Confers Temozolomide Resistance Research Articles

High expression of LncRNA HOTAIR is a risk factor for temozolomide resistance in glioblastoma via activation of the miR-214/β-catenin/MGMT pathway

HOX transcript antisense RNA (HOTAIR) is upregulated in glioblastoma (GBM) and associated with temozolomide (TMZ) resistance. However, the mechanisms underlying HOTAIR-mediated TMZ resistance remains poorly understood. HOTAIR expression in glioma-related public datasets and drug response estimation were analyzed using bioinformatics. These findings were verified by overexpressing HOTAIR in TMZ-sensitive U251 cells and/or silencing HOTAIR in resistant U251 cells (U251R). The cytotoxic effects were evaluated using cell viability assay and flow cytometry analysis of cell cycle and apoptosis. In this study, we found that HOTAIR was upregulated in TMZ-resistant GBM cell lines and patients with high HOTAIR expression responded poorly to TMZ therapy. HOTAIR knockdown restored TMZ sensitivity in U251R cells, while HOTAIR overexpression conferred TMZ resistance in U251 cells. Wnt/β-catenin signaling was enriched in patients with high HOTAIR expression; consistently, HOTAIR positively regulated β-catenin expression in U251 cells. Moreover, HOTAIR-mediated TMZ resistance was associated with increased MGMT protein level, which resulted from the HOTAIR/miR-214-3p/β-catenin network. Besides, GBM with high HOTAIR expression exhibited sensitivity to methotrexate. Methotrexate enhanced TMZ sensitivity in U251R cells, accompanied by reduced expression of HOTAIR and β-catenin. Thus, we conlcude that HOTAIR is a risk factor for TMZ resistance and methotrexate may represent a potential therapeutic drug for patients with high HOTAIR expression level.

USP19 regulates DNA methylation damage repair and confers temozolomide resistance through MGMT stabilization.

To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance. Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management. USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients. The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.

Abstract 5439: Metastasis-enhancing protein KITENIN confers temozolomide resistance in glioblastoma with unmethylated MGMT via upregulation of cancer stem cell makers

Abstract Background: Elevated expression of cancer stem cell (CSC) markers is linked with progression, poor survival, and treatment resistance in glioblastoma (GBM). Our prior work identified KAI1 COOH-terminal interacting tetraspanin (KITENIN) as key to GBM progression. We investigated its influence on temozolomide (TMZ) resistance in GBM through modulation of CSC markers. Methods: We examined KITENIN and CSC markers in TMZ-resistant GBM cells and patient-derived GBM cells, and assessed therapeutic responses to TMZ in mice implanted with KITENIN-modulated GBM cells. The correlation of these markers with clinical outcomes was analyzed using our cohort and The Cancer Genome Atlas (TCGA) data. Results: High KITENIN expression correlated positively with elevated CSC marker levels in human GBM samples and KITENIN-modulated GBM cells. Inhibition of KITENIN using usnic acid reduced tumor progression by decreasing CSC marker expression. Our data and TCGA analyses linked co-directional increase of these factors with tumor recurrence and shorter survival in GBM patients with unmethylated MGMT. This increase was also observed in TMZ-resistant cell lines with unmethylated MGMT. Implantation of KITENIN-overexpressing cells led to TMZ resistance in our mouse model, associated with co-directionally increased CD44 and ALDH1A1 expression. The link between KITENIN and CD44/ALDH1A1 could be inhibited by DKC2511, a novel KITENIN inhibitor, and T5244, a c-Fos/AP-1 inhibitor. Conclusion: KITENIN might contribute to TMZ resistance in GBM patients with unmethylated MGMT, potentially through AP-1 activation and subsequent upregulation of CSC markers. Hence, targeting KITENIN could provide a novel strategy to overcome TMZ resistance in GBM patients with unmethylated MGMT. Citation Format: Eun-Jeong Ahn, Yeong Jin Kim, Tae-Young Jung, Jae-Hyuk Lee, Joon Haeng Rhee, Kyung-Keun Kim, Sung Sun Kim, Nah Ihm Kim, Hangun Kim, Kyung-Sub Moon, Kyung-Hwa Lee. Metastasis-enhancing protein KITENIN confers temozolomide resistance in glioblastoma with unmethylated MGMT via upregulation of cancer stem cell makers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5439.

Histone H3K9 Lactylation Confers Temozolomide Resistance in Glioblastoma via LUC7L2-Mediated MLH1 Intron Retention.

Temozolomide (TMZ) resistance remains the major obstacle in the treatment of glioblastoma (GBM). Lactylation is a novel post-translational modification that is involved in various tumors. However, whether lactylation plays a role in GBM TMZ resistance remains unclear. Here it is found that histone H3K9 lactylation (H3K9la) confers TMZ resistance in GBM via LUC7L2-mediated intron 7 retention of MLH1. Mechanistically, lactylation is upregulated in recurrent GBM tissues and TMZ-resistant cells, and is mainly concentrated in histone H3K9. Combined multi-omics analysis, including CUT&Tag, SLAM-seq, and RNA-seq, reveals that H3K9 lactylation is significantly enriched in the LUC7L2 promoter and activates LUC7L2 transcription to promote its expression. LUC7L2 mediates intron 7 retention of MLH1 to reduce MLH1 expression, and thereby inhibit mismatch repair (MMR), ultimately leading to GBM TMZ resistance. Of note, it is identified that a clinical anti-epileptic drug, stiripentol, which can cross the blood-brain barrier and inhibit lactate dehydrogenase A/B (LDHA/B) activity, acts as a lactylation inhibitor and renders GBM cells more sensitive to TMZ in vitro and in vivo. These findings not only shed light on the mechanism of lactylation in GBM TMZ resistance but also provide a potential combined therapeutic strategy for clinical GBM treatment.

Suppression of ITPKB degradation by Trim25 confers TMZ resistance in glioblastoma through ROS homeostasis

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 for TMZ-resistant GBM.

ADAM17 Confers Temozolomide Resistance in Human Glioblastoma Cells and miR-145 Regulates Its Expression.

Glioblastoma (GBM) is a malignant brain tumor, commonly treated with temozolomide (TMZ). Upregulation of A disintegrin and metalloproteinases (ADAMs) is correlated to malignancy; however, whether ADAMs modulate TMZ sensitivity in GBM cells remains unclear. To explore the role of ADAMs in TMZ resistance, we analyzed changes in ADAM expression following TMZ treatment using RNA sequencing and noted that ADAM17 was markedly upregulated. Hence, we established TMZ-resistant cell lines to elucidate the role of ADAM17. Furthermore, we evaluated the impact of ADAM17 knockdown on TMZ sensitivity in vitro and in vivo. Moreover, we predicted microRNAs upstream of ADAM17 and transfected miRNA mimics into cells to verify their effects on TMZ sensitivity. Additionally, the clinical significance of ADAM17 and miRNAs in GBM was analyzed. ADAM17 was upregulated in GBM cells under serum starvation and TMZ treatment and was overexpressed in TMZ-resistant cells. In in vitro and in vivo models, ADAM17 knockdown conferred greater TMZ sensitivity. miR-145 overexpression suppressed ADAM17 and sensitized cells to TMZ. ADAM17 upregulation and miR-145 downregulation in clinical specimens are associated with disease progression and poor prognosis. Thus, miR-145 enhances TMZ sensitivity by inhibiting ADAM17. These findings offer insights into the development of therapeutic approaches to overcome TMZ resistance.

DDDR-12. THE ROLE OF CASEIN KINASE 1 EPSILON IN TEMOZOLOMIDE SENSITIVITY IN GLIOBLASTOMA STEM CELLS

Abstract A major hurdle in effectively treating glioblastoma is the resistance to current chemotherapy temozolomide (TMZ), exhibited intrinsically in glioblastoma stem cells (GSCs). GSCs are a population of tumor cells that are self-renewing, highly resilient and capable of forming genetically heterogeneous and difficult-to-treat tumors. Therefore, overcoming TMZ resistance holds significant promise in the treatment of glioblastoma. Casein kinase 1 ε (CK1ε) has been identified as a stem cell factor that is important for GSCs to self-renew. However, whether CK1ε confers TMZ resistance in GSCs remains elusive. METHODS To test the role of CK1ε in the chemoresistance of GSCs, we treated GSC adherent cell lines with TMZ, CK1ε inhibitor IC261, and a combination of TMZ plus IC261. Cell viability was assessed with MTS viability assay or CellTiter-Blue viability assay. RESULTS Cell viability was significantly decreased in GSCs treated with 0.8μM IC261 combined with 100μM TMZ as compared to cells treated with either 0.8μM IC261 or 100μM TMZ alone (p < 0.05). We assessed synergistic drug effects using the Bliss independence model to yield Excess Over Bliss (EOB) scores. EOB > 0% indicates a synergistic effect; EOB = 0% indicates additive effect; and EOB < 0% indicates antagonistic effect. Cells treated with 0.8μM IC261 together with 100μM TMZ yielded EOB of > 0%. CONCLUSIONS Previous work has identified CK1ε as an important stem cell factor in the survival GSCs, but there lacked studies testing the role of CK1ε in TMZ chemoresistance in vitro. We demonstrate synergistic inhibition of cell viability in GSCs treated with CK1ε inhibitor IC261 in combination with TMZ. Based on these results, CK1ε is suggested to play an important role in the chemoresistance of GSCs to TMZ.

FOXM1-mediated NUF2 expression confers temozolomide resistance to human glioma cells by regulating autophagy via the PI3K/AKT/mTOR signaling pathway.

Glioma is the most common malignant tumor in the central nervous system and has a high mortality rate. Temozolomide (TMZ) is a widely used chemotherapeutic drug for glioma. NDC80 kinetochore complex (NUF2) is suggested to play a regulatory role in different cancers, but its specific function and mechanism in glioblastoma TMZ resistance remain unknown. NUF2, assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), was highly expressed in glioma cell lines. TMZ was used to treat cells to establish a TMZ-resistant cell line. The potential functions of NUF2 in glioma were assessed using cell counting kit-8 (CCK-8) assays, colony formation assays, 5-Ethynyl-2'-deoxyuridine (EdU) assays, flow cytometry, Western blotting, and a tumor xenograft model. The results showed that NUF2 knockdown attenuated malignant phenotypes of TMZ-resistant cells and prevented tumor growth. Mechanistically, as luciferase reporter assays and chromatin immunoprecipitation (ChIP) as showed, Fox transcription factor M1 (FOXM1) had binding sites on the NUF2 promoter. Rescue assays demonstrated that FOXM1 upregulation counteracted the inhibitory effects of NUF2 depletion on the malignancies of TMZ-resistant cells. This study demonstrates that FOXM1-activated NUF2 promotes TMZ to human glioma cells by regulating proliferation, apoptosis, and autophagy.

Reprogramming of arachidonate metabolism confers temozolomide resistance to glioblastoma through enhancing mitochondrial activity in fatty acid oxidation

BackgroundSp1 is involved in the recurrence of glioblastoma (GBM) due to the acquirement of resistance to temozolomide (TMZ). Particularly, the role of Sp1 in metabolic reprogramming for drug resistance remains unknown.MethodsRNA-Seq and mass spectrometry were used to analyze gene expression and metabolites amounts in paired GBM specimens (primary vs. recurrent) and in paired GBM cells (sensitive vs. resistant). ω-3/6 fatty acid and arachidonic acid (AA) metabolism in GBM patients were analyzed by targeted metabolome. Mitochondrial functions were determined by Seahorse XF Mito Stress Test, RNA-Seq, metabolome and substrate utilization for producing ATP. Therapeutic options targeting prostaglandin (PG) E2 in TMZ-resistant GBM were validated in vitro and in vivo.ResultsAmong the metabolic pathways, Sp1 increased the prostaglandin-endoperoxide synthase 2 expression and PGE2 production in TMZ-resistant GBM. Mitochondrial genes and metabolites were obviously increased by PGE2, and these characteristics were required for developing resistance in GBM cells. For inducing TMZ resistance, PGE2 activated mitochondrial functions, including fatty acid β-oxidation (FAO) and tricarboxylic acid (TCA) cycle progression, through PGE2 receptors, E-type prostanoid (EP)1 and EP3. Additionally, EP1 antagonist ONO-8713 inhibited the survival of TMZ-resistant GBM synergistically with TMZ.ConclusionSp1-regulated PGE2 production activates FAO and TCA cycle in mitochondria, through EP1 and EP3 receptors, resulting in TMZ resistance in GBM. These results will provide us a new strategy to attenuate drug resistance or to re-sensitize recurred GBM.

Oncogenic Forkhead box D3 antisense RNA 1 promotes cell survival and confers temozolomide resistance in glioblastoma cells through the miR-128-3p/WEE1 G2 checkpoint kinase axis

ABSTRACT Although temozolomide (TMZ) is recommended for glioblastoma (GBM) treatment, patients treated with TMZ usually develop TMZ resistance. Thus, there is an urgent need to elucidate the mechanism through which GBM cells acquire TMZ resistance. FOXD3-AS1, a recently discovered lncRNA, shows high expression in diverse cancer types. Nonetheless, its role in GBM remains unclear. This study found that FOXD3-AS1 was overexpressed in GBM cells and associated with dismal prognostic outcome in GBM patients. Functional studies revealed that depletion of FOXD3-AS1 inhibited cell growth and induced apoptosis of GBM cells. Results also showed that FOXD3-AS1 participates in the tolerance of GBM cells to TMZ. Specifically, TMZ-resistant cells exhibited higher FOXD3-AS1 expression compared to parental cells. Overexpression of FOXD3-AS1 increased TMZ tolerance in TMZ sensitive cells, whereas depletion of FOXD3-AS1 sensitized TMZ-resistant cells to TMZ treatment. Mechanistically, WEE1 was positively expressed with FOXD3-AS1. Given that both FOXD3-AS1 and WEE1 contain a binding site for miR-128-3p, FOXD3-AS1 could act as a competing endogenous RNA (ceRNA) to promote WEE1 expression by sponging miR-128-3p. Furthermore, we demonstrated that WEE1 was upregulated in TMZ-resistant GBM cells. Overexpression of WEE1 increased TMZ tolerance in TMZ sensitive cells, whereas deletion of FOXD3-AS1 promoted TMZ-resistant cells to be more sensitive to TMZ. Importantly, depletion of WEE1 could reverse TMZ resistant phenotype in FOXD3-AS1-overexpressed GBM cells. Collectively, our findings reveal a critical role of FOXD3-AS1 in the survival of GBM cells and TMZ resistance, which suggests that FOXD3-AS1 is a potential biomarker for the diagnosis and treatment of GBM.

Multiple Pathway-Dephosphorylated ASK-1 Confers Temozolomide-Resistance to Human Glioma Cells.

To elucidate the function of ASK-1 and the role of its modulators in the induction of Temozolamide (TMZ) resistance in glioma and the underlying mechanism. ASK-1 phosphorylation, the IC50 of TMZ, cell viability, and apoptosis were assessed in the U87 and U251 glioma cell lines and the derived TMZ-resistant cell lines U87-TR and U251-TR. We then blocked ASK-1 function, either with an inhibitor or by overexpression of multiple ASK-1 upstream modulators, to further explore the role of ASK-1 in TMZ-resistant glioma. TMZ-resistant glioma cells showed high IC50 values of TMZ, high survival, and low levels of apoptosis following the TMZ challenge. ASK-1 phosphorylation, but not protein expression, was higher in U87 and U251 cells than in TMZ-resistant glioma cells exposed to TMZ. The addition of the ASK-1 inhibitor selonsertib (SEL) resulted in the dephosphorylation of ASK-1 in U87 and U251 cells after the TMZ challenge. SEL treatment increased the TMZ resistance of U87 and U251 cells, as evidenced by the increased IC50 and cell survival rate and low apoptosis rate. Overexpression of some ASK-1 upstream suppressors [Thioredoxin (Trx), protein phosphatase 5 (PP5), 14-3-3, and cell division cycle 25C (Cdc25C)] led to various degrees of ASK-1 dephosphorylation and a TMZresistant phenotype in U87 and U251 cells. Dephosphorylation of ASK-1 induced TMZ resistance in human glioma cells, and several ASK-1 upstream suppressors, including Trx, PP5, 14-3-3, and Cdc25C, are involved in this phenotypic change induced by dephosphorylation of ASK-1.

JCI-20679 suppresses autophagy and enhances temozolomide-mediated growth inhibition of glioblastoma cells

Glioblastoma, a type of brain cancer, is one of the most aggressive and lethal types of malignancy. The present study shows that JCI-20679, an originally synthesized mitochondrial complex I inhibitor, enhances the anti-proliferative effects of suboptimal concentrations of the clinically used chemotherapeutic drug temozolomide in glioblastoma cells. Analysis of the effects of temozolomide combined with JCI-20679 using isobologram and combination index methods demonstrated that the combination had synergistic effects in murine and human glioblastoma cells. We found that JCI-20679 inhibited the temozolomide-mediated induction of autophagy that facilitates cellular survival. The autophagy induced by temozolomide increased ATP production, which confers temozolomide resistance in glioblastoma cells. JCI-20679 blocked temozolomide-mediated increases in ATP levels and increased the AMP/ATP ratio. Furthermore, JCI-20679 enhanced the therapeutic effects of temozolomide in an orthotopic transplantation model of glioblastoma. These results indicate that JCI-20679 may be promising as a novel agent for enhancing the efficacy of temozolomide against glioblastoma.

RUNX1 (RUNX family transcription factor 1), a target of microRNA miR-128-3p, promotes temozolomide resistance in glioblastoma multiform by upregulating multidrug resistance-associated protein 1 (MRP1)

ABSTRACT Glioblastoma multiform (GBM) is the most frequent type of malignant brain tumor with a poor prognosis. After optimal surgery, radiotherapy plus temozolomide (TMZ) is the standard treatment for GBM patients. However, the development of TMZ resistance limits its efficacy in GBM management. Runt Related Transcription Factor 1 (RUNX1) and microRNAs have been implicated in drug resistance of TMZ in GBM. In this study, we revealed the underlying mechanism of TMZ resistance and identified miR-128-3p/RUNX1 axis as a novel target for TMZ resistance in GBM. RUNX1 expression was significantly upregulated in GBM tissues as compared to normal tissues, and its expression was even higher in recurrent GBM tissues and TMZ-resistant GBM cells. RUNX1 depletion inhibited the viability, proliferation, migration, invasion and TMZ resistance of GBM cells, which could be rescued by RUNX1 overexpression. We further identified miR-128-3p as a tumor-suppressor whose overexpression restored the sensitivity of TMZ in GBM cells. miR-128-3p negatively regulated RUNX1 and subsequently downregulated multidrug resistance-associated protein 1 (MRP1). Together, the present study indicates that RUNX1 confers TMZ resistance in GBM by upregulating MRP1, which is negatively regulated by miR-128-3p. Targeting miR-128-3p/RUNX1/MRP1 axis provides a potential strategy to overcome TMZ resistance in GBM.

A combined treatment regimen of MGMT-modified \u03b3\u03b4 T cells and temozolomide chemotherapy is effective against primary high grade gliomas

Chemotherapeutic drugs such as the alkylating agent Temozolomide (TMZ), in addition to reducing tumor mass, can also sensitize tumors to immune recognition by transient upregulation of multiple stress induced NKG2D ligands (NKG2DL). However, the potential for an effective response by innate lymphocyte effectors such as NK and γδ T cells that recognize NKG2DL is limited by the drug’s concomitant lymphodepleting effects. We have previously shown that modification of γδ T cells with a methylguanine DNA methyltransferase (MGMT) transgene confers TMZ resistance via production of O6-alkylguanine DNA alkyltransferase (AGT) thereby enabling γδ T cell function in therapeutic concentrations of TMZ. In this study, we tested this strategy which we have termed Drug Resistant Immunotherapy (DRI) to examine whether combination therapy of TMZ and MGMT-modified γδ T cells could improve survival outcomes in four human/mouse xenograft models of primary and refractory GBM. Our results confirm that DRI leverages the innate response of γδ T cells to chemotherapy-induced stress associated antigen expression and achieves synergies that are significantly greater than either individual approach.

Exosome-mediated transfer of MIF confers temozolomide resistance by regulating TIMP3/PI3K/AKT axis in gliomas

Temozolomide (TMZ) resistance is an important cause of clinical treatment failure and poor prognosis in gliomas. Increasing evidence indicates that cancer-derived exosomes contribute to chemoresistance; however, the specific contribution of glioma-derived exosomes remains unclear. The aim of this study was to explore the role and underlying mechanisms of exosomal macrophage migration inhibitory factor (MIF) on TMZ resistance in gliomas. We first demonstrated that MIF was upregulated in the exosomes of TMZ-resistant cells, engendering the transfer of TMZ resistance to sensitive cells. Our results indicated that exosomal MIF conferred TMZ resistance to sensitive cells through the enhancement of cell proliferation and the repression of cell apoptosis upon TMZ exposure. MIF knockdown enhanced TMZ sensitivity in resistant glioma cells by upregulating Metalloproteinase Inhibitor 3 (TIMP3) and subsequently suppressing the PI3K/AKT signaling pathway. Additionally, exosomal MIF promoted tumor growth and TMZ resistance of glioma cells in vivo, while IOS-1 (MIF inhibitor) promotes glioma TMZ sensitive in vivo. Taken together, our study demonstrated that exosome-mediated transfer of MIF enhanced TMZ resistance in glioma through downregulating TIMP3 and further activating the PI3K/AKT signaling pathway, highlighting a prognostic biomarker and promising therapeutic target for TMZ treatment in gliomas.

Heparanase confers temozolomide resistance by regulation of exosome secretion and circular RNA composition in glioma.

Temozolomide (TMZ) resistance is the main challenge in the management of glioma patients. Heparanase can mediate the secretion and function of exosomes, which are considered to be a promising molecular delivery system for cancer therapy. Therefore, this study aimed to investigate whether heparanase‐mediated delivery of exosomes was related to TMZ resistance of glioma. Heparanase was upregulated in TMZ‐resistant glioma cells, and overexpression of heparanase led to increased resistance of U87 cells to TMZ. Knockdown of heparanase led to increased sensitivity of TMZ‐resistant U251 cells (U251R) cells to TMZ. Heparanase promoted the secretion of exosomes from glioma cells, and coculture with exosomes derived from heparanase knockdown U251R cells partly restored the sensitivity of U251 cells to TMZ compared with exosomes derived from si‐control transfected U251R cells. It was identified by circular RNA microarrays that hsa_circ_0042003 was upregulated in exosomes derived from U251R, which could be positively regulated by heparanase. U251R cell‐derived exosomal hsa_circ_0042003 conferred the resistance of U251 cells to TMZ. In vivo studies also showed that U251R cell‐derived exosomes induced resistance of U251 cells to TMZ, and the combination of tail‐injected exosomal si‐heparanase or exosomal si‐hsa_circ_0042003 and intraperitoneal TMZ applied to nude mice abolished TMZ resistance. Heparanase mediated the transfer of exosomal hsa_circ_0042003 from TMZ‐resistant glioma cells to drug‐sensitive cells, which contributed to the chemoresistance of glioma to TMZ.

Long Noncoding RNA KCNQ1OT1 Confers Gliomas Resistance to Temozolomide and Enhances Cell Growth by Retrieving PIM1 From miR-761.

Many studies have found that the dysregulation of long noncoding RNA (lncRNA) contributed to cancer initiation, progression, and recurrence via multiple signaling pathways. However, the underlying mechanisms of lncRNA in temozolomide (TMZ)-resistant gliomas were not well understood, hindering the improvement of TMZ-based therapies. The present study demonstrated that the lncRNA KCNQ1OT1 increased in TMZ-resistant glioma cells compared to the TMZ-sensitive cells. The introduction of KCNQ1OT1 promoted cell viability, clonogenicity, and rhodamine 123 efflux while hampering TMZ-induced apoptosis. Moreover, KCNQ1OT1 directly sponged miR-761, which decreased in TMZ-resistant sublines. The overexpression of miR-761 attenuated cell viability and clonogenicity, while triggering apoptosis and rhodamine 123 accumulation post-TMZ exposure, leading to a response to TMZ. The interaction between miR-761 and 3'-untranslated region of PIM1 attenuated PIM1-mediated signaling cascades. Furthermore, the knockdown of KCNQ1OT1 augmented the TMZ-induced tumor regression in TMZ-resistant U251 mouse models. Briefly, the present study evaluated that KCNQ1OT1 conferred TMZ resistance by releasing PIM1 expression from miR-761, resulting in the upregulation of PIM-mediated MDR1, c-Myc, and Survivin. The present findings demonstrated that the interplay of KCNQ1OT1: miR-761: PIM1 regulated chemoresistance in gliomas and provided a promising therapeutic target for TMZ-resistant glioma patients.

TGF-β1 modulates temozolomide resistance in glioblastoma via altered microRNA processing and elevated MGMT.

BackgroundOur previous studies have indicated that miR-198 reduces cellular methylguanine DNA methyltransferase (MGMT) levels to enhance temozolomide sensitivity. Transforming growth factor beta 1 (TGF-β1) switches off miR-198 expression by repressing K-homology splicing regulatory protein (KSRP) expression in epidermal keratinocytes. However, the underlying role of TGF-β1 in temozolomide resistance has remained unknown.MethodsThe distribution of KSRP was detected by western blotting and immunofluorescence. Microarray analysis was used to compare the levels of long noncoding RNAs (lncRNAs) between TGF-β1–treated and untreated cells. RNA immunoprecipitation was performed to verify the relationship between RNAs and KSRP. Flow cytometry and orthotopic and subcutaneous xenograft tumor models were used to determine the function of TGF-β1 in temozolomide resistance.ResultsOverexpression of TGF-β1 contributed to temozolomide resistance in MGMT promoter hypomethylated glioblastoma cells in vitro and in vivo. TGF-β1 treatment reduced cellular MGMT levels through suppressing the expression of miR-198. However, TGF-β1 upregulation did not affect KSRP expression in glioma cells. We identified and characterized 2 lncRNAs (H19 and HOXD-AS2) that were upregulated by TGF-β1 through Smad signaling. H19 and HOXD-AS2 exhibited competitive binding to KSRP and prevented KSRP from binding to primary miR-198, thus decreasing miR-198 expression. HOXD-AS2 or H19 upregulation strongly promoted temozolomide resistance and MGMT expression. Moreover, KSRP depletion abrogated the effects of TGF-β1 and lncRNAs on miR-198 and MGMT. Finally, we found that patients with low levels of TGF-β1 or lncRNA expression benefited from temozolomide therapy.ConclusionsOur results reveal an underlying mechanism by which TGF-β1 confers temozolomide resistance. Furthermore, our findings suggest that a novel combination of temozolomide with a TGF-β inhibitor may serve as an effective therapy for glioblastomas.

The CXCL12/CXCR4 axis confers temozolomide resistance to human glioblastoma cells via up-regulation of FOXM1

Glioblastoma multiforme (GBM) is the most common primary malignancy in the adult central nervous, and is characterized by high aggressiveness and a high mortality rate. The high mortality rate is largely due to the development of drug resistance. Temozolomide (TMZ) resistance is considered to be one of the major reasons responsible for GBM therapy failure. CXCL12/CXCR4 has been demonstrated to be involved in cell proliferation, migration, invasion, angiogenesis, and radioresistance in GBM. However, its role in TMZ resistance in GBM is unknown. In this study, we aimed to evaluate the role of CXCL12/CXCR4 in mediating the TMZ resistance to GBM cells and explore the underlying mechanisms. We found that the CXCL12/CXCR4 axis enhanced TMZ resistance in GBM cells. Further study showed that CXCL12/CXCR4 conferred TMZ resistance and promoted the migration and invasion of GBM cells by up-regulating FOXM1. This resistance was partially reversed by suppressing CXCL12/CXCR4 and FOXM1 silencing. Our study revealed the vital role of CXCL12/CXCR4 in mediating the resistance of GBM cells to TMZ, and suggested that targeting CXCL12/CXCR4 axis may attenuate the resistance to TMZ in GBM.

Exosome-mediated transfer of circRNA CircNFIX enhances temozolomide resistance in glioma.

Development of chemotherapy resistance remains a major obstacle for glioma management. Exosome-mediated transfer of circular RNAs (circRNAs) are being found to have relevance to many human cancers, including glioma. The purpose of this study is to explore the effect and underlying mechanism of exosomal circRNA nuclear factor I X (CircNFIX) on temozolomide (TMZ) chemoresistance in glioma. Our results indicated that exosomal CircNFIX was up-regulated in the serum of TMZ-resistant patients and predicted poor prognosis. Exosomal CircNFIX from TMZ-resistant cells conferred TMZ resistance to recipient sensitive cells through the enhancement of cell migration and invasion and the repression of cell apoptosis under TMZ exposure. CircNFIX directly interacted with miR-132 by binding to miR-132. CircNFIX knockdown enhanced TMZ sensitivity in resistant glioma cells by up-regulating miR-132. Additionally, exosomal CircNFIX promoted tumor growth and its depletion enhanced TMZ sensitivity in glioma cells in vivo. Taken together, our study suggests that exosome-mediated transfer of CircNFIX enhances TMZ resistance in glioma at least partially through sponging miR-132, highlighting a potentially prognostic biomarker and therapeutic target for improving the clinical benefits of TMZ treatment in patients with glioma.