RESUMO
BACKGROUND: Improving our ability to predict cancer progression and response to conservative or radical intent therapy is critical if we are to prevent under or over treatment of individual patients. Whereas the majority of solid tumors now have a range of molecular and/or immunological markers to help define prognosis and treatment options, prostate cancer still relies mainly on histological grading and clinical parameters. We have recently reported that androgen receptor (AR) expression in stroma inversely associates with prostate cancer-specific survival, and that stromal AR reduces metastasis. For this paper, we tested the hypothesis that the AR-regulated gene FKBP51 could be used as a marker of AR activity to better predict outcome. METHODS: Using immunohistochemistry on a cohort of 64 patient-matched benign and malignant prostate tissues, we assessed patient outcome by FKBP51 and AR levels. Immunoblot and RT-qPCR were used to demonstrate androgen regulation of FKBP51 in primary and primary human prostatic fibroblasts and fibroblast cell-lines. RESULTS: As predicted by FKBP51 level, high AR activity in cancer stroma was associated with longer median survival (1,306 days) compared with high AR alone (699 days), whereas those with low AR and/or low FKBP51 did poorly (384 and 338 days, respectively). Survival could not be predicted on the basis cancer epithelial AR levels or activity, and was not associated with immunoreactivity in patient matched benign tissues. CONCLUSION: FKBP51 improves the ability of stromal AR to predict prostate cancer-specific mortality. By adding additional immunological assessment, similar to what is already in place in a number of other cancers, we could better serve patients with prostate cancer in prognosis and informed treatment choices. Prostate 77:185-195, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Humanos , Masculino , Prognóstico , Células Estromais/metabolismo , Células Estromais/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Esophageal adenocarcinoma is a male-dominant disease, but the role of androgens is unclear. AIMS: To examine the expression and clinical correlates of the androgen receptor (AR) and the androgen-responsive gene FK506-binding protein 5 (FKBP5) in esophageal adenocarcinoma. METHODS: Expression of AR and FKBP5 was determined by immunohistochemistry. The effect of the AR ligand 5α-dihydrotestosterone (DHT) on the expression of a panel of androgen-responsive genes was measured in AR-positive and AR-negative esophageal adenocarcinoma cell lines. Correlations in expression between androgen-responsive genes were analyzed in an independent cohort of esophageal adenocarcinoma tissues. RESULTS: There was AR staining in 75 of 77 cases (97 %), and FKBP5 staining in 49 (64 %), all of which had nuclear AR. Nuclear AR with FKBP5 expression was associated with decreased median survival (451 vs. 2800 days) and was an independent prognostic indicator (HR 2.894, 95 % CI 1.3966.002, p = 0.0043) in multivariable Cox proportional hazards models. DHT induced a significant increase in expression of the androgen-responsive genes FKBP5, HMOX1, FBXO32, VEGFA, WNT5A, and KLK3 only in AR-positive cells in vitro. Significant correlations in expression were observed between these androgen-responsive genes in an independent cohort of esophageal adenocarcinoma tissues. CONCLUSION: Nuclear AR and expression of FKBP5 is associated with decreased survival in esophageal adenocarcinoma.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Predisposição Genética para Doença , Receptores Androgênicos/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Adenocarcinoma/genética , Linhagem Celular Tumoral , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Prognóstico , Receptores Androgênicos/genética , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
BACKGROUND: In breast cancer, progesterone receptor (PR) positivity or abundance is positively associated with survival and treatment response. It was initially believed that PR was a useful diagnostic marker of estrogen receptor activity, but increasingly PR has been recognised to play an important biological role in breast homeostasis, carcinogenesis and metastasis. Although PR expression is almost exclusively observed in estrogen receptor positive tumors, few studies have investigated the cellular mechanisms of PR action in the context of ongoing estrogen signalling. METHODS: In this study, we contrast PR function in estrogen pretreated ZR-75-1 breast cancer cells with vehicle treated ZR-75-1 and T-47D breast cancer cells using expression microarrays and chromatin immunoprecipitation-sequencing. RESULTS: Estrogen cotreatment caused a dramatic increase in the number of genes regulated by progesterone in ZR-75-1 cells. In T-47D cells that have naturally high levels of PR, estrogen and progesterone cotreatment resulted in a reduction in the number of regulated genes in comparison to treatment with either hormone alone. At a genome level, estrogen pretreatment of ZR-75-1 cells led to a 10-fold increase in the number of PR DNA binding sites detected using ChIP-sequencing. Time course assessment of progesterone regulated genes in the context of estrogen pretreatment highlighted a series of important regulatory pathways, including those driven by epithelial growth factor receptor (EGFR). Importantly, progesterone applied to cells pretreated with estradiol resulted in switching of the PAM50-determined intrinsic breast cancer subtype from Luminal A to Basal-like, and increased the Oncotype DX® Unscaled Recurrence Score. CONCLUSION: Estrogen pretreatment of breast cancer cells increases PR steady state levels, resulting in an unequivocal progesterone response that upregulates key members of growth factor pathways. The transformative changes progesterone exerts on the breast cancer subtype suggest that these subtyping tools should be used with caution in premenopausal women.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptores ErbB/biossíntese , Estrogênios/administração & dosagem , Progesterona/administração & dosagem , Ativação Transcricional/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Receptores de Progesterona/biossíntese , Ativação Transcricional/fisiologia , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
It is well established that the development and homeostasis of the mammary gland are highly dependent upon the actions of ovarian hormones progesterone and estrogen, as well as the availability of prolactin for the pregnant and lactating gland. More recently it has become apparent that immune system cells and cytokines play essential roles in both mammary gland development as well as breast cancer. Here, we review hormonal effects on mammary gland biology during puberty, menstrual cycling, pregnancy, lactation and involution, and dissect how hormonal control of the immune system may contribute to mammary development at each stage via cytokine secretion and recruitment of macrophages, eosinophils, mast cells and lymphocytes. Collectively, these alterations may create an immunotolerant or inflammatory immune environment at specific developmental stages or phases of the menstrual cycle. Of particular interest for further research is investigation of the combinatorial actions of progesterone and estrogen during the luteal phase of the menstrual cycle and key developmental points where the immune system may play an active role both in mammary development as well as in the creation of an immunotolerant environment, thereby affecting breast cancer risk.
Assuntos
Microambiente Celular/imunologia , Hormônios/imunologia , Hormônios/metabolismo , Sistema Imunitário/metabolismo , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Feminino , Humanos , Sistema Imunitário/imunologia , Lactação/imunologia , Lactação/metabolismo , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Humanas/imunologia , Neoplasias Mamárias Animais/imunologia , Neoplasias Mamárias Animais/metabolismo , GravidezRESUMO
Solid tumors have an increased reliance on Hsp70/Hsp90 molecular chaperones for proliferation, survival and maintenance of intracellular signaling systems. An underinvestigated component of the chaperone system is the tetratricopeptide repeat (TPR)-containing cochaperone, which coordinates Hsp70/Hsp90 involvement on client proteins as well as having diverse individual actions. A potentially important cochaperone in prostate cancer (PCa) is small glutamine-rich TPR-containing protein alpha (SGTA), which interacts with the androgen receptor (AR) and other critical cancer-related client proteins. In this study, the authors used small interfering RNA coupled with genome-wide expression profiling to investigate the biological significance of SGTA in PCa and its influence on AR signaling. Knockdown of SGTA for 72 hr in PCa C4-2B cells significantly altered expression of >1,900 genes (58% decreased) and reduced cell proliferation (p < 0.05). The regulation of 35% of 5α-dihydrotestosterone (DHT) target genes was affected by SGTA knockdown, with gene-specific effects on basal or DHT-induced expression or both. Pathway analysis revealed a role for SGTA in p53, generic PCa and phosphoinositol kinase (PI3K) signaling pathways; the latter evident by a reduction in PI3K subunit p100ß levels and decreased phosphorylated Akt. Immunohistochemical analysis of 64 primary advanced PCa samples showed a significant increase in the AR:SGTA ratio in cancerous lesions compared to patient-matched benign prostatic hyperplasia tissue (p < 0.02). This study not only provides insight into the biological actions of SGTA and its effect on genome-wide AR transcriptional activity and other therapeutically targeted intracellular signaling pathways but also provides evidence for PCa-specific alterations in SGTA expression.
Assuntos
Proteínas de Transporte/fisiologia , Proliferação de Células , Fosfatidilinositol 3-Quinases/fisiologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptores Androgênicos/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/antagonistas & inibidores , Di-Hidrotestosterona/farmacologia , Humanos , Masculino , Camundongos , Chaperonas Moleculares , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/análise , Transcriptoma , Proteína Supressora de Tumor p53/análiseRESUMO
Diamide linked γ-cyclodextrin (γ-CD) dimers are proposed as molecular-scale delivery agents for the anticancer agent curcumin. N,N'-Bis(6(A)-deoxy-γ-cyclodextrin-6(A)-yl)succinamide (66γCD2su) and N,N'-bis(6(A)-deoxy-γ-cyclodextrin-6(A)-yl)urea (66γCD2ur) markedly suppress the degradation of curcumin by forming a strong 1:1 cooperative binding complexes. The results presented in this study describe the potential efficacy of 66γCD2su and 66γCD2ur for intracellular curcumin delivery to cancer cells. Cellular viability assays demonstrated a dose-dependent antiproliferative effect of curcumin in human prostate cancer (PC-3) cells that was preserved by the curcumin-66γCD2su complex. In contrast, delivery of curcumin by 66γCD2ur significantly delayed the antiproliferative effect. We observed similar patterns of gene regulation in PC-3 cells for curcumin complexed with either 66γCD2su or 66γCD2ur in comparison to curcumin alone, although curcumin delivered by either 66γCD2su or 66γCD2ur induces a slightly higher up-regulation of heme oxygenase-1. Highlighting their nontoxic nature, neither 66γCD2su nor 66γCD2ur carriers alone had any measurable effect on cell proliferation or candidate gene expression in PC-3 cells. Finally, confocal fluorescence imaging and uptake studies were used to demonstrate the intracellular delivery of curcumin by 66γCD2su and 66γCD2ur. Overall, these results demonstrate effective intracellular delivery and action of curcumin when complexed with 66γCD2su and 66γCD2ur, providing further evidence of their potential applications to deliver curcumin effectively in cancer and other treatment settings.
Assuntos
Curcumina/química , Curcumina/farmacologia , Diamida/química , Neoplasias da Próstata/tratamento farmacológico , gama-Ciclodextrinas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
OBJECTIVE: To assess if a cell-based readout of androgen action in serum demonstrates a closer association with recognized classical parameters of androgen action in men than current measures of serum testosterone (T). DESIGN: To develop, validate and utilize a mammalian cell-based assay to measure specifically bioactive T and determine if this measure is a physiologically relevant fraction of serum T. MEASUREMENTS AND PARTICIPANTS: We have developed a specific serum T bioassay using human prostate cancer cells. A rapid 5-min exposure to 100% serum followed by serum withdrawal confers specificity of the assay to serum T and provides sufficient sensitivity to measure T in male serum samples. Matrix effects were experimentally discounted as a confounding issue. A total of 960 male serum samples from the Florey Adelaide Male Ageing Study (FAMAS) with previous comprehensive cohort data and serum measurements were utilized. RESULTS: Bioassay T measurement in the 960 FAMAS serum samples returned a median of 10.7 nmol/l (1.7-45.4), and was most closely related to immunoassayed total T, but not immunoassayed bioavailable T or calculated free T. Immunoassayed total T demonstrated a positive association with isometric grip-strength (R(2) = 0.127, P < 0.001), self-reported sexual desire (R(2) = 0.113, P < 0.001) and erectile function (R(2) = 0.085, P < 0.05) while bioassay T did not. CONCLUSIONS: While cellular bioassays offer a rapid and sensitive means of identifying the androgenic potential of complex environmental compounds, the utility of such assays in defining a clinically relevant fraction of serum T distinct from total T needs further investigation.
Assuntos
Bioensaio/métodos , Testosterona/análise , Testosterona/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Bioensaio/normas , Células CHO , Células COS , Células Cultivadas , Chlorocebus aethiops , Estudos de Coortes , Cricetinae , Cricetulus , Técnicas de Diagnóstico Endócrino/normas , Humanos , Masculino , Pessoa de Meia-Idade , Testosterona/normasRESUMO
Fibroblasts express androgen receptor (AR) in the normal prostate and during prostate cancer development. We have reported that loss of AR expression in prostate cancer-associated fibroblasts is a poor prognostic indicator. Here we report outcomes of direct and indirect co-cultures of immortalised AR-positive (PShTert-AR) or AR-negative (PShTert) myofibroblasts with prostate cancer cells. In the initial co-cultures the AR-negative PC3 cell line was used so AR expression and signalling were restricted to the myofibroblasts. In both direct and indirect co-culture with PShTert-AR myofibroblasts, paracrine signalling to the PC3 cells slowed proliferation and induced apoptosis. In contrast, PC3 cells proliferated with PShTert myofibroblasts irrespective of the co-culture method. In direct co-culture PC3 cells induced apoptosis in and destroyed PShTerts by direct signalling. Similar results were seen in direct co-cultures with AR-negative DU145 and AR-positive LNCaP and C4-2B prostate cancer cell lines. The AR ligand 5α-dihydrotestosterone (DHT) inhibited the proliferation of the PShTert-AR myofibroblasts, thereby reducing the extent of their inhibitory effect on cancer cell growth. These results suggest loss of stromal AR would favour prostate cancer cell growth in vivo, providing an explanation for the clinical observation that reduced stromal AR is associated with a poorer outcome.
RESUMO
Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechanisms underpinning stromal AR contribution to disease development and progression remain unclear. Using patient-matched benign and malignant prostate samples, we show a significant association between low AR levels in cancer associated stroma and increased prostate cancer-related death at one, three and five years post-diganosis, and in tissue recombination models with primary prostate cancer cells that low stromal AR decreases castration-induced apoptosis. AR-regulation was found to be different in primary human fibroblasts isolated from adjacent to cancerous and non-cancerous prostate epithelia, and to represent altered activation of myofibroblast pathways involved in cell cycle, adhesion, migration, and the extracellular matrix (ECM). Without AR signaling, the fibroblast-derived ECM loses the capacity to promote attachment of both myofibroblasts and cancer cells, is less able to prevent cell-matrix disruption, and is less likely to impede cancer cell invasion. AR signaling in prostate cancer stroma appears therefore to alter patient outcome by maintaining an ECM microenvironment inhibitory to cancer cell invasion. This paper provides comprehensive insight into AR signaling in the non-epithelial prostate microenvironment, and a resource from which the prognostic and therapeutic implications of stromal AR levels can be further explored.
Assuntos
Miofibroblastos/patologia , Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Células Estromais/patologia , Microambiente Tumoral , Idoso , Idoso de 80 Anos ou mais , Androgênios/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Estudos de Casos e Controles , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Gradação de Tumores , Invasividade Neoplásica , Orquiectomia , Prognóstico , Hiperplasia Prostática/tratamento farmacológico , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Análise Serial de Tecidos , Células Tumorais CultivadasRESUMO
There is extensive knowledge of androgen receptor (AR) signaling in cancer cells, but less regarding androgen action in stromal cells of the tumor microenvironment. We report here the genome-wide effects of a stromal cell specific molecular adapter and AR coregulator, hydrogen peroxide-inducible gene 5 (Hic-5/TGFB1I1), on AR function in prostate myofibroblasts. Following androgen stimulation, Hic-5 rapidly translocates to the nucleus, coincident with increased phosphorylation of focal adhesion kinase. As a coregulator, Hic-5 acted to amplify or inhibit regulation of approximately 50% of AR target genes, affected androgen regulation of growth, cell adhesion, motility and invasion. These data suggest Hic-5 as a transferable adaptor between focal adhesions and the nucleus of prostate myofibroblasts, where it acts a key mediator of the specificity and sensitivity of AR signaling. We propose a model in which Hic-5 coordinates AR signaling with adhesion and extracellular matrix contacts to regulate cell behavior in the tumor microenvironment.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Miofibroblastos/metabolismo , Receptores Androgênicos/genética , Células Estromais/metabolismo , Androgênios/farmacologia , Adesão Celular , Linhagem Celular Transformada , Movimento Celular , Núcleo Celular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Masculino , Anotação de Sequência Molecular , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Fosforilação , Próstata/metabolismo , Próstata/patologia , Transporte Proteico , Receptores Androgênicos/metabolismo , Transdução de Sinais , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Testosterona/farmacologia , Microambiente TumoralRESUMO
Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and is associated with an increased risk of breast cancer, possibly due to disruption of androgen receptor (AR) signaling. In contrast, the synthetic HRT Tibolone does not increase breast density, and is rapidly metabolized to estrogenic 3α-OH-tibolone and 3ß-OH-tibolone, and a delta-4 isomer (Δ(4)-TIB) that has both androgenic and progestagenic properties. Here, we show that 5α-dihydrotestosterone (DHT) and Δ(4)-TIB, but not MPA, stabilize AR protein levels, initiate specific AR intramolecular interactions critical for AR transcriptional regulation, and increase proliferation of AR positive MDA-MB-453 breast cancer cells. Structural modeling and molecular dynamic simulation indicate that Δ(4)-TIB induces a more stable AR structure than does DHT, and MPA a less stable one. Microarray expression analyses confirms that the molecular actions of Δ(4)-TIB more closely resembles DHT in breast cancer cells than either ligand does to MPA.
Assuntos
Androgênios/farmacologia , Di-Hidrotestosterona/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/genética , Norpregnenos/farmacologia , Receptores Androgênicos/genética , Androgênios/química , Androgênios/metabolismo , Biotransformação , Linhagem Celular Tumoral , Di-Hidrotestosterona/química , Di-Hidrotestosterona/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Acetato de Medroxiprogesterona/química , Acetato de Medroxiprogesterona/farmacologia , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/metabolismo , Norpregnanos/metabolismo , Norpregnenos/química , Norpregnenos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Androgênicos/metabolismo , Relação Estrutura-AtividadeRESUMO
The androgen receptor (AR) signaling pathway is a common therapeutic target for prostate cancer, because it is critical for the survival of both hormone-responsive and castrate-resistant tumor cells. Most of the detailed understanding that we have of AR transcriptional activation has been gained by studying classical target genes. For more than two decades, Kallikrein 3 (KLK3) (prostate-specific antigen) has been used as a prototypical AR target gene, because it is highly androgen responsive in prostate cancer cells. Three regions upstream of the KLK3 gene, including the distal enhancer, are known to contain consensus androgen-responsive elements required for AR-mediated transcriptional activation. Here, we show that KLK3 is one of a specific cluster of androgen-regulated genes at the centromeric end of the kallikrein locus with enhancers that evolved from the long terminal repeat (LTR) (LTR40a) of an endogenous retrovirus. Ligand-dependent recruitment of the AR to individual LTR-derived enhancers results in concurrent up-regulation of endogenous KLK2, KLK3, and KLKP1 expression in LNCaP prostate cancer cells. At the molecular level, a kallikrein-specific duplication within the LTR is required for maximal androgen responsiveness. Therefore, KLK3 represents a subset of target genes regulated by repetitive elements but is not typical of the whole spectrum of androgen-responsive transcripts. These data provide a novel and more detailed understanding of AR transcriptional activation and emphasize the importance of repetitive elements as functional regulatory units.
Assuntos
Androgênios/metabolismo , Calicreínas/metabolismo , Antígeno Prostático Específico/biossíntese , Sequências Repetidas Terminais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Centrômero/metabolismo , Células Epiteliais/citologia , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Retroviridae/genéticaRESUMO
The cellular response to circulating sex steroids is more than the sum of individual hormone actions, instead representing an interplay between activities of the evolutionarily related steroid hormone receptors. An example of this interaction is in breast cancer, where the risk of dying from estrogen receptor-α (ERα)-positive disease decreases approximately 4-fold when androgen receptor (AR) expression is high. In this study, we used chromatin immunoprecipitation sequencing (ChIP-seq) and microarray expression profiling to investigate the genomic and transcriptional cross talk between AR and ERα signaling in a luminal breast cancer cell line model, ZR-75-1. Expression profiling demonstrated reciprocal interference between 5α-dihydrotestosterone (DHT)- and 17ß-estradiol (E(2))-induced transcriptional programs. Specifically, regulation of 26% of E(2) and 15% of DHT target genes was significantly affected by cotreatment with the other hormone, in the majority of cases (78-83%) antagonistically. Pathway analysis suggested that DHT cotreatment, for example, depleted E(2)-regulated pathways in cell survival and proliferation. ChIP-seq identified substantial overlap between the steroid receptor cistromes in ZR-75-1 cells, with 10-13% of AR- and ERα-binding sites located within 10 kb of the other receptor. Enrichment of androgen response elements in ERα-binding sites and vice versa was revealed by motif analysis, and AR-binding sites were enriched about E(2)-responsive genes affected by DHT cotreatment. Targeted ChIP and expression analysis revealed locus-specific outcomes when AR and ERα bind to the same DNA region. This work provides the first cistrome data for two steroid receptors in the same cell, insight into the antagonistic interplay between estrogens and androgens in luminal breast cancer, and an important resource for future work aimed at evaluating interrelated steroid receptors in different cellular systems.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Redes Reguladoras de Genes/genética , Genoma Humano/genética , Receptores Androgênicos/genética , Androgênios/farmacologia , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Feminino , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Receptores Androgênicos/metabolismoRESUMO
Ligand-dependent activity of steroid receptors is affected by tetratricopeptide repeat (TPR)-containing co-chaperones, such as small glutamine-rich tetratricopeptide repeat-containing alpha (SGTA). However, the precise mechanisms by which the predominantly cytoplasmic TPR proteins affect downstream transcriptional outcomes of steroid signaling remain unclear. In this study, we assessed how SGTA affects ligand sensitivity and action of the androgen receptor (AR) using a transactivation profiling approach. Deletion mapping coupled with structural prediction, transcriptional assays, and in vivo regulation of AR-responsive promoters were used to assess the role of SGTA domains in AR responses. At subsaturating ligand concentrations of ≤ 0.1 nM 5α-dihydrotestosterone, SGTA overexpression constricted AR activity by an average of 32% (P<0.002) across the majority of androgen-responsive loci tested, as well as on endogenous promoters in vivo. The strength of the SGTA effect was associated with the presence or absence of bioinformatically predicated transcription factor motifs at each site. Homodimerizaion of SGTA, which is thought to be necessary for chaperone complex formation, was found to be dependent on the structural integrity of amino acids 1-80, and a core evolutionary conserved peptide within this region (amino acids 21-40) necessary for an effect of SGTA on the activity of both exogenous and endogenous AR. This study provides new insights into the subdomain structure of SGTA and how SGTA acts as a regulator of AR ligand sensitivity. A change in AR:SGTA ratio will impact the cellular and molecular response of prostate cancer cells to maintain androgenic signals, which may influence tumor progression.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Chaperonas Moleculares/metabolismo , Receptores Androgênicos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , Sequência Conservada , Di-Hidrotestosterona/metabolismo , Genes Reporter , Ligantes , Multimerização Proteica , Estrutura Terciária de Proteína , Receptores Androgênicos/genética , Fatores de Transcrição/metabolismoRESUMO
The response of prostate cells to androgens reflects a combination of androgen receptor (AR) transactivation and transrepression, but how these two processes differ mechanistically and influence prostate cancer risk and disease outcome remain elusive. Given recent interest in targeting AR transrepressive processes, a better understanding of AR/corepressor interaction and responses is warranted. Here, we used transactivation and interaction assays with wild-type and mutant ARs, and deletion AR fragments, to dissect the relationship between AR and the corepressor, silencing mediator for retinoic acid and thyroid hormone receptors (SMRT). We additionally tested how these processes are influenced by AR agonist and antagonist ligands, as well as by variation in the polyglutamine tract in the AR amino terminal domain (NTD), which is encoded by a polymorphic CAG repeat in the gene. SMRT was recruited to the AR ligand binding domain by agonist ligand, and as determined by the effect of strategic mutations in activation function 2 (AF-2), requires a precise conformation of that domain. A distinct region of SMRT also mediated interaction with the AR-NTD via the transactivation unit 5 (TAU5; residues 315-538) region. The degree to which SMRT was able to repress AR increased from 17% to 56% as the AR polyglutamine repeat length was increased from 9 to 42 residues, but critically this effect could be abolished by increasing the SMRT:AR molar ratio. These data suggest that the extent to which the CAG encoded polyglutamine repeat influences AR activity represents a balance between corepressor and coactivator occupancy of the same ligand-dependent and independent AR interaction surfaces. Changes in the homeostatic relationship of AR to these molecules, including SMRT, may explain the variable penetrance of the CAG repeat and the loss of AR signaling flexibility in prostate cancer progression.
Assuntos
Proteínas Correpressoras/metabolismo , Correpressor 2 de Receptor Nuclear/metabolismo , Peptídeos/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Sequências Repetitivas de Ácido Nucleico , Antagonistas de Receptores de Andrógenos/metabolismo , Androgênios/análise , Animais , Linhagem Celular , Chlorocebus aethiops , Proteínas Correpressoras/genética , Masculino , Mutação , Correpressor 2 de Receptor Nuclear/genética , Peptídeos/genética , Próstata/metabolismo , Neoplasias da Próstata/genética , Domínios e Motivos de Interação entre Proteínas , Receptores Androgênicos/genética , Ativação TranscricionalRESUMO
IMPORTANCE OF THE FIELD: The TGF-beta's are pleiotropic cytokines that regulate multiple cellular functions. Their role in the prostate is important for normal prostate development and also in prostate tumourigenesis. AREAS COVERED IN THIS REVIEW: The interactions TGF-beta-mediated signalling has with maintaining prostate health, as well as its role in prostate tumourigenesis and prostate tumour immune evasion, with emphasis on how a breakdown in these interactions may influence disease progression. WHAT THE READER WILL GAIN: That TGF-beta influences normal prostate growth and differentiation by regulating the balance between epithelial cell proliferation and apoptosis, and involving the androgen receptor pathway. That TGF-beta protects and maintains prostate stem cells and a review of the contrasting role TGF-beta has in prostate tumourigenesis and tumour development, where TGF-beta acts as a tumour suppressor and then switches roles to become a tumour promoter, and creates a local immunosuppressive niche leading to systemic tumour tolerance. TAKE HOME MESSAGE: TGF-beta signalling in prostate cancer is a valid target for the treatment of this disease; however any therapeutic regimen will require an understanding of all aspects of the TGF-beta-signalling nexus, otherwise by the very pleiotrophic nature of TGF-beta, limited clinical benefits may result.
Assuntos
Neoplasias da Próstata/etiologia , Neoplasias da Próstata/imunologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Antagonistas de Androgênios/farmacologia , Animais , Humanos , Ativação Linfocitária , Masculino , Proteoglicanas/fisiologia , Receptores Androgênicos/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Células-Tronco/fisiologia , Linfócitos T/imunologia , Evasão TumoralRESUMO
The androgen receptor (AR) is an important signaling molecule in multiple tissues, yet its mode of action and cell-specific activities remain enigmatic. AR function has been best studied in the prostate, in which it is essential for growth and homeostasis of the normal organ as well as each stage of cancer development. Investigation of mechanisms responsible for continued AR action that evolve during prostate cancer progression or after hormonal management of the disease have been instructive in defining AR signaling pathways. In the current paper, we use sequence similarity and the collocation of somatic mutations in prostate cancer to define residues 501-535 of the AR amino-terminal domain as an important mediator of receptor function. Specifically, the 501-535 region is required for optimal interaction of the amino-terminal domain with both the p160 coactivator, nuclear receptor coactivator-2, and the AR-ligand binding domain in the amino/carboxyl (N/C) interaction. The N/C interaction is decreased by deletion of the 501-535 region but is distinct from deletion of the (23)FQNLF(27) peptide in that it does not affect the capacity of the AR to activate transcription from a chromatin integrated reporter or recruitment of the receptor to androgen-responsive loci in vivo. Collectively, we have been able to outline two classes of N/C-deficient AR variant that are divergent in their capacity to act in a chromatin context, thereby further defining the interplay between N/C interaction and coregulator recruitment via multiple receptor domains. These mechanisms are likely to be key determinants of the cell and promoter specific activities of the AR.
Assuntos
Cromatina/genética , Variação Genética/genética , Neoplasias da Próstata/genética , Domínios e Motivos de Interação entre Proteínas/genética , Receptores Androgênicos/genética , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromatina/fisiologia , Progressão da Doença , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/fisiopatologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Receptores Androgênicos/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The androgen receptor signaling axis plays an essential role in the development, function and homeostasis of male urogenital structures including the prostate gland although the mechanism by which the AR axis contributes to the initiation, progression and metastatic spread of prostate cancer remains somewhat enigmatic. A number of molecular events have been proposed to act at the level of the AR and associated coregulators to influence the natural history of prostate cancer including deregulated expression, somatic mutation, and post-translational modification. The purpose of this article is to review the evidence for deregulated expression and function of the AR and associated coactivators and corepressors and how such events might contribute to the progression of prostate cancer by controlling the selection and expression of AR targets.