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Brain and spinal tumors affect 1 in 1000 people by 25 years of age, and have diverse histological, biological, anatomical and dissemination characteristics. A mortality of 30-40% means the majority are cured, although two-thirds have life-long disability, linked to accumulated brain injury that is acquired prior to diagnosis, and after surgery or chemo-radiotherapy. Only four drugs have been licensed globally for brain tumors in 40 years and only one for children. Most new cancer drugs in clinical trials do not cross the blood-brain barrier (BBB). Techniques to enhance brain tumor drug delivery are explored in this review, and cover those that augment penetration of the BBB, and those that bypass the BBB. Developing appropriate delivery techniques could improve patient outcomes by ensuring efficacious drug exposure to tumors (including those that are drug-resistant), reducing systemic toxicities and targeting leptomeningeal metastases. Together, this drug delivery strategy seeks to enhance the efficacy of new drugs and enable re-evaluation of existing drugs that might have previously failed because of inadequate delivery. A literature review of repurposed drugs is reported, and a range of preclinical brain tumor models available for translational development are explored.
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The overall survival for patients with primary glioblastoma is very poor. Glioblastoma contains a subpopulation of glioma stem cells (GSC) that are responsible for tumour initiation, treatment resistance and recurrence. PPARα is a transcription factor involved in the control of lipid, carbohydrate and amino acid metabolism. We have recently shown that PPARα gene and protein expression is increased in glioblastoma and has independent clinical prognostic significance in multivariate analyses. In this work, we report that PPARα is overexpressed in GSC compared to foetal neural stem cells. To investigate the role of PPARα in GSC, we knocked down its expression using lentiviral transduction with short hairpin RNA (shRNA). Transduced GSC were tagged with luciferase and stereotactically xenografted into the striatum of NOD-SCID mice. Bioluminescent and magnetic resonance imaging showed that knockdown (KD) of PPARα reduced the tumourigenicity of GSC in vivo. PPARα-expressing control GSC xenografts formed invasive histological phenocopies of human glioblastoma, whereas PPARα KD GSC xenografts failed to establish viable intracranial tumours. PPARα KD GSC showed significantly reduced proliferative capacity and clonogenic potential in vitro with an increase in cellular senescence. In addition, PPARα KD resulted in significant downregulation of the stem cell factors c-Myc, nestin and SOX2. This was accompanied by downregulation of the PPARα-target genes and key regulators of fatty acid oxygenation ACOX1 and CPT1A, with no compensatory increase in glycolytic flux. These data establish the aberrant overexpression of PPARα in GSC and demonstrate that this expression functions as an important regulator of tumourigenesis, linking self-renewal and the malignant phenotype in this aggressive cancer stem cell subpopulation. We conclude that targeting GSC PPARα expression may be a therapeutically beneficial strategy with translational potential as an adjuvant treatment. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Neoplasias Encefálicas/patología , Glioblastoma/patología , PPAR alfa/metabolismo , ARN Interferente Pequeño/farmacología , Animales , Biomarcadores de Tumor/metabolismo , Transformación Celular Neoplásica , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen/métodos , Humanos , Lentivirus , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/patología , Fenotipo , Transducción de Señal/fisiología , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a lethal type of pediatric brain tumor that is resistant to conventional chemotherapies. Palbociclib is a putative novel DIPG treatment that restricts the proliferation of rapidly dividing cancer cells via selective inhibition of cyclin-dependent kinase (CDK) 4 and CDK6. However, implementing palbociclib as a monotherapy for DIPG is unfeasible, as CDK4/6 inhibitor resistance is commonplace and palbociclib does not readily cross the blood-brain barrier (BBB) or persist in the central nervous system. To inhibit the growth of DIPG cells, we aimed to use palbociclib in combination with the rapamycin analog temsirolimus, which is known to ameliorate resistance to CDK4/6 inhibitors and inhibit BBB efflux. MATERIALS AND METHODS: We tested palbociclib and temsirolimus in three patient-derived DIPG cell lines. The expression profiles of key proteins in the CDK4/6 and mammalian target of rapamycin (mTOR) signaling pathways were assessed, respectively, to determine feasibility against DIPG. Moreover, we investigated effects on cell viability and examined in vivo drug toxicity. RESULTS: Immunoblot analyses revealed palbociclib and temsirolimus inhibited CDK4/6 and mTOR signaling through canonical perturbation of phosphorylation of the retinoblastoma (RB) and mTOR proteins, respectively; however, we observed noncanonical downregulation of mTOR by palbociclib. We demonstrated that palbociclib and temsirolimus inhibited cell proliferation in all three DIPG cell lines, acting synergistically in combination to further restrict cell growth. Flow cytometric analyses revealed both drugs caused G1 cell cycle arrest, and clonogenic assays showed irreversible effects on cell proliferation. Palbociclib did not elicit neurotoxicity in primary cultures of normal rat hippocampi or when infused into rat brains. CONCLUSION: These data illustrate the in vitro antiproliferative effects of CDK4/6 and mTOR inhibitors in DIPG cells. Direct infusion of palbociclib into the brain, in combination with systemic delivery of temsirolimus, represents a promising new approach to developing a much-needed treatment for DIPG.
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OBJECTIVE The pan-histone deacetylase inhibitor panobinostat has preclinical efficacy against diffuse intrinsic pontine glioma (DIPG), and the oral formulation has entered a Phase I clinical trial. However, panobinostat does not cross the blood-brain barrier in humans. Convection-enhanced delivery (CED) is a novel neurosurgical drug delivery technique that bypasses the blood-brain barrier and is of considerable clinical interest in the treatment of DIPG. METHODS The authors investigated the toxicity, distribution, and clearance of a water-soluble formulation of panobinostat (MTX110) in a small- and large-animal model of CED. Juvenile male Wistar rats (n = 24) received panobinostat administered to the pons by CED at increasing concentrations and findings were compared to those in animals that received vehicle alone (n = 12). Clinical observation continued for 2 weeks. Animals were sacrificed at 72 hours or 2 weeks following treatment, and the brains were subjected to neuropathological analysis. A further 8 animals received panobinostat by CED to the striatum and were sacrificed 0, 2, 6, or 24 hours after infusion, and their brains explanted and snap-frozen. Tissue-drug concentration was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Large-animal toxicity was investigated using a clinically relevant MRI-guided translational porcine model of CED in which a drug delivery system designed for humans was used. Panobinostat was administered at 30 µM to the ventral pons of 2 juvenile Large White-Landrace cross pigs. The animals were subjected to clinical and neuropathological analysis, and findings were compared to those obtained in controls after either 1 or 2 weeks. Drug distribution was determined by LC-MS/MS in porcine white and gray matter immediately after CED. RESULTS There were no clinical or neuropathological signs of toxicity up to an infused concentration of 30 µM in both small- and large-animal models. The half-life of panobinostat in rat brain after CED was 2.9 hours, and the drug was observed to be distributed in porcine white and gray matter with a volume infusion/distribution ratio of 2 and 3, respectively. CONCLUSIONS CED of water-soluble panobinostat, up to a concentration of 30 µM, was not toxic and was distributed effectively in normal brain. CED of panobinostat warrants clinical investigation in patients with DIPG.
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Antineoplásicos/administración & dosificación , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Convección , Glioma/tratamiento farmacológico , Panobinostat/administración & dosificación , Animales , Antineoplásicos/farmacocinética , Neoplasias del Tronco Encefálico/diagnóstico por imagen , Proteínas de Unión al Calcio/metabolismo , Cromatografía Liquida , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Proteínas de Microfilamentos/metabolismo , Panobinostat/farmacocinética , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Wistar , Porcinos , Espectrometría de Masas en Tándem , Factores de Tiempo , Distribución Tisular/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIMS: Histopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses. METHODS: Total RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes, 18S and GAPDH. RESULTS: Reference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene. CONCLUSIONS: This study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.
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Neoplasias Encefálicas/genética , Epilepsia del Lóbulo Temporal/genética , Fijadores/química , Formaldehído/química , Perfilación de la Expresión Génica , Glioblastoma/genética , Adhesión en Parafina , Estabilidad del ARN , ARN Neoplásico/genética , Fijación del Tejido/métodos , Neoplasias Encefálicas/cirugía , Estudios de Casos y Controles , Epilepsia del Lóbulo Temporal/cirugía , Perfilación de la Expresión Génica/normas , Glioblastoma/cirugía , Humanos , Adhesión en Parafina/normas , Valor Predictivo de las Pruebas , Control de Calidad , Reproducibilidad de los Resultados , Factores de Tiempo , Fijación del Tejido/normasRESUMEN
Targeting epigenetic changes in diffuse intrinsic pontine glioma (DIPG) may provide a novel treatment option for patients. This report demonstrates that sodium valproate, a histone deacetylase inhibitor (HDACi), can increase the cytotoxicity of carboplatin in an additive and synergistic manner in DIPG cells in vitro. Sodium valproate causes a dose-dependent decrease in DIPG cell viability in three independent ex vivo cell lines. Furthermore, sodium valproate caused an increase in acetylation of histone H3. Changes in cell viability were consistent with an induction of apoptosis in DIPG cells in vitro, determined by flow cytometric analysis of Annexin V staining and assessment of apoptotic markers by western blotting. Subsequently, immunofluorescent staining of neuronal and glial markers was used to determine toxicity in normal rat hippocampal cells. Pre-treatment of cells with sodium valproate enhanced the cytotoxic effects of carboplatin, in three DIPG cell lines tested. These results demonstrate that sodium valproate causes increased histone H3 acetylation indicative of HDAC inhibition, which is inversely correlated with a reduction in cell viability. Cell viability is reduced through an induction of apoptosis in DIPG cells. Sodium valproate potentiates carboplatin cytotoxicity and prompts further work to define the mechanism responsible for the synergy between these two drugs and determine in vivo efficacy. These findings support the use of sodium valproate as an adjuvant treatment for DIPG.
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Adyuvantes Farmacéuticos/farmacología , Anticonvulsivantes/farmacología , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Glioma/tratamiento farmacológico , Ácido Valproico/farmacología , Acetilación/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Neoplasias del Tronco Encefálico/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Reposicionamiento de Medicamentos/métodos , Epigénesis Genética/efectos de los fármacos , Glioma/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Humanos , RatasRESUMEN
AIMS: PPARα agonists are in current clinical use as hypolipidaemic agents and show significant antineoplastic effects in human glioblastoma models. To date however, the expression of PPARα in large-scale glioblastoma datasets has not been examined. We aimed to investigate the expression of the transcription factor PPARα in primary glioblastoma, the relationship between PPARα expression and patients' clinicopathological features and other molecular markers associated with gliomagenesis. METHODS AND RESULTS: With protein immunoblotting techniques and reverse transcription quantitative real-time PCR, PPARα was found to be significantly overexpressed in glioblastoma compared with control brain tissue (P = 0.032 and P = 0.005). PPARA gene expression was found to be enriched in the classical glioblastoma subtype within The Cancer Genome Atlas (TCGA) dataset. Although not associated with overall survival when assessed by immunohistochemistry, cross-validation with the TCGA dataset and multivariate analyses identified PPARA gene expression as an independent prognostic marker for overall survival (P = 0.042). Finally, hierarchical clustering revealed novel, significant associations between high PPARA expression and a putative set of glioblastoma molecular mediators including EMX2, AQP4, and NTRK2. CONCLUSIONS: PPARα is overexpressed in primary glioblastoma and high PPARA expression functions as an independent prognostic marker in the glioblastoma TCGA dataset. Further studies are required to explore genetic associations with high PPARA expression and to analyse the predictive role of PPARα expression in glioblastoma models in response to PPARα agonists.
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Biomarcadores de Tumor/análisis , Neoplasias Encefálicas/patología , Glioblastoma/patología , PPAR alfa/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Niño , Femenino , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Humanos , Isocitrato Deshidrogenasa/genética , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , PPAR alfa/análisis , Pronóstico , Modelos de Riesgos ProporcionalesRESUMEN
Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332.
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Carcinoma de Células Escamosas/metabolismo , Polaridad Celular , Colágeno Tipo VII/fisiología , Epidermólisis Ampollosa Distrófica/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Neoplasias Cutáneas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos CD , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Técnicas de Cocultivo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Queratinocitos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Trasplante de Neoplasias , Transportadores de Anión Orgánico Sodio-Independiente/genética , Regiones Promotoras Genéticas , Transporte de Proteínas , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos , Transcripción Genética , Células Tumorales Cultivadas , beta Catenina/genética , beta Catenina/metabolismo , KalininaRESUMEN
Desmoplakin is a ubiquitous component of desmosomes and desmosome-like structures, such as the cardiomyocyte area composita. Two major isoforms, desmoplakin I (DSPI) and desmoplakin II (DSPII) are encoded by alternative mRNA transcripts differentially spliced from the same gene. The resulting proteins are identical in amino acid sequence with the exception that DSPII contains only one third of the central alpha-helical rod domain present in DSPI. Here we describe a novel minor isoform of desmoplakin that is also produced by alternative splicing of the desmoplakin gene and that we name desmoplakin Ia (DSPIa). DSPIa is an alternatively spliced DSPI mRNA with a unique splice donor site that is 90% homologous to and downstream of the DSPII specific donor. The resulting DSPIa mRNA is in-frame and encodes a protein that has a central alpha-helical rod domain of intermediate size and that is 156 amino acids larger than DSPII and 443 amino acids smaller than DSPI. We demonstrate, through recombinant expression and short interfering RNA knockdown, that the DSPIa protein is readily detectable, albeit at substantially lower levels than the dominant isoforms, DSPI and DSPII. DSPIa mRNA has a similar tissue distribution to that of DSPI and of DSPII.
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Desmoplaquinas/metabolismo , Empalme Alternativo/genética , Secuencia de Bases , Línea Celular Tumoral , ADN Complementario/genética , Desmoplaquinas/genética , Células Epidérmicas , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Queratinocitos/metabolismo , Masculino , Datos de Secuencia Molecular , Miocardio/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sitios de Empalme de ARN/genética , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
Glioblastomas are highly malignant brain tumours; they have been described as one of the most deadly human cancers. Two conceptual classifications of the condition exist: primary (de novo), which does not exhibit prior disease and secondary glioblastoma, which develops from a pre-existing glioma. This study investigates whether GPR26 is differentially transcribed in glioblastoma tissue from patients of different ages, in order to define a candidate genetic marker. The transcriptional profile of GPR26 was compared in nine samples: seven glioblastoma tissues and two normal brain tissues using PCR. Despite GPR26 being present in the glioblastoma tissues, it was not transcribed in any of the four cell lines tested. GPR26 transcription ratios were compared between normal and cancerous samples, also age categories <50 and >60 years were compared. Results suggested differential transcription of GPR26, which is significantly less transcribed in tissues from older patients, implied by a p-value of 0.03. This study has identified GPR26 to be a genetic indicator of primary glioblastoma, suggesting that it could be a suppressor of primary glioblastoma development.