RESUMO
Androgen deprivation therapy (ADT) is the core treatment for advanced prostate cancer (PCa), with a proven survival benefit. ADT lowers circulating testosterone levels throughout the body, but with it comes a variety of reported side effects including fatigue, muscle wastage, weight gain, hot flushes and importantly cognitive impairment, depression, and mood swings. Testosterone has a key role in brain masculinization, but its direct effects are relatively poorly understood, due both to the brain's extreme complexity and the fact that some of testosterone activities are driven via local conversion to oestrogen, especially during embryonic development. The exact roles, function, and location of the androgen receptor (AR) in the adult male brain are still being discovered, and therefore the cognitive side effects of ADT may be unrecognized or under-reported. The age of onset of several neurological diseases overlap with PCa, therefore, there is a need to separate ADT side effects from such co-morbidities. Here we analysed the activity and expression level of the AR in the adult mouse brain, using an ARE-Luc reporter mouse and immunohistochemical staining for AR in all the key brain regions via coronal slices. We further analysed our data by comparing to the Allen Mouse Brain Atlas. AR-driven luciferase activity and distinct nuclear staining for AR were seen in several key brain areas including the thalamus, hypothalamus, olfactory bulb, cerebral cortex, Purkinje cells of the cerebellum and the hindbrain. We describe and discuss the potential role of AR in these areas, to inform and enable extrapolation to potential side effects of ADT in humans.
Assuntos
Encéfalo , Receptores Androgênicos , Receptores Androgênicos/metabolismo , Animais , Camundongos , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , MasculinoRESUMO
BACKGROUND/AIM: MiR-221, often described both as an oncogenic microRNA and as a tumour suppressor, targets mRNAs involved in carcinogenesis. While other oncogenic microRNAs showed correlations with prostate cancer cell lines' aggressiveness, miR-221 showed an unusual overexpression in PC3. MATERIALS AND METHODS: CRISPR was used to delete miR-221 from PC3 cells. Analysing the characteristics of PC3miR-221del cells, a reduced growth rate and expression of cell-cycle genes was observed. In global gene expression/ontology analysis of PC3miR-221del cells, cell-cell and cell-substrate adhesion pathways were found to be greatly affected. In addition, reduced levels of adhesion, invasion and motility for PC3miR-221del cells, a change in F-actin localisation and a reduction of EMT markers were observed. RESULTS: The tumour suppressor gene, DIRAS3, was a predicted target of miR-221. In PC3miR-221del cells DIRAS3 was up-regulated at the gene and protein level. Ectopic expression of DIRAS3 in PC3wt cells recapitulated the cellular morphology changes seen in PC3miR-221del cells. DIRAS3 3'UTR was more stable in PC3miR-221del cells, as measured by semi-quantitative PCR and luciferase fusion reporter assays. CONCLUSION: MiR-221 promotes aggressiveness of PC3 cells by down-regulating DIRAS3, and promoting epithelial-to-mesenchymal transition.
Assuntos
Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , MicroRNAs/genética , Deleção de Sequência/genética , Regiões 3' não Traduzidas/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Oncogenes/genética , Células PC-3 , Neoplasias da Próstata/genética , Regulação para Cima/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Deshydroxy propioanilides were synthesised by Michael addition reaction between substituted thiophenols onto four different phenylacrylamide derivatives to give twenty-three novel deshydroxy bicalutamide derivatives lacking the central hydroxyl group. The antiproliferative activities of these compounds were evaluated against human prostate cancer cell lines and thirteen compounds showed better inhibitory activities (IC50â¯=â¯2.67-13.19 µM) compared to bicalutamide (IC50â¯=â¯20.44 µM) in LNCaP. Remarkably, novel double branched bicalutamide analogues (27 and 28) were isolated as major by-products and found to have the best activity across three human prostate cancer cell lines (LNCaP, VCaP and PC3). The most active compound 28 shows sub-micromolar activity (IC50â¯=â¯0.43 µM in LNCaP), which represents more than 40-fold improvement over the clinical anti-androgen bicalutamide (IC50â¯=â¯20.44 µM) and a more than 3 fold improvement over enzalutamide (IC50â¯=â¯1.36 µM). Moreover, strong reduction of PSA expression in LNCaP cells upon treatment with compounds 27, 28 and 33 was observed during qPCR analysis, confirming their AR antagonist activity. Molecular modelling studies revealed a novel binding mode of these structurally distinct double branched analogues within the ligand binding domain (LBD) of the androgen receptor.
Assuntos
Antagonistas de Androgênios/síntese química , Anilidas/farmacologia , Descoberta de Drogas , Nitrilas/farmacologia , Compostos de Tosil/farmacologia , Antagonistas de Androgênios/farmacologia , Anilidas/síntese química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Modelos Moleculares , Nitrilas/síntese química , Neoplasias da Próstata/tratamento farmacológico , Compostos de Tosil/síntese químicaRESUMO
Prostate cancer often develops antiandrogen resistance, possibly via androgen receptor (AR) mutations, which change antagonists to agonists. Novel therapies with increased anticancer activity, while overcoming current drug resistance are urgently needed. Enobosarm has anabolic effects on muscle and bone while having no effect on the prostate. Here, we describe the activity of novel chemically modified enobosarm analogues. The rational addition of bis-trifluoromethyl groups into ring B of enobosarm, profoundly modified their activity, pharmacokinetic and tissue distribution profiles. These chemical structural modifications resulted in an improved AR binding affinity-by increasing the molecular occupational volume near helix 12 of AR. In vitro, the analogues SK33 and SK51 showed very potent antiandrogenic activity, monitored using LNCaP/AR-Luciferase cells where growth, PSA and luciferase activity were used as AR activity measurements. These compounds were 10-fold more potent than bicalutamide and 100-fold more potent than enobosarm within the LNCaP model. These compounds were also active in LNCaP/BicR cells with acquired bicalutamide resistance. In vivo, using the AR-Luc reporter mice, these drugs showed potent AR inhibitory activity in the prostate and other AR-expressing tissues, e.g., testes, seminal vesicles, and brain. These compounds do not inhibit AR activity in the skeletal muscle, and spleen, thus indicating a selective tissue inhibitory profile. These compounds were also active in vivo in the Pb-Pten deletion model. SK33 and SK51 have significantly different and enhanced activity profiles compared with enobosarm and are ideal candidates for further development for prostate cancer therapy with potentially fewer side effects. Mol Cancer Ther; 17(9); 1846-58. ©2018 AACR.
Assuntos
Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Antagonistas de Androgênios/química , Antagonistas de Androgênios/farmacocinética , Anilidas/química , Anilidas/farmacocinética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Estrutura Molecular , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Baço/metabolismo , Imagem com Lapso de Tempo/métodosRESUMO
PTen loss is one of the most frequent events in prostate cancer both at the initiation stage and during late stage metastatic development. The mouse model of prostate-specific probasin-mediated Pten deletion leads to prostate intraepithelial neoplasia (PIN) leading to adenocarcinoma. Using this model, we analysed the miR and mRNA transcriptome profile of Pten -/- PIN versus wild type age-matched prostate tissues and analysed the effects of Pten loss on miR expression in the early neoplastic process. At the PIN stage, Pten loss significantly changed the expression of over 20 miRNAs and over 4000 genes. The observed miR expression indicated a strong immunological cohort, which is seen in many human and mouse cancers and is thought to derive from infiltrating B and T immune cells. However, upon in situ hybridisation, these immunologically related miRs did not correlate with immune cell location, and emanated from the prostate epithelium itself and not from the associated immune cells present. Growing Pten -/- prostate cells in culture showed that the overexpressed miRNAs seen in Pten -/- were directly in response to the overactive PI3 kinase pathway and were in part responsible in reducing target gene expression levels. Inhibition of PI3 kinase downstream regulators, or re-introducing wild type Pten cDNA reduced miR overexpression resulting in increased miR target gene expression. MiR inhibitors also showed this pattern, and synergised with an mTORC1 inhibitor. Overall, Pten deletion in the prostate epithelium activated a cohort of inflammation-related miRs usually associated with immune responses from B and T cells. These oncomiRs may then accelerate carcinogenesis.
RESUMO
AIM: To examine the expression and function of activin receptor-like kinase 7 (ALK7) in breast cancer, its association with disease prognosis, and its impact on breast cancer cell function. PATIENTS AND METHODS: A cohort of patients with breast cancer were examined for ALK7 expression in association with pathological and clinical aspects. In vitro cell assays of ALK7 were investigated using an expression plasmid. RESULTS: Overall higher levels of ALK7 transcripts were seen in the breast cancer samples vs. normal tissue. However, within the cancer cohort, lower levels of ALK7 transcript were associated with poor prognosis. Patients with lower expression of ALK7 also had shorter survival. Overexpression of ALK7 reduced proliferation and adhesion of breast cancer cells in vitro. We found that overexpressed ALK7 had complex effects on the MCF-7 cell sensitivity to chemotherapy drugs. CONCLUSION: Decreased expression of ALK7 in breast cancer is correlated with poor prognosis. ALK7 is a negative regulator of adhesion and proliferation of breast cancer cells. This suggests that ALK7 is a potential tumor suppressor in breast cancer.
Assuntos
Receptores de Ativinas Tipo I/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular/genética , Proliferação de Células/genética , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , PrognósticoRESUMO
Nuclear receptors drive key processes during development, reproduction, metabolism, and disease. In order to understand and analyze, as well as manipulate, their actions it is imperative that we are able to study them in whole animals and in a spatiotemporal manner. The increasing repertoire of transgenic animals, expressing reporter genes driven by a specific nuclear receptor, enables us to do this. Use of luciferase reporter genes is the method of choice of many researchers as it is well tolerated, relatively easy to use, and robust. Further, luciferase lends itself to the process as it can penetrate tissue and can be manipulated to degrade rapidly thus allowing a dynamic response. However, limited resolution, lack of quantitation, and the largely two-dimensional images acquired make it desirable to support results using ex vivo imaging and enzymatic and/or immunohistochemical analysis of dissected tissue. As well as enabling the visualization of nuclear receptor signaling in wild-type animals, crossing these mouse models with models of disease will provide invaluable information on how such signaling is dysregulated during disease progression, and how we may manipulate nuclear receptor signaling in therapy. The use of in vivo imaging therefore provides the power to determine where and when in development, aging, and disease nuclear receptors are active and how ligands or receptor modulators affect this.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Luciferases/metabolismo , Neoplasias da Próstata/genética , Receptores Citoplasmáticos e Nucleares/genética , Transcrição Gênica , Animais , Regulação Neoplásica da Expressão Gênica , Hormônios/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , Neoplasias da Próstata/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recently, research has shed new light on the role of Prohibitin (PHB) in cancer pathogenesis across an array of cancer types. Important mechanisms for PHB have been unveiled in several cancers, especially with regard to the androgen independent state of prostate cancer (PC) and oestrogen dependent breast cancer. However, PHB is often overlooked due to its complex but subtle roles within the cell. Having gathered both historical and current research exploring PHB's role in different cancer types including prostate and breast, here we aim to pair this information with its molecular properties in the hope of translating this information into a clinical perspective, thus discussing its possible use in future cancer therapy.
Assuntos
Antineoplásicos/metabolismo , Neoplasias da Mama/genética , Ciclo Celular/genética , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Feminino , Humanos , Masculino , ProibitinasRESUMO
Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic responses dependent upon cellular context.
Assuntos
Antagonistas de Androgênios/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/isolamento & purificação , Próstata/efeitos dos fármacos , Proteoma/isolamento & purificação , Receptores Androgênicos/química , Sequência de Aminoácidos , Antagonistas de Androgênios/química , Anilidas/química , Anilidas/farmacologia , Linhagem Celular Tumoral , Acetato de Ciproterona/química , Acetato de Ciproterona/farmacologia , Flutamida/análogos & derivados , Flutamida/química , Flutamida/farmacologia , Humanos , Masculino , Anotação de Sequência Molecular , Dados de Sequência Molecular , Mutação , Nandrolona/análogos & derivados , Nandrolona/química , Nandrolona/farmacologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Nitrilas/química , Nitrilas/farmacologia , Próstata/metabolismo , Próstata/patologia , Proteoma/genética , Proteoma/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais , Compostos de Tosil/química , Compostos de Tosil/farmacologiaRESUMO
Hormones are key drivers of cancer development. To date, interest has largely been focussed on the classical model of hormonal gene regulation, but there is increasing evidence for a role of hormone signalling pathways in post-translational regulation of gene expression. In particular, a complex and dynamic network of bi-directional interactions with microRNAs (miRs) at all stages of biogenesis and during target gene repression is emerging. miRs, which act mainly by negatively regulating gene expression through association with 3'-UTRs of mRNA species, are increasingly understood to be important in development, normal physiology and pathogenesis. Given recent demonstrations of altered miR profiles in a diverse range of cancers, their ability to function as oncogenes or tumour suppressors, and hormonal regulation of miRs, understanding mechanisms by which miRs are generated and regulated is vitally important. miRs are transcribed by RNA polymerase II and then processed in the nucleus by the Drosha-containing Microprocessor complex and in the cytoplasm by Dicer, before mature miRs are incorporated into the RNA-induced silencing complex. It is increasingly evident that multiple cellular signalling pathways converge upon the miR biogenesis cascade, adding further layers of regulatory complexity to modulate miR maturation. This review summarises recent advances in identification of novel components and regulators of the Microprocessor and Dicer complexes, with particular emphasis on the role of hormone signalling pathways in regulating their activity. Understanding hormone regulation of miR production and how this is perturbed in cancer are critical for the development of miR-based therapeutics and biomarkers.
Assuntos
MicroRNAs/metabolismo , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Esteroides/metabolismo , Animais , Receptor alfa de Estrogênio/metabolismo , Hormônios/metabolismo , Humanos , Receptores Androgênicos/metabolismo , Transdução de SinaisRESUMO
Prostate cancer, the most common cancer of western men, requires new biomarkers, especially given that the benefits of PSA testing remain uncertain. Nucleic acids can now be accurately and sensitively detected in human blood. Over the last decade, investigations into utility of circulating cell-free miRNA, DNA and mRNA as novel biomarkers have expanded exponentially. In the near future, they may be routinely used to accurately diagnose cancers, stratify indolent from aggressive disease and inform treatment decisions. However, advancement of such tests into clinical settings is hampered by technical problems with assay specificity and sensitivity, and small study sizes. This review highlights the different forms of circulating nucleic acids and those that show the most potential as viable biomarkers for prostate cancer.
Assuntos
Biomarcadores Tumorais/sangue , Ácidos Nucleicos/sangue , Neoplasias da Próstata/diagnóstico , Biomarcadores Tumorais/genética , DNA/sangue , DNA/metabolismo , Metilação de DNA , DNA Mitocondrial/sangue , Humanos , Masculino , MicroRNAs/sangue , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/metabolismo , RNA Mensageiro/sangueRESUMO
Androgens, required for normal development and fertility of males and females, have vital roles in the reproductive tract, brain, cardiovascular system, smooth muscle and bone. Androgens function via the androgen receptor (AR), a ligand-dependent transcription factor. To assay and localise AR activity in vivo we generated the transgenic "ARE-Luc" mouse, expressing a luciferase reporter gene under the control of activated endogenous AR. In vivo imaging of androgen-mediated luciferase activity revealed several strongly expressing tissues in the male mouse as expected and also in certain female tissues. In males the testes, prostate, seminal vesicles and bone marrow all showed high AR activity. In females, strong activity was seen in the ovaries, uterus, omentum tissue and mammary glands. In both sexes AR expression and activity was also found in salivary glands, the eye (and associated glands), adipose tissue, spleen and, notably, regions of the brain. Luciferase protein expression was found in the same cell layers as androgen receptor expression. Additionally, mouse AR expression and activity correlated well with AR expression in human tissues. The anti-androgen bicalutamide reduced luciferase signal in all tissues. Our model demonstrates that androgens can act in these tissues directly via AR, rather than exclusively via androgen aromatisation to estrogens and activation of the estrogen receptor. Additionally, it visually demonstrates the fundamental importance of AR signalling outside the normal role in the reproductive organs. This model represents an important tool for physiological and developmental analysis of androgen signalling, and for characterization of known and novel androgenic or antiandrogenic compounds.
Assuntos
Receptores Androgênicos/fisiologia , Ativação Transcricional , Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Animais , Células COS , Chlorocebus aethiops , Feminino , Genes Reporter , Humanos , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Células MCF-7 , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Nitrilas/farmacologia , Especificidade de Órgãos , Compostos de Tosil/farmacologiaRESUMO
MicroRNAs (miRs) play an important role in the development of many complex human diseases and may have tumour suppressor or oncogenic (oncomir) properties. Prostate cancer is initially an androgen-driven disease, and androgen receptor (AR) remains a key driver of growth even in castration-resistant tumours. However, AR-mediated oncomiR pathways remain to be elucidated. We demonstrate that miR-27a is an androgen-regulated oncomir in prostate cancer, acting via targeting the tumour suppressor and AR corepressor, Prohibitin (PHB). Increasing miR-27a expression results in reduced PHB mRNA and protein levels, and increased expression of AR target genes and prostate cancer cell growth. This involves a novel mechanism for androgen-mediated miR regulation, whereby AR induces a transient increase in miR-23a27a24-2 transcription, but more significantly accelerates processing of the primiR-23a27a24-2 cluster. Androgens therefore regulate miR-27a expression both transcriptionally (via AR binding to the cluster promoter) and post-transcriptionally (accelerating primiR processing to the mature form). We further show that a miR-27a anti-sense oligonucleotide, by opposing the effects of mir-27a, has therapeutic potential in prostate cancer.
Assuntos
Androgênios/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Proteínas Oncogênicas/genética , Neoplasias da Próstata/genética , Processamento Pós-Transcricional do RNA , Proteínas Repressoras/genética , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Masculino , MicroRNAs/metabolismo , Dados de Sequência Molecular , Proteínas Oncogênicas/metabolismo , Proibitinas , Neoplasias da Próstata/metabolismo , Ligação Proteica , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismoRESUMO
Progression of prostate cancer is highly dependent upon the androgen receptor pathway, such that knowledge of androgen-regulated proteins is vital to understand and combat this disease. Using a proteomic screen, we found the RNA-binding protein FUS/TLS (Fused in Ewing's Sarcoma/Translocated in Liposarcoma) to be downregulated in response to androgen. FUS has recently been shown to be recruited by noncoding RNAs to the regulatory regions of target genes such as cyclin D1, in which it represses transcription by disrupting complex formation. Here we show that FUS has some characteristics of a putative tumor suppressor, as its overexpression promoted growth inhibition and apoptosis of prostate cancer cells, whereas its knockdown increased cell proliferation. This effect was reproducible in vivo, such that increasing FUS levels in tumor xenografts led to dramatic tumor regression. Furthermore, FUS promoted conditions that favored cell-cycle arrest by reducing the levels of proliferative factors such as cyclin D1 and Cdk6 and by increasing levels of the antiproliferative Cdk inhibitor p27. Immunohistochemical analysis revealed that FUS expression is inversely correlated with Gleason grade, demonstrating that patients with high levels of FUS survived longer and were less likely to have bone metastases, suggesting that loss of FUS expression may contribute to cancer progression. Taken together, our results address the question of how androgens regulate cell-cycle progression, by demonstrating that FUS is a key link between androgen receptor signaling and cell-cycle progression in prostate cancer.
Assuntos
Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteína FUS de Ligação a RNA/biossíntese , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Imuno-Histoquímica , Masculino , Nandrolona/análogos & derivados , Nandrolona/farmacologia , Neoplasias Hormônio-Dependentes/genética , Neoplasias da Próstata/genética , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Receptores Androgênicos/metabolismoRESUMO
Current hormonal therapies for prostate cancer are effective initially, but inevitably tumours progress to an advanced, metastatic stage, often referred to as 'androgen independent'. However, the androgen receptor (AR) signalling pathway is still key for their growth. It is speculated that tumours escape hormonal control via reduction in corepressor proteins. Manipulating such proteins is thus a potential therapeutic strategy to halt or even reverse tumour progression. We aimed to elucidate the effects of altering levels of the AR corepressor and androgen-target protein prohibitin (PHB) on prostate tumour growth. Prostate cancer cells incorporating an integrated androgen-responsive reporter gene and stably expressing vectors to inducibly overexpress or knockdown PHB were generated and used to assess effects on androgen signalling (by real time imaging) and tumour growth both in culture and in vivo. PHB overexpression inhibited AR activity and prostate-specific antigen (PSA) expression as well as androgen-dependent growth of cells, inducing rapid accumulation in G(0)/G(1). Conversely, reduction in PHB increased AR activity, PSA expression, androgen-mediated growth and S-phase entry. In vivo, doxycycline-induced PHB regulation resulted in marked changes in AR activity, and showed significant effects upon tumour growth. Overexpression led to tumour growth arrest and protection from hormonal starvation, whereas RNAi knockdown resulted in accelerated tumour growth, even in castrated mice. This study provides proof of principle that i) reduction in PHB promotes both androgen-dependent and 'androgen-independent' tumour growth, and ii) altering AR activity via increasing levels or activity of corepressors is a valid therapeutic strategy for advanced prostate cancer.
Assuntos
Androgênios/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Doxiciclina/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proibitinas , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testosterona/sangue , Transfecção , Células Tumorais CultivadasRESUMO
A panel of tumour models used extensively for in vivo evaluation of new drugs was characterised for their p53 status. Basal p53 protein levels were measured by immunodetection on both formalin-fixed tumour tissue and from protein extracts of fresh tumours. High levels of nuclear-specific staining, indicative of p53 mutation, was seen in 15/25 tumours, with the remainder showing intermittent or no staining. The functional status of p53 cDNA from these tumours was assayed within the functional analysis of separated alleles in yeast (F.A.S.A.Y.) reporter system. The cDNA from those tumours with high levels of p53 protein showed 14/15 failing to activate the reporter gene. The cDNA from tumours with low or non-detectable p53 levels showed 8/10 with wild-type p53. Tumours were grown subcutaneously in mice (n=10). Each mouse was given maximum tolerated doses for either doxorubicin, 5-fluorouracil or cisplatin. Tumour volumes were measured daily, alongside untreated controls. The specific growth delay values for each tumour were separated into two groups, those with functional p53 (wild-type) and those without (mutant and null status). The Mann-Whitney U test was performed on the groups of data, to evaluate differences in their response on the basis of p53 status. Cisplatin was moderately active against tumours with wild-type and mutant p53 genes with no significant difference seen between both groups. However, a significant difference in specific growth delay was seen between the two groups when treated with doxorubicin or 5-fluorouracil (P=0.05), indicating a role for p53 protein in modulating the in vivo efficacy of these agents.