Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells

Glioblastoma multiforme (GBM) is a malignant primary brain tumor. The 5-year relative survival rate of patients with GBM remains <30% on average despite aggressive treatments, and secondary therapy fails in 90% of patients. In chemotherapeutic failure, detoxification proteins are crucial to the activity of chemotherapy drugs. Usually, glutathione S-transferase (GST) superfamily members act as detoxification enzymes by activating xenobiotic metabolites through conjugation with glutathione in healthy cells. However, some overexpressed GSTs not only increase GST activity but also trigger chemotherapy resistance and tumorigenesis-related signaling transductions. Whether GSTM3 is involved in GBM chemoresistance remains unclear. In the current study, we found that T98G, a GBM cell line with pre-existing temozolomide (TMZ) resistance, has high glycolysis and GSTM3 expression. GSTM3 knockdown in T98G decreased glycolysis ability through lactate dehydrogenase A activity reduction. Moreover, it increased TMZ toxicity and decreased invasion ability. Furthermore, we provide next-generation sequencing–based identification of significantly changed messenger RNAs of T98G cells with GSTM3 knockdown for further research. GSTM3 was downregulated in intrinsic TMZ-resistant T98G with a change in the expression levels of some essential glycolysis-related genes. Thus, GSTM3 was associated with glycolysis in chemotherapeutic resistance in T98G cells. Our findings provide new insight into the GSTM3 mechanism in recurring GBM.     

Extracellular Sphingosine-1-Phosphate Downstream of EGFR Increases Human Glioblastoma Cell Survival

Sphingosine-1-phosphate (S1P) is a crucial mediator involved in the progression of different cancers, including glioblastoma multiforme (GBM), the most frequent and deadly human brain tumor, characterized by extensive invasiveness and rapid cell growth. Most of GBMs overexpress the epidermal growth factor receptor (EGFR), and we investigated the possible link between S1P and EGFR signaling pathways, focusing on its role in GBM survival, using the U87MG human cell line overexpressing EGFR (EGFR+). We previously demonstrated that EGFR+ cells have higher levels of extracellular S1P and increased sphingosine kinase-1 (SK1) activity than empty vector expressing cells. Notably, we demonstrated that EGFR+ cells are resistant to temozolomide (TMZ), the standard chemotherapeutic drug in GBM treatment, and the inhibition of SK1 or S1P receptors made EGFR+ cells sensitive to TMZ; moreover, exogenous S1P reverted this effect, thus involving extracellular S1P as a survival signal in TMZ resistance in GBM cells. In addition, both PI3K/AKT and MAPK inhibitors markedly reduced cell survival, suggesting that the enhanced resistance to TMZ of EGFR+ cells is dependent on the increased S1P secretion, downstream of the EGFR-ERK-SK1-S1P pathway. Altogether, our study provides evidence of a functional link between S1P and EGFR signaling pathways enhancing the survival properties of GBM cells.     

Ex Vivo Expanded and Activated Natural Killer Cells Prolong the Overall Survival of Mice with Glioblastoma-like Cell-Derived Tumors

Glioblastoma (GBM) is the leading malignant intracranial tumor and is associated with a poor prognosis. Highly purified, activated natural killer (NK) cells, designated as genuine induced NK cells (GiNKs), represent a promising immunotherapy for GBM. We evaluated the anti-tumor effect of GiNKs in association with the programmed death 1(PD-1)/PD-ligand 1 (PD-L1) immune checkpoint pathway. We determined the level of PD-1 expression, a receptor known to down-regulate the immune response against malignancy, on GiNKs. PD-L1 expression on glioma cell lines (GBM-like cell line U87MG, and GBM cell line T98G) was also determined. To evaluate the anti-tumor activity of GiNKs in vivo, we used a xenograft model of subcutaneously implanted U87MG cells in immunocompromised NOG mice. The GiNKs expressed very low levels of PD-1. Although PD-L1 was expressed on U87MG and T98G cells, the expression levels were highly variable. Our xenograft model revealed that the retro-orbital administration of GiNKs and interleukin-2 (IL-2) prolonged the survival of NOG mice bearing subcutaneous U87MG-derived tumors. PD-1 blocking antibodies did not have an additive effect with GiNKs for prolonging survival. GiNKs may represent a promising cell-based immunotherapy for patients with GBM and are minimally affected by the PD-1/PD-L1 immune evasion axis in GBM.     

Temozolomide Induces the Acquisition of Invasive Phenotype by O6-Methylguanine-DNA Methyltransferase (MGMT)+ Glioblastoma Cells in a Snail-1/Cx43-Dependent Manner

Glioblastoma multiforme (GBM) recurrences after temozolomide (TMZ) treatment result from the expansion of drug-resistant and potentially invasive GBM cells. This process is facilitated by O6-Methylguanine-DNA Methyltransferase (MGMT), which counteracts alkylating TMZ activity. We traced the expansion of invasive cell lineages under persistent chemotherapeutic stress in MGMT^low (U87) and MGMT^high (T98G) GBM populations to look into the mechanisms of TMZ-induced microevolution of GBM invasiveness. TMZ treatment induced short-term, pro-invasive phenotypic shifts of U87 cells, in the absence of Snail-1 activation. They were illustrated by a transient induction of their motility and followed by the hypertrophy and the signs of senescence in scarce U87 sub-populations that survived long-term TMZ stress. In turn, MGMT^high T98G cells reacted to the long-term TMZ treatment with the permanent induction of invasiveness. Ectopic Snail-1 down-regulation attenuated this effect, whereas its up-regulation augmented T98G invasiveness. MGMT^low and MGMT^high cells both reacted to the long-term TMZ stress with the induction of Cx43 expression. However, only in MGMT^high T98G populations, Cx43 was directly involved in the induction of invasiveness, as manifested by the induction of T98G invasiveness after ectopic Cx43 up-regulation and by the opposite effect after Cx43 down-regulation. Collectively, Snail-1/Cx43-dependent signaling participates in the long-term TMZ-induced microevolution of the invasive GBM front. High MGMT activity remains a prerequisite for this process, even though MGMT-related GBM chemoresistance is not necessary for its initiation.     

Somatostatin Receptor Splicing Variant sst5TMD4 Overexpression in Glioblastoma Is Associated with Poor Survival,Increased Aggressiveness Features,and Somatostatin Analogs Resistance

Glioblastoma (GBM) is the most malignant and lethal brain tumor. Current standard treatment consists of surgery followed by radiotherapy/chemotherapy; however, this is only a palliative approach with a mean post-operative survival of scarcely ~12–15 months. Thus, the identification of novel therapeutic targets to treat this devastating pathology is urgently needed. In this context, the truncated splicing variant of the somatostatin receptor subtype 5 (sst5TMD4), which is produced by aberrant alternative splicing, has been demonstrated to be overexpressed and associated with increased aggressiveness features in several tumors. However, the presence, functional role, and associated molecular mechanisms of sst5TMD4 in GBM have not been yet explored. Therefore, we performed a comprehensive analysis to characterize the expression and pathophysiological role of sst5TMD4 in human GBM. sst5TMD4 was significantly overexpressed (at mRNA and protein levels) in human GBM tissue compared to non-tumor (control) brain tissue. Remarkably, sst5TMD4 expression was significantly associated with poor overall survival and recurrent tumors in GBM patients. Moreover, in vitro sst5TMD4 overexpression (by specific plasmid) increased, whereas sst5TMD4 silencing (by specific siRNA) decreased, key malignant features (i.e., proliferation and migration capacity) of GBM cells (U-87 MG/U-118 MG models). Furthermore, sst5TMD4 overexpression in GBM cells altered the activity of multiple key signaling pathways associated with tumor aggressiveness/progression (AKT/JAK-STAT/NF-κB/TGF-β), and its silencing sensitized GBM cells to the antitumor effect of pasireotide (a somatostatin analog). Altogether, these results demonstrate that sst5TMD4 is overexpressed and associated with enhanced malignancy features in human GBMs and reveal its potential utility as a novel diagnostic/prognostic biomarker and putative therapeutic target in GBMs.     

Functional In Vitro Assessment of VEGFA/NOTCH2 Signaling Pathway and pRB Proteasomal Degradation and the Clinical Relevance of Mucolipin TRPML2 Overexpression in Glioblastoma Patients

Glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high vascularization and its growth depends on the formation of new blood vessels. We have previously demonstrated that TRPML2 mucolipin channel expression increases with the glioma pathological grade. Herein by ddPCR and Western blot we found that the silencing of TRPML2 inhibits expression of the VEGFA/Notch2 angiogenic pathway. Moreover, the VEGFA/Notch2 expression increased in T98 and U251 cells stimulated with the TRPML2 agonist, ML2-SA1, or by enforced-TRPML2 levels. In addition, changes in TRPML2 expression or ML2-SA1-induced stimulation, affected Notch2 activation and VEGFA release. An increased invasion capability, associated with a reduced VEGF/VEGFR2 expression and increased vimentin and CD44 epithelial-mesenchymal transition markers in siTRPML2, but not in enforced-TRPML2 or ML2-SA1-stimulated glioma cells, was demonstrated. Furthermore, an increased sensitivity to Doxorubicin cytotoxicity was demonstrated in siTRPML2, whereas ML2-SA1-treated GBM cells were more resistant. The role of proteasome in Cathepsin B-dependent and -independent pRB degradation in siTRPML2 compared with siGLO cells was studied. Finally, through Kaplan-Meier analysis, we found that high TRPML2 mRNA expression strongly correlates with short survival in GBM patients, supporting TRPML2 as a negative prognostic factor in GBM patients.     

Golgi Phosphoprotein 3 Regulates the Physical Association of Glycolipid Glycosyltransferases

Glycolipid glycosylation is an intricate process that mainly takes place in the Golgi by the complex interplay between glycosyltransferases. Several features such as the organization, stoichiometry and composition of these complexes may modify their sorting properties, sub-Golgi localization, enzymatic activity and in consequence, the pattern of glycosylation at the plasma membrane. In spite of the advance in our comprehension about physiological and pathological cellular states of glycosylation, the molecular basis underlying the metabolism of glycolipids and the players involved in this process remain not fully understood. In the present work, using biochemical and fluorescence microscopy approaches, we demonstrate the existence of a physical association between two ganglioside glycosyltransferases, namely, ST3Gal-II (GD1a synthase) and β3GalT-IV (GM1 synthase) with Golgi phosphoprotein 3 (GOLPH3) in mammalian cultured cells. After GOLPH3 knockdown, the localization of both enzymes was not affected, but the fomation of ST3Gal-II/β3GalT-IV complex was compromised and glycolipid expression pattern changed. Our results suggest a novel control mechanism of glycolipid expression through the regulation of the physical association between glycolipid glycosyltransferases mediated by GOLPH3.     

Up-Regulation of Cyclooxygenase-2 (COX-2) Expression by Temozolomide (TMZ) in Human Glioblastoma (GBM) Cell Lines

TMZ-resistance remains a main limitation in glioblastoma (GBM) treatment. TMZ is an alkylating agent whose cytotoxicity is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. The inflammatory marker COX-2 has been implicated in GBM tumorigenesis, progression, and stemness. COX-2 inhibitors are considered a GBM add-on treatment due to their ability to increase TMZ-sensitivity. We investigated the effect of TMZ on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG). β-catenin, MGMT, and SOX-2 expression was analyzed. The effects of NS398, COX-2 inhibitor, alone or TMZ-combined, were studied evaluating cell proliferation by the IncuCyte^® system, cell cycle/apoptosis, and clonogenic potential. COX-2, β-catenin, MGMT, and SOX-2 expression was evaluated by RT-PCR, Western blotting, and immunofluorescence and PGE2 by ELISA. Our findings, sustaining the role of COX-2/PGE2 system in TMZ-resistance of GBM, show, for the first time, a relevant, dose-dependent up-regulation of COX-2 expression and activity in TMZ-treated T98G that, in turn, correlated with chemoresistance. Similarly, all the COX-2-dependent signaling pathways involved in TMZ-resistance also resulted in being up-modulated after treatment with TMZ. NS398+TMZ was able to reduce cell proliferation and induce cell cycle arrest and apoptosis. Moreover, NS398+TMZ counteracted the resistance in T98G preventing the TMZ-induced COX-2, β-catenin, MGMT, and SOX-2 up-regulation.     

CRISPR-Cas9–Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.     

C3G Protein,a New Player in Glioblastoma

C3G (RAPGEF1) is a guanine nucleotide exchange factor (GEF) for GTPases from the Ras superfamily, mainly Rap1, although it also acts through GEF-independent mechanisms. C3G regulates several cellular functions. It is expressed at relatively high levels in specific brain areas, playing important roles during embryonic development. Recent studies have uncovered different roles for C3G in cancer that are likely to depend on cell context, tumour type, and stage. However, its role in brain tumours remained unknown until very recently. We found that C3G expression is downregulated in GBM, which promotes the acquisition of a more mesenchymal phenotype, enhancing migration and invasion, but not proliferation. ERKs hyperactivation, likely induced by FGFR1, is responsible for this pro-invasive effect detected in C3G silenced cells. Other RTKs (Receptor Tyrosine Kinases) are also dysregulated and could also contribute to C3G effects. However, it remains undetermined whether Rap1 is a mediator of C3G actions in GBM. Various Rap1 isoforms can promote proliferation and invasion in GBM cells, while C3G inhibits migration/invasion. Therefore, other RapGEFs could play a major role regulating Rap1 activity in these tumours. Based on the information available, C3G could represent a new biomarker for GBM diagnosis, prognosis, and personalised treatment of patients in combination with other GBM molecular markers. The quantification of C3G levels in circulating tumour cells (CTCs) in the cerebrospinal liquid and/or circulating fluids might be a useful tool to improve GBM patient treatment and survival.     

Stearoyl CoA Desaturase-1 Silencing in Glioblastoma Cells: Phospholipid Remodeling and Cytotoxicity Enhanced upon Autophagy Inhibition

Modulation of lipid metabolism is a well-established cancer hallmark, and SCD1 has been recognized as a key enzyme in promoting cancer cell growth, including in glioblastoma (GBM), the deadliest brain tumor and a paradigm of cancer resistance. The central goal of this work was to identify, by MS, the phospholipidome alterations resulting from the silencing of SCD1 in human GBM cells, in order to implement an innovative therapy to fight GBM cell resistance. With this purpose, RNAi technology was employed, and low serum-containing medium was used to mimic nutrient deficiency conditions, at which SCD1 is overexpressed. Besides the expected increase in the saturated to unsaturated fatty acid ratio in SCD1 silenced-GBM cells, a striking increase in polyunsaturated chains, particularly in phosphatidylethanolamine and cardiolipin species, was noticed and tentatively correlated with an increase in autophagy (evidenced by the increase in LC3BII/I ratio). The contribution of autophagy to mitigate the impact of SCD1 silencing on GBM cell viability and growth, whose modest inhibition could be correlated with the maintenance of energetically associated mitochondria, was evidenced by using autophagy inhibitors. In conclusion, SCD1 silencing could constitute an important tool to halt GBM resistance to the available treatments, especially when coupled with a mitochondria disrupter chemotherapeutic.     

Mechanisms of EGFR Resistance in Glioblastoma

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Despite numerous efforts to target epidermal growth factor receptor (EGFR), commonly dysregulated in GBM, approaches directed against EGFR have not achieved the same degree of success as seen in other tumor types, particularly as compared to non-small cell lung cancer (NSCLC). EGFR alterations in glioblastoma lie primarily in the extracellular domain, unlike the kinase domain alterations seen in NSCLC. Small molecule inhibitors are difficult to develop for the extracellular domain. Monoclonal antibodies can be developed to target the extracellular domain but must contend with the blood brain barrier (BBB). We review the role of EGFR in GBM, the history of trialed treatments, and the potential paths forward to target the pathway that may have greater success.     

A Novel Role of BIRC3 in Stemness Reprogramming of Glioblastoma

Stemness reprogramming remains a largely unaddressed principal cause of lethality in glioblastoma (GBM). It is therefore of utmost importance to identify and target mechanisms that are essential for GBM stemness and self-renewal. Previously, we implicated BIRC3 as an essential mediator of therapeutic resistance and survival adaptation in GBM. In this study, we present novel evidence that BIRC3 has an essential noncanonical role in GBM self-renewal and stemness reprogramming. We demonstrate that BIRC3 drives stemness reprogramming of human GBM cell lines, mouse GBM cell lines and patient-derived GBM stem cells (GSCs) through regulation of BMP4 signaling axis. Specifically, BIRC3 induces stemness reprogramming in GBM through downstream inactivation of BMP4 signaling. RNA-Seq interrogation of the stemness reprogramming hypoxic (pseudopalisading necrosis and perinecrosis) niche in GBM patient tissues further validated the high BIRC3/low BMP4 expression correlation. BIRC3 knockout upregulated BMP4 expression and prevented stemness reprogramming of GBM models. Furthermore, siRNA silencing of BMP4 restored stemness reprogramming of BIRC3 knockout in GBM models. In vivo silencing of BIRC3 suppressed tumor initiation and progression in GBM orthotopic intracranial xenografts. The stemness reprograming of both GSCs and non-GSCs populations highlights the impact of BIRC3 on intra-tumoral cellular heterogeneity GBM. Our study has identified a novel function of BIRC3 that can be targeted to reverse stemness programming of GBM.     

CCL18 Expression Is Higher in a Glioblastoma Multiforme Tumor than in the Peritumoral Area and Causes the Migration of Tumor Cells Sensitized by Hypoxia

Glioblastoma multiforme (GBM) is a brain tumor with a very poor prognosis. For this reason, researchers worldwide study the impact of the tumor microenvironment in GBM, such as the effect of chemokines. In the present study, we focus on the role of the chemokine CCL18 and its receptors in the GBM tumor. We measured the expression of CCL18, CCR8 and PITPNM3 in the GMB tumor from patients (16 men and 12 women) using quantitative real-time polymerase chain reaction. To investigate the effect of CCL18 on the proliferation and migration of GBM cells, experiments were performed using U-87 MG cells. The results showed that CCL18 expression was higher in the GBM tumor than in the peritumoral area. The women had a decreased expression of PITPNM3 receptor in the GBM tumor, while in the men a lower expression of CCR8 was observed. The hypoxia-mimetic agent, cobalt chloride (CoCl₂), increased the expression of CCL18 and PITPNM3 and thereby sensitized U-87 MG cells to CCL18, which did not affect the proliferation of U-87 MG cells but increased the migration of the test cells. The results indicate that GBM cells migrate from hypoxic areas, which may be important in understanding the mechanisms of tumorigenesis.     

EGFR-Targeted Pentacyclic Triterpene Analogues for Glioma Therapy

Glioma, particularly its most malignant form, glioblastoma multiforme (GBM), is the most common and aggressive malignant central nervous system tumor. The drawbacks of the current chemotherapy for GBM have aroused curiosity in the search for targeted therapies. Aberrantly overexpressed epidermal growth factor receptor (EGFR) in GBM results in poor prognosis, low survival rates, poor responses to therapy and recurrence, and therefore EGFR-targeted therapy stands out as a promising approach for the treatment of gliomas. In this context, a series of pentacyclic triterpene analogues were subjected to in vitro and in silico assays, which were conducted to assess their potency as EGFR-targeted anti-glioma agents. In particular, compound 10 was the most potent anti-glioma agent with an IC₅₀ value of 5.82 µM towards U251 human glioblastoma cells. Taking into account its low cytotoxicity to peripheral blood mononuclear cells (PBMCs), compound 10 exerts selective antitumor action towards Jurkat human leukemic T-cells. This compound also induced apoptosis and inhibited EGFR with an IC₅₀ value of 9.43 µM compared to erlotinib (IC₅₀ = 0.06 µM). Based on in vitro and in silico data, compound 10 stands out as a potential orally bioavailable EGFR-targeted anti-glioma agent endowed with the ability to cross the blood–brain barrier (BBB).     

Radiolabeled Gold Nanoseeds Decorated with Substance P Peptides: Synthesis,Characterization and In Vitro Evaluation in Glioblastoma Cellular Models

Despite some progress, the overall survival of patients with glioblastoma (GBM) remains extremely poor. In this context, there is a pressing need to develop innovative therapy strategies for GBM, namely those based on nanomedicine approaches. Towards this goal, we have focused on nanoparticles (AuNP-SP and AuNP-SPTyr8) with a small gold core (ca. 4 nm), carrying DOTA chelators and substance P (SP) peptides. These new SP-containing AuNPs were characterized by a variety of analytical techniques, including TEM and DLS measurements and UV-vis and CD spectroscopy, which proved their high in vitro stability and poor tendency to interact with plasma proteins. Their labeling with diagnostic and therapeutic radionuclides was efficiently performed by DOTA complexation with the trivalent radiometals ⁶⁷Ga and ¹⁷⁷Lu or by electrophilic radioiodination with ¹²⁵I of the tyrosyl residue in AuNP-SPTyr8. Cellular studies of the resulting radiolabeled AuNPs in NKR1-positive GBM cells (U87, T98G and U373) have shown that the presence of the SP peptides has a crucial and positive impact on their internalization by the tumor cells. Consistently, ¹⁷⁷Lu-AuNP-SPTyr8 showed more pronounced radiobiological effects in U373 cells when compared with the non-targeted congener ¹⁷⁷Lu-AuNP-TDOTA, as assessed by cell viability and clonogenic assays and corroborated by Monte Carlo microdosimetry simulations.     

The E3 Ubiquitin Ligase NEDD4-1 Mediates Temozolomide-Resistant Glioblastoma through PTEN Attenuation and Redox Imbalance in Nrf2–HO-1 Axis

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. It is highly resistant to chemotherapy, and tumor recurrence is common. Neuronal precursor cell-expressed developmentally downregulated 4-1 (NEDD4-1) is an E3 ligase that controls embryonic development and animal growth. NEDD4-1 regulates the tumor suppressor phosphatase and tensin homolog (PTEN), one of the major regulators of the PI3K/AKT/mTOR signaling axis, as well as the response to oxidative stress. Methods: The expression levels of NEDD4-1 in GBM tissues and different cell lines were determined by quantitative real-time polymerase chain reaction and immunohistochemistry. In vitro and in vivo assays were performed to explore the biological effects of NEDD4-1 on GBM cells. Temozolomide (TMZ)-resistant U87MG and U251 cell lines were specifically established to determine NEDD4-1 upregulation and its effects on the tumorigenicity of GBM cells. Subsequently, miRNA expression in TMZ-resistant cell lines was investigated to determine the dysregulated miRNA underlying the overexpression of NEDD4-1. Indole-3-carbinol (I3C) was used to inhibit NEDD4-1 activity, and its effect on chemoresistance to TMZ was verified. Results: NEDD4-1 was significantly overexpressed in the GBM and TMZ-resistant cells and clinical samples. NEDD4-1 was demonstrated to be a key oncoprotein associated with TMZ resistance, inducing oncogenicity and tumorigenesis of TMZ-resistant GBM cells compared with TMZ-responsive cells. Mechanistically, TMZ-resistant cells exhibited dysregulated expression of miR-3129-5p and miR-199b-3p, resulting in the induced NEDD4-1 mRNA-expression level. The upregulation of NEDD4-1 attenuated PTEN expression and promoted the AKT/NRF2/HO-1 oxidative stress signaling axis, which in turn conferred amplified defense against reactive oxygen species (ROS) and eventually higher resistance against TMZ treatment. The combination treatment of I3C, a known inhibitor of NEDD4-1, with TMZ resulted in a synergistic effect and re-sensitized TMZ-resistant tumor cells both in vitro and in vivo. Conclusions: These findings demonstrate the critical role of NEDD4-1 in regulating the redox imbalance in TMZ-resistant GBM cells via the degradation of PTEN and the upregulation of the AKT/NRF2/HO-1 signaling pathway. Targeting this regulatory axis may help eliminate TMZ-resistant glioblastoma.