Breast Cancer Res Treat (2015) 151:335–345 DOI 10.1007/s10549-015-3398-z PRECLINICAL STUDY AroER tri-screenTM is a novel functional assay to estimate both estrogenic and estrogen precursor activity of chemicals or biological specimens 1 1 1 1 Noriko Kanaya • Duc M. Nguyen • Hannah Lu • Yuan-Zhong Wang • 1 2 3 2 3 Li-Yu Hsin • Myrto Petreas • David Nelson • Weihong Guo • Peggy Reynolds • 1 1 Tim Synold • Shiuan Chen Received: 16 April 2015 / Accepted: 18 April 2015 / Published online: 12 May 2015 Ó Springer Science+Business Media New York 2015 Abstract The purpose of the study is to define AroER tri- estrogenic activities in biological samples as a potential screen’s utility for identifying endocrine-disrupting che- intermediate risk factor for breast cancer. micals (EDCs) that target aromatase and/or estrogen re- ceptor (ER), and to measure the total estrogenic activity in Keywords AroER tri-screen Á Endocrine-disrupting biological specimens. ER-positive, aromatase-expressing chemicals (EDCs) Á Aromatase Á Estrogenic MCF-7 breast cancer cells were stably transfected with an estrogen responsive element (ERE)-driven luciferase re- porter plasmid to yield a new high-throughput screening Introduction platform—the AroER tri-screen. AroER tri-screen was capable of identifying estrogen precursors, such as corto- By interfering with hormone biosynthesis, metabolism, or doxone, which function as estrogens through a two-step action, EDCs can cause deviations in both normal conversion process in aromatase-expressing tissue. Fur- homeostatic control and reproduction [1]. Through multi- thermore, the system proved useful for assessing EDC ple mechanisms, EDC exposure (during development or in activity in biologically relevant samples. Estimating these later life) may perturb the physiological actions of hor- activities is critical because natural estrogens and estro- mones [2]. We are exposed to thousands of chemicals in genic EDCs are important factors in ER-positive breast food, water, and the general environment every day; and cancer risk. As our research demonstrates, incorporating some of them can act as EDCs. Therefore, high-throughput functionally active aromatase into the AroER tri-screen screening systems must be established to identify EDCs in produces a powerful and unique tool to (1) identify new these locales. With this in mind, we developed the AroER EDCs targeting aromatase and/or ER; (2) discover novel tri-screen assay: estrogen receptor (ER)-positive, aro- EDCs activated by aromatase; and (3) estimate overall matase-expressing MCF-7 breast cancer cells were stably transfected with an estrogen responsive element (ERE)- driven luciferase reporter plasmid. The AroER tri-screen Electronic supplementary material The online version of this can identify several types of EDCs: estrogenic ones, which article (doi:10.1007/s10549-015-3398-z) contains supplementary material, which is available to authorized users. increase luciferase signal without 17b-estradiol (E2); antiestrogenic EDCs, which inhibit the E2-induced lu- & Shiuan Chen ciferase signal; and aromatase inhibitor (AI)-like EDCs, [email protected] which suppress a testosterone (T)-induced luciferase sig- nal. This high quality assay has an excellent signal-to- 1 Department of Cancer Biology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, background ratio (6.9-fold); a low coefficient of variation CA 91010, USA (5.4 %); and an impressive screening window coefficient 2 Environmental Chemistry Branch, California Department of (Z-factor of 0.78) [3]. The technique was first optimized in Toxic Substances Control, Sacramento, CA 94710, USA a 96-well plate format; it was then miniaturized into a 3 The Cancer Prevention Institute of California, Fremont, 1536-well plate format to screen both the NIH Clinical CA 94538, USA Collection [Evotec (US) Inc, San Francisco, CA] and the 123 336 Breast Cancer Res Treat (2015) 151:335–345 Tox-21 10 K library [4]. The Food and Drug Administra- can assess both estrogenic activity and (aromatizable) tion (FDA) announced the availability of a draft guidance estrogen precursor activity simultaneously in tested biolo- for industry entitled ‘‘Endocrine Disruption Potential of gical samples. As such, we demonstrate the extended utility Drugs: Nonclinical Evaluation’’ on September 20, 2013. As of the AroER tri-screen: the presence of functionally active stated in this draft guidance, additional studies are needed aromatase makes it a unique tool to identify novel EDCs to characterize potential endocrine-disrupting properties of targeting aromatase and/or ER; it is also a versatile method currently used drugs. AroER tri-screen identified novel to estimate the overall activity of female hormones in EDCs from 446 drugs in the NIH Clinical Collection, biological samples, which can be correlated to risks asso- which included FDA-approved drugs [3]. Since these drugs ciated with developing breast cancer. (with hitherto unknown estrogen-disrupting function) are currently used in the clinic or serve as precursors for the development of new drugs, people can be exposed to their Materials and methods endocrine-disrupting activities. In this regard, in vitro high- throughput screening of EDCs can help inform decisions Samples about both drug development and treatment, provided the screen is highly reproducible and renders a minimal num- Whole blood was centrifuged and serum was aliquoted and ber of false-positives (for example through rescreening and frozen at -80 °C until analysis. The study protocol was validation) [5]. Herein, we disclose positive-hit chemicals approved by the Committee for the Protection of Human identified by the AroER tri-screen and show novel Subjects (CPHS). mechanistic actions of chemicals as estrogenic compounds. Luciferase assay In most cases during an EDC exposure event, multiple compounds are simultaneously encountered (i.e., most Prior to the assay, AroER tri-screen cells were cultured materials are mixtures). In this regard, the effects of dif- in phenol red-free MEM containing 10 % charcoal- ferent classes of EDCs may be additive or even synergistic treated FBS for 2 days and then seeded into 96-well [1]. In biological samples, direct analytical chemistry ap- plates at 2 9 105 cells per well. After 24 h, the cells proaches can (with high sensitivity) quantify levels of were individually treated with the extract or compound known EDCs. However, using this approach, contributions alone and/or with ICI (100 nM) or Let (200 nM) in a from compounds not typically considered EDCs cannot be total volume of 100 lL. After an overnight incubation at anticipated and included in the analysis. Furthermore, the 37 °C, 100 lL of 1X One-Glo reagent (Promega Cor- combined activities of a mixture of EDCs can be over- poration) was added to each well and incubated for looked. To compensate for these disadvantages, a func- 5 min. Luciferase signal was measured using SpectraMax tional assay must be used to determine the sample’s overall M5 microplate reader (Molecular Devices, Sunnyvale, biological activity. Using mammalian cells transfected CA) and normalized by protein concentration. Each as- with ERE-luciferase reporter vector, conventional ER say was performed in triplicate. For the transient trans- bioassays have been used to detect bioactiviy in human fection experiment using C4-12/ERa or C4-12/ERb samples [6–9]. Especially for breast cancer studies in stable cell line, cells were seeded into 24-well plates at postmenopausal women, ER bioassays can predict an in- 1 9 105 cells per well. After 24 h of seeding, the cells creased risk of postmenopausal breast cancer indepen- were individually transfected with pGL4.26 [luc2/minP/ dently from hormone levels [10–12]. In this study, we Hygro] (ERE) plasmid [3]. An additional 24 h later, demonstrated directly that the detectable estrogenic activity 3 cells were treated with each compound. After an over- in tested human serum samples correlated with the sam- night incubation at 37 °C, cells were lysed with 200 lL ple’s ability to promote the proliferation of breast cancer of passive lysis buffer. 20 lL of supernatant was mixed cell line MCF-7aro. Furthermore, by employing both in- with 20 lL of luciferase reagent (Promega Corporation). hibitors [ICI 182,780 (ICI) and Letrozole (Let)] and The luciferase signal was measured using a TD-20/20 estrogenic chemicals (including both natural and synthetic luminometer (Turner designs, Sunnyvale, CA) and nor- chemicals) during the assay, we can also define the action malized by protein concentration. Each assay was per- of the active constituents. To isolate compounds for this formed in triplicate. assay, steroid hormones and potential EDCs are extracted using a C-18 solid-phase extraction (SPE) column; they are Reagents and cell culture then subjected to our AroER tri-screen procedure. Below, Generation of the C4-12/ERa and C4-12/ERb cells we present data indicating that the unique AroER tri-screen Extraction by solid-phase extraction (SPE) column 123 Breast Cancer Res Treat (2015) 151:335–345 337 Cell proliferation assay Cortodoxone functionally acting like estrogens Liquid chromatography and mass spectrometry through a two-step conversion process Please see supplementary materials for procedures. Among these drugs, cortodoxone, ethylestrenol, and mes- tanolone maximized luciferase activity at 10 lM. While Statistical analysis Let should not affect the activities of ER agonists, corto- doxone activity was nevertheless inhibited by Let. A report Data are presented as mean ± SD. One-way analysis of by Azueby et al. suggests that cortodoxone can be con- variance (ANOVA) with Dunnett’s post hoc test was used verted into androstenedione by an uncharacterized enzyme to determine significant difference in human sample ana- [14]. To account for our observed sensitivity of cortodox- lysis. Unpaired t tests were used for the rest of the analysis. one to Let, we hypothesize that cortodoxone (through the We used GraphPad Prism 6.01 (GraphPad Software Inc., unidentified enzyme) is first converted to androstenedione; San Diego, CA) to perform statistical analysis.