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Clinical Cancer Research 13, 4882, August 15, 2007. doi: 10.1158/1078-0432.CCR-06-3093
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Hydroxamic Acid Analogue Histone Deacetylase Inhibitors Attenuate Estrogen Receptor-{alpha} Levels and Transcriptional Activity: A Result of Hyperacetylation and Inhibition of Chaperone Function of Heat Shock Protein 90

Warren Fiskus1, Yuan Ren2, Alex Mohapatra3, Purva Bali1, Aditya Mandawat1, Rekha Rao1, Bryan Herger1, Yonghua Yang1, Peter Atadja4, Jie Wu2 and Kapil Bhalla1

Authors' Affiliations: 1 Medical College of Georgia Cancer Center, Augusta, Georgia; 2 H. Lee Moffitt Cancer Center, Tampa, Florida; 3 Yale University, New Haven, Connecticut; and 4 Novartis Pharmaceuticals, Inc., Cambridge, Massachusetts

Requests for reprints: Kapil Bhalla, MCG Cancer Center, Medical College of Georgia, 1120 15th Street, CN-2101 Augusta, GA 30912. Phone: 706-721-0566; Fax: 706-721-0469; E-mail: kbhalla{at}mcg.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The molecular chaperone heat shock protein (hsp)-90 maintains estrogen receptor (ER)-{alpha} in an active conformation, allowing it to bind 17ß-estradiol (E2) and transactivate genes, including progesterone receptor (PR)-ß and the class IIB histone deacetylase HDAC6. By inhibiting HDAC6, the hydroxamic acid analogue pan-HDAC inhibitors (HA-HDI; e.g., LAQ824, LBH589, and vorinostat) induce hyperacetylation of the HDAC6 substrates {alpha}-tubulin and hsp90. Hyperacetylation of hsp90 inhibits its chaperone function, thereby depleting hsp90 client proteins. Here, we determined the effect of HA-HDIs on the levels and activity of ER{alpha}, as well as on the survival of ER{alpha}-expressing, estrogen-responsive human breast cancer MCF-7 and BT-474 cells.

Experimental Design: Following exposure to HA-HDIs, hsp90 binding, polyubiquitylation levels, and transcriptional activity of ER{alpha}, as well as apoptosis and loss of survival, were determined in MCF-7 and BT-474 cells.

Results: Treatment with HA-HDI induced hsp90 hyperacetylation, decreased its binding to ER{alpha}, and increased polyubiquitylation and depletion of ER{alpha} levels. HA-HDI treatment abrogated E2-induced estrogen response element-luciferase expression and attenuated PRß and HDAC6 levels. Exposure to HA-HDI also depleted p-Akt, Akt, c-Raf, and phospho-extracellular signal–regulated kinase-1/2 levels, inhibited growth, and sensitized ER{alpha}-positive breast cancer cells to tamoxifen.

Conclusions: These findings show that treatment with HA-HDI abrogates ER{alpha} levels and activity and could sensitize ER{alpha}-positive breast cancers to E2 depletion or ER{alpha} antagonists.

17ß-Estradiol (E2, estrogen) regulates the growth and survival of breast cancer cells (1, 2). Estrogen receptor {alpha} (ER{alpha}) and ERß belong to the nuclear receptor super family and serve as ligand-activated transcription factors and signal transducers for E2 (2–4). Transcriptional activation by ER{alpha} is governed by two separate activation functions: the NH2-terminal, hormone-independent, constitutively active AF-1 and the ligand-dependent COOH-terminal AF-2 (2). On engagement of the COOH-terminal, ligand-binding and dimerization domain, ER{alpha} dissociates from heat shock protein (hsp)-90, dimerizes, and binds to the estrogen response elements (ERE) through the central DNA binding domain to the promoters of the ER target genes (2–5). This triggers the activation/repression of several downstream target genes including progesterone receptor (PR), pS2, cathepsin D, and histone deacetylase (HDAC)-6 (2, 6). In E2-responsive breast cancer cells, transcriptional regulation by ER{alpha} is regulated by multiple factors. At the ERE, it is governed by the interaction of ER{alpha} with p160 family of coactivators and corepressors (e.g., NCoR), which in turn recruit histone acetyl transferases (HAT; e.g., p300 and HDACs) to the EREs (7). Through membrane-bound ER{alpha}, E2 can also induce cross talk and activation of epidermal growth factor receptor and insulin-like growth factor-I receptor signaling (8, 9). Conversely, ligand-independent activation of ER{alpha} by phosphorylation mediated by the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways has also been shown (10–12). E2-complexed ER{alpha} also induces proliferation of breast cancer cells by transcriptional up-regulation of cyclin D1 through cyclic AMP response elements in the cyclin D1 promoter, whereas ER{alpha}-mediated transactivation in breast cancer cells is facilitated by cyclin D1 (13, 14).

Hsp90 is an important ATP-dependent molecular chaperone, playing a critical role in maintaining nascent or refolding denatured polypeptides in functionally mature conformation under the stressful tumor microenvironment defined by hypoxia, acidosis, and reactive oxygen species (15). Hsp90 is abundantly expressed in transformed cells (e.g., ER-positive or ER-negative breast cancer cells), where ER{alpha} and ERß, several constitutively overexpressed or mutated signaling oncoprotein kinases, or other critical signaling proteins, known as client proteins, are dependent on the proper folding capacity of the hsp90-based chaperone machine (15–17). ER{alpha} is part of a multiprotein complex containing hsp90 and other chaperones that are required to maintain the receptor in a conformation capable of ligand binding (5, 16). Small-molecule inhibitors of ATP have been shown to bind more efficiently and disrupt the chaperone function of hsp90, thereby promoting the degradation of the client proteins in breast cancer cells (e.g., ER{alpha}, c-Raf, Akt, and cyclin D1) via the ubiquitin proteasome system (15, 18, 19). Treatment with hsp90 inhibitors has also been shown to induce growth arrest, differentiation, and apoptosis of human breast cancer cells (19). Therefore, hsp90 is an important and emerging target in breast cancer therapy (18, 19). Recently, the predominantly cytosolic HDAC6, a member of the class IIB HDACs, has been shown to be a deacetylase for the molecular chaperone hsp90 and for {alpha}-tubulin (20–24). Consistent with this, the hydroxamic acid analogue pan-HDAC inhibitors [HA-HDI; e.g., suberoylanilide hydroxamic acid or vorinostat (SAHA), LAQ824, and LBH589], which also inhibit HDAC6, were shown to induce hyperacetylation of hsp90 and {alpha}-tubulin (23, 25, 26). This was not seen after treatment with short-chain fatty acid class of HDIs, which do not inhibit HDAC6 (23). Acetylation of hsp90 inhibited its ATP binding and chaperone association with its client proteins, including glucocorticoid receptor (23, 24). This impaired the ligand binding and transactivation by glucocorticoid receptor (24, 27). Additionally, the disruption of the chaperone function of hsp90 led to misfolding, polyubiquitylation, and proteasomal degradation of hsp90 client oncoproteins (e.g., Her-2, Bcr-Abl, Akt, c-Raf, and mutant FLT-3; refs. 23, 25, 26). However, in these studies, the effect of treatment with HA-HDI on chaperone association of ER{alpha} with hsp90, as well as on the levels and transcriptional activity of ER{alpha}, was not determined. In the present studies, we have determined that treatment with HA-HDI disrupts the chaperone binding of hsp90 with ER{alpha}, resulting in polyubiquitylation, proteasomal degradation, and depletion of ER{alpha} with its transcriptional activity in E2-responsive breast cancer cells. We also determined that by mediating concomitant depletion of the other progrowth and prosurvival hsp90 client proteins (e.g., Akt, c-Raf, and cyclin D1), HA-HDI treatment also induces growth arrest and apoptosis of ER{alpha}-expressing breast cancer cells.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents. LAQ824 and LBH589 were kindly provided by Novartis Corp. Vorinostat (SAHA) was a gift from Merck. The proteasome inhibitor N-acetyl leucyl-leucyl norlucinal (ALLnL) and valproic acid were purchased from Sigma, whereas bortezomib was a gift from Millenium Pharmaceuticals. Monoclonal anti-ER{alpha} and polyclonal anti-PR were purchased from Santa Cruz Biotechnology. Anti-hsp90 antibody was purchased from Stressgen Biotechnologies Corp. Anti-ubiquitin antibody was purchased from Covance. Anti–cyclin D1 was obtained from Cell Signaling. Polyclonal anti–poly(ADP-ribose) polymerase (PARP), anti–p-Akt, anti-Akt, anti–phospho-extracellular signal–regulated kinase (ERK), anti-ERK1/2, anti–acetylated histone 3 antibody, anti–c-Raf antibody, anti-p21, and anti–ß-actin antibodies were obtained as previously described (25, 28).

Cell culture. The human breast cancer cells lines MDA-MB-231, MCF-7, and BT-474 were acquired from the American Type Culture Collection and maintained in culture as previously described (25, 28).

Assessment of cytotoxicity. Untreated and drug-treated cells were stained with trypan blue (Sigma). The numbers of nonviable cells were determined by counting the cells that showed trypan blue uptake in a hemocytometer and reported as percentage of untreated control cells (23, 25, 28). Additionally, 3-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was also used to determine the cytotoxic effects of drugs, as previously described (23, 25, 28). Cells were seeded at 2 x 104 per well of a 96-well plate in 100 µL of complete medium and cultured overnight at 37°C. The medium was replenished and HA-HDI was added and incubated for 48 h at 37°C. Three hours before the end of the incubation period, 20 µL of PBS containing 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were added to the wells and incubated at 37°C for 3 h. Following this incubation, the plate was centrifuged at 200 x g, the supernatant was removed, and 200 µL of DMSO were added to the wells to dissolve the crystals. The absorbance of each well was measured on a Benchmark Plus plate reader (Bio-Rad) at 540 nm.

Immunoprecipitation. Following treatment with HA-HDI, cells were trypsinized, washed twice with 1x PBS, and cell lysates were prepared by incubation for 30 min on ice in fresh lysis buffer [1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 1 µg/mL pepstin A, 2 µg/mL aprotinin, 20 mmol/L p-nitrophenyl phosphate, 0.5 mmol/L sodium orthovanadate, and 1 mmol/L 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride] as previously described (23, 25). The resulting cell lysates were centrifuged at 12,000 rpm in a tabletop centrifuge for 15 min to remove the nuclear and cellular debris. For immunoprecipitation reactions, 300 to 500 µg of each cell lysate were mixed gently with 5 µg of anti-Hsp90 or anti-ER{alpha} and incubated on ice for 1 to 2 h. For polyubiquitylation studies of ER{alpha}, 1,000 µg of total cell lysate were used for the immunoprecipitation reactions. Protein G beads were washed twice with fresh lysis buffer and added to the protein/antibody mixture. The lysate/bead mixtures were incubated on a rotator overnight at 4°C. The following day, the immunoprecipitates were separated from unbound protein by brief centrifugation at 8,000 rpm and washed thrice in fresh lysis buffer. Immunoprecipitates were eluted from the beads by boiling in 1x SDS sample buffer before loading.

Western blot analyses. Western blot analyses of acetylated histone H3, ER{alpha}, PR, hsp90, acetylated lysine, ubiquitin, p-Akt, Akt, p-ERK1/2, ERK1/2, c-Raf, p21, PARP, hsp70, HDAC6, acetylated {alpha}-tubulin, and ß-actin were done with specific antisera or monoclonal antibodies as previously described (23, 25, 26). Western blots were scanned for densitometry analysis using Adobe Photoshop (Adobe Systems, Inc.). Densitometry was done using ImageQuant 5.2 (Molecular Dynamics). The expression levels of ß-actin were used as a loading control in all Western blot analyses.

Dual luciferase reporter assay. Cells were seeded at 3 x 104 per well in 12-well plates. Cells were cotransfected with 0.36 µg pERELuc (29) and 0.04 µg pRL-TK (Promega) using Lipofectamine 2000 reagents (Invitrogen) according to the supplier's protocol. Twenty-four hours after transfection, the cells were serum starved and treated with 100 nmol/L LAQ824 or mock treated for 18 h, followed by E2 (Sigma) treatment for 6 h. Firefly luciferase and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega) according to the supplier's protocol. The firefly luciferase activity was normalized to Renilla luciferase activity as the internal control for transfection.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Treatment with HA-HDIs attenuates the expression of ER{alpha} and HDAC6 in cultured breast cancer cell lines. We first determined the effects of the HA-HDI LAQ824 on ER{alpha} levels in MCF-7 and BT-474 cells. Figure 1 shows that exposure to LAQ824 for 24 h depleted the levels of ER{alpha} in a dose-dependent manner in both cell types (Fig. 1A and B). This was associated with attenuation of the levels of PRß and HDAC6, two of the gene expressions transactivated by ER{alpha}. Additionally, LAQ824-mediated depletion of ER{alpha} was also associated with attenuation of the progrowth c-Myc and cyclin D1 proteins (Fig. 1A and B), a finding consistent with these proteins being downstream targets of ER{alpha} and that treatment with LAQ824 abrogates ER{alpha} transactivation in breast cancer cells. For all of these effects of LAQ824, BT-474 cells were relatively less sensitive than MCF-7 cells. As previously reported by us, similar to the other HA-pan-HDIs that inhibit class I and II HDACs including HDAC6, treatment with LAQ824 induced acetylation of histone H3 and up-regulated p21 levels, as well as induced {alpha}-tubulin acetylation and increased hsp70 levels in a dose-dependent manner (Fig. 1A and B). We also determined the effects of another HA-HDI, vorinostat (SAHA), on the levels of ER{alpha}, PRß, HDAC6, and downstream target proteins c-Myc and cyclin D1 in MCF-7 and BT-474. Similar to treatment with LAQ824, vorinostat treatment, in a dose-dependent manner, also induced p21 and acetylation of {alpha}-tubulin, as well as increased hsp70 levels and attenuated ER{alpha}, PRß, HDAC6, cyclin D1, and c-Myc levels (Fig. 1C and D). Similar effects on ER{alpha}, PRß, HDAC6, cyclin D1, and c-Myc levels were also observed following treatment of MCF-7 and BT-474 cells with 50 nmol/L LBH589 for 24 h (data not shown).


Figure 1
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  		Fig. 1. Effects of pan-HDAC inhibitors LAQ824 and vorinostat on ER{alpha}, PRß, and HDAC6 in cultured ER{alpha}-positive breast cancer cells. A, MCF-7 cells were treated with the indicated doses of LAQ824 for 24 h. Following this, histones were acid extracted or total cell lysates were harvested and Western blot analysis was done for p21, ER{alpha}, PRß, HDAC6, hsp70, c-Myc, cyclin D1, and acetylated {alpha}-tubulin. The levels of ß-actin in the cell lysates served as the loading control. B, BT-474 cells were treated with the indicated doses of LAQ824 for 24 h. Following this, histones were extracted or cell lysates were harvested and Western blot analysis was done for acetylated histone H3, p21, ER{alpha}, PRß, HDAC6, hsp70, and acetylated {alpha}-tubulin. The levels of ß-actin in the cell lysates served as the loading control. C, MCF-7 cells were treated with the indicated doses of vorinostat for 24 h. Following this, immunoblot analysis was done for p21, ER{alpha}, PRß, HDAC6, hsp70, c-Myc, cyclin D1, and acetylated {alpha}-tubulin. D, BT-474 cells were treated with the indicated doses of vorinostat for 24 h. Following this, cell lysates were harvested and immunoblot analysis was done for p21, ER{alpha}, PRß, HDAC6, hsp70, c-Myc, cyclin D1, and acetylated {alpha}-tubulin. The levels of ß-actin in the cell lysates served as the loading control.

 
LAQ824 inhibits transcriptional activity and cellular proliferation mediated by estrogen. We next determined the effect of LAQ824 treatment on the transcriptional activity of E2 in MCF-7 cells, which had been transfected with vectors containing an ERE-luciferase reporter construct (pERELuc) and the control pRL (Renilla luciferase)-TK luciferase reporter. Following serum starvation, treatment with as little as 1 nmol/L E2 for 6 h showed a marked increase in luciferase transcription and activity (Fig. 2A ). A modest increase in luciferase activity was observed when MCF-7 cells were stimulated with 10 nmol/L E2 (Fig. 2A). Next, the transfected MCF-7 cells were cotreated with 100 nmol/L LAQ824 in the presence or absence of E2 stimulation. E2-induced luciferase activity in the MCF-7 cells was completely inhibited by cotreatment with LAQ824. Transcriptional inhibition of luciferase reporter was observed even when MCF-7 cells were stimulated with 10 nmol/L E2 (Fig. 2A). Similar results were obtained following cotreatment with 50 nmol/L LBH589 (data not shown). We next determined the effects of LAQ824 treatment on estrogen-dependent cell proliferation. E2-starved MCF-7 cells were treated with 1 nmol/L E2 and/or 100 nmol/L LAQ824. Treatment with E2 alone showed marked increase in cell numbers over 120 h (Fig. 2B). Cotreatment with LAQ824 abrogated E2-induced proliferation of MCF-7 cells, although LAQ824 also inhibited proliferation of the control non–E2-stimulated MCF-7 cells (Fig. 2B). We next determined the levels of ER{alpha} following treatment with 1 or 10 nmol/L E2 and/or 100 nmol/L LAQ824. Whereas treatment with E2 (1 or 10 nmol/L) or LAQ824 alone decreased ER{alpha} levels, cotreatment with LAQ824 and E2 reduced ER{alpha} to an almost undetectable level (Fig. 2C). This may explain the abrogation of E2-mediated increased reporter gene transcription and increased loss of cell viability seen after treatment with LAQ824 and E2 versus E2 alone (Fig. 2A and B).


Figure 2
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  		Fig. 2. LAQ824 inhibits estrogen (E2)-stimulated ER{alpha} transcription and E2-dependent cell proliferation. A, MCF-7 cells were cotransfected with an ERE luciferase reporter construct (pERELuc) and the control vector pRL-TK. Transfected cells were serum starved, treated with or without LAQ824 (100 nmol/L), and then stimulated with E2 as indicated for 6 h. The firefly luciferase activity was determined and normalized to Renilla luciferase activity. Columns, mean of three experiments done in duplicate; bars, SE. B, MCF-7 cells (1.5 x 105 per well) were plated in six-well plates in triplicates and incubated in phenol-free DMEM containing 10% charcoal-dextran–treated fetal bovine serum. Twenty-four hours after plating, cells were treated with 1 nmol/L E2, 50 nmol/L LAQ824, or both, as indicated. Cell viability was determined by trypan blue exclusion every 24 h following the addition of E2 and/or LAQ824. Points, mean of two experiments done in triplicate. C, MCF-7 cells were plated in E2-stripped medium and allowed to adhere. Following this, the cells were treated with the indicated concentrations of E2 and LBH589 for 18 h and Western blot analysis of ER{alpha} was done. The levels of ß-actin in the lysates served as the loading control.

 
Attenuation of ER{alpha} and HDAC6 levels by HA-HDIs is reversible. Next, we determined whether the effects of HA-HDI on ER{alpha} and HDAC6 are reversible. Protein levels of ER{alpha}, PRß, HDAC6, and p21 were determined following treatment of MCF-7 cells with vorinostat, LAQ824, or LBH589 for 24 h and following incubation of the drug-treated cells in drug-free culture medium. As shown in Fig. 3 , HA-HDI–depleted ER{alpha}, PRß, and HDAC6 levels were partially restored on incubation of the cells for 24 h in the HA-HDI–free medium. In contrast, HA-HDI–induced p21 levels returned to baseline following incubation in the drug-free medium. These findings indicate that the effects of HA-HDI on ER{alpha} and its transcriptional activity are reversible in breast cancer cells.


Figure 3
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  		Fig. 3. Sustained depletion of ER{alpha} levels by pan-HDAC inhibitors in MCF-7 cells. MCF-7 cells were treated with the indicated doses of vorinostat, LAQ824, or LBH589 for 24 h. Following this, cells were washed free of the drug and incubated an additional 8 and 24 h. Total cell lysates were harvested and Western blot analysis was done for ER{alpha}, PRß, HDAC6, and p21. The levels of ß-actin served as the loading control.

 
HA-HDIs induce hsp90 acetylation and disrupt chaperone association of hsp90 with ER{alpha}. We had previously reported that treatment with HA-HDIs inhibits HDAC6, which induces hyperacetylation of hsp90 but inhibits its ATP binding and chaperone association with Her-2, Akt, and c-Raf in breast cancer cells (25, 28). Here, we determined whether HA-HDI treatment also disrupts the chaperone association of hsp90 with ER{alpha} in breast cancer cells. Exposure to 250 nmol/L LAQ824 for 4 to 8 h induced hyperacetylation of hsp90 in MCF-7 cells (Fig. 4A ). Consistent with this, LAQ824 treatment completely abrogated the levels of ER{alpha} and c-Raf bound to the immunoprecipitates of hsp90 from MCF-7 cells (Fig. 4B). This was associated with an increase in polyubiquitylated ER{alpha} in LAQ824-treated MCF-7 cells (Fig. 4C). LAQ824-induced polyubiquitylation of ER{alpha} was associated with proteasomal degradation of ER{alpha} in MCF-7 cells, as indicated by the restoration of ER{alpha} levels by cotreatment with the proteasome inhibitor ALLnL and LAQ824 (Fig. 4D). Similar observations were made following treatment of MCF-7 cells with vorinostat (1.0-2.0 µmol/L) or LBH589 (50 nmol/L) and bortezomib (50 nmol/L). These results show that treatment with HA-HDI, by inhibiting chaperone association of ER{alpha} with hsp90, induces polyubiquitylation and proteasomal degradation of ER{alpha} in breast cancer cells.


Figure 4
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  		Fig. 4. LAQ824 induces hsp90 acetylation and disrupts its binding to ER{alpha}, resulting in increased polyubiquitylation and proteosomal degradation of ER{alpha}. A, MCF-7 cells were treated with 250 nmol/L LAQ824 for the indicated times. Following this, cell lysates were harvested and immunoprecipitated with anti-hsp90 mouse monoclonal antibody. Immunoprecipitates were immunoblotted for acetylated hsp90. Blots were stripped and probed for total hsp90. B, MCF-7 cells were treated with 250 nmol/L LAQ824 for 8 h. Following this, cell lysates were harvested and immunoprecipitated with anti-hsp90 rat monoclonal antibody. Western blots were done for hsp90, ER{alpha}, and c-Raf in the immunoprecipitates. C, MCF-7 cells were treated with the indicated doses of LAQ824 for 4 h. Following this, cell lysates were harvested and ER{alpha} was immunoprecipitated from the samples. Immunoblot analysis was done for polyubiquitylated ER{alpha} and hsp90. The ubiquitin blot was stripped and probed for total ER{alpha}. D, MCF-7 cells were treated for 8 h with 250 nmol/L LAQ824 alone or following a 1-h pretreatment with ALLnL. Following this, cell lysates were harvested and immunoblot analysis was done for ER{alpha}. The levels of ß-actin served as the loading control.

 
Treatment with LAQ824 depletes progrowth and prosurvival signaling proteins in ER{alpha}-expressing breast cancer cells. We next determined the effects of LAQ824 on the downstream progrowth and prosurvival signaling pathways in breast cancer cells. As shown in Fig. 5A , treatment with LAQ824 for 24 h caused significant depletion of p-Akt, p-ERK1/2, and c-Raf in MCF-7 cells. LAQ824 treatment also resulted in depletion of c-Raf, p-Akt, and p-ERK1/2 in BT-474 cells (Fig. 5B). Depletion of c-Raf was associated with decline in the levels of p-ERK1/2 but not total levels of ERK1/2. Depletion of c-Raf and Akt levels by LAQ824 is consistent with HDAC inhibitor–induced acetylation of hsp90 and inhibition of ATP binding and chaperone function of hsp90 with its client proteins Akt and c-Raf, leading to their polyubiquitylation and subsequent proteasomal degradation.


Figure 5
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  		Fig. 5. LAQ824 treatment depletes p-Akt, c-Raf, and p-ERK1/2 levels in MCF-7 and BT-474 cells. A, MCF-7 cells were treated with the indicated doses of LAQ824 for 24 h. Following this, cell lysates were harvested and immunoblot analysis was done for p-Akt, Akt, c-Raf, p-ERK1/2, and ERK1/2 on the total cell lysates. The levels of ß-actin served as the loading control. B, BT-474 cells were treated with the indicated doses of LAQ824 for 24 h. Following this, cell lysates were harvested and immunoblot analysis was done for p-Akt, Akt, c-Raf, p-ERK1/2, and ERK1/2 on the total cell lysates. The levels of ß-actin served as the loading control.

 
Treatment with HA-HDIs induces PARP cleavage and decreases cell viability of ER{alpha}-positive breast cancer cells. We have previously reported that treatment with HA-HDI induces the accumulation of breast cancer cells in the G2-M phase of the cell cycle (25, 28). Here, we determined the effects of HA-HDIs on the viability of MCF-7 and BT-474. Exposure to LAQ824 for 48 h caused a dose-dependent decrease in cell viability of MCF-7 and BT-474 cells, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Fig. 6A ). Similar results were obtained when viability was assessed with the trypan blue exclusion method (data not shown). Treatment with vorinostat (0.5-2 µmol/L for 48 h) also inhibited cell survival of MCF-7 and BT-474 cells in a dose-dependent manner (data not shown). As shown in Fig. 6B, treatment with LAQ824 also induced PARP cleavage, consistent with induction of caspase-dependent apoptosis in MCF-7 and BT-474 cells. We have reported similar findings following treatment of BT-474 cells with vorinostat (25). Collectively, these data indicate that exposure to HA-HDIs could significantly lower the threshold for apoptosis by modulating the levels of several regulators of cell survival in E2-responsive ER{alpha}-positive breast cancer cells. We also determined the effects of cotreatment with HA-HDI and 4-hydroxytamoxifen, a well-known selective ER modulator on MCF-7 cells. Whereas treatment with 4-hydroxytamoxifen (1.0 µmol/L) did not induce cell death, similar to LAQ824 (Fig. 2B), LBH589 treatment for 48 h markedly induced loss of cell viability of MCF-7 cells (Fig. 6C). Cotreatment with LBH589 and 4-hydroxytamoxifen versus LBH589 alone induced significantly more loss of viability of MCF-7 cells (P < 0.05; Fig. 6C). This was also associated with marked depletion of ER{alpha} levels (data not shown).


Figure 6
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  		Fig. 6. HA-HDI and 4-hydroxytamoxifen treatment diminishes cell viability and HA-HDI induces PARP cleavage in cultured breast cancer cell lines. A, MCF-7 and BT-474 cells were treated with the indicated doses of LAQ824 for 48 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Points, mean of three experiments; bars, SE. B, MCF-7 and BT-474 cells were treated with the indicated doses of LAQ824 for 24 h. Following this, cell lysates were harvested and Western blot analysis was done for full-length and cleaved PARP. The levels of ß-actin in the cell lysates served as the loading control. C, MCF-7 cells were plated and allowed to adhere. Following this, the cells were treated with the indicated concentrations of 4-hydroxytamoxifen (4-HT) and/or LBH589 for 48 h. Cell death was assessed by trypan blue dye uptake in a hemocytometer.

 
HA-HDI treatment sensitizes ER{alpha}-negative cells to 4-hydroxytamoxifen but does not induce ER{alpha} expression. We next determined the effects of the HA-HDI LBH589 on the sensitivity of human breast cancer cells that lack ER{alpha} and Her-2 amplification. Treatment with LBH589 (100 nmol/L) for up to 48 h did not derepress or induce ER{alpha} in MB-231 cells, although LBH589 treatment depleted cyclin D1 levels (Fig. 7A ). Neither 4-hydroxytamoxifen alone nor cotreatment with 4-hydroxytamoxifen and LBH589 induced ER{alpha} levels. 4-Hydroxytamoxifen cotreatment also did not affect LBH589-mediated depletion of cyclin D1 (Fig. 7A). Consistent with these findings, cotreatment with 4-hydroxytamoxifen did not augment either the cell growth inhibition or loss of cell viability of MB-231 cells induced by LBH589 (Fig. 7B and C).


Figure 7
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  		Fig. 7. Treatment with LBH589 neither derepresses ER{alpha} nor cotreatment with 4-hydroxytamoxifen augments cell growth inhibition or loss of viability of ER{alpha}-negative MDA-MB-231 cells. A, MDA-MB-231 cells were treated with the indicated doses of 4-hydroxytamoxifen and LBH589 for 24 h. Following this, total cell lysates were harvested and immunoblot analysis was done for ER{alpha} and cyclin D1. The levels of ß-actin in the cell lysates served as the loading control. B, MDA-MB-231 cells were treated with the indicated concentrations of 4-hydroxytamoxifen and/or LBH589 for 48 and 72 h. Cells were trypsinized and total cell counts were taken with a Coulter counter. C, MDA-MB-231 cells were treated with the indicated concentrations of 4-hydroxytamoxifen and/or LBH589 for 48 h. Cell death was assessed with the trypan blue dye uptake method.

 
Effect of class I HDAC inhibitor on ER{alpha} levels. We next determined the effects of the class I HDAC specific inhibitor valproic acid on both ER{alpha} levels and loss of cell survival of MCF-7 cells. Treatment with the safely achievable levels of valproic acid (0.5 mmol/L) did not lower ER{alpha} levels (30) and only minimally increased cell death (Fig. 8A and B ). However, higher levels of valproic acid known to be clinically toxic partially reduced ER{alpha} levels without significantly increasing the loss of cell viability of MCF-7 cells (Fig. 8A and B).


Figure 8
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  		Fig. 8. Valproic acid minimally depletes ER{alpha} and cyclin D1 and induces minimal loss of viability in ER{alpha}-positive breast cancer cells. A, MCF-7 cells were treated with the indicated concentrations of valproic acid for 24 h. Following this, cell lysates were harvested and immunoblot analysis was done for ER{alpha} and cyclin D1. The levels of ß-actin in the cell lysates served as the loading control. B, MCF-7 cells were plated and allowed to adhere. Following this, the cells were treated with the indicated concentrations of valproic acid for 48 h. Cell viability was determined with the trypan blue dye exclusion method.

 

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In the present studies, we show that by inducing acetylation and disruption of the chaperone function of hsp90, treatment with HA-HDI depletes the levels and transcriptional activity of ER{alpha}, resulting in attenuation of ER{alpha}-targeted gene products including PRß, HDAC6, c-Myc, and cyclin D1. This is accompanied by depletion of other hsp90 client proteins (e.g., p-Akt, c-Raf, p-ERK1/2, and cyclin D1). Taken together with down-modulation of the levels of c-Myc and induction of p21, also mediated by HA-HDI treatment, these molecular perturbations may be responsible for HA-HDI–induced growth arrest and apoptosis of E2-responsive human breast cancer cells. In HA-HDI–treated cells, dose-dependent induction of hsp70 also suggests that HA-HDI treatment disrupts the chaperone association of heat shock factor-1 with hsp90, which is further evidence that HA-HDIs inhibit hsp90 chaperone function in breast cancer cells (29). In a previous report, we had shown that HA-HDI treatment inhibits ATP binding of hsp90, which is known to abrogate the chaperone function of hsp90 (15, 23). Previous reports have also clearly shown that treatment with hsp90 inhibitor geldanamycin and its analogues, 17-allylamino-demethoxygeldanamycin or radicicol, depletes ER{alpha} levels and ER transcriptional activity (31, 32). Consistent with this, hsp90 inhibition by HA-HDI also resulted in abrogation of ER{alpha} levels and activity.

Hypermethylation of the ER{alpha} gene is known to be an important mechanism responsible for the absence of ER{alpha} expression and de novo resistance to endocrine therapy in breast cancer cells (33). Functional ER{alpha} expression was shown to be restored in breast cancer cells lacking ER{alpha} expression by treatment with DNA demethylating agents combined with HDAC inhibitors (34, 35). This was also shown to restore tamoxifen sensitivity in ER{alpha}-negative breast cancer cells (35). However, in these reports the effect of further treatment with HA-HDI on the derepressed ER{alpha} levels and transactivation was not documented. Other reports have noted that treatment with HA-HDI depletes levels of ER{alpha} in MCF-7 cells, but unlike the findings presented here, in these reports the acetylation and disruption of hsp90 function was not elucidated as the underlying mechanism (36). Recently, HDAC6 has been shown to be an E2-responsive gene and overexpression of HDAC6 is commonly seen in ER{alpha}-expressing breast cancer cells (6, 37). This suggests that HDAC6 is a potential target in E2-responsive breast cancer cells. Because HDAC6 is the hsp90 deacetylase and inhibition of HDAC6 is responsible for HA-HDI–mediated hsp90 inhibition (23, 24), targeting HDAC6 may potentially be a useful anti-ER{alpha} treatment strategy in E2-responsive breast cancers. Our findings clearly show that treatment with HA-HDI depletes ER{alpha} levels in the ER{alpha}-positive MCF-7 cells and does not induce ER{alpha} levels in ER{alpha}-negative MB-231 cells. These results are consistent with our findings that cotreatment with 4-hydroxytamoxifen and LBH589 induces more cell death in ER{alpha}-positive but not ER{alpha}-negative cells. Accordingly, we envision that benefits of the combination of a HA-HDI and selective ER modulator would be mostly limited against the ER{alpha}-positive breast cancer model. This also needs to be preclinically confirmed in the in vivo xenograft models of human breast cancers.

It is clear that during intrinsic and/or acquired resistance to endocrine therapy with either aromatase inhibitors or selective ER modulators such as tamoxifen, ER{alpha} remains functionally active and, therefore, a target for therapy (38). Interaction or cross-talk of ER{alpha} with insulin-like growth factor-I receptor and phosphatidylinositol 3-kinase or epidermal growth factor receptor may lead to increased signaling through Akt and mitogen-activated protein kinase pathways, providing progrowth and prosurvival signaling to breast cancer cells (10–12). Additionally, activation of mitogen-activated protein kinase in ER{alpha}-positive breast cancer cells has been shown to induce the molecular phenotype of ER{alpha}-negative breast cancers (39). Hence, treatment with HA-HDI, which down-regulates p-Akt, Akt, c-Raf, and p-ERK1/2 levels, could potentially overcome intrinsic resistance to selective ER modulators. During prolonged exposure, the acquired resistance to tamoxifen has been shown to be due to up-regulation of epidermal growth factor receptor and Her-2 pathways (40). In addition, Her-2 amplification can result in phosphorylation and activation ER{alpha} and the coactivator AIB1, conferring growth stimulatory agonistic activity on tamoxifen (12, 38). In this setting again, HA-HDI–mediated depletion of Her-2 and p-ERK1/2 could potentially overcome resistance to tamoxifen (25, 28). In the acquired resistance to long-term estrogen suppression by aromatase inhibition, ER{alpha}-positive breast cancer cells display enhanced sensitivity to E2-induced apoptosis (41, 42). Here, again, enhanced cross-talk between ER{alpha}, Her-2, and mitogen-activated protein kinase is operative, and treatment with HA-HDI could override resistance due to E2 deprivation by aromatase inhibitors (43). Collectively, these studies have elucidated mechanisms underlying de novo resistance and emergence of acquired resistance to endocrine therapies. Our findings presented here, along with the previously published reports, strengthen the rationale for determining the activity of nonendocrine, targeted agents such as HA-HDIs, alone or in combination with antiestrogens or aromatase inhibitors, in the treatment of ER{alpha}-positive breast cancer.


   Footnotes
 
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.

Received 12/29/06; revised 5/14/07; accepted 5/23/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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