title: Development of a novel molecular sensor for imaging estrogen receptor-coactivator protein-protein interactions.
abstract: Anti-estrogens, in particular tissue selective anti-estrogens, have been the bedrock of adjuvant therapy for patients with estrogen receptor alpha (ERα) positive breast cancer. Though current therapies have greatly enhanced patient prognosis, there continues to be an impetus for the development of improved anti-estrogens. ERα is a nuclear receptor transcription factor which activates gene expression through the recruitment of transcriptional coactivator proteins. The SRC family of coactivators, which includes AIB1, has been shown to be of particular importance for ERα mediated transcription. ERα-AIB1 interactions are indicative of gene expression and are inhibited by anti-estrogen treatment. We have exploited the interaction between ERα and AIB1 as a novel method for imaging ERα activity using a split luciferase molecular sensor. By producing a range of ERα ligand binding domain (ER-LBD) and AIB1 nuclear receptor interacting domain (AIB-RID) N- and C-terminal firefly luciferase fragment fusion proteins, constructs which exhibited more than a 10-fold increase in luciferase activity with E2 stimulation were identified. The specificity of the E2-stimulated luciferase activity to ERα-AIB1 interaction was validated through Y537S and L539/540A ER-LBD fusion protein mutants. The primed nature of the split luciferase assay allowed changes in ERα activity, with respect to the protein-protein interactions preceding transcription, to be assessed soon after drug treatment. The novel assay split luciferase detailed in this report enabled modulation of ERα activity to be sensitively imaged in vitro and in living subjects and potentially holds much promise for imaging the efficacy of novel ERα specific therapies.
authors.affiliation: Comprehensive Cancer Imaging Centre, Imperial College London, London, United Kingdom.
authors.forename: Madryn C
authors.initials: MC
authors.lastname: Lake
authors.forename: Quang-Dé
authors.initials: QD
authors.lastname: Nguyen
authors.forename: Simak
authors.initials: S
authors.lastname: Ali
authors.forename: Eric O
authors.initials: EO
authors.lastname: Aboagye
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D020847:Estrogen Receptor Modulators / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology; D005260:Female; D000077267:Fulvestrant; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation / Q000235:genetics; D056921:Nuclear Receptor Coactivator 3 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors
chemical_list: C506487:ESR1 protein, human; D020847:Estrogen Receptor Modulators; D047628:Estrogen Receptor alpha; D004967:Estrogens; D011993:Recombinant Fusion Proteins; D013629:Tamoxifen; C016601:afimoxifene; D000077267:Fulvestrant; D004958:Estradiol; D008156:Luciferases; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3