Primary Human Glioma Research Articles

PO-020 Functional characterisation of variant of unknown significate in familial breast cancer

IntroductionGlioblastoma (GBM), the common and aggressive human primary brain tumour (WHO grade IV), is highly resistant to standard radio- and chemotherapy. This is partly due to numerous genetic alterations in oncogenes and DNA damage repair systems. Despite progress in understanding the molecular background of GBM and advances in treatment modalities, survival of GMB patients is only 14 months post-diagnosis. RECQL4 belongs to RecQ family of ATP-dependent DNA helicases and plays an important role in genomic integrity and stability maintenance via involvement in DNA replication, repair, recombination, transcription and telomere maintenance. Mutations in the human RecQ genes are linked with cancer predisposition and/or premature ageing. Of all five human helicases, only RECQL4 is found in mitochondria. We explored if alterations of RECQL4 expression or functions contribute to pathogenesis of human GBM.Material and methodsWe determined the RECQL4 expression in various tumour specimens (tumour samples, human primary and established glioma cell cultures) by qPCR and Western Blotting. We determined the effect of RECQL4 depletion on cell viability, proliferation and GBM sphere formation using MTT metabolism, BrdU incorporation and tumour sphere forming assays, respectively.Results and discussionsWe found the upregulated expression of RECQL4 in GBM at mRNA and protein levels when compared to non-transformed human astrocytes. This finding was corroborated by TCGA data analysis. Fractionation of mitochondrial and cytosolic fractions from human glioma cells revealed the presence of RECQL4 in mitochondria. Downregulation (by siRNA) or genetic depletion of RECQL4 (by CRISPRCas9 knockout) in human glioma LN18 and U87-MG cells impaired cell viability and proliferation. We found upregulation of RECQL4 expression in GBM sphere cultures, enriched in glioma stem cells. Transient knock-down of RECQL4 significantly affected tumour sphere formation as evidenced by decreased numbers and sizes of cultured spheres.ConclusionThese data indicate that deregulation of RECQL4 expression or function may play an important role in GBM pathobiology. Our results provide a rationale for further studies of RECQL4 role in gliomagenesis.Supported by National Science Centre grants 2015/19/N/NZ3/02374 (SKK) and 2016/22/M/NZ3/00679 (BK) and the Foundation for Polish Science TEAM-TECH Core Facility project „NGS platform for comprehensive diagnostics and personalised therapy in neuro-oncology’ (AK).

MiR‑141 inhibits glioma vasculogenic mimicry by controlling EphA2 expression.

Human glioma is a pernicious tumor from the central nervous system; it has been reported that microRNAs (miRs) may have carcinogenic or tumor suppressor effects on human glioma. The aim of the present study was to assess miR-141 expression and functional role in human primary glioma, as well as in tumor-derived cell lines. The expression of miR-141 in primary human glioma tissues and cell lines was assessed by employing reverse transcription-quantitative polymerase chain reaction. Next, its role in cellular growth, migration, invasion and vasculogenic mimicry (VM) regulation was determined using various in vitro and in vivo assays, and on the identification its target gene(s) using luciferase assays. The results demonstrated that miR-141 expression was downregulated, and Ephrin type-A receptor 2 (EphA2) was upregulated in the primary human gliomas and human glioma-derived cell lines tested. In addition, a negative correlation existed between miR-141 and EphA2 expression levels in glioma grades II, III and IV. Furthermore, exogenous miR-141 expression resulted in decreased proliferation, migration and invasion, as well as in apoptosis and cell cycle arrest in vitro. It was also revealed that exogenous miR-141 expression resulted in in vivo inhibition of tumor growth and inhibition of the development of VM. Finally, the present study successfully confirmed that EphA2 was a direct target of miR-141 in glioma-derived cells using luciferase assays. Based on these results, it was concluded that miR-141 may regulate cell proliferation, migration, invasion and VM formation by controlling EphA2 expression; also, its target EphA2 may be a novel diagnostic/prognostic biomarker and a potential anti-VM therapeutic target.

PAM-OBG: A monoamine oxidase B specific prodrug that inhibits MGMT and generates DNA interstrand crosslinks, potentiating temozolomide and chemoradiation therapy in intracranial glioblastoma.

Via extensive analyses of genetic databases, we have characterized the DNA-repair capacity of glioblastoma with respect to patient survival. In addition to elevation of O6-methylguanine DNA methyltransferase (MGMT), down-regulation of three DNA repair pathways; canonical mismatch repair (MMR), Non-Homologous End-Joining (NHEJ), and Homologous Recombination (HR) are correlated with poor patient outcome.We have designed and tested both in vitro and in vivo, a monoamine oxidase B (MAOB) specific prodrug, PAM-OBG, that is converted by glioma MAOB into the MGMT inhibitor O6-benzylguanine (O6BG) and the DNA crosslinking agent acrolein. In cultured glioma cells, we show that PAM-OBG is converted to O6BG, inhibiting MGMT and sensitizing cells to DNA alkylating agents such as BCNU, CCNU, and Temozolomide (TMZ). In addition, we demonstrate that the acrolein generated is highly toxic in glioma treated with an inhibitor of Nucleotide Excision Repair (NER).In mouse intracranial models of primary human glioma, we show that PAM-OBG increases survival of mice treated with either BCNU or CCNU by a factor of six and that in a chemoradiation model utilizing six rounds of TMZ/2Gy radiation, pre-treatment with PAM-OBG more than doubled survival time.

Methotrexate-Loaded Extracellular Vesicles Functionalized with Therapeutic and Targeted Peptides for the Treatment of Glioblastoma Multiforme.

Despite promising in vitro evidence for effective glioblastoma treatment, most drugs are hindered from entering the central nervous system because of the presence of the blood-brain barrier (BBB). Thus, successful modification of drug delivery and novel therapeutic strategies are needed to overcome this obstacle. Extracellular vesicles (EVs), cell-derived membrane-encapsulated structures with diameters ranging from 50 to 1000 nm, have been explored as the drug delivery system to deliver their cargo to the brain tissue. Moreover, tumor targeting and selective drug delivery has been facilitated by engineering their parent cells to secrete modified EVs. However, the method suffers from many shortcomings including poor repeatability and complex and time-consuming operations. In this context, we present an easy-to-adapt and highly versatile methodology to modify EVs with an engineered peptide capable of recognition and eradication of glioma. On the basis of molecular recognition between phospholipids on EV lipid bilayer membranes and ApoA-I mimetic peptides, we have developed methotrexate (MTX)-loaded EVs functionalized with therapeutic [Lys-Leu-Ala (KLA)] and targeted [low-density lipoprotein (LDL)] peptides. In vitro experiments demonstrated that EVs decorated with LDL or KLA-LDL could obviously ameliorate their uptake by human primary glioma cell line U87 and permeation into three-dimensional glioma spheroids in contrast to blank EVs, and consequently, the treatment outcome of the payload is improved. Both ex vivo and in vivo imaging experiments revealed that peptide LDL could obviously promote EV extravasation across the BBB and distribution in the glioma site. Furthermore, compared with the mice administrated with MTX and MTX@EVs, MTX@EVs-KLA-LDL-treated mice showed the longest median survival period. In conclusion, functionalizing with the peptide onto EV surfaces may provide a substantial advancement in the application of EVs for selective target binding as well as therapeutic effects for brain tumor treatment.

MicroRNA-200a downregulation in human glioma leads to Gαi1 over-expression, Akt activation, and cell proliferation.

We previously identified a pivotal role for G protein α inhibitory subunit 1 (Gαi1) in mediating PI3K-Akt signaling by receptor tyrosine kinases (RTKs). Here, we examined the expression and biological function of Gαi1 in human glioma. Gαi1 mRNA and protein expression were significantly upregulated in human glioma tissues, which correlated with downregulation of an anti-Gαi1 miRNA: microRNA-200a ("miR-200a"). Forced-expression of miR-200a in established (A172/U251MG lines) and primary (patient-derived) human glioma cells resulted in Gαi1 downregulation, Akt inactivation and proliferation inhibition. Reduction of Gαi1 expression by shRNA, dominant negative mutant interference, or complete Gαi1 depletion inhibited Akt activation and cell proliferation. Notably, miR-200a was unable to inhibit glioma cell proliferation when Gαi1 was silenced or mutated. Co-immunoprecipitation studies, in human glioma cells and tissues, show that Gαi1 forms a complex with multiple RTKs (EGFR, PDGFRα, and FGFR) and the adapter protein Gab1. In vivo, the growth of subcutaneous and orthotopic glioma xenografts in nude mice was largely inhibited by expression of Gαi1 shRNA or miRNA-200a. Collectively, miR-200a downregulation in human glioma leads to Gαi1 over-expression, Akt activation and glioma cell proliferation.

The melatonin-MT1 receptor axis modulates tumor growth in PTEN-mutated gliomas

More than 40% of glioma patients have tumors that harbor PTEN (phosphatase and tensin homologue deleted on chromosome ten) mutations; this disease is associated with poor therapeutic resistance and outcome. Such mutations are linked to increased cell survival and growth, decreased apoptosis, and drug resistance; thus, new therapeutic strategies focusing on inhibiting glioma tumorigenesis and progression are urgently needed. Melatonin, an indolamine produced and secreted predominantly by the pineal gland, mediates a variety of physiological functions and possesses antioxidant and antitumor properties. Here, we analyzed the relationship between PTEN and the inhibitory effect of melatonin in primary human glioma cells and cultured glioma cell lines. The results showed that melatonin can inhibit glioma cell growth both in culture and in vivo. This inhibition was associated with PTEN levels, which significantly correlated with the expression level of MT1 in patients. In fact, c-fos-mediated MT1 was shown to be a key modulator of the effect of melatonin on gliomas that harbor wild type PTEN. Taken together, these data suggest that melatonin-MT1 receptor complexes represent a potential target for the treatment of glioma.

Novel Cancer Stem Marker and Its Applicability for Grading Primary Human Gliomas.

Poorly differentiated cell populations including tumor-initiating stem cells have been demonstrated to display a unique ability to natively internalize fragmented double-stranded DNA. Using this feature as a marker, we show that 0.1% to 6% of human glioblastoma cells from the bioptates can effectively internalize a fluorescently labeled DNA probe. Of these, using samples from 3 patients, 66% to 100% cells are also positive for CD133, a well-established surface marker of tumor-initiating glioma stem cells. Using the samples from primary malignant brain lesions (33 patients), we demonstrate that tumor grading significantly correlates (R = .71) with the percentage of DNA-internalizing cells. No such correlation is observed for relapse samples (18 patients).

MicroRNA-29a-3p Downregulation Causes Gab1 Upregulation to Promote Glioma Cell Proliferation

Background/Aims: Glioma causes significant human mortalities annually. Molecularly-targeted therapy is a focus of glioma research. Methods: Grb2-associated binding 1 (Gab1) expression and microRNA-29a-3p (“miR-29a-3p”) expression in human glioma cells and tissues were tested by Western blotting assay and qRT-PCR assay. shRNA/siRNA strategy was applied to silence Gab1 in human glioma cells. miR-29a or anti-sense miR-29a construct was transfected to human glioma cells. Cell proliferation was tested by BrdU ELISA assay and cell counting assay. Results: We show that expression of Gab1 was significantly elevated in human glioma tissues and cells, which correlated with downregulation of its putative microRNA: miR-29a-3p. In A172 glioma cells and primary human glioma cells, Gab1 shRNA/siRNA inhibited Akt-Erk activation and cell proliferation. Forced-expression of miR-29a-3p downregulated Gab1, inhibiting glioma cell proliferation, whereas miR-29a-3p was in-effective on cell proliferation in Gab1-silenced A172 cells. Furthermore, introduction of a 3’-untranslated region (3’-UTR) mutant Gab1 (UTR-G160A) blocked miR-29a-3p-induced inhibition on Akt signaling and A172 cell proliferation. Conclusions: miR-29a-3p downregulation leads to Gab1 upregulation to promote glioma cell proliferation.

Association of aquaporin‑1 with tumor migration, invasion and vasculogenic mimicry in glioblastoma multiforme.

The present study aimed to assess the expression and functional role of aquaporin-1 (AQP1) in glioblastoma multiforme (GBM) migration, invasion and vasculogenic mimicry (VM). In the primary human gliomas and human glioma‑derived cell lines tested, it was observed that the expression of AQP1 was upregulated. In addition, it was demonstrated that silencing of AQP1 expression resulted in decreased migration and invasion, in addition to vasculogenic mimicry invitro. It was additionally observed that silencing of AQP1 expression resulted in invivo inhibition of tumor growth, a decrease in the expression of invasion‑associated protein, and suppression of VM formation. Based on these data, it was concluded that AQP1 may serve a role in GBM migration, invasion and VM formation, and that it may serve as a novel diagnostic/prognostic biomarker and a potential therapeutic target.

PDGFA/PDGFR\u03b1-regulated GOLM1 promotes human glioma progression through activation of AKT

BackgroundGolgi Membrane Protein 1 (GOLM1), a protein involved in the trafficking of proteins through the Golgi apparatus, has been shown to be oncogenic in a variety of human cancers. Here, we examined the role of GOLM1 in the development of human glioma.MethodsqRT-PCR, immunohistochemistry, and western blot analysis were performed to evaluate GOLM1 levels in cell lines and a cohort of primary human glioma and non-neoplastic brain tissue samples. Glioma cell lines were modified with lentiviral constructs expressing short hairpin RNAs targeting GOLM1 or overexpressing the protein to assess function in proliferation, viability, and migration and invasion in vitro using EdU, CCK8, clone-forming, Transwell assays, 3D tumor spheroid invasion assay and in vivo in orthotopic implantations. Protein lysates were used to screen a membrane-based antibody array to identify kinases mediated by GOLM1. Specific inhibitors of PDGFRα (AG1296) and AKT (MK-2206) were used to examine the regulation of PDGFA/PDGFRα on GOLM1 and the underlying pathway respectively.ResultsqRT-PCR, immunohistochemistry and western blot analysis revealed GOLM1 expression to be elevated in glioma tissues and cell lines. Silencing of GOLM1 attenuated proliferation, migration, and invasion of U251, A172 and P3#GBM (primary glioma) cells, while overexpression of GOLM1 enhanced malignant behavior of U87MG cells. We further demonstrated that activation of AKT is the driving force of GOLM1-promoted glioma progression. The last finding of this research belongs to the regulation of PDGFA/PDGFRα on GOLM1, while GOLM1 was also a key element of PDGFA/PDGFRα-mediated activation of AKT, as well as the progression of glioma cells.ConclusionsPDGFA/PDGFRα-regulated GOLM1 promotes glioma progression possibly through activation of a key signaling kinase, AKT. GOLM1 interference may therefore provide a novel therapeutic target and improve the efficacy of glioma treatment, particularly in the case of the proneural molecular subtype of human glioma.

Cofilin-1 and Phosphoglycerate Kinase-1 Up Regulated the Radiation Resistance in Primary Human Glioma Cells

Cofilin-1 and Phosphoglycerate Kinase-1 Up Regulated the Radiation Resistance in Primary Human Glioma Cells

Autophagy inhibition sensitizes LY3023414-induced anti-glioma cell activity in vitro and in vivo.

PI3K-AKT-mTOR signaling is a valuable treatment target for human glioma. LY3023414 is a novel, highly-potent and pan PI3K-AKT-mTOR inhibitor. Here, we show that LY3023414 efficiently inhibited survival and proliferation of primary and established human glioma cells. Meanwhile, apoptosis activation was observed in LY3023414-treated glioma cells. LY3023414 blocked AKT-mTOR activation in human glioma cells. Further studies show that LY3023414 induced feedback activation of autophagy in U251MG cells. On the other hand, autophagy inhibition via adding pharmacological inhibitors or silencing Beclin-1/ATG-5 significantly potentiated LY3023414-induced glioma cell apoptosis. In vivo studies demonstrated that U251MG xenograft tumor growth in mice was suppressed by oral administration of LY3023414. Remarkably, LY3023414's anti-tumor activity was further augmented against the Beclin-1-silenced U251MG tumors. Together, our results suggest that targeting PI3K-AKT-mTOR cascade by LY3023414 inhibits human glioma cell growth in vitro and in vivo. Autophagy inhibition could further sensitize LY3023414 against human glioma cells.

Identification of genes and pathways potentially related to PHF20 by gene expression profile analysis of glioblastoma U87 cell line

BackgroundGlioblastoma is the most common and aggressive brain tumor associated with a poor prognosis. Plant homeodomain finger protein 20 (PHF20) is highly expressed in primary human gliomas and its expression is associated with tumor grade. However, the molecular mechanism by which PHF20 regulates glioblastoma remains poorly understood.MethodsGenome wide gene expression analysis was performed to identify differentially expressed genes (DEGs) in U87 cells with PHF20 gene knockdown. Gene ontology (GO) and pathway enrichment analyses were performed to investigate the functions and pathways of DEGs. Pathway-net and signal-net analyses were conducted to identify the key genes and pathways related to PHF20.ResultsExpression of 540 genes, including FEN1 and CCL3, were significantly altered upon PHF20 gene silencing. GO analysis results showed that DEGs were significantly enriched in small molecule metabolic and apoptotic processes. Pathway analysis indicated that DEGs were mainly involved in cancer and metabolic pathways. The MAPK, apoptosis and p53 signaling pathways were identified as the hub pathways in the pathway network, while PLCB1, NRAS and PIK3 s were hub genes in the signaling network.ConclusionsOur findings indicated that PHF20 is a pivotal upstream regulator. It affects the occurrence and development of glioma by regulating a series of tumor-related genes, such as FEN1, CCL3, PLCB1, NRAS and PIK3s, and activation of apoptosis signaling pathways. Therefore, PHF20 might be a novel biomarker for early diagnosis, and a potential target for glioblastoma therapies.

Glioma cell fate decisions mediated by Dll1-Jag1-Fringe in Notch1 signaling pathway

BackgroundThe Notch family of proteins plays a vital role in determining cell fates, such as proliferation, differentiation, and apoptosis. It has been shown that Notch1 and its ligands, Dll1 and Jag1, are overexpressed in many glioma cell lines and primary human gliomas. The roles of Notch1 in some cancers have been firmly established, and recent data implicate that it plays important roles in glioma cell fate decisions. This paper focuses on devising a specific theoretical framework that incorporates Dll1, Jag1, and Fringe in Notch1 signaling pathway to explore their functional roles of these proteins in glioma cells in the tumorigenesis and progression of human gliomas, and to study how glioma cell fate decisions are modulated by both trans-activation and cis-inhibition.ResultsThis paper presents a computational model for Notch1 signaling pathway in glioma cells. Based on the bifurcation analysis of the model, we show that how the glioma cell fate decisions are modulated by both trans-activation and cis-inhibition mediated by the Fringe protein, providing insight into the design and control principles of the Notch signaling system and the gliomas.ConclusionsThis paper presents a computational model for Notch1 signaling pathway in glioma cells based on intertwined dynamics with cis-inhibition and trans-activation involving the proteins Notch1, Dll1, Jag1, and Fringe. The results show that how the glioma cell fate transitions are performed by the Notch1 signaling. Transition from grade III ∼ IV with significantly high Notch1 to grade I ∼ II with high Notch1, and then to normal cells by repressing the Fringe levels or decreasing the strength of enhancement induced by Fringe.

Abstract 1524: BCL6 modulates the TP53 and STAT pathways in glioma

Abstract Glioblastoma multiforme (GBM) remains the most aggressive brain malignancy with little improvement in prognosis or therapy for decades. Recently, we identified BCL6, also known as ZBTB27, to be a novel oncogene in GBM. In this study, we performed IHC analysis of 153 primary human glioma specimens and 8 normal brain samples. BCL6 expression is robustly elevated in tumor samples and positively correlated with glioma pathological grade. High BCL6 expression strongly predicts a worse prognosis of GBM patients. Depletion of BCL6 in human GBM cells reduced the incorporation of BrdU, promoted the cellular senescence and inhibited the growth of human GBM cells in vivo. Next, genome-wide occupancy of BCL6 in GBM cells was characterized by ChIP-seq assay. Genomic regions centered on BCL6 peaks are co-enriched with RNA-Pol II and flanked with strong H3K27ac and H3K4me3 modifications. MYC and two long non-coding RNAs MALAT1 and NEAT1 were identified as novel BCL6 targets in GBM. Moreover, pathway enrichment analysis of BCL6 peak-associated genes reveals a significant enrichment of JAK-STAT, TP53, ERBB and MAPK pathways. We demostrated further that BCL6 represses the TP53 pathway and promotes the JAK-STAT pathway activation in GBM cells. Together, our findings uncover potential downstream targets and provide a better understanding of BCL6 function in GBM. Citation Format: Ye Chen, Liang Xu, Marina Dutra-Clarke, Anand Mayakonda, De-Chen Lin, Lynnette Koh, Yuk Kien Chong, Edwin Sandanaraj, Vikas Madan, Henry Yang, Ngan Doan, Jonathan W. Said, William H. Yong, Markus Müschen, Beng Ti Ang, Carol Tang, Joshua J. Breunig, Phillip Koeffler. BCL6 modulates the TP53 and STAT pathways in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1524. doi:10.1158/1538-7445.AM2017-1524

CD70, a novel target of CAR T-cell therapy for gliomas.

Cancer immunotherapy represents a promising treatment approach for malignant gliomas but is hampered by the limited number of ubiquitously expressed tumor antigens and the profoundly immunosuppressive tumor microenvironment. We identified cluster of differentiation (CD)70 as a novel immunosuppressive ligand and glioma target. Normal tissues derived from 52 different organs and primary and recurrent low-grade gliomas (LGGs) and glioblastomas (GBMs) were thoroughly evaluated for CD70 gene and protein expression. The association between CD70 and patients' overall survival and its impact on T-cell death was also evaluated. Human and mouse CD70-specific chimeric antigen receptors (CARs) were tested respectively against human primary GBMs and murine glioma lines. The antitumor efficacies of these CARs were also examined in orthotopic xenograft and syngeneic models. CD70 was not detected in peripheral and brain normal tissues but was constitutively overexpressed by isocitrate dehydrogenase (IDH) wild-type primary LGGs and GBMs in the mesenchymal subgroup and recurrent tumors. CD70 was also associated with poor survival in these subgroups, which may link to its direct involvement in glioma chemokine productions and selective induction of CD8+ T-cell death. To explore the potential for therapeutic targeting of this newly identified immunosuppressive axis in GBM tumors, we demonstrate that both human and mouse CD70-specific CAR T cells recognize primary CD70+ GBM tumors in vitro and mediate the regression of established GBM in xenograft and syngeneic models without illicit effect. These studies identify a previously uncharacterized and ubiquitously expressed immunosuppressive ligand CD70 in GBMs that also holds potential for serving as a novel CAR target for cancer immunotherapy in gliomas.

Glioma Cell Proliferation Promoted by AK4 Regulated mTORC1 Pathway Activation

A hallmark of cancer cell biology is the dysregulation of signaling pathways that manage cancer‐associated phenotypes such as proliferation and survival. One dysfunctional pathway in cancer cells is the mechanistic target of rapamycin (mTOR). The mTOR pathway, involving both mTOR Complex 1 (mTORC1) and mTORC2, functions as a central regulator of cell metabolism, growth, proliferation, and survival. mTORC1 kinase signaling allows for transitions between metabolic states by responding to environmental signals and controlling processes that regulate energy. Recently, the expression of an Adenylate Kinase 4 (AK4) protein has been identified as modulating cellular bioenergetics through the activation of pathways such as mTORC1, resulting in both enhanced proliferation and activation of stress responses. Additionally, reduced AK4 expression has been observed in primary human glioma cells, while the mTORC1 signaling pathway is highly activated in identical primary glioma cells. We hypothesize that AK4 expression regulates mTORC1 signaling and induces downstream effects that promote glioma cell biology. To determine if AK4 expression affects mTORC1 signaling, we knocked down AK4 expression using siRNA in U87‐MG glioma cell lines. Phospho‐specific modifications of activators and inhibitors upstream of the mTORC1 pathway and its downstream targets were assessed using Western blot (WB). AK4 knockdown (KD) and WB analysis revealed over‐active mTORC1 phosphorylation and inactivation of the downstream translational repressor, 4E‐BP1, compared to control glioma cell lines. Likewise, WB analysis displayed increased mTORC1 phosphorylation and activation of the downstream protein ribosomal S6, a regulator of protein translation. Reduced phosphorylation of upstream inhibitors and regulatory proteins of mTORC1, Tuberous Sclerosis Complex 2 (TSC2) and Regulatory‐associated protein of mTOR (RPTOR), was observed when compared to controls. Additionally, reduced mTORC1 phosphorylation of the autophagy inducing serine/threonine protein kinase ULK1 was evident by WB. Furthermore, bioluminescence analysis of AK4 KD U87‐MG glioma cells shows an increase in cell proliferation under nutrient stressed conditions compared to control. Overall, these data suggest that AK4 reduction may contribute to over‐active mTORC1 kinase signaling in glioma cells. Studies characterizing the role of AK4 reduction on glioma cell proliferation could further explain the contribution of endogenously reduced AK4 expression in glioma cell biology. By identifying the regulatory role of AK4, we hope to elucidate the therapeutic potential of modulating AK4 levels in cancer cells.Support or Funding InformationNSF# HRD‐1463889.

Gab3 overexpression in human glioma mediates Akt activation and tumor cell proliferation

This current study tested expression and potential biological functions of Gab3 in human glioma. Gab3 mRNA and protein expression was significantly elevated in human glioma tissues and glioma cells. Its level was however low in normal brain tissues and primary human astrocytes. In both established (U251MG cell line) and primary human glioma cells, Gab3 knockdown by shRNA/siRNA significantly inhibited Akt activation and cell proliferation. Reversely, forced Gab3 overexpression in U251MG cells promoted Akt activation and cell proliferation. In vivo, the growth of U251MG tumors in nude mice was inhibited following expressing Gab3 shRNA. Akt activation in cancer tissues was also suppressed by Gab3 shRNA. Together, we conclude that Gab3 overexpression in human glioma mediates Akt activation and cancer cell proliferation.

In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway

We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G2M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy.

ANGI-10. GENETIC DOWN REGULATION OF CXCR4 IN GLIOMA CELLS REDUCES INVASION, REDUCES TUMOR PROGRESSION, AND INCREASES SENSITIVITY TO RADIATION

Glioblastoma multiforme (GBM) is a lethal brain tumor. Radiation therapy has been an essential part of treatment for glioma patients. Despite high radiation therapy along with anticancer drugs, GBMs invariably recur. Glioma Stem Cells (GSCs) resistant to chemo and radiation therapies are mainly responsible for GBM recurrence. Therefore, increase sensitivity of GBM cells to radiation and/chemo therapies could be of therapeutic value. We have recently described that GBM derived glioma stem cells grow along blood vessels (Baker et al, 2014). In this study we observed that different primary human glioma stem cell lines, HF2303, MSP-12, IN859 and IN2045, and a mouse glioma Gl26-cit cell line, showed significant directional migration towards human and mouse brain-derived endothelial (MBVE) cells. To uncover the mechanism of GBM cell migration, we tested the role of various chemokines and inhibitors utilizing in vitromigration assays. Notably, migration of Gl26-cit and HF2303 towards MBVE was significantly inhibited by AMD3100 (CXCR4 inhibitor). We further confirmed these findings by knocking down of CXCR4 in GL26-cit cells using shRNA. The migration of CXCR4 knock-down in GL26-cit-shCXCR4 cells towards MBVE was significantly reduced compared to control shRNA treated Gl26-cit cells (Gl26-cit-NT). We next established an orthotopic brain tumor in mice using GL26-cit-shCXCR4 and Gl26-cit-NT cells. Mice implanted with down regulated CXCR4 GL26-cit-shCXCR4 cells exhibited prolonged survival compared to Gl26-cit-NT mice. Brain section analysis showed that GL26-cit-shCXCR4 cells are less invasive. Lastly, we tested the effect of radiation on mice implanted with GL26-cit-shCXCR4 or Gl26-cit-NTcells. Mice implanted with GL26-cit-shCXCR4 cells showed further improvement in survival compared to Gl26-cit-NT tumor-bearing mice upon radiation. In summary, CXCR4-CXCL12 signaling is critical for the perivascular invasion of GBM cells and targeting this axis makes tumors less invasive and more sensitive to radiation therapy. Targeting CXCR4-CXCL12 signaling could be a potential therapeutic strategy for treatment of GBM.