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Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes1. Here we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence predicted loss-of-function variants in this cohort after filtering for artefacts caused by sequencing and annotation errors. Using an improved model of human mutation rates, we classify human protein-coding genes along a spectrum that represents tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve the power of gene discovery for both common and rare diseases.
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Exoma/genética , Genes Essenciais/genética , Variação Genética/genética , Genoma Humano/genética , Adulto , Encéfalo/metabolismo , Doenças Cardiovasculares/genética , Estudos de Coortes , Bases de Dados Genéticas , Feminino , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Mutação com Perda de Função/genética , Masculino , Taxa de Mutação , Pró-Proteína Convertase 9/genética , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Metastasis is the main cause of death for patients with breast cancer. Many studies have characterized the genomic landscape of breast cancer during its early stages. However, there is evidence that genomic alterations are acquired during the evolution of cancers from their early to late stages, and that the genomic landscape of early cancers is not representative of that of lethal cancers1-7. Here we investigated the landscape of somatic alterations in 617 metastatic breast cancers. Nine driver genes (TP53, ESR1, GATA3, KMT2C, NCOR1, AKT1, NF1, RIC8A and RB1) were more frequently mutated in metastatic breast cancers that expressed hormone receptors (oestrogen and/or progesterone receptors; HR+) but did not have high levels of HER2 (HER2-; n = 381), when compared to early breast cancers from The Cancer Genome Atlas. In addition, 18 amplicons were more frequently observed in HR+/HER2- metastatic breast cancers. These cancers showed an increase in mutational signatures S2, S3, S10, S13 and S17. Among the gene alterations that were enriched in HR+/HER2- metastatic breast cancers, mutations in TP53, RB1 and NF1, together with S10, S13 and S17, were associated with poor outcome. Metastatic triple-negative breast cancers showed an increase in the frequency of somatic biallelic loss-of-function mutations in genes related to homologous recombination DNA repair, compared to early triple-negative breast cancers (7% versus 2%). Finally, metastatic breast cancers showed an increase in mutational burden and clonal diversity compared to early breast cancers. Thus, the genomic landscape of metastatic breast cancer is enriched in clinically relevant genomic alterations and is more complex than that of early breast cancer. The identification of genomic alterations associated with poor outcome will allow earlier and better selection of patients who require the use of treatments that are still in clinical trials. The genetic complexity observed in advanced breast cancer suggests that such treatments should be introduced as early as possible in the disease course.
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Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Evolução Molecular , Genoma Humano/genética , Genômica , Mutação , Metástase Neoplásica/genética , Análise Mutacional de DNA , Progressão da Doença , Feminino , Humanos , Masculino , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The female mammary gland is a very dynamic organ that undergoes continuous tissue remodeling during adulthood. Although it is well established that the number of menstrual cycles and pregnancy (in this case transiently) increase the risk of breast cancer, the reasons are unclear. Growing clinical and experimental evidence indicates that improper involution plays a role in the development of this malignancy. Recently, we described the miR-424(322)/503 cluster as an important regulator of mammary epithelial involution after pregnancy. Here, through the analysis of â¼3000 primary tumors, we show that miR-424(322)/503 is commonly lost in a subset of aggressive breast cancers and describe the genetic aberrations that inactivate its expression. Furthermore, through the use of a knockout mouse model, we demonstrate for the first time that loss of miR-424(322)/503 promotes breast tumorigenesis in vivo. Remarkably, we found that loss of miR-424(322)/503 promotes chemoresistance due to the up-regulation of two of its targets: BCL-2 and insulin-like growth factor-1 receptor (IGF1R). Importantly, targeted therapies blocking the aberrant activity of these targets restore sensitivity to chemotherapy. Overall, our studies reveal miR-424(322)/503 as a tumor suppressor in breast cancer and provide a link between mammary epithelial involution, tumorigenesis, and the phenomenon of chemoresistance.
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Neoplasias da Mama/genética , MicroRNAs/genética , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Deleção de Genes , Genes Supressores de Tumor , Humanos , Neoplasias Mamárias Experimentais/genética , Camundongos , Gravidez , Complicações Neoplásicas na Gravidez/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Fosfatases cdc25/genéticaRESUMO
BACKGROUND: Inflammatory breast cancer (IBC) is the most pro-metastatic form of BC. Better understanding of its enigmatic pathophysiology is crucial. We report here the largest whole-exome sequencing (WES) study of clinical IBC samples. METHODS: We retrospectively applied WES to 54 untreated IBC primary tumor samples and matched normal DNA. The comparator samples were 102 stage-matched non-IBC samples from TCGA. We compared the somatic mutational profiles, spectra and signatures, copy number alterations (CNAs), HRD and heterogeneity scores, and frequencies of actionable genomic alterations (AGAs) between IBCs and non-IBCs. The comparisons were adjusted for the molecular subtypes. RESULTS: The number of somatic mutations, TMB, and mutational spectra were not different between IBCs and non-IBCs, and no gene was differentially mutated or showed differential frequency of CNAs. Among the COSMIC signatures, only the age-related signature was more frequent in non-IBCs than in IBCs. We also identified in IBCs two new mutational signatures not associated with any environmental exposure, one of them having been previously related to HIF pathway activation. Overall, the HRD score was not different between both groups, but was higher in TN IBCs than TN non-IBCs. IBCs were less frequently classified as heterogeneous according to heterogeneity H-index than non-IBCs (21% vs 33%), and clonal mutations were more frequent and subclonal mutations less frequent in IBCs. More than 50% of patients with IBC harbored at least one high-level of evidence (LOE) AGA (OncoKB LOE 1-2, ESCAT LOE I-II), similarly to patients with non-IBC. CONCLUSIONS: We provide the largest mutational landscape of IBC. Only a few subtle differences were identified with non-IBCs. The most clinically relevant one was the higher HRD score in TN IBCs than in TN non-IBCs, whereas the most intriguing one was the smaller intratumor heterogeneity of IBCs.
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Neoplasias da Mama , Neoplasias Inflamatórias Mamárias , Humanos , Feminino , Neoplasias Inflamatórias Mamárias/genética , Neoplasias Inflamatórias Mamárias/patologia , Neoplasias da Mama/genética , Estudos Retrospectivos , Mutação/genética , GenômicaRESUMO
Cancer cell heterogeneity is a major driver of therapy resistance. To characterize resistant cells and their vulnerabilities, we studied the PLZF-RARA variant of acute promyelocytic leukemia, resistant to retinoic acid (RA), using single-cell multiomics. We uncovered transcriptional and chromatin heterogeneity in leukemia cells. We identified a subset of cells resistant to RA with proliferation, DNA replication, and repair signatures that depend on a fine-tuned E2F transcriptional network targeting the epigenetic regulator enhancer of zeste homolog 2 (EZH2). Epigenomic and functional analyses validated the driver role of EZH2 in RA resistance. Targeting pan-EZH2 activities (canonical/noncanonical) was necessary to eliminate leukemia relapse-initiating cells, which underlies a dependency of resistant cells on an EZH2 noncanonical activity and the necessity to degrade EZH2 to overcome resistance. Our study provides critical insights into the mechanisms of RA resistance that allow us to eliminate treatment-resistant leukemia cells by targeting EZH2, thus highlighting a potential targeted therapy approach. Beyond RA resistance and acute promyelocytic leukemia context, our study also demonstrates the power of single-cell multiomics to identify, characterize, and clear therapy-resistant cells.
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Leucemia Promielocítica Aguda , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Tretinoína/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Receptor alfa de Ácido Retinoico/genética , Receptores do Ácido Retinoico/genética , Fatores de Transcrição/genética , Proteínas Nucleares/genéticaRESUMO
BACKGROUND: Anti-PD1/PDL1 immune checkpoint inhibitors (ICI) transformed the prognosis of patients with advanced non-small cell lung cancer (NSCLC). However, the response rate remains disappointing and toxicity may be life-threatening, making urgent identification of biomarkers predictive for efficacy. Immunologic Constant of Rejection signature (ICR) is a 20-gene expression signature of cytotoxic immune response with prognostic value in some solid cancers. Our objective was to assess its predictive value for benefit from anti-PD1/PDL1 in patients with advanced NSCLC. METHODS: We retrospectively profiled 44 primary tumors derived from NSCLC patients treated with ICI as single-agent in at least the second-line metastatic setting. Transcriptomic analysis was performed using the nCounter® analysis system and the PanCancer Immune Profiling Panel. We then pooled our data with clinico-biological data from four public gene expression data sets, leading to a total of 162 NSCLC patients treated with single-agent anti-PD1/PDL1. ICR was applied to all samples and correlation was searched between ICR classes and the Durable Clinical Benefit (DCB), defined as stable disease or objective response according to RECIST 1.1 for a minimum of 6 months after the start of ICI. RESULTS: The DCB rate was 29%; 22% of samples were classified as ICR1, 30% ICR2, 22% ICR3, and 26% ICR4. These classes were not associated with the clinico-pathological variables, but showed enrichment from ICR1 to ICR4 in quantitative/qualitative markers of immune response. ICR2-4 class was associated with a 5.65-fold DCB rate when compared with ICR1 class. In multivariate analysis, ICR classification remained associated with DCB, independently from PDL1 expression and other predictive immune signatures. By contrast, it was not associated with disease-free survival in 556 NSCLC TCGA patients untreated with ICI. CONCLUSION: The 20-gene ICR signature was independently associated with benefit from anti-PD1/PDL1 ICI in patients with advanced NSCLC. Validation in larger retrospective and prospective series is warranted.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Estudos Retrospectivos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , BiomarcadoresRESUMO
BACKGROUND: Soft tissue sarcomas (STS) are heterogeneous and pro-metastatic tumors. Identification of accurate prognostic factors and novel therapeutic targets are crucial. CSPG4 is a cell surface proteoglycan with oncogenic functions. It recently emerged as a potential target for immunotherapy, including cell therapy based on CSPG4-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CSPG4-CAR.CIKs) in STS. However, expression of CSPG4 is poorly known in STS so far. METHODS: We analyzed CSPG4 gene expression in 1378 localized STS clinical samples, and searched for correlations with clinicopathological data, including disease-free survival (DFS), and with tumor immune features. RESULTS: CSPG4 expression was heterogeneous across samples. High expression was associated with younger patients' age, more frequent undifferentiated pleomorphic sarcoma and myxofibrosarcoma pathological subtypes, more frequent internal trunk tumor site, and more CINSARC high-risk samples. No correlation existed with pathological tumor size and grade, and tumor depth. Patients with high CSPG4 expression displayed 49% (95% CI 42-57) 5-year DFS versus 61% (95% CI 56-68) in patients with low expression (p = 3.17E-03), representing a 49% increased risk of event in the "CSPG4-high" group (HR = 1.49, 95% CI 1.14-1.94). This unfavorable prognostic value persisted in multivariate analysis, independently from other variables. There were significant differences in immune variables between "CSPG4-high" and "CSPG4-low" tumors. The "CSPG4-low" tumors displayed profiles suggesting higher anti-tumor cytotoxic immune response and higher potential vulnerability to immune checkpoint inhibitors (ICI). By contrast, the "CSPG4-high" tumors displayed profiles implying an immune-excluded tumor microenvironment, potentially induced by hypoxia, resulting from an immature chaotic microvasculature, and/or the presence of contractile myofibroblasts. CONCLUSIONS: Patients with "CSPG4-high" STS, theoretically candidate for CAR.CIKs, display shorter DFS and an immune environment unfavorable to vulnerability to CAR.CIKs, which could be improved by combining anti-angiogenic drugs able to normalize the tumor vasculature. By contrast, "CSPG4-low" STS are better candidates for immune therapy involving ICI.
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Antineoplásicos , Receptores de Antígenos Quiméricos , Sarcoma , Neoplasias de Tecidos Moles , Adulto , Inibidores da Angiogênese , Proteoglicanas de Sulfatos de Condroitina , Citocinas , Humanos , Inibidores de Checkpoint Imunológico , Imunidade , Proteínas de Membrana , Prognóstico , Proteoglicanas/metabolismo , Sarcoma/genética , Sarcoma/terapia , Neoplasias de Tecidos Moles/patologia , Microambiente TumoralRESUMO
The oncogenic events involved in breast implant-associated anaplastic large cell lymphoma (BI-ALCL) remain elusive. To clarify this point, we have characterized the genomic landscape of 34 BI-ALCLs (15 tumor and 19 in situ subtypes) collected from 54 BI-ALCL patients diagnosed through the French Lymphopath network. Whole-exome sequencing (n = 22, with paired tumor/germline DNA) and/or targeted deep sequencing (n = 24) showed recurrent mutations of epigenetic modifiers in 74% of cases, involving notably KMT2C (26%), KMT2D (9%), CHD2 (15%), and CREBBP (15%). KMT2D and KMT2C mutations correlated with a loss of H3K4 mono- and trimethylation by immunohistochemistry. Twenty cases (59%) showed mutations in ≥1 member of the JAK/STAT pathway, including STAT3 (38%), JAK1 (18%), and STAT5B (3%), and in negative regulators, including SOCS3 (6%), SOCS1 (3%), and PTPN1 (3%). These mutations were more frequent in tumor-type samples than in situ samples (P = .038). All BI-ALCLs expressed pSTAT3, regardless of the mutational status of genes in the JAK/STAT pathway. Mutations in the EOMES gene (12%) involved in lymphocyte development, PI3K-AKT/mTOR (6%), and loss-of-function mutations in TP53 (12%) were also identified. Copy-number aberration (CNA) analysis identified recurrent alterations, including gains on chromosomes 2, 9p, 12p, and 21 and losses on 4q, 8p, 15, 16, and 20. Regions of CNA encompassed genes involved in the JAK/STAT pathway and epigenetic regulators. Our results show that the BI-ALCL genomic landscape is characterized by not only JAK/STAT activating mutations but also loss-of-function alterations of epigenetic modifiers.
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Implantes de Mama/efeitos adversos , Epigênese Genética , Janus Quinases/metabolismo , Linfoma Anaplásico de Células Grandes/etiologia , Linfoma Anaplásico de Células Grandes/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Adulto , Idoso , Idoso de 80 Anos ou mais , Variações do Número de Cópias de DNA , Feminino , Genoma Humano , Humanos , Linfoma Anaplásico de Células Grandes/patologia , Pessoa de Meia-Idade , Mutação/genéticaRESUMO
Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.
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Leucemia Eritroblástica Aguda/genética , Proteínas de Neoplasias/fisiologia , Fatores de Transcrição/fisiologia , Transcriptoma , Adulto , Animais , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Dioxigenases , Eritroblastos/metabolismo , Eritropoese/genética , Feminino , Fator de Transcrição GATA1/deficiência , Fator de Transcrição GATA1/genética , Técnicas de Introdução de Genes , Heterogeneidade Genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , RNA-Seq , Quimera por Radiação , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição/genética , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/fisiologia , Sequenciamento do Exoma , Adulto JovemRESUMO
Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human 'knockout' variants in protein-coding genes.
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Exoma/genética , Variação Genética/genética , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Humanos , Fenótipo , Proteoma/genética , Doenças Raras/genética , Tamanho da AmostraRESUMO
Clinico-pathological factors fail to consistently predict the outcome after pancreatic resection for pancreatic ductal adenocarcinoma (PDAC). PDACs show a high level of inter- and intra- tumor genetic heterogeneity. A molecular classification should help sort patients into less heterogeneous and more appropriate groups regarding the metastatic risk and the therapeutic response, with the consequences of better predicting evolution and better orienting the treatment. PDAC can be classified based on mutational subtypes and 18gene alterations. Whole-genome sequencing identified mutational signatures, mutational burden and hyper-mutated tumors with specific DNA repair defects. Their overlap/similarities allow the definition of molecular subtypes. DNA and RNA classifications can be used in prognosis assessment. They are useful in therapeutic choice for they allow the design of approaches that can predict the respective drug sensitivity of each molecular subtype. This review provides a comprehensive analysis of available molecular classifications in PDAC and how this can help guide clinical decisions.
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Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Técnicas de Diagnóstico Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Medicina de Precisão , Animais , Carcinoma Ductal Pancreático/classificação , Carcinoma Ductal Pancreático/patologia , Tomada de Decisão Clínica , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Heterogeneidade Genética , Predisposição Genética para Doença , Humanos , Terapia de Alvo Molecular , Mutação , Neoplasias Pancreáticas/classificação , Neoplasias Pancreáticas/patologia , Fenótipo , Valor Preditivo dos Testes , TranscriptomaRESUMO
INTRODUCTION: In BCR-ABL1-negative myeloproliferative neoplasms, myelofibrosis (MF) is either primary (PMF) or secondary (SMF) to polycythemia vera or essential thrombocythemia. MF is characterized by an increased risk of transformation to acute myeloid leukemia (AML) and a shortened life expectancy. METHODS: Because natural histories of PMF and SMF are different, we studied by targeted next generation sequencing the differences in the molecular landscape of 86 PMF and 59 SMF and compared their prognosis impact. RESULTS: PMF had more ASXL1 (47.7%) and SRSF2 (14%) gene mutations than SMF (respectively 27.1% and 3.4%, P = .04). Poorer survival was associated with RNA splicing mutations (especially SRSF2) and TP53 in PMF (P = .0003), and with ASXL1 and TP53 mutations in SMF (P < .0001). These mutations of poor prognosis were associated with biological features of scoring systems (DIPSS and MYSEC-PM score). Mutations in TP53/SRSF2 in PMF or TP53/ASXL1 in SMF were more frequent as the risk of these scores increased. This allowed for a better stratification of MF patients, especially within the DIPSS intermediate-1 risk group (DIPSS) or the MYSEC-PM high risk group. AML transformation occurred faster in SMF than in PMF and patients who transformed to AML were more SRSF2-mutated and less CALR-mutated at MF sampling. CONCLUSIONS: PMF and SMF have different but not specific molecular profiles and different prognosis depending on the molecular profile. This may be due to differences in disease history. Combining mutations and existing scores should improve prognosis assessment.
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The presence of nuclear ERBB2 receptor-type tyrosine kinase is one of the causes of the resistance to membrane ERBB2-targeted therapy in breast cancers. It has been previously reported that this nuclear location arises through at least two different mechanisms: proteolytic shedding of the extracellular domain of the full-length receptor and translation of the messenger RNA (mRNA)-encoding ERBB2 from internal initiation codons. Here, we report a new mechanism and function where a significant portion of nuclear ERBB2 results from the translation of the variant ERBB2 mRNA under the transcriptional control of a distal promoter that is actively used in breast cancer cells. We show that both membrane ERBB2a and nuclear ERBB2b isoforms are prevalently expressed in breast cancer cell lines and carcinoma samples. The ERBB2b isoform, which is translated from mRNA variant 2, can directly translocate into the nucleus due to the lack of the signal peptide which is required for an intermediate membrane location. Small interfering RNA-mediated gene silencing showed that ERBB2b can repress ERBB2a expression, encoded by variant 1, whereas ERBB2a activates ERBB2b. Nuclear ERBB2 binding to its own promoter was revealed by chromatin immunoprecipitation assay. Altogether, our results provide new insights into the origin and function of nuclear ERBB2 where it can participate at the same time in a positive or a negative feedback autoregulatory loop, dependent on which of its promoters this bona fide transcription factor is acting. They also provide a new understanding for the resistance to therapies targeting the membrane-anchored ERBB2 in breast cancer.
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Skeletal dysplasias are a clinically and genetically heterogeneous group of bone and cartilage disorders. A total of 436 skeletal dysplasias are listed in the 2015 revised version of the nosology and classification of genetic skeletal disorders, of which nearly 20% are still genetically and molecularly uncharacterized. We report the clinical and molecular characterization of a lethal skeletal dysplasia of the short-rib group caused by mutation of the mouse Fop gene. Fop encodes a centrosomal and centriolar satellite (CS) protein. We show that Fop mutation perturbs ciliogenesis in vivo and that this leads to the alteration of the Hedgehog signaling pathway. Fop mutation reduces CSs movements and affects pericentriolar material composition, which probably participates to the ciliogenesis defect. This study highlights the role of a centrosome and CSs protein producing phenotypes in mice that recapitulate a short rib-polydactyly syndrome when mutated.
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Ciliopatias/genética , Proteínas Proto-Oncogênicas/genética , Síndrome de Costela Curta e Polidactilia/genética , Fatores de Transcrição/genética , Animais , Centríolos/genética , Centrossomo/metabolismo , Centrossomo/patologia , Cílios/genética , Cílios/patologia , Ciliopatias/fisiopatologia , Humanos , Camundongos , Mutação , Síndrome de Costela Curta e Polidactilia/fisiopatologiaRESUMO
BACKGROUND: Triple-negative breast cancers (TNBC) are poor-prognosis tumours candidate to chemotherapy as only systemic treatment. We previously found that PRICKLE1, a prometastatic protein involved in planar cell polarity, is upregulated in TNBC. We investigated the protein complex associated with PRICKLE1 in TNBC to identify proteins possibly involved in metastatic dissemination, which might provide new prognostic and/or therapeutic targets. METHODS: We used a proteomic approach to identify protein complexes associated with PRICKLE1. The mRNA expression levels of the corresponding genes were assessed in 8982 patients with invasive primary breast cancer. We then characterised the molecular interaction between PRICKLE1 and the guanine nucleotide exchange factor ECT2. Finally, experiments in Xenopus were carried out to determine their evolutionarily conserved interaction. RESULTS: Among the PRICKLE1 proteins network, we identified several small G-protein regulators. Combined analysis of the expression of PRICKLE1 and small G-protein regulators had a strong prognostic value in TNBC. Notably, the combined expression of ECT2 and PRICKLE1 provided a worst prognosis than PRICKLE1 expression alone in TNBC. PRICKLE1 regulated ECT2 activity and this interaction was evolutionary conserved. CONCLUSIONS: This work supports the idea that an evolutionarily conserved signalling pathway required for embryogenesis and activated in cancer may represent a suitable therapeutic target.
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Proteínas com Domínio LIM/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Evolução Molecular , Feminino , Humanos , Proteínas com Domínio LIM/genética , Pessoa de Meia-Idade , Prognóstico , Proteoma/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transcriptoma , Neoplasias de Mama Triplo Negativas/genética , Proteínas Supressoras de Tumor/genética , Xenopus laevis , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Curli are amyloid proteins that are assembled into extracellular polymeric fibers by bacteria during biofilm formation. The beta-sheet-rich protein CsgA, the primary structural component of the fibers, is secreted through dedicated machinery and self-assembles into cell-anchored fibers many times longer than the cell. Here, we have developed an in situ fluorescence assay for curli production that exploits the fluorescent properties of Congo red (CR) dye when bound to amyloid, allowing for rapid and robust curli quantification. We initially evaluated three amyloid-binding dyes for the fluorescent detection of curli in bacterial culture and found only Congo red compatible with in situ quantification. We further characterized the fluorescent properties of the dye directly in bacterial culture and calibrated the fluorescence using purified CsgA protein. We then used the Congo red assay to rapidly develop and characterize inducible curli-producing constructs in both an MC4100-derived lab strain of Escherichia coli and a derivative of the probiotic strain E. coli Nissle. This technique can be used to evaluate curli production in a minimally invasive manner using a range of equipment, simplifying curli quantification and the development of novel engineered curli systems.IMPORTANCE Curli are proteins produced by many bacteria as a structural component of biofilms, and they have recently emerged as a platform for fabrication of biological materials. Curli fibers are very robust and resistant to degradation, and the curli subunits can tolerate many protein fusions, facilitating the biosynthesis of novel functional materials. A serious bottleneck in the development of more sophisticated engineered curli systems is the rapid quantification of curli production by the bacteria. In this work we address this issue by developing a technique to monitor curli production directly in bacterial cultures, allowing for rapid curli quantification in a manner compatible with many powerful high-throughput techniques that can be used to engineer complex biological material systems.
Assuntos
Proteínas Amiloidogênicas/química , Vermelho Congo/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas Amiloidogênicas/metabolismo , Biofilmes/crescimento & desenvolvimento , Escherichia coli/química , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fluorescência , Coloração e RotulagemRESUMO
AIMS: This study sought to clarify the molecular pathways underlying the putative evolution from lymphomatoid papulosis (LyP) to cutaneous anaplastic large-cell lymphoma (c-ALCL) and lymph node invasion (LNI). METHODS AND RESULTS: We analysed nine sequential tumours from the same patient presenting with parallel evolution of LyP (n = 3) and c-ALCL (n = 1) with LNI (n = 1), combined with systemic diffuse large B-cell lymphoma (DLBCL) (n = 4). Clonality analysis showed a common clonal T-cell origin in the five CD30+ lesions, and a common clonal B-cell origin in the four DLBCL relapses. Array-comparative genomic hybridisation and targeted next-generation sequencing analysis demonstrated relative genomic stability of LyP lesions as compared with clonally related anaplastic large-cell lymphoma (ALCL) tumours, which showed 4q and 22q13 deletions involving the PRDM8 and TIMP3 tumour suppressor genes, respectively. The three analysed CD30+ lesions showed mostly private (specific to each sample) genetic alterations, suggesting early divergence from a common precursor. In contrast, DLBCL tumours showed progressive accumulation of private alterations, indicating late divergence. CONCLUSIONS: Sequential cutaneous and nodal CD30+ tumours were clonally related. This suggests that LyP, c-ALCL and LNI represent a continuous spectrum of clonal evolution emerging from a common precursor of cutaneous CD30+ lymphoproliferations. Therefore, nodal ALCL tumours in the context of LyP should be considered as a form of transformation rather than composite lymphoma.