TMZ-resistant Glioblastoma Multiforme Research Articles

Abstract 2984: Developing a new combinational therapy to combat temozolomide resistance in glioblastoma multiforme

Abstract Background: Glioblastoma multiforme (GBM) is the most lethal central nervous system malignancy, and recurrence is seen in nearly all cases. Temozolomide (TMZ), a DNA alkylating agent, shows initial promise in eradicating primary GBM, but has little effect on recurrent GBM. Our previous work identified the Phosphatidylinositol 3-kinase Catalytic Subunit Beta (PIK3CB) of the PI3K pathway to be selectively important for the survival and progression of GBM (1). Other previous research also shows that a gap-junction protein, Connexin 43 (Cx43), promotes TMZ resistance in GBM by activating PI3K signaling and that this resistance can be reversed by treatment with a peptide compound named αCT1 (2). More recently, it was discovered that G-protein coupled receptors (GPCRs), specifically the Gβγ subunit, are upstream activators of Cx43/PI3K/AKT signaling in TMZ-resistant GBM cells. We believe that selectively targeting and blocking the activators of these signaling pathways will help to restore and enhance sensitivity to TMZ, yielding successful eradication of GBM cells. We therefore propose to test the effect of a quadruple combination of TMZ, αCT1, TGX-221, and gallein, a selective inhibitor of the Gβγ subunit of GPCRs, on GBM tumor growth. Hypothesis: Gβγ subunit inhibition will substantially increase GBM cytotoxicity in combination with αCT1, TGX-221 (a PIK3CB inhibitor), and TMZ. Methods: MTS cell viability assay was used to determine optimum gallein dosage both alone and in combinational therapies in experimental cell lines with variable Cx43 expression rates and TMZ sensitivities. This assay was also used to determine cell viability after treatment with an optimized quadruple combination therapy in these same lines. Current work is focused on quantifying Cx43/PIK3CB/AKT signaling inhibition via Western blot, as well as applying the optimized quadruple combination treatment in GBM stem cells, three-dimensional GBM tumor models, primary GBM patient samples, and healthy astrocytes. Results: Significant inhibitory effects have been shown with an optimized quadruple combination therapy compared to TMZ alone in TMZ-resistant experimental cell lines. Conclusions: While results in experimental lines have been favorable, additional work is still needed to elucidate the efficacy of the optimized quadruple combination therapy, which will include testing primary GBM patient samples, GBM stem cells, healthy astrocytes and three-dimensional tumor models.

STAT3 inhibition induced temozolomide-resistant glioblastoma apoptosis via triggering mitochondrial STAT3 translocation and respiratory chain dysfunction

Recent evidence has demonstrated that the signal transducer and activator of transcription 3 (STAT3) gene are abnormally active in glioblastoma multiforme (GBM), and this change is crucial for the tumor survival and chemotherapy-resistant. Certain preclinical pharmacology studies have focused on STAT3 phosphorylation and homodimerization, and have developed a class of salicylic acid-based inhibitors, which blocks the nuclear translocation-dependent canonical STAT3 signaling. In the present study, we demonstrated that the salicylic acid-based compound SH-4-54 was quite toxic to temozolomide (TMZ)-resistant GBM cells and could trigger apoptosis in these cells via enhancing mitochondrial translocation–dependent non-canonical STAT3 pathway. We demonstrated that incubation of TMZ-resistant GBM cells with SH-4-54 led to mitochondrial STAT3 (mitoSTAT3) activation and respiratory dysfunction reflected by disrupted (or suppressed) activities of oxidative phosphorylation complexes and oxygen consumption rate. Mechanistically, we proved that SH-4-54 could increase mitoSTAT3 transmembrane import via GRIM-19 and reinforce the association between mitoSTAT3 and mitochondrial transcription factor A (TFAM), indicating that SH-4-54 could facilitate the binding of mitoSTAT3 to mitochondria DNA (mtDNA) and negatively regulate mitochondrial-encoded genes, thus leading to the abnormal oxidation respiratory. Lastly, using GRIM-19 knockout cell line and subcutaneous xenotransplanted tumor model, we elaborately showed the enrichment of SH-4-54 in mitochondria by LC-MS/MS analysis. In conclusion, our data demonstrate thatthe salicylic acid-based compound SH-4-54 is quite effective in killing TMZ-resistant GBM cells and this cytotoxicity is attributed to mitoSTAT3 activation.

Downregulation of SNRPG induces cell cycle arrest and sensitizes human glioblastoma cells to temozolomide by targeting Myc through a p53-dependent signaling pathway.

Objective: Temozolomide (TMZ) is commonly used for glioblastoma multiforme (GBM) chemotherapy. However, drug resistance limits its therapeutic effect in GBM treatment. RNA-binding proteins (RBPs) have vital roles in posttranscriptional events. While disturbance of RBP-RNA network activity is potentially associated with cancer development, the precise mechanisms are not fully known. The SNRPG gene, encoding small nuclear ribonucleoprotein polypeptide G, was recently found to be related to cancer incidence, but its exact function has yet to be elucidated.Methods: SNRPG knockdown was achieved via short hairpin RNAs. Gene expression profiling and Western blot analyses were used to identify potential glioma cell growth signaling pathways affected by SNRPG. Xenograft tumors were examined to determine the carcinogenic effects of SNRPG on glioma tissues.Results: The SNRPG-mediated inhibitory effect on glioma cells might be due to the targeted prevention of Myc and p53. In addition, the effects of SNRPG loss on p53 levels and cell cycle progression were found to be Myc-dependent. Furthermore, SNRPG was increased in TMZ-resistant GBM cells, and downregulation of SNRPG potentially sensitized resistant cells to TMZ, suggesting that SNRPG deficiency decreases the chemoresistance of GBM cells to TMZ via the p53 signaling pathway. Our data confirmed that SNRPG suppression sensitizes GBM cells to TMZ by targeting Myc via the p53 signaling cascade.Conclusions: These results indicated that SNRPG is a probable molecular target of GBM and suggested that suppressing SNRPG in resistant GBM cells might be a substantially beneficial method for overcoming essential drug resistance.

Decreased APE-1 by Nitroxoline Enhances Therapeutic Effect in a Temozolomide-resistant Glioblastoma: Correlation with Diffusion Weighted Imaging

Glioblastoma multiforme (GBM) is one of the most aggressive human tumors with poor survival rates. The current standard treatment includes chemotherapy with temozolomide (TMZ), but acquisition of resistance is a persistent clinical problem limiting the successful treatment of GBM. The purpose of our study was to investigate therapeutic effects of nitroxoline (NTX) against TMZ-resistant GBM in vitro and in vivo in TMZ-resistant GBM-bearing mouse model, which was correlated with diffusion-weighted imaging (DWI). For in vitro study, we used TMZ-resistant GBM cell lines and evaluated therapeutic effects of NTX by clonogenic and migration assays. Quantitative RT-PCR was used to investigate the expression level of TMZ-resistant genes after NTX treatment. For in vivo study, we performed 9.4 T MR imaging to obtain T2WI for tumor volume measurement and DWI for assessment of apparent diffusion coefficient (ADC) changes by NTX in TMZ-resistant GBM mice (n = 8). Moreover, we performed regression analysis for the relationship between ADC and histological findings, which reflects the changes in cellularity and apurinic/apyrimidinic endonuclease-1 (APE-1) expression. We observed the recovery of TMZ-induced morphological changes, a reduced number of colonies and a decreased rate of migration capacity in TMZ-resistant cells after NTX treatment. The expression of APE-1 was significantly decreased in TMZ-resistant cells after NTX treatment compared with those without treatment. In an in vivo study, NTX reduced tumor growth in TMZ-resistant GBM mice (P = 0.0122). Moreover, ADC was increased in the NTX-treated TMZ-resistant GBM mice compared to the control group (P = 0.0079), which was prior to a tumor volume decrease. The cellularity and APE-1 expression by histology were negatively correlated with the ADC value, which in turn resulted in longer survival in NTX group. The decreased expression of APE-1 by NTX leads to therapeutic effects and is inversely correlated with ADC in TMZ-resistant GBM. Therefore, NTX is suggested as potential therapeutic candidate against TMZ-resistant GBM.

SNAP reverses temozolomide resistance in human glioblastoma multiforme cells through down‐regulation of MGMT

Glioblastoma multiforme (GBM) is the most frequently occurring and gravest primary tumor of the CNS in adults. The development of chemoresistance to temozolomide (TMZ), the first-line chemotherapy for GBM, is an important factor contributing to poor treatment outcomes. Down-regulation of O-6-methylguanine-DNA methyltransferase (MGMT) expression in GBM cells is an attractive strategy for overcoming TMZ resistance and improving outcomes. This study revealed that the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) exerts antitumorigenic effects on TMZ-sensitive and TMZ-resistant (TMZ-R) glioma cells. Pretreatment with SNAP not only induced apoptosis, mitochondrial dysfunction, and hypoxia-inducing factor 1, but also resensitized TMZ-R GBM cells to TMZ through down-regulation of MGMT expression. SNAP acted principally through post-translational modification of p53, phosphorylated N-myc downstream regulated gene 1, and MGMT protein stability in TMZ-R GBM cells. Additionally, when applied together, SNAP and TMZ enhanced the inhibition of tumor growth in vitro and in vivo. This study sheds new light on a potential strategy to overcome TMZ resistance in GBM and thus possesses the potential for prolonging survival of patients with GBM.-Tsai, C.-K., Huang, L.-C., Wu, Y.-P., Kan, I.-Y., Hueng, D.-Y. SNAP reverses temozolomide resistance in human glioblastoma multiforme cells through down-regulation of MGMT.

Enhanced Copper-Temozolomide Interactions by Protein for Chemotherapy against Glioblastoma Multiforme.

Current treatment of recurrent glioblastoma multiforme (GBM) demands dose-intense temozolomide (TMZ), a prodrug of 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC), based on the spontaneous hydrolysis of TMZ at basic pH. However, how to control the activity of MTIC remains unknown, which poses a particular challenge to search a reliable MTIC receptor. We reported that copper, for the first time, is found to recognize and bind MTIC in the process of TMZ degradation, which means copper can play an important role in enhancing the bioavailability of MTIC derived from TMZ. Using apoferritin as a model copper-bound protein, we studied the copper-TMZ interaction in protein and observed efficient MTIC immobilization with high binding efficiency (up to 92.9% based on original TMZ) and capacity (up to 185 MTIC moieties per protein). The system was stable against both alkaline and acidic pH and could be activated by glutathione to liberate MTIC, which paves a way to deliver a DNA-alkylating agent for both TMZ-sensitive and TMZ-resistant GBM chemotherapy. Our study provides a new insight for understanding the potential relationship between the special GBM microenvironment (specific copper accumulation) and the therapeutic effect of TMZ.

Preclinical Evidence of STAT3 Inhibitor Pacritinib Overcoming Temozolomide Resistance via Downregulating miR-21-Enriched Exosomes from M2 Glioblastoma-Associated Macrophages.

Background: The tumor microenvironment (TME) plays a crucial role in virtually every aspect of tumorigenesis of glioblastoma multiforme (GBM). A dysfunctional TME promotes drug resistance, disease recurrence, and distant metastasis. Recent evidence indicates that exosomes released by stromal cells within the TME may promote oncogenic phenotypes via transferring signaling molecules such as cytokines, proteins, and microRNAs. Results: In this study, clinical GBM samples were collected and analyzed. We found that GBM-associated macrophages (GAMs) secreted exosomes which were enriched with oncomiR-21. Coculture of GAMs (and GAM-derived exosomes) and GBM cell lines increased GBM cells’ resistance against temozolomide (TMZ) by upregulating the prosurvival gene programmed cell death protein 4 (PDCD4) and stemness markers SRY (sex determining region y)-box 2 (Sox2), signal transducer and activator of transcription 3 (STAT3), Nestin, and miR-21-5p and increasing the M2 cytokines interleukin 6 (IL-6) and transforming growth factor beta 1(TGF-β1) secreted by GBM cells, promoting the M2 polarization of GAMs. Subsequently, pacritinib treatment suppressed GBM tumorigenesis and stemness; more importantly, pacritinib-treated GBM cells showed a markedly reduced ability to secret M2 cytokines and reduced miR-21-enriched exosomes secreted by GAMs. Pacritinib-mediated effects were accompanied by a reduction of oncomiR miR-21-5p, by which the tumor suppressor PDCD4 was targeted. We subsequently established patient-derived xenograft (PDX) models where mice bore patient GBM and GAMs. Treatment with pacritinib and the combination of pacritinib and TMZ appeared to significantly reduce the tumorigenesis of GBM/GAM PDX mice as well as overcome TMZ resistance and M2 polarization of GAMs. Conclusion: In summation, we showed the potential of pacritinib alone or in combination with TMZ to suppress GBM tumorigenesis via modulating STAT3/miR-21/PDCD4 signaling. Further investigations are warranted for adopting pacritinib for the treatment of TMZ-resistant GBM in clinical settings.

Assessment of Early Therapeutic Response to Nitroxoline in Temozolomide-Resistant Glioblastoma by Amide Proton Transfer Imaging: A Preliminary Comparative Study with Diffusion-weighted Imaging

Amide proton transfer (APT) imaging is a novel molecular MRI technique to detect endogenous mobile proteins and peptides through chemical exchange saturation transfer. In this preliminary study, the purpose was to evaluate the feasibility of APT imaging in monitoring the early therapeutic response to nitroxoline (NTX) in a temozolomide (TMZ)-resistant glioblastoma multiforme (GBM) mouse model, which was compared with diffusion-weighted imaging (DWI). Here, we prepared TMZ-resistant GBM mouse model (n = 12), which were treated with 100 mg/kg/day of NTX (n = 4) or TMZ (n = 4), or saline (n = 4) for 7 days for the evaluation of short-term treatment by using APT imaging and DWI sequentially. The APT signal intensities and apparent diffusion coefficient (ADC) values were calculated and compared before and after treatment. Moreover, immunohistological analysis was also employed for the correlation between APT imaging and histopathology. The association between the APT value and Ki-67 labeling index was evaluated by using simple linear regression analysis. The short-term NTX treatment resulted in significant decrease in APT value as compared to untreated and TMZ group, in which APT signals were increased. However, we did not observe significantly increased mean ADC value following short-term NTX treatment. The Ki-67 labeling index shows a correlation with APT value. APT imaging could show the earlier response to NTX treatment as compared to ADC values in a TMZ-resistant mouse model. We believe that APT imaging can be a useful imaging biomarker for the early therapeutic evaluation in GBM patients.

Cycling Quiescence in Temozolomide Resistant Glioblastoma Cells Is Partly Explained by microRNA-93 and -193-Mediated Decrease of Cyclin D.

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.

20(S)-ginsenoside-Rg3 reverses temozolomide resistance and restrains epithelial-mesenchymal transition progression in glioblastoma.

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 O6‐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.

Aldoxorubicin and Temozolomide combination in a xenograft mice model of human glioblastoma.

Glioblastoma Multiforme (GBM) is still an incurable disease. The front-line Temozolomide (TMZ)-based therapy suffers from poor efficacy, underlining the need of new therapies.Preclinically, Aldoxorubicin (Aldox), a novel prodrug of Doxorubicin (Dox), has been successfully tested against GBM, encouraging the study of its association with other agents.For the first time, we evaluated the effectiveness of Aldox combined to TMZ in preclinical models of GBM.Our in vitro results demonstrated that the anti–glioma effect of Aldox was more marked than TMZ and their combination increased the killing effect of the anthracycline in TMZ-resistant GBM cells. Moreover, unlike Dox, Aldox was able to accumulate in P-glycoprotein (P-gp)-overexpressed cells due to a negative regulation of the P-gp function.We also compared efficacy and safety of weekly administrations of Aldox (16 mg/kg), with or without TMZ (0.9 mg/kg, daily injections), in the U87 xenograft mouse model.Aldox therapy induced a moderate tumor volume inhibition (TVI) and an increased survival rate (+12.5% vs vehicle). On the other hand, when combined to TMZ, Aldox caused a significant TVI (P=0.0175 vs vehicle) and delayed the mortality during the experimental period, although TVI and endpoint survival percentage (+37.5% vs vehicle) were not significantly different from TMZ alone.Our preliminary data showed that Aldox exerts anti–glioma effects in vitro and in vivo. It also enhances its antitumor activity when combined with TMZ, resulting in a superior efficacy compared to the single agents, without adverse side effects.

Silencing SATB1 overcomes temozolomide resistance by downregulating MGMT expression and upregulating SLC22A18 expression in human glioblastoma cells.

Glioblastoma multiforme (GBM) is the most common malignant tumor of the central nervous system and has a very poor prognosis. Currently, patients were treated by resection followed by radiotherapy plus concurrent temozolomide (TMZ) chemotherapy. However, many patients are resistant to TMZ-induced DNA damage because of upregulated expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). In this study, upregulation of SATB1 and MGMT, and downregulation of SLC22A18 resulted in acquisition of TMZ resistance in GBM U87 cells. Inactivation of special AT-rich sequence-binding protein 1 (SATB1) using short hairpin RNA (shRNA) downregulated MGMT expression and upregulated solute carrier family 22 member 18 (SLC22A18) expression in GBM cells. This suggested SATB1-mediated posttranscriptional regulation of the MGMT and SLC22A18 protein levels. Immunohistochemical analysis of malignant glioma specimens demonstrated a significant positive correlation between the levels of MGMT and SATB1, and a negative correlation between the levels of SLC22A18 and SATB1. Importantly, in recurrent, compared with the primary, lesions in 15 paired identical tumors, the SATB1 and MGMT protein levels were increased and the SLC22A18 levels were decreased. Finally, in TMZ-resistant GBM, SATB1 knockdown enhanced TMZ efficacy. Consequently, SATB1 inhibition might be a promising strategy combined with TMZ chemotherapy to treat TMZ-resistant GBM.

Exosomal transfer of miR-151a enhances chemosensitivity to temozolomide in drug-resistant glioblastoma

Chemoresistance blunts the effect of Temozolomide (TMZ) in the treatment of glioblastoma multiforme (GBM). Whether exosomal transfer of miRNAs derived from TMZ-resistant GBM cells could confer TMZ resistance remains to be determined. qPCR was used to determine miR-151a expression in two TMZ-resistant GBM cell lines. The direct targets of miR-151a were identified by microarray assays, bioinformatics and further RNA chromatin immunoprecipitation (RNA-ChIP) assay. We characterized exosomes from TMZ-resistant cell lines, serum and cerebrospinal fluid (CSF) and determined the effect of exosomes from TMZ-resistant cells on recipient GBM cells. miR-151a loss drove the acquisition of TMZ resistance. Restored miR-151a expression sensitized TMZ-resistant GBM cells via inhibiting XRCC4-mediated DNA repair. TMZ-resistant GBM cells conferred TMZ chemoresistance to recipient TMZ-sensitive cells in an exosomal miR-151a loss-dependent manner. Restoration of exosomal miR-151a from donor TMZ-resistant cells abolished the chemoresistance dissemination that was directed by donor TMZ-resistant cells. CSF-derived exosomes contained miRNA signatures reflective of the underlying chemoresistant status of GBMs in terms of miR-151a expression levels. Exosomal miR-151a is not only essentially a less-invasive ‘liquid biopsy’ that might predict chemotherapy response, but also represents a promising therapeutic target for therapy-refractory GBMs.

β-catenin contributes to cordycepin-induced MGMT inhibition and reduction of temozolomide resistance in glioma cells by increasing intracellular reactive oxygen species

Glioblastoma multiforme (GBM) is one of the most aggressive human tumors, and it has a poor prognosis. Temozolomide (TMZ) is the primary alkylating agent used to treat GBM. Nevertheless, a number of GBM patients are resistant to TMZ. Therefore, there is an urgent need for more effective therapeutic options. Cordycepin (COR) is a natural chemical with anti-tumor effects, although its mechanism of action is poorly understood. Several lines of evidence suggest that O6-methylguanine DNA methyltransferase (MGMT) repairs damaged DNA and contributes to drug resistance to TMZ in gliomas. The Wnt/β-catenin pathway regulates MGMT gene expression. However, whether cordycepin inhibits MGMT expression by downregulating the β catenin pathway and augmenting chemosensitivity to TMZ in glioma cells remains unclear. In the present study, we found that cordycepin inhibited the viability of glioma cells and induced apoptosis, cell cycle arrest, overproduction of reactive oxygen species (ROS) and reduction of glutathione (GSH) in vitro. Moreover, cordycepin significantly reduced tumor volume and prolonged median survival of tumor-bearing rats in vivo. We also found that cordycepin inhibited MGMT expression and augmented chemosensitivity to TMZ in glioma cells in vitro and in vivo, accompanied by downregulation of p-GSK-3β and β-catenin. Moreover, overexpression of MGMT reversed the synergistic effect of cordycepin and TMZ. Pharmacological inhibition of GSK-3β with CHIR-99021 or overexpression of β-catenin reversed cordycepin-induced reduction of cell viability, downregulation of β-catenin and MGMT, increase of apoptosis and reduction of TMZ resistance. Furthermore, we found that β-catenin regulated cordycepin-induced overproduction of ROS by decreasing GSH. Inhibition of ROS production with N-acetyl-l-cysteine (NAC) not only rescued the reduction of cell viability but also eliminated β-catenin and MGMT inhibition, prevented glioma cells apoptosis and reversed the synergistic effect of cordycepin and TMZ. Taken together, we demonstrated that β-catenin contributed to cordycepin-induced MGMT inhibition and reduction of TMZ resistance in glioma cells via increasing intracellular ROS. These results indicate that cordycepin may be a novel agent to improve GBM treatment, especially in TMZ-resistant GBM with high MGMT expression.

P.038 Investigating the role of long non-coding RNAs in glioblastoma multiforme

Background: Malignant gliomas are the most common and deadly brain tumors. Mean survival rate for a patient diagnosed with a glioblastoma multiforme (GBM) remains slightly over one year. Standard of care consists of treatment with temozolomide (TMZ) and radiotherapy. Recent work has highlighted functions of long non-coding RNAs (lncRNAs) in GBM progression and TMZ response even though the information regarding these newly discovered molecules is sparse. The overarching objective of this project was thus to assess the expression of select lncRNAs in GBM tumor samples and in models of TMZ resistance. Methods: A qRT-PCR-based approach was undertaken to measure six lncRNAs in 19 primary GBM samples, four GBM cell lines and in-house developed TMZ-resistant GBM cells. Results: Elevated levels of Hotair and H19 were observed in primary GBM tumors while decreased expression of MEG3 was recorded in the same samples. Interestingly, levels of PANDA increased 3.4-fold in GBM cells resistant to TMZ when compared with their sensitive counterparts. Conclusions: Overall, this work provides evidence of lncRNA deregulation in GBM tumors and reveals a previously unexplored lncRNA potentially involved in TMZ resistance. Modulation of lncRNA targets via RNAi-mediated approaches is envisioned to clarify their function and to strengthen their position as therapeutic options in GBMs.

LncRNA NEAT1 is involved in temozolomide resistance by regulating MGMT in glioblastoma multiforme

Introduction: As one of the most aggressive and lethal tumors, glioblastoma multiforme (GBM) is commonly treated by surgical resection combined with radiotherapy and chemotherapy. Temozolomide (TMZ) is the preferred chemotherapy medicine against GBM. However, recurrent GBM patients exhibit TMZ resistance. As reported in previous studies, NEAT1 is over-expressed in glioma cells; thus, we explored the relationship between NEAT1 and TMZ resistance in GBM. Materials and Methods: The expression levels of NEAT1 and O -6-methylguanine-DNA methyltransferase (MGMT) in GBM tissues and cells were determined by quantitative real-time PCR. Si-RNA and the over-expression vector were transfected into GBM cells to modulate the level of related molecules. Western blot analysis was used to investigate the protein expression of MGMT. The cell viability and IC-50 were analyzed by CCK-8, and apoptosis was detected by flow cytometry assay. Result: The expression level of NEAT1 in the TMZ-sensitive GBM tissues and cells was lower than that in the TMZ-resistant GBM tissues and cells. Furthermore, due to the down-regulation of NEAT1 in TMZ-resistant GBM cells transfected with Si-RNA, the viability and IC-50 value of the GBM cell lines were decreased, and the knockdown of NEAT1 significantly enhanced TMZ-induced cell apoptosis in GBM cells. We also determined that the mRNA and protein level of MGMT were up-regulated in TMZ-resistant GBM cells. Interference with MGMT expression led to a decrease in viability and IC-50 value in the GBM cells, and the knockdown of NEAT1 suppressed the transcription and translation levels of MGMT. However, the over-expression of MGMT enhanced TMZ resistance in NEAT1-silenced U87 and U251 cells. Conclusion: NEAT1 participates in the TMZ resistance of GBM cells by regulating MGMT.

Quinacrine enhances temozolomide cytotoxicity in temozolomide-sensitive and -resistant glioblastoma cells

Background: The alkylating agent temozolomide (TMZ) is widely used in glioblastoma multiforme (GBM) therapy. Unfortunately, TMZ-resistance frequently occurs in recurrent GBM and is the major cause of treatment failure. The anti-malarial drug quinacrine (QC) harbors antitumor and chemosensitivity properties, but its interactions with TMZ in GBM remain unclear. This study aimed to investigate whether QC would sensitize TMZ in TMZ-sensitive and TMZ-resistant GBM cells as well as the underlying mechanisms. Materials and Methods: The cytotoxicity of QC and TMZ in TMZ-sensitive and TMZ-resistant GBM cells was evaluated using in vitro cell viability assay and colony formation assay. Cellular apoptosis and protein expression levels were determined using TUNEL assay and immunoblotting, respectively. Results: QC substantially enhanced TMZ cytotoxicity in both TMZ-sensitive and TMZ-resistant cells. Such cytotoxic effect was accompanied by changes in the expression levels of LC3II, p62 and cleaved caspase 3, and increased cellular apoptosis. The results suggested that QC could sensitize GBM cells to TMZ at least partially through apoptosis induction, in which autophagy inhibition might be involved. Conclusion: The antimalarial drug QC may hold promise as a potentiation of TMZ treatment in GBM, especially in cases of TMZ-resistance.

CBIO-23. CHARACTERIZATION OF BMP SYSTEM MUTATIONS IN RECURRENT PRIMARY GLIOBLASTOMA PRIMARY CELL CULTURES IN RELATION TO GLIOBLASTOMA INITIATING STEM-LIKE CELLS AND TEMOZOLOMIDE RESISTANCE

Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary brain tumor. Its recurrence and rapidly acquired resistance to current therapies, including treatment with temozolomide (TMZ), results from the rapid expansion of re-emerging Glioblastoma Tumor Initiating stem cell-like Cells (GTICs). Targeted Exome Sequencing (TES) revealed that in such recurrent human GBM missense mutations arise in genes of the Bone Morphogenetic Protein (BMP) system in addition to those of other molecular systems found in primary GBM. To determine in which subpopulation BMP system mutations accumulate, we subjected two GBM primary cultures with a known mutations in ZFYVE16 (A290S) or BMPR1B (K222E) to targeted sequencing after FAC-sorting these cells based on the acknowledged stem cell markers CD44 and CD133. We found that ZFYVE16 (A290S) and BMPR1B (K222E) mutations accumulate within the CD133+/CD44- GTICs. Treatment of the primary culture with the latter mutation, with BMP4 suggests that the response to BMP signals is markedly reduced. Cells from these cultured were also cultured again after FAC-sorting for CD133 and CD44. Surprisingly, after three passages all subpopulations acquired a variation in expression of these markers specific for each patient. This shows that a 2D culture is able to loose and acquire stem cell markers and might resemble the original heterogeneity of the parental tumor more closely than we originally expected. In a separate set of experiments, RNA-sequencing of a TMZ-resistant primary GBM culture (GS295), created during the course of this work, compared to the TMZ-sensitive GS295, shows that several genes of the BMP system (NOGGIN, BMP7, ID1 and GATA3) are differentially expressed. Our results suggest that BMP system mutations tend to accumulate in CD133+ GTICs, that BMP signaling is altered within GBM primary cultures and that several mutated genes of the BMP system may correlate with acquisition of TMZ resistance.

Acquired temozolomide resistance in human glioblastoma cell line U251 is caused by mismatch repair deficiency and can be overcome by lomustine.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. While the alkylating agent temozolomide (TMZ) has prolonged overall survival, resistance evolution represents an important clinical problem. Therefore, we studied the effectiveness of radiotherapy and CCNU in an in vitro model of acquired TMZ resistance. We studied the MGMT-methylated GBM cell line U251 and its in vitro derived TMZ-resistant subline, U251/TMZ-R. Cytotoxicity of TMZ, CCNU, and radiation was tested. Both cell lines were analyzed for MGMT promotor status and expression of mismatch repair genes (MMR). The influence of MMR inhibition by cadmium chloride (CdCl2) on the effects of both drugs was evaluated. During the resistance evolution process in vitro, U251/TMZ-R developed MMR deficiency, but MGMT status did not change. U251/TMZ-R cells were more resistant to TMZ than parental U251 cells (cell viability: 92.0% in U251/TMZ-R/69.2% in U251; p=0.032) yet more sensitive to CCNU (56.4%/80.8%; p=0.023). The effectiveness of radiotherapy was not reduced in the TMZ-resistant cell line. Combination of CCNU and TMZ showed promising results for both cell lines and overcame resistance. CdCl2-induced MMR deficiency increased cytotoxicity of CCNU. Our results confirm MMR deficiency as a crucial process for resistance evolution to TMZ. MMR-deficient TMZ-resistant GBM cells were particularly sensitive to CCNU and to combined CCNU/TMZ. Effectiveness of radiotherapy was preserved in TMZ-resistant cells. Consequently, CCNU might be preferentially considered as a treatment option for recurrent MGMT-methylated GBM and may even be suitable for prevention of resistance evolution in primary treatment.

GADD45A plays a protective role against temozolomide treatment in glioblastoma cells

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 (GADD45Akd) enhanced the cell growth arrest and cell death induced by TMZ, even in natural (T98) and adapted (TR-U373) TMZ-resistant cells. Interestingly, GADD45Akd decreased the expression of O6-methylguanine-DNA methyltransferase (MGMT) in TMZ-resistant cells (T98 and TR-U373). In MGMT-deficient/TMZ-sensitive cells (U87 and U373), GADD45Akd 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.