Glioblastoma stem cells (GSC) are a significant cell model for explaining brain tumor recurrence. However, mechanisms underlying their radiochemoresistance remain obscure. Here we show that most clonogenic cells in GSC cultures are sensitive to radiation treatment (RT) with or without temozolomide (TMZ). Only a few single cells survive treatment and regain their self-repopulating capacity. Cells re-populated from treatment-resistant GSC clones contain more clonogenic cells compared to those grown from treatment-sensitive GSC clones, and repeated treatment cycles rapidly enriched clonogenic survival. When compared to sensitive clones, resistant clones exhibited slower tumor development in animals. Upregulated genes identified in resistant clones via comparative expression microarray analysis characterized cells under metabolic stress, including blocked glucose uptake, impaired insulin/Akt signaling, enhanced lipid catabolism and oxidative stress, and suppressed growth and inflammation. Moreover, many upregulated genes highlighted maintenance and repair activities, including detoxifying lipid peroxidation products, activating lysosomal autophagy/ubiquitin-proteasome pathways, and enhancing telomere maintenance and DNA repair, closely resembling the anti-aging effects of caloric/glucose restriction (CR/GR), a nutritional intervention that is known to increase lifespan and stress resistance in model organisms. Although treatment–introduced genetic mutations were detected in resistant clones, all resistant and sensitive clones were subclassified to either proneural (PN) or mesenchymal (MES) glioblastoma subtype based on their expression profiles. Functional assays demonstrated the association of treatment resistance with energy stress, including reduced glucose uptake, fatty acid oxidation (FAO)-dependent ATP maintenance, elevated reactive oxygen species (ROS) production and autophagic activity, and increased AMPK activity and NAD+ levels accompanied by upregulated mRNA levels of SIRT1/PGC-1α axis and DNA repair genes. These data support the view that treatment resistance may arise from quiescent GSC exhibiting a GR-like phenotype, and suggest that targeting stress response pathways of resistant GSC may provide a novel strategy in combination with standard treatment for glioblastoma.

BackgroundTemozolomide (TMZ) is an oral DNA-alkylating agent used for treating patients with glioblastoma. However, therapeutic benefits of TMZ can be compromised by the expression of O6-methylguanine methyltransferase (MGMT) in tumor tissue. Here we used MGMT-expressing glioblastoma stem cells (GSC) lines as a model for investigating the molecular mechanism underlying TMZ resistance, while aiming to explore a new treatment strategy designed to possibly overcome resistance to the clinically relevant dose of TMZ (35 μM).MethodsMGMT-expressing GSC cultures are resistant to TMZ, and IC50 (half maximal inhibitory concentration) is estimated at around 500 μM. Clonogenic GSC surviving 500 μM TMZ (GSC-500 μM TMZ), were isolated. Molecular signatures were identified via comparative analysis of expression microarray against parental GSC (GSC-parental). The recombinant protein of top downregulated signature was used as a single agent or in combination with TMZ, for evaluating therapeutic effects of treatment of GSC.ResultsThe molecular signatures characterized an activation of protective stress responses in GSC-500 μM TMZ, mainly including biotransformation/detoxification of xenobiotics, blocked endoplasmic reticulum stress-mediated apoptosis, epithelial-to-mesenchymal transition (EMT), and inhibited growth/differentiation. Bone morphogenetic protein 7 (BMP7) was identified as the top down-regulated gene in GSC-500 μM TMZ. Although augmenting BMP7 signaling in GSC by exogenous BMP7 treatment did not effectively stop GSC growth, it markedly sensitized both GSC-500 μM TMZ and GSC-parental to 35 μM TMZ treatment, leading to loss of self-renewal and migration capacity. BMP7 treatment induced senescence of GSC cultures and suppressed mRNA expression of CD133, MGMT, and ATP-binding cassette drug efflux transporters (ABCB1, ABCG2), as well as reconfigured transcriptional profiles in GSC by downregulating genes associated with EMT/migration/invasion, stemness, inflammation/immune response, and cell proliferation/tumorigenesis. BMP7 treatment significantly prolonged survival time of animals intracranially inoculated with GSC when compared to those untreated or treated with TMZ alone (p = 0.0017), whereas combination of two agents further extended animal survival compared to BMP7 alone (p = 0.0489).ConclusionsThese data support the view that reduced endogenous BMP7 expression/signaling in GSC may contribute to maintained stemness, EMT, and chemoresistant phenotype, suggesting that BMP7 treatment may provide a novel strategy in combination with TMZ for an effective treatment of glioblastoma exhibiting unmethylated MGMT.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0459-1) contains supplementary material, which is available to authorized users.

Glioblastoma stem cells (GSC) co-exhibiting a tumor-initiating capacity and a radio-chemoresistant phenotype, are a compelling cell model for explaining tumor recurrence. We have previously characterized patient-derived, treatment-resistant GSC clones (TRGC) that survived radiochemotherapy. Compared to glucose-dependent, treatment-sensitive GSC clones (TSGC), TRGC exhibited reduced glucose dependence that favor the fatty acid oxidation pathway as their energy source. Using comparative genome-wide transcriptome analysis, a series of defense signatures associated with TRGC survival were identified and verified by siRNA-based gene knockdown experiments that led to loss of cell integrity. In this study, we investigate the prognostic value of defense signatures in glioblastoma (GBM) patients using gene expression analysis with Probeset Analyzer (131 GBM) and The Cancer Genome Atlas (TCGA) data, and protein expression with a tissue microarray (50 GBM), yielding the first TRGC-derived prognostic biomarkers for GBM patients. Ribosomal protein S11 (RPS11), RPS20, individually and together, consistently predicted poor survival of newly diagnosed primary GBM tumors when overexpressed at the RNA or protein level [RPS11: Hazard Ratio (HR) = 11.5, p<0.001; RPS20: HR = 4.5, p = 0.03; RPS11+RPS20: HR = 17.99, p = 0.001]. The prognostic significance of RPS11 and RPS20 was further supported by whole tissue section RPS11 immunostaining (27 GBM; HR = 4.05, p = 0.01) and TCGA gene expression data (578 primary GBM; RPS11: HR = 1.19, p = 0.06; RPS20: HR = 1.25, p = 0.02; RPS11+RPS20: HR = 1.43, p = 0.01). Moreover, tumors that exhibited unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) or wild-type isocitrate dehydrogenase 1 (IDH1) were associated with higher RPS11 expression levels [corr (IDH1, RPS11) = 0.64, p = 0.03); [corr (MGMT, RPS11) = 0.52, p = 0.04]. These data indicate that increased expression of RPS11 and RPS20 predicts shorter patient survival. The study also suggests that TRGC are clinically relevant cells that represent resistant tumorigenic clones from patient tumors and that their properties, at least in part, are reflected in poor-prognosis GBM. The screening of TRGC signatures may represent a novel alternative strategy for identifying new prognostic biomarkers.

Glioblastoma is incurable. Glioblastoma stem cells (GSC) became a model for explaining tumor recurrence due to their tumorigenic capacity, migratory nature, and radio-chemoresistant phenotype. To prevent tumor recurrence, a strategy targeting of GSC must be identified and added into the treatment. To identify vital genes that promote GSC migration and sustain self-renewal and anti-apoptotic features we have established tumorigenic CD133+ GSC lines (n=3 patients), which are capable of clonal self-renewal and asymmetric division for producing fast-growing CD133- daughter cells that form tumor spheres. CD133+ GSC can migrate outward from primary spheres and form spheres again, which provide an visible, functional model for identifying essential genes of CD133+GSC via loss-of-function RNA interference (RNAi) screen. To identify genes and pathways that confer the migration nature and super longevity of GSC, we constructed a siRNA library consisting of siRNA clones targeting 2079 genes overexpressed in tumorigenic CD133+ GSC when compared to non-tumorigenic, autologous tumor cell lines, normal brain tissue, human embryonic stem cells, normal neural stem cells, fetal neural progenitor cells and glioblastoma tumors, respectively, as determined by the comparative expression microarray analysis (E/B&gt;2X, E-B&gt;100, p value&lt;0.05). An automated high-throughput siRNA screen was performed in 384-well plates using a reverse transfection procedures and the functional consequence were scored in 3 categories: score 1- degree of cell migration and size of tumor spheres were slightly decreased, score 2- cell migration was interrupted, and score 3-cell apoptosis was detected thereby no tumor spheres were formed. The siRNA targeting endothelin 3 served as positive siRNA control, which suppress GSC migration and induction of GSC apoptosis. Our primary screen has identified 193 genes whose inhibition by RNAi had led to impaired cell migration while knockdown of 128 genes led to the cell apoptosis and loss of self-repopulating capacity. Overlapped gene functions in regulation of both GSC migration and survival were detected, and genes which have never been reported to be associated with cell migration and cell survival were identified. Genes that maintain GSC survival and self-renewal capacity (score 3) mainly include those with roles in stemness maintenance, tumor suppressor/antigrowth/antidifferentiation, DNA repair, cell cycle checkpoint, While genes involved in GSC migration mainly include those with a role in cell adhesion, cell spreading, chemotaxis, axon guidance, neural crest cell migration. A high confidence gene list will be presented in the meeting. The development of an innovative therapy targeting essential genes of GSC that promote cell migration and/or cell survival could attenuate the infiltrating nature of GBM tumor, prolong treatment sensitivity, and prevent tumor recurrence. Citation Format: Yue Liu, Yibei Zhang, Jonathan L. Tso, Jimmy C. Menjivar, Jane Y. Tian, Linda Liau, William McBride, Cho-Lea Tso. High-throughput RNAi screening identifies genes controlling glioblastoma stem cell migration and survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3765. doi:10.1158/1538-7445.AM2013-3765

Glioblastoma remain resistant to radiotherapy (RT) and chemotherapy. To elucidate the cellular and molecular basis of glioblastoma resistance to standard treatment, we isolated and characterized cellular and molecular properties of glioblastoma stem cell clones (GC) resistance to RT (4Gy x 3) with and without 10 μM temozolomide (TMZ) (TRGC-RT, TRGC-RT+TMZ) and compared to those of treatment-sensitive GSC clones (TSGC). To explore the protective strategies which drive the super-survivability of TRGC, we performed comparative analysis of genome-wide gene expression profiles in TRGC and TSGC (n=3 patients) and have identified 56 defense genes significantly overexpressed in TRGC that mainly associated with metabolic stress, suppression of growth, differentiation and inflammation, and activation of cellular and genomic maintenance and repair networks. Functional assays confirmed the association of treatment resistance with reduced glucose uptake, fatty acid oxidation-dependent ATP maintenance, and increased AMPK activation, NAD+ levels and autophagic activity accompanied by upregulated mRNA levels of SIRT1/PGC-1α axis and DNA repair genes. The association of metabolic gene pathways and radiochemoresisatnce of TRGC were further confirmed by the treatment of TRGC with three potent inhibitors that blocking of SIRT1/SIRT2 pathway (tenovin-6), autophagy (3-MA) and fatty acid oxidation (Etomoxir). Uniquely, all three inhibitors promoted cell apoptosis in a higher degree in TRGC than that of TSGC, and the combination of inhibitors with RT further enhance treatment efficacy when compared to RT or RT+TMZ. To further explore additional gene pathways which contribute to radiochemoresistance, we performed a loss-of-function RNA interference (RNAi) screen against 56 genes and had discovered that knockdown of ribosomal proteins, RPL27A, RPS11, or RPL38, or a spliceosome-associated protein, SF3B1 alone without RT showed the significantly higher apoptosis levels by which render in to loss of tumor sphere-initiating ability. Moreover, combined siRNA therapy with RT further showed the enhancement of cell depletion, in contrast to RT or RT+TMZ which showed the return of full-grown tumor spheres. Coincidently, both RPL38 and SF3B1 have a role in repression of Hox gene expression. These data therefore support the view that treatment resistance may arise from GSC exhibiting glucose restriction, lipid catabolism-dependent and cellular quiescent phenotype and suggest that targeting metabolic pathways and stemness of TRGC may provide a novel strategy in combination with standard treatment for glioblastoma. Citation Format: yibei zhang, Yue Liu, Jimmy Christian Menjivar, Jonathan Tso, Jane Tian, William H. Yong, Linda M. Liau, William McBride, Cho-Lea Tso. Overcoming of radiochemoresistance in glioblastoma stem cells via inhibition of lipid catabolism and SIRT1 deacetylase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3727. doi:10.1158/1538-7445.AM2013-3727

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