RESUMEN
Germ cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell-intrinsic ß-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in ß-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of ß-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of Znrf3, a negative regulator of WNT/ß-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that ß-catenin is a central gatekeeper in ovarian differentiation and gametogenesis.
Asunto(s)
Diferenciación Celular , Células Germinativas/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Femenino , Células Germinativas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/metabolismo , Proteínas Wnt/genética , beta Catenina/genéticaRESUMEN
Glioblastomas are the most common primary brain tumors, highly vascularized, infiltrating, and resistant to current therapies. This cancer leads to a fatal outcome in less than 18 months. The aggressive behavior of glioblastomas, including resistance to current treatments and tumor recurrence, has been attributed to glioma stemlike/progenitor cells. The transcription factor EGR1 (early growth response 1), a member of a zinc finger transcription factor family, has been described as tumor suppressor in gliomas when ectopically overexpressed. Although EGR1 expression in human glioblastomas has been associated with patient survival, its precise location in tumor territories as well as its contribution to glioblastoma progression remain elusive. In the present study, we show that EGR1-expressing cells are more frequent in high grade gliomas where the nuclear expression of EGR1 is restricted to proliferating/progenitor cells. We show in primary cultures of glioma stemlike cells that EGR1 contributes to stemness marker expression and proliferation by orchestrating a PDGFA-dependent growth-stimulatory loop. In addition, we demonstrate that EGR1 acts as a positive regulator of several important genes, including SHH, GLI1, GLI2, and PDGFA, previously linked to the maintenance and proliferation of glioma stemlike cells.
Asunto(s)
Comunicación Autocrina , Neoplasias Encefálicas/metabolismo , Proliferación Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Neoplasias Encefálicas/patología , Femenino , Glioblastoma/patología , Humanos , Masculino , Células Madre Neoplásicas/patología , Células Tumorales CultivadasRESUMEN
Cell populations with differing proliferative, stem-like and tumorigenic states co-exist in most tumors and especially malignant gliomas. Whether metabolic variations can drive this heterogeneity by controlling dynamic changes in cell states is unknown. Metabolite profiling of human adult glioblastoma stem-like cells upon loss of their tumorigenicity revealed a switch in the catabolism of the GABA neurotransmitter toward enhanced production and secretion of its by-product GHB (4-hydroxybutyrate). This switch was driven by succinic semialdehyde dehydrogenase (SSADH) downregulation. Enhancing GHB levels via SSADH downregulation or GHB supplementation triggered cell conversion into a less aggressive phenotypic state. GHB affected adult glioblastoma cells with varying molecular profiles, along with cells from pediatric pontine gliomas. In all cell types, GHB acted by inhibiting α-ketoglutarate-dependent Ten-eleven Translocations (TET) activity, resulting in decreased levels of the 5-hydroxymethylcytosine epigenetic mark. In patients, low SSADH expression was correlated with high GHB/α-ketoglutarate ratios, and distinguished weakly proliferative/differentiated glioblastoma territories from proliferative/non-differentiated territories. Our findings support an active participation of metabolic variations in the genesis of tumor heterogeneity.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Carcinogénesis/metabolismo , Glioma/metabolismo , Hidroxibutiratos/metabolismo , Células Madre Neoplásicas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Anciano , Animales , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/cirugía , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/cirugía , Carcinogénesis/patología , Muerte Celular/fisiología , Proliferación Celular/fisiología , Niño , Preescolar , Femenino , Glioma/patología , Glioma/cirugía , Humanos , Masculino , Ratones Desnudos , Persona de Mediana Edad , Trasplante de Neoplasias , Células Madre Neoplásicas/patología , Succionato-Semialdehído Deshidrogenasa/metabolismoRESUMEN
Stem cell-like properties of glioma initiating cells (GiCs) fuel glioblastoma (GBM) development by providing the different cell types that comprise the tumor. It is therefore likely that the molecular circuitries that regulate their decision to self-renew or commit to a more differentiated state may offer targets for future innovative therapies. In previous micro-RNA profiling studies to search for regulators of stem cell plasticity, we identified miR-18a* as a potential candidate and its expression correlated with the stemness state. Here, using human GiCs we found that miR-18a* expression promotes clonal proliferation in vitro and tumorigenicity in vivo. Mechanistically, ERK-dependent induction of miR-18a* directly represses expression of DLL3, an autocrine inhibitor of NOTCH, thus enhancing the level of activated NOTCH-1. Activated NOTCH-1 in turn is required for sustained ERK activation. This feed-forward loop, driven by miR-18a*, is required to turn on the SHH-GLI-NANOG network, essential for GiC self-renewal. Hence, by tightly regulating expression of DLL3, miR-18a* constitutes an important signaling mediator for fine tuning the level of GiC self-renewal.
Asunto(s)
Glioma/genética , MicroARNs/genética , Receptor Notch1/metabolismo , Anciano , Animales , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Regulación hacia Abajo , Glioma/metabolismo , Glioma/patología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos NOD , MicroARNs/biosíntesis , MicroARNs/metabolismo , Persona de Mediana Edad , Receptor Notch1/genética , TransfecciónRESUMEN
Glioblastomas (GBs) are incurable brain tumors. The persistence of aggressive stem-like tumor cells after cytotoxic treatments compromises therapeutic efficacy, leading to GBM recurrence. Forcing the GBM cells to irreversibly abandon their aggressive stem-like phenotype may offer an alternative to conventional cytotoxic treatments. Here, we show that the RNA binding protein CELF2 is strongly expressed in mitotic and OLIG2-positive GBM cells, while it is downregulated in differentiated and non-mitotic cells by miR-199a-3p, exemplifying GBM intra-tumor heterogeneity. Using patient-derived cells and human GBM samples, we demonstrate that CELF2 plays a key role in maintaining the proliferative/OLIG2 cell phenotype with clonal and tumorigenic properties. Indeed, we show that CELF2 deficiency in patient-derived GSCs drastically reduced tumor growth in the brains of nude mice. We further show that CELF2 promotes TRIM28 and G9a expression, which drive a H3K9me3 epigenetic profile responsible for the silencing of the SOX3 gene. Thus, CELF2, which is positively correlated with OLIG2 and Ki67 expression in human GBM samples, is inversely correlated with SOX3 and miR-199a-3p. Accordingly, the invalidation of SOX3 in CELF2-deficient patient-derived cells rescued proliferation and OLIG2 expression. Finally, patients expressing SOX3 above the median level of expression tend to have a longer life expectancy. CELF2 is therefore a crucial target for the malignant potential of GBM and warrants attention when developing novel anticancer strategies.
RESUMEN
Glioblastomas (GBM) are heterogeneous tumors with high metabolic plasticity. Their poor prognosis is linked to the presence of glioblastoma stem cells (GSC), which support resistance to therapy, notably to temozolomide (TMZ). Mesenchymal stem cells (MSC) recruitment to GBM contributes to GSC chemoresistance, by mechanisms still poorly understood. Here, we provide evidence that MSCs transfer mitochondria to GSCs through tunneling nanotubes, which enhances GSCs resistance to TMZ. More precisely, our metabolomics analyses reveal that MSC mitochondria induce GSCs metabolic reprograming, with a nutrient shift from glucose to glutamine, a rewiring of the tricarboxylic acid cycle from glutaminolysis to reductive carboxylation and increase in orotate turnover as well as in pyrimidine and purine synthesis. Metabolomics analysis of GBM patient tissues at relapse after TMZ treatment documents increased concentrations of AMP, CMP, GMP, and UMP nucleotides and thus corroborate our in vitro analyses. Finally, we provide a mechanism whereby mitochondrial transfer from MSCs to GSCs contributes to GBM resistance to TMZ therapy, by demonstrating that inhibition of orotate production by Brequinar (BRQ) restores TMZ sensitivity in GSCs with acquired mitochondria. Altogether, these results identify a mechanism for GBM resistance to TMZ and reveal a metabolic dependency of chemoresistant GBM following the acquisition of exogenous mitochondria, which opens therapeutic perspectives based on synthetic lethality between TMZ and BRQ. Significance: Mitochondria acquired from MSCs enhance the chemoresistance of GBMs. The discovery that they also generate metabolic vulnerability in GSCs paves the way for novel therapeutic approaches.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Células Madre Mesenquimatosas , Humanos , Glioblastoma/tratamiento farmacológico , Resistencia a Antineoplásicos , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Temozolomida/farmacología , Mitocondrias , Células Madre NeoplásicasRESUMEN
Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/terapia , Glioblastoma/tratamiento farmacológico , Microambiente Tumoral/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/patologíaRESUMEN
Cell motility is critical for tumor malignancy. Metabolism being an obligatory step in shaping cell behavior, we looked for metabolic weaknesses shared by motile cells across the diverse genetic contexts of patients' glioblastoma. Computational analyses of single-cell transcriptomes from thirty patients' tumors isolated cells with high motile potential and highlighted their metabolic specificities. These cells were characterized by enhanced mitochondrial load and oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). Functional assays with patients' tumor-derived cells and -tissue organoids, and genetic and pharmacological manipulations confirmed that the cells depend on enhanced ROS production and MPST activity for their motility. MPST action involved protection of protein cysteine residues from damaging hyperoxidation. Its knockdown translated in reduced tumor burden, and a robust increase in mice survival. Starting from cell-by-cell analyses of the patients' tumors, our work unravels metabolic dependencies of cell malignancy maintained across heterogeneous genomic landscapes.
Asunto(s)
Glioblastoma , Ratones , Animales , Glioblastoma/genética , Cisteína/metabolismo , Sulfurtransferasas/genética , Sulfurtransferasas/metabolismo , Estrés Oxidativo , Movimiento Celular/genéticaRESUMEN
Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Sistema Nervioso/citología , Neuronas/citología , Técnicas de Cultivo de Tejidos/métodos , Línea Celular , Electrofisiología , Células Madre Embrionarias/fisiología , Células Madre Embrionarias/ultraestructura , Humanos , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Sistema Nervioso/ultraestructura , Neuronas/ultraestructura , Reacción en Cadena de la PolimerasaRESUMEN
There is great interest in understanding how the cancer stem cell population may be maintained in solid tumors. Here, we show that tumor cells exhibiting stem-like properties and expression of pluripotency markers NANOG and OCT4 can arise from original differentiated tumor cells freshly isolated from human glioblastomas (GBM) and that have never known any serum culture conditions. Induction of EGR1 by EGFR/ERK signaling promoted cell conversion from a less aggressive, more differentiated cellular state to a self-renewing and strongly tumorigenic state, expressing NANOG and OCT4. Expression of these pluripotency markers occurred before the cells re-entered the cell cycle, demonstrating their capacity to change and dedifferentiate without any cell divisions. In differentiated GBM cells, ERK-mediated repression of miR-199a-3p induced EGR1 protein expression and triggered dedifferentiation. Overall, this signaling pathway constitutes an ERK-mediated "toggle switch" that promotes pluripotency marker expression and stem-like features in GBM cells. SIGNIFICANCE: This study defines an ERK-mediated molecular mechanism of dedifferentiation of GBM cells into a stem-like state, expressing markers of pluripotency.See related commentary by Koncar and Agnihotri, p. 3195.
Asunto(s)
Glioblastoma , MicroARNs , Desdiferenciación Celular , Diferenciación Celular , Proteína 1 de la Respuesta de Crecimiento Precoz , Glioblastoma/genética , Humanos , MicroARNs/genética , Proteína Homeótica Nanog/genética , Células Madre NeoplásicasRESUMEN
Ubiquitylation of RhoA has emerged as an important aspect of both the virulence of Escherichia coli producing cytotoxic necrotizing factor (CNF) 1 toxin and the establishment of the polarity of eukaryotic cells. Owing to the molecular activity of CNF1, we have investigated the relationship between permanent activation of RhoA catalyzed by CNF1 and subsequent ubiquitylation of RhoA by Smurf1. Using Smurf1-deficient cells and by RNA interference (RNAi)-mediated Smurf1 knockdown, we demonstrate that Smurf1 is a rate-limiting and specific factor of the ubiquitin-mediated proteasomal degradation of activated RhoA. We further show that the cancer cell lines HEp-2, human embryonic kidney 293 and Vero are specifically deficient in ubiquitylation of either activated Rac, Cdc42, or Rho, respectively. In contrast, CNF1 produced the cellular depletion of all three isoforms of Rho proteins in the primary human cell types we have tested. We demonstrate that ectopic expression of Smurf1 in Vero cells, deficient for RhoA ubiquitylation, restores ubiquitylation of the activated forms of RhoA. We conclude here that Smurf1 ubiquitylates activated RhoA and that, in contrast to human primary cell types, some cancer cell lines have a lower ubiquitylation capacity of specific Rho proteins. Thus, both CNF1 and transforming growth factor-beta trigger activated RhoA ubiquitylation through Smurf1 ubiquitin-ligase.
Asunto(s)
Toxinas Bacterianas/farmacología , Proteínas de Escherichia coli/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Línea Celular , Humanos , Riñón , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitina/efectos de los fármacos , Ubiquitina-Proteína Ligasas/genética , Proteína de Unión al GTP rhoA/efectos de los fármacosRESUMEN
BACKGROUND: The impact of bariatric surgery (BS) on the sexual functioning of patients is poorly studied. Our aim was to analyze the sexual function, depressive symptoms, and self-esteem of morbidly obese women (MOW) undergoing BS. PATIENTS AND METHODS: Quality of sexual life was prospectively evaluated in 43 consecutive MOW (18-50 years) who underwent BS. Female sexual function index (FSFI), Beck depression inventory (BDI), and Rosenberg self-esteem scale (RSES) questionnaires were administered to evaluate sexual satisfaction, depressive symptoms, and self-esteem, respectively. A control group of 36 healthy, non-obese, female patients (HW) was recruited for comparison. Results of questionnaires were compared between three periods (before BS and at 3- and 6-month follow-up) and between MOW and HW. RESULTS: Before BS, the FSFI score was significantly lower in MOW compared to HW (17 ± 12 vs 27 ± 8, p = 0.0001) while at 3- and 6-month post-BS, a significant amelioration (p = 0.01) occurred. In particular, after BS, all components of the FSFI score (sexual desire, excitement, lubrification, orgasm, satisfaction, and pain) were ameliorated. The pre-BS BDI score was higher in MOW than in HW (8 ± 6 vs 5 ± 5, p = 0.004) while at postoperative months 3 and 6, a significant amelioration was found (p = 0.025 and 0.005, respectively). Before BS, no significant differences occurred in the RSES score between MOW and HW (30 ± 7 vs 32 ± 6, p = 0.014), whereas the MOW RSES scores at 6-month post-BS were improved when compared with the HW RSES scores. CONCLUSIONS: BS results in a significant improvement in the quality of sexual life, depressive symptoms, and self-esteem in MOW.
Asunto(s)
Cirugía Bariátrica , Obesidad Mórbida/cirugía , Adulto , Depresión/psicología , Femenino , Humanos , Estudios Longitudinales , Persona de Mediana Edad , Obesidad Mórbida/fisiopatología , Obesidad Mórbida/psicología , Estudios Prospectivos , Escalas de Valoración Psiquiátrica , Calidad de Vida , Autoimagen , Conducta Sexual/fisiología , Conducta Sexual/psicología , Encuestas y Cuestionarios , Adulto JovenRESUMEN
BACKGROUND: Obesity is a well-known risk factor for female pelvic floor disorders (PFD). This study assessed the effects of bariatric surgery (BS) on pelvic organ prolapse symptoms (POPs) and urinary (UI) and anal incontinence (AI) in morbidly obese women undergoing either sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB). METHODS: Morbidly obese women undergoing BS from June 2016 to May 2017 were prospectively included. POPs, UI, and AI were compared at baseline and at 1 year after surgery using validated questionnaires. RESULTS: Seventy-two consecutive women were enrolled, 54 (75%) (30 (56%) RYBP and 24 (44%) SG) completed the study at 1 year and were considered for the final analysis. The mean age and mean preoperative BMI were 43 ± 11.8 years (range, 20-65) and 41 ± 5.4 kg/m2 (range, 35-56), respectively. At baseline, 30 (56%), 32 (59%), and 27 (50%) patients, respectively, had AI (flatus only 72%), UI, and POPs. The mean TBWL% at 1 year was 33%. In the whole study population, weight loss was associated with a significant improvement in UI (p < 0.001) but there was no significant difference in terms of AI and POPs. In the subgroups analysis, AI increased significantly 1 year after the RYGB (p = 0.02) due to an increase in flatus incontinence (p = 0.04). No significant difference in AI was found 1 year after the SG. CONCLUSION: BS is associated with a significant improvement in UI but not in POPs. RYBP seems to increase AI, mainly flatus incontinence, compared to SG.
Asunto(s)
Incontinencia Fecal/cirugía , Gastrectomía , Derivación Gástrica , Prolapso de Órgano Pélvico/cirugía , Incontinencia Urinaria/cirugía , Adulto , Anciano , Incontinencia Fecal/etiología , Femenino , Flatulencia , Humanos , Laparoscopía , Persona de Mediana Edad , Obesidad Mórbida/complicaciones , Obesidad Mórbida/cirugía , Prolapso de Órgano Pélvico/etiología , Proyectos Piloto , Estudios Prospectivos , Incontinencia Urinaria/etiología , Adulto JovenRESUMEN
BACKGROUND: Recent evidence has indicated an increased risk of Barrett's esophagus (BE) in the long term after sleeve gastrectomy (SG). AIM: The aim of the study is to investigate the spectrum of gastroesophageal reflux disease (GERD) symptoms as well as the prevalence of BE, at minimum 5 years after SG in patients who underwent SG in different bariatric centers of two countries: France and Italy. PATIENTS AND METHODS: Five high volume outpatient centers dedicated to bariatric surgery that routinely perform upper GI endoscopy before any bariatric procedures were invited to participate in the study. From January 2017 to June 2018, each center during scheduled postoperative evaluation after surgery asked a minimum 10 consecutive patients, which had performed SG at least 5 years before and with no evidence of BE preoperatively, to undergo another upper GI endoscopy. RESULTS: Ninety (66 F) consecutive patients were enrolled. The mean follow-up was 78 ± 15 months, and the mean total body weight loss was 25 ± 12%. The prevalence of BE was 18.8% with no significant difference among centers. Weight loss failure was significantly associated with BE (p < 0.01). The prevalence of GERD symptoms, erosive esophagitis, and the usage of PPIs increased from 22%, 10%, and 22% before the SG to 76%, 41%, and 52% at the time of follow-up, respectively (p < 0.05). CONCLUSIONS: This multicenter study show a high rate of BE at least 5 years after SG. Weight loss failure was significantly associated with BE. We suggest to provide systematic endoscopy in these patients to rule out this condition.
Asunto(s)
Cirugía Bariátrica/efectos adversos , Esófago de Barrett/etiología , Gastrectomía/efectos adversos , Adulto , Cirugía Bariátrica/métodos , Esófago de Barrett/epidemiología , Endoscopía del Sistema Digestivo/métodos , Esofagitis/epidemiología , Esofagitis/etiología , Femenino , Estudios de Seguimiento , Francia/epidemiología , Gastrectomía/métodos , Reflujo Gastroesofágico/epidemiología , Reflujo Gastroesofágico/etiología , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Obesidad Mórbida/epidemiología , Obesidad Mórbida/cirugía , Úlcera Péptica/epidemiología , Úlcera Péptica/etiología , Prevalencia , Inhibidores de la Bomba de Protones/uso terapéutico , Pérdida de PesoRESUMEN
Glioblastoma (GBM) is the brain tumor with the worst prognosis composed of a cell subpopulation called Glioblastoma Stem-like Cells (GSCs) responsible for tumor recurrence mediated by cell invasion. GSCs persist in a hypoxic microenvironment which promotes extracellular adenosine production and activation of the A3 Adenosine Receptor (A3AR), therefore, the aim of this study was to determine the role of extracellular adenosine and A3AR on GSCs invasion under hypoxia. GSCs were obtained from a U87MG cell line and primary cultures of GBM patients, and then incubated under normoxia or hypoxia. Gene expression was evaluated by RNAseq, RT-qPCR, and western blot. Cell migration was measured by spreading and transwell boyden chamber assays; cell invasion was evaluated by Matrigel-coated transwell, ex vivo brain slice, and in vivo xenograft assays. The contribution of A3AR on cell migration/invasion was evaluated using the A3AR antagonist, MRS1220. Extracellular adenosine production was higher under hypoxia than normoxia, mainly by the catalytic action of the prostatic acid phosphatase (PAP), promoting cell migration/invasion in a HIF-2-dependent process. A3AR blockade decreased cell migration/invasion and the expression of Epithelial-Mesenchymal Transition markers. In conclusion, high levels of extracellular adenosine production enhance cell migration/invasion of GSCs, through HIF-2/PAP-dependent activation of A3AR under hypoxia.
Asunto(s)
Adenosina/metabolismo , Neoplasias Encefálicas/metabolismo , Movimiento Celular , Glioblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Receptor de Adenosina A3/metabolismo , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Células Madre Neoplásicas/patología , Receptor de Adenosina A3/genética , Transducción de Señal , Células Tumorales Cultivadas , Hipoxia Tumoral , Microambiente TumoralRESUMEN
Glioblastomas are incurable primary brain tumors that affect patients of all ages. The aggressiveness of this cancer has been attributed in part to the persistence of treatment-resistant glioblastoma stem-like cells. We have previously discovered the tumor-suppressor properties of the microRNA cluster miR-302-367, representing a potential treatment for glioblastoma. Here, we attempted to develop a cell-based therapy by taking advantage of the capability of glioma cells to secrete exosomes that enclose small RNA molecules. We engineered primary glioma cells to stably express the miR-302-367. Remarkably, these cells altered, in a paracrine-dependent manner, the expression of stemness markers, the proliferation and the tumorigenicity of neighboring glioblastoma cells. Further characterization of the secretome derived from miR-302-367 expressing cells showed that a large amount of miR-302-367 was enclosed in exosomes, which were internalized by the neighboring glioblastoma cells. This miR-302-367 cell-to-cell transfer resulted in the inhibition of its targets such as CXCR4/SDF1, SHH, cyclin D, cyclin A and E2F1. Orthotopic xenograft of miR-302-367-expressing cells together with glioblastoma stem-like cells efficiently altered the tumor development in mice brain.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , MicroARNs/biosíntesis , Familia de Multigenes , Proteínas de Neoplasias/biosíntesis , ARN Neoplásico/biosíntesis , Animales , Tratamiento Basado en Trasplante de Células y Tejidos , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Ratones , MicroARNs/genética , Proteínas de Neoplasias/genética , ARN Neoplásico/genéticaRESUMEN
Cell migration and extracellular matrix remodeling are two essential processes of wound healing, regulated by extracellular metalloproteinases such as matrix metalloproteinase-2 (Gelatinase A) and matrix metalloproteinase-9 (Gelatinase B). Expression of matrix metalloproteinase-9 is deregulated in numerous wound healing pathologies. To date the mechanisms regulating matrix metalloproteinase-9 during normal wound healing are poorly documented. Using both primary cultures of normal human keratinocytes and a wounding device especially designed to dissect the molecular events during the healing process in vitro, we show that matrix metalloproteinase-9 is stimulated by injury in normal human keratinocytes. This upregulation results from the mechanical stress created by injury and not from a soluble factor, secreted by wounded normal human keratinocytes. We also demonstrate that the Rho family of small GTPases, p38[MAPK] and JNK together play a key part in the signaling pathways controlling the stimulation of matrix metalloproteinase-9 in wounded cells. We provide lines of evidence indicating that in wounded keratinocytes, upregulation of matrix metalloproteinase-9 depends on two distinct pathways. The first involves Rac1 and/or Cdc42 that control the activation of p38[MAPK]. The second depends on RhoA activation that is required for stimulation of JNK.
Asunto(s)
Queratinocitos/fisiología , Metaloproteinasa 9 de la Matriz/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Piel/lesiones , Proteínas de Unión al GTP rho/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Queratinocitos/citología , Metaloproteinasa 9 de la Matriz/metabolismo , Proteína Quinasa 8 Activada por Mitógenos , ARN Mensajero/análisis , Transducción de Señal/fisiología , Piel/citología , Solubilidad , Cicatrización de Heridas/fisiología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The aim of this study was to characterize some of the molecular events stimulated in vitro in response to injury within a confluent culture of normal epidermal keratinocytes as a model to understand the mechanisms of wound healing. To this end, an original device was developed specifically designed to perform calibrated injuries of great lengths within mono-stratified or pluri-stratified keratinocyte cultures. The experiments performed in this study validate this device as an appropriate tool for studying epidermal wound healing; this is because it performs mechanical injuries that stimulate the expression of multiple healing markers also known to be upregulated during wound healing in vivo (growth factors, cytokines, proteinases, extracellular matrix proteins). Using this device, it was demonstrated in human keratinocytes: mechanical injuries (i) immediately stimulate the tyrosine phosphorylation of numerous cellular proteins; (ii) induce molecular cascades leading to the activation of p21ras, mitogen-activated protein kinases, extracellular signal-regulated kinases 1/2, c-Jun NH2 terminal kinase, and p38 mitogen-activated protein kinase; and (iii) increase the phosphorylation of their respective substrates, c-jun and activator transcription factor 1. Wounding of these cells also results in increases in the DNA binding activities of several jun/fos activator protein-1 transcription factor complexes. It is important to note that the development of an appropriate wounding system was essential for performing this study, as use of a classical wounding procedure did not enable the detection of the biologic parameters reported above. In conclusion, these data indicate that using the appropriate system, it is possible to identify the signaling pathways activated in normal human keratinocyte cells after injury. In this study, it was shown that the mitogen-activated protein kinase pathways and activator protein-1 are stimulated in response to physical injury, and may be involved in regulating the expression of healing markers.
Asunto(s)
Células Epidérmicas , Queratinocitos/enzimología , Cicatrización de Heridas/fisiología , Células Cultivadas , Técnicas Citológicas/métodos , Técnicas Citológicas/normas , Epidermis/lesiones , Epidermis/fisiología , Humanos , Técnicas In Vitro , Queratinocitos/citología , Sistema de Señalización de MAP Quinasas/fisiología , Metaloendopeptidasas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Validación de Programas de Computación , Factor de Transcripción AP-1/metabolismo , Tirosina/metabolismoRESUMEN
BACKGROUND: The differential diagnosis between infiltrative glioma (IG) and benign or curable glial lesions, such as gliosis, pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor, ganglioglioma, or demyelinating disease, may be challenging for the pathologist because specific markers are lacking. Recently, we described a strong EGFR immunolabelling pattern in cells with a high nuclear to cytoplasmic ratio that enables the discrimination of low-grade IG from gliosis. The aim of this study was to extend our observation to high-grade glioma to assess whether EGFR expression pattern is of value in the discrimination of all IG from noninfiltrative glial lesions (NIG), including gliosis, benign tumors, and demyelinating disease. METHODS: One hundred one IG and 58 NIG were compared for immunohistochemical expression of EGFR with use of an antibody that recognizes an epitope in the extracellular domain of both EGFRwt and EGFRvIII. Highly EGFR-positive cells with a high nuclear to cytoplasmic ratio were isolated and further characterized. RESULTS: Cells with intense EGFR staining and a high nuclear to cytoplasmic ratio were significantly associated with the diagnosis of IG (P < .0001). The sensitivity and specificity of this staining pattern for the diagnosis of IG were 95% and 100%, respectively. EGFR expression was independent of IDH1 mutations and EGFR amplification. Finally, we showed that these particular cells displayed the phenotype and properties of glial progenitors and coexpressed CXCR4, a marker of invasiveness. CONCLUSIONS: We demonstrate that cells with intense EGFR staining and a high nuclear to cytoplasmic ratio are specific criteria for the diagnosis of IG, irrespective of grade, histological subtype, and progression pathway, and their identification represents a tool to discriminate IG from benign or curable glial lesions.