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1.
Cell ; 173(3): 595-610.e11, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29656894

RESUMO

The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with >10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance.


Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Biomarcadores Tumorais , Cromossomos , Evolução Clonal , Progressão da Doença , Evolução Molecular , Feminino , Heterogeneidade Genética , Variação Genética , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Mutação , Metástase Neoplásica , Fenótipo , Filogenia , Prognóstico , Estudos Prospectivos , Análise de Sequência de DNA
2.
Cell ; 173(3): 581-594.e12, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29656895

RESUMO

Clear-cell renal cell carcinoma (ccRCC) exhibits a broad range of metastatic phenotypes that have not been systematically studied to date. Here, we analyzed 575 primary and 335 metastatic biopsies across 100 patients with metastatic ccRCC, including two cases sampledat post-mortem. Metastatic competence was afforded by chromosome complexity, and we identify 9p loss as a highly selected event driving metastasis and ccRCC-related mortality (p = 0.0014). Distinct patterns of metastatic dissemination were observed, including rapid progression to multiple tissue sites seeded by primary tumors of monoclonal structure. By contrast, we observed attenuated progression in cases characterized by high primary tumor heterogeneity, with metastatic competence acquired gradually and initial progression to solitary metastasis. Finally, we observed early divergence of primitive ancestral clones and protracted latency of up to two decades as a feature of pancreatic metastases.


Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Mutação , Metástase Neoplásica , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Biópsia , Mapeamento Cromossômico , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 9 , Progressão da Doença , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fenótipo , Estudos Prospectivos , Trombose , Resultado do Tratamento
3.
Nature ; 616(7957): 534-542, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37046095

RESUMO

Metastatic disease is responsible for the majority of cancer-related deaths1. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Evolução Clonal , Células Clonais , Evolução Molecular , Neoplasias Pulmonares , Metástase Neoplásica , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Clonais/patologia , Estudos de Coortes , Progressão da Doença , Neoplasias Pulmonares/patologia , Metástase Neoplásica/diagnóstico , Metástase Neoplásica/patologia , Recidiva Local de Neoplasia
4.
Nature ; 607(7917): 163-168, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35768509

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) shows pronounced epithelial and mesenchymal cancer cell populations1-4. Cellular heterogeneity in PDAC is an important feature in disease subtype specification3-5, but how distinct PDAC subpopulations interact, and the molecular mechanisms that underlie PDAC cell fate decisions, are incompletely understood. Here we identify the BMP inhibitor GREM16,7 as a key regulator of cellular heterogeneity in pancreatic cancer in human and mouse. Grem1 inactivation in established PDAC in mice resulted in a direct conversion of epithelial into mesenchymal PDAC cells within days, suggesting that persistent GREM1 activity is required to maintain the epithelial PDAC subpopulations. By contrast, Grem1 overexpression caused an almost complete 'epithelialization' of highly mesenchymal PDAC, indicating that high GREM1 activity is sufficient to revert the mesenchymal fate of PDAC cells. Mechanistically, Grem1 was highly expressed in mesenchymal PDAC cells and inhibited the expression of the epithelial-mesenchymal transition transcription factors Snai1 (also known as Snail) and Snai2 (also known as Slug) in the epithelial cell compartment, therefore restricting epithelial-mesenchymal plasticity. Thus, constant suppression of BMP activity is essential to maintain epithelial PDAC cells, indicating that the maintenance of the cellular heterogeneity of pancreatic cancer requires continuous paracrine signalling elicited by a single soluble factor.


Assuntos
Transição Epitelial-Mesenquimal , Peptídeos e Proteínas de Sinalização Intercelular , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/patologia , Camundongos , Neoplasias Pancreáticas/patologia , Fatores de Transcrição da Família Snail
5.
Immunity ; 49(2): 353-362.e5, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30119997

RESUMO

The epithelium and immune compartment in the intestine are constantly exposed to a fluctuating external environment. Defective communication between these compartments at this barrier surface underlies susceptibility to infections and chronic inflammation. Environmental factors play a significant, but mechanistically poorly understood, role in intestinal homeostasis. We found that regeneration of intestinal epithelial cells (IECs) upon injury through infection or chemical insults was profoundly influenced by the environmental sensor aryl hydrocarbon receptor (AHR). IEC-specific deletion of Ahr resulted in failure to control C. rodentium infection due to unrestricted intestinal stem cell (ISC) proliferation and impaired differentiation, culminating in malignant transformation. AHR activation by dietary ligands restored barrier homeostasis, protected the stem cell niche, and prevented tumorigenesis via transcriptional regulation of of Rnf43 and Znrf3, E3 ubiquitin ligases that inhibit Wnt-ß-catenin signaling and restrict ISC proliferation. Thus, activation of the AHR pathway in IECs guards the stem cell niche to maintain intestinal barrier integrity.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Epiteliais/fisiologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Células-Tronco/citologia , Junções Íntimas/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinogênese/patologia , Diferenciação Celular/imunologia , Linhagem Celular , Proliferação de Células , Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Hidrocarboneto Arílico/genética , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética , Via de Sinalização Wnt/fisiologia
6.
Cell ; 149(3): 642-55, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22541434

RESUMO

Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide; nearly half contain mutations in the receptor tyrosine kinase/RAS pathway. Here we show that RAS-pathway mutant NSCLC cells depend on the transcription factor GATA2. Loss of GATA2 reduced the viability of NSCLC cells with RAS-pathway mutations, whereas wild-type cells were unaffected. Integrated gene expression and genome occupancy analyses revealed GATA2 regulation of the proteasome, and IL-1-signaling, and Rho-signaling pathways. These pathways were functionally significant, as reactivation rescued viability after GATA2 depletion. In a Kras-driven NSCLC mouse model, Gata2 loss dramatically reduced tumor development. Furthermore, Gata2 deletion in established Kras mutant tumors induced striking regression. Although GATA2 itself is likely undruggable, combined suppression of GATA2-regulated pathways with clinically approved inhibitors caused marked tumor clearance. Discovery of the nononcogene addiction of KRAS mutant lung cancers to GATA2 presents a network of druggable pathways for therapeutic exploitation.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Fator de Transcrição GATA2/metabolismo , Redes Reguladoras de Genes , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ras/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Fator de Transcrição GATA2/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Proteínas ras/genética
7.
Immunity ; 47(6): 1083-1099.e6, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29246442

RESUMO

The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3' UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers.


Assuntos
Antígeno B7-H1/imunologia , Neoplasias Colorretais/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/imunologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Tristetraprolina/imunologia , Evasão Tumoral , Animais , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Clivagem do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transdução de Sinais , Tristetraprolina/genética
8.
Mol Cell ; 70(4): 707-721.e7, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29754823

RESUMO

DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4-/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4-/-p53+/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.


Assuntos
Carcinogênese/patologia , DNA Polimerase II/química , DNA Polimerase II/fisiologia , Replicação do DNA , Deficiências do Desenvolvimento/etiologia , Transtornos do Crescimento/etiologia , Leucopenia/etiologia , Animais , Carcinogênese/genética , Células Cultivadas , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Humanos , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteína Supressora de Tumor p53/fisiologia
10.
Clin J Sport Med ; 33(1): 33-44, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36111996

RESUMO

OBJECTIVE: The purpose of this study was to explore primary care sports medicine physicians' comfort, competence, education, and scope of training in caring for transgender and gender nonconforming (TGNC) patients/athletes. DESIGN: Mixed-methods, cross-sectional survey. SETTING: Online. PATIENTS OR PARTICIPANTS: In total, 4300 e-mails were successfully sent with 252 eligible responses received from the American Medical Society for Sports Medicine members. INDEPENDENT VARIABLES: Previous relationships with TGNC persons; previous relationships with TGNC patients/athletes; frequency of care for TGNC patients/athletes. MAIN OUTCOME MEASURES: The participants completed a 38-item tool used to assess perceived comfort and competence treating TGNC patients/athletes. Physicians defined "transgender" and described their thoughts on unfair competitive advantage of transgender athletes. RESULTS: Most participants had worked with a TGNC patient (70.2%, n = 177), but far fewer worked with a TGNC athlete (n = 26.6%, n = 67). Among the participants who provided a definition of transgender (n = 183), only 28.4% (n = 52) of participants were able to correctly define the term, whereas most were able to partially (57.9%, n = 106) characterize the term. The most common mechanisms identified for learning about TGNC patients were reading peer-reviewed journal articles (44.8%, n = 113) and CME (41.3%, n = 104). Those with previous TGNC friend/family, patient, and athlete relationships had a significantly different level of comfort and competence treating TGNC patients/athletes. CONCLUSIONS: Previous care relationships with TGNC strongly influences comfort and perceived competence of primary care sports medicine physicians. Training, from unbiased peer-reviewed sources of data, is critical to improve care for TGNC patients/athletes.


Assuntos
Médicos , Medicina Esportiva , Pessoas Transgênero , Humanos , Estudos Transversais , Atletas
11.
Gastroenterology ; 161(4): 1179-1193, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34197832

RESUMO

BACKGROUND & AIMS: Colorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy. METHODS: We selected multiple regions per tumor showing variable T-cell infiltration for a total of 738 regions from 29 patients, divided into discovery and validation cohorts. We performed multiregional whole-exome and RNA sequencing of the tumor cells and integrated these with T-cell receptor sequencing, high-dimensional imaging mass cytometry, detection of programmed death-ligand 1 (PDL1) interaction in situ, multiplexed immunofluorescence, and computational spatial analysis of the TME. RESULTS: In hypermutated CRCs, response to anti-PD1 immunotherapy was not associated with TMB but with high clonality of immunogenic mutations, clonally expanded T cells, low activation of Wnt signaling, deregulation of the interferon gamma pathway, and active immune escape mechanisms. Responsive hypermutated CRCs were also rich in cytotoxic and proliferating PD1+CD8 T cells interacting with PDL1+ antigen-presenting macrophages. CONCLUSIONS: Our study clarified the limits of TMB as a predictor of response of CRC to anti-PD1 immunotherapy. It identified a population of antigen-presenting macrophages interacting with CD8 T cells that consistently segregate with response. We therefore concluded that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages to promote cytotoxic antitumor activity.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Fenômenos Imunogenéticos , Imunogenética , Nivolumabe/uso terapêutico , Microambiente Tumoral , Anticorpos Monoclonais Humanizados/efeitos adversos , Biomarcadores Tumorais/genética , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Ensaios Clínicos como Assunto , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Mutação , Nivolumabe/efeitos adversos , Valor Preditivo dos Testes , Receptor de Morte Celular Programada 1/antagonistas & inibidores , RNA-Seq , Reprodutibilidade dos Testes , Fatores de Tempo , Transcriptoma , Resultado do Tratamento , Macrófagos Associados a Tumor/efeitos dos fármacos , Macrófagos Associados a Tumor/imunologia , Sequenciamento do Exoma
12.
Gastroenterology ; 159(4): 1328-1341.e3, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32553763

RESUMO

BACKGROUND & AIMS: Notch signaling maintains intestinal stem cells (ISCs). When ISCs exit the niche, Notch signaling among early progenitor cells at position +4/5 regulates their specification toward secretory vs enterocyte lineages (binary fate). The transcription factor ATOH1 is repressed by Notch in ISCs; its de-repression, when Notch is inactivated, drives progenitor cells to differentiate along the secretory lineage. However, it is not clear what promotes transition of ISCs to progenitors and how this fate decision is established. METHODS: We sorted cells from Lgr5-GFP knockin intestines from mice and characterized gene expression patterns. We analyzed Notch regulation by examining expression profiles (by quantitative reverse transcription polymerase chain reaction and RNAscope) of small intestinal organoids incubated with the Notch inhibitor DAPT, intestine tissues from mice given injections of the γ-secretase inhibitor dibenzazepine, and mice with intestine-specific disruption of Rbpj. We analyzed intestine tissues from mice with disruption of the RUNX1 translocation partner 1 gene (Runx1t1, also called Mtg8) or CBFA2/RUNX1 partner transcriptional co-repressor 3 (Cbfa2t3, also called Mtg16), and derived their organoids, by histology, immunohistochemistry, and RNA sequencing (RNA-seq). We performed chromatin immunoprecipitation and sequencing analyses of intestinal crypts to identify genes regulated by MTG16. RESULTS: The transcription co-repressors MTG8 and MTG16 were highly expressed by +4/5 early progenitors, compared with other cells along crypt-villus axis. Expression of MTG8 and MTG16 were repressed by Notch signaling via ATOH1 in organoids and intestine tissues from mice. MTG8- and MTG16-knockout intestines had increased crypt hyperproliferation and expansion of ISCs, but enterocyte differentiation was impaired, based on loss of enterocyte markers and functions. Chromatin immunoprecipitation and sequencing analyses showed that MTG16 bound to promoters of genes that are specifically expressed by stem cells (such as Lgr5 and Ascl2) and repressed their transcription. MTG16 also bound to previously reported enhancer regions of genes regulated by ATOH1, including genes that encode Delta-like canonical Notch ligand and other secretory-specific transcription factors. CONCLUSIONS: In intestine tissues of mice and human intestinal organoids, MTG8 and MTG16 repress transcription in the earliest progenitor cells to promote exit of ISCs from their niche (niche exit) and control the binary fate decision (secretory vs enterocyte lineage) by repressing genes regulated by ATOH1.


Assuntos
Proteínas Correpressoras/fisiologia , Proteínas de Ligação a DNA/fisiologia , Enterócitos/citologia , Enterócitos/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Repressoras/fisiologia , Células-Tronco/citologia , Fatores de Transcrição/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem da Célula , Camundongos , Nicho de Células-Tronco , Células-Tronco/metabolismo
13.
PLoS Genet ; 14(5): e1007383, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29746474

RESUMO

Down Syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and results in a spectrum of phenotypes including learning and memory deficits, and motor dysfunction. It has been hypothesized that an additional copy of a few Hsa21 dosage-sensitive genes causes these phenotypes, but this has been challenged by observations that aneuploidy can cause phenotypes by the mass action of large numbers of genes, with undetectable contributions from individual sequences. The motor abnormalities in DS are relatively understudied-the identity of causative dosage-sensitive genes and the mechanism underpinning the phenotypes are unknown. Using a panel of mouse strains with duplications of regions of mouse chromosomes orthologous to Hsa21 we show that increased dosage of small numbers of genes causes locomotor dysfunction and, moreover, that the Dyrk1a gene is required in three copies to cause the phenotype. Furthermore, we show for the first time a new DS phenotype: loss of motor neurons both in mouse models and, importantly, in humans with DS, that may contribute to locomotor dysfunction.


Assuntos
Síndrome de Down/genética , Atividade Motora/genética , Neurônios Motores/metabolismo , Degeneração Neural/genética , Adulto , Idoso , Animais , Autopsia , Modelos Animais de Doenças , Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Medula Espinal/metabolismo , Medula Espinal/patologia , Quinases Dyrk
14.
Circ Res ; 122(2): 231-245, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29233846

RESUMO

RATIONALE: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. OBJECTIVE: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. METHODS AND RESULTS: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and ß-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/ß-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. CONCLUSIONS: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.


Assuntos
Antígenos CD/biossíntese , Caderinas/biossíntese , Endotélio Vascular/metabolismo , Epigênese Genética/fisiologia , Animais , Antígenos CD/genética , Caderinas/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Endotélio Vascular/ultraestrutura , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Proteínas do Grupo Polycomb/metabolismo , Ligação Proteica/fisiologia
15.
Nature ; 514(7523): 498-502, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25341788

RESUMO

After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion--driven by lymphocyte influx and proliferation--that is the critical hallmark of adaptive immunity.


Assuntos
Células Dendríticas/fisiologia , Fibroblastos/citologia , Linfonodos/citologia , Células Estromais/citologia , Actomiosina/metabolismo , Animais , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Células Dendríticas/imunologia , Feminino , Fibroblastos/fisiologia , Inflamação/imunologia , Lectinas Tipo C/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Células Estromais/fisiologia , Proteínas ras/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP , Proteína de Ligação a GTP rhoC
16.
Gastroenterology ; 151(3): 526-39, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27215660

RESUMO

BACKGROUND & AIMS: Pancreatitis is the most important risk factor for pancreatic ductal adenocarcinoma (PDAC). Pancreatitis predisposes to PDAC because it induces a process of acinar cell reprogramming known as acinar-to-ductal metaplasia (ADM)-a precursor of pancreatic intraepithelial neoplasia lesions that can progress to PDAC. Mutations in KRAS are found at the earliest stages of pancreatic tumorigenesis, and it appears to be a gatekeeper to cancer progression. We investigated how mutations in KRAS cooperate with pancreatitis to promote pancreatic cancer progression in mice. METHODS: We generated mice carrying conditional alleles of Yap1 and Taz and disrupted Yap1 and Taz using a Cre-lox recombination strategy in adult mouse pancreatic acinar cells (Yap1fl/fl;Tazfl/fl;Ela1-CreERT2). We crossed these mice with LSL-KrasG12D mice, which express a constitutively active form of KRAS after Cre recombination. Pancreatic tumor initiation and progression were analyzed after chemically induced pancreatitis. We analyzed pancreatic tissues from patients with pancreatitis or PDAC by immunohistochemistry. RESULTS: Oncogenic activation of KRAS in normal, untransformed acinar cells in the pancreatic tissues of mice resulted in increased levels of pancreatitis-induced ADM. Expression of the constitutive active form of KRAS in this system led to activation of the transcriptional regulators YAP1 and TAZ; their function was required for pancreatitis-induced ADM in mice. The JAK-STAT3 pathway was a downstream effector of KRAS signaling via YAP1 and TAZ. YAP1 and TAZ directly mediated transcriptional activation of several genes in the JAK-STAT3 signaling pathway; this could be a mechanism by which acinar cells that express activated KRAS become susceptible to inflammation. CONCLUSIONS: We identified a mechanism by which oncogenic KRAS facilitates ADM and thereby generates the cells that initiate neoplastic progression. This process involves activation of YAP1 and TAZ in acinar cells, which up-regulate JAK-STAT3 signaling to promote development of PDAC in mice.


Assuntos
Carcinogênese/genética , Neoplasias Pancreáticas/genética , Pancreatite/complicações , Transdução de Sinais/genética , Células Acinares/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Janus Quinases/metabolismo , Camundongos , Mutação , Pâncreas/patologia , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/patologia , Pancreatite/induzido quimicamente , Pancreatite/patologia , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Risco , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteínas de Sinalização YAP
17.
Proc Natl Acad Sci U S A ; 110(27): E2490-9, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23766371

RESUMO

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO(-)) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis.


Assuntos
Adenoma/patologia , Linhagem da Célula/genética , Neoplasias Colorretais/patologia , Células-Tronco Multipotentes/patologia , Células-Tronco Neoplásicas/patologia , Adenoma/genética , Adenoma/metabolismo , Diferenciação Celular/genética , Células Clonais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , DNA Mitocondrial/genética , DNA de Neoplasias/genética , Epigênese Genética , Humanos , Modelos Biológicos , Células-Tronco Multipotentes/metabolismo , Mutação , Células-Tronco Neoplásicas/metabolismo
18.
Blood ; 121(24): 4930-7, 2013 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-23645836

RESUMO

Neutrophil recruitment is an important early step in controlling tissue infections or injury. Here, we report that this influx depends on both tissue-resident mast cells and macrophages. Mice with mast cell deficiency recruit reduced numbers of neutrophils in the first few hours of intraperitoneal lipopolysaccharide (LPS) stimulation. Conversely, in mice with clodronate-ablated macrophages, neutrophils extravasate, but have limited ability to reach the peritoneal fluid. Tissue macrophages synthesize neutrophil chemoattractants CXCL1/CXCL2 (CXC chemokine ligands 1/2) in response to LPS. Mast cells also produce these chemokines of which a proportion are preformed in granules. Release of the granules and new CXCL1/CXCL2 synthesis is Toll-like receptor 4-dependent. Both in vivo studies with blocking monoclonal antibodies and in vitro chemotaxis experiments show the neutrophil response to mast cells and macrophages to be CXCL1/CXCL2-dependent. The data are in keeping with the model that mast cells, optimally positioned in close proximity to the vasculature, initiate an early phase of neutrophil recruitment by releasing the chemoattractants CXCL1/CXCL2. Having arrived within the stimulated tissue, neutrophils penetrate further in a macrophage-dependent manner. Therefore, we demonstrate a positive role for mast cells in tissue inflammation and define how this comes about with contribution from a second tissue cell, the macrophage.


Assuntos
Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Macrófagos Peritoneais/metabolismo , Mastócitos/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Animais , Líquido Ascítico/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Macrófagos Peritoneais/patologia , Mastócitos/patologia , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
19.
Cancer Cell ; 11(4): 311-9, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17418408

RESUMO

Germline mutations in the fumarate hydratase (FH) tumor suppressor gene predispose to leiomyomatosis, renal cysts, and renal cell cancer (HLRCC). HLRCC tumors overexpress HIF1alpha and hypoxia pathway genes. We conditionally inactivated mouse Fh1 in the kidney. Fh1 mutants developed multiple clonal renal cysts that overexpressed Hif1alpha and Hif2alpha. Hif targets, such as Glut1 and Vegf, were upregulated. We found that Fh1-deficient murine embryonic stem cells and renal carcinomas from HLRCC showed similar overexpression of HIF and hypoxia pathway components to the mouse cysts. Our data have shown in vivo that pseudohypoxic drive, resulting from HIF1alpha (and HIF2alpha) overexpression, is a direct consequence of Fh1 inactivation. Our mouse may be useful for testing therapeutic interventions that target angiogenesis and HIF-prolyl hydroxylation.


Assuntos
Carcinoma de Células Renais/etiologia , Fumarato Hidratase/genética , Inativação Gênica/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Doenças Renais Císticas/etiologia , Neoplasias Renais/etiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Hipóxia Celular , Proliferação de Células , Feminino , Transportador de Glucose Tipo 1/metabolismo , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Cancer Discov ; 14(6): 1018-1047, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38581685

RESUMO

Understanding the role of the tumor microenvironment (TME) in lung cancer is critical to improving patient outcomes. We identified four histology-independent archetype TMEs in treatment-naïve early-stage lung cancer using imaging mass cytometry in the TRACERx study (n = 81 patients/198 samples/2.3 million cells). In immune-hot adenocarcinomas, spatial niches of T cells and macrophages increased with clonal neoantigen burden, whereas such an increase was observed for niches of plasma and B cells in immune-excluded squamous cell carcinomas (LUSC). Immune-low TMEs were associated with fibroblast barriers to immune infiltration. The fourth archetype, characterized by sparse lymphocytes and high tumor-associated neutrophil (TAN) infiltration, had tumor cells spatially separated from vasculature and exhibited low spatial intratumor heterogeneity. TAN-high LUSC had frequent PIK3CA mutations. TAN-high tumors harbored recently expanded and metastasis-seeding subclones and had a shorter disease-free survival independent of stage. These findings delineate genomic, immune, and physical barriers to immune surveillance and implicate neutrophil-rich TMEs in metastasis. SIGNIFICANCE: This study provides novel insights into the spatial organization of the lung cancer TME in the context of tumor immunogenicity, tumor heterogeneity, and cancer evolution. Pairing the tumor evolutionary history with the spatially resolved TME suggests mechanistic hypotheses for tumor progression and metastasis with implications for patient outcome and treatment. This article is featured in Selected Articles from This Issue, p. 897.


Assuntos
Neoplasias Pulmonares , Microambiente Tumoral , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Microambiente Tumoral/imunologia , Linfócitos T/imunologia , Células Mieloides/imunologia , Feminino , Masculino , Evasão da Resposta Imune
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