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BACKGROUND: Temozolomide (TMZ) treatment efficacy in glioblastoma is determined by various mechanisms such as TMZ efflux, autophagy, base excision repair (BER) pathway, and the level of O6-methylguanine-DNA methyltransferase (MGMT). Here, we reported a novel small-molecular inhibitor (SMI) EPIC-1042 (C20H28N6) with the potential to decrease TMZ efflux and promote PARP1 degradation via autolysosomes in the early stage. METHODS: EPIC-1042 was obtained from receptor-based virtual screening. Co-immunoprecipitation and pull-down assays were applied to verify the blocking effect of EPIC-1042. Western blotting, co-immunoprecipitation, and immunofluorescence were used to elucidate the underlying mechanisms of EPIC-1042. In vivo experiments were performed to verify the efficacy of EPIC-1042 in sensitizing glioblastoma cells to TMZ. RESULTS: EPIC-1042 physically interrupted the interaction of PTRF/Cavin1 and caveolin-1, leading to reduced secretion of small extracellular vesicles (sEVs) to decrease TMZ efflux. It also induced PARP1 autophagic degradation via increased p62 expression that more p62 bound to PARP1 and specially promoted PARP1 translocation into autolysosomes for degradation in the early stage. Moreover, EPIC-1042 inhibited autophagy flux at last. The application of EPIC-1042 enhanced TMZ efficacy in glioblastoma in vivo. CONCLUSION: EPIC-1042 reinforced the effect of TMZ by preventing TMZ efflux, inducing PARP1 degradation via autolysosomes to perturb the BER pathway and recruitment of MGMT, and inhibiting autophagy flux in the later stage. Therefore, this study provided a novel therapeutic strategy using the combination of TMZ with EPIC-1042 for glioblastoma treatment.
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Glioblastoma , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/genética , Dacarbazina/uso terapêutico , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Caveolina 1/metabolismo , Caveolina 1/farmacologia , Caveolina 1/uso terapêutico , Linhagem Celular Tumoral , Enzimas Reparadoras do DNA/genética , Metilases de Modificação do DNA/genética , Autofagia , Resistencia a Medicamentos Antineoplásicos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/farmacologia , Poli(ADP-Ribose) Polimerase-1/uso terapêuticoRESUMO
BACKGROUND: Metabolism reprogramming plays a vital role in glioblastoma (GBM) progression and recurrence by producing enough energy for highly proliferating tumor cells. In addition, metabolic reprogramming is crucial for tumor growth and immune-escape mechanisms. Epidermal growth factor receptor (EGFR) amplification and EGFR-vIII mutation are often detected in GBM cells, contributing to the malignant behavior. This study aimed to investigate the functional role of the EGFR pathway on fatty acid metabolism remodeling and energy generation. METHODS: Clinical GBM specimens were selected for single-cell RNA sequencing and untargeted metabolomics analysis. A metabolism-associated RTK-fatty acid-gene signature was constructed and verified. MK-2206 and MK-803 were utilized to block the RTK pathway and mevalonate pathway induced abnormal metabolism. Energy metabolism in GBM with activated EGFR pathway was monitored. The antitumor effect of Osimertinib and Atorvastatin assisted by temozolomide (TMZ) was analyzed by an intracranial tumor model in vivo. RESULTS: GBM with high EGFR expression had characteristics of lipid remodeling and maintaining high cholesterol levels, supported by the single-cell RNA sequencing and metabolomics of clinical GBM samples. Inhibition of the EGFR/AKT and mevalonate pathways could remodel energy metabolism by repressing the tricarboxylic acid cycle and modulating ATP production. Mechanistically, the EGFR/AKT pathway upregulated the expressions of acyl-CoA synthetase short-chain family member 3 (ACSS3), acyl-CoA synthetase long-chain family member 3 (ACSL3), and long-chain fatty acid elongation-related gene ELOVL fatty acid elongase 2 (ELOVL2) in an NF-κB-dependent manner. Moreover, inhibition of the mevalonate pathway reduced the EGFR level on the cell membranes, thereby affecting the signal transduction of the EGFR/AKT pathway. Therefore, targeting the EGFR/AKT and mevalonate pathways enhanced the antitumor effect of TMZ in GBM cells and animal models. CONCLUSIONS: Our findings not only uncovered the mechanism of metabolic reprogramming in EGFR-activated GBM but also provided a combinatorial therapeutic strategy for clinical GBM management.
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Glioblastoma , Animais , Linhagem Celular Tumoral , Metabolismo Energético , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ácidos Graxos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Ligases/metabolismo , Ácido Mevalônico/antagonistas & inibidores , Ácido Mevalônico/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Temozolomida/farmacologia , Temozolomida/uso terapêuticoRESUMO
Rationale: Glioblastoma (GBM) displays a complex metabolic reprogramming in cancer cells. Adenosine triphosphate (ATP) is one of the central mediators of cell metabolism and signaling. GBM cells generate ATP by glycolysis and the tricarboxylic acid (TCA) cycle associated with oxidative phosphorylation (OXPHOS) through the breaking-down of pyruvate or fatty acids to meet the growing energy demand of cancer cells. Therefore, it's urgent to develop novel treatments targeting energy metabolism to hinder tumor cell proliferation in GBM. Methods: Non-targeted metabolomic profiling analysis was utilized to evaluate cell metabolic reprogramming using a small molecule inhibitor (SMI) EPIC-0412 treatment. Cellular oxygen consumption rate (OCR) and the total proton efflux rate (PER), as well as ATP concentration, were tracked to study metabolic responses to specifically targeted inhibitors, including EPIC-0412, arachidonyl trifluoromethyl ketone (AACOCF3), and 2 deoxy-D-glucose (2-DG). Cancer cell proliferation was assessed by CCK-8 measurements and colony formation assay. Additionally, flow cytometry, immunoblotting (IB), and immunofluorescence (IF) analyses were performed with GBM cells to understand their tumorigenic properties under treatments. Finally, the anticancer effects of this combination therapy were evaluated in the GBM mouse model by convection-enhanced delivery (CED). Results: We found that SMI EPIC-0412 could effectively perturb the TCA cycle, which participated in the combination therapy of cytosolic phospholipase A2 (cPLA2)-inhibitor AACOCF3, and hexokinase II (HK2)-inhibitor 2-DG to disrupt the GBM energy metabolism for targeted metabolic treatments. ATP production was significantly declined in glioma cells when treated with monotherapy (EPIC-0412 or AACOCF3), dual therapy (EPIC-0412 + AACOCF3), or triple therapy (EPIC-0412 + AACOCF3 +2-DG) regimen. Our experiments revealed that these therapies hindered glioma cell proliferation and growth, leading to the reduction in ATP production and G0/G1 cell cycle arrest. We demonstrated that the combination therapy effectively extended the survival of cerebral tumor-bearing mice. Conclusion: Our findings indicate that the TCA-phospholipid-glycolysis metabolism axis can be blocked by specific inhibitors that significantly disrupt the tumor energy metabolism and suppress tumor proliferation in vitro and in vivo, suggesting that targeting ATP synthesis inhibition in cancer cells might be an attractive therapeutic avenue in GBM management.
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Glioblastoma , Glioma , Fosfolipídeos , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Ácidos Graxos , Glioblastoma/metabolismo , Glucose/metabolismo , Glicólise/fisiologia , Hexoquinase/antagonistas & inibidores , Fosfolipases A2/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Fosfolipídeos/metabolismo , Prótons , Piruvatos/metabolismo , Ácidos Tricarboxílicos/uso terapêuticoRESUMO
Glioblastoma (GBM) is the most common and lethal type of primary malignant central nervous system (CNS) tumor with an extremely poor prognosis, and the mesenchymal subtype of GBM has the worst prognosis. Here, we found that lncRNA PRADX was overexpressed in the mesenchymal GBM and was transcriptionally regulated by RUNX1-CBFß complex, overexpressed PRADX suppressed BLCAP expression via interacting with EZH2 and catalyzing trimethylation of lysine 27 on histone H3 (H3K27me3). Moreover, we showed that BLCAP interacted with STAT3 and reduced STAT3 phosphorylation, overexpressed PRADX activated STAT3 phosphorylation, and promoted ACSL1 expression via suppressing BLCAP expression, accelerating tumor metabolism. Finally, we determined that combined of ACSL1 and CPT1 inhibitors could reverse the accelerated cellular metabolism and tumor growth induced by PRADX overexpression in vivo and in vitro. Collectively, PRADX/PRC2 complex activated the STAT3 pathway and energy metabolism in relation to mesenchymal GBM progression. Furthermore, our findings provided a novel therapeutic strategy targeting the energy metabolism activity of GBM.
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BACKGROUND: Targeting glioblastoma (GBM) energy metabolism through multiple metabolic pathways has emerged as an effective therapeutic approach. Dual inhibition of phospholipid and mitochondrial metabolism with cytoplasmic phospholipase A2 (cPLA2) knockdown and metformin treatment could be a potential strategy. However, the strategic prerequisite is to explore a carrier capable of co-delivering the therapeutic combination to cross the blood-brain barrier (BBB) and preferentially accumulate at the GBM site. METHODS: Blood exosomes (Exos) were selected as the combination delivery carriers. The cellular uptake of Exos and the therapeutic effects of the combination strategy were evaluated in primary GBM cells. In vivo GBM-targeted delivery efficiency and anti-GBM efficacy were tested in a patient-derived xenograft (PDX) model. RESULTS: Here, we showed that the Exos-mediated cPLA2 siRNA/metformin combined strategy could regulate GBM energy metabolism for personalized treatment. Genomic analysis and experiments showed that polymerase 1 and transcript release factor (PTRF, a biomarker of GBM) positively regulated the uptake of Exos by GBM cells, confirming the feasibility of the delivery strategy. Further, Exos could co-load cPLA2 siRNA (sicPLA2) and metformin and co-deliver them across the BBB and into GBM tissue. The mitochondrial energy metabolism of GBM was impaired with this combination treatment (Exos-Met/sicPLA2). In the PDX GBM model, systemic administration of Exos-Met/sicPLA2 reduced tumor growth and prolonged survival. CONCLUSIONS: Our findings demonstrated that Exos-based combined delivery of sicPLA2 and metformin selectively targeted the GBM energy metabolism to achieve antitumor effects, showing its potential as a personalized therapy for GBM patients.
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Exossomos , Glioblastoma , Metformina , Humanos , Linhagem Celular Tumoral , Metabolismo Energético , Exossomos/metabolismo , Glioblastoma/patologia , Fosfolipases A2/metabolismo , Fosfolipases A2/uso terapêutico , Fosfolipases A2 Citosólicas/metabolismo , RNA Interferente Pequeno , Ensaios Antitumorais Modelo de Xenoenxerto , AnimaisRESUMO
Exosomes are small extracellular vehicles which could transport genetic materials and proteins between cells. Although there are reports about exosomes crossing the blood-brain barrier (BBB), the underlying mechanisms still need further study. We found that exosomes from primary brain tumors could upregulate the expression of Lipocalin-2 (LCN2) in bEnd.3 brain microvascular endothelial cells (BMVECs). Furthermore, exosomes increased the membrane fluidity of bEnd.3 cells in an LCN2 dependent manner. Both intraperitoneal injection and caudal vein injection of LCN2 increased the number of nanocapsules crossing the BBB. Evans Blue staining revealed that LCN2 does not interrupt the integrity of the BBB, as observed in the traumatic brain injury model. Tandem mass tags quantitative proteomics and bioinformatics analysis revealed that LCN2 is upregulated by exosomes via the JAK-STAT3 pathway, but not delivered from exosomes. Knocking down LCN2 in bEnd.3 cells significantly abrogated the effect of exosomes on BMVEC membrane fluidity. Previously, we have reported that 2-methacryloyloxyethyl phosphorylcholine (MPC) and a peptide crosslinker could encapsulate mAbs to achieve nanocapsules. The nanocapsules containing choline analogs could effectively penetrate the BBB to deliver therapeutic monoclonal antibodies (tAbs) to the glioma. However, the delivered tAbs could be significantly reduced by blocking the release of exosomes from the gliomas. Application of tAb nanocapsules prior to treatment with MK2206, an AKT pathway inhibitor that has been shown to inhibit the production of exosomes, resulted in a better combination. Insights from this study provide a mechanistic framework with regard to how glioblastomas hijack BMVECs using exosomes. In addition, we provide a strategy for maximizing the effect of the choline-containing nanocapsules and MK2206 combination. These results also demonstrate the therapeutic role of tAbs in glioblastoma and brain tumor metastasis, by shedding new light on strategies that can be used for BBB-penetrating therapies.
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Exossomos , Glioblastoma , Glioma , Nanocápsulas , Barreira Hematoencefálica/metabolismo , Colina , Células Endoteliais/metabolismo , Exossomos/metabolismo , Glioblastoma/metabolismo , Glioma/metabolismo , Humanos , Lipocalina-2/metabolismoRESUMO
Liposomes have been developed as drug delivery carriers to enhance the antitumor efficiency of therapeutic agents. Lipusu® (Lip), a paclitaxel (PTX) liposome, has been widely used in the treatment of breast cancer. Compared with PTX, Lip could change the biodistribution and reduce the systemic toxicity. However, there was no positive effect on the entry of PTX into tumor cells, and thus the therapeutic effect was not significantly improved. Therefore, it is meaningful to engineer Lip for improving tumor cellular uptake efficiency. Here, lysophosphatidylcholine (LPC)-engineered Lip (LPC-Lip) was constructed via inserting single chain lipid tails into liposomal lipid bilayers, which was realized by simple incubation. Compared with Lip, the better cellular uptake of liposomes modified with LPC resulted in enhanced cytotoxic activity of LPC-Lip in 4T1 cells. Furthermore, stronger tumor growth inhibition was observed in LPC-Lip treated 4T1 tumor-bearing mice without significant side effects. In conclusion, by modulating the lipid composition of Lip, the antitumor efficacy can be improved, and LPC engineered Lip may serve as a promising formulation of PTX for future cancer therapy.
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Neoplasias da Mama , Lipossomos , Animais , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Lipossomos/farmacologia , Lisofosfatidilcolinas/uso terapêutico , Camundongos , Paclitaxel/uso terapêutico , Distribuição TecidualRESUMO
Background: Checkpoint blockade therapies targeting programmed death ligand 1 (PD-L1) and its receptor programmed cell death 1 promote T cell-mediated immune surveillance against tumors and have been associated with significant clinical benefit in cancer patients. The long-stranded non-coding RNA HOTAIR is highly expressed and associated with metastasis in a variety of cancer types and promotes tumor metastasis at least in part through association with the PRC2 complex that induces redirection to hundreds of genes involved in tumor metastasis. Here, we report that HOTAIR is an activator lncRNA of the NF-κB pathway and demonstrate that its apparent upregulation promotes inflammatory signaling and immune escape in glioma cells. Methods: Bioinformatics analysis was used to elucidate the relationship between HOTAIR and NF-κB pathway in HOTAIR knockdown glioma cells. At the cytological level, protein hybridization and immunofluorescence were used to detect the response of proteins in the NF-κB signaling pathway to HOTAIR regulation. ChIP and ChIRP experiments identified HOTAIR target genes. Animal experiments verified alterations in inflammation and immune escape following HOTAIR knockdown and activity inhibition. Results: HOTAIR activated the expression of proteins involved in NF-κB, TNFα, MAPK and other inflammatory signaling pathways. In addition, HOTAIR induced various proteins containing protein kinase structural domains and promoted the enrichment of proteins and complexes of important inflammatory signaling pathways, such as the TNFα/NF-κB signaling protein complex, the IκB kinase complex, and the IKKA-IKKB complex. In addition, HOTAIR aberrantly activated biological processes involved in glioma immune responses, T-cell co-stimulation and transcription initiation by RNA polymerase II. HOTAIR facilitated the induction of IκBα phosphorylation by suppressing the expression of the NF-κB upstream protein UBXN1, promoting NF-κB phosphorylation and nuclear translocation. In vivo, reduction of HOTAIR decreased PD-L1 protein expression, indicating that cells are more likely to be targeted by immune T cells. Conclusion: In conclusion, our results provide convincing evidence that lncRNA HOTAIR drives aberrant gene transcription and immune escape from tumor cells through the NF-κB pathway.
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Neoplasias Encefálicas/genética , Glioma/genética , NF-kappa B/metabolismo , RNA Longo não Codificante/metabolismo , Evasão Tumoral/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Glioma/imunologia , Glioma/patologia , Humanos , Camundongos , RNA Longo não Codificante/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glioblastoma (GBM) is the most common primary central nervous system tumor and has a poor prognosis, with a median survival time of only 14 months from diagnosis. Abnormally expressed long noncoding RNAs (lncRNAs) are important epigenetic regulators of chromatin modification and gene expression regulation in tumors, including GBM. We previously showed that the lncRNA HOTAIR is related to the cell cycle progression and can be used as an independent predictor in GBM. Lysine-specific demethylase 1 (LSD1), binding to 3' domain of HOTAIR, speciï¬cally removes mono- and di-methyl marks from H3 lysine 4 (H3K4) and plays key roles during carcinogenesis. In this study, we combined a HOTAIR-EZH2 disrupting agent and an LSD1 inhibitor, AC1Q3QWB (AQB) and GSK-LSD1, respectively, to block the two functional domains of HOTAIR and potentially provide therapeutic benefit in the treatment of GBM. Using an Agilent Human ceRNA Microarray, we identified tumor suppressor genes upregulated by AQB and GSK-LSD1, followed by Chromatin immunoprecipitation (ChIP) assays to explore the epigenetic mechanisms of genes activation. Microarray analysis showed that AQB and GSK-LSD1 regulate cell cycle processes and induces apoptosis in GBM cell lines. Furthermore, we found that the combination of AQB and GSK-LSD1 showed a powerful effect of inhibiting cell cycle processes by targeting CDKN1A, whereas apoptosis promoting effects of combination therapy were mediated by BBC3 in vitro. ChIP assays revealed that GSK-LSD1 and AQB regulate P21 and PUMA, respectively via upregulating H3K4me2 and downregulating H3K27me3. Combination therapy with AQB and GSK-LSD1 on tumor malignancy in vitro and GBM patient-derived xenograft (PDX) models shows enhanced anti-tumor efficacy and appears to be a promising new strategy for GBM treatment through its effects on epigenetic regulation.
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Benzofuranos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Histona Desmetilases/antagonistas & inibidores , RNA Longo não Codificante/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Benzofuranos/farmacologia , Neoplasias Encefálicas/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Humanos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
Exosomes derived from non-tumor cells hold great potential as drug delivery vehicles because of their good biosafety and natural transference of bioactive cargo between cells. However, compared to tumor-derived exosomes, efficient delivery is limited by their weak interactions with tumor cells. It is essential to engineer exosomes that improve tumor cellular internalization efficiency. A simple and effective strategy to enhance tumor cell uptake by engineering the exosome membrane lipids can be established by drawing on the role of lipids in tumor exosomes interacting with tumor cells. Amphiphilic phosphatidylcholine (PC) molecules are inserted into the membrane lipid layer of reticulocyte-derived exosomes (Exos) by simple incubation to construct PC-engineered exosomes (PC-Exos). It is demonstrated that PC-Exos showed significantly enhanced tumor cell internalization and uptake rate compared to native Exos, up to a twofold increase. After therapeutic agent loading, PC-Exos remarkably promotes intracellular drug or RNA accumulation in cancer cells, thus showing enhanced in vitro anti-tumor activity. This work demonstrates the crucial role of engineering exosomal lipids in modulating cancer cellular uptake, which may shed light on the design of high-efficiency exosome-based drug delivery carriers.
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Antineoplásicos , Exossomos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Exossomos/metabolismo , FosfatidilcolinasRESUMO
Background: Immunotherapy, especially checkpoint inhibitors targeting PD-1 or PD-L1, has revolutionized cancer therapy. However, PD-1/PD-L1 inhibitors have not been investigated thoroughly in glioblastoma (GBM). Studies have shown that polymerase 1 and transcript release factor (PTRF/Cavin-1) has an immune-suppressive function in GBM. Thus, the relationship between PTRF and PD-L1 and their role in immune suppression requires further investigation in GBM. Methods: We used public databases and bioinformatics analysis to investigate the relationship between PTRF and PD-L1. We next confirmed the predicted relationship between PTRF and PD-L1 in primary GBM cell lines by using different experimental approaches. RIP-Seq, RIP, ChIP, and qRT-PCR were conducted to explore the molecular mechanism of PTRF in immunosuppression. Results: We found that PTRF stabilizes lncRNA NEAT1 to induce NF-κB and PD-L1 and promotes immune evasion in GBM. PTRF was found to correlate with immunosuppression in the public GBM databases. PTRF increased the level of PD-L1 in primary cell lines from GBM patients. We carried out RIP-Seq of GBM cells and found that PTRF interacts with lncRNA NEAT1 and stabilizes its mRNA. PTRF also promoted the activity of NF-κB by suppressing UBXN1 expression via NEAT1 and enhanced the transcription of PD-L1 through NF-κB activation. Finally, PTRF promoted immune evasion in GBM cells by regulating PD-1 binding and PD-L1 mediated T cell cytotoxicity. Conclusions: In summary, our study identified the PTRF-NEAT1-PD-L1 axis as a novel immune therapeutic target in GBM.
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Antígeno B7-H1/metabolismo , Glioblastoma/etiologia , Glioblastoma/metabolismo , NF-kappa B/metabolismo , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/mortalidade , Glioblastoma/patologia , Xenoenxertos , Humanos , Estimativa de Kaplan-Meier , Camundongos , Prognóstico , Estabilidade de RNA , Evasão TumoralRESUMO
BACKGROUND: Metabolism remodeling is a hallmark of glioblastoma (GBM) that regulates tumor proliferation and the immune microenvironment. Previous studies have reported that increased polymerase 1 and transcript release factor (PTRF) levels are associated with a worse prognosis in glioma patients. However, the biological role and the molecular mechanism of PTRF in GBM metabolism remain unclear. METHODS: The relationship between PTRF and lipid metabolism in GBM was detected by nontargeted metabolomics profiling and subsequent lipidomics analysis. Western blotting, quantitative real-time PCR, and immunoprecipitation were conducted to explore the molecular mechanism of PTRF in lipid metabolism. A sequence of in vitro and in vivo experiments (both xenograft tumor and intracranial tumor mouse models) were used to detect the tumor-specific impacts of PTRF. RESULTS: Here, we show that PTRF triggers a cytoplasmic phospholipase A2 (cPLA2)-mediated phospholipid remodeling pathway that promotes GBM tumor proliferation and suppresses tumor immune responses. Research in primary cell lines from GBM patients revealed that cells overexpressing PTRF show increased cPLA2 activity-resulting from increased protein stability-and exhibit remodeled phospholipid composition. Subsequent experiments revealed that PTRF overexpression alters the endocytosis capacity and energy metabolism of GBM cells. Finally, in GBM xenograft and intracranial tumor mouse models, we showed that inhibiting cPLA2 activity blocks tumor proliferation and prevents PTRF-induced reduction in CD8+ tumor-infiltrating lymphocytes. CONCLUSIONS: The PTRF-cPLA2 lipid remodeling pathway promotes tumor proliferation and suppresses immune responses in GBM. In addition, our findings highlight multiple new therapeutic targets for GBM.
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Glioblastoma , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Humanos , Imunidade , Metabolismo dos Lipídeos , Camundongos , Fosfolipases A2 , Fosfolipases A2 Citosólicas , Fosfolipídeos , Proteínas de Ligação a RNA/metabolismo , Microambiente TumoralRESUMO
Up-to-date knowledge regarding the biogenesis and functioning of microRNAs (miRNAs) has provided a much more comprehensive and concrete view of miRNA biology than anyone ever expected. Diverse genetic origins and biogenesis pathways leading to functional miRNAs converge on the synthesis of ≈21-nucleotide RNA duplex, almost all of which are processed from long premature sequences in a DICER- and/or DROSHA-dependent manner. Formerly, it was assumed that one mature strand of the duplex is preferentially selected for entry into the silencing complex, and the paired passenger strands (miRNA*) are subjected to degradation. However, given the consolidated evidence of substantial regulatory activity of miRNA* species, currently, this preconception has been overturned. Here, we see the caveat and opportunity toward exogenously manipulating the expression of premature miRNA, leading to simultaneous upregulation or downregulation of dual regulatory strands due to altered expressions. The caveat is the overlooked miRNA* interference while manipulating the expression of a target miRNA at the premature stage, wherein lies the opportunity. If the dual strands of a pre-miRNA function synergistically, the overlooked miRNA* interference may inversely optimize the therapeutic performance. Insightfully, targeting the premature miRNAs may serve as the "one-two punch" against diseases, especially cancers, and this has been discussed in detail in this review.
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The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) system holds great promise for the cancer gene therapy. However, due to complicated signal networks and various compensatory mechanisms in tumors, adjusting a single molecular pathway has limited effects on cancer treatments. Herein, a virus-like nanoparticle (VLN) was reported as a versatile nanoplatform to co-deliver CRISPR/Cas9 system and small molecule drugs for effective malignant cancer treatment. VLN has a core-shell structure, in which small molecule drugs and CRISPR/Cas9 system are loaded in the mesoporous silica nanoparticle (MSN)-based core, which is further encapsulated with a lipid shell. This structure allows VLN maintaining stable during blood circulation. As reaching tumors, VLN releases the CRISPR/Cas9 system and small molecule drugs in response to the reductive microenvironment, resulting in the synergistic regulation of multiple cancer-associated pathways. By loading a single guide RNA (sgRNA) targeting programmed death-ligand 1 and axitinib, VLN achieved to disrupt multiple immunosuppressive pathways and suppress the growth of melanoma in vivo. More importantly, VLN can co-deliver almost any combination of sgRNAs and small molecule drugs to tumors, suggesting the great potential of VLN as a general platform for the development of advanced combination therapies against malignant tumors.
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Nanopartículas , Neoplasias , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Imunoterapia , Neoplasias/genética , Neoplasias/terapiaRESUMO
Rationale: Developing an effective nanoplatform to realize 'multi-in-one' is essential to broaden the therapeutic potential of combination therapy. Exosomes are ideal candidates since their intrinsic abilities of integrating multiple contents and functions. However, only limited efforts have been devoted to engineering exosomes to integrate the needed properties, also considering the safety and yield, for tumor-targeted and efficient gene/chemo combination therapy. Methods: Herein, by manipulating the exosome membrane, blood exosomes with high abundance and safety are engineered as a versatile combinatorial delivery system, where the doxorubicin (Dox) and cholesterol-modified miRNA21 inhibitor (miR-21i) are co-embedded into the lipid bilayer of exosomes, and the magnetic molecules and endosomolytic peptides L17E are bind to the exosome membrane through ligand-receptor coupling and electrostatic interactions, respectively. Results: It is proved that such engineering strategy not only preserves their intrinsic features, but also readily integrates multiple properties of tumor targeting, efficient transfection and gene/chemo combination therapy into blood exosomes. The lipid bilayer structure of exosomes allows them to co-load Dox and miR-21i with high-payloads. Moreover, profiting from the integration of magnetic molecules and L17E peptides, the engineered exosomes exhibit an enhanced tumor accumulation and an improved endosome escape ability, thereby specifically and efficiently delivering encapsulated cargos to tumor cells. As a result, a remarkable inhibition of tumor growth is observed in the tumor-bearing mice, and without noticeable side effects. Conclusions: This study demonstrates the potential of engineered blood exosomes as feasible co-delivery nanosystem for tumor-targeted and efficient combination therapy. Further development by replacing the drugs combined regimens can potentially make this engineered exosome become a general platform for the design of safe and effective combination therapy modality.
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Engenharia Química/métodos , Sistemas de Liberação de Medicamentos/métodos , Exossomos/química , Terapia Genética/métodos , Neoplasias/terapia , Animais , Linhagem Celular Tumoral , Terapia Combinada/efeitos adversos , Terapia Combinada/métodos , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Feminino , Regulação Neoplásica da Expressão Gênica , Terapia Genética/efeitos adversos , Humanos , Nanopartículas Magnéticas de Óxido de Ferro , Camundongos , MicroRNAs/antagonistas & inibidores , Neoplasias/genética , Neoplasias/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The EGFR-vIII mutation is the most common malignant event in GBM. Epigenetic reprogramming in EGFR-activated GBM has recently been suggested to downregulate the expression of tumour suppressor genes. Histone acetylation is important for chromatin structure and function. However, the role and biological function of H2AZK4/7AC in tumours have not yet been clarified. RESULTS: In our study, we found that EGFR-vIII negatively regulated H2AZK4/7AC expression though the PI3K/AKT-HDAC2 axis. Because HDAC1 and HDAC2 are highly homologous enzymes that usually form multi-protein complexes for transcriptional regulation and epigenetic landscaping, we simultaneously knocked out HDAC1 and HDAC2 and found that H2AZK4/7AC and H3K27AC were upregulated, which partially released EGFR-vIII-mediated inhibition of USP11, negative regulator of cell cycle. In addition, we demonstrated in vitro and in vivo that FK228 induced G1/S transition arrest in GBM with EGFR-vIII mutation. FK228 could enhance anti-tumour activity by upregulating expression of the tumour suppressor USP11 in GBM cells. CONCLUSIONS: EGFR-vIII mutation downregulates H2AZK4/7AC and H3K27AC, inhibiting USP11 expression though the PI3K/AKT-HDAC1/2 axis. FK228 is an effective and promising treatment for GBM with EGFR-vIII mutation.
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Immunosuppression is a key factor leading to a low therapeutic efficiency of the currently used immunotherapies. Monotherapies are unable to overcome immunosuppression because of the complex interplay of immune cells in tumors. Herein, we report a multifunctional nanomodulator (MFNM) as a carrier to deliver different types of immune modulators for comodulating multiple pathways. An MFNM has a core-shell structure, in which small-molecule drugs are encapsulated in a mesoporous silica nanoparticle (MSN) core with a pH-responsive polymer layer. Further, the polymeric shell provides active sites that are readily modifiable by multiple types of antibodies to regulate the immune-related processes. By codelivering cyclophosphamide (CTX), αPD-L1 (B7-H1), and α4-1BB (CD137L) monoclonal antibodies (mAbs) to tumors, an MFNM has been shown to regulate multiple immune pathways and enhance an antitumor immunity. As antibodies and small-molecule drugs loaded in an MFNM can be modified based on the tumor type, the MFNM provides a feasible platform for the development of advanced immunotherapies that require simultaneous modulation of multiple biological processes.
RESUMO
Runt-Related Transcription Factor 1 (RUNX1) is highly expressed in the Mesenchymal (Mes) subtype of glioblastoma (GBM). However, the specific molecular mechanism of RUNX1 in Mes GBM remains largely elusive. In this study, cell and tumor tissue typing were performed by RNA-sequencing. Co-immunoprecipitation (co-IP) and immunofluorescence (IF) were employed to identify members of the RUNX1 transcriptional protein complex. Bioinformatics analysis, chromatin immunoprecipitation (ChIP), and luciferase reporter experiments were utilized to verify target genes. Analyses of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) verified the expression levels and prognoses associated with RUNX1/p-SMAD3/SUV39H1 target genes. In vivo patient-derived xenograft (PDX) studies and in vitro functional studies verified the impact of RUNX1 on the occurrence and development of GBM. The results showed that RUNX1 was upregulated in Mes GBM cell lines, tissues and patients and promoted proliferation and invasion in GBM in a TGFß pathway-dependent manner in vivo and in vitro. We found and verified that BCL3 and MGP are transcriptionally activated by p-SMAD3 /RUNX1, while MXI1 is transcriptionally suppressed by the RUNX1/SUV39H1-H3K9me3 axis. This finding offers a theoretical rationale for using molecular markers and choosing therapeutic targets for the Mes type of GBM.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Progressão da Doença , Glioblastoma/genética , Células HEK293 , Xenoenxertos , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prognóstico , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Regulação para CimaRESUMO
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) enzyme, Cas13a, holds great promise in cancer treatment due to its potential for selective destruction of tumor cells via collateral effects after target recognition. However, these collateral effects do not specifically target tumor cells and may cause safety issues when administered systemically. Herein, a dual-locking nanoparticle (DLNP) that can restrict CRISPR/Cas13a activation to tumor tissues is described. DLNP has a core-shell structure, in which the CRISPR/Cas13a system (plasmid DNA, pDNA) is encapsulated inside the core with a dual-responsive polymer layer. This polymer layer endows the DLNP with enhanced stability during blood circulation or in normal tissues and facilitates cellular internalization of the CRISPR/Cas13a system and activation of gene editing upon entry into tumor tissue. After carefully screening and optimizing the CRISPR RNA (crRNA) sequence that targets programmed death-ligand 1 (PD-L1), DLNP demonstrates the effective activation of T-cell-mediated antitumor immunity and the reshaping of immunosuppressive tumor microenvironment (TME) in B16F10-bearing mice, resulting in significantly enhanced antitumor effect and improved survival rate. Further development by replacing the specific crRNA of target genes can potentially make DLNP a universal platform for the rapid development of safe and efficient cancer immunotherapies.
Assuntos
Antígeno B7-H1/genética , Terapia Genética/métodos , Imunoterapia/métodos , Nanopartículas , Receptor de Morte Celular Programada 1/genética , Animais , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Camundongos , Terapia de Alvo Molecular , Nanopartículas/química , Plasmídeos/genética , Polietilenoglicóis/química , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
RNA is rarely used as a therapeutic target due to its flexible structure and instability. CRISPR-Cas13a is a powerful tool for RNA knockdown, and the potential application of CRISPR-Cas13a in cancer cells should be further studied. In this study, overexpression of LwCas13a by lentivirus in glioma cells reveals that crRNA-EGFP induces a "collateral effect" after knocking down the target gene in EGFP-expressing cells. EGFRvIII is a unique EGFR mutant subtype in glioma, and the CRISPR-Cas13a system induces death in EGFRvIII-overexpressing glioma cells. Bulk and single-cell RNA sequencing analysis in U87-Cas13a-EGFRvIII cells confirm the collateral effect of the CRISPR-Cas13a system. Furthermore, CRISPR-Cas13a inhibits the formation of glioma intracranial tumors in mice. The results demonstrate the collateral effect of the CRISPR-Cas13a system in cancer cells and the powerful tumor-eliminating potential of this system.