Glioma Radioresistance Research Articles

RDNA-03. AUTOPHAGY AS A NOVEL THERAPEUTIC TARGET IN MUTANT IDH1 GLIOMAS

Abstract Mutated Isocitrate dehydrogenase 1 (IDH1R132H; mIDH1) is found in 50% of all adult gliomas and in 80% of lower-grade gliomas. IDH1R132H promotes 2-hydroxyglutarate production which inhibits histone and DNA demethylases, inducing epigenetic reprogramming of the tumor transcriptome. We recently demonstrated that epigenetic reprogramming enhances DNA-damage response and confers radioresistance in mIDH1 glioma, harboring p53 and ATRX loss. Furthermore, several studies indicate that IDH1R132H triggers cellular metabolism reprogramming. In this study, RNA-seq data revealed that mIDH1 glioma neurospheres (mIDH1-NS) have downregulated gene ontologies related to mitochondrial metabolism. Analysis of mitochondrial and glycolytic activity, using a Seahorse Analyzer, showed that mIDH1-NS have decreased levels of oxygen consumption and extracellular acidification rates, characteristic of quiescent state. Nevertheless, mIDH1 glioma cells can grow and generate tumors, suggesting that they might maintain self-renewal via autophagy. To investigate this hypothesis, we studied autophagy in wild-type and mIDH1 glioma cells analyzing LC3 conversion, p62 expression, and ULK1 protein phosphorylation. Mouse and human mIDH1 glioma cells exhibited increased expression of pULK1-S555, enhanced conversion of LC3I to LC3II, and decreased expression of p62, indicating that mIDH1 cells have an augmented autophagy. Also, key proteins involved in autophagy, including MST4 and UVRAG, are upregulated in mIDH1-NS. We also studied autophagy flux using LC3-mCherry-GFP reporter to identify LC3 recruitment at the autophagosome membranes and the autolysosome activity. Our results showed that mIDH1 cells have increased LC3 recruitment and enhanced autolysosome activity. Interestingly, autophagy has been related with glioma radioresistance. In our study, mouse and human mIDH1 glioma cells are sensitive to autophagy inhibition. In addition, autophagy inhibition restored radiosensitivity in mouse and human mIDH1 glioma cells, and in our mIDH1 glioma model, enhancing median survival compared with radiation alone. In conclusion, our results indicate that mIDH1 glioma cells maintain self-renewal via activation of autophagy, which represents a novel therapeutic target.

Combined elevation of TRIB2 and MAP3K1 indicates poor prognosis and chemoresistance to temozolomide in glioblastoma.

IntroductionGlioblastoma (GBM) is the most lethal primary malignant brain tumor in adults with poor survival due to acquired therapeutic resistance and rapid recurrence. Currently, the standard clinical strategy for glioma includes maximum surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy; however, the median survival of patients with GBM remains poor despite these comprehensive therapies. Therefore, the identification of new prognostic biomarkers is urgently needed to evaluate the malignancy and long‐term outcome of glioma.AimsTo further investigate prognostic biomarkers and potential therapeutic targets for GBM.ResultsIn this study, we identified tribbles pseudokinase 2 (TRIB2) as one of the genes that is most correlated with pathological classification, radioresistance, and TMZ resistance in glioma. Additionally, the expression of mitogen‐activated protein kinase kinase kinase 1 (MAP3K1) showed a strong correlation with TRIB2. Moreover, a combined increase in TRIB2 and MAP3K1 was observed in GBM and indicated a poor prognosis of patients with glioma. Finally, enriched TRIB2 expression and MAP3K1 expression were shown to be associated with resistance to TMZ and radiotherapy.ConclusionCombined elevation of TRIB2 and MAP3K1 could be novel prognostic biomarkers and potential therapeutic targets to evaluate the malignancy and long‐term outcomes of GBM.

FGFR2-mediated phosphorylation of PTEN at tyrosine 240 contributes to the radioresistance of glioma.

Ionizing radiation (IR) is a standard-of-care treatment for glioma patients; however, the clinical efficacy is limited due to therapeutic resistance. A recent study published by Ma et al. (Cancer Cell 35:504-518, 2019) reported that the phosphorylation of phosphatase and tensin homolog (PTEN) at tyrosine 240 (pY240-PTEN) promotes the radioresistance of human glioma cells. After treatment with IR, the fibroblast growth factor receptor 2 (FGFR2)-mediated phosphorylation that generated pY240-PTEN could effectively promote the decondensation of chromatin through an interaction with Ki-67, leading to DNA damage repair and radioresistance. However, such promising findings need to be addressed in detail after considering the following points. (1) The authors should take into consideration whether or not the nuclear-cytoplasmic translocalization of PTEN occurs. (2) The roles of FGFR2-PTEN downregulation should be validated using both genetic and pharmacological inhibition models. (3) Some of the data shown by the authors are confusing and did not support the conclusion that patients with higher PTEN and FGFR2 expression were relatively IR resistant.

Carbon irradiation overcomes glioma radioresistance by eradicating stem cells and forming an antiangiogenic and immunopermissive niche.

Tumor radioresistance leading to local therapy failure remains a major obstacle for successful treatment of high-grade glioma. We hypothesized that distinct radiobiological features of particle therapy with carbon ions may circumvent glioma radioresistance. We demonstrate that carbon irradiation (CIR) efficiently eradicates radioresistant patient-derived glioma stem cells (GSCs), leading to growth inhibition and prolonged survival. The impact of CIR at the tumor-stroma interface was further investigated in 2 syngeneic mouse and 2 orthotopic GSC xenograft models. Intriguingly, tumor regressions and long-term local controls were observed at doses greater than or equal to 15-Gy CIR. Fractionated CIR further prolonged survival. The enhanced relative biological effectiveness of CIR in vivo was attributed to its potent antiangiogenic effects and eradication of radioresistant hypoxic tumor cells. Blockade of the HIF1-α/stromal cell-derived factor 1/CXCR4 axis by CIR reduced the recruitment of microglia and myeloid-derived suppressor cells (CD11b+Gr1+). Consequently, CIR abrogated M2-like immune polarization and enhanced the influx of CD8+ cells, generating an immunopermissive niche. We report that radiotherapy with carbon ions could surmount several central glioma resistance mechanisms by eradicating hypoxic and stem cell-like tumor cells, as well as modulating the glioma niche toward an antiangiogenic and less immunosuppressive state. Conclusively, potentially novel rationales for CIR in conquering glioma radioresistance are provided.

Expression profiles and potential functions of circular RNAs in extracellular vesicles isolated from radioresistant glioma cells

The molecular mechanisms of circular RNAs (circRNAs) in extracellular vesicles (EVs) associated with glioma radioresistance remain unknown. The aim of the present study was to assess the differential circRNA expression profiles between EVs isolated from U251 cells and EVs isolated from radioresistant U251 (RR‑U251) cells. Identified circRNAs in EVs isolated from RR‑U251 cells (RR‑EVs) act as a U251 microRNA (miRNA) sponge. The circRNA expression was determined using RNA sequencing (RNA‑seq) technique. A total of 1,235 circRNAs were detected. We identified 63 upregulated and 48 downregulated circRNAs in RR‑EVs compared with those from U251 cells (Nor‑EVs). The expression level of candidate circATP8B4 was confirmed using real‑time quantitative PCR. It was significantly higher in RR‑EVs than in Nor‑EVs. Expression profile of RR‑U251 and U251 miRNAs was conducted. miRanda and RNAhybrid softwares was used to predict the U251 downregulated miRNAs interacting with circATP8B4. CircATP8B4 from RR‑EVs may be transferred to normal glioma U251 cells and act as an miR‑766 sponge to promote cell radioresistance. In conclusion, using RNA‑seq and bioinformatics, it was found that circATP8B4 in RR‑EVs acts as a U251 miR‑766 sponge, which may be involved in glioma radioresistance.

MicroRNA-153-3p enhances cell radiosensitivity by targeting BCL2 in human glioma

BackgroundGlioma is the most prevalent malignant tumor in human central nervous systems. Recently, the development of resistance to radiotherapy in glioma patients markedly vitiates the therapy outcome. MiR-153-3p has been reported to be closely correlated with tumor progression, but its effect and molecular mechanism underlying radioresistance remains unclear in glioma.MethodsThe expression of miR-153-3p was determined in radioresistant glioma clinical specimens as well as glioma cell lines exposed to irradiation (IR) using quantitative real-time PCR. Cell viability, proliferation and apoptosis were then evaluated by MTT assay, colony formation assay, Flow cytometry analysis and caspase-3 activity assay in glioma cells (U87 and U251). Tumor forming was evaluated by nude mice model in vivo. TUNEL staining was used to detect cell apoptosis in nude mice model. The target genes of miR-153-3p were predicted and validated using integrated bioinformatics analysis and a luciferase reporter assay.ResultsHere, we found that miR-153-3p was down-regulated in radioresistant glioma clinical specimens as well as glioma cell lines (U87 and U251) exposed to IR. Enhanced expression of miR-153-3p promoted the radiosensitivity, promoted apoptosis and elevated caspase-3 activity in glioma cells in vitro, as well as the radiosensitivity in U251 cell mouse xenografs in vivo. Mechanically, B cell lymphoma-2 gene (BCL2) was identified as the direct and functional target of miR-153-3p. Moreover, restoration of BCL2 expression reversed miR-153-3p-induced increase of radiosensitivity, apoptosis and caspase-3 activity in U251 cells in vitro. In addition, clinical data indicated that the expression of miR-153-3p was significantly negatively associated with BCL2 in radioresistance of glioma samples.ConclusionsOur findings suggest that miR-153-3p is a potential target to enhance the effect of radiosensitivity on glioma cells, thus representing a new potential therapeutic target for glioma.

TGF-β/Smads Signaling Affects Radiation Response and Prolongs Survival by Regulating DNA Repair Genes in Malignant Glioma.

To understand the molecular mechanism underlying the causal relationship between aberrant upregulation of transforming growth factor beta (TGF-β) and radio-resistance in glioma. The mouse glioma cell GL261 was irradiated, and relative expression of TGF-β/Smad signaling genes was determined by real-time PCR and western blotting. The DNA repair response on exogenous TGF-β or LY2109761 was evaluated by quantification of diverse genes by real-time PCR and western blotting. Xenograft mice were employed for in vivo investigation to assess the response to irradiation and LY2109761 either alone or in combination. The expression of DNA repair genes was further determined in the xenograft tumor. The TGF-β/Smad signaling pathway was activated by radiation in the GL261 cell line. The exogenous complement of TGF-β significantly stimulated DNA repair response. Administration of LY2109761 suppressed DNA repair genes. Simultaneous treatment with LY2109761 abrogated the upregulation of DNA repair genes in GL261. In the xenograft tumor model, LY2109761 synergistically improved the therapeutic effect of radiation via improvement of sensitivity. Our data suggested that LY2109761 treatment re-sensitized glioma to radiation via antagonizing TGF-β/Smad-induced DNA repair.

GPR56/ADGRG1 Inhibits Mesenchymal Differentiation and Radioresistance in Glioblastoma

A mesenchymal transition occurs both during the natural evolution of glioblastoma (GBM) and in response to therapy. Here, we report that the adhesion G-protein-coupled receptor, GPR56/ADGRG1, inhibits GBM mesenchymal differentiation and radioresistance. GPR56 is enriched in proneural and classical GBMs and is lost during their transition toward a mesenchymal subtype. GPR56 loss of function promotes mesenchymal differentiation and radioresistance of glioma initiating cells both invitro and invivo. Accordingly, a low GPR56-associated signature isprognostic of a poor outcome in GBM patients even within non-G-CIMP GBMs. Mechanistically, we reveal GPR56 as an inhibitor of the nuclear factor kappaB (NF-κB) signaling pathway, thereby providing the rationale by whichthis receptor prevents mesenchymal differentiation and radioresistance. A pan-cancer analysis suggests that GPR56 might be an inhibitor of the mesenchymal transition across multiple tumor types beyond GBM.

A molecular view of the radioresistance of gliomas.

Gliomas originate from glial cells and are the most frequent primary brain tumors. High-grade gliomas occur ∼4 times more frequently than low-grade gliomas, are highly malignant, and have extremely poor prognosis. Radiotherapy, sometimes combined with chemotherapy, is considered the treatment of choice for gliomas and is used after resective surgery. Despite great technological improvements, the radiotherapeutic effect is generally limited, due to the marked radioresistance exhibited by gliomas cells, especially glioma stem cells (GSCs). The mechanisms underlying this phenomenon are multiple and remain to be fully elucidated. This review attempts to summarize current knowledge on the molecular basis of glioma radioresistance by focusing on signaling pathways, microRNAs, hypoxia, the brain microenvironment, and GSCs. A thorough understanding of the complex interactions between molecular, cellular, and environmental factors should provide new insight into the intrinsic radioresistance of gliomas, potentially enabling improvement, through novel concurrent therapies, of the clinical efficacy of radiotherapy.

Identification of a gene signature associated with radiotherapy and prognosis in gliomas

Glioma is one of the most common primary brain tumors with poor prognosis. Although radiotherapy is an important treatment method for gliomas, the efficacy is still limited by the high occurrence of radioresistance and the underlying molecular mechanism is unclear. Here, we performed a data mining work based on four glioma expression datasets. These datasets were classified into training set and validation set. Radiotherapy-induced differential expressed genes and prognosis-associated genes were screened using different classifiers. The Kaplan-Meier curves along with the two-sided Log Rank (Mantel-Cox) test were used to evaluate overall survival. We found the gene expression profiles of gliomas between those patients received radiotherapy and those patients without received radiotherapy were quite different. A 20-gene signature was identified, which was associated with radiotherapy.Furthermore, a novel 5-gene signature (HOXC10, LOC101928747, CYB561D2, RPL36A and RPS4XP2) as an independent predictor of glioma patients’ prognosis was further derived from the 20-gene signature. These findings provided a new insight into the molecular mechanism of radioresistance in gliomas. The 5-gene signature might represent therapeutic target for gliomas.

Cofilin-1 and phosphoglycerate kinase 1 as promising indicators for glioma radiosensibility and prognosis.

Glioma is a primary malignancy in central nervous system. Radiotherapy has been used as one of the standard treatments for glioma for decades. Since radioresistance can reduce the curative efficacy of radiotherapy in glioma, investigating the cause of radioresistance and predicting the tumour radiosensibility appeared particularly important. We previously reported that CFL1 and PGK1 are over-expressed in radioresistant U251 glioma cells. In this study, the level of CFL1 and PGK1 of 113 glioma tissues were measured by ELISA method. The relevance of the expression of these two proteins to radiosensibility was analyzed by mean test and multivariate logistic regression. The survival analysis was carried out in 85 irradiated patients and 105 followed-up patients respectively. The relationship between protein expression and clinical parameters was explored in overall 113 patients, and the correlation between CFL1 and PGK1 were determined as well. Our results showed that the expression of CFL1 and PGK1 were significantly higher (P < 0.001) in radioresistant patients than others. The multivariate Logistic regression demonstrated that the expression of CFL1 (p < 0.001) and PGK1 (p < 0.001) were associated with radioresistance in glioma. The multivariate Cox regression in overall survival suggested that CFL1 level or PGK1 level could be the independent prognosis factor for poor prognosis in 113 glioma patients. In addition, CFL1 expression was positively correlated with PGK1 expression in glioma. The results suggested that as promising indicators, CFL1 and PGK1 could be used to evaluate glioma radiosensibility and prognosis. These two proteins could also be the potential therapeutic targets of glioma.

MicroRNA-590-3p enhances the radioresistance in glioblastoma cells by targeting LRIG1.

microRNA (miR)-590 has been found to serve potential roles in cancer development; however, the expression and function of miR-590 in human gliomas remains to be elucidated. The present study aimed to investigate the expression of miR-590 in human glioma tissues and radioresistant human glioblastoma cells (U251R), and to determine the effect and related molecular mechanism of miR-590-3p on the radiosensitivity of U251R cells in vitro. The results from reverse transcription-quantitative polymerase chain reaction indicated that miR-590-3p was upregulated in human glioma tissues and radioresistant human glioblastoma cells, and that miR-590-3p expression was higher in high grade than in low grade gliomas. In vitro experiments revealed that the miR-590-3p inhibitor enhanced the radiosensitivity of U251R cells by suppressing cell viability, decreasing colony formation capacity and increasing cell apoptosis rate, as demonstrated by MTT, colony formation and flow cytometry analyses. A luciferase reporter assay demonstrated that leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) was a direct target of miR-590-3p. Furthermore, it was demonstrated that the effect of miR-590-3p suppression on cell viability, colony formation capacity and cell apoptosis rate was attenuated by the knockdown of LRIG1 in the U251R cells. In conclusion, the present study revealed that miR-590-3p was upregulated in human glioma tissues and radioresistant human glioblastoma cells, and miR-590-3p contributes to the radioresistance of human glioblastoma cells by directly targeting LRIG1. These findings may provide potential therapeutic strategies to prevent radioresistance in human gliomas.

A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

Tumor recurrence in glioblastoma (GBM) is, in part, attributed to increased epithelial-to-mesenchymal transition (EMT) and enhanced tumor cell dissemination in adjacent brain parenchyma after ionizing radiation (IR). EMT is associated with aggressive behavior, increased stem-like characteristics and treatment resistance in malignancies; however, the underlying signaling mechanisms that regulate EMT are poorly understood. We identified grade-dependent PAK4 upregulation in gliomas and further determined its role in mesenchymal transition and radioresistance. IR treatment significantly elevated expression and nuclear localization of PAK4 in correlation with induction of reactive oxygen species (ROS) and mesenchymal transition in GBM cells. Stable PAK4 overexpression promoted mesenchymal transition by elevating EMT marker expression in these cells. Of note, transcription factor-DNA binding arrays and chromatin immunoprecipitation experiments identified the formation of a novel nuclear PAK4/PPARγ complex which was recruited to the promoter of Nox1, a PPARγ target gene. In addition, IR further elevated PAK4/PPARγ complex co-recruitment to Nox1 promoter, and increased Nox1 expression and ROS levels associated with mesenchymal transition in these cells. Conversely, specific PAK4 downregulation decreased PPARγ-mediated Nox1 expression and suppressed EMT in IR-treated cells. In vivo orthotopic tumor experiments showed inhibition of growth and suppression of IR-induced PPARγ and Nox1 expression by PAK4 downregulation in tumors. Our results provide the first evidence of a novel role for PAK4 in IR-induced EMT and suggest potential therapeutic efficacy of targeting PAK4 to overcome radioresistance in gliomas.

Silencing of ATM expression by siRNA technique contributes to glioma stem cell radiosensitivity in vitro and in vivo.

Evidence has shown that both high expression of the ataxia-telangiectasia mutated (ATM) gene and glioma stem cells (GSCs) are responsible for radioresistance in glioma. Thus, we hypothesized that brain tumor radiosensitivity may be enhanced via silencing of the ATM gene in GSCs. In the present study we successfully induced GSCs from two cell lines and used CD133 and nestin to identify GSCs. A lentivirus was used to deliver siRNA-ATMPuro (Agroup) to GSCs prior to radiation, while siRNA-HKPuro (Ngroup) and GSCs (Cgroup) were used as negative and blank controls, respectively. RT-qPCR and western blotting were performed to verify the efficiency of the siRNA-ATM technique. The expression of the ATM gene and ATM protein were significantly downregulated post-transfection. Cell Counting Kit-8 (CCK-8) and colony formation assays revealed that the Agroup demonstrated weak cell proliferation and lower survival fractions post-irradiation compared to the C/Ngroups. Flow cytometry was used to examine the percentage of cell apoptosis and G2phase arrest, which were both higher in the Agroup than in the C/Ngroups. We found that the comet tail percentage evaluated by comet assay was higher in the Agroup than in the C/Ngroups. After radiation treatment, three radiosensitive genes [p53, proliferating cell nuclear antigen (PCNA), survivin] exhibited a decreasing tendency as determined by RT-qPCR. Mice underwent subcutaneous implantation, followed by radiation, and the resulting necrosis and hemorrhage were more obvious in the Agroup than in the Ngroups. In conclusion, silencing of ATM via the siRNA technique improved radiosensitivity of GSCs both invitro and invivo.

PS2 - 201 Bone Marrow-Derived Mesenchymal Stem Cells-Mediated Radioresistance in Glioma

Ionizing radiation (IR) is one of the conventional post-surgical treatments for Glioblastoma Multiforme (GBM). Mesenchymal stem cells (MSCs) constitute a subpopulation of bone marrow derived cells which are actively recruited to the site of radiation and/or tumour microenvironment (TME), both of which have important implications for neovascularization and tumor progression. The goal of this project is to investigate the functional contribution of MSCs in the TME. We postulate that Bone Marrow-MSCs promote radio-resistance in GBM via cell cycle arrest. We tested the effect of MSC on U87 glioblastoma cell line in response to IR. We found that MSC co-culture, MSC-conditioned media (MSCCM) and irradiated MSC-conditioned media (MSCIRCM) did not reduce IR-induced p53 (ser15) phosphorylation, signifying intact p53-dependent DNA damage pathway in all conditions. However, both MSCCM and MSCIRCM temporally increased phospho-Chk2, a kinase involved in ATM-dependent cascade and cell cycle arrest. This increase occurred at 24 hours and reverted to baseline levels by 48 hours. Interestingly, IR (15Gy) caused transiently heightened metabolic rate under MSC and MSC IRCM as opposed to IR-null treatment at 48 hours elevated cell proliferation. MSCCM, but not MSCIRCM, marginally reduced caspase 3/7-dependent apoptotic levels. The combination of IR and MSCCM as well as MSCIRCM first increased protein level of phospho-Chk2 at 24 hours; followed by increased metabolic rate at 48 hours; and lastly, boosted proliferation at 72 hours. This data combined proposes plausible machinery for BM-MSC mediated radio-resistance by initiating cell cycle arrest in tumour cells for DNA damage repair.

C-Fos over-expression promotes radioresistance and predicts poor prognosis in malignant glioma.

c-Fos is a major component of activator protein (AP)-1 complex. It has been implicated in cell differentiation, proliferation, angiogenesis, invasion, and metastasis. To investigate the role of c-Fos in glioma radiosensitivity and to understand the underlying molecular mechanisms, we downregulated c-Fos gene expression by lentivirus-mediated shRNA in glioma cell lines and subsequently analyzed the radiosensitivity, DNA damage repair capacity, and cell cycle distribution. Finally, we explored its prognostic value in 41 malignant glioma patients by immunohistochemistry. Our results showed that silencing c-Fos sensitized glioma cells to radiation by increasing radiation-induced DNA double strand breaks (DSBs), disturbing the DNA damage repair process, promoting G2/M cell cycle arrest, and enhancing apoptosis. c-Fos protein overexpression correlated with poor prognosis in malignant glioma patients treated with standard therapy. Our findings provide new insights into the mechanism of radioresistance in malignant glioma and identify c-Fos as a potentially novel therapeutic target for malignant glioma patients.

Upregulation of Long Noncoding RNA Small Nucleolar RNA Host Gene 18 Promotes Radioresistance of Glioma by Repressing Semaphorin 5A

Although increasing evidence has shown that long noncoding RNAs play an important regulatory role in carcinogenesis and tumor progression, little is known about the role of small nucleolar RNA host gene 18 (SNHG18) in cancer. The goal of this study was to investigate the expression of SNHG18 and its clinical significance in glioma. Differences in the lncRNA expression profile between M059K and M059J cells were assessed by lncRNA expression microarray analysis. The expression and localization of SNHG18 in glioma cells or tissues was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH), respectively. the clinical associations of SNHG18 in glioma was evaluated by qRT-PCR, ISH and immunohistochemistry. The role of SNHG18 in glioma radiosensitivity was evaluated by colony formation assays, immunofluorescence, Western blot and tumor growth inhibition study. The present study investigated the clinical associations of SNHG18 and its role in glioma. Our results showed that the expression of SNHG18 was remarkably upregulated in clinical glioma tissues compared with normal brain tissues. SNHG18 expression was associated with the clinical tumor grade and correlated negatively with isocitrate dehydrogenase 1 mutation. In addition, knockdown of SNHG18 with short hairpin RNA suppressed the radioresistance of glioma cells, and transgenic expression of SNHG18 had the opposite effect. Furthermore, xenograft tumors grown from cells with SNHG18 deletion were more radiosensitive than tumors grown from control cells. Further studies revealed that SNHG18 promotes radioresistance by inhibiting semaphorin 5A and that inhibition of semaphorin 5A expression abrogated the radiosensitizing effect caused by SNHG18 deletion. Our findings provide new insights into the role of SNHG18 in glioma and suggest its potential as a target for glioma therapy.

Abstract 2525: Sema3C promotes glioma stem cell survival and radioresistance

Abstract Introduction: Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs) are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the non-stem tumor cell (NSTC) population is not well defined. Methods/Materials: GSCs were enriched from patient specimens and functionally validated. We used a candidate approach to screen for secreted factors that were preferentially used by GSCs but not NSTCs. Expression was assessed by Western blot, and confirmed by immunohistochemistry and immunofluorescence in vitro and in human and mouse GBM tissues. Ligand-receptor interactions were confirmed by co-immunoprecipitation. Rac activity was assessed by Rac-GTP pulldown. Co-culture epistasis experiments were performed using knockdown and recombinant protein approaches. Glioma stem cell marker analysis (CD133, Sox2, Olig2), cell viability (ATP-dependent assays), apoptosis (TUNEL, PI/AnnexinV, cleaved caspase 3/7), self-renewal (tumorsphere formation and limiting dilution assay) and invasion (transwell migration assay) were performed. GSCs were separated in PlexinD1 hi and lo populations by FACS. Radiation (3 Gy) was delivered by Cs-137 irradiator. GSCs were used to establish orthotopic xenograft models of glioblastoma and tumor growth and animal survival were assessed. Results: We have found that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/NF-κB signaling in an autocrine and paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs) or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype in vivo. Furthermore, GSCs that expressed high levels of Plexin receptors were more resistant to radiation, and Sema3C and Plexin receptors were upregulated after irradiation. Together, these data support that Sema3C signaling contributes to radiation resistance. Conclusions: Our study supports targeting Sema3C to break this GSC-specific autocrine/paracrine loop to improve glioblastoma treatment, potentially with a high therapeutic index. Citation Format: Jianghong Man, Jocelyn Shoemake, Qiulian Wu, Jennifer Yu. Sema3C promotes glioma stem cell survival and radioresistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2525.

Silencing of ataxia-telangiectasia mutated by siRNA enhances the in vitro and in vivo radiosensitivity of glioma.

It is reported that high expression of the ataxia-telangiectasia mutated (ATM) gene is linked with radioresistance in glioma. We hypothesized that the radiosensitivity of this brain tumor is enhanced by silencing of the ATM gene. We transfected the glioma cell line U251 with the siRNA-ATMpuro (groupA) lentivirus or the siRNA-HKpuro (groupN, negative control) lentivirus before irradiation. RT-qPCR and western blotting were performed to verify the efficiency of siRNA‑mediated ATM silencing. Expression levels of the ATM gene and protein were obviously downregulated after transfection. Moreover, the expression of the p53, PCNA and survivin genes, which are related to radiosensitivity, was also decreased. CCK-8 and colony formation assays showed lower cell proliferation and survival in groupA than in groupsN andC (control group that was not transfected with any siRNA). The level of double-stranded DNA breaks was also greater in groupA, as determined by the comet tail assay. Flow cytometry showed a higher rate of cell apoptosis and a higher number of cells in the G2 phase in groupA. Furthermore, caspase-3, caspase-8 and caspase-9 activity was also higher in group A. Invivo analysis in mouse models created by implantation of the transfected cell lines showed that the amount of necrosis and hemorrhage was higher in groupA than that in the control groups. In conclusion, silencing of ATM via the siRNA technique could improve the invitro and invivo radiosensitivity of glioma cells.

Inhibition of the Rac1-WAVE2-Arp2/3 signaling pathway promotes radiosensitivity via downregulation of cofilin-1 in U251 human glioma cells.

The Ras-related C3 botulinum toxin substrate1 (Rac1)-WASP-family verprolin-homologous protein-2 (WAVE2)-actin-related protein 2/3 (Arp2/3) signaling pathway has been identified to be involved in cell migration and invasion in various types of cancer cell. Cofilin‑1 (CFL‑1), which is regulated by the Rac1‑WAVE2‑Arp2/3 signaling pathway, may promote radioresistance in glioma. Therefore, the present study aimed to investigate the potential role of the Rac1‑WAVE2‑Arp2/3 signaling pathway in radioresistance in U251 human glioma cells and elucidate its affect on CFL‑1 expression. Western blot analysis was performed to evaluate the protein expression of CFL‑1. In the present study, Rac1 was inhibited by NSC23766, WAVE2 was inhibited by transfection with short hairpin (sh)RNA‑WAVE2 using Lipofectamine™2000 and Arp2/3 was inhibited by CK‑666. Cell viability was measured using the 3‑(4,5‑dimethylthiazol‑2‑yl)-2,5‑diphenyltetrazolium bromide assay, the cell migration ability was examined by a wound‑healing assay, and the cell invasion ability was assessed using a Transwell culture chamber system. The results showed that inhibition of the Rac1‑WAVE2‑Arp2/3 signaling pathway using NSC23766, shRNA‑WAVE2 or CK‑666 reduced the cell viability, migration and invasion abilities in U251 human glioma cells, concordant with a reduced expression of CFL‑1. Furthermore, the expression of CFL‑1 was significantly increased in radioresistant U251 glioma cells when compared with normal U251 human glioma cells. These findings indicate that inhibition of the Rac1‑WAVE2‑Arp2/3 signaling pathway may promote radiosensitivity, which may partially result from the downregulation of CFL‑1 in U251 human glioma cells.