RESUMO
RNA variants that emerge from editing and alternative splicing form important regulatory stages in protein signalling. In this report, we apply an integrated DNA and RNA variant detection workbench to define the range of RNA variants that deviate from the reference genome in a human melanoma cell model. The RNA variants can be grouped into (i) classic ADAR-like or APOBEC-like RNA editing events and (ii) multiple-nucleotide variants (MNVs) including three and six base pair in-frame non-canonical unmapped exons. We focus on validating representative genes of these classes. First, clustered non-synonymous RNA edits (A-I) in the CDK13 gene were validated by Sanger sequencing to confirm the integrity of the RNA variant detection workbench. Second, a highly conserved RNA variant in the MAP4K5 gene was detected that results most likely from the splicing of a non-canonical three-base exon. The two RNA variants produced from the MAP4K5 locus deviate from the genomic reference sequence and produce V569E or V569del isoform variants. Low doses of splicing inhibitors demonstrated that the MAP4K5-V569E variant emerges from an SF3B1-dependent splicing event. Mass spectrometry of the recombinant SBP-tagged MAP4K5V569E and MAP4K5V569del proteins pull-downs in transfected cell systems was used to identify the protein-protein interactions of these two MAP4K5 isoforms and propose possible functions. Together these data highlight the utility of this integrated DNA and RNA variant detection platform to detect RNA variants in cancer cells and support future analysis of RNA variant detection in cancer tissue.
Assuntos
Processamento Alternativo , DNA/genética , Éxons , Proteínas Serina-Treonina Quinases/genética , RNA/genética , Humanos , Isoenzimas , Edição de RNARESUMO
Colorectal cancer (CRC) is characterized by major inter-tumor diversity that complicates the prediction of disease and treatment outcomes. Recent efforts help resolve this by sub-classification of CRC into natural molecular subtypes; however, this strategy is not yet able to provide clinicians with improved tools for decision making. We here present an extended framework for CRC stratification that specifically aims to improve patient prognostication. Using transcriptional profiles from 1,100 CRCs, including >300 previously unpublished samples, we identify cancer cell and tumor archetypes and suggest the tumor microenvironment as a major prognostic determinant that can be influenced by the microbiome. Notably, our subtyping strategy allowed identification of archetype-specific prognostic biomarkers that provided information beyond and independent of UICC-TNM staging, MSI status, and consensus molecular subtyping. The results illustrate that our extended subtyping framework, combining subtyping and subtype-specific biomarkers, could contribute to improved patient prognostication and may form a strong basis for future studies.
Assuntos
Biomarcadores Tumorais/classificação , Neoplasias Colorretais/genética , Transcriptoma , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Neoplasias Colorretais/classificação , Neoplasias Colorretais/patologia , Humanos , Microbiota , Microambiente TumoralRESUMO
The cis-regulatory effects responsible for cancer development have not been as extensively studied as the perturbations of the protein coding genome in tumorigenesis. To better characterize colorectal cancer (CRC) development we conducted an RNA-sequencing experiment of 103 matched tumour and normal colon mucosa samples from Danish CRC patients, 90 of which were germline-genotyped. By investigating allele-specific expression (ASE) we show that the germline genotypes remain important determinants of allelic gene expression in tumours. Using the changes in ASE in matched pairs of samples we discover 71 genes with excess of somatic cis-regulatory effects in CRC, suggesting a cancer driver role. We correlate genotypes and gene expression to identify expression quantitative trait loci (eQTLs) and find 1,693 and 948 eQTLs in normal samples and tumours, respectively. We estimate that 36% of the tumour eQTLs are exclusive to CRC and show that this specificity is partially driven by increased expression of specific transcription factors and changes in methylation patterns. We show that tumour-specific eQTLs are more enriched for low CRC genome-wide association study (GWAS) P values than shared eQTLs, which suggests that some of the GWAS variants are tumour specific regulatory variants. Importantly, tumour-specific eQTL genes also accumulate more somatic mutations when compared to the shared eQTL genes, raising the possibility that they constitute germline-derived cancer regulatory drivers. Collectively the integration of genome and the transcriptome reveals a substantial number of putative somatic and germline cis-regulatory cancer changes that may have a role in tumorigenesis.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/genética , Sequências Reguladoras de Ácido Nucleico/genética , Alelos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/patologia , Metilação de DNA , Perfilação da Expressão Gênica , Genes Neoplásicos , Estudo de Associação Genômica Ampla , Genótipo , Mutação em Linhagem Germinativa/genética , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Locos de Características Quantitativas/genética , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.
Assuntos
Apoptose/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ensaios de Triagem em Larga Escala , MicroRNAs/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Proliferação de Células , Metilação de DNA/genética , Regulação para Baixo/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Mucosa Intestinal/patologia , Microdissecção e Captura a Laser , Masculino , Camundongos , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Estadiamento de Neoplasias , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos TestesRESUMO
In order to establish a successful infection, it is of crucial importance for invading viruses to alter the activities of the regulatory protein p53. Beta-herpesviruses stabilize p53 and likely direct its activities towards generation of a replication-friendly environment. We here study the mechanisms behind HHV-6B-induced stabilization and inactivation of p53. Stable transgene expression of the HHV-6B protein U19 was sufficient to achieve upregulation of p53. U19 bound directly to the p53-regulating protein HDM2 in vitro, co-precipitated together with HDM2 in lysates, and co-localized with HDM2 in the nucleus when overexpressed. U19 contained a sequence with a putative p53 BOX I-motif for HDM2 binding. Mutation of the two key amino acids within this motif was sufficient to inhibit all the described U19 functions. Our study provides further insight into p53-modulating strategies used by herpesviruses and elucidates a mechanism used by HHV-6B to circumvent the antiviral response.
Assuntos
Herpesvirus Humano 6/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Infecções por Roseolovirus/metabolismo , Transativadores/química , Transativadores/metabolismo , Proteína Supressora de Tumor p53/química , Proteínas Virais/química , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Herpesvirus Humano 6/química , Herpesvirus Humano 6/genética , Humanos , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/genética , Infecções por Roseolovirus/genética , Infecções por Roseolovirus/virologia , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/genéticaRESUMO
Keratin 23 (KRT23) is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression in vitro. Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed that KRT23 depletion affected molecules of the cell cycle and DNA replication, recombination and repair. In vitro analyses confirmed that KRT23 depletion significantly decreased the cellular proliferation of SW948 and LS1034 cells and markedly decreased the expression of genes involved in DNA damage response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced proliferation of the KRT23 depleted cells compared to irradiated control cells.
Assuntos
Proliferação de Células , Reparo do DNA/genética , Queratinas Tipo I/genética , Interferência de RNA , Sequência de Bases , Western Blotting , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dano ao DNA , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Raios gama , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Células HEK293 , Humanos , Queratinas Tipo I/metabolismo , Proteína Homóloga a MRE11 , Microscopia de Fluorescência , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/genéticaRESUMO
Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein.
Assuntos
Herpesvirus Humano 6/genética , Interações Hospedeiro-Patógeno , Proteína Supressora de Tumor p53/genética , Proteínas Virais/genética , Morte Celular/efeitos da radiação , Dano ao DNA , Raios gama , Regulação da Expressão Gênica/efeitos da radiação , Células HCT116 , Herpesvirus Humano 6/metabolismo , Humanos , Análise em Microsséries , Transdução de Sinais/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/metabolismoRESUMO
Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan(®) Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR-362-3p, miR-570, miR-148 a* and miR-944) were expressed at a higher level in tumors from patients with no recurrence (p<0.015), compared with tumors from patients with recurrence. A significant association with increased disease free survival was confirmed for miR-362-3p in a second independent cohort of 43 CRC patients, using single TaqMan(®) microRNA assays. In vitro functional analysis showed that over-expression of miR-362-3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR-362-3p targets by mRNA profiling of HCT116 cells over-expressing miR-362-3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR-362-3p over-expression. We conclude that miR-362-3p may be a novel prognostic marker in CRC, and hypothesize that the positive effects of augmented miR-362-3p expression may in part be mediated through the targets E2F1, USF2 and PTPN1.
Assuntos
Biomarcadores Tumorais/metabolismo , Pontos de Checagem do Ciclo Celular , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Fator de Transcrição E2F1/metabolismo , MicroRNAs/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Fatores Estimuladores Upstream/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Proliferação de Células , Sobrevivência Celular , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/genética , Fator de Transcrição E2F1/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Prognóstico , Modelos de Riscos Proporcionais , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Recidiva , Regulação para Cima , Fatores Estimuladores Upstream/genéticaRESUMO
Gene silencing by DNA hypermethylation of CpG islands is a well-characterized phenomenon in cancer. The effect of hypomethylation in particular of non-CpG island genes is much less well described. By genome-wide screening, we identified 105 genes in microsatellite stable (MSS) colorectal adenocarcinomas with an inverse correlation (Spearman's ρ ≤ -0.40) between methylation and expression. Of these, 35 (33%) were hypomethylated non-CpG island genes and two of them, APOLD1 (Spearman's ρ = -0.82) and SRPX2 (Spearman's ρ = -0.80) were selected for further analyses. Hypomethylation of both genes were localized events not shared by adjacent genes. A set of 662 FFPE DNA samples not only confirmed that APOLD1 and SRPX2 are hypomethylated in CRC but also revealed hypomethylation to be significantly (p < 0.01) associated with tumors being localized in the left side, CpG island methylator phenotype negative, MSS, BRAF wt, undifferentiated and of adenocarcinoma histosubtype. Demethylation experiments supported SRPX2 being epigenetically regulated via DNA methylation, whereas other mechanisms in addition to DNA methylation seem to be involved in the regulation of APOLD1. We further identified miR-149 as a potential novel post-transcriptional regulator of SRPX2. In carcinoma tissue, miR-149 was downregulated and inversely correlated to SRPX2 (ρ = -0.77). Furthermore, ectopic expression of miR-149 significantly reduced SRPX2 transcript levels. Our study highlights that in colorectal tumors, hypomethylation of non-CpG island-associated promoters deregulate gene expression nearly as frequent as do CpG-island hypermethylation. The hypomethylation of SRPX2 is focal and not part of a large block. Furthermore, it often translates to an increased expression level, which may be modulated by miR-149.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Metilação de DNA , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Adenoma/genética , Apolipoproteínas/metabolismo , Ilhas de CpG , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana , Instabilidade de Microssatélites , Proteínas de Neoplasias , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica , TranscriptomaRESUMO
BACKGROUND: Approximately half of all human genes use alternative transcription start sites (TSSs) to control mRNA levels and broaden the transcriptional output in healthy tissues. Aberrant expression patterns promoting carcinogenesis, however, may arise from alternative promoter usage. RESULTS: By profiling 108 colorectal samples using exon arrays, we identified nine genes (TCF12, OSBPL1A, TRAK1, ANK3, CHEK1, UGP2, LMO7, ACSL5, and SCIN) showing tumor-specific alternative TSS usage in both adenoma and cancer samples relative to normal mucosa. Analysis of independent exon array data sets corroborated these findings. Additionally, we confirmed the observed patterns for selected mRNAs using quantitative real-time reverse-transcription PCR. Interestingly, for some of the genes, the tumor-specific TSS usage was not restricted to colorectal cancer. A comprehensive survey of the nine genes in lung, bladder, liver, prostate, gastric, and brain cancer revealed significantly altered mRNA isoform ratios for CHEK1, OSBPL1A, and TCF12 in a subset of these cancer types.To identify the mechanism responsible for the shift in alternative TSS usage, we antagonized the Wnt-signaling pathway in DLD1 and Ls174T colorectal cancer cell lines, which remarkably led to a shift in the preferred TSS for both OSBPL1A and TRAK1. This indicated a regulatory role of the Wnt pathway in selecting TSS, possibly also involving TP53 and SOX9, as their transcription binding sites were enriched in the promoters of the tumor preferred isoforms together with their mRNA levels being increased in tumor samples. Finally, to evaluate the prognostic impact of the altered TSS usage, immunohistochemistry was used to show deregulation of the total protein levels of both TCF12 and OSBPL1A, corresponding to the mRNA levels observed. Furthermore, the level of nuclear TCF12 had a significant correlation to progression free survival in a cohort of 248 stage II colorectal cancer samples. CONCLUSIONS: Alternative TSS usage in colorectal adenoma and cancer samples has been shown for nine genes, and OSBPL1A and TRAK1 were found to be regulated in vitro by Wnt signaling. TCF12 protein expression was upregulated in cancer samples and correlated with progression free survival.
Assuntos
Neoplasias Colorretais/genética , Éxons , Sítio de Iniciação de Transcrição , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Processamento Alternativo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Quinase 1 do Ponto de Checagem , Estudos de Coortes , Neoplasias Colorretais/patologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Mensageiro/metabolismo , Receptores de Esteroides , Via de Sinalização WntRESUMO
BACKGROUND: Patterns of genome-wide methylation vary between tissue types. For example, cancer tissue shows markedly different patterns from those of normal tissue. In this paper we propose a beta-mixture model to describe genome-wide methylation patterns based on probe data from methylation microarrays. The model takes dependencies between neighbour probe pairs into account and assumes three broad categories of methylation, low, medium and high. The model is described by 37 parameters, which reduces the dimensionality of a typical methylation microarray significantly. We used methylation microarray data from 42 colon cancer samples to assess the model. RESULTS: Based on data from colon cancer samples we show that our model captures genome-wide characteristics of methylation patterns. We estimate the parameters of the model and show that they vary between different tissue types. Further, for each methylation probe the posterior probability of a methylation state (low, medium or high) is calculated and the probability that the state is correctly predicted is assessed. We demonstrate that the model can be applied to classify cancer tissue types accurately and that the model provides accessible and easily interpretable data summaries. CONCLUSIONS: We have developed a beta-mixture model for methylation microarray data. The model substantially reduces the dimensionality of the data. It can be used for further analysis, such as sample classification or to detect changes in methylation status between different samples and tissues.
Assuntos
Neoplasias do Colo/genética , Metilação de DNA , Modelos Estatísticos , Algoritmos , Ilhas de CpG , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente PrincipalRESUMO
In our study, whole-genome methylation arrays were applied to identify novel genes with tumor specific DNA methylation of promoter CpG islands in pre-malignant and malignant colorectal lesions. Using a combination of Illumina HumanMethylation27 beadchips, Methylation-Sensitive High Resolution Melting (MS-HRM) analysis, and Exon arrays (Affymetrix) the DNA methylation pattern of â¼14,000 genes and their transcript levels were investigated in six normal mucosas, six adenomas and 30 MSI and MSS carcinomas. Sixty eight genes with tumor-specific hypermethylation were identified (p < 0.005). Identified hypermethylated sites were validated in an independent sample set of eight normal mucosas, 12 adenomas, 40 MSS and nine MSI cancer samples. The methylation patterns of 15 selected genes, hypermethylated in adenomas and carcinomas (FLI1, ST6GALNAC5, TWIST1, ADHFE1, JAM2, IRF4, CNRIP1, NRG1 and EYA4), in carcinomas only (ABHD9, AOX1 and RERG), or in MSI but not MSS carcinomas (RAMP2, DSC3 and MLH1) were validated using MS-HRM. Four of these genes (MLH1, AOX1, EYA4 and TWIST1) had previously been reported to be hypermethylated in CRC. Eleven genes, not previously known to be affected by CRC specific hypermethylation, were identified and validated. Inverse correlation to gene expression was observed for six of the 15 genes with Spearman correlation coefficients ranging from -0.39 to -0.60. For six of these genes the altered methylation patterns had a profound transcriptional association, indicating that methylation of these genes may play a direct regulatory role. The hypermethylation changes often occurred already in adenomas, indicating that they may be used as biomarkers for early detection of CRC.
Assuntos
Adenoma/genética , Carcinoma/genética , Neoplasias Colorretais/genética , Ilhas de CpG/genética , Metilação de DNA , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Neuregulina-1/metabolismoRESUMO
Genomic alterations play important roles in colorectal cancer (CRC) carcinogenesis. Here, we aimed to identify and characterize recurrent copy-number alterations (CNAs) associated with clinical outcome of CRC by the use of single nucleotide polymorphism arrays, genomic quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH). Colorectal neoplasia specimens and paired germline samples from 144 patients (40 adenomas and 104 carcinomas) as well as 40 CRC cell lines were investigated. This large dataset revealed frequent loss, including homozygous loss, at chr16p13.2 (from 5.9 to 7.42Mb). The loss was observed in 30% of adenomas and even more frequently in carcinomas, 56%, indicating that the loss define a subset of adenomas with a propensity for invasion. Consistent with this, the loss occurred twice as frequent in villous (40%) as in tubular adenomas (20%). The loss occurred independently of microsatellite stability and could be validated by qPCR in an independent sample cohort (n = 71). In Stage II/III, microsatellite stable (MSS) CRC it was associated with poor recurrence free survival (hazard ratio 2.4; p = 0.02; Multivariate Cox regression analysis). No transcriptional consequences of the losses were observed, and the only gene, A2BP1, located in the region showed no mutations. Correlation with other CNAs was established for chr3p22 in carcinomas and chr20p (inverse) in adenomas. FISH documented the chr16p13.2 region to be involved in complex structural rearrangements that included translocation to chr3p22 in some cases. The findings indicate that structural rearrangements involving chr16p13.2 are very frequent in colorectal neoplasia, often lead to homozygous deletion, and are associated with poor clinical outcome.
Assuntos
Adenoma/genética , Carcinoma , Cromossomos Humanos Par 16 , Neoplasias Colorretais/genética , Variações do Número de Cópias de DNA , Idoso , Carcinoma/genética , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , PrognósticoRESUMO
Alternative splicing is a crucial step in the generation of protein diversity and its misregulation is observed in many human cancer types. By analyzing 143 colorectal samples using exon arrays, SLC39A14, a divalent cation transporter, was identified as being aberrantly spliced in tumor samples. SLC39A14 contains two mutually exclusive exons 4A and 4B and the exon 4A/4B ratio was significantly altered in adenomas (p = 3.6 × 10(-10)) and cancers (p = 9.4 × 10(-11)), independent of microsatellite stability status. The findings were validated in independent exon array data sets and by quantitative real-time reverse-transcription PCR (qRT-PCR). Aberrant Wnt signaling is a hallmark of colorectal tumorigenesis and is characterized by nuclear ß-catenin. Experimental inactivation of Wnt signaling in DLD1 and Ls174T cells by knockdown of ß-catenin or overexpression of dominant negative TCFs (TCF1 and TCF4) altered the 4A/4B ratio, indicating that SLC39A14 splicing is regulated by the Wnt pathway. An altered 4A/4B ratio was also observed in gastric and lung cancer where Wnt signaling is also known to be aberrantly activated. The splicing factor SRSF1 and its regulator, the kinase SRPK1, were found to be deregulated upon Wnt inactivation in colorectal carcinoma cells. SRPK1 was also found up-regulated in both adenoma samples (p = 1.5 × 10(-5)) and cancer samples (p = 5 × 10(-4)). In silico splicing factor binding analysis predicted SRSF1 to bind predominantly to the cancer associated exon 4B, hence, it was hypothesized that SRPK1 activates SRSF1 through phosphorylation, followed by SRSF1 binding to exon 4B and regulation of SLC39A14 splicing. Indeed, siRNA-mediated knockdown of SRPK1 and SRSF1 in DLD1 and SW480 colorectal cancer cells led to a change in the 4A/4B isoform ratio, supporting a role of these factors in the regulation of SLC39A14 splicing. In conclusion, alternative splicing of SLC39A14 was identified in colorectal tumors and found to be regulated by the Wnt pathway, most likely through regulation of SRPK1 and SRSF1.
Assuntos
Processamento Alternativo/genética , Proteínas de Transporte de Cátions/genética , Neoplasias Colorretais/genética , Transdução de Sinais , Proteínas Wnt/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Éxons/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Íntrons/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-ArgininaRESUMO
The tumor suppressor protein p53 is activated by distinct cellular stresses including radiation, hypoxia, type I interferon, and DNA/RNA virus infection. The transactivation domain of p53 contains a phosphorylation site at Ser20 whose modification stabilizes the binding of the transcriptional co-activator p300 and whose mutation in murine transgenics induces B-cell lymphoma. Although the checkpoint kinase CHK2 is implicated in promoting Ser20 site phosphorylation after irradiation, the enzyme that triggers this phosphorylation after DNA viral infection is undefined. Using human herpesvirus 6B (HHV-6B) as a virus that induces Ser20 site phosphorylation of p53 in T-cells, we sought to identify the kinase responsible for this virus-induced p53 modification. The p53 Ser20 kinase was fractionated and purified using cation, anion, and dye-ligand exchange chromatography. Mass spectrometry identified casein kinase 1 (CK1) and vaccinia-related kinase 1 (VRK1) as enzymes that coeluted with virus-induced Ser20 site kinase activity. Immunodepletion of CK1 but not VRK1 removed the kinase activity from the peak fraction, and bacterially expressed CK1 exhibited Ser20 site kinase activity equivalent to that of the virus-induced native CK1. CK1 modified p53 in a docking-dependent manner, which is similar to other known Ser20 site p53 kinases. Low levels of the CK1 inhibitor D4476 selectively inhibited HHV-6B-induced Ser20 site phosphorylation of p53. However, x-ray-induced Ser20 site phosphorylation of p53 was not blocked by D4476. These data highlight a central role for CK1 as the Ser20 site kinase for p53 in DNA virus-infected cells but also suggest that distinct stresses may selectively trigger different protein kinases to modify the transactivation domain of p53 at Ser20.
Assuntos
Caseína Quinase I/fisiologia , Herpesvirus Humano 6/metabolismo , Infecções por Roseolovirus/virologia , Serina/química , Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Antivirais/farmacologia , Caseína Quinase I/química , Catálise , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Fosforilação , Estrutura Terciária de Proteína , Ativação TranscricionalRESUMO
Human herpesvirus 6B (HHV-6B) induces significant accumulation of p53 in both the nucleus and cytoplasm during infection. Activation of p53 by DNA damage is known to induce either growth arrest or apoptosis; nevertheless, HHV-6B-infected cells are arrested in their cell cycle independently of p53, and only a minor fraction of the infected cells undergoes apoptosis. Using pifithrin-alpha, a p53 inhibitor, and p53-null cells, this study showed that infected epithelial cells accumulated viral transcripts and proteins to a significantly higher degree in the absence of active p53. Moreover, HHV-6B-induced cytopathic effects were greatly enhanced in the absence of p53. This suggests that, in epithelial cells, some of the functions of p53 leading to cell-cycle arrest and apoptosis are restrained by HHV-6B infection, whereas other cellular defences, causing inhibition of virus transcription, are partially retained.
Assuntos
Herpesvirus Humano 6/imunologia , Proteína Supressora de Tumor p53/fisiologia , Replicação Viral/imunologia , Benzotiazóis/farmacologia , Linhagem Celular , Efeito Citopatogênico Viral , Inibidores Enzimáticos/farmacologia , Células Epiteliais/virologia , Humanos , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/deficiênciaRESUMO
Human herpesvirus (HHV) 6A induce fusion from without (FFWO), whereas HHV-6B is believed to be ineffective in this process. Here, we demonstrate that HHV-6B induces rapid fusion in both epithelial cells and lymphocytes. The fusion was identified 1 h postinfection, could be inhibited by antibodies to HHV-6B gH and to the cellular receptor CD46, and was dependent on virus titer but independent of de novo protein synthesis and UV inactivation of the virus. Comparisons indicate that HHV-6A is only 10-fold more effective in inducing FFWO than HHV-6B. These data demonstrate that HHV-6B can induce FFWO in epithelial cells and lymphocytes.
Assuntos
Herpesvirus Humano 6/fisiologia , Fusão Celular , Linhagem Celular , Humanos , Proteína Cofatora de Membrana/metabolismo , Ligação Proteica , Proteínas do Envelope Viral/metabolismoRESUMO
BACKGROUND: Various forms of cellular stress can activate the tumour suppressor protein p53, an important regulator of cell cycle arrest, apoptosis, and cellular senescence. Cells infected by human herpesvirus 6B (HHV-6B) accumulate aberrant amounts of p53. OBJECTIVES: The aim of this study was to investigate the role of p53 accumulation in the HHV-6B-induced cell cycle arrest. STUDY DESIGN: The role of p53 was studied using the p53 inhibitor pifithrin-a, and cells genetically deficient in functional p53 by homologous recombination. RESULTS: In response to HHV-6B infection, epithelial cells were arrested in the G1/S phase of the cell cycle concomitant with an aberrant accumulation of p53. However, the known p53-induced mediator of cell cycle arrest, p21, was not upregulated. Approximately 90% of the cells expressed HHV-6B p41, indicative of viral infection. The presence of pifithrin-a, a p53 inhibitor, did not reverse the HHV-6B-induced cell cycle block. In support of this, HHV-6B infection of p53(-/-) cells induced a cell cycle block before S-phase with kinetics similar to or faster than that observed by infection in wt cells. CONCLUSIONS: HHV-6B infection inhibited host cell proliferation concomitantly with p53 accumulation, but importantly the block in cell cycle occurred by a pathway independent of p53.
Assuntos
Proliferação de Células , Herpesvirus Humano 6/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Benzotiazóis/farmacologia , Ciclo Celular , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Proteínas de Ligação a DNA/biossíntese , Humanos , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/biossíntese , Proteínas Virais/biossíntese , Replicação ViralRESUMO
We studied the interactions between human herpesvirus 6B (HHV-6B) and its host cell. Productive infections of T-cell lines led to G1/S- and G2/M-phase arrest in the cell cycle concomitant with an increased level and enhanced DNA-binding activity of p53. More than 70% of HHV-6B-infected cells did not bind annexin V, indicating that the majority of cells were not undergoing apoptosis. HHV-6B infection induced Ser20 and Ser15 phosphorylation on p53, and the latter was inhibited by caffeine, an ataxia telangiectasia mutated kinase inhibitor. Thus, a productive HHV-6B infection suppresses T-cell proliferation concomitant with the phosphorylation and accumulation of p53.