RESUMEN
Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein by promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting by promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function.
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Núcleo Celular , Proteínas Oncogénicas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Oncogénicas/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Transporte de Proteínas , Biosíntesis de Proteínas , Proteínas de la Membrana/metabolismoRESUMEN
The neuroepithelial cell transforming gene 1 (Net1) is a guanine nucleotide exchange factor for the small GTPase RhoA that promotes cancer cell motility and metastasis. Two isoforms of Net1 exist, Net1 and Net1A, both of which are sequestered in the nucleus in quiescent cells to prevent aberrant RhoA activation. Many cell motility stimuli drive cytosolic relocalization of Net1A, but mechanisms controlling this event are not fully understood. Here, we demonstrate that epithelial growth factor stimulates protein kinase Src- and Abl1-dependent phosphorylation of Net1A to promote its cytosolic localization. We show that Abl1 efficiently phosphorylates Net1A on Y373, and that phenylalanine substitution of Y373 prevents Net1A cytosolic localization. Furthermore, we found that Abl1-driven cytosolic localization of Net1A does not require S52, which is a phosphorylation site of a different kinase, c-Jun N-terminal kinase, that inhibits nuclear import of Net1A. However, we did find that MKK7-stimulated cytosolic localization of Net1A does require Y373. We also demonstrate that aspartate substitution at Y373 is sufficient to promote Net1A cytosolic accumulation, and expression of Net1A Y373D potentiates epithelial growth factor-stimulated RhoA activation, downstream myosin light chain 2 phosphorylation, and F-actin accumulation. Moreover, we show that expression of Net1A Y373D in breast cancer cells also significantly increases cell motility and Matrigel invasion. Finally, we show that Net1A is required for Abl1-stimulated cell motility, which is rescued by expression of Net1A Y373D, but not Net1A Y373F. Taken together, this work demonstrates a novel mechanism controlling Net1A subcellular localization to regulate RhoA-dependent cell motility and invasion.
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Factores de Intercambio de Guanina Nucleótido , Proteínas Proto-Oncogénicas c-abl , Proteína de Unión al GTP rhoA , Movimiento Celular , Citosol/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fosforilación , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismoRESUMEN
BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with high intratumoral heterogeneity. There is a lack of effective therapeutics for PDAC. Entosis, a form of nonapoptotic regulated cell death mediated by cell-in-cell structures (CICs), has been reported in multiple cancers. However, the role of entosis in PDAC progression remains unclear. METHODS: CICs were evaluated using immunohistochemistry and immunofluorescence staining. The formation of CICs was induced by suspension culture. Through fluorescence-activated cell sorting and single-cell RNA sequencing, entosis-forming cells were collected and their differential gene expression was analyzed. Cell functional assays and mouse models were used to investigate malignant phenotypes. Clinical correlations between entosis and PDAC were established by retrospective analysis. RESULTS: Entosis was associated with an unfavorable prognosis for patients with PDAC and was more prevalent in liver metastases than in primary tumors. The single-cell RNA sequencing results revealed that several oncogenes were up-regulated in entosis-forming cells compared with parental cells. These highly entotic cells demonstrated higher oncogenic characteristics in vitro and in vivo. NET1, neuroepithelial cell transforming gene 1, is an entosis-related gene that plays a pivotal role in PDAC progression and is correlated with poor outcomes. CONCLUSIONS: Entosis is correlated with PDAC progression, especially in liver metastasis. NET1 is a newly validated entosis-related gene and a molecular marker of poor outcomes. PDAC cells generate a highly aggressive subpopulation marked by up-regulated NET1 via entosis, which may drive PDAC progression.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ratones , Animales , Humanos , Entosis , Estudios Retrospectivos , Línea Celular Tumoral , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Regulación Neoplásica de la Expresión Génica , Proliferación Celular/genética , Neoplasias PancreáticasRESUMEN
BACKGROUND: Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily guanine nucleotide exchange factor that governs a wide array of biological processes. However, its roles in meiotic oocyte remain unclear. We herein demonstrated that the NET1-HACE1-RAC1 pathway mediates meiotic defects in the progression of oocyte maturation. METHODS: NET1 was reduced using a specific small interfering RNA in mouse oocytes. Spindle assembly, chromosomal alignment, the actin cap, and chromosomal spreads were visualized by immunostaining and analyzed under confocal microscopy. We also applied mass spectroscopy, and western blot analysis for this investigation. RESULTS: Our results revealed that NET1 was localized to the nucleus at the GV stage, and that after GVBD, NET1 was localized to the cytoplasm and predominantly distributed around the chromosomes, commensurate with meiotic progression. NET1 resided in the cytoplasm and significantly accumulated on the spindle at the MI and MII stages. Mouse oocytes depleted of Net1 exhibited aberrant first polar body extrusion and asymmetric division defects. We also determined that Net1 depletion resulted in reduced RAC1 protein expression in mouse oocytes, and that NET1 protected RAC1 from degradation by HACE1, and it was essential for actin dynamics and meiotic spindle formation. Importantly, exogenous RAC1 expression in Net1-depleted oocytes significantly rescued these defects. CONCLUSIONS: Our results suggest that NET1 exhibits multiple roles in spindle stability and actin dynamics during mouse oocyte meiosis.
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Actinas , Huso Acromático , Animales , Ratones , Actinas/metabolismo , Meiosis , Oncogenes , Oocitos/metabolismo , Huso Acromático/metabolismoRESUMEN
Mechanosensitive hair cells (HCs) in the cochlear sensory epithelium are critical for sound detection and transduction. Mammalian HCs in the cochlea undergo cytogenesis during embryonic development, and irreversible damage to hair cells postnatally is a major cause of deafness. During the development of the organ of Corti, HCs and supporting cells (SCs) originate from the same precursors. In the neonatal cochlea, damage to HCs activates adjacent SCs to act as HC precursors and to differentiate into new HCs. However, the plasticity of SCs to produce new HCs is gradually lost with cochlear development. Here, we delineate an essential role for the guanine nucleotide exchange factor Net1 in SC trans-differentiation into HCs. Net1 overexpression mediated by AAV-ie in SCs promoted cochlear organoid formation and HC differentiation under two and three-dimensional culture conditions. Also, AAV-Net1 enhanced SC proliferation in Lgr5-EGFPCreERT2 mice and HC generation as indicated by lineage tracing of HCs in the cochleae of Lgr5-EGFPCreERT2/Rosa26-tdTomatoloxp/loxp mice. We further found that the up-regulation of Wnt/ß-catenin and Notch signaling in AAV-Net1-transduced cochleae might be responsible for the SC proliferation and HC differentiation. Also, Net1 overexpression in SCs enhanced SC proliferation and HC regeneration and survival after HC damage by neomycin. Taken together, our study suggests that Net1 might serve as a potential target for HC regeneration and that AAV-mediated gene regulation may be a promising approach in stem cell-based therapy in hearing restoration.
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Transdiferenciación Celular , Células Ciliadas Auditivas , Animales , Ratones , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Cóclea , Ratones TransgénicosRESUMEN
Adaptation to environmental changes is crucial for cell fitness. In Saccharomyces cerevisiae, variations in external osmolarity trigger the activation of the stress-activated protein kinase Hog1 (high-osmolarity glycerol 1), which regulates gene expression, metabolism, and cell-cycle progression. The activation of this kinase leads to the regulation of G1, S, and G2 phases of the cell cycle to prevent genome instability and promote cell survival. Here we show that Hog1 delays mitotic exit when cells are stressed during metaphase. Hog1 phosphorylates the nucleolar protein Net1, altering its affinity for the phosphatase Cdc14, whose activity is essential for mitotic exit and completion of the cell cycle. The untimely release of Cdc14 from the nucleolus upon activation of Hog1 is linked to a defect in ribosomal DNA (rDNA) and telomere segregation, and it ultimately delays cell division. A mutant of Net1 that cannot be phosphorylated by Hog1 displays reduced viability upon osmostress. Thus, Hog1 contributes to maximizing cell survival upon stress by regulating mitotic exit.
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Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , ADN Ribosómico/metabolismo , Mutación , Proteínas Nucleares/genética , Presión Osmótica/fisiología , Fosforilación/genética , Proteínas Tirosina Fosfatasas/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Homeostasis del Telómero/fisiologíaRESUMEN
Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusion-enriched RNAs encode factors linked to cancer progression, such as the RAB13 GTPase and the NET1 guanine nucleotide exchange factor, and are regulated by the tumor-suppressor protein APC. However, tumor cells in vivo often do not move as single cells but rather utilize collective modes of invasion and dissemination. Here, we developed an inducible system of three-dimensional (3D) collective invasion to study the behavior and importance of protrusion-enriched RNAs. We find that, strikingly, both the RAB13 and NET1 RNAs are enriched specifically at the invasive front of leader cells in invasive cell strands. This localization requires microtubules and coincides with sites of high laminin concentration. Indeed, laminin association and integrin engagement are required for RNA accumulation at the invasive front. Importantly, perturbing RNA accumulation reduces collective 3D invasion. Examination of in vivo tumors reveals a similar localization of the RAB13 and NET1 RNAs at potential invasive sites, suggesting that this mechanism could provide a targeting opportunity for interfering with collective cancer cell invasion.
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Movimiento Celular/genética , Invasividad Neoplásica/genética , Neoplasias/patología , ARN Mensajero/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Microscopía Intravital , Ratones , Microscopía Confocal , Invasividad Neoplásica/prevención & control , Neoplasias/genética , Proteínas Oncogénicas/genética , ARN Interferente Pequeño , Esferoides Celulares , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión al GTP rab/genéticaRESUMEN
The neuroepithelial cell transforming gene 1A (Net1A, an isoform of Net1) is a RhoA subfamily guanine nucleotide exchange factor (GEF) that localizes to the nucleus in the absence of stimulation, preventing it from activating RhoA. Once relocalized in the cytosol, Net1A stimulates cell motility and extracellular matrix invasion. In the present work, we investigated mechanisms responsible for the cytosolic relocalization of Net1A. We demonstrate that inhibition of MAPK pathways blocks Net1A relocalization, with cells being most sensitive to JNK pathway inhibition. Moreover, activation of the JNK or p38 MAPK family pathway is sufficient to elicit Net1A cytosolic localization. Net1A relocalization stimulated by EGF or JNK activation requires nuclear export mediated by CRM1. JNK1 (also known as MAPK8) phosphorylates Net1A on serine 52, and alanine substitution at this site prevents Net1A relocalization caused by EGF or JNK activation. Glutamic acid substitution at this site is sufficient for Net1A relocalization and results in elevated RhoA signaling to stimulate myosin light chain 2 (MLC2, also known as MYL2) phosphorylation and F-actin accumulation. Net1A S52E expression stimulates cell motility, enables Matrigel invasion and promotes invadopodia formation. These data highlight a novel mechanism for controlling the subcellular localization of Net1A to regulate RhoA activation, cell motility, and invasion.
Asunto(s)
Movimiento Celular , Carioferinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Oncogénicas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Estrés Fisiológico , Movimiento Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Ácido Glutámico/metabolismo , Humanos , Células MCF-7 , Mutación/genética , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Podosomas/efectos de los fármacos , Podosomas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal , Estrés Fisiológico/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Proteína Exportina 1RESUMEN
The aim of this study was to establish a novel targeting nanobubble (TNB) conjugated with small interfering RNA (siRNA)-cyanine 5 (Cy5) and to validate its theranostic ability in vivo. The TNB conjugated with neuroepithelial transforming gene 1 (NET-1) siRNA-Cy5 was prepared by thin-film hydration and mechanical sonication method. A hepatocellular carcinoma (HCC) xenograft model was established by subcutaneously injecting SMMC-7721 cells in BALB/c nude mice. The NET-1 siRNA-conjugated TNB was utilized for accurate contrast-enhanced ultrasound in vivo imaging, which was enabled by the target ligand GPC-3 antibody and specific gene transfection with the aid of low-frequency ultrasound (LFUS) irradiation. BALB/c nude mice bearing tumors were randomized into 5 groups and irradiated with LFUS for 5 min after TNB administration; mice were treated twice a week for a total of 60 d. The mean particle size of TNB was <500 nm. Mice treated with NET-1 siRNA-conjugated TNB showed a significant decrease in tumor growth and the highest survival rate. Our findings offer an effective and safe gene vehicle and probe for molecular imaging in vivo. It may improve the early diagnosis and treatment effects of HCC.-Wu, B., Shang, H., Liang, X., Sun, Y., Jing, H., Han, X., Cheng, W. Preparation of novel targeting nanobubbles conjugated with small interfering RNA for concurrent molecular imaging and gene therapy in vivo.
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Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Imagen Molecular , Nanoestructuras , ARN Interferente Pequeño , Animales , Línea Celular , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales , Distribución AleatoriaRESUMEN
BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as important roles in gastric cancer (GC). However, the role of the dysregulated lncRNAs in GC remained large unknown. We investigated the clinical significance, biological function and mechanism of CTC-497E21.4 in GC. METHODS: Firstly, RTFQ-PCR was used to detect the expression of CTC-497E21.4 in GC. Furthermore, knockdown of CTC-497E21.4 was conducted to assess the effect of CTC-497E21.4 in vitro and vivo. Subcellular localization of CTC-497E21.4 was determined by nuclear plasmolysis PCR and FISH. We also predicted CTC-497E21.4 binding miRNAs and downstream target genes and evaluated its regulation of miR-22 by acting as a ceRNA. RESULT: CTC-497E21.4 was upregulated in GC tissues and GC cell lines (P < 0.05), and the expression was associated with depth of invasion, lymph node metastasis, and neurological invasion. Besides, knockdown of CTC-497E21.4 inhibited cell proliferation, invasion and promoted cell cycle arrest in vitro and inhibited tumorigenesis in vivo. Mechanistic investigations indicated that CTC-497E21.4 acted as a ceRNA for miR-22 and regulated NET1 expression. CTC-497E21.4/miR-22-3p/NET1 participated in the RhoA signaling pathway in the GC progression. CONCLUSION: CTC-497E21.4 competed with miR-22 to regulate the expression of NET1 and regulated the malignant progression of GC through RhoA signaling pathway.
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Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteínas Oncogénicas/metabolismo , ARN Largo no Codificante/genética , Neoplasias Gástricas/patología , Proteína de Unión al GTP rhoA/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Ciclo Celular , Proliferación Celular , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Metástasis Linfática , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas Oncogénicas/genética , Pronóstico , Transducción de Señal , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína de Unión al GTP rhoA/genéticaRESUMEN
The GTPase RhoA is a major player in many different regulatory pathways. RhoA catalyzes GTP hydrolysis, and its catalysis is accelerated when RhoA forms heterodimers with proteins of the guanine nucleotide exchange factor (GEF) family. Neuroepithelial cell transforming gene 1 (Net1) is a RhoA-interacting GEF implicated in cancer, but the structural features supporting the RhoA/Net1 interaction are unknown. Taking advantage of a simple production and purification process, here we solved the structure of a RhoA/Net1 heterodimer with X-ray crystallography at 2-Å resolution. Using a panel of several techniques, including molecular dynamics simulations, we characterized the RhoA/Net1 interface. Moreover, deploying an extremely simple peptide-based scanning approach, we found that short peptides (penta- to nonapeptides) derived from the protein/protein interaction region of RhoA could disrupt the RhoA/Net1 interaction and thereby diminish the rate of nucleotide exchange. The most inhibitory peptide, EVKHF, spanning residues 102-106 in the RhoA sequence, displayed an IC50 of â¼100 µm without further modifications. The peptides identified here could be useful in further investigations of the RhoA/Net1 interaction region. We propose that our structural and functional insights might inform chemical approaches for transforming the pentapeptide into an optimized pseudopeptide that antagonizes Net1-mediated RhoA activation with therapeutic anticancer potential.
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Proteínas Oncogénicas/química , Proteína de Unión al GTP rhoA/química , Secuencia de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacología , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Simulación de Dinámica Molecular , Terapia Molecular Dirigida , Proteínas Oncogénicas/metabolismo , Péptidos/química , Péptidos/farmacología , Conformación Proteica/efectos de los fármacos , Mapas de Interacción de Proteínas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Alineación de Secuencia , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Ulp1 and Ulp2, in the yeast Saccharomyces cerevisiae, are the founding members of deSUMOylating enzymes. These enzymes remove small ubiquitin-like modifier (SUMO) from proteins and are conserved in all eukaryotes. Previous studies have shown that Ulp1 deSUMOylates the bulk of intracellular SUMOylated proteins, whereas Ulp2 is a highly specific enzyme. However, the mechanism for Ulp2's substrate specificity has been insufficiently understood. Here we show that the C-terminal regulatory domain of Ulp2 contains three distinct, yet conserved, motifs that control its in vivo substrate specificity and cell growth. Among them, a SUMO-interacting motif (SIM) was found to coordinate with the domain of Ulp2 that binds to the nucleolar protein Csm1 to ensure maximal deSUMOylation of Ulp2's nucleolar substrates. We found that whereas the Csm1-binding domain of Ulp2 recruits this enzyme to the nucleolus, Ulp2's C-terminal SIM promotes its SUMO protease activity and plays a key role in mediating the in vivo specificity of Ulp2. Thus, the substrate specificity of Ulp2 is controlled by both its subcellular localization and the SUMOylation status of its substrates. These findings illustrate the highly coordinated and dynamic nature of the SUMO pathways in maintaining homeostasis of intracellular SUMOylation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Endopeptidasas/química , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Regulación Fúngica de la Expresión Génica , Cinética , Proteínas Nucleares/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad por Sustrato , Sumoilación , Ubiquitina/genética , UbiquitinaciónRESUMEN
Net1 is a well-characterized oncoprotein with RhoA-specific GEF activity. Oncogenic Net1, which lacks the first 145 amino acids, is present in the cytosol and contributes to the efficient activation of RhoA and the formation of actin stress fibers in a number of tumor cell types. Meanwhile, wild-type Net1 is predominantly localized in the nucleus at steady state due to its N-terminal nuclear localization sequences, where the function of nuclear Net1 has not been fully determined. Here, we find that zebrafish net1 is expressed specifically in mesendoderm precursors during gastrulation. Endogenous Net1 is located in the nucleus during early embryonic development. Gain- and loss-of-function experiments in zebrafish embryos and mammalian cells demonstrate that, regardless of its GEF activity, nuclear Net1 is critical for zebrafish mesendoderm formation and Nodal signal transduction. Detailed analyses of protein interactions reveal that Net1 associates with Smad2 in the nucleus in a GEF-independent manner, and then promotes Smad2 activation by enhancing recruitment of p300 (also known as EP300) to the transcriptional complex. These findings describe a novel genetic mechanism by which nuclear Net1 facilitates Smad2 transcriptional activity to guide mesendoderm development.
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Núcleo Celular/metabolismo , Endodermo/embriología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mesodermo/embriología , Proteína Nodal/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Embrión no Mamífero/metabolismo , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Mesodermo/metabolismo , Modelos Biológicos , Unión Proteica , Transducción de Señal , Proteína Smad2/metabolismo , Regulación hacia Arriba/genética , Pez Cebra/genéticaRESUMEN
OBJECTIVE: The influence of perioperative factors, such as anaesthetic and analgesic techniques, on metastatic spread following surgery for primary cancer removal is of growing interest. The present study investigated the effects of sevoflurane on canine mammary tumour cell proliferation (MTT colorimetric assay) and on the expression of neuroepithelial transforming gene 1 (NET1). STUDY DESIGN: Prospective controlled in vitro trial. STUDY MATERIAL: Primary (CIPp) and metastatic canine tubular adenocarcinoma (CIPm) cells. METHODS: To perform MTT tests, cell lines were seeded at a density of 3000 cells per well and incubated with sevoflurane (1, 2.5 or 4 mM) or only with the culture medium (control). Sevoflurane was added to the cell cultures every hour to avoid changes in drug concentration. MTT assays were performed after 6 hours of exposure obtaining absolute values of absorbance. The RNA isolated from the lysates of the same cell lines underwent quantitative polymerase chain reaction to evaluate NET1 gene expression changes compared with controls. One- or two-way analysis of variance was used as appropriate (p < 0.05). RESULTS: A significant increase in cell proliferation compared with controls was observed in CIPp treated with lower sevoflurane concentrations, whereas a significant decrease in cell proliferation was found in CIPm treated with all the sevoflurane concentrations. All CIPp treatments did not induce changes in gene expression compared with controls, whereas a significant increase in gene expression was observed in CIPm between controls and the higher sevoflurane concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Sevoflurane treatments modified the cell proliferation rate in both cell lines showing an increase or decrease when applied to CIPp or CIPm, respectively. Expression of the NET1 gene increased after treatment with sevoflurane 4 mM in metastatic cells. The role of sevoflurane on cancer recurrence should be further investigated.
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Adenocarcinoma/veterinaria , Anestésicos/farmacología , Proliferación Celular/efectos de los fármacos , Enfermedades de los Perros/patología , Neoplasias Mamarias Animales/patología , Proteínas Oncogénicas/genética , Sevoflurano/farmacología , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Colorimetría/veterinaria , Perros , Femenino , Expresión Génica/efectos de los fármacos , Neoplasias Mamarias Animales/genética , Estudios ProspectivosRESUMEN
BACKGROUND: The RhoA activating protein Net1 contributes to breast cancer cell proliferation, motility, and invasion in vitro, yet little is known about its roles in mammary gland tumorigenesis and metastasis. METHODS: Net1 knockout (KO) mice were bred to mice with mammary gland specific expression of the polyoma middle T antigen (PyMT) oncogene. Mammary gland tumorigenesis and lung metastasis were monitored. Individual tumors were assessed for proliferation, apoptosis, angiogenesis, RhoA activation, and activation of PyMT-dependent signaling pathways. Primary tumor cells from wild-type and Net1 KO mice were transplanted into the mammary glands of wild-type, nontumor-bearing mice, and tumor growth and metastasis were assessed. Gene expression in wild-type and Net1 KO tumors was analyzed by gene ontology enrichment and for relative activation of gene expression signatures indicative of signaling pathways important for breast cancer initiation and progression. A gene expression signature indicative of Net1 function was identified. Human breast cancer gene expression profiles were screened for the presence of a Net1 gene expression signature. RESULTS: We show that Net1 makes fundamental contributions to mammary gland tumorigenesis and metastasis. Net1 deletion delays tumorigenesis and strongly suppresses metastasis in PyMT-expressing mice. Moreover, we observe that loss of Net1 reduces cancer cell proliferation, inhibits tumor angiogenesis, and promotes tumor cell apoptosis. Net1 is required for maximal RhoA activation within tumors and for primary tumor cell motility. Furthermore, the ability of PyMT to initiate oncogenic signaling to ERK1/2 and PI3K/Akt1 is inhibited by Net1 deletion. Primary tumor cell transplantation indicates that the reduction in tumor angiogenesis and lung metastasis observed upon Net1 deletion are tumor cell autonomous effects. Using a gene expression signature indicative of Net1 activity, we show that Net1 signaling is activated in 10% of human breast cancers, and that this correlates with elevated proliferation and PI3K pathway activity. We also demonstrate that human breast cancer patients with a high Net1 gene expression signature experience shorter distant metastasis-free survival. CONCLUSIONS: These data indicate that Net1 is required for tumor progression in the PyMT mouse model and suggest that Net1 may contribute to breast cancer progression in humans.
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Antígenos Transformadores de Poliomavirus/genética , Neoplasias de la Mama/genética , Neoplasias Pulmonares/genética , Neoplasias Mamarias Animales/genética , Proteínas Oncogénicas/genética , Animales , Neoplasias de la Mama/patología , Carcinogénesis/genética , Movimiento Celular , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Glándulas Mamarias Humanas/patología , Neoplasias Mamarias Animales/patología , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Dysregulation of neuroepithelial transforming gene-1 (NET-1) has been shown in hepatocellular carcinoma (HCC) patients. We aimed to evaluate the influence of NET-1 on HCC invasion, adhesion and growth. In vitro cellular functional assays including invasion and adhesion were performed to evaluate the effects of knockdown and overexpression of NET-1. HCC cells were transplanted into nude mice, and tumor growth was assessed. BAX, caspase 3, caspase 8 and BCL2 protein levels were detected by western blot. After transfection with NET-1 siRNA, NET-1 positive ratio in HCC cells significantly decreased. Cell invasion and adhesion assay showed that knockdown of NET-1 reduced the invasion and adhesion ability of HCC cells, whereas overexpression of NET-1 increased the ability. The evaluation of tumor growth revealed that NET-1 knockdown significantly decreased tumor volume and weight, while NET-1 overexpression promoted tumor growth in nude mice. Western blot showed that NET-1 knockdown increased BAX, caspase 3 and caspase 8 expression but decreased BCL2 expression, whereas NET-1 overexpression significantly down-regulated BAX, caspase 3 and caspase 8 expression but increased BCL2 expression. Our data suggest that NET-1 promotes HCC invasion, adhesion and growth by regulating BAX, caspase 3, caspase 8 and BCL2 expression.
Asunto(s)
Carcinoma Hepatocelular/fisiopatología , Caspasa 3/genética , Caspasa 8/genética , Neoplasias Hepáticas/fisiopatología , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína X Asociada a bcl-2/genética , Western Blotting , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/genética , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Adhesión Celular/genética , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Net1 isoform A (Net1A) is a RhoA GEF that is required for cell motility and invasion in multiple cancers. Nuclear localization of Net1A negatively regulates its activity, and we have recently shown that Rac1 stimulates Net1A relocalization to the plasma membrane to promote RhoA activation and cytoskeletal reorganization. However, mechanisms controlling the subcellular localization of Net1A are not well understood. Here, we show that Net1A contains two nuclear localization signal (NLS) sequences within its N-terminus and that residues surrounding the second NLS sequence are acetylated. Treatment of cells with deacetylase inhibitors or expression of active Rac1 promotes Net1A acetylation. Deacetylase inhibition is sufficient for Net1A relocalization outside the nucleus, and replacement of the N-terminal acetylation sites with arginine residues prevents cytoplasmic accumulation of Net1A caused by deacetylase inhibition or EGF stimulation. By contrast, replacement of these sites with glutamine residues is sufficient for Net1A relocalization, RhoA activation and downstream signaling. Moreover, the N-terminal acetylation sites are required for rescue of F-actin accumulation and focal adhesion maturation in Net1 knockout MEFs. These data indicate that Net1A acetylation regulates its subcellular localization to impact on RhoA activity and actin cytoskeletal organization.
Asunto(s)
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas Oncogénicas/metabolismo , Acetilación , Transporte Activo de Núcleo Celular/fisiología , Animales , Membrana Celular/genética , Núcleo Celular/genética , Citoesqueleto/genética , Células HeLa , Humanos , Ratones , Ratones Noqueados , Neuropéptidos/genética , Neuropéptidos/metabolismo , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Proteínas Oncogénicas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD) often coexist and shared some genetic influences. Evidence from the existing literature indicated that comorbid with ODD may increase the heterogeneity of ADHD genetics. Our present study sought to investigate the role of norepinephrine transporter gene (NET1) for ADHD comorbid with ODD. METHODS: Six single nucleotide polymorphisms (SNPs) of NET1 were genotyped for a total of 1,815 ADHD cases, including 587 subjects (32.3%) with ODD. Chi-square tests were conducted for pseudo case-control study comparing allelic and genotypic distributions between ADHD with and without ODD. Among them, there were 1,249 probands together with their parents composing trios for family-based association studies using transmission disequilibrium tests (TDTs). In addition, 1,337 ADHD probands have detailed information of ODD symptoms and were included for quantitative analyses with genotypes using analyses of covariance (ANCOVA). To consider the overlap and correlation of other comorbidities with ODD and eliminate their potential confounding effect, we further repeated above analyses for 'pure ADHD+ODD' versus 'ADHD-only' after excluding other comorbidities except for ODD. RESULTS: The pseudo case-control study showed different allelic and genotypic distributions of SNP rs3785143 between ADHD with ODD and those without ODD. Family-based association tests indicated overtransmission of the T allele of rs3785143 in ADHD with ODD trios, but no biased transmission in those without ODD. ANCOVA showed association between genotypes of rs3785143 with ODD symptoms in ADHD probands, especially with 'Argumentative/Defiant Behavior (ADB)' dimension after controlling gender, age, clinical subtypes and intelligence. Above association still existed after removing the samples with other comorbidities. CONCLUSION: NET1 was associated with comorbidity of ODD and ODD symptoms in ADHD probands. Our findings emphasize the importance of considering the comorbidity of ODD in ADHD genetic studies, especially ADHD with ADB. However, further replication in independent sample or different populations is still needed.
Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/genética , Déficit de la Atención y Trastornos de Conducta Disruptiva/genética , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/genética , Adolescente , Adulto , Trastorno por Déficit de Atención con Hiperactividad/epidemiología , Déficit de la Atención y Trastornos de Conducta Disruptiva/epidemiología , Niño , Comorbilidad , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Padres , Polimorfismo de Nucleótido SimpleRESUMEN
A significant number of the genetic alterations observed in cancer patients lie within nonprotein-coding segments of the genome, including regions coding for long noncoding RNAs (lncRNAs). LncRNAs display aberrant expression in breast cancer (BrCa), but the functional implications of this altered expression remain to be elucidated. By performing transcriptome screen in a triple negative BrCa (TNBC) isogenic 2D and 3D spheroid model, we observed aberrant expression of >1000 lncRNAs during BrCa progression. The chromatin-associated lncRNA MANCR shows elevated expression in metastatic TNBC. MANCR is upregulated in response to cellular stress and modulates DNA repair and cell proliferation. MANCR promotes metastasis as MANCR-depleted cells show reduced cell migration, invasion, and wound healing in vitro, and reduced metastatic lung colonization in xenograft experiments in vivo. Transcriptome analyses reveal that MANCR modulates expression and pre-mRNA splicing of genes, controlling DNA repair and checkpoint response. MANCR promotes the transcription of NET1A, a Rho-GEF that regulates DNA damage checkpoint and metastatic processes in cis, by differential promoter usage. Experiments suggest that MANCR regulates the expression of cancer-associated genes by modulating the association of various transcription factors and RNA-binding proteins. Our results identified the metastasis-promoting activities of MANCR in TNBC by cis-regulation of gene expression.
RESUMEN
Colon cancer (CC) is a common malignancy over the world and its morbidity and mortality significantly went up in China in recent years. Molecular functions in cancers have gradually been the pivot subject in cancer research. Neuroepithelial cell transforming 1 (NET1) was reported to contribute to prostate cancer and gastric cancer. Our study figured out that NET1 was overexpressed in CC cells. Then, loss-of-function assays revealed that NET1 facilitated CC cell proliferation and repressed CC cell apoptosis. Next, miR-338-3p was confirmed to target NET1. After that, we verified that circ_0017552 which originates from NET1 could positively modulate NET1 expression. Besides, circ_0017552 was a sponge of miR-338-3p. Rescue assays' results demonstrated that circ_0017552 could regulate CC cell proliferation and apoptosis through up-regulation of NET1. A transcription factor named Sp1 (SP1) was found to be present in circ_0017552. SP1 induced transcription of circ_0017552 to facilitate CC cell proliferation and inhibit CC cell apoptosis. In a word, SP1-induced circ_0017552 regulated CC cell proliferation and apoptosis through miR-338-3p/NET1 axis.