RESUMO
In the evolution of species, the karyotype changes with a timescale of tens to hundreds of thousand years. In the development of cancer, the karyotype often is modified in cancerous cells over the lifetime of an individual. Characterizing these changes and understanding the mechanisms leading to them has been of interest in a broad range of disciplines including evolution, cytogenetics, and cancer genetics. A central issue relates to the relative roles of random vs deterministic mechanisms in shaping the changes. Although it is possible that all changes result from random events followed by selection, many results point to other non-random factors that play a role in karyotype evolution. In cancer, chromosomal instability leads to characteristic changes in the karyotype, in which different individuals with a specific type of cancer display similar changes in karyotype structure over time. Statistical analyses of chromosome lengths in different species indicate that the length distribution of chromosomes is not consistent with models in which the lengths of chromosomes are random or evolve solely by simple random processes. A better understanding of the mechanisms underlying karyotype evolution should enable the development of quantitative theoretical models that combine the random and deterministic processes that can be compared to experimental determinations of the karyotype in diverse settings.
Assuntos
Cariótipo , Humanos , Animais , Evolução Molecular , Modelos Genéticos , Neoplasias/genética , Evolução BiológicaRESUMO
The basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and displays consistent large chromosomal deletions. Here, we characterize evolution and maintenance of chromosome 4p (chr4p) loss in basal breast cancer. Analysis of The Cancer Genome Atlas data shows recurrent deletion of chr4p in basal breast cancer. Phylogenetic analysis of a panel of 23 primary tumor/patient-derived xenograft basal breast cancers reveals early evolution of chr4p deletion. Mechanistically we show that chr4p loss is associated with enhanced proliferation. Gene function studies identify an unknown gene, C4orf19, within chr4p, which suppresses proliferation when overexpressed-a member of the PDCD10-GCKIII kinase module we name PGCKA1. Genome-wide pooled overexpression screens using a barcoded library of human open reading frames identify chromosomal regions, including chr4p, that suppress proliferation when overexpressed in a context-dependent manner, implicating network interactions. Together, these results shed light on the early emergence of complex aneuploid karyotypes involving chr4p and adaptive landscapes shaping breast cancer genomes.
Assuntos
Neoplasias da Mama , Redes Reguladoras de Genes , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Animais , Camundongos , Cromossomos Humanos Par 4/genética , Proliferação de Células/genética , Aberrações Cromossômicas , Linhagem Celular Tumoral , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Cancer can develop from an accumulation of alterations, some of which cause a nonmalignant cell to transform to a malignant state exhibiting increased rate of cell growth and evasion of growth suppressive mechanisms, eventually leading to tissue invasion and metastatic disease. Triple-negative breast cancers (TNBC) are heterogeneous and are clinically characterized by the lack of expression of hormone receptors and human epidermal growth factor receptor 2 (HER2), which limits its treatment options. Since tumor evolution is driven by diverse cancer cell populations and their microenvironment, it is imperative to map TNBC at single-cell resolution. Here, we describe an experimental procedure for isolating a single-cell suspension from a TNBC patient-derived xenograft, subjecting it to single-cell RNA sequencing using droplet-based technology from 10× Genomics and analyzing the transcriptomic data at single-cell resolution to obtain inferred copy number aberration profiles, using scCNA. Data obtained using this single-cell RNA sequencing experimental and analytical methodology should enhance our understanding of intratumor heterogeneity which is key for identifying genetic vulnerabilities and developing effective therapies.
Assuntos
Variações do Número de Cópias de DNA , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Genômica , Xenoenxertos , Humanos , Neoplasias de Mama Triplo Negativas/genética , Microambiente TumoralRESUMO
Understanding how forces orchestrate tissue formation requires technologies to map internal tissue stress at cellular length scales. Here, we develop ultrasoft mechanosensors that visibly deform under less than 10 Pascals of cell-generated stress. By incorporating these mechanosensors into multicellular spheroids, we capture the patterns of internal stress that arise during spheroid formation. We experimentally demonstrate the spontaneous generation of a tensional 'skin', only a few cell layers thick, at the spheroid surface, which correlates with activation of mechanobiological signalling pathways, and balances a compressive stress profile within the tissue. These stresses develop through cell-driven mechanical compaction at the tissue periphery, and suggest that the tissue formation process plays a critically important role in specifying mechanobiological function. The broad applicability of this technique should ultimately provide a quantitative basis to design tissues that leverage the mechanical activity of constituent cells to evolve towards a desired form and function.
Assuntos
Hidrogéis/química , Microfluídica , Esferoides Celulares/fisiologia , Estresse Mecânico , Técnicas de Cultura de Células , Proliferação de Células , Humanos , Engenharia Tecidual , Células Tumorais CultivadasRESUMO
Triple-negative breast cancers (TNBCs) display a complex spectrum of mutations and chromosomal aberrations. Chromosome 5q (5q) loss is detected in up to 70% of TNBCs, but little is known regarding the genetic drivers associated with this event. Here, we show somatic deletion of a region syntenic with human 5q33.2-35.3 in a mouse model of TNBC. Mechanistically, we identify KIBRA as a major factor contributing to the effects of 5q loss on tumor growth and metastatic progression. Re-expression of KIBRA impairs metastasis in vivo and inhibits tumorsphere formation by TNBC cells in vitro. KIBRA functions co-operatively with the protein tyrosine phosphatase PTPN14 to trigger mechanotransduction-regulated signals that inhibit the nuclear localization of oncogenic transcriptional co-activators YAP/TAZ. Our results argue that the selective advantage produced by 5q loss involves reduced dosage of KIBRA, promoting oncogenic functioning of YAP/TAZ in TNBC.
Assuntos
Anemia Macrocítica/genética , Genes Supressores de Tumor , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Mamárias Experimentais/genética , Fosfoproteínas/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Modelos Animais de Doenças , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Metástase Neoplásica , Fosfoproteínas/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Genome-sequencing efforts have led to great strides in the annotation of protein-coding genes and other genomic elements. The current challenge is to understand the functional role of each gene and how genes work together to modulate cellular processes. Genetic interactions define phenotypic relationships between genes and reveal the functional organization of a cell. Synthetic genetic array (SGA) methodology automates yeast genetics and enables large-scale and systematic mapping of genetic interaction networks in the budding yeast,Saccharomyces cerevisiae SGA facilitates construction of an output array of double mutants from an input array of single mutants through a series of replica pinning steps. Subsequent analysis of genetic interactions from SGA-derived mutants relies on accurate quantification of colony size, which serves as a proxy for fitness. Since its development, SGA has given rise to a variety of other experimental approaches for functional profiling of the yeast genome and has been applied in a multitude of other contexts, such as genome-wide screens for synthetic dosage lethality and integration with high-content screening for systematic assessment of morphology defects. SGA-like strategies can also be implemented similarly in a number of other cell types and organisms, includingSchizosaccharomyces pombe,Escherichia coli, Caenorhabditis elegans, and human cancer cell lines. The genetic networks emerging from these studies not only generate functional wiring diagrams but may also play a key role in our understanding of the complex relationship between genotype and phenotype.
Assuntos
Automação Laboratorial/métodos , Análise Mutacional de DNA , Redes Reguladoras de Genes , Biologia Molecular/métodos , Anotação de Sequência Molecular , Saccharomyces cerevisiae/genética , Animais , Caenorhabditis elegans/genética , Linhagem Celular Tumoral , Escherichia coli/genética , Humanos , Schizosaccharomyces/genéticaRESUMO
Synthetic lethal interactions enable a novel approach for discovering specific genetic vulnerabilities in cancer cells that can be exploited for the development of therapeutics. Despite successes in model organisms such as yeast, discovering synthetic lethal interactions on a large scale in human cells remains a significant challenge. We describe a comparative genomic strategy for identifying cancer-relevant synthetic lethal interactions whereby candidate interactions are prioritized on the basis of genetic interaction data available in yeast, followed by targeted testing of candidate interactions in human cell lines. As a proof of principle, we describe two novel synthetic lethal interactions in human cells discovered by this approach, one between the tumor suppressor gene SMARCB1 and PSMA4, and another between alveolar soft-part sarcoma-associated ASPSCR1 and PSMC2. These results suggest therapeutic targets for cancers harboring mutations in SMARCB1 or ASPSCR1 and highlight the potential of a targeted, cross-species strategy for identifying synthetic lethal interactions relevant to human cancer.
Assuntos
Neoplasias/genética , Animais , Técnicas de Cultura de Células , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Genômica , Humanos , Neoplasias/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína SMARCB1 , Fatores de Transcrição/genéticaRESUMO
Dosage suppression is a genetic interaction in which overproduction of one gene rescues a mutant phenotype of another gene. Although dosage suppression is known to map functional connections among genes, the extent to which it might illuminate global cellular functions is unclear. Here we analyze a network of interactions linking dosage suppressors to 437 essential genes in yeast. For 424 genes, we curated interactions from the literature. Analyses revealed that many dosage suppression interactions occur between functionally related genes and that the majority do not overlap with other types of genetic or physical interactions. To confirm the generality of these network properties, we experimentally identified dosage suppressors for 29 genes from pooled populations of temperature-sensitive mutant cells transformed with a high-copy molecular-barcoded open reading frame library, MoBY-ORF 2.0. We classified 87% of the 1,640 total interactions into four general types of suppression mechanisms, which provided insight into their relative frequencies. This work suggests that integrating the results of dosage suppression studies with other interaction networks could generate insights into the functional wiring diagram of a cell.
Assuntos
Redes Reguladoras de Genes , Saccharomyces cerevisiae/genética , Supressão Genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Árvores de Decisões , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Genes Essenciais , Genes Fúngicos , Sequenciamento de Nucleotídeos em Larga Escala , Cinetocoros/metabolismo , Mutação , Fases de Leitura Aberta , Fenótipo , Mapeamento de Interação de Proteínas/métodos , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
rIPC (remote ischaemic preconditioning) is a phenomenon whereby short periods of ischaemia and reperfusion of a tissue or organ (e.g. mesentery, kidney) can protect a distant tissue or organ (e.g. heart) against subsequent, potentially lethal, ischaemia. We, and others, have shown that transient limb ischaemia can provide potent myocardial protection experimentally and clinically during cardiac surgery. Nonetheless, our understanding of the signal transduction from remote stimulus to local effect remains incomplete. The aim of the present study was to define the humoral nature of rIPC effector(s) from limb ischaemia and to study their local effects in isolated heart and cardiomyocyte models. Using a Langendorff preparation, we show that infarct size after coronary artery ligation and reperfusion was substantially reduced by rIPC in vivo, this stimulus up-regulating the MAPKs (mitogen-activating protein kinases) p42/p44, and inducing PKCepsilon (protein kinase Cepsilon) subcellular redistribution. Pre-treatment with the plasma and dialysate of plasma (obtained using 15 kDa cut-off dialysis membrane) from donor rabbits subjected to rIPC similarly protected against infarction. The effectiveness of the rIPC dialysate was abrogated by passage through a C18 hydrophobic column, but eluate from this column provided the same level of protection. The dialysate of rIPC plasma from rabbits and humans was also tested in an isolated fresh cardiomyocyte model of simulated ischaemia and reperfusion. Necrosis in cardiomyocytes treated with rIPC dialysate was substantially reduced compared with control, and was similar to cells pre-treated by 'classical' preconditioning. This effect, by rabbit rIPC dialysate, was blocked by pre-treatment with the opiate receptor blocker naloxone. In conclusion, in vivo transient limb ischaemia releases a low-molecular-mass (<15 kDa) hydrophobic circulating factor(s) which induce(s) a potent protection against myocardial ischaemia/reperfusion injury in Langendorff-perfused hearts and isolated cardiomyocytes in the same species. This cardioprotection is transferable across species, independent of local neurogenic activity, and requires opioid receptor activation.