The relevance of cysteine metabolism in cancer has gained considerable interest in recent years, largely focusing on its role in generating the antioxidant glutathione. Through metabolomic profiling ...using a combination of high-throughput liquid and gas chromatography-based mass spectrometry on a total of 69 patient-derived glioma specimens, this report documents the discovery of a parallel pathway involving cysteine catabolism that results in the accumulation of cysteine sulfinic acid (CSA) in glioblastoma. These studies identified CSA to rank as one of the top metabolites differentiating glioblastoma from low-grade glioma. There was strong intratumoral concordance of CSA levels with expression of its biosynthetic enzyme cysteine dioxygenase 1 (CDO1). Studies designed to determine the biologic consequence of this metabolic pathway identified its capacity to inhibit oxidative phosphorylation in glioblastoma cells, which was determined by decreased cellular respiration, decreased ATP production, and increased mitochondrial membrane potential following pathway activation. CSA-induced attenuation of oxidative phosphorylation was attributed to inhibition of the regulatory enzyme pyruvate dehydrogenase. Studies performed in vivo abrogating the CDO1/CSA axis using a lentiviral-mediated short hairpin RNA approach resulted in significant tumor growth inhibition in a glioblastoma mouse model, supporting the potential for this metabolic pathway to serve as a therapeutic target. Collectively, we identified a novel, targetable metabolic pathway involving cysteine catabolism contributing to the growth of aggressive high-grade gliomas. These findings serve as a framework for future investigations designed to more comprehensively determine the clinical application of this metabolic pathway and its contributory role in tumorigenesis.
Although considerable progress has been made toward understanding glioblastoma biology through large-scale genetic and protein expression analyses, little is known about the underlying metabolic ...alterations promoting their aggressive phenotype. We conducted global metabolomic profiling on patient-derived glioma specimens and identified specific metabolic programs differentiating low- and high-grade tumors, with the metabolic signature of glioblastoma reflecting accelerated anabolic metabolism. When coupled with transcriptional profiles, we identified the metabolic phenotype of the mesenchymal subtype to consist of accumulation of the glycolytic intermediate phosphoenolpyruvate and decreased pyruvate kinase activity. Unbiased hierarchical clustering of metabolomic profiles identified three subclasses, which we term energetic, anabolic, and phospholipid catabolism with prognostic relevance. These studies represent the first global metabolomic profiling of glioma, offering a previously undescribed window into their metabolic heterogeneity, and provide the requisite framework for strategies designed to target metabolism in this rapidly fatal malignancy.
Rapidly growing tumors require efficient means to allow them to adapt to fluctuating microenvironments consisting of hypoxia, nutrient deprivation, and acidosis. The unfolded protein response (UPR) ...represents a defense mechanism allowing cells to respond to these adverse conditions. The chaperone protein GRP78 serves as a master UPR regulator that is aberrantly expressed in a variety of cancers, including glioma. Therefore, cancer cells may be particularly reliant upon the adaptive mechanisms offered by the UPR and targeting GRP78 may represent a unique therapeutic strategy. Here we report that diffuse expression of GRP78 protein is present in Grade III-IV, but not Grade I-II glioma. To determine the role GRP78 plays in glioblastoma tumorigenesis, we explored the anti-tumor activity of the novel fusion protein EGF-SubA, which combines EGF with the cytotoxin SubA that has been recently shown to selectively cleave GRP78. EGF-SubA demonstrated potent tumor-specific proteolytic activity and cytotoxicity in glioblastoma lines and potentiated the anti-tumor activity of both temozolomide and ionizing radiation. To determine if the tumor microenvironment influences EGF-SubA activity, we maintained cells in acidic conditions that led to both UPR activation and increased EGF-SubA induced cytotoxicity. EGF-SubA was well tolerated in mice and led to a significant tumor growth delay in a glioma xenograft mouse model. The UPR is emerging as an important adaptive pathway contributing to glioma tumorigenesis. Targeting its primary mediator, the chaperone protein GRP78, through specific, proteolytic cleavage with the immunotoxin EGF-SubA represents a novel and promising multi-targeted approach to cancer therapy.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Histone deacetylase (HDAC) inhibitors represent an emerging class of anticancer agents progressing through clinical trials. Although their primary target is thought to involve acetylation of core ...histones, several nonhistone substrates have been identified, including heat shock protein (HSP) 90, which may contribute towards their antitumor activity. Glucose-regulated protein 78 (GRP78) is a member of the HSP family of molecular chaperones and plays a central role in regulating the unfolded protein response (UPR). Emerging data suggest that GRP78 is critical in cellular adaptation and survival associated with oncogenesis and may serve as a cancer-specific therapeutic target. On the basis of shared homology with HSP family proteins, we sought to determine whether GRP78 could serve as a molecular target of the HDAC inhibitor vorinostat. Vorinostat treatment led to GRP78 acetylation, dissociation, and subsequent activation of its client protein double-stranded RNA-activated protein-like endoplasmic reticulum kinase (PERK). Investigations in a panel of cancer cell lines identified that UPR activation after vorinostat exposure is specific to certain lines. Mass spectrometry performed on immunoprecipitated GRP78 identified lysine-585 as a specific vorinostat-induced acetylation site of GRP78. Downstream activation of the UPR was confirmed, including eukaryotic initiating factor 2α phosphorylation and increase in ATF4 and C/EBP homologous protein expression. To determine the biologic relevance of UPR activation after vorinostat, RNA interference of PERK was performed, demonstrating significantly decreased sensitivity to vorinostat-induced cytotoxicity. Collectively, these findings indicate that GRP78 is a biologic target of vorinostat, and activation of the UPR through PERK phosphorylation contributes toward its antitumor activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Azurin, a member of a family of copper-containing proteins involved in electron transfer called cupredoxins, demonstrates structural features similar to the variable domains of the immunoglobulin ...superfamily members and to various mammalian cellsurface receptors or extracellular domains of intercellular adhesion molecules. An azurin-like protein called Laz with an additional N-terminal 39 amino acid peptide known as H.8 epitope is present on the surface of gonnococci and meningococci.We demonstrate that azurin, Laz and H.8-azurin can bind with the C-terminal cleavage product MSP1-19 of merozoite surface protein 1 (MSP1) of the malarial parasite Plasmodium falciparum and significantly reduce parasitemia. Azurin and Laz alsobound strongly to HIV-1 gp120. Interestingly, azurin could not only bind to gp120 but also to the intercellular adhesion molecule ICAM-3 and the CD4 receptors of T cells, mimicking the functionality of DC-SIGN with which it also binds avidly. Furthermore,these three proteins significantly suppressed HIV-1 growth in peripheral blood mononuclear cells and such suppression appeared to be occurring at an entry stage in the infection process. The presence of both antimalarial and antiretroviral activityin azurin, H.8-azurin and Laz makes these proteins, or peptides derived from them, potential therapeutic agents in the treatment of malaria, HIV-1 infections or co-infections with both P. falciparum and HIV-1.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Post‐translational modification through protein acetylation is emerging as an important mode of cellular regulation. We have previously demonstrated the role that glucose‐regulated protein 78 kDa ...(GRP78) acetylation and subsequent activation of the unfolded protein response (UPR) play in the antitumor activity of class I histone deacetylase (HDAC) inhibitors, which primarily target class I HDACs. In this study, we explored the contributory role these class I HDACs may play in UPR regulation. Binding studies were performed using immunoprecipitation/immunoblotting following dual‐transfection with HA‐tagged GRP78 and FLAG‐tagged HDACs. Subcellular localization was performed using Western blot of fractionated cell lysates and confocal microscopy. Individual HDACs were inhibited using RNA interference. We identified the potential of HDACs 1, 2, and 3 to bind to GRP78. These HDACs colocalized with GRP78 in the endoplasmic reticulum (ER). Inhibition of individual HDACs resulted in GRP78 acetylation and selective activation of the UPR. Although traditionally viewed as nuclear enzymes, we demonstrate that Class I HDACs localize to the ER, bind to GRP78, and selectively activate the UPR, representing a novel mode of UPR regulation and mechanism of action of HDAC inhibitors.—Kahali, S., Sarcar, B., Prabhu, A., Seto, E., Chinnaiyan, P. Class I histone deacetylases localize to the endoplasmic reticulum and modulate the unfolded protein response. FASEB J. 26, 2437‐2445 (2012). www.fasebj.org
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Histone deacetylase (HDAC) inhibitors represent a promising class of anti-cancer agents that are actively being evaluated in the context of clinical trials in solid tumors, including glioblastoma. ...What makes these agents particularly attractive is their capacity to enhance the activity of commonly used cytotoxics in cancer therapy, including both chemotherapy and ionizing radiation. As recent investigations suggest HDAC inhibitors may potentiate the cytotoxicity of topoisomerase inhibitors, which continue to be a commonly used class of agents in the treatment of glioblastoma, we performed preclinical studies to determine if this combination may be a promising strategy in glioblastoma. The effects of the HDAC inhibitor vorinostat and SN38, which is the active metabolite of the topoisomerase I inhibitor CPT-11, was evaluated using the clonogenic assay. Various treatment schedules were tested to determine optimum drug sequencing. Induction of DNA double strand breaks (DSBs) with the combination of vorinostat and SN38 was evaluated using the neutral comet assay, and their subsequent repair was evaluated by γH2AX foci kinetics using immunofluorescent cytochemistry. Vorinostat enhanced the cytotoxicity of SN38 in glioblastoma cell lines. Optimal treatment schedules involved maximal concurrent administration of agents. Pretreatment with either agent did not enhance cytotoxicity. Vorinostat potentiated SN38-induced DNA DSBs and attenuated their subsequent repair. These results indicate vorinostat enhances the cytotoxicity of SN38 in glioblastoma cell lines, suggesting this combination may be a worthwhile strategy to test in the context of a clinical trial.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The purpose of this study was to determine the capacity of MK-1775, a potent Wee-1 inhibitor, to abrogate the radiation-induced G(2) checkpoint arrest and modulate radiosensitivity in glioblastoma ...cell models and normal human astrocytes. The radiation-induced checkpoint response of established glioblastoma cell lines, glioblastoma neural stem (GNS) cells, and astrocytes were determined in vitro by flow cytometry and in vivo by mitosis-specific staining using immunohistochemistry. Mechanisms underlying MK-1775 radiosensitization were determined by mitotic catastrophe and γH2AX expression. Radiosensitivity was determined in vitro by the clonogenic assay and in vivo by tumor growth delay. MK-1775 abrogated the radiation-induced G(2) checkpoint and enhanced radiosensitivity in established glioblastoma cell lines in vitro and in vivo, without modulating radiation response in normal human astrocytes. MK-1775 appeared to attenuate the early-phase of the G(2) checkpoint arrest in GNS cell lines, although the arrest was not sustained and did not lead to increased radiosensitivity. These results show that MK-1775 can selectively enhance radiosensitivity in established glioblastoma cell lines. Further work is required to determine the role Wee-1 plays in checkpoint activation of GNS cells.
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