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Introduction: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. This study integrates common genetic association results from the latest ALS genome-wide association study (GWAS) summary statistics with functional genomic annotations with the aim of providing mechanistic insights into ALS risk loci, inferring drug repurposing opportunities, and enhancing prediction of ALS risk and clinical characteristics. Methods: Genes associated with ALS were identified using GWAS summary statistic methodology including SuSiE SNP-based fine-mapping, and transcriptome- and proteome-wide association study (TWAS/PWAS) analyses. Using several approaches, gene associations were integrated with the DrugTargetor drug-gene interaction database to identify drugs that could be repurposed for the treatment of ALS. Furthermore, ALS gene associations from TWAS were combined with observed blood expression in two external ALS case-control datasets to calculate polytranscriptomic scores and evaluate their utility for prediction of ALS risk and clinical characteristics, including site of onset, age at onset, and survival. Results: SNP-based fine-mapping, TWAS and PWAS identified 118 genes associated with ALS, with TWAS and PWAS providing novel mechanistic insights. Drug repurposing analyses identified six drugs significantly enriched for interactions with ALS associated genes, though directionality could not be determined. Additionally, drug class enrichment analysis showed gene signatures linked to calcium channel blockers may reduce ALS risk, whereas antiepileptic drugs may increase ALS risk. Across the two observed expression target samples, ALS polytranscriptomic scores significantly predicted ALS risk (R 2 = 5.1 %; p-value = 3.2 × 10-27) and clinical characteristics. Conclusions: Functionally-informed analyses of ALS GWAS summary statistics identified novel mechanistic insights into ALS aetiology, highlighted several therapeutic research avenues, and enabled statistically significant prediction of ALS risk.
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Single-cell multiomic analysis of the epigenome, transcriptome, and proteome allows for comprehensive characterization of the molecular circuitry that underpins cell identity and state. However, the holistic interpretation of such datasets presents a challenge given a paucity of approaches for systematic, joint evaluation of different modalities. Here, we present Panpipes, a set of computational workflows designed to automate multimodal single-cell and spatial transcriptomic analyses by incorporating widely-used Python-based tools to perform quality control, preprocessing, integration, clustering, and reference mapping at scale. Panpipes allows reliable and customizable analysis and evaluation of individual and integrated modalities, thereby empowering decision-making before downstream investigations.
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Análisis de la Célula Individual , Programas Informáticos , Transcriptoma , Análisis de la Célula Individual/métodos , Perfilación de la Expresión Génica/métodos , Humanos , Flujo de TrabajoRESUMEN
Lymph node (LN) fine needle aspiration (LN FNA) represents a powerful technique for minimally invasive sampling of human LNs in vivo and has been used effectively to directly study aspects of the human germinal center response. However, systematic deep phenotyping of the cellular populations and cell-free proteins recovered by LN FNA has not been performed. Thus, we studied human cervical LN FNAs as a proof-of-concept and used single-cell RNA-sequencing and proteomic analysis to benchmark this compartment, define the purity of LN FNA material, and facilitate future studies in this immunologically pivotal environment. Our data provide evidence that LN FNAs contain bone-fide LN-resident innate immune populations, with minimal contamination of blood material. Examination of these populations reveals unique biology not predictable from equivalent blood-derived populations. LN FNA supernatants represent a specific source of lymph- and lymph node-derived proteins, and can, aided by transcriptomics, identify likely receptor-ligand interactions. This represents the first description of the types and abundance of immune cell populations and cell-free proteins that can be efficiently studied by LN FNA. These findings are of broad utility for understanding LN physiology in health and disease, including infectious or autoimmune perturbations, and in the case of cervical nodes, neuroscience.
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Ganglios Linfáticos , Humanos , Ganglios Linfáticos/inmunología , Biopsia con Aguja Fina/métodos , Proteómica/métodos , Inmunidad Innata , Femenino , Análisis de la Célula Individual/métodos , Centro Germinal/inmunología , MasculinoRESUMEN
Introduction: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. This study integrates the latest ALS genome-wide association study (GWAS) summary statistics with functional genomic annotations with the aim of providing mechanistic insights into ALS risk loci, inferring drug repurposing opportunities, and enhancing prediction of ALS risk and clinical characteristics. Methods: Genes associated with ALS were identified using GWAS summary statistic methodology including SuSiE SNP-based fine-mapping, and transcriptome- and proteome-wide association study (TWAS/PWAS) analyses. Using several approaches, gene associations were integrated with the DrugTargetor drug-gene interaction database to identify drugs that could be repurposed for the treatment of ALS. Furthermore, ALS gene associations from TWAS were combined with observed blood expression in two external ALS case-control datasets to calculate polytranscriptomic scores and evaluate their utility for prediction of ALS risk and clinical characteristics, including site of onset, age at onset, and survival. Results: SNP-based fine-mapping, TWAS and PWAS identified 117 genes associated with ALS, with TWAS and PWAS providing novel mechanistic insights. Drug repurposing analyses identified five drugs significantly enriched for interactions with ALS associated genes, with directional analyses highlighting α-glucosidase inhibitors may exacerbate ALS pathology. Additionally, drug class enrichment analysis showed calcium channel blockers may reduce ALS risk. Across the two observed expression target samples, ALS polytranscriptomic scores significantly predicted ALS risk (R2 = 4%; p-value = 2.1×10-21). Conclusions: Functionally-informed analyses of ALS GWAS summary statistics identified novel mechanistic insights into ALS aetiology, highlighted several therapeutic research avenues, and enabled statistically significant prediction of ALS risk.
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Alzheimer's disease (AD) is the most common form of dementia, and risk-influencing genetics implicates microglia and neuroimmunity in the pathogenesis of AD. Induced pluripotent stem cell (iPSC)-derived microglia (iPSC-microglia) are increasingly used as a model of AD, but the relevance of historical immune stimuli to model AD is unclear. We performed a detailed cross-comparison over time on the effects of combinatory stimulation of iPSC-microglia, and in particular their relevance to AD. We used single-cell RNA sequencing to measure the transcriptional response of iPSC-microglia after 24â h and 48â h of stimulation with prostaglandin E2 (PGE2) or lipopolysaccharide (LPS)+interferon gamma (IFN-γ), either alone or in combination with ATPγS. We observed a shared core transcriptional response of iPSC-microglia to ATPγS and to LPS+IFN-γ, suggestive of a convergent mechanism of action. Across all conditions, we observed a significant overlap, although directional inconsistency to genes that change their expression levels in human microglia from AD patients. Using a data-led approach, we identify a common axis of transcriptomic change across AD genetic mouse models of microglia and show that only LPS provokes a transcriptional response along this axis in mouse microglia and LPS+IFN-γ in human iPSC-microglia. This article has an associated First Person interview with the first author of the paper.
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Enfermedad de Alzheimer , Microglía , Enfermedad de Alzheimer/metabolismo , Animales , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Humanos , Interferón gamma/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Ratones , Microglía/patología , Transcriptoma/genéticaRESUMEN
Thirst emerges from a range of cellular changes that ultimately motivate an animal to consume water. Although thirst-responsive neuronal signals have been reported, the full complement of brain responses is unclear. Here, we identify molecular and cellular adaptations in the brain using single-cell sequencing of water-deprived Drosophila. Water deficiency primarily altered the glial transcriptome. Screening the regulated genes revealed astrocytic expression of the astray-encoded phosphoserine phosphatase to bi-directionally regulate water consumption. Astray synthesizes the gliotransmitter D-serine, and vesicular release from astrocytes is required for drinking. Moreover, dietary D-serine rescues aay-dependent drinking deficits while facilitating water consumption and expression of water-seeking memory. D-serine action requires binding to neuronal NMDA-type glutamate receptors. Fly astrocytes contribute processes to tripartite synapses, and the proportion of astrocytes that are themselves activated by glutamate increases with water deprivation. We propose that thirst elevates astrocytic D-serine release, which awakens quiescent glutamatergic circuits to enhance water procurement.
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Serina , Transmisión Sináptica , Animales , Astrocitos/metabolismo , Drosophila/metabolismo , Ácido Glutámico/metabolismo , N-Metilaspartato/metabolismo , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismo , Transmisión Sináptica/fisiología , Sed , Agua/metabolismoRESUMEN
For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.
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Drosophila melanogaster/citología , Drosophila melanogaster/genética , Transcriptoma , Animales , Núcleo Celular/metabolismo , Bases de Datos Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Femenino , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genes de Insecto , Masculino , RNA-Seq , Caracteres Sexuales , Análisis de la Célula Individual , Factores de Transcripción/genéticaRESUMEN
α-Synuclein is critical in the pathogenesis of Parkinson's disease and related disorders, yet it remains unclear how its aggregation causes degeneration of human dopaminergic neurons. In this study, we induced α-synuclein aggregation in human iPSC-derived dopaminergic neurons using fibrils generated de novo or amplified in the presence of brain homogenates from Parkinson's disease or multiple system atrophy. Increased α-synuclein monomer levels promote seeded aggregation in a dose and time-dependent manner, which is associated with a further increase in α-synuclein gene expression. Progressive neuronal death is observed with brain-amplified fibrils and reversed by reduction of intraneuronal α-synuclein abundance. We identified 56 proteins differentially interacting with aggregates triggered by brain-amplified fibrils, including evasion of Parkinson's disease-associated deglycase DJ-1. Knockout of DJ-1 in iPSC-derived dopaminergic neurons enhance fibril-induced aggregation and neuronal death. Taken together, our results show that the toxicity of α-synuclein strains depends on aggregate burden, which is determined by monomer levels and conformation which dictates differential interactomes. Our study demonstrates how Parkinson's disease-associated genes influence the phenotypic manifestation of strains in human neurons.
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Neuronas Dopaminérgicas/patología , Atrofia de Múltiples Sistemas/patología , Enfermedad de Parkinson/patología , alfa-Sinucleína/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Muerte Celular , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas , Atrofia de Múltiples Sistemas/metabolismo , Enfermedad de Parkinson/metabolismo , Fenotipo , Agregado de Proteínas , Agregación Patológica de Proteínas , Conformación Proteica , Proteína Desglicasa DJ-1/metabolismo , Mapeo de Interacción de Proteínas , alfa-Sinucleína/química , alfa-Sinucleína/toxicidadRESUMEN
Current protocols for producing cerebellar neurons from human pluripotent stem cells (hPSCs) often rely on animal co-culture and mostly exist as monolayers, limiting their capability to recapitulate the complex processes in the developing cerebellum. Here, we employed a robust method, without the need for mouse co-culture to generate three-dimensional cerebellar organoids from hPSCs that display hallmarks of in vivo cerebellar development. Single-cell profiling followed by comparison to human and mouse cerebellar atlases revealed the presence and maturity of transcriptionally distinct populations encompassing major cerebellar cell types. Encapsulation with Matrigel aimed to provide more physiologically-relevant conditions through recapitulation of basement-membrane signalling, influenced both growth dynamics and cellular composition of the organoids, altering developmentally relevant gene expression programmes. We identified enrichment of cerebellar disease genes in distinct cell populations in the hPSC-derived cerebellar organoids. These findings ascertain xeno-free human cerebellar organoids as a unique model to gain insight into cerebellar development and its associated disorders.
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Diferenciación Celular , Cerebelo/citología , Células Madre Pluripotentes Inducidas/metabolismo , Organoides/citología , Anciano , Animales , Biomarcadores , Técnicas de Cultivo de Célula , Línea Celular , Colágeno , Biología Computacional/métodos , Combinación de Medicamentos , Femenino , Perfilación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Laminina , Proteoglicanos , Células de Purkinje/metabolismoRESUMEN
BACKGROUND: TREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer's disease (AD), including R47H. TREM2 signalling via SYK aids phagocytosis, chemotaxis, survival, and changes to microglial activation state. In AD mouse models, knockout (KO) of TREM2 impairs microglial clustering around amyloid and prevents microglial activation. The R47H mutation is proposed to reduce TREM2 ligand binding. We investigated cell phenotypes of the R47H mutant and TREM2 KO in a model of human microglia, and compared their transcriptional signatures, to determine the mechanism by which R47H TREM2 disrupts function. METHODS: We generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines, with homozygous R47H mutation or TREM2 knockout (KO). We firstly validated the effect of the R47H mutant on TREM2 surface and subcellular localization in pMac. To assess microglial phenotypic function, we measured phagocytosis of dead neurons, cell morphology, directed migration, survival, and LPS-induced inflammation. We performed bulk RNA-seq, comparing significant differentially expressed genes (DEGs; p < 0.05) between the R47H and KO versus WT, and bioinformatically predicted potential upstream regulators of TREM2-mediated gene expression. RESULTS: R47H modified surface expression and shedding of TREM2, but did not impair TREM2-mediated signalling, or gross phenotypes that were dysregulated in the TREM2 KO (phagocytosis, motility, survival). However, altered gene expression in the R47H TREM2 pMac overlapped by 90% with the TREM2 KO and was characterised by dysregulation of genes involved with immunity, proliferation, activation, chemotaxis, and adhesion. Downregulated mediators of ECM adhesion included the vitronectin receptor αVß3, and consequently, R47H TREM2 pMac adhered weakly to vitronectin compared with WT pMac. To counteract these transcriptional defects, we investigated TGFß1, as a candidate upstream regulator. TGFß1 failed to rescue vitronectin adhesion of pMac, although it improved αVß3 expression. CONCLUSIONS: The R47H mutation is not sufficient to cause gross phenotypic defects of human pMac under standard culture conditions. However, overlapping transcriptional defects with TREM2 KO supports the hypothesised partial loss-of-function effects of the R47H mutation. Furthermore, transcriptomics can guide us to more subtle phenotypic defects in the R47H cells, such as reduced cell adhesion, and can be used to predict targets for therapeutic intervention.
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Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Enfermedad de Alzheimer/genética , Encéfalo , Humanos , Macrófagos , Glicoproteínas de Membrana/genética , Microglía , Fenotipo , Receptores Inmunológicos/genéticaRESUMEN
We describe a human single-nuclei transcriptomic atlas for the substantia nigra (SN), generated by sequencing approximately 17,000 nuclei from matched cortical and SN samples. We show that the common genetic risk for Parkinson's disease (PD) is associated with dopaminergic neuron (DaN)-specific gene expression, including mitochondrial functioning, protein folding and ubiquitination pathways. We identify a distinct cell type association between PD risk and oligodendrocyte-specific gene expression. Unlike Alzheimer's disease (AD), we find no association between PD risk and microglia or astrocytes, suggesting that neuroinflammation plays a less causal role in PD than AD. Beyond PD, we find associations between SN DaNs and GABAergic neuron gene expression and multiple neuropsychiatric disorders. Conditional analysis reveals that distinct neuropsychiatric disorders associate with distinct sets of neuron-specific genes but converge onto shared loci within oligodendrocytes and oligodendrocyte precursors. This atlas guides our aetiological understanding by associating SN cell type expression profiles with specific disease risk.
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Expresión Génica , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , Enfermedad de Alzheimer/metabolismo , Astrocitos/metabolismo , Encéfalo , Neuronas Dopaminérgicas/metabolismo , Humanos , Microglía/metabolismo , Mitocondrias/metabolismo , Enfermedades del Sistema Nervioso/patología , Sustancia Negra/patología , TranscriptomaRESUMEN
RECQL1, a key member of the RecQ family of DNA helicases, is required for DNA replication and DNA repair. Two recent studies have shown that germline RECQL1 mutations are associated with increased breast cancer susceptibility. Whether altered RECQL1 expression has clinicopathologic significance in sporadic breast cancers is unknown. We evaluated RECQL1 at the transcriptomic level (METABRIC cohort, n = 1,977) and at the protein level [cohort 1, n = 897; cohort 2, n = 252; cohort 3 (BRCA germline deficient), n = 74]. In RECQL1-depleted breast cancer cells, we investigated anthracycline sensitivity. High RECQL1 mRNA was associated with intClust.3 (P = 0.026), which is characterized by low genomic instability. On the other hand, low RECQL1 mRNA was linked to intClust.8 [luminal A estrogen receptor-positive (ER+) subgroup; P = 0.0455] and intClust.9 (luminal B ER+ subgroup; P = 0.0346) molecular phenotypes. Low RECQL1 expression was associated with shorter breast cancer-specific survival (P = 0.001). At the protein level, low nuclear RECQL1 level was associated with larger tumor size, lymph node positivity, high tumor grade, high mitotic index, pleomorphism, dedifferentiation, ER negativity, and HER-2 overexpression (P < 0.05). In ER+ tumors that received endocrine therapy, low RECQL1 was associated with poor survival (P = 0.008). However, in ER- tumors that received anthracycline-based chemotherapy, high RECQL1 was associated with poor survival (P = 0.048). In RECQL1-depleted breast cancer cell lines, we confirmed doxorubicin sensitivity, which was associated with DNA double-strand breaks accumulation, S-phase cell-cycle arrest, and apoptosis. We conclude that RECQL1 has prognostic and predictive significance in breast cancers. Mol Cancer Ther; 16(1); 239-50. ©2016 AACR.
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Biomarcadores de Tumor , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , RecQ Helicasas/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Reparación del ADN , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética , Humanos , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , RecQ Helicasas/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Análisis de Supervivencia , Carga TumoralRESUMEN
AIMS: Multigene assay is recommended currently for prognostic stratification of the clinically indeterminate group of breast cancer (BC) patients defined as lymph node (LN)-negative, oestrogen receptor (ER)-positive, HER2-negative (LN- /ER+ /HER2- ) to determine the use of chemotherapy. However, this cohort, comprising approximately 40% of BC, is not a homogeneous group and shows variable outcome. This study aims to determine the prognostic value of routinely assessed variables, singly and in combination, in LN- /ER+ /HER2- BC patients. METHODS AND RESULTS: A total of 830 LN- /ER+ /HER2- chemotherapy-naive BCs were investigated. The prognostic value of histological grade, tumour size, lymphovascular invasion (LVI), progesterone receptor (PgR) and Ki67 labelling index (Ki67LI) was assessed. In this series, only 25% of patients received hormone therapy. Median follow-up was 172 months. In the whole cohort, tumour grade, size, LVI, PgR and Ki67LI were correlated highly with outcome in a time-dependent manner. The outcome of this group varied widely from 97% (20% of cases) to 50% survival rate after 10-year follow-up using a combination of these markers. A prognostic index (Nottingham Px) incorporating grade, size, PgR and Ki67LI, was developed. The index can stratify the whole cohort robustly as well as the higher-risk subgroup (NPI score >3.4) into distinct prognostic classes. CONCLUSION: Current routinely assessed variables can provide additional prognostic information in LN- /ER+ /HER2- BC. The proposed (Nottingham Px) index can stratify the BC clinically indeterminate group of patients into excellent and poor prognostic subgroups and can be used to identify reliably patients for systemic chemotherapy or further multigene prognostic testing. Performance of prognostic variables in these tumours is time-dependent, and should be considered in future studies.
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Neoplasias de la Mama/patología , Receptores de Estrógenos/biosíntesis , Adulto , Anciano , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/mortalidad , Femenino , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Receptor ErbB-2/análisis , Análisis de Matrices TisularesRESUMEN
BACKGROUND: Proliferation markers and profiles have been recommended for guiding the choice of systemic treatments for breast cancer. However, the best molecular marker or test to use has not yet been identified. We did this study to identify factors that drive proliferation and its associated features in breast cancer and assess their association with clinical outcomes and response to chemotherapy. METHODS: We applied an artificial neural network-based integrative data mining approach to data from three cohorts of patients with breast cancer (the Nottingham discovery cohort (n=171), Uppsala cohort (n=249), and Molecular Taxonomy of Breast Cancer International Consortium [METABRIC] cohort; n=1980). We then identified the genes with the most effect on other genes in the resulting interactome map. Sperm-associated antigen 5 (SPAG5) featured prominently in our interactome map of proliferation and we chose to take it forward in our analysis on the basis of its fundamental role in the function and dynamic regulation of mitotic spindles, mitotic progression, and chromosome segregation fidelity. We investigated the clinicopathological relevance of SPAG5 gene copy number aberrations, mRNA transcript expression, and protein expression and analysed the associations of SPAG5 copy number aberrations, transcript expression, and protein expression with breast cancer-specific survival, disease-free survival, distant relapse-free survival, pathological complete response, and residual cancer burden in the Nottingham discovery cohort, Uppsala cohort, METABRIC cohort, a pooled untreated lymph node-negative cohort (n=684), a multicentre combined cohort (n=5439), the Nottingham historical early stage breast cancer cohort (Nottingham-HES; n=1650), Nottingham early stage oestrogen receptor-negative breast cancer adjuvant chemotherapy cohort (Nottingham-oestrogen receptor-negative-ACT; n=697), the Nottingham anthracycline neoadjuvant chemotherapy cohort (Nottingham-NeoACT; n=200), the MD Anderson taxane plus anthracycline-based neoadjuvant chemotherapy cohort (MD Anderson-NeoACT; n=508), and the multicentre phase 2 neoadjuvant clinical trial cohort (phase 2 NeoACT; NCT00455533; n=253). FINDINGS: In the METABRIC cohort, we detected SPAG5 gene gain or amplification at the Ch17q11.2 locus in 206 (10%) of 1980 patients overall, 46 (19%) of 237 patients with a PAM50-HER2 phenotype, and 87 (18%) of 488 patients with PAM50-LumB phenotype. Copy number aberration leading to SPAG5 gain or amplification and high SPAG5 transcript and SPAG5 protein concentrations were associated with shorter overall breast cancer-specific survival (METABRIC cohort [copy number aberration]: hazard ratio [HR] 1·50, 95% CI 1·18-1·92, p=0·00010; METABRIC cohort [transcript]: 1·68, 1·40-2·01, p<0·0001; and Nottingham-HES-breast cancer cohort [protein]: 1·68, 1·32-2·12, p<0·0001). In multivariable analysis, high SPAG5 transcript and SPAG5 protein expression were associated with reduced breast cancer-specific survival at 10 years compared with lower concentrations (Uppsala: HR 1·62, 95% CI 1·03-2·53, p=0·036; METABRIC: 1·27, 1·02-1·58, p=0·034; untreated lymph node-negative cohort: 2·34, 1·24-4·42, p=0·0090; and Nottingham-HES: 1·73, 1·23-2·46, p=0·0020). In patients with oestrogen receptor-negative breast cancer with high SPAG5 protein expression, anthracycline-based adjuvant chemotherapy increased breast cancer-specific survival overall compared with that for patients who did not receive chemotherapy (Nottingham-oestrogen receptor-negative-ACT cohort: HR 0·37, 95% CI 0·20-0·60, p=0·0010). Multivariable analysis showed high SPAG5 transcript concentrations to be independently associated with longer distant relapse-free survival after receiving taxane plus anthracycline neoadjuvant chemotherapy (MD Anderson-NeoACT: HR 0·68, 95% CI 0·48-0·97, p=0·031). In multivariable analysis, both high SPAG5 transcript and high SPAG5 protein concentrations were independent predictors for a higher proportion of patients achieving a pathological complete response after combination cytotoxic chemotherapy (MD Anderson-NeoACT: OR 1·71, 95% CI, 1·07-2·74, p=0·024; Nottingham-ACT: 8·75, 2·42-31·62, p=0·0010). INTERPRETATION: SPAG5 is a novel amplified gene on Ch17q11.2 in breast cancer. The transcript and protein products of SPAG5 are independent prognostic and predictive biomarkers that might have clinical utility as biomarkers for combination cytotoxic chemotherapy sensitivity, especially in oestrogen receptor-negative breast cancer. FUNDING: Nottingham Hospitals Charity and the John and Lucille van Geest Foundation.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Genómica/métodos , Proteoma/análisis , Adulto , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Terapia Neoadyuvante , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Tasa de Supervivencia , TranscriptomaRESUMEN
BACKGROUND: MYC is amplified in approximately 15% of breast cancers (BCs) and is associated with poor outcome. c-MYC protein is multi-faceted and participates in many aspects of cellular function and is linked with therapeutic response in BCs. We hypothesised that the functional role of c-MYC differs between molecular subtypes of BCs. METHODS: We therefore investigated the correlation between c-MYC protein expression and other proteins involved in different cellular functions together with clinicopathological parameters, patients' outcome and treatments in a large early-stage molecularly characterised series of primary invasive BCs (n=1106) using immunohistochemistry. The METABRIC BC cohort (n=1980) was evaluated for MYC mRNA expression and a systems biology approach utilised to identify genes associated with MYC in the different BC molecular subtypes. RESULTS: High MYC and c-MYC expression was significantly associated with poor prognostic factors, including grade and basal-like BCs. In luminal A tumours, c-MYC was associated with ATM (P=0.005), Cyclin B1 (P=0.002), PIK3CA (P=0.009) and Ki67 (P<0.001). In contrast, in basal-like tumours, c-MYC showed positive association with Cyclin E (P=0.003) and p16 (P=0.042) expression only. c-MYC was an independent predictor of a shorter distant metastases-free survival in luminal A LN+ tumours treated with endocrine therapy (ET; P=0.013). In luminal tumours treated with ET, MYC mRNA expression was associated with BC-specific survival (P=0.001). In ER-positive tumours, MYC was associated with expression of translational genes while in ER-negative tumours it was associated with upregulation of glucose metabolism genes. CONCLUSIONS: c-MYC function is associated with specific molecular subtypes of BCs and its overexpression confers resistance to ET. The diverse mechanisms of c-MYC function in the different molecular classes of BCs warrants further investigation particularly as potential therapeutic targets.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/genética , Células Endocrinas/fisiología , Proteínas Proto-Oncogénicas c-myc/genética , Anciano , Proteínas de la Ataxia Telangiectasia Mutada/genética , Biomarcadores de Tumor/genética , Fosfatidilinositol 3-Quinasa Clase I , Ciclina B1/genética , Ciclina E/genética , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Fosfatidilinositol 3-Quinasas/genética , Pronóstico , ARN Mensajero/genética , Receptor ErbB-2/genéticaRESUMEN
Werner syndrome protein (WRN) is a RecQ helicase that participates in DNA repair, genome stability and cellular senescence. The five human RecQ helicases, RECQL1, Bloom, WRN, RECQL4 and RECQL5 play critical roles in DNA repair and cell survival after treatment with the anticancer drug camptothecin (CPT). CPT derivatives are widely used in cancer chemotherapy to inhibit topoisomerase I and generate DNA double-strand breaks during replication. Here we studied the effects of CPT on the stability and expression dynamics of human RecQ helicases. In the cells treated with CPT, we observed distinct effects on WRN compared to other human RecQ helicases. CPT altered the cellular localization of WRN and induced its degradation by a ubiquitin-mediated proteasome pathway. WRN knockdown cells as well as CPT treated cells became senescent and stained positive for senescence-associated ß-galactosidase at a higher frequency compared to control cells. However, the senescent phenotype was attenuated by ectopic expression of WRN suggesting functional implication of WRN degradation in CPT treated cells. Approximately 5-23% of breast cancer tumors are known to respond to CPT-based chemotherapy. Interestingly, we found that the extent of CPT-induced WRN degradation correlates with increasing sensitivity of breast cancer cells to CPT. The abundance of WRN decreased in CPT-treated sensitive cells; however, WRN remained relatively stable in CPT-resistant breast cancer cells. In a large clinical cohort of breast cancer patients, we find that WRN and topoisomerase I expression correlate with an aggressive tumor phenotype and poor prognosis. Our novel observations suggest that WRN abundance along with CPT-induced degradation could be a promising strategy for personalizing CPT-based cancer chemotherapeutic regimens.
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Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Camptotecina/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Helicasa del Síndrome de Werner/metabolismo , Apoptosis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , ADN-Topoisomerasas de Tipo I/genética , Femenino , Humanos , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Helicasa del Síndrome de Werner/genéticaRESUMEN
Signal transducer and activator of transcription (STAT) transcription factors family are involved in diverse cellular biological functions. Reports regarding the prognostic impact of STAT3 expression in breast cancer (BC) are variable whether being a factor of poor or good prognosis. Immunohistochemical expression of phospho-STAT3 (pSTAT3) was studied in large series of invasive BC (n = 1270). pSTAT3 and STAT3 were quantified using reverse phase protein array (RPPA) on proteins extracted from macro-dissected FFPE tissues (n = 49 cases). STAT3 gene expression in the METABRIC cohort was also investigated. STAT3 gene expression prognostic impact was externally validated using the online BC gene expression data (n = 26 datasets, 4.177 patients). pSTAT3 was expressed in the nuclei and cytoplasm of invasive BC cells. Nuclear pSTAT3 overexpression was positively associated with smaller tumour size, lower grade, good NPI, negative lymphovascular invasion (LVI), ER+, PgR+, p53-, HER2-, and low Ki67LI and an improved breast cancer-specific survival (BCSS), independently of other factors. On RPPA, the mean pSTAT3 and STAT3 expressions were higher in ER+, PgR+, and smaller size tumours. Higher STAT3 transcripts in the METABRIC cohort were observed in cases with favourable prognostic criteria and as well as improved BCSS within the whole cohort, ER+ cohort with and without hormonal therapy, and ER- cohort including those who did not receive adjuvant chemotherapy. Pooled STAT3 gene expression data in the external validation cohort showed an association with improved patients' outcome (P < 0.001, HR = 0.84, 95 % CI 0.79-0.90). Results of this study suggest nuclear localisation of pSTAT3 as favourable prognostic marker in invasive BC, results re-enforced by analysis of STAT3 gene expression data. This good prognostic advantage was maintained in patients who received and who did not receive adjuvant therapy. Therefore, STAT3 could have context-dependent molecular roles of in BC, results which warrant further prospective verification in clinical trials.
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Neoplasias de la Mama/patología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Núcleo Celular/metabolismo , Estudios de Cohortes , Citoplasma/metabolismo , Bases de Datos Genéticas , Femenino , Humanos , Fosforilación , Pronóstico , Análisis de SupervivenciaRESUMEN
PURPOSE: The expression of HAGE as a novel prognostic and predictive tool was assessed in 1,079 triple-negative breast cancers (TNBC). EXPERIMENTAL DESIGN: HAGE protein expression was investigated in an early primary TNBC (EP-TNBC; n = 520) cohort who received adjuvant chemotherapy (ACT) and in a locally advanced primary TNBC cohort who received anthracycline combination Neo-ACT (n = 110; AC-Neo-ACT). HAGE-mRNA expression was evaluated in the METABRIC-TNBC cohort (n = 311) who received ACT and in a cohort of patients with TNBC who received doxorubicin/cyclophosphamide Neo-ACT, followed by 1:1 randomization to ixabepilone (n = 68) or paclitaxel (n = 64) as part of a phase II clinical trial. Furthermore, a cohort of 128 tumors with integrated HAGE gene copy number changes, mRNA, and protein levels were analyzed. RESULTS: In patients with EP-TNBC, who were chemotherapy-naïve, high HAGE protein expression (HAGE(+)) was associated with a higher risk of death [HR, 1.3; 95% confidence interval (CI), 1.2-1.5; P = 0.000005] when compared with HAGE(-) cases. Patients who received ACT and expressed mRNA-HAGE(+) were at a lower risk of death than those who were mRNA-HAGE(-) (P = 0.004). The expression of HAGE was linked to the presence of tumor-infiltrating lymphocytes (TIL), and both features were found to be independent predictors for pathologic complete response (pCR, P < 0.001) and associated with prolonged survival (P < 0.01), following AC-Neo-ACT. In patients with residual disease, HAGE(+) had a 2-fold death risk increase (P = 0.018) compared with HAGE(-). CONCLUSIONS: HAGE expression is a potential prognostic marker and a predictor of response to anthracycline treatment in TNBC. A prospective clinical trial to examine the therapeutic value of HAGE for TNBC cases is warranted.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal de Mama/metabolismo , ARN Helicasas DEAD-box/metabolismo , Proteínas de Neoplasias/metabolismo , Transcriptoma , Neoplasias de la Mama Triple Negativas/metabolismo , Adulto , Biomarcadores de Tumor/genética , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/mortalidad , Carcinoma Ductal de Mama/terapia , Quimioterapia Adyuvante , Hibridación Genómica Comparativa , ARN Helicasas DEAD-box/genética , Variaciones en el Número de Copia de ADN , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Linfocitos Infiltrantes de Tumor/inmunología , Persona de Mediana Edad , Análisis Multivariante , Terapia Neoadyuvante , Proteínas de Neoplasias/genética , Pronóstico , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/terapiaRESUMEN
RECQL5 is a member of the RecQ family of DNA helicases and has key roles in homologous recombination, base excision repair, replication and transcription. The clinicopathological significance of RECQL5 expression in breast cancer is unknown. In this study, we have evaluated RECQL5 mRNA expression in 1977 breast cancers, and RECQL5 protein level in 1902 breast cancers [Nottingham Tenovus series (n = 1650) and ER- cohort (n = 252)]. Expression levels were correlated to aggressive phenotypes and survival outcomes. High RECQL5 mRNA expression was significantly associated with high histological grade (P = 0.007), HER2 overexpression (P = 0.032), ER+/HER2-/high proliferation genefu subtype (P < 0.0001), integrative molecular clusters (intClust 1and 9) (P < 0.0001) and poor survival (P < 0.0001). In subgroup analysis, high RECQL5 mRNA level remains significantly associated with poor BCSS in ER+ cohort (P < 0.0001) but not in ER- cohort (P = 0.116). At the protein level, in tumours with low RAD51, high RECQL5 level was significantly associated with high histological grade (P < 0.0001), higher mitotic index (P = 0.008), dedifferentiation (P = 0.025), pleomorphism (P = 0.027) and poor survival (P = 0.003). In subgroup analysis, high RECQL5/low RAD51 remains significantly associated with poor BCSS in ER+ cohort (P = 0.010), but not in ER- cohort (P = 0.628). In multivariate analysis, high RECQL5 mRNA and high RECQL5/low RAD51 nuclear protein coexpression independently influenced survival (P = 0.022) in whole cohort and in the ER+ subgroup. Preclinically, we show that exogenous expression of RECQL5 in MCF10A cells can drive proliferation supporting an oncogenic function for RECQL5 in breast cancer. We conclude that RECQL5 is a promising biomarker in breast cancer.
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Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , RecQ Helicasas/biosíntesis , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , ADN-Topoisomerasas de Tipo II/biosíntesis , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Células MCF-7 , Invasividad Neoplásica , Pronóstico , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Recombinasa Rad51/biosíntesis , Recombinasa Rad51/genética , RecQ Helicasas/genética , Análisis de Matrices TisularesRESUMEN
RECQL4 helicase is a molecular motor that unwinds DNA, a process essential during DNA replication and DNA repair. Germ-line mutations in RECQL4 cause type II Rothmund-Thomson syndrome (RTS), characterized by a premature ageing phenotype and cancer predisposition. RECQL4 is widely considered to be a tumour suppressor, although its role in human breast cancer is largely unknown. As the RECQL4 gene is localized to chromosome 8q24, a site frequently amplified in sporadic breast cancers, we hypothesized that it may play an oncogenic role in breast tumourigenesis. To address this, we analysed large cohorts for gene copy number changes (n = 1977), mRNA expression (n = 1977) and protein level (n = 1902). Breast cancer incidence was also explored in 58 patients with type II RTS. DNA replication dynamics and chemosensitivity was evaluated in RECQL4-depleted breast cancer cells in vitro. Amplification or gain in gene copy number (30.6%), high-level mRNA expression (51%) and high levels of protein (23%) significantly associated with aggressive tumour behaviour, including lymph node positivity, larger tumour size, HER2 overexpression, ER-negativity, triple-negative phenotypes and poor survival. RECQL4 depletion impaired the DNA replication rate and increased chemosensitivity in cultured breast cancer cells. Thus, although recognized as a 'safe guardian of the genome', our data provide compelling evidence that RECQL4 is tumour promoting in established breast cancers. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.