expected cells of cleavable complexes involving topoisomerase IIh rather than (untreated control) with a range of 0 to 1. topoisomerase IIa leads to topoisomerase II poison–induced Microarray. Expression levels of topoisomerase IIa mRNA were cell death. This may broaden the clinical applicability of evaluated by microarray analysis using Affymetrix Genechips (Affyme- trix, Santa Clara, CA) by standard protocols (Moffitt, Molecular topoisomerase II poisons and overcome drug resistance due to Biology Core). Hybridization to Affymetrix chips was analyzed acquired depletion and mutations of topoisomerase IIa. using Affymetrix Microarray Suite 5.0 software. Signal intensity was scaled to an average intensity of 500 before comparison analysis. The MAS 5.0 software uses a statistical algorithm to assess changes in Materials and Methods mRNA abundance in a direct comparison between two samples (Statistical algorithms description document. http://www.affymetrix. Chemicals and antibodies. Trichostatin A, sodium butyrate (NaB), com/support/technical/whitepapers.affx). This analysis is based on the valproic acid, mitoxantrone, and teniposide (VM-26) were purchased behavior of 16 different oligonucleotide probes designed to detect the from Sigma Chemical Co. (St. Louis, MO). SAHA was provided by Aton same gene. Using the programmed default values, probe sets that Pharma (Merck, Whitehouse Station, NJ). Epirubicin was purchased yielded a change P < 0.04 were identified as changed (increased or from Pfizer, Inc. (New York, NY). XK469 was a kind gift from Dr. decreased), and those that yielded a P between 0.04 and 0.06 were Robert Snapka. All other reagents were of analytic grade and purchased identified as marginally changed. from standard suppliers. Antibodies used were as follows: Ki-S1 Western blot analysis. Samples were prepared using SDS lysis buffer (monoclonal, Chemicon, Temecula, CA) and 454 (polyclonal, devel- [2% SDS, 10% glycerol, 0.06 mol/L Tris (pH 6.8)] and evaluated for oped by Dr. Dan Sullivan) for topoisomerase IIa, anti-topoisomerase protein concentration using the bicinchoninic acid method (Pierce, IIh (monoclonal, BD Biosciences, San Jose, CA) and JAB (polyclonal, Rockford, IL). Proteins (50 Ag) were separated on 8% SDS-PAGE gels developed by Dr. Dan Sullivan), and acetylated histone H3 antibody and transferred to nitrocellulose membranes. Membranes were blocked (Upstate Biotechnology, Chicago, IL). in tris-buffered saline containing 0.05% Tween 20 (TBST), 5% nonfat Cell lines. SKBr-3, MCF-7, KM12C, A375, BT-474, and MDA-MB- milk and incubated with primary antibody in TBST, 5% nonfat milk, 361 cells were purchased from the American Type Culture Collection overnight at 4jC. Membranes were washed thrice for 10 minutes with (Manassas, VA). Doxorubicin-resistant MCF-7 cells (MCF:Dox) were TBST and incubated with the appropriate secondary antibody in TBST, a kind gift from Dr. Fred Hausheer. Cell lines were maintained in 5% nonfat milk for 90 minutes at room temperature. Antibody binding DMEM supplemented with 10% heat-inactivated fetal bovine serum was visualized by chemiluminescence on autoradiography film. (FBS), 2 mmol/L glutamine, and 50 units/mL penicillin, and 50 Ag/mL Relative expression of proteins was determined by densitometry streptomycin (Life Technologies Bethesda Research Laboratories, analysis of at least two scanned autoradiography films from indepen- Carlsbad, CA). Cells were incubated in a humidified atmosphere with dent experiments using Photoshop software.

5% CO2 at 37jC. Immunofluorescence. MCF-7 cells were treated with 2 mmol/L Cytotoxicity assays. Topoisomerase II poison cytotoxicity was valproic acid for 48 hours. Cells were harvested by trypsinization, evaluated in the presence of HDACi by apoptotic and clonogenic washed in PBS, and adhered to glass slide using Cytospin Funnels assays. (Shandon, Pittsburgh, PA) at a density of 1  105/mL. Slides were Apoptosis was scored by the presence of nuclear chromatin blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour and condensation and DNA fragmentation and evaluated with fluorescence probed with antibodies specific for topoisomerase IIa, KiS1 (mono- microscopy using bis-benzimide staining. Briefly, cells were treated clonal, 1:50), and acetylated histone H3 (polyclonal, 1:100) in 1% with an HDACi for 48 hours, after which the media was removed and BSA/PBS for 1 hour at room temperature in a humidified chamber. replaced with media containing the indicated concentrations of a Slides were washed in PBS and incubated with anti-rabbit Alexa Fluor topoisomerase II poison. After 4 hours, the topoisomerase II poison 488 and anti-mouse Alexa Fluor 546 (Molecular Probes) diluted 1:100 was removed, and the cells were cultured for an additional 48 hours. in 1% BSA/PBS containing goat serum for 1 hour at room temperature Cells were harvested using a Cell Scraper (Fisher, Hampton, NH), fixed in a humidified chamber. Slides were washed with PBS and in 4% paraformaldehyde for 10 minutes at room temperature, and coverslipped using Prolong Gold mounting media (Molecular Probes). washed with PBS. Cell nuclei were stained with 0.5 Ag/mL of bis- Images were acquired by confocal microscopy. The square pixel surface benzimide trihydrochloride (Hoechst #33258, Molecular Probes, area for the respective protein expression was analyzed in at least 50 Eugene, OR). Two hundred cells were counted for each experiment cells per treatment using Photoshop software. For each measured and evaluated for apoptotic scores (apoptotic nuclei/all nuclei  100). nucleus, the background staining was measured in the immediate Each experiment was repeated thrice, and the SE was calculated. vicinity and subtracted from the average square pixel surface area. For clonogenic assays, cells were plated on six-well dishes at a Evaluation of secondary antibody staining only served as internal density of 150 per well and allowed to adhere for 24 hours. For drug staining control. Levels for the fluorescence detection were set combination studies, cells were incubated with medium containing 0.5 accordingly. Experiments were repeated at least twice. mmol/L valproic acid. At this concentration of valproic acid, valproic Antisense/small interfering RNA. Topoisomerase IIa and top- acid had no discernable effects on growth or apoptosis. After a 48-hour oisomerase IIh protein expression was blocked with antisense treatment with valproic acid, the medium was removed and replaced oligonucleotides and small interfering RNA (siRNA) duplexes. with medium containing 0, 1, 10, 25, 50, or 100 nmol/L epirubicin for Antisense: The DNA sequences for the oligonucleotide probes were

ClinCancerRes2005;11(23)December1,2005 8468 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Interaction of HDAC and Topoisomerase II Inhibitors as follows: 5V-CTGCAATGGTGACACTTCCAT-3V for topoisomerase IIa BSA for 10 minutes, dried, and counterstained with 0.5 Ag/mL of and 5V-TTTGTAGTGGACAGAAACACAGTA-3V for topoisomerase IIh as bisbenzimide trihydrochloride (Hoechst #33258, Molecular Probes). described by Towatari et al. (21). Oligonucleotide (1 Ag) was Images were acquired by confocal microscopy and the square pixel suspended in 100 AL Optimem (Life Technologies Bethesda Research surface area of the respective protein was analyzed for a minimum of Laboratories) containing 2 AL Superfect transfection reagent (Qiagen, 50 cells per treatment group. Statistical analysis was done by ANOVA. Valencia, CA). The oligonucleotide suspension was raised to a total Experiments were repeated at least twice. volume of 700 AL by the addition of DMEM containing 10% fetal Band depletion. The band depletion assay was done as described by bovine serum and incubated on cell monolayers (1 Â 105 cells) for Xiao et al. (8). Briefly, 5 Â 105 cells were lysed in alkaline lysis solution 24 hours. The oligonucleotide mixture was replaced the following (200 mmol/L NaOH, 2 mmol/L EDTA), and the lysate was neutralized day, and the cells were incubated for an additional 4 hours before [neutralization buffer: 1 mol/L HCl, 600 mmol/L Tris (pH 8.0)]. The experimental procedures. Controls included topoisomerase IIa and neutralized lysate was then mixed with 3Â SDS sample buffer [150 topoisomerase IIh sense oligonucleotides as well as incubation of mmol/L Tris-HCl (pH 6.8), 6 mmol/L EDTA, 45% sucrose, 9% SDS, cells with Superfect in the absence of oligonucleotide. siRNA: RNA 10% h-mercaptoethanol], and the lysates were separated on 8% SDS- duplexes for topoisomerase IIa (sense, GGUAUUCCUGUUGUU- PAGE gels. GAAC) and topoisomerase IIh (sense, GGUUACCUUUGUGC- CAGGU) were purchased from Ambion (Austin, TX). Cells were suspended in 0.1 mL siPort electroporation buffer (3 Â 106/mL, Results Ambion), mixed with 1 Ag siRNA, and pulsed with 300 V for 0.5 milliseconds. Pulsed cells were incubated at 37jC for 15 minutes Interactions of topoisomerase II poisons and histone deacetylase before experimentation. The Silencer Negative Control #2 siRNA inhibitors. We previously reported an increased sensitivity of (Ambion), a nonsense siRNA duplex, was used as a control. tumor cells to topoisomerase II–targeting agents after pretreat- Trapped-in-agarose DNA immunostaining assay. The trapped-in- ment with an HDACi in vitro and in vivo (14, 20). Treatment agarose DNA immunostaining assay (TARDIS) was done as described with HDACi led to dose- and time-dependent histone 4 by Willmore et al. (22) with modifications. Cells (2 Â 10 per slide) acetylation and subsequent modulation of genes and proteins were imbedded in agarose and lysed [2.5 mol/L NaCl, 100 mmol/L essential for the maintenance of heterochromatin. The ensuing EDTA (pH 10), 10 mmol/L Tris, 1% sarkosyl, 1% Triton X-100] in the chromatin decondensation was associated with an increased presence of protease inhibitors (2 Ag/mL pepstatin, 10 Ag/mL binding of topoisomerase II poisons to the DNA substrate and aprotinin, 10 Ag/mL leupeptin, 0.2 mmol/L Na3VO4, and 1 mmol/L phenylmethylsulfonyl fluoride) for 30 minutes. Imbedded cells were in the presence of topoisomerase II, with increased DNA washed for 20 minutes with 1 mol/L NaCl in the presence of protease damage and cell death. inhibitors and incubated in 2% BSA in PBS for 1 hour. Immunostain- The sensitivity of cancer cells to topoisomerase II–targeting ing consisted of anti-topoisomerase IIa (454, polyclonal, 1:200) and agents has been linked to expression of the topoisomerase IIa anti-topoisomerase IIh (monoclonal, 1:100) in 0.5% BSA for 1 hour at isoform, whereas studies on the role of topoisomerase IIh room temperature in a humidified chamber. Slides were washed twice have been more limited. Our experimental data indicate that for 10 minutes in 0.5% BSA and incubated with the appropriate preexposure of tumor cells to HDACi potentiates the apoptosis secondary antibodies (anti-rabbit 546 and anti-mouse 488; Molecular induced by the topoisomerase II poisons epirubicin and Probes) at a concentration of 1:200 in 0.5% BSA containing goat serum mitoxantrone (Fig. 1A). Potentiation was not limited to a (1:100) for 1 hour at room temperature. Slide were washed with 0.5% certain class of HDACi and was observed with selective (SAHA and trichostatin A) as well as with nonselective (valproic acid) HDACi. Similarly, potentiation occurred with other topoiso- merase II–targeting agents, such as doxorubicin, etoposide, and VM-26 (see below; data not shown; and refs. 14, 20). Densitometry analysis of Western blots (n = 3) evaluating cancer cells derived from different organs, including breast, colon, and melanoma, showed that the examined cell lines with decreased or mutated topoisomerase IIa, required higher concentration of epirubicin for IC50 (Table 1) as assessed by colony-forming assays. As reported by other investigators, these data suggest that not organ specificity but rather expression of the topoisomerase II isoforms was predictive for response (13). A 48-hour preexposure of these cells to the HDACi valproic acid resulted in a decrease in the IC50 of epirubicin. The concentration of valproic acid used for these experiments affected <10% of cells, resulting in a fractional inhibition of cell growth by valproic acid alone of Fi < 0.1 compared with untreated cells (Table 1). Isobologram analysis using the CalcuSyn software suggested the interaction between valproic acid and epirubicin was synergistic in SKBr3, MCF-7, A375, BT- 474, KM12C, and MCF:Dox cells (data not shown). A decrease

Fig. 1. HDACi potentiate topoisomerase II inhibitor cytotoxicity. MCF_7 in IC50 was not observed in MDA-361 cells with a near breast cancer cells were treated with 2 mmol/L valproic acid (VPA )or0.5Amol/L depletion of both topoisomerase II isoforms topoisomerase IIa SAHA for 48 hours followed by exposure to 0.5 Amol/L epirubicin or mitoxantrone and topoisomerase IIh. In contrast, in the epirubicin-resistant for 4 hours. Cells were harvested 24 hours later and evaluated for nuclear condensation and fragmentation. Columns, mean from at least three replicate cell line MCF:Dox, the IC50 of epirubicin was lowered by experiments; bars, SE. valproic acid despite a mutation and down-regulation of the

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Ta b l e 1. Percentage expression of topoisomerase IIa and topoisomerase IIh relative to MCF-7 cells and the IC50 dose (95% confidence intervals) of epirubicin by clonogenic analysis with and without a 48-hour pretreatment with the indicated valproic acid dose in the breast cancer celllines SKBr-3, MCF-7,BT-474, MDA-361,and MCF:Dox; the colon cancer cell line KM12C; and 0.25 mmol/L valproic acid in the melanoma cell lineA375

Cell lines % Expression (95% confidence interval) IC50, nmol/L (95% confidence interval) Fractional inhibition by VPA

To p o I I A To p o I I B Epi VPA:Epi Dose (mmol/L) Fi SKBr-3 138 (125-151) 99 (95-103) 22 (19-25) 10 (7-15) 0.5 0.00 MCF-7 100 100 22 (17-27) 7 (4-15) 0.5 0.00 A375 52 (49-55) 87 (85-89) 22 (10-45) 13(9-18) 0.25 0.09 BT-474 83 (69-97) 38 (16-60) 38 (35-40) 16 (12-22) 0.5 0.00 KM12C 76(73-79) 31(12-50) 36(20-65) 21(18-24) 0.5 0.00 MDA-361 37 (26-48) 9 (2-16) 132 (101-174) 129(67-265) 0.5 0.00 MCF-7:Dox 23 (19-27) 64 (56-72) 2308 (1584-3363) 519(133-2031) 0.5 0.00

NOTE: Effects on cell growth byVPA alone on the respective cell lines were depicted as fractional inhibition (Fi). Abbreviations: topo II, topoisomerase II;VPA, valproic acid; Epi, epirubicin.

topoisomerase IIa protein. Although a potential role of isomerase IIa depletion are independent events (data not multidrug resistance genes in the potentiation of epirubicin shown). Furthermore, a SAHA-induced depletion of top- by valproic acid cannot be ruled out in the scope of these oisomerase IIa was observed at concentration of SAHA (>5 experiments, limited microarray experiments do not suggest a Amol/L) that were shown to result in a G2-M arrest, the cell change in the expression of MDR and MDR-related proteins cycle phase where topoisomerase IIa is increased (23). (data not shown). Relevance of topoisomerase IIb as a target. Potentiation of Topoisomerase IIa expression is down-regulated by histone topoisomerase II targeting agents by HDACi in the setting of deacetylase inhibitor. It is thought that sensitivity of cancer topoisomerase IIa depletion argued for the involvement of the cells to topoisomerase II–targeting agents is predominantly alternative topoisomerase II isoform topoisomerase IIh. Syner- mediated through topoisomerase IIa (13–17). However, we gy between an HDACi and a topoisomerase II–targeting agent observed an HDACi-induced sensitization in cells with was preserved when using the topoisomerase IIh–specific depleted topoisomerase IIa but not in cells depleted of both poison, XK469, signifying a potential role of topoisomerase topoisomerase II isoforms. The effects of HDACi on the IIh as a target for drug therapy. A potentiation of XK469 by expression of topoisomerase IIa and topoisomerase IIh were preexposure to valproic acid was found in several examined evaluated across sensitive and resistant cell lines. As reported breast cancer cell lines, including SKBr-3, MCF-7, and the previously, we found that HDACi-induced sensitization of topoisomerase IIa–depleted MCF:Dox cells (Fig. 3A). To cells to topoisomerase II poisons was maximal after a 24- to further define the involvement of topoisomerase IIh in the 48-hour preexposure to the HDACi (14). Time course interaction between HDACi and topoisomerase II poisons, evaluating the protein expression of topoisomerase IIa, top- both topoisomerase II isoforms were alternatively depleted by oisomerase IIh, and topoisomerase I showed that after a siRNA or antisense and exposed to HDACi before administra- 48-hour exposure to 2 Amol/L SAHA, rather than an up- tion of a topoisomerase II poison. We have shown above that regulation, a depletion of topoisomerase IIa protein was treatment with HDACi resulted in a down-regulation of observed (Fig. 2A). The depletion of topoisomerase IIa topoisomerase IIa (Fig. 2). Treatment of MCF-7 cells with expression was not limited to SAHA but was observed with siRNA resulted in significant depletion of topoisomerase IIa the short-chain fatty acids valproic acid and NaB as well as the and topoisomerase IIh protein levels without affecting viability hydroxamic acid trichostatin A (Fig. 2B). The decrease in (Fig. 3B; data not shown). Topoisomerase IIa was further nuclear protein expression corresponded to a time-dependent depleted with the addition of valproic acid (Fig. 3B). Depletion reduction in the expression of topoisomerase IIa mRNA of topoisomerase IIa by siRNA did not effect the potentiation (Fig. 2C) and not a change in cellular location (Fig. 2D). of epirubicin, mitoxantrone, or VM-26 by valproic acid (Fig. Furthermore, HDACi had no significant effects on the 3C). In contrast, the synergistic activity between HDACi and expression of topoisomerase I and topoisomerase IIh (Fig. 2A these topoisomerase II–targeting drugs was abrogated by the and B). Expression levels of topoisomerase IIa vary with depletion of topoisomerase IIh (Fig. 3C). These findings were alterations in cell cycle distribution with peak expression in mirrored when antisense was used to deplete topoisomerase G2-M. To rule out a causal link between HDACi-induced cell IIa or topoisomerase IIh or when SAHA was used in lieu of cycle block and the observed reduction in topoisomerase IIa valproic acid (data not shown). expression, topoisomerase IIa levels were studied over a wide Although the topoisomerase IIa siRNA was exquisitely range of HDACi concentrations. Topoisomerase IIa was specific, the topoisomerase IIh siRNA showed a slight off- depleted at concentrations of HDACi that did not perturb cell target effect on the expression of topoisomerase IIa. This off- cycle progression (Fig. 2B; data not shown), suggesting that target effect unlikely contributed to the abrogation of synergy HDACi-induced cell cycle arrest and HDACi-induced topo- between valproic acid and topoisomerase II poisons because

ClinCancerRes2005;11(23)December1,2005 8470 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Interaction of HDAC and Topoisomerase II Inhibitors the direct depletion of topoisomerase IIa by siRNA in the absence of HDACi effects and the further depletion of topoisomerase IIa by the addition of valproic acid did not sensitize cells to topoisomerase II poisons. Hence, in the presence of an HDACi, topoisomerase II poison cytotoxicity seems mediated through topoisomerase IIh. Histone deacetylase inhibitor increases the formation of DNA/ topoisomerase IIb cleavable complexes. Treatment with HDACi result in chromatin decondensation and increased access of topoisomerase II–targeting agents to the DNA substrate

Fig. 3. Topoisomerase IIh (topo IIb) is the prime target of topoisomerase II inhibitors. A, % apoptotic nuclei in cells treated with 2 mmol/L valproic acid (VPA ) for 48 hours followed by 0, 0.1, and 0.5 mmol/L of the topoisomerase IIh ^ specific inhibitor XK469 for 48 hours in the cell lines MCF-7 and SKBr-3 and the topoisomerase IIa ^ depleted MCF:Dox. B, expression of topoisomerase IIa and topoisomerase IIh in MCF-7 cells after exposure to siRNA and 2 mmol/L valproic acid for 48 hours. Densitometry analysis of the protein bands is listed as % expression relative to control samples.C, % apoptotic nuclei in cells treated with 2 mmol/L valproic acid followed by 0.5 Amol/L epirubicin, 0.5 Amol/L mitoxantrone, or1 Amol/L VM-26 for 4 hours, after depletion of topoisomerase IIa and topoisomerase IIh by siRNA. Columns, mean from atleastthree differentreplicate experiments; bars, SE.

(14, 20). Topoisomerase II poisons are thought to confer cytotoxicity through the stabilization of topoisomerase II/DNA cleavable complexes. We evaluated whether the presence of HDACi resulted in the involvement of a specific topoisomerase II isoform in the cleavable complexes stabilized by top- oisomerase II poisons. The cleavable complexes involving topoisomerase IIa or topoisomerase IIh induced by epirubicin in the presence and absence of a 48-hour pretreatment with 2 mmol/L valproic acid were evaluated by trapped-in-agarose DNA immunostaining Fig. 2. HDACi exposure depletes topoisomerase IIa (topo IIa) butnot assay. topoisomerase IIh. A, Western blot depicting changes in the expression of topoisomerase IIa in log-phase MCF-7 cells after exposure to 2 Amol/L SAHA for Immunofluorescence analysis of confocal microscopy images the indicated times. B, expression of topoisomerase IIa and topoisomerase IIh in showed that at concentrations of epirubicin with minimal log-phase MCF-7 cells treated with 2 mmol/L valproic acid (VPA ), 0.5 Amol/L SAHA, 0.05 Amol/L trichostatin A (TSA), and 2 mmol/L NaB for 48 hours. effect on the stabilization of cleavable complexes involving C, microarray analysis showing the relative topoisomerase IIa mRNA expression topoisomerase IIa (P > 0.05), there was a 3-fold increase in the after 0, 4, 24, and 48 hours of incubation with 2 mmol/L valproic acid.D, expression accumulation of topoisomerase IIh–containing cleavable and location of topoisomerase IIa and acetylation of histone H3 in MCF-7 cells after exposure to 2 mmol/L valproic acid for 48 hours using confocal microscopy at a complexes stabilized by epirubicin in the presence of valproic Â63 magnification. acid (Fig. 4A and B). This difference reached statistical

www.aacrjournals.org 8471 Clin Cancer Res 2005;11(23) December 1, 2005 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Therapy: Preclinical significance (*, P < 0.001). Similar results were observed when addition, topoisomerase IIh may compensate, albeit only mitoxantrone was used in lieu of epirubicin (data not shown). partially, for topoisomerase IIa during chromatin condensa- Valproic acid by itself did not induce the formation of tion and cell segregation (9, 32). Despite similar catalytic cleavable complexes. As described above, valproic acid treat- activities of the topoisomerase II isoenzymes, cell survival ment results in depletion of topoisomerase IIa. has been correlated with the expression of topoisomerase IIa To further establish whether these findings were intrinsic to (12, 33). The role of topoisomerase II as drug targets for the anthracycline family members (intercalating topoisomerase topoisomerase II poisons has been evaluated in several studies; II poisons), band depletion assays were performed using the and it has been suggested that topoisomerase IIa is the relevant nonintercalating topoisomerase II poison, VM-26. This drug target of topoisomerase II poisons. Disruption or mutation of has been reported to induce cell death with the preferential topoisomerase IIa was associated with drug resistance formation of topoisomerase IIa–containing cleavable com- (34–36). In particular, there are several reports suggesting plexes (24). The band pattern of topoisomerase IIa and topoisomerase IIa to be an important target for epirubicin, the topoisomerase IIh was evaluated in MCF-7 cells exposed to drug used predominantly for this study; however, much less is VM-26 and the HDACi valproic acid (Fig. 4C). Band depletion known about the role of topoisomerase IIh as a drug target for assays allow detection of topoisomerase IIa and topoisomerase epirubicin (17). There currently are several drugs approved for IIh complexed with DNA. The topoisomerase enzymes that are clinical use that predominantly target topoisomerase IIa, covalently bound to DNA (cleavable complexes) are prohibited whereas the clinical utility of topoisomerase IIh–specific drugs from entering the acrylamide gel resulting in a band depletion, is still under investigation (37). whereas topoisomerase II enzymes not bound to DNA readily Here, we provide evidence that HDACi render topoisomerase enter the gel. Exposure of cells to VM-26 resulted in a 52% IIh a relevant target and effector substrate for topoisomerase II depletion of topoisomerase IIh band, whereas no effects were poisons. We have shown that preexposure of tumor cells to seen with valproic acid alone. In cells preexposed to 2 mmol/L HDACi led to histone acetylation and down-regulation of valproic acid, VM-26 induced an 81% reduction in the proteins essential for the maintenance of heterochromatin topoisomerase IIh band, suggesting an increase in cleavable (20). The ensuing chromatin decondensation was associated complex formation Fig. 4C (bottom). with increased binding of topoisomerase II poisons to the DNA VM-26 induced a 51% depletion in the topoisomerase IIa substrate (14, 20). The kinetics of the HDACi-induced band. There was a 46% decrease in the topoisomerase IIa band chromatin decondensation suggested that a 48-hour preexpo- after treatment with valproic acid alone. In cells treated with sure was optimal for synergistic activity. valproic acid before VM-26, there was a 92% depletion of the We now show that exposure of tumor cells to HDACi topoisomerase IIa band (Fig. 4C, top). However, as discussed significantly reduced the concentrations of topoisomerase II above, treatment with valproic acid resulted in a down- poisons required for growth inhibition, even in cells regulation of topoisomerase IIa protein expression (Fig. 2A manipulated for target-specific resistance to topoisomerase and B and Fig. 3B). The further reduction in the topoisomerase II–targeting agents. Sensitization was not tissue specific but IIa band by VM-26 in the presence of valproic acid resulted was observed in breast, colon, leukemia, melanoma, and from the additive effects of the HDACi-induced down- sarcoma cell lines. We observed a time-dependent reduction regulation of the protein and the loss of topoisomerase IIa in the expression of topoisomerase IIa mRNA and protein in band due to cleavable complex formation induced by VM-26. the presence of HDACi that was maximal at 48 hours. In This is further supported by the trapped-in-agarose DNA contrast, the effects of HDACi on topoisomerase IIh were immunostaining assay showing no increase in the topoisomer- minimal. Modulation of the topoisomerase II isoforms with ase IIa–cleavable complexes induced by valproic acid alone. siRNA and antisense showed that depletion of topoisomerase These findings suggest that treatment of cell with valproic acid IIh abrogated the HDACi-induced potentiation of topoiso- enhanced the formation of cleavable complexes involving merase II poisons, whereas the depletion of topoisomerase topoisomerase IIh but not topoisomerase IIa in the presence of IIa did not affect this synergistic interaction. Furthermore, VM-26. topoisomerase II poison cytotoxicity in the presence of HDACi was associated with an accumulation of cleavable Discussion complexes involving topoisomerase IIh. The depletion of topoisomerase IIa and increase in topoisomerase IIh– Topoisomerase II is an essential enzyme for cell survival, and containing cleavable complexes provide a strong argument the increased expression of topoisomerase II in many cancer for a more prominent role of topoisomerase IIh in the cells has made these enzymes attractive targets for cancer synergistic interaction between HDACi and topoisomerase therapy. Topoisomerase II exists in two isoforms, topoisomer- II–targeting agents. ase IIa and topoisomerase IIh, with partial functional Combinations of HDACi and topoisomerase II–targeting redundancy (9, 25–28). In yeast, either isoform may substitute agents have been reported by several investigators. Although for the endogenous topoisomerase II enzyme (29). Murine Johnson et al. observed a decrease in sensitivity of leukemia transgenic studies have shown that although topoisomerase cells to etoposide after preexposure to an HDACi (38), Tsai IIa may compensate for a loss of topoisomerase IIh during et al. observed a sensitization of leukemia cells to etoposide embryogenesis (30), the loss of topoisomerase IIa leads to (18). This discrepancy is most like explained by differences in embryonic death (31). However, topoisomerase IIh seems HDACi preexposure and drug concentration. Where Johnson essential for neuromuscular development after birth (30). In et al. preexposed HL-60 cells to 100 nmol/L trichostatin A for adult mammalian cells, disruption of topoisomerase IIh by 0.5 hours, Tsai et al. used 400 nmol/L trichostatin A for 4 siRNA has no detrimental effects on cell cycle progression. In hours. Since then, other groups have reported increased

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sensitivity of several tumor cell lines to topoisomerase II Fraser et al. reported an initial activation of the topoisomer- targeting agents after preexposure to an HDACi (14, 19, 20, 39, ase IIa gene promoter with a transient up-regulation of 40). Synergy was dependent on sequence and timing of drug topoisomerase IIa protein expression in leukemia cells treated administration and associated with HDACi-induced chromatin with the HDACi NaB for 18 to 24 hours. This promoter decondensation (14, 19). activation was reversed with longer HDACi exposure, resulting Although topoisomerase IIa has been proposed as the in topoisomerase IIa promoter activity repression (41). In effector molecule for cytotoxicity for topoisomerase II poison, contrast, Kim et al. found no change in the expression or the specific roles of topoisomerase IIa and topoisomerase IIh activity of topoisomerase IIa in glioblastoma cells exposed to in the interaction between HDACi and topoisomerase II the HDACi SAHA (19); however, the exposure times may poisons have not been defined. have differed. We observed an HDACi-induced depletion of

Fig. 4. Topoisomerase IIh (topo IIb) is the effector protein. A, trapped-in-agarose DNA immunostaining analysis of cleavable complexes involving topoisomerase IIa (red)and topoisomerase IIh (green)in cells treated with 0.5 Amol/L epirubicin (Epi) for 4 hours in the presence and absence of a 48-hour pretreatment with 2 mmol/L valproic acid (VPA ) by confocal microscopy. B, immunofluorescence analysis of square pixel surface area per replicate sample depicting changes in topoisomerase IIa and topoisomerase IIh cleavable complexes for the indicated treatment groups. Squares, mean; square brackets, 95% confidence interval. *, P V 0.001. C, band depletion assay of topoisomerase IIa and topoisomerase IIh in MCF-7 cells incubated with 50 Amol/L VM-26 after pretreatment with 0 or 2 mmol/L valproic acid and densitometry analysis relative to untreated cells.Topoisomerase IIa and topoisomerase IIh were depleted in the presence of VM-26, suggesting the formation of cleavable complexes. Topoisomerase IIh was further depleted when cells were exposed to valproic acid beforeVM-26, suggesting an enhancement of cleavable complex formation.The decrease in topoisomerase IIa induced by valproic acid was not due to the formation of cleavable complexes butdue to down-regulation of protein expression (see Fig. 2B and Fig. 3B).Therefore, the depletion of topoisomerase IIa in the VPA:VM-26 sample was a reflection of protein down-regulation by valproic acid and cleavable complex formation induced byVM-26 alone and did notsignify an enhancement ofVM-26-induced cleavable complex formation.

www.aacrjournals.org 8473 Clin Cancer Res 2005;11(23) December 1, 2005 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Therapy: Preclinical topoisomerase IIa mRNA and protein. Maximal effects only topoisomerase II–targeting agents. This is further supported occurred after a prolonged exposure to the HDACi (48 hours). by the findings that depletion of topoisomerase IIh abrogated Topoisomerase IIa depletion was not limited to a specific the synergistic interaction, whereas depletion of topoisomerase HDACi class but was observed with both short-chain fatty acid IIa did not affect synergy. and hydroxamic acid HDACi. In part, the differences between Although topoisomerase IIh may be a target of selected the findings presented in this report and those presented by topoisomerase II poisons (44–46), most reports suggest that others may be explained by differences in the duration of the cytotoxicity for the clinically relevant topoisomerase II– HDACi exposure. More importantly, these findings may also targeting agents is determined by topoisomerase IIa levels suggest a limited role of topoisomerase IIa in the observed (47–50). The data presented here show that HDACi poten- synergy between an HDACi and a topoisomerase II poison. tiates the cytotoxicity of topoisomerase IIa and topoisomerase This is further supported by our findings showing that near- IIh–specific topoisomerase II poisons by recruiting topoiso- complete depletion of topoisomerase IIa by siRNA or antisense merase IIh as a target. The results of this study may have in the presence of an HDACi did not inhibit the potentiation several clinical implications. HDACi may lower the concen- of topoisomerase II poisons by HDACi. The HDACi-induced tration of topoisomerase II targeting agents required for depletion of topoisomerase IIa mRNA and protein expression activity, thereby limiting adverse effects. Emerging drug was maximal at 48 hours, which correlated to maximal HDACi- resistance due to a loss or mutation of topoisomerase IIa induced chromatin decondensation and the optimal timing of may be circumvented by engaging topoisomerase IIh as an synergy between HDACi and topoisomerase II–targeting alternative target. A potentiation of cytotoxicity was not agents. Although topoisomerase IIa was reported to be observed in fibroblast exposed to an HDACi before exposure involved in chromatin condensation (42), a mechanistic link to a topoisomerase II poison (data not shown). Furthermore, between the topoisomerase IIa depletion and chromatin preliminary results from an ongoing phase I trial studying a decondensation cannot be established by the presented data. combination of valproic acid and epirubicin did not suggest More evidence suggesting topoisomerase IIh as a relevant a potentiation of epirubicin-induced toxicity (Munster et al, target in the potentiation of topoisomerase II–targeting agents ASCO 2005 #A3084). The differential effects of this combina- by HDACi emerges from studies using the topoisomerase IIh– tion on somatic versus tumor cells may limit tissue-specific specific poison XK469. XK469 was shown to induce cleavable toxicities, such as anthracycline-associated cardiotoxicity. In complexes in several cell lines in vitro; however, meaningful addition, HDACi may increase the clinical utility of h-specific effects on tumor burden in vivo were not achievable at tolerable drugs. doses (43). We found that XK469 by itself only caused minimal apoptosis in breast cancer cells; however, when administered in a sequence-specific combination with HDACi, we observed a Acknowledgments potentiation of XK469 induced apoptosis by HDACi. These We thank Dr. Fred Hausheer (Bionumerick Pharmaceuticals Inc., San Antonio, experiments collectively imply that topoisomerase IIh is a TX) for the MCF:Dox cell line and Dr. Robert Snapka (Department of Radiology, relevant target in the interaction between HDACi and Ohio State University, Columbus, OH) for XK469.

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