RESUMO
Colorectal malignancies are a leading cause of cancer-related death1 and have undergone extensive genomic study2,3. However, DNA mutations alone do not fully explain malignant transformation4-7. Here we investigate the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,370 samples from 30 primary cancers and 8 concomitant adenomas and generated 1,207 chromatin accessibility profiles, 527 whole genomes and 297 whole transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent somatic chromatin accessibility alterations, including in regulatory regions of cancer driver genes that were otherwise devoid of genetic mutations. Genome-wide alterations in accessibility for transcription factor binding involved CTCF, downregulation of interferon and increased accessibility for SOX and HOX transcription factor families, suggesting the involvement of developmental genes during tumourigenesis. Somatic chromatin accessibility alterations were heritable and distinguished adenomas from cancers. Mutational signature analysis showed that the epigenome in turn influences the accumulation of DNA mutations. This study provides a map of genetic and epigenetic tumour heterogeneity, with fundamental implications for understanding colorectal cancer biology.
Assuntos
Neoplasias Colorretais , Epigenoma , Genoma Humano , Mutação , Humanos , Adenoma/genética , Adenoma/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cromatina/genética , Cromatina/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Epigenoma/genética , Oncogenes/genética , Fatores de Transcrição/metabolismo , Genoma Humano/genética , InterferonsRESUMO
Genetic and epigenetic variation, together with transcriptional plasticity, contribute to intratumour heterogeneity1. The interplay of these biological processes and their respective contributions to tumour evolution remain unknown. Here we show that intratumour genetic ancestry only infrequently affects gene expression traits and subclonal evolution in colorectal cancer (CRC). Using spatially resolved paired whole-genome and transcriptome sequencing, we find that the majority of intratumour variation in gene expression is not strongly heritable but rather 'plastic'. Somatic expression quantitative trait loci analysis identified a number of putative genetic controls of expression by cis-acting coding and non-coding mutations, the majority of which were clonal within a tumour, alongside frequent structural alterations. Consistently, computational inference on the spatial patterning of tumour phylogenies finds that a considerable proportion of CRCs did not show evidence of subclonal selection, with only a subset of putative genetic drivers associated with subclone expansions. Spatial intermixing of clones is common, with some tumours growing exponentially and others only at the periphery. Together, our data suggest that most genetic intratumour variation in CRC has no major phenotypic consequence and that transcriptional plasticity is, instead, widespread within a tumour.
Assuntos
Adaptação Fisiológica , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Fenótipo , Humanos , Adaptação Fisiológica/genética , Células Clonais/metabolismo , Células Clonais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Mutação , Sequenciamento do Exoma , Transcrição GênicaRESUMO
BACKGROUND: Previous studies have provided a comprehensive picture of genomic alterations in primary and metastatic Hormone Receptor (HR)-positive, Human Epidermal growth factor Receptor 2 (HER2)-negative breast cancer (HR+ HER2- BC). However, the evolution of the genomic landscape of HR+ HER2- BC during adjuvant endocrine therapies (ETs) remains poorly investigated. METHODS AND FINDINGS: We performed a genomic characterization of surgically resected HR+ HER2- BC patients relapsing during or at the completion of adjuvant ET. Using a customized panel, we comprehensively evaluated gene mutations and copy number variation (CNV) in paired primary and metastatic specimens. After retrieval and quality/quantity check of tumor specimens from an original cohort of 204 cases, 74 matched tumor samples were successfully evaluated for DNA mutations and CNV analysis. Along with previously reported genomic alterations, including PIK3CA, TP53, CDH1, GATA3 and ESR1 mutations/deletions, we found that ESR1 gene amplification (confirmed by FISH) and MAP3K mutations were enriched in metastatic lesions as compared to matched primary tumors. These alterations were exclusively found in patients treated with adjuvant aromatase inhibitors or LHRH analogs plus tamoxifen, but not in patients treated with tamoxifen alone. Patients with tumors bearing MAP3K mutations in metastatic lesions had significantly worse distant relapse-free survival (hazard ratio [HR] 3.4, 95% CI 1.52-7.70, p value 0.003) and worse overall survival (HR 5.2, 95% CI 2.10-12.8, p-value < 0.001) independently of other clinically relevant patient- and tumor-related variables. CONCLUSIONS: ESR1 amplification and activating MAP3K mutations are potential drivers of acquired resistance to adjuvant ETs employing estrogen deprivation in HR+ HER2- BC. MAP3K mutations are associated with worse prognosis in patients with metastatic disease.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Variações do Número de Cópias de DNA/genética , Amplificação de Genes , Mutação , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Receptor ErbB-2/genética , TamoxifenoRESUMO
Understanding the biological and clinical impact of copy number aberrations (CNAs) on the development of precision therapies in cancer remains an unmet challenge. Genetic amplification of chromosome 1q (chr1q-amp) is a major CNA conferring an adverse prognosis in several types of cancer, including in the blood cancer multiple myeloma (MM). Although several genes across chromosome 1 (chr1q) portend high-risk MM disease, the underpinning molecular etiology remains elusive. Here, with reference to the 3-dimensional (3D) chromatin structure, we integrate multi-omics data sets from patients with MM with genetic variables to obtain an associated clinical risk map across chr1q and to identify 103 adverse prognosis genes in chr1q-amp MM. Prominent among these genes, the transcription factor PBX1 is ectopically expressed by genetic amplification and epigenetic activation of its own preserved 3D regulatory domain. By binding to reprogrammed superenhancers, PBX1 directly regulates critical oncogenic pathways and a FOXM1-dependent transcriptional program. Together, PBX1 and FOXM1 activate a proliferative gene signature that predicts adverse prognosis across multiple types of cancer. Notably, pharmacological disruption of the PBX1-FOXM1 axis with existing agents (thiostrepton) and a novel PBX1 small molecule inhibitor (T417) is selectively toxic against chr1q-amp myeloma and solid tumor cells. Overall, our systems medicine approach successfully identifies CNA-driven oncogenic circuitries, links them to clinical phenotypes, and proposes novel CNA-targeted therapy strategies in MM and other types of cancer.
Assuntos
Mieloma Múltiplo , Cromossomos Humanos Par 1/metabolismo , Proteína Forkhead Box M1/genética , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Prognóstico , Análise de Sistemas , Fatores de Transcrição/genéticaRESUMO
The effects of excitability, refractoriness, and impulse conduction have been independently related to enhanced arrhythmias in the aged myocardium in experimental and clinical studies. However, their combined arrhythmic effects in the elderly are not yet completely understood. Hence, the aim of the present work is to relate relevant cardiac electrophysiological parameters to enhanced arrhythmia vulnerability in the in vivo senescent heart. We used multiple-lead epicardial potential mapping in control (9-month-old) and aged (24-month-old) rat hearts. Cardiac excitability and refractoriness were evaluated at numerous epicardial test sites by means of the strength-duration curve and effective refractory period, respectively. During sinus rhythm, durations of electrogram intervals and waves were prolonged in the senescent heart, compared with control, demonstrating a latency in tissue activation and recovery. During ventricular pacing, cardiac excitability, effective refractory period, and dispersion of refractoriness increased in the aged animal. This scenario was accompanied by impairment of impulse propagation. Moreover, both spontaneous and induced arrhythmias were increased in senescent cardiac tissue. Histopathological evaluation of aged heart specimens revealed connective tissue deposition and perinuclear myocytolysis in the atria, while scattered microfoci of interstitial fibrosis were mostly present in the ventricular subendocardium. This work suggests that enhanced arrhythmogenesis in the elderly is a multifactorial process due to the joint increase in excitability and dispersion of refractoriness in association with enhanced conduction inhomogeneity. The knowledge of these electrophysiological changes will possibly contribute to improved prevention of the age-associated increase in cardiac arrhythmias.
Assuntos
Arritmias Cardíacas , Sistema de Condução Cardíaco , Masculino , Ratos , Animais , Miocárdio , Ventrículos do Coração , Átrios do CoraçãoRESUMO
BACKGROUND: Current popular variant calling pipelines rely on the mapping coordinates of each input read to a reference genome in order to detect variants. Since reads deriving from variant loci that diverge in sequence substantially from the reference are often assigned incorrect mapping coordinates, variant calling pipelines that rely on mapping coordinates can exhibit reduced sensitivity. RESULTS: In this work we present GeDi, a suffix array-based somatic single nucleotide variant (SNV) calling algorithm that does not rely on read mapping coordinates to detect SNVs and is therefore capable of reference-free and mapping-free SNV detection. GeDi executes with practical runtime and memory resource requirements, is capable of SNV detection at very low allele frequency (<1%), and detects SNVs with high sensitivity at complex variant loci, dramatically outperforming MuTect, a well-established pipeline. CONCLUSION: By designing novel suffix-array based SNV calling methods, we have developed a practical SNV calling software, GeDi, that can characterise SNVs at complex variant loci and at low allele frequency thus increasing the repertoire of detectable SNVs in tumour genomes. We expect GeDi to find use cases in targeted-deep sequencing analysis, and to serve as a replacement and improvement over previous suffix-array based SNV calling methods.
Assuntos
Variação Genética , Genoma , Neoplasias/genética , Software , Algoritmos , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sequenciamento Completo do GenomaRESUMO
OBJECTIVES: To investigate serum levels of a panel of angiogenic inducers (VEGF, FGF-2, Angiopoietin 1, -2, soluble VCAM-1) and inhibitors (angiostatin, endostatin, pentraxin-3) in patients with giant cell arteritis (GCA) and Takayasu's arteritis (TAK), in order to gain further insights into the molecular mechanisms driving angiogenesis dysregulation in large-vessel vasculitis (LVV). METHODS: Sera were obtained from 33 TAK patients and 14 GCA patients and from two groups of age-matched normal controls (NC). Disease activity was assessed using 18F-FDG PET/CT and clinical indices including NIH/Kerr criteria and ITAS. Angiogenic and anti-angiogenic factor serum levels were evaluated using commercial ELISA kits. Pentraxin 3 (PTX3) serum levels were evaluated by non-commercial ELISA, as already described. RESULTS: Among the angiogenic factors, only VEGF serum levels were significantly higher in TAK patients compared to NC. No difference was found between angiogenic factor levels in GCA patients compared to those detected in NC. Anti-angiogenic factor (Angiostatin, Endostatin, PTX3) serum levels were significantly higher in both GCA and TAK patients compared to NC. Significant associations were observed between VEGF and PTX3 levels and disease activity evaluated using PET scan and clinical indices. Cluster analysis based on PET scan scores in TAK patients showed significant ordered differences in VEGF and angiostatin serum levels. Indeed, we noted a progressive increase of VEGF and angiostatin from NC to the cluster including patients with the highest and more diffuse scan positivity. CONCLUSIONS: Our overall results demonstrate a circulating molecular profile characterised by a prevailing expression of anti-angiogenic soluble factors.
Assuntos
Proteínas Angiogênicas/sangue , Proteínas Angiostáticas/sangue , Arterite de Células Gigantes/sangue , Arterite de Takayasu/sangue , Angiopoietina-1 , Angiopoietina-2 , Angiostatinas , Proteína C-Reativa , Endostatinas , Fator 2 de Crescimento de Fibroblastos , Humanos , Neovascularização Patológica/sangue , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia por Emissão de Pósitrons , Componente Amiloide P Sérico , Molécula 1 de Adesão de Célula Vascular , Fator A de Crescimento do Endotélio VascularRESUMO
OBJECTIVES: Systemic sclerosis (SSc) is a severe multiple-organ disease characterised by unpredictable clinical course, inadequate response to treatment, and poor prognosis. National SSc registries may provide large and representative patients cohorts required for descriptive and prognostic studies. Therefore, the Italian Society for Rheumatology promoted the registry SPRING (Systemic sclerosis Progression INvestiGation). METHODS: The SPRING is a multi-centre rheumatological cohort study encompassing the wide scleroderma spectrum, namely the primary Raynaud's phenomenon (pRP), suspected secondary RP, Very Early Diagnosis of Systemic Sclerosis (VEDOSS), and definite SSc. Here we describe the demographic and clinical characteristics of a population of 2,028 Italian patients at the initial phase of enrolment, mainly focusing on the cohort of 1,538 patients with definite SSc. RESULTS: Definite SSc showed a significantly higher prevalence of digital ulcers, capillaroscopic 'late' pattern, oesophageal and cardio-pulmonary involvement compared to VEDOSS, as expected on the basis of the followed classification criteria. The in-depth analysis of definite SSc revealed that male gender, diffuse cutaneous subset, and anti-Scl70 seropositivity were significantly associated with increased prevalence of the most harmful disease manifestations. Similarly, patients with very short RP duration (≤1 year) at SSc diagnosis showed a statistically increased prevalence of unfavourable clinico-serological features. CONCLUSIONS: Nationwide registries with suitable subsetting of patients and follow-up studies since the prodromal phase of the disease may give us valuable insights into the SSc natural history and main prognostic factors.
Assuntos
Doença de Raynaud , Escleroderma Sistêmico , Estudos de Coortes , Humanos , Itália , Masculino , Angioscopia Microscópica , Sistema de RegistrosRESUMO
Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.
Assuntos
Código das Histonas , Histonas/metabolismo , Neoplasias/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Xenoenxertos , Código das Histonas/genética , Histonas/genética , Humanos , Camundongos , Camundongos Nus , Modelos Biológicos , Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Células Tumorais CultivadasRESUMO
Chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-seq) has greatly improved the reliability with which transcription factor binding sites (TFBSs) can be identified from genome-wide profiling studies. Many computational tools are developed to detect binding events or peaks, however the robust detection of weak binding events remains a challenge for current peak calling tools. We have developed a novel Bayesian approach (ChIP-BIT) to reliably detect TFBSs and their target genes by jointly modeling binding signal intensities and binding locations of TFBSs. Specifically, a Gaussian mixture model is used to capture both binding and background signals in sample data. As a unique feature of ChIP-BIT, background signals are modeled by a local Gaussian distribution that is accurately estimated from the input data. Extensive simulation studies showed a significantly improved performance of ChIP-BIT in target gene prediction, particularly for detecting weak binding signals at gene promoter regions. We applied ChIP-BIT to find target genes from NOTCH3 and PBX1 ChIP-seq data acquired from MCF-7 breast cancer cells. TF knockdown experiments have initially validated about 30% of co-regulated target genes identified by ChIP-BIT as being differentially expressed in MCF-7 cells. Functional analysis on these genes further revealed the existence of crosstalk between Notch and Wnt signaling pathways.
Assuntos
Imunoprecipitação da Cromatina/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Modelos Estatísticos , Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo , Teorema de Bayes , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Células K562 , Células MCF-7 , Fator de Transcrição 1 de Leucemia de Células Pré-B , Proteínas Proto-Oncogênicas/metabolismo , Receptor Notch3 , Receptores Notch/metabolismoRESUMO
The hallmarks of cancer capture the most essential phenotypic characteristics of malignant transformation and progression. Although numerous factors involved in this multi-step process are still unknown to date, an ever-increasing number of mutated/altered candidate genes are being identified within large-scale cancer genomic projects. Therefore, investigators need to be aware of available and appropriate techniques capable of determining characteristic features of each hallmark. We review the methods tailored to experimental cancer researchers to evaluate cell proliferation, programmed cell death, replicative immortality, induction of angiogenesis, invasion and metastasis, genome instability, and reprogramming of energy metabolism. Selecting the ideal method is based on the investigator's goals, available equipment and also on financial constraints. Multiplexing strategies enable a more in-depth data collection from a single experiment - obtaining several results from a single procedure reduces variability and saves time and relative cost, leading to more robust conclusions compared to a single end point measurement. Each hallmark possesses characteristics that can be analyzed by immunoblot, RT-PCR, immunocytochemistry, immunoprecipitation, RNA microarray or RNA-seq. In general, flow cytometry, fluorescence microscopy, and multiwell readers are extremely versatile tools and, with proper sample preparation, allow the detection of a vast number of hallmark features. Finally, we also provide a list of hallmark-specific genes to be measured in transcriptome-level studies. Although our list is not exhaustive, we provide a snapshot of the most widely used methods, with an emphasis on methods enabling the simultaneous evaluation of multiple hallmark features.
Assuntos
Neoplasias/patologia , Apoptose , Caspases/análise , Proliferação de Células , Metabolismo Energético , Epigênese Genética , Instabilidade Genômica , Guias como Assunto , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neovascularização Patológica/etiologia , TelômeroRESUMO
OBJECTIVES: To investigate serum levels of IL- 6 and soluble IL-6 receptor (sIL-6R) in patients with large-vessel vasculitis and their relationship with disease activity. METHODS: Sera were obtained from 33 Takayasu's arteritis (TAK) patients and 14 giant cell arteritis (GCA) patients, and from 60 age-matched normal controls (NCs). Disease activity was assessed using 18F-FDG PET/CT and clinical indices including NIH/Kerr criteria and ITAS. Among TAK patients with active disease at baseline, clinical records and serum samples from 11 TAK patients were available for the longitudinal study. IL-6 and sIL-6R serum levels were evaluated using commercial ELISA kits. RESULTS: IL-6 and sIL-6R serum levels were significantly higher in both GCA and TAK patients compared to NCs. IL-6 levels in TAK patients were significantly increased irrespective of disease phase, while a significant increase in sIL-6R concentrations was only found in TAK patients with active disease. Conversely, in GCA, IL-6 levels were significantly raised only in patients with active diseases, whereas sIL-6R levels appeared to be significantly higher irrespective of disease activity. Longitudinal analysis showed that levels of sIL-6R in TAK patients were significantly higher only at baseline, compared to NCs, whereas IL-6 levels were found to be significantly increased at each follow-up time point. CONCLUSIONS: These overall results might suggest a role for sIL-6R as a potential biomarker for disease activity in TAK patients, whereas in GCA, modifications of IL-6 might better identify patients with active disease.
Assuntos
Arterite de Células Gigantes/sangue , Interleucina-6/sangue , Receptores de Interleucina-6/sangue , Arterite de Takayasu/sangue , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Análise por Conglomerados , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Fluordesoxiglucose F18/administração & dosagem , Arterite de Células Gigantes/diagnóstico por imagem , Arterite de Células Gigantes/imunologia , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Valor Preditivo dos Testes , Prognóstico , Compostos Radiofarmacêuticos/administração & dosagem , Índice de Gravidade de Doença , Arterite de Takayasu/diagnóstico por imagem , Arterite de Takayasu/imunologia , Fatores de Tempo , Regulação para CimaRESUMO
Chromatin constitutes a repressive barrier to the process of ligand-dependent transcriptional activity of nuclear receptors. Nucleosomes prevent the binding of estrogen receptor α (ERα) in absence of ligand and thus represent an important level of transcriptional regulation. Here, we show that in breast cancer MCF-7 cells, TLE3, a co-repressor of the Groucho/Grg/TLE family, interacts with FoxA1 and is detected at regulatory elements of ERα target genes in absence of estrogen. As a result, the chromatin is maintained in a basal state of acetylation, thus preventing ligand-independent activation of transcription. In absence of TLE3, the basal expression of ERα target genes induced by E2 is increased. At the TFF1 gene, the recruitment of TLE3 to the chromatin is FoxA1-dependent and prevents ERα and RNA polymerase II recruitment to TFF1 gene regulatory elements. Moreover, the interaction of TLE3 with HDAC2 results in the maintenance of acetylation at a basal level. We also provide evidence that TLE3 is recruited at several other regulatory elements of ERα target genes and is probably an important co-regulator of the E2 signaling pathway. In sum, our results describe a mechanism by which TLE3 affects ligand dependency in ERα-regulated gene expression via its binding restricting function and its role in gene regulation by histone acetylation.
Assuntos
Proteínas Correpressoras/metabolismo , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica , Linhagem Celular , Cromatina/metabolismo , Proteínas Correpressoras/fisiologia , Fator 3-alfa Nuclear de Hepatócito/fisiologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Células MCF-7 , Elementos Reguladores de Transcrição , Transdução de Sinais , Transcrição GênicaRESUMO
The estrogen receptor (ER)α drives growth in two-thirds of all breast cancers. Several targeted therapies, collectively termed endocrine therapy, impinge on estrogen-induced ERα activation to block tumor growth. However, half of ERα-positive breast cancers are tolerant or acquire resistance to endocrine therapy. We demonstrate that genome-wide reprogramming of the chromatin landscape, defined by epigenomic maps for regulatory elements or transcriptional activation and chromatin openness, underlies resistance to endocrine therapy. This annotation reveals endocrine therapy-response specific regulatory networks where NOTCH pathway is overactivated in resistant breast cancer cells, whereas classical ERα signaling is epigenetically disengaged. Blocking NOTCH signaling abrogates growth of resistant breast cancer cells. Its activation state in primary breast tumors is a prognostic factor of resistance in endocrine treated patients. Overall, our work demonstrates that chromatin landscape reprogramming underlies changes in regulatory networks driving endocrine therapy resistance in breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Montagem e Desmontagem da Cromatina/fisiologia , Epigênese Genética/fisiologia , Receptor alfa de Estrogênio/metabolismo , Redes Reguladoras de Genes/fisiologia , Transdução de Sinais/fisiologia , Western Blotting , Neoplasias da Mama/metabolismo , Imunoprecipitação da Cromatina , Epigênese Genética/genética , Feminino , Redes Reguladoras de Genes/genética , Humanos , Estimativa de Kaplan-Meier , Células MCF-7 , Análise em Microsséries , Receptores Notch/metabolismo , Transdução de Sinais/genéticaRESUMO
INTRODUCTION: Resistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ERα)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells. METHODS: We used two models of endocrine therapies resistant (ETR) breast cancer: tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF7 cells. We evaluated the migratory and invasive capacity of these cells by Transwell assays. Expression of epithelial to mesenchymal transition (EMT) regulators as well as Notch receptors and targets were evaluated by real-time PCR and western blot analysis. Moreover, we tested in vitro anti-Nicastrin monoclonal antibodies (mAbs) and gamma secretase inhibitors (GSIs) as potential EMT reversal therapeutic agents. Finally, we generated stable Nicastrin overexpessing MCF7 cells and evaluated their EMT features and response to tamoxifen. RESULTS: We found that ETR cells acquired an epithelial to mesenchymal transition (EMT) phenotype and displayed increased levels of Nicastrin and Notch targets. Interestingly, we detected higher level of Notch4 but lower levels of Notch1 and Notch2 suggesting a switch to signalling through different Notch receptors after acquisition of resistance. Anti-Nicastrin monoclonal antibodies and the GSI PF03084014 were effective in blocking the Nicastrin/Notch4 axis and partially inhibiting the EMT process. As a result of this, cell migration and invasion were attenuated and the stem cell-like population was significantly reduced. Genetic silencing of Nicastrin and Notch4 led to equivalent effects. Finally, stable overexpression of Nicastrin was sufficient to make MCF7 unresponsive to tamoxifen by Notch4 activation. CONCLUSIONS: ETR cells express high levels of Nicastrin and Notch4, whose activation ultimately drives invasive behaviour. Anti-Nicastrin mAbs and GSI PF03084014 attenuate expression of EMT molecules reducing cellular invasiveness. Nicastrin overexpression per se induces tamoxifen resistance linked to acquisition of EMT phenotype. Our finding suggest that targeting Nicastrin and/or Notch4 warrants further clinical evaluation as valid therapeutic strategies in endocrine-resistant breast cancer.
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Neoplasias da Mama/tratamento farmacológico , Transição Epitelial-Mesenquimal/fisiologia , Glicoproteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Receptores Notch/genética , Tamoxifeno/farmacologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/biossíntese , Anticorpos Monoclonais/farmacologia , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/patologia , Antígeno CD24/metabolismo , Movimento Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/biossíntese , Invasividade Neoplásica , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Receptor Notch4 , Receptores Notch/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Esferoides Celulares , Tetra-Hidronaftalenos/farmacologia , Células Tumorais Cultivadas , Valina/análogos & derivados , Valina/farmacologiaRESUMO
Chromatin is a well-known obstacle to transcription as it controls DNA accessibility, which directly impacts the recruitment of the transcriptional machinery. The recent burst of functional genomic studies provides new clues as to how transcriptional competency is regulated in this context. In this review, we discuss how these studies have shed light on a specialized subset of transcription factors, defined as pioneer factors, which direct recruitment of downstream transcription factors to establish lineage-specific transcriptional programs. In particular, we present evidence of an interplay between pioneer factors and the epigenome that could be central to this process. Finally, we discuss how pioneer factors, whose expression and function are altered in tumors, are also being considered for their prognostic value and should therefore be regarded as potential therapeutic targets. Thus, pioneer factors emerge as key players that connect the epigenome and transcription in health and disease.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Animais , Montagem e Desmontagem da Cromatina/genética , Humanos , Modelos Biológicos , Ligação Proteica/fisiologia , Transporte Proteico/genética , Fatores de Tempo , Fatores de Transcrição/genética , Transcrição Gênica/fisiologiaRESUMO
OBJECTIVE: Chronic periaortitis (CP) usually responds to glucocorticoids, but some patients have glucocorticoid-refractory disease or contraindications to glucocorticoid therapy. This study was undertaken to evaluate treatment with the anti-interleukin-6 receptor (anti-IL-6R) antibody tocilizumab in 2 patients with CP, one with refractory disease and the other with contraindications to glucocorticoids, and to assess IL-6 levels in an additional cohort of patients with CP. METHODS: Both patients were given intravenous tocilizumab (8 mg/kg) once every 4 weeks for 6 months. Serum IL-6 was measured in 22 patients with active CP and 16 healthy controls. Tissue IL-6 expression was assessed by confocal microscopy in biopsy specimens obtained from 6 patients with CP. RESULTS: In the first patient, whose disease was refractory to various immunosuppressive treatments, tocilizumab added to ongoing therapy with prednisone and methotrexate allowed prednisone withdrawal and induced resolution of symptoms, acute-phase reactant normalization, and reduction in (18) F-fluorodeoxyglucose ((18) F-FDG) uptake on positron emission tomography. The patient experienced a relapse 7 months later and was successfully retreated with tocilizumab. In the second patient, who was unable to tolerate glucocorticoids because of psychiatric side effects, tocilizumab monotherapy induced sustained clinical and laboratory remission, (18) F-FDG uptake disappearance, and CP shrinkage. Serum IL-6 levels were significantly higher in patients with active CP than in controls (P < 0.0001), and IL-6 was abundantly expressed in biopsy specimens from CP patients, particularly by T cells, B cells, histiocytes, fibroblasts, and vascular smooth muscle cells. CONCLUSION: Tocilizumab may be a therapeutic option for CP. The systemic and tissue up-regulation of IL-6 in CP, together with the clinical benefit of IL-6R blockade observed in our 2 patients, suggest that IL-6 may contribute to CP pathogenesis.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Receptores de Interleucina-6/antagonistas & inibidores , Fibrose Retroperitoneal/metabolismo , Idoso , Feminino , Humanos , Imunossupressores/uso terapêutico , Mediadores da Inflamação/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Fibrose Retroperitoneal/sangue , Fibrose Retroperitoneal/tratamento farmacológico , Resultado do TratamentoRESUMO
Altered transcriptional programs are a hallmark of diseases, yet how these are established is still ill-defined. PBX1 is a TALE homeodomain protein involved in the development of different types of cancers. The estrogen receptor alpha (ERα) is central to the development of two-thirds of all breast cancers. Here we demonstrate that PBX1 acts as a pioneer factor and is essential for the ERα-mediated transcriptional response driving aggressive tumors in breast cancer. Indeed, PBX1 expression correlates with ERα in primary breast tumors, and breast cancer cells depleted of PBX1 no longer proliferate following estrogen stimulation. Profiling PBX1 recruitment and chromatin accessibility across the genome of breast cancer cells through ChIP-seq and FAIRE-seq reveals that PBX1 is loaded and promotes chromatin openness at specific genomic locations through its capacity to read specific epigenetic signatures. Accordingly, PBX1 guides ERα recruitment to a specific subset of sites. Expression profiling studies demonstrate that PBX1 controls over 70% of the estrogen response. More importantly, the PBX1-dependent transcriptional program is associated with poor-outcome in breast cancer patients. Correspondingly, PBX1 expression alone can discriminate a priori the outcome in ERα-positive breast cancer patients. These features are markedly different from the previously characterized ERα-associated pioneer factor FoxA1. Indeed, PBX1 is the only pioneer factor identified to date that discriminates outcome such as metastasis in ERα-positive breast cancer patients. Together our results reveal that PBX1 is a novel pioneer factor defining aggressive ERα-positive breast tumors, as it guides ERα genomic activity to unique genomic regions promoting a transcriptional program favorable to breast cancer progression.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/metabolismo , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas/metabolismo , Sítios de Ligação , Proliferação de Células , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Estimativa de Kaplan-Meier , Fator de Transcrição 1 de Leucemia de Células Pré-B , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Ativação Transcricional , Resultado do TratamentoRESUMO
Pathological studies have demonstrated that the adventitial layer is markedly thickened in Takayasu (TAK) as compared to large vessel giant cell arteritis (LV-GCA). An ultrasound (US) examination of the arterial vessels allows the determination of intima media thickness (IMT) and of adventitial layer thickness (extra media thickness (EMT)). No previous study has evaluated if there are differences in EMT thickness between TAK and LV-GCA. In this cross-sectional retrospective study of stored ultrasound (US) imaging, we have compared common carotid artery (CCA) EMT and IMT in a series of consecutive TAK and LV-GCA patients. US examination CCA IMT and EMT were significantly higher in TAK as compared to LV-GCA. With ROC curve analysis, we have found that an EMT > 0.76 mm has high sensitivity and specificity for TAK CCA examination. The percentage of CCA at EMT > 0.76 mm and the total arterial wall thickening were significantly higher in TAK group examinations. EMT thickness correlated with disease duration and IMT in the TAK group, as well as with the IMT and ESR values in the LV-GCA group. Upon multivariate logistic regression analysis, factors independently associated with TAK CCA were EMT > 0.76 mm and age. No significant variation in IMT and EMT could be demonstrated in subsequent US CCA examinations.
RESUMO
OBJECTIVES: The current knowledge about the role of comorbidities in systemic sclerosis (SSc) is limited. Therefore, the aim of this study was to evaluate the prevalence of comorbidities and their impact on disease activity and prognosis in the Systemic sclerosis PRogression INvestiGation (SPRING) registry. METHODS: SSc patients from the SPRING registry, fulfilling the ACR/EULAR 2013 classification criteria, with complete data on baseline comorbidities were enrolled. The Charlson comorbidity index (CCI) was used to quantify the overall comorbidity burden. The disease activity was calculated using the revised EUSTAR activity index (AI). The impact of SSc features on CCI, the effect of CCI on SSc disease activity and mortality were tested with multivariable regression models. RESULTS: Among 1910 SSc patients enrolled, 67.3 % had at least one comorbidity at baseline. The most frequent comorbidities were systemic arterial hypertension (23.7 %), osteoporosis (12.9 %) and dyslipidemia (11 %). The mean value of CCI score was 2.0 ± 1.8. When patients were grouped according to increasing levels of CCI, a clear separation in the distribution of SSc-related clinical features could be observed. Among over 900 patients with available follow-up, no association between baseline CCI and changes in disease activity was observed. Conversely, the risk of death over time was independently predicted by both CCI and AI. CONCLUSIONS: Comorbidities and disease activity independently impact on the prognosis of SSc patients. This suggests that the management of comorbidities, together with the reduction of disease activity, is fundamental to improve patient survival.