Rational discovery of novel type-III FTF antagonists to competitively suppress TIF-2 coactivation in liver cancer.

The α-fetoprotein transcription factor (FTF) is a member of the nuclear receptor NR5A subfamily, which is involved in the pathogenesis of liver cancer and some other gastrointestinal cancers. The protein's transcriptional activity is regulated by binding TIF-2 coactivator at its coactivator-interacting site (CIS); suppression of the transcriptional activity has been recognized as a potential therapeutic strategy against cancer. Previously, small-molecule antagonists have been developed to target the ligand-binding site (LBS) of FTF ligand-binding domain, which simply occupy the site to exclusively block natural ligand entry (type-I antagonists) or destabilize the agonist conformation of activation helix 12 of the domain (type-II antagonists). Here, we describe the use of small-molecule competitors (type-III antagonists) to directly disrupt FTF-TIF-2 interaction by competitively targeting FTF CIS site. High-throughput virtual screening is performed against a structurally diverse, commercially available compound library to identify FTF CIS binders as competitor candidates, from which 12 hits are manually selected and their competitive potency with TIF-2 core binding sequence for FTF CIS site is tested with CC50 values up to 2.5 µM. Structural modeling analysis revealed that the competitive ligands can form a complicated network of noncovalent interactions to specifically or nonspecifically pack against FTF CIS site, thus preventing TIF-2 from binding to the site.

Steroid Receptor Coactivator-2 Controls the Pentose Phosphate Pathway through RPIA in Human Endometrial Cancer Cells.

Steroid receptor coactivator-2 (SRC-2) is a transcriptional coregulator that modulates the activity of many transcription factors. Levels of SRC-2 are elevated in endometrial biopsies from polycystic ovary syndrome patients, a population predisposed to endometrial cancer (EC). Increased expression of SRC-2 is also detected in neoplastic endometrium suggesting a causal link between elevated SRC-2 expression and the emergence of endometrial disorders that can lead to cancer. Here, we reveal that SRC-2 knockdown reduces EC cell proliferation and anchorage-independence. Additionally, SRC-2 is required to maintain cellular glycolytic capacity and oxidative phosphorylation, processes essential for EC cell proliferation. Importantly, SRC-2 is critical for the normal performance of the pentose phosphate pathway (PPP). Perturbation of the PPP due to loss of SRC-2 expression may result from the depletion of ribose-5-P isomerase (RPIA), a key enzyme of the PPP. As with SRC-2, RPIA knockdown reduces EC cell proliferation, which is accompanied by a decrease in glycolytic capacity and oxidative phosphorylation. Glucose metabolite tracking experiments confirmed that knockdown of SRC-2 and RPIA downregulates the metabolic rate of both glycolysis and the PPP, highlighting a novel regulatory cross-talk between glycolysis and the PPP modulated by SRC-2.

High-Content Screening Campaign to Identify Compounds That Inhibit or Disrupt Androgen Receptor-Transcriptional Intermediary Factor 2 Protein-Protein Interactions for the Treatment of Prostate Cancer.

Twenty percent of prostate cancer (PCa) patients develop a noncurable drug-resistant form of the disease termed castration-resistant prostate cancer (CRPC). Overexpression of Androgen Receptor (AR) coactivators such as transcriptional intermediary factor 2 (TIF2) is associated with poor CRPC patient outcomes. We describe the implementation of the AR-TIF2 protein-protein interaction biosensor (PPIB) assay in a high-content screening (HCS) campaign of 143,535 compounds. The assay performed robustly and reproducibly and enabled us to identify compounds that inhibited dihydrotestosterone (DHT)-induced AR-TIF2 protein-protein interaction (PPI) formation or disrupted preexisting AR-TIF2 PPIs. We used multiparameter HCS data z-scores to identify and deprioritize cytotoxic or autofluorescent outliers and confirmed the resulting qualified actives in triplicate. None of the confirmed AR-TIF2 PPIB inhibitors/disruptors exhibited activity in a p53-hDM2 PPIB counter screen, indicating that they were unlikely to be either nonselective PPI inhibitors or to interfere with the biosensor assay format. However, eight confirmed AR-TIF2 PPIB actives also inhibited the glucocorticoid receptor (GR) nuclear translocation counter screen by >50%. These compounds were deprioritized because they either lacked AR specificity/selectivity, or they inhibited a shared component of the AR and GR signaling pathways. Twenty-nine confirmed AR-TIF2 PPIB actives also inhibited the AR nuclear localization counter screen, suggesting that they might indirectly inhibit the AR-TIF2 PPIB assay rather than directly blocking/disrupting PPIs. A total of 62.2% of the confirmed actives inhibited the DHT-induced AR-TIF2 PPI formation in a concentration-dependent manner with IC50s < 40 µM, and 59.4% also disrupted preexisting AR-TIF2 PPI complexes. Overall, the hit rate for the AR-TIF2 PPIB HCS campaign was 0.12%, and most hits inhibited AR-TIF2 PPI formation and disrupted preexisting AR-TIF2 complexes with similar AR-red fluorescent protein distribution phenotypes. Further secondary and tertiary hit characterization assays are underway to select AR-TIF2 PPI inhibitor/disruptor hits suitable for medicinal chemistry lead optimization and development into novel PCa/CRPC therapeutics.

Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization.

STUDY QUESTION: Is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) required for progesterone-driven endometrial stromal cell decidualization? SUMMARY ANSWER: GREB1 is a novel progesterone-responsive gene required for progesterone-driven human endometrial stromal cell (HESC) decidualization. WHAT IS KNOWN ALREADY: Successful establishment of pregnancy requires HESCs to transform from fibroblastic to epithelioid cells in a process called decidualization. This process depends on the hormone progesterone, but the molecular mechanisms by which it occurs have not been determined. STUDY DESIGN, SIZE, DURATION: Primary and transformed HESCs in which GREB1 expression was knocked down were decidualized in culture for up to 6 days. Wild-type and progesterone receptor (PR) knockout mice were treated with progesterone, and their uteri were assessed for levels of GREB1 expression. PARTICIPANTS/MATERIALS, SETTING, METHODS: Analysis of previous data included data mining of expression profile data sets and in silico transcription factor-binding analysis. Endometrial biopsies obtained from healthy women of reproductive age during the proliferative phase (Days 8-12) of their menstrual cycle were used for isolating HESCs. Experiments were carried out with early passage (no more than four passages) HESCs isolated from at least three subjects. Transcript levels of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were detected by quantitative RT-PCR as readouts for HESC decidualization. Cells were also imaged by phase-contrast microscopy. To assess the requirement for GREB1, PR and SRC-2, cells were transfected with specifically targeted small interfering RNAs. Results are shown as mean and SE from three replicates of one representative patient-derived primary endometrial cell line. Experiments were also conducted with transformed HESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Progesterone treatment of mice and transformed HESCs led to an ~5-fold (5.6 ± 0.81, P < 0.05, and 5.2 ± 0.26, P < 0.01, respectively) increase in GREB1 transcript levels. This increase was significantly reduced in the uteri of PR knock-out mice (P < 0.01), in HESCs treated with the PR antagonist RU486 (P < 0.01), or in HESCs in which PR expression was knocked down (P < 0.05). When GREB1 expression was knocked down, progesterone-driven decidualization markers in both immortalized and primary HESCs was significantly reduced (P < 0.05 and P < 0.01). Finally, GREB1 knock down signficantly reduced expression of the PR target genes WNT4 and FOXOA1 (P < 0.05 and P < 0.01, respectively). LARGE SCALE DATA: This study used the Nuclear Receptor Signaling Atlas. LIMITATIONS, REASONS FOR CAUTION: Although in vitro cell culture studies indicate that GREB1 is required for endoemtrial decidualization, the in vivo role of GREB1 in endometrial function and dysfunction should be assessed by using knock-out mouse models. WIDER IMPLICATIONS OF THE FINDINGS: Identification and functional analysis of GREB1 as a key molecular mediator of decidualization may lead to improved diagnosis and clinical management of women with peri-implantation loss due to inadequate endometrial decidualization. STUDY FUNDING AND COMPETING INTEREST(S): This research was funded in part by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O.M., and a NIH/NICHD grant (R01 HD-042311) to J.P.L. The authors declare no conflicts of interests.

Steroid receptor coactivators present a unique opportunity for drug development in hormone-dependent cancers.

Steroid receptor coactivators (SRCs) are essential regulators of nuclear hormone receptor function. SRCs coactivate transcription mediated by hormone stimulation of nuclear receptors and other transcription factors and have essential functions in human physiology and health. The SRCs are over expressed in a number of cancers such as breast, prostate, endometrial and pancreatic cancers where they promote tumor growth, invasion, metastasis and chemo-resistance. With their multiple roles in cancer, the SRCs are promising targets for the development of small molecule agents that can interfere with their function. For instance, perturbing SRC function with small molecule inhibitors and stimulators has been shown to be effective in reducing tumor growth in vivo. These early studies demonstrate that targeting the SRCs might prove effective for cancer treatment and more effort should be made to realize the untapped potential of developing drugs designed to target these coactivators.

The N-terminal domain of the androgen receptor drives its nuclear localization in castration-resistant prostate cancer cells.

Androgen-independent nuclear localization is required for androgen receptor (AR) transactivation in castration-resistant prostate cancer (CRPC) and should be a key step leading to castration resistance. However, mechanism(s) leading to androgen-independent AR nuclear localization are poorly understood. Since the N-terminal domain (NTD) of AR plays a role in transactivation under androgen-depleted conditions, we investigated the role of the NTD in AR nuclear localization in CRPC. Deletion mutagenesis was used to identify amino acid sequences in the NTD essential for its androgen-independent nuclear localization in C4-2, a widely used CRPC cell line. Deletion mutants of AR tagged with green fluorescent protein (GFP) at the 5'-end were generated and their signal distribution was investigated in C4-2 cells by fluorescent microscopy. Our results showed that the region of a.a. 294-556 was required for androgen-independent AR nuclear localization whereas a.a. 1-293 mediates Hsp90 regulation of AR nuclear localization in CRPC cells. Although the region of a.a. 294-556 does not contain a nuclear import signal, it was able to enhance DHT-induced import of the ligand binding domain (LBD). Also, transactivation of the NTD could be uncoupled from its modulation of AR nuclear localization in C4-2 cells. These observations suggest an important role of the NTD in AR intracellular trafficking and androgen-independent AR nuclear localization in CRPC cells.

Development of stapled short helical peptides capable of inhibiting vitamin D receptor (VDR)-coactivator interactions.

We synthesized stapled helical leucine-based peptides (DPI-01-07) containing 2-aminoisobutyric acid and a covalent cross-linked unit as inhibitors of vitamin D receptor (VDR)-coactivator interactions. The effects of these peptides on the human VDR were examined in an inhibition assay based on the receptor cofactor assay system, and one of them, DPI-07, exhibited potent inhibitory activity (IC50: 3.2 µM).

Anti-estrogenic activity of a human resveratrol metabolite.

BACKGROUND AND AIMS: Resveratrol, the most investigated dietary compound in studies aimed at linking wine consumption to human health, is an extremely minor component of this beverage and it is generally studied in vitro as the unconjugated aglycone at concentrations largely exceeding those found in the human circulatory system after dietary intake. Moreover, following intestinal absorption, trans-resveratrol and its glucoside, which are naturally present in wine and other food sources, are converted to sulphate and glucuronide metabolites. An estrogenic activity has previously been documented for resveratrol, yet nothing is known about the activity of its blood-circulating metabolic derivatives. METHODS AND RESULTS: Using a yeast two-hybrid detection system relying on the interaction between the ligand-binding domain of the human oestrogen receptors α and ß and the human coactivator Tif2, we have systematically examined the oestrogen agonist and antagonist activities of the two main resveratrol forms present in planta (trans-resveratrol and trans-resveratrol-3-O-glucoside) and of the three main metabolites found in human plasma (trans-resveratrol-3-O-sulphate, trans-resveratrol-3-O-glucuronide and trans-resveratrol-4'-O-glucuronide). Only resveratrol-3-O-sulphate was found to display a fairly strong and oestrogen receptor α-preferential antagonistic activity, which was confirmed in a human breast adenocarcinoma cell line containing a luciferase reporter gene under the control of an oestrogen-responsive promoter. CONCLUSIONS: We show, for the first time, that resveratrol-3-O-sulphate, but neither of its metabolites, is endowed with anti-estrogenic activity and how human metabolism of phenolic substances plays a pivotal role in modulating their biological effect.

Discovery of the first irreversible small molecule inhibitors of the interaction between the vitamin D receptor and coactivators.

The vitamin D receptor (VDR) is a nuclear hormone receptor that regulates cell proliferation, cell differentiation, and calcium homeostasis. The receptor is activated by vitamin D analogues that induce the disruption of VDR-corepressor binding and promote VDR-coactivator interactions. The interactions between VDR and coregulators are essential for VDR-mediated transcription. Small molecule inhibition of VDR-coregulator binding represents an alternative method to the traditional ligand-based approach in order to modulate the expression of VDR target genes. A high throughput fluorescence polarization screen that quantifies the inhibition of binding between VDR and a fluorescently labeled steroid receptor coactivator 2 peptide was applied to discover the new small molecule VDR-coactivator inhibitors, 3-indolylmethanamines. Structure-activity relationship studies with 3-indolylmethanamine analogues were used to determine their mode of VDR-binding and to produce the first VDR-selective and irreversible VDR-coactivator inhibitors with the ability to regulate the transcription of the human VDR target gene TRPV6.

A quantitative high-throughput screen identifies novel inhibitors of the interaction of thyroid receptor beta with a peptide of steroid receptor coactivator 2.

The thyroid hormone receptors (TR) are members of the nuclear hormone receptor (NHR) superfamily that regulate development, growth, and metabolism. Upon ligand binding, TR releases bound corepressors and recruits coactivators to modulate target gene expression. Steroid receptor coactivator 2 (SRC2) is an important coregulator that interacts with TRß to activate gene transcription. To identify novel inhibitors of the TRß and SRC2 interaction, the authors performed a quantitative high-throughput screen (qHTS) of a TRß-SRC2 fluorescence polarization assay against more than 290 000 small molecules. The qHTS assayed compounds at 6 concentrations up to 92 µM to generate titration-response curves and determine the potency and efficacy of all compounds. The qHTS data set enabled the characterization of actives for structure-activity relationships as well as for potential artifacts such as fluorescence interference. Selected qHTS actives were tested in the screening assay using fluoroprobes labeled with Texas Red or fluorescein. The retest identified 19 series and 4 singletons as active in both assays with 40% or greater efficacy, free of compound interference, and not toxic to mammalian cells. Selected compounds were tested as independent samples, and a methylsulfonylnitrobenzoate series inhibited the TRß-SRC2 interaction with 5 µM IC(50). This series represents a new class of thyroid hormone receptor-coactivator modulators.

A role for the transcription intermediary factor 2 in zebrafish myelopoiesis.

OBJECTIVE: TIF2 is fused with MOZ in the inv(8)(p11q13) acute myeloid leukemia. TIF2, member of the p160 family, is a histone acetyl transferase (HAT). Deletion of p160 genes were performed in mice. Some observations suggest that p160 family members may perform overlapping functions in mice. Therefore, we decided to choose the zebrafish model to study TIF2. The aim of this study was to characterize the role of this HAT during embryonic development. MATERIAL AND METHODS: We use antisense, morpholino-modified oligomers to transiently knockdown tif2 gene, thus determining whether TIF2 plays a role in zebrafish early development. RESULTS: We show that tif2 is involved in embryogenesis and in primitive hematopoiesis. tif2-knockdown zebrafish embryos are smaller than controls, they demonstrate shorter tails, they display notochord deformation and they exhibit U-shaped tail somites. A synthetic RNA encoding human TIF2 rescues the tif2-knockdown phenotype. Analysis of fli1 expression by whole-mount in situ hybridization indicates normal angioblast specification, but altered localization of intersomitic vessels. The posterior intermediate cell mass, in which a part of primitive hematopoiesis occurs, is altered in tif2 morphants and whole-mount in situ hybridization analyses of l-plastin and mpx expression suggest a specific inhibition of granulocytic and macrophagic differentiation at late stages. CONCLUSION: These data indicate an important role for TIF2 in zebrafish primitive myelopoiesis.

Differential recruitment of p160 coactivators by glucocorticoid receptor between Schwann cells and astrocytes.

In the nervous system, glucocorticoids can exert beneficial or noxious effects, depending on their concentration and the duration of hormonal stimulation. They exert their effects on neuronal and glial cells by means of their cognate receptor, the glucocorticoid receptor (GR), which recruits the p160 coactivator family members SRC-1 (steroid receptor coactivator 1), SRC-2, and SRC-3 after hormone binding. In this study, we investigated the molecular pathways used by the GR in cultured glial cells of the central and the peripheral nervous systems, astrocytes and Schwann cells (MSC80 cells), respectively. We performed functional studies based on transient transfection of a minimal glucocorticoid-sensitive reporter gene into the glial cells to test the influence of overexpression or selective inhibition by short interfering RNA of the three p160 coactivator family members on GR transactivation. We demonstrate that, depending on the glial cell type, GR differentially recruits p160 family members: in Schwann cells, GR recruited SRC-1a, SRC-1e, or SRC-3, whereas in astrocytes, SRC-1e and SRC-2, and to a lesser extent SRC-3, were active toward GR signaling. The C-terminal nuclear receptor-interacting domain of SRC-1a participates in its exclusion from the GR transcriptional complex in astrocytes. Immunolocalization experiments revealed a cell-specific intracellular distribution of the p160s, which was dependent on the duration of the hormonal induction. For example, within astrocytes, SRC-1 and SRC-2 were mainly nuclear, whereas SRC-3 unexpectedly localized to the lumen of the Golgi apparatus. In contrast, in Schwann cells, SRC-1 showed a nucleocytoplasmic shuttling depending on hormonal stimulation, whereas SRC-2 remained strictly nuclear and SRC-3 remained predominantly cytoplasmic. Altogether, these results highlight the cell specificity and the time dependence of p160s recruitment by the activated GR in glial cells, revealing the complexity of GR-p160 assembly in the nervous system.