RESUMEN
BACKGROUND: As the number of RNA-seq datasets that become available to explore transcriptome diversity increases, so does the need for easy-to-use comprehensive computational workflows. Many available tools facilitate analyses of one of the two major mechanisms of transcriptome diversity, namely, differential expression of isoforms due to alternative splicing, while the second major mechanism-RNA editing due to post-transcriptional changes of individual nucleotides-remains under-appreciated. Both these mechanisms play an essential role in physiological and diseases processes, including cancer and neurological disorders. However, elucidation of RNA editing events at transcriptome-wide level requires increasingly complex computational tools, in turn resulting in a steep entrance barrier for labs who are interested in high-throughput variant calling applications on a large scale but lack the manpower and/or computational expertise. RESULTS: Here we present an easy-to-use, fully automated, computational pipeline (Automated Isoform Diversity Detector, AIDD) that contains open source tools for various tasks needed to map transcriptome diversity, including RNA editing events. To facilitate reproducibility and avoid system dependencies, the pipeline is contained within a pre-configured VirtualBox environment. The analytical tasks and format conversions are accomplished via a set of automated scripts that enable the user to go from a set of raw data, such as fastq files, to publication-ready results and figures in one step. A publicly available dataset of Zika virus-infected neural progenitor cells is used to illustrate AIDD's capabilities. CONCLUSIONS: AIDD pipeline offers a user-friendly interface for comprehensive and reproducible RNA-seq analyses. Among unique features of AIDD are its ability to infer RNA editing patterns, including ADAR editing, and inclusion of Guttman scale patterns for time series analysis of such editing landscapes. AIDD-based results show importance of diversity of ADAR isoforms, key RNA editing enzymes linked with the innate immune system and viral infections. These findings offer insights into the potential role of ADAR editing dysregulation in the disease mechanisms, including those of congenital Zika syndrome. Because of its automated all-inclusive features, AIDD pipeline enables even a novice user to easily explore common mechanisms of transcriptome diversity, including RNA editing landscapes.
Asunto(s)
Programas Informáticos , Transcriptoma , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Expresión Génica , Ontología de Genes , Humanos , Análisis de Componente Principal , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Edición de ARN , RNA-Seq , Células Madre/citología , Células Madre/metabolismo , Células Madre/virología , Virus Zika/fisiologíaRESUMEN
BACKGROUND: One key step in the development of prostate cancer (PCa) metastasis is the loss of E-cadherin expression associated with increased cellular motility and tumor invasion. This loss of E-cadherin expression is also required during normal embryogenesis and similar transcriptional repressors have been identified in both processes. We have previously reported the presence of one such transcription factor, WT1 in high Gleason grade prostate tumor tissues, and its absence in non-neoplastic or benign prostatic hyperplasia tissues. RESULTS: To better understand the effect of WT1 on E-cadherin expression and migration of PCa cells we quantified WT1 and E-cadherin mRNA levels in normal prostate epithelial and PCa cell lines with varying migratory potential. In WT1 transfected cells E-cadherin transcript levels were decreased, while they were increased in siWT1-RNA transfected PCa cells, suggesting that elevated WT1 expression was sufficient to dampen E-cadherin levels and potentially enhance migratory ability. To delineate the mechanism of WT1-mediated repression of E-cadherin, potential WT1 binding sites were tested in vitro and in vivo binding of WT1 to the E-cadherin promoter in the chromatin of LNCaP and PC3 cells was assessed by Chromatin Immunoprecipitation. The effect of WT1 binding was measured in reporter assays; in PC3 and DU145 cells WT1 decreased the activity of the proximal E-cadherin promoter. Using site-directed mutagenesis, a newly identified WT1 binding site located 146 bp from the transcription start site was shown to be required for this repression by WT1. Transwell migration and wound healing assays revealed that in LNCaP cells with low migratory potential, over-expression of WT1 was sufficient to enhance migration, conversely, in the highly migratory PC3 cells silencing of WT1 decreased migration. CONCLUSIONS: These findings suggested that WT1 expression in high grade prostate cancer may contribute to migration and metastasis. Thus, in prostate cancer WT1 may function as a novel oncogene facilitating development of the lethal metastatic phenotype.
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Cadherinas/genética , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Genes del Tumor de Wilms , Antígenos CD , Secuencia de Bases , Cadherinas/metabolismo , Línea Celular Tumoral , Cartilla de ADN/genética , Expresión Génica , Humanos , Masculino , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Neoplasias de la Próstata , Unión Proteica , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMEN
BACKGROUND: Vascular Endothelial Growth Factor (VEGF) is regulated by a number of different factors, but the mechanism(s) behind androgen-mediated regulation of VEGF in prostate cancer are poorly understood. RESULTS: Three novel androgen receptor (AR) binding sites were discovered in the VEGF promoter and in vivo binding of AR to these sites was demonstrated by chromatin immunoprecipitation. Mutation of these sites attenuated activation of the VEGF promoter by the androgen analog, R1881 in prostate cancer cells. The transcription factors AR and Sp1 were shown to form a nuclear complex and both bound the VEGF core promoter in chromatin of hormone treated CWR22Rv1 prostate cancer cells. The importance of the Sp1 binding site in hormone mediated activation of VEGF expression was demonstrated by site directed mutagenesis. Mutation of a critical Sp1 binding site (Sp1.4) in the VEGF core promoter region prevented activation by androgen. Similarly, suppression of Sp1 binding by Mithramycin A treatment significantly reduced VEGF expression. CONCLUSIONS: Our mechanistic study of androgen mediated induction of VEGF expression in prostate cancer cells revealed for the first time that this induction is mediated through the core promoter region and is dependent upon a critical Sp1 binding site. The importance of Sp1 binding suggests that therapy targeting the AR-Sp1 complex may dampen VEGF induced angiogenesis and, thereby, block prostate cancer progression, helping to maintain the indolent form of prostate cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Factor de Transcripción Sp1/metabolismo , Congéneres de la Testosterona/fisiología , Activación Transcripcional , Factor A de Crecimiento Endotelial Vascular/genética , Antagonistas de Receptores Androgénicos/farmacología , Anilidas/farmacología , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Cromatina/metabolismo , Expresión Génica , Humanos , Masculino , Metribolona/farmacología , Nitrilos/farmacología , Regiones Promotoras Genéticas , Neoplasias de la Próstata , Unión Proteica , Receptores Androgénicos/metabolismo , Elementos de Respuesta , Congéneres de la Testosterona/farmacología , Compuestos de Tosilo/farmacología , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The prostate gland represents a multifaceted system in which prostate epithelia and stroma have distinct physiological roles. To understand the interaction between stroma and glandular epithelia, it is essential to delineate the gene expression profiles of these two tissue types in prostate cancer. Most studies have compared tumor and normal samples by performing global expression analysis using a mixture of cell populations. This report presents the first study of prostate tumor tissue that examines patterns of differential expression between specific cell types using laser capture microdissection (LCM). METHODS: LCM was used to isolate distinct cell-type populations and identify their gene expression differences using oligonucleotide microarrays. Ten differentially expressed genes were then analyzed in paired tumor and non-neoplastic prostate tissues by quantitative real-time PCR. Expression patterns of the transcription factors, WT1 and EGR1, were further compared in established prostate cell lines. WT1 protein expression was also examined in prostate tissue microarrays using immunohistochemistry. RESULTS: The two-step method of laser capture and microarray analysis identified nearly 500 genes whose expression levels were significantly different in prostate epithelial versus stromal tissues. Several genes expressed in epithelial cells (WT1, GATA2, and FGFR-3) were more highly expressed in neoplastic than in non-neoplastic tissues; conversely several genes expressed in stromal cells (CCL5, CXCL13, IGF-1, FGF-2, and IGFBP3) were more highly expressed in non-neoplastic than in neoplastic tissues. Notably, EGR1 was also differentially expressed between epithelial and stromal tissues. Expression of WT1 and EGR1 in cell lines was consistent with these patterns of differential expression. Importantly, WT1 protein expression was demonstrated in tumor tissues and was absent in normal and benign tissues. CONCLUSIONS: The prostate represents a complex mix of cell types and there is a need to analyze distinct cell populations to better understand their potential interactions. In the present study, LCM and microarray analysis were used to identify novel gene expression patterns in prostate cell populations, including identification of WT1 expression in epithelial cells. The relevance of WT1 expression in prostate cancer was confirmed by analysis of tumor tissue and cell lines, suggesting a potential role for WT1 in prostate tumorigenesis.
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Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Rayos Láser , Microdisección/instrumentación , Próstata/metabolismo , Neoplasias de la Próstata/genética , Células del Estroma/metabolismo , Línea Celular Tumoral , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Células Epiteliales/patología , Perfilación de la Expresión Génica/métodos , Humanos , Inmunohistoquímica , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Mensajero/metabolismo , Células del Estroma/patología , Análisis de Matrices Tisulares , Proteínas WT1/genéticaRESUMEN
Interactions of proteins with DNA play an important role in regulating the biological functions of DNA. Here we propose and demonstrate the detection of protein-DNA binding using surface-enhanced Raman scattering (SERS). In this method, double-stranded DNA molecules with potential protein-binding sites are labeled with dye molecules and immobilized on metal nanoparticles. The binding of proteins protects the DNA from complete digestion by exonuclease and can be detected by measuring the SERS signals before and after the exonuclease digestion. As a proof of concept, this SERS-based protein-DNA interaction assay is validated by studying the binding of a zinc finger transcription factor WT1 with DNA sequences derived from the promoter of the human vascular endothelial growth factor.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas WT1/metabolismo , Secuencia de Bases , ADN/química , ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Agar , Humanos , Datos de Secuencia Molecular , Espectrometría Raman , Proteínas WT1/química , Proteínas WT1/genéticaRESUMEN
Signal intensity in fluorescence microscopy is often measured relative to arbitrary standards. We propose a calibration method based on a solution of the same fluorophore, whose binding to cells needs to be quantified. The method utilizes the low sensitivity of intensity to the object distance in wide-field imaging of uniform materials. Liquid layers of slowly varying depth were prepared by immersing a spherical lens into a drop of a fluorophore placed on a slide. Flatfield-corrected images of the contact and surrounding areas showed linear dependence of the gray level on the depth of fluorescent liquid. This allowed conversion of the measured intensity into the number of molecules per unit area. The method was applied to different cell types stained by WGA-Alexa 488 and WGA-TRITC. Consistent results were obtained by comparing microscopy with flow cytometry, comparing imaging through different objectives and comparing different WGA conjugates. Reproducibility of calibration was within 97% when low magnification was used. Fluorescence of free and bound WGA was found to be different, however, and therefore precise measurement of the number of cell-bound molecules was problematic in this particular system. We conclude that the method achieves reliable measurement of cellular staining in the units of soluble fluorophore. For probes whose fluorescent properties are unaffected by binding, quantification of staining in true molecular units should be possible.
Asunto(s)
Microscopía Fluorescente/métodos , Aglutininas del Germen de Trigo/metabolismo , Calibración , Eritrocitos/metabolismo , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Células HeLa , Humanos , Unión Proteica , Rodaminas/metabolismo , Solubilidad , Especificidad por SustratoRESUMEN
The restricted expression of Wilms tumor 1 (WT1) and cyclin A1 (CCNA1) in normal tissues, as opposed to their abnormal expression in leukemia demonstrates the applicability of WT1 and CCNA1 as cancer antigens for immunotherapy, and as markers for prognosis and relapse. In this study, the WT1 and CCNA1 mRNA levels were found to be elevated in bone marrow samples from pediatric acute promyelocytic leukemia (APL or AMLM3) patients, and to be quite varied in pediatric acute lymphocytic leukemia (ALL) patients, compared to nonleukemic bone marrow controls. Consistent with the observed upregulation of both WT1 and CCNA1 in APL, WT1 overexpression elevated the CCNA1 mRNA levels in K562 leukemia cells. Treatment with curcumin decreased the WT1 levels in K562 cells, and also decreased CCNA1 protein expression. The examination of the CCNA1 promoter identified potential canonical WT1 binding sites within the 3kb region upstream of the transcription start site. Chromatin immunoprecipitation and luciferase reporter assays confirmed WT1 binding and the activation of the CCNA1 promoter. Furthermore, the GCrich core CCNA1 promoter region provided additional noncanonical WT1 activation sites, as revealed by promoter assays. The importance of the GCrich core region of the CCNA1 promoter was confirmed by treating the K562 cells with mithramycin A, which blocks the binding of zinc finger transcription factors to GCrich sequences. Mithramycin A subsequently suppressed both CCNA1 promoter activity and protein expression in the K562 cells. Taken together, the data from the WT1 overexpression, and curcumin and mithramycin A treatment experiments, as well as those from chromatin binding assays, along with inferences from patient RNA analyses, establish a plausible link between WT1 and CCNA1, and support the functional significance of an elevated WT1 expression in leukemia, which may also affect CCNA1 expression.
Asunto(s)
Ciclina A1/genética , Leucemia Promielocítica Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas WT1/genética , Adolescente , Sitios de Unión , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Niño , Preescolar , Curcumina/farmacología , Ciclina A1/química , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lactante , Células K562 , Leucemia Promielocítica Aguda/metabolismo , Masculino , Plicamicina/análogos & derivados , Plicamicina/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Proteínas WT1/química , Proteínas WT1/metabolismoRESUMEN
BACKGROUND: Gene expression analyses have led to a better understanding of growth control of prostate cancer cells. We and others have identified the presence of several zinc finger transcription factors in the neoplastic prostate, suggesting a potential role for these genes in the regulation of the prostate cancer transcriptome. One of the transcription factors (TFs) identified in the prostate cancer epithelial cells was the Wilms tumor gene (WT1). To rapidly identify coordinately expressed prostate cancer growth control genes that may be regulated by WT1, we used an in silico approach. RESULTS: Evolutionary conserved transcription factor binding sites (TFBS) recognized by WT1, EGR1, SP1, SP2, AP2 and GATA1 were identified in the promoters of 24 differentially expressed prostate cancer genes from eight mammalian species. To test the relationship between sequence conservation and function, chromatin of LNCaP prostate cancer and kidney 293 cells were tested for TF binding using chromatin immunoprecipitation (ChIP). Multiple putative TFBS in gene promoters of placental mammals were found to be shared with those in human gene promoters and some were conserved between genomes that diverged about 170 million years ago (i.e., primates and marsupials), therefore implicating these sites as candidate binding sites. Among those genes coordinately expressed with WT1 was the kallikrein-related peptidase 3 (KLK3) gene commonly known as the prostate specific antigen (PSA) gene. This analysis located several potential WT1 TFBS in the PSA gene promoter and led to the rapid identification of a novel putative binding site confirmed in vivo by ChIP. Conversely for two prostate growth control genes, androgen receptor (AR) and vascular endothelial growth factor (VEGF), known to be transcriptionally regulated by WT1, regulatory sequence conservation was observed and TF binding in vivo was confirmed by ChIP. CONCLUSION: Overall, this targeted approach rapidly identified important candidate WT1-binding elements in genes coordinately expressed with WT1 in prostate cancer cells, thus enabling a more focused functional analysis of the most likely target genes in prostate cancer progression. Identifying these genes will help to better understand how gene regulation is altered in these tumor cells.
Asunto(s)
Evolución Biológica , Secuencia Conservada , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Proteínas WT1/genética , Dedos de Zinc/genética , Animales , Secuencia de Bases , Sitios de Unión , Inmunoprecipitación de Cromatina , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Humanos , Masculino , Antígeno Prostático Específico/genética , Receptores Androgénicos/genética , Alineación de Secuencia , Factores de Transcripción/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
As S-phase checkpoints play critical roles in maintaining genomic integrity and replicating the human genome correctly, understanding the molecular mechanism by which they regulate the therapeutic response is of great interest. Previously, we reported that the cytotoxic effect of a zinc-bound form of Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL), which is currently evaluated in clinical trials, in combination with low-dose CPT-11, induces apoptosis of C4-2 human prostate cancer cells and tissues. Here, we show that apoptosis, induced synergistically by this combination treatment, was associated with accumulation of cells in early S phase, indicated by cell cycle analyses, increased proliferating cell nuclear antigen, and Chk2-Thr(68) phosphorylation in tumors xenografted in mice. The combination treatment induced an S-phase checkpoint response through activation of Chk2 and Chk1 by the ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3 related kinases, leading to phosphorylation and decreased Cdc25A levels. Cdc25A-dependent regulation of cyclin-dependent kinase 2 (Cdk2) and changes in association of p21(WAF1/CIP1) and hSpy1 with Cdk2 resulted in inhibition of Cdk2-associated kinase activity. Knockdown of ataxia telangiectasia mutated/Chk2 and ataxia telangiectasia mutated and Rad3 related/Chk1 by small inhibitory RNAs abrogated the S-phase checkpoint and accelerated apoptosis, resulting in caspase-3 activation and poly(ADP-ribose) polymerase 1 cleavage following combination treatment. Thus, Apo2L/TRAIL + CPT-11 treatment-induced apoptosis is regulated through an S-phase checkpoint controlled by the Chk2-Cdc25A and Chk1-Cdc25A pathways and inhibition of Cdk2-associated kinase activity. Low-dose CPT-11 and aphidicolin increased the proportion of S-phase cells and sensitized cells to Apo2L/TRAIL, by inducing phosphatidylserine externalization, caspase activation, and poly(ADP-ribose) polymerase 1 cleavage. Combinations with S-phase arrest-inducing chemotherapeutic drugs may represent promising avenues for clinical development of Apo2L/TRAIL.
Asunto(s)
Apoptosis/efectos de los fármacos , Camptotecina/análogos & derivados , Neoplasias de la Próstata/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Fase S/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Afidicolina/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Camptotecina/administración & dosificación , Camptotecina/farmacología , Camptotecina/uso terapéutico , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Humanos , Irinotecán , Ligandos , Masculino , Ratones , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , Trasplante Heterólogo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Understanding angiogenesis and growth control is central for elucidating prostate tumorigenesis. However, the mechanisms of activation of the angiogenic gene, vascular endothelial growth factor (VEGF) are complex and its regulation in prostate cancer is not well understood. In previous studies, VEGF expression levels were correlated with altered levels of the zinc finger transcription factor, WT1. Since the VEGF promoter has several potential WT1 binding sites and WT1 regulates many growth control genes, here we assessed whether WT1 might also regulate VEGF transcription. Using transfection and DNA binding assays, functional WT1 binding sites were localized within the proximal VEGF promoter. Transfection of the DDS-WT1 (R394W) zinc finger mutant had no significant effect on VEGF-luciferase reporter activity, suggesting that an intact zinc finger DNA binding domain was required. Interestingly, WT1-mediated regulation of VEGF reporter constructs varied in different cell types. In androgen-responsive, LNCaP prostate cancer cells, hormone treatment enhanced WT1-mediated activation of the VEGF promoter constructs. Overall, these results suggest that WT1 transcriptionally regulates VEGF through interaction of its zinc finger DNA binding domain with the proximal GC-rich VEGF promoter. These findings may shed light on the role of WT1 in angiogenesis and prostate cancer progression.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Factor A de Crecimiento Endotelial Vascular/genética , Proteínas WT1/metabolismo , Sitios de Unión , Línea Celular Tumoral , Humanos , Masculino , Metribolona/farmacología , Congéneres de la Testosterona/farmacología , Activación TranscripcionalRESUMEN
The zinc finger transcription factor, WT1, regulates many growth control genes, repressing or activating transcription depending on the gene and cell type. Based on earlier analyses of the effect of WT1 on androgen responsive genes, we hypothesized that there may be an interaction between the androgen signaling pathway and WT1, such that the commonly used Renilla luciferase control vectors were activated in LNCaP prostate cancer cells. Using cotransfection assays we tested the effects of WT1 and/or the androgen analog, R1881, on two Renilla luciferase vectors, pRL-SV40 and the promoter-less pRL-null. To determine whether the zinc finger DNA binding domain was required, the zinc finger mutant DDS-WT1 (R394W) was tested; but it had no significant effect on the Renilla luciferase vectors. To determine whether the androgen signaling pathway was required, WT1 was co-transfected with Renilla vectors in cells with varied hormone responsiveness. The WT1 effect on pRL-null varied from no significant effect in 293 and PC3 cells to very strong enhancement in LNCaP cells treated with 5 nM R1881. Overall, these results suggest that hormone enhanced WT1 mediated activation of Renilla luciferase and that these interactions require an intact WT1 zinc finger DNA binding domain.
Asunto(s)
Luciferasas de Renilla/análisis , Congéneres de la Testosterona/farmacología , Activación Transcripcional , Proteínas WT1/metabolismo , Línea Celular Tumoral , Genes Reporteros , Humanos , Luciferasas de Renilla/genética , Metribolona/farmacología , Activación Transcripcional/efectos de los fármacosRESUMEN
To identify physiologically relevant WT1 transcriptional target genes in prostate cancer cells, we have established stably transfected LNCaP cell lines expressing either WT1(A), its mutant counterpart DDS(R384W), or vector control. Microarray analyses of these cells revealed that vascular endothelial growth factor (VEGF) was differentially expressed in the engineered lines. Regulation of VEGF by WT1 likely contributes to kidney angiogenesis during development and WT1 mutants such as DDS(R384W) are associated with the Denys-Drash syndrome (DDS), characterized by renal abnormalities. Recent mechanistic studies have demonstrated that the WT1(A) isoform binds VEGF promoter sequences and transcriptionally regulates VEGF reporter constructs. However, regulation of VEGF is complex, involving both transcriptional and post-transcriptional processes. This study examined the ability of hormone and Actinomycin D treatment to alter VEGF mRNA levels in stably transfected WT-LNCaP, DDS-LNCaP, or V-LNCaP prostate cancer cells. The rationale of this study was based on a previous finding that enhancement of VEGF expression in DDS-LNCaP cells occurred only in the presence of the androgen analog, R1881. One possible explanation for these results was that DDS-WT1 stabilized VEGF mRNA so that it accumulated to higher levels. This hypothesis was tested by treating engineered LNCaP cells with Actinomycin D (Act D) and then measuring VEGF mRNA levels by quantitative real-time PCR. The combined effects of WT1 or DDS(R384W) and hormone were tested in these message stability assays and also in transcription assays of transiently transfected LNCaP cells. The results indicated that DDS-WT1 is unable to regulate VEGF transcription or stabilize VEGF mRNA in LNCaP prostate cancer cells. However our observations are also consistent with wild-type WT1(A) having both transcriptional and post-transcriptional effects on VEGF mRNA levels in the presence of hormone. These studies of VEGF regulation by WT1 and dysregulation by DDS(R384W) suggest an important role for WT1 in both normal and tumor-related angiogenesis.
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Regulación Neoplásica de la Expresión Génica , Neovascularización Patológica/genética , Neoplasias de la Próstata/irrigación sanguínea , Estabilidad del ARN , Factor A de Crecimiento Endotelial Vascular/genética , Proteínas WT1/fisiología , Línea Celular Tumoral , Dactinomicina/farmacología , Síndrome de Denys-Drash/genética , Hormonas/farmacología , Humanos , Masculino , Mutación , Neovascularización Fisiológica/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Transcripción Genética/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMEN
Prostate cancer progression is controlled by the androgen receptor and new blood vessel formation, or angiogenesis, which promotes metastatic prostate cancer growth. Angiogenesis is induced by elevated expression of vascular endothelial growth factor (VEGF). VEGF is regulated by many factors in the tumor microenvironment including lowered oxygen levels and elevated androgens. Here we review evidence delineating hormone mediated mechanisms of VEGF regulation, including novel interactions between the androgen receptor (AR), epigenetic and zinc-finger transcription factors, AR variants and the hypoxia factor, HIF-1. The relevance of describing the impact of both hormones and hypoxia on VEGF expression and angiogenesis is revealed in recent reports of clinical therapies targeting both VEGF and AR signaling pathways. A better understanding of the complexities of VEGF expression could lead to improved targeting and increased survival time for a subset of patients with metastatic castration-resistant prostate cancer.
RESUMEN
The Wilms' tumor suppressor gene product (WT1) regulates expression of growth control genes. Microarray analysis of gene expression profiles of hormone-treated LNCaP prostate cancer cell lines transfected with either wild-type WT1 or a zinc finger mutant form, DDS (R394W), revealed significantly altered patterns of expression. Validation studies using quantitative real-time PCR confirmed the differential expression of the tumor progression gene, vascular endothelial growth factor (VEGF). WT1-LNCaP cells had significantly reduced levels of VEGF mRNA when compared to vector control cells; in contrast, DDS-LNCaP cells showed elevated levels of VEGF transcripts. To address a functional role for WT1 overexpression, we investigated whether induction of VEGF expression, by the synthetic androgen R1881, would be disrupted in wild-type or mutant WT1-transfected LNCaP cells. Hormone treatment failed to elevate VEGF transcript levels above uninduced baseline levels in WT1-LNCaP cells, despite 48 h of treatment with 5 nM R1881. Consistent with our quantitative real-time PCR analysis, immunofluorescent staining of VEGF protein was reduced in WT1-LNCaP cells in both the presence and absence of R1881 treatment. Conversely, VEGF levels increased in vector control and DDS-LNCaP cells treated with 5 nM R1881. Not only do these studies point out the regulatory potential of WT1 for VEGF, but they also indicate an altered function for the mutant DDS isoform. Because VEGF is associated with neovascularization and promotion of metastasis in a variety of solid tumors including prostate cancer, a better understanding of the regulation of VEGF expression by transcription factors, such as WT1, is important for halting disease progression.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Transfección , Factor A de Crecimiento Endotelial Vascular/genética , Proteínas WT1/genética , Expresión Génica/fisiología , Perfilación de la Expresión Génica , Humanos , Masculino , Metribolona/farmacología , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Próstata/patología , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas WT1/metabolismoRESUMEN
Impaired expression of connexins, the gap junction subunits that facilitate direct cell-cell communication, have been implicated in prostate cancer growth. To elucidate the crucial role of connexins in prostate cancer progression, we performed a systematic quantitative RT-PCR screening of connexin expression in four representative prostate cancer cell lines across the spectrum of malignancy. Transcripts of several connexin subunits were detected in all four cell lines, and connexin 43 (Cx43) showed marked elevation at both RNA and protein levels in cells with increased metastatic potential. Analysis of gap-junction-mediated intercellular communication revealed homocellular coupling in PC-3 cells, which had the highest C x 43 expression, with minimal coupling in LNCaP cells where C x 43 expression was very low. Treatment with the gap junction inhibitor carbenoxolone or connexin mimetic peptide ACT-1 did not impair cell growth, suggesting that growth is independent of functional gap junctions. PC-3 cells with C x 43 expression reduced by shRNA showed decreased migration in monolayer wound healing assay, as well as decreased transwell invasion capacities when compared to control cells expressing non-targeting shRNA. These results, together with the correlation between C x 43 expression levels and the metastatic capacity of the cell lines, suggest a role of C x 43 in prostate cancer invasion and metastasis.
Asunto(s)
Movimiento Celular , Conexina 43/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Conexina 43/genética , Uniones Comunicantes/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Interferencia de ARN , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
The primary form of therapy for prostate cancer is androgen ablation resulting in apoptosis and expression of apoptotic genes (i.e. par-4). Prostate cancer cells that survive androgen ablation therapy express pro-survival genes (i.e. bcl-2) permitting these androgen independent (AI) cells to overcome apoptotic signals and proliferate in the absence of normal growth signals. To disrupt tumor growth and progression to AI, we expressed the tumor suppressor gene, WT1 in LNCaP prostate tumor cells. The WT1 transcription factor modulates expression and activity of several prostate growth control genes (i.e. par-4, bcl-2 and AR) in vitro. To provide insight into potential mechanisms of prostate cancer growth suppression both the transcriptionally active form of wild-type WT1 (D) and an inactive WT1 (D) R394W mutant form were stably transfected in LNCaP cells. Surprisingly both transfected lines underwent apoptosis and were growth suppressed in nude mice. A 3-fold reduction in overall tumor incidence and volume was associated with increased apoptosis, as evidenced by DNA fragmentation and par-4 expression, and was reduced or absent in early forming LNCaP tumors. After several months the indolent WT1-LNCaP cells became proliferative forming small tumors lacking par-4 protein. Although bcl-2 protein was present in all LNCaP tumors at this late-stage, it was detected in only a minority of WT1-LNCaP tumors, suggesting that pro-survival signals continued to be reduced in WT1-suppressed tumor cells. While the mechanisms of WT1-mediated growth suppression and apoptosis in LNCaP tumor cells are unknown, our results argue against simple transcriptional regulation since the mutant WT1 (D) R394W suppressed tumor formation similarly to wild-type WT1. This suggests that the mechanism of WT1-mediated growth suppression does not rely upon DNA binding at known WT1 recognition sites.
Asunto(s)
Genes Supresores de Tumor , Neoplasias de la Próstata/metabolismo , Proteínas WT1/biosíntesis , Animales , Apoptosis , Western Blotting , División Celular , Línea Celular Tumoral , Fragmentación del ADN , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Desnudos , Microscopía Fluorescente , Trasplante de Neoplasias , Plásmidos/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/terapia , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores Androgénicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética , Transfección , Proteínas WT1/genéticaRESUMEN
Chromosomal aneuploidy is associated with invasive bladder cancer and one of the genes implicated in these changes is Aurora-A/STK15/BTAK, that is localized on chromosome 20q13 and encodes a centrosome-associated serine/threonine kinase. To better understand the association between Aurora-A/STK15 expression, tumor aneuploidy and clinical prognosis, we sought to determine whether overexpression of Aurora-A/STK15 in cultured urothelial cells facilitated chromosomal instability. Using immunofluorescence staining, Northern and Western blot analyses, we verified that overexpression of Aurora-A/STK15 in bladder tumor cell lines enhanced chromosomal instability. Additionally, we observed that some bladder tumor cell lines expressed more Aurora-A/STK15 than cultured normal urothelial cells and that Aurora-A/STK15 expression was higher in an immortalized E7 urothelial cell line having 20q amplification than in an E6 line lacking 20q amplification. These results were consistent with our observations of higher mRNA levels in some T3 invasive bladder tumors than in T1 superficial tumors and adjacent normal bladder tissue. Overall our results suggest that overexpression of Aurora-A/STK15 in bladder tumor cells contributes to tumor progression by promoting chromosomal instability leading to aneuploidy.
Asunto(s)
Aneuploidia , Regulación de la Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/farmacología , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Aurora Quinasa A , Aurora Quinasas , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente , Humanos , Pronóstico , Regulación hacia Arriba , Urotelio/citologíaRESUMEN
The early growth response gene 1, EGR1, is an important transcriptional regulator and acts as the convergent point between a variety of extracellular stimuli and activation of target genes. Unlike other tumor types, prostate tumors express high levels of EGR1 relative to normal tissues. However, the mechanism of EGR1 regulation in prostate tumor cells is unknown. As EGR1 expression and epidermal growth factor (EGF) signaling are frequently upregulated in prostate tumors, we tested the hypothesis that EGF induces EGR1 expression in prostate cancer cells. Using RT-PCR to quantify EGR1 transcripts, we found that EGF induced EGR1 expression in a dose- and time-dependent manner and the ERK pathway inhibitor, PD98059, abrogated the EGF-mediated EGR1 response in LNCaP and PC3 cells. Analysis of the EGR1 promoter using deletion constructs identified an EGF-responsive region in the proximal promoter (-771 to -245 bp) containing 3 potential serum response element (SRE) sites. In vivo chromatin immunoprecipitation assays demonstrated that Elk-1 binding at the SRE sites of the EGR1 promoter was enhanced by EGF treatment in PC3 cells. Overexpression of Elk-1 was sufficient to activate the EGF-responsive region of EGR1 promoter in PC3 cells and, similarly, a dominant-negative Elk-1 suppressed EGR1 promoter activity. Taken together, these results demonstrate for the first time that EGR1 expression in PC3 cells is mediated through an EGF-ERK-Elk-1 signaling cascade.