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1.
Mol Cancer Ther ; 23(6): 791-808, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38412481

ABSTRACT

Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2-associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited "on-target" toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2-mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.


Subject(s)
Disease Progression , Prostatic Neoplasms, Castration-Resistant , Signal Transduction , Male , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/pathology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Humans , Animals , Mice , Signal Transduction/drug effects , Receptors, Androgen/metabolism , Cell Line, Tumor , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Cell Proliferation , Xenograft Model Antitumor Assays , Gene Expression Regulation, Neoplastic/drug effects
2.
Lab Invest ; 103(11): 100245, 2023 11.
Article in English | MEDLINE | ID: mdl-37652207

ABSTRACT

BCL-2-associated athanogene-1L (BAG-1L) is a critical co-regulator that binds to and enhances the transactivation function of the androgen receptor, leading to prostate cancer development and progression. Studies investigating the clinical importance of BAG-1L protein expression in advanced prostate cancer have been limited by the paucity of antibodies that specifically recognize the long isoform. In this study, we developed and validated a new BAG-1L-specific antibody using multiple orthogonal methods across several cell lines with and without genomic manipulation of BAG-1L and all BAG-1 isoforms. Following this, we performed exploratory immunohistochemistry to determine BAG-1L protein expression in normal human, matched castration-sensitive prostate cancer (CSPC) and castration-resistant prostate cancer (CRPC), unmatched primary and metastatic CRPC, and early breast cancer tissues. We demonstrated higher BAG-1L protein expression in CRPC metastases than in unmatched, untreated, castration-sensitive prostatectomies from men who remained recurrence-free for 5 years. In contrast, BAG-1L protein expression did not change between matched, same patient, CSPC and CRPC biopsies, suggesting that BAG-1L protein expression may be associated with more aggressive biology and the development of castration resistance. Finally, in a cohort of patients who universally developed CRPC, there was no association between BAG-1L protein expression at diagnosis and time to CRPC or overall survival, and no association between BAG-1L protein expression at CRPC biopsy and clinical outcome from androgen receptor targeting therapies or docetaxel chemotherapy. The limitations of this study include the requirement to validate the reproducibility of the assay developed, the potential influence of pre-analytical factors, timing of CRPC biopsies, relatively small patient numbers, and heterogenous therapies on BAG-1L protein expression, and the clinical outcome analyses performed. We describe a new BAG-1L-specific antibody that the research community can further develop to elucidate the biological and clinical significance of BAG-1L protein expression in malignant and nonmalignant diseases.


Subject(s)
Prostatic Neoplasms, Castration-Resistant , Receptors, Androgen , Male , Humans , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Receptors, Androgen/therapeutic use , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Reproducibility of Results , Transcription Factors , Antibodies
3.
Cancer Res Commun ; 3(7): 1378-1396, 2023 07.
Article in English | MEDLINE | ID: mdl-37520743

ABSTRACT

The pro-oncogenic activities of estrogen receptor alpha (ERα) drive breast cancer pathogenesis. Endocrine therapies that impair the production of estrogen or the action of the ERα are therefore used to prevent primary disease metastasis. Although recent successes with ERα degraders have been reported, there is still the need to develop further ERα antagonists with additional properties for breast cancer therapy. We have previously described a benzothiazole compound A4B17 that inhibits the proliferation of androgen receptor-positive prostate cancer cells by disrupting the interaction of the cochaperone BAG1 with the AR. A4B17 was also found to inhibit the proliferation of estrogen receptor-positive (ER+) breast cancer cells. Using a scaffold hopping approach, we report here a group of small molecules with imidazopyridine scaffolds that are more potent and efficacious than A4B17. The prototype molecule X15695 efficiently degraded ERα and attenuated estrogen-mediated target gene expression as well as transactivation by the AR. X15695 also disrupted key cellular protein-protein interactions such as BAG1-mortalin (GRP75) interaction as well as wild-type p53-mortalin or mutant p53-BAG2 interactions. These activities together reactivated p53 and resulted in cell-cycle block and the induction of apoptosis. When administered orally to in vivo tumor xenograft models, X15695 potently inhibited the growth of breast tumor cells but less efficiently the growth of prostate tumor cells. We therefore identify X15695 as an oral selective ER degrader and propose further development of this compound for therapy of ER+ breast cancers. Significance: An imidazopyridine that selectively degrades ERα and is orally bioavailable has been identified for the development of ER+ breast cancer therapeutics. This compound also activates wild-type p53 and disrupts the gain-of-function tumorigenic activity of mutant p53, resulting in cell-cycle arrest and the induction of apoptosis.


Subject(s)
Breast Neoplasms , Estrogen Antagonists , Female , Humans , Breast Neoplasms/drug therapy , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha/genetics , Estrogens , Receptors, Estrogen/genetics , Tumor Suppressor Protein p53/genetics
4.
iScience ; 25(5): 104175, 2022 May 20.
Article in English | MEDLINE | ID: mdl-35479411

ABSTRACT

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.

5.
ACS Chem Biol ; 16(11): 2103-2108, 2021 11 19.
Article in English | MEDLINE | ID: mdl-34506104

ABSTRACT

All current clinically approved androgen deprivation therapies for prostate cancer target the C-terminal ligand-binding domain of the androgen receptor (AR). However, the main transactivation function of the receptor is localized at the AR N-terminal domain (NTD). Targeting the AR NTD directly is a challenge because of its intrinsically disordered structure and the lack of pockets for drugs to bind. Here, we have taken an alternative approach using the cochaperone BAG1L, which interacts with the NTD, to develop a novel AR inhibitor. We describe the identification of 2-(4-fluorophenyl)-5-(trifluoromethyl)-1,3-benzothiazole (A4B17), a small molecule that inhibits BAG1L-AR NTD interaction, attenuates BAG1L-mediated AR NTD activity, downregulates AR target gene expression, and inhibits proliferation of AR-positive prostate cancer cells. This compound represents a prototype of AR antagonists that could be key in the development of future prostate cancer therapeutics.


Subject(s)
Androgen Receptor Antagonists/pharmacology , Antineoplastic Agents/pharmacology , Benzothiazoles/pharmacology , Prostatic Neoplasms/drug therapy , Receptors, Androgen/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , DNA-Binding Proteins/metabolism , Down-Regulation/drug effects , Humans , Male , Prostatic Neoplasms/metabolism , Protein Binding/drug effects , Protein Domains , Receptors, Androgen/chemistry , Transcription Factors/metabolism
6.
J Mol Endocrinol ; 62(4): R289-R299, 2019 05.
Article in English | MEDLINE | ID: mdl-30913537

ABSTRACT

Androgens are important determinants of normal and malignant prostate growth. They function by binding to the C-terminal ligand-binding domain (LBD) of the androgen receptor (AR). All clinically approved AR-targeting antiandrogens for prostate cancer therapy function by competing with endogenous androgens. Despite initial robust responses to androgen deprivation therapy, nearly all patients with advanced prostate cancer relapse with lethal castration-resistant prostate cancer (CRPC). Progression to CRPC is associated with ongoing AR signaling, which in part, is due to the expression of constitutively active AR splice variants that contain the N-terminus of the receptor but lack the C-terminus. Currently, there are no approved therapies specifically targeting the AR N-terminus. Current pharmacologic targeting strategies for inhibiting the AR N-terminal region have proven difficult, due to its intrinsically unstructured nature and lack of enzymatic activity. An alternative approach is to target key molecules such as the cochaperone BAG1L that bind to and enhance the activity of the AR AF1. Here, we review recent literature that suggest Bag-1L is a promising target for AR-positive prostate cancer.


Subject(s)
Biomarkers, Tumor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Prostatic Neoplasms/etiology , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , Disease Susceptibility , Humans , Male , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Targeted Therapy , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/etiology , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Protein Binding , Protein Interaction Domains and Motifs , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry
7.
Cancer Cell ; 35(3): 401-413.e6, 2019 03 18.
Article in English | MEDLINE | ID: mdl-30773341

ABSTRACT

Androgen deprivation therapy for prostate cancer (PCa) benefits patients with early disease, but becomes ineffective as PCa progresses to a castration-resistant state (CRPC). Initially CRPC remains dependent on androgen receptor (AR) signaling, often through increased expression of full-length AR (ARfl) or expression of dominantly active splice variants such as ARv7. We show in ARv7-dependent CRPC models that ARv7 binds together with ARfl to repress transcription of a set of growth-suppressive genes. Expression of the ARv7-repressed targets and ARv7 protein expression are negatively correlated and predicts for outcome in PCa patients. Our results provide insights into the role of ARv7 in CRPC and define a set of potential biomarkers for tumors dependent on ARv7.


Subject(s)
Alternative Splicing , Prostatic Neoplasms, Castration-Resistant/genetics , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Male , Prostatic Neoplasms, Castration-Resistant/metabolism , Tissue Array Analysis , Transcription, Genetic
8.
J Allergy Clin Immunol ; 144(4S): S31-S45, 2019 10.
Article in English | MEDLINE | ID: mdl-30772496

ABSTRACT

Mast cells (MCs), which are best known for their detrimental role in patients with allergic diseases, act in a diverse array of physiologic and pathologic functions made possible by the plurality of MC types. Their various developmental avenues and distinct sensitivity to (micro-) environmental conditions convey extensive heterogeneity, resulting in diverse functions. We briefly summarize this heterogeneity, elaborate on molecular determinants that allow MCs to communicate with their environment to fulfill their tasks, discuss the protease repertoire stored in secretory lysosomes, and consider different aspects of MC signaling. Furthermore, we describe key MC governance mechanisms (ie, the high-affinity receptor for IgE [FcεRI]), the stem cell factor receptor KIT, the IL-4 system, and both Ca2+- and phosphatase-dependent mechanisms. Finally, we focus on distinct physiologic functions, such as chemotaxis, phagocytosis, host defense, and the regulation of MC functions at the mucosal barriers of the lung, gastrointestinal tract, and skin. A deeper knowledge of the pleiotropic functions of MC mediators, as well as the molecular processes of MC regulation and communication, should enable us to promote beneficial MC traits in physiology and suppress detrimental MC functions in patients with disease.


Subject(s)
Chemotaxis/immunology , Intestinal Mucosa/immunology , Mast Cells/immunology , Phagocytosis , Respiratory Mucosa/immunology , Signal Transduction/immunology , Animals , Calcium/immunology , Humans , Interleukin-4/immunology , Intestinal Mucosa/pathology , Lysosomes/immunology , Lysosomes/pathology , Mast Cells/pathology , Proto-Oncogene Proteins c-kit/immunology , Receptors, IgE/immunology , Respiratory Mucosa/pathology
9.
Data Brief ; 20: 1177-1183, 2018 Oct.
Article in English | MEDLINE | ID: mdl-30238026

ABSTRACT

Protein tyrosine phosphatases and glucocorticoids are known to regulate allergic and antiallergic action in activated mast cells. Here we provide RNA sequencing and quantitative real-time PCR data from bone marrow derived mast cells, for wild-type and PEST-domain-enriched tyrosine phosphatase (PEP) null mice, activated by immunoglobulin E sensitization and dinitrophenol treatment, and additionally treated with the glucocorticoid dexamethasone. The transcriptomics experiment was performed in duplicate with a total of 16 samples (GSE108972).

10.
Biomolecules ; 7(2)2017 06 19.
Article in English | MEDLINE | ID: mdl-28629183

ABSTRACT

The human androgen receptor (AR) is a ligand inducible transcription factor that harbors an amino terminal domain (AR-NTD) with a ligand-independent activation function. AR-NTD is intrinsically disordered and displays aggregation properties conferred by the presence of a poly-glutamine (polyQ) sequence. The length of the polyQ sequence as well as its adjacent sequence motifs modulate this aggregation property. AR-NTD also contains a conserved KELCKAVSVSM sequence motif that displays an intrinsic property to form amyloid fibrils under mild oxidative conditions. As peptide sequences with intrinsic oligomerization properties are reported to have an impact on the aggregation of polyQ tracts, we determined the effect of the KELCKAVSVSM on the polyQ stretch in the context of the AR-NTD using atomic force microscopy (AFM). Here, we present evidence for a crosstalk between the amyloidogenic properties of the KELCKAVSVSM motif and the polyQ stretch at the AR-NTD.


Subject(s)
Amyloid/chemistry , Peptides/chemistry , Receptors, Androgen/chemistry , Amino Acid Sequence , Amyloid/metabolism , Polymerization , Receptors, Androgen/metabolism
11.
Sci Transl Med ; 8(370): 370ra181, 2016 12 21.
Article in English | MEDLINE | ID: mdl-28003546

ABSTRACT

Spinobulbar muscular atrophy (SBMA) is an X-linked neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. SBMA belongs to the family of polyQ diseases, which are fatal neurodegenerative disorders mainly caused by protein-mediated toxic gain-of-function mechanisms and characterized by deposition of misfolded proteins in the form of aggregates. The neurotoxicity of the polyQ proteins can be modified by phosphorylation at specific sites, thereby providing the rationale for the development of disease-specific treatments. We sought to identify signaling pathways that modulate polyQ-AR phosphorylation for therapy development. We report that cyclin-dependent kinase 2 (CDK2) phosphorylates polyQ-AR specifically at Ser96 Phosphorylation of polyQ-AR by CDK2 increased protein stabilization and toxicity and is negatively regulated by the adenylyl cyclase (AC)/protein kinase A (PKA) signaling pathway. To translate these findings into therapy, we developed an analog of pituitary adenylyl cyclase activating polypeptide (PACAP), a potent activator of the AC/PKA pathway. Chronic intranasal administration of the PACAP analog to knock-in SBMA mice reduced Ser96 phosphorylation, promoted polyQ-AR degradation, and ameliorated disease outcome. These results provide proof of principle that noninvasive therapy based on the use of PACAP analogs is a therapeutic option for SBMA.


Subject(s)
Muscular Disorders, Atrophic/metabolism , Peptides/chemistry , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , Receptors, Androgen/metabolism , Animals , Cell Proliferation , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclin-Dependent Kinase 2/metabolism , Glutamine/metabolism , HEK293 Cells , Humans , Ligands , Membrane Potential, Mitochondrial , Mice , Mice, Transgenic , PC12 Cells , Phosphorylation , Protein Denaturation , Protein Folding , Rats , Rats, Sprague-Dawley , Signal Transduction
12.
Small ; 12(38): 5330-5338, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27511293

ABSTRACT

The profiling of allergic responses is a powerful tool in biomedical research and in judging therapeutic outcome in patients suffering from allergy. Novel insights into the signaling cascades and easier readouts can be achieved by shifting activation studies of bulk immune cells to the single cell level on patterned surfaces. The functionality of dinitrophenol (DNP) as a hapten in the induction of allergic reactions has allowed the activation process of single mast cells seeded on patterned surfaces to be studied following treatment with allergen specific Immunoglobulin E antibodies. Here, a click-chemistry approach is applied in combination with polymer pen lithography (PPL) to pattern DNP-azide on alkyne-terminated surfaces to generate arrays of allergen. The large area functionalization offered by PPL allows an easy incorporation of such arrays into microfluidic chips. In such a setup, easy handling of cell suspension, incubation process, and read-out by fluorescence microscopy will allow immune cell activation screening to be easily adapted for diagnostics and biomedical research.


Subject(s)
Allergens/chemistry , Click Chemistry/methods , Mast Cells/metabolism , Polymers/chemistry , Printing/methods , Animals , Cell Count , Cell Line , Immunoglobulin E/metabolism , Mice, Inbred C57BL , Microscopy, Fluorescence , Phosphotyrosine/metabolism , Rats , Receptors, Fc/metabolism , Rhodamines/chemistry
13.
Cancer Cell ; 29(6): 846-858, 2016 06 13.
Article in English | MEDLINE | ID: mdl-27238081

ABSTRACT

Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in speckle-type POZ protein (SPOP) stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly upregulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP mutant and CRPC patients.


Subject(s)
Carrier Proteins/genetics , Nuclear Proteins/genetics , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms/genetics , Receptors, Androgen/genetics , Repressor Proteins/genetics , Animals , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Proliferation , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Male , Neoplasm Transplantation , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Receptors, Androgen/chemistry , Receptors, Androgen/metabolism , Signal Transduction
14.
Small ; 12(29): 3985-94, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27240250

ABSTRACT

The physical and mechanical properties of the tumor microenvironment are crucial for the growth, differentiation and migration of cancer cells. However, such microenvironment is not found in the geometric constraints of 2D cell culture systems used in many cancer studies. Prostate cancer research, in particular, suffers from the lack of suitable in vitro models. Here a 3D superporous scaffold is described with thick pore walls in a mechanically stable and robust architecture to support prostate tumor growth. This scaffold is generated from the cryogelation of poly(ethylene glycol) diacrylate to produce a defined elastic modulus for prostate tumor growth. Lymph node carcinoma of the prostate (LNCaP) cells show a linear growth over 21 d as multicellular tumor spheroids in such a scaffold with points of attachments to the walls of the scaffold. These LNCaP cells respond to the growth promoting effects of androgens and demonstrate a characteristic cytoplasmic-nuclear translocation of the androgen receptor and androgen-dependent gene expression. Compared to 2D cell culture, the expression or androgen response of prostate cancer specific genes is greatly enhanced in the LNCaP cells in this system. This scaffold is therefore a powerful tool for prostate cancer studies with unique advantages over 2D cell culture systems.


Subject(s)
Cryogels/chemistry , Elastic Modulus , Polyethylene Glycols/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dihydrotestosterone/pharmacology , Humans , Male , Microscopy, Electron, Scanning , Prostatic Neoplasms
15.
Adv Exp Med Biol ; 872: 217-33, 2015.
Article in English | MEDLINE | ID: mdl-26215996

ABSTRACT

Glucocorticoids (GCs) are steroid hormones with widespread effects. They control intermediate metabolism by stimulating gluconeogenesis in the liver, mobilize amino acids from extra hepatic tissues, inhibit glucose uptake in muscle and adipose tissue, and stimulate fat breakdown in adipose tissue. They also mediate stress response. They exert potent immune-suppressive and anti-inflammatory effects particularly when administered pharmacologically. Understanding these diverse effects of glucocorticoids requires a detailed knowledge of their mode of action. Research over the years has uncovered several details on the molecular action of this hormone, especially in immune cells. In this chapter, we have summarized the latest findings on the action of glucocorticoids in immune cells with a view of identifying important control points that may be relevant in glucocorticoid therapy.


Subject(s)
Glucocorticoids/physiology , Immune System/physiology , Humans
16.
Nucl Recept Signal ; 12: e005, 2014.
Article in English | MEDLINE | ID: mdl-25422595

ABSTRACT

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.


Subject(s)
DNA-Binding Proteins/metabolism , Genomics/methods , Intramolecular Oxidoreductases/metabolism , Molecular Chaperones/metabolism , Receptors, Steroid/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Cell Nucleus/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Humans , Molecular Sequence Data , Prostaglandin-E Synthases , Transcription Factors/chemistry , Transcription Factors/deficiency , Transcription Factors/genetics
17.
Oncotarget ; 5(18): 8681-9, 2014 Sep 30.
Article in English | MEDLINE | ID: mdl-25237833

ABSTRACT

Anterior gradient 2 (AGR2) is a gene predominantly expressed in mucus-secreting tissues or in endocrine cells. Its expression is drastically increased in tumors including prostate cancer. Here we investigated whether AGR2 transcript levels can be used as a biomarker to detect prostate cancer (PCa). Using a PCR-based approach, we could show that in addition to the wild-type (AGRwt long and short) transcripts, five other AGR2 splice variants (SV) (referred to as AGR2 SV-C, -E, -F, -G and -H) were present in cancer cell lines. In tissue biopsies, SV-H and AGR2wt (short) distinguished between benign and PCa (p ≤ 0.05 n = 32). In urine exosomes, AGR2 SV-G and SV-H outperformed serum PSA. Receiver operating characteristic (ROC) curves showed the highest discriminatory power of SV-G and SV-H in predicting PCa. AGR2 SV-G and SV-H are potential diagnostic biomarkers for the non-invasive detection of PCa using urine exosomes.


Subject(s)
Alternative Splicing , Biomarkers, Tumor/genetics , Prostatic Neoplasms/genetics , Proteins/genetics , Aged , Area Under Curve , Biomarkers, Tumor/blood , Biomarkers, Tumor/urine , Biopsy , Cell Line, Tumor , Exosomes/metabolism , Gene Expression Regulation, Neoplastic , Humans , Kallikreins/blood , Male , Middle Aged , Mucoproteins , Oncogene Proteins , Pilot Projects , Predictive Value of Tests , Prognosis , Prostate-Specific Antigen/blood , Prostatic Neoplasms/blood , Prostatic Neoplasms/pathology , Prostatic Neoplasms/urine , RNA, Messenger/urine , ROC Curve
18.
Methods Mol Biol ; 1204: 197-204, 2014.
Article in English | MEDLINE | ID: mdl-25182772

ABSTRACT

Spinal bulbar muscular atrophy (SBMA) (also known as Kennedy's disease) is a motor degenerative disease caused by an amplification of the polyglutamine stretch at the N-terminus of the human androgen receptor (AR). Amplifications larger than 40 glutamine residues are thought to lead to the disease. A characteristic feature of this disease is a ligand-dependent misfolding and aggregation of the mutant receptor that lead to the death of motor neurons. Initially, large cytoplasmic and nuclear aggregates reaching sizes of 6 µm were thought to be the pathogenic agents. Later studies have suggested that oligomeric species with sizes of less than 1 µm that occur prior to the formation of the larger aggregates are the toxic agents. However, there have been disagreements regarding the shape of these oligomers, as most studies have been carried out with peptide fragments of the androgen receptor containing different lengths of polyglutamine stretch. We have isolated the wild-type AR with a polyglutamine stretch of 22 (ARQ22) and a mutant receptor with a stretch of 65 (ARQ65) using a baculovirus system and have analyzed the oligomeric structures formed by these receptors with atomic force microscopy. This method has allowed us to determine the conformations of the full-length wild-type and mutant AR and revealed the conformation of the mutant AR that causes SBMA.


Subject(s)
Microscopy, Atomic Force/methods , Muscular Atrophy, Spinal/genetics , Receptors, Androgen/chemistry , Receptors, Androgen/genetics , Animals , Baculoviridae/genetics , Cell Culture Techniques/methods , Cell Line , Humans , Insecta , Mutation , Peptides/chemistry , Peptides/genetics , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Software
19.
Small ; 10(10): 1991-8, 2014 May 28.
Article in English | MEDLINE | ID: mdl-24616258

ABSTRACT

In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non-nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip-pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid-independent and -dependent recruitment of the GR-GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane-bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid-mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid-mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling.


Subject(s)
Cell Membrane/metabolism , Cell Membrane/ultrastructure , Mast Cells/metabolism , Microscopy, Fluorescence/methods , Molecular Imaging/methods , Receptors, Glucocorticoid/metabolism , Animals , Cell Line , Rats
20.
J Biol Chem ; 289(13): 8839-51, 2014 Mar 28.
Article in English | MEDLINE | ID: mdl-24523409

ABSTRACT

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.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Receptors, Androgen/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Allosteric Regulation , Amino Acid Motifs , Amino Acid Sequence , Cell Line , DNA-Binding Proteins/genetics , Humans , Mutation , Oligopeptides/chemistry , Oligopeptides/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, Androgen/chemistry , Receptors, Androgen/genetics , Repetitive Sequences, Amino Acid , Transcription Factors/genetics , Transcriptional Activation
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