RESUMO
MYC promotes tumor growth through multiple mechanisms. Here, we show that, in human glioblastomas, the variant MYC transcript encodes a 114-amino acid peptide, MYC pre-mRNA encoded protein (MPEP), from the upstream open reading frame (uORF) MPEP. Secreted MPEP promotes patient-derived xenograft tumor growth in vivo, independent of MYC through direct binding, and activation of tropomyosin receptor kinase B (TRKB), which induces downstream AKT-mTOR signaling. Targeting MPEP through genetic ablation reduced growth of patient-derived 4121 and 3691 glioblastoma stem cells. Administration of an MPEP-neutralizing antibody in combination with a small-molecule TRKB inhibitor reduced glioblastoma growth in patient-derived xenograft tumor-bearing mice. The overexpression of MPEP in surgical glioblastoma specimens predicted a poor prognosis, supporting its clinical relevance. In summary, our results demonstrate that tumor-specific translation of a MYC-associated uORF promotes glioblastoma growth, suggesting a new therapeutic strategy for glioblastoma.
Assuntos
Glioblastoma , Fases de Leitura Aberta , Proteínas Proto-Oncogênicas c-myc , Receptor trkB , Glioblastoma/patologia , Glioblastoma/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Animais , Humanos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fases de Leitura Aberta/genética , Camundongos , Linhagem Celular Tumoral , Receptor trkB/metabolismo , Proliferação de Células/efeitos dos fármacos , Ligação Proteica , Transdução de Sinais , Peptídeos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
The mechanisms underlying stimuli-induced dynamic structural remodeling of RNAs for the maintenance of cellular physiological function and survival remain unclear. Here, we showed that in MGMT promoter-methylated glioblastoma (GBM), the RNA helicase DEAD-box helicase 46 (DDX46) is phosphorylated by temozolomide (TMZ)-activated checkpoint kinase 1 (CHK1), resulting in a dense-to-loose conformational change and an increase in DDX46 helicase activity. DDX46-mediated tertiary structural remodeling of LINC01956 exposes the binding motifs of LINC01956 to the 3' untranslated region of O6-methylguanine DNA methyltransferase (MGMT). This accelerates recruitment of MGMT mRNA to the RNA export machinery and transportation of MGMT mRNA from the nucleus to the cytoplasm, leading to increased MGMT abundance and TMZ resistance. Using patient-derived xenograft (PDX) and tumor organoid models, we found that treatment with the CHK1 inhibitor SRA737abolishes TMZ-induced structural remodeling of LINC01956 and subsequent MGMT up-regulation, resensitizing TMZ-resistant MGMT promoter-methylated GBM to TMZ. In conclusion, these findings highlight a mechanism underlying temozolomide-induced RNA structural remodeling and may represent a potential therapeutic strategy for patients with TMZ-resistant MGMT promoter-methylated GBM.
Assuntos
RNA Helicases DEAD-box , Metilases de Modificação do DNA , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , RNA Longo não Codificante , Temozolomida , Proteínas Supressoras de Tumor , Glioblastoma/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Metilases de Modificação do DNA/metabolismo , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Regiões Promotoras Genéticas/genética , Metilação de DNA/genética , Metilação de DNA/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Quinase 1 do Ponto de Checagem/genética , Linhagem Celular Tumoral , Camundongos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fosforilação/efeitos dos fármacosRESUMO
Objective. Carboxypeptidase E (CPE) plays an important role in the biosynthesis of neurotransmitters and peptide hormones including insulin. It also promotes cell proliferation, survival, and invasion of tumor cells. The endoplasmic reticulum stress, hypoxia, and nutrient supply are significant factors of malignant tumor growth including glioblastoma. There are data indicating that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) suppressed glioblastoma cell proliferation and increased invasiveness of these cells. The present study aims to investigate the regulation of the CPE gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in the regulation of this gene expression and function in tumorigenesis. Methods. Human glioblastoma cells U87MG (transfected by an empty vector; control) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine; for glucose and glutamine deprivations, the cells were cultured in DMEM medium without glucose or glutamine for 16 h, respectively. The expression level of the CPE gene was studied by quantitative RT-PCR and normalized to ACTB. Results. It was found that inhibition of endoribonuclease and protein kinase activities of ERN1 led to a strong up-regulation of CPE gene expression in glioblastoma cells. The expression of this gene also increased in glioblastoma cells after silencing ERN1. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease only. The expression of the CPE gene was resistant to hypoxia in control U87MG cells, but increased in cells with ERN1 knockdown. The expression of this gene was up-regulated under glutamine deprivation in control glioblastoma cells, but decreased upon ERN1 knockdown. However, glucose deprivation decreased the expression of CPE gene in both types of used cells, but ERN1 inhibition enhanced this effect. Conclusion. The results of the present study demonstrate that inhibition of ERN1 strongly up-regulated the expression of pro-oncogenic CPE gene through protein kinase activity of ERN1 and that increased CPE gene expression possibly participates in ERN1 knockdown-mediated invasiveness of glioblastoma cells.
Assuntos
Carboxipeptidase H , Estresse do Retículo Endoplasmático , Endorribonucleases , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Proteínas Serina-Treonina Quinases , Humanos , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/patologia , Carboxipeptidase H/metabolismo , Carboxipeptidase H/genética , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Endorribonucleases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Glucose/metabolismo , Técnicas de Silenciamento de Genes , Hipóxia Celular/fisiologia , Transdução de Sinais/fisiologiaRESUMO
CD2-associated protein (CD2AP) is a scaffolding/adaptive protein that regulates intercellular adhesion and multiple signaling pathways. Although emerging evidence suggests that CD2AP is associated with several malignant tumors, there is no study investigating the expression and biological significance of CD2AP in glioblastoma multiforme (GBM). Here by studying public datasets, we found that CD2AP expression was significantly elevated in GBM and that glioma patients with increased CD2AP expression had a worse prognosis. We also confirmed the increase of CD2AP expression in clinical GBM samples and GBM cell lines. CD2AP overexpression in GBM cells promoted their proliferation, colony formation, migration, and invasion in vitro and their tumorigenesis in vivo, and reduced cell apoptosis both at basal levels and in response to temozolomide. While CD2AP knockdown had the opposite effects. Mechanistically, we revealed that CD2AP interacted with TRIM5, an NF-κB modulator. CD2AP overexpression and knockdown increased and decreased TRIM5 levels as well as the NF-κB activity, respectively. Moreover, downregulation of TRIM5 reversed elevated NF-κB activity in GBM cells with CD2AP overexpression; and inhibition of the NF-κB activity attenuated malignant features of GBM cells with CD2AP overexpression. Our findings demonstrate that CD2AP promotes GBM progression through activating TRIM5-mediated NF-κB signaling and that downregulation of CD2AP can attenuate GBM malignancy, suggesting that CD2AP may become a biomarker and the CD2AP-TRIM5-NF-κB axis may become a therapeutic target for GBM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Glioblastoma , NF-kappa B , Transdução de Sinais , Glioblastoma/patologia , Glioblastoma/metabolismo , Glioblastoma/genética , Humanos , NF-kappa B/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Progressão da Doença , Proliferação de Células , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Camundongos , Camundongos Nus , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Apoptose , Masculino , Feminino , Proteínas do CitoesqueletoRESUMO
Glycolytic metabolic reprogramming in cancer is regulated by both cancer intrinsic variations like isocitrate dehydrogenase 1 (IDH1) status and non-cancerous microenvironment components like tumor associated macrophages (TAMs). However, the detailed mechanism remains elusive. Here, we identify hexosaminidase B (HEXB) as a key regulator for glycolysis in glioblastoma (GBM). HEXB intercellularly manipulates TAMs to promote glycolysis in GBM cells, while intrinsically enhancing cancer cell glycolysis. Mechanistically, HEXB elevation augments tumor HIF1α protein stability through activating ITGB1/ILK/YAP1; Subsequently, HIF1α promotes HEXB and multiple glycolytic gene transcription in GBM cells. Genetic ablation and pharmacological inhibition of HEXB elicits substantial therapeutic effects in preclinical GBM models, while targeting HEXB doesn't induce significant reduction in IDH1 mutant glioma and inhibiting IDH1 mutation-derived 2-hydroxyglutaric acid (2-HG) significantly restores HEXB expression in glioma cells. Our work highlights a HEXB driven TAMs-associated glycolysis-promoting network in GBM and provides clues for developing more effective therapies against it.
Assuntos
Neoplasias Encefálicas , Carcinogênese , Glioblastoma , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia , Isocitrato Desidrogenase , beta-N-Acetil-Hexosaminidases , Humanos , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Linhagem Celular Tumoral , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Carcinogênese/genética , Camundongos , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/genética , Microambiente Tumoral/imunologia , Regulação Neoplásica da Expressão Gênica , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/imunologia , Integrina beta1/metabolismo , Integrina beta1/genética , Glutaratos/metabolismo , Mutação , Proteínas de Sinalização YAP/metabolismoRESUMO
BACKGROUND: Glioblastoma (GBM) stands as the most prevalent and aggressive form of adult gliomas. Despite the implementation of intensive therapeutic approaches involving surgery, radiation, and chemotherapy, Glioblastoma Stem Cells contribute to tumor recurrence and poor prognosis. The induction of Glioblastoma Stem Cells differentiation by manipulating the transcriptional machinery has emerged as a promising strategy for GBM treatment. Here, we explored an innovative approach by investigating the role of the depolarized resting membrane potential (RMP) observed in patient-derived GBM sphereforming cell (GSCs), which allows them to maintain a stemness profile when they reside in the G0 phase of the cell cycle. METHODS: We conducted molecular biology and electrophysiological experiments, both in vitro and in vivo, to examine the functional expression of the voltage-gated sodium channel (Nav) in GSCs, particularly focusing on its cell cycle-dependent functional expression. Nav activity was pharmacologically manipulated, and its effects on GSCs behavior were assessed by live imaging cell cycle analysis, self-renewal assays, and chemosensitivity assays. Mechanistic insights into the role of Nav in regulating GBM stemness were investigated through pathway analysis in vitro and through tumor proliferation assay in vivo. RESULTS: We demonstrated that Nav is functionally expressed by GSCs mainly during the G0 phase of the cell cycle, suggesting its pivotal role in modulating the RMP. The pharmacological blockade of Nav made GBM cells more susceptible to temozolomide (TMZ), a standard drug for this type of tumor, by inducing cell cycle re-entry from G0 phase to G1/S transition. Additionally, inhibition of Nav substantially influenced the self-renewal and multipotency features of GSCs, concomitantly enhancing their degree of differentiation. Finally, our data suggested that Nav positively regulates GBM stemness by depolarizing the RMP and suppressing the ERK signaling pathway. Of note, in vivo proliferation assessment confirmed the increased susceptibility to TMZ following pharmacological blockade of Nav. CONCLUSIONS: This insight positions Nav as a promising prognostic biomarker and therapeutic target for GBM patients, particularly in conjunction with temozolomide treatment.
Assuntos
Diferenciação Celular , Glioblastoma , Células-Tronco Neoplásicas , Canais de Sódio Disparados por Voltagem , Glioblastoma/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Diferenciação Celular/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Animais , Temozolomida/farmacologia , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Proliferação de Células/efeitos dos fármacos , CamundongosRESUMO
SUMMARY: Imagine if we could simultaneously predict spatial protein expression in tissues from their routine Hematoxylin and Eosin (H&E) stained images, and create tissue images given protein expression profiles thus enabling virtual simulations of how protein expression alterations impact histology in complex diseases like cancer. Such an approach could lead to more informed diagnostic and therapeutic decisions for precision medicine at lower costs and shorter turnaround times, more detailed insights into underlying disease pathology as well as improvement in predictive and generative performance. In this study, we investigate the intricate correlation between protein expressions obtained from Hyperion mass cytometry and histopathological microstructures in conventional H&E stained glioblastoma (GBM) samples, unveiling morphological patterns and cellular-level spatial alterations associated with protein expression changes. To model these complex relationships, we propose a novel generative-predictive framework called Ouroboros for producing H&E images from protein expressions and simultaneously predicting protein expressions from H&E images. Our comprehensive sample-independent validation over 9920 tissue spots from 4 GBM samples encompassing visual image analysis, quantitative analysis, subspace alignment and perturbation experiments shows that the proposed generative-predictive approach offers significant improvements in predicting protein expression from images in comparison to baseline methods as well as accurate generation of virtual GBM sample images. This proof of concept study can contribute to advancing our understanding of histological responses to protein expression perturbations and lays the foundations for further developments in this area. AVAILABILITY AND IMPLEMENTATION: Implementation and associated data for the proposed approach are available at the URL: https://github.com/Srijay/Ouroboros.
Assuntos
Glioblastoma , Humanos , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Biologia Computacional/métodosRESUMO
Glioblastoma remains one of the deadliest brain malignancies. First-line therapy consists of maximal surgical tumor resection, accompanied by chemotherapy and radiotherapy. Malignant cells escape surgical resection by migrating into the surrounding healthy brain tissue, where they give rise to the recurrent tumor. Based on gene expression, tumor cores can be subtyped into mesenchymal, proneural, and classical tumors, each being associated with differences in genetic alterations and cellular composition. In contrast, the adjacent brain parenchyma where infiltrating malignant cells escape surgical resection is less characterized in patients. Using spatial transcriptomics (n = 11), we show that malignant cells within proneural or mesenchymal tumor cores display spatially organized differences in gene expression, although such differences decrease within the infiltrated brain tissue. Malignant cells residing in infiltrated brain tissue have increased expression of genes related to neurodevelopmental pathways and glial cell differentiation. Our findings provide an updated view of the spatial landscape of glioblastomas and further our understanding of the malignant cells that infiltrate the healthy brain, providing new avenues for the targeted therapy of these cells after surgical resection.
Assuntos
Neoplasias Encefálicas , Encéfalo , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Receptores Notch , Transdução de Sinais , Humanos , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Receptores Notch/metabolismo , Receptores Notch/genética , Encéfalo/metabolismo , Encéfalo/patologia , Transcriptoma , Sinapses/metabolismo , Masculino , Feminino , Linhagem Celular Tumoral , Neuroglia/metabolismo , Neuroglia/patologia , Diferenciação Celular/genéticaRESUMO
Glioblastoma (GBM) represents an aggressive brain tumor, characterized by intra- and inter-tumoral heterogeneity and therapy resistance, leading to unfavourable prognosis. An increasing number of studies pays attention on the regulation of ferroptosis, an iron-dependent cell death, as a strategy to reverse drug resistance in cancer. However, the debate on whether this strategy may have important implications for the treatment of GBM is still ongoing. In the present study, we used ferric ammonium citrate and erastin to evaluate ferroptosis induction effects on two human GBM cell lines, U-251 MG, with proneural characteristics, and T98-G, with a mesenchymal profile. The response to ferroptosis induction was markedly different between cell lines, indeed T98-G cells showed an enhanced antioxidant defence, with increased glutathione levels, as compared to U-251 MG cells. Moreover, using bioinformatic approaches and analysing publicly available datasets from patients' biopsies, we found that GBM with a mesenchymal phenotype showed an up-regulation of several genes involved in antioxidant mechanisms as compared to proneural subtype. Thus, our results suggest that GBM subtypes differently respond to ferroptosis induction, emphasizing the significance of further molecular studies on GBM to better discriminate between various tumor subtypes and progressively move towards personalized therapy.
Assuntos
Antioxidantes , Ferroptose , Glioblastoma , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Humanos , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , Linhagem Celular Tumoral , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Compostos Férricos/farmacologia , Compostos de Amônio Quaternário/farmacologia , Glutationa/metabolismo , PiperazinasRESUMO
Background: Glioblastoma (GBM) is characterized by abundant neovascularization as an essential hallmark. Vasculogenic mimicry (VM) is a predominant pattern of GBM neovascularization. However, the biological functions of circRNAs prompting VM formation in GBM remains unclarified. Methods: The circular RNA circCMTM3 was identified through high-throughput sequencing and bioinformatics analysis. The expression of circCMTM3 in exosomes in glioma tissues and cells was verified via RT-qPCR and FISH. In vitro and in vivo assays, such as EdU, MTS, Transwell, and tube formation assays were performed to investigate functional roles of circCMTM3. Meanwhile, in situ tumorigenesis assay were implemented to explore the influences of circCMTM3 on the GBM progression. Additionally, RNA pull-down, RIP, ChIP, and dual-luciferase reporter gene assays were executed to confirm the underlying regulation mechanism of circCMTM3. Results: CircCMTM3, as a novel circular RNA, was packaged into exosomes derived from glioblastoma stem cells (GSCs), which facilitates the phenotypic transition of differentiated glioma cells (DGCs) to VM. Mechanistically, exosomal circCMTM3 is internalized by DGCs and disrupt the ubiquitination degradation of STAT5A and STAT5B by E3 ubiquitin ligase CNOT4. Additionally, through molecular scaffold function of circCMTM3, STAT5A is activated and triggers transcriptional regulation of target genes including the pro-vasculogenic factor CHI3L2 and the RNA-binding protein SRSF1. Subsequently, circCMTM3/STAT5A/SRSF1 positive feedback loop sustainably enhances VM formation and accelerates tumor progression in GBM. Conclusion: Exosomal circCMTM3 possessing growth factor-mimetic property activates the JAK2/STAT5A pathway via non-canonical manner, and promotes VM formation in GBM. The molecular communications between GSCs and DGCs offers a therapeutic strategy for targeting the neovascularization of GBM.
Assuntos
Neoplasias Encefálicas , Exossomos , Glioblastoma , Células-Tronco Neoplásicas , Neovascularização Patológica , RNA Circular , Fator de Transcrição STAT5 , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , RNA Circular/metabolismo , RNA Circular/genética , Humanos , Fator de Transcrição STAT5/metabolismo , Exossomos/metabolismo , Neovascularização Patológica/metabolismo , Linhagem Celular Tumoral , Animais , Células-Tronco Neoplásicas/metabolismo , Camundongos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Fosforilação , Camundongos Nus , Regulação Neoplásica da Expressão Gênica , Camundongos Endogâmicos BALB C , Proteínas Supressoras de TumorRESUMO
Glioblastoma (GBM) is the most aggressive glial tumor of the adult brain, associated with invariably fatal outcome, and a deeper understanding of the underlying malignant mechanisms is necessary to address the current therapeutic failure. We previously demonstrated the role of the CXCL12/CXCR4 axis in GBM cell migration and resistance to ionizing radiation. The atypical chemokine receptor ACKR3, responsible for CXCL12 scavenging, was previously suggested as additional important player in the context of GBM. Following validation of the detection tools, we observed that ACKR3 is expressed within GBM patient tumor tissue, distributed in diverse cell types. In contrast to CXCR4, ACKR3 expression in patient-derived stem-like cells (GSCs) remains however low, while ACKR3 gene expression by tumor cells appears to be modulated by the in-vivo environment. Using overexpression models, we also showed that in vitro ACKR3 had no significant direct effect on cell proliferation or invasion. Altogether, these results suggest that in vitro ACKR3 plays a minor role in malignant GBM cell biology and that its expression is possibly regulated by in-vivo influences. The subtle and multifaceted functions ACKR3 could exert in GBM should therefore only be tackled within a comprehensive tumor microenvironment considering tumoral but also non-tumoral cells.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Receptores CXCR , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Receptores CXCR/metabolismo , Receptores CXCR/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Linhagem Celular Tumoral , Movimento Celular/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genética , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Microambiente Tumoral/genéticaRESUMO
Glioblastoma (GBM) remains one of the most aggressive and lethal brain tumours, characterized by rapid progression and limited treatment options. This study investigated the regulatory roles of circular RNA circJUN, and its functional interaction with microRNA miR-3064 in GBM pathogenesis. We employed bioinformatic analyses and clinical sample validation to identify circJUN as a potential target in GBM. Subsequently, we engineered GBM cell lines with stable circJUN knockout or overexpression, and transfected them with miR-3064 mimic/inhibitor or IGFBP5 small interfering RNA (siRNA)/expression vector to elucidate the molecular mechanisms governing GBM proliferation and invasion. To investigate the in vivo effects, xenograft tumour models were established in nude mice using engineered cells to assess the roles of circJUN in tumour growth regulation. Our analyses revealed significant overexpression of circJUN in GBM tissues compared to healthy controls, which strongly correlated with poor patient prognosis. In vitro and in vivo experiments demonstrated that circJUN overexpression could enhance GBM cell proliferation and invasion. Mechanistic investigations uncovered EIF4A3 as an interacting factor of circJUN which promotes circJUN expression, and circJUN modulates miR-3064 activity to regulate the malignancy of GBM cells. Furthermore, we identified IGFBP5, a crucial regulator of cell growth, as a direct target of miR-3064, thereby establishing an additional layer of control over GBM proliferation and invasion. Our study unveils a complex regulatory network involving circJUN, miR-3064 and IGFBP5 in GBM pathogenesis, underscoring their potential as novel therapeutic targets for improving patient outcomes. Our findings not only contribute to the understanding of GBM biology but also pave the way for innovative therapeutic approaches in the management of this malignancy.
Assuntos
Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina , Camundongos Nus , MicroRNAs , Invasividade Neoplásica , RNA Circular , MicroRNAs/genética , MicroRNAs/metabolismo , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/metabolismo , Humanos , Proliferação de Células/genética , RNA Circular/genética , RNA Circular/metabolismo , Animais , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Linhagem Celular Tumoral , Camundongos , Movimento Celular/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Masculino , Feminino , Ensaios Antitumorais Modelo de Xenoenxerto , PrognósticoRESUMO
Despite considerable improvements in understanding the biology of glioblastoma (GB), it still remains the most lethal type of brain tumor in adults. The role of innate immune cells in the development of GB was recently described. In particular, the tumor-immune cell interactions are thought to be critical in enabling tumor tolerance and even protection against therapeutics. Interestingly, the GB cells express proteins belonging to the family of intracellular pattern-recognition receptors, namely the NOD-like receptors (NLRs). Their activation may trigger the formation of the inflammasome complex leading to the secretion of mature IL-1ß and IL-18 and thus resulting in cell death. Intrudingly, the expression of most NLRs was found to be correlated with tumor progression and poor prognosis. We speculate that recognizing the role of NOD-like receptors in GB has the potential to improve the effectiveness of diagnostic tools and prognosis, while also encouraging the development of novel precision medicine-based therapies.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/metabolismo , Glioblastoma/imunologia , Glioblastoma/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/genética , Prognóstico , Animais , Proteínas NLR/metabolismo , Inflamassomos/metabolismo , Inflamassomos/imunologiaRESUMO
Tumor-associatedmacrophages (TAMs) exhibit remarkable heterogeneity in glioblastoma. Spatially resolved single-cell transcriptomic studies identified a monocyte-derived TAM subset localized in the peri-necrotic niche, driven by hypoxic cues to acquire ahypoxia response signature. These hypoxia-TAMs destabilize endothelial adherens junctions through adrenomedullin paracrine signaling, promoting the formation of hyperpermeable neovasculature that impedes drug delivery. Blocking adrenomedullin produced by hypoxia-TAMs restores vascular integrity, increases drug deliveryinto tumors, and provides combinatorial therapeutic benefits. Here we discuss the heterogeneity of TAMs regarding functional states and locations in glioblastomas, and propose future directions for studying the temporospatial dynamics of multifaceted TAM. HIGHLIGHTS: Single-cell omics reveal a functionally and spatially distinct hypoxia-TAM subset in glioblastoma. Adrenomedullin secreted by hypoxia-TAM destabilizes tumor vasculature and its blockade enhances vessel integrity and drug delivery.
Assuntos
Glioblastoma , Macrófagos , Microambiente Tumoral , Glioblastoma/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Humanos , Macrófagos/metabolismo , Macrófagos Associados a Tumor/metabolismoRESUMO
In tumors with WT p53, alternate mechanisms of p53 inactivation are reported. Here, we have identified a long noncoding RNA, PITAR (p53 Inactivating TRIM28 Associated RNA), as an inhibitor of p53. PITAR is an oncogenic Cancer/testis lncRNA and is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). We establish that TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase, is a direct target of PITAR. PITAR interaction with TRIM28 RNA stabilized TRIM28 mRNA, which resulted in increased TRIM28 protein levels and reduced p53 steady-state levels due to enhanced p53 ubiquitination. DNA damage activated PITAR, in addition to p53, in a p53-independent manner, thus creating an incoherent feedforward loop to inhibit the DNA damage response by p53. While PITAR silencing inhibited the growth of WT p53 containing GSCs in vitro and reduced glioma tumor growth in vivo, its overexpression enhanced the tumor growth in a TRIM28-dependent manner and promoted resistance to Temozolomide. Thus, we establish an alternate way of p53 inactivation by PITAR, which maintains low p53 levels in normal cells and attenuates the DNA damage response by p53. Finally, we propose PITAR as a potential GBM therapeutic target.
Assuntos
Dano ao DNA , RNA Longo não Codificante , Proteína 28 com Motivo Tripartido , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/genética , Proteína 28 com Motivo Tripartido/metabolismo , Proteína 28 com Motivo Tripartido/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Linhagem Celular Tumoral , Animais , Masculino , Glioma/metabolismo , Glioma/genética , Glioma/patologia , Regulação Neoplásica da Expressão Gênica , Estabilidade de RNA , Ligação Proteica , Camundongos , Glioblastoma/genética , Glioblastoma/metabolismoRESUMO
BACKGROUND: Patients with glioblastoma (GBM) have a poor prognosis and limited treatment options. The mRNA decapping enzyme scavenger (DCPS) is a cap-hydrolyzing enzyme. The DCPS inhibitor RG3039 exhibited excellent central nervous system bioavailability in vivo and was safe and well tolerated in healthy volunteers in a phase 1 clinical trial. In this study, we investigated the expression of DCPS in GBM and the anti-tumor activity of RG3039 in various preclinical models of GBM. METHODS: DCPS expression was examined in human GBM and paired peritumoral tissues. Its prognostic role was evaluated together with clinicopathological characteristics of patients. The anti-GBM effect of RG3039 was determined using GBM cell lines, patient-derived organoids, and orthotopic mouse models. The therapeutic mechanisms of DCPS inhibition were explored. RESULTS: DCPS is overexpressed in GBM and is associated with poor survival of patients with GBM. The DCPS inhibitor RG3039 exhibited robust anti-GBM activities in GBM cell lines, patient-derived organoids and orthotopic mouse models, with drug exposure achievable in humans. Mechanistically, RG3039 downregulated STAT5B expression, thereby suppressing proliferation, survival and colony formation of GBM cells. CONCLUSIONS: DCPS is a promising target for GBM. Inhibition of DCPS with RG3039 at doses achievable in humans downregulates STAT5B expression and reduces proliferation, survival and colony formation of GBM cells. Given the excellent anti-cancer activity and central nervous system bioavailability in vivo and good tolerance in humans, RG3039 warrants further study as a potential GBM therapy.
Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Animais , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Endorribonucleases/metabolismo , Endorribonucleases/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Feminino , Masculino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Camundongos Nus , Camundongos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To explore the inhibitory effect ORY-1001, a lysine-specific histone demethylase 1 (LSD1) inhibitor, on growth of glioblastoma (GBM) and the underlying mechanism. METHODS: We analyzed LSD1 expressions in GBM and normal brain tissues based on data from TCGA and HPA databases. Female BALB/c mouse models bearing xenografts derived from U87 cells or cells with lentivirus-mediated LSD1 silencing or Notch overexpression were treated with saline or 400 µg/kg ORY-1001 by gavage every 7 days, and GBM formation and survival time of the mice were recorded. The effect of ORY-1001 on GBM cell viability was assessed, and its effect on LSD1 expression was analyzed with Western blotting. The genes and pathways associated with LSD1 were analyzed using bioinformatics methods. Western blotting and qRT-PCR were used to detect Notch/HES1 pathway expression after LSD1 silencing and ORY-1001 treatment. The impact of ORY-1001 on viability of U87 cells with Notch1 silencing or overexpression was assessed, and the regulatory effects of ORY-1001 on Notch/HES1 pathway were analyzed using chromatin immunoprecipitation assay. RESULTS: A high expression of LSD1 in GBM was negatively correlated with patient survival (P < 0.001). ORY-1001 and LSD1 silencing obviously reduced tumor burden and prolonged the survival time of GBM-bearing mice. ORY-1001 treatment significantly inhibited the viability and dose-dependently decreased LSD1 expression in GBM cells, and such inhibitory effect of ORY-1001 was attenuated by LSD1 silencing. The Notch pathway enriched the differential genes related to LSD1, and Notch/HES1 pathway expression was significantly down-regulated after LSD1 silencing and ORY-1001 treatment. Notch1 overexpression significantly attenuated the anti-tumor effect of ORY-1001 on GBM. Mechanistically, ORY-1001 disrupted the interaction between LSD1 and the Notch pathway target genes including Notch3, HES1 and CR2. CONCLUSION: ORY-1001 down-regulates the Notch/HES1 pathway by inhibiting LSD1 expression to suppress the growth of GBM in mice.
Assuntos
Proliferação de Células , Glioblastoma , Histona Desmetilases , Camundongos Endogâmicos BALB C , Fatores de Transcrição HES-1 , Histona Desmetilases/metabolismo , Histona Desmetilases/genética , Animais , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , Camundongos , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição HES-1/metabolismo , Fatores de Transcrição HES-1/genética , Humanos , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais , Receptores Notch/metabolismo , Regulação para Baixo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologiaRESUMO
Antipsychotic drugs have been shown to have antitumor effects but have had limited potency in the clinic. Here, we unveil that pimozide inhibits lysosome hydrolytic function to suppress fatty acid and cholesterol release in glioblastoma (GBM), the most lethal brain tumor. Unexpectedly, GBM develops resistance to pimozide by boosting glutamine consumption and lipogenesis. These elevations are driven by SREBP-1, which we find upregulates the expression of ASCT2, a key glutamine transporter. Glutamine, in turn, intensifies SREBP-1 activation through the release of ammonia, creating a feedforward loop that amplifies both glutamine metabolism and lipid synthesis, leading to drug resistance. Disrupting this loop via pharmacological targeting of ASCT2 or glutaminase, in combination with pimozide, induces remarkable mitochondrial damage and oxidative stress, leading to GBM cell death in vitro and in vivo. Our findings underscore the promising therapeutic potential of effectively targeting GBM by combining glutamine metabolism inhibition with lysosome suppression.
Assuntos
Glioblastoma , Glutamina , Metabolismo dos Lipídeos , Lisossomos , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glutamina/metabolismo , Humanos , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Linhagem Celular Tumoral , Animais , Sistema ASC de Transporte de Aminoácidos/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Camundongos , Glutaminase/metabolismo , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Antígenos de Histocompatibilidade MenorRESUMO
Given the infiltrative nature of human glioblastoma (GBM), cocktail drug therapy will remain a vital tool for the treatment of the disease. We investigated fluspirilene, perphenazine, and sulpiride, three classic anti-schizophrenic drugs, as possible anti-GBM agents. The CCK-8 assay demonstrated that fluspirilene possesses the most outstanding anti-GBM effect. We performed molecular mechanisms studies in vitro and an orthotopic xenograft model in mice. Fluspirilene inhibited proliferation and migration in vitro in U87MG and U251 GBM cell lines. Flow cytometry demonstrated that treatment increased apoptosis and cells accumulated in the G2/M phase. Our analysis of publicly available expression data for several cell lines treated with the drug led to the identification of several genes, including KIF20A, that are downregulated by fluspirilene and lead to growth inhibition/apoptosis. We also demonstrated that siRNA knockdown of KIF20A, a member of the kinesin family, attenuated cell proliferation in GBM cells and an orthotopic xenograft model in mice. A regulator of KIF20A, the oncogenic transcription factor FOXM1, was identified using the String database, which harbors protein interaction networks. In fluspirilene-treated cells, FOXM1 protein was decreased, indicating that KIF20A was downregulated in the presence of the drug due to decreased FOXM1 protein. These results demonstrate that fluspirilene is an effective anti-GBM agent that works by suppressing the FOXM1-KIF20A oncogenic axis.
Assuntos
Apoptose , Proliferação de Células , Proteína Forkhead Box M1 , Glioblastoma , Cinesinas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Forkhead Box M1/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Humanos , Animais , Camundongos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Cinesinas/antagonistas & inibidores , Cinesinas/metabolismo , Movimento Celular/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Rationale: Glioma stem cells (GSCs) have emerged as pivotal drivers of tumor malignancy, sustained by various microenvironmental factors, including immune molecules and hypoxia. In our previous study, we elucidated the significant role of transforming growth factor beta-induced protein (TGFBI), a protein secreted by M2-like tumor-associated macrophages, in promoting the malignant behavior of glioblastoma (GBM) under normoxic conditions. Building upon these findings, the objective of this study was to comprehensively explore the crucial role and underlying mechanisms of autocrine TGFBI in GSCs under hypoxic conditions. Methods: We quantified TGFBI expression in glioma specimens and datasets. In vitro and in vivo assays were employed to investigate the effects of TGFBI on sustaining self-renewal and tumorigenesis of GSCs under hypoxia. RNA-seq and LC-MS/MS were conducted to explore TGFBI signaling mechanisms. Results: TGFBI is preferentially expressed in GSCs under hypoxic conditions. Targeting TGFBI impair GSCs self-renewal and tumorigenesis. Mechanistically, TGFBI was upregulated by HIF1α in GSCs and predominantly activates the AKT-c-MYC signaling pathway in GSCs by stabilizing the EphA2 protein through preventing its degradation. Conclusion: TGFBI plays a crucial role in maintaining the stem cell properties of GSCs in the hypoxic microenvironment. Targeting the TGFBI/EphA2 axis emerges as a promising and innovative strategy for GBM treatment, with the potential to improve the clinical outcomes of patients.