The HDAC inhibitor LBH589 (panobinostat) is an inhibitory modulator of aromatase gene expression

Abstract

Aromatase converts androgens to estrogens. Although third-generation aromatase inhibitors (AIs) are important drugs in hormonal therapy for breast cancer in postmenopausal women, there are concerns about the side effects associated with the estrogen deprivation achieved with AIs. Expression of aromatase in breast cancer tissue is driven by different promoters than those in noncancer tissues; thus, suppression of aromatase expression in cancer tissues through the down-regulation of breast tumor-specific promoters would reduce the side effects associated with whole-body suppression of estrogen biosynthesis by AIs. We report that histone deacetylase inhibitor LBH589 (panobinostat) is a potent inhibitor of aromatase expression (with an IC(50) value < 25 nM). LBH589 selectively suppresses human aromatase gene promoters I.3/II, which are preferentially used in breast cancer tissue. Furthermore, using the H295R cell culture model, we found that achieving the same degree of inhibition of aromatase activity required only one-fifth as much letrozole (an AI) in the presence of 25 nM LBH589 as in the absence of LBH589. We also used an H295R/MCF7 coculture model to demonstrate the synergistic interaction of LBH589 + letrozole in suppressing the proliferation of hormone-responsive breast cancer cells. Finally, our results also indicate that LBH589 down-regulates the activity of promoters I.3/II in an epigenetic fashion. LBH589 reduces the levels of C/EBPdelta, decreases the binding of C/EBPdelta, and increases the levels and binding of acetyl-histones to the promoters I.3/II. These findings provide an important basis for future clinical evaluations of LBH589 in hormone-dependent breast cancer.

Fig. 1.

Suppression of aromatase activity/expression in H295R cells by LBH589. (A) H295R cells were treated with LBH589 for 24 h. After treatment, the cells were washed with PBS, and aromatase activity was measured by the [³H] H₂O release assay. Student's t test was used for statistical analysis compared with the vehicle control. **P < 0.001. (B) MCF7 and MCF7/Her2 cells were treated with LBH589 (50 nM) or with DMSO as a control for 24 h. After treatment, total RNA was isolated; 5 μg of total RNA was used for real-time qPCR for quantifying aromatase gene expression. β-actin mRNA was amplified as an internal control. All samples were run in triplicate, and SDs were calculated. (C) After treatment with LBH589 (50 nM) for 24 h, H295R cells were lysed and applied for Western blot analysis using aromatase antiserum.

Fig. 2.

Suppression of promoters I.3/II luciferase reporter activity by HDAC inhibitors. (A) Promoters I.3/II stably transfected HeLa cells were treated with LBH589 at indicated concentrations for 24 h. Cells were then lysed and harvested, and luciferase activity was determined and normalized with protein concentration in triplicate for each treatment condition. Student's t test was used for statistical analysis compared with the vehicle control. **P < 0.001. (B) Promoters I.3/II stably transfected HeLa cells were treated with TSA, SAHA, or SBHA at the indicated concentrations for 24 h. Cells were lysed and harvested. The luciferase activity was determined and normalized with protein concentration.

Fig. 3.

Effects of LBH589 on the expression of key proteins. (A) Western blot analyses were performed with H295R cells that had treated with LBH589 (50 nM) for 24 h. After treatment, cells were lysed and harvested, and cell lysates were analyzed by immunoblotting with indicated antibodies. (B) ChIP analysis was performed to determine binding of C/EBPδ to the aromatase promoter. H295R cells were treated with LBH589 (25 nM or 50 nM) for 24 h. Cells were harvested after cross-linking with 1% formaldehyde. Immunoprecipitation was conducted with normal rabbit IgG, anti–acetyl-histone H3, and anti-C/EBPδ antibody. PCR was performed using primers to amplify the C/EBPδ-binding region of promoters I.3/II. (C) Quantitive ChIP analysis was performed to quantify the binding of C/EBPδ to the aromatase promoter. H295R cells were treated with LBH589 (50 nM) for 24 h. The cells were then harvested after cross-linking with 1% formaldehyde. Real-time qPCR was performed using primers to amplify the C/EBPδ-binding region of promoters I.3/II.

Fig. 4.

(A) Synergistic inhibitory effect of LBH589 and letrozole on aromatase activity in H295R cells. The cells were treated with LBH589 and letrozole at indicated concentrations for 24 h. After treatment, the cells were washed with PBS, and aromatase activity was measured using the [³H] H₂O release assay. (B) Proliferation assay of H295R/MCF7 coculture model. MCF-7 and H295R cells were cocultured in 96-well plates with hormone-deprived media at a concentration of 1 × 10³ and 5 × 10² per well, respectively. The next day, hormone-deprived media containing 10 nM T or E2 was added to the cells to induce proliferation, with DMSO as a vehicle control. Cells were cultured for 9 d, and media were replaced every 72 h. Cell viability was assessed by the MTT assay and measured at 570 nm on a SpectraMax M5 microplate reader. Three replicates were performed for each measurement, and the mean and SD were calculated. (C) Combined effect of LBH589 and letrozole on the H295R/MCF7 coculture model. With the conditions described above, cells were cultured for 9 d with hormones with or without the two drugs. Three replicates were performed for each measurement, and the mean and SD were calculated. (D) Isobologram analysis of the combined effect of LBH589 and letrozole on the H295R/MCF7 coculture model. The cells were cultured with the two drugs at different concentrations at conditions described above. Normalized isobolograms were produced by CalcuSyn software. Values below the threshold line represent synergistic combination.

Fig. 5.

Different treatment strategies for hormone-dependent breast cancer.