Control of steroid receptor dynamics and function by genomic actions of the cochaperones p23 and Bag-1L

Molecular chaperones encompass a group of unrelated proteins that facilitate the correct assembly and disassembly of other macromolecular structures, which they themselves do not remain a part of. They associate with a large and diverse set of coregulators termed cochaperones that regulate their function and specificity. Amongst others, chaperones and cochaperones regulate the activity of several signaling molecules including steroid receptors, which upon ligand binding interact with discrete nucleotide sequences within the nucleus to control the expression of diverse physiological and developmental genes. Molecular chaperones and cochaperones are typically known to provide the correct conformation for ligand binding by the steroid receptors. While this contribution is widely accepted, recent studies have reported that they further modulate steroid receptor action outside ligand binding. They are thought to contribute to receptor turnover, transport of the receptor to different subcellular localizations, recycling of the receptor on chromatin and even stabilization of the DNA-binding properties of the receptor. In addition to these combined effects with molecular chaperones, cochaperones are reported to have additional functions that are independent of molecular chaperones. Some of these functions also impact on steroid receptor action. Two well-studied examples are the cochaperones p23 and Bag-1L, which have been identified as modulators of steroid receptor activity in nuclei. Understanding details of their regulatory action will provide new therapeutic opportunities of controlling steroid receptor action independent of the widespread effects of molecular chaperones

A chemical probe for BAG1 targets androgen receptor-positive prostate cancer through oxidative stress signaling pathway

BAG1 is a family of polypeptides with a conserved C-terminal BAG domain that functions as a nucleotide exchange factor for the molecular chaperone HSP70. BAG1 proteins also control several signaling processes including proteostasis, apoptosis and transcription. The largest isoform, BAG1L, controls the activity of the androgen receptor (AR) and is upregulated in prostate cancer. Here, we show that BAG1L regulates AR dynamics in the nucleus and its ablation attenuates AR target gene expression especially those involved in oxidative stress and metabolism. We show that a small molecule, A4B17 that targets the BAG domain downregulates AR target genes similar to a complete BAG1L knockout and upregulates the expression of oxidative stress-induced genes involved in cell death. Furthermore, A4B17 outperformed the clinically approved antagonist enzalutamide in inhibiting cell proliferation and prostate tumor development in a mouse xenograft model. BAG1 inhibitors therefore offer unique opportunities for antagonizing AR action and prostate cancer growth

Clinical Utility of Circulating Tumour Cell Androgen Receptor Splice Variant-7 Status in Metastatic Castration-resistant Prostate Cancer.

Abstract Background Detection of androgen receptor splice variant-7 (AR-V7) mRNA in circulating tumour cells (CTCs) is associated with worse outcome in metastatic castration-resistant prostate cancer (mCRPC). However, studies rarely report comparisons with CTC counts and biopsy AR-V7 protein expression. Objective To determine the reproducibility of AdnaTest CTC AR-V7 testing, and associations with clinical characteristics, CellSearch CTC counts, tumour biopsy AR-V7 protein expression and overall survival (OS). Design, setting, and participants CTC AR-V7 status was determined for 227 peripheral blood samples, from 181 mCRPC patients with CTC counts (202 samples; 136 patients) and matched mCRPC biopsies (65 samples; 58 patients). Outcome measurements and statistical analysis CTC AR-V7 status was associated with clinical characteristics, CTC counts, and tissue biopsy AR-V7 protein expression. The association of CTC AR-V7 status and other baseline variables with OS was determined. Results and limitations Of the samples, 35% were CTC+/AR-V7+. CTC+/AR-V7+ samples had higher CellSearch CTC counts (median CTC; interquartile range [IQR]: 60, 19–184 vs 9, 2–64; Mann-Whitney test p Conclusions Studies reporting the prognostic relevance of CTC AR-V7 status must account for CTC counts. Discordant CTC AR-V7 results and AR-V7 protein expression in matched, same-patient biopsies are reported. Patient summary Liquid biopsies that determine circulating tumour cell androgen receptor splice variant-7 status have the potential to impact treatment decisions in metastatic castration-resistant prostate cancer patients. Robust clinical qualification of these assays is required before their routine use

Coregulator Control of Androgen Receptor Action by a Novel Nuclear Receptor-Binding Motif

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR

CD24 Is Not Required for Tumor Initiation and Growth in Murine Breast and Prostate Cancer Models

CD24 is a small, heavily glycosylated, GPI-linked membrane protein, whose expression has been associated with the tumorigenesis and progression of several types of cancer. Here, we studied the expression of CD24 in tumors of MMTV-PyMT, Apc1572/T+ and TRAMP genetic mouse models that spontaneously develop mammary or prostate carcinoma, respectively. We found that CD24 is expressed during tumor development in all three models. In MMTV-PyMT and Apc1572T/+ breast tumors, CD24 was strongly but heterogeneously expressed during early tumorigenesis, but decreased in more advanced stages, and accordingly was increased in poorly differentiated lesions compared with well differentiated lesions. In prostate tumors developing in TRAMP mice, CD24 expression was strong within hyperplastic lesions in comparison with non-hyperplastic regions, and heterogeneous CD24 expression was maintained in advanced prostate carcinomas. To investigate whether CD24 plays a functional role in tumorigenesis in these models, we crossed CD24 deficient mice with MMTV-PyMT, Apc1572T/+ and TRAMP mice, and assessed the influence of CD24 deficiency on tumor onset and tumor burden. We found that mice negative or positive for CD24 did not significantly differ in terms of tumor initiation and burden in the genetic tumor models tested, with the exception of Apc1572T/+ mice, in which lack of CD24 reduced the mammary tumor burden slightly but significantly. Together, our data suggest that while CD24 is distinctively expressed during the early development of murine mammary and prostate tumors, it is not essential for the formation of tumors developing in MMTV-PyMT, Apc1572T/+ and TRAMP mice

Neuroligin: Characterization of a neuronal transmembrane protein

Synapsen sind hoch spezialisierte Kontaktstellen, an welchen die Kommunikation zwischen Nervenzellen stattfindet. Bei der Steuerung von Entstehung und Modulation der synaptischen Kontakte sind maßgeblich Zelladhäsionsproteine beteiligt. Neuroligine sind postssynaptisch lokalisierte Transmembranproteine. Sie bilden mit beta-Neurexinen transsynaptische Zelladhäsionssysteme, deren Lokalisation und Eigenschaften darauf hindeuten, dass diese an der Steuerung der Synapsenentstehung beteiligt sind. Während die extrazellulären Bindungen der Neuroligine recht gut untersucht worden sind, waren bisher nur wenige intrazelluläre Bindungspartner bekannt. Die vorliegende Studie beschreibt die Identifizierung und Charakterisierung dreier Proteine, welche an den intrazellularen C-Terminus von Neuroligin binden: die Ubiquitinligase Nedd4.1 sowie die Strukturproteine 4.1N und 4.1B. Ubiquitinligasen wie Nedd4.1 regulieren die Lebensdauer von Proteinen durch Ubiquitinierung. Auf diese Weise wird eine Vielzahl zellulärer Prozesse, unter anderem auch die Modulation von Synapsen gesteuert. Die vorliegende Studie zeigt die Interaktion zwischen einer als PY-Motiv benannten konservierten Sequenz am C-Terminus von Neuroligin und den WW-Domänen von Nedd4.1. Neuroligin und Nedd4.1 sind gemeinsam in den postsynaptischen Kompartimenten lokalisiert. Die Interaktion resultiert jedoch nicht in der Ubiquitinierung von Neuroligin, fixiert Nedd4.1 jedoch in der Postsynapse und versetzt es so in die Lage, die Verweildauer anderer Proteine in dieser Region zu regulieren. Um weiteren Aufschluss über die Funktion und Zielproteine von Nedd4.1 zu erhalten, wurde eine Nedd4.1-Deletionsmutante in der Maus generiert. Diese Mutanten sterben bereits im Embryonalstadium ab. Auf diese Weise wird belegt, dass Nedd4.1 tatsächlich essentiell für die Embryonalentwicklung ist. Die Proteine 4.1N und 4.1B koordinieren die Integration von Proteinen in das Zytoskelett. Die Ergebnisse der vorliegenden Arbeit geben Hinweise auf die Bildung von Proteinkomplexen zwischen Neuroligin, 4.1-Proteinen und Proteinen der Familie der membran assoziierten Guanylatkinasen (MAGuKs) in der submembranen Matrix. Die Interaktion zwischen Neuroligin 1 und Protein 4.1, nicht jedoch die MAGuK-Interaktion scheint entscheidend für die Zielsteuerung von Neuroligin zur postsynaptischen Plasmamembran und dessen Verankerung mit dem dortigen Aktin-Zytoskelett zu sein