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1.
Urol Oncol ; 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32057596

RESUMO

PURPOSE: Following radical nephroureterectomy for upper urinary tract urothelial carcinoma (UTUC), intravesical recurrence (IVR) is found in 22% to 47% of patients. Patients with a primary urothelial carcinoma of the bladder (UCB) have an increased risk of a future UTUC (1%-5%). Paired UTUC and UCB might represent clonally related tumors due to intraluminal seeding of tumor cells or might be separate entities of urothelial carcinoma caused by field cancerization. We systematically reviewed all the relevant literature to address the possible clonal relation of UTUC and paired UCB. MATERIALS AND METHODS: MEDLINE, EMBASE, and COCHRANE databases were systematically searched for relevant citations published between January 2000 and July 2019. This study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Of 5038 citations identified, 86 full papers were screened, and 9 studies met the inclusion criteria. RESULTS: The populations studied and the molecular techniques used to assess clonality of UTUC and paired UCB differed largely over time. Eight studies reported on primary UTUC and meta- or synchronous IVR without a history of UCB. A total of 118 tumors (55 UTUC and 63 IVR) from 49 patients were included, of which 94% seemed to be clonally related. Five studies reported on primary UCB and subsequent UTUC with a total of 61 tumors (30 UCB and 31 UTUC) from 14 patients; a possible clonal origin was identified for 85% of the tumors. CONCLUSION: Taking into account the limitations of microsatellite technology in comparison to Next Generation Sequencing and currently accepted concepts of tumor heterogeneity and evolution, this systematic review shows that most, if not all, UTUC and paired UCB likely are clonally related.

2.
Nat Commun ; 10(1): 5251, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31748536

RESUMO

Metastatic castration-resistant prostate cancer (mCRPC) has a highly complex genomic landscape. With the recent development of novel treatments, accurate stratification strategies are needed. Here we present the whole-genome sequencing (WGS) analysis of fresh-frozen metastatic biopsies from 197 mCRPC patients. Using unsupervised clustering based on genomic features, we define eight distinct genomic clusters. We observe potentially clinically relevant genotypes, including microsatellite instability (MSI), homologous recombination deficiency (HRD) enriched with genomic deletions and BRCA2 aberrations, a tandem duplication genotype associated with CDK12-/- and a chromothripsis-enriched subgroup. Our data suggests that stratification on WGS characteristics may improve identification of MSI, CDK12-/- and HRD patients. From WGS and ChIP-seq data, we show the potential relevance of recurrent alterations in non-coding regions identified with WGS and highlight the central role of AR signaling in tumor progression. These data underline the potential value of using WGS to accurately stratify mCRPC patients into clinically actionable subgroups.

3.
Nat Genet ; 51(10): 1450-1458, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570896

RESUMO

The whole-genome sequencing of prospectively collected tissue biopsies from 442 patients with metastatic breast cancer reveals that, compared to primary breast cancer, tumor mutational burden doubles, the relative contributions of mutational signatures shift and the mutation frequency of six known driver genes increases in metastatic breast cancer. Significant associations with pretreatment are also observed. The contribution of mutational signature 17 is significantly enriched in patients pretreated with fluorouracil, taxanes, platinum and/or eribulin, whereas the de novo mutational signature I identified in this study is significantly associated with pretreatment containing platinum-based chemotherapy. Clinically relevant subgroups of tumors are identified, exhibiting either homologous recombination deficiency (13%), high tumor mutational burden (11%) or specific alterations (24%) linked to sensitivity to FDA-approved drugs. This study provides insights into the biology of metastatic breast cancer and identifies clinically useful genomic features for the future improvement of patient management.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Neoplasias da Mama/genética , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/genética , Mutação , Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Estudos de Coortes , Feminino , Genômica , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Prognóstico
4.
Cell Rep ; 28(10): 2704-2714.e5, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484079

RESUMO

The human ether-a-go-go-related gene KCNH2 encodes the voltage-gated potassium channel underlying IKr, a current critical for the repolarization phase of the cardiac action potential. Mutations in KCNH2 that cause a reduction of the repolarizing current can result in cardiac arrhythmias associated with long-QT syndrome. Here, we investigate the regulation of KCNH2 and identify multiple active enhancers. A transcribed enhancer ∼85 kbp downstream of Kcnh2 physically contacts the promoters of two Kcnh2 isoforms in a cardiac-specific manner in vivo. Knockdown of its ncRNA transcript results in reduced expression of Kcnh2b and two neighboring mRNAs, Nos3 and Abcb8, in vitro. Genomic deletion of the enhancer, including the ncRNA transcription start site, from the mouse genome causes a modest downregulation of both Kcnh2a and Kcnh2b in the ventricles. These findings establish that the regulation of Kcnh2a and Kcnh2b is governed by a complex regulatory landscape that involves multiple partially redundantly acting enhancers.

5.
Invest Ophthalmol Vis Sci ; 60(10): 3595-3605, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31425584

RESUMO

Purpose: Uveal melanoma (UM) is characterized by multiple chromosomal rearrangements and recurrent mutated genes. The aim of this study was to investigate if copy number variations (CNV) alone and in combination with other genetic and clinico-histopathological variables can be used to stratify for disease-free survival (DFS) in enucleated patients with UM. Methods: We analyzed single nucleotide polymorphisms (SNP) array data of primary tumors and other clinical variables of 214 UM patients from the Rotterdam Ocular Melanoma Study (ROMS) cohort. Nonweighted hierarchical clustering of SNP array data was used to identify molecular subclasses with distinct CNV patterns. The subclasses associate with mutational status of BAP1, SF3B1, or EIF1AX. Cox proportional hazard models were then used to study the predictive performance of SNP array cluster-, mutation-, and clinico-histopathological data, and their combination for study endpoint risk. Results: Five clusters with distinct CNV patterns and concomitant mutations in BAP1, SF3B1, or EIF1AX were identified. The sample's cluster allocation contributed significantly to mutational status of samples in predicting the incidence of metastasis during a median of 45.6 (interquartile range [IQR]: 24.7-81.8) months of follow-up (P < 0.05) and vice versa. Furthermore, incorporating all data sources in one model yielded a 0.797 C-score during 100 months of follow-up. Conclusions: UM has distinct CNV patterns that correspond to different mutated driver genes. Incorporating clinico-histopathological, cluster and mutation data in the analysis results in good performance for UM-related DFS prediction.


Assuntos
Fator de Iniciação 1 em Eucariotos/genética , Enucleação Ocular , Melanoma/genética , Melanoma/cirurgia , Fosfoproteínas/genética , Polimorfismo de Nucleotídeo Único , Fatores de Processamento de RNA/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Neoplasias Uveais/genética , Neoplasias Uveais/cirurgia , Idoso , Variações do Número de Cópias de DNA , DNA de Neoplasias/genética , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Melanoma/diagnóstico , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Neoplasias Uveais/diagnóstico
7.
PLoS One ; 14(3): e0208659, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30921348

RESUMO

Krüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the KLF1 Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the KLF1 Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the 782 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We propose that reduced expression of XPO7 is partially responsible for the erythroid defects observed in KLF1 Nan erythroid cells.


Assuntos
Anemia Hemolítica/genética , Células Eritroides/citologia , Fatores de Transcrição Kruppel-Like/genética , Proteína ran de Ligação ao GTP/genética , Substituição de Aminoácidos , Animais , Diferenciação Celular , Células Cultivadas , Cromatina/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Células Eritroides/metabolismo , Eritropoese , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Análise de Sequência de RNA/métodos , Proteína ran de Ligação ao GTP/metabolismo
8.
Br J Cancer ; 120(4): 444-452, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30739914

RESUMO

BACKGROUND: Testicular germ cell cancer (TGCC), being the most frequent malignancy in young Caucasian males, is initiated from an embryonic germ cell. This study determines intratumour heterogeneity to unravel tumour progression from initiation until metastasis. METHODS: In total, 42 purified samples of four treatment-resistant nonseminomatous (NS) TGCC were investigated, including the precursor germ cell neoplasia in situ (GCNIS) and metastatic specimens, using whole-genome and targeted sequencing. Their evolution was reconstructed. RESULTS: Intratumour molecular heterogeneity did not correspond to the supposed primary tumour histological evolution. Metastases after systemic treatment could be derived from cancer stem cells not identified in the primary cancer. GCNIS mostly lacked the molecular marks of the primary NS and comprised dominant clones that failed to progress. A BRCA-like mutational signature was observed without evidence for direct involvement of BRCA1 and BRCA2 genes. CONCLUSIONS: Our data strongly support the hypothesis that NS is initiated by whole-genome duplication, followed by chromosome copy number alterations in the cancer stem cell population, and accumulation of low numbers of somatic mutations, even in therapy-resistant cases. These observations of heterogeneity at all stages of tumourigenesis should be considered when treating patients with GCNIS-only disease, or with clinically overt NS.


Assuntos
Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Testiculares/genética , Evolução Molecular , Genes BRCA1 , Genes BRCA2 , Humanos , Perda de Heterozigosidade , Masculino , Mutação , Metástase Neoplásica , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Testiculares/patologia , Sequenciamento Completo do Genoma
9.
Mol Cancer Res ; 17(2): 521-531, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30257989

RESUMO

Aberrant activation of Wnt/ß-catenin signaling plays a key role in the onset and development of hepatocellular carcinomas (HCC), with about half of them acquiring mutations in either CTNNB1 or AXIN1. The serine/threonine kinase receptor-associated protein (STRAP), a scaffold protein, was recently shown to facilitate the aberrant activation of Wnt/ß-catenin signaling in colorectal cancers. However, the function of STRAP in HCC remains completely unknown. Here, increased levels of STRAP were observed in human and mouse HCCs. RNA sequencing of STRAP knockout clones generated by gene editing of Huh6 and Huh7 HCC cells revealed a significant reduction in expression of various metabolic and cell-cycle-related transcripts, in line with their general slower growth observed during culture. Importantly, Wnt/ß-catenin signaling was impaired in all STRAP knockout/down cell lines tested, regardless of the underlying CTNNB1 or AXIN1 mutation. In accordance with ß-catenin's role in (cancer) stem cell maintenance, the expressions of various stem cell markers, such as AXIN2 and LGR5, were reduced and concomitantly differentiation-associated genes were increased. Together, these results show that the increased STRAP protein levels observed in HCC provide growth advantage among others by enhancing Wnt/ß-catenin signaling. These observations also identify STRAP as a new player in regulating ß-catenin signaling in hepatocellular cancers. IMPLICATIONS: Elevated STRAP levels in hepatocellular cancers provide a growth advantage by enhancing Wnt/ß-catenin signaling.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/genética
10.
Cell Rep ; 24(9): 2312-2328.e7, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30157426

RESUMO

IBD syndromes such as Crohn's disease and ulcerative colitis result from the inflammation of specific intestinal segments. Although many studies have reported on the regenerative response of intestinal progenitor and stem cells to tissue injury, very little is known about the response of differentiated lineages to inflammatory cues. Here, we show that acute inflammation of the mouse small intestine is followed by a dramatic loss of Lgr5+ stem cells. Instead, Paneth cells re-enter the cell cycle, lose their secretory expression signature, and acquire stem-like properties, thus contributing to the tissue regenerative response to inflammation. Stem cell factor secretion upon inflammation triggers signaling through the c-Kit receptor and a cascade of downstream events culminating in GSK3ß inhibition and Wnt activation in Paneth cells. Hence, the plasticity of the intestinal epithelium in response to inflammation goes well beyond stem and progenitor cells and extends to the fully differentiated and post-mitotic Paneth cells.


Assuntos
Inflamação/metabolismo , Intestino Delgado/fisiopatologia , Regeneração Nervosa/fisiologia , Celulas de Paneth/metabolismo , Animais , Diferenciação Celular , Modelos Animais de Doenças , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais
11.
Clin Cancer Res ; 24(24): 6277-6287, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30139880

RESUMO

PURPOSE: Tumors of germline BRCA1/2 mutated carriers show homologous recombination (HR) deficiency (HRD), resulting in impaired DNA double-strand break (DSB) repair and high sensitivity to PARP inhibitors. Although this therapy is expected to be effective beyond germline BRCA1/2 mutated carriers, a robust validated test to detect HRD tumors is lacking. In this study, we therefore evaluated a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh breast cancer tissue: the recombination REpair CAPacity (RECAP) test. EXPERIMENTAL DESIGN: Fresh samples of 170 primary breast cancer were analyzed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by exploring BRCA deficiencies, mutational signatures, tumor-infiltrating lymphocytes (TIL), and microsatellite instability (MSI). RESULTS: RECAP was completed successfully in 148 of 170 samples (87%). Twenty-four tumors showed HRD (16%), whereas six tumors were HR intermediate (HRi; 4%). HRD was explained by BRCA deficiencies (mutations, promoter hypermethylation, deletions) in 16 cases, whereas seven HRD tumors were non-BRCA related. HRD tumors showed an increased incidence of high TIL counts (P = 0.023) compared with HR proficient (HRP) tumors and MSI was more frequently observed in the HRD group (2/20, 10%) than expected in breast cancer (1%; P = 0.017). CONCLUSIONS: RECAP is a robust functional HR assay detecting both BRCA1/2-deficient and BRCA1/2-proficient HRD tumors. Functional assessment of HR in a pseudo-diagnostic setting is achievable and produces robust and interpretable results.

13.
BMC Bioinformatics ; 19(1): 236, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29929481

RESUMO

BACKGROUND: Current normalization methods for RNA-sequencing data allow either for intersample comparison to identify differentially expressed (DE) genes or for intrasample comparison for the discovery and validation of gene signatures. Most studies on optimization of normalization methods typically use simulated data to validate methodologies. We describe a new method, GeTMM, which allows for both inter- and intrasample analyses with the same normalized data set. We used actual (i.e. not simulated) RNA-seq data from 263 colon cancers (no biological replicates) and used the same read count data to compare GeTMM with the most commonly used normalization methods (i.e. TMM (used by edgeR), RLE (used by DESeq2) and TPM) with respect to distributions, effect of RNA quality, subtype-classification, recurrence score, recall of DE genes and correlation to RT-qPCR data. RESULTS: We observed a clear benefit for GeTMM and TPM with regard to intrasample comparison while GeTMM performed similar to TMM and RLE normalized data in intersample comparisons. Regarding DE genes, recall was found comparable among the normalization methods, while GeTMM showed the lowest number of false-positive DE genes. Remarkably, we observed limited detrimental effects in samples with low RNA quality. CONCLUSIONS: We show that GeTMM outperforms established methods with regard to intrasample comparison while performing equivalent with regard to intersample normalization using the same normalized data. These combined properties enhance the general usefulness of RNA-seq but also the comparability to the many array-based gene expression data in the public domain.


Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Humanos
14.
Nat Protoc ; 13(3): 459-477, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29419817

RESUMO

Chromosome conformation capture (3C) and its derivatives (e.g., 4C, 5C and Hi-C) are used to analyze the 3D organization of genomes. We recently developed targeted chromatin capture (T2C), an inexpensive method for studying the 3D organization of genomes, interactomes and structural changes associated with gene regulation, the cell cycle, and cell survival and development. Here, we present the protocol for T2C based on capture, describing all experimental steps and bio-informatic tools in full detail. T2C offers high resolution, a large dynamic interaction frequency range and a high signal-to-noise ratio. Its resolution is determined by the resulting fragment size of the chosen restriction enzyme, which can lead to sub-kilobase-pair resolution. T2C's high coverage allows the identification of the interactome of each individual DNA fragment, which makes binning of reads (often used in other methods) basically unnecessary. Notably, T2C requires low sequencing efforts. T2C also allows multiplexing of samples for the direct comparison of multiple samples. It can be used to study topologically associating domains (TADs), determining their position, shape, boundaries, and intra- and inter-domain interactions, as well as the composition of aggregated loops, interactions between nucleosomes, individual transcription factor binding sites, and promoters and enhancers. T2C can be performed by any investigator with basic skills in molecular biology techniques in ∼7-8 d. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments.


Assuntos
Biologia Computacional/métodos , Mapeamento Físico do Cromossomo/métodos , Análise de Sequência de DNA/métodos , Animais , Cromatina/ultraestrutura , Montagem e Desmontagem da Cromatina/fisiologia , Mapeamento Cromossômico/métodos , DNA , Regulação da Expressão Gênica , Genoma/genética , Genoma Humano/genética , Genoma Humano/fisiologia , Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Nucleossomos , Software
15.
J Mol Diagn ; 20(2): 166-176, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29305224

RESUMO

Exploration and visualization of next-generation sequencing data are crucial for clinical diagnostics. Software allowing simultaneous visualization of multiple regions of interest coupled with dynamic heuristic filtering of genetic aberrations is, however, lacking. Therefore, the authors developed the web application SNPitty that allows interactive visualization and interrogation of variant call format files by using B-allele frequencies of single-nucleotide polymorphisms and single-nucleotide variants, coverage metrics, and copy numbers analysis results. SNPitty displays variant alleles and allelic imbalances with a focus on loss of heterozygosity and copy number variation using genome-wide heterozygous markers and somatic mutations. In addition, SNPitty is capable of generating predefined reports that summarize and highlight disease-specific targets of interest. SNPitty was validated for diagnostic interpretation of somatic events by showcasing a serial dilution series of glioma tissue. Additionally, SNPitty is demonstrated in four cancer-related scenarios encountered in daily clinical practice and on whole-exome sequencing data of peripheral blood from a Down syndrome patient. SNPitty allows detection of loss of heterozygosity, chromosomal and gene amplifications, homozygous or heterozygous deletions, somatic mutations, or any combination thereof in regions or genes of interest. Furthermore, SNPitty can be used to distinguish molecular relationships between multiple tumors from a single patient. On the basis of these data, the authors demonstrate that SNPitty is robust and user friendly in a wide range of diagnostic scenarios.


Assuntos
Tomada de Decisão Clínica/métodos , Visualização de Dados , Frequência do Gene/genética , Sequenciamento de Nucleotídeos em Larga Escala , Internet , Navegador , Algoritmos , Alelos , Composição de Bases , Biomarcadores Tumorais , Variações do Número de Cópias de DNA , Dosagem de Genes , Humanos , Perda de Heterozigosidade , Neoplasias/diagnóstico , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Exoma
16.
Cell Rep ; 20(1): 61-75, 2017 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-28683324

RESUMO

The Nucleosome Remodeling and Deacetylase (NURD) complex is a key regulator of cell differentiation that has also been implicated in tumorigenesis. Loss of the NURD subunit Deleted in Oral Cancer 1 (DOC1) is associated with human oral squamous cell carcinomas (OSCCs). Here, we show that restoration of DOC1 expression in OSCC cells leads to a reversal of epithelial-mesenchymal transition (EMT). This is caused by the DOC1-dependent targeting of NURD to repress key transcriptional regulators of EMT. NURD recruitment drives extensive epigenetic reprogramming, including eviction of the SWI/SNF remodeler, formation of inaccessible chromatin, H3K27 deacetylation, and binding of PRC2 and KDM1A, followed by H3K27 methylation and H3K4 demethylation. Strikingly, depletion of SWI/SNF mimics the effects of DOC1 re-expression. Our results suggest that SWI/SNF and NURD function antagonistically to control chromatin state and transcription. We propose that disturbance of this dynamic equilibrium may lead to defects in gene expression that promote oncogenesis.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Transição Epitelial-Mesenquimal , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Neoplasias Bucais/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Células Cultivadas , Montagem e Desmontagem da Cromatina , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Metilação , Neoplasias Bucais/genética , Processamento de Proteína Pós-Traducional
17.
Genome Res ; 27(6): 922-933, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28341771

RESUMO

The spatial arrangement of chromatin is linked to the regulation of nuclear processes. One striking aspect of nuclear organization is the spatial segregation of heterochromatic and euchromatic domains. The mechanisms of this chromatin segregation are still poorly understood. In this work, we investigated the link between the primary genomic sequence and chromatin domains. We analyzed the spatial intranuclear arrangement of a human artificial chromosome (HAC) in a xenospecific mouse background in comparison to an orthologous region of native mouse chromosome. The two orthologous regions include segments that can be assigned to three major chromatin classes according to their gene abundance and repeat repertoire: (1) gene-rich and SINE-rich euchromatin; (2) gene-poor and LINE/LTR-rich heterochromatin; and (3) gene-depleted and satellite DNA-containing constitutive heterochromatin. We show, using fluorescence in situ hybridization (FISH) and 4C-seq technologies, that chromatin segments ranging from 0.6 to 3 Mb cluster with segments of the same chromatin class. As a consequence, the chromatin segments acquire corresponding positions in the nucleus irrespective of their chromosomal context, thereby strongly suggesting that this is their autonomous property. Interactions with the nuclear lamina, although largely retained in the HAC, reveal less autonomy. Taken together, our results suggest that building of a functional nucleus is largely a self-organizing process based on mutual recognition of chromosome segments belonging to the major chromatin classes.


Assuntos
Núcleo Celular/genética , Cromossomos Artificiais Humanos/metabolismo , Eucromatina/metabolismo , Fibroblastos/metabolismo , Heterocromatina/metabolismo , Retina/metabolismo , Animais , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cromossomos Artificiais Humanos/ultraestrutura , Eucromatina/classificação , Eucromatina/ultraestrutura , Fibroblastos/ultraestrutura , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Heterocromatina/classificação , Heterocromatina/ultraestrutura , Humanos , Hibridização in Situ Fluorescente , Camundongos , Cultura Primária de Células , Retina/ultraestrutura
18.
PLoS One ; 12(3): e0174039, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28319171

RESUMO

BACKGROUND: Neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (MIBC) provides a small but significant survival benefit. Nevertheless, controversies on applying NAC remain because the limited benefit must be weight against chemotherapy-related toxicity and the delay of definitive local treatment. Therefore, there is a clear clinical need for tools to guide treatment decisions on NAC in MIBC. Here, we aimed to validate a previously reported 20-gene expression signature that predicted lymph node-positive disease at radical cystectomy in clinically node-negative MIBC patients, which would be a justification for upfront chemotherapy. METHODS: We studied diagnostic transurethral resection of bladder tumors (dTURBT) of 150 MIBC patients (urothelial carcinoma) who were subsequently treated by radical cystectomy and pelvic lymph node dissection. RNA was isolated and the expression level of the 20 genes was determined on a qRT-PCR platform. Normalized Ct values were used to calculate a risk score to predict the presence of node-positive disease. The Cancer Genome Atlas (TCGA) RNA expression data was analyzed to subsequently validate the results. RESULTS: In a univariate regression analysis, none of the 20 genes significantly correlated with node-positive disease. The area under the curve of the risk score calculated by the 20-gene expression signature was 0.54 (95% Confidence Interval: 0.44-0.65) versus 0.67 for the model published by Smith et al. Node-negative patients had a significantly lower tumor grade at TURBT (p = 0.03), a lower pT stage (p<0.01) and less frequent lymphovascular invasion (13% versus 38%, p<0.01) at radical cystectomy than node-positive patients. In addition, in the TCGA data, none of the 20 genes was differentially expressed in node-negative versus node-positive patients. CONCLUSIONS: We conclude that a 20-gene expression signature developed for nodal staging of MIBC at radical cystectomy could not be validated on a qRT-PCR platform in a large cohort of dTURBT specimens.


Assuntos
Carcinoma/genética , Carcinoma/patologia , Cistectomia , Metástase Linfática/diagnóstico , Transcriptoma , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma/metabolismo , Carcinoma/cirurgia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Excisão de Linfonodo , Masculino , Pessoa de Meia-Idade , Neoplasias Musculares/metabolismo , Neoplasias Musculares/patologia , Gradação de Tumores , Estadiamento de Neoplasias , Estudos Retrospectivos , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/cirurgia
20.
Artigo em Inglês | MEDLINE | ID: mdl-28035242

RESUMO

BACKGROUND: The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function-the storage, expression, and replication of genetic information-is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture (T2C), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence. RESULTS: The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. CONCLUSIONS: This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow "architectural sequencing" at a genome mechanics level to understand the inseparable systems genomic properties.

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