RESUMO
OBJECTIVE: Recently, single nucleotide polymorphisms (SNPs) associated with esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE) were identified; rs10419226 (CRTC1), rs11789015 (BARX1), rs2687201 (FOXP1), rs2178146 (FOXF1), rs3111601 (FOXF1), and rs9936833 (FOXF1). These findings indicate that genetic susceptibility could play a role in the initiation of EAC in BE patients. The aim of this study was to validate the association between these previously identified SNPs and the risk of EAC in an independent and large case-control study. DESIGN: Six SNPs found to be associated with EAC and BE were genotyped by a multiplex SNaPshot analysis in 1071 EAC patients diagnosed and treated in the Netherlands. Allele frequencies were compared to a control group derived from the Rotterdam Study, a population-based prospective cohort study (n = 6206). Logistic regression analysis and meta-analysis were performed to calculate odds ratios (OR). RESULTS: Rs10419226 (CRTC1) showed a significantly increased EAC risk for the minor allele (OR = 1.17, P = 0.001), and rs11789015 (BARX1) showed a significantly decreased risk for the minor allele (OR = 0.85, P = 0.004) in the logistic regression analysis. The meta-analysis of the original GWAS and the current study revealed an improved level of significance for rs10419226 (CRTC1) (OR = 1.18, P = 6.66 × 10(-10)) and rs11789015 (BARX1) (OR = 0.83, P = 1.13 × 10(-8)). CONCLUSIONS: This independent and large Dutch case-control study confirms the association of rs10419226 (CRTC1) and rs11789015 (BARX1) with the risk of EAC. These findings suggest a contribution of the patient genetic make-up to the development of EAC and might contribute to gain more insight in the etiology of this cancer.
RESUMO
BACKGROUND: The molecular basis of the clinical heterogeneity of prostate cancer (PCa) is not well understood. OBJECTIVE: The purpose of our study was to identify and characterize genes in a clinically relevant gene expression signature in a subgroup of primary PCa positive for transmembrane protease, serine 2 (TMPRSS2)-v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG). DESIGN, SETTING, AND PARTICIPANTS: We studied gene expression profiles by unsupervised hierarchical clustering in 48 primary PCas from patients with a long clinical follow-up. Results were correlated with clinical outcome and validated in an independent patient cohort. Selected genes from a defined classifier were tested in vitro for biologic properties. INTERVENTION: Initial treatment of primary tumors was radical prostatectomy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Associations between clinical and histopathologic variables were evaluated by the Pearson χ(2) test, Mann-Whitney U test, or Kruskal-Wallis test, where appropriate. The log-rank test or Breslow method was used for statistical analysis of Kaplan-Meier survival curves. RESULTS AND LIMITATIONS: Most tumors that overexpressed ERG clustered separately from other primary PCas. No differences in any clinical end points between ERG-positive and ERG-negative cancers were detected. Importantly, within the ERG-positive samples, two subgroups were identified, which differed significantly in prostate-specific antigen recurrence-free survival, and cancer-specific and overall survival. From our findings, we defined a gene expression classifier of 36 genes. In a second, completely independent tumor set, the classifier also distinguished ERG-positive subgroups with different clinical outcome. In both patient cohorts, the classifier was not predictive in ERG-negative tumors. Biologic processes regulated by genes in the classifier included cell adhesion and bone remodeling. Tumor growth factor-ß signaling was indicated as the main differing signaling pathway between the two ERG subgroups. In vitro biologic assays of two selected genes from the classifier (inhibin, beta A [INHBA] and cadherin 11, type 2, OB-cadherin (osteoblast) [CDH11]) supported a functional role in PCa progression. Possible multifocality and limited number of PCa samples can be limitations of the study. CONCLUSIONS: The classifier identified can contribute to prediction of tumor progression in ERG-positive primary prostate tumors and might be instrumental in therapy decisions.
Assuntos
Neoplasias da Próstata/genética , Transcriptoma , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Prognóstico , Neoplasias da Próstata/química , Transativadores/análise , Regulador Transcricional ERGRESUMO
ETV1 is overexpressed in a subset of clinical prostate cancers as a fusion transcript with many different partners. However, ETV1 can also be overexpressed as a full-length transcript. Full-length ETV1 protein functions differently from truncated ETV1 produced by fusion genes. In this study we describe the genetic background of full-length ETV1 overexpression and the biological properties of different full-length ETV1 isoforms in prostate cancer. Break-apart FISH showed in five out of six patient samples with overexpression of full-length ETV1 a genomic rearrangement of the gene, indicating frequent translocation. We were able to study the rearrangements in more detail in two tumors. In the first tumor 5'-RACE on cDNA showed linkage of the complete ETV1 transcript to the first exon of a prostate-specific two exon ncRNA gene that maps on chromosome 14 (EST14). This resulted in the expression of both full-length ETV1 transcripts and EST14-ETV1 fusion transcripts. In chromosome spreads of a xenograft derived from the second prostate cancer we observed a complex ETV1 translocation involving a chromosome 7 fragment that harbors ETV1 and fragments of chromosomes 4 and 10. Further studies revealed the overexpression of several different full-length transcripts, giving rise to four protein isoforms with different N-terminal regions. Even the shortest isoform synthesized by full-length ETV1 stimulated in vitro anchorage-independent growth of PNT2C2 prostate cells. This contrasts the lack of activity of even shorter N-truncated ETV1 produced by fusion transcripts. Our findings that in clinical prostate cancer overexpression of full-length ETV1 is due to genomic rearrangements involving different chromosomes and the identification of a shortened biologically active ETV1 isoform are highly relevant for understanding the mechanism of ETV1 function in prostate cancer.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , RNA Neoplásico/análise , Fatores de Transcrição/genética , Translocação Genética , Cromossomos Humanos , Humanos , Masculino , Isoformas de ProteínasRESUMO
In this study, we describe the properties of novel ETV1 fusion genes, encoding N-truncated ETV1 (dETV1), and of full-length ETV1, overexpressed in clinical prostate cancer. We detected overexpression of novel ETV1 fusion genes or of full-length ETV1 in 10% of prostate cancers. Novel ETV1 fusion partners included FOXP1, an EST (EST14), and an endogenous retroviral repeat sequence (HERVK17). Like TMPRSS2, EST14 and HERVK17 were prostate-specific and androgen-regulated expressed. This unique expression pattern of most ETV1 fusion partners seems an important determinant in prostate cancer development. In transient reporter assays, full-length ETV1 was a strong transactivator, whereas dETV1 was not. However, several of the biological properties of dETV1 and full-length ETV1 were identical. On stable overexpression, both induced migration and invasion of immortalized nontumorigenic PNT2C2 prostate epithelial cells. In contrast to dETV1, full-length ETV1 also induced anchorage-independent growth of these cells. PNT2C2 cells stably transfected with dETV1 or full-length ETV1 expression constructs showed small differences in induced expression of target genes. Many genes involved in tumor invasion/metastasis, including uPA/uPAR and MMPs, were up-regulated in both cell types. Integrin beta3 (ITGB3) was clearly up-regulated by full-length ETV1 but much less by dETV1. Based on the present data and on previous findings, a novel concept of the role of dETV1 and of full-length ETV1 overexpression in prostate cancer is proposed.
Assuntos
Proteínas de Ligação a DNA/genética , Fusão Gênica , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Animais , Adesão Celular/genética , Movimento Celular/genética , Proteínas de Ligação a DNA/biossíntese , Etiquetas de Sequências Expressas , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/biossínteseRESUMO
Recently, fusion of ERG to the androgen-regulated, prostate-specific TMPRSS2 gene has been identified as the most frequent genetic alteration in prostate cancer. At low frequency, TMPRSS2-ETV1 and TMPRSS2-ETV4 fusion genes have been described. In this study, we report two novel ETV4 fusion genes in prostate cancer: KLK2-ETV4 and CANT1-ETV4. Both gene fusions have important unique aspects. KLK2 is a well-established androgen-induced and prostate-specific gene. Fusion of KLK2 to ETV4 results in the generation of an additional ETV4 exon, denoted exon 4a. This novel exon delivers an ATG for the longest open reading frame, in this way avoiding translation start in KLK2 exon 1. Although wild-type CANT1 has two alternative first exons (exons 1 and 1a), only exon 1a was detected in CANT1-ETV4 fusion transcripts. We show that CANT1 transcripts starting at exon 1a have an androgen-induced and prostate-specific expression pattern, whereas CANT1 transcripts starting at exon 1 are not prostate specific. So, the two novel ETV4 fusion partners possess as predominant common characteristics androgen-induction and prostate-specific expression.
Assuntos
Proteínas E1A de Adenovirus/genética , Androgênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas/genética , Proteínas E1A de Adenovirus/metabolismo , Animais , Sequência de Bases , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Biológicos , Dados de Sequência Molecular , Neoplasias Hormônio-Dependentes/genética , Nucleotidases/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Especificidade de Órgãos/genética , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Distribuição Tecidual , Calicreínas Teciduais/genética , Calicreínas Teciduais/metabolismoRESUMO
BACKGROUND: Specificity of transgene expression is important for safety during gene therapeutical applications. For prostate cancer, transcriptional targeting has been applied but was hampered by loss of specificity and low activity. We constructed a small chimeric promoter for high and prostate-specific transgene expression from adenoviral vectors. METHODS: A chimeric promoter, composed of the prostate-specific antigen (PSA) enhancer and the rat probasin promoter, was cloned into an adenoviral vector and its activity was compared to vectors containing conventional prostate-specific promoters and the constitutive Cytomegalovirus (CMV) promoter in in vitro and in vivo prostate cancer models. RESULTS: The chimeric PSA-probasin promoter was the most active prostate-specific promoter reaching up to 20% of CMV promoter activity while maintaining prostate-specificity. CONCLUSIONS: The chimeric PSA-probasin promoter is a small promoter that can be utilized in viral vectors for high prostate-specific transgene expression.
Assuntos
Adenocarcinoma/terapia , Terapia Genética/métodos , Regiões Promotoras Genéticas , Neoplasias da Próstata/terapia , Transgenes , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenoviridae/genética , Proteína de Ligação a Androgênios/genética , Animais , Linhagem Celular Tumoral , Citomegalovirus/genética , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Plasmídeos/genética , Antígeno Prostático Específico/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Recombinantes de Fusão/genéticaRESUMO
The PTEN tumor suppressor gene is frequently inactivated in human tumors, including prostate cancer. Based on the Cre/loxP system, we generated a novel mouse prostate cancer model by targeted inactivation of the Pten gene. In this model, Cre recombinase was expressed under the control of the prostate-specific antigen (PSA) promoter. Conditional biallelic and monoallelic Pten knock-out mice were viable and Pten recombination was prostate-specific. Mouse cohorts were systematically characterized at 4 to 5, 7 to 9, and 10 to 14 months. A slightly increased proliferation rate of epithelial cells was observed in all prostate lobes of monoallelic Pten knock-out mice (PSA-Cre;Pten-loxP/+), but minimal pathologic changes were detected. All homozygous knock-out mice (PSA-Cre;Pten-loxP/loxP) showed an increased size of the luminal epithelial cells, large areas of hyperplasia, focal prostate intraepithelial neoplasia lesions and an increased prostate weight at 4 to 5 months. More extensive prostate intraepithelial neoplasia and focal microinvasion occurred at 7 to 9 months; invasive prostate carcinoma was detected in all male PSA-Cre;Pten-loxP/loxP mice at 10 to 14 months. At 15 to 16 months, a rare lymph node metastasis was found. In hyperplastic cells and in tumor cells, the expression of phospho-AKT was up-regulated. In hyperplastic and tumor cells, expression of luminal epithelial cell cytokeratins was up-regulated; tumor cells were negative for basal epithelial cell cytokeratins. Androgen receptor expression remained detectable at all stages of tumor development. The up-regulation of phospho-AKT correlated with an increased proliferation rate of the epithelial cells, but not with a reduced apoptosis.
Assuntos
Apoptose/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Tirosina Fosfatases/genética , Proteínas Supressoras de Tumor/genética , Fatores Etários , Alelos , Animais , Processos de Crescimento Celular/genética , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Masculino , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase , Fosforilação , Hiperplasia Prostática/genética , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Supressoras de Tumor/biossínteseRESUMO
We characterized the specifically androgen-regulated gene (SARG), which is expressed in the androgen receptor (AR) and glucocorticoid receptor (GR) positive cell line lymph node carcinoma of the prostate-1F5 (LNCaP-1F5). SARG mRNA expression can be up-regulated by androgens, but not by glucocorticoids. SARG mRNA expression is high in prostate tissue. SARG is composed of four exons and spans a region of 14.5 kbp on chromosome 1q32.2. Transcripts of 5.5, 3.3 and 2.3 kb are the result of alternative polyadenylation. SARG mRNA splice variants lack exon 2 and vary in length of exon 1. The SARG protein has a length of 601 amino acids and is located in the cytoplasm. By screening the 18 kbp genomic sequence flanking the transcription start site we identified the imperfect direct repeat 5'-TGTGCTaacTGTTCT-3'in intron 1 as an active androgen response element (ARE-SARG+4.6). A 569 bp genomic DNA fragment containing this element functioned as an androgen-specific enhancer in transiently transfected LNCaP-1F5 cells. ARE-SARG+4.6 cooperated with flanking sequences for optimal activity. Inactivation of ARE-SARG+4.6 completely abolished the androgen response of the enhancer. Chromatin immunoprecipitation (ChIP) experiments showed chromatin structural changes of the enhancer in the presence of R1881. ARE-SARG+4.6 was able to bind to the androgen receptor, but not to the glucocorticoid receptor, correlating with its androgen-specific activity in transfections.
Assuntos
Proteínas Nucleares/genética , Elementos de Resposta , Fatores de Transcrição/genética , Processamento Alternativo , Sequência de Aminoácidos , Androgênios/metabolismo , Sequência de Bases , Carcinoma/genética , Cromossomos Humanos Par 1 , Biologia Computacional/métodos , Elementos Facilitadores Genéticos/genética , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Poliadenilação , Neoplasias da Próstata/genética , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Among nuclear receptors, the androgen receptor (AR) is unique in that its ligand-binding domain (LBD) interacts with the FXXLF motif in the N-terminal domain, resembling coactivator LXXLL motifs. We compared AR- and estrogen receptor alpha-LBD interactions of the wild-type AR FXXLF motif and coactivator transcriptional intermediary factor 2 LXXLL motifs and variants of these motifs. Random mutagenesis revealed a key role for the F residues in FXXLF motifs in high-affinity and selective AR LBD interaction. The FXXLF motif in full-length AR and transcriptional intermediary factor 2 LXXLL motifs competed for an overlapping binding site. A computer model of the AR LBD/AR FXXLF complex showed that the bulky F residues are buried in a deep coactivator-binding groove. The corresponding groove in estrogen receptor alpha LBD is considerably shallower, explaining lack of binding of any of the FXXLF motifs tested. FXXLF and LXXLL motif interaction depended on different charged amino acid residues in the AR LBD present at opposite ends of the coactivator groove. In conclusion, our data demonstrate the importance of a deep hydrophobic groove and alternative usage of charged amino acids in specifying peptide binding to the AR LBD.
Assuntos
Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Fatores de Transcrição/química , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sítios de Ligação , Ligação Competitiva , Análise Mutacional de DNA , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Coativador 2 de Receptor Nuclear , Peptídeos/química , Conformação Proteica , Receptores Androgênicos/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
PTEN is a tumour suppressor gene involved in cell cycle control, apoptosis and mediation of adhesion and migration signalling. Germline mutations of PTEN in humans are associated with familial tumour syndromes, among them Cowden disease. Glioblastomas, highly malignant glial tumours of the central nervous system frequently show loss of PTEN. Recent reports have outlined some aspects of PTEN function in central nervous system development. Using a conditional gene disruption approach, we inactivated Pten in mice early during embryogenesis locally in a region specific fashion and later during postnatal development in a cell-specific manner, to study the role of PTEN in differentiation, migration and neoplastic transformation. We show that PTEN is required for the realisation of normal cerebellar architecture, for regulation of cell and organ size, and for proper neuronal and glial migration. However, PTEN is not required for cell differentiation and lack of PTEN is not sufficient to induce neoplastic transformation of neuronal or glial cells