C-Myc negatively controls the tumor suppressor PTEN by upregulating miR-26a in glioblastoma multiforme cells

The c-Myc oncogene is amplified in many tumor types. It is an important regulator of cell proliferation and has been linked to altered miRNA expression, suggesting that c-Myc-regulated miRNAs might contribute to tumor progression. Although miR-26a has been reported to be upregulated in glioblastoma multiforme (GBM), the mechanism has not been established. We have shown that ectopic expression of miR-26a influenced cell proliferation by targeting PTEN, a tumor suppressor gene that is inactivated in many common malignancies, including GBM. Our findings suggest that c-Myc modulates genes associated with oncogenesis in GBM through deregulation of miRNAs via the c-Myc–miR-26a–PTEN signaling pathway. This may be of clinical relevance.     

Hypercalcemia in glioblastoma multiforme

Hypercalcemia is commonly associated with cancer, occurring in around 10-20% of cancer patients. Hypercalcemia is usually related to solid and non-solid malignancies specifically breast cancer, lung cancer and multiple myeloma. Hypercalcemia has been reported to occur in association with astrocytomas, and uncommonly in gliomas. We report a case of a previously healthy man presenting with glioblastoma multiforme . He was found to have persistently elevated serum calcium and calcitriol with normal parathyroid function. This is the first reported case of hypercalcemia associated with glioblastoma multiforme.     

Deciphering the signaling pathways of cancer stem cells of glioblastoma multiforme: role of Akt/mTOR and MAPK pathways

These findings emphasize that the mTOR pathway may contribute to maintenance of quiescence of CSCs, and provide a basis for manipulating CSCs in the treatment of GBM. Future research should focus on further defining the PI3K/Akt/mTOR molecular network in the regulation of stem cell quiescence and provide rationale for targeting the cancer-initiating cells of GBM.     

Equine herpesvirus type 1-mediated oncolysis of human glioblastoma multiforme cells

The cytolytic animal virus equine herpesvirus type 1 (EHV-1) was evaluated for its oncolytic potential against five human glioblastoma cell lines. EHV-1 productively infected four of these cell lines, and the degree of infection was positively correlated with glioma cell death. No human major histocompatibility complex class 1 (MHC-I) was detected in the resistant glioma line, while infection of the susceptible glioma cell lines, which expressed human MHC-I, were blocked with antibody to MHC-I, indicating that human MHC-I acts as an EHV-1 entry receptor on glioma cells.     

PARP-1 protein expression in glioblastoma multiforme

One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8,000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker.     

mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells

Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase–Akt–mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes.     

Rapamycin-mediated mTOR inhibition attenuates survivin and sensitizes glioblastoma cells to radiation therapy

Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation resistance in tumors including glioblastoma multiforme. The downregulation of survivin could sensitize glioblastoma cells to radiation therapy. In this study, we investigated the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), in attenuating survivin and enhancing the therapeutic efficacy for glioblastoma cells, and elucidated the underlying mechanisms. Here we tested various concentrations of rapamycin (1-8 nM) in combination with radiation dose 4 Gy. Rapamycin effectively modulated the protein kinase B (Akt)/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, and this inhibition was further enhanced by radiation. The expression level of survivin was decreased in rapamycin pre-treatment glioblastoma cells followed by radiation; meanwhile, the phosphorylation of H2A histone family member X (H2AX) at serine-139 (γ-H2AX) was increased. p21 protein was also induced on radiation with rapamycin pre-treatment, which enhanced G1 arrest and the accumulation of cells at G0/subG1 phase. Furthermore, the clonogenic cell survival assay revealed a significant dose-dependent decrease in the surviving fraction for all three cell lines pre-treated with rapamycin. Our studies demonstrated that targeting survivin may be an effective approach for radiosensitization of malignant glioblastoma.     

Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion.     

Pathologic anatomy and CT correlations in the glioblastoma multiforme

6 cases of glioblastoma multiforme, 1 anaplastic astrocytoma, and 1 astrocytoma were studied pathologically post mortem. The specimens were compared to antemortem CT scans, when available. By use of whole brain sections, each glioblastoma was divided into a central area of necrosis, a surrounding rim of hypercellular neoplasm, and a peripheral zone of infiltration. The dimensions and areas of these three zones were quantified. The striking findings of the study were the often small diameter of the hypercellular region and the frequently narrow peripheral zone of detectable infiltrating cells. Comparison of the autopsy specimens with CT scans confirmed previous studies of the glioblastoma. The radiographic central area of low density is necrosis; the enhancing rim is a markedly hypercellular region, and the perilesional low density encompasses a population of infiltrating tumor cells.     

Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL

Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non-small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine-galactose-sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.     

The treatment of glioblastoma multiforme through activation of microglia and TRAIL induced by rAAV2-mediated IL-12 in a syngeneic rat model

Background

Microglial cells are the predominant immune cells in malignant brain tumors, but tumors may release some factors to reduce their defensive functions. Restoration of the anti-cancer function of microglia has been proposed as a treatment modality for glioblastoma. We examined the effect of intra-cranially administered recombinant adeno-associated virus encoding interleukin-12 (rAAV2/IL12) on transfection efficiency, local immune activity and survival in a rat model of glioblastoma multiforme.

Methods

F344 rats were injected with rAAV2/IL12 and implanted with syngeneic RG2 cells (glioblastoma cell line). Intracerebral interleukin-12 and interferon-γ concentrations were determined by ELISA. Activation of microglia was determined by expressions of ED1 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) which were evaluated by Western blotting and immunohistochemistry. The proliferation of cancer cells was evaluated with Ki67 immunohistochemistry and apoptosis of cancer cells with TUNEL.

Results

The brains treated with rAAV2/IL-12 maintained high expression of interleukin-12 and interferon-γ for at least two months. In syngeneic tumor model, brains treated with rAAV2/IL12 exhibited more infiltration of activated microglia cells as examined by ED1 and TRAIL stains in the tumor. In addition, the volume of tumor was markedly smaller in AAV2/IL12-treated group and the survival time was significantly longer in this group too.

Conclusion

The intra-cerebrally administered rAAV2/IL-12 efficiently induces long lasting expression of IL-12, the greater infiltration of activated microglia cells in the tumor associated improved immune reactions, resulting in the inhibited growth of implanted glioblastoma and the increased survival time of these rats.     

Implementation of a combined CDK inhibition and arginine-deprivation approach to target arginine-auxotrophic glioblastoma multiforme cells

Constitutive activation of cyclin-dependent kinases (CDKs) or arginine auxotrophy are hallmarks of Glioblastoma multiforme (GBM). The latter metabolic defect renders tumor cells vulnerable to arginine-depleting substances, such as arginine deiminase from Streptococcus pyogenes (SpyADI). Previously, we confirmed the susceptibility of patient-derived GBM cells towards SpyADI as well as CDK inhibitors (CDKis). To improve therapeutic effects, we here applied a combined approach based on SpyADI and CDKis (dinaciclib, abemaciclib). Three arginine-auxotrophic patient-derived GBM lines with different molecular characteristics were cultured in 2D and 3D and effects of this combined SpyADI/CDKi approach were analyzed in-depth. All CDKi/SpyADI combinations yielded synergistic antitumoral effects, especially when given sequentially (SEQ), i.e., CDKi in first-line and most pronounced in the 3D models. SEQ application demonstrated impaired cell proliferation, invasiveness, and viability. Mitochondrial impairment was demonstrated by increasing mitochondrial membrane potential and decreasing oxygen consumption rate and extracellular acidification rate after SpyADI/abemaciclib monotherapy or its combination regimens. The combined treatment even induced autophagy in target cells (abemaciclib/SpyADI > dinaciclib/SpyADI). By contrast, the unfolded protein response and p53/p21 induced senescence played a minor role. Transmission electron microscopy confirmed damaged mitochondria and endoplasmic reticulum together with increased vacuolization under CDKi mono- and combination therapy. SEQ-abemaciclib/SpyADI treatment suppressed the DSB repair system via NHEJ and HR, whereas SEQ-dinaciclib/SpyADI treatment increased γ-H2AX accumulation and induced Rad51/Ku80. The latter combination also activated the stress sensor GADD45 and β-catenin antagonist AXIN2 and induced expression changes of genes involved in cellular/cytoskeletal integrity. This study highlights the strong antitumoral potential of a combined arginine deprivation and CDK inhibition approach via complex effects on mitochondrial dysfunction, invasiveness as well as DNA-damage response. This provides a good starting point for further in vitro and in vivo proof-of-concept studies to move forward with this strategy.Subject terms: CNS cancer, DNA damage and repair, Cell death

The complex effects of combined CDki/SPyADI application on arginine-auxotrophic glioblastoma multiforme cells. CDki/SPyADI combination therapy impairs cell proliferation, invasiveness, gene expression, induces mitochondrial impairment, and vacuole formation. Abemaciclib-SPyADI-treatment suppresses DNA repair, dinaciclib-SpyADI-treatment enhances γ-H2AX accumulation and activates the stress sensor GADD45 and β-catenin antagonist AXIN2. Both CDKi/SpyADI combinations significantly boost cell death.     

Investigation of serum proteome alterations in human glioblastoma multiforme

Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most common and lethal adult malignant brain tumor. The present study was conducted to investigate the alterations in the serum proteome in GBM patients compared to healthy controls. Comparative proteomic analysis was performed employing classical 2DE and 2D‐DIGE combined with MALDI TOF/TOF MS and results were further validated through Western blotting and immunoturbidimetric assay. Comparison of the serum proteome of GBM and healthy subjects revealed 55 differentially expressed and statistically significant (p <0.05) protein spots. Among the identified proteins, haptoglobin, plasminogen precursor, apolipoprotein A‐1 and M, and transthyretin are very significant due to their functional consequences in glioma tumor growth and migration, and could further be studied as glioma biomarkers and grade‐specific protein signatures. Analysis of the lipoprotein pattern indicated elevated serum levels of cholesterol, triacylglycerol, and low‐density lipoproteins in GBM patients. Functional pathway analysis was performed using multiple software including ingenuity pathway analysis (IPA), protein analysis through evolutionary relationships (PANTHER), database for annotation, visualization and integrated discovery (DAVID), and GeneSpring to investigate the biological context of the identified proteins, which revealed the association of candidate proteins in a few essential physiological pathways such as intrinsic prothrombin activation pathway, plasminogen activating cascade, coagulation system, glioma invasiveness signaling, and PI3K signaling in B lymphocytes. A subset of the differentially expressed proteins was applied to build statistical sample class prediction models for discrimination of GBM patients and healthy controls employing partial least squares discriminant analysis (PLS‐DA) and other machine learning methods such as support vector machine (SVM), Decision Tree and Naïve Bayes, and excellent discrimination between GBM and control groups was accomplished.