RESUMEN
Disaggregation of amyloid fibrils is a fundamental biological process required for amyloid propagation. However, due to the lack of experimental systems, the molecular mechanism of how amyloid is disaggregated by cellular factors remains poorly understood. Here, we established a robust in vitro reconstituted system of yeast prion propagation and found that heat-shock protein 104 (Hsp104), Ssa1 and Sis1 chaperones are essential for efficient disaggregation of Sup35 amyloid. Real-time imaging of single-molecule fluorescence coupled with the reconstitution system revealed that amyloid disaggregation is achieved by ordered, timely binding of the chaperones to amyloid. Remarkably, we uncovered two distinct prion strain conformation-dependent modes of disaggregation, fragmentation and dissolution. We characterized distinct chaperone dynamics in each mode and found that transient, repeated binding of Hsp104 to the same site of amyloid results in fragmentation. These findings provide a physical foundation for otherwise puzzling in vivo observations and for therapeutic development for amyloid-associated neurodegenerative diseases.
Asunto(s)
Priones , Proteínas de Saccharomyces cerevisiae , Amiloide/química , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Prion diseases are transmissible fatal neurodegenerative disorders affecting humans and other mammals. The disease transmission can occur between different species but is limited by the sequence homology between host and inoculum. The crucial molecular event in the progression of this disease is prion formation, starting from the conformational conversion of the normal, membrane-anchored prion protein (PrPC) into the misfolded, ß-sheet-rich and aggregation-prone isoform (PrPSc), which then self-associates into the infectious amyloid form called prion. Amyloid is the aggregate formed from one-dimensional protein association. As amyloid formation is a key hallmark in prion pathogenesis, studying which segments in prion protein are involved in the amyloid formation can provide molecular details in the cross-species transmission barrier of prion diseases. However, due to the difficulties of studying protein aggregates, very limited knowledge about prion structure or prion formation was disclosed by now. In this study, cross-seeding assay was used to identify the segments involved in the amyloid fibril formation of full-length hamster prion protein, SHaPrP(23-231). Our results showed that the residues in the segments 108-127, 172-194 (helix 2 in PrPC) and 200-227 (helix 3 in PrPC) are in the amyloid core of hamster prion fibrils. The segment 127-143, but not 107-126 (which corresponds to hamster sequence 108-127), was previously reported to be involved in the amyloid core of full-length mouse prion fibrils. Our results indicate that hamster prion protein and mouse prion protein use different segments to form the amyloid core in amyloidogenesis. The sequence-dependent core formation can be used to explain the seeding barrier between mouse and hamster.
Asunto(s)
Amiloide/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Priónicas/metabolismo , Animales , Cricetinae , Ratones , Multimerización de ProteínaRESUMEN
Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10(-30)), OSECs (P = 2.4 × 10(-23)) and HMECs (P = 6.7 × 10(-15)) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer.
Asunto(s)
Predisposición Genética a la Enfermedad , Neoplasias Ováricas/genética , Cromatina/genética , Cromatina/metabolismo , Femenino , Estudio de Asociación del Genoma Completo , Histonas/genética , Histonas/metabolismo , Humanos , Especificidad de Órganos , Neoplasias Ováricas/metabolismo , Polimorfismo de Nucleótido Simple , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Amyloidogenesis, self-propagation of protein or peptide monomers to amyloid fibrils, has been linked to incurable pathogenesis of neurodegenerative diseases such as Alzheimer's disease and prion diseases. Investigations of amyloid structures and how monomers are transformed through seeding are therefore crucial for developing therapeutics toward these diseases. Here we describe a cross-seeding method to explore the amyloid core in prion fibrils that uses preformed amyloid fibrils as a seed to induce the transformation of other protein or peptide monomers to amyloid fibrils.
Asunto(s)
Amiloidosis , Enfermedades por Prión , Priones , Humanos , Amiloide/química , Priones/química , Proteínas AmiloidogénicasRESUMEN
Prostate cancers (PCas) become resistant to hormone withdrawal through increased androgen receptor (AR) signaling. Here we show increased AR-mediated transcription efficiency in PCa cells that have acquired the ability to grow in low concentrations of androgen. Compared to androgen-dependent PCa cells, these cells showed increased activity of transiently transfected reporters and increased mRNA synthesis relative to levels of AR occupancy of the prostate-specific antigen (PSA) gene. The locus also displayed up to 10-fold-higher levels of histone H3-K9/K14 acetylation and H3-K4 methylation across the entire body of the gene. Although similar increased mRNA expression and locus-wide histone acetylation were also observed at another kallikrein locus (KLK2), at a third AR target locus (TMPRSS2) increased gene expression and locus-wide histone acetylation were not seen in the absence of ligand. Androgen-independent PCa cells have thus evolved three distinctive alterations in AR-mediated transcription. First, increased RNA polymerase initiation and processivity contributed to increased gene expression. Second, AR signaling was more sensitive to ligand. Third, locus-wide chromatin remodeling conducive to the increased gene expression in the absence of ligand was apparent and depended on sustained AR activity. Therefore, increased AR ligand sensitivity as well as locus-specific chromatin alterations contribute to basal gene expression of a subpopulation of specific AR target genes in androgen-independent PCa cells. These features contribute to the androgen-independent phenotype of these cells.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromatina/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismo , Transcripción Genética , Acetilación/efectos de los fármacos , Andrógenos/metabolismo , Animales , Cromatina/efectos de los fármacos , Dihidrotestosterona/farmacología , Genes Reporteros/genética , Histonas/metabolismo , Humanos , Ligandos , Luciferasas/genética , Masculino , Metilación/efectos de los fármacos , Ratones , Ratones Desnudos , Antígeno Prostático Específico/genética , Elementos de Respuesta/efectos de los fármacos , Elementos de Respuesta/genética , Células Tumorales CultivadasRESUMEN
Androgen receptor (AR) recruitment of transcriptional corepressors NCoR and SMRT can be enhanced by antagonists such as mifepristone. This study shows that enhanced NCoR binding to the mifepristone-liganded AR is mediated by the NCoR COOH-terminal N1 CoRNR box and that this selectivity is due to charged residues unique to the COOH-terminal CoRNR boxes of NCoR and SMRT. Significantly, these residues are on a helical face adjacent to oppositely charged residues in helix 4 of the AR ligand-binding domain. Mutagenesis of these AR residues in helix 4, as well as mutation of lysine 720 in helix 3 (predicted to interact with the CoRNR box), markedly impaired AR recruitment of NCoR, indicating that N1 CoRNR box binding is being stabilized by these ionic interactions in the AR ligand-binding domain coactivator/corepressor binding site. Finally, results using a helix 12-deleted AR indicate that mifepristone induces allosteric changes in addition to helix 12 displacement that are critical for NCoR binding. These findings show that AR antagonists can enhance corepressor recruitment by stabilizing a distinct antagonist conformation of the AR coactivator/corepressor binding site and support the development of additional antagonists that may be able to further enhance AR recruitment of corepressors.
Asunto(s)
Antagonistas de Receptores Androgénicos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Mifepristona/farmacología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Secuencia Conservada , Humanos , Isoleucina/metabolismo , Ligandos , Datos de Secuencia Molecular , Co-Represor 1 de Receptor Nuclear , Co-Represor 2 de Receptor Nuclear , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Androgénicos/químicaRESUMEN
Genome-wide association studies have identified 40 ovarian cancer risk loci. However, the mechanisms underlying these associations remain elusive. In this study, we conducted a two-pronged approach to identify candidate causal SNPs and assess underlying biological mechanisms at chromosome 9p22.2, the first and most statistically significant associated locus for ovarian cancer susceptibility. Three transcriptional regulatory elements with allele-specific effects and a scaffold/matrix attachment region were characterized and, through physical DNA interactions, BNC2 was established as the most likely target gene. We determined the consensus binding sequence for BNC2 in vitro, verified its enrichment in BNC2 ChIP-seq regions, and validated a set of its downstream target genes. Fine-mapping by dense regional genotyping in over 15,000 ovarian cancer cases and 30,000 controls identified SNPs in the scaffold/matrix attachment region as among the most likely causal variants. This study reveals a comprehensive regulatory landscape at 9p22.2 and proposes a likely mechanism of susceptibility to ovarian cancer. SIGNIFICANCE: Mapping the 9p22.2 ovarian cancer risk locus identifies BNC2 as an ovarian cancer risk gene.See related commentary by Choi and Brown, p. 439.
Asunto(s)
Carcinoma Epitelial de Ovario/genética , Cromosomas Humanos Par 9 , Neoplasias Ováricas/genética , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Mapeo Cromosómico , Cistadenocarcinoma Seroso/genética , ADN de Neoplasias/genética , Proteínas de Unión al ADN/genética , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido SimpleRESUMEN
Alzheimer's disease (AD) is the most common neurodegenerative disease. Imbalance between the production and clearance of amyloid ß (Aß) peptides is considered to be the primary mechanism of AD pathogenesis. This amyloid hypothesis is supported by the recent success of the human anti-amyloid antibody aducanumab, in clearing plaque and slowing clinical impairment in prodromal or mild patients in a phase Ib trial. Here, a peptide combining polyarginines (polyR) (for charge repulsion) and a segment derived from the core region of Aß amyloid (for sequence recognition) was designed. The efficacy of the designed peptide, R8-Aß(25-35), on amyloid reduction and the improvement of cognitive functions were evaluated using APP/PS1 double transgenic mice. Daily intranasal administration of PEI-conjugated R8-Aß(25-35) peptide significantly reduced Aß amyloid accumulation and ameliorated the memory deficits of the transgenic mice. Intranasal administration is a feasible route for peptide delivery. The modular design combining polyR and aggregate-forming segments produced a desirable therapeutic effect and could be easily adopted to design therapeutic peptides for other proteinaceous aggregate-associated diseases.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/uso terapéutico , Encéfalo/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Péptidos/uso terapéutico , Administración Intranasal , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/patología , Amiloide/antagonistas & inhibidores , Amiloide/ultraestructura , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/ultraestructura , Animales , Encéfalo/patología , Línea Celular , Cognición/efectos de los fármacos , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/patología , Modelos Animales de Enfermedad , Femenino , Trastornos de la Memoria/complicaciones , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/patología , Ratones Endogámicos C57BL , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/ultraestructura , Péptidos/administración & dosificación , Péptidos/químicaRESUMEN
Ligand-activated androgen receptors (ARs) occupy target genes and recruit histone modifiers that influence transcriptional competency. In LNCaP prostate cancer cells, the natural ligand 5alpha-dihydrotestosterone (DHT) activates transiently transfected AR-responsive promoter constructs; concurrent treatment with the protein kinase A activator forskolin enhanced AR stimulation induced by DHT. Additional treatment with the cytokine IL-6, purportedly an AR activator, markedly inhibited receptor activity. To assess AR activity on natural chromatin-integrated promoters/enhancers, we determined AR occupancy of the endogenous prostate specific antigen (PSA) promoter/enhancer as well as PSA expression in LNCaP cells treated with DHT; AR occupancy of the PSA enhancer was rapid (within 1 h of stimulation), robust (10-fold over background), and sustained (8-16 h). In contrast, AR occupancy of the PSA promoter was only increased by 2-fold. Histone H3 acetylation at both the enhancer and promoter was evident 1-2 h after DHT treatment. Detectable pre- and mature PSA mRNA levels appeared after 1 and 6 h treatment, respectively. Substantial qualitative and quantitative differences in PSA expression and AR occupancy of the PSA enhancer were observed when DHT-induced and ligand-independent activations of the AR were compared; forskolin stimulated PSA mRNA and protein expression, whereas IL-6 inhibited both DHT- and forskolin-stimulated expression. IL-6 did not diminish DHT-dependent AR occupancy of the PSA enhancer but inhibited CBP/p300 recruitment, histone H3 acetylation, and cell proliferation. These findings provide a contextual framework for interpreting the contribution of non-steroidal activation of the AR to signaling in vivo, and have implications for prostate cancer cell growth.
Asunto(s)
Dihidrotestosterona/farmacología , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata , Receptores Androgénicos/metabolismo , Acetilación , Adenilil Ciclasas/metabolismo , División Celular/fisiología , Colforsina/farmacología , Dihidrotestosterona/metabolismo , Genes Reporteros , Histonas/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-6/farmacología , Ligandos , Luciferasas/genética , Masculino , Regiones Promotoras Genéticas , Antígeno Prostático Específico/genética , Receptores Androgénicos/genética , Células Tumorales CultivadasRESUMEN
Genome-wide association studies (GWAS) have identified 12 epithelial ovarian cancer (EOC) susceptibility alleles. The pattern of association at these loci is consistent in BRCA1 and BRCA2 mutation carriers who are at high risk of EOC. After imputation to 1000 Genomes Project data, we assessed associations of 11 million genetic variants with EOC risk from 15,437 cases unselected for family history and 30,845 controls and from 15,252 BRCA1 mutation carriers and 8,211 BRCA2 mutation carriers (3,096 with ovarian cancer), and we combined the results in a meta-analysis. This new study design yielded increased statistical power, leading to the discovery of six new EOC susceptibility loci. Variants at 1p36 (nearest gene, WNT4), 4q26 (SYNPO2), 9q34.2 (ABO) and 17q11.2 (ATAD5) were associated with EOC risk, and at 1p34.3 (RSPO1) and 6p22.1 (GPX6) variants were specifically associated with the serous EOC subtype, all with P < 5 × 10(-8). Incorporating these variants into risk assessment tools will improve clinical risk predictions for BRCA1 and BRCA2 mutation carriers.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo/métodos , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Alelos , Carcinoma Epitelial de Ovario , Femenino , Genes Reporteros , Genotipo , Heterocigoto , Humanos , Mutación , Sitios de Carácter Cuantitativo , Riesgo , Adulto JovenRESUMEN
Homeobox genes comprise a nearly ubiquitous and highly conserved superfamily of developmental regulatory genes that encode transcription factors involved in the determination of axis and tissue identity. While homeobox gene expression has been well characterized in a variety of embryonic tissues, their expression has not been extensively studied in lymphoid progenitor cells or in sites of lymphogenesis. To examine homeobox gene expression in the developing thymus, we screened an embryonic day 13.5 thymus cDNA library by polymerase chain reaction (PCR) using degenerate oligonucleotides within the highly conserved homeodomain region of eight homeobox gene families. The resulting PCR products were then cloned and sequenced. Transcripts for multiple Dlx family members and Lhx2 were repeatedly detected in this screen. Screening of embryonic day 16.5 and adult murine thymus and Thy1+ thymocytes was performed for selected members of these homeobox gene families. Transcripts encoding Lhx2, Lhx3, and Lhx9, as well as Dlx1 and Dlx2 were detected in both thymus and purified thymocytes. Dlx1 is a member of the distal-less homeobox gene family that has been shown to regulate embryonic craniofacial development. Significantly, Dlx1 is expressed in the third branchial arch, which contributes to the thymus. Although Dlx1 knockout mice did not display any obvious developmental defects in thymus or thymocyte development, the expression of these homeobox genes in neural crest derivatives suggests a possible role in cell migration and development that may overlap with other homeobox genes.
Asunto(s)
Genes Homeobox/genética , Timo/embriología , Animales , Desarrollo Embrionario y Fetal , Feto/metabolismo , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/citología , Timo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: Galeterone inhibits the enzyme CYP17A1 and is currently in phase II clinical trials for castration-resistant prostate cancer (CRPC). Galeterone is also a direct androgen receptor (AR) antagonist and may enhance AR degradation. This study was undertaken to determine the molecular basis for AR effects and their therapeutic potential. EXPERIMENTAL DESIGN: Effects of galeterone on AR expression and activities were examined in prostate cancer cell lines. RESULTS: Similar to the AR antagonist enzalutamide, but in contrast to bicalutamide, galeterone did not induce binding of a constitutively active VP16-AR fusion protein to reporter genes and did not induce AR recruitment to endogenous androgen-regulated genes based on chromatin immunoprecipitation. Galeterone at low micromolar concentrations that did not induce cellular stress responses enhanced AR protein degradation in LNCaP and C4-2 cells, which express a T878A mutant AR, but not in prostate cancer cells expressing wild-type AR. Further transfection studies using stable LNCaP and PC3 cell lines ectopically expressing wild-type or T878A-mutant ARs confirmed that galeterone selectively enhances degradation of the T878A-mutant AR. CONCLUSIONS: Similar to enzalutamide, galeterone may be effective as a direct AR antagonist in CRPC. It may be particularly effective against prostate cancer cells with the T878A AR mutation but may also enhance degradation of wild-type AR in vivo through a combination of direct and indirect mechanisms. Finally, these findings show that conformational changes in AR can markedly enhance its degradation and thereby support efforts to develop further antagonists that enhance AR degradation.
Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Androstadienos/farmacología , Bencimidazoles/farmacología , Cromatina/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Neoplasias de la Próstata/tratamiento farmacológico , Proteolisis/efectos de los fármacos , Receptores Androgénicos/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromatina/genética , Inmunoprecipitación de Cromatina , Humanos , Masculino , Proteínas Mutantes/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Unión Proteica/efectos de los fármacos , Receptores Androgénicos/genética , Células Tumorales CultivadasRESUMEN
The presence of regulatory T cells (Treg) in solid tumors is known to play a role in patient survival in ovarian cancer and other malignancies. We assessed inherited genetic variations via 749 tag single-nucleotide polymorphisms (SNP) in 25 Treg-associated genes (CD28, CTLA4, FOXP3, IDO1, IL10, IL10RA, IL15, 1L17RA, IL23A, IL23R, IL2RA, IL6, IL6R, IL8, LGALS1, LGALS9, MAP3K8, STAT5A, STAT5B, TGFB1, TGFB2, TGFB3, TGFBR1, TGRBR2, and TGFBR3) in relation to ovarian cancer survival. We analyzed genotype and overall survival in 10,084 women with invasive epithelial ovarian cancer, including 5,248 high-grade serous, 1,452 endometrioid, 795 clear cell, and 661 mucinous carcinoma cases of European descent across 28 studies from the Ovarian Cancer Association Consortium (OCAC). The strongest associations were found for endometrioid carcinoma and IL2RA SNPs rs11256497 [HR, 1.42; 95% confidence interval (CI), 1.22-1.64; P = 5.7 × 10(-6)], rs791587 (HR, 1.36; 95% CI, 1.17-1.57; P = 6.2 × 10(-5)), rs2476491 (HR, = 1.40; 95% CI, 1.19-1.64; P = 5.6 × 10(-5)), and rs10795763 (HR, 1.35; 95% CI, 1.17-1.57; P = 7.9 × 10(-5)), and for clear cell carcinoma and CTLA4 SNP rs231775 (HR, 0.67; 95% CI, 0.54-0.82; P = 9.3 × 10(-5)) after adjustment for age, study site, population stratification, stage, grade, and oral contraceptive use. The rs231775 allele associated with improved survival in our study also results in an amino acid change in CTLA4 and previously has been reported to be associated with autoimmune conditions. Thus, we found evidence that SNPs in genes related to Tregs seem to play a role in ovarian cancer survival, particularly in patients with clear cell and endometrioid epithelial ovarian cancer.
Asunto(s)
Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Variación Genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/mortalidad , Linfocitos T Reguladores/metabolismo , Femenino , Expresión Génica , Mutación de Línea Germinal , Humanos , Subunidad alfa del Receptor de Interleucina-2/genética , Clasificación del Tumor , Invasividad Neoplásica , Neoplasias Ováricas/patología , Evaluación del Resultado de la Atención al Paciente , Polimorfismo de Nucleótido Simple , PronósticoRESUMEN
TERT-locus SNPs and leukocyte telomere measures are reportedly associated with risks of multiple cancers. Using the Illumina custom genotyping array iCOGs, we analyzed â¼480 SNPs at the TERT locus in breast (n = 103,991), ovarian (n = 39,774) and BRCA1 mutation carrier (n = 11,705) cancer cases and controls. Leukocyte telomere measurements were also available for 53,724 participants. Most associations cluster into three independent peaks. The minor allele at the peak 1 SNP rs2736108 associates with longer telomeres (P = 5.8 × 10(-7)), lower risks for estrogen receptor (ER)-negative (P = 1.0 × 10(-8)) and BRCA1 mutation carrier (P = 1.1 × 10(-5)) breast cancers and altered promoter assay signal. The minor allele at the peak 2 SNP rs7705526 associates with longer telomeres (P = 2.3 × 10(-14)), higher risk of low-malignant-potential ovarian cancer (P = 1.3 × 10(-15)) and greater promoter activity. The minor alleles at the peak 3 SNPs rs10069690 and rs2242652 increase ER-negative (P = 1.2 × 10(-12)) and BRCA1 mutation carrier (P = 1.6 × 10(-14)) breast and invasive ovarian (P = 1.3 × 10(-11)) cancer risks but not via altered telomere length. The cancer risk alleles of rs2242652 and rs10069690, respectively, increase silencing and generate a truncated TERT splice variant.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/etiología , Sitios Genéticos/genética , Neoplasias Ováricas/etiología , Polimorfismo de Nucleótido Simple/genética , Telomerasa/genética , Telómero/genética , Empalme Alternativo , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Cromatina/genética , Metilación de ADN , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Luciferasas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de RiesgoRESUMEN
Epithelial ovarian cancer (EOC) has a heritable component that remains to be fully characterized. Most identified common susceptibility variants lie in non-protein-coding sequences. We hypothesized that variants in the 3' untranslated region at putative microRNA (miRNA)-binding sites represent functional targets that influence EOC susceptibility. Here, we evaluate the association between 767 miRNA-related single-nucleotide polymorphisms (miRSNPs) and EOC risk in 18,174 EOC cases and 26,134 controls from 43 studies genotyped through the Collaborative Oncological Gene-environment Study. We identify several miRSNPs associated with invasive serous EOC risk (odds ratio=1.12, P=10(-8)) mapping to an inversion polymorphism at 17q21.31. Additional genotyping of non-miRSNPs at 17q21.31 reveals stronger signals outside the inversion (P=10(-10)). Variation at 17q21.31 is associated with neurological diseases, and our collaboration is the first to report an association with EOC susceptibility. An integrated molecular analysis in this region provides evidence for ARHGAP27 and PLEKHM1 as candidate EOC susceptibility genes.
Asunto(s)
Cromosomas Humanos Par 17 , Predisposición Genética a la Enfermedad , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Carcinoma Epitelial de Ovario , Femenino , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
Genome-wide association studies (GWAS) have identified four susceptibility loci for epithelial ovarian cancer (EOC), with another two suggestive loci reaching near genome-wide significance. We pooled data from a GWAS conducted in North America with another GWAS from the UK. We selected the top 24,551 SNPs for inclusion on the iCOGS custom genotyping array. We performed follow-up genotyping in 18,174 individuals with EOC (cases) and 26,134 controls from 43 studies from the Ovarian Cancer Association Consortium. We validated the two loci at 3q25 and 17q21 that were previously found to have associations close to genome-wide significance and identified three loci newly associated with risk: two loci associated with all EOC subtypes at 8q21 (rs11782652, P = 5.5 × 10(-9)) and 10p12 (rs1243180, P = 1.8 × 10(-8)) and another locus specific to the serous subtype at 17q12 (rs757210, P = 8.1 × 10(-10)). An integrated molecular analysis of genes and regulatory regions at these loci provided evidence for functional mechanisms underlying susceptibility and implicated CHMP4C in the pathogenesis of ovarian cancer.
Asunto(s)
Cistadenocarcinoma Seroso/etiología , Sitios Genéticos/genética , Predisposición Genética a la Enfermedad , Neoplasias Ováricas/etiología , Polimorfismo de Nucleótido Simple/genética , Estudios de Casos y Controles , Conducta Cooperativa , Cistadenocarcinoma Seroso/patología , Femenino , Interacción Gen-Ambiente , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Metaanálisis como Asunto , Invasividad Neoplásica , Neoplasias Ováricas/patología , Factores de RiesgoRESUMEN
Previously available androgen receptor (AR) antagonists (bicalutamide, flutamide, and nilutamide) have limited activity against AR in prostate cancers that relapse after castration [castration resistant prostate cancer (CRPC)]. However, recent AR competitive antagonists such as MDV3100, generated through chemical modifications to the current AR ligands, appear to have increased activity in CRPC and have novel mechanisms of action. Using pharmacophore models and a refined homology model of the antagonist-liganded AR ligand binding domain, we carried out in silico screens of small molecule libraries and report here on the identification of a series of structurally distinct nonsteroidal small molecule competitive AR antagonists. Despite their unique chemical architectures, compounds representing each of six chemotypes functioned in vitro as pure AR antagonists. Moreover, similarly to MDV3100 and in contrast to previous AR antagonists, these compounds all prevented AR binding to chromatin, consistent with each of the six chemotypes stabilizing a similar AR antagonist conformation. Additional studies with the lead chemotype (chemotype A) showed enhanced AR protein degradation, which was dependent on helix 12 in the AR ligand binding domain. Significantly, chemotype A compounds functioned as AR antagonists in vivo in normal male mice and suppressed AR activity and tumor cell proliferation in human CRPC xenografts. These data indicate that certain ligand-induced structural alterations in the AR ligand binding domain may both impair AR chromatin binding and enhance AR degradation and support continued efforts to develop AR antagonists with unique mechanisms of action and efficacy in CRPC.
Asunto(s)
Antagonistas de Receptores Androgénicos/análisis , Antagonistas de Receptores Androgénicos/uso terapéutico , Biología Computacional/métodos , Descubrimiento de Drogas , Orquiectomía , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/cirugía , Antagonistas de Receptores Androgénicos/química , Antagonistas de Receptores Androgénicos/farmacología , Animales , Células COS , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , ADN de Neoplasias/metabolismo , Humanos , Masculino , Ratones , Modelos Biológicos , Modelos Moleculares , Neoplasias de la Próstata/patología , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Receptores Androgénicos/química , Reproducibilidad de los Resultados , Homología Estructural de Proteína , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Androgen receptor (AR) continues to play a central role in prostate cancers that relapse after androgen deprivation therapy, but these tumors are refractory to available AR antagonists. In a recent issue of Science, Tran et al. describe an antagonist that prevents AR recruitment to chromatin and shows efficacy in relapsed prostate cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/fisiología , Antagonistas de Receptores Androgénicos , Andrógenos , Antineoplásicos/farmacología , Benzamidas , Cromatina/metabolismo , Humanos , Masculino , Recurrencia Local de Neoplasia , Nitrilos , Orquiectomía , Feniltiohidantoína/farmacología , Feniltiohidantoína/uso terapéutico , Neoplasias de la Próstata/patología , Transducción de Señal , Activación Transcripcional/efectos de los fármacosRESUMEN
BACKGROUND: The androgen receptor (AR) plays a pivotal role in prostate cancer (PCa) initiation and progression. To date, studies have focused disproportionately on androgen-stimulated genes such as prostate-specific antigen (PSA), while repressed genes have gained little attention, even though they too may be involved in regulating cell growth, differentiation, and apoptosis. METHODS: ChIP Display was used to identify putative AR target genes in the ablation-resistant human PCa cell line, C4-2B. Quantitative real-time reverse transcription-PCR analysis was used to measure gene expression in cells subjected to dihydrotestosterone (DHT) timecourse and dose-response, as well as AR knock-down and bicalutamide-treatments. RESULTS: We report on three genes, KIAA1217, CHRM1, and WBSCR28, which were newly identified in a screen for AR-occupied regions in C4-2B PCa cells, and which were repressed by treatment with DHT. AR knock-down resulted in increased KIAA1217, CHRM1, and WBSCR28 mRNA, indicating that, like PSA stimulation, AR represses these three genes even in the absence of added ligand. DHT decreased KIAA1217 and CHRM1 pre-mRNA levels, suggesting AR-mediated transcriptional inhibition. Cycloheximide attenuated DHT-mediated repression of CHRM1, suggesting the requirement of new protein synthesis. Furthermore, bicalutamide treatment did not mimic, but rather antagonized DHT-mediated KIAA1217 repression. Unlike the handful of androgen-repressed genes studied thus far, AR occupancy at KIAA1217, CHRM1, and WBSCR28 was mapped outside their respective 5'-promoter regions. CONCLUSIONS: Many more genes likely share AR-mediated gene repression through distal regulatory elements. Further study of such targets and their transcriptional regulation may help explain the receptor's tumorigenicity in PCa.
Asunto(s)
Neoplasias de la Próstata/genética , Receptores Androgénicos/fisiología , Antagonistas de Andrógenos/farmacología , Anilidas/farmacología , Línea Celular Tumoral , Cicloheximida/farmacología , Dihidrotestosterona/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Nitrilos/farmacología , Neoplasias de la Próstata/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Receptor Muscarínico M1 , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Receptores Muscarínicos/biosíntesis , Receptores Muscarínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Compuestos de Tosilo/farmacología , TransfecciónRESUMEN
Whereas the androgen receptor (AR) protein shares similarities in the structure of its DNA- and hormone-binding domains with other members of the steroid nuclear receptor family, the molecule in its unliganded form has a seemingly unordered amino-terminal transactivation domain unique to the AR. A comprehensive understanding of the specific sub-structures and protein-protein interactions inherent to this domain in both its inactive and activated states remains un-achieved. Therefore, the malleability of this peptide region in accommodating the diverse repertoire of transcription-modulating AR cofactors creates a great challenge for those intent on generating relevant three-dimensional molecular models. The AR transactivation domain achieves this flexibility through a series of conformational steps dependent on the presence of cofactors that induce allosteric changes, and thus has evolved several conserved peptide motifs representing key protein-protein interaction surfaces. Elucidation of these signaling regions, including their involvement in inducing AR transactivation domain structural changes, is of foremost interest in understanding how the AR achieves its pivotal role in regulating the androgen signaling axis particularly during the progression of prostate cancer.