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. 2009 Aug;116(3):461-70.
doi: 10.1007/s10549-008-0151-x. Epub 2008 Aug 3.

Characterization of the weak estrogen receptor alpha agonistic activity of exemestane

Selma Masri  1 , Ki Lui, Sheryl Phung, Jingjing Ye, Dujin Zhou, Xin Wang, Shiuan Chen
Affiliations

Characterization of the weak estrogen receptor alpha agonistic activity of exemestane

Selma Masri et al. Breast Cancer Res Treat. 2009 Aug.

Abstract

Third generation aromatase inhibitors (AI) have shown good clinical efficacy in comparison to the anti-estrogen tamoxifen. The steroidal AI, exemestane (EXE) has previously been shown to act as an androgen, but this report demonstrates the estrogen-like activity of EXE. Based on genome-wide microarray analysis, high correlation was seen between EXE-Only (EXE O, hormone-free) and hormone-containing AI-resistant lines. In addition, the top regulated genes in the EXE O lines were mostly estrogen-responsive genes. This estrogen-like activity of EXE was further validated using estrogen receptor (ER) activity assays, where in comparison to 17beta-estradiol (E2), EXE was able to induce ER activity, though at a higher concentration. Also, this EXE-mediated ER activity was blocked by the ER antagonist ICI as well as the ERalpha-specific antagonist methyl-piperidino-pyrazole (MPP). Similarly, EXE was able to induce proliferation of breast cancer cell lines, MCF-7 and MCF-7aro, as well as activate transcription of known estrogen-responsive genes, i.e., PGR, pS2 and AREG. These results suggest that EXE does have weak estrogen-like activity.

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Figures

Fig. 1
Fig. 1
Similarity matrix of resistant cell lines. (a) EXE and ANA-resistant lines, as well as T-Only (T) and LTEDaro controls, were normalized with parental MCF-7aro and the correlation coefficients of 5007 significant genes were displayed as a similarity matrix, using Pearson's correlation. Significant genes were selected based on a fold-change criteria of ±1.2-fold and a P-value < 0.05. Correlation coefficients ranged between 0.5 to 0.98, with red indicating good correlation and green representing less correlated lines. Additionally, correlation coefficients were displayed graphically using Partek Genomics Suite for T + EXE R versus EXE O (b) and T + ANA R versus ANA O (c). Correlation between T + EXE R and EXE O is 0.901017 and correlation between T+ANA R and ANA O lines is 0.703406
Fig. 2
Fig. 2
EXE-mediated ER activity in MCF-7 and MCF-7aro lines. (a) ER activity, measured by endogenous ER binding to a pGL3-ERE luciferase reporter, was performed in the MCF-7 and T47D cell lines, using DMSO as a vehicle control for E2 and EXE treatment. Luciferase activity was normalized with total protein levels and indicated as relative luciferase units (RLU). All samples were performed in triplicate with standard deviation shown. For statistical analysis, samples were compared to DMSO treated using Student's T-test, ** indicates P-value ≤ 0.01 and *** indicates statistical significance at a P-value ≤ 0.001. (b) EXE-mediated ER activity in the presence of the ER antagonist ICI, as well as the ERα-specific antagonist (MPP) and ERβ-selective antagonist (PHTPP). For statistical analysis, samples were compared to 1 μM EXE or DMSO control as shown using Student's T-test, ** indicates P-value ≤ 0.01 and *** indicates statistical significance at a P-value ≤ 0.001
Fig. 3
Fig. 3
Dose-response curves of T, E2 and EXE in MCF-7aro/ERE lines. Dose–response was determined for T, E2 and EXE in the MCF-7 aromatase and ERE stable luciferase reporter cell line, MCF-7aro/ERE. Cells were treated with T, E2 and EXE at indicated concentrations for 24 h. Luciferase substrate (Promega, Madison, WI) was mixed with cell lysate using a robotic injector and luciferase units were determined using the Victor V plate reader (Perkin Elmer, Waltham, MA). Luciferase activity was standardized based on the protein concentration and shown as relative luciferase units (RLU)
Fig. 4
Fig. 4
EXE-dependent functional analysis in MCF-7 and MCF-7aro lines. (a) Cell proliferation assays were performed in the MCF-7 and MCF-7aro cell lines using the CellTiter 96 aqueous one proliferation assay kit in 96-well plate format. After being cultured in hormone-deprived media for 2 days, 5000 cells were cultured for 4 days in the presence of DMSO vehicle control, E2 or EXE at indicated concentrations. All samples shown were performed in triplicate, with standard deviation shown. For statistical analysis, samples were compared to DMSO treated using Student's T-test, ** indicates P-value ≤ 0.01 and *** indicates statistical significance at a P-value ≤ 0.001. Quantitative real-time PCR was performed with E2 and EXE treated MCF-7aro cells. Cells were hormone depleted and treated with DMSO, E2 or EXE for 2 days prior to harvesting total RNA. Expression of estrogen-responsive genes, PGR (b) and pS2 (c), was normalized with the beta-actin housekeeping gene. All samples were performed in triplicate with standard deviation shown. For statistical analysis, samples were compared to DMSO treated using Student's T-test, ** indicates P-value ≤ 0.01 and *** indicates statistical significance at a P-value ≤ 0.001
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