Alterations in Cell Motility, Proliferation, and Metabolism in Novel Models of Acquired Temozolomide Resistant Glioblastoma.
ABSTRACT: Glioblastoma (GBM) is an aggressive and incurable tumor of the brain with limited treatment options. Current first-line standard of care is the DNA alkylating agent temozolomide (TMZ), but this treatment strategy adds only ~4 months to median survival due to the rapid development of resistance. While some mechanisms of TMZ resistance have been identified, they are not fully understood. There are few effective strategies to manage therapy resistant GBM, and we lack diverse preclinical models of acquired TMZ resistance in which to test therapeutic strategies on TMZ resistant GBM. In this study, we create and characterize two new GBM cell lines resistant to TMZ in vitro, based on the 8MGBA and 42MGBA cell lines. Analysis of the TMZ resistant (TMZres) variants in conjunction with their parental, sensitive cell lines shows that acquisition of TMZ resistance is accompanied by broad phenotypic changes, including increased proliferation, migration, chromosomal aberrations, and secretion of cytosolic lipids. Importantly, each TMZ resistant model captures a different facet of the "go" (8MGBA-TMZres) or "grow" (42MGBA-TMZres) hypothesis of GBM behavior. These in vitro model systems will be important additions to the available tools for investigators seeking to define molecular mechanisms of acquired TMZ resistance.
Project description:Glioblastoma multiforme (GBM) is one of the most aggressive cancers. Despite recent advances in multimodal therapies, high-grade glioma remains fatal. Temozolomide (TMZ) is an alkylating agent used worldwide for the clinical treatment of GBM; however, the innate and acquired resistance of GBM limits its application. Here, we found that TMZ inhibited the proliferation and induced the G2/M arrest of GBM cells. Therefore, we performed microarrays to identify the cell cycle- and apoptosis-related genes affected by TMZ. Notably, GADD45A was found to be up-regulated by TMZ in both cell cycle and apoptosis arrays. Furthermore, GADD45A knockdown (GADD45A<sup>kd</sup>) enhanced the cell growth arrest and cell death induced by TMZ, even in natural (T98) and adapted (TR-U373) TMZ-resistant cells. Interestingly, GADD45A<sup>kd</sup> decreased the expression of O<sup>6</sup>-methylguanine-DNA methyltransferase (MGMT) in TMZ-resistant cells (T98 and TR-U373). In MGMT-deficient/TMZ-sensitive cells (U87 and U373), GADD45A<sup>kd</sup> decreased TMZ-induced TP53 expression. Thus, in this study, we investigated the genes influenced by TMZ that were important in GBM therapy, and revealed that GADD45A plays a protective role against TMZ treatment which may through TP53-dependent and MGMT-dependent pathway in TMZ-sensitive and TMZ-resistant GBM, respectively. This protective role of GADD45A against TMZ treatment may provide a new therapeutic strategy for GBM treatment.
Project description:Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.
Project description:Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O?-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O?-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions N³-methyladenine and N?-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.
Project description:Glioblastoma multiforme (GBM) is a chemotherapy-resistant brain tumor with limited treatment options. Temozolomide (TMZ), an alkylating agent, is a front-line chemotherapeutic drug currently employed in GBM. Although it is currently the most promising chemotherapy for GBM, resistance to TMZ is also common and accounts for many treatment failures. Therefore, understanding the underlying mechanisms that generate resistance is essential to develop more effective chemotherapies. Here, we show that microRNA-101 (miR-101) was significantly downregulated in TMZ-resistant GBM cells and human specimens. Instead, over-expression of miR-101 could sensitize resistant GBM cells to TMZ through downregulation of glycogen synthase kinase 3? (GSK3?). Moreover, we found that GSK3? inhibition could enhance TMZ effect through repression of MGMT via promoter methylation. Importantly, decreased expression of miR-101 is related to poor prognosis in patients with GBM, suggesting its potential role as a new prognostic marker in GBM. In conclusion, our study demonstrates that miR-101 can reverse TMZ resistance by inhibition of GSK3? in GBM, thus offer a novel and powerful strategy for GBM therapy.
Project description:Glioblastoma multiforme (GBM) is the most frequent and aggressive primary malignant brain tumor in adults. Current standard radiotherapy and adjuvant chemotherapy with the alkylating agent temozolomide (TMZ) yield poor clinical outcome. This is due to the stem-like properties of tumor cells and genetic abnormalities in GBM, which contribute to resistance to TMZ and progression. In this study, we used cold atmospheric plasma (CAP) to enhance the sensitivity to TMZ through inhibition of antioxidant signaling (linked to TMZ resistance). We demonstrate that CAP indeed enhances the cytotoxicity of TMZ by targeting the antioxidant specific glutathione (GSH)/glutathione peroxidase 4 (GPX4) signaling. We optimized the threshold concentration of TMZ on five different GBM cell lines (U251, LN18, LN229, U87-MG and T98G). We combined TMZ with CAP and tested it on both TMZ-sensitive (U251, LN18 and LN229) and TMZ-resistant (U87-MG and T98G) cell lines using two-dimensional cell cultures. Subsequently, we used a three-dimensional spheroid model for the U251 (TMZ-sensitive) and U87-MG and T98G (TMZ-resistant) cells. The sensitivity of TMZ was enhanced, i.e., higher cytotoxicity and spheroid shrinkage was obtained when TMZ and CAP were administered together. We attribute the anticancer properties to the release of intracellular reactive oxygen species, through inhibiting the GSH/GPX4 antioxidant machinery, which can lead to DNA damage. Overall, our findings suggest that the combination of CAP with TMZ is a promising combination therapy to enhance the efficacy of TMZ towards the treatment of GBM spheroids.
Project description:BACKGROUND:Although temozolomide (TMZ) resistance is a significant clinical problem in glioblastoma (GBM), its underlying molecular mechanisms are poorly understood. In this study, we identified the role of exosomal microRNAs (miRNAs) from TMZ-resistant cells as important mediators of chemoresistance in GBM cells. METHODS:Exosomes were isolated from TMZ-resistant GBM cells and characterized via scanning electron microscopy (SEM). Expression levels of miR-1238 in GBM cell lines and their exosomes, clinical tissues, and sera were evaluated by RT-qPCR. In vitro and in vivo experiments were performed to elucidate the function of exosomal miR-1238 in TMZ resistance in GBM cells. Co-immunoprecipitation assays and western blot analysis were used to investigate the potential mechanisms of miR-1238/CAV1 that contribute to TMZ resistance. FINDINGS:MiR-1238 levels were higher in TMZ-resistant GBM cells and their exosomes than in sensitive cells. Higher levels of miR-1238 were found in the sera of GBM patients than in healthy people. The loss of miR-1238 may sensitize resistant GBM cells by directly targeting the CAV1/EGFR pathway. Furthermore, bioactive miR-1238 may be incorporated into the exosomes shed by TMZ-resistant cells and taken up by TMZ-sensitive cells, thus disseminating TMZ resistance. INTERPRETATION:Our findings establish that miR-1238 plays an important role in mediating the acquired chemoresistance of GBM and that exosomal miR-1238 may confer chemoresistance in the tumour microenvironment. These results suggest that circulating miR-1238 serves as a clinical biomarker and a promising therapeutic target for TMZ resistance in GBM. FUND: This study was supported by the National Natural Science Foundation of China (No·81402056, 81472362, and 81772951) and the National High Technology Research and Development Program of China (863) (No·2012AA02A508).
Project description:Glioblastoma multiforme (GBM) is one of the most malignant human intracranial tumors. Temozolomide (TMZ) is the primary alkylating agent for GBM patients. However, many GBM patients are resistant to TMZ. Therefore, patients with GBM urgently need more effective therapeutic options. 20(S)-ginsenoside-Rg3 (20(S)-Rg3) is a natural chemical with anti-tumor effects, but at present there is little understanding of its functional mechanism. Several research reports have demonstrated that O<sup>6</sup> -methylguanine DNA-methyltransferase (MGMT) repairs damaged DNA and contributes to TMZ resistance in gliomas. In addition, recent studies have shown that MGMT gene expression could be regulated by the Wnt/β-catenin pathway. However, whether 20(S)-Rg3 inhibits MGMT expression and augments chemosensitivity to Temozolomide (TMZ) in glioma cells remains unclear. In this study, we explored the modulating effects of 20(S)-Rg3 on MGMT. We used glioma cell lines, primary cell strain (including T98G, U118 and GBM-XX; all of them are MGMT-positive glioma cell lines) and xenograft glioma models to examine whether 20(S)-Rg3 increased the sensitivity to TMZ and to reveal the underlying mechanisms. We found that the MGMT expression was effectively downregulated by 20(S)-Rg3 via the Wnt/β-catenin pathway in glioma cell lines, and TMZ resistance was significantly reversed by 20(S)-Rg3. Meanwhile, 20(S)-Rg3 shows no obvious cytotoxicity at its effective dose and is well tolerated in vivo. In addition, we found that 20(S)-Rg3 significantly restrains the epithelial-mesenchymal transition (EMT) progression of glioma cells. Taken together, these results indicate that 20(S)-Rg3 may be a novel agent to use in treatment of GBM, especially in TMZ-resistant GBM with high MGMT expression.
Project description:Glioblastoma multiforme (GBM) is a fatal malignancy of the central nervous system, commonly associated with chemoresistance. The alkylating agent Temozolomide (TMZ) is the front-line chemotherapeutic agent and has undergone intense studies on resistance. These studies reported on mismatch repair gene upregulation, ABC-targeted drug efflux, and cell cycle alterations. The mechanism by which TMZ induces cell cycle arrest has not been well-established. TMZ-resistant GBM cells have been linked to microRNA (miRNA) and exosomes. A cell cycle miRNA array identified distinct miRNAs only in exosomes from TMZ-resistant GBM cell lines and primary spheres. We narrowed the miRs to miR-93 and -193 and showed in computational analyses that they could target Cyclin D1. Since Cyclin D1 is a major regulator of cell cycle progression, we performed cause-effect studies and showed a blunting effects of miR-93 and -193 in Cyclin D1 expression. These two miRs also decreased cell cycling quiescence and induced resistance to TMZ. Taken together, our data provide a mechanism by which GBM cells can exhibit TMZ-induced resistance through miRNA targeting of Cyclin D1. The data provide a number of therapeutic approaches to reverse chemoresistance at the miRNA, exosomal and cell cycle points.
Project description:Glioblastoma multiforme (GBM) represents the most common and deadly primary brain malignancy, particularly due to temozolomide (TMZ) and radiation (RT) resistance. To better understand resistance mechanisms, we examined global kinase activity (kinomic profiling) in both treatment sensitive and resistant human GBM patient-derived xenografts (PDX or "xenolines").Thirteen orthotopically-implanted xenolines were examined including 8 with known RT sensitivity/resistance, while 5 TMZ resistant xenolines were generated through serial TMZ treatment in vivo. Tumors were harvested, prepared as total protein lysates, and kinomically analyzed on a PamStation®12 high-throughput microarray platform with subsequent upstream kinase prediction and network modeling.Kinomic profiles indicated elevated tyrosine kinase activity associated with the radiation resistance phenotype, including FAK and FGFR1. Furthermore, network modeling showed VEGFR1/2 and c-Raf hubs could be involved. Analysis of acquired TMZ resistance revealed more kinomic variability among TMZ resistant tumors. Two of the five tumors displayed significantly altered kinase activity in the TMZ resistant xenolines and network modeling indicated PKC, JAK1, PI3K, CDK2, and VEGFR as potential mediators of this resistance.GBM xenolines provide a phenotypic model for GBM drug response and resistance that when paired with kinomic profiling identified targetable pathways to inherent (radiation) or acquired (TMZ) resistance.
Project description:Effective sensitizing strategies potentially can extend the benefit of temozolomide (TMZ) therapy in patients with glioblastoma (GBM). We previously demonstrated that robust TMZ-sensitizing effects of the [poly (ADP-ribose) polymerase] (PARP) inhibitor veliparib (ABT-888) are restricted to TMZ-sensitive GBM xenografts. The focus of this study is to provide an understanding for the differential sensitization in paired TMZ-sensitive and -resistant GBM models.The impact of veliparib on TMZ-induced cytotoxicity and DNA damage was evaluated in vitro and in vivo in models of acquired TMZ resistance (GBM12TMZ-mgmt(High), GBM12TMZ-mgmt(Low), and U251TMZ), inherent TMZ resistance (T98G), and TMZ-sensitive (U251 and GBM12). In vivo drug efficacy, pharmacokinetics, and pharmacodynamics were analyzed using clinically relevant dosing regimens.Veliparib enhanced TMZ cytotoxicity and DNA-damage signaling in all GBM models in vitro with more pronounced effects in TMZ-resistant lines at 3 to 10 ?mol/L veliparib. In vivo, combined TMZ/veliparib, compared with TMZ alone, significantly delayed tumor growth and enhanced DNA-damage signaling and ?H2AX levels in the sensitive GBM12 xenograft line but not in the resistant GBM12TMZ lines. The pharmacokinetic profile of veliparib was similar for GBM12 and GBM12TMZ tumors with Cmax (?1.5 ?mol/L) in tissue significantly lower than concentrations associated with optimal in vitro sensitizing effects for resistant tumors. In contrast, robust suppression of PARP-1 expression by shRNA significantly increased TMZ sensitivity of U251TMZ in vitro and in vivo.In vitro cytotoxicity assays do not adequately model the therapeutic index of PARP inhibitors, as concentrations of veliparib and TMZ required to sensitize TMZ-resistant cancer cells in vivo cannot be achieved using a tolerable dosing regimen.