Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standa...Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standard chemotherapeutic for GBM,but TMZ treatment benefits are compromised by chemoresistance.This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM.Western blot,realtime PCR,flow cytometry,and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1(RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells.Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment.Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells.Knockdown of E2F1 increased sensitivity to TMZ.Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1,mediates TMZ resistance,and has a potential E2F1 binding site on the promoter.Knockdown of RAD51AP1 enhanced the sensitivity of TMZ;however,overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells.Furthermore,RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase(MGMT)expression.The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated,but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment.RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair.Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.展开更多
目的：探讨胶质瘤细胞接受联合X线辐射及T M Z化疗干预后其干细胞标志物的变化情况。方法将51例人脑胶质瘤标本作为观察对象，将标本分为22例脑胶质瘤Ⅰ～Ⅱ级的低级别组及29例脑胶质瘤Ⅲ～Ⅳ级的高级别组，所有入选标本均给予联合X线...目的：探讨胶质瘤细胞接受联合X线辐射及T M Z化疗干预后其干细胞标志物的变化情况。方法将51例人脑胶质瘤标本作为观察对象，将标本分为22例脑胶质瘤Ⅰ～Ⅱ级的低级别组及29例脑胶质瘤Ⅲ～Ⅳ级的高级别组，所有入选标本均给予联合X线辐射及 TMZ 化疗干预。对比分析2组干预前后胶质细胞中CD133及 ABCG2的表达情况，并对CD133及ABCG2改善情况与细胞恶性程度的相关性进行分析。结果2组间CD133及ABCG2的表达均存在显著差异，高级别组表达程度均明显高于低级别组（P＜0.05）。2组在接受联合X线辐射及TMZ化疗干预后，CD133及ABCG2均明显改善，以低级别组改善更为明显（ P＜0.05），且治疗前后CD133及ABCG2改善度与细胞恶性程度存在负向直线相关性（ P＜0.05）。结论不同恶性程度的胶质瘤细胞中CD133及ABCG2的表达存在显著差异。联合X线辐射及TMZ化疗可有效改善CD133及ABCG2表达，且与细胞恶性程度存在负向直线相关性。展开更多
Temozolomide(TMZ)resistance is a major obstacle in glioma treatment.Nuclear protein-1(NUPR1)is a regulator of glioma progression.This study investigated the mechanism of NUPR1 in TMZ resistance in hypoxiatreated gliom...Temozolomide(TMZ)resistance is a major obstacle in glioma treatment.Nuclear protein-1(NUPR1)is a regulator of glioma progression.This study investigated the mechanism of NUPR1 in TMZ resistance in hypoxiatreated glioma cells and its mechanism in modulating autophagy.We treated TMZ-resistant cells U251-TMZ and T98G-TMZ to normoxia or hypoxia and silenced NUPR1 in hypoxia-treated U251-TMZ and T98G-TMZ cells to assess cell viability,proliferation,apoptosis,LC3-II/LC3-I and p62 expressions,and autophagic flux under different concentrations of TMZ.We found that hypoxia upregulated NUPR1 expression and autophagy while NUPR1 silencing suppressed hypoxia-induced TMZ resistance and autophagy in glioma cells.We also investigated the interaction between NUPR1 and lysine demethylase 3A(KDM3A),as well as the enrichments of KDM3A and H3 lysine 9 dimethylation(H3K9me2)in the transcription factor EB(TFEB)promoter region.Our results suggest that hypoxia-induced NUPR1 promotes TFEB transcription by binding to KDM3A and reducing H3K9me2 levels,thereby augmenting glioma cell autophagy and TMZ resistance.Moreover,the overexpression of KDM3A or TFEB promoted glioma cell autophagy.In a xenograft tumor model,silencing NUPR1 suppressed TMZ resistance in glioma cells in vivo.Overall,our findings highlight a mechanism by which NUPR1 enhances glioma cell autophagy and TMZ resistance via the KDM3A/TFEB axis.展开更多
Temozolomide(TMZ)represents a standard-of-care chemotherapeutic agent in glioblastoma(GBM).However,the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma.Although spec...Temozolomide(TMZ)represents a standard-of-care chemotherapeutic agent in glioblastoma(GBM).However,the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma.Although specific innovative approaches,such as immunotherapy,have shown favorable clinical outcomes,the inherent invasiveness of most gliomas continues to make them challenging to treat.Consequently,there is an urgent need to identify effective therapeutic targets for gliomas to overcome chemoresistance and facilitate drug development.This investigation used mass spectrometry to examine the proteomic profiles of six pairs of GBM patients who underwent standard-of-care treatment and surgery for both primary and recurrent tumors.A total of 648 proteins exhibiting significant differential expression were identified.Gene Set Enrichment Analysis(GSEA)unveiled notable alterations in pathways related to METABOLISM_OF_LIPIDS and BIOLOGICAL_OXIDATIONS between the primary and recurrent groups.Validation through glioma tissue arrays and the Xiangya cohort confirmed substantial upregulation of inositol 1,4,5-triphosphate(IP3)kinase B(ITPKB)in the recurrence group,correlating with poor survival in glioma patients.In TMZ-resistant cells,the depletion of ITPKB led to an increase in reactive oxygen species(ROS)related to NADPH oxidase(NOX)activity and restored cell sensitivity to TMz.Mechanistically,the decreased phosphorylation of the E3 ligase Trim25 at the S100 position in recurrent GBM samples accounted for the weakened ITPKB ubiquitination.This,in turn,elevated ITPKB stability and impaired ROS production.Furthermore,ITPKB depletion or the ITPKB inhibitor GNF362 effectively overcome TMZ chemoresistance in a glioma xenograft mouse model.These findings reveal a novel mechanism underlying TMZ resistance and propose ITPKB as a promising therapeutic target forTMZ-resistant GBM.展开更多
基金supported by the Science and Technology Project of Tianjin Municipal Health Commission(Grant Nos.TJWJ2022MS003 and TJWJ2021ZD008)the Tianjin Science and Technology Plan Project(Grant Nos.21JCYBJC01520 and 20JCYBJC01070)。
文摘Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standard chemotherapeutic for GBM,but TMZ treatment benefits are compromised by chemoresistance.This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM.Western blot,realtime PCR,flow cytometry,and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1(RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells.Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment.Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells.Knockdown of E2F1 increased sensitivity to TMZ.Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1,mediates TMZ resistance,and has a potential E2F1 binding site on the promoter.Knockdown of RAD51AP1 enhanced the sensitivity of TMZ;however,overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells.Furthermore,RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase(MGMT)expression.The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated,but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment.RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair.Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.
基金supported by Scientific Research Project of Education Department of Liaoning Province(Grant Number LJKZ0854).
文摘Temozolomide(TMZ)resistance is a major obstacle in glioma treatment.Nuclear protein-1(NUPR1)is a regulator of glioma progression.This study investigated the mechanism of NUPR1 in TMZ resistance in hypoxiatreated glioma cells and its mechanism in modulating autophagy.We treated TMZ-resistant cells U251-TMZ and T98G-TMZ to normoxia or hypoxia and silenced NUPR1 in hypoxia-treated U251-TMZ and T98G-TMZ cells to assess cell viability,proliferation,apoptosis,LC3-II/LC3-I and p62 expressions,and autophagic flux under different concentrations of TMZ.We found that hypoxia upregulated NUPR1 expression and autophagy while NUPR1 silencing suppressed hypoxia-induced TMZ resistance and autophagy in glioma cells.We also investigated the interaction between NUPR1 and lysine demethylase 3A(KDM3A),as well as the enrichments of KDM3A and H3 lysine 9 dimethylation(H3K9me2)in the transcription factor EB(TFEB)promoter region.Our results suggest that hypoxia-induced NUPR1 promotes TFEB transcription by binding to KDM3A and reducing H3K9me2 levels,thereby augmenting glioma cell autophagy and TMZ resistance.Moreover,the overexpression of KDM3A or TFEB promoted glioma cell autophagy.In a xenograft tumor model,silencing NUPR1 suppressed TMZ resistance in glioma cells in vivo.Overall,our findings highlight a mechanism by which NUPR1 enhances glioma cell autophagy and TMZ resistance via the KDM3A/TFEB axis.
基金supported by grants from the National Natural Science Foundation of China (82272659,82030087)the Science and Technology Innovation Program of Hunan Province (2022RC1210,2021RC3029)the Hunan Provincial Natural Science Foundation Project (2022JJ30073,2021JC0002).
文摘Temozolomide(TMZ)represents a standard-of-care chemotherapeutic agent in glioblastoma(GBM).However,the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma.Although specific innovative approaches,such as immunotherapy,have shown favorable clinical outcomes,the inherent invasiveness of most gliomas continues to make them challenging to treat.Consequently,there is an urgent need to identify effective therapeutic targets for gliomas to overcome chemoresistance and facilitate drug development.This investigation used mass spectrometry to examine the proteomic profiles of six pairs of GBM patients who underwent standard-of-care treatment and surgery for both primary and recurrent tumors.A total of 648 proteins exhibiting significant differential expression were identified.Gene Set Enrichment Analysis(GSEA)unveiled notable alterations in pathways related to METABOLISM_OF_LIPIDS and BIOLOGICAL_OXIDATIONS between the primary and recurrent groups.Validation through glioma tissue arrays and the Xiangya cohort confirmed substantial upregulation of inositol 1,4,5-triphosphate(IP3)kinase B(ITPKB)in the recurrence group,correlating with poor survival in glioma patients.In TMZ-resistant cells,the depletion of ITPKB led to an increase in reactive oxygen species(ROS)related to NADPH oxidase(NOX)activity and restored cell sensitivity to TMz.Mechanistically,the decreased phosphorylation of the E3 ligase Trim25 at the S100 position in recurrent GBM samples accounted for the weakened ITPKB ubiquitination.This,in turn,elevated ITPKB stability and impaired ROS production.Furthermore,ITPKB depletion or the ITPKB inhibitor GNF362 effectively overcome TMZ chemoresistance in a glioma xenograft mouse model.These findings reveal a novel mechanism underlying TMZ resistance and propose ITPKB as a promising therapeutic target forTMZ-resistant GBM.
