Research Article Selective Inhibition of Histone Deacetylase 2 Silences Progesterone Receptor–Mediated Signaling Elona Bic¸aku, Douglas C. Marchion, Morgen L. Schmitt, and Pamela N. Mu¨nster Department of Interdisciplinary Oncology, Experimental Therapeutics Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida Abstract and LBH589, convey tamoxifen sensitivity to the ER-negative cell line, MDA-231, by inducing the release of HDAC1 from the ERa Several histone deacetylases (HDAC) are involved in estrogen a receptor (ER)–mediated gene transactivation, and HDAC promoter, thus restoring ER expression (3, 4) or the activation of ERh, as suggested by others (5). In ER-positive cell lines, HDAC inhibitors have been reported to restore sensitivity to a antihormonal therapy. The modulation of ER is the most inhibitors have been shown to repress ER expression (6, 7) and sensitize cells to tamoxifen (5, 8), which may involve the up- promising approach to ER-expressing breast cancers. Recent h studies further suggest a critical role of the progesterone regulation or translocation of ER (9, 10). Although these studies receptor (PR) on ER signaling. Although HDAC inhibitors suggest a benefit of adding an HDAC inhibitor to antiestrogen modulate ER, little is known about their effects on PR. We therapy, the differential effects on ERsuggest that the modulation of ERmay not entirely explain the synergy between HDAC evaluated the roles of specific HDAC isoenzymes and their inhibition on both ER and PR signaling and their importance inhibitors and antiestrogen therapy. in response to endocrine therapy. The roles of individual About 70% of women with breast cancer present with tumors HDAC isoenzymes on ER and PR expression and their that express ERand the majority of those coexpress progesterone functions were evaluated by depletion of select HDAC enzymes receptor (PR; ref. 11). Most of the currently used antihormonal using siRNA or pharmacologic inhibition. Cotreatment of therapies predominantly target ER. Antiprogestins and other breast cancer cell lines with HDAC inhibitors and the modalities targeting PRare now rarely used, not due to inefficacy antiestrogen, tamoxifen, resulted in synergistic antitumor but due to undesirable side effects such as profound weight gain activity with simultaneous depletion of both ER and PR. (12, 13). Although it seems that the coexpression of PRin patients, Selective inhibition of HDAC2, but not HDAC1 or HDAC6, whose tumors express ER, predicts a better outcome, the role of PRas a predictive factor for antihormone therapy remains under was sufficient to potentiate tamoxifen-induced apoptosis in ER/PR-positive cells. Depletion of HDAC1 and HDAC6 was debate (14–16). Furthermore, there are very few reported associated with down-regulation of ER but not PR. Only the modalities to address the potential benefits of simultaneous selective depletion of HDAC2 siRNA down-regulated both ER down-regulation of ERand PR,or the predictive value of the and PR expression, and was sufficient to potentiate tamoxifen. pharmacologic modulation of PRin addition to ER.Although PR Selective depletion of HDAC2 resulted in simultaneous levels are induced by ER, PR may become hormone independent depletion of ER and PR, and potentiated the effects of upon transformation to a more aggressive phenotype (17–19). antihormonal therapy in ER-positive cells. A more effective The role of PRas a target for endocrine therapy is still under debate pharmacologic inhibition of HDAC2 and evaluation of HDAC2 and very little is known about the interaction between HDAC and PR as therapeutic targets or as predictive markers in enzymes and PR. hormonal therapy may be considered when combining HDAC In this study, we show that the selective inhibition of HDAC2 by inhibitors and hormonal therapy. [Cancer Res 2008;68(5):1513–9] siRNAresulted in simultaneous depletion of ERas well as PRand potentiated the antitumor effects of tamoxifen in ER-positive cell lines. Introduction Histone acetyl-transferases and histone deacetylases (HDAC) Materials and Methods play a crucial role in gene regulation. Specific interference with histone acetylation and methylation by drugs has shown to be Chemicals and antibodies. Valproic acid was purchased from Sigma- Aldrich. Vorinostat (suberoylanilide hydroxamic acid) was provided by Aton promising in the treatment and prevention of cancer. Several Pharma. 4-OH-Hydroxytamoxifen (tamoxifen) and MS-275 were purchased reports suggest a potential role of HDACs and their inhibitors in from Calbiochem. ERa, PR, and HER2-neu–directed antibodies were breast cancer. A direct interaction of HDAC1 and estrogen receptor purchased from Santa Cruz Biotechnology, Inc. HDAC1 and HDAC2 (ER) may be involved in the response to antiestrogen therapy antibodies were purchased from Upstate Biotechnology. HDAC6 was (1). Experimental overexpression of HDAC1 was associated with purchased from Santa Cruz Biotechnology, Inc. Glyceraldehyde-3-phosphate repression of ERin MCF-7 cells and reactivation of ERupon dehydrogenase antibody was purchased from Chemicon. exposure to the HDAC inhibitor, trichostatin A (TSA; ref. 2). Further Cell lines. All cell lines were purchased from the American Type Culture reports suggest that the hydroxamic acid HDAC inhibitors, TSA Collection and maintained in DMEM (Fisher Scientific) with 10% heat- inactivated fetal bovine serum (Sigma-Aldrich), 2 mmol/L glutamine, and 50 unit/mL penicillin and 50 Ag/mL streptomycin (Fisher Scientific). Cells were incubated in a humidified atmosphere with 5% CO2 at 37jC. Requests for reprints: Pamela N. Mu¨nster, H. Lee Moffitt Cancer Center, 12901 Microarray. Alterations in gene expression induced by valproic acid and Magnolia Drive, SRC 22007, Tampa, FL 33612. Phone: 813-745-8948; Fax: 813-745-1984; vorinostat were evaluated by microarray using Affymetrix U133-plus E-mail: [email protected]. I2008 American Association for Cancer Research. 2.0 Genechips by standard protocols (H. Lee Moffitt Cancer Center and doi:10.1158/0008-5472.CAN-07-2822 Research Institute, Bill and Beverly Young National Functional Genomics www.aacrjournals.org 1513 Cancer Res 2008; 68: (5). March 1, 2008 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2008 American Association for Cancer Research. Cancer Research Figure 1. HDAC inhibitors synergize with tamoxifen in breast cancer cell lines. A, differential expression of HDAC1, 2 and 6, ER, and HER2 in a limited breast cancer cell line panel by Western blot analysis. B, treatment of breast cancer cell lines with different levels of ERand HER2expression with tamoxifen ( tam)inthe presence of the HDAC inhibitor, valproic acid (VPA), resulted in increased cell death compared with tamoxifen alone by MTS assays and potentiation of tamoxifen-induced apoptosis in MCF-7 cells (C) treated for 48 h with three different classes of HDAC inhibitors (MS-275, 2 Amol/L; vorinostat, 1 Amol/L; or valproic acid, 2 mmol/L) either alone or in combination with tamoxifen (10 Amol/L). Center), with duplicate samples for each condition run on separate 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo- Genechips. Hybridization to Affymetrix chips was analyzed using Affymetrix phenyl)2H-tetrazolium, inner salt assay. Equal numbers of cells (1 Â 104 Microarray Suite 5.0 software. Signal intensity was scaled to an average cells per well for MCF-7, T-47D, and SKBr-3; 5 Â 103 cells per well for intensity of 500 before comparison analysis. The MAS 5.0 software uses a MDA-231) were cultured in 96-well plates, followed by a 24-hour incubation statistical algorithm to assess increases or decreases in mRNA abundance at 37jC, 5% CO2. Cells were then incubated with 2 mmol/L valproic acid, in a direct comparison between two samples (Statistical Algorithms 10 Amol/L tamoxifen, or the combination for 48 h and evaluated for description document).1 proliferation using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy- SiRNA. RNA duplexes for HDAC1, HDAC2, and HDAC6 were purchased phenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay (Promega). from Ambion. Cells were transfected by electroporation using the Western blot analysis. Cells were harvested in tissue culture medium Nucleofector transfection kit according to manufacturer’s recommenda- using a Cell Lifter, washed in PBS, and solubilized using SDS lysis buffer tions (Amaxa). Cells (4 Â 106) were suspended in 0.1 mL electroporation [2% SDS, 10% glycerol, and 0.06 mol/L Tris (pH 6.8)]. The protein buffer containing 1 Amol/L siRNA and pulsed with a cell line–specific concentration of the SDS lysates was determined by the bicinchoninic acid protocol as described by the manufacturer. Pulsed cells were resuspended method (Pierce). Proteins (50 Ag) were separated on 8% SDS-PAGE gels and in 0.5 mL complete medium without antibiotics and incubated at 37jCfor transferred to nitrocellulose membranes. Membranes were blocked in tris- 15 min before experimentation. The Silencer Negative Control #2 siRNA buffered saline containing 0.05% Tween 20 (TBST), 5% nonfat milk; and (Ambion), a nonsense siRNA duplex, was used as a control. incubated with primary antibody in TBST, 5% nonfat milk, overnight at Apoptosis assay. Cells (5 Â 105) were treated with drugs (MS-275, 4jC. Membranes were washed thrice for 10 min with TBST and incubated 1 Amol/L; vorinostat, 1 Amol/L; valproic acid, 2 mmol/L; tamoxifen, with the appropriate secondary antibody in TBST, 5% nonfat milk for 10 Amol/L; or combination) for 48 h. Cells were harvested in medium using 60 min at room temperature. Antibody binding was visualized by chemi- a Cell Lifter (Fisher Scientific) and washed in PBS. Cell nuclei were stained luminescence on autoradiography film. with 0.5 Ag/mL bis-benzimide (Hoechst 33258). Apoptosis was scored by the presence of nuclear chromatin condensation and DNA fragmentation, Results and evaluated by fluorescence microscopy. Two hundred cells were counted per experiment in at least six different fields and scored for apoptosis HDAC inhibitors potentiate the antiestrogen, tamoxifen, (apoptotic nuclei/all nuclei Â100).