Invasion Of Glioma Cell Lines Research Articles

High ECM2 Expression Predicts Poor Clinical Outcome and Promotes the Proliferation, Migration, and Invasiveness of Glioma.

Glioma is the most prevalent and fatal intracranial malignant tumor. Extracellular matrix protein 2 (ECM2) has rarely been studied in gliomas. Therefore, we explored the role of ECM2 in lower-grade gliomas (LGGs). The RNA-seq and clinicopathology data were obtained from the TCGA database. The immunohistochemical (IHC) staining was used to verify the expression of ECM2. Functional enrichment analyses, immune-related analyses, drug sensitivity, and mutation profile analyses were further conducted. Cox regression and Kaplan-Meier curves were utilized for survival analyses, while four external datasets were used to validate the prognostic role of ECM2. Furthermore, qRT-PCR, CCK-8, wound healing, and transwell assays were performed to confirm the function of ECM2 in gliomas. The study found a significant upregulation of ECM2 expression with increasing glioma grades and a significant association between ECM2 expression and tumor immune infiltration. Cox regression verified the prognostic role of ECM2 in LGG patients (HR = 1.656, 95%CI = 1.055-2.600, p = 0.028). High ECM2 expression was significantly associated with poor outcome (p < 0.001). Four external datasets validated its prognostic value. After the knockdown of ECM2, the functional experiments showed a significant decrease in proliferation, migration, and invasion in glioma cell lines. The study suggested the potential of ECM2 as a novel immune-associated prognostic biomarker and therapeutic target for glioma patients.

Calponin 3 Acts as a Potential Diagnostic and Prognostic Marker and Promotes Glioma Cell Proliferation, Migration, and Invasion

Calponin 3 (CNN3) is involved in the proliferation and invasion of cervical cancer and osteosarcoma cells. However, the role of CNN3 in glioma tumorigenesis remains to be elucidated. CNN3 mRNA expression in normal brain tissue and gliomas, including glioblastoma multiforme and lower-grade glioma, was analyzed using GEPIA 2 and Oncomine. CNN3 levels in glioma tissues were identified using immunohistochemical data provided by the Human Protein Atlas website. The relationship between CNN3 mRNA expression and clinical characteristics of patients with glioma was analyzed using the Oncomine database and The Cancer Genome Atlas. The diagnostic value of CNN3 expression in glioma was analyzed using receiver operating characteristic analysis according to The Cancer Genome Atlas and Genotype-Tissue Expression data. The relationship between CNN3 and prognosis was analyzed using GEPIA 2. The function of CNN3 knockdown in glioma cell lines was analyzed using cell proliferation, Transwell, and Western blot assays. Both mRNA and protein levels of CNN3 were distinctly higher in lower-grade glioma and glioblastoma multiforme tissues than those in normal brain tissues, particularly glioblastoma. A higher CNN3 mRNA level had significant relationship with higher World Health Organization grade, isocitrate dehydrogenase wild-type status, and 1p/19q noncodeletion. CNN3 mRNA expression is a highly accurate marker for the diagnosis of glioma. Patients with glioma in the high-CNN3 group had significantly lower disease-free survival and survival rates. In addition, CNN3 silencing significantly inhibited cell proliferation, migration, invasion, and the phosphorylation of P65 NF-κB. CNN3 expression is increased in glioma, particularly glioblastoma. Silencing CNN3 expression inhibited the proliferation, migration, and invasion of glioma cell lines.

Circular RNA circBFAR promotes glioblastoma progression by regulating a miR-548b/FoxM1 axis.

Glioblastoma multiforme (GBM) is the most common and aggressive type of tumor of the primary nervous system. Treatment options for GBM include surgery, chemotherapy, and radiation therapy; however, the clinical outcomes are poor, with a high rate of recurrence. An increasing number of studies have shown that circular RNAs (circRNAs) serve important roles in several types of cancer. Gene Expression Omnibus (GEO) database was utilized to identify the differentially expressed circRNAs and their biological functions. Then, we detected the circular RNA bifunctional apoptosis regulator (circBFAR) was significantly increased in three GEO datasets. However, the role of circBFAR has not been reported in GBM. In this study, the expression of circBFAR was significantly increased both in GBM tissues or cell lines and was negatively correlated with overall survival in patients with GBM. Knockdown of circBFAR inhibited proliferation and invasion both in vitro and in vivo. Increased expression of circBFAR resulted in a reduction of miR-548b expression in glioma cells. A luciferase reporter and RIP assay indicated that miR-548b was a direct target of circBFAR, and miR-548b may negatively regulate the expression of FoxM1. Rescue experiments showed that overexpression of FoxM1 could counter the effect of circBFAR silencing on the proliferation and invasion of glioma cell lines. Moreover, we identified that circBFAR regulates FoxM1 by interacting with miR-548b in glioma cells. In conclusion, the present study demonstrated that a circBFAR/miR-548b/FoxM1 axis regulates the development of GBM and highlights potentially novel therapeutic targets for the treatment of GBM.

LncRNA WEE2-AS1 Knockdown Inhibits the Proliferation, Migration and 3 Invasion of Glioma Cells via Regulating miR-29b-2-5p/TPM3 Axis.

Glioma is a general malignant tumor with a dismal prognosis. Long noncoding RNAs (lncRNAs) have been implicated in the initiation and processes of tumors. An investigation of the GEPIA database revealed that long noncoding RNA WEE2 antisense RNA 1 (WEE2-AS1) is upregulated in glioma tissues compared to normal brain tissues, and validation with quantitative real-time polymerase chain reaction (qRT-PCR) revealed that WEE2-AS1 expression was consistent with the database prediction. Fluorescence in situ hybridization (FISH) assays revealed that WEE2-AS1 was localized primarily in the cytoplasm. Clone formation experiment and EDU assay were used to detect cell proliferation ability, and Transwell assay was used to detect cell migration and invasion ability, Western-blot assay and immunofluorescence were used to determine TPM3 protein level. Functional experiments revealed that the downregulation of WEE2-AS1 impeded cell proliferation, migration, and invasion in glioma cell lines. Furthermore, downregulation of WEE2-AS1 suppressed tumor growth in vivo. Bioinformatics predictions and integrated experiments indicated that WEE2-AS1 promoted tropomyosin 3 (TPM3) expression by sponging miR-29b-2-5p. A dual-luciferase reporter assay was conducted to uncover the binding of WEE2-AS1 and miR-29b-2-5p and that of miR-29b-2-5p and TPM3. Additionally, a series of rescue assays showed that WEE2-AS1 promotes proliferation, migration, and invasion by targeting miR-29b-2-5p to regulate TPM3 expression. Ultimately, the results of this study indicate that WEE2-AS1 plays an oncogenic role in glioma and may promote further investigations of the diagnostic and prognostic value of WEE2-AS1 in glioma.

LncRNA ADAMTS9-AS1 knockdown suppresses cell proliferation and migration in glioma through downregulating Wnt/β-catenin signaling pathway.

The long non-coding RNA antisense 1 ADAMTS9-AS1 has been reported to serve as an oncogene or tumor suppressor in several tumors, including colorectal cancer and hepatocellular carcinoma. Nevertheless, the clinical significance and biological behaviors of ADAMTS9-AS1 in glioma still remain unclear. Therefore, the goal of this study was to evaluate the functional roles and potential mechanisms of ADAMTS9-AS1 in glioma cells. Using quantitative real-time polymerase chain reaction analysis, we found that ADAMTS9-AS1 was upregulated in glioma tissues and cells in comparison to corresponding controls. ADAMTS9-AS1 expression level was correlated to tumor size (p = 0.005) and the World Health Organization grade (p = 0.002). Kaplan–Meier analysis and Cox multivariate analysis showed that ADAMTS9-AS1 could serve as an independent prognostic factor affecting the overall survival of glioma patients. Functionally, depletion of ADAMTS9-AS1 significantly suppressed the proliferation, migration, and invasion in glioma cell lines (U251 and U87), as shown through cell counting kit-8 assay, Edu corporation assay, wound healing assay, and transwell assay. Furthermore, we demonstrated that knockdown of ADAMTS9-AS1 suppressed Wnt1, β-catenin, c-myc, and proliferating cell nuclear antigen, while upregulating E-cadherin expression. In conclusion, our data revealed that ADAMTS9-AS1 confers oncogenic function in the progression of glioma, thus targeting ADAMTS9-AS1 might be a promising therapeutic strategy for this disease.

The COX10-AS1/miR-641/E2F6 Feedback Loop Is Involved in the Progression of Glioma

Glioma is the most common primary tumour of the central nervous system and is considered one of the greatest challenges for neurosurgery. Mounting evidence has shown that lncRNAs participate in various biological processes of tumours, including glioma. This study aimed to reveal the role and relevant mechanism of COX10-AS1 in glioma. The expression of COX10-AS1, miR-641 and E2F6 was measured by qRT-PCR and/or western blot. Clone formation assays, EdU assays, Transwell assays and tumour xenograft experiments were performed to evaluate the effects of COX10-AS1, miR-641 and E2F6 on glioma proliferation, migration and invasion. Luciferase reporter assays, RNA pull-down assays and ChIP assays were conducted to analyse the relationship among COX10-AS1, miR-641 and E2F6. We demonstrated that COX10-AS1 was upregulated in glioma tissues and cell lines, which was related to the grade of glioma and patient survival. Next, through functional assays, we found that COX10-AS1 influenced the proliferation, migration and invasion of glioma cell lines. Then, with the help of bioinformatics analysis, we confirmed that COX10-AS1 regulated glioma progress by acting as a sponge of miR-641 to regulate E2F6. Moreover, further study indicated that E2F6 could promote COX10-AS1 expression by binding to its promoter region. Taken together, the data indicated that COX10-AS1 acts as an oncogene in combination with COX10-AS1/miR-641/E2F6 in glioma, which may be beneficial to the diagnosis and treatment of glioma.

Overexpression of microRNA-939-5p Contributes to Cell Proliferation and Associates Poor Prognosis in Glioma.

Glioma is the main brain tumor worldwide and has a worse prognosis. MicroRNAs (miRNAs) are proved to involve in massive malignant tumors including glioma. In this study, we tried to detect the microRNA-939-5p (miR-939-5p) expression pattern and explore its prognostic significance in glioma. We performed the quantitative real-time PCR to examine the relative expression of miR-939-5p in glioma. The Kaplan-Meier method and Cox regression analysis were used to reveal the prognostic importance of miR-939-5p. The influence of miR-939-5p on cell proliferation, migration, and invasion was investigated by the Cell Counting kit-8 (CCK-8), colony formation assay, and Transwell assay. Besides, the target gene of miR-939-5p was provided by luciferase reporter assay. Our data substantiated the expression of miR-939-5p was obviously increased in glioma tissues and cell lines. The upregulation of miR-939-5p predicted a poor survival rate and might act as an alternative prognostic indicator in glioma. The elevated expression of miR-939-5p boosted proliferation, migration, and invasion in glioma cell lines. The alternation of miR-939-5p changed the protein expression of TIMP metallopeptidase inhibitor 2 (TIMP2). These findings indicated the overexpression of miR-939-5p was associated with the poor prognosis of glioma patients. MiR-939-5p may function as an oncogene by targeting TIMP2.

LncRNA WEE2-AS1 knockdown inhibits the proliferation, migration and invasion of glioma cells via regulating miR-29b-2-5p/TPM3 axis.

Glioma is a general malignant tumor with a dismal prognosis. Long noncoding RNAs (lncRNAs) have been implicated in the initiation and processes of tumors. An investigation of the GEPIA database revealed that long noncoding RNA WEE2 antisense RNA 1 (WEE2-AS1) is upregulated in glioma tissues compared to normal brain tissues, and validation with quantitative real-time polymerase chain reaction (qRT-PCR) revealed that WEE2-AS1 expression was consistent with the database prediction. Fluorescence in situ hybridization (FISH) assays revealed that WEE2-AS1 was localized primarily in the cytoplasm. Clone formation experiment and EDU assay were used to detect cell proliferation ability, and Transwell assay was used to detect cell migration and invasion ability, Western-blot assay and immunofluorescence were used to determine TPM3 protein level. Functional experiments revealed that the downregulation of WEE2-AS1 impeded cell proliferation, migration, and invasion in glioma cell lines. Furthermore, downregulation of WEE2-AS1 suppressed tumor growth in vivo. Bioinformatics predictions and integrated experiments indicated that WEE2-AS1 promoted tropomyosin 3 (TPM3) expression by sponging miR-29b-2-5p. A dual-luciferase reporter assay was conducted to uncover the binding of WEE2-AS1 and miR-29b-2-5p and that of miR-29b-2-5p and TPM3. Additionally, a series of rescue assays showed that WEE2-AS1 promotes proliferation, migration, and invasion by targeting miR-29b-2-5p to regulate TPM3 expression. Ultimately, the results of this study indicate that WEE2-AS1 plays an oncogenic role in glioma and may promote further investigations of the diagnostic and prognostic value of WEE2-AS1 in glioma.

Co-overexpression of RIOK1 and AKT1 as a prognostic risk factor in glioma.

Glioblastoma multiforme (GBM) is one of the most frequent primary malignancies of the brain. Although the treatment strategy has significantly improved, patient prognosis remains poor. In vitro studies have shown that the right open reading frame kinase 1/protein kinase B (RIOK1-AKT) signaling pathway plays an important role in the malignant phenotype of glioma cells. This study aimed to investigate the co-expression of RIOK1 and ATK in glioma tissues and its clinical significance. Compared with normal tissues, RIOK1 and AKT1 expression were significantly upregulated in glioma tissues. In addition, patients with higher World Health Organization staging grades had increased RIOK1 and AKT1 expression levels, and RIOK1 and AKT1 expression were positively correlated. Notably, both RIOK1 and AKT1 expressions were correlated with poor prognosis. In vitro experiments showed that silencing RIOK1 inhibited the proliferation, migration, and invasion of glioma cell lines by suppressing AKT and c-Myc expression. These results indicate that the RIOK1-AKT1 axis could play an important role in GBM progression.

Identification of 2-Fluoropalmitic Acid as a Potential Therapeutic Agent Against Glioblastoma.

Glioblastomas (GBMs) are aggressive malignant brain tumors. Although chemotherapy with temozolomide (TMZ) can extend patient survival, most patients eventually demonstrate resistance. Therefore, novel therapeutic agents that overcome TMZ chemoresistance are required to improve patient outcomes. Drug screening is an efficient method to find new therapeutic agents from existing drugs. In this study, we explored a novel anti-glioma agent by drug screening and analyzed its function with respect to GBM treatment for future clinical applications. Drug libraries containing 1,301 diverse chemical compounds were screened against two glioma stem cell (GSC) lines for drug candidate selection. The effect of selected agents on GSCs and glioma was estimated through viability, proliferation, sphere formation, and invasion assays. Combination therapy was performed to assess its ability to enhance TMZ cytotoxicity against GBM. To clarify the mechanism of action, we performed methylation-specific polymerase chain reaction, gelatin zymography, and western blot analysis. The acyl-CoA synthetase inhibitor 2-fluoropalmitic acid (2-FPA) was selected as a candidate anti-glioma agent. 2-FPA suppressed the viability and stem-like phenotype of GSCs. It also inhibited proliferation and invasion of glioma cell lines. Combination therapy of 2-FPA with TMZ synergistically enhanced the efficacy of TMZ. 2-FPA suppressed the expression of phosphor-ERK, CD133, and SOX-2; reduced MMP-2 activity; and increased methylation of the MGMT promoter. 2-FPA was identified as a potential therapeutic agent against GBM. To extend these findings, physiological studies are required to examine the efficacy of 2-FPA against GBM in vivo.

The COX10-AS1/miR-641/E2F6 Feedback Loop Is Involved in the Progression of Glioma

Background: Glioma is the most common primary tumour of the central nervous system and is considered one of the greatest challenges for neurosurgery. Mounting evidence has shown that lncRNAs participate in various biological processes of tumours, including glioma. This study aimed to reveal the role and relevant mechanism of COX10-AS1 in glioma. Methods: The expression of COX10-AS1, miR-641 and E2F6 was measured by qRT-PCR and/or western blot. Clone formation assays, EdU assays and Transwell assays were performed to evaluate the effects of COX10-AS1, miR-641 and E2F6 on glioma proliferation, migration and invasion. Luciferase reporter assays, RNA pull-down assays and ChIP assays were conducted to analyse the relationship among COX10-AS1, miR-641 and E2F6. Findings: COX10-AS1 was upregulated in glioma tissues and cell lines, which was related to the grade of glioma and patient survival. Next, through functional assays, we found that COX10-AS1 influenced the proliferation, migration and invasion of glioma cell lines. Then, with the help of bioinformatics analysis, we confirmed that COX10-AS1 regulated glioma by acting as a sponge of miR-641 to regulate E2F6. Moreover, further study indicated that E2F6 could promote COX10-AS1 expression by binding to its promoter region. Interpretation: COX10-AS1 acts as an oncogene in combination with COX10-AS1/miR-641/E2F6 in glioma, which may be beneficial to the diagnosis and treatment of glioma. Funding Statement: This study was supported by grants from National Natural Science Foundation of China (No.81502168) and Nanjing Medical Science and technique Development Foundation. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All the participants signed written informed consent forms. This research was approved by the Ethics Committee of the Nanjing First Hospital.

Avasimibe inhibits the proliferation, migration and invasion of glioma cells by suppressing linc00339

Glioma is one of the most destructive human tumours. Although standard treatment has improved the prognosis for glioma patients, the survival of glioma patients is still unsatisfactory. Avasimibe, an effective inhibitor of cholesterol acyltransferase 1 (ACAT1), has shown anti-tumour efficacy in many kinds of tumours. However, its role and related mechanism in glioma has not been fully elucidated. In the present study, we show that avasimibe effectively inhibits the proliferation, migration and invasion of glioma cell lines. Through LncRNA microarrays, we found that linc00339 levels were closely related to the anti-tumour effect of avasimibe. With the help of a series of functional assays, we show that avasimibe inhibits the proliferation, migration and invasion of glioma cell lines by suppressing linc00339 in vitro and in vivo. Our findings may provide a new approach for glioma therapy.

LncRNA NNT-AS1 promote glioma cell proliferation and metastases through miR-494-3p/PRMT1 axis

ABSTRACT Long noncoding RNAs (lncRNAs) are key players in cancer progression. However, the function of lncRNA NNT-AS1 on glioma is unclear. In the present study, a total of 73 tumor tissues and matched adjacent non-tumor tissues were collected, and glioma cell lines were cultured in vitro. mRNA expression was tested using RT-qPCR. The protein expression level was determined using the western blot assay, cell proliferation was measured using the CCK-8 and BrdU proliferation assay, and the cell cycle, cell migration and invasion were determined using flow cytometry analysis, the wound healing assay and transwell, respectively. The results showed that lncNNT-AS1 is significantly up-regulated during the early stages of glioma. In particular, high levels of NNT-AS1 are observed in glioma cell lines compared to human astrocyte (HA) cells. Furthermore, the inhibition of lnc-NNT-AS1 by siRNA interfere attenuates the cell viability, proliferation, migration and invasion of glioma cell lines. Mechanistically, the inhibition of NNT-AS1 directly interacted with miRNA-494-3p, and positively regulated the downstream target PRMT1 in vitro. Further study proved that the overexpression of miRNA-494-3p and the inhibition of PRMT1 could attenuate both glioma cell proliferation and metastases. Collectively, our results indicated that the miR-494-3p-PRMT1 axis is involved the tumor-suppressive effects of NNT-AS1 inhibition, which sheds new light on lncRNA-directed diagnostics and the therapeutics of glioma.

Knockdown of lncRNA KCNQ1OT1 inhibits glioma progression by regulating miR-338-3p/RRM2

AbstractThe dysregulated lncRNA play essential roles in glioma development. This study aimed to investigate the role and mechanism of lncRNA potassium voltage-gated channel subfamily Q member 1 opposite strand/ antisense transcript 1 (KCNQ1OT1) in glioma progression. Tumor tissues and adjacent normal samples were collected from 30 glioma patients. The expression levels of lncRNA KCNQ1OT1, microRNA (miR)-338-3p and ribonucleotide reductase M2 (RRM2) were detected by quantitative real-time polymerase chain reaction or western blot analyses. Levels of cell viability, apoptosis, cell migration and invasion in glioma cell lines were determined using cell counting kit-8, flow cytometry with annexin V-FITC and trans-well assays, respectively. The role of KCNQ1OT1 in glioma development in vivo was investigated using a xenograft model. The target association between miR-338-3p and KCNQ1OT1 or RRM2 was validated by luciferase reporter assay. The results found that expression of KCNQ1OT1 was enhanced in glioma tissues and cells, and KCNQ1OT1 knockdown inhibited cell viability, migration and invasion, and xenograft tumor growth, but promoted apoptosis. miR-338-3p was targeted via KCNQ1OT1 and could reverse the effect of KCNQ1OT1 on glioma progression. RRM2 was targeted via miR-338-3p and attenuated the suppressive effect of miR-338-3p on glioma cell viability, migration and invasion. Besides, KCNQ1OT1 overexpression increased RRM2 expression, and this event was weakened via miR-338-3p up-regulation. In conclusion, the present finding suggest that silencing of KCNQ1OT1 can suppress the development and progression of glioma by up-regulating miR-338-3p and down-regulating RRM2.

Circular RNA TTBK2 promotes the development of human glioma cells via miR-520b/EZH2 axis.

Glioma is a highly malignant human disease characterized by limited response to clinical therapy. Evidence indicated that circular RNA Tau tubulin kinase 2 circular RNAs (circ-TTBK2) participated in glioma pathogenesis. However, the precise effect of circ-TTBK2 on glioma progression is needed to be highlighted. The levels of circ-TTBK2, microRNA-520b (miR-520b), and enhancer of zeste homologue 2 (EZH2) were detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) or Western blot. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was performed to determine cell proliferation in vitro. Besides, a flow cytometry assay was conducted to examine apoptosis of A172 and U251 cells. Cell invasion was identified using the transwell assay. Moreover, Dual-Luciferase reporter assay was used to confirm the interaction between miR-520b and circ-TTBK2 or EZH2. The role of circ-TTBK2 in glioma progression was exposed using xenograft tumor experiments. The levels of circ-TTBK2 and EZH2 were markedly augmented, whereas miR-520b expression level was notably reduced in glioma tissues and cell lines. Either circ-TTBK2 or EZH2 detection could clearly facilitate cell apoptosis and block proliferation and invasion in A172 and U251 cells, while the effect of circ-TTBK2 or EZH2 deficiency was reverted by co-transfecting with miR-520 inhibitor. Moreover, circ-TTBK2 exerted its roles via miR-520b/EZH2 axis in glioma cells, and the knockdown of circ-TTBK2 could hinder the progression of glioma. Circ-TTBK2/miR-520b/EZH2 axis modulated cell proliferation, apoptosis, and invasion in glioma cell lines, and might serve as potential targets for glioma diagnosis and therapy.

DDIS-05. IDENTIFICATION OF 2-FLUORO PALMITIC ACID AS A POTENTIAL THERAPEUTIC AGENT AGAINST GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) represents the most aggressive and frequent type of primary malignant brain tumors with a dismal clinical outcome. Though adjuvant temozolomide (TMZ) chemotherapy followed by surgical resection can extend patient`s post-operative survival, a considerable number of GBM cases are refractory to TMZ. Novel therapeutic agents that can overcome chemoresistance against TMZ are urgent. Drug repositioning is a process of identifying new indications for existing drugs and provides potential possibilities to discover new drugs. In this study, we explored novel anti-glioma agents which enhances the effect of TMZ with the strategy of drug repositioning. METHODS Drug library which contains 1300 diverse chemical compounds was screened using 2 kinds of glioma stem cell (GSC) lines to select novel therapeutic candidate. The effect of candidate drug on the proliferation of GSCs was estimated by sphere formation assay. To evaluate its efficacy against glioma cell biology, proliferation assay, matrigel invasion assay were performed. To clarify the mechanism of drug effects, we investigated target molecules by gelatin zymography and western blot. RESULTS Acyl CoA synthetase inhibitor named 2-fluoropalmitic acid (2-FPA) was selected as a novel candidate. 2-FPA suppressed proliferation of glioma cell lines by single administration and/or combination with TMZ. The sphere formation of GSCs was suppressed by combination therapy. Combination therapy enhanced TMZ effect. 2-FPA also suppressed invasion of glioma cell lines in a dose dependent manner. 2-FPA suppressed MMP-2 activity and phosphorylation of STAT3. CONCLUSION 2-FPA was identified as a novel potential therapeutic agent against GBM.

CBMT-18. THE ROLE OF BIOMARKER CANDIDATE GELSOLIN AND ITS MICRORNAS IN GLIOBLASTOMA

Abstract BACKGROUND Among potential glioblastoma (GBM) blood biomarkers that we identified recently, we focused on gelsolin (GSN), a key regulator of actin filament disassembly. GSN was significantly lower in the blood of patients with GBM than in that of healthy controls. In this study, we analyzed the function of GSN and identified microRNAs (miRs) involved in GSN expression in GBM. METHODS QRT-PCR and western blot were introduced to evaluate the expression level of GSN in normal brain and GBM tissue. The localization of GSN was examined by immunohistochemistry and immunocytochemistry using human samples. The association between the expression level of GSN and progression free survival (PFS) /overall survival (OS) in GBM was assessed by Kaplan-Meier analysis. The function of GSN and its signal transduction in glioma cell lines were analyzed using small interfering RNA (siRNA) knockdown of GSN. Additionally, miRs controlling GSN expression were retrieved from databases of miRs, and miRs related to GSN expression were identified in GBM tissues. RESULTS The expression level of GSN was significantly lower in GBM tissues compared to normal brains. Normal astrocytes mainly expressed GSN. High expressor of GSN showed longer PFS and OS than low expressor. Proliferation and invasion in glioma cell lines were significantly promoted by siRNA for GSN accompanied with the activation of glycogen synthase kinase 3β. In GBM tissues, the expression levels of GSN and miR-654-5p, 450b-5p showed an inverse correlation. The inhibitor for miR-654-5p and miR-450b-5p accelerated GSN expression resulting reduction of proliferation. CONCLUSION GSN plays a role as suppressor of proliferation and invasion in GBM. miR-654-5p and miR-450b-5p which control GSN expression can be targets against GBM.

Abstract 3930: Oligosaccharyltransferase inhibition reduces glioma cell invasion and migration

Abstract Purpose: Glioblastoma (GBM) is the most common and aggressive primary brain neoplasm. Cell migration, invasion, and resistance to therapy lead to a high rate of recurrence in GBM. Glycoprotein receptor overexpression or activation caused by mutation is critical for the development and progression of many tumors including GBM. N-linked glycosylation is an endoplasmic reticulum co- and post-translational modification common to RTKs and other glycoproteins that is crucial for receptor activation. We hypothesized that altering N-linked glycosylation with an oligosaccharyltransferase (OST) inhibitor (NGI-1), could effectively disrupt GBM cell proliferation, migration, and invasion. Experimental Procedures: The effects of OST inhibition on migration and invasion were assessed in vitro by wound healing assay, Boyden chamber assay and 3D Matrigel assay in glioma cell lines (D54, SKMG3, U251, T98G and 42-MG) and in patient-derived cell lines (GBM6, GBM26 and GBM76) in the presence or the absence of NGI-1. Cell proliferation was determined by the MTT assay over 5 days of drug treatment. Radiation survival was evaluated by clonogenic assay. Cell adhesion molecules were measured by phospho-blot assay. Results: OST inhibition reduced cell proliferation in a dose-response manner with a significant reduction of 50% or greater over a time course of 5 days. These responses were observed in 6 of the 8 glioma and patient-derived cell lines tested while only small effects were found in the less sensitive U251 and GBM6 cell lines. A 70 to 80% reduction of tumor cell invasion was observed after NGI-1 treatment in T98G, SKMG3 and GBM76 cells with a 40 to 50% reduction in U251, D54 and GBM6 cells. Interestingly, neither 42-MG or GBM26 cells showed a decrement in invasion with the treatment of NGI-1, suggesting this effect is mediated by discrete cell signaling pathways. These results also correspond to a significant reduction of radiation clonogenic survival caused by OST inhibition in SKMG3 and D54 cells. In matrigel assays significant changes in 3D tumor growth and morphology were also observed over a time course of 72h following NGI-1 treatment. Together this work shows that cell lines with ErbB-driven signaling (D54 and SKMG3) and patient-derived cell lines with the A289T EGFR activating mutation (GBM26 and GBM76) are sensitive to OST inhibtion with NGI-1 treatment. Changes in glycosylation of cell adhesion molecules, such as integrins and NCAMs, were also observed and studies to define the glycoprotein targets that regulate glioma migration and invasion are underway. Conclusion: This study suggests that the inhibition of N-glycosylation with a small molecule OST inhibitor suppresses migration and invasion of glioma cell lines and GBM patient-derived cell lines and supports further preclinical investigation in malignant glioma. Citation Format: Marta Baro, Jann N. Sarkaria, Joseph N. Contessa. Oligosaccharyltransferase inhibition reduces glioma cell invasion and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3930.

The miR-26a/AP-2α/Nanog signaling axis mediates stem cell self-renewal and temozolomide resistance in glioma.

Aberrant expression of transcription factor AP-2α has been functionally associated with various cancers, but its clinical significance and molecular mechanisms in human glioma are largely elusive.Methods: AP-2α expression was analyzed in human glioma tissues by immunohistochemistry (IHC) and in glioma cell lines by Western blot. The effects of AP-2α on glioma cell proliferation, migration, invasion and tumor formation were evaluated by the 3-(4,5-dimethyNCthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) and transwell assays in vitro and in nude mouse models in vivo. The influence of AP-2α on glioma cell stemness was analyzed by sphere-formation, self-renewal and limiting dilution assays in vitro and in intracranial mouse models in vivo. The effects of AP-2α on temozolomide (TMZ) resistance were detected by the MTT assay, cell apoptosis, real-time PCR analysis, western blotting and mouse experiments. The correlation between AP-2α expression and the expression of miR-26a, Nanog was determined by luciferase reporter assays, electrophoretic mobility shift assay (EMSA) and expression analysis.Results: AP-2α expression was downregulated in 58.5% of glioma tissues and in 4 glioma cell lines. AP-2α overexpression not only reduced the proliferation, migration and invasion of glioma cell lines but also suppressed the sphere-formation and self-renewal abilities of glioma stem cells in vitro. Moreover, AP-2α overexpression inhibited subcutaneous and intracranial xenograft tumor growth in vivo. Furthermore, AP-2α enhanced the sensitivity of glioma cells to TMZ. Finally, AP-2α directly bound to the regulatory region of the Nanog gene, reduced Nanog, Sox2 and CD133 expression. Meanwhile, AP-2α indirectly downregulated Nanog expression by inhibiting the interleukin 6/janus kinase 2/signal transducer and activator of transcription 3 (IL6/JAK2/STAT3) signaling pathway, consequently decreasing O6-methylguanine methyltransferase (MGMT) and programmed death-ligand 1 (PD-L1) expression. In addition, miR-26a decreased AP-2α expression by binding to the 3' untranslated region (UTR) of AP-2α and reversed the tumor suppressive role of AP-2α in glioma, which was rescued by a miR-26a inhibitor. TMZ and the miR-26a inhibitor synergistically suppressed intracranial GSC growth.Conclusion: These results suggest that AP-2α reduces the stemness and TMZ resistance of glioma by inhibiting the Nanog/Sox2/CD133 axis and IL6/STAT3 signaling pathways. Therefore, AP-2α and miR-26a inhibition might represent a new target for developing new therapeutic strategies in TMZ resistance and recurrent glioma patients.

Knockdown of HDAC1 expression suppresses invasion and induces apoptosis in glioma cells.

Glioma is the most common malignant tumor of the central nervous system, with a low survival rate of five years worldwide. Although high expression and prognostic value of histone deacetylase 1 (HDAC1) have been recently reported in various types of human tumors, the molecular mechanism underlying the biological function of HDAC1 in glioma is still unclear. We found that HDAC1 was elevated in glioma tissues and cell lines. HDAC1 expression was closely related with pathological grade and overall survival of patients with gliomas. Downregulation of HDAC1 inhibited cell proliferation, prevented invasion of glioma cell lines, and induced cell apoptosis. The expression of apoptosis and metastasis related molecules were detected by RT-PCR and Western blot, respectively, in U251 and T98G cells with HDAC1 knockdown. We found that HDAC1 knockdown upregulated expression of BIM, BAX, cleaved CASPASE3 and E-CADHERIN, and decreased expression of TWIST1, SNAIL and MMP9 in U251 and T98G cells with HDAC1 knockdown. In vivo data showed that knockdown of HDAC1 inhibited tumor growth in nude mice. In summary, HDAC1 may therefore be considered an unfavorable progression indicator for glioma patients, and may also serve as a potential therapeutic target.