Aromatase-overexpressing Cells Research Articles

Inhibition of the aromatase enzyme by exemestane cysteine conjugates.

Exemestane (EXE) is an aromatase inhibitor used to treat hormone-dependent breast cancer. EXE is extensively metabolized, with unchanged EXE and its active metabolite 17-dihydroexemestane (17-DHE) accounting for 17 and 12%, respectively, of total plasma EXE in vivo The major circulating EXE metabolites are the cysteine conjugates of EXE and 17-DHE, and the 17-DHE glucuronide, which together account for 70% of total plasma EXE in vivo The goal of the present study was to examine the inhibition potential of major metabolites of EXE through inhibition assays using aromatase-overexpressing cells and pooled ovarian tissues. Estrone formation was used as a measure of aromatase activity and was detected and quantified using UPLC-MS. EXE-cys, 17β-DHE, and 17β-DHE-cys all exhibited inhibition of estrone formation at both 1 µM and 10 µM concentrations, with 17β-DHE and EXE-cys showing significant inhibition of estrone formation (63% each) at 10 µM. In contrast, 17β-DHE-Gluc displayed minimal inhibition (5-8%) at both concentrations. In ovarian tissue, EXE-cys and 17β-DHE showed similar patterns of inhibition, with 49% and 47% inhibition, respectively, at 10 µM. The IC50 value for EXE-cys (16 {plus minus} 10 µM) was similar to 17β-DHE (9.2 {plus minus} 2.7 µM) and higher than EXE (1.3 {plus minus} 0.28 µM), and all three compounds showed time-dependent inhibition with IC50 shifts of 13 {plus minus} 10, 5.0 {plus minus} 2.5 and 36 {plus minus} 12-fold, respectively. Given its high circulating levels in patients taking EXE, these results suggest that EXE-cys may contribute to the pharmacologic effect of EXE in vivo Significance Statement The current study is the first to examine the major phase II metabolites of EXE (EXE-cys, 17β-DHE-cys, and 17β-DHE-Gluc) for inhibition potential against the target enzyme, aromatase (CYP19A1). EXE-cys was found to significantly inhibit aromatase in a time dependent manner. Given its high circulating levels in patients taking EXE, this phase II metabolite may play an important role in reducing circulating estrogen levels in vivo.

Abstract 3275: Development of a new ex-vivo orthotopic model-Human breast cancer cells in mouse mammary gland organ culture

Abstract Mouse mammary organ culture (MMOC) has been classically employed for evaluating the efficacy of chemopreventive agents against development of carcinogen-induced preneoplastic lesions. Efficacy of chemopreventive agents observed in MMOC correlates well with that observed in in-vivo carcinogenesis models. In the present study, we developed a new ex-vivo Human in Mouse organ culture model which mimics in-vivo orthotopic breast cancer model. Since we introduced human breast cancer cells in mouse mammary gland, this model is termed as human Breast cancer (BCA) in Mouse Mammary Organ Culture (BCa-in-MMOC)). Three to four week old female BALB/c mice were sensitized with estradiol (1μg) + progesterone (1mg) for 9 days. On the 10th day animals were sacrificed and 2.5x104 T47Dparental or T47D aromatase overexpressing cells were injected into the fourth pair of thoracic mammary glands. The glands were excised then cultured at 37°C under 95% O2 / 5% CO2 in hMEM medium containing 10% charcoal stripped FBS/supplemented with Testosterone (1nM) and progesterone (1uM) and growth promoting hormones (5 µg insulin, 5 μg prolactin per ml medium). At the end of the experiment, the glands were fixed in formalin. The paraffin embedded sections (longitudinal) of entire glands were processed for histopathological examination (H and E stain) and immnohistochemical staining of various proteins. Mammary glands were evaluated for the presence of T47D cells, their growth pattern and their molecular responsiveness to estradiol. T47D cells (both types) injected into mammary glands were easily identified against mouse cells by intense human specific Ck-18 immunofluorescence staining. Histopathological observation of mammary gland sections showed that growth pattern of injected cancer cells was identical to that observed of breast cancer cells injected in vivo in athymic mice. Interestingly, clusters of cancer cells in the mammary gland stroma appear similar to those observed in breast tumors in women. Cancer cells injected into glands survived and continued to grow (as evident from Ki-67 immunostaining) after 15 days in culture. Cancer cells maintained their original characteristics (ER+, PR+, EGFR+, and aromatase). T47D cells with enhanced aromatase expression growing in the MMOC could metabolize testosterone to estrogen, which resulted in enhanced cell proliferation and induction of estrogen target genes such as ER and PR-B. Mouse mammary glands with T47D aromatase overexpressing cells also showed changes typical to estrogenic milieu. In summary this model provides a novel, inexpensive ex-vivo model, which could be used to study effects of therapeutic agents on the cancer cells growing in orthotopic micromilieu. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3275. doi:1538-7445.AM2012-3275

P2-17-05: Antiprogestin Proellex Suppresses Proliferation of Aromatase Overexpressing and Letrozole Resistant T47D Breast Cancer Cells.

Abstract Background: Aromatase is a cytochrome P450 CYP19A1 enzyme responsible for the conversion of C19 androgens to C18 estrogen. Aromatase overexpression leads to increase in local estrogen concentration in post menopausal women diagnosed for breast cancer. Aromatase inhibitors (AI) are used as one of the first line therapies for the treatment of ER+PR+ breast cancers. However, many patients acquire resistance to AI treatment. Therefore alternative approaches are being sought for patients with AI resistance. Previous reports suggest that upregulation of progesterone receptor can lead to enhanced expression of EGFR/ERK/MAPK involved in acquiring resistance. In this study, we evaluated the effect of a selective progesterone receptor modulator, CDB4124 (Proellex) with low glucocorticoid activity on aromatase overexpressing and Letrozole resistant T47D cells. Methods: Aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines were generated by stable transfection with plasmid containing CYP19A1 coding region, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10μM Letrozole. Cell proliferation was determined by MTT or Crystal violet assays. Gene expressions were quantitated by qRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's ‘T’ test or ANOVA followed by Bonferroni's post hoc test. Results: T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA and protein) and as a result exhibited increased conversion of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone. In T47Darom cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex, Letrozole and other known AI (Anastrozole, Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. The inhibition of cell proliferation was significantly greater when cells were treated with Proellex in combination to other AIs. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex may be due to PR-B/EGFR modulation in ER+PR+, aromatase overexpressing cells. Conclusion: Overall these results suggest that antiprogestin, Proellex could be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for AI resistant breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-17-05.

Abstract 3589: Characterization of 17-dihydroexemestane glucuronidation. A role for the UGT2B17 deletion in exemestane pharmacogenetics

Abstract Exemestane is a 3rd-generation aromatase inhibitor (AI) used in the treatment of breast cancer in postmenopausal women. Reduction to form 17-dihydroexemestane and subsequent glucuronidation to hydroexemestane-17-O-glucuronide is a major pathway for exemestane metabolism. In the present study, homogenates from a newly-developed HEK293 aromatase-overexpressing cell line (HEK293-aro) were used to examine the activity of exemestane versus 17-dihydroexemestane in vitro. 17-dihydroexemestane exhibited an inhibition of estrone formation in HEK293-aro cell homogenates (IC50=2.3±0.83 μM) that was similar to that observed for exemestane (IC50=1.4±0.42 μM). UGT-overexpressing cell lines were screened for activity against 17-dihydroexemestane and UGTs 2B17>1A10>1A8>1A4 were the only UGTs that exhibited activity. Of the hepatic UGTs, UGT2B17 exhibited a 17-fold higher Vmax/KM than UGT1A4. In human liver microsome (HLM) specimens, the rate of hydroexemestane-17-O-glucuronide formation using 17-dihydroexemestane as substrate was shown to be significantly (p<0.001) decreased (14-fold) in HLMs from subjects with the UGT2B17 (0/0) deletion genotype as compared with HLMs from subjects wild type (+/+) for UGT2B17. A significant (r2=0.72) correlation was observed between HLM hydroexemestane-17-O-glucuronide formation and liver UGT2B17 expression in the same subjects, and the KM for UGT2B17(+/+) HLMs against 17-dihydroexemestane was 7.3 μM, ∼ 2-fold lower than that observed for UGT2B17 against 17-dihydroexemestane in vitro (14.5 μM). UGT2B17 (0/0) HLMs exhibited a 1.7-fold higher KM and a 36-fold lower Vmax/KM than UGT2B17 (+/+) HLMs. These data suggest that UGT2B17 is important in the metabolism of 17-dihydroexemestane, a major active metabolite of exemestane, and that the UGT2B17 deletion could have an important role in overall patient response to exemestane. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3589.

Intermittent treatment of hormone-dependent breast cancer using exemestane: translational research from lab bench to bedside.

Abstract Abstract #3040 Background and Rationale: Endocrine therapy agents such as aromatase inhibitors (AIs) have shown good clinical efficacy in the treatment of post-menopausal breast cancer patients. Currently, the major clinical hindrance with endocrine therapy is disease progression or acquired resistance. Intermittent treatment of endocrine therapy agents has been proposed in several clinical trials to address whether this treatment strategy can delay the onset of acquired resistance in hormone-dependent breast cancers. To support these clinical trials, we have performed a pre-clinical study using the steroidal AI exemestane (EXE) to understand potential molecular features that differentiate continuous treatment of EXE from intermittent administration.
 Methods: The ER+ and aromatase-overexpressing cell culture model system (MCF-7aro) was used in this pre-clinical study. The cells were treated either continuously (T+EXE Cont) or intermittently (T+EXE Interm) for 2 weeks with EXE and 1 week without EXE, in the presence of testosterone (T). In addition, a non-steroidal AI letrozole, was used in an identical intermittent manner.
 Results: Based on cell culture studies, acquired resistance was reached at 14 weeks in the T+EXE Cont treated cells, while T+EXE Interm cells were still responsive to EXE. At 34 weeks, acquired resistance developed in the T+EXE Interm cell lines, which demonstrates a delay in time to progression. The non-steroidal AI letrozole did not demonstrate a delay in acquired resistance using this intermittent model. Based on microarray analysis, we observed that gene expression of certain estrogen-responsive genes was elevated in the T+EXE Cont cells, while select genes showed lower levels of up-regulation in T+EXE Interm lines.
 Discussion: Based on our previous studies, up-regulation of a key estrogen-responsive gene, amphiregulin (AREG), is responsible for activating EGFR and mediating resistance to EXE. Expression of genes such as AREG are decreased in the T+EXE Interm cell lines, suggesting the suppression of growth factor signaling implicated in driving resistance. In addition, we have reported that EXE acts as an aromatase destabilizer at the protein level. Unlike letrozole or anastrozole, EXE-mediated protein degradation of aromatase may play a key role in reducing estrogen levels that are responsible for driving breast cancer survival and proliferation. Since these pre-clinical studies provide an important mechanistic basis for intermittent endocrine therapy, a clinical trial to evaluate the intermittent use of exemestane has been discussed and is planned to initiate in the near future. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3040.