Geoffrey L. Greene, Ph.D.

Mechanism of Action of Female Steroid Hormones and Nuclear Receptors; Development and Characterization of Novel SERMs.

Research Summary

Mechanism of action of female steroid hormones and nuclear receptors. Development and Characterization of novel SERMs.

The overall goal of my research is to determine the molecular mechanisms by which female steroid hormones regulate development, differentiation and/or cellular proliferation and survival in hormone responsive tissues and cancers. Our primary model system is cultured human breast cancer cells, in which estrogens are mitogens and in which the expression or activities of diverse enzymes, regulatory proteins and growth factors are regulated by estrogens via one or both of the two human estrogen receptor subtypes (ERa & ERb). We are actively studying several aspects of steroid receptor action, including the role of phosphorylation in transcriptional activation of ER, the roles of ER-associated proteins in receptor-mediated responses, the molecular nature of transcriptional activation and/or repression in the regulation of target gene expression, nongenomic actions of estrogens and the detailed structural requirements for ligand binding in the estrogen receptor, especially in regard to discrimination between estrogen agonists and antagonists (SERMs). We have also used RNA display and gene array techniques to identify genes that are differentially suppressed or induced by SERMs in breast cancer cells. Monocyte chemoattractant protein-1 (MCP-1) was identified as an estrogen-suppressed gene by this approach. The current major focus of the lab is 1) to elucidate the molecular mechanisms by which SERMs elicit tissue-selective agonist or antagonist responses via one or both ER subtypes and 2) to create novel ER subtype-selective SERMs via a combination of structure-based drug design and de novo drug discovery.

3D crystallographic structural information for ERa and ERb ligand binding domains complexed with receptor-selective and activity-selective ligands is being determined both to understand and to design tissue- and ER subtype-selective estrogen receptor modulators (TSERMs). These complexes also include peptides that represent interaction domains for various co-activators, co-repressors or phage display-generated peptides to better understand the structural and molecular nature of ER/effector protein interactions in response to diverse natural and synthetic compounds. An additional interest is to develop a mouse model in which ERa is replaced with a mutant ERa that does not recognize endogenous estradiol but will respond normally to a synthetic estrogen such as DES. An appropriate gene-targeting construct has now been made and will be introduced into mouse ES cells to generate mice that express ligand-selective mutant ERa. This model should prove useful for studying estrogen-regulated development of the reproductive tract, bone, cardiovasculature and CNS, and will also be used for studying the genesis and progression of hormone dependent mammary cancers. We are also trying to determine the role of ERa and several coactivators as suppressors of NF-kB induced cytokine responses, especially MCP-1 recruitment of macrophages in tumors, bone and atherosclerotic lesions. Progress has been made determining the underlying molecular mechanisms for this anti-inflammatory activity of estrogens. An additional project involves the development of monoclonal antibodies to ERb that can be used to assess the prognostic and therapeutic value of ERb expression in breast tumors as well as in the progression of normal breast epithelium to cancer. All of these projects have direct relevance and application to breast and uterine cancer genesis, progression, treatment and prevention, as well as to the development of compounds that can be used for hormone replacement therapy in postmenopausal women.

Technical description of Recent Progress with ERa/b Structure Determinations

Insight into the molecular basis of estrogen agonism and antagonism was revealed by the crystal structures of ERa and ERb ligand binding domains (LBDs) complexed with several ligands, including estradiol (E2), diethylstilbestrol (DES), raloxifene (RAL), 4-hydroxy-tamoxifen (OHT), and genistein (GEN). For agonists like DES, inclusion of a peptide derived from an essential LXXLL interaction motif (NR box) found in several related p160 nuclear receptor transcriptional co-activators helped define the AF-2/co-activator interface. Although agonists and antagonists bind at the same site within the core of the LBD, each induces distinct conformations in the transactivation domain (AF-2) of the LBD, especially in the positioning of helix 12, providing structural evidence for multiple mechanisms of selective antagonism in the nuclear receptor family. Interestingly, the OHT/RAL and DES/E2 structures collectively reveal and define a multipurpose docking site on ERa that can accommodate either helix 12 or one of several coregulators. In addition, a comparison of the two structures reveals that there are at least two distinct mechanisms by which structural features of OHT promote an "autoinhibitory" helix 12 conformation. Helix 12 positioning can be determined both by steric considerations, such as the presence of an extended side chain in the ligand, and by local structural distortions in and around the ligand binding pocket. Thus, one would predict that effective estrogen antagonists do not necessarily require bulky or extended side chains, which has now been demonstrated by the development and characterization of the ER subtype-selective compound R,R-THC) described below.

As part of a search for ER subtype-selective ligands, the synthetic compound, R,R-5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (R,R-THC), was identified as a selective estrogen agonist when bound to ERa and as an antagonist when bound to ERb. To better understand this selective behavior, a major goal of our work was to determine the crystallographic structures of human ERa and ERb ligand binding domains (LBDs) complexed with R,R-THC. These structures have now been solved and refined, suggesting mechanisms by which this compound can act as an ERa agonist and as an ERb antagonist. Interestingly, R,R-THC stabilizes a conformation of the ERa LBD that favors coactivator association and a conformation of the ERb LBD that prevents coactivator association. A comparison of the two structures, combined with functional data, reveals that R,R-THC does not act on ERb through the same mechanisms used by other known ER antagonists that have bulky or extended side chains. Instead, R,R-THC antagonizes ERb through a novel mechanism we term “passive antagonism”. Paradoxically, the R,R-THC-ERb structure is very similar to the structure induced by genistein, which acts as a partial estrogen through both ER subtypes. Ongoing mutagenesis studies should help define the molecular and structural differences that are responsible for these unanticipated results. In addition, the passive antagonism mechanism suggests a novel approach to the design of ligands that selectively antagonize the two ER subtypes. Such ligands may have novel therapeutic properties that can be exploited to prevent or treat breast cancer.

Selected Papers

Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA and Carlquist M. (1997). Molecular basis of agonism and antagonism in the oestrogen receptor. Nature, 389(6652): p. 753-8.

Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA and Greene GL. (1998). The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell, 95(7): p. 927-37.

Razandi M, Pedram A, Greene GL and Levin ER. (1999). Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells. Mol Endocrinol, 13(2): p. 307-19.

Kushner PJ, Agard D, Feng WJ, Lopez G, Schiau A, Uht R, Webb P and Greene G. (2000). Oestrogen receptor function at classical and alternative response elements. Novartis Found Symp, 230: p. 20-6; discussion 27-40.

Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM and Webb P. (2000). Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol, 74(5): p. 311-7.

Griffin C, Flouriot G, Sharp P, Greene G and Gannon F. (2001). Distribution analysis of the two chicken estrogen receptor-alpha isoforms and their transcripts in the hypothalamus and anterior pituitary gland. Biol Reprod, 65(4): p. 1156-63.

Benz CC, Hilakivi-Clarke L, Conzen S, Dorn RV, Fleming GF, Grant K, Greene G, Hellman S, Henderson C, Hoover R, Hryniuk W, Jeffrey S, Lippman M, Lung J, Mitchell M and Pike M. (2001). Expedition inspiration consensus 2001. Breast Cancer Res Treat, 70(3): p. 213-9.

Janulis M, Trakul N, Greene G, Schaefer EM, Lee JD and Rosner MR. (2001). A novel mitogen-activated protein kinase is responsive to Raf and mediates growth factor specificity. Mol Cell Biol, 21(6): p. 2235-47.

Shiau AK, Barstad D, Radek JT, Meyers MJ, Nettles kW, Katzenellenbogen BS, Katzenellenbogen JA, Agard DA and Greene GL. (2002). Structural characterization of a subtype-selective ligand reveals a novel mode of estrogen receptor antagonism. Nat Struct Biol, 9(5): p. 359-64.

Benz C, Clark G, Conzen S, Dorn R, Fuqua S, Gralow J, Greene G, Heimann R, Hellman S, Lippman M, Rosen N and Weiner L. (2003). Consensus statement: Expedition Inspiration fund for breast cancer research meeting 2002. Breast Cancer Res Treat, 78(1): p. 127-31.

Greene GL. (2003). In vivo imaging reveals estrogen receptor's hidden personality. Nat Med, 9(1): p. 22-3.

Nettles KW and Greene GL. (2003). Nuclear receptor ligands and cofactor recruitment. Is there a coactivator "On Deck"? Mol Cell, 11(4): p. 850-1.