RESUMO
Short-read sequencing is the workhorse of cancer genomics yet is thought to miss many structural variants (SVs), particularly large chromosomal alterations. To characterize missing SVs in short-read whole genomes, we analyzed 'loose ends'-local violations of mass balance between adjacent DNA segments. In the landscape of loose ends across 1,330 high-purity cancer whole genomes, most large (>10-kb) clonal SVs were fully resolved by short reads in the 87% of the human genome where copy number could be reliably measured. Some loose ends represent neotelomeres, which we propose as a hallmark of the alternative lengthening of telomeres phenotype. These pan-cancer findings were confirmed by long-molecule profiles of 38 breast cancer and melanoma cases. Our results indicate that aberrant homologous recombination is unlikely to drive the majority of large cancer SVs. Furthermore, analysis of mass balance in short-read whole genome data provides a surprisingly complete picture of cancer chromosomal structure.
Assuntos
Neoplasias da Mama , Genômica , Humanos , Feminino , Genômica/métodos , Análise de Sequência de DNA/métodos , Genoma Humano/genética , Aberrações Cromossômicas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Variação Estrutural do Genoma/genéticaRESUMO
Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations1. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure2. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These data revealed a distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocal pair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.
Assuntos
Proteína BRCA1 , Proteína BRCA2 , Aberrações Cromossômicas , Reparo do DNA , Neoplasias , Humanos , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Inversão Cromossômica , Reparo do DNA/genética , Neoplasias/genética , Translocação Genética/genética , Recombinação Homóloga , Análise Citogenética , Aberrações Cromossômicas/classificaçãoRESUMO
Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.
Assuntos
Reparo do DNA , Anemia de Fanconi , Genômica , Neoplasias de Cabeça e Pescoço , Humanos , Aldeídos/efeitos adversos , Aldeídos/metabolismo , Reparo do DNA/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Neoplasias de Cabeça e Pescoço/induzido quimicamente , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Infecções por Papillomavirus , Carcinoma de Células Escamosas de Cabeça e Pescoço/induzido quimicamente , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Dano ao DNA/efeitos dos fármacosRESUMO
High-order three-dimensional (3D) interactions between more than two genomic loci are common in human chromatin, but their role in gene regulation is unclear. Previous high-order 3D chromatin assays either measure distant interactions across the genome or proximal interactions at selected targets. To address this gap, we developed Pore-C, which combines chromatin conformation capture with nanopore sequencing of concatemers to profile proximal high-order chromatin contacts at the genome scale. We also developed the statistical method Chromunity to identify sets of genomic loci with frequencies of high-order contacts significantly higher than background ('synergies'). Applying these methods to human cell lines, we found that synergies were enriched in enhancers and promoters in active chromatin and in highly transcribed and lineage-defining genes. In prostate cancer cells, these included binding sites of androgen-driven transcription factors and the promoters of androgen-regulated genes. Concatemers of high-order contacts in highly expressed genes were demethylated relative to pairwise contacts at the same loci. Synergies in breast cancer cells were associated with tyfonas, a class of complex DNA amplicons. These results rigorously link genome-wide high-order 3D interactions to lineage-defining transcriptional programs and establish Pore-C and Chromunity as scalable approaches to assess high-order genome structure.
Assuntos
Sequenciamento por Nanoporos , Nanoporos , Androgênios , Cromatina/genética , Humanos , Fatores de Transcrição/genéticaRESUMO
Telomere crisis contributes to cancer genome evolution, yet only a subset of cancers display breakage-fusion-bridge (BFB) cycles and chromothripsis, hallmarks of experimental telomere crisis identified in previous studies. We examine the spectrum of structural variants (SVs) instigated by natural telomere crisis. Eight spontaneous post-crisis clones did not show prominent patterns of BFB cycles or chromothripsis. Their crisis-induced genome rearrangements varied from infrequent simple SVs to more frequent and complex SVs. In contrast, BFB cycles and chromothripsis occurred in MRC5 fibroblast clones that escaped telomere crisis after CRISPR-controlled telomerase activation. This system revealed convergent evolutionary lineages altering one allele of chromosome 12p, where a short telomere likely predisposed to fusion. Remarkably, the 12p chromothripsis and BFB events were stabilized by independent fusions to chromosome 21. The data establish that telomere crisis can generate a wide spectrum of SVs implying that a lack of BFB patterns and chromothripsis in cancer genomes does not indicate absence of past telomere crisis.
Assuntos
Cromotripsia , Neoplasias/genética , Telômero/química , Linhagem Celular , Instabilidade Cromossômica , Fibroblastos , Genoma , Instabilidade Genômica , Humanos , Pulmão , Metáfase , Modelos Biológicos , Telômero/ultraestruturaRESUMO
Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.
Assuntos
Variação Estrutural do Genoma/genética , Genômica/métodos , Neoplasias/genética , Inversão Cromossômica/genética , Cromotripsia , Variações do Número de Cópias de DNA/genética , Rearranjo Gênico/genética , Genoma Humano/genética , Humanos , Mutação/genética , Sequenciamento Completo do Genoma/métodosRESUMO
Small GTPases play a critical role in membrane traffic. Among them, Arf6 mediates transport to and from the plasma membrane, as well as phosphoinositide signalling and cholesterol homeostasis. Here we delineate the molecular basis for the link between Arf6 and cholesterol homeostasis using an inducible knockout (KO) model of mouse embryonic fibroblasts (MEFs). We find that accumulation of free cholesterol in the late endosomes/lysosomes of Arf6 KO MEFs results from mistrafficking of Niemann-Pick type C protein NPC2, a cargo of the cation-independent mannose-6-phosphate receptor (CI-M6PR). This is caused by a selective increase in an endosomal pool of phosphatidylinositol-4-phosphate (PI4P) and a perturbation of retromer, which controls the retrograde transport of CI-M6PR via sorting nexins, including the PI4P effector SNX6. Finally, reducing PI4P levels in KO MEFs through independent mechanisms rescues aberrant retromer tubulation and cholesterol mistrafficking. Our study highlights a phosphoinositide-based mechanism for control of cholesterol distribution via retromer.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Colesterol/metabolismo , Lisossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Fator 6 de Ribosilação do ADP , Animais , Endossomos/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos Knockout , Receptor IGF Tipo 2/metabolismoRESUMO
Expression of the Wnt target gene phospholipase D1 (PLD1) is up-regulated in various carcinomas, including colorectal cancer (CRC). However, the mechanistic significance of its elevated expression in intestinal tumorigenesis remains unknown. In this study, we show that genetic and pharmacological targeting of PLD1 disrupts spontaneous and colitis-associated intestinal tumorigenesis in Apc(Min/+) and azoxymethane/dextran sodium sulfate mice models. Intestinal epithelial cell-specific PLD1 overexpression in Apc(Min/+) mice accelerated tumorigenesis with increased proliferation and nuclear ß-catenin levels compared with Apc(Min/+) mice. Moreover, PLD1 inactivation suppressed the self-renewal capacity of colon cancer-initiating cells (CC-ICs) by decreasing expression of ß-catenin via E2F1-induced microRNA (miR)-4496 up-regulation. Ultimately, low expression of PLD1 coupled with a low level of CC-IC markers was predictive of a good prognosis in CRC patients, suggesting in vivo relevance. Collectively, our data reveal that PLD1 has a crucial role in intestinal tumorigenesis via its modulation of the E2F1-miR-4496-ß-catenin signaling pathway. Modulation of PLD1 expression and activity represents a promising therapeutic strategy for the treatment of intestinal tumorigenesis.
Assuntos
Carcinogênese/metabolismo , Neoplasias Intestinais/fisiopatologia , Fosfolipase D/metabolismo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Animais , Apoptose/fisiologia , Azoximetano , Western Blotting , Primers do DNA/genética , Sulfato de Dextrana , Citometria de Fluxo , Células HEK293 , Humanos , Imuno-Histoquímica , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Neoplasias Intestinais/metabolismo , Camundongos , MicroRNAs/metabolismo , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase em Tempo Real , Análise Serial de Tecidos , beta Catenina/genéticaRESUMO
The CA125 antigen is found in the serum of many patients with serous ovarian cancer and has been widely used as a disease marker. CA125 has been shown to be an independent factor for clinical outcome in this disease. In The Cancer Genome Atlas ovarian cancer project, MUC16 expression levels are frequently increased, and the highest levels of MUC16 expression are linked to a significantly worse survival. To examine the biologic effect of the proximal portion of MUC16/CA125, NIH/3T3 (3T3) fibroblast cell lines were stably transfected with the carboxy elements of MUC16. As few as 114 amino acids from the carboxy-terminal portion of MUC16 were sufficient to increase soft agar growth, promote matrigel invasion, and increase the rate of tumor growth in athymic nude mice. Transformation with carboxy elements of MUC16 was associated with activation of the AKT and ERK pathways. MUC16 transformation was associated with up-regulation of a number of metastases and invasion gene transcripts, including IL-1ß, MMP2, and MMP9. All observed oncogenic changes were exclusively dependent on the extracellular "ectodomain" of MUC16. The biologic impact of MUC16 was also explored through the creation of a transgenic mouse model expressing 354 amino acids of the carboxy-terminal portion of MUC16 (MUC16c354). Under a CMV, early enhancer plus chicken ß actin promoter (CAG) MUC16c354 was well expressed in many organs, including the brain, colon, heart, kidney, liver, lung, ovary, and spleen. MUC16c354 transgenic animals appear to be viable, fertile, and have a normal lifespan. However, when crossed with p53-deficient mice, the MUC16c354:p53+/- progeny displayed a higher frequency of spontaneous tumor development compared to p53+/- mice alone. We conclude that the carboxy-terminal portion of the MUC16/CA125 protein is oncogenic in NIH/3T3 cells, increases invasive tumor properties, activates the AKT and ERK pathways, and contributes to the biologic properties of ovarian cancer.
Assuntos
Antígeno Ca-125/genética , Antígeno Ca-125/metabolismo , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Animais , Antígeno Ca-125/química , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/química , Camundongos , Camundongos Nus , Células NIH 3T3 , Invasividade Neoplásica , Neoplasias Experimentais , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Myocardial inflammation is critical for ventricular remodeling after ischemia. Phospholipid mediators play an important role in inflammatory processes. In the plasma membrane they are degraded by phospholipase D1 (PLD1). PLD1 was shown to be critically involved in ischemic cardiovascular events. Moreover, PLD1 is coupled to tumor necrosis factor-α signaling and inflammatory processes. However, the impact of PLD1 in inflammatory cardiovascular disease remains elusive. Here, we analyzed the impact of PLD1 in tumor necrosis factor-α-mediated activation of monocytes after myocardial ischemia and reperfusion using a mouse model of myocardial infarction. PLD1 expression was highly up-regulated in the myocardium after ischemia/reperfusion. Genetic ablation of PLD1 led to defective cell adhesion and migration of inflammatory cells into the infarct border zone 24 hours after ischemia/reperfusion injury, likely owing to reduced tumor necrosis factor-α expression and release, followed by impaired nuclear factor-κB activation and interleukin-1 release. Moreover, PLD1 was found to be important for transforming growth factor-ß secretion and smooth muscle α-actin expression of cardiac fibroblasts because myofibroblast differentiation and interstitial collagen deposition were altered in Pld1(-/-) mice. Consequently, infarct size was increased and left ventricular function was impaired 28 days after myocardial infarction in Pld1(-/-) mice. Our results indicate that PLD1 is crucial for tumor necrosis factor-α-mediated inflammation and transforming growth factor-ß-mediated collagen scar formation, thereby augmenting cardiac left ventricular function after ischemia/reperfusion.
Assuntos
Inflamação/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Fosfolipase D/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Movimento Celular/fisiologia , Cicatriz/metabolismo , Cicatriz/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/enzimologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization.
Assuntos
Citoesqueleto/fisiologia , Macrófagos/imunologia , Infiltração de Neutrófilos/imunologia , Fagocitose/imunologia , Fosfolipase D/deficiência , Animais , Western Blotting , Citoesqueleto/imunologia , Camundongos , Neuropeptídeos/metabolismo , Pancreatite/imunologia , Ácidos Fosfatídicos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
Angiogenesis inhibitors, which target tumor cells, confer only short-term benefits on tumor growth. We report that ablation of the lipid signaling enzyme phospholipase D1 (PLD1) in the tumor environment compromised the neovascularization and growth of tumors. PLD1 deficiency suppressed the activation of Akt and mitogen-activated protein kinase signaling pathways by vascular endothelial growth factor in vascular endothelial cells, resulting in decreased integrin-dependent cell adhesion to, and migration on, extracellular matrices, as well as reduced tumor angiogenesis in a xenograft model. In addition, mice lacking PLD1 incurred fewer lung metastases than did wild-type mice. Bone marrow transplantation and binding studies identified a platelet-derived mechanism involving decreased tumor cell-platelet interactions, in part because of impaired activation of αIIbß3 integrin in platelets, which decreased the seeding of tumor cells into the lung parenchyma. Treatment with a small-molecule inhibitor of PLD1 phenocopied PLD1 deficiency, efficiently suppressing both tumor growth and metastasis in mice. These findings reveal that PLD1 in the tumor environment promotes tumor growth and metastasis and, taken together with previous reports on the roles of PLD in tumor cell-intrinsic adaptations to stress, suggest the potential use of PLD inhibitors as cancer therapeutics.
Assuntos
Neoplasias da Mama/enzimologia , Células Endoteliais/enzimologia , Neoplasias Pulmonares/enzimologia , Neovascularização Patológica/enzimologia , Fosfolipase D/metabolismo , Transdução de Sinais , Microambiente Tumoral , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Endoteliais/patologia , Feminino , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Knockout , Metástase Neoplásica , Transplante de Neoplasias , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Fosfolipase D/genética , Transplante HeterólogoRESUMO
Growing evidence implicates aberrant lipid signaling in Alzheimer's disease (AD). While phospholipases A2 and C have been recently shown to mediate key actions of amyloid ß-peptide (Aß) through a dysregulation of arachidonic acid and phosphatidylinositol-4,5-bisphosphate metabolism, respectively, the role of phospholipase D (PLD) has so far remained elusive. PLD produces phosphatidic acid (PA), a bioactive lipid involved in multiple aspects of cell physiology, including signaling and membrane trafficking processes. Here we show that oligomeric Aß enhances PLD activity in cultured neurons and that this stimulatory effect does not occur upon ablation of PLD2 via gene targeting. Aß fails to suppress long-term potentiation in PLD2-deficient hippocampal slices, suggesting that PLD2 is required for the synaptotoxic action of this peptide. In vivo PLD activity, as assessed by detection of phosphatidylethanol levels using mass spectrometry (MS) following ethanol injection, is also increased in the brain of a transgenic mouse model of AD (SwAPP). Furthermore, Pld2 ablation rescues memory deficits and confers synaptic protection in SwAPP mice despite a significant Aß load. MS-based lipid analysis of Pld2 mutant brains in the presence or absence of the SwAPP transgene unmasks striking crosstalks between different PA species. This lipid analysis shows an exquisite acyl chain specificity and plasticity in the perturbation of PA metabolism. Collectively, our results point to specific molecular species of PA as key modulators of AD pathogenesis and identify PLD2 as a novel potential target for therapeutics.