RESUMO
Copy number variants (CNVs) are a recently recognized class of human germ line polymorphisms and are associated with a variety of human diseases, including cancer. Because of the strong genetic influence on prostate cancer, we sought to identify functionally active CNVs associated with susceptibility of this cancer type. We queried low-frequency biallelic CNVs from 1,903 men of Caucasian origin enrolled in the Tyrol Prostate Specific Antigen Screening Cohort and discovered two CNVs strongly associated with prostate cancer risk. The first risk locus (P = 7.7 × 10(-4), odds ratio = 2.78) maps to 15q21.3 and overlaps a noncoding enhancer element that contains multiple activator protein 1 (AP-1) transcription factor binding sites. Chromosome conformation capture (Hi-C) data suggested direct cis-interactions with distant genes. The second risk locus (P = 2.6 × 10(-3), odds ratio = 4.8) maps to the α-1,3-mannosyl-glycoprotein 4-ß-N-acetylglucosaminyltransferase C (MGAT4C) gene on 12q21.31. In vitro cell-line assays found this gene to significantly modulate cell proliferation and migration in both benign and cancer prostate cells. Furthermore, MGAT4C was significantly overexpressed in metastatic versus localized prostate cancer. These two risk associations were replicated in an independent PSA-screened cohort of 800 men (15q21.3, combined P = 0.006; 12q21.31, combined P = 0.026). These findings establish noncoding and coding germ line CNVs as significant risk factors for prostate cancer susceptibility and implicate their role in disease development and progression.
Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 15 , Dosagem de Genes , Predisposição Genética para Doença , Neoplasias da Próstata/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proliferação de Células , Estudos de Coortes , Humanos , Masculino , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/patologiaRESUMO
Mosaicism with an isodicentric 8 with a breakpoint at p23.3 [idic(8)(p23.3)] is very rare. We report the first prenatal case on a male fetus, in which obstetric ultrasound revealed multiple congenital anomalies at 28 weeks of gestation. Cytogenetic analysis of amniocytes showed mos 45,XY,-8,psu idic(8)(p23.3)[16]/46,XY,psu idic(8)(p23.3)[4], and that of cord blood lymphocytes revealed mos 46,XY, psu idic(8)(p23.3)[37]/45,XY,-8,psu idic(8)(p23.3)[13]. Fluorescence in situ hybridization studies revealed that the break-reunion occurred at the cytoband 8p23.3 within the physical position 2.08 Mb from the 8p telomere. Chromosomal microarray analyses further assigned the duplication/deletion breakpoint at 2.16 Mb (Agilent 244K) and at 2.19 Mb (Affymetrix SNP6.0). Analysis of microsatellite DNA indicated that the psu idic(8)(p23.3) was derived from the maternal chromosome 8. Together, these findings indicate that the fetus was nullisomic for ~2.2 Mb from 8pter, trisomic for the rest of chromosome 8 in mosaic condition, and likely had breaks in MYOM2 repeats of the maternal chromosome 8.
Assuntos
Anormalidades Múltiplas/diagnóstico , Recombinação Homóloga/genética , Diagnóstico Pré-Natal , Trissomia/diagnóstico , Dissomia Uniparental/diagnóstico , Adulto , Cromossomos Humanos Par 8 , Análise Citogenética , Feminino , Idade Gestacional , Humanos , Hibridização in Situ Fluorescente , Imageamento por Ressonância Magnética , Mosaicismo , GravidezRESUMO
The detection of copy number variants (CNV) by array-based platforms provides valuable insight into understanding human diversity. However, suboptimal study design and data processing negatively affect CNV assessment. We quantitatively evaluate their impact when short-sequence oligonucleotide arrays are applied (Affymetrix Genome-Wide Human SNP Array 6.0) by evaluating 42 HapMap samples for CNV detection. Several processing and segmentation strategies are implemented, and results are compared to CNV assessment obtained using an oligonucleotide array CGH platform designed to query CNVs at high resolution (Agilent). We quantitatively demonstrate that different reference models (e.g. single versus pooled sample reference) used to detect CNVs are a major source of inter-platform discrepancy (up to 30%) and that CNVs residing within segmental duplication regions (higher reference copy number) are significantly harder to detect (P < 0.0001). After adjusting Affymetrix data to mimic the Agilent experimental design (reference sample effect), we applied several common segmentation approaches and evaluated differential sensitivity and specificity for CNV detection, ranging 39-77% and 86-100% for non-segmental duplication regions, respectively, and 18-55% and 39-77% for segmental duplications. Our results are relevant to any array-based CNV study and provide guidelines to optimize performance based on study-specific objectives.
Assuntos
Variações do Número de Cópias de DNA , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Algoritmos , Sequência de Bases , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Sondas de Oligonucleotídeos/química , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Approximately 10% of patients with chronic lymphocytic leukaemia (CLL) have a family history of the disease or a related lymphoproliferative disorder, yet the relationship of familial CLL to genomic abnormalities has not been characterized in detail. We therefore studied 75 CLL patients, half familial and half sporadic, using high-resolution array comparative genomic hybridization (CGH), in order to better define the relationship of genomic abnormalities to familial disease and other biological prognostic factors. Our results showed that the most common high-risk deletion in CLL, deletion 11q, was significantly associated with sporadic disease. Comparison of familial to sporadic disease additionally identified a copy number variant region near the centromere on 14q, proximal to IGH@, in which gains were associated both with familial CLL, and with mutated IGHV and homozygous deletion of 13q. Homozygous deletion of 13q was also found to be associated with mutated IGHV and low expression of ZAP-70, and a significantly longer time to first treatment compared to heterozygous deletion or lack of alteration. This study is the first high resolution effort to investigate and report somatic genetic differences between familial and sporadic CLL.
Assuntos
Aberrações Cromossômicas , Leucemia Linfocítica Crônica de Células B/genética , Adulto , Idoso , Deleção Cromossômica , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 14/genética , Hibridização Genômica Comparativa/métodos , Feminino , Predisposição Genética para Doença , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-IdadeRESUMO
Emerging molecular and clinical data suggest that ETS fusion prostate cancer represents a distinct molecular subclass, driven most commonly by a hormonally regulated promoter and characterized by an aggressive natural history. The study of the genomic landscape of prostate cancer in the light of ETS fusion events is required to understand the foundation of this molecularly and clinically distinct subtype. We performed genome-wide profiling of 49 primary prostate cancers and identified 20 recurrent chromosomal copy number aberrations, mainly occurring as genomic losses. Co-occurring events included losses at 19q13.32 and 1p22.1. We discovered three genomic events associated with ERG rearranged prostate cancer, affecting 6q, 7q, and 16q. 6q loss in nonrearranged prostate cancer is accompanied by gene expression deregulation in an independent dataset and by protein deregulation of MYO6. To analyze copy number alterations within the ETS genes, we performed a comprehensive analysis of all 27 ETS genes and of the 3 Mbp genomic area between ERG and TMPRSS2 (21q) with an unprecedented resolution (30 bp). We demonstrate that high-resolution tiling arrays can be used to pin-point breakpoints leading to fusion events. This study provides further support to define a distinct molecular subtype of prostate cancer based on the presence of ETS gene rearrangements.
Assuntos
Aberrações Cromossômicas , Rearranjo Gênico , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-ets/genética , Transativadores/genética , Linhagem Celular Tumoral , Mapeamento Cromossômico , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Regulador Transcricional ERGRESUMO
Germline copy number variants (CNVs) and single-nucleotide polymorphisms (SNPs) form the basis of inter-individual genetic variation. Although the phenotypic effects of SNPs have been extensively investigated, the effects of CNVs is relatively less understood. To better characterize mechanisms by which CNVs affect cellular phenotype, we tested their association with variable CpG methylation in a genome-wide manner. Using paired CNV and methylation data from the 1000 genomes and HapMap projects, we identified genome-wide associations by methylation quantitative trait locus (mQTL) analysis. We found individual CNVs being associated with methylation of multiple CpGs and vice versa. CNV-associated methylation changes were correlated with gene expression. CNV-mQTLs were enriched for regulatory regions, transcription factor-binding sites (TFBSs), and were involved in long-range physical interactions with associated CpGs. Some CNV-mQTLs were associated with methylation of imprinted genes. Several CNV-mQTLs and/or associated genes were among those previously reported by genome-wide association studies (GWASs). We demonstrate that germline CNVs in the genome are associated with CpG methylation. Our findings suggest that structural variation together with methylation may affect cellular phenotype.
RESUMO
BACKGROUND: MicroRNAs have recently emerged as promising circulating biomarkers in diverse cancer types, including ovarian cancer. We utilized conditional, doxycycline-induced fallopian tube (FT)-derived cancer models to identify changes in miRNA expression in tumors and plasma, and further validated the murine findings in high-grade ovarian cancer patient samples. METHODS: We analyzed 566 biologically informative miRNAs in doxycycline-induced FT and metastatic tumors as well as plasma samples derived from murine models bearing inactivation of Brca, Tp53, and Pten genes. We identified miRNAs that showed a consistent pattern of dysregulated expression and validated our results in human patient serum samples. RESULTS: We identified six miRNAs that were significantly dysregulated in doxycycline-induced FTs (P < .05) and 130 miRNAs differentially regulated in metastases compared to normal fallopian tissues (P < .05). Furthermore, we validated miR-21a-5p, miR-146a-5p, and miR-126a-3p as dysregulated in both murine doxycycline-induced FT and metastatic tumors, as well as in murine plasma and patient serum samples. CONCLUSIONS: In summary, we identified changes in miRNA expression that potentially accompany tumor development in murine models driven by commonly found genetic alterations in cancer patients. Further studies are required to test both the function of these miRNAs in driving the disease and their utility as potential biomarkers for diagnosis and/or disease progression.
Assuntos
Doxiciclina/efeitos adversos , Tubas Uterinas/patologia , Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , Neoplasias Ovarianas/genética , Animais , Biomarcadores Tumorais/genética , Tubas Uterinas/química , Tubas Uterinas/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Neoplasias Ovarianas/patologiaRESUMO
PURPOSE: Chronic lymphocytic leukemia (CLL) with 17p deletion typically progresses quickly and is refractory to most conventional therapies. However, some del(17p) patients do not progress for years, suggesting that del(17p) is not the only driving event in CLL progression. We hypothesize that other concomitant genetic abnormalities underlie the clinical heterogeneity of del(17p) CLL. EXPERIMENTAL DESIGN: We profiled the somatic mutations and copy number alterations (CNA) in a large group of del(17p) CLLs as well as wild-type CLL and analyzed the genetic basis of their clinical heterogeneity. RESULTS: We found that increased somatic mutation number associates with poor overall survival independent of 17p deletion (P = 0.003). TP53 mutation was present in 81% of del(17p) CLL, mostly clonal (82%), and clonal mutations with del(17p) exhibit shorter overall survival than subclonal mutations with del(17p) (P = 0.019). Del(17p) CLL has a unique driver mutation profile, including NOTCH1 (15%), RPS15 (12%), DDX3X (8%), and GPS2 (6%). We found that about half of del(17p) CLL cases have recurrent deletions at 3p, 4p, or 9p and that any of these deletions significantly predicts shorter overall survival. In addition, the number of CNAs, but not somatic mutations, predicts shorter time to treatment among patients untreated at sampling. Indolent del(17p) CLLs were characterized by absent or subclonal TP53 mutation and few CNAs, with no difference in somatic mutation number. CONCLUSIONS: We conclude that del(17p) has a unique genomic profile and that clonal TP53 mutations, 3p, 4p, or 9p deletions, and genomic complexity are associated with shorter overall survival. Clin Cancer Res; 23(3); 735-45. ©2016 AACR.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 17/ultraestrutura , Leucemia Linfocítica Crônica de Células B/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Quebra Cromossômica , Cromossomos Humanos Par 17/genética , Células Clonais , Progressão da Doença , Feminino , Dosagem de Genes , Mutação em Linhagem Germinativa , Humanos , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Mutação , Polimorfismo de Nucleotídeo Único , Saliva/química , Proteína Supressora de Tumor p53/genética , Sequenciamento do ExomaRESUMO
The platelet-derived growth factor receptor (PDGFR) is a receptor tyrosine kinase overexpressed in a subset of solid tumors and therefore is the target of drugs inhibiting this function such as imatinib mesylate (Gleevec). Thus far, drug therapy has played a limited role in the treatment of localized prostate cancer (PCa). This study characterizes PDGFR-beta expression in a wide spectrum of PCa samples to provide empirical data as part of a rational treatment strategy. A survey of five published prostate expression array studies, including 100 clinically localized PCa, did not identify tumors with increased PDGFR-beta expression level. Protein expression of PDGFR-beta, as determined by immunohistochemistry, revealed 5% of clinically localized PCa and 16% of metastatic PCa cases to show moderate or strong expression. To develop a strategy to detect patients most likely to profit from Gleevec treatment, we analyzed cDNA expression array data from 10,000 transcripts for PDGFR-beta expression and divided tumors in groups based on PDGFR-beta expression level. Performing a supervised analysis to identify potential comarkers of PDGFR-beta in PCa, we identified a set of genes whose expression was associated with PDGFR-beta status including early growth response 1 (Egr1), an upstream effector of PDGF (4.2-fold upregulation), alpha-methylacyl-CoA racemase, as well as v-Maf and neuroblastoma suppressor of tumorigenicity (both with a 2.2-fold downregulation). Taken together, this study suggests that only a small subset of PCas may be amenable to tyrosine kinase inhibitors specific for PDGFR.
Assuntos
Neoplasias da Próstata/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Antineoplásicos/uso terapêutico , Benzamidas , Linhagem Celular Tumoral , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mesilato de Imatinib , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Fator de Crescimento Derivado de Plaquetas/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/análise , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Regulação para Cima/genéticaRESUMO
High-grade serous ovarian carcinoma presents significant clinical and therapeutic challenges. Although the traditional model of carcinogenesis has focused on the ovary as a tumor initiation site, recent studies suggest that there may be additional sites of origin outside the ovary, namely the secretory cells of the fallopian tube. Our study demonstrates that high-grade serous tumors can originate in fallopian tubal secretory epithelial cells and also establishes serous tubal intraepithelial carcinoma as the precursor lesion to high-grade serous ovarian and peritoneal carcinomas in animal models targeting the Brca, Tp53, and Pten genes. These findings offer an avenue to address clinically important questions that are critical for cancer prevention and early detection in women carrying BRCA1 and BRCA2 mutations.
Assuntos
Transformação Celular Neoplásica , Cistadenocarcinoma Seroso/etiologia , Neoplasias das Tubas Uterinas/patologia , Genes BRCA1 , Genes BRCA2 , Neoplasias Ovarianas/etiologia , Lesões Pré-Cancerosas/patologia , Animais , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Epitélio/patologia , Feminino , Genes p53 , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Gradação de Tumores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fator de Transcrição PAX8 , PTEN Fosfo-Hidrolase/genética , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/fisiologiaRESUMO
To elucidate the role of ETS gene fusions in castration-resistant prostate cancer (CRPC), we characterized the transcriptome of 54 CRPC tumor samples from men with locally advanced or metastatic disease. Trefoil factor 3 (TFF3) emerged as the most highly differentially regulated gene with respect to ERG rearrangement status and resistance to hormone ablation therapy. Conventional chromatin immunoprecipitation (ChIP)-polymerase chain reaction and ChIP followed by DNA sequencing (ChIP-seq) revealed direct binding of ERG to ETS binding sites in the TFF3 promoter in ERG-rearranged prostate cancer cell lines. These results were confirmed in ERG-rearranged hormone-naive prostate cancer (HNPC) and CRPC tissue samples. Functional studies demonstrated that ERG has an inhibitory effect on TFF3 expression in hormone-naive cancer but not in the castration-resistant state. In addition, we provide evidence suggesting an effect of androgen receptor signaling on ERG-regulated TFF3 expression. Furthermore, TFF3 overexpression enhances ERG-mediated cell invasion in CRPC prostate cancer cells. Taken together, our findings reveal a novel mechanism for enhanced tumor cell aggressiveness resulting from ERG rearrangement in the castration-resistant setting through TFF3 gene expression.
Assuntos
Peptídeos/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transativadores/metabolismo , Imunoprecipitação da Cromatina , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Invasividade Neoplásica/genética , Neoplasias Hormônio-Dependentes , Peptídeos/metabolismo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Transdução de Sinais , Transativadores/genética , Fatores de Transcrição , Regulador Transcricional ERG , Fator Trefoil-3RESUMO
PURPOSE: Dihydrotestosterone (DHT) is an important factor in prostate cancer (PCA) genesis and disease progression. Given PCA's strong genetic component, we evaluated the possibility that variation in genes involved in DHT metabolism influence PCA risk. EXPERIMENTAL DESIGN: We investigated copy number variants (CNV) and single nucleotide polymorphisms (SNP). We explored associations between CNV of uridine diphospho-glucuronosyltransferase (UGT) genes from the 2B subclass, given their prostate specificity and/or involvement in steroid metabolism and PCA risk. We also investigated associations between SNPs in genes (HSD3B1, SRD5A1/2, and AKR1C2) involved in the conversion of testosterone to DHT, and in DHT metabolism and PCA risk. The population consisted of 426 men (205 controls and 221 cases) who underwent prostate-specific antigen screening as part of a PCA early detection program in Tyrol, Austria. RESULTS: No association between CNV in UGT2B17 and UGT2B28 and PCA risk was identified. Men carrying the AA genotype at SNP rs6428830 (HSD3B1) had an odds ratio (OR) of 2.0 [95% confidence intervals (95% CI), 1.1-4.1] compared with men with GG, and men with AG or GG versus AA in rs1691053 (SRD5A1) had an OR of 1.8 (95% CI, 1.04-3.13). Individuals carrying both risk alleles had an OR of 3.1 (95% CI, 1.4-6.7) when compared with men carrying neither (P = 0.005). Controls with the AA genotype on rs7594951 (SRD5A2) tended toward higher serum DHT levels (P = 0.03). CONCLUSIONS: This is the first study to implicate the 5alpha-reductase isoform 1 (SRD5A1) and PCA risk, supporting the rationale of blocking enzymatic activity of both isoforms of 5alpha-reductase for PCA chemoprevention.
Assuntos
Di-Hidrotestosterona/metabolismo , Predisposição Genética para Doença , Neoplasias da Próstata/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Adulto , Idoso , Biomarcadores Tumorais/genética , Proteínas de Ligação a DNA/genética , Dosagem de Genes , Genótipo , Glucuronosiltransferase/genética , Humanos , Hidroxiesteroide Desidrogenases/genética , Masculino , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor , Polimorfismo de Nucleotídeo Único , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/metabolismo , Fatores de Risco , Testosterona/metabolismoRESUMO
BACKGROUND: Current prostate cancer prognostic models are based on pre-treatment prostate specific antigen (PSA) levels, biopsy Gleason score, and clinical staging but in practice are inadequate to accurately predict disease progression. Hence, we sought to develop a molecular panel for prostate cancer progression by reasoning that molecular profiles might further improve current clinical models. METHODS: We analyzed a Swedish Watchful Waiting cohort with up to 30 years of clinical follow up using a novel method for gene expression profiling. This cDNA-mediated annealing, selection, ligation, and extension (DASL) method enabled the use of formalin-fixed paraffin-embedded transurethral resection of prostate (TURP) samples taken at the time of the initial diagnosis. We determined the expression profiles of 6100 genes for 281 men divided in two extreme groups: men who died of prostate cancer and men who survived more than 10 years without metastases (lethals and indolents, respectively). Several statistical and machine learning models using clinical and molecular features were evaluated for their ability to distinguish lethal from indolent cases. RESULTS: Surprisingly, none of the predictive models using molecular profiles significantly improved over models using clinical variables only. Additional computational analysis confirmed that molecular heterogeneity within both the lethal and indolent classes is widespread in prostate cancer as compared to other types of tumors. CONCLUSIONS: The determination of the molecularly dominant tumor nodule may be limited by sampling at time of initial diagnosis, may not be present at time of initial diagnosis, or may occur as the disease progresses making the development of molecular biomarkers for prostate cancer progression challenging.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Biomarcadores Tumorais/genética , Progressão da Doença , Humanos , MasculinoRESUMO
Chromosomal rearrangements account for all erythroblast transformation-specific (ETS) family member gene fusions that have been reported in prostate cancer and have clinical, diagnostic, and prognostic implications. Androgen-regulated genes account for the majority of the 5' genomic regulatory promoter elements fused with ETS genes. TMPRSS2-ERG, TMPRSS2-ETV1, and SLC45A3-ERG rearrangements account for roughly 90% of ETS fusion prostate cancer. ELK4, another ETS family member, is androgen regulated, involved in promoting cell growth, and highly expressed in a subset of prostate cancer, yet the mechanism of ELK4 overexpression is unknown. In this study, we identified a novel ETS family fusion transcript, SLC45A3-ELK4, and found it to be expressed in both benign prostate tissue and prostate cancer. We found high levels of SLC45A3-ELK4 mRNA restricted to a subset of prostate cancer samples. SLC45A3-ELK4 transcript can be detected at high levels in urine samples from men at risk for prostate cancer. Characterization of the fusion mRNA revealed a major variant in which SLC45A3 exon 1 is fused to ELK4 exon 2. Based on quantitative PCR analyses of DNA, unlike other ETS fusions described in prostate cancer, the expression of SLC45A3-ELK4 mRNA is not exclusive to cases harboring a chromosomal rearrangement. Treatment of LNCaP cancer cells with a synthetic androgen (R1881) revealed that SLC45A3-ELK4, and not endogenous ELK4, mRNA expression is androgen regulated. Altogether, our findings show that SLC45A3-ELK4 mRNA expression is heterogeneous, highly induced in a subset of prostate cancers, androgen regulated, and most commonly occurs through a mechanism other than chromosomal rearrangement (e.g., trans-splicing).
Assuntos
Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , RNA Mensageiro/genética , Linhagem Celular Tumoral , Deleção Cromossômica , Cromossomos Humanos Par 1 , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hibridização in Situ Fluorescente , Masculino , Metribolona/farmacologia , Proteínas de Fusão Oncogênica/biossíntese , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-ets/biossíntese , Proteínas Proto-Oncogênicas c-ets/genética , RNA Mensageiro/biossíntese , Congêneres da Testosterona/farmacologia , Transcrição GênicaRESUMO
Early detection and diagnosis of prostate cancer is key to designing effective treatment strategies. Microarrays have resulted in the discovery of hepsin (HPN) as a biomarker for detection of prostate cancer. In this study, we explore the development of HPN imaging probes for detection of prostate cancer. We used phage display to isolate HPN binding peptides with 190 + 2.2 nmol/L affinity in monomeric form and high specificity. The identified peptides were able to detect human prostate cancer on tissue microarrays and in cell-based assays. HPN-targeted imaging agents were synthesized by conjugating multiple peptides to fluorescent nanoparticles to further improve avidity through multivalency and to improve pharmacokinetics. When injected into mouse xenograft models, HPN-targeted nanoparticles bound specifically to HPN-expressing LNCaP xenografts compared with non-HPN-expressing PC3 xenografts. HPN imaging may provide a new method for detection of prostate cancer.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/enzimologia , Serina Endopeptidases/análise , Animais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Nus , Biblioteca de Peptídeos , Neoplasias da Próstata/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , TransfecçãoRESUMO
BACKGROUND: The majority of prostate cancers harbor gene fusions of the 5'-untranslated region of the androgen-regulated transmembrane protease serine 2 (TMPRSS2) promoter with erythroblast transformation-specific transcription factor family members. The common fusion between TMPRESS2 and v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) is associated with a more aggressive clinical phenotype, implying the existence of a distinct subclass of prostate cancer defined by this fusion. METHODS: We used complementary DNA-mediated annealing, selection, ligation, and extension to determine the expression profiles of 6144 transcriptionally informative genes in archived biopsy samples from 455 prostate cancer patients in the Swedish Watchful Waiting cohort (1987-1999) and the United States-based Physicians(') Health Study cohort (1983-2003). A gene expression signature for prostate cancers with the TMPRSS2-ERG fusion was determined using partitioning and classification models and used in computational functional analysis. Cell proliferation and TMPRSS2-ERG expression in androgen receptor-negative (NCI-H660) prostate cancer cells after treatment with vehicle or estrogenic compounds were assessed by viability assays and quantitative polymerase chain reaction, respectively. All statistical tests were two-sided. RESULTS: We identified an 87-gene expression signature that distinguishes TMPRSS2-ERG fusion prostate cancer as a discrete molecular entity (area under the curve = 0.80, 95% confidence interval [CI] = 0.792 to 0.81; P < .001). Computational analysis suggested that this fusion signature was associated with estrogen receptor (ER) signaling. Viability of NCI-H660 cells decreased after treatment with estrogen (viability normalized to day 0, estrogen vs vehicle at day 8, mean = 2.04 vs 3.40, difference = 1.36, 95% CI = 1.12 to 1.62) or ERbeta agonist (ERbeta agonist vs vehicle at day 8, mean = 1.86 vs 3.40, difference = 1.54, 95% CI = 1.39 to 1.69) but increased after ERalpha agonist treatment (ERalpha agonist vs vehicle at day 8, mean = 4.36 vs 3.40, difference = 0.96, 95% CI = 0.68 to 1.23). Similarly, expression of TMPRSS2-ERG decreased after ERbeta agonist treatment (fold change over internal control, ERbeta agonist vs vehicle at 24 hours, NCI-H660, mean = 0.57- vs 1.0-fold, difference = 0.43-fold, 95% CI = 0.29- to 0.57-fold) and increased after ERalpha agonist treatment (ERalpha agonist vs vehicle at 24 hours, mean = 5.63- vs 1.0-fold, difference = 4.63-fold, 95% CI = 4.34- to 4.92-fold). CONCLUSIONS: TMPRSS2-ERG fusion prostate cancer is a distinct molecular subclass. TMPRSS2-ERG expression is regulated by a novel ER-dependent mechanism.
Assuntos
Antineoplásicos Hormonais/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/agonistas , Estrogênios/metabolismo , Neoplasias Hormônio-Dependentes/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias da Próstata/metabolismo , Transdução de Sinais , Antineoplásicos Hormonais/uso terapêutico , Área Sob a Curva , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , DNA Complementar/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inquéritos Epidemiológicos , Humanos , Masculino , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/patologia , Nitrilas/farmacologia , Fenóis , Médicos/estatística & dados numéricos , Reação em Cadeia da Polimerase , Propionatos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Pirazóis/farmacologia , Serina Endopeptidases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suécia/epidemiologia , TransfecçãoRESUMO
The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the alpha and beta subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGalphabeta) was used to investigate roles of critical residues of the alpha subunit in hormone receptor interaction and biological activity. The alpha subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type beta subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCGalpha P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCGalpha T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the alpha subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormone-receptor interactions.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Mutação/genética , Anticorpos Monoclonais , Gonadotropina Coriônica Humana Subunidade beta/análise , Fermentação , Subunidade alfa de Hormônios Glicoproteicos/análise , Subunidade alfa de Hormônios Glicoproteicos/genética , Humanos , Soros Imunes , Proteínas Mutantes/isolamento & purificação , Radioimunoensaio , Receptores da Gonadotropina/metabolismo , Relação Estrutura-AtividadeRESUMO
Microarrays have been used to identify genes involved in cancer progression. We have now developed an algorithm that identifies dysregulated pathways from multiple expression array data sets without a priori definition of gene expression thresholds. Integrative microarray analysis of pathways (IMAP) was done using existing expression array data from localized and metastatic prostate cancer. Comparison of metastatic cancer and localized disease in multiple expression array profiling studies using the IMAP approach yielded a list of about 100 pathways that were significantly dysregulated (P < 0.05) in prostate cancer metastasis. The pathway that showed the most significant dysregulation, HIV-I NEF, was validated at both the transcript level and the protein level by quantitative PCR and immunohistochemical analysis, respectively. Validation by unsupervised analysis on an independent data set using the gene expression signature from the HIV-I NEF pathway verified the accuracy of our method. Our results indicate that this pathway is especially dysregulated in hormone-refractory prostate cancer.