1526 Classification of anti-estrogens according to intramolecular FRET effects on phospho-mutants of estrogen receptor A Wilbert Zwart, Alexander Griekspoor, Introduction Mariska Rondaij, Desiree Verwoerd, Three quarter of breast cancer patients have estrogen Jacques Neefjes, and Rob Michalides receptor (ER)–positive disease and are commonly treated with anti-estrogen tamoxifen. Despite being a successful Department of Tumor Biology, the Netherlands Cancer Institute, drug, almost 50% reduction in recurrence during 10 years Amsterdam, the Netherlands of follow-up of ER-positive patients and a reduction in mortality by a third, still, a substantial proportion of breast Abstract cancer patients who are treated with tamoxifen develop a Anti-estrogen resistance is a major clinical problem in the relapse and are to be treated with different anti-estrogens treatment of breast cancer. In this study, fluorescence and/or aromatase inhibitors (1–3). Early diagnosis of anti- resonance energy transfer (FRET) analysis, a rapid and estrogen resistance could therefore lead to a proper patient direct way to monitor conformational changes of estrogen selection for adequate therapy. receptor A (ERA) upon anti-estrogen binding, was used to A lead to early diagnosis of resistance to anti-estrogens is characterize resistance to anti-estrogens. Nine different provided by the molecular mechanism of resistance to anti- anti-estrogens all induced a rapid FRET response within estrogens. Anti-estrogens that bind the receptor inhibit its minutes after the compounds have liganded to ERA in live activity by modulating transactivation capacities of either cells, corresponding to an inactive conformation of the the NH2-terminally located AF-1 and/or AF-2 at the COOH a a ERA. Phosphorylation of Ser305 and/or Ser236 of ERA by terminus of ER (4). The most carboxyl-terminal -helix protein kinase A (PKA) and of Ser118 by mitogen-activated (H12) of the ER-ligand binding domain (ER-LBD) acts as a protein kinase (MAPK) influenced the FRET response dif- molecular switch for transactivation to occur. Its orientation ferently for the various anti-estrogens. PKA and MAPK are determines the transcriptional readout of the receptor. both associated with resistance to anti-estrogens in breast Binding of the different anti-estrogens to the LBDreorients cancer patients. Their respective actions can result in H12 and conceals the coactivator-binding groove that a seven different combinations of phospho-modifications in consists of a pocket formed by -helices 3, 4, 5, and 12 ERA where the FRET effects of particular anti-estrogen(s) (5, 6). This distortion of H12 is not fixed, but occurs to are nullified. The FRET response provided information on various extents, depending on the side chain and polarity the activity of ERA under the various anti-estrogen con- of the anti-estrogen applied (7). The conformational state of a ditions as measured in a traditional reporter assay. ER can be measured using biophysical methods such as Tamoxifen and EM-652 were the most sensitive to kinase fluorescence resonance energy transfer (FRET; ref. 8). Using activities, whereas ICI-182,780 (Fulvestrant) and ICI- FRET, we have shown that anti-estrogens induce a 164,384 were the most stringent. The different responses conformational change that is overridden by phosphoryla- a of anti-estrogens to the various combinations of phospho- tion of particular target sites on ER , resulting in resistance modifications in ERA elucidate why certain anti-estrogens to that anti-estrogen (9). For instance, resistance to 305 a are more prone than others to develop resistance. These tamoxifen is caused by phosphorylation of Ser of ER data provide new insights into the mechanism of action by protein kinase A (PKA). Tamoxifen binds but then a of anti-hormones and are critical for selection of the fails to induce the inactive conformation, invoking ER - correct individual patient-based endocrine therapy in dependent transactivation instead. PKA activity thus breast cancer. [Mol Cancer Ther 2007;6(5):1526–33] induces a switch from antagonistic to agonistic effects of tamoxifen on ERa. In a retrospective clinical study, we confirmed that an elevated PKA level is associated with tamoxifen resistance in ER-positive breast cancer (9). Ser305 is also the target of p21-activated kinase, PAK-1 (10), and Received 12/5/06; revised 2/21/07; accepted 3/27/07. overexpression of PAK-1 is in a similar way associated with Grant support: Dutch Cancer Organization, Koningin Wilhelmina Fonds resistance to tamoxifen (11). In addition, resistance to anti- 2005-3388. estrogens is also associated with modification of ERa by The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked mitogen-activated protein kinase (MAPK; refs. 12–14) and advertisement in accordance with 18 U.S.C. Section 1734 solely to by the expression levels and/or phosphorylation status of indicate this fact. cofactors such as SRC-1 (15) and SRC-3 (14, 16). Aberrant Requests for reprints: Rob Michalides, Division of Tumor Biology, the activation of other signaling pathways in ER-positive breast Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, the Netherlands. Phone: 31-20-5122022; Fax: 31-205122029. cancer cells will result in post-translational modification(s) E-mail: [email protected] on the ER that affect resistance to anti-estrogens. In this Copyright C 2007 American Association for Cancer Research. way, and of clinical relevance, resistance to two different doi:10.1158/1535-7163.MCT-06-0750 anti-estrogens used in the clinic, tamoxifen and ICI-182,780 Mol Cancer Ther 2007;6(5). May 2007 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Molecular Cancer Therapeutics 1527 (Fulvestrant), was distinguishable: resistance to tamoxifen 8-Br-cyclic AMP (cAMP) and analyzed by Western blotting was due to PKA-mediated phosphorylation of Ser305, using antibodies against GFP (23) or against phospho-S118- whereas resistance to ICI-182,780 (Fulvestrant) required ERa (Cell Signaling Technology) or against phospho-S305- additional overexpression of cofactors cyclin D1 and SRC-1. ERa (Upstate). Anti-estrogen characteristic requirements for resistance are Fluorescence Resonance Energy Transfer also foreseen by a different binding profile of randomly Before FRET experiments, cells on coverslips were generated peptides to ERa in the presence of various anti- mounted in bicarbonate-buffered saline [containing estrogens (17–19). Moreover, the three-dimensional struc- 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, tures of the LBDof ER a bound to different anti-estrogens 1mmol/LCaCl2, 23 mmol/L NaHCO3,10mmol/L indicate anti-estrogen–specific distortions of ERa (20). glucose, and 10 mmol/L HEPES (pH, 7.2)] in a heated j In the present study, we investigated the requirements for tissue culture chamber at 37 C under 5% CO2. Cells were resistance to nine different anti-estrogens using a FRET analyzed on an inverted Zeiss Axiovert 135 microscope  approach and related these to consecutive ER transactiva- equipped with a dry Achroplan 63 objective. FRET equip- tion events. This led to seven different combinations of ment was as described previously (24). Cyan fluorescent phospho-modifications in ERa, each of which is associated protein (CFP) was excited at 432 F 5 nm, and emission of with a FRET-related resistance to particular anti-estrogen(s). YFP was detected at 527 nm and CFP at 478 nm. FRET was This anti-estrogen–specific profile reveals a mechanism expressed as the ratio of YFP to CFP signals. The ratio was for anti-estrogen resistance and provides a molecular expla- arbitrarily set as 1.0 at the onset of the experiment. Changes nation for the outcome of anti-estrogen therapy. are expressed as percent deviation from this initial value of 1.0. For data acquisition, Felix software (PTI Inc.) was used. Experimental Procedures Data were plotted using proFit (QuantumSoft). ERE-Luciferase Reporter Assays Cell Culture and Transfection Luciferase assays were done as described previously (25). Human osteosarcoma U2OS cells were cultured in DMEM 4 In short, 8  10 U2OS cells were plated in a 24-well plate in the presence of 10% FCS and standard antibiotics. U2OS a culture dish and cultured overnight in CTS, after which cells containing ER constructs were cultured in phenol cells were transfected with 10 ng of pcDNA3-YFP-ER-CFP red–free DMEM containing 5% charcoal-treated serum or mutants, 0.2 Ag ERE-tk-Firefly luciferase (25) and 1 ng of (CTS, Hyclone) 48 h before analysis. For the FRET experi- SV40 Renilla luciferase construct using PEI. Directly after À À ments, cells were cultured overnight on 2-cm round glass transfection, 10 8 mol/L estrogen or 10 7 mol/L anti- coverslips. Twenty-four hours before analysis, cells were estrogen was added to the cells that were cultured for a transfected with pcDNA3-YFP-ER -CFP or mutants using 48 h before harvesting. Membrane-permeable 8-Br-cAMP polyethylenimine (PEI, Mw 25 kDa, Polysciences; ref. 21). (30) was present during the last 16 h at a final concentra- Estradiol (Sigma), 4-OH-tamoxifen (Sigma), raloxifene (Sig- tion of 0.1 mmol/L. ma), EM-652 (kindly provided by Dr. C. Labrie, University of Quebec, Canada), toremifene (Schering), arzoxifene, laso- foxifene, ICI-164,384 (the last three kindly provided by Results Organon, Oss, the Netherlands), GW7604, the active form of Characterization of the Phospho-Variants of ERA GW5638 (ref. 22; kindly provided by GlaxoSmithKline), or In this study we used phospho-mutants of ERa that were ICI-182,780 (Tocris) were added at the concentrations characterized by Western blotting using phospho-ERa– indicated. Forskolin (Sigma) was added 15 min before specific antibodies (Fig. 1B). U2OS cells containing either À5 measurements at a final concentration of 10 mol/L.