RESUMO
The blood-brain barrier (BBB) acquires unique properties to regulate neuronal function during development. The formation of the BBB, which occurs in tandem with angiogenesis, is directed by the Wnt/ß-catenin signaling pathway. Yet the exact molecular interplay remains elusive. Our study reveals the G protein-coupled receptor GPR126 as a critical target of canonical Wnt signaling, essential for the development of the BBB's distinctive vascular characteristics and its functional integrity. Endothelial cell-specific deletion of the Gpr126 gene in mice induced aberrant vascular morphogenesis, resulting in disrupted BBB organization. Simultaneously, heightened transcytosis in vitro compromised barrier integrity, resulting in enhanced vascular permeability. Mechanistically, GPR126 enhanced endothelial cell migration, pivotal for angiogenesis, acting through an interaction between LRP1 and ß1 integrin, thereby balancing the levels of ß1 integrin activation and recycling. Overall, we identified GPR126 as a specifier of an organotypic vascular structure, which sustained angiogenesis and guaranteed the acquisition of the BBB properties during development.
Assuntos
Barreira Hematoencefálica , Integrina beta1 , Receptores Acoplados a Proteínas G , Animais , Camundongos , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar , Movimento Celular , Células Endoteliais/metabolismo , Integrina beta1/metabolismo , Integrina beta1/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos Knockout , Neovascularização Fisiológica , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Via de Sinalização Wnt , Masculino , FemininoRESUMO
Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors.
Assuntos
Movimento Celular , Amplificação de Genes , Proteínas Proto-Oncogênicas c-myc , Estresse Mecânico , Humanos , Movimento Celular/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Linhagem Celular Tumoral , Camundongos , Mitose/genética , Instabilidade Cromossômica , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismoRESUMO
Glioblastomas exhibit remarkable heterogeneity at various levels, including motility modes and mechanoproperties that contribute to tumor resistance and recurrence. In a recent study using gridded micropatterns mimicking the brain vasculature, glioblastoma cell motility modes, mechanical properties, formin content, and substrate chemistry are linked. Now is presented, SP2G (SPheroid SPreading on Grids), an analytic platform designed to identify the migratory modes of patient-derived glioblastoma cells and rapidly pinpoint the most invasive sub-populations. Tumorspheres are imaged as they spread on gridded micropatterns and analyzed by this semi-automated, open-source, Fiji macro suite that characterizes migration modes accurately. SP2G can reveal intra-patient motility heterogeneity with molecular correlations to specific integrins and EMT markers. This system presents a versatile and potentially pan-cancer workflow to detect diverse invasive tumor sub-populations in patient-derived specimens and offers a valuable tool for therapeutic evaluations at the individual patient level.
RESUMO
Cancers feature substantial intratumoral heterogeneity of genetic and phenotypically distinct lineages. Although interactions between coexisting lineages are emerging as a potential contributor to tumor evolution, the extent and nature of these interactions remain largely unknown. We postulated that tumors develop ecological interactions that sustain diversity and facilitate metastasis. Using a combination of fluorescent barcoding, mathematical modeling, metabolic analysis, and in vivo models, we show that the Allee effect, i.e., growth dependency on population size, is a feature of tumor lineages and that cooperative ecological interactions between lineages alleviate the Allee barriers to growth in a model of triple-negative breast cancer. Soluble metabolite exchange formed the basis for these cooperative interactions and catalyzed the establishment of a polyclonal community that displayed enhanced metastatic dissemination and outgrowth in xenograft models. Our results highlight interclonal metabolite exchange as a key modulator of tumor ecology and a contributing factor to overcoming Allee effect-associated growth barriers to metastasis.
Assuntos
Corantes , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Modelos Animais de Doenças , Densidade DemográficaRESUMO
Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4,5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localisation. Here, we unveil the role of PIP4K2B as a mechanoresponsive enzyme. PIP4K2B protein level strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to softness, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.
Assuntos
Heterocromatina , Neoplasias , Humanos , 1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Núcleo Celular/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Neoplasias/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.
Assuntos
Actinas , Neoplasias , DNA , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Citosol/metabolismo , Transdução de SinaisRESUMO
Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.
Assuntos
Neoplasias Colorretais , Neoplasias Peritoneais , Humanos , Matriz Extracelular Descelularizada , Peritônio , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/terapia , Organoides , Neoplasias Colorretais/metabolismoRESUMO
Immunotherapy is improving the prognosis and survival of cancer patients, but despite encouraging outcomes in different cancers, the majority of tumors are resistant to it, and the immunotherapy combinations are often accompanied by severe side effects. Here, we show that a periodic fasting-mimicking diet (FMD) can act on the tumor microenvironment and increase the efficacy of immunotherapy (anti-PD-L1 and anti-OX40) against the poorly immunogenic triple-negative breast tumors (TNBCs) by expanding early exhausted effector T cells, switching the cancer metabolism from glycolytic to respiratory, and reducing collagen deposition. Furthermore, FMD reduces the occurrence of immune-related adverse events (irAEs) by preventing the hyperactivation of the immune response. These results indicate that FMD cycles have the potential to enhance the efficacy of anti-cancer immune responses, expand the portion of tumors sensitive to immunotherapy, and reduce its side effects.
Assuntos
Jejum , Neoplasias de Mama Triplo Negativas , Antígeno B7-H1/metabolismo , Glicólise , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Microambiente TumoralRESUMO
Primary Epstein-Barr virus (EBV)-positive nodal T/NK-cell lymphoma (PTCL-EBV) is a poorly understood disease which shows features resembling extranodal NK/T-cell lymphoma (ENKTL) and is currently not recognized as a distinct entity but categorized as a variant of primary T-cell lymphoma not otherwise specified (PTCL-NOS). Herein, we analyzed copynumber aberrations (n=77) with a focus on global measures of genomic instability and homologous recombination deficiency and performed gene expression (n=84) and EBV miRNA expression (n=24) profiling as well as targeted mutational analysis (n=16) to further characterize PTCL-EBV in relation to ENKTL and PTCL-NOS. Multivariate analysis revealed that patients with PTCL-EBV had a significantly worse outcome compared to patients with PTCL-NOS (P=0.002) but not to those with ENKTL. Remarkably, PTCL-EBV exhibited significantly lower genomic instability and homologous recombination deficiency scores compared to ENKTL and PTCL-NOS. Gene set enrichment analysis revealed that many immune-related pathways, interferon α/γ response, and IL6_JAK_STAT3 signaling were significantly upregulated in PTCLEBV and correlated with lower genomic instability scores. We also identified that NFκB-associated genes, BIRC3, NFKB1 (P50) and CD27, and their proteins are upregulated in PTCL-EBV. Most PTCL-EBV demonstrated a type 2 EBV latency pattern and, strikingly, exhibited downregulated expression of most EBV miRNA compared to ENKTL and their target genes were also enriched in immune-related pathways. PTCL-EBV also showed frequent mutations of TET2, PIK3CD and STAT3, and are characterized by microsatellite stability. Overall, poor outcome, low genomic instability, upregulation of immune pathways and downregulation of EBV miRNA are distinctive features of PTCL-EBV. Our data support the concept that PTCL-EBV could be considered as a distinct entity, provide novel insights into the pathogenesis of the disease and offer potential new therapeutic targets for this tumor.
Assuntos
Infecções por Vírus Epstein-Barr , Linfoma Extranodal de Células T-NK , Linfoma de Células T Periférico , MicroRNAs , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Instabilidade Genômica , Herpesvirus Humano 4/genética , Humanos , Linfoma Extranodal de Células T-NK/diagnóstico , Linfoma Extranodal de Células T-NK/genética , Linfoma de Células T Periférico/diagnóstico , Linfoma de Células T Periférico/genética , MicroRNAs/genética , Regulação para CimaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19), known to be more common in the elderly, who also show more severe symptoms and are at higher risk of hospitalization and death. Here, we show that the expression of the angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 cell receptor, increases during aging in mouse and human lungs. ACE2 expression increases upon telomere shortening or dysfunction in both cultured mammalian cells and in vivo in mice. This increase is controlled at the transcriptional level, and Ace2 promoter activity is DNA damage response (DDR)-dependent. Both pharmacological global DDR inhibition of ATM kinase activity and selective telomeric DDR inhibition by the use of antisense oligonucleotides prevent Ace2 upregulation following telomere damage in cultured cells and in mice. We propose that during aging telomere dysfunction due to telomeric shortening or damage triggers DDR activation and this causes the upregulation of ACE2, the SARS-CoV-2 cell receptor, thus contributing to make the elderly more susceptible to the infection.
Assuntos
Envelhecimento , Enzima de Conversão de Angiotensina 2/genética , COVID-19 , Dano ao DNA , Telômero , Idoso , Envelhecimento/genética , Animais , Humanos , Camundongos , SARS-CoV-2 , Telômero/genéticaRESUMO
In tumor-bearing mice, cyclic fasting or fasting-mimicking diets (FMD) enhance the activity of antineoplastic treatments by modulating systemic metabolism and boosting antitumor immunity. Here we conducted a clinical trial to investigate the safety and biological effects of cyclic, five-day FMD in combination with standard antitumor therapies. In 101 patients, the FMD was safe, feasible, and resulted in a consistent decrease of blood glucose and growth factor concentration, thus recapitulating metabolic changes that mediate fasting/FMD anticancer effects in preclinical experiments. Integrated transcriptomic and deep-phenotyping analyses revealed that FMD profoundly reshapes anticancer immunity by inducing the contraction of peripheral blood immunosuppressive myeloid and regulatory T-cell compartments, paralleled by enhanced intratumor Th1/cytotoxic responses and an enrichment of IFNγ and other immune signatures associated with better clinical outcomes in patients with cancer. Our findings lay the foundations for phase II/III clinical trials aimed at investigating FMD antitumor efficacy in combination with standard antineoplastic treatments. SIGNIFICANCE: Cyclic FMD is well tolerated and causes remarkable systemic metabolic changes in patients with different tumor types and treated with concomitant antitumor therapies. In addition, the FMD reshapes systemic and intratumor immunity, finally activating several antitumor immune programs. Phase II/III clinical trials are needed to investigate FMD antitumor activity/efficacy.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Jejum , Antineoplásicos/administração & dosagem , Neoplasias da Mama/dietoterapia , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias Colorretais/dietoterapia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do TratamentoRESUMO
Metastatic tumors remain lethal due to primary/acquired resistance to therapy or cancer stem cell (CSC)-mediated repopulation. We show that a fasting-mimicking diet (FMD) activates starvation escape pathways in triple-negative breast cancer (TNBC) cells, which can be identified and targeted by drugs. In CSCs, FMD lowers glucose-dependent protein kinase A signaling and stemness markers to reduce cell number and increase mouse survival. Accordingly, metastatic TNBC patients with lower glycemia survive longer than those with higher baseline glycemia. By contrast, in differentiated cancer cells, FMD activates PI3K-AKT, mTOR, and CDK4/6 as survival/growth pathways, which can be targeted by drugs to promote tumor regression. FMD cycles also prevent hyperglycemia and other toxicities caused by these drugs. These data indicate that FMD has wide and differential effects on normal, cancer, and CSCs, allowing the rapid identification and targeting of starvation escape pathways and providing a method potentially applicable to many malignancies.
Assuntos
Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Jejum , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
In some organs, such as the brain, endothelial cells form a robust and highly selective blood-to-tissue barrier. However, in other organs, such as the intestine, endothelial cells provide less stringent permeability, to allow rapid exchange of solutes and nutrients where needed. To maintain the structural and functional integrity of the highly dynamic blood-brain and gut-vascular barriers, endothelial cells form highly specialized cell-cell junctions, known as adherens junctions and tight junctions. Claudins are a family of four-membrane-spanning proteins at tight junctions and they have both barrier-forming and pore-forming properties. Tissue-specific expression of claudins has been linked to different diseases that are characterized by barrier impairment. In this review, we summarize the more recent progress in the field of the claudins, with particular attention to their expression and function in the blood-brain barrier and the recently described gut-vascular barrier, under physiological and pathological conditions.Abbreviations: 22q11DS 22q11 deletion syndrome; ACKR1 atypical chemokine receptor 1; AD Alzheimer disease; AQP aquaporin; ATP adenosine triphosphate; Aß amyloid ß; BAC bacterial artificial chromosome; BBB blood-brain barrier; C/EBP-α CCAAT/enhancer-binding protein α; cAMP cyclic adenosine monophosphate (or 3',5'-cyclic adenosine monophosphate); CD cluster of differentiation; CNS central nervous system; DSRED discosoma red; EAE experimental autoimmune encephalomyelitis; ECV304 immortalized endothelial cell line established from the vein of an apparently normal human umbilical cord; EGFP enhanced green fluorescent protein; ESAM endothelial cell-selective adhesion molecule; GLUT-1 glucose transporter 1; GVB gut-vascular barrier; H2B histone H2B; HAPP human amyloid precursor protein; HEK human embryonic kidney; JACOP junction-associated coiled coil protein; JAM junctional adhesion molecules; LYVE1 lymphatic vessel endothelial hyaluronan receptor 1; MADCAM1 mucosal vascular addressin cell adhesion molecule 1; MAPK mitogen-activated protein kinase; MCAO middle cerebral artery occlusion; MMP metalloprotease; MS multiple sclerosis; MUPP multi-PDZ domain protein; PATJ PALS-1-associated tight junction protein; PDGFR-α platelet-derived growth factor receptor α polypeptide; PDGFR-ß platelet-derived growth factor receptor ß polypeptide; RHO rho-associated protein kinase; ROCK rho-associated, coiled-coil-containing protein kinase; RT-qPCR real time quantitative polymerase chain reactions; PDGFR-ß soluble platelet-derived growth factor receptor, ß polypeptide; T24 human urinary bladder carcinoma cells; TG2576 transgenic mice expressing the human amyloid precursor protein; TNF-α tumor necrosis factor α; WTwild-type; ZO zonula occludens.
Assuntos
Claudinas , Células Endoteliais , Peptídeos beta-Amiloides , Animais , Encéfalo , Camundongos , Junções ÍntimasRESUMO
Tumors undergo dynamic immunoediting as part of a process that balances immunologic sensing of emerging neoantigens and evasion from immune responses. Tumor-infiltrating lymphocytes (TIL) comprise heterogeneous subsets of peripheral T cells characterized by diverse functional differentiation states and dependence on T-cell receptor (TCR) specificity gained through recombination events during their development. We hypothesized that within the tumor microenvironment (TME), an antigenic milieu and immunologic interface, tumor-infiltrating peripheral T cells could reexpress key elements of the TCR recombination machinery, namely, Rag1 and Rag2 recombinases and Tdt polymerase, as a potential mechanism involved in the revision of TCR specificity. Using two syngeneic invasive breast cancer transplantable models, 4T1 and TS/A, we observed that Rag1, Rag2, and Dntt in situ mRNA expression characterized rare tumor-infiltrating T cells. In situ expression of the transcripts was increased in coisogenic Mlh1-deficient tumors, characterized by genomic overinstability, and was also modulated by PD-1 immune-checkpoint blockade. Through immunolocalization and mRNA hybridization analyses, we detected the presence of rare TDT+RAG1/2+ cells populating primary tumors and draining lymph nodes in human invasive breast cancer. Analysis of harmonized single-cell RNA-sequencing data sets of human cancers identified a very small fraction of tumor-associated T cells, characterized by the expression of recombination/revision machinery transcripts, which on pseudotemporal ordering corresponded to differentiated effector T cells. We offer thought-provoking evidence of a TIL microniche marked by rare transcripts involved in TCR shaping.
Assuntos
Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Recombinação Genética/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Mama/imunologia , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linfócitos T CD8-Positivos/metabolismo , Dano ao DNA/imunologia , DNA Nucleotidilexotransferase/genética , DNA Nucleotidilexotransferase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteínas Nucleares/metabolismo , RNA-Seq , Receptores de Antígenos de Linfócitos T , Análise de Célula Única , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSBs) and promotes their resolution via the DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). We and others have shown that DDR activation requires DROSHA; however, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment, and how DROSHA influences DNA repair remains poorly understood. Here, we show that DROSHA associates with DSBs independently of transcription. Neither H2AX, nor ATM or DNA-PK kinase activities are required for recruitment of DROSHA to break sites. Rather, DROSHA interacts with RAD50, and inhibition of the MRN complex by mirin treatment abolishes this interaction. MRN complex inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Dano ao DNA/genética , Reparo do DNA/genética , Recombinação HomólogaRESUMO
We applied digital spatial profiling for 87 immune and stromal genes to lymph node germinal center (GC) dark- and light-zone (DZ/LZ) regions of interest to obtain a differential signature of these two distinct microenvironments. The spatially resolved 53-genes signature, comprising key genes of the DZ mutational machinery and LZ immune and mesenchymal milieu, was applied to the transcriptomes of 543 GC-related diffuse large B cell lymphomas and double-hit (DH) lymphomas. According to the DZ/LZ signature, the GC-related lymphomas were sub-classified into two clusters. The subgroups differed in the distribution of DH cases and survival, with most DH displaying a distinct DZ-like profile. The clustering analysis was also performed using a 25-genes signature composed of genes positively enriched in the non-B, stromal sub-compartments, for the first time achieving DZ/LZ discrimination based on stromal/immune features. The report offers new insight into the GC microenvironment, hinting at a DZ microenvironment of origin in DH lymphomas.
RESUMO
BACKGROUND: Intra-tumour heterogeneity in lymphoid malignancies encompasses selection of genetic events and epigenetic regulation of transcriptional programs. Clonal-related neoplastic cell populations are unsteadily subjected to immune editing and metabolic adaptations within different tissue microenvironments. How tissue-specific mesenchymal cells impact on the diversification of aggressive lymphoma clones is still unknown. METHODS: Combining in situ quantitative immunophenotypical analyses and RNA sequencing we investigated the intra-tumour heterogeneity and the specific mesenchymal modifications that are associated with A20 diffuse large B-cell lymphoma (DLBCL) cells seeding of different tissue microenvironments. Furthermore, we characterized features of lymphoma-associated stromatogenesis in human DLBCL samples using Digital Spatial Profiling, and established their relationship with prognostically relevant variables, such as MYC. FINDINGS: We found that the tissue microenvironment casts a relevant influence over A20 transcriptional landscape also impacting on Myc and DNA damage response programs. Extending the investigation to mice deficient for the matricellular protein SPARC, a stromal prognostic factor in human DLBCL, we demonstrated a different immune imprint on A20 cells according to stromal Sparc proficiency. Through Digital Spatial Profiling of 87 immune and stromal genes on human nodal DLBCL regions characterized by different mesenchymal composition, we demonstrate intra-lesional heterogeneity arising from diversified mesenchymal contextures and impacting on the stromal and immune milieu. INTERPRETATION: Our study provides experimental evidence that stromal microenvironment generates topological determinants of intra-tumour heterogeneity in DLBCL involving key transcriptional pathways such as Myc expression, damage response programs and immune checkpoints. FUNDING: This study has been supported by the Italian Foundation for Cancer Research (AIRC) (grants 15999 and 22145 to C. Tripodo) and by the University of Palermo.
Assuntos
Biomarcadores Tumorais , Heterogeneidade Genética , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Células Estromais/metabolismo , Microambiente Tumoral/genética , Animais , Linhagem Celular Tumoral , Biologia Computacional/métodos , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Imunofenotipagem , Hibridização In Situ , Camundongos , Modelos Biológicos , Fenótipo , Prognóstico , Análise de Sequência de RNA , Células Estromais/patologia , TranscriptomaRESUMO
DNA double-strand breaks (DSBs) are toxic DNA lesions, which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homologous recombination (HR) by contributing to the recruitment of the HR proteins BRCA1, BRCA2, and RAD51, without affecting DNA-end resection. In S/G2-phase cells, dilncRNAs pair to the resected DNA ends and form DNA:RNA hybrids, which are recognized by BRCA1. We also show that BRCA2 directly interacts with RNase H2, mediates its localization to DSBs in the S/G2 cell-cycle phase, and controls DNA:RNA hybrid levels at DSBs. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , RNA Longo não Codificante/metabolismo , Reparo de DNA por Recombinação , Ribonuclease H/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Fase G2/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Fase S/genéticaRESUMO
Identification of important, functional small RNA (sRNA) species is currently hampered by the lack of reliable and sensitive methods to isolate and characterize them. We have developed a method, termed target-enrichment of sRNAs (TEsR), that enables targeted sequencing of rare sRNAs and diverse precursor and mature forms of sRNAs not detectable by current standard sRNA sequencing methods. It is based on the amplification of full-length sRNA molecules, production of biotinylated RNA probes, hybridization to one or multiple targeted RNAs, removal of nontargeted sRNAs and sequencing. By this approach, target sRNAs can be enriched by a factor of 500-30,000 while maintaining strand specificity. TEsR enriches for sRNAs irrespective of length or different molecular features, such as the presence or absence of a 5' cap or of secondary structures or abundance levels. Moreover, TEsR allows the detection of the complete sequence (including sequence variants, and 5' and 3' ends) of precursors, as well as intermediate and mature forms, in a quantitative manner. A well-trained molecular biologist can complete the TEsR procedure, from RNA extraction to sequencing library preparation, within 4-6 d.