Cellular and Molecular Medicine projects

If you are interested in any of the projects below, contact the project supervisor in the first instance.

Do cancers recapitulate embryonic processes? If so, how?

Contact: Dr Abdelkader Essafi

The processes driving embryonic development are fundamentally different from those observed in healthy adult cells.  That is why most master regulators of cell fate determination and tissue remodeling in the embryo are silenced in the adult. However, in cancer, we observe an abnormal reactivation of some of these master regulators at different stages during disease progression.

Remarkably the early, rather than the late recapitulation of the epithelial-to-mesenchymal transition (EMT), is sufficient to generate migratory and/or chemo-resistant cancer stem-like cells (CSCs). EMT and the reverse process mesenchymal-to-epithelial transition (MET) are major tissue remodeling processes critical for the development of many embryonic tissues such as the kidney and the vasculature. The multifunctional Wilms’ tumour protein (WT1) is a major regulator of the EMT-MET balance in those same tissues. In this project, we are interested in understanding the molecular mechanisms underlying WT1 function as an EMT-MET regulator upon abnormal re-activation in cancers.

Therefore, the candidate will build on our previous findings to a) delineate and b) functionally characterize the molecular mechanisms downstream of WT1 in epithelial cancer. Promisingly, WT1-based immunotherapy clinical trials are underway as WT1 has topped a National Cancer Institute (NCI) antigen prioritization list. This is because as a developmental master regulator, WT1’s well-documented expression in adult tissues is restricted to few non-epithelial cell types yet it is abnormally reactivated uniquely in epithelial cancer cells. Here, we will use a well-established inducible oncogene-transformed epithelial cell model in order to a) investigate the role of Wt1’s multiple isoforms in driving cell fate. To this end we will use sequencing and proteomics-based approaches. Then, we will b) validate a selected and prioritized list of key WT1 targets. To achieve the latter, we will focus on gain and loss of function experiments of those prioritized targets using CRISPR-based tools in in vitro 3D cell culture as well as in vivo models.

Because the abnormal reactivation of the embryonic isoforms of WT1 will hijack embryonic cellular remodelling processes that may directly contribute to cancer aggressiveness; the investigation of mechanisms that are activated upon WT1 misexpression could have major implications for cancer therapy.

References

  • Essafi A, et al., A wt1-controlled chromatin switching mechanism underpins tissue-specific wnt4 activation and repression. Developmental Cell. (2011) Sep 13;21(3):559
  • Essafi A, Hastie ND. WT1 the oncogene: a tale of death and HtrA. Molecular Cell. (2010) Jan 29;37(2):153-5. Preview
  • Martínez-Estrada OM, et al, Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin. Nature Genetics. (2010) Jan;42(1):89-93

How to apply: Please make an online application for this project at www.bris.ac.uk/pg-howtoapply. Please select Cellular and Molecular Medicine on the Programme Choice page and enter details of the studentship when prompted in the Funding and Research Details sections of the form.

Candidate requirements: Upper-second (2.1) class degree or equivalent in a relevant discipline (Cellular or Molecular Biology, Immunology, Biochemistry). Prior interest or research experience in cell culture and molecular biology techniques will be advantageous.

Funding: This is a 3 year funded PhD studentship; funding includes a standard stipend. Funds are also available for tuition fees at 'home' (UK/EEA) rates. Note that UK/EEA Residency legal requirements apply.

"Inducing tolerance to blood group antigens in sickle cell disease"

Contact Dr Blair or Professor Anstee

Sickle-cell disease (SCD) is a hereditary blood disorder, caused by an abnormality in haemoglobin S (Hb S). Almost 300,000 children are born with a form of sickle-cell disease every year. Chronic blood transfusions are often used in the management of sickle-cell disease to alleviate anaemia and to prevent complications by decreasing the number of red blood cells (RBC) that can sickle by adding normal red blood cells. However, development of autoantibodies and alloantibodies to red cells is a major complication in SCD, due in part to donor–recipient genetic or racial disparity. Many patients will go on to develop additional antibodies, complicating the search for compatible donor cells and leading to delays in treatment. New strategies are required to reduce alloimmunisation and increase the availability of red cells for the treatment of these patients.

Alloimunisation has been shown to be greater in individuals with thalassemia who received their first transfusion after 3 years of age compared to infants transfused prior to this age. In the first year of life infants are sufficiently immature for the induction of immune tolerance. Chronic transfusions begun during this time may lead to lower immunisation rates. Consequently, it may be possible to induce tolerance to red blood cell (RBC) antigens by exposing young infants to these cells; however, this has not been tested in SCD patients. RBC shed cell-membrane-derived vesicles containing damaged cellular components. The vesicles are taken up by immune cells that digest and dispose of this cellular debris. Patients with SCD shed more vesicles and it may be possible to use these as a means of inducing immune tolerance to blood group antigens. This project will investigate induction of tolerance using plasma from SCD patients and the mechanisms underlying it.

The PhD student will join the research team of Dr Allison Blair and work closely with the research team of Professor David Anstee. 

Please make an online application for this project at http://www.bris.ac.uk/study/postgraduate/apply. Please select Cellular and Molecular Medicine on the Programme Choice page and enter details of the studentship when prompted in the Funding and Research Details sections of the form.

References

  • Badami KG. Induction of immune tolerance to RBC, platelet, and neutrophil antigens and IgA. Med Hypotheses. 2015;84(6):586-588. 
  • 2. Smith-Whitley K, Thompson AA. Indications and complications of transfusions in sickle cell disease. Pediatr Blood Cancer. 2012;59(2):358-364. 
  • Verduzco LA, Nathan DG. Sickle cell disease and stroke. Blood. 2009;114(25):5117-5125. 
  • Velilla PA, Rugeles MT, Chougnet CA. Defective antigen-presenting cell function in human neonates. Clin Immunol. 2006;121(3):251-259.

"Systems Virology for studying new viruses and old"

Supervisor: Dr David Matthews

We are interested in the application of high throughput proteomics and deep sequencing techniques to the study of viral infections. We have established a strong lead in this area and are currently working with world leading international partners to look at important zoonotic viruses such as MERS and Hendra virus as well as more established diseases such as Dengue, influenza, Herpes Viruses and gene therapy vectors such as adenovirus. There are several projects available in our laboratory to study how viruses interact with their hosts (human, animal or insect) using state of the art proteomics and deep sequencing base techniques several of which also involve using the University of Bristol’s supercomputer facility.

The development and application of these systems-wide approaches provides opportunities for cutting edge training in a range of techniques including big data biology, deep sequencing, high throughput proteomics, application of bioinformatics to the study of important viral pathogens (and their hosts) as well as practical training in molecular biology and basic virology techniques. Thus we have scope for training bioinformaticians in practical laboratory application of bioinformatics techniques to real world problems as well as for students with a more traditional biological sciences based background who want to engage with these exciting high throughput technologies that continue to revolutionise biological research.

References

  • Evans, VC, Barker, G, Heesom, K.J., Fan, J. Bessant, C, Matthews DA (2012) De novo derivation of proteomes from transcriptomes for transcript and protein identification. Nature Methods (9) 1207-1211.
  • Lam, Y. W., Evans, V. C., Heesom, K. J., Lamond, A. I. and Matthews, D. A. (2010). Proteomic analysis of the nucleolus in adenovirus infected cells. Mol Cell Proteomics, 9, 117-130.

"The mechanism of action of the Wilms’ tumour 1 protein"

Supervisor: Prof Stefan Roberts

Since its discovery the Wilms’ tumour 1 protein WT1 has proved to be a fascinating protein that has roles both in transcriptional regulation and RNA processing. WT1 was originally identified as a gene product that is frequently mutated or abnormally expressed in Wilms’ tumours, the most common solid childhood malignancy. However, WT1 has since been found to play a role in several other malignancies, including leukemia, breast and lung cancer. Our interests concern the mechanisms by which WT1 regulates transcription and its role in both development and disease.

We discovered that the transcriptional activity of WT1 is regulated by interaction with a transcriptional co-repressor. We identified one of the major components of the co-repressor complex (BASP1) and have recently identified candidates for the remaining factors. The BASP1 complex exhibits tumour suppressor activity and its intracellular distribution can be regulated by myristoylation, phosphorylation and sumoylation of BASP1. How these modifications regulate the WT1-BASP1 dynamic and their role in transcriptional regulation by WT1 in differentiation and disease are a current focus of our work.

The project will involve the analysis of novel WT1-interactiong proteins and BASP1 complex components using a combination of cell and molecular techniques including immunocytochemistry, RNAi, chromatin immunoprecipitation and genomics.

References

  • Toska, E., Campbell, H.A., Shandilya, J., Goodfellow, S.J., Shore, P., Medler, K.F. and Roberts, S.G.E. (2012). Repression of transcription by WT1-BASP1 requires the myristoylation of BASP1 and the PIP2-dependent recruitment of histone deacetylase.  Cell Reports 2, 462-469.
  • Shandilya, J., Wang, Y. and Roberts, S.G.E. (2012). TFIIB dephosphorylation links transcription inhibition with the p53-dependent DNA damage response. Proceedings of the National Academy of Sciences USA 109, 18797-18802. 
  • Goodfellow, S. J., Rebello, M. R., Zeef, L. A. H., Toska, E., Rudd, S. G., Medler, K. F. and Roberts, S. G. E. (2011). WT1 and its transcriptional cofactor BASP1 redirect the differentiation pathway of an established blood cell line. Biochemical Journal 435, 113-125.
  • Hartkamp, J., Carpenter, B. and Roberts, S. G. E. (2010). The Wilms' Tumour Suppressor Protein WT1 is processed by the Serine Protease HtrA2/Omi. Molecular Cell  37, 159-171.

"Imaging signalling to understand T cell function in health and disease"

Supervisor: Prof Christoph Wuelfing

By observing signalling as it occurs over time inside live T cells we learn how T cells function. How does that work? Cellular function is regulated in complex signalling networks with dozens of interacting proteins. Importantly, each protein enriches at a particular location at a particular time inside a cell as we determine with large-scale live cell imaging of T cell activation (ref 1, 2) (http://www.bristol.ac.uk/cellmolmed/infect-immune/wuelfing/spatiotemporal-patterning/). The spatiotemporal organization of many signalling intermediates together then determines how regulatory information flows through the T cell signalling network. With our imaging data we gain unique access to this regulatory information and thus generate insight into T cell function in health and disease.

Our scientific focus is on the regulation of the efficiency of T cell activation. We study individual molecules such as the costimulatory receptor CD28 or the tyrosine kinase Itk (ref 3) and have begun to apply our approaches to models of autoimmune disease (systemic lupus erythematosus and multiple sclerosis) and the killing of tumour cells by cytotoxic T cells.  Any project will combine large-scale live cell imaging of T cell activation (including advanced image analysis) with various T cell functional assays.

References

  • Singleton, K. L., Roybal, K. T., Sun, Y., Fu, G., Gascoigne, N. R. J., van Oers, N. S. C., and Wülfing, C. (2009) Spatiotemporal patterning during T cell activation is highly diverse. Sci. Signal., 2(65), ra15.
  • Roybal, K.T., Sinai, P., Murphy, R.F., Verkade, P., and Wülfing, C. (2013) The actin-driven spatiotemporal organization of T cell signaling at the systems scale. Immunol. Rev.  256, 133-147.
  • Singleton, K. L., Gosh, M., Dandekar, R. D., Au-Yeung, B. B., Ksionda, O., Tybulewicz, V. J. L., Altman, A., Fowell, D. J., Wülfing, C. (2011) Itk controls the spatiotemporal organization of T cell activation. Sci. Signal., 4(193), ra66.

"Function and structure of the type III secretion system of Shigella flexneri"

Supervisor: Dr Ariel Blocker

Type III secretion systems (T3SS) are widely distributed, essential virulence determinants of many Gram-negative bacteria. T3SS serve to translocate, upon physical contact with eukaryotic host cells, proteins from the bacterial cytoplasm into the host cytoplasm. Our laboratory studies the function and structure of the T3SS of Shigella flexneri, the agent of human bacillary dysentery, using a variety of cell biological, biochemical, genetic and structural approaches. Presently, we mainly focus on how the T3SS apparatus, and hence the bacterium, first senses and thus interacts with host cells. However, MRes and PhD projects will be tailored to student interest and expertise as much as possible. The project range is very broad and stretches from how Shigella uses its T3SS to invade and replicate inside host epithelial cells (very cell biological) to detailed functional and structural biochemistry and high-resolution electron microscopy imaging of the T3SS machine from "tip to toe" (quite biochemical, molecular and structural), to translational research into the application of our recent findings to development of a vaccine against shigellosis (fairly medical) or small molecules inhibiting T3SS function (chemical and biochemical).

References

  • Roehrich, A.D., Guillossou, E., Blocker*, A.J. and Martinez-Argudo, I. (2013) Shigella IpaD has a dual role: signal transduction from the T3SS needle tip and intracellular secretion regulation. Mol. Micro. 87, 690-706. *Corresponding author.
  • Martinez-Argudo, I., A.K.J. Veenendaal, X. Liu, A.D. Roehrich, G. Franzoni, K.N. van Rietschoten, Y.V. Morimoto, Y. Saijo-Hamano, M.B. Avison, D.J. Studholme, K. Namba, T. Minamino, and A.J. Blocker. (2013) Isolation ofSalmonella enterica sv. Typhimurium mutants resistant to the inhibitory effect of salicylidene acylhydrazides on flagella-mediated motility. PLoS One8(1): e52179. doi:10.1371/journal.pone.0052179.    
  • Fujii, T., Cheung, M., Blanco, A., Kato, T., Blocker*, A.J. and Namba*, K. (2012) The structure of a type III secretion needle at 7Å resolution provides insights into its assembly and signaling mechanisms. Proc. Natl. Acad. Sci. USA. 109, 4461-4466.*Joint corresponding authors.
  • Martinez-Argudo, I. and Blocker, A.J. (2010) The Shigella T3SS needle transmits a signal for MxiC release, which controls secretion of effectors. Mol. Microbiol. 78, 1365–1378.
  • Hodgkinson, J.L., Horsley, A., Stabat, D., Simon, M., Johnson, S., da Fonseca, P.C.A, Morris, E.P., Wall, J.S., Lea, S.M. and Blocker, A.J. (2009) 3D reconstruction of the Shigella flexneri T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout. Nat. Struct. Mol. Biol.16, 477-485.

"The importance of the atypical NF-κB signalling pathway in colorectal carcinogenesis"

Supervisor: Prof Ann Williams

Bowel cancer remains the second highest cause of cancer deaths in the UK: despite recent progress there remains an urgent need to find new ways for the prevention and/or treatment of advanced disease. Understanding the molecular mechanisms that allow tumour cell survival will allow us to identify novel targets for both the prevention and treatment of colorectal cancer.  As joint PI of the Cancer Research UK Bristol Colorectal Tumour Biology Group in the Department of Cellular and Molecular Medicine, my research interests include chemoprevention and the link between inflammation and colorectal cancer; we are currently working on regulators of NF-κB signalling and their role in promoting the colorectal cancer stem cell niche and tumour invasion and metastasis.

Recently our lab has shown that two regulators of the NF-κB pathway, Bcl-3 and the newly identified cofactor BAG-1, are highly expressed in pre-malignant and colorectal cancer tissue and act as potent survival factors particularly under conditions relevant to the tumour microenvironment.  Targets identified to date include genes central to the development of colorectal cancer including COX-2 and EGFR, as well as the β-catenin and AKT signalling pathways. Projects in my lab propose to elucidate the function of these NF-κB complexes within the context not only of the colorectal tumour microenvironment but also in the metastatic process, and hope to assess their use as biomarkers for assessing malignant disease risk in inflammatory bowel disease and/or prognosis in colorectal cancer patients.

References

  • Perkins ND. (2007). Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 8: 49-62.
  • Palmer S, Chen YH (2008) Bcl‐3, a multifaceted modulator of NF‐κB‐mediated gene transcription. Immunol Res. 42, 210-218.
  • Townsend PA, Stephanou A, Packham G, and Latchman DS. (2005). BAG-1: a multi-functional pro-survival molecule. Int J Biochem Cell Biol 37: 251-259.
  • Clemo NK, Collard TJ, Southern S, Edwards KD, Moorghen M, Packham G, Hague A, Paraskeva C and Williams AC. (2008) BAG-1 is up-regulated in colorectal tumour progression and promotes colorectal tumour cell survival through increased NF-kB activity, Carcinogenesis 29(4): 849-57.
  • Kaidi A, Williams AC, Paraskeva C.(2007) Interaction between beta-catenin and HIF-1 promotes cellular adaptation to hypoxia. Nat Cell Biol. 9(2):210-7.
  • Samantha L. Southern, Tracey J. Collard, Bettina Urban, Victoria Skeen, Helena Smartt, Angela Hague, Fiona Oakley , Neil D. Perkin, Christos Paraskeva and Ann C. Williams. (2012) BAG-1 interacts with the p50-p50 homodimeric NF-κB complex: implications for colorectal carcinogenesis Oncogene. 31;31(22):2761-72.
  • Puvvada, S. D., Funkhouser, W. K., Greene, K., Deal, A., Chu, H., Baldwin, A. S., Tepper, J. E., and O'Neil, B. H. (2010). NF-kB and Bcl-3 activation are prognostic in metastatic colorectal cancer. Oncology 78, 181-188.

"Epigenetics of cancer"

Supervisor: Dr Keith Brown

My group’s interests are in the molecular genetics of cancer, in particular epigenetic changes in childhood cancers and cancer stem cells.  Epigenetic alterations are heritable modifications of DNA that do not affect the primary sequence, such as methylation of cytosines in CpG dinucleotides, which is usually associated with transcriptional repression.  Cancers often have an overall hypomethylation of the genome, leading to chromosomal instability, accompanied by gene-specific hypermethylation that inactivates tumour suppressor genes.  In childhood cancers our current projects are characterizing genome-wide DNA methylation changes in clinically important tumours, such as Wilms' tumour of the kidney and neuroblastoma, a cancer of the sympathetic nervous system.  This has identified genes that are abnormally methylated in childhood cancers, which are being further studied using functional analyses in standard tissue culture and more sophisticated systems designed to replicate early fetal development.  In the stem cell work, our current projects aim to determine how stem cell markers are regulated in normal tissues and during tumorigenesis, with special reference to epigenetic modifications that play a pivotal role in modulating stemness.  This work is using colorectal cancer cell lines to study epigenetic marks and how dietary factors and the tumour microenvironment regulate them.

References

  • Charlet, J., M. Schnekenburger, K. W. Brown and M. Diederich (2012). "DNA demethylation increases sensitivity of neuroblastoma cells to chemotherapeutic drugs." Biochemical Pharmacology 83(7): 858-865.
  • Dallosso, A. R., A. L. Hancock, M. Szemes, K. Moorwood, L. Chilukamarri, H. H. Tsai, A. Sarkar, J. Barasch, R. Vuononvirta, C. Jones, K. Pritchard-Jones, B. Royer-Pokora, S. B. Lee, C. Owen, S. Malik, Y. Feng, M. Frank, A. Ward, K. W. Brown and K. Malik (2009). "Frequent long-range epigenetic silencing of protocadherin gene clusters on chromosome 5q31 in Wilms' tumor." PLoS Genetics 5(11): e1000745.
  • Brown, K. W., F. Power, B. Moore, A. K. Charles and K. T. Malik (2008). "Frequency and timing of loss of imprinting at 11p13 and 11p15 in Wilms' tumor development." Molecular Cancer Research 6(7): 1114-1123.

"Novel carcinogenic modes regulated by the WT1 oncoprotein"

Supervisor: Dr Karim Malik

Normal development requires stringently regulated orchestration of gene expression programmes. Epigenetic modifications such as DNA and histone methylation are critical in determining the plasticity and expressivity of the genome, thereby defining cell phenotypes. Aberrant epigenetic control is associated with disease, in particular carcinogenesis, where tumour suppressor gene silencing is a common pathogenic factor. However, although epigenetic regulation is critical in regulating gene expression programmes in normal development, as well as aberrant programmes in disease, very little is known about how the epigenetic machinery is regulated or directed.

The Wilms' tumour suppressor protein, WT1, is a transcription factor involved in the early development of several tissue systems. In many cancers, WT1 is over-expressed and considered to be an oncogene. These pivotal roles in normal and cancer cells suggested to us that WT1 may influence epigenetic control of gene expression. This project will utilise advanced molecular biology techniques to examine whether WT1 can alter the levels of specific epigenetic modulators, and thereby influence growth of normal and cancer cells.

References

  • Lotem, J. & Sachs, L. Epigenetics and the plasticity of differentiation in normal and cancer stem cells. Oncogene 25, 7663-7672 (2006).
  • Yang, L., Han, Y., Suarez Saiz, F. & Minden, M. D. A tumor suppressor and oncogene: the WT1 story. Leukemia 21, 868-76 (2007).
  • Miyoshi, Y. et al. High expression of Wilms' tumor suppressor gene predicts poor prognosis in breast cancer patients. Clin Cancer Res 8, 1167-71 (2002).
  • Roberts, S. G. Transcriptional regulation by WT1 in development. Curr.Opin.Genet.Dev. 15, 542-547 (2005).

Funding: This is a 3 year funded PhD studentship; funding includes a standard stipend. Funds are also available for tuition fees at 'home' (UK/EEA) rates. Note that UK/EEA Residency legal requirements apply.

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