Cancer Research Annual Meeting 2010 Protein Translation and Cancer
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Cancer Research 68, 1513, March 1, 2008. doi: 10.1158/0008-5472.CAN-07-2822
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Selective Inhibition of Histone Deacetylase 2 Silences Progesterone Receptor–Mediated Signaling

Elona Biçaku, Douglas C. Marchion, Morgen L. Schmitt and Pamela N. Münster

Department of Interdisciplinary Oncology, Experimental Therapeutics Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida

Requests for reprints: Pamela N. Münster, H. Lee Moffitt Cancer Center, 12901 Magnolia Drive, SRC 22007, Tampa, FL 33612. Phone: 813-745-8948; Fax: 813-745-1984; E-mail: pamela.munster{at}moffitt.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several histone deacetylases (HDAC) are involved in estrogen receptor (ER)–mediated gene transactivation, and HDAC inhibitors have been reported to restore sensitivity to antihormonal therapy. The modulation of ER is the most promising approach to ER-expressing breast cancers. Recent studies further suggest a critical role of the progesterone receptor (PR) on ER signaling. Although HDAC inhibitors modulate ER, little is known about their effects on PR. We evaluated the roles of specific HDAC isoenzymes and their inhibition on both ER and PR signaling and their importance in response to endocrine therapy. The roles of individual HDAC isoenzymes on ER and PR expression and their functions were evaluated by depletion of select HDAC enzymes using siRNA or pharmacologic inhibition. Cotreatment of breast cancer cell lines with HDAC inhibitors and the antiestrogen, tamoxifen, resulted in synergistic antitumor activity with simultaneous depletion of both ER and PR. Selective inhibition of HDAC2, but not HDAC1 or HDAC6, was sufficient to potentiate tamoxifen-induced apoptosis in ER/PR-positive cells. Depletion of HDAC1 and HDAC6 was associated with down-regulation of ER but not PR. Only the selective depletion of HDAC2 siRNA down-regulated both ER and PR expression, and was sufficient to potentiate tamoxifen. Selective depletion of HDAC2 resulted in simultaneous depletion of ER and PR, and potentiated the effects of antihormonal therapy in ER-positive cells. A more effective pharmacologic inhibition of HDAC2 and evaluation of HDAC2 and PR as therapeutic targets or as predictive markers in hormonal therapy may be considered when combining HDAC inhibitors and hormonal therapy. [Cancer Res 2008;68(5):1513–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Histone acetyl-transferases and histone deacetylases (HDAC) play a crucial role in gene regulation. Specific interference with histone acetylation and methylation by drugs has shown to be promising in the treatment and prevention of cancer. Several reports suggest a potential role of HDACs and their inhibitors in breast cancer. A direct interaction of HDAC1 and estrogen receptor (ER) may be involved in the response to antiestrogen therapy (1). Experimental overexpression of HDAC1 was associated with repression of ER in MCF-7 cells and reactivation of ER upon exposure to the HDAC inhibitor, trichostatin A (TSA; ref. 2). Further reports suggest that the hydroxamic acid HDAC inhibitors, TSA and LBH589, convey tamoxifen sensitivity to the ER-negative cell line, MDA-231, by inducing the release of HDAC1 from the ER{alpha} promoter, thus restoring ER{alpha} expression (3, 4) or the activation of ERβ, as suggested by others (5). In ER-positive cell lines, HDAC inhibitors have been shown to repress ER{alpha} expression (6, 7) and sensitize cells to tamoxifen (5, 8), which may involve the up-regulation or translocation of ERβ (9, 10). Although these studies suggest a benefit of adding an HDAC inhibitor to antiestrogen therapy, the differential effects on ER suggest that the modulation of ER may not entirely explain the synergy between HDAC inhibitors and antiestrogen therapy.

About 70% of women with breast cancer present with tumors that express ER and the majority of those coexpress progesterone receptor (PR; ref. 11). Most of the currently used antihormonal therapies predominantly target ER. Antiprogestins and other modalities targeting PR are now rarely used, not due to inefficacy but due to undesirable side effects such as profound weight gain (12, 13). Although it seems that the coexpression of PR in patients, whose tumors express ER, predicts a better outcome, the role of PR as a predictive factor for antihormone therapy remains under debate (14–16). Furthermore, there are very few reported modalities to address the potential benefits of simultaneous down-regulation of ER and PR, or the predictive value of the pharmacologic modulation of PR in addition to ER. Although PR levels are induced by ER, PR may become hormone independent upon transformation to a more aggressive phenotype (17–19). The role of PR as a target for endocrine therapy is still under debate and very little is known about the interaction between HDAC enzymes and PR.

In this study, we show that the selective inhibition of HDAC2 by siRNA resulted in simultaneous depletion of ER as well as PR and potentiated the antitumor effects of tamoxifen in ER-positive cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and antibodies. Valproic acid was purchased from Sigma-Aldrich. Vorinostat (suberoylanilide hydroxamic acid) was provided by Aton Pharma. 4-OH-Hydroxytamoxifen (tamoxifen) and MS-275 were purchased from Calbiochem. ER{alpha}, PR, and HER2-neu–directed antibodies were purchased from Santa Cruz Biotechnology, Inc. HDAC1 and HDAC2 antibodies were purchased from Upstate Biotechnology. HDAC6 was purchased from Santa Cruz Biotechnology, Inc. Glyceraldehyde-3-phosphate dehydrogenase antibody was purchased from Chemicon.

Cell lines. All cell lines were purchased from the American Type Culture 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 µg/mL streptomycin (Fisher Scientific). Cells were incubated in a humidified atmosphere with 5% CO2 at 37°C.

Microarray. Alterations in gene expression induced by valproic acid and vorinostat were evaluated by microarray using Affymetrix U133-plus 2.0 Genechips by standard protocols (H. Lee Moffitt Cancer Center and Research Institute, Bill and Beverly Young National Functional Genomics Center), with duplicate samples for each condition run on separate Genechips. Hybridization to Affymetrix chips was analyzed 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 increases or decreases in mRNA abundance in a direct comparison between two samples (Statistical Algorithms description document).1

SiRNA. RNA duplexes for HDAC1, HDAC2, and HDAC6 were purchased from Ambion. Cells were transfected by electroporation using the Nucleofector transfection kit according to manufacturer's recommendations (Amaxa). Cells (4 x 106) were suspended in 0.1 mL electroporation buffer containing 1 µmol/L siRNA and pulsed with a cell line–specific protocol as described by the manufacturer. Pulsed cells were resuspended in 0.5 mL complete medium without antibiotics and incubated at 37°C for 15 min before experimentation. The Silencer Negative Control #2 siRNA (Ambion), a nonsense siRNA duplex, was used as a control.

Apoptosis assay. Cells (5 x 105) were treated with drugs (MS-275, 1 µmol/L; vorinostat, 1 µmol/L; valproic acid, 2 mmol/L; tamoxifen, 10 µmol/L; or combination) for 48 h. Cells were harvested in medium using a Cell Lifter (Fisher Scientific) and washed in PBS. Cell nuclei were stained with 0.5 µg/mL bis-benzimide (Hoechst 33258). Apoptosis was scored by the presence of nuclear chromatin condensation and DNA fragmentation, and evaluated by fluorescence microscopy. Two hundred cells were counted per experiment in at least six different fields and scored for apoptosis (apoptotic nuclei/all nuclei x100). Error bars depict SE.

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt assay. Equal numbers of cells (1 x 104 cells per well for MCF-7, T-47D, and SKBr-3; 5 x 103 cells per well for MDA-231) were cultured in 96-well plates, followed by a 24-hour incubation at 37°C, 5% CO2. Cells were then incubated with 2 mmol/L valproic acid, 10 µmol/L tamoxifen, or the combination for 48 h and evaluated for proliferation using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay (Promega).

Western blot analysis. Cells were harvested in tissue culture medium using a Cell Lifter, washed in PBS, and solubilized using SDS lysis buffer [2% SDS, 10% glycerol, and 0.06 mol/L Tris (pH 6.8)]. The protein concentration of the SDS lysates was determined by the bicinchoninic acid method (Pierce). Proteins (50 µg) were separated on 8% SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked in tris-buffered saline containing 0.05% Tween 20 (TBST), 5% nonfat milk; and incubated with primary antibody in TBST, 5% nonfat milk, overnight at 4°C. Membranes were washed thrice for 10 min with TBST and incubated with the appropriate secondary antibody in TBST, 5% nonfat milk for 60 min at room temperature. Antibody binding was visualized by chemiluminescence on autoradiography film.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HDAC inhibitors potentiate the antiestrogen, tamoxifen, independent of class and structure. Synergistic or additive effects between HDAC inhibitors and tamoxifen have been reported in ER-positive and ER-negative cells (1–10, 20). However, the mechanism(s) by which HDAC inhibitors synergize with antiestrogens in ER-positive and ER-negative breast cancer cells remain under debate. In ER-positive cells, treatment with an HDAC inhibitor, such as TSA, vorinostat, LBH529, and MS-275, has been shown to reduce the expression of ER{alpha} while increasing the sensitivity of cancer cells to tamoxifen. It has further been proposed that select HDAC inhibitors may reactivate the expression of ER{alpha} or ERβ in ER-negative cell lines, thereby increasing the sensitivity of these cells to an antiestrogen (4, 5, 9, 21).

The effects of HDAC inhibitors on tamoxifen-induced cell growth arrest and cell death were evaluated in cells with different levels of ER and HER2 expression. Figure 1A shows the expression of ER{alpha} and HER2 in a panel of breast cancer cell lines as well as the expression of several known relevant targets of HDAC inhibitors: HDAC1, HDAC2, and HDAC6. The cell lines, SKBr-3 (HER2+, ER–), MCF-7 (HER2-, ER+), BT-474 (HER2+, ER+), MDA-231 (HER2–, ER–), MDA-361 (HER2+, ER+), and T-47D (HER2–, ER+) were evaluated for cell survival after simultaneous treatment with 0, 0.25, 0.5, 1, or 2 mmol/L of the HDAC inhibitor, valproic acid, and 10 µmol/L of the antiestrogen, tamoxifen, for 48 h by MTS assays (Fig. 1B). An enhancement of the antiproliferative effects of tamoxifen was observed in all examined cell lines irrespective of their ER and HER2 status, suggesting that ER and HER2 may not be sole contributors to the synergy. These effects were synergistic by isobologram analyses (data not shown).


Figure 1
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  		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 ER and HER2 expression with tamoxifen (tam) in the 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 µmol/L; vorinostat, 1 µmol/L; or valproic acid, 2 mmol/L) either alone or in combination with tamoxifen (10 µmol/L).

 
To determine whether the synergistic activity between an HDAC inhibitor and an antiestrogen was dependent on the structural type of HDAC inhibitor, MCF-7 breast cancer cells were treated with 10 µmol/L tamoxifen for 48 h in the presence of the benzamide, MS-275 (2 µmol/L); the hydroxamic acid, vorinostat (1 µmol/L); or the short-chain fatty acid, valproic acid (2 mmol/L), and evaluated for apoptotic nuclei (Fig. 1C). A greater than additive or synergistic interaction was seen with all of the HDAC inhibitors when added to tamoxifen. Similar to the MTS assays, HDAC inhibitors sensitized all evaluated cell lines to tamoxifen-induced apoptosis, independent of their ER or HER2 status.

The effects of different classes of HDAC inhibitors on ER. Although synergistic effects of HDAC inhibitors and tamoxifen have been described in ER-positive and ER-negative cells (1–10, 20), differential effects on ER were noted. In ER-positive cells, HDAC inhibitors have been reported to down-regulate ER, whereas in ER-negative cells, HDAC inhibitors, such as the hydroxamic acids, TSA, and LBH589 may up-regulate the expression of ER. To rule out a differential effect on ER expression depending on the type of HDAC inhibitor, the ER-positive MCF-7 cells were exposed to various HDAC inhibitors representing three structurally different classes at clinically relevant concentrations, including the fatty acids (valproic acid), the hydroxamic acids (vorinostat), and the amides (MS-275; refs. 22–24). Tamoxifen was used as a positive control as its effects on ER protein expression are well described. Treatment with all three HDAC inhibitors as well as tamoxifen resulted in depletion of ER protein in the ER-positive MCF-7 cells (Fig. 2A ). The effects of HDAC inhibitors on ER were then evaluated in cell lines with various degrees of ER expression using the HDAC inhibitor, valproic acid. As seen in Fig. 2B, ER was down-regulated in all examined ER-positive cell lines. In contrast, protein expression of ER was observed neither at baseline nor after treatment with valproic acid at 0.5 or 2 or 5 mmol/L, or with vorinostat 1 µmol/L in the ER-negative cell lines, MDA-231 and SKBr-3 cells (Fig. 2B; data not shown).


Figure 2
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  		Figure 2. HDAC inhibitors down-regulate the expression of ER{alpha} in ER-positive cell lines. A, treatment of the ER-positive cell line MCF-7 with the structurally distinct HDAC inhibitors, valproic acid, vorinostat (Vor), and MS-275 for 48 h led to reduced ER{alpha} protein levels. As a positive control, cells were also treated with tamoxifen. B, effects of valproic acid (2 mmol/L) on ER{alpha} protein expression in ER-positive cells (MCF-7 and T47D) and ER-negative cells (MD-231 and SKBr-3). C, mean fluorescence signal intensity (MAS5 generated) of ER{alpha} and ERβ mRNA in two replicates of MCF-7 and MDA-231 cells after exposure to either vehicle (control), 1µmol/L vorinostat, or 2 mmol/L valproic acid for 48 h. Columns, mean; bars, SE. Analyses by Student’s t test suggested a significant change in the expression of ER{alpha} after treatment with vorinostat (P = 0.005) and valproic acid (P = 0.015) in MCF-7 cells but no significant changes on ER{alpha} in the MDA-231 cells or on ERβ in either cell line.

 
To further delineate the effects of HDAC inhibitors on ER, HDAC inhibitor-induced changes in ER mRNA levels were analyzed by microarray analysis. Exposure to an HDAC inhibitor for 48 h was associated with a down-regulation of ER{alpha} mRNA levels compared with baseline in the ER-positive MCF-7 cell line. The reduction in ER{alpha} expression was seen with 1 µmol/L vorinostat [47% (46–48%)] as well as with 2 mmol/L valproic acid [60% (58–62%); Fig. 2C]. In contrast, baseline levels of ER{alpha} mRNA expression were low in MDA-231, and at the concentrations used to elicit synergy with tamoxifen, no changes in ER{alpha} mRNA expression were observed with either valproic acid or vorinostat (Fig. 2C). In addition, no significant changes in ERβ mRNA expression were seen in either MCF-7 or MDA-231 cells after exposure to the respective HDAC inhibitors (Fig. 2C).

The synergistic interaction between tamoxifen and HDAC inhibitors involves down-regulation of PR. The synergistic interaction between HDAC inhibitors and tamoxifen in ER-positive as well as ER-negative tumor cells in the absence of an effect of ER expression in the ER-negative tumor cells suggested the possible involvement of an additional or an alternative effect on a tamoxifen-regulated protein such as the PR. Exposure of cells to an HDAC inhibitor was associated with a down-regulation of PR (Fig. 3 ). The HDAC inhibitor effects on PR were not linked to a certain class of HDAC inhibitors but occurred with both a fatty acid (valproic acid) and a hydroxamic acid (vorinostat; Fig. 3A and B). The effects of valproic acid and vorinostat on PR were observed at the gene transcript level as well as at the protein level. Furthermore, the simultaneous exposure of cells to valproic acid and tamoxifen was associated with a more pronounced effect on PR. As seen in Fig. 3C, the combination of tamoxifen and valproic acid affected both isoforms of PR, PR-A, and PR-B (25). In contrast, no effects on PR mRNA were observed in the MDA-231 cells; however, these cells showed minimal expression of PR (Fig. 3A). Furthermore, compared with the protein expression shown for T47D and MCF-7, protein expression in SKBR-3 and MDA-231 was only minimally detectable (Fig. 3B). These cells are typically considered PR-negative.


Figure 3
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  		Figure 3. Synergistic modulation of PR levels by HDAC inhibitors and tamoxifen. A, mean fluorescence signal intensity (MAS5 generated) indicating relative mRNA expression of the PR in MCF-7 and MDA-231 cells by microarray after a 48-h exposure to vehicle (control), 1 µmol/L vorinostat, or 2 mmol/L valproic acid. Two biological replicates for each condition were run on two separate Genechips. Columns, mean; bars, SE. B, Western blot analysis showing PR protein levels in MCF-7 (PR-B), T47D (PR-A and PR-B), MDA-231 (PR-B), and SKBr-3 (PR-B) cells in the presence and absence of 2 mmol/L valproic acid or 1 µmol/L vorinostat for 48 h. Top, relative expression of PR in these cell lines. C, enhanced depletion of PR protein levels by valproic acid was seen in cells cotreated with tamoxifen (10 µmol/L).

 
Cooperative down-regulation of ER and PR is mediated through HDAC2. Although there are several HDAC inhibitors under development, there is currently no consensus on whether the development of isotype-specific HDAC inhibitors will result in more effective drugs for the treatment of any one particular or several tumor types, or whether the expression of select HDAC enzymes may serve as a prognostic or predictive factor. To determine a potential role of select HDAC enzymes in the synergy between an HDAC inhibitor and tamoxifen, the known relevant targets of HDAC inhibitors, HDAC1, HDAC2, and HDAC6 were selectively depleted using siRNA.

HDAC6 is not a target of valproic acid when used at physiologic concentrations (26), whereas the hydroxamic acids such as vorinostat affect HDAC6 at clinically relevant concentrations. The selective depletion of HDAC1, 2, and 6 were all associated with the reduced expression of ER in T47D cells (Fig. 4A ). In contrast, siRNA depletion of HDAC2 resulted in the down-regulation of ER as well as both isoforms of PR, PR-A, and PR-B (Fig. 4A). The effects of select HDAC depletion on tamoxifen-induced apoptosis and growth were evaluated after selective depletion of each, HDAC1, HDAC2, or HDAC6. In the absence of tamoxifen, no significant effects on growth by MTS assay or apoptosis were observed with depletion of either three HDAC enzymes (data not shown; Fig. 4B). However, although the effects of HDAC1 and HDAC6 depletion showed no additional effects, the selective depletion of HDAC2 was associated with a significant enhancement of tamoxifen-induced apoptosis (Fig. 4B). The effects of HDAC2 depletion by siRNA on ER and PR were similar to those seen with 2 mmol/L valproic acid (Fig. 4C). No effects of valproic acid on HDAC1 and HDAC2 protein expression were observed (Fig. 4C). Selective depletion of HDAC1 and HDAC2 did not result in potentiation of tamoxifen in the ER- and PR-negative cell line, MDA-231 (Fig. 4D). Expression of ER and PR in MDA-231 cells are considered absent, and treatment of these cells with an HDAC inhibitor did not affect ER or PR mRNA or protein expression (Figs. 2B and C; 3A and B).


Figure 4
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  		Figure 4. Role of HDAC enzymes in the synergy between valproic acid and tamoxifen in ER-positive cell lines. A, selective depletion of HDAC2 but not HDAC1 or HDAC6 by siRNA in T47D cells resulted in reduced expression of both the ER{alpha} and the PR, and sensitized cells to apoptosis induced by 10 µmol/L tamoxifen (B). C, modulation of ER{alpha} and PR expression after siRNA depletion of HDAC2 was comparable with the effects on ER and PR by a 48-h valproic acid (2 mmol/L) treatment of MCF-7 cells. No effects of valproic acid on HDAC1 and HDAC2 were seen. D, HDAC2 depletion by siRNA potentiated the apoptosis induced by 10 µmol/L tamoxifen in MCF-7 and T47D cells but not MDA-231 cells. No sensitization to tamoxifen was observed when cells were depleted of HDAC1. A nonsilencing siRNA was used as control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The potential role of HDAC inhibitors to enhance the efficacy of tamoxifen by modulation of ER has been described previously (2–10). In this study, we present the putative role of the PR in the synergistic interaction between HDAC inhibitors and antiestrogen therapy in ER- or PR-expressing cells. Similar to the findings reported by others, we found that the HDAC inhibitor effects on tamoxifen-induced apoptosis (Fig. 1) were associated with a depletion of ER mRNA and protein expression in ER-positive cells (Fig. 2). However, we found minimal or no changes in ER expression in ER-negative cells at concentrations of valproic acid or vorinostat used to elicit a synergistic interaction with tamoxifen, suggesting that at least for the ER-negative cell lines, modulation of ER alone may not completely explain the synergy between an HDAC inhibitor and tamoxifen. Our data suggest that in ER-positive cell lines, exposure to an HDAC inhibitor was associated with a depletion of PR expression. The effects on PR were further enhanced in the presence of tamoxifen. HDAC inhibitors were associated with a depletion of both ER and PR mRNA as well as protein levels (Fig. 3). Valproic acid affected both PR isoforms in T47-D cells.

Our data further suggested that the effects of valproic acid on ER and PR could also be elicited by the selective down-regulation of HDAC2 (Fig. 4). Depletion of HDAC1, HDAC2, or HDAC6 by siRNA resulted in depletion of ER, but only the selective depletion of HDAC2 resulted in reduced expression of both ER and PR. The selective depletion of HDAC2 but not HDAC1 or HDAC6 was further associated with enhanced tamoxifen-induced apoptosis, similar to the effects seen with valproic acid in cells expressing either ER or PR (Fig. 4). Several investigators have reported a link between HDAC enzymes and hormonal regulation of breast cancer. HDAC1 mRNA levels by reverse transcription-PCR as well as HDAC1 and HDAC3 protein expression by immunohistochemistry were statistically associated with smaller, ER- and PR-positive tumors, whereas HDAC1 mRNA, but not HDAC1 protein expression, was linked to node-negative tumors (27, 28). In addition, increased HDAC1 mRNA and protein expression were linked to better outcome, whereas HDAC3 expression did not seem to affect overall or disease-free survival (27, 28). However, the reports were equivocal in whether the expression of HDAC1 mRNA was an independent prognostic indicator or linked to other features (27). Increased expression of HDAC6 has been associated with improved disease-free and overall survival in patients with hormone-sensitive breast tumors treated with tamoxifen (20). A direct interaction of HDAC1 and the ER has been implicated in the response to antiestrogen therapy (1). Genetic overexpression of HDAC1 in MCF-7 cells has been associated with repression of ER{alpha}, with subsequent reactivation upon exposure to the HDAC inhibitor, TSA (2). Other investigators have proposed that the hydroxamic acid HDAC inhibitors, TSA and LBH589, may convey sensitivity to tamoxifen in the ER-negative cell line, MDA-231, by inducing release of HDAC1 from the ER{alpha} promoter, thus, restoring ER{alpha} expression (3, 4). Although HDAC2 has been associated with poorer outcomes in colon cancer and may be overexpressed in APC gene–related colon polyps, the role of HDAC2 in breast cancer has not been determined (29).

In contrast to reports by other investigators, the concentrations and the agents used to potentiate tamoxifen in our studies, valproic acid and vorinostat, were not associated with an increase in ER protein expression as reported for the hydroxamic acids, TSA and LBH589, in ER-negative tumor cells (3, 4). The induction of ER reported in these studies may be due to a more potent inhibition of HDAC6 induced by the use of these compounds (TSA and LBH589). HDAC inhibitor treatment resulted in depletion of ER in ER-positive cell lines; however, although the selective depletion of HDAC1 and HDAC6 was associated with a decrease in ER protein expression, this was not sufficient to potentiate tamoxifen. The depletion of HDAC2 by siRNA was sufficient to reduce ER and PR expression and to potentiate the apoptosis induced by tamoxifen to a similar degree as valproic acid and vorinostat. These findings suggest that for the interaction between HDAC inhibitors and antiestrogens, HDAC2 may be the relevant target. Selective depletion of HDAC1, HDAC2, or HDAC6 did not potentiate the effects of tamoxifen in the ER- and PR-negative cell line, MDA231. Furthermore, HDAC inhibitors did not affect either ER or PR mRNA or protein expression in these cells, which are commonly considered ER- and PR-negative. The synergy seen with tamoxifen and HDAC inhibitors in these cells are likely due to a different mechanism than the synergy seen in the ER- or PR-positive cell lines and may require the inhibition of another select HDAC enzyme or the simultaneous interference of several HDAC enzymes.

The role of HDAC2 rather than HDAC1 or HDAC6 is further supported by the findings that not only the hydroxamic acids but also the more class I–specific HDAC inhibitors such as valproic acid and MS-275 potentiated tamoxifen. Although vorinostat has been associated with inhibition of HDAC6 at the concentrations used for the presented studies, valproic acid requires supratherapeutic doses (>15 mmol/L) for HDAC6 inhibition (26). This is further supported by the absence of {alpha}-tubulin acetylation seen with valproic acid in this study (data not shown). Our data suggest that the effects of HDAC inhibitors on the potentiation of tamoxifen-induced apoptosis are predominantly mediated through HDAC2 and involve the presence and modulation of PR. The role of PR in breast cancer as a prognostic and predictive marker remains under debate, and therapies targeting PR are not commonly used. Several studies suggest that tumors that express both ER and PR may respond better to antiestrogen therapy; hence, the effects of HDAC inhibitors on PR may open several new venues for drug development.

Despite the link between HDAC enzymes and hormone receptor expression, to date, no studies have determined the relevance of individual HDAC enzymes as targets for HDAC inhibitors. Our data suggest that the selective depletion of HDAC2 by siRNA potentiated the effects of tamoxifen, whereas the selective depletion of HDAC1 and HDAC6 did not affect the apoptosis or the growth arrest induced by tamoxifen in ER- and/or PR-expressing breast cancer cells. The effects on apoptosis were similar to those seen with 2 mmol/L valproic acid. In contrast to reports by other investigators, no effects of valproic acid on HDAC2 protein expression were seen at the concentrations used in the MCF-7 and T47D cells (30, 31).

In summary, although HDAC inhibitors seem to have antitumor activity as single agents in hematologic malignancies and select solid tumors, their efficacy in breast cancer may be limited. However, HDAC inhibitors may potentiate the effects of tamoxifen in ER- or PR-positive breast cancers by modulation of not only ER but also PR signaling mediated by HDAC2. Given the emerging development of HDAC inhibitors and the still controversial role of PR as a predictive marker of response, these studies may provide insight into the mechanism by which HDAC inhibitors potentiate antiestrogen therapy and may provide guidance in the further development of more selective means to inhibit HDAC2.


   Acknowledgments
 
Grant support: Department of Defense Concept Award (W81XWH-06-1-0622).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Anita Bruce and Connie Schmitt for editorial assistance, and Bill and Beverly Young National Functional Genomics Center for their contribution.


   Footnotes
 
1 http://www.affymetrix.com/support/technical/whitepapers.affx Back

Received 7/24/07. Revised 11/ 9/07. Accepted 1/ 3/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Varshochi R, Halim F, Sunters A, et al. ICI182,780 induces p21Waf1 gene transcription through releasing histone deacetylase 1 and estrogen receptor {alpha} from Sp1 sites to induce cell cycle arrest in MCF-7 breast cancer cell line. J Biol Chem 2005;280:3185–96.[Abstract/Free Full Text]
  2. Kawai H, Li H, Avraham S, Jiang S, Avraham HK. Overexpression of histone deacetylase HDAC1 modulates breast cancer progression by negative regulation of estrogen receptor {alpha}. Int J Cancer 2003;107:353–8.[CrossRef][Medline]
  3. Yang X, Phillips DL, Ferguson AT, Nelson WG, Herman JG, Davidson NE. Synergistic activation of functional estrogen receptor (ER)- {alpha} by DNA methyltransferase and histone deacetylase inhibition in human ER-{alpha}-negative breast cancer cells. Cancer Res 2001;61:7025–9.[Abstract/Free Full Text]
  4. Zhou Q, Atadja P, Davidson NE. Histone deacetylase inhibitor LBH589 reactivates silenced estrogen receptor {alpha} (ER) gene expression without loss of DNA hypermethylation. Cancer Biol Ther 2007;6:64–9.[Medline]
  5. Hodges-Gallagher L, Valentine CD, Bader SE, Kushner PJ. Inhibition of histone deacetylase enhances the anti-proliferative action of antiestrogens on breast cancer cells and blocks tamoxifen-induced proliferation of uterine cells. Breast Cancer Res Treat 2006;105:297–309.[CrossRef][Medline]
  6. Reid G, Metivier R, Lin CY, et al. Multiple mechanisms induce transcriptional silencing of a subset of genes, including oestrogen receptor {alpha}, in response to deacetylase inhibition by valproic acid and trichostatin A. Oncogene 2005;24:4894–907.[CrossRef][Medline]
  7. Rocha W, Sanchez R, Deschenes J, et al. Opposite effects of histone deacetylase inhibitors on glucocorticoid and estrogen signaling in human endometrial Ishikawa cells. Mol Pharmacol 2005;68:1852–62.[Abstract/Free Full Text]
  8. Hirokawa Y, Arnold M, Nakajima H, Zalcberg J, Maruta H. Signal therapy of breast cancers by the HDAC inhibitor FK228 that blocks the activation of PAK1 and abrogates the tamoxifen-resistance. Cancer Biol Ther 2005;4:956–60.[CrossRef][Medline]
  9. Duong V, Licznar A, Margueron R, et al. ER{alpha} and ERβ expression and transcriptional activity are differentially regulated by HDAC inhibitors. Oncogene 2006;25:1799–806.[CrossRef][Medline]
  10. Jang ER, Lim SJ, Lee ES, et al. The histone deacetylase inhibitor trichostatin A sensitizes estrogen receptor {alpha}-negative breast cancer cells to tamoxifen. Oncogene 2004;23:1724–36.[CrossRef][Medline]
  11. Pichon MF, Broet P, Magdelenat H, et al. Prognostic value of steroid receptors after long-term follow-up of 2257 operable breast cancers. Br J Cancer 1996;73:1545–51.[Medline]
  12. Kaufmann M, Bajetta E, Dirix LY, et al. Exemestane is superior to megestrol acetate after tamoxifen failure in postmenopausal women with advanced breast cancer: results of a phase III randomized double-blind trial. The Exemestane Study Group. J Clin Oncol 2000;18:1399–411.[Abstract/Free Full Text]
  13. Stuart NS, Warwick J, Blackledge GR, et al. A randomised phase III cross-over study of tamoxifen versus megestrol acetate in advanced and recurrent breast cancer. Eur J Cancer 1996;32A:1888–92.[CrossRef]
  14. Yu KD, Liu GY, Di GH, et al. Progesterone receptor status provides predictive value for adjuvant endocrine therapy in older estrogen receptor-positive breast cancer patients. Breast 2007;16:307–15.[CrossRef][Medline]
  15. Stendahl M, Ryden L, Nordenskjold B, Jonsson PE, Landberg G, Jirstrom K. High progesterone receptor expression correlates to the effect of adjuvant tamoxifen in premenopausal breast cancer patients. Clin Cancer Res 2006;12:4614–8.[Abstract/Free Full Text]
  16. Dowsett M, Houghton J, Iden C, et al. Benefit from adjuvant tamoxifen therapy in primary breast cancer patients according oestrogen receptor, progesterone receptor, EGF receptor and HER2 status. Ann Oncol 2006;17:818–26.[Abstract/Free Full Text]
  17. Lin VC, Eng AS, Hen NE, Ng EH, Chowdhury SH. Effect of progesterone on the invasive properties and tumor growth of progesterone receptor-transfected breast cancer cells MDA-MB-231. Clin Cancer Res 2001;7:2880–6.[Abstract/Free Full Text]
  18. McGowan EM, Saad S, Bendall LJ, Bradstock KF, Clarke CL. Effect of progesterone receptor a predominance on breast cancer cell migration into bone marrow fibroblasts. Breast Cancer Res Treat 2004;83:211–20.[CrossRef][Medline]
  19. Kato S, Pinto M, Carvajal A, et al. Progesterone increases tissue factor gene expression, procoagulant activity, and invasion in the breast cancer cell line ZR-75–1. J Clin Endocrinol Metab 2005;90:1181–8.[Abstract/Free Full Text]
  20. Saji S, Kawakami M, Hayashi S, et al. Significance of HDAC6 regulation via estrogen signaling for cell motility and prognosis in estrogen receptor-positive breast cancer. Oncogene 2005;24:4531–9.[CrossRef][Medline]
  21. Sharma D, Saxena NK, Davidson NE, Vertino PM. Restoration of tamoxifen sensitivity in estrogen receptor-negative breast cancer cells: tamoxifen-bound reactivated ER recruits distinctive corepressor complexes. Cancer Res 2006;66:6370–8.[Abstract/Free Full Text]
  22. Kelly WK, O'Connor OA, Krug LM, et al. Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer. J Clin Oncol 2005;23:3923–31.[Abstract/Free Full Text]
  23. Kelly WK, Richon VM, O'Connor O, et al. Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously. Clin Cancer Res 2003;9:3578–88.[Abstract/Free Full Text]
  24. Munster P, Marchion D, Bicaku E, et al. Phase I trial of histone deacetylase inhibition by valproic acid followed by the topoisomerase II inhibitor epirubicin in advanced solid tumors: a clinical and translational study. J Clin Oncol 2007;25:1979–85.[Abstract/Free Full Text]
  25. Kastner P, Krust A, Turcotte B, et al. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J 1990;9:1603–14.[Medline]
  26. Gurvich N, Tsygankova OM, Meinkoth JL, Klein PS. Histone deacetylase is a target of valproic acid-mediated cellular differentiation. Cancer Res 2004;64:1079–86.[Abstract/Free Full Text]
  27. Zhang Z, Yamashita H, Toyama T, et al. Quantitation of HDAC1 mRNA Expression in Invasive Carcinoma of the Breast. Breast Cancer Res Treat 2005;94:11–6.[CrossRef][Medline]
  28. Krusche CA, Wulfing P, Kersting C, et al. Histone deacetylase-1 and -3 protein expression in human breast cancer: a tissue microarray analysis. Breast Cancer Res Treat 2005;90:15–23.[CrossRef][Medline]
  29. Zhu P, Martin E, Mengwasser J, Schlag P, Janssen KP, Gottlicher M. Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis. Cancer Cell 2004;5:455–63.[CrossRef][Medline]
  30. Kramer OH, Zhu P, Ostendorff HP, et al. The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2. EMBO J 2003;22:3411–20.[CrossRef][Medline]
  31. Atmaca A, Al-Batran SE, Maurer A, et al. Valproic acid (VPA) in patients with refractory advanced cancer: a dose escalating phase I clinical trial. Br J Cancer 2007;97:177–82.[CrossRef][Medline]



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