RESUMO
Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-ß/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence.
Assuntos
Senescência Celular , Desenvolvimento Embrionário , Saco Endolinfático/embriologia , Mesonefro/embriologia , Animais , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Saco Endolinfático/citologia , Feminino , Humanos , Rim/embriologia , Masculino , Mesonefro/citologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Genomics studies routinely confront researchers with long lists of tumor alterations detected in patients. Such lists are difficult to interpret since only a minority of the alterations are relevant biomarkers for diagnosis and for designing therapeutic strategies. PanDrugs is a methodology that facilitates the interpretation of tumor molecular alterations and guides the selection of personalized treatments. To do so, PanDrugs scores gene actionability and drug feasibility to provide a prioritized evidence-based list of drugs. Here, we introduce PanDrugs2, a major upgrade of PanDrugs that, in addition to somatic variant analysis, supports a new integrated multi-omics analysis which simultaneously combines somatic and germline variants, copy number variation and gene expression data. Moreover, PanDrugs2 now considers cancer genetic dependencies to extend tumor vulnerabilities providing therapeutic options for untargetable genes. Importantly, a novel intuitive report to support clinical decision-making is generated. PanDrugs database has been updated, integrating 23 primary sources that support >74K drug-gene associations obtained from 4642 genes and 14 659 unique compounds. The database has also been reimplemented to allow semi-automatic updates to facilitate maintenance and release of future versions. PanDrugs2 does not require login and is freely available at https://www.pandrugs.org/.
Assuntos
Multiômica , Neoplasias , Humanos , Variações do Número de Cópias de DNA , Genômica/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Medicina de Precisão/métodosRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known as cancer stem cells (CSCs), are implicated in PDAC aggressiveness. Thus, comprehending how this subset of cells evades the immune system is crucial for advancing novel therapies. DESIGN: We used the KPC mouse model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) and primary tumour cell lines to investigate putative CSC populations. Transcriptomic analyses were conducted to pinpoint new genes involved in immune evasion. Overexpressing and knockout cell lines were established with lentiviral vectors. Subsequent in vitro coculture assays, in vivo mouse and zebrafish tumorigenesis studies, and in silico database approaches were performed. RESULTS: Using the KPC mouse model, we functionally confirmed a population of cells marked by EpCAM, Sca-1 and CD133 as authentic CSCs and investigated their transcriptional profile. Immune evasion signatures/genes, notably the gene peptidoglycan recognition protein 1 (PGLYRP1), were significantly overexpressed in these CSCs. Modulating PGLYRP1 impacted CSC immune evasion, affecting their resistance to macrophage-mediated and T-cell-mediated killing and their tumourigenesis in immunocompetent mice. Mechanistically, tumour necrosis factor alpha (TNFα)-regulated PGLYRP1 expression interferes with the immune tumour microenvironment (TME) landscape, promoting myeloid cell-derived immunosuppression and activated T-cell death. Importantly, these findings were not only replicated in human models, but clinically, secreted PGLYRP1 levels were significantly elevated in patients with PDAC. CONCLUSIONS: This study establishes PGLYRP1 as a novel CSC-associated marker crucial for immune evasion, particularly against macrophage phagocytosis and T-cell killing, presenting it as a promising target for PDAC immunotherapy.
Assuntos
Carcinoma Ductal Pancreático , Células-Tronco Neoplásicas , Neoplasias Pancreáticas , Animais , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Camundongos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Humanos , Linhagem Celular Tumoral , Evasão Tumoral/imunologia , Modelos Animais de Doenças , Evasão da Resposta Imune , Microambiente Tumoral/imunologiaRESUMO
SUMMARY: bollito is an automated, flexible and parallelizable computational pipeline for the comprehensive analysis of single-cell RNA-seq data. Starting from FASTQ files or preprocessed expression matrices, bollito performs both basic and advanced tasks in single-cell analysis integrating >30 state-of-the-art tools. This includes quality control, read alignment, dimensionality reduction, clustering, cell-marker detection, differential expression, functional analysis, trajectory inference and RNA velocity. bollito is built using the Snakemake workflow management system, which easily connects each execution step and facilitates the reproducibility of results. bollito's modular design makes it easy to incorporate other packages into the pipeline enabling its expansion with new functionalities. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at https://gitlab.com/bu_cnio/bollito under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Análise da Expressão Gênica de Célula Única , Software , Reprodutibilidade dos Testes , RNA , Fluxo de TrabalhoRESUMO
The initiating oncogenic event in almost half of human lung adenocarcinomas is still unknown, a fact that complicates the development of selective targeted therapies. Yet these tumours harbour a number of alterations without obvious oncogenic function including BRAF-inactivating mutations. Inactivating BRAF mutants in lung predominate over the activating V600E mutant that is frequently observed in other tumour types. Here we demonstrate that the expression of an endogenous Braf(D631A) kinase-inactive isoform in mice (corresponding to the human BRAF(D594A) mutation) triggers lung adenocarcinoma in vivo, indicating that BRAF-inactivating mutations are initiating events in lung oncogenesis. Moreover, inactivating BRAF mutations have also been identified in a subset of KRAS-driven human lung tumours. Co-expression of Kras(G12V) and Braf(D631A) in mouse lung cells markedly enhances tumour initiation, a phenomenon mediated by Craf kinase activity, and effectively accelerates tumour progression when activated in advanced lung adenocarcinomas. We also report a key role for the wild-type Braf kinase in sustaining Kras(G12V)/Braf(D631A)-driven tumours. Ablation of the wild-type Braf allele prevents the development of lung adenocarcinoma by inducing a further increase in MAPK signalling that results in oncogenic toxicity; this effect can be abolished by pharmacological inhibition of Mek to restore tumour growth. However, the loss of wild-type Braf also induces transdifferentiation of club cells, which leads to the rapid development of lethal intrabronchiolar lesions. These observations indicate that the signal intensity of the MAPK pathway is a critical determinant not only in tumour development, but also in dictating the nature of the cancer-initiating cell and ultimately the resulting tumour phenotype.
Assuntos
Adenocarcinoma/genética , Mutação com Perda de Função , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas B-raf/genética , Adenocarcinoma/patologia , Alelos , Animais , Carcinogênese/genética , Progressão da Doença , Feminino , Genes da Neurofibromatose 1 , Humanos , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
KRAS mutant lung adenocarcinomas remain intractable for targeted therapies. Genetic interrogation of KRAS downstream effectors, including the MAPK pathway and the interphase CDKs, identified CDK4 and RAF1 as the only targets whose genetic inactivation induces therapeutic responses without causing unacceptable toxicities. Concomitant CDK4 inactivation and RAF1 ablation prevented tumor progression and induced complete regression in 25% of KRAS/p53-driven advanced lung tumors, yet a significant percentage of those tumors that underwent partial regression retained a population of CDK4/RAF1-resistant cells. Characterization of these cells revealed two independent resistance mechanisms implicating hypermethylation of several tumor suppressors and increased PI3K activity. Importantly, these CDK4/RAF1-resistant cells can be pharmacologically controlled. These studies open the door to new therapeutic strategies to treat KRAS mutant lung cancer, including resistant tumors.
Assuntos
Adenocarcinoma de Pulmão/genética , Quinase 4 Dependente de Ciclina/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/metabolismo , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Inativação Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
MOTIVATION: Drug immunomodulation modifies the response of the immune system and can be therapeutically exploited in pathologies such as cancer and autoimmune diseases. RESULTS: DREIMT is a new hypothesis-generation web tool, which performs drug prioritization analysis for immunomodulation. DREIMT provides significant immunomodulatory drugs targeting up to 70 immune cells subtypes through a curated database that integrates 4960 drug profiles and â¼2600 immune gene expression signatures. The tool also suggests potential immunomodulatory drugs targeting user-supplied gene expression signatures. Final output includes drug-signature association scores, FDRs and downloadable plots and results tables. AVAILABILITYAND IMPLEMENTATION: http://www.dreimt.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Reposicionamento de Medicamentos , Transcriptoma , Bases de Dados Factuais , Bases de Dados de Produtos Farmacêuticos , ImunomodulaçãoRESUMO
BACKGROUND: Autoimmune diseases are heterogeneous pathologies with difficult diagnosis and few therapeutic options. In the last decade, several omics studies have provided significant insights into the molecular mechanisms of these diseases. Nevertheless, data from different cohorts and pathologies are stored independently in public repositories and a unified resource is imperative to assist researchers in this field. RESULTS: Here, we present Autoimmune Diseases Explorer ( https://adex.genyo.es ), a database that integrates 82 curated transcriptomics and methylation studies covering 5609 samples for some of the most common autoimmune diseases. The database provides, in an easy-to-use environment, advanced data analysis and statistical methods for exploring omics datasets, including meta-analysis, differential expression or pathway analysis. CONCLUSIONS: This is the first omics database focused on autoimmune diseases. This resource incorporates homogeneously processed data to facilitate integrative analyses among studies.
Assuntos
Doenças Autoimunes , Biologia Computacional , Doenças Autoimunes/epidemiologia , Doenças Autoimunes/genética , Bases de Dados Factuais , HumanosRESUMO
Success in precision medicine depends on accessing high-quality genetic and molecular data from large, well-annotated patient cohorts that couple biological samples to comprehensive clinical data, which in conjunction can lead to effective therapies. From such a scenario emerges the need for a new professional profile, an expert bioinformatician with training in clinical areas who can make sense of multi-omics data to improve therapeutic interventions in patients, and the design of optimized basket trials. In this review, we first describe the main policies and international initiatives that focus on precision medicine. Secondly, we review the currently ongoing clinical trials in precision medicine, introducing the concept of 'precision bioinformatics', and we describe current pioneering bioinformatics efforts aimed at implementing tools and computational infrastructures for precision medicine in health institutions around the world. Thirdly, we discuss the challenges related to the clinical training of bioinformaticians, and the urgent need for computational specialists capable of assimilating medical terminologies and protocols to address real clinical questions. We also propose some skills required to carry out common tasks in clinical bioinformatics and some tips for emergent groups. Finally, we explore the future perspectives and the challenges faced by precision medicine bioinformatics.
Assuntos
Biologia Computacional , Medicina de Precisão , Estudos de Coortes , HumanosRESUMO
MOTIVATION: Genetic alterations lead to tumor progression and cell survival but also uncover cancer-specific vulnerabilities on gene dependencies that can be therapeutically exploited. RESULTS: vulcanSpot is a novel computational approach implemented to expand the therapeutic options in cancer beyond known-driver genes unlocking alternative ways to target undruggable genes. The method integrates genome-wide information provided by massive screening experiments to detect genetic vulnerabilities associated to tumors. Then, vulcanSpot prioritizes drugs to target cancer-specific gene dependencies using a weighted scoring system based on well known drug-gene relationships and drug repositioning strategies. AVAILABILITY AND IMPLEMENTATION: http://www.vulcanspot.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Neoplasias , Biologia Computacional , Reposicionamento de Medicamentos , Humanos , Mutação , SoftwareRESUMO
Organization of epithelial cells during follicular lumen formation is crucial for thyroid morphogenesis and function of the thyroid gland; however, the molecular mechanisms underlying this are poorly understood. To investigate this process, we established three-dimensional (3D) epithelial culture model systems using Fischer rat thyroid (FRT) cells or murine primary thyrocytes that developed polarized spherical structures with a central lumen, mimicking thyroid follicles. Using microarray-based differential expression analysis of FRT cells grown under 2D or 3D conditions, followed by RNA-mediated interference (RNAi) and morphogenetic analysis, we identified a key role for the thyroid transcription factor Pax8 and its target cadherin-16 (Cdh16) in the generation of polarized follicle-like structures. Silencing Pax8 expression inhibited the acquisition of apical-basal membrane polarity and impaired lumen formation. Both laminin and ß1-integrin (Itgb1) expression was reduced, and cell cytoskeleton polarized distribution was altered. Silencing Cdh16 expression also led to the formation of defective structures characterized by very low laminin expression at the follicle-matrix interface, downregulation of Itgb1, and unpolarized distribution of cell cytoskeleton. Our results demonstrate that Pax8 controls apical-basal follicular polarization and follicle formation through Cdh16.
Assuntos
Polaridade Celular , Células Epiteliais da Tireoide/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/farmacologia , Regulação para Baixo/efeitos dos fármacos , Combinação de Medicamentos , Integrina beta1/metabolismo , Laminina/metabolismo , Laminina/farmacologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fator de Transcrição PAX8 , Proteoglicanas/farmacologia , Ratos , Células Epiteliais da Tireoide/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
OBJECTIVE: Colorectal cancer (CRC) is a major health concern. Vitamin D deficiency is associated with high CRC incidence and mortality, suggesting a protective effect of vitamin D against this disease. Given the strong influence of tumour stroma on cancer progression, we investigated the potential effects of the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on CRC stroma. DESIGN: Expression of vitamin D receptor (VDR) and two 1,25(OH)2D3 target genes was analysed in 658 patients with CRC with prolonged clinical follow-up. 1,25(OH)2D3 effects on primary cultures of patient-derived colon normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were studied using collagen gel contraction and migration assays and global gene expression analyses. Publicly available data sets (n=877) were used to correlate the 1,25(OH)2D3-associated gene signature in CAFs with CRC outcome. RESULTS: High VDR expression in tumour stromal fibroblasts was associated with better overall survival (OS) and progression-free survival in CRC, independently of its expression in carcinoma cells. 1,25(OH)2D3 inhibited the protumoural activation of NFs and CAFs and imposed in CAFs a 1,25(OH)2D3-associated gene signature that correlated with longer OS and disease-free survival in CRC. Furthermore, expression of two genes from the signature, CD82 and S100A4, correlated with stromal VDR expression and clinical outcome in our cohort of patients with CRC. CONCLUSIONS: 1,25(OH)2D3 has protective effects against CRC through the regulation of stromal fibroblasts. Accordingly, expression of VDR and 1,25(OH)2D3-associated gene signature in stromal fibroblasts predicts a favourable clinical outcome in CRC. Therefore, treatment of patients with CRC with VDR agonists could be explored even in the absence of VDR expression in carcinoma cells.
Assuntos
Calcitriol/farmacologia , Fibroblastos Associados a Câncer/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Receptores de Calcitriol/metabolismo , Vitaminas/farmacologia , Carcinoma/química , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/efeitos dos fármacos , Neoplasias Colorretais/química , Intervalo Livre de Doença , Expressão Gênica/efeitos dos fármacos , Humanos , Proteína Kangai-1/genética , Receptores de Calcitriol/análise , Proteína A4 de Ligação a Cálcio da Família S100/genética , Taxa de Sobrevida , TranscriptomaRESUMO
Chronic lymphocytic leukemia (CLL) has heterogeneous clinical and biological behavior. Whole-genome and -exome sequencing has contributed to the characterization of the mutational spectrum of the disease, but the underlying transcriptional profile is still poorly understood. We have performed deep RNA sequencing in different subpopulations of normal B-lymphocytes and CLL cells from a cohort of 98 patients, and characterized the CLL transcriptional landscape with unprecedented resolution. We detected thousands of transcriptional elements differentially expressed between the CLL and normal B cells, including protein-coding genes, noncoding RNAs, and pseudogenes. Transposable elements are globally derepressed in CLL cells. In addition, two thousand genes-most of which are not differentially expressed-exhibit CLL-specific splicing patterns. Genes involved in metabolic pathways showed higher expression in CLL, while genes related to spliceosome, proteasome, and ribosome were among the most down-regulated in CLL. Clustering of the CLL samples according to RNA-seq derived gene expression levels unveiled two robust molecular subgroups, C1 and C2. C1/C2 subgroups and the mutational status of the immunoglobulin heavy variable (IGHV) region were the only independent variables in predicting time to treatment in a multivariate analysis with main clinico-biological features. This subdivision was validated in an independent cohort of patients monitored through DNA microarrays. Further analysis shows that B-cell receptor (BCR) activation in the microenvironment of the lymph node may be at the origin of the C1/C2 differences.
Assuntos
Linfócitos B , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Linfocítica Crônica de Células B/genética , Idoso , Sequência de Bases , Feminino , Perfilação da Expressão Gênica , Humanos , Região Variável de Imunoglobulina , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Ribossomos/genética , Spliceossomos/genéticaRESUMO
Vertebrates have two cohesin complexes that consist of Smc1, Smc3, Rad21/Scc1 and either SA1 or SA2, but their functional specificity is unclear. Mouse embryos lacking SA1 show developmental delay and die before birth. Comparison of the genome-wide distribution of cohesin in wild-type and SA1-null cells reveals that SA1 is largely responsible for cohesin accumulation at promoters and at sites bound by the insulator protein CTCF. As a consequence, ablation of SA1 alters transcription of genes involved in biological processes related to Cornelia de Lange syndrome (CdLS), a genetic disorder linked to dysfunction of cohesin. We show that the presence of cohesin-SA1 at the promoter of myc and of protocadherin genes positively regulates their expression, a task that cannot be assumed by cohesin-SA2. Lack of SA1 also alters cohesin-binding pattern along some gene clusters and leads to dysregulation of genes within. We hypothesize that impaired cohesin-SA1 function in gene expression underlies the molecular aetiology of CdLS.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica , Subunidades Proteicas/metabolismo , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Síndrome de Cornélia de Lange/genética , Embrião de Mamíferos , Fibroblastos , Camundongos , Camundongos Knockout , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Proteínas Proto-Oncogênicas c-myc/genética , CoesinasRESUMO
Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of primary cutaneous T-cell lymphoproliferative processes, mainly composed of mycosis fungoides and Sézary syndrome, the aggressive forms of which lack an effective treatment. The molecular pathogenesis of CTCL is largely unknown, although neoplastic cells show increased signaling from T-cell receptors (TCRs). DNAs from 11 patients with CTCL, both normal and tumoral, were target-enriched and sequenced by massive parallel sequencing for a selection of 524 TCR-signaling-related genes. Identified variants were validated by capillary sequencing. Multiple mutations were found that affected several signaling pathways, such as TCRs, nuclear factor κB, or Janus kinase/signal transducer and activator of transcription, but PLCG1 was found to be mutated in 3 samples, 2 of which featured a redundant mutation (c.1034T>C, S345F) in exon 11 that affects the PLCx protein catalytic domain. This mutation was further analyzed by quantitative polymerase chain reaction genotyping in a new cohort of 42 patients with CTCL, where it was found in 19% of samples. Immunohistochemical analysis for nuclear factor of activated T cells (NFAT) showed that PLCG1-mutated cases exhibited strong NFAT nuclear immunostaining. Functional studies demonstrated that PLCG1 mutants elicited increased downstream signaling toward NFAT activation, and inhibition of this pathway resulted in reduced CTCL cell proliferation and cell viability. Thus, increased proliferative and survival mechanisms in CTCL may partially depend on the acquisition of somatic mutations in PLCG1 and other genes that are essential for normal T-cell differentiation.
Assuntos
Linfoma de Células T/genética , Mutação , Fosfolipase C gama/genética , Neoplasias Cutâneas/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfoma de Células T/patologia , Masculino , Camundongos , Células NIH 3T3 , Neoplasias Cutâneas/patologiaRESUMO
OBJECTIVES: The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance. DESIGN: Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology. RESULTS: We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-ß1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R. CONCLUSIONS: Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Ativinas/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinoma Ductal Pancreático/genética , Autorrenovação Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/genética , Análise Serial de Proteínas , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Formil Peptídeo/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise Serial de Tecidos , Fator de Crescimento Transformador beta1/farmacologia , CatelicidinasRESUMO
Screening for germline mutations in breast cancer-associated genes BRCA1 and BRCA2 is indicated for patients with breast cancer from high-risk breast cancer families and influences both treatment options and clinical management. However, only 25% of selected patients test positive for BRCA1/2 mutation, indicating that additional diagnostic biomarkers are necessary. We analyzed 124 formalin-fixed paraffin-embedded (FFPE) tumor samples from patients with hereditary (104) and sporadic (20) invasive breast cancer, divided into two series (A and B). Microarray expression profiling of 829 human miRNAs was performed on 76 samples (Series A), and bioinformatics tool Prophet was used to develop and test a microarray classifier. Samples were stratified into a training set (n = 38) for microarray classifier generation and a test set (n = 38) for signature validation. A 35-miRNA microarray classifier was generated for the prediction of BRCA1/2 mutation status with a reported 95% (95% CI = 0.88-1.0) and 92% (95% CI: 0.84-1.0) accuracy in the training and the test set, respectively. Differential expression of 12 miRNAs between BRCA1/2 mutation carriers versus noncarriers was validated by qPCR in an independent tumor series B (n = 48). Logistic regression model based on the expression of six miRNAs (miR-142-3p, miR-505*, miR-1248, miR-181a-2*, miR-25* and miR-340*) discriminated between tumors from BRCA1/2 mutation carriers and noncarriers with 92% (95% CI: 0.84-0.99) accuracy. In conclusion, we identified miRNA expression signatures predictive of BRCA1/2 mutation status in routinely available FFPE breast tumor samples, which may be useful to complement current patient selection criteria for gene testing by identifying individuals with high likelihood of being BRCA1/2 mutation carriers.
Assuntos
Neoplasias da Mama/genética , Genes BRCA1 , Genes BRCA2 , MicroRNAs/análise , Mutação , Feminino , Formaldeído , Humanos , Modelos Logísticos , Inclusão em ParafinaRESUMO
BRCA1 germline mutations increase the lifetime risk of developing breast and ovarian cancers. However, taking into account the differences in disease manifestation among mutation carriers, it is probable that different BRCA1 mutations have distinct haploinsufficiency effects and lead to the formation of different phenotypes. Using lymphoblastoid cell lines derived from heterozygous BRCA1 mutation carriers and non-carriers, we investigated the haploinsufficiency effects of various mutation types using qPCR, immunofluorescence, and microarray technology. Lymphoblastoid cell lines carrying a truncating mutation showed significantly lower BRCA1 mRNA and protein levels and higher levels of gamma-H2AX than control cells or those harboring a missense mutation, indicating greater spontaneous DNA damage. Cells carrying either BRCA1 mutation type showed impaired RAD51 foci formation, suggesting defective repair in mutated cells. Moreover, compared to controls, cell lines carrying missense mutations displayed a more distinct expression profile than cells with truncating mutations, which is consistent with different mutations giving rise to distinct phenotypes. Alterations in the immune response pathway in cells harboring missense mutations point to possible mechanisms of breast cancer initiation in carriers of these mutations. Our findings offer insight into how various heterozygous mutations in BRCA1 could lead to impairment of BRCA1 function and provide strong evidence of haploinsufficiency in BRCA1 mutation carriers.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Reparo do DNA , Heterozigoto , Mutação , Alelos , Proteína BRCA1/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Análise por Conglomerados , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Haploinsuficiência , Histonas/metabolismo , Humanos , Rad51 Recombinase/metabolismo , TranscriptomaRESUMO
BACKGROUND: The identification of novel biomarkers for early breast cancer detection would be a great advance. Because of their role in tumorigenesis and stability in body fluids, microRNAs (miRNAs) are emerging as a promising diagnostic tool. Our aim was to identify miRNAs deregulated in breast tumors and evaluate the potential of circulating miRNAs in breast cancer detection. METHODS: We conducted miRNA expression profiling of 1919 human miRNAs in paraffin-embedded tissue from 122 breast tumors and 11 healthy breast tissue samples. Differential expression analysis was performed, and a microarray classifier was generated. The most relevant miRNAs were analyzed in plasma from 26 healthy individuals and 83 patients with breast cancer (36 before and 47 after treatment) and validated in 116 healthy individuals and 114 patients before treatment. RESULTS: We identified a large number of miRNAs deregulated in breast cancer and generated a 25-miRNA microarray classifier that discriminated breast tumors with high diagnostic sensitivity and specificity. Ten miRNAs were selected for further investigation, of which 4 (miR-505-5p, miR-125b-5p, miR-21-5p, and miR-96-5p) were significantly overexpressed in pretreated patients with breast cancer compared with healthy individuals in 2 different series of plasma. MiR-505-5p and miR-96-5p were the most valuable biomarkers (area under the curve 0.72). Moreover, the expression levels of miR-3656, miR-505-5p, and miR-21-5p were decreased in a group of treated patients. CONCLUSIONS: Circulating miRNAs reflect the presence of breast tumors. The identification of deregulated miRNAs in plasma of patients with breast cancer supports the use of circulating miRNAs as a method for early breast cancer detection.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , MicroRNAs/sangue , MicroRNAs/genética , Detecção Precoce de Câncer , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Valores de Referência , Reprodutibilidade dos TestesRESUMO
MOTIVATION: RUbioSeq has been developed to facilitate the primary and secondary analysis of re-sequencing projects by providing an integrated software suite of parallelized pipelines to detect exome variants (single-nucleotide variants and copy number variations) and to perform bisulfite-seq analyses automatically. RUbioSeq's variant analysis results have been already validated and published. AVAILABILITY: http://rubioseq.sourceforge.net/.