RESUMO
The androgen receptor (AR) plays a central role in prostate cancer progression to the castration-resistant (CR) lethal state. L-Dopa decarboxylase (DDC) is an AR coactivator that increases in expression with disease progression and is coexpressed with the receptor in prostate adenocarcinoma cells, where it may enhance AR activity. Here, we hypothesize that the DDC enzymatic inhibitor, carbidopa, can suppress DDC-coactivation of AR and retard prostate tumor growth. Treating LNCaP prostate cancer cells with carbidopa in transcriptional assays suppressed the enhanced AR transactivation seen with DDC overexpression and decreased prostate-specific antigen (PSA) mRNA levels. Carbidopa dose-dependently inhibited cell growth and decreased survival in LNCaP cell proliferation and apoptosis assays. The inhibitory effect of carbidopa on DDC-coactivation of AR and cell growth/survival was also observed in PC3 prostate cancer cells (stably expressing AR). In vivo studies demonstrated that serum PSA velocity and tumor growth rates elevated â¼2-fold in LNCaP xenografts, inducibly overexpressing DDC, were reverted to control levels with carbidopa administration. In castrated mice, treating LNCaP tumors, expressing endogenous DDC, with carbidopa delayed progression to the CR state from 6 to 10 weeks, while serum PSA and tumor growth decreased 4.3-fold and 5.4-fold, respectively. Our study is a first time demonstration that carbidopa can abrogate DDC-coactivation of AR in prostate cancer cells and tumors, decrease serum PSA, reduce tumor growth and delay CR progression. Since carbidopa is clinically approved, it may be readily used as a novel therapeutic strategy to suppress aberrant AR activity and delay prostate cancer progression.
Assuntos
Carbidopa/farmacologia , Dopa Descarboxilase/metabolismo , Neoplasias da Próstata/fisiopatologia , Receptores Androgênicos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Masculino , Camundongos , Orquiectomia , Antígeno Prostático Específico/biossíntese , Antígeno Prostático Específico/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , RNA Mensageiro/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Response to bicalutamide after castration failure is not durable and treatment options at this stage are limited. Carbidopa, an L-dopa decarboxylase (AR-coactivator) inhibitor, has been shown to retard prostate tumor growth/PSA production in xenografts. Here, we hypothesize that pharmacological targeting of the AR-axis by combination treatment with bicalutamide plus carbidopa significantly enhances antitumoral activity in vitro and in vivo compared to monotherapy with either drug. METHODS: Carbidopa was tested for its ability to enhance the effects of bicalutamide on cell viability, apoptosis and PSA transactivation in LNCaP and C4-2 cells. The castration-resistant prostate cancer (CRPC) LNCaP xenograft tumor model was used in vivo. After CRPC progression, mice were treated with carbidopa (50 mg/kg) and bicalutamide (50 mg/kg) as monotherapy or in combination. Tumor volume and serum PSA were evaluated weekly. RESULTS: Combination treatment of carbidopa plus bicalutamide significantly inhibited cell viability in both cell lines and induced apoptosis. The combination treatment also decreased androgen-induced PSA transactivation by 62.6% in LNCaP cells and by 55.6% in C4-2 cells compared to control, while bicalutamide monotherapy reduced PSA levels by 27.5% and 29.1% in LNCaP and C4-2 cells. In vivo, bicalutamide monotherapy delayed LNCaP CRPC tumor growth rate by 72.2%, while combination treatment reduced tumor growth by 84.4% compared to control. Serum PSA was also reduced 70.6% with bicalutamide monotherapy, while combination therapy reduced PSA levels by 76.7% compared to control. CONCLUSIONS: This study demonstrates preclinical proof-of-principle that pharmacological targeting of prostate tumors by combination treatment of bicalutamide plus carbidopa significantly reduces AR activity, and thereby delays CRPC tumor progression in vivo.
Assuntos
Adenocarcinoma/patologia , Anilidas/farmacologia , Antineoplásicos/farmacologia , Carbidopa/farmacologia , Nitrilas/farmacologia , Orquiectomia , Neoplasias da Próstata/patologia , Receptores Androgênicos/efeitos dos fármacos , Compostos de Tosil/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Anilidas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carbidopa/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Progressão da Doença , Dopaminérgicos/farmacologia , Dopaminérgicos/uso terapêutico , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Nus , Nitrilas/uso terapêutico , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Compostos de Tosil/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Progression to the castration-resistant state is the incurable and lethal end stage of prostate cancer, and there is strong evidence that androgen receptor (AR) still plays a central role in this process. We hypothesize that knocking down AR will have a major effect on inhibiting growth of castration-resistant tumors. EXPERIMENTAL DESIGN: Castration-resistant C4-2 human prostate cancer cells stably expressing a tetracycline-inducible AR-targeted short hairpin RNA (shRNA) were generated to directly test the effects of AR knockdown in C4-2 human prostate cancer cells and tumors. RESULTS: In vitro expression of AR shRNA resulted in decreased levels of AR mRNA and protein, decreased expression of prostate-specific antigen (PSA), reduced activation of the PSA-luciferase reporter, and growth inhibition of C4-2 cells. Gene microarray analyses revealed that AR knockdown under hormone-deprived conditions resulted in activation of genes involved in apoptosis, cell cycle regulation, protein synthesis, and tumorigenesis. To ensure that tumors were truly castration-resistant in vivo, inducible AR shRNA expressing C4-2 tumors were grown in castrated mice to an average volume of 450 mm(3). In all of the animals, serum PSA decreased, and in 50% of them, there was complete tumor regression and disappearance of serum PSA. CONCLUSIONS: Whereas castration is ineffective in castration-resistant prostate tumors, knockdown of AR can decrease serum PSA, inhibit tumor growth, and frequently cause tumor regression. This study is the first direct evidence that knockdown of AR is a viable therapeutic strategy for treatment of prostate tumors that have already progressed to the castration-resistant state.
Assuntos
Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Antagonistas de Receptores de Andrógenos , Animais , Castração , Linhagem Celular Tumoral , Marcação de Genes , Humanos , Masculino , Camundongos , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/patologia , Transplante HeterólogoRESUMO
BACKGROUND: The androgen receptor is a ligand-induced transcriptional factor, which plays an important role in normal development of the prostate as well as in the progression of prostate cancer to a hormone refractory state. We previously reported the identification of a novel AR coactivator protein, L-dopa decarboxylase (DDC), which can act at the cytoplasmic level to enhance AR activity. We have also shown that DDC is a neuroendocrine (NE) marker of prostate cancer and that its expression is increased after hormone-ablation therapy and progression to androgen independence. In the present study, we generated tetracycline-inducible LNCaP-DDC prostate cancer stable cells to identify DDC downstream target genes by oligonucleotide microarray analysis. RESULTS: Comparison of induced DDC overexpressing cells versus non-induced control cell lines revealed a number of changes in the expression of androgen-regulated transcripts encoding proteins with a variety of molecular functions, including signal transduction, binding and catalytic activities. There were a total of 35 differentially expressed genes, 25 up-regulated and 10 down-regulated, in the DDC overexpressing cell line. In particular, we found a well-known androgen induced gene, TMEPAI, which wasup-regulated in DDC overexpressing cells, supporting its known co-activation function. In addition, DDC also further augmented the transcriptional repression function of AR for a subset of androgen-repressed genes. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time RT-PCR. CONCLUSION: Taken together, our results provide evidence for linking DDC action with AR signaling, which may be important for orchestrating molecular changes responsible for prostate cancer progression.
Assuntos
Adenocarcinoma/enzimologia , Androgênios , Dopa Descarboxilase/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/fisiologia , Neoplasias Hormônio-Dependentes/enzimologia , Neoplasias da Próstata/enzimologia , Receptores Androgênicos/fisiologia , Adenocarcinoma/metabolismo , Western Blotting , Linhagem Celular Tumoral/metabolismo , Dopa Descarboxilase/genética , Indução Enzimática/efeitos dos fármacos , Perfilação da Expressão Gênica , Vetores Genéticos/farmacologia , Humanos , Masculino , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraciclina/farmacologia , TransfecçãoRESUMO
Current hormone withdrawal therapies used for treatment of advanced prostate cancer lead to androgen-independent tumor growth. Increased prostatic neuroendocrine (NE) cell density has been implicated in promoting progression of prostate cancer, but the process by which this occurs remains unclear. The aim of this study was to determine whether there is an association of increased NE differentiation with neoadjuvant hormone therapy and Gleason grade. Using adjacently sectioned tissue microarrays, the expression profile of novel and known NE markers were monitored. L-Dopa decarboxylase (DDC), a catecholamine synthesis enzyme and androgen receptor (AR) coregulator protein, was identified as an additional NE marker of prostate cancer. Immunohistochemical analysis of DDC with the established NE markers, chromogranin A and bombesin, revealed a significant increase in NE differentiation after 6 months of hormone therapy and after progression to androgen independence but no apparent correlation with Gleason grade. In addition, dual immunofluorescence analysis revealed that approximately 55% of the mixed population of DDC- and chromogranin A-expressing NE cells continue to express AR. Taken together, these results suggest that the increase of NE differentiation in prostate cancers depends specifically on duration of hormone therapy. This increase may be due to the transdifferentiation of AR-expressing epithelial-derived adenocarcinoma cells into an NE cell phenotype.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/biossíntese , Dopa Descarboxilase/biossíntese , Sistemas Neurossecretores/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Idoso , Bombesina/análise , Diferenciação Celular , Cromogranina A/análise , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Estadiamento de Neoplasias , Sistemas Neurossecretores/metabolismo , Sistemas Neurossecretores/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/análise , Índice de Gravidade de DoençaRESUMO
The AR (androgen receptor) is a ligand-regulated transcription factor, which belongs to the steroid receptor family and plays an essential role in growth and development of the prostate. Transcriptional activity of steroid receptors is modulated by interaction with co-regulator proteins and yeast two-hybrid analysis is commonly used to identify these steroid receptor-interacting proteins. However, a limitation of conventional two-hybrid systems for detecting AR protein partners has been that they only allow for analysis of the ligand- and DNA-binding domains of the receptor, as its NTD (N-terminal domain) possesses intrinsic transactivation activity. To identify AR N-terminus-interacting proteins, its NTD was used in the RTA (repressed transactivator) system, which is specifically designed for transactivator bait proteins and was shown to be suitable for two-hybrid analysis with the AR NTD. DDC (L-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, which was subsequently confirmed in vitro and in vivo. Furthermore, transient transfection of DDC in prostate cancer cells strongly enhanced ligand-dependent AR transcriptional activity, an effect that was antagonized using high concentrations of the anti-androgen bicalutamide. Glucocorticoid receptor activity was also strongly enhanced with DDC co-transfection, while oestrogen receptor activity was only mildly affected. Together, our data demonstrate that DDC interacts with AR to enhance steroid receptor transactivation, which may have important implications in prostate cancer progression.
Assuntos
Dopa Descarboxilase/metabolismo , Receptores Androgênicos/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Anilidas/farmacologia , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Dopa Descarboxilase/genética , Receptor alfa de Estrogênio , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Dados de Sequência Molecular , Nitrilas , Ligação Proteica , Ratos , Receptores Androgênicos/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Compostos de Tosil , Ativação Transcricional/efeitos dos fármacos , Técnicas do Sistema de Duplo-HíbridoRESUMO
Aberrant expression of androgen receptor (AR) coregulators has been linked to progression of prostate cancers to castration resistance. Using the repressed transactivator yeast two-hybrid system, we found that TATA binding protein-associated factor 1 (TAF1) interacted with the AR. In tissue microarrays, TAF1 was shown to steadily increase with duration of neoadjuvant androgen withdrawal and with progression to castration resistance. Glutathione S-transferase pulldown assays established that TAF1 bound through its acetylation and ubiquitin-activating/conjugating domains (E1/E2) directly to the AR N terminus. Coimmunoprecipitation and ChIP assays revealed colocalization of TAF1 and AR on the prostate-specific antigen promoter/enhancer in prostate cancer cells. With respect to modulation of AR activity, overexpression of TAF1 enhanced AR activity severalfold, whereas small interfering RNA knockdown of TAF1 significantly decreased AR transactivation. Although full-length TAF1 showed enhancement of both AR and some generic gene transcriptional activity, selective AR coactivator activity by TAF1 was demonstrated in transactivation experiments using cloned N-terminal kinase and E1/E2 functional domains. In keeping with AR coactivation by the ubiquitin-activating and -conjugating domain, TAF1 was found to greatly increase the cellular amount of polyubiquitinated AR. In conclusion, our results indicate that increased TAF1 expression is associated with progression of human prostate cancers to the lethal castration-resistant state. Because TAF1 is a coactivator of AR that binds and enhances AR transcriptional activity, its overexpression could be part of a compensatory mechanism adapted by cancer cells to overcome reduced levels of circulating androgens.
Assuntos
Receptores Androgênicos/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Western Blotting , Linhagem Celular Tumoral , Histona Acetiltransferases , Humanos , Imunoprecipitação , Técnicas In Vitro , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Ligação Proteica , RNA Interferente Pequeno , Receptores Androgênicos/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Ativação Transcricional , Técnicas do Sistema de Duplo-HíbridoRESUMO
The androgen receptor (AR), a steroid receptor family member, is a ligand-dependent transcription factor that has an integral role in normal prostate development. Alterations in AR-mediated activity can result in abnormal gene expression, dysregulated cell growth and prostate cancer. Coregulator proteins that interact with AR to influence activity and specificity of the AR-response may also have an important role in prostate cancer progression. Since the NH(2)-terminal domain (NTD) of AR encodes the ligand-independent activation function (AF)-1, this domain is incompatible with conventional yeast two-hybrid systems. Therefore, we have used the Tup1 repressed transactivator (RTA) system, which exploits the intrinsic transactivation properties of AR.NTD, for identification of novel AR-interacting proteins. Using this system, cyclin G-associated kinase (GAK) was identified as an AR interacting protein, and GST pull-down assays were used to confirm the interaction. GAK was shown to enhance the AF-1 function of AR activity in a ligand-dependent manner. Additionally, GAK enhanced the AR transcriptional response even at low concentrations of androgens, which is relevant to AR activity in androgen-independent prostate cancer. Finally, neo-adjuvant hormone therapy (NHT) tissue microarray analysis demonstrated that GAK expression increased significantly with prostate cancer progression to androgen independence, which suggests a prognostic role for GAK in advanced disease.
Assuntos
Androgênios/uso terapêutico , Ciclinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Ciclinas/genética , Progressão da Doença , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Receptores Androgênicos/genética , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Análise Serial de Tecidos , Ativação Transcricional/genéticaRESUMO
BACKGROUND: Relaxin is a potent peptide hormone normally secreted by the prostate. This study characterized relaxin expression during prostate cancer progression to androgen independence (AI), and in response to androgens. METHODS: The prostate cancer cell line, LNCaP, was assayed by microarrays and confirmatory Northern analysis to assess changes in relaxin levels due to androgen treatment and in LNCaP xenografts following castration. Relaxin protein levels were examined by immunohistochemistry (IHC) in tissue microarrays of human prostate cancer samples following androgen ablation. RESULTS: Relaxin levels decreased in a time and concentration-dependent manner due to androgens in vitro, and increased in xenografts post-castration. Relaxin increased in radical prostatectomy specimens after 6 months of androgen ablation and in AI tumors, was highest in bone metastases. CONCLUSIONS: Relaxin is negatively regulated by androgens in vitro and in vivo, which correlates to clinical prostate cancer specimens following androgen ablation. The role of relaxin in angiogenesis and tissue remodeling suggests it may contribute to prostate cancer progression.