RESUMO
Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.
Assuntos
Autofagia/fisiologia , Glucose/metabolismo , Homeostase , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Condicionamento Físico Animal/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Adiponectina/sangue , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína Beclina-1 , Células Cultivadas , Gorduras na Dieta/efeitos adversos , Privação de Alimentos/fisiologia , Técnicas de Introdução de Genes , Intolerância à Glucose/induzido quimicamente , Intolerância à Glucose/prevenção & controle , Teste de Tolerância a Glucose , Homeostase/efeitos dos fármacos , Leptina/sangue , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Mutação , Miocárdio/citologia , Fosforilação/genética , Resistência Física/genética , Resistência Física/fisiologia , Esforço Físico/genética , Esforço Físico/fisiologia , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Corrida/fisiologiaRESUMO
Liquid biopsies enable early detection and monitoring of diseases such as cancer, but their sensitivity remains limited by the scarcity of analytes such as cell-free DNA (cfDNA) in blood. Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. We sought to transiently augment the level of circulating tumor DNA (ctDNA) in a blood draw by attenuating its clearance in vivo. We report two intravenous priming agents given 1 to 2 hours before a blood draw to recover more ctDNA. Our priming agents consist of nanoparticles that act on the cells responsible for cfDNA clearance and DNA-binding antibodies that protect cfDNA. In tumor-bearing mice, they greatly increase the recovery of ctDNA and improve the sensitivity for detecting small tumors.
Assuntos
Ácidos Nucleicos Livres , Neoplasias , Animais , Camundongos , Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/sangue , DNA Tumoral Circulante/sangue , Biópsia Líquida , Mutação , Neoplasias/sangue , Neoplasias/diagnóstico , Humanos , Feminino , Camundongos Endogâmicos BALB C , Sensibilidade e EspecificidadeRESUMO
Blood-based, or "liquid," biopsies enable minimally invasive diagnostics but have limits on sensitivity due to scarce cell-free DNA (cfDNA). Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo . Here, we sought to augment the level of circulating tumor DNA (ctDNA) detected in a blood draw by attenuating the clearance of cfDNA in vivo . We report a first-in-class intravenous DNA-binding priming agent given 2 hours prior to a blood draw to recover more cfDNA. The DNA-binding antibody minimizes nuclease digestion and organ uptake of cfDNA, decreasing its clearance at 1 hour by over 150-fold. To improve plasma persistence and limit potential immune interactions, we abrogated its Fc-effector function. We found that it protects GC-rich sequences and DNase-hypersensitive sites, which are ordinarily underrepresented in cfDNA. In tumor-bearing mice, priming improved tumor DNA recovery by 19-fold and sensitivity for detecting cancer from 6% to 84%. These results suggest a novel method to enhance the sensitivity of existing DNA-based cancer testing using blood biopsies.
RESUMO
Liquid biopsies are enabling minimally invasive monitoring and molecular profiling of diseases across medicine, but their sensitivity remains limited by the scarcity of cell-free DNA (cfDNA) in blood. Here, we report an intravenous priming agent that is given prior to a blood draw to increase the abundance of cfDNA in circulation. Our priming agent consists of nanoparticles that act on the cells responsible for cfDNA clearance to slow down cfDNA uptake. In tumor-bearing mice, this agent increases the recovery of circulating tumor DNA (ctDNA) by up to 60-fold and improves the sensitivity of a ctDNA diagnostic assay from 0% to 75% at low tumor burden. We envision that this priming approach will significantly improve the performance of liquid biopsies across a wide range of clinical applications in oncology and beyond.
RESUMO
Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules ('single duplexes') to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10-8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.
Assuntos
Neoplasias , Sêmen , Masculino , Humanos , Adulto , Mutação/genética , Neoplasias/genética , Neoplasias/diagnóstico , Análise de Sequência de DNA , DNA , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.
Assuntos
Endopeptidases , Via de Sinalização Wnt , beta Catenina , Animais , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Ligases/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores Wnt , Ubiquitina , Peixe-Zebra/metabolismo , Endopeptidases/metabolismoRESUMO
With great sadness, the scientific community received the news of the loss of Beth Levine on 15 June 2020. Dr. Levine was a pioneer in the autophagy field and work in her lab led not only to a better understanding of the molecular mechanisms regulating the pathway, but also its implications in multiple physiological and pathological conditions, including its role in development, host defense, tumorigenesis, aging or metabolism. This review does not aim to provide a comprehensive view of autophagy, but rather an outline of some of the discoveries made by the group of Beth Levine, from the perspective of some of her own mentees, hoping to honor her legacy in science.
RESUMO
BACKGROUND: The transcription factor signal transducer and activator of transcription 3 (STAT3) drives progression in glioblastoma (GBM), suggesting STAT3 as a therapeutic target. Surprisingly however, GBM cells generally show primary resistance to STAT3 blockade. METHODS: Human glioblastoma cell lines LN229, U87, SF767, and U373, and patient-derived xenografts (PDXs) GBM8 and GBM43 were used to evaluate epidermal growth factor receptor (EGFR) activation during STAT3 inhibition. Protein and gene expression experiments, protein stability assays, cytokine arrays, phospho-tyrosine arrays and EGFR-ligand protein arrays were performed on STAT3 inhibitor-treated cells. To evaluate antitumor activity, we administered a betacellulin (BTC)-neutralizing antibody alone and in combination with STAT3 inhibition. BTC is an EGFR ligand. We therefore treated mice with orthotopic xenografts using the third-generation EGFR inhibitor osimertinib, with or without STAT3 knockdown. RESULTS: We demonstrate that both small-molecule inhibitors and knockdown of STAT3 led to expression and secretion of the EGFR ligand BTC, resulting in activation of EGFR and subsequent downstream phosphorylation of nuclear factor-kappaB (NF-κB). Neutralizing antibody against BTC abrogated activation of both EGFR and NF-κB in response to inhibition of STAT3; with combinatorial blockade of STAT3 and BTC inducing apoptosis in GBM cells. Blocking EGFR and STAT3 together inhibited tumor growth, improving survival in mice bearing orthotopic GBM PDXs in vivo. CONCLUSION: These data reveal a feedback loop among STAT3, EGFR, and NF-κB that mediates primary resistance to STAT3 blockade and suggest strategies for therapeutic intervention.
Assuntos
Glioblastoma , Animais , Betacelulina , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Camundongos , NF-kappa B/metabolismo , Fosforilação , Fator de Transcrição STAT3/metabolismoRESUMO
The mTOR signaling is dysregulated prominently in human cancers including glioblastoma, suggesting mTOR as a robust target for therapy. Inhibitors of mTOR have had limited success clinically, however, in part because their mechanism of action is cytostatic rather than cytotoxic. Here, we tested three distinct mTOR kinase inhibitors (TORKi) PP242, KU-0063794, and sapanisertib against glioblastoma cells. All agents similarly decreased proliferation of glioblastoma cells, whereas PP242 uniquely induced apoptosis. Apoptosis induced by PP242 resulted from off-target cooperative inhibition of JAK2 and protein kinase C alpha (PKCα). Induction of apoptosis was also decreased by additional on-target inhibition of mTOR, due to induction of autophagy. As EGFR inhibitors can block PKCα, EGFR inhibitors erlotinib and osimertinib were tested separately in combination with the JAK2 inhibitor AZD1480. Combination therapy induced apoptosis of glioblastoma tumors in both flank and in patient-derived orthotopic xenograft models, providing a preclinical rationale to test analogous combinations in patients. SIGNIFICANCE: These findings identify PKCα and JAK2 as targets that drive apoptosis in glioblastoma, potentially representing a clinically translatable approach for glioblastoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Feminino , Glioblastoma/patologia , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Purinas/farmacologia , Purinas/uso terapêutico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Macrófagos/metabolismo , Microambiente Tumoral/fisiologia , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Glioblastoma/imunologia , Glioblastoma/patologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/imunologia , Macrófagos/patologia , Reprodutibilidade dos TestesRESUMO
Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, representative of cerebellar progenitors. We transduced NES cells with MYCN, observing medulloblastoma upon orthotopic implantation in mice. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome patients, who are predisposed to medulloblastoma due to germline-mutated PTCH1, also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3X or loss of GSE1 both accelerating tumorigenesis. These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma.
Assuntos
Síndrome do Nevo Basocelular/genética , Neoplasias Encefálicas/genética , Meduloblastoma/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Células-Tronco Neurais/fisiologia , Células Neuroepiteliais/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Síndrome do Nevo Basocelular/metabolismo , Síndrome do Nevo Basocelular/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/genética , RNA Helicases DEAD-box/genética , Modelos Animais de Doenças , Engenharia Genética , Predisposição Genética para Doença , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Camundongos , Camundongos SCID , Proteína Proto-Oncogênica N-Myc/genética , Proteínas de Neoplasias/genética , Receptor Patched-1/genética , Transplante de Células-Tronco , Transplante HeterólogoRESUMO
Amplification of epidermal growth factor receptor (EGFR) and its active mutant EGFRvIII occurs frequently in glioblastoma (GBM). While EGFR and EGFRvIII play critical roles in pathogenesis, targeted therapy with EGFR-tyrosine kinase inhibitors (TKIs) or antibodies has only shown limited efficacy in patients. Here we discuss signaling pathways mediated by EGFR/EGFRvIII, current therapeutics, and novel strategies to target EGFR/EGFRvIII-amplified GBM.
Assuntos
Neoplasias Encefálicas/terapia , Receptores ErbB/fisiologia , Glioblastoma/terapia , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Neoplasias Encefálicas/genética , Receptores ErbB/antagonistas & inibidores , Glioblastoma/genética , Humanos , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
: Amplification of the EGFR gene and its truncation mutant EGFRvIII are hallmarks of glioblastoma. Although coexpression of EGFR and EGFRvIII confers a growth advantage, how EGFR and EGFRvIII influence the tumor microenvironment remains incompletely understood. Here, we show that EGFR and EGFRvIII cooperate to induce macrophage infiltration via upregulation of the chemokine CCL2. EGFRvIII was significantly enriched in glioblastoma patient samples with high CCL2, and knockout of CCL2 in tumors coexpressing EGFR and EGFRvIII led to decreased infiltration of macrophages. KRAS was a critical signaling intermediate for EGFR- and EGFRvIII-induced expression of CCL2. Our results illustrate how EGFR and EGFRvIII direct the microenvironment in glioblastoma. SIGNIFICANCE: Full-length EGFR and truncated EGFRvIII work through KRAS to upregulate the chemokine CCL2 and drive macrophage infiltration in glioblastoma.
Assuntos
Neoplasias Encefálicas/metabolismo , Quimiocina CCL2/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Citocinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Microglia/metabolismo , Transplante de Neoplasias , Fosforilação , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Microambiente Tumoral , Regulação para CimaRESUMO
In this issue of Cancer Cell, Villa et al. report that survival of glioblastoma cells is dependent on uptake of cholesterol. A synthetic agonist of the Liver X receptor depleted cholesterol in GBM cells, slowing growth of GBM xenografts.
Assuntos
Colesterol , Glioblastoma , HumanosRESUMO
Autophagy is a fundamental adaptive response to amino acid starvation orchestrated by conserved gene products, the autophagy (ATG) proteins. However, the cellular cues that activate the function of ATG proteins during amino acid starvation are incompletely understood. Here we show that two related stress-responsive kinases, members of the p38 mitogen-activated protein kinase (MAPK) signaling pathway MAPKAPK2 (MK2) and MAPKAPK3 (MK3), positively regulate starvation-induced autophagy by phosphorylating an essential ATG protein, Beclin 1, at serine 90, and that this phosphorylation site is essential for the tumor suppressor function of Beclin 1. Moreover, MK2/MK3-dependent Beclin 1 phosphorylation (and starvation-induced autophagy) is blocked in vitro and in vivo by BCL2, a negative regulator of Beclin 1. Together, these findings reveal MK2/MK3 as crucial stress-responsive kinases that promote autophagy through Beclin 1 S90 phosphorylation, and identify the blockade of MK2/3-dependent Beclin 1 S90 phosphorylation as a mechanism by which BCL2 inhibits the autophagy function of Beclin 1.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteína Beclina-1 , Linhagem Celular Tumoral , Meios de Cultura , Genes Supressores de Tumor , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologiaRESUMO
In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G1/G0 in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G2/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G2/M transition and arrest in G1/G 00 autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G1/G0 quiescent state.
Assuntos
Autofagia/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Nitrogênio/deficiência , Nitrogênio/farmacologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Saccharomyces cerevisiae/citologia , Fenótipo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Aberrant signaling through the class I phosphatidylinositol 3-kinase (PI3K)-Akt axis is frequent in human cancer. Here, we show that Beclin 1, an essential autophagy and tumor suppressor protein, is a target of the protein kinase Akt. Expression of a Beclin 1 mutant resistant to Akt-mediated phosphorylation increased autophagy, reduced anchorage-independent growth, and inhibited Akt-driven tumorigenesis. Akt-mediated phosphorylation of Beclin 1 enhanced its interactions with 14-3-3 and vimentin intermediate filament proteins, and vimentin depletion increased autophagy and inhibited Akt-driven transformation. Thus, Akt-mediated phosphorylation of Beclin 1 functions in autophagy inhibition, oncogenesis, and the formation of an autophagy-inhibitory Beclin 1/14-3-3/vimentin intermediate filament complex. These findings have broad implications for understanding the role of Akt signaling and intermediate filament proteins in autophagy and cancer.