Identifying Therapeutic Targets In Schwannomas And Meningiomas Biology Essay

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Meningiomas and schwannomas are common tumours of the brain caused by mutations in the gene for merlin. They occur spontaneously as well as early in life when inherited as part of the tumour prone disease Neurofibromatosis 2. 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 especially in certain meningiomas which are difficult to operate. The extensive tumour load when these tumours are inherited is an additional and major therapeutic dilemma, leading to further increased 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. We have over the years identified a number of features in the schwannoma cells that are relevant to tumourigenesis 4-8. Using these we showed 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 PDGF-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 7,9-12 (Ammoun et al. in press}. This allowed us to identify potential therapeutic targets re-profiling modern drugs already available for use in other tumours and led to successful testing of a variety of new kinase inhibitors drug AZD6244, Sorafenib, Lapatinib, Imatinib as well as Nilotinib, 10-12 (Ammoun et al. in press}. Sorafenib was 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, 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 13. Additionally we have only studied schwannomas. The number of meningiomas however are a crucial determinant in the survival when these tumours are inherited i.e. in Neurofibromatosis type 2 (NF2) patients. Recently a method to culture human meningioma cells developed by V Ramesh laboratory at Harvard was published 14, with whom we have started a collaboration. Crucially initial but isolated data from immunhistochemistry indicate common aberrant pathways like the PDGF-R, which seems relevant in these tumours as it is overexpressed in schwannomas and meningiomas.

Hypothesis

We hypothesize that in Merlin’s genetically well defined and stable tumours i.e. schwannomas and meningiomas there ought to be 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.

Aims

1. Using proteomics 15 looking specifically at the phosphoproteome/kinases we want to identify common therapeutic targets in schwannomas and meningiomas as these common targets are the relevant targets for our patients. The existing knowledge we have on schwannomas will help to verify proteomic results from this project.

2. Additionally 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

Common targets

We plan to compare the phosphoproteome of 5 different human primary schwannoma culture and 5 different human primary meningioma cultures. We will use cells in the second 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 in the UK and one in Germany.

In the likely event that we identify common potential therapeutic target, which we already have investigated in schwannomas functional assays, we will validate their relevance in meningioma cell culture model and test their functional relevance using inhibitors and read outs we have established in schwannomas.

If we identify 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. 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 identify a common target for which we cannot find a pharmaceutical inhibitor we will not pursue this molecule in this PhD thesis but will follow up later.

We will not analyse drug effects as part of this PhD project but this will be done subsequently

From our preliminary work we know that sorafenib and nilotinib reduce schwannoma cell proliferation likely by inhibiting PDGF-R resulting in decreased activation of Raf/Mek/Erk and Akt signalling. We will analyse the phosphoproteome/kinases +/- nilotinib. This will help to get a more complete picture and help to understand any potential drug effect/failure better. Thus we plan to analyse 5 schwannoma samples with and without Nilotinib. This will add to a phase 0 clinical trial we are currently doing in NF2 patients with cutaneous schwannomas.

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 and schwannoma cultures) 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 [12C]) amino acids for 5 doubling times. The proteins will metabolically 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 enrichment for phosphorylated proteins Phopshoproteins will be enriched by Titanium oxide columns online before the LC-MS/MS at the new proteomic suite at UoP. To control for total protein expression we will simultaneously run a non enriched samples. 1ug loading is needed for the LC MS. The isotopic differences will be distinguished using a Thermo LTQ Orbitrap Velos mass spectrometer (MS) or Waters Synapt G2 at UoP (tender currently out). Each mass spectrometer is ideally suited to identifying multiple components in complex protein mixtures, identifying post translational modifications and comparative quantification using SILAC. Based on the relative peak intensity of the isotopic peptide pairs, we shall quantify 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 quantifying hundreds to several thousand proteins - depends on complexity and dynamic range of our samples 16. We will compare the phosphoproteome of schwannoma vs. meningioma and later of treated vs. untreated cells. Common activated protein will be verified by Western blotting. In addition we have started a collaboration with T Joos (NMI, Reutlingen) who has established a sequential Luminex based assay measuring total and phosphoprotein of EGFR, Her2, HGFR, IGF-1R, PDGFβR, VEGFR2 and Tie 2 17. He has agreed to run samples from schwannoma and meningiomas for free to fill empty slots in experimental runs for a different project.

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 introduces a mass difference between two proteomes. Further by performing an enrichment step prior to mass spectrometry analysis, this will allow for the phosphoproteome to be analysed in a less complicated and more comprehensive manner. We will use the built in software and/or the free Mascot software package 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 enriched proteins will be further analysed by looking at 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. Although the SILAC technique can quantify changes as small as 10% between samples, we shall chose a 1.5 fold increase or decrease as a cut-off 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 RAF/Mek/Erk, Akt, PDGFR, IGF1R, ErbB2/ErbB3 in schwannomas and Mek/Erk and PDGFR that are inhibited in schwannomas. Once all the phosphoproteins have been identified that is up or down regulated by 1.5 fold or more they will be analysed by ‘principle components analyses. 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.

Verification using of newly identified phospohproteins as potential drug targets will be down as previously described 10-12

Time scales and milestones

Milestones for completion of project

Research Activity of PhD Student

Estimated timescales

Scientific Outcomes

Project familiarization

Training in cell culture and maintenance of tumour cells, basic biochemistry training SDS-PAGE and western blotting,

Months 1-6

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

Months 4-8

Preparation of menigioma and schwannoma cultures and treated tumour cells for phophoproteome analysis

Training in mass spectroscopy and MS on meningioma and schwannoma cultures

Month 6-20

Training in bioinformatics/mass spec analysis and analysis of data

Months 8-24

Identification and quantification of protein targets and their quantities in schwannoma and meningioma cells

Translation research: identification of potential novel targets in schwannoma and meningioma cells,

Months 24-32

To identify the effects of novel compounds on tumour cell signalling

Write and submit thesis; final report and manuscripts to be submitted.

Months 32-36

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 the UoP team (T Madgett, N Avent) with expertise in clinical neurobiology. Furthermore it is a 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 drug candidates 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 the UoP proteomics team and OH. Director of studies is OH. The PhD student will be supported by a B Clin Sci placement student who will concentrate on meningioma cell cultures.

Additional consumable support

Consumables costs for the proteomic work will be covered the School of Biomedical Sciences at the University of Plymouth (Head of School: Prof Neil Avent).

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