RESUMEN
The MYC oncoprotein globally affects the function of RNA polymerase II (RNAPII). The ability of MYC to promote transcription elongation depends on its ubiquitylation. Here, we show that MYC and PAF1c (polymerase II-associated factor 1 complex) interact directly and mutually enhance each other's association with active promoters. PAF1c is rapidly transferred from MYC onto RNAPII. This transfer is driven by the HUWE1 ubiquitin ligase and is required for MYC-dependent transcription elongation. MYC and HUWE1 promote histone H2B ubiquitylation, which alters chromatin structure both for transcription elongation and double-strand break repair. Consistently, MYC suppresses double-strand break accumulation in active genes in a strictly PAF1c-dependent manner. Depletion of PAF1c causes transcription-dependent accumulation of double-strand breaks, despite widespread repair-associated DNA synthesis. Our data show that the transfer of PAF1c from MYC onto RNAPII efficiently couples transcription elongation with double-strand break repair to maintain the genomic integrity of MYC-driven tumor cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Elongación de la Transcripción Genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Línea Celular Tumoral , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-myc/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Obesity-induced diabetes affects >400 million people worldwide. Uncontrolled lipolysis (free fatty acid release from adipocytes) can contribute to diabetes and obesity. To identify future therapeutic avenues targeting this pathway, we performed a high-throughput screen and identified the extracellular-regulated kinase 3 (ERK3) as a hit. We demonstrated that ß-adrenergic stimulation stabilizes ERK3, leading to the formation of a complex with the cofactor MAP kinase-activated protein kinase 5 (MK5), thereby driving lipolysis. Mechanistically, we identified a downstream target of the ERK3/MK5 pathway, the transcription factor FOXO1, which promotes the expression of the major lipolytic enzyme ATGL. Finally, we provide evidence that targeted deletion of ERK3 in mouse adipocytes inhibits lipolysis, but elevates energy dissipation, promoting lean phenotype and ameliorating diabetes. Thus, ERK3/MK5 represents a previously unrecognized signaling axis in adipose tissue and an attractive target for future therapies aiming to combat obesity-induced diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Metabolismo Energético/genética , Lipólisis/genética , Proteína Quinasa 6 Activada por Mitógenos/genética , Proteína Quinasa 6 Activada por Mitógenos/metabolismo , Obesidad/complicaciones , Células 3T3 , Tejido Adiposo/enzimología , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Proteína Forkhead Box O1/metabolismo , Eliminación de Gen , Células HEK293 , Humanos , Hipoglucemiantes/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipasa/genética , Lipasa/metabolismo , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/genéticaRESUMEN
RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Humanos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Etopósido/farmacología , Precursores del ARN/metabolismo , Factores de Transcripción/metabolismo , ADN , Proteínas de Unión al ARN/metabolismoRESUMEN
MYC is an oncogenic transcription factor that binds globally to active promoters and promotes transcriptional elongation by RNA polymerase II (RNAPII)1,2. Deregulated expression of the paralogous protein MYCN drives the development of neuronal and neuroendocrine tumours and is often associated with a particularly poor prognosis3. Here we show that, similar to MYC, activation of MYCN in human neuroblastoma cells induces escape of RNAPII from promoters. If the release of RNAPII from transcriptional pause sites (pause release) fails, MYCN recruits BRCA1 to promoter-proximal regions. Recruitment of BRCA1 prevents MYCN-dependent accumulation of stalled RNAPII and enhances transcriptional activation by MYCN. Mechanistically, BRCA1 stabilizes mRNA decapping complexes and enables MYCN to suppress R-loop formation in promoter-proximal regions. Recruitment of BRCA1 requires the ubiquitin-specific protease USP11, which binds specifically to MYCN when MYCN is dephosphorylated at Thr58. USP11, BRCA1 and MYCN stabilize each other on chromatin, preventing proteasomal turnover of MYCN. Because BRCA1 is highly expressed in neuronal progenitor cells during early development4 and MYC is less efficient than MYCN in recruiting BRCA1, our findings indicate that a cell-lineage-specific stress response enables MYCN-driven tumours to cope with deregulated RNAPII function.
Asunto(s)
Proteína BRCA1/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Regulación de la Expresión Génica , Humanos , Neuroblastoma/genética , Neuroblastoma/patología , Estabilidad Proteica , Tioléster Hidrolasas/metabolismoRESUMEN
YAP, the key protein effector of the Hippo pathway, is a transcriptional co-activator that controls the expression of cell cycle genes, promotes cell growth and proliferation and regulates organ size. YAP modulates gene transcription by binding to distal enhancers, but the mechanisms of gene regulation by YAP-bound enhancers remain poorly understood. Here we show that constitutive active YAP5SA leads to widespread changes in chromatin accessibility in untransformed MCF10A cells. Newly accessible regions include YAP-bound enhancers that mediate activation of cycle genes regulated by the Myb-MuvB (MMB) complex. By CRISPR-interference we identify a role for YAP-bound enhancers in phosphorylation of Pol II at Ser5 at MMB-regulated promoters, extending previously published studies that suggested YAP primarily regulates the pause-release step and transcriptional elongation. YAP5SA also leads to less accessible 'closed' chromatin regions, which are not directly YAP-bound but which contain binding motifs for the p53 family of transcription factors. Diminished accessibility at these regions is, at least in part, a consequence of reduced expression and chromatin-binding of the p53 family member ΔNp63 resulting in downregulation of ΔNp63-target genes and promoting YAP-mediated cell migration. In summary, our studies uncover changes in chromatin accessibility and activity that contribute to the oncogenic activities of YAP.
Asunto(s)
Proteínas de Ciclo Celular , Movimiento Celular , Cromatina , Genes cdc , Factores de Transcripción , Transcripción Genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/genética , Cromatina/genética , Cromatina/metabolismo , Genes cdc/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Señalizadoras YAP/química , Proteínas Señalizadoras YAP/metabolismo , Humanos , Línea Celular , Elementos de Facilitación Genéticos , ADN Polimerasa II/química , ADN Polimerasa II/metabolismo , FosforilaciónRESUMEN
The Hippo signalling pathway and its central effector YAP regulate proliferation of cardiomyocytes and growth of the heart. Using genetic models in mice we show that the increased proliferation of embryonal and postnatal cardiomyocytes due to loss of the Hippo-signaling component SAV1 depends on the Myb-MuvB (MMB) complex. Similarly, proliferation of postnatal cardiomyocytes induced by constitutive active YAP requires MMB. Genome studies revealed that YAP and MMB regulate an overlapping set of cell cycle genes in cardiomyocytes. Protein-protein interaction studies in cell lines and with recombinant proteins showed that YAP binds directly to B-MYB, a subunit of MMB, in a manner dependent on the YAP WW domains and a PPXY motif in B-MYB. Disruption of the interaction by overexpression of the YAP binding domain of B-MYB strongly inhibits the proliferation of cardiomyocytes. Our results point to MMB as a critical downstream effector of YAP in the control of cardiomyocyte proliferation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/genética , Miocitos Cardíacos/citología , Transactivadores/química , Transactivadores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Animales Recién Nacidos , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proliferación Celular , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Miocitos Cardíacos/química , Regiones Promotoras Genéticas , Ratas , Proteínas Señalizadoras YAPRESUMEN
Nutrient overload in combination with decreased energy dissipation promotes obesity and diabetes. Obesity results in a hormonal imbalance, which among others activates G protein-coupled receptors utilizing diacylglycerol (DAG) as secondary messenger. Protein kinase D1 (PKD1) is a DAG effector, which integrates multiple nutritional and hormonal inputs, but its physiological role in adipocytes is unknown. Here, we show that PKD1 promotes lipogenesis and suppresses mitochondrial fragmentation, biogenesis, respiration, and energy dissipation in an AMP-activated protein kinase (AMPK)-dependent manner. Moreover, mice lacking PKD1 in adipocytes are resistant to diet-induced obesity due to elevated energy expenditure. Beiging of adipocytes promotes energy expenditure and counteracts obesity. Consistently, deletion of PKD1 promotes expression of the ß3-adrenergic receptor (ADRB3) in a CCAAT/enhancer binding protein (C/EBP)-α- and δ-dependent manner, which leads to the elevated expression of beige markers in adipocytes and subcutaneous adipose tissue. Finally, deletion of PKD1 in adipocytes improves insulin sensitivity and ameliorates liver steatosis. Thus, depletion of PKD1 in adipocytes increases energy dissipation by several complementary mechanisms and might represent an attractive strategy to treat obesity and its related complications.
Asunto(s)
Adipocitos/metabolismo , Adiposidad , Metabolismo Energético , Hígado Graso/metabolismo , Obesidad/metabolismo , Proteína Quinasa C/metabolismo , Grasa Subcutánea/metabolismo , Células 3T3-L1 , Adipocitos/patología , Animales , Proteína delta de Unión al Potenciador CCAAT/genética , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Hígado Graso/genética , Hígado Graso/patología , Femenino , Humanos , Masculino , Ratones , Ratones Mutantes , Obesidad/genética , Obesidad/patología , Proteína Quinasa C/genética , Receptores Adrenérgicos beta 3/genética , Receptores Adrenérgicos beta 3/metabolismo , Sistemas de Mensajero Secundario/genética , Grasa Subcutánea/fisiologíaRESUMEN
Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on glutamine, inducing "glutamine addiction". Surprisingly, colon cancer cells that express high levels of MYC due to WNT pathway mutations are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses translation of endogenous MYC via the 3'-UTR of the MYC mRNA, enabling escape from apoptosis. This regulation is mediated by glutamine-dependent changes in adenosine-nucleotide levels. Glutamine deprivation causes a global reduction in promoter association of RNA polymerase II (RNAPII) and slows transcriptional elongation. While activation of MYC restores binding of MYC and RNAPII function on most promoters, restoration of elongation is imperfect and activation of MYC in the absence of glutamine causes stalling of RNAPII on multiple genes, correlating with R-loop formation. Stalling of RNAPII and R-loop formation can cause DNA damage, arguing that the MYC 3'-UTR is critical for maintaining genome stability when ribonucleotide levels are low.
Asunto(s)
Regiones no Traducidas 3' , Regulación Enzimológica de la Expresión Génica , Glutamina/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , Ribonucleótidos/metabolismo , Línea Celular , Humanos , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Epigenetic changes have likely contributed to the large size and enhanced cognitive abilities of the human brain which evolved within the last 2 million years after the human-chimpanzee split. Using reduced representation bisulfite sequencing, we have compared the methylomes of neuronal and non-neuronal cells from 3 human and 3 chimpanzee cortices. Differentially methylated regions (DMRs) with genome-wide significance were enriched in specific genomic regions. Intraspecific methylation differences between neuronal and non-neuronal cells were approximately 3 times more abundant than interspecific methylation differences between human and chimpanzee cell types. The vast majority (>90%) of human intraspecific DMRs (including DMRs in retrotransposons) were hypomethylated in neurons, compared with glia. Intraspecific DMRs were enriched in genes associated with different neuropsychiatric disorders. Interspecific DMRs were enriched in genes showing human-specific brain histone modifications. Human-chimpanzee methylation differences were much more frequent in non-neuronal cells (n. DMRs = 666) than in neurons (n. DMRs = 96). More than 95% of interspecific DMRs in glia were hypermethylated in humans. Although without an outgroup we cannot assign whether a change in methylation occurred in the human or chimpanzee lineage, our results are consistent with a wave of methylation affecting several hundred non-neuronal genes during human brain evolution.
Asunto(s)
Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Metilación de ADN/genética , Neuronas/metabolismo , Pan troglodytes/fisiología , Anciano , Animales , Evolución Molecular , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Trastornos Mentales/genética , Trastornos Mentales/patología , Metaboloma , Neuroglía/metabolismo , Especificidad de la EspecieRESUMEN
Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non-small cell tumors and secondary transitions from non-small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of "small cell-ness" based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT-signaling in the context of mutual bi-allelic RB1 and TP53 lesions. Additionally, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH-ASCL1-RB-p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon-based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well.
Asunto(s)
Carcinoma Neuroendocrino/genética , Carcinoma Neuroendocrino/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Transducción de Señal , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Análisis Mutacional de ADN , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Receptores Notch/genética , Receptores Notch/metabolismo , Proteína de Retinoblastoma/genética , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Using a functional genomics approach, four candidate genes (PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda. These genes encode CAZy family GT34 glycosyltransferases that are involved in the synthesis of cell-wall xyloglucans and heteromannans. The full-length coding sequences of three orthologs (PrGT34A, B and C) were isolated from a xylem-specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1â6)-α-xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a galactosyltransferase from fenugreek (Trigonella foenum-graecum) that is involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell-culture system. The heterologous proteins were purified, and in vitro assays showed that, when incubated with UDP-xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and, when incubated with UDP-galactose and the same cello-oligosaccharides, PrGT34B showed some galactosyltransferase activity. The ratio of xylosyltransferase to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products using galactosidases showed the linkages formed were α-linkages. Analysis of the products of PrGT34B by MALDI-TOF MS showed that up to three xylosyl residues were transferred from UDP-xylose to cellohexaose. The heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity.
Asunto(s)
Glicosiltransferasas/genética , Pinus taeda/genética , Pinus/genética , Proteínas de Plantas/genética , Pared Celular/metabolismo , Genómica , Glucanos/biosíntesis , Glicosiltransferasas/química , Mananos/biosíntesis , Espectrometría de Masas , Datos de Secuencia Molecular , Filogenia , Pinus taeda/enzimología , Proteínas de Plantas/química , Xilanos/biosíntesisRESUMEN
YAP activation in cancer is linked to poor outcomes, making it an attractive therapeutic target. Previous research focused on blocking the interaction of YAP with TEAD transcription factors. Here, we took a different approach by disrupting YAP's binding to the transcription factor B-MYB using MY-COMP, a fragment of B-MYB containing the YAP binding domain fused to a nuclear localization signal. MY-COMP induced cell cycle defects, nuclear abnormalities, and polyploidization. In an AKT and YAP-driven liver cancer model, MY-COMP significantly reduced liver tumorigenesis, highlighting the importance of the YAP-B-MYB interaction in tumor development. MY-COMP also perturbed the cell cycle progression of YAP-dependent uveal melanoma cells but not of YAP-independent cutaneous melanoma cell lines. It counteracted YAP-dependent expression of MMB-regulated cell cycle genes, explaining the observed effects. We also identified NIMA-related kinase (NEK2) as a downstream target of YAP and B-MYB, promoting YAP-driven transformation by facilitating centrosome clustering and inhibiting multipolar mitosis.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
In pancreatic ductal adenocarcinoma (PDAC), endogenous MYC is required for S-phase progression and escape from immune surveillance. Here we show that MYC in PDAC cells is needed for the recruitment of the PAF1c transcription elongation complex to RNA polymerase and that depletion of CTR9, a PAF1c subunit, enables long-term survival of PDAC-bearing mice. PAF1c is largely dispensable for normal proliferation and regulation of MYC target genes. Instead, PAF1c limits DNA damage associated with S-phase progression by being essential for the expression of long genes involved in replication and DNA repair. Surprisingly, the survival benefit conferred by CTR9 depletion is not due to DNA damage, but to T-cell activation and restoration of immune surveillance. This is because CTR9 depletion releases RNA polymerase and elongation factors from the body of long genes and promotes the transcription of short genes, including MHC class I genes. The data argue that functionally distinct gene sets compete for elongation factors and directly link MYC-driven S-phase progression to tumor immune evasion.
Asunto(s)
Fenómenos Bioquímicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas c-myc , Animales , Ratones , Carcinoma Ductal Pancreático/patología , Proliferación Celular , ARN Polimerasas Dirigidas por ADN/metabolismo , Evasión Inmune , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
MYC family oncoproteins regulate the expression of a large number of genes and broadly stimulate elongation by RNA polymerase II (RNAPII). While the factors that control the chromatin association of MYC proteins are well understood, much less is known about how interacting proteins mediate MYC's effects on transcription. Here, we show that TFIIIC, an architectural protein complex that controls the three-dimensional chromatin organisation at its target sites, binds directly to the amino-terminal transcriptional regulatory domain of MYCN. Surprisingly, TFIIIC has no discernible role in MYCN-dependent gene expression and transcription elongation. Instead, MYCN and TFIIIC preferentially bind to promoters with paused RNAPII and globally limit the accumulation of non-phosphorylated RNAPII at promoters. Consistent with its ubiquitous role in transcription, MYCN broadly participates in hubs of active promoters. Depletion of TFIIIC further increases MYCN localisation to these hubs. This increase correlates with a failure of the nuclear exosome and BRCA1, both of which are involved in nascent RNA degradation, to localise to active promoters. Our data suggest that MYCN and TFIIIC exert an censoring function in early transcription that limits promoter accumulation of inactive RNAPII and facilitates promoter-proximal degradation of nascent RNA.
Asunto(s)
Cromatina , Proteína Proto-Oncogénica N-Myc , Regiones Promotoras Genéticas , ARN Polimerasa II , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Proteína Proto-Oncogénica N-Myc/genética , Humanos , Cromatina/metabolismo , Unión Proteica , Factores de Transcripción TFII/metabolismo , Factores de Transcripción TFII/genética , Transcripción Genética , Línea Celular TumoralRESUMEN
Protein regulator of cytokinesis 1 (PRC1) is a microtubule-binding protein with essential roles in mitosis and cytokinesis. PRC1 is frequently overexpressed in cancer cells where it could contribute to chromosomal instability. Due to its nuclear localization in interphase, it has been speculated that PRC1 has additional functions that are involved in its pro-tumorigenic functions. In this study we investigated the potential nuclear functions of PRC1 in a lung cancer cell line. Genome wide expression profiling by RNA sequencing revealed that the expression of PRC1 results in activation of the p53 pathway and inhibition of the pro-proliferative E2F-dependent gene expression. A mutant of PRC1 that is unable to enter into the nucleus regulated the same gene sets as wildtype PRC1, suggesting that PRC1 has no nuclear-exclusive functions in A549 cells. Instead, induction of p53 by PRC1 correlates with multinucleation and depends on the localization of PRC1 to the midbody, suggesting that the induction of p53 is a consequence of overexpressed PRC1 to interfere with the normal function of PRC1 during cytokinesis. Activation of p53 by PRC1 results in cellular senescence but not in apoptosis. In conclusion, while PRC1 is frequently overexpressed in many cancers, the p53 pathways may initially protect cancer cells from the negative effects of PRC1 overexpression on cytokinesis. Because depletion of PRC1 also results in p53-pathway activation and senescence, levels of PRC1 need to be tightly regulated to allow unperturbed proliferation. Targeting the expression or function of PRC1 could create a therapeutic vulnerability for the treatment of cancer.
Asunto(s)
Proteínas de Ciclo Celular , Citocinesis , Proteína p53 Supresora de Tumor , Humanos , Células A549 , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Citocinesis/genética , Expresión Génica , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Ribosomal biogenesis and protein synthesis are deregulated in most cancers, suggesting that interfering with translation machinery may hold significant therapeutic potential. Here, we show that loss of the tumor suppressor adenomatous polyposis coli (APC), which constitutes the initiating event in the adenoma carcinoma sequence for colorectal cancer (CRC), induces the expression of RNA polymerase I (RNAPOL1) transcription machinery, and subsequently upregulates ribosomal DNA (rDNA) transcription. Targeting RNAPOL1 with a specific inhibitor, CX5461, disrupts nucleolar integrity, and induces a disbalance of ribosomal proteins. Surprisingly, CX5461-induced growth arrest is irreversible and exhibits features of senescence and terminal differentiation. Mechanistically, CX5461 promotes differentiation in an MYC-interacting zinc-finger protein 1 (MIZ1)- and retinoblastoma protein (Rb)-dependent manner. In addition, the inhibition of RNAPOL1 renders CRC cells vulnerable towards senolytic agents. We validated this therapeutic effect of CX5461 in murine- and patient-derived organoids, and in a xenograft mouse model. These results show that targeting ribosomal biogenesis together with targeting the consecutive, senescent phenotype using approved drugs is a new therapeutic approach, which can rapidly be transferred from bench to bedside.
Asunto(s)
Neoplasias Colorrectales , ARN Polimerasa I , Animales , Nucléolo Celular/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Humanos , Ratones , ARN Polimerasa I/genética , Proteínas Ribosómicas/metabolismo , SenoterapéuticosRESUMEN
Deregulated expression of the MYC oncoprotein enables tumor cells to evade immune surveillance, but the mechanisms underlying this surveillance are poorly understood. We show here that endogenous MYC protects pancreatic ductal adenocarcinoma (PDAC) driven by KRASG12D and TP53R172H from eradication by the immune system. Deletion of TANK-binding kinase 1 (TBK1) bypassed the requirement for high MYC expression. TBK1 was active due to the accumulation of double-stranded RNA (dsRNA), which was derived from inverted repetitive elements localized in introns of nuclear genes. Nuclear-derived dsRNA is packaged into extracellular vesicles and subsequently recognized by toll-like receptor 3 (TLR3) to activate TBK1 and downstream MHC class I expression in an autocrine or paracrine manner before being degraded in lysosomes. MYC suppressed loading of dsRNA onto TLR3 and its subsequent degradation via association with MIZ1. Collectively, these findings suggest that MYC and MIZ1 suppress a surveillance pathway that signals perturbances in mRNA processing to the immune system, which facilitates immune evasion in PDAC. SIGNIFICANCE: This study identifies a TBK1-dependent pathway that links dsRNA metabolism to antitumor immunity and shows that suppression of TBK1 is a critical function of MYC in pancreatic ductal adenocarcinoma.
Asunto(s)
Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Evasión Inmune , Factores de Transcripción de Tipo Kruppel/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Bicatenario , Adenocarcinoma/inmunología , Animales , Transporte Biológico , Carcinoma Ductal Pancreático/inmunología , Núcleo Celular/metabolismo , Eliminación de Gen , Células HEK293 , Humanos , Sistema Inmunológico , Intrones , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Desnudos , Neoplasias Pancreáticas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Análisis de Secuencia de ADN , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Amplification of MYCN is the driving oncogene in a subset of high-risk neuroblastoma. The MYCN protein and the Aurora-A kinase form a complex during S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription-replication conflicts and activates the Ataxia telangiectasia and Rad3 related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription-replication conflicts is an effective therapy for MYCN-driven neuroblastoma (141 words).
Asunto(s)
Aurora Quinasa A , Neuroblastoma , Animales , Apoptosis/genética , Aurora Quinasa A/genética , Línea Celular Tumoral , Ratones , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/tratamiento farmacológicoRESUMEN
The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca2+ increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca2+ concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca2+ rise led to an increase in mitochondrial Ca2+ concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca2+ homeostasis and induces cytoplasmic Ca2+ overload, which results in both apoptotic and necrotic cell death in parallel or succession.IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca2+ overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca2+ homeostasis.
Asunto(s)
Calcio/metabolismo , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/fisiopatología , Staphylococcus aureus/fisiología , Apoptosis , Muerte Celular , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Células HeLa , Homeostasis , Interacciones Huésped-Patógeno , Humanos , Viabilidad Microbiana , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
The transcription factor ∆Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ∆Np63 and maintains elevated ∆NP63 levels in SCC by counteracting its proteasome-mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ∆Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.