RESUMEN
BACKGROUND: Precision treatment of glioblastoma is increasingly focused on molecular subtyping, with the mesenchymal subtype particularly resistant to temozolomide. Here, we aim to develop a targeted therapy for temozolomide resensitization in the mesenchymal subtype. METHODS: We integrated kinomic profiles and kinase inhibitor screens from patient-derived proneural and mesenchymal glioma-propagating cells and public clinical datasets to identify key protein kinases implicated in temozolomide resistance. RNAseq, apoptosis assays, and comet assays were used to examine the role of p38MAPK signaling and adaptive chemoresistance in mesenchymal cells. The efficacy of dual p38MAPK and MEK/ERK inhibition using ralimetinib (selective orally active p38MAPK inhibitor; phase I/II for glioblastoma) and binimetinib (approved MEK1/2 inhibitor for melanoma; phase II for high-grade glioma) in primary and recurrent mesenchymal tumors was evaluated using an intracranial patient-derived tumor xenograft model, focusing on survival analysis. RESULTS: Our transcriptomic-kinomic integrative analysis revealed p38MAPK as the prime target whose gene signature enables patient stratification based on their molecular subtypes and provides prognostic value. Repurposed p38MAPK inhibitors synergize favorably with temozolomide to promote intracellular retention of temozolomide and exacerbate DNA damage. Mesenchymal cells exhibit adaptive chemoresistance to p38MAPK inhibition through a pH-/calcium-mediated MEK/ERK pathway. Dual p38MAPK and MEK inhibition effectively maintain temozolomide sensitivity in primary and recurrent intracranial mesenchymal glioblastoma xenografts. CONCLUSIONS: Temozolomide resistance in mesenchymal glioblastoma is associated with p38MAPK activation. Adaptive chemoresistance in p38MAPK-resistant cells is mediated by MEK/ERK signaling. Adjuvant therapy with dual p38MAPK and MEK inhibition prolongs temozolomide sensitivity, which can be developed into a precision therapy for the mesenchymal subtype.
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Neoplasias Encefálicas , Resistencia a Antineoplásicos , Glioblastoma , Temozolomida , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos , Temozolomida/farmacología , Humanos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Ratones , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Antineoplásicos Alquilantes/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células Tumorales Cultivadas , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , PronósticoRESUMEN
Glioblastoma (GBM) is the most common primary malignant brain cancer in adults with a dismal prognosis. Temozolomide (TMZ) is the first-in-line chemotherapeutic; however, resistance is frequent and multifactorial. While many molecular and genetic factors have been linked to TMZ resistance, the role of the solid tumor morphology and the tumor microenvironment, particularly the blood-brain barrier (BBB), is unknown. Here, the authors investigate these using a complex in vitro model for GBM and its surrounding BBB. The model recapitulates important clinical features such as a dense tumor core with tumor cells that invade along the perivascular space; and a perfusable BBB with a physiological permeability and morphology that is altered in the presence of a tumor spheroid. It is demonstrated that TMZ sensitivity decreases with increasing cancer cell spatial organization, and that the BBB can contribute to TMZ resistance. Proteomic analysis with next-generation low volume sample workflows of these cultured microtissues revealed potential clinically relevant proteins involved in tumor aggressiveness and TMZ resistance, demonstrating the utility of complex in vitro models for interrogating the tumor microenvironment and therapy validation.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Temozolomida/farmacología , Temozolomida/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Barrera Hematoencefálica/metabolismo , Microambiente Tumoral , Proteómica , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Current technologies to subtype glioblastoma (GBM), the most lethal brain tumor, require highly invasive brain biopsies. Here, we develop a dedicated analytical platform to achieve direct and multiplexed profiling of circulating RNAs in extracellular vesicles for blood-based GBM characterization. The technology, termed 'enzyme ZIF-8 complexes for regenerative and catalytic digital detection of RNA' (EZ-READ), leverages an RNA-responsive transducer to regeneratively convert and catalytically enhance signals from rare RNA targets. Each transducer comprises hybrid complexes - protein enzymes encapsulated within metal organic frameworks - to configure strong catalytic activity and robust protection. Upon target RNA hybridization, the transducer activates directly to liberate catalytic complexes, in a target-recyclable manner; when partitioned within a microfluidic device, these complexes can individually catalyze strong chemifluorescence reactions for digital RNA quantification. The EZ-READ platform thus enables programmable and reliable RNA detection, across different-sized RNA subtypes (miRNA and mRNA), directly in sample lysates. When clinically evaluated, the EZ-READ platform established composite signatures for accurate blood-based GBM diagnosis and subtyping.
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Neoplasias Encefálicas , Glioblastoma , MicroARNs , Humanos , MicroARNs/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , ARN Mensajero , Hibridación de Ácido Nucleico , Glioblastoma/genética , Glioblastoma/patologíaRESUMEN
This review describes recent technological advances applied to glioblastoma (GBM), a brain tumor with dismal prognosis. International consortial efforts suggest the presence of molecular subtypes within histologically identical GBM tumors. This emphasizes that future treatment decisions should no longer be made based solely on morphological analyses, but must now take into consideration such molecular and cellular heterogeneity. The use of single-cell technologies has advanced our understanding and assignation of functional subtypes revealing therapeutic vulnerabilities. Our team has developed stratification approaches in the past few years, and we have been able to identify patient cohorts enriched for various signaling pathways. Importantly, our Glioportal brain tumor resource has been established under the National Neuroscience Institute Tissue Bank in 2021. This resource offers preclinical capability to validate working hypotheses established from patient clinical datasets. This review highlights recent developments with the ultimate goal of assigning functional meaning to molecular subtypes, revealing therapeutic vulnerabilities.
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Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Humanos , Terapia Molecular Dirigida , Medicina de Precisión , PronósticoRESUMEN
Glioblastoma are heterogeneous tumors composed of highly invasive and highly proliferative clones. Heterogeneity in invasiveness could emerge from discrete biophysical properties linked to specific molecular expression. We identified clones of patient-derived glioma propagating cells that were either highly proliferative or highly invasive and compared their cellular architecture, migratory, and biophysical properties. We discovered that invasiveness was linked to cellular fitness. The most invasive cells were stiffer, developed higher mechanical forces on the substrate, and moved stochastically. The mechano-chemical-induced expression of the formin FMN1 conferred invasive strength that was confirmed in patient samples. Moreover, FMN1 expression was also linked to motility in other cancer and normal cell lines, and its ectopic expression increased fitness parameters. Mechanistically, FMN1 acts from the microtubule lattice and promotes a robust mechanical cohesion, leading to highly invasive motility.
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Movimiento Celular/fisiología , Forminas/metabolismo , Glioblastoma/metabolismo , Invasividad Neoplásica/patología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proteínas Fetales/metabolismo , Glioblastoma/patología , Humanos , Proteínas de Microfilamentos/metabolismoRESUMEN
Molecular profiling of the most aggressive brain tumor glioblastoma (GBM) on the basis of gene expression, DNA methylation, and genomic variations advances both cancer research and clinical diagnosis. The enhancer architectures and regulatory circuitries governing tumor-intrinsic transcriptional diversity and subtype identity are still elusive. Here, by mapping H3K27ac deposition, we analyze the active regulatory landscapes across 95 GBM biopsies, 12 normal brain tissues, and 38 cell line counterparts. Analyses of differentially regulated enhancers and super-enhancers uncovered previously unrecognized layers of intertumor heterogeneity. Integrative analysis of variant enhancer loci and transcriptome identified topographies of transcriptional enhancers and core regulatory circuitries in four molecular subtypes of primary tumors: AC1-mesenchymal, AC1-classical, AC2-proneural, and AC3-proneural. Moreover, this study reveals core oncogenic dependency on super-enhancer-driven transcriptional factors, long noncoding RNAs, and druggable targets in GBM. Through profiling of transcriptional enhancers, we provide clinically relevant insights into molecular classification, pathogenesis, and therapeutic intervention of GBM.
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Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Cromatina/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , HumanosRESUMEN
Hypoxic microenvironment is a hallmark of solid tumors, especially glioblastoma. The strong reliance of glioma-propagating cells (GPCs) on hypoxia-induced survival advantages is potentially exploitable for drug development. Methods: To identify key signaling pathways for hypoxia adaptation by patient-derived GPCs, we performed a kinase inhibitor profiling by screening 188 small molecule inhibitors against 130 different kinases in normoxia and hypoxia. Potential kinase candidates were prioritized for in vitro and in vivo investigations using a ranking algorithm that integrated information from the kinome connectivity network and estimated patients' survival based on expression status. Results: Hypoxic drug screen highlighted extensive modifications of kinomic landscape and a crucial functionality of c-MET-PI3K. c-MET inhibitors diminished phosphorylation of c-MET and PI3K in GPCs subjected to hypoxia, suggesting its role in the hypoxic adaptation of GPCs. Mechanistically, the inhibition of c-MET and PI3K impaired antioxidant defense, leading to oxidative catastrophe and apoptosis. Repurposed c-MET inhibitors PF04217903 and tivantinib exhibited hypoxic-dependent drug synergism with temozolomide, resulting in reduced tumor load and growth of GPC xenografts. Detailed analysis of bulk and single-cell glioblastoma transcriptomes associates the cellular subpopulation over-expressing c-MET with inflamed, hypoxic, metastatic, and stem-like phenotypes. Conclusions: Thus, our "bench to bedside (the use of patient-derived GPCs and xenografts for basic research) and back (validation with independent glioblastoma transcriptome databases)" analysis unravels the novel therapeutic indications of c-MET and PI3K/Akt inhibitors for the treatment of glioblastoma, and potentially other cancers, in the hypoxic tumor microenvironment.
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Glioma/genética , Hipoxia/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-met/genética , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioma/tratamiento farmacológico , Humanos , Hipoxia/tratamiento farmacológico , Masculino , Ratones , Fosforilación/efectos de los fármacos , Fosforilación/genética , Pirazinas/farmacología , Pirrolidinonas/farmacología , Quinolinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Temozolomida/farmacología , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Triazoles/farmacologíaRESUMEN
Recent genomic studies on the glioblastoma (GBM) subtypes (e.g., mesenchymal, proneural, and classical) pave a way for effective clinical treatments of the recurrent brain tumor. However, identification of the GBM subtype is complicated by the intratumoral heterogeneity that results in coexistence of multiple subtypes within the tissue specimen. Here, we present the use of hyperspectral stimulated Raman scattering (SRS) microscopy for rapid, label-free molecular assessment of GBM intratumoral heterogeneity with submicron resolution. We develop a unique label-free Raman imaging diagnostic platform consisting of the spectral focusing hyperspectral SRS imaging of the large-area GBM tissue specimens, SRS images, and spectrum retrieval using the multivariate curve resolution algorithm and subtype classification based on the quadratic support vector machine model for rapid molecular subtyping of GBMs. Both the stain-free SRS histological images and 2D subtype maps can be obtained within 20-30 min which is superior to the days of the conventional single-cell RNA sequencing. While the SRS histology assesses the demyelination status as a new diagnostic feature, the SRS mapping provides a new insight into intratumoral heterogeneity across GBM tissue specimens. We find that the major proportions of the GBM tissues agree with the diagnostic results of the genomic analysis, but nontrivial portions of the remaining SRS image tiles in the specimens are found to belong to other molecular subtypes, implying the substantial degree of GBM heterogeneity. The rapid SRS imaging diagnostic platform developed has shown the ability of unveiling tumor heterogeneity in GBM tissues accurately, which would promote the improvement of the GBM-targeted therapy in near future.
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Neoplasias Encefálicas/patología , Glioblastoma/patología , Técnicas Histológicas , Microscopía Óptica no Lineal/métodos , Neoplasias Encefálicas/diagnóstico por imagen , Glioblastoma/diagnóstico por imagen , Humanos , Sensibilidad y EspecificidadRESUMEN
Parkinson's disease (PD) is an age-dependent neurodegenerative condition. Leucine-rich repeat kinase 2 (LRRK2) mutations are the most frequent cause of sporadic and autosomal dominant PD. The exact role of LRRK2 protective variants (R1398H, N551K) together with a pathogenic mutant (G2019S) in aging and neurodegeneration is unknown. We generated the following myc-tagged UAS-LRRK2 transgenic Drosophila: LRRK2 (WT), N551K, R1398H, G2019S single allele, and double-mutants (N551K/G2019S or R1398H/G2019S). The protective variants alone were able to suppress the phenotypic effects caused by the pathogenic LRRK2 mutation. Next, we conducted RNA-sequencing using mRNA isolated from dopaminergic neurons of these different groups of transgenic Drosophila. Using pathway enrichment analysis, we identified the top 10 modules (p < 0.05), with "LRRK2 in neurons in Parkinson's disease" among the candidates. Further dissection of this pathway identified the most significantly modulated gene nodes such as eEF1A2, ACTB, eEF1A, and actin cytoskeleton reorganization. The induction of the pathway was successfully restored by the R1398H protective variant and R1398H-G2019S or N551K-G2019S rescue experiments. The oxidoreductase family of genes was also active in the pathogenic mutant and restored in protective and rescue variants. In summary, we provide in vivo evidence supporting the neuroprotective effects of LRRK2 variants. RNA sequencing of dopaminergic neurons identified upregulation of specific gene pathways in the Drosophila carrying the pathogenic variant, and this was restored in the rescue phenotypes. Using protective gene variants, our study identifies potential new targets and provides proof of principle of a new therapeutic approach that will further our understanding of aging and neurodegeneration in PD.
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Proteínas de Drosophila/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Mutación , Enfermedad de Parkinson/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Organismos Modificados Genéticamente , Enfermedad de Parkinson/genéticaRESUMEN
The original version of the article unfortunately contained an error.
RESUMEN
Intratumoral heterogeneity is a hallmark of glioblastoma (GBM) tumors, thought to negatively influence therapeutic outcome. Previous studies showed that mesenchymal tumors have a worse outcome than the proneural subtype. Here we focus on STAT3 as its activation precedes the proneural-mesenchymal transition. We first establish a STAT3 gene signature that stratifies GBM patients into STAT3-high and -low cohorts. STAT3 inhibitor treatment selectively mitigates STAT3-high cell viability and tumorigenicity in orthotopic mouse xenograft models. We show the mechanism underlying resistance in STAT3-low cells by combining STAT3 signature analysis with kinome screen data on STAT3 inhibitor-treated cells. This allows us to draw connections between kinases affected by STAT3 inhibitors, their associated transcription factors and target genes. We demonstrate that dual inhibition of IGF-1R and STAT3 sensitizes STAT3-low cells and improves survival in mice. Our study underscores the importance of serially profiling tumors so as to accurately target individuals who may demonstrate molecular subtype switching.
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Predisposición Genética a la Enfermedad/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Animales , Supervivencia Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Imidazoles/farmacología , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Ratones , Pirazinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor IGF Tipo 1/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Temozolomida/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma (GBM) is the most aggressive tumor of the brain. NF1, a tumor suppressor gene and RAS-GTPase, is one of the highly mutated genes in GBM. Dysregulated NF1 expression promotes cell invasion, proliferation, and tumorigenesis. Loss of NF1 expression in glioblastoma is associated with increased aggressiveness of the tumor. Here, we show that NF1-loss in patient-derived glioma cells using shRNA increases self-renewal, heightens cell invasion, and promotes mesenchymal subtype and epithelial mesenchymal transition-specific gene expression that enhances tumorigenesis. The neurofibromin protein contains at least four major domains, with the GAP-related domain being the most well-studied. In this study, we report that the leucine-rich domain (LRD) of neurofibromin inhibits invasion of human glioblastoma cells without affecting their proliferation. Moreover, under conditions tested, the NF1-LRD fails to hydrolyze Ras-GTP to Ras-GDP, suggesting that its suppressive function is independent of Ras signaling. We further demonstrate that rare variants within the NF1-LRD domain found in a subset of the patients are pathogenic and reduce NF1-LRD's invasion suppressive function. Taken together, our results show, for the first time, that NF1-LRD inhibits glioma invasion, and provides evidence of a previously unrecognized function of NF1-LRD in glioma biology.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Leucina/metabolismo , Mutación , Invasividad Neoplásica/genética , Neurofibromina 1/genética , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/patología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neurofibromina 1/metabolismoRESUMEN
The transient receptor potential melastatin 4 (TRPM4) channel has been suggested to play a key role in the treatment of ischemic stroke. However, in vivo evaluation of TRPM4 channel, in particular by direct channel suppression, is lacking. In this study, we used multimodal imaging to assess edema formation and quantify the amount of metabolically functional brain salvaged after a rat model of stroke reperfusion. TRPM4 upregulation in endothelium emerges as early as 2 h post-stroke induction. Expression of TRPM4 channel was suppressed directly in vivo by treatment with siRNA; scrambled siRNA was used as a control. T2-weighted MRI suggests that TRPM4 inhibition successfully reduces edema by 30% and concomitantly salvages functionally active brain, measured by 18F-FDG-PET. These in vivo imaging results correlate well with post-mortem 2,3,5-triphenyltetrazolium chloride (TTC) staining which exhibits a 34.9% reduction in infarct volume after siRNA treatment. Furthermore, in a permanent stroke model, large areas of brain tissue displayed both edema and significant reductions in metabolic activity which was not shown in transient models with or without TRPM4 inhibition, indicating that tissue salvaged by TRPM4 inhibition during stroke reperfusion may survive. Evans Blue extravasation and hemoglobin quantification in the ipsilateral hemisphere were greatly reduced, suggesting that TRPM4 inhibition can improve BBB integrity after ischemic stroke reperfusion. Our results support the use of TRPM4 blocker for early stroke reperfusion.
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Regulación de la Expresión Génica/fisiología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Imagen Multimodal/métodos , Daño por Reperfusión/tratamiento farmacológico , Canales Catiónicos TRPM/metabolismo , Animales , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/fisiopatología , Edema Encefálico , Modelos Animales de Enfermedad , Fluorodesoxiglucosa F18/farmacocinética , Lateralidad Funcional , Procesamiento de Imagen Asistido por Computador , Infarto de la Arteria Cerebral Media/complicaciones , Masculino , Análisis por Micromatrices , Fosfopiruvato Hidratasa/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/uso terapéutico , Ratas , Ratas Wistar , Daño por Reperfusión/complicaciones , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/genética , Factor de von Willebrand/metabolismoRESUMEN
We report the development and implementation of an epi-detected spectral-focusing hyperspectral stimulated Raman scattering (SRS) imaging technique for label-free biomolecular subtyping of glioblastomas (GBMs). The hyperspectral SRS imaging technique developed generates SRS image stacks (from 2800 to 3020 cm-1 at 7 cm-1 intervals) within 30 s through controlling the time delay between the chirped pump and Stokes beams. SRS images at representative Raman shifts (e.g., 2845, 2885, and 2935 cm-1) delineate the biochemical variations and morphological differences between proneural and mesenchymal subtypes of GBMs. Multivariate curve resolution (MCR) analysis on hyperspectral SRS images enables the quantification of major biomolecule distributions in mesenchymal and proneural GBMs. Further principal component analysis (PCA) and linear discriminant analysis (LDA) together with leave-one SRS spectrum-out, cross-validation (LOOCV) yields a diagnostic sensitivity of 96.7% (29/30) and specificity of 88.9% (28/36) for differentiation between mesenchymal and proneural subtypes of GBMs. This study shows great potential of applying hyperspectral SRS imaging technique developed for rapid, label-free molecular subtyping of GBMs in neurosurgery.
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Neoplasias Encefálicas/clasificación , Glioblastoma/clasificación , Microscopía Óptica no Lineal/métodos , Espectrometría Raman/métodos , Humanos , Análisis Multivariante , Análisis de Componente PrincipalRESUMEN
Glioma tumors constitute a significant portion of microglial cells, which are known to support tumor progression. The present study demonstrates that transforming growth factor-ß (TGFß) signaling pathway in microglia in a glioma environment is involved in tumor progression and pathogenesis. It has been shown that the TGFß level is elevated in higher grades of gliomas and its signaling pathway regulates tumor progression through phosphorylation of SMAD2 and SMAD3, which form a complex with SMAD4 to regulate target gene transcription. In an in vitro cell line-based model increased protein levels of pSMAD2/3, total SMAD2/3 and SMAD4 were observed in murine BV2 microglia cultured in glioma conditioned medium (GCM), indicative of the activated TGFß signaling pathway in microglia associated with glioma environment. Immunofluorescence labeling further revealed the expression of SMAD4 in microglial and non-microglial cells of human glioblastomas tissue in vivo. Functional analysis through shRNA-mediated stable knockdown of SMAD4 in microglia revealed the downregulation of the expression of matrix metalloproteinase 9 (MMP9), which has been shown to be involved in tumor progression and cell migration. Further, knockdown of SMAD4 in microglia decreased the migration of microglial cells towards GCM, indicating that SMAD4 promotes microglial migration in glioma environment. In addition, SMAD4 has been shown to be post-transcriptionally regulated by microRNA-146a, which was downregulated in microglia treated with GCM. Overexpression of miR-146a resulted in decreased expression of SMAD4 together with tumor supportive gene MMP9 in microglia, and subsequently suppressed microglial migration towards GCM, possibly through regulation of SMAD4. On the other hand, the cell viability assay revealed decreased viability of glioma cells when they were treated with conditioned medium derived from SMAD4 knockdown microglia or miR-146a overexpressed microglia as compared to glioma cells treated with the medium from control microglial cells. Taken together, the present study suggests that microglial SMAD4 which is epigenetically regulated by miR-146a promotes microglial migration in gliomas and glioma cell viability.
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Competitive BET bromodomain inhibitors (BBIs) targeting BET proteins (BRD2, BRD3, BRD4, and BRDT) show promising preclinical activities against brain cancers. However, the BET protein-dependent glioblastoma (GBM)-promoting transcriptional network remains elusive. Here, with mechanistic exploration of a next-generation chemical degrader of BET proteins (dBET6), we reveal a profound and consistent impact of BET proteins on E2F1- dependent transcriptional program in both differentiated GBM cells and brain tumor-initiating cells. dBET6 treatment drastically reduces BET protein genomic occupancy, RNA-Pol2 activity, and permissive chromatin marks. Subsequently, dBET6 represses the proliferation, self-renewal, and tumorigenic ability of GBM cells. Moreover, dBET6-induced degradation of BET proteins exerts superior antiproliferation effects compared to conventional BBIs and overcomes both intrinsic and acquired resistance to BBIs in GBM cells. Our study reveals crucial functions of BET proteins and provides the rationale and therapeutic merits of targeted degradation of BET proteins in GBM.
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Antineoplásicos/farmacología , Factor de Transcripción E2F1 , Glioblastoma , Proteínas Serina-Treonina Quinasas , Proteínas de Unión al ARN , Proteínas de Ciclo Celular , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Factor de Transcripción E2F1/antagonistas & inhibidores , Factor de Transcripción E2F1/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismoRESUMEN
Chromosomal rearrangements are common in cancer. More than 50% occur in common fragile sites and disrupt tumor suppressors. However, such rearrangements are not known in gastric cancer. Here we report recurrent 18q2 breakpoints in 6 of 17 gastric cancer cell lines. The rearranged chromosome 18, t(9;18), in MKN7 cells was flow sorted and identified by reverse chromosome painting. High-resolution tiling array hybridization mapped breakpoints to DOK6 (docking protein 6) intron 4 in FRA18C (18q22.2) and an intergenic region in 9q22.2. The same rearrangement was detected by FISH in 22% of 99 primary gastric cancers. Intron 4 truncation was associated with reduced DOK6 transcription. Analysis of The Cancer Genome Atlas stomach adenocarcinoma cohort showed significant correlation of DOK6 expression with histological and molecular phenotypes. Multiple oncogenic signaling pathways (gastrin-CREB, NGF-neurotrophin, PDGF, EGFR, ERK, ERBB4, FGFR1, RAS, VEGFR2 and RAF/MAP kinase) known to be active in aggressive gastric cancers were strikingly diminished in gastric cancers with low DOK6 expression. Median survival of patients with low DOK6-expressing tumors was 2100 days compared with 533 days in patients with high DOK6-expressing tumors (log-rank P = 0.0027). The level of DOK6 expression in tumors predicted patient survival independent of TNM stage. These findings point to new functions of human DOK6 as an adaptor that interacts with diverse molecular components of signaling pathways. Our data suggest that DOK6 expression is an integrated biomarker of multiple oncogenic signals in gastric cancer and identify FRA18C as a new cancer-associated fragile site.
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The Cancer Genome Atlas effort has generated significant interest in a new paradigm shift in tumor tissue analysis, patient diagnosis and subsequent treatment decision. Findings have highlighted the limitation of sole reliance on histology, which can be confounded by inter-observer variability. Such studies demonstrate that histologically similar grade IV brain tumors can be divided into four molecular subtypes based on gene expression, with each subtype demonstrating unique genomic aberrations and clinical outcome. These advances indicate that curative therapeutic strategies must now take into account the molecular information in tumor tissue, with the goal of identifying molecularly stratified patients that will most likely to receive treatment benefit from targeted therapy. This in turn spares non-responders from chemotherapeutic side effects and financial costs. In advancing clinical stage drug candidates, the banking of brain tumor tissue necessitates the acquisition of not just tumor tissue with clinical history and robust follow-up, but also high quality molecular information such as somatic mutation, transcriptomic and DNA methylation profiles which have been shown to predict patient survival independent of current clinical indicators. Additionally, the derivation of cell lines from such tumor tissue facilitates the development of clinically relevant patient-derived xenograft mouse models that can prospectively reform the tumor for further studies, yet have retrospective clinical history to associate bench and in vivo findings with clinical data. This represents a core capability of Precision Medicine where the focus is on understanding inter- and intra-tumor heterogeneity so as to best tailor therapies that will result in improved treatment outcomes.
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Bancos de Muestras Biológicas/estadística & datos numéricos , Neoplasias Encefálicas/terapia , Regulación Neoplásica de la Expresión Génica , Glioblastoma/terapia , Proteínas de Neoplasias/genética , Transcriptoma , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Biología Computacional/métodos , Metilación de ADN , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Mutación , Clasificación del Tumor , Proteínas de Neoplasias/metabolismo , Medicina de Precisión , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastomas are extremely aggressive brain tumors with highly invasive properties. Brain linear tracks such as blood vessel walls constitute their main invasive routes. Here we analyze rat C6 and patient-derived glioma cell motility in vitro using micropatterned linear tracks to mimic blood vessels. On laminin-coated tracks (3-10 µm), these cells used an efficient saltatory mode of migration similar to their in vivo migration. This saltatory migration was also observed on larger tracks (50-400 µm in width) at high cell densities. In these cases, the mechanical constraints imposed by neighboring cells triggered this efficient mode of migration, resulting in the formation of remarkable antiparallel streams of cells along the tracks. This motility involved microtubule-dependent polarization, contractile actin bundles and dynamic paxillin-containing adhesions in the leading process and in the tail. Glioma linear migration was dramatically reduced by inhibiting formins but, surprisingly, accelerated by inhibiting Arp2/3. Protein expression and phenotypic analysis indicated that the formin FHOD3 played a role in this motility but not mDia1 or mDia2. We propose that glioma migration under confinement on laminin relies on formins, including FHOD3, but not Arp2/3 and that the low level of adhesion allows rapid antiparallel migration.
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Neoplasias Encefálicas/patología , Ensayos de Migración Celular/métodos , Glioma/patología , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Fenómenos Biomecánicos , Neoplasias Encefálicas/irrigación sanguínea , Adhesión Celular , Recuento de Células , Movimiento Celular , Forminas , Glioblastoma/patología , Glioma/irrigación sanguínea , Humanos , Laminina/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Paxillin/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
BACKGROUND: Cell surface sialylation is associated with tumor cell invasiveness in many cancers. Glioblastoma is the most malignant primary brain tumor and is highly infiltrative. ST3GAL1 sialyltransferase gene is amplified in a subclass of glioblastomas, and its role in tumor cell self-renewal remains unexplored. METHODS: Self-renewal of patient glioma cells was evaluated using clonogenic, viability, and invasiveness assays. ST3GAL1 was identified from differentially expressed genes in Peanut Agglutinin-stained cells and validated in REMBRANDT (n = 390) and Gravendeel (n = 276) clinical databases. Gene set enrichment analysis revealed upstream processes. TGFß signaling on ST3GAL1 transcription was assessed using chromatin immunoprecipitation. Transcriptome analysis of ST3GAL1 knockdown cells was done to identify downstream pathways. A constitutively active FoxM1 mutant lacking critical anaphase-promoting complex/cyclosome ([APC/C]-Cdh1) binding sites was used to evaluate ST3Gal1-mediated regulation of FoxM1 protein. Finally, the prognostic role of ST3Gal1 was determined using an orthotopic xenograft model (3 mice groups comprising nontargeting and 2 clones of ST3GAL1 knockdown in NNI-11 [8 per group] and NNI-21 [6 per group]), and the correlation with patient clinical information. All statistical tests on patients' data were two-sided; other P values below are one-sided. RESULTS: High ST3GAL1 expression defines an invasive subfraction with self-renewal capacity; its loss of function prolongs survival in a mouse model established from mesenchymal NNI-11 (P < .001; groups of 8 in 3 arms: nontargeting, C1, and C2 clones of ST3GAL1 knockdown). ST3GAL1 transcriptomic program stratifies patient survival (hazard ratio [HR] = 2.47, 95% confidence interval [CI] = 1.72 to 3.55, REMBRANDT P = 1.92 x 10â»8; HR = 2.89, 95% CI = 1.94 to 4.30, Gravendeel P = 1.05 x 10⻹¹), independent of age and histology, and associates with higher tumor grade and T2 volume (P = 1.46 x 10â»4). TGFß signaling, elevated in mesenchymal patients, correlates with high ST3GAL1 (REMBRANDT gliomacor = 0.31, P = 2.29 x 10⻹°; Gravendeel gliomacor = 0.50, P = 3.63 x 10⻲°). The transcriptomic program upon ST3GAL1 knockdown enriches for mitotic cell cycle processes. FoxM1 was identified as a statistically significantly modulated gene (P = 2.25 x 10â»5) and mediates ST3Gal1 signaling via the (APC/C)-Cdh1 complex. CONCLUSIONS: The ST3GAL1-associated transcriptomic program portends poor prognosis in glioma patients and enriches for higher tumor grades of the mesenchymal molecular classification. We show that ST3Gal1-regulated self-renewal traits are crucial to the sustenance of glioblastoma multiforme growth.