Meningiomas and schwannomas are common tumours of the brain caused by mutations in the gene for merlin and occur early in life when inherited. Population based studies suggest that up to 1:300 people will develop a tumour in their lifetime (spontaneous or inherited) caused by a mutation in the merlin gene 1, 2. As these tumours grow slowly classical chemotherapeutic treatments are not effective. The only treatment options currently available are local therapies, surgery and radiosurgery, leaving patients with considerable morbidity. The extensive tumour load when these tumours are inherited is an additional and major therapeutic dilemma, leading to further increased morbidity, disability and reduced life expectancy. Approaches to develop an effective and systemic treatment are therefore of particular importance and of broad relevance 3. The fact that these tumours are genetically stable and homogenous allowed us to develop a unique model of human schwannoma cell culture, derived from patients at surgery, with the aim to identify and test new targeted therapies. This model includes primary pure schwannoma cultures which we compared to primary human Schwann cells. We have over the years identified a number of features in the schwannoma cells that are relevant to tumourigenesis 4-6. Using these we showed as proof of principle that this model is indeed useful to identify new therapeutic targets. More precisely we have successfully showed activation of a variety of growth factor receptors PDFG-R, ErbB2/ErbB3, IGF1-R and downstream intracellular signalling pathways i.e. the GTPase, Pi3K/Akt and Raf/Mek/Erk signalling pathways and their role in tumourigenesis 7-9. This allows us to identify potential therapeutic targets re-profiling modern drugs already available for use in these tumours and led to successful testing of a new kinase inhibitor drug Sorafenib, which inhibits PDGF-R and Raf and proliferation in our schwannoma model 10. This is the first drug published to be successful in an NF2 in vitro model. The signalling pathways we have studied so far were however driven by our knowledge of their role in normal Schwann cell physiology, i.e. Schwann cell proliferation and survival. Crucially, however many potentially relevant pathways have only been studied in a linear fashion and not methodically enough and some have not been studied in Schwann cells at all. One needs to take a broader view to understand the relevant signalling network alterations in Merlin's tumours. The usefulness of a broader more systematic approach is also nicely illustrated in glioblastomas by McLendon et al 11. In addition we have only studied schwannomas. The number of meningiomas are a crucial determinant in the survival of Neurofibromatosis type 2 (NF2) patients. Recently a method to culture human meningioma cells and normal arachnoidal cells developed by V Ramesh laboratory at Harvard was published 12, which whom we have started a collaboration. PDGFR is overexpressed in schwannomas and meningiomas.
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We hypothesize that in Merlin's genetically well defined and stable schwannomas and meningiomas there are common therapeutic targets that need to be identified. We also hypothesize that an attempt to understand complex signalling networks/matrices is necessary in order to find and better understand suitable therapeutic targets.
1. Using proteomics looking specifically at the phosphoproteome/kinases we want to identify common therapeutic targets in schwannomas and meningiomas as these are the relevant targets for our patients. The knowledge we have on schwannomas will help to verify proteomic results.
2. We want to use a proteomic approach to understand the complex matrix of signalling pathway activation in these tumours. Subsequently we will use our in vitro models to verify functional relevance of what we find in the proteomic approach.
Experimental design and methods
We plan to use 5 different human primary schwannoma culture and 5 different human primary meningioma cultures and compare their phopshoproteom. We will use cells in the second to third passage in which we have cell cultures more than 99% purity 4. We have a collection of schwannoma and meningioma cultures frozen down ready to use and have a continuous supply via established collaboration with two different neurosurgery centres. From our preliminary work we know that Sorafenib reduces schwannoma cell proliferation likely by inhibiting PDGF-R resulting in decreased activation of Raf/Merk/Erk and Akt. Analysing the phosphoproteome/kinases +/- Sorafenib would however help to get a more complete picture and help to understand effect. We plan to analyse 5 samples with and without Sorafenib.
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If comparing menigiomas and schwannomas we identify a signalling molecule for which we cannot find a pharmaceutical inhibitor we will not pursue this molecule in this PHD thesis. We have established contacts with the pharmaceutical industry namely GSK, Astra Zeneca, Pfizer, Bayer and Novartis thus we are confident that we will find a suitable inhibitor for most of the interesting targets.
If we find potential therapeutic targets, which we already have investigated in schwannomas we will validate them in meningioma cell culture model and test their functional relevance using inhibitors and read outs we have established i.e proliferation, survival and adhesion.
If we identify a new targets we will validate them in both in vitro models (schwannoma and meningioma) and test the functional relevance using inhibitors and functional read outs established in our lab.
Quantitative Proteomics. Protein phosphorylation is an important regulatory pathway in the targeting of drugs to treat schwannomas and meningiomas. 2 x 106 (from one tumour) cells will be grown in the presence of defined culture medium containing stable isotopic amino acids employing SILAC (stable isotopic labelling by amino acid in cell culture) technology. This will enable the above cell preparations to be quantitatively analysed for differential phosphoprotein expression. Briefly, two cell populations (e.g. meningioma and schwannoma cultures or cultures with and without drug) will be grown in identical cell culture media deficient in some essential amino acids. One cell population will be grown with heavy (isotopic L-arginine-U-13C614N4) amino acids, while the other cell population will be grown in medium with light (normal L-arginine) amino acids. The cells proteins will incorporate the amino acids (heavy and light). Approximately 2 x 106 cells will be lysed in buffer containing 1% NP-40, 0.1% sodium orthovanadate and protease inhibitors (complete tablets, Roche diagnostics). The lysates will be centrifuged at 14,000g to pellet cell debris. A Bradford assay will be performed to equalize protein concentrations for subsequent 1:1 mixing of the light and heavy lysates before the immunoprecipitation step. Mixed lysates will be pre-cleaned on protein A beads for 1 h to reduce nonspecific binding of abundant proteins. Precleaned lysates will then undergo immunoprecipitation with generic anti-phosphotryrosine and anti-phosphoserine antibody. Then the phosphorylated proteins and their binding partners will be run on 1D SDS-PAGE gels and stained with Colloidal Blue to visualize the gel lanes. The entire gel lanes will be cut into 10 or 20 equal sized gel pieces and trypsin digested. The resulting peptides containing the light and heavy peptides from each cell culture will be chemically identical but isotopically distinct and will be processed together. The isotopic differences will be distinguished using a Thermo LTQ Orbitrap mass spectrometer (MS) or Agilent 6510 Quadrupole Time-of-Flight LC/MS based in Exeter. Each mass spectrometer is ideally suited to identifying multiple components in complex protein mixtures, identifying post translational modifications and comparative quantitation using SILAC. Based on the relative peak intensity of the isotopic peptide pairs, we shall quantitate differential protein expression and identify common/differential post-translational modifications between schwannoma cells and meningimoma cells and cells with and without inhibitors. This method is capable of identifying and quantitating hundreds to several thousand proteins - depends on complexity and dynamic range of our samples. We will compare the phosphoproteome of schwannoma vs meningioma and of treated vs untreated cells.
Immunoprecipitation and Western blot analysis. For blotting experiments we use 1 x 105 schwannomas and meningiomas in serum starved medium. This allows 2 Western Blots, which can each be reprobed 5 times. Cells will then be treated with and without Sorafenib and other potential phosphorylaton inhibitors. The cells will be scraped into lysis buffer and the cleared lysates incubated with agarose conjugated 4G10 Anti-Phosphotyrosine antibody (which is capable of detecting 30 phosphorylated tyrosine kinases) for 6 h. After 3 washed with lysis buffer, immunoprecipitated proteins will be resolved on SDS-PAGE. After transfer, the PVDF membrane will be blocked with 2% BSA and incubated with specific phosphotase/kinase antibodies, followed by HRP-conjugated secondary antibodies. The membranes will be subjected to chemiluminescent detection and images captured digitally at fixed time points.
Data analysis of phosphoproteins; Researchers are increasingly confronted with long lists of proteins results from mass spectrometry analysis that may or may not be associated with a biological phenotype. Our approach is different, in that it is a method based on stable isotope labelling that introduce a mass difference between two proteomes. Further by performing a immunoprecipitation step prior to mass spectrometry analysis, this will allow for the phosphoproteome to be analysed in a less complicated and more comprehensive manner. Data files of completed LC-MS runs will be converted to mascot generic format files. The Mascot software package will be used for database search and protein identification with IPI human protein database. Peptide mass tolerance and fragment mass tolerance will be set at 0.2 Da for each initial search. An iterative calibration algorithm will be applied to raw data to provide mass accuracies within 20 p.p.m. All protein identifications will be based on at least two matching peptides and tandem mass spectra will be additional validated manually. Identification of the proteins precipitated by anti-phosphotyrosine antibodies will be further analysed by looking at the the fold increase or decrease at any given time point. For example, the peptide maps of phosphoproteins in a sample encoded with 13C-labelled arginine and the control sample with the standard 12C form of arginine will be distinguished by a mass difference of 6 Da, making it possible to determine the ratio of a particular protein in the two samples quantitatively. If a protein is more abundant in the 'heavy' form, the protein will have been tyrosine-phosphorylated and thus will be isolated in a greater amount by the affinity purification. Although the SILAC technique can quantify changes as small as 10% between samples, we shall chose a 1.5 fold increase or decrease in phosphorylation as a cutoff for biological significance. Furthermore we shall compare the profiles obtained by quantitative proteomics with those of our standard western blot analysis of specific signalling pathway candidate proteins e.g. activation of PAK, RAF/Mek/Erk, Akt, PDGFR, IGF1R, ErbB2/ErbB3 in schwannomas and Mek/Erk and PDGFR that are inhibited in schwannomas after Sorafenib treatment. Once the all the phosphoproteins have been identified that are up or down regulated by 1.5 fold or more they will be analysed by 'principle components analysis'. This is a mathematical procedure that will be used to transform a number of correlated increased or decreased phosphorylated proteins into a smaller cluster of uncorrelated variable e.g. grouping seemingly unrelated proteins that all give rise to a 2-fold decrease in phosphorylation after treatment with a specific drug treatment. This will enable us to determine if specific identified proteins are altered in a similar manner and form part of a known or novel signaling pathway, rather than just identifying a number of unrelated phosphorylation events. Prof Tim Frayling of IBCS has agreed to help design the analysis model for this part of the project.
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Schematic outline of tumour cells preparation for identification and quantificationof phosphoproteins targets
Time scales and milestones
Milestones for completion of project
Research Activity of PhD Student
Training in cell culture and maintenance of tumour cells, basic biochemistry training SDS-PAGE and western blotting, immunoprecipitation
Training in, stable isotopic labelling by amino acid in cell culture and fluorescent labelling of cells and use of microscope. Labelleling of menigioma and schwannoma cells
Preparation of menigioma and schwannoma cultures and treated tumour cells for phophoproteome analysis
Training in mass spectroscopy and MS on meningioma and schwannoma cultures
Training in bioinformatics/mass spec analysis and analysis of data
Identification and quantification of protein targets and their quantities in schwannoma and meningioma cells
Translation research: study the effect of novel inhibitors on phosphorylaton/kinase expression in schwannoma and meningioma cells,
To identify the effects of novel compounds on tumour cell signalling
Write and submit thesis; final report and manuscripts to be submitted.
Write up thesis
How this project fits into current research activities
This project is truly interdisciplinary. It combines the protein chemistry and proteomics expertise of PE with expertise in clinical neurobiology. Furthermore it is a truly translational neuroscience project as it is specifically focused on defining novel signalling pathway targets to inhibit by known and novel therapeutics, thus in line with one of PCMDs research focuses. OH has vast expertise in this field of research both at the clinical and biomedical research level. Our unique approach of using a human cell culture model and new modern drugs for which side effects are known and minor, allows us to translate successful drugs rather quickly into clinical trials. To this end, Peninsula Medical School has a fully accredited clinical trials unit. The PhD student will be jointly supervised by PE and OH. Director of studies is PE. The first 8 month the student will mainly work in Plymouth, thereafter mainly in Exeter and in year 3 likely at both localities.
Additional consumable support
This work will require the use of contracted MS services and additional consumable money will be made available to cover the costs up of the analysis from the 'children's tumour foundation'.