Glioblastoma Multiforme Tumor Cells Research Articles

ET-34 * HIGHLY SELECTIVE HSV VIROTHERAPY FOR TREATMENT OF GLIOBLASTOMA

Glioblastoma Multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSV) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals. Although proven safe for treatment of GBM in patients, oHSVs efficacy has been limited, a consequence of poor intra-tumoral virus replication and spread. To counter these limitations, we have developed oHSVs whose selective replication in GBM cells does not rely on defective genes. This was accomplished by (i) full retargeting of oHSV to utilize the epidermal growth factor receptor (EGFR) for infection of human GBM tumor cells and (ii) further vector engineering to modify the essential HSV immediate early gene (ICP4) for sensitivity to repression by the microRNA mir-124. Mir-124 is highly expressed in neurons but virtually absent in GBM and highly conserved among different species. The mir-124-regulated vector was unable to replicate in nude mice following intracranial inoculation supporting vector safety and was shown to be effective (50-60% long term survival) in the treatment of human GBM in nude mice. To enhance vector intra-tumor vector spread, our EGFR retargeted-mir-124 controlled vector was further modified by vector arming with the matrix metalloproteinase gene encoding MMP9. MMP9 degrades collagen type IV, a major component of the extracellular matrix (ECM) and basement membranes of glioblastomas but absent in normal brain tissue. Studies are ongoing to determine whether MMP9 expression enhances vector spread in GBM neurospheres and as a therapeutic agent for enhanced treatment of human GBM in animals.

CD44-mediated adhesion to hyaluronic acid contributes to mechanosensing and invasive motility.

The high-molecular-weight glycosaminoglycan, hyaluronic acid (HA), makes up a significant portion of the brain extracellular matrix. Glioblastoma multiforme (GBM), a highly invasive brain tumor, is associated with aberrant HA secretion, tissue stiffening, and overexpression of the HA receptor CD44. Here, transcriptomic analysis, engineered materials, and measurements of adhesion, migration, and invasion were used to investigate how HA/CD44 ligation contributes to the mechanosensing and invasive motility of GBM tumor cells, both intrinsically and in the context of Arg-Gly-Asp (RGD) peptide/integrin adhesion. Analysis of transcriptomic data from The Cancer Genome Atlas reveals upregulation of transcripts associated with HA/CD44 adhesion. CD44 suppression in culture reduces cell adhesion to HA on short time scales (0.5-hour postincubation) even if RGD is present, whereas maximal adhesion on longer time scales (3 hours) requires both CD44 and integrins. Moreover, time-lapse imaging demonstrates that cell adhesive structures formed during migration on bare HA matrices are more short lived than cellular protrusions formed on surfaces containing RGD. Interestingly, adhesion and migration speed were dependent on HA hydrogel stiffness, implying that CD44-based signaling is intrinsically mechanosensitive. Finally, CD44 expression paired with an HA-rich microenvironment maximized three-dimensional invasion, whereas CD44 suppression or abundant integrin-based adhesion limited it. These findings demonstrate that CD44 transduces HA-based stiffness cues, temporally precedes integrin-based adhesion maturation, and facilitates invasion. This study reveals that the CD44 receptor, which is commonly overexpressed in GBM tumors, is critical for cell adhesion, invasion, and mechanosensing of an HA-based matrix.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Live attenuated measles virus vaccine therapy for locally established malignant glioblastoma tumor cells.

Glioblastoma multiforme is the most aggressive malignant primary brain tumor in humans, with poor prognosis. A new glioblastoma cell line (ANGM5) was established from a cerebral glioblastoma multiforme in a 72-year-old Iraqi man who underwent surgery for an intracranial tumor. This study was carried out to evaluate the antitumor effect of live attenuated measles virus (MV) Schwarz vaccine strain on glioblastoma multiforme tumor cell lines in vitro. Live attenuated MV Schwarz strain was propagated on Vero, human rhabdomyosarcoma, and human glioblastoma-multiform (ANGM5) cell lines. The infected confluent monolayer appeared to be covered with syncytia with granulation and vacuolation, as well as cell rounding, shrinkage, and large empty space with cell debris as a result of cell lysis and death. Cell lines infected with virus have the ability for hemadsorption to human red blood cells after 72 hours of infection, whereas no hemadsorption of uninfected cells is seen. Detection of MV hemagglutinin protein by monoclonal antibodies in infected cells of all cell lines by immunocytochemistry assay gave positive results (brown color) in the cytoplasm of infected cells. Cell viability was measured after 72 hours of infection by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results showed a significant cytotoxic effect for MV (P≤0.05) on growth of ANGM5 and rhabdomyosarcoma cell lines after 72 hours of infection. Induction of apoptosis by MV was assessed by measuring mitochondrial membrane potentials in tumor cells after 48, 72, and 120 hours of infection. Apoptotic cells were counted, and the mean percentage of dead cells was significantly higher after 48, 72, and 120 hours of infection compared with control cells. This study concludes that live attenuated MV Schwarz vaccine induces the oncolytic effect in Iraqi tumor cell line ANGM5 and in the rhabdomyosarcoma cell line through syncytia in tumor cells, which is one of the causes of cell death. The MV vaccine strain has the ability to insert its hemagglutinin protein into the tumor cell surface, leading to modification of the antigenic surface of tumor cells that may induce an antitumor immune response, MV vaccine strain induced cell killing by direct cytolysis and apoptosis induction. These antitumor features may indicate the use of MV in the treatment of glioblastoma.

Targeting endocannabinoid signaling in tumor-associated macrophages as treatment for glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common form of primary brain tumor and is diagnosed in approximately 15,000 people each year in the United States alone. No cure for this type of cancer exists, and the current standard of care treatments provide little benefit and are associated with debilitating side effects. Recent evidence shows that a large number of tumor-associated macrophages (TAMs) invade the GBM tumor mass and secrete factors that directly and indirectly promote tumor growth. TAMs express a panel of unique receptors that could be targeted for therapeutic benefit. One such receptor, cannabinoid receptor 2 (CB2), is a member of the endocannabinoid (eCB) signaling system, and its activation has been shown to tightly control the migration and phenotype of both macrophages and microglia. Additional receptors, also engaged by eCBs and cannabinoid-like compounds, are expressed by macrophages and microglia. These receptors also control cell migration and phenotype, but exhibit distinct pharmacological profiles and operate through a different mechanism of action. Strong evidence accumulated over the past decade indicates that membrane receptors expressed by TAMs represent novel targeting opportunities to treat GBM tumor progression. Here we review studies that significantly increased our understanding of the molecular mechanism of action of receptors engaged by eCBs and cannabinoid-like compounds expressed by GBM tumor cells and TAMs. This evidence provides a strong rationale for developing new therapeutics that target the eCB signaling of TAMs for the treatment of GBM while minimizing the typical side effects associated with standard care. WIREs Membr Transp Signal 2014,3:39–51. doi: 10.1002/wmts.101 Conflict of interest: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.

CD44-Based Adhesion and Mechanotransductive Signaling on Engineered Hyaluronic Acid Matrices

Glioblastoma multiforme (GBM) is the most clinically aggressive primary brain tumor and is characterized by diffuse infiltration of glioma cells into brain parenchyma, which is rich in the glycosaminoglycan hyaluronic acid (HA). The extracellular matrix of GBM tumors is associated with aberrant HA secretion, overexpression of the HA receptor CD44, and tissue stiffening. Here we combine matrix engineering and genetic manipulation of GBM tumor cells to determine the role of HA/CD44 adhesion in the outputs of cell adhesion, migration, and invasion. We show that HA/CD44 adhesion contributes to the mechanosensing and invasive motility of GBM tumor cells, both intrinsically and in the context of RGD/integrin adhesion. We also find that three-dimensional cell invasion is maximized in an HA-rich environment, but reduction of CD44-based adhesion or addition of integrin-adhesive peptides to the matrix suppresses invasion. While comparatively much is known about cell-matrix mechanosensing through integrins and the role it may play in tumor progression, our findings reveal a previously under-appreciated role of CD44 and possibly other HA-specific receptors in this process. These findings have broad implications for the field's fundamental understanding of how cancer cells interact with the tumor microenvironment and suggest new therapeutic strategies.

Identification of Novel Human Leukocyte Antigen-A*0201-Restricted, Cytotoxic T Lymphocyte Epitopes on CD133 for Cancer Stem Cell Immunotherapy

Targeting cancer stem cells (CSCs) with immunotherapy may be an effective means to prevent recurrences in glioblastoma multiforme (GBM). It is well established that CD133 is expressed in the population of GBM tumor cells representing CSCs. This raises a possibility that CD133 could serve as a potential target for cytotoxic T cells (CTLs) to target glioblastoma cancer stem cells. Two potential human leukocyte antigen (HLA)-A*0201-restricted CD133 epitopes, ILSAFSVYV (CD133-405) and YLQWIEFSI (CD133-753), showed strong binding to HLA-A*0201 molecules. In vitro immunogenicity studies generated peptide-specific CD8(+) CTLs from normal donors. Autologous monocyte-derived dendritic cells pulsed with the CD133-405 or CD133-753 peptides generated CTLs that efficiently recognized the CD133 epitopes presented in T2 HLA-A*0201 cells and specifically lysed CD133+ HLA-A*0201(+) GBM CSCs. These studies demonstrated natural processing and subsequent presentation of these epitopes in GBM CSCs and the ability of CTLs to kill CSCs bearing the antigen. Immunization studies in mice using the mouse homolog CD133 epitopes demonstrated immunogenicity in the absence of autoimmune damage. The results presented in this study support the use of CD133-specific epitope vaccines to target CSCs in glioblastoma and other cancers.

MiR-124 inhibits the growth of glioblastoma through the downregulation of SOS1

Glioblastoma multiforme (GBM) is a lethal brain tumor in adults. Despite advances in treatments, such as surgery, radiotherapy and chemotherapy, high‑grade glioma remains fatal. The molecular and cellular mechanisms for GBM are not entirely clear and further studies are required to elucidate these. MicroRNAs (miRNAs) are small, non‑coding, endogenous RNAs that are involved in cell differentiation and proliferation, and have been suggested to play a role in a variety of types of cancer. In this study, we investigated the role of miR-124 in the inhibition of proliferation of GBM cells. The downregulation of miR-124 in human GBM tumor cell lines was detected using quantitative RT-PCR. To assess the function of miR-124, we constructed stable cell lines, U87-124 and U373-124, which overexpressed miR-124 using lentiviral vectors. Overexpression of miR-124 inhibited the proliferation of GBM cancer cells in vitro. Using integrated bioinformatics analysis, SOS1 was found to be a direct target for miR-124, which is frequently upregulated in gliomas. Dual‑luciferase reporter assays confirmed that the SOS1 mRNA 3'‑untranslated regions (UTR) was directly targeted by miR-124 and that the mutated 3'UTR was not affected. This was revealed to be mechanistically associated with the induction of SOS/Ras/Raf/ERK and the suppression of ERK activity, which was achieved by silencing SOS1. This study therefore indicates an important role for miR-124 in the regulation of growth in the molecular etiology of GBM, and offers a potential strategy for the use of miR-124 in cancer treatment.

Abstract 2493: Identification of MGMT-binding proteins involved in the negative regulation of angiogenesis and invasion.

Abstract Background: Glioblastoma multiforme (GBM) is the most frequent and most aggressive form of primary malignant brain tumors in adults. The dismal prognosis of GBM patients stems from the highly angiogenic and invasive behavior of GBM tumor cells. O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein ubiquitously expressed in normal tissues has been extensively characterized for its role in resistance to alkylating agents used in GBM treatment. We reported for the first time an inverse relationship between expression of MGMT and the angiogenic and invasive profile of GBM cell lines. The mechanisms by which MGMT affects angiogenesis and invasion are unknown. We hypothesized that interactions of MGMT with binding proteins (BPs) may account for additional functions beyond its known role as a DNA repair protein. Methods: As a first screening to identify MGMT-BPs with a functional relevance for invasion and angiogenesis, we performed affinity purification of MGMT-BPs following overexpression of FLAG-tagged MGMT and mass spectrometry analysis using 293T-Flag/MGMT and control Flag-tagged empty vector (293T-Flag/EV). Lysates were subjected to affinity purification using an anti-Flag monoclonal antibody covalently attached to agarose resin. The affinity bound FLAG fusion proteins were eluted and separated on SDS-PAGE. Coomassie Blue staining enabled the identification of 6 bands including Flag-MGMT in 293T-Flag/MGMT but not the Flag/EV control. The bands were excised from the gel, subjected to trypsin digestion and identified by liquid chromatography-tandem mass spectrometry. The resultant MS/MS spectra were searched against a proteome database for peptide matching and protein identification. Proteins were identified with high confidence using Scaffold software. Results: Our analysis provided evidence for binding of MGMT to 120 BPs. Using gene ontology (GO) database to search for functional categories, we identified proteins involved in DNA repair, ubiquitin pathway, DNA replication and transcription, RNA metabolism and processing, cell cycle and division, response to stress and cell death. Importantly, we identified proteins involved in cell motility and/or angiogenesis, cytoskeletal-related proteins (15 proteins), small GTPases family and their regulators (10 proteins, such as Rho guanine nucleotide exchange factor 2) and two proteins involved in angiogenesis (Endoribonuclease Dicer and Ribonuclease inhibitor). We also used T98G a human GBM cell line with constitutive expression of MGMT to perform immunoprecipitation of endogenous MGMT (anti-MGMT antibody or the IgG1 isotype control). Mass spectrometry and proteomic analysis of MGMT-BPs in T98G is underway. Conclusion: Our data provide new structural aspects of MGMT and shed light into the multifaceted role of MGMT, which may lead to the identification of novel therapeutic targets in GBM. Citation Format: Siham Sabri, Yaoxian Xu, Nicolas Stifani, Bassam S. Abdulkarim. Identification of MGMT-binding proteins involved in the negative regulation of angiogenesis and invasion. [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 2493. doi:10.1158/1538-7445.AM2013-2493

Treatment of brain glioblastoma multiforme with pcDNA3.1-Egr. 1p-p16 combined with gamma knife radiation: An experimental study on nude mice

High post-operative recurrence and poor prognosis are likely to be related to the infiltrative growth of the glioblastoma multiforme (GBM). The primary objective of this study is to investigate the possible synergistic effect of the combined treatment of gamma knife radio-surgery (GKRS) and gene therapy for GBM and secondary objective is to explore the role of GKRS for the temporal and spatial regulation of the gene expression. The study performed on 70 nude mice and randomly divided into seven groups. Subcutaneous injection of human GBM tumor cells (T98G) was carried out to establish the animal models. Various doses of liposome-mediated pcDNA3.1-Egr. 1p-p16 recombinant plasmid were transfected through intra-tumor injection. GKRS was scheduled following the plasmid transfection. Tumor volumes were measured every 4 days after the treatment. Subcutaneous tumor nodule specimens were collected to analyze the cell apoptosis and p16 gene expression using terminal-deoxynucleoitidyl transferase mediated nick end labeling staining and reverse transcription-polymerase chain reaction. Tumor volumes, levels of cell apoptosis and p16 gene expression were compared between groups. Rates of tumor growth were significantly lower in the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS groups than that in the remaining groups 28 days following the GKRS management. The p16mRNA expression was noted in both of the pcDNA3.1-Egr. 1p-p16 plasmid group and the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS with marginal-dose of 20 Gy group. The level of messenger ribonucleic acid expression was higher in the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS with the marginal-dose of 20 Gy group, with a markedly increased apoptotic and necrotic cells, than that in the pcDNA3.1-Egr. 1p-p16 plasmid group. In animal studies, pcDNA3.1-Egr. 1p-p16 in combination with GKRS is a preferable management option for the GBM to the sole use of GKRS or gene therapy. It may be a novel approach for the treatment of human patient with GBM.

Vaccination with Irradiated Tumor Cells Pulsed with an Adjuvant That Stimulates NKT Cells Is an Effective Treatment for Glioma

The prognosis for patients with glioblastoma multiforme (GBM) remains extremely poor despite recent treatment advances. There is an urgent need to develop novel therapies for this disease. We used the implantable GL261 murine glioma model to investigate the therapeutic potential of a vaccine consisting of intravenous injection of irradiated whole tumor cells pulsed with the immuno-adjuvant α-galactosylceramide (α-GalCer). Vaccine treatment alone was highly effective in a prophylactic setting. In a more stringent therapeutic setting, administration of one dose of vaccine combined with depletion of regulatory T cells (Treg) resulted in 43% long-term survival and the disappearance of mass lesions detected by MRI. Mechanistically, the α-GalCer component was shown to act by stimulating "invariant" natural killer-like T cells (iNKT cells) in a CD1d-restricted manner, which in turn supported the development of a CD4(+) T-cell-mediated adaptive immune response. Pulsing α-GalCer onto tumor cells avoided the profound iNKT cell anergy induced by free α-GalCer. To investigate the potential for clinical application of this vaccine, the number and function of iNKT cells was assessed in patients with GBM and shown to be similar to age-matched healthy volunteers. Furthermore, irradiated GBM tumor cells pulsed with α-GalCer were able to stimulate iNKT cells and augment a T-cell response in vitro. Injection of irradiated tumor cells loaded with α-GalCer is a simple procedure that could provide effective immunotherapy for patients with high-grade glioma.

O(6)-Methylguanine-DNA Methyltransferase Is a Novel Negative Effector of Invasion in Glioblastoma Multiforme

The dismal prognosis of glioblastoma multiforme (GBM) is mostly due to the high propensity of GBM tumor cells to invade. We reported an inverse relationship between GBM angiogenicity and expression of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), which has been extensively characterized for its role in resistance to alkylating agents used in GBM treatment. In the present study, given the major role of angiogenesis and invasion in GBM aggressiveness, we aimed to investigate the relationship between MGMT expression and GBM invasion. Stable overexpression of MGMT in the U87MG cell line significantly decreased invasion, altered expression of invasion-related genes, decreased expression of α(5)β(1) integrin and focal adhesion kinase, and reduced their spindle-shaped morphology and migration compared with the empty vector control. Conversely, short hairpin RNA-mediated stable knockdown of MGMT or its pharmacologic depletion in the MGMT-positive T98G cell line were required for increased invasion. The inverse relationship between MGMT and invasion was further validated in primary GBM patient-derived cell lines. Using paraffin-embedded tumors from patients with newly diagnosed GBM (n = 59), tumor MGMT promoter hypermethylation (MGMT gene silencing) was significantly associated with increased immunohistochemical expression of the proinvasive matricellular protein secreted protein acidic and rich in cysteine (SPARC; P = 0.039, χ(2) test). Taken together, our findings highlight for the first time the role of MGMT as a negative effector of GBM invasion. Future studies are warranted to elucidate the role of SPARC in the molecular mechanisms underlying the inverse relationship between MGMT and GBM invasion and the potential use of MGMT and SPARC as biomarkers of GBM invasion.

Real-time dynamic and sequential tracking of tumor propagation and associated immune responses in the CNS microenvironment.

9520 Background: Ex vivo experimental systems are often unable to fully capture complex intercellular communication between tumor cells and surrounding tissues - a critical feature in understanding cancer development and immune evasion. Imaging modality such as bioluminescence lacks the resolution necessary to discern subtle structural differences and heterogeneity in the tumor niche. Microscopic examination of fixed specimens is devoid of the 3-dimensional context or evolution of tumor progression within the same host. Methods: New insights have come from studies involving the use of intravital 2-photon laser scanning microscopy (2P-LSM), which allows deep visualization (>300um) with single-cell resolution (<1um), thus enables direct observation of cellular behavior in intact tissues at a suitable dynamic spatial-time resolution. We study the role of tumor niche in shaping immune repertoire and develop strategies to modify tumor niche to enhance anti-tumor immunity. Results: One example is our study of glioblastoma multiforme (GBM), which contains a cellular hierarchy with a CD133+ sub-population representing self-renewing and tumorigenic GBM stem cells (GSCs). In a xeno-transplant model, GSC was capable of tumor initiation in the mouse brain. To directly test the relative tumorigenic potential of GSCs (CD133+) and non-GSCs (CD133-), we inoculated paired tumor populations from the same primary GBM tumor cells and monitored tumor competition by serial 2P-LSM through implanted cranial windows. Serial 2P-LSM imaging shows that after 35 days, GBM formation was driven exclusively by GSCs but not non-GSCs. To interrogate tumor-associated immune responses, we inoculated syngeneic mouse glioma tumors into C57BL/6 mice. Using this and CNS-inflammatory models, we have begun to undercover the role of perivascular antigen-presenting cells and microglia in guiding the recruitment of CNS-bound lymphocytes. Conclusions: Our data provide the first direct functional evidence that CSCs are responsible for tumor propagation in GBM, and represent an in vivo experimental platform to monitor immunotherapeutic interventions.

Protein tyrosine phosphatase mu regulates glioblastoma cell growth and survival in vivo

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor. Extensive proliferation and dispersal of GBM tumor cells within the brain limits patient survival to approximately 1 year. Hence, there is a great need for the development of better means to treat GBM. Receptor protein tyrosine phosphatase (PTP)µ is proteolytically cleaved in GBM to yield fragments that promote dispersal of GBM cells. While normal brain tissue retains expression of full-length PTPµ, low-grade human astrocytoma samples have varying amounts of full-length PTPµ and cleaved PTPµ. In the highest-grade astrocytomas (i.e., GBM), PTPµ is completely proteolyzed into fragments. We demonstrate that short hairpin RNA mediated knockdown of full-length PTPµ and PTPµ fragments reduces glioma cell growth and survival in vitro. The reduction in growth and survival following PTPµ knockdown is enhanced when cells are grown in the absence of serum, suggesting that PTPµ may regulate autocrine signaling. Furthermore, we show for the first time that reduction of PTPµ protein expression decreases the growth and survival of glioma cells in vivo using mouse xenograft flank and i.c. tumor models. Inhibitors of PTPµ could be used to reduce the growth and survival of GBM cells in the brain, representing a promising therapeutic target for GBM.

Protein Phosphatase 2A Mediates Dormancy of Glioblastoma Multiforme-Derived Tumor Stem-Like Cells during Hypoxia

PurposeThe hypoxic microenvironment of glioblastoma multiforme (GBM) is thought to increase resistance to cancer therapies. Recent evidence suggests that hypoxia induces protein phosphatase 2A (PP2A), a regulator of cell cycle and cell death. The effects of PP2A on GBM tumor cell proliferation and survival during hypoxic conditions have not been studied.Experimental DesignExpression of PP2A subunits and HIF-α proteins was measured in 65 high-grade astrocytoma and 18 non-neoplastic surgical brain specimens by western blotting. PP2A activity was measured by an immunoprecipitation assay. For in vitro experiments, GBM-derived tumor stem cell-like cells (TSCs) were exposed to severe hypoxia produced by either CoCl2 or 1% O2. PP2A activity was inhibited either by okadaic acid or by shRNA depletion of the PP2A C subunit. Effects of PP2A activity on cell cycle progression and cell survival during hypoxic conditions were assessed using flow cytometry.ResultsIn our patient cohort, PP2A activity was positively correlated with HIF-1∝ protein expression (P = 0.002). Patients with PP2A activity levels above 160 pMP had significantly worse survival compared to patients with levels below this threshold (P = 0.002). PP2A activity was an independent predictor of survival on multivariable analysis (P = 0.009). In our in vitro experiments, we confirmed that severe hypoxia induces PP2A activity in TSCs 6 hours after onset of exposure. PP2A activity mediated G1/S phase growth inhibition and reduced cellular ATP consumption in hypoxic TSCs. Conversely, inhibition of PP2A activity led to increased cell proliferation, exhaustion of intracellular ATP, and accelerated P53-independent cell death of hypoxic TSCs.ConclusionsOur results suggest that PP2A activity predicts poor survival in GBM. PP2A appears to reduce the metabolic demand of hypoxic TSCs and enhances tumor cell survival. Modulation of PP2A may be a potential target for cancer therapy.

Abstract A105: A small RNA interference screen of genes involved in DNA repair identifies genes mediating radiosensitivity in glioblastoma.

Abstract Purpose: Glioblastoma multiforme (GBM) is the most common primary brain tumor in the USA. Standard treatment for primary GBM includes surgical resection, followed by concurrent chemotherapy and radiotherapy (RT). As a result of the major role RT plays in the treatment of GBM, there is much interest in identifying the determinants of radiosensitivity to improve the efficacy of clinically tolerable radiation doses. Also, presence of GBM stem cells within tumor makes it remarkably resistant to current radio- and chemotherapies. Here we have studied DNA repair genes as a determinant of radiosensitivity in GBM. Materials and Methods: We performed a small interfering RNA (siRNA) screen of 206 genes involved in DNA damage repair to identify genes whose knockdown can increase tumor sensitivity to RT. siRNA screens were performed in two different GBM tumor cells; U251 and U87-MG and in one GBM stem cell line, GBAM1. At 72 hrs, after transfection, cells were irradiated for 24 hrs. As a marker of DNA damage and repair, γH2AX activity was analyzed by ELISA after 24 hrs in unirradiated and irradiated cells. Results: We identified several genes which upon downregulation increased radiosenstiivity of GBM tumor cells. The various genes includes DNA double strand break repair genes, genes involved in translesion DNA synthesis and DNA damage signaling genes. Nevertheless, we observed differences between GBM tumor cells and stem cells. In tumor cells, some of the radiosensitizer genes were BRCA2, PHB, PTCH, SMC5, ERCC5 and POL G. On the other hand, GBM stem cells showed radiosensitization with downregulation of RFC1, UBE2A, RPA1 RAD54L, XPA, FANCL and TOP2A genes. There was one gene common between GBM tumor cells and stem cells, REV1L. Rev1-like protein is as a scaffold that recruits DNA polymerases involved in translesion DNA synthesis (TLS) of damaged DNA. This is an ongoing study. We are further validating role of some of these targets in both tumor cells and stem cells. Conclusions: We observed inherent differences in radiosensitivity between GBM tumor cells and stem cells. Identification of tumor cell specific and stem cell specific targets may help us in determining better treatment options for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A105.

Acquired MET expression confers resistance to EGFR inhibition in a mouse model of glioblastoma multiforme.

Glioblastoma Multiforme (GBM) is an aggressive brain tumor for which there is no cure. Overexpression of wild-type EGFR and loss of the tumor suppressor genes Ink4a/Arf and PTEN are salient features of this deadly cancer. Surprisingly, targeted inhibition of EGFR has been clinically disappointing, demonstrating an innate ability for GBM to develop resistance. Efforts at modeling GBM in mice using wild-type EGFR have proven unsuccessful to date, hampering endeavors at understanding molecular mechanisms of therapeutic resistance. Here, we describe a unique genetically engineered mouse model of EGFR-driven gliomagenesis that uses a somatic conditional overexpression and chronic activation of wild-type EGFR in cooperation with deletions in the Ink4a/Arf and PTEN genes in adult brains. Using this model, we establish that chronic activation of wild-type EGFR with a ligand is necessary for generating tumors with histopathological and molecular characteristics of GBMs. We show that these GBMs are resistant to EGFR kinase inhibition and we define this resistance molecularly. Inhibition of EGFR kinase activity using tyrosine kinase inhibitors in GBM tumor cells generates a cytostatic response characterized by a cell cycle arrest, which is accompanied by a substantial change in global gene expression levels. We demonstrate that a key component of this pattern is the transcriptional activation of the MET receptor tyrosine kinase and that pharmacological inhibition of MET overcomes the resistance to EGFR inhibition in these cells. These findings provide important new insights into mechanisms of resistance to EGFR inhibition and suggest that inhibition of multiple targets will be necessary to provide therapeutic benefit for GBM patients.

Targeting the AKT Pathway in Glioblastoma

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. The treatment options for patients diagnosed with GBM are limited and the current median survival is 14-16 months following diagnosis. Genetic mutations have been identified that act as drivers of GBM growth and these should be considered as a basis for identifying novel therapeutic strategies. AKT is a downstream serine/threonine kinase in the RTK/PTEN/PI3K pathway and large scale genomic analysis of GBM has demonstrated that this pathway is mutated in the majority of GBMs. This RTK/PTEN/PI3K pathway leads to activated AKT and phospho-AKT levels are elevated in the majority of GBM tumor samples and cell lines, which studies show help glioma cells grow uncontrolled, evade apoptosis, and enhance tumor invasion. AKT represents a nodal point in this pathway which allows for amplification of growth signals, thereby making inhibition of AKT an attractive target for GBM therapy. Many different classes of AKT inhibitors exist, however, few have been tested sufficiently to demonstrate in vivo efficacy. This article will summarize the key components of the Akt pathway with special attention to gliomas, the genetic alterations driving this pathway in gliomas, and the studies evaluating inhibitors of this pathway. Inhibitors of the Akt pathway represent a potential treatment option against GBM and additional research efforts are required to fully explore and develop this possible treatment strategy.

CTL recognition of a novel HLA-A*0201-binding peptide derived from glioblastoma multiforme tumor cells.

Genetic instability of tumor cells can result in translation of proteins that are out of frame, resulting in expression of neopeptides. These neopeptides are not self-proteins and therefore should be immunogenic. By eluting peptides from human glioblastoma multiforme (GBM) tumor cell surfaces and subjecting them to tandem mass spectrometry, we identified a novel peptide (KLWGLTPKVTPS) corresponding to a frameshift in the 3' beta-hydroxysteroid dehydrogenase type 7 (HSD3B7) gene. HLA-binding algorithms predicted that a 9-amino acid sequence embedded in this peptide would bind to HLA-A*0201. We confirmed this prediction using an HLA-A*0201 refolding assay followed by live cell relative affinity assays, but also showed that the 12-mer binds to HLA-A*0201. Based on the 9-mer sequence, optimized peptide ligands (OPL) were designed and tested for their affinities to HLA-A*0201 and their abilities to elicit anti-peptide and CTL capable of killing GBM in vitro. Wild-type peptides as well as OPL induced anti-peptide CTL as measured by IFN-γ ELISPOTS. These CTL also killed GBM tumor cells in chromium-51 release assays. This study reports a new CTL target in GBM and further substantiates the concept that rational design and testing of multiple peptides for the same T-cell epitope elicits a broader response among different individuals than single peptide immunization.

Oncolytic Vesicular Stomatitis Virus Induces Apoptosis in U87 Glioblastoma Cells by a Type II Death Receptor Mechanism and Induces Cell Death and Tumor Clearance In Vivo

Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-X(L) had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-X(L) overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-X(L) regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-X(L)-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-X(L) overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.