RESUMEN
Anti-spike neutralizing antibodies (S NAbs) have been developed for prevention and treatment against COVID-19. The nanoscopic characterization of the dynamic interaction between spike proteins and S NAbs remains difficult. By using high-speed atomic force microscopy (HS-AFM), we elucidate the molecular property of an S NAb and its interaction with spike proteins. The S NAb appeared as monomers with a Y conformation at low density and formed hexameric oligomers at high density. The dynamic S NAb-spike protein interaction at RBD induces neither RBD opening nor S1 subunit shedding. Furthermore, the interaction was stable at endosomal pH. These findings indicated that the S NAb could have a negligible risk of antibody-dependent enhancement. Dynamic movement of spike proteins on small extracellular vesicles (S sEV) resembled that on SARS-CoV-2. The sensitivity of variant S sEVs to S NAb could be evaluated using HS-AFM. Altogether, we demonstrate a nanoscopic assessment platform for evaluating the binding property of S NAbs.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Antivirales , Anticuerpos NeutralizantesRESUMEN
The p53 family remains a captivating focus of an extensive number of current studies. Accumulating evidence indicates that p53 abnormalities rank among the most prevalent in cancer. Given the numerous existing studies, which mostly focus on the mutations, expression profiles, and functional perturbations exhibited by members of the p53 family across diverse malignancies, this review will concentrate more on less explored facets regarding p53 activation and stabilization by the nuclear pore complex (NPC) in cancer, drawing on several studies. p53 integrates a broad spectrum of signals and is subject to diverse regulatory mechanisms to enact the necessary cellular response. It is widely acknowledged that each stage of p53 regulation, from synthesis to degradation, significantly influences its functionality in executing specific tasks. Over recent decades, a large body of data has established that mechanisms of regulation, closely linked with protein activation and stabilization, involve intricate interactions with various cellular components. These often transcend canonical regulatory pathways. This new knowledge has expanded from the regulation of genes themselves to epigenomics and proteomics, whereby interaction partners increase in number and complexity compared with earlier paradigms. Specifically, studies have recently shown the involvement of the NPC protein in such complex interactions, underscoring the further complexity of p53 regulation. Furthermore, we also discuss therapeutic strategies based on recent developments in this field in combination with established targeted therapies.
Asunto(s)
Neoplasias , Poro Nuclear , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Poro Nuclear/metabolismo , Poro Nuclear/genética , Animales , Regulación Neoplásica de la Expresión GénicaRESUMEN
The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow; however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution. Here, using a stromal cell line from the zebrafish kidney, zebrafish kidney stromal (ZKS), we uncover that an inhibitor of canonical Wnt signaling, IWR-1-endo, is a potent regulator of HSCs. Coculture assays revealed that ZKS cells were in part supportive of maintenance, but not expansion, of gata2a:GFP+runx1:mCherry+ (gata2a+runx1+) HSCs. Transcriptome analysis revealed that, compared with candidate niche cells in the kidney, ZKS cells weakly expressed HSC maintenance factor genes, thpo and cxcl12, but highly expressed canonical Wnt ligand genes, wnt1, 7bb, and 9a. Thpo supplementation in ZKS culture slightly increased, but inhibition of canonical Wnt signaling by IWR-1-endo treatment largely increased the number of gata2a+runx1+ cells (>2-fold). Moreover, we found that gata2a+runx1+ cells can be maintained by supplementing both IWR-1-endo and Thpo without stromal cells. Collectively, our data provide evidence that IWR-1-endo can be used as a novel supporting factor for HSCs.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal , Pez Cebra , Animales , Proliferación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Ligandos , Ratones , Vía de Señalización Wnt/genética , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
To reconstruct systematically hyperactive transcription factor (TF)-dependent transcription networks in squamous cell carcinomas (SCCs), a computational method (ELMER) was applied to 1293 pan-SCC patient samples, and 44 hyperactive SCC TFs were identified. As a top candidate, DLX5 exhibits a notable bifurcate re-configuration of its bivalent promoter in cancer. Specifically, DLX5 maintains a bivalent state in normal tissues; its promoter is hypermethylation, leading to DLX5 transcriptional silencing in esophageal adenocarcinoma (EAC). In stark contrast, DLX5 promoter gains active histone marks and becomes transcriptionally activated in ESCC, which is directly mediated by SOX2. Functionally, silencing of DLX5 substantially inhibits SCC viability both in vitro and in vivo. Mechanistically, DLX5 cooperates with TP63 in regulating â¼2000 enhancers and promoters, which converge on activating cancer-promoting pathways. Together, our data establish a novel and strong SCC-promoting factor and elucidate a new epigenomic mechanism - bifurcate chromatin re-configuration - during cancer development.
Asunto(s)
Adenocarcinoma/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Adenocarcinoma/patología , Animales , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Metilación de ADN/genética , Neoplasias Esofágicas/patología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Xenoinjertos , Humanos , Masculino , Ratones , Persona de Mediana Edad , Regiones Promotoras Genéticas/genéticaRESUMEN
Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.
Asunto(s)
COVID-19/metabolismo , COVID-19/virología , Interacciones Microbiota-Huesped/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/metabolismo , Transporte Activo de Núcleo Celular , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Modelos Biológicos , Membrana Nuclear/metabolismo , Membrana Nuclear/virología , Proteínas de Complejo Poro Nuclear/antagonistas & inhibidores , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas no Estructurales Virales/genéticaRESUMEN
The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11-7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Células Epiteliales/metabolismo , Subunidad p40 de la Interleucina-12/metabolismo , Subunidad p19 de la Interleucina-23/metabolismo , Mucosa Intestinal/metabolismo , Sistema de Señalización de MAP Quinasas , Sustitución de Aminoácidos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Células Epiteliales/patología , Células HCT116 , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Subunidad p40 de la Interleucina-12/genética , Subunidad p19 de la Interleucina-23/genética , Mucosa Intestinal/patología , Mutación Missense , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
BACKGROUND & AIMS: We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins. METHODS: We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase-accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extraterminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured. RESULTS: We identified super-enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone. CONCLUSIONS: In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.
Asunto(s)
Epigénesis Genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción/genética , Transcripción Genética , Proteínas Supresoras de Tumor/genética , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Ensamble y Desensamble de Cromatina , Epigénesis Genética/efectos de los fármacos , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones Endogámicos NOD , Ratones SCID , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transcriptoma , Carga Tumoral , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The novel human betacoronavirus SARS-CoV-2 has caused an unprecedented pandemic in the 21st century. Several studies have revealed interactions between SARS-CoV-2 viral proteins and host nucleoporins, yet their functions are largely unknown. Here, we demonstrate that the open-reading frame 6 (ORF6) of SARS-CoV-2 can directly manipulate localization and functions of nucleoporins. We found that ORF6 protein disrupted nuclear rim staining of nucleoporins RAE1 and NUP98. Consequently, this disruption caused aberrant nucleocytoplasmic trafficking and led to nuclear accumulation of mRNA transporters such as hnRNPA1. Ultimately, host cell nucleus size was reduced and cell growth was halted.
Asunto(s)
Tamaño del Núcleo Celular , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas Virales/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/virología , Células HEK293 , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Humanos , SARS-CoV-2RESUMEN
The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.
Asunto(s)
Roturas del ADN de Doble Cadena , Histona Acetiltransferasas/fisiología , Histona Desacetilasas/fisiología , Recombinación Homóloga , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Acetilación , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Histona Acetiltransferasas/genética , Histona Desacetilasas/genética , Humanos , Microscopía Fluorescente , Técnicas del Sistema de Dos HíbridosRESUMEN
Influenza A hemagglutinin (HA) is one of the crucial virulence factors that mediate host tropism and viral infectivity. Presently, the mechanism of the fusogenic transition of HA remains elusive. Here, we used high-speed atomic force microscopy (HS-AFM) to decipher the molecular dynamics of HA and its interaction with exosomes. Our data reveal that the native conformation of HA in the neutral buffer is ellipsoidal, and HA undergoes a conformational change in an acidic buffer. Real-time visualization of the fusogenic transition by HS-AFM suggests that the mechanism is possibly fit to the "uncaging" model, and HA intermediate appears as Y-shaped. A firm interaction between the HA and exosome in an acidic buffer indicates the insertion of a fusion peptide into the exosomal layer and subsequently destabilizes the layer, resulting in the deformation or rupture of exosomes, releasing exosomal contents. In contrast, the HA-exosome interaction is weak in a neutral buffer because the interaction is mediated by weak bonds between the HA receptor-binding site and receptors on the exosome.
Asunto(s)
Exosomas , Gripe Humana , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas , Humanos , Concentración de Iones de Hidrógeno , Simulación de Dinámica MolecularRESUMEN
p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type-specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation-inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine-glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC.
Asunto(s)
Carcinoma de Células Escamosas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Proteínas de Complejo Poro Nuclear/genética , Neoplasias Cutáneas/genética , Neoplasias del Cuello Uterino/genética , Quinasas Asociadas a rho/genética , Transporte Activo de Núcleo Celular/genética , Secuencia de Aminoácidos , Atlas como Asunto , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Diferenciación Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Biología Computacional , Citosol/metabolismo , Femenino , Células HEK293 , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Fosforilación , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Quinasas Asociadas a rho/metabolismoRESUMEN
ZBTB transcription factors orchestrate gene transcription during tissue development. However, their roles in glioblastoma (GBM) remain unexplored. Here, through a functional screening of ZBTB genes, we identify that BCL6 is required for GBM cell viability and that BCL6 overexpression is associated with worse prognosis. In a somatic transgenic mouse model, depletion of Bcl6 inhibits the progression of KrasG12V-driven high-grade glioma. Transcriptome analysis demonstrates the involvement of BCL6 in tumor protein p53 (TP53), erythroblastic leukemia viral oncogene homolog (ErbB), and MAPK signaling pathways. Indeed, BCL6 represses the expression of wild-type p53 and its target genes in GBM cells. Knockdown of BCL6 augments the activation of TP53 pathway in response to radiation. Importantly, we discover that receptor tyrosine kinase AXL is a transcriptional target of BCL6 in GBM and mediates partially the regulatory effects of BCL6 on both MEK-ERK (mitogen-activated protein/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase) and S6K-RPS6 (ribosomal protein S6 kinase-ribosomal protein S6) axes. Similar to BCL6 silencing, depletion of AXL profoundly attenuates GBM proliferation both in vitro and in vivo. Moreover, targeted inhibition of BCL6/nuclear receptor corepressor 1 (NCoR) complex by peptidomimetic inhibitor not only significantly decreases AXL expression and the activity of MEK-ERK and S6K-RPS6 cascades but also displays a potent antiproliferative effect against GBM cells. Together, these findings uncover a glioma-promoting role of BCL6 and provide the rationale of targeting BCL6 as a potential therapeutic approach.
Asunto(s)
Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Gefitinib , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones Mutantes , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-6/genética , Quinazolinas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa del Receptor AxlRESUMEN
OBJECTIVES: Oesophageal squamous cell carcinoma (OSCC) is an aggressive malignancy and the major histological subtype of oesophageal cancer. Although recent large-scale genomic analysis has improved the description of the genetic abnormalities of OSCC, few targetable genomic lesions have been identified, and no molecular therapy is available. This study aims to identify druggable candidates in this tumour. DESIGN: High-throughput small-molecule inhibitor screening was performed to identify potent anti-OSCC compounds. Whole-transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) were conducted to decipher the mechanisms of action of CDK7 inhibition in OSCC. A variety of in vitro and in vivo cellular assays were performed to determine the effects of candidate genes on OSCC malignant phenotypes. RESULTS: The unbiased high-throughput small-molecule inhibitor screening led us to discover a highly potent anti-OSCC compound, THZ1, a specific CDK7 inhibitor. RNA-Seq revealed that low-dose THZ1 treatment caused selective inhibition of a number of oncogenic transcripts. Notably, further characterisation of the genomic features of these THZ1-sensitive transcripts demonstrated that they were frequently associated with super-enhancer (SE). Moreover, SE analysis alone uncovered many OSCC lineage-specific master regulators. Finally, integrative analysis of both THZ1-sensitive and SE-associated transcripts identified a number of novel OSCC oncogenes, including PAK4, RUNX1, DNAJB1, SREBF2 and YAP1, with PAK4 being a potential druggable kinase. CONCLUSIONS: Our integrative approaches led to a catalogue of SE-associated master regulators and oncogenic transcripts, which may significantly promote both the understanding of OSCC biology and the development of more innovative therapies.
Asunto(s)
Acrilamidas/farmacología , Aminopiridinas/farmacología , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Expresión Génica/efectos de los fármacos , Fenilendiaminas/farmacología , Pirimidinas/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Esofágicas/tratamiento farmacológico , Femenino , Perfilación de la Expresión Génica , Proteínas del Choque Térmico HSP40/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Trasplante de Neoplasias , Oncogenes/genética , Fosfoproteínas/genética , Análisis de Secuencia de ARN , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Factores de Transcripción , Transcriptoma , Proteínas Señalizadoras YAP , Quinasas p21 Activadas/genética , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome-cell interactions are crucial, but they are not well understood. Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.
Asunto(s)
Exosomas/efectos de la radiación , Integrina beta1/metabolismo , Células Madre Mesenquimatosas/efectos de la radiación , Tetraspanina 28/metabolismo , Línea Celular , Dinamina II/metabolismo , Exosomas/metabolismo , Rayos gamma , Técnicas de Silenciamiento del Gen , Humanos , Cadenas alfa de Integrinas/metabolismo , Integrina beta1/análisis , Integrina beta1/genética , Sistema de Señalización de MAP Quinasas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Estrés Oxidativo , Tetraspanina 28/análisis , Tetraspanina 28/genéticaRESUMEN
The size of the nuclear pore should, in principle, prevent HIV-1 entry. However, HIV-1 capsid is able to gain nuclear pore entry. In a recent issue of Nature, Fu et al. and Dickson et al. demonstrate that the HIV-1 capsid mimics the nuclear transport protein karyopherins to access host nuclei.
Asunto(s)
Infecciones por VIH , Poro Nuclear , Humanos , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Infecciones por VIH/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismoRESUMEN
Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC3C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DSion) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC3C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC3C)_DSion(100%)/1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect.
Asunto(s)
Proliferación Celular , Criopreservación , Crioprotectores , Polímeros , Criopreservación/métodos , Polímeros/química , Polímeros/farmacología , Crioprotectores/farmacología , Crioprotectores/química , Proliferación Celular/efectos de los fármacos , Animales , Humanos , Supervivencia Celular/efectos de los fármacos , Congelación , RatonesRESUMEN
Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.
Asunto(s)
Daño del ADN , Tolerancia a Radiación , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , ADN/efectos de la radiación , ADN/química , Línea Celular Tumoral , Proteínas de Ciclo Celular/metabolismo , Elementos de Facilitación Genéticos , Genoma Humano , Proteínas Nucleares/metabolismo , Proteínas que Contienen BromodominioRESUMEN
Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.
Asunto(s)
Proteínas , Imagen Individual de Molécula , Animales , Ratones , Microscopía de Fuerza Atómica/métodos , Proteínas/química , Núcleo Celular , Encéfalo/diagnóstico por imagen , MamíferosRESUMEN
Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level.
RESUMEN
Squamous cell carcinomas (SCCs) are common and aggressive malignancies. Immune check point blockade (ICB) therapy using PD-1/PD-L1 antibodies has been approved in several types of advanced SCCs. However, low response rate and treatment resistance are common. Improving the efficacy of ICB therapy requires better understanding of the mechanism of immune evasion. Here, we identify that the SCC-master transcription factor TP63 suppresses interferon-γ (IFNγ) signaling. TP63 inhibition leads to increased CD8+ T cell infiltration and heighten tumor killing in in vivo syngeneic mouse model and ex vivo co-culture system, respectively. Moreover, expression of TP63 is negatively correlated with CD8+ T cell infiltration and activation in patients with SCC. Silencing of TP63 enhances the anti-tumor efficacy of PD-1 blockade by promoting CD8+ T cell infiltration and functionality. Mechanistically, TP63 and STAT1 mutually suppress each other to regulate the IFNγ signaling by co-occupying and co-regulating their own promoters and enhancers. Together, our findings elucidate a tumor-extrinsic function of TP63 in promoting immune evasion of SCC cells. Over-expression of TP63 may serve as a biomarker predicting the outcome of SCC patients treated with ICB therapy, and targeting TP63/STAT/IFNγ axis may enhance the efficacy of ICB therapy for this deadly cancer.