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BACKGROUND In glioblastoma (GBM),Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance. METHODS Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2),a stabilized form of PGE2,to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1,a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting,quantitative real-time (qRT)-PCR,and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM. RESULTS In GBM cells,dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads,in turn,to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage,which are dependent on Id1. CONCLUSIONS In GBM,Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively,these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients. View Publication

We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells,and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse,we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality. View Publication

MicroRNA-10b (miR-10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes,while absent in normal neuroglial cells of the brain. miR-10b inhibition strongly impairs proliferation and survival of cultured glioma cells,including glioma-initiating stem-like cells (GSC). Although several miR-10b targets have been identified previously,the common mechanism conferring the miR-10b-sustained viability of GSC is unknown. Here,we demonstrate that in heterogeneous GSC,miR-10b regulates cell cycle and alternative splicing,often through the non-canonical targeting via 5'UTRs of its target genes,including MBNL1-3,SART3,and RSRC1. We have further assessed the inhibition of miR-10b in intracranial human GSC-derived xenograft and murine GL261 allograft models in athymic and immunocompetent mice. Three delivery routes for the miR-10b antisense oligonucleotide inhibitors (ASO),direct intratumoral injections,continuous osmotic delivery,and systemic intravenous injections,have been explored. In all cases,the treatment with miR-10b ASO led to targets' derepression,and attenuated growth and progression of established intracranial GBM. No significant systemic toxicity was observed upon ASO administration by local or systemic routes. Our results indicate that miR-10b is a promising candidate for the development of targeted therapies against all GBM subtypes. View Publication

The potential ability to differentiate dedifferentiated adipocytes into a neural lineage is attracting strong interest as an emerging method of producing model cells for the treatment of a variety of neurological diseases. Here,we describe the efficient conversion of dedifferentiated adipocytes into a neural-like cell population. These cells grew in neurosphere-like structures and expressed a high level of the early neuroectodermal marker Nestin. These neurospheres could proliferate and express stemness genes,suggesting that these cells could be committed to the neural lineage. After neural induction,NeuroD1,Sox1,Double Cortin,and Eno2 were not expressed. Patch clamp data did not reveal different electrophysiological properties,indicating the inability of these cells to differentiate into mature neurons. In contrast,the differentiated cells expressed a high level of CLDN11,as demonstrated using molecular method,and stained positively for the glial cell markers CLDN11 and GFAP,as demonstrated using immunocytochemistry. These data were confirmed by quantitative results for glial cell line-derived neurotrophic factor production,which showed a higher secretion level in neurospheres and the differentiated cells compared with the untreated cells. In conclusion,our data demonstrate morphological,molecular,and immunocytochemical evidence of initial neural differentiation of mature adipocytes,committing to a glial lineage. View Publication

Glioblastoma multiforme (GBM),the most common and lethal tumor of the adult brain,generally shows chemo- and radioresistance. MicroRNAs (miRs) regulate physiological processes,such as resistance of GBM cells to temozolomide (TMZ). Although miRs are attractive targets for cancer therapeutics,the effectiveness of this approach requires targeted delivery. Mesenchymal stem cells (MSCs) can migrate to the sites of cancers,including GBM. We report on an increase in miR-9 in TMZ-resistant GBM cells. miR-9 was involved in the expression of the drug efflux transporter,P-glycoprotein. To block miR-9,methods were developed with Cy5-tagged anti-miR-9. Dye-transfer studies indicated intracellular communication between GBM cells and MSCs. This occurred by gap junctional intercellular communication and the release of microvesicles. In both cases,anti-miR-9 was transferred from MSCs to GBM cells. However,the major form of transfer occurred with the microvesicles. The delivery of anti-miR-9 to the resistant GBM cells reversed the expression of the multidrug transporter and sensitized the GBM cells to TMZ,as shown by increased cell death and caspase activity. The data showed a potential role for MSCs in the functional delivery of synthetic anti-miR-9 to reverse the chemoresistance of GBM cells.Molecular Therapy-Nucleic Acids (2013) 2,e126; doi:10.1038/mtna.2013.60; published online 1 October 2013. View Publication

The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein folding. ER Ca2+ depletion and accumulation of unfolded proteins activate the molecular chaperone GRP78 (glucose-regulated protein 78) which in turn triggers the ER stress response (ERSR) pathway aimed to restore ER homeostasis. Failure to adapt to stress,however,results in apoptosis. We and others have shown that malignant cells are more susceptible to ERSR-induced apoptosis than their normal counterparts,implicating the ERSR as a potential target for cancer therapeutics. Predicated on these findings,we developed an assay that uses a GRP78 biosensor to identify small molecule activators of ERSR in glioma cells. We performed a quantitative high-throughput screen (qHTS) against a collection of ˜425,000 compounds and a comprehensive panel of orthogonal secondary assays was formulated for stringent compound validation. We identified novel activators of ERSR,including a compound with a 2,9-diazaspiro[5.5]undecane core,which depletes intracellular Ca2+ stores and induces apoptosis-mediated cell death in several cancer cell lines,including patient-derived and 3D cultures of glioma cells. This study demonstrates that our screening platform enables the identification and profiling of ERSR inducers with cytotoxic activity and advocates for characterization of these compound in in vivo models. View Publication

BACKGROUND Glioma is the most common form of primary malignant brain tumor in adults,with approximately 4 cases per 100 000 people each year. Gliomas,like many tumors,are thought to primarily metabolize glucose for energy production; however,the reliance upon glycolysis has recently been called into question. In this study,we aimed to identify the metabolic fuel requirements of human glioma cells. METHODS We used database searches and tissue culture resources to evaluate genotype and protein expression,tracked oxygen consumption rates to study metabolic responses to various substrates,performed histochemical techniques and fluorescence-activated cell sorting-based mitotic profiling to study cellular proliferation rates,and employed an animal model of malignant glioma to evaluate a new therapeutic intervention. RESULTS We observed the presence of enzymes required for fatty acid oxidation within human glioma tissues. In addition,we demonstrated that this metabolic pathway is a major contributor to aerobic respiration in primary-cultured cells isolated from human glioma and grown under serum-free conditions. Moreover,inhibiting fatty acid oxidation reduces proliferative activity in these primary-cultured cells and prolongs survival in a syngeneic mouse model of malignant glioma. CONCLUSIONS Fatty acid oxidation enzymes are present and active within glioma tissues. Targeting this metabolic pathway reduces energy production and cellular proliferation in glioma cells. The drug etomoxir may provide therapeutic benefit to patients with malignant glioma. In addition,the expression of fatty acid oxidation enzymes may provide prognostic indicators for clinical practice. View Publication

OBJECTIVE To develop in vitro adenoid cystic carcinoma cell line as a surrogate for functional studies. MATERIALS AND METHODS Cells obtained from a primary ACC of the base of tongue were cultivated in vitro and immortalized with h-TERT. Morphologic,cytogenetic and functional studies were performed. RESULTS Tumor cells were verified by positive reactions to keratin and smooth muscle actin and phenotypic cellular and nuclear features. In-vitro cell growth and colony formation assay supported their tumor nature. CONCLUSION We authenticated an ACC cell line with hybrid epithelial-myoepithelial feature as a resource for functional experimentation. View Publication

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation,migration,and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme,a highly aggressive brain cancer,suggesting that ion channel expression may be perturbed in this population. However,little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing,we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance,expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally,genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes,gene mutations,survival outcomes,regional tumor expression,and experimental responses to loss-of-function. Together,the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance. View Publication

Glioblastoma (GBM) is a common and malignant tumor with a poor prognosis. Glioblastoma stem cells (GSCs) have been reported to be involved in tumorigenesis,tumor maintenance and therapeutic resistance. Thus,to discover novel candidate therapeutic drugs for anti-GBM and anti-GSCs is an urgent need. We hypothesized that if treatment with a drug could reverse,at least in part,the gene expression signature of GBM and GSCs,this drug may have the potential to inhibit pathways essential in the formation of GBM and thereby treat GBM. Here,we collected 356 GBM gene signatures from public databases and queried the Connectivity Map. We systematically evaluated the in vitro antitumor effects of 79 drugs in GBM cell lines. Of the drugs screened,thioridazine was selected for further characterization because it has potent anti-GBM and anti-GSCs properties. When investigating the mechanisms underlying the cytocidal effects of thioridazine,we found that thioridazine induces autophagy in GBM cell lines,and upregulates AMPK activity. Moreover,LC3-II was upregulated in U87MG sphere cells treated with thioridazine. In addition,thioridazine suppressed GBM tumorigenesis and induced autophagy in vivo. We not only repurposed the antipsychotic drug thioridazine as a potent anti-GBM and anti-GSCs agent,but also provided a new strategy to search for drugs with anticancer and anticancer stem cell properties. View Publication

The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID,celecoxib,to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. View Publication

Glioblastomas (GBMs) are highly aggressive,invasive brain tumors with bad prognosis and unmet medical need. These tumors are heterogeneous being constituted by a variety of cells in different states of differentiation. Among these,cells endowed with stem properties,tumor initiating/propagating properties and particularly resistant to chemo- and radiotherapies are designed as the real culprits for tumor maintenance and relapse after treatment. These cells,termed cancer stem-like cells,have been designed as prominent targets for new and more efficient cancer therapies. G-protein coupled receptors (GPCRs),a family of membrane receptors,play a prominent role in cell signaling,cell communication and crosstalk with the microenvironment. Their role in cancer has been highlighted but remains largely unexplored. Here,we report a descriptive study of the differential expression of the endo-GPCR repertoire in human glioblastoma cancer stem-like cells (GSCs),U-87 MG cells,human astrocytes and fetal neural stem cells (f-NSCs). The endo-GPCR transcriptome has been studied using Taqman Low Density Arrays. Of the 356 GPCRs investigated,138 were retained for comparative studies between the different cell types. At the transcriptomic level,eight GPCRs were specifically expressed/overexpressed in GSCs. Seventeen GPCRs appeared specifically expressed in cells with stem properties (GSCs and f-NSCs). Results of GPCR expression at the protein level using mass spectrometry and proteomic analysis are also presented. The comparative GPCR expression study presented here gives clues for new pathways specifically used by GSCs and unveils novel potential therapeutic targets. View Publication