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1.
Genet Med ; 22(10): 1653-1666, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32665703

RESUMO

PURPOSE: We assessed the associations between population-based polygenic risk scores (PRS) for breast (BC) or epithelial ovarian cancer (EOC) with cancer risks for BRCA1 and BRCA2 pathogenic variant carriers. METHODS: Retrospective cohort data on 18,935 BRCA1 and 12,339 BRCA2 female pathogenic variant carriers of European ancestry were available. Three versions of a 313 single-nucleotide polymorphism (SNP) BC PRS were evaluated based on whether they predict overall, estrogen receptor (ER)-negative, or ER-positive BC, and two PRS for overall or high-grade serous EOC. Associations were validated in a prospective cohort. RESULTS: The ER-negative PRS showed the strongest association with BC risk for BRCA1 carriers (hazard ratio [HR] per standard deviation = 1.29 [95% CI 1.25-1.33], P = 3×10-72). For BRCA2, the strongest association was with overall BC PRS (HR = 1.31 [95% CI 1.27-1.36], P = 7×10-50). HR estimates decreased significantly with age and there was evidence for differences in associations by predicted variant effects on protein expression. The HR estimates were smaller than general population estimates. The high-grade serous PRS yielded the strongest associations with EOC risk for BRCA1 (HR = 1.32 [95% CI 1.25-1.40], P = 3×10-22) and BRCA2 (HR = 1.44 [95% CI 1.30-1.60], P = 4×10-12) carriers. The associations in the prospective cohort were similar. CONCLUSION: Population-based PRS are strongly associated with BC and EOC risks for BRCA1/2 carriers and predict substantial absolute risk differences for women at PRS distribution extremes.

2.
Fam Cancer ; 18(4): 381-388, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31435815

RESUMO

The Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) calculates the probability that a woman carries a pathogenic variant in BRCA1 or BRCA2 based on her pedigree and the population frequencies of pathogenic alleles of BRCA1 (0.0006394) and BRCA2 (0.00102) in the United Kingdom (UK). BOADICEA allows the clinician to define the population frequencies of pathogenic alleles of BRCA1 and BRCA2 for other populations but only includes preset values for the Ashkenazy Jewish and Icelandic populations. Among 173 early-onset breast cancer pedigrees in Denmark, BOADICEA discriminated well between carriers and non-carriers of pathogenic variants (area under the receiver operating characteristics curve: 0.81; 95% CI 0.74-0.86) but underestimated the frequency of carriers of pathogenic variants in BRCA1 or BRCA2 as measured by the observed-to-expected ratio (O/E 1.83; 95% CI 1.18-2.84). This reflects findings from older studies of BOADICEA in UK, German, Italian, and Chinese populations, all accounting for the different calibration for different carrier probabilities. To improve the performance of BOADICEA for non-UK populations, we developed a method to derive population frequencies of pathogenic alleles of BRCA1 and BRCA2. Compared to the UK population frequencies, we estimated the Danish population frequencies of pathogenic alleles to be higher for BRCA1 (0.0015; 95% CI 0.00064-0.0034) and lower for BRCA2 (0.00052; 95% CI 0.00018-0.0017) after adjusting for the different calibration of BOADICEA for different carrier probabilities. Incorporating additional population frequencies into BOADICEA could improve its performance for non-UK populations.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Frequência do Gene , Modelos Genéticos , Adulto , Idade de Início , Algoritmos , Neoplasias da Mama/epidemiologia , Calibragem , Dinamarca , Feminino , Heterozigoto , Humanos , Incidência , Linhagem , Reino Unido/epidemiologia
3.
NPJ Genom Med ; 2: 36, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29263845

RESUMO

The functions and biomarker potential of circular RNAs (circRNAs) in various cancer types are a rising field of study, as emerging evidence relates circRNAs to tumorigenesis. Here, we profiled the expression of circRNAs in 457 tumors from patients with non-muscle-invasive bladder cancer (NMIBC). We show that a set of highly expressed circRNAs have conserved core splice sites, are associated with Alu repeats, and enriched with Synonymous Constraint Elements as well as microRNA target sites. We identified 113 abundant circRNAs that are differentially expressed between high and low-risk tumor subtypes. Analysis of progression-free survival revealed 13 circRNAs, among them circHIPK3 and circCDYL, where expression correlated with progression independently of the linear transcript and the host gene. In summary, our results demonstrate that abundant circRNAs possess multiple biological features, distinguishing them from low-expressed circRNAs and non-circularized exons, and suggest that circRNAs might serve as a new class of prognostic biomarkers in NMIBC.

4.
Sci Rep ; 7(1): 395, 2017 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-28341852

RESUMO

Aberrant expression of long non-coding RNAs (lncRNAs) has been regarded as a critical component in bladder cancer (BC) and lncRNAs have been associated with BC development and progression although their overall expression and functional significance is still unclear. The aim of our study was to identify novel lncRNAs with a functional role in BC carcinogenesis. RNA-sequencing was used to identify aberrantly expressed lncRNAs in 8 normal and 72 BC samples. We identified 89 lncRNAs that were significantly dys-regulated in BC. Five lncRNAs; LINC00958, LINC01296, LINC00355, LNC-CMC1-1 and LNC-ALX1-2 were selected for further analyses. Silencing of LINC00958 or LINC01296 in vitro reduced both cell viability and migration. Knock-down of LINC00958 also affected invasion and resistance to anoikis. These cellular effects could be linked to direct/indirect regulation of protein coding mRNAs involved in cell death/survival, proliferation and cellular movement. Finally, we showed that LINC00958 binds proteins involved in regulation and initiation of translation and in post-transcriptional modification of RNA, including Metadherin, which has previously been associated with BC. Our analyses identified novel lncRNAs in BC that likely act as oncogenic drivers contributing to an aggressive cancerous phenotype likely through interaction with proteins involved in initiation of translation and/or post-transcriptional modification of RNA.


Assuntos
Regulação Neoplásica da Expressão Gênica , Oncogenes , RNA Longo não Codificante/genética , Neoplasias da Bexiga Urinária/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Perfilação da Expressão Gênica , Humanos , Transcriptoma , Regulação para Cima
5.
Nat Commun ; 7: 13875, 2016 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-28004750

RESUMO

We currently have limited knowledge of the involvement of long non-coding RNAs (lncRNAs) in normal cellular processes and pathologies. Here, we identify and characterize SNHG5 as a stable cytoplasmic lncRNA with up-regulated expression in colorectal cancer. Depletion of SNHG5 induces cell cycle arrest and apoptosis in vitro and limits tumour outgrowth in vivo, whereas SNHG5 overexpression counteracts oxaliplatin-induced apoptosis. Using an unbiased approach, we identify 121 transcript sites interacting with SNHG5 in the cytoplasm. Importantly, knockdown of key SNHG5 target transcripts, including SPATS2, induces apoptosis and thus mimics the effect seen following SNHG5 depletion. Mechanistically, we suggest that SNHG5 stabilizes the target transcripts by blocking their degradation by STAU1. Accordingly, depletion of STAU1 rescues the apoptosis induced after SNHG5 knockdown. Hence, we characterize SNHG5 as a lncRNA promoting tumour cell survival in colorectal cancer and delineate a novel mechanism in which a cytoplasmic lncRNA functions through blocking the action of STAU1.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas do Citoesqueleto/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Apoptose , Células CACO-2 , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Neoplasias Colorretais/genética , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Técnicas de Silenciamento de Genes , Células HCT116 , Células HT29 , Humanos , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas/metabolismo , Estabilidade de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Regulação para Cima
6.
J Extracell Vesicles ; 5: 31488, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27576678

RESUMO

Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. These contain biomolecules, including proteins and microRNAs (miRNAs). Both circulating EVs and miRNAs have received much attention as biomarker candidates for non-invasive diagnostics. Here we describe a sensitive analytical method for isolation and subsequent miRNA profiling of epithelial-derived EVs from blood samples of patients with colorectal cancer (CRC). The epithelial-derived EVs were isolated by immunoaffinity-capture using the epithelial cell adhesion molecule (EpCAM) as marker. This approach mitigates some of the specificity issues observed in earlier studies of circulating miRNAs, in particular the negative influence of miRNAs released by erythrocytes, platelets and non-epithelial cells. By applying this method to 2 small-scale patient cohorts, we showed that blood plasma isolated from CRC patients prior to surgery contained elevated levels of 13 EpCAM(+)-EV miRNAs compared with healthy individuals. Upon surgical tumour removal, the plasma levels of 8 of these were reduced (miR-16-5p, miR-23a-3p, miR-23b-3p, miR-27a-3p, miR-27b-3p, miR-30b-5p, miR-30c-5p and miR-222-3p). These findings indicate that the miRNAs are of tumour origin and may have potential as non-invasive biomarkers for detection of CRC. This work describes a non-invasive blood-based method for sensitive detection of cancer with potential for clinical use in relation to diagnosis and screening. We used the method to study CRC; however, it is not restricted to this disease. It may in principle be used to study any cancer that release epithelial-derived EVs into circulation.

7.
Mol Oncol ; 10(8): 1266-82, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27396952

RESUMO

It is well established that lncRNAs are aberrantly expressed in cancer where they have been shown to act as oncogenes or tumor suppressors. RNA profiling of 314 colorectal adenomas/adenocarcinomas and 292 adjacent normal colon mucosa samples using RNA-sequencing demonstrated that the snoRNA host gene 16 (SNHG16) is significantly up-regulated in adenomas and all stages of CRC. SNHG16 expression was positively correlated to the expression of Wnt-regulated transcription factors, including ASCL2, ETS2, and c-Myc. In vitro abrogation of Wnt signaling in CRC cells reduced the expression of SNHG16 indicating that SNHG16 is regulated by the Wnt pathway. Silencing of SNHG16 resulted in reduced viability, increased apoptotic cell death and impaired cell migration. The SNHG16 silencing particularly affected expression of genes involved in lipid metabolism. A connection between SNHG16 and genes involved in lipid metabolism was also observed in clinical tumors. Argonaute CrossLinking and ImmunoPrecipitation (AGO-CLIP) demonstrated that SNHG16 heavily binds AGO and has 27 AGO/miRNA target sites along its length, indicating that SNHG16 may act as a competing endogenous RNA (ceRNA) "sponging" miRNAs off their cognate targets. Most interestingly, half of the miRNA families with high confidence targets on SNHG16 also target the 3'UTR of Stearoyl-CoA Desaturase (SCD). SCD is involved in lipid metabolism and is down-regulated upon SNHG16 silencing. In conclusion, up-regulation of SNHG16 is a frequent event in CRC, likely caused by deregulated Wnt signaling. In vitro analyses demonstrate that SNHG16 may play an oncogenic role in CRC and that it affects genes involved in lipid metabolism, possible through ceRNA related mechanisms.


Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Metabolismo dos Lipídeos/genética , RNA Longo não Codificante/metabolismo , Via de Sinalização Wnt/genética , Apoptose/genética , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Neoplasias Colorretais/patologia , Citoplasma/metabolismo , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Motivos de Nucleotídeos/genética , Polirribossomos/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/genética , RNA Nucleolar Pequeno/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima/genética
8.
Cell ; 158(6): 1281-1292, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-25215487

RESUMO

A dichotomous choice for metazoan cells is between proliferation and differentiation. Measuring tRNA pools in various cell types, we found two distinct subsets, one that is induced in proliferating cells, and repressed otherwise, and another with the opposite signature. Correspondingly, we found that genes serving cell-autonomous functions and genes involved in multicellularity obey distinct codon usage. Proliferation-induced and differentiation-induced tRNAs often carry anticodons that correspond to the codons enriched among the cell-autonomous and the multicellularity genes, respectively. Because mRNAs of cell-autonomous genes are induced in proliferation and cancer in particular, the concomitant induction of their codon-enriched tRNAs suggests coordination between transcription and translation. Histone modifications indeed change similarly in the vicinity of cell-autonomous genes and their corresponding tRNAs, and in multicellularity genes and their tRNAs, suggesting the existence of transcriptional programs coordinating tRNA supply and demand. Hence, we describe the existence of two distinct translation programs that operate during proliferation and differentiation.


Assuntos
Diferenciação Celular , Proliferação de Células , Biossíntese de Proteínas , RNA de Transferência/genética , Anticódon , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Códon , Histonas/metabolismo , Humanos , Neoplasias/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Transcriptoma
9.
PLoS One ; 9(6): e96767, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24892549

RESUMO

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 Testes
10.
Int J Cancer ; 133(1): 67-78, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23280316

RESUMO

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/genética
11.
Int J Cancer ; 132(10): 2303-15, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23115050

RESUMO

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 , Instabilidade de Microssatélites , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Genética , Transcriptoma
12.
Fam Cancer ; 8(4): 489-500, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19697156

RESUMO

Recently, we have performed a population based study to analyse the frequency of colorectal cancer related MLH1 and MSH2 missense mutations in the Danish population. Half of the analyzed mutations were rare and most likely only present in the families where they were identified originally. Some of the missense mutations were located in conserved regions in the MLH1 and MSH2 proteins indicating a relation to disease development. In the present study, we functionally characterized 10 rare missense mutations in MLH1 and MSH2 identified in 13 Danish CRC families. To elucidate the pathogenicity of the missense mutations, we carried out in vitro functional analyses. The missense mutations were analyzed for their effect on protein expression and repair efficiency. The results of the functional analysis were correlated with clinical data on the families carrying these mutations. Eight missense mutations resulted in proteins with expression and repair efficiency similar to the wild type. One missense mutation (MSH2 p.Met688Val) caused reduced protein expression and one (MSH2 p.Leu187Arg) caused both reduced protein expression and repair deficiency. The MSH2 p.Leu187Arg mutation was found in an Amsterdam II family presenting with high microsatellite instability and loss of MSH2 and MSH6 proteins in tumours. In conclusion, only 1/10 missense mutations displayed repair deficiency and could be classified as pathogenic. No final conclusion can be drawn on the MSH2 p.Met688Val mutation, which caused reduced protein expression. Although, no deficiencies have been identified in the proteins harbouring the other missense mutations, pathogenicity of these variants cannot be unambiguously excluded.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/genética , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Adulto , Western Blotting , Dinamarca , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Linhagem
13.
BMC Med Genet ; 9: 52, 2008 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-18547406

RESUMO

BACKGROUND: Mutations in the mismatch repair genes hMLH1 and hMSH2 predispose to hereditary non-polyposis colorectal cancer (HNPCC). Genetic screening of more than 350 Danish patients with colorectal cancer (CRC) has led to the identification of several new genetic variants (e.g. missense, silent and non-coding) in hMLH1 and hMSH2. The aim of the present study was to investigate the frequency of these variants in hMLH1 and hMSH2 in Danish patients with sporadic colorectal cancer and in the healthy background population. The purpose was to reveal if any of the common variants lead to increased susceptibility to colorectal cancer. METHODS: Associations between genetic variants in hMLH1 and hMSH2 and sporadic colorectal cancer were evaluated using a case-cohort design. The genotyping was performed on DNA isolated from blood from the 380 cases with sporadic colorectal cancer and a sub-cohort of 770 individuals. The DNA samples were analyzed using Single Base Extension (SBE) Tag-arrays. A Bonferroni corrected Fisher exact test was used to test for association between the genotypes of each variant and colorectal cancer. Linkage disequilibrium (LD) was investigated using HaploView (v3.31). RESULTS: Heterozygous and homozygous changes were detected in 13 of 35 analyzed variants. Two variants showed a borderline association with colorectal cancer, whereas the remaining variants demonstrated no association. Furthermore, the genomic regions covering hMLH1 and hMSH2 displayed high linkage disequilibrium in the Danish population. Twenty-two variants were neither detected in the cases with sporadic colorectal cancer nor in the sub-cohort. Some of these rare variants have been classified either as pathogenic mutations or as neutral variants in other populations and some are unclassified Danish variants. CONCLUSION: None of the variants in hMLH1 and hMSH2 analyzed in the present study were highly associated with colorectal cancer in the Danish population. High linkage disequilibrium in the genomic regions covering hMLH1 and hMSH2, indicate that common genetic variants in the two genes in general are not involved in the development of sporadic colorectal cancer. Nevertheless, some of the rare unclassified variants in hMLH1 and hMSH2 might be involved in the development of colorectal cancer in the families where they were originally identified.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/genética , Variação Genética , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Estudos de Coortes , Neoplasias Colorretais Hereditárias sem Polipose/genética , Dinamarca , Feminino , Frequência do Gene , Genótipo , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Mutação de Sentido Incorreto , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
14.
Mol Oncol ; 1(2): 181-95, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19383294

RESUMO

Transcript profiling of 27 normal colon mucosas and 258 adenocarcinomas showed Keratin23 to be increased in 78% microsatellite-stable tumors, while microsatellite-instable tumors showed low transcript levels, comparable to normal mucosas. Immunohistochemical analyses demonstrated that 88% of microsatellite-instable tumors were negative for Keratin23 protein, while 70% of MSS tumors and metastases derived from MSS-tumors showed high Keratin23 levels. Immunofluorescence analysis localized Keratin23 in the Golgi-apparatus. Golgi accumulation was unique for gastrointestinal adenocarcinomas. Immunoprecipitation and 2D-blot analysis revealed Keratin23 to be a 46.8 kDa phosphoprotein. Keratin23 impaired the proliferation of human colon cancer cells significantly, leading to cell death in microsatellite-instable but not microsatellite-stable cell lines, while COS7 cells experienced multiple nuclei and apoptosis. Keratin23 expression correlated significantly with transcription factor CEBPB. In conclusion, Keratin23 expression is a novel and important difference between microsatellite-stable and microsatellite-instable colon cancers.


Assuntos
Adenocarcinoma/metabolismo , Proliferação de Células , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Queratinas Tipo I/biossíntese , Instabilidade de Microssatélites , Proteínas de Neoplasias/biossíntese , Fosfoproteínas/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Apoptose/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células COS , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Sobrevivência Celular/genética , Chlorocebus aethiops , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Perfilação da Expressão Gênica , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Queratinas Tipo I/genética , Masculino , Transcrição Genética/genética , Células Tumorais Cultivadas
15.
Cancer Res ; 65(1): 34-45, 2005 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-15665277

RESUMO

Bladder cancer is a common disease characterized by multiple recurrences and an invasive disease course in more than 10% of patients. It is of monoclonal or oligoclonal origin and genomic instability has been shown at certain loci. We used a 10,000 single nucleotide polymorphism (SNP) array with an average of 2,700 heterozygous SNPs to detect allelic imbalances (AI) in 37 microdissected bladder tumors from 17 patients. Eight tumors represented upstaging from Ta to T1, eight from T1 to T2+, and one from Ta to T2+. The AI was strongly stage-dependent as four chromosomal arms showed AI in > 50% of Ta samples, eight in T1, and twenty-two in T2+ samples. The tumors showed stage-dependent clonality as 61.3% of AIs were reconfirmed in later T1 tumors and 84.4% in muscle-invasive tumors. Novel unstable chromosomal areas were identified at chromosomes 6q, 10p, 16q, 20p, 20q, and 22q. The tumors separated into two distinct groups, highly stable tumors (all Ta tumors) and unstable tumors (2/3 muscle-invasive). All 11 unstable tumors had lost chromosome 17p areas and 90% chromosome 8 areas affecting Netrin-1/UNC5D/MAP2K4 genes as well as others. AI was present at the TP53 locus in 10 out of 11 unstable tumors, whereas 6 had homozygous TP53 mutations. Tumor distribution pattern reflected AI as seven out of eight patients with additional upper urinary tract tumors had genomic stable bladder tumors (P < 0.05). These data show the power of high-resolution SNP arrays for defining clinically relevant AIs.


Assuntos
Desequilíbrio Alélico/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único/genética , Neoplasias da Bexiga Urinária/genética , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Éxons , Regulação Neoplásica da Expressão Gênica , Heterozigoto , Homozigoto , Humanos , Repetições de Microssatélites , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Proteína Supressora de Tumor p53/genética , Neoplasias da Bexiga Urinária/patologia
16.
Mol Cell Proteomics ; 4(4): 534-44, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15671042

RESUMO

We initiated the present study to identify new genes associated with colorectal cancer. In a previously published microarray study an EST (W80763), later identified as the gene hFKBP10 (NM_021939), was found to be strongly expressed in tumors while absent in the normal mucosa. Here we describe this gene hFKBP10 together with its encoded protein hFKBP65 as a novel marker associated with colorectal cancer. Analysis of 31 colorectal adenocarcinomas and 14 normal colorectal mucosa by RealTime PCR for hFKBP10 showed a significant up-regulation in tumors, when compared with normal mucosa. Immunohistochemical analysis of 26 adenocarcinomas and matching normal mucosa, as well as benign hyperplastic polyps and adenomas, using a monoclonal anti-hFKBP65 antibody, showed that the protein was not present in normal colorectal epithelial cells, but strongly expressed in the tumor cells of colorectal cancer. The protein was also expressed in fibroblasts of both normal mucosa and tumor tissue. Western blot analysis of matched tumors and normal mucosa supported the finding of increased hFKBP65 expression in tumors compared with normal mucosa, in addition to identifying the molecular mass of hFKBP65 to approximately 72 kDa. Cellular localization and glycosylation studies revealed the hFKBP65 protein to be localized in the endoplasmic reticulum, and to be N-glycosylated. In conclusion, the protein hFKBP65 is associated with colorectal cancer, and we hypothesize the protein to be involved in fibroblast and transformed epithelial cell-specific protein synthesis in the endoplasmic reticulum.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Adenocarcinoma/patologia , Adenoma/patologia , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Western Blotting , Células COS , Chlorocebus aethiops , Pólipos do Colo/patologia , Neoplasias Colorretais/patologia , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hiperplasia , Imuno-Histoquímica , Mucosa Intestinal/citologia , Ponto Isoelétrico , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas de Ligação a Tacrolimo/análise , Proteínas de Ligação a Tacrolimo/química , Transfecção
17.
Cancer Res ; 62(15): 4352-63, 2002 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12154040

RESUMO

Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each pool) of total RNA from left-sided sporadic colorectal carcinomas. We compared normal tissue to carcinoma tissue from Dukes' stages A-D (noninvasive to distant metastasis) and identified 908 known genes and 4,155 ESTs that changed remarkably from normal to tumor tissue. Based on intensive filtering 226 known genes and 157 ESTs were found to be highly relevant for CRC. The alteration of known genes was confirmed in >70% of the cases by array analysis of 25 single samples. Two-way hierarchical average linkage cluster analysis clustered normal tissue together with Dukes' A, clustered Dukes' B with Dukes' C, and clustered Dukes' D separately. Real-time PCR of 10 known genes and 5 ESTs demonstrated excellent reproducibility of the array-based findings. The most frequently altered genes belonged to functional categories of metabolism (22%), transcription and translation (11%), and cellular processes (9%). Fifteen nuclear encoded mitochondrial proteins were all down-regulated in CRC. We identified several chromosomal locations with clusters of either potential oncogenes or potential tumor suppressors. Some of these, such as aminopeptidase N/CD13 and sigma B3 protein on chromosome 15q25, coincided with a high frequency of loss of heterozygosity. The genes and ESTs presented in this study encode new potential tumor markers as well as potential novel therapeutic targets for prevention or therapy of CRC.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 6/genética , Análise por Conglomerados , Neoplasias Colorretais/patologia , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Ligação Genética , Humanos , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Oncogenes
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