Discovery of Potent, Selective, and Orally Bioavailable Estrogen-Related Receptor-γ Inverse Agonists To Restore the Sodium Iodide Symporter Function in Anaplastic Thyroid Cancer.

An inverse agonist of estrogen-related receptor-γ (ERRγ), an orphan nuclear receptor encoded by E srrg, enhances sodium iodide symporter-mediated radioiodine uptake in anaplastic thyroid cancer (ATC) cells, thereby facilitating responsiveness to radioiodine therapy in vitro. We synthesized potent, selective, and orally bioavailable ERRγ-inverse agonists and evaluated their activity by analyzing in vitro pharmacology and absorption, distribution, metabolism, excretion, and toxicity profiles. X-ray crystallographic analysis of the ligand and ERRγ complex showed that 35 completely binds to the target protein (PDB 6A6K ). Our results showed improved radioiodine avidity in ATC cells through compound 35-mediated upregulation of iodide-handling genes, leading to enhanced responsiveness to radioiodine therapy in vitro. Importantly, in vivo 124I-positron emission tomography/computed tomography imaging revealed that 35 increases radioiodine avidity in CAL62 tumors. Collectively, these results demonstrated that 35 can be developed as a promising treatment for ERRγ-related cancer in the future.

Activation function-1 domain of estrogen receptor regulates the agonistic and antagonistic actions of tamoxifen.

The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.

Molecular changes associated with the agonist activity of hydroxy-tamoxifen and the hyper-response to estradiol in hydroxy-tamoxifen-resistant breast cancer cell lines.

The aim of this study was to explore the pharmacological response to 4-hydroxy-tamoxifen (OH-Tam) and to estradiol (E2) in three cell lines: MVLN, a human breast carcinoma cell line derived from MCF-7, and two MVLN-derived OH-Tam-resistant (OTR) cell lines, called CL6.8 and CL6.32. The OH-Tam response in the OTR cells was associated with the development of both an agonist activity of the drug on cell proliferation and the resistance of the cells to OH-Tam-induced apoptosis. The OTR cells also developed an increased sensitivity to the E2 growth-stimulating activity. To delineate the genes that determine such responses, we combined a mini-array-based gene-selection approach and an extensive real-time quantitative PCR exploration in the MVLN and OTR cell lines exposed to three pharmacological conditions: a 4-day treatment with E2, OH-Tam or both E2 and OH-Tam. Compiled data revealed a hyper-response to E2 and a modification of the OH-Tam pharmacological response (loss of antagonist action and agonist activity) at the gene-expression level. The proteins encoded by the genes selected in this study have been reported to be involved in the regulation of cell proliferation, cell transformation, DNA repair and apoptosis, or belong to the ErbB/epidermal growth factor receptor-driven pathway. Our data also provide evidence of changes in transcriptional co-regulator expression, elevated mitogen-activated protein kinase activity and increase in the phosphorylation status of estrogen receptor alpha on serine residue 118 in the OTR cell lines, suggesting the possible involvement of such mechanisms in the agonist activity of OH-Tam and/or the hyper-response of cells to E2. Taken together, our study should enhance our knowledge of the multifactorial events associated with the development of Tam resistance in two independent cell lines issued from the same selection process and should help in the identification of potential molecular targets for diagnosis or therapy.

The Src kinase pathway promotes tamoxifen agonist action in Ishikawa endometrial cells through phosphorylation-dependent stabilization of estrogen receptor (alpha) promoter interaction and elevated steroid receptor coactivator 1 activity.

Tamoxifen is the most widely used selective estrogen receptor modulator for breast cancer in clinical use today. However, tamoxifen agonist action in endometrium remains a major hurdle for tamoxifen therapy. Activation of the nonreceptor tyrosine kinase src promotes tamoxifen agonist action, although the mechanisms remain unclear. To examine these mechanisms, the effect of src kinase on estrogen and tamoxifen signaling in tamoxifen-resistant Ishikawa endometrial adenocarcinoma cells was assessed. A novel connection was identified between src kinase and serine 167 phosphorylation in estrogen receptor (ER)-alpha via activation of AKT kinase. Serine 167 phosphorylation stabilized ER interaction with endogenous ER-dependent promoters. Src kinase exhibited the additional function of potentiating the transcriptional activity of Gal-steroid receptor coactivator 1 (SRC-1) and Gal-cAMP response element binding protein-binding protein in endometrial cancer cells while having no effect on Gal-p300-associated factor and Gal fusions of the other p160 coactivators glucocorticoid-interacting protein 1 (transcriptional intermediary factor 2/nuclear coactivator-2/SRC-2) and amplified in breast cancer 1 (receptor-associated coactivator 3/activator of transcription of nuclear receptor/SRC-3). Src effects on ER phosphorylation and SRC-1 activity both contributed to tamoxifen agonist action on ER-dependent gene expression in Ishikawa cells. Taken together, these data demonstrate that src kinase potentiates tamoxifen agonist action through serine 167-dependent stabilization of ER promoter interaction and through elevation of SRC-1 and cAMP response element binding protein-binding protein coactivation of ER.

SRA coactivation of estrogen receptor-alpha is phosphorylation-independent, and enhances 4-hydroxytamoxifen agonist activity.

The ability of steroid receptor RNA activator (SRA), an AF-1 coactivator, to contribute to differences in estrogen receptor (ER)-alpha and ERbeta transcriptional activity was tested. In transient transfections, SRA expression increased ERalpha- and ERbeta-dependent gene expression. However, when the receptors' amino-terminal A/B regions were examined as GAL4 DNA binding domain fusions, SRA enhanced the activity of GAL-ABalpha but not GAL-ABbeta. Exogenous SRA also enhanced AF-2 activity for both receptors, indicating that SRA effects are not limited to AF-1. Simultaneously mutating three phosphorylation sites within GAL-ABalpha domain only modestly reduced SRA coactivation of GAL-ABalpha, suggesting that phosphorylation does not play a major role in SRA function relative to this domain. SRA enhanced ERalpha activity stimulated by 4-hydroxytamoxifen, but was unable to convert this mixed antiestrogen to an ERbeta agonist. Thus, SRA is an ERalpha AF-1-specific coactivator that enhances the agonist activity of tamoxifen-bound ERalpha and may contribute to tamoxifen resistance.

Phase III randomized trial of droloxifene and tamoxifen as first-line endocrine treatment of ER/PgR-positive advanced breast cancer.

PURPOSE: This trial was designed to demonstrate equivalence between droloxifene 40 mg/d and tamoxifen 20 mg/d as first-line treatment in pre- and post-menopausal women with ER+ and/or PgR+ advanced breast cancer based on time to disease progression and tumor response. MATERIALS AND METHODS: One thousand three hundred fifty four women with measurable disease, previously untreated by hormonal or chemotherapy for advanced or recurrent breast cancer, were enrolled by 179 institutions in 35 countries. Patients were stratified at baseline for menopausal status. Patients receiving adjuvant hormonal therapy within I year were excluded. All patients gave written informed consent, were randomized to 40mg droloxifene or 20 mg tamoxifen daily as single-agent therapy and underwent tumor assessment every 3 months. A central committee reviewed digitized images for all cases of tumor progression or objective response. RESULTS: The hazard ratio (droloxifene/tamoxifen) for the primary endpoint, time to disease progression, was 1.287 favoring tamoxifen (95% C.I.: 1.114-1.487; p <.001). The objective response rate (CR+PR) was 22.4% for droloxifene and 28.6% for tamoxifen (p = .02). Tamoxifen was superior to droloxifene overall, among both pre- and postmenopausal patients and among patients < or =65 years; there was no difference among women >65 years. The hazard ratio for all-cause mortality was 0.871 (95% C.I.: 0.672-1.129; p = .29), favoring droloxifene but not statistically significant. CONCLUSIONS: Droloxifene was significantly less effective than tamoxifen overall and particularly among women under 65 years. Tamoxifen and droloxifene were both less effective in pre-menopausal women with receptor-positive disease compared to post-menopausal women. Further clinical development of droloxifene was stopped.