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1.
J Urol ; 193(2): 690-8, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25132238

ABSTRACT

PURPOSE: Spliced variant forms of androgen receptor were recently identified in castration resistant prostate cancer cell lines and clinical samples. We identified the cistrome and gene signature of androgen receptor splice variants in castration resistant prostate cancer cell lines and determined the clinical significance of androgen receptor splice variant regulated genes. MATERIALS AND METHODS: The castration resistant prostate cancer cell line 22Rv1, which expresses full-length androgen receptor and androgen receptor splice variants endogenously, was used as the research model. We established 22Rv1-ARFL(-)/ARV(+) and 22Rv1-ARFL(-)/ARV(-) through RNA interference. Chromatin immunoprecipitation coupled with next generation sequencing and microarray techniques were used to identify the cistrome and gene expression profiles of androgen receptor splice variants in the absence of androgen. RESULTS: Androgen receptor splice variant binding sites were identified in 22Rv1-ARFL(-)/ARV(+). A gene set was regulated uniquely by androgen receptor splice variants but not by full-length androgen receptor in the absence of androgen. Integrated analysis revealed that some genes were directly modulated by androgen receptor splice variants. Unsupervised clustering analysis showed that the androgen receptor splice variant gene signature differentiated benign from malignant prostate tissue as well as localized prostate cancer from metastatic castration resistant prostate cancer specimens. Some genes that were modulated uniquely by androgen receptor splice variants also correlated with histological grade and biochemical failure. CONCLUSIONS: Androgen receptor splice variants can bind to DNA independent of full-length androgen receptor in the absence of androgen and modulate a unique set of genes that is not regulated by full-length androgen receptor. The androgen receptor splice variant gene signature correlates with disease progression. It distinguishes primary cancer from castration resistant prostate cancer specimens and benign from malignant prostate specimens.


Subject(s)
Gene Expression Regulation, Neoplastic , Prostatic Neoplasms, Castration-Resistant/genetics , Receptors, Androgen/genetics , Transcriptome , High-Throughput Nucleotide Sequencing , Humans , Male , Protein Isoforms , Tumor Cells, Cultured
2.
Curr Drug Targets ; 14(4): 401-7, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23565753

ABSTRACT

Androgens and the androgen receptor have been the focus of prostate cancer research since the early 1940s, when Huggins and Hodges demonstrated that removal of androgens caused advanced prostate cancer to regress. Since that time, a large number of androgen deprivation therapies have been developed in an effort to cure this disease, but prostate cancer remains one of the leading causes of cancer death in males worldwide. This is due in part to the emergence of castration- recurrent prostate cancer in patients with advanced disease who have failed androgen deprivation therapy. The androgen receptor is still a major player in castration-recurrent disease, and though much has been discovered since the early work of Huggins and Hodges regarding how prostate cancer cells manage to avoid the effects of androgen deprivation, survival times for men with advanced prostate cancer have changed only modestly. Research is now directed toward delineating the mechanisms of action of the androgen receptor under castrate conditions, whether through amplification of the AR, mutation, expression of splice variants, use of alternate signaling pathways, aberrant expression and activation of coregulators, or intratumoral androgen biosynthesis. Genome-wide association studies are also adding to the wealth of knowledge surrounding the androgen receptor, and with this knowledge comes the ability to design new drug therapies directed toward eradication of this disease.


Subject(s)
Androgen Antagonists/therapeutic use , Prostatic Neoplasms/drug therapy , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Androgens/genetics , Androgens/metabolism , Animals , Genome-Wide Association Study/methods , Humans , Male , Mutation , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Signal Transduction
3.
Mol Endocrinol ; 26(5): 716-35, 2012 May.
Article in English | MEDLINE | ID: mdl-22456196

ABSTRACT

Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate, correlate with aggressive disease, and are associated with biochemical recurrence. Thus, understanding the mechanism(s) by which SRF conveys androgen regulation to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we show that the small GTPase ras homolog family member A (RhoA) mediates androgen-responsiveness of more than half of SRF target genes. Interference with expression of RhoA, activity of the RhoA effector Rho-associated coiled-coil containing protein kinase 1 (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor megakaryocytic acute leukemia (MAL) prevented full androgen regulation of SRF target genes. Androgen treatment induced RhoA activation, increased the nuclear content of MAL, and led to MAL recruitment to the promoter of the SRF target gene FHL2. In clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival after radical prostatectomy. Overexpression of RhoA markedly increased the androgen-responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. The use of isogenic cell lines and a xenograft model that mimics the transition from androgen-stimulated to castration-recurrent PCa indicated that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Androgen-responsiveness of SRF target genes in castration-recurrent PCa cells continued to rely on AR, RhoA, SRF, and MAL and the presence of intact SRF binding sites. Silencing of RhoA, use of Rho-associated coiled-coil containing protein kinase 1 inhibitors, or an inhibitor of SRF-MAL interaction attenuated (androgen-regulated) cell viability and blunted PCa cell migration. Taken together, these studies demonstrate that the RhoA signaling axis mediates clinically relevant AR action in PCa.


Subject(s)
Androgens/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Neoplasm Proteins/metabolism , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Signal Transduction/drug effects , rhoA GTP-Binding Protein/metabolism , Androgens/adverse effects , Animals , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Male , Mice , Mice, Nude , Muscle Proteins/genetics , Muscle Proteins/metabolism , Neoplasm Proteins/agonists , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Recurrence, Local , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Promoter Regions, Genetic/drug effects , Prostate/drug effects , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/pathology , Prostatic Neoplasms/surgery , Protein Kinase Inhibitors/pharmacology , Protein Transport/drug effects , Receptors, Androgen/genetics , Recombinant Proteins/agonists , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Trans-Activators , Transcription Factors/genetics , Transcription Factors/metabolism , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/agonists , rhoA GTP-Binding Protein/antagonists & inhibitors , rhoA GTP-Binding Protein/genetics
4.
Cancer Res ; 71(5): 1978-88, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21324924

ABSTRACT

The androgen receptor (AR) is the principal target for treatment of non-organ-confined prostate cancer (PCa). Androgen deprivation therapies (ADT) directed against the AR ligand-binding domain do not fully inhibit androgen-dependent signaling critical for PCa progression. Thus, information that could direct the development of more effective ADTs is desired. Systems and bioinformatics approaches suggest that considerable variation exists in the mechanisms by which AR regulates expression of effector genes, pointing to a role for secondary transcription factors. A combination of microarray and in silico analyses led us to identify a 158-gene signature that relies on AR along with the transcription factor SRF (serum response factor), representing less than 6% of androgen-dependent genes. This AR-SRF signature is sufficient to distinguish microdissected benign and malignant prostate samples, and it correlates with the presence of aggressive disease and poor outcome. The AR-SRF signature described here associates more strongly with biochemical failure than other AR target gene signatures of similar size. Furthermore, it is enriched in malignant versus benign prostate tissues, compared with other signatures. To our knowledge, this profile represents the first demonstration of a distinct mechanism of androgen action with clinical relevance in PCa, offering a possible rationale to develop novel and more effective forms of ADT.


Subject(s)
Biomarkers, Tumor/genetics , Gene Expression Profiling , Prostatic Neoplasms/genetics , Receptors, Androgen/genetics , Serum Response Factor/genetics , Humans , Lasers , Male , Microdissection , Neoplasm Staging , Oligonucleotide Array Sequence Analysis , Prostatic Neoplasms/pathology , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , Transfection
5.
J Steroid Biochem Mol Biol ; 125(1-2): 32-8, 2011 May.
Article in English | MEDLINE | ID: mdl-20883781

ABSTRACT

Steroid 5 alpha-reductase inhibitors (5ARIs) have been approved for use clinically in treatment of benign prostate hyperplasia (BPH) and accompanying lower urinary tract symptoms (LUTS) and have also been evaluated in clinical trials for prevention and treatment of prostate cancer. There are currently two steroidal inhibitors in use, finasteride and dutasteride, both with distinct pharmacokinetic properties. This review will examine the evidence presented by various studies supporting the use of these steroidal inhibitors in the prevention and treatment of prostate disease. Article from the Special issue on Targeted Inhibitors.


Subject(s)
5-alpha Reductase Inhibitors/therapeutic use , Clinical Trials as Topic , Prostatic Hyperplasia/drug therapy , Prostatic Neoplasms/drug therapy , 5-alpha Reductase Inhibitors/chemistry , Azasteroids/chemistry , Azasteroids/therapeutic use , Dutasteride , Finasteride/chemistry , Finasteride/therapeutic use , Humans , Male , Molecular Structure
6.
Prostate ; 70(9): 959-70, 2010 Jun 15.
Article in English | MEDLINE | ID: mdl-20166126

ABSTRACT

BACKGROUND: Deregulated androgen receptor (AR) action is critical for prostate cancer (PCa) progression. Aberrant expression of AR-associated coregulators contributes to AR activity in PCa. The mechanisms underlying coregulator expression in PCa are under intense investigation as they may lead to alternative means of targeting AR activity in PCa cells. We have recently shown that over 30% of coregulator expression in the PCa cell line LNCaP is subject to androgen regulation. METHODS: Using multiple PCa cell lines as well as xenograft models, non-malignant prostate epithelial cell lines and androgen-responsive tissues derived from a male Wistar rat model system, we explored the effect of androgen stimulation and androgen deprivation on the expression of the core coactivators SRC1, SRC2, SRC3, CBP, and p300. RESULTS: Androgen stimulation of model systems representing PCa led to a decrease in the expression of SRC1, SRC2, SRC3, CBP, and p300, whereas androgen deprivation induced the expression of these coactivators. In contrast, expression of these coregulators remained largely unaffected following changes in the androgenic milieu in AR-positive models representing non-malignant prostate cells and tissues. CONCLUSIONS: Our data indicate differences in the regulation of coregulator expression between neoplastic and normal prostate cells. These findings emphasize the important potential of targeting the mechanisms regulating coregulator expression for therapeutic intervention in PCa.


Subject(s)
Nuclear Receptor Coactivators/genetics , Prostate/metabolism , Prostatic Neoplasms/genetics , Receptors, Androgen/genetics , p300-CBP Transcription Factors/genetics , Animals , Blotting, Western , Cell Fractionation , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cells, Cultured , Male , Nuclear Receptor Coactivators/metabolism , Prostatic Neoplasms/metabolism , Rats , Rats, Wistar , Receptors, Androgen/metabolism , Reverse Transcriptase Polymerase Chain Reaction , p300-CBP Transcription Factors/metabolism
7.
Cancer Res ; 69(24): 9448-56, 2009 Dec 15.
Article in English | MEDLINE | ID: mdl-19934328

ABSTRACT

By using LNCaP and its derivative cell lines, we first observed an association between tumor necrosis factor-alpha (TNF-alpha) resistance and hormone independence. Moreover, we found that the expression of tumor necrosis factor receptor-associated death domain (TRADD) was reduced in androgen deprivation-independent cells compared with that in androgen deprivation-dependent cells. TRADD is a crucial transducer for TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation. Knocking down TRADD expression in LNCaP cells impaired TNF-alpha-induced NF-kappaB activation and androgen receptor repression, whereas overexpression of TRADD in C4-2B cells restored their sensitivity to TNF-alpha. Finally, we found that androgen deprivation reduces TRADD expression in vitro and in vivo, suggesting that androgen deprivation therapy may promote the development of TNF-alpha resistance by reducing TRADD expression during prostate cancer progression.


Subject(s)
Prostatic Neoplasms/metabolism , TNF Receptor-Associated Death Domain Protein/biosynthesis , Aged , Aged, 80 and over , Androgen Receptor Antagonists , Androgens/deficiency , Cell Line, Tumor , Cell Survival/drug effects , Disease Progression , Humans , Male , Middle Aged , NF-kappa B/metabolism , Neoplasms, Hormone-Dependent/genetics , Neoplasms, Hormone-Dependent/metabolism , Neoplasms, Hormone-Dependent/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Receptors, Androgen/metabolism , TNF Receptor-Associated Death Domain Protein/genetics , Transfection , Tumor Necrosis Factor-alpha/pharmacology
8.
Prostate ; 69(16): 1730-43, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19676081

ABSTRACT

BACKGROUND: In the prostate, androgens play a crucial role in normal and cancerous growth; hence the androgenic pathway has become a target of therapeutic intervention. Dutasteride is a 5 alpha-reductase (5AR) inhibitor currently being evaluated both for chemoprevention and treatment of prostate cancer. Dutasteride inhibits both 5AR I and II enzymes, effectively blocking conversion of testosterone to dihydrotestosterone (DHT) in the prostate. This greatly reduces the amount of the active ligand DHT available for binding to the androgen receptor (AR) and stimulating proliferation, making this a good candidate for chemoprevention of prostate cancer. In this study, we sought to determine how dutasteride is functioning at the molecular level, using a prostate cancer xenograft model. METHODS: Androgen-responsive LuCaP 35 xenograft tumors were grown in Balb/c mice. Subcutaneously implanted time-release pellets were used for drug delivery. Microarray analysis was performed using the Affymetrix HG-U133Av2 platform to examine changes in gene expression in tumors following dutasteride treatment. RESULTS: Dutasteride significantly reduced tumor growth in LuCaP 35 xenografts by affecting genes involved in apoptotic, cytoskeletal remodeling, and cell cycle pathways among others. Notably, genes in the Rho GTPase signaling pathway, shown to be important in androgen-deprivation conditions, were significantly up-regulated. CONCLUSION: We have identified multiple pathways outside of the androgenic pathway in prostate cancer xenografts affected by treatment with dutasteride. These findings provide insights into the function of dutasteride within the tumor microenvironment, potentially allowing for development of agents that can be used in combination with this drug to further enhance its effectiveness.


Subject(s)
Azasteroids/administration & dosage , Cholestenone 5 alpha-Reductase/antagonists & inhibitors , Enzyme Inhibitors/administration & dosage , Neoplasm Transplantation , Prostatic Neoplasms/metabolism , Transplantation, Heterologous , Androgens/deficiency , Animals , Cell Division/drug effects , Delayed-Action Preparations , Drug Implants , Dutasteride , Gene Expression/drug effects , Male , Mice , Mice, Inbred BALB C , Microarray Analysis , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Signal Transduction/drug effects , Up-Regulation , rho GTP-Binding Proteins/metabolism
9.
Mol Endocrinol ; 23(4): 572-83, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19164447

ABSTRACT

Aberrant coregulator expression that occurs during prostate cancer (PCa) progression correlates with poor prognosis and aggressive disease. This has been attributed to the ability to regulate androgen receptor-mediated transcription. We have shown previously that the androgenic milieu regulates the expression of the coactivators p300 and FHL2, with severe consequences for PCa cell proliferation and androgen receptor transcriptional activity. To determine the extent of androgen dependency of coregulator genes, we designed a cDNA-mediated annealing, selection, extension, and ligation RNA profiling array that probes the expression of 186 coregulators. Using this assay, we demonstrated androgen control over approximately 30% of coregulator genes in PCa cells. For a subset of 15 functionally diverse coregulators, androgen regulation was confirmed using real-time RT-PCR and immunoblotting. The extent, dose dependency, and kinetics by which androgens affect coregulator expression differed widely, indicating diverse molecular mechanisms underlying these effects. Moreover, differences in coregulator expression were observed between isogenic androgen-dependent and castration-recurrent PCa cells. Small interfering RNA-mediated changes in coregulator expression had profound effects on cell proliferation, which were most pronounced in castration-recurrent cells. Taken together, our integrated approach combining expression profiling, characterization of androgen-dependent coregulator expression, and validation of the importance of altered coregulator expression for cell proliferation identified several potential novel therapeutic targets for PCa treatment.


Subject(s)
Androgens/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Oligonucleotide Array Sequence Analysis/methods , Prostatic Neoplasms/metabolism , Transcription Factors , Cell Line, Tumor , Cell Proliferation , Humans , Male , Prostatic Neoplasms/pathology , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
10.
Endocr Relat Cancer ; 16(1): 123-37, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18845648

ABSTRACT

Urothelial carcinoma (UC) of the bladder is approximately three times more common in men than women. While the etiology for this gender difference in incidence remains unknown, a role for androgen receptor (AR) signaling has been suggested. The mechanisms by which AR activity is regulated in UC cells, however, are largely elusive. Here, we explore the significance of coregulators that are critical for the formation of a functional AR transcriptional complex, in UC cells. Using two AR-positive UC cell lines, TCC-SUP and UMUC3, we demonstrate the expression of the coactivators NCOA1, NCOA2, NCOA3, CREBBP, and EP300 in UC cells. small interfering RNA-mediated knockdown of the AR or any of these coactivators markedly impacted cell viability and abrogated androgen-dependent cell proliferation. Noteworthy, contrary to AR-positive prostate cancer cells, expression of these AR-associated coactivators was not androgen regulated in UC cells. To assess the clinical relevance of coactivator expression, we performed immunohistochemistry on paraffin-embedded sections from 55 patients with UC of the bladder. We found that while 24 out of 55 (44%) of tumors expressed the AR, each of the coactivators was expressed by 85-100% of the bladder cancers. Moreover, we noted a significant downregulation of NCOA1 expression in tumors versus adjacent, non-tumor bladder urothelium, with a mean of 68% (range 0-100) of tumor cells demonstrating NCOA1 staining versus a mean of 81% (range 0-90) of non-tumor cells (P=0.03). Taken together, our data suggest an important role for AR-associated coactivators in UC and point toward differences in the regulation of AR activity between bladder and prostate cancer cells.


Subject(s)
Histone Acetyltransferases/genetics , Nuclear Receptor Coactivator 2/genetics , Receptors, Androgen/metabolism , Trans-Activators/genetics , Transcription Factors/genetics , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/physiopathology , Androgens/metabolism , CREB-Binding Protein/genetics , CREB-Binding Protein/metabolism , Cell Line, Tumor , E1A-Associated p300 Protein/genetics , E1A-Associated p300 Protein/metabolism , Female , Gene Expression Regulation, Neoplastic , Histone Acetyltransferases/metabolism , Humans , Immunohistochemistry , Male , Nuclear Receptor Coactivator 1 , Nuclear Receptor Coactivator 2/metabolism , Nuclear Receptor Coactivator 3 , RNA, Small Interfering , Signal Transduction/physiology , Trans-Activators/metabolism , Transcription Factors/metabolism , Urinary Bladder Neoplasms/metabolism , Urothelium/physiology
11.
Cancer Res ; 68(13): 5469-77, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18593950

ABSTRACT

The standard systemic treatment for prostate cancer (PCa) is androgen ablation, which causes tumor regression by inhibiting activity of the androgen receptor (AR). Invariably, PCa recurs with a fatal androgen-refractory phenotype. Importantly, the growth of androgen-refractory PCa remains dependent on the AR through various mechanisms of aberrant AR activation. Here, we studied the 22Rv1 PCa cell line, which was derived from a CWR22 xenograft that relapsed during androgen ablation. Three AR isoforms are expressed in 22Rv1 cells: a full-length version with duplicated exon 3 and two truncated versions lacking the COOH terminal domain (CTD). We found that CTD-truncated AR isoforms are encoded by mRNAs that have a novel exon 2b at their 3' end. Functionally, these AR isoforms are constitutively active and promote the expression of endogenous AR-dependent genes, as well as the proliferation of 22Rv1 cells in a ligand-independent manner. AR mRNAs containing exon 2b and their protein products are expressed in commonly studied PCa cell lines. Moreover, exon 2b-derived species are enriched in xenograft-based models of therapy-resistant PCa. Together, our data describe a simple and effective mechanism by which PCa cells can synthesize a constitutively active AR and thus circumvent androgen ablation.


Subject(s)
Alternative Splicing/physiology , Drug Resistance, Neoplasm/genetics , Exons , Prostatic Neoplasms/genetics , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Animals , Antineoplastic Agents, Hormonal/therapeutic use , Base Sequence , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred BALB C , Mice, SCID , Molecular Sequence Data , Prostatic Neoplasms/drug therapy , Protein Isoforms/genetics , Protein Isoforms/physiology , Transfection , Transplantation, Heterologous
12.
Cancer Res ; 67(20): 10067-77, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-17942941

ABSTRACT

Systemic prostate cancer therapy requires androgen ablation, which inhibits the production or action of androgens. Prostate cancer ultimately relapses during androgen ablation, and an androgen depletion-independent (ADI) phenotype emerges. Aberrant androgen receptor (AR) activation underlies therapy resistance at this stage of the disease, and mounting evidence implicates the large and highly disordered AR NH2-terminal domain (NTD) as a key mediator of this activity. In this study, we investigated the role of the NTD transactivation unit 5 (TAU5) domain in mediating AR transcriptional activity in cell-based models of prostate cancer progression. AR replacement and Gal4-based promoter tethering experiments revealed that AR TAU5 had a dichotomous function, inhibiting ligand-dependent AR activity in androgen-dependent prostate cancer cells, while enhancing ligand-independent AR activity in ADI prostate cancer cells. Molecular dissection of TAU5 showed that a WxxLF motif was fully responsible for its ligand-independent activity. Mechanistically, WxxLF did not rely on an interaction with the AR ligand-binding domain to mediate ligand-independent AR activity. Rather, WxxLF functioned as an autonomous transactivation domain. These data show that ligand-dependent and ligand-independent AR activation rely on fundamentally distinct mechanisms, and define WxxLF as the major transactivation motif within the AR TAU5 domain.


Subject(s)
Androgens/deficiency , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Amino Acid Motifs , Amino Acid Sequence , Androgen Receptor Antagonists , Animals , Base Sequence , Humans , Ligands , Male , Mice , Molecular Sequence Data , Prostatic Neoplasms/genetics , Protein Structure, Tertiary , Receptors, Androgen/genetics , Transcriptional Activation
13.
Prostate ; 67(10): 1111-20, 2007 Jul 01.
Article in English | MEDLINE | ID: mdl-17477363

ABSTRACT

PURPOSE: With malignant progression to androgen independence, prostate cancer cells develop resistance to apoptosis and exhibit a variety of gene expression changes, including increased fatty acid synthase (FASN) expression. Increased FASN expression has been shown to correlate with poor prognosis, and correspondingly, the FASN gene has been proposed as a therapeutic target. Because FASN is an androgen regulated gene in the prostate, we have examined the effects of dutasteride on FASN in prostate cancer cells in vitro. Dutasteride is a novel dual inhibitor of the 5 alpha-reductase enzymes and is currently in use both for treatment of benign prostate hyperplasia (BPH) and in the reduction by dutasteride of prostate cancer events (REDUCE) prostate cancer prevention trial. METHODS: Microarray analysis was used to identify genes affected by treatment with dutasteride, followed by real time PCR confirmation. FASN expression at the protein level was examined using Western blotting and immunocytochemistry. Enzymatic activity of FASN was assayed by (14)C-labeled malonyl-CoA incorporation. Viability after dutasteride treatment was assayed by MTS (Promega) and apoptosis via caspase 3/7 by DEVD cleavage assay. RESULTS: We have demonstrated that the 5 alpha-reductase inhibitor dutasteride, at clinically relevant levels, inhibits FASN mRNA, protein expression and enzymatic activity in prostate cancer cells. CONCLUSIONS: This is the first study to examine the effects of clinically relevant levels of dutasteride on prostate cancer cells at the molecular level and specifically, demonstrating the inhibition of FASN in these cells.


Subject(s)
Azasteroids/pharmacology , Enzyme Inhibitors/pharmacology , Fatty Acid Synthases/antagonists & inhibitors , Fatty Acid Synthases/metabolism , Prostatic Neoplasms/enzymology , Apoptosis/drug effects , Apoptosis/physiology , Caspase 3/metabolism , Caspase 7/metabolism , Cell Line, Tumor , Cholestenone 5 alpha-Reductase/genetics , Cholestenone 5 alpha-Reductase/metabolism , Dose-Response Relationship, Drug , Dutasteride , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Humans , Male , Prostatic Neoplasms/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Sterol Regulatory Element Binding Proteins/physiology
14.
Genes Chromosomes Cancer ; 45(7): 702-16, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16615098

ABSTRACT

Studies on the genetic basis of prostate cancer (PCa) have lead to mixed results with the only consensus being that PCa is a complex disease. Our goal was to gain insight into potential events involved in the acquisition of the androgen-refractory phenotype in PCa cells regardless of DNA-change dependence. To this end, we examined two LNCaP PCa cell line models of progression-one developed in vivo and one developed in vitro-using molecular cytogenetic and microarray gene expression analyses and extended this investigation of specific events into PCa tumors. The chromosomal changes observed in both in vivo and in vitro androgen-independent cell lines are similar to those seen in PCa during tumor progression. Correspondingly, gene expression analysis showed significant heterogeneity in the genes expressed among androgen-independent cells, but with some common gene expression changes that correlated with the acquired androgen-independent phenotype. Thus, growth conditions under which the cells progress appeared to impact the mechanisms used for progression, albeit within tumor-type-specific pathways. Our findings suggest that a dynamic and adaptable combination of epigenetic and DNA-change-dependent events can be used by PCa cells for the acquisition of the androgen-independent phenotype. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.


Subject(s)
Androgens/metabolism , Epigenesis, Genetic , Prostatic Neoplasms/genetics , Animals , Cell Line, Tumor , Chromosome Banding , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Humans , In Situ Hybridization, Fluorescence , Loss of Heterozygosity , Male , Mice , Oligonucleotide Array Sequence Analysis , Phenotype , Prostatic Neoplasms/metabolism , Spectral Karyotyping
15.
Cancer Res ; 65(13): 5965-73, 2005 Jul 01.
Article in English | MEDLINE | ID: mdl-15994976

ABSTRACT

Prostate cancer is the most frequent non-skin cancer in men. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor has been shown to play an important role. Androgen receptor is expressed in both early and late-stage prostate cancer. Also, androgen-regulated pathways are thought to be active as evidenced by elevated levels of prostate-specific antigen (PSA). In addition, several androgen receptor coactivators and cytokines are involved in prostate cancer progression. In this regard, we have shown previously that the coactivator p300 plays a major role in the androgen-independent activation of PSA by interleukin 6 (IL-6), a cytokine involved in late-stage prostate cancer. In this study, we investigated the role of p300 and its homologue CREB-binding protein in prostate cancer cells treated chronically with IL-6. We found that p300 but not CREB-binding protein induced activation of PSA in these cells and that the histone acetyltransferase activity of p300 was critical. This effect was independent of the presence of androgens or antiandrogens. Moreover, we found markedly reduced levels of androgen receptor in these cells and p300 transfection did not affect those levels, suggesting that the p300 effect on PSA could be bypassing the androgen receptor. Transfection with exogenous androgen receptor showed minimal response of PSA to androgens but higher response to p300. We found similar effects of p300 on the androgen response element III, which mediates the androgen receptor-dependent activation of PSA. Finally, we showed that p300 alone regulates expression of the endogenous PSA gene in the IL-6-treated cells. These findings reveal a new insight in the progression of prostate cancer, suggesting that coactivators, such as p300, play more important roles in late-stage prostate cancer, and could regulate androgen-dependent genes in the absence or with very low levels of androgen receptor.


Subject(s)
Interleukin-6/pharmacology , Nuclear Proteins/physiology , Prostate-Specific Antigen/biosynthesis , Receptors, Androgen/physiology , Trans-Activators/physiology , Androgens/deficiency , Androgens/physiology , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Interleukin-6/physiology , Male , Nuclear Proteins/genetics , Prostate-Specific Antigen/genetics , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/immunology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Trans-Activators/genetics , Transfection
16.
J Androl ; 25(6): 944-53, 2004.
Article in English | MEDLINE | ID: mdl-15477368

ABSTRACT

We sought preclinical data on the cellular and molecular effects of dutasteride in androgen-responsive, human prostate cancer (PCa) cells to better understand the mechanisms of action of 5 alpha-reductase inhibition in these cells. We used the human prostate cancer cell line LNCaP, which exhibits most features of PCa cells including androgen responsiveness. Our findings show that dutasteride kills PCa cells in vitro; it dramatically reduced viability and proliferation and disrupted genes and cellular pathways involved in metabolic, cell cycle, and apoptotic responses besides those expected in androgen-signaling pathways. Microchip gene array expression analysis revealed activation of genes in the FasL/tumor necrosis factor alpha (TNF-alpha) apoptotic and cell-survival pathways, correlating with the growth and survival effects in the LNCaP cells. Real-time polymerase chain reaction confirmed expression level changes seen by microarray analysis of candidate genes such as PLA2G2A, CDK8, CASP7, MDK, and NKX3.1. Collectively, our findings delineate the cellular and molecular effects of dutasteride in androgen-responsive PCa cells in vitro and may lead to its better therapeutic and chemopreventive use in PCa.


Subject(s)
Azasteroids/pharmacology , Cholestenone 5 alpha-Reductase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Gene Expression , Prostatic Neoplasms/genetics , Apoptosis/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Dutasteride , Humans , Male , Oligonucleotide Array Sequence Analysis , Prostatic Neoplasms/pathology , Prostatic Neoplasms/physiopathology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics
17.
Cancer Res ; 62(20): 5632-6, 2002 Oct 15.
Article in English | MEDLINE | ID: mdl-12384515

ABSTRACT

Prostate cancer (PCa) begins as an androgen-dependent tumor that will eventually progress to an androgen-independent stage after androgen ablation. Although little is understood about this transition to androgen independence, the androgen receptor (AR) appears to be involved. The coactivator p300 has been shown to interact with the AR during its androgen-dependent transactivation. We show that p300 is involved downstream of the mitogen-activated protein kinase pathway during transactivation of the AR by interleukin-6 (IL-6). Furthermore, we demonstrate that sequestration of p300 with E1A inhibits the IL-6-dependent transactivation of the AR, and that increasing amounts of p300 reverse this inhibition. A mutant p300 that lacks histone acetyltransferase (HAT) activity did not reverse E1A-mediated inhibition. By using small-interference RNA designed to target p300 transcripts, we demonstrate that, after silencing p300, there was no induction of AR activity by IL-6. These findings reveal a unique role for p300 and its HAT activity, indicating that it is necessary for the ligand-independent transactivation of the AR in androgen-independent PCa cells.


Subject(s)
Acetyltransferases/physiology , Cell Cycle Proteins/physiology , Interleukin-6/pharmacology , Neoplasms, Hormone-Dependent/metabolism , Prostatic Neoplasms/metabolism , Receptors, Androgen/physiology , Transcriptional Activation/physiology , Acetyltransferases/antagonists & inhibitors , Acetyltransferases/biosynthesis , Acetyltransferases/genetics , Androgens/physiology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/genetics , Histone Acetyltransferases , Humans , Immunohistochemistry , Interleukin-6/antagonists & inhibitors , Interleukin-6/physiology , MAP Kinase Signaling System/physiology , Male , Neoplasms, Hormone-Dependent/enzymology , Neoplasms, Hormone-Dependent/genetics , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/genetics , RNA, Small Interfering/genetics , Receptors, Androgen/genetics , Transcription Factors , Transfection , Tumor Cells, Cultured , p300-CBP Transcription Factors
18.
Cancer Res ; 62(4): 1008-13, 2002 Feb 15.
Article in English | MEDLINE | ID: mdl-11861374

ABSTRACT

Prostate cancer cells depend on androgens and the androgen receptor (AR) for survival. However, after androgen ablation therapy, tumors relapse to an androgen-refractory state. To determine whether the androgen receptor is critical for proliferation of androgen-refractory prostate cancer cells, we disrupted the activity of the androgen receptor with an antibody and an AR mRNA hammerhead ribozyme in the following cell lines: LNCaP (androgen-sensitive), LNCaP-Rf and LNCaP-C4 (androgen-refractory), and DU-145 (androgen-insensitive). Microinjection of either antibody or ribozyme inhibited proliferation of androgen-refractory cells. These findings demonstrate that the AR is critical for proliferation of androgen-refractory cells, even in the absence of androgens.


Subject(s)
Androgen Receptor Antagonists , Prostatic Neoplasms/pathology , Androgen Antagonists/pharmacology , Antibodies, Neoplasm/pharmacology , Cell Division/physiology , Drug Resistance, Neoplasm , Humans , Male , Neoplasms, Hormone-Dependent/pathology , RNA, Catalytic/pharmacology , Receptors, Androgen/genetics , Receptors, Androgen/immunology , Receptors, Androgen/physiology
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