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Glioblastoma multiform (GBM) is the most frequent primitive brain tumor with a high recurrence and mortality. Histone deacetylase inhibitors (HDACi) have evoked great interest because they are able to change transcriptomic profiles to promote tumor cell death but also induce side effects due to the lack of selectivity. We show in this paper new anticancer properties and mechanisms of action of low concentrations of vorinostat on various GBM cells which acts by affecting microtubule cytoskeleton in a non-histone 3 (H3) manner. Indeed, vorinostat induces tubulin acetylation and detyrosination, affects EB stabilizing cap on microtubule plus ends and suppresses microtubule dynamic instability. We previously identified EB1 overexpression as a marker of bad prognostic in GBM. Interestingly, we show for the first time to our knowledge, a strong decrease of EB1 expression in GBM cells by a drug. Altogether, our results suggest that low dose vorinostat, which is more selective for HDAC6 inhibition, could therefore represent an interesting therapeutic option for GBM especially in patients with EB1 overexpressing tumor with lower expected side effects. A validation of our hypothesis is needed during future clinical trials with this drug in GBM.

Immunotherapies targeting tumour-infiltrating lymphocytes (TILs) that express the immune checkpoint molecule programmed cell death-1 (PD-1) have shown promise in preclinical glioblastoma models but have had limited success in clinical trials. To assess when glioblastoma is most likely to benefit from immune checkpoint inhibitors we determined the density of TILs in primary and recurrent glioblastoma. Thirteen cases of matched primary and recurrent glioblastoma tissue were immunohistochemically labelled for CD3, CD8, CD4 and PD-1, and TIL density assessed. CD3+ TILs were observed in all cases, with the majority of both primary (69.2%) and recurrent (61.5%) tumours having low density of TILs present. CD8+ TILs were observed at higher densities than CD4+ TILs in both tumour groups. PD-1+ TILs were sparse and present in only 25% of primary and 50% of recurrent tumours. Quantitative analysis of TILs demonstrated significantly higher CD8+ TIL density at recurrence (p = 0.040). No difference was observed in CD3+ (p = 0.191), CD4+ (p = 0.607) and PD-1+ (p = 0.070) TIL density between primary and recurrent groups. This study shows that TILs are present at low densities in both primary and recurrent glioblastoma. Furthermore, PD-1+ TILs were frequently absent, which may provide evidence as to why anti-PD-1 immunotherapy trials have been largely unsuccessful in glioblastoma.

Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system with a dismal prognosis. Locoregional failure is common despite high doses of radiation therapy, which has prompted great interest in developing novel strategies to radiosensitize these cancers. Our group previously identified a calcium channel blocker (CCB), mibefradil, as a potential GBM radiosensitizer. We discovered that mibefradil selectively inhibits a key DNA repair pathway, alternative non-homologous end joining. We then initiated a phase I clinical trial that revealed promising initial efficacy of mibefradil, but further development was hampered by dose-limiting toxicities, including CCB-related cardiotoxicity, off-target hERG channel and cytochrome P450 enzymes (CYPs) interactions. Here, we show that mibefradil inhibits DNA repair independent of its CCB activity, and report a series of mibefradil analogues which lack CCB activity and demonstrate reduced hERG and CYP activity while retaining potency as DNA repair inhibitors. We present in vivo pharmacokinetic studies of the top analogues with evidence of brain penetration. We also report a targeted siRNA-based screen which suggests a possible role for mTOR and Akt in DNA repair inhibition by this class of drugs. Taken together, these data reveal a new class of mibefradil-based DNA repair inhibitors which can be further advanced into pre-clinical testing and eventually clinical trials, as potential GBM radiosensitizers.

Glioblastoma is one of the most common malignant brain tumors, with which patients have a mean survival of 24 months. Glypican-1 has been previously shown to be overexpressed in human glioblastoma and to be negatively correlated with patient’s survival. This study aimed to investigate how glypican-1 influences the tumoral profile of human glioblastoma using in vitro cell line models. By downregulating the expression of glypican-1 in U-251 MG cells, we observed that the cellular growth and proliferation were highly reduced, in which cells were significantly shifted towards G0 as opposed to G1 phases. Cellular migration was severely affected, and glypican-1 majorly impacted the affinity towards laminin-binding of glioblastoma U-251 MG cells. This proteoglycan was highly prevalent in glioblastoma cells, being primarily localized in the cellular membrane and extracellular vesicles, occasionally with glypican-3. Glypican-1 could also be found in cell-cell junctions with syndecan-4 but was not identified in lipid rafts in this study. Glypican-1-silenced cells were much more susceptible to temozolomide than in U-251 MG itself. Therefore, we present evidence not only to support facts that glypican-1 is an elementary macromolecule in glioblastoma tumoral microenvironment but also to introduce this proteoglycan as a promising therapeutic target for this lethal tumor.

// Lubov Mitrofanova 1 , Anton Hazratov 1 , Boris Galkovsky 1 , Andrey Gorshkov 1 , 2 , Danila Bobkov 2 , 3 , Dmitry Gulyaev 4 and Evgeny Shlyakhto 5 1 Almazov National Medical Research Centre, Pathomorphology Research Laboratory, St. Petersburg, Russia 2 Smorodintsev Research Institute of Influenza, Laboratory of Intracellular Signaling and Transport, St. Petersburg, Russia 3 Institute of Cytology of the Russian Academy of Science, Laboratory of Cell Biology in Culture, St. Petersburg, Russia 4 Almazov National Medical Research Centre, Research Department of Neurosurgery, St. Petersburg, Russia 5 Almazov National Medical Research Centre, General Director, St. Petersburg, Russia Correspondence to: Lubov Mitrofanova, email: lubamitr@yandex.ru Keywords: telocytes; pericytes; confocal microscopy; primary culture of glioma telocytes; glioblastoma Received: June 05, 2019 Accepted: October 26, 2019 Published: January 28, 2020 ABSTRACT Telocytes (Tcs) and pericytes (Pcs) are two types of perivascular interstitial cell known to be widespread in various organs and tissues, including the brain. We postulated that Tcs and Pcs may be involved in glioblastoma (GBM) neovascularization. Objective Morphological study of Tc and Pc roles in GBM. Materials and Methods Samples from 15 GBM, 10 diffuse astrocytoma, as well as 5 control samples were studied. We used immunohistochemistry (IHC) with antibodies (Abs) to GFAP, Ki-67, CD117, NeuroD1, NG2, CD34, and SMA. Confocal laser scanning microscopy (CLSM) of 4 glioma tissue cultures and 4 GBM sections was performed with GFAP, CD117, CD34/connexin43, NeuroD1/connexin43, CD34/NG2 and CD13/CD117 Abs. Electron microscopy (EM) of GBM was performed in 4 cases. Results The presence of Tcs and Pcs was shown in GBM (IHC, EM, CLSM) and glioma cultures (CLSM). The Tc immunophenotype was CD117 + /CD34 + /connexin43 + /NeuroD1 + . The Pc immunophenotype was SMA + /NG2 + /CD13 + . The number of Tcs in GBM specimens was 10 times higher than in astrocytoma. We also identified CD13/CD117 and CD34/NG2 co-expressing cells in GBM blood vessels. Conclusion Four immunophenotypes were found in GBM vessels, corresponding to endotheliocytes, Pcs, Tcs, and a mixed Pc/Tc immunophenotype. These and forthcoming improvements in our understanding of the origin and function of Tcs, including their relationship with Pcs, are necessary steps in oncology. Study of these cell types (Tcs, Pcs) and their roles in brain tumor oncogenesis will likely enable improved targeted therapies and support development of new forms of anti-neoplastic drugs.

Targeting solid tumor antigens with chimeric antigen receptor (CAR) T cell therapy requires tumor specificity and tolerance toward variability in antigen expression levels. Given the relative paucity of unique cell surface proteins on tumor cells for CAR targeting, we have focused on identifying tumor-specific epitopes that arise as a consequence of target protein posttranslational modification. We designed a CAR using a mAb806-based binder, which recognizes tumor-specific untethered EGFR. The mAb806 epitope is also exposed in the EGFRvIII variant transcript. By varying spacer domain elements of the CAR, we structurally tuned the CAR to recognize low densities of EGFR representative of non-gene amplified expression levels in solid tumors. The appropriately tuned short-spacer 2nd generation EGFR806-CAR T cells showed efficient in vitro cytokine secretion and glioma cell lysis, which was competitively blocked by a short peptide encompassing the mAb806 binding site. Unlike the nonselective Erbitux-based CAR, EGFR806-CAR T cells did not target primary human fetal brain astrocytes expressing wild-type EGFR, but showed a similar level of activity compared to Erbitux-CAR when the tumor-specific EGFRvIII transcript variant was overexpressed in astrocytes. EGFR806-CAR T cells successfully treated orthotopic U87 glioma implants in NSG mice, with 50% of animals surviving to 90 days. With additional IL-2 support, all tumors were eradicate without recurrence after 90 days. In a novel human induced pluripotent stem cell (iPSC)-derived teratoma xenograft model, EGFR806-CAR T cells infiltrated but were not activated in EGFR+ epidermal cell nests as assessed by Granzyme B expression. These results indicate that EGFR806-CAR T cells effectively and selectively target EGFR-expressing tumor cells.

Glioblastoma are highly invasive and associated with limited therapeutic options and a grim prognosis. Using stem cells to extend current therapeutic strategies by targeted drug delivery to infiltrated tumors cells is highly attractive. This study analyzes the tumor homing and therapeutic abilities of clinical grade human mesenchymal stem cells (MSCs) in an orthotopic glioblastoma mouse model. Our time course analysis demonstrated that MSCs display a rapid targeted migration to intracerebral U87 glioma xenografts growing in the contralateral hemisphere within the first 48h hours after application as assessed by histology and 7T magnetic resonance imaging. MSCs accumulated predominantly peritumorally but also infiltrated the main tumor mass and targeted distant tumor satellites while no MSCs were found in other regions of the brain. Intratumoral application of MSCs expressing herpes simplex virus thymidine kinase followed by systemic prodrug application of ganciclovir led to a significant tumor growth inhibition of 86% versus the control groups (p

Glioblastoma multiforme is an aggressive cancer type with poor patient outcomes. Interestingly, we reported previously a novel association between the little studied paucimannosidic N-linked glycoepitope and glioblastoma. Paucimannose has only recently been detected in vertebrates where it exhibits a very restricted tumor-specific expression. Herein, we demonstrate for the first time a very high protein paucimannosylation in human grade IV glioblastoma and U-87MG and U-138MG glioblastoma cells. Furthermore, we revealed the involvement of paucimannosidic epitopes in tumorigenic processes including cell proliferation, migration, invasion and adhesion. Finally, we identified AHNAK which is discussed as a tumor suppressor as the first paucimannose-carrying protein in glioblastoma and show the involvement of AHNAK in the observed paucimannose-dependent effects. This study is the first to provide evidence of a protective role of paucimannosylation in glioblastoma, a relationship that with further in vivo support may have far reaching benefits for patients suffering from this often fatal disease.

// Dorte Schou Noroxe 1 , 2 , Olga Ostrup 3 , Christina Westmose Yde 3 , Lise Barlebo Ahlborn 3 , Finn Cilius Nielsen 3 , Signe Regner Michaelsen 1 , Vibeke Andree Larsen 4 , Jane Skjoth-Rasmussen 5 , Jannick Brennum 5 , Petra Hamerlik 6 , Hans Skovgaard Poulsen 1 , 2 and Ulrik Lassen 2 1 Department of Radiation Biology, Rigshospitalet, 2100 Copenhagen, Denmark 2 Department of Oncology, Rigshospitalet, 2100 Copenhagen, Denmark 3 Center for Genomic Medicine, Rigshospitalet, 2100 Copenhagen, Denmark 4 Department of Neuroradiology, Rigshospitalet, 2100 Copenhagen, Denmark 5 Department of Neurosurgery, Rigshospitalet, 2100 Copenhagen, Denmark 6 Danish Cancer Society, 2100 Copenhagen, Denmark Correspondence to: Dorte Schou Noroxe, email: anne.dorte.schou.noeroexe@regionh.dk Keywords: glioblastoma; liquid biopsy; cell-free DNA; fragment length; base-pair Received: March 10, 2019 Accepted: May 29, 2019 Published: July 09, 2019 ABSTRACT Background: Glioblastoma (GB) is an incurable brain cancer with limited treatment options. The aim was to test the feasibility of using cell-free DNA (cfDNA) to support evaluation of treatment response, pseudo-progression and whether progression could be found before clinical and/or radiologic progression. Results: CfDNA fluctuated during treatment with the highest levels before diagnostic surgery and at progression. An increase was seen in 3 out of 4 patients at the time of progression while no increase was seen in 3 out of 4 patients without progression. CfDNA levels could aid in 3 out of 3 questionable cases of pseudo-progression. Methods: Eight newly diagnosed GB patients were included. Blood samples were collected prior to diagnosis, before start and during oncologic treatment until progression. Seven patients received concurrent radiotherapy/Temozolomide with adjuvant Temozolomide with one of the patients included in a clinical trial with either immunotherapy or placebo as add-on. One patient received radiation alone. CfDNA concentration was determined for each blood sample. Conclusions: It was feasible to measure cfDNA concentration. Despite the limited cohort size, there was a good tendency between cfDNA and treatment course and -response, respectively with the highest levels at progression.

Grade IV astrocytoma, also known as glioblastoma multiforme (GBM), is the most common and aggressive intracranial glial tumor. GBM is associated with very poor survival and effective treatments have remained elusive so far. Mounting evidence indicates that CD164 contributes to stemness and tumorigenesis in normal cells and is overexpressed in various tumor types, including glioblastoma. Using tissue microarray immunohistochemistry, we show that there is a significant correlation between CD164 expression and glioma type and grade. Depletion of CD164 expression in human glioblastoma cells with siRNA reduced proliferation, migration, and invasiveness. In parallel, immunoblotting showed that downregulation of CD164 expression decreased Akt activation and modified the expression of autophagy markers by upregulating Beclin-1 and LC3B and downregulating p62. These effects were mimicked by inhibition of Akt with MK2206, which suggests that CD164 induces autophagy via Akt/Beclin-1 signaling. We propose that CD164 may serve as a GBM molecular marker and a potential target in therapeutic strategies aimed to improve outcomes for this devastating brain tumor.

Receptor tyrosine kinase (RTK) pathways are known to play an important role in tumor cell proliferation of glioblastoma (GBM). Cellular determinants of RTK-inhibitor sensitivity are important to optimize and tailor treatment strategies. The stress response gene activating transcription factor 4 (ATF4) is involved in homeostasis and cellular protection. However, little is known about its function in GBM. We found that the ATF4/p-eIF2α pathway is activated in response to Sunitinib in primary tumor initiating progenitor cell cultures (BTICs). Furthermore, lysosome entrapment of RTK-inhibitors (RTK-Is) leads to accumulation of autophagosomes. In case of Sunitinib treated cells, autophagy is additionally increased by ATF4 mediated upregulation of autophagy genes. Inhibition of ATF4 by small interfering RNA (siRNA) reduced autophagy and cell proliferation after Sunitinib treatment in a subset of BTIC cultures. Overall, this study suggests a pro-survival role of the ATF4/p-eIF2α pathway in a cell type and treatment specific manner.

Purpose: Glioblastoma multiforme (GBM) or astrocytoma grade IV is the most common type of primary brain tumor in adults. In the present study, we investigate the role of the complement system in the glioblastoma situation in an experimental model, since we have previously been able to show a blockade of this system in the glioblastoma setting. Technique and results: A GFP-positive glioblastoma cell line was used to induce glioblastomas subcutaneously in rats (n=42). Antibodies against C1-Inactivator (C1-IA) were used to try to re-activate the complement system. We were able to demonstrate an increased survival in rats treated with anti-C1-IA with an intratumoral route, and we could establish the same the results in a second series. Serum analyses revealed decreased levels of IL-1b and GM-CSF in animals 24 days after tumor cell inoculation in the anti-C1-IA group when compared to controls. Immunohistochemistry revealed decreased expression of C1-IA following treatment. Interpretation: These results are in line with our previous work showing an upregulation of C1-IA, which is able to block the classical complement pathway, in glioblastomas. Treatment with antibodies against C1-IA seems to be beneficial in the glioblastoma situation, and no side effects could be seen in our experiments. (Less)

CD4+ helper T (Th) cells play a critical role in shaping anti-tumor immunity by virtue of their ability to differentiate into multiple lineages in response to environmental cues. Various CD4+ lineages can orchestrate a broad range of effector activities during the initiation, expansion, and memory phase of endogenous anti-tumor immune response. In this clinical corelative study, we found that Glioblastoma (GBM) induces multi- and mixed-lineage immune response in the tumor microenvironment. Whole-genome bisulfite sequencing of tumor infiltrating and blood CD4+ T-cell from GBM patients showed 13571 differentially methylated regions and a distinct methylation pattern of methylation of tumor infiltrating CD4+ T-cells with significant inter-patient variability. The methylation changes also resulted in transcriptomic changes with 341 differentially expressed genes in CD4+ tumor infiltrating T-cells compared to blood. Analysis of specific genes involved in CD4+ differentiation and function revealed differential methylation status of TBX21, GATA3, RORC, FOXP3, IL10 and IFNG in tumor CD4+ T-cells. Analysis of lineage specific genes revealed differential methylation and gene expression in tumor CD4+ T-cells. Interestingly, we observed dysregulation of several ligands of T cell function genes in GBM tissue corresponding to the T-cell receptors that were dysregulated in tumor infiltrating CD4+ T-cells. Our results suggest that GBM might induce epigenetic alterations in tumor infiltrating CD4+ T-cells there by influencing anti-tumor immune response by manipulating differentiation and function of tumor infiltrating CD4+ T-cells. Thus, further research is warranted to understand the role of tumor induced epigenetic modification of tumor infiltrating T-cells to develop effective anti-GBM immunotherapy.

// Hisham S. Alhajala 1 , John L. Markley 2 , Jin Hae Kim 2 , Mona M. Al-Gizawiy 3 , Kathleen M. Schmainda 3 , John S. Kuo 4 and Christopher R. Chitambar 1 , 3 1 Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin, USA 2 Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA 3 Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA 4 Department of Neurosurgery and Mulva Clinic for the Neurosciences, Dell Medical School, Austin, Texas, USA Correspondence to: Christopher R. Chitambar, email: chitambr@mcw.edu Keywords: glioblastoma; iron; gallium; metformin; mitochondrial complex I Received: February 01, 2020 Accepted: April 03, 2020 Published: April 28, 2020 ABSTRACT New drugs are needed for glioblastoma, an aggressive brain tumor with a dismal prognosis. We recently reported that gallium maltolate (GaM) retards the growth of glioblastoma in a rat orthotopic brain tumor model by inhibiting mitochondrial function and iron-dependent ribonucleotide reductase (RR). However, GaM’s mechanism of action at the mitochondrial level is not known. Given the interaction between gallium and iron metabolism, we hypothesized that gallium might target iron-sulfur (Fe-S) cluster-containing mitochondrial proteins. Using Extracellular Flux Analyzer technology, we confirmed that after a 24-h incubation, GaM 50 μmol/L inhibited glioblastoma cell growth by

Radiation is utilized in the therapy of more than 50% of cancer patients. Unfortunately, many malignancies become resistant to radiation over time. We investigated the hypothesis that one method of a cancer cell's ability to survive radiation occurs through cellular communication via exosomes. Exosomes are cell-derived vesicles containing DNA, RNA, and protein. Three properties were analyzed: 1) exosome function, 2) exosome profile and 3) exosome uptake/blockade. To analyze exosome function, we show radiation-derived exosomes increased proliferation and enabled recipient cancer cells to survive radiation in vitro. Furthermore, radiation-derived exosomes increased tumor burden and decreased survival in an in vivo model. To address the mechanism underlying the alterations by exosomes in recipient cells, we obtained a profile of radiation-derived exosomes that showed expression changes favoring a resistant/proliferative profile. Radiation-derived exosomes contain elevated oncogenic miR-889, oncogenic mRNAs, and proteins of the proteasome pathway, Notch, Jak-STAT, and cell cycle pathways. Radiation-derived exosomes contain decreased levels of tumor-suppressive miR-516, miR-365, and multiple tumor-suppressive mRNAs. Ingenuity pathway analysis revealed the most represented networks included cell cycle, growth/survival. Upregulation of DNM2 correlated with increased exosome uptake. To analyze the property of exosome blockade, heparin and simvastatin were used to inhibit uptake of exosomes in recipient cells resulting in inhibited induction of proliferation and cellular survival. Because these agents have shown some success as cancer therapies, our data suggest their mechanism of action could be limiting exosome communication between cells. The results of our study identify a novel exosome-based mechanism that may underlie a cancer cell's ability to survive radiation.

High grade gliomas, including glioblastoma (GB), are devastating malignancies with very poor prognosis. Over the course of the last decade, there has been a failure to develop new treatments for GB. Reasons for this failure include the lack of validation of novel molecular targets, which are often characterized in animal models and directly transposed to human trials. Here we build on our previous findings, which describe how the multi-functional co-receptor Neuropilin-1 (NRP1) signals through glioma associated microglia/macrophages (GAMS) to promote murine glioma, and investigate NRP1 expression in human glioma. Clinical and gene expression data were obtained via The Cancer Genome Atlas (TCGA), and analyzed using R statistical software. Additionally, CIBERSORT in silico deconvolution was used to determine fractions of immune cell sub-populations within the gene expression datasets. We find that NRP1 expression is correlated with poor prognosis, glioma grade, and associates with the mesenchymal GB subtype. In human GB, NRP1 expression is highly correlated with markers of monocytes/macrophages, as well as genes that contribute to the pro-tumorigenic phenotype of these cells.

// William P. Katt 1 , Nicolas J. Blobel 1 , Svetlana Komarova 2 , Marc A. Antonyak 1 , Ichiro Nakano 2 and Richard A. Cerione 1, 3 1 Department of Molecular Medicine, Cornell University, Ithaca, NY, USA 2 Department of Neurosurgery, University of Alabama at Birmingham, Birmingham, AL, USA 3 Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA Correspondence to: Richard A. Cerione, email: rac1@cornell.edu Keywords: transglutaminase; glioblastoma; signaling; glioma stem cells; cancer Received: July 25, 2018 Accepted: September 15, 2018 Published: September 28, 2018 ABSTRACT The protein crosslinking enzyme tissue transglutaminase (tTG) is an acyltransferase which catalyzes transamidation reactions between two proteins, or between a protein and a polyamine. It is frequently overexpressed in several different types of human cancer cells, where it has been shown to contribute to their growth, survival, and invasiveness. tTG is capable of adopting two distinct conformational states: a protein crosslinking active (“open”) state, and a GTP-bound, crosslinking inactive (“closed”) state. We have previously shown that the ectopic expression of mutant forms of tTG, which constitutively adopt the open conformation, are toxic to cells. This raises the possibility that strategies directed toward causing tTG to maintain an open state could potentially provide a therapeutic benefit for cancers in which tTG is highly expressed. Here, we report the identification of a small molecule, TTGM 5826, which stabilizes the open conformation of tTG. Treatment of breast and brain cancer cell lines, as well as glioma stem cells, with this molecule broadly inhibits their transformed phenotypes. Thus, TTGM 5826 represents the lead compound for a new class of small molecules that promote the toxicity of cancer cells by stabilizing the open state of tTG.

In glioblastoma multiforme (GBM), cancer stem cells (CSCs) are thought to be responsible for gliomagenesis, resistance to treatment and recurrence. Unfortunately, the prognosis for GBM remains poor and recurrence frequently occurs in the peritumoral tissue within 2 cm from the tumor edge. In this area, a population of CSCs has been demonstrated which may recapitulate the tumor after surgical resection. In the present study, we aimed to characterize CSCs derived from both peritumoral tissue (PCSCs) and GBM (GCSCs) in order to deepen their significance in GBM development and progression. The stemness of PCSC/GCSC pairs obtained from four human GBM surgical specimens was investigated by comparing the expression of specific stem cell markers such as Nestin, Musashi-1 and SOX2. In addition, the growth rate, the ultrastructural features and the expression of other molecules such as c-Met, pMet and MAP kinases, involved in cell migration/invasion, maintenance of tumor stemness and/or resistance to treatments were evaluated. Since it has been recently demonstrated the involvement of the long non-coding RNAs (lncRNAs) in the progression of gliomas, the expression of H19 lncRNA, as well as of one of its two mature products miR-675-5p was evaluated in neurospheres. Our results show significant differences between GCSCs and PCSCs in terms of proliferation, ultrastructural peculiarities and, at a lower extent, stemness profile. These differences might be important in view of their potential role as a therapeutic target.

There is a clear unmet need for novel therapeutic agents for management of primary and secondary brain tumors. Novel therapeutic agents with excellent central nervous system (CNS) penetration and therapeutic activity are urgently needed. EDO-S101 is a novel alkylating and histone deacetylase inhibiting agent created by covalent fusion of bendamustine and vorinostat. We used murine models to perform CNS pharmacokinetic analysis and preclinical therapeutic evaluation of EDO-S101 for CNS lymphoma, metastatic triple-negative breast cancer of the brain, and glioblastoma multiforme. EDO-S101 has excellent CNS penetration of 13.8% and 16.5% by intravenous infusion and bolus administration respectively. It shows promising therapeutic activity against CNS lymphoma, metastatic triple-negative breast cancer of the brain, and glioblastoma multiforme with significant prolongation of survival compared to no-treatment controls. Therapeutic activity was higher with IV infusion compared to IV bolus. It should be evaluated further for therapeutic use in brain tumors.

Therapy resistance and recurrence in Glioblastoma is due to the presence of residual radiation resistant cells. However, because of their inaccessibility from patient biopsies, the molecular mechanisms driving their survival remain unexplored. Residual Radiation Resistant (RR) and Relapse (R) cells were captured using cellular radiation resistant model generated from patient derived primary cultures and cell lines. iTRAQ based quantitative proteomics was performed to identify pathways unique to RR cells followed by in vitro and in vivo experiments showing their role in radio-resistance. 2720 proteins were identified across Parent (P), RR and R population with 824 and 874 differential proteins in RR and R cells. Unsupervised clustering showed proteasome pathway as the most significantly deregulated pathway in RR cells. Concordantly, the RR cells displayed enhanced expression and activity of proteasome subunits, which triggered NFkB signalling. Pharmacological inhibition of proteasome activity led to impeded NFkB transcriptional activity, radio-sensitization of RR cells in vitro, and significantly reduced capacity to form orthotopic tumours in vivo. We demonstrate that combination of proteasome inhibitor with radio-therapy abolish the inaccessible residual resistant cells thereby preventing GBM recurrence. Furthermore, we identified first proteomic signature of RR cells that can be exploited for GBM therapeutics.

Glioblastoma (GBM) is the most common and the deadliest type of primary brain tumor, with a median survival time of only 15 months despite aggressive treatment. Although most patients have an extremely poor prognosis, a relatively small number of patients survive far beyond the median survival time. Investigation of these exceptional responders has sparked a great deal of interest and is becoming an important focus in the field of cancer research. To investigate the molecular differences between typical and exceptional responders in GBM, comparative analyses of somatic mutations, copy number, methylation, and gene expression datasets from The Cancer Genome Atlas were performed, and the results of these analyses were integrated via gene ontology and pathway analyses to assess the functional significance of the differential aberrations. Less severe copy number loss of CDKN2A, lower expression of CXCL8, and FLG mutations are all associated with an exceptional response. Typical responders are characterized by upregulation of NF-κB signaling and of pro-inflammatory cytokines, while exceptional responders are characterized by upregulation of Alzheimer's and Parkinson's disease pathways as well as of genes involved in synaptic transmission. The upregulated pathways and processes in typical responders are consistently associated with more aggressive tumor phenotypes, while those in the exceptional responders suggest a retained ability in tumor cells to undergo cell death in response to treatment. With the upcoming launch of the National Cancer Institute's Exceptional Responders Initiative, similar studies with much larger sample sizes will likely become possible, hopefully providing even more insight into the molecular differences between typical and exceptional responders.

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.

// Sara Franceschi 1 , Francesca Lessi 1 , Paolo Aretini 1 , Valerio Ortenzi 2 , Cristian Scatena 2 , Michele Menicagli 1 , Marco La Ferla 1 , Prospero Civita 1 , Katia Zavaglia 3 , Claudia Scopelliti 1 , Alessandro Apollo 4 , Francesco Giovanni Carbone 2 , Riccardo Vannozzi 5 , Generoso Bevilacqua 6 , Francesco Pasqualetti 7 , Antonio Giuseppe Naccarato 2 and Chiara Maria Mazzanti 1 1 Fondazione Pisana per la Scienza, Onlus, Pisa, Italy 2 Department of Translational Research and New Technologies in Medicine, University Hospital of Pisa, Pisa, Italy 3 SOD Molecular Genetics, University Hospital of Pisa, Pisa, Italy 4 Tumor Cell Biology Unit, Istituto Tumori Toscana, Florence, Italy 5 Neurosurgery Department, University Hospital of Pisa, Pisa, Italy 6 Pathological Anatomy Section, San Rossore Clinic, Pisa, Italy 7 Radiotherapy Department, University Hospital of Pisa, Pisa, Italy Correspondence to: Chiara Maria Mazzanti, email: c.mazzanti@fpscience.it Keywords: glioblastoma; next generation sequencing (NGS); recurrence free survival (RFS); onco-drivers; tumor suppressors Received: November 17, 2017 Accepted: April 02, 2018 Published: May 08, 2018 ABSTRACT Glioblastoma is a devastating disease that despite all the information gathered so far, its optimal management remains elusive due to the absence of validated targets from clinical studies. A better clarification of the molecular mechanisms is needed. In this study, having access to IDH1 wild-type glioblastoma of patients with exceptionally long recurrence free survival (RFS), we decided to compare their mutational and gene expression profile to groups of IDH1 wild-type glioblastoma of patients with shorter RFS, by using NGS technology. The exome analysis revealed that Long-RFS tumors have a lower mutational rate compared to the other groups. A total of 158 genes were found differentially expressed among the groups, 112 of which distinguished the two RFS extreme groups. Overall, the exome data suggests that shorter RFS tumors could be, chronologically, in a more advanced state in the muli-step tumor process of sequential accumulation of mutations. New players in this kind of cancer emerge from the analysis, confirmed at the RNA/DNA level, identifying, therefore, possible oncodrivers or tumor suppressor genes.

The absence of major progress in the treatment of glioblastoma (GBM) is partly attributable to our poor understanding of both GBM tumor biology and the acquirement of treatment resistance in recurrent GBMs. Recurrent GBMs are characterized by their resistance to radiation. In this study, we used an established stable U87 radioresistant GBM model and total RNA sequencing to shed light on global mRNA expression changes following irradiation. We identified many genes, the expressions of which were altered in our radioresistant GBM model, that have never before been reported to be associated with the development of radioresistant GBM and should be concertedly further investigated to understand their roles in radioresistance. These genes were enriched in various biological processes such as inflammatory response, cell migration, positive regulation of epithelial to mesenchymal transition, angiogenesis, apoptosis, positive regulation of T-cell migration, positive regulation of macrophage chemotaxis, T-cell antigen processing and presentation, and microglial cell activation involved in immune response genes. These findings furnish crucial information for elucidating the molecular mechanisms associated with radioresistance in GBM. Therapeutically, with the global alterations of multiple biological pathways observed in irradiated GBM cells, an effective GBM therapy may require a cocktail carrying multiple agents targeting multiple implicated pathways in order to have a chance at making a substantial impact on improving the overall GBM survival.

Glioma-Initiating Cells (GICs) are thought to be responsible for tumor initiation, progression and recurrence in glioblastoma (GBM). In previous studies, we reported the constitutive phosphorylation of the STAT3 transcription factor in GICs derived from GBM patient-derived xenografts, and that STAT3 played a critical role in GBM tumorigenesis. In this study, we show that CRISPR/Cas9-mediated deletion of STAT3 in an established GBM cell line markedly inhibited tumorigenesis by intracranial injection but had little effect on cell proliferation in vitro. Tumorigenesis was rescued by the enforced expression of wild-type STAT3 in cells lacking STAT3. In contrast, GICs were highly addicted to STAT3 and upon STAT3 deletion GICs were non-viable. Moreover, we found that STAT3 was constitutively activated in GICs by phosphorylation on both tyrosine (Y705) and serine (S727) residues. Therefore, to study STAT3 function in GICs we established an inducible system to knockdown STAT3 expression (iSTAT3-KD). Using this approach, we demonstrated that Y705-STAT3 phosphorylation was critical and indispensable for GIC-induced tumor formation. Both phosphorylation sites in STAT3 promoted GIC proliferation in vitro. We further showed that S727-STAT3 phosphorylation was Y705-dependent. Targeted microarray and RNA sequencing revealed that STAT3 activated cell-cycle regulator genes, and downregulated genes involved in the interferon response, the hypoxia response, the TGFβ pathway, and remodeling of the extracellular matrix. Since STAT3 is an important oncogenic driver of GBM, the identification of these STAT3 regulated pathways in GICs will inform the development of better targeted therapies against STAT3 in GBM and other cancers.

Glioblastoma (GBM) represents the most common and lethal brain tumor. High vascularization, necrosis and invasiveness are hallmarks of GBM aggressiveness with recent data suggesting the important role of glioblastoma stem cells (GSCs) in these processes. It is now well established that cancer cells employ specialized structures termed invadosomes to potentiate invasion. However, the role of these structures in GBM dissemination remains poorly investigated. In this study, we showed that GBM-isolated GSCs form invadopodia-like protrusions endowed with degradative action. Interestingly, their formation depends on extracellular matrix (ECM) sensing via the CD44 receptor. We also found that GSCs invasive migration occurring during tubes assembly is promoted through invadopodia-mediated-ECM remodeling and LIM kinases signaling. Moreover, our study demonstrates that GSCs are highly adaptable cells that are able not only to restore damaged endothelial-derived tubes but also to generate in cooperation with normal endothelial cells (ECs) intact vascular channels. Taken together, our data provide new insights in GBM microvasculature and suggest that GSCs targeting in combination with anti-VEGF therapy may block tumor progression.

Common cancer treatments include radiation therapy, chemotherapy including molecular targeted drugs and anticancer drugs, and surgical treatment. Recent studies have focused on investigating the mechanisms by which immune cells attack cancer cells and produce immune tolerance-suppressing cytokines, as well as on their potential application in cancer immunotherapy. We conducted expression profiling of CD274 (PD-L1), GATA3, IFNG, IL12R, IL12RB2, IL4, PDCD1 (PD-1), PDCD1LG2 (PD-L2), and TBX21 (T-bet) using data of 158 glioblastoma multiforme (GBM) patients with clinical information available at The Cancer Genome Atlas. Principal component analysis of the expression profiling data was used to derive an equation for evaluating the status of Th1 and Th2 cells. GBM specimens were divided based on the median of the Th scores. The results revealed that Th1HighTh2Low and Th1LowTh2Low statuses indicated better prognosis than Th1HighTh2High, and were evaluated based on the downregulation of PD-L1, PD-L2, and PD-1. Furthermore, Th2Low divided based on the threshold, as well as CD274Low and PDCD1Low, were associated with good prognosis. In the Th2Low subgroup, 14 genes were identified as potential prognostic markers. Of these, SLC11A1Low, TNFRSF1BLow, and LTBRLow also indicated good prognosis. These results suggest that low Th2 balance and low activity of the PD-L1/PD-1 axis predict good prognosis in GBM. The set of genes identified in the present study could reliably predict survival in GBM patients and serve as useful molecular markers. Furthermore, this set of genes could prove to be novel targets for cancer immunotherapy.

// Thomas Lindner 1, * , Hajrullah Ahmeti 2, * , Julia Juhasz 1 , Michael Helle 3 , Olav Jansen 1 , Michael Synowitz 2 and Stephan Ulmer 1 1 Clinic for Radiology and Neuroradiology, UKSH Kiel, Kiel, Germany 2 Clinic for Neurosurgery, UKSH Kiel, Kiel, Germany 3 Philips Research Laboratories, Hamburg, Germany * These authors contributed equally to this work Correspondence to: Thomas Lindner, email: Thomas.Lindner@uksh.de Keywords: magnetic resonance; imaging; perfusion; glioblastoma; in-vivo Received: November 19, 2017 Accepted: March 18, 2018 Published: April 06, 2018 ABSTRACT Resection control using magnetic resonance imaging during neurosurgical interventions increases confidence regarding the extent of tumor removal already during the procedure. In addition to morphological imaging, functional information such as perfusion might become an important marker of the presence and extent of residual tumor mass. The aim of this study was to implement arterial spin labeling (ASL) perfusion imaging as a noninvasive alternative to dynamic susceptibility contrast (DSC) perfusion imaging in patients suffering from intra-axial tumors for resection control already during surgery. The study included 15 patients suffering from glioblastoma multiforme in whom perfusion imaging using DSC and ASL was performed before, during, and after surgery. The data obtained from intraoperative scanning were analyzed by two readers blinded to any clinical information, and the presence of residual tumor mass was evaluated using a ranking scale. Similarity of results was analyzed using the intraclass correlation coefficient and Pearson’s correlation coefficient. The results show that intraoperative ASL is as reliable as DSC when performing intraoperative perfusion imaging. According to the results of this study, intraoperative imaging using ASL represents an attractive alternative to contrast agent-based perfusion imaging.

Proteomics aids to the discovery and expansion of protein-protein interaction networks, which are key to understand molecular mechanisms in physiology and physiopathology, but also to infer protein function in a guilt-by-association fashion. In this study we use a systematic protein-protein interaction membrane yeast two-hybrid method to expand the interactome of TRPV2, a cation channel related to nervous system development. After validation of the interactome in silico, we define a TRPV2-interactome signature combining proteomics with the available physio-pathological data in Disgenet to find interactome-disease associations, highlighting nervous system disorders and neoplasms. The TRPV2-interactome signature against available experimental data is capable of discriminating overall risk in glioblastoma multiforme prognosis, progression, recurrence, and chemotherapy resistance. Beyond the impact on glioblastoma physiopathology, this study shows that combining systematic proteomics with in silico methods and available experimental data is key to open new perspectives to define novel biomarkers for diagnosis, prognosis and therapeutics in disease. This project has been carried out with funding from Spanish Government (MICINN-SAF2010-21385 and BFU2017-87843-R to A.P.-M), a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (PIOF-GA-2009-237120 to A.P.-M.), the Generalitat de Catalunya research program (AGAUR, 2014-SGR-1628 to A.P.-M.). P.D.-M. was the recipient of a FI fellowship from Generalitat de Catalunya (FI-2013FIB00251); A.P.-M. was the recipient of the Universitat Autònoma de Barcelona-Programa Banco de Santander Fellowship

// Diana Horvath-Rizea 1, * , Alexey Surov 2, * , Karl-Titus Hoffmann 3 , Nikita Garnov 4 , Cathrin Vorkel 2 , Patricia Kohlhof-Meinecke 5 , Oliver Ganslandt 6 , Hansjorg Bazner 7 , Georg Alexander Gihr 1 , Marcell Kalman 1 , Elina Henkes 1 , Hans Henkes 1 and Stefan Schob 3 1 Clinic for Neuroradiology, Katharinenhospital Stuttgart, Stuttgart, Germany 2 Clinic for Diagnostic and Interventional Radiology, University Hospital Leipzig, Leipzig, Germany 3 Department for Neuroradiology, University Hospital Leipzig, Leipzig, Germany 4 Eichamt Leipzig, Leipzig, Germany 5 Department for Pathology, Katharinenhospital Stuttgart, Stuttgart, Germany 6 Clinic for Neurosurgery, Katharinenhospital Stuttgart, Stuttgart, Germany 7 Clinic for Neurology, Katherinenhospital Stuttgart, Stuttgart, Germany * These authors contributed equally to this work Correspondence to: Stefan Schob, email: Stefan.Schob@Medizin.Uni-Leipzig.de Keywords: ring enhancing lesion; glioblastoma, brain abscess; diffusion weighted imaging; histogram analysis Received: December 26, 2017 Accepted: January 30, 2018 Published: April 06, 2018 ABSTRACT Background: Morphologically similar appearing ring enhancing lesions in the brain parenchyma can be caused by a number of distinct pathologies, however, they consistently represent life-threatening conditions. The two most frequently encountered diseases manifesting as such are glioblastoma multiforme (GBM) and brain abscess (BA), each requiring disparate therapeutical approaches. As a result of their morphological resemblance, essential treatment might be significantly delayed or even ommited, in case results of conventional imaging remain inconclusive. Therefore, our study aimed to investigate, whether ADC histogram profiling reliably can distinguish between both entities, thus enhancing the differential diagnostic process and preventing treatment failure in this highly critical context. Methods: 103 patients (51 BA, 52 GBM) with histopathologically confirmed diagnosis were enrolled. Pretreatment diffusion weighted imaging (DWI) was obtained in a 1.5T system using b values of 0, 500, and 1000 s/mm 2 . Whole lesion ADC volumes were analyzed using a histogram-based approach. Statistical analysis was performed using SPSS version 23. Results: All investigated parameters were statistically different in comparison of both groups. Most importantly, ADCp10 was able to differentiate reliably between BA and GBM with excellent accuracy (0.948) using a cutpoint value of 70 x 10 -5 mm 2 × s -1 . Conclusions: ADC whole lesion histogram profiling provides a valuable tool to differentiate between morphologically indistinguishable mass lesions. Among the investigated parameters, the 10th percentile of the ADC volume distinguished best between GBM and BA.

Glioblastoma (GBM) is a highly aggressive brain cancer with poor prognosis and low survival rate. Invasive cancer stem-like cells (CSCs) are responsible for tumor recurrence because they escape current treatments. Our main goal was to study the proteome of three GBM subpopulations to identify key molecules behind GBM cell phenotypes and potential cell markers for migrating cells. We used SuperQuant-an enhanced quantitative proteome approach-to increase proteome coverage. We found 148 proteins differentially regulated in migrating CSCs and 199 proteins differentially regulated in differentiated cells. We used Ingenuity Pathway Analysis (IPA) to predict upstream regulators, downstream effects and canonical pathways associated with regulated proteins. IPA analysis predicted activation of integrin-linked kinase (ILK) signaling, actin cytoskeleton signaling, and lysine demethylase 5B (KDM5B) in CSC migration. Moreover, our data suggested that microRNA-122 (miR-122) is a potential upstream regulator of GBM phenotypes as miR-122 activation was predicted for differentiated cells while its inhibition was predicted for migrating CSCs. Finally, we validated transferrin (TF) and procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) as potential markers for migrating cells.

// Leland J. Dunwoodie 1 , William L. Poehlman 1 , Stephen P. Ficklin 2 and Frank Alexander Feltus 1 1 Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA 2 Department of Horticulture, Washington State University, Pullman, WA 99164, USA Correspondence to: Frank Alexander Feltus, email: ffeltus@clemson.edu Keywords: glioblastoma; systems biology; gene co-expression networks; complement system; cancer Received: July 28, 2017 Accepted: January 09, 2018 Published: January 13, 2018 ABSTRACT Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, and available experimental and routine therapies result in limited survival benefits. A vulnerability of GBM cells to catastrophic vacuolization and cell death, a process termed methuosis, induced by Vacquinol-1 (VQ-1) has been described earlier. In the present study, we investigate the efficacy of VQ-1 treatment in two syngeneic rat GBM models, RG2 and NS1. VQ-1 treatment affected growth of both RG2 and NS1 cells in vitro. Intracranially, significant reduction in RG2 tumor size was observed, although no effect was seen on overall survival. No survival advantage or effect on tumor size was seen in animals carrying the NS1 models compared to untreated controls. Furthermore, immunological staining of FOXP3, CD4 and CD8 showed no marked difference in immune cell infiltrate in tumor environment following treatment. Taken together, a survival advantage of VQ-1 treatment alone could not be demonstrated here, even though some effect upon tumor size was seen. Staining for immune cell markers did not indicate that VQ-1 either reduced or increased host anti-tumor immune response.

Several classification systems have been proposed to address genomic heterogeneity of glioblastoma multiforme, but they either showed limited prognostic value and/or are difficult to implement in routine diagnostics. Here we propose a prognostic stratification model for these primary tumors based on tumor gene amplification profiles, that might be easily implemented in routine diagnostics, and potentially improve the patients management. Gene amplification profiles were prospectively evaluated in 80 primary glioblastoma multiforme tumors using single-nucleotide polymorphism arrays and the results obtained validated in publicly available data from 267/347 cases. Gene amplification was detected in 45% of patients, and chromosome 7p11.2 including the EGFR gene, was the most frequently amplified chromosomal region – either alone (18%) or in combination with amplification of DNA sequences in other chromosomal regions (10% of cases). Other frequently amplified DNA sequences included regions in chromosomes 12q(10%), 4q12(7%) and 1q32.1(4%). Based on their gene amplification profiles, glioblastomas were subdivided into: i) tumors with no gene amplification (55%); ii) tumors with chromosome 7p/EGFR gene amplification (with or without amplification of other chromosomal regions) (38%); and iii) glioblastoma multiforme with a single (11%) or multiple (6%) amplified DNA sequences in chromosomal regions other than chromosome 7p. From the prognostic point of view, these amplification profiles showed a significant impact on overall survival of glioblastoma multiforme patients (p>0.001). Based on these gene amplification profiles, a risk-stratification scoring system was built for prognostic stratification of glioblastoma which might be easily implemented in routine diagnostics, and potentially contribute to improved patient management., This work was supported by RETICC RD06/0020/0035, RD06/0020/0059 and RD12/0036/0048 grants from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, (Madrid, Spain and FONDOS FEDER), AES PI16/000476 (Instituto de Salud Carlos III, Madrid, Spain and FONDOS FEDER), GRS909A14 (JCYL) and CB16/12/00400 grant (CIBERONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER).

// Margaret Pain 1, * , Huaien Wang 1, * , Eunjee Lee 2 , Maya Strahl 2 , Wissam Hamou 2 , Robert Sebra 2 , Jun Zhu 2 and Raymund L. Yong 1 1 Departments of Neurosurgery and Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA 2 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA * These authors have contributed equally to this work Correspondence to: Raymund L. Yong, email: raymund.yong@mountsinai.org Keywords: glioblastoma; gliosarcoma; TP53 mutation; temozolomide; mutagenesis Received: October 11, 2017 Accepted: December 11, 2017 Published: December 20, 2017 ABSTRACT Background: Gliosarcoma is a rare variant of glioblastoma (GBM) that exhibits frequent mutations in TP53 and can develop in a secondary fashion after chemoradiation of a primary GBM. Whether temozolomide (TMZ)-induced mutagenesis of the TP53 DNA-binding domain (DBD) can drive the pathogenesis of gliosarcoma is unclear. Methods: We identified a case of a primary GBM that rapidly progressed into secondary gliosarcoma shortly after chemoradiation was initiated. Bulk tumor was collected and gliomasphere cultures derived from both the pre- and post-treatment tumors. We performed targeted DNA sequencing and transcriptome analyses of the specimens to understand their phylogenetic relationship and identify differentially expressed gene pathways. Gliomaspheres from the primary GBM were treated with TMZ and then analyzed to compare patterns of mutagenesis in vivo and ex vivo . Results: The pre- and post-treatment tumors shared EGFR , CDKN2A , and PTEN mutations, but only the secondary gliosarcoma exhibited TP53 DBD missense mutations. Two mutations, R110C, and R175H, were identified, each in distinct clones. Both were base transitions characteristic of TMZ mutagenesis. Gene expression analysis identified increased JAK-STAT signaling in the gliosarcoma, together with reduced expression of microRNAs known to regulate epithelial-mesenchymal transition. Ex vivo treatment of the GBM spheres with TMZ generated numerous variants in cancer driver genes, including TP53 and CDH1 , which were mutated in the post-treatment tumor. Conclusions: TMZ-induced TP53 gain-of-function mutations can have a driving role in secondary gliosarcoma pathogenesis. Analysis of variants identified in ex vivo TMZ-treated gliomaspheres may have utility in predicting GBM evolutionary trajectories in vivo during standard chemoradiation.

Glioblastoma presents as a heterogeneous disease with poor prognosis despite the use of multimodal therapy. Analysis of genomic DNA changes between initial diagnosis and recurrence in response to standard treatment protocols would enhance understanding of disease progression and better inform new treatment strategies. A cohort of 21 patients with primary glioblastoma were examined between diagnosis and first recurrence. This study presented a rare opportunity to characterize molecular alterations in tumors observed in three patients who received no therapeutic intervention, other than surgery, offering a unique control. We focused this study by comparing the dynamic mutation profiles between the primary tumors and their matched recurrent counterparts. Molecular profiling of tumors was performed using multiplexed targeted deep sequencing of 409 well characterized cancer-associated genes, achieving a mean read depth of 1272 x. Three levels of evidence suggested an evolutionary pattern consistent with a response to therapy-mediated selection pressures exists in treated patients: 1) variant burden was reduced in recurrent tumors, 2) neutral evolutionary dynamics apparent in untreated tumors shifted toward a non-neutral mode of evolution in treated patients at recurrence, and 3) the recurrent tumor of one patient displayed an increased mutation rate attributable to a temozolomide-associated hypermutator phenotype. Our observations suggest that current treatment modalities are likely to fail in achieving long term remission with the majority of relapse samples containing distinct mutations when compared to primary diagnostic samples.

// Jia Wang 1 , Yuchen Xie 2 , Xiaobin Bai 1 , Ning Wang 1 , Hai Yu 1, 2 , Zhong Deng 1, 2 , Minxue Lian 1 , Shuo Yu 2 , Hao Liu 1 , Wanfu Xie 1 and Maode Wang 1 1 Department of Neurosurgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China 2 School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China Correspondence to: Wanfu Xie, email: wanfu67@aliyun.com Maode Wang, email: maodewang@163.com Keywords: glioma stem-like cells; glioblastoma; TTK; MTFR2 Received: June 15, 2017 Accepted: November 21, 2017 Published: December 11, 2017 ABSTRACT Accumulating evidence has proved that glioma stem-like cells (GSCs) are responsible for tumorigenesis, treatment resistance, and subsequent tumor recurrence in glioblastoma (GBM). In this study, we identified dual specificity protein kinase TTK (TTK) as the most up-regulated and differentially expressed kinase encoding genes in GSCs. Functionally, TTK was essential for in vitro clonogenicity and in vivo tumor propagation in GSCs. Clinically, TTK expression was highly enriched in GBM, moreover, was inversely correlated with a poor prognosis in GBM patients. Mechanistically, mitochondrial fission regulator 2 (MTFR2) was identified as one of the most correlated genes to TTK and transcriptionally regulated TTK expression via activation of TTK promoter. Collectively, MTFR2-dependent regulation of TTK plays a key role in maintaining GSCs in GBM and is a potential novel druggable target for GBM.

Glioblastoma resists chemoradiotherapy, then, recurs to be a fatal space-occupying lesion. The recurrence is caused by re-growing cell populations such as glioma stem cells (GSCs), suggesting that GSC populations should be targeted. This study addressed whether a novel anti-cancer drug, OTS964, an inhibitor for T-LAK cell originated protein kinase (TOPK), is effective in reducing the size of the heterogeneous GSC populations, a power-law coded heterogeneous GSC populations consisting of glioma sphere (GS) clones, by detailing quantitative growth properties. We found that OTS964 killed GS clones while suppressing the growth of surviving GS clones, thus identifying clone-eliminating and growth-disturbing efficacies of OTS964. The efficacies led to a significant size reduction in GS populations in a dose-dependent manner. The surviving GS clones reconstructed GS populations in the following generations; the recovery of GS populations fits a recurrence after the chemotherapy. The recovering GS clones resisted the clone-eliminating effect of OTS964 in sequential exposure during the growth recovery. However, surprisingly, the resistant properties of the recovered-GS clones had been plastically canceled during self-renewal, and then the GS clones had become re-sensitive to OTS964. Thus, OTS964 targets GSCs to eliminate them or suppress their growth, resulting in shrinkage of the power-law coded GSC populations. We propose a therapy focusing on long-term control in recurrence of glioblastoma via reducing the size of the GSC populations by OTS964.

Glioblastoma (GBM) is a deadly and incurable brain tumor. Although microRNAs (miRNAs) play critical roles in regulating the cancer cell phenotype, the underlying mechanisms of how they regulate tumorigenesis are incompletely understood. We previously showed that miR-203a is expressed at relatively low levels in GBM patients, and ectopic miR-203a expression in GBM cell lines inhibited cell proliferation and migration, increased sensitivity to apoptosis induced by interferon (IFN) or temozolomide in vitro, and inhibited GBM tumorigenesis in vivo. Here we show that ectopic expression of miR-203a in GBM cell lines promotes the IFN response pathway as evidenced by increased IFN production and IFN-stimulated gene (ISG) expression, and high basal tyrosine phosphorylation of multiple STAT proteins. Importantly, we identified that miR-203a directly suppressed the protein levels of ataxia-telangiectasia mutated (ATM) kinase that negatively regulates IFN production. We found that high ATM expression in GBM correlates with poor patient survival and that ATM expression is inversely correlated with miR-203a expression. Knockout of ATM expression and inhibition of ATM function in GBM cell lines inhibited cell proliferation and migration, increased sensitivity to apoptosis induced by therapeutic agents in vitro, and markedly suppressed GBM tumor growth and promoted animal survival. In contrast, restoring ATM levels in GBM cells ectopically expressing miR-203a increased tumorigenicity and decreased animal survival. Our study suggests that low miR-203a expression in GBM suppresses the interferon response through an ATM-dependent pathway.

Glioblastomas (GBMs) are among the most malignant of all human tumors and have poor prognosis. The current standard of care (SOC) includes maximal surgical tumor resection followed by adjuvant temozolomide (TMZ) and concomitant radiotherapy (RT). However, even with this treatment, the 5-year survival rate is less than 10%, and thus, follow-up treatment is required to improve efficacy. In GBMs as well as many other solid cancers, PI3K/mTOR signaling is overactivated. Therefore, multiple tumor-based PI3K inhibitors have been studied in various cancers. In the current study, we investigated the effect of the dual PI3K/mTOR inhibitor dactolisib on TMZ+RT treatment in three human GBM cell lines and a orthotopic xenograft model. Dactolisib alone induced cytotoxicity and pro-apoptotic effects, which act as antitumor factors. Combined with SOC treatment, dactolisib inhibited cell viability, induced enhanced pro-apoptotic effect, and attenuated migration/invasion in all three cell lines, thereby enhancing the SOC therapeutic effect. Protein microarray analysis showed that A172 cells treated with TMZ+RT+dactolisib had higher p27 and lower Bcl-2 expression than other groups. Moreover, in the xenograft model, oral dactolisib combined with TMZ+RT inhibited tumor growth and prolonged survival. Thus, SOC combined with dactolisib shows potent anti-tumor activity and has promising potential for solid tumor treatment.

Glioma stem cell (GSC)-targeted therapy is expected to be one of the most innovative approaches to treat patients with glioblastoma (GBM). A number of the drugs that restrain the signaling pathway essential for GSC maintenance have been under clinical trials. Here, we identified fluspirilene, a traditional antipsychotic drug, as a GSC-targeting agent, selected from thousands of existing drugs, and investigated its therapeutic effects against GBM with the purpose of drug repositioning. To develop novel therapeutics targeting GSCs, we initially screened drug libraries for small-molecule compounds showing a greater efficacy, compared to that of controls, in inhibiting the proliferation and survival of different GSC lines using cell proliferation assay. Drugs already reported to show therapeutic effects against GBM or those under clinical trials were excluded from subsequent screening. Finally, we found three drugs showing remarkable antiproliferative effects on GSCs at low concentrations and investigated their therapeutic effects on GSCs, glioma cell lines, and in a GBM mouse model. Of the three compounds, fluspirilene demonstrated a significant inhibitory effect on the proliferation and invasion of glioma cells as well as in the model mice treated with the drug. These effects were associated with the inactivation of the signal transducer and activator of transcription 3 (STAT3). Redeveloping of fluspirilene is a promising approach for the treatment of GBM.

Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults. In spite of advances in diagnosis and therapy, the prognosis is still relatively poor. The invasive property of GBM is the major cause of death in patients. Epithelial-to-mesenchymal transition-like process (EMT-like process) is considered to play an important role in the invasive property. Metformin has been reported as a regulator of EMT-like process. In this study, we confirmed that metformin inhibited TGF-β1-induced EMT-like process and EMT-associated migration and invasion in LN18 and U87 GBM cells. Our results also showed that metformin significantly suppressed self-renewal capacity of glioblastoma stem cells (GSCs), and expression of stem cell markers Bmi1, Sox2 and Musashi1, indicating that metformin can inhibit cancer stem-like properties of GBM cells. We further clarified that metformin specifically inhibited TGF-β1 activated AKT, the downstream molecular mTOR and the leading transcription factor ZEB1. Taken together, our data demonstrate that metformin inhibits TGF-β1-induced EMT-like process and cancer stem-like properties in GBM cells via AKT/mTOR/ZEB1 pathway and provide evidence of metformin for further clinical investigation targeted GBM.

Glioblastoma (GBM) show significant inter- and intra-tumoral heterogeneity, impacting response to treatment and overall survival time of 12-15 months. To study glioblastoma phenotypic heterogeneity, multi-parametric magnetic resonance images (MRI) of 85 glioblastoma patients from The Cancer Genome Atlas were analyzed to characterize tumor-derived spatial habitats for their relationship with outcome (overall survival) and to identify their molecular correlates (i.e., determine associated tumor signaling pathways correlated with imaging-derived habitat measurements). Tumor sub-regions based on four sequences (fluid attenuated inversion recovery, T1-weighted, post-contrast T1-weighted, and T2-weighted) were defined by automated segmentation. From relative intensity of pixels in the 3-dimensional tumor region, "imaging habitats" were identified and analyzed for their association to clinical and genetic data using survival modeling and Dirichlet regression, respectively. Sixteen distinct tumor sub-regions ("spatial imaging habitats") were derived, and those associated with overall survival (denoted "relevant" habitats) in glioblastoma patients were identified. Dirichlet regression implicated each relevant habitat with unique pathway alterations. Relevant habitats also had some pathways and cellular processes in common, including phosphorylation of STAT-1 and natural killer cell activity, consistent with cancer hallmarks. This work revealed clinical relevance of MRI-derived spatial habitats and their relationship with oncogenic molecular mechanisms in patients with GBM. Characterizing the associations between imaging-derived phenotypic measurements with the genomic and molecular characteristics of tumors can enable insights into tumor biology, further enabling the practice of personalized cancer treatment. The analytical framework and workflow demonstrated in this study are inherently scalable to multiple MR sequences.

Glioblastoma multiforme (GBM) is the most aggressive CNS tumor and is characterized by a very high frequency of clinical relapse after therapy and thus by a dismal prognosis, which strongly compromises patients survival. We have recently identified the small molecule SI113, as a potent and selective inhibitor of SGK1, a serine/threonine protein kinase, that modulates several oncogenic signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation and perturbs cell cycle progression by modulating SGK1-related substrates. SI113 is also able to strongly and consistently block, in vitro and in vivo, growth and survival of human hepatocellular-carcinomas, either used as a single agent or in combination with ionizing radiations. In the present paper we aim to study the effect of SI113 on human GBM cell lines with variable p53 expression. Cell viability, cell death, caspase activation and cell cycle progression were then analyzed by FACS and WB-based assays, after exposure to SI113, with or without oxidative stress and ionizing radiations. Moreover, autophagy and related reticulum stress response were evaluated. We show here, that i) SGK1 is over-expressed in highly malignant gliomas and that the treatment with SI113 leads to ii) significant increase in caspase-mediated apoptotic cell death in GBM cell lines but not in normal fibroblasts; iii)enhancement of the effects of ionizing radiations; iv) modulation of the response to oxidative reticulum stress; v) induction of cytotoxic autophagy. Evidence reported here underlines the therapeutic potential of SI113 in GBM, suggesting a new therapeutic strategy either alone or in combination with radiotherapy.

// Maria Oikonomaki 1 , Pierre Bady 1, 2, 3 and Monika E. Hegi 1 1 Department of Clinical Neurosciences, University Hospital Lausanne, Lausanne, Switzerland 2 Department of Research and Education, University Hospital Lausanne, Lausanne, Switzerland 3 Bioinformatics Core Facility, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland Correspondence to: Monika E. Hegi, email: Monika.Hegi@CHUV.ch Keywords: glioblastoma; USP15; HECTD1; tumor suppressor; WNT pathway Received: September 12, 2017 Accepted: November 13, 2017 Published: November 30, 2017 ABSTRACT Expression based prediction of new genomic alterations in glioblastoma identified the de-ubiquitinase Ubiquitin Specific Peptidase 15 ( USP15 ) as potential tumor suppressor gene associated with genomic deletions (11%). Ectopic expression of USP15 in glioblastoma cell-lines reduced colony formation and growth in soft agar, while overexpression of its functional mutant had the opposite effect. Evaluation of the protein binding network of USP15 by Mass Spectrometry in glioblastoma cells uncovered eight novel interacting proteins, including HECT Domain Containing E3 Ubiquitin Protein Ligase 1 (HECTD1), whose mouse homologue has been associated with an inhibitory effect on the WNT-pathway. USP15 de-ubiquitinated and thereby stabilized HECTD1 in glioblastoma cells, while depletion of USP15 led to decreased HECTD1 protein levels. Expression of USP15 in glioblastoma cells attenuated WNT-pathway activity, while expression of the functional mutant enhanced the activity. Modulation of HECTD1 expression pheno-copied the effects observed for USP15. In accordance, human glioblastoma display a weak but significant negative correlation between USP15 and AXIN2 expression. Taken together, the data provide evidence that USP15 attenuates the canonical WNT pathway mediated by stabilization of HECTD1, supporting a tumor suppressing role of USP15 in a subset of glioblastoma.

// In Young Kim 1, 2, * , MiRi Kwon 1, 2, * , Min-Koo Choi 3 , Dongjoo Lee 4 , Dong Min Lee 1, 2 , Min Ji Seo 1, 2 and Kyeong Sook Choi 1, 2 1 Department of Biochemistry, Ajou University School of Medicine, Suwon, Korea 2 BK21 Plus Program, Department of Biomedical Sciences, Ajou University School of Medicine, Suwon, Korea 3 College of Pharmacy, Dankook University, Cheonan, Korea 4 College of Pharmacy, Ajou University, Suwon, Korea * These authors contributed equally to this work Correspondence to: Kyeong Sook Choi, email: kschoi@ajou.ac.kr Keywords: ophiobolin A; endoplasmic reticulum stress; proteostasis; thiol; paraptosis-like cell death Received: August 05, 2017 Accepted: October 28, 2017 Published: November 20, 2017 ABSTRACT Ophiobolin A (OP-A), a fungal sesterterpene from Bipolaris oryzae , was recently shown to have anti-glioma activity. We show here that OP-A induces paraptosis-like cell death accompanied by dilation of the endoplasmic reticulum (ER) in glioma cells, and that CHOP-mediated ER stress plays a critical role in this process. OP-A-induced ER-derived dilation and cell death were found to be independent of reactive oxygen species, but were effectively blocked by various thiol antioxidants. We observed that OP-A can react with cysteinyl thiols to form Michael adducts, suggesting that the ability of OP-A to covalently modify free sulfhydryl groups on proteins may cause protein misfolding and the accumulation of misfolded proteins, leading to paraptosis-like cell death. Taken together, these results indicate that the disruption of thiol proteostasis may critically contribute to the anti-glioma activity of OP-A.

// Ying-Chun Song 1, * , Gai-Xia Lu 1, * , Hong-Wei Zhang 2, * , Xiao-Ming Zhong 3, * , Xian-Ling Cong 4, * , Shao-Bo Xue 1, * , Rui Kong 1, * , Dan Li 1 , Zheng-Yan Chang 5 , Xiao-Feng Wang 6 , Yun-Jie Zhang 6 , Ran Sun 4 , Li Chai 1 , Ru-Ting Xie 5 , Ming-Xiang Cai 1 , Ming Sun 1 , Wei-Qing Mao 1 , Hui-Qiong Yang 5 , Yun-Chao Shao 6 , Su-Yun Fan 1 , Ting-Miao Wu 1 , Qing Xia 6 , Zhong-Wei Lv 1 , David A. Fu 6 and Yu-Shui Ma 1, 7 1 Department of Nuclear Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China 2 Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China 3 Department of Radiology, Jiangxi Provincial Tumor Hospital/Ganzhou City People’s Hospital, Nanchang 330029, China 4 Department of Biobank, China-Japan Unoin Hospital, Jilin University, Changchun 130033, China 5 Department of Pathology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China 6 Department of Orthopedic Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China 7 Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, College of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China * These authors have contributed equally to this work Correspondence to: Zhong-Wei Lv, email: shtjnmd@163.com David A. Fu, email: 061101021@fudan.edu.cn Yu-Shui Ma, email: mayushui2015@126.com Keywords: GBM, glioma, proteomics analysis, gene expression analysis, Bioinformatics Analysis Received: May 24, 2017 Accepted: August 26, 2017 Published: October 19, 2017 ABSTRACT Glioblastoma multiforme (GBM), the most aggressive and lethal primary brain tumor, is characterized by very low life expectancy. Understanding the genomic and proteogenomic characteristics of GBM is essential for devising better therapeutic approaches.Here, we performed proteomic profiling of 8 GBM and paired normal brain tissues. In parallel, comprehensive integrative genomic analysis of GBM was performed in silico using mRNA microarray and sequencing data. Two whole transcript expression profiling cohorts were used - a set of 3 normal brain tissues and 22 glioma tissue samples and a cohort of 5 normal brain tissues and 49 glioma tissue samples. A validation cohort included 529 GBM patients from The Cancer Genome Atlas datasets. We identified 36 molecules commonly changed at the level of the gene and protein, including up-regulated TGFBI and NES and down-regulated SNCA and HSPA12A. Single amino acid variant analysis identified 200 proteins with high mutation rates in GBM samples. We further identified 14 differentially expressed genes with high-level protein modification, among which NES and TNC showed differential expression at the protein level. Moreover, higher expression of NES and TNC mRNAs correlated with shorter overall survival, suggesting that these genes constitute potential biomarkers for GBM.

Anti-VEGF therapy with Bevacizumab is approved for glioblastoma treatment, however, it is known that tumors acquired resistance and eventually became even more aggressive and infiltrative after treatment. In the present study we aimed to unravel the potential cellular mechanisms of resistance to Bevacizumab in glioblastoma in vitro models. Using a panel of glioblastoma cell lines we found that Bevacizumab is able to block the secreted VEGF by the tumor cells and be internalized to the cytoplasm, inducing cytotoxicity in vitro. We further found that Bevacizumab increases the expression of hypoxic (HIF-1α and CAIX) and glycolytic markers (GLUT1 and MCT1), leading to higher glucose uptake and lactate production. Furthermore, we showed that part of the consumed glucose by the tumor cells can be stored as glycogen, hampering cell dead following Bevacizumab treatment. Importantly, we found that this change on the glycolytic metabolism occurs independently of hypoxia and before mitochondrial impairment or autophagy induction. Finally, the combination of Bevacizumab with glucose uptake inhibitors decreased in vivo tumor growth and angiogenesis and shift the expression of glycolytic proteins. In conclusion, we reported that Bevacizumab is able to increase the glucose metabolism on cancer cells by abrogating autocrine VEGF in vitro. Define the effects of anti-angiogenic drugs at the cellular level can allow us to discover ways to revert acquired resistance to this therapeutic approaches in the future, This study was partially developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), and through the Competitiveness Factors Operational Programme (COMPETE), by Portuguese funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038, and by Brazilian MCTI/CNPq No 73/2013. VMG was recipient from a PhD fellowship (SFRH/BD/51997/2012) from Fundação para a Ciência e Tecnologia (FCT), Portugal. FC was recipient of a master FAPESP fellowship (n° 2014/03684-0) and “BEPE - Bolsa Estágio de Pesquisa no Exterior” (n° 2015/02691-6). OM is recipient of a post-doc fellowship (SFRH/BPD/108351/2015) from FCT, Portugal, info:eu-repo/semantics/publishedVersion

Anti-angiogenic therapy induces the apparent normalization of vascular structure, decreases microvessel density (MVD), and improves tumor oxygenation in glioblastomas (GBMs). Six initial and recurrent tumor pairs after bevacizumab (Bev) treatment were compared with GBMs from nine patients resected under neoadjuvant Bev treatment with regard to histological characteristics; MVD; MIB-1 index; and expression of vascular endothelial growth factor (VEGF) and its receptors, hypoxia markers (hypoxia-inducible factor 1 alpha, carbonic anhydrase 9), and nestin as a marker of glioma stem-like cells. In recurrent tumors post-Bev treatment, while the MVD remained low compared with the paired initial tumors (pre-Bev tumors), the expression of hypoxic markers were increased and were even higher in expression compared with the paired pre-Bev tumors in three of the six cases. MIB-1 indices were similar among the initial GBMs, neoadjuvant group, and recurrent tumors post-Bev treatment. The nestin-positive cell ratio of the post-Bev recurrent tumors was as high as that of the pre-Bev tumors. The expression of VEGF and VEGFR1 was increased in the post-Bev recurrent tumors in three and four cases, respectively, compared with the paired pre-Bev tumors. In the majority of Bev-refractory GBMs, tumor hypoxia was present with a paradoxical decrease in MVD. These findings suggest that re-activation of tumor angiogenesis is not initially involved in the acquisition of resistance to Bev.

// Liqiang Liu 1 , Xiaowei Zhang 1 , Chengrui Nan 1 , Zongmao Zhao 1 , Shucheng Ma 1 , Wenhua Li 2 , Hongchao Hu 1 and Zhaohui Liang 1 1 Neurosurgical Department, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China 2 Neurosurgical Department, Dongying People’s Hospital of Shandong Province, Dongying, Shandong, China Correspondence to: Liqiang Liu, email: llq74@sina.com Keywords: MicroRNA-182; glioblastoma; PPP1R1C; protein phosphatase 1 regulatory inhibitor subunit 1C Received: October 27, 2016 Accepted: August 06, 2017 Published: September 27, 2017 ABSTRACT Glioblastoma (GBM) is an incurable cancer, with mean post-diagnosis survival time of 14-16 months. Metagenomic analysis by The Cancer Genome Atlas (TCGA) program has identified microRNA-182-5p (miR-182-5p or miR-182) as the only miRNA associated with favorable disease prognosis and temozolomide (TMZ) susceptibility. Previous reports have indicated that miR-182 down regulates expression of BCL2L12, c-MET , and HIF2A . However, other messenger RNA (mRNA) targets of miR-182 have not been validated which would explain its association with a favorable disease prognosis. In situ analysis revealed that protein phosphatase 1 regulatory inhibitor subunit 1C ( PPP1R1C ) is a putative target of miR-182. PPP1R1C protein and RNA expression as assessed by tissue microarray and quantitative real time PCR, respectively, was inversely correlated to miR-182 expression in glioblastoma patients and in the metastatic glioblastoma cell line U87-MG. Reporter assays using PPP1R1C 3’ untranslated region (UTR) showed that miR-182 can interact with the wild-type but not a miR-182-5-seed mutant. Ectopic expression of miR-182 mimic in the U87-MG cell line significantly decreased proliferation as well as suppressed in vitro migration and invasion. Opposite observations were made when the non-malignant neuronal cell line HCN-2 was transfected with anti-miR-182 antagomir. The miR-182 mimic or siRNA targeting PPP1R1C induced TMZ susceptibility indicating that decreased susceptibility to TMZ in GBM patients might be attributed to high expression of PPP1R1C. Inverse correlation of PPP1R1C mRNA and miR-182 levels in 20 GBM patients confirmed the same. Cumulatively, our results indicate that loss of miR-182 leads to increased expression of PPP1R1C which in part explain disease progression and resistance to TMZ therapy.