RESUMEN
Primary, glioblastoma, and secondary brain tumors, from metastases outside the brain, are among the most aggressive and therapeutically resistant cancers. A physiological barrier protecting the brain, the blood-brain barrier (BBB), functions as a deterrent to effective therapies. To enhance cancer therapy, we developed a cancer terminator virus (CTV), a unique tropism-modified adenovirus consisting of serotype 3 fiber knob on an otherwise Ad5 capsid that replicates in a cancer-selective manner and simultaneously produces a potent therapeutic cytokine, melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24). A limitation of the CTV and most other viruses, including adenoviruses, is an inability to deliver systemically to treat brain tumors because of the BBB, nonspecific virus trapping, and immune clearance. These obstacles to effective viral therapy of brain cancer have now been overcome using focused ultrasound with a dual microbubble treatment, the focused ultrasound-double microbubble (FUS-DMB) approach. Proof-of-principle is now provided indicating that the BBB can be safely and transiently opened, and the CTV can then be administered in a second set of complement-treated microbubbles and released in the brain using focused ultrasound. Moreover, the FUS-DMB can be used to deliver the CTV multiple times in animals with glioblastoma growing in their brain thereby resulting in a further enhancement in survival. This strategy permits efficient therapy of primary and secondary brain tumors enhancing animal survival without promoting harmful toxic or behavioral side effects. Additionally, when combined with a standard of care therapy, Temozolomide, a further increase in survival is achieved. The FUS-DMB approach with the CTV highlights a noninvasive strategy to treat brain cancers without surgery. This innovative delivery scheme combined with the therapeutic efficacy of the CTV provides a novel potential translational therapeutic approach for brain cancers.
Asunto(s)
Adenoviridae , Barrera Hematoencefálica , Neoplasias Encefálicas , Animales , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/virología , Humanos , Adenoviridae/genética , Interleucinas/genética , Línea Celular Tumoral , Microburbujas/uso terapéutico , Ratones , Glioblastoma/terapia , Glioblastoma/virología , Glioblastoma/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Viroterapia Oncolítica/métodos , Vectores Genéticos/administración & dosificación , Temozolomida/uso terapéutico , Ratones DesnudosRESUMEN
Glioblastoma is the most common form of primary brain cancer in adults, and the disease has a serious prognosis. Although great progress has been made in molecular characteristics, no major breakthroughs in treatment have been achieved for many years. In this article we present a clinical review of current diagnostics and treatment, as well as the challenges and opportunities inherent in developing improved and more personalised treatment.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Adulto , Glioblastoma/diagnóstico , Glioblastoma/terapia , Pronóstico , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/terapiaRESUMEN
Long non-coding RNAs play critical roles in tumour progression. Through analysis of publicly available genomic datasets, we found that MIR22HG, the host gene of microRNAs miR-22-3p and miR-22-5p, is ranked among the most dysregulated long non-coding RNAs in glioblastoma. The main purpose of this work was to determine the impact of MIR22HG on glioblastoma growth and invasion and to elucidate its mechanistic function. The MIR22HG/miR-22 axis was highly expressed in glioblastoma as well as in glioma stem-like cells compared to normal neural stem cells. In glioblastoma, increased expression of MIR22HG is associated with poor prognosis. Through a number of functional studies, we show that MIR22HG silencing inhibits the Wnt/ß-catenin signalling pathway through loss of miR-22-3p and -5p. This leads to attenuated cell proliferation, invasion and in vivo tumour growth. We further show that two genes, SFRP2 and PCDH15, are direct targets of miR-22-3p and -5p and inhibit Wnt signalling in glioblastoma. Finally, based on the 3D structure of the pre-miR-22, we identified a specific small-molecule inhibitor, AC1L6JTK, that inhibits the enzyme Dicer to block processing of pre-miR-22 into mature miR-22. AC1L6JTK treatment caused an inhibition of tumour growth in vivo. Our findings show that MIR22HG is a critical inducer of the Wnt/ß-catenin signalling pathway, and that its targeting may represent a novel therapeutic strategy in glioblastoma patients.
Asunto(s)
Glioblastoma/genética , MicroARNs/genética , Vía de Señalización Wnt/genética , beta Catenina/genética , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Glioma/genética , Masculino , Ratones Desnudos , ARN Largo no Codificante/genéticaRESUMEN
Proteolipid protein 2 (PLP2) is an integral ion channel membrane protein of the endoplasmic reticulum. The protein has been shown to be highly expressed in many cancer types, but its importance in glioma progression is poorly understood. Using publicly available datasets (Rembrandt, TCGA and CGGA), we found that the expression of PLP2 was significantly higher in high-grade gliomas than in low-grade gliomas. We confirmed these results at the protein level through IHC staining of high-grade (n = 56) and low-grade glioma biopsies (n = 16). Kaplan-Meier analysis demonstrated that increased PLP2 expression was associated with poorer patient survival. In functional experiments, siRNA and shRNA PLP2 knockdown induced ER stress and increased apoptosis and autophagy in U87 and U251 glioma cell lines. Inhibition of autophagy with chloroquine augmented apoptotic cell death in U87- and U251-siPLP2 cells. Finally, intracranial xenografts derived from U87- and U251-shPLP2 cells revealed that loss of PLP2 reduced glioma growth in vivo. Our results therefore indicate that increased PLP2 expression promotes GBM growth and that PLP2 represents a potential future therapeutic target.
Asunto(s)
Apoptosis/genética , Autofagia/genética , Neoplasias Encefálicas/genética , Estrés del Retículo Endoplásmico/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Proteínas con Dominio MARVEL/genética , Proteolípidos/genética , Animales , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/ultraestructura , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/genética , Técnicas de Silenciamiento del Gen , Glioblastoma/ultraestructura , Humanos , Proteínas con Dominio MARVEL/metabolismo , Masculino , Ratones , Pronóstico , Proteolípidos/metabolismo , Factor de Transcripción CHOP/metabolismoRESUMEN
Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Xenoinjertos/inmunología , Organoides/patología , Temozolomida/uso terapéutico , Animales , Neoplasias Encefálicas/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioma/genética , Xenoinjertos/efectos de los fármacos , Humanos , Ratones , Recurrencia Local de Neoplasia/genética , Organoides/inmunología , Medicina de Precisión/métodos , RatasRESUMEN
Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14-15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome-wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late-stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy-lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Autofagia/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Temozolomida/administración & dosificación , Tioridazina/administración & dosificación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Autofagosomas/efectos de los fármacos , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Glioblastoma/genética , Humanos , Lisosomas/efectos de los fármacos , Ratones , Mutaciones Letales Sintéticas , Temozolomida/uso terapéutico , Tioridazina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Malignant melanoma is the most aggressive type of skin cancer and is closely associated with the development of brain metastases. Despite aggressive treatment, the prognosis has traditionally been poor, necessitating improved therapies. In melanoma, the mitogen activated protein kinase and the phosphoinositide 3-kinase signaling pathways are commonly altered, and therapeutically inhibiting one of the pathways often upregulates the other, leading to resistance. Thus, combined treatment targeting both pathways is a promising strategy to overcome this. Here, we studied the in vitro and in vivo effects of the PI3K inhibitor buparlisib and the MEK1/2 inhibitor trametinib, used either as targeted monotherapies or in combination, on patient-derived melanoma brain metastasis cell lines. Scratch wound and trans-well assays were carried out to assess the migratory capacity of the cells upon drug treatment, whereas flow cytometry, apoptosis array and Western blots were used to study apoptosis. Finally, an in vivo treatment experiment was carried out on NOD/SCID mice. We show that combined therapy was more effective than monotherapy. Combined treatment also more effectively increased apoptosis, and inhibited tumor growth in vivo. This suggests a clinical potential of combined treatment to overcome ceased treatment activity which is often seen after monotherapies, and strongly encourages the evaluation of the treatment strategy on melanoma patients with brain metastases.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Encefálicas/prevención & control , Melanoma/tratamiento farmacológico , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológico , Aminopiridinas/administración & dosificación , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Melanoma/metabolismo , Melanoma/patología , Ratones Endogámicos NOD , Ratones SCID , Morfolinas/administración & dosificación , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Low-grade, pilocytic astrocytomas are treated by resection, but additional therapy is necessary for those tumors with anaplastic features. Arsenic trioxide (As2O3) is emerging as an effective chemotherapeutic agent for treatment of malignant glioblastoma multiforme, where Cathepsin L silencing enables lower, less harmful As2O3 concentrations to achieve the desired cytotoxic effect. Here, we evaluated the effects of As2O3 combined with stable Cathepsin L shRNA silencing on cell viability/metabolic activity, and apoptosis in primary cultures of recurrent malignantly transformed pilocytic astrocytoma (MPA). These cells expressed high Cathepsin L levels, and when grown as monolayers and spheroids, they were more resistant to As2O3 than the U87MG glioblastoma cell line. Caspases 3/7 activity in MPA58 spheroids was not significantly affected by As2O3, possibly due to higher chemoresistance of primary biopsy tissue of less malignant astrocytoma versus the malignant U87MG cell line. However, As2O3 treatment was cytotoxic to MPA spheroids after silencing of Cathepsin L expression. While Cathepsin L silencing only slightly decreased the live/dead cell ratio in As2O3-treated MPA-si spheroids under our experimental conditions, there was an increase in As2O3-mediated apoptosis in MPA-si spheroids, as indicated by elevated caspases 3/7 activity. Therefore, Cathepsin L silencing by gene manipulation can be applied when a more aggressive approach is needed in treatment of pilocytic astrocytomas with anaplastic features.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Astrocitoma/tratamiento farmacológico , Neoplasias Encefálicas/tratamiento farmacológico , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Catepsina L/genética , Óxidos/farmacología , Neoplasias de la Médula Espinal/tratamiento farmacológico , Animales , Apoptosis/genética , Trióxido de Arsénico , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Activación Enzimática/inmunología , Glioblastoma/tratamiento farmacológico , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Óxidos/toxicidad , Interferencia de ARN , ARN Interferente Pequeño/genética , Esferoides Celulares , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future analysis in clinical GBM samples.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Glioblastoma/genética , Proteínas de Neoplasias/biosíntesis , Neovascularización Patológica/genética , Proteómica , Animales , Apoptosis/efectos de los fármacos , Bevacizumab/administración & dosificación , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Masculino , Ratones , Proteínas de Neoplasias/genética , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.
Asunto(s)
Neoplasias Encefálicas/genética , Redes Reguladoras de Genes/genética , Glioma/genética , Invasividad Neoplásica/genética , Proteínas de Unión al GTP rho/genética , Apoptosis/genética , Neoplasias Encefálicas/patología , Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Variaciones en el Número de Copia de ADN , Regulación Neoplásica de la Expresión Génica/genética , Glioma/patología , Células HEK293 , Humanos , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
BACKGROUND: Gene therapeutic strategies with suicide genes are currently investigated in clinical trials for brain tumors. Previously, we have shown that lentiviral vectors delivering the suicide gene HSV-Tk to experimental brain tumors promote a highly significant treatment effect and thus are promising vectors for clinical translation. METHODS: In the present study, we tested lentiviral vectors delivering the suicide gene HSV-Tk.007, a highly active mutant of HSV-Tk, to rat brains as a preclinical toxicity study. We injected 10(6) vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped functional lentiviral particles harboring the suicide gene HSV-Tk.007 into the brain of healthy, immunocompetent rats. During prodrug treatment with ganciclovir (GCV), we measured weight and assessed the behavior of the rats in an open field test. After 14 days of GCV treatment, we analyzed HSV-Tk.007 expression in different brain cell populations, as well as inflammatory responses and apoptosis. RESULTS: During prodrug treatment with GCV, behavior experiments did not reveal differences between the treated rats and the control groups. Analysis of HSV-Tk expression in different brain cell populations showed that transduced normal brain cells survived GCV treatment. There were no statistically significant differences in the number of transduced cells between treatment and control groups. Furthermore, inflammatory responses and apoptosis of brain cells were not observed. CONCLUSIONS: We show that HSV-Tk.007-mediated suicide gene therapy is not toxic to normal brain cells. This observation is of high relevance for the translation of lentivirus-mediated suicide gene therapies into the clinic for the treatment of brain tumor patients. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Encéfalo/metabolismo , Genes Transgénicos Suicidas/genética , Terapia Genética/métodos , Simplexvirus/enzimología , Timidina Quinasa/metabolismo , Animales , Antivirales/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Encéfalo/citología , Encéfalo/efectos de los fármacos , Línea Celular Tumoral , Ganciclovir/farmacología , Humanos , Lentivirus/genética , Microscopía Confocal , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Mutación , Ratas , Simplexvirus/genética , Timidina Quinasa/genéticaRESUMEN
Intercellular communication between cancer cells, especially between cancer and stromal cells, plays an important role in disease progression. We examined the intercellular transfer of organelles and proteins in vitro and in vivo and the role of tunneling nanotubes (TNTs) in this process. TNTs are membrane bridges that facilitate intercellular transfer of organelles of unclear origin. Using 3-dimensional quantitative and qualitative confocal microscopy, we showed that TNTs contain green fluorescent protein (GFP)-early endosome antigen (EEA) 1, GFP Rab5, GFP Rab11, GFP Rab8, transferrin (Tf), and Tf receptor (Tf-R) fused to mCherry (Tf-RmCherry). Tf-RmCherry was transferred between cancer cells by a contact-dependent but secretion-independent mechanism. Live cell imaging showed TNT formation preceding the transfer of Tf-RmCherry and involving the function of the small guanosine triphosphatase (GTPase) Rab8, which colocalized with Tf-RmCherry in the TNTs and was cotransferred to acceptor cells. Tf-RmCherry was transferred from cancer cells to fibroblasts, a noteworthy finding that suggests that this process occurs between tumor and stromal cells in vivo. We strengthened this hypothesis in a xenograft model of breast cancer using enhanced (e)GFP-expressing mice. Tf-RmCherry transferred from tumor to stromal cells and this process correlated with an increased opposite transfer of eGFP from stromal to tumor cells, together pointing toward complex intercellular communication at the tumor site.
Asunto(s)
Neoplasias de la Mama/metabolismo , Fibroblastos/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Transferrina/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Neoplasias de la Mama/genética , Fibroblastos/patología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microscopía Confocal , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Transporte de Proteínas/genética , Receptores de Transferrina/genética , Células del Estroma/metabolismo , Células del Estroma/patología , Proteínas de Unión al GTP rab/genéticaRESUMEN
Diffuse gliomas comprise a group of primary brain tumors that originate from glial (precursor) cells and present as a variety of malignancy grades which have in common that they grow by diffuse infiltration. This phenotype complicates treatment enormously as it precludes curative surgery and radiotherapy. Furthermore, diffusely infiltrating glioma cells often hide behind a functional blood-brain barrier, hampering delivery of systemically administered therapeutic and diagnostic compounds to the tumor cells. The present review addresses the biological mechanisms that underlie the diffuse infiltrative phenotype, knowledge of which may improve treatment strategies for this disastrous tumor type. The invasive phenotype is specific for glioma: most other brain tumor types, both primary and metastatic, grow as delineated lesions. Differences between the genetic make-up of glioma and that of other tumor types may therefore help to unravel molecular pathways, involved in diffuse infiltrative growth. One such difference concerns mutations in the NADP(+)-dependent isocitrate dehydrogenase (IDH1 and IDH2) genes, which occur in >80% of cases of low grade glioma and secondary glioblastoma. In this review we present a novel hypothesis which links IDH1 and IDH2 mutations to glutamate metabolism, possibly explaining the specific biological behavior of diffuse glioma.
Asunto(s)
Neoplasias Encefálicas/patología , Quimiotaxis , Glioma/patología , Ácido Glutámico/fisiología , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Transformación Celular Neoplásica/genética , Quimiotaxis/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Glioma/tratamiento farmacológico , Glioma/genética , Ácido Glutámico/farmacología , Glutamina/metabolismo , Humanos , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismoRESUMEN
Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing (13)C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of (13)C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, L-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Adulto , Anciano , Animales , Bevacizumab , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Neoplasias Encefálicas/diagnóstico por imagen , Femenino , Glioblastoma/diagnóstico por imagen , Glutamina/metabolismo , Glutatión/metabolismo , Glucólisis/efectos de los fármacos , Humanos , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/metabolismo , Masculino , Ratones SCID , Ratones Transgénicos , Persona de Mediana Edad , Trasplante de Neoplasias , Estrés Oxidativo/efectos de los fármacos , Cintigrafía , Ratas DesnudasRESUMEN
Arsenic in the form of arsenic trioxide (ATO) is used as a therapeutic drug for treatment of acute promyelocytic leukemia (APL). The mechanism by which this agent cures this disease was previously shown to involve direct interactions between ATO and the promyelocytic leukemia protein (PML), as well as accelerated degradation of the APL-associated fusion oncoprotein PML/retinoic acid receptor α (RARA). Here we investigated the fate of PML-generated nuclear structures called PML bodies in ATO-treated cells. We found that ATO inhibits formation of progeny PML bodies while it stabilizes cytoplasmic precursor compartments, referred to as cytoplasmic assemblies of PML and nucleoporins (CyPNs), after cell division. This block in PML body recycling is readily detected at pharmacologic relevant ATO concentrations (0.02-0.5µM) that do not cause detectable cell-cycle defects, and it does not require modification of PML by SUMOylation. In addition, PML and PML/RARA carrying mutations previously identified in ATO-resistant APL patients are impeded in their ability to become sequestered within CyPNs. Thus, ATO may inhibit nuclear activities of PML and PML/RARA in postmitotic cells through CyPN-dependent cytoplasmic sequestration.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Citoplasma/metabolismo , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Óxidos/farmacología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Trióxido de Arsénico , Ciclo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Leucemia Promielocítica Aguda/genética , Mutación/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteína de la Leucemia Promielocítica , Reciclaje , Sumoilación/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the understanding of intra-tumoural heterogeneity at the cellular and the genetic level is mandatory to improve treatment and clinical outcome. By combining ploidy-based flow sorting with array-comparative genomic hybridization we show that primary GBMs present as either mono- or polygenomic tumours (64 versus 36%, respectively). Monogenomic tumours were limited to a pseudodiploid tumour clone admixed with normal stromal cells, whereas polygenomic tumours contained multiple tumour clones, yet always including a pseudodiploid population. Interestingly, pseudodiploid and aneuploid fractions carried the same aberrations as defined by identical chromosomal breakpoints, suggesting that evolution towards aneuploidy is a late event in GBM development. Interestingly, while clonal heterogeneity could be recapitulated in spheroid-based xenografts, we find that genetically distinct clones displayed different tumourigenic potential. Moreover, we show that putative cancer stem cell markers including CD133, CD15, A2B5 and CD44 were present on genetically distinct tumour cell populations. These data reveal the clonal heterogeneity of GBMs at the level of DNA content, tumourigenic potential and stem cell marker expression, which is likely to impact glioma progression and treatment response. The combined knowledge of intra-tumour heterogeneity at the genetic, cellular and functional level is crucial to assess treatment responses and to design personalized treatment strategies for primary GBM.
Asunto(s)
Carcinogénesis/patología , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/patología , Glioblastoma/genética , Glioblastoma/patología , Células Madre Neoplásicas/patología , Fenotipo , Animales , Biopsia , Carcinogénesis/genética , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN/genética , ADN de Neoplasias/genética , Citometría de Flujo , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ploidias , Estudios Retrospectivos , Análisis de la Célula IndividualRESUMEN
The identification and significance of cancer stem-like cells in malignant gliomas remains controversial. It has been proposed that cancer stem-like cells display increased drug resistance, through the expression of ATP-binding cassette transporters that detoxify cells by effluxing exogenous compounds. Here, we investigated the 'side population' phenotype based on efflux properties of ATP-binding cassette transporters in freshly isolated human glioblastoma samples and intracranial xenografts derived thereof. Using fluorescence in situ hybridization analysis on sorted cells obtained from glioblastoma biopsies, as well as human tumour xenografts developed in immunodeficient enhanced green fluorescence protein-expressing mice that allow an unequivocal tumour-stroma discrimination, we show that side population cells in human glioblastoma are non-neoplastic and exclusively stroma-derived. Tumour cells were consistently devoid of efflux properties regardless of their genetic background, tumour ploidy or stem cell associated marker expression. Using multi-parameter flow cytometry we identified the stromal side population in human glioblastoma to be brain-derived endothelial cells with a minor contribution of astrocytes. In contrast with their foetal counterpart, neural stem/progenitor cells in the adult brain did not display the side population phenotype. Of note, we show that CD133-positive cells often associated with cancer stem-like cells in glioblastoma biopsies, do not represent a homogenous cell population and include CD31-positive endothelial cells. Interestingly, treatment of brain tumours with the anti-angiogenic agent bevacizumab reduced total vessel density, but did not affect the efflux properties of endothelial cells. In conclusion our findings contribute to an unbiased identification of cancer stem-like cells and stromal cells in brain neoplasms, and provide novel insight into the complex issue of drug delivery to the brain. Since efflux properties of endothelial cells are likely to compromise drug availability, transiently targeting ATP-binding cassette transporters may be a valuable therapeutic strategy to improve treatment effects in brain tumours.
Asunto(s)
Neoplasias Encefálicas/patología , Células Endoteliales/patología , Glioblastoma/patología , Células Madre Neoplásicas/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Neoplasias Encefálicas/química , Línea Celular Tumoral , Células Endoteliales/química , Femenino , Glioblastoma/química , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Células Madre Neoplásicas/química , Fenotipo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As2O3) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.
Asunto(s)
Apoptosis , Neoplasias Encefálicas/metabolismo , Catepsina L/antagonistas & inhibidores , Silenciador del Gen , Glioblastoma/metabolismo , Óxidos/toxicidad , Esferoides Celulares/efectos de los fármacos , Trióxido de Arsénico , Arsenicales , Neoplasias Encefálicas/patología , Catepsina L/genética , Catepsina L/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Glioblastoma/patología , Humanos , Esferoides Celulares/metabolismo , Células Tumorales CultivadasRESUMEN
Most tumours are derived from a single cell that is transformed into a cancer-initiating cell (cancer stem cell) that has the capacity to proliferate and form tumours in vivo. However, the origin of the cancer stem cell remains elusive. Interestingly, during development and tissue repair the fusion of genetic and cytoplasmic material between cells of different origins is an important physiological process. Such cell fusion and horizontal gene-transfer events have also been linked to several fundamental features of cancer and could be important in the development of the cancer stem cell.
Asunto(s)
Transformación Celular Neoplásica/patología , Neoplasias/patología , Células Madre/patología , Animales , Fusión Celular , Transformación Celular Neoplásica/genética , Transferencia de Gen Horizontal/fisiología , Humanos , Neoplasias/genéticaRESUMEN
Bevacizumab, an antibody against vascular endothelial growth factor (VEGF), is a promising, yet controversial, drug in human glioblastoma treatment (GBM). Its effects on tumor burden, recurrence, and vascular physiology are unclear. We therefore determined the tumor response to bevacizumab at the phenotypic, physiological, and molecular level in a clinically relevant intracranial GBM xenograft model derived from patient tumor spheroids. Using anatomical and physiological magnetic resonance imaging (MRI), we show that bevacizumab causes a strong decrease in contrast enhancement while having only a marginal effect on tumor growth. Interestingly, dynamic contrast-enhanced MRI revealed a significant reduction of the vascular supply, as evidenced by a decrease in intratumoral blood flow and volume and, at the morphological level, by a strong reduction of large- and medium-sized blood vessels. Electron microscopy revealed fewer mitochondria in the treated tumor cells. Importantly, this was accompanied by a 68% increase in infiltrating tumor cells in the brain parenchyma. At the molecular level we observed an increase in lactate and alanine metabolites, together with an induction of hypoxia-inducible factor 1α and an activation of the phosphatidyl-inositol-3-kinase pathway. These data strongly suggest that vascular remodeling induced by anti-VEGF treatment leads to a more hypoxic tumor microenvironment. This favors a metabolic change in the tumor cells toward glycolysis, which leads to enhanced tumor cell invasion into the normal brain. The present work underlines the need to combine anti-angiogenic treatment in GBMs with drugs targeting specific signaling or metabolic pathways linked to the glycolytic phenotype.