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BACKGROUND & AIMS: Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation. METHODS: Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, in vitro, and in vivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues. RESULTS: Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases. CONCLUSIONS: Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site.
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Neoplasias del Colon , Neoplasias Hepáticas , Neoplasias Pulmonares , Ratones , Animales , Humanos , gamma Catenina/metabolismo , Neoplasias Pulmonares/patología , Neoplasias del Colon/genética , Neoplasias Hepáticas/patologíaRESUMEN
A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity.
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Neoplasias Colorrectales , Neoplasias Primarias Secundarias , Neoplasias Peritoneales , Neoplasias Colorrectales/patología , Humanos , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/secundario , Peritoneo/metabolismo , Calidad de VidaRESUMEN
Histopathological studies have revealed key processes of atherosclerotic plaque thrombosis. However, the diversity and complexity of lesion types highlight the need for improved sub-phenotyping. Here we analyze the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These plaque phenotypes were associated with clinical presentation and showed differences in cellular compositions. Validation in coronary segments showed that the molecular signature of these plaques was linked to coronary ischemia. One of the plaque types with the most severe clinical symptoms pointed to both inflammatory and fibrotic cell lineages. Further, we did a preliminary analysis of potential circulating biomarkers that mark the different plaques phenotypes. In conclusion, the definition of the plaque at risk for a thrombotic event can be fine-tuned by in-depth transcriptomic-based phenotyping. These differential plaque phenotypes prove clinically relevant for both carotid and coronary artery plaques and point to distinct underlying biology of symptomatic lesions.
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Evasion of apoptosis is a hallmark of cancer, which is frequently mediated by upregulation of the antiapoptotic BCL-2 family proteins. In colorectal cancer (CRC), previous work has highlighted differential antiapoptotic protein dependencies determined by the stage of the disease. While intestinal stem cells (ISCs) require BCL-2 for adenoma outgrowth and survival during transformation, ISC-specific MCL1 deletion results in disturbed intestinal homeostasis, eventually contributing to tumorigenesis. Colon cancer stem cells (CSCs), however, no longer require BCL-2 and depend mainly on BCL-XL for their survival. We therefore hypothesized that a shift in antiapoptotic protein reliance occurs in ISCs as the disease progresses from normal to adenoma to carcinoma. By targeting antiapoptotic proteins with specific BH3 mimetics in organoid models of CRC progression, we found that BCL-2 is essential only during ISC transformation while MCL1 inhibition did not affect adenoma outgrowth. BCL-XL, on the other hand, was crucial for stem cell survival throughout the adenoma-to-carcinoma sequence. Furthermore, we identified that the limited window of BCL-2 reliance is a result of its downregulation by miR-17-5p, a microRNA that is upregulated upon APC-mutation driven transformation. Here we show that BCL-XL inhibition effectively impairs adenoma outgrowth in vivo and enhances the efficacy of chemotherapy. In line with this dependency, expression of BCL-XL, but not BCL-2 or MCL1, directly correlated to the outcome of chemotherapy-treated CRC patients. Our results provide insights to enable the rational use of BH3 mimetics in CRC management, particularly underlining the therapeutic potential of BCL-XL targeting mimetics in both early and late-stage disease.
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Adenoma/genética , Neoplasias Colorrectales/genética , Proteína bcl-X/genética , Adenoma/mortalidad , Adenoma/patología , Animales , Apoptosis , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Análisis de SupervivenciaRESUMEN
Survival rates of cancer patients vary widely within and between malignancies. While genetic aberrations are at the root of all cancers, individual genomic features cannot explain these distinct disease outcomes. In contrast, intra-tumour heterogeneity (ITH) has the potential to elucidate pan-cancer survival rates and the biology that drives cancer prognosis. Unfortunately, a comprehensive and effective framework to measure ITH across cancers is missing. Here, we introduce a scalable measure of chromosomal copy number heterogeneity (CNH) that predicts patient survival across cancers. We show that the level of ITH can be derived from a single-sample copy number profile. Using gene-expression data and live cell imaging we demonstrate that ongoing chromosomal instability underlies the observed heterogeneity. Analysing 11,534 primary cancer samples from 37 different malignancies, we find that copy number heterogeneity can be accurately deduced and predicts cancer survival across tissues of origin and stages of disease. Our results provide a unifying molecular explanation for the different survival rates observed between cancer types.
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Variaciones en el Número de Copia de ADN , Heterogeneidad Genética , Modelos Genéticos , Neoplasias/mortalidad , Microambiente Tumoral/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Simulación por Computador , Conjuntos de Datos como Asunto , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genómica , Humanos , Masculino , Persona de Mediana Edad , Mutación , Neoplasias/genética , Neoplasias/patología , Pronóstico , Supervivencia sin Progresión , Medición de Riesgo/métodos , Tasa de Supervivencia , Adulto JovenRESUMEN
Most human cancers are aneuploid, due to a chromosomal instability (CIN) phenotype. Despite being hallmarks of cancer, however, the roles of CIN and aneuploidy in tumor formation have not unequivocally emerged from animal studies and are thus still unclear. Using a conditional mouse model for diverse degrees of CIN, we find that a particular range is sufficient to drive very early onset spontaneous adenoma formation in the intestine. In mice predisposed to intestinal cancer (ApcMin/+), moderate CIN causes a remarkable increase in adenoma burden in the entire intestinal tract and especially in the distal colon, which resembles human disease. Strikingly, a higher level of CIN promotes adenoma formation in the distal colon even more than moderate CIN does, but has no effect in the small intestine. Our results thus show that CIN can be potently oncogenic, but that certain levels of CIN can have contrasting effects in distinct tissues.
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Carcinogénesis/genética , Inestabilidad Cromosómica , Oncogenes/genética , Adenoma/genética , Aneuploidia , Animales , Proliferación Celular , Segregación Cromosómica , Colon/patología , Modelos Animales de Enfermedad , Femenino , Neoplasias Gastrointestinales/genética , Neoplasias Intestinales/genética , Intestinos/patología , Cariotipo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , OrganoidesRESUMEN
Pyridox(am)ine 5'-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. PNPO deficiency results in neonatal/infantile seizures and neurodevelopmental delay. To gain insight into this disorder we generated Pnpo deficient (pnpo-/-) zebrafish (CRISPR/Cas9 gene editing). Locomotion analysis showed that pnpo-/- zebrafish develop seizures resulting in only 38% of pnpo-/- zebrafish surviving beyond 20â¯days post fertilization (dpf). The age of seizure onset varied and survival after the onset was brief. Biochemical profiling at 20 dpf revealed a reduction of PLP and pyridoxal (PL) and accumulation of PMP and pyridoxamine (PM). Amino acids involved in neurotransmission including glutamate, γ-aminobutyric acid (GABA) and glycine were decreased. Concentrations of several, mostly essential, amino acids were increased in pnpo-/- zebrafish suggesting impaired activity of PLP-dependent transaminases involved in their degradation. PLP treatment increased survival at 20 dpf and led to complete normalization of PLP, PL, glutamate, GABA and glycine. However, amino acid profiles only partially normalized and accumulation of PMP and PM persisted. Taken together, our data indicate that not only decreased PLP but also accumulation of PMP may play a role in the clinical phenotype of PNPO deficiency.
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Encefalopatías Metabólicas/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Enfermedades Metabólicas/etiología , Enfermedades Metabólicas/metabolismo , Piridoxaminafosfato Oxidasa/deficiencia , Convulsiones/etiología , Convulsiones/metabolismo , Pez Cebra/metabolismo , Aminoácidos/metabolismo , Animales , Encefalopatías Metabólicas/etiología , Oxidorreductasas/metabolismo , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/metabolismo , Piridoxamina/metabolismo , Piridoxaminafosfato Oxidasa/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
BACKGROUND AND AIMS: Women who develop preeclampsia during pregnancy are at a higher risk for developing cardiovascular disease. As platelets are affected by preeclampsia, we set out to identify whether platelets carry information in their transcriptome on cardiovascular risk in women with former preeclampsia. METHODS: Platelets were isolated from asymptomatic women with previous preeclampsia, who underwent screening with coronary computed tomography angiography. Platelet RNA was isolated and used to construct gene networks using an unbiased approach. Platelet gene modules assembled from the network were related to risk factors and clinical traits of these women, including coronary artery calcium scores (CACS). RESULTS: We found multiple gene modules which correlated with CACS (correlation coefficients: 0.44 to 0.59, pâ¯=â¯0.05 to 0.007). The genes from two clinically relevant modules were expressed at a higher level in the group with calcifications (pâ¯=â¯3.9â¯×â¯10-10 and 0.02) and enriched for platelet-related gene-sets such as platelet activation. The first of these modules was also enriched (ppermutationâ¯=â¯0.0546) for genes mapped to known coronary artery disease susceptibility loci. Additional unbiased network analyses in platelet RNA of patients with overt cardiovascular disease underlined the importance of the identified modules for disease by high preservation. (pâ¯=â¯1.6â¯×â¯10-9 to 1.7â¯×â¯10-47). CONCLUSIONS: We found platelet RNA modules that correlated with CACS in asymptomatic women with previous preeclampsia. Whether or not platelets directly contribute to this disease trajectory, or reflect the underlying plaque substrate remains to be determined, but enrichment for coronary artery disease susceptibility genes emphasizes the importance for the disease.
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Plaquetas/metabolismo , Enfermedad de la Arteria Coronaria/genética , Redes Reguladoras de Genes , Preeclampsia/genética , ARN/genética , Transcriptoma , Calcificación Vascular/genética , Enfermedades Asintomáticas , Coagulación Sanguínea/genética , Estudios de Casos y Controles , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/diagnóstico , Diagnóstico Precoz , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Fenotipo , Activación Plaquetaria/genética , Preeclampsia/sangre , Preeclampsia/diagnóstico , Embarazo , ARN/sangre , Factores de Riesgo , Calcificación Vascular/sangre , Calcificación Vascular/diagnósticoRESUMEN
Vasculature performs a critical function in tissue homeostasis, supply of oxygen and nutrients, and the removal of metabolic waste products. Vascular problems are implicated in a large variety of pathologies and accurate in vitro models resembling native vasculature are of great importance. Unfortunately, existing in vitro models do not sufficiently reflect their in vivo counterpart. The complexity of vasculature requires the examination of multiple cell types including endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), as well as vessel location in the body from which they originate. The use of canine blood vessels provides a way to study vasculature with similar vessel size and physiology compared to human vasculature. We report an isolation procedure that provides the possibility to isolate both the endothelial and smooth muscle cells from the same vessels simultaneously, enabling new opportunities in investigating vasculature behavior. Canine primary ECs and VSMCs were isolated from the vena cava, vena porta and aorta. All tissue sources were derived from three donors for accurate comparison and to reduce inter-animal variation. The isolation and purification of the two distinct cell types was confirmed by morphology, gene- and protein-expression and function. As both cell types can be derived from the same vessel, this approach allows accurate modeling of vascular diseases and can also be used more widely, for example, in vascular bioreactors and tissue engineering designs. Additionally, we identified several new genes that were highly expressed in canine ECs, which may become candidate genes for novel EC markers. In addition, we observed transcriptional and functional differences between arterial- and venous-derived endothelium. Further exploration of the transcriptome and physiology of arteriovenous differentiation of primary cells may have important implications for a better understanding of the fundamental behavior of the vasculature and pathogenesis of vascular disease.
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Genome-wide association studies (GWASs) have identified many genetic risk factors for CKD. However, linking common variants to genes that are causal for CKD etiology remains challenging. By adapting self-transcribing active regulatory region sequencing, we evaluated the effect of genetic variation on DNA regulatory elements (DREs). Variants in linkage with the CKD-associated single-nucleotide polymorphism rs11959928 were shown to affect DRE function, illustrating that genes regulated by DREs colocalizing with CKD-associated variation can be dysregulated and therefore, considered as CKD candidate genes. To identify target genes of these DREs, we used circular chromosome conformation capture (4C) sequencing on glomerular endothelial cells and renal tubular epithelial cells. Our 4C analyses revealed interactions of CKD-associated susceptibility regions with the transcriptional start sites of 304 target genes. Overlap with multiple databases confirmed that many of these target genes are involved in kidney homeostasis. Expression quantitative trait loci analysis revealed that mRNA levels of many target genes are genotype dependent. Pathway analyses showed that target genes were enriched in processes crucial for renal function, identifying dysregulated geranylgeranyl diphosphate biosynthesis as a potential disease mechanism. Overall, our data annotated multiple genes to previously reported CKD-associated single-nucleotide polymorphisms and provided evidence for interaction between these loci and target genes. This pipeline provides a novel technique for hypothesis generation and complements classic GWAS interpretation. Future studies are required to specify the implications of our dataset and further reveal the complex roles that common variants have in complex diseases, such as CKD.
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Cromatina/química , ADN/química , Conformación de Ácido Nucleico , Insuficiencia Renal Crónica/genética , Animales , Vías Biosintéticas/genética , Células Cultivadas , Bases de Datos Genéticas , Células Endoteliales , Predisposición Genética a la Enfermedad/genética , Genotipo , Homeostasis/genética , Humanos , Túbulos Renales , Ratones , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN/métodosRESUMEN
The biogenesis of ribosomes and their coordination of protein translation consume an enormous amount of cellular energy. As such, it has been established that the inhibition of either process can extend eukaryotic lifespan. Here, we used next-generation sequencing to compare ribosome-associated RNAs from normal strains of Caenorhabditis elegans to those carrying the life-extending daf-2 mutation. We found a long noncoding RNA (lncRNA), transcribed telomeric sequence 1 (tts-1), on ribosomes of the daf-2 mutant. Depleting tts-1 in daf-2 mutants increases ribosome levels and significantly shortens their extended lifespan. We find tts-1 is also required for the longer lifespan of the mitochondrial clk-1 mutants but not the feeding-defective eat-2 mutants. In line with this, the clk-1 mutants express more tts-1 and fewer ribosomes than the eat-2 mutants. Our results suggest that the expression of tts-1 functions in different longevity pathways to reduce ribosome levels in a way that promotes life extension.
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Genomic rearrangements are a common cause of human congenital abnormalities. However, their origin and consequences are poorly understood. We performed molecular analysis of two patients with congenital disease who carried de novo genomic rearrangements. We found that the rearrangements in both patients hit genes that are recurrently rearranged in cancer (ETV1, FOXP1, and microRNA cluster C19MC) and drive formation of fusion genes similar to those described in cancer. Subsequent analysis of a large set of 552 de novo germline genomic rearrangements underlying congenital disorders revealed enrichment for genes rearranged in cancer and overlap with somatic cancer breakpoints. Breakpoints of common (inherited) germline structural variations also overlap with cancer breakpoints but are depleted for cancer genes. We propose that the same genomic positions are prone to genomic rearrangements in germline and soma but that timing and context of breakage determines whether developmental defects or cancer are promoted.
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Aberraciones Cromosómicas , Cromosomas Humanos/genética , Anomalías Congénitas/genética , Reordenamiento Génico , Genoma Humano , Mutación de Línea Germinal , Animales , Puntos de Rotura del Cromosoma , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead/genética , Células HEK293 , Humanos , MicroARNs/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Pez CebraRESUMEN
Understanding the complexity of the human brain and its functional diversity remain a major challenge. Distinct anatomical regions are involved in an array of processes, including organismal homeostasis, cognitive functions, and susceptibility to neurological pathologies, many of which define our species. Distal enhancers have emerged as key regulatory elements that acquire histone modifications in a cell- and species-specific manner, thus enforcing specific gene expression programs. Here, we survey the epigenomic landscape of promoters and cis-regulatory elements in 136 regions of the adult human brain. We identify a total of 83,553 promoter-distal H3K27ac-enriched regions showing global characteristics of brain enhancers. We use coregulation of enhancer elements across many distinct regions of the brain to uncover functionally distinct networks at high resolution and link these networks to specific neuroglial functions. Furthermore, we use these data to understand the relevance of noncoding genomic variations previously linked to Parkinson's disease incidence.
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Encéfalo/metabolismo , Elementos de Facilitación Genéticos , Redes Reguladoras de Genes , Enfermedad de Parkinson/genética , Epigénesis Genética , Genoma Humano , Histonas/genética , Histonas/metabolismo , Humanos , Especificidad de Órganos , Enfermedad de Parkinson/metabolismoRESUMEN
Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.
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Ansiedad/genética , Mapeo Cromosómico/métodos , Cardiopatías/genética , Esclerosis Múltiple/genética , Análisis de Secuencia de ADN/métodos , Animales , Animales no Consanguíneos , Variación Genética/genética , Genotipo , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Fenotipo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo/genética , RatasRESUMEN
BACKGROUND: Paired-tag sequencing approaches are commonly used for the analysis of genome structure. However, mammalian genomes have a complex organization with a variety of repetitive elements that complicate comprehensive genome-wide analyses. RESULTS: Here, we systematically assessed the utility of paired-end and mate-pair (MP) next-generation sequencing libraries with insert sizes ranging from 170 bp to 25 kb, for genome coverage and for improving scaffolding of a mammalian genome (Rattus norvegicus). Despite a lower library complexity, large insert MP libraries (20 or 25 kb) provided very high physical genome coverage and were found to efficiently span repeat elements in the genome. Medium-sized (5, 8 or 15 kb) MP libraries were much more efficient for genome structure analysis than the more commonly used shorter insert paired-end and 3 kb MP libraries. Furthermore, the combination of medium- and large insert libraries resulted in a 3-fold increase in N50 in scaffolding processes. Finally, we show that our data can be used to evaluate and improve contig order and orientation in the current rat reference genome assembly. CONCLUSIONS: We conclude that applying combinations of mate-pair libraries with insert sizes that match the distributions of repetitive elements improves contig scaffolding and can contribute to the finishing of draft genomes.
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Biblioteca de Genes , Genoma , Ratas/genética , Análisis de Secuencia de ADN/métodos , Animales , Secuencia de Bases , Mapeo Contig/métodos , Secuencias Repetitivas Esparcidas/genéticaRESUMEN
BACKGROUND: With the advent of next generation sequencing it has become possible to detect genomic variation on a large scale. However, predicting which genomic variants are damaging to gene function remains a challenge, as knowledge of the effects of genomic variation on gene expression is still limited. Recombinant inbred panels are powerful tools to study the cis and trans effects of genetic variation on molecular phenotypes such as gene expression. RESULTS: We generated a comprehensive inventory of genomic differences between the two founder strains of the rat HXB/BXH recombinant inbred panel: SHR/OlaIpcv and BN-Lx/Cub. We identified 3.2 million single nucleotide variants, 425,924 small insertions and deletions, 907 copy number changes and 1,094 large structural genetic variants. RNA-sequencing analyses on liver tissue of the two strains identified 532 differentially expressed genes and 40 alterations in transcript structure. We identified both coding and non-coding variants that correlate with differential expression and alternative splicing. Furthermore, structural variants, in particular gene duplications, show a strong correlation with transcriptome alterations. CONCLUSIONS: We show that the panel is a good model for assessing the genetic basis of phenotypic heterogeneity and for providing insights into possible underlying molecular mechanisms. Our results reveal a high diversity and complexity underlying quantitative and qualitative transcriptional differences.
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Variaciones en el Número de Copia de ADN , Ratas Endogámicas BN/genética , Ratas Endogámicas SHR/genética , Recombinación Genética , Transcriptoma , Animales , Codón de Terminación/genética , Duplicación de Gen , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Técnicas de Genotipaje , Mutación INDEL , Hígado/citología , Modelos Genéticos , Fenotipo , Sitios de Empalme de ARN , Empalme del ARN , Ratas , Análisis de Secuencia de ARN/métodosRESUMEN
PURPOSE: In the era of DNA-guided personalized cancer treatment, it is essential to conduct predictive analysis on the tissue that matters. Here, we analyzed genetic differences between primary colorectal adenocarcinomas (CRC) and their respective hepatic metastasis. EXPERIMENTAL DESIGN: The primary CRC and the subsequent hepatic metastasis of 21 patients with CRC were analyzed using targeted deep-sequencing of DNA isolated from formalin-fixed, paraffin-embedded archived material. RESULTS: We have interrogated the genetic constitution of a designed "Cancer Mini-Genome" consisting of all exons of 1,264 genes associated with pathways relevant to cancer. In total, 6,696 known and 1,305 novel variations were identified in 1,174 and 667 genes, respectively, including 817 variants that potentially altered protein function. On average, 83 (SD = 69) potentially function-impairing variations were gained in the metastasis and 70 (SD = 48) variations were lost, showing that the primary tumor and hepatic metastasis are genetically significantly different. Besides novel and known variations in genes such as KRAS, BRAF, KDR, FLT1, PTEN, and PI3KCA, aberrations in the up/downstream genes of EGFR/PI3K/VEGF-pathways and other pathways (mTOR, TGFß, etc.) were also detected, potentially influencing therapeutic responsiveness. Chemotherapy between removal of the primary tumor and the metastasis (N = 11) did not further increase the amount of genetic variation. CONCLUSION: Our study indicates that the genetic characteristics of the hepatic metastases are different from those of the primary CRC tumor. As a consequence, the choice of treatment in studies investigating targeted therapies should ideally be based on the genetic properties of the metastasis rather than on those of the primary tumor.
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Adenocarcinoma/genética , Adenocarcinoma/secundario , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Medicina de PrecisiónRESUMEN
Routine methods for assaying steady-state mRNA levels such as RNA-seq and micro-arrays are commonly used as readouts to study the role of transcription factors (TFs) in gene expression regulation. However, cellular RNA levels do not solely depend on activity of TFs and subsequent transcription by RNA polymerase II (Pol II), but are also affected by RNA turnover rate. Here, we demonstrate that integrated analysis of genome-wide TF occupancy, Pol II binding and steady-state RNA levels provide important insights in gene regulatory mechanisms. Pol II occupancy, as detected by Pol II ChIP-seq, was found to correlate better with TF occupancy compared to steady-state RNA levels and is thus a more precise readout for the primary transcriptional mechanisms that are triggered by signal transduction. Furthermore, analysis of differential Pol II occupancy and RNA-seq levels identified genes with high Pol II occupancy and relatively low RNA levels and vice versa. These categories are strongly enriched for genes from different functional classes. Our results demonstrate a complementary value in Pol II chip-seq and RNA-seq approaches for better understanding of gene expression regulation.
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Regulación de la Expresión Génica , ARN Polimerasa II/metabolismo , ARN/metabolismo , Factores de Transcripción/análisis , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Genómica/métodos , Humanos , Análisis de Secuencia de ARN , Vía de Señalización WntRESUMEN
The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3-5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5-6 d for array-based enrichment and 3-4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussed.
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ADN/química , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia de ADN/métodos , Código de Barras del ADN Taxonómico , Biblioteca de Genes , Genoma , Genómica , Técnicas de Amplificación de Ácido NucleicoRESUMEN
To identify novel proteins required for receptor-mediated endocytosis, we have developed an RNAi-based screening method in Drosophila S2 cells, based on uptake of a scavenger receptor ligand. Some known endocytic proteins are essential for endocytosis in this assay, including clathrin and alpha-adaptin; however, other proteins important for synaptic vesicle endocytosis are not required. In a small screen for novel endocytic proteins, we identified the Drosophila homologue of Vps35, a component of the retromer complex, involved in endosome-to-Golgi trafficking. Loss of Vps35 inhibits scavenger receptor ligand endocytosis, and causes mislocalisation of a number of receptors and endocytic proteins. Vps35 has tumour suppressor properties because its loss leads to overproliferation of blood cells in larvae. Its loss also causes signalling defects at the neuromuscular junction, including upregulation of TGFbeta/BMP signalling and excessive formation of synaptic terminals. Vps35 negatively regulates actin polymerisation, and genetic interactions suggest that some of the endocytic and signalling defects of vps35 mutants are due to this function.