Adam T. Szafran

Estrogen-receptor-α exchange and chromatin dynamics are ligand- and domain-dependent

We report a mammalian-based promoter chromosomal array system developed for single-cell studies of transcription-factor function. Designed after the prolactin promoter-enhancer, it allows for the direct visualization of estrogen receptor α (ERα) and/or Pit-1 interactions at a physiologically regulated transcription locus. ERα- and ligand-dependent cofactor recruitment, large-scale chromatin modifications and transcriptional activity identified a distinct fingerprint of responses for each condition. Ligand-dependent transcription (more than threefold activation compared with vehicle, or complete repression by mRNA fluorescent in situ hybridization) at the array correlated with its state of condensation, which was assayed using a novel high throughput microscopy approach. In support of the nuclear receptor hit-and-run model, photobleaching studies provided direct evidence of very transient ER-array interactions, and revealed ligand-dependent changes in koff. ERα-truncation mutants indicated that helix-12 and interactions with co-regulators influenced both large-scale chromatin modeling and photobleaching recovery times. These data also showed that the ERα DNA-binding domain was insufficient for array targeting. Collectively, quantitative observations from this physiologically relevant biosensor suggest stochastic-based dynamics influence gene regulation at the promoter level.

Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility.

Using manual and automated high throughput microscopy (HTM), ligand‐dependent trafficking of green fluorescent protein‐androgen receptor (GFP‐AR) was analyzed in fixed and living cells to determine its spatial distribution, solubility, mobility, and co‐activator interactions. Within minutes, addition of the agonist R1881 resulted translocation of GFP‐AR from the cytoplasm to the nucleus, where it displayed a hyperspeckled pattern and extraction resistance in low expressing cells. AR antagonists (Casodex, hydroxyflutamide) also caused nuclear translocation, however, the antagonist‐bound GFP‐AR had a more diffuse nuclear distribution, distinct from the agonist‐bound GFP‐AR, and was completely soluble; overexpressed GFP‐AR in treated cells was extraction resistant, independent of ligand type. To more dramatically show the different effects of ligand on AR distribution, we utilized an AR with a mutation in the DNA binding domain (ARC619Y) that forms distinct foci upon exposure to agonists but retains a diffuse nuclear distribution in the presence of antagonists. Live‐cell imaging of this mutant demonstrated that cytoplasmic foci formation occurs immediately upon agonist but not antagonist addition. Fluorescence recovery after photobleaching (FRAP) revealed that agonist‐bound GFP‐AR exhibited reduced mobility relative to unliganded or antagonist‐bound GFP‐AR. Importantly, agonist‐bound GFP‐AR mobility was strongly affected by protein expression levels in transiently transfected cells, and displayed reduced mobility even in slightly overexpressing cells. Cyan fluorescent protein‐AR (CFP‐AR) and yellow fluorescent protein‐CREB binding protein (YFP‐CBP) in the presence of agonists and antagonists were used to demonstrate that CFP‐AR specifically co‐localizes with YFP‐CBP in an agonist dependent manner. Dual FRAP experiments demonstrated that CBP mobility mirrored AR mobility only in the presence of agonist. HTM enabled simultaneous studies of the sub‐cellular distribution of GFP‐AR and ARC619Y in response to a range of concentrations of agonists and antagonists (ranging from 10−12 to 10−5) in thousands of cells. These results further support the notion that ligand specific interactions rapidly affect receptor and co‐factor organization, solubility, and molecular dynamics, and each can be aberrantly affected by mutation and overexpression. J. Cell. Biochem. 98: 770–788, 2006.

Regulation of SRC-3 Intercompartmental Dynamics by Estrogen Receptor and Phosphorylation

ABSTRACT The steroid receptor coactivator 3 gene (SRC-3) (AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family transcription coactivator and a known oncogene. Despite its importance, the functional regulation of SRC-3 remains poorly understood within a cellular context. Using a novel combination of live-cell, high-throughput, and fluorescent microscopy, we report SRC-3 to be a nucleocytoplasmic shuttling protein whose intracellular mobility, solubility, and cellular localization are regulated by phosphorylation and estrogen receptor α (ERα) interactions. We show that both chemical inhibition and small interfering RNA reduction of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by epidermal growth factor signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known participants in the phosphocode that regulates SRC-3 activity. Accordingly, the cytoplasmic localization of a nonphosphorylatable SRC-3 mutant further supported these results. In the presence of ERα, U0126 also dramatically reduces (i) ligand-dependent colocalization of SRC-3 and ERα, (ii) the formation of ER-SRC-3 complexes in cell lysates, and (iii) SRC-3 targeting to a visible, ERα-occupied and -regulated prolactin promoter array. Taken together, these results indicate that phosphorylation coordinates SRC-3 coactivator function by linking the probabilistic formation of transient nuclear receptor-coactivator complexes with its molecular dynamics and cellular compartmentalization. Technically and conceptually, these findings have a new and broad impact upon evaluating mechanisms of action of gene regulators at a cellular system level.

The activity of the androgen receptor variant AR-V7 is regulated by FOXO1 in a PTEN-PI3K-AKT-dependent way

The androgen receptor (AR) AR‐V7 splice isoform is a constitutively active outlaw transcription factor. Transition of prostate cancer (PC) to the castration‐resistant phenotype correlates with AR‐V7 accumulation, suggesting that PC progression in patients refractory to conventional therapy is due to the activity of this AR isoform. The mechanism of AR‐V7 constitutive activation is not known.

Discovery and Mechanistic Characterization of a Novel Selective Nuclear Androgen Receptor Exporter for the Treatment of Prostate Cancer

Despite the success of medical strategies to reduce androgen levels in the treatment of prostate cancer, this disease invariably relapses to a castrate-resistant state that is generally fatal. Although it had been thought that androgen-insensitive cancers no longer relied on the androgen receptor (AR) for growth and survival, it is now clear that this is not the case. Because relapses are known to occur by many mechanisms that keep the AR functionally active, strategies to block AR accumulation in the nucleus may be therapeutically useful. Here, we report the discovery of a selective nuclear androgen receptor exporter (SNARE) that functions to exclude AR from the nucleus. SNARE-1 binds wild-type and mutant ARs and efficiently inhibits their transactivation activity and ability to induce PSA gene expression. SNARE-1 inhibits the androgen-sensitive growth of LNCaP cells and tumor xenografts. Quantitative subcellular localization studies suggest that SNARE-1 inhibits nuclear translocation of AR, but also facilitates export of nuclear AR that has been translocated by an agonist. Mechanistic studies indicate that SNARE-1 rapidly phosphorylates p38 mitogen-activated protein kinase (MAPK) and Ser(650) of the AR. Additionally, SNARE-1 was found to promote ubiquitination of AR in LNCaP cells. Lastly, SNARE-1 functions as a tissue-selective AR inhibitor, as it fails to phosphorylate p38 MAPK in U2OS bone cells that are stably transfected with AR. In summary, SNARE-1 inhibits AR function by a mechanism that is distinct from clinically available antiandrogens, such that it might inform novel methods to block AR function in androgen-independent prostate cancer.

Androgen Receptor Mutations Associated with Androgen Insensitivity Syndrome: A High Content Analysis Approach Leading to Personalized Medicine

Androgen insensitivity syndrome (AIS) is a rare disease associated with inactivating mutations of AR that disrupt male sexual differentiation, and cause a spectrum of phenotypic abnormalities having as a common denominator loss of reproductive viability. No established treatment exists for these conditions, however there are sporadic reports of patients (or recapitulated mutations in cell lines) that respond to administration of supraphysiologic doses (or pulses) of testosterone or synthetic ligands. Here, we utilize a novel high content analysis (HCA) approach to study AR function at the single cell level in genital skin fibroblasts (GSF). We discuss in detail findings in GSF from three historical patients with AIS, which include identification of novel mechanisms of AR malfunction, and the potential ability to utilize HCA for personalized treatment of patients affected by this condition.

Inhibition of the hexosamine biosynthetic pathway promotes castration-resistant prostate cancer

The precise molecular alterations driving castration-resistant prostate cancer (CRPC) are not clearly understood. Using a novel network-based integrative approach, here, we show distinct alterations in the hexosamine biosynthetic pathway (HBP) to be critical for CRPC. Expression of HBP enzyme glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is found to be significantly decreased in CRPC compared with localized prostate cancer (PCa). Genetic loss-of-function of GNPNAT1 in CRPC-like cells increases proliferation and aggressiveness, in vitro and in vivo. This is mediated by either activation of the PI3K-AKT pathway in cells expressing full-length androgen receptor (AR) or by specific protein 1 (SP1)-regulated expression of carbohydrate response element-binding protein (ChREBP) in cells containing AR-V7 variant. Strikingly, addition of the HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) to CRPC-like cells significantly decreases cell proliferation, both in-vitro and in animal studies, while also demonstrates additive efficacy when combined with enzalutamide in-vitro. These observations demonstrate the therapeutic value of targeting HBP in CRPC.

High‐Resolution, High‐Throughput Microscopy Analyses of Nuclear Receptor and Coregulator Function

Steroid nuclear receptors are ligand-dependent transcription factors that have been studied since the early 1960s by principally biochemical and reporter assay approaches. From these studies an elegant and complex model of nuclear receptor transcription regulation has been developed. Inherent to both biochemical and reporter assay approaches is the generation of averaged responses and it is not generally considered that individual cells could exhibit quite varied responses. In some cases, recent microscopic single-cell analyses provide markedly different responses relative to traditional approaches based on population averaging and underscore the need to continue refinement of the current model of nuclear receptor-regulated transcription. While single-cell analyses of nuclear receptor action have been hindered by the predominantly qualitative nature of the approach, high-throughput microscopy is now available to resolve this issue. This chapter demonstrates the utility of high-throughput microscopic analyses of nuclear receptor and nuclear receptor coregulator function. The ability of high-throughput microscopy to generate physiologically appropriate test populations by filtering based on morphological and protein of interest expression criteria is demonstrated. High-resolution, high-throughput microscopy is illustrated that provides quantitative subcellular information for both androgen and estrogen receptors. Efforts are ongoing to develop model systems that provide additional multiplex data and with refined image analyses to achieve true high-content imaging screens.

Automated Microscopy and Image Analysis for Androgen Receptor Function

Systems-level approaches have emerged that rely on analytical, microscopy-based technology for the discovery of novel drug targets and the mechanisms driving AR signaling, transcriptional activity, and ligand independence. Single cell behavior can be quantified by high-throughput microscopy methods through analysis of endogenous protein levels and localization or creation of biosensor cell lines that can simultaneously detect both acute and latent responses to known and unknown androgenic stimuli. The cell imaging and analytical protocols can be automated to discover agonist/antagonist response windows for nuclear translocation, reporter gene activity, nuclear export, and subnuclear transcription events, facilitating access to a multiplex model system that is inherently unavailable through classic biochemical approaches. In this chapter, we highlight the key steps needed for developing, conducting, and analyzing high-throughput screens to identify effectors of AR signaling.

The myImageAnalysis Project: A Web-Based Application for High-Content Screening

A major challenge faced by screening centers developing image-based assays is the wide range of assays needed compared to the limited resources that are available to effectively analyze and manage them. To overcome this limitation, we have developed the web-based myImageAnalysis (mIA) application, integrated with an open database connectivity compliant database and powered by Pipeline Pilot (PLP) that incorporates dataset tracking, scheduling and archiving, image analysis, and data reporting. For system administrators, mIA provides automated methods for managing and archiving data. For the biologist, this application allows those without any programming or image analysis experience to quickly develop, validate, and share results of complex image-based assays. Further, the structure of the application within PLP allows those with experience in PLP programming to easily add additional analysis tools as required. The tools within mIA allow users to assess basic (cell count, protein per cell, protein subcellular localization) and more advanced (engineered cell lines analysis, cell toxicity) biological image-based assays that employ advanced statistics and provides key assay performance metrics.