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The protooncoprotein N-Myc, which is overexpressed in approximately 25% of neuroblastomas as the consequence of MYCN gene amplification, has long been postulated to regulate DNA double-strand break (DSB) repair in neuroblastoma cells, but experimental evidence of this function is presently scant. Here, we show that N-Myc transcriptionally activates the long noncoding RNA MILIP to promote nonhomologous end-joining (NHEJ) DNA repair through facilitating Ku70-Ku80 heterodimerization in neuroblastoma cells. High MILIP expression was associated with poor outcome and appeared as an independent prognostic factor in neuroblastoma patients. Knockdown of MILIP reduced neuroblastoma cell viability through the induction of apoptosis and inhibition of proliferation, retarded neuroblastoma xenograft growth, and sensitized neuroblastoma cells to DNA-damaging therapeutics. The effect of MILIP knockdown was associated with the accumulation of DNA DSBs in neuroblastoma cells largely due to decreased activity of the NHEJ DNA repair pathway. Mechanistical investigations revealed that binding of MILIP to Ku70 and Ku80 increased their heterodimerization, and this was required for MILIP-mediated promotion of NHEJ DNA repair. Disrupting the interaction between MILIP and Ku70 or Ku80 increased DNA DSBs and reduced cell viability with therapeutic potential revealed where targeting MILIP using Gapmers cooperated with the DNA-damaging drug cisplatin to inhibit neuroblastoma growth in vivo. Collectively, our findings identify MILIP as an N-Myc downstream effector critical for activation of the NHEJ DNA repair pathway in neuroblastoma cells, with practical implications of MILIP targeting, alone and in combination with DNA-damaging therapeutics, for neuroblastoma treatment.
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Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Neuroblastoma , ARN Largo no Codificante , Humanos , ADN/genética , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , ARN Largo no Codificante/genéticaRESUMEN
Climate has critical roles in the origin, pathogenesis and transmission of infectious zoonotic diseases. However, large-scale epidemiologic trend and specific response pattern of zoonotic diseases under future climate scenarios are poorly understood. Here, we projected the distribution shifts of transmission risks of main zoonotic diseases under climate change in China. First, we shaped the global habitat distribution of main host animals for three representative zoonotic diseases (2, 6, and 12 hosts for dengue, hemorrhagic fever, and plague, respectively) with 253,049 occurrence records using maximum entropy (Maxent) modeling. Meanwhile, we predicted the risk distribution of the above three diseases with 197,098 disease incidence records from 2004 to 2017 in China using an integrated Maxent modeling approach. The comparative analysis showed that there exist highly coincident niche distributions between habitat distribution of hosts and risk distribution of diseases, indicating that the integrated Maxent modeling is accurate and effective for predicting the potential risk of zoonotic diseases. On this basis, we further projected the current and future transmission risks of 11 main zoonotic diseases under four representative concentration pathways (RCPs) (RCP2.6, RCP4.5, RCP6.0, and RCP8.5) in 2050 and 2070 in China using the above integrated Maxent modeling with 1,001,416 disease incidence records. We found that Central China, Southeast China, and South China are concentrated regions with high transmission risks for main zoonotic diseases. More specifically, zoonotic diseases had diverse shift patterns of transmission risks including increase, decrease, and unstable. Further correlation analysis indicated that these patterns of shifts were highly correlated with global warming and precipitation increase. Our results revealed how specific zoonotic diseases respond in a changing climate, thereby calling for effective administration and prevention strategies. Furthermore, these results will shed light on guiding future epidemiologic prediction of emerging infectious diseases under global climate change.
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Epidemias , Zoonosis , Animales , Incidencia , Zoonosis/epidemiología , Ecosistema , Cambio Climático , China/epidemiologíaRESUMEN
Long noncoding RNAs (lncRNAs) have recently been identified to be tightly linked to diverse human diseases. However, our knowledge of Systemic Lupus Erythematosus (SLE)-related lncRNAs remains limited. In the present study we investigated the contribution of the lncRNA NEAT1 to the pathogenesis of SLE. Here, we found NEAT1 expression was abnormally increased in SLE patients and predominantly expressed in human monocytes. Additionally, NEAT1 expression was induced by LPS via p38 activation. Silencing NEAT1 significantly reduced the expression of a group of chemokines and cytokines, including IL-6, CXCL10, etc., which were induced by LPS continuously and in late stages. Furthermore, it was identified the involvement of NEAT1 in TLR4-mediated inflammatory process was through affecting the activation of the late MAPK signaling pathway. Importantly, there was a positive correlation between NEAT1 and clinical disease activity in SLE patients. In conclusion, the increased NEAT1 expression may be a potential contributor to the elevated production of a number of cytokines and chemokines in SLE patients. Our findings suggest lncRNA contributes to the pathogenesis of lupus and provides potentially novel target for therapeutic intervention.
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Citocinas/inmunología , Mediadores de Inflamación/inmunología , Lupus Eritematoso Sistémico/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , ARN Largo no Codificante/inmunología , Western Blotting , Línea Celular Tumoral , Análisis por Conglomerados , Citocinas/genética , Citocinas/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Ontología de Genes , Humanos , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Monocitos/inmunología , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
The relationship between bacteria and tumors has been the hot spot of clinical research in recent years. Pseudoglutamicibacter cumminsii is an aerobic Gram-positive bacterium commonly found in soil. Recent studies have identified P. cumminsii in patients with cutaneous and urinary tract infections. However, little is known on its pathogenesis as well as involvement in other clinical symptoms. In this study, we first report the isolation of P. cumminsii in blood of an epithelial mesothelioma patient. The clinical and laboratory characteristics of P. cumminsii were first described and evaluated. The pure colony of P. cumminsii was then identified using automated microorganism identification system and mass spectrum. The whole genome of the newly identified strain was sequenced with third generation sequencing (TGS). The assembled genome was further annotated and analyzed. Whole genomic and comparative genomic analysis revealed that the isolated P. cumminsii strain in this study had a genome size of 2,179,930 bp and had considerable unique genes compared with strains reported in previous findings. Further phylogenetic analysis showed that this strain had divergent phylogenetic relationship with other P. cumminsii strains. Based on these results, the newly found P. cumminsii strain was named P. cumminsii XJ001 (PC1). Virulence analysis identified a total of 71 pathogenic genes with potential roles in adherence, immune modulation, nutrition/metabolism, and regulation in PC1. Functional analysis demonstrated that the annotated genes in PC1 were mainly clustered into amino acid metabolism (168 genes), carbohydrate metabolism (107 genes), cofactor and vitamin metabolisms (98 genes), and energy metabolism (68 genes). Specifically, six genes including yodJ, idh, katA, pyk, sodA, and glsA were identified within cancer pathways, and their corresponding homologous genes have been documented with precise roles in human cancer. Collectively, the above results first identified P. cumminsii in the blood of tumor patients and further provide whole genomic landscape of the newly identified PC1 strain, shedding light on future studies of bacteria in tumorigenesis.
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BACKGROUND: Sporadic parathyroid adenoma (PA) is the most common cause of hyperparathyroidism, yet the mechanisms involved in its pathogenesis remain incompletely understood. METHODS: Surgically removed PA samples, along with normal parathyroid gland (PG) tissues that were incidentally dissected during total thyroidectomy, were analysed using single-cell RNA-sequencing with the 10× Genomics Chromium Droplet platform and Cell Ranger software. Gene set variation analysis was conducted to characterise hallmark pathway gene signatures, and single-cell regulatory network inference and clustering were utilised to analyse transcription factor regulons. Immunohistochemistry and immunofluorescence were performed to validate cellular components of PA tissues. siRNA knockdown and gene overexpression, alongside quantitative polymerase chain reaction, Western blotting and cell proliferation assays, were conducted for functional investigations. RESULTS: There was a pervasive increase in gene transcription in PA cells (PACs) compared with PG cells. This is associated with high expression of histone-lysine N-methyltransferase 2A (KMT2A). High KMT2A levels potentially contribute to promoting PAC proliferation through upregulation of the proto-oncogene CCND2, which is mediated by the transcription factors signal transducer and activator of transcription 3 (STAT3) and GATA binding protein 3 (GATA3). PA tissues are heavily infiltrated with myeloid cells, while fibroblasts, endothelial cells and macrophages in PA tissues are commonly enriched with proinflammatory gene signatures relative to their counterparts in PG tissues. CONCLUSIONS: We revealed the previously underappreciated involvement of the KMT2AâSTAT3/GATA3âCCND2 axis and chronic inflammation in the pathogenesis of PA. These findings underscore the therapeutic promise of KMT2A inhibition and anti-inflammatory strategies, highlighting the need for future investigations to translate these molecular insights into practical applications. HIGHLIGHTS: Single-cell RNA-sequencing reveals a transcriptome catalogue comparing sporadic parathyroid adenomas (PAs) with normal parathyroid glands. PA cells show a pervasive increase in gene expression linked to KMT2A upregulation. KMT2A-mediated STAT3 and GATA3 upregulation is key to promoting PA cell proliferation via cyclin D2. PAs exhibit a proinflammatory microenvironment, suggesting a potential role of chronic inflammation in PA pathogenesis.
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Adenoma , N-Metiltransferasa de Histona-Lisina , Inflamación , Neoplasias de las Paratiroides , Humanos , Neoplasias de las Paratiroides/genética , Neoplasias de las Paratiroides/metabolismo , Neoplasias de las Paratiroides/patología , Adenoma/genética , Adenoma/metabolismo , Adenoma/patología , Inflamación/genética , Inflamación/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proto-Oncogenes Mas , Proliferación Celular/genéticaRESUMEN
P53 inactivation occurs in about 50% of human cancers, where p53-driven p21 activity is devoid and p27 becomes essential for the establishment of the G1/S checkpoint upon DNA damage. Here, this work shows that the E2F1-responsive lncRNA LIMp27 selectively represses p27 expression and contributes to proliferation, tumorigenicity, and treatment resistance in p53-defective colon adenocarcinoma (COAD) cells. LIMp27 competes with p27 mRNA for binding to cytoplasmically localized hnRNA0, which otherwise stabilizes p27 mRNA leading to cell cycle arrest at the G0/G1 phase. In response to DNA damage, LIMp27 is upregulated in both wild-type and p53-mutant COAD cells, whereas cytoplasmic hnRNPA0 is only increased in p53-mutant COAD cells due to translocation from the nucleus. Moreover, high LIMp27 expression is associated with poor survival of p53-mutant but not wild-type p53 COAD patients. These results uncover an lncRNA mechanism that promotes p53-defective cancer pathogenesis and suggest that LIMp27 may constitute a target for the treatment of such cancers.
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Adenocarcinoma , Neoplasias del Colon , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , ARN Largo no Codificante , Humanos , Daño del ADN/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismoRESUMEN
BACKGROUND: Distant metastasis is the major cause of clear cell renal cell carcinoma (ccRCC)-associated mortality. However, molecular mechanisms involved in ccRCC metastasis remain to be fully understood. With the increasing appreciation of the role of long non-coding RNAs (lncRNAs) in cancer development, progression, and treatment resistance, the list of aberrantly expressed lncRNAs contributing to ccRCC pathogenesis is expanding rapidly. METHODS: Bioinformatics analysis was carried out to interrogate publicly available ccRCC datasets. In situ hybridization and qRT-PCR assays were used to test lncRNA expression in human ccRCC tissues and cell lines, respectively. Chromatin immunoprecipitation and luciferase reporter assays were used to examine transcriptional regulation of gene expression. Wound healing as well as transwell migration and invasion assays were employed to monitor ccRCC cell migration and invasion in vitro. ccRCC metastasis was also examined using mouse models in vivo. RNA pulldown and RNA immunoprecipitation were performed to test RNA-protein associations, whereas RNA-RNA interactions were tested using domain-specific chromatin isolation by RNA purification. RESULTS: MILIP expression was upregulated in metastatic compared with primary ccRCC tissues. The increased MILIP expression in metastatic ccRCC cells was driven by the transcription factor AP-2 gamma (TFAP2C). Knockdown of MILIP diminished the potential of ccRCC cell migration and invasion in vitro and reduced the formation of ccRCC metastatic lesions in vivo. The effect of MILIP on ccRCC cells was associated with alterations in the expression of epithelial-to-mesenchymal transition (EMT) hallmark genes. Mechanistically, MILIP formed an RNA-RNA duplex with the snail family transcriptional repressor 1 (Snai1) mRNA and bound to Y-box binding protein 1 (YBX1). This promoted the association between the YBX1 protein and the Snai1 mRNA, leading to increased translation of the latter. Snai1 in turn played an important role in MILIP-driven ccRCC metastasis. CONCLUSIONS: The TFAP2C-responsive lncRNA MILIP drives ccRCC metastasis. Targeting MILIP may thus represent a potential avenue for ccRCC treatment.
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Carcinoma de Células Renales , Neoplasias Renales , ARN Largo no Codificante , Factores de Transcripción de la Familia Snail , Proteína 1 de Unión a la Caja Y , Animales , Carcinoma de Células Renales/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/genética , Ratones , ARN Largo no Codificante/genética , ARN Mensajero , Factores de Transcripción de la Familia Snail/genética , Proteína 1 de Unión a la Caja Y/genéticaRESUMEN
Active crosstalk between the nervous system and breast cancer cells has been experimentally demonstrated in vitro and in animal models. However, low frequencies of peripheral nerve presence in human breast cancers reported in previous studies (~30% of cases) potentially negate a major role of the nervous system in breast cancer development and progression. This study aimed to clarify the incidence of nerves within human breast cancers and to delineate associations with clinicopathological features. Immunohistochemical staining was conducted in formalin-fixed paraffin-embedded breast cancer tissue sections using antibodies against the pan-neuronal markers protein gene product 9.5 and growth-associated protein 43, and the sympathetic nerve-specific marker tyrosine hydroxylase. Nerve trunks and isolated nerve fibers were quantitated. The chi-squared test was used to determine the associations between nerve counts and clinicopathological parameters. The log-rank test was used to compare differences in patient progression-free survival (PFS) and overall survival (OS). The overall frequency of peripheral nerves in breast cancers was 85%, a markedly higher proportion than reported previously. Of note, most nerves present in breast cancers were of the sympathetic origin. While high density of nerve trunks or isolated nerve fibers was associated with poor PFS and OS of patients, high nerve trunk density appeared also to predict poor patient PFS independently of lymph node metastasis. Innervation of breast cancers is a common event correlated with poor patient outcomes. These findings support the notion that the nervous system plays an active role in breast cancer pathogenesis.
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In recent years, the plant morphology has been well studied by multiple approaches at cellular and subcellular levels. Two-dimensional (2D) microscopy techniques offer imaging of plant structures on a wide range of magnifications for researchers. However, subcellular imaging is still challenging in plant tissues like roots and seeds. Here we use a three-dimensional (3D) imaging technology based on the X-ray microscope (XRM) and analyze several plant tissues from different plant species. The XRM provides new insights into plant structures using non-destructive imaging at high-resolution and high contrast. We also utilized a workflow aiming to acquire accurate and high-quality images in the context of the whole specimen. Multiple plant samples including rice, tobacco, Arabidopsis and maize were used to display the differences of phenotypes. Our work indicates that the XRM is a powerful tool to investigate plant microstructure in high-resolution scale. Our work also provides evidence that evaluate and quantify tissue specific differences for a range of plant species. We also characterize novel plant tissue phenotypes by the XRM, such as seeds in Arabidopsis, and utilize them for novel observation measurement. Our work represents an evaluated spatial and temporal resolution solution on seed observation and screening.
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Arabidopsis/ultraestructura , Imagenología Tridimensional , Nicotiana/ultraestructura , Orgánulos/ultraestructura , Oryza/ultraestructura , Semillas/ultraestructura , Zea mays/ultraestructura , Oryza/anatomía & histología , Tomografía Computarizada por Rayos XRESUMEN
Rationale: Recurrent and metastatic cancers often undergo a period of dormancy, which is closely associated with cellular quiescence, a state whereby cells exit the cell cycle and are reversibly arrested in G0 phase. Curative cancer treatment thus requires therapies that either sustain the dormant state of quiescent cancer cells, or preferentially, eliminate them. However, the mechanisms responsible for the survival of quiescent cancer cells remain obscure. Methods: Dual genome-editing was carried out using a CRISPR/Cas9-based system to label endogenous p27 and Ki67 with the green and red fluorescent proteins EGFP and mCherry, respectively, in melanoma cells. Analysis of transcriptomes of isolated EGFP-p27highmCherry-Ki67low quiescent cells was conducted at bulk and single cell levels using RNA-sequencing. The extracellular acidification rate and oxygen consumption rate were measured to define metabolic phenotypes. SiRNA and inducible shRNA knockdown, chromatin immunoprecipitation and luciferase reporter assays were employed to elucidate mechanisms of the metabolic switch in quiescent cells. Results: Dual labelling of endogenous p27 and Ki67 with differentiable fluorescent probes allowed for visualization, isolation, and analysis of viable p27highKi67low quiescent cells. Paradoxically, the proto-oncoprotein c-Myc, which commonly drives malignant cell cycle progression, was expressed at relatively high levels in p27highKi67low quiescent cells and supported their survival through promoting mitochondrial oxidative phosphorylation (OXPHOS). In this context, c-Myc selectively transactivated genes encoding OXPHOS enzymes, including subunits of isocitric dehydrogenase 3 (IDH3), whereas its binding to cell cycle progression gene promoters was decreased in quiescent cells. Silencing of c-Myc or the catalytic subunit of IDH3, IDH3α, preferentially killed quiescent cells, recapitulating the effect of treatment with OXPHOS inhibitors. Conclusion: These results establish a rigorous experimental system for investigating cellular quiescence, uncover the high selectivity of c-Myc in activating OXPHOS genes in quiescent cells, and propose OXPHOS targeting as a potential therapeutic avenue to counter cancer cells in quiescence.
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Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Antígeno Ki-67/metabolismo , Melanoma/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Senescencia Celular , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Isocitrato Deshidrogenasa/metabolismo , Neoplasias/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Fase de Descanso del Ciclo Celular , Transcriptoma/genéticaRESUMEN
Genomic amplification of the distal portion of chromosome 3q, which encodes a number of oncogenic proteins, is one of the most frequent chromosomal abnormalities in malignancy. Here we functionally characterise a non-protein product of the 3q region, the long noncoding RNA (lncRNA) PLANE, which is upregulated in diverse cancer types through copy number gain as well as E2F1-mediated transcriptional activation. PLANE forms an RNA-RNA duplex with the nuclear receptor co-repressor 2 (NCOR2) pre-mRNA at intron 45, binds to heterogeneous ribonucleoprotein M (hnRNPM) and facilitates the association of hnRNPM with the intron, thus leading to repression of the alternative splicing (AS) event generating NCOR2-202, a major protein-coding NCOR2 AS variant. This is, at least in part, responsible for PLANE-mediated promotion of cancer cell proliferation and tumorigenicity. These results uncover the function and regulation of PLANE and suggest that PLANE may constitute a therapeutic target in the pan-cancer context.
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Empalme Alternativo/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/genética , ARN Largo no Codificante/genética , Células A549 , Línea Celular Tumoral , Proliferación Celular/genética , Cromosomas Humanos Par 3/genética , Variaciones en el Número de Copia de ADN/genética , Factor de Transcripción E2F1/metabolismo , Células HCT116 , Ribonucleoproteína Heterogénea-Nuclear Grupo M/genética , Humanos , Células MCF-7 , Neoplasias/patología , Co-Represor 2 de Receptor Nuclear/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
PURPOSE: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. EXPERIMENTAL DESIGN: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. RESULTS: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. CONCLUSIONS: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.
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Acetanilidas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Reordenamiento Génico , Compuestos Heterocíclicos con 3 Anillos/farmacología , Terapia Molecular Dirigida/métodos , Neuroblastoma/tratamiento farmacológico , Oligopéptidos/farmacología , Telomerasa/genética , Factores de Transcripción/antagonistas & inhibidores , Animales , Apoptosis , Proliferación Celular , Quimioterapia Combinada , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neuroblastoma/metabolismo , Neuroblastoma/patología , Inhibidores de Proteasoma/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The deubiquitinase cylindromatosis (CYLD) functions as a tumor suppressor inhibiting cell proliferation in many cancer types including melanoma. Here we present evidence that a proportion of melanoma cells are nonetheless addicted to CYLD for survival. The expression levels of CYLD varied widely in melanoma cell lines and melanomas in vivo, with a subset of melanoma cell lines and melanomas displaying even higher levels of CYLD than melanocyte lines and nevi, respectively. Strikingly, although short hairpin RNA (shRNA) knockdown of CYLD promoted, as anticipated, cell proliferation in some melanoma cell lines, it reduced cell viability in a fraction of melanoma cell lines with relatively high levels of CYLD expression and did not impinge on survival and proliferation in a third type of melanoma cell lines. The decrease in cell viability caused by CYLD knockdown was due to induction of apoptosis, as it was associated with activation of the caspase cascade and was abolished by treatment with a general caspase inhibitor. Mechanistic investigations demonstrated that induction of apoptosis by CYLD knockdown was caused by upregulation of receptor-interacting protein kinase 1 (RIPK1) that was associated with elevated K63-linked polyubiquitination of the protein, indicating that CYLD is critical for controlling RIPK1 expression in these cells. Of note, microRNA (miR) profiling showed that miR-99b-3p that was predicted to target the 3-untranslated region (3-UTR) of the CYLD mRNA was reduced in melanoma cell lines with high levels of CYLD compared with melanocyte lines. Further functional studies confirmed that the reduction in miR-99b-3p expression was responsible for the increased expression of CYLD in a highly cell line-specific manner. Taken together, these results reveal an unexpected role of CYLD in promoting survival of a subset of melanoma cells and uncover the heterogeneity of CYLD expression and its biological significance in melanoma.
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Proliferación Celular/genética , Enzima Desubiquitinante CYLD/metabolismo , Melanoma/enzimología , Melanoma/genética , Regiones no Traducidas 3'/genética , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Enzima Desubiquitinante CYLD/genética , Técnicas de Silenciamiento del Gen , Humanos , Melanoma/patología , MicroARNs/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Regulación hacia ArribaRESUMEN
The functions of the proto-oncoprotein c-Myc and the tumor suppressor p53 in controlling cell survival and proliferation are inextricably linked as "Yin and Yang" partners in normal cells to maintain tissue homeostasis: c-Myc induces the expression of ARF tumor suppressor (p14ARF in human and p19ARF in mouse) that binds to and inhibits mouse double minute 2 homolog (MDM2) leading to p53 activation, whereas p53 suppresses c-Myc through a combination of mechanisms involving transcriptional inactivation and microRNA-mediated repression. Nonetheless, the regulatory interactions between c-Myc and p53 are not retained by cancer cells as is evident from the often-imbalanced expression of c-Myc over wildtype p53. Although p53 repression in cancer cells is frequently associated with the loss of ARF, we disclose here an alternate mechanism whereby c-Myc inactivates p53 through the actions of the c-Myc-Inducible Long noncoding RNA Inactivating P53 (MILIP). MILIP functions to promote p53 polyubiquitination and turnover by reducing p53 SUMOylation through suppressing tripartite-motif family-like 2 (TRIML2). MILIP upregulation is observed amongst diverse cancer types and is shown to support cell survival, division and tumourigenicity. Thus our results uncover an inhibitory axis targeting p53 through a pan-cancer expressed RNA accomplice that links c-Myc to suppression of p53.
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Neoplasias/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Carcinogénesis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , ARN Largo no Codificante/genética , Sumoilación , Proteína p53 Supresora de Tumor/genética , UbiquitinaciónRESUMEN
Protein products of the regenerating islet-derived (REG) gene family are important regulators of many cellular processes. Here we functionally characterise a non-protein coding product of the family, the long noncoding RNA (lncRNA) REG1CP that is transcribed from a DNA fragment at the family locus previously thought to be a pseudogene. REG1CP forms an RNA-DNA triplex with a homopurine stretch at the distal promoter of the REG3A gene, through which the DNA helicase FANCJ is tethered to the core promoter of REG3A where it unwinds double stranded DNA and facilitates a permissive state for glucocorticoid receptor α (GRα)-mediated REG3A transcription. As such, REG1CP promotes cancer cell proliferation and tumorigenicity and its upregulation is associated with poor outcome of patients. REG1CP is also transcriptionally inducible by GRα, indicative of feedforward regulation. These results reveal the function and regulation of REG1CP and suggest that REG1CP may constitute a target for cancer treatment.
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Carcinogénesis/genética , Elementos de Facilitación Genéticos/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas Asociadas a Pancreatitis/genética , ARN Helicasas/genética , ARN Largo no Codificante/genética , Transcripción Genética , Biomarcadores de Tumor/genética , Línea Celular , Línea Celular Tumoral , ADN/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Células HT29 , Humanos , Neoplasias/genética , Neoplasias/patología , Proteínas Asociadas a Pancreatitis/metabolismo , Regiones Promotoras Genéticas/genética , ARN Helicasas/metabolismoRESUMEN
: Cancer cells in quiescence (G0 phase) are resistant to death, and re-entry of quiescent cancer cells into the cell-cycle plays an important role in cancer recurrence. Here we show that two p53-responsive miRNAs utilize distinct but complementary mechanisms to promote cancer cell quiescence by facilitating stabilization of p27. Purified quiescent B16 mouse melanoma cells expressed higher levels of miRNA-27b-3p and miRNA-455-3p relative to their proliferating counterparts. Induction of quiescence resulted in increased levels of these miRNAs in diverse types of human cancer cell lines. Inhibition of miRNA-27b-3p or miRNA-455-3p reduced, whereas its overexpression increased, the proportion of quiescent cells in the population, indicating that these miRNAs promote cancer cell quiescence. Accordingly, cancer xenografts bearing miRNA-27b-3p or miRNA-455-3p mimics were retarded in growth. miRNA-27b-3p targeted cyclin-dependent kinase regulatory subunit 1 (CKS1B), leading to reduction in p27 polyubiquitination mediated by S-phase kinase-associated protein 2 (Skp2). miRNA-455-3p targeted CDK2-associated cullin domain 1 (CAC1), which enhanced CDK2-mediated phosphorylation of p27 necessary for its polyubiquitination. Of note, the gene encoding miRNA-27b-3p was embedded in the intron of the chromosome 9 open reading frame 3 gene that was transcriptionally activated by p53. Similarly, the host gene of miRNA-455-3p, collagen alpha-1 (XXVII) chain, was also a p53 transcriptional target. Collectively, our results identify miRNA-27b-3p and miRNA-455-3p as important regulators of cancer cell quiescence in response to p53 and suggest that manipulating miRNA-27b-3p and miRNA-455-3p may constitute novel therapeutic avenues for improving outcomes of cancer treatment. SIGNIFICANCE: Two novel p53-responsive microRNAs whose distinct mechanisms of action both stabilize p27 to promote cell quiescence and may serve as therapeutic avenues for improving outcomes of cancer treatment.
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Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs/genética , Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Senescencia Celular/genética , Genes Reporteros , Genes cdc , Humanos , Ratones , Modelos Biológicos , Fosforilación , Interferencia de ARN , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismoRESUMEN
The expression of CD47 on the cancer cell surface transmits "don't eat me" signalling that not only inhibits phagocytosis of cancer cells by phagocytes but also impairs anti-cancer T cell responses. Here we report that oncogenic activation of ERK plays an important role in transcriptional activation of CD47 through nuclear respiratory factor 1 (NRF-1) in melanoma cells. Treatment with BRAF/MEK inhibitors upregulated CD47 in cultured melanoma cells and fresh melanoma isolates. Similarly, melanoma cells selected for resistance to the BRAF inhibitor vemurafenib expressed higher levels of CD47. The increase in CD47 expression was mediated by ERK signalling, as it was associated with rebound activation of ERK and co-knockdown of ERK1/2 by siRNA diminished upregulation of CD47 in melanoma cells after exposure to BRAF/MEK inhibitors. Furthermore, ERK1/2 knockdown also reduced the constitutive expression of CD47 in melanoma cells. We identified a DNA fragment that was enriched with the consensus binding sites for NRF-1 and was transcriptionally responsive to BRAF/MEK inhibitor treatment. Knockdown of NRF-1 inhibited the increase in CD47, indicating that NRF-1 has a critical role in transcriptional activation of CD47 by ERK signalling. Functional studies showed that melanoma cells resistant to vemurafenib were more susceptible to macrophage phagocytosis when CD47 was blocked. So these results suggest that NRF-1-mediated regulation of CD47 expression is a novel mechanism by which ERK signalling promotes the pathogenesis of melanoma, and that the combination of CD47 blockade and BRAF/MEK inhibitors may be a useful approach for improving their therapeutic efficacy.
RESUMEN
MTH1 helps prevent misincorporation of ROS-damaged dNTPs into genomic DNA; however, there is little understanding of how MTH1 itself is regulated. Here, we report that MTH1 is regulated by polyubiquitination mediated by the E3 ligase Skp2. In melanoma cells, MTH1 was upregulated commonly mainly due to its improved stability caused by K63-linked polyubiquitination. Although Skp2 along with other components of the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex was physically associated with MTH1, blocking the SCF function ablated MTH1 ubiquitination and expression. Conversely, overexpressing Skp2-elevated levels of MTH1 associated with an increase in its K63-linked ubiquitination. In melanoma cell lines and patient specimens, we observed a positive correlation of Skp2 and MTH1 expression. Mechanistic investigations showed that Skp2 limited DNA damage and apoptosis triggered by oxidative stress and that MAPK upregulated Skp2 and MTH1 to render cells more resistant to such stress. Collectively, our findings identify Skp2-mediated K63-linked polyubiquitination as a critical regulatory mechanism responsible for MTH1 upregulation in melanoma, with potential implications to target the MAPK/Skp2/MTH1 pathway to improve its treatment. Cancer Res; 77(22); 6226-39. ©2017 AACR.
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Enzimas Reparadoras del ADN/genética , Melanoma/genética , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Enzimas Reparadoras del ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas/genética , Melanoma/metabolismo , Melanoma/patología , Monoéster Fosfórico Hidrolasas/metabolismo , Poliubiquitina/metabolismo , Estabilidad Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas Asociadas a Fase-S/metabolismo , UbiquitinaciónRESUMEN
Accumulating evidence suggests significant biological effects caused by extremely low frequency electromagnetic fields (ELF-EMF). Although exo-endocytosis plays crucial physical and biological roles in neuronal communication, studies on how ELF-EMF regulates this process are scarce. By directly measuring calcium currents and membrane capacitance at a large mammalian central nervous synapse, the calyx of Held, we report for the first time that ELF-EMF critically affects synaptic transmission and plasticity. Exposure to ELF-EMF for 8 to 10 days dramatically increases the calcium influx upon stimulation and facilitates all forms of vesicle endocytosis, including slow and rapid endocytosis, endocytosis overshoot and bulk endocytosis, but does not affect the RRP size and exocytosis. Exposure to ELF-EMF also potentiates PTP, a form of short-term plasticity, increasing its peak amplitude without impacting its time course. We further investigated the underlying mechanisms and found that calcium channel expression, including the P/Q, N, and R subtypes, at the presynaptic nerve terminal was enhanced, accounting for the increased calcium influx upon stimulation. Thus, we conclude that exposure to ELF-EMF facilitates vesicle endocytosis and synaptic plasticity in a calcium-dependent manner by increasing calcium channel expression at the nerve terminal.
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Canales de Calcio/fisiología , Calcio/química , Campos Electromagnéticos , Endocitosis , Terminales Presinápticos/fisiología , Sinapsis/fisiología , Animales , Comunicación Celular , Exocitosis , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal , Neuronas/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas SNARE/fisiologíaRESUMEN
Oncogenic mutations of BRAF occur in approximately 10% of colon cancers and are associated with their resistance to clinically available therapeutic drugs and poor prognosis of the patients. Here we report that colon cancer cells with mutant BRAF are also resistant to the heat shock protein 90 (HSP90) inhibitor AUY922, and that this is caused by rebound activation of ERK and Akt. Although AUY922 triggered rapid reduction in ERK and Akt activation in both wild-type and mutant BRAF colon cancer cells, activation of ERK and Akt rebounded shortly in the latter leading to resistance of the cells to AUY922-induced apoptosis. Reactivation of ERK was associated with the persistent expression of mutant BRAF, which, despite being a client of HSP90, was only partially degraded by AUY922, whereas reactivation of Akt was related to the activity of the HSP90 co-chaperone, cell division cycle 37 (CDC37), in that knockdown of CDC37 inhibited Akt reactivation in mutant colon cancer cells treated with AUY922. In support, as a HSP90 client protein, Akt was only diminished by AUY922 in wild-type but not mutant BRAF colon cancer cells. Collectively, these results reveal that reactivation of ERK and Akt associated respectively with the activity of mutant BRAF and CDC37 renders mutant BRAF colon cancer cells resistant to AUY922, with implications of co-targeting mutant BRAF and/or CDC37 and HSP90 in the treatment of mutant BRAF colon cancers.