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1.
Nat Methods ; 14(10): 1003-1009, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28869758

RESUMO

GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Guanosina Trifosfato/metabolismo , Proteínas Luminescentes/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Guanosina Trifosfato/genética , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/genética , Mutação
2.
Prostate ; 76(4): 359-68, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26642837

RESUMO

BACKGROUND: CWR22 is a human xenograft model of primary prostate cancer (PCa) that is often utilized to study castration recurrent (CR) PCa. CWR22 recapitulates clinical response to androgen deprivation therapy (ADT), in that tumors regress in response to castration, but can recur after a period of time. METHODS: Two cohorts of mice, totaling 117 mice were implanted with CWR22, allowed to develop tumors, castrated by pellet removal and followed for a period of 32 and 50 weeks. Mice presenting with tumors >2.0 cm(3) at the primary site, moribund appearance, or palpable masses other than the primary tumor were sacrificed prior to the endpoint of the study. Tumor tissue, serum, and abnormal lesions were collected upon necropsy and analyzed by IHC, H&E, and PCR for presence of metastatic lesions arising from CWR22. RESULTS: Herein, we report that CWR22 progresses after castration from a primary, hormonal therapy-naïve tumor to metastatic disease in 20% of castrated nude mice. Histological examination of CWR22 primary tumors revealed distinct pathologies that correlated with metastatic outcome after castration. CONCLUSION: This is the first report and characterization of spontaneous metastasis in the CWR22 model, thus, CWR22 is a bona-fide model of clinical PCa representing the full progression from androgen-sensitive, primary PCa to metastatic CR-PCa.


Assuntos
Metástase Neoplásica , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias da Próstata/patologia , Androgênios , Animais , Biomarcadores Tumorais/análise , Modelos Animais de Doenças , Xenoenxertos , Humanos , Imuno-Histoquímica , Metástase Linfática/patologia , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Recidiva Local de Neoplasia/patologia , Transplante de Neoplasias , Neoplasias Hormônio-Dependentes , Orquiectomia , Fenótipo , Neoplasias da Próstata/cirurgia , Testosterona/sangue
3.
Carcinogenesis ; 34(2): 248-56, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23087083

RESUMO

The current study investigated transcriptional distortion in prostate cancer cells using the vitamin D receptor (VDR) as a tool to examine how epigenetic events driven by corepressor binding and CpG methylation lead to aberrant gene expression. These relationships were investigated in the non-malignant RWPE-1 cells that were 1α,25(OH)(2)D(3) responsive (RWPE-1) and malignant cell lines that were 1α,25(OH)(2)D(3) partially responsive (RWPE-2) and resistant (PC-3). These studies revealed that selective attenuation and repression of VDR transcriptional responses in the cancer cell lines reflected their loss of antiproliferative sensitivity. This was evident in VDR target genes including VDR, CDKN1A (encodes p21( (waf1/cip1) )) and GADD45A; NCOR1 knockdown alleviated this malignant transrepression. ChIP assays in RWPE-1 and PC-3 cells revealed that transrepression of CDKN1A was associated with increased NCOR1 enrichment in response to 1α,25(OH)(2)D(3) treatment. These findings supported the concept that retained and increased NCOR1 binding, associated with loss of H3K9ac and increased H3K9me2, may act as a beacon for the initiation and recruitment of DNA methylation. Overexpressed histone methyltransferases (KMTs) were detectable in a wide panel of prostate cancer cell lines compared with RWPE-1 and suggested that generation of H3K9me2 states would be favored. Cotreatment of cells with the KMT inhibitor, chaetocin, increased 1α,25(OH)(2)D(3)-mediated induction of CDKN1A expression supporting a role for this event to disrupt CDKN1A regulation. Parallel surveys in PC-3 cells of CpG methylation around the VDR binding regions on CDKN1A revealed altered basal and VDR-regulated DNA methylation patterns that overlapped with VDR-induced recruitment of NCOR1 and gene transrepression. Taken together, these findings suggest that sustained corepressor interactions with nuclear-resident transcription factors may inappropriately transform transient-repressive histone states into more stable and repressive DNA methylation events.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Correpressor 1 de Receptor Nuclear/metabolismo , Neoplasias da Próstata/genética , Receptores de Calcitriol/metabolismo , Apoptose/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Calcitriol/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Imunoprecipitação da Cromatina , Epigênese Genética , Humanos , Masculino , Correpressor 1 de Receptor Nuclear/genética , Regiões Promotoras Genéticas/genética , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Calcitriol/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
4.
Am J Physiol Endocrinol Metab ; 305(7): E785-94, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23900419

RESUMO

Overnutrition during the suckling period (small litter, SL) results in the development of adult-onset obesity. Our aim was to investigate whether two levels of caloric restriction (CR) in the early postweaning period can reverse obese phenotype in SL rats. The normal litter (NL) had 12 pups/dam and SL had 3 male pups/dam from the postnatal day 3 until day 21. After weaning, rats consumed lab chow as indicated: 1) NL and SL groups were on ad libitum regimen up to day 140, 2) another SL group was pair-fed (SL/PF) to NL(∼14% reduction), 3) SL/PF/AL group was pair-fed up to day 94 and then switched to ad libitum feeding, 4) SL/CR group received 24% reduction (moderate CR) in food intake compared with SL, and 5) SL/CR/AL group was on 24% CR up to day 94 and then switched to ad libitum feeding. Pair-feeding reduced body weight gains and serum insulin and leptin levels compared with SL rats, but these parameters were restored to SL levels in the SL/PF/AL rats after switching to ad libitum feeding. Interestingly, the moderate CR normalized these parameters in SL/CR and SL/CR/AL rats compared with NL. The expression of neuropeptide Y, proopiomelanocortin, and leptin receptor returned to control levels in hypothalami from SL/CR and SL/CR/AL rats. These results indicate that appropriate manipulation of energy intake during the early postweaning period could lead to longer-lasting effects on the regulation of body weight homeostasis via reversal of the early preweaning programming effects on the hypothalamic appetite regulation mechanism.


Assuntos
Restrição Calórica , Lactação/fisiologia , Obesidade/terapia , Hipernutrição/complicações , Animais , Animais Lactentes , Peso Corporal , Feminino , Insulina/sangue , Leptina/sangue , Masculino , Obesidade/etiologia , Obesidade/metabolismo , Hipernutrição/metabolismo , Ratos , Ratos Sprague-Dawley
5.
Bioinformatics ; 28(5): 729-30, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22253290

RESUMO

UNLABELLED: The Illumina Infinium HumanMethylation450 BeadChip is a newly designed high-density microarray for quantifying the methylation level of over 450 000 CpG sites within human genome. Illumina Methylation Analyzer (IMA) is a computational package designed to automate the pipeline for exploratory analysis and summarization of site-level and region-level methylation changes in epigenetic studies utilizing the 450K DNA methylation microarray. The pipeline loads the data from Illumina platform and provides user-customized functions commonly required to perform exploratory methylation analysis for individual sites as well as annotated regions. AVAILABILITY: IMA is implemented in the R language and is freely available from http://www.rforge.net/IMA.


Assuntos
Ilhas de CpG , Metilação de DNA , Análise de Sequência com Séries de Oligonucleotídeos , Software , Genoma Humano , Humanos
6.
Mol Cancer Res ; 21(1): 24-35, 2023 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-36166196

RESUMO

Identifying and leveraging unique points of metabolic dysregulation in different disease settings is vital for safe and effective incorporation of metabolism-targeted therapies in the clinic. In addition, it has been shown identification of master metabolic transcriptional regulators (MMTR) of individual metabolic pathways, and how they relate to the disease in question, may offer the key to understanding therapeutic response. In prostate cancer, we have previously demonstrated polyamine biosynthesis and the methionine cycle were targetable metabolic vulnerabilities. However, the MMTRs of these pathways, and how they affect treatment, have yet to be explored. We sought to characterize differential sensitivity of prostate cancer to polyamine- and methionine-targeted therapies by identifying novel MMTRs. We began by developing a gene signature from patient samples, which can predict response to metabolic therapy, and further uncovered a MMTR, JAZF1. We characterized the effects of JAZF1 overexpression on prostate cancer cells, basally and in the context of treatment, by assessing mRNA levels, proliferation, colony formation capability, and key metabolic processes. Lastly, we confirmed the relevance of our findings in large publicly available cohorts of prostate cancer patient samples. We demonstrated differential sensitivity to polyamine and methionine therapies and identified JAZF1 as a MMTR of this response. IMPLICATIONS: We have shown JAZF1 can alter sensitivity of cells and its expression can segregate patient populations into those that do, or do not highly express polyamine genes, leading to better prediction of response to a polyamine targeting therapy.


Assuntos
Poliaminas , Neoplasias da Próstata , Masculino , Humanos , Poliaminas/metabolismo , Poliaminas/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Metionina/metabolismo , Redes e Vias Metabólicas , Proteínas de Ligação a DNA/metabolismo , Proteínas Correpressoras/metabolismo
7.
Cell Rep ; 42(2): 112120, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36774550

RESUMO

Intestinal colonization of the oral bacterium Haemophilus parainfluenzae has been associated with Crohn's disease (CD) severity and progression. This study examines the role of periodontal disease (PD) as a modifier for colonization of H. parainfluenzae in patients with CD and explores the mechanisms behind H. parainfluenzae-mediated intestinal inflammation. Fifty subjects with and without CD were evaluated for the presence of PD, and their oral and fecal microbiomes were characterized. PD is associated with increased levels of H. parainfluenzae strains in subjects with CD. Oral inoculation of H. parainfluenzae elicits strain-dependent intestinal inflammation in murine models of inflammatory bowel disease, which is associated with increased intestinal interferon-γ (IFN-γ)+ CD4+ T cells and disruption of the host hypusination pathway. In summary, this study establishes a strain-specific pathogenic role of H. parainfluenzae in intestinal inflammation and highlights the potential effect of PD on intestinal colonization by pathogenic H. parainfluenzae strains in patients with CD.


Assuntos
Doença de Crohn , Doenças Periodontais , Humanos , Animais , Camundongos , Haemophilus parainfluenzae , Doença de Crohn/complicações , Doença de Crohn/metabolismo , Inflamação
8.
iScience ; 26(11): 108303, 2023 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-38026169

RESUMO

Neuroblastoma is the most common extracranial solid tumor in children. MYCN amplification is detected in almost half of high-risk cases and is associated with poorly differentiated tumors, poor patient prognosis and poor response to therapy, including retinoids. We identify the aryl hydrocarbon receptor (AhR) as a transcription factor promoting the growth and suppressing the differentiation of MYCN-amplified neuroblastoma. A neuroblastoma specific AhR transcriptional signature reveals an inverse correlation of AhR activity with patients' outcome, suggesting AhR activity is critical for disease progression. AhR modulates chromatin structures, reducing accessibility to regions responsive to retinoic acid. Genetic and pharmacological inhibition of AhR results in induction of differentiation. Importantly, AhR antagonism with clofazimine synergizes with retinoic acid in inducing differentiation both in vitro and in vivo. Thus, we propose AhR as a target for MYCN-amplified neuroblastoma and that its antagonism, combined with current standard-of-care, may result in a more durable response in patients.

9.
Cancers (Basel) ; 14(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36230673

RESUMO

The survival of patients with solid tumors, such as prostate cancer (PCa), has been limited and fleeting with anti-angiogenic therapies. It was previously thought that the mechanism by which the vasculature regulates tumor growth was driven by a passive movement of oxygen and nutrients to the tumor tissue. However, previous evidence suggests that endothelial cells have an alternative role in changing the behavior of tumor cells and contributing to cancer progression. Determining the impact of molecular signals/growth factors released by endothelial cells (ECs) on established PCa cell lines in vitro and in vivo could help to explain the mechanism by which ECs regulate tumor growth. Using cell-conditioned media collected from HUVEC (HUVEC-CM), our data show the stimulated proliferation of all the PCa cell lines tested. However, in more aggressive PCa cell lines, HUVEC-CM selectively promoted migration and invasion in vitro and in vivo. Using a PCa-cell-line-derived xenograft model co-injected with HUVEC or preincubated with HUVEC-CM, our results are consistent with the in vitro data, showing enhanced tumor growth, increased tumor microvasculature and promoted metastasis. Gene set enrichment analyses from RNA-Seq gene expression profiles showed that HUVEC-CM induced a differential effect on gene expression when comparing low versus highly aggressive PCa cell lines, demonstrating epigenetic and migratory pathway enrichments in highly aggressive PCa cells. In summary, paracrine stimulation by HUVEC increased PCa cell proliferation and tumor growth and selectively promoted migration and metastatic potential in more aggressive PCa cell lines.

10.
BMC Biol ; 8: 6, 2010 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-20092614

RESUMO

BACKGROUND: Folate (vitamin B9) is essential for cellular proliferation as it is involved in the biosynthesis of deoxythymidine monophosphate (dTMP) and s-adenosylmethionine (AdoMet). The link between folate depletion and the genesis and progression of cancers of epithelial origin is of high clinical relevance, but still unclear. We recently demonstrated that sensitivity to low folate availability is affected by the rate of polyamine biosynthesis, which is prominent in prostate cells. We, therefore, hypothesized that prostate cells might be highly susceptible to genetic, epigenetic and phenotypic changes consequent to folate restriction. RESULTS: We studied the consequences of long-term, mild folate depletion in a model comprised of three syngenic cell lines derived from the transgenic adenoma of the mouse prostate (TRAMP) model, recapitulating different stages of prostate cancer; benign, transformed and metastatic. High-performance liquid chromatography analysis demonstrated that mild folate depletion (100 nM) sufficed to induce imbalance in both the nucleotide and AdoMet pools in all prostate cell lines. Random oligonucleotide-primed synthesis (ROPS) revealed a significant increase in uracil misincorporation and DNA single strand breaks, while spectral karyotype analysis (SKY) identified five novel chromosomal rearrangements in cells grown with mild folate depletion. Using global approaches, we identified an increase in CpG island and histone methylation upon folate depletion despite unchanged levels of total 5-methylcytosine, indicating a broad effect of folate depletion on epigenetic regulation. These genomic changes coincided with phenotype changes in the prostate cells including increased anchorage-independent growth and reduced sensitivity to folate depletion. CONCLUSIONS: This study demonstrates that prostate cells are highly susceptible to genetic and epigenetic changes consequent to mild folate depletion as compared to cells grown with supraphysiological amounts of folate (2 microM) routinely used in tissue culture. In addition, we elucidate for the first time the contribution of these aspects to consequent phenotype changes in epithelial cells. These results provide a strong rationale for studying the effects of folate manipulation on the prostate in vivo, where cells might be more sensitive to changes in folate status resulting from folate supplementation or antifolate therapeutic approaches.


Assuntos
Epigênese Genética/genética , Deficiência de Ácido Fólico/genética , Deficiência de Ácido Fólico/fisiopatologia , Neoplasias da Próstata/genética , 5-Metilcitosina/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Ilhas de CpG/genética , Dano ao DNA/genética , Metilação de DNA/genética , Masculino , Camundongos , Neoplasias da Próstata/metabolismo
11.
Sci Rep ; 11(1): 11405, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075163

RESUMO

Understanding the epigenetic control of normal differentiation programs might yield principal information about critical regulatory states that are disturbed in cancer. We utilized the established non-malignant HPr1-AR prostate epithelial cell model that upon androgen exposure commits to a luminal cell differentiation trajectory from that of a basal-like state. We profile the dynamic transcriptome associated with this transition at multiple time points (0 h, 1 h, 24 h, 96 h), and confirm that expression patterns are strongly indicative of a progressive basal to luminal cell differentiation program based on human expression signatures. Furthermore, we establish dynamic patterns of DNA methylation associated with this program by use of whole genome bisulfite sequencing (WGBS). Expression patterns associated with androgen induced luminal cell differentiation were found to have significantly elevated DNA methylation dynamics. Shifts in methylation profiles were strongly associated with Polycomb repressed regions and to promoters associated with bivalency, and strongly enriched for binding motifs of AR and MYC. Importantly, we found that dynamic DNA methylation patterns observed in the normal luminal cell differentiation program were significant targets of aberrant methylation in prostate cancer. These findings suggest that the normal dynamics of DNA methylation in luminal differentiation contribute to the aberrant methylation patterns in prostate cancer.


Assuntos
Próstata , Neoplasias da Próstata/metabolismo , Diferenciação Celular , Linhagem Celular , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Próstata/citologia , Próstata/metabolismo , Próstata/patologia
12.
Cell Rep ; 37(11): 110109, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34910907

RESUMO

This study addresses the roles of nuclear receptor corepressor 2 (NCOR2) in prostate cancer (PC) progression in response to androgen deprivation therapy (ADT). Reduced NCOR2 expression significantly associates with shorter disease-free survival in patients with PC receiving adjuvant ADT. Utilizing the CWR22 xenograft model, we demonstrate that stably reduced NCOR2 expression accelerates disease recurrence following ADT, associates with gene expression patterns that include neuroendocrine features, and induces DNA hypermethylation. Stably reduced NCOR2 expression in isogenic LNCaP (androgen-sensitive) and LNCaP-C4-2 (androgen-independent) cells revealed that NCOR2 reduction phenocopies the impact of androgen treatment and induces global DNA hypermethylation patterns. NCOR2 genomic binding is greatest in LNCaP-C4-2 cells and most clearly associates with forkhead box (FOX) transcription factor FOXA1 binding. NCOR2 binding significantly associates with transcriptional regulation most when in active enhancer regions. These studies reveal robust roles for NCOR2 in regulating the PC transcriptome and epigenome and underscore recent mutational studies linking NCOR2 loss of function to PC disease progression.


Assuntos
Antagonistas de Androgênios/farmacologia , Androgênios/deficiência , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Recidiva Local de Neoplasia/patologia , Correpressor 2 de Receptor Nuclear/antagonistas & inibidores , Neoplasias da Próstata/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Masculino , Camundongos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Correpressor 2 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/metabolismo , Prognóstico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Commun Biol ; 3(1): 158, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242058

RESUMO

The retinoblastoma tumor suppressor gene (RB1) plays a critical role in coordinating multiple pathways that impact cancer initiation, disease progression, and therapeutic responses. Here we probed molecular features associated with the RB-pathway across 31 tumor-types. While the RB-pathway has been purported to exhibit multiple mutually exclusive genetic events, only RB1 alteration is mutually exclusive with deregulation of CDK4/6 activity. An ER+ breast cancer model with targeted RB1 deletion was used to identify signatures of CDK4/6 activity and RB-dependency (CDK4/6-RB integrated signature). This signature was prognostic in tumor-types with gene expression features indicative of slower growth. Single copy loss on chromosome 13q encompassing the RB1 locus is prevalent in many cancers, yielding reduced expression of multiple genes in cis, and is inversely related to the CDK4/6-RB integrated signature supporting a cause-effect relationship. Genes that are positively and inversely correlated with the CDK4/6-RB integrated signature define new tumor-specific pathways associated with RB-pathway activity.


Assuntos
Biomarcadores Tumorais/genética , Cromossomos Humanos Par 13 , Neoplasias da Retina/genética , Retinoblastoma/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Bases de Dados Genéticas , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologia , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Transdução de Sinais , Transcriptoma , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
14.
Cancer Epidemiol Biomarkers Prev ; 29(2): 443-451, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31848205

RESUMO

BACKGROUND: Nicotine-containing electronic cigarette (e-cig) use has become widespread. However, understanding the biological impact of e-cigs compared with smoking on the lung is needed. There are major gaps in knowledge for chronic effects and for an etiology to recent acute lung toxicity leading to death among vapers. METHODS: We conducted bronchoscopies in a cross-sectional study of 73 subjects (42 never-smokers, 15 e-cig users, and 16 smokers). Using bronchoalveolar lavage and brushings, we examined lung inflammation by cell counts, cytokines, genome-wide gene expression, and DNA methylation. RESULTS: There were statistically significant differences among never-smokers, e-cig users, and smokers for inflammatory cell counts and cytokines (FDR q < 0.1). The e-cig users had values intermediate between smokers and never-smokers, with levels for most of the biomarkers more similar to never-smokers. For differential gene expression and DNA methylation, e-cig users also more like never-smokers; many of these genes corresponded to smoking-related pathways, including those for xenobiotic metabolism, aryl hydrocarbon receptor signaling, and oxidative stress. Differentially methylated genes were correlated with changes in gene expression, providing evidence for biological effects of the methylation associations. CONCLUSIONS: These data indicate that e-cigs are associated with less toxicity than cigarettes for smoking-related pathways. What is unknown may be unique effects for e-cigs not measured herein, and a comparison of smokers completely switching to e-cigs compared with former smokers. Clinical trials for smokers switching to e-cigs who undergo serial bronchoscopy and larger cross-sectional studies of former smokers with and without e-cig use, and for e-cigs who relapse back to smoking, are needed. IMPACT: These data can be used for product regulation and for informing tobacco users considering or using e-cigs. What is unknown may be unique effects for e-cigs not measured herein, and clinical trials with serial bronchoscopy underway can demonstrate a direct relationship for changes in lung biomarkers.


Assuntos
Broncoscopia/estatística & dados numéricos , Fumar Cigarros/efeitos adversos , Sistemas Eletrônicos de Liberação de Nicotina , Pulmão/patologia , não Fumantes/estatística & dados numéricos , Fumantes/estatística & dados numéricos , Adulto , Biomarcadores/análise , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Contagem de Células , Fumar Cigarros/patologia , Citocinas/análise , Citocinas/metabolismo , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Pulmão/diagnóstico por imagem , Pulmão/imunologia , Masculino , Adulto Jovem
15.
Nat Commun ; 11(1): 52, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911608

RESUMO

Prostatic luminal epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen, sensitizing them to perturbations of connected metabolic pathways. Enhanced flux is driven by spermidine/spermine N1-acetyltransferase (SSAT) activity, which acetylates polyamines leading to their secretion and drives biosynthetic demand. The methionine salvage pathway recycles one-carbon units lost to polyamine biosynthesis to the methionine cycle to overcome stress. Prostate cancer (CaP) relies on methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme, to relieve strain. Here, we show that inhibition of MTAP alongside SSAT upregulation is synergistic in androgen sensitive and castration recurrent CaP models in vitro and in vivo. The combination treatment increases apoptosis in radical prostatectomy ex vivo explant samples. This unique high metabolic flux through polyamine biosynthesis and connected one carbon metabolism in CaP creates a metabolic dependency. Enhancing this flux while simultaneously targeting this dependency in prostate cancer results in an effective therapeutic approach potentially translatable to the clinic.


Assuntos
Metionina/metabolismo , Poliaminas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Acetiltransferases/genética , Acetiltransferases/metabolismo , Adenina/administração & dosagem , Adenina/análogos & derivados , Animais , Apoptose , Linhagem Celular Tumoral , Quimioterapia Combinada , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Pirrolidinas/administração & dosagem , Terapia de Salvação , Espermina/administração & dosagem , Espermina/análogos & derivados , Espermina/metabolismo
16.
Mol Cancer Res ; 6(8): 1365-74, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18667590

RESUMO

We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, including prostatic intraepithelial neoplasia, well-differentiated tumors, early poorly differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and Dnmt3b protein expression were increased in all stages; however, after normalization to cyclin A to account for cell cycle regulation, Dnmt proteins remained overexpressed in prostatic intraepithelial neoplasia and well-differentiated tumors, but not in poorly differentiated tumors. Restriction landmark genomic scanning analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (early and late) tumors. Several genes identified by restriction landmark genomic scanning showed hypermethylation of downstream regions correlating with mRNA overexpression, including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both early and late stages of prostate cancer. DNA hypermethylation and DNA hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not correlate with either variable, with the exception of a borderline significant association between Dnmt1 expression and DNA hypermethylation. In summary, our data reveal the relative timing of and relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression in an in vivo model system.


Assuntos
Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Modelos Animais de Doenças , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Adenocarcinoma/genética , Animais , Canais de Cálcio Tipo N , Canais de Cálcio Tipo P/genética , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/genética , Canais de Cálcio Tipo Q/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Genoma/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estadiamento de Neoplasias , Neoplasias da Próstata/genética
17.
Neuro Oncol ; 11(4): 414-22, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19001526

RESUMO

Both genetic and epigenetic mechanisms contribute to meningioma development by altering gene expression and protein function. To determine the relative contribution of each mechanism to meningioma development, we used an integrative approach measuring copy number and DNA methylation changes genomewide. We found that genetic alterations affected 1.9%, 7.4%, and 13.3% of the 691 loci studied, whereas epigenetic mechanisms affected 5.4%, 9.9%, and 10.3% of these loci in grade I, II, and III meningiomas, respectively. Genetic and epigenetic mechanisms rarely involved the same locus in any given tumor. The predilection for epigenetic rather than genetic silencing was exemplified at the 5' CpG island of WNK2, a serine-threonine kinase gene on chromosome 9q22.31. WNK2 is known to negatively regulate epidermal growth factor receptor signaling via inhibition of MEK1 (mitogen-activated protein kinase kinase 1), and point mutations have been reported in WNK1, WNK2, WNK3, and WNK4. In meningiomas, WNK2 was aberrantly methylated in 83% and 71% of grade II and III meningiomas, respectively, but rarely in a total of 209 tumors from 13 other tumor types. Aberrant methylation of the CpG island was associated with decreased expression in primary tumors. WNK2 could be reactivated with a methylation inhibitor in IOMM-Lee, a meningioma cell line with a densely methylated WNK2 CpG island and lack of WNK2 expression. Expression of exogenous WNK2 inhibited colony formation, implicating it as a potential cell growth suppressor. These findings indicate that epigenetic mechanisms are common across meningiomas of all grades and that for specific genes such as WNK2, epigenetic alteration may be the dominant, grade-specific mechanism of gene inactivation.


Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Neoplasias Meníngeas/genética , Meningioma/genética , Proteínas Serina-Treonina Quinases/genética , Western Blotting , Ensaio de Unidades Formadoras de Colônias , Hibridização Genômica Comparativa , Ilhas de CpG , Citometria de Fluxo , Imunofluorescência , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/metabolismo , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Neoplasias Meníngeas/classificação , Neoplasias Meníngeas/patologia , Meningioma/classificação , Meningioma/patologia , Antígenos de Histocompatibilidade Menor , Invasividade Neoplásica , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas , Proteína Quinase 1 Deficiente de Lisina WNK
18.
Methods Mol Biol ; 507: 131-48, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18987812

RESUMO

Restriction landmark genomic scanning (RLGS) is a method that provides a quantitative genetic and epigenetic (cytosine methylation) assessment of thousands of CpG islands in a single gel without prior knowledge of gene sequence. The method is based on two-dimensional separation of radiolabeled genomic DNA into nearly 2,000 discrete fragments that have a high probability of containing gene sequences. Genomic DNA is digested with an infrequently cutting restriction enzyme, such as NotI or AscI, radiolabeled at the cleaved ends, digested with a second restriction enzyme, and then electrophoresed through a narrow, 60-cm-long agarose tube-shaped gel. The DNA in the tube gel is then digested by a third, more frequently cutting restriction enzyme and electrophoresed, in a direction perpendicular to the first separation, through a 5% nondenaturing polyacrylamide gel, and the gel is autoradiographed. Radiolabeled NotI or AscI sites are frequently used as "landmarks" because NotI or AscI cannot cleave methylated sites and since an estimated 89% and 83% of the recognition sites, respectively, are found within CpG islands. Using a methylation-sensitive enzyme, the technique has been termed RLGS-M. The resulting RLGS profile displays both the copy number and methylation status of the CpG islands. Integrated with high-resolution gene copy-number analyses, RLGS enables one to define genetic or epigenetic alteration in cells. These profiles are highly reproducible and are therefore amenable to inter- and intraindividual DNA sample comparisons. RLGS was the first of many technologies to allow large-scale DNA methylation analysis of CpG islands.


Assuntos
Ilhas de CpG , Metilação de DNA , Eletroforese em Gel Bidimensional/métodos , Animais , Neoplasias Encefálicas/química , Neoplasias Encefálicas/genética , DNA/análise , DNA/química , DNA/genética , Impressões Digitais de DNA/métodos , DNA de Neoplasias/química , DNA de Neoplasias/genética , Desoxirribonucleases de Sítio Específico do Tipo II , Genoma , Humanos , Reação em Cadeia da Polimerase
19.
Oncogene ; 38(3): 421-444, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30120411

RESUMO

Expression levels of retinoic acid receptor gamma (NR1B3/RARG, encodes RARγ) are commonly reduced in prostate cancer (PCa). Therefore, we sought to establish the cellular and gene regulatory consequences of reduced RARγ expression, and determine RARγ regulatory mechanisms. RARG shRNA approaches in non-malignant (RWPE-1 and HPr1-AR) and malignant (LNCaP) prostate models revealed that reducing RARγ levels, rather than adding exogenous retinoid ligand, had the greatest impact on prostate cell viability and gene expression. ChIP-Seq defined the RARγ cistrome, which was significantly enriched at active enhancers associated with AR binding sites. Reflecting a significant genomic role for RARγ to regulate androgen signaling, RARγ knockdown in HPr1-AR cells significantly regulated the magnitude of the AR transcriptome. RARγ downregulation was explained by increased miR-96 in PCa cell and mouse models, and TCGA PCa cohorts. Biochemical approaches confirmed that miR-96 directly regulated RARγ expression and function. Capture of the miR-96 targetome by biotin-miR-96 identified that RARγ and a number of RARγ interacting co-factors including TACC1 were all targeted by miR-96, and expression of these genes were prominently altered, positively and negatively, in the TCGA-PRAD cohort. Differential gene expression analyses between tumors in the TCGA-PRAD cohort with lower quartile expression levels of RARG and TACC1 and upper quartile miR-96, compared to the reverse, identified a gene network including several RARγ target genes (e.g., SOX15) that significantly associated with worse disease-free survival (hazard ratio 2.23, 95% CI 1.58 to 2.88, p = 0.015). In summary, miR-96 targets a RARγ network to govern AR signaling, PCa progression and disease outcome.


Assuntos
Adenocarcinoma/patologia , Androgênios , MicroRNAs/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias Hormônio-Dependentes/patologia , Neoplasias da Próstata/patologia , RNA Neoplásico/fisiologia , Receptores do Ácido Retinoico/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Animais , Linhagem Celular Tumoral , Progressão da Doença , Elementos Facilitadores Genéticos , Proteínas Fetais/metabolismo , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/mortalidade , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais , Receptor gama de Ácido Retinoico
20.
Cancer Epidemiol Biomarkers Prev ; 17(3): 571-7, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18349274

RESUMO

The aim of this study was to determine the role of prostate-specific membrane antigen (PSMA) as a prognostic marker in endometrial adenocarcinoma (EAC) and to explore whether its down-regulation could be due to epigenetic mechanism. First, we examined the expression and the prognostic value of PSMA by semiquantitative reverse transcription-PCR and immunohistochemistry in EAC tissue samples. Second, to explore the role of CpG methylation in down-regulation PSMA in EAC, we evaluated PSMA CpG island methylation using methylation-specific PCR in cells lines and in a subset of patients' samples. Furthermore, association of the status of tumor methylation to the clinical and histologic variables was also evaluated. Higher PSMA mRNA levels were associated with stage I (P = 0.046) and PSMA protein intensity by immunohistochemistry (P = 0.032). In multivariate analysis, loss of PSMA expression was associated with a worse disease-free survival (P = 0.02). PSMA was methylated in prostate cell lines (DU145 and PC3) and endometrial cell lines. In addition, PSMA was methylated in 5 of 18 samples (all 5 had low PSMA mRNA value). There was a significant association between PSMA methylation and loss of protein expression by immunohistochemistry and PSMA-RNA level with P value of 0.036 and 0.011, respectively. In addition, there was an association between PSMA methylation and tumor size (P = 0.025). In summary, (a) PSMA is underexpressed in advanced stage EAC, (b) loss of PSMA expression can be considered as a prognostic marker in patients with EAC, and (c) loss of PSMA expression in a subset of EAC cases could be due to epigenetic silencing.


Assuntos
Antígenos de Superfície/metabolismo , Neoplasias do Endométrio/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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