RESUMO
In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity.
Assuntos
Adenosina Trifosfatases/metabolismo , Ciclinas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Proliferação de Células , Ciclina D1/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Fosforilação , Saccharomyces cerevisiae/citologiaRESUMO
The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1Psy-triggered immunity. HopZ3 acetylates AvrPto1Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3's targets such as AvrPto1Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3's unusual capacity to modify histidine in addition to serine, threonine and lysine residues.
Assuntos
Acetiltransferases/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Proteínas de Bactérias/antagonistas & inibidores , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Pseudomonas syringae/patogenicidade , Solanum lycopersicum/imunologia , Acetilação , Acetiltransferases/genética , Acetiltransferases/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismoRESUMO
Therapy-related myeloid neoplasms (t-MNs) are high-risk late effects with poorly understood pathogenesis in cancer survivors. It has been postulated that, in some cases, hematopoietic stem and progenitor cells (HSPCs) harboring mutations are selected for by cytotoxic exposures and transform. Here, we evaluate this model in the context of deficiency of CUX1, a transcription factor encoded on chromosome 7q and deleted in half of t-MN cases. We report that CUX1 has a critical early role in the DNA repair process in HSPCs. Mechanistically, CUX1 recruits the histone methyltransferase EHMT2 to DNA breaks to promote downstream H3K9 and H3K27 methylation, phosphorylated ATM retention, subsequent γH2AX focus formation and propagation, and, ultimately, 53BP1 recruitment. Despite significant unrepaired DNA damage sustained in CUX1-deficient murine HSPCs after cytotoxic exposures, they continue to proliferate and expand, mimicking clonal hematopoiesis in patients postchemotherapy. As a consequence, preexisting CUX1 deficiency predisposes mice to highly penetrant and rapidly fatal therapy-related erythroleukemias. These findings establish the importance of epigenetic regulation of HSPC DNA repair and position CUX1 as a gatekeeper in myeloid transformation.
Assuntos
Cromossomos de Mamíferos , Reparo do DNA , Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio , Leucemia Eritroblástica Aguda , Proteínas de Neoplasias , Segunda Neoplasia Primária , Proteínas Nucleares , Proteínas Repressoras , Animais , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Hematopoiese Clonal , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
For decades genotoxic therapy has been a mainstay in the treatment of cancer, based on the understanding that the deregulated growth and genomic instability that drive malignancy also confer a shared vulnerability. Although chemotherapy and radiation can be curative, only a fraction of patients benefit, while nearly all are subjected to the harmful side-effects. Drug repurposing, defined here as retooling existing drugs and compounds as chemo or radiosensitizers, offers an attractive route to identifying otherwise non-toxic agents that can potentiate the benefits of genotoxic cancer therapy to enhance the therapeutic ratio. This review seeks to highlight recent progress in defining cellular mechanisms of the DNA damage response including damage sensing, chromatin modification, DNA repair, checkpoint signaling, and downstream survival and death pathways, as a framework to determine which drugs and natural products may offer the most potential for repurposing as chemo- and/or radiosensitizers. We point to classical examples and recent progress that have identified drugs that disrupt cellular responses to DNA damage and may offer the greatest clinical potential. The most important next steps may be to initiate prospective clinical trials toward translating these laboratory discoveries to benefit patients.
Assuntos
Antineoplásicos/uso terapêutico , Enzimas Reparadoras do DNA/antagonistas & inibidores , Descoberta de Drogas , Reposicionamento de Medicamentos/métodos , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Dano ao DNA , Reparo do DNA , HumanosRESUMO
The binding of DNA-dependent protein kinase catalytic subunit (DNA-PKcs, also known as PRKDC) to Ku proteins at DNA double-strand breaks (DSBs) has long been considered essential for non-homologous end joining (NHEJ) repair, providing a rationale for use of DNA-PKcs inhibitors as cancer therapeutics. Given lagging clinical translation, we reexamined mechanisms and observed instead that DSB repair can proceed independently of DNA-PKcs. While repair of radiation-induced DSBs was blocked in cells expressing shRNAs targeting Ku proteins or other NHEJ core factors, DSBs were repaired on schedule despite targeting DNA-PKcs. Although we failed to observe a DSB repair defect, the γH2AX foci that formed at sites of DNA damage persisted indefinitely after irradiation, leading to cytokinesis failure and accumulation of binucleated cells. Following this mitotic slippage, cells with decreased DNA-PKcs underwent accelerated cellular senescence. We identified downregulation of ataxia-telangiectasia mutated kinase (ATM) as the critical role of DNA-PKcs in recovery from DNA damage, insofar as targeting ATM restored γH2AX foci resolution and cytokinesis. Considering the lack of direct impact on DSB repair and emerging links between senescence and resistance to cancer therapy, these results suggest reassessing DNA-PKcs as a target for cancer treatment.
Assuntos
Senescência Celular , Citoproteção , Reparo do DNA/efeitos da radiação , Proteína Quinase Ativada por DNA/metabolismo , Mitose , Radiação Ionizante , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Aurora Quinase B/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citocinese/efeitos dos fármacos , Citocinese/efeitos da radiação , Citoproteção/efeitos dos fármacos , Citoproteção/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Histonas/metabolismo , Humanos , Células MCF-7 , Camundongos , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Pironas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/efeitos da radiação , Quinase 1 Polo-LikeRESUMO
P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.
Assuntos
Antígenos Nucleares/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas Associadas à Matriz Nuclear/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Reparo do DNA por Junção de Extremidades/genética , Regulação para Baixo , Feminino , Células HEK293 , Humanos , Ligação ProteicaRESUMO
The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Fúngico , Histona Acetiltransferases , Proteínas de Saccharomyces cerevisiae , Acetilação , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Diverse stresses including genotoxic therapy can induce proliferating cancer cells to undergo cellular senescence and take on the characteristic phenotypes of replicative cellular aging. This accelerated or therapy-induced senescence has been alternatively proposed to contribute to therapeutic efficacy or resistance. Toward better understanding this cell state, we sought to define the core transcriptome of accelerated senescence in cancer cells. RESULTS: We examined senescence induced by ionizing irradiation or ectopic overexpression of the stoichiometric cyclin-dependent kinase (CDK) inhibitor p21CIP/WAF1/SDI1 in the human breast cancer cell line MCF7. While radiation produces a strong DNA damage response, ectopic expression of p21 arrests cell cycle progression independently of DNA damage. Both conditions promoted senescence within 5 days. Microarray analysis revealed 378 up- and 391 down-regulated genes that were shared between the two conditions, representing a candidate signature. Systems analysis of the shared differentially expressed genes (DEGs) revealed strong signals for cell cycle control and DNA damage response pathways and predicted multiple upstream regulators previously linked to senescence. Querying the shared DEGs against the Connectivity Map (cmap) database of transcriptional responses to small molecules yielded 20 compounds that induce a similar gene expression pattern in MCF7 cells. Of 16 agents evaluated, six induced senescence on their own. Of these, the selective estrogen receptor degrader fulvestrant and the histone acetyltransferase inhibitor vorinostat did so without causing chromosomal damage. CONCLUSIONS: Using a systems biology approach with experimental validation, we have defined a core gene expression signature for therapy-induced senescence.
Assuntos
Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Biologia Computacional/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Transcriptoma/efeitos dos fármacos , Humanos , Células MCF-7 , Quinases Ativadas por p21/metabolismoRESUMO
Enumeration of tumor-infiltrating lymphocytes (TILs) in H&E stained tissue sections has demonstrated limited value in predicting immune responses to cancer immunotherapy, likely reflecting the diversity of cell types and immune activation states among tumor infiltrates. Multiparametric flow cytometry enables robust phenotypic and functional analysis to distinguish suppression from activation, but tissue dissociation eliminates spatial context. Multiplex methods for immunohistochemistry (IHC) are emerging, but these interrogate only a single tissue section at a time. Here, we report transparent tissue tomography (T3) as a tool for three-dimensional (3D) imaging cytometry in the complex architecture of the tumor microenvironment, demonstrating multiplexed immunofluorescent analysis in core needle biopsies. Using T3 imaging, image processing and machine learning to map CD3+CD8+ cytotoxic T cells (CTLs) in whole core needle biopsies from Her2+ murine mammary tumors and human head and neck surgical specimens revealed marked inhomogeneity within single needle cores, confirmed by serial section IHC. Applying T3 imaging cytometry, we discovered a strong spatial correlation between CD3+CD8+ CTLs and microvasculature in the EGFR+ parenchyma, revealing significant differences among head and neck cancer patients. These results show that T3 offers simple and rapid access to three-dimensional and quantitative maps of the tumor microenvironment and immune infiltrate, offering a new diagnostic tool for personalized cancer immunotherapy.
Assuntos
Biópsia com Agulha de Grande Calibre/métodos , Imageamento Tridimensional/métodos , Linfócitos do Interstício Tumoral/imunologia , Tomografia Computadorizada por Raios X/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Citometria por Imagem/métodos , Imuno-Histoquímica/métodos , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Neoplasias Mamárias Animais/diagnóstico por imagem , Neoplasias Mamárias Animais/imunologia , Neoplasias Mamárias Animais/patologia , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
Current nanoparticle (NP) drug carriers mostly depend on the enhanced permeability and retention (EPR) effect for selective drug delivery to solid tumors. However, in the absence of a persistent EPR effect, the peritumoral endothelium can function as an access barrier to tumors and negatively affect the effectiveness of NPs. In recognition of the peritumoral endothelium as a potential barrier in drug delivery to tumors, poly(lactic-co-glycolic acid) (PLGA) NPs are modified with a quinic acid (QA) derivative, synthetic mimic of selectin ligands. QA-decorated NPs (QA-NP) interact with human umbilical vein endothelial cells expressing E-/P-selectins and induce transient increase in endothelial permeability to translocate across the layer. QA-NP reach selectin-upregulated tumors, achieving greater tumor accumulation and paclitaxel (PTX) delivery than polyethylene glycol-decorated NPs (PEG-NP). PTX-loaded QA-NP show greater anticancer efficacy than Taxol or PTX-loaded PEG-NP at the equivalent PTX dose in different animal models and dosing regimens. Repeated dosing of PTX-loaded QA-NP for two weeks results in complete tumor remission in 40-60% of MDA-MB-231 tumor-bearing mice, while those receiving control treatments succumb to death. QA-NP can exploit the interaction with selectin-expressing peritumoral endothelium and deliver anticancer drugs to tumors to a greater extent than the level currently possible with the EPR effect.
Assuntos
Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Células Endoteliais/metabolismo , Nanopartículas/química , Ácido Quínico/química , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Nus , Microscopia Confocal , Polímeros/química , Selectinas/química , Microambiente Tumoral/fisiologiaRESUMO
Enzyme-dependent post-translational modifications (PTMs) mediate the cellular regulation of proteins and can be discovered using proteomics. However, even where the peptides of interest can be enriched for analysis with state-of-the-art LC-MS/MS tools and informatics, only a fraction of peptide ions can be identified confidently. Thus, many PTM sites remain undiscovered and unconfirmed. In this minireview, we use a case study to discuss how the use of inclusion lists, turning off isotopic exclusion, and manual validation significantly increased depth of coverage, facilitating discovery of acetylation sites in targets of an acetyltransferase virulence factor. These underutilized strategies have the potential to help answer many mechanistic biological questions that large-scale proteomic studies cannot.
Assuntos
Peptídeos/análise , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem/métodos , Acetilação , Animais , Cromatografia Líquida/métodos , Humanos , Peptídeos/química , Peptídeos/metabolismoRESUMO
The nuclear face of the nuclear membrane is enriched with the intermediate filament protein lamin A. Mutations in LMNA, the gene encoding lamin A, lead to a diverse set of inherited conditions including myopathies that affect both the heart and skeletal muscle. To gain insight about lamin A protein interactions, binding proteins associated with the tail of lamin A were characterized. Of 130 nuclear proteins found associated with the lamin A tail, 17 (13%) were previously described lamin A binding partners. One protein not previously linked to lamin A, matrin-3, was selected for further study, because like LMNA mutations, matrin-3 has also been implicated in inherited myopathy. Matrin-3 binds RNA and DNA and is a nucleoplasmic protein originally identified from the insoluble nuclear fraction, referred to as the nuclear matrix. Anti-matrin-3 antibodies were found to co-immunoprecipitate lamin A, and the lamin-A binding domain was mapped to the carboxy-terminal half of matrin-3. Three-dimensional mapping of the lamin A-matrin-3 interface showed that the LMNA truncating mutation Δ303, which lacks the matrin-3 binding domain, was associated with an increased distance between lamin A and matrin-3. LMNA mutant cells are known to have altered biophysical properties and the matrin-3-lamin A interface is positioned to contribute to these defects.
Assuntos
Lamina Tipo A/metabolismo , Músculo Esquelético/metabolismo , Doenças Musculares/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Anticorpos Anti-Idiotípicos , Sítios de Ligação , Humanos , Lamina Tipo A/genética , Músculo Esquelético/patologia , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Mutação , Proteínas Associadas à Matriz Nuclear/genética , Ligação Proteica , Proteínas de Ligação a RNA/genéticaRESUMO
Current strategies to target tumors with nanomedicines rely on passive delivery via the enhanced permeability and retention effect, leveraging the disorganized tumor microvasculature to promote macromolecule extravasation and the reduced lymphatic and venous drainage that favor retention. Nonetheless, FDA approvals and clinical use of nanomedicines have lagged, reflecting failure to display superiority over conventional formulations. Here, we have exploited image-guided X-irradiation to augment nanoparticle accumulation in tumors. A single 5 Gy dose of radiation, below that required to significantly delay tumor growth, can markedly enhance delivery of macromolecules and nanoparticles. The radiation effect was independent of endothelial cell integrity, suggesting a primary role for damage to microvascular pericytes and/or interstitial extracellular matrix. Significantly, radiation-guided delivery potentiated the therapeutic effects of PEGylated liposomal doxorubicin on experimental tumors. Applied to patients, these results suggest repurposing image-guided radiotherapy as a tool to guide cancer nanomedicine delivery, enhancing local control for primary tumors and metastatic disease while limiting systemic toxicity.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/radioterapia , Nanomedicina/métodos , Radioterapia Guiada por Imagem/métodos , Animais , Feminino , Humanos , Imuno-Histoquímica , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Radiação Ionizante , Microambiente Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multiparametric flow cytometry offers a powerful approach to single-cell analysis with broad applications in research and diagnostics. Despite advances in instrumentation, progress in methodology has lagged. Currently there is no simple and efficient method for antibody labeling or quantifying the number of antibodies bound per cell. Herein, we describe a DNA-directed assembly approach to fluorescent labeling that overcomes these barriers. Oligonucleotide-tagged antibodies and microparticles can be annealed to complementary oligonucleotides bearing fluorophores to create assay-specific labeling probes and controls, respectively. The ratio of the fluorescence intensity of labeled cells to the control particles allows direct conversion of qualitative data to quantitative units of antibody binding per cell. Importantly, a single antibody can be labeled with any fluorophore by using a simple mix-and-match labeling strategy. Thus, any antibody can provide a quantitative probe in any fluorescent channel, thus overcoming major barriers to the use of flow cytometry as a technique for systems biology and clinical diagnostics.
Assuntos
Anticorpos/metabolismo , DNA/metabolismo , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Antígenos/metabolismo , Oligonucleotídeos/metabolismo , Espectrometria de FluorescênciaRESUMO
Cellular senescence has been increasingly recognized as a hallmark of cancer, reflecting its association with aging and inflammation, its role as a response to deregulated proliferation and oncogenic stress, and its induction by cancer therapies. While therapy-induced senescence (TIS) has been linked to resistance, recurrence, metastasis, and normal tissue toxicity, TIS also has the potential to enhance therapy response and stimulate anti-tumor immunity. In this review, we examine the Jekyll and Hyde nature of senescent cells (SnCs), focusing on how their persistence while expressing the senescence-associated secretory phenotype (SASP) modulates the tumor microenvironment through autocrine and paracrine mechanisms. Through the SASP, SnCs can mediate both resistance and response to cancer therapies. To fulfill the unmet potential of cancer immunotherapy, we consider how SnCs may influence tumor inflammation and serve as an antigen source to potentiate anti-tumor immune response. This new perspective suggests treatment approaches based on TIS to enhance immune checkpoint blockade. Finally, we describe strategies for mitigating the detrimental effects of senescence, such as modulating the SASP or targeting SnC persistence, which may enhance the overall benefits of cancer treatment.
Assuntos
Senescência Celular , Resistencia a Medicamentos Antineoplásicos , Neoplasias , Humanos , Neoplasias/patologia , Neoplasias/imunologia , Microambiente Tumoral/imunologia , Fenótipo Secretor Associado à Senescência , Animais , Imunoterapia/métodos , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologiaRESUMO
Autophagy is known to suppress tumor initiation by removing genotoxic stresses in normal cells. Conversely, autophagy is also known to support tumor progression by alleviating metabolic stresses in neoplastic cells. Centered on this pro-tumor role of autophagy, there have been many clinical trials to treat cancers through systemic blocking of autophagy. Such systemic inhibition affects both tumor cells and non-tumor cells, and the consequence of blocked autophagy in non-tumor cells in the context of tumor microenvironment is relatively understudied. Here, we examined the effect of autophagy-deficient myeloid cells on the progression of autophagy-competent tumors. We found that blocking autophagy only in myeloid cells modulated tumor progression markedly but such effects were context dependent. In a tumor implantation model, the growth of implanted tumor cells was substantially reduced in mice with autophagy-deficient myeloid cells; T cells infiltrated deeper into the tumors and were responsible for the reduced growth of the implanted tumor cells. In an oncogene-driven tumor induction model, however, tumors grew faster and metastasized more in mice with autophagy-deficient myeloid cells. These data demonstrate that the autophagy status of myeloid cells plays a critical role in tumor progression, promoting or suppressing tumor growth depending on the context of tumor-myeloid cell interactions. This study indicates that systemic use of autophagy inhibitors in cancer therapy may have differential effects on rates of tumor progression in patients due to effects on myeloid cells and that this warrants more targeted use of selective autophagy inhibitors in a cancer therapy in a clinical setting.
RESUMO
CUX1 is a homeodomain-containing transcription factor that is essential for the development and differentiation of multiple tissues. CUX1 is recurrently mutated or deleted in cancer, particularly in myeloid malignancies. However, the mechanism by which CUX1 regulates gene expression and differentiation remains poorly understood, creating a barrier to understanding the tumor-suppressive functions of CUX1. Here, we demonstrate that CUX1 directs the BAF chromatin remodeling complex to DNA to increase chromatin accessibility in hematopoietic cells. CUX1 preferentially regulates lineage-specific enhancers, and CUX1 target genes are predictive of cell fate in vivo. These data indicate that CUX1 regulates hematopoietic lineage commitment and homeostasis via pioneer factor activity, and CUX1 deficiency disrupts these processes in stem and progenitor cells, facilitating transformation.
Assuntos
Cromatina , Células-Tronco Hematopoéticas , Proteínas de Homeodomínio , Proteínas Repressoras , Humanos , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Cromatina/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Animais , Camundongos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Linhagem da Célula , Montagem e Desmontagem da Cromatina , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genéticaRESUMO
Radiotherapy offers an effective treatment for advanced cancer but local and distant failures remain a significant challenge. Here, we treated melanoma and pancreatic carcinoma in syngeneic mice with ionizing radiation (IR) combined with the poly(ADP-ribose) polymerase inhibitor (PARPi) veliparib to inhibit DNA repair and promote accelerated senescence. Based on prior work implicating cytotoxic T lymphocytes (CTLs) as key mediators of radiation effects, we discovered that senescent tumor cells induced by radiation and veliparib express immunostimulatory cytokines to activate CTLs that mediate an effective antitumor response. When these senescent tumor cells were injected into tumor-bearing mice, an antitumor CTL response was induced which potentiated the effects of radiation, resulting in elimination of established tumors. Applied to human cancers, radiation-inducible immunotherapy may enhance radiotherapy responses to prevent local recurrence and distant metastasis.
Assuntos
Benzimidazóis/farmacologia , Vacinas Anticâncer/uso terapêutico , Imunoterapia/métodos , Melanoma Experimental/terapia , Neoplasias Pancreáticas/terapia , Radiossensibilizantes/farmacologia , Animais , Vacinas Anticâncer/imunologia , Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Terapia Combinada , Citocinas/biossíntese , Citocinas/imunologia , Citotoxicidade Imunológica , Feminino , Humanos , Ativação Linfocitária , Melanoma Experimental/imunologia , Melanoma Experimental/mortalidade , Camundongos , Transplante de Neoplasias , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/mortalidade , Inibidores de Poli(ADP-Ribose) Polimerases , Taxa de Sobrevida , Linfócitos T Citotóxicos/imunologia , Células Tumorais CultivadasRESUMO
Therapy-induced senescence (TIS) may contribute to therapy resistance; however, evidence also suggests that senescent cells (SnCs) may promote anti-tumor immunity. Here, we present a protocol for examining the capability of TIS to stimulate type 1 conventional CD103+ dendritic cells (DCs). We describe steps for isolating and differentiating CD103+ DCs from murine bone marrow, inducing senescence in murine colon carcinoma cell line CT26, and coculturing DCs with SnCs. We then detail the flow cytometric analysis of DC maturation and activation. For complete details on the use and execution of this protocol, please refer to Liu et al. (2022)1 and Liu et al. (2023).2.