RESUMEN
Glioblastoma stem cells (GSCs) are implicated in tumor neovascularization, invasiveness, and therapeutic resistance. To illuminate mechanisms governing these hallmark features, we developed a de novo glioblastoma multiforme (GBM) model derived from immortalized human neural stem/progenitor cells (hNSCs) to enable precise system-level comparisons of pre-malignant and oncogene-induced malignant states of NSCs. Integrated transcriptomic and epigenomic analyses uncovered a PAX6/DLX5 transcriptional program driving WNT5A-mediated GSC differentiation into endothelial-like cells (GdECs). GdECs recruit existing endothelial cells to promote peritumoral satellite lesions, which serve as a niche supporting the growth of invasive glioma cells away from the primary tumor. Clinical data reveal higher WNT5A and GdECs expression in peritumoral and recurrent GBMs relative to matched intratumoral and primary GBMs, respectively, supporting WNT5A-mediated GSC differentiation and invasive growth in disease recurrence. Thus, the PAX6/DLX5-WNT5A axis governs the diffuse spread of glioma cells throughout the brain parenchyma, contributing to the lethality of GBM.
Asunto(s)
Glioblastoma/genética , Glioblastoma/patología , Invasividad Neoplásica/genética , Proteína Wnt-5a/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Epigenómica , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Células-Madre Neurales/metabolismo , Factor de Transcripción PAX6/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Cohesin plays a crucial role in the organization of topologically-associated domains (TADs), which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here, we discover that the histone demethylase PHF2 associates with RAD21, a core subunit of cohesin, to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably, the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient, active replication origins, and can act as boundaries to separate adjacent domains. Accordingly, PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore, we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation, and that PHF2's histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops, where cohesin represses dormant replication origins directly or indirectly, to sustain DNA replication in NSC.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Cohesinas , Replicación del ADN , Proteínas de Unión al ADN , Células-Madre Neurales , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Ratones , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Cromatina/metabolismo , Origen de Réplica , Histona Demetilasas/metabolismo , Histona Demetilasas/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Genoma/genética , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Ratones NoqueadosRESUMEN
Telomere shortening is a well-established hallmark of cellular aging. Telomerase reverse transcriptase (TERT) plays a crucial role in maintaining the length of telomeres, which are specialised protective caps at the end of chromosomes. The lack of in vitro aging models, particularly for the central nervous system (CNS), has impeded progress in understanding aging and age-associated neurodegenerative diseases. In this study, we aimed to explore the possibility of inducing aging-associated features in cell types of the CNS using hiPSC (human induced pluripotent stem cell) technology. To achieve this, we utilised CRISPR/Cas9 to generate hiPSCs with a loss of telomerase function and shortened telomeres. Through directed differentiation, we generated motor neurons and astrocytes to investigate whether telomere shortening could lead to age-associated phenotypes. Our findings revealed that shortened telomeres induced age-associated characteristics in both motor neurons and astrocytes including increased cellular senescence, heightened inflammation, and elevated DNA damage. We also observed cell-type specific age-related morphology changes. Additionally, our study highlighted the fundamental role of TERT and telomere shortening in neural progenitor cell (NPC) proliferation and neuronal differentiation. This study serves as a proof of concept that telomere shortening can effectively induce aging-associated phenotypes, thereby providing a valuable tool to investigate age-related decline and neurodegenerative diseases.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Telomerasa , Humanos , Acortamiento del Telómero , Células Madre Pluripotentes Inducidas/metabolismo , Astrocitos/metabolismo , Telomerasa/genética , Telómero , Neuronas Motoras/metabolismo , FenotipoRESUMEN
The relatively quiet mutational landscape of rhabdomyosarcoma (RMS) suggests that epigenetic deregulation could be central to oncogenesis and tumour aggressiveness. Histone variants have long been recognised as important epigenetic regulators of gene expression. However, the role of histone variants in RMS has not been studied hitherto. In this study, we show that histone variant H3.3 is overexpressed in alveolar RMS (ARMS), an aggressive subtype of RMS. Functionally, knockdown of H3F3A, which encodes for H3.3, significantly impairs the ability of ARMS cells to undertake migration and invasion and reduces Rho activation. In addition, a striking reduction in metastatic tumour burden and improved survival is apparent in vivo. Using RNA-sequencing and ChIP-sequencing analyses, we identified melanoma cell adhesion molecule (MCAM/CD146) as a direct downstream target of H3.3. Loss of H3.3 resulted in a reduction in the presence of active marks and an increase in the occupancy of H1 at the MCAM promoter. Cell migration and invasion were rescued in H3F3A-depleted cells through MCAM overexpression. Moreover, we identified G9a, a lysine methyltransferase encoded by EHMT2, as an upstream regulator of H3F3A. Therefore, this study identifies a novel H3.3 dependent axis involved in ARMS metastasis. These findings establish the potential of MCAM as a therapeutic target for high-risk ARMS patients. © 2022 The Pathological Society of Great Britain and Ireland.
Asunto(s)
Histonas , Rabdomiosarcoma Alveolar , Humanos , Línea Celular Tumoral , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/metabolismo , Regiones Promotoras Genéticas , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Alveolar/patologíaRESUMEN
SETDB1 is a key regulator of lineage-specific genes and endogenous retroviral elements (ERVs) through its deposition of repressive H3K9me3 mark. Apart from its H3K9me3 regulatory role, SETDB1 has seldom been studied in terms of its other potential regulatory roles. To investigate this, a genomic survey of SETDB1 binding in mouse embryonic stem cells across multiple libraries was conducted, leading to the unexpected discovery of regions bereft of common repressive histone marks (H3K9me3, H3K27me3). These regions were enriched with the CTCF motif that is often associated with the topological regulator Cohesin. Further profiling of these non-H3K9me3 regions led to the discovery of a cluster of non-repeat loci that were co-bound by SETDB1 and Cohesin. These regions, which we named DiSCs (domains involving SETDB1 and Cohesin) were seen to be proximal to the gene promoters involved in embryonic stem cell pluripotency and lineage development. Importantly, it was found that SETDB1-Cohesin co-regulate target gene expression and genome topology at these DiSCs. Depletion of SETDB1 led to localized dysregulation of Cohesin binding thereby locally disrupting topological structures. Dysregulated gene expression trends revealed the importance of this cluster in ES cell maintenance as well as at gene 'islands' that drive differentiation to other lineages. The 'unearthing' of the DiSCs thus unravels a unique topological and transcriptional axis of control regulated chiefly by SETDB1.
Asunto(s)
Retrovirus Endógenos , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Retrovirus Endógenos/metabolismo , Genómica , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Ratones , CohesinasRESUMEN
Loss-of-function diseases are often caused by a mutation in a protein traversing the secretory pathway that compromises the normal balance between protein folding, trafficking, and degradation. We demonstrate that the innate cellular protein homeostasis, or proteostasis, capacity can be enhanced to fold mutated enzymes that would otherwise misfold and be degraded, using small molecule proteostasis regulators. Two proteostasis regulators are reported that alter the composition of the proteostasis network in the endoplasmic reticulum through the unfolded protein response, increasing the mutant folded protein concentration that can engage the trafficking machinery, restoring function to two nonhomologous mutant enzymes associated with distinct lysosomal storage diseases. Coapplication of a pharmacologic chaperone and a proteostasis regulator exhibits synergy because of the former's ability to further increase the concentration of trafficking-competent mutant folded enzymes. It may be possible to ameliorate loss-of-function diseases by using proteostasis regulators alone or in combination with a pharmacologic chaperone.
Asunto(s)
Enfermedades por Almacenamiento Lisosomal/metabolismo , Pliegue de Proteína , Proteínas/metabolismo , Línea Celular , Fibroblastos/metabolismo , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/metabolismo , Humanos , Leupeptinas/farmacología , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Chaperonas Moleculares/farmacología , Triterpenos Pentacíclicos , Enfermedad de Tay-Sachs/tratamiento farmacológico , Enfermedad de Tay-Sachs/metabolismo , Triterpenos/farmacologíaRESUMEN
An integrated genomic and functional analysis to elucidate DNA damage signaling factors promoting self-renewal of glioma stem cells (GSCs) identified proliferating cell nuclear antigen (PCNA)-associated factor (PAF) up-regulation in glioblastoma. PAF is preferentially overexpressed in GSCs. Its depletion impairs maintenance of self-renewal without promoting differentiation and reduces tumor-initiating cell frequency. Combined transcriptomic and metabolomic analyses revealed that PAF supports GSC maintenance, in part, by influencing DNA replication and pyrimidine metabolism pathways. PAF interacts with PCNA and regulates PCNA-associated DNA translesion synthesis (TLS); consequently, PAF depletion in combination with radiation generated fewer tumorspheres compared with radiation alone. Correspondingly, pharmacological impairment of DNA replication and TLS phenocopied the effect of PAF depletion in compromising GSC self-renewal and radioresistance, providing preclinical proof of principle that combined TLS inhibition and radiation therapy may be a viable therapeutic option in the treatment of glioblastoma multiforme (GBM).
Asunto(s)
Neoplasias Encefálicas/radioterapia , Proteínas Portadoras/genética , Glioblastoma/radioterapia , Células Madre Neoplásicas/efectos de la radiación , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Proteínas Portadoras/metabolismo , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Glioblastoma/genética , Glioblastoma/mortalidad , Glioblastoma/patología , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Pirimidinas/biosíntesis , Tolerancia a Radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inactivation of tumour-suppressor genes by homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighbouring genes. We propose that homozygous deletions in such passenger genes can expose cancer-specific therapeutic vulnerabilities when the collaterally deleted gene is a member of a functionally redundant family of genes carrying out an essential function. The glycolytic gene enolase 1 (ENO1) in the 1p36 locus is deleted in glioblastoma (GBM), which is tolerated by the expression of ENO2. Here we show that short-hairpin-RNA-mediated silencing of ENO2 selectively inhibits growth, survival and the tumorigenic potential of ENO1-deleted GBM cells, and that the enolase inhibitor phosphonoacetohydroxamate is selectively toxic to ENO1-deleted GBM cells relative to ENO1-intact GBM cells or normal astrocytes. The principle of collateral vulnerability should be applicable to other passenger-deleted genes encoding functionally redundant essential activities and provide an effective treatment strategy for cancers containing such genomic events.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Genes Esenciales/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Terapia Molecular Dirigida/métodos , Eliminación de Secuencia/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/deficiencia , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular , Cromosomas Humanos Par 1/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Supresores de Tumor , Glioblastoma/patología , Homocigoto , Humanos , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/uso terapéutico , Ratones , Trasplante de Neoplasias , Ácido Fosfonoacético/análogos & derivados , Ácido Fosfonoacético/farmacología , Ácido Fosfonoacético/uso terapéutico , Fosfopiruvato Hidratasa/antagonistas & inhibidores , Fosfopiruvato Hidratasa/deficiencia , Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/metabolismo , ARN Interferente Pequeño/genética , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
Epitranscriptomic RNA modifications are crucial for the maintenance of glioma stem cells (GSCs), the most malignant cells in glioblastoma (GBM). 3-methylcytosine (m3C) is a new epitranscriptomic mark on RNAs and METTL8 represents an m3C writer that is dysregulated in cancer. Although METTL8 has an established function in mitochondrial tRNA (mt-tRNA) m3C modification, alternative splicing of METTL8 can also generate isoforms that localize to the nucleolus where they may regulate R-loop formation. The molecular basis for METTL8 dysregulation in GBM, and which METTL8 isoform(s) may influence GBM cell fate and malignancy remain elusive. Here, we investigated the role of METTL8 in regulating GBM stemness and tumorigenicity. In GSC, METTL8 is exclusively localized to the mitochondrial matrix where it installs m3C on mt-tRNAThr/Ser(UCN) for mitochondrial translation and respiration. High expression of METTL8 in GBM is attributed to histone variant H2AZ-mediated chromatin accessibility of HIF1α and portends inferior glioma patient outcome. METTL8 depletion impairs the ability of GSC to self-renew and differentiate, thus retarding tumor growth in an intracranial GBM xenograft model. Interestingly, METTL8 depletion decreases protein levels of HIF1α, which serves as a transcription factor for several receptor tyrosine kinase (RTK) genes, in GSC. Accordingly, METTL8 loss inactivates the RTK/Akt axis leading to heightened sensitivity to Akt inhibitor treatment. These mechanistic findings, along with the intimate link between METTL8 levels and the HIF1α/RTK/Akt axis in glioma patients, guided us to propose a HIF1α/Akt inhibitor combination which potently compromises GSC proliferation/self-renewal in vitro. Thus, METTL8 represents a new GBM dependency that is therapeutically targetable.
Asunto(s)
Glioblastoma , Subunidad alfa del Factor 1 Inducible por Hipoxia , Metiltransferasas , Células Madre Neoplásicas , Proteínas Proto-Oncogénicas c-akt , Metilación de ARN , Animales , Humanos , Ratones , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Carcinogénesis/genética , Carcinogénesis/patología , Carcinogénesis/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Ratones Desnudos , Mitocondrias/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Transducción de Señal , Metilación de ARN/genética , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
MAD2 is a spindle assembly checkpoint protein that participates in the formation of mitotic checkpoint complex, which blocks mitotic progression. RNF8, an established DNA damage response protein, has been implicated in mitotic checkpoint regulation but its exact role remains poorly understood. Here, RNF8 proximity proteomics uncovered a role of RNF8-MAD2 in generating the mitotic checkpoint signal. Specifically, RNF8 competes with a small pool of p31comet for binding to the closed conformer of MAD2 via its RING domain, while CAMK2D serves as a molecular scaffold to concentrate the RNF8-MAD2 complex via transient/weak interactions between its p-Thr287 and RNF8's FHA domain. Accordingly, RNF8 overexpression impairs glioma stem cell (GSC) mitotic progression in a FHA- and RING-dependent manner. Importantly, low RNF8 expression correlates with inferior glioma outcome and RNF8 overexpression impedes GSC tumorigenicity. Last, we identify PLK1 inhibitor that mimics RNF8 overexpression using a chemical biology approach, and demonstrate a PLK1/HSP90 inhibitor combination that synergistically reduces GSC proliferation and stemness. Thus, our study has unveiled a previously unrecognized CAMK2D-RNF8-MAD2 complex in regulating mitotic checkpoint with relevance to gliomas, which is therapeutically targetable.
Asunto(s)
Proteínas de Ciclo Celular , Glioma , Proteínas Mad2 , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Glioma/genética , Glioma/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Mitosis , Proteínas Nucleares/metabolismo , Huso Acromático/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Altering intracellular calcium levels is known to partially restore mutant enzyme homeostasis in several lysosomal storage diseases, but why? We hypothesized that endoplasmic reticulum (ER) calcium increases enhance the folding, trafficking and function of these mutant misfolding- and degradation-prone lysosomal enzymes by increasing chaperone function. Here we report that increasing ER calcium levels by reducing ER calcium efflux through the ryanodine receptor, using antagonists or RNAi, or by promoting ER calcium influx by SERCA2b overexpression enhances mutant glucocerebrosidase (GC) homeostasis in cells derived from individuals with Gaucher's disease. Post-translational regulation of the calnexin folding pathway by an elevated ER calcium concentration seems to enhance the capacity of this chaperone system to fold mutant misfolding-prone enzymes, increasing the folded mutant GC population that can engage the trafficking receptor at the expense of ER-associated degradation, increasing the lysosomal GC concentration and activity.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Glucosilceramidasa/genética , Sustitución de Aminoácidos , Bloqueadores de los Canales de Calcio/farmacología , Diltiazem/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/enzimología , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/metabolismo , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/metabolismo , Humanos , Lisosomas/enzimología , Pliegue de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Vasodilatadores/farmacología , Verapamilo/farmacologíaRESUMEN
Cancer cells adopt metabolic reprogramming to promote cell survival under metabolic stress. A key regulator of cell metabolism is AMP-activated protein kinase (AMPK) which promotes catabolism while suppresses anabolism. However, the underlying mechanism of AMPK in handling metabolic stress in cancer remains to be fully understood. In this study, by performing a proteomics screening of AMPK-interacting proteins in non-small-cell lung cancer (NSCLC) cells, we discovered the platelet isoform of phosphofructokinase 1 (PFKP), a rate-limiting enzyme in glycolysis. Moreover, PFKP was found to be highly expressed in NSCLC patients associated with poor survival. We demonstrated that the interaction of PFKP and AMPK was greatly enhanced upon glucose starvation, a process regulated by PFKP-associated metabolites. Notably, the PFKP-AMPK interaction promoted mitochondrial recruitment of AMPK which subsequently phosphorylated acetyl-CoA carboxylase 2 (ACC2) to enhance long-chain fatty acid oxidation, a process helping maintenance of the energy and redox homeostasis and eventually promoting cancer cell survival under glucose starvation. Collectively, we revealed a critical non-glycolysis-related function of PFKP in regulating long-chain fatty acid oxidation via AMPK to alleviate glucose starvation-induced metabolic stress in NSCLC cells.
RESUMEN
The histone variant H2AZ is overexpressed in diverse cancer types where it facilitates the accessibility of transcriptional regulators to the promoters of cell cycle genes. However, the molecular basis for its dysregulation in cancer remains unknown. Here, we report that glioblastomas (GBM) and glioma stem cells (GSCs) preferentially overexpress H2AZ for their proliferation, stemness and tumorigenicity. Chromatin accessibility analysis of H2AZ2 depleted GSC revealed that E2F1 occupies the enhancer region within H2AZ2 gene promoter, thereby activating H2AZ2 transcription. Exploration of other H2AZ2 transcriptional activators using a customized "anti-H2AZ2" query signature for connectivity map analysis identified STAT3. Co-targeting E2F and STAT3 synergistically reduced the levels of H2AZ, histone 3 lysine 27 acetylation (H3K27ac) and cell cycle gene transcription, indicating that E2F1 and STAT3 synergize to activate H2AZ gene transcription in GSCs. Remarkably, an E2F/STAT3 inhibitor combination durably suppresses GSC tumorigenicity in an orthotopic GBM xenograft model. In glioma patients, high STAT3 signaling is associated with high E2F1 and H2AZ2 expression. Thus, GBM has uniquely opted the use of E2F1- and STAT3-containing "enhanceosomes" that integrate multiple signaling pathways to achieve H2AZ gene activation, supporting a translational path for the E2F/STAT3 inhibitor combination to be applied in GBM treatment.
Asunto(s)
Neoplasias Encefálicas , Factor de Transcripción E2F1 , Glioblastoma , Glioma , Histonas , Factor de Transcripción STAT3 , Acetilación , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Cromatina/genética , Cromatina/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioma/genética , Glioma/metabolismo , Histonas/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Nanoparticles have been explored in glioblastomas as they can traverse the blood-brain barrier and target glioblastoma selectively. However, direct observation of nanoparticle trafficking into glioblastoma cells and their underlying intracellular fate after systemic administration remains uncharacterized. Here, based on high-resolution transmission electron microscopy experiments of an intracranial glioblastoma model, it is shown that ligand-modified nanoparticles can traverse the blood-brain barrier, endocytose into the lysosomes of glioblastoma cells, and undergo endolysosomal escape upon photochemical ionization. Moreover, an optimal dose of metronomic chemotherapy using dual-drug-loaded nanocarriers can induce an augmented antitumor effect directly on tumors, which has not been recognized in previous studies. Metronomic chemotherapy enhances antitumor effects 3.5-fold compared with the standard chemotherapy regimen using the same accumulative dose in vivo. This study provides a conceptual framework that can be used to develop metronomic nanoparticle regimens as a safe and viable therapeutic strategy for treating glioblastomas and other advanced-stage solid tumors.
Asunto(s)
Glioblastoma , Nanopartículas , Barrera Hematoencefálica , Endocitosis , Glioblastoma/tratamiento farmacológico , Humanos , Nanopartículas/químicaRESUMEN
The neural stem cells (NSCs) are essential for normal brain development and homeostasis. The cell state (i.e. quiescent versus activated) and fate (i.e. the cell lineage of choice upon differentiation) of NSCs are tightly controlled by various redox and epigenetic regulatory mechanisms. There is an increasing appreciation that redox and epigenetic regulations are intimately linked, but how this redox-epigenetics crosstalk affects NSC activity remains poorly understood. Another unresolved topic is whether the NSCs actually contribute to brain ageing and neurodegenerative diseases. In this review, we aim to 1) distill concepts that underlie redox and epigenetic regulation of NSC state and fate; 2) provide examples of the redox-epigenetics crosstalk in NSC biology; and 3) highlight potential redox- and epigenetic-based therapeutic opportunities to rescue NSC dysfunctions in ageing and neurodegenerative diseases.
Asunto(s)
Epigénesis Genética , Células-Madre Neurales , Diferenciación Celular , Linaje de la Célula , Oxidación-ReducciónRESUMEN
Glioblastoma (GBM) is a uniformly lethal disease driven by glioma stem cells (GSCs). Here, we use a chemical biology approach to unveil previously unknown GBM dependencies. By studying sulconazole (SN) with anti-GSC properties, we find that SN disrupts biotin distribution to the carboxylases and histones. Transcriptomic and metabolomic analyses of SN-treated GSCs reveal metabolic alterations that are characteristic of biotin-deficient cells, including intracellular cholesterol depletion, impairment of oxidative phosphorylation, and energetic crisis. Furthermore, SN treatment reduces histone biotinylation, histone acetylation, and expression of superenhancer-associated GSC critical genes, which are also observed when biotin distribution is genetically disrupted by holocarboxylase synthetase (HLCS) depletion. HLCS silencing impaired GSC tumorigenicity in an orthotopic xenograft brain tumor model. In GBM, high HLCS expression robustly indicates a poor prognosis. Thus, the dependency of GBM on biotin distribution suggests that the rational cotargeting of biotin-dependent metabolism and epigenetic pathways may be explored for GSC eradication.
RESUMEN
We describe a non-fluorescent, second generation stilbene that very selectively binds to transthyretin in complex biological environments and remains dark until it chemoselectively reacts with the pK(a)-perturbed Lys-15 ε-amino group of transthyretin to form a bright blue fluorescent conjugate. Stilbene A2 is mechanistically unusual in that it remains non-fluorescent in cell lysates lacking transthyretin, even though there is likely some proteome binding. Thus, it is especially useful for cellular imaging, as background fluorescence is undetectable until A2 reacts with transthyretin. The mechanistic basis for the effective lack of environment-sensitive fluorescence of A2 when bound to, but before reacting with, transthyretin is reported. Stilbene A2 exhibits sufficiently rapid transthyretin conjugation kinetics at 37 °C to enable pulse-chase experiments to be performed, in this case demonstrating that transthyretin is secreted from HeLa cells. As the chase compound, we employed C1, a cell-permeable, highly selective, non-covalent, transthyretin-binding dihydrostilbene that cannot become fluorescent. The progress reported is viewed as a first and necessary step toward our long-term goal of creating a one-chain, one-binding-site transthyretin tag, whose fluorescence can be regulated by adding A2 or an analogous molecule. Fusing proteins of interest to a one-chain, one-binding-site transthyretin tag regulated by A2 should be useful for studying folding, trafficking, and degradation in the cellular secretory pathway, utilizing pulse-chase experiments. Immediate applications of A2 include utilizing its conjugate fluorescence to quantify transthyretin concentration in human plasma, reflecting nutritional status, and determining the binding stoichiometry of kinetic stabilizer drugs to transthyretin in plasma.
Asunto(s)
Prealbúmina/metabolismo , Estilbenos/química , Estilbenos/metabolismo , Fibroblastos/metabolismo , Fluorescencia , Células HeLa , Humanos , Prealbúmina/análisis , Prealbúmina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND AND AIM: Reduction of short-chain poorly absorbed carbohydrates (FODMAPs) in the diet reduces symptoms of irritable bowel syndrome (IBS). In the present study, we aimed to compare the patterns of breath hydrogen and methane and symptoms produced in response to diets that differed only in FODMAP content. METHODS: Fifteen healthy subjects and 15 with IBS (Rome III criteria) undertook a single-blind, crossover intervention trial involving consuming provided diets that were either low (9 g/day) or high (50 g/day) in FODMAPs for 2 days. Food and gastrointestinal symptom diaries were kept and breath samples collected hourly over 14 h on day 2 of each diet. RESULTS: Higher levels of breath hydrogen were produced over the entire day with the high FODMAP diet for healthy volunteers (181 +/- 77 ppm.14 h vs 43 +/- 18; mean +/- SD P < 0.0001) and patients with IBS (242 +/- 79 vs 62 +/- 23; P < 0.0001), who had higher levels during each dietary period than the controls (P < 0.05). Breath methane, produced by 10 subjects within each group, was reduced with the high FODMAP intake in healthy subjects (47 +/- 29 vs 109 +/- 77; P = 0.043), but was not different in patients with IBS (126 +/- 153 vs 86 +/- 72). Gastrointestinal symptoms and lethargy were significantly induced by the high FODMAP diet in patients with IBS, while only increased flatus production was reported by healthy volunteers. CONCLUSIONS: Dietary FODMAPs induce prolonged hydrogen production in the intestine that is greater in IBS, influence the amount of methane produced, and induce gastrointestinal and systemic symptoms experienced by patients with IBS. The results offer mechanisms underlying the efficacy of the low FODMAP diet in IBS.
Asunto(s)
Dieta Baja en Carbohidratos , Carbohidratos de la Dieta/administración & dosificación , Flatulencia/prevención & control , Mucosa Intestinal/metabolismo , Síndrome del Colon Irritable/dietoterapia , Dolor Abdominal/etiología , Dolor Abdominal/prevención & control , Adulto , Anciano , Pruebas Respiratorias , Estudios Cruzados , Registros de Dieta , Carbohidratos de la Dieta/efectos adversos , Carbohidratos de la Dieta/metabolismo , Espiración , Femenino , Fermentación , Flatulencia/etiología , Flatulencia/metabolismo , Flatulencia/fisiopatología , Humanos , Hidrógeno/metabolismo , Intestinos/fisiopatología , Síndrome del Colon Irritable/complicaciones , Síndrome del Colon Irritable/metabolismo , Síndrome del Colon Irritable/fisiopatología , Letargia/etiología , Letargia/prevención & control , Masculino , Metano/metabolismo , Persona de Mediana Edad , Método Simple Ciego , Factores de Tiempo , Resultado del Tratamiento , Victoria , Adulto JovenRESUMEN
Significance: The epigenomic/metabolic landscape in cancer has been studied extensively in the past decade and forms the basis of various drug targets. Yet, cancer treatment remains a challenge, with clinical trials exhibiting limited efficacy and high relapse rates. Patients respond differently to therapy, which is fundamentally attributed to tumor heterogeneity, both across and within tumors. This review focuses on the interactions between the heterogeneous tumor microenvironment (TME) and the epigenomic/metabolic axis in cancer, as well as the emerging technologies under development to aid heterogeneity studies. Recent Advances: Interlinks between epigenetics and metabolism in cancer have been reported. Emerging studies have unveiled interactions between the TME and cancer cells that play a critical role in regulating epigenetics and reprogramming cancer metabolism, suggesting a three-way cross talk. Critical Issues: This cross talk accentuates the multiplex nature of cancer, and the importance of considering tumor heterogeneity in various epigenomic/metabolic cancer studies. Future Directions: With the advancement in single-cell profiling, it may be possible to identify cancer subclones and their unique vulnerabilities to develop a multimodal therapy. Drugs targeting the TME are currently being studied, and a better understanding of the TME in regulating cancer epigenetics and metabolism may hold the key to identifying novel therapeutic targets.