Sveva Grande

Metabolic / Proteomic Signature Defines Two Glioblastoma Subtypes With Different Clinical Outcome

Glioblastoma (GBM) is one of the deadliest human cancers. Because of the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies based on biologically and clinically relevant subclassification systems. Analyzing a collection of seventeen patient-derived glioblastoma stem-like cells (GSCs) by gene expression profiling, NMR spectroscopy and signal transduction pathway activation, we identified two GSC clusters, one characterized by a pro-neural-like phenotype and the other showing a mesenchymal-like phenotype. Evaluating the levels of proteins differentially expressed by the two GSC clusters in the TCGA GBM sample collection, we found that SRC activation is associated with a GBM subgroup showing better prognosis whereas activation of RPS6, an effector of mTOR pathway, identifies a subgroup with a worse prognosis. The two clusters are also differentiated by NMR spectroscopy profiles suggesting a potential prognostic stratification based on metabolic evaluation. Our data show that the metabolic/proteomic profile of GSCs is informative of the genomic/proteomic GBM landscape, which differs among tumor subtypes and is associated with clinical outcome.

1H NMR detects different metabolic profiles in glioblastoma stem-like cells

The metabolic profiles of glioblastoma stem‐like cells (GSCs) growing in neurospheres were examined by 1H NMR spectroscopy. Spectra of two GSC lines, labelled 1 and 83, from tumours close to the subventricular zone of the temporal lobe were studied in detail and compared with those of neural stem/progenitor cells from the adult olfactory bulb (OB‐NPCs) and of the T98G glioblastoma cell line. In both GSCs, signals from myoinositol (Myo‐I), UDP‐hexosamines (UDP‐Hex) and glycine indicated an astrocyte/glioma metabolism. For line 1, the presence of signals from N‐acetyl aspartate, GABA and creatine pointed to a neuronal fingerprint. These metabolites were almost absent from line 83 spectra, whereas lipid signals, absent from normal neural lineages, were intense in line 83 spectra and remained low in those of line 1, irrespective of apoptotic fate. Spectra of OB‐NPC cells displayed strong similarities with those from line 1, with low lipid signals and clearly detectable neuronal signals. In contrast, the spectral profile of line 83 was more similar to that of T98G, displaying high lipids and nearly complete absence of the neuronal markers. A mixed neural–astrocyte metabolic phenotype with a strong neuronal fingerprint was therefore found in line 1, while an astrocytic/glioma‐like metabolism prevailed in line 83. We found a signal assigned to the amide proton of N‐acetyl galactosamine in GSC lines and in OB‐NPC spectra, whereas it was absent from those of T98G cells. This signal may be related to a stem‐cell‐specific protein glycosylation pattern and is therefore suggested as a marker of cell multipotency.

Characterization of 1H NMR detectable mobile lipids in cells from human adenocarcinomas

Magnetic resonance spectroscopy studies are often carried out to provide metabolic information on tumour cell metabolism, aiming for increased knowledge for use in anti‐cancer treatments. Accordingly, the presence of intense lipid signals in tumour cells has been the subject of many studies aiming to obtain further insight on the reaction of cancer cells to external agents that eventually cause cell death. The present study explored the relationship between changes in neutral lipid signals during cell growth and after irradiation with gamma rays to provide arrest in cell cycle and cell death. Two cell lines from human tumours were used that were differently prone to apoptosis following irradiation. A sub‐G1 peak was present only in the radiosensitive HeLa cells. Different patterns of neutral lipids changes were observed in spectra from intact cells, either during unperturbed cell growth in culture or after radiation‐induced growth arrest. The intensities of triglyceride signals in the spectra from extracted total lipids changed concurrently. The increase in lipid peak intensities did not correlate with the apoptotic fate. Modelling to fit the experimental data revealed a dynamic equilibrium between the production and depletion of neutral lipids. This is observed for the first time in cells that are different from adipocytes.

Radiation effects on soluble metabolites in cultured HeLa cells examined by 1H MRS: Changes in concentration of glutathione and of lipid catabolites induced by gamma rays and proton beams

Cultured HeLa cells were irradiated with a single acute dose of either gamma rays (40 Gy) or low‐energy proton beams (20 Gy). 1H magnetic resonance spectra of intact cells harvested at different times after irradiation and of the correspondent perchloric acid (PCA) extracts prepared at different times after irradiation were run. Selected signals from glutathione and lactate were examined with the aim of investigating effects of irradiation on antioxidative stores and on mitochondrial activity. An increase of signal intensity of glutathione (GSH) takes place at 15 and 24 hr after irradiation, while a decrease of its signal intensity, accompanied by an increase of that of free glutamate, starts appearing 48 hr after irradiation. Lactate signal increases 48 hr after irradiation. Signals from lipid catabolites were also examined to explore their sensitivity in predicting the response to radiotherapy. Intensity ratios of signals of glycerophosphorylcholine and choline to that of phosphorylcholine increase with time after irradiation. Irradiating cells with gamma rays or proton beams at half a dose produces effects comparable to the metabolic variations presented here. The present experiments allow more insight into the complex pattern of the changes of GSH by irradiation and indicate that magnetic resonance spectroscopy signals from GSH, glutamate, lactate, and lipid catabolites are affected by irradiation. Finally, these data represent a first indication that the relative biological efficiency for some metabolic damage of low‐energy proton beams with respect to gamma rays can reach a value of 2.

Role of Glutathione in Apoptosis Induced by Radiation as Determined by 1H MR Spectra of Cultured Tumor Cells

Abstract Rosi, A., Grande, S., Luciani, A. M., Palma, A., Giovannini, C., Guidoni, L., Sapora, O. and Viti, V. Role of Glutathione in Apoptosis Induced by Radiation as Determined by 1H MR Spectra of Cultured Tumor Cells. Radiat. Res. 167, 268–282 (2007). The relationship between apoptosis induced by γ radiation and glutathione in cells of two human cancer cell lines, HeLa from cervix carcinoma and MCF-7 from mammary carcinoma, was examined. MCF-7 cells appeared to be more radioresistant than HeLa cells, and radiation-induced apoptosis, which was monitored by assessing phosphatidylserine externalization, was observed in HeLa cells but not in MCF-7 cells. Glutathione levels monitored by 1H MRS were higher in MCF-7 cells than in HeLa cells, while the opposite was true for the free glu signals. MCF-7 cells became more radiosensitive when treated with 0.1 mM buthionine sulfoximine, which inhibits GSH synthesis through inactivation of γ-glutamylcysteine synthetase, with the concomitant appearance of radiation-induced apoptosis. We can thus reasonably associate, at least in part, the resistance of MCF-7 cells to apoptosis with a high level of glutathione and probably with a high activity of γ-glutamylcysteine synthetase. A late decrease in glutathione concentration after irradiation was observed in MCF-7 cells, but not in HeLa cells and to a lesser degree in buthionine sulfoximine-treated MCF-7 cells. This would indicate that the radiation-induced decrease in glutathione concentration is not related to the onset of apoptosis, but it is more likely related to glutathione consumption as a result of detoxification reactions.

Glycosidic intermediates identified in 1H MR spectra of intact tumour cells may contribute to the clarification of aspects of glycosylation pathways.

The glycosylation process, through the addition of carbohydrates, is a major post‐translational modification of proteins and glycolipids. Proteins may be glycosylated in either the secretory pathway leading to N‐linked or O‐linked glycoproteins or as nucleocytoplasmic glycosylation that targets only single proteins involving a single β‐linked N‐acetylglucosamine. In both cases, the key precursors are the uridine diphospho‐N‐acetylhexosamines synthesised by the hexosamine biosynthetic pathway. Furthermore, uridine diphospho‐N‐acetylglucosamine participates in the biosynthesis of sialic acid. In this work, we propose MRS for the detection of uridine diphospho‐N‐acetylhexosamines visible in high‐resolution MR spectra of intact cells from different human tumours. Signals from the nucleotide and amino sugar moieties, including amide signals observed for the first time in whole cells, are assigned, also taking advantage of spectral changes that follow cell treatment with ammonium chloride. Finally, parallel changes in uridine diphospho‐N‐acetylhexosamines and glutamine pools, observed after pH changes induced by ammonium chloride in the different tumour cell lines, may provide more details on the glycosylation processes. Copyright

Identification of amide protons of glutathione in MR spectra of tumour cells

Signals attributable to amide protons and used in previous studies to measure intracellular pH were observed in the low‐field region of the 1H‐MR spectra of four tumour cell lines: T98G, MCF‐7, A172 and HeLa. The signals were more intense in the spectra of the two cell lines (T98G and MCF‐7) characterised by higher concentrations of glutathione (GSH). After comparison with 1H‐MR spectra of GSH in solution at different pH values, the peaks were attributed to NHs of the Cys and Gly residues of GSH. Modification of the intracellular concentration of GSH by treatment with buthionine sulfoximine produced comparable decreases in the intensity of aliphatic signals of GSH and NHs under examination. The assignment was therefore confirmed. Copyright

Metabolism of glutathione in tumour cells as evidenced by 1H MRS

1H MRS signals of glutathione and of free glutamate were examined in samples from cultured tumour cells, namely MCF‐7 from mammary carcinoma and TG98 from malignant glioma, with the aim of relating signal intensities to aspects of GSH metabolism. Spectra of cells harvested at different cell densities suggest that GSH and glu signal intensities are related to cell density and proliferation and their ratio is dependent on the activity of the γ‐glutamyl cysteine synthetase. The hypothesis is confirmed by experiments performed on cells treated with buthionine sulfoximine that inhibits the enzyme activity.

1H NMR spectroscopy of glioblastoma stem-like cells identifies alpha-aminoadipate as a marker of tumor aggressiveness

Patients suffering from glioblastoma multiforme (GBM) face a poor prognosis with median survival of about 14 months. High recurrence rate and failure of conventional treatments are attributed to the presence of GBM cells with stem‐like properties (GSCs). Metabolite profiles of 42 GSC lines established from the tumor tissue of adult GBM patients were screened with 1H NMR spectroscopy and compared with human neural progenitor cells from human adult olfactory bulb (OB‐NPCs) and from the developing human brain (HNPCs).

A three-microRNA signature identifies two subtypes of glioblastoma patients with different clinical outcomes

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults, characterized by aggressive growth, limited response to therapy, and inexorable recurrence. Because of the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies based on biologically and clinically relevant patient stratification systems. Analyzing a collection of patient‐derived GBM stem‐like cells (GSCs) by gene expression profiling, nuclear magnetic resonance spectroscopy, and signal transduction pathway activation, we identified two GSC clusters characterized by different clinical features. Due to the widely documented role played by microRNAs (miRNAs) in the tumorigenesis process, in this study we explored whether these two GBM patient subtypes could also be discriminated by different miRNA signatures. Global miRNA expression pattern was analyzed by oblique principal component analysis and principal component analysis. By a combined inferential strategy on PCA results, we identified a reduced set of three miRNAs – miR‐23a, miR‐27a, and miR‐9* (miR‐9‐3p) – able to discriminate the proneural‐ and mesenchymal‐like GSC phenotypes as well as mesenchymal and proneural subtypes of primary GBM included in The Cancer Genome Atlas (TCGA) data set. Kaplan–Meier analysis showed a significant correlation between the selected miRNAs and overall survival in 429 GBM specimens from TCGA‐identifying patients who had an unfavorable outcome. The survival prognostic capability of the three‐miRNA signatures could have important implications for the understanding of the biology of GBM subtypes and could be useful in patient stratification to facilitate interpretation of results from clinical trials.