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

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.

CCAAT/enhancer binding protein delta up-regulates aromatase promoters I.3/II in breast cancer epithelial cells.

Aromatase is the enzyme responsible for the last step of estrogen synthesis. The female hormone, estrogen, is known to stimulate breast cancer cell growth. Because the expression of aromatase in breast cancer tissues is driven by unique promoters I.3 and II, a more complete understanding of the regulatory mechanism of aromatase expression through promoters I.3/II in breast tumors should be valuable in developing targeted therapies, which selectively suppress estrogen production in breast tumor tissue. Results from in vivo footprinting analyses revealed several protein binding sites, numbered 1 to 5. When site 2 (-124/-112 bp, exon I.3 start site as +1) was mutated, promoters I.3/II activity was dramatically reduced, suggesting that site 2 is a positive regulatory element. Yeast one-hybrid screening revealed that a potential protein binding to site 2 was CCAAT/enhancer binding protein delta (C/EBP delta). C/EBP delta was shown to bind to site 2 of aromatase promoters I.3/II in vitro and in vivo. C/EBP delta up-regulated promoters I.3/II activity through this site and, as a result, it also up-regulated aromatase transcription and enzymatic activity. p65, a subunit of nuclear factor-kappaB (NF-kappaB) transcription factor, inhibited C/EBP delta-up-regulated aromatase promoters I.3/II and enzymatic activity. This inhibitory effect of p65 was mediated, in part, through prevention of the C/EBP delta binding to site 2. This C/EBP delta binding site in aromatase promoters I.3/II seems to act as a positive regulatory element in non-p65-overexpressing breast cancer epithelial cells, whereas it is possibly inactive in p65 overexpressing cancer epithelial cells, such as estrogen receptor-negative breast cancer cells.

Grape seed extract is an aromatase inhibitor and a suppressor of aromatase expression.

Aromatase is the enzyme that converts androgen to estrogen. It is expressed at higher levels in breast cancer tissues than normal breast tissues. Grape seed extract (GSE) contains high levels of procyanidin dimers that have been shown in our laboratory to be potent inhibitors of aromatase. In this study, GSE was found to inhibit aromatase activity in a dose-dependent manner and reduce androgen-dependent tumor growth in an aromatase-transfected MCF-7 (MCF-7aro) breast cancer xenograft model, agreeing with our previous findings. We have also examined the effect of GSE on aromatase expression. Reverse transcription-PCR experiments showed that treatment with 60 mug/mL of GSE suppressed the levels of exon I.3-, exon PII-, and exon I.6-containing aromatase mRNAs in MCF-7 and SK-BR-3 cells. The levels of exon I.1-containing mRNA, however, did not change with GSE treatment. Transient transfection experiments with luciferase-aromatase promoter I.3/II or I.4 reporter vectors showed the suppression of the promoter activity in a dose-dependent manner. The GSE treatment also led to the down-regulation of two transcription factors, cyclic AMP-responsive element binding protein-1 (CREB-1) and glucocorticoid receptor (GR). CREB-1 and GR are known to up-regulate aromatase gene expression through promoters I.3/II and I.4, respectively. We believe that these results are exciting in that they show GSE to be potentially useful in the prevention/treatment of hormone-dependent breast cancer through the inhibition of aromatase activity as well as its expression.

Growth inhibition of estrogen receptor-positive and aromatase-positive human breast cancer cells in monolayer and spheroid cultures by letrozole, anastrozole, and tamoxifen.

Two third-generation aromatase inhibitors, letrozole and anastrozole, and the antiestrogen tamoxifen, were compared for growth-inhibiting activity in two estrogen receptor (ER)-positive aromatase-overexpressing human breast cancer cell lines, MCF-7aro and T-47Daro. Inhibition of hormone (1 nM testosterone)-stimulated proliferation was evaluated in both monolayer cultures and in three-dimensional spheroid cultures. Letrozole and anastrozole were also compared for effectiveness of aromatase inhibition, and relative affinity for aromatase, under both monolayer and spheroid growth conditions. Letrozole was an effective inhibitor of MCF-7aro monolayer cell proliferation, with an estimated 50% inhibitory concentration (IC50) of 50-100 nM, whereas an IC50 was not reached with anastrozole at any concentration tested (100-500 nM). An IC50 of tamoxifen was 1000 nM. Proliferation of T-47Daro monolayer cells was more sensitive to inhibition by all three agents; as with MCF-7aro cells, letrozole was the most effective inhibitor. MCF-7aro spheroids were slightly less sensitive than monolayer cells proliferation-inhibiting effects of letrozole (IC50 about 200 nM), and there was no significant inhibition with 100-200 nM anastrozole or 200-1000 nM tamoxifen. Letrozole and anastrozole significantly inhibited T-47Daro spheroid cell proliferation, at 15-25 and 50 nM, respectively, consistent with the greater sensitivity of T-47Daro monolayer cells to inhibition of proliferation by these agents. Tamoxifen failed to significantly inhibit T-47Daro spheroid cell proliferation over a 100-500 nM concentration range. Determination of aromatase inhibition in monolayers of both cell lines by a direct-access microsomal assay and an intact-cell assay revealed that letrozole was more active than anastrozole in monolayers of both cell lines and in both assays. In MCF-7aro spheroids following cell lysis, only letrozole significantly inhibited aromatase activity, supporting the conclusion that letrozole binds stronger to aromatase than anastrozole does. Our results demonstrate that MCF-7aro and T-47Daro spheroids could be a suitable model for evaluation of growth-inhibitory effects of agents used in hormonal therapy of breast cancer.

Positive and negative transcriptional regulation of aromatase expression in human breast cancer tissue.

By performing primer-specific RT-PCR analyses, three laboratories including ours have found that exons I.3 and PII are the two major exon Is present in aromatase mRNAs isolated from breast tumors. These results suggest that promoters I.3 and II are the major promoters directing aromatase expression in breast tumors. The characterization of transcription factors that interact with the two elements near promoters I.3 and II, i.e., S1 and CREaro, helps us better understand the mechanism of the switch of promoter usage between normal breast tissue and cancer tissue. The positions of the two regulatory regions were mapped by DNase I footprinting and DNA deletion analyses. We applied the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library for genes encoding the proteins binding to these regions. Our results suggest that in normal breast tissue, the function of promoters I.3 and II is suppressed through the binding of EAR-2, COUP-TFI, and RARgamma to S1, and through the binding of Snail/Slug proteins to their binding site that quenches the CREaro activity. In cancer tissue, the expression levels of EAR-2, COUP-TF1, EARgamma, Snail, and Slug decrease, and aromatase expression is then up-regulated through the binding of ERRalpha to S1 and the binding of CREB1 or related factors to CREaro. In a separate study, we found that estrogen could up-regulate aromatase expression in breast cancer cells by a non-genomic action of ERalpha via cross-talk with growth factor-mediated pathways. Our preliminary results suggest that protein kinase C delta participates in this ERalpha-growth factor mediated regulation. To further understand the regulatory mechanism, we have recently initiated an in vivo footprinting analysis of the -260/+76 bp region of promoter I.3. The experiments were conducted with both MCF-7 and MDA-MB-231 breast cancer cells. Our results revealed several footprinted sites. Five regions (sites 1-5) were then selected for functional analysis through DNA site-directed mutagenesis experiments. This analysis has also confirmed the promoter I.3 TATA site and Snail/Slug binding site. These mutants showed higher luciferase activity when compared to the wild-type, indicating that the proteins binding to these sites were acting as repressors. By reviewing findings from our laboratory and other laboratories, a detailed mechanism for the transcriptional regulation of aromatase expression in breast cancer tissue is summarized and discussed.

Letrozole-, anastrozole-, and tamoxifen-responsive genes in MCF-7aro cells: a microarray approach.

Antiestrogens and aromatase inhibitors are important drugs in the treatment of estrogen-dependent breast cancer. To investigate the effects of these drugs on gene expression in breast cancer cells, we treated estrogen receptor-positive MCF-7 cells stably transfected with the aromatase gene (known as MCF-7aro cells) with testosterone, 17 beta-estradiol, two aromatase inhibitors (letrozole and anastrozole), and an antiestrogen (tamoxifen). We found that testosterone or 17 beta-estradiol induced the proliferation of MCF-7aro cells at a rate six times faster than the untreated cells. In addition, the testosterone-induced proliferation of MCF-7aro cells was effectively suppressed by letrozole, anastrozole, or tamoxifen. Microarray analyses on Affymetrix Human Genome U133A GeneChips (Affymetrix, Santa Clara, CA) were carried out using total RNA isolated from the control and treated cells. At the false discovery rate of 0.05 and a minimum fold-change criteria of 1.5, 104 genes were identified that were up-regulated and 109 genes were identified that were down-regulated by both androgen and estrogen. More than 50% of these hormone-regulated genes were counter-regulated by all three inhibitors and >90% were counter-regulated by at least one of the inhibitors. Comparing the effect of each inhibitor on gene expression, we observed that letrozole and anastrozole are more similar in terms of the genes they affect compared with treatment with tamoxifen. To validate the gene expression profiles identified from microarray analyses, the expression patterns of 13 representative genes were examined by Northern analysis. Finally, the genes identified as statistically significant were classified based on their expression patterns and biological function/pathways. The results of this study provide us with a better understanding of the actions of both aromatase inhibitors and antiestrogens at the molecular level. We believe that the results of this study serve as the first step in identifying unique expression patterns following drug treatment, and that this will ultimately be useful in customizing patient treatment strategies for hormone-dependent breast cancer.

Structure-function studies of aromatase and its inhibitors: a progress report.

The utilization of computer modeling, site-directed mutagenesis, inhibition kinetic analysis and reaction metabolite analysis allows us to better understand the structure-function relationship between aromatase and its inhibitors. Our results have helped in determining how steroidal and nonsteriodal aromatase inhibitors bind to the active site of the enzyme. This information has also aided in the understanding of the reaction mechanism of aromatase. Furthermore, our structure-function studies of aromatase have generated important information for predicting how environmental chemicals interact with the enzyme. During the last 2 years, a new aromatase computer model based on the X-ray structure of rabbit cytochrome P450 2C5 has been generated and used to evaluate the results obtained from new aromatase mutants produced in this laboratory. In addition, we have succeeded in the expression and purification of functionally active aromatase using an Escherichia coli expression method. The catalytic properties of this recombinant aromatase are similar to those properties exhibited by the human placental aromatase preparation and the mammalian cell-expressed enzyme. The E. coli expressed aromatase will be very useful for further structure-function studies of aromatase. Our laboratory has also evaluated the growth-inhibiting activity of aromatase inhibitors in estrogen receptor-positive breast cancer using three-dimensional cell cultures of aromatase-over expressing MCF-7 and T-47D cell lines (i.e. MCF-7aro and T-47Daro). Our results demonstrate that these three-dimensional cultures are valuable approaches to assess the growth-inhibiting activity of aromatase inhibitors. Finally, we have identified several phytochemicals to be potent inhibitors of aromatase. To demonstrate the impact of the phytochemicals on estrogen formation in vivo, we showed that the intake of anti-aromatase chemicals from red wine was capable of suppressing MCF-7aro-mediated tumor formation in nude mice and aromatase-induced hyperplasia in a transgenic mouse model in which aromatase is over-expressed in the mammary tissue.