RESUMEN
BACKGROUND: Expression of epithelial cell adhesion molecule (EpCAM) is deregulated in epithelial malignancies. Beside its role in cell adhesion, EpCAM acts as signalling molecule with tumour-promoting functions. Thus, EpCAM is part of the molecular network of oncogenic receptors and considered an interesting therapeutic target. METHODS: Here, we thoroughly characterised EpCAM expression on mRNA and protein level in comprehensive tissue studies including non-cancerous prostate specimens, primary tumours of different grades and stages, metastatic lesions, and therapy-treated tumour specimens, as well as in prostate cancer cell lines. RESULTS: Epithelial cell adhesion molecule was overexpressed at mRNA and at protein level in prostate cancer tissues and cell lines. Altered EpCAM expression was an early event in prostate carcinogenesis with an upregulation in low-grade cancers and further induction in high-grade tumours and metastatic lesions. Interestingly, EpCAM was repressed upon induction of epithelial-to-mesenchymal transition (EMT) following chemotherapeutic treatment with docetaxel. Oppositely, re-induction of the epithelial phenotype through miRNAs miR-200c and miR-205, two inducers of mesenchymal-to-epithelial transition (MET), led to re-induction of EpCAM in chemoresistant cells. Furthermore, we prove that EpCAM cleavage, the first step of EpCAM signalling takes place in prostate cancer cells but in contrast to other cancer entities, EpCAM has no measurable impact on the proliferative behaviour of prostate cells, in vitro. CONCLUSIONS: In conclusion, our data confirm that EpCAM overexpression is an early event during prostate cancer progression. Epithelial cell adhesion molecule displays a dynamic, heterogeneous expression and associates with epithelial cells rather than mesenchymal, chemoresistant cells along with processes of EMT and MET.
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Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Transición Epitelial-Mesenquimal/genética , MicroARNs/genética , Neoplasias de la Próstata/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Molécula de Adhesión Celular Epitelial , Humanos , Masculino , MicroARNs/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , ARN Mensajero/genética , Taxoides/farmacologíaRESUMEN
BACKGROUND: Class 3 semaphorins are secreted proteins that act as guidance cues for migrating cells via their transmembrane receptors plexins and neuropilins. Semaphorins have a role in cancer affecting tumor progression both directly, and indirectly by affecting angiogenesis. METHODS: The expression of semaphorins and their receptors in prostate cancer cell lines and tissue was determined by RT-PCR, Western blotting and immunohistochemistry. The effect of Sema3E on prostate cancer cell lines was determined by adhesion assays and transwell migration assays. RESULTS: Semaphorins and their receptors, plexins and neuropilins, are widely co-expressed in prostate cancer cell lines and tissue with a significant overexpression of Sema3E in tumor tissue. Sema3E affected integrin-mediated adhesion to fibronectin of prostate cancer cells, and inhibited their motility. Expression of Sema3C was upregulated and Sema3A and Sema3E were down regulated in prostate cells by hypoxia, consistent with an additional role for Sema3A and 3E as anti-angiogenic factors in prostate cancer. CONCLUSIONS: Semaphorin 3E is aberrantly expressed in prostate cancer and affects adhesion and motility of prostate cancer cells, indicating a role for the Sema3E/PlexinD1 signaling pathway in prostate cancer and identifying a new possible target for therapy.
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Moléculas de Adhesión Celular/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Neuropilinas/biosíntesis , Neoplasias de la Próstata/metabolismo , Semaforinas/biosíntesis , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Humanos , Inmunohistoquímica , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuropilinas/genética , Neuropilinas/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Neoplásico/química , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semaforinas/genética , Semaforinas/metabolismo , Transducción de SeñalRESUMEN
PURPOSE: Disorders of sex (DSD) development represent a serious condition. Most of the underlying mechanisms remain unclear. Disturbances within the androgen receptor (AR) pathway frequently account for 46 XY-DSDs. The individual gender-related outcome often is unsatisfactory. We present a long-term AR gene-mutation-associated follow-up in a group of 46 XY-DSD patients. METHODS: Twenty patients (46 XY) who underwent genitoplasty in infancy or early childhood were retrospectively identified. Median follow-up after surgery was 16 years. All were undervirilized at initial presentation. Thirteen had female gender assignment, and 7 were raised as males. A genital skin biopsy and subsequent fibroblast cultures were done. The specific binding of dihydrotestosterone, the thermostability of the receptor hormone complex, and 5-α-reductase activity were measured. AR gene mutations were detected by direct sequencing. The individual outcome was correlated with specific AR mutations. RESULTS: AR point mutations were detected in 12, 7 were previously unknown. There was no specific androgen binding in 3, reduced affinity in 9, and normal binding in 8 patients. 5-α-Reductase activity was normal in 15, reduced in 4 and completely absent in 1 patient. CONCLUSIONS: Retrospective evaluation revealed previously unknown and established AR gene mutations being associated with a distinct long-term outcome. Identification of the molecular mechanisms causing DSD will likely improve timely diagnosis and therapy. Exact characterization of AR activation and function may offer a treatment modality in affected patients. These data may allow us to give prognostic estimations on the individual outcome adding objective criteria for gender assignment in 46 XY-DSD patients.
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Trastornos del Desarrollo Sexual/genética , Mutación , Receptores Androgénicos/genética , Adolescente , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Masculino , Estudios Retrospectivos , Factores de Tiempo , Adulto JovenRESUMEN
Interleukin (IL)-12 is a proinflammatory cytokine that contributes to innate resistance and to the development of antigen-specific T cell responses. Among other effects, prostaglandin E2 (PGE2) inhibits the production of IL-12 by macrophages activated with lipopolysaccharide (LPS). Here we investigated the effects of PGE2 on human dendritic cells (DCs) which develop in the presence of GM-CSF and IL-4. We demonstrate that in the absence of LPS, PGE2 dose dependently stimulated the production of IL-12 by DCs. Although PGE2 alone stimulated the production of low amounts of IL-12 only, it synergized with tumor necrosis factor (TNF)-alpha to induce high levels of IL-12 production by DCs. Addition of TNF-alpha in the absence of PGE2 had no effect on IL-12 production. Conversely, in the presence of LPS, PGE2 inhibited IL-12 production by DCs in a dose-dependent manner. The combination of PGE2 and TNF-alpha efficiently silenced mannose receptor-mediated endocytosis in DCs and readily induced neo-expression of the CD83 antigen. In addition, the expression of various surface antigens such as major histocompatibility complex class I and II, adhesion, as well as costimulatory molecules was upregulated by this treatment. The effects of PGE2 on IL-12 synthesis and CD83 expression could be mimicked by dibutyryl-cAMP and forskolin, indicating that they were due to the intracellular elevation of cAMP levels. DC treated with PGE2 and TNF-alpha were most potent in stimulating allogeneic T cell proliferation. Our data demonstrate that PGE2 contributes to the maturation of human DCs and that PGE2 can be a potent enhancer of IL-12 production by human DCs.
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Células Dendríticas/inmunología , Dinoprostona/farmacología , Interleucina-12/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Presentación de Antígeno/efectos de los fármacos , Bucladesina/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , AMP Cíclico/fisiología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Sinergismo Farmacológico , HumanosRESUMEN
Insulin-like growth factor (IGF) and insulin (INS) proteins regulate key cellular functions through a complex interacting multi-component molecular network, known as the IGF/INS axis. We describe how dynamic and multilayer interactions give rise to the multifunctional role of the IGF/INS axis. Furthermore, we summarise the importance of the regulatory IGF/INS network in cancer, and discuss the possibilities and limitations of therapies targeting the IGF/INS axis with reference to ongoing clinical trials concerning the blockage of IGF1R in several types of cancer.
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Homeostasis/fisiología , Insulina/fisiología , Neoplasias/fisiopatología , Somatomedinas/fisiología , Aminoácidos/metabolismo , Progresión de la Enfermedad , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Glucógeno/metabolismo , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/fisiología , Neoplasias/genética , Neoplasias/terapia , Receptor IGF Tipo 2/fisiología , Transducción de SeñalRESUMEN
During the 10(th) week of gestation human prostate development is about to start. Androgens are the crucial factors to stimulate the initial interactions between the epithelium and mesenchyme. One of the key events in androgen metabolism is the transformation of circulating testosterone to 5alpha-dihydrotestosterone (DHT) by tissue-linked 5alpha-reductase. Both, the formation of a male phenotype and the androgen-mediated growth of the prostate are mediated by DHT. To date the function of 5alpha-reductase 1 (5alphaR1) still remains unclear whereas 5alpha-reductase 2 (5alphaR2) is supposed to be the predominant isoenzyme in human accessory sex tissue. Only little data are available on the detection, distribution, and effects of both isoenzymes during fetal life and infancy. Recently, immunohistochemical investigations of serial sections from fetuses and infants using specific antibodies directed against 5alphaR1 and 5alphaR2 seem to shed light on that issue. Moreover, the detection of downstream products of androgen synthesis using RT-PCR analyses for 17-beta hydroxysteroid dehydrogenase Type 2 (17 betaHSD 2), 17 betaHSD Type 3 and 17 betaHSD Type 7 adds to discovering the molecular biological background. New studies confirm that both isoenzymes are present throughout fetal development. On the transcriptional level RT-PCR for 5alphaR1 and 5alphaR2 certifies these findings. 17 betaHSD 2, 3 and 7 representing the most relevant enzymatic downstream products of cellular androgen synthesis were revealed by RT-PCR as well. Current studies discovered the expression and distribution of both 5alpha-reductase isoenzymes as well as the potential contribution of 5alphaR1 during fetal human prostate development.
RESUMEN
Reifenstein syndrome is an eponymic term that describes partial androgen-insensitive disorders. Androgen receptor isolated from five patients with this syndrome contains a specific mutation in the DNA binding domain of the receptor. This mutation converts an alanine to a threonine at position 596 next to the zinc catenation site at the second finger. The threonine 596 mutant receptor mediated normal androgen response at promoters with closely positioned multiple regulatory elements for the androgen receptor and other transcription factors. Promoters with single isolated androgen response elements were not transactivated by the mutant receptor. In in vitro receptor-DNA binding studies, interaction with DNA by the mutant receptor was achieved only in the presence of an anti-androgen receptor antibody. Exchanging alanine 596 in the wild-type androgen receptor with serine or valine produced mutants with properties indistinguishable from those of the naturally occurring threonine 596 mutant receptor. These results indicate that an alanine residue at position 596 contributes important structural and functional activities to the androgen receptor. In the androgen receptor from the patients with Reifenstein syndrome, in which this alanine is converted to a threonine, wild-type receptor properties can be restored by exchanging an additional threonine at position 602 to an alanine. An alanine residue at position 596 or 602 in the DNA binding domain of the androgen receptor is therefore important for the full function of this receptor. In all steroid receptors that bind the core sequence AGAACANNNTGTTCT, an alanine residue is also present at a position equivalent to alanine 596 in the androgen receptor.
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Hipogonadismo/genética , Hipogonadismo/metabolismo , Mutación , Receptores Androgénicos/deficiencia , Receptores Androgénicos/genética , Alanina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Secuencia Conservada , ADN/genética , ADN/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Síndrome , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The action of the glucocorticoid receptor (GR) on beta-casein gene transcription serves as a well-studied example of a case where the action of the GR is dependent on the activity of another transcription factor, STAT5. We have investigated the domain-requirement of the GR for this synergistic response in transfection experiments employing GR mutants and CV-1 or COS-7 cells. The results were influenced by the expression levels of the GR constructs. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and the antiglucocorticoid RU486 exhibited a weak agonistic activity. When the N-terminal region of the GR was exchanged with the respective domain of the progesterone receptor, STAT5-dependent transactivation was reduced at low and high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the beta-casein gene promoter. Furthermore, at high GR expression levels RU486 was nearly as efficient as dexamethasone in activating transcription via the STAT5 dependent beta-casein gene promoter. The results reconcile the controversial issue regarding the DNA binding-independent action of the GR together with STAT5 and provide evidence that the mode of action of the GR depends not only on the type of the particular promoter at which it acts but also on the concentration of the GR. GR DNA binding function appears to be mandatory for beta-casein gene expression in mammary epithelial cells, since the promoter function is completely dependent on the integrity of GR binding sites in the promoter.
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Proteínas de Unión al ADN/metabolismo , Proteínas de la Leche , Receptores de Glucocorticoides/genética , Transactivadores/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células COS , Proteínas Portadoras/genética , Caseínas/genética , Línea Celular , Chlorocebus aethiops , ADN/metabolismo , Dimerización , Proteína HMGB1 , Proteínas del Grupo de Alta Movilidad/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factor de Transcripción STAT5 , Dedos de ZincRESUMEN
BACKGROUND: One of the major challenges in prostate cancer (PCa) treatment is distinguishing insignificant PCa from those forms that need active treatment. We evaluated the impact of PSA isoforms on risk stratification in patients with low-risk PCa as well as in active surveillance (AS) candidates who underwent radical prostatectomy. METHODS: A total of 112 patients with biopsy confirmed Gleason score (GS) 6 PCa of four different international institutions were prospectively enrolled in the study. Blood withdrawal was performed the day before radical prostatectomy. In addition, patients were classified according to the EAU and NCCN criteria for AS candidates. PSA, free PSA (fPSA) and proPSA were measured using dual monoclonal antibody sandwich immunoassays. In addition, the Prostate Health Index (PHI=proPSA/fPSA × âPSA) was calculated. Final histology of the radical prostatectomy specimens was correlated to PSA, its isoforms and PHI. RESULTS: Serum proPSA levels were significantly elevated in those patients with an upgrade in final histology (GS⩾7). In addition, higher proPSA levels were predictive for extraprostatic extension (⩾pT3a) as well as for positive surgical margins. Interestingly, PHI had an even higher predictive power when compared with proPSA alone concerning GS upgrading, extraprostatic extension and surgical margins in both the total and the AS patient group. CONCLUSION: We showed in a multicenter study that proPSA is a valuable biomarker to detect patients with aggressive PCa in a cohort of GS 6 patients, who would benefit from active tumor therapy. Combining proPSA with the standard markers PSA and fPSA using PHI further increases the predictive accuracy significantly. Moreover, our data support the use of PHI for monitoring PCa patients under AS.
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Calicreínas/sangre , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/sangre , Precursores de Proteínas/sangre , Adulto , Anciano , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estudios Prospectivos , Próstata/patología , Próstata/cirugía , Prostatectomía , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Resultado del TratamientoRESUMEN
Nearly all primary prostatic carcinomas have been found to express the androgen receptor (AR) protein, which is the intracellular mediator of androgen action. To gain a better insight into the mechanisms of androgen independence of advanced prostatic carcinoma, it is important to know whether the AR is also present in metastases of androgen-independent tumors. We have assessed the status of the AR and the prostate-specific antigen in 22 metastases of 18 patients with progressive prostate cancer. In 18 cases, the metastases were localized in bone, in 3 cases in the epidural space, and in 1 case in the periosteum. All but one patient had received some kind of endocrine treatment for prostatic carcinoma. Paraffin-embedded tissue sections were stained for the AR following a streptavidinbiotin-peroxidase protocol with the polyclonal antibody PG-21, which is directed against amino acids 1 through 21 of the rat and the human AR. The percentage of AR-positive cells was evaluated on the basis of an arbitrary 4-point scale. All 22 tumor metastases displayed AR positivity. One AR-positive metastatic lesion did not stain for prostate-specific antigen, but in all other metastases, this protein was detected by means of immunohistochemistry. The present study provides evidence that, unlike androgen-independent prostatic carcinoma cell lines, distant prostatic carcinoma metastases do express the AR. These findings indicate that the AR may be involved in the progression of prostate cancer.
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Neoplasias Óseas/secundario , Neoplasias Óseas/ultraestructura , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/ultraestructura , Receptores Androgénicos/análisis , Neoplasias Óseas/química , Espacio Epidural/patología , Humanos , Inmunohistoquímica , Masculino , Metástasis de la Neoplasia/diagnóstico , Periostio/patología , Antígeno Prostático Específico/análisis , Neoplasias de la Próstata/químicaRESUMEN
Aberrant activation of the androgen receptor (AR) may be one of the mechanisms which contribute to progression of prostatic carcinoma to an androgen-independent stage. We investigated effects of growth factors on stimulation of the AR-mediated gene transcription in human prostatic tumor cell lines. DU-145 cells, which do not contain endogenous AR, were cotransfected with an androgen-inducible chloramphenicol acetyltransferase (CAT) reporter gene and an AR expression vector. The reporter gene (CAT) was driven either by artificial promoters consisting of one or two androgen-responsive elements in front of a TATA box or by the promoter of the prostate-specific antigen (PSA) gene, a naturally occurring androgen-inducible promoter. Insulin-like growth factor-I (IGF-I), at a concentration of 50 ng/ml, stimulated AR-mediated reporter gene transcription to the same extent as the synthetic androgen methyltrienolone. This growth factor was effective irrespective of the nature of the androgen-inducible promoter. Keratinocyte growth factor (KGF) and epidermal growth factor (EGF), at concentrations of 50 ng/ml, activated CAT reporter gene transcription only in experiments in which the artificial promoter with two androgen-responsive elements was used. Insulin-like growth factor-II and basic fibroblast growth factor displayed no effect on AR-mediated gene transcription. None of the growth factors stimulated reporter gene activity in control experiments when added to cells cotransfected with the CAT gene and an empty expression vector. AR activation by IGF-I, KGF, and EGF was completely inhibited by the pure AR antagonist casodex, showing that these effects are AR mediated. Activation of endogenous AR by growth factors was studied in the LNCaP cell line by determination of PSA secretion. IGF-I, at a concentration of 50 ng/ml, increased the PSA level in the supernatant of this cell line 5-fold. Again, the IGF-I effect on PSA secretion was blocked by casodex. Our results provide evidence that IGF-I, KGF, and EGF directly activate the AR in the absence of androgens, which means that the androgen-signaling chain may be activated by growth factors in an androgen-depleted environment. These findings may have implications for endocrine therapy for metastatic prostatic carcinoma.
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Factor de Crecimiento Epidérmico/farmacología , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Antagonistas de Andrógenos/farmacología , Anilidas/farmacología , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Genes Reporteros , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Masculino , Metribolona/farmacología , Nitrilos , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/genética , Transducción de Señal , Compuestos de Tosilo , Transfección , Células Tumorales CultivadasRESUMEN
Interleukin-6 (IL-6) levels are frequently elevated in sera of patients with metastatic prostate cancer. IL-6 receptors are expressed in prostate cancer cell lines, as well as in benign prostate hyperplasia and prostate cancer tissue specimens. The androgen receptor (AR) is a key transcription factor that is present in all stages of prostate carcinoma, even in therapy-refractory tumors. In an attempt to investigate possible cross-talk between IL-6 and androgen signal transduction cascades, we tested the effects of this cytokine on AR transcriptional activity. The regulation of AR activity by IL-6 was studied in DU-145 cells, which were cotransfected with the androgen-responsive reporter plasmid ARE2TATACAT and the AR expression vector pSG5AR. We show that IL-6 up-regulates AR activity in a ligand-independent manner, as well as synergistically, with very low doses of the synthetic androgen methyltrienolone (5-10 pM). Therefore, AR activation by IL-6 may be operative in prostate cancer patients who have decreased androgen levels because of androgen ablation therapy. The maximal induction of reporter gene activity by IL-6 alone (50 ng/ml) was 67% of that stimulated by 1 nM of methyltrienolone. The nonsteroidal antiandrogen bicalutamide (Casodex) nearly completely inhibited AR activation by IL-6. IL-6 effects on AR activity were also abolished or greatly reduced by inhibitors of protein kinase A and C and mitogen-activated protein kinase pathways. In concordance with the results obtained in DU-145 cells, IL-6 induced AR-regulated prostate-specific antigen mRNA and protein in LNCaP cells. Stimulation of prostate-specific antigen protein secretion by IL-6 was antagonized by bicalutamide and inhibitors of protein kinase A and mitogen-activated protein kinase signaling pathways. Taken together, our data show for the first time that IL-6 is a nonsteroidal activator of the AR and that this activation is implicated in the regulation of prostate-specific proteins. Keeping in mind that IL-6, its receptor, and the AR are expressed in prostate cancers, cross-talk between IL-6 and AR signaling pathways may have clinical significance.
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Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Interleucina-6/farmacología , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/efectos de los fármacos , Regulación hacia Abajo , Humanos , Masculino , Antígeno Prostático Específico/biosíntesis , Inhibidores de Proteínas Quinasas , Transfección , Células Tumorales CultivadasRESUMEN
Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) are potent mitogens that regulate proliferation of prostate cancer cells via autocrine and paracrine loops and promote tumor metastasis. They exert their action through binding to the corresponding cell surface receptors that initiate an intracellular phosphorylation cascade, leading to the activation of mitogen-activated protein kinases (MAPKs), which recruit transcription factors. We have studied the effects of EGF, IGF-I, and the protein kinase A (PKA) activator forskolin on the activation of p42/ extracellular signal-regulated kinase (ERK)2, which is a key kinase in mediation of growth factor-induced mitogenesis in prostate cancer cells. The activity of p42/ERK2 was determined by immune complex kinase assays and by immunoblotting using a phospho p44/p42 MAPK-specific antibody. EGF, IGF-I, and forskolin-induced PKA activity stimulate intracellular signaling pathways converging at the level of p42/ERK2. In the androgen-insensitive DU145 cell line, there is a constitutive basal p42/ ERK2 activity that is not present in androgen-sensitive LNCaP cells. Constitutive p42/ERK2 activity is abrogated by blockade of the EGF receptor. Hence, it is obviously caused by an autocrine loop involving this receptor. The effects of EGF on p42/ERK2 are potentiated by forskolin in both cell lines. The blockade of PKA by the specific inhibitor H89 attenuates this synergism. This finding is in contrast to those obtained in several other systems studied thus far, in which PKA activators inhibited MAPKs. p42/ERK2 in DU145 cells is highly responsive to IGF-I stimulation, whereas no effect of IGF-I on p42/ERK2 can be measured in LNCaP cells. Moreover, our results demonstrate that selective blockade of the EGF receptor in prostate cancer cells does not only inhibit the action of EGF, but also IGF-I-induced activation of the MAPK pathway and the interaction with the PKA pathway. In conclusion, these findings offer new possibilities for a therapeutical intervention in prostate cancer by targeting signaling pathways of growth factors and PKA.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Neoplasias de la Próstata/metabolismo , Transducción de Señal/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Humanos , Masculino , Células Tumorales CultivadasRESUMEN
Novel drugs like Abiraterone or Enzalutamide, which target androgen receptor (AR) signaling to improve androgen deprivation therapy (ADT), have been developed during the past years. However, the application of these drugs is limited because of occurrence of inherent or acquired therapy resistances during the treatment. Thus, identification of new molecular targets is urgently required to improve current therapeutic prostate cancer (PCa) treatment strategies. PIAS1 (protein inhibitor of activated STAT1 (signal transducer and activator of transcription-1)) is known to be an important cell cycle regulator and PIAS1-mediated SUMOylation is essential for DNA repair. In this context, elevated PIAS1 expression has already been associated with cancer initiation. Thus, in the present study, we addressed the question of whether PIAS1 targeting can be used as a basis for an improved PCa therapy in combination with anti-androgens. We show that PIAS1 significantly correlates with AR expression in PCa tissue and in cell lines and demonstrate that high PIAS1 levels predict shorter relapse-free survival. Our patient data are complemented by mechanistic and functional in vitro experiments that identify PIAS1 as an androgen-responsive gene and a crucial factor for AR signaling via prevention of AR degradation. Furthermore, PIAS1 knockdown is sufficient to decrease cell proliferation as well as cell viability. Strikingly, Abiraterone or Enzalutamide treatment in combination with PIAS1 depletion is even more effective than single-drug treatment in multiple PCa cell models, rendering PIAS1 as a promising target protein for a combined treatment approach to improve future PCa therapies.
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Retroalimentación Fisiológica , Neoplasias de la Próstata/patología , Proteínas Inhibidoras de STAT Activados/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal , Andrógenos/farmacología , Androstenos/farmacología , Benzamidas , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Retroalimentación Fisiológica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Nitrilos , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Inhibidoras de STAT Activados/deficiencia , Proteínas Inhibidoras de STAT Activados/genética , Estabilidad Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Androgénicos/genética , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Transcripción Genética/efectos de los fármacosRESUMEN
Androgens are important growth regulators in prostate cancer. Their known mode of action in target cells requires binding to a cytoplasmic androgen receptor followed by a nuclear translocation event and modulation of the expression of specific genes. Here, we report another mode of action of this receptor. Treatment of androgen responsive prostate cancer cells with dihydrotestosterone leads to a rapid and reversible activation of mitogen-activated protein kinases MAPKs (also called extracellular signal-regulated kinases or Erks). Transient transfection assays demonstrated that the androgen receptor-mediated activation of MAP kinase results in enhanced activity of the transcription factor Elk-1. This action of the androgen receptor differs from its known transcriptional activity since it is rapid and insensitive to androgen antagonists such as hydroxyflutamide or casodex. Biochemical studies as well as analyses with dominant negative mutants showed the involvement of kinases such as MAPK/Erk kinase, phosphatidyl-inositol 3-kinase and protein kinase C in the androgen receptor-mediated activation of MAP kinase. These results demonstrate a novel regulatory action of the androgen receptor and prove that in addition to its known transcriptional effects, it also uses non-conventional means to modulate several cellular signalling processes.
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Adenocarcinoma/patología , Proteínas de Unión al ADN , Dihidrotestosterona/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/fisiología , Neoplasias Hormono-Dependientes/patología , Neoplasias de la Próstata/patología , Receptores Androgénicos/fisiología , Testosterona , Factores de Transcripción , Adenocarcinoma/metabolismo , Antagonistas de Andrógenos/farmacología , Núcleo Celular/enzimología , Citoplasma/enzimología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Indoles/farmacología , Ligandos , Masculino , Maleimidas/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/efectos de los fármacos , Neoplasias Hormono-Dependientes/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Receptores Androgénicos/efectos de los fármacos , Proteínas Recombinantes de Fusión/fisiología , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales Cultivadas , Proteína Elk-1 con Dominio etsRESUMEN
PURPOSE: The levels of interleukin-6 (IL-6) are frequently elevated in sera from patients with advanced prostate carcinoma. Our main objective was to investigate changes in responsiveness to IL-6 and/or androgen that occur in LNCaP cells after long-term treatment with IL-6. This in vitro model could be of clinical relevance because of its similarity with late-stage prostate carcinoma. EXPERIMENTAL DESIGN: LNCaP human prostate cancer cells were treated with IL-6 at a concentration of 5 ng/ml. After 20 passages, the new subline LNCaP-IL-6+ has been established. Passages 20-40 are referred to as low passages (LP) and passages 41-73 as high passages (HP). LNCaP cells passaged at the same time in the absence of IL-6 were used as controls (LNCaP-IL-6-). Cells were counted after treatment with either IL-6 or the synthetic androgen methyltrienolone (R1881), and cell cycle analysis was performed. Binding of IL-6 or R1881 was assessed by radioligand binding assays. Reporter gene activity was measured by chloramphenicol acetyltransferase assay. Prostate-specific antigen in LNCaP-IL-6+ supernatants was measured by an enzyme immunoassay. Expression of IL-6 mRNA and protein was assessed by reverse transcription-PCR and ELISA, respectively. RESULTS: The basal proliferation rate in HP LNCaP-IL-6+ cells was higher than that in LNCaP-IL-6- cells. IL-6 inhibited proliferation of LNCaP-IL-6- cells but not that of either LP or HP of LNCaP-IL-6+ cells. This inability to elicit a growth-inhibitory response was associated with lack of effect on cell cycle distribution in the LNCaP-IL-6+ subline. In parallel, IL-6 binding decreased gradually during long-term IL-6 treatment and, in HP, reached only 33% of the levels measured in controls. Binding of radiolabeled androgen increased 2-fold in HP LNCaP-IL-6+ cells. Reporter gene assays revealed that R1881, at nanomolar concentrations, was a more potent androgen receptor activator in LNCaP-IL-6+ than in LNCaP-IL-6- cells. However, androgen- and IL-6-induced prostate-specific antigen secretion decreased in long-term IL-6-treated cells. IL-6 cDNA fragments were detected by reverse transcription-PCR in HP LNCaP-IL-6+ cells but not in controls or LP. IL-6 protein was first detected in passage 36 of LNCaP-IL-6+ cells, and it increased in HP. CONCLUSIONS: Long-term treatment of LNCaP human prostate cancer cells with IL-6 leads to abolishment of inhibitory growth response. In contrast to control cells, the LNCaP-IL-6+ subline expresses IL-6 mRNA and protein.
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Interleucina-6/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Andrógenos/metabolismo , Andrógenos/farmacología , Unión Competitiva , Recuento de Células , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Metribolona/metabolismo , Metribolona/farmacología , Antígeno Prostático Específico/efectos de los fármacos , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Tritio , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Antihormones are by definition antagonists of steroid hormone action. They interact with the ligand binding domains of steroid hormone receptors and competitively inhibit the action of the receptors by mechanisms that are not quite understood. In certain cases antihormones also exhibit agonistic activity especially in connection with certain naturally occurring receptor mutants. These observations together with findings of indiscriminate interaction of antihormones with several classes of steroid receptors have necessitated a search of more effective and reliable antihormones. Recent advances in the resolution of the crystal structure of the ligand binding domains of certain members of the steroid receptor family and identification of non-liganded activation of steroid receptors have produced considerable information that can be harnessed into a fruitful search for a new generation of antihormones.
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Antagonistas de Hormonas/metabolismo , Receptores de Esteroides/antagonistas & inhibidores , Receptores de Esteroides/metabolismo , Esteroides/antagonistas & inhibidores , Animales , Antagonistas de Hormonas/química , Humanos , Ligandos , Esteroides/química , Esteroides/metabolismoRESUMEN
Structural changes of the androgen receptor (AR) may contribute to the development of resistance to endocrine therapy in prostatic carcinoma. We have isolated AR cDNA fragments from seven tumor specimens derived from patients with advanced metastatic prostatic tumors. In one specimen obtained from a patient who failed to respond to endocrine and cytotoxic therapy we have detected a point mutation in the hormone-binding domain of the receptor. This AR mutation is a guanine-to-adenine transition at nucleotide 2671 that leads to substitution of methionine for the wild type valine at position 715. It is a somatic mutation because it was not present in the AR genomic DNA fragments isolated from prostatic and testicular tissues of the same patient. The mutant AR was recreated in an expression vector and transiently expressed in COS-7 and CV-1 cells. Hormone-binding assays revealed that the mutant receptor does not differ from the wild type receptor in its ability to bind androgen. The dissociation constant for the synthetic androgen mibolerone was 3 nM for both receptors. There was also no significant difference in binding of other steroids and nonsteroidal antiandrogens as revealed by competition binding assays. However, transfection experiments to determine the trans-activation potential of the mutant receptor produced differences in the action of this receptor compared to the wild type receptor. Dihydrotestosterone and the synthetic androgens methyltrienolone (R1881) and mibolerone were equally proficient in conferring trans-activation activity to both the mutant and wild type receptors. Adrenal androgens such as dehydroepiandrosterone and androstenedione, as well as progesterone mediated a higher trans-activation through the mutant than through the wild type receptor. These data demonstrate that the exchange of a single valine into methionine at position 715 in the AR promoters trans-activation not only by testicular but also by adrenal androgens and progesterone. This pattern of ligand-dependent trans-activation may have significance in the process controlling the progression of prostatic carcinoma.
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Androstenodiona/farmacología , Carcinoma/genética , Deshidroepiandrosterona/farmacología , Dihidrotestosterona/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/genética , Mutación Puntual , Progesterona/farmacología , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Glándulas Suprarrenales/metabolismo , Anciano , Secuencia de Aminoácidos , Antagonistas de Andrógenos/metabolismo , Androstenodiona/metabolismo , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/genética , ADN de Neoplasias/genética , Deshidroepiandrosterona/metabolismo , Dihidrotestosterona/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Hormonas Esteroides Gonadales/farmacología , Humanos , Masculino , Datos de Secuencia Molecular , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Receptores Androgénicos/efectos de los fármacos , Receptores Androgénicos/metabolismo , Esteroides/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
There is increasing clinical interest in dendritic cells that are capable of initiating antitumor immune responses. Dendritic cells cultured from human blood mononuclear cells using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) are competent for antigen uptake but express relatively low levels of costimulatory molecules and thus correspond to immature resident tissue dendritic cells. In this study we took advantage of the new dendritic cell-specific marker CD83, which is expressed by mature dendritic cells, to delineate the maturation of cultured human blood dendritic cells. Although dendritic cells cultured with GM-CSF and IL-4 contained transcripts for CD83 as determined by reverse transcription PCR, CD83 protein was barely detectable by flow cytometry, confirming that dendritic cells obtained with this system are immature. However, treatment of dendritic cells with tumor necrosis factor-alpha (TNF-alpha) significantly increased the levels of CD83 transcripts and induced CD83 protein expression in dendritic cells. In contrast to the initiation of dendritic cell culture, which was facilitated by high cell density (5 x 10(6) cells/mL), differentiation into CD83+ dendritic cells required a low cell concentration (0.5 x 10(6) cells/mL). At higher cell density (1 x 10(6) cells/mL), CD83 expression was suppressed and was almost completely prevented at 2 x 10(6) cells/mL. Induction of CD83 expression was accompanied by a strong upregulation of the costimulator B7-2 (CD86) on dendritic cells. While untreated CD83(-) dendritic cells efficiently internalized fluoresceinated Dextran, TNF-alpha treated CD83+ dendritic cells excluded these molecules, confirming that maturation of dendritic cells was associated with the silencing of the antigen-capturing machinery. Morphologically, CD83+ dendritic cells presented with pronounced cytoplasmic projections (veils) characteristic of mature dendritic cells. In summary, we show that cell density critically regulates dendritic cell development. Knowledge of the appropriate conditions for dendritic cell generation and maturation will be important in clinical immunotherapy settings.