Fellows who have recently completed projects in the Clinical Research Training Programme
Supervisors: Dr Matthew Murray and Professor Nick Coleman
Targeting oncogenic microRNA clusters in malignant germ cell tumours using tiny locked nucleic acids
Germ cell tumours (GCTs) affect babies, children and young people. They occur in the gonads (ovaries/testes), but also in other sites, including the brain. At present, treatment regimes include a combination of surgery, chemotherapy and/or radiotherapy. Although most patients with malignant GCTs do well, some patients still have poor outcomes. For those that survive, many suffer long-term effects of treatment, including permanent damage to hearing, the kidneys, lungs and heart.
MicroRNAs are short, non-coding RNAs that regulate the expression of genes in cells, so abnormal microRNA levels may lead to cancer. Dr Matthew Murray and the Coleman group have identified two ‘clusters’ of microRNAs that are at very high levels in all malignant GCTs. These particular microRNAs are present at very low or undetectable levels in healthy human cells. We aim to block the effects of these microRNAs using specific inhibitors called tiny LNAs.
We ultimately aim to develop a novel therapy to improve clinical outcomes for patients with malignant GCTs by improving survival of patients with high-risk tumours and reducing toxic effects of chemotherapy for patients with low-risk disease.
Supervisors: Dr Nitzan Rosenfeld - Cancer Research UK Cambridge Institute, Dr Pippa Corrie& Mr Amer Durrani - Cambridge University Hospitals
Melanoma, the most aggressive form of skin cancer, results in 2,203 deaths per year in the United Kingdom and its incidence has increased faster than any other cancer. Melanoma invades and metastasizes early and is resistant to conventional chemotherapy. There are no reliable biomarkers of disease response to treatment. The development of targeted therapies, including BRAF and MEK inhibitors, has provided increasing evidence that genetic events are responsible for response and resistance to therapy. With this in mind, the MelResist study is exploring the genetic basis of resistance to these therapies.
Circulating tumour DNA (ctDNA) is emerging as a non-invasive method to monitor tumour changes. The Rosenfeld lab at the Cancer Research UK Cambridge Institute (CRUK CI) have demonstrated the potential of cell free circulating tumour DNA (ctDNA) as a non-invasive liquid biopsy for personalized cancer therapy in a number of advanced cancers, including breast, ovarian and lung cancer. The group have developed a low-cost, high-throughput method called tagged-amplicon deep sequencing (TAm-Seq) which allows identification of cancer mutations in plasma at allele frequencies as low as 2%, with sensitivity and specificity of >97%. (Forshew et al. Sci Transl Med 2012).
Developing a technique of detecting ctDNA in the plasma and/or urine of melanoma patients has the potential to revolutionize the management of this life-threatening disease. To be able to measure tumour burden and specific mutations during the course of treatment could guide clinicians regarding whom to continue targeted therapy and equally predict patients likely to relapse. Detection of mutations responsible for resistance to targeted therapies will aid the development of second line therapies. In addition, detection of ctDNA may be of prognostic value in melanoma patients undergoing resection of their primary or locoregional disease who are at high risk of recurrence, so that targeted therapies could be utilised at an earlier stage of their treatment.
Supervisor: Dr Nitzan Rosenfeld - Cancer Research Uk Cambridge Institute
Prostate cancer is the most common male cancer in the UK affecting 50% of 50-year-old men. Many men have indolent disease that would not limit their life span. Some however, have aggressive disease that will progress if not treated. Current monitoring methods e.g. blood tests or prostatic biopsies, are unable to distinguish accurately between indolent and aggressive disease. This is partly because, in the prostate gland of a single man there can be multiple cancer foci.
Patients with cancer have mutations in their DNA and, recent studies show that these mutations can be tracked non-invasively through simple blood tests (circulating tumour DNA or ctDNA). This project will investigate the ability of ctDNA to display and monitor the complexity of prostate cancer in men.
We hope that in the future, ctDNA analysis could be used to non-invasively predict aggressive cancers and stratify men to early aggressive treatment to improve outcomes.
Supervisor: James Brenton
More details to follow
Supervisor: Paul Lehner
Identification of the cellular components involved in the quality control and regulation of MHC class I molecules
The aim of this project is to identify the cellular components involved in the quality control and regulation of Major Histocompatibility Complex (MHC) class I molecules.
MHC class I plays a critical role in immune surveillance for viral infections and tumours via presentation of intracellular protein-derived peptides to cytotoxic T lymphocytes. MHC class I molecules are heterotrimers consisting of a transmembrane heavy chain, '2-microglobulin and a peptide ligand. The folding and assembly of these complexes is subject to stringent quality control within the endoplasmic reticulum (ER). However, accumulation of misfolded MHC class I molecules is observed in a variety of pathological settings, for example aggregation of HLA-B27 in ankylosing spondylitis. In addition, many viruses exploit these cellular quality control mechanisms in order to down-regulate MHC class I expression at the cell surface and thus evade the host immune system. Read more..
Under normal circumstances, class I molecules which fail to achieve their native conformation due to protein misfolding, or the absence of bound peptide, are targeted for elimination via the ER-associated degradation (ERAD) pathway. This requires recognition by the ERAD machinery, ubiquitination and retrograde translocation from the ER to the cytosol for proteasome-mediated degradation. The critical enzyme in this process is the ubiquitin E3 ligase which confers substrate specificity.
An initial flow cytometry based 'loss of function' siRNA screen has identified an E3 ligase required for endogenous MHC class I regulation and has highlighted other potential components of the MHC class I ERAD pathway. Further aims of this project include biochemical characterisation of these components, elucidation of their mode of action and analysis of their potential role in disease.
Supervisor: Kevin Brindle
Mutimodality imaging of early response to treatment in murine breast cancer models
This PhD project will further develop molecular imaging methods established in Professor Brindle's lab for assessment of early drug response in cancer. The work will focus on breast cancer and will predominantly use two different approaches. Read more'
The first approach involves imaging of apoptosis using a variety of agents and imaging methods both in vitro and in vivo. One of the main agents of interest is the C2A fragment of synaptotagmin which will be used with a fluorophore for in vitro work. It will also be used with radioactive labels for use with in vivo PET and SPECT imaging.
The second approach is imaging of intracellular biochemical changes, such as pH, using hyperpolarised 13C with MRI for both in vitro and in vivo studies.
The aim of the project is to develop these methods so they can be applied to predict response to treatment in cancer after just a single dose.
Supervisor: John Gurdon & Jerome Jullien
Real time monitoring of transcriptional reprogramming. Embryonic stem cells have the remarkable property of being able to differentiate into every different kind of cell-type. This makes them an ideal cell type for replacement therapy, if such cells can be derived from the accessible cells of adult humans. Nuclear reprogramming is the process by which the transcriptional profile of a somatic nucleus is switched to that of an embryonic stem cell (ESC) nucleus. One way to achieve this is to perform nuclear transfer whereby somatic nuclei are transplanted into eggs or oocytes.
By transplanting the nuclei of mouse somatic cells into Xenopus oocytes, we plan to develop a real time monitoring system of transcriptional reprogramming. Our real time system involves tagging the gene of interest and its RNA products with various nucleotide sequences that can be recognized by fluorescent proteins. We are aiming to apply this technique to the pluripotency genes Sox2, Oct4 and Nanog. These genes are characteristic of ES cells, and their reactivation from somatic nuclei is a good indication of successful reprogramming.
Supervisors: Edwin Chilvers & Phillip Hawkins
Regulation of neutrophil apoptosis by the phosphoinositide 3-kinases. The aim of my research is to define the precise phosphoinositide 3-kinase (PI3-K) isoforms and downstream effector pathways involved in regulating survival signalling in neutrophils. Neutrophils are essential to the innate immune defence against invading pathogens, however aberrant neutrophil activation or survival mediates secondary tissue damage. This is associated with clinically important inflammatory lung disorders including acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Apoptosis is the key determinant of neutrophil longevity and plays a critical role in the resolution of granulocytic inflammation. Despite the cardinal importance of this process, the molecular pathways involved in regulating neutrophil survival/apoptosis remain poorly defined.
Our laboratory has demonstrated the importance of phosphoinositide 3-kinases in cytokine modulated neutrophil survival. The PI3-Ks regulate fundamental cellular processes related to cell growth, survival and migration through the generation of key phospholipid second messengers. PI3-K activation may be stimulated by tyrosine kinase-linked receptors (Class IA PI3-Ks a, ' and d) or by G-protein-coupled receptors (Class IB PI3-K). As yet the PI3-K isoform(s) responsible for the regulation of neutrophil apoptosis are unknown.
My studies have demonstrated (1) that the rate of constitutive apoptosis in human neutrophils is markedly delayed by GM-CSF and C5a in a concentration- and time-dependent manner, (2) the cytoprotective effect of GM-CSF and C5a is >80% PI3-K-dependent (3) GM-CSF treatment period of 120 minutes is fully sufficient to confer its long term cytoprotective effect and (4) both GM-CSF and C5a induce rapid and transient AKT phosphorylation in a PI3-K dependent manner. More recently, I have also obtained the first reported effects of a range of selective PI3-K Class I inhibitors on both constitutive and modulated human neutrophil apoptosis rates and their impact on downstream AKT signalling pathways.
These studies will inform our understanding of the molecular mechanisms controlling the balance of neutrophil survival/apoptosis and aid development of novel therapeutic strategies designed to limit the extent and duration of neutrophil-dominated inflammation without impairing host immunity.
Supervisor: Ian Mills
Neuroendocrine differentiation and stem cells in prostate cancer.The existence of a stem cell niche in the basal layer of prostate epithelia has been proposed, which harbours cells demonstrating both self-renewal and differentiation potential into luminal, basal and neuroendocrine cell types. Some aggressive and treatment resistant prostate cancers contain cells that exhibit a neuroendocrine phenotype. Transcription factors upregulated in these cancers (e.g. Hes6) have been linked to regulation of recognised stem cell pathways. Isolation and culture of prostate stem cells has proved difficult.
By contrast, there are established techniques in mammalian skin for the study of cell lineage and the role of transcription factors in this process. c-Myc has been shown to regulate self-renewal and differentiation of epidermal stem cells and it is also a recognised oncogene in prostate cancer. My project has therefore begun by characterising Hes6 in skin cells and I have found that its expression is increased in response to Myc activation.
The next step has been to translate these experiments to prostate cancer cells. The key questions will be, is Hes6 expression regulated by c-Myc, What is the functional effect of Hes6 expression, and does expression of Hes6 drive androgen sensitive prostate cancer cells into an androgen resistant phenotype? Understanding mechanisms by which stem cells in the prostate commit to specific differentiated progeny, and particularly the means by which prostate cancer stem cells produce cancer of varying phenotype that is resistant to conventional therapy, should deliver targets for improved treatment of prostate cancer.
Supervisor: Ken Smith
MicroRNAs in the immune system
MicroRNAs (miRNAs) have only been discovered about ten years ago. They are approximately 22 nucleotide long, non-coding RNAs, that are highly conserved across different species. Despite being non-coding. miRNAs efficiently regulate the gene expression in multiple cellular processes. MiRNAs silence their targets either by inhibiting gene translation or by destabilizing the mRNA transcript. Read more..
The miRNA mir210 is expressed at baseline levels in resting B- and T- cells, but is strongly up regulated in activated lymphocytes. Interestingly mir210 is also highly expressed in several human malignancies and its over expression has been reported to correlate with low survival rate in pancreatic adenocarcinomas.
However, the function of mir210 in the immune system has not been described yet. To study this miRNA in more details, the laboratory of Professor Ken GC Smith has established a transgenic mouse line that over expresses mir210 in lymphocytes. In collaboration with the Sanger Centre, we also plan to generate a mir210 knockout mouse model.
The aim of my research project is to study the effects of mir210 on T- and NK-cell development as well as T-cell trafficking. Additionally we would like to investigate the role of mir210 in T-cell malignancies and its function in cancerogenesis.
Supervisor: Austin Smith
Self renewal and cell fate mechanisms in human neural stem cells
Self-renewal is the process by which a stem cell divides to repeatedly generate identical copies of itself. Cancer is thought to arise from mutations that inappropriately activate self-renewal programs. Despite the importance of neural stem cell self-renewal, we are only beginning to understand how it is regulated. Read more..
The primary goal of my research is to use pluripotent stem cell-derived neuro-epithelial stem (NES) cells as an in vitro model system to uncover the mechanisms of self-renewal and cell fate specification of primitive neuroepithelial cells. I will characterise the gene expression profile of human NES cells and their differentiating progeny, and relate this to genes that regulate the undifferentiated state and proliferation of neural stem cells in other organisms as well as genes implicated in the formation of primitive neuroectodermal tumours (PNETs).
I will test the role of candidate transcriptional regulators by developing a robust, efficient and reliable assay of differentiating NES cells using RNA interference (RNAi) and gene overexpression techniques. Ultimately, my goal is to determine how the inappropriate activation of self-renewal programs in NES cells may relate to the formation of PNETs, a heterogeneous group of tumours occurring in children that are histologically composed of undifferentiated or poorly-differentiated neuroepithelial cells. I will genetically manipulate NES cells to over-activate self-renewal programs and transplant these cells into immunodeficient mouse brain to examine whether this recapitulates the development of PNETs.
In parallel, I will attempt to isolate and characterise NES cells from human foetal brain in order to investigate the normal counterpart of the in vitro iPS-derived NES cells. I will also endeavour to derive tumour initiating stem cell lines from PNETs to provide a better understanding of how this aggressive tumour may develop.