NIHRDataInsights/HRCSData huggingface dataset:
Title: Regulation of the Rac activator Tiam1 by ubiquitylation
Funder: Medical Research Council

Abstract: RAS genes are frequently mutated in human cancer. The proteins they encode drive cell proliferation, survival, invasion and metastasis. To develop therapeutic agents targeting RAS function, focus has been applied to its downstream effector pathways. RAC1 is a key mediator of RAS transformation, as demonstrated by the resistance of Rac1 knockout mice to develop various Ras-induced tumours. TIAM1 is a selective activator of RAC that couples RAC and RAS activation. Like Rac1, Tiam1 is required for Ras-induced tumour formation in mice. However, mouse tumours lacking Tiam1 progress more frequently to malignancy, potentially due to TIAM1-RAC's ability to stimulate intercellular adhesion and thereby suppress invasion. Nonetheless, it is apparent that TIAM1 can also promote single cell motility and invasion. To target therapeutically the TIAM1-RAC signalling module requires increased understanding of its diverse cellular roles and also of its intricate spatio-temporal control. In unpublished data, we have identified an interaction between TIAM1 and the E3 Ubiqutin ligase HUWE1 and shown that TIAM1 is ubiquitylated by HUWE1 and degraded by the proteasome during stimulation of cell migration by the cytokine HGF/SF. HUWE1 is overexpressed in a spectrum of human tumours and this has been correlated with malignant progression. We hypothesise that through spatio-temporal regulation of TIAM1 expression, HUWE1 diverts TIAM1-RAC from promoting intercellular adhesion, to allow malignant progression. We now propose to map the interaction sites of TIAM1 with HUWE1 and identify the specific residues that are ubiquitylated. We then propose to determine whether HUWE1 regulates TIAM1 expression in a panel of cancer cell lines and whether manipulating the ubiquitylation of TIAM1 by HUWE1 impacts on cancer cell motility, invasiveness, proliferation or survival. Finally, we shall address the significance of the HUWE1-TIAM1 interaction for tumourigenesis using mouse tumour models.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Human SNM1A - a novel DNA repair role and the development of inhibitors.
Funder: Medical Research Council

Abstract: Here we propose a combined biological and chemistry-based approach to characterise the role of the human SNM1A in DNA double-strand break repair, validating it as a therapeutic target for radio and chemo-sensitisation in cancer. We have recently discovered that, in addition to its established role in interstrand cross-link repair, SNM1A plays a role in the repair of DNA double-strand breaks (DSBs) induced by radiation and radiomimetic drugs. In one arm of this work, we will define the relationship of SNM1A to the other major DSB repair pathways, and discover how it is recruited to sites of damage, a process mediated, at least in part, through interactions with poly-ADP-ribose chains. We wil also undertake detailed biochemical and structural studies to define at the molecular level how SNM1A digests damaged DNA, including DNA containing oxidised bases, relevant to its role in the repair of DSBs induced by chemo- and radio-therapeutics. We will combine this analysis with further development of small molecule inhibitors of SNM1A. We have recently discovered that several beta-lactam antibiotics are able to competitively inhibit DNA hydrolysis by SNM1A in vitro, within the low micromolar range, indicating that beta-lactam family molecules might provide an excellent starting point for the rational development of SNM1A inhibitors. We will employ focussed screens employing existing libraries of several thousand beta-lactam compounds and their structural analogues to inhibit SNM1A. In parallel with this we will further develop a cell-based assays that allow us to test the ability of these compounds to induce cellular sensitisation to DNA damage. Cellular specificity will be defined by the ability of the inhibitory compounds to selectively increase damage sensitisation and the hallmarks of repair defects in human cells, but not in isogenic lines disrupted for SNM1A.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: The synthesis and evaluation of new fluorescent-labelled P2Y2 receptor chemical probes.
Funder: Medical Research Council

Abstract: Activation of purinergic P2Y2 receptors by ATP released from damaged cells plays a significant role in inflammatory conditions and as such, the receptor is an important therapeutic target in many areas of clinical need. However, the elucidation of the receptor signalling and drug discovery has lagged behind that of other GPCRs due in part to the lack of commercially available P2Y2R radio labelled ligands and stable high affinity P2Y2R ligands. We will initially evaluate and expand on the known structure activity relationship (SAR) in a series of stable P2Y2R antagonists, where the antagonist activity has been recovered through the serendipitous discovery of the molecular recognition invoked by a single nitrogen atom, thereby giving a series of stable non acidic, non-phosphate containing P2Y2R antagonists. We will aim to synthesize a set of P2Y2R antagonist chemical scaffolds with suitable attachment points and groups suitable to carry out high speed synthetic chemistry where we will explore linking groups from the P2Y2R antagonist chemical scaffold to a BODIPY fluorescent probe. The resulting high affinity selective P2Y2R antagonist probes will be used to visualize, through for example confocal microscopy, cell populations where the P2Y2R is expressed, gaining valuable insight into cell signalling processes in both healthy and diseased cells. The resulting high affinity selective P2Y2R antagonist probes will be used to develop a novel fluorescence based ligand binding assay for future fragment-based lead generation programs to discover novel P2Y2R ligands. In combination with the chemistry-driven design programs, structural work will be explored through a combination of the emerging P2Y2R homology model and the SAR to be generated within this research project. Finally, valuable insights gleaned from this research could be applied to other purinergic receptors, offering insights into cell signalling and ultimately the design of new ligands for therapeutic benefit.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Dysregulation of intestinal morphogen gradients leads to ectopic stem-cell properties in intestinal neoplasia and inflammation/restitution
Funder: Medical Research Council

Abstract: Aims and objectives: To assess the consequences of disrupted morphogen gradients in the intestinal epithelium and the consequences of this disruption on stem-cell dynamics. To do this we will test the following hypotheses: 1. That genetic disruption of tissue-specific GREM1 expression causes ectopic stem-cell properties resulting in ectopic crypt formation in human familial and sporadic polyps and an existing mouse model. 2. That the uniform media morphogen supplementation in intestinal organoid culture systems disrupts endogenous morphogen gradients promoting crypt budding. 3. That the dysregulated morphogen balance that results from an altered cellular context in intestinal inflammation and regeneration promotes crypt budding and fission as part of the intestinal repair process. Methodology: Hypotheses are generated on the back of observations made in human tissue. We will then use state of the art in vitro culture systems and animal models to functionally test these hypotheses. All animal models are currently available at the host institute thus there is no requirement for time consuming transgenic animal generation. Exploitation: Little is understood about the development of serrated polyp- and colitis- associated carcinogenesis. An understanding of the stem-cell dynamics that underlie these conditions will help understand tumour pathogenesis, aid in the development of biomarkers and may provide insight into genetic heterogeneity and response to therapy. Functional insight into morphogen signalling also remains an area of significant therapeutic interest as potentially druggable targets may harness the considerable biological effects of manipulation of these pathways in established tumours.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Tensin regulation of tumour cell movement: a link between metabolism and motility
Funder: Medical Research Council

Abstract: Background: Cell metabolism is likely to influence many cellular processes, and we have recently identified a novel relationship between a metabolic sensor (CtBP2) and Tensin-4, a structural adaptor protein. Our preliminary data show that increased glycolysis modulates CtBP2-dependent expression of Tensin-4. Tensin-3 and -4 have a key role in regulating integrin-dependent interactions with the extracellular matrix, including adhesion, motility and also modulating TGF-beta activation. Objectives/Methodology: 1) What are the functional roles of the Tensin isoforms in vitro? We will utilise gene knockdown of the Tensins in integrin-dependent functional assays. 2) How does the metabolic sensor CtBP2 regulate Tensin-4 to control a cell's motile/invasive phenotype? We will initially perform time course experiments with the temporal analysis of PTEN/Talin/Tensin-4 Mr.NA levels after CtBP knockdown, then further dissect these through specific signallling pathway inhibitor studies. To address whether CtBP2 is directly recruited to the Tensin-4 promoter, a chromatin immunoprecipitation (ChIP) analysis will be performed. 3) Do Tensin-3 and -4 contribute to a signaling complex regulating TGF-B 1 activation? We will use TGF-B1 activation assays utilising gene knockdown and specific inhibitors. 4) What is the expression of Tensin isoforms in human tumours? Tissue microarrays of multiple tumour types will undergo immunhistochemisty analysis. 5) Do Tensins promote tumour progression in vivo? Scientific/Medical Opportunities: We will explore how metabolism regulates cell-ECM interactions, which may uncover new targets of diagnostic and therapeutic importance. Importantly, the project will determine the role of Tensins in cancer and determine how cell metabolism may be regulating a tumour's invasive phenotype. Long-term the simultaneous inhibition of both Tensin-4 and CtBP may lead to potent effects, especially in the realm of inhibiting cancer cell invasion and metastasis.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Investigation of local brain invasion by cerebral metastases and implications for clinical management
Funder: Medical Research Council

Abstract: Aims: 1. Identify Mr.I markers of brain metastasis invasion 2. Histopathology assessment of the tumour/brain boundary 3. Identify biological markers of brain metastasis invasion and prognosis Resource: I will use a historical cohort of 121 patients with paraffin embedded tissue from operated brain metastases in the Walton Research Tissue Bank (WRTB) (approval granted - see Ethical Information) as well as obtaining tissue from new patients undergoing image guided surgical resection of brain metastases at Walton and being entered into the bank. Methods: 1. Preoperative diffusion Mr.I scans will be used to record a number of measures both from within the tumour and across the brain-tumour interface. Pilot data has already confirmed the validity of this method (presented and submitted for publication) and is suggestive of relationships between these measures and patient outcomes. Suitable types of scans identified so far in 76 cases. 2. & 3. Using histology and immunocytochemistry prospectively compare cellularity, extraceullar matrix density, vascularity and expression of the known metastasis inducing proteins AGR2, S100A4, S100P, osteopontin & FANCD2 in tissue samples taken at the tumour boundary during routine image-guided surgery with Mr.I measures in (1) from co-localised preoperative advanced Mr.I scans. Method developed and 6 cases performed in this manner in 2 month pilot, we operate on c. 70 metastases per year and will recruit at least 40 cases in 18 months. Outcomes: Actuarial survival analyses stratified for established oncology predictors (age, number of metastases, whole brain radiotherapy) in addition to the radiological and biological markers identified. Correlation of radiological and biological markers of invasion with different primary cancers. Conclusion: The proposed research will increase understanding of how invasive brain metastases are and what the markers of this are, informing future clinical treatment.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: SIP1 induced epithelial to mesenchymal transition (EMT) promotes metastasis and chemoresistance in colorectal cancer.
Funder: Medical Research Council

Abstract: The aim of my study is to investigate the biological consequence of SIP1 induced EMT to metastasis and chemoresistance. My objectives are to 1) Characterise the transcriptional regulation of ERCC1 in epithelial and mesenchymal CRC cells, 2) Investigate the contribution of ERCC1 up regulation by SIP1 to chemoresistance, 3) Perform in-vivo experiments to validate SIP1 induced chemoresistance by modulating ERCC1 expression and 4) Validate SIP1 expression as a predictive biomarker of outcome in an independent patient cohort. Objective 1 will be achieved by cloning the promoter region of ERCC1 in pGL3 (luciferase reporter) plasmid and conducting luciferase assays in induced and un-induced cells. I will also change critical nucleotides in the E-box regulatory sequence by site directed mutagenesis to identify motifs facilitating ERCC1 transactivation. To investigate the influence of ERCC1 up regulation by SIP1 to chemoresistance, I will generate stable knock down and ERCC1 transfected DLD-SIP1 cells. I will then undertake apoptosis assays using Oxaliplatin before and after induction of EMT. I will also use an established orthotopic mouse model to analyse the influence of SIP1 mediated up regulation of ERCC1 and monitor real time tumour growth and chemotherapy response using IVIS Lumina III in vivo system. Additionally, after final treatment I will cull and undertake immunohistochemistry (IHC) to assess for the presence of SIP1 expressing metastatic cells. Finally, I will validate the role of SIP1 as a predictive biomarker in an independent patient cohort. The results of this study will provide scientific and clinical insight into mechanisms mediating chemoresistance in CRC. Demonstrating ERCC1 mediates chemoresistance may highlight the necessity to manufacture specific ERCC1 inhibitors, a potential chemosensitiser. Validating, SIP1 as a biomarker in an independent patient cohort will facilitate its progress towards a prospective study and clinical use in the future.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Quatitative proteomic analysis of castrate-resistant prostate cancer
Funder: Medical Research Council

Abstract: Aims: To study the proteome of castrate resistant prostate cancer (CRPC) and identify novel important proteins that can be exploited as potential therapeutic targets. Objectives: To apply available in vitro and in vivo pre-clinical CRPC models to an unbiased proteomic analysis to identify novel proteins associated with CRPC. Methods: Three Human PC cell lines and their CR isogenic derivatives will be selected and transfected with luciferase expressing constructs. Orthograft in vivo models will be used with castration to create a physiological environment for CRPC, compared to hormone naïve (HN) tumours. Tumours will be removed at the same stage as determined by luciferase signal comparing HN and CR tumours (all 3 pairs of cells). Experiments will be performed in triplicates to ensure accurate quantification and identification of proteins. SILAC-based analysis of the tumours will be performed with peptides fractionated using filter-aided sample preparation and strong anion exchange chromatography. High-resolution mass spectrometry (MS) will be performed and data will be analysed for differential abundance of proteins common to the three HN/CR tumour pairs. Candidate proteins will be selected for validation. Western blot and qRT-PCR, will test the abundance of candidate proteins in the CR cell lines. Knockdown of the candidate protein(s) in CR cell lines and orthografting will test reversibility of the CR phenotype. Immuno-staining of human tissue microarrays will attempt to confirm the protein(s) in clinical samples. Contingency plan: If cells cannot be labelled by SILAC, a label-free approach may be used requiring quadruplicate orthograft models as quantification is less accurate than with SILAC. Scientific Opportunities: Proteomic analysis of CRPC will generate large quantities of data, which may aid other research groups. Medical Opportunities: Identification of commonly expressed important proteins in CRPC may provide ideal therapeutic drug targets.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS
Title: Making cone beam CT imaging fit for aggressive targeting & adaptive re-planning of photon and proton radiotherapy
Funder: Medical Research Council

Abstract: In-treatment cone-beam CT (CBCT) imaging is widely used in radiotherapy (RT) to provide 3D anatomical data for patient positioning and tumour targeting. However, because of X-ray scatter, non-ideal detectors and large regions of interest, CBCT cannot be used for essential re-calculation of delivered X-ray or proton dose when anatomical changes are observed following pre-treatment planning and during daily delivery. The proposers have previously developed and patented a software post-processing method to restore accurate tissue attenuation values in CBCT images by generation of an adaptive filter based on prior imaging. The method has proved successful for pelvic and head and neck images, which show marked improvements in visual image quality and numerical integrity for photon dose calculation. However this has not yet been achieved for thorax images, where it is particularly important to correctly account for differences in tissue attenuation due to the presence of highly variable tissue densities and of course physiological motion, primarily respiratory effects. First we will consolidate these advances, specifically by developing streamlined software tools that make the method suitable for implementation in the clinical environment and subsequent commercialization. Then we will perform Monte Carlo modelling of patient and imaging processes to advance our methodology, particularly for challenging lung imaging. Finally, we will pilot the use of corrected in-room images to address the pressing but unmet need for in-treatment proton therapy monitoring and re-planning. The resulting tools for correction of CBCT images will significantly reduce the time required to investigate the clinical impact of anatomical changes observed with CBCT (currently consuming a large amount of RT physics staff resource), and will pave the way for CBCT based adaptive photon and proton RT, where treatments are individually optimized mid-course based on imaging acquired on-treatment.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS
Title: Modulating human alloresponses with lenalidomide to improve the outcome of allogeneic haematopoietic stem cell transplantation
Funder: Medical Research Council

Abstract: A major toxicity after allogeneic haematopoieteic stem cell transplant (AHSCT) is Graft-versus-Host-Disease (GvHD), mediated by alloreactive donor T cells that damage healthy recipient tissues. Donor T cells also kill malignant cells via the Graft-versus-Tumour (GvT) effect. Treatment failure after AHSCT results from either too little alloreactivity (no GvT, disease relapse) or too much (severe GvHD). Lenalidomide is a new drug with immunostimulatory capacity and therefore potential to strengthen GvT effects after AHSCT. However, early clinical experience of lenalidomide maintenence after AHSCT has resulted in increased GvHD limiting this approach. Little is known about how lenalidomide affects human alloreactivity and a better mechanistic understanding is needed for the drug to be used more effectively post-transplant. We have already shown in the laboratory that lenalidomide selectively increases proliferation of alloreactive CD8+ T cells, and that these cells have a unique gene expression pattern. Thus we have already identified the donor alloreactive cell subset potentiated by lenalidomide and several candidate cellular pathways likely to mediate this effect. This project now seeks to build on these important findings with a series of labaratory experiments aimed at identifying the best way to use lenalidomide to maximize GvT without causing excess GvHD. Using T cells from both adult volunteer donors and umbilical cord blood we will phenotypically and functionally characterize the potentiated alloreactive CD8+ cells in HLA-mismatched and -matched settings. We will use models of different ways to give lenalidomide (e.g before, during or after the transplant) Finally we will measure the effect of lenalidomide on several immune parameters that can potentiate GvT but not GvHD (tissue-restricted minor antigen-specific responses, alloreactive NK reposnses). These studies have direct clinical and translational potential to reduce treatment failure after AHSCT


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.2 CELLULAR AND GENE THERAPIES
Health categories: CANCER AND NEOPLASMS